2018

USP 41 | THE UNITED STATES PHARMACOPEIA

N F 36 THE NATIONAL FORMULARY

Volume 3 By authority of the United States Pharmacopeial Convention

Prepared by the Council of Experts and its Expert Committees

Official from May 1, 2018

The designation on the cover of this publication, “USP NF 2018,” is for ease of
identification only. The publication contains two separate compendia: The United
oars Pharmacopeia, Forty-First Revision, and The National Formulary, Thirty-Sixth
Edition.

THE UNITED STATES PHARMACOPEIAL CONVENTION

12601 Twinbrook Parkway, Rockville, MD 20852
 SIX-MONTH IMPLEMENTATION GUIDELINE

The United States Pharmacopeia—National Formulary and its supplements become official six months after being released to
the public. The USP-NF, whic is released on November 1 of each year, becomes official on May 1 of the following year. This
six-month implementation timing gives users more time to bring their methods and proceduresinto compliance with new
and revised USP-NF requirements.
The table below describes the official dates of the USP-NF and its supplements. The 2017 USP 40-NF 35, and its supple-
ments, Interim Revision Announcements (IRAs) and Revision Bulletins to that edition, will be official until May 1, 2018, at which
time the USP 41—-NF 36 becomes official.

Publication Release Date Official Date Official Until

USP 41-NF 36 November 1, 2017 May 1, 2018 May 1, 2019 (except as superseded by supplements, /RAs, and
Revision Bulletins)
First Supplement to the February 1, 2018 August 1, 2018 May 1, 2019 (except as superseded by Second Supplement, IRAs,
USP 41-NF 36 and Revision Bulletins)
Second Supplement to the June 1, 2018 December 1, 2018 May 1, 2019 (except as superseded by /RAs and Revision Bulletins)
USP 41-NF 36
USP 42-NF 37 November 1, 2018 May 1, 2019 May 1, 2020 (except as superseded by supplements, /RAs, and
Revision Bulletins)

The table below gives the details of the /RAs that will apply to USP 41—-NF 36,

IRA. PF Posting Date Comment Due Date IRA Posting Date IRA Official Date
4401) January 2, 2018 March 31, 2018 May 25, 2018 july 1, 2018
44(2) March 1, 2018 May 31, 2018 July 27, 2018 September 1, 2018
44(3) May 1, 2018 july 31, 2018 September 28, 2018 November 1, 2018
44(4) July 2, 2018 September 30, 2018 November 23, 2018 January
1, 2019
44(5) September
4, 2018 November 30, 2018 January 25, 2019 March 1, 2019
44(6) November 1, 2018 January 31, 2019 March 29, 2019 May 1, 2019

Revision Bulletins published on the USP website become official on the date specified in the Revision Bulletin.

NOTICE AND WARNING

Concerning U.S. Patent or Trademark Rights—The inclusion in The United States Pharmacopeia or in the National Formulary of a
monograph on any drug in respect to which patent or trademark rights may exist shall not be deemed, and is not intended
as, a grant of, or authority to exercise, any right or privilege protected by such patent or trademark. All such rights and
privileges are vested in the patent or trademark owner, and no other person may exercise the same without express
permission, authority, or license secured from such patent or trademark owner.
Concerning Use of USP or NF Text—Attention is called to the fact that USP and NF text is fully copyrighted. Authors and
others wishing to use portions of the text should request permission to do so from the Secretary of the USPC Board of
Trustees.

Copyright © 2017 The United States Pharmacopeial Convention 12601 Twinbrook Parkway, Rockville, MD 20852

All rights reserved.

ISSN: 0195-7996
ISBN: 978-1-936424-70-2

Printed in the United States by United Book Press, Inc., Baltimore, MD

USP 41-NF 36 Contents iii

Contents

VOLUME 1 Guide to General Chapters .......... 13

USP 41
Mission Statement and Preface...... vii

People 2015-2020 Revision Cycle ..... xi Monographs

Officers «ss iss 4 104 saweweememevee
senateae xi Official Monographs for USP 41, A-l......... 19
Board (Of Trustees«a's ow sxscmssnsienmonsattey
MEWw9 xi
COUNEII OF EXPerts:.. 2. ncinaincomveaycraintls
sara,we xi Index
FADGIE COMINITEES (5oe ccmerernar
ee64 mmaS xii Combined Index to USP 41 and NF 36....... I-1
In Memoriam 1... 0.0... cece
eee xviii

Members of the United States

Pharmacopeial Convention, VOLUME 2
as of May 31, 2017................ xix
2016 Recognition of Monograph
and Reference Material Donors ... xxvi Notices
General Notices and Requirements ........... ix
Articles of Incorporation ........... xxviii
USP Governance ................... xxix Guide to General Chapters .......... xix
Bylaws 2.6... eee ees xxix
Rules and Procedures .............-.000- xxix
Monographs
USP Policies: «2s 44.4 2 2 ¢%'s B92 paaethtandllinals Xxix
Official Monographs for USP 41, J-Z....... 2303

Admissions .....................055 Xxxiii

Index
Articles Admitted to USP 47 by
SUDPIEMIENE, cnn cee ene ee ee ee a wo ieeneiassenns xxxiii Combined Index to USP 47 and NF 36....... I-1
New Articles Appearing in USP 47 That Were Not

VOLUME 3
Included in USP 40 Including
Supplements 0 cee svcevssa3 54seseRey XXxiv
Articles Included in USP 40 But Not
Included in USP 47 2.0... cece eee nee XXxiV
Annotated List. sci css eeecssaeaassssawe XXXVi Notices
General Notices and Requirements ........... ix
Notices
General Notices and Requirements ........... 3 Guide to General Chapters .......... xix
iv Contents
Global Health
Official Monographs ................00. 4415
Dietary Supplements
Official Monographs’ s iu. ss eis cease enemys 4417
NF 36
Admissions
Articles Admitted to NF 36 by Supplement .. 5167
New Articles Appearing in NF 36 That Were Not
Included in NF 35 Including
Supplements ........ 000-00. e eee 5167
New Articles Appearing in NF 36 ......... 5167
Annotated List o oo.¢ ean ws ane a svicacwmesnnaes 5168
Excipients
USP and NF Excipients, Listed by
CAMQOOIY «seu eeu s e485 8s semeewRwee
¢ 5169
Monographs
Official Monographs for NF 36 ........... 5179
Index
Combined Index to USP 41 and NF 36....... I-1
VOLUME 4
Notices
General Notices and Requirements ........... ix
Guide to General Chapters .......... xix
Reagents, Indicators, and
Solutions ....................00-
Reagent Specifications...............005 5664
Indicators and Indicator Test Papers ....... 5745
USP 41-NF 36
SOWWUONS: ss sweemeeng
ekeRea Lbabasa4ae 5748
Butler SolWHONS vcvwowwew erences es auas 5748
Colorimetric Solutions ..............-. 5749
Test SOlUHONS acccweee
ye eeeegeeryusaas 5750
Volumetric Solutions ..............005 5761
Chromatographic Columns.............. 5774
Reference Tables
Containers for Dispensing Capsules and
Tablets... eee eee eee 5781
Description and Relative Solubility of USP
and NF AMIGIES! siscncay
¢ecoygoaaaERs 5791
Approximate Solubilities of USP and
NF Articles... 0... cece 5851
Atomic Weights cuqwaessee
deebesescde 5859
Half-Lives of Selected Radionuclides ....... 5860
Alcoholometric Table...............0005 5861
Intrinsic Viscosity Table ..............04. 5863
General Chapters
See page xix for detailed contents
General Tests and Assays..............45 5915
General Requirements for Tests and Assays .. 5915
Apparatus for Tests and Assays ........... 5954
Microbiological Tests « so coi.
sewssGusssens 5959
Biological Tests and Assays .............. 5991
Chemical Tests and Assays ............-. 6094
Physical Tests and Determinations......... 6327
Index
Combined Index to USP 41 and NF 36....... 1-1
VOLUME 5
Notices
General Notices and Requirements ........... ix
USP 41-NF 36 Contents v

7
am]

Guide to General Chapters .......... xix Dietary Supplements.............-.0005 8153 $

General Chapters Index

=2
See page 63 for detailed contents Combined Index to USP 47 and NF 36....... 1 a

General Information ................000. 6699

USP 41 General Notices vii

General Notices and

Requirements

NM SELICID)
cepIbte
Applying to Standards, Tests,
Assays, and Other Specifications
of the United States Pharmacopeia

1. Title and Revision....................... ix 6.50. Preparation of Solutions ................8. xiv

£200, Units Necessary to Complete a Test ......... xiv
2. Reco
Official Status and Legal 680: Equipment 2222220 xiv
70. WUSince ne «uae oo en MemREEONECA
6a TF i

ne AGS TB GAG + eeanene 2 « comme # © omnes ww ix

2.10. Official Text... 22.2... eee eee ix
2.20. Official Articles . 2... 0... ccc eee eee eee ix 7 Ae etanee Requirements comeueaa sees OF
2.30. Legal RECOQNIBION) w.00 06 ee a ee warned
aao ix 7:20. Rounding Rules........---.
002c lll, sa

3. Conformance to Standards............. ix —
3.10. ppplcgeileg Of Standards « «2 «x aexcowveraig
46%& ix 8. ea nie eee Det ineithons iE 2 SUH Ao beled 2 nv
3.20. Indicating Conformance..............00005 x 820. About ........
8.30. Alcohol Content
4. Monographs and General Chapters .... xi 8.40. Atomic Weights
4.10. Monographs ...... 2.0.0... ceeeee eee xi 8.50. Blank Determinations ..............00005 xv
4.20. General Chapters... 0... 0.0.0.0
eeeeeeee xi 8.60, Concomitantly «sss en 22 eeoemmey
nes902es xvi
8.70. Desiccator ... i
5. Monograph Components ............... ect aeeit :
5.10. Molecular Formula... ........ 0200000000. 8.100 Nel “ol aa
5.20. Added Substances ...........
000.0eseas 3.110. NUM
5.30. Description and Solubility. ..............4. 3.120. Odor -
S-A0. Identificationinns. «.35 6 i naa osepeene
9 6oe 8.130. Percent...
BBO. ASSAY, woronenean 342313,8 §Hees 8.140.Pe “ hE ge Concenttations...........20
00. ;
5.60. Impurities and Foreign Substances 3.150.Pressure CNCCNMSUCNS etree
s EEGRTEEY “v
5.70. Performance Tests .. 0... 0... eee eee eee iii 3.160.Re ton Tine ccc tee a
5.80. USP Reference Standards ..............00. iii rag Specific
Reece rcp
8.170. Gravity... © FH 0...
EN sian ae aa ene ey aM
ee eee xvi
8.180. Temperatures 1... eee eee xvi
6. Testing Practices and Procedures ..... xiii 8.190. Time... i
6.10. Safe Laboratory Practices .........-...000.xiii 8.200. Transfer. . .
6.20. Automated Procedures. ..........-..0.00.xiii 8.210. Vacuum
6.30. Alternative and Harmonized Methods and 8.220. Vacuum Desiccator ..............-00005 xvi
Procedures:s.< <3 24274 cuswmeeees bedaeesxiii 8.230. Water 2... ee eee xvi
6.40. Dried, Anhydrous, Ignited, or Solvent- 8.240. Weights and Measures ............0.0005 xvi
Free Basis... 2... ee eee
eee xiii
 viii General Notices USP 41

9. Prescribing and Dispensing............ xvi 10. Preservation, Packaging, Storage,

9.10 Use of Metric Units... 2. eeeeeu ea eee anes xvii and Labeling ........................
9.20 Changes in Volume.... 0.2.06. ceceeeeeexviii 10.10, Packaging and Storage a .
10.20. Labeling»... 2... 2. eee ee eee eee

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USP 41
GENERAL NOTICES AND
REQUIREMENTS
The General Notices and Requirements section (the General
Notices) presents the basic assumptions, definitions, and de-
fault conditions for the interpretation and application of the
United States Pharmacopeia(USP) and the National Formulary
(NF).
Requirements stated in these General Notices apply to all
articles recognized in the USP and NF (the “compendia”)
and to all general chapters unless specifically stated
otherwise.
1. TITLE AND REVISION
The full title of this publication (consisting of five volumes
and including its Supplements), is The Pharmacopeia of the
United States of America, Forty-First Revision and the Na-
tional Formulary, Thirty-Sixth Edition. These titles may be ab-
breviated to USP 41, to NF 36, and to USP 41-NF 36. The
United States Pharmacopeia, Forty-First Revision, and the Na-
tional Formulary, Thirty-Sixth Edition, supersede all earlier re-
visions. Where the terms “USP,” “NF,” or “USP—NF’ are used
without further qualification during the period in which
these compendia are official, they refer only to USP 41, NF
36, and any Supplement(s) thereto. The same titles, with no
further distinction, apply equally to print or electronic pres-
entation of these contents. Although USP and NF are pub-
lished under one cover and share these General Notices, they
are separate compendia.
This revision is official beginning May 1, 2018 unless oth-
erwise indicated in specific text.
Supplements to USP and NF are published periodically.
Accelerated Revisions, published periodically on the Offi-
cial Text section of USP’s website (http://www.usp.org/usp-
nf/official-text), are designed to make revisions official more
quickly than through the routine process for publishing
standards in the USP-NF. Interim Revision Announcements are
Accelerated Revisions to USP and NF that contain official re-
visions and their effective dates.
Revision Bulletins are Accelerated Revisions to official text
or postponements that require expedited publication. They
generally are official immediately unless otherwise specified
in the Revision Bulletin.
Errata are Accelerated Revisions representing corrections
to items erroneously published. Announcements of the avail-
ability of new USP Reference Standards and announcements
of tests or procedures that are held in abeyance pending
availability of required USP Reference Standards are also
available on the “Official Text” tab of USP’s website.
2. OFFICIAL STATUS AND LEGAL RECOGNITION
2.10. Official Text
Official text of the USP and NF is published in the USP-NF
Online (www.uspnf.com) in the edition identified as “CUR-
RENTLY OFFICIAL” and in Accelerated Revisions that super-
sede the USP-NF Online as described below.
Routine revisions are published in the USP-NF Online and
become official on the date indicated, usually six months
after publication. Accelerated Revisions supersede the
USP-NF Online and become official on the date indicated.
Links to Accelerated Revisions on the USP website can be
found in any superseded monograph or general chapter in
the USP-NF Online.
Print and USB flash drive versions of the USP and NF also
are available. Routine revisions are provided with the same
timing as the USP-NF Online. Official text published in Sup-
plements supersedes that in the previously published print or
General Notices ix
NM SEIU ID)
USB flash drive versions of USP-NF. These versions also are
superseded by Accelerated Revisions as described above.
In the event of anyclisparity between the print or USB
flash drive versions and the USP-NF Online, the USP-NF On-
line will be deemed to apply.
PEPE
2.20. Official Articles
An official article is an article that is recognized in USP or
NF. An article is deemed to be recognized and included in a
compendium when a monograph for the article is published
in the compendium and an official date is generally or spe-
cifically assigned to the monograph.
The title specified in a monograph is the official title for
such article. Other names considered to be synonyms of the
official titles may not be used as substitutes for official titles.
Official articles include both official substances and official
products. An official substance is a drug substance, excipient,
dietary ingredient, other ingredient, or component ofa fin-
ished device for which the monograph title includes no indi-
cation of the nature of the finished form.
An official product is a drug product, dietary supplement,
compounded preparation, or finished device for which a
monograph is provided.
2.30. Legal Recognition
The USP and NF are recognized in the laws and regula-
tions of many countries throughout the world. Regulatory
authorities may enforce the standards presented in the USP
and NF, but because recognition of the USP and NF may
vary by country, users should understand applicable laws
and regulations. In the United States under the Federal
Food, DruG), and Cosmetic Act (FDCA), both USP and NF are
recognized as official compendia. A drug with a name rec-
ognized in USP-NF must comply with compendial identit
standards or be deemed adulterated, misbranded, or both.
See, eg FDCA § 501(b) and 502(e)(3)(b); also FDA regula-
tions, 21 CFR § 299.5(a&b). To avoid being deemed
adulterated, such drugs must also comply with compendial
standards for strength, quality, and purity, unless labeled to
show all respects in which the drug differs. See, e.g., FDCA
§ 501(b) and 21 CFR § 299.5(c). In addition, to avoid being
deemed misbranded, drugs recognized in USP-NF must also
be packaged and labeled in compliance with compendial
standards. See FDCA § 502(g).
A dietary supplement represented as conforming to speci-
fications in USP will be deemed a misbranded food if it fails
to so conform. See FDCA § 403(s)(2)(D).
Enforcement of USP standards is the responsibility of FDA
and other government authorities in the U.S. and elsewhere.
USP has no role in enforcement.
Change to read:
3. CONFORMANCE TO STANDARDS
3.10. Applicability of Standards
Standards for an article recognized in the compendia
(USP-NF) are expressed in the article’s monograph, applica-
ble general chapters, and General Notices. The identity,
strength, quality, and purity of an article are determined by
the official tests, procedures, and acceptance criteria, and
other requirements incorporated in the monograph, in ap-
plicable general chapters, or in the General Notices. “Appli-
cable general chapters” means general chapters numbered
 x General Notices
below 1000 or above 2000 that are made applicable to an
article through reference in General Notices, a monograph,
or another applicable general chapter numbered below
1000. Where the requirements of a monograph differ from
the requirements specified in these General Notices or an
applicable general chapter, the monograph requirements
apply and supersede the requirements of the General Notices
or applicable general chapters, whether or not the mono-
graph explicitly states the difference.
General chapters numbered 1000 to 1999 are for infor-
mational purposes only. They contain no mandatory tests,
ww assays, or other requirements applicable to any official arti-
Ro=]
C7
cle, regardless of citation in a general chapter numbered
below 1000, a monograph, or these General Notices. Gen-
°
ra eral chapters numbered above 2000 apply only to articles
that are intended for use as dietary ingredients and dietary
3-
supplements. General chapter citations in NF monographs
v refer to USP general chapters.
= Early adoption of revised standards in advance of the offi-
Vv
fe) cial date is allowed by USP unless specified otherwise at the
time of publication. Where revised standards for anexisting
article have been published as final approved “official text”
(as approved in section 2.10 Official Text) but have not yet
reached the official date (six months after publication, un-
less otherwise specified; see “official date”, section 2.20. Of-
ficial Articles), compliance with the revised standard shall not
preclude a finding or indication of conformance with com-
pendial standards, unless USP specifies otherwise by prohib-
iting early adoption in a particular standard.
The standards in the relevant monograph, general chap-
ter(s), and General Notices apply at all times in the life of the
article from production to expiration. It is also noted that
the manufacturer’s specifications, and manufacturing prac-
tices (e.g., Quality by Design, Process Analytical Technology,
and Real Time Release Testing initiatives), generally are fol-
lowed to ensure that the article will comply with com-
pendial standards until its expiration date, when stored as
directed. Every compendial article in commerce shall be so
constituted that when examined in accordance with these
assays and test procedures, it meets all applicable pharma-
copeial requirements (General Notices, monographs, and
general chapters). Thus, any official article is expected to
meet the compendial standards if tested, and any official
article actually tested as directed in the relevant monograph
must meet such standards to demonstrate compliance.
Some tests, such as those for Dissolution and Uniformity of
Dosage Units, require multiple dosage units in conjunction
with a decision scheme. These tests, albeit using a number
of dosage units, are in fact one determination. These proce-
dures should not be confused with statistical sampling
plans. The similarity to statistical procedures may seem to
suggest an intent to make inference to some larger group of
units, but in all cases, statements about whether the com-
pendial standard is met apply only to the units tested. Re-
peats, replicates, statistical rejection of outliers, or extrapola-
tions of results to larger populations, as well as the necessity
and appropriate requency of batch testing, are neither
specified nor proscribed by the compendia; such decisions
are based on the objectives of the testing. Frequency of
testing and sampling are left to the preferences or direction
of those performing compliance testing, and other users of
USP-NF, including manufacturers, buyers, or regulatory
authorities.
Official products are prepared according to recognized
prne/ples of good manufacturing practice and from ingredi-
ents that meet USP or NF standards, where standards for
such ingredients exist (for dietary supplements, see section
3.10.20 Applicability of Standards to Medical Devices, Dietary
Supplements, and Their Components and Ingredients).
Official substances a pepaed according to recognized
principles of good manufacturing practice and from ingredi-
ents complying with specifications designed to ensure that
the resultant substances meet the requirements of the com-
pendial monographs.
USP 41
3.10.10. Applicability of Standards to Drug Products,
Drug Substances, and Excipients
The applicable USP or NF standard applies to any article
marketed in the United States that (1) is recognized in the
compendium and (2) is intended or labeled for use as a
drug or as an ingredient in a drug. Such articles (drug prod-
ucts, drug substances, and excipients) include both human
drugs (whether dispensed by prescription, “over the
counter,” or otherwise), as well as animal drugs. The appli-
cable standard applies to such articles whether or not the
added designation “USP” or “NF” is used. The standards
apply equally to articles bearing the official titles or names
derived by transposition of the definitive words of official
titles or transposition in the order of the names of two or
more 4drug substancesausrs; in official titles, or where there
is use of synonyms with the intent or effect of suggesting a
significant degree of identity with the official title or name.
3.10.20. Applicability of Standards to Medical Devices,
Dietary Supplements, and Their Components and
Ingredients
An article recognized in USP or NF shall comply with the
compendial standards if the article is a medical devter com-
ponent intended for a medical device, dietary supplement,
dietary ingredient, or other ingredient that is intended for
incorporation into a dietary supplement, and is labeled as
conforming to the USP or NF.
Generally, dietary supplements are prepared from ingredi-
ents that meet USP, NF, or Food Chemicals Codex standards.
Where such standards do not exist, substances may be used
in dietary supplements if they have been shown to be of
acceptable food grade quality using other suitable
procedures.
3.10.30. Applicability of Standards to the Practice of
Compounding (New)
USP compounding practice standards, Pharmaceutical
Compounding—Nonsterile Preparations (795) and Pharmaceu-
tical Compounding—Sterile Preparations (797), as appropriate,
apply to compounding practice or activity regardless of
whether a monograph exists for the compounded prepara-
tion or these chapters are referenced in such a monograph.
In the United States, (795) and (797) are not applicable to
drugs compounded by entities registered with FDA as out-
sourcing facilities as defined ty FDCA § 503B, because such
facilities are required to comply with FDA’s current good
manufacturing practice requirements. pent te prepa-
rations, including drug products compounded by outsourc-
ing facilities, may also be subject to applicable monographs;
see section 2.20 Official Articles and section 4.10
Monographs.
3.20. Indicating Conformance
A drug product, drug substance, or excipient may use the
designation “USP” or “NF” in conjunction with its official
title or elsewhere on the label only when (1) a monograph
is provided in the specified compendium and (2) the article
complies with the identity prescribed in the specified
compendium.
When a drug product, drug substance, compounded
preparation, or excipient differs from the relevant USP or NF
standard of strength, quality, or purity, as determined by
the application of the tests, procedures, and acceptance cri-
teria set forth in the relevant compendium, its difference
shall be plainly stated on its label.
When a drug product, drug substance, compounded
preparation, or excipient fails to comply with the identity
prescribed in USP or NF or contains an added substance that
interferes with the prescribed tests and procedures, the arti-
cle shall be designated by a name that is clearly distinguish-
ing and differentiating from any name recognized in USP or
NF.
A medical device, dietary supplement, or ingredient or
component of a medicaldevice or dietary supplement may
use the designation “USP” or “NF” in conjunction with its
official title or elsewhere on the label only when (1) a mon-
ograph is provided in the specified compendium and (2)
USP 41
the article complies with the monograph standards and
other applicable standards in that compertut
The designation “USP” or “NF” on the label may not and
does not constitute an endorsement by USP and does not
represent assurance by USP that the article is known to
comply with the relevant standards. USP may seek legal re-
dress if an article purports to be or is represented as an
official article in one of USP’s compendia and such claim is
determined by USP not to be made in good faith.
The designation “USP-NF” may be used on the label of
an article provided that the label also bears a statement
such as “Meets NF standards as published by USP,” indicat-
ing the particular compendium to which the article purports
to apply.
When the letters “USP,” “NF,” or “USP—NF” are used on
the label of an article to indicate compliance with com-
pendial standards, the letters shall appeal in conjunction
with the official title of the article. The letters are not to be
enclosed in any symbol such as a circle, square, etc., and
shall appear in capital letters.
If a dietary supplement does not comply with all applica-
ble compendial requirements but contains one or more die-
tary ingredients or other ingredients that are recognized in
USP or NF, the individual ingredient(s) may be designated as
complying with USP or NF standards or being of USP or NF
quality provided that the designation is limited to the indi-
vidual ingredient(s) and does not suggest that the dietary
supplement complies with USP standards.
4, MONOGRAPHS AND GENERAL CHAPTERS
4.10. Monographs
Monographs set forth the article’s name, definition, speci-
fication, and other requirements related to packaging, stor-
age, and labeling. The specification consists of tests, proce-
dures, and acceptance criteria that help ensure the identity,
strength, quality, and purity of the article. For general re-
quirements relating to specific monograph sections, see sec-
tion 5 Monograph Components.
Because monographs may not provide standards for all
relevant characteristics, some official substances may con-
form to the USP or NF standard but differ with regard to
nonstandardized properties that are relevant to their use in
specific preparations. To assure substitutability in such in-
stances, users may wish to ascertain functional equivalence
or determine such characteristics before use.
4.10.10. Applicability of Test Procedures
A single monograph may include more than one test,
Bessa and/or acceptance criterion for the same attri-
ute. Unless otherwise specified in the monograph, all tests
are requirements. In some cases, monograph instructions al-
low the selection of tests that reflect attributes of different
manufacturers’ articles, such as different polymorphic forms,
impurities, hydrates, and dissolution. Monograph instruc-
tions indicate the tests, procedures, and/or acceptance crite-
ria to be used and the required labeling.
The order in which the tests are listed in the monograph
is based on the order in which they are approved by the
relevant Expert Committee for inclusion in the monograph.
Test 1 is not necessarily the test for the innovator or for the
reference product. Depending on monograph instructions, a
labeling statement is not typically required if Test 1 is used.
4.10.20. Acceptance Criteria
The acceptance criteria allow for analytical error, for una-
voidable variations in manufacturing and compounding, and
for deterioration to an extent considered acceptable under
practical conditions. The existence of compendial accep-
tance criteria does not constitute a basis for a claim that an
official substance that more nearly approaches 100% purity
“exceeds” compendial quality. Similarly, the fact that an ar-
ticle has been prepared to tighter criteria than those speci-
fied in the monograph does not constitute a basis for a
claim that the article “exceeds” the compendial
requirements.
An official product shall be formulated with the intent to
provide 100% of the quantity of each ingredient declared
General Notices xi
on the label. Where the minimum amount of a substance
present in a dietary supplement is required by law to be
igher than the lower acceptance criterion allowed for in
the monograph, the upper acceptance criterion contained
in the monograph may be increased by a corresponding
amount.
The acceptance criteria specified in individual monographs
and in the general chapters for compounded preparations
are based on such attributes of quality as might be ex-
tes to characterize an article compounded from suitable
ulk drug substances and ingredients, using the procedures
provided or recognized principles of good compounding
NM ACL LD)
practice, as described in these compendia.
4.20. General Chapters
Each general chapter is assigned a number that appears in
angle brackets adjacent to the chapter name (eg. Chroma-
tography (621)). General chapters may contain the
cepIPCe
following:
° Descriptions of tests and procedures for application
through individual monographs,
° Descriptions and specifications of conditions and prac-
tices for pharmaceutical compounding,
° General information for the interpretation of the com-
pendial requirements,
e Descriptions of general pharmaceutical storage, dispens-
ing, and packaging practices, or
° General guidance to manufacturers of official substances
or official products.
When a general chapter is referenced in a monograph,
acceptance criteria may be presented after a colon.
Some chapters may serve as introductory overviews of a
test or of analytical techniques. They may reference other
general chapters that contain techniques, details of the pro-
cedures, and, at times, acceptance criteria.
Change to read:
5. MONOGRAPH COMPONENTS
5.10. Molecular Formula
The use of the molecular formula for the 4official sub-
stance(s)auses) named in defining the required strength of a
compendial article is intended to designate the chemical en-
tity or entities, as given in the complete chemical name of
the article, having absolute (100%) purity.
5.20. Added Substances
Added substances are presumed to be unsuitable for in-
clusion in an official article and therefore prohibited, if their
presence impairs the bioavailability, therapeutic efficacy, or
safety of the official article; or they interfere with the assays
and tests prescribed for determining compliance with the
ae standards (see section 3.20 Indicating
Conformance).
The air in a container of an official article may, where
appron at be evacuated or be replaced by carbon diox-
ide, helium, argon, or nitrogen, or by a mixture of these
jases. The use of such gas need not be declared in the
jabeling.
5.20.10. Added Substances in Official Substances
Official substances may contain only the specific added
substances that are permitted by the individual monograph.
Such added substances shall not exceed the quantity re-
quired for providing their intended effect. Where such addi-
tion is permitted, the label shall indicate the name(s) and
amount(s) of any added substance(s).
5.20.20. Added Substances (Excipients and Ingredients)
in Official Products
Suitable substances and excipients such as antimicrobial
agents, pharmaceutical bases, carriers, coatings, flavors, pre-
servatives, stabilizers, and vehicles may be added to an offi-
cial product to enhance its stability, usefulness, or elegance,
or to facilitate its preparation, unless otherwise specified in
the individual monograph.
 xii General Notices
Added substances and excipients employed solely to im-
part color may be incorporated into official products other
than those intended for parenteral or ophthalmic use, in
accordance with the regulations pertaining to the use of
colors issued by the U.S. Food and Drug Administration
(FDA), provided such added substances or excipients are
otherwise appropriate in all respects. (See also Injections
and Implanted Drugs Products (1), Product Quality Tests Com-
mon to Parenteral Dosage Forms, Specific Tests, Vehicles and
added substances, Added substances.)
The proportions of the substances constituting the base in
”
v ointment and suppository products and preparations may
Be
y be varied to maintain a suitable consistency under different
climatic conditions, provided that the concentrations of
°
va Adrug substancesause;: are not varied and provided that the
bioavailability, therapeutic efficacy, and safety of the prepa-
rc
-
ration are not impaired.
o 5.20.20.1. In Compounded Preparations
= Compounded preparations for which a complete compo-
o
O sition is given shall contain only the ingredients named in
the formulas unless specifically exempted herein or in the
individual monoraph. Deviation from the specified
processes or methods of compounding, although not from
the ingredients or proportions thereof, may occur provided
that the finished preparation conforms to the relevant stan-
dards and to preparations produced by following the speci-
fied process.
Where a monograph for a compounded preparation calls
for an ingredient in an amount expressed on the dried ba-
sis, the ingredient need not be dried before use if due al-
lowance is made for the water or other volatile substances
present in the quantity taken.
Specially denatured alcohol formulas are available for use
in accordance with federal statutes and regulations of the
Internal Revenue Service. A suitableformula of specially de-
natured alcohol may be substituted for Alcohol in the manu-
facture of official preparations intended for internal or topi-
cal use, provided that the denaturant is volatile and does
not remain in the finished product. A preparation that is
intended for topical application to the skin may contain spe-
cially denatured alcohol, provided that the denaturant is ei-
ther a usual ingredient in the preparation or a permissible
added substance; in either case the denaturant shall be
identified on the label of the topical preparation. Where a
process is given in the individual monedtaRny any prepara-
tion compounded using denatured alcohol shall be identical
to that prepared by the monograph process.
5.20.20.2. In Dietary Supplements
Additional ingredients may be added to dietary supple-
ment products provided that the additional ingredients: (1)
comply with applicable regulatory requirements; and (2) do
not interfere with the assays and tests prescribed for deter-
mining compliance with compendial standards.
5.30. Description and Solubility
Only where a quantitative solubility test is given in a
monograph and is designated as such is it a test for purity.
A monograph may include information regarding the arti-
cle’s description. Information about an article’s “description
and solubility” also is provided in the reference table
Description and Relative Solubility of USP and NF Articles. The
reference table merely denotes the properties of articles that
compywith eee standards. The reference table is
inten echigpanely or those who use, prepare, and dispense
drugs and/or related articles. Although the information pro-
vided in monographs and the information in the reference
table may indirectly assist in the preliminary evaluation of an
article, it is not intended to serve as a standard or test for
purity.
The approximate solubility of a compendial substance is
indicated by one of the following descriptive terms:
USP 41
Parts of Solvent Required
Descriptive Term for 1 Part of Solute
Very soluble Less than 1
Freely soluble From 1 to 10
Soluble From 10 to 30
Sparingly soluble From 30 to 100
Slightly soluble From 100 to 1,000
Very slightly soluble From 1,000 to 10,000
Greater than or equal to
Practically insoluble, or Insoluble 10,000
5.40. 4ldentificationausrs;
A compendial test titled 4auses; [dentification is provided as
an aid in verifying the identity of articles as they are pur-
ported to be, e.g., those taken from labeled containers, and
to establish whether it is the article named in USP-NF. The
4,usrar Identification test for a particular article may consist of
one or more procedures. When a compendial 4ausra: Identifi-
cation Atestausra; is undertaken, all requirements of all speci-
fied procedures in the test must be met to satisfy the re-
quirements of the test. Failure of an article to meet all the
requirements of a prescribed 4auses; Identification test (i.e.,
failure to meet the requirements of all of the specified pro-
cedures that are components of that test) indicates that the
article is mislabeled and/or adulterated.
5.50. Assay
Assay tests for compounded preparations are not in-
tended for evaluating a tompavinded preparation before
dispensing, but instead are intended to serve as the official
test in the event of a question or dispute regarding the
preparation’s conformance to official standards.
5.50.10. Units of Potency (Biological)
For substances that cannot be completely characterized
by chemical or physical means or that need confirmation of
functionality or tertiary structure, it may be necessary to ex-
press quantities of biological activity in units of biological
potency, each defined by an authoritative, designated refer-
ence standard. In cases where international reference mater-
ials have been discontinued, international units of potency
may be defined in terms of molecular mass, such as in the
cases of vitamins A, D, and E.
Where available, World Health Organization (WHO) inter-
national biological standards define the International Units
(IU). USP monographs refer to the units assigned by USP
Reference Standardseither directly as International Units (IU)
or as “USP Units.” For some biological products, units of
potency are value assigned against a corresponding U.S.
Standard established by FDA, whether or not International
Units or USP Units have been defined (see Biologics (1041)).
Note that product-related labeling, e.g., on containers, need
not use the full phrase “USP [product name] Units” that
appears in many USP monograph labeling sections. The
term “USP Units” can be used on product labeling consis-
tent with USP compendial requirements, provided it is clear
from the context that the Apotencyausrs: is stated in terms
of USP [product name] Units. In such circumstances it
should be clear that “USP Units” and “USP [product name]
Units” share the same meaning.
5.60. Impurities and Foreign Substances
Tests for the presence of impurities and foreign substances
are provided to limit such substances to amounts that are
unobjectionable under conditions in which the article is cus-
tomarily employed (see also Impurities in Drug Substances
and Drug Products (1086)).
Nonmonograph tests and acceptance criteria suitable for
detecting and controlling impurities that may result from a
change in the processing methods or that may be intro-
duced from external sources should be employed in addi-
tion to the tests provided in the individual monograph,
where the presence of the impurity is inconsistent with ap-
plicable good manufacturing practices or good pharmaceu-
tical practices.
USP 41
5.60.10. Other Impurities in USP and NF Articles
If a USP or NF monograph includes an assay or organic
impurity test based on chromatography, other than a test
for residual solvents, and that monograph procedure does
not detect an impurity present in the substance, the amount
and identity of the impurity, where both are known, shall
be stated in the labeling (certificate of analysis) of the offi-
cial substance, under the heading Other Impurity(ies).
The presence of any unlabeled other impurity in an offi-
cial substance is a variance from the standard if the content
is 0.1% or greater. The sum of all Other Impurities combined
with the monograph-detected impurities may not exceed
2.0% (see Ordinary Impurities (466)), unless otherwise stated
in the monograph.
The following categories of drug substances are excluded
from Other Impurities requirements:
Fermentation products and semi-synthetics derived
therefrom,
Radiopharmaceuticals,
Biologics,
Biotechnology-derived products,
Peptides,
Herbals, and
Crude products of animal or plant origin.
Any substance known to be toxic shall not be listed under
Other Impurities.
5.60.20. Residual Solvents in USP and NF Articles
All USP and NF articles are subject to relevant control of
residual solvents, even when no test is specified in the indi-
vidual monograph. If solvents are used during production,
they must be of suitable quality. In addition, the toxicity
and residual level of each solvent shall be taken into consid-
eration, and the solvents limited according to the principles
defined and the requirements specified in Residual Solvents
(467), using the general methods presented therein or other
suitable methods.
5.60.30. Elemental Impurities in USP Drug Products and
Dietary Supplements
Agus) Elemental impurities Aauses in official drug prod-
ucts Aare controlledauses: according to the principles defined
and requirements specified in Elemental Impurities—Limits
(232). Aausra: Elemental contaminants ausps1 in official die-
tary supplements Aare controlledauses; according to the prin-
ciples defined and requirements specified in Elemental Con-
taminants in Dietary Supplements (2232). Sauspsi
5.70. Performance Tests
Where content uniformity determinations have been
made using the same analytical methodology specified in
the Assay, with appropriate allowances made for differences
in sample preparation, the average of all of the individual
content uniformity determinations may be used as the Assay
value.
5.80. USP Reference Standards
USP Reference Standards are authentic specimens that
have been approved as suitable for use as comparison stan-
dards in USP or NF tests and assays. (See USP Reference Stan-
dards (11).) Where USP or NF tests or assays call for the use
of a USP Reference Standard, only those results obtained
using the specified USP Reference Standard are conclusive.
Where a procedure calls for the use of a compendial article
rather than for a USP Reference Standard as a material stan-
dard of reference, a substance meeting all of the com-
pendial monograph requirements for that article shall be
used. If any new USP or NF standard requires the use of a
new USP Reference Standard that is not yet available, that
portion of the standard containing the requirement shall not
e official until the specified USP reference material is
available.
Unless a Reference Standard label bears a specific potency
or content, assume the Reference Standard is 100.0% pure
in the official application. Unless otherwise directed in the
procedure in the individual monograph or in a general
chapter, USP Reference Standards are to be used in accor-
General Notices xiii
dance with the instructions on the label of the Reference
Standard.
Change to read:
6. TESTING PRACTICES AND PROCEDURES
6.10. Safe Laboratory Practices
In performing compendial procedures, safe laboratory
practices shall be followed, including precautionary meas-
ures, protective equipment, and work practices consistent
with the chemicals and procedures used. Before undertaking 9)
any procedure described in the compendia, the analyst @
=)
should be aware of the hazards associated with the chemi- ®
cals and the techniques and means of protecting against =
them. These compendia are not designed to describe such f=
hazards or protective measures. ra
6.20. Automated Procedures )
m7
Automated and manual procedures employing the same a
basic chemistry are considered equivalent “provided the au- ©
a)
tomated system is properly qualified as being suitable to
execute the compendial manual method and the analytical
procedure is verified under the new equipment conditions.
AUSPAT
6.30. Alternative and Harmonized Methods and
Procedures
4An alternative method or procedure is defined as any
method or procedure other than the compendial method or
procedure for the article in question. The alternative method
or procedure must be fully validated (see Validation of Com-
pendial Procedures (1225)) and must produce comparable re-
sults to the compendia! method or procedure within allowa-
ble limits established on a case-by-case basis. Alternative
methods or procedures can be developed for any one of a
number of reasons not limited to simplification of sample
preparation, enhanced precision and accuracy, improved
(shortened) run time, or being better suited to automation
than the compendial method or procedure. uses; Only those
results obtained by the methods and procedures given in
the compendia are conclusive.
4For evaluation as a potential replacement or addition to
the standard, usps: alternative Amethods andauses: proce-
dures should be submitted to USP 4auspy: (see section 4.70.
Monographs).
Certain general chapters contain a statement that the text
in question is harmonized with the corresponding text of
the European Pharmacopoeia and/or the Japanese Pharmaco-
poeia and that these texts are interchangeable. Therefore, if
a substance or preparation is found to comply with a re-
quirement using an interchangeable method or procedure
from one of these pharmacopeias, it should comply with the
requirements of the USP-NF. Whena difference appears, or
in the event of dispute, only the result obtained by the
method and/or procedure given in the USP-NF is conclusive.
6.40. Dried, Anhydrous, Ignited, or Solvent-Free Basis
All calculations in the compendia assume an “as-is” basis
unless otherwise specified.
Test procedures may be performed on the undried or
unignited substance and the results calculated on the dried,
anhydrous, or ignited basis, provided a test for Loss on Dry-
ing, or Water Determination, or Loss on Ignition, respectively,
is given in the pansiee Where the presence of moisture
or other volatile material may interfere with the procedure,
previous drying of the substance is specified in the individ-
ual monograph and is obligatory.
The term “solvent-free” signifies that the calculation shall
be corrected for the presence of known solvents as deter-
mined using the methods described in (467) unless a test
for limit of organic solvents is provided in the monograph.
The term “previously dried” without qualification signifies
that the substance shall be dried as directed under Loss on
Drying (731) or Water Determination (921) (gravimetric
determination).
 xiv General Notices
Where drying in vacuum over a desiccant is directed, a
vacuum desiccator, a vacuum drying pistol, or other suitable
vacuum drying apparatus shall be used.
6.40.10. Ignite to Constant Weight
“Ignite to constant weight” means that ignition shall be
continued at 800 + 25°, unless otherwise indicated, until
two consecutive weighings, the second of which is taken
after an additional period appropriate to the nature and
quantity of the residue, do not differ by more than 0.50 mg
per g of substance taken.
”
6.40.20. Dried to Constant Weight
v “Dried to constant weight” means that drying shall be
ce continued until two consecutive weighings, the second of
=
° which is taken after an additional drying period appropriate
re to the nature and quantity of the residue, do not differ by
more than 0.50 mg per g of substance taken.
Ts7
o 6.50. Preparation of Solutions
i 6.50.10. Filtration
o Where a procedure gives direction to “filter” without fur-
Le) ther qualification, the liquid shall be passed through suitable
filter paper or equivalent device until the filtrate is clear.
Due to the possibility of filter effects, the initial volumes of a
filtrate may be discarded.
6.50.20. Solutions
Unless otherwise specified, all solutions shall be prepared
with Purified Water. Solutions for quantitative measures shall
be prepared using accurately weighed or accurately meas-
ured analytes (see section 8.20 About).
An expression such as “(1 in 10)” means that 1 part by
volume of a liquid shall be diluted with, or 1 part by weight
of a solid shall be dissolved in, a sufficient quantity of the
diluent or solvent to make the volume of the finished solu-
tion 10 parts by volume. 4For example, a 1 in 10 solution is
prey diluting 1 mL of a liquid or dissolving 1 g of a
solid in sufficient solvent to make 10 mL of the solution.
usps; AN expression such as “(20:5:2)” means that the re-
spective numbers of parts, by volume, of the designated
liquids shall be mixed, unless otherwise indicated.
6.50.20.1. Adjustments to Solutions
Whena specified concentration is called for in a proce-
dure, a solution of other normality or molarity may be used,
provided that allowance is made for the difference in con-
centration and that the change does not increase the error
of measurement.
Proportionately larger or smaller quantities than the speci-
fied weights and volumes of assay or test substances and
Reference Standards may be taken, provided the measure-
ment is made with at least equivalent accuracy.
Unless otherwise indicated, analyte concentrations shall be
prepared to within ten percent (10%) of the indicated
value. In the case in which a procedure is adapted to the
working range of an instrument, solution concentrations
may differ from the indicated value by more than ten per-
cent (10%), with appropriate changes in associated calcula-
tions. Any changes shall fall within the validated range of
the instrument.
When adjustment of pH is indicated with either an acid or
base and the concentration is not indicated, appropriate
concentrations of that acid or base may be used.
6.50.20.2. Test Solutions
Information on Test Solutions (TS) is provided in the Test
Solutions portion of the Reagents, Indicators, and Solutions
section of the USP-NF. Use of an alternative Test Solution or
a change in the Test Solution used may require validation.
6.50.20.3. Indicator Solutions
Where a procedure specifies the use of an indicator TS,
approximately 0.2 mL, or 3 drops, of the solution shall be
added unless otherwise directed.
6.60. Units Necessary to Complete a Test
Unless otherwise specified, a sufficient number of units to
ensure a suitable analytical result shall be taken.
USP 41
6.60.10. Tablets
Where the procedure of a Tablet monograph directs to
weigh and finely powder not fewer than a given number of
Tablets, a counted number of Tablets shall be weighed and
reduced to a powder. The portion of the powdered Tablets
taken shall be representative of the whole Tablets and shall,
in turn, be weighed accurately.
6.60.20. Capsules
Where the procedure of a Capsule mesegraph gives di-
rection to remove, as completely as possible, the contents of
not fewer than a given number of the Capsules, a counted
number of Capsules shall be carefully opened and the con-
tents quantitatively removed, combined, mixed, and
weighed accurately. The portion of mixed Capsules contents
taken shall be representative of the contents of the Capsules
and shall, in turn, be weighed accurately.
6.70. Reagents
The proper conduct of the compendial procedures and
the reliability of the results depend, in part, upon the quality
of the reagents used in the performance of the procedures.
Unless otherwise specified, reagents conforming to the spec-
ifications set forth in the current edition of Reagent Chemi-
cals published by the American Chemical Society (ACS) shall
be used. Where such ACS reagent specifications are not
available or where the required purity differs, compendial
specifications for reagents of acceptable quality are provided
(see the Reagents, Indicators, and Solutions section of the
USP-NF). Reagents not covered by any of these specifica-
tions should be of a grade suitable to the proper perfor-
mance of the method of assay or test involved.
Listing of these reagents, including the indicators and so-
lutions employed as reagents, in no way implies that they
have therapeutic utility; furthermore, any reference to USP
or NF in their labeling shall include also the term “reagent”
or “reagent grade.” USP may supply reagents if they other-
wise may not be generally commercially available.
6.80. Equipment
Unless otherwise specified, a specification for a definite
size or type of container or apparatus in a procedure is
given solely as a recommendation. Other dimensions or
types may be used if they are suitable for the intended use.
6.80.10. Apparatus for Measurement
Where volumetric flasks or other exact measuring, weigh-
ing, or sorting devices are specified, this or other equipment
of at least equivalent accuracy shall be employed.
6.80.10.1. Pipet/Pipette
Where a Pipe pipeie is specified, a suitable buret may be
substituted. Where a “to contain” pipet/pipette is specified,
a suitable volumetric flask may be substituted.
6.80.10.2. Light Protection
Where low-actinic or light-resistant containers are speci-
fied, either containers specially treated to protect contents
from light or clear containers that have been rendered
opaque by application of a suitable coating or wrapping
may be used.
6.80.20. Instrumental Apparatus
An instrument may be substituted for the specified instru-
ment if the substitute uses the same fundamental principles
of operation and is of equivalent or greater sensitivity and
accuracy. These characteristics shall be qualified as appropri-
ate. Where a particular brand or source of a material, instru-
ment, or piece of equipment, or the name and address of a
manufacturer or distributor, is mentioned (ordinarily in a
footnote), this identification is furnished solely for informa-
poral puipases as a matter of convenience, without implica-
tion of approval, endorsement, or certification.
6.80.20.1. Chromatographic Tubes and Columns
The term “diameter” refers to internal diameter (ID).
6.80.20.2. Tubing
The term “diameter” refers to outside diameter (OD).
USP 41 General Notices xv

6.80.20.3. Steam Bath 7.20. Rounding Rules

Where use of a steam bath is directed, use actively flow-
ing steam or another regulated heat source controlled at an
equivalent temperature.
6.80.20.4. Water Bath
A water bath requires vigorously boiling water unless oth-
erwise specified.
6.80.30. Temperature Reading Devices
Temperature reading devices suitable for pharmacopeial
tests conform to specifications that are traceable to a Na-
tional Institute of Standards and Technology (NIST) standard
or equivalent. Temperature reading devices may be of the
liquid-in-glass type or an analog or digital temperature indi-
cator type, such as a resistance temperature device, thermis-
tor, or thermocouple. Standardization of thermometers is
performed on an established testing frequency with a tem-
perature standard traceable to NIST. For example, refer to
the current issue of American Society of Testing and Materi-
als (ASTM) standards E1 for liquid-in-glass thermometers.
7. TEST RESULTS
7.10. Interpretation of Requirements
Analytical results observed in the laboratory (or calculated
from experimental measurements) are compared with stated
acceptance criteria to determine whether the article con-
forms to compendial requirements.
The reportable value, which often is a summary value for
several individual determinations, is compared with the ac-
ceptance criteria. The reportable value is the end result of a
completed measurement procedure, as documented.
Where acceptance criteria are expressed numerically
herein through specification of an upper and/or lower limit,
ermitted values include the specified values themselves,
ut no values outside the limit(s). Acceptance criteria are
considered significant to the last digit shown.
7.10.5. Nominal Concentrations in Equations
Where a “nominal concentration” is specified, calculate
the concentration based on the label claim. In assay proce-
dures, water correction is typically stated in the Definition
and on the label of the USP Reference Standard. For other
procedures, correction for assayed content, potency, or both
is made prior to using the concentration in the equation
provided in the monograph.
7.10.10. Equivalence Statements in Titrimetric
Procedures
The directions for titrimetric procedures conclude with a
statement of the weight of the analyte that is equivalent to
each mL of the standardized titrant. In such an equivalence
statement, the number of significant figures in the concen-
tration of the titrant should Be understood to correspond to
the number of significant figures in the weight of the
analyte. Corrections to calculations based on the blank de-
termination are to be made for all titrimetric assays where
appropriate (see Titrimetry (541)).
The observed or calculated values shall be rounded off to
the number of decimal places that is in agreement with the
limit expression. Numbers should not be rounded until the
final calculations for the reportable value have been com-
pleted. Intermediate calculations (e.g., slope for linearity)
may be rounded for reporting purposes, but the original
(not rounded) value should be used for any additional re-
quired calculations. Acceptance criteria are fixed numbers
and are not rounded.
When rounding is required, consider only one digit in the
decimal place to the right of the last place in the limit ex- a
pression. If this digit is smaller than 5, it is eliminated and i)
Fo)
the preceding digit is unchanged. If this digit is equal to or ®
=
greater than 5, it is eliminated and the preceding digit is
increased by 1. =
8. TERMS AND DEFINITIONS Zz
°
8.10. Abbreviations am
RS refers to a USP Reference Standard. a
J
© CS refers to a Colorimetric Solution. al
e TS refers to a Test Solution.
¢ VS refers to a Volumetric Solution that is standardized in
accordance with directions given in the individual mon-
ograph or in the Reagents, Indicators, and Solutions sec-
tion of USP-NF.
8.20. About
“About” indicates a quantity within 10%.
If the measurement is stated to be “accurately measured”
or “accurately weighed,” follow the statements in Volumetric
Apparatus (31) and Balances (41), respectively.
8.30. Alcohol Content
Percentages of alcohol, such as those under the heading
Alcohol Content, refer to percentage by volume of C,HsOH
at 15.56°. Where a formula, test, or assay calls for alcohol,
ethyl alcohol, or ethanol, the USPmonograph article Alcohol
shall be used. Where reference is made to “C2HsOH,” abso-
lute (100%) ethanol is intended. Where a procedure calls for
dehydrated alcohol, alcohol absolute, or anhydrous alcohol,
aa monograph article Dehydrated Alcohol shall be
used.
8.40. Atomic Weights
Atomic weights used in computing molecular weights and
the factors in the assays and elsewhere are those established
by the IUPAC Commission on Isotopic Abundances and
Atomic Weights.
8.50. Blank Determinations
Where it is directed that “any necessary correction” be
made by a blank determination, the determination shall be
conducted using the same quantities of the same reagents
treated in the same manner as the solution or mixture con-
taining the portion of the substance under assay or test, but
with the substance itself omitted.

Illustration of Rounding Numerical Values

for Comparison with Requirements
Compendial Requirement Unrounded Value Rounded Result Conforms
Assay limit 298.0% 97.96% 98.0% Yes
97.92% 97.9% No
97.95% 98.0% Yes
Assay limit <101.5% 101.55% 101.6% No
101.46% 101.5% Yes
101.45% 101.5% Yes
Limit test <0.02% 0.025% 0.03% No
0.015% 0.02% Yes
0.027% 0.03% No
Limit test <3 ppm 3.5 ppm 4 ppm No
3.4 ppm 3 ppm Yes
2.5 ppm 3 ppm Yes
 xvi General Notices USP 41

8.60. Concomitantly 8.210. Vacuum

“Concomitantly” denotes that the determinations or
measurements are to be performed in immediate
succession.
8.70. Desiccator
The instruction “in a desiccator” indicates use of a tightly
closed container of suitable size and design that maintains
an atmosphere of low moisture content by means of a suita-
ble desiccant such as anhydrous calcium chloride, magne-
sium perchlorate, phosphorus pentoxide, or silica gel. See
also section 8.220 Vacuum Desiccator.
General Notices
“Vacuum” denotes exposure to a pressure of less than
20 mm of mercury (2.67 kPas), unless otherwise indicated.
8.220. Vacuum Desiccator
“Vacuum desiccator” indicates a desiccator that maintains
a low-moisture atmosphere at a reduced pressure of not
more than 20 mm of mercury (2.67 kPas) or at the pressure
designated in the individual monograph.
8.230. Water
8.230.10. Water as an Ingredient in an Official Product
As an ingredient in an official product, water meets the

8.80. Logarithms requirements of the appropriate water monograph in USP or

Logarithms are to the base 10. NF.
8.90. Microbial Strain 8.230.20. Water in the Manufacture of Official
A microbial strain cited and identified by its American Substances
Type Culture Collection (ATCC) catalog number shall be When used in the manufacture of official substances,
used directly or, if subcultured, shall be used not more than water shall meet the requirements for drinking water as set
five passages removed from the original strain. forth in the U.S. Environmental Protection Agency National
8.100. Negligible Primary Drinking Water Regulations or in the drinking water
“Negligible” indicates a quantity not exceeding 0.50 mg. regulations of the European Union or of Japan, or in the
8.110. NLT/NMT World Health Organization’s Guidelines for Drinking Water
“NLT” means “not less than.” “NMT” means “not more Quality. Additional specifications may be required in
than.” monographs.
8.120. Odor 8.230.30. Water in a Compendial Procedure
“Odorless,” “practically odorless,” wu “a faint characteristic When water is called for in a compendial procedure, the
odor,” and variations thereof indicate evaluation of a suita- USP monograph article Purified Water shall be used unless
ble quantity of freshly opened material after exposure to the otherwise specified. Definitions for other types of water are
air for 15 minutes. An odor designation is descriptive only provided in Reagents, Indicators, and Solutions and in Water
and should not be regarded as a standard of purity for a for Pharmaceutical Purposes (1231).
particular lot of an article. 8.240. Weights and Measures
8.130. Percent In general, weights and measures are expressed in the
“Percent” used without qualification means: International System of Units (SI) as established and revised
¢ For mixtures of solids and semisolids, percent weight in by the Conférence générale des poids et mesures. For com-
weight; pendial purposes, the term “weight” is considered to be
e For solutions or suspensions of solids in liquids, percent synonymous with “mass.”
weight in volume; Molality is designated by the symbol m preceded by a
¢ For solutions of liquids in liquids, percent volume in number that represents the number of moles of the desig:
volume; nated solute contained in 1 kilogram of the designated
e For solutions of gases in liquids, percent weight in solvent.
volume. Molarity is designated by the symbol M preceded by a
For example, a 1 percent solution is prepared by dissolv- number that represents the number of moles of the desig-
ing 1g of a solid or semisolid, or 1 mL of a liquid, in suffi- nated solute contained in an amount of the designated sol-
cient solvent to make 100 mL of the solution. vent that is sufficient to prepare 1 liter of solution.
8.140. Percentage Concentrations Normality is designated by the symbol N preceded by a
Percentage concentrations are expressed as follows: number that represents the number of equivalents of the
e Percent Weight in Weight (w/w) is defined as the num- designated solute contained in an amount of the designated
ber of g of a solute in 100g of solution. solvent that is sufficient to prepare 1 liter of solution.
° Percent Weight in Volume (w/v) is defined as the number The symbol for degrees &) without a qualifying unit of
of g of a solute in 100 mL of solution. measure represents degrees Celsius.
© Percent Volume in Volume (v/v) is defined as the number Chart of Symbols and Prefixes commonly employed for S|
of mL of a solute in 100 mL of solution. metric units and other units:
8.150. Pressure
Pressure is determined by use of a suitable manometer or Units Symbol Notes
barometer calibrated in terms of the pressure exerted by a Length
column of mercury of the stated height. meter, m
8.160. Reaction Time centimeter cm
Reaction time is 5 minutes unless otherwise specified. millimeter mm.
8.170. Specific Gravity Previously referred to
Specific gravity is the weight of a substance in air at 25° micrometer um as a micron
divided by the weight of an equal volume of water at the Previously the symbol
same temperature. mu (for millimicron)
8.180. Temperatures nanometer am was used
Temperatures are expressed in centigrade (Celsius) de- Angstrom A Equal to 0.1 nm
grees, and all measurements are made at 25° unless other- Mass
wise indicated. Where moderate heat is specified, any tem-
kilogram kg
perature not higher than 45° (113° F) is indicated.
gram g
8.190. Time
Unless otherwise specified, rounding rules, as described in milligram mg
section 7.20 Rounding Rules, apply to any time specified.
8.200. Transfer
“Transfer” indicates a quantitative manipulation.
USP 41 General Notices xvii

Units Symbol Notes Units Symbol Notes

The symbol 1g is used pounds per
in the USP and NF to square
represent micrograms, inch psi
but micrograms may millimeter
be represented as of mercu-
“meg”for labeling ry mmHg Equal to 133.322 Pa
and prescribing pur- Electrical
poses. The term units
‘gamma, symbolized STinere A
by y, frequently is
used to represent mi- volt NV. (2)
crograms in biochemi- millivolt mV a
microgram Lg cal literature. hertz Hz. Unit of frequenc' iy
nanogram ng kilohertz kHz aa
picogram _pq megahertz MHz z
Also referred to as the electron °
unified atomic mass volt eV =
unit and is equal to kilo-elec- a
1/12 times the mass tron volt keV 9
of the free carbon 12 mega-elec-
dalton Da atom. tron‘volt MeV
kilodalton kDa Radiation
Time SI unit of activity for
second Ss becquerel Bq radionuclides
minute min kilobec-
hour h querel kBq
Volume megabec-
1 Lis equal to 1000 querel MBq
cm3 (cubic centime- gigabec-
liter L ters) querel GBq
deciliter dL Non-S! unit of activity
1 mL is equal to 1 cm3, curie Ci for radionuclides
sometimes referred to millicurie mCi
milliliter mL as cc microcurie uCi
microliter nL nanocurie nCi
Tempera- Other
une acceleration
Celsius C due to Used to express rate of
Amount of gravity g centrifugation
Substance revolutions
Historically referred to per min- Used to express rate of
as gram-molecular ute rpm centrifugation
weight or gram-atom-
mole mol ic weight
millimole mmol Selected SI Prefixes
micromole umol Name Symbol Factor
femtomole fmol giga G 109
Also referred ioas mega M 106
gram-equivalen' =
weight. It is used in Kilo. i 10»
the calculation of sub- deci a 105
stance concentration centi c 107
in units of normality. milli m 103
This unit is no longer micro Lu 10-6
preferred for use in nano n 10-9
; analytical chemistry or ico p 10-12
equivalent Eq metrology. Feviites f 10-5
milli equiv-
alent mea aaa : 9. PRESCRIBING AND DISPENSING
anor Poona
lution, related to
9.10. UseAT
of Metric Units m . «,
spbetance coricentras Prescriptions for compendialarticles shall be written to
osmole Oemel tion, state the quantity and/or strength desired in metric units
=o unless otherwise indicated in the individual monograph [see
failhiosmole mOsmol also section 5.50.70 Units of Potency (Biological) above]. If an
Pressure amount is prescribed by any other system of measurement,
pascal Pa only an amount that is the metric equivalent of the pre-
kilopascal kPa scribed amount shall be dispensed. Abbreviations for the
terms “Units” or “International Units” shall not be used for
 xviii General Notices USP 41

labeling or prescribing purposes. Apothecary unit designa- quirements (659), unless different requirements are provided
tions i labels and abElIng shall not be used. 7 in an individual monograph.
9.20. Changes in Volume 10.20. Labeling
In the dispensing of prescription medications, slight All articles in USP or NF are subject to the labeling re-
changes in volume owing to variations in room tempera- quirements specified in Labeling (7), unless different require-
tures may be disregarded. ments are provided in an individual monograph.
10, PRESERVATION, PACKAGING, STORAGE, AND
LABELING
10.10. Packaging and Storage
All articles in USP or NF are subject to the packaging and
al storage requirements specified in Packaging and Storage Re-
v
x
=o
re
3
~~
cv
i
v
e)
USP 41 Guide to General Chapters xix

Guide to General Chapters

(For complete alphabetical list of all general chapters in this Pharmacopeia, see under “General chapters” in the index.)

GENERAL TESTS AND ASSAYS (124) Erythropoietin Bioassays..............4. 6061

(126) Somatropin Bioidentity Tests ............ 6063
(127) Flow Cytometric Enumeration of CD34+
Cells: so 5k oo waitionrumeateels
UPD?MEUM,(Se 6065
General Requirements (129) Analytical Procedures for Recombinant
for Tests and Assays Therapeutic Monoclonal Antibodies ....... 6070
(130) Protein A Quality Attributes ............. 6076
(1) Eee and Implanted Drug Products (Parenter- 4151) Pyrogen: Test «0. .MGRhiug
yell.eile’ 6083
als)—Product Quality Tests .................. 5915 (161) Medical Devices—Bacterial Endotoxin and (9)
(2) Oral Drug Products—Product Quality Tests ... 5921 Pyrogen: Tests ecscems
a=Sie) N le NUiekabd 6085 oO
(3) Topical and Transdermal Drug Products— J
(162) Diphtheria Antitoxin Potency Testing for Human oO
Product, Otaliity, Tests cnc gnettcreatco tue}n ryesene 5926 Immune Globulins:s..0:0:63 49 FE OL PE AS 6088 ~
(4) Mucosal Drug Products—Product Quality (165) Prekallikrein Activator... .............00. 6070 =
TeSts! soci ois by bb Binkeahtaarae
aAen85 al yin 5933 (171) Vitamin By2 Activity Assay... 6...
ee 6091 a
(5) Inhalation and Nasal Drug Products—General a
oy
Information and Product Quality Tests 5938 To
C7) LADEUING, 5 os 5 ss dutsmeboomnsceety-duewe't
tabi aatanen ». 5945 Chemical Tests and Assays pa
oO
(11) USP Reference Standards .............004 5951 i
Identification Tests “

Apparatus for Tests and Assays
(17) Prescription Container Labeling ........... 5954
(31) Volumetric Apparatus
(41): Balances’; s zarciineint aesmusmewet}ecdclig tee ials 5958
Microbiological Tests
(51) Antimicrobial Effectiveness Testing ......... 5959
(55) Biological Indicators—Resistance Performance
Tests SO Oh Re RCLRODE alas of, 5962
(61) Microbiological Examination of Nonsterile
Products: Microbial Enumeration Tests ...... 5965
(62) Microbiological Examination of Nonsterile
Products: Tests for Specified Microorganisms. . 5971
(63) Mycoplasma Tests . . 5978
(71) Sterility: Tests; ss'nxkRawew
hsav8U4 ys 2eave 5984
Biological Tests and Assays
(81) Antibiotics—Microbial Assays .............
(85) Bacterial Endotoxins Test ........
(87) Biological Reactivity Tests, In Vitro .
(88) Biological Reactivity Tests, In Vivo
(89) Enzymes Used as Ancillary Materials in
Pharmaceutical Manufacturing
(89.1) Collagenase] ............
(89.2) Collagenase I]. . 1... eee
(90) Fetal Bovine Serum—Quiality Attributes and
Functionality: Testsicncisa «use 6 bo uu eseeriwn
(91) Calcium Pantothenate Assay...........005 6041
(92) Growth Factors and Cytokines Used in Cell
Therapy Manufacturing .............
(111) Design and Analysis of Biological Assays .
(115) Dexpanthenol Assay
(121) Insulin Assays 2.2...
eeeeeee ee eee
(121.1) Physicochemical Analytical Procedures
for lnsuliins: « - ceomeucesd
24s 5,5292pS 6056
(181) Identification—Organic Nitrogenous Bases . . 6094
(191) Identification Tests—General............. 6094
(193) Identification—Tetracyclines ........... 6100
255957 (197) Spectrophotometric Identification Tests ..... 6101
(201) Thin-Layer Chromatographic Identification
Testi a. s a5 3 neem as 22 ss ohare eS 6102
(202) Identification of Fixed Oils By Thin-Layer
Chromatography ...........0
00 eeeee 6103
(203) High-Performance Thin-Layer Chromatography
Procedure for Identification of Articles
OF Botanical OFIGIM seus isiven «aya % dno n, mpavanaieys 6105
Limit Tests
(206) AMIRI. « ceamseues
6sea8s8eeREG 6107
(207) Test for 1,6-Anhydro Derivative for Enoxaparin
Sodium ........ 0.02. ceeeee eee 6108
(208) Anti-Factor Xa and Anti-Factor Ila Assays for
Unfractionated and Low Molecular Weight
Heparins « « « axssssansag
ss35525.5 peceneaN 6113
(209) Low Molecular Weight Heparin Molecular
Weight Determinations
(210) Monosaccharide Analysis
CQTAD ARSENIC 9 5 Brice npo'uginceis
&o°S'>alo
(212) Oligosaccharide Analysis
(221) Chloride and Sulfate .........
(223) Dimethylaniline.............
(226) 4-Epianhydrotetracycline
(227) 4-Aminophenol in Acetaminophen-Containing
6038 Dig PrOGUCES ii... 0iscbisi0i odo oe oe» saline 6141
(228) Ethylene Oxide and Dioxane ... - - 6142
(231) Heavy Metals .............. .. 6145
(232) Elemental Impurities—Limits....
. .. 6147
(233) Elemental Impurities—Procedures . 6151
AL) WON: «= swe wemtiowmse a25558s an O1SS
(251) L6Ad s ss va kecesayug.sg 2... 6155
(261) Mercury .........-. 0-020 .... 6157
(267) Porosimetry by Mercury Intrusion......... 6160
 xx Guide to General Chapters USP 41

(268) Porosity by Nitrogen Adsorption— (661.2) PlasticPackaging Systems for

Desorption... 0.0... cece
eee eee Pharmaceutical Use: « « essuionnoa
se4aa4eK8 6424
(271) Readily Carbonizable Substances Test ...... (670) Auxiliary Packaging Components ......... 6428
(281) :Residue On IGNIOM 6 Lerccombicecten
dat 6ey (671) Containers—Performance Testing ......... 6436
291) SelenUM 3. . o 8's co Phe tion Sus wells Me (GOVEGOUON B28 45 ss ketenes’ aoeosOSs 6443
(695) Crystallinity... 6.0...
eeeeeeeee eeeee 6445
(696) Characterization of Crystalline Solids by
Other Tests and Assays Microcalorimetry and Solution Calorimetry .. 6445
(697) Container Content for Injections.......... 6449
(301) Acid-Neutralizing Capacity .............. (698) Deliverable Volume ............0000005 6450
(311) Alginates Assay... 2.0... ee eee (699) Density of Solids .............22.0000. 6453
(341) Antimicrobial Agents—Content (701) Disintegration... 2.2.2...
eee eeeeee 6455
(345) Assay for Citric Acid/Citrate and Phosphate . . 6176 (705) Quality Attributes of Tablets Labeled
(351) Assay for.Stenoids: ...« ccgusememn
oc oss 6177 as Having a Functional Score ............ 6457
(381) Elastomeric Closures for Injections . . . 6178 711) Dissolution: « « « « = + caasrenvena
sacsaewane 6459
(391) Epinephrine Assay ............. . 6183 (721) Distilling Range « x «, cssisansass
ao3seoo98K 6469
(401) Fats and Fixed Oils ............ . 6184 (724) Drtig'Release:. irs oe setosisannts
aebe'e o'swacay 6471
(411) Folic Acid Assay... 0... eee 6197 (729) Globule Size Distribution in Lipid Injectable
(413) Impurities Testing in Medical Gases
. - 6201 EMUISIONS 5 s < 5 s = SeaieRpe id oo ge 8 meee
(415) Medical Gases Assay ........... . 6202 (730) Plasma Spectrochemistry ...............
(425) lodometric Assay—Antibiotics............ 6205 (731) Loss on Drying ..........0-0
eeee eee
(429) Light Diffraction Measurement of Particle (733) LOSS ON IGNITION: *o xpaceinevenece
aeoe 6xmeA
SIZE oussas i warehndewgiongics sunsahd 6206 (735) X-Ray Fluorescence Spectrometry
(431) Methoxy Determination ...... is 6212 (736) Mass Spectrometry oiiisa
5 93's3fete aie
a) (441) Niacin or Niacinamide Assay .. 2« 6213 (741) Melting Rane or Temperature. ..........
-
oy (451) Nitrite Titration ............ . . 6218 4755) MIDINUEA, RIL (&: 3.ssn voce os atce augue. seennteoyencites
~~
Q (461) Nitrogen Determination
. . 6219 (761) Nuclear Magnetic Resonance Spectroscopy . . 6500
J (466)Ordina Impurities ...... 6220 (771) Ophthalmic Products—Quality Tests ....... 6510
p=
1S)
ash Residual Solvents... 0.2.00 seess cee 46222 (776) Op
pacal MIChOSCOPY” cts nce ocaais og = 8 eupeiRIT 6516
(469) Ethylene Glycol, Diethylene Glycol, and (781) O
Ptical Rotation, <csnc puna gs )5,3, oe tae ee Pe 6519
s
-
Triethylene Glycol in Ethoxylated (782) Vibrational Circular Dichroism
© SUDStANCES nk. cies cite Medenen terme See ay ene 6237 SPECtrOSCOPY’ Or Sass 34eeHEiog 6520
= (471) Oxygen Flask Combustion ............5- 6238 (785) Osmolality and Osmolarity.............. 6527
o (481)Ribo NAVIN ASSAY. wn e-scanmmynnaciee
so axon2 © 6239
1S) (786) Particle Size Distribution Estimation by
(501) Salts of ne Nitrogenous Bases........ 6245 Analytical Sieving ...........-..-02045 6530
(503) Acetic Acid in Peptides................. 6246 (787) Subvisible Particulate Matter in Therapeutic
(503.1) Trifluoroacetic‘Acid (TFA) in Peptides ..... 6247 Protein INjeCHONs ccsmus:evere
sew 2 0 2454roncaees 6534
(507) Protein Determination Procedures......... 6248 (788) Particulate Matter in Injections ........... 6537
2 1) anole. Steroid Ne, Si ereupetne Be tie aaa ee 6253 {789) Particulate Matter in Ophthalmic Solutions . . 6540
(525) Sulfur Dioxide... eeebeee tee 6254 (790) Visible Particulates in Injections........... 6542
(531) Thiamine Assay . (791) BH sas 6s 7s he RES Bs EG EEE BRET 6543
ASAT) TCIMIOEY fas wo Heo alenetnw idienn 6268
«HoBoh,8Oy (795) Pharmaceutical Compounding—Nonsterile
(551) Vitamin E Assay. ......... 4 6272 Preparations .... 2.2...0.0eeeeeeeeee 6546
(561) Articles of Botanical Origin. ............. 6279 (797) Pharmaceutical Compounding—Sterile
(563) Identification of Articles of Botanical Origin . . 6293 Preparations:-.-..ieusisacaamanen
iocMattie! 6554
(565) Botanical Extracts 6305 (800) Hazardous Drugs—Handling In Healthcare
(571) Vitamin A Assay.. . 6307 Setting
Se ab(eion waiter &
(580) Vitamin C Assay . . (801) Polarography ....
(581) Vitamin D Assay ... (811) Powder Fineness . . off
ssaeseeee 6325
(591) Zine: Determination: « «cise (821)-Radioactivity;).), ssikacct.wid
agin) calasaorQ.
(823) Positron Emission Tomography
Physical Tests and Determinations Drugs for Compounding, Investigational,
and Research) USS. sins a soa ov a 2 0 aces 6629
(601) Inhalation and Nasal Drug Products: Aerosols, (831) Refractive Index...
Sprays, and Powders—Performance Quality (841) Specific Gravity
TOSSiessnvere 0 6 a 8 08 & & wore ecmmvedte RTMTONTS o'g 6327 (846) Specific Surface Area .... 6... 22 eee eee
(602) Propellants ....... (852) Atomic Absorption Spectroscopy
(603) TopcalAerosols . P MEMHA (853) Fluorescence Spectroscopy... ...
(604) Leak Rate: sca sf celitratunmee eres (854) Mid-Infrared Spectroscopy ............-.
(610) Alternative Microbiological Sampling Methods (855) Nephelometry, Turbidimetry, and Visual
for Nonsterile Inhaled and Nasal Products
. . . 6356 GCOMPpariSON: 3a.cequantcin'ys
ph48835,8Lone 6658
(611) Alcohol Determination. . 2.6.0.0... .000s 6358 (857) Ultraviolet-Visible Spectroscopy........... 6660
(616) Bulk Density and Tapped Density of (861) Sutures—Diameter........0...0..-2.000. 6666
POWGEES 4 56 4.» 5 DU Stain UO RAIGESSB 6360 (871) Sutures—Needle Attachment ............ 6667
(621): Ghromatography tases sie Mae, . . 6363 (881) Tensile Soeur tueasbionsnanens aersasaahesncsk autres 6668
(631) Color and Ac rernicity aussutseseaatitia s Satay « 4 6375 (891) Thermal Analysis ............-200-2005 6669
(641) Completeness of Solution............ . . 6376 (905) Uniformity of De MES. oss 5-3, oes groreyoes 6673
(643) Total Organic Carbon ........... . . 6377 e 1) Viscosity—Capillary Methods ............ 6677
645) Water Conductivity ........... 912) Viscosity—Rotational Methods ........... 6679
651) Congealing Temperature ......... (913) Viscosity—Rolling Ball Method ........... 6684
(659) Packaging and Storage Requirements . . . 6384 (914) Viscosity—Pressure Driven Methods ....... 6686
(660) Containers—Glass ...........022.0008- 6390 (921) Water Determination ...............-.. 6687
(661) Plastic Packaging Systems and Their Materials (941) Characterization of Crystalline and Partially
Of ConstUGtion esis sh WatstFied aSEIT 6396 ee Solids by X-Ray Powder
(661.1) Plastic Materials of Construction ........ 6403 Diffraction: (KRPD) vcsceisseweoess
6 64sFosresatmnan 6692
USP 41
GENERAL INFORMATION
(1004) Mucosal Drug Products—
Performance Tests fbn
sasSTL dk 6699
(1005) Acoustic Emission .... 06...
eee eee 6702
(1010) Analytical Data—tinterpretation and
TREAEMEN Giese ao acctsmmanawe
wisSGleePie ave
(1024) Bovine Serum .
(1025) Pancreatin
(1027) Flow:Cytometty’s os scans
aysiiee22% 6744
(1029) Good Documentation Guidelines ........ 6760
(1030) Biological Assay Chapters—Overview and
GlOSsaty se 8 S575 5 ecststcanncsiel
se ogFE ELSear 6764
(1031) The Biocompatibility of Materials Used
in Drug Containers, Medical Devices, and
limiplantsss)-2 Nr esas,
Bot ETSwile 6775
(1032) Design and Development of Biological
ASSAYSITSO SET! SIN RlyPPR yal 6785
(1033) Biological ey Validation: SQN esd «oa 6803
(1034) Analysis of Biological Assays ............ 6818
(1039)'@hemometries: 3 {1 S22 TS eee Le 6831
(1041) Biologics: 3.622 ke EES wee 6849
(1043) Ancillary Materials for Cell, Gene, and Tissue-
Engineered-Products: 4:25 80552
645.02h scans
(1044) Cryopreservation of Cells ..............
(1046) Cellular and Tissue-Based Products . .
(1047) Gene Therapy Products .............
(1048) Quality of Biotechnological Products: Anal
of the Expression Construct in Cells Used for
Production of r-DNA Derived Protein
Produjetsso2 Pv OUD Iie 2 adel 6928
(1049) Quality of Biotechnological Products: Stability
Testing of Biotechnological/Biological
Products2:9 Jicny te Cetily Ri ee 6930
(1050) Viral Safety Evaluation of Biotechnology
Products Derived from Cell Lines of Human
orAnimal Origin eiel2 Vs Ae eet ak, 6935
(1050.1) Design, Evaluation, And Characterization
of Viral Clearance Procedures ........... 6950
(1051) Cleaning Glass Apparatus.............. 6960
(1052) Biotechnology-Derived Articles—Amino Acid
Anialysis! COS Doss os Ya Oe Tad 6961
(1053) Capillary Electrophoresis............... 6973
(1054) Biotechnology-Derived Articles—Isoelectric
Focusing’s (eu taths. Wein, Sb sh 6981
(1055) Biotechnology-Derived Articles—Peptide
Mapping 8 T tite aes. OL eed eect w 6984
(1056) Biotechnology-Derived Articles—Polyacrylamide
Gel Electrophoresis 4. 2... 5 6991
(1057) Biotechnology-Derived Articles—Total Protein
ASSAY x & 4 5 suetsennamaigice
wfoon«0SAdamitle Hocwnaege 6998
(1058) Analytical Instrument Qualification . . . 7005
(1059) Excipient Performance ........... «a ZO11
(1061) Color—Instrumental Measurement . . . . 7040
(1062) Tablet Compression Characterization... ... 7042
(1063) Shear Cell Methodology for Powder
Flow Testing .... 0.02.40.
e ceeeeeeee 7054
(1064) Identification of Articles of Botanical Origin
by High-Performance Thin-Layer
Chromatography Procedure ............ 7065
(1065) lon Chromatography ................. 7075
(1066) Physical Environments That Promote Safe
Medication Use 0:6 seer seeccceseues 7078
(1072) Disinfectants and Antiseptics ........... 7090
(1074) Excipient Biological Safety Evaluation
GUIGDCTIMESiccsmsscese ue me o U8 & eyscenetegepinnees
wt 7095
(1078) Good Manufacturing Practices for Bulk
Pharmaceutical Excipients.............. 7100
(1079) Good Storage and Distribution Practices for
Drug Products’, ss ss tec de eee eee 7120
(1079.1) Storage and Transportation of Investigational
Drug PROdUEES).. «4» os 9.5 % © yonecmenereye
saoo 7130
(1080) Bulk Pharmaceutical Excipients—Certificate of
ANALYSIS scomuesen ao ov 02 2s mo HS GZ 7133
Guide to General Chapters xxi
(1084) Glycoprotein and Glycan Analysis—General
Considerations silstsecadonx
tos.3a zesraCuca 7141
(1086) Impurities in Drug Substances and Drug
PrOduats: + + ccagactiemay
ay|Dasma: 7152
(1087) Apparent Intrinsic Dissolution—Dissolution
Testing Procedures for Rotating Disk and
Stationary Disk ..........-...0.0005. 7155
(1088) In Vitro and In Vivo Evaluation of Dosage
FORMS 0 « «0 « qncknciihitin
dsWeide:Tiewtalleodtenmenm 7159
{1090) Assessment of Drug Product
Performance—Bioavailability, Bioequivalence,
anid Dissolution aweenon's cakes oe weariness 7170
(1091) Labeling of Inactive Ingredients ......... 7178
(1092) The Dissolution Procedure: Development
and Validation: e:c.c.sce wi ate etleee weds 7178
(1094) Capsules—Dissolution Testing and Related
Quality Attributessc, £01135 1088s gies d hadedeiveredt 7198
(1097) Bulk Powder Sampling Procedures ....... 7206
(1102) Immunological Test Methods—General
Considerations ziw00 34 53452 EAR a NS 7219
{1103) Immunological Test Methods—Enzyme-
Linked Immunosorbent Assay (ELISA) ..... 7226
(1104) Immunological Test Methods—Immunoblot
ANALYSIS... 0s, scergsecnconmsp
a. PelesPabcbarkesade 7237
(1105) Immunological Test Methods—Surface 9)
o
Plasmon:Resonamee o1s.(tiesi,
derhareiewies 7248 |
(1106) Immunogenicity Assays—Design and o
=
Validation of Immunoassays to Detect Ines
Anti-Drug Antibodies ...............-. 7264
(1106.1) Immunogenicity Assays—Design and fa)
a
Validation of Assays to Detect Anti-Drug iy
Neutralizing Antibody me]
o
(1111) Microbiological Examination of Nonsterile oO
5
Products: Acceptance Criteria for Pharmaceutical “
Preparations and Substances for Pharmaceutical
USCS ss a na taemseeiaa8lesathle 7297
(1112) Application of Water Activity Determination to
Nonsterile Pharmaceutical Products....... 7298
(1113) Microbial Characterization, Identification, and
Strain: LY PING edsncsctirefda does alacatatveatonals 7301
(1115) Bioburden Control of Nonsterile Drug
Substances and Products .............. 7305
(1116) Microbiological Control and Monitoring of
Aseptic Processing Environments......... 7312
(1117) Microbiological Best Laboratory Practices... 7325
(1118) Monitoring Devices—Time, Temperature, and
Humniditysriersitsiew
dial alecDetebeehaie O's 7331
(1119) Near-Infrared Spectroscopy ......... 361/337
(1120) Raman Spectroscopy ........... 7343
(1121) Nomenclature ............-. Siiadesen 7351
(1125) Nucleic Acid-Based Techniques—General . . . 7353
(1126) Nucleic Acid-Based Techniques—Extraction,
Detection, and Sequencing ............ 7359
(1127) Nucleic Acid-Based Techniques—
Amplificationenes::44-<)z
suze 8BseuEraee's = 7369
(1128) Nucleic Acid-Based Techniques—
MicrOatray® acini: 3 96 dee 6 4 8 gy ectueneusnene
4 1379
(1129) Nucleic Acid-Based Techniques—
GEPIGEV ING, auubct xen te! 9b, modes prod proritlamnersog 7385
(1130) Nucleic Acid-Based Techniques—Approaches for
Detecting Trace Nucleic Acids (Residual DNA
TOSUNG) ewsncsen ss vhs 4,7 6 ¢ Peewee 6¥ 7389
(1132) Residual Host Cell Protein Measurement in
Biopharmaceuticals ...............04. 7393
(1136) Packaging and Repackaging—Single-Unit
Containers ..... 2... cee eee 7414
(1151) Pharmaceutical Dosage Forms .......... 7425
(1152) Animal Drugs for Use in Animal Feeds... .. 7450
(1160) Pharmaceutical Calculations in Pharmacy
PACH CR csnyanse< 5 3 24 4 HERE RESTA ee @ 0 7451
(1163) Quality Assurance in Pharmaceutical
Compounding....
(1174): Powder FIOW..... 6... 6 2 ee eo seers
oo
(1176) Prescription Balances and Volumetric
Apparatus won ss sss 0s oo oeeeoweNS
&gS 7485
 xxii Guide to General Chapters
(1177) Good Packaging Practices ............. 7492
(1178) Good Repackaging Practices............ 7495
(1180) Human Plasma ...... 0.20.20 .00 00005 7497
(1181) Scanning Electron Microscopy .......... 7519
(1184) Sensitization Testing.............254-. 7529
{1191) Stability Considerations in Dispensing
PraCUGOnesaucsse 4 b § 3525 HG eR MORERRER
GE 7540
(1195) Significant Change Guide for Bulk
Pharmaceutical Excipients.............. 7545
(1197) Good Distribution Practices for Bulk
Pharmaceutical Excipients.............. 7556
(1207) Sterile Product Packaging—Integrity
Evalationi csi. o 360 6 4 oy 2 aeteawaren << 7578
(1207.1) Package Integrity Testing in the Product
Life Cycle—Test Method Selection
and Validation ...............002.004 7585
(1207.2) Package Integrity Leak Test Technologies . . 7597
(1207.3) Package Seal Quality Test Technologies. . . 7614
(1208) Sterility Testing—Validation of Isolator
SYSTEMS! cameos waa oo ok re kredamenes WX 7617
(1210) Statistical Tools for Procedure Validation ... 7622
(1211) Sterilization and Sterility Assurance of
Compendial Articles... ...........00-. 7633
(1216) Tablet Friability ..............000000. 7634
“
- (121.7): Tablet Breaking Forces: 615. 06 Sehavereitee
a 7635
2a (1222) Terminally Sterilized Pharmaceutical Products—
2 Parametric Releases 0. b 4 PU he. ed sas 7638
J (1223) Validation of Alternative Microbiological
&
1) Methods ccavasicea 2a'd'd 2s betel ecishisais
ga3 7642
(1223.1) Validation of Alternative Methods to Antibiotic
Ss
~ Microbial Assays ............-200-008 7656
a (1224) Transfer of Analytical Procedures......... 7663
= Delivery Systems ersiaac sceattereraarsiaeet
o (1225) Validation of Compendial Procedures ..... 7665
oO (1226) Verification of Compendial Procedures... .. 7671
(1227) Validation of Microbial Recovery from
Pharmacopeial Articles ..........-..... 7672
(1228) Depyrogenation ........ 0.0.ceeeeee 7676
(1228.1) Dry Heat Depyrogenation ............
(1228.3) Depyrogenation by Filtration. .........
(1228.5) Endotoxin Indicators For
Depyrogenation!? 02.3 tae miiitinidei
s&
(1229) Sterilization of Compendial Articles.......
(1229.1) Steam Sterilization by Direct Contact ....
(1229.2) Moist Heat Sterilization of Aqueous
Miquids". satire
Lett bee,ee
(1229.3) Monitoring of Bioburden.............
(1229.4) Sterilizing Filtration of Liquids .........
(1229.5) Biological Indicators for Sterilization .....
(1229.6) Liquid-Phase Sterilization .........
(1229.7) Gaseous Sterilization ............ 2 Matter in Injections and Ophthalmic
(1229.8) Dry Heat Sterilization ...............
(1229.9) Physicochemical Integrators And
Indicators for Sterilization ..........
(1229.10) Radiation Sterilization ..... i (1823) Positron Emission Tomography Drugs—
(1229.11) Vapor Phase Sterilization ....
. Informationiscasa
(1229.12) New Sterilization Methods... .
(1229.13) Sterilization-In-Place .........
(1229.14) Sterilization Cycle Development . . .
(1229.15) Sterilizing Filtration Of Gases ....
. and. Practice:).csis!} samiivieateetestois
(1230) Water for Hemodialysis Applications . . : (1854) Mid-Infrared Spectroscopy—Theory
(1231) Water for Pharmaceutical Purposes .......
(1234) Vaccines for Human Use—Polysaccharide
and Glycoconjugate Vaccines ........... 7778
(1235) Vaccines for Human Use—General
Considerations:2% 8230. 80 9 Baigent 7795
USP 41
(1237) Virology Test Methods ................ 7812
(1238) Vaccines for Human Use—Bacterial
VACCINESresciviegs a6 vo 3 ve + HereNNS,S
sa 7833
(1240) Virus Testing of Human Plasma for Further
Manufacture «53sec gseneeeaansgas 7846
(1241) Water-Solid Interactions in Pharmaceutical
Systems ....... 00-00.ecece eeeeee 7856
(1251) Weighing on an Analytical Balance ....... 7860
(1265) Written Prescription Drug Information—
GuideliNeS.ccisse:s z.2 xo vals Rear ae aa 7866
(1285) Preparation of Biological Specimens for
Histologic and Immunohistochemical
AtialYSiscscguses oa x 2 5 5 FEE E essence
oo» 7868
(1285.1) Hematoxylin and Eosin Staining of Sectioned
Tissue for Microscopic Examination ....... 7872
(1601) Products for Nebulization—Characterization
TOSS searnicntodie se 2 «as suey welageememangeeral
aewn 7874
(1602) Spacers and Valved Holding Chambers
Used With Inhalation Aerosols—
Characterization Tests, 5 ¢ csi essen 7878
(1644) Theory and Practice of Electrical Conductivity
Measurements of Solutions............. 7890
(1660) Evaluation of the Inner Surface Durability
of Glass Containers )2 06.6 d)deapnmcowns
«5 7897
(1661) Evaluation of Plastic Packaging Systems and
Their Materials of Construction with Respect
to Their User Safety Impact ............ 7902
(1663) Assessment of Extractables Associated with
Pharmaceutical Packaging/Delivery
SYSLEMSiaikieaiathAlcea « « 0 7910
(1664) Assessment of Drug Product Leachables
Associated with Pharmaceutical Packaging/
+ 69 7924
(1664.1) Orally Inhaled and Nasal Drug Products . . 7937
(1724) Semisolid Drug Products—Performance
TOStS 25) vari. dg a sy dasigkdesn
oss4»» 7944
(1730) Plasma Spectrochemistry—
Theory; and-Practice picicisi iisindeerauerene
deda fd 7956
(1735) X-Ray Fluorescence Spectrometry—
Theory and Practicejrnt. sent penjsghhs«bey 7963
(1736) Applications of Mass Spectrometry ....... 7982
(1761) Applications of Nuclear Magnetic Resonance
Spectroscopy. « sis<tsleicdagidaenitae)
1c 8004
(1771) Ophthalmic Products—Performance Tests . . 8024
(1782) Vibrational Circular Dichroism Spectroscopy—
Theory<and, Practice; i0.G iencadasiae
(oc 8025
(1787) Measurement of Subvisible Particulate Matter in
Therapeutic Protein Injections........... 8038
(1788) Methods for the Determination of Particulate
Solutions. 2... 6... eee eee eee 8052
(1790) Visual Inspection of Injections........... 8066
(1821) Radioactivity—Theory and Practice ....... 8084
2 nejeresnezed
aglelgb8634 8098
(1852) Atomic Absorption Spectroscopy—Theory
and Practice’... :s. ee secerteat
sacl?«a 8109
(1853) Fluorescence Spectroscopy—Theory
vd=«+ 8118
and::Practice: «<<» «aparoteniigtd sw) aes 8127
(1857) Ultraviolet-Visible Spectroscopy—Theory
and: Practice: ss 4. << & meblmeigenius
A a 6 8136
(1911): Rheometty’ os ewieccen
nenessaet 5G 8145
USP 41 Guide to General Chapters xxiii

DIETARY SUPPLEMENTS (2040) Disintegration and Dissolution of Dietary

SUDPICMENES3 ce: a= oo woo yvineeess @ao 8178
(2021) Microbial Enumeration Tests—Nutritional and (2091) Weight Variation of Dietary Supplements. . . 8185
Dietary:Supplements: «sis 42 ceases 8153 (2232) Elemental Contaminants in Dietary
(2022) Microbiological Procedures for Absence of Supplemenitsiss 35 oso a6 eo nememay
«aa 8186
Specified Microorganisms—Nutritional and (2250) Detection of Irradiated Dietary
Dietary Supplements ...............4. 8158 Supplements ...........
0-0eeeeeee 8190
(2023) Microbiological Attributes of Nonsterile {2251) Screening for Undeclared Drugs and
Nutritional and Dietary Supplements...... 8164 Drug Analogs . 12. r eee ene geene eens 8193
(2030) Supplemental Information for Articles of (2750) Manufacturing Practices for Dietary
Botanical O1igifisiss ass eee ae v geameeerowes 8168 SupPleMeNtSis sss a ee een are 6 aw SF 8210

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General Chapters
USP 41 Global Health Monographs / Chlorhexidine 4415

Global Health Monographs

Preface
This section contains monographs for articles which are not currently legally marketed in the United States, but which have
been approved by a stringent regulatory authority as defined by the World Health Organization and are used for essential
purposes in other parts of the world. Selection and prioritization of new entries to this section will be accomplished in close
collaboration with stakeholders throughout the global health community. These monographs are not applicable to articles
marketed for use in the United States.

Analysis
Chlorhexidine Gluconate Topical Gel Samples: Standard solution and Sample solution
Develop the chromatogram in a Developing solvent sys-
DEFINITION
tem until the solventfront has moved 10 cm from the
Chlorhexidine Gluconate Topical Gel is prepared from point of spotting. Remove the plate from the chamber
Chlorhexidine Gluconate Solution. It contains NLT 90.0% and dry at 110° for 20 min. Allow to cool and spray
and NMT 110.0% of the labeled amount of chlorhexidine with Spray reagent. Heat the plate at 110° for 10 min.
gluconate (C22H30Cl2Ni0 - 2C6H1207). Acceptance criteria: The principal spot of the Sample
[NoTte—The U.S. Food and Drug Administration has not re- solution corresponds in color, size, and R; value to that
viewed the safety and efficacy of Chlorhexidine Gluconate of the Standard solution.
Topical Gel and it is not approved for marketing in the ASSAY
United States.] ¢ PROCEDURE
IDENTIFICATION Buffer: Dissolve 27.6 g of sodium dihydrogen phos- fa}
e A, ULTRAVIOLET ABSORPTION (197U) phate and 10 mL of triethylamine in 1.5 L of water. Ad- [Ss
Wavelength range: 200-400 nm Just with phosphoric acid to a pH of 3.0 and dilute with
water to 2000 mL. c
Sample solution: Nominally 0.01 mg/mL of chlorhex-
idine gluconate from Topical Gel, prepared as follows. Solution A: Acetonitrile and Buffer (30:70) o
Transfer a suitable amount of Topical Gel to an appro- Solution B: Acetonitrile ro)
priate volumetric flask and dilute with water to volume. Mobile phase: See Table 7. @
Acceptance criteria: The UV absorption spectrum of 2
the Sample solution exhibits two maxima at 231 and Table 1 Ss
255 nm and two minima at 222 and 242 nm. Time Solution A Solution B a
e B. The retention time of the major peak of the Sample (min) (%) (%)
solution corresponds to that of the Standard solution, as
obtained in the Assay. 0 100 0
° . ee CHROMATOGRAPHIC IDENTIFICATION TEST 9 100 oO
10 45 55
Diluent: Acetonitrile and water (1:1) 15 45 55
Standard solution: 10 mg/mL of USP Potassium Gluco- 16 100 0
nate RS in Diluent 21 100 0
Sample solution: Nominally 20 mg/mL of chlorhexidine
gluconate from Topical Gel, prepared as follows. Trans- Standard solution: 50 g/mL of USP Chlorhexidine
fer a suitable amount of Topical Gel, equivalent to Acetate RS in Solution A
500 mg of chlorhexidine gluconate, to a 25-mL volu- Sample stock solution: Nominally 0.4 mg/mL of
metric flask. Add a sufficient quantity of Diluent, soni- chlorhexidine gluconate from Topical Gel, prepared as
cate with intermittent shaking for 30 min, and dilute follows. Transfer a suitable amount of Topical Gel,
with Diluent to volume. Centrifuge the solution for 5 equivalent to 40 mg of chlorhexidine gluconate, to a
min at 3000 rpm. 100-mL volumetric flask. Add about 70 mL of Solution
Chromatographic system A, sonicate with intermittent shaking for 30 min, and
AR SIDENE: 0.25-mm layer of chromatographic silica dilute with Solution A to volume.
ge Sample solution: Nominally 80 ug/mL of chlorhexidine
Application volume: 10 WL gis from the Sample stock solution in Solution A
Developing solvent system: Alcohol, ethyl acetate, Chromatographic system
ammonium hydroxide, and water (5:1:1:3) (See Chromatography (621), System Suitability.)
Spray reagent: Dissolve 2.5 g of ammonium molyb-
date in 50 mL of 2 N sulfuric acid in a 100-mL volu-
metric flask. Add 1.0g of ceric sulfate, swirl to dis-
solve, and dilute with 2 N sulfuric acid to volume.
 4416 Chlorhexidine / Global Health Monographs USP 41

Mode: LC metric flask. Add about 70 mL of Solution A, sonicate

Detector: UV 239 nm with intermittent shaking for 30 min, and dilute with
Column: 4.6-mm x 25-cm; 5-um packing L1 Solution A to volume. Centrifuge the solution.
Column temperature: 40° System suitability
Flow rate: 1.5 mL/min Samples: System suitability solution and Standard
Injection volume: 50 uL solution
System suitability Suitability requirements
Sample: Standard solution Resolution: NLT 3.0 between chlorhexidine and p-
Suitability requirements chloroaniline, System suitability solution
Tailing factor: NMT 2.0 Relative standard deviation: NMT 5.0%, Standard
Relative standard deviation: NMT 2.0% solution
Analysis Analysis
Samples: Standard solution and Sample solution Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of Calculate the percentage of p-chloroaniline in the por-
chlorhexidine gluconate (Cz2H30Cl2Nio - 2CeH1207) in tion of Topical Gel taken:
the portion of Topical Gel taken:
Result = (ru/rs) x (Cs/Cu) x 100
Result = (ru/rs) x (Cs/Cu) < (Mra/Mz) x 100
ru = peak response of p-chloroaniline from the
ry = peak area of chlorhexidine from the Sample Sample solution
solution Is = peak response of p-chloroaniline from the
Is = peak area of chlorhexidine from the Standard Standard solution
solution Cs = concentration of USP p-Chloroaniline RS in the
Cs = concentration of USP Chlorhexidine Acetate Standard solution (mg/mL)
RS in the Standard solution (g/mL) Cy = nominal concentration of chlorhexidine
Cu = nominal concentration of chlorhexidine gluconate in the Sample solution (mg/mL)
gluconate in the Sample solution (ug/mL) Acceptance criteria: NMT 0.35%
My = molecular weight of chlorhexidine gluconate,
897.76 Pee ieee
Mz = molecular weight of chlorhexidine acetate, e PH (791
5 625.55 ¢ sili Sample solution: Nominally 1% of chlorhexidine gluco-
Acceptance criteria: 90.0%-110.0% nate from Topical Gel in water
Acceptance criteria: 5.0-7.0
IMPURITIES
e LIMIT OF p-CHLOROANILINE ADDITIONAL REQUIREMENTS
Solution A, Solution B, Mobile phase, and Chromato- © PACKAGING AND STORAGE: Preserve in well-closed contain-
graphic system: Proceed as directed in the Assay. ers, protected from light. Store at controlled room
System suitability solution: 50 g/mL of USP temperature.
Chlorhexidine Acetate RS and 1 ug/mL of USP p- ¢ USPREFERENCESTANDARDS (11)
GH Monographs

Chloroaniline RS in Solution A USP Chlorhexidine Acetate RS

lution: 1.0 Lof USP p-Chl ili USP p-Chloroaniline RS
gelagetacs ie ueIm!. © Hae ae USP Potassium Gluconate RS
Sample solution: Nominally 0.4 mg/mL of chlorhex-
idine gluconate from Topical Gel, prepared as follows.
Transfer a suitable amount of Topical Gel, equivalent to
40 mg of chlorhexidine gluconate, to a 100-mL volu-
USP 41 Dietary Supplements / N-Acetylglucosamine 4417

Dietary Supplements
Official Monographs

[Nott—The relative retention times for N-acetyl-

N-Acetylglucosamine glucosamine and glucosamine are 1.0 and about 2.8,
respectively.]
oH Suitability requirements
Signal-to-noise ratio: NLT 10 for the glucosamine
peak, System suitability solution
Resolution: NLT 5.0 between the N-acetyl-
glucosamine and glucosamine peaks, System suitabil-
ity solution
Tailing factor: NMT 2.0, Standard solution
Relative standard deviation: NMT 2.0%, Standard
solution
CgHisNOc 221.21 Analysis
2-(Acetylamino)-2-deoxy-D-glucose; Samples: Standard solution and Sample solution
N-Acetyl-D-Glucosamine [7512-17-6]. Calculate the percentage of N-acetylglucosamine
(attigNOe) in the portion of N-Acetylglucosamine
DEFINITION taken:
N-Acetylglucosamine contains NLT 98.0% and NMT 102.0%
of N-acetylglucosamine (CsHisNO¢), calculated on the Result = (ru/rs) x (Cs/Cu) x 100
dried basis.
tu = peak response from the Sample solution
IDENTIFICATION Is = peak response from the Standard solution
A. INFRARED ABSORPTION (197K) Cs = concentration of USP N-Acetylglucosamine RS
e B. It meets the requirements in the test for Optical Rota- in the Standard solution (mg/mL)
tion (7815), Specific Rotation. Cu = concentration of N-Acetylglucosamine in the
e C. The retention time of the major peak of the Sample Sample solution (mg/mL)
solution corresponds to that of the Standard solution, as Acceptance criteria: 98.0%-102.0% on the dried basis
obtained in the Assay.
IMPURITIES
ASSAY © RESIDUE ON IGNITION (281): NMT 0.1%
© PROCEDURE e CHLORIDE AND SULFATE, Chloride (221): NMT 0.1%
Buffer: Transfer 3.5 g of dibasic potassium phosphate to e ELEMENTAL IMPURITIES—PROCEDURES (233)
a 1-L volumetric flask, and add sufficient water to dis- Acceptance criteria
solve. Add 0.25 mL of ammonium hydroxide, dilute Arsenic: NMT 1 ug/g
with water to volume, and mix. Adjust with phosphoric Lead: NMT 10ug/g
sydesbouo-: sq

acid to a pH of 7.5. e RELATED COMPOUNDS

Mobile phase: Acetonitrile and Buffer (75:25) Buffer, Mobile phase, Diluent, System suitability solu-
Diluent: Acetonitrile and water (50:50) tion, Chromatographic system, and System suitabil-
System suitability solution: 1.0 mg/mL of USP N- ity: Proceed as directed in the Assay.
Acetylglucosamine RS and 0.6 mg/mL of USP Glucosa- Sample solution: 2.5 mg/mL of N-Acetylglucosamine in
mine Hydrochloride RS in Diluent Diluent
Standard solution: 1.0 mg/mL of USP N-Acetyl- Analysis
glucosamine RS in Diluent Sample: Sample solution
Sample solution: 1.0 mg/mL of N-Acetylglucosamine in Calculate the percentage of each impurity in the por-
Diluent tion of N-Acetylglucosamine taken:
Chromatographic system
(See Chromatography (621), System Suitability.) Result = (ru/r7) x 100
Mode: LC
Detector: UV 195 nm ty = peak response of each impurity from the
Column: 4.6-mm x 15-cm; 3-m packing L8& Sample solution
Column temperature: 35° nr = sum of the peak responses from the Sample
Flow rate: 1.5 mL/min solution
Injection volume: 10 uL
System suitability
Samples: System suitability solution and Standard
solution
 4418 N-Acetylglucosamine / Dietary Supplements USP 41

Acceptance criteria DEFINITION

Individual impurity: NMT 0.5% N-Acetyltyrosine contains NLT 98.5% and NMT 101.0% of
Total impurities: NMT 2.0% N-acetyltyrosine (Ci7Hi3NO,), as N-acetyl-L-tyrosine, calcu-
e Limit OF GLUCOSAMINE lated on the dried basis.
Buffer, Mobile phase, Diluent, System suitability solu-
tion, Chromatographic system, and System suitabil- IDENTIFICATION
ity: Proceed as directed in the Assay. e A. INFRARED ABSORPTION (197K)
Standard solution: 0.6 mg/mL of USP Glucosamine Hy- e B. OPTICAL ROTATION, Specific Rotation (781S)
drochloride RS in Diluent Sample solution: 10 mg/mL
Sample solution: 50 mg/mL of N-Acetylglucosamine in Acceptance criteria: NLT +46.0° and NMT +49.0°, de-
Diluent termined at 20°
Analysis e C. The R; value of the principal spot of the Sample solu-
Samples: Standard solution and Sample solution tion in the test for Organic Impurities corresponds to that
Calculate the percentage of glucosamine in the portion of Standard solution 1.
of N-Acetylglucosamine taken:
ASSAY
Result = (ru/rs) x (Cs/Cu) * (Mi/M2) x 100 © PROCEDURE
Sample solution: Dissolve about 180 mg of N-
ru = peak response of glucosamine from the Acetyltyrosine, weighed, in 50 mL of carbon dioxide-
Sample solution free water.
rs = peak response of glucosamine from the Titrimetric system
Standard solution (See Titrimetry (541).)
Cs = concentration of USP Glucosamine Mode: Direct titration
Hydrochloride RS in the Standard solution Titrant: 0.1 N sodium hydroxide VS
(mg/mL) Endpoint detection: Potentiometric
Cu = concentration of N-Acetylglucosamine in the Equivalency: Each mL of 0.1 N sodium hydroxide VS
Sample solution (mg/mL) is equivalent to 22.32 mg of N-acetyltyrosine
M; molecular weight of glucosamine, 179.17 (CiiHi3NO,).
M2 molecular weight of glucosamine
hydrochloride, 215.63 IMPURITIES
Acceptance criteria: NMT 1.0% e RESIDUE ON IGNITION (281): NMT 0.1%
e CHLORIDE AND SULFATE, Chloride (221)
SPECIFIC TESTS Sample: 0.7g
e OPTICAL ROTATION, Specific Rotation (781S) Standard: 0.40 mL of 0.01 N hydrochloric acid
Sample solution: 20 mg/mL in water, perform the Acceptance criteria: NMT 200 ppm
measurement 3 h after sample preparation. e CHLORIDE AND SULFATE, Sulfate (221)
Acceptance criteria: +39.0° to +43.0° Sample: 1.2g
PH (791) Standard: 0.25 mL of 0.020N sulfuric acid
Sample solution: 10 mg/mL in water Acceptance criteria: NMT 200 ppm
Acceptance criteria: 6.0-8.0 © IRON (241): NMT 20 ppm
Loss ON DRYING (731)
Analysis: Dry a sample at 105° for 2 h. Delete the following:
Acceptance criteria: NMT 0.5%
MELTING RANGE OR TEMPERATURE (741): 196°-205° °e HEAVY METALS, Method 1 (231): NMT 10 ppme coriiai1-
MICROBIAL ENUMERATION TESTS (2021): The total aerobic Jan-2018)
bacterial count does not exceed 10? cfu/g; the total com- © ORGANIC IMPURITIES
bined molds and yeasts count does not exceed 103 cfu/ Adsorbent: 0.25-mm layer of chromatographic silica
g. gel mixture
ABSENCE OF SPECIFIED MICROORGANISMS (2022): Meets the Standard stock solution 1: 8 mg/mL of USP N-Acetyl-L-
requirements of the tests for absence of Salmonella spe- tyrosine RS in a mixture of water, glacial acetic acid,
cies and Escherichia coli and alcohol (3:3:94)
DS Monographs

ADDITIONAL REQUIREMENTS Standard solution 1: Dilute Standard stock solution 1

© PACKAGING AND STORAGE: Preserve in tight, light-resistant with alcohol to obtain a solution having a known con-
containers. centration of about 0.4 mg/mL.
e USP REFERENCE STANDARDS (11) Standard solution 2: 0.8 mg/mL of USP L-Tyrosine RS
USP N-Acetylglucosamine RS dissolved in a mixture of glacial acetic acid and water
USP Glucosamine Hydrochloride RS (1:1), and diluted with alcohol
Sample solution: Transfer 0.8 g of N-Acetyltyrosine to a
10-mL volumetric flask, dissolve in 6 mL of a mixture of
glacial acetic acid and water (1:1), and dilute with alco-
hol to volume.
Application volume: 5 ul
N-Acetyltyrosine Developing solvent system: A mixture of ammonia
and 2-propanol (3:7)
Spray reagent: Dissolve 0.2 g of ninhydrin in 100 mL
of a mixture of butanol and 2 N acetic acid (95:5).
Analysis: Proceed as directed for Chromatography (621),
ox

Thin-Layer Chromatography. After air-drying the plate,

g

repeat the development process. After air-drying a sec-

ond time, examine the plate under short-wave UV light,
GQ H
1 i 3N O4 223.2 and record principal and secondary spots. Spray the
N-Acetyl-L-tyrosine; plate with Spray reagent, and heat between 100° and
(25)-2-(Acetylamino)-3-(4-hydroxyphenyl)propanoic acid) 105° for about 15 min. Examine the plate under white
[537-55-3]. light, and record the principal and secondary spots.
USP 41
Acceptance criteria: Under the short-wave UV light,
any secondary spot observed from the Sample solution
is not larger or more intense than the principal spot
from Standard solution 1. After applying the Spray rea-
gent, under white light, any secondary spot at the locus
of tyrosine from the Sample solution is not larger or
more intense than the principal spot from Standard so-
lution 2.
Individual impurities: NMT 0.5%
Limit of tyrosine: NMT 1.0%
SPECIFIC TESTS
e Loss ON DRYING (731)
Analysis: Dry a sample at 105° for 3 h.
Acceptance criteria: NMT 0.1%
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in well-closed contain-
ers, and store at controlled room temperature.
e USP REFERENCE STANDARDS (11)
USP N-Acetyl-L-tyrosine RS
USP L-Tyrosine RS
Ademetionine Disulfate Tosylate—see S-
Adenosyl--methionine Disulfate Tosylate
S-Adenosyl-L-methionine Disulfate
Tosylate
Former Title: Ademetionine Disulfate Tosylate
NH
j 4 ° 2
WL ST AL x ALA
slteeta‘ NH | A i"a.o
8 on, b ao ellsA N
halos ne Pe :
Co2H3aNoO16Sa
5-(Adenosyl)-L-methionine disulfate tosylate;
(35)-5’-[(3-Amino-3-carboxypropyl)methylsulfonio]-5’-deoxy-
adenosine, disulfate-methylbenzenesulfonate
[97540-22-2].
DEFINITION
5-Adenosyl-L-methionine Disulfate Tosylate is the
disulfate-tosylate mixed salt of a mixture of diaster-
eoisomers of the S-adenosyl-L-methionine ion. It contains
NLT 95.0% and NMT 105.0% of S-adenosyl-t-methionine
disulfate tosylate (C22H34N6O16S4) calculated through the
content of S-adenosyl-l-methionine (CisH23NeOsS*), calcu-
lated on the anhydrous basis.
IDENTIFICATION
e A. INFRARED ABSORPTION (197K)
e B. The retention time of the major peak of the Sample
solution corresponds to that of S-adenosyl-L-methionine in
the System suitability solution, as obtained in the CONTENT
OF S-ADENOSYL-L-METHIONINE.
COMPOSITION
e CONTENT OF S-ADENOSYL-L-METHIONINE
Solution A: 10 mL of glacial acetic acid in 500 mL of
water. Add 2.06 g of sodium 1-hexanesulfonate, and di-
lute with water to 1000 mL.
Mobile phase: Acetronitrile and Solution A (15:85)
System suitability solution: 400 ug/mL each of USP S-
Adenosyl-L-methionine Disulfate Tosylate RS and USP S-
Adenosyl-L-homocysteine RS
Standard solution A: 400 j1g/mL of USP S-Adenosyl-L-
homocysteine RS
Dietary Supplements / S-Adenosyl 4419
Standard solution B: 200 g/mL from Standard solution
A
Standard solution C: 80 g/mL from Standard solution
A
Sample solution: 20mg of S-Adenosyl-L-methionine Di-
sulfate Tosylate in 40 mL of water. Stir for 30 min, then
dilute with water to 50.0 mL. Transfer 1.0 mL of the
solution to a 1.5-mL microcentrifuge tube, and centri-
fuge for 1 min. Use a portion of the supernatant.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 260 nm
Column: 4.6-mm x 15-cm; 3-um packing L1
Flow rate: 1 mL/min
Injection size: 10 pL
System suitability
Samples: System suitability solution and Standard solu-
tion B
[NoTte—The relative retention times for S-adenosyl-L-
homocysteine and S-adenosyl-t-methionine disulfate
tosylate are about 0.68 and 1.0, respectively.]
Suitability requirements
Resolution: NLT 1.5 between S-adenosyl-L-homocys-
teine and S-adenosyl-L-methionine
Tailing factor: NMT 1.5, Standard solution B
Relative standard deviation: NMT 2.0% for S-ade-
nosyl-L-homocysteine, Standard solution B
Analysis
Samples: Standard solution A, Standard solution B,
Standard solution C, and Sample solution
[Note—Record the chromatograms, and measure the
area of the S-adenosyl-L-homocysteine peak in all three
Standard solutions and the 5-adenosyl-L-methionine di-
sulfate tosylate peak in the Sample solution.]
Plot a calibration curve of the peak area of the Stan-
dard solutions versus the corresponding S-adenosyl-L-
homocysteine concentration, in mg/mL, and draw
the straight line best fitting the three points. From
the calibration curve, and using the peak area of S-
adenosyl-L-methionine from the chromatogram from
766.80 the Sample solution, determine the concentration, C,
in mg/mL, of S-adenosyl-L-methionine as S-adenosyl-
L-homocysteine in the Sample solution.
Calculate the percentage of CisH23NeOsS* in the portion
of S-Adenosyl-t-methionine Disulfate Tosylate taken:
Result = (C/Cy) x (Ma/Mi2) x 100
Cc = concentration of S-adenosyl-t-methionine as S-
adenosyl-L-homocysteine obtained from the
linear regression line (mg/mL)
sydeabouow sa
Cy = concentration of S-Adenosyl-L-Methionine
Disulfate Tosylate in the Sample solution
(mg/mL)
Mn = molecular weight of S-adenosyl-L-methionine,
399.44
Mz = molecular weight of S-adenosyl-L-
homocysteine, 384.41
Acceptance criteria: 49.5%-54.7% on the anhydrous
basis, equivalent to 95.0%-105% of S-adenosyl-L-methi-
onine disulfate tosylate on the anhydrous basis
¢ CONTENT OF SULFATE
Mobile phase: 8.0 mM sodium carbonate and 1.0 mM
sodium bicarbonate in water
Standard solution: 0.18 mg/mL of potassium sulfate
Sample solution: 0.5 mg/mL of S-Adenosyl-L-methio-
nine Disulfate Tosylate
Chromatographic system
(See Chromatography (621), System Suitability.)
 4420 S-Adenosyl / Dietary Supplements USP 41

Mode: LC Iss = areas of the peaks corresponding to the S,5-

Detector: lon detector with suppressed conductivity isomer in the Sample solution
Column: 4.0-mm x 25-cm; 7-1um packing L74 Trs = areas of the peaks corresponding to the R,5S-
Column temperature: 30° isomer in the Sample solution
Flow rate: 1 mL/min Acceptance criteria: NLT 60% and NLT the labeled
Injection size: 25 uL amount of the S,S-isomer
System suitability
Sample: Standard solution ADDITIONAL REQUIREMENTS
Suitability requirements ¢ PACKAGING AND STORAGE: Preserve in tight, light-resistant
Column efficiency: NLT 8200 theoretical plates containers, and store in a refrigerator.
Tailing factor: NMT 1.5 e LABELING: Label it to indicate the minimum content of S,
Relative standard deviation: NMT 1.0% S-isomer, as a percentage.
Analysis e USP REFERENCE STANDARDS (11)
Samples: Standard solution and Sample solution USP S-Adenosyl-L-methionine Disulfate Tosylate RS
[Notre—Measure the area of the sulfate peak.] USP S-Adenosyl-l-homocysteine RS
Calculate the percentage of sulfate (SO) in the portion
of S-Adenosyl-L-methionine Disulfate Tosylate taken:
Result = (ru/rs) x (Cs/Cu) x 100
Tu = peak response of sulfate from the Sample
Alanine—see Alanine General Monographs
solution
ls = peak response of sulfate from the Standard
solution L-Alanyl-L-glutamine
Cs = concentration of sulfate (SO4) in the Standard
solution (mg/mL)
Cy = concentration of S-Adenosyl-L-methionine Nie
e °
I
N,
Disulfate Tosylate in the Sample solution Hc” Yr or
(mg/mL)
Acceptance criteria: 23.5%-26.5%
Oo %

2 NH
IMPURITIES
CsHisN304 217.23
Delete the following: L-2-(1-Oxo-2-amino-propylamino)-4-amino-4-oxobutanoic
acid [39537-23-0].
°e HEAVY METALS, Method / (231): NMT 20 ppme (oricai1-
jan-2018) DEFINITION
L-Alanyl-L-glutamine contains NLT 98.0% and NMT 101.5%
SPECIFIC TESTS of L-alanyl-L-glutamine (CgHisN3Ox), calculated on the an-
e PH (791): 1.0-2.0, in an aqueous solution (1 in 20) ao and solvent-free basis, and excluding alanine and
© WATER DETERMINATION, Method Ia (921): NMT 3.0% glutamine.
e ISOMERIC RATIO
Buffer: Transfer 4.2 g of citric acid monohydrate and IDENTIFICATION
2.03 g of sodium dihydrogen phosphate dihydrate to a e A. INFRARED ABSORPTION (197A)
1-L volumetric flask, and dissolve in and dilute with e B. It meets the requirements for Optical Rotation, Specific
water to volume. Rotation (7815) in Specific Tests.
Mobile phase: 4.0 g of sodium dodecyl sulfate and
440 mL of acetonitrile. Dilute with Buffer to 1 L. ASSAY
© PROCEDURE
Standard solution: 1.0 mg/mL of USP S-Adenosyl-L-me-
thionine Disulfate Tosylate RS Sample: 300mg
Sample solution: 1.0 mg/mL of S-Adenosyl-L-methio- Blank: Mix 5 mL of formic acid with 50 mL of glacial
acetic acid.
DS Monographs

nine Disulfate Tosylate

Chromatographic system Titrimetric system
(See Titrimetry (541).)
(See Chromatography (621), System Suitability.)
Mode: Direct titration
Mode: LC
Detector: UV 254 nm Titrant: 0.1 N perchloric acid VS
Column: 4.6-mm x 25-cm; 5-um packing L1 Endpoint detection: Potentiometric
Flow rate: 1.2 mL/min Analysis: Dissolve the Sample in 5 mL of formic acid,
Injection size: 20 pL add 50 mL of glacial acetic acid, and titrate with the
System suitability Titrant. Perform a Blank determination, and make any
Sample: Standard solution necessary correction.
[Note—The relative retention times for R,S-isomers and Calculate the percentage of L-alanyl-t-glutamine
5,S-isomers are about 0.94 and 1.0, respectively.] (CgHisN3Ox) in the Sample taken:
Suitability requirements Result; = [(Vs — Vs) x N x (F/W)] x 100
Resolution: NLT 1.0 between the S,S-isomer and the
R,S-isomer Vs = Titrant volume consumed by the Sample (mL)
Analysis Ve = Titrant volume consumed by the Blank (mL)
Samples: Standard solution and Sample solution N = actual normality of the Titrant (mEq/mL)
Identify the peaks of the S,S- and R,S-isomers of the F = equivalency factor, 217.2 mg/mEq
Sample solution by comparison with the Standard solu- Ww = Sample weight (mg)
tion, and calculate the percentage of the S,5-isomer:
Result = [rss/(rss + frs)] x 100
USP 41 Dietary Supplements / Alanyl 4421

Calculate the percentage of L-alanyl-l-glutamine
(CsHisN3O4) in the Sample taken, excluding alanine
and glutamine:
Resultz = [(Result; — a — b)]/[(100 — Ala — Gin)] x 100
a= Ala x (Mn/Mr2)
b= Gin x (Ma/Mz)
Ala = percentage of alanine from the Related
Compounds test
Gin = percentage of glutamine from the Related
Compounds test
Mai = Mee weight of L-alanyl-i-glutamine,
21712
Mz = molecular weight of alanine, 89.1
M3 = molecular weight of glutamine, 146.1
Acceptance criteria: 98.0%-101.5% on the anhydrous
and solvent-free basis, and excluding alanine an
glutamine
IMPURITIES
e RESIDUE ON IGNITION (281); NMT 0.1%
e CHLORIDE AND SULFATE, Chloride (221)
Sample: 0.89g
sae solution: 0.50 mL of 0.010 N hydrochloric
aci
Acceptance criteria: NMT 200 ug/g
CHLORIDE AND SULFATE, Sulfate (221)
Sample: 0.98g
Standard solution: 0.20 mL of 0.010 N sulfuric acid
Acceptance criteria: NMT 200 pg/g
IRON (241): NMT 10 ptg/g
RESIDUAL SOLVENTS (467,
Acceptance criteria
Isopropanol: NMT 0.5%
[Note—For the Acceptance criteria for any other residual
solvents, see Residual Solvents (467).]
Limit oF AMMONIUM
Standard stock solution: Dissolve 1.486 g of ammo-
nium chloride in 500.0 mL of water.
Standard calibration solutions: Transfer 0.01, 0.1, 1.0,
and 10.0 mL of Standard stock solution into separate
100-mL volumetric flasks, and dilute with water to vol-
ume. The final concentrations are 0.1, 1, 10, and
100 g/mL of ammonium ions (NH¢*), respectively.
Sample solution: Transfer 1.0 g of L-Alanyl-L-glutamine
to a 150-mL beaker containing a plastic-coated stirring
bar, add 100.0 mL of water, and stir until dissolved.
the pH, which must be above 11; if not, adjust with
10.N sodium hydroxide. After 3 min, measure the po-
tential, and determine the corresponding ammonium
ion concentration from the calibration curve.
Calculate the content of ammonium in the portion of
the Sample taken:
Result = (Vx ©/W
Vv = volume of the Sample solution (mL)
G = concentration of ammonium ions in the
Sample solution determined from the
Standard response line (ug/mL)
Ww = weight of L-Alanyl-L-glutamine taken to
prepare the Sample solution (g)
Acceptance criteria: NMT 200 g/g
e RELATED COMPOUNDS
Buffer solution: Dissolve 6.84 g of monobasic potas-
sium phosphate in 1000 mL of water.
Mobile phase: Acetonitrile and Buffer solution (650:350)
System suitability solution: Transfer 25 mg of USP L-
Alanyl-L-glutamine RS and 5 mg of USP L-Alanyl-t-ala-
nine RS into a 25-mL volumetric flask, and dilute with
water to volume. Transfer 1.0 mL of this solution into a
10-mL volumetric flask, and dilute with Mobile phase to
volume.
Standard solution 1: 0.025 mg/mL of USP L-Alanine RS
in Mobile phase
Standard solution 2: 0.1 mg/mL of USP Glutamine RS
in Mobile phase
Sample solution: 2.5 mg/mL of t-Alanyl-L-glutamine in
Mobile phase
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode:
Detector: UV 215 nm
Column: 4.6-mm x 25-cm; 5-\um packing L8
Flow rate: 0.7 mL/min
Injection volume: 20 UL
System suitability
Sample: System suitability solution
[Note—The relative retention times for L-alanyl-L-alanine
and L-alanyl-t-glutamine are 0.86 and 1.0,
respectively.]
Suitability requirements
Column efficiency: NLT 8000 theoretical plates for
the L-alanyl-L-glutamine peak
Resolution: NLT 2.0 between L-alanyl-l-glutamine
and L-alanyl-L-alanine
Analysis
Samples: Standard solution 1, Standard solution 2, and
sydesbouow sa

Electrode system: Use a gas-sensing, ammonia-specific Sample solution

indicating electrode with internal reference connected Calculate the percentage of alanine and glutamine in
to a pH meter capable of measuring potentials with a the portion of the Sample taken:
minimum reproducibility of 0.1 mV (see pH (791)).
Condition the electrode according to the manufactur- Result = (ru/rs) x (Cs/Cu) x 100
er’s instructions. ru = peak response of alanine or glutamine from
Analysis the Sample solution
Standard response line: Transfer 100 mL of water into rs = peak response of alanine from Standard
a 150-mL beaker containing a plastic-coated stirring solution 1 or glutamine from Standard
bar, insert the electrode into the water, stir, and meas- solution 2
ure the potential. Add 1 mL of 10 N sodium hydroxide concentration of USP L-Alanine RS in Standard
Cs
i)

solution, stir, and measure the potential after stabiliza-
tion (about 3 min). The potential difference must be
below 20 mV. Transfer 100.0 mL of each of Standard Cu
calibration solutions (0.1, 1, 10, 100 ng/mL of ammo-
nium ions) into separate 150-mL beakers, and add
1 mL of 10 N sodium hydroxide. Insert the electrode
into each solution, stir, and measure the potential after
stabilization (about 3 min). Plot a curve (four calibra-
tion points) of the potential (mV) as function of am-
monium ion concentrations (ug/mL).
Sample: Sample solution fu
Rinse the electrode, insert it into the Sample solution,
add 1 mL of 10 N sodium hydroxide, and stir. Check
solution 1 (mg/mL) or concentration of USP
Glutamine RS in Standard solution 2 gin)
= concentration of L-Alanyl-L-glutamine in the
Sample solution (mg/mL)
Calculate the percentage of any other specified and
uispeetticnd impurities in the portion of the Sample
taken:
Result = (ru/rr) x 100
= peak response of each individual impurity
 4422 Alanyl / Dietary Supplements USP 41

rr = sum of the responses of all the ee Analysis

excluding peak responses of alanine and Samples: Standard solution A, Standard solution B, and
glutamine Sample solution
Acceptance criteria: See Table 1. Develop in a chamber containing Developing solvent sys-
tem A until the solvent front has move 10.5 cm from
Table 1 the origin. Remove the plates, and allow to dry. Turn
the plates 90°, and develop in a chamber containing
Relative Acceptance Developing solvent system B until the solvent front has
Retention Criteria, moved 10.5 cm from the origin. Remove the plates,
Name Time NMT (%) and allow to dry. Spray with Derivatization reagent.
Cyclo(ala-gin) 0.27 0.2 Heat the plates at 105°-110° for 10 min, and examine
Alanine 0.55 1.0 under white light.
Glutamine 0.59 0.5 Suitability requirements: The order, from top to bot-
Ala-ala-gin 1.10 0.3 tom, of ginsenosides on the chromatographic plates is
Rgz (on left) and Rg; (onrigh, Rf, Re, Rd, Rc, Rb2 (on
Ala-glu 2.20 0.2 left) and Rb; (on right), an Ro, Ginsenosides Rg2, Roi,
Any unspecified impurity _— 0.1 Rf, Re, and Rd are found on the upper half of the
Total unspecified impurities = 0.5 lates; the remaining ginsenosides are found on the
lower half after chromatographing with Developing sol-
SPECIFIC TESTS
vent system B. Standard solution A does not exhibit a
spot for ginsenoside Rf. Standard solution B exhibits a
© OPTICAL ROTATION, Specific Rotation (7815S) spot for ginsenoside Rf.
Sample solution: 50 mg/mL in water. Perform the Acceptance criteria: The spots from the Sample solution
measurement at 20°. correspond to those from Standard solution A.
Acceptance criteria: +9.0° to +11.0° ¢ B. The retention times of the peaks for ginsenosides Rgi,
e WATER DETERMINATION, Method Ia (921): NMT 1.0% Re, Rb;, Rb2, Rc2, and Rd of the Sample solution corre-
ADDITIONAL REQUIREMENTS spond to those of Standard solution A, as obtained in the
© PACKAGING AND STORAGE: Preserve in well-closed, tight, test for Content of Ginsenosides. The ratio of the peak
light-resistant containers. responses for ginsenosides Rb2 to Rb; is less than 0.4,
e USP REFERENCE STANDARDS (11) and the ratio of the eakresporise for ginsenosides Rg;
USP L-Alanine RS to Rb; is less than 0.3. The cl romatogram shows no sig-
USP L-Alanyl-L-alanine RS nificant peak at the retention time corresponding to that
USP L-Alanyl-L-glutamine RS for ginsenoside Rf of Standard solution B, as obtained in
USP Glutamine RS the test for Content of Ginsenosides.
COMPOSITION
e CONTENT OF GINSENOSIDES
Solution A: Water
Solution B: Acetonitrile and water (4:1)
Alpha Lipoic Acid—see Alpha Lipoic Acid Mobile phase: See Table 1.
under L.
Table 1
Time Solution A Solution B
(min) (%) (%)
American Ginseng 0 76 24
12 76- 24
DEFINITION
American Ginseng consists of the dried roots of Panax quin- 28 65 35
quefolius L. (Fam. Araliaceae). It contains NLT 4.0% of to- 51.5 56.5 43.5
tal ginsenosides, calculated on the dried basis. 5255, 0 100
64.5 76 24
DS Monographs

IDENTIFICATION
e A. THIN-LAYER CHROMATOGRAPHY
Standard solution A: 20mafmt of USP Powdered
American Ginseng Extract RS in methanol
Standard solution B: 20 mg/mL of USP Powdered
Asian Ginseng Extract RS in methanol
Sample solution: Transfer about 1.0g of finely pow-
dered American Ginseng to a 25-mlL flask fitted with a
reflux condenser. Add 10.0 mL of a mixture of metha-
nol and water (7:13), and heat under reflux for 15 min.
Cool, filter, and dilute the filtrate with methanol to
10.0 mL.
Adsorbent: 0.25-mm layer of silica gel, typically 20 cm
long (TLC plates)
Application volume: 20 pL
Developing solvent system A: Chloroform, methanol,
and water (13:7:2). Use the lower phase.
Developing solvent system B: Butyl alcohol, ethyl ace-
tate, and water (4:1:5). Use the upper phase.
Derivatization reagent: Dissolve 0.5 mL of
anisaldehyde in 10 mL of glacial acetic acid, add 85 mL
of methanol, mix, and carefully add 5 mL of sulfuric
acid.
77 76 24
Diluent: Alcohol and water (4:6)
Standard solution A: Transfer a quantity of USP Pow-
dered American Ginseng Extract RS, equivalent to about
2.mg of ginsenoside Rbj, to a suitable container, and
dissolve in 10.0 mL of Diluent.
Standard solution B: Transfer a quantity of USP Pow-
dered Asian Ginseng Extract RS, equivalent to about
2 mg of ginsenoside Rg;, to a suitable container, and
dissolve in 10.0 mL of Diluent.
Sample solution: Reduce 100 g of American Ginseng to
a powder, and transfer about 7.0 g of the powder, ac-
curately weighed, to a 100-mL round-bottom flask fit-
ted with a reflux condenser. Add 50 mL of Diluent and
a few grains of pumice, boil on a water bath under
reflux for 1 h, cool, and filter. Wash the flask and the
residue with 20 mL of Diluent, and pass through the
same filter. Combine the filtrates, and evaporate in a
rotary evaporator at 50° to dryness. Dissolve the residue
in 10.0 mL of Diluent.
Chromatographic system
(See Chromatography (621), System Suitability.)
USP 41
Mode: LC
Detector: UV 203 nm
Analytical column: 4.6-mm x 15-cm; 3-um packing
L1
Guard column: 4.6-mm x 2.0-cm; packing L1
Column temperature: 25°
Flow rate: 1.5 mL/min
Injection size: 10 uL
System suitability
Sample: Standard solution B
Suitability requirements
Chromatogram similarity: The chromatogram is sim-
ilar to the reference chromatogram provided with the
lot of USP Powdered American Ginseng Extract RS
being used.
Relative standard deviation: NMT 2.0%, determined
for the sum of the peak areas for the six major ginse-
nosides, in replicate injections
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Identify ginsenosides Rgi, Re, Rbi, Rc, Rbz, and Rd in
the Standard solutions and the Sample solution by com-
paring the chromatograms with the reference chro-
matogram provided with USP Powdered American
Ginseng Extract RS, and measure the peak responses.
Calculate the percentages of individual ginsenosides in
the portion of American Ginseng taken:
Result = (ru/rs) x Cs x (V/W) x 100
tu = peak response of ginsenoside Rai, Re, Rb, Rc,
Rb2, or Rd from the Sample solution
Is = peak response of ginsenoside Rgi, Re, Rb:, Rc,
Rb, or Rd from the appropriate Standard
solution
Cs = concentration of ginsenoside Rgi, Re, Rbi, Rc,
Rbz, or Rd in the appropriate Standard
solution (mg/mL)
Vv = volume of the Sample solution (mL)
w = weight of American Ginseng taken to prepare
the Sample solution (mg)
Calculate the percentage of total ginsenosides in the
portion of American Ginseng taken by adding the
individual percentages.
Acceptance criteria: NLT 4.0% of total ginsenosides on
the dried basis
CONTAMINANTS
© ARTICLES OF BOTANICAL ORIGIN, Limits of Elemental Impuri-
ties (561): Meets the requirements
e ARTICLES OF BOTANICAL ORIGIN, Pesticide Residue Analysis
(561): Meets the requirements
e MICROBIAL ENUMERATION TESTS (2021): The total aerobic
microbial count does not exceed 104 cfu/g. The total
combined molds and yeasts count does not exceed 102
u/g.
¢ ABSENCE OF SPECIFIED MICROORGANISMS (2022): It meets
the requirements of the tests for absence of Salmonella
species and Escherichia coli.
SPECIFIC TESTS
© BOTANICAL CHARACTERISTICS
Macroscopic: Fusiform or cylindrical roots, sometimes
branched, typically 1-10 cm, sometimes up to 20 cm,
in length and up to 2.5 cm in diameter at the crown,
with one or more stem scars. Externally pale yellow to
golden, rough-textured, with prominent horizontal
rings and fine longitudinal ridges as a result of drying.
Root scars or fine rootlets are present. If stem base is
present, scales are thin and perishing (differs from P.
ginseng, in which scales at base of stem are fleshy and
persistent). Fracture is short; fractured surface is white
to ivory, with distinct aromatic odor and rings of secre-
tory canals present in secondary phloem.
Dietary Supplements / American Ginseng 4423
Microscopic
Transverse section of root: Multiple layers of thin-
walled cork cells are present. Secondary phloem is
characterized by conspicuous air lacunae; abundant,
starch-containing storage parenchyma; few sieve ele-
ments, found in small groupings; and rings of
schizogenous secretory canals. Each secretory canal is
lined with 6-8 epithelial cells that lack starch. Xylem is
characterized by abundant starch-containing storage
parenchyma and a few tracheary elements, composed
of nonlignified tracheids and slightly lignified spiral or
reticulated vessels lacking secretory canals and found
in isolation or in small groupings. Druse crystals are
sometimes present within vascular parenchyma cells.
Diarch or triarch primary xylem is in center of root.
e ARTICLES OF BOTANICAL ORIGIN, Foreign Organic Matter
(561): NMT 2.0%
e Loss ON DRYING (731)
Sample: 1.0 g of American Ginseng, finely powdered
Analysis: Dry the Sample at 105° for 2 h.
Acceptance criteria: NMT 10.0%
e ARTICLES OF BOTANICAL ORIGIN, Total Ash (561): NMT
8.0%
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in tight, light-resistant
containers, and store protected from heat.
e LABELING: The label states the Latin binomial and, follow-
ing the official name, the parts of the plant contained in
the article.
e USP REFERENCE STANDARDS (11)
USP Powdered American Ginseng Extract RS
USP Powdered Asian Ginseng Extract RS
Powdered American Ginseng
DEFINITION
Powdered American Ginseng is American Ginseng reduced
to a fine or a very fine powder. It contains NLT 4.0% of
total ginsenosides, calculated on the dried basis.
IDENTIFICATION
e A. THIN-LAYER CHROMATOGRAPHY
Standard solution A: 20 mg/mL of USP Powdered
American Ginseng Extract RS in methanol
Standard solution B: 20 mg/mL of USP Powdered
Asian Ginseng Extract RS in methanol
Sample solution: Transfer about 1.0 g of Powdered
American Ginseng to a 25-mL flask fitted with a reflux \~]
“
condenser. Add 10.0 mL of a mixture of methanol and
water (7:13), and heat under reflux for 15 min. Cool, =
filter, and dilute the filtrate with methanol to 10.0 mL. °
]
Adsorbent: 0.25-mm layer of silica gel, typically 20 cm °
long (TLC plates) ©
ca
Application volume: 20 uL 2
Developing solvent system A: Chloroform, methanol, mo]
and water (13:7:2). Use the lower phase. a
ry
Developing solvent system B: Butyl alcohol, ethyl ace-
tate, and water (4:1:5). Use the upper phase.
Derivatization reagent: Dissolve 0.5 mL of
anisaldehyde in 10 mL of glacial acetic acid, add 85 mL
of methanol, mix, and carefully add 5 mL of sulfuric
acid.
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Develop in a chamber containing Developing solvent sys-
tem A until the solvent front has moved 10.5 cm from
the origin. Remove the plates, and allow to dry. Turn
the plates 90°, and develop in a chamber containing
Developing solvent system B until the solvent front has
moved 10.5 cm from the origin. Remove the plates,
 4424 American Ginseng / Dietary Supplements
and allow to dry. Spray with Derivatization reagent.
Heat the plates at 105°-110° for 10 min, and examine
under white light.
Suitability requirements: The order, from top to bot-
tom, of ginsenosides on the chromatographic plates is:
Rgz (on left) and Rg; (on right), Rf, Re, Rd, Rc, Rbz (on
left) and Rb; (on right), and Ro. Ginsenosides Rgz, Rai,
Rf, Re, and Rd are found on the upper half of the
lates; the remaining ginsenosides are found on the
ower half after chromatographing with Developing sol-
vent system B. Standard solution A does not exhibit a
spot for ginsenoside Rf. Standard solution B exhibits a
spot for ginsenoside Rf.
Acceptance criteria: The spots from the Sample solution
correspond to those from Standard solution A.
e B. The retention times of the peaks for ginsenosides Rgu,
Re, Rbi, Rbz, Rc2, and Rd of the Sample solution corre-
spond to those of Standard solution A, as obtained in the
test for Content of Ginsenosides. The ratio of the peak
responses for ginsenosides Rbz to Rb; is less than 0.4,
and the ratio of the peak atna for ginsenosides Roi
to Rb; is less than 0.3. The chromatogram shows no sig-
nificant peak at the retention time corresponding to that
for ginsenoside Rf of Standard solution B, as obtained in
the test for Content of Ginsenosides.
COMPOSITION
@ CONTENT OF GINSENOSIDES
Solution A: Water
Solution B: Acetonitrile and water (4:1)
Mobile phase: See Table 1.
Table 1
Time Solution A Solution B
(min) (%) (%)
0 76 24
12 76 24
28 65 35)
51.5 56.5 43.5
52:5. 0 100
64.5 76 24
77 76 24
Diluent: Alcohol and water (4:6)
Standard solution A: Transfer a quantity of USP Pow-
dered American Ginseng Extract RS, equivalent to about
2 mg of ginsenoside Rb;, to a suitable container, and
dissolve in 10.0 mL of Diluent.
Standard solution B: Transfer a quantity of USP Pow-
dered Asian Ginseng Extract RS, equivalent to about
DS Monographs
2 mg of ginsenoside Rg:, to a suitable container, and
dissolve in 10.0 mL of Diluent.
Sample solution: Transfer about 1.0 g of Powdered
American Ginseng, accurately weighed, to a 100-mL
round-bottom flask fitted with a reflux condenser. Add
50 mL of Diluent and a few grains of pumice, boil on a
water bath under reflux for 1 h, cool, and filter. Wash
the flask and the residue with 20 mL of Diluent, and
pass through the same filter. Combine the filtrates, and
evaporate in a rotary evaporator at 50° to dryness. Dis-
solve the residue in 10.0 mL of Diluent.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 203 nm
Analytical column: 4.6-mm x 15-cm; 3-um packing
u Acceptance criteria: NMT 10.0%
USP 41
Guard column: 4.6-mm x 2.0-cm; packing L1
Column temperature: 25°
Flow rate: 1.5 mL/min
Injection size: 10 uL
System suitability
Sample: Standard solution B
Suitability requirements
Chromatogram similarity: The chromatogram is sim-
ilar to the reference chromatogram provided with the
lot of USP Powdered American Ginseng Extract RS
being used.
Relative standard deviation: NMT 2.0%, determined
for the sum of the peak areas for the six major ginse-
nosides, in replicate injections
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Identify ginsenosides Rg;, Re, Rb:, Rc, Rbz, and Rd in
the Standard solutions and the Sample solution by com-
paring the chromatograms with the reference chro-
matogram provided with USP Powdered American
Ginseng Extract RS, and measure the peak responses.
Calculate the percentages of individual ginsenosides in
the portion of Powdered American Ginseng taken:
Result = (ru/rs) x Cs x (V/W) x 100
ru = peak response of ginsenoside Rai, Re, Rb, Rc,
Rb2, or Rd from the Sample solution
rs = peak response of ginsenoside Raj, Re, Rb, Rc,
Rb, or Rd from the appropriate Standard
solution
Cs = concentration of ginsenoside Rg:, Re, Rbi, Rc,
Rb2, or Rd in the appropriate Standard
solution (mg/mL)
Vv = volume of the Sample solution (mL)
w = weight of Powdered American Ginseng taken
to prepare the Sample solution (mg)
Calculate the percentage of total ginsenosides in the
portion of Powdered American Ginseng taken by
adding the individual percentages.
Acceptance criteria: NLT 4.0% of total ginsenosides on
the dried basis
CONTAMINANTS
¢ ARTICLES OF BOTANICAL ORIGIN, Limits of Elemental Impuri-
ties (561): Meets the requirements
e ARTICLES OF BOTANICAL ORIGIN, Pesticide Residue Analysis
(561): Meets the requirements
e MICROBIAL ENUMERATION TESTS (2021): The total aerobic
microbial count does not exceed 104 cfu/g. The total
ones molds and yeasts count does not exceed 10?
cfu/g.
° ABSENCE OF SPECIFIED MICROORGANISMS (2022): {t meets
the requirements of the tests for absence of Salmonella
species and Escherichia coli.
SPECIFIC TESTS
¢ BOTANICAL CHARACTERISTICS: Pale yellowish-brown pow-
der with a slightly aromatic odor; oval parenchymatous
cells packed with starch granules and occasional druse
crystals of calcium oxalate; yellowish-brown secretory
vessels with yellowish-brown contents
e ARTICLES OF BOTANICAL ORIGIN, Foreign Organic Matter
(561): NMT 2.0%
e Loss ON DRYING (731)
Sample: 1.0 g of Powdered American Ginseng
Analysis: Dry the Sample at 105° for 2 h.
e ARTICLES OF BOTANICAL ORIGIN, Total Ash (561)
Sample: 1.0 g of Powdered American Ginseng
Acceptance criteria: NMT 8%
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Store in tight containers, pro-
tected from light, moisture, and heat.
USP 41 Dietary Supplements / American Ginseng 4425

e LABELING: The label states the Latin binomial and, follow- COMPOSITION
ing the official name, the parts of the plant contained in e@ CONTENT OF GINSENOSIDES
the article. Solution A: Water
e USP REFERENCE STANDARDS (11) Solution B: Acetonitrile and water (4:1)
USP Powdered American Ginseng Extract RS Mobile phase: See Table 7.
USP Powdered Asian Ginseng Extract RS
Table 1
Time Solution A Solution B
(min) (%) (%)
Powdered American Ginseng Extract 0 76 24
12 76 24
DEFINITION 28 65 35
Powdered American Ginseng Extract is prepared from the 515. 56.5 43.5
pulverized dried roots of Panax quinquefolius L. (Fam. 52.5 oO 100
Araliaceae), using suitable solvents, and dried to a pow- 64.5 76 24
der. It contains NLT 10.0% of total ginsenosides, calcu-
77 76 24
lated on the anhydrous basis. The ratio of starting crude
plage material to Powdered American Ginseng Extract is Diluent: Alcohol and water (4:6)
etween 3:1 and 7:1. Standard solution A: Transfer a quantity of USP Pow-
IDENTIFICATION dered American Ginseng Extract RS, equivalent to about
e A. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST 2 mg of ginsenoside Rb;, to a suitable container, and
Standard solution A: 20 pee of USP Powdered dissolve in 10.0 mL of Diluent.
American Ginseng Extract RS in methanol Standard solution B: Transfer a quantity of USP Pow-
Standard solution B: 20 mg/mL of USP Powdered dered Asian Ginseng Extract RS, equivalent to about
Asian Ginseng Extract RS in methanol 2 mg of ginsenoside Rgi, to a suitable container, and
Sample solution: 20 mg/mL in methanol dissolve in 10.0 mL of Diluent.
Adsorbent: 0.25-mm layer of chromatographic silica Sample solution: Transfer a quantity of Powdered
gel, typically 20 cm long (TLC plates) American Ginseng Extract, equivalent to about 5 mg of
Application volume: 20 uL ginsenosidles, to a suitable container. Dissolve in
Developing solvent system A: Chloroform, methanol, 0.0 mL of Diluent, sonicating for 10 min, and filter.
and water (13:7:2). Use the lower phase. Chromatographic system
Developing solvent system B: Butyl alcohol, ethyl ace- (See Chromatography (621), System Suitability.)
tate, and water (4:1:5). Use the upper phase. Mode: LC
Spray reagent: Dissolve 0.5 mL of anisaldehyde in Detector: UV 203 nm
10 mL of glacial acetic acid, add 85 mL of methanol, Analytical column: 4.6-mm x 15-cm; 3-Lm packing
mix, and carefully add 5 mL of sulfuric acid. L1
Analysis Guard column: 4.6-mm x 2.0-cm; packing L1
Samples: Standard solution A, Standard solution B, and Column temperature: 25°
Sample solution Flow rate: 1.5 mL/min
Develop in a chamber containing Developing solvent sys- Injection size: 10 uL
tem A until the solvent front has moved 10.5 cm from System suitability
the origin. Remove the plates, and allow to dry. Turn Sample: Standard solution B
the plates 90°, and develop in a chamber containing Suitability requirements
Developing solvent system B until the solvent front has Chromatogram similarity: The chromatogram is sim-
moved 10.5 cm from the origin. Remove the plates, ilar to the Reference Chromatogram provided with
and allow to dry. Spray with Spray reagent. Heat the the lot of USP Powdered Asian Ginseng Extract RS
plates at 105°-110° for 10 min, and examine. being used.
Suitability requirements: The order, from top to bot- Relative standard deviation: NMT 2.0%, determined
tom, of ginsenosides on the poraloa aphie plates is: for the sum of the peak areas for the 6 major ginse-
nosides, in replicate injections
sydesbouo-; sa

Roe (on left) and Rg; (on right), Rf, Re, Rd, Rc, Rb2 (on
left) and Rb; (on right), and Ro. Ginsenosides Rgz, Rai,
Rf, Re, and Rd are found on the upper half of the
lates; the remaining ginsenosides are found on the
jower half after chromatographing with Developing sol-
vent system B. Standard solution A does not exhibit a
spot for ginsenoside Rf. Standard solution B exhibits a
spot for ginsenoside Rf.
Acceptance criteria: The spots from the Sample solution
correspond to those from Standard solution A.
¢ B. The retention times of the peaks for ginsenosides Rgi,
Re, Rb;, Rbz, Rc, and Rd of the Sample solution corre-
spond to those of Standard solution A, as obtained in the
test for Content of Ginsenosides. The ratio of the peak
responses for ginsenosides Rbz to Rb; is less than 0.4,
and the ratio of the peak responses for ginsenosides Rg:
to Rb; is less than 0.3. The Sample solution shows no
significant peak at the retention time corresponding to
that for ginsenoside Rf of Standard solution B, as obtained
in the test for Content of Ginsenosides.
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Identify ginsenosides Rg:, Re, Rb:, Rc, Rbz, and Rd in
the Standard solutions and the Sample solution by com-
paring the chromatograms with the Reference Chro-
matogram provided with USP Powdered American
Ginseng Extract RS, and measure the peak responses.
Calculate the percentages of individual ginsenosides in
mG portion of Powdered American Ginseng Extract
taken:
Result = (ru/rs) x (Cs/Cu) x P
ru = peak response of ginsenosides Rgi, Re, Rbi,
Rc, Rb2, or Rd from the Sample solution
rs = peak response of ginsenosides Rg:, Re, Rbi,
Rc, Rb2, or Rd from the appropriate Standard
solution
Cs = concentration of ginsenosides Rgi, Re, Rbi, Rc,
Rb, or Rd in the appropriate Standard
solution (mg/mL)
 4426 American Ginseng / Dietary Supplements USP 41

Cu = concentration of Powdered American Ginseng Powdered Extract, to a conical flask. Extract at 55°
Extract in the Sample solution (mg/mL) with three 20-mL portions of a mixture of methanol
P = labeled amount, in percentage, of each and water (2:8). Evaporate the combined extracts to
relevant ginsenoside in USP Powdered dryness under vacuum at 45°-50°. Dissolve the residue
American Ginseng Extract RS in 5 mL of methanol.
Calculate the percentage of total ginsenosides in the Standard solution A: 20 mg/mL of USP Powdered
poten of Powdered American Ginseng Extract taken American Ginseng Extract RS in methanol
y adding the individual percentages. Standard solution B: 20 mg/mL of USP Powdered
Acceptance criteria: NLT 10.0% of total ginsenosides Asian Ginseng Extract RS in methanol
on the anhydrous basis Application volume: 20 wL
Developing solvent system A: The lower phase of a
CONTAMINANTS mixture of chloroform, methanol, and water (13:7:2)
Developing solvent er B: The upper phase of a
Delete the following: mixture of butyl alcohol, ethyl acetate, and water
(4:1:5)
©. HEAVY METALS, Method II (231): NMT 20 ppm ee Spray reagent: Dissolve 0.5 mL of anisaldehyde in
SRE aa) PEO 10 mL oralacial acetic acid, add 85 mL of methanol,
e ARTICLES OF BOTANICAL ORIGIN, General Method for Pesti- ele Sere ully add 5 mL of sulfuric acid, and mix.
cide Residues Analysis (561): Meets the requirements Sy yr 1S TesGniti ard ehh
© MICROBIAL ENUMERATION TESTS (2021): The total aerobic amples: Sample solution, Standard solution A, and
Standard solution B
microbial count does not exceed 104 cfu/g. The total
combined molds and yeasts count does not exceed 103 Develop the chromatograms in a chamber containing
cfu/g. Davsioniag solvent system A until the solvent front has
e MICROBIOLOGICAL PROCEDURES FOR ABSENCE OF SPECIFIED MI- moved 10.5 cm from the origin. Remove the plate
CROORGANISMS (2022): It meets the requirements of the from the chamber, and allow to dry. Turn the plate
tests for absence of Salmonella species and Escherichia 90°, and develop in a chamber containing Developing
solvent system B until the solvent front has moved
coll 10.5 cm from the origin. Remove the plate from the
SPECIFIC TESTS chamber, and allow to dry. Spray with Spray reagent.
¢ WATER DETERMINATION, Method | (921): NMT 7.0% Heat the plate at 105°-110° for 10 min, and ex-
e BOTANICAL EXTRACTS, Residue on Evaporation (565): Meets amine. The order, from top to bottom, of ginseno-
the requirements sides on the plates is Rgz (on left) and Rg; (on right),
© ALCOHOL DETERMINATION, Method |/ (611): NMT 0.25% iy Re, Rd, Rc, Rbz (on left) and Rb; (on right), and
‘0.
ADDITIONAL REQUIREMENTS Ginsenosides Rgz, Rgi, Rf, Re, and Rd are found on the
© PACKAGING AND STORAGE: Preserve in tight, light-resistant upper half of the eee the remaining ginsenosides
containers. are found on the lower half after chromatographing
e LABELING: The label states the Latin binomial and, follow- with Developing solvent system B.
ing the official name, the Ba of the plant from which Acceptance criteria: Standard solution A does not ex-
the article was derived. Label it to indicate the content of hibit a spot for ginsenoside Rf. Standard solution B ex-
total ginsenosides, the extracting solvent used for prepa- hibits a spot for ginsenoside Rf. The spots from the
ration, and the ratio of the starting crude plant material Sample solution correspond to those from Standard solu-
to the Powdered Extract. It meets the labeling require- tion A.
ments under Botanical Extracts (565). e B. The retention times of the peaks for ginsenosides Rgi,
e USP REFERENCE STANDARDS (11) Re, Rb;, Rbz, Rcz, and Rd in the chromatogram of the
USP Powdered American Ginseng Extract RS Sample solution correspond to those from the Standard
USP Powdered Asian Ginseng Extract RS solution, as obtained in the test for Content of Ginseno-
sides. The ratio of the peak response for Rb2 to the peak
response for Rb, is less than 0.4; and the ratio of the
peak response for Rg; to the peak response for Rb, is less
than 0.3. There is no significant peak at the retention
DS Monographs

American Ginseng Capsules time corresponding to that of ginsenoside Rf in the Sys-

tem pete solution, as obtained in the test for Content
DEFINITION of Ginsenoside.
American Ginseng Capsules contain Powdered American
Ginseng Extract. Capsules contain NLT 90.0% and NMT ane GINSENOSIDES
110.0% of the labeled amount of Extract, calculated as Method 1
the sum of ginsenosides Rgi, Re, Rbi, Rc, Rbz, and Rd. Diluent: Water and alcohol (3:2)

IDENTIFICATION Solution A: Water

© A.(201)
THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST Solution B: Acetonitrile and water (4:1)
Mobile phase: See the gradient table below.
Sample solution
Soft-shell gelatin Capsules: Transfer a portion of the Time Solution A Solution B
contents of the Capsules, equivalent to 100 mg of (min) (%) (%)
Powdered Extract, to a separatory funnel containing 0 76 24
30 mL of a mixture of hexanes, methanol, and water 12 76 24
(20:15:10), dissolve in this mixture, and collect the
lower layer. Wash the upper layer with three 15-mL ze 85 35.
portions of a mixture of methanol and water (15:10), 51.5 56.5 43.5
and combine the washings with the lower layer. Evap- 52.5 0 100
orate to dryness under vacuum at 45°-50°. Dissolve 64.5 76 24
the residue in 5 mL of methanol. 77 76 24
Hard-shell gelatin Capsules: Transfer a portion of the
contents of the Capsules, equivalent to 100 mg of
USP 41
Standard solution: A solution of USP Powdered Amer-
ican Ginseng Extract RS in Diluent containing the
equivalent of 0.2 mg/mL of ginsenoside Rb;
Sample solution (soft-gelatin Capsules): Open NLT 20
Capsules, transfer the contents to a suitable container,
and mix to homogenize. Transfer a portion, expected
to contain an amount of Extract equivalent to 12 mg
of ginsenosides, to a suitable flask with a stopper. Add
5.0 mL of tetrahydrofuran, and sonicate for 5 min.
Add 25.0 mL of a mixture of methanol and water
(4:6), and shake for 50 min in an automatic shaker.
Transfer 15.0 mL of the obtained emulsion to a centri-
fuge tube with a stopper, add 800 mg of sodium chlo-
ride, shake for 30 s, and centrifuge to obtain a clear
upper phase. Dilute 1.0 mL of the upper phase with
4 mL of water in a suitable tube, and transfer the solu-
tion to a column containing 360 mg of packing L2
that has been previously treated with 3.0 mL of meth-
anol followed by 8.0 mL of water. [NoTE—Elute slowly,
not faster than 1 drop/s, in all elution steps. Do not
use vacuum,] Rinse the tube with 5 mL of water, trans-
fer to the column taking the precaution of slow elu-
tion, and discard the eluate. Repeat the elution with
5 mL of a mixture of methanol and water (4:6), and
discard the eluate. Elute the ginsenosides with 5.0 mL
of methanol. Evaporate the solution under a stream of
nitrogen at 40° (50 min), and dissolve the residue with
1.0 mL of a solution of acetonitrile and water (1:4).
System suitability solution: 24 mg/mL of USP Pow-
dered Asian Ginseng Extract RS in Diluent. Filter.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 203 nm
Column
Guard column: 4.6-mm x 2.0-cm; packing L1
Aratytteal column: 4.6-mm x 15-cm; 3-um packing
Column temperature: 25°
Flow rate: 1.5 mL/min
Injection size: 10 ul
System suitability
Sample: System suitability solution (inject 20 wL)
Suitability requirements
Chromatogram similarity: The System suitability solu-
tion chromatogram is similar to the Reference Chro-
matogram provided with the lot of USP Powdered
Asian Ginseng Extract RS being used.
Relative standard deviation: NMT 2.0%, determined
for the sum of the peak areas for the six major ginse-
nosides, in repeated injections
Analysis
Samples: Standard solution and Sample solution
Identify ginsenosides Rg:, Re, Rbi, Rc, Rb2, and Rd in
the Standard solution and the Sample solution by
comparing the chromatograms with the Reference
Chromatogram provided with USP Powdered Ameri-
can Ginseng Extract RS being used, and measure the
peak responses.
Calculate the quantity, in mg, of each relevant ginse-
noside (Rg, Re, Rbi, Rc, Rbz, and Rd) in the portion
of Capsule contents taken:
Result = 0.3 x (ru/ts) x Cs x P
tu = peak area for each relevant ginsenoside from
the Sample solution
rs = peak area for each relevant ginsenoside from
the Standard solution
Cs = concentration of USP Powdered American
Ginseng Extract RS in the Standard solution
rain)
P = labeled amount, in percentage, of each
relevant ginsenoside in the USP Powdered
American Ginseng Extract RS lot being used
Dietary Supplements / American Ginseng 4427
Calculate the content of total a T, in mg,
by adding the amounts of individual ginsenoside.
Calculate the percentage of Powdered Extract with
respect to the label claim:
Result = T x (Aws/W) x (100/L) x (100/L)
T = content of total ginsenosides in the portion of
Capsule contents taken (mg)
Awr = average weight of Capsule contents (mg/
Capsule)
Ww = weight of the portion of Capsule contents
taken (mg)
Le = content of total ginsenosides, mg, in 100 mg
of the Extract used to prepare the Capsules
L: = amount of Extract per Capsule according to
label claim (mg/Capsule)
Method 2
Diluent, Solution A, Solution B, Mobile phase,
System suitability solution, Chromatographic
system, and Suitability requirements: Proceed as di-
rected under Method 7.
Solvent A: Upper phase of a mixture consisting of
hexane, methanol, and water (4:3:2)
Solvent B: Lower phase of a mixture consisting of
hexane, methanol, and water (4:3:2)
Standard solution: A solution of USP Powdered Amer-
ican Ginseng Extract RS in Diluent containing the
equivalent of 1 mg/mL of ginsenoside Rb;
Sample solution A (for soft-gelatin Capsules): Open
NLT 20 Capsules and transfer the contents to a suita-
ble container. Mix to homogenize and transfer a por-
tion, expected to contain an amount of Extract equiva-
lent to 15 mg of total ginsenosides, to a 50-mL flask.
Add 10.0 mL of Solvent A, and sonicate for 3-5 min at
25°-30°. Transfer the solution to a 125-mL separatory
funnel. To the residue add 10 mL of Solvent B, and
sonicate for 3-5 min at 25°-30°. Transfer the solution
to the same separatory funnel. Repeat the above pro-
cedure twice (the total volume will be about 60 mL).
Shake, and then allow the phases to separate. Collect
the combined lower phase in a round-bottom flask,
and wash the combined upper phase twice with
10 mL of Solvent B. Evaporate the combined lower
phase to dryness under vacuum at 45°-50°. Transfer
the residue to a 10-mL volumetric flask using small
volumes of methanol, and dilute with methanol to
volume.
Sample solution B (for hard-gelatin Capsules): Weigh
the contents of NLT 20 Capsules, and composite the
contents. Transfer a portion of the composite, ex-
pected to contain an amount of Extract equivalent to
sydesBouow: sa
15 mg of total ginsenosides, to a conical flask. Add
15 mL of methanol, and shake to mix. Sonicate the
mixture at 25°-30° for 30 min. Cool, pass through
filter paper, and return the residue to the conical flask.
Add another 15 mL of methanol, sonicate the mixture
at 25°-30° for 30 min, and filter. Wash the residue
with three 15-mL portions of methanol. Evaporate the
combined extracts and washing to dryness under vac-
uum at 45°-50°. Transfer the residue to a 10-mL volu-
metric flask using small volumes of methanol, and di-
lute with methanol to volume.
Analysis
Samples: Standard solution and Sample solution
Identify ginsenosides Rg:, Re, Rbi, Rc, Rb2, and Rd in
the Standard solution and the Sample solution by
comparing the chromatograms with the Reference
Chromatogram provided with USP Powdered Ameri-
can Ginseng Extract RS, and measure the peak
responses.
 4428 American Ginseng / Dietary Supplements
Calculate the quantity, in mg, of each relevant ginse-
noside (Rgi, Re, Rb:, Rc, Rbz, and Rd) in the portion
of Capsule contents taken:
Result = 0.1 x (ru/rs) x Cs x P
tu = peak area for each relevant ginsenoside from
the Sample solution
Is = peak area for each relevant ginsenoside from
the Standard solution
Gs = concentration of USP Powdered American
Ginseng Extract RS in the Standard solution
(mg/ml)
RP = labeled amount, in percentage, of each
relevant ginsenoside in the USP Powdered
American Ginseng Extract RS lot being used
Calculate the content of total ginsenosides; T, in mg,
by adding the amounts of individual ginsenoside.
Calculate the percentage of Powdered Extract with
respect to the label claim:
Result = T x (Awr/W) x (100/Le) x (100/L)
T = content of total ginsenosides in the portion of
Capsule contents taken (mg)
Awr = average weight of Capsule contents (mg/
Capsule)
Ww = weight of the portion of Capsule contents
taken (mg)
Le = content of total ginsenosides, mg, in 100 mg
of the Extract used to prepare the Capsules
L = amount of Extract per Capsule according to
label claim (agie eules
Acceptance criteria: 90.0%-110.0% of Extract, calcu-
lated as the sum of ginsenosides Rgi, Re, Rbi, Rc, Rbz,
and Rd
PERFORMANCE TESTS
e DISINTEGRATION AND DISSOLUTION OF DIETARY SUPPLEMENTS
(2040): Meet the requirements for Disintegration
e WEIGHT VARIATION OF DIETARY SUPPLEMENTS (2091): Meet
the requirements
CONTAMINANTS
e MICROBIAL ENUMERATION TESTS (2021): The total aerobic
microbial count does not exceed 104 cfu/g. The total
combined molds and yeasts count does not exceed 103
cfu/g.
° Anseice OF SPECIFIED MICROORGANISMS (2022): Meet the
requirements of the tests for absence of Salmonella spe-
cies and Escherichia coli.
DS Monographs
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in tight containers,
protected from light. Store at controlled room
temperature.
e LABELING: The label states the Latin binomial and, follow-
ing the official name, the article from which the Capsules
were prepared. The label also indicates the amount of
Extract, in mg/Capsule. Label the Capsules to indicate
the percentage of ginsenosides in the Extract contained
in the Capsules. For soft-gelatin Capsules, state the
method for Content of Ginsenosides with which the prod-
uct complies only if Method 7 is not used.
USP 41
o USP REFERENCE STANDARDS (11)
USP Powdered American Ginseng Extract RS
USP Powdered Asian Ginseng Extract RS
American Ginseng Tablets
DEFINITION
American Ginseng Tablets contain Powdered American Gin-
seng Extract. Tablets contain NLT 90.0% and NMT
110.0% of Extract, calculated as the sum of ginsenosides
Rgi, Re, Rb;, Rc, Rbz, and Rd.
IDENTIFICATION
° a CHROMATOGRAPHIC IDENTIFICATION TEST
Standard solution A: 20 mg/mL of USP Powdered
American Ginseng Extract RS in methanol
Standard solution B: 20 mg/mL of USP Powdered
Asian Ginseng Extract RS in methanol
Sample solution: Transfer a quantity of finely powdered
Tablets, equivalent to 100 mg of Extract, to a conical
flask. Extract at 55° with three 20-mL portions of a mix-
ture of methanol and water (2:8). Evaporate the com-
bined extracts to dryness under vacuum at 45°-50°.
Dissolve the residue in 5 mL of methanol.
Application volume: 20 uL
ay solvent system A: The lower phase of a
mixture of chloroform, methanol, and water (13:7:2)
eoing solvent Seren B: The upper phase of a
as). of butyl alcohol, ethyl acetate, ond-wates
4:1:5
Spray reagent: Dissolve 0.5 mL of anisaldehyde in
10 mL of glacial acetic acid, add 85 mL of methanol,
mix, carefully add 5 mL of sulfuric acid, and mix.
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Proceed as directed in the chapter. Develop in a
chamber containing Developing solvent system A until
the solvent front has moved 10.5 cm from the origin.
Remove the plates from the chamber, and allow to
dry. Turn the plates 90°, and develop in a chamber
containing Developing solvent system B until the sol-
vent front has moved 10.5 cm from the origin. Re-
move the plates from the chamber, and allow to dry.
Spray with Spray reagent. Heat the plates at
105°-110° for 10 min, and examine. The order, from
top to bottom, of ginsenosides on the plates is Rgz
(on left) and Rg: (on right), Rf, Re, Rd, Rc, Rb2 (on
left) and Rb; (on right), and Ro. Ginsenosides Rgz,
Rgi, Rf, Re, and Rd are found on the upper half of
the plates; the remaining ginsenosides are found on
the lower half after chromatographing with Develop-
ing solvent system B
Acceptance criteria: The chromatogram of Standard so-
lution A does not exhibit a spot for ginsenoside Rf. Stan-
dard solution B exhibits a spot for ginsenoside Rf. The
spots from the Sample solution correspond to those
from Standard solution A.
e B. The retention times of the peaks for ginsenosides Rgi,
Re, Rb;, Rbz, Rez, and Rd in the chromatogram of the
Sample solution correspond to those from the Standard
solution, as obtained in the test for Content of Ginseno-
sides. The ratio of the peak response for Rb2 to the peak
response for Rb; is less than 0.4; and the ratio of the
peak response forRa to the peak response for Rb; is less
than 0.3. The Sample solution chromatogram shows no
significant peak at the retention time corresponding to
that of ginsenoside Rf in the System suitability solution, as
obtained in the test for Content of Ginsenosides.
USP 41 Dietary Supplements / Andrographis 4429

STRENGTH rs = peak area for each relevant ginsenoside from

e CONTENT OF GINSENOSIDES the Standard solution
Diluent: Water and alcohol (3:2) Cs = concentration of USP Powdered American
Solution A: Water Ginseng Extract RS in the Standard solution
Solution B: Acetonitrile and water (4:1) (mg/mL)
Mobile phase: See the gradient table below. P = labeled amount, in percentage, of each
relevant ginsenoside in the USP Powdered
Time Solution A Solution B American Ginseng Extract RS lot being used
(min) (%) (%) Calculate the content of total ginsenosides, T, in mg,
0 76 24
by adeig i amounts of individual ginsenoside.
Calculate the percentage of Powdered Extract with
12 76 24 respect to the label claim:
28 65 35
5135 56.5 43.5 Result = T x (Awr/W) x (100/Le) x (100/L)
$2.5 0 100
a = content of total ginsenosides in the portion of
64.5 76 24
Tablets taken (mg)
77 76 24 Awr = average weight of Tablets (mg/Tablet)
Ww = weight of the portion of Tablets taken (mg)
Standard solution: A solution of USP Powdered Ameri- Le = content of total ginsenosides, mg, in 100 mg
can Ginseng Extract RS in Diluent containing the equiv- of the Extract used to prepare the Tablets
alent of 1 mg/mL of ginsenoside Rb; L = amount of Extract per Tablet according to
Sample solution: Accurately weigh and finely powder label claim (mg/Tablet)
NLT 20 Tablets. Transfer to a conical flask an accurately Acceptance criteria: 90.0%-110.0% of Powdered Ex-
weighed portion of the powder expected to contain an tract, calculated as the sum of ginsenosides Rg;, Re,
amount of Extract equivalent to 15 mg of total ginseno- Rb;, Rc, Rbz, and Rd
sides, add 15 mL of methanol, and shake to mix. Soni-
cate the mixture at 25°-30° for 30 min. Cool, pass PERFORMANCE TESTS
through filter paper, and return the residue to the coni- © DISINTEGRATION AND DISSOLUTION OF DIETARY SUPPLEMENTS
cal flask. Add another 15 mL of methanol, sonicate the (2040): Meet the requirements for Disintegration
mixture at 25°-30° for 30 min, and filter. Wash the e WEIGHT VARIATION OF DIETARY SUPPLEMENTS (2091): Meet
residue with three 15-mL portions of methanol. Evapo- the requirements
rate the combined extracts and washings to dryness
under vacuum at 45°-50°. Transfer the residue to a CONTAMINANTS
10.0-mL volumetric flask, using small volumes of meth- ¢ MICROBIAL ENUMERATION TESTS—NUTRITIONAL AND DIETARY
anol, and dilute with methanol to volume. SUPPLEMENTS (2021): The total aerobic microbial count
System suitability solution: 24 mg/mL of USP Pow- does not exceed 104 cfu/g. The total combined molds
dered Asian Ginseng Extract RS in Diluent. Filter. and yeasts count does not exceed 103 cfu/g. Meet the
Chromatographic system requirements of the tests for absence of Salmonella spe-
(See Chromatography (621), System Suitability.) cies and Escherichia coli.
Mode: LC
Detector: UV 203 nm ADDITIONAL REQUIREMENTS
Column © PACKAGING AND STORAGE: Preserve in tight containers,
Guard column: 4.6-mm x 2.0-cm; packing L1 protected from light. Store at controlled room
i need column: 4.6-mm x 15-cm; 3-um packing temperature.
L e LABELING: The label states the Latin binomial and, follow-
Column temperature: 25° ing the official name, the article from which the Tablets
Flow rate: 1.5 mL/min were prepared. The label also indicates the amount of
Injection size: 10 ul Extract, in mg/Tablet. Label the Tablets to indicate the
System suitability percentage of total ginsenosides in the Extract contained
Sample: System suitability solution (inject 20 wL) in the Tablets.
Suitability requirements e USP REFERENCE STANDARDS (11)
Chromatogram similarity: The System suitability solu- USP Powdered American Ginseng Extract RS Ss
tion chromatogram is similar to the Reference Chro- USP Powdered Asian Ginseng Extract RS Fd
matogram provided with the lot of USP Powdered °
Asian Ginseng Extract RS being used. |
Relative standard deviation: NMT 2.0%, determined °
for the sum of the peak areas for the six major ginse- =
nosides, in repeated injections Andrographis 3
Analysis
Samples: Standard solution and Sample solution DEFINITION wv
Identify ginsenosides Rgi, Re, Rb;, Rc, Rbz, and Rd in Andrographis consists of the dried stems and leaves of An-
the Standard solution and ape solution by compar- drographis paniculata (Burm. f.) Nees (Fam. Acanthaceae).
ing the chromatograms with the Reference Chromat- It contains NLT 1.0% of diterpene lactones, calculated on
ogram provided with USP Powdered American Gin- the dried basis as the sum of andrographolide, neoandro-
seng Extract RS lot being used, and measure the peak grapholide, 14-deoxy-11,12-didehydroandrographolide,
responses. andrograpanin.
Calculate the quantity, in mg, of each relevant ginse-
noside (Rg, Re, Rbi, Rc, Rbz, and Rd) in the portion IDENTIFICATION
of Tablets taken: ° * lalla CHROMATOGRAPHIC IDENTIFICATION TEST
201
Result = 0.1 x (ru/rs) x Cs x P Standard solution 1: Use Standard solution A, prepared
as directed in the test for Content of Diterpene Lactones.
tu = peak area for each relevant ginsenoside from Standard solution 2: Sonicate an amount of USP Pow-
the Sample solution dered Andrographis Extract RS, equivalent to about
 4430 Andrographis / Dietary Supplements USP 41

15 mg of diterpene lactones, for 10-15 min in 25 mL of Table 1

methanol, centrifuge, and use the supernatant. Ti Solution Soluti
Sample solution: Use Sample stock solution, prepared as aan sa vob)” e avon B
directed in the test for Content of Diterpene Lactones. — ae (%)
Adsorbent: Chromatographic silica gel mixture with an 9 as 7
average particle size of 10-15 um (TLC plates) 18 35 45
Application volume: 10 UL, as 5-10 mm bands 25 20 80
Developing solvent system: Chloroform, acetone, and 28 20 80
toluene (2:2:1) 35 55 45
Derivatization reagent: A mixture of 1% vanillin in al- 40 95
cohol and 10% sulfuric acid in alcohol (1:1) 3
Analysis 45 95 $
Samples: Standard solution 1, Standard solution 2, and Chromatographic system
Sample solution (See Chromatography (621), System Suitability.)
Use a saturated chamber. Develop until the solvent Mode: LC
front has moved up about 90% of the length of the Detector: UV 223 nm
plate. Remove the plate from the chamber, dry, treat Column: 4.6-mm x 25-cm; 5-um packing L1
with Derivatization reagent, heat for 5-10 min at 100°, Flow rate: 1.5 mL/min
and examine under white light. Injection volume: 20 pL
Acceptance criteria: The Sample solution exhibits three System suitability
main grayish-blue zones with R; values of approximately Samples: Standard solution A and Standard solution B
0.4, 0.6, and 0.8 that correspond in position and color Suitability requirements
to zones in Standard solution 2. Standard solution 1 ex- The chromatogram of Standard solution B is similar to
hibits a grayish-blue zone due to andrographolide at an the reference chromatogram provided with the lot of
R; of about 0.4. The Sample solution exhibits a zone USP Powdered Andrographis Extract RS being used.
similar in color and R- value to that due to andrograph- Column efficiency: NLT 5000 theoretical plates,
olide in Standard solution 1. Standard solution A
e B. The retention time of the main peak of the Sample Tailing factor: NMT 1.5 for the andrographolide
solution obtained in the test for Content of Diterpene Lac- peak, Standard solution A
tones corresponds to that of andrographolide in Standard Relative standard deviation: NMT 2.0%, determined
solution A. \dentify other diterpene lactone peaks in the for the andrographolide peak in replicate injections,
Sample solution by comparison with Standard solution B Standard solution A
and the reference chromatogram provided with the lot Resolution: NLT 5 between the neoandrographolide
of USP Powdered Andrographis Extract RS being used. and 14-deoxy-11,12-didehydroandrographolide
The Sample solution shows additional peaks correspond- peaks, Standard solution B
ing to neoandrographolide, 14-deoxy-11,12- Analysis
didehydroandrographolide, and andrograpanin. Samples: Standard solution A, Standard solution B, and
Sample solution
COMPOSITION Using the chromatogram of Standard solution A, Stan-
e CONTENT OF DITERPENE LACTONES
Solution A: Dissolve 0.14 g of potassium dihydrogen dard solution B, and the reference chromatogram pro-
phosphate in 900 mL of water, add 0.5 mL of phos- vided with the lot of USP Powdered Andrographis Ex-
phoric acid, dilute with water to 1000 mL, mix, filter, tract RS being used, identify the retention times of the
and degas. peaks corresponding to the different diterpene lac-
Solution B: Acetonitrile, filtered and degassed tones. The approximate relative retention times of the
Standard solution A: Dissolve a weighed quantity of different diterpene lactones are provided in Table 2.
USP Andrographolide RS in methanol to obtain a 1.0-
mg/mL solution. Transfer 5.0 mL of this solution to a Table 2
10-mL volumetric flask, dilute with acetonitrile to vol- Relative
ume, and mix. Retention
Standard solution B: Transfer an amount of USP Pow- Analyte Time
dered Andrographis Extract RS, equivalent to about
DS Monographs

Andrographolide 1.00
25 mg of diterpene lactones, to a 50-mL volumetric
flask, add 25 mL of methanol, heat gently for 15-20 Neoandrographolide 1.16
min, dilute with acetonitrile to volume, and mix. Before 14-Deoxy-11,12-didehydroandrographolide 31
injection, pass through a membrane filter of 0.45-um or Andrograpanin 150
finer pore size, discarding the first 5 mL of the filtrate.
Sample stock solution: Transfer about 209 of finel Separately calculate the percentages of andrographo-
powdered Andrographis to a 250-mlL flask fitted with a lide, neoandrographolide, 14-deoxy-11,12-
reflux condenser. Add 50 mL of methanol, reflux for 15 didehydroandrographolide, and andrograpanin in the
min, cool to room temperature, and decant the super- portion of Andrographis taken:
natant. Repeat until the extract is colorless. Combine
the extracts, filter, concentrate under vacuum, and ad- Result = (ru/rs) x (Cs/W) x 10F
just the volume to 50.0 mL using methanol.
Sample solution: Transfer 25.0 mL of Sample stock solu- ru = peak area of each identified diterpene lactone
tion to a 50-mL volumetric flask, dilute with acetonitrile in the Sample solution
to volume, and mix. Before injection, pass through a ls = peak area of andrographolide in Standard
membrane filter of 0.45-um or finer pore size, discard- solution A
ing the first 5 mL of the filtrate. Cs = concentration of USP Andrographolide RS in
Mobile phase: See Table 1. Standard solution A (mg/ml)
w = weight of Andrographis taken to prepare the
Sample solution (g)
USP 41
F = conversion factor: 1.00 for andrographolide,
3.90 for neoandrographolide, 1.45 for
14-deoxy-11,12-didehydroandrographolide,
and 2.65 for andrograpanin
Acceptance criteria: NLT 1.0% for the sum of the per-
centages of andrographolide, neoandrographolide,
14-deoxy-11,1 ee eden ide, and an-
drograpanin, on the dried basis
IMPURITIES
e ARTICLES OF BOTANICAL ORIGIN, Limits of Elemental Impuri-
ties (561): Meets the requirements
© ARTICLES OF BOTANICAL ORIGIN, Foreign Organic Matter
(561): NMT 2.0%
e ARTICLES OF BOTANICAL ORIGIN, Pesticide Residue Analysis
(561): Meets the requirements
SPECIFIC TESTS
© BOTANICAL CHARACTERISTICS
Macroscopic: Stem is dark green, woody, 2-6 mm in
diameter, peabng numerous branches, showing slightly
swollen nodes, the upper part is distinctly quadrangular
with four bulges in the four corners, and the lower part
is somewhat rounded; texture is fragile, easily broken;
branches are quadrangular, often narrowly winged in
the upper part. Leaves are simple, opposite, short, peti-
olated or nearly sessile; lamina is crumpled and easily
broken, lanceolate or ovate-lanceolate when whole,
2-7 cm long, 1-3 cm wide, with acuminate apex, retic-
ulate venation, and cuneate-decurrent base, margin en-
tire or undulate; the upper surface is green, the lower
surface grayish-green; both surfaces are glabrous. Phar-
macopeial article consists of dry mixtures of crisp, dark-
em broken leaves and quadrangular stems; leaves are
rittle; stems are fracture short, fibrous.
Microscopic
Transverse section of stems: Epidermal layer shows
cells containing round, long-elliptical or clavate cal-
cium carbonate deposits (cystoliths), 1-4 celled non-
glandular hairs and multicellular, disk-shaped glandular
airs; collenchyma is below the epidermis and in the
bulges; endodermis is distinct; vascular bundles sur-
round the parenchyma of the central pith; small acicu-
lar crystals of calcium oxalate are present in the cortex
and pith.
Transverse section of leaves: Subsquare or rectangu-
lar upper and lower epidermal cells; lower epidermal
cells are relatively smaller; both epidermal layers show
cells containing cystoliths, nonglandular hairs and
glandular hairs similar to those of the stem; mesophyll
is composed of 1-2 layers of palisade parenchyma and
spongy parenchyma; loosely arranged spongy paren-
chyma ar across the upper part of the midrib;
vascular bundles of midrib are collateral and grooved;
cells containing cystoliths appear above the xylem.
Loss ON DRYING (731)
Sample: 1.0g of finely powdered Andrographis
Analysis: Dry the Sample at 105° for 3 h.
Acceptance criteria: NMT 12.0%
ARTICLES OF BOTANICAL ORIGIN, Total Ash (561)
Sample: 1.0 of finely powdered Andrographis
Acceptance criteria: NMT 15%
e ARTICLES OF BOTANICAL ORIGIN, Acid-Insoluble Ash (561):
NMT 3.0%
¢ ARTICLES OF BOTANICAL ORIGIN, Alcohol-Soluble Extractives,
Method 2 (561): NLT 8.0%
MICROBIAL ENUMERATION TESTS (2021): The total aerobic
bacterial count does not exceed 105 cfu/g; the total com-
bined molds and yeasts count does not exceed 103 cfu/
g; and the bile-tolerant Gram-negative bacterial count
does not exceed 103 cfu/g.
ABSENCE OF SPECIFIED MICROORGANISMS (2022): Meets the
requirements of the tests for absence of Salmonella spe-
cies and Escherichia coli
Dietary Supplements / Andrographis 4431
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in well-closed contain-
ers, protected from light and moisture, and store at
room temperature.
e LABELING: The label states the Latin binomial and, follow-
ing the official name, the parts of the plant contained in
the article.
e USP REFERENCE STANDARDS (11)
USP Andrographolide RS
USP Powdered Andrographis Extract RS
Powdered Andrographis
DEFINITION
Powdered Andrographis is Andrographis reduced to a fine
or very fine powder. It contains NLT 1.0% of diterpene
lactones, calculated on the dried basis as the sum of the
andrographolide, neoandrographolide, 14-deoxy-11,12-
didehydroandrographolide, and andrograpanin.
IDENTIFICATION
oA. eee CHROMATOGRAPHIC IDENTIFICATION TEST
(201
Standard solution 1: Use Standard solution A, prepared
as directed in the test for Content of Diterpene Lactones.
Standard solution 2: Sonicate an amount of USP Pow-
dered Andrographis Extract RS, equivalent to about
15 mg of diterpene lactones, for 10-15 min in 25 mL of
methanol, centrifuge, and use the supernatant.
Sample solution: Use Sample stock solution, prepared as
directed in the test for Content of Diterpene Lactones.
Adsorbent: Chromatographic silica gel mixture with an
average particle size of 10-15 um (TLC plates)
Application volume: 10 iL, as 5-10 mm bands
Developing solvent system: Chloroform, acetone, and
toluene (2:2:1)
Derivatization reagent: A mixture of 1% vanillin in al-
cohol and 10% sulfuric acid in alcohol (1:1)
Analysis
Samples: Standard solution 1, Standard solution 2, and
Sample solution
Use a saturated chamber. Develop until the solvent
front has moved up about 90% of the length of the
plate. Remove the plate from the chamber, dry, treat
with Derivatization reagent, heat for 5-10 min at 100°,
and examine under white light.
Acceptance criteria: The Sample solution exhibits three
main grayish-blue zones with R; values of approximately
sydesbouow sa
0.4, 0.6, and 0.8 that correspond in position and color
to the main zones of Standard solution 2. Standard solu-
tion 1 exhibits a grayish-blue zone due to andrographo-
lide at an Rr of about 0.4. The Sample solution exhibits a
zone similar in color and Rr value to that due to andro-
grapholide in Standard solution 1.
B. The retention time of the main peak of the Sample
solution obtained in the test for Content of Diterpene Lac-
tones corresponds to that of andrographolide in Standard
solution A. \dentify other diterpene lactone peaks in the
Sample solution by comparison with Standard solution B
and the reference chromatogram provided with the lot
of USP Powdered Andrographis Extract RS being used.
The Sample solution shows additional peaks correspond-
ing to neoandrographolide, 14-deoxy-11,12-
didehydroandrographolide, and andrograpanin.
COMPOSITION
e CONTENT OF DITERPENE LACTONES
Solution A: Dissolve 0.14 g of potassium dihydrogen
phosphate in 900 mL of water, add 0.5 mL of phos-
phoric acid, dilute with water to 1000 mL, mix, filter,
and degas.
 4432 Andrographis / Dietary Supplements
Solution B: Acetonitrile, filtered and degassed
Standard solution A: Dissolve a weighed quantity of
USP Andrographolide RS in methanol to obtain a 1.0-
mg/mL solution. Transfer 5.0 mL of this solution to a
10-mL volumetric flask, dilute with acetonitrile to vol-
ume, and mix.
Standard solution B: Transfer an amount of USP Pow-
dered Andrographis Extract RS, equivalent to about
25 mg of diterpene lactones, to a 50-mL volumetric
flask, add 25 mL of methanol, heat gently for 15-20
min, dilute with acetonitrile to volume, and mix. Before
injection, pass through a membrane filter of 0.45-11m or
finer pore size, discarding the first 5 mL of the filtrate.
Sample stock solution: Transfer about 2.0 g of Pow-
dered Andrographis to a 250-mlL flask fitted with a re-
flux condenser. Add 50 mL of methanol, reflux for 15
min, cool to room temperature, and decant the super-
natant. Repeat until the extract is colorless. Combine
the extracts, filter, concentrate under vacuum, and ad-
just the volume to 50.0 mL using methanol.
Sample solution: Transfer 25.0 mL of Sample stock solu-
tion to a 50-mL volumetric flask, dilute with acetonitrile
to volume, and mix. Before injection, pass through a
membrane filter of 0.45-uum or finer pore size discard-
ing the first 5 mL of the filtrate.
Mobile phase: See Table 1.
Table 1
Time Solution A Solution B
(min) (%) (%)
0 95 5 14-deoxy-11,12-didehydroandrographolide, and an-
18 55 45
25 20 80
28 20 80
35 55 45
40 95 3 e ARTICLES OF BOTANICAL ORIGIN, Pesticide Residue Analysis
45 95 5
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 223 nm
Column: 4.6-mm x 25-cm; 5-um packing L1
Flow rate: 1.5 mL/min
Injection volume: 20 uL
System suitability
Samples: Standard solution A and Standard solution B
Suitability requirements
The chromatogram of Standard solutionB is similar to
DS Monographs
the reference chromatogram provided with the lot of
USP Powdered Andrographis Extract RS being used.
Column efficiency: NLT 5000 theoretical plates,
Standard solution A
Tailing factor: NMT 1.5 for the andrographolide
peak, Standard solution A
Relative standard deviation: NMT 2.0%, determined
for the andrographolide peak in replicate injections,
Standard solution A
Resolution: NLT 5 between the neoandrographolide
and 14-deoxy-11,12-didehydroandrographolide
peaks, Standard solution B
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Using the chromatogram of Standard solution A, Stan-
dard solution B, and the reference chromatogram pro-
vided with the lot of USP Powdered Andrographis Ex-
tract RS being used, identify the retention times of the
peaks corresponding to the different diterpene lac-
tones. The approximate relative retention times of the
different diterpene lactones are provided in Table 2.
USP 41
Table 2
Relative
Retention
Analyte Time
Andrographolide 1.00
Neoandrographolide 1.16
14-Deoxy-11,12-didehydroandrographolide 1.31
Andrograpanin 1.50
Separately calculate the percentages of andrographo-
lide, neoandrographolide, 14-deoxy-11,12-
didehydroandrographolide, and andrograpanin in the
portion of Powdered Andrographis taken:
Result = (ru/rs) x (Cs/W) x 10F
ty = peak area of each identified diterpene lactone
from the Sample solution
Is = peak area of andrographolide from Standard
solution A
Cs = concentration of USP Andrographolide RS in
Standard solution A (mg/mL)
w = weight of Powdered Andrographis taken to
prepare the Sample solution @)
E = conversion factor: 1.00 for andrographolide,
3.90 for neoandrographolide, 1.45 for
14-deoxy-11,12-didehydroandrographolide,
and 2.65 for andrograpanin
Acceptance criteria: NLT 1.0% for the sum of the per-
centages of andrographolide, neoandrographolide,
drograpanin, on the dried basis
IMPURITIES
e ARTICLES OF BOTANICAL ORIGIN, Limits of Elemental Impuri-
ties (561): Meets the requirements
(561): Meets the requirements
SPECIFIC TESTS
BOTANICAL CHARACTERISTICS
Macroscopic: It is a grayish-brown powder.
Microscopic: It reveals cells of the upper and lower epi-
dermis of the leaves, some cells containing large cysto-
liths, up to 36 um in diameter and 180 um long, with
a hilum-shaped scar in the large end; 1- to 4-celled
nonglandular hairs; disk-shaped glandular hairs, 8-celled
head and very short stalk; dngic stomata mostly on
the lower epidermis; stem epidermal cells, some cells
containing cystoliths, stomata, nonglandular hairs and
glandular hairs similar to those of the leaves; thin-
walled parenchyma cells; collenchyma cells; acicular
hloem fibers; tracheids; vessels, with spiral and scalari-
orm thickening.
Loss ON DRYING (731)
Sample: 1.0 g of Powdered Andrographis
Analysis: Dry the Sample at 105° for 3 h.
Acceptance criteria: NMT 12.0%
ARTICLES OF BOTANICAL ORIGIN, Total Ash (561)
Sample: 1.0g of Powdered Andrographis
Acceptance criteria: NMT 15%
ARTICLES OF BOTANICAL ORIGIN, Acid-/nsoluble Ash (561):
NMT 3.0%
ARTICLES OF BOTANICAL ORIGIN, Alcoho/-Soluble Extractives,
Method 2 (561): NLT 8.0%
MICROBIAL ENUMERATION TESTS (2021): The total aerobic
bacterial count does not exceed 105 cfu/g; the total com-
bined molds and yeasts count does not exceed 103 cfu/
g; and the bile-tolerant Gram-negative bacterial count
does not exceed 103 cfu/g.
ABSENCE OF SPECIFIED MICROORGANISMS (2022): Meets the
requirements of the tests for absence of Salmonella spe-
cies and Escherichia coli
USP 41 Dietary Supplements / Andrographis 4433

ADDITIONAL REQUIREMENTS COMPOSITION

e PACKAGING AND STORAGE: Preserve in well-closed contain- © CONTENT OF DITERPENE LACTONES
ers, protected from light and moisture, and store at Solution A: Dissolve 0.14 g of potassium dihydrogen
room temperature. phosphate in 900 mL of water, add 0.5 mL of phos-
e LABELING: The label states the Latin binomial and, follow- phoric acid, dilute with water to 1000 mL, mix, filter,
ing the official name, the parts of the plant contained in and degas.
the article. Solution B: Use filtered and degassed acetonitrile.
e USP REFERENCE STANDARDS (11) Standard solution A: Dissolve a weighed quantity of
USP Andrographolide RS USP Andrographolide RS in methanol to obtain a solu-
USP Powdered Andrographis Extract RS tion having a known concentration of about 1.0 mg/
mL. Transfer 5.0 mL of this solution to a 10-mL volu-
metric flask, dilute with acetonitrile to volume, and mix.
Standard solution B: Transfer an amount of USP Pow-
dered Andrographis Extract RS, equivalent to about
Powdered Andrographis Extract 25 mg of diterpene lactones, to a 50-mL volumetric
flask, add 25 mL of methanol, heat gently for 15-20
DEFINITION min, dilute with acetonitrile to volume, and mix. Before
Powdered Andrographis Extract is prepared from An- injection, pass through a membrane filter of 0.45-um or
oli pel by extraction with methanol or alcohol. The finer pore size, discarding the first 5 mL of the filtrate.
ratio of plant material to extract is between 15:1 and Sample solution: Transfer a weighed amount of Pow-
10:1. It contains NLT 90.0% and NMT 110.0% of the dered Andrographis Extract, equivalent to about 25 mg
labeled amount of diterpene lactones, calculated on the of diterpene lactones, to a 50-mL volumetric flask, ad
dried basis as the sum of andrographolide, neoandro- 25 mL of methanol, heat gently for 15-20 min, dilute
grapholide, 14-deoxy-11,12-didehydroandrographolide, with acetonitrile to volume, and mix. Before injection,
and andrograpanin. The content of 14-deoxy-11,12- pass through a membrane filter of 0.45-4m or finer
didehydroandrographolide is NMT 15% of the total pore size, discarding the first 5 mL of the filtrate.
diterpene lactones. It may contain suitable added sub- Mobile phase: See the gradient table below.
stances as carriers.
Time Solution A Solution B
IDENTIFICATION (min) (%) (%)
e * oo CHROMATOGRAPHIC IDENTIFICATION TEST 0 95 5
201
18 55 45
Standard solution 1: Use Standard solution A, prepared
as directed in the test for Content of Diterpene Lactones. 25 20 80
Standard solution 2: Sonicate for 10-15 min a quan- 28 20 80
tity of USP Powdered Andrographis Extract RS, equiva- 35 55 45
lent to about 15 mg of diterpene lactones, in 25 mL of 40 95 5.
methanol. Centrifuge, and use the supernatant. 45 95 5
Sample solution: Sonicate for 10-15 min a quantity of
Powdered Andrographis Extract, equivalent to about Chromatographic system
15 mg of diterpene lactones, in 25 mL of methanol. (See Chromatography (621), System Suitability.)
Centrifuge, and use the supernatant. Mode: LC
Adsorbent: Chromatographic silica gel mixture with Detector: UV 223 nm
an average particle size of 10-15 tum (TLC plates) Column: 4.6-mm x 25-cm; 5-um packing L1
Application volume: 10 wL, as 5-10 mm bands Flow rate: 1.5 mL/min
Developing solvent system: Chloroform, acetone, and Injection size: 20 wL
toluene (2:2:1) System suitability
Spray reagent: A mixture containing 1% vanillin in al- Samples: Standard solution A and Standard solution B
cohol and 10% sulfuric acid in alcohol (1:1) Suitability requirements
Analysis The chromatogram from Standard solutionB is similar
Samples: Standard solution 1, Standard solution 2, and to the reference chromatogram provided with the lot
va!

Sample solution of USP Powdered Andrographis Extract RS.

Use a saturated chamber. Develop until the solvent Column efficiency: NLT 5000 theoretical plates,
front has moved up about 90% of the plate. Remove Standard solution A
the plate from the chamber. Dry, and spray with
itrelB] olel ble

Tailing factor: NMT 1.5 for the andrographolide

Spray reagent. Heat for 5-10 min at 100°, and ex-
amine under visible light.
Acceptance criteria: The Sample solution exhibits three
main grayish blue zones with R; values of approximately
0.4, 0.6, and 0.8 that correspond in position and color
to zones in Standard solution 2. Standard solution 1 ex-
hibits a grayish blue zone due to andrographolide at an
Re of about 0.4. The Sample solution exhibits a zone
similar in color and R- value to that due to andrograph-
olide in Standard solution 1.
¢ B. The Sample solution in the test for Content of Diterpene
Lactones shows a main peak at a retention time corre-
sponding to that of andrographolide in Standard solution
A. Identify other diterpene lactone peaks in the Sample
solution by comparison with Standard solution B and the
reference chromatogram provided with the lot of USP
Powdered Andrographis Extract RS. The Sample solution
shows additional peaks corresponding to neoandrograph-
olide, 14-deoxy-11,12-didehydroandrographolide, and
andrograpanin.
peak, Standard solution A
Relative standard deviation: NMT 2.0%, determined
from the andrographolide peak for replicate injec-
tions, Standard solution A
Resolution: NLT 5 between the neoandrographolide
and 14-deoxy-11,12-didehydroandrographolide
peaks, Standard solution B
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Using Standard solution A, Standard solution B, and the
reference chromatogram provided with the lot of USP
Powdered Andrographis Extract RS being used, iden-
tify the retention times of the peaks corresponding to
different diterpene lactones. The approximate relative
retention times of the different diterpene lactones are
provided in the following table:
 4434 Andrographis / Dietary Supplements USP 41

Analyte
Relative
Retention Time
Arginine—see Arginine General Monographs
Andrographolide 1.00
Neoandrographolide 1.16
14-Deoxy-11,12-didehydroan- Arginine Hydrochloride—see Arginine
drographolide
Andrograpanin
131
150
Hydrochloride General Monographs
Separately calculate the percentages of andrographo-
lide, neoandrographolide, 14-deoxy-11,12-
didehydroandrographolide, and andrograpanin in the Arginine Capsules
portion of Powdered Andrographis Extract taken:
DEFINITION
Result = (ru/rs) x (Cs/W) « SF Arginine Capsules contain NLT 90.0% and NMT 110.0% of
the labeled amount of arginine or arginine hydrochloride
Tu = peak response for each diterpene lactone from in an amount equivalent to arginine (CeHi4N4O2).
the Sample solution
rs = peak response for andrographolide from IDENTIFICATION
Standard solution A e Oy CHROMATOGRAPHIC IDENTIFICATION TEST
Cs = concentration of USP Andrographolide RS in
Standard solution A (mg/mL) Standard solution: 1.5 mg/mL of USP L-Arginine RS or
Ww = weight of Powdered Andrographis Extract USP Arginine Hydrochloride RS in water
taken to prepare the Sample solution (g) Sample solution: Weigh the content of NLT 20 Cap-
F = conversion factor for each analyte (1.00 for sules, mix, and transfer a portion of the content, equiv-
andrographolide, 3.90 for alent to about 150 mg of arginine, to a 100-mL volu-
neoandrographolide, 1.45 for 14-deoxy- metric flask, add 80 mL of water, sonicate for 15 min,
11,12-didehydroandrographolide, and 2.65 dilute with water to volume, mix, and filter.
for andrograpanin) Application volume: 5 wiL
Acceptance criteria: 90.0%-110.0%, on the dried ba- Developing solvent system: Isopropyl alcohol and am-
sis, of the labeled amount of diterpene lactones calcu- monium hydroxide (7:3)
lated as the sum of the percentages of andrographo- Spray reagent: 2 mg/mL of ninhydrin in a mixture of
lide, neoandrographolide, 14-deoxy-11,12- utyl alcohol and 2 N acetic acid (95:5)
didehydroandrographolide, and andrograpanin Analysis: Proceed as directed for Chromatography (621),
Thin-Layer Cron ipteg Pe Dry the plate at 100°-105°
IMPURITIES until the ammonia disappears completely. Spray with
Inorganic Impurities the Spray reagent, and heat at 100°-105° for about 15
min. Examine the plate under white light.
Acceptance criteria: The principal spot from the Sam-
Delete the following: ple solution corresponds in appearance and R; value to
that of the Standard solution.
°e HEAVY METALS, Method I! (231): NMT 20 ppme coment. e B. The retention time of the major peak of the Sample
Jan-2018) solution corresponds to that from the Standard solution,
Organic Impurities as obtained in the Strength.
e PROCEDURE: ARTICLES OF BOTANICAL ORIGIN, General
Method for Pesticide Residues Analysis (561): Meets the STRENGTH
requirements © PROCEDURE
Buffer: 6.9 mg/mL of monobasic sodium phosphate in
SPECIFIC TESTS water. Adjust with phosphoric acid to a pH of 3.5.
© Loss ON DRYING (731): Dry 2.0g at 105° for 3 h: it loses Solution A: 0.5 mg/mL of 1-octanesulfonic acid sodium
NMT 5.0% of its weight. salt in Buffer
e MICROBIAL ENUMERATION TESTS (2021): The total aerobic Mobile phase: Solution A and acetonitrile (95:5)
microbial count does not exceed 104 cfu/g. The total Standard solution: 1.5 mg/mL of USP L-Arginine RS or
DS Monographs

eapnes yeasts and molds count does not exceed 103 USP Arginine Hydrochloride RS in Buffer
cfu/g. Sample solution: Weigh the content of NLT 20 Cap-
e MICROBIOLOGICAL PROCEDURES FOR ABSENCE OF SPECIFIED MI- sules, mix, and transfer a portion of the content, equiv-
CROORGANISMS (2022): It meets the requirements of the alent to about 150 mg of arginine, to a 100-mL volu-
tests for absence of Salmonella species and Escherichia metric flask, add 80 mL of Buffer, sonicate for 15 min,
coli. dilute with Buffer to volume, mix, and filter.
Chromatographic system
ADDITIONAL REQUIREMENTS (See Chromatography (621), System Suitability.)
© PACKAGING AND STORAGE: Preserve in well-closed contain- Mode: LC
ers, protected from light and moisture, and store at con- Detector: UV 215 nm
trolled room temperature. Column: 4.6-mm x 25-cm; packing L7
e LABELING: The label states the Latin binomial and, follow- Flow rate: 0.8 mL/min
ing the official name, the part of the plant from which Injection size: 10 wl
the article was prepared. It meets other labeling require- System suitability
ments under Botanical Extracts (565). Sample: Standard solution
e USP REFERENCE STANDARDS (11) Suitability requirements
USP Andrographolide RS Column efficiency: NLT 1500 theoretical plates
USP Powdered Andrographis Extract RS Relative standard deviation: NMT 2.0% from the ar-
ginine peak, in repeated injections
USP 41
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of ar-
ginine in the portion of Capsules taken:
Result = (ru/ts) x (Cs/Cu) x 100
ty = peak response from the Sample solution
Ts = peak response from the Standard solution
Cs = concentration of the Standard solution
(mg/mL)
Cu = nominal concentration of Arginine in the
Sample solution (mg/mL)
Acceptance criteria: 90.0%-110.0% of the labeled
amount of arginine (CsHi4N4O2)
PERFORMANCE TESTS
© DISINTEGRATION AND DISSOLUTION OF DIETARY SUPPLEMENTS
(2040): Meet the requirements for Dissolution
Medium: 0.1 N hydrochloric acid; 900 mL
Apparatus 2: 100 rpm
Time: 60 min
Standard solution: Proceed as directed in the Procedure
for Strength.
Sample solution: Sample per Disintegration and Dissolu-
tion of Dietary Supplements (2040). Dilute with Medium
to a concentration similar to that of the Standard
solution.
Analysis: Determine the amount of arginine dissolved
in the Procedure for Strength, making any necessary
modifications.
Tolerances: NLT 75% of the labeled amount of argi-
nine (CéHi4N4Oz) is dissolved.
© WEIGHT VARIATION OF DIETARY SUPPLEMENTS (2091): Meet
the requirements
ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Preserve in tight, light-resistant
containers.
e LABELING: The label states the form of arginine that is
used and the equivalent amount of arginine.
e USP REFERENCE STANDARDS (11)
USP L-Arginine RS
USP Arginine Hydrochloride RS
Arginine Tablets
DEFINITION
Arginine Tablets contain NLT 90.0% and NMT 110.0% of
the labeled amount of arginine or arginine hydrochloride
in an amount equivalent to arginine (CsHiaN4O2).
IDENTIFICATION
° “a CHROMATOGRAPHIC IDENTIFICATION TEST
Standard solution: 1.5 mg/mL of USP L-Arginine RS or
USP Arginine Hydrochloride RS in water
sample solution: Weigh and finely powder NLT 20
Tablets, mix, and transfer a portion of the powder,
equivalent to about 150 mg of arginine, to a 100-mL
volumetric flask, add 80 mL of water, sonicate for 15
min, dilute with water to volume, mix, and filter.
Application volume: 5 ul
Developing solvent system: Isopropyl alcohol and am-
monium hydroxide (7:3)
Spray reagent: 2 mg/mL of ninhydrin in a mixture of
utyl alcohol and 2 N acetic acid (95:5)
Analysis: Proceed as directed for Chromatography (621),
Thin-Layer Chromatography. Dry the plate at 100°-105°
until the ammonia disappears completely. Spray with
the Spray reagent, and heat at 100°-105° for about 15
min. Examine the plate under white light.
Dietary Supplements / Arginine 4435
Acceptance criteria: The principal spot from the Sam-
ple solution corresponds in appearance and R; value to
that from the Standard solution.
e B. The retention time of the major peak of the Sample
solution corresponds to that of the Standard solution, as
obtained in Strength.
STRENGTH
© PROCEDURE
Buffer: 6.9 mg/mL of monobasic sodium phosphate in
water. Adjust with phosphoric acid to a pH of 3.5.
Solution A: 0.5 mg/mL of 1-octanesulfonic acid sodium
salt in Buffer
Mobile phase: Solution A and acetonitrile (95:5)
Standard solution: 1.5 mg/mL of USP L-Arginine RS or
USP Arginine Hydrochloride RS in Buffer
Sample solution: Weigh and finely powder NLT 20
Tablets, mix, and transfer a portion of the powder,
equivalent to about 150 mg of arginine, to a 100-mL
volumetric flask, add 80 mL of Buffer, sonicate for 15
min, dilute with Buffer to volume, mix, and filter.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 215 nm
Column: 4.6-mm x 25-cm; packing L7
Flow rate: 0.8 mL/min
Injection size: 10pL
System suitability
Sample: Standard solution
Suitability requirements
Column efficiency: NLT 1500 theoretical plates
Relative standard deviation: NMT 2.0% from the ar-
ginine peak, in repeated injections
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of ar-
ginine in the portion of Tablets taken:
Result = (ru/rs) x (Cs/Cu) x 100
tu = peak response from the Sample solution
ls = peak response from the Standard solution
Cs = concentration of the Standard solution
(mg/mL)
Cu = nominal concentration of Arginine in the
Sample solution (mg/mL)
Acceptance criteria: 90.0%-110.0% of the labeled
amount of arginine (CsHi4N4O2)
PERFORMANCE TESTS
¢ DISINTEGRATION AND DISSOLUTION OF DIETARY SUPPLEMENTS
sydesbouow sa
(2040): Meet the requirements for Dissolution
Medium: 0.1 N hydrochloric acid; 900 mL
Apparatus 2: 100 rpm
Time: 60 min
Standard solution: Proceed as directed in the Procedure
for Strength.
Sample solution: Sample per Disintegration and Dissolu-
tion of Dietary Supplements (2040). Dilute with Medium
to a concentration similar to that of the Standard
solution.
Analysis: Determine the amounts of arginine dissolved
in the Procedure for Strength, making any necessary
modifications.
Tolerances: NLT 75% of the labeled amount of argi-
nine (Cs6Hi4N4Oz) is dissolved.
e@ WEIGHT VARIATION OF DIETARY SUPPLEMENTS (2091): Meet
the requirements
ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Preserve in tight, light-resistant
containers.
e LABELING: The label states the form of arginine that is
used and the equivalent amount of arginine.
 4436 Arginine / Dietary Supplements
e USP REFERENCE STANDARDS (11)
USP L-Arginine RS
USP Arginine Hydrochloride RS
Ascorbic Acid—see Ascorbic Acid General
Monographs
Ascorbic Acid Oral Solution—see Ascorbic
Acid Oral Solution General Monographs
Ascorbic Acid Tablets—see Ascorbic Acid
Tablets General Monographs
Ashwagandha Root
DEFINITION
Ashwagandha Root is the dried mature root of Withania
somnifera (L.) Dunal (Fam. Solanaceae). It contains NLT
0.3% of withanolides, calculated on the dried basis as the
sum of withanolide aglycones calculated as withanolide A,
and withanolide glycosides calculated as withanoside IV.
IDENTIFICATION
e A. HPTLC FoR ARTICLES OF BOTANICAL ORIGIN (203)
Standard solution A: 0.2 mg/mL each of USP B-Sitos-
terol RS and USP Withanolide A RS in methanol
Standard solution B: 20 mg/mL of USP Powdered
Ashwagandha Root Extract RS in methanol. Sonicate for
10 min, centrifuge, and use the supernatant. [NOTE—
Retain the supernatant for use in the test for Content of
Withanolides|
Sample solution: ie about 1 g of Ashwagandha
Root, finely powdered, in 10 mL of methanol, and soni-
cate for 15 min. Centrifuge, and use the supernatant.
Chromatographic system
Adsorbent: Chromatographic silica gel with an aver-
age particle size of 5 um (HPTLC plate)’
Application volume: 2 UL each of Standard solution A
and Standard solution B, and 10 uL of the Sample solu-
tion, as 8-mm bands
DS Monographs
Relative humidity: Condition the plate toa relative
humidity of 33%.
Temperature: Ambient, not to exceed 30°
Developing solvent system: Toluene, ethyl acetate,
and glacial acetic acid (55:45:3)
Developing distance: 6 cm
Derivatization reagent: 20 mL of sulfuric acid com-
bined with 180 mL of ice-cold methanol
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Apply the Samples as bands and dry in air. Develop in a
saturated chamber and dry in air. Treat the plate with
the Derivatization reagent, heat at 100° for 5 min, and
ars under UV light at 365 nm and under white
ight.
System suitability: Under UV light at 365 nm, the der-
ivatized chromatogram of Standard solution A displays
in its lower third a blue band due to withanolide A and
in its middle third a grayish-blue band due to B-sitos-
1Suitable commercially available plates are HPTLC Silica Gel 60 Fass from EMD
Millipore (e.g., Part No. 1.05642.0001).
USP 41
terol. The chromatogram of Standard solution B displays
a light-gray to whitish band due to withanone below
the withanolide A band, and a faint light-gray band
above the B-sitosterol band; there is also a light-gra'
band close to the solvent front. A reddish band gightly
above the application line is due to withaferin A. Under
white light, the bands due to B-sitosterol and withano-
lide A appear violet-gray. Additional faint bands may
appear.
Acceptance criteria: Under UV light at 365 nm and
under white light, the chromatogram of the Sample so-
lution displays the bands similar in position and color to
those seen in Standard solution B. Additional bands may
be observed, in particular a band just above that due to
withanolideA (light-brown under UV light at 365 nm,
dark-brown under white light), and a thin band below
that due to B-sitosterol (light-blue under UV light at
365 nm, gray-violet under white light). Bands vary in
intensity, and some of those seen in the chromatogram
of Standard solution B may be very faint or absent from
the Sample solution.
e B. HPLC
Analysis: Proceed as directed in the test for Content of
Withanolides.
Acceptance criteria: The Sample solution shows main
peaks at retention times corresponding to those of
withanolide A and withanoside IV in Standard solution A.
The Sample solution shows some of the withanolides
listed in Table 2.
COMPOSITION
e CONTENT OF WITHANOLIDES
Solution A: Dissolve 0.14 g of potassium dihydrogen
phosphate in 900 mL of water, add 0.5 mL of phos-
phoric acid, dilute with water to 1000 mL, and mix.
Solution B: Acetonitrile, filtered and degassed
Standard solution A: A composite solution containing
0.1 mg/mL of USP Withanolide A RS and 0.1 mg/mL of
USP Withanoside IV RS in methanol, accurately calcu-
lated. Use gentle heat to aid dissolution.
Standard solution B: Dilute two-fold a portion of Stan-
dard solution B from Identification A with methanol, and
mix well. Before injection, pass through a membrane
filter of 0.45-um or finer pore size.
Sample solution: Transfer about 5.00 g of Ashwa-
gandha Root, finely powdered and accurately weighed,
to a 250-mL round-bottom flask fitted with a reflux
condenser. Add 50 mL of methanol, reflux on a water
bath for 15 min, cool to room temperature, decant,
and retain the solvent. Repeat until the solvent is color-
less. Combine the retained solvents, filter, concentrate
under vacuum to about 40 mL, transfer to a 50-mL vol-
umetric flask, and adjust the volume with methanol.
Before injection, pass througha filter of 0.45-um or
finer pore size, discarding the first few milliliters of the
filtrate.
Mobile phase: See Table 1.
Table 1
Time Solution A Solution B
(min) (%) (%)
0 95 5
18 55 45,
25 20 80
28 20 80
30 95 5
40 95 5
Chromatographic system
(See Chromatography (621), System Suitability.)
USP 41 Dietary Supplements / Ashwagandha 4437

Mode: LC Cs = concentration of USP Withanoside IV RS in

Detector: UV 227 nm Standard solution A (mg/mL)
Column: 4.6-mm x 25-cm, end-capped; packing L1 v = volume of the Sample solution (mL)
Column temperature: 27° Ww = weight of Ashwagandha Root taken to prepare
Flow rate: 1.5 mL/min the Sample solution (mg)
Injection volume: 20 pL Acceptance criteria: The sum of the percentages of
System suitability withanolide aglycones and withanolide glycosides is
Samples: Standard solution A and Standard solution B NLT 0.3%, calcined on the dried basis. [NOTE—Be-
Using the chromatogram of Standard solution B and the cause of inherent variation, some of the withanolides
reference chromatogram provided with the lot of USP mentioned in this test may be present in minor quanti-
Powdered Ashwagandha Root Extract RS being used, ties or may be totally absent. The sample will be
identify the retention times of the peaks corresponding osei compliant if the sum of the withanolides is NLT
to withanolide aglycones and glycosides. The approxi- 3%.
mate relative retention times are provided in Table 2.
IMPURITIES
e ARTICLES OF BOTANICAL ORIGIN (561), Limits of Elemental
Table 2
Impurities: Meets the requirements
Relative ARTICLES OF BOTANICAL ORIGIN (561), Pesticide Residue
Analyte Retention Time Analysis: Meets the requirements
Withanoside IV 0.70 ARTICLES OF BOTANICAL ORIGIN (561), Methods of Analysis,
Physagulin D 0.75 Foreign Organic Matter. NMT 2.0%
ARTICLES OF BOTANICAL ORIGIN (561), Test for Aflatoxins:
27-Hydroxywithanone 0.80
Meets the requirements
Withanoside V 0.89 MICROBIAL ENUMERATION TESTS (2021): The total aerobic
Withanoside VI 0.89 bacterial count does not exceed 105 cfu/g, the total com-
Withaferin A 0.92 bined molds and yeasts count does not exceed 103 cfu/
Withastramonolide 0.96 g, and the bile-tolerant Gram-negative bacterial count
Withanolide A 1.00 does not exceed 103 cfu/g.
ABSENCE OF SPECIFIED MICROORGANISMS (2022), Test Proce-
Withanone 1.01
dures, Test for Absence of Salmonella Species and Test for
Withanolide B 1.14 Absence of Escherichia coli: Meets the requirements
Suitability requirements SPECIFIC TESTS
The chromatogram of Standard solution B is similar to e BOTANICAL CHARACTERISTICS
the reference chromatogram provided with the lot of Macroscopic: Primary roots are not branched and are
Use: Rawesied Ashwagandha Root Extract RS being straight, conical, or finger-like in shape and variable in
used. thickness with age; some carry a crown, consisting of a
Resolution: NLT 1.0 between the withanolide A and number of remains of stem base; the outer surface is
withanone peaks, and NLT 3.0 between the buff to Bart velow with longitudinal wrinkles; frac-
withaferin A peak and the peak corresponding to ture is short and uneven; secondary roots are thin and
coeluting withanoside V and withanoside VI, Standard fibrous.
solution B Microscopic: Transverse section of root shows a narrow
Tailing factor: NMT 1.5 for the withanolide A peak, band of yellowish crumpled cork, moderate-size cortex,
Standard solution A and a wide wood. The cork cells are rectangular, radi-
Relative standard deviation: NMT 2.0% for replicate ally flattened, nonlignified, and filled with starch grains
injections, withanolide A peak, Standard solution A and reddish brown content; cork cambium is 2-4 dif-
Analysis fused rows of cells; secondary cortex is formed of
Samples: Standard solution A, Standard solution B, and 20-25 rows of thin-walled parenchymatous cells, filled
Sample solution with starch grains, and shows occasional microsphe-
Calculate the percentage of withanolide aglycones in noidal crystals of calcium oxalate; phloem consists of
the portion of Ashwagandha Root taken: sieve tubes, companion cells, and phloem parenchyma;
vascular cambium consists of ae elongated
sydeibouo=: sa

Result = (ru/rs) x Cs x (V/W) x 100
ty = sum of the peak areas of withaferin A,
withastramonolide, withanolide A,
withanone, and withanolide B from the
Sample solution
ls = peak area of withanolide A from Standard
solution A
Cs = concentration of USP Withanolide A RS in
Standard solution A (mg/mL)
Vv = volume of the Sample solution (mL)
Ww = weight of Ashwagandha Root taken to prepare
the Sample solution (mg)
Calculate the percentage of withanolide glycosides in
the portion of Ashwagandha Root taken:
Result = (ru/rs) x Cs x (V/W) x 100
ru = sum of the peak areas of withanoside IV,
withanoside V, and withanoside VI from the
Sample solution
rs = peak area of withanoside IV from Standard
solution A
parenchymatous cells; vessels and tracheids are in radial
rows toward the periphery of the wood; medullary rays
are uniseriate to 2- to 3-seriate, and are filled with
starch grains; scattered vessels in groups are embedded
in the parenchyma; vessels have pitted and scalariform
thickenings, and generally the end walls are perforated;
a few fibers with thick lignified walls are also found
scattered in the wood.
Loss ON DRYING (731)
Sample: 1.0g of finely pone? Ashwagandha Root
Analysis: Dry the Sample at 105° for 3 h.
Acceptance criteria: NMT 12.0%
ARTICLES OF BOTANICAL ORIGIN (561), Methods of Analysis,
Total Ash
Sample: 1.0g of finely powdered Ashwagandha Root
Acceptance criteria: NMT 7.0%
ARTICLES OF BOTANICAL ORIGIN (561), Methods of Analysis,
Acid-Insoluble Ash: NMT 1.0%
ARTICLES OF BOTANICAL ORIGIN (561), Methods of Analysis,
Alcohol-Soluble Extractives, Method 2: NLT 10.0%
 4438 Ashwagandha / Dietary Supplements USP 41

ADDITIONAL REQUIREMENTS Acceptance criteria: Under UV light at 365 nm and

© PACKAGING AND STORAGE: Preserve in well-closed contain- under white light, the chromatogram of the Sample so-
ers, protected from light and moisture, and store at lution displays the bands similar in position and color to
room temperature. those seen in Standard solution B. Additional bands may
e LABELING: The label states the Latin binomial and, follow- be observed, in particular a band just above that due to
ing the official name, the part of the plant contained in withanolide A (light-brown under UV light at 365 nm,
the article. dark-brown under white light), and a thin band below
© USP REFERENCE STANDARDS (11) that due to B-sitosterol (light-blue under UV light at
USP Powdered Ashwagandha Root Extract RS 365 nm, gray-violet under white light). Bands vary in
USP B-Sitosterol RS intensity, and some of those seen in the chromatogram
USP Withanolide A RS of Standard solution B may be very faint or absent from
USP Withanoside IV RS the Sample solution.
e B. HPLC
Analysis: Proceed as directed in the test for Content of
Withanolides.
Acceptance criteria: The Sample solution shows main
Powdered Ashwagandha Root peaks at retention times corresponding to those of
withanolide A and withanoside IV in Standard solution A.
DEFINITION The Sample solution shows some of the withanolides
Powdered Ashwagandha Root is Ashwagandha Root reduced listed in Table 2.
to a fine or very fine powder. It contains NLT 0.3% of
withanolides, calculated on the dried basis as the sum of
COMPOSITION
e CONTENT OF WITHANOLIDES
withanolide aglycones calculated as withanolide A, and
withanolide glycosides calculated as withanoside IV. Solution A: Dissolve 0.14 g of potassium dihydrogen
phosphate in 900 mL of water, add 0.5 mL of phos-
IDENTIFICATION phoric acid, dilute with water to 1000 mL, and mix.
e A. HPTLC FOR ARTICLES OF BOTANICAL ORIGIN (203) Solution B: Acetonitrile, filtered and degassed
Standard solution A: 0.2 mg/mL each of USP B-Sitos- Standard solution A: A composite solution containing
terol RS and USP Withanolide A RS in methanol 0.1 mg/mL of USP Withanolide A RS and 0.1 mg/mL of
Standard solution B: 20 mg/mL of USP Powdered USP Withanoside IV RS in methanol, accurately calcu-
Ashwagandha Root Extract RS in methanol. Sonicate for lated. Use gentle heat to aid dissolution.
10 min, centrifuge, and use the supernatant. [NoTE— Standard solution B: Dilute two-fold a portion of the
Retain the supernatant for use in the test for Content of Standard solution B from Identification A with methanol,
Withanolides| and mix well. Before injection, pass through a mem-
Sample solution: Suspend about 1 g of Powdered brane filter of 0.45-11m or finer pore size.
Ashwagandha Root in 10 mL of methanol, and sonicate Sample solution: Transfer about 5.00 g of accurately
for 15 min. Centrifuge, and use the supernatant. weighed Powdered Ashwagandha Root to a 250-mL
Chromatographic system round-bottom flask fitted with a reflux condenser. Add
Adsorbent: Chromatographic silica gel with an aver- 50 mL of methanol, reflux on a water bath for 15 min,
age particle size of 5 um (HPTLC plate)! cool to room temperature, decant, and retain the sol-
Application volume: 2 uL each of Standard solution A vent. Repeat until the solvent is colorless. Combine the
and Standard solution B, and 10 uL of the Sample solu- retained solvents, filter, concentrate under vacuum to
tion, as 8-mm bands about 40 mL, transfer to a 50-mL volumetric flask, and
Relative humidity: Condition the plate to a relative adjust the volume with methanol. Before injection, pass
humidity of 33%. throughafilter of 0.45-um or finer pore size, discarding
Temperature: Ambient, not to exceed 30° the first few milliliters of the filtrate.
Developing solvent system: Toluene, ethyl acetate, Mobile phase: See Table 1.
and glacial acetic acid (55:45:3)
Developing distance: 6 cm Table 1
Derivatization reagent: 20 mL of sulfuric acid com- Time Solution A Solution B
bined with 180 mL of ice-cold methanol
DS Monographs

(min) (%) (%)

Analysis
Samples: Standard solution A, Standard solution B, and 0 95 5
Sample solution 18 55 45,
Apply the Samples as bands and dry in air. Develop in a 25 20 80
saturated chamber and dry in air. Treat the plate with 28 20 80
the Derivatization reagent, heat at 100° for 5 min, and 30 95 5
ae under UV light at 365 nm and under white 40 25! 5
ight.
system suitability: Under UV light at 365 nm, the der- Chromatographic system
ivatized chromatogram of Standard solution A displays (See Chromatography (621), System Suitability.)
in its lower third a blue band due to withanolide A and Mode: LC
in its middle third a grayish-blue band due to B-sitos- Detector: UV 227 nm
terol. The chromatogram of Standard solution B displays Column: 4.6-mm x 25-cm, end-capped; packing L1
a light-gray to whitish band due to withanone below Column temperature: 27°
the withanolide A band, anda faint light-gray band Flow rate: 1.5 mL/min
above the f-sitosterol band; there is a6 alight gray Injection volume: 20 wL
band close to the solvent front. A reddish band slightly System suitability
above the application line is due to withaferin A. Under Samples: Standard solution A and Standard solution B
white light, the bands due to B-sitosterol and withano- Using the chromatogram of Standard solution B and the
lide A appear violet-gray. Additional faint bands may reference chromatogram provided with the lot of USP
appear. Powdered Ashwagandha Root Extract RS being used,
1Suitable commercially available plates are HPTLC Silica Gel 60 Fas4 from EMD identify the retention times of the peaks corresponding
Millipore (e g., Part No. 1.05642.0001).
USP 41 Dietary Supplements / Ashwagandha 4439

to withanolide aglycones and glycosides. The approxi- IMPURITIES

mate relative retention times are provided in Table 2. © ARTICLES OF BOTANICAL ORIGIN (561), Limits of Elemental
Impurities: Meets the requirements
Table 2 e ARTICLES OF BOTANICAL ORIGIN (561), Pesticide Residue
Analysis: Meets the requirements
Relative e ARTICLES OF BOTANICAL ORIGIN (561), Test for Aflatoxins:
Analyte Retention Time Meets the requirements
Withanoside IV 0.70 e MICROBIAL ENUMERATION TESTS (2021): The total aerobic
Physagulin D 0.75 bacterial count does not exceed 10° cfu/g, the total com-
27-Hydroxywithanone 0.80 bined molds and yeasts count does not exceed 103 cfu/
Withanoside V 0.89
g, and the bile-tolerant Gram-negative bacteria count
does not exceed 103 cfu/g.
Withanoside VI 0.89 e ABSENCE OF SPECIFIED MICROORGANISMS (2022), Test Proce-
Withaferin A 0.92 dures, Test for Absence of Salmonella Species and Test for
Withastramonolide 0.96 Absence of Escherichia coli: Meets the requirements
Withanolide A 1.00
SPECIFIC TESTS
Withanone 1.01
e BOTANICAL CHARACTERISTICS
Withanolide B 1.14 Macroscopic: Dusty white or grey to light brown pow-
der with a characteristic odor and a mucilaginous, bit-
Suitability requirements ter, acrid taste
The chromatogram of Standard solution B is similar to Microscopic: Collapsed cork cells filled with starch
the reference chromatogram provided with the lot of grains and reddish-brown content; thin-walled cortex
Yep pawretre Ashwagandha Root Extract RS being parenchyma cells filled with starch grains and occa-
used. sional microsphenoidal crystals of calcium oxalate; ves-
Resolution: NLT 1.0 between the withanolide A and sels have pitted and scalariform thickenings, and gener-
withanone peaks, and NLT 3.0 between the
ally with end walls perforated; a few fibers with thick
withaferin A peak and the peak corresponding to lignified walls and simple pits; abundant starch grains,
coeluting withanoside V and withanoside VI, Standard mostly simple, sometimes compound, spherical, reni-
solution B form-oval with central hilum.
Tailing factor: NMT 1.5 for the withanolide A peak, Loss ON DRYING (731)
Standard solution A Sample: 1.0 g of Powdered Ashwagandha Root
Relative standard deviation: NMT 2.0% for replicate Analysis: Dry the Sample at 105° for 3 h.
injections, withanolide A peak, Standard solution A Acceptance criteria: NMT 12.0%
Analysis ARTICLES OF BOTANICAL ORIGIN (561), Methods of Analysis,
Samples: Standard solution A, Standard solution B, and Total Ash
Sample solution Sample: 1.0 g of Powdered Ashwagandha Root
Calculate the percentage of withanolide aglycones in Acceptance criteria: NMT 7.0%
the portion of Powdered Ashwagandha Root taken: ARTICLES OF BOTANICAL ORIGIN (561), Methods of Analysis,
Result = (ru/rs) x Cs x (V/W) x 100 Acid-Insoluble Ash: NMT 1.0%
ARTICLES OF BOTANICAL ORIGIN (561), Methods of Analysis,
tu = sum of the peak areas of withaferin A, Alcohol-Soluble Extractives, Method 2: NLT 10.0%
withastramonolide, withanolide A,
ADDITIONAL REQUIREMENTS
withanone, and withanolide B from the
¢ PACKAGING AND STORAGE: Preserve in well-closed contain-
Sample solution ers, protected from light and moisture, and store at
Is = peak area of withanolide A from Standard room temperature.
solution A
e LABELING: The label states the Latin binomial and, follow-
Cs = concentration of USP Withanolide A RS in
ing the official name, the part of the plant contained in
Standard solution A (mg/mL)
Vv = volume of the Sample solution (mL) the article.
e USP REFERENCE STANDARDS (11)
Ww = weight of Powdered Ashwagandha Root taken USP Powdered Ashwagandha Root Extract RS
a

to prepare the Sample solution (mg) USP B-Sitosterol RS

Calculate the percentage of withanolide Shtosiies in
the portion of Powdered Ashwagandha Root taken: USP Withanolide A RS
USP Withanoside IV RS
Etre FB ofl UC

Result = (ru/rs) x Cs x (V/W) x 100

tu = sum of the peak areas of withanoside IV,
withanoside V, and withanoside VI from the
Sample solution
rs = peak area of withanoside IV from Standard
solution A
Cs = concentration of USP Withanoside IV RS in
Standard solution A (mg/mL)
V = volume of the Sample solution (mL)
Ww = weight of Powdered Ashwagandha Root taken
to prepare the Sample solution (mg)
Acceptance criteria: The sum of the percentages of
withanolide aglycones and withanolide glycosides is
NLT 0.3%, calculated on the dried basis. [NoTE—Be-
cause of inherent variation, some of the withanolides
mentioned in this test may be present in minor quanti-
ties or may be totally absent. The sample will be
deemed compliant if the sum of the withanolides is NLT
0.3%.]
Powdered Ashwagandha Root Extract
DEFINITION
Powdered Ashwagandha Root Extract is prepared from
Ashwagandha Root using methanol, alcohol, water, or
mixtures of these solvents. It contains NLT 2.5% of with-
anolides, calculated on the dried basis as the sum of with-
anolide aglycones calculated as withanolide A and withan-
olide glycosides calculated as withanoside IV. It may
contain suitable added substances.
IDENTIFICATION
e A. HPTLC FOR ARTICLES OF BOTANICAL ORIGIN (203)
Standard solution A: 0.2 mg/mL each of USP B-Sitos-
terol RS and USP Withanolide A RS in methanol
 4440 Ashwagandha / Dietary Supplements USP 41

Standard solution B: 20 mg/mL of USP Powdered USP Withanoside IV RS in methanol, accurately calcu-
Ashwagandha Root Extract RS in methanol. Sonicate for lated. Use gentle heat to aid dissolution.
10 min, centrifuge, and use the supernatant. [NoTE— Standard solution B: Dilute two-fold a portion of Stan-
Retain the supernatant for use in the test for Content of dard solution B from Identification A with methanol, and
Withanolides| mix well. Before injection, pass through a membrane
Sample solution: Suspend about 200 mg of Powdered filter of 0.45-um or finer pore size.
Ashwagandha Root Extract in 10 mL of methanol, and Sample solution: Transfer about 100 mg of Powdered
sonicate for 10 min. Centrifuge, and use the Ashwagandha Root Extract, accurately weighed, to a
supernatant. 10-mL volumetric flask, add about 7 mL of methanol,
Chromatographic system heat gently on a water bath for 20 min, cool, dilute
Adsorbent: Chromatographic silica gel with an aver- with methanol to volume, and mix. Before injection,
age particle size of 5 um (HPTLC plate)’ pass through a membrane filter of 0.45-1m or finer
Application volume: 2 uL each of Standard solution A, pote size, discarding the first few milliliters of the
Standard solution B, and Sample solution, as 8-mm iltrate.
bands Mobile phase: See Table 1.
Relative humidity: Condition the plate toa relative
humidity of 33%. Table 1
Temperature: Ambient, not to exceed 30°
Developing solvent system: Toluene, ethyl acetate, Time Solution A Solution B
and glacial acetic acid (55:45:3) (min) (%) (%)
Developing distance: 6 cm oO 95: 5
Derivatization reagent: 20 mL of sulfuric acid com- 18 55 45
bined with 180 mL of ice-cold methanol 25 20 80
Analysis
28 20 80
Samples: Standard solution A, Standard solution B, and
Sample solution 30 95 5
Apply the Samples as bands and dry in air. Develop in a 40 95 5
saturated chamber and dry in air. Treat the plate with
the Derivatization reagent, heat at 100° for 5 min, and Chromatographic system
examine under UV light at 365 nm and under white (See Chromatography (621), System Suitability.)
light. Mode: LC
System suitability: Under UV light at 365 nm, the der- Detector: UV 227 nm
ivatized chromatogram of Standard solution A displays Column: 4.6-mm x 25-cm, end-capped; packing L1
in its lower third, a blue band due to withanolide A and Column temperature: 27°
in its middle third a grayish-blue band due to B-sitos- Flow rate: 1.5 mL/min
terol. The chromatogram of Standard solution B displays Injection volume: 20 pL
a light-gray to whitish band due to withanone below System suitability
the withanolide A band, anda faint light-gray band Samples: Standard solution A and Standard solution B
above the f-sitosterol band; there is also a light stay Using the chromatogram of Standard solution B and
band close to the solvent front. A reddish band slightly the reference chromatogram provided with the lot of
above the application line is due to withaferin A. Under USP Powdered Ashwagandha Root Extract RS being
white light, the bands due to f-sitosterol and withano- used, identify the retention times of the peaks corre-
lide A appear violet-gray. Additional faint bands may sponding to withanolide aglycones and glycosides.
appear. The approximate relative retention times are provided
Acceptance criteria: Under UV light at 365 nm and in Table 2.
under white light, the chromatogram of the Sample so-
lution displays the bands similar in position and color to Table 2
those seen in Standard solution B. Additional bands may Relative
be observed, in particular a band just above that due to Analyte Retention Time
withanolide A (light-brown under UV light at 365 nm,
dark-brown under white light), and a thin band below Withanoside IV 0.70
DS Monographs

that due to B-sitosterol ‘light-blue under UV light at Physagulin D. 0.75

365 nm, gray-violet under white light). Bands vary in 27-Hydroxywithanone 0.80
intensity, and some of those seen in the chromatogram Withanoside V 0.89
of Standard solution B may be very faint or absent from Withanoside VI 0.89
the Sample solution. Withaferin A 0.92
e B. HPLC
Analysis: Proceed as directed in the test for Content of Withastramonolide 0.96
Withanolides. Withanolide A 1.00
Acceptance criteria: The Sample solution shows main Withanone 1.01
peaks at retention times corresponding to those of Withanolide B 1,14
withanolide A and withanoside IV in Standard solution A.
The Sample solution may show some of the withanolides Suitability requirements
listed in Table 2. The chromatogram of Standard solution B is similar to
the reference chromatogram provided with the lot of
COMPOSITION USP Powdered Ashwagandha Root Extract RS being
@ CONTENT OF WITHANOLIDES used.
Solution A: Dissolve 0.14 g of potassium dihydrogen Resolution: NLT 1.0 between the withanolide A and
phosphate in 900 mL of water, add 0.5 mL of phos- withanone peaks, and NLT 3.0 between the
phoric acid, dilute with water to 1000 mL, and mix. withaferin A peak and the peak corresponding to
Standard solution A: A composite solution containing coeluting withanoside V and withanoside VI, Standard
0.1 mg/mL of USP Withanolide A RS and 0.1 mg/mL of solution B
‘Suitable commercially available plates are HPTLC Silica Gel 60 Fas4 from EMD Tailing factor: NMT 1.5 for the withanolide A peak,
Millipore (e.g., Part No. 1.05642.0001). Standard solution A
USP 41
Relative standard deviation: NMT 2.0% for replicate
injections, withanolide A peak, Standard solution A
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Calculate the percentage of withanolide aglycones in
we portion of Powdered Ashwagandha Root Extract
taken:
Result = (ru/rs) x Cs x (V/W) x 100
tu = sum of the peak areas of withaferin A,
withastramonolide, withanolide A, withanone
and withanolide B from the Sample solution
peak area of withanolide A from Standard
a
solution A
concentration of USP Withanolide A RS in
Standard solution A (mg/mL)
Vv = volume of the Sample solution (mL)
Ww = weight of Powdered Ashwagandha Root
io taken to prepare the Sample solution
mg)
Calculate he percentage of withanolide aon in
the portion of Powdered Ashwagandha Root taken:
Result = (ru/rs) x Cs x (V/W) x 100
tu = sum of the peak areas of withanoside IV,
withanoside V, and withanoside VI from the
Sample solution
Is = peak area of withanoside IV from Standard
solution A
Cs = concentration of USP Withanoside IV RS in
Standard solution A (mg/mL)
Vv = volume of the Sample solution (mL)
w = weight of Powdered Ashwagandha Root
aa taken to prepare the Sample solution
mg
Acceptance criteria: The sum of the percentages of the
withanolide aglycones and withanolide glycosides is
NLT 2.5%, calculated on the dried basis. [NoTE—Be-
cause of inherent variation, some of the withanolides
mentioned in this test may be present in minor quanti-
ties or may be totally absent. The sample will be
deemed compliant if the sum of the total withanolides
is NLT 2.5%.
IMPURITIES
Delete the following:
°e HEAVY METALs (231), Method Il: NMT 20 ppme omar.
Jan-2018)
e ARTICLES OF BOTANICAL ORIGIN (561), Pesticide Residue
Analysis: Meets the requirements
BOTANICAL EXTRACTS (565), Preparations, General Pharma-
copeial Requirements, Residual Solvents: Meets the
requirements
ARTICLES OF BOTANICAL ORIGIN (561), Test for Aflatoxins:
Meets the requirements
MICROBIAL ENUMERATION TESTS (2021): The total aerobic
bacterial count does not exceed 104 cfu/g, and the total
combined molds and yeasts count does not exceed 103
cfu/g.
ABSENCE OF SPECIFIED MICROORGANISMS (2022), Test Proce-
dures, Test for Absence of Salmonella Species and Test for
Absence of Escherichia coli: Meets the requirements
Dietary Supplements / Asian Ginseng 4441
SPECIFIC TESTS
e Loss ON DRYING (731)
Sample: 2,0 g of Powdered Ashwagandha Root Extract
Analysis: Dry the Sample at 105° for 3 h.
Acceptance criteria: NMT 6.0%
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in well-closed contain-
ers, protected from light and moisture, and store at con-
trolled room temperature.
e LABELING: The label states the Latin binomial and, follow-
ing the official name, the part of the plant from which
the article was prepared. It meets other labeling require-
ments under Botanical Extracts (565).
e USP REFERENCE STANDARDS (11)
USP Powdered Ashwagandha Root Extract RS
USP B-Sitosterol RS
USP Withanolide A RS
USP Withanoside IV RS
Asian Ginseng
DEFINITION
Asian Ginseng consists of the dried roots of Panax ginseng
C.A. Mey. (Fam. Araliaceae). It contains NLT 0.2% of gin-
senoside Rg; and NLT 0.1% of ginsenoside Rb;, both cal-
culated on the dried basis.
IDENTIFICATION
e A. THIN-LAYER CHROMATOGRAPHY
Standard solution: 5 mg/mL each of arbutin and escin,
in methanol
Sample solution: LO of finely powdered Asian Gin-
eng in a 25-mL flask fitted with a reflux condenser.
Add 10.0 mL of a mixture of methanol and water (7:3),
and heat under reflux for 15 min. Cool, filter, and di-
lute the filtrate with methanol to 10.0 mL.
Adsorbent: 0.25-mm layer of chromatographic silica
gel, typically 20 cm long (TLC plates)
Application volume: 20 uL, as bands
Developing solvent system: The upper layer of a mix-
ture of butyl alcohol, ethyl acetate, and water
(10: 2.5: 5) in an unsaturated chamber
Spray reagent: Dissolve 0.5 mL of anisaldehyde in
10 mL of glacial acetic acid, add 85 mL of methanol,
mix, and carefully add 5 mL of sulfuric acid, and mix.
Analysis
Samples: Standard solution and Sample solution
sydeibouow: Sa
Develop the chromatograms until the solvent front has
moved up about three-fourths of the length of the
plate. Remove the plate from the chamber, mark the
solvent front, and allow the plate to dry. Spray with
Spray reagent. Heat the plate at 105°-110° for 10 min,
and examine the plate under white light.
System suitability: The Standard solution chromato-
gram shows, in the upper third, a brown zone corre-
sponding to arbutin, and in the lower third, a gray
zone corresponding to escin.
Acceptance criteria: The Sample solution exhibits violet-
gray zones corresponding to ginsenoside Rg; in the up-
pe portion and to ginsenoside Re in the middle and in
etween the zones corresponding to arbutin and escin
in the Standard solution. A violet-gray zone correspond-
ing to ginsenoside Rb; is located at the same R; value as
the gray zone corresponding to escin in the Standard
solution. Other, less intense bands may be observed be-
tween the zones due to ginsenosides Rb, and Re, and
the zone closest to the origin corresponds to ginseno-
side Rc. Other spots may be visible in the lower third of
the chromatogram.
e B. The retention times of the peaks for ginsenosides Rgi,
Re, Rf, Rbi, Rc, and Rd in the Sample solution chromato-
 4442 Asian Ginseng / Dietary Supplements USP 41

gram correspond to those in the Standard solution, as Gs = concentration of ginsenoside Rg; or

obtained in the test for Content of Ginsenosides Rb; and ginsenoside Rb, in the Standard solution
Rgi. The ratio of the peak area for ginsenoside Rb2 to the (mg/mL)
eak area for ginsenoside Rb; is NLT 0.4 (differentiation Vv = final volume of the Sample solution (mL)
rom American ginseng). w = weight of Asian Ginseng taken to prepare the
Sample solution (mg)
COMPOSITION Acceptance criteria
e CONTENT OF GINSENOSIDES RB, AND RG, Ginsenoside Rg;: NLT 0.2% on the dried basis
Solution A: Water Ginsenoside Rb;: NLT 0.1% on the dried basis
Solution B: Acetonitrile and water (4:1)
Mobile phase: See Table 7. CONTAMINANTS
e ARTICLES OF BOTANICAL ORIGIN, Limits of Elemental Impuri-
Table 1 ties (561): Meets the requirements
e ARTICLES OF BOTANICAL ORIGIN, Pesticide Residue Analysis
Time Solution A Solution B (561): Meets the requirements
(min) (%) (%) e MICROBIAL ENUMERATION TESTS (2021): The total aerobic
0 76 24 microbial count does not exceed 10* cfu/g. The total
12: 76 24 Se molds and yeasts count does not exceed 10?
28 65 35 cfu/g.
51.5 56.5 43.5
e ABSENCE OF SPECIFIED MICROORGANISMS (2022): It meets
the requirements of the tests for absence of Salmonella
52.5. 0 100 species and Escherichia coli.
64.5 76 24
77 76 24 SPECIFIC TESTS
BOTANICAL CHARACTERISTICS
Diluent: Alcohol and water (4:6) Macroscopic: Fusiform or cylindrical roots, with distinct
Standard solution: Transfer a quantity of USP Pow- aromatic odor, sometimes branched, typically 1-10 cm,
dered Asian Ginseng Extract RS, equivalent to 2 mg of sometimes up to 20 cm in length and up to 2.5 cm in
ginsenoside Rg;, to a suitable container, and dissolve in diameter at the crown, with one or more stem scars.
10.0 mL of Diluent. [NoTE—The concentrations of ginse- Externally pale yellow to golden, rough textured in the
noside Rg; and ginsenoside Rb, in this solution are not lower part, with prominent horizontal rings and fine
expected to be equal and are determined on the basis longitudinal ridges as a result of drying. Root scars or
of the labeled quantities present in USP Powdered Asian fine rootlets are present. Fractures are short, with the
Ginseng Extract RS.] fractured surface, white to ivory, exposing a ring of se-
Sample solution: Reduce 100 g of Asian Ginseng to a cretory canals present in secondary phloem.
powder, and transfer about 1.0 g of the powder, accu- Microscopic: Transverse section of root presents multi-
rately weighed, to a 100-mL, round-bottom flask fitted ple layers of thin-walled cork cells. Secondary phloem
with a reflux condenser. Add 50 mL of Diluent and a characterized by conspicuous air lacunae, abundant
few grains of pumice, and boil on a water bath under starch-containing storage parenchyma, few sieve ele-
reflux for 1 h. Cool, and filter. Wash the flask and the ments, and rings of schizogenous secretory canals. Xy-
residue with 20 mL of Diluent, and pass through the lem characterized by abundant starch-containing stor-
same filter. Combine the filtrates, and evaporate in a age parenchyma, few tracheary elements, and a lack of
rotary evaporator at 50° to dryness. Dissolve the residue secretory canals. Druse crystals are sometimes present
in 10.0 mL of Diluent. with vascular parenchyma cells.
Chromatographic system ARTICLES OF BOTANICAL ORIGIN, Foreign Organic Matter
(See Chromatography (621), System Suitability.) (561): NMT 2.0%
Mode: LC ARTICLES OF BOTANICAL ORIGIN, Alcohol-Soluble Extractives,
Detector: UV 203 nm Method 2 (561): NLT 14.0%
Analytical column: 4.6-mm x 15-cm; 3-4um packing Loss ON DRYING (731)
u1 Sample: 1.0g of Asian Ginseng, finely powdered
Guard column: 4.6-mm x 2.0-cm; packing L1 Analysis: Dry the Sample at 105° for 2 h.
Column temperature: 25° Acceptance criteria: NMT 12.0%
DS Monographs

Flow rate: 1.5 mL/min
Injection volume: 10 uL
System suitability
Sample: Standard solution
Suitability requirements
Chromatogram similarity: The chromatogram is sim-
ilar to the reference chromatogram provided with the
lot of USP Powdered Asian Ginseng Extract RS being
used,
Relative standard deviation: NMT 2.0%, determined
for the sum of the peak areas for the 6 major ginse-
nosides, in replicate injections
Analysis
Samples: Standard solution and Sample solution
Calculate the percentages of ginsenosides Rb; and Rai
in the portion of Asian Ginseng taken:
Result = (ru/rs) x Cs x (V/W) x 100
ry = peak response ofGinsonoside Rgi or
ginsenoside Rb, from the Sample solution
rs = peak response of geotoee Rg; or
ginsenoside Rb; from the Standard solution
ARTICLES OF BOTANICAL ORIGIN, Total Ash (561)
Sample: 1.0g of Asian Ginseng, finely powdered
Acceptance criteria: NMT 8.0%
ARTICLES OF BOTANICAL ORIGIN, Acid-/nsoluble Ash (561):
NMT 1.0%
ADDITIONAL REQUIREMENTS
PACKAGING AND STORAGE: Preserve in well-closed contain-
ers, and store in a cool, dry place.
LABELING: The label states the Latin binomial and, follow-
ing the official name, the part of the plant contained in
the article.
USP REFERENCE STANDARDS (11)
USP Powdered Asian Ginseng Extract RS
Powdered Asian Ginseng
DEFINITION
Powdered Asian Ginseng is Asian Ginseng reduced toa fine
or very fine powder. It contains NLT 0.2% of ginsenoside
USP 41
Rg: and NLT 0.1% of ginsenoside Rb, both calculated on
the dried basis.
IDENTIFICATION
e A. THIN-LAYER CHROMATOGRAPHY
Standard solution: 5 mg/mL each of arbutin and escin,
in methanol
Sample solution: 1.0 g of Powdered Asian Ginseng in a
25-mL flask fitted with a reflux condenser. Add 10.0 mL
of a mixture of methanol and water (7:3), and heat
under reflux for 15 min. Cool, filter, and dilute the fil-
trate with methanol to 10.0 mL.
Adsorbent: 0.25-mm layer of chromatographic silica
gel, typically 20 cm long (TLC plates)
Application volume: 20 uL, as bands
Developing solvent system: The upper layer of a mix-
ture of butyl alcohol, ethyl acetate, and water
(10: 2.5: 5) in an unsaturated chamber
Spray reagent: Dissolve 0.5 mL of anisaldehyde in
10 mL of glacial acetic acid, and add 85 mL of metha-
nol, mix, and carefully add 5 mL of sulfuric acid to this
mixture.
Analysis
Samples: Standard solution and Sample solution
Develop the chromatograms until the solvent front has
moved up about three-fourths of the length of the
plate. Remove the plate from the chamber, mark the
solvent front, and allow the plate to dry. Spray with
Spray reagent. Heat the plate at 105°-110° for 10 min,
and examine the plate under white light.
System suitability: The Standard solution chromato-
gram shows, in the upper third, a brown zone corre-
sponding to arbutin, and in the lower third, a gray
zone corresponding to escin.
Acceptance criteria: The Sample solution exhibits violet-
gray zones corresponding to ginsenoside Rg; in the up-
et portion and to ginsenoside Re in the middle and in
tween the zones corresponding to arbutin and escin
in the Standard solution. A violet-gray zone correspond-
ing to ginsenoside Rb; is located at the same R, value as
the gray zone corresponding to escin in the Standard
solution. Other, less intense bands may be observed be-
tween the zones due to ginsenosides Rb; and Re, and
the zone closest to the origin corresponds to ginseno-
side Rc. Other spots may be visible in the lower third of
the chromatogram.
e B. The retention times of the peaks forgipsenasicles Rgi,
Re, Rf, Rb:, Rc, and Rd in the Sample solution chromato-
gram correspond to those in the Standard solution, as
obtained in the test for Content of Ginsenosides Rb, and
Rg. The ratio of the peak area for ginsenoside Rb2 to the
eak area for ginsenoside Rb; is NLT 0.4 (differentiation
tom American ginseng).
COMPOSITION
© CONTENT OF GINSENOSIDES RB, AND RG,
Solution A: Water
Solution B: Acetonitrile and water (4:1)
Mobile phase: See Table 7.
Table 1
Time Solution A Solution B
(min) (%) (%)
0 76 24
12 76 24
28 65 35,
STS. 56.5 43.5
52.5 oO 100
64.5 76 24
77 76 24
Dietary Supplements / Asian Ginseng 4443
Diluent: Alcohol and water (4:6)
Standard solution: Transfer a quantity of USP Pow-
dered Asian Ginseng Extract RS, equivalent to 2 mag of
ginsenoside Rg;, to a suitable container, and dissolve in
10.0 mL of Diluent. [NotE—The concentrations of ginse-
noside Rg; and ginsenoside Rb; in this solution are not
expected to be equal and are determined on the basis
of the labeled quantities present in USP Powdered Asian
Ginseng Extract RS.]
Sample solution: Transfer about 1.0 g of Powdered
Asian Ginseng, accurately weighed, to a 100-mL,
round-bottom flask fitted with a reflux condenser. Add
50 mL of a mixture of Diluent and a few grains of pum-
ice, and boil on a water bath under reflux for 1 h. Cool,
and filter. Wash the flask and the residue with 20 mL of
Diluent, and pass through the same filter. Combine the
filtrates, and evaporate in a rotary evaporator at 50° to
dryness. Dissolve the residue in 10.0 mL of Diluent.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 203 nm
Analytical column: 4.6-mm x 15-cm; 3-um packing
U1
Guard column: 4.6-mm x 2.0-cm; packing L1
Column temperature: 25°
Flow rate: 1.5 mL/min
Injection volume: 10 uL
System suitability
Sample: Standard solution
Suitability requirements
Chromatogram similarity: The eno is sim-
ilar to the reference chromatogram provided with the
lot of USP Powdered Asian Ginseng Extract RS being
used.
Relative standard deviation: NMT 2.0%, determined
for the sum of the peak areas for the 6 major ginse-
nosides, in replicate injections
Analysis
Samples: Standard solution and Sample solution
Calculate the percentages of ginsenosides Rb; and Rgi
in the portion of Powdered Asian Ginseng taken:
Result = (ru/rs) x Cs x (V/W) x 100
ru = peak response of ginsenoside Rg; or
ginsenoside Rb; from the Sample solution
rs = peak response of ginsenoside Rg; or
ginsenoside Rb; from the Standard solution
Cs = concentration of ginsenoside Rg; or
ginsenoside Rb; in the Standard solution
(mg/mL)
sydesbouow Sa
Vv = final volume of the Sample solution (mL)
w = weight of Powdered Asian Ginseng used to
prepare the Sample solution (mg)
Acceptance criteria
Ginsenoside Rg: NLT 0.2% on the dried basis
Ginsenoside Rb;: NLT 0.1% on the dried basis
CONTAMINANTS
© ARTICLES OF BOTANICAL ORIGIN, Limits of Elemental Impuri-
ties (561): Meets the requirements
e ARTICLES OF BOTANICAL ORIGIN, Pesticide Residue Analysis
(561): Meets the requirements
e MICROBIAL ENUMERATION TESTS (2021): The total aerobic
microbial count does not exceed 104 cfu/g. The total
oa molds and yeasts count does not exceed 10?
cfu/g.
¢ ABSENCE OF SPECIFIED MICROORGANISMS (2022): It meets
the requirements of the tests for absence of Salmonella
species and Escherichia coli.
SPECIFIC TESTS
¢ BOTANICAL CHARACTERISTICS: Pale yellowish-brown pow-
der with a slightly aromatic odor. Under a microscope,
 4444 Asian Ginseng / Dietary Supplements USP 41

the powder shows traces of cork composed of thin-
walled polygonal cells but mainly with phelloderm on
the outside; wide cortex of parenchymatous cells with
numerous secretory canals arranged in concentric zones;
parenchymatous xylem with nonlignified tracheids and
slightly lignified vessels with spiral and reticulate thicken-
ing, isolated or in small groups; small granules of starch
0.5-1.0 um in diameter inallef the parenchymatous
cells; and occasional cluster crystals of calcium oxalate in
the cells of the central region.
ARTICLES OF BOTANICAL ORIGIN, Foreign Organic Matter
(561): NMT 2.0%
ARTICLES OF BOTANICAL ORIGIN, Alcohol-Soluble Extractives,
Method 2 (561): NLT 14.0%
Loss ON DRYING (731)
Sample: 1.0 g of Powdered Asian Ginseng
Analysis: Dry the Sample at 105° for 2 h.
Acceptance criteria: NMT 12.0%
ARTICLES OF BOTANICAL ORIGIN, Total Ash (561)
Sample: 1.0 g of Powdered Asian Ginseng
Acceptance criteria: NMT 8.0%
ARTICLES OF BOTANICAL ORIGIN, Acid-insoluble Ash (561):
NMT 1.0%
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in well-closed contain-
ers, and store in a cool, dry place.
e LABELING: The label states the Latin binomial and, follow-
ing the official name, the part of the plant source from
which the article was derived.
e USP REFERENCE STANDARDS (11)
USP Powdered Asian Ginseng Extract RS
=o reagent: Alcohol, acetic anhydride, and sulfuric
acid (18:1:1)
Analysis
Samples: Standard solution and Sample solution
Saturate the chamber with Developing solvent system
for 2 h. Develop the chromatograms until the solvent
front has moved up about three-fourths of the length
of the plate. Remove the plate from the chamber,
mark the solvent front, and allow the plate to dry.
Spray with Spray reagent, and heat in an oven at 105°
tot1 min. Immediately examine the plate in white
ight.
Acceptance criteria: The Sample solution exhibits,
among other spots, eight brown-violet spots at the Rr
values of about 0.70, 0.60, 0.50, 0.36, 0.30, 0.28,
0.20, and 0.18, corresponding in color and Ry values to
those obtained for the Standard solution.
e B. Add 2 mL of glacial acetic acid to 0.1 g of Powdered
Asian Ginseng Extract, warm for 5 min in a hot water
bath, and filter. Gently add 0.5 mL of sulfuric acid to
1.0 mL of the filtrate.
Acceptance criteria: A red-brown color develops at the
zone of contact.
e C. The retention times of the peaks for ginsenosides Rai,
Re, Rf, Rb;, Rbz, Rc, and Rd in the Sample solution chro-
matogram correspond to those in the Standard solution,
as obtained in the test for Content of Ginsenosides. The
ratio of the peak area of Rb2 to the peak area of Rb; is
NLT 0.4 (differentiation from American Ginseng).
COMPOSITION
e CONTENT OF GINSENOSIDES
Solution A: Water
Solution B: Acetonitrile and water (4:1)
Mobile phase: See Table 7.

Powdered Asian Ginseng Extract Table 1

Time Solution A Solution B
DEFINITION (min) (%) (%)
Powdered Asian Ginseng Extract is prepared from Asian Gin-
0 76 24
seng by maceration, percolation, or both processes per-
formed at room temperature with suitable solvents such 12 76 24
as alcohol, methanol, water, or mixtures of these solvents, 28 65 35
and by concentrating the fluidextract at temperatures be- S135. 56.5 43.5
low 50°. The ratio of the starting crude plant material to 52.5 0 100
Powdered Asian Ginseng Extract is between 3:1 and 7:1. 64.5 76 24
It contains NLT 3.0% of ginsenosides Rgi, Re, Rbi, Rc,
77 76 24
Rbz, and Rd combined, calculated on the anhydrous basis.
It may contain other added substances. Diluent: Alcohol and water (4:6)
IDENTIFICATION Standard solution: 24 mg/mL of USP Powdered Asian
”
in e A. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST Ginseng Extract RS in Diluent. Dissolve by sonicating for
5 Extraction column: Use a solid-phase extraction col- 10 min, mix, and filter.
a
s
umn that contains C18 packing with 55- to 105-m Sample solution: Proceed as directed for Standard solu-
i) particle size and a ratio of sorbent mass to column vol- tion, except use Powdered Asian Ginseng Extract.
i} Chromatographic system
S ume of 360 mg/0.85 mL, or equivalent. Condition the
(See Chromatography (621), System Suitability.)
S column before use by washing with 3 mL of methanol
Mode: LC
= and 8 mL of water.
Detector: UV 203 nm
” Standard solution: Transfer about 0.1 g of USP Pow-
dered Asian Ginseng Extract RS to a 5-mL volumetric Analytical column: 4.6-mm x 15-cm; 3-1um packing
a L1
flask, and proceed as directed for the Sample solution,
beginning with “Dissolve in water”. Guard column: 4.6-mm x 2.0-cm; packing L1
Sample solution: About 1.0 g of Powdered Asian Gin- Column temperature: 25°
seng Extract in a 25-mL volumetric flask. Dissolve in Flow rate: 1.5 mL/min
water, sonicating if necessary. Dilute with water to vol- Injection size: 20 uL
ume. Transfer 4.0 mL of this solution to the Extraction System suitability
column, wash with 10 mL of water, and discard the elu- Sample: Standard solution
ate. Elute the column with 2 mL of methanol. [NoTE— Suitability requirements
Do not use vacuum, elute manually and slowly.] Collect Chromatogram similarity: The chromatogram is sim-
the eluate in a suitable vial. ilar to the Reference Chromatogram provided with
Adsorbent: 0.2-mm layer of chromatographic silica gel the lot of USP Powdered Asian Ginseng Extract RS
mixture on a high-performance thin-layer plate being used.
Application volume: 10 wL, as bands Relative standard deviation: NMT 2.0%, determined
Developing solvent system: Chloroform, methanol, for the sum of the peak areas for the 6 major ginse-
and water (65:35:10). Use the lower phase. nosides, in replicate injections
USP 41
Analysis
Samples: Standard solution and Sample solution
Identify the peaks for the ginsenosides by comparison
with the Reference Chromatogram provided with the
lot of USP Powdered Asian Ginseng Extract RS being
used, and measure the peak areas for the 6 major
ginsenosides.
Calculate the percentage of each relevant ginsenoside
(Rgi, Re, Rb;, Rc, Rb2, and Rd) in the portion of Pow-
dered Asian Ginseng Extract taken:
Result = (ru/ts) x (Cs/Cu) x P
ty = peak area for each relevant ginsenoside from
the Sample solution
Is = peak area for each relevant ginsenoside from
the Standard solution
Cs = concentration of USP Powdered Asian Ginseng
Extract RS in the Standard solution (mg/mL)
Cu concentration of Powdered Asian Ginseng
il}
Extract in the Sample solution (mg/mL)
P = labeled amount, in percentage, of each
relevant ginsenoside in the USP Powdered
Asian Ginseng Extract RS
Calculate the percentage of ginsenosides by adding the
percentages of each relevant ginsenoside.
Acceptance criteria: NLT 3.0% on the anhydrous basis
CONTAMINANTS
Delete the following:
°e HEAVY METALS (231): NMT 30 ppme coffciat 1-jan-2018)
e ARTICLES OF BOTANICAL ORIGIN, General Method for Pesti-
cide Residues Analysis (561): Meets the requirements
¢ MICROBIAL ENUMERATION TESTS (2021): The total aerobic
microbial count does not exceed 300 cfu/g. The total
one molds and yeasts count does not exceed 100
u/g.
° MICROBIOLOGICAL PROCEDURES FOR ABSENCE OF SPECIFIED MI-
CROORGANISMS (2022): It meets the requirements of the
tests for absence of Salmonella species, Escherichia coli,
and Staphylococcus aureus.
SPECIFIC TESTS
e@ WATER DETERMINATION, Method | (921): NMT 7.0%, de-
termined on a 0.15-g specimen
e ALCOHOL DETERMINATION, Method II (611): NMT 0.25%
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Meets the requirements in Bo-
tanical Extracts (565)
© LABELING: Meets the requirements in Botanical Extracts
(565)
© USP REFERENCE STANDARDS (11)
USP Powdered Asian Ginseng Extract RS
Asian Ginseng Tablets
DEFINITION
Asian Ginseng Tablets are prepared from Powdered Asian
Ginseng Extract. They contain NLT 90.0% and NMT
110.0% of Powdered Extract, calculated as the sum of
ginsenosides Rg:, Re, Rbi, Rc, Rbz, and Rd.
IDENTIFICATION
° ‘on CHROMATOGRAPHIC IDENTIFICATION TEST
01
Standard solution: 5 mg/ml each of arbutin and escin,
in methanol
Sample solution: Transfer the equivalent of 100 mg of
Powdered Extract from powdered Tablets to a conical
Dietary Supplements / Asian Ginseng 4445
flask, and extract three times, each with a 20-mL por-
tion of a mixture of methanol and water (4:1), in a 55°
bath for 30 min, stirring with a magnetic stirrer. Evapo-
rate the combined extracts to dryness in vacuum be-
tween 45° and 50°, and dissolve the residue in 10 mL
of a mixture of methanol and water (3:2).
Application volume: 20 uL, as bands
Developing solvent system: The upper layer of a mix-
ture of butyl alcohol, ethyl acetate, and water (4:1:2) in
an unsaturated chamber
Spray reagent: 0.5 mL of anisaldehyde in 10 mL of gla-
cial acetic acid. Add 85 mL of methanol, carefully add
5 mL of sulfuric acid, and mix.
Analysis
Samples: Standard solution and Sample solution
Proceed as directed in the chapter. Remove the plate
from the developing chamber, and allow it to dry.
Spray with Spray reagent. Heat the plate at
105°-110° for 10 min, and examine the plate.
Acceptance criteria: The chromatogram of the Stan-
dard solution shows, in the upper third, a brown zone
corresponding to arbutin and, in the lower third, a gray
zone corresponding to escin. Between these two zones,
the chromatogram of the Sample solution exhibits vio-
let-gray zones corresponding to ginsenoside Rg; in the
upper portion and to ginsenoside Re in the middle. A
violet-gray zone corresponding to ginsenoside Rb; is lo-
cated at the same Rr value as the gray zone corre-
sponding to escin in the chromatogram of the Standard
solution. Other, less intense bands may be observed be-
tween the zones due to ginsenosides Rb; and Re, and
the zone closest to the origin corresponds to ginseno-
side Rc. Other spots may be visible in the lower third of
the chromatogram.
e B. The retention times of the relevant analytes of the
Sample solution correspond to those of the Standard solu-
tion, as obtained in the test for Content of Ginsenosides.
The retention time of the peak for ginsenoside Rf of the
Sample solution corresponds to that of the Standard solu-
tion, as obtained in the test for Content of Ginsenosides.
STRENGTH
© CONTENT OF GINSENOSIDES
Diluent: Water and alcohol (3:2)
Solution A: Water
Solution B: Acetonitrile and water (4:1)
Mobile phase: See the gradient table below.
Time Solution A Solution B
(min) (%) (%)
0 76 24 im
12 76 24 As
28 65 35 =
51.5, 56.5 43.5, 3
52.5 0 100 3
64.5 76 24 b=)
77 76 24 a
Standard solution: 40 mg/mL of USP Powdered Asian a
Ginseng Extract RS in Diluent. Filter.
Sample solution: Weigh and finely powder NLT 20
Tablets. Transfer a quantity of the powder, equivalent to
200 mg of Powdered Extract to a conical flask, and ex-
tract three times, each with a 20-mL portion of a mix-
ture of methanol and water (4:1), in a 55° bath for 30
min, stirring with a magnetic stirrer. Evaporate the
combined extracts to dryness in a vacuum between 45°
ane 50°. Dissolve the residue in 5.0 mL of Diluent, and
ilter.
Chromatographic system
(See Chromatography (621), System Suitability.)
 4446 Asian Ginseng / Dietary Supplements
Mode: LC
Detector: UV 203 nm
Column
Guard: 4.6-mm x 2.0-cm; packing L1
Analytical: 4.6-mm x 15-cm; 3-um packing L1
Column temperature: 25°
Flow rate: 1.5 mL/min
Injection size: 20 uL
System suitability
Sample: Standard solution
Suitability requirements
Chromatogram similarity: The Standard solution
chromatogram is similar to the Reference Chromato-
gram provided with the lot of USP Powdered Asian
Ginseng Extract RS being used.
Relative standard deviation: NMT 2.0%, determined
for the sum of the peak areas for the six major ginse-
nosides, in repeated injections
Analysis
Samples: Standard solution and Sample solution
Record the chromatograms, identify the peaks for the
ginsenosides by comparison with the Reference Chro-
matogram provided with the lot of USP Powdered
Asian Ginseng Extract RS being used, and measure
the peak areas for the six major ginsenosides.
Calculate the quantity, in mg, of each relevant ginse-
noside (Rgi, Re, Rb:, Rc, Rbz, and Rd) in the portion
of Tablets taken:
Result = 0.05 x (ru/ts) x Cs x P
Tu = peak areas for each relevant ginsenoside from
the Sample solution
rs = peak areas for each relevant ginsenoside from
the Standard solution
Cs = concentration of USP Powdered Asian Ginseng
Extract RS in the Standard solution (mg/mL)
Pp = labeled amount, in percentage, of each
relevant ginsenoside in the USP Powdered
Asian Ginseng Extract RS lot being used
Calculate the content of total ginsenosides, T, in mg,
by adding the amounts of individual ginsenoside.
Calculate the percentage of Powdered Extract with
respect to the label claim:
Result = T x (Awr/W) x (100/Le) x (100/L)
T = content of total ginsenosides in the portion of
Tablets taken (mg)
Awr = average weight of Tablets (mg/Tablet)
Ww = weight of the portion of Tablets taken (mg)
Le = content of total ginsenosides in 100 mg of the
DS Monographs
Extract used to prepare the Tablets (mg)
L = amount of Extract per Tablet according to
label claim (mg/Tablet)
Acceptance criteria: 90.0%-110.0% of Powdered Ex-
tract, calculated as the sum of ginsenosides Rgi, Re,
Rb,, Rc, Rbz, and Rd
PERFORMANCE TESTS
e DISINTEGRATION AND DISSOLUTION OF DIETARY SUPPLEMENTS
(2040): Meet the requirements for Disintegration
e@ WEIGHT VARIATION OF‘Dierary SUPPLEMENTS (2091): Meet
the requirements
CONTAMINANTS
e MICROBIAL ENUMERATION TESTS (2021): The total aerobic
microbial count does not exceed 104 cfu/g, and the total
combined molds and yeasts count does not exceed 1000
cfu/g. Tablets meet the requirements of the tests for ab-
sence of Salmonella species and Escherichia coli.
ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Preserve in tight containers,
protected from light.
USP 41
© LABELING: The label states the Latin binomial and, follow-
ing the official name, the article from which the Tablets
were prepared. The label also indicates the amount of
Powdered Extract, in mg/Tablet, and the content, in mg,
of ginsenosides per 100 mg of Powdered Extract.
e USP REFERENCE STANDARDS (11)
USP Powdered Asian Ginseng Extract RS
Aspartic Acid—see Aspartic Acid General
Monographs
Astaxanthin Esters
Astaxanthin esters;
Astaxanthin fatty acid esters;
tae acid esters of (35,3'S)-3,3’-dihydroxy-B,B-carotene-4,4’-
jone.
DEFINITION
Astaxanthin Esters is obtained by extraction with either
supercritical carbon dioxide or acetone from cultures of
Haematococcus pluvialis. It consists mainly of 35,3’S stereo-
isomers of astaxanthin in the monoester, diester, and free
forms. The monoester form is the most abundant, fol-
lowed by the diester form. The free form is a minor com-
ponent. Suitable antioxidants may be added. It contains
NLT 5% of total astaxanthin, calculated as free astax-
anthin on the anhydrous basis.
IDENTIFICATION
e A. THIN-LAYER CHROMATOGRAPHY
Standard solution: 10 mg/mL of USP Astaxanthin Es-
ters from Haematococcus pluvialis RS in acetone
Sample solution: 10 mg/mL of Astaxanthin Esters in
acetone
Chromatographic system
(See Chromatography (621), Thin-Layer Chromato-
graphy.) Me eg
Adsorbent: 0.25-mm layer of chromatographic silica
gel mixture. Dry the adsorbent at 110° for 1 h before
use.
Application volume: 5 ul
Developing solvent system: Hexane and acetone
(70:30,
System suitabilit
Suitability requirement: The chromatogram from the
Standard solution exhibits three clearly separated
zones, with astaxanthin diester having the highest Rr
value, followed by astaxanthin monoester (the most
intense) and free astaxanthin (the least intense).
Analysis
Samples: Standard solution and Sample solution
Develop the chromatogram in thepeeled solvent
system until the solvent front has moved about three-
fourths of the length of the plate. Remove the plate
from the chamber, and dry in a current of air. Ex-
amine the plates under white light.
Acceptance criteria: The Sample solution exhibits three
main zones corresponding in R; value to those obtained
from the Standard solution. The zone in the middle
(monoester) is the most intense, and the zone with the
lower Rr is the least intense.
e B. HPLC: The Sample solution exhibits three major peaks
with the retention times corresponding to those of
13-cis-astaxanthin, all-trans-astaxanthin, and 9-cis-astax-
anthin peaks in the Standard solution, as obtained in the
test for Content of Total Astaxanthin.
USP 41 Dietary Supplements / Astaxanthin 4447

ASSAY inert gas at room temperature, add 3 mL of acetone,

© CONTENT OF TOTAL ASTAXANTHIN sonicate, and filter the mixture. The filtered solution is
[Note—Astaxanthin determined by this method is total the Sample solution.
astaxanthin, including the free astaxanthin, the Chromatographic system
monoester, and the diester.] (See Chromatography (621), System Suitability.)
Buffer solution: Dissolve 6.06 g of tris(hydroxymethyl) Mode: LC
aminomethane in 750 mL of water, adjust with 1 N Detector: 474 nm
hydrochloric acid to a pH of 7.0, and dilute with water Column: YMC Carotenoid, 4.6-mm x 25-cm, 5-uum
to 1000 mL. acking L62
Cholesterol esterase solution: 4 U/mL of cholesterol Flow rate: 1.0 mL/min
esterase! in Buffer solution. Prepare fresh daily. Injection volume: 20 uL
Solution A: Methanol System suitability
Solution B: tButylmethylether Sample: Standard solution
Solution C: Phosphoric acid, 1% aqueous [Note—The approximate relative retention times for
Mobile phase: See Table 1. 13-cis-astaxanthin, all-trans-astaxanthin, 9-cis-astax-
anthin, and apocarotenal (trans-beta-apo-8’-carotenal)
Table 1 are listed in Table 2.]
Time Solution A Solution B Solution C
Table 2
(min) (%) (%) (%)
0 81 15: 4 Relative Relative
15 66 30 4 Retention Response
Name of Compound Time Factor
23 16 80 4
13-cis-Astaxanthin 0.9 133:
27 16 80 4
all-trans-Astaxanthin 1.0 1.0
27.1 81 15 4
9-cis-Astaxanthin 1.4 V3)
35: 81 1s 4
Apocarotenal (internal _
Internal standard solution: 37.5 ug/mL of USP Apo- standard) Az
carotenal RS in acetone
Standard stock solution: Transfer 30 mg of USP Astax- Suitability requirements
anthin Esters from Haematococcus pluvialis RS to a Chromatogram similarity: The chromatogram from
100-mL volumetric flask. Dissolve in 30 mL of acetone, the Standard solution is similar to the Reference
shake by mechanical means, and dilute with acetone to Chromatogram provided with the USP Astaxanthin
volume. Esters from Haematococcus pluvialis RS being used.
Standard solution: Combine 2.0 mL of the Standard Resolution: NLT 2.0 between 13-cis-astaxanthin and
stock solution and 1.0 mL of the /nternal standard solu- all-trans-astaxanthin
tion in a glass centrifuge tube. Add 3.0 mL of Choles- Relative standard deviation: NMT 2.0% for the all-
terol esterase solution to the tube, and mix gently by trans-astaxanthin peak
inversion. Place the tube in a block heater set to 37°, Analysis
and allow the reaction to continue for 45 min, gently Samples: Standard solution and Sample solution
and slowly inering the tube every 10 min. After 45 Calculate the percentage of total astaxanthin content in
min, add 1g of sodium sulfate and 2 mL of petroleum the portion of sample taken:
ether to the tube. Mix on a vortex mixer for 30 s, then
centrifuge at 3000 rpm for 3 min. Carefully transfer the Result = (Ru/Rs) x (Cs/Cu) x P
petroleum ether layer to a 10-mL glass centrifuge tube Ruy = [(1.3 x peak area of 13-cis-astaxanthin +peak
containing 1 g of anhydrous sodium sulfate. Be careful area of all-trans-astaxanthin + 1.1 x pea
to avoid pipetting the intermediate emulsive layer. area of 9-cis-astaxanthin)/peak area of the
Evaporate the petroleum ether layer using a vacuum or internal standard] from the Sample solution
a stream of inert gas at room temperature, add 3 mL of Rs = [(1.3 x peak area of 13-cis-astaxanthin + pa
acetone, sonicate, and filter the mixture. The filtered
area of all-trans-astaxanthin + 1.1 x peal
solution is the Standard solution.
sydesbouow sa

area of 9-cis-astaxanthin)/peak area of the

Sample stock solution: Warm a quantity of the sample internal standard] from the Standard solution
in a water bath at 50°-60° for 30 min. Shake the sam- Cs = concentration of USP Astaxanthin Esters from
ple well at 10-min intervals. After 30 min, transfer Haematococcus pluvialis RS in the Standard
30 mg of the sample to a 100-mL volumetric flask. Dis- solution (mg/mL)
solve in 30 mL of acetone, shake by mechanical means, Cu = concentration of the Sample solution (mg/mL)
and dilute with acetone to volume. P = labeled amount of total astaxanthin as free
Sample solution: Combine 2.0 mL of the Sample stock astaxanthin in the USP Astaxanthin Esters
solution and 1.0 mL of the Internal standard solution in a from Haematococcus pluvialis RS (%)
glass centrifuge tube. Add 3.0 mL of Cholesterol esterase Acceptance criteria: NLT 5% of total astaxanthin, cal-
solution to the tube, and mix gently by inversion. Place culated as free astaxanthin on the anhydrous basis
the tube in a block heater set to 37°, and allow the
reaction to continue for 45 min, gently and slowly in- CONTAMINANTS
verting the tube every 10 min. After 45 min, add 1g of e ELEMENTAL IMPURITIES—PROCEDURES (233)
sodium sulfate and 2 mL of petroleum ether to the Acceptance criteria
tube. Mix on a vortex mixer for 30 s, then centrifuge at Arsenic: NMT 2.0 ug/g
3000 rpm for 3 min. Carefully transfer the petroleum Cadmium: NMT 1.0 ug/g
ether layer to a 10-mL glass centrifuge tube containing Lead: NMT 1.0 ug/g
1 g of anhydrous sodium sulfate. Be careful to avoid Mercury: NMT 1.0 ug/g
pipetting the intermediate emulsive layer. Evaporate the © MICROBIAL ENUMERATION TESTS (2021): The total aerobic
petroleum ether layer using a vacuum or a stream of bacterial count does not exceed 103 cfu/g, and the total
1 Use Wako Pure Chemicals catalog no. 037-11221, available from www. coe molds and yeasts count does not exceed 10?
wakousa.com; Sigma catalog no. C9281, available from www.sigmaaldrich. cfu/g.
com; or equivalent.
 4448 Astaxanthin / Dietary Supplements
e ABSENCE OF SPECIFIED MICROORGANISMS (2022): Meets the
requirements of the tests for absence of Salmonella spe-
cies and Escherichia coli
¢ PHEOPHORBIDE CONTENT
Solution A: 50 mg/mL of sodium sulfate
Solution B: Saturated solution of sodium sulfate
Sample stock solution: Transfer 100 mg of the sample
to a 10-mL test tube, add 10 mL of acetone, and dis-
solve with sonication. Quantitatively transfer this solu-
tion to a separatory funnel, rinsing the test tube three
times with 10-mL portions of acetone and adding the
rinsings to the funnel. Add 30 mL of ethyl ether to the
separatory funnel, followed by 50 mL of Solution A. Mix
the contents of the separatory funnel by shaking gently,
then draw off and discard the lower layer. Repeat wash-
ing with Solution A three times. Dehydrate the remain-
ing extract with anhydrous sodium sulfate, then transfer
the extract to a 50-mL volumetric flask, and dilute with
ethyl ether to volume.
Sample solution: Transfer 20 mL of the Sample stock
solution to a small beaker. Add 20 mL of 17% hydro-
chloric acid, and mix the solution vigorously. Transfer
the hydrochloric acid layer to a separatory funnel, and
repeat the extraction with a second 10-mL portion of
17% hydrochloric acid, adding the hydrochloric acid
layer to the separatory funnel. Add 150 mL of Solution
B, 20 mL ofethyl ether, and mix the contents of the
separatory funnel by shaking. Transfer the ethyl ether
layer to a 20-mL volumetric flask, and dilute with ethyl
ether to volume.
Instrumental conditions
(See Ultraviolet-Visible Spectroscopy (857).)
Analytical wavelength: 667 nm
Cell path: 1 cm
Blank: Ethyl ether
Analysis
Sample: Sample solution
Calculate the percentage of pheophorbide in the por-
tion of sample taken:
Result = A/(C x F)
A = absorbance of the Sample solution
Cc = concentration of the Sample solution (g/mL)
F = coefficient of extinction (E'™) of pure
pheophorbide in ethyl ether (100 mL: g +1 -
cm-1), 702
Acceptance criteria: NMT 0.02%
SPECIFIC TESTS
e WATER DETERMINATION, Method | (921): NMT 0.5%
DS Monographs
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in well-closed
containers.
e USP REFERENCE STANDARDS (11)
USP Astaxanthin Esters from Haematococcus pluvialis RS
USP Apocarotenal RS
trans-beta-Apo-8’-carotenal.
C30H40O
Astragalus Root
DEFINITION
Astragalus Root consists of the dried root of Astragalus mem-
branaceus var. mongholicus (Bunge) P.K.Hsiao or Astragalus
membranaceus (Fisch.) Bunge (Fam. Fabaceae). Astragalus
root is typically harvested from a 2- to 3-year-old plant in
early fall. It contains NLT 0.04% of cycloartane saponins
and NLT 0.03% of isoflavonoids calculated on the dried
basis.
USP 41
IDENTIFICATION
e A. HPTLC FoR ARTICLES OF BOTANICAL ORIGIN (203)
Standard solution A: 1 mg/mL of USP Astragaloside IV
RS in methanol
Standard solution B: 2 mg/mL of USP Daidzin RS and
1 mg/mL of USP Daidzein RS in methanol
Standard solution C: 50 mg/mL of USP Astragalus Root
Dry Extract RS in methanol. Sonicate for about 10 min,
centrifuge, and use the supernatant.
Sample solution: Heat 3 g of Astragalus Root, finely
powdered, in 50 mL of methanol for 50 min under re-
flux. Centrifuge, withdraw the supernatant, and evapo-
rate to dryness under reduced pressure. Dissolve the
residue in 1.0 mL of water. Transfer the resulting solu-
tion onto a 6-mL solid-phase extraction column con-
taining 500 mg of sorbent previously conditioned with
3 mL of methanol and 3 mL of water.’ Wash with
15 mL of water followed by 15 mL of 30% methanol,
and discard the rinsate. Elute with 20 mL of methanol,
collect the eluate, evaporate to dryness under reduced
pressure, and dissolve the residue in 2 mL of methanol.
Chromatographic system
Adsorbent: Chromatographic silica gel with an aver-
age particle size of 5 um (HPTLC plate)?
Application volume: 3 tL each of Standard solution A,
Standard solution B, Standard solution C, and Sample
solution as 8-mm bands
Relative humidity: Condition the plate toa relative
humidity of 33%.
Temperature: Ambient, not to exceed 30°
Developing solvent system: Ethyl acetate, methanol,
and water (100: 13.5: 10)
Developing distance: 6 cm
Derivatization reagent: 10% Sulfuric acid in metha-
nol. [NoTe—Prepare fresh. Slowly and gradually add
sulfuric acid to ice-cold methanol, and mix well.]
System suitability
Samples: Standard solution A, Standard solution B, and
Standard solution C
Suitability requirements
Chromatographic pattern: Under long-wave UV
light (365 nm), following derivatization, Standard so-
lution A exhibits an orange band in the middle of the
lower third of the plate due to astragaloside IV, with
a retardation factor (R) of approximately 0.15. In
Standard solution B, daidzin and daidzein form bluish-
grey bands with R; of spprosmarny, 0.34 and 0.76,
respectively; the proximal band is sharper, while the
distal is somewhat diffuse. In Standard solution C, four
orange bands are seen in the lower third of the plate,
corresponding to astragalosides IV, Ill, Il, and | with Rr
of approximately 0.15, 0.18, 0.24, and 0.34, respec-
tively. The Rr of the astragaloside | band approxi-
mates that of daidzin in Standard solution B. The up-
per two-thirds of the plate typically display a number
of bluish, greenish, and pinkish diffuse bands, one of
which corresponds to that of daidzein in Standard so-
lution B.
Analysis
Samples: Standard solution A, Standard solution B,
Standard solution C, and Sample solution
Apply the Samples as bands and dry in air. Develop in a
saturated chamber. Air-dry, treat with Derivatization re-
agent, heat for 5 min at 100°, and examine under
long-wave UV light (365 nm).
Acceptance criteria: Under long-wave UV light (365
nm), the Sample solution exhibits bands corresponding
in color and R; to similar bands from Standard solution
C, In the lower third of the chromatogram, a number
of orange bands are present; the most prominent ones
corresponding to astragalosides | and Il, with Rr of ap-
proximately 0.34 and 0.24, respectively. In the upper
1Suitable commercially available SPE columns are Bakerbond Octadecy! Cis.
2Suitable commercially available plates are HPTLC Silica Gel 60 Fas« from EMD
Millipore (e.g., part no. 1.05642.0001).
USP 41 Dietary Supplements / Astragalus 4449

part of the chromatogram, a number of diffuse bands Column: 4.6-mm x 25-cm; 5-yum packing L1
are present, and additional weak bands may appear Column temperature: 25°
with respect to those seen in Standard solution C. Flow rate: 0.8 mL/min
[Note—The root of Hedysarum polybotros, a common Injection volume: 15 pL
adulterant, does not show orange bands corresponding System suitability
to astragalosides | and II.] Samples: Standard solutions A-E and Standard solution
e B. HPLC F

Analysis: Proceed as directed in the test for Content of Suitability requirements

Isoflavonoids and Saponins. Chromatographic similarity: The chromatogram of
Acceptance criteria: The Sample solution exhibits peaks Standard solutionFis similar to the reference chro-
at the retention times corresponding to those of matogram provided with the lot of USP Astragalus
calycosin 7-O-B-D-glucopyranoside, ononin, calycosin, Root Dry Extract RS being used.
formononetin, astragaloside IV, astragaloside |, and as- Theoretical plates: NLT 3,000 for calycosin 7-O-B-D-
tragaloside || from Standard solution F. glucopyranoside (UV) and astragaloside IV (ELSD)
peaks, Standard solution A
COMPOSITION Correlation coefficient: NLT 0.995 for each regres-
¢@ CONTENT OF ISOFLAVONOIDS AND SAPONINS sion line as determined in Analysis
Solution A: 0.3% Formic acid Analysis
Solution B: Acetonitrile Samples: Standard solutions A-F and Sample solution
Mobile phase: See Table 7. Using the UV absorbance chromatograms of Standard
solutions A-E, Standard solution F, and the reference
Table 1 chromatogram provided with the lot of USP Astragalus
Root Dry Extract RS being used, identify the specified
Time Solution A Solution B
isoflavonoids in the Sample solution chromatogram.
(min) (%) (%) Measure the areas of the isoflavonoid peaks. Plot the
0 80 20 areas of the relevant peaks against the respective con-
15 80 20 centrations (mg/mL) of each analyte in Standard solu-
25: 68 32 tions A-E, and determine the equations of the resulting
35 66 34 least-squares regression lines.
45 55 45 Using the equations of the relevant least-squares lines,
determine the concentrations of each specified
55 50 50 isoflavonoid (calycosin 7-O-B-D-glucopyranoside,
75 25 75 ononin, calycosin, and forttononetinyvin the Sample
80 80 20 solution.
100 80 20 Separately calculate the percentages of each isoflavo-
noid in the portion of Astragalus Root taken:
Standard solution A: Prepare a composite solution
containing 0.4 mg/mL of USP Astragaloside IV RS, Result = Cx (V/W) x 100
0.1 mg/mL of USP Calycosin RS, 0.2 mg/mL of USP
Calycosin 7-O-B-D-Glucopyranoside RS, 0.05 mg/mL of G = concentration of the relevant isoflavonoid in
USP Formononetin RS, and 0.1 mg/mL of USP Ononin the Sample solution (mg/mL)
RS in methanol. Vv = volume of the Sample solution (mL)
Standard solutions B, C, D, E: Prepare four consecutive Ww = weight of Astragalus Root taken to prepare the
two-fold serial dilutions of Standard solution A in Sample solution (mg)
methanol. Calculate the sum of percentages of isoflavonoids.
Standard solution F: Sonicate 150 mg of USP Astraga- Using the ELSD chromatograms of Standard solutions
lus Root Dry Extract RS in 5 mL of methanol. Pass A-£, Standard solution F, and the reference
through a nylon filter of 0.45-um pore size, and discard chromatogram, provided with the lot of USP
the initial 1 mL of the filtrate. Astragalus Root Dry Extract RS being used, identify all
Sample solution: Accurately weigh 1.5 g of Astragalus specified saponins in the Sample solution
Root reduced to fine powder, and transfer into a chromatogram. The approximate relative retention
100-mL round-bottomed flask. Attach the condenser times for astragalosides | and Il, with respect to 4
and reflux in 60 mL of methanol for 3 h. Filter and astragaloside IV, are provided in Table 2. z
evaporate methanol to dryness under reduced pressure.
Dissolve the residue in a small amount of methanol, Table 2 3
and transfer quantitatively into a 5-mL volumetric flask.
Adjust with methanol to volume and mix well. Pass Analyte Relative Retention Time &
through a nylon filter of 0.45-um pore size, and discard Astragaloside IV 1.00 PY
the initial 1 mL of the filtrate. Astragaloside Il 1.10 —
Chromatographic system Astragaloside | 1.28 wa
(See Chromatography (621), System Suitability.)
Mode: HPLC Measure the areas of the saponin peaks. Plot the
Detectors: UV 280 nm and ELSD, connected in series logarithms of astragaloside IV peak areas against the
ELSD drift tube temperature: Optimize according to logarithms of their respective concentrations (mg/mL)
the manufacturer’s recommendations to achieve opti- in Standard solutions A-E, and determine the equation
mal signal-to-noise ratio, typically 105°. of a least-squares regression line. Using the equation of
ELSD carrier gas flow: Optimize according to the the least-squares line for astragaloside IV, calculate the
manufacturer's recommendations to achieve optimal concentrations of each specified saponin (astragaloside
signal-to-noise ratio, typically 2.70 L/min. |, astragaloside II, and astragaloside IV) in the Sample
solution.
Separately calculate the percentages of each saponin in
the portion of Astragalus Root taken:
Result = Cs x (V/W) x 100
 4450 Astragalus / Dietary Supplements
Cs = concentration of the relevant saponin in the
Sample solution (mg/mL)
Vv = volume of the Sample solution (mL)
w = weight of Astragalus Root taken to prepare the
Sample solution (mg)
Calculate the sum of percentages of saponins.
Acceptance criteria
Sum of isoflavonoids: NLT 0.03% on the dried basis
Sum of saponins: NLT 0.04% on the dried basis
CONTAMINANTS
e ELEMENTAL IMPURITIES—PROCEDURES (233)
Acceptance criteria
Arsenic: NMT 1.5 ug/g
Cadmium: NMT 0.3 ug/g
Lead: NMT 5.0 ug/g
Mercury: NMT 0.1 ug/g
e ARTICLES OF BOTANICAL ORIGIN (561), Pesticide Residue
Analysis: Meets the requirements
e MICROBIAL ENUMERATION TESTS (2021): The total aerobic
bacterial count does not exceed 105 cfu/g, total com-
bined yeasts and molds count does not exceed 103 cfu/
g, and the bile-tolerant Gram-negative bacteria count
does not exceed 103 cfu/g.
e ABSENCE OF SPECIFIED MICROORGANISMS (2022), Test Proce-
dures, Test for Absence of Salmonella Species and Test for
Absence of Escherichia coli: Meets the requirements
SPECIFIC TESTS
¢ BOTANICAL CHARACTERISTICS
Macroscopic: Astragalus Root is cylindrical, some upper
branches relatively thick, 30-90 cm long, 0.5-3.5 cm in
diameter. Externally pale brownish yellow or pale
brown (but not red), with irregular, longitudinal wrin-
kles or furrows. Texture hard and tenacious, broken
with difficulty, fracture highly fibrous and starchy; bark
yellowish-white; wood pale yellow, with radiate stria-
tions and fissures, the center part of old root occasion-
ally looking like rotten wood, blackish brown or
hollowed.
Microscopic: The transverse section shows cork consist-
ing of many rows of tangentially elongated cells. Phello-
derm, 3-7 rows of collenchymatous cells. Outer part of
hloem rays often curved and fissured, fibers in bundles
tom 6-22 um in diameter, with longitudinal fissures
and truncate or brush-like ends. The walls are thickened
and lignified or slightly lignified, arranged alternately
with sieve tube groups; stone cells sometimes visible
near phelloderm. Cambium ina ring. Xylem vessels
scattered singly or 2-3 aggregated in groups; wood fi-
bers among vessel stone cells singly or 2-4 in groups,
sometimes visible in rays. Parenchymatous cells contain
DS Monographs
starch granules. In the longitudinal section, no solitary
calcium oxalate crystals are seen outside the fiber bun-
dle (a distinction from Hedysarum polybotros and other
Hedysarum species, common adulterants).
ARTICLES OF BOTANICAL ORIGIN (561), Foreign Organic Mat-
ter. NMT 2.0%
Loss ON DRYING (731)
Sample: 1.0g of finely powdered Astragalus Root
Analysis: Dry the Sample at 105° for 3 h.
Acceptance criteria: NMT 10.0%
ARTICLES OF BOTANICAL ORIGIN (561), Total Ash
Sample: 1.0 g of powdered Astragalus Root
Acceptance criteria: NMT 5.0%
ARTICLES OF BOTANICAL ORIGIN (561), Acid-/nsoluble Ash
Sample: 1.0 g of powdered Astragalus Root
Acceptance criteria: NMT 1.0%
e ARTICLES OF BOTANICAL ORIGIN (561), Alcohol-Soluble Ex-
tractives, Method 1
Sample: 2-4 g of powdered Astragalus Root
Acceptance criteria: NLT 2.0%
ARTICLES OF BOTANICAL ORIGIN (561), Water-Soluble Extrac-
tives, Method 1
USP 41
Sample: 2-4g of powdered Astragalus Root
Acceptance criteria: NLT 17.0%
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in well-closed contain-
ers, protected from light and moisture, and store at
room temperature.
e LABELING: The label states the Latin binomial of the spe-
cies from which the article was derived.
e USP REFERENCE STANDARDS (11)
USP Astragaloside IV RS
USP Astragalus Root Dry Extract RS
USP Calycosin RS
USP Calycosin 7-O-8-D-Glucopyranoside RS
USP Daidzein RS
USP Daidzin RS
USP Formononetin RS
USP Ononin RS
Astragalus Root Powder
DEFINITION
Astragalus Root Powder consists of the dried root of Astraga-
lus membranaceus var. mongholicus (Bunge) P.K.Hsiao or
Astragalus membranaceus (Fisch.) Bunge (Fam. Fabaceae)
reduced to powder or very fine powder. Astragalus root is
ppleally harvested from a 2- to 3-year-old plant in earl
fall. It contains NLT 0.04% of cycloartane saponins one
NLT 0.03% of isoflavonoids calculated on the dried basis.
IDENTIFICATION
e A. HPTLC For ARTICLES OF BOTANICAL ORIGIN (203)
Standard solution A: 1 mg/mL of USP Astragaloside IV
RS in methanol
Standard solution B: 2mg/mL of USP Daidzin RS and
1 mg/mL of USP Daidzein RS in methanol
Standard solution C: 50 mg/mL of USP Astragalus Root
Dry Extract RS in methanol. Sonicate for about 10 min,
centrifuge, and use the supernatant.
Sample solution: Heat 3 g of Astragalus Root Powder
in 50 mL of methanol for 50 min under reflux. Centri-
fuge, withdraw the supernatant, and evaporate to dry-
ness under reduced pressure. Dissolve the residue in
1.0 mL of water. Transfer the resulting solution onto a
6-mL solid-phase extraction column containing 500 mg
of sorbent previously conditioned with 3 mL of metha-
nol and 3 mL of water.’ Wash with 15 mL of water fol-
lowed by 15 mL of 30% methanol, and discard the rin-
sate. Elute with 20 mL of methanol, collect the eluate,
evaporate to dryness under reduced pressure, and dis-
solve the residue in 2 mL of methanol.
Chromatographic system
Adsorbent: Chromatographic silica gel with an aver-
age particle size of 5 um (HPTLC plate)?
Application volume: 3 wL each of Standard solution A,
Standard solution B, Standard solution C, and Sample
solution as 8-mm bands
Relative humidity: Condition the plate toa relative
humidity of 33%.
Temperature: Ambient, not to exceed 30°
Developing solvent system: Ethyl acetate, methanol,
and water (100: 13.5: 10)
Developing distance: 6 cm
Derivatization reagent: 10% Sulfuric acid in metha-
nol. [NoTe—Prepare fresh. Slowly and gradually add
sulfuric acid to ice-cold methanol, and mix well.]
System suitability
Samples: Standard solution A, Standard solution B, and
Standard solution C
1 Suitable commercially available SPE columns are Bakerbond Octadecy! Cis.
2 Suitable commercially available plates are HPTLC Silica Gel 60 Fas, from
EMD Millipore (e.g., part no. 1.05642.0001).
USP 41 Dietary Supplements / Astragalus 4451

Suitability requirements Standard solutions B, C, D, E: Prepare four consecutive

Chromatographic pattern: Under long-wave UV
light (365 nm), following derivatization, Standard so-
lution A exhibits an orange band in the middle of the
lower third of the plate due to astragaloside IV, with
a retardation factor (Rr) of approximately 0.15. In
Standard solution B, daidzin and daidzein form bluish-
grey bands with R; of approximately 0.34 and 0.76,
respectively; the proximal band is sharper, while the
distal is somewhat diffuse. In Standard solution C, four
orange bands are seen in the lower third of the plate,
corresponding to astragalosides IV, III, Il, and | with Rr
of approximately 0.15, 0.18, 0.24, and 0.34, respec-
tively. The Rr of the astragaloside | band approxi-
mates that of daidzin in Standard solution B. The up-
per two-thirds of the plate typically display a number
of bluish, greenish, and pinkish diffuse bands, one of
which corresponds to that of daidzein in Standard so-
lution B.
Analysis
Samples: Standard solution A, Standard solution B,
Standard solution C, and Sample solution
Apply the Samples as bands and dry in air. Develop in a
saturated chamber. Air-dry, treat with Derivatization re-
agent, heat for 5 min at 100°, and examine under
long-wave UV light (365 nm).
Acceptance criteria: Under long-wave UV light (365
nm), the Sample solution exhibits bands corresponding
in color and Rr to similar bands from Standard solution
C. In the lower third“of the chromatogram, a number
of orange bands are present; the most prominent ones
corresponding to astragalosides | and Il, with Rr of ap-
proximately 0.34 and 0.24, respectively. In the upper
part of the chromatogram, a number of diffuse bands
two-fold serial dilutions of Standard solution A in
methanol.
Standard solution F: Sonicate 150 mg of USP Astraga-
lus Root Dry Extract RS in 5 mL of methanol. Pass
through a nylon filter of 0.45-4m pore size, and discard
the initial 1 mL of the filtrate.
Sample solution: Accurately weigh 1.5 g of Astragalus
Root Powder, and transfer into a 100-mL round-bot-
tomed flask. Attach the condenser and reflux in 60 mL
of methanol for 3 h. Filter and evaporate methanol to
dryness under reduced pressure, dissolve the residue in
a small amount of methanol, and transfer quantitatively
into a 5-mL volumetric flask. Adjust with methanol to
volume and mix well. Pass aa a nylon filter of
0.45-um pore size, and discard the initial 1 mL of the
filtrate.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: HPLC
Detectors: UV 280 nm and ELSD, connected in series
ELSD drift tube temperature: Optimize according to
the manufacturer’s recommendations to achieve opti-
mal signal-to-noise ratio, typically 105°.
ELSD carrier gas flow: Optimize according to the
manufacturer’s recommendations to achieve optimal
Saree oer ratio, typically 2.70 L/min.
Column: 4.6-mm x 25-cm; 5-um packing L1
Column temperature: 25°
Flow rate: 0.8 mL/min
Injection volume: 15 wL
System suitability
Samples: Standard solutions A-E and Standard solution
F

are present, and additional weak bands may appear
with respect to those seen in Standard solution C.
{[Note—The root of Hedysarum polybotros, a common
adulterant, does not show orange bands corresponding
to astragalosides | and II.]
e B. HPLC
Analysis: Proceed as directed in the test for Content of
Isoflavonoids and Saponins.
Acceptance criteria: The Sample solution exhibits peaks
at the retention times corresponding to those of
calycosin 7-O-B-D-glucopyranoside, ononin, calycosin,
formononetin, astragaloside IV, astragaloside |, and as-
tragaloside II from Standard solution F.
COMPOSITION
© CONTENT OF ISOFLAVONOIDS AND SAPONINS
Solution A: 0.3% Formic acid
Solution B: Acetonitrile
Suitability requirements
Chromatographic similarity: The chromatogram of
Standard solutionF is similar to the reference chro-
matogram provided with the lot of USP Astragalus
Root Dry Extract RS being used.
Theoretical plates: NLT 3,000 for calycosin 7-O-B-p-
glucopyranoside (UV) and astragaloside IV (ELSD)
peaks, Standard solution A
Correlation coefficient: NLT 0.995 for each regres-
sion line as determined in Analysis
Analysis
Samples: Standard solutions A-F and Sample solution
Using the UV absorbance chromatograms of Standard
solutions A-E, Standard solution F, and the reference
chromatogram, provided with the lot of USP Astraga-
lus Root Dry Extract RS being used, identify the speci-
fied isoflavonoids in the Sample solution chromato-
sydeibouo= Sa

gram. Measure the areas of the isoflavonoid peaks.

Mobile phase: See Table 1. Plot the areas of the relevant peaks against the respec-
tive concentrations (mg/mL) of each analyte in Stan-
Table 1 dard solutions A-E, and determine the equations of the
resulting least-squares regression lines.
Time Solution A Solution B
Using the equations of the relevant least-squares lines,
(min) (%) (%)
determine the concentrations of each specified
0 80 20 isoflavonoid (calycosin 7-O-B-D-glucopyranoside,
15 80 20 ononin, calycosin, and formononetin) in the Sample
25 68 32 solution.
35 66 34 Separately calculate the percentages of each isoflavo-
45 55 AS noid in the portion of Astragalus Root Powder taken:
55) 50 50 Result = Cx (V/W) x 100
75 25 75
80 80 20 G = concentration of the relevant isoflavonoid in
100 80 20 the Sample solution (mg/mL)
V = volume of the Sample solution (mL)
Standard solution A: Prepare a composite solution w = weight of Astragalus Root Powder taken to
containing 0.4 mg/mL of USP Astragaloside IV RS, prepare the Sample solution (mg)
0.1 mg/mL of USP Calycosin RS, 0.2 mg/mL of USP Calculate the sum of percentages of isoflavonoids.
Calycosin 7-O-B-D-Glucopyranoside RS, 0.05 mg/mL of Using the ELSD chromatograms of Standard solutions
USP Formononetin RS, and 0.1 mg/mL of USP Ononin A-£, Standard solution F, and the reference
RS in methanol. chromatogram, provided with the lot of USP
 4452 Astragalus / Dietary Supplements
manage Root Dry Extract RS being used, identify all
specified saponins in the Sample solution
chromatogram. The approximate relative retention
times for astragalosides | and II, with respect to
astragaloside IV, are provided in Table 2.
Table 2
Analyte Relative Retention Time
Astragaloside IV 1.00
Astragaloside II 1.10
Astragaloside | 1.28
Measure the areas of the saponin peaks. Plot the
logarithms of astragaloside IV peak areas against the
logarithms of their respective concentrations (mg/mL)
in Standard solutions A-E, and determine the equation
of least-squares regression line. Using the equation of
the least-squares line for astragaloside IV, calculate the
concentrations of each specified saponin (astragaloside
|, astragaloside II, and astragaloside IV) in the Sample
solution.
Separately calculate the percentages of each saponin in
the portion of Astragalus Root Powder taken:
Result = Cs x (V/W) x 100
Cs = concentration of the relevant saponin in the
Sample solution (mg/mL)
Vv = volume of the Sample solution (mL)
Ww = weight of Astragalus Root Powder taken to
prepare the Sample solution (mg)
Calculate the sum of percentages of saponins.
Acceptance criteria
Sum of isoflavonoids: NLT 0.03% on the dried basis
Sum of saponins: NLT 0.04% on the dried basis
CONTAMINANTS
e ELEMENTAL IMPURITIES—PROCEDURES (233)
Acceptance criteria
Arsenic: NMT 1.5 yg/g
Cadmium: NMT 0.3 ug/g
Lead: NMT 5.0 ug/g
Mercury: NMT 0.1 ug/g
e ARTICLES OF BOTANICAL ORIGIN (561), Pesticide Residue
Analysis: Meets the requirements
e MICROBIAL ENUMERATION TESTS (2021): The total aerobic
bacterial count does not exceed 105 cfu/g, total com-
bined yeasts and molds count does not exceed 103 cfu/
g, and the bile-tolerant Gram-negative bacteria count
does not exceed 103 cfu/g.
DS Monographs
o ABSENCE OF SPECIFIED MICROORGANISMS (2022), Test Proce-
dures, Test for Absence of Salmonella Species and Test for
Absence of Escherichia coli: Meets the requirements
SPECIFIC TESTS
e BOTANICAL CHARACTERISTICS
Macroscopic: White to pale-yellow powder, splintery
and fibrous
Microscopic: Fibers occur in bundles or scattered, 3-30
um in diameter, walls thickened, the primary walls of-
ten separated from secondary, with longitudinal fissures
and truncate or brush-like ends, polychromatic in po-
larized light. Stone cells occasionally visible, sub-
rounded, oblong or irregular, slightly thick-walled,
bright yellowish-white in polarized light. Cork cells ir-
regular or polygonal, sometimes with sinuous anticlinal
walls. In Astragalus membranaceus var. mongholicus
(Bunge) P.K.Hsiao, reticulated vessels abundant, bor-
dered-pitted vessels few, 16-150 um in diameter. In As-
tragalus membranaceus (Fisch.) Bunge, bordered-pitted
vessels abundant, bordered pits arranged closely, up to
200 um in diameter. Simple starch granules spheroidal
or ellipsoid, 3-23 um in diameter, with visible linear or
punctate hilum. Occasional compound starch granules
USP 41
composed of 2-4 components. Observed in polarized
light, starch granules are black and cruciate. Calcium
oxalate crystals are absent.
ARTICLES OF BOTANICAL ORIGIN (561), Foreign Organic Mat-
ter. NMT 2.0%
Loss ON DRYING (731)
Sample: 1.0g of Astragalus Root Powder
Analysis: Dry the Sample at 105° for 3 h.
Acceptance criteria: NMT 10.0%
ARTICLES OF BOTANICAL ORIGIN (561), Total Ash
Sample: 1.0 of Astragalus Root Powder
Acceptance criteria: NMT 5.0%
e ARTICLES OF BOTANICAL ORIGIN (561), Acid-Insoluble Ash
Sample: 1.0 of Astragalus Root Powder
Acceptance criteria: NMT 1.0%
ARTICLES OF BOTANICAL ORIGIN (561), Alcohol-Soluble Ex-
tractives, Method 1
Sample: 2-4g of Astragalus Root Powder
Acceptance criteria: NLT 2.0%
ARTICLES OF BOTANICAL ORIGIN (561), Water-Soluble Extrac-
tives, Method 1
Sample: 2-4g of Astragalus Root Powder
Acceptance criteria: NLT 17.0%
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in well-closed contain-
ers, protected from light and moisture, and store at
room temperature.
© LABELING: The label states the Latin binomial of the spe-
cies from which the article was derived.
e USP REFERENCE STANDARDS (11)
USP Astragaloside IV RS
USP Astragalus Root Dry Extract RS
USP Calycosin RS
USP Calycosin 7-O-B-D-Glucopyranoside RS
USP Daidzein RS
USP Daidzin RS
USP Formononetin RS
USP Ononin RS
Astragalus Root Dry Extract
DEFINITION
Astragalus Root Dry Extract is prepared from the dried root
of Astragalus membranaceus var. mongholicus (Bunge) P.K.
Hsiao or Astragalus membranaceus (Fisch.) Bunge (Fam.
Fabaceae) subjected to aqueous or hydroalcoholic extrac-
tion. It contains NLT 90.0% and NMT 110.0% of the la-
beled amounts of cycloartane saponins and isoflavonoids
calculated on the anhydrous basis. It may contain suitable
substances added as carriers.
IDENTIFICATION
e A. HPTLC For ARTICLES OF BOTANICAL ORIGIN (203)
Standard solution A: 1 mg/mL of USP Astragaloside IV
RS in methanol
Standard solution B: 2mg/mlL of USP Daidzin RS and
1 mg/mL of USP Daidzein RS in methanol
Standard solution C: 50 mg/mL of USP Astragalus Root
Dry Extract RS in methanol. Sonicate for about 10 min,
centrifuge, and use the supernatant.
Sample solution: 50 mg/mL of Astragalus Root Dry Ex-
tract in methanol. Sonicate for about 10 min, centri-
fuge, and use the supernatant.
Chromatographic system
Adsorbent: Chromatographic silica gel with an aver-
age particle size of 5 um (HPTLC plate)!
Application volume: 3 wL each of Standard solution A,
Standard solution B, Standard solution C, and Sample
solution as 8-mm bands
1 Suitable commercially available plates are HPTLC Silica Gel 60 Fasq from
EMD Millipore (e.g., part no. 1.05642.0001).
USP 41 Dietary Supplements / Astragalus 4453

Relative humidity: Condition the plate to a relative
humidity of 33%.
Temperature: Ambient, not to exceed 30°
Developing solvent system: Ethyl acetate, methanol,
and water (100: 13.5: 10)
Developing distance: 6 cm
Derivatization reagent: 10% Sulfuric acid in metha-
nol. [NoTte—Prepare fresh. Slowly and gradually add
sulfuric acid to ice-cold methanol, and mix well.]
System suitability
Samples: Standard solution A, Standard solution B, and
Standard solution C
Suitability requirements
Chromatographic pattern: Under long-wave UV
light (365 nm), following derivatization, Standard so-
lution A exhibits an orange band in the middle of the
lower third of the plate due to astragaloside IV, with
a retardation factor (R) of approximately 0.15. In
Standard solution B, daidzin and daidzein form bluish-
grey bands with R; of approximately 0.34 and 0.76,
respectively; the proximal band is sharper, while the
distal is somewhat diffuse. In Standard solution C, four
orange bands are seen in the lower third of the plate,
corresponding to astragalosides IV, Ill, Il, and | with Rr
of approximately 0.15, 0.18, 0.24, and 0.34, respec-
tively. The Rr of the astragaloside | band approxi-
mates that of daidzin in Standard solution B. The up-
per two-thirds of the plate typically display a number
of bluish, greenish, and pinkish bands, one of which
corresponds to that of daidzein in Standard solution B.
Analysis
Samples: Standard solution A, Standard solution B,
Standard solution C, and Sample solution
Apply the Samples as bands and dry in air. Develop in a
saturated chamber. Air-dry, treat with Derivatization re-
agent, heat for 5 min at 105°, and examine under
long-wave UV light (365 nm).
Acceptance criteria: Under long-wave UV light (365
nm), the Sample solution exhibits bands corresponding
in color and R; to similar bands from Standard solution
C, at the Rr values listed in Chromatographic pattern.
[Note—The extract of Hedysarum polybotros, a common
adulterant, does not show orange bands corresponding
to astragalosides | and II.]
e B. HPLC
Analysis: Proceed as directed in the test for Content of
Isoflavonoids and Saponins.
Acceptance criteria: The Sample solution exhibits peaks
at the retention times corresponding to those of
calycosin 7-O-B-D-glucopyranoside, ononin, calycosin,
formononetin, astragaloside |V, astragaloside |, and as-
Standard solution A: Prepare a composite solution
containing 0.4 mg/mL of USP Astragaloside IV RS,
0.1 mg/mL of USP Calycosin RS, 0.2 mg/mL of USP
Calycosin 7-O-B-D-Glucopyranoside RS, 0.05 mg/mL of
USP Formononetin RS, and 0.1 mg/mL of USP Ononin
RS in methanol.
Standard solutions B, C, D, E: Prepare four consecutive
two-fold serial dilutions of Standard solution A in
methanol.
Standard solution F: Sonicate 150 mg of USP Astraga-
lus Root Dry Extract RS in 5 mL of methanol. Pass
through a nylon filter of 0.45-4m pore size, and discard
the initial 1 mL of the filtrate.
Sample solution: Accurately weigh about 300 mg of
Astragalus Root Dry Extract into a 10-mL volumetric
flask. Add 5 mL of methanol and sonicate for 10 min.
Cool, adjust with methanol to volume, and mix well.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: HPLC
Detectors: UV 280 nm and ELSD, connected in series
ELSD drift tube temperature: Optimize according to
the manufacturer’s recommendations to achieve opti-
mal signal-to-noise ratio, typically 105°.
ELSD carrier gas flow: Optimize according to the
manufacturer’s recommendations to achieve optimal
signal-to-noise ratio, typically 2.70 L/min.
Column: 4.6-mm x 25-cm; 5-4m packing L1
Column temperature: 25°
Flow rate: 0.8 mL/min
Injection volume: 15 pL
System suitability
Samples: Standard solutions A-E and Standard solution
FE
Suitability requirements
Chromatographic similarity: The chromatogram of
Standard solution F is similar to the reference chro-
matogram provided with the lot of USP Astragalus
Root Dry Extract RS being used.
Theoretical plates: NLT 3,000 for calycosin 7-O-B-D-
glucopyranoside (UV) and astragaloside IV (ELSD)
peaks, Standard solution A
Correlation coefficient: NLT 0.995 for each regres-
sion line as determined in Analysis
Analysis
Samples: Standard solutions A-F and Sample solution
Using the UV absorbance chromatograms of Standard
solutions A-E, Standard solution F, and the reference
chromatogram, provided with the lot of USP Astraga-
lus Root Dry Extract RS being used, identify the speci-
fied isoflavonoids in the Sample solution chromato-
sydesbouow sa

tragaloside II from Standard solution F. gram. Measure the areas of the isoflavonoid peaks.
Plot the areas of the relevant peaks against the respec-
COMPOSITION tive concentrations (mg/mL) of each analyte in Stan-
© CONTENT OF ISOFLAVONOIDS AND SAPONINS dard solutions A-E, and determine the equations of the
Solution A: 0.3% Formic acid resulting least-squares regression lines.
Solution B: Acetonitrile Using the equations of the relevant least-squares lines,
Mobile phase: See Table 7. determine the concentrations of each specified
isoflavonoid (calycosin 7-O-B-D-glucopyranoside,
Table 1 ononin, calycosin, and formononetin) in the Sample
solution.
Time Solution A Solution B
Separately calculate the percentages of each isoflavo-
(min) (%) (%) ee in the portion of Astragalus Root Dry Extract
0 80 20 taken:
15 80 20
25 68 32 Result = C, x (V/W) x 100
35 66 34
G = concentration of the relevant isoflavonoid in
45 55 45
the Sample solution (mg/mL)
55 50 50 Vv = volume of the Sample solution (mL)
75 25 75 w = weight of Astragalus Root Dry Extract taken to
80 80 20 prepare the Sample solution (mg)
100 80 20 Calculate the sum of percentages of isoflavonoids.
 4454 Astragalus / Dietary Supplements USP 41

Calculate the percentage of the labeled amount of i yeasts and molds count does not exceed 103
isoflavonoids in the portion of Astragalus Root Dry cfu/g.
Extract taken: © ABSENCE OF SPECIFIED MICROORGANISMS (2022), Test Proce-
dures, Test for Absence of Salmonella Species and Test for
Result = (P/L) x 100 Absence of Escherichia coli: Meets the requirements
P = sum of percentages of isoflavonoids in the SPECIFIC TESTS
Astragalus Root Dry Extract, as calculated © RESIDUE ON IGNITION (281)
above (%) Sample: 1.0 of Astragalus Root Dry Extract
L = labeled amount of isoflavonoids in the Acceptance criteria: NMT 5.0%
Astragalus Root Dry Extract (%) e BOTANICAL EXTRACTS (565), Residual Solvents: Meets the
Using the ELSD chromatograms of Standard solutions requirements
A-E, Standard solution F, and the reference @ WATER DETERMINATION (921), Method la
chromatogram, provided with the lot of USP Acceptance criteria: NMT 6.0%
Astragalus Root Dry Extract RS being used, identify all
specified saponins in the Sample solution ADDITIONAL REQUIREMENTS
chromatogram. The approximate relative retention © PACKAGING AND STORAGE: Preserve in well-closed contain-
times for astragalosides | and Il, with respect to ers, protected from light and moisture, and store at
astragaloside IV, are provided in Table 2. room temperature.
e LABELING: The label states the Latin binomial of the spe-
cies from which the article was derived. The label also
Table 2
indicates the content of isoflavonoids and cycloartane sa-
Analyte Relative Retention Time ponins, the solvent used in extract preparation, and the
Astragaloside IV 1.00 ratio of the starting crude plant material to dry extract. It
Astragaloside II 1.10 bes the labeling requirements of Botanical Extracts
Astragaloside | 1.28
565).
e USP REFERENCE STANDARDS (11)
Measure the areas of the saponin peaks. Plot the USP Astragaloside IV RS
logarithms of Astragaloside !V peak areas against the USP Astragalus Root Dry Extract RS
logarithms of their respective concentrations (mg/mL) USP Calycosin RS
in Standard solutions A-E, and determine the equation USP Calycosin 7-O-B-D-Glucopyranoside RS
of a least-squares regression line. Using the equation of USP Daidzein RS
the least-squares line for astragaloside IV, calculate the USP Daidzin RS
concentrations of each specified saponin (astragaloside USP Formononetin RS
|, astragaloside II, and astragaloside IV) in the Sample USP Ononin RS
solution.
Separately calculate the percentages of each saponin in
the portion of Astragalus Root Dry Extract taken:
Result = Cs x (V/W) x 100 Aztec Marigold Zeaxanthin Extract
Gs = concentration of the relevant saponin in the He on
Sample solution (mg/mL) HyC
VV
CHs CH CH

Vv = volume of the Sample solution (mL) wR ‘oO AIR

Se i eI SS al
w = weight of Astragalus Root Dry Extract taken to oH, oy va “cry
prepare the Sample solution (mg) HO" ‘CHs

Calculate the sum of percentages of saponins.

Calculate thepercentage of the labeled amount of C4oHs6O2 568.87
saponins in the portion of Astragalus Root Dry Extract (all-E)-1,1'-(3,7,12,16-Tetramethyl-1,3,5,7,9,11,13,15,
taken: 17-octadecanonaene-1,18-diyl)bis[2,6,6-trimethylcyclohex-
ene-3-ol];
Result = (P/L) x 100 3R,3’R-B,B-Carotene-3,3’-diol [148-68-3].
DS Monographs

P = sum of percentages of saponins inAstragalus DEFINITION

Root Dry Extract, as calculated above (%) Aztec Marigold Zeaxanthin Extract is a purified extract, de-
L = labeled amount of saponins in Astragalus Root rived from the flowers of Tagetes erecta L., grown from
Dry Extract (%) seeds of varieties of the Scarletade cultivar rich in zeaxan-
Acceptance criteria thin. The extract contains NLT 36.0% of total carotenoids
Sum of isoflavonoids: 90.0%-110.0% of the labeled calculated as zeaxanthin (C4oHs6O2), NLT 30.0% of all-
amount of isoflavonoids on the anhydrous basis trans-zeaxanthin, and NMT 8.0% of lutein, calculated on
Sum of saponins: 90.0%-110.0% of the labeled the dried basis.
amount of saponins on the anhydrous basis
IDENTIFICATION
CONTAMINANTS oA.
e ELEMENTAL IMPURITIES—PROCEDURES (233) Sample solution: Use the Sample solution from the test
Acceptance criteria for Content of Total Carotenoids.
Arsenic: NMT 1.5 ug/g Analysis: Record the UV-Vis spectrum from 300-600
Cadmium: NMT 0.3 g/g nm.
Lead: NMT 5.0 ug/g Acceptance criteria: The Sample solution shows a shoul-
Mercury: NMT 0.1 ug/g der at about 428 nm, an absorption maximum at about
o ARTICLES OF BOTANICAL ORIGIN (561), Pesticide Residue 450 nm, and another maximum at about 478 nm.
Analysis: Meets the requirements e B. The retention time of the major peak of the Sample
e MICROBIAL ENUMERATION TESTS (2021): The total aerobic solution corresponds to that of the Standard solution, as
bacterial count does not exceed 104 cfu/g, and total obtained in the test for Content of Zeaxanthin.
USP 41 Dietary Supplements / Aztec Marigold Zeaxanthin 4455

e C. The retention time of the major peak of the Sample wash down the product into the flask using Diluent.]
solution corresponds to that of 3R,3'R-B,B-carotene-3,3’- Dilute with Diluent to volume, and mix well. Allow the
diol from the Standard solution, as obtained in the test insolubles to settle for at least 10 min. Pass the super-
for Stereoisomeric Composition. natant through a membrane filter of 0.45-m pore size.
Sample solution: Transfer 0.5 mL of the Sample stock
COMPOSITION solution to an 8-mL vial, and evaporate to dryness with
© CONTENT OF TOTAL CAROTENOIDS the aid of a stream of nitrogen. Dissolve the residue in
[NoteE—Use low-actinic glassware.] a 4.0-mL mixture of methyl tert-butyl ether and metha-
Sample stock solution: Use the Sample stock solution nol (5:95).
from the test for Content of Zeaxanthin. Chromatographic system
Sample solution: Transfer 2.0 mL of the Sample stock (See Chromatography (621), System Suitability.)
solution to a 100-mL volumetric flask, dilute with etha- Mode: HPLC
nol to volume, and mix well. Detector: 450 nm
Instrumental conditions Column: 2.0-mm x 15-cm; 3-4um packing L62
(See Ultraviolet-Visible Spectroscopy (857).) Flow rate: 0.4 mL/min
Analytical wavelength: 450 nm Injection volume: 10 uL
Cell path: 1cm System suitability
Blank: Ethanol Sample: Standard solution
Analysis [NotE—The approximate relative retention times for lu-
Sample: Sample solution tein and zeaxanthin are 0.87 and 1.0, respectively.]
[Note—The absorbance reading should be between 0.2 Suitability requirements
AU to 1.0 AU. If not, readjust the dilution of the Chromatographic similarity: The chromatogram
solution.] from the Standard solution is similar to the reference
Calculate the percentage of the total carotenoids as ze- chromatogram provided with the USP Aztec Marigold
axanthin (CaoHs6O2): Zeaxanthin Extract RS being used.
Resolution: NLT 1.0 between zeaxanthin and lutein
Result = A/(C x F) Tailing factor: NMT 2.0 for the zeaxanthin peak
Relative standard deviation: NMT 2.0% for the zea-
A = absorbance of the Sample solution xanthin peak
Gc = concentration of the Sample solution (g/mL) Analysis
F = coefficient of extinction (E'%) of zeaxanthin in Sample: Sample solution
ethanol (100 mL - g-'- cm-"), 2480 Calculate the percentage of all-trans-zeaxanthin
Acceptance criteria: NLT 36.0% of total carotenoids (C4oHssO2) in the portion of the sample taken:
(1) as zeaxanthin (C4oHs6O2) on the dried basis
@ CONTENT OF ZEAXANTHIN Result = (ru/rr) x T
[Note—Use low-actinic glassware.]
Solution A: Methyl tert-butyl! ether tu = peak response of all-trans-zeaxanthin from the
Solution B: Water Sample solution
Solution C: Methanol nr = sum of the responses for all the peaks from
Diluent: Mixture of hexane, ethanol, acetone, and tolu- the Sample solution
ene (10:6:7:7) T = percentage of total carotenoids as determined
Mobile phase: Gradient elution. See Table 7. in the test for Content of Total Carotenoids
Acceptance criteria: NLT 30.0% of all-trans-zeaxanthin
Table 1 on the dried basis
e LUTEIN AND OTHER RELATED COMPOUNDS
Time Solution A Solution B Solution C [Note—Use low-actinic glassware.]
(min) (%) (%) (%) Mobile phase, Standard solution, Sample solution,
0.0 5 7 88 Chromatographic system, and System suitability:
15 15 7 78 Proceed as directed in the test for Content of
30 45 7 48 Zeaxanthin.
60 75 6.5 18.5 Analysis
66 75: 6 1S: Sample: Sample solution 4
Calculate the percentage of lutein in the portion of the
76 5 a 88
sample taken: ES
86 5 Z 838
Result = (ru/r) x T 2
Standard stock solution: Transfer 20.0 mg of USP Az-
tec Marigold Zeaxanthin Extract RS into a 100-mL volu- ru = peak response of lutein from the Sample $
metric flask, add 75 mL of Diluent to the flask to sus- Solution Se]
pend the sample, and sonicate for 5 min. Dilute with tt = sum of the responses for all the peaks from ms
Diluent to volume, and mix well. Allow the insolubles to the Sample solution 2
settle for at least 10 min. Pass the supernatant through Fe = percentage of total carotenoids as determined
a membrane filter of 0.45-11m pore size. in the test for Content of Total Carotenoids
Standard solution: Transfer 0.5 mL of the Standard Calculate the percentage of other related compounds in
stock solution to an 8-mL vial, evaporated to dryness the portion of the sample taken:
with an aid of a stream of nitrogen. Dissolve the residue
in 4.0 mL of a mixture of methyl tert-butyl ether and Result = (ru/r) x 100
methanol (5:95).
Sample stock solution: Transfer 20.0 mg of Extract to a tu = peak response of individual related
100-mL volumetric flask, add 75 mL of Diluent to the compounds peaks from the Sample solution
flask to suspend the sample, and sonicate for 5 min. rr = sum of the responses for all the peaks from
[CauTIoN—Electrostatic charges may cause the sample the Sample solution
to sputter and stick to the sides of the flask. Carefully
 4456 Aztec Marigold Zeaxanthin / Dietary Supplements USP 41

Acceptance criteria Acceptance criteria

Lutein: NMT 8.0% on the dried basis (3R,3’R)-Zeaxanthin: NLT 99.0%
Other related compounds: NMT 2.0% (3R,3’S meso)-Zeaxanthin: NMT 1.0%
© STEREOISOMERIC COMPOSITION
[NoT&—Use low-actinic glassware.] CONTAMINANTS
Mobile phase: Gradient elution. See Table 2. e ELEMENTAL IMPURITIES—PROCEDURES (233)
Acceptance criteria
Arsenic: NMT 0.5 ug/g
Table 2
Cadmium: NMT 1.0 ug/g
Time n-Hexane 2-Propanol Lead: NMT 5.0 g/g
(min) (%) (%) Mercury: NMT 0.1 ug/g
0.0 95 5
50 95 Si
SPECIFIC TESTS
e Loss ON DRYING (731)
55 50 50 Analysis: Dry a sample under vacuum at 105° for 3 h.
63 50 50 Acceptance criteria: NMT 25.0%
65 95 5
7S. 95 5
ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Preserve in tight, light-resistant
Standard stock solution: 0.1 mg/mL of USP Aztec Mar- containers.
igold Zeaxanthin Extract RS in methylene chloride. Son- e USP REFERENCE STANDARDS (11)
icate if necessary to dissolve the sample. USP Aztec Marigold Zeaxanthin Extract RS
Standard solution: Transfer 1.0 mL of the Standard
stock solution into a 15-mL test tube, and evaporate to
dryness with the aid of a stream of nitrogen. Dissolve
the residue in a 10.0-mL mixture of 2-propanol and
hexane (5:95). Pass through a membrane filter of 0.45- Bacopa
_
lum pore size.
Sample stock solution: Weigh 10 mg of Extract into a DEFINITION
100-mL volumetric flask, add 85 mL of methylene chlo- Bacopa consists of the dried stems and leaves of Bacopa
ride, sonicate, and swirl to dissolve. Dilute with methyl- monnieri (L.) Pennell (Fam. Scrophulariaceae). It contains
ene chloride to volume. NLT 2.5% of triterpene glycosides, calculated on the dried
Sample solution: Transfer 1.0 mL of the Sample stock basis as the sum of bacopaside |, bacoside Az, bacopaside
solution into a 15-mL test tube, and evaporate to dry- II, the jujubogenin isomer of bacopasaponin C, and
ness with the aid of a nitrogen stream. Dissolve the bacopasaponin C.
residue in a 10.0-mL mixture of 2-propanol and hexane
(5:95). Pass the solution through a membrane filter of IDENTIFICATION
0.45-um pore size. e A. Bacopa meets the requirements for Specific Tests, Bo-
Chromatographic system tanical Characteristics.
(See Chromatography (621), System Suitability.) ° e ee CHROMATOGRAPHIC IDENTIFICATION TEST
Mode: HPLC 201
Detector: 450 nm Standard solution: Transfer about 10 mg of USP Pow-
Column: 4.6-mm x 25-cm; 5-14m packing L51 dered Bacopa Extract RS to a 10-mL volumetric flask,
Flow rate: 0.8 mL/min and add about 8 mL of methanol. Sonicate and heat
Injection volume: 20 uL gently for 15-20 min, dilute with methanol to volume,
System suitability mix, centrifuge, and use the supernatant.
Sample: Standard solution Sample solution: Use the Sample solution, prepared as
[NoTe—The approximate relative retention times for directed in the test for Content of Triterpene Glycosides.
(3R,3’S TETRA (3R,3’R)-zeaxanthin, and (3R, Adsorbent: Chromatographic silica gel mixture with an
3’R,6’R)-lutein are 0.92, 1.00, and 1.12, respectively.] average particle size of 10-15 um (TLC plates)
Suitability requirements Application volume: 15 wL, as 5-10 mm bands
Resolution: The resolution between each pair of Developing solvent system: Ethyl acetate, methanol,
DS Monographs

peaks due to (3R,3’S meso)-zeaxanthin, (3R,3’R)-zea- and water (7:2:1)

xanthin, and (3R,3’R,6’R)-lutein is NLT 1.0.
Chromatogram similarity: The chromatogram from
the Standard solution is similar to the reference chro-
malgor provided with the USP Aztec Marigold Ze-
axanthin Extract RS being used.
Analysis
Samples: Standard solution and Sample solution
Identify the peaks of the relevant analytes in the chro-
matogram of the Sample solution by comparing with
those in the chromatogram of the Standard solution
obtained from the System suitability.
Calculate the percentages of (3R,3’S meso)-zeaxanthin
and (3R,3’R)-zeaxanthin in the portion of the sample
taken:
Result = (ru/rr) x 100
ru = peak response of the corresponding analyte
tr = sum of the responses for two peaks
e C. HPLC IDENTIFICATION TEST: The Sample solution from
Spray reagent: 1% Vanillin in alcohol and 10% sulfuric
acid in alcohol (1:1)
Analysis
Samples: Standard solution and Sample solution
Apply the samples as bands (see Chromatography (621)).
Use a saturated chamber. Develop the chromatograms
until the solvent front has moved up about three-
fourths of the plate. Remove the plate from the cham-
ber, dry, spray with Spray reagent, heat for 5-10 min at
70°, and examine under white light.
Acceptance criteria: The Sample solution exhibits a
main dark blue zone due to a mixture of bacoside As,
bacopaside Il, the jujubogenin isomer of bacopasaponin
C, and bacopasaponin C at an Rr value of approxi-
mately 0.6 and a faint pink spot due to bacopaside | at
an R; value of approximately 0.4, both of which corre-
spond in position and color to zones in the chromato-
gram of the Standard solution. Other zones are ob-
served for the Sample solution and Standard solution.
the test for Content of Triterpene Glycosides shows a main
peak at the retention time corresponding to that of
USP 41
bacoside A3 in the chromatogram of Standard solution A.
Identify other triterpene glycoside peaks in the Sample
solution by comparison with the chromatogram of Stan-
dard solution B and the reference chromatogram pro-
vided with the lot of USP Powdered Bacopa Extract RS.
The Sample solution shows additional peaks correspond-
ing to bacopaside |, bacopaside II, the jujubogenin iso-
mer of bacopasaponin C, and bacopasaponin C.
COMPOSITION
© CONTENT OF TRITERPENE GLYCOSIDES
Solution A: Dissolve 0.14 g of anhydrous potassium
dihydrogen phosphate in 900 mL of water, add 0.5 mL
of phosphoric acid, dilute with water to 1000 mL, mix,
filter, and degas.
Solution B: Use filtered and degassed acetonitrile.
Mobile phase: See the gradient table below.
Time Solution A Solution B
(min) (%) (%)
0 70 30
25 60 40
26 70 30
30 70 30
Standard solution A: Sonicate an accurately weighed
quantity of USP Bacoside A3 RS in methanol to obtain a
solution with a concentration of about 0.5 mg/mL.
Standard solution B: Transfer about 10 mg of USP
Powdered Bacopa Extract RS to a 10-mL volumetric
flask, and add about 8 mL of methanol. Sonicate and
heat gently for 15-20 min, dilute with methanol to vol-
ume, and mix. Before injection, pass through a mem-
brane filter of 0.45-um or finer pore size, discarding the
first 5 mL of the filtrate.
Sample solution: Transfer about 2.5 g of Bacopa, finely
powdered, to a 100-mL round-bottom flask fitted with
a reflux condenser. Add 25 mL of methanol, reflux on a
water bath for 10 min, cool to room temperature, and
decant the supernatant. Repeat until the last extract is
colorless. Combine the extracts, filter, concentrate
under vacuum, and adjust the volume to 100 mL using
methanol. Before injection, pass through a membrane
filter of 0.45-11m or finer pore size, discarding the first
5 mL of the filtrate.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 205 nm
Column: 4.6-mm x 25-cm; 5-um, endcapped, base-
deactivated packing L1
Column temperature: 27°
Flow rate: 1.5 mL/min
Injection volume: 20 uL
System suitability
Samples: Standard solution A and Standard solution B
Suitability requirements
Chromatogram similarity: The chromatogram from
Standard solution B is similar to the reference chro-
matogram provided with the lot of USP Powdered
Bacopa Extract RS being used.
Resolution: NLT 1.0 between the bacopaside II and
bacoside A3 peaks, Standard solution B
Tailing factor: NMT 1.5 for the bacoside A3 peak,
Standard solution A
Relative standard deviation: NMT 2% determined
from the bacoside A3 peak for replicate injections,
Standard solution A
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Using the chromatograms of Standard solution A and
Standard solution B and the reference chromatogram
provided with the lot of USP Powdered Bacopa Extract
Dietary Supplements / Bacopa 4457
RS being used, identify the retention times of the
peaks corresponding to different triterpene glycosides.
The approximate relative retention times of the rele-
vant triterpene glycosides are provided in the follow-
ing table.
Relative
Retention
Analyte Time
Bacopaside | 0.73
Bacoside A3 1.00
Bacopaside I! 1.04
The jujubogenin isomer of bacopasaponin C V5
Bacopasaponin C W322:
Separately calculate the percentages of bacopaside |,
bacoside A3, bacopaside Il, the jujubogenin isomer of
bacopasaponin C, and bacopasaponin C in the portion
of Bacopa taken:
Result = (ru/rs) x Cs x (V/W) x F x 100
ru = peak response for each triterpene glycoside
from the Sample solution
rs = peak response for bacoside A3 from Standard
solution A
G = concentration of USP Bacoside A; RS in
Standard solution A (mg/mL)
Vv = final volume of the Sample solution (mL)
Ww = weight of Bacopa used to prepare the Sample
solution (mg)
F = conversion factor for each analyte: 1.00 for
bacoside A3, 1.03 for bacopaside |, 0.81 for
bacopaside Il, 0.99 for the jujubogenin
isomer of bacopasaponin C, and 0.75 for
bacopasaponin C
Acceptance criteria: Add the percentages of bacopa-
side |, bacoside A3, bacopaside Il, the jujubogenin iso-
mer of bacopasaponin C, and bacopasaponin C: NLT
2.5% is found, calculated on the dried basis.
IMPURITIES
© ARTICLES OF BOTANICAL ORIGIN, Acid-Insoluble Ash (561):
NMT 6.0%
e ARTICLES OF BOTANICAL ORIGIN, Limits of Elemental Impuri-
ties (561): Meets the requirements
¢ ARTICLES OF BOTANICAL ORIGIN, Foreign Organic Matter
(561): NMT 2.0%
¢ ARTICLES OF BOTANICAL ORIGIN, Pesticide Residue Analysis
(561): Meets the requirements
sydeibouo-: sa
SPECIFIC TESTS
© BOTANICAL CHARACTERISTICS
Macroscopic: Stem is creeping, succulent, glabrous,
soft, obtuse-angular; with long internodes, rooting at
nodes; devoid of leaves toward the base; branches as-
cending. Leaves are simple, sessile or short petiolate,
opposite, succulent, 1-2 mm thick; oblong-obovate or
spatulate, margin entire or rarely dentate, apex
rounded, midrib indistinct, 0.6-2.5 cm long, 3-8 mm
wide; the upper surface is green, the lower surface is
green and dotted. Pharmacopeial article is yellowish in
color; consists of dry mixtures of broken leaves and
stems, with majority of leaves detached; mild and hay-
like odor, and very bitter taste.
Microscopic
Transverse section of stems: Epidermal layer; a wide
cortex composed of thin-wall parenchyma cells and
large intercellular spaces; xylem vessels radially ar-
ranged, uniseriate medullary rays; pith composed of
thin-wall, round or isodiametric cells with distinct in-
tercellular spaces. Resin canals and pericyclic sclereids
are absent.
 4458 Bacopa / Dietary Supplements
Transverse section of leaves: Shows a more or less
isobilateral structure; epidermis with glandular hair and
stomata; upper surface has more hairs and less sto-
mata than the lower surface; mesophyll composed of
spongy tissue, a few prisms of calcium oxalate, and
vascular bundles are present.
Loss ON DRYING (731)
Sample: 1.0g of finely powdered paehpe
Analysis: Dry the Sample at 105° for 3 h.
Acceptance criteria: NMT 12.0%
ARTICLES OF BOTANICAL ORIGIN, Total Ash (561)
Sample: 1.0g of finely powdered Bacopa
Acceptance criteria: NMT 18%
ARTICLES OF BOTANICAL ORIGIN, Alcohol-Soluble Extractives,
Method 2 (561): NLT 6.0%
MICROBIAL ENUMERATION TESTS (2021): The total aerobic
bacterial count does not exceed 10° cfu/g, the total com-
bined molds and yeasts count does not exceed 103 cfu/
g, and the bile-tolerant Gram-negative bacteria does not
exceed 103 cfu/g.
ABSENCE OF SPECIFIED MICROORGANISMS (2022): Meets the
requirements of the tests for absence of Salmonella spe-
cies and Escherichia coli
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in well-closed contain-
ers, protected from light and moisture, and store at
room temperature.
e LABELING: The label states the Latin binomial and, follow-
ing the official name, the parts of the plant contained in
the article.
e USP REFERENCE STANDARDS (11)
USP Bacoside A3 RS
USP Powdered Bacopa Extract RS
Powdered Bacopa
DEFINITION
Powdered Bacopa is Bacopa reduced to a powder or very
fine powder. It contains NLT 2.5% of triterpene glyco-
sides, calculated on the dried basis as the sum of bacopa-
side |, bacoside A3, bacopaside II, the jujubogenin isomer
of bacopasaponin C, and bacopasaponin C.
IDENTIFICATION
e A. Powdered Bacopa meets the requirements under Spe-
cific Tests, Botanical Characteristics.
e B. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST
DS Monographs
(201)
Standard solution: Transfer about 10 mg of USP Pow-
dered Bacopa Extract RS to a 10-mL volumetric flask,
and add about 8 mL of methanol. Sonicate and heat
gently for 15-20 min, dilute with methanol to volume,
mix, centrifuge, and use the supernatant.
Sample solution: Use the Sample solution, prepared as
directed in the test for Content of Triterpene Glycosides.
Adsorbent: Chromatographic silica gel mixture with an
average particle size of 10-15 jum (TLC plates)
Application volume: 15 ul, as 5-10 mm bands
Developing solvent system: Ethyl acetate, methanol,
and water (7:2:1)
Spray reagent: 1% Vanillin in alcohol and 10% sulfuric
acid in alcohol (1:1)
Analysis
Samples: Standard solution and Sample solution
Apply the samples as bands (see Chromatography
(ea )). Use a saturated chamber. Develop the chro-
matograms until the solvent front has moved up about
three-fourths of the plate. Remove the plate from the
chamber, dry, spray with Spray reagent, heat for 5-10
min at 70°, and examine under white light.
USP 41
Acceptance criteria: The Sample solution exhibits a
main dark blue zone due to mixture of bacoside A3,
bacopaside Il, the jujubogenin isomer of bacopasaponin
C, and bacopasaponin C at an R; value of approxi-
mately 0.6 and a faint pink spot due to bacopaside | at
an R; value of approximately 0.4, both of which corre-
spond in position and color to zones in the chromato-
gram of the Standard solution. Other zones are ob-
served for the Sample solution and Standard solution.
e C, HPLC IDENTIFICATION TEST: The Sample solution from
the test for Content of Triterpene Glycosides shows a main
peak at a retention time corresponding to that of baco-
side A3 in the chromatogram of Standard solution A. Iden-
tify other triterpene glycoside peaks in the Sample solu-
tion by comparison with the chromatogram of Standard
solution B and the reference chromatogram provided
with the lot of USP Powdered Bacopa Extract RS being
used. The Sample solution shows additional peaks corre-
sponding to bacopaside |, bacopaside Il, the jujubogenin
isomer of bacopasaponin C, and bacopasaponin C.
COMPOSITION
e@ CONTENT OF TRITERPENE GLYCOSIDES
Solution A: Dissolve 0.14 g of anhydrous potassium
dihydrogen phosphate in 900 mL of water, add 0.5 mL
of phosphoric acid, dilute with water to 1000 mL, mix,
filter, and degas.
Solution B: Use filtered and degassed acetonitrile.
Mobile phase: See the gradient table below.
Time Solution A Solution B
(min) (%) (%)
0 70 30
25 60 40
26 70 30
30 70 30
Standard solution A: Sonicate an act rately weighed
quantity of USP Bacoside A3 RS in methanol to obtain a
solution having a known concentration of about
0.5 mg/mL.
Standard solution B: Transfer about 10 mg of USP
Powdered Bacopa Extract RS to a 10-mL volumetric
flask, and add about 8 mL of methanol. Sonicate and
heat gently for 15-20 min, dilute with methanol to vol-
ume, and mix. Before injection, pass through a mem-
brane filter of 0.45-tum or finer pore size, discarding the
first 5 mL of the filtrate.
Sample solution: Transfer about 2.5 g of Powdered
Bacopa, accurately weighed, to a 100-mL round-bot-
tom flask fitted with a reflux condenser. Add 25 mL of
methanol, reflux on a water bath for 10 min, cool to
room temperature, and decant the supernatant. Repeat
until the last extract is colorless. Combine the extracts,
filter, concentrate under vacuum, and adjust the vol-
ume to 100 mL using methanol. Before injection, pass
through a membrane filter of 0.45-1m or finer pore
size, discarding the first 5 mL of the filtrate.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 205 nm
Column: 4.6-mm x 25-cm; 5-um, endcapped, base-
deactivated packing L1
Column temperature: 27°
Flow rate: 1.5 mL/min
Injection volume: 20 uL
System suitability
Samples: Standard solution A and Standard solution B
Suitability requirements
Chromatogram similarity: The chromatogram from
Standard solution B is similar to the reference chro-
matogram provided with the lot of USP Powdered
Bacopa Extract RS being used.
USP 41
Resolution: NLT 1.0 between the bacopaside II and
bacoside A3 peaks, Standard solution B
Tailing factor: NMT 1.5 for the bacoside A3 peak,
Standard solution A
Relative standard deviation: NMT 2% determined
from the bacoside A3 peak for replicate injections,
Standard solution A
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Using the chromatograms of Standard solution A and
Standard solution B and the reference chromatogram
provided with the lot of USP Powdered Bacopa Extract
RS being used, identify the retention times of the
peaks corresponding to different triterpene glycosides.
The approximate relative retention times of the rele-
vant triterpene glycosides are provided in the follow-
ing table.
Relative
Retention
Analyte Time
Bacopaside | 0.73
Bacoside A3 1.00
Bacopaside II 1.04
The jujubogenin isomer of bacopasaponin C 1.15
Bacopasaponin C 1.22
Separately calculate the percentages of bacopaside |,
bacoside As, bacopaside II, the jujubogenin isomer of
bacopasaponin C, and bacopasaponin Cin the portion
of Powdered Bacopa taken:
Result = (ru/rs) x Cs x (V/W) x Fx 100
ty = peak response for each triterpene glycoside
from the Sample solution
rs = peak response for bacoside A3 from Standard
solution A
Cs = concentration of USP Bacoside A3 RS in
Standard solution A (mg/mL)
Vv = final volume of the Sample solution (mL)
w = weight of Powdered Bacopa used to prepare
the Sample solution (mg)
F = conversion factor for each analyte: 1.00 for
bacoside A3, 1.03 for bacopaside |, 0.81 for
bacopaside II, 0.99 for the jujubogenin
isomer of bacopasaponin C, and 0.75 for
bacopasaponin C
Acceptance criteria: Add the percentages of bacopa-
side |, bacoside A3, bacopaside II, the jujubogenin iso-
mer of bacopasaponin C, and bacopasaponin C: NLT
2.5% is found on the dried basis.
IMPURITIES
e ARTICLES OF BOTANICAL ORIGIN, Acid-Insoluble Ash (561):
NMT 6.0%
e ARTICLES OF BOTANICAL ORIGIN, Limits of Elemental Impuri-
ties (561): Meets the requirements
e ARTICLES OF BOTANICAL ORIGIN, Pesticide Residues Analysis
(561): Meets the requirements
SPECIFIC TESTS
¢ BOTANICAL CHARACTERISTICS: Yellowish in color; mild and
hay-like odor, and very bitter taste. Under a microscope,
it shows fragments of upper and lower epidermal cells of
the leaves in surface view, having sessile glandular
trichomes with 4-8 cells and diacytic or anomocytic sto-
mata; upper epidermis has more trichomes and less sto-
mata than the lower epidermis; lower epidermis cells
with sinuous anticlinal walls and at places striated cuticle;
fragments of epidermal cells of the stem in surface view;
parenchyma cells enclosing air cavities and some contain
rosette and prismatic crystals of calcium oxalate; frag-
Dietary Supplements / Bacopa 4459
ments of longitudinally cut annular and spiral vessels;
fragments of cortical cells of the stem; and crystals of
calcium oxalate.
e Loss ON DRYING (731)
Sample: 1.0 g of Powdered Bacopa
Analysis: Dry the Sample at 105° for 3 h.
Acceptance criteria: 12.0%
ARTICLES OF BOTANICAL ORIGIN, Total Ash (561)
Sample: 1.0g of Powdered Bacopa
Acceptance criteria: NMT 18%
ARTICLES OF BOTANICAL ORIGIN, Alcohol-Soluble Extractives,
Method 2 (561): NLT 6.0%
MICROBIAL ENUMERATION TESTS (2021): The total aerobic
bacterial count does not exceed 105 cfu/g, the total com-
bined molds and yeasts count does not exceed 103 cfu/
g, and the bile-tolerant Gram-negative bacteria does not
exceed 103 cfu/g.
° ABSENCE OF SPECIFIED MICROORGANISMS (2022): Meets the
requirements of the tests for absence of Salmonella spe-
cies and Escherichia coli
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in well-closed contain-
ers, protected from light and moisture, and store at
room temperature.
e LABELING: The label states the Latin binomial and, follow-
ing the official name, the parts of the plant contained in
the article.
e USP REFERENCE STANDARDS (11)
USP Bacoside A3 RS
USP Powdered Bacopa Extract RS
Powdered Bacopa Extract
DEFINITION
Powdered Bacopa Extract is prepared from Bacopa by ex-
traction with water, alcohol, methanol, or a mixture of
these solvents. The ratio of plant material to extract is
between 20:1 and 10:1. It contains NLT 90.0% and NMT
110.0% of the labeled amount of triterpene glycosides,
calculated on the dried basis as the sum of bacopaside |,
bacoside A3, bacopaside Il, the jujubogenin isomer of
bacopasaponin C, and bacopasaponin C. It may contain
suitable added substances as carriers.
IDENTIFICATION
e A. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST
Standard solution: Transfer about 10 mg of USP Pow-
sydeibouo-: Sa
dered Bacopa Extract RS to a 10-mL volumetric flask,
and add about 8 mL of methanol. Sonicate and heat
gently for 15-20 min, dilute with methanol to volume,
mix, centrifuge, and use the supernatant.
Sample solution: Sonicate for about 10 min an amount
of Powdered Bacopa Extract equivalent to about 40 mg
of triterpene glycosides in 10 mL of methanol, centri-
fuge, and use the supernatant.
Adsorbent: Chromatographic silica gel mixture with an
average particle size of 10-15 um (ILC plates)
Application volume: 15 ul, as 5-10 mm bands
Developing solvent system: Ethyl acetate, methanol,
and water (7:2:1)
Spray reagent: 1% vanillin in alcohol and 10% sulfuric
acid in alcohol (1:1)
Analysis
Samples: Standard solution and Sample solution
Apply the samples as bands to a suitable thin-layer
chromatographic plate (see Chromatography (621)).
Use a saturated chamber. Develop the chromato-
grams until the solvent front has moved up about
three-fourths of the plate. Remove the plate from the
chamber, dry, spray with Spray reagent, heat for 5-10
min at about 70°, and examine under visible light.
 4460 Bacopa / Dietary Supplements
Acceptance criteria: The Sample solution exhibits a
main dark blue zone due to a mixture of bacoside As,
bacopaside II, the jujubogenin isomer of bacopasaponin
C, and bacopasaponin C at an R; value of approxi-
mately 0.6 and a faint pink spot due to bacopaside | at
an R; value of approximately 0.4, both of which corre-
spond in position and color to zones in the chromato-
gram of the Standard solution. Other zones are ob-
served for the Sample solution and Standard solution.
e B. HPLC IDENTIFICATION TEST: The Sample solution from
the test for Content of Triterpene Glycosides shows a main
peak at a retention time corresponding to that of baco-
side A3 in the chromatogram of Standard solution A. |den-
tify other triterpene glycoside peaks in the Sample solu-
tion by comparison with the chromatogram of Standard
solution B and the reference chromatogram provided
with the lot of USP Powdered Bacopa Extract RS being
used. The Sample solution shows additional peaks corre-
sponding to bacopaside |, bacopaside Il, the jujubogenin
isomer of bacopasaponin C, and bacopasaponin C.
COMPOSITION
e CONTENT OF TRITERPENE GLYCOSIDES
Solution A: Dissolve 0.14 g of anhydrous potassium
dihydrogen phosphate in 900 mL of water, add 0.5 mL
of phosphoric acid, dilute with water to 1000 mL, mix,
filter, and degas.
Solution B: Use filtered and degassed acetonitrile.
Mobile phase: See the gradient table below.
Time Solution A Solution B
(min) (%) (%)
0 70 30
25 60 40
26 70 30
30 70 30
Standard solution A: Sonicate a weighed quantity of
USP Bacoside A3 RS in methanol to opti a solution
with a concentration of about 0.5 mg/mL.
Standard solution B: Transfer about 10 mg of USP
Powdered Bacopa Extract RS to a 10-mL volumetric
flask, and add about 8 mL of methanol. Sonicate and
heat gently for 15-20 min, dilute with methanol to vol-
ume, and mix. Before injection, pass through a mem-
brane filter of 0.45-uum or finer pore size, discarding the
first 5 mL of the filtrate.
Sample solution: Transfer an amount of Powdered
Bacopa Extract, equivalent to about 25 mg triterpene
glycosides, to a 25-mL volumetric flask, and add 15 mL
of methanol. Sonicate and heat gently for 15-20 min,
DS Monographs
dilute with methanol to volume, and mix. Before injec-
tion, pass through a membrane filter of 0.45-4m or
finer pore size, discarding the first 5 mL of the filtrate.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 205 nm
Organic Impurities
Column: 4.6-mm x 25-cm; 5-um, endcapped, base-
deactivated packing L1
Column temperature: 27+1°
Flow rate: 1.5 mL/min
Injection size: 20 uL
System suitability
Samples: Standard solution A and Standard solution B
Suitability requirements
Chromatogram similarity: The chromatogram from
Standard solution B is similar to the reference chro-
matogram provided with the lot of USP Powdered
Bacopa Extract RS being used.
Resolution: NLT 1.0 between the bacopaside Il and
bacoside A3 peaks, Standard solution B
Tailing factor: NMT 1.5 for the bacoside A3 peak,
Standard solution A
USP 41
Relative standard deviation: NMT 2% determined
from the bacoside A3 peak for replicate injections,
Standard solution A
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Using the chromatograms of Standard solution A and
Standard solution B and the reference chromatogram
provided with the lot of USP Powdered Bacopa Ex-
tract RS being used, identify the retention times of
the peaks corresponding to different triterpene glyco-
sides. The approximate relative retention times of the
different triterpene glycosides are provided in the fol-
lowing table.
Relative
Retention
Analyte Time
Bacopaside | 0.73
Bacoside A3 1.00
Bacopaside II 1.04
The jujubogenin isomer of bacopasaponin C A315)
Bacopasaponin C 1.22
Separately calculate the percentages of bacopaside |,
bacoside A3, bacopaside II, jujubogenin isomer of
bacopasaponin C, and bacopasaponinCin the por-
tion of Powdered Bacopa Extract taken:
Result = (ru/rs) x (Cs/Cu) x F x 100
tu = peak response for each triterpene glycoside
from the Sample solution
Ts = peak response for bacoside A3 in Standard
solution A
Cs = concentration of USP Bacoside A3 RS in
Standard solution A (mg/mL)
Cu = concentration of Powdered Bacopa Extract in
the Sample solution (mg/mL)
F = conversion factor for each analyte: 1.00 for
bacoside A3, 1.03 for bacopaside |, 0.81 for
bacopaside II, 0.99 for the jujubogenin
isomer of bacopasaponin C, and 0.75 for
bacopasaponin C
Acceptance criteria: Add the percentages of bacopa-
side |, bacoside Az, bacopaside II, the jujubogenin Iso-
mer of bacopasaponin C, and bacopasaponin C: NLT
90.0%-NMT 110.0% of the labeled amount of
triterpene glycosides is found on the dried basis.
IMPURITIES
Inorganic Impurities
Delete the following:
®o HEAVY METALS, Method II! (231): NMT 20 ppme citiciai-
Jan-2018)
e PROCEDURE: ARTICLES OF BOTANICAL ORIGIN, General
Method for Pesticide Residues Analysis (561): Meets the
requirements
SPECIFIC TESTS
e Loss ON DRYING (731): Dry 1.0 g of Powdered Bacopa
Extract at 105° for 3 h: it loses NMT 5% of its Seat
e MICROBIAL ENUMERATION TESTS (2021): The total aerobic
microbial count does not exceed 104 cfu/g. The total
nee molds and yeasts count does not exceed 103
cfu/g.
e MICROBIOLOGICAL PROCEDURES FOR ABSENCE OF SPECIFIED MI-
CROORGANISMS (2022): It meets the requirements of the
Sa for absence of Salmonella species and Escherichia
CoH.
USP 41
© OTHER REQUIREMENTS: It meets the requirements of the
test for Residual Solvents under Botanical Extracts (565).
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in well-closed contain-
ers, protected from light and moisture, and store at con-
trolled room temperature.
e LABELING: The label states the Latin binomial and, follow-
ing the official name, the part of the plant from which
the article was derived. It meets other labeling require-
ments under Botanical Extracts (565).
e USP REFERENCE STANDARDS (11)
USP Bacoside A3 RS
USP Powdered Bacopa Extract RS
Banaba Leaf
DEFINITION
Banaba Leaf consists of the dried leaves of Lagerstroemia
Speciosa (L.) Pers. (Fam. Lythraceae). It contains NLT 0.2%
of corosolic acid (C3oH4gO4), calculated on the dried basis.
IDENTIFICATION
e A. Meets the requirements for Specific Tests, Botanic
Characteristics
e B. THIN-LAYER CHROMATOGRAPHY
Standard solution A: 0.2 mg/mL of USP Corosolic Acid
RS in methanol
Standard solution B: 10 mg/mL of USP Lagerstroemia
speciosa Leaf Dry Extract RS in methanol. Sonicate for
10 min, centrifuge, and use the supernatant.
Sample solution: About 0.2 g of Banaba Leaf, finely
powdered, in 10 mL of methanol. Sonicate for 15 min,
centrifuge, and use supernatant.
Chromatographic system
(See Chromatography (621), Thin-Layer Chromato-
graphy.)
Mode: HPTLC
Adsorbent: Chromatographic silica gel mixture with
an average particle size of 5 um (HPTLC plates)
Application volume: 6 tL each of Standard solution A
and Standard solution B and 8 wL of the Sample solution
as 8-mm bands
Relative humidity: Condition the plate to a relative
humidity of about 33% usinga suitable device.
Developing solvent system: A mixture of toluene,
ethyl acetate, and acetic acid (55: 45: 0.5)
Derivatization reagent: 85 mL of ice-cooled methanol
mixed with 10 mL of glacial acetic acid, 5 mL of sulfu-
tic acid, and 0.5 mL of p-anisaldehyde
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Apply the samples as bands to a suitable HPTLC plate,
and dry in air. Develop the chromatograms in an un-
saturated chamber, remove the plate from the cham-
ber, and dry. Treat with Derivatization reagent, heat for
3 min at 100°, and examine under visible light.
System suitability: Under visible light, the chromato-
ge of Standard solution B exhibits the most intense
and, a violet or blue band, with similar Re and color as
the corosolic acid band in the chromatogram of Stan-
dard solution A; a blue band close to the start (about Rr
0.1), consistent with asiatic acid; two minor blue bands
in between the corosolic and asiatic bands; a minor
blue band due to virgatic acid, above the band due to
corosolic acid; and just below the asiatic band, a minor
brown band. Standard solution B also exhibits two mi-
nor violet bands, separated, at about three-fourths of
the chromatogram; the band with the lower R; corre-
sponds to oleanolic acid.
Dietary Supplements / Banaba 4461
Acceptance criteria: Under visible light, the chromato-
gram of the Sample solution exhibits the most intense
band as a violet band corresponding in color and R- to
the band due to corosolic acid in the chromatogram of
Standard solution A, as well as the following bands cor-
responding to similar bands of Standard solution B: a
minor blue band close to the start (about R; 0.1), a
minor brownish band above the corosolic acid, and a
minor violet band at about three-fourths of the
chromatogram.
e C. HPLC
Analysis: Proceed as directed in Content of Corosolic
Acid.
Acceptance criteria: The chromatogram of the Sample
solution exhibits a group of three peaks. The one in the
center is the most intense of the grou and occurs at a
retention time corresponding to that of corosolic acid in
the chromatogram of Standard solution A. The peak that
elutes before corosolic acid has about one-half to one-
third of the intensity of that of corosolic acid, and the
peak that elutes after corosolic acid has the lesser inten-
sity of the three and is consistent with virgatic acid. A
minor peak due to oleanolic acid elutes later in the
chromatogram.
COMPOSITION
e CONTENT OF CoROSOLIC ACID
Solution A: Dilute 0.1% phosphoric acid in water.
Solution B: Acetonitrile
Mobile phase: A mixture of Solution A and Solution B
(4:6)
Standard solution A: 0.1 mg/mL of USP Corosolic Acid
RS in methanol
Standard solution B: 5.0 mg/mL of USP Lagerstroemia
speciosa Leaf Dry Extract RS in methanol. Sonicate if
necessary. Before injection, pass through a membrane
filter of 0.45-uum or finer pore size. Discard the first few
mL of the filtrate.
Sample solution: Transfer about 5.0 g of Banaba Leaf,
finely powdered and accurately weighed, to a round-
bottom flask. Add 75 mL of methanol, reflux for 15
min, set aside to settle, and decant the supernatant.
Repeat the extraction three more times, then combine
the extracts. Filter, and concentrate under reduced
pressure. Transfer to a 100-mL volumetric flask, adjust
with methanol to volume, and mix. Before injection,
pass through a membrane filter of 0.45-um or finer
pore size. Discard the first few mL of the filtrate.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 205 nm
sydeibouo-=w sa
Column: 4.6-mm x 25-cm; 5-um packing L1
Column temperature: 25°
Flow rate: 1.6 mL/min
Injection volume: 20 uL
System suitability
Samples: Standard solution A and Standard solution B
[NotE—The approximate relative retention times of the
individual peaks for corosolic acid, virgatic acid, and
oleanolic acid are 1.0, 1.1, and 3.2, respectively.]
Suitability requirements
Chromatogram similarity: The chromatogram from
Standard solution B is similar to the reference chro-
matogram provided with the lot of USP Lagerstroemia
speciosa Leaf Dry Extract RS being used.
Resolution: NLT 1.5 between the corosolic acid peak
and the preceding peak, Standard solution B
Tailing factor: NMT 2.0 for the corosolic acid peak,
Standard solution A
Relative standard deviation: NMT 2.0% determined
from the corosolic acid peak in repeated injections,
Standard solution A
 4462 Banaba / Dietary Supplements
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Identify the relative retention times of the peaks for
corosolic acid, virgatic acid, and oleanolic acid in the
Sample solution.
Calculate the percentage of corosolic acid in the por-
tion of Banaba Leaf taken:
Result = (ru/rs) x Cs x (V/W) x 100
tu = peak area of corosolic acid from Sample
solution
ls = peak area of corosolic acid from Standard
Solution A
Cs = concentration of corosolic acid in Standard
solution A (mg/mL)
Vv = volume of Sample solution (mL)
w = weight of Banaba Leaf taken to prepare the
Sample solution (mg)
Acceptance criteria: NLT 0.2% of corosolic acid on the
dried basis
CONTAMINANTS
e ELEMENTAL IMPURITIES—PROCEDURES (233)
Acceptance criteria
Arsenic: NMT 2.0 ug/g
Cadmium: NMT 0.5 yg/g
Lead: NMT 5 ug/g
Mercury: NMT 0.2 ug/g
e ARTICLES OF BOTANICAL ORIGIN, General Method for Pesti-
cide Residues Analysis (561): Meets the requirements
e MICROBIAL ENUMERATION TESTS (2021): The total aerobic
bacterial count does not exceed 105 cfu/g, the total com-
bined molds and yeasts count does not exceed 103 cfu/
g, and the bile-tolerant Gram-negative bacteria count
does not exceed 103 cfu/g.
e ABSENCE OF SPECIFIED MICROORGANISMS (2022): Meets the
requirements of the tests for absence of Salmonella spe-
cies and Escherichia coli
SPECIFIC TESTS
e BOTANIC CHARACTERISTICS
Macroscopic: Banaba leaves vary in shape, including
lanceolate, oblong-lanceolate, oblong, and elliptic
ovate. They are up to 34cm long and 11 cm wide; ol-
ivegreen to yellowish brown; entirely or slightly wavy
at the margins; base acute; apex acute to acuminate;
leathery in texture; and petiolate with petioles up to
1cm long.
Microscopic
Transverse section of the midrib: A layer of upper
DS Monographs
epidermis composed of rectangular to round cells cov-
ered with thin cuticle; a few layers of collenchyma
cells; numerous layers of parenchyma cells, some con-
taining cluster crystals of calcium oxalate, with large
intercellular spaces; groups of lignified fiber bundles;
and secretory canals scattered in the parenchyma
zone. Bicollateral vascular bundle encircled by a con-
tinuous sheath of fibers accompanied by sclerenchyma
and cells containing cluster crystals of calcium oxalate;
secretory canals between the vascular bundles; numer-
ous layers of parenchyma cells, some containing clus-
ter crystals of calcium oxalate, with large intercellular
spaces; a few layers of collenchyma; a layer of lower
epidermal cells.
Transverse section of the lamina: A layer of upper
epidermis composed of rectangular cells about twice
as large as those of the lower epidermis. Some cells
are secretory cells, which tend to protrude into the
mesophyll and sometimes appear to be below the a
per epidermis. Two layers of rectangular palisade cells;
4-6 layers of parenchyma cells, some containing
prisms of calcium oxalate and others containing clus-
ters crystals of calcium oxalate, with large intercellular
USP 41
spaces; groups of vascular bundles scattered in the pa-
renchyma zone; lower epidermis showing stomata.
ARTICLES OF BOTANICAL ORIGIN, Foreign Organic Matter
(561): NMT 2.0%
HO38 a Dryinc (731)
nalysis: Dry 2.0 g of Banaba Leaf, finely powdered, at
105° for 2 . i oP
Acceptance criteria: NMT 10%
ARTICLES OF BOTANICAL ORIGIN, Total Ash (561)
Analysis: 2.0 9 of Banaba Leaf, finely powdered
Acceptance criteria: NMT 7.0%
ARTICLES OF BOTANICAL ORIGIN, Alcohol-Soluble Extractives,
Method 7 (561): NMT 10.0%
ARTICLES OF BOTANICAL ORIGIN, Acid-/nsoluble Ash (561)
Analysis: 4.0 g of Banaba Leaf, finely powdered
Acceptance criteria: NMT 2%
e ARTICLES OF BOTANICAL ORIGIN, Water-Soluble Extractives,
Method 1 (561): NLT 18.0%
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in well-closed contain-
ers, protected from light and moisture, and store at
room temperature.
e LABELING: The label states the Latin binomial and, follow-
ing the official name, the part(s) of the plant contained
in the article.
e USP REFERENCE STANDARDS (11)
USP Corosolic Acid RS
USP Lagerstroemia speciosa Leaf Dry Extract RS
Banaba Leaf Powder
DEFINITION
Banaba Leaf Powder consists of the dried leaves of Lager-
stroemia speciosa (L.) Pers. (Fam. Lythraceae) reduced to
owder or very fine powder. It contains NLT 0.2% of the
labeled amount of corosolic acid (C3oH4gO4), calculated on
the dried basis.
IDENTIFICATION
e A. Meets the requirements for Specific Tests, Botanic
Characteristics
e B. THIN-LAYER CHROMATOGRAPHY
Standard solution A: 0.2 mg/mL of USP Corosolic Acid
RS in methanol
Standard solution B: 10 mg/mL of USP Lagerstroemia
speciosa Leaf Dry Extract RS in methanol. Sonicate for
10 min, centrifuge, and use the supernatant.
Sample solution: About 0.2 g of Banaba Leaf Powder
in 10 mL of methanol. Sonicate for 15 min, centrifuge,
and use the supernatant.
Chromatographic system
(See Chromatography (621), Thin-Layer Chromato-
graphy.)
Mode: HPTLC
Adsorbent: Chromatographic silica gel mixture with
an average particle size of 5 um (HPTLC plates)
Application volume: 6 LL each of Standard solution A
and Standard solution B and 8 uL of the Sample solution
as 8-mm bands
Relative humidity: Condition the plate to a relative
humidity of about 33% using a suitable device.
Column temperature: 25°
Developing solvent system: A mixture of toluene,
ethyl acetate, and acetic acid (55: 45: 0.5)
Derivatization reagent: 85 mL of ice-cooled methanol
mixed with 10 mL of glacial acetic acid, 5 mL of sulfu-
ric acid, and 0.5 mL of p-anisaldehyde
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
USP 41
Apply the samples as bands to a suitable HPTLC plate,
and dry in air. Develop the chromatograms in an un-
saturated chamber, remove the plate from the cham-
ber, and dry. Treat with Derivatization reagent, heat for
3 min at 100°, and examine under visible light.
System suitability: Under visible light, the chromato-
gram of Standard solution B exhibits the most intense
and, a violet or blue band, with similar Re and color as
the corosolic acid band in the chromatogram of Stan-
dard solution A; a blue band close to the start (about Rr
0.1), consistent with asiatic acid; two minor blue bands
in between the corosolic and asiatic bands; a minor
blue band due to virgatic acid, above the band due to
corosolic acid; and just below the asiatic band, a minor
brown band. Standard solution B also exhibits two mi-
nor violet bands, separated, at about three-fourths of
the chromatogram; the band with the lower R, corre-
sponds to oleanolic acid.
Acceptance criteria: Under visible light, the chromato-
gram of the Sample solution exhibits the most intense
band as a violet band corresponding in color and R; to
the band due to corosolic acid in the chromatogram of
Standard solution A, as well as the following bands cor-
responding to similar bands of Standard solution B: a
minor blue band close to the start (about Ry 0.1), a
minor brownish band above the corosolic acid, and a
minor violet band at about three-fourths of the
chromatogram.
e C. HPLC
Analysis: Proceed as directed in Content of Corosolic
Acid.
Acceptance criteria: The chromatogram of the Sample
solution exhibits a group of three peaks. The one in the
center is the most intense of the group and occurs at a
retention time eerespencing to that of corosolic acid in
the chromatogram of Standard solution A. The peak that
elutes before corosolic acid has about one-half to one-
third of the intensity of that of corosolic acid, and the
peak that elutes after corosolic acid has the lesser inten-
sity of the three and is consistent with virgatic acid. A
minor peak due to oleanolic acid elutes later in the
chromatogram.
COMPOSITION
© CONTENT OF COROSOLIC ACID
Solution A: Dilute 0.1% phosphoric acid in water.
Solution B: Acetonitrile
ee phase: A mixture of Solution A and Solution B
(4:
Standard solution A: 0.1 mg/mL of USP Corosolic Acid
RS in methanol
Standard solution B: 5.0 mg/mL of USP Lagerstroemia
speciosa Leaf Dry Extract RS in methanol. Sonicate if
necessary. Before injection, pass through a membrane
filter of 0.45-m or finer pore size. Discard the first few
mL of the filtrate.
Sample solution: Transfer about 5.0 g of Banaba Leaf
Powder, accurately weighed, to a round-bottom flask.
Add 75 mL of methanol, reflux for 15 min, set aside to
settle, and decant the supernatant. Repeat the extrac-
tion three more times, then combine the extracts. Filter,
and concentrate under reduced pressure. Transfer to a
100-mL volumetric flask, adjust with methanol to vol-
ume, and mix. Before injection, pass through a mem-
brane filter of 0.45-um or finer pore size. Discard the
first few mL of the filtrate.
Chromatographic system
(See Chromatography (621), System Suitability.)
Dietary Supplements / Banaba 4463
Mode: LC
Detector: UV 205 nm
Column: 4.6-mm x 25-cm; 5-m packing L1
Flow rate: 1.6 mL/min
Injection volume: 20 ul
System suitability
Samples: Standard solution A and Standard solution B
[NoTte—The approximate relative retention times of the
individual peaks for corosolic acid, virgatic acid, and
oleanolic acid are 1.0, 1.1, and 3.2 min, respectively.]
Suitability requirements
Chromatogram similarity: The chromatogram from
Standard solution B is similar to the reference chro-
matogram provided with the lot of USP Lager-
stroemia speciosa Leaf Dry Extract RS being used.
Resolution: NLT 1.5 between the corosolic acid
peak and the preceding peak, Standard solution B
Tailing factor: NMT 2.0 for the corosolic acid peak,
Standard solution A
Relative standard deviation: NMT 2.0% deter-
mined from the corosolic acid peak in repeated in-
jections, Standard solution A
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Identify the relative retention times of the peaks for
corosolic acid, virgatic acid, and oleanolic acid in the
Sample solution.
Calculate the percentage of the labeled amount of
corosolic acid in the portion of Banaba Leaf Powder
taken:
Result = (ru/rs) x Cs x (V/W) x 100
ru = peak area of corosolic acid from the Sample
solution
fs = peak area of corosolic acid from Standard
solution A
Gs = concentration of corosolic acid in Standard
solution A (mg/mL)
Vv = volume of the Sample solution (mL)
w = weight of Banaba Leaf Powder taken to
prepare the Sample solution (mg)
Acceptance criteria: NLT 0.2% of the labeled amount
of corosolic acid on the dried basis
CONTAMINANTS
© ELEMENTAL IMPURITIES—PROCEDURES (233)
Acceptance criteria
Arsenic: NMT 2.0 ug/g
Cadmium: NMT 0.5 ug/g
Lead: NMT 5 ug/g
sydesbouo=: sa
Mercury: NMT 0.2 ug/g
© ARTICLES OF BOTANICAL ORIGIN, General Method for Pesti-
cide Residues Analysis (561): Meets the requirements
e MICROBIAL ENUMERATION TESTS (2021): The total aerobic
bacterial count does not exceed 104 cfu/g, and the total
arenes molds and yeasts count does not exceed 102
cfu/g.
e MICROBIAL PROCEDURES FOR ABSENCE OF SPECIFIED MICROOR-
GANISMS (2022): Meets the requirements of the tests for
absence of Salmonella species and Escherichia coli
SPECIFIC TESTS
e BOTANIC CHARACTERISTICS
Macroscopic: Grayish-green powder
ahi lr Fragments of upper epidermis with poly-
gonal cells, some containing calcium oxalate crystals,
and no stomata; fragments of lower epidermis cells
with irregular shapes and slightly wavy walls, anomo-
cytic stomata; fragments of upper epidermal cells with
underlying palisade cells; fragments of parenchyma
cells, some containing prisms of calcium oxalate and
others containing cluster crystals of calcium oxalate; oil
cells; fragments of lignified fibers; fragments of spiral
 4464 Banaba / Dietary Supplements
vessels; fragments of pitted vessels associated with
fibers
ARTICLES OF BOTANICAL ORIGIN, General Method for Pesti-
cide Residues Analysis (561): Meets the requirements
Loss ON DRYING (731)
Sample: 2.0 g of Banaba Leaf, finely powdered
Analysis: Dry the Sample at 105° for 2 h.
Acceptance criteria: NMT 10%
ARTICLES OF BOTANICAL ORIGIN, Total Ash (561)
Analysis: 2.0 g of Banaba Leaf, finely powdered
Acceptance criteria: NMT 7.0%
ARTICLES OF BOTANICAL ORIGIN, Acid-insoluble Ash (561)
Analysis: 4.0 g of Banaba Leaf, finely powdered
Acceptance criteria: NMT 2%
ARTICLES OF BOTANICAL ORIGIN, A/cohol-Soluble Extractives,
Method 1 (561): NLT 10.0%
ARTICLES OF BOTANICAL ORIGIN, Water-Soluble Extractives,
Method 1 (561): NLT 18.0%
ADDITIONAL REQUIREMENTS
PACKAGING AND STORAGE: Preserve in well-closed contain-
ers, protected from light and moisture, and store at
room temperature.
LABELING: The label states the Latin binomial and, follow-
ing the official name, the part(s) of the plant contained
in the article.
USP REFERENCE STANDARDS (11)
USP Corosolic Acid RS
USP Lagerstroemia speciosa Leaf Dry Extract RS
Banaba Leaf Dry Extract
DEFINITION
Banaba Leaf Dry Extract consists of the dried leaves of Lage-
troemia speciosa (L.) Pers. (Fam. Lythraceae) by extraction
with hydroalcoholic mixtures. It contains NLT 90.0% and
NMT 110.0% of the labeled amount of corosolic acid
(C3oH4g0.), calculated on the dried basis.
IDENTIFICATION
A. THIN-LAYER CHROMATOGRAPHY
Standard solution A: 0.2 mg/mL of USP Corosolic Acid
RS in methanol
Standard solution B: 10 mg/mL of USP Lagerstroemia
speciosa Leaf Dry Extract RS in methanol. Sonicate for
10 min, centrifuge, and use the supernatant.
Sample solution: Banaba Leaf Dry Extract in methanol
at a concentration equivalent to 0.2 mg/mL of corosolic
DS Monographs
acid according to the label claim. Sonicate if necessary.
Chromatographic system
(See Chromatography (621), Thin-Layer Chromato-
graphy.)
Mode: HPTLC
Adsorbent: Chromatographic silica gel mixture with
an average particle size of 5 um CAPTLGplates)
Application volume: 6 uL as 8-mm bands
Relative humidity: Condition the plate to a relative
humidity of about 33% using a suitable device.
Column temperature: 25°
Developing solvent system: A mixture of toluene,
ethyl acetate, and acetic acid (55: 45: 0.5)
Developing distance: 6 cm
Derivatization reagent: 85 mL of ice-cooled methanol
mixed with 10 mL of glacial acetic acid, 5 mL of sulfu-
ric acid, and 0.5 mL of p-anisaldehyde
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Apply the samples as bands to a suitable HPTLC plate,
and dry in air. Develop the chromatograms in an un-
saturated chamber, remove the plate from the cham-
USP 41
ber, and dry. Treat with Derivatization reagent, heat for
3 min at 100°, and examine under visible light.
System suitability: Under visible light, the chromato-
gram of Standard solution B exhibits the most intense
and, a violet or blue band, with similar Re and color as
the corosolic acid band in the chromatogram of Stan-
dard solution A; a blue band close to the start (about R;
0.1), consistent with asiatic acid; two minor blue bands
in between the corosolic and asiatic bands; a minor
blue band due to virgatic acid, above the band due to
corosolic acid; and just below the asiatic band, a minor
brown band. Standard solution B also exhibits two mi-
nor violet bands, separated, at about three-fourths of
the chromatogram; the band with the lower R, corre-
sponds to oleanolic acid.
Acceptance criteria: Under visible light, the chromato-
ram of the Sample solution exhibits the most intense
and as a violet band corresponding in color and Rr to
the band due to corosolic acid in the chromatogram of
Standard solution A, as well as the following bands cor-
responding to similar bands of Standard solution B: a
minor blue band close to the start (about R; 0.1), two
minor purple bands below the corosolic acid, two mi-
nor blue bands above the corosolic acid, and two mi-
nor violet bands, which are separated, at about three-
fourths of the chromatogram.
e B. HPLC
Analysis: Proceed as directed in Content of Corosolic
Acid.
Acceptance criteria: The chromatogram of the Sample
solution exhibits a group of three peaks. The one in the
center is the most intense of the group and occurs at a
retention time corresponding to that of corosolic acid in
the chromatogram ofstandard solution A. The peak that
elutes before corosolic acid has about one-half to one-
third of the intensity of that of corosolic acid, and the
peak that elutes after corosolic acid has the lesser inten-
sity of the three and is consistent with virgatic acid. A
minor peak due to oleanolic acid elutes later in the
chromatogram.
COMPOSITION
© CONTENT OF COROSOLIC ACID
Solution A: Dilute 0.1% phosphoric acid in water.
Solution B: Acetonitrile
Mobile phase: A mixture of Solution A and Solution B
(4:6)
Standard solution A: 0.1 mg/mL of USP Corosolic Acid
RS in methanol
Standard solution B: 5.0 mg/mL of USP Lagerstroemia
speciosa Leaf Dry Extract RS in methanol. Sonicate if
necessary. Before injection, pass through a membrane
filter of 0.45-~m or finer pore size. Discard the first few
mL of the filtrate.
Sample solution: Banaba Leaf Dry Extract in methanol
at a concentration equivalent to 0.1 mg/mL of corosolic
acid according to the label claim. Sonicate if necessary.
Before injection, pass through a membrane filter of
0.45-um or finer pore size. Discard the first few mL of
the filtrate.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 205 nm
Column: 4.6-mm x 25-cm; 5-um packing L1
Flow rate: 1.6 mL/min
Injection volume: 20 uL
System suitability
Samples: Standard solution A and Standard solution B
[Note—The approximate relative retention times of the
individual peaks for corosolic acid, virgatic acid, and
oleanolic acid are 1.00, 1.1, and 3.2, respectively.]
Suitability requirements
Chromatogram similarity: The chromatogram from
Standard solution B is similar to the reference chro-
USP 41
matogram provided with the lot of USP Lager-
stroemia speciosa Leaf Dry Extract RS being used.
Resolution: NLT 1.5 between the corosolic acid
peak and the preceding peak, Standard solution B
Tailing factor: NMT 2.0 for the corosolic acid peak,
Standard solution A
Relative standard deviation: NMT 2.0%, deter-
mined from the corosolic acid peak in repeated in-
jections, Standard solution A
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Identify the relative retention times of the peaks for
corosolic acid, virgatic acid, and oleanolic acid of the
Sample solution.
Calculate the percentage of the labeled amount of
corosolic acid in the portion of Banaba Leaf Dry Ex-
tract taken:
Result = (ru/rs) x (Cs/Cy) x 100
ry = peak area of corosolic acid from the Sample
solution
Is = peak area of corosolic acid from Standard
solution A
Cs = concentration of corosolic acid in Standard
solution A (mg/mL)
Cy = concentration of Banaba Leaf Dry Extract in
the Sample solution (ergime)
Calculate the percentage of the labeled amount of
corosolic acid in the portion of Extract taken:
Result = (P/L) x 100
P = content of corosolic acid as determined above
(%)
L = labeled amount of corosolic acid (%)
Acceptance criteria: 90.0%-110.0% of the labeled
amount of corosolic acid on the dried basis
CONTAMINANTS
e ELEMENTAL IMPURITIES—PROCEDURES (233)
Acceptance criteria
Arsenic: NMT 2.0 ug/g
Cadmium: NMT 0.5 ug/g
Lead: NMT 5u0/g
Mercury: NMT 0.2 g/g
e ARTICLES OF BOTANICAL ORIGIN, General Method for Pesti-
cide Residues Analysis (561): Meets the requirements
e MICROBIAL ENUMERATION TESTS (2021): The total aerobic
microbial count does not exceed 104 cfu/g, and the total
coi inssa molds and yeasts count does not exceed 103
cfu/g.
e MICROBIAL PROCEDURES FOR ABSENCE OF SPECIFIED MICROOR-
GANISMS (2022): Meets the requirements of the tests for
absence of Salmonella species and Escherichia coli
SPECIFIC TESTS
e Loss ON DRYING (731)
Sample: 2.0g of Banaba Leaf Dry Extract
Analysis: Dry the Sample at 105° for 2 h.
Acceptance criteria: NMT 8%
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in well-closed contain-
ers, protected from light and moisture, and store at
room temperature.
e LABELING: The label states the Latin binomial and, follow-
ing the official name, the parts of the plant from which
the article was derived. It meets other labeling require-
ments in Botanical Extracts (565).
Dietary Supplements / Beta Carotene 446!
e USP REFERENCE STANDARDS (11)
USP Corosolic Acid RS
USP Lagerstroemia speciosa Leaf Dry Extract RS
Beta Carotene—see Beta Carotene General
Monographs
Beta Carotene Preparation
DEFINITION
Beta Carotene Preparation is a combination of beta carotene
with one or more inert substances. It may be in a solid or
a liquid form. It contains NLT 95.0% and NMT 130.0% of
the labeled amount of total beta carotene (C4oHs6) on the
anhydrous basis.
IDENTIFICATION
cA.
Sample solution: Transfer 5.0 mL of Sample stock solu-
tion A or Sample stock solution B from the test for Con-
tent of Beta Carotene to a 100-mL volumetric flask, and
dilute with cyclohexane to volume. Pass the solution
through a membrane filter of 0.45-m pore size.
Analysis: Record the UV-Vis spectrum from 300 to 600
nm.
Acceptance criteria: The Sample solution shows a shoul-
der at about 427 nm, an absorption maximum at about
455 nm, and another maximum at about 483 nm. The
absorbance ratio A4ss/Aagz is between 1.14 and 1.18.
e B. The retention time of the major peak of the Sample
solution corresponds to that of the Standard solution, as
obtained in the test for Content of Beta Carotene.
COMPOSITION
© CONTENT OF BETA CAROTENE
[NoTe—Use low-actinic glassware.]
Mobile phase: Transfer 50 mg of butylated hydroxytol-
uene to a 1-L volumetric flask, and dissolve with 20 mL
of 2-propanol. Add 0.2 mL of N-ethyldiisopropylamine,
25 mL of 0.2% ammonium acetate solution, 455 mL of
acetonitrile, and about 450 mL of methanol. Allow the
solution to reach room temperature, and dilute with
methanol to volume.
Diluent: 50 g/mL of butylated hydroxytoluene in
alcohol
System suitability solution: Transfer 20 mg of USP Beta
sydesbouo-: sa
Carotene System Suitability RS to a 50-mL volumetric
flask. Add 1 mL of water and 4 mL of tetrahydrofuran,
and sonicate for 5 min. Dilute with Diluent to volume,
and sonicate for 5 min. Cool to room temperature, pass
the suspension through a membrane filter of 0.45-um
pore size, and use the clear filtrate.
Standard stock solution: 60 g/mL of USP Beta Caro-
tene RS in tetrahydrofuran
Standard solution A: Transfer 5.0 mL of the Standard
stock solution to a 100-mL volumetric flask, add 5.0 mL
of tetrahydrofuran, and dilute with Diluent to volume.
The concentration of the all-trans-beta carotene in this
solution will be determined by the spectrophotometric
procedure using Standard solution B as follows.
Standard solution B: Transfer 5.0 mL of the Standard
stock solution to a 100-mL volumetric flask, and dilute
with cyclohexane to volume. Prepare in triplicate.
Instrumental conditions
(See Ultraviolet-Visible Spectroscopy (857).)
 4466 Beta Carotene / Dietary Supplements USP 41

Analytical wavelength: 456 nm
Cell path: 1.cm
Blank: Cyclohexane
Analysis
Sample: Standard solution B
Calculate the concentration of total beta carotene
(mg/mL) as all-trans-beta carotene (C4oHse) in Standard
solution B:
Result = A/F
A = average absorbance of the three preparations
of Standard solution B
F = absorptivity of pure all-trans-beta carotene in
cyclohexane, 250.5
Sample stock solution A (for solid Beta Carotene Prepa-
rations): Transfer a quantity of Preparation, equivalent
to 10 mg of beta carotene, to a 250-mL volumetric
flask. Add 250 mg of butylated hydroxytoluene, 0.5 mL
of alkaline protease R, and 15 mL of water. Tilt the flask
gently to wet the entire contents. Sonicate the solution
in an ultrasonic bath at about 50° for 30 min, and swirl
at 10-min intervals. Add 100 mL of alcohol to the warm
suspension, and shake vigorously. Add 135 mL of meth-
ylene chloride, and shake again. Let the mixture stand
in the dark until it reaches room temperature (about 2
h). Dilute with methylene chloride to volume, shake
vigorously, and allow solids to settle in the dark.
Sample stock solution B (for liquid Beta Carotene sus-
pensions in oil Preparations): Transfer a quantity of
Preparation, equivalent to 20 mg of beta carotene, to a
250-mL volumetric flask. Add 250 mg of butylated
hydroxytoluene, 120 mL of methylene chloride, and
100 mL of alcohol. Shake the flask until the sample is
completely dissolved or suspended. Let the mixture
stand in the dark until it reaches room temperature
(about 2 h). Add methylene chloride to volume, and
shake again vigorously.
Sample solution: Transfer 5.0 mL of Sample stock solu-
tion A or Sample stock solution B to a 50-mL volumetric
flask, and dilute with a mixture of methylene chloride
and Diluent (1:1) to volume. Pass through a membrane
filter of 0.45-um pore size.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 448 nm
Column: 4.6-mm x 25-cm; 5-um packing L68
Column temperature: 30°
Flow rate: 0.6 mL/min
Injection volume: 20 pL
¢ ALPHA CAROTENE AND OTHER RELATED COMPOUNDS
DS Monographs
Tailing factor: NMT 2.0 for the beta carotene peak,
Standard solution A
Relative standard deviation: NMT 2.0% for the beta
carotene peak from replicate injections, Standard solu-
tion A
Chromatogram similarity: The chromatogram from
the System suitability solution is similar to the refer-
ence chromatogram provided with the lot of USP
Beta Carotene System Suitability RS being used.
Analysis
Samples: Standard solution A and Sample solution
Record the chromatograms, and identify the peaks of
the relevant analytes of the Sample solution by compar-
ing with those of the System suitability solution. Meas-
ure the peak area responses.
Calculate the percentage of the labeled amount of total
beta carotene in the portion of Preparation taken:
Result = (ru/rs) x (Cs/Cu) x 100
tu = [(peak area of all-trans-beta carotene) + (peak
area of 9-cis-beta carotene) + (peak area of
13-cis-beta carotene x 1.2) + (peak area of
15-cis-beta carotene x 1.4) + (sum of peak
areas of other cis-isomers of beta carotene)]
in the Sample solution
peak area of all-trans-beta carotene in
Standard solution A
Cs = concentration of all-trans-beta carotene in
Standard solution A as determined by
spectrometric procedure (mg/mL)
Cu = nominal concentration of Preparation in the
Sample solution (mg/mL)
Calculate the percentage of the labeled amount of all-
ven Bela carotene in the portion of Preparation
taken:
Result = (ru/rs) x (Cs/Cu) x 100
ty = peak area of all-trans-beta carotene in the
Sample solution
ls = peak area of all-trans-beta carotene in
Standard solution A
Cs = concentration of all-trans-beta carotene in
Standard solution A as determined by
spectrometric procedure (mg/mL)
cy = nominal concentration of Preparation in the
Sample solution (mg/mL)
Acceptance criteria: The Preparation contains
95.0%
-1 30.0% of the labeled amount of total beta car-
otene, calculated as (C4oHs) on the anhydrous basis.

System suitability
Samples: System suitability solution and Standard solu- Mobile phase, System suitability solution, Sample so-
tion A lution, and Chromatographic system: Proceed as di-
The approximate relative retention times of the compo- rected in the test for Content of Beta Carotene.
nents in the System suitability solution are listed in Ta- Injection volume: 20 ul
ble 1. Analysis
Sample: Sample solution
Calculate the percentage of alpha carotene and other
Table 1
individual related compounds relative to total beta car-
Relative Relative otene in the portion of Preparation taken:
Retention Response
Name Time Factor Result = (ru/rr) x 100
all-trans-Alpha carotene 0.93 10
ty = peak area of alpha carotene or other
all-trans-Beta carotene 1.00 1.0
individual related compounds from the
9-cis-Beta carotene 1.07 1.0 Sample solution
13-cis-Beta carotene TAZ V2: nr = sum of the peak areas of all the peaks from
15-cis-Beta carotene 1.21 1.4 the Sample solution
Acceptance criteria
Suitability requirements Alpha carotene: NMT 1.0%
Resolution: NLT 1.2 between beta carotene and al- Any other individual related compound: NMT 1.0%
pha carotene and between beta carotene and 9-cis- Total related compounds (including alpha carotene):
eta carotene, System suitability solution NMT 5%
USP 41
IMPURITIES
e RESIDUE ON IGNITION (281): NMT 2.0%
Delete the following:
°o HEAVY METALS, Method II (231): NMT 10 ppme cofticiat1-
Jan-2018)
SPECIFIC TESTS
e WATER DETERMINATION (921), Method |: NMT 8.0% for
solid Preparations; NMT 1.0% for liquid Preparations
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in tightly sealed, light-
and oxygen-resistant containers. Store in a cool place.
e LABELING: The label states the name and content of any
carriers and antioxidants added to the formulation, the
content of total carotenoids as beta carotene, and the
percentages of cis- and all-trans-isomers in the total beta
carotene at the time of product manufacture and release.
e USP REFERENCE STANDARDS (11)
USP Beta Carotene RS
(all-E)-1,1’-(3,7,12,16-Tetramethyl-1,3,5,7,9,11,13,15,
17-octadecanonaene-1,18-
diyl)bis[2,6,6-trimethylcyclohexene].
USP Beta Carotene System Suitability RS
Beta Carotene Capsules—see Beta
Carotene Capsules General Monographs
Beta Glucan
DEFINITION
Beta Glucan is obtained by extraction from the cell wall of
fermented and thermally processed Baker’s yeast
(Saccharomyces cervisiae). \t is comprised mainly of B-
1,3)/(1,6) branched glucan polymers. Small amounts of
-(1,6)-glucan and chitin are also expected to be present
in the final product. It contains NLT 70% beta glucan,
calculated as glucose after enzymatic hydrolysis, on the
dried basis.
IDENTIFICATION
e NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY (761)
Standard solution: Dissolve 10 mg of USP Beta Glucan
RS in 0.6 mL of dimethyl sulfoxide—ds, and heat at 100°
for 1 h. Then add 0.1 mL of D20, mix the solution, and
transfer to an NMR tube.
Sample solution: Dissolve 10 mg of Beta Glucan in
0.6 mL of dimethyl sulfoxide-ds, and heat at 100° for 1
h. Then add 0.1 mL of D20, mix the solution, and
transfer to an NMR tube.
Analysis: Collect 'H NMR spectra at 80°, and compare
individual resonances from the Sample solution to those
from the Standard solution. The major signals associated
with Beta Glucan are shown in Table 1.
Table 1
1H NMR Major Signals USP Beta Glucan RS
H-1 (1,3)-glucan 4.52, d, | = 7.5 Hz, 1H
H-2, 4, and 5 (1,3-) 3.27-3.33, m, 3H
H-3 and 6b (1,3-) 3.45-3.48, m, 2H
H-6a (1,3-) 3.71, d, | = 11 Hz, 1H
H-1 (1,6)-glucan 4.27, d, | = 7.7 Hz, 1H
Integrate the area under the peaks five times for each
sample, and average. Determine the relative percentage
Dietary Supplements / Beta Glucan 4467
of (1,6) linked glucan in the portion of Beta Glucan
taken:
Result = {A/(A + B)} x 100
A = integration values for the signal at 4.27 ppm,
corresponding to H-1 from (1,6) glucan
B = integration values for the si nal at 4.52 ppm
corresponding) to H-1 (1,3) glucan
Acceptance criteria: The 'H spectrum of the Sample
solution exhibits a chemical shift pattern with signal lo-
cations and relative intensities that correspond to those
of the Standard solution. In addition, the relative per-
centage of (1,6) linked glucan is 10%-18% of the total
linkages.
COMPOSITION
e CONTENT OF BETA GLUCAN
Buffer A: Dissolve 11.6 mL of glacial acetic acid in ap-
proximately 900 mL of water. Adjust the solution with
20% sodium hydroxide solution to a pH of 5, and di-
lute with water to 1000 mL.
Buffer B: Dissolve 69.6 mL of glacial acetic acid in ap-
peoeately 800 mL of water. pe with 20% sodium
ydroxide solution to a pH of 3.8, and dilutewith
water to 1000 mL.
Buffer C: Dissolve 12.12 g of tris(hydroxymethyl)-
aminomethane (TRIS), 11.69 g sodium chloride, and
4.16 g of ethylenediaminetetraacetic acid (EDTA) tetra-
sodium salt dihydrate in approximately 900 mL of
water. Adjust with concentrated hydrochloric acid or
20% sodium hydroxide solution to a pH of 7.5, and
dilute with water to 1000 mL. [Note—Buffer C can be
stored for 1 year at 2°-8°.]
Buffer D!: Transfer 45.287 g of dibasic potassium phos-
phate, 30.382 g of p-hydroxybenzoic acid, and 4g of
sodium azide into a 1000-mL volumetric flask, and care-
fully add 800 mL of water. Mix witha stirring bar and
mild heat until completely dissolved. Allow the solution
to cool, adjust with 16.7% potassium hydroxide solu-
tion to a pH of 7.4, and dilute with water to volume.
[NoTte—Store Buffer D in an amber bottle with an expi-
ration date of 3 years at 4°.]
Lyticase solution: Prepare the required volume of lyti-
case from Arthrobacter luteus? at a concentration of 10
U/uL by dissolving the quantity stated by the manufac-
turer (U/mg) in a solution containing 10% (v/v) Buffer
Cc. [NoTte—Unused solution can be stored at NMT -15°
with an expiration date of 1 year. Every time a different
lot of lyticase is used, the concentration of lyticase solu-
tion required needs to be qualified.]
(1,6)-Glucanase solution: 1U/300 uL solution of lyophi-
sydeibouow= sa
lized (1,6)-glucanase3 in Buffer A. [NoTE—Solids may not
fully dissolve; therefore, this solution should be handled
as a homogeneous suspension. The solution is stable for
at least 60 days at NMT -15°.]
Polishing enzyme mix: Mix 2000U of exo-beta-glu-
canase* and 400 U of beta-glucosidase’ in 100.0 mL of
Buffer A. A premix of the enzymes may be used as an
alternative’. [NoTE—Store on ice during the procedure,
and for use in a same-day assay. Unused Polishing en-
zyme mix can be refrozen once at NMT -15° with an
expiration date of 2 years.]
Glucose oxidase/peroxidase reagent: Dissolve the
contents of the Glucose Determination Reagent? in 1 L
of water containing 50 mL of Buffer D. [NoTE—Store
the reagent in an amber bottle, and label with an expi-
1 This buffer is also available as Bottle #3 of the K-YBGL kit (Megazyme), or
Bottle #1 of the GOPOD kit (Megazyme).
2iiusese from Arthrobacter luteus, Sigma 14025, or equivalent.
> Commercially available as Pustulanase, Cel136, Prokazyme, or equivalent.
4 E-EXBGL 200 U/mL, 200 U/bottle, Megazyme, or equivalent.
5 200 U/bottle, Megazyme, or equivalent.
6 E-EXBGOS, Megazyme, or equivalent.
7 Bottle #4 of K-YBGL kit, or Bottle 2 of GOPOD kit, Megazyme, or equiva-
lent.
 4468 Beta Glucan / Dietary Supplements
ration date of 3 months at a temperature between 2°
and 8° or 1 year at NMT -17°. Minimize the time spent
at room temperature.]
Sample solution: Transfer 15-20 mg of Beta Glucan
into a 16- x 100-mm glass vial. Place the vial in an ice
bath. Add a 0.4-mL aliquot of cold potassium hydroxide
(1 in 6) while mixing on a vortex mixer to disperse the
powder. Return the vial to the ice bath. Continue cy-
cling through mixing on a vortex mixer and placing the
vials in the ice bath for 20 min. The mixture should
turn into a homogenous, translucent dispersion.
Standard solution: Proceed as directed for the Sample
solution except replace Beta Glucan with USP Beta Glu-
can RS. [NoTE—Prepare the Sample solution and Stan-
dard solution in triplicate. It is critical for the success of
the assay that the sample is well dispersed.]
Lyticase digestion: Upon removal of all vials containing
the Sample solution or the Standard solution from the ice
bath, add 1.6 mL of Buffer B and 600 uL of Lyticase solu-
tion to each vial. Incubate the mixture at 50° for 12-18
h, and cool to room temperature.
(1,6)-Glucanase digestion: After cooling of all vials, re-
move a 130-uL aliquot of each vial, and digest further
by adding 25 uL of 16.7% potassium hydroxide solu-
tion and 300 uL of (7,6)-Glucanase solution. Incubate vi-
als at 80° for 15 min, and cool to room temperature.
Beta glucanase/glucosidase digestion: After cooling of
all vials, add 390 uL of the Polishing enzyme mix to each
vial, and incubate the vials at 40° for 1 h. Cool them to
room temperature, centrifuge, and transfer 50-uL ali-
quots (in dupa) to new vials.
Enzyme blank solution: Prepare enzyme blanks in trip-
licate by combining all the reagents used during the
digestion steps except the Sample solution or Standard
solution.
Instrumental conditions
(See Ultraviolet-Visible Spectroscopy (857).)
Mode: Vis
Analytical wavelength: 510 nm
Analysis: Dilute the 50-uL aliquots obtained after the
Beta glucanase/glucosidase digestion with 50 wL of water,
and then add 3 mL of Glucose oxidase/peroxidase rea-
gent. Incubate the vials for 20 min at 40°. Determine
the absorbance of each vial of the Sample solution or
Standard solution against the Enzyme blank solution. Pre-
pare a standard curve using the absorbance of similarly
treated series of glucose standards (0, 0.1, 0.25, 0.5,
and 1.0 mg/mL). From the slope of the standard curve
and the absorbance of the digested Sample solution and
Standard solutions, determine the concentration, C, in
mg/mL, of liberated glucose in the cuvette:
DS Monographs
Result = (As — As)/slope
As = average absorbance of the sample or USP Beta
Glucan RS
As = average absorbance of the Enzyme blank
solution
Calculate the percentage of beta glucan as glucose in
the portion of Beta Glucan taken:
Result = 100 x C/{{(WTs/F1) x (F2/F3)]/2}
WTs = original weight of the sample or USP Beta
Glucan RS (mg)
FI = total volume in the vial during Lyticase
digestion, 2.6 mL
F2 = volume of the sample or USP Beta Glucan RS
transferred to a new vial during (1,6)-
Glucanase digestion, 0.130 mL
F3 = total volume during Beta glucanase/glucosidase
digestion, 0.845 mL
Acceptance criteria: NLT 70% beta glucan, calculated
as glucose after enzymatic hydrolysis, on the dried basis
USP 41
e CONTENT OF PROTEIN
Sample: 1.0g of Beta Glucan
Analysis: Proceed as directed in Nitrogen Determination
(461), and multiply the nitrogen content by 6.25.
Acceptance criteria: NMT 10.0%
© CONTENT OF FAT
Sample: 2g of Beta Glucan, previously dried
Analysis: Transfer the Sample to an extraction thimble,
mix with an equivalent quantity of dry, clean sand.
Placea fat-free cotton or glass wool Blue on top of the
thimble. Place the thimble in a continuous-extraction
apparatus provided with a tared collection flask. Pour
75 mL of solvent hexane through the sample into the
collection flask. Extract at a condensation rate of
5-6 drops/s for 4 h, then at a rate of 2-3 drops/s for
the next 16 h. Detach the collection flask, carefully
evaporate the solvent, and dry the collection flask and
its contents in a drying oven at 100° for 30 min to
constant weight. Calculate the percentage of the ex-
tract (crude fat) in the portion of Beta Glucan taken.
Acceptance criteria: NMT 20.0%
CONTAMINANTS
e ELEMENTAL IMPURITIES—PROCEDURES (233)
Acceptance criteria
Arsenic: NMT 0.5 ug/g
Cadmium: NMT 0.5 ug/g
Lead: NMT 0.5 ug/g
Mercury: NMT 0.1 ug/g
e MICROBIAL ENUMERATION TESTS (2021): The total aerobic
bacterial count does not exceed 2 x 104 cfu/g, the total
combined molds and yeasts count does not exceed 2.5
x10' cfu/g, and the bile-tolerant Gram-negative bacteria
does not exceed 10 cfu/g.
¢ ABSENCE OF SPECIFIED MICROORGANISMS (2022): Meets the
requirements of the tests for the absence of Salmonella
species and Escherichia coli
SPECIFIC TESTS
© GLYCOGEN
Buffer B: Prepare as directed in Content of Beta Glucan.
Amyloglucosidase/invertase solution’: A mixture of
1630 U/mL of amyloglucosidase and 500 U/mL of in-
vertase in glycerol solution (50% v/v)
Sample: 100mg
Analysis: Transfer the Sample in triplicate into individual
16- x 150-mm glass screw-cap vials. Place the vials in
an ice bath, and add to each vial a 2-mL aliquot of cold
potassium hydroxide (1 in 6) while mixing on a vortex
mixer to disperse the powder. Return the vial to the ice
bath. Continue cycling through mixing on a vortex
mixer and placing vials in the ice bath for 20 min. The
mixture should turn into a homogenous, translucent
dispersion. Add 8 mL of Buffer B. Mix thoroughly on a
vortex mixer, and immediately add 200 uL of Amy-
loglucosidase/invertase solution, and mix again on a vor-
tex mixer. Incubate the mixture at 40° for 30-35 min.
Cool to room temperature. Mix again on a vortex
mixer, transfer to a suitable centrifuge tube, and centri-
fuge until a clear supernatant is obtained. Transfer du-
plicate 50-uL aliquots of supernatant into new vials, and
proceed as directed for the Analysis in Content of Beta
Glucan.
Acceptance criteria: NMT 1.0%
© MANNOSE
Solution A: 100% purified water
Solution B: 956 mM sodium hydroxide
Mobile phase: See Table 2.
8Alternatively, Bottle #2 of K-YBGL kit (Megazyme, or equivalent) could be
used directly.
USP 41 Dietary Supplements / Bifidobacterium 446°

Table 2 Column: 4-mm x 25-cm; packing L47

Time Solution A Solution B
Guard column: 4-mm x 5-cm; packing L47
(min) (%) (%)
Column temperature: 30°
Flow rate: 0.4 mL/min
0 36.0 64.0 Injection size: 10 uL
15.0 36.0 64.0 System suitability
35.0 (sample injec- Sample: Standard Identification #5
tion) 59.4 40.6 [Note—The relative retention times for inositol, man-
80.0 59.4 40.6 nose, and glucose are 0.68, 0.95 and 1.0,
respectively.]
[Nott—The run time typically required is 80 min.] Suitability requirements
Internal standard solution: 0.8 mg/mL of USP Inositol Resolution: NLT 1.5 between mannose and glucose
RS in water Analysis
Sample solution: Weigh 2.0-4.0 mg of Beta Glucan in Samples: Standard solutions and Sample solution
duplicate into vials with stir bars. Add 500 uL of pure Calculate the ratios of the peak area response of the
trifluoroacetic acid (TFA), and allow the mixture to form lucose and mannose to the peak area response of the
a uniform dispersion by stirring for 1 h at room temper- internal standard from the Standard solutions. Make
ature. Incubate in a 80° water bath for 2 h with stirring, two standard response lines by plotting the peak area
and then cool to room temperature. Add 100 ul of the response ratio versus the amount (ug) of the glucose
Internal standard solution to each vial, and incubate and mannose in the Standard solutions. Calculate the
with stirring in a boiling water bath for 15 min. Cool ratio of the peak area response of the glucose and
again to room temperature, then add 1.07 mL of water mannose to the peak area response of the internal
to each vial, and incubate with stirring in a boiling standard from the Sample solution. From the calculated
water bath for 1 h. Cool the solutions to room temper- ratios of peak responses for glucose and mannose and
ature, and dry overnight on a SpeedVac, or equivalent, their respective standard response lines, determine the
at low heat with the cryopumping system off. Dissolve content of glucose, Ce, and mannose, Cy, both in jg,
the dried preparation in 2.5 mL of deionized water, and in the Sample solution.
pass through a PTFE syringe filter of 0.2-14m pore size. Calculate the percentage of mannose in the portion of
Dilute with an equal volume of water before injection. Beta Glucan taken:
Standard solutions: Prepare a 4-mg/mL solution of
USP Dextrose RS and a 80-1g/mL solution of USP Man- Result = Cw/(Cw + Cc) x 100
nose RS. Separately transfer aliquots of these solutions
to individual vials (see Table 3). Prepare each standard Cu = content of mannose in the Sample solution
in duplicate, and freeze-dry them. Treat the freeze-dried from the mannose regression line (ug)
vials as directed in the Sample solution, beginning with (€ = content of glucose in the Sample solution from
“Add 500 pL of pure trifluoroacetic acid”. the glucose regression line (11g)
Acceptance criteria: NMT 1.0% mannose, as a func-
tion of total hexose recovered (glucose and mannose)
Table 3
e RESIDUE ON IGNITION (281): NMT 2.5%
Standard uL/Vial Content pL/Vial Content e Loss ON DRYING (731)
Identi- of 4 mg/ of Glu- of 80 11g/ of Man- Sample: 1g
fication mL cose mL nose Analysis: Dry the Sample at 105° for 3 h.
Number Glucose (ug) Mannose (ug) Acceptance criteria: NMT 8.0%
0 0 0 0 0
1 100 400 25 Z
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in tight and light-resis-
2 200 800 50 4 tant containers.
3 300 1200 100 8 e USP REFERENCE STANDARDS (11)
4 500 2000 200 16 USP Beta Glucan RS
5 1000 4000 400 32 USP Dextrose RS
USP Inositol RS
Chromatographic system USP Mannose RS
sydesbouo; sq

(See Chromatography (621), System Suitability.)

Mode: LC
Detector: Electrochemical detector
Detector mode: Pulsed amperometric detection
Detector range: 3000 pC (may be modified if Bifidobacterium animalis subsp. lactis
needed)
Working electrode: Gold DEFINITION
Reference electrode: pH, silver-silver chloride Bifidobacterium animalis subsp. lactis is a lactic-acid produc-
Electrochemical waveform: See Table 4. ing, Gram-positive, rod-shaped, non-spore-forming, anaer-
obic bacterium that is pleomorphic. Various rod shapes
Table 4 may be observed, but they are typically curved/clubbed,
Time Potential and often branched. Bifidobacterium animalis subsp. lactis
Integration occurs as a white- to cream-colored powder that is pro-
(s) (Vv)
duced via fermentation of one of three strains of
0.00 0.10 Bifidobacterium animalis subsp. lactis: Bi-O7 (ATCC
0.20 0.10 Start $D5220), BI-04 (ATCC $D5219), and HNO19 (ATCC
0.40 0.10 End SD5674). Suitable cryoprotectants may be added to the
0.41 2.00 concentrated bacteria following fermentation, after which
0.42 =2.00 the product is frozen and then freeze-dried. The formu-
0.43 0.60 lated product may be blended with food-grade diluents
and/or bulking agents. It contains NLT 100% of the la-
0.44 —0.10
beled viable cell count of one of the three Bifidobacterium
0.50 -0.10 animalis subsp. /actis strains.
 4470 Bifidobacterium / Dietary Supplements
IDENTIFICATION
e A. NUCLEIC ACID-BASED IDENTIFICATION
[Note—In all cases for Identification A, “sterile water” re-
fers to sterile, nuclease-free water acceptable for use in
molecular biology."]
Buffer: Use a molecular biology-grade 10 mM tris-hy-
drochloride, 1 mM EDTA sodium buffer.2
Sample solution: 100 mg/mL of the freeze-dried probi-
otic powder in Buffer
Primer set: Use primer(s) set per strain (see Table 1).3
Primers should be diluted in Buffer to a stock concentra-
tion of 100 uM, then further diluted to 25 uM in Buffer,
and stored at —20°. A positive test for each Primer set to
the strain is expected to give an amplification product
specified in Table 7.
Table 1. Primer Set
Strain Primer
Set 1: Forward (5’-3’) CATCGCAACTT-
CACCCACATTG and Reverse (5’-3’)
ATGCCGTACCCCTGAATGAAG. The
annealing temperature of set 1: 57.0°.
An amplification product of this set is
533 base pairs.
Set 2: Forward (5’-3’) ACG-
GATATGTATAGGTGGCATGC and
Reverse (5’-3’) GTATGTT-
CAATCGTATGCAGCCC. The annealing
temperature of set 2: 56.0°. An ampli-
fication product of this set is 492 base
pairs with a single nucleotide poly-
Bi-07 morphism (SNP).
Forward (5’-3’) CATCGCAACTTCACC-
CACATTG and Reverse (5’-3’)
ATGCCGTACCCCTGAATGAAG., An
amplification product of this set is
BI-04 479 base pairs.
Set 1: Forward (5’-3’)
ACTCTTCTGTGTCGTTCTGCTTIC and
Reverse (5’-3’) CAAGGATTGGAACGC-
GAGAAAAC. The annealing tempera-
ture of set 1: 56.0°. An amplification
product of this set is 351 base pairs
with an SNP.
Set 2: Forward (5’-3’)
CCTGCTGTGGTGAATACGAAGAA and
Reverse (5’-3’) TTGCATCTTGTA-
CAGTTCGGCAT. The annealing
temperature of set 2: 57.0°. An ampli-
fication product of this set is 531 base
DS Monographs
HNO19 pairs with an SNP.
Polymerase chain reaction (PCR) sample prepara-
tions: For each Primer set, prepare a solution contain-
ing 1 wL of the Sample solution, 10 wL of mastermix
polymerase,4 1 wL of diluted forward primer (25 UM),
1 ul of diluted reverse primer (25 uM), and 12 uL of
sterile water.
PCR negative control: Prepare as directed for the PCR
sample preparations, replacing the 1 wL of Sample solu-
tion with 1 uL of sterile water.
1 Suitable PCR-Certified Waters, RNase and DNase Free, are available from
www.teknova.com.
2 Suitable buffers (e.g., TE Buffer 1X, Molecular Biology Grade) are available
from www.promega.com.
3 DNA primers are commercially available (custom manufacture) from Inte-
grated DNA Technologies (www.idtdna.com) and other commercial sources.
45 Prime MasterMix polymerase from 5 Prime.
USP 41
PCR amplification: Perform PCR on each PCR sample
preparation and the PCR negative control using an ap-
propriate thermal cycler (see Table 2).5 Perform the PCR
amplification cycle per primer and per strain as speci-
fied in Table 2.
Table 2. PCR Amplification Cycle
Strain PCR Amplification Cycle
Incubate at 95° for 7 min (step 1); 95°
for 30 s (step 2); the Primer set
annealing temperature for 30 s (step
3); and at 72° for 1 min (for Primer
Set 1) or 30 s (for Primer set 2; step
4). Repeat steps 2-4 for 34 cycles,
then incubate at 72° for 5 min and
Bi-07 hold at 4°.
Incubate at 95° for 7 min (step 1); 95°
for 30 s (step 2); 57.0° for 30 s (step
3); and at 72° for 1 min (step 4).
Repeat steps 2-4 for 34 cycles, then
incubate at 72° for 5 min and hold
BI-04 at 4°.
Incubate at 95° for 7 min (step 1); 95°
for 30 s (step 2); the Primer set
annealing temperature for 30 s (step
3); and at 72° for 30 s (for Primer set
1) or 1 min (for Primer set 2; step 4).
Repeat steps 2-4 for 34 cycles, then
incubate at 72° for 5 min and hold
HNO19 at 4°.
Analysis: Analyze the products of the PCR amplification
for each PCR sample preparation and for the PCR nega-
tive control using an automated on-chip electrophoresis
system with a DNA kit.6 Follow the manufacturer's in-
structions for analysis. Alternatively, anaes and visuali-
zation may be accomplished using gel electrophoresis.
Prepare or use a commercially available 1% (w/v)
agarose gel in a 1X tris-acetic acid-EDTA buffer (40 mM
tris-hydrochloride, 1% glacial acetic acid, and 1 mM
EDTA). Stain the gel with 0.5 mg/mL of ethidium bro-
mide in water and de-stain with deionized water. [CAu-
TION—Ethidium bromide is considered a toxic substance
and a potential mutagen. Use appropriate personal pro-
tective equipment (including nitrile gloves) when han-
dling this reagent.] Use a DNA ladder standard (1 KB
plus)” suitable for determining the size of linear double-
stranded DNA fragments between 100 and 12,000 base
pairs. The ladder standard should be used in the first
and last lanes on the gel to allow for proper compari-
son of onplicons.
Analysis of the PCR negative contro! must result in the
absence of any amplification products or the prepara-
tion of the PCR sample preparations, and the PCRnega-
tive control must be repeated, followed by PCR amplifi-
cation and Analysis.
Acceptance criteria: The PCR sample preparation pre-
pared with strain-specific Primer set gives an acceptable
amplification product per strain (see Table 3).
5 Suitable thermal cyclers are available from Eppendorf® (www.eppendorf.
com).
6 Suitable automated on-chip electrophoresis systems with a DNA kit are
available from Agilent (Agilent 2100 Bioanalyzer with Agilent DNA 1000 Kit
www.genomics.agilent.com).
’ Suitable 1 KB plus DNA ladders are available from www.thermofisher.com.
USP 41
Table 3. Acceptance Criteria of PCR Amplification Products
Strain Acceptance Criteria per Strain
The PCR sample preparation prepared
with Primer set 1 gives an amplifica-
tion product of 533 base pairs. There
should be no amplification product of
479 base pairs. The PCR sample prepa-
ration prepared with Primer set 2 gives
an amplification product of 492 base
pairs with an SNP identified as
(underlined in the following 15 base
pair sequence) GCGGG-
CAAGTGCTGG. The SNP location is
218 base pairs from the 5’ end of the
forward primer described in Primer set
2. The sequence of the amplicon
should be determined by validated,
Bi-07 standard sequencing technologies.
The PCR sample preparation prepared
with the Primer set gives an amplifica-
tion product of 479 base pairs. There
should be no amplification product of
BI-04 533 base pairs.
The PCR sample preparation prepared
with Primer set 1 gives an amplifica-
tion product of 351 base pairs with
an SNP identified as (underlined in
the following 15 base pair sequence)
CTTCAGATTTTAGGC. The SNP loca-
tion is 44 base pairs from the 5’ end
of the forward primer described in
Primer set 1. The PCR sample prepara-
tion prepared with Primer set 2 gives
an amplification product of 531 base
pairs with an SNP identified as
(underlined in the following 15 base
pair sequence) GCCCGCTCAAACGAA.
The SNP location is 279 base pairs
from the 5’ end of the forward primer
described in Primer set 2. The
sequence of the amplicon should be
determined by validated, standard
HNO19. sequencing technologies.
ASSAY
¢ ENUMERATION
Agar medium: Prepare as follows or use a suitable
commercially available agar (see Table 4).8
Table 4. Lactobacilli MRS Agar
Quantity
Reagent (g)
Proteose peptone no. 3 10.0
Beef extract 10.0
Yeast extract 5.0
Dextrose 20.0
Polysorbate 80 1.0
Ammonium citrate 2.0
Sodium acetate 5.0
Magnesium sulfate 0.1
Manganese sulfate 0.05
Dipotassium phosphate 2.0
Agar 15.0
Suspend Lactobacilli MRS Agar in 1 L of purified water
in an appropriately sized conical flask or beaker (suffi-
ciently large to not boil over). Cover the flask or
beaker with aluminum foil and heat with stirring to
8 Difco™ Lactobacilli MRS Agar, or equivalent. Suitable Lactobacilli MRS Agars
are available from www.vwr.com or other chemical/microbiological suppliers.
Dietary Supplements / Bifidobacterium 4471
boiling on a hot plate. Allow to boil for 1 min to com-
pletely dissolve the medium, then autoclave the solu-
tion at 121° for 15 min. Cool to 45° and use immedi-
ately. Boiled Agar medium may also be aseptically
transferred into individual media bottles in 100- or
200-mL aliquots before sterilizing, and then autoclaved
and stored for later use. [NoTE—Can be stored at 4°
(heat gently to 45° to melt the agar before use).] Im-
mediately before use, aseptically add 1.0 mL of a ster-
ile 5% (w/v) cysteine hydrochloride solution for each
100 mL of Agar medium prepared, such that the final
concentration of cysteine hydrochloride in the Agar
medium is 0.05%.
Sample broth: Prepare as follows or use a suitable
commercially available broth (see Table 5).9
Table 5. Lactobacilli MRS Broth
Quantity
Reagent (g)
Proteose peptone no. 3 10.0
Beef extract 10.0
Yeast extract 5.0
Dextrose 4 20.0
Polysorbate 80 1.0
Ammonium citrate 2.0
Sodium acetate 5.0
Magnesium sulfate 0.1
Manganese sulfate 0.05
Dipotassium phosphate 2.0
Suspend Lactobacilli MRS Broth in 1 L of purified water
in an appropriately sized conical flask or beaker (suffi-
ciently large to not boil over). Cover the flask or
beaker with aluminum foil and heat with stirring to
boiling on a hot plate. Allow to boil for 1 min to com-
pletely dissolve the broth ingredients, then autoclave
the solution at 121° for 15 min. Broth may also be
aseptically transferred into individual media bottles in
100- or 200-mL aliquots before sterilizing, and then
autoclaved and stored for later use. [NoTE—Can be
stored at 4° (allow broth to come to room tempera-
ture before use).]
Peptone diluent: Prepare a solution of 0.1% peptone’?
in water (w/v) and adjust to a pH of 7.0 with a solution
of lactic acid. Using an autoclave, steam sterilize the
solution at 121° for NLT 15 min, then allow to cool in
the unopened autoclave. Dispense into sterile contain-
ers as needed for preparing samples.
Sample preparation: Aseptically transfer 11.0 g of
sydeibouo-: sa
freeze-dried probiotic powder into a sterile stomacher
bag. Add 99 mL of previously sterilized (room tempera-
ture) Sample broth to the bag and blend at 230 rpm for
30 s in a stomacher. Hold the mixture at room temper-
ature for 30 min to allow rehydration of the freeze-
dried sample, then blend in the stomacher for an addi-
tional 30 s at 230 rpm. This is the primary 10-1 dilution.
Using sterilized, filtered pipet tips, make seria! dilutions
by aseptically transferring 1.0 mL of the primary 10~
dilution to sterile media bottles, each containing
99.0 mL of Peptone diluent (10-3 dilution). Repeat this
operation until the desired dilution series is obtained.
[Nott—The dilutions used in the Analysis should be
expected to contain 25-250 cfu/mL.] Shake the media
bottles for complete mixing before proceeding with
the Analysis.
Analysis: For each Sample preparation to be plated, pre-
pare Petri plates as follows. Using three sterile, filtered
‘1-mL pipet tips, aseptically transfer 1.0 mL of the Sam-
ple preparation separately into three appropriately la-
° Difco™ Lactobacilli MRS Broth, or equivalent are available from www.vwwr.
com or other chemical/microbiological suppliers.
1° Suitable peptone for microbiological analysis is available from BD Bacto™
(www.bd.com).
 4472 Bifidobacterium / Dietary Supplements USP 41

beled sterile 15- x 100-mm Petri plates, then pour IDENTIFICATION

about 15 mL of the 45° Agar medium into each plate,
flaming the lip of the bottle between pours. Place the
lid on each plate after adding the Agar medium, then
gently swirl the plates to mix the Sample preparation
and the Agar medium. [NoTe—Be careful to avoid spill-
age onto the lid of the dish when swirling the plates.]
Repeat this procedure for additional dilutions of the
Sample preparation. Prepare one blank plate that con-
tains only the Agar medium and a second blank plate in
which 1.0 mL of Peptone diluent has been mixed with
the Agar medium. Allow the plates to sit at room tem-
perature onalevel surface until the Agar medium solidi-
fies, then incubate the plates at 38° for 72 h under
anaerobic conditions."!
After 72 h of incubation, count the colonies and record
the results as viable cfu/g, taking into account the ap-
propriate dilution factor of the Sample preparation.
Only count plates containing 25-250 colonies. Deter-
mine the average plate count, in cfu/g.
Acceptance criteria: NLT 100% of the labeled viable
cell count, in cfu/g
CONTAMINANTS
[NoTe—The methods of microbial analysis included in this
section as examples represent currently accepted methods
commonly used in industry. Users may substitute other vali-
dated test methods for the methods in this section.]
e MICROBIAL ENUMERATION TESTS (2021): The total com-
bined molds and yeasts count does not exceed 10? cfu/
°Non-Lacic AciD BACTERIA: ISO International Standard
number 13559 (IDF 153), available from the International
Organization for Standardization (www.iso.org). The total
non-lactic acid bacteria count is less than 5 x 103 cfu/g.
e ABSENCE OF SPECIFIED MICROORGANISMS (2022), Test Proce-
dures, Test for Absence of Escherichia coli: |t meets the
requirements of the tests for absence of Escherichia coli. It
meets the requirements of the tests for absence of Salmo-
nella species in 28,
e Listeria: (See Food Chemicals Codex, Appendix XV.) It
meets the requirements of the tests for absence of Listeria
in 25g.
ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Preserve in high-barrier foil
laminate bags and store at or below 4°.
e LABELING: This ingredient should be labeled with the ge-
nus and species names, or genus, species, and strain
names and with the formulated enumeration in cfu/g (or
similar units). This monograph applies only to three
strains of Bifidobacterium animalis subsp. lactis—Bi-07, BI-
DS Monographs
© A. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST
Standard solution: 4 mg/mL of USP Powdered Bilberry
Extract RS in methanol. Centrifuge, and use the clear
supernatant.
Sample solution: 4 mg/mL of Powdered Bilberry Ex-
tract in methanol. Centrifuge, and use the clear
supernatant.
Adsorbent: Use suitable thin-layer chromatographic
plates coated with a layer of cellulose.
Application volume: 10 pL
Developing solvent system A: Glacial acetic acid, hy-
drochloric acid, and water (15:3:82)
Developing solvent system B: Glacial acetic acid and
water (6:4)
Analysis
Samples: Standard solution and Sample solution
Use a saturated chamber. Develop the chromatograms
using Developing solvent system A, and dry the plate
with the aid of a current of warm air. Develop the
chromatograms in the same direction using Develop-
pa some system B. Examine the plate under visible
light.
Acceptance criteria: The chromatogram of the Sample
solution exhibits three main red bands with R; values of
approximately 0.55, 0.65, and 0.70 that are similar in
position and color to the corresponding main bands in
the chromatogram of the Standard solution.
¢ B. The retention times of the anthocyanoside peaks in
the chromatogram of the Sample solution correspond to
those in the chromatogram of Standard solution C, as ob-
tained in the test for Content of Anthocyanosides and
Anthocyanidins. The peaks due to delphinidin-3-O-galac-
toside chloride and delphinidin-3-O-glucoside chloride
are the most intense peaks and are of similar intensity,
and each is more intense than the peak due to cyanidin-
3-O-glucoside chloride. The peaks due to cyanidin-3-O-
galactoside chloride, delphinidin-3-O-arabinoside chlo-
ride, and cyanidin-3-O-glucoside chloride are of similar
intensity. Each of the remaining anthocyanoside peaks is
of lower intensity than the peak due to cyanidin-3-O-
glucoside chloride.
COMPOSITION
e CONTENT OF ANTHOCYANOSIDES AND ANTHOCYANIDINS
Solvent: Methanol and hydrochloric acid (49:1)
Diluent: 85% phosphoric acid and water (1:9)
Solution A: Formic acid and water (1:9)
Solution B: Acetonitrile, methanol, formic acid, and
water (45:45:20:80)
Mobile phase: See Table 7.

04, and HNO19—and no other strains of Bifidobacterium

animalis subsp. lactis cultures. fable"
Time Solution A Solution B
(min) (%) (%)
0 93 Z
. 35 75 25
Powdered Bilberry Extract 45 35 Zz
46 0 100
700
DEFINITION 50 0
Powdered Bilberry Extract is prepared from the ripe fruits of
Vaccinium myrtillus L. (Fam. Ericaceae) using suitable sol- 31 93 Z
vents such as alcohol, methanol, or water or mixtures of 60 93 7
these solvents. The ratio of the starting plant material to
Powdered Extract is between 153:1 and 76:1. It contains Standard stock solution A: 0.4 mg/mL of USP Cy-
NLT 36.0% of anthocyanosides, calculated as cyanidin- anidin-3-O-glucoside Chloride RS in Solvent. [NoTE—Dis-
3-O-glucoside chloride, and NMT 1.0% of anthocyanidins, solve using sonication.]
calculated as cyanidin chloride; both are calculated on the Standard solution A: 0.08 mg/mL of USP Cyanidin-
anhydrous basis. 3-O-glucoside Chloride RS from Standard stock solution
A " Diluent. [NoTteE—This solution is stable for 48 h at
1! Suitable anaerobic systems are available from BD GasPak™ EZ Container 4°.
System (www.bd.com).
Standard stock solution B: 0.5 mg/mL of USP Cyanidin
Chloride RS in Solvent. [NoTE—Dissolve using
sonication.]
USP 41 Dietary Supplements / Bilberry 4473

Standard solution B: 0.01 mg/mL of USP Cyanidin Table 2 (Continued)

Chloride RS from Standard stock solution B in Diluent. Relative
[Nott—This solution is stable for 36 h at 4°] Retention
Standard solution C: Transfer 125 mg of USP Pow- Analyte Time
dered Bilberry Extract RS to a 100-mL volumetric flask,
add 25 mL of Solvent, sonicate to dissolve, and dilute Petunidin-3-O-galactoside chloride 1.08
with Diluent to volume. [NoTE—This solution is stable Cyanidin-3-O-arabinoside chloride 1.11
for 48 h at 4°.] Petunidin-3-O-glucoside chloride 1.24
Sample solution: Transfer 125 mg of Powdered Bilberry Peonidin-3-O-galactoside chloride 1.36
Extract to a 100-mL volumetric flask, add 25 mL of Sol- Petunidin-3-O-arabinoside chloride 139)
vent, sonicate to dissolve, and dilute with Diluent to
Peonidin-3-O-glucoside chloride 1.55
volume.
Chromatographic system Malvidin-3-O-galactoside chloride 1.58
(See Chromatography (621), System Suitability.) Peonidin-3-O-arabinoside chloride 1.67
[Note—Use deactivated silanized HPLC vials.] Malvidin-3-O-glucoside chloride 1.76
Mode: LC Malvidin-3-O-arabinoside chloride 1.91
Detector: UV-Vis 535 nm
Column: 4.6-mm x 25-cm; 5-m packing L1 Separately calculate the percentages of anthocyanidins
Temperature (see Table 3) in the portion of Powdered Extract taken:
Refrigerated autosampler: 4°
Column: 30+1° Result = (ru/rs) x (Cs/Cu) x 100
Flow rate: 1 mL/min
Injection size: 10 uL tu = peak response of each of the anthocyanidins
System suitability in the Sample solution
Samples: Standard solution A and Standard solution C ts = peak response of cyanidin chloride in Standard
Suitability requirements solution B
Chromatogram similarity: The chromatogram from Gs = concentration of USP Cyanidin Chloride RS in
Standard solution C is similar to the Reference Chro- Standard solution B (mg/mL)
matogram provided with USP Powdered Bilberry Ex- Cy = concentration of Powdered Extract in the
tract RS. Sample solution (mg/mL)
Resolution: NLT 0.8 between the delphinidin-3-O-
arabinoside, malvidin-3-O-galactoside, and petunidin- Table 3
3-O-arabinose peaks and NLT 1.0 for other compo- Relative
nents, Standard solution C
Retention
Tailing factor range: 0.8-2.0 for the cyanidin-3-O-
Analyte Time
glucoside chloride peak, Standard solution A
Relative standard deviation: NMT 2.0% for cy- Delphinidin chloride 1.28
anidin-3-O-glucoside chloride peak in repeated injec- Cyanidin chloride 1.82
tions, Standard solution A Petunidin chloride 2.08
Analysis Peonidin chloride 2.27
Samples: Standard solution A, Standard solution B, Malvidin chloride 2.30
Standard solution C, and Sample solution
Using the chromatogram of Standard solution C and the Acceptance criteria
Reference Chromatogram, identify the retention times Sum of all anthocyanosides: NLT 36.0% on the anhy-
of the peaks corresponding to the different anthocya- drous basis
nosides and anthocyanidins. The approximate relative Sum of all anthocyanidins: NMT 1.0% on the anhy-
retention times, relative to cyanidin-3-O-glucoside drous basis
chloride, are provided for the anthocyanosides in Table
2 and for the anthocyanidins in Table 3. CONTAMINANTS
Separately calculate the percentages of each anthocya-
noside (see Table 2) in the portion of Powdered Extract
taken: Delete the following: \~]
a)

Result = (ru/rs) x (Cs/Cu) x 100 °o HEAVY METALS, Method Ii (231): NMT 20 ppme (oriat1- =
Jan-2018) i)
ry = peak response of each of the anthocyanosides e ARTICLES OF BOTANICAL ORIGIN, General Method for Pesti- 3
re}
in the Sample solution cide Residues Analysis (561): Meets the requirements eo}=
rs = peak response of cyanidin-3-O-glucoside e MICROBIAL ENUMERATION TESTS (2021) i}
chloride in Standard solution A Total aerobic microbial count: NMT 104 cfu/g a}
Cs = concentration of USP Cyanidin-3-O-glucoside a combined yeasts and molds count: NMT 103 a
Pe
Chloride RS in Standard solution A (mg/mL) cfu/g
Cy = concentration of Powdered Extract in the e MICROBIOLOGICAL PROCEDURES FOR ABSENCE OF SPECIFIED MI-
Sample solution (mg/mL) CROORGANISMS (2022): It meets the requirements of the
= for absence of Salmonella species and Escherichia
Table 2
coli.
Relative SPECIFIC TESTS
Retention e ACID INSOLUBLE FRACTION
Analyte Time Sample: 5g of Powdered Extract finely ground
Delphinidin-3-O-galactoside chloride 0.61 Analysis: Transfer about 1 g to a 500-ml flask, add
Delphinidin-3-O-glucoside chloride 0.73
200 mL of 0.1 N hydrochloric acid, and shake vigor-
ously for 2 h. Pass the solution through a previously
Cyanidin-3-O-galactoside chloride 0.84 tared sintered-glass filter, wash the flask with 30 mL of
Delphinidin-3-O-arabinoside chloride 0.86 0.1 N hydrochloric acid, and transfer the washings to
Cyanidin-3-O-glucoside chloride 1.00 the filter. Wash the filter with 30 mL of 0.1 N hydro-
 4474 Bilberry / Dietary Supplements
chloric acid in 5-mL portions. Dry the filter for 3 h at
105°, cool in a desiccator, and weigh. Calculate the
percentage of the acid insoluble fraction.
Acceptance criteria: NMT 5%
e WATER DETERMINATION, Method Ia (921): NMT 4.5%, de-
termined on 0.5g
e RESIDUE ON IGNITION (281): NMT 3.0%, determined on
1.0g
© BOTANICAL EXTRACTS, Residual Solvents (565): Meets the
requirements
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in well-closed contain-
ers, protected from light and moisture, and store at con-
trolled room temperature.
e LABELING: The label states the Latin binomial and, follow-
ing the official name, the part of the plant from which
the article was prepared. It meets other labeling require-
ments under Botanical Extracts (565).
e USP REFERENCE STANDARDS (11)
USP Powdered Bilberry Extract RS
USP Cyanidin Chloride RS
USP Cyanidin-3-O-glucoside Chloride RS
Biotin—see Biotin General Monographs
Biotin Capsules—see Biotin Capsules
General Monographs
Biotin Tablets—see Biotin Tablets General
Monographs
Black Cohosh
DEFINITION
Black Cohosh consists of the dried rhizome and roots of
Actaea racemosa L. [Cimicifuga racemosa (L.) Nutt.]
(Ranunculaceae). It is harvested in the summer. It contains
NLT 0.4% of triterpene glycosides, calculated as 23-epi-
26-deoxyactein! (C37Hs6O10) on the dried basis.
DS Monographs
IDENTIFICATION
° A. THIN-LAYER CHROMATOGRAPHY
Standard solution A: 100 mg/mL of USP Powdered
Black Cohosh Extract RS in methanol
Standard solution B: 1 mg/mL each of USP Actein RS,
USP 2s-epi-26-Deaxyacietn RS, and isoferulic acid in
methano
Sample solution: Transfer 5 g of finely powdered Black
Cohosh to a screw-cap centrifuge tube, add 10 mL of a
mixture of alcohol and water (7:3), and heat on a
steam bath for 10 min. Centrifuge, and use the clear
supernatant.
Adsorbent: Chromatographic silica gel mixture with an
average particle size of 10-15 um (TLC plates)
Application volume: 10 ul
Developing solvent system: Use the upper phase of a
mixture of butyl alcohol, glacial acetic acid, and water
(5:1:4).
Derivatization reagent: Methanol, glacial acetic acid,
sulfuric acid, and p-anisaldehyde (85: 10: 5: 0.5).
[Note—Store in a refrigerator. The reagent is colorless;
discard if color appears.]
1 23-epi-26-Deoxyactein is sometimes referred to as 27-deoxyactein.
USP 41
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Develop the chromatograms until the solvent front has
moved about 15 cm, and dry the plate in a stream of
air. Examine the plate under UV light at 365 nm. Treat
the plate with Derivatization reagent, heat at 100° for
5 min, and examine in white light.
Acceptance criteria: Under UV light at 365 nm, the
chromatogram of the Sample solution exhibits main
zones similar in position and color to the main zones of
Standard solution A. In the upper third of the plate, the
Sample solution exhibits a blue fluorescent zone at the
level of the zone due to isoferulic acid of Standard solu-
tion B. Under white light, the Sample solution exhibits
main zones similar in position and color to the main
zones of Standard solution A. Standard solution B exhibits
red-violet zones due to actein and 23-epi-26-deoxy-
actein. The Sample solution exhibits several greenish-
brown spots in the lower third of the plate and several
violet zones above; two of these violet zones occur at Rr
values similar to those due to actein and 23-epi-26-de-
oxyactein of Standard solution B.
e B. HPTLC For ARTICLES OF BOTANICAL ORIGIN (203)
Adsorbent: Chromatographic silica gel mixture with an
average particle size of 5 um (HPTLC plates)
Standard solution A: 0.5 mL of Standard solution A pre-
pared in Identification test A, diluted with methanol to
2mL
Standard solution B: 1.0 mL of Standard solution B pre-
pared in Identification test A, diluted with methanol to
smb
Sample solution: Transfer 0.5 g of Black Cohosh, finely
powdered, to a screw-cap tube, add 5 mL of methanol,
sonicate for 10 min, and filter into a 10-mL volumetric
flask. Wash the residue on the filter paper four times,
using 1 mL of methanol for each washing; add the
washings to the volumetric flask; and dilute with meth-
anol to volume.
Application volume: 2 uL as 8-mm bands
Developing solvent system: Toluene, ethyl formate,
and formic acid (5:3:2)
Derivatization reagent: Proceed as directed for /dentifi-
cation test A.
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Develop the chromatograms until the solvent front has
moved about two-thirds of the length of the plate,
and dry the plate in a current of air. Treat the plate
with Derivatization reagent, heat at 100° for 5 min,
and examine in white light.
Acceptance criteria: The Sample solution exhibits main
zones similar in position and color to the main zones of
Standard solution A. Standard solution B exhibits red-vio-
let zones due to actein and 23-epi-26-deoxyactein at Rr
values of about 0.5 and 0.4, respectively. The Sample
solution exhibits zones similar in color and Rr values to
those due to actein and 23-epi-26-deoxyactein of Stan-
dard solution B.
e C. The Sample solution exhibits peaks for cimiracemoside
A, 26-deoxycimicifugoside, (265)-actein, 23-epi-26-deoxy-
actein, cimigenol-arabinoside, and cimigenol-xyloside at
retention times corresponding to these compounds in
the Standard solution, as obtained in the test for Content
of Triterpene Glycosides. The ratio of the peak areas of
cimigenol-arabinoside to cimigenol-xyloside is NLT 0.4
(distinction from Cimicifuga foetida).
COMPOSITION
@ CONTENT OF TRITERPENE GLYCOSIDES
Standard solution: Dissolve a quantity of USP Pow-
dered Black Cohosh Extract RS in methanol with shak-
ing for 1 min, and dilute with methanol to obtain a
solution having a known concentration of 30 mg/mL.
USP 41 Dietary Supplements / Black Cohosh 4475

Pass through a membrane filter of 0.45-um or finer identify the retention times of the peaks corresponding
pore size. to the triterpene glycosides. The approximate relative
23-epi-26-Deoxyactein standard solutions: Dissolve retention times of the triterpene glycosides are pro-
USP 23-epi-26-Deoxyactein RS in methanol with shaking vided in Table 2.
for 1 min. Dilute quantitatively, and stepwise if neces-
sary, to obtain solutions having concentrations of 500, Table 2
100, 50, 25, and 12.5 ug/mL. Pass through a mem-
brane filter of 0.45-um or finer pore size. Relative
System suitability solution: 0.1 mg/mL each of USP Retention
Actein RS and USP 23-epi-26-Deoxyactein RS in Compound Time
methanol Cimicifugoside H-1 0.61
Sample solution: Accurately weigh about 750 mg of Cimiracemoside A 0.78
Black Cohosh, finely powdered, and place in a 20-mL (26R)-Actein 0.94
PaEScapped centrifuge tube. Add 15 mL of methanol, 26-Deoxycimicifugoside 0.96
sonicate for 30 min, centrifuge, and retain the superna-
tant. Repeat the extraction twice. Evaporate the com- (265S)-Actein 0.98
bined extracts under vacuum at 45°-50°. Dissolve the 23-epi-26-Deoxyactein 1.00
residue in methanol, and quantitatively transfer to a Acetyl-shengmanol-xyloside 1.03
10-mL volumetric flask. Dilute with methanol to vol- Cimigenol-arabinoside 1.08
ume, and pass through a membrane filter of 0.45-1m Cimigenol-xyloside (cimicifugoside) 1.13
or finer pore size. 26-Deoxyactein 1.22
Solution A: 0.05% Trifluoroacetic acid in water
Solution B: Acetonitrile 25-Acetyl-cimigenol-arabinoside 1.60
Mobile phase: See Table 7. (245)-25-Acetyl-cimigenol-xyloside 1.64
25-O-Methyl-cimigenol-arabinoside 1.90
Table 1 25-O-Methyl-cimigenol-xyloside 1.93

Time Water Solution A Solution B Plot the logarithms of the peak areas against the loga-
(min) (%) (%) (%) rithms of the concentrations, in g/mL, of the 23-epi-
0 0 80 20 26-Deoxyactein standard solutions, and establish the
8 0 80 20 calibration curve by least-squares regression. The cor-
15 68 0 32 relation coefficient for the regression line is NLT 0.995.
From the plot, determine the concentration, C, in
55 36 0 64
g/mL, of the relevant analytes in the Sample solution.
65 3: 0 95 Separately calculate the percentages of the individual
70 5 0 95 triterpene glycosides in Table 2 as 23-epi-26-deoxy-
85 0 80 20 cee (C37Hs6O10) in the portion of Black Cohosh
taken:
Chromatographic system
(See Chromatography (621), System Suitability.) Result = (Vx O/(F
x W) x 100
Mode: LC
Detector: Evaporative light-scattering Vv final volume of the Sample solution (mL)
oil

[Note—The Detector is set up according to the manu- Cc concentration of the relevant analyte in the
facturer’s instructions in order to achieve a signal-to- Sample solution (g/mL)
noise ratio of NLT 10 for the 12.5-g/mL 23-epi-26-De- fe = factor to convert mg to pg, 1000 g/mg
oxyactein standard solution.] Ww = weight of Black Cohosh taken to prepare the
Column: 4.6-mm x 25-cm; 5-uum packing L1 Sample solution (mg)
Column temperature: 35° Calculate the percentage of triterpene glycosides in the
Flow rate: 1.6 mL/min portion of Black Cohosh taken by adding the
Injection volume: 20 uL percentages of the individual analytes.
System suitability Acceptance criteria: NLT 0.4% on the dried basis
sydeibouo-: Sa

Samples: System suitability solution, Standard solution,

and 100-11g/mL 23-epi-26-Deoxyactein standard solution
Suitability requirements
Chromatogram similarity: The chromatogram of the
Standard solution is similar to the reference chromato-
gram provided with the lot of USP Powdered Black
Cohosh Extract RS being used.
Resolution: NLT 1.0 between the (26S)-actein and
the 23-epi-26-deoxyactein peaks, System suitability
solution
Tailing factor: NMT 2.0 for the 23-epi-26-deoxy-
actein peak, 100-1g/mL 23-epi-26-Deoxyactein stan-
dard solution
Relative standard deviation: NMT 2.0% of the loga-
rithm of the area response of the 23-epi-26-deoxy-
actein peak in repeated injections, 100-ug/mL 23-epi-
26-Deoxyactein standard solution
Analysis
Samples: System suitability solution, Standard solution,
23-epi-26-Deoxyactein standard solutions, and Sample
solution
Using the chromatogram of the Standard solution and
the reference chromatogram provided with the lot of
USP Powdered Black Cohosh Extract RS being used,
CONTAMINANTS
© ARTICLES OF BOTANICAL ORIGIN, Limits of Elemental Impuri-
ties (561): Meets the requirements
e ARTICLES OF BOTANICAL ORIGIN, Pesticide Residue Analysis
(561): Meets the requirements
© MICROBIAL ENUMERATION TESTS (2021): The total aerobic
microbial count does not exceed 105 cfu/g, the total
combined molds and yeasts count does not exceed 103
cfu/g, and the bile-tolerant Gram-negative bacteria count
does not exceed 103 cfu/g.
© ABSENCE OF SPECIFIED MICROORGANISMS (2022): Meets the
requirements of the tests for the absence of Salmonella
species and Escherichia coli
SPECIFIC TESTS
e BOTANICAL CHARACTERISTICS
Macroscopic: The Black Cohosh rhizome is dark brown,
longitudinally grooved, rough, strongly knotty, and
somewhat curled and irregular. It is 15 cm long and up
to 2.5 cm thick. The upper surface is covered with nu-
merous round scars of the earlier stalks; laterally, it is
clearly curled, and the lower surface is covered with
thin, longitudinally grooved, dark brown, easily breaka-
 4476 Black Cohosh / Dietary Supplements
ble roots. The fracture is horny and fibrous. The trans-
verse cut shows a thin outer bark surrounding a ring of
numerous pale, narrow wedges of vascular tissue alter-
nating with dark medullary rays and a large central
pith. Black Cohosh roots are dark brown, between 1
and 3 mm in diameter, brittle, nearly cylindrical or ob-
tusely quadrangular, and longitudinally wrinkled. The
fracture is short. The transverse cut shows a distinct
cambium line separating a wide outer bark from a cen-
tral region composed of three to six wedges of lignified
xylem tissue united by their apices and separated by
broad nonlignified medullary rays.
Microscopic: In a surface view, suberous epidermal cells
are tabular with moderately thickened walls. The paren-
chymatous cortex is filled with starch. Xylem wedges
are as and composed of numerous small vessels
with bordered pits or reticulately thickened walls, thin-
walled fibers, and xylem parenchyma. The parenchyma
of the pith is unlignified. Medullary rays are filled with
starch granules, which are spherical or polygonal and
are mostly simple or two to three compounded but can
be up to six compounded. Individual starch granules
are between 3 and 15 um in diameter, each with a
somewhat central slit-shaped hilum.
ARTICLES OF BOTANICAL ORIGIN, Foreign Organic Matter
(561): NMT 2.0% of foreign organic matter, and NMT
5.0% of stem bases
ARTICLES OF BOTANICAL ORIGIN, A/cohol-Soluble Extractives,
Method 2 (561): NLT 8.0%, using a mixture of alcohol
and water (1:1) instead of alcohol.
Loss ON DRYING (731)
Sample: 1.0g of Black Cohosh
Analysis: Dry the Sample at 105° for 2 h.
Acceptance criteria: NMT 12.0%
ARTICLES OF BOTANICAL ORIGIN, Total Ash (561): NMT
10.0%
e ARTICLES OF BOTANICAL ORIGIN, Acid-/nsofuble Ash (561):
NMT 4.0%
e B. HPTLC FoR ARTICLES OF BOTANICAL ORIGIN (203)
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in a well-closed, light-
resistant container. Protect from moisture, and store at
room temperature.
e LABELING: The label states the Latin binomial and, follow-
ing the official name, the parts of the plant contained in
the article. Dosage forms prepared with this article
should bear the following statement: Discontinue use
and consult a healthcare practitioner if you havea liver
disorder or develop symptoms of liver trouble, such as
abdominal pain, dark urine, or jaundice.
e USP REFERENCE STANDARDS (11)
DS Monographs
USP Actein RS
USP Powdered Black Cohosh Extract RS
USP 23-epi-26-Deoxyactein RS
Powdered Black Cohosh
DEFINITION
Powdered Black Cohosh is Black Cohosh reduced to a pow-
der or a very fine powder. It contains NLT 0.4% of
triterpene glycosides, calculated as 23-epi-26-deoxyactein!
(C37Hs6O10) on the dried basis.
IDENTIFICATION
e A. THIN-LAYER CHROMATOGRAPHY
Standard solution A: 100 mg/mL of USP Powdered
Black Cohosh Extract RS in methanol
Standard solution B: 1 mg/ml each of USP Actein RS,
USP pene RS, and isoferulic acid in
methano
1 23-epi-26-Deoxyactein is sometimes referred to as 27-deoxyactein.
USP 41
Sample solution: Transfer 5 g of Powdered Black Co-
hosh to a screw-cap centrifuge tube, add 10 mL of a
mixture of alcohol and water (7:3), and heat on a
steam bath for 10 min. Centrifuge, and use the clear
supernatant.
Adsorbent: Chromatographic silica gel mixture with an
average particle size of 10-15 um (TLC plates)
Application volume: 10 pL
Developing solvent systems Use the upper phase of a
Gaye of butyl alcohol, glacial acetic acid, and water
5:1:4).
Derivatization reagent: Methanol, glacial acetic acid,
sulfuric acid, and p-anisaldehyde (85: 10: 5: 0.5).
[Note—Store in a refrigerator. The reagent is colorless;
discard if color appears.]
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Develop the chromatograms until the solvent front has
moved about 15 cm, and dry the plate in a stream of
air. Examine the plate under UV light at 365 nm. Treat
the plate with Derivatization reagent, heat at 100° for
5 min, and examine in white light.
Acceptance criteria: Under UV light at 365 nm, the
cnromatograny of the Sample solution exhibits main
zones similar in position and color to the main zones of
Standard solution A. In the upper third of the plate, the
Sample solution exhibits a blue fluorescent zone at the
level of the zone due to isoferulic acid of Standard solu-
tion B. Under white light, the Sample solution exhibits
main zones similar in position and color to the main
zones of Standard solution A. Standard solution B exhibits
red-violet zones due to actein and 23-epi-26-deoxy-
actein. The Sample solution exhibits several greenish-
brown spots in the lower third of the plate and several
violet zones above; two of these violet zones occur at Rr
values similar to those due to actein and 23-epi-26-de-
oxyactein of Standard solution B.
Standard solution A: 0.5 mL of Standard solution A pre-
pared in Identification test A, diluted with methanol to
2mL
Standard solution B: 1.0 mL of Standard solution B pre-
pared in Identification test A, diluted with methanol to
smb
Sample solution: Transfer 0.5 g of Powdered Black Co-
hosh to a screw-cap tube, add 5 mL of methanol, soni-
cate for 10 min, and filter into a 10-mL volumetric flask.
Wash the residue on the filter paper four times, using
1 mL of methanol for each washing; add the washings
to the volumetric flask; and dilute with methanol to
volume.
Adsorbent: Chromatographic silica gel mixture with an
average particle size of 5 um (HPTLC plates)
Application volume: 2 wl as 8-mm bands
Developing solvent system: Toluene, ethyl formate,
and formic acid (5:3:2)
Derivatization reagent: Proceed as directed for Identifi-
cation test A.
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Develop the chromatograms until the solvent front has
moved about two-thirds of the length of the plate,
and dry the plate with in a current of air. Treat the
plate with Derivatization reagent, heat at 100° for 5
min, and examine in white light.
Acceptance criteria: The Sample solution exhibits main
zones similar in position and color to the main zones of
Standard solution A. Standard solution B exhibits red-vio-
let zones due to actein and 23-epi-26-deoxyactein at R-
values of about 0.5 and 0.4, respectively. The Sample
solution exhibits zones similar in color and Ry values to
those due to actein and 23-epi-26-deoxyactein of Stan-
dard solution B.
USP 41 Dietary Supplements / Black Cohosh 4477

e C. The Sample solution exhibits peaks for cimiracemoside Resolution: NLT 1.0 between the (26S)-actein and
A, 26-deoxycimicifugoside, (265)-actein, 23-epi-26-deoxy- the 23-epi-26-deoxyactein peaks, System suitability
actein, cimigenol-arabinoside, and cimigenol-xyloside at solution
retention times corresponding to those compounds in Tailing factor: NMT 2.0 for the 23-epi-26-deoxy-
the Standard solution, as obtained in the test for Content actein peak, 100-41g/mL 23-epi-26-Deoxyactein stan-
of Triterpene Glycosides. The ratio of the peak areas of dard solution
cimigenol-arabinoside to cimigenol-xyloside is NLT 0.4 Relative standard deviation: NMT 2.0% of the loga-
(distinction from Cimicifuga foetida). rithm of the area responses for replicate injections,
100-ug/mL 23-epi-26-Deoxyactein standard solution
COMPOSITION Analysis
© CONTENT OF TRITERPENE GLYCOSIDES Samples: System suitability solution, Standard solution,
Standard solution: Dissolve a quantity of USP Pow- 23-epi-26-Deoxyactein standard solutions, and Sample
dered Black Cohosh Extract RS in methanol with shak- solution
ing for 1 min, and dilute with methanol to obtain a Using the chromatogram of the Standard solution and
solution having a known concentration of 30 mg/mL. the reference chromatogram provided with the lot of
Pass through a membrane filter of 0.45-um or finer USP Powdered Black Cohosh Extract RS being used,
pore size. identify the retention times of the peaks corresponding
23-epi-26-Deoxyactein standard solutions: Dissolve to the triterpene glycosides. The approximate relative
USP 23-epi-26-Deoxyactein RS in methanol with shaking retention times of the triterpene glycosides are pro-
for 1 min. Dilute quantitatively, and stepwise if neces- vided in Table 2.
sary, to obtain solutions having concentrations of 500,
100, 50, 25, and 12.5 g/mL. Pass through a mem-
Table 2
brane filter of 0.45-um or finer pore size.
System suitability solution: 0.1 mg/mL each of USP Relative
Actein RS and USP 23-epi-26-Deoxyactein RS in Retention
methanol Name Time
Sample solution: Accurately weigh about 750 mg of Cimicifugoside H-1 0.61
Powdered Black Cohosh, and place in a 20-mL PTFE- Cimiracemoside A 0.78
capped centrifuge tube. Add 15 mL of methanol, soni- (26R)-Actein 0.94
cate for 30 min, centrifuge, and retain the supernatant.
Repeat the extraction twice. Evaporate the combined 26-Deoxycimicifugoside 0.96
extracts under vacuum at 45°-50°. Dissolve the residue (265)-Actein 0.98
in methanol, and quantitatively transfer to a 10-mL vol- 23-epi-26-Deoxyactein 1.00
umetric flask. Dilute with methanol to volume, and pass Acetyl-shengmanol-xyloside 1.03
through a membrane filter of 0.45-11m or finer pore Cimigenol-arabinoside 1.08
size. Cimigenol-xyloside (cimicifugoside) 1.13
Solution A: 0.05% Trifluoroacetic acid in water
Solution B: Acetonitrile 26-Deoxyactein 1.22
Mobile phase: See Table 1. 25-Acetyl-cimigenol-arabinoside 1.60
(245)-25-Acetyl-cimigenol-xyloside 1.64
Table 1 25-O-Methyl-cimigenol-arabinoside 1.90
25-O-Methyl-cimigenol-xyloside 1.93
Time Water Solution A Solution B
(min) (%) (%) (%) Plot the logarithms of the peak areas against the loga-
0 oO 80 20 rithms of the concentrations, in g/mL, of the 23-epi-
8 0 80. 20 26-Deoxyactein standard solution, and establish the cali-
15 68 0 32 bration curve by least-squares regression. The correla-
55 36 0 64 tion coefficient for the regression line is NLT 0.995.
65 5 0 95 From the plot, determine the concentration, C, in
lig/mL, of the relevant analytes in the Sample solution.
70 5 0 95 Separately calculate the percentages of the individual
NRG!

85 0 80 20 triterpene glycosides in Table 2 as 23-epi-26-deoxy-

actein (C37Hs6O10) in the portion of Powdered Black
Chromatographic system Cohosh taken:
(See Chromatography (621), System Suitability.)
SiTe #1olel blo

Mode: LC Result = (Vx C/(F

Detector: Evaporative light-scattering
[Nott—The Detector is set up according to the manu-
facturer’s instructions in order to achieve a signal-to-
noise ratio of NLT 10 for the 12.5-~g/mL 23-epi-26-De-
oxyactein standard solution.]
Column: 4.6-mm x 25-cm; 5-"um packing L1
Column temperature: 35°
Flow rate: 1.6 mL/min
Injection volume: 20 uL
System suitability
Samples: System suitability solution, Standard solution,
and 100-ug/mL 23-epi-26-Deoxyactein standard solution
Suitability requirements
Chromatogram similarity: The chromatogram of the
Standard solution is similar to the reference chromato-
gram provided with the lot of USP Powdered Black
Cohosh Extract RS being used.
x W) x 100
Vv = volume of the Sample solution (mL)
Cc = concentration of the relevant analyte in the
Sample solution (ug/mL)
F = factor to convert mg to ug, 1000 1g/mg
w = weight of Powdered Black Cohosh taken to
prepare the Sample solution (mg)
Calculate the percentage of triterpene glycosides in the
portion of Powdered Black Cohosh taken by adding
the percentages of the individual analytes.
Acceptance criteria: NLT 0.4% on the dried basis
CONTAMINANTS
¢ ARTICLES OF BOTANICAL ORIGIN, Limits of Elemental Impuri-
ties (561): Meets the requirements
e ARTICLES OF BOTANICAL ORIGIN, Pesticide Residue Analysis
(561): Meets the requirements
e MICROBIAL ENUMERATION TESTS (2021): The total aerobic
microbial count does not exceed 105 cfu/g; the total
 4478 Black Cohosh / Dietary Supplements
combined molds and yeasts count does not exceed 103
cfu/g; and the bile-tolerant Gram-negative bacteria count
does not exceed 103 cfu/g.
e ABSENCE OF SPECIFIED MICROORGANISMS (2022): Meets the
requirements of the tests for the absence of Salmonella
species and Escherichia coli
SPECIFIC TESTS
© BOTANICAL CHARACTERISTICS
Macroscopic: The material is a light to dark brown
powder, is odorless or has a slight odor, and has an
acrid or bitter taste.
Microscopic: It shows numerous starch granules with
concentric striations,simple or compound. The individ-
ual granules are spherical or more or less polygonal and
are between 3 and 15 um in diameter, each with a
somewhat central slit-shaped hilum. Vessels with bor-
dered pits occur, as do lignified fibers. Reddish to
brown fragments of suberized epidermis with more or
less tabular cells occur.
ARTICLES OF BOTANICAL ORIGIN, Alcohol-Soluble Extractives,
Method 2 (561): NLT 8.0%, using a mixture of alcohol
and water (1:1) instead of alcohol
Loss ON DRYING (731)
Sample: 1g of Powdered Black Cohosh
Analysis: Dry the Sample at 105° for 2 h.
Acceptance criteria: NMT 12.0%
ARTICLES OF BOTANICAL ORIGIN, Tota! Ash (561): | NMT
10.0%
ARTICLES OF BOTANICAL ORIGIN, Acid-insoluble Ash (561):
NMT 4.0%
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in well-closed, light-
resistant containers, and protect from moisture.
e LABELING: The label states the Latin binomial and, follow-
ing the official name, the parts of the plant from which
the article was derived. Dosage forms prepared with this
article should bear the following statement: Discontinue
use and consult a healthcare practitioner if you have a
liver disorder ordevelop symptoms of liver trouble, such
as abdominal pain, dark urine, or jaundice.
e USP REFERENCE STANDARDS (11)
USP Actein RS
USP Powdered Black Cohosh Extract RS
USP 23-epi-26-Deoxyactein RS
Powdered Black Cohosh Extract
DS Monographs
DEFINITION
Powdered Black Cohosh Extract is prepared from Black Co-
hosh by extraction with hydroalcoholic mixtures or other
suitable solvents. It contains NLT 90.0% and NMT
110.0% of the labeled amount of triterpene glycosides,
calculated as 23-epi-26-deoxyactein (C37Hs6O10) on the
dried basis.
IDENTIFICATION
e A. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST
Standard solution A: 100 mg/mL of USP Powdered
Black Cohosh Extract RS in methanol
Standard solution B: 1 mg/mL each of USP Actein RS,
USP 23-epi-26-Deoxyactein RS, and isoferulic acid in
methano
Sample solution: Shake a quantity of Powdered Ex-
tract, equivalent to 25 mg of triterpene glycosides, in
10 mL of methanol. Allow to stand for 15 min before
use.
Adsorbent: Chromatographic silica gel mixture with an
average particle size of 10-15 um (TLC plates)
USP 41
Application volume: 10 wL
Developing solvent system: Use the upper phase of a
Cie. of butylalechal, glacial acetic acid, and water
5:1:4).
Spray reagent: Methanol, glacial acetic acid, sulfuric
acid, and p-anisaldehyde (85:10:5:0.5). [NoTE—Store in
a refrigerator. The reagent is colorless; discard if color
appears.]
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Develop the chromatograms until the solvent front has
moved about 15 cm, and dry the plate with the aid
of a current of air.
Acceptance criteria: Examine the plate under UV light
at 365 nm. The chromatogram of the Sample solution
exhibits main zones similar in position and color to the
main zones in the chromatogram of Standard solution A.
In the upper third of the plate, the chromatogram of
the Sample solution exhibits a blue fluorescent zone at
the level of the zone due to isoferulic acid in the chro-
matogram of Standard solution B, Spray the plate with
Spey. reagent, heat at 100° for 5 min, and examine in
aylight. The chromatogram of the Sample solution ex-
hibits main zones similar in position and color to the
main zones in the chromatogram of Standard solution A.
The chromatogram of Standard solution B exhibits red-
violet zones due to actein and 23-epi-26-deoxyactein.
The chromatogram of the Sample solution exhibits sev-
eral greenish-brown spots in the lower third of the plate
and several violet zones above; two of these violet
zones occur at Rr values similar to those due to actein
and 23-epi-26-deoxyactein in the chromatogram of
Standard solution B.
e B. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST
Standard solution A: 0.5 mL of Standard solution A pre-
pared in Identification test A, diluted with methanol to
2mL
Standard solution B: 1.0 mL of Standard solution B pre-
pared in Identification test A, diluted with methanol to
Smt
Sample solution: Dilute 1 mL of the Sample solution
prepared in Identification test A with methanol to
10 mL.
Adsorbent: Chromatographic silica gel mixture with an
average particle size of 5 um (HPTLC plates)
Application volume: 2 ul as an 8-mm band
Developing solvent system: Toluene, ethyl formate,
and formic acid (5:3:2)
Spray reagent: Proceed as directed for Identification
test A.
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Develop the chromatograms until the solvent front has
moved about two-thirds of the length of the plate,
and dry the plate with the aid of a current of air.
Spray the plate with Spray reagent, heat at 100° for 5
min, and examine in daylight.
Acceptance criteria: The chromatogram of the Sample
solution exhibits main zones similar in position and
color to the main zones in the chromatogram of Stan-
dard solution A. The chromatogram of Standard solution
B exhibits red-violet zones due to actein and 23-epi-
26-deoxyactein at Rr values of about 0.5 and 0.4, re-
spectively. The chromatogram of the Sample solution
exhibits zones similar in color and R; values to those
due to actein and 23-epi-26-deoxyactein in the chro-
matogram of Standard solution B.
e C. The chromatogram of the Sample solution exhibits
peaks for cimiracemoside A, 26-deoxycimicifugoside,
(265) actein, 23-epi-26-deoxyactein, cimigenol-arabino-
side, and cimigenol-xyloside at retention times corre-
sporeing to those compounds in the chromatogram of
the Standard solution, as obtained in the test for Content
USP 41 Dietary Supplements / Black Cohosh 4479

of Triterpene Glycosides. The ratio of the peak areas of Analysis

cimigenol-arabinoside to cimigenol-xyloside is NLT 0.4 Samples: System suitability solution, Standard solution,
(distinction from Cimicifuga foetida). 23-epi-26-Deoxyactein standard solutions, and Sample
solution
COMPOSITION Using the chromatogram of the Standard solution and
© CONTENT OF TRITERPENE GLYCOSIDES the Reference Chromatogram provided with the lot
Standard solution: Dissolve a quantity of USP Pow- of USP Powdered Black Cohosh Extract RS, identify
dered Black Cohosh Extract RS in methanol with shak- the retention times of the peaks corresponding to the
ing for 1 min, and dilute with methanol to obtain a triterpene glycosides. The approximate relative reten-
solution having a known concentration of 30 mg/mL. tion times of the triterpene glycosides are provided in
Pass through a membrane filter of 0.45-m or finer Table 2.
pore size.
23-epi-26-Deoxyactein standard solutions: Dissolve
USP 23-epi-26-Deoxyactein RS in methanol with shaking Table 2
for 1 min. Dilute quantitatively, and stepwise if neces- Relative
sary, to obtain solutions having known concentrations Retention
of 500, 100, 50, 25, and 12.5 g/mL. Pass through a Name Time
membrane filter of 0.45-11m or finer pore size. Cimicifugoside H-1 0.61
System suitability solution: 0.1 mg/mL each of USP Cimiracemoside A 0.78
Actein RS and USP 23-epi-26-Deoxyactein RS in
(26R)-Actein 0.94
methanol
Sample solution: Transfer a quantity of Powdered Ex- 26-Deoxycimicifugoside 0.96
tract, equivalent to 7.5 mg of triterpene glycosides, to a (265)-Actein 0.98
10-mL volumetric flask, add 7 mL of methanol, and 23-epi-26-Deoxyactein 1.00
sonicate for 30 min. Dilute with methanol to volume. Acetyl-shengmanol-xyloside 1.03
Centrifuge, or pass througha filter of 0.45-~m or finer Cimigenol-arabinoside 1.08
pore size.
Cimigenol-xyloside (cimicifugoside) 1.13
Solution A: 0.05% trifluoroacetic acid in water
Solution B: Acetonitrile 26-Deoxyactein 1.22
Mobile phase: See Table 7. 25-Acetyl-cimigenol-arabinoside 1.60
(245)-25-Acetyl-cimigenol-xyloside 1.64
Table 1 25-O-Methyl-cimigenol-arabinoside 1.90
25-O-Methyl-cimigenol-xyloside 1:93
Time Water Solution A Solution B
(min) (%) (%) (%) Plot the logarithms of the peak area responses versus
0 9 80 20 the logarithms of the concentrations, in tug/mL, of
8 0 80 20 the 23-epi-26-Deoxyactein standard solutions, and de-
15 68 ‘0 32 termine the regression lineusing a least-squares anal-
55 36 0 64 ysis. The correlation coefficient for the regression line
65 5 0 95 is NLT 0.995. From the graphs so obtained, deter-
mine the concentration, C, in g/mL, of the relevant
70 5 0 95;
analyte in the Sample solution. Separately calculate
85 oO 80 20 the percentages of cimicifugoside H-1, cimiracemo-
side A, (26R)-actein, 26-deoxycimicifugoside, (265)-
Chromatographic system actein, 23-epi-26-deoxyactein, acetyl-shengmanol-x-
(See Chromatography (621), System Suitability.) yloside, cimigenol-arabinoside, cimigenol-xyloside
Mode: LC
Detector: Evaporative light-scattering
(cimicifugoside), 26-deoxyactein, 25-acetyl-cimigeno-
l-arabinoside, (245)-25-acetyl-cimigenol-xyloside,
[Note—The detector is set up according to the manu- 25-O-methyl-cimigenol-arabinoside, and 25-O-
facturer’s instruction in order to achieve a signal-to- methyl-cimigenol-xyloside as 23-epi-26-deoxyactein
noise ratio of NLT 10 for the 12.5-ug/mL 23-epi-26-De-
(C37Hs6Or0) in the portion of Extract taken:
oxyactein standard solution.]
sydesbouo=: sa

Column: 4.6-mm x 25-cm; 5-um packing L1 Result = (Vx O/(F

Column temperature: 35°
Flow rate: 1.6 mL/min
Injection size: 20 uL
System suitability
Samples: System suitability solution, Standard solution,
and 100-ug/mL 23-epi-26-Deoxyactein standard solution
Suitability requirements
Chromatogram similarity: The chromatogram of the
Standard solution is similar to the Reference Chromat-
ogram provided with the lot of USP Powdered Black
Cohosh Extract RS being used.
Resolution: NLT 1.0 between the (265)-actein and
the 23-epi-26-deoxyactein peaks, System suitability
solution
Tailing factor: NMT 2.0 for the 23-epi-26-deoxy-
actein peak, 100-ug/mL 23-epi-26-Deoxyactein stan-
dard solution
Relative standard deviation: NMT 2.0% of the loga-
rithm of the area response of the 23-epi-26-deoxy-
actein peak in repeated injections, 100-11g/mL 23-epi-
26-Deoxyactein standard solution
x W) x 100
Vv = volume of the Sample solution (mL)
C = concentration of the relevant analyte in the
Sample solution (ug/mL)
Mi = factor to convert mg to jug, 1000 j1g/mg
= weight of the Powdered Extract taken to
prepare the Sample solution (mg)
Calculate the percentage of the labeled amount of
triterpene aiysesides in the portion of Extract taken
by adding all of the percentages calculated for
individual analytes.
Acceptance criteria: 90.0%-110.0% on the dried basis
CONTAMINANTS
Delete the following:
°o HEAVY METALs (231): NMT 10 PPMe CFfciai t-jan-2018)
e MICROBIAL ENUMERATION TESTS (2021): It meets the re-
quirements of the tests for absence of Salmonella species
and Escherichia coli. The total bacterial count does not
 4480 Black Cohosh / Dietary Supplements
exceed 104 cfu/g, and the total combined molds and
yeasts count does not exceed 103 cfu/g.
e OTHER REQUIREMENTS: It meets the requirements under
Botanical Extracts (565), Pesticide Residues.
SPECIFIC TESTS
e Loss ON DRYING (731); NMT 5.0%
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in tight, light-resistant
containers, and store in a cool place.
e LABELING: It meets the requirements under Botanical Ex-
tracts (565). Label it to indicate the content of triterpene
glycosides, in percentage, calculated as 23-epi-26-deoxy-
actein. Dosage forms prepared with this article should
bear the following statement: “Discontinue use and con-
sult a healthcare practitioner if you havea liver disorder
or develop symptoms of liver trouble, such as abdominal
pain, dark urine, or jaundice.”
oe USP REFERENCE STANDARDS (11)
USP Actein RS
USP Powdered Black Cohosh Extract RS
USP 23-epi-26-Deoxyactein RS
Black Cohosh Fluidextract
DEFINITION
Black Cohosh Fluidextract is prepared from Black Cohosh by
extraction with hydroalcoholic mixtures or isopropa-
nol-water mixtures. Each mL contains the extracted con-
stituents of 1 g of the plant material. It contains NLT
90.0% and NMT 110.0% of the labeled amount of
triterpene glycosides, calculated as 23-epi-26-deoxyactein
(C37Hs6O10).
IDENTIFICATION
e A. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST
Standard solution A: 100 mg/mL of USP Powdered
Black Cohosh Extract RS in methanol
Standard solution B: 1 mg/ml each of USP Actein RS,
USP 23-epi-26-Deoxyactein RS, and isoferulic acid in
methano!
Sample solution: Fluidextract
Adsorbent: Chromatographic silica gel mixture with an
average particle size of 10-15 um (TLC plates)
Application volume: 10 pL
Developing solventsystem: Use the upper phase of a
mixture of butyl alcohol, glacial acetic acid, and water
DS Monographs
(5:1:4).
Spray reagent: Methanol, glacial acetic acid, sulfuric
acid, and p-anisaldehyde (85:10:5:0.5). [NoTE—Store in
a refrigerator. The reagent is colorless; discard if color
appears.]
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Develop the chromatograms until the solvent front has
moved about 15 cm, and dry the plate with the aid
of a current of air.
Acceptance criteria: Examine the pate under UV light
at 365 nm. The Sample solution exhibits main zones
similar in position and color to the main zones of Stan-
dard solution A. In the upper third of the plate, the
Sample solution exhibits a blue fluorescent zone at the
level of the zone due to isoferulic acid of Standard solu-
tion B. Spray the plate with Spray reagent, heat at 100°
for 5 min, and examine in daylight. The Sample solution
exhibits main zones similar in position and color to the
main zones of Standard solution A. Standard solution B
exhibits red-violet zones due to actein and 23-epi-
USP 41
26-deoxyactein. The Sample solution exhibits several
greenish-brown spots in the lower third of the plate
and several violet zones above; two of these violet
zones occur at Ry values similar to those due to actein
and 23-epi-26-deoxyactein of Standard solution B.
e B. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST
Standard solution A: 0.5 mL of Standard solution A pre-
Sen in Identification test A, diluted with methanol to
-O mL
Standard solution B: 1.0 mL of Standard solution B pre-
pared in Identification test A, diluted with methanol to
5.0 mL
Sample solution: Use the Fluidextract, diluting if neces-
sary with a suitable solvent to obtain a concentration of
0.25 mg/mL of triterpene glycosides.
Adsorbent: Chromatographic silica gel mixture with an
average particle size of 5 um (HPTLC plates)
Application volume: 2 uL as an 8-mm band
Developing solvent system: Toluene, ethyl formate,
and formic acid (5:3:2)
Spray reagent: Proceed as directed for Identification
test A.
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Develop until the solvent front has moved two-thirds
of the length of the plate, and dry the plate with the
aid of a current of air. Spray the plate with Spray
reagent, heat at 100° for 5 min, and examine in
daylight.
Acceptance criteria: The Sample solution exhibits main
zones similar in position and color to the main zones of
Standard solution A. Standard solution B exhibits red-vio-
let zones due to actein and 23-epi-26-deoxyactein at Rr
values of about 0.5 and 0.4, respectively. The Sample
solution exhibits zones similar in color and Rr values to
those due to actein and 23-epi-26-deoxyactein of Stan-
dard solution B.
e C. The Sample solution exhibits peaks for cimiracemoside
A, 26-deoxycimicifugoside, (26S)-actein, 23-epi-26-deoxy-
actein, cimigenol-arabinoside, and cimigenol-xyloside at
retention times corresponding to those compounds in
the Standard solution, as obtained in the test for Content
of Triterpene Glycosides. The ratio of the peak areas of
cimigenol-arabinoside to cimigenol-xyloside is NLT 0.4
(distinction from Cimicifuga foetida).
COMPOSITION
e CONTENT OF TRITERPENE GLYCOSIDES
Standard solution: Dissolve a quantity of USP Pow-
dered Black Cohosh Extract RS in methanol with shak-
ing for 1 min, and dilute with methanol to obtain a
solution having a known concentration of 30 mg/mL.
Pass through a membrane filter of 0.45-um or finer
pore size.
23-epi-26-Deoxyactein standard solutions: Dissolve
USP 23-epi-26-Deoxyactein RS in methanol with shaking
for 1 min. Dilute quantitatively, and stepwise if neces-
sary, to obtain solutions having known concentrations
of 500, 100, 50, 25, and 12.5 ug/mL. Pass through a
membrane filter of 0.45-j1m or finer pore size.
System suitability solution: 0.1 mg/mL each of USP
Actein RS and USP 23-epi-26-Deoxyactein RS in
methanol
Sample solution: Use the Fluidextract, diluting if neces-
sary with methanol to obtain a concentration of about
0.75 mg/mL of triterpene glycosides. Centrifuge, or
pass througha filter of 0.45-um or finer pore size.
Solution A: 0.05% trifluoroacetic acid in water
Solution B: Acetonitrile
Mobile phase: See Table 1.
USP 41 Dietary Supplements / Black Cohosh 4481

Table 1 Table 2 (Continued)

Time Water Solution A Solution B Relative
(min) (%) (%) (%) Retention
0 0 80 20 Compound Time
8 0 80 20 25-O-Methyl-cimigenol-arabinoside 1.90
15 68 0 32 25-O-Methyl-cimigenol-xyloside 1.93

22 28 2 fe Plot the logarithms of the peak area responses versus

SS 5 o 95 the logarithms of the concentrations, in g/mL, of
70 5 0 95 the 23-epi-26-Deoxyactein standard solutions, and de-
85 0 80 20 termine the regression lineusing a least-squares anal-
ysis. The coheaten coefficient for the regression line
Chromatographic system is NLT 0.995. From the graphs so obtained, deter-
(See Chromatography (621), System Suitability.) mine the concentration, C, in ug/mL, of the relevant
Mode: LC analyte in the Sample solution. Separately calculate
Detector: Evaporative light-scattering the concentrations, in ug/mL, of cimicifugoside H-1,
[Note—The detector is set up according to the manu- cimiracemoside A, (26R)-actein, 26-deoxycimicifugo-
facturer’s instruction in order to achieve a signal-to- side, (265)-actein, 23-epi-26-deoxyactein, acetyl-
noise ratio of NLT 10 for the 12.5-g/mL 23-epi-26-De- shengmanol-xyloside, cimigenol-arabinoside, cimige-
oxyactein standard solution.] nol-xyloside (cimicifugoside), 26-deoxyactein, 25-ace-
Column: 4.6-mm x 25-cm; 5-4um packing L1 tyl-cimigenol-arabinoside, (245)-25-acetyl-cimigenol-
Column temperature: 35° xyloside, 25-O-methy|-cimigenol-arabinoside, and
Flow rate: 1.6 mL/min 25-O-methyl-cimigenol-xyloside as 23-epi-26-deoxy-
Injection size: 20 uL actein (C37HssOi0) in the portion of Fluidextract taken:
System suitability
Samples: System suitability solution, Standard solution, Result = (D x C/V)
and 100-\1g/mL 23-epi-26-Deoxyactein standard solution
Suitability requirements D = dilution factor for the Sample solution, if
Chromatogram similarity: The chromatogram of the applicable: final volume of Sample solution/
Standard solution is similar to the Reference Chromat- volume of aliquot of Fluidextract taken
ogram provided with the lot of USP Powdered Black (mL/mL)
Cohosh Extract RS being used. Cc = concentration of the relevant analyte in the
Resolution: NLT 1.0 between the (265)-actein and Sample solution (g/mL)
the 23-epi-26-deoxyactein peaks, System suitability = volume of the Fluidextract taken to prepare
solution the Sample solution (mL)
Tailing factor: NMT 2.0 for the 23-epi-26-deoxy- Calculate the percentage of the labeled amount of
actein peak, 100-ug/mL 23-epi-26-Deoxyactein stan- triterpene glycosides in the portion of Fluidextract
dard solution taken:
Relative standard deviation: NMT 2.0% of the loga-
rithm of the area response of the 23-epi-26-deoxy- Result = ZC/L x 100
actein peak in repeated injections, 100-1g/mL 23-epi-
26-Deoxyactein standard solution =C = sum of concentrations of the individual
Analysis triterpene glycosides (mg/mL)
Samples: System suitability solution, Standard solution, i = labeled concentration of triterpene glycosides
23-epi-26-Deoxyactein standard solutions, and Sample of the Fluidextract (mg/mL)
solution Acceptance criteria: 90.0%-110.0%
Using the chromatogram of the Standard solution and CONTAMINANTS
the Reference Chromatogram provided with the lot
of USP Powdered Black Cohosh Extract RS, identify
the retention times of the peaks corresponding to the Delete the following:
triterpene glycosides. The approximate relative reten-
sydeabouo-=: sa

tion times of the triterpene glycosides are provided in °o HEAVY METALS (231): NMT 10 ppme (oficial 1-jan-2018)
Table 2. e MICROBIAL ENUMERATION TESTS (2021): The total bacterial
count does not exceed 104 cfu/g, and the total com-
Table 2 bined molds and yeasts count does not exceed 103 cfu/
g.
Relative e OTHER REQUIREMENTS: It meets the requirements under
Retention Botanical Extracts (565), Residual Solvents and Pesticide
Compound Time Residues.
Cimicifugoside H-1 0.61
Cimiracemoside A 0.78 ADDITIONAL REQUIREMENTS
(26R)-Actein 0.94 © PACKAGING AND STORAGE: Preserve in tight, light-resistant
containers, and store in a cool place.
26-Deoxycimicifugoside 0.96
e LABELING: It meets the requirements for Labeling under
(265)-Actein 0.98 Botanical Extracts (565). Label it to indicate the content,
23-epi-26-Deoxyactein 1.00 in percentage, of triterpene glycosides, calculated as
Acetyl-shengmanol-xyloside 1.03 Sp a Dosage forms prepared with this
Cimigenol-arabinoside 1.08 article should bear the following statement: Discontinue
Cimigenol-xyloside (cimicifugoside) 1.13 use and consult a healthcare practitioner if you have a
26-Deoxyactein 1.22
liver disorder or develop symptoms of liver trouble, such
as abdominal pain, dark urine, or jaundice.
25-Acetyl-cimigenol-arabinoside 1.60
(245)-25-Acetyl-cimigenol-xyloside 1.64
 4482 Black Cohosh / Dietary Supplements
e USP REFERENCE STANDARDS (11)
USP Actein RS
USP Powdered Black Cohosh Extract RS
USP 23-epi-26-Deoxyactein RS
Black Cohosh Tablets
DEFINITION
Black Cohosh Tablets contain Powdered Black Cohosh Ex-
tract or Black Cohosh Fluidextract. Tablets contain NLT
90.0% and NMT 110.0% of the labeled amount of Pow-
dered Extract or Fluidextract, represented by the content
of triterpene glycosides, calculated as 23-epi-26-deoxy-
actein (C37Hs6O10).
IDENTIFICATION
e oe CHROMATOGRAPHIC IDENTIFICATION TEST
Adsorbent: Chromatographic Misael mixture with an
average particle size of 10-15 «um (TLC plates)
Sample solution: 10 mL of the Sample solution pre-
pared for Identification test B. Evaporate to dryness, and
redissolve in 1 mL of methanol.
Standard solution A: 100 mg/mL of USP Powdered
Black Cohosh Extract RS in methanol
Standard solution B: 1 mg/mL each of USP Actein RS,
USP 23-epi-26-Deoxyactein RS, and isoferulic acid in
methano
Application volume: 10 uL
Developing solvent system: Use the upper phase of a
oa of butyl alcohol, glacial acetic acid, and water
5:1:4).
Spray reagent: Methanol, glacial acetic acid, sulfuric
acid, and p-anisaldehyde (85:10:5:0.5)
[Note—Store in a refrigerator. The reagent is colorless;
discard if color appears.]
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Develop until the solvent front has moved 15 cm, and
dry the plate with the aid of a current of air. Examine
the plate under UV light at a wavelength of 365 nm.
Spray the plate with Spray reagent, heat at 100° for 5
min, and examine in daylight.
Acceptance criteria: The Sample solution exhibits main
zones similar in position and color to the main zones of
Standard solution A. Examined under UV light, the Sam-
ple solution exhibits a blue fluorescent zone at the level
DS Monographs
of the zone due to isoferulic acid in Standard solution B,
in the Upper third of the plate. Examined after treat-
ment with Spray reagent, the Sample solution exhibits
several greenish-brown spots in the lower third of the
plate and several violet zones above; two of these violet
zones occur at R- values similar to those due to actein
and 23-epi-26-deoxyactein in Standard solution B.
e B. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST
(201)
Adsorbent: Chromatographic silica gel mixture with an
average particle size of 5 um (HPTLC plates)
Sample solution: Transfer the equivalent of the labeled
amount of Powdered Extract or Fluidextract, containing
25 mg of triterpene glycosides from a portion of pow-
dered Tablets, to 25 mL of water; shake to disperse; and
sonicate for 10 min. Add 75 mL of methanol, and soni-
cate for 10 min. Allow to stand for 15 min, and use the
clear supernatant.
Standard solution A: Methanol and Standard solution A
prepared in Identification test A (3:1)
Standard solution B: Methanol and Standard solution B
prepared in /dentification test A (4:1)
USP 41
Application volume: 2 iL as an 8-mm band
Developing solvent system: Toluene, ethyl formate,
and formic acid (5:3:2)
Spray reagent: Methanol, glacial acetic acid, sulfuric
acid, and p-anisaldehyde (85:10:5:0.5)
[NoTt—Store in a refrigerator. The reagent is colorless;
discard if color appears.]
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Develop until the solvent front has moved two-thirds
of the length of the plate, and dry the plate with the
aid of a current of air. Spray the plate with Spray
reagent, heat at 100° for 5 min, and examine in
daylight.
Acceptance criteria: The Sample solution exhibits main
zones similar in position and color to the main zones of
Standard solution A, two of which are red-violet zones at
R; values of 0.5 and 0.4, similar in color and R; values
to those due to actein and 23-epi-26-deoxyactein in
Standard solution B.
e C. The Sample solution exhibits peaks for cimiracemoside
A, 26-deoxycimicifugoside, (265) actein, 23-epi-26-deoxy-
actein, cimigenol-arabinoside, and cimigenol-xyloside at
retention times corresponding to those compounds in
the Standard solution, as obtained in the test for Content
of Triterpene Glycosides. The ratio of the peak areas of
cimigenol-arabinoside to cimigenol-xyloside is NLT 0.4
(distinction from Cimicifuga foetida).
STRENGTH
© CONTENT OF TRITERPENE GLYCOSIDES
Solution A: Filtered and degassed 0.05% trifluoroacetic
acid in water
Solution B: Filtered and degassed acetonitrile
Mobile phase: See the gradient table below.
Time Water Solution A Solution B
(min) (%) (%) (%)
0 0 80 20
8 0 80 20
15 68 0 32
55 36 0 64
65 5 oO 95
70 5 o 95
85 0 80 20
System suitability solution: 0.1 mg/mL each of USP
Actein RS and USP 23-epi-26-Deoxyactein RS in
methanol
Standard solution: Dissolve a quantity of USP Pow-
dered Black Cohosh Extract RS in methanol with shak-
ing for 1 min, and dilute with methanol to obtain a
solution having a known concentration of 30 mg/mL.
Pass through a membrane filter having a 0.45-11m or
finer porosity.
23-epi-26-Deoxyactein standard solutions: Dissolve
USP 23-epi-26-Deoxyactein RS in methanol with shaking
for 1 min. Dilute quantitatively, and stepwise if neces-
sary, to obtain solutions having known concentrations
of 500, 100, 50, 25, and 12.5 g/mL. Pass through a
membrane filter having a 0.45-um or finer a
Sample solution: Weigh NLT 20 Tablets, and finely
powder. Transfer a quantity of the powder, equivalent
to 8 mg of triterpene glycosides, to a suitable polytef-
capped centrifuge tube. Add 3 mL of water, shake to
disperse, and sonicate for 10 min at 60°. Add 3 mL of
methanol, and sonicate for 10 min. Centrifuge, and
transfer the clear supernatant to a 10-mL volumetric
flask. Wash the residue twice with 1.5 mL of a mixture
of methanol and water (1:1), and transfer the washings
to the volumetric flask. Dilute with a mixture of metha-
USP 41 Dietary Supplements / Black Pepper 4483

nol and water (1:1) to volume, and pass through a Calculate theSuantity, in mg, of triterpene glycosides
membrane filter having a 0.45-um or finer porosity. in the portion of Tablets taken:
Chromatographic system
(See Chromatography (621), System Suitability.) Result = C+/100
Mode: LC
Detector: Evaporative light-scattering Ce = sum of the concentrations C, in g/mL, of all
[Note—Detector is set up according to the manufactur- the relevant triterpene glycosides, calculated
er’s instruction in order to achieve a signal-to-noise ra- as 23-epi-26-deoxyactein
tio of NLT 10 for the 12.5 ug/mL 23-epi-26-Deoxy- Calculate the percentage of the labeled amount of
actein standard solution.] Extract in the portion of Tablets taken:
Column: 4.6-mm x 25-cm; 5-um packing L1
Column temperature: 35° Result = (Awr/W) x (100/Le) x (100/L) x Cr
Flow rate: 1.6 mL/min Awr = average weight of Tablets
Injection size: 20 pL WwW = weight of sample
System suitability Le = labeled content, as percentage, of triterpenes
Samples: System suitability solution and 100 g/mL of in the Extract used to prepare the Tablets
23-epi-26-Deoxyactein standard solution L = labeled amount of Extract per Tablet
Suitability requirements Cr = content, in mg, of triterpenes in the sample
Chromatographic profile: The chromatogram of the Acceptance criteria: 90.0%-110.0% of the labeled
Standard solution is similar to the Reference Chromat- amount of Powdered Extract or Fluidextract, repre-
ogram provided with the lot of USP Powdered Black sented by the content of triterpene glycosides
Cohosh Extract RS.
Resolution: NLT 1.0 between the (265)-actein and PERFORMANCE TESTS
the 23-epi-26-deoxyactein peaks, System suitability e DISINTEGRATION AND DISSOLUTION OF DIETARY SUPPLEMENTS
solution (2040): Meet the requirements for Disintegration
Tailing factor: NMT 2.0 for the 23-epi-26-deoxy- © WEIGHT VARIATION OF DIETARY SUPPLEMENTS (2091): Meet
actein peak, 100 ug/mL 23-epi-26-Deoxyactein stan- the requirements
dard solution
Relative standard deviation: NMT 2.0% for the log- CONTAMINANTS
arithm of the area responses for replicate injections, ¢ MICROBIAL ENUMERATION TESTS—NUTRITIONAL AND DIETARY
100 g/mL 23-epi-26-Deoxyactein standard solution SUPPLEMENTS (2021): The total bacterial count does not
Analysis exceed 104 cfu/g, and the total combined molds and
Samples: System suitability solution, Standard solution, yeasts count does not exceed 103 cfu/g.
23-epi-26-Deoxyactein standard solutions, and Sample @ MICROBIAL PROCEDURES FOR ABSENCE OF SPECIFIED MiICROOR-
solution GANISMS—NUTRITIONAL AND DIETARY SUPPLEMENTS
Using the chromatogram of the Standard solution and (2022): Tablets meet the requirements of the tests for
the Reference Chromatogram provided with the lot absence of Salmonella species and Escherichia coli.
of USP Powdered Black Cohosh Extract RS, identify
the retention times of the peaks corresponding to the ADDITIONAL REQUIREMENTS
triterpene glycosides. The approximate relative reten- e PACKAGING AND STORAGE: Preserve in tight, light-resistant
tion times of the triterpene glycosides are provided in containers, and store at room temperature.
the following table. © LABELING: The label states the Latin binomial and, follow-
ing the official name, the article from which the Tablets
Relative were prepared. The label also indicates the amount, in
Rete ntion mg/Tablet, of Powdered Extract or Fluidextract; the. sol-
Navies Time vents used to prepare the Powdered Extract or Fluid-
aa ; extract; and the ratio of starting crude plant material to
Cimicifuaoside H-1 oon Powdered Extract or Fluidextract. Label it to indicate the
Cimiracemoside A 0.78 content, in percentage, of triterpene glycosides as 23-epi-
(26R)-Actein 0.94 26-deoxyactein in the Powdered Extract or Fluidextract
26-Deoxycimicifugoside 0.96 used to prepare the Tablets.
sydesbouo= sa

(265)-Actein 0.98 The label bears the following statement: Discontinue use
23-epi-26-Deoxyactein 1.00 and consult a healthcare practitioner if you havealiver
Aceh i xviosldl : disorder or develop symptoms of liver trouble, such as
cetyl-shengmanol-xyloside 1:03 abdominal pain, dark urine, or jaundice.
Cimigenol-arabinoside 1.08 o USP REFERENCE STANDARDS (11)
Cimigenol-xyloside (cimicifugoside) 1.13 USP Actein RS
26-Deoxyactein 1.22 USP Powdered Black Cohosh Extract RS
25-Acetyl-cimigenol-arabinoside 1.60 USP 23-epi-26-Deoxyactein RS
(245S)-25-Acetyl-cimigenol-xyloside 1.64
25-O-Methyl-cimigenol-arabinoside 1.90
25-O-Methyl-cimigenol-xyloside 1.93

Plot the logarithms of the peak area responses versus
the logarithms of the concentrations, in g/mL, of
the 23-epi-26-Deoxyactein standard solutions, and de-
termine the regression line using a least-squares anal-
ysis. The correlation coefficient for the regression line
is NLT 0.995. From the graphs so obtained, deter-
mine the concentration, C, in g/mL, of the relevant
analyte in the Sample solution.
Black Pepper
DEFINITION
Black Pepper consists of the dried fully developed unripe
fruits of Piper nigrum L. (Fam. Piperaceae). It contains NLT
2.5% of piperine, calculated on the dried basis.
IDENTIFICATION
e A. Black Pepper meets the requirements under Specific
Tests, Botanical Characteristics.
 4484 Black Pepper / Dietary Supplements USP 41

e B. HPTLC FoR ARTICLES OF BOTANICAL ORIGIN (203) 0.5 mL of phosphoric acid. Dilute with water to
Standard solution A: 0.9 mg/mL of USP Piperine RS in 1000 mL, mix, filter, and degas.
methanol Solution B: Acetonitrile
Standard solution B: 2.0 mg/mL of borneol in Mobile phase: See Table 7.
methanol
Standard solution C: 5 mg/mL of USP Powdered Black Table 1
Pepper Extract RS in methanol. Sonicate for about 10
min, centrifuge, and use the supernatant. Time Solution A Solution B
Sample solution: Sonicate for 10 min about 0.5 g of (min) (%) (%)
Black Pepper, finely powdered, in 5 mL of methanol, 0 95 5
centrifuge, and use the supernatant. 18 55 45
Chromatographic system 25 20 80
Adsorbent: Chromatographic silica gel mixture with 28 20 80
an average particle size of 5 um (HPTLC plates)
Application volume: 15 ul of Standard solution C, 7 wL 35 55 45
of the Sample solution, and 3 L of the Standard solu- 40 95 5
tion A and Standard solution B, as bands 45 95 5
Developing solvent system: Hexanes and ethyl ace-
tate (5:3) [Note—Proceed under subdued light or using low-ac-
Derivatization reagent: A mixture of 17 mL of ice- tinic glassware.]
cooled methanol, 2 mL of acetic acid, 1 mL of sulfuric Standard solution A: 0.1 mg/mL of USP Piperine RS in
acid, and 0.1 mL of anisaldehyde, mixed in this order methanol
Analysis Standard solution B: Sonicate a portion of USP Pow-
Samples: Standard solution A, Standard solution B, dered Black Pepper Extract RS in methanol to obtain a
Standard solution C, and Sample solution solution having a concentration of about 0.5 mg/mL.
Apply the Samples as bands. Use a saturated chamber, Before injection, pass through a membrane filter of
and condition the plate to a relative humidity of about 0.45-um or finer pore size, discarding the first few mL
33% using a suitable device. Develop until the solvent of the filtrate.
front has moved up about 7 cm from the lower edge Sample solution: Transfer about 2.0 g of Black Pepper,
of the plate. Remove the plate from the chamber, nie finely powdered and accurately weighed, to a 250-mL
and examine under UV light at 254 nm. Treat with the flask fitted with a reflux condenser. Add 50 mL of meth-
Derivatization reagent, heat for 5 min at 100°, and ex- anol, reflux on a water bath for about 20 min, allow to
amine under white light. settle, and decant the supernatant. Repeat until the last
Acceptance criteria: Under UV 254 nm, the chromato- extract is colorless. Combine the extracts, concentrate
gram of the Sample solution exhibits an intense quench- under vacuum, and adjust the volume to 100 mL using
ing band at R; of about 0.15 corresponding to the pip- methanol. Before injection, pass through a membrane
erine band in the chromatogram of Standard solution A, filter of 0.45-\1m or finer pore size, discarding the first
a quenching band at Rr of about 0.02, and three few mL of the filtrate.
quenching bands located between R; of about 0.3 and Chromatographic en
0.5. Under white light, the derivatized chromatogram (See Chromatography (621), System Suitability.)
of the Sample solution exhibits main bands similar in Mode: LC
position and color to the main bands in the chromato- Detector: UV 343 nm and 270 nm “
gram of Standard solution C. These bands include a dark Column: 4.6-mm x 25-cm; 5-um, 100 A packing L1
green band of the same color and R; as the piperine Flow rate: 1.5 mL/min
band in Standard solution A (Rr of about 0.15), a weak Injection volume: 20 pL
violet band at R- of about 0.47 below the position of System suitability
the band due to borneol in Standard solution B, and a Samples: Standard solution A and Standard solution B
greenish band in the lower part of the chromatogram Suitability requirements
at Rr of about 0.07. Other minor bands may be ob- Chromatogram similarity: The chromatogram ob-
served in the Sample solution and Standard solution C tained from Standard solution B is similar to the refer-
chromatograms. No blue bands are detected in the ence chromatogram provided with the lot of USP
Powdered Black Pepper Extract RS being used.
DS Monographs

chromatogram of the Sample solution at Rr of about

0.10 and 0.58 (distinction from long pepper).
e C. HPLC
Analysis: Proceed as directed in the test for Content of
Piperine.
Acceptance criteria: The chromatogram of the Sample
solution obtained at 343 nm exhibits a major peak at
the retention time corresponding to piperine. Identify
other piperamide peaks in the Sample solution chromat-
ogram by comparison with the chromatogram of Stan-
dard solution B and the reference chromatogram pro-
vided with the lot of USP Powdered Black Pepper
Extract RS being used. The Sample solution chromato-
gram shows an additional peak corresponding to piper-
yline. The chromatogram of the Sample solution ob-
tained at 270 nm does not exhibit a peak due to (2E,
46)-N-isobutyldecadienamide at a relative retention time
of 1.14 to the piperine peak (distinction from long
pepper).
COMPOSITION
o CONTENT OF PIPERINE
Solution A: Dissolve 0.14 g of anhydrous potassium
dihydrogen phosphate in 900 mL of water, and add
Tailing factor: NMT 1.5 for the piperine peak, Stan-
dard solution A
Relative standard deviation: NMT 2.5% determined
from the piperine peak in replicate injections, Stan-
dard solution A
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
[Note—Standard solution A, Standard solution B, and the
Sample solution are stable for 6 h at room
temperature.]
Using the chromatograms of Standard solution A, Stan-
dard solution B, and the reference chromatogram pro-
vided with the lot of USP Powdered Black Pepper Ex-
tract RS being used, identify the retention times of the
peaks corresponding to piperine and piperyline in the
Sample solution chromatogram.
Calculate the percentage of piperine in the portion of
Black Pepper taken:
Result = (ru/rs) x Cs x (V/W) x 100
USP 41
tu = peak area of piperine from the Sample solution
chromatogram at 343 nm
rs = peak area of piperine from the Standard
solution A chromatogram at 343 nm
Cs = concentration of piperine in Standard solution
A (mg/ml)
Vv = final volume of the Sample solution (mL)
w = weight of Black Pepper used to prepare the
Sample solution (mg)
Acceptance criteria: NLT 2.5% on the dried basis
CONTAMINANTS
e ARTICLES OF BOTANICAL ORIGIN, Limits of Elemental Impuri-
ties (561): Meets the requirements
e ARTICLES OF BOTANICAL ORIGIN, Pesticide Residue Analysis
(561): Meets the requirements
© MICROBIAL ENUMERATION TESTS (2021): The total aerobic
bacterial count does not exceed 105 cfu/g, the total com-
bined molds and yeasts count does not exceed 103 cfu/
g, and the bile-tolerant Gram-negative bacterial count
does not exceed 103 cfu/g.
e ABSENCE OF SPECIFIED MICROORGANISMS (2022): Meets the
requirements of the tests for absence of Salmonella spe-
cies and Escherichia coli
SPECIFIC TESTS
e BOTANICAL CHARACTERISTICS
Macroscopic: Fruit is an indehiscent, one-seeded berry,
globose, ovoid to oblong, 3.5-6 mm in diameter, hard;
surface is greyish-black to brownish-black, rough, with
raised reticulate wrinkles, shows remains of sessile
stigma on the tip and a basal scar showing point of
attachment to the axis; characteristic aromatic odor;
characteristic pungent taste; seed white and hollow.
Microscopic
Transverse cut: Circular in outline with corrugated
margin; shows outer narrow brownish pericarp; a seed
coat encircling the wide central whitish perisperm,
hollow in center; a narrow vertically running channel
connecting the hollow center of the fruit to a small
endosperm adherent to the remains of the stigma; a
small embryo is present in the endosperm; a conical
short projection at the base showing point of attach-
ment to the axis.
Transverse section: Shows a well-differentiated peri-
carp, testa and perisperm; pericarp consists of a layer
of epicarp, a wide mesocarp, and a layer of endocarp;
the epicarp layer is covered with thick cuticle contain-
ing a few stomata and small prisms of calcium oxalate;
mesocarp composed of 2-3 layers of parenchyma cells
showing groups of rectangular to circular lignified
sclereids, a broad zone (12-15 layers) of tangentially
running parenchyma cells containing starch grains and
showing isolated oval oil cells, a broad zone (10-15
layers) of compactly arranged parenchyma cells
smaller than those of the outer zone and showing
groups of fibrovascular bundles, 1-2 layers of tangen-
tially running oil cells, and 2-3 layers of thick-walled
parenchyma cells; endocarp is composed of one layer
of three-sided thickened pitted stone cells (beaker-
shape cells); testa is composed of one layer of cells
filled with brown pigments; perisperm is very wide,
composed of cells full of starch grains, some aleurone
grains, and oil cells.
¢ ARTICLES OF BOTANICAL ORIGIN, Foreign Organic Matter
(561): NMT 2.0%
e ARTICLES OF BOTANICAL ORIGIN, Alcohol-Soluble Extractives,
Method 1 (561): NLT 10.0%
e ARTICLES OF BOTANICAL ORIGIN, Water-Soluble Extractives,
Method 1 (561): NLT 9.0%
Dietary Supplements / Black Pepper 4485
e Loss ON DRYING (731)
Sample: 1.0 of finely paced Black Pepper
Analysis: Dry the Sample at 105° for 2 h.
Acceptance criteria: NMT 12.0%
e ARTICLES OF BOTANICAL ORIGIN, Tota! Ash (561)
Sample: 1.0 of finely powdered Black Pepper
Acceptance criteria: NMT 5.0%
e ARTICLES OF BOTANICAL ORIGIN, Acid-/nsoluble Ash (561)
Sample: 1.0g of finely powdered Black Pepper
Acceptance criteria: NMT 1.0%
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in well-closed contain-
ers, protected from light and moisture, and store at
room temperature.
e LABELING: The label states the Latin binomial and, follow-
ing the official name, the part of the plant contained in
the article.
e USP REFERENCE STANDARDS (11)
USP Piperine RS
USP Powdered Black Pepper Extract RS
Powdered Black Pepper
DEFINITION
Powdered Black Pepper is Black Pepper reduced to powder
or very fine powder. It contains NLT 2.5% of piperine,
calculated on the dried basis.
IDENTIFICATION
e A. Powdered Black Pepper meets the requirements under
Specific Tests, Botanical Characteristics.
e B. HPTLC For ARTICLES OF BOTANICAL ORIGIN (203)
Standard solution A: 0.9 mg/mL of USP Piperine RS in
methanol
Standard solution B: 2.0 mg/mL of borneol in
methanol
Standard solution C: 5 mg/mL of USP Powdered Black
Pepper Extract RS in methanol. Sonicate for about 10
min, centrifuge, and use the supernatant.
Sample solution: Sonicate for 10 min about 0.5 g of
Powdered Black Pepper in 5 mL of methanol, centri-
fuge, and use the supernatant.
Chromatographic system
Adsorbent: Chromatographic silica gel mixture with
an average particle size of 5 zm (HPTLC plates)
Application volume: 15 ul of Standard solution C, 7 wL
of the Sample solution, and 3 wL of Standard solution A
sydesbouo-: sa
and Standard solution B, as bands, 8 mm
Developing solvent system: Hexanes and ethyl ace-
tate (5:3)
Derivatization reagent: A mixture of 17 mL of ice-
cooled methanol, 2 mL of acetic acid, 1 mL of sulfuric
acid, and 0.1 mL of anisaldehyde, mixed in this order
Analysis
Samples: Standard solution A, Standard solution B,
Standard solution C, and Sample solution
Apply the Samples as bands. Use a saturated chamber,
and condition the plate to a relative humidity of about
33% using a suitable device. Develop until the solvent
front has moved up about 7 cm from the lower edge
of the plate. Remove the plate from the chamber, dry,
and examine under UV light at 254 nm. Treat with the
Derivatization reagent, heat for 5 min at 100°, and ex-
amine under white light.
Acceptance criteria: Under UV 254 nm, the chromato-
ram of the souiple solution exhibits an intense quench-
ing band at R; of about 0.15 corresponding to the pip-
erine band in the chromatogram of Standard solution A,
a quenching band at R; of about 0.02, and three
quenching bands of similar intensity equally spaced lo-
cated between R; of about 0.3 and 0.5. Under white
 4486 Black Pepper / Dietary Supplements
light, the derivatized chromatogram of the Sample solu-
tion exhibits main bands similar in position and color to
the main bands in the chromatogram of Standard solu-
tion C. These bands include a dark green band of the
same color and R; as the piperine band in Standard so-
lution A (Re of about 0.15), a weak violet band at Ry of
about 0.47 below the position of the band due to bor-
neol in Standard solution B, and a greenish band in the
lower part of the chromatogram at R; of about 0.07.
Other minor bands may be observed in the Sample so-
lution and Standard solution C chromatograms. No blue
bands are detected in the chromatogram of the Sample
solution at Rr of about 0.10 and 0.58 (distinction from
long pepper).
° C. HPLC
Analysis: Proceed as directed in the test for Content of
Piperine.
Acceptance criteria: The chromatogram of the Sample
solution obtained at 343 nm exhibits a major peak at
the retention time corresponding to piperine. Identify
other piperamide peaks in the Sample solution chromat-
ogram by comparison with the chromatogram of Stan-
dard solution B and the reference chromatogram pro-
vided with the lot of USP Powdered Black Pepper
Extract RS being used. The Sample solution chromato-
gram shows an additional peak corresponding to piper-
yline. The chromatogram of the Sample solution Ass
tained at 270 nm does not exhibit a peak due to (2E,
46)-N-isobutyldecadienamide ata relative retention time
of 1.14 to the piperine peak (distinction from long
pepper).
COMPOSITION
© CONTENT OF PIPERINE
Solution A: Dissolve 0.14g of anhydrous potassium
dihydrogen phosphate in 900 mL of water, and add
0.5 mL of phosphoric acid. Dilute with water to
1000 mL, mix, filter, and degas.
Solution B: Acetonitrile
Mobile phase: See Table 1.
Table 1
Time Solution A Solution B
(min) (%) (%)
0 95 5 Acceptance criteria: NLT 2.5% on the
18 55 45
25 20 80
28 20 80
35 55 45
40 95 5 (561): Meets the requirements
DS Monographs
45 95 5 ¢ MICROBIAL ENUMERATION TESTS (2021): The total aerobic
[Note—Proceed under subdued light or using low-ac-
tinic glassware.]
Standard solution A: 0.1 mg/mL of USP Piperine RS in
methanol
Standard solution B: Sonicate a portion of USP Pow-
dered Black Pepper Extract RS in methanol to obtain a
solution having a concentration of about 0.5 mg/mL.
Before injection, pass through a membrane filter of
0.45-um or finer pore size, discarding the first few mL
of the filtrate.
Sample solution: Transfer about 2.0 g of Powdered
Black Pepper, accurately weighed, to a 250-mL flask fit-
ted with a reflux condenser. Add 50 mL of methanol,
reflux on a water bath for about 20 min, allow to settle,
and decant the supernatant. Repeat until the last ex-
tract is colorless. Combine the extracts, concentrate
under vacuum, and adjust the volume to 100 mL using
methanol. Before injection, pass through a membrane
filter of 0.45-um or finer pore size, discarding the first
few mL of the filtrate.
USP 41
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 343 nm and 270 nm i
Column: 4.6-mm x 25-cm; 5-um, 100 A packing L1
Flow rate: 1.5 mL/min
Injection volume: 20 uL
System suitability
Samples: Standard solution A and Standard solution B
Suitability requirements
Chromatogram similarity: The chromatogram ob-
tained from Standard solutionB is similar to the refer-
ence chromatogram provided with the lot of USP
Powdered Black Pepper Extract RS being used.
Tailing factor: NMT 1.5 for the piperine peak, Stan-
dard solution A
Relative standard deviation: NMT 2.5% determined
from the piperine peak in replicate injections, Stan-
dard solution A
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
[Note—Standard solution A, Standard solution B, and the
Sample solution are stable for 6 h at room
temperature.]
Using the chromatograms of Standard solution A, Stan-
dard solution B, and the reference chromatogram pro-
vided with the lot of USP Powdered Black Pepper Ex-
tract RS being used, identify the retention times of the
peaks corresponding to piperine and piperyline in the
Sample solution chromatogram.
Calculate the percentage of piperine in the portion of
Powdered Black Pepper taken:
Result = (ru/rs) x Cs x (V/W) x 100
tu = peak area of piperine from the Sample solution
chromatogram at 343 nm
rs = peak area of piperine from the Standard
solution A chromatogram at 343 nm
Cs = concentration of piperine in Standard solution
A (mg/mL)
Vv = final volume of the Sample solution (mL)
w = weight of Powdered Black Pepper used to
prepare the Sample solution (ms)
dried basis
CONTAMINANTS
© ARTICLES OF BOTANICAL ORIGIN, Limits of Elemental Impuri-
ties (S61): Meets the requirements
e ARTICLES OF BOTANICAL ORIGIN, Pesticide Residue Analysis
bacterial count does not exceed 105 cfu/g, the total com-
bined molds and yeasts count does not exceed 103 cfu/
g, and the bile-tolerant Gram-negative bacterial count
does not exceed 103 cfu/g.
© ABSENCE OF SPECIFIED MICROORGANISMS (2022): Meets the
requirements of the tests for absence of Salmonella spe-
cies and Escherichia coli
SPECIFIC TESTS
e BOTANICAL CHARACTERISTICS
Macroscopic: Blackish-grey powder; characteristic aro-
matic odor; characteristic pungent taste
Microscopic: Fragments of polygonal epicarp cells,
some containing small prisms of calcium oxalate; paren-
chyma cells containing starch grains; oil cells; lignified
sclereids; three-sided thickened pitted stone cells
(beaker-shape cells) both in surface and side view; yel-
lowish-brown polygonal cells of the testa; fragments of
spiral vessels; parenchyma cells full of starch grains;
aleurone grains; oil drops; and starch grains
e ARTICLES OF BOTANICAL ORIGIN, Alcohol-Soluble Extractives,
Method 1 (561): NLT 10.0%
USP 41
e ARTICLES OF BOTANICAL ORIGIN, Water-Soluble Extractives,
Method 1 (561): NLT 9.0%
Loss ON DRYING (731)
Sample: 1.0g of Powdered Black Pepper
Analysis: Dry the Sample at 105° for 2 h.
Acceptance criteria: NMT 12.0%
ARTICLES OF BOTANICAL ORIGIN, Total Ash (561)
Sample: 1.0 g of Powdered Black Pepper
Acceptance criteria: NMT 5.0%
ARTICLES OF BOTANICAL ORIGIN, Acid-Insoluble Ash (561)
Sample: 1.0 g of Powdered Black Pepper
Acceptance criteria: NMT 1.0%
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in well-closed contain-
ers, protected from light and moisture, and store at
room temperature.
e LABELING: The label states the Latin binomial and, follow-
ing the official name, the part of the plant from which
the article was derived.
e USP REFERENCE STANDARDS (11)
USP Piperine RS
USP Powdered Black Pepper Extract RS
Powdered Black Pepper Extract
DEFINITION
Powdered Black Pepper Extract is prepared from Black Pep-
per using suitable solvents such as ethyl acetate, metha-
nol, or a mixture of these solvents. The ratio of plant ma-
terial to extract is about 15:1. It contains NLT 90.0% and
NMT 110.0% of the labeled amount of piperine, calcu-
lated on the dried basis. It may contain suitable added
substances as carriers.
IDENTIFICATION
e A. THIN-LAYER CHROMATOGRAPHY
Standard solution A: 0.9 mg/mL of USP Piperine RS in
methanol
Standard solution B: 2.0 mg/mL of borneol in
methanol
Standard solution C: 5 mg/mL of USP Powdered Black
Pepper Extract RS in methanol. Sonicate for about 10
min, centrifuge, and use the supernatant.
Sample solution: Sonicate for about 10 min an amount
of Powdered Extract, equivalent to about 10 mg of pip-
erine, in 10 mL of methanol, centrifuge, and use the
supernatant.
Chromatographic system
(See ae (621), Thin-Layer Chromato-
raphy.
adsorbent Chromatographic silica gel mixture with
an average particle size of 5 um (HPTLC plates)
Application volume: 15 wl of Standard solution C, 7 uL
of the Sample solution, and 3 ul of Standard solution A
and Standard solution B, as bands, 8 mm
Developing solvent system: A mixture of hexanes
and ethyl acetate (5:3)
Derivatization reagent: A mixture of 17 mL of ice-
cooled methanol, 2 mL of acetic acid, 1 mL of sulfuric
acid, and 0.1 mL of anisaldehyde mixed in this order.
Analysis
Samples: Standard solution A, Standard solution B,
Standard solution C, and Sample solution
Apply the Samples as bands to a suitable high perfor-
mance thin-layer chromatographic plate (see Chroma-
tography (621). Use a saturated chamber, and condi-
tion the plate to a relative humidity of about 33%
using a suitable device. Develop the chromatograms
until the solvent front has moved up about 7 cm
from the lower edge of the plate. Remove the plate
from the chamber, dry, and examine under UV light
Dietary Supplements / Black Pepper 4487
at 254 nm. Derivatize with the Derivatization reagent,
heat for 5 min at 100°, and examine under visible
light.
Accertance criteria: Under UV 254 nm, the chromato-
gram of the Sample solution exhibits an intense quench-
ing band at R; of about 0.15 corresponding in R; to the
piperine band in the chromatogram of Standard solution
A, a quenching band at Rr of about 0.02, and three
quenching bands of similar intensity equally spaced lo-
cated between R; of about 0.3 and 0.5. Under visible
light and after derivatization, the chromatogram of the
Sample solution exhibits main bands similar in position
and color to the main bands in the chromatogram of
Standard solution C. These bands include a dark green
band of the same color and R; as the piperine band in
Standard solution A (R- of about 0.15), a weak violet
band at R; of about 0.47 below the position of the
band due to borneol in Standard solution B, and a
greenish band in the lower part of the chromatogram
at Rr of about 0.07. Other minor bands may be ob-
served in the Sample solution and Standard solution C
chromatograms. No blue bands are detected in the
chromatogram of the Sample solution at Rr of about
0.10 and 0.58 (distinction from Long Pepper).
e B. HPLC
Analysis: Proceed as directed in the test for Content of
Piperine.
Acceptance criteria: The chromatogram of the Sample
solution obtained at 343 nm exhibits a major peak at
the retention time corresponding to piperine. Identify
other piperamide peaks in the Sample solution chromat-
ogram by comparison with the chromatogram of Stan-
dard solution B and the reference chromatogram pro-
vided with the lot of USP Powdered Black Pepper
Extract RS being used. The Sample solution chromato-
gram shows an additional peak corresponding topipet:
yline. The chromatogram of the Sample solution ob-
tained at 270 nm does not exhibit a peak due to (2E,
46)-N-isobutyldecadienamide at a relative retention time
of 1.14 to the piperine peak (distinction from Long
Pepper).
COMPOSITION
© CONTENT OF PIPERINE
Solution A: Dissolve 0.14 g of anhydrous potassium
dihydrogen phosphate in 900 mL of water, and add
0.5 mL of phosphoric acid. Dilute with water to
1000 mL, mix, filter, and degas.
Solution B: Acetonitrile
Mobile phase: See Table 7.
Table 1 we
Time Solution A Solution B =
(min) (%) (%) °
0 95 5 =
18 55 45 no]
25 20 80 ey
28 20 80 =
35 55 45 4
40 95 5
45 95 5
[NotE—Proceed under subdued light or using low-actinic
glassware.]
Standard solution A: 0.1 mg/mL of USP Piperine RS in
methanol
Standard solution B: Sonicate a portion of USP Pow-
dered Black Pepper Extract RS in methanol to obtain a
solution having a concentration of about 0.5 mg/mL.
Before injection, pass through a membrane filter of
0.45-um or finer pore size, discarding the first few mL
of the filtrate.
 4488 Black Pepper / Dietary Supplements USP 41

Sample solution: Transfer an accurately weighed
amount of Powdered Extract, equivalent to about
25 mg of piperine, to a 25-mL volumetric flask, add
15 mL of methanol, and sonicate for 10 min. Cool to
room temperature, dilute with methanol to volume,
and mix. Dilute the obtained solution in methanol
(1:10). Before injection, pass through a membrane filter
of 0.45-um or finer pore size, discarding the first few
mL of the filtrate.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 343 nm and 270 nm : ers, protected from light and moisture, and store at con-
Column: 4.6-mm x 25-cm; 5-um, 100 A packing L1
Flow rate: 1.5 mL/min
Injection volume: 20 pL
System suitability
Samples: Standard solution A and Standard solution B
Suitability requirements
Chromatogram similarity: The chromatogram ob-
tained from Standard solution B is similar to the refer-
ence chromatogram provided with the lot of USP
Powdered Black Pepper Extract RS being used.
Tailing factor: NMT 1.5 for the piperine peak, Stan-
dard solution A
Relative standard deviation: NMT 2.5% determined
from the piperine peak in repeated injections, Stan-
dard solution A
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
[Note—Standard solution A, Standard solution B, and the
Sample solution are stable for 6 hours at room
temperature.]
Using the chromatograms of Standard solution A, Stan-
dard solution B, and the reference chromatogram pro-
vided with the lot of USP Powdered Black Pepper Ex-
tract RS being used, identify the retention time of the
peaks corresponding to piperine and piperyline in the
Sample solution chromatogram.
Calculate the percentage of piperine in the portion of
Powdered Extract taken:
P = (rulrs) x (Cs/Cu) x 100
tu = peak area for piperine from the Sample
solution chromatogram at 343 nm
ls = peak area for piperine from the Standard
solution A chromatogram at 343 nm
Gs = concentration of piperine in Standard solution
ia A (mg/mL)
Re Cy = concentration of Powdered Extract in the
= Sample solution (mg/mL)
fd Calculate the percentage of the labeled amount of
e piperine in the portion of Powdered Extract taken:
5 Result = (P/L) x 100
= P = content of piperine as determined above (%)
Fan) L = labeled amount of piperine (%)
Acceptance criteria: 90%-110% on the dried basis
e ABSENCE OF SPECIFIED MICROORGANISMS (2022): Meets the
requirements of the tests for absence of Salmonella spe-
cies and Escherichia coli
SPECIFIC TESTS
e Loss ON DRYING (731)
Sample: 1.0 g of Powdered Extract
Analysis: Dry at 105° for 2 h.
Acceptance criteria: NMT 7.0%
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in well-closed contain-
trolled room temperature.
e LABELING: The label states the Latin binomial and, follow-
ing the official name, the part of the plant from which
the article was derived. It meets other labeling require-
ments in Botanical Extracts (565).
e USP REFERENCE STANDARDS (11)
USP Piperine RS
USP Powdered Black Pepper Extract RS
Borage Seed Oil
[84012-16-8].
DEFINITION
Borage Seed Oil is derived from seeds of Borago officinalis L.
The oil is extracted by cold press or supercritical fluid ex-
traction and then refined. A suitable antioxidant may be
added.
IDENTIFICATION
e A. It meets the requirements in Specific Tests for Fats and
Fixed Oils (401), Fatty Acid Composition.
e B. IDENTIFICATION OF FIXED OILS BY THIN-LAYER CHROMA-
TOGRAPHY (202): The R; values of the principal spots of
me Sample solution correspond to those of the Standard
solution.
IMPURITIES
Delete the following:
°o HEAVY METALS, Method II (231): NMT 10 ug/ge conical 1-
Jan-2038)
SPECIFIC TESTS
e FATS AND FIXED OILS, Acid Value (401): NMT 1.0
FATS AND FIXED OlLs, Peroxide Value (401): NMT 5.0
FATS AND FIXED OILS, Saponification Value (401): 184-194
FATS AND FIXED OILS, Unsaponifiable Matter (401): NMT
2.0%
FATS AND FIXED OILS, Fatty Acid Composition (401): Bor-
age Seed Oil exhibits the composition profile of fatty
acids in Table 1.
Table 1

CONTAMINANTS Shorthand Percentage

Fatty Acid Notation (%)
Palmitic acid 16:0 8.0-11.0
Delete the following: Stearic acid 18:0 2.0-5.0

°e HEAVY MeTALs, Method II) (231): NMT 20 ug/ge circa 1- Oleic acid 18:1 14.0-19.0
Jan-2018)
Gammaz-linolenic
e ARTICLES OF BOTANICAL ORIGIN, General Method for Pesti- acid 18:3 18.0-24.0
cide Residues Analysis (561): Meets the requirements Linoleic acid 18:2 34.0-42.0
e MicROBIAL ENUMERATION TESTS (2021): The total aerobic Arachidic acid 20:0 NMT 0.5
bacterial count does not exceed 104 cfu/g, and the total Gadoleic acid 20:1 2.0-6.0
oes molds and yeasts count does not exceed 103 Behenic acid 22:0 NMT 0.8
cfu/g.
USP 41 Dietary Supplements / Borage 4489

Table 1 (Continued) Internal standard: Methyl pentadecanoic acid

Shorthand Percentage Sample solution: Weigh NLT 10 Capsules. With a sharp
Fatty Acid Notation (%)
blade, carefully slice open the Capsules, avoiding loss of
shell material. Combine the Capsule contents in a suita-
Erucic acid 22:1 NMT 5.0 ble container, and mix well. Remove any adhering sub-
Nervonic acid 24:1 NMT 4.5 stance from the al ip Capsules by washing with sev-
eral portions of diethyl ether, and discard the washings.
e REFRACTIVE INDEX (831): 1.474-1.478 at 20° Allow the empty Capsule shells to air-dry over a period
of NMT 30 min, aking precautions to avoid uptake or
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in tight containers, loss of moisture. Weigh the empty Capsule shells, and
preferably under an atmosphere of an inert gas, pro- calculate the average fill weight/Capsule (A,). Transfer
tected from light. 80 mg of the accurately weighed combined Capsule
e LABELING: Label it to indicate the name and quantity of contents directly into a tared 30-mL screw-top glass
centrifuge tube. Re-tare, and accurately weigh about
any added antioxidants. Where Borage Seed Oil is in-
tended for use in the manufacture of dosage forms, it is 40 mg of Internal standard. Add 2 mL of 0.5 N metha-
so labeled. nolic sodium hydroxide solution, tightly cap, and transfer
to a heating block or another appropriate heating de-
vice. Reflux the solution until fat globules disappear
Delete the following: (usually 5-10 min). Add 2 mL of 0.14 g/mL boron
trifluoride in methanol, cap, and reflux for 2 min. Add
°e USP REFERENCE STANDARDS (11) 4 mL of chromatographic n-heptane, cap, and reflux for
USP Borage Seed Oil RS 1 min. Cool, add about 8 mL of saturated sodium chlo-
© (CN I-May-2018) ride solution, shake, and centrifuge to separate the lay-
ers. Dilute an aliquot of the upper (heptane) layer 1:8
with chromatographic n-heptane, and mix well.
System suitability solution: Using about 80 mg of USP
Borage Seed Oil RS, proceed as directed for the Sample
Borage Seed Oil Capsules solution, beginning with “Transfer 80 mg” without the
addition of the Internal standard.
DEFINITION
Standard solution: Directly into a tared 30-mL screw-
Borage Seed Oil Capsules are prepared with Borage Oil de- top glass centrifuge tube accurately weigh about 20 mg
rived from seeds of Borago officinalis L. by cold pressin of USP Methyl Linolenate RS, 40 mg of USP Methyl Li-
supercritical fluid extraction and contain NLT 95.0% of noleate RS, and 20 mg of USP Methy! Oleate RS. Pro-
the labeled amounts of y-linolenic (C18:3 n-6), linoleic ceed as directed for the Sample solution, beginning with
(C18:2 n-6), and oleic (C18:1 n-9) acids. “Re-tare”.
Chromatographic system
IDENTIFICATION (See Chromatography (621), System Suitability.)
o A. FATTY ACID PROFILE Mode: GC
System suitability solution and Chromatographic sys- Detector: Flame ionization
tem: Proceed as directed in Strength. Column: 0.53-mm x 30-m fused silica capillary; coated
Sample solution: Proceed as directed for the Sample with a 1.0-1m film of G16
solution in Strength, beginning with “Transfer 80 mg” Temperatures
without the addition of the /nternal standard. Injection port: 220°
Analysis: Identify the specified fatty acid methyl ester Detector: 260°
peaks by comparing them to the reference chromato- Column: See Table 2.
paw provided with the lot of USP Borage Seed Oil RS
eing used, Determine the percentage of each constitu- Table 2
ent relative to the total integrated area.
Acceptance criteria: The Sample solution conforms to Hold Time
the fatty acid composition profile in Table 1. Initial Temperature Final at Final
Temperature Ramp Temperature | Temperature
sydeabouo; sq

©) (¢/min) © (min)
Table 1
70 0 70. 2
Shorthand Percentage 70 5. 240 5
Fatty Acid Notation (%)
Palmitic acid 16:0 8.0-11.0 Carrier gas: Helium
Stearic acid 18:0 2.0-5.0 Linear velocity: 50 cm/s
Oleic acid 18:1 n-9 14.0-19.0
Split mode: Splitless
Injection volume: 1 pL
y-Linolenic acid 18:3 n-6 18.0-24.0 System suitability
Linoleic acid 18:2 n-6 34.0-42.0 Samples: System suitability solution and Standard
Arachidic acid 20:0 NMT 0.5 solution
Gondoic acid 20:1 n-9 NMT 6.0 Suitability requirements
Behenic acid 22:0 NMT 0.8 Chromatogram similarity: The System suitability solu-
Erucic acid 22:1 n-9 NMT 5.0
tion chromatogram is similar to the reference chro-
matogram provided with the lot of USP Borage Seed
Nervonic acid 24:1 n-9 NMT 4.5 Oil RS being used.
Resolution: NLT 1.5 between methyl oleate and
STRENGTH methyl stearate, System suitability solution
© CONTENT OF y-LINOLENIC, LINOLEIC, AND OLEIC ACIDS Relative standard deviation: NMT 2% for the peak
[Note—y-Linolenic acid is quantitated against USP area ratios of analytes to internal standard, Standard
Methyl Linolenate RS in this procedure.] solution
0.5 N methanolic sodium hydroxide solution: Dis-
solve 2 g of sodium hydroxide in 100 mL of methanol.
 5390 Hydroxypropyl / Official Monographs
Delete the following:
°e HEAVY METALS, Method II (231): NMT 10 j1g/ge cotticat 1.
Jan-2018)
SPECIFIC TESTS
e Loss ON DRYING (731)
Analysis: Dry at 105° for 1 h.
Acceptance criteria: NMT 5.0%
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in tight containers.
Hydroxypropyl Methylcellulose—see
Hypromellose General Monographs
Hymetellose
Methylhydroxyethylcellulose;
Cellulose 2-hydroxyethyl methyl ether [9032-42-2].
DEFINITION
Hymetellose is a partly O-(methylated) and O-(2-hydroxy-
ethylated) cellulose.
IDENTIFICATION
oA.
Sample solution: Use the Sample solution prepared in
the test for Color of Solution.
Analysis: Heat the Sample solution in a water bath while
stirring.
Acceptance criteria; At a temperature above 50°, the
solution becomes cloudy or a flocculent precipitate is
formed. The solution becomes clear again on cooling.
° B.
Sample solution: 1 mL of the solution from Identifica-
tion test A
Analysis: Transfer the Sample solution to a glass plate,
and allow the water to evaporate.
Acceptance criteria: A thin film is formed.
eC.
Sample solution: 10 mL of the solution from Identifica-
tion test A
Analysis: To the Sample solution add 0.3 mL of 2 N ace-
tic acid and 2.5 mL of tannic acid TS.
Acceptance criteria: A yellowish-white, flocculent pre-
cipitate is formed that dissolves in ammonia TS.
° D.
Sample: 1g
Diethanolamine-sodium nitroprusside solution:
50 mg/mL of sodium nitroprusside solution adjusted
with 1 N hydrochloric acid to a pH of 9.8. Mix 11 mL
of this solution with 1 mL of a diethanolamine solution
(200 mg/mL) in water.
Analysis: In a test tube about 160 mm long, thoroughly
mix the Sample with 2 g of ney powdered manganese
sulfate. Introduce, to a depth of 2 cm into the upper
part of the tube, a strip of filter paper impregnated
with a freshly prepared Diethanolamine-sodium ni-
NF Monographs
troprusside solution. Insert the tube 8 cm intoa silicone-
oil bath at 190°-200°. Perform a blank test without the
addition of Hymetellose.
Acceptance criteria: The filter paper becomes blue
within 10 min.
o E.
Sample: 0.2g
Analysis: Dissolve the Sample completely, without heat-
ing, in 15 mL of 70% sulfuric acid. Pour the solution
while stirring into 100 mL of ice water, and dilute with
ice water to 250 mL. Transfer 1 mL of this solution to a
NF 36
test tube, and while cooling in ice water, add dropwise
8 mL of sulfuric acid, and mix thoroughly. Heat in a
water bath for exactly 3 min, and immediately cool in
ice water. While the mixture is cold, carefully add
pe mL of ninhydrin TS, and mix well. Allow to stand at
Acceptance criteria: A pink color is produced immedi-
ately and does not become violet within 100 min.
IMPURITIES
e RESIDUE ON IGNITION (281)
Sample: 1.0g
Acceptance criteria: NMT 1.0%
¢ CHLORIDE AND SULFATE, Chloride (221)
Sample solution: Dilute 1.0 mL of the Sample solution
from the test for Color of Solution with water to 20 mL,
and add 1 mL of nitric acid and 1 mL of silver nitrate
TS. Mix, and allow to stand for 5 min, protected from
direct sunlight.
Control solution: Dilute 0.71 mL of 0.020 N hydro-
chloric acid to 100 mL. Mix 10 mL of this solution with
water to 20 mL, and add 1 mL of nitric acid and 1 mL
of silver nitrate TS. Mix, and allow to stand for 5 min,
protected from direct sunlight.
Analysis
(See Nephelometry, Turbidimetry, and Visual Comparison
(855), Visual Comparison.)
Compare the turbidity of the Sample solution and Con-
trol solution, if any.
Acceptance criteria: 0.5%. Any turbidity produced by
a Sample solution does not exceed that of the Contro/
solution.
Delete the following:
°o HEAVY METALS, Method /I (231): NMT 20 ,tg/Ge vorrei.
Jan-2018)
SPECIFIC TESTS
e PH (791)
Sample solution: Use the Sample solution from the test
for Color of Solution.
Acceptance criteria: 5.5-8.0
e Loss ON DRYING (731)
Sample: 1.0g
Analysis: Dry the Sample at 105° to constant weight.
Acceptance criteria: NMT 10.0%
e ViIsCcOsITY—ROTATIONAL METHODS (912)
Sample: An amount equivalent to 6.0 g of dried
Hymetellose
Analysis: While stirring, add the Sample to 150 g of car-
bon dioxide-free water heated to 90°. Stir with a pro-
peller-type stirrer for 10 min, place the flask in a bath of
ice water, continue the stirring, and allow to remain in
the bath of ice water for 40 min to ensure that dissolu-
tion is complete. Adjust the mass of the solution to
300 g, and centrifuge the solution to expel any en-
trapped air. Adjust the temperature of the solution to
20+0.1°, and determine the viscosity using a rotational
viscometer with a shear rate of 10/s.
Acceptance criteria: The apparent viscosity is
75%-140% of the value stated on the label.
e COLOR OF SOLUTION
Diluent: 27.5 mL of hydrochloric acid in 1000 mL of
water
Standard solution: Prepare immediately before use.
Mix 2.4 mL of ferric chloride CS and 0.6 mL of cobal-
tous chloride CS with Diluent to make 10 mL, and dilute
5 mL of this solution with Diluent to make 100 mL.
Sample solution: While stirring, add a portion equiva-
lent to 1.0 g of dried Hymetellose to 50g of carbon
dioxide-free water heated to 90°. Allow to cool, adjust
the weight of the solution to 100 g with carbon diox-
ide-free water, and stir until dissolution is complete.
 4490 Borage / Dietary Supplements
Analysis
Samples: Sample solution and Standard solution
[Note—The relative retention times for gamma methyl
linolenate and alpha methyl linolenate are about 0.98
and 1.0, respectively.]
Identify the retention times of the relevant fatty acid
methyl esters by comparing the peaks in the chromat-
ogram of the System suitability solution with those in
the reference chromatogram. Identify the locus for the
internal standard peak by comparison of the chromat-
ograms of the Standard solution and System suitability
solution.
Calculate the content, in mg/g, of y-linolenic, linoleic,
and oleic acids in the portion of Capsules taken:
Result = (Ru/Rs) x (Au/As) x (ms/W) x (Mii/M,2)
Ru = peak area ratio of the relevant methyl ester to
the internal standard from the Sample
solution
Rs = peak area ratio of the relevant methyl ester to
the internal standard from the Standard
solution
Au = weight of the Internal standard in the Sample
solution (mg)
As = weight of the Internal standard in the Standard
solution (mg)
ms = weight of the relevant USP Methyl Ester RS in
the Standard solution (mg)
w = sample weight used to prepare the Sample
solution (g)
Mi = neat weight of the relevant fatty acid (g/
mol)
Mz = molecular weight of the relevant fatty acid
methyl ester (g/mol)
Calculate the percentage of the labeled amounts of
each (y-linolenic, linoleic, and oleic) acid in the portion
of Capsules taken:
Result = A x Ar x (100/L)
A = content of the relevant fatty acid in the
portion of Capsule content taken (mg/g)
Ar = average fill weight (g)
L = labeled content of the relevant fatty acid (mg/
Capsule)
Acceptance criteria: 95.0% of the labeled amounts of
y-linolenic, linoleic, and oleic acids
PERFORMANCE TESTS
© DISINTEGRATION AND DISSOLUTION (2040): Meet the re-
quirements in Rupture Test for Soft Shell Capsules
DS Monographs
© WEIGHT VARIATION OF DIETARY SUPPLEMENTS (2091): Meet
the requirements
SPECIFIC TESTS
e FATS AND FIXED OILS, Peroxide Value (401): NMT 10.0
CONTAMINANTS
e MICROBIAL ENUMERATION TESTS (2021): The total aerobic
microbial count does not exceed 1 x 103 cfu/g, and the
combined molds and yeasts count does not exceed
3 x 102 cfu/g.
e ABSENCE OF SPECIFIED MICROORGANISMS (2022): Meet the
requirements of the tests for absence of Escherichia coli
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in tightly-closed, light-
resistant containers.
e LABELING: The label states the article which the Capsules
were prepared with and content of y-linolenic, linoleic,
and oleic acids in mg/Capsule.
USP 41
e USP REFERENCE STANDARDS (11)
USP Borage Seed Oil RS
USP Methyl Linoleate RS
USP Methyl Linolenate RS
USP Methyl Oleate RS
Boswellia serrata
DEFINITION
Boswellia serrata is the oleogum resin obtained by incision or
puduced by spontaneous exudation from the stem and
ranches of Boswellia serrata Roxb. (Fam. Burseraceae). It
contains NLT 1.0% of the keto derivatives of B-boswellic
acid, calculated on the dried basis as the sum of 11-keto-
B-boswellic acid and 3-acetyl-11-keto-B-boswellic acid.
IDENTIFICATION
© A. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST
(201)
Standard solution: Treat a quantity of USP Boswellia
serrata Extract RS with gentle heating in methanol to
obtain a solution having a known concentration of
30 mg/mL, cool, centrifuge, and use the supernatant.
Sample solution: Use the Sample solution, prepared as
directed in the test below for Content of Keto-Derivatives
of B-Boswellic Acids, and concentrate to 10% of the
volume.
Asner 0.25-mm layer of chromatographic silica
ge
Developing solvent system: A mixture of hexane and
ethyl acetate (6:4)
Derivatization reagent: Prepare a solution of 10% sul-
furic acid in methanol. [NoTE—Prepare immediately
before use.]
Application volume: 10 uL
Analysis
Samples: Standard solution and Sample solution
Apply the samples as bands to a suitable thin-layer
chromatographic plate (see Chromatography (621)).
Use a saturated chamber. Develop until the solvent
front has moved up about 90% of the plate. Remove,
dry, and examine under UV light at 254 nm. Dip in
the Derivatization reagent, heat for 5-10 min at 100°,
and examine under white light.
Acceptance criteria: Under UV light at 254 nm, the
Sample solution exhibits two main zones due to 11-keto-
B-boswellic acid and 3-acetyl-11-keto-B-boswellic acid at
Re values of about 0.30 and 0.36, respectively, corre-
sponding to zones from the Standard solution. Under
white light, the Sample solution exhibits two additional
zones due to B-boswellic acid and 3-acetyl-B-boswellic
acid at Rr values of about 0.49 and 0.58, respectively,
corresponding to zones from the Standard solution.
Other, less intense zones are observed for the Sample
solution and the Standard solution.
e B. The 210-nm chromatogram of the Sample solution, in
the test for Content of Keto-Derivatives of B-Boswellic Acids,
exhibits peaks for 11-keto-B-boswellic acid, 3-acetyl-
1 1-keto-B-boswellic acid, B-boswellic acid, and 3-acety|-B-
boswellic acid at retention times that correspond to
those in the 210-nm chromatogram of Standard solution
Band the 210-nm reference chromatogram provided
with the USP Boswellia serrata Extract RS.
COMPOSITION
e CONTENT OF KETO-DERIVATIVES OF B-BOSWELLIC ACIDS
Standard solution A: Dissolve a quantity of USP 3-Ace-
tyl-11-keto-B-Boswellic Acid RS in methanol to obtain a
solution having a known concentration of 0.1 mg/mL.
Standard solution B: Treat a quantity of USP Boswellia
serrata Extract RS with gentle heating in methanol to
obtain a solution having a known concentration of
NF 36
Analysis: Make the comparison by viewing the sub-
stance and the solution downward in matched color-
comparison tubes against a white surface (see Color and
Achromicity (631)).
Acceptance criteria: The Sample solution is not more
intensely colored than the Standard solution.
© CLARITY OF SOLUTION
Hydrazine sulfate solution: 10 mg/mL of hydrazine
sulfate. Allow to stand for 4-6 h before use.
[CauTion—Hydrazine sulfate is highly toxic. Avoid skin
contact.]
Methenamine solution: Transfer 2.5 g of methenamine
to a 100-mL glass-stoppered flask, add 25.0 mL of
water, insert the glass stopper, and mix to dissolve.
Primary opalescent mixture: To the flask containing
Methenamine solution add 25.0 mL of Hydrazine sulfate
solution, mix, and allow to stand for 24 h. This suspen-
sion is stable for 2 months. Mix before use, and do not
use if it adheres to the container.
Opalescence standard: Dilute 15.0 mL of Primary opal-
escent mixture with water to 1000.0 mL. Use this sus-
pension within 24 h after preparation.
Reference suspension: Transfer 30.0 mL of Opalescence
Standard to a 100-mL volumetric flask, and dilute with
water to volume.
Sample solution: Use the solution from the test for
Color of Solution.
Analysis: Transfer a sufficient portion of the Sample so-
lution to a test tube of colorless, transparent, neutral
glass with a flat base and an internal diameter of
15-25 mm to obtain a depth of 40 mm. Similarly trans-
fer a portion of the Reference suspension to a separate
matching test tube. Compare the Sample solution and
the Reference suspension in diffused daylight, viewing
vertically against a black background (see Nephelometry,
Turbidimetry, and Visual Comparison (855), Visual Com-
parison) 5 min after preparation of the Reference
Suspension.
Acceptance criteria: The Sample solution is not more
opalescent than the Reference suspension.
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in well-closed contain-
ers. No storage requirements are specified.
e LABELING: Label it to indicate the viscosity of the solution
(1 in 50) at 20°.
Hypophosphorous Acid
H3PO2 66.00
Phosphinic acid;
Hypophosphorous acid [6303-21-5].
DEFINITION
Hypophosphorous Acid contains NLT 30.0% and NMT
2.0% of H3POz.
IDENTIFICATION
e A. Hypophosphorous Acid yields a white precipitate with
mercuric chloride TS. This precipitate becomes gray
when an excess of hypophosphite is present.
e B. Hypophosphorous Acid acidified with sulfuric acid and
warmed with cupric sulfate TS yields a red precipitate.
ASSAY
© PROCEDURE
Sample solution: Pour 7 mL of Hypophosphorous Acid
into a tared, glass-stoppered flask, and weigh. Dilute
with about 25 mL of water, and add phenolphthalein
TS.
Official Monographs / Hypromellose 5391
Analysis: Titrate with 1 N sodium hydroxide VS. Each
mL of 1 N sodium hydroxide is equivalent to 66.00 mg
of H3PO2.
Acceptance criteria: 30.0%-32.0%
IMPURITIES
Inorganic Impurities
Delete the following:
°e HEAVY METALS, Method |/ (231)
Analysis: Place 0.90 mL (1 Q of Hypophosphorous Acid
in a small beaker, and add 3 mL of water. Add 1 mL of
nitric acid, and evaporate on a steam bath to about
1 mL. Again add | mL of nitric acid, and evaporate on a
steam bath. Dissolve the residue in 3 mL of water, add
6 N ammonium hydroxide until the solution is distinctly
alkaline to litmus, then boil gently until the odor of
ammonia disappears. Add 2 mL of 1 N acetic acid and
15 mL of warm water, filter, and dilute the filtrate with
water to 25 mL.
Acceptance criteria: NMT 20 pome (oiticia 1-jen-2018)
SPECIFIC TESTS
e Limit OF BARIUM AND OXALATE
Sample solution: Hypophosphorous Acid and water
(1:3)
Analysis 1: Neutralize 30 mL of the Sample solution with
6 N ammonium hydroxide: the mixture exhibits little or
recipitation. Filter, acidify 10 mL of the filtrate with
Me rochloric acid, and add 2 mL of potassium sulfate
TS;
Acceptance criteria 1: No turbidity is produced
(barium).
Analysis 2: To a 10-mL portion of the filtrate obtained
in Analysis 1, add 1 mL of calcium chloride TS.
Acceptance criteria 2: The filtrate shows no turbidity
upon the addition of the calcium chloride TS (oxalate).
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in tight containers.
Hypromellose Acetate Succinate
Hydroxypropyl methylcellulose acetate succinate;
Cellulose, 2-hydroxypropyl methyl ether, acetate hydrogen
butanedioate;
Cellulose, 2-hydroxypropyl methyl ether, acetate succinate
[71138-97-1].
DEFINITION
Hypromellose Acetate Succinate is a mixture of acetic acid
and monosuccinic acid esters of hydroxypropyl methylcel-
lulose. It contains NLT 12.0% and NMT 28.0% of
methoxy groups (-OCH3), NLT 4.0% and NMT 23.0% of
hydroxypropoxy groups (-OCH2CHOHCHs), NLT 2.0%
and NMT 16.0% of acetyl groups (-COCHs), and NLT
4.0% and NMT 28.0% of succinoyl groups
(-COC2H4COOh), calculated on the dried basis.
IDENTIFICATION
e A. INFRARED ABSORPTION (197A)
ytrelFL Lele AN BEN
Sample: Neat. Do not dry specimen.
Analysis: Use a Fourier transform IR spectrophotometer
fitted with a suitable accessory for single bounce atten-
uated total reflectance (see Mid-Infrared Spectroscopy
{854)) with a diamond or germanium crystal. Acquire a
background single-beam spectrum with a clean dia-
mond or germanium crystal sampling plate in place.
Place the sample on the diamond or germanium crystal
sampling surface with a microspatula or equivalent. For
best results, the sample should cover the crystal surface
 5392 Hypromellose / Official Monographs NF 36

under the pressure point tip. Using the pressure device, Analysis
apply pressure to the sample, making sure the sample Samples: Standard solution 1 and Sample solution
remains centered under the pressure tip. Acquire a sin- Calculate the percentage of acetic acid, A, in the por-
gle-beam spectrum of the sample, and make the neces- tion of Hypromellose Acetate Succinate taken:
sary corrections for the background. Release the pres-
sure device, and clear it from the sample area. Wipe the A = (tualtsa) x (Ca/Cu) x 100
sample off the crystal and pressure device tip, and rinse
both with acetone. Tua = peak response for acetic acid from the Sample
Acceptance criteria: The IR spectrum of the Sample ex- solution
hibits maxima only at the same wavelengths as a simi- rsa = peak response for acetic acid from Standard
larly obtained spectrum of USP Hypromellose Acetate solution 1
Succinate RS. Ca = concentration of acetic acid in Standard
solution 1 (mg/mL)
ASSAY Cu = concentration of So ereurnos Acetate
e ACETYL AND SUCCINOYL GROUPS Succinate in the Sample solution (mg/mL)
Phosphoric acid solution: 1.25 M phosphoric acid and Calculate the percentage of acetyl groups (-COCHs) in
water (2:98) the portion of Hypromellose Acetate Succinate taken:
Buffer: 2.72 g/L of monobasic potassium phosphate
Diluent: Adjust the Buffer with 1 N sodium hydroxide to Result = (A — Afe) (Mi1/Mi2)
a pH of 7.5.
Acetic acid stock solution: Add approximately 20 mL A = defined above
of water to a stoppered, 100-mL volumetric flask, place Aree = percentage of free acetic acid, as determined
the flask on a balance, and tare. Transfer 2.0 mL of gla- in the test for Limit of Free Acetic and Succinic
cial acetic acid to the flask, and record the weight of Acids
the acid added. Fill the flask with water to volume. Mn = molecular weight of the acetyl group, 43.04
Transfer 6 mL of the resulting solution into a 100-mL M2 = molecular weight of acetic acid, 60.05
volumetric flask, and dilute with water to volume. Calculate the percentage of succinic acid, S, in the
AUS acid stock solution: 1.3 mg/mL of succinic portion of Hypromellose Acetate Succinate taken:
aci
Mobile phase: Adjust the Buffer to a pH of 2.8 by the S = (rus/rss) x (Cs/Cu) x 100
dropwise addition of 6 M phosphoric acid. Pass through
Tus = peak response for succinic acid from the
a 0.22-um nylon filter.
Standard solution 1: Transfer 4.0 mL of the Acetic acid Sample solution
stock solution and 4.0 mL of the Succinic acid stock solu- rss = peak response for succinic acid from Standard
solution 1
tion to a 25-mL volumetric flask. Dilute with Mobile
phase to volume, and mix. Cs = concentration of succinic acid in Standard
solution 1 (mg/mL)
Standard solution 2: Prepare as directed for Standard
solution 1. This solution is prepared as a duplicate. Cy = concentration of Hypromellose Acetate
Succinate in the Sample solution (mg/mL)
Sample solution: Weigh 12.4 mg of Hypromellose Ace-
tate Succinate into a glass vial. Transfer 4.0 mL of 1.0 N Calculate the percentage of succinoy! groups
sodium hydroxide to the vial, and stir the solution for 4 (-COC,H4COOH) in the portion of Hypromellose
h. Then, add 4.0 mL of 1.25 M phosphoric acid to the Acetate Succinate taken:
same vial to bring the pH of the solution to 3 or less. Result = (S — Stee) x (Mri/Mrz)
Invert the test Sample solution vial several times to en-
sure complete mixing, and pass throughafilter of 0.22- iS = defined above
um pore size. Use the clear filtrate. Stee | = percentage of free succinic acid, as
Chromatographic system determined in the test for Limit of Free Acetic
(See Chromatography (621), System Suitability.) and Succinic Acids
Mode: LC My = molecular weight of the succinoyl group,
Detector: UV 215 nm 101.08
Column: 4.6-mm x 15-cm; 5-um packing L1 Mz = molecular weight of succinic acid, 118.09
Column temperature: 20°-30° Acceptance criteria
Flow rate: 1 mL/min Acetyl groups (-COCHs): 2.0%-16.0% on the dried
Run time: 15 min basis
Injection volume: 10 uL Succinoyl groups (-COC,H,COOH): 4.0%-28.0% on
System suitability the dried basis
Samples: Standard solution 1 and Standard solution 2 e@ CONTENT OF METHOXY AND 2-HYDROXYPROPOXY GROUPS
Suitability requirements [CauTIoN—Hydriodic acid and its reaction byproducts are
Column efficiency: NLT 8000 theoretical plates, de- highly toxic. Perform all steps in the preparation of the
termined from the succinic acid peak, Standard solu- Sample solution and the Standard solution in a properly
tion 1 functioning hood. Specific safety practices to be followed
Tailing factor: 0.9-1.5 for the succinic acid peak, are to be identified to the analyst performing this test.]
Standard solution 1 Hydriodic acid: Use a reagent having a specific gravity
Relative standard deviation: NMT 2.0% for each of at least 1.69, equivalent to 55% hydrogen iodide.
peak from six replicate injections, Standard solution 1
NF Monographs

Solution A: Methanol and water (10:90)

Peak difference: The difference in peak areas be- Solution B: Methanol and water (85:15)
tween Standard solution 1 and Standard solution 2 for Mobile phase: See Table 1.
both acetic and succinic acids peaks does not exceed
2%.
([Note—After each run sequence, the column should be Table 1
flushed first by 50% water and 50% acetonitrile for 60 Time Solution A Solution B
min and then by 100% methanol for 60 min. The col- (min) (%) (%)
umn should be stored in 100% methanol.] 0 70 30
8 40 60
NF 36
Table 1 (Continued)
Time Solution A Solution B
(min) (%) (%)
10 15 85
17 15 85
[Note—These gradient elution times are established on
an HPLC system with a dwell volume of approximately
2.0 mL. The injection time can be adjusted relative to
the start of a run to accommodate the changein
dwell volume from one HPLC system to another to
achieve the separation described.]
Standard stock solution: Transfer 2 mL of o-xylene into
a stoppered, 10-mL volumetric flask, place the flask on
a balance, and tare. Add 200 pL of methyl iodide, insert
the stopper into the flask, and accurately weigh: the
weight of methyl iodide is about 350 mg. Tare the flask
again, add 34 LL of isopropyl iodide, and weigh the
flask: the recorded weight of isopropyl iodide is 50 mg.
Dilute with o-xylene to volume, and mix.
Standard solution: Transfer 85 mg of adipic acid into
an 8-mL vial (or other suitable container), add 2 mL of
Hydriodic acid, and add 2.0 mL of the Standard stock
solution. Shake and allow the phases to separate. Care-
fully transfer approximately 1.5 mL of the o-xylene (top)
layer to a small vial, making sure that the bottom aque-
ous layer is not disturbed. Transfer 1.0 mL of the result-
ing solution to a 10-mL volumetric flask, and dilute
with methanol to volume. [NOTE—This solution is stable
for 8 h at 5°.]
Sample solution: [CAuTIoN—Use a cap that has a top
safety relief valve, such as a Minniert valve, to prevent
accidental explosion of the vial under high pressure
when heated.] Weigh 65 mg of Hypromellose Acetate
Succinate into a 5-mL reaction vial, and add 2.0 mL of
o-xylene and about 100 mg of ae acid. Add 2.0 mL
of Hydriodic acid, and close the vial tightly with a cap.
Weigh the vial before heating, and place the vial into a
heating block at 150°. Shake the vial after 5 min and
after 30 min of heating. Remove the vial from the
heating block after 1h of heating, and cool. Weigh
the vial. If the weight loss is greater than 10 mg, dis-
card the mixture, and prepare another reaction solu-
tion. Carefully transfer approximately 1.5 mL of the
top o-xylene layer into a small glass vial, making sure
that the bottom aqueous layer is not disturbed. Trans-
fer 1.0 mL of this solution into a 10-mL volumetric
flask, and dilute with methanol to volume. [NoTE—This
solution is stable for 8 h at 5°.]
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 254 nm
Column: 4.6-mm x 15-cm; 5-um packing L1
Column temperature: 30°
Flow rate: 1 mL/min
Injection volume: 10 LL
System suitability
Sample: Standard solution
Suitability requirements
Column efficiency: NLT 10,000 theoretical plates,
determined from the methyl iodide peak
Tailing factor: 0,.9-1.5 for the methyl iodide peak
Relate standard deviation: NMT2.0% for each
pea
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of methoxy groups (-OCHs) in
the portion of Hypromellose Acetate Succinate taken:
Result = (rum/tsm) x (Cs/Cu) x (Mr/Mr2)
tum = peak response for methyl iodide from the
Sample solution
Official Monographs / Hypromellose 5393
Ts = peak response for methyl iodide from the
Standard solution
Gs = concentration of methyl iodide in the Standard
solution (mg/mL)
Cu = concentration of Hypromellose Acetate
Succinate in the Sample solution (mg/mL)
Ma = molecular weight of the methoxy group,
31.03
Mz = molecular weight of methyl iodide, 141.94
Calculate the percentage of 2-hydroxypropoxy groups
(-OCH2CHOHCHs) in the portion of Hypromellose
Acetate Succinate taken:
Result = (ru/rsi) < (Cs/Cu) & (Ma/My2)
Tu = peak response for isopropyl iodide from the
Sample solution
rs = peak response for isopropyl iodide from the
Standard solution
Cs = concentration of isopropyl iodide in the
Standard solution (mg/mL)
Cu = concentration of Hypromellose Acetate
Succinate in the Sample solution (mg/mL)
M1 = molecular weight of the 2-hydroxypropoxy
group, 75.09
Mz = molecular weight of isopropyl iodide, 169.99
Acceptance criteria
Methoxy groups (-OCHs): 12.0%-28.0% on the
dried basis
Hydroxypropoxy groups (-OCH2CHOHCHs3):
4.0%-23.0% on the dried basis
IMPURITIES
e RESIDUE ON IGNITION (281)
Analysis: Determine at 600 + 50°.
Acceptance criteria: NMT 0.20%
Delete the following:
°e HEAVY METALS, Method i! (231): NMT 10 ug/ge corticia 1-
Jan-2018)
e LIMIT OF FREE ACETIC AND SUCCINIC ACIDS
Phosphoric acid solution, Buffer, Diluent, Acetic acid
stock solution, Succinic acid stock solution, Mobile
phase, Standard solution, and Chromatographic sys-
tem: Proceed as directed in the Assay for Acetyl and
Succinoyl Groups.
Sample solution: Weigh 102 mg of Hypromellose Ace-
tate Succinate into a glass vial. Transfer 4.0 mL of Dilu-
ent to the vial, and stir the content for 2 h. Then, trans-
fer 4.0 mL of the Phosphoric acid solution to the same
vial to bring the pH of the Sample solution to 3 or less.
Invert the vial several times to ensure complete mixing,
centrifuge, and use the clear supernatant.
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of free acetic acid, Aree, in the
portion of Hypromellose Acetate Succinate taken:
Arce = (rua! rsa) x (CalCu) x 100
Tua = peak response for acetic acid from the Sample
solution
Isa = peak response for acetic acid from the
sydeibouo- 4N
Standard solution
Ga = concentration of acetic acid in the Standard
solution (mg/mL)
Cu = concentration of Hypromellose Acetate
Succinate in the Sample solution i
Calculate the percentage of free succinic acid, See, in
the portion of Hypromellose Acetate Succinate taken:
Stee = (rus/rss) x (Cs/Cy) x 100
 5394 Hypromellose / Official Monographs NF 36

Tus = peak response for succinic acid from the IMPURITIES

Sample solution ¢ RESIDUE ON IGNITION (281): NMT 0.20%
rss = peak response for succinic acid from the e CHLORIDE AND SULFATE, Chloride (221)
Standard solution Sample solution: Dissolve 1.0 g in 40 mL of 0.2 N so-
Cs = concentration of succinic acid in the Standard dium hydroxide, add 1 drop Gf phenelphthaleis TS,
solution (mg/mL) and add 2N nitric acid dropwise, with stirring, until the
Cu = concentration of Hypromellose Acetate red color is discharged. Add an additional 20 mL of 2N
Succinate in the Sample solution (mg/mL) nitric acid with stirring. Heat on a water bath, with stir-
Acceptance criteria: The sum of free acetic acid and ting, until the gel-like precipitate formed becomes gran-
free succinic acid is NMT 1.0%. ular. Cool the mixture, and centrifuge. Separate the liq-
uid phase, and wash the residue with three successive
SPECIFIC TESTS 20-mL portions of water, separating the washings by
e Loss ON DRYING (731) centrifuging. Dilute the combined liquids with water to
Analysis: Dry at 105° for 1 h. 200 mL, mix, and filter.
Acceptance criteria: NMT 5.0% Standard solution: Treat 0.50 mL of 0.01 N hydrochlo-
e Viscosity—CAPILLARY METHODS (911) ric acid with 10 mL of 0.2 N sodium hydroxide, add
Sodium hydroxide solution: Immediately before use, 7 mL of 2.N nitric acid, and dilute with water to 50 mL.
prepare 4.3 mg/mL of sodium hydroxide in carbon di- Acceptance criteria: A 50-mL portion of the Sample so-
oxide-free water. lution shows no more chloride than the Standard solu-
Analysis: To 2.00 g of Hypromellose Acetate Succinate, tion (0.07%).
previously dried, add Sodium hydroxide solution to make
100.0 g, insert a stopper into the vessel, and dissolve b'
constant shaking for 30 min. Adjust the temperature o Delete the following:
the solution to 20 +0.1°, and determine the viscosity in
a suitable viscometer. °e HEAVY METALS, Method I! (231): NMT 10 'g/ge coma.
eens criteria: 80%-120% of that stated on the Jen-2018)
abe e LIMIT OF FREE PHTHALIC ACID
Pec ee Acetonitrile and 0.1 M cyanoacetic acid
ADDITIONAL REQUIREMENTS 785
© PACKAGING AND STORAGE: Preserve in tight containers. No Standard solution: Transfer 12.5 mg of phthalic acid to
storage requirements specified. a 250-mL volumetric flask, and add 125 mL of acetoni-
e LABELING: Label it to indicate its nominal viscosity type. trile. Add 25 mL of water, and dilute with acetonitrile to
e USP REFERENCE STANDARDS (11) volume.
USP Hypromellose Acetate Succinate RS Sample solution: Transfer 200 mg of Hypromellose
Phthalate to a 100-mL volumetric flask. Add 50 mL of
acetonitrile, and sonicate to dissolve partially. Add
10 mL of water, and sonicate to dissolve. Cool to room
temperature, and dilute with acetonitrile to volume.
Hypromellose Phthalate Chromatographic system
(See Chromatography (621), System Suitability.)
DEFINITION Mode:
Hypromellose Phthalate is a monophthalic acid ester of Detector: UV 235 nm
hydroxypropyl methylcellulose. It contains methoxy Column: 4.6-mm x 25-cm; packing L1 with a high
(-OCH3), hydroxypropoxy (-OCH2CHOHCHs), and carbon load
phthalyl (o-carboxybenzoyl, CgHsO3) groups. It contains Flow rate: 2 mL/min
NLT 21.0% and NMT 35.0% of phthalyl groups, calcu- Injection volume: 10 pL
lated on the anhydrous basis. System suitability
Sample: Standard solution
IDENTIFICATION Suitability requirements
e A. INFRARED ABSORPTION (197K): Do not dry specimens. Relative standard deviation: NMT 1.0% for replicate
injections
ASSAY Analysis
e PHTHALYL CONTENT Samples: Standard solution and Sample solution
Sample: 1g Calculate the percentage of phthalic acid in the por-
Analysis: Transfer the Sample to a conical flask, dissolve tion of Hypromellose Phthalate taken:
in 50 mL of a mixture of alcohol, acetone, and water
(2:2:1), add phenolphthalein TS, and titrate with 0.1 N Result = (ru/rs) x (Cs/Cu) x 100
sodium hydroxide VS. Perform a blank determination
(see Titrimetry (541)). ty = peak response of phthalic acid from the
Calculate the percentage of phthalyl taken: Sample solution
rs = peak response of phthalic acid from the
Result = [0.01 x Ma x (V/W)] — [2 x (Ma/Mr2) x PI Standard solution
Gs = concentration of phthalic acid in the Standard
Mn molecular weight of the phthalyl group, 149.1 Solution (mg/mL)
”“ V volume of 0.1 N sodium hydroxide consumed Cu = concentration of Hypromellose Phthalate in
= after correction for the blank (mL) the Sample solution (mg/mL)
a Ww weight of Hypromellose Phthalate taken, Acceptance criteria: NMT 1.0%
i
i}

calculated on the anhydrous basis (g)

a
=

i) Ma molecular weight of phthalic acid, 166.1 SPECIFIC TESTS

fe P percentage of free phthalic acid found as e WATER DETERMINATION, Method | (921): NMT 5.0%
5 directed in the test for Limit of Free Phthalic e ViscosiTy—CAPILLARY METHODS (911)
= Acid Sample solution: Dissolve 10 g, previously dried at
J Acceptance criteria: 21.0%-35.0% of phthalyl groups 105° for 1 h, in 90 g of a mixture of methanol and
re on the anhydrous basis methylene chloride (1:1 w/w) by mixing and shaking.
NF 36 Official Monographs / Inositol 5395

Analysis: Determine the viscosity at 20 + 0.1°. IMPURITIES

Acceptance criteria: 80%-120% of that indicated by e RESIDUE ON IGNITION (281): NMT 3.0%
the label
ADDITIONAL REQUIREMENTS Delete the following:
© PACKAGING AND STORAGE: Preserve in tight containers.
e LABELING: Label it to indicate its viscosity and nominal ®o HEAVY MetaLs, Method I/ (231): NMT 10 19/9e cotficia 1-
phthaly! content. Jan-2018)
e USP REFERENCE STANDARDS (11)
USP Hypromellose Phthalate RS SPECIFIC TESTS
© PH (791)
Sample solution: 10 mg/mL
Acceptance criteria: 6.0-7.5
e Loss ON DRYING (731)
Analysis: Dry under vacuum over phosphorus pentox-
Imidurea ide for 48 h.
Acceptance criteria: NMT 3.0%
9.
"9 on
if go e COLOR AND CLARITY OF SOLUTION
Sample: 3.0g
wT J fh Tn Analysis: Dissolve the Sample in 7.0 mL of water in a
ONO
a TY8 test tube.
Acceptance criteria: The solution is clear and colorless.
CiHigNgOg 388.29 ADDITIONAL REQUIREMENTS
N,N’-Methylenebis[N’-[3-(hydroxymethyl)-2,5-dioxo- © PACKAGING AND STORAGE: Preserve in tight containers.
4-imidazolidinyl]urea]; e USP REFERENCE STANDARDS (11)
1,1’-Methylenebis[3-[3-(hydroxymethyl)-2,5-dioxo- USP Imidurea RS
4-imidazolidiny!]urea] [39236-46-9].
DEFINITION
Imidurea contains NLT 26.0% and NMT 28.0% of nitrogen
(N), calculated on the dried basis. Inositol
IDENTIFICATION
pH
e A. INFRARED ABSORPTION (197K) Vf
OTHER COMPONENTS tom (on
© NITROGEN CONTENT ud ‘OH
Sample: 150mg
Titrimetric system
(See Titrimetry (541).) C6Hi206 180.16
Mode: Direct titration cis-1,2,3,5-trans-4,6-Cyclohexanehexol;
Titrant: 0.1 N hydrochloric acid VS myo-Inositol [87-89-8].
Endpoint detection: Visual DEFINITION
Analysis: Place the Sample in a 500-mL Kjeldahl flask, Inositol contains NLT 97.0% and NMT 102.0% of Inositol
add 8.0g of anhydrous sodium sulfate, 0.5 g of (CsH120¢), calculated on the anhydrous basis.
anhydrous cupric sulfate, 0.1 g of yellow mercuric ox-
ide, and 11 mL of sulfuric acid. Incline the flask at an IDENTIFICATION
angle of 45°, and gently heat the mixture, keeping the e A. INFRARED ABSORPTION (197K)
temperature below the boiling point until tara has ¢ B. The retention time of the major peak of the Sample
ceased. Increase the heat until the acid boils briskly, solution corresponds to that of the Standard solution, as
and continue the heating until the solution has become obtained in the Assay.
clear green in color or practically colorless for 30 min.
Allow to cool, cautiously add 225 mL of water, mix the ASSAY
contents of the flask, and again cool. Add cautiously © PROCEDURE
1.0g of zinc metal dust, 2.0 g of sodium thiosulfate, Mobile phase: Water
and 18.0 g of sodium hydroxide pellets, and without System suitability solution: 0.05 mg/mL of USP Inosi-
delay connect the flask to a Kjeldahl connecting bulb tol RS and 0.05 mg/mL of USP Mannitol RS
(trap), previously attached to a condenser, the delivery Standard solution: 50 mg/mL of USP Inositol RS
tube of which dips beneath the surface of 50 mL of Sample solution: 50 mg/mL of Inositol
boric acid solution (40 mg/mL) in a 300-mL conical Chromatographic system
flask. Mix the contents of the Kjeldahl flask by gentle (See Chromatography (621), System Suitability.)
rotation, and distill 125 mL into the receiver. Add NLT Mode: LC
3 drops of methyl red-methylene blue TS to the con- Detector: Refractive index
tents of the receiving vessel, and determine the ammo- Column: 7.8-mm x 30-cm or equivalent; packing L19
nia by titration with Titrant. Perform a blank determina- Temperature
LeyABETN]

tion, and make any necessary correction. Column: 85°

Calculate the percentage of nitrogen in the portion of Detector: Constant temperature of 30°-35°
ExtTe l-BLele

Imidurea taken. Each mL of 0.1 N hydrochloric acid is Flow rate: 0.5 mL/min
equivalent to 1.401 mg of nitrogen. Injection volume: 10 uL
Acceptance criteria: 26.0%-28.0% of nitrogen (N) on System suitability
the dried basis Samples: System suitability solution and Standard
solution
[Note—The relative retention times for inositol and
mannitol are 1.0 and 1.3, respectively.]
 5396 Inositol / Official Monographs
Suitability requirements
Resolution: NLT 4.0 between inositol and mannitol,
System suitability solution
Relative standard deviation: NMT 2.0%, Standard
solution
Analysis
Samples: Standard solution and Sample solution
[NotE—Record the chromatograms over a period of
two times the retention time of inositol, and measure
the peak responses.]
Calculate the percentage of Inositol (CséH:206¢) in the
portion of sample taken:
Result = (ru/rs) x (Cs/Cu) x 100
tu = peak response of inositol from the Sample
solution
Is = peak response of inositol from the Standard
solution
Cs = concentration of USP Inositol RS in the
Standard solution (mg/mL)
Cu = concentration of Inositol in the Sample solution
(mg/mL)
Acceptance criteria: 97.0%-102.0% on the anhydrous
basis
IMPURITIES
e BARIUM
Sample solution: Use the Sample solution prepared in
the test for Clarity of Solution. To 10 mL of the Sample
Solution add 1 mL of diluted sulfuric acid.
Acceptance criteria: When examined immediately and
after 1 h, any opalescence in the solution is not more
intense than that in a mixture of 1 mL of water and
10 mL of the Sample solution from the test for Clarity of
Solution.
e Limit oF LEAD
Lead nitrate stock solution: Dissolve 159.8 mg of lead
nitrate in 100 mL of water to which has been added
1 mL of nitric acid, then dilute with water to 1000 mL.
Prepare and store this solution in glass containers free
from soluble lead salts.
Standard lead solution: On the day of use, dilute
10.0 mL of the Lead nitrate stock solution with water to
100.0 mL. Each mL of the Standard lead solution con-
tains the equivalent of 10 ug of lead. A comparison so-
lution prepared on the basis of 100 uL of the Standard
lead solution per g of substance being tested contains
the equivalent of 1 part of lead per million parts of sub-
stance being tested.
Sample solution: Dissolve 20.0 g of Inositol in diluted
acetic acid, and dilute with diluted acetic acid to
100 mL. Add 2.0 mL of a saturated ammonium pyrroli-
dinedithiocarbamate solution (containing about 10 g of
ammonium pyrrolidinedithiocarbamate per L) and
10.0 mL of methyl isobutyl ketone, and shake for 30 s.
Protect from bright light. Allow the two layers to sepa-
rate, and use the methyl isobutyl ketone layer.
Blank solution: Prepare as directed for the Sample solu-
tion, except omit the use of Inositol.
Standard solutions: Prepare as directed for the Sample
solution, except prepare three Standard solutions by
adding 0.5, 1.0, and 1.5 mL, respectively, of the Stan-
dard lead solution in addition to the 20.0 g of Inositol to
be examined.
NF Monographs
Instrumental conditions
(See Atomic Absorption Spectroscopy (852).)
Mode: Atomic absorption
Analytical wavelength: 283.3 nm
Lamp: Lead hollow-cathode
Flame: Air-acetylene
Analysis: Set the atomic absorption spectrometer to
zero, using the Blank solution. Introduce the Sample so-
lution and each of the three Standard solutions into the
instrument, and record the steady absorbance reading.
NF 36
Plot the absorbance readings against the known con-
centrations of added lead (in jug), and draw a straight
line. Extrapolate the line until it meets the concentra-
tion axis to obtain the concentration, in mg/kg, of lead
in the sample.
Acceptance criteria: NMT 0.5 mg/kg
© ORGANIC IMPURITIES
Mobile phase, System suitability solution, and Sample
solution: Proceed as directed in the Assay.
Standard solution: Transfer 2.0 mL of the Standard so-
lution, prepared as directed in the Assay, to a 100-mL
volumetric flask, and dilute with water to volume.
[Note—This solution contains 1 mg/mL of inositol.]
Chromatographic system: Proceed as directed in the
Assay, except use an injection volume of 20 pL.
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of each impurity in the por-
tion of Inositol taken:
Result = (ru/rs) x (Cs/Cu) x 100
tu = peak response of any impurity from the
Sample solution
rs = peak response of inositol from the Standard
solution
Cs = concentration of USP Inositol RS in the
Standard solution (mg/mL)
Cu = concentration of Inositol in the Sample solution
(mg/mL)
Acceptance criteria
Individual impurities: NMT 0.3%
Total impurities: NMT 1.0%. [NoTE—Disregard any
impurity peak that is less than 0.05%.]
SPECIFIC TESTS
© CLARITY OF SOLUTION
[NoteE—The Sample solution is to be compared to Refer-
ence suspension A in diffused daylight 5 min after prepa-
ration of Reference suspension A.
Solution A: 10 mg/mL of hydrazine sulfate. Allow to
stand for 4-6 h before use.
Solution B: 100 mail of methenamine prepared in a
glass-stoppered flask.
Primary opalescent suspension: [NOTE—This suspen-
sion is stable for 2 months, provided it is stored in a
glass container free from surface defects. The suspen-
sion must not adhere to the glass and must be well
mixed before use.] Solution A and Solution B (1:1). Allow
to stand for 24 h.
Opalescence standard: Transfer 15.0 mL of the Primary
opalescent suspension to a 1000-mL volumetric flask, di-
lute with water to volume. [NoTE—This suspension
should not be used beyond 24hafter preparation.]
Reference suspensions
Reference suspension A: Transfer 5.0 mL of the Opal-
escence standard to a 100-mL volumetric flask, and di-
lute with water to volume.
Reference suspension B: Transfer 10.0 mL of the
Opalescence standard to a 100-mL volumetric flask,
and dilute with water to volume.
Sample solution: 100 mg/mL of Inositol
Analysis: Transfer a sufficient portion of the Sample so-
lution to a sample tube of colorless, transparent, neutral
glass, with a flat base and an internal diameter of
15-25 mm, to obtain a depth of 40 mm. Similarly
transfer portions of Reference suspension A, Reference sus-
pension B, and water to separate matching sample
tubes. Compare the Sample solution, Reference suspen-
sion A, Reference suspension B, and water in diffused
daylight, viewing vertically against a black background
(see Nephelometry, Turbidimetry, and Visual Comparison
(855)). [NoTE—The diffusion of light must be such that
Reference suspension A can readily be distinguished from
NF 36
water, and that Reference suspension B can readily be
distinguished from Reference suspension A.]
Acceptance criteria: The Sample solution shows the
same clarity as that of water.
© COLOR OF SOLUTION
Standard stock solutions: Prepare three solutions, A, B,
and C, containing, respectively, the following parts of
ferric chloride CS, cobaltous chloride CS, cupric sulfate
CS, and diluted hydrochloric acid.
Standard stock solution A: 2.4: 0.6: 0: 7.0
Standard stock solution B: 2.4: 1.0: 0.4: 6.2
Standard stock solution C: 9.6: 0.2: 0.2:0
Standard solutions: [NoTE—Prepare the Standard solu-
tions immediately before use.]
Standard solution A: Transfer 2.5 mL of Standard
stock solution A to a 100-mL volumetric flask, dilute
with diluted hydrochloric acid to volume, and mix.
Standard solution B: Transfer 2.5 mL of Standard stock
solution B to a 100-mL volumetric flask, dilute with di-
luted hydrochloric acid to volume, and mix.
Standard solution C: Transfer 0.75 mL of Standard
Stock solution C to a 100-mL volumetric flask, dilute
with diluted hydrochloric acid to volume, and mix.
Sample solution: Use the Sample solution prepared in
Clarity of Solution.
Analysis: Transfer a sufficient portion of the Sample so-
lution to a sample tube of colorless, transparent, neutral
glass, witha flat base and an internal diameter of
15-25 mm, to obtain a depth of 40 mm. Similarly
transfer portions of Standard solution A, Standard solu-
tion B, Standard solution C, and water to separate
matching sample tubes. Compare the Sample solution,
Standard solution A, Standard solution B, Standard solu-
tion C, and water in diffused daylight, viewing vertically
against a white background (see Nephelometry, Turbi-
dimetry, and Visual Comparison (855)).
Acceptance criteria: The Sample solution is not more
intensely colored than Standard solution A, Standard so-
lution B, Standard solution C, or water.
@ CONDUCTIVITY
Sample solution: 0.2 g/mL of Inositol in water (previ-
ously boiled and cooled to room temperature).
Apparatus: Use a conductivity meter ora resistivity
meter that measures the resistance of the column of
liquid between the electrodes of the immersed measur-
ing device. The apparatus is supplied with alternating
current to avoid the effects of electrode polarization. It
is equipped with a temperature compensation device or
a precision thermometer.
Reagents: Prepare three Standard solutions of potassium
chloride containing 0.7455, 0.0746, and 0.0149 g, re-
spectively, of potassium chloride per 1000.0 g of solu-
tion. These solutions should be prepared with water
that has been previously boiled and cooled to room
temperature and whose conductivity does not exceed 2
uS/cm. The conductivity and resistivity of these three
solutions at 20° are provided in Table 7.
Table 1
Concentration of
Solution Conductivity Resistivity
(g/1000.0 g) (uS/em) (Q-cm)
0.7455 1330 752
0.0746 133.0 7519
0.0149 26.6 37,594
Calibration: Choose a conductivity cell that is appropri-
ate for the conductivity of the solution to be examined.
The higher the expected conductivity, the higher the
cell constant that must be chosen. Commonly used
conductivity cells have cell constants of the order 0.1,
1, and 10 cm. Use a standard solution of potassium
chloride that is appropriate for the measurement. The
Official Monographs / Invert 5397
conductivity value of the standard solution of potassium
chloride should be near the expected conductivity value
of the Sample solution. Rinse the cell several times with
water that has been previously boiled and cooled to
room temperature, and rinse at least twice with the po-
tassium chloride solution used for the determination of
the cell constant of the conductivity cell. Measure the
resistance of the conductivity cell, using the potassium
chloride solution at 20 +0.1°.
Calculate the constant, in cm-, of the conductivity cell:
Result = Rec X Kxer
Rxc) + = measured resistance, expressed in mega-ohms
Kx ~~ = conductivity of the standard solution of
potassium chloride used, expressed in US/
cm. The measured constant of the
conductivity cell must be within 5% of the
given value.
Analysis: Rinse the conductivity cell several times with
water that has been previously boiled and cooled to
room temperature, and rinse at least twice with the
Sample solution. Measure the conductivity of the Sample
solution, while gently stirring with a magnetic stirrer.
Acceptance criteria’. NMT 20 uS/cm
¢@ WATER DETERMINATION, Method | (921): NMT 0.5% de-
termined on a 1.0-g sample
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in well-closed contain-
ers, and store at room temperature.
e USP REFERENCE STANDARDS (11)
USP Inositol RS
USP Mannitol RS
Invert Sugar
Invert Sugar Syrup [8013-17-0].
DEFINITION
Invert Sugar is an aqueous solution of inverted or partly in-
verted, refined or partly refined sucrose containing dex-
trose (glucose), fructose, and sucrose. It is produced by
the hydrolysis or partial hydrolysis of sucrose with suitable
acids or enzymes. Total Invert Sugar contains NLT 90.0%
of dextrose and fructose, and medium Invert Sugar con-
tains NLT 45.0% and NMT 55.0% of dextrose and fruc-
tose, both calculated on the total solids basis.
IDENTIFICATION
oA.
Mobile phase: Acetonitrile and water (80:20, v/v)
Standard solution: 10 mg/mL each of USP Fructose RS,
USP Dextrose RS, and USP Sucrose RS
Sample solution: 50 mg/mL of Invert Sugar
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: Refractive index
Column: 4.6-mm x 15-cm; 5-um packing L&
Temperatures
Column: 45°
sydesbouow 4N
Detector: 40°
Flow rate: 2.0 mL/min
Injection volume: 15 uL
Analysis
Samples: Standard solution and Sample solution
[NotE—The relative retention times for fructose, dex-
trose, and sucrose are about 0.5, 0.6, and 1.0,
respectively.]
Compare the retention times for frucose, dextrose, and
sucrose given in the two chromatograms.
 5398 Invert / Official Monographs
Acceptance criteria: The retention times of the fruc-
tose, dextrose, and sucrose peaks of the Sample solution
correspond to those of the Standard solution.
ASSAY
© PROCEDURE
Apparatus: Mount a ring support on a ring stand 1-2
in above a gas burner and mount a second ring 6-7 in
above the first. Place a 6-in open-wire gauze on the
lower ring to support a 400-mL conical flask, and place
a 4-in watch glass with a center hole on the upper rin
to deflect heat. Attach a 50-mL buret to the ring stan
so that the tip just passes through the watch glass cen-
tered above the flask. Place an indirectly lighted white
surface behind the assembly for observing the
endpoint.
Sample solution: 2.5-4.0 mg/mL of Invert Sugar
Analysis: Conduct a preliminary test to ascertain the
volume of water to be added to the 20.0 mL of stan-
dardized cupric tartrate, alkaline, solution VS to obtain a
final total volume of 75.0 mL when the endpoint of the
titration is reached. The invert sugar content of the
Sample solution should be between 250 and 400 mg
per 100.0 mL so that a titer between 25 and 40 mL is
needed to achieve the endpoint.
Calculate the amount of water to be added to the cu-
pric tartrate, alkaline, solution VS as the difference:
Result = 75.0 — (20.0 mL of cupric tartrate, alkaline, so-
lution VS + number of mL of preliminary titer)
Transfer 20.0 mL of cupric tartrate, alkaline, solution VS
to a 400-mL flask containing a few glass beads or boil-
ing chips. Rapidly add the Sample solution to within
0.5 mL of the endpoint, and mix by swirling at ambi-
ent temperature. Immediately place the flask on the
wire gauze of the Apparatus, and adjust the burner
flame so that the boiling point of the solution is
reached in about 2 min. Boil gently but steadily for 2
min. As boiling continues, add 3-4 drops of methylene
blue solution (1 in 100). Complete the titration within
1 min by adding the Sample solution dropwise until
the blue color disappears. Allow a 5-s reaction time
between drops at the end of titration.
Calculate the percentage of invert sugar in the sample
taken:
P, = [W/(Cs x Vs)] x 100
Ww = amount of invert sugar equivalent to 20.0 mL
of cupric tartrate, alkaline, solution VS,
100 mg
Gs = concentration of the Sample solution (mg/mL)
Vs = volume of the Sample solution used in the
titration (mL)
Calculate the percentage of invert sugar on the total
solids basis:
Po = (P;/D) x 100
NF Monographs
NF 36
D = percentage of total solids corrected for the
percentage of invert sugar and temperature
as obtained in Total Solids
Acceptance criteria
Total Invert Sugar: NLT 90.0% of dextrose and fruc-
tose on the total solids basis
Medium Invert Sugar: 45.0%-55.0% of dextrose and
fructose on the total solids basis
IMPURITIES
e RESIDUE ON IGNITION (281)
Analysis: Weigh a portion of Invert Sugar in a platinum
dish. Heat gently until the sample ignites, then allow
the sample to burn until it self-extinguishes. Cool, then
wet the residue with 2 mL of concentrated sulfuric acid,
and heat the sample over a low flame until dry. Ignite
to constant weight in a muffle furnace at 800 + 25° for
30 min, or longer if necessary for complete ignition;
cool in a desiccator; and weigh.
Acceptance criteria: NMT 0.2%
Delete the following:
®e HEAVY METALS (231): NMT 10 U9/Ge cosicial 1-4an.2018)
SPECIFIC TESTS
© PH (791): 3.0-5.5
e TOTAL SOLIDS
Instrumental conditions
(See Refractive Index (831).)
Mode: Refractometer, operating at 589 nm, equipped
with a jacket for water circulation or some other
mechanism for maintaining the sample at 20 + 0.1° or
some other fixed temperature. Before proceeding with
measurements, ensure that the sample and the prism
have reached the equilibrium temperature and that
the instrument has been properly checked and cali-
brated against a standard provided by the
manufacturer.
Analysis: Measure the refractive index of Invert Sugar,
and convert the value to the approximate percentage
of solids (uncorrected for invert sugar) by using Table 1.
Correct for invert sugar and temperature:
D=(S+T)+(Pi:x
FD
S = approximate percent solids determined from
the refractive index table (see Table 1)
Tt = temperature correction derived from the
temperature correction table (see Table 2) if
the refractometer was operated at other than
20°
Py = percentage of invert sugar determined as
directed in the Assay
F = deWhalley factor, 0.022
NF 36 Official Monographs / Invert 5399

Table 1°
Sucrose
(g/100
g)> 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
56 1.4329 4332 4334 4336 4338 4340 4343 4345 4347 4349
57 1.4352 4354 4356 4358 4360 4363 4365 4367 4369 4372
58 1.4374 4376 4378 4380 4383 4385 4387 4389 4392 4394
59 1.4396 4398 4401 4403 4405 4407 4410 4412 4414 A417
60 1.4419 4421 4423 4426 4428 4430 4432 4435 4437 4439
61 1.4442 4444 4446 4448 4451 4453 4455 4458 4460 4462
62 1.4464 4467 4469 4471 4474 4476 4478 4481 4483 4485
63 1.4488 4490 4492 4495 4497 4499 4502 4504 4506 4509
64 1.4511 4513 4516 4518 4520 4523 4525 4527 4530 4532
65 1.4534 4537 4539 4541 4544 4546 4548 4551 4553 4556
66 1.4558 4560 4563 4565 A567 4570 4572 4575 4577. 4579
67 1.4582 4584 4586 4589 4591 4594 4596 4598 4601 4603
68 1.4606 4608 4610 4613 4615 4618 4620 4623 4625 4627
69 1.4630 4632 4635 4637 4639 4642 4644 4647 4649 4652
70 1.4654 4657 4659 4661 4664 4666 4669 4671 4674 4676
7\ 1.4679 4681 4683 4686 4688 4691 4693 4696 4698 4701
72 1.4703 4706 4708 4711 4713 4716 4718 4721 4723 4726
73 1.4728 4730 4733 4735 4738 4740 4743 4745 4748 4750
74 1.4753 4756 4758 4761 4763 4766 4768 4771 4773 4776
75 1.4778 4781 4783 4786 4788 4791 4793 4796 4798 4801
76 1.4804 4806 4809 4811 4814 4816 4819 4821 4824 4826
77 1.4829 4832 4834 4837 4839 4842 4844 4847 4850 4852
78 1.4855 4857 4860 4862 4865 4868 4870 4873 4875 4878
79 1.4881 4883 4886 4888 4891 4894 4896 4899 4901 4904
80 1.4907 4909 4912 4914 4917 4920 4922 4925 4928 4930
81 1.4933 4935 4938 4941 4943 4946 4949 4951 4954 4957
82 1.4959 4962 4964 4967 4970 4972 4975 4978 4980 4983
83 1.4986 4988 4991 4994 4996 A999 5002 5004 5007 5010
84 1.5012 5015 5018 5020 5023 5026 5029 5031 5034 5037
85 1.5039 = _— = _— =— = _ a =
2 SouRCE: “International Refractive Index Scale of ICUMSA (1974) for Pure Sucrose Solutions at 20°C and 589 nm.” Adapted from “Refractometry and Tables—
Official” (ICUMSA SPS-3 1994), International Commission for Uniform Methods of Sugar Analysis (ICUMSA), c/o British Sugar Technical Centre, Colney,
Norwich NR4 7UB, England. No rounding has been carried out; therefore, values given may be too low by a maximum of 1 x 10+.
> The refractive index of sugar solutions is used as a rapid method for the approximate determination of dry substance content. For the determination of dry
substance content in aqueous solutions of mixtures of sucrose and invert sugar the Refractive Index Scale for Pure Sucrose Solutions is typically used. The
sucrose content is considered to be an equivalent to the approximate dry substance content.

sydesbouow: 4N
 NF Monographs

sydoiBouoy jorIYyO / WeAUL OOS

Table 2?
Temperature Measured Sucrose (% solids)
@) oO 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85
Subtract from the measured value
15 0.29 0.30 0.32 0.33 0.34 | 0.35 0.36 0.37 0.37 0.38 0.38 0.38 0.38 0.38 0.38 0.38 0.37 0.37
16 0.24 0.25 0.26 0.27 0.28 0.28 0.29 0.30 0.30 0.30 0.31 0.31 Q:31 0.31 0.31 0.30 0.30 0.30
17 0.18 0.19 0.20 0.20 0.21 0.21 0.22 0.22 0.23 0.23 0.23 0.23 0.23 0.23 0.23 0.23 0.23 0.22
18 0.12 0.13 0.13 0.14 0.14 0.14 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15
19 0.06 0.06 0.07 0.07 0.07__| 0.07 0.07 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.07
Add to the measured value
21 0.06 0.07 0.07 0.07 0.07 0.07 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.07
22 0.13 0.14 0.14 0.14 0.15 0.15 0.15 0.15 0.16 0.16 0.16 0.16 0.16 0.16 0.15 0.15 0.15 0.15
23 0.20 0.21 0.21 0.22 0.22 | 0.23 0.23 0.23 0.23 0.24 0.24 0.24 0.24 0.23 0.23 0.23 0.23 0.22
24 0.27 0.28 0.29 0.29 0.30 0.30 0.31 0.31 0.31 0.32 0.32 0.32 0.32 0.31 0.31 0.31 0.30 0.30
25 0.34 0.35 0.36 0.37 0.38 0.38 0.39 0.39 0.40 0.40 0.40 0.40 0.40 0.39 0.39 0.38 0.38 0.37
26 0.42 0.43 0.44 0.45 0.46 0.46 0.47 0.47 0.48 0.48 0.48 0.48 0.48 0.47 0.47 0.46 0.46 0.45
27 0.50 0.51 0.52 0.53 0.54 0.55 0.55 0.56 0.56 0.56 0.56 0.56 0.56 0.55 0.55 0.54 0.53 0.52
28 0.58 0.59 0.60 0.61 0.62 | 0.63 0.64 0.64 0.64 0.65 0.65 0.64 0.64 0.63 0.63 0.62 0.61 0.60
29 0.66 0.67 0.68 0.70 0.71 0.71 0.72 0.73 0.73 0.73 0.73 0.73 0.72 0.72 0.71 0.70 0.69 0.67
30. 0.74 0.76 0.77 0.78 0.79 0.80 0.81 0.81 0.82 0.82 0.81 0.81 0.80 0.80 0.79 0.78 0.76 0.75
31 0.83 0.84 0.85 0.87 0.88 0.89 0.89 0.90 0.90 0.90 0.90 0.89 089 0.88 0.87 0.86 0.84 0.82
32 0.92 0.93 0.94 0.96 0.97 0.98 0.98 0.99 0.99 0.99 0.99 0.98 0.97 0.96 0.95 0.93 0,92 0.90
33 1.01 1.02 1.03 1.05 1.06 _| 1.07 1.07 1.08 1.08 1.08 1.07 1.07 1.06 1.04 1.03 1.01 1.00 0.98
34 1.10 11 TA3 1.14 1.15 1.16 1.16 TEA 1.17 1.16 1.16 1.15 1.14 1.13 1.11 1.09 1.07 1.05
35 1.19 1.21 1.22 1.23 1.24 25 1,25: 1.26 1.26 1.25 1,25 1.24 1.23 1.21 Ast9: ATZ: 1.15 1.13
36 1.29 1.30 1.31 1.33. 1.34 1.34 1.35 135. 35. 1.34 1.34 1.33 1.31 1.29. 1.28 1.25 1,23 1.20
37 1.39 1.40 1.41 1.42 1.43 1.44 1.44 1.44 1.44 1.43 1.43 1.41 1.40 1.38 1.36 1.33 1.31 1.28
38 1.49 1.50 1.51 1.52 1.53 1L.53. 1.54 1.54 4253 1.53 1.52 1.50 1.48 1.46 1.44 1.42 139 1.36
39 1.59 1.60 1.61 1.62 1.63 | 1.63 1.63 1.63 1.63 1.62 1.61 1.59 1:57. 1.55 Ag5Z 1.50 1.47 1.43
40 1.69 1.70 1.71 1.72 1.73 1.73 1.73 1.733 1.72 LA 1.70 1.68 1.66 1.63 1.61 1.58 1,54 1.51
2 Source: Adapted from “Refractometry and Tables—Official” (ICUMSA SPS-3 1994), International Commission for Uniform Methods of Sugar Analysis (ICUMSA), c/o British Sugar Technical Centre, Colney,
Norwich NR4 7UB, England.

9¢ IN
USP 41
10 mg/mL. Before injection, pass througha filter of
0.45-um pore size.
Sample solution: Transfer about 2.0 g of crushed Bos-
wellia serrata to a round-bottom flask, and reflux in
50 mL of methanol on a water bath for 15 min, stirring
magnetically. Repeat until the extract is colorless. Evap-
orate the combined extracts to about 50 mL, transfer to
a 100-mL volumetric flask, and dilute with methanol to
volume. Before injection, pass throughafilter of 0.45-
lum pore size, and discard the first few mL of the
filtrate.
Mobile phase: Preparea filtered and degassed mixture
of acetonitrile, water, and glacial acetic acid
(900: 100: 0.1). Make adjustments if necessary.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 254 nm
Column: 4.6-mm x 25-cm; packing L1
Flow rate: See Table 7.
Table 1
Time Flow Rate
(min) (mL/min)
0 1
5 13S
10 2 NMT 0.5%
30 2 ARTICLES OF BOTANICAL ORIGIN, Alcoho!-Soluble Extractives,
32 1 Method 2 (561):
45 1 ADDITIONAL REQUIREMENTS
Injection volume: 20 uL
System suitability
Samples: Standard solution A and Standard solution B
[NotE—The relative retention times for 11-keto-B-bos-
wellic acid and 3-acetyl-11-keto-B-boswellic acid are
about 1.0 and 1.4, respectively.]
Suitability requirements: The chromatogram of Stan-
dard solution B is similar to the 254-nm reference chro-
matogram provided with the lot of USP Boswellia ser-
rata Extract RS being used.
Relative standard deviation: NMT 2.0% for the
3-acetyl-11-keto-B-boswellic acid peak in replicate in-
jections, Standard solution A
Tailing factor: NMT 1.5, 3-acetyl-11-keto-B-boswellic
acid peak, Standard solution A
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Using the chromatogram of Standard solution B and the
reference chromatogram provided with the lot of USP
Boswellia serrata Extract RS being used, identify the re-
tention times of the peaks of 11-keto-B-boswellic acid
and 3-acetyl-11-keto-B-boswellic acid in the Sample
solution.
Separately calculate the percentages of the two analytes
in the portion of Boswellia serrata taken:
Result = (ru/rs) x (Cs/W) x 10F
fu = peak area of each analyte from the Sample
solution
rs = peak area of 3-acetyl-11-keto-B-boswellic acid
from Standard solution A
Gs = concentration of USP 3-Acetyl-11-keto-B-
Boswellic Acid RS in Standard solution A
(mg/mL)
w = weight of Boswellia serrata taken to prepare
the Sample solution (g)
F = conversion factor for each analyte: 0.93 for
11-keto-B-boswellic acid and 1.0 for 3-acetyl-
11-keto-B-boswellic acid
Dietary Supplements / Boswellia 4491
Acceptance criteria: Add the percentages calculated for
11-keto-B-boswellic acid and 3-acetyl-11-keto-B-boswel-
lic acid; NLT 1.0% on the dried basis.
IMPURITIES
e ARTICLES OF BOTANICAL ORIGIN, Limits of Elemental Impuri-
ties (561): Meets the requirements
© ARTICLES OF BOTANICAL ORIGIN, Foreign Organic Matter
(561): NMT 2.0%
© ARTICLES OF BOTANICAL ORIGIN, Pesticide Residue Analysis
(561): Meets the requirements
SPECIFIC TESTS
e BOTANICAL CHARACTERISTICS
Macroscopic: |t occurs as small ovoid tears, sometimes
forming agglomerated masses up to 5 cm long and
2.cm thick; whitish to golden yellow; fracture is brittle,
and fractured surface is waxy and translucent character-
istic aromatic odor; aromatic, slightly mucilaginous
taste.
Loss ON DRYING (731)
Sample: 1.0g of Boswellia serrata, finely powdered
Analysis: Dry the Sample at 105° for 2 h.
Acceptance criteria: NMT 12.0%
ARTICLES OF BOTANICAL ORIGIN, Total Ash (561)
Sample: 2.0g of Boswellia serrata, finely powdered
Acceptance criteria: NMT 2.0%
ARTICLES OF BOTANICAL ORIGIN, Acid-Insoluble Ash (561):
NLT 56%
© PACKAGING AND STORAGE: Preserve in well-closed contain-
ers, protected from light and moisture, and store in a
cool place.
e LABELING: The label states the Latin binomial of the spe-
cies of Boswellia from which the oleogum resin was
obtained.
e USP REFERENCE STANDARDS (11)
USP 3-Acetyl-11-keto-B-Boswellic Acid RS
USP Boswellia serrata Extract RS
Boswellia serrata Extract
DEFINITION
Boswellia serrata Extract is prepared from pulverized Boswel-
lia serrata, using suitable solvents such as isopropanol, al-
cohol, methanol, hexanes, or mixtures of these solvents.
sydesbouo-= sa
The ratio of starting plant material to Extract is approxi-
mately 6:1. It contains NLT 90.0% and NMT 110.0% of
the labeled amount of Extract, calculated, on the dried
basis, as the sum of 11-keto-B-boswellic acid and 3-acetyl-
11-keto-B-boswellic acid; it may contain suitable added
substances.
IDENTIFICATION
° a CHROMATOGRAPHIC IDENTIFICATION TEST
Standard solution: Treat a quantity of USP Boswellia
serrata Extract RS with gentle heating in methanol to
obtain a solution having a known concentration of
30 mg/mL, cool, centrifuge, and use the supernatant.
Sample solution: Treat a quantity of Extract with gentle
heating in methanol to obtain a solution having a
known concentration of 30 mg/mL, cool, centrifuge,
and use the supernatant.
> ie 0.25-mm layer of chromatographic silica
ge
Developing solvent system: A mixture of hexane and
ethyl acetate (6:4)
NF 36 Official Monographs / \sobutyl 5401

Acceptance criteria ¢ HIGH-BOILING RESIDUES: NMT 5 ug/mL, determined as di-

Total Invert Sugar: 71.7%-77.3% rected in Propellants (602)
Medium Invert Sugar: 76.2%-77.2% ¢ ACIDITY OF RESIDUE
Sample: Residue from the test for High-Boiling Residues
ADDITIONAL REQUIREMENTS Analysis: Add 10 mL of water to the Sample, mix by
© LABELING: Label to indicate the percentage of total solids swirling for about 30 s, add 2 drops of methyl orange
and Invert Sugar. TS, insert the stopper in the tube, and shake vigorously.
¢ PACKAGING AND STORAGE: Preserve in tight, light-resistant Acceptance criteria: No pink or red color appears in
containers. the aqueous layer.
e USP REFERENCE STANDARDS (11)
USP Dextrose RS ADDITIONAL REQUIREMENTS
USP Fructose RS e PACKAGING AND STORAGE: Preserve in tight cylinders, and
USP Sucrose RS prevent exposure to excessive heat.

Isobutane lsobutyl Alcohol

CH, iia
net 0H
H.c7 “CH,

CH3CH(CH3)CH2OH 74.12
Git 38.12 2-Methyl-1-propanol;
DEFINITION 2-Methylpropyl alcohol;
Isobutane contains NLT 95.0% of isobutane (C4Hio). 1-Isobutanol [78-83-1].
[CauTioN—lsobutane is highly flammable and explosive.] DEFINITION
IDENTIFICATION lsobutyl Alcohol contains NLT 98.0% of 2-methyl-1-propa-
e A. IR ABsorPTION: Exhibits maxima, among others, at nol (C4Hi00).
about the following wavelengths (4m): 3.4 (vs), 6.8 (s),
7.2 (m), 8.5 (m), and 10.9 (m).
IDENTIFICATION
© A. INFRARED ABSORPTION (197F)
e B. The vapor pressure of a test specimen obtained as e B. The retention time of the major peak of the Sample
directed for Propellants (602), and determined at 21° by solution corresponds to that of the 2-methyl-1-propanol
means of a suitable pressure gauge, is between 303 and peak of the System suitability solution, as obtained in the
331 kPa absolute (44 and 48 ose. Assay.
ASSAY ASSAY
© PROCEDURE
Chromatographic system © PROCEDURE
System suitability solution: USP 1-Butanol RS and USP
(See Chromatography (621), System Suitability.) 2-Methyl-1-Propanol RS (1:1)
Mode: GC Reference solution: 0.1% of Isobutyl Alcohol in water
Detector: Thermal conductivity Sample solution: Isobutyl Alcohol (neat)
Column: 3-mm x 6-m aluminum; packed with 10
weight percent of liquid phase G30 on support $1D Chromatographic system
Column temperature: 33° (See Chromatography (621), System Suitability.)
Carrier gas: Helium Mode: GC
Detector: Flame ionization
Flow rate: 50 mL/min
Column: 0,53-mm x 30-m; coated with a 3.0-um
Injection volume: 2 UL
System suitability
layer of thickness phase G43
Temperatures
Sample: Isobutane
Detector: 250°
Suitability requirements
Injection port: 140°
Sample response, comparison: Peak responses for
Column: See Table 7.
isobutane from duplicate injections agree within 1%.
Analysis
Sample: Isobutane Table 1
Connect one Isobutane cylinder to the chromatograph Hold Time at
through a suitable sampling valve and a flow control Initial Temperature Final Final
valve downstream from the sampling valve. Flush the Temperature Ramp Temperature | Temperature
liquid specimen through the sampling valve, taking @) (¢/min) @) (min)
cals to avoid entrapment of gas or air in the sampling 40 = 40 20
valve.
40 10 240 20
Calculate the percentage purity of Isobutane:
sydeibouow 4N

Result = (ru/rz) x 100 Carrier gas: Helium

Flow rate: 4.8—4.9 mL/min
ru = peak response of isobutane Injection volume: 1 wL
rr = sum of all the peak responses Injection type: Split injection. The split ratio is 30:1.
Acceptance criteria: NLT 95.0% [Note—A needle wash with the Sample solution is rec-
ommended to minimize the carry over.]
SPECIFIC TESTS System suitability
© WaTER: NMT 0.001%, determined as directed in Propel- Sample: System suitability solution
lants (602) [Note—The 2-methyl-1-propanol peak typically elutes at
about 11 min, and 1-butanol at about 15 min. The
 5402 Isobutyl / Official Monographs NF 36

relative retention times for 2-methyl-1-propanol and Ir = sum of all the peaks from the Sample solution,
1-butanol are 0.7 and 1.0, respectively.] except those each of which with an area less
Suitability requirements than 0.1 times the area of the major peak
Resolution: NLT 2.0 between 2-methyl-1-propanol from the Reference solution)
and 1-butanol Acceptance criteria: See Table 3. Disregard any peak
Relative standard deviation: NMT 2.0% with an area less than 0.1 times the area of the major
Analysis peak from the Reference solution, corresponding to
Samples: Reference solution and Sample solution 0.01%.
Calculate thepercentage of 2-methyl-1-propanol
(C4Hi0O) in the portion of fsabubyt Alea taken: Table 3
Result = (ru/r7) x 100 Impurity Percentage (%)
The area of any peak of the Sample solution
ty = peak response of isobutyl! alcohol corresponding to isobutyraldehyde, ry, is
ls = sum of all the peaks except those each of NMT half of the difference (Ar) between the
which with an area less than 0.1 times the area of the peak due to isobutyraldehyde in
area of the major peak from the Reference the Standard solution and the area of the
solution peak due to isobutyraldehyde in the Sample
Acceptance criteria: NLT 98.0% lsobutyraldehyde Solution, corresponding to NMT 0.1%.
IMPURITIES The area of any peak of the Sample solution
© LIMIT OF ISOBUTYRALDEHYDE, BUTYRALDEHYDE, 2-BUTANOL, corresponding to butyraldehyde, ri, is NMT
half of the difference (An) between the area
1-BUTANOL, AND OTHER VOLATILE IMPURITIES
Sample solution and Chromatographic system: Pro- of the peak due to butyraldehyde in the
ceed as directed in the Assay. Standard solution and the area of the peak
due to butyraldehyde in the Sample solution,
Reference solution: 0.1% of lsobutyl Alcohol in water
Standard solution: 0.2% of USP Isobutyraldehyde RS, Butyraldehyde corresponding to NMT 0.1%.
0.2% of USP Butyraldehyde RS, 0.1% of USP 1-Butanol The area of any peak of the Sample solution
RS, and 0.1% of USP 2-Butanol RS in the Sample corresponding to 2-butanol, ry, is NMT the
solution difference (Ar) between the area of the peak
System suitability due to 2-butanol in the Standard solution
Sample: Standard solution and the area of the peak due to 2-butanol in
[Note—See Table 2 for relative retention times.] the Sample solution, corresponding to NMT
2-Butanol 0.1%.
The area of any peak of the Sample solution
Table 2
corresponding to 1-butanol, ry, is NMT the
Relative difference (An) between the area of the peak
Retention due to 1-butanol in the Standard solution
Component Time and the area of the peak due to 1-butanol in
lsobutyraldehyde 0.4 the Sample solution, corresponding to NMT
Butyraldehyde 0.5 1-Butanol 0.1%.
2-Butanol 0.6 Total impurities NMT 2.0%
2-Methyl-1-propanol 0.8 @ LIMIT OF NONVOLATILE RESIDUE
1-Butanol 1.0 Sample: 100 mL
Analysis: Evaporate the Sample in a tared porcelain dish
Suitability requirements on a steam bath, and dry at 105° for 30 min.
Resolution: NLT 1.5 between all adjacent peaks Acceptance criteria: The weight of the residue does
Analysis not exceed 4 mg, corresponding to NMT 0.004%.
Samples: Sample solution, Reference solution, and Stan-
dard solution SPECIFIC TESTS
If any peaks of the Sample solution have the same reten- e ACIDITY
tion times as the peaks due to isobutyraldehyde, Sample: 74 mL (60g)
butyraldehyde, 2-butanol, and 1-butanol, subtract the Analysis: Titrate the Sample with 0.020 N alcoholic po-
areas of any such peaks from the peak areas of the tassium hydroxide, using phenolphthalein TS as the in-
Standard solution at these retention times. The differ- dicator, until a pink color persists for NLT 15 s.
ence is calculated below: Acceptance criteria: NMT 2.5 mL is consumed.
e@ WATER DETERMINATION, Method | (921): NMT 0.5%
Result (Ar) = rs — ru
ADDITIONAL REQUIREMENTS
rs = peak response of each individual impurity e PACKAGING AND STORAGE: Preserve in tight containers,
(isobutyraldehyde, butyraldehyde, 2-butanol, and prevent exposure to excessive heat.
or 1-butanol) from the Standard solution e USP REFERENCE STANDARDS (11)
tu = peak response of each individual impurity USP 1-Butanol RS
(isobutyraldehyde, butyraldehyde, 2-butanol, USP 2-Butanol RS
or 1-butanol), if present, from the Sample
NF Monographs

USP Butyraldehyde RS
solution USP Sea lehyde RS
Calculate the percentage of each impurity other than USP 2-Methyl-1-Propanol RS
isobutyraldehyde, butyraldehyde, 2-butanol, and
1-butanol in the portion of Isobutyl Alcohol taken:
Result = (ru/rr) x 100
ru = peak response of each impurity other than
isobutyraldehyde, butyraldehyde, 2-butanol,
and 1-butanol from the Sample solution
NF 36 Official Monographs / |somalt 5403

Columns
Isomalt Guard: 4.6-mm x 3-cm; packing L19
Analytical: 7.8-mm x 30-cm; packing L19
Portions of this monograph that are national USP text, and Column temperature: 80 + 3°
are not part of the harmonized text, are marked with Flow rate: 0.5 mL/min
symbols (*s) to specify this fact. Injection volume: 20 uL
System suitability
fOH Sample: Standard solution
\. OH OH [Note—The relative retention times for 1,1-GPM and
Hom (yo. Soa 1,6-GPS are about 1.0 and 1.2, respectively.]
Suitability requirements
Resolution: NLT 2.0 between 1,1-GPM and 1,6-GPS
Relative standard deviation: NMT 2.0% for the 1,6-
GPS and 1,1-GPM peaks
ed ah : ue’ + 2H,0 Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of 1,6-GPS in the portion of
lsomalt taken:
CHO 344.31 Result = (ru/rs) x (Cs/Cu) x 100
Cy2H24O11 - 2H20 380.32
6-0-o.-Glucopyranosy|-D-sorbitol and 1-O-a-D-glucopyra- ty = peak response of 1,6-GPS from the Sample
nosyl-D-mannitol dihydrate; solution
6-O-c-D-Glucopyranosyl-D-glucitol and 1-O-c-D-glucopyra- Is = peak response of 1,6-GPS from the Standard
nosyl-D-mannitol dihydrate [64519-82-0]. solution
Cs = concentration of 1,6-GPS in the Standard
DEFINITION solution, with calculation based on the
lsomalt contains NLT 98.0% and NMT 102.0% of a mixture declared 1,6-GPS content of USP Isomalt RS
of 6-O-a-D-glucopyranosyl-D-sorbitol (1,6-GPS) and 1-O-a- (mg/mL)
D-glucopyranosyl-D-mannitol (1,1-GPM), and neither of Cu = concentration of lsomalt in the Sample solution
the two components is less than 3.0% of the mixture, (mg/mL) :
calculated on the anhydrous basis. Calculate the percentage of 1,1-GPM in the portion of
lsomalt taken:
IDENTIFICATION
e a aa CHROMATOGRAPHIC IDENTIFICATION TEST Result = (ru/rs) x (Cs/Cy) x 100
201
Standard solution: 5 mg/mL of USP Isomalt RS ty = peak response of 1,1-GPM from the Sample
Sample solution: 5 mg/mL solution
Chromatographic system rs = peak response of 1,1-GPM from the Standard
Adsorbent: 0.25-mm layer of chromatographic silica solution
gel mixture containing a fluorescent indicator having Cs = concentration of 1,1-GPM in the Standard
optimal intensity at 254 nm solution, with calculation based on the
Application volume: 1 uL declared 1,1-GPM content of USP Isomalt RS
Developing solvent system: Ethyl acetate, pyridine, (mg/mL)
water, acetic acid, and propionic acid (10:10:2:1:1) Cu = concentration of lsomalt in the Sample solution
Analysis (mg/mL)
Samples: Standard solution and Sample solution Acceptance criteria: 98.0%-102.0% of a mixture of
Proceed as directed in the chapter. Thoroughly dry the 6-O-a.-D-glucopyranosyl-D-sorbitol (1,6-GPS) and 1-O-a-
starting points in warm air. Develop over 10 cm using D-glucopyranosyl-D-mannitol (1,1-GPM), and neither of
the Developing solvent system, dry the plate in a cur- the two components is less than 3.0% of the mixture,
rent of hot air, and dip for 3s in a 1-mg/mL solution calculated on the anhydrous basis
of sodium periodate. Dip the plate for 3 s in a mixture
of dehydrated alcohol, sulfuric acid, acetic acid, and IMPURITIES
anisaldehyde (90:5:1:1). Dry the plate in a current of
hot air until colored spots become visible. The back- Delete the following:
ground color may be brightened by exposure to warm
steam. Examine in daylight. °e HEAVY METALS, Method | (231): NMT 10 fig/ge cca
-
Acceptance criteria: The principal spots of the Sample Jan-2018)
solution are similar in position and color to those of the o Limit OF NICKEL
Standard solution.» [Nott—The purity of the reagents and the water used
e B. The retention times of the two principal peaks of the must be suitable for trace analysis, and the reagents
Sample solution correspond to those of the Standard solu- and water must be free of nickel.]
tion, as obtained in the Assay. Sample solution: Dissolve 10.0 g of lsomalt in 30 mL of
ASSAY dilute acetic acid (115-125 g/L), add water, and shake
to dissolve. Dilute with water to 100.0 mL. Add 2.0 mL ra
¢ PROCEDURE nn
Mobile phase: Water of saturated ammonium pyrrolidinedithiocarbamate TS
Standard solution: 20 mg/mL of USP Isomalt RS and 10.0 mL of water-saturated methyl isobutyl ketone Es
(CoHi20, 4-methyl-2-pentanone), and then shake for 30 CS}
Sample solution: 20 mg/mL of lsomalt =
Chromatographic system s, protected from bright light. Allow the layers to sepa- °
(See Chromatography (621), System Suitability.) rate and use the methyl isobutyl ketone layer. iro)=
Mode: LC Standard solutions: Prepare three reference solutions in 2
the same manner as the Sample solution except add 3
Detector: Refractive index, maintained at a constant
temperature (40° for example) 0.5 mL, 1.0 mL, and 1.5 mL, respectively, of nickel stan- s
a)
dard solution TS (10 ppm Ni) in addition to the 10.0 g
of the substance to be examined.
 5404 Isomalt / Official Monographs NF 36

Blank solution: Treat water-saturated methyl isobutyl Table 1

ketone as described for preparation of the Sample solu-
Acceptance
tion omitting the Isomalt.
Criteria,
Instrumental conditions
Name NMT (%)
(See Atomic Absorption Spectroscopy (852).)
Mode: Atomic a pee spectrophotometry Mannitol 0.5
Analytical wavelength: 232.0 nm Sorbitol 0.5
Lamp: Nickel hollow-cathode Any unknown impurity 0.5
Flame: Air-acetylene Total impurities 2.0
Analysis
Samples: Sample solution, Standard solutions, and Blank e REDUCING SUGARS
solution Sample solution: Dissolve 3.3 g in 10 mL of Purified
Set the zero of the instrument using the Blank solution. Water with the aid of gentle heat. Cool and add 20 mL
Record the average of the steady readings for each of of cupric citrate TS and a few glass beads. Heat so that
the Standard solutions and the Sample solution. Be- boiling begins after 4 min, and maintain boiling for 3
tween each measurement, rinse with water and ascer- min. Cool rapidly, and add 100 mL of a 2.4% (v/v) so-
tain that the reading returns to zero with the Blank lution of glacial acetic acid and 20 mL of 0.025 M io-
solution. Plot the absorbances of the Standard solutions dine VS. With continuous shaking, add 25 mL of a mix
and the Sample solution versus the added quantity of ture of hydrochloric acid and water (6:94).
nickel. Extrapolate the line joining the points on the Analysis: After the precipitate has dissolved, titrate the
graph until it meets the concentration axis. The dis- excess iodine with 0.05 N sodium thiosulfate VS, using
tance between this point and the intersection of the 1 mL of starch TS, added toward the end of the titra-
axes represents the concentration of nickel in the Sam- tion as an indicator.
ple solution. Acceptance criteria: NLT 12.8 mL of 0.05 N sodium
Acceptance criteria: NMT 1 ug/g, calculated on the thiosulfate VS is required, corresponding to NMT 0.3%
anhydrous basis of reducing sugars, determined on the anhydrous basis
© ORGANIC IMPURITIES as glucose.
Mobile phase, Sample solution, and Chromatographic
system: Proceed as directed in the Assay. SPECIFIC TESTS
System suitability solution: 20 mg/mL of USP Isomalt oe WATER DETERMINATION (921), Method
|
RS and 0.1 mg/mL each of USP Mannitol RS and USP Sample: 0.3 g
Sorbitol RS in water Analysis: Add the Sample to a mixture of anhydrous
Standard solution: 0.1 mg/mL each of USP Sorbitol RS methanol and formamide (1:1) at 50+ 5°.
and USP Mannitol RS Acceptance criteria: NMT 7.0%
System suitability e@ CONDUCTIVITY
Sample: System suitability solution Sample solution: Dissolve 20g in carbon dioxide-free
[Note—The relative retention times for 1,1-GPM, 1,6- water with gentle heating (40°-50°), cool, and dilute
GPS, mannitol, and sorbitol are about 1.0, 1.2, 1.6, with the same solvent to 100 mL.
and 2.0, respectively. The typical retention time for Analysis: yang an papier nate conductivity meter that
1,1-GPM is about 12.3 mind has been standardized with a potassium chloride con-
Suitability requirements ductivity calibration standard, measure the conductivity
Resolution: NLT 2.0 between 1,1-GPM and 1,6-GPS of the Sample solution while gently stirring with a mag-
Analysis netic stirrer.
Samples: Sample solution and Standard solution Acceptance criteria: NMT 20 yS/cm
Calculate the percentage of mannitol or sorbitol in the ADDITIONAL REQUIREMENTS
portion of Isomalt taken: e *PACKAGING AND STORAGE: Preserve in well-closed con-
Result = (ru/rs) x (Cs/Cy) x 100 tainers. No storage requirements are specified.»
e LABELING: Label it to indicate the percentage content of
ty = peak response of mannitol or sorbitol from the 1,6-GPS and 1,1-GPM.
Sample solution e USP REFERENCE STANDARDS (11)
fs a peak response of mannitol or sorbitol from the USP Isomalt RS
Standard solution USP Mannitol RS
Gs = concentration of USP Mannitol RS or USP USP Sorbitol RS
Sorbitol RS in the Standard solution (mg/mL)
Cy = concentration of Isomalt in the Sample solution
(mg/ml)
Calculate the percentage of any unknown impurity in
the portion of Isomalt taken: Isopropyl Alcohol—see Isopropyl! Alcohol
Result = (ru/rs) x (Cs/Cy) x 100
General Monographs
tu = peak response of each unknown impurity from
the Sample solution
L rs = peak response of sorbitol from the Standard Isopropyl Myristate
roe solution
a Cs = concentration of USP Sorbitol RS in the
ro] Standard solution (mg/mL)
° Cu = concentration of Isomalt in the Sample solution
EB
>
(mg/ml)
Acceptance criteria: See Table 1. [NotE—Disregard any
; Ci7H3402 270.45
7 impurity peak that is less than 0.1%.] Tetradecanoic acid, 1-methylethyl ester;
Isopropyl myristate [110-27-0].
rs
NF 36 Official Monographs / \sopropy! 5405

DEFINITION Acceptance criteria: NLT 90.0%

Isopropyl Myristate consists of esters of isopropyl alcohol
and saturated high molecular weight fatty acids, princi- IMPURITIES
pally myristic acid. It contains NLT 90.0% of isopropyl e RESIDUE ON IGNITION (281): NMT 0.1%
myristate (Ci7H3402).
SPECIFIC TESTS
IDENTIFICATION e SPECIFIC GRAVITY (841): 0.846-0.854
e A. The retention times of the major peaks of the Sample REFRACTIVE INDEX (831): 1.432-1.436 at 20°
solution correspond to those of the System suitability solu- FATS AND FIXED OILS, Acid Value (401): NMT 1
tion, as obtained in the Assay. FATS AND FIXED OILS, /odine Value (401): NMT 1
FATS AND FIXED OiLs, Saponification Value (401): 202-212
ASSAY
¢ PROCEDURE ADDITIONAL REQUIREMENTS
System suitability solution: 5.0mara of USP Isopro- e PACKAGING AND STORAGE: Preserve in tight, light-resistant
pyl Myristate RS and 0.5 mg/mL of USP Isopropyl Pal- containers.
mitate RS in n-hexane e USP REFERENCE STANDARDS (11)
Sample solution: 5.0 mg/mL of Isopropyl Myristate in USP Isopropyl Myristate RS
n-hexane USP Isopropyl Palmitate RS
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: GC
Detector: Flame ionization
Column: 0.32-mm x 15-m fused silica coated with a Isopropyl Palmitate
1.0-uum layer of stationary phase G27
Temperatures
Injection port: 240° Wye NNN
Detector: 280°
Column: See Table 7.
Ci9H3gO2 298.50
Table 1 Hexadecanoic acid, 1-methylethyl ester;
Isopropyl palmitate [142-97-6].
Hold Time
Initial Temperature Final at Final DEFINITION
Temperature Ramp Temperature | Temperature Isopropyl Palmitate consists of esters of isopropyl alcohol
() (¢/min) «) (min) and saturated high molecular weight fatty acids. It con-
150 =_ 150 1 tains NLT 90.0% of isopropyl palmitate (CisH3sO2).
150 6 230 8
IDENTIFICATION
Carrier gas: Helium e A. The retention times of the major peaks of the Sample
Flow rate: 1.5 mL/min solution correspond to those of the System suitability solu-
Injection volume: 2.0 wL tion, as obtained in the Assay.
Injection type: Split injection, split ratio 10:1 ASSAY
Run time: 22 min e PROCEDURE
System suitability System suitability solution: 5.0 mg/mL of USP Isopro-
Sample: System suitability solution py! Palmitate RS and 0.5 mg/mL of USP Isopropyl My-
[Note—The relative retention times for isopropyl myris- ristate RS in n-hexane
tate and isopropyl palmitate are 1.0 and 1.3, Sample solution: 5.0 mg/mL of Isopropyl Palmitate in
respectively. n-hexane
Suitability requirements Chromatographic system
Resolution: NLT 6.0 between the isopropyl myristate (See Chromatography (621), System Suitability.)
and isopropyl palmitate peaks Mode: GC
Tala actor: NMT 2 for the isopropyl myristate Detector: Flame ionization
pea Column: 0.32-mm x 15-m fused silica; coated with a
Relative standard deviation: NMT 2.0% 1.0-um layer of stationary phase G27
Analysis Temperatures
Sample: Sample solution Injection port: 240°
Calculate the percentage of isopropyl myristate Detector: 280°
(Ci7H3402) in the portion of Isopropyl Myristate taken: Column: See Table 7.
Result = (ru/rr) x 100
Table 1
tu peak area of isopropyl myristate Hold Time
tr sum of the peak areas of all the peaks, except Initial Temperature Final at Final
the solvent peak Temperature Ramp Temperature | Temperature
@) (¢/min) «) (min)
EXtTe[-B] oLelUCo AMET]

150 = 150 1
150 6 230 8
 5406 Isopropyl / Official Monographs NF 36

Carrier gas: Helium Sample: Sample amount (see Fats and Fixed Oils (401),
Flow rate: 1.5 mL/min Table 1)
Injection volume: 2.0 uL Acceptance criteria: NMT 6.0
Injection type: Split injection, split ratio 10:1 e B. CHROMATOGRAPHIC IDENTITY
Run time: 22 min Analysis: Proceed as directed in the Assay.
System suitability Acceptance criteria: The retention time of the major
Sample: System suitability solution peak of the Sample solution corresponds to that of the
[Note—The relative retention times for isopropyl myris- Standard solution, as obtained in the Assay.
tate and isopropyl! palmitate are 0.8 and 1.0,
respectively.] ASSAY
Suitability requirements © PROCEDURE
Resolution: NLT 6.0 between the isopropyl myristate Mobile phase: Tetrahydrofuran
and isopropyl palmitate peaks Reference solution: 2 mg/mL of isostearic acid in Mo-
Tailing factor: NMT 2 for the isopropyl palmitate bile phase
peak Standard solution: 40 mg/mL of USP Isosteary! !sos-
Relative standard deviation: NMT 2.0% tearate RS in Mobile phase
Analysis Sample solution: 40 mg/mL of Isostearyl Isostearate in
Sample: Sample solution Mobile phase
Calculate the percentage of isopropyl palinitate Chromatographicsytem
(CigH3gO2z) in the portion of Isopropyl Palmitate taken: (See Chromatography {621}, System Suitability.)
Mode: LC
Result = (ru/rr) x 100 Detector: Differential refractive index
Column: Two 7.5-mm x 30-cm columns in tandem;
fu peak area of isopropyl palmitate
= 3-um packing L21
Ir = sum of the peak areas of all the peaks, except Temperatures
the solvent peak Column: 35°
Acceptance criteria: NLT 90.0% Detector: 35°
Flow rate: 1.0 mL/min
IMPURITIES Injection volume: 20 uL
e RESIDUE ON IGNITION (281): NMT 0.1% Run time: 30 min
System suitability
SPECIFIC TESTS Samples: Reference solution and Standard solution
e SPECIFIC GRAVITY (841): 0.850-0.855 [NotE—The relative retention times for isostearyl isos-
e REFRACTIVE INDEX (831): 1.435-1.438 tearate, isostearic acid, and isosteary! alcohol are 1.00,
e FATS AND FIXED OILS, Acid Value (401): NMT 1 1.07, and 1.09, respectively.]
e FATS AND FIXED OILS, lodine Value (401): NMT 1 Suitability requirements
e FATS AND FIXED OILS, Saponification Value (401): 183-193 Resolution: NLT 1.3 between the isosteary! isos-
ADDITIONAL REQUIREMENTS tearate peak and the adjacent peak at the retention
¢ PACKAGING AND STORAGE: Preserve in tight, light-resistant time 1.09 relative to isostearyl isostearate, Standard
containers. Solution
e USP REFERENCE STANDARDS (11) Relative standard deviation: NMT 2.0%, determined
USP Isopropyl Myristate RS from the isostearyl isostearate peak, Standard solution
USP Isopropyl! Palmitate RS Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of isostearyl isostearate in the
portion of Isostearyl Isostearate taken:
Result = (ru/rs) x (G/Cu) x 100
Add the following:
ty = peak response of isostearyl isostearate from
the Sample solution
alsosteary! Isostearate Is = peak response of isosteary! isostearate from
the Standard solution
G = concentration of USP Isostearyl isostearate RS
DEFINITION in the Standard solution
lsosteary! Isostearate is obtained by esterification of isostearic Cu = concentration of Isostearyl Isostearate in the
acid which is a mixture mainly of saturated branched 18 Sample solution
carbon-chain (C18) fatty acids, linear hexadecanoic Acceptance criteria: NLT 85.0%
(C16:0) and octadecanoic acids (C18:0), with isostearyl
alcohol. It contains NLT 85.0% of isostearyl isostearate. IMPURITIES
© RESIDUE ON IGNITION (281): NMT 0.2%
IDENTIFICATION
o A. ESTER SPECIFIC TESTS
Analysis 1: Proceed as directed in Fats and Fixed Oils e FATS AND FIXED OILS (401), Procedures, Hydroxyl Value
”“
(401), Procedures, Saponification Value. Sample: 4g
a= Sample: 3 Analysis: Proceed as directed in the chapter. A sand
is
Ss
Analysis: Proceed as directed in the chapter. Heat bath can be used.
under reflux for 1h.
=.)
—
Acceptance criteria: 90-110
} Analysis 2: Proceed as directed in Fats and Fixed Oils
i
Sj 4401), Procedures, Acid Value.
=
J
vs
NF 36
Acceptance criteria: NMT 25.0
e FATS AND FIXED Olis (401), Procedures, lodine Value
Sample: 3g
Acceptance criteria: NMT 5.0
e FATS AND FIXED OILS (401), Procedures, Peroxide Value
Sample: 39
Acceptance criteria: NMT 5.0
e@ WATER DETERMINATION (921), Method |
Sample: 3g
Acceptance criteria: NMT 0.5%
e REFRACTIVE INDEX (831): 1.454-1.464 at 25°
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in tight containers,
protected from light and moisture. Store at room tem-
Ea and avoid exposure to excessive heat.
e USP REFERENCE STANDARDS (11)
USP Isostearyl Isostearate RS
ANF30
Juniper Tar—see Juniper Tar General
Monographs
Kaolin—see Kaolin General Monographs
Alpha-Lactalbumin
Co2eHosaNie20196S9 14178
[9051-29-0].
DEFINITION
Alpha-Lactalbumin is a lyophilized or spray-dried powder of
compact globular metalloprotein that may containa sin-
gle bound calcium ion and is capable of binding zinc and
other metals. Alpha-Lactalbumin is isolated either from
bovine milk or from whey, both of which should be from
edible sources suitable for human use. All materials de-
rived from bovine sources must originate from countries
free of bovine spongiform encephalopathy. It contains al-
pha-lactalbumin at NLT 90.0% of the labeled total protein
content. The remainder consists mostly of beta-lactoglob-
ulin. It may contain suitable stabilizers.
IDENTIFICATION
© A. SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS
Gel fixing solution: In a 1-L Pyrex bottle, thoroughly
mix 500 mL of water, 400 mL of alcoholic TS, and
100 mL of glacial acetic acid.
Gel staining solution: Prepare a solution of Coomassie
blue G-250 having a concentration of 0.25 g/L in a
10.0% (v/v) acetic acid solution.’ Store at room tem-
perature.
Destaining solution: 10.0% (v/v) acetic acid in water
[Note—This solution may be stored at room tempera-
ture for up to 6 months from the date prepared.
Sample buffer: Prepare a solution containing 200 mM
tris(hydroxymethyl)aminomethane hydrochloride (Tris-
HCl), 2% (w/v) sodium dodecyl sulfate (SDS), 40% (v/
v) glycerol, and 0.04% (w/v) Coomassie blue G-250. If
necessary, adjust with hydrochloric acid or sodium hy-
droxide to a pH of 6.8.2
Running buffer: Prepare a solution containing 100 mM
tris(hydroxymethyl)aminomethane, 100 mM N-
1A suitable gel staining solution is available from, e.g., Bio-Rad. Coomassie
brilliant blue G-250 is available from Bio-Rad, Cat. # 161-0406.
2A suitable sample buffer is available as Tricine sample buffer from Bio-Rad,
Cat. # 161-0739.
Official Monographs / Lactalbumin 5407
tris(hydroxymethyl)methyiglycine (tricine), and 0.1%
(w/v) SDS in water. If necessary, adjust with hydrochlo-
ric acid or sodium hydroxide to a pH of 8.3.3 Ina
400-mL beaker, thoroughly mix 35 mL of the solution
so obtained (or 10x Tris/Tricine/SDS buffer)* with
315 mL of water.
Molecular weight marker: Use a suitable molecular
weight marker containing protein bands between 3.5
and 27 kDa.
Molecular weight standard solution: Transfer 16 ul of
the Sample buffer into a 0.5-mL microcentrifuge tube.
Pipet 4 uL of the Molecular weight marker into the
microcentrifuge tube, and mix. Incubate the mixture in
the closed microcentrifuge tube for 5 min at 95°. After
incubation, allow the tube to stand for 5 min at room
temperature. Centrifuge at 5000 rpm for 1 min.
Alpha-Lactalbumin standard stock solution: 1.0%
(w/v) of USP Alpha-Lactalbumin RS in water in a 2-mL
centrifuge tube.
Alpha-Lactalbumin standard working solution: Pipet
21 ul of the Sample buffer and 3 uL of the Alpha-Lactal-
bumin standard stock solution into a 0.5-mL microcen-
trifuge tube, and mix. Proceed as directed for Molecular
weight standard solution beginning with “Incubate the
mixture”.
Sample stock solution: 1.0% (w/v) of Alpha-Lactalbu-
min in water in a 2-mL centrifuge tube.
Sample solution: Pipet 21 uL of the Sample buffer and
3 pl of the Sample stock solution into a 0.5-mL
microcentrifuge tube, and mix. Proceed as directed for
Molecular weight standard solution beginning with “In-
cubate the mixture”.
SDS-PAGE gel and apparatus set-up: Following the
manufacturer’s instructions, assemble and fill a 16.5%
Tris-Tricine Ready GelS in the Mini-Protean Ill Electro-
phoresis Module,é or in an equivalent module. Add Run-
ning buffer appropriately to this apparatus.
Analysis
Gel loading: Load 10 ul of the Molecular weight stan-
dard solution, 2.5 wL of the Alpha-Lactalbumin standard
working solution, and 2.5 wL of the Sample solution, re-
spectively, into the 16.5% Tris-Tricine SDS-PAGEgel.
[Note—The loaded samples contain approximately
3 ug of protein based on the sample weight.]
Running the gel: Set the voltage to 100 V, and run at
a constant voltage. Run the gel until the tracking dye
front is approximately 10 mm from the bottom of the
gel (approximately 80-90 min).
Gel fixing: Remove the gel, transfer to a peas con-
tainer, and soak in the Gel fixing solution for 30 min on
a shaking rack. Decant the Gel fixing solution. Rinse
with water, and decant.
Gel staining: Pour pany 100 mL of the Gel
staining solution into the staining container. Place the
gel into the staining container, and allow the stain to
completely cover the gel. Place the staining container
on an appropylate shaker, and stain the gel for 60-90
min with gentle shaking.
Destaining: Drain the Gel staining solution into an ap-
propriate waste container, and add 100 mL of De-
stag, solution to the container to cover the gel.
Place the container on an appropriate shaker, and
shake with gentle agitation for 30 min. Discard the
used Destaining solution, and repeat destaining as nec-
sydesBouo- 4N
essary. Repeat rinsing with Destaining solution three to
four times at 30-min intervals or until the gel is de-
stained to the desired clarity.
Acceptance criteria: The Alpha-Lactalbumin has one
major band at 14 kDa, a minor band at 16 kDa, and a
3 An undiluted suitable running buffer is available as 10x Tris/Tricine/SDS
buffer from Bio-Rad, Cat. # 161-0744.
4 Available as 10x Tris/Tricine/SDS buffer from Bio-Rad, Cat. # 161-0744.
5 Available from Bio-Rad, Cat. # 161-1107 or 161-1179.
6 Available from Bio-Rad, Cat. # 165-3302.
 5408 Lactalbumin / Official Monographs
molecular weight that is similar to that of USP Alpha-
Lactalbumin RS.
e B. The retention time of the major peak for alpha-lactal-
bumin from the Sample solution corresponds to that of
the Standard solution, as obtained in the test for Content
of Alpha-Lactalbumin in the Assay.
ASSAY
© CONTENT OF ALPHA-LACTALBUMIN
Mobile phase: Prepare a solution of 0.02 M Tris-HCl,
0.5% SDS, and 0.1 N sodium chloride. Adjust the pH of
the solution to 5.95 + 0.05. Pass this solution through a
filter having a 0.5-um or finer porosity, and degas.
Standard solution: 1.0 mg/mL of USP Alpha-Lactalbu-
min RS in Mobile phase. [NotE—Prepare it immediately
before use.]
System suitability solutuion: 0.5 mg/mL of USP Alpha-
Lactalbumin RS and 0.5 mg/mL of beta-lactoglobulin in
Mobile phase
Sample solution: 1.0 mg/mL of Alpha-Lactalbumin in
Mobile phase
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 280 nm
Column: 7.8-mm x 30-cm analytical column, packing
L33
[NoTe—Equilibrate the column for approximately 90
min at 0.6 mL/min of Mobile phase or until a stable
baseline is achieved.]
Flow rate: 0.6 mL/min
Injection size: 20 uL
System suitability
Sample: System suitability solution
[Note—The relative retention times for beta-lactoglobu-
lin and alpha-lactalbumin are 0.91 and 1.00,
respectively.]
Suitability requirements
Resolution: NLT 1.65 between beta-lactoglobulin and
alpha-lactaloumin
Tailing factor: Not greater than 1.1 for the alpha-
lactalbumin peak
Analysis
Samples: Standard solution and Sample solution
Calculate the purity of Alpha-Lactalbumin as a percent-
age of total protein:
Result = (ru/rs) x [Cs/(Cu x P)] x 100
tu = peak response for alpha-lactalbumin from the
Sample solution
Is = peak response for alpha-lactalbumin from the
Standard solution
Cs = concentration of USP Alpha-Lactalbumin RS in
the Standard solution (mg/mL)
Cu = concentration of Alpha-Lactalbumin in the
Sample solution (mg/mL)
P = percentage of total protein content
Acceptance criteria: Content of alpha-lactalbumin is
NLT 90.0% of the labeled total protein content.
© TOTAL PROTEIN CONTENT
Sample: 250 mg of Alpha-Lactalbumin
Analysis: Combust the Sample in the presence of pure
oxygen (99.9%) in an airtight oven at 950° with a suit-
a)
aol
able nitrogen analyzer. The components such as carbon
re dioxide, sulfur dioxide, and moisture are absorbed by
sS various in-line chemical filters. All nitrogenous matter is
Dd
—
converted into nitrogen in the presence of catalytic
° converters. The weight percent of nitrogen is measured
¢ by a thermal conductivity detector. Blank the system by
iS analyzing a suitable nitrogen blank material, such as
= powered cellulose, and obtaining a zero reading. Cali-
J rate and qualify the system by using EDTA. The rela-
CA tive standard deviation for replicate runs is NMT 0.5%.
Calculate the weight percent of total protein content in
NF 36
Alpha-Lactalbumin by multiplying the percentage of ni-
trogen found by 6.23.
Acceptance criteria: Total protein content is NLT
90.0%.
OTHER COMPONENTS
© LIMIT OF BETA-LACTOGLOBULIN
Mobile phase, System suitability solution, Sample so-
lution, Chromatographic system, and System suitabil-
ity: Prepare as directed in the test for Content of Alpha-
Lactalbumin.
Standard solution: 1.0 mg/mL of beta lactoglobulin in
a phase. [Note—Prepare it immediately before
use.
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of beta-lactoglobulin as a
percentage of total protein:
Result = (ru/ts) x [Cs/(Cu x P)] x 100
tu = peak response for beta-lactoglobulin from the
Sample solution
Is = peak response for beta-lactoglobulin from the
Standard solution
Cs = concentration of beta-lactoglobulin in the
Standard solution (mg/mL)
Cu = concentration of Alpha-Lactalbumin in the
Sample solution (mg/mL)
B = percentage of total protein content
Acceptance criteria: NMT 6.5%, calculated on the to-
tal protein basis
© CONTENT OF CALCIUM
Standard stock solution: Dissolve 1.249 g of calcium
carbonate in 270 mL of 3 N hydrochloric acid (dilute
250 mL of hydrochloric acid with water to 1000 mL) in
a 1000-mL volumetric flask. Dilute with water to vol-
ume, and mix. Dilute 50 mL of the solution so obtained
to 1000 mL. The Standard stock solution contains 25 j1g/
mL of calcium.”
Lanthanum chloride solution: Weigh 11.7 g (+
100 mg) of lanthanum oxide, and transfer to a
1000-mL volumetric flask. Add enough water to wet
the powder, and then slowly add 50 mL of hydrochloric
acid. [Cautlon—Exothermic reaction.] Let the test speci-
men dissolve, dilute with water to volume, and mix.
This solution contains 1% (w/v) of lanthanum and is
stable for up to 6 months when stored at room
temperature.
Blank solution: 10-fold dilution of Lanthanum chloride
solution
Working standard solutions: To five identical 25-mL
volumetric flasks add 0, 5, 10, 15, and 20 mL, respec-
tively, of Standard stock solution. Add 2.5 mL of Lantha-
num chloride solution, and dilute with water to volume.
The Working standard solutions contain 0, 5, 10, 15, and
20 g/mL of calcium, each containing 0.1% (w/v) of
lanthanum.
Sample solution: Transfer 1.0 g of Alpha-Lactalbumin
to a 100-mL volumetric flask, add 10 mL of Lanthanum
chloride solution, and dilute with water to volume.
Spectrometric conditions
See Atomic crap Spectroscopy (852).)
Mode: Atomic absorption spectrophotometry
Analytical wavelength: 422.7 nm
Lamp: Calcium hollow-cathode lamp
Flame: Reduced air—acetylene
Analysis
Samples: Working standard solutions and Sample
solution
7 A commercially prepared, certificated AA standard is available as Calcium
AA, ICP standards, 1000 ppm Ca in dilute hydrochloric acid, Cat. # ACATKH,
from RICCA. Make an appropriate dilution to obtain a final concentration of
25 ug/mL of calcium.
NF 36
Concomitantly determine the absorbances of the Sam-
ples using the Blank solution. [NoTE—Optimize flame
arameters in accordance with the instrument manu-
acturer’s instructions.]
Plot the absorbances of the Working standard solutions
versus the concentration, in g/mL, of calcium, and
draw the straight line best fitting the five plotted
points. From the graph so obtained, determine the
concentration, C, in g/mL, of calcium in the Sample
solution.
Calculate the quantity of calcium (Ca), in mg, in each g
of Alpha-Lactalbumin taken:
Result
= (V x C)/W
x F x 100
Vv = volume of the Sample solution, 100 mL
Cc = as determined above
Ww = weight of Alpha-Lactalbumin taken to prepare
the Sample solution (g)
F = conversion factor for ig to mg (103 mg/ug)
Acceptance criteria: NMT 1 mg/g of calcium
IMPURITIES
Inorganic Impurities
e ASH: Ignite 1g of Alpha-Lactalbumin at NMT 550° until
free from carbon. Cool in a desiccator, and weigh: NMT
3.5% is found.
Delete the following:
°e HEAVY METALS, Method [I (231): NMT 10 ppme coincial 1-
Jan-2018)
e Limit OF PHOSPHORUS
Hydrochloric acid solution: Pipet 250 mL of hydrochlo-
ric acid into a 1000-mL volumetric flask, dilute with
water to volume, and mix.
Molybdovanadate reagent: Dissolve 20 g of ammo-
nium molybdate in 200 mL of water with the aid of
heat, and then allow the molybdate solution to cool.
Dissolve 1.0 g of ammonium vanadate in 125 mL of
water with the aid of heat, cool, and add 160 mL of
hydrochloric acid. Gradually add, with stirring, the mo-
lybdate solution to the vanadate solution, and dilute
with water to 1000 mL.
Phosphorus standard stock solution |: Transfer 8.8 g
of monobasic potassium phosphate (KH2PO,), previ-
ously dried for 2 h at 10s, to a 1000-mL volumetric
flask, and add 750 mL of water to dissolve. Dilute with
water to volume. This solution contains 2 mg/mL of
hosphorus. [NoTE—Store the solution in a refrigerator.]
Phosphorus standard stock solution II: Immediately
before use, dilute 50 mL of Phosphorus standard stock
solution | with water to 1000 mL. [NoTE—Store in a
refrigerator.]
Standard solutions: Transfer 0.0 mL, 5.0 mL, 8.0 mL,
10.0 mL, and 15.0 mL of Phosphorus standard stock solu-
tion Il, respectively, to five identical 100-mL volumetric
flasks. Proceed as directed in the Analysis: after treat-
ment with the Molybdovanadate reagent, the resulting
final He ehelys concentrations for the Standard solu-
tions are 0.0, 5.0, 8.0, 10.0, and 15.0 ug/mL,
respectively.
Sample solution: Transfer 4.0 g of Alpha-Lactalbumin
to an ashing dish. Dry the test specimen on a hot plate
or steam bath. Ignite in a muffle furnace at a maximum
temperature of 600° until free of carbon. Cool, add
40 mL of Hydrochloric acid solution and several drops of
nitric acid, and bring to boil on a hot plate. Cool, trans-
fer to a 100-mL volumetric flask by rinsing the ashing
dish with water, dilute with water to volume, and mix.
Pipet 20.0 mL of the Sample solution into a 100-mL vol-
umetric flask.
Spectrometric conditions
(See Ultraviolet-Visible Spectroscopy (857).)
Official Monographs / Lactaloumin 5409
Mode: UV-Vis
Analytical Wavelength: 400 nm
Analysis
Samples: Standard solutions and the Sample solution
To each of the flasks containing the Samples, add
20.0 mL of Molybdovanadate reagent, dilute with water
to volume, mix, and allow to stand for exactly 10 min
for maximum color development. The Standard solu-
tions and the Sample solution are treated identically.
Concomitantly determine the absorbances of each of
the Samples in 1-cm cells using the Spectrometric condi-
tions described above. Use one of the Standard solu-
tions with phosphorus concentration at 0.0 ug/mL to
zero the spectrophotometer. Plot the absorbances of
the Standard solutions versus concentration, in g/mL,
of phosphorus, and draw the straight line best fittin:
the four plotted points. From the graph so obtained,
determine the concentration, C, in g/mL, of phos-
phorus in the Sample solution.
Calculate the quantity, in ug, of phosphorus in each g
of Alpha-Lactalbumin taken:
Result = (V x C)/W x D
Vv = volume of the Sample solution, 100 mL
C = as determined above
Ww = weight of Alpha-Lactalbumin taken to prepare
the Sample solution (g)
D = dilution factor, 5
Acceptance criteria: NMT 700 g/g of phosphorus
Organic Impuriti ies
© PROCEDURE 1: LimiT OF LIPID (FAT)
Weighing dish preparation: bret the clean dishes
under the same conditions that will be used for final
drying after fat extraction. Ensure that all surfaces
where weighing dishes will be placed (i.e., hot plate,
desiccator, etc.) are clean and free of particulates. At
the end of oven drying, place the weighing dishes in a
desiccator, and cool to room temperature. Immediately
before use, weigh the dishes to the nearest 0.1 mg,
and record the weights. Check the balance zero after
weighing each dish. Protect the weighed dishes from
contamination with extraneous matter.
Analysis
Transfer 0.5 g of Alpha-Lactalbumin to a Mojonnier-
style ether extraction flask that has the capacity to
hold a volume of 21-23 mL in the lower bulb plus
neck at the bottom of the flask. The flask has a
smooth, round opening at the top that can be sealed
when closed with cork. Add 10 mL of water at a tem-
erature of 40°, and mix. Add 1.5 mL of ammonium
ydroxide to the Alpha-Lactalbumin, and mix thor-
oughly. Add 3 drops of phenolphthalein TS to help
sharpen the visualappeiance of the interface be-
tween the ether and the aqueous layers during ex-
traction. Add 10 mL of alcohol, close with the cork
stopper that has been water soaked, and shake the
flask for 15 s.
For the first extraction, add 25 mL of ether, replace
the cork stopper, and shake the flask very vigorously
for approximately 1 min, releasing built-up pressure
by loosening the stopper as necessary. Add 25 mL of
petroleum ether, replace the cork stopper, and repeat
vigorous shaking for about 1 min. Centrifuge the
flask at about 600 rpm for NLT 30 s to obtain a clean
sydesBbouow 4N
separation of the aqueous (bright pink) and the ether
phases. Decant the ether solution into a suitable
weighing dish prepared as directed for Weighing dish
preparation. When the ether solution is decanted into
the dish, be careful not to pour any Ssuspeniced solids
or aqueous phase into the weighing dish. Ether can
be evaporated at NMT 100° from the dish while con-
ducting the second extraction.
For the second extraction, add 5 mL of alcohol to the
original flask, close with the cork stopper, and shake
 5410 Lactalbumin / Official Monographs
vigorously for 15 s. Add 15 mL of ether, replace the
cork, and shake the flask vigorously for about 1 min.
Add 15 mL of petroleum ether, replace the cork stop-
per, and repeat vigorous shaking for about 1 min.
Centrifuge the flask at about 600 rpm for NLT 30 s to
obtain a clean separation of the aqueous (bright
pink) and the ether phases. If the interface is below
the neck of the flask, add water to bring the level
about halfway up to the neck. Add water slowly
down the inside surface of the flask so that there is
minimum disturbance of the interface. Decant the
ether solution for the second extraction into the same
weighing dish used for the first extraction.
For the third extraction, omit addition of the alcohol
and repeat the procedure used for the second extrac-
tion. Completely evaporate the solvents in a hood on
a hot plate at NMT 100°, and avoid spattering. Dry
the extracted fat and the weighing dish to constant
weight in a forced air oven at 100° + 1° for NLT 30
min or in a vacuum oven at 70° to 75° at more than
50.8 cm (20 inches) of vacuum for NLT 7 min. Re-
move the weighing dish from the oven, and place in
a desiccator to cool to room temperature. Record the
weight of the weighing dish containing the fat.
Run a blank determination using water, and record the
weight of any dry residue collected. The reagent
blank should be less than 2.0 mg of residue. [NoTE—
A negative number is not accep]
Calculate the weight percent of lipid (fat) in the por-
tion of Alpha-Lactalbumin taken:
Result = [(W2 — W1) — Ws]/W x 100
W, ~~ = weight of empty weighing dish (g)
W.2 == weight of the weighing dish containing fat
(g)
W3 = weight of the reagent blank residue (g)
WwW = weight of the Alpha-Lactalbumin taken for the
fat extraction (g)
Acceptance criteria: NMT 1.0% is found. The differ-
ence between duplicate runs is NMT 0.03% fat.
e PROCEDURE 2: LIMIT OF LACTOSE
Carrez | solution: Transfer 3.60 g of potassium ferrocy-
anide [K4Fe(CN)¢- 3H2O] to a 100-mL volumetric flask,
dissolve in and dilute with water to volume, and mix.
Carrez Il solution: Transfer 7.20 g of zinc sulfate
heptahydrate (ZnSO,- 7H2O) to a 100-mL volumetric
flask, dissolve in and dilute with water to volume, and
mix.
[Note—The following four test reagents are included in
a test kit.8]
Test reagent 1: About 600 mg of lyophilisate consist-
ing of a mixture of citrate buffer (pH 6.6), nicotin-
amide adenine dinucleotide (NAD) (35 mg), anhydrous
magnesium sulfate, and stabilizers (added if necessary).
Dissolve lyophilisate in 7.0 mL of water before use.
Test reagent 2: About 1.7 mL of an enzyme suspen-
sion of B-galactosidase (approximately 100 Units).
Test reagent 3: 34 mL of a solution consisting of 0.51
M potassium diphosphate buffer (pH 8.6), and stabiliz-
ers (added if necessary).
Test reagent 4: About 1.7 mL of an enzyme suspen-
sion of galactose dehydrogenase (about 40 Units).
Sample solution: Transfer 1.0
g of Alpha-Lactalbumin
”
to a 100-mL volumetric flask, add about 60 mL of
a=
is water, and mix. Add 5 mL of Carrez | solution, and mix.
S Add 5 mL of Carrez II solution, and mix. Add 10 mL of
—
Dp 0.1 N sodium hydroxide solution, and mix vigorously.
2) Dilute with water to volume, and mix. Pass through a
= min in the holo form is between 58° and 61°.
S filter paper, and use the clear filtrate. Pe erst
= cedure Breaks emulsions, absorbs some colors, and pre-
cipitates proteins.]
J at 100°, at a pressure of
ra 8 Available from Boehringer-Mannheim (R-Biopharm, Inc., 7950 Old US 27S,
Marshall, Ml 49068 USA; Tel: +1-877-789-3033 or +1-269-789-3033; Fax:
+1-269-789-3070; www.r-biopharm.com).
NF 36
Analysis
Label one glass or disposable plastic cuvet as “blank”
and the second glass or disposable plastic cuvet as
“test”. [NoTE—These two cuvets should be equiva-
lent.] To each cuvet, pipet 0.20 mL of Test reagent 1
and 0.05 mL of Test reagent 2. Pipet 0.10 mL of the
Sample solution into the cuvet that is labeled “test”.
Mix both cuvets with their stirrers, and incubate at
20°-25° for 20 min. Pipet 1.00 mL of Test reagent 3
into each cuvet. Pipet 2.00 mL of water into the
cuvet that is labeled “blank” and 1.90 mL of water
into the cuvet containing the Sample solution. Mix,
and incubate at 20°-25° for about 2 min.
Determine the absorbances, As: and Asi, at 340 nm,
for the Sample solution and the blank, respectively.
Add 0.05 mL of Test reagent 4 to each cuvet. Mix and
incubate at 20°-25° until the reaction has stopped
(about 10-15 min). Determine the absorbances As2
and Ag2, at 340 nm, again for the Sample solution and
the blank, respectively. If the reaction has not
stopped after 15 min, continue to read the ab-
sorbances at 2-min intervals until the absorbance for
the Sample solution remains constant for two succes-
sive measurements.
Calculate the percentage of lactose in the portion of
Alpha-Lactalbumin taken:
Result = Vi x V2 x M, x [(As2 — Asi) — (As2 — Asi)]/(e x L
x V3 x W) x 100
Vy = volume of the Sample solution, 0.1L
Vz = volume of the final sample solution in the
cuvet, 3.30 mL
M, = molecular weight of lactose monohydrate,
360.32 g/mol
& = absorption coefficient of nicotinamide adenine
dinucleotide reduced form (NADH) at 340
nm, 6300 L- mol-!- cm"?
L = light path of the cuvet, 1.0 cm
V3 = volume of the Sample solution taken into the
cuvet, 0.1 mL
Ww = weight of Alpha-Lactalbumin taken to prepare
the Sample solution (g)
Acceptance criteria: NMT 1.0% of lactose
SPECIFIC TESTS
e MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI-
FIED MICROORGANISMS (62): The total aerobic bacterial
count does not exceed 1000 cfu/g. The total combined
molds and yeasts count does not exceed 100 cfu/g. It
meets the requirements of the tests for absence of Salmo-
nella species and Escherichia coli.
¢ DENATURATION TEMPERATURE
Sample solution: Prepare a protein dough by mixing
3 g of Alpha-Lactalbumin powder with 2 g of water.
Place the dough into a well-sealed sample container.
Analysis: Perform two measurements on the dough
sample ueitig a differential scanning calorimeter. Heat
to 140°, and scan. Cool rapidly to below room temper-
ature, and rescan. Apply a scan rate of 10°/min. Weigh
pans before and after scanning to verify that no mois-
ture loss occurs during the scanning process. Measure
and record the denaturation temperatures as peak tem-
peratures. The formation of two peaks indicates the
presence of both the apo form and the holo form of
Alpha-Lactalbumin.
Acceptance criteria: The denaturation temperature for
Apne tartare in the apo form is between 50° and
52°; the denaturation temperature for Alpha-Lactalbu-
° PH (791): NMT 7.5, in a solution (1 in 10)
e Loss ON DRYING (731): Dry 1.0-1.5 g in a vacuum oven
660 mm of mercury, and with
i j i 59 j
continuous dry air feed for 5 h: it loses NMT 6.5% of its
weight.
 4492 Boswellia / Dietary Supplements
Dipping reagent: Prepare a solution of 10% sulfuric
acid in methanol. [NoTE—Prepare fresh immediately
before use.]
Application volume: 10 pL
Analysis
Samples: Standard solution and Sample solution
Apply the Samples as bands to a suitable thin-layer
chromatographic plate (see Chromatography (621)).
Use a saturated chamber. Develop until the solvent
front has moved up about 90% of the plate. Remove,
dry, and examine under UV light at 254 nm. Dip in
the Dipping reagent, heat for 5-10 min at 100°, and
examine under visible light.
Acceptance criteria: Under UV light at 254 nm, the
Sample solution exhibits two main zones due to 11-keto-
B-boswellic acid and 3-acetyl-11-keto-B-boswellic acid at
R; values of about 0.30 and 0.36, respectively, corre-
sponding to zones from the Standard solution. Under
visible light, the Sample solution exhibits two additional
zones due to B-boswellic acid and 3-acetyl--boswellic
acid at R¢ values of about 0.49 and 0.58, respectively,
corresponding to zones from the Standard solution.
Other, less intense zones are observed for the Sample
solution and the Standard solution.
e B. The 210-nm chromatogram of the Sample solution, in
the test for Content of Keto-Derivatives of B-Boswellic Acids,
exhibits peaks for 11-keto-B-boswellic acid, 3-acetyl-
11-keto-B-boswellic acid, B-boswellic acid, and 3-acetyl-B-
boswellic acid at retention times that correspond to
those in the 210-nm chromatogram of Standard solution
B and the 210-nm Reference Chromatogram provided
with the USP Boswellia serrata Extract RS.
COMPOSITION
© CONTENT OF KETO-DERIVATIVES OF B-BOSWELLIC ACIDS
Standard solution A: Dissolve a quantity of USP 3-Ace-
tyl-11-keto-B-Boswellic Acid RS in methanol to obtain a
solution having a known concentration of 0.1 mg/mL.
Standard solution B: Treat a quantity of USP Boswellia
serrata Extract RS with gentle heating in methanol to
obtain a solution having a known concentration of
10 mg/mL. Before injection, pass throughafilter of
0.45-uum pore size.
Sample solution: Treat a quantity of Extract with gentle
heating in methanol to obtain a solution having a
known concentration of 10 mg/mL. Before injection,
pass througha filter of 0.45-uum pore size, and discard
the first few mL of the filtrate.
Mobile phase: Preparea filtered and degassed mixture
of acetonitrile, water, and glacial acetic acid
(900:100:0.1). Make adjustments if necessary.
2 Chromatographic system
Q (See Chromatography (621), System Suitability.)
s Mode: LC
3 Detector: UV 254 nm
rd Column: 4.6-mm x 25-cm; packing L1
cS Flow rate: See the gradient table below.
ww Time Flow Rate
a (min) (mL/min)
0 1
5. 15
10 2 CROORGANISMS (2022):
30 2 tests for absence of Salmonella species and Escherichia
32 1 coli.
45 1
Injection size: 20 WL
System suitability
Samples: Standard solution A and Standard solution B
[NoTE—The relative retention times for 11-keto-B-bos-
wellic acid and 3-acetyl-11-keto-B-boswellic acid are
about 1.0 and 1.4, respectively.]
USP 41
Suitability requirements: The chromatogram of Stan-
dard solution B is similar to the 254-nm Reference
Chromatogram provided with the USP Boswellia serrata
Extract RS.
Tailing factor: NMT 1.5, 3-acetyl-11-keto-B-boswellic
acid peak, Standard solution A
Relative standard deviation: NMT 2.0% of the
3-acetyl-11-keto-B-boswellic acid peak response for
replicate injections, Standard solution A
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Using the chromatogram of Standard solution B and
the 254-nm Reference Chromatogram provided with
the lot of USP Boswellia serrata Extract RS, identify the
retention times of the peaks of 11-keto-B-boswellic
acid and 3-acetyl-11-keto-B-boswellic acid in the Sam-
ple solution chromatogram.
Separately calculate the percentages of 11-keto-B-bos-
wellic acid and 3-acetyl-11-keto-B-boswellic acid in
the portion of Extract taken:
Result = (ru/ts) x (CsV/W) x 100F
tu = peak area of each analyte from the Sample
solution
rs = peak area of 3-acetyl-11-keto-B-boswellic acid
from Standard solution A
Cs = concentration of USP 3-Acetyl-11-keto-B-
Boswellic Acid RS in Standard solution A
(mg/mL)
= final volume of the Sample solution (mL)
Ww = weight of Extract taken to prepare the Sample
solution (mg)
F = conversion factor for each analyte (0.93 for
11-keto-B-boswellic acid and 1.0 for 3-acetyl-
11-keto-B-boswellic acid)
Acceptance criteria: Add the percentages of the two
analytes. It contains NLT 90.0% and NMT 110.0% of
the labeled amount of Extract, calculated on the dried
basis, as the sum of 11-keto-B-boswellic acid and 3-ace-
tyl-11-keto-B-boswellic acid.
IMPURITIES
Inorganic Impurities
Delete the following:
°e HEAVY METALS, Method II (231): NMT 20 ppme coticia 1-
Jan-2048)
Organic Impurities
© PROCEDURE: ARTICLES OF BOTANICAL ORIGIN, General
Method for Pesticide Residues Analysis (561): Meets the
requirements
SPECIFIC TESTS
e Loss ON DRYING (731): Dry 1.0g of Extract at 105° for 2
h: it loses NMT 5.0% of its weight.
e MICROBIAL ENUMERATION TESTS (2021): The total aerobic
bacterial count does not exceed 104 cfu/g, and the total
combined molds and yeasts count does not exceed 103
cfu/g.
°MICROBIOLOGICAL PROCEDURES FOR ABSENCE OF SPECIFIED Mi-
Meets the requirements of the
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in well-closed contain-
ers, protected from light and moisture, and store in a
cool place.
e LABELING: The label states the Latin binomial and, follow-
ing the official name, the part of the plant from which
the article was prepared. It meets other labeling require-
ments under Botanical Extracts (565).
NF 36 Official Monographs / Lactitol 5411

ADDITIONAL REQUIREMENTS Cs = concentration of USP Lactitol RS in the

© PACKAGING AND STORAGE: Preserve in tight containers, Standard solution (mg/mL)
and store at the temperature indicated on the label. Cu = concentration of Lactitol in the Sample solution
e LABELING: Label it to state the protein content, expressed (mg/mL)
as a total protein percentage. Indicate the type of source Acceptance criteria: 98.0%-101.0% on the anhydrous
material, expressed as bovine milk, whey, or both, used asis
to manufacture the final product. Label it to indicate the
storage conditions, the expiration date, and the name IMPURITIES
and concentration of any added stabilizers. e RESIDUE ON IGNITION (281): NMT 0.5%
e USP REFERENCE STANDARDS (11)
USP Alpha-Lactalbumin RS Delete the following:

°e HEAVY IMETALS (231)

Test preparation: Dissolve 4 g in 25 mL of water.
Acceptance criteria: NMT 5 ppMe <ottcie! }-jan-2018)
Lactic Acid—see Lactic Acid General e RELATED COMPOUNDS
Monographs Standard solution: 0.3 mg/mL of USP Lactitol RS
Sample solution: Prepare as directed in the Assay.
Chromatographic system: Proceed as directed in the
Assay.
Lactitol System suitability
Sample: Standard solution
HO HH OH [NotE—The relative retention times for lactose, glucose,
galactose, lactulitol, lactitol, galactitol, and sorbitol are
about 0.53, 0.58, 0.67, 0.72, 1.0, 1.55, and 1.68,
respectively.]
Analysis
Samples: Standard solution and Sample solution
Calculate the percentages of galactitol, sorbitol, lactu-
litol, lactose, glucose, and galactose in the portion of
Lactitol taken:
Ci2H24O11 344,31 Result = (ru/rs) x (Cs/Cu) x 100
Cy2H4O11 - H2O 362.34
Tu = peak response of the relevant related
Ci2H24011 - 2H20 380.35 compound, if observed, from the Sample
4-0-B-D-Galactopyranosyl-D-glucitol [585-86-4]. solution
Monohydrate [81025-04-9]. Is = peak response of lactitol from the Standard
Dihydrate [81025-03-8].
solution
DEFINITION Cs = concentration of USP Lactitol RS in the
Lactitol contains NLT 98.0% and NMT 101.0% of Cy2H24011, Standard solution (mg/mL)
calculated on the anhydrous basis. Cy = concentration of Lactitol in the Sample solution
(mg/mL)
IDENTIFICATION Acceptance criteria: The total of the percentages of all
A. INFRARED ABSORPTION (197K) related compounds is NMT 1.5%.
e REDUCING SUGARS
ASSAY Standard solution: Pipet 2 mL of a dextrose solution
© PROCEDURE containing 0.5 mg/mL into a 10-mL conical flask.
Mobile phase: Water Sample solution: Disolve 500 mg in 2.0 mL of water in
Standard solution: 10.0 mg/mL of USP Lactitol RS a 10-mL conical flask.
Sample solution: 10.0 mg/mL of Lactitol Analysis: Concomitantly add 1 mL of alkaline cupric tar-
Chromatographic system trate TS to each solution, heat to boiling, and cool.
(See Chromatography (621), System Suitability.) Acceptance criteria: NMT 0.2%, calculated as dex-
Mode: LC trose. The Sample solution shows no more turbidity than
Detector: Refractive index that produced in the Standard solution, in which a red-
Column: 7.8-mm x 30-cm; packing L34 dish brown precipitate forms.
Column temperature: 85°
Flow rate: 0.7 mL/min SPECIFIC TESTS
Injection size: 25 wL © WATER DETERMINATION, Method | (921): For the monohy-
System suitability drate form, 4.5%-5.5%; for the dihydrate form,
Sample: Standard solution 9.5%-10.5%; and for the anhydrous form, NMT 0.5%.
Suitability requirements
Relative standard deviation: NMT 1.0% for lactitol ADDITIONAL REQUIREMENTS
Analysis ¢ PACKAGING AND STORAGE: Preserve in well-closed
containers.
sydeabouo=: 4N

Samples: Standard solution and Sample solution

Calculate the percentage of lactitol (Ci2H24011) in the e LABELING: Label it to indicate whether it is the monohy-
portion of Lactitol taken: drate, the dihydrate, or the anhydrous form.
e USP REFERENCE STANDARDS (11)
Result = (ru/rs) x (Cs/Cu) x 100 USP Lactitol RS

Ty = peak response from the Sample solution

rs = peak response from the Standard solution
 5412 Lactobionic Acid / Official Monographs
Lactobionic Acid
CiaH22012 (acid form) 358.30
[96-82-2].
Ho
on \=o
Ho)
a Thioacetamide reagent: To 0.2 ml of thioacetamide
es dno * TS add 1 mL of a mixture of 5 mL of water, 15 mL of
fom Co
"
Ci2H20011 (S-lactone) 340.28
[5965-65-1].
on on
\.
“ve ‘re
Ho OOH OH J ‘oH
4-O-B-Galactopyranosyl-D-gluconic acid.
DEFINITION
Lactobionic Acid is a mixture in variable proportions of 4-O-
B-D-galactopyranosyl-D-gluconic acid and 4-O-B-pD-
galactopyranosyl-D-glucono-1,5-lactone. It contains NLT
98.0% and NMT 102.0%, on the anhydrous basis.
IDENTIFICATION
© A. INFRARED ABSORPTION (197K): [NOTE—If the spectra
obtained show differences, dissolve the test substance
and USP Lactobionic Acid RS separately in water, dry at
105°, and record new spectra using the residues.]
e B. THIN-LAYER CHROMATOGRAPHY (621)
Standard solution: 10 mg/mL of USP Lactobionic Acid
RS
Sample solution: 10 mg/mL of Lactobionic Acid
Adsorbent: Chromatographic silica gel mixture with an
average particle size of 10-15 um (TLC plates)
Developing solvent: Methanol, ethyl acetate, ammo-
nium hydroxide, and water (2:1:1:1)
Application volume: 5 wL
Spray reagent: Slowly add 10 mL of sulfuric acid to
about 40 mL of water. Mix, and allow to cool. Dilute
with water to 100 mL, and mix. Add 2.5 g of ammo-
nium molybdate and 1 g of ceric sulfate, and shake for
15 min to dissolve.
Analysis: Develop the chromatograms until the solvent
front has moved about three-fourths the length of the
plate, and allow to dry. Spray the plate with Spray rea-
gent, and allow to dry. Repeat two more times, heat at
110° for 15 min, and examine.
Acceptance criteria: The principal spot from the Sam-
ple solution is similar intposition and color to the princi-
pal spot from the Standard solution.
ASSAY
© PROCEDURE
Sample: 0.350g of Lactobionic Acid
Analysis: Dissolve the Sample in 50 mL of carbon
dioxide-free water, previously heated to 30°. Immedi-
ately titrate with 0.1 N sodium hydroxide, and deter-
NF Monographs
mine the two equivalence points potentiometrically.
(See Titrimetry (541).)
Each mL of 0.1 N sodium hydroxide consumed to the
first equivalency point is equivalent to 35.83 mg of
Ci2H22012 (corresponds to the acid form), and each mL
of 0.1 N sodium hydroxide consumed between the
first and second equivalency points is equivalent to
ine mg of Ci2H20O11 (corresponds to the 6-lactone
orm).
Calculate the content, expressed as a percentage, of the
lactobionic acid as the sum of both results.
NF 36
Acceptance criteria: 98.0%-102.0% on the anhydrous
basis
IMPURITIES
Delete the following:
°o HEAVY METALS (231)
1M sodium hydroxide, and 20 mL of glycerin. Heat in
a water bath for 20 s. [NoTe—Prepare immediately
before use.]
Lead nitrate stock solution: Prepare as directed for
Special Reagents in Heavy Metals (231).
Standard solution: On the day of use, dilute 2.0 mL of
the Lead nitrate stock solution (10 ppm Pb) in water to
30 mL.
Sample solution: Dissolve 1 g of Lactobionic Acid in
water to 30 mL.
Prepare the filtration Cis by adapting the barrel
of a 50-mL aD je without its piston to a support con-
taining, on the plate, a membrane filter of 3-11m pore
size, and above it a prefilter.
Transfer the Sample solution into the syringe barrel, put
the piston in place, and then apply an even pressure
on it until the whole of the liquid has been filtered.
When opening the support and removing the prefilter,
check that the membrane filter remains uncontami-
nated with impurities. If this is not the case, replace it
with another membrane filter, and repeat the opera-
tion under the same conditions.
Analysis: To the prefiltrate, add 2 mL. of pH 3.5 Acetate
Buffer, Mix, and add 1.2 mL of Thioacetamide reagent.
Mix immediately, allow to stand for 10 min, and again
filter as described above, but inverting the order of the
filters, the liquid passing first through the membrane
filter before passing through the prefilter. The filtration
must be carried out slowly and uniformly by applying
moderate and constant pressure to the piston of the
syringe. After complete filtration, open the support, re-
move the membrane filter, and dry using filter paper. In
parallel, treat the Standard solution in the same manner
as the Sample solution.
Acceptance criteria: The color of the spot from the
Sample solution is not more intense than that from the
Standard solution (NMT 20 ppm).e (rticiat 1-Jan-2018)
SPECIFIC TESTS
e WATER DETERMINATION, Method Ia (921)
Sample solution: 0.50 g in a mixture of methanol and
formamide (2:1)
Acceptance criteria: NMT 5.0%
e APPEARANCE OF SOLUTION
Sample solution: 120 mg/mL of Lactobionic Acid
Standard stock solution: Pipet 24.0 mL of ferric chlo-
ride CS and 6.0 mL of cobaltous chloride CS into a
100-mL volumetric flask. Dilute with 1% (w/v) hydro-
chloric acid to volume.
Reference solution: Pipet 12.5 mL of the Standard stock
solution into a 100-mL volumetric flask. Dilute with 1%
(w/v) hydrochloric acid to volume.
Acceptance criteria: The Sample solution is clear and
not more intensely colored than the Reference solution.
© OPTICAL ROTATION, Specific Rotation (781S)
Sample solution: 10 mg/mL of Lactobionic Acid. Allow
to stand for 24 h.
Acceptance criteria: +23.0° to +29.0° (anhydrous
substance)
¢ REDUCING SUGARS
Sample solution: Dissolve 5.0 g of Lactobionic Acid in
25 mL of water with the aid of gentle heat, and cool.
Analysis: To the Sample solution add 20 mL of cupric
citrate TS and a few glass beads. Heat so that boiling
NF 36 Official Monographs / Lactose 5413

begins after 4 min, and maintain boiling for 3 min. plate from the chamber, mark the solvent front, and
Cool rapidly, and add 100 mL of a 2.4% solution of dry the plate in a current of warm air. Spray the plate
glacial acetic acid and 20.0 mL of 0.025 M iodine VS. evenly with Spray reagent. Heat the plate at 130° for
With continuous shaking, add 25 mL of a mixture of 10 min.
6 mL of hydrochloric acid and 94 mL of water. When System suitability: The test is not valid unless Standard
the precipitate has dissolved, titrate the excess iodine solution B shows four clearly discernible spots, disregard-
with 0.05 M sodium thiosulfate VS using 1 mL of starch ing any spots at the origin.
TS, added toward the end of the titration as an Acceptance criteria: The principal spot from the Sam-
indicator. ple solution corresponds in appearance and R; value to
Acceptance criteria: NLT 12.8 mL of 0.05 M sodium that from Standard solution A.«
thiosulfate VS is required, corresponding to NMT 0.2%
of reducing sugars, as glucose. OTHER COMPONENTS
e Ams OF BOTANICAL ORIGIN, Total Ash (561): NMT © *CONTENT OF ALPHA AND BETA ANOMERS
” oO Silylation reagent: Dimethyl sulfoxide, pyridine, and
trimethylsilylimidazole (19.5: 58.5: 22)
ADDITIONAL REQUIREMENTS Standard solution: Prepare a mixture of alpha-lactose
© PACKAGING AND STORAGE: Preserve in well-closed monohydrate and beta-lactose having an anomeric ratio
containers. of about 1:1 based on the labeled anomeric contents of
e USP REFERENCE STANDARDS (11) the alpha-lactose monohydrate and the beta-lactose. In-
USP Lactobionic Acid RS troduce 10 mg of this mixture into a vial with a screw
cap. Add 4 mL of Silylation reagent. Sonicate for 20 min
at room temperature. Transfer 400 pL to an injection
vial. Add 1 mL of pyridine. Close the vial, and mix well.
Sample solution: Introduce 10 mg of Anhydrous Lac-
Anhydrous Lactose tose into a vial with a screw cap. Add 4 mL of Silylation
reagent. Sonicate for 20 min at room temperature.
Portions of the monograph text that are national USP text, Transfer 400 UL to an injection vial. Add 1 mL of pyri-
and are not part of the harmonized text, are marked with dine. Close the vial, and mix well.
symbols (*¢) to specify this fact. Chromatographic system
HO, (See Chromatography (621), System Suitability.)
Mode: GC
L° Detector: Flame ionization
HO, (ee
OH Columns
Hoo) on Precolumn:'’ 0.53-mm x 2-m intermediate polarity
‘oH deactivated fused silica
(elpha-Lactose) Analytical:2_ 0.25-mm x 15-m G27 on fused silica;
OH film thickness 0.25 um
Temperatures
DEFINITION Detector: 325°
Anhydrous Lactose is O-8-D-galactopyranosyl-(1—4)-B-D- Injection port: 275° or use cold on-column injection
glucopyranose (B-lactose), or a mixture of O-B-D- Column: See Table 1.
galactopyranosyl-(14)-B-D-glucopyranose and O-B-D-
galactopyranosyl-(1 >4)-c.-D-glucopyranose (c-lactose). Table 1

IDENTIFICATION Hold Time at

e A. INFRARED ABSORPTION (197K) Initial Temperature Final Final
° ‘on CHROMATOGRAPHIC IDENTIFICATION TEST Temperature Ramp Temperature | Temperature
©) (¢/min) «@) (min)
ae erent 0.25-mm layer of chromatographic silica 80 = 80. 1
je 80 35 150 =
Diluent: Methanol and water (3:2) 150 12 300 2
Standard solution A: 0.5 mg/mL of USP Anhydrous
Lactose RS in Diluent Carrier gas: Helium
Standard solution B: Contains 0.5 mg/mL of USP Dex- Flow rate: 2.8 mL/min
trose RS, 0.5 mg/mL of USP Anhydrous Lactose RS, Injection volume: 0.5 uL
0.5 mg/mL of USP Fructose RS, and 0.5 mg/mL of USP Injection type: Splitless or by cold on-column
Sucrose RS in Diluent injection
Sample solution: 0.5 mg/mL of Anhydrous Lactose in System suitability
Diluent Sample: Standard solution
Application volume: 2 uL Suitability requirements
Developing solvent Sister Ethylene dichloride, glacial Resolution: NLT 3.0 between the peaks due to alpha-
acetic acid, methanol, and water (10:5:3:2) lactose and beta-lactose
Spray reagent: 5 mg/mL of thymol in a mixture of al- Analysis
cohol and sulfuric acid (19:1) Sample: Sample solution
Analysis [Note—The relative retention time with reference to ra
Samples: Standard solution A, Standard solution B, and beta-lactose is about 0.9 for alpha-lactose (retention
Sample solution time = about 12 min).] i
Allow the spots to dry, and develop the plate in a pa- Calculate the percentage content of alpha-lactose: <
per-lined chromatographic chamber equilibrated with
the Developing solvent system for about 1 h prior to Result = So/(So + Ss) x 100 S|
use. Allow the chromatogram to develop until the sol-
vent front has moved about three-quarters of the Sa = area of the peak due to alpha-lactose eS
length of the plate. Remove the plate from the cham- So = area of the peak due to beta-lactose a
ber, dry in a current of warm air, and redevelop the ' Restek Guard column is suitable.
plate in fresh Developing solvent system. Remove the 2Varian CP-Sil 8 CB is suitable.
 5414 Lactose / Official Monographs
Calculate the percentage content of beta-lactose:
Result = Sp/(Sa + Se) x 100
Si = area of the peak due to alpha-lactose
So = area of the peak due to beta-lactose
a4
IMPURITIES
Delete the following:
°o *HEAVY IMETALS, Method Il (231): NMT 5 ppmee cors1
fan-2018)
e RESIDUE ON IGNITION (281): NMT 0.1%
SPECIFIC TESTS
e CLARITY AND COLOR OF SOLUTION
Hydrazine sulfate solution: Dissolve 1.0 2 of hydrazine
sulfate in water, and dilute to 100.0 mL. Allow to stand
for 4-6 h.
Hexamethylenetetramine solution: In a 100-mL
ground-glass stoppered flask dissolve 2.5 g of hexa-
methylenetetramine in 25.0 mL of water.
Primary opalescent suspension: To the Hexamethylene-
tetramine solution in the flask add 25.0 mL of the Hydra-
zine sulfate solution. Mix and allow to stand for 24 h.
This suspension is stable for 2 months, provided it is
stored in a glass container free from surface defects.
The suspension must not adhere to the glass and must
be well mixed before use.
Standard opalescence: Dilute 15.0 mL of the Primary
opalescent suspension to 1000.0 mL with water. This sus-
pawion is freshly prepared and may be stored for up to
24h.
Reference suspension: To 5.0 mL of the Standard opal-
escence add 95.0 mL of water. Mix and shake before
use.
Reference solution: To 6.0 mL of ferric chloride CS,
2.5 mL of cobaltous chloride CS, and 1.0 mL of cupric
sulfate CS add hydrochloric acid (10 g/L HCl) to make
1000 mL.
Sample solution: 1g in 10 mL of boiling water. Allow
to cool.
Instrumental conditions
Mode: Vis
Analytical wavelength: 400 nm
Acceptance criteria: NMT 0.04 for the absorbance di-
vided by the path length in centimeters; and the clarity
of the Sample solution is the same as that of water or its
opalescence is not more pronounced than that of the
Reference suspension, and it is not more colored than
the Reference solution.
e Loss ON DRYING (731)
Analysis: Dry a sample at 80° for 2 h.
Acceptance criteria: NMT 0.5%
WATER DETERMINATION, Method | (921)
Sample solution: Anhydrous Lactose in a mixture of
methanol and formamide (2:1)
Acceptance criteria: NMT 1.0%
e MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI-
FIED MICROORGANISMS (62): The total aerobic microbial
count is NMT 102 cfu/g and *the total combined molds
and yeasts count is NMT 50 cfu/ge. It meets the require-
NF Monographs
ments of the test for absence of Escherichia coli.
PROTEIN AND LIGHT-ABSORBING IMPURITIES
(See Ultraviolet-Visible Spectroscopy (857).)
Sample solution: 1% solution (w/v)
Instrumental conditions
Mode: UV
Wavelength range: 210-300 nm
Acceptance criteria: NMT 0.25 for the absorbance di-
vided by the path length in centimeters at 210-220
nm; NMT 0.07 for the absorbance divided by the path
length in centimeters at 270-300 nm
NF 36
e ACIDITY OR ALKALINITY
Sample solution: Dissolve 6 g by heating in 25 mL of
carbon dioxide-free water, cool, and add 0.3 mL of
phenolphthalein TS.
Acceptance criteria: The solution is colorless, and NMT
0.4 mL of 0.1 N sodium hydroxide is required to pro-
duce a pink or red color.
© OPTICAL ROTATION, Specific Rotation (781S)
Sample solution: Dissolve 10 g by heating in 80 mL of
water to 50°. Allow to cool, and add 0.2 mL of 6N
ammonium hydroxide. Allow to stand for 30 min, and
dilute with water to 100 mL.
Acceptance criteria: +54.4° to +55.9°, calculated on
the anhydrous basis, at 20°
ADDITIONAL REQUIREMENTS
© *PACKAGING AND STORAGE: Preserve in tight containers.
e LABELING: Where the labeling indicates the relative quan-
tities of alpha- and beta-lactose, determine compliance
using Content of Alpha and Beta Anomers. Where the la-
beling states the particle size distribution, it also indicates
the dio, dso, and dso values and the range for each.
e@ USP REFERENCE STANDARDS (11)
USP Dextrose RS
USP Fructose RS
USP Anhydrous Lactose RS
USP Sucrose RS»
Lactose Monohydrate
Portions of the monograph text that are national USP text,
and are not part of the harmonized text, are marked with
symbols (%) to specify this fact.
DEFINITION
Lactose Monohydrate is the monohydrate of O-B-p-
galactopyranosyl-(1 >4)-a-D-glucopyranose. [NOoTE—Lac-
tose Monohydrate may be modified as to its physical
characteristics. It may contain varying proportions of
amorphous lactose.]
IDENTIFICATION
A. INFRARED ABSORPTION (197K)
° "a iene CHROMATOGRAPHIC IDENTIFICATION TEST
201
Diluent: Methanol and water (3:2)
Standard solution A: 0.5 mg/mL of USP Lactose Mono-
hydrate RS in Diluent
Standard solution B: 0.5 mg/mL each of USP Dextrose
RS, USP Lactose Monohydrate RS, USP Fructose RS, and
USP Sucrose RS in Diluent
Sample solution: 0.5 mg/mL of Lactose Monohydrate
in Diluent
Pe 0.25-mm layer of chromatographic silica
ge
Application volume: 2 uL
Developing solvent a Ethylene dichloride, glacial
acetic acid, methanol, and water (10:5:3:2)
Spray reagent: 5 mg/mL of thymol in a mixture of al-
cohol and sulfuric acid (19:1)
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Allow the spots to dry, and develop the plate in a pa-
per-lined chromatographic chamber equilibrated with
Developing solvent system for about 1 h prior to use.
Allow the chromatogram to develop until the solvent
front has moved about three-quarters of the length of
the plate. Remove the plate from the chamber, ny in
a current of warm air, and redevelop the plate in fresh
Developing solvent system. Remove the plate from the
chamber, mark the solvent front, and dry the plate in
NF 36
a current of warm air. Spray the plate evenly with
Spray reagent. Heat the plate at 130° for 10 min.
System suitability: The test is not valid unless the chro-
matogram of Standard solution B shows four clearly dis-
cernible spots, disregarding any spots at the origin.
Acceptance criteria: The principal spot from the Sam-
ple solution corresponds in appearance and Rr value to
that from Standard solution A.*
IMPURITIES
© RESIDUE ON IGNITION (281)
Analysis: A sample ignited at a temperature of
600 + 50°
Acceptance criteria: NMT 0.1%
Delete the following:
°o *HEAVY METALS (231)
Sample solution: 4g in 20 mL of warm water. Add
1 mL of 0.1 N hydrochloric acid, and dilute with water
to 25 mL.
Acceptance criteria: NMT 5 1g/Qee6 ‘official 1-42-2018)
SPECIFIC TESTS
© CLARITY AND COLOR OF SOLUTION
Sample solution: 1g in 10 mL of boiling water
Analysis: The Sample solution is clear and nearly color-
less. Determine the absorbance of this solution at a
wavelength of 400 nm.
Acceptance criteria: The absorbance divided by the
path length, in cm, is NMT 0.04.
© *IVIICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI-
FIED MICROORGANISMS (62): The total aerobic microbial
count does not exceed 1 x 10? cfu/g, the total combined
molds and yeasts count does not exceed 5 x 10! cfu/g,
and it meets the requirements of the test for absence of
Escherichia coli.¢
© OPTICAL ROTATION, Specific Rotation (781S)
Sample solution: Dissolve 10 g by heating in 80 mL of
water to 50°. Allow to cool, and add 0.2 mL of 6N
ammonium hydroxide. Allow to stand for 30 min, and
dilute with water to 100 mL.
Acceptance criteria: +54.4° to +55.9°, calculated on
the anhydrous basis, determined at 20°
¢ ACIDITY OR ALKALINITY
Sample solution: Dissolve 6 g by heating in 25 mL of
carbon dioxide-free water, cool, and add 0.3 mL of
phenolphthalein TS.
Acceptance criteria: The solution is colorless, and NMT
0.4 mL of 0.1 N sodium hydroxide VS is required to
produce a pink or red color.
e *Loss ON DRYING (731)
Analysis: Dry a sample at 80° for 2 h.
Acceptance criteria
Monohydrate: NMT 0.5%
Monohydrate, modified: NMT 1.0%.
e WATER DETERMINATION, Method | (921)
Sample solution: Prepare a preparation containing Lac-
tose Monohydrate in a mixture of methanol and forma-
mide (2:1).
Acceptance criteria: 4.5%-5.5%
¢ PROTEIN AND LIGHT-ABSORBING IMPURITIES
(See Ultraviolet-Visible Spectroscopy (857).)
Sample solution: 1% (w/v)
Analysis: Measure the light absorption of the Sample
solution in the range of 210-300 nm.
Acceptance criteria: The absorbance divided by the
path length, in cm, is NMT 0.25 in the range of
210-220 nm and is NMT 0.07 in the range of 270-300
nm.
ADDITIONAL REQUIREMENTS
¢ *PACKAGING AND STORAGE: Preserve in tight containers.
e LABELING: Where the labeling states the particle size dis-
tribution, it also indicates the dio, dso, and doo values and
Official Monographs / Lanolin 5415
the range for each. For modified Lactose Monohydrate,
also label it to indicate the method of modification.
e USP REFERENCE STANDARDS (11)
USP Dextrose RS
USP Fructose RS
USP Lactose Monohydrate RS
USP Sucrose RSe
Lanolin, Anhydrous—see Lanolin General
Monographs
Lanolin, Modified—see Modified Lanolin
General Monographs
Lanolin Alcohols
[8027-33-6].
DEFINITION
Lanolin Alcohols is a mixture of aliphatic alcohols, triterpe-
noid alcohols, and sterols, obtained by the hydrolysis of
Lanolin. It may contain NMT 0.1% of a suitable
antioxidant.
IDENTIFICATION
cA.
Sample: 0.5g
Analysis: Dissolve the Sample in 5 mL of chloroform,
and add 1 mL of acetic anhydride and 2 drops of sulfu-
ric acid.
Acceptance criteria: A green color is produced.
ASSAY
e CONTENT OF STEROLS (as cholesterol)
Sample: 20g
Analysis: Melt the Sample on a water bath, mix, and
allow to cool. Dissolve 100 mg in 12 mL of warm (60°)
90% alcohol. Allow to stand for 18 h, pass through a
medium-porosity, sintered-glass filter, and wash the res-
idue with two 15-mL portions of 90% alcohol. Com-
bine the filtrate and washings, add 20 mL ofa freshly
ete ale 1-in-100 solution of digitonin in 90% alcohol,
and warm to 60°. Allow to cool, pass through a me-
dium-porosity, sintered-glass filter with the aid of gentle
vacuum, wash the residue with 10 mL of 90% alcohol,
and dry at 105° to constant weight. Each g of residue is
equivalent to 0.239 g of cholesterol.
Acceptance criteria: NLT 30.0% of sterols, calculated
as cholesterol
IMPURITIES
e RESIDUE ON IGNITION (281): NMT 0.15%
e COPPER
Solution A: 1 mg/mL of sodium diethyldithiocarbamate
Sample: 5.0
Control: Add 1 mL of Solution A and a few drops of 6 N
ammonium hydroxide to 2.5 mL of a 39.3-ppm solu-
sydeibouow 4N
tion of cupric sulfate. Dilute with water to 50 mL.
Analysis: Heat the Sample over a small flame until char-
red, ignite the residue at 550°, and dissolve the ash in
5 mL of hydrochloric acid, with the aid of heat. Cool,
dilute with water, render alkaline with ammonium hy-
droxide, boil to remove the excess ammonia, add a few
drops of bromine TS, boil again, and filter. To the fil-
trate add 1 mL of Solution A, a few drops of 6 N ammo-
nium hydroxide, and sufficient water to bring the vol-
ume to 50 mL.
 5416 Lanolin / Official Monographs NF 36

Acceptance criteria: The Sample is not darker than the Standard solution B: 0.5 mg/mL of cholestane,
Control (5 ppm). 0.5 mg/mL of cholestanol, and 0.25 mg/mL of 24,25-
dihydrolanosterol in dehydrated alcohol
SPECIFIC TESTS Standard solution C: 0.5 mg/mL of USP Cetyl Alcohol
e MELTING RANGE OR TEMPERATURE, Class /] (741): NLT 56° RS in dehydrated alcohol. [NoTE—Vortex or sonification
e ACIDITY AND ALKALINITY helps standard preparation.]
Analysis: Boil 10 g with 100 mL of water for 5 min, Standard solution D: 0.5 mg/mL of USP Stearyl Alco-
with frequent stirring. Remove the source of heat, add hol RS in dehydrated alcohol. [NoTE—Vortex or sonifica-
0.5 mL of phenolphthalein TS, and stir. tion helps standard preparation.]
Acceptance criteria: No pink color is produced. Add Sample solution: 2.5 mg/mL of Hydrogenated Lanolin
0.5 mL of methyl orange TS, and stir: no red color is hydrated alcohol
produced. Chromatographic system
Loss ON DRYING (731) (See Chromatography (621), System Suitability.)
Analysis: Dry at 105° for 1 h. Mode: GC
Acceptance criteria: NMT 0.5% Detector: Flame ionization
FATS AND FIXED OILS, Acid Value (401): NMT 2.0. Column: 0.25-mm x 30-m fused-silica capillary; 0.25-
FATS AND FIXED OILS, Hydroxyl Value (401): 120-180 um layer of phase G2
FATS AND FIXED OILS, Peroxide Value (401) Temperatures
Sample: Take wedge-shaped pieces with bases that Detector: 350°
contain part of the surface. Injection port: 325°
Analysis: Melt the pieces before carrying out the deter- Column: See Table 1.
mination. Before adding the 0.5 mL of saturated potas-
sium iodide solution, cool the solution obtained to
Table 1
room temperature.
Acceptance criteria: NMT 15 Hold Time
FATS AND FIXED OILS, Saponification Value (401) Initial Temperature Final at Final
e

Sample: 5g of molten Lanolin Alcohols Temperature Ramp Temperature | Temperature

Analysis: Reflux the Sample with the alcoholic potas- © (¢/min) @) (min)
sium hydroxide for 4 h. 100 = 100 5.
Acceptance criteria: NMT 12 100 5. 300 15
ADDITIONAL REQUIREMENTS Carrier gas: Helium
e PACKAGING AND STORAGE: Preserve in well-closed, light- Flow rate: 1 mL/min
resistant containers, and store at controlled room Injection volume: 1 uL
temperature. System suitability
e LABELING: Label it to indicate the name and quantity of Samples: Standard solution A, Standard solution B,
any antioxidant added. Standard solution C, and Standard solution D
[Note—See Table 2 for relative retention times.]

Table 2
Hydrogenated Lanolin Relative
Retention
[8031-44-5]. Component Time
Cetyl alcohol 1.00
DEFINITION
A mixture of higher aliphatic alcohols, hydrocarbons, and Stearyl alcohol TAS
sterols is obtained from the direct high-pressure, high- Cholestane 1.63
temperature hydrogenation of lanolin. During the hydro- Cholestanol Te
genation process, the esters and acids present are re- 24,25-Dihydrolanosterol 1.85
duced to the corresponding alcohols. Alcoholic derivatives
may be further reduced to hydrocarbons. It may contain System suitability requirements
antioxidants. Resolution: NLT 30 between cetyl alcohol and stearyl
alcohol, Standard solution A
IDENTIFICATION Analysis
cA. Samples: Standard solution A, Standard solution B,
Sample: 50mg Standard solution C, Standard solution D, and Sample
Analysis: Dissolve the Sample in 5 mL of methylene solution
chloride, and add 1 mL of acetic anhydride and 0.1 mL Identify the peaks from Standard solution A, Standard
of sulfuric acid. solution B, Standard solution C, and Standard solution D.
Acceptance criteria: A green color is produced. Calculate the percentage of cetyl alcohol (stearyl alco-
e B. CHROMATOGRAPHIC IDENTITY hol, cholestane, cholestanol, or 24,25-dihydrolanos-
Analysis: Proceed as directed in the test for Chromato- terol) in the portion of Hydrogenated Lanolin taken:
graphic Profile of Fatty Alcohols, Hydrocarbons, and Sterols
in the Assay. Result = (ru/rs) x (Cs/Cu) x 100
NF Monographs

Acceptance criteria: The retention times of the cetyl

alcohol, stearyl alcohol, cholestane, cholestanol, and
24,25-dihydrolanosterol peaks of the Sample solution
correspond to those of Standard solution A, as obtained
in the Assay.
ASSAY
© CHROMATOGRAPHIC PROFILE OF FATTY ALCOHOLS, HYDROCAR-
BONS, AND STEROLS
Standard solution A: 2.5 mg/mL of USP Hydrogenated
Lanolin RS in dehydrated alcohol
ru = peak response of cetyl alcohol (stearyl alcohol,
cholestane, cholestanol, or 24,25-
dihydrolanosterol) from the Sample solution
rs = peak response of cetyl alcohol (stearyl alcohol,
cholestane, cholestanol, or 24,25-
dihydrolanosterol) from Standard solution C,
Standard solution D, or Standard solution B
NF 36 Official Monographs / Lauroyl 5417

Gs = concentration of USP Cetyl Alcohol RS (USP IDENTIFICATION

Stearyl Alcohol RS, cholestane, cholestanol, e A. INFRARED ABSORPTION (197K)
or 24,25-dihydrolanosterol) in Standard e B. The retention time of the major peak of the Sample
solution C, Standard solution D, or Standard solution corresponds to that of the Standard solution, as
solution B (mg/mL) obtained in the Assay.
Cy = concentration of Hydrogenated Lanolin in the
Sample solution (mg/mL) ASSAY
Acceptance criteria e PROCEDURE
Sample solution: Chromatogram exhibits a profile Solvent A: 0.1% (v/v) trifluoroacetic acid in water
similar to that in the chromatogram of anda solu- Mobile phase: Acetonitrile and Solvent A (85:15, v/v)
tion A. Standard solution: 15 mg/mL of USP Lauric Acid RS in
Cetyl alcohol, stearyl alcohol, cholestane, cholesta- Mobile phase
nol, and 24,25-dihydrolanosterol: See Table 3. Sample solution: 15 mg/mL of Lauric Acid in Mobile
phase
Chromatographic system
Table 3 (See Chromatography (621), System Suitability.)
Acceptance Mode: LC
Name Criteria (%) Detector: UV 212 nm
Cetyl_ alcohol 2-15 Column: 4.6-mm x 15-cm analytical; 5-um packing L1
Stearyl alcohol 0.5-10 Column temperature: Ambient
Flow rate: 1.0 mL/min
Cholestane 2-13
Injection volume: 10 pL
Cholestanol 3-11 System suitability
24,25-Dihydrolanosterol 3-15 Sample: Standard solution
Suitability requirements
IMPURITIES Relative standard deviation: NMT 3.0%, Standard
e RESIDUE ON IGNITION (281): NMT 0.1%, determined on solution
5.0g Analysis
Samples: Standard solution and Sample solution
SPECIFIC TESTS Calculate the percentage of lauric acid (Ci2H24O2) in the
e [MELTING RANGE OR TEMPERATURE (741): 45°-55°. Allow to portion of Lauric Acid taken:
stand at 20° for 16 h.
FATS AND FIXED OILS, Acid Value (401): NMT 1.0, deter- Result = (ru/rs) x (Cs/Cu) x 100
mined on 5.0g
FATS AND FIXED O1Ls, Hydroxy! Value (401): 140-180 tu = peak response of lauric acid from the Sample
FATS AND FIXED OILS, Saponification Value (401): NMT solution
8.0. Heat under reflux for 4 h. rs = peak response of lauric acid from the Standard
Loss ON DRYING (731) solution
Sample: 2.0g Cs = concentration of USP Lauric Acid RS in the
Standard solution (mg/mL)
Analysis: Dry the Sample in an oven at 105° for 1 h.
Acceptance criteria: NMT 3.0% Cu = concentration of Lauric Acid in the Sample
solution (mg/mL)
ADDITIONAL REQUIREMENTS Acceptance criteria: 98.0%-102.0% on the anhydrous
¢ PACKAGING AND STORAGE: Preserve in well-closed contain- basis
ers, protected from light. Do not store above 45°.
© LABELING: Label it to indicate the name and amount of IMPURITIES
the antioxidant added. e RESIDUE ON IGNITION (281)
e USP REFERENCE STANDARDS (11) Sample: 10g
USP Cetyl Alcohol RS Acceptance criteria: NMT 0.1%
USP Hydrogenated Lanolin RS SPECIFIC TESTS
USP Steary! Alcohol RS © FATS AND FIXED OILS, /odine Value (401): NMT 3.0
© FATS AND FIXED OILS, Peroxide Value (401): NMT 10.0
¢ WATER DETERMINATION, Method Ia (921): NMT 0.2%
ADDITIONAL REQUIREMENTS
Lauric Acid © PACKAGING AND STORAGE: Preserve in well-closed contain-
ers. Store at room temperature.
° e USP REFERENCE STANDARDS (11)
Be Ne vA, USP Lauric Acid RS

Cy2H2402 200,32
Dodecanoic acid;
1-Dodecanoic acid; Lauroy! Polyoxylglycerides Pa
1-Undecanecarboxylic acid; al
n-Dodecanoic acid [143-07-7]. DEFINITION
Lauroyl Polyoxylglycerides is a mixture of monoesters, dies- a
DEFINITION ters, and triesters of glycerol and monoesters and diesters °
Lauric Acid contains NLT 98.0% and NMT 102.0% of ]
of polyethylene glycols. The pee glycols used °
godecanele acid (Ci2H2402), calculated on the anhydrous have a mean molecular weight between 300 and 1500. Ko}
4
aSIS.
The article is produced by pe alcoholysis of saturated Sy
oils, mainly containing vee es of lauric acid with pol- mo]
=>
roger glycols, by esterification of glycerol and poly- a)
ethylene glycols with fatty acids, or as a mixture of glyc-
 5418 Lauroyl / Official Monographs NF 36

erol esters and ethylene oxide condensate with the fatty Calculate the percentage of glycerol in the sample
acids of the hydrogenated oils. It may contain free poly- taken:
ethylene glycols.
Result = {[(Vs — Vs) x N x FJ/W} x 100
IDENTIFICATION
e A. INFRARED ABSORPTION (197K) Titrant volume consumed by the Blank (mL)

nud
°a: THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST Titrant volume consumed by the Sample (mL)

=
201) actual normality of the Titrant (mEq/mL)
Standard solution: 50 mg/mL of USP Lauroy! Polyoxyl- equivalency factor, 23.0 mg/mEq
glycerides RS in methylene chloride Ww = Sample weight (mg)
Sample solution: 50 mg/mL of Lauroy! Polyoxylglycer- Acceptance criteria: NMT 5.0%
ides in methylene chloride
Application volume: 10 pL SPECIFIC TESTS
Developing solvent system: Ether and hexanes (70:30) e FATS AND FIXED OILS, Acid Value (401)
Spray reagent: 0.1 mg/mL of rhodamineBin alcohol Sample: 2.0g
Analysis Acceptance criteria: NMT 2.0
Samples: Standard solution and Sample solution © FATS AND FIXED OWS, Fatty Acid Composition (401):
Proceed as directed in the chapter. Then spray the plate Lauroyl Polyoxylglycerides exhibits the composition pro-
with Spray reagent, and locate the spots on the plate file of fatty acids shown in Table 1.
by examination under UV light at a wavelength of 365
nm. Table 1
meeepionce criteria: The R; values of the principal spots
Carbon-Chain Number of Percentage
of the Sample solution correspond to those of the Stan-
dard solution. Length Double Bonds (%)
e C. It meets the requirements in Specific Tests (see Table 1) 8 0 $15.0
for Fats and Fixed Oils, Fatty Acid Composition (401). 10 0 $12.0
12 0 30.0-50.0
IMPURITIES 14 0 5.0-25.0
16 0 4.0-25.0
Delete the following: 18 oO 5.0-35.0

°e HEAVY METALS, Method II (231): NMT 10 19/ge cortical 1- e FATS AND FIXED OlLs, Hydroxy! Value (401)
Jan-2018) Sample: 1.0g
e ARTICLES OF BOTANICAL ORIGIN, Total Ash (561): NMT Acceptance criteria: Within the range specified in Table
0.1% 2 for the labeled type
© ALKALINE IMPURITIES
Sample: 5.0g Table 2
Analysis: Heat the Sample slightly until the test sub-
stance melts, add 10 mL of alcohol and 0.05 mL of bro- Type of Hydroxyl
mophenol blue TS, and mix well. While the solution is Polyethylene Glycols Value
still warm, titrate with 0.01 N hydrochloric acid VS to 300 65-85
change the color to yellow. 400 60-80
Acceptance criteria: NMT 1.0 mL of 0.01 N hydrochlo- 600 50-70
ric acid is required. 1500 36-56
e LIMIT OF FREE ETHYLENE OXIDE AND DIOXANE
Analysis: Proceed as directed in Ethylene Oxide and Di- e FATS AND FIXED OILS, lodine Value (401): NMT 2.0
oxane (228), Method I. e FATS AND FIXED OILS, Peroxide Value (401)
Acceptance criteria Sample: 2.0g
Ethylene oxide: NMT 1 ug/g Acceptance criteria: NMT 6.0
Dioxane: NMT 10 ug/g e FATS AND FIXED OILS, Saponification Value (401)
© LIMIT OF FREE GLYCEROL Sample: 2.0g
Sample: 1.29 Acceptance criteria: Within the range specified in Table
Periodic acetic acid solution: Dissolve 0.446 g of so- 3 for the labeled type
dium periodate in 2.5 mL of a 25% (v/v) solution of
es acid, diluting to 100.0 mL with glacial acetic
Table 3
acid.
Potassium iodide solution: 75 mg/mL of potassium Type of Saponification
iodide Polyethylene Glycols Value
Blank: 25 mL of methylene chloride 300 190-204
Titrimetric system 400 170-190
(See Titrimetry (541).) 600 150-170
Mode: Residual titration
Titrant: 0.1 M sodium thiosulfate VS 1500 79-93
Endpoint detection: Visual e WATER DETERMINATION, Method | (921)
NF Monographs

Analysis: Dissolve the Sample in 25 mL of methylene Sample: 1.0g

chloride, heating if necessary. Cool, and add 100 mL of Analysis: Instead of using methanol as the solvent, one
water and 25.0 mL of Periodic acetic acid solution. Shake,
of two solvent systems can be used: a mixture of meth-
and allow to stand for 30 min. Add 40 mL of Potassium ylene chloride and anhydrous methanol (70:30 v/v), or
iodide solution, and allow to stand for 1 min. Add 1 mL anhydrous pyridine.
of starch TS, and titrate the liberated iodine with 0.1 M
sodium thiosulfate VS. Perform a blank determination,
and make any necessary correction.
NF 36 Official Monographs / Lecithin 5419

Acceptance criteria: NMT 1.0% Acceptance criteria: The R- values of the spots for
phosphatidylcholine, phosphatidylethanolamine, phos-
ADDITIONAL REQUIREMENTS phatidic acid, and lysophosphatidylcholine from the
© PACKAGING AND STORAGE: Preserve in tight containers, Sample solution correspond to those from Standard solu-
protected from light and moisture. Store at controlled tion A and Standard solution B. [NotE—Depending on
room temperature. the sample tested, if a phospholipid component
e LABELING: Label it to indicate thetype and the average presents in a low amount in the sample, the corre-
nominal molecular weight of polyethylene glycols used as sponding spot in the Sample solution on the TLC may
part of the official title. not be visualized.]
e USP REFERENCE STANDARDS (11)
USP Lauroyl Polyoxylglycerides RS ASSAY
© CONTENT OF PHOSPHOLIPIDS
[Note—Perform the test for lysophosphatidylcholine for
Lecithin intended for use in the manufacture of inject-
able dosage forms.]
Lecithin Solution A: Mix 1342g (2.0L) of n-hexane, 334.1 g
(425 mL) of isopropanol (2-propanol), 39.4 g (38 mL)
[8002-43-5]. of glacial acetic acid, and 2.0 mL of triethylamine.
Solution B: Mix 663.5 g (850 mL) of isopropanol,
DEFINITION 15.8 g (15 mL) of glacial acetic acid, 140 g (140 mL) of
Lecithin is a complex mixture of acetone-insoluble phospha- water, and 0.8 mL of triethylamine.
tides, which consist chiefly of phosphatidylcholine, phos- Solvent: n-Hexane, isopropanol, and water (46:46:8, v/
phatidylethanolamine, phosphatidylinositol, and phospha- v/v). [NOTE—To avoid the formation of two phases, mix
tidic acid, present in conjunction with various amounts of the isopropanol and water first, and then add the n-
other substances such as triglycerides, fatty acids, and car- hexane.]
bohydrates, as separated from the crude vegetable oil Mobile phase: See Table 1.
source. The content of each of the phospholipids
(phosphatidylcholine, phosphatidylethanolamine,
Table 1
phosphatidylinositol, and phosphatidic acid) is indicated
on the certificate of analysis. Flow Solution Solution
Program Time Rate A B
IDENTIFICATION Step (min) (mL/min) (%). (%)_
¢ A. IDENTIFICATION OF PHOSPHOLIPIDS BY THIN-LAYER 1 0 1.0 95 5
CHROMATOGRAPHY
2 5.0 1.0 80 20
Mobile phase: Chloroform, methanol, water (65:25:4,
v/v/v) 3 8.5 1.0 60 40
Standard solution A: 10 mg/mL of USP Phosphatidic 4 14.0 1.0 55 45
Acid (Soy) Monosodium RS and 10 mg/mL of USP 5 15.0 1.0 0 100
Phosphatidylcholine (Soy) RS in Mobile phase 6 AS: 1.0 0 100
Standard solution B: 7 mg/mL of USP Phosphatidyleth- Z 17.6 1.0 95 5
anolamine (Soy) RS and 7 mg/mL of USP Lysophospha-
8 21.0 1.0 95, 5
tidylcholine (Soy) RS in Ol pies
Sample solution: 20 mg/mL of Lecithin in Mobile phase 9 22.0 2.0 95 5
Chromatographic system 10 27.0 2.0 95: 5
(See Chromatography (621), Thin-Layer Chromato- u 29.0 1.0 95 5
graphy.)
Mode: TLC Phospholipids standard stock solution (2X): 0.8 mg/
Plate: 20-cm x 20-cm, silica gel 60 on aluminum foil, mL of USP Phosphatidylcholine (Soy) RS, 0.4 mg/mL of
0.2-mm layer USP Phosphatidylethanolamine (Soy) RS, 0.4 mg/mL of
Application volume: 20 pL phosphatidylinositol prepared from USP Phosphatidyli-
Spray reagent: Dilute 80 mL of phosphoric acid nositol (Soy) Sodium RS, and 0.2 mg/mL of Sy he
(85%) with 600 mL of water in a 1-L volumetric flask. tidic acid prepared from USP Phosphatidic Acid (Soy)
[Note—Add water to the flask first.] While stirring, add Monosodium RS in Solvent. [NoTe—Due to the highly
100 g of anhydrous cupric sulfate. After stirring for 10 hydroscopic nature of phospholipids, take special pre-
min, most of the cupric sulfate is dissolved. Add water caution in the Standard preparation.]
to volume and continue stirring until the solid com- Phospholipids standard solutions: Prepare as directed
pletely dissolves. in Table 2.
Analysis
Samples: Standard solution A, Standard solution B, and Table 2
Sample solution
Fill the chromatography chamber with the Mobile phase Phospholipids
to a height of about 0.5 cm. Place a fat-free, U-shaped Standard Stock Solution
filter paper in the glass trough and press it against the (2X): Solvent
wall. Sufficient saturation is reached once the Mobile Concentration (v/v)
phase has permeated to the upper rim of the filter 0.6X BiZ
sydesbouo;= iN

paper. Apply the Samples in different bands to the pre- 0.8X 4:6
viously marked starting point on a TLC plate. Place the 1.0X 5:5,
TLC plate in the saturated chromatography chamber. 1.2X 6:4
When the Mobile phase front has reached the mark
1.4X 73
(12 cm above the starting point), remove the TLC
plate, and dry it using a dryer. Spray or immerse the System suitability solution: Phospholipids standard solu-
‘TLC plate in the Spray reagent, and dry it again with a tion 1.0X
dryer (a current of hot air). Heat the plate to 170° for Lysophosphatidylcholine standard stock solution
10 min. Develop all lipids by charring as dark brown (2X): 60 j1g/mL of USP Lysophosphatidylcholine (Soy)
spots. RS in Solvent
 5420 Lecithin / Official Monographs
Resolution solution: Phospholipids standard stock solu-
tion (2X) and Lysophosphatidylcholine standard stock so-
lution (2X) (1:1)
Lysophosphatidylcholine standard solution: 30 ug/mL
of USP Lysophosphatidylcholine oy) RS in Solvent
Sample solution: 1 mg/mL of Lecithin in Solvent.
[Note—lIf necessary, adjust the concentration of the
Sample solution to obtain the concentration of each of
the phospholipids within the calibration range.]
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: Evaporative light-scattering
Column: 4-mm x 12.5-cm; 5-4um packing L20
Temperatures
Detector: 50°
Column: 55°
Flow rate: 1.0 mL/min with step gradient at 2.0 mL/
min (see Table 1)
Injection volume: 20 uL
[Note—Depending on the different settings of the De-
tector, the Detector temperature and Flow rate can be
ae as long as system suitability requirements are
met.
System suitability
Samples: System suitability solution and Resolution
solution
[NoTte—The relative retention times for phosphatidic
acid, phosphatidylethanolamine, phosphatidylcholine,
phosphatidylinositol, and lysophosphatidylcholine are
0.4, 0.9, 1.0, 1.2, and 1.3, respectively, for the Resolu-
tion solution.]
Suitability requirements
Resolution: NLT 2.0, System suitability solution
Relative standard deviation: NMT 5.0%, System suit-
ability solution
Analysis
Samples: Phospholipids standard solutions, Lysophospha-
tidylcholine standard solution, and Sample solution
Identify the peaks of the relevant phospholipids from
the Sample solution by comparison with the
Phospholipids standard solutions. Measure the areas of
the phospholipid peaks. Plot the logarithms of the rel-
evant responses versus the logarithms of the concen-
trations, in mg/mL, of each of the phospholipids from
the Standard solutions, and determine the linear regres-
sion line using a least-squares analysis. The correlation
coefficient for the linear regression line is NLT 0.995.
From the graphs, determine the concentration (QO, in
mg/mL, of the relevant phospholipid in the Sample
solution.
Calculate the percentage of each of the phospholipids
(phosphatidic acid, phosphatidyiethang amine,
phosphatidylcholine, and phosphatidylinositol) in the
portion of Lecithin taken:
Result = (Cu/Cs) x 100
Cu = concentration of each of the phospholipids in
the Sample solution (mg/mL)
Cs = concentration of Lecithin (mg/mL)
Based on the Lysophosphatidylcholine standard solution,
identify the peak of lysophosphatidylcholine. Compare
the peak area of lysophosphatidylcholine from the
Lysophosphatidylcholine standard solution and the
NF Monographs
Sample solution, respectively.
Acceptance criteria
Content of each of the phospholipids
(phosphatidylcholine, phosphatidylethanolamine,
phosphatidylinositol, and phosphatidic acid): Within
the respective ranges stated on the label
For Lecithin intended for use in the manufacture of
injectable dosage forms
Content of Dea tss aiikioeies The peak area
of lysophosphatidylcholine in the Sample solution is
NF 36
NMT the peak area of lysophosphatidylcholine in the
Lysophosphatidylcholine standard solution, correspond-
ing to NMT 3.0% of lysophosphatidylcholine in
Lecithin.
Content of phosphatidylcholine: NLT 70.0%
IMPURITIES
Delete the following:
°e HEAVY METALS, Method II (231): NMT 20 ppme cotfical1-
Jan-2018)
e LEAD (251): NMT 10 ppm
e HEXANE-INSOLUBLE MATTER
Sample: If the substance under test is plastic or semi-
solid, soften the Lecithin by warming it at a tempera-
ture not exceeding 60°, and then mix. Weigh 10.0 g
into a 250-mL conical flask.
Analysis: To the Sample add 100 mL of hexane. Shake
until solution is apparently complete or until no more
residue seems to be dissolving. Pass through a coarse-
porosity filtering funnel that previously has been heated
at 105° for 1 h, cooled, and weighed. Wash the flask
with two 25-mL portions of hexane, and pour both
washings through the funnel. Dry the funnel at 105° for
1 h. [CAUTION—Hexane is flammable.] Cool to room
temperature, and determine the gain in weight.
Acceptance criteria: NMT 0.3%
For Sunflower Lecithin: NMT 1.0%
SPECIFIC TESTS
e CONTENT OF ACETONE-INSOLUBLE MATTER
Sample: If the substance under test is plastic or semi-
solid, soften the Lecithin by warming it briefly at a tem-
perature not exceeding 60°, and then mix. Transfer 2 g
to a 40-mL centrifuge tube that previously has been
tared along withastirring rod, cool, and weigh.
Analysis: To the Sample add 15.0 mL of acetone, and
warm carefully in a water bath to melt the test speci-
men without evaporating the acetone. Stir to help dis-
solve completely, and place in an ice-water bath for 5
min. De-oiled lecithin and fractions are suspended in
acetone by stirring. Add acetone that has been previ-
ously chilled to 0°-5° to the 40-mL mark on the tube,
stirring during the addition. Cool in an ice-water bath
for 15 min, stir, remove the rod, clarify by centrifuging
at about 2000 rpm for 5 min, and decant. Break up the
residue with the stirring rod, and refill the centrifuge
tube to the 40-mL ra with chilled acetone, while stir-
ting. Cool in an ice-water bath for 15 min, stir, remove
the rod, centrifuge, and decant. Break up the residue
with the stirring rod. Place the tube in a horizontal po-
sition until most of the acetone has evaporated. Mix
again, and heat the tube containing the acetone-insolu-
ble residue and the stirring rod at 105° to constant
weight. [CAUTION—Acetone is flammable.]
Determine the weight of the residue, and calculate the
percentage of acetone-insoluble matter.
Acceptance criteria: NLT 50.0%
For Lecithin intended for use in the manufacture of
injectable dosage forms: NLT 80.0%
e FATS AND FIXED OILS (401), Acid Value
Sample: If the substance under test is plastic or semi-
solid, soften the Lecithin by warming it briefly at a tem-
perature not exceeding 60°, and then mix. Transfer 2 g
to a 250-mL conical flask.
Analysis: Dissolve the Sample in 50 mL of petroleum
ether with 100°-120° boiling range. To this solution
add 50 mL of alcohol, previously neutralized to phenol-
phthalein with 0.1 N sodium hydroxide, and mix. Add
phenolphthalein TS. Titrate with 0.1 N sodium hydrox-
ide VS to a pink endpoint that persists for 5 s.
USP 41
e USP REFERENCE STANDARDS (11)
USP 3-Acetyl-11-keto-B-Boswellic Acid RS
USP Boswellia serrata Extract RS
Calcifediol—see Calcifedio! General
Monographs
Calcifediol Capsules—see Calcifediol
Capsules General Monographs
Calcium Ascorbate—see Calcium Ascorbate
General Monographs
Calcium Carbonate—see Calcium
Carbonate General Monographs
Calcium Carbonate Oral Suspension—
see Calcium Carbonate Oral Suspension
General Monographs
Calcium Carbonate Tablets—see Calcium
Carbonate Tablets General Monographs
Calcium Citrate—see Calcium Citrate
General Monographs
Calcium Citrate Tablets
DEFINITION
Calcium Citrate Tablets contain NLT 90.0% and NMT
110.0% of the labeled amount of calcium (Ca).
IDENTIFICATION
e A. The Sample solution from the test for Strength pro-
duces line emissions or absorptions at the characteristic
wavelengths for calcium.
© B. IDENTIFICATION TESTS—GENERAL, Calcium (191) and Cit-
rate (191)
Analysis: Grind a Tablet to a fine powder in a mortar.
Transfer the powder to a centrifuge tube, add 2-5 mL
of water, sonicate for 1 min, shake, and centrifuge.
Acceptance criteria: The supernatant meets the re-
quirements of the tests.
STRENGTH
[NoTe—A standard stock solution is commercially available
at different calcium concentrations. Necessary volumetric
adjustment can be made in the Standard solution. Concen-
trations of the Standard solution and the Sample solution
may be modified to fit the linear or working range of the
instrument.]
Dietary Supplements / Calcium 4493
© CONTENT OF CALCIUM, Procedure 1
Standard stock solution: Weigh about 1.001 g of cal-
cium carbonate, previously dried at 300° for 3 h and
cooled in a desiccator for 2 h, and dissolve in 25 mL of
1.N hydrochloric acid. Boil to expel carbon dioxide, and
dilute with water to 100 mL to obtain a solution having
a known concentration of about 4000 pg/mL of
calcium.
Standard solution: To a 200-mL volumetric flask add
100 mL of water and 4 mL of nitric acid, and mix thor-
oughly. Pipet 25.0 mL of the Standard stock solution
into the volumetric flask, and dilute with water to vol-
ume to obtain a solution having a known concentration
of about 500 g/mL of calcium.
ou solution: Weigh and finely powder NLT 20
Tablets. Transfer a weighed portion of the powdered
Tablets, ae to about 0.1 g of calcium, to a
50-mL flask. Add 4 mL of nitric acid, and heat the solu-
tion to boil gently, during which fuming evolves. Boil
the solution for an additional 30 min with constant
swirling, during which no fuming should be observed.
Cool the solution to room temperature, quantitatively
transfer all of the solution to a 200-mL volumetric flask,
dilute with water to volume, mix, and filter.
Instrumental conditions
(See Plasma Spectrochemistry (730).)
Mode: {ICP-AES
Analytical wavelength: 317.93 nm. [NoTeE—The oper-
ating conditions may be developed and optimized
based on the manufacturer’s recommendation. A typi-
cal a includes radio frequency (RF) power of
about 1300 watts, argon torch flow of about 15 L/
min, argon auxiliary flow of about 0.2 L/min, and a
nebulizer flow rate of about 0.8 L/min.]
Analysis: Determine the emission of the Standard solu-
tion, the Sample solution, and a 2% nitric acid solution
as the blank at the wavelength indicated above.
Calculate the percentage of the labeled amount of cal-
cium (Ca) in the portion of Tablets taken:
Result = (ru/rs) x (Cs/Cu) x 100
ty = peak response of calcium from the Sample
solution
rs = peak response of calcium from the Standard
solution
Gs = concentration of calcium in the Standard
solution (g/mL)
Cu = nominal concentration of calcium in the
Sample solution (g/mL)
Acceptance criteria: 90.0%-110.0%
sydesHbouow Sa
e CONTENT OF CALCIUM, Procedure 2
Lanthanum chloride solution: 267 mg/mL of lantha-
num chloride heptahydrate in 0.125 N hydrochloric
acid
Calcium standard solution: Dissolve 1.001 g of calcium
carbonate, previously dried at 300° for 3 h and cooled
in a desiccator for 2 h, in 25 mL of 1 N hydrochloric
acid. Boil to expel carbon dioxide, and dilute with
water to 1000 mL to obtain a concentration of 400 ug/
mL of calcium.
Standard stock solution: 100 j1g/mL of calcium from
Calcium standard solution in 0.125 N hydrochloric acid
Standard solutions: Into separate 100-mL volumetric
flasks pipet 1.0, 1.5, 2.0, 2.5, and 3.0 mL of the Stan-
dard stock solution. To each flask add 1.0 mL of Lantha-
num chloride solution, and dilute with water to volume
to obtain concentrations of 1.0, 1.5, 2.0, 2.5, and
3.0 g/mL of calcium.
NF 36
Calculate the amount, in mg, of potassium hydroxide
required to neutralize the free acids in 1.0 g of
Lecithin:
Result = (M, x N x V)/W
M, molecular weight of potassium hydroxide,
11
N normality of the sodium hydroxide VS
V volume of the sodium hydroxide VS
consumed in the titration (mL)
Ww = weight of Lecithin taken (g)
Acceptance criteria: NMT 36
e PEROXIDE VALUE
Sample: 5g of Lecithin
Analysis: Transfer the Sample into a 250-mL Erlenmeyer
flask with a ground-glass stopper, add 35 mL of a mix-
ture of chloroform and glacial acetic acid (2:1), and
mix. Completely dissolve the test specimen while shak-
ing gently. The solution becomes transparent. Com-
pletely replace the air in the flask with nitrogen. While
purging with nitrogen, add 1 mL of potassium iodide
solution (165 mg/mL of potassium iodide), then stop
the flow of the nitrogen, and immediately place a stop-
per in the flask. Shake for 1 min, and allow to stand in
a dark place for 5 min. Add 75 mL of water, replace the
stopper again, and shake vigorously. Titrate with 0.01
N sodium thiosulfate VS, adding starch TS as the
endpoint is approached, and continue the titration until
the blue starch color has just disappeared. Perform a
blank determination (see Titrimetry (541)), and make
any necessary correction.
Calculate the peroxide value, as mEq of peroxide per
1000 g of Lecithin:
Result = (S x N/W) x 1000
S = net volume of sodium thiosulfate solution
required for titration (mL)
N = normality of the sodium thiosulfate solution
Ww = weight of Lecithin taken (g)
Acceptance criteria: NMT 10
For Lecithin intended for use in the manufacture of
injectable dosage forms: NMT 3
© MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI-
FIED MICROORGANISMS (62): The total aerobic microbial
count does not exceed 103 cfu/g, and the total com-
bined molds and yeasts count does not exceed 102 cfu/
g. It meets the requirements of the tests for absence of
Salmonella species and Escherichia coli.
© WATER DETERMINATION (921), Method I: NMT 2.0%
ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Preserve in well-closed, light-
resistant containers. Store at the temperature indicated
on the label. Protect from excess heat and moisture.
e LABELING: Label to indicate the content of each of the
phospholipids (phosphatidylcholine, phosphatidylethanol-
amine, phosphatidylinositol, and phosphatidic acid). The
labeling also indicates the natural source of lecithin.
Where Lecithin is intended for use in the manufacture of
injectable dosage forms, it is so labeled. Label it to indi-
cate the storage conditions.
Official Monographs / Lemon 5421
e USP REFERENCE STANDARDS (11)
USP Lysophosphatidylcholine (Soy) RS
USP Phosphatidic Acid (Soy) Monosodium RS
USP Phosphatidylcholine (Soy) RS
USP Phosphatidylethanolamine (Soy) RS
USP Phosphatidylinosito! (Soy) Sodium RS
Lemon Oil
DEFINITION
Lemon Oil is the volatile oil obtained by expression, without
the aid of heat, from the fresh peel of the fruit of Citrus x
limon (L.) Osbeck (Fam. Rutaceae), with or without the
previous separation of the pulp and the peel. The total
aldehyde content, calculated as citral (CioHi6O), is NLT
2.2% and NMT 3.8% for California-type Lemon Oil, and
NLT 3.0% and NMT 5.5% for Italian-type Lemon Oil.
[NotE—Do not use Lemon Oil that has a terebinthine odor.]
ASSAY
© TOTAL ALDEHYDE CONTENT
Reagent solution: Dissolve 4.5 g of hydroxylamine hy-
drochloride in 13 mL of water. Add 85 mL of tertiary
pue alcohol, mix, and adjust with 0.5 N potassium
hydroxide to a pH of 3.4.
Sample: 5 mL
Analysis: Pipet 50 mL of the Reagent solution into a
conical flask containing the Sample. Insert the stopper
in the flask, and allow to stand at room temperature for
30 min, with occasional shaking. Titrate the liberated
hydrochloric acid with 0.5 N alcoholic potassium hy-
droxide VS to a pH of 3.4. Each mL of 0.5 N alcoholic
potassium hydroxide consumed in the titration is equiv-
alent to 76.12 mg of total aldehydes, calculated as citral
(CioH16O).
Acceptance criteria: The total aldehyde content, calcu-
lated as citral (CioHi6O), is 2.2%-3.8% for California-
type Lemon Oil or 3.0%-5.5% for Italian-type Lemon
Oil.
IMPURITIES
Delete the following:
°o HEAVY METALS, Method !/ (231): NMT 40 119/Ge ‘otic! 1-
Jan-2018)
SPECIFIC TESTS
e SPECIFIC GRAVITY (841): 0.849-0.855
e OPTICAL ROTATION, Angular Rotation (781A): +57° to
+65.6°
© REFRACTIVE INDEX (831): 1.473-1.476 at 20°
e ULTRAVIOLET ABSORBANCE
Sample solution: Dilute 250 mg of Oil to 100 mL with
alcohol
Blank: Alcohol
Instrumental conditions
(See Ultraviolet-Visible Spectroscopy (857).)
Mode: UV-Vis
Spectral range: 260-400 nm
Analysis
sydeibouo= 4N
Samples: Sample solution and Blank
Record the spectrum in a 1-cm cell, and determine the
absorbance at the wavelength of maximum absorb-
ance at about 315 nm using the line drawn tangent
to the curves appearing as minima in the spectrum in
wavelength regions above and below the maximum
wavelength as the baseline.
Acceptance criteria: The absorbance, calculated on the
basis of a 250-mg specimen, is NLT 0.20 for California-
type Lemon Oil or NLT 0.49 for Italian-type Lemon Oil.
 5422 Lemon / Official Monographs
e FOREIGN OlLs: Place 50 mL of Oil in a four-bulb
Ladenburg flask having the following dimensions: the
lower or main bulb is about 6 cm in diameter, and the
smaller condensing bulbs are about 3.5, 3.0, and 2.5 cm
in diameter; the distance from the bottom of the flask to
the side-arm is about 20 cm. Distill Oil at a rate of 1
drop/s until the distillate measures 5 mL: the angular ro-
tation of the first 5 mL is NMT 6° less than that of the
original Oil. The refractive index at 20° of this same por-
tion is 0.001—-0.003 lower than that of the original Oil.
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in well-filled, tight
containers, and avoid exposure to excessive heat.
e LABELING: The label states the Latin binomial and, follow-
ing the official name, the part of the plant source from
which the article was derived. Label it to also indicate
whether it is California-type or Italian-type Lemon Oil.
The label indicates that Oil is not to be used if it has a
terebinthine odor.
Lemon Tincture
DEFINITION
Lemon Tincture is Pecpated from lemon peel, which is the
outer yellow rind of the fresh, ripe fruit of Citrus x Limon
Osbeck (Fam. Rutaceae).
Prepare Lemon Tincture as follows.
Lemon Peel 500 q
Alcohol 900 mL
Alcohol, a sufficient quantity to make 1000 mL.
Macerate the Lemon Peel in 900 mL of Alcohol in a closed
container, and store in a warm place. Agitate the con-
tainer frequently for 3 days or until the soluble matter is
dissolved. Transfer the mixture to a filter, using talc as the
filtering medium, and when most of the liquid has
drained away, wash the residue on the filter with a suffi-
cient amount of Alcohol, and combine the filtrates so that
the preparation is brought to a final volume of 1000 mL.
OTHER COMPONENTS
e ALCOHOL DETERMINATION, Method | (611): 62%-72% of
the labeled amount
IMPURITIES
Delete the following:
®o HEAVY METALS, Method Ii (231): NMT 40 Lg/mLe cottcial
J-lan-2018)
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Package in tight, light-resistant
containers, and avoid exposure to direct sunlight and to
excessive heat. Store at controlled room temperature.
e LABELING: The label states the Latin binomial and, follow-
ing the official name, the part of the plant source from
which the article was derived.
NF Monographs
Licorice Fluidextract
DEFINITION / Jan-2018°
Prepare Licorice Fluidextract as follows (see Pharmaceutical
Compounding—Nonsterile Preparations (795)). To 1000 g of
coarsely ground Licorice, add about 3000 mL of boiling
NF 36
Purified Water, mix, and allow to macerate in a suitable,
covered percolator for 2 h. Allow the percolation to pro-
ceed at a rate of 1-3 mL/min, gradually adding boiling
Purified Water until the Licorice is exhausted. Add enough
diluted ammonia solution to the percolate to impart a
distinctly ammoniacal odor, and boil the liquid actively
under normal atmospheric pressure until it is reduced in
volume to about 1500 mL. Filter the liquid, evaporate the
filtrate on a steam bath until the residue measures
750 mL, cool, gradually add 250 mL of Alcohol and
enough Purified Water to make the product measure
1000 mL, and mix.
OTHER COMPONENTS
¢ ALCOHOL DETERMINATION, Method | (611): 20.0%-24.0%
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in tight, light-resistant
containers, and avoid exposure to direct sunlight and to
excessive heat.
e LABELING: The label states the Latin binomial and, follow-
ing the official name, the part of the plant source from
which the article was derived.
Linoleoyl Polyoxylglycerides
DEFINITION
Linoleoy! Polyoxylglycerides is a mixture of monoesters, dies-
ters, and triesters of glycerol and monoesters and diesters
of polyethylene glycols. The poleryens glycols used
have a mean molecular weight between 300 and 400.
The article is produced by partial alcoholysis of unsatu-
rated oils, mainly containing triglycerides of linoleic acid
with polyethylene glycol, by esterification of glycerol and
polyethylene glycol with fatty acids, or as a mixture of
lycerol esters and ethylene oxide condensate with the
atty acids of the unsaturated oils. It may contain free pol-
yethylene glycols.
IDENTIFICATION
e A. INFRARED ABSORPTION (197F)
e B. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST
(201)
Standard solution: 50 mg/mL of USP Linoleoy! Polyox-
ylglycerides RS in methylene chloride
Sample solution: 50 mg/mL of Linoleoyl Polyoxylgly-
cerides in methylene chloride
Application volume: 10 uL
Developing solvent system: Ether and hexanes (70:30)
Spray reagent: 0.1 mg/mL of rhodamineBin alcohol
Analysis
Samples: Standard solution and Sample solution
Proceed as directed in the chapter. Then spray the plate
with Spray reagent, and locate the spots on the plate
by examination under UV light at a wavelength of 365
nam.
Acceptance criteria: The R, values of the principal spots
of the Sample solution correspond to those of the Stan-
dard solution.
e C. It meets the requirements in Specific Tests (see Table 1)
for Fats and Fixed Oils, Fatty Acid Composition (401).
IMPURITIES
Delete the following:
°e HEAVY METALS, Method /I (231): NMT 10 19/Ge cotta1
° ARTICLES OF BOTANICAL ORIGIN, Total Ash (561): NMT
0.1%
NF 36 Official Monographs / Magnesium 5423

© ALKALINE IMPURITIES e FATS AND FIXED OILS, Hydroxy! Value (401)

Sample: 5.0g
Analysis: To the Sample add 10 mL of alcohol and
0.05 mL of bromophenol blue TS, and mix well. Titrate
with 0.01 N hydrochloric acid VS to change the color
to yellow.
Acceptance criteria: NMT 1.0 mL of 0.01 N hydrochlo-
tic acid is required.
e Limit OF FREE ETHYLENE OXIDE AND DIOXANE
Analysis: Proceed as directed in Ethylene Oxide and Di-
oxane (228), Method |.
Acceptance criteria
Ethylene oxide: NMT 1 ug/g
Dioxane: NMT 10 ug/g
e Limit OF FREE GLYCEROL
Sample: 1.2g
Periodic acetic acid solution: Dissolve 0.446 g of so-
dium periodate in 2.5 mL of a 25% (v/v) solution of
sulfuric acid. Dilute with glacial acetic acid to 100.0 mL.
Potassium iodide solution: 75 mg/mL of potassium
iodide
Blank: 25 mL of methylene chloride
Titrimetric system
(See Titrimetry (541).)
Mode: Residual titration
Titrant: 0.1 M sodium thiosulfate VS
Endpoint detection: Visual
Analysis: Dissolve the Sample in 25 mL of methylene
chloride, heating if a Cool, and add 100 mL of
water and 25.0 mL of Periodic acetic acid solution. Shake,
and allow to stand for 30 min. Add 40 mL of Potassium
iodide solution, and allow to stand for 1 min. Add 1 mL
of starch TS, and titrate the liberated iodine with 0.1 M
sodium thiosulfate VS. Perform a blank determination,
and make any necessary correction.
Calculate the percentage of glycerol in the sample
taken:
Result = {[(Vs — Vs) x N x FJ/W} x 100
Ve = Titrant volume consumed by the Blank (mL)
Vs Titrant volume consumed by the Sample (mL)
ou
Sample: 1.0g
Acceptance criteria: 45-65
FATS AND FIXED OILS, /odine Value (401): 90-110
e FATS AND FIXED OILS, Peroxide Value (401)
Sample: 2.0g
Acceptance criteria: NMT 12.0
FATS AND FIXED OlLs, Saponification Value (401)
Sample: 2.0g
Acceptance criteria: 150-170
WATER DETERMINATION, Method | (921)
Sample: 1.0g
Analysis: Instead of using methanol as the solvent, one
of two solvent systems can be used: a mixture of meth-
ylene chloride and anhydrous methanol (70:30 v/v), or
anhydrous pyridine.
Acceptance criteria: NMT 1.0%
ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Preserve in tight containers,
protected from light and moisture. Store at controlled
room temperature.
e LABELING: Label it to indicate thetype and the average
nominal molecular weight of polyethylene glycol used as
part of the official title.
e USP REFERENCE STANDARDS (11)
USP Linoleoyl Polyoxylglycerides RS
Magnesium Aluminometasilicate
DEFINITION
Magnesium Aluminometasilicate is a synthetic material that
exists in two forms, Type I-A and Type I-B, having differ-
ent pH requirements. The required contents for both
forms are the same: NLT 29.1% and NMT 35.5% of alu-
minum oxide (Al2O3), NLT 11.4% and NMT 14.0% of
magnesium oxide (MgO), and NLT 29.2% and NMT
ae of silicon dioxide (SiOz), calculated on the dried
asis.

N actual normality of the Titrant (mEq/mL)

F = equivalency factor, 23.0 mg/mEq IDENTIFICATION
Ww = Sample weight (mg) e A, IDENTIFICATION TESTS—GENERAL, Aluminum (191)
Acceptance criteria: NMT 5.0% Sample solution: Transfer 0.5 g of Magnesium Alumi-
nometasilicate to a suitable container, add 5 mL of a
SPECIFIC TESTS sulfuric acid solution (1 in 3), and heat until white
e FATS AND FIXED OILS, Acid Value (401) fumes are observed. Cool, add 20 mL of water, and fil-
Sample: 2.0g ter. Neutralize the filtrate with ammonia TS, and retain
Acceptance criteria: NMT 2.0 for use in Identification test B. Collect the precipitate,
e FATS AND FIXED OILS, Fatty Acid Composition (401): _Li- and dissolve in 3 N hydrochloric acid.
Hee Polyoxylglycerides exhibits the composition pro- Acceptance criteria: The Sample solution meets the
file of fatty acids shown in Table 1. requirements.
© B. IDENTIFICATION TESTS—GENERAL, Magnesium (191)
Table 1 Sample solution: Use the filtrate retained from /dentifi-
Carbon-Chain Number of Percentage
cation test A.
Length Double Bonds (%) Acceptance criteria: The Sample solution meets the
requirements.
16 0 4.0-20.0 «:C:
18 0 $6.0 Analysis: Prepare a bead by fusing a few crystals of
18 1 20.0-35.0 sodium ammonium phosphate on a platinum loop in
18 2 50.0-65.0 the flame of a Bunsen burner. Place the hot, transpar-
18 3 $2.0 ent bead in contact with Magnesium Aluminometasili-
20 0 <1.0 cate, and again fuse. ra
Acceptance criteria: Silica floats about in the bead pro-
20 1 $1.0
ducing, upon cooling, an opaque bead with a web-like <4
structure. =
ASSAY S4
e ALUMINUM OXIDE
2
Edetate disodium titrant: Prepare a solution with a me]
concentration of 18.6 g/L of edetate disodium in water a
Aa)
and standardize as follows. Weigh 2 g of aluminum
wire, transfer to a 1000-mL volumetric flask, and add
 5424 Magnesium / Official Monographs
50 mL of a mixture of hydrochloric acid and water
(1:1). Swirl the flask to ensure contact of the aluminum
and the acid, and allow the reaction to proceed until all
of the aluminum has dissolved. Dilute with water to
volume. Pipet 10 mL of this solution into a 250-mL
beaker and add, in the order named and with continu-
ous stirring, 25.0 mL of Edetate disodium titrant and
20 mL of acetic acid-ammonium acetate buffer TS, and
boil gently for 5 min. Cool, and add 50 mL of alcohol
and 2 mL of dithizone TS. Titrate with 0.05 M zinc sul-
fate VS to a bright rose-pink color. Perform a blank de-
termination, substituting 10 mL of water for the alumi-
num solution, and make any necessary correction.
Calculate the molarity of the solution taken:
Result = W/(A, x V)
Ww = weight of aluminum in the portion of solution
taken (mg)
Ar = atomic weight of aluminum, 26.98
V 7 ven’ of Edetate disodium titrant consumed
mL
Sample solution: Transfer 1.25 g of Magnesium Alumi-
nometasilicate to a conical flask, add 10 mL of 3 N hy-
drochloric acid and 50 mL of water, and heat on a
water bath for 15 min. To this solution add 8 mL of
hydrochloric acid, and heat on a water bath for 10 min.
After cooling, transfer the solution to a 250-mL volu-
metric flask, rinse the conical flask with water, and add
the washings to the volumetric flask. Dilute with water
to volume. Centrifuge, and use the supernatant as the
Sample solution. Retain a portion for use in the Assay for
Magnesium Oxide.
Analysis: Transfer 20.0 mL of the Sample solution to a
beaker and add 20.0 mL of Edetate disodium titrant. To
this solution add 15 mL of acetic acid-ammonium ace-
tate buffer TS and 20 mL of water, and boil for 5 min.
After cooling, add 50 mL of alcohol and 2 mL of
dithizone TS, and titrate with 0.05 M zinc sulfate VS
until the color of the solution changes from green-violet
to rose-pink. Perform a blank determination. Each mL of
0.05 M Edetate disodium titrant is equivalent to
2.5490 mg of Al,O3.
Acceptance criteria: 29.1%-35.5% of aluminum oxide
(Al2O3) on the dried basis
Change to read:
© MAGNESIUM OXIDE
Sample solution: Use the Sample solution prepared for
use in the Assay for Aluminum Oxide.
Analysis: Transfer 50.0 mL of the Sample solution to a
suitable container, add 50 mL of water and 25 mL of a
trolamine solution (1 in 2), and shake well. Add 25 mL
of ammonia-ammonium chloride buffer TS and 0.04g
of °eriochrome black T trituratione cere apr2017 as the
indicator. Titrate with 0.05 M edetate disodium VS until
the red-purple color changes to blue and persists for 30
s. Each mL of 0.05 M edetate disodium VS is equivalent
to 2.0152 mg of MgO.
Acceptance criteria: 11.4%-14.0% of magnesium ox-
ide (MgO) on the dried basis
SILICON DIOXIDE
Sample: 19
NF Monographs
Analysis: To the Sample add 30 mL of 3 N hydrochloric
acid, and evaporate on a water bath to dryness. Mois-
ten the residue with hydrochloric acid, and evaporate
again on a water bath to dryness. To the residue add
8 mL of hydrochloric acid and 25 mL of hot water, and
stir. Allow to stand, and then decant the supernatant
through an ashless filter paper. To the residue in the
container add 10 mL of hot water, stir, and decant the
supernatant through the filter paper. Wash the residue
NF 36
in the container with three additional 10-mL portions of
hot water, stir, and decant as described above. Treat
the residue in the container with 50 mL of water, and
heat on a water bath for 15 min. Filter, and rinse the
residue on the filter paper with hot water until no pre-
cipitate is obtained when 1 mL of silver nitrate TS is
added to 5 mL of the washing. Transfer the filter paper
and its contents to a tared platinum crucible, heat to
dryness, incinerate, and continue to heat at 800 + 25°
for 1 h. Cool, and weigh. Moisten the residue with
6 mL of hydrofluoric acid, evaporate to dryness, and ig-
nite for 5 min. Cool, and weigh. The loss in weight
represents the weight of silicon dioxide (SiO2).
Acceptance criteria: 29.2%-35.6% of silicon dioxide
(SiO) on the dried basis
IMPURITIES
¢ CHLORIDE AND SULFATE, Chloride (221)
Sample: A 20-mL portion of the diluted filtrate retained
from the test for Soluble Salts
Control: 0.75 mL of 0.020 N hydrochloric acid
Acceptance criteria: NMT 0.053%; the Sample shows
no more chloride than corresponds to the Control.
© CHLORIDE AND SULFATE, Sulfate (221)
Sample: A 2-mL portion of the diluted filtrate retained
from the test for Soluble Salts
Control: 0.5 mL of 0.020N sulfuric acid
Acceptance criteria: NMT 0.480%; the Sample shows
no more sulfate than corresponds to the Control.
© ARSENIC, Method | (211): NMT 3 g/g
e IRON (241)
Sample solution: To 0.11 g of Magnesium Alumino-
metasilicate add 8 mL of 2N nitric acid, boil for 1 min,
and cool. Dilute with water to 100 mL, and centrifuge.
Dilute 30 mL of the supernatant with water to 45 mL.
Acceptance criteria: NMT 0.03%
Delete the following:
°o HEAVY METALS, Method | (231)
Test preparation: Transfer 2.67 g of Magnesium Alumi-
nometasilicate to a suitable container, add 20 mL of
water and 8 mL of hydrochloric acid, and evaporate to
dryness on a water bath. To the residue add5 mL of
1N acetic acid and 20 mL of water, boil for 2 min, add
0.4 g of hydroxylamine hydrochloride, and heat to boil-
Ing. Cool, dilute with water to 100 mL, and filter. Use
25 mL of the filtrate as the Test preparation.
Monitor preparation: Transfer another 25 mL of the di-
luted filtrate to a suitable container, and add 2.0 mL of
Standard Lead Solution.
Standard solution: Transfer 2 mL of hydrochloric acid
to a suitable container, and evaporate to dryness on a
water bath. To the residue add 2.0 mL of Standard Lead
Solution and 0.1 g of hydroxylamine hydrochloride. Di-
lute with water to 25 mL.
Acceptance criteria: NMT 30 119/ge circa 1-1an-2018)
SPECIFIC TESTS
e ACID-CONSUMING CAPACITY
Sample solution: Transfer 0.2 g of Magnesium Alumi-
nometasilicate to a glass-stoppered flask, and add
100.0 mL of 0.1 N hydrochloric acid VS. Stopper the
flask tightly, shake at 37
+2° for 1 h, and filter. Use the
filtrate.
Analysis: Transfer 50.0 mL of the Sample solution to a
beaker, and while stirring, titrate the excess hydrochlo-
ric acid with 0.1 N sodium hydroxide VS to attain a pH
of 3.5. Perform a blank determination.
Acceptance criteria: NLT 210 mL of 0.1 N hydrochloric
acid is consumed per g of Magnesium Aluminometasili-
cate, calculated on the dried basis.
NF 36 Official Monographs / Magnesium 5425

© PH (791) ASSAY
Sample: 29 e ALUMINUM OXIDE
Analysis: Add 50 mL of water to the Sample. While stir- Edetate disodium titrant: Prepare a solution with a
ring, immerse the pH electrodes in the suspension, and concentration of 18.6 g/L of edetate disodium in water,
after 2 min, record the pH. and standardize as follows. Weigh 2 g of aluminum
Acceptance criteria wire, transfer to a 1000-mL volumetric flask, and add
Type I-A: 6.5-8.5 50 mL of a mixture of hydrochloric acid and water
Type I-B: 8.5-10.5 (1:1). Swirl the flask to ensure contact of the aluminum
e LOSS ON DRYING (731) and the acid, and allow the reaction to proceed until all
Analysis: Dry at 110° for 7 h. the aluminum has dissolved. Dilute with water to vol-
Acceptance criteria: NMT 20.0% ume. Pipet 10 mL of this solution into a 250-mL beaker,
© SOLUBLE SALTS and add, in the order named and with continuous stir-
Sample: 10.0g ring, 25.0 mL of Edetate disodium titrant and 20 mL of
Analysis: Transfer the Sample to a suitable container, acetic acid-ammonium acetate buffer TS. Boil gently for
add 150 mL of water, and boil gently for 15 min, with 5 min. Cool, and add 50 mL of alcohol and 2 mL of
shaking. After cooling, dilute with water to 150 mL, and dithizone TS. Titrate with 0.05 M zinc sulfate VS to a
centrifuge. Dilute 75 mL of the clear filtrate with water bright rose-pink color. Perform a blank determination,
to 100 mL, and retain the diluted filtrate for use in the substituting 10 mL of water for the aluminum solution,
tests for Alkalinity, Chloride, and Sulfate. Evaporate and make any necessary correction.
25 mL of the diluted filtrate on a water bath, and heat Calculate the molarity of the solution taken:
at 700° for 2 h.
Acceptance criteria: NMT 0.020 g (NMT 1.6%) Result = W/(A, x V)
@ ALKALINITY
Sample: A 20-mL portion of the diluted filtrate retained Ww = weight of aluminum in the portion of solution
from the test for Soluble Salts taken (g)
Analysis: Add 2 drops of phenolphthalein TS to the A, = atomic weight of aluminum, 26.98 g/mol
Sample. Vv = volume of Edetate disodium titrant consumed
Acceptance criteria: If a pink color is produced, NMT (mL)
0.50 mL of 0.1 N hydrochloric acid is required to dis- Sample solution: Transfer 1.25 g of Magnesium Alumi-
charge it. nosilicate to a conical flask, add 10 mL of 3 N hydro-
chloric acid and 50 mL of water, and heat on a water
ADDITIONAL REQUIREMENTS bath for 15 min. To this solution add 8 mL of hydro-
© PACKAGING AND STORAGE: Preserve in tight containers, chloric acid, and heat on a water bath for 10 min. After
and prevent exposure to excessive heat. cooling, transfer the solution to a 250-mL volumetric
e LABELING: Label it to indicate whether it is Type I-A or flask, rinse the conical flask with water, and add the
Type I-B. washings to the volumetric flask. Dilute with water to
volume. Centrifuge, and use the supernatant as the
Sample solution.
[NoTe—Retain a portion of the Sample solution for use
in the Assay for Magnesium Oxide.
Magnesium Aluminosilicate Blank: 10 mL of 3N hydrochloric acid and 50 mL of
water
DEFINITION Titrimetric system
Magnesium Aluminosilicate is a synthesized material that (See Titrimetry (541).)
contains NLT 20.5% and NMT 27.7% of magnesium ox- Mode: Residual titration
ide (MgO), NLT 27.0% and NMT 34.3% of aluminum Titrant: Edetate disodium titrant
oxide (AlzO3), and NLT 14.4% and NMT 21.7% of silicon Back-titrant: 0.05 M zinc sulfate VS
dioxide (SiOz), calculated on the dried basis. Endpoint detection: Visual
Analysis: Transfer 20.0 mL of the Sample solution to a
IDENTIFICATION beaker, and add 20.0 mL of Titrant. To this solution add
e A. IDENTIFICATION TESTS—GENERAL, Aluminum (191) 15 mL of acetic acid-ammonium acetate buffer TS and
Sample: 0.59 20 mL of water, and boil for 5 min. After cooling, add
Analysis: Transfer the Sample to a suitable container, 50 mL of alcohol and 2 mL of dithizone TS, and titrate
add 5 mL ofa sulfuric acia solution (1 in 3), and heat with the Back-titrant until the color of the solution
until white fumes are observed. Cool, add 20 mL of changes from green-violet to rose-pink. Perform a blank
water, and filter. Neutralize the filtrate with ammonia determination, and make the necessary correction. Each
TS, and retain for use in Identification test B. Collect the mL of 0.05 M Edetate disodium titrant is equivalent to
precipitate, and dissolve in 3 N hydrochloric acid. 2.5490 mg of aluminum oxide (Al203).
Acceptance criteria: Meets the requirements Acceptance criteria: 27.0%-34.3% on the dried basis
o B. IDENTIFICATION TESTS—GENERAL, Magnesium (191) © MAGNESIUM OXIDE
Sample solution: The filtrate retained from Identification Sample solution: Use the portion retained from the
test A Sample solution prepared in the Assay for Aluminum
Acceptance criteria: Meets the requirements Oxide.
eC Titrimetric system
Analysis: Prepare a bead by fusing a few crystals of (See Titrimetry (541).)
sydeiBbouow 4N

sodium ammonium phosphate on a platinum loop in Mode: Direct titration

the flame of a Bunsen burner. Place the hot, transpar-
ent bead in contact with Magnesium Aluminosilicate,
and again fuse.
Acceptance criteria: The silica floats about in the bead,
roducing, upon cooling, an opaque bead with a web-
ike structure.
Titrant: 0.05 M edetate disodium VS
Endpoint detection: Visual
Analysis: Transfer 50.0 mL of the Sample solution to a
suitable container, add 50 mL of water and 25 mL of a
trolamine solution (500 mg/mL), and shake well. Add
25 mL of ammonia—ammonium chloride buffer TS and
0.04 g of eriochrome black TS trituration as the indica-
tor. Titrate with Titrant until the red-purple color
changes to blue and persists for 30 s. Each mL of 0.05
 5426 Magnesium / Official Monographs NF 36

M edetate disodium VS is equivalent to 2.0152 mg of
magnesium oxide (MgO).
Acceptance criteria: 20.5%-27.7% on the dried basis
© SILICON DIOXIDE
Sample: 1
Analysis: To the Sample add 30 mL of 3 N hydrochloric
acid, and evaporate on a water bath to dryness. Mois-
ten the residue with hydrochloric acid, and again evap-
orate on a water bath to dryness. To the residue add
8 mL of hydrochloric acid and 25 mL of hot water, and
stir. Allow to stand, then decant the supernatant
through an ashless filter paper. To the residue in the
container add 10 mL of hot water, stir, and decant the
supernatant through the filter paper. Wash the residue
in the container with three additional 10-mL portions of
hot water, stir, and decant as described above. Treat
the residue in the container with 50 mL of water, and
heat on a water bath for 15 min. Filter, and rinse the
residue on the filter paper with hot water until no pre-
cipitate is obtained when 1 mL of silver nitrate TS is
added to 5 mL of the washing. Transfer the filter paper
and its contents to a tared platinum crucible, heat to
dryness, incinerate, and continue to heat at 800 + 25°
for 1 h. Cool, and weigh. Moisten the residue with
6 mL of hydrofluoric acid, evaporate to dryness, and ig-
nite for 5 min. Cool, and weigh. The loss in weight
represents the weight of SiOz.
Acceptance criteria: 14.4%-21.7% on the dried basis
IMPURITIES
e CHLORIDE AND SULFATE, Chloride (221)
Analysis: A 20-mL portion of the diluted filtrate re-
tained from the test for Soluble Salts shows no more
chloride than corresponds to 0.75 mL of 0.020 N hy-
drochloric acid.
Acceptance criteria: NMT 0.053%
e CHLORIDE AND SULFATE, Sulfate (221)
Analysis: A 2-mL portion of the diluted filtrate retained
from the test for Soluble Salts shows no more sulfate
than corresponds to 0.5 mL of 0.020 N sulfuric acid.
Acceptance criteria: NMT 0.480%
© ARSENIC, Method | (211): NMT 3 ug/g
e IRON (241)
Sample: 0.11g
Analysis: To the Sample add 8 mL of 2 N nitric acid,
boil for 1 min, and cool. Dilute with water to 100 mL,
and centrifuge. Dilute 30 mL of the supernatant with
water to 45 mL.
Acceptance criteria: NMT 0.03%
Acceptance criteria: NMT 30 19/Qe cortcial 1 Jan-2018)
SPECIFIC TESTS
¢ ACID-CONSUMING CAPACITY
Sample solution: Transfer 029 of Magnesium Alumi-
nosilicate to a glass-stoppered flask, and add 100.0 mL
of 0.1 N hydrochloric acid VS. Stopper the flask tightly,
shake at 37+ 2° for 1 h, and filter. Use the filtrate.
Analysis: Transfer 50.0 mL of the filtrate from the Sam-
ple solution to a beaker, and while stirring, titrate the
excess hydrochloric acid with 0.1 N sodium hydroxide
VS to a pH of 3.5. Perform a blank determination, and
make any necessary corrections.
Acceptance criteria: NLT 250 mL of 0.1 N hydrochloric
acid is consumed perg of Magnesium Aluminosilicate,
calculated on the dried basis.
PH (791)
Sample: 2g
Analysis: Add 50 mL of water to the Sample. While stir-
ring, immerse the pH electrodes in the suspension, and
after 2 min, record the pH.
Acceptance criteria: 8.5-10.5
e LOSS ON DRYING (731)
Analysis: Dry at 110° for 7 h.
Acceptance criteria: NMT 20.0%
© SOLUBLE SALTS
Sample: 10.0g
Analysis: Transfer the Sample to a suitable container,
add 150 mL of water, and boil gently for 15 min, with
shaking. After cooling, dilute with water to 150 mL, and
centrifuge. Dilute 75 mL of the clear filtrate with water
to 100 mL, and retain the diluted filtrate for use in the
tests for Chloride, Sulfate, and Alkalinity. Evaporate
25 mL of the diluted filtrate on a water bath, and heat
at 700° for 2 h.
Acceptance criteria: NMT 1.6%; the residue weighs
NMT 0.020 g.
e ALKALINITY
Sample: 20 mL of diluted filtrate retained from the test
for Soluble Salts
Analysis: Add 2 drops of phenolphthalein TS to the
Sample, containing 1 g of Magnesium Aluminosilicate.
Acceptance criteria: If a pink color is produced, NMT
0.50 mL of 0.1 N hydrochloric acid is required to dis-
charge it.
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in tight containers,
and prevent exposure to excessive heat.

Delete the following:

°e HEAVY METALS, Method | (231) Magnesium Aluminum Silicate

Standard preparation: Transfer 2 mL of hydrochloric
acid to a suitable container, and evaporate to dryness DEFINITION
on a water bath. To the residue add 2.0 mL of Standard Magnesium Aluminum Silicate is a blend of colloidal
Lead Solution and 0.1 g of hydroxylamine hydrochlo- montmorillonite and saponite that has been processed to
ride. Dilute with water to 25 mL. remove grit and nonswellable ore components.
Sample: 2.679 The requirements for viscosity and ratio of aluminum con-
Test preparation: Transfer the Sample to a suitable con- tent to magnesium content differ for the several types of
tainer, add 20 mL of water and 8 mL of hydrochloric Magnesium Aluminum Silicate, as set forth in the table
acid, and evaporate to dryness on a water bath. To the jelow.
residue add 5 mL of 1 N acetic acid and 20 mL of
al water. Boil for 2 min, add 0.4 g of hydroxylamine hy-
ao Viscosity Al Content/ Appear-
rs drochloride, and heat to boiling. Cool, dilute with water
to 100 mL, and filter. Use 25 mi of the filtrate as the Type (mPa:s) Mg Content ance
woSs
Dp Test preparation. Min. Max. Min. Max.
i) Monitor preparation: Transfer 25 mL of the filtrate Fine
= from the Test preparation to a suitable container, and granules
3 add 2.0 mL of Standard Lead Solution. IA 225 600 0.5 1.2 or flakes
= Microfine
J
1B 150 450 0.5 1.2 powder
Zz
NF 36
Viscosity Al Content/ Appear-
Type (mPa:s) Mg Content ance
Min. Max. Min. Max.
Fine
granules
IC 800 2200 0.5 12 or flakes
Fine
granules
IA 100 300 14 2.8 or flakes
IDENTIFICATION
eA.
Sample: 2g
Analysis 1: “Add the Sample in small portions to 100 mL
of water with intense agitation. Allow to stand for 12 h
to ensure complete hydration. Place 2 mL of the result-
ing mixture on a suitable glass slide, and allow to air-
dry at room temperature to produce an oriented film.
Place the slide in a vacuum desiccator over a free sur-
face of ethylenegical. Evacuate the desiccator, and
close the stopcock so that the ethylene glycol saturates
the desiccator chamber. Allow to stand for 12 h. Record
the X-ray diffraction pattern (see X-Ray Diffraction
(941)), and calculate the d values.
Acceptance criteria 1: The largest, peak corresponds to
advalue between 15.0 and 17.2 A.
Analysis 2: Prepare a random powder specimen of
Magnesium Aluminum Silicate, record the X-ray diffrac-
tion pattern, and determine the dvalues in the region
between 1.48 and 1.54 A.
Acceptance criteria 2: Peaks are found at 1.492-1.504
A and at 1.510-1.540 A.
e B. It meets the requirements of the test for Viscosity in
Specific Tests.
e C. It meets the requirements for Ratio of aluminum con-
tent to magnesium content in the test for Aluminum Con-
tent and Magnesium Content.
e D. Its ar corresponds to the description in the
table in the Definition.
ASSAY
¢ ALUMINUM CONTENT AND MAGNESIUM CONTENT
Aluminum content
Aluminum standard stock solution: Dissolve 1.000 g
of aluminum in a mixture of 10 mL of hydrochloric
acid and 10 mL of water by gentle heating. Transfer
the solution to a 1000-mL volumetric flask, dilute with
water to volume, and mix. This solution contains the
equivalent of 1 mg/mL of aluminum.
Aluminum standard solutions: Transfer 2-, 5-, and
10-mL aliquots of the Aluminum standard stock solution
to separate 100-mL volumetric flasks containing
200 mg of sodium chloride, dilute each with water to
volume, and mix.
Sample stock solution: Transfer 0.200 g of Magne-
sium Aluminum Silicate to a 25-mL platinum crucible
containing 1.0 g of lithium metaborate, and mix. Us-
ing a muffle furnace or a suitable burner, heat slowly
at first, and ignite at 1000°-1200° for 15 min. Cool,
place the crucible in a 100-mL beaker containing
25 mL of dilute nitric acid (50 mg/mL), and add an
additional 50 mL of the dilute acid, filling and sub-
merging the upright crucible. Place a polyfluoro-
carbon-coated meareee stirring bar into the crucible,
and stir gently with a magnetic stirrer to dissolve. Pour
the contents into a 250-mL beaker, and remove the
crucible. Warm the solution, transfer through a rapid-
flow filter paper with the aid of water into a 200-mL
volumetric flask, dilute with water to volume, and mix.
Sample solution: Pipet 20 mL of the Sample stock solu-
tion into a 100-mL volumetric flask. Add 20 mL of a
solution of sodium chloride (10 mg/mL), dilute with
water to volume, and mix.
Official Monographs / Magnesium 5427
Instrumental conditions
(See Atomic Absorption Spectroscopy (852).)
Mode: Atomic absorption spectrophotometer
equipped with a single-slot burner
Analytical wavelength: 309 nm
Lamp: Aluminum hollow-cathode
Flame: Oxidizing acetylene—air—nitrous oxide
Analysis
Samples: Aluminum standard solutions and Sample
solution
Determine the absorbances of the Aluminum standard
solutions and the Sample solution. Fromalinear re-
gression equation calculated from the absorbances
and concentrations of the Aluminum standard solu-
tions, determine the aluminum content in the magne-
sium aluminum silicate.
Magnesium content
Lanthanum solution: Stir 88.30 g of lanthanum chlo-
ride (LaClz) with 500 mL of 6 N hydrochloric acid to
dissolve, transfer with the aid of water to a 1000-mL
volumetric flask, dilute with water to volume, and mix.
Magnesium standard stock solution: Place 1.000g of
magnesium in a 250-mL beaker containing 20 mL of
water, and carefully add 20 mL of hydrochloric acid,
warming, if necessary, to complete the reaction. Trans-
fer the solution to a 1000-mL volumetric flask, dilute
with water to volume, and mix. This solution contains
the equivalent of 1 mg/mL of magnesium. Transfer
10.0 mL of this solution to a 1000-mL volumetric flask,
dilute with water to volume, and mix.
Magnesium standard solutions: Transfer 5-, 10-, 15-,
and 20-mL aliquots of the Magnesium standard stock
solution to separate 100-mL volumetric flasks. To each
flask add 20.0 mL of Lanthanum solution, dilute with
water to volume, and mix.
Sample stock solution: Use the Sample stock solution
prepared as directed for Aluminum content.
Sample solution: Transfer 25 mL of the Sample stock
solution to a 50-mL volumetric flask, dilute with water
to volume, and mix. Transfer 5.0 mL of this solution to
a 100-mL volumetric flask, add 20.0 mL of Lanthanum
solution, dilute with water to volume, and mix.
Instrumental conditions
(See Atomic Absorption Spectroscopy (852).)
Mode: Atomic absorption
Analytical wavelength: 285 nm
Lamp: Magnesium hollow-cathode
Flame: Reducing flame of acetylene—air
Analysis
Samples: Magnesium standard solutions and Sample
solution
Determine the absorbances of the Sample solution and
the Magnesium standard solutions. Fromalinear re-
gression equation calculated from the absorbances
and concentrations of the Magnesium standard solu-
tions, determine the magnesium content in the mag-
nesium aluminum silicate.
Ratio of aluminum content to magnesium content
Analysis: Using the results from the Aluminum content
and the Magnesium content, determine the ratio of alu-
minum content to magnesium content.
Acceptance criteria
Type IA: 0.5-1.2
Type IB: 0.5-1.2
Type IC: 0.5-1.2 cA
a]
Type IIA: 1.4-2.8
=
IMPURITIES °
S
e ARSENIC (211), Method
| °
Standard preparation: Transfer 5.0 mL (5 wg of arse- oy
nic) of the Standard Arsenic Solution to a 25-mL volu- =
i)
metric flask, and add dilute hydrochloric acid (1:25) to me]
volume. a
=>
Test preparation: Transfer 13.3 g of Magnesium Alumi-
num Silicate to a 250-mL beaker containing 100 mL of
 5428 Magnesium / Official Monographs
dilute hydrochloric acid (1:25), mix, cover with a watch
glass, and boil gently with occasional stirring for 15 min
without allowing excessive foaming. Allow the insoluble
material to settle, and decant the hot supernatant
through a rapid-flow filter paper into a 200-mL volu-
metric flask, retaining as much sediment as possible in
the beaker. Add 25 mL of hot dilute hydrochloric acid
(1:25) to the residue in the beaker, stir, and heat to
boiling. Allow the insoluble material to settle, and de-
cant the a through the filter into the 200-mL
volumetric flask. Repeat the extraction with four addi-
tional 25-mL portions of hot dilute hydrochloric acid
(1:25), decanting each hot supernatant through the fil-
ter into the volumetric flask. At the last extraction,
transfer as much of the insoluble material as possible
onto the filter. Cool the combined filtrates to room
temperature, add dilute hydrochloric acid (1:25) to vol-
ume, and mix. Use 25 mL for the test.
Acceptance criteria: NMT 3 \1g/g; the absorbance due
to any red color from the Test preparation does not ex-
ceed that produced by the Standard preparation.
e LEAD
Standard preparation: On the day of use, dilute
3.0 mL of lead nitrate stock solution TS with water to
ey mL. Each mL contains the equivalent of 3 wg of
lead.
Sample: 10.0g
Test preparation: Transfer the Sample to a 250-mL
beaker containing 100 mL of dilute hydrochloric acid
(1:25), stir, cover with a watch glass, and boil for 15
min. Cool to room temperature, and allow the insoluble
matter to settle. Decant the supernatant through a
rapid-flow filter paper into a 400-mL beaker. Add 25 mL
of hot water to the insoluble matter in the 250-mL
beaker, and stir. Allow the insoluble matter to settle,
and decant the supernatant through the filter into the
400-mL beaker. Repeat the extraction with two addi-
tional 25-mL portions of water, decanting each super-
natant portion through the filter into the 400-mL
beaker. Wash the filter with 25 mL of hot water, collect-
ing this filtrate in the 400-mL beaker. Concentrate the
combined extracts by gentle bali to approximately
20 mL. If a precipitate appears, add 2-3 drops of nitric
acid, heat to boiling, and cool to room temperature.
Filter the concentrated extracts through a rapid-flow fil-
ter paper into a 50-mL volumetric flask. Transfer the
remaining contents of the 400-mL beaker through the
filter paper and into the flask with water. Dilute with
water to volume.
Instrumental conditions
(See Atomic Absorption Spectroscopy (852).)
Mode: Atomic absorption spectrophotometer
equipped with a deuterium arc background correction
and a single-slot burner
Analytical wavelength: 284 nm
Lamp: Lead hollow-cathode
Flame: Oxidizing flame of air and acetylene
Acceptance criteria: The absorbance of the Test prepa-
ration is NMT that of the Standard preparation (15 ug/
9).
SPECIFIC TESTS
e MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI-
FIED MICROORGANISMS (62): Its total aerobic microbial
count does not exceed 103 cfu/g, and it meets the re-
NF Monographs
quirements of the test for absence of Escherichia coli.
NF 36
e PH (791)
Sample suspension: 50 mg/mL
Acceptance criteria: 9.0-10.0
e Loss ON DRYING (731)
Analysis: Dry at 110° to constant weight.
Acceptance criteria: NMT 8.0%
Change to read:
© VISCOSITY
Sample: After determining the Loss on Drying, weigh a
quantity of Magnesluts Aluminum Silicate, equivalent to
25.0g on the dried basis. Over a period of a few
seconds, transfer the undried test specimen to a suita-
ble 1-L blender jar containing an amount of water,
maintained at a temperature of 25 + 2°, that is sufficient
to produce a mixture weighing 500 g. Blend for 3 min,
accurately timed, at 14,000-15,000 rpm (high speed).’
[Note—Heat generated during blending causes a tem-
perature rise to above 30°.]
Analysis: Transfer the contents of the blender to a
600-mL beaker, and allow to stand for 5 min. The sam-
ple temperature should be 33 + 3°. Using a suitable ro-
tational viscometer? equipped with a spindle as speci-
fied below, operate the viscometer at 60 rpm for 6 min,
accurately timed, and record the scale reading.
For Type IA, use a spindle with a cylinder 1.87 cm in
diameter and 0.69 cm high attached to a shaft
0.32 cm in diameter, the distance from the top of the
cylinder to the lower tip of the shaft being 2.54 cm,
and the immersion depth being 5.00 cm (No. 2 spin-
dle). If the scale reading is greater than 90% of full
scale, repeat the measurement, using a spindle similar
to the No, 2 spindle but with the cylinder 1.27 cm in
diameter and 0.16 cm high instead (No. 3 spindle).
For Type IC, use a No. 3 spindle. If the scale reading is
greater than 90% of full scale, repeat the measure-
ment using a spindle consisting of a cylindrical shaft
0.32 cm in diameter and with an immersion depth of
4.05 cm (No. 4 spindle).
For Types IB and IIA, use a No. 2 spindle.
Acceptance criteria
Type IA: 225-600 ®mPa- Se ccar i-apr2017)
Type IB: 150-450 ®mPa - Se ear 1-apr-2017)
Type IC: 800-2200 ®mPa - Se cre i.apr-2017)
Type IIA: 100-300 ®mPa - Secexr 1491-20177
e AciD DEMAND
Sample: After determining the Loss on Drying, weigh a
quantity of Magnesium Aluminum Silicate equivalent to
5.00 g.
Analysis: Disperse the Sample in 500 mL of water with
the aid of a suitable blender fitted with a 1-L jar. Using
a stopwatch, designate zero time. With constant mix-
ing, add 3.0-mL portions of 0.100 N hydrochloric acid
at 5, 65, 125, 185, 245, 305, 365, 425, 485, 545, 605,
665, and 725 s, and add a 1.0-mL portion at 785 s.
Determine the pH potentiometrically at 840 s.
Acceptance criteria: NMT 4.0
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in tight containers.
e LABELING: Label it to indicate its type.
1A suitable blender is available from Waring as Waring Commercial Blender
Model 7009G or equivalent with 1-L glass jar andtachometer adapter, Model
CAC24 or equivalent.
2A suitable viscometer is available from Brookfield as Viscometer Model LVF or
LVT, or equivalent.
NF 36
Magnesium Silicate
DEFINITION
Magnesium Silicate is a compound of magnesium oxide and
silicon dioxide. It contains NLT 15.0% of magnesium ox-
ide (MgO) and NLT 67.0% of silicon dioxide (SiO), calcu-
lated on the ignited basis.
IDENTIFICATION
© A. IDENTIFICATION TESTS—GENERAL, Magnesium (191)
Sample: 500mg
Analysis: Mix the Sample with 10 mL of 3 N hydrochlo-
ric acid. Filter, and neutralize the filtrate to litmus paper
with 6 N ammonium hydroxide.
Acceptance criteria: The neutralized filtrate meets the
requirements.
eB.
Analysis: Prepare a bead by fusing a few crystals of
sodium ammonium phosphate on a platinum loop in
the flame of a Bunsen burner. Place the hot, transpar-
ent bead in contact with Magnesium Silicate, and again
fuse.
Acceptance criteria: Silica floats about in the bead,
roducing, upon cooling, an opaque bead with a web-
ike structure.
ASSAY
© MAGNESIUM OXIDE
Sample: 1.5g
Titrimetric system
Mode: Residual titration
Titrant: 1.N sodium hydroxide VS
Endpoint detection: Visual
Analysis: Dissolve the Sample in 50.0 mL of 1.N sulfuric
acid VS. Digest on a steam bath for 1 h, cool to room
temperature, and add methyl orange TS. Titrate the ex-
cess acid in the sample with Titrant. Each mL of 1N
sulfuric acid is equivalent to 20.15 mg of MgO.
Acceptance criteria: NLT 15.0% on the ignited basis
© SILICON DIOXIDE
Sample: 700mg
Analysis: Transfer the Sample to a small platinum dish.
Add 10 mL of 1 N sulfuric acid, and heat on a steam
bath to dryness, leaving the dish uncovered. Treat the
residue with 25 mL of water, and digest on a steam
bath for 15 min. Decant the supernatant through an
ashless filter paper, with the aid of suction, and wash
the residue, by decantation, three times with hot water,
passing the washings through the filter paper. Finally,
transfer the residue to the filter, and wash thoroughly
with hot water. Transfer the filter paper and its contents
to the platinum dish previously used. Heat to dryness,
incinerate, ignite strongly for 30 min, cool, and weigh.
Moisten the residue with water, and add 6 mL of hy-
drofluoric acid and 3 drops of sulfuric acid. Evaporate to
dryness, ignite for 5 min, cool, and weigh. The loss in
weight represents the weight of SiOz.
Acceptance criteria: NLT 67.0% on the ignited basis
IMPURITIES
© FLUORIDE
Indicator solution: 100 mg/mL of lanthanum alizarin
complexan mixture in 60% isopropyl alcohol. Filter the
solution if it is not clear.
Standard solution: 2.21 1g/mL of sodium fluoride in
0.1 N hydrochloric acid
Sample solution: Preparea slurry consisting of 5.0 g of
Magnesium Silicate and 45 mL of 0.1 N hydrochloric
acid. Stir at room temperature for 15 min, and pass
throughafilter of 0.45-11m pore size into a 50-mL volu-
metric flask. Wash the filter with five 1-mL portions of
0.1 N hydrochloric acid, collecting the washings in the
flask. Dilute with 0.1 N hydrochloric acid to volume.
Official Monographs / Magnesium 5429
Instrumental conditions
Mode: Vis
Analytical wavelength: About 620 nm
Cell: 1.cm
Blank: 0.1 N hydrochloric acid, Indicator solution, and
water (5:5:15)
Analysis: Transfer 5.0 mL of the Standard solution and
Sample solution to separate 25-mL volumetric flasks, add
5.0 mL of Indicator solution, dilute with water to vol-
ume, and allow to stand for 1 h in diffuse light at ambi-
ent temperature. Determine the absorbance of the solu-
tions against the Blank.
Acceptance criteria: 10 ug/g; the absorbance of the
Sample solution is NMT than that of the Standard
solution.
© SOLUBLE SALTS
Sample: 10.0g
Analysis: Boil the Sample with 150 mL of water for 15
min. Cool to room temperature, and allow the mixture
to stand for 15 min. Filter with the aid of suction, trans-
fer the filtrate to a 200-mL volumetric flask, and dilute
with water to volume. Evaporate 50.0 mL of this solu-
tion, representing 2.5 g of the Silicate, in a tared plati-
num dish to dryness. Ignite gently to constant weight.
Retain the remaining diluted solution for the test for
Free Alkali.
Acceptance criteria: 3.0%; NMT 75.0 mg
e FREE ALKALI
Sample: 20 mL of the retained diluted filtrate prepared
in the test for Soluble Salts
Analysis: Add 2 drops of phenolphthalein TS to the
Sample, representing 1 g of Magnesium Silicate.
Acceptance criteria: If a pink color is produced, NMT
2.5 mL of 0.1 N hydrochloric acid is required to dis-
charge it.
Delete the following:
°o HEAVY METALS (231)
Solution A: Hydrochloric acid in water (1 in 100)
Test preparation: Boil 4.0 g of Magnesium Silicate with
a mixture of 50 mL of water and 10 mL of hydrochloric
acid for 20 min, and add water to maintain the volume
during the boiling. Add ammonium hydroxide until the
mixture is only slightly acid to litmus paper. Filter with
the aid of suction, and wash with 15-20 mL of water,
combining the washings with the original filtrate. Add
2 drops of phenolphthalein TS, then addaslight excess
of 6 N ammonium hydroxide. Discharge the pink color
with Solution A, then add 8 mL of Sofution A. Dilute
with water to 100 mL, and use 25 mL of the solution
for the test.
Acceptance criteria: NMT 20 2g/Qe ‘oiicial 14an-2018)
e LEAD (251)
Test preparation: Dissolve 1.0 g of Magnesium Silicate
in 20 mL of 3 N hydrochloric acid, evaporate on a
steers bath to 10 mL, dilute with water to 20 mL, and
cool.
Acceptance criteria: NMT 10 ug/g
SPECIFIC TESTS
e RATIO OF SIO, TO McO
Analysis: Divide the percentage of SiOz obtained in the
Assay for Silicon Dioxide by the percentage of MgO ob-
sydeibouow: 4N
tained in the Assay for Magnesium Oxide.
Acceptance criteria: 2.50-4,50
e Loss ON DRYING (731)
[NoTe—Retain the dried specimen for the test for Loss on
Ignition.]
Analysis: Dry at 105° for 2 h.
Acceptance criteria: NMT 15.0%
e Loss ON IGNITION (733)
Sample: The specimen retained from the test for Loss
on Drying
 5430 Magnesium / Official Monographs
Analysis: Ignite the Sample at 900°-1000° for 20 min.
Acceptance criteria: NMT 15%, previously dried
e PH (791)
Sample solution: A well-mixed aqueous suspension (1
in 10)
Acceptance criteria: 7.0-10.8
ADDITIONAL REQUIREMENTS . [Note—For the preparation of all aqueous solutions and
ainers. AND/STOHAGE:: Bresehve.in Well-closed
. aa
Magnesium Stearate
Portions of the monograph text that are national USP text,
and are not part of the harmonized text, are marked with
symbols (%) to specify this fact.
Octadecanoic acid, magnesium salt;
Magnesium stearate [557-04-0].
DEFINITION
Magnesium Stearate is a compound of magnesium with a
mixture of solid ergatie acids, and consists chiefly of vari-
able proportions of magnesium stearate and magnesium
palmitate. The fatty acids are derived from edible sources.
It contains NLT 4.0% and NMT 5.0% of magnesium
(Mg), calculated on the dried basis.
IDENTIFICATION
e A. IDENTIFICATION TESTS—GENERAL (191), Magnesium
Sample solution: Mix 5.0 g with 50 mL of peroxide-
free ether, 20 mL of diluted nitric acid, and 20 mL of
water in a round-bottom flask. Connect the flask to a
reflux condenser, and reflux until dissolution is com-
plete. Allow to cool, and transfer the contents of the
flask to a separator. Shake, allow the layers to separate,
and transfer the aqueous layer toa flask. Extract the
ether layer with two 4-mLpotions of water, and add
these aqueous extracts to the main aqueous extract.
Wash the aqueous extract with 15 mL of peroxide-free
ether, transfer the aqueous extract to a 50-mL volumet-
ric flask, and dilute with water to volume. Retain the
unused portion of this solution for the chloride and sul-
fate impurity tests.
Acceptance criteria: The Sample solution meets the
requirements.
e B. The retention times of the peaks corresponding to ste-
aric acid and palmitic acid of the Sample solution corre-
spond to those of the System suitability solution, as ob-
tained in the test for Relative Content of Stearic Acid and
Palmitic Acid.
ASSAY
e PROCEDURE
Buffer: Dissolve 5.4 g of ammonium chloride in water,
add 20 mL of ammonium hydroxide, and dilute with
water to 100 mL.
Sample: 500mg
Analysis: To the Sample add 50 mL of a mixture of bu-
tyl alcohol and dehydrated alcohol (1:1), 5 mL of am-
monium hydroxide, 3 mL of Buffer, 30.0 mL of 0.1 M
edetate disodium VS, and 1 or 2 drops of eriochrome
black TS. Heat at 45°-50° until the solution is clear.
NF Monographs
Cool, and titrate the excess edetate disodium with 0.1
M zinc sulfate VS until the solution color changes from
blue to violet (see Titrimetry (541)). Perform a blank de-
termination, and make any necessary correction. Each
mL of 0.1 M edetate disodium is equivalent to
2.431 mg of magnesium (Mg).
Acceptance criteria: 4.0%-5.0% on the dried basis
IMPURITIES fee . Ramp time
¢ CHLORIDE AND SULFATE (221), Chloride: A 10.0-mL portion
of the Sample solution prepared in Identification test A
NF 36
shows no more chloride than corresponds to 1.4 mL of
0.020 N hydrochloric acid (0.1%).
e CHLORIDE AND SULFATE (221), Sulfate: A 6.0-mL portion of
the Sample solution prepared in Identification test A shows
no more sulfate than corresponds to 3.0 mL of 0.020 M
sulfuric acid (1.0%).
e Limit oF CADMIUM
for the rinsing of glassware before use, use water that
has been passed through a strong-acid, strong-base,
mixed-bed ion-exchange resin. Select all reagents to
have as low a content of cadmium, lead, and nickel as
practicable, and store all reagent solutions in containers
of borosilicate glass. Cleanse glassware before use by
soaking in warm 8N nitric acid for 30 min, and rinse
with deionized water.]
Matrix modifier solution: Prepare a solution containing
200 mg/mL of monobasic ammonium phosphate and
10 mg/mL of magnesium nitrate. Alternatively, use an
appropriate matrix modifier as recommended by the
manufacturer of the graphite furnace atomic absorption
(GFAA) spectrophotometer.
Blank: Nitric acid in water (1 in 4)
Standard solution: 0.00825 wg/mL of cadmium nitrate
tetrahydrate in Blank, corresponding to a known con-
centration of 0.0030 g/mL of cadmium
Sample stock solution: Transfer 0.100 g of Magnesium
Stearate to a suitable pe eee (PTFE)-
lined acid-digestion bomb, and add 2.5 mL of nitric
acid. Close and seal the bomb according to the manu-
facturer’s operating instructions. [CAUTION—When using
an acid-digestion bomb, be thoroughly familiar with
thesafety and operating instructions. Carefully follow
the bomb manufacturer's instructions regarding care
and maintenance of these se euicestor bombs. Do
not use metal-jacketed bombs or liners that have been
used with hydrochloric acid because of contamination
from corrosion of the metal jacket by hydrochloric
acid.] Heat the bomb in an oven at 170° for 3 h. Air
cool the bomb slowly to room temperature as per the
bomb manufacturer’s instructions. Place the bomb in a
hood, and open carefully because corrosive gases may
be expelled. Dilute the residue with water to 10.0 mL in
a volumetric flask.
Sample solutions: Dilute the Sample stock solution with
Blank (1 in 10). Prepare mixtures of this solution, the
Standard solution, and the Blank with the following pro-
portional compositions, by volume (mL): 1.0/0/1.0, 1.0/
0.5/0.5, and 1.0/1.0/0. Add 50 ul of Matrix modifier so-
lution to each mixture. These Sample solutions contain,
respectively, 0, 0.00075, and 0.0015 g/mL of cad-
mium from the Standard solution. [NoTE—Retain the re-
maining Sample stock solution for use in the tests for
Limit of Lead and Limit of Nickel.]
Instrumental conditions
(See Atomic Absorption Spectroscopy (852).)
Mode: Atomic absorption spectrophotometry (using a
suitable GFAA spectrophotometer equipped with a py-
rolytic tube with platform)
Analytical wavelength: Cadmium emission line at
228.8 nm
Lamp: Cadmium hollow-cathode
Temperature: Use the temperature programming rec-
ommended for cadmium by the GFAA manufacturer
(for Sane of temperature parameters for GFAA
analysis of cadmium, see Table 1).
Table 1
= 7
Drying Ashing Atomization
Stage Stage Stage
Temperature 110° 600° 1800°
10s 10s Os
Hold ‘tine 20 5 30 s 55
 4494 Calcium / Dietary Supplements USP 41

Sane solution: [NoTt—Finely powder NLT 20 Calcium Glubionate Syrup—see Calcium

Tablets.]
Transfer an equivalent to 5 Tablets from powdered Tab- Glubionate Syrup General Monographs
lets to a porcelain crucible. Heat the crucible in a muf-
fle furnace maintained at 550° for 6-12 h, and cool.
Add 60 mL of hydrochloric acid, and boil gently on a
hot plate or steam bath for 30 min, intermittently rins- Calcium Gluceptate—see Calcium
ing the inner surface of the crucible with 6 N hydro- Gluceptate General Monographs
chloric acid. Cool, and quantitatively transfer the con-
tents of the crucible to a 100-mL volumetric flask.
Rinse the crucible with small portions of 6 N hydro-
chloric acid, and add the rinsings to the flask. Dilute Calcium Gluconate—see Calcium
with water to volume, and filter, discarding the first
5 mL of the filtrate. Dilute this solution quantitatively, Gluconate General Monographs
with 0.125 N hydrochloric acid to obtain a concentra-
tion of 2 g/mL of calcium, adding 1 mL of Lanthanum
chloride solution per 100 mL of the final volume.
Instrumental conditions Calcium Gluconate Tablets—see Calcium
(See Atomic Absorption Spectroscopy (852).)
Mode: Atomic a sae spectrophotometry
Gluconate Tablets General Monographs
Analytical wavelength: Calcium emission line at
422.7 nm
Lamp: Calcium hollow-cathode Calcium Glycerophosphate
Flame: Nitrous oxide-acetylene
Blank: 0.125 N hydrochloric acid containing 1 mL of
Lanthanum chloride solution per 100 mL cat
Analysis
Samples: Standard solutions and the Sample solution
Determine the absorbances of the solutions, using the
Blank. From a linear regression equation, calculated C3H7CaOcP 210.14
using the absorbance of the Standard solutions versus 1,2,3-Propanetriol, mono(dihydrogen phosphate) calcium
concentrations, determine the concentration, C, in salt (1:1);
Lig/mL of calcium in the Sample solution. Calcium glycerophosphate [27214-00-2].
Calculate the percentage of the labeled amount of cal-
cium (Ca) in the portion of Tablets taken: DEFINITION
Calcium Glycerophosphate is a mixture, in variable propor-
Result = (C/Cy) x 100 tions, of calcium (RS)-2,3-dihydroxypropyl phosphate and
calcium 2-h crony tycrog mene phosphate,
Cc = determined concentration of calcium in the which may be hydrated. Calcium Glycerophosphate con-
Sample solution tains NLT 18.6% and NMT 19.4% of calcium (Ca), calcu-
Cy = nominal concentration of calcium in the lated on the dried basis.
Sample solution
Acceptance criteria: 90.0%-110.0% IDENTIFICATION
eA.
CONTAMINANTS Analysis: Ignite 0.1 g in a crucible. Take up the residue
e MICROBIAL ENUMERATION TESTS (2021): The total aerobic with 5 mL of nitric acid, heat on a water bath for 1
microbial count does not exceed 1000 cfu/g. The total min, and filter. Mix 1 mL of the filtrate with 2 mL of
ayulnee yeasts and molds count does not exceed 100 ammonium molybdate TS.
cfu/g. Acceptance criteria: A yellow color develops.
e ABSENCE OF SPECIFIED MICROORGANISMS (2022): Meet the eB.
requirements of the test for absence of Escherichia coli Analysis: Dissolve 20 mg of the substance being ex-
DS Monographs

amined in 5 mL of 5 M acetic acid, and add 0.5 mL of

PERFORMANCE TESTS
e DISINTEGRATION AND DISSOLUTION OF DIETARY SUPPLEMENTS
(2040): Meet the requirements for Disintegration, 15
min
o WEIGHT VARIATION OF DIETARY SUPPLEMENTS (2091):
the requirements
potassium ferrocyanide solution (53 mg/mL). The result-
ing solution remains clear. To the clear solution, add
50 mg of ammonium chloride.
Meet Acceptance criteria: A white crystalline precipitate is
produced.

ADDITIONAL REQUIREMENTS ASSAY

© PACKAGING AND STORAGE:
containers.
e LABELING: The label states the quantity of calcium in
terms of mg/Tablet.
© PROCEDURE
Preserve in well-closed
Sample: 200mg
Titrimetric system
(See Titrimetry (541).)
Mode: Direct titration
Titrant: 0.1 M edetate disodium VS
Endpoint detection: Colorimetric
Blank: 300 mL of water. Add 6 mL of 10M sodium
hydroxide and 15 mg of calconcarboxylic acid
triturate.
Analysis: Dissolve the Sample in 300 mL of water, add
6 mL of 10M sodium hydroxide and 15 mg of calcon-
carboxylic acid triturate. Titrate with Titrant until the so-
lution is a distinct blue color.
NF 36
Analysis: Use the Blank to set the instrument to zero.
Plot the absorbances of the Sample solutions versus their
contents of cadmium, in tg/mL, as furnished by the
Standard solution, draw the straight line best fitting the
three points, usinga linear least-squares fit, and extra-
polate the line until it intercepts the concentration axis
on the negative side. From the intercept determine the
concentration, C, in g/mL, of cadmium in the Sample
solution containing 0 mL of the Standard solution.
Calculate the content, in ppm, of cadmium in the spec-
imen taken:
Result = (C/W) x F
e Limit OF NICKEL
Ww = weight of Magnesium Stearate taken to
prepare the Sample stock solution (g)
F = dilution factor for the sample, 200
Alternatively, the GFAA software can be used to
calculate the cadmium content of the sample. For
either calculation, the correlation coefficient (r) of the
standard additions plot must be at least 0.99.
Acceptance criteria: NMT 3 ppm
e Limit OF LEAD
[Note—For the preparation of all aqueous solutions and
for the rinsing of glassware before use, use water that
has been passed through a strong-acid, strong-base,
mixed-bed ion-exchange resin. Select all reagents to
have as low a content of cadmium, lead, and nickel as
practicable, and store all reagent solutions in containers
of borosilicate glass. Cleanse glassware before use by
soaking in warm 8 N nitric acid for 30 min, and rinse
with deionized water.]
Matrix modifier solution: Prepare as directed for Ma-
trix modifier solution in Limit of Cadmium.
Blank: Prepare as directed for Blank in Limit of
Cadmium.
Standard solution: 0.1598 g/mL of lead nitrate in
Blank, corresponding to a known concentration of
0.100 g/mL of lead. Prepare and store any solutions of
ieee nitrate in glass containers free from soluble lead
salts.
Sample stock solution: Use a portion of the Sample
stock solution retained from the test for Limit of
Cadmium.
Sample solutions: Prepare mixtures of the Sample stock
solution, the Standard solution, and the Blank with the
following proportional compositions, by volume (mL):
1.0/0/1.0, 1.0/0.5/0.5, and 1.0/1.0/0. Add 50 uL of the
Matrix modifier solution to each mixture. These Sample
solutions contain, respectively, 0, 0.025, and 0.05 y1g/
mL of lead from the Standard solution.
Instrumental conditions
(See Atomic Absorption Spectroscopy (852).)
Mode: Atomic absorption spectrophotometry (using a
suitable GFAA spectrophotometer equipped with a py-
rolytic tube with platform)
Analytical wavelength: Lead emission line at 283.3
nm
Lamp: Lead hollow-cathode
Temperature: Use the temperature pega rec-
ommended for lead by the GFAA manufacturer (for
examples of temperature parameters for GFAA analysis
of lead, see Table 1).
Analysis: Use the Blank to set the instrument to zero.
Plot the absorbances of the Sample solutions versus their
contents of lead, in g/mL, as furnished by the Stan-
dard solution, draw the straight line best fitting the
three points, using a linear least-squares fit, and extra-
polate the line until it intercepts the concentration axis
on the negative side. From the intercept determine the
concentration, C, in jug/mL, of lead in the Sample solu-
tion containing 0 mL of the Standard solution.
Official Monographs / Magnesium 5431
Calculate the content, in ppm, of lead in the specimen
taken:
Result = (C/W) x F
w = weight of Magnesium Stearate taken to
repare the Sample stock solution (g)
F = dilution factor for the sample, 20
Alternatively, the GFAA software can be used to calculate
the lead content of the sample. For either calculation,
the correlation coefficient (r) of the standard additions
plot must be at least 0.99.
Acceptance criteria: NMT 10 ppm
[Note—For the preparation of all aqueous solutions and
for the rinsing of glassware before use, use water that
has been passed through a strong-acid, strong-base,
mixed-bed ion-exchange resin. Select all reagents to
have as low a content of cadmium, lead, and nickel as
practicable, and store all reagent solutions in containers
of borosilicate glass. Cleanse glassware before use by
soaking in warm 8N nitric acid for 30 min, and rinse
with deionized water.]
Matrix modifier solution: Prepare as directed for Ma-
trix modifier solution in Limit of Cadmium.
Blank: Prepare as directed for Blank in Limit of
Cadmium.
Standard solution: 0.2477 ug/mL of nickel nitrate hex-
ahydrate in Blank, corresponding to a known concentra-
tion of 0.050 g/mL of nickel
Sample stock solution: Use a portion of the Sample
stock solution retained from the test for Limit of
Cadmium.
Sample solutions: Prepare mixtures of the Sample stock
solution, the Standard solution, and the Blank with the
following proportional compositions, by volume (mL):
1.0/0/1.0, 1.0/0.5/0.5, and 1.0/1.0/0. Add 50 uL of the
Matrix modifier solution to each mixture. These Sample
solutions contain, respectively, 0, 0.0125, and 0.025 y1g/
mL of nickel from the Standard solution.
Instrumental conditions
(See Atomic Absorption Spectroscopy (852).)
Mode: Atomic absorption spectrophotometry (using a
suitable GFAA spectrophotometer equipped with a py-
rolytic tube with platform)
Analytical wavelength: Nickel emission line at 232.0
nm
Lamp: Nickel hollow-cathode
Temperature: Use the temperature programming rec-
ommended for nickel by the GFAA manufacturer (for
examples of temperature parameters for GFAA analysis
of nickel, see Table 7).
Analysis: Use the Blank to set the instrument to zero.
Plot the absorbances of the Sample solutions versus their
contents of nickel, in ug/mL, as furnished by the Stan-
dard solution, draw the straight line best fitting the
three points, using a linear least-squares fit, and extra-
polate the line until it intercepts the concentration axis
on the negative side. From the intercept determine the
concentration, C, in ug/mL, of nickel in the Sample solu-
tion containing 0 mL of the Standard solution.
Calculate the content, in ppm, of nickel in the speci-
men taken:
Result = (C/W) x F 74
n
Ww = weight of Magnesium Stearate taken to x
prepare the Sample stock solution (g) fo)
F = dilution factor for the sample, 20 =]
Alternatively, the GFAA software can be used to °
Ko}
calculate the nickel content of the sample. For either =.
cy
calculation, the correlation coefficient (r) of the mo}
standard additions plot must be at least 0.99. s
7)
 5432 Magnesium / Official Monographs NF 36

Acceptance criteria: NMT 5 ppm Carrier gas: Helium

SPECIFIC TESTS
e *MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI-
FIED MICROORGANISMS (62): The total aerobic microbial
count does not exceed 103 cfu/g, the total combined
molds and yeasts count does not exceed 5 x 102 cfu/g. It
meets the requirements of the tests for absence of Salmo-
nella species and Escherichia coli.e
© ACIDITY OR ALKALINITY
Sample solution: To 1.0 g add 20 mL of carbon diox-
ide-free water, boil on a steam bath for 1 min with
continuous shaking, cool, and filter. Add 0.05 mL of
bromothymol blue TS to 10 mL of the filtrate.
Acceptance criteria: NMT 0.05 mL of 0.1 N hydrochlo-
ric acid or 0.1 N sodium hydroxide is required to
change the color of the indicator.
© *SPECIFIC SURFACE AREA (846)
[Note—In cases where there are no functionality-related
concerns regarding the specific surface area of this arti-
cle, this test may be omitted.]
Where the labeling states the specific surface area, deter-
mine the specific surface area value as directed in the
chapter in the P/Po range of 0.05-0.15, and using out-
gan conditions of 2 h at 40°. If the plot deviates
rom linearity for P/Po values of 0.05-0.15, then a suita-
ble range of P/Py values should be validated for linear-
ity. In this case, it is necessary to state the range of
validated P/Po values, the increment of the P/Po values,
and the outgassing conditions used.»
¢ Loss ON DRYING (731)
Analysis: Dry at 105° to constant weight.
Acceptance criteria: NMT 6.0%
© RELATIVE CONTENT OF STEARIC ACID AND PALMITIC ACID
System suitability solution: Transfer 50 mg each of
USP Stearic Acid RS and USP Palmitic Acid RS to a small
conical flask fitted with a suitable reflux condenser. Add
5.0 mL of a solution prepared by dissolving 14 g of bo-
ron trifluoride in methanol to make 100 mL, swirl to
mix, and reflux for 10 min until the solids have dis-
solved. Add 4 mL of chromatographic n-heptane
through the condenser, and reflux for 10 min. Cool,
add 20 mL of saturated sodium chloride solution, shake, ADDITIONAL REQUIREMENTS
and allow the layers to separate. Pass the n-heptane © PACKAGING AND STORAGE: Preserve in tight containers.
layer through 0.1 g of anhydrous sodium sulfate (previ- e *LABELING: Where the labeling states the specific surface
ously washed with chromatographic n-heptane) into a
suitable flask. Transfer 1.0 mL of this solution to a
10-mL volumetric flask, and dilute with chromato-
e USP REFERENCE STANDARDS (11)
graphic n-heptane to volume.
Sample solution: Transfer 100 mg of Magnesium Stea-
rate to a small conical flask fitted with a suitable reflux
condenser, and proceed as directed for System suitability
solution, beginning with “Add 5.0 mL of a solution pre-
pared by dissolving”.
Chromatographic system
(See Chromatography (621), System Suitability.) Maleic Acid
Mode: GC
Detector: Flame ionization
Flow rate: 2.4 mL/min
Injection volume: 1 wL
Injection type: Splitless injection system
System suitability
Sample: System suitability solution
[Note—The relative retention times for methyl palmitate
and methyl stearate are about 0.9 and 1.0,
respectively.]
Suitability requirements
Resolution: NLT 5.0 between methy! palmitate and
methyl stearate
Relative standard deviation: NMT 3.0% for the pal-
mitate and stearate peak areas from six replicate in-
jections; NMT 1.0% for the peak area ratio of palmi-
tate to stearate from six replicate injections
Analysis
Sample: Sample solution
Measure the peak areas for all the fatty acid esters in
the chromatogram.
Calculate the percentage of stearic acid in the fatty acid
fraction of the portion of Magnesium Stearate taken:
Result = (ru/rr) x 100
tu = peak area of methyl stearate from the Sample
solution
ia = sum of the peak areas of all the fatty acid
esters from the Sample solution
Similarly, calculate the percentage of palmitic acid in
the portion of Magnesium Stearate taken.
Result = (re/r;) x 100
Ip = peak area of methyl palmitate from the
Sample solution
Ir = sum of the peak areas of all the fatty acid
esters from the Sample solution
Acceptance criteria: NLT 40% for the stearate peak.
The sum of the stearate and palmitate peaks is NLT
90% of the total peak areas of all the fatty acids.
area, it also indicates which method specified in Specific
Surface Area (846) is used. Label to indicate that the fatty
acids are derived from edible sources.
USP Palmitic Acid RS
USP Stearic Acid RS
=o

Column; 0.32-mm x 30-m fused silica capillary, ( ‘OH

bonded with a 0.5-um layer of phase G16 en
Temperatures
oy

Injector: 220°
Detector: 260°
Column: See Table 2. C4H4Ox 116.07
“
<= (Z)-Butenedioic acid;
a Table 2 cis-Butenedioic acid [110-16-7].
S
i]
a) Hold Time DEFINITION
ro} Initial Temperature Final at Final Maleic Acid contains NLT 99.0% and NMT 101.0% of
r=
Sj Temperature Ramp Temperature | Temperature C4H4Oq, calculated on the anhydrous basis.
= ©)
70
(¢/min)
=
(°)
70
(min)
2 IDENTIFICATION
—
e A, PROCEDURE
re 70 5 240 5
Analysis: Dissolve 500 mg of Maleic Acid in 10 mL of
water.
NF 36
Acceptance criteria: The pH of the solution is less than
2 solution shows a light brown color. Dilute each of the
e B. The principal spot from Sample solution B corresponds
in color, size, and R; value to that from Standard solution
A, as obtained in the procedure for Limit of Fumaric Acid.
e C. INFRARED ABSORPTION (197K)
ASSAY
© PROCEDURE
Sample solution: 10 mg/mL of Maleic Acid
Analysis: Titrate the Sample solution with 1 N sodium
hydroxide VS, using phenolphthalein TS as the indica-
tor. Each mL of 1 N sodium hydroxide is equivalent to
58.04 mg of C4H4Ou.
Hccep tance: criteria: 99.0%-101.0% on the anhydrous
asis
IMPURITIES
Inorganic Impurities
© RESIDUE ON IGNITION (281): NMT 0.1%, determined on a
1.0-g portion
© LIMIT OF IRON
Solution A: Dissolve 9.7 g of potassium thiocyanate in
100 mL of water.
Diluted standard iron solution: Immediately before
use, dilute 1 volume of Standard Iron Solution, prepared
as directed in Iron (241), with 9 volumes of water.
[Note—This solution contains the equivalent of 1 tg/
mL of iron.]
Standard solution: Add 6 mL of water to 5 mL of Di-
luted standard iron solution. Add 1 mL of diluted hydro-
chloric acid and 0.05 mL of bromine TS. After 5 min,
remove the excess of bromine with the aid of a current
of air, add 3 mL of Solution A, and shake well.
Sample solution: Dissolve 1 g of Maleic Acid in 10 mL
of water. Add 2 mL of diated: hydrochloric acid and
0.05 mL of bromine TS. After 5 min, remove the excess
of bromine with the aid of a current of air, add 3 mL of
Solution A, and shake well.
Analysis: Allow the Standard solution and Sample solu-
tion to stand for 5 min.
Acceptance criteria: Any red color in the Sample solu-
tion is not more intense than that in the Standard solu-
tion (NMT 5 ppm).
Delete the following:
®e HEAVY MeTALs, Method I/ (231)
Test preparation: Transfer 1.0 g of Maleic Acid to a
quartz crucible, and add 0.5 g of magnesium oxide. Ig-
nite the crucible to dull redness until a homogeneous
white or grayish-white mass is obtained. Ignite at 800°
for 1 h, cool, and dissolve the residue by adding two
5-mL portions of diluted hydrochloric acid. Add 0.1 mL
of phenolphthalein TS, then add ammonium hydroxide
until a pink color is obtained. Cool, add glacial acetic
acid until the solution is decolorized, then add 0.5 mL
of glacial acetic acid in excess, and dilute with water to
20.0 mL.
Standard solution: To 0.5 9 of magnesium oxide add
1,0 mL of Standard Lead Solution, and evaporate to dry-
ness at 105° for 1 h. Following the procedure described
for preparation of the Test preparation, ignite, dissolve
in diluted hydrochloric acid, add ammonia and then
acetic acid, and dilute with water to 20.0 mL.
Blank: Water and Test preparation (10:2)
Analysis: To 12 mL of the Test preparation add 2.0 mL
of pH 3.5 Acetate Buffer, mix, add to 1.2 mL of
thioacetamide-glycerin base TS, and mix immediately.
To 10 mL. of the Standard solution add 2.0 mL of the
Test preparation and 2.0 mL of pH 3.5 Acetate Buffer,
mix, add 1.2 mL of thioacetamide-glycerin base TS,
and mix immediately.
Official Monographs / Maleic 5433
Compared to the Blank, the solution from the Standard
solutions from the Test preparation and Standard solu-
tion with water to 50 mL. Allow to stand for 2 min,
and view downward over a white surface.
Acceptance criteria: The color of the solution from the
Test preparation is not darker than that of the solution
from the Standard solution (NMT 10 ppm)... cotticiat 1-jan-
2018)
Organic Impurities
¢ PROCEDURE: LIMIT OF FUMARIC ACID
Adsorbent: 0.25-mm layer of chromatographic silica
gel mixture
Standard solution A: 2 mg/mL of USP Maleic Acid RS
in acetone
Standard solution B: 1.5 mg/mL of USP Fumaric Acid
RS in acetone
System suitability solution: Equal volumes of Standard
solution A and Standard solution B
Sample solution A: 100 mg/mL of Maleic Acid in
acetone
Sample solution B: 2 mg/mL of Maleic Acid in acetone
from Sample solution A
Developing solvent system: Heptane, butanol, chloro-
form, and anhydrous formic acid (44:36:16:16)
Appleton volume: 10 lL for the System suitability so-
lution and 5 wl each for Standard solution A, Standard
solution B, Sample solution A, and Sample solution B
Analysis
Samples: Standard solution A, Standard solution B, Sys-
tem suitability solution, Sample solution A, and Sample
solution B
Proceed as directed in Chromatography (621), Thin-
Layer Chromatography, using an unsaturated cham-
ber. Dry the plate at 100° for 15 min, and examine
the plate under short-wavelength UV light at 254
nm.
Acceptance criteria: The chromatogram from the Sys-
tem suitability solution exhibits two clearly separated
Spats, and any spot corresponding to fumaric acid in
the chromatogram from Sample solution A does not ex-
ceed, in size or intensity, the principal spot in the chro-
matogram from Standard solution B (NMT 1.5% of fu-
maric acid).
SPECIFIC TESTS
e COLOR AND CLARITY OF SOLUTION
Dilute hydrochloric acid solution: Dilute 27.5 mL of
hydrochloric acid with water to 1000 mL.
Reference solution: Mix 2.4 mL of ferric chloride CS
and 0.6 mL of cobaltous chloride CS with Dilute hydro-
chloric acid solution to make 10 mL. Dilute 5 mL of this
solution with Dilute hydrochloric acid solution to make
100 mL.
Sample solution: 100 mg/mL of Maleic Acid
Analysis: Place the Reference solution and the Sample
solution in matched color-comparison tubes, and com-
pare the solutions by viewing them downward against
a white surface (see Color and Achromicity (631)).
Acceptance criteria: The Sample solution is clear and
not more intensely colored than the Reference solution.
© WATER DETERMINATION, Method | (921): NMT 2.0%
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in tight glass contain-
sydesBouo-: 4N
ers, protected from light. Store at room temperature.
e USP REFERENCE STANDARDS (11)
USP Fumaric Acid RS
USP Maleic Acid RS
 5434 Malic / Official Monographs NF 36

Suitability requirements
Malic Acid Resolution: NLT 2.5 for the maleic acid and malic
acid peaks; NLT 7.0 for the malic acid and fumaric
acid peaks
Relative standard deviation: NMT 2.0% for the ma-
leic acid peak
Analysis
Samples: Standard solution and Sample solution
C4HeOs 134.09 Calculate the percentage of maleic acid and of fumaric
Hydroxybutanedioic acid, (+)-; acid in the portion of Malic Acid taken:
(£)-Malic acid;
(+)-Hydroxysuccinic acid [617-48-1]. Result = (ru/rs) x (Cs/Cu) x 100
DEFINITION tu = peak response of maleic acid or fumaric acid
Malic Acid contains NLT 99.0% and NMT 100.5% of from the Sample solution
C4HeOs. Is = peak response of maleic acid or fumaric acid
from the Standard solution
IDENTIFICATION Gs = concentration of USP Maleic Acid RS or USP
e A. INFRARED ABSORPTION (197K): On undried sample Fumaric Acid RS in the Standard solution
(mg/mL)
ASSAY Cu = concentration of Malic Acid in the Sample
¢ PROCEDURE solution (mg/mL)
Sample: 2g Acceptance criteria
Titrimetric system Fumaric acid: NMT 1.0%
(See Titrimetry (541).) Maleic acid: NMT 0.05%
Mode: Direct titration © WATER-INSOLUBLE SUBSTANCES
Titrant: 1.N sodium hydroxide VS Sample: 25g
Endpoint detection: Visual Analysis: Dissolve the Sample in 100 mL of water, filter
Analysis: Dissolve the Sample in 40 mL of recently the solution through a tared filtering crucible, wash the
boiled and cooled water. Add phenolphthalein TS, and filter with hot water, and dry at 100° to constant
titrate to the first appearance of a faint pink color that weight.
persists for NLT 30 s. Perform a blank determination. Acceptance criteria: The increase in weight is NMT
Calculate the percentage of malic acid (C4HeOs) in the 25 mg (0.1%).
Sample taken:
ADDITIONAL REQUIREMENTS
Result
= [(V- B) x Nx Fx 100]/W © PACKAGING AND STORAGE: Preserve in well-closed
containers.
Vv = volume of Titrant consumed by the Sample e USP REFERENCE STANDARDS (11)
(mL) USP Fumaric Acid RS
B = volume of Titrant consumed by the Blank (mL) USP Maleic Acid RS
N = actual normality of the Titrant (mEq/mL) USP Malic Acid RS
F = equivalency factor, 67.04 mg/mEq
w = weight of the Sample (mg)
Acceptance criteria: 99.0%-100.5%
IMPURITIES
e RESIDUE ON IGNITION (281): NMT 0.1% Maltitol

Delete the following: pH HO

Se Non
HOM

°e HEAVY METALS, Method iI (231): NMT 20 ppme cotticial1- Ho {Some

Jan-2018) i ‘OH
e FUMARIC AND MALEIc ACIDS wh on
Mobile phase: 0.01 N sulfuric acid ‘oH
System suitability solution: 1 mg/mL of Malic Acid,
10 pg/mL of USP Fumaric Acid RS, and 4 ug/mL of USP Ci2H24011 344.31
Maleic Acid RS in Mobile phase D-Glucopyranosyl-D-glucitol [585-88-6].
Standard solution: 5.0 g/mL of USP Fumaric Acid RS
and 2.0 g/mL of USP Maleic Acid RS in Mobile phase DEFINITION
Sample solution: 1.0 mg/mL of Malic Acid in Mobile Maltitol contains NLT 92.0% and NMT 100.5% of D-maltitol
phase (Ci2H24011), calculated on the anhydrous basis. The
Chromatographic system amounts of total sugars, other polyhydric alcohols, and
(See Chromatography (621), System Suitability.) any polyol anhydrides, if detected, are not included in the
Mode: LC requirements or in the calculated amount in General No-
Detector: UV 210 nm
NF Monographs

tices and Requirements, 5.60.10 Other Impurities in USP and

Column: 6.5-mm x 30-cm; packing L17 NF Articles.
Column temperature: 37+1°
Flow rate: 0.6 mL/min IDENTIFICATION
Injection size: 20 uL e A. INFRARED ABSORPTION (197K)
System suitability e B. The retention time of the major peak of the Sample
Sample: System suitability solution solution corresponds to that of the Standard solution, as
[NoTe—The relative retention times for maleic acid, ma- obtained in the Assay.
lic acid, and fumaric acid are 0.6, 1.0, and 1.5,
respectively.]
NF 36 Official Monographs / Maltitol 5435

ASSAY Set the instrument to zero using the organic layer

e PROCEDURE from the Blank solution. Concomitantly determine the
Mobile phase: Water. [NoTE—Degas the Mobile phase absorbances of the organic layer from the Samples at
before use.] least three times each. Record the average of the
System suitability solution: 4.8 mg/g of USP Maltitol steady readings for each of the Standard solutions and
RS and 4.8 mg/g of sorbitol the Sample solution. Between each measurement, as-
Standard solution: 10 mg/g of USP Maltitol RS and pirate the organic layer from the Blank solution, and
1.6 mg/g of sorbitol ascertain that the reading returns to zero. Plot the
Sample solution: Dissolve 0.20 g of Maltitol in water, absorbances of the Standard solutions and the Sample
and dilute with water to 20 g. Record the final solution solution versus the added quantity of nickel. Extrapo-
een: and mix thoroughly. The solution is 10 mg/g of late the line joining the points on the graph until it
altitol. meets the concentration axis. The distance between
Chromatographic system this point and the intersection of the axes represents
(See Chromatography (621), System Suitability.) the concentration of nickel in the Sample solution.
Mode: LC Acceptance criteria: NMT 1 ug/g
Detector: Refractive index e REDUCING SUGARS
Column: 7.8-mm x 10-cm; packing L34 Sample: 3.3g
Temperatures Titrimetric system
Column; 60+ 2° Mode: Residual titration
Detector: 35° Titrant: 0.05 N sodium thiosulfate VS
Flow rate: 0.5 mL/min Endpoint detection: Visual
Injection volume: 10 pL Analysis: Dissolve the Sample in 3 mL of water with the
System suitability aid of gentle heat. Cool, and add 20.0 mL of cupric
Samples: System suitability solution and Standard citrate TS and a few glass beads. Heat so that boiling
solution begins after 4 min, and maintain boiling for 3 min.
[Note—The relative retention times for maltitol and sor- Cool rapidly, and add 40 mL of diluted acetic acid,
bitol are 0.48 and 1.0, respectively] 60 mL of water, and 20.0 mL of 0.05 N iodine VS. With
Suitability requirements continuous shaking, add 25 mL of hydrochloric acid in
Resolution: NLT 2.0 between maltitol and sorbitol, water solution (6:94). When the precipitate has dis-
System suitability solution solved, titrate the excess of iodine with Titrant using
Relative standard deviation: NMT 2.0%, Standard 2 mL of starch TS, added towards the end of the titra-
solution tion, as an indicator.
Analysis Acceptance criteria: NLT 12.8 mL of Titrant is required,
Samples: Standard solution and Sample solution corresponding to NMT 0.3% of reducing sugars, as glu-
Calculate the percentage of D-maltito! (Ci2H24011) in cose. [NoTE—The amount determined in this test is not
the portion of Maltitol taken: included in the calculated amount in General Notices
and Requirements, 5.60.10 Other Impurities in USP and
Result = (ru/rs) x (Cs/Cu) x [100/(100 — W)] x 100 NF Articles.]
tu = peak response from the Sample solution SPECIFIC TESTS
rs = peak response from the Standard solution e MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI-
Gs = concentration of USP Maltitol RS in the FIED MICROORGANISMS (62): The total aerobic microbial
Standard solution (mg/g) count using the Plate Method does not exceed 103 cfu/g,
G = concentration ofMaltivor in the Sample and the total combined molds and yeasts count does not
solution (mg/g) exceed 10? cfu/g.
Ww = percentage obtained in the test for Water e CONDUCTIVITY
Determination Sample solution: 200 mg/mL
Acceptance criteria: 92.0%-100.5% on the anhydrous Analysis: Using an appropriate conductivity meter,
basis choose a conductivity cell that is appropriate for the
properties and conductivity of the solution to be ex-
IMPURITIES amined. Use a certified reference material, for example
e LIMIT OF NICKEL a solution of potassium chloride, that is appropriate for
Sample solution: Dissolve 20.0 g of Maltitol in diluted the measurement."
acetic acid, and dilute with diluted acetic acid to The conductivity value of the certified reference mate-
150 mL. tial should be near the expected conductivity value of
Blank solution: 150 mL of diluted acetic acid the solution to be examined. After calibrating the ap-
Standard solutions: Prepare three solutions by adding paratus with a certified reference material solution,
0.5, 1.0, and 1.5 mL of nickel standard solution TS to rinse the conductivity cell several times with water and
20.0 g of Maltitol dissolved in diluted acetic acid, and at least twice with the aqueous solution to be ex-
dilute with the same solvent to 150 mL. amined. Measure the conductivity of the Sample solu-
Instrumental conditions tion at a temperature of 20°, while gently stirring with
(See Atomic Absorption Spectroscopy (852).) a magnetic stirrer.
Mode: Atomic absorption spectrophotometry Acceptance criteria: NMT 20 uS/cm
Analytical wavelength: Maxima at about 232.0 nm e WATER DETERMINATION, Method | (921): NMT 1.0%
Lamp: Nickel hollow-cathode
sydeibouo- 4N

Flame: Air-acetylene ADDITIONAL REQUIREMENTS

Analysis
Samples: Standard solutions and Sample solution
To each sample, add 2.0 mL of a saturated ammonium
pyrrolidinedithiocarbamate solution (containing 10 g/
L of ammonium Eye el eg eecetadtee and
10.0 mL of methyl isobutyl ketone, and shake for 30
s. Protect from bright light. Allow the two layers to
separate, and use the methyl isobutyl ketone layer.
e PACKAGING AND STORAGE: Preserve in well-closed contain-
ers. No storage requirements are specified.
1 Commercially available conductivity calibration solutions for conductivity
meter standardization, standardized by methods traceable to the National
Institute of Standards and Technology (NIST), may be used. Solutions pre-
pared according to the instructionsgiven in ASTM Standard D1125 may be
used, provided the conductivity of tl e resultant solution is the same as that
of the solution prepared from the NIST-certified material.
 5436 Maltitol / Official Monographs NF 36

¢ USP REFERENCE STANDARDS (11) Carrier gas: Helium

USP Maltito] RS Flow rate: 3.0 mL/min
Injection size: 1.0 uL
Injection type: Split injection. The split ratio is about
10:1. [NoOTE—A general purpose split/splitless, taper,
glass wool, and deactivated liner is used.]
Maltitol Solution System suitability
Sample: Standard solution
DEFINITION [Note—See the relative retention time table below. Rel-
Maltitol Solution is a water solution containing, on the an- ative retention times are provided for information only,
hydrous basis, NLT 50.0% of D-maltitol (Ci2H24011) (w/w) and the standards should be used to ensure appropri-
and NMT 8.0% of D-sorbitol (CsH14O¢) (w/w). The ate peak identification.]
amounts of total sugars, other polyhydric alcohols, and
any polyol anhydrides, if detected, are not included in the Relative
requirements nor in the calculated amount under Other Retention
Impurities. Name Time
Ethylene glycol 1.0
IDENTIFICATION
1,3-Butanediol (internal stan-
© A. PROCEDURE
Sample: 1.4 g of Maltitol Solution dard) 2.2
Analysis: Dissolve the Sample in 75 mL of water. Trans- Diethylene glycol 2.8
fer 3 mL of this solution to a 15-cm test tube, add 3 mL
of freshly prepared catechol (1 in 10), and mix. Add Suitability requirements
6 mL of sulfuric acid, and mix. Gently heat the tube in Resolution: NLT 15 between ethylene glycol and 1,3-
a flame for about 30 s. butanediol
Acceptance criteria: A deep pink or wine-red color Analysis
appears. Samples: Standard solution and Sample solution
e B. The retention time of the major peak of the Sample Based on the Standard solution, identify the peaks of
solution corresponds to that of the Standard solution, as ethyleneglycol, 1,3-butanediol (internal standard),
obtained in the Assay. and diethylene glycol. Compare peak area ratios of
© C. LIMIT OF DIETHYLENE GLYCOL AND ETHYLENE GLYCOL ethylene glycol to the internal standard and of dieth-
Diluent: Acetone and water (96:4) ylene glycol to the internal standard in the Standard
Standard stock solution: 0.5 mg/mL of USP Diethylene solution and Sample solution, respectively.
Glycol RS and 0.5 mg/mL of USP Ethylene Glycol RS in Acceptance criteria
Diluent Diethylene glycol: The peak area ratio of diethylene
Internal standard stock solution: 0.5 mg/mL of 1,3- glycol to the internal standard in the Sample solution is
butanediol (internal standard) in Diluent NMT the peak area ratio of diethylene glycol to the
Standard solution: 0.04 mg/mL of USP Diethylene Gly- internal standard in the Standard solution, correspond-
col RS, 0.04 moira of USP Ethylene Glycol RS, and ing to NMT 0.10% of diethylene glycol in Maltitol
0.04 mg/mL of 1,3-butanediol, in Diluent, prepared Solution.
from the Standard stock solution and Internal standard Ethylene glycol: The peak area ratio of ethylene glycol
stock solution to the internal standard in the Sample solution is NMT
Sample solution: Transfer 1.0 g of Maltitol Solution to the peak area ratio of ethylene glycol to the internal
a 25-mL volumetric flask. Add 1.0 mL of water to the standard in the Standard solution, corresponding to
flask, and mix on a vortex mixer for 3 min. Add 2.0 mL NMT 0.10% of ethylene glycol in Maltitol Solution.
of the Internal standard stock solution and 5 mL of Dilu- ASSAY
ent, and mix on a vortex mixer for 3 min. Add the e PROCEDURE
remaining Diluent to the flask to volume in two equal Mobile phase: Water
portions. Mix the contents for about 3 min after each Standard solution: 10 mg/g of USP Maltitol RS and
addition of Diluent. Pass a portion of the supernatant 1.6 mg/g of USP Sorbitol RS
layer through a nylon filter of 0.45-um pore size. Dis- Sample solution: 20 mg/g of Maltitol Solution in water
card the first 2 mL of the filtrate, and collect the rest of Chromatographic system
the filtrate for analysis. [NoTE—Acetone is used to pre- (See Chromatography (621), System Suitability.)
cipitate maltitol.] Mode: LC
Chromatographic system Detector: Refractive index
(See Chromatography (621), System Suitability.) Column: 7.8-mm x 10-cm; packing L34
Mode: GC Temperature
Detector: Flame ionization Column: 60+2°
Column: 0.32-mm x 15-m fused-silica capillary col- Detector: 35°
umn; 0.25-um layer of phase G46 Flow rate: 0.5 mL/min
Temperature Injection size: 10 uL
Detector: 300° System suitability
Injection port: 240° Sample: Standard solution
Column: See temperature program table below. [Note—The relative retention times for maltotriitol, mal-
eed
”

rs
i} Hold Time
titol, and sorbitol are 0.38, 0.48, and 1.0,
respectively]
—
Dp Initial Temperature Final at Final
i} Temperature Ramp Temperature | Temperature
=
3 &) (¢/min) () (min)
= 70
70
=
50
70
300
2
5
Ss
ne
NF 36
Suitability requirements
Tailing factor: NMT 1.2 for maltitol and sorbitol
Relative standard deviation: NMT 2.0%
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage, on the anhydrous basis, of
Cy2H24O11 and CeHi4O¢ in the portion taken:
Result = (ru/ts) x (Cs/Cu) x [100/(100 — W)] x 100
tu = peak response of D-maltito! or D-sorbitol from
the Sample solution
Is = peak response of D-maltitol or D-sorbitol from
the Standard solution
Gs = concentration of the appropriate USP
Reference Standard in the Standard solution
(mg/g) . __.
Cu = concentration of Maltitol Solution in the
Sample solution (mg/g)
Ww = percentage in the test for Water Determination
Acceptance criteria: NLT 50.0% of D-maltitol (w/w)
and NMT 8.0% of D-sorbitol (w/w), on the anhydrous
basis
IMPURITIES
Inorganic Impurities
e RESIDUE ON IGNITION (281): NMT 0.1%, calculated on the
anhydrous basis, determined on a 2-g portion
¢ LIMIT OF NICKEL
Solution A: 10 mg/mL of ammonium pyrrolidine
dithiocarbamate
Sample solution: Dissolve and dilute 20.0 g of Maltitol
Solution with diluted acetic acid to 100 mL. Add 2.0 mL
of Solution A and 10.0 mL of methyl isobutyl ketone,
and shake for 30 s. Protect from bright light. Allow the
two layers to separate, and use the methyl isobutyl ke-
tone layer.
Standard solutions: Prepare as directed for the Sample
solution, except to prepare three solutions by adding
0.5, 1.0, and 1.5 mL of nickel standard solution TS.
Blank solution: Prepare as directed for the Sample solu-
tion, except to omit the use of the Maltitol Solution.
Spectrometric conditions
(See Atomic Absorption Spectroscopy (852).)
Mode: Atomic absorption spectrophotometry
Analytical wavelength: 232.0 nm (maximum
absorbance)
Lamp: Nickel hollow-cathode
Flame: Air-acetylene
Analysis
Samples: Sample solution, Standard solutions, and Blank
solution
Set the instrument to zero using the Blank solution.
Concomitantly determine the absorbances of the
Standard solutions and the Sample solution at least
three times each. Record the average of the steady
readings for each of the Standard solutions and the
Sample solution. Between each measurement, aspirate
the Blank solution, and ascertain that the reading
turns to zero. Plot the absorbances of the Standard
solutions and the Sample solution versus the added
quantity of nickel. Extrapolate the line joining the
points on the graph until it meets the concentration
axis. The distance between this point and the inter-
section of the axes represents the concentration of
nickel in the Sample solution.
Acceptance criteria: NMT 1 ppm, calculated on the an-
hydrous basis
Organic impurities
© PROCEDURE: REDUCING SUGARS
Sample: An amount of Maltitol Solution equivalent to
3.3 g on the anhydrous basis
Analysis: To the Sample add 3 mL of water, 20.0 mL of
cupric citrate TS, and a few glass beads. Heat so that
Official Monographs / Maltodextrin 5437
boiling begins after 4 min, and maintain boiling for 3
min. Cool rapidly, and add 40 mL of diluted acetic
acid, 60 mL of water, and 20.0 mL of 0.05 N iodine
VS. With continuous shaking, add 25 mL of a mixture
of 6 mL of hydrochloric acid and 94 mL of water.
When the precipitate has dissolved, titrate the excess of
iodine with 0.05 N sodium thiosulfate VS using 2 mL of
starch TS, added toward the end of the titration, as an
indicator.
[Note—The amount determined in this test is not in-
cluded in the calculated amount under Other
Impurities.]
Acceptance criteria: NLT 12.8 mL of 0.05 N sodium thio-
sulfate VS is required, corresponding to NMT 0.3% of re-
ducing sugars, on the anhydrous basis, as glucose.
SPECIFIC TESTS
© MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECIFIC
MICROORGANISMS (62): The total aerobic microbial count
using the Plate Method is NMT 1000 cfu/mL, and the
total combined molds and yeasts count is NMT 100 cfu/
mL.
e PH (791): 5.0-7.5, in a 14% (w/w) solution of Maltitol
Solution in carbon dioxide-free water
e WATER DETERMINATION, Method | (921): NMT 31.5%
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in well-closed contain-
ers. No storage requirements are specified.
© USP REFERENCE STANDARDS (11)
USP Diethylene Glycol RS
USP Ethylene Glycol RS
USP Maltitol RS
USP Sorbitol RS
Maltodextrin
DEFINITION
Maltodextrin is a nonsweet, nutritive saccharide mixture of
polymers that consists of D-glucose units, with a Dextrose
Equivalent less than 20. It is prepared by the partial hy-
drolysis of a food grade starch with suitable acids and/or
enzymes. It may be physically modified to improve its
physical and functional characteristics.
ASSAY
e DEXTROSE EQUIVALENT
Standard solution: 10 mg/mL of USP Dextrose RS
Sample solution: Transfer 5 g of Maltodextrin with the
aid of hot water to a 100-mL volumetric flask, cool, add
water to volume, and mix.
Analysis: Transfer 25.0-mL portions of alkaline cupric
tartrate TS to each of two boiling flasks. Bring the con-
tents of one flask to boiling within 2 min while titrating
with the Standard solution to within 0.5 mL of the antic-
re- ipatedpol aBoil gently for 2 min. Continue to boil
gently, add 2 drops of methylene blue solution (1 in
100), and complete the titration within 1 min by add-
ing the Standard solution dropwise or in small incre-
ments until the blue color disappears, determined by
viewing against a white background in daylight or
under equivalent illumination. If more than 0.5 mL of ra
the titrant was required after the addition of the indica- a]
tor, repeat the titration, adding the necessary volume of E
titrant before adding the indicator. Bring the contents i}
of the second flask to boiling, and similarly titrate with =
ry
=oy
the Sample solution.
Calculate the Dextrose Equivalent, on the dried basis, in
i)
the portion of Maltodextrin taken: a}
=3
Result = [100/(1 — 0.01 x A)] x (Cs/Cu) x (Vs/Vu) al
 5438 Maltodextrin / Official Monographs NF 36

A = percentage Loss on Drying of the Maltodextrin to the unavoidable pressure due to the height of the
taken Hydrogen peroxide solution above the tip of the bubbler,
Cs = concentration of USP Dextrose RS in the F. Keeping the backpressure as low as possible reduces
Standard solution (mg/mL) the likelihood that sulfur dioxide will be lost through
Cy = concentration of Maltodextrin in the Sample leaks. Preboil vinyl and silicone tubing. Apply a thin film
solution (mg/mL) of stopcock grease to the sealing surfaces of all of the
Vs = titrated volume of the Standard solution (mL) joints except uejot between the separatory funnel
Vu = titrated volume of the Sample solution (mL) and the flask, and clamp the joints to ensure tightness.
Acceptance criteria: Less than 20 The separatory funnel, B, has a capacity of 100 mL or
[Note—This is a limit test. For Maltodextrins with lower greater. The inlet adapter, A, with a hose connector
reducing values, other procedures may give other rovides a means of applying headpressure over the so-
results.] ution. [NOTE—A pressure-equalizing dropping funnel is
not recommended because condensate, whic!
IMPURITIES contain sulfur dioxide, is deposited in the funnel and
e RESIDUE ON IGNITION (281): NMT 0.5% the side arm.]

Delete the following:

°e HEAVY METALS, Method I! (231): NMT 5 ppme (offic! t4an-

2018) -—_>
e LIMIT OF PROTEIN
Sample: 10g .
Analysis: Transfer the Sample to an 800-mL Kjeldahl ||
flask, and add 10 g of anhydrous potassium sulfate or
sodium sulfate, 300 mg of copper selenite or mercuric ||
oxide, and 60 mL of sulfuric acid. Gently heat the mix-
ture, keeping the flask inclined at about a 45° angle,
and after frothing has ceased, boil briskly until the solu- A=
tion has remained clear for about 1 h. Cool, and very
cautiously add about 50 mL of water while swirling to Care

dissipate the resulting heat. Add an additional

150-250 mL of water, mix, and cool again. Cautiously
pour 75 ml (or enough to make the mixture strongly
alkaline) of sodium hydroxide solution (2 in 5) down
the inside of the flask so that it forms a layer under the —&—"

acid solution, and then add a few pieces of granular

zinc. Immediately connect the flask to a distillation ap- SS

paratus consisting of a Kjeldahl connecting bulb and a

condenser, the delivery tube of which extends well be-
neath the surface of an accurately measured excess of D
0.1 N sulfuric acid contained in a 500-mL flask. Gently
rotate the contents of the Kjeldahl flask to mix, and
distill until all ammonia has passed into the absorbing
acid solution (about 250 mL of distillate). To the receiv-
ing flask add 0.25 mL of methyl red-methylene blue TS,
and titrate the excess acid with 0.1 N sodium hydrox- H
ide. Perform a blank determination, substituting pure
by f bs
nh ty j q i

sucrose or dextrose for the test specimen, and make

any necessary correction. Each mL of 0.1 N sulfuric acid Vt At Py
consumed is equivalent to 1.401 mg of nitrogen (N). Cc ——>}
v’
Calculate the percentage of N in the specimen taken,
and then calculate the percentage of protein by multi-
plying the percentage of N by 6.25.
Acceptance criteria: NMT 0.1%
e Limit OF SULFUR DIOXIDE
Hydrogen peroxide solution: Dilute 30% hydrogen
peroxide with water to obtain a 3% solution. Just
efore use, add 3 drops of methyl red TS, and neutral-
ize to a yellow endpoint with 0.01 N sodium hydrox- Figure 1. Apparatus for the Sulfur Dioxide Test.
ide. Do not exceed the endpoint. The round-bottomed flask, C, is a 1000-mL flask with
Nitrogen: Use high-purity nitrogen, with a flow regula- three 24/40 tapered joints. The gas inlet tube, D, is
tor that will maintain a flow of 200 +10 mL/min. Guard long enough to permit introduction of the nitrogen
against the presence of oxygen by passing the nitrogen within 2.5 cm of the bottom of the flask. The Allihn
through a scrubber, such as alkaline pyrogallel, pre- condenser, £, has a jacket length of 300 mm. The bub-
NF Monographs

pared as follows. Add 4.5 g of pyrogallol to a gas-wash-
ing bottle, purge the bottle with nitrogen for3 min,
and add a solution containing 85 mL of water and 65g
of potassium hydroxide, while maintaining an atmos-
phere of nitrogen in the bottle.
Apparatus: The apparatus (see Figure 1) is designed to
effect the selective transfer of sulfur dioxide from the
specimen in boiling aqueous hydrochloric acid to the
Hydrogen peroxide solution. The backpressure is limited
bler, F, is fabricated from glass according to the
dimensions given in Figure 2. TheHydrogen peroxide
solution is contained in a vessel, G, having an inside
diameter of about 2.5 cm and a depth of about
18cm. Circulate coolant, such as a mixture of water
and methanol (4:1) maintained at 5°, to chill the
condenser.
NF 36 Official Monographs / Maltol 5439

I 160 mm >| e PH (791): 4.0-7.0, in a 0.2-g/mL solution in carbon di-

oxide-free water.
e Loss ON DRYING (731): Dry a sample at 105° for 2 hina
forced-air oven: it loses NMT 6.0% of its weight.
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in tight containers, or
in well-closed containers, at a temperature not exceeding
30° and a relative humidity not exceeding 50%.
e USP REFERENCE STANDARDS (11)
USP Dextrose RS
324/40

ih Maltol
185 mm

L\
wt yr
é

C6H6O3 126.11
Figure 2. Bubbler (F) for the Sulfur Dioxide Apparatus. 3-Hydroxy-2-methyl-4-pyrone [118-71-8].

Analysis: Position the Apparatus in a heating mantle DEFINITION

controlled by a power-regulating device. Add 400 mL of Maltol contains NLT 99.0% of maltol (CsH6O3), calculated
water to the flask. Close the stopcock of the separatory on the anhydrous basis.
funnel, and add 90 mL of 4 N hydrochloric acid to the IDENTIFICATION
separatory funnel. Begin the flow of Nitrogen at a rate e A. INFRARED ABSORPTION (197K)
of 200 + 10 mL/min. Start the condenser coolant flow. e B. ULTRAVIOLET ABSORPTION (197U)
Add 30 mL of the Hydrogen peroxide solution to vessel sample solution: 0.01 mg/mL in 0.1 N hydrochloric
G. After 15 min, remove the separatory funnel, and aci
transfer a mixture of 50.0 g of Maltodextrin and Blank: 0.1 N hydrochloric acid
100 mL of alcohol solution (5 in 100). Aepy stopcock
grease to the outer joint of the separatory unnel, re- ASSAY
turn the separatory funnel to the tapered joint flask, © PROCEDURE
and concomitantly resume the nitrogen flow. Apply Standard solution: 0.01 mg/mL of USP Maltol RS in
headpressure above the hydrochloric acid solution in 0.1 N hydrochloric acid
the separatory funnel with a rubber bulb equipped with Sample solution: 0.01 mg/mL of Maltol in 0.1 N hy-
a valve. Open the stopcock of the separatory funnel to drochloric acid
Bering the hydrochloric acid solution to flow into the Instrumental conditions
lask. Continue to maintain sufficient pressure above the Mode: UV
hydrochloric acid solution to force it into the flask. Analytical wavelength: Maximum at about 274 nm
[Not&—The stopcock may be temporarily closed, if nec- Blank: 0.1 N hydrochloric acid
essary, to pump up the pressure.] Analysis
To guard against escape of sulfur dioxide (SOz) into the Samples: Standard solution and Sample solution
separatory funnel, close the stopcock before the last Calculate the percentage of maltol (CéH6O3) in the por-
few mL of hydrochloric acid drain out. Apply power to tion of Maltol taken:
the heating mantle sufficient to cause about 85 drops
of reflux/min. After refluxing for 1.75 h, remove vessel Result = (Au/As) x (CGs/Cu) x 100
G, add 3 drops of methyl red TS, and titrate the con-
tents with 0.01 N sodium hydroxide VS, using a Au = absorbance of the Sample solution
10-mL buret with an overflow tube and a hose con- As = absorbance of the Standard solution
nection to a carbon dioxide-absorbing tube, to a yel- G = concentration of USP Maltol RS in the
low endpoint that persists for at least 20 s. Perform a Standard solution (mg/mL)
blank determination, and make any necessary correc- Cy = concentration of the Sample solution (mg/mL)
tion (see Titrimetry (541)). Acceptance criteria: NLT 99.0% on the anhydrous
Calculate the quantity, in g/g, of SO2 in the portion of basis
Maltodextrin taken:
IMPURITIES
Result = 1000 x Fx Vx N/W e RESIDUE ON IGNITION (281): NMT 0.2%, determined on
1.0g
F milliequivalent weight of sulfur dioxide, 32.03 e LEAD (251): NMT 10 ppm
a

Vv titrant volume consumed (mL)

ABE]
Vou

N actual normality of the titrant

ExtTe |BLstelerol

Ww = weight of Maltodextrin taken (g) Delete the following:

Acceptance criteria: NMT 40 ug/g (ppm)
°e HEAVY METALS, Method II (231): NMT 20 ppme cotticat 1-
SPECIFIC TESTS Jan-2018)
e MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI-
FIED MICROORGANISMS (62): It meets the requirements of SPECIFIC TESTS
the tests for absence of Salmonella species and Escherichia e MELTING RANGE OR TEMPERATURE, Class Ia (741):
coli. 160°-164°
 5440 Maltol / Official Monographs NF 36

e@ WATER DETERMINATION, Method | (921): NMT 0.5% Injection volume: 20 pL

System suitability
ADDITIONAL REQUIREMENTS Samples: System suitability solution and Standard
e PACKAGING AND STORAGE: Preserve in tight containers, solution
protected from light. No storage requirements are [Note—The relative retention times for maltotriose,
specified. maltose, and glucose are about 0.9, 1.0, and 1.2,
e@ USP REFERENCE STANDARDS (11) respectively.]
USP Maltol RS Suitability requirements
Resolution: NLT 1.6 between maltotriose and malt-
ose, System suitability solution
Relative standard deviation: NMT 2.0%, Standard
solution
Maltose Analysis
Samples: Standard solution and Sample solution
on Calculate, on the anhydrous basis, the percentage of
Maltose taken:
0
Ho yO Result = [(ru/rs) x (Cs/Cu)]/[(100 — W)/100] x 100
wh on Son
ty = peak response from the Sample solution
ts = peak response from the Standard solution
CiaH22011 + H20 360.31 Cs = concentration of USP Maltose Monohydrate
Ci2H2011 342.30 RS in the Standard solution, on the
4-O-0-D-Glucopyranosyl-B-D-glucopyranose [69-79-4]. anhydrous basis (mg/g)
4-O-o.-D-Glucopyranosyl--D-glucopyranose monohydrate Cu = concentration of Maltose in the Sample
[6363-53-7]. solution (mg/g)
Ww = percentage of water from the test for Water
DEFINITION Determination
Acceptance criteria: NLT 92.0% on the anhydrous
basis
Change to read:
IMPURITIES
Maltose is a sugar. It contains one molecule of water of e RESIDUE ON IGNITION (281)
hydration or is anhydrous. It contains NLT 92.0% of malt- Sample: 2g
ose, calculated on the anhydrous basis. The amounts of Acceptance criteria: NMT 0.05%
other sugars, if detected, are not included in the require-
ments or the calculated amount in General Notices, ~e (cw
1-May-2018) 5.60.10 Other Impurities in USP and NF Articles. Delete the following:

IDENTIFICATION ®e HEAVY METALS, Method | (231): NMT 5 U1g/ge corres 1-Jan-

e A. Perform this test for Anhydrous Maltose only. 2018)
Sample solution: 50 mg/mL
Analysis: Add 2-3 drops of the Sample solution to 5 mL SPECIFIC TESTS
of hot alkaline cupric tartrate TS. e DEXTRIN, STARCH, AND SULFITE
Acceptance criteria: A red precipitate is formed. Sample solution: 1.0 g of Maltose in 10 mL of water
e B. The retention time of the major peak of the Sample Analysis: Add 1 drop of iodine TS to the Sample
solution corresponds to that of the Standard solution, as solution.
obtained in the Assay. Acceptance criteria: A yellow color develops. Then add
e C. INFRARED ABSORPTION (197K): Perform the test for 1 drop of starch TS; a blue color develops.
Maltose Monohydrate only. Use the undried sample and © PH (791)
USP Maltose Monohydrate RS. Sample solution: 100 mg/mL in carbon dioxide-free
water
ASSAY Acceptance criteria
e PROCEDURE Anhydrous form: 3.7—4.7
Mobile phase: Water Monohydrate form: 4.0-5.5
System suitability solution: 10 mg/g each of maltotri- e@ WATER DETERMINATION, Method | (921)
ose, maltose, and glucose Anhydrous: NMT 1.5%
Standard solution: Dissolve USP Maltose Monohydrate Monohydrate: 4.5%-6.5%
RS in water to obtain a solution having a concentration
of about 10 mg/g. Calculate the exact concentration on ADDITIONAL REQUIREMENTS
the anhydrous basis. © PACKAGING AND STORAGE: Preserve in well-closed contain-
Sample solution: Dissolve 0.10 g of Maltose in water, ers. No storage requirements specified.
and dilute with water to 10 g. Record the final solution e USP REFERENCE STANDARDS (11)
weight, and mix thoroughly. USP Maltose Monohydrate RS
Chromatographic system
NF Monographs

(See Chromatography (621), System Suitability.)

Mode: LC
Detector: Refractive index
Column: 7.8-mm x 30-cm; packing L58
Temperatures
Column: 80+ 2°
Detector: 40°
Flow rate: 0.35 mL/min; adjust so that the resolution
between maltotriose and maltose is NLT 1.6.
USP 41
Calculate the percentage of calcium (Ca) in the portion
of Calcium Glycerophosphate taken:
Result
= [(V— B) x Mx Fx 100]/W
Vv = Sample titrant volume (mL)
B = Blank titrant volume (mL)
M = titrant molarity (mM/mL)
F = equivalency factor, 40.08 mg/mM
w = weight of the Sample (mg)
Acceptance criteria: 18.6%-19.4% on the dried basis
IMPURITIES
e LEAD (251): NMT 4 ppm
e IRON (241)
Standard solution: Dilute 1 volume of Standard Iron
Solution, prepared as directed in the chapter, with
water to 10 volumes (1 t1g/mL).
Analysis: Dissolve 0.20 g in 10 mL of water. Add 2 mL
of a 20% (w/v) solution of citric acid, 0.1 mL of thiogly-
colic acid, and mix. Make alkaline with 10 M ammonia,
dilute with water to 20 mL, and allow to stand for 5
min. Any pink color produced is not more intense than
that obtained by treating 4 mL of the Standard solution
in the same manner.
Acceptance criteria: NMT 20 ppm
Delete the following:
°e HEAVY METALS, Method | (231): NMT 20 ppme cra t.
Jan-2018)
e LIMIT OF CHLORIDE
Standard solution: 8.24 ug/mL of sodium chloride in
water
Sample solution: Dissolve 125 mg in a 10-mL mixture
of 5 M acetic acid and water (2:8), and dilute with
water to 15 mL.
Analysis: To the Sample solution add 1 mL of 2 M nitric
acid, then add 1 mL ofa silver nitrate solution (17rig
mL), and allow to stand for 5 min protected from light.
To 10 mL of the Standard solution add 5 mL of water,
1 mL of 2M nitric acid, and 1 mL of silver nitrate solu-
tion (17 mg/mL), and allow to stand for 5 min pro-
tected from light. When viewed against a dark back-
ground, the Sample solution is not more turbid than the
Standard solution.
Acceptance criteria: NMT 0.04%
e LIMIT OF SULFATE
Standard solution: 36.2 g/mL of potassium sulfate in
water
Sample solution: Use the Sample solution prepared as
directed in the test for Appearance of Solution.
Analysis: To 15 mL of the Standard solution add 0.5 mL
of 5 M acetic acid and 1 mL of barium chloride solution
(250 mg/mL). Repeat, using 15 mL of the Sample solu-
tion. Allow the solutions to stand for 5 min protected
from light. When viewed against a dark background,
the Sample solution is not more turbid than the Stan-
dard solution.
Acceptance criteria: NMT 0.2%
e Limit OF ARSENIC
Standard stock solution: In a 250-mL volumetric flask
dissolve 330 mg of arsenic trioxide in 5 mL of 2 N so-
dium hydroxide, and dilute with water to volume.
Transfer 1 mL of this solution to a 100-mL volumetric
flask, and dilute with water to volume.
Standard solution: Transfer 1 mL of Standard stock solu-
tion into a 10-mL volumetric flask, and dilute with
water volume.
Tin(Il) chloride solution: Heat 20g of tin with 85 mL
of hydrochloric acid until no more hydrogen is released.
Allow to cool. Dilute 1 volume of this solution with 10
volumes of dilute hydrochloric acid (200 mg/mL of hy-
drochloric acid in water).
Dietary Supplements / Calcium 4495
Sample solution: Transfer 330 mg of Calcium Glycer-
ophosphate to a 25.0-mL volumetric flask, and dilute
with water to volume.
Apparatus: Prepare a 100-mL side-arm conical flask
containing a magnetic stirring bar. Attach to the conical
flask a ground-glass stopper through which passes a
glass tube 20-cm long, with an internal diameter of
mm. The lower end of the tube is inside the conical
flask and has been drawn to a tip with an internal di-
ameter of 1 mm. An orifice 3 mm in diameter is 15 mm
from the tip and at least 3 mm below the lower surface
of the stopper. The upper end of the tube hasa flat
fend surface at a right angle to the axis of the tube.
second glass tube of the same internal diameter and
30 mm long, witha similar flat ground surface, is
placed in contact with the ground surface of the first
tube and is held in position by a clamp and springs.
Into the lower tube, insert 55 mg of loosely packed lead
acetate cotton. Place a disk of mercuric bromide paper
between the flat surfaces of the tubes.
Analysis: Before placing the tube assembly into the
flask, transfer the Sample solution to the flask, and add
15.0 mL of hydrochloric acid, 0.1 mL of Tin(Il) chloride
solution, and 5 mL of potassium iodide TS. Allow to
stand for 15 min, and add 5g of activated zinc. Assem-
ble the apparatus immediately, and immerse the flask in
a water bath at a temperature such that a uniform
evolution of gas is maintained. After not less than 2 h,
examine the stain produced on the mercuric bromide
paper. Perform the same procedure using 1.0 mL of the
Standard solution. The stain produced on the mercuric
bromide paper by the Sample solution is not more in-
tense than that produced by the Standard solution.
Acceptance criteria: NMT 3 ppm
© LIMIT OF PHOSPHATES
Sulfomolybdic solution: Dissolve with heating 2.5 g of
ammonium molybdate in 20 mL of water. Dilute 28 mL
of sulfuric acid with 50 mL of water, and cool. Mix the
two solutions, and dilute with water to 100 mL.
Tin(Il) chloride solution: Prepare as directed in the test
for Limit of Arsenic.
Standard stock solution: 14.3 g/mL of monobasic po-
tassium phosphate in water.
Standard solution: Transfer 1.0 mL of Standard stock
Solution to a 100-mL volumetric flask, and dilute with
water to volume.
Sample solution: Transfer 2.5 mL of the Sample solution
from the test for Appearance of Solution to a 100-mL
volumetric flask, and dilute with water to volume.
Analysis: To 100 mL of the Sample solution add 4 mL of
Sulfomolybdic solution, mix, and add 0.1 mL of Tin(Il)
sydesBouow-w Sa
chloride solution. To 100 mL of the Standard solution,
add 4 mL of Sulfomolybdic solution, mix, and add
0.1 mL of Tin(II) chloride solution. Allow the preparations
to stand for 10 min, then examine 20 mL of each prep-
aration. Any color produced by the Sample solution is
not more intense than that produced by the Standard
solution.
Acceptance criteria: NMT 0.04%
e Citric AciD
Mercury(Il) sulfate solution: 1g of mercuric oxide in
20 mL of water and 4 mL of sulfuric acid
Analysis: Mix 5 g of Calcium Glycerophosphate with
20 mL of carbon dioxide-free water, and filter. To the
filtrate add 0.15 mL of sulfuric acid, and filter. To the
filtrate add 5 mL of Mercury(II) sulfate solution, and heat
to boiling. Add 0.5 mL of 0.2 M potassium permanga-
nate, and heat to boiling.
Acceptance criteria: No precipitate is formed.
e GLYCEROL AND ALCOHOL-SOLUBLE SUBSTANCES
Analysis: Mix 1g with 25 mL of alcohol, and shake for
1 min. Filter, evaporate the filtrate to dryness on a
water bath, and dry the residue at 70° for 1 h.
Acceptance criteria: The residue weighs NMT 5 mg
(0.5%).
NF 36 Official Monographs / Mandelic 5441

Table 1 (Continued)
Mandelic Acid Relative
Retention
Component Time
Benzaldehyde 3.6
(7 J Acetophenone 4.8

CeHeO3 152.15
Benzeneacetic acid, a-hydroxy-;
(RS)-2-Hydroxy-2-phenylacetic acid;
(4)-o.-Hydroxyphenylacetic acid;
2-Hydroxy-2-phenylacetic acid [90-64-2].
DEFINITION
Mandelic Acid contains NLT 98.0% and NMT 102.0% of a-
hydroxyphenylacetic acid (CgHsO3), calculated on the an-
hydrous basis.
IDENTIFICATION
© A. INFRARED ABSORPTION (197K)
¢ B. CHROMATOGRAPHIC IDENTITY
Analysis: Proceed as directed in the Assay.
Acceptance criteria: The retention time of the major
peak of the Sample solution corresponds to that of the
Standard solution, as obtained in the Assay.
ASSAY
© PROCEDURE
Solvent A: 0.01 M phosphoric acid
Mobile pase: Acetonitrile, methanol, and Solvent A
(17:3:80
Standard stock solution A: Prepare a solution having
known concentrations of 0.2 mg/mL of acetophenone,
0.5 mg/mL of benzoylformic acid, and 0.25 mg/mL of
USP Benzaldehyde RS, respectively, in Mobile phase.
Standard stock solution B: Transfer 25 mg of USP Ben-
zoic Acid RS to a 250-mL volumetric flask, add 5.0 mL
of Standard stock solution A, dilute with Mobile phase to
volume, and mix.
Standard stock solution C: Use USP Mandelic Acid RS
to prepare a solution having a known concentration of
5 mg/mL in Mobile phase.
System suitability solution: Transfer 5.0 mL of Standard
stock solution B and 20.0 mL of Standard stock solution C
to a 100-mL volumetric flask, dilute with Mobile phase
to volume, and mix. The solution contains 1 mg/mL of
USP Mandelic Acid RS, 0.2 g/mL of acetophenone,
0.5 ug/mL of benzoylformic acid, 0.25 g/mL of USP
Benzaldehyde RS, and 5 t1g/mL of USP Benzoic Acid RS.
Standard solution: 1 mg/mL of USP Mandelic Acid RS
in Mobile phase prepared from Standard stock solution C
re solution: 1 mg/mL of Mandelic Acid in Mobile
phase
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 240 nm
Column: 4.6-mm x 25-cm; 5-um packing L1
Column temperature: Ambient
Flow rate: 0.8 mL/min
Injection volume: 20 yl
System suitability
Sample: System suitability solution
[Note—See Table 1 for relative retention times.]
Suitability requirements
Resolution: NLT 3.0, between the benzoylformic acid
eak and the mandelic acid peak and between the
enzoic acid peak and the benzaldehyde peak
Relative standard deviation: NMT 1% for the man-
delic acid peak
Tailing factor: NMT 2.0 for each peak
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of mandelic acid (CgHgOs) in
the portion of Mandelic Acid taken:
Result = (ru/rs) x (Cs/Cu) x 100
ru = peak response of mandelic acid from the
Sample solution
rs = peak response of mandelic acid from the
Standard solution
Cs = concentration of USP Mandelic Acid RS in the
Standard solution (mg/mL)
Cu = concentration of Mandelic Acid in the Sample
solution (mg/mL)
Acceptance criteria: 98.0%-102.0% on the anhydrous
basis
IMPURITIES
e RESIDUE ON IGNITION (281): NMT 0.1%
Delete the following:
°e HEAVY MeTALs, Method I! (231): NMT 20 ug/ge cic 1-
Jan-2018)
e Limit OF BENZOYLFORMIC ACID, BENZALDEHYDE, BENZOIC
ACID, AND ACETOPHENONE
Standard stock solution A, Standard stock solution B,
Chromatographic system, and System suitability:
Proceed as directed in the Assay.
Standard solution: Transfer 5.0 mL of Standard stock
solution B to a 100-mL volumetric flask, dilute with Mo-
bile phase to volume, and mix. The Standard solution
contains 0.2 g/mL of acetophenone, 0.5 tug/mL of
benzoylformic acid, 0.25 g/mL of USP Benzaldehyde
RS, and 5 ug/mL of USP Benzoic Acid RS.
Sample solution: 2.5 mg/mL of Mandelic Acid in Mo-
bile phase
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of each related compound
(benzoylformic acid, benzaldehyde, benzoic acid, or
acetophenone) in the portion of Mandelic Acid taken:
Result = (ru/rs) x (Cs/Cu) x 100
tu = peak response of the relevant related
compound (benzoylformic acid,
benzaldehyde, benzoic acid, or
acetophenone) from the Sample solution
sydesbouow 4N

rs = peak response of the relevant related

compound (benzoylformic acid,
Table 1
benzaldehyde, benzoic acid, or
Relative acetophenone) from the Standard solution
Retention Cs = concentration of the relevant related
Component Time compound (benzoylformic acid, USP
Benzoylformic acid 0.9 Benzaldehyde RS, USP Benzoic Acid RS, or
Mandelic acid 1.0 acetophenone) in the Standard solution
Benzoic acid 3.2 (mg/mL)
 5442 Mandelic / Official Monographs NF 36

GC = concentration of Mandelic Acid in the Sample Poly(methacrylic acid, ethyl acrylate);

solution (mg/mL) Methacrylic acid-ethyl acrylate copolymer [25212-88-8].
Acceptance criteria
Benzoylformic acid: NMT 0.1% DEFINITION
Benzoic acid: NMT 1.0% Methacrylic Acid and Ethyl Acrylate Copolymer consists of
Benzaldehyde: NMT 0.05% methacrylic acid and ethyl acrylate monomers arranged in
Acetophenone: NMT 0.01% a random distribution. Methacrylic acid units in Metha-
© LIMIT OF CHLORIDE crylic Acid and Ethyl Acrylate Copolymer, previously dried,
Sample: 1g are NLT 46.0% and NMT 50.6%. It may contain suitable
Analysis: Proceed as directed for Chloride and Sulfate surface-active agents.
(221), Chloride.
Acceptance criteria: 0.01%; a 1-g portion shows no IDENTIFICATION
more than corresponds to 0.15 mL of 0.020 N hydro- e A. INFRARED ABSORPTION (197K): Use USP Methacrylic
chloric acid. Acid and Ethyl Acrylate Copolymer (1:1) RS for Metha-
crylic Acid and Ethyl Acrylate Copolymer having a range
SPECIFIC TESTS of 46.0%-50.6% for methacrylic acid units.
¢ MELTING RANGE OR TEMPERATURE (741): 118°-121° e B. It meets the requirements of the Assay.
© WATER DETERMINATION, Method la (921): NMT 0.5%
© TURBIDITY ASSAY
© PROCEDURE
(See Nephelometry, Turbidimetry, and Visual Comparison
(855), Visual Comparison.) Sample: 1g, previously dried
Sample solution: 50 mg/mL in water Analysis: Dissolve the Sample in 100 mL of neutralized
Blank: Reserve a portion of the water used to prepare acetone, and titrate with 0.1 N sodium hydroxide VS,
the Sample solution. determining the endpoint potentiometrically (see Ti-
Fixed reproducible standards: See Determination of trimetry (541)). Each mL of 0.1 N sodium hydroxide is
Turbidity in Elastomeric Closures for Injections (381). equivalent to 8.609 mg of methacrylic acid (C4H6O2)
Analysis: Measure the turbidity of the Sample solution units.
and Blank as directed in Nephelometry, Turbidimetry, and Acceptance criteria: 46.0%-50.6%
Visual Comparison (855), Visual Comparison against the IMPURITIES
Fixed reproducible standards. © RESIDUE ON IGNITION (281): NMT 0.4%
Acceptance criteria: The turbidity is the difference be-
tween the values obtained for the Blank and the Sample
solution, expressed in Nephelos units. The Sample solu- Delete the following:
tion shows no more turbidity than the 10 Nephelos
units standard. °e HEAVY METALS, Method il (231): NMT 20 ug/Ge corte! 1-
Jan-2018)
ADDITIONAL REQUIREMENTS e Limit OF METHACRYLIC ACID AND ETHYL ACRYLATE
© PACKAGING AND STORAGE: Preserve in well-closed contain- Sodium perchlorate solution: 35 mg/mL of sodium
es Store in a dry and well-ventilated place. Protect from perchlorate. This solution has a concentration of 0.25
ight. M.
° usp REFERENCE STANDARDS (11) Mobile phase: Add phosphoric acid dropwise to water
USP Benzaldehyde RS to obtain a solution with a pH of 2.0. Prepare a mixture
USP Benzoic Acid RS of this acidified water and methanol (80:20), and
USP Mandelic Acid RS degas.
Standard solution: Dissolve 0.01 g of methacrylic acid
and 0.01 g of ethyl acrylate in 5 mL of butanol, and
add methanol to exactly 100 mL. Transfer 1.0 mL of this
solution to a 100-mL volumetric flask. Dilute with meth-
Mannitol—see Mannitol General anol to volume. Mix 5.0 mL of this solution with 5.0 mL
of Sodium perchlorate solution. This solution contains
Monographs about 0.5 g/mL each of methacrylic acid and ethyl
acrylate.
Sample solution: Transfer about 3 g of Methacrylic
Acid and Ethyl Acrylate Copolymer to a 50-mL volumet-
Meglumine—see Meglumine General ric flask, dilute with methanol to volume, and mix. Add
Monographs 5.0 mL of this solution dropwise, while continuously
stirring, to a beaker that contains 5.0 mL of Sodium per-
chlorate solution. Remove the precipitated polymer to
obtain a clear supernatant by centrifugation (e.g., NLT
Menthol—see Menthol General Monographs 5000 x g for NLT 5 min). Use the clear supernatant.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Methacrylic Acid and Ethyl Acrylate Detector: UV 202 nm
Column: 4.0-mm x 12.5-cm; 7-1um packing L1
NF Monographs

Copolymer Flow rate: 2 mL/min

2|
Injection volume: 20 yl
System suitability
Sample: Standard solution
{Note—The relative retention times for methacrylic acid
and ethyl acrylate are 1.0 and 2.6, respectively.]
Ri Re Suitability requirements
CH oH Resolution: NLT 2.0 between methacrylic acid and
or HO City ethyl acrylate
NF 36
Relative standard deviation: NMT 5.0%
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of each monomer (metha-
crylic acid or ethyl acrylate) in the portion of Metha-
crylic Acid and Ethyl Acrylate Copolymer taken:
Result = (ru/rs) x (C/W) x Vex Dx Fx 100
Tu = peak response of the monomer (methacrylic
acid or ethyl acrylate) from the Sample
solution
ls = peak response of the monomer (methacrylic
acid or ethyl acrylate) from the Standard
solution
Cc = concentration of the monomer (methacrylic
acid or ethyl acrylate) in the Standard
solution (4ug/mL)
Ww = weight of Methacrylic Acid and Ethyl Acrylate
Copolymer taken to prepare the Sample
solution (g)
Ve = final volume of the Sample solution, 10 mL
D = dilution factor for preparation of the Sample
solution, 10
F = conversion factor, 10-° g/ug
Acceptance criteria: NMT 0.01% for the total amount
of monomers
SPECIFIC TESTS
e ViIScosITY—ROTATIONAL METHODS (912)
Analysis: Place 254.6 g of isopropyl alcohol and 7.9 g
of water in a test flask. Add a quantity of Methacrylic
Acid and Ethyl Acrylate Copolymer, equivalent to 37.5 g
of solids on the dried basis, while stirring by means of a
magnetic stirrer. Close the flask, and continue stirring
until the polymer has dissolved completely. Adjust the
temperature to 20 +0.1°. Equip a rotational rheometer"
following Method I]. The shear rate under the test con-
dition is NLT 1 s+ and NMT 100 s~. Validations
demonstrate that equivalent viscosity value is also ob-
tained using a rotational viscometer with a cylindrical
spindle 1.9 cm in diameter and 6.5 cm high, attached
to a shaft 0.3 cm in diameter.2 The spindle rotates at 30
rpm at an immersion depth of 8.15 cm (see Method /).
Follow the instrument manufacturer's directions to
measure the apparent veo
Acceptance criteria: 100-200 mPa-s, for Methacrylic
Acid and Ethyl Acrylate Copolymer with a range of
46.0%-50.6% for methacrylic acid units
e Loss ON DRYING (731)
Analysis: Dry at 110° for 6 h.
Acceptance criteria: NMT 5.0%
ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Preserve in tight containers,
and store at controlled room temperature.
e LABELING: Label it to indicate the range of methacrylic
acid units. The labeling also indicates the name and
quantity of any added surface-active agent.
e USP REFERENCE STANDARDS (11)
USP Methacrylic Acid and Ethyl Acrylate Copolymer (1:1)
RS (USP Methacrylic Acid Copolymer, Type C RS)
1A suitable rheometer is available from Physica Messtechnik GmbH as the
coaxial Cylinder 27 or the Double-Gap-Cylinder 26.7, or any other equivalent
rheometer.
2A suitable spindle is available from Brookfield as an LV1 spindle, or the
equivalent.
Official Monographs / Methacrylic 5443
Methacrylic Acid and Ethyl Acrylate
Copolymer Dispersion
DEFINITION
Methacrylic Acid and Ethyl Acrylate Copolymer Dispersion is
an aqueous dispersion of Methacrylic Acid and Ethyl! Acry-
late Copolymer. It contains, on the basis of the calculated
amount of dry substance in the Dispersion, NLT 46.0%
and NMT 50.6% of methacrylic acid units. It may contain
suitable surface-active agents.
IDENTIFICATION
e A. INFRARED ABSORPTION (197K): Proceed as directed in
the chapter, except use the residue obtained in the test
for Loss on Drying as the sample.
e B. It meets the requirements in the Assay.
ASSAY
© PROCEDURE
Sample: 2.5 g of the Dispersion
Titrimetric system
(See Titrimetry (541).)
Mode: Direct titration
Titrant: 0.1 N sodium hydroxide VS
Endpoint detection: Potentiometric
Analysis: Dissolve the Sample in 100 mL of neutralized
acetone. Titrate the solution as directed in Titrimetric
system. Each mL of 0.1 N sodium hydroxide is equiva-
lent to 8.609 mg of methacrylic acid (C,HeO2) units.
Calculate, on the dried basis, he, percentage of metha-
crylic acid units in the portion of Dispersion taken:
Result = (V
x N)/[W
x (100 — L)] x 860.9
Vv = volume of Titrant consumed (mL)
N = normality of the Titrant
w = weight of Dispersion taken (g)
L = percentage of the Loss on Drying value for the
Dispersion
Acceptance criteria: 46.0%-50.6% based on the calcu-
lated amount of dry substance in the Dispersion
IMPURITIES
e RESIDUE ON IGNITION (281)
Analysis: Using mild heating conditions (e.g., steam
bath, sand bath) to avoid loss of material, evaporate
the Dispersion to dryness prior to ignition.
Acceptance criteria: NMT 0.2% residue is obtained,
calculated on the undried Dispersion basis.
Delete the following:
°e HEAVY METALS, Method // (231)
Analysis: Using miid heating conditions (e.g., steam
bath, sand bath) to avoid loss of material, evaporate
the Dispersion to dryness prior to wetting with sulfuric
acid and ignition.
Acceptance criteria: The color of the solution from the
Test Preparation is not darker than that of the solution
from the Standard Preparation (20 19/Q).e(orficiat 1-Jan-2018)
e Limit OF MONOMERS
Mobile phase: Add phosphoric acid dropwise to water
to obtain a solution with a pH of 2.0. Prepare a mixture
sydesbouo- 4IN
of this acidified water and methanol (80:20), and
degas.
Sodium perchlorate solution: Dissolve 3.5 g of sodium
perchlorate in 100 mL of water. This solution has a con-
centration of 0.25 M.
Standard solution: Dissolve 0.01 g of methacrylic acid
and 0.01 g of ethyl! acrylate in 5 mL of butanol, and
add methanol to make exactly 100 mL. Transfer 1.0 mL
of this solution to a 100-mL volumetric flask, and dilute
with methanol to volume. Mix 10.0 mL of this solution
 5444 Methacrylic / Official Monographs
with 5.0 mL of Sodium perchlorate solution, accurately
measured. This solution contains about 0.67 tug/mL
each of methacrylic acid and ethyl acrylate.
Sample solution: Transfer a quantity of Dispersion,
equivalent to 3 g of solids on the dried basis, to a
50-mL volumetric flask, dilute with methanol to volume,
and mix. Add 10.0 mL of this solution dropwise while
continuously stirring into a beaker that contains 5.0 mL
of Sodium perchlorate solution, accurately measured. Re-
move the precipitated polymer by centrifugation (e.g.,
NLT 5000 x g for NLT 5 min). Use the clear
supernatant.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 202 nm
Column: 4.0-mm x 12.5-cm; 7-um packing L1
Flow rate: 2 mL/min
Injection volume: 20 pL
System suitability
Sample: Standard solution
[Note—The relative retention times for methacrylic acid
and ethyl acrylate are 1.0 and 2.6, respectively.]
Suitability requirements
Resolution: NLT 2.0 between methacrylic acid and
ethyl acrylate
Relative standard deviation: NMT 5.0%, determined
for each analyte
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of each monomer in the
weight of the Dispersion taken:
Result = (ru/rs) x (C/W) x Ve x Dx Fx 100
ru = peak response of the monomer (methacrylic
acid or ethyl acrylate) from the Sample
solution
rs = peak response of the monomer (methacrylic
acid or ethyl acrylate) from the Standard
solution
c = concentration of the monomer (methacrylic
acid or ethyl acrylate) in the Standard
solution (g/mL)
w = weight of the Dispersion taken to prepare the
Sample solution (g)
Ve = final volume of the Sample solution, 15 mL
D = dilution factor for preparation of the Sample
solution, 5
F = conversion factor, 10-¢ g/ug
Acceptance criteria: NMT 0.01% of total monomers,
based on the weight of the Dispersion taken
SPECIFIC TESTS
e COAGULUM CONTENT
Analysis: Weigh a stainless steel sieve having 90-um
openings or a suitable single-woven wire cloth with a
mesh width of 90 um, and filter 100 g of the Dispersion
through it. [NoTE—Suitable single-woven wire cloth
mesh meets the requirements set in ISO 9044.] Wash
the sieve or the cloth with distilled water until a clear
filtrate is obtained, and dry the sieve or the cloth to
constant weight at 110°.
Acceptance criteria: The weight of the residue does
” not exceed 1000 mg (1%).
Ea
a e Loss ON DRYING (731)
ct
_
Analysis: Dry at 110° for 6h.
Dd Acceptance criteria: 68.5%-71.5%
2) e MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI-
e FIED MICROORGANISMS (62): The total aerobic microbial
iS count does not exceed 103 cfu/g, and the total com-
= bined molds and yeasts count does not exceed 10? cfu/
Ll
g.
PA
NF 36
° PH (791): 2.0-3.0
© VISCOSITY—ROTATIONAL METHODS, Method | (912)
Analysis: Equip a suitable rotational viscometer with an
adapter comprising a cylindrical spindle rotating within
an accurately machined chamber (or tube).1 Mix the
Dispersion, pipet the volume of test specimen recom-
mended by the instrument manufacturer into the
chamber (or tube), and ensure that the temperature of
the test specimen is at 20
+ 0.1°. The shear rate under
the test condition is NLT 1 s-! and NMT 100 s-1.2 Meas-
ure the apparent viscosity following the instrument
manufacturer’s directions.
Acceptance criteria: The viscosity is between 2 and 15
mPa -s.
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in tight containers.
Store at controlled room temperature. Protect from
freezing.
e LABELING: The label indicates the name and amount of
any substance added as a surface-active agent.
e USP REFERENCE STANDARDS (11)
USP Methacrylic Acid and Ethyl Acrylate Copolymer (1:1)
RS (USP Methacrylic Acid Copolymer, Type C RS)
Methacrylic Acid and Methyl
Methacrylate Copolymer
Pe]
Ri= Hor CH;
(Ratio of H to CHs is either 1:1 or 1:2)
Poly(methacrylic acid, methyl methacrylate);
Methacrylic acid-methyl methacrylate copolymer
[25086-15-1].
DEFINITION
Methacrylic Acid and Methyl Methacrylate Copolymer con-
sists of methacrylic acid and methyl methacrylate mono-
mers arranged in a random distribution. Methacrylic acid
units in Methacrylic Acid and Methyl Methacrylate Co-
polymer, previously dried, are NLT 27.6% and NMT
50.6%. It may contain suitable surface-active agents.
IDENTIFICATION
e A. INFRARED ABSORPTION (197K)
Use USP Methacrylic Acid and Methyl Methacrylate Co-
polymer (1:1) RS for Methacrylic Acid and Methyl
Methacrylate Copolymer, with a range of 46.0%-50.6%
for methacrylic acid units.
Use USP Methacrylic Acid and Methyl Methacrylate Co-
polymer (1:2) RS for Methacrylic Acid and Methyl
Methacrylate Copolymer, with a range of 27.6%-30.7%
for methacrylic acid units.
e B. It meets the requirements of the Assay.
ASSAY
¢ PROCEDURE
Sample: 1g, previously dried
Analysis: Dissolve the Sample in 100 mL of neutralized
acetone, and titrate with 0.1 N sodium hydroxide VS,
determining the endpoint potentiometrically (see Ti-
trimetry (541)). Each mL of 0.1 N sodium hydroxide is
1A commercial device is available from Brookfield as an ultra-low (UL) viscos-
ity adapter. The adapter comprises a 0.4-cm diameter shaft, an accurately
machined chamber (or tube) with an internal diameter of 2.8 cm and a
depth of 13.5 cm, and a cylindrical spindle 2.5 cm in diameter and 9.1 cm in
eight.
2 The cylindrical spindle rotates at 30 rpm, which corresponds to a shear rate
of approximately 37 s”'.
NF 36
equivalent to 8.609 mg of methacrylic acid (C4H6O2)
units.
Acceptance criteria
Methacrylic acid and methyl methacrylate copolymer
(1:2): 27.6%-30.7%
Methacrylic acid and methyl methacrylate copolymer
(1:1): 46.0%-50.6%
IMPURITIES
e RESIDUE ON IGNITION (281): NMT 0.1%
Delete the following:
°e HEAVY MeTALs, Method // (231): NMT 20 ug/ge corciai +.
Jan-2018)
e Limit OF METHACRYLIC ACID AND METHYL METHACRYLATE
Phosphate buffer: Prepare an aqueous solution con-
taining 17.8 g/L of anhydrous dibasic sodium phos-
phate and 17.0 g/L of monobasic potassium phosphate.
Adjust with phosphoric acid to a pH of 2.0. This buffer
has a concentration of 0.125 M.
Mobile phase: Add phosphoric acid dropwise to water
to obtain a solution with a pH of 2.0. Prepare a mixture
of this acidified water and methanol (80:20), and
degas.
Standard solution: Dissolve 0.05 g of methacrylic acid
and 0.05 g of methyl methacrylate in 5 mL of butanol,
and add methanol to exactly 100 mL. Transfer 1.0 mL
of this solution to a 100-mL volumetric flask. Dilute
with methanol to volume. Mix 3.0 mL of this solution
with 10.0 mL of Phosphate buffer. This solution contains
1.15 g/mL each of methacrylic acid and methyl meth-
acrylate. [NoTe—Due to volatility of monomers, tightly
closed vials should be used.]
Sample solution: Transfer 1 g of Methacrylic Acid and
Methyl Methacrylate Copolymer to a 50-mL volumetric
flask, dilute with methanol to volume, and mix. Add
3 mL of this solution dropwise, while continuously stir-
ring to a beaker that contains 10.0 mL of Phosphate
buffer. Remove the precipitated polymer to obtain a
clear supernatant by centrifugation (e.g., NLT 5000 x g
for NLT 5 min). Use the clear supernatant. [NoTE—Due
to ay of monomers, tightly closed vials should be
used.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 202 nm
Column: 4.0-mm x 12.5-cm; 7-um packing L1
Flow rate: 2 mL/min
Injection volume: 20 uL
System suitability
Sample: Standard solution
[Nott—The relative retention times for methacrylic acid
and methyl methacrylate are 1.0 and 2.8,
respectively.]
Suitability requirements
Resolution: NLT 2.0 between methacrylic acid and
methyl methacrylate
Relative standard deviation: NMT 5.0%
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of each monomer (metha-
crylic acid or methyl methacrylate) in the portion of
Meteciylle Acid and Methyl Methacrylate Copolymer
taken:
Result = (ru/rs) x (C/W) x Vex Dx Fx 100
Official Monographs / Methacrylic 5445
Ty = peak response of the monomer (methacrylic
acid or methyl methacrylate) from the
Sample solution
rs = peak response of the monomer (methacrylic
acid or methyl methacrylate) from the
Standard solution
G = concentration of the monomer (methacrylic
acid or methyl methacrylate) in the Standard
solution (ug/mL)
Ww = weight of Methacrylic Acid and Methy!
Methacrylate Copolymer taken to prepare
the Sample solution (g)
Ve = final volume of the Sample solution, 13 mL
D = dilution factor for preparation of the Sample
solution, 16.7
F = conversion factor, 10-* g/w
Acceptance criteria: NMT 0.05% for the total amount
of monomers
SPECIFIC TESTS
e ViscosiITy—ROTATIONAL METHODs (912)
Analysis: Place 254.6 g of isopropyl alcohol and 7.9 g
of water in a test flask. Add a quantity of Methacrylic
Acid and Methyl Methacrylate Copolymer, equivalent to
37.5 g of solids on the dried basis, while stirring by
means of a magnetic stirrer. Close the flask, and con-
tinue stirring until the polymer has dissolved com-
pletely. Adjust the temperature to 20+ 0.1°. Equip a ro-
tational rheometer’ following Method II. The shear rate
under the test condition is NLT 1 s-' and NMT 100 s".
Validations demonstrate that an equivalent viscosity
value is also obtained using a rotational viscometer with
a cylindrical spindle 1.9 cm in diameter and 6.5 cm
high, attached to a shaft 0.3 cm in diameter.? The spin-
dle rotates at 30 rpm at an immersion depth of
8.15 cm (see Method /). Follow the instrument manufac-
turer’s directions to measure the apparent viscosity.
Acceptance criteria
Methacrylic acid and methyl methacrylate co-
polymer, with a range of 46.0%-50.6% for metha-
crylic acid units: 60-120 mPa-s
Methacrylic acid and methyl methacrylate co-
pent with a range of 27.6%-30.7% for metha-
crylic acid units: 50-200 mPa-s
© Loss ON DRYING (731)
Analysis: Dry at 110° for 6 h.
Acceptance criteria: NMT 5.0%
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in tight containers,
and store at controlled room temperature.
e LABELING: Label it to indicate the range of methacrylic
acid units. The labeling also indicates the name and
quantity of any added surface-active agent.
e USP REFERENCE STANDARDS (11)
USP Methacrylic Acid and Methyl Methacrylate Co-
polymer (1:1) RS (USP Methacrylic Acid Copolymer,
‘Type A RS)
USP Methacrylic Acid and Methyl Methacrylate Co-
polymer (1:2) RS (USP Methacrylic Acid Copolymer,
‘Type B RS)
1A suitable rheometer is available from Physica Messtechnik GmbH as the
Coaxial-Cylinder 27 or the Double-Gap-Cylinder 26.7, or any other equivalent
rheometer.
2A suitable spindle is available from Brookfield as an LV1 spindle, or the
sydesbouo= 4N
equivalent.
 5446 Methacrylic / Official Monographs
Partially-Neutralized Methacrylic Acid
and Ethyl Acrylate Copolymer
n by the precipitation 1s negligible for contents in the
R, = CHg, Ro =H or
R, = CHg, Ro = Na or
R,=H, Ry=CoHs
Partially-neutralized poly(methacrylic acid, ethyl acrylate);
Partially-neutralized methacrylic acid—ethyl acrylate co-
polymer [25212-88-8].
DEFINITION
Partially-Neutralized Methacrylic Acid and Ethyl Acrylate Co-
polymer consists of methacrylic acid and ethyl acrylate
monomers arranged in a random distribution, some units
of methacrylic acid in the copolymer are neutralized by
sodium hydroxide. The non-neutralized methacrylic acid
units in the partially-neutralized methacrylic acid and
ethyl acrylate copolymer are NLT 43.2% and NMT 47.6%,
calculated on the dried basis. It may contain suitable
emulsifiers.
IDENTIFICATION
e A. INFRARED ABSORPTION
Sample: 100 mg of Partially-Neutralized Methacrylic
Acid and Ethyl Acrylate Copolymer
Analysis: Dissolve the Sample in 1 mL of dehydrated al-
cohol, and place 2 drops of the solution on a sodium
chloride (or potassium bromide) plate. Dry to evaporate
the solvent and allow to form a film and cover with
another sodium chloride (or potassium bromide) plate.
Acceptance criteria: The IR absorption spectrum of Par-
tially-Neutralized Methacrylic Acid and Ethyl Acrylate
Copolymer exhibits maxima corresponding to the same
wavelengths as that of a similar preparation of USP Par-
tially-Neutralized Methacrylic Acid and Ethyl Acrylate
Copolymer (1:1) RS, treated in the same manner.
e B. It meets the requirements of the Assay.
ASSAY
e PROCEDURE
Sample: 1g, calculated on the dried basis
Analysis: Dissolve the Sample in 40 mL of water and
60 mL of 2-propanol, and titrate with 0.1 N sodium
hydroxide VS, determining the endpoint potentiometri-
cally (see Titrimetry (541)). Each mL of 0.1 N sodium
hydroxide is equivalent to 8.609 mg of methacrylic acid
(C4H6O2) units.
Acceptance criteria: 43.2%-47.6% for Partially-Neutral-
ized Methacrylic Acid and Ethyl Acrylate Copolymer
IMPURITIES
Inorganic impurities
e RESIDUE ON IGNITION (281): 2.0%-3.5%
NF Monographs
Delete the following:
®. HEAvy METALS, Method Il (231): NMT 20 ppme cotta
1-
Jon-2018)
Organic Impurities
e PROCEDURE: LIMIT OF METHACRYLIC ACID AND ETHYL
ACRYLATE
Phosphoric acid solution: 0.1% phosphoric acid pre-
pared from phosphoric acid
Mobile phase: Methanol and Phosphoric acid solution
(3:7)
NF 36
Standard solution: 1.0 j1g/mL each of methacrylic acid
and ethyl acrylate in methanol
Sample solution: Transfer 0.5 g of Partially-Neutralized
Methacrylic Acid and Ethyl Acrylate Copolymer to a
25-mL volumetric flask, and dissolve in 20 mL of meth-
anol. Add Phosphoric acid solution dropwise to precipi-
tate the polymer while continuously stirring until the
volume of 25 mL is reached. [NoTE—Stir with a mag-
netic stirrer for 10 min. Any volume deviation caused
ppm range. Use a magnetic stirrer of appropriate size
to avoid a large variance from the final volume of the
Sample solution.] As soon as the solid matter has set-
tled, pass the supernatant throughafilter of 0.45--4m
pore size. [NoTE—Solution that cannot be filtered is
centrifuged at NLT 20,000 x g for NLT 30 min.] Use
the clear supernatant.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 205 nm
Column: 4.0-mm x 12.5-cm analytical column, 5-um
packing L1 or 4.6-mm x 15.0-cm analytical column,
5-um packing L1
Flow rate: 1.2 mL/min
Injection size: 20 uL
[NotE—Where appropriate, the volume must be
adapted to the sensitivity of the detector.]
[Note—Column switching system may be used for
extension of column lifetime.]
System aay
Sample: Standard solution
[NoTtt—The relative retention times for methacrylic
acid and ethyl acrylate are 1.0 and 2.2,
res| cave,
Suitability requirements
Resolution: NLT 5.0 between methacrylic acid and
ethyl acrylate
Relative standard deviation: NMT 5.0%
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of each monomer (metha-
crylic acid or ethyl acrylate) in the pouien of Metha-
crylic Acid and Ethyl Acrylate Copolymer taken:
Result = (ru/ts) x (Cs/W) x Ve x F x 100
ty = peak response from the Sample solution
rs = peak response from the Standard solution
Cs = concentration of the Standard solution
(ug/ml) : :
W = weight of Partially-Neutralized Motec ale
Acid and Ethyl Acrylate Copolymer taken to
prepare the Sample solution (g)
Ve = final volume of the Sample solution, 25 mL
F = conversion factor, 10-§ g/g
Acceptance criteria: NMT 0.01% for the total amount
of monomers
SPECIFIC TESTS
© Loss ON DRYING (731): Dry a sample at 110° for 6 h: it
loses NMT 5.0% of its weight.
e VIsSCcosITY—ROTATIONAL METHODS (912): Weigh 400 g of
water into a short form, 600-mL beaker (internal diame-
ter about 80 mm and height 120 mm). After determining
the Loss on Drying, weigh a quantity of undried Partially-
Neutralized Methacrylic Acid and Ethyl Acrylate Co-
iolymer, equivalent to 100 g on the dried basis. Transfer
he sample to the beaker very slowly while effectively stir-
ring (avoid lumps). Ensure that the stirring is very effec-
tive at the beginning and that the powder is immersed
very slowly at the same time. Once the powder is dis-
persed and no lumps are visible, gentle stirring is then
sufficient. Ensure a colloidal dispersion (milky white liq-
uid) by stirring at room temperature for 3 h and taking
NF 36 Official Monographs / Methyl 5447

care to avoid mixing in excess air. Afterwards allow the Table 1 (Continued)
container to stand for 1 h, control the temperature to Hold Time at|
23 40.19, and let the entrapped air dissipate. [NOTE— Initial Temperature Final Final
Ensure that the concentration of this solution is 20% (w/ Temperature Ramp Temperature | Temperature
w).] Determine the viscosity of this solution at 23 +0.1° (¢/min) «@) (min)
@)
using a suitable rotational viscometer with a cylindrical
40 20 240 =
spindle 1.9 cm in diameter and 6.5 cm high, attached to
a shaft 0.3 cm in diameter.’ The spindle rotates at 50 Carrier gas: Helium
rpm at an immersion depth of 8.1 cm. Follow the instru- Linear velocity: 35 cm/s
ment manufacturer’s directions to measure the apparent Injection type: Split ratio, 20:1
viscosity. Injection size: 1 pL
Acceptance criteria: 20-100 mPa-s System suitability
ADDITIONAL REQUIREMENTS Samples: System suitability solution and Standard
© PACKAGING AND STORAGE: Preserve in tight containers, solution
and store at controlled room temperature. [Note—The relative retention times for methyl alcohol,
e LABELING: Label it to indicate the range of non-neutral- acetone, and acetonitrile are 1.0, about 1.6, and about
ized methacrylic acid units. The labeling also indicates 1,8, respectively.]
the name and quantity of any emulsifier if the content is Suitability requirements
0.10% or greater. Resolution: NLT 15 between methyl alcohol and ace-
e USP REFERENCE STANDARDS (11) tone, System suitability solution
USP Partially-Neutralized Methacrylic Acid and Ethyl Ac- Tailing factor: NMT 1.5 for methyl alcohol, System
rylate Copolymer (1:1) RS suitability solution
Relative standard deviation: NMT 2.0% for the ratio
of the peak area of methyl alcohol to acetonitrile,
Standard solution
Analysis
Samples: Standard solution and Sample solution
Methyl Alcohol Calculate the percentage of methyl alcohol (CH3OH) in
the portion of Methyl Alcohol taken:
Hye"
Result = (Ru/Rs) x (Cs/Cu) x 100
CH,O 32.04 Ru = peak area ratio from the Sample solution
Methanol [67-56-1]. Rs = peak area ratio from the Standard solution
Cs = concentration of USP Methyl Alcohol RS in the
DEFINITION Standard solution (mg/mL)
Methyl Alcohol contains NLT 99.5% of CH3OH. Cu = concentration of Methyl Alcohol in the Sample
[CautTion—Methyl Alcohol is poisonous.] solution (mg/mL)
IDENTIFICATION Acceptance criteria: NLT 99.5%
e A. INFRARED ABSORPTION (197F) IMPURITIES
e B. The retention time of the major peak of the Sample © NONVOLATILE RESIDUE
solution corresponds to that of the Standard solution, as Sample: 250 mL of Methyl Alcohol
obtained in the Assay. Analysis: Evaporate the Sample in a 600-mL beaker on
ASSAY a steam bath, in a well-ventilated hood, until the vol-
° PROCEDURE ume is reduced to about 100 mL. Cool, transfer a por-
System suitability solution: Dilute 1.0 mL of USP tion of the liquid to a suitable, tared 50-mL platinum
Methyl Alcohol RS and 1.0 mL of USP Acetone RS with dish on a steam bath, and evaporate. Repeat the pro-
tetrahydrofuran to 50 mL. cess until all of the liquid has been transferred, and
Internal standard solution: 2% (v/v) acetonitrile in then Svaporate to dryness. Dry at 105° for 30 min,
tetrahydrofuran cool, an weigh.
Standard solution: 15.8 mg/mL of USP Methyl Alcohol Acceptance criteria: The weight of the residue does
RS in Internal standard solution not exceed 2 mg, corresponding to NMT 0.001% (w/
Sample solution: 15.8 mg/mL of Methyl Alcohol in In- w).
ternal standard solution e ACETONE AND ALDEHYDES (as acetone)
Chromatographic system Standard solution: Dilute 1.9 mL (1.5 g) of acetone
(See Chromatography (621), System Suitability.) with water to 1000 mL, then dilute 1.0 mL of this solu-
Detector: Flame ionization tion with water to 100 mL. Dilute 2 mL of the resulting
Column: 0.32-mm x 30-m fused-silica capillary col- solution with water to 5 mL. The Standard solution con-
umn, coated with a 1.8-m layer of phase G43 tains 30 ug of acetone and is freshly prepared.
Temperature Sample solution: Dilute 1.25 mL (1 g) of Methyl Alco-
Injector: 200° hol with water to 5 mL.
Detector: 280° Analysis: Adjust to and maintain each solution at 20°.
Column: See Table 1. Add 5 mL of alkaline mercuric—potassium iodide TS to
sydeibouo- 4N

each of the Standard solution and Sample solution.

Acceptance criteria: Any turbidity produced in the
Table 1 Sample solution is not greater than that produced in the
Hold Time at! Standard solution (NMT 0.003%).
Initial Temperature Final Final e READILY CARBONIZABLE SUBSTANCES (271)
Temperature Ramp Temperature | Temperature Sample: 5 mL
@) (¢/min) C). _(min) Analysis: Cool 5 mL of sulfuric acid, contained in a
40 — 40 5 small conical flask, to 10°, and add the Sample drop-
wise with constant mixing, maintaining the temperature
1A suitable spindle is available from Brookfield as an LV1 spindle, or the below 20° throughout the test.
equivalent.
 5448 Methyl / Official Monographs NF 36

Acceptance criteria: No discoloration develops. e ACIDITY

e READILY OXIDIZABLE SUBSTANCES Sample: 15.0 mL
Sample: 20 mL of Methyl Alcohol Analysis: Mix the Sample with 15 mL of neutralized al-
Analysis: Cool the Sample to 15°, add 0.1 mL of 0.1 N cohol, add phenolphthalein TS, and titrate with 0.050
potassium permanganate, and allow to stand at 15°. N sodium hydroxide.
Acceptance criteria: The pink color does not com- Acceptance criteria: NMT 0.40 mL is required for
pletely disappear within 5 min. neutralization.
SPECIFIC TESTS ADDITIONAL REQUIREMENTS
e ACIDITY ¢ PACKAGING AND STORAGE: Preserve in tight containers.
Sample solution: Mix 25 mL of water with 10 mL of
alcohol and 0.5 mL of phenolphthalein TS, and add
0.02 N sodium hydroxide until a slight pink color per-
sists after shaking for 30 s. Taking precautions to avoid
absorption of carbon dioxide, add 19 mL (15 g) of Methyl Salicylate
Methyl! Alcohol.
Analysis: Titrate the Sample solution with 0.020 N so-

co
dium hydroxide.
Acceptance criteria: NMT 0.45 mL of 0.020 N sodium
hydroxide is required to produce a pink color. "OH
e ALKALINITY (as ammonia)
Sample: 28.6 mL (22.6 g) of Methyl Alcohol
Analysis: Mix the Sample with 25 mL of water, add 1 CsHsO3 152.15
aoe of methyl red TS, and titrate with 0.020 N sulfuric Benzoic acid, 2-hydroxy-, methyl ester;
acia. Methyl salicylateti 19-36-8].
Acceptance criteria: NMT 0.20 mL of 0.020 N sulfuric
acid is required to produce a pink color (3 ppm). DEFINITION
e WATER DETERMINATION, Method | (921): NMT 0.1% Methyl Salicylate is produced synthetically or is obtained by
maceration and subsequent distillation with steam from
ADDITIONAL REQUIREMENTS the leaves of Gaultheria procumbens L. (Fam. Ericaceae) or
e PACKAGING AND STORAGE: Preserve in tight containers, re- from the bark of Betula lenta L. (Fam. Betulaceae). It con-
mote from heat, sparks, and open flames. tains NLT 98.0% and NMT 100.5% of methyl salicylate
e USP REFERENCE STANDARDS (11) (CsHsOs).
USP Acetone RS
USP Methyl! Alcohol RS IDENTIFICATION
e A. INFRARED ABSORPTION (197F)
e B, CHROMATOGRAPHIC IDENTITY
Analysis: Proceed as directed in the Assay.
Acceptance criteria: The retention time of the major
Methyl lsobutyl Ketone peak of the Sample solution corresponds to that of the
Standard solution.
ASSAY
¢ PROCEDURE
Mobile phase: Methanol and 0.1% phosphoric acid
(55:45)
C6H120 100.16 Diluent: Methanol
2-Pentanone, 4-methyl-; System suitability solution: 150 g/mL of USP Methyl
4-Methyl-2-pentanone [108-10-1]. Salicylate RS and 3 ug/mL of USP Methyl Salicylate Re-
lated CompoundA RS in Diluent
DEFINITION Standard solution: 150 g/mL of USP Methyl Salicylate
Methyl Isobutyl Ketone contains NLT 99.0% of methyl
RS in Diluent
isobutyl ketone (CeH120).
Sample solution: 150 g/mL of Methyl Salicylate in
IDENTIFICATION Diluent
e A. The IR absorption spectrum of a thin film of it be- Chromatographic system
tween sodium chloride crystals exhibits maxima, among (See Chromatography (621), System Suitability.)
others, at the following wavelengths, in um: 5.81 (vs), Mode: LC
6.80 (m), 7.00 (m), 7.09 (m), 7.29 (vs), 7.72 (m), 8.06 Detector: UV 237 nm
(m), 8.31 (sh), 8.53 (s), and 8.91 (m). Column: 4.6-mm x 7.5-cm; 3.5-\um packing L7
Column temperature: Ambient
IMPURITIES Flow rate: 1.0 mL/min
e LIMIT OF NONVOLATILE RESIDUE Injection volume: 10 uL
Sample: 50 mL Run time: 7 min
Analysis: Evaporate the Sample in a tared porcelain dish System suitability
one steam bath, and dry at 105° for 1 h. Weigh the Samples: System suitability solution and Standard
NF Monographs

residue. solution
Acceptance criteria: NMT 4 mg (0.008%) [Nott—The relative retention times for methyl salicylate
and dimethyl! 4-hydroxyisophthalate are 1.0 and 1.2,
SPECIFIC TESTS respectively.]
© SPECIFIC GRAVITY (841): NMT 0.799, indicating NLT Suitability requirements
99.0% of methyl isobutyl ketone (CsH120) Resolution: NLT 1.5 between methyl salicylate and
e DISTILLING RANGE, Method | (721): Between 114° and dimethy! 4-hydroxyisophthalate, System suitability
117°, a correction factor of 0.046°/mm Hg being applied solution
as necessary Tailing factor: NMT 1.5 for the methyl salicylate
peak, Standard solution
NF 36 Official Monographs / Methylene 5449

Relative standard deviation: NMT 0.5% for the slightly levorotatory, the angular rotation not exceeding
methy! salicylate peak, Standard solution -1.5° in a 100-mm tube.
Analysis
Samples: Standard solution and Sample solution ADDITIONAL REQUIREMENTS
Calculate the percentage of methyl salicylate in the por- e PACKAGING AND STORAGE: Preserve in tight containers.
tion of Methyl Salicylate taken: e LABELING: Label it to indicate whether it was made syn-
thetically or distilled from either of the plants of
Result = (ru/rs) x (Cs/Cy) x 100 Gaultheria procumbens or Betula lenta.
e USP REFERENCE STANDARDS (11)
ru = peak response from the Sample solution USP Methyl Salicylate RS
rs = peak response from the Standard solution USP Methyl Salicylate Related Compound A RS
Cs = concentration of USP MethylSalicylate RS in Dimethyl 4-hydroxyisophthalate.
the Standard solution (\1g/mL) GioHi0Os 210.18
Cu = concentration of Methyl Salicylate in the USP Salicylic Acid RS
Sample solution (g/mL)
Acceptance criteria: 98.0%-100.5%
IMPURITIES
Methylcellulose—see Methylcellulose
Delete the following: General Monographs
°e HEAVY METALS, Method II (231): NMT 20 19/Qe cortici 1.
Jan-2018)
e LIMIT OF SALICYLIC ACID AND DIMETHYL Methylene Chloride
4-HYDROXYISOPHTHALATE
Mobile phase, Diluent, Sample solution, and Chromat-
ographic system: Proceed as directed in the Assay. CH2Cl2 84.93
Standard solution: 0.15 g/mL of USP Salicylic Acid RS, Methane, dichloro-;
0.15 g/mL of USP Methyl Salicylate RS, and 0.75 pg/ Dichloromethane [75-09-2].
mL of USP Methyl Salicylate Related Compound A RS in DEFINITION
Diluent Methylene Chloride contains NLT 99.0% of methylene chlo-
System suitability ride (CH2Cl2). [CAUTION—Perform all steps involving evap-
Sample: Standard solution oration of methylene chloride in a well-ventilated fume
[Nott—The relative retention times for salicylic acid, hood.]
methyl salicylate, and dimethyl 4-hydroxyisophthalate
are 0.6, 1.0, and 1.2, respectively.] IDENTIFICATION
Suitability requirements eA.
Resolution: NLT 4 between salicylic acid and methyl Sample: 5 mL
salicylate; NLT 2 between methyl salicylate and di- Analysis: Place the Sample into a glass-stoppered,
methyl 4-hydroxyisophthalate 10-mL conical flask, and shake for several min. Remove
Relative standard deviation: NMT 3% for all three the stopper, quickly withdraw a portion of the vapor
peaks into a 50-mL syringe that is not fitted with a needle,
Analysis and inject the vapor into a suitable evacuated gas cell.
Samples: Sample solution and Standard solution Acceptance criteria: The IR absorption spectrum of the
Calculate the percentage of each individual impurity in vapor shows strong doublet peaks at 7.8 and 7.9 um
the portion of Methyl Salicylate taken: and at 13.2 and 13.4 um, and relatively few minor
peaks.
Result = (ru/rs) x (Cs/Cu) x 100
ASSAY
Ty = peak response of salicylic acid or dimethyl e PROCEDURE
4-hydroxyisophthalate from the Sample System suitability solution: Methylene chloride and
solution chloroform (3:7)
fs = peak response of salicylic acid or dimethyl Chromatographic system bility)
4-hydroxyisophthalate from the Standard (See Chromatogra (621), System Suitability.
solution Mode: GC a
Cs = concentration of USP Salicylic Acid RS or USP Detector: Thermal conductivity (under typical
Methyl Salicylate Related Compound A RS in conditions)
the Standard solution (g/mL) Column: 4-mm x 1.8-m; packed with 15% liquid
Cu = concentration of Methyl Salicylate in the phase G18 on 30- to 60-mesh S1C unsilanized support
Sample solution (g/mL) Temperatures
Acceptance criteria Injection port: 200°
Salicylic acid: NMT 0.1% Detector: 250°
Dimethyl! 4-hydroxyisophthalate: NMT 0.5% Column: 60°
Carrier gas: Helium
SPECIFIC TESTS
sydesBouo- 4N

Flow rate: 20 mL/min

¢ SOLUBILITY IN 70% ALCOHOL: One volume of synthetic Injection volume: 1 uL
MethylSalicylate dissolves in seven volumes of 70% alco- System suitability
hol. One volume of natural Methyl Salicylate dissolves in Sample: System suitability solution
seven volumes of 70% alcohol, the solution shows NMT Suitability requirements
a slight cloudiness. Resolution: NLT 4.0 between methylene chloride and
e SPECIFIC GRAVITY (841): 1.180-1.185 for the synthetic va- chloroform
riety; 1.176-1.182 for the natural variety Tailing factor: NMT 1.4
e OPTICAL ROTATION, Angular Rotation (781A): Synthetic Relative standard deviation: The peak response ratio
Methyl Salicylate and that from Betula lenta are optically does not exceed 2% for five replicate injections.
inactive. Methyl Salicylate from Gaultheria procumbens is
 5450 Methylene / Official Monographs NF 36

Analysis ADDITIONAL REQUIREMENTS

Sample: Methylene Chloride e PACKAGING AND STORAGE: Preserve in tight containers.
Inject the Sample, and determine the peak responses
by any convenient means. [NoTE—The order of elu-
tion is amylenes (5 or 6 peaks), if present, and then
methylene chloride.]
Calculate the percentage of methylene chloride Methylparaben
(CH2Cl,) in the portion of sample taken:
Result = (ru/rr) x 100
ry = peak response of methylene chloride
rr = sum of all the peak responses
Acceptance criteria: NLT 99.0%
CgHgO3 152.15
IMPURITIES Benzoic acid, 4-hydroxy-, methyl ester;
e LIMIT OF NONVOLATILE RESIDUE Methyl p-hydroxybenzoate [99-76-3].
Sample: 50g
Analysis: Evaporate the Sample in a platinum or porce- DEFINITION
lain dish on a steam bath, and dry at 105° for 30 min. Methylparaben contains NLT 98.0% and NMT 102.0% of
Acceptance criteria: NMT 0.002%; NMT 1 mg of CsHsO3.
residue
IDENTIFICATION
e A. INFRARED ABSORPTION (197M)
Delete the following: e B. MELTING RANGE OR TEMPERATURE (741): 125°-128°
®o HEAVY METALS, Method | (231) ASSAY
Test preparation: 15 mL (20g) e PROCEDURE
Analysis: Evaporate the Test preparation in a glass evap- Mobile phase, Sample solution, Standard solution B,
orating dish on a steam bath to dryness. Cool, add and Chromatographic system: Proceed as described
2 mL of hydrochloric acid, and slowly evaporate again in the procedure for Related Substances.
on a steam bath to dryness. Dissolve the residue in System suitability
1 mL of 1 N acetic acid, and add 24 mL of water. Sample: Standard solution B
Acceptance criteria: NMT 1 6/9e‘oificiat 14an.2018) Suitability requirements
Relative standard deviation: NMT 0.85% for 6
SPECIFIC TESTS injections
e LIMIT OF HYDROGEN CHLORIDE Analysis
Sample: 20.0 mL Samples: Sample solution and Standard solution B
Analysis: Into each of twoGlass-stoppere, 50-mL Calculate the percentage of Methylparaben in the Sam-
color-comparison cylinders having an internal diameter ple solution:
of 20 mm, place 10 mL of water, 2 drops of phenol-
phthalein TS, and sufficient 0.010 N sodium hydroxide Result = P x (ru x Cs)/(rs X Cu)
to produce a pink color that persists after vigorous
shaking for 30 s and is of equal intensity in each cylin- P = labeled purity of USP Methylparaben RS
der. expressed as a percentage
[NoTe—In the following step, take special care to avoid tu = peak area of methylparaben from the Sample
contamination with carbon dioxide.] solution
Into one of the cylinders, place the Sample and 0.70 mL Cs = concentration of methylparaben in Standard
of 0.010 N sodium hydroxide, and shake again. solution B
Acceptance criteria: NMT 0.001%; the pink color in Ts = peak area of methylparaben from Standard
the sample cylinder is at least as intense as that in the solution B
comparison cylinder, and the color persists for NLT 15 Cu = concentration of Methylparaben in the Sample
min. solution
© SpEciFIC GRAVITY (841): 1.318-1.322 Acceptance criteria: 98.0%-102.0%
e WATER DETERMINATION, Method | (921): NMT 0.02%
e FREE CHLORINE IMPURITIES
Sample: 10 mL Inorganic Impurities
Analysis: To the Sample add 10 mL of water and 0.1 mL e RESIDUE ON IGNITION (281): NMT 0.1%, determined on
of potassium iodide TS, shake for 2 min, and allow the 1.0g
liquids to separate. Organic Impurities
Acceptance criteria: The lower layer does not show a e PROCEDURE: RELATED SUBSTANCES
violet tint. Mobile phase: Methanol and a 6.8 g/L solution of po-
tassium dihydrogen phosphate (65:35 v/v)
Sample solution: Dissolve 50.0 mg of Methylparaben
in 2.5 mL of methanol, and dilute with Mobile phase to
50.0 mL. Dilute 10.0 mL of this solution with Mobile
NF Monographs

phase to 100.0 mL.

Standard solution A: 5.0 g/mL each of p-hydroxy-
benzoic acid and USP Methylparaben RS in Mobile
phase
Standard solution B: Dissolve 50.0 mg of USP Methyl-
paraben RS in 2.5 mL of methanol, and dilute with Mo-
bile phase to 50.0 mL. Dilute 10.0 mL of this solution
with Mobile phase to 100.0 mL.
 4496 Calcium / Dietary Supplements USP 41

SPECIFIC TESTS © B. IDENTIFICATION TESTS—GENERAL, Calcium (191): A

e APPEARANCE OF SOLUTION 5-mg/mL solution meets the requirements.
Opalescent suspension: Dissolve 1g of hydrazine sul- e C. HPLC: The retention time of the major peak of the
fate in 100 mL of water and allow to stand for 4-6 h. Sample solution corresponds to that of the Standard solu-
Add 25 mL of this solution to 25 mL of a solution con- tion, as obtained in the Assay. It complies with the accep-
taining 100mg/mL of methenamine in water, and allow tance criteria of the test for Enantiomeric Purity.
to stand for 24 h.
Primary reference suspension: Dilute 15.0 mL of the ASSAY
Opalescent suspension with water to 1000 mL. [NoTE— © PROCEDURE
This suspension is freshly prepared and may be stored Buffer: 7.8 g/L of sodium dihydrogen phosphate dihy-
for NMT 24 h.] drate in water
Reference suspension: Primary reference suspension and Solution A: Adjust the Buffer with 32% (w/v) sodium
water (30:70). [NoTE—Shake before using.] hydroxide solution to a pH of 6.5.
Sample solution: Dissolve 1.5 g at room temperature in Solution B: Methanol and Buffer (35:65). Adjust with
150 mL of carbon dioxide-free water. 32% (w/v) sodium hydroxide solution to a pH of 8.0.
Analysis: Compare the epalescence of equal volumes of Mobile phase: Gradient elution. See Table 1.
the Sample solution and the Reference suspension.
Acceptance criteria: The Sample solution is not more Table 1
opalescent than the Reference suspension.
e ACIDITY OR ALKALINITY Time Solution A Solution B
Analysis: Dissolve 1 g of Calcium Glycerophosphate in (min) (%) (%)
100 mL of water. Add 0.1 mL of 1.0% (w/v) phenol- 0 100 0
phthalein solution. 14 45 55
Acceptance criteria: NMT 0.5 mL of 0.1 M sodium hy- 17 0 100
droxide or 0.1 M hydrochloric acid is required to 24 0 100
change the color of the indicator. 24.01 100 0
© Loss ON DRYING (731): Dry a sample at 150° for 4 h: it
33 100 0
loses NMT 12.0% of its weight.
ADDITIONAL REQUIREMENTS [NoTte—After analysis the column should be flushed and
© PACKAGING AND STORAGE: Preserve in well-closed contain- stored in a mixture of methanol and water (85:15).]
ers, and store at controlled room temperature. System suitability solution: Transfer 25 mg of USP
Folic Acid RS and 25 mg of USP 4-Aminobenzoyl-
glutamic Acid RS to a 100-mL volumetric flask. Add
about 15 mg each of sodium hydrogen carbonate and
sodium carbonate to the flask, add sufficient water, son-
icate to dissolve, and dilute with water to volume.
Calcium L-5-Methyltetrahydrofolate Transfer 1.0 mL of this solution to a second 100-mL vol-
umetric flask containing 50 mg of USP Calcium DL-
eA 5-Methyltetrahydrofolate RS, dissolve, and dilute with
[ x ANi water to volume.
x , 4 [NotE—The following Standard and Sample solutions
cat? a
oH Ls
Z A
ee must be injected immediately after preparation and in-
jected only once.]
Standard solution: 0.5 mg/mL of USP Calcium DL-
5-Methyltetrahydrofolate RS in water
Sample solution: 0.5 mg/mL of Calcium L-5-Methylte-
trahydrofolate in water
CaoH23CaN7O¢ - xH20 Chromatographic system
C29H23CaN7O¢ (anhydrous) 497.52 (See Chromatography (621), System Suitability.)
N-[4-[[(2-Amino-1,4,5,6, ec senalyaro metNyie 1 xe 25) Mode: LC
Detector: UV 280 nm
DS Monographs

pteridinyl)methyl]amino]benzoyl]-t-glutamic acid, calcium

salt (1:1);
N-{4-[[((65)-2-Amino-1,4,5,6,7,8-hexahydro-5-methyl-4-oxo-
6-pteridinyl)methy!]amino]-benzoy)}-i-glutamic acid, cal-
cium salt (1:1) [151533-22-1].
System suitability
DEFINITION
Calcium L-5-Methyltetrahydrofolate contains NLT 95.0%
and NMT 102.0% of calcium 5-methyltetrahydrofolate
(C2oH23CaN7O6¢), the sum of the L- and D-diastereoisomers,
calculated on the anhydrous and solvent-free basis, of
which NMT 1.0% corresponds to calcium D-
5-methyltetrahydrofolate.
IDENTIFICATION
e A. INFRARED ABSORPTION (197K)
[NoTe—If the spectra obtained show differences, dissolve
the substance to be examined and the USP Calcium DL-
5-Methyltetrahydrofolate RS separately in the minimum
quantity of water, and add dropwise sufficient acetone
to produce a precipitate. Allow to stand for 15 min,
centrifuge to collect the precipitate, wash the precipi-
tate twice with a minimum quantity of acetone, and
dry. Record new spectra using the residues.]
Column: 4.6-mm x 25-cm; 5-um packing L1
Column temperature: 32°
Flow rate: 1.1 mL/min
Injection volume: 10 LL
Samples: System suitability solution and Standard
solution
[Note—For the System suitability solution the relative re-
tention times of the component peaks are listed in Ta-
ble 2. The L- and D-isomers of 5-methyltetrahydrofolate
co-elute as a single peak. The 4a-hydroxy-5-methylte-
trahydrofolic acid, 5-methyltetrahydropteroic acid, and
dimethyltetrahydrofolic acid are included as minor
components in USP Calcium DL-5-Methyltetrahydrofo-
late RS.]
Suitability requirements
Resolution: System suitability solution
NLT 6 between 4-aminobenzoylglutamic acid
and 4athycroxy semethyltattanyarsialic acid
NLT 8 between folic acid and 5-methylte-
trahydrofolic acid
NLT 15 between 5-methyltetrahydrofolic acid
and dimethyltetrahydrofolic acid
NF 36
Standard solution C: Dilute 1.0 mL of the Sample solu-
tion with Mobile phase to 20.0 mL. Dilute 1.0 mL of this
solution with Mobile phase to 10.0 mL.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 272 nm
Column: 4.6-mm x 15-cm; 5-{1m packing L1
Flow rate: 1.3 mL/min
Injection size: 10 pL
Run time: About 5 times the retention time of
methylparaben
System a
Sample: Standard solution A
[Note—The retention time of methylparaben is about
2.3 min, the relative retention time for p-hydroxy-
benzoic acid is about 0.6.]
Suitability requirements
Resolution: NLT 2.0 between the p-hydroxybenzoic
acid and methylparaben peaks
Analysis
Samples: Sample solution and Standard solution C
[Not&—Disregard any limit that is 0.2 times the area
of the principal peak in the chromatogram obtained
with Standard solution C (0.1%).]
Acceptance criteria
Lo aeho acid: The peak area in the Sample
solution, multiplied by 1.4 to correct for the calcula-
tion of content, is NMT the area of the principal peak
in Standard solution C (0.5%).
Unspecified impurities: The peak area of each impu-
rity in the Sample solution is NMT the area of the
principal peak in Standard solution C (0.5%).
Total impurities: The total peak area for all impurities
in the Sample solution is NMT twice the area of the
principal peak in Standard solution C (1.0%).
SPECIFIC TESTS
e COLOR OF SOLUTION
Sample solution: 100 mg/mL in alcohol
Comparison solution: Mix 2.4 mL of ferric chloride CS,
1.0 mL of cobaltous chloride CS, and 0.4 mL of cupric
sulfate CS with 0.3 N hydrochloric acid to make 10 mL.
Dilute 5 mL of this solution with 0.3 N hydrochloric
acid to make 100 mL. [NoTe—Prepare and use this solu-
tion immediately.]
Analysis
Samples: Alcohol, Sample solution, and Comparison
solution
Make the comparison by viewing the solutions down-
ward in matched color-comparison tubes against a
white surface (see Color and Achromicity (631)).
Acceptance criteria: The Sample solution is clear and
not more intensely colored than alcohol or the Compari-
son solution.
e ACIDITY
Sample solution: To 2 mL of the Sample solution pre-
ared in the test for Color of Solution, add 3 mL of alco-
ol, 5 mL of carbon dioxide-free water, and 0.1 mL of
bromocresol green TS.
Analysis: Titrate with 0.10 N sodium hydroxide.
Acceptance criteria: NMT 0.1 mL is required to pro-
duce a blue color.
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in well-closed
containers.
Official Monographs / Methylparaben 5451
e USP REFERENCE STANDARDS (11)
USP Methylparaben RS
Methylparaben Sodium
Nat J Ja
CegH7NaO3 174.13
Benzoic acid, 4-hydroxy-, methyl ester, sodium salt;
Methyl p-hydroxybenzoate, sodium salt;
Sodium 4-methoxycarbonylphenolate [5026-62-0].
DEFINITION
Methylparaben Sodium contains NLT 95.0% and NMT
103.0% of methylparaben sodium (CsH7NaOs), calculated
on the anhydrous basis.
IDENTIFICATION
cA.
Standard: 0.5 g of USP Methylparaben RS
Sample: 0.5g
Analysis: Dissolve the Sample in 5 mL of water. Acidify
with hydrochloric acid, and filter the resulting precipi-
tate. Wash the precipitate with water, and dry it over
silica gel for 5 h. Repeat with the Standard.
Acceptance criteria: The IR absorption spectrum of a
mineral oil dispersion of the Sample exhibits maxima
only at the same wavelengths as those of a similar
preparation of the Standard.
eB.
Sample solution: Ignite 0.3 g of Methylparaben So-
dium, cool, and dissolve the residue in about 3 mL of
3 N hydrochloric acid.
Acceptance criteria: A platinum wire dipped in the
Sample solution imparts an intense, persistent yellow
color to a nonluminous flame.
ASSAY
© PROCEDURE
Mobile phase: Methanol and a 6.8 g/L solution of po-
tassium dihydrogen phosphate (65:35, v/v)
System suitability solution: 5.0 ug/mL each of p-hy-
Bpesjpencon acid and USP Methylparaben RS in Mobile
jase
standard solution: Dissolve 50.0 mg of USP Methylpar-
aben RS in 2.5 mL of methanol, and dilute with Mobile
phase to 50.0 mL. Dilute 10.0 mL of this solution with
Mobile phase to 100.0 mL.
Sample solution: Dissolve 50.0 mg of Methylparaben
Sodium in 2.5 mL of methanol, and dilute with Mobile
phase to 50.0 mL. Dilute 10.0 mL of this solution with
Mobile phase to 100.0 mL.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 272 nm
Column: 4.6-mm x 15-cm; 5-um packing L1
Flow rate: 1.3 mL/min
sydesbouo- 4N
Injection volume: 10 pL
Run time: About 5 times the retention time of the
methylparaben peak
System suitability
Samples: System suitability solution and Standard
solution
[Note—The retention time for methylparaben is about
2.2 min; the relative retention times for p-hydroxyben-
zoic acid and methylparaben are about 0.7 and 1.0,
respectively.]
 5452 Methylparaben / Official Monographs NF 36

Suitability requirements SPECIFIC TESTS

Resolution: NLT 2.0 between the p-hydroxybenzoic e COMPLETENESS OF SOLUTION (641)
acid and methylparaben peaks, System suitability Sample solution: 1g of Methylparaben Sodium dis-
solution solved in water
Relative standard deviation: NMT 0.85% for six in- Acceptance criteria: Meets the requirements
jections, Standard solution © PH (791)
Analysis Sample solution: 1 mg/mL
Samples: Standard solution and Sample solution Acceptance criteria: 9.5-10.5
Calculate the percentage of methylparaben sodium e@ WATER DETERMINATION, Method | (921): NMT 5.0%
ee Nan) in the portion of Methylparaben Sodium
taken: ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Preserve in tight containers.
Result = P x (ru/rs) x (Cs/Cu) x (Mr/Mi2) e USP REFERENCE STANDARDS (11)
USP Methylparaben RS
P = labeled purity of USP Methylparaben RS
expressed as a percentage
tu = peak area of methylparaben from the Sample
solution
rs = peak area of methylparaben from the Standard Methylpyrrolidone
solution
G = concentration of methylparaben in the
Standard solution *
Cu = concentration of Methylparaben Sodium in Neo
\/
the Sample solution
Mn = molecular weight of methylparaben sodium,
174.13 CsHsNO 99.1
M2 = molecular weight of methylparaben, 152.15 1-Methyl-2-pyrrolidinone;
Acceptance criteria: 95.0%-102.0% on the anhydrous N-Methyl-2-pyrrolidone;
basis N-Methylpyrrolidone;
1-Methyl-2-pyrrolidone;
IMPURITIES Pyrrolidin, 1-methyl-2-one-;
e RELATED COMPOUNDS 1 Met vipyiroball2-onie:
Mobile phase, System suitability solution, Sample so- N-Methyl-y-butyrolactam;
lution, and Chromatographic system: Proceed as di- N-Methyl tetrahydropyrrolone;
rected in the Assay. 1-Methyl-2-oxopyrrolidine;
Standard solution: Dilute 1.0 mL of the Sample solution N-Methyl-1-oxotetramethyleneamine;
with Mobile phase to 20.0 mL. Dilute 1.0 mL of this so- 2-Methyl-2-azacyclopentanone [872-50-4].
lution with Mobile phase to 10.0 mL.
System suitability IDENTIFICATION
Sample: System suitability solution © A, INFRARED ABSORPTION (197F)
[Note—The retention time for methylparaben is about
2.2 min; the relative retention times for p-hydroxyben- IMPURITIES
zoic acid and methylparaben are about 0.7 and 1.0,
respectively.] Delete the following:
Suitability requirements
Resolution: NLT 2.0 between the p-hydroxybenzoic °e HEAVY METALS (231): NMT 10 ppMe (otc t-jen-2018)
acid and methylparaben peaks © ORGANIC IMPURITIES
Analysis Standard solution: To 1 mL of USP Methylpyrrolidone
Samples: Standard solution and Sample solution RS, add 1 mL of pyrrolidone, and dilute with methylene
Acceptance criteria chloride to 20 mL.
p-Hydroxybenzoic acid: NMT 3.0%; the peak area in Sample solution: Methylpyrrolidone (neat)
the Sample solution, multiplied by 1.4 to correct for Chromatographic system
the calculation of content, is NMT 6 times the area of (See Chromatography (621), System Suitability.)
the principal peak in the Standard solution. Mode: GC
Unspecified impurities: NMT 0.5%; the peak area of Detector: Flame ionization
each impurity in the Sample solution is NMT the area Column: 0.32-mm x 30-m fused-silica capillary; 5-um
of the principal peak in the Standard solution. layer of phase G2
Total impurities: NMT 1.0%; the total peak area for Temperatures
all unspecified impurities in the Sample solution is NMT Injector: 280°
twice the area of the principal peak in the Standard Detector: 280°
solution. Column: See Table 1.
© CHLORIDE AND SULFATE, Chloride (221)
Sample: 0.2
Control: 0.10 mL of 0.020 N hydrochloric acid Table 1
Acceptance criteria: 0.035%; the Sample shows no
NF Monographs

Hold Time at
more chloride than the Control. Initial Temperature Final Final
e CHLORIDE AND SULFATE, Sulfate (221) Temperature Ramp Temperature | Temperature
Sample: 0.25g (@) (¢/min) ©) (min)
Control: 0.30 mL of 0.020 N sulfuric acid 100 _ 100 o
Acceptance criteria: 0.12%; the Sample shows no more
100 3 170 30
sulfate than the Control.
NF 36
Carrier gas: Nitrogen
Linear velocity: 20 cm/s
Injection type: Split ratio about 100:1
Injection volume: 1 uL
System suitability
Sample: Standard solution
Suitability requirements
Resolution: NLT 2.0 between pyrrolidone and
methylpyrrolidone
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of each impurity, excluding
any solvent peaks and peaks NMT 0.02%, in the por-
tion of Methylpyrrolidone taken:
Result = (ru/rz) x 100
tu = peak response of each individual impurity
from the Sample solution
tr = sum of the responses of all the peaks from the
Sample solution
Acceptance criteria: NMT 0.1% of any individual im-
purity; and NMT 0.3% of total impurities
SPECIFIC TESTS
e ALKALINITY
Bromothymol blue solution: Dissolve 50 mg of
bromothymol blue in a mixture of 4 mL of 0.02 M so-
dium hydroxide and 20 mL of alcohol, and dilute with
water to 100 mL.
Sample: Methylpyrrolidone (neat)
Analysis: Add 0.5 mL of Bromothymol blue solution as
indicator to 50 mL of water, and adjust with 0.02 M
potassium hydroxide or 0.02 M hydrochloric acid until
a yellow color is obtained. Add 50 mL of the Sample.
Titrate with 0.02 M hydrochloric acid to the initial
coloration.
Acceptance criteria: NMT 8.0 mL of 0.02 M hydrochlo-
ric acid is required.
© CLARITY OF SOLUTION
[Note—The Sample is to be compared to the Reference
suspension in diffused daylight 5 min after preparation
of the Reference sspensirst
Hydrazine solution: 10 mg/mL of hydrazine sulfate.
Note—Allow to stand 4-6h before use.]
Methenamine solution: Transfer 2.5 g of methenamine
to a 100-mL glass-stoppered flask, add 25.0 mL of
water, insert the glass stopper, and mix to dissolve.
Primary ee suspension
[Note—This suspension is stable for 2 months, provided
it is stored in a glass container free from surface de-
fects. The suspension must not adhere to the glass and
must be well mixed before use.]
Transfer 25.0 mL of the Hydrazine solution to the Methe-
namine solution in the 100-mL glass-stoppered flask.
[NoTte—Allow to stand for 24 h.]
Opalescence standard: Transfer 15.0 mL of the Primary
opalescent suspension to a 1000-mL volumetric flask,
and dilute with water to volume. [NoTE—This suspen-
sion should not be used beyond 24hafter
preparation.]
Reference suspension: Transfer 5.0 mL of the Opales-
cence standard to a 100-mL volumetric flask, and dilute
with water to volume.
Sample: Methylpyrrolidone (neat)
Analysis: Transfer a sufficient portion of the Sample to a
test tube of colorless, transparent, neutral glass with a
flat base and an internal diameter of 15-25 mm to ob-
tain a depth of 40 mm. Similarly transfer portions of the
Reference suspension and water to separate matching
test tubes. Compare the Sample, Reference suspension,
and water in diffused daylight, viewing vertically against
a black background (see Nephelometry, Turbidimetry,
Official Monographs / Mineral 5453
and Visual Comparison (855)). [NoTe—The diffusion of
light must be such that the Reference suspension can
readily be distinguished from water.]
Acceptance criteria: The Sample shows the same clarity
as that of water, or its opalescence is not more pro-
nounced than that of the Reference suspension.
© COLOR OF SOLUTION
Comparison solution: Mix 3.0 mL of ferric chloride CS,
3.0 mL of cobaltous chloride CS, and 2.4 mL of cupric
sulfate CS with 0.3 N hydrochloric acid to make 10 mL.
Dilute 1.0 mL of this solution with 0.3 N hydrochloric
acid to make 100 mL. [NoTE—Prepare and use this solu-
tion immediately)
Sample: Methylpyrrolidone (neat)
Analysis: Transfer a sufficient portion of the Sample to a
test tube of colorless, transparent, neutral glass with a
flat base and an internal diameter of 15-25 mm to ob-
tain a depth of 40 mm. Similarly transfer a portion of
the Comparison solution to a separate matching test
tube. Compare the color of the Sample with that of the
Comparison solution in diffused daylight, viewing verti-
cally against a white background (see Nephelometry,
Turbidimetry, and Visual Comparison (855)).
Acceptance criteria: The Sample is not more intensely
colored than the Comparison solution.
e WATER DETERMINATION, Method Ic (921): NMT 0.1%, de-
termined on 1.0g
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in light-resistant
containers.
oe USP REFERENCE STANDARDS (11)
USP Methylpyrrolidone RS
Mineral Oil—see Mineral Oil General
Monographs
Mineral Oil, Rectal—see Mineral Oil, Rectal
General Monographs
Light Mineral Oil
DEFINITION
Light Mineral Oil is a purified mixture of liquid hydrocar-
ions obtained from petroleum. It may contain a suitable
stabilizer.
IDENTIFICATION
@ A. INFRARED ABSORPTION (197F)
e B. It meets the requirements in Specific Tests for Viscos-
ity—Capillary Methods (911).
IMPURITIES
e LIMIT OF POLYCYCLIC AROMATIC HYDROCARBONS
nak od sulfoxide: Use spectrophotometric grade di-
methyl sulfoxide.
n-Hexane: Use n-hexane that has been washed by be-
sydesbouow 4N
ing shaken previously twice with one-fifth its volume of
Dimethyl! sulfoxide.
Standard solution: 7.0 pai of USP Naphthalene RS
in isooctane (2,2,4-trimethylpentane)
Standard blank: 2,2,4-Trimethylpentane
Sample solution: Transfer 25.0 mL of Light Mineral Oil
and 25 mL of n-Hexane to a 125-mL separator, and
mix. [NoTe—Use no lubricants other than water on the
stopcock, or use a separator equipped with a suitable
polymeric stopcock.]
 5454 Mineral / Official Monographs NF 36

Add 5.0 mL of Dimethyl! sulfoxide, and shake the mixture Sample: 4.0 mL
vigorously for 1 min. Allow to stand until the lower Analysis: Combine the Sample, 2 mL of dehydrated al-
layer is clear, transfer the lower layer to another cohol, and 2 drops of Solution A, heat at 70° for 10 min
125-mL separator, add 2 mL of n-Hexane, and shake with frequent shaking, and cool.
vigorously. Use the lower layer. Acceptance criteria: No dark brown color develops.
Sample blank: Dimethy! sulfoxide that has been shaken
previously vigorously for 1 min with n-Hexane in the ADDITIONAL REQUIREMENTS
ratio of 5 mL of Dimethyl sulfoxide to 25 mL of n-Hexane e PACKAGING AND STORAGE: Preserve in tight, light-resistant
Instrumental conditions containers. No storage requirements specitied.
(See Ultraviolet-Visible Spectroscopy (857).) ¢ LABELING: Label it to indicate the name and quantity of
Mode: UV any substance added as a stabilizer, and label packages
Analytical wavelengths intended for direct use by the public to indicate that it is
Standard solution: 275 nm not intended for internal use.
Sample solution: 260-350 nm e USP REFERENCE STANDARDS (11)
Cell: 1cm USP Mineral Oil RS
Analysis USP Naphthalene RS
Samples: Standard solution, Standard blank, Sample so-
lution, and Sample blank
Acceptance criteria: The absorbance at any wavelength
in the specified range of the Sample solution is NMT
one-third of the absorbance of the Standard solution. Topical Light Mineral Oil—see Topical
SPECIFIC TESTS Light Mineral Oil General Monographs
e SPECIFIC GRAVITY (841): 0.818-0.880
© VISCOSITY—CAPILLARY METHODS (911): 3.0-34.4 mm2-
s" for kinematic viscosity, measured with a capillary vis-
cometer at 40 +0.1° Mono- and Di-glycerides
e ACIDITY
Sample solution: Combine 10 mL of Light Mineral Oil DEFINITION
and 20 mL of boiling water, shake vigorously for 1 min, Mono- and Di-glycerides is a mixture of glycerol mono- and
and allow to cool. Remove, and filter the aqueous layer. di-esters, with minor amounts of tri-esters, of fatty acids
Analysis: To 10 mL of the Sample solution add 0.1 mL of from edible oils. It contains NLT 40.0% of monoglycer-
phenolphthalein TS. ides. The monoglyceride content is NLT 90.0% and NMT
Acceptance criteria: The solution does not produce a 110.0% of the value indicated in the labeling. It may con-
pink color. NMT 1.0 mL of 0.01 N sodium hydroxide is tain suitable stabilizers.
required to producea pink color.
e READILY CARBONIZABLE SUBSTANCES TEST (271) ASSAY
e MONOGLYCERIDES
Sample: 5 mL
Standard solution: In a glass-stoppered test tube that Mobile phase: Tetrahydrofuran
previously has been rinsed with hot nitric acid (see Sample solution: 40 mg/mL of Mono- and Di-glycer-
Cleaning Glass Apparatus (1051)), mix 3 mL of ferric ides in tetrahydrofuran
chloride CS, 1.5 mL of cobaltous chloride CS, and Chromatographic system
0.5 mL of cupric sulfate CS then overlaid with 5 mL of (See Chromatography (621), System Suitability.)
Light Mineral Oil. Mode: LC
Analysis: Place the Sample in a Glas stoppeted test Detector: Refractive index >
tube that previously has been rinsed with hot nitric acid Column: 7-mm x 60-cm; 5-11m packing L21 (100 A)
(see Cleaning Glass Apparatus (1051)), then rinsed with [NoteE—Two or three 7.5-mm x 30-cm L21 columns
water, and dried. Add 5 mL of sulfuric acid containing may be used in place of one 60-cm column provided
94.5%-94.9% of H2SO., and heat in a boiling water that System suitability requirements are met.]
bath for 10 min. After the test tube has been in the Temperatures
bath for 30 s, remove it quickly, and, while holding the Column: 40°
stopper in place, give three vigorous, vertical shakes Detector: 40°
over an amplitude of about 5 in. Repeat every 30 s. Do Flow rate: 1 mL/min
not keep the test tube out of the bath longer than 3 s Injection volume: 40 wL
for each shaking period. At the end of 10 min from the System suitabilit
time when first placed in the water bath, remove the Sample: Sample solution
test tube. [Note—The order of elution is triglycerides, diglycerides,
Acceptance criteria: The oil portion of the Sample may monoglycerides, and glycerin.]
turn hazy, but it remains colorless or shows a slight Suitability requirements
pink or yellow color, and the acid portion of the Sample Relative standard deviation: NMT 1.0%, determined
does not become darker than the Standard solution. from the monoglycerides peak
e SOLID PARAFFIN Analysis
Samples Light Mineral Oil that has been dried previ- Sample: Sample solution
ously in a beaker at 105° for 2 h and cooled to room Calculate the percentage of monoglycerides in the
temperature in a desiccator over silica gel portion of Mono- and Di-glycerides taken:
NF Monographs

Analysis: Fill a tall, cylindrical, standard oil-sample bot- Result = (ru/r) x 100
tle of colorless glass of 120-mL capacity with the Sam-
ple. Insert the stopper, and immerse the bottle in a mix- ru = peak response for monoglycerides
ture of ice and water for 4 h. tr = sum of the responses of all the peaks, except
Acceptance criteria: The Sample is sufficiently clear that the solvent peak
a black line 0.5 mm in width, on a white background, Acceptance criteria: 90.0%-110.0% of the value indi-
held vertically behind the bottle, is clearly visible. cated in the labeling
e Limit OF SULFUR COMPOUNDS
Solution A: Saturated solution of lead(II) oxide in so-
dium hydroxide (200 mg/mL)
NF 36 Official Monographs / Monoglyceride 5455

IMPURITIES IDENTIFICATION
e RESIDUE ON IGNITION (281): NMT 0.1% e A. INFRARED ABSORPTION (197F)
© ARSENIC, Method I] (211): NMT 3 ug/g
ASSAY
e PROCEDURE
Delete the following: Sample solution: Weigh a glass-stoppered weighing
bottle containing 25 mL of water. Add 1 g of
°e HEAVY METALS, Method 1 231): NMT 10 ug/ge cortical. Monoethanolamine, and reweigh.
Jan-2018) Blank: 25 mL of water
e LIMIT OF FREE GLYCERIN Titrimetric system
Mobile phase, Sample solution, and Chromatographic (See Titrimetry (541).)
system: Proceed as directed in the Assay for Mode: Direct titration
Monoglycerides. Titrant: 0.5 N hydrochloric acid VS
Standard solutions: 0.5, 1.0, 2.0, and 4.0 mg/mL of Endpoint detection: Visual
USP Glycerin RS in tetrahydrofuran Analysis: Transfer the Sample solution to a suitable flask,
Analysis add a mixed indicator of 5 parts bromocresol green TS
Samples: Sample solution and Standard solutions and 6 parts methyl red TS for a total of approximately
Measure the responses for the glycerin peaks. Plot the 11 parts of solution. Titrate the Sample solution with Ti-
concentration, in mg/mL, of USP Glycerin RS in the trant. Perform a blank determination.
Standard solutions versus the glycerin peak responses Calculate the percentage of monoethanolamine
obtained. From the standard curve so obtained, de- (C2H7NO) in the portion of sample taken:
termine the glycerin concentration in the Sample
solution. Result ={[(Vs — Vs) x N x F]/W} x 100
Calculate the percentage of glycerin in the portion of
Mono- and Di-glycerides taken: Vs = Titrant volume consumed by the Sample
solution (mL)
Result = (Cy/Cs) x 100 Ve = Titrant volume consumed by the Blank (mL)
N = actual normality of the Titrant (mEq/mL)
Cu = gfycerin concentration in the Sample solution F = equivalency factor, 61.08 mg/mEq
tom the standard curve (mg/mL) Ww = sample weight (mg)
& = concentration of the Sample solution (mg/mL) Acceptance criteria: 98.0%-100.5%
Acceptance criteria: NMT 7.0%
IMPURITIES
SPECIFIC TESTS e RESIDUE ON IGNITION (281): NMT 0.1%
e FATS AND FIXED Os, Acid Value (401): NMT 4
e FATS AND FIXED OILS, Hydroxyl Value (401): SPECIFIC TESTS
90.0%-110.0% of the value indicated in the labeling e SPECIFIC GRAVITY (841): 1.013-1.016
e FATS AND FIXED OILS, /odine Value (401): 90.0%-110.0% e DISTILLING RANGE, Method II (721): NLT 95% of it distills
of the value indicated in the labeling. If the value stated between 167° and 173°, a correction factor of 0.052°
in the labeling is less than 10, the iodine value is NMT per mm applied as necessary.
10.
e FATS AND FIXED OtLs, Saponification Value (401): ADDITIONAL REQUIREMENTS
90.0%-110.0% of the value indicated in the labeling © PACKAGING AND STORAGE: Preserve in tight, light-resistant
containers.
ADDITIONAL REQUIREMENTS e USP REFERENCE STANDARDS (11)
e PACKAGING AND STORAGE: Preserve in tight, light-resistant USP Monoethanolamine RS
containers.
e LABELING: The labeling indicates the monoglyceride con-
tent, hydroxyl value, iodine value, saponification value,
and name and quantity of any stabilizers.
e USP REFERENCE STANDARDS (11) Monoglyceride Citrate
USP Glycerin RS
Citric acid ester of glyceryl monooleate [36291-32-4].
DEFINITION
Monoglyceride Citrate is a mixture of glyceryl monooleate
and its citric acid monoester, manufactured by the reac-
Monoethanolamine tion of glyceryl monooleate with citric acid under con-
trolled conditions. It contains NLT 14.0% and NMT
woo ae of total citric acid, calculated on the anhydrous
asis.
CoH7NO 61.08 IDENTIFICATION
Ethanol, 2-amino-; eA.
2-Aminoethanol [141-43-5]. Sample: 1g
DEFINITION Analysis: Reflux the Sample with 15 mL of 0.5 N potas-
sydeibouo- 4N

Monoethanolamine contains NLT 98.0% and NMT 100.5%
by weight of monoethanolamine (C2H7NO).
sium hydroxide solution in dehydrated alcohol for 1 h.
Add 15 mL of water, and acidify with diluted hydro-
chloric acid (about 6 mL). Dissolve any oil drops or solid
produced in 5 mL of hexane. Remove the hexane layer,
extract again with 5 mL of hexane, and again remove
the hexane layer.
[Note—Keep the resulting aqueous layer for /dentifica-
tion tests B and C.]
 5456 Monoglyceride / Official Monographs NF 36

Acceptance criteria: Oil drops or a white to yellowish- Au = absorbance of the Sample solution
white solid are produced that are soluble in 5 mL of As = absorbance of the Standard solution
hexane. Vv = volume of the Sample solution (mL)
e B. IDENTIFICATION TESTS—GENERAL, Citrate (191) Cs = concentration of USP Citric Acid RS in the
Sample: 1 mL of the aqueous layer resulting from /den- Standard solution (mg/mL)
tification test A w = weight of Monoglyceride Citrate taken to
Analysis: Evaporate the Sample in a porcelain dish. prepare the Sample solution (mg)
Acceptance criteria: The residue meets the Acceptance criteria: 14.0%-17.0% on the anhydrous
requirements. asis
ec.
Sample: 5 mL of the aqueous layer resulting from /den- IMPURITIES
tification test A ° Renu ON IGNITION (281): NMT 0.3%, determined on
Analysis: Transfer the Sample to a test tube. Add excess 9
calcium hydroxide as a powder, place in boiling water
for 5 min, shaking several times, cool, and filter. Trans- Delete the following:
fer one drop of the filtrate into a test tube, and add
about 50 mg of potassium hydrogen sulfate. On top of °e HEAVY METALS, Method I! (231): NMT 10 ppme comal1-
the test tube, place a filter paper moistened with a rea- Jan-2018)
gent for acrolein consisting of a mixture of 5% ni-
troprusside solution in water and 20% piperidine solu- SPECIFIC TESTS
tion in water (1:1). Heat the test tube. e FATS AND FIXED OILS, Acid Value (401): 70-100
Aceptance criteria: The filter paper turns blue (pres- © FATS AND FIXED OILS, Saponification Value (401): 260-265
ence of glycerin). The color changes to light red after © WATER DETERMINATION, Method | (921): NMT 0.2%
addition of sodium hydroxide TS.
ADDITIONAL REQUIREMENTS
ASSAY © PACKAGING AND STORAGE: Preserve in well-closed contain-
e CONTENT OF CITRIC ACID ers. No storage requirements specified.
Standard solution: 0.23 mg/mL of USP Citric Acid RS e USP REFERENCE STANDARDS (11)
Sample solution: Transfer 150 mg of Monoglyceride USP Citric Acid RS
Citrate into a saponification flask, add 50 mL of 4% po-
tassium hydroxide solution in dehydrated alcohol, and
reflux for 1 h. Acidify the reaction mixture with hydro-
chloric acid to a pH of 2.8-3.2, transfer into a 400-mL
beaker, and evaporate to dryness on a steam bath. Monosodium Glutamate
Quantitatively transfer the contents of the beaker into a
separator, using NMT 50 mL of water, and extract with
three 50-mL portions of petroleum ether, discarding the
extracts. Transfer the water layer to a 100-mL volumet- HO" ‘O07 Nat + HO
ric flask, and dilute with water to volume.
Blank: Water
Instrumental conditions CsHgNNaQOz - H20 187.13
Mode: UV-Vis t-Glutamic acid, sodium salt, hydrate;
Analytical wavelength: 450 nm Monosodium L-glutamate, hydrate [6106-04-3].
Cell; 1.cm
Analysis DEFINITION
Samples: Standard solution, Sample solution, and Blank Monosodium Glutamate contains NLT 99.0% and NMT
Pipet 2.0 mL each of the Standard solution, Sample solu- 100.5% of monosodium glutamate (CsHsNNaQO,- H20).
tion, and Blank into separate 40-mL graduated centri-
fuge tubes. Add 2 mL of a 1 in 2 sulfuric acid solution IDENTIFICATION
and 11 mL of water to each tube. Boil for 3 min, cool, e A. INFRARED ABSORPTION (197A)
and add 5 mL of bromine TS to each tube. Dilute to
20 mL, allow to stand for 10 min, and centrifuge.
Transfer 4.0 mL of the supernatant from each tube into Change to read:
separate 19- x 110-mm test tubes, add 1 mL of water,
0.5 mL of a 1 in 2 sulfuric acid solution, and 0.3 mL of e B. IDENTIFICATION TESTS—GENERAL (191), Sodium: It meets
1M potassium bromide, and shake. Add 0.3 mL of 1.5 the requirements of test Ave (cw i-itey-2018)
N potassium permanganate, shake, and allow to stand ASSAY
for 2 min. Add 1 mL of a saturated solution of ferrous © PROCEDURE
sulfate, shake, allow to stand for 2 min, and then di- Sample: 250mg
lute with water to 10 mL. Add 10.0 mL of n-hexane Titrimetric system
(previously washed with sulfuric acid, followed by a (See Titrimetry (541).)
water wash, and then dried over anhydrous sodium Mode: Direct titration
sulfate), shake vigorously for 2 min, and centrifuge at Titrant: 0.1 N perchloric acid VS
low speed for 1 min. Transfer 5.0 mL of the hexane Blank: 100 mL of glacial acetic acid with a few drops
extract into a 20- x 145-mm tube containing 10.0 mL
NF Monographs

of 4% sodium sulfide solution, and briefly shake vigor-
ously (three oscillations only). Centrifuge the mixture
at low speed for 1 min. Immediately determine the
absorbance of each aqueous layer from the Standard
solution and Sample solution against the aqueous layer
from the Blank.
Calculate the percentage of citric acid in the portion of
Monoglyceride Citrate taken:
Result = (Au/As) x (V x Cs/W) x 100
of water
Endpoint detection: Potentiometric
Analysis: Wet the Sample with a few drops of water.
Dissolve in 100 mL of glacial acetic acid. Titrate with
0.1 N perchloric acid VS. Perform a blank
determination.
Calculate the percentage of monosodium glutamate
(CsHsNNaO, - H2O) in the Sample taken:
Result = [(Vs — Vs) x Na x Fx 100]/W
 Official Monographs / Myristic 5457

Vs = Titrant volume consumed by the Sample (mL) Acceptance criteria: 97.0%-101.0% on the anhydrous
Vp = Titrant volume consumed by the Blank (mL) basis
Na = actual normality of the Titrant (mEq/mL)
F = equivalency factor, 93.56 mg/mEq IMPURITIES
w = Sample weight (mg) e RESIDUE ON IGNITION (281): NMT 0.1%
Acceptance criteria: 99.0%-100.5% e SELENIUM (291)
Test solution: 200 uL
IMPURITIES Acceptance criteria: 30 g/g
e CHLORIDE AND SULFATE (221), Chloride: A 280-mg portion
shows no more chloride than corresponds to 1.0 mL of
0.020 N hydrochloric acid (0.25%). Delete the following:
e LEAD (251): NMT 10 ug/g
°o HEAVY METALS, Method I/ (231): NMT 20 L1g/ge corticiat 1-
Jan-2018)
Delete the following:
SPECIFIC TESTS
°e HEAVY METALS, Method ] (231): NMT 20 ug/ge coriciat1- e SPECIFIC GRAVITY (841): 1.241-1.250
Jan-2013) e REFRACTIVE INDEX (831): 1.521-1.526
e PH (791)
SPECIFIC TESTS Sample solution: 1 in 10
e CLARITY AND COLOR OF SOLUTION Acceptance criteria: 3.5-7.0
Sample solution: 1.0g in 10 mL of water e WATER DETERMINATION, Method II (921): NMT 5.0%
Standard solution: To 0.2 mL of a solution of sodium
chloride containing 10 g/mL of chloride ion (Cl), add ADDITIONAL REQUIREMENTS
20 mL of water and mix. Then add 1 mL of 5 N nitric © PACKAGING AND STORAGE: Preserve in tight containers.
acid, 0.2 mL of dextrin solution (1 in 50), and 1 mL of
silver nitrate TS, and allow to stand for 15 min.
Analysis: Compare the Sample solution with the Stan-
dard solution (see Nephelometry, Turbidimetry, and Visual
Comparison (855)). Myristic Acid
Acceptance criteria: The Sample solution is colorless
and has no more turbidity than the Standard solution. °
© OPTICAL ROTATION (781S), Procedures, Specific Rotation Wee Ax,
Sample solution: 100 mg/mL in 2 N hydrochloric acid
Acceptance criteria: +24.8° to +25.3°, determined at
20° Cy4H2gO2 228.37
e PH (791): 6.7-7.2, in a solution (1 in 20) Tetradecanoic acid;
e Loss ON DRYING (731) 1-Tetradecanoic acid;
Analysis: Dry at 100° for 5 h. 1-Tridecanecarboxylic acid [544-63-8].
Acceptance criteria: NMT 0.5%
DEFINITION
ADDITIONAL REQUIREMENTS Myristic Acid is obtained from coconut oil and other fats. It
¢ PACKAGING AND STORAGE: Preserve in tight containers. contains NLT 97.0% of myristic acid (Ci4H23O2).
e USP REFERENCE STANDARDS (11)
USP Monosodium Glutamate RS IDENTIFICATION
© A. INFRARED ABSORPTION (197D) or (197K)
Sample: Undried specimen
Acceptance criteria: Meets the requirements
e B. The retention time of the major peak of the Sample
Monothioglycerol solution corresponds to that of the Standard solution, as
obtained in the test for Fats and Fixed Oils, Fatty Acid
Composition in the Assay.
ws Sou
ASSAY
e FATS AND FIXED OILS, Fatty Acid Composition (401)
System suitability solution: Prepare as directed in the
C3Hg02S 108.16 chapter, except that only stearic acid and palmitic acid
1,2-Propanediol, 3-mercapto-; are used.
3-Mercapto-1,2-propanedio] [96-27-5]. Sample solution: Prepare as directed for the Test Solu-
tion in the chapter.
DEFINITION Standard solution: Prepare as directed for the Sample
Monothioglycerol contains NLT 97.0% and NMT 101.0% of solution, using 100 mg of USP Myristic Acid RS instead
monothioglycerol (C3HsO2S), calculated on the anhydrous of the substance to be examined.
basis. Chromatographic system: Prepare as directed in the
chapter.
ASSAY Injection size: 1 uL
sydeibouo-= 4N

© PROCEDURE System suitability

Sample: 400 mg (See Chromatography (621), System Suitability.)
Titrimetric system Sample: System suitability solution
(See Titrimetry (541).) Suitability requirements
Mode: Direct titration Resolution: NLT 1.5 between methyl stearate and
Titrant: 0.1 N iodine VS methyl palmitate
Endpoint detection: Visual Analysis
Analysis: Dissolve the Sample in 50 mL of water. Titrate Samples: Standard solution and Sample solution
with Titrant, adding 3 mL of starch TS as the endpoint Identify the methyl myristate peak from the Sample so-
is approached. Each mL of Titrant is equivalent to lution by comparing the retention times of the peaks
10.82 mg of monothioglycerol (C3HgO2S).
 5458 Myristic / Official Monographs
with those from the Standard solution. Measure the re-
sponses for all the peaks from the Sample solution, ex-
cluding the solvent peak.
Calculate the percentage of myristic acid (C,4H2gO2) in
the portion of Myristic Acid taken:
Result = (A/B) x 100
A = peak response for methyl! myristate from the
Sample solution
B = sum of all the peak responses in the Sample
solution except the solvent peak
Acceptance criteria: NLT 97.0%
IMPURITIES
© RESIDUE ON IGNITION (281): NMT 0.1%
e LIMIT OF LEAD
[Note—Select reagents with as low a lead content as
practicable, and store all solutions in high-density poly-
ethylene containers. Rinse all plastic and glassware thor-
oughly with warm 8N nitric acid followed by deionized
water.]
Standard stock solution: Dissolve 160 mg of lead ni-
trate in 100 mL of water containing 1 mL of nitric acid.
Dilute with water to 1000 mL.
Standard solutions: [NotT&—Prepare these solutions on
the day of use.] Transfer 10.0 mL of Standard stock solu-
tion to a 100-mL volumetric flask, and dilute with water
to volume. Each mL of this solution contains the equiv-
alent of about 10 jg of lead. Dilute accurately meas-
ured volumes of the diluted Standard stock solution with
water to obtain solutions with known concentrations of
1, 2, and 5 ug/mL of lead.
Sample solution: Transfer 5 g of Myristic Acid to an
evaporating dish. Add 5 mL of a 25% sulfuric acid solu-
tion, and distribute the sulfuric acid uniformly through
the sample. Within a hood, place the dish on a steam
bath to evaporate most of the water. Place the dish on
a burner, and slowly pre-ash the sample by expelling
most of the sulfuric acid. Place the dish in a muffle
furnace that has been set at 525°, and ash the sample
until the residue appears free from carbon. Prepare a
blank by ashing 5 mL of a 25% sulfuric acid solution.
Cool, and cautiously wash down the inside of each
evaporation dish with water. Treat both the sample and
the blank as follows. Add 5 mL of 1 N hydrochloric
acid. Place each dish on a steam bath, and evaporate to
dryness. To each dish add 1.0 mL of 3 N hydrochloric
acid and about 5 mL of water, and heat briefly on a
steam bath to dissolve any residue. Transfer each solu-
tion quantitatively to a 10-mL volumetric flask, and di-
lute with water to volume.
Instrumental conditions
(See Atomic Absorption Spectroscopy (852).)
Mode: Atomic a sorption spectrophotometry
Analytical wavelength: 283.3 nm at the lead emission
line
Lamp: Lead electrodeless discharge
Flame: Air—acetylene with a suitable burner head
Slit width: 0.7 nm
Blank: Water. [NoTE—Perform a blank determination
following the manufacturer’s operating instructions.]
Analysis
Samples: Standard solutions, Sample solution, and Blank
Determine the corrected absorbance values by sub-
NF Monographs
tracting the absorbance of the Blank from the absorb-
ance of each of the Standard solutions and from the
absorbance of the Sample solution. Prepare a standard
curve by plotting the corrected absorbance values of
the Standard solutions versus their corresponding con-
centration, in g/mL. From the calibration curve, de-
termine the lead concentration in the Sample solution.
NF 36
Calculate the lead content, in ppm, in the portion of
Myristic Acid taken:
Result = (C/Ws) x V
€ = measured concentration of lead in the Sample
solution from the standard curve (ug/mL)
Ws = welget of Myristic Acid taken (g)
Vv = final volume of the Sample solution, 10 mL
Acceptance criteria: NMT 2 ppm
e Limit OF MINERAL AcIDs
Sample: 5g of melted Myristic Acid
Analysis: Shake the Sample with an equal volume of
hot water for 2 min, cool, and filter.
Acceptance criteria: The filtrate is not reddened by the
addition of 1 drop of methyl orange TS.
SPECIFIC TESTS
© CONGEALING TEMPERATURE (651): 48°-55.5°
FATS AND FIXED OILS, Acid Value (401): 242-249
FATS AND FIXED OILS, /odine Value (401): NMT 1.0
FATS AND FIXED OILS, Peroxide Value (401): NMT 10.0
ltd AND FIXED Os, Unsaponifiable Matter (401): NMT
© WATER DETERMINATION, Method | (921): NMT 0.2%
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in well-closed contain-
ers. No storage requirements specified.
e USP REFERENCE STANDARDS (11)
USP Myristic Acid RS
Myristyl Alcohol
Hye NNN
Ci4H300 214,39
n-Tetradecan-1-ol;
1-Tetradecanol;
1-Hydroxytetradecane;
1-Tetradecyl alcohol [112-72-1].
DEFINITION
Myristyl Alcohol contains NLT 90.0% and NMT 102.0% of
myristyl alcohol (C;4H300), the remainder consisting
chiefly of related alcohols. It is obtained from sources of
vegetable, animal, or synthetic origin.
IDENTIFICATION
© A. CHROMATOGRAPHIC IDENTITY
Analysis: Proceed as directed in the Assay.
Acceptance criteria: The retention time of the major
peak of the Sample solution, excluding the solvent and
internal standard peaks, corresponds to the myristyl al-
cohol peak of the Standard solution.
ASSAY
e PROCEDURE
Internal standard solution: 1 mg/mL of 1-pentadeca-
nol (internal standard) in ethanol
System suitability solution: pare 1 mg/mL of USP
Cetyl Alcohol RS, 1 mg/mL of USP Stearyl Alcohol RS,
and 1 mg/mL of USP Oley! Alcohol RS in Internal stan-
dard solution, and heat the solution in a sealed con-
tainer in a 50° water bath until all fatty alcohols are
dissolved. Allow the solution to cool to room tempera-
ture, and mix well.
Standard solution: 1.0 mg/mL of USP Myristyl Alcohol
RS in Internal standard solution
Sample solution: 1.0 mg/mL of Myristyl Alcohol in In-
ternal standard solution
NF 36 Official Monographs / Myristyl 5459

Chromatographic system Cu = concentration of Myristyl Alcohol in the

(See Chromatography (621), System Suitability.) Sample solution (mg/mL)
Mode: GC Acceptance criteria: 90.0%-102.0%
Detector: Flame ionization
Column: 0.25-mm x 30-m fused-silica capillary col- IMPURITIES
umn, coated with a 0.25-um layer of phase G7 e RESIDUE ON IGNITION (281): NMT 0.1%, determined on
Temperatures 29
Injection port: 270° © LIMIT OF RELATED FATTY ALCOHOLS
Detector: 280° Solution A: 1 mg/mL of 1-pentadecanol in ethanol
Column: See Table 1. Resolution solution: Prepare 1 mg/mL of USP Lauryl Al-
cohol RS, 1 mg/mL of USP Myristy! Alcohol RS, 1 mg/
mL of USP Cetyl Alcohol RS, 1 mg/mL of USP Stearyl
Table 1 Alcohol RS, and 1 mg/mL of USP Oleyl Alcohol RS in
Hold Time at Solution A. Heat the solution in a sealed container in a
Initial Temperature Final Final 50° water bath until all fatty alcohols are dissolved. Al-
Temperature Ramp Temperature | Temperature low the solution to cool to room temperature, and mix
©) (¢/min) (cy (min) well. Dilute the solution with ethanol to obtain a solu-
60 20 180 — tion containing 0.05 mg/mL each of USP Lauryl Alcohol
180 10 220 5 RS, USP Myristyl Alcohol RS, USP Cetyl Alcohol RS,
1-pentadecanol, USP Stearyl Alcohol RS, and USP Oleyl
Carrier gas: Hydrogen Alcohol RS.
Flow rate: 2.0 mL/min, constant flow mode Sample solution: 1 mg/mL of Myristyl Alcohol in
Injection volume: 1 uL ethanol
Injection ae Split injection; split ratio is 100:1 Chromatographic system: Proceed as directed in the
Liner: Single taper, low pressure drop liner with deac- Assay, except for the split ratio.
tivated wool Injection eee: Split injection; split ratio is 5:1
Run time: 15 min System suitability
System suitability Sample: Resplatlen solution
Samples: System suitability solution and Standard [Note—See Table 3 for the relative retention times.]
solution
[Note—See Table 2 for the relative retention times.] Table 3
Relative
Table 2 Retention
Relative Component Time
Retention Lauryl alcohol 0.79
Component Time Myristyl alcohol 0.92
Myristyl alcohol 0.92 1-Pentadecanol 1.00
1-Pentadecanol Cetyl alcohol 1.08
(internal standard) 1.00 Stearyl alcohol 225
Cetyl alcohol 1.08 Oleyl alcohol V27:
Stearyl alcohol 1,25.
Oley! alcohol 1:27. Suitability requirements
Resolution: NLT 15 between myristyl alcohol and
Suitability requirements 1-pentadecanol peaks; NLT 30 between the cetyl al-
Resolution: NLT 30 between the cetyl alcohol and cohol and stearyl alcohol peaks; NLT 2.0 between the
stearyl alcohol ae NLT 2.0 between the stearyl al- stearyl alcohol and oleyl alcohol peaks
cote and oley! alcohol peaks, System suitability Analysis
solution Samples: Resolution solution and Sample solution
Tailing factor: 0.8-1.8 for the myristyl alcohol and Identify each related fatty alcohol peak in the Sample
1-pentadecanol peaks, Standard solution solution based on that in the Resolution solution.
Relative standard deviation: NMT 1%, using the Calculate the percentage of each related fatty alcohol or
area ratio of myristyl alcohol to 1-pentadecanol, Stan- any unspecified impurity in the portion of Myristyl Al-
dard solution cohol taken:
Analysis
Samples: Standard solution and Sample solution Result = (ru/r7) x 100
Calculate the percentage of myristyl alcohol (Ci4H30O)
in the portion of Myristyl Alcohol taken: ty = peak response of each related fatty alcohol (or
any unspecified impurity) from the Sample
Result = (Ru/Rs) x (Cs/Cu) x 100 solution
nr = sum of all the peak responses excluding peak
Ru peak feapanse ratio of myristyl alcohol to the responses due to solvent from the Sample
Minter standard (peak response of myristyl solution
Sie response of the internal Acceptance criteria: Disregard peaks that are less than
standard) from the Sample solution 0.05% for any unspecified impurities, and any peaks
ExtTels]oelereyAMET

Rs = peak fesponse ratio of myristyl alcohol to the due to solvent.

internal standard (peak response of myristy!
geonal pen response of the internal
standard) from the Standard solution
Cs = concentration of USP Myristyl Alcohol RS in
the Standard solution (mg/mL)
Sum of unspecified impurities: NUT 1%
Sum of related fatty alcohols and unspecified impuri-
ties: NMT 10.0%
 5460 Myristyl / Official Monographs NF 36

SPECIFIC TESTS Mode: LC

e FATS AND FIXED OILS, Acid Value (401): NMT 2 Detector: UV 210 nm
© FATS AND FIXED OILS, /odine Value (401): NMT 1 Column: 4.6-mm x 10-cm; packing L1
¢ FATS AND FIXED OILS, Hydroxyl Value (401): 250-267 Column temperature: 45°
e@ WATER DETERMINATION, Method | (921): NMT 0.5% Flow rate: 1.5 mL/min
Injection volume: 25 uL
ADDITIONAL REQUIREMENTS System suitability
e PACKAGING AND STORAGE: Preserve in well-closed Sample: Standard solution
containers. Suitability requirements
e LABELING: Label it to indicate whether it is derived from Tailing factor: NMT 2.0
vegetable, animal, or synthetic sources. Relative standard deviation: NMT 2.0%
e USP REFERENCE STANDARDS (11) Analysis
USP Cetyl Alcohol RS Samples: Standard solution and Sample solution
USP Lauryl Alcohol RS Calculate the percentage of neotame (C2oH30N20s) in
USP Myristyl Alcohol RS the portion of Neotame taken:
USP Oleyl Alcohol RS
USP Stearyl Alcohol RS Result = (ru/rs) « (Cs/Cy) x 100

tu = peak response from the Sample solution

rs = peak response from the Standard solution
Cs = concentration of USP Neotame RS in the
Neotame Standard solution (mg/mL)
Cu = concentration of Neotame in the Sample
solution (mg/mL)
tJJ ‘
Acceptance criteria: 97.0%-102.0% on the anhydrous
basis
we aL oon IMPURITIES
wad ‘os | e RESIDUE ON IGNITION (281): NMT 0.2%
bif e LEAD
[Note—Use acid-cleaned (mixture of 5% nitric acid and
5% hydrochloric acid followed by rinsing with water)
Ca0H30N20s 378.46 autosampler cups and volumetric glassware to avoid
L-Phenylalanine, N-[N-(3,3-dimethylbutyl)-l-c-aspartyl]- contamination. For the preparation of all aqueous solu-
1-methyl ester; tions and for the rinsing of glassware before use, use
N-[N-(3,3-Dimethylbutyl)-L-c-aspartyl]-L-phenylalanine water that has been passed througha strong-acid,
j-methyl ester [165450-17-9]. strong-base, mixed-bed ion-exchange resin. Select all
DEFINITION reagents to have as low a content of lead as practica-
Neotame contains NLT 97.0% and NMT 102.0% of ne- ble. Store standards and samples in acid-cleaned poly-
otame (C20H30N20s), calculated on the anhydrous basis. ethylene containers.]
Diluent: Transfer 2 mL of lead-free nitric acid into a
IDENTIFICATION 1000-mL volumetric flask, dilute with water to volume,
e A. INFRARED ABSORPTION (197K) and mix.
Standard stock solution: 79.9 mg of lead nitrate in
ASSAY 100 mL of Diluent in a 500-mL volumetric flask, and
e PROCEDURE dilute with Diluent to volume. Transfer 10.0 mL of the
Mobile phase: Dissolve 3.0 g of sodium 1-heptanesul- resulting solution into a 100-mL volumetric flask, and
fonate in 740 mL of water in a suitable 1000-mL vessel, dilute with Diluent to volume. Each mL of the Standard
and add 3.8 mL of triethylamine. Adjust the resulting stock solution contains the equivalent of 10 tg of lead.
solution with phosphoric acid to a pH of 3.5, and dilute Standard solution A: Dilute an aliquot of the Standard
with water to 750 mL. Add 250 mL of acetonitrile, and stock solution with Diluent to obtain a solution having a
adjust with phosphoric acid to an apparent pH of 3.7. concentration of 0.03 ug/mL.
Standard solution: 1.0 mg/mL of USP Neotame RS in Standard solution B: Dilute an aliquot of the Standard
Mobile phase stock solution with Diluent to obtain a solution having a
Sample solution: 1.0 mg/mL of Neotame in Mobile concentration of 0.015 g/mL.
phase. [NoTE—This solution is stable for up to 32 h Sample solution: Transfer 160 mg of Neotame to a
when stored at a temperature of 0°-10°.] 10-mL volumetric flask. Dissolve in and dilute with Dilu-
Chromatographic system ent to volume.
(See Chromatography (621), System Suitability.) Blank: Diluent
Instrumental conditions
(See Atomic Absorption Spectroscopy (852).)
[Note—Optimize the instrument program as recom-
mended by the manufacturer for lead, using a char
temperature of 500° and an atomization temperature
of 2000°.]
NF Monographs

Mode: Atomic absorption spectrophotometer with a

graphite furnace, ie ly coated graphite tubes, a
solid pyrolytic graphite platform, and a background
compensation system
USP 41 Dietary Supplements / Calcium 4497

Relative standard deviation: Prepare three separate Analysis

Standard solutions, and inject each immediately and Samples: Standard solution and Sample solution
only one time. NMT 2.0%; peak response factor from [Note—The impurities are listed in Table 2.]
three injections Calculate the percentage of each impurity, as free acid,
Analysis Ce portion of Calcium L-5-Methyltetrahydrofolate
Samples: Standard solution and Sample solution taken:
Calculate the percentage of calcium 5-methylte-
trahydrofolate (C2oH23CaN7O6¢), the sum of the L- and Result = (ru/rs) x (Cs/Cu) x Fx (Mr/M,2) x 100
D-diastereoisomers, in the portion of Calcium L-
5-Methyltetrahydrofolate taken: tu = peak response of the corresponding impurity
from the Sample solution
Result = (ru/rs) x (Cs/Cu) x 100 rs = peak response of the principal peak from the
Standard solution
Ty = peak response from the Sample solution Cs = concentration of USP Calcium DL-
rs = peak response from the Standard solution 5-Methyltetrahydrofolate RS in the Standard
G = concentration of USP Calcium D1L- solution (mg/mL)
5-Methyltetrahydrofolate RS in the Standard Cy = concentration of Calcium L-5-Methyltetra-
solution (mg/mL) hydrofolate in the Sample solution (mg/mL)
Cu = concentration of Calcium L-5-Methyltetra- F = relative response factor for the corresponding
hydrofolate in the Sample solution (mg/mL) impurity peak (see Table 2)
Acceptance criteria: 95.0%-102.0% on the anhydrous Mn = molecular weight of L-5-methyltetrahydrofolic
and solvent-free basis acid, 459.46
Mr2 = molecular weight of calcium L-5-methyltetra-
IMPURITIES hydrofolate, 497.52
© CHLORIDE Acceptance criteria
Sample: 300mg [Note—Disregard any impurity peak less than 0.05%.]
Blank: Mix 1 mL of nitric acid with 75 mL of water. Individual impurities: See Table 2.
Titrimetric system
(See Titrimetry (541).)
Table 2
Mode: Direct titration
Titrant: 0.005 M silver nitrate VS Relative Relative Acceptance
Endpoint detection: Potentiometric Retention Response Criteria,
Analysis: Dissolve the Sample in 75 mL of water (heat Name Time Factor NMT (%)
to maximum of 40°), add 1 mL of nitric acid, and ti- 4-Aminobenzoyl-
trate with the Titrant. Perform a Blank determination, glutamic acidé 0.29 0.91 0.5
and make any necessary correction. 4a-Hydroxy-5-methyl-
Calculate the percentage of chloride (Cl) in the Sample tetrahydrofolic acid’ 0.37 1.09 1.0
taken:
(6R)-Mefoxs4 0.49 1.05 _
Result = [(Vs — Vs) x M x F/W] x 100 1.0 (sum of
(65)-Mefox«s 0.50 1.05 6R and 6S)
Vs = volume of Titrant consumed by the Sample Tetrahydrofolic acide 0.65 1.00* 0.5
(ml) 7,8-Dihydrofolic acidt 0.83 0.95 0.5
Ve = volume of Titrant consumed by the Blank (mL) Folic acids 0.85 0.83 0.5
M = actual molarity of the Titrant pene ae
F = equivalency factor, 35.45 mg/mmol 5,10-Methylenete-
w = Sample weight (ng) trahydrofolic acid’ 0.88 1.00* 0.5
Acceptance criteria: NMT 0.5% 5-Methyltetrahydrop-
e ELEMENTAL IMPURITIES—PROCEDURES (233) teroic acid’ 1.10 0.67 0.5
Acceptance criteria Dimethylte-
Boron: NMT 50 ug/g trahydrofolic acidi 1:25 1.00« 0.15
Platinum: NMT 10 yg/g Total impurities — = 2.5)
sydesBouo-= sa

Arsenic: NMT 1.5 ae 2 N-(4-Aminobenzoyl)-t-glutamic acid.

Cadmium: NMT 0.5 ug/g > N-[4-({[(65)-2-Amino-4a-hydroxy-5-methyl-4-oxo-1,4,4a,5,6,7,8,8a-
Lead: NMT 1.0 ug/g octahydropteridin-6-yl]methyl}amino)benzoyl]-\-glutamic acid.
Mercury: NMT 1.5 ug/g ¢ 2-Amino-8-methyl-4,9-dioxo-7-methyl-p-aminobenzoyl-glutamate-6,7,8,
e RESIDUAL SOLVENTS (467) 9-tetrahydro-4H-pyrazino-(1,2-a)-s-triazine.
Acceptance criteria 4 Report the impurity Mefox as the sum of 65- and 6R-Mefox.
Ethanol: NMT 0.5% © N-[4-({[(S)-2-Amino-4-0xo-1,4,5,6,7,8-hexahydropteridin-6-yl]meth-
2-Propanol: NMT 0.5% yl}amino)benzoyl]-l-glutamic acid.
¥ N-(4-{[(2-Amino-4-oxo-1,4,7,8-tetrahydropteridin-6-yl)methyl]ami-
[Note—For acceptance criteria for any other residual sol- no}benzoyl)-L-glutamic acid.
vents, see Residual Solvents (467).] 9 N-(4-{[(2-Amino-4-oxo-1 ,4-dihydropteridin-6-yl)methylJamino}benzoyl)-L-
e RELATED COMPOUNDS glutamic acid.
Solution A, Solution B, Mobile phase, System suitabil- N-(4-(3-Amino-1-oxo-5,6,6a,7-tetrahydroimidazo[1,5-flpteridin-8(1H,4H,
ee solution, Standard solution, Sample solution, 9H)-yl)benzyl)-L-glutamic acid.
Chromatographic system, System suitability, and 1 ($)-4-{[(2-Amino-5-methyl-4-oxo-1,4,5,6,7,8-hexahydropteridin-6-
any requirements: Proceed as directed in the yl)methylJamino}benzoic acid.
\ssay. i EE ies) 5 Meth 2 (enetiylamino) 4-oXo-1,4,5, 6/7, tiexehyaropter
idin-6-yl]methyl}amino)benzoy!]-L-glutamic acid.
* Estimated factor.

e ENANTIOMERIC PURITY
Buffer: 4.54 g/L of sodium dihydrogen phosphate dihy-
drate in water
Mobile phase: Acetonitrile and Buffer (3:97). Adjust
with 32% (w/v) sodium hydroxide to a pH of 6.8.
NF 36 Official Monographs / Nitric 5461

Analytical wavelength: 283.3 nm Cs = concentration of USP Neotame RS in Standard

Lamp: Lead hollow-cathode solution B (mg/mL)
Purge gas: Argon Cu = concentration of Neotame in the Sample
Alternate gas: Breathing-quality air solution (mg/mL)
Volume: 15 pL Acceptance criteria
Analysis Neotame related compound A: NMT 1.5%
Samples: Standard solution A, Standard solution B, Other impurities: NMT 2.0%
Sample solution, and Blank
Correct the peak areas of the Sample solution, Standard SPECIFIC TESTS
solution A, and Standard solution B for the Blank peak e OPTICAL ROTATION (7815S), Procedures, Specific Rotation
area. Generate the appropriate lead calibration al- Sample solution: 5 mg/mL in water
gorithm, and determine the lead concentration in the Acceptance criteria: —40.0° to —43.4°, at 20°
Sample solution, in g/mL. © WATER DETERMINATION (921), Method |, Method la
Calculate the amount of lead, in ug/g, in the portion of Sample: 0.50g
Neotame taken: Acceptance criteria: NMT 5.0%

Result = (Cx V)/(Wx FA) ADDITIONAL REQUIREMENTS

e PACKAGING AND STORAGE: Preserve in well-closed contain-
cE = blank-corrected lead concentration in the ers, store in a dry place, and avoid exposure to excessive
Sample solution (g/mL) heat.
Vv = volume of the Sample solution, 10 mL e USP REFERENCE STANDARDS (11)
w = weight of Neotame taken to prepare the USP Neotame RS
Sample solution (mg) USP Neotame Related Compound A RS
F = conversion factor, mg/g N-[3,3-Dimethylbutyl)-L-c-asparty!]-L-phenylalanine.
Acceptance criteria: NMT 2 ug/g
e RELATED COMPOUNDS
Mobile phase and Chromatographic system: Proceed
as directed in the Assay.
Standard solution A: 0.03 mg/mL of USP Neotame Re- Nitric Acid
lated Compound A RS in Mobile phase
Standard solution B: Prepare as directed for the Stan- HNO3 63.01
dard solution in the Assay. Nitric acid [7697-37-2].
Detector sensitivity solution: Transfer 2 mL of Standard
solution A to a 50-mL volumetric flask, and dilute with DEFINITION
Mobile phase to volume. Nitric Acid contains NLT 69.0% and NMT 71.0%, by
Sample solution: 2 mg/mL of Neotame in Mobile weight, of nitric acid (HNOs). [CAuTION—Avoid contact,
phase. [NoTtE—This solution is stable for up to 32 h because Nitric Acid rapidly destroys tissues.]
when stored at a temperature of 0°-10°.]
System suitability IDENTIFICATION
Samples: Standard solution A and Detector sensitivity A. IDENTIFICATION TESTS—GENERAL, Nitrate (191): Meets
solution the requirements
Suitability requirements
Relative standard deviation: NMT 5.0%, Standard ASSAY
solution A e PROCEDURE
Signal-to-noise ratio: NLT 10, Detector sensitivity Sample solution: Weigh 2 mL of Nitric Acid in a glass-
solution stoppered conical flask, and add 25 mL of water. Add
Analysis methyl red TS.
Samples: Standard solution A, Standard solution B, and Analysis: Titrate the Sample solution with 1 N sodium
Sample solution hydroxide VS. Each mL of 1 N sodium hydroxide is
Calculate the percentage of neotame related compound equivalent to 63.01 mg of HNOs (see Titrimetry (541)).
A in the portion of Neotame taken: Acceptance criteria: 69.0%-71.0%
IMPURITIES
Result = (ru/rs) x (Cs/Cu) x 100 ¢ RESIDUE ON IGNITION (281)
tu = peak response of neotame related compound Sample: 70 mL (100 g)
A from the Sample solution Analysis: Place the Sample in a tared crucible, add
rs = peak response of neotame related compound 2 drops of sulfuric acid, and evaporate to dryness. Ignite
A from Standard solution A for 15 min.
Gs = concentration of USP Neotame Related Acceptance criteria: NMT 0.5 me (5 ppm)
Compound A RS in Standard solution A e CHLORIDE AND SULFATE, Chloride (221)
Sample: 35 mL (50g)
(mg/mL) Control: 35 wl of 0.020 N hydrochloric acid
Cu = concentration of Neotame in the Sample
solution (mg/mL) Acceptance criteria: NMT 0.5 ppm; the Sample shows
Calculate the percentage of other impurities in the no more chloride than corresponds to the Control.
portion of Neotame taken: © CHLORIDE AND SULFATE, Sulfate (221)
sydeiBbouow 4N

Sample: 28 mL
Result = (rz/rs) x (Cs/Cy) x 100 Control: 40 ul of 0.020 N sulfuric acid in an equal vol-
ume of solution containing the quantities of reagents
tr = sum of the peak responses of all impurities used in the analysis
(except those of neotame related compound Analysis: Add 10 mg of sodium carbonate to the Sam-
A and the solvent, if observed) from the ple. Evaporate to dryness, dissolve in a mixture of 4 mL
Sample solution of water and 1 mL of dilute hydrochloric acid (50 mg/
ls = peak response of neotame from Standard mL), and filter if necessary. Wash with two 2-mL por-
solution B tions of water, dilute with water to 10 mL, and add
1 mL of barium chloride TS. Observe 10 min after add-
ing the barium chloride.
 5462 Nitric / Official Monographs NF 36

Acceptance criteria: 1 ppm; any turbidity produced by SPECIFIC TESTS

the Sample is not greater than that produced by the [Note—Reduce the container pressure by means of a regula-
Control. tor. Measure the gases with a gas volume meter down-
e IRON (241) stream from the detector tube to minimize contamination
Sample: 35 mL (50g) or change of the specimens.]
Analysis: Evaporate the Sample to dryness, dissolve the e CARBON MONOXIDE
residue in 2 mL of hydrochloric acid, and dilute with Sample: 1000+50 mL
water to 47 mL. Analysis: Pass the Sample through a carbon monoxide
Acceptance criteria: NMT 0.2 ug/g detector tube (see Reagents, Indicators, and Solutions) at
the rate specified for the tube.
Acceptance criteria: NMT 10 ppm
Delete the following: e LIMIT OF OXYGEN
Analysis: Determined as directed in the Assay
®o HEAVY METALS, Method
| (231) Acceptance criteria: NMT 1.0%
Test preparation: To 70 ml (100 g) of Nitric Acid in a e ODOR
250-mL beaker add 10 mg of sodium carbonate, and Analysis: Carefully open the container valve to produce
evaporate on a steam bath to dryness. Add 25 mL of a moderate flow of gas. Do not direct the gas stream
water. toward the face, but deflect a portion of the stream
Acceptance criteria: NMT 0.2 ppMe coicial 1-Jan-2018) toward the nose.
SPECIFIC TESTS Acceptance criteria: No appreciable odor is discernible.
e CLARITY AND COLOR OF SOLUTION ADDITIONAL REQUIREMENTS
Analysis: Mix it in its original container, and transfer ¢ PACKAGING AND STORAGE: Preserve in cylinders.
10 mL to a 20- x 150-mm test tube. Compare with
water in a similar test tube.
Acceptance criteria: The liquids are equally clear and
free from suspended matter, and when viewed transver-
sely by transmitted light, exhibit no apparent difference
in color. Nitrogen 97 Percent
ADDITIONAL REQUIREMENTS DEFINITION
¢ PACKAGING AND STORAGE: Preserve in tight containers. Nitrogen 97 Percent is Nitrogen produced from air by phys-
ical separation methods. It contains NLT 97.0%, by vol-
ume, of nitrogen (Nz).
IDENTIFICATION
e A. The flame of a burning wood splinter is extinguished
Nitrogen when inserted into a test tube filled with Nitrogen 97
Percent. [NoTE—Exercise caution.]
No 28.01
Nitrogen [7727-37-9]. ASSAY
© PROCEDURE
DEFINITION Standard: Oxygen-helium certified standard (see Re-
Nitrogen contains NLT 99.0%, by volume, of nitrogen (Nz). agents, Indicators, and Solutions)
Sample: Nitrogen 97 Percent
IDENTIFICATION Chromatographic system
e A. The flame of a burning wood splinter is extinguished (See Chromatography (621), System Suitability.)
when inserted into a test tube filled with Nitrogen. Mode: GC
[Note—Exercise caution.] Detector: Thermal conductivity
ASSAY Column: 3-m length x 4-mm inside diameter: molecu-
e@ PROCEDURE lar sieve prepared from a synthetic alkali-metal alumi-
Sample: Nitrogen nosilicate capable of absorbing molecules having
Standard: Oxygen-helium certified standard (see Re- diameters of up to 0.5 nm and completely separating
agents, Indicators, and Solutions) oxygen from nitrogen
Chromatographic system Carrier gas: Helium (99.99%)
(See Chromatography (621), System Suitability.) Temperature: Thermostatically controlled
Mode: GC Analysis
Detector: Thermal conductivity Samples: Standard and Sample
Column: 3-m length x 4-mm inside diameter: molecu- Introduce the Samples separately into the gas chromat-
lar sieve prepared from a synthetic alkali-metal alumi- ograph by means of a gas sampling valve.
nosilicate capable of absorbing molecules having Acceptance criteria: The peak response produced by
diameters of up to 0.5 nm and completely separating the Sample exhibits a retention time corresponding to
oxygen from nitrogen that produced by the Standard and is equivalent to
Carrier gas: Helium (99.99%) NMT 3.0% of oxygen when compared to the peak re-
Temperature: Thermostatically controlled sponse of the Standard, indicating NLT 97.0%, by vol-
ume, of nitrogen (Na).
NF Monographs

Analysis
Samples: Standard and Sample IMPURITIES
Introduce the Samples separately into the gas chromat- [Note—Reduce the container pressure by means of a regula-
ograph by means of a gas sampling valve. tor. Measure the gases with a gas volume meter down-
Acceptance criteria: The peak response produced by stream from the detector tube to minimize contamination
the Sample exhibits a retention time corresponding to or change of the specimens.]
that produced by the Standard and is equivalent to
NMT 1.0% of oxygen when compared to the peak re-
sponse of the Standard, indicating NLT 99.0%, by vol-
ume, of No.
NF 36
e CARBON DIOXIDE
Sample: 1000+50 mL
Analysis: Pass the Sample through a carbon dioxide de-
tector tube (see Reagents, Indicators, and Solutions) at
the rate specified for the tube.
Acceptance criteria: The indicator change corresponds
to NMT 300 ppm
CARBON MONOXIDE
Sample: 1000+50 mL
Analysis: Pass the Samplerhrongl a carbon monoxide
detector tube (see Reagents, Indicators, and Solutions) at
the rate specified for the tube.
Acceptance criteria: NMT 10 ppm
© SULFUR DIOXIDE
Sample: 1000+50 mL
Analysis: Pass the Sample through a sulfur dioxide de-
tector tube (see Reagents, Indicators, and Solutions) at
the rate specified for the tube.
Acceptance criteria: NMT 5 ppm
Limit OF NITRIC OXIDE AND NITROGEN DIOXIDE
Sample: 500+50 mL
Analysis: Pass the Sample through a nitric oxide-ni-
trogen dioxide detector tube (see Reagents, Indicators,
and Solutions) at the rate specified for the tube.
Acceptance criteria: NMT 2.5 ppm
Limit OF OXYGEN
Analysis: Determined as directed in the Assay
Acceptance criteria: NMT 3.0%
SPECIFIC TESTS
e ODOR
Analysis: Carefully open the container valve to produce
a moderate flow of gas. Do not direct the gas stream
toward the face, but deflect a portion of the stream
toward the nose.
Acceptance criteria: No appreciable odor is discernible.
ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Preserve in cylinders or in a
low-pressure collecting tank.
© LABELING: Where it is piped directly from the collecting
tank to the point of use, label each outlet “Nitrogen 97
Percent”.
Nonoxynol 9—see Nonoxynol 9 General
Monographs
Octoxynol 9
Poly(oxy-1,2-ethanediyl), «-[4-(1,1,3,3-tetramethylbutyl)
phenyl]-a-hydroxy-;
a-[4-(1,1,3,3,-Tetramethylbutyl)phenyl]-o-hydroxypoly(oxy-
1,2-ethanediyl);
Polyethylene glycol mono[p-(1,1,3,3-tetramethylbutyl)
henyl] ether;
Polyethylene glycol mono(4-tert-octylphenyl) ether
[9002-93-1].
DEFINITION
Octoxynol 9 is an anhydrous liquid mixture consisting
chiefly of mono[p-(1,1,3,3-tetramethylbutyl)]-
phenyl ethers of polyethylene glycols, corresponding to:
(CH3)3C(CH2)C(CH3)2C6H4(OCH2CH2)nOH
Official Monographs / Octoxynol 5463
in which the average value of n is about 9. It contains NLT
90.0% and NMT 110.0% of Octoxynol 9.
IDENTIFICATION
e A. INFRARED ABSORPTION (197F): On undried specimen
e B. The retention time of the major peak of the Sample
solution corresponds to that of the Standard solution, as
obtained in the Assay.
ASSAY
e PROCEDURE
Mobile phase: Methanol:water (4:1)
Standard solution: 25 mg/mL of USP Octoxynol 9 RS
in Mobile phase
System suitability solution: 25 mg/mL of USP Octoxy-
nol 9 RS and 25 mg/mL of USP Nonoxynol 9 RS in
Mobile phase
Somels solution: 25 mg/mL of Octoxynol 9 in Mobile
phase
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 280 nm
Column: 4.6-mm x 25-cm, 5-um packing L1
Column temperature: Ambient
Flow rate: 1.0 mL/min
Injection size: 10 uL
System suitability
Samples: System suitability solution and Standard
solution
[NotE—The relative retention times for octoxynol 9 and
nonoxynol 9 are 1.0 and 1.4, respectively.]
Suitability requirements
Resolution: NLT 2.0 between octoxynol 9 and no-
noxynol 9, System suitability solution
Relative standard deviation: NMT 2.0%, Standard
solution
Analysis
Samples: Standard solution and Sample solution
Record the chromatograms, and measure the re-
sponses for octoxynol 9, including any shoulders and
bum S.
Calculate the percentage of octoxynol 9 in the portion
of test specimen taken:
Result = (ru/rs) x (Cs/Cu) x 100
tu = peak response of octoxynol 9 from the Sample
solution
fs = peak response of octoxynol 9 from the
Standard solution
Cs = concentration of USP Octoxynol 9 RS in the
Standard solution (mg/mL)
Gu = concentration of Octoxynol 9 in the Sample
solution (mg/mL)
Acceptance criteria: 90.0%-110.0%
¢ CONTENT OF FREE POLYETHYLENE GLYCOLS
Sample: 10g
Analysis: Transfer the Sample to a 250-mL beaker. Add
100 mL of ethyl acetate, and stir on a magnetic stirrer
to make a solution. Transfer, with the aid of 100 mL of
5.N sodium chloride, to a pear-shaped, 500-mL
separator fitted with a glass stopper. Insert the stopper,
and shake vigorously for 1 min. Remove the stopper
carefully to release the pressure. Immerse a thermome-
sydeibouo-: 4IN
ter in the mixture, and support the separator so that it
is partially immersed in a water bath maintained at 50°.
Swirl the separator gently while letting the internal tem-
perature rise to 40°-45°. Immediately remove the
separator from the bath, dry the outside surface, and
drain the salt (lower) layer into another pear-shaped,
500-mL separator. In the same manner, extract the
ethyl acetate layer a second time with 100 mL of fresh
5.N sodium chloride, combining the two aqueous ex-
tracts. Discard the ethyl acetate layer.
 5464 Octoxynol / Official Monographs
Wash the combined aqueous layers with 100 mL of
ethyl acetate, using the same technique, and drain the
salt (lower) layer into a clean pear-shaped, 500-mL
separator. Discard the ethyl acetate layer.
Extract the aqueous layer with two successive 100-mL
portions of chloroform, draining the chloroform
(lower) layers through Whatman folded filter paper 2V,
and combining them into a 250-mL beaker.
Evaporate on a steam bath or with a rotary evaporator
to dryness, and continue heating to remove chloro-
form. Allow the beaker to cool. Add 25 mL of acetone,
and dissolve the residue on a magnetic stirrer. Pass
through Whatman folded filter paper 2V into a tared
250-mL beaker, rinsing with two 25-mL portions of
acetone. Evaporate on a steam bath or with a rotary
evaporator to dryness. Dry in vacuum at 60° for 1 h.
Allow the beaker to cool, andweigh.
Acceptance criteria: NMT 1.0% of polyethylene glycol
IMPURITIES
e RESIDUE ON IGNITION (281): NMT 0.4%
Delete the following:
°o HEAVY METALS (231):
NMT 20 PPMee (iticial 1-jan-2015)
o LIMIT OF FREE ETHYLENE OXIDE
Stripped octoxynol 9: Maintain Octoxynol 9 at a tem-
perature of 150° with constant stirring in an open ves-
sel until it no longer displays a peak for ethylene oxide
when chromatographed as directed below.
Standard stock solution: [Note—Ethylene oxide is
toxic and flammable. Prepare these solutions in a well-
ventilated hood, using great care.] Chill all apparatus
and reagents used in the preparation of standards in a
refrigerator or freezer before use. Fill a chilled pressure
bottle with liquid ethylene oxide from a lecture bottle,
and store in a freezer when not in use. Use a small
piece of polyethylene film to protect the liquid from
contact with the rubber gasket. Transfer about 100 mL
of chilled isopropyl alcohol to a 500-mL volumetric
flask. Using a chilled graduated cylinder, transfer 25 mL
of ethylene oxide to the isopropyl alcohol, and swirl
gently to mix. Dilute with additional chilled isopropyl
alcohol to volume, replace the stopper, and swirl gently
to mix. This stock solution contains about 43.6 mg/mL
of ethylene oxide.
Standard solutions: Pipet 25 mL of 0.5 N alcoholic hy-
drochloric acid, prepared by mixing 45 mL of hydro-
chloric acid with 1 L of alcohol, into a 500-mL conical
flask containing 40 g of magnesium chloride hexahy-
drate. Shake the mixture to effect saturation. Pipet
10 mL of the Standard stock solution into the flask, and
add 20 drops of bromocresol green TS. If the solution is
not yellow (acid), add an additional volume, accurately
measured, of 0.5 N alcoholic hydrochloric acid to give
an excess of about 10 mL. Record the total volume of
0.5 N alcoholic hydrochloric acid added. Insert the
stopper into the flask, and allow to stand for 30 min.
Titrate the excess acid with 0.5 N alcoholic potassium
hydroxide VS. Performa blank titration, using 10.0 mL
of isopropyl alcohol instead of Standard stock solution,
adding the same total volume of 0.5 N alcoholic hydro-
chloric acid, and note the difference in volumes re-
m) quired. Each mL of the difference in volumes of 0.5 N
=
alcoholic potassium hydroxide consumed is equivalent
Qa
i to 22.02 i of ethylene oxide. Calculate the concentra-
fee tion, in mg/mL, of ethylene oxide in the Standard stock
aD
solution. Standardize daily. Store in a refrigerator. Pre-
io}
Cc pare a 1000-ppm standard by pipeting into a container
fo}
the calculated volume (about 2 mL) of cold Standard
= stock solution that on the basis of the standardization
re contains 88.6 mg of ethylene oxide, and adding 87.0g
Fe
of Stripped octoxynol 9. Prepare 10-, 5-, and 0.5-ppm
NF 36
standards by quantitatively diluting the 1000-ppm stan-
dard with additional Stripped octoxynol 9.
Standard solution 0.5 ppm: Transfer 5+ 0.01 g of the
Standard solution containing 0.5 ppm ethylene oxide to
suitable serum vials equipped with pressure-tight sep-
tum closures designed to relieve any excessive pressure,
and seal them.
Standard solution 5 ppm: Transfer 5+0.01 g of the
Standard solution containing 5 ppm ethylene oxide to
suitable serum vials equipped with pressure-tight sep-
tum closures designed to relieve any excessive pressure,
and seal them.
Standard solution 10 ppm: Transfer 5+ 0.01 g of the
Standard solution containing 10 hen ethylene oxide to
suitable serum vials equipped with pressure-tight sep-
tum closures designed to relieve any excessive pressure,
and seal them.
System suitability solution: 10 g/mL of ethylene ox-
ide and 10 ug/mL of acetaldehyde in Stripped octoxynol
9
Sample solution: Transfer 5 + 0.01 g of Octoxynol 9 to
a serum vial of the same kind as the vials used for Stan-
dard solution A.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: GC
Detector: Flame ionization
Column: 2.1-mm x 6.4-m nickel; 60- to 80-mesh sup-
port S9 (under typical conditions)
Temperature
Column: 100°
Injector: 160°
Detector: 200°
Carrier gas: Helium
Flow rate: 30 mL/min
System suitability
Samples: System suitability solution, Standard solution
0.5 ppm, Standard solution 5 ppm, and Standard solu-
tion 10 ppm
Suitability requirements
Resolution: NLT 1.5 between ethylene oxide and ac-
etaldehyde, System suitability solution
Calibration: None of the points used for constructing
the straight line Calibration curve deviates from the
line by more than 10%, Standard solution 0.5 ppm,
Standard solution 5 ppm, Standard solution 10 ppm.
Analysis
Samples: System suitability solution, Standard solution
0.5 ppm, Standard solution 5 ppm, Standard solution
10 ppm, and Sample solution
Calibration: Place the vial containing Standard solution
10 ppm in an oven, and heat at 90° for 30 min. Re-
move the vial from the oven. Using a gas-tight sy-
ringe, immediately inject a 100-uL aliquot of the head-
space gas into the gas chromatograph. Obtain the
area for the ethylene oxide peak (retention time ap-
proximately 8 min). Raise the temperature of the col-
umn to 200° after ethylene oxide elutes to volatilize
heavy components. Re-equilibrate the column at 100°.
Repeat the foregoing steps, using the vials containing
Standard solution 0.5 ppm and Standard solution 5 ppm.
On linear graph paper, plot area units versus ppm eth-
ylene oxide for the standards, and draw the best
straight line through the points.
Place the vial containing the Sample solution in an oven,
and heat at 90° for 30 min. Remove the vial from the
oven. Immediately inject a 100-uL aliquot of the head-
space gas into the gas chromatograph, and obtain the
area for the ethylene oxide peak.
Calculate the concentration of ethylene oxide in the
sample, in ppm:
Result = ry x S$
ru = peak area from the Sample solution
NF 36
S = slope of the standard curve (ppm/peak area
unit)
Acceptance criteria: NMT 5 ppm
© LIMIT OF DIOXANE
Apparatus: Assemble a closed-system vacuum distilla-
tion apparatus, using glass vacuum stopcocks (A, B, and
©, as shown in Figure 1. The concentrator tube (D)' is
made of borosilicate or quartz (not flint) glass, gradu-
ated precisely enough to measure the 0.9 mL or more
of distillate collected and marked so that the analyst
can dilute accurately to 2.0 mL.
TO McLEOD
GAUGE
VENT C the ice to melt without heating. After the ice has
TO
Re ‘ and repeat the sequence of chilling and thawing until
PUMP a tube once again for about 2 min, and release the vac-
$24/40 10/30
VACUUM
TRAP
Figure 1. Closed-system vacuum distillation apparatus for
dioxane.
Standard solution: 100 g/mL of dioxane in water. Use
a freshly prepared solution.
Sample solution: Transfer 20.0 g to a 50-mL round-
bottom flask (6) having a 24/40 ground-glass neck joint.
Add 1.0 mL of water. Place a small polytef-covered stir-
ting bar in the flask, insert the stopper, and stir to mix.
Immerse the flask in an ice bath, and chill for 1 min.
Wrap heating tape around the tube connecting the
concentrator tube (D) and the round-bottom flask, and
apply 10V to the tape. Apply a light coating of high-
vacuum silicone grease to the ground-glass joints, and
connect the concentrator tube to the 10/30 joint and
the round-bottom flask to the 24/40 joint. Immerse the
vacuum trap in a Dewar flask filled with liquid nitrogen,
close stopcocks A and B, open stopcock C, and begin
evacueng the system with a vacuum pump. Prepare a
slurry bath from powdered dry ice and methanol, and
raise the bath to the neck of the round-bottom flask.
After freezing the contents of the flask for 10 min, and
when the vacuum system is operating at a 0.05-mm
pressure or lower, open stopcock A for 20 s, then close
it. Remove the slurry bath, and allow the flask to warm
in air for 1 min, Immerse the flask in a water bath
maintained at a temperature of 20°-25°, and after
about 5 min warm the water bath to 35°-40° (sufficient
to liquefy most specimens) while stirring slowly but
constantly with the magnetic bar. Cool the water in the
bath by adding ice, and chill for about 2 min. Replace
the water bath with the slurry bath, freeze the contents
of the round-bottom flask for 10 min, open stopcock A
for 20 s, and then close it. Remove the slurry bath, and
repeat the heating steps as before, this time reaching a
final temperature of 45°-50° or a temperature neces-
sary to melt the Pa completely. If there is any
condensation in the tube connecting the round-bottom
1A suitable tube is available as Chromaflex concentrator tube, Kontes Glass
Co., Vineland, Nj (Catalog No. K42560-0000).
Official Monographs / Octoxynol 5465
flask to the concentrator tube, slowly increase the volt-
age to the heating tape, and heat until the condensa-
tion disappears.
Stir with the magnetic stirrer throughout the following
steps. Very slowly immerse the concentrator tube in a
Dewar flask containing liquid nitrogen.
[CauTion—When there is liquid distillate in the concentra-
tor tube, immerse the tube in the liquid nitrogen very
slowly, or the tube will break.]
Water will begin to distill into the concentrator tube. As
ice forms in the concentrator tube, raise the Dewar
flask to keep the liquid nitrogen level only slightly be-
low the level of ice in the ube, When water begins to
freeze in the neck of the 10/30 joint, or when liquid
nitrogen reaches the 2.0-mL graduation mark on the
concentrator tube, remove the Dewar flask, and allow
melted, check the volume of water that has distilled,
NLT 0.9 mL of water has been collected. Freeze the
uum first by opening stopcock B, followed by opening
stopcock A. Remove the concentrator tube from the
apparatus, close it with a greased stopper, and allow
the ice to melt without heating. Mix the contents of
the concentrator tube by swirling, note the volume of
distillate, and dilute with water to 2.0 mL, if necessary.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: GC
Detector: Flame ionization
Column: 2-mm x 1.8-m glass; support S10 (under
typical conditions)
Temperature
Column: 140°
Injector: 200°
Detector: 250°
Carrier gas: Nitrogen or helium
Flow rate: 35 mL/min
Install an oxygen scrubber between the carrier gas line
and the column. Condition the column for 72h at
230° with 30-40 mL/min carrier flow. [NoTE—Sup-
port S10 is oxygen-sensitive. Each time a column is
installed, flush with carrier gas for 30-60 min before
heating.]
Injection size: 2-4 uL
Analysis
Samples: Standard solution and Sample solution
Acceptance criteria: The height of the peak from the
Sample solution is NMT that from the Standard solution:
NMT 10 pg/g (ppm).
SPECIFIC TESTS
FATS AND FIXED OILS, Acid Value (401): NMT 0.2
FATS AND FIXED O1Ls, Hydroxyl Value (401): 85-101
FATS AND FIXED OILS, Peroxide Value (401): NMT 10.0
WATER DETERMINATION, Method | (921): NMT 0.5%
ADDITIONAL REQUIREMENTS
PACKAGING AND STORAGE: Preserve in tight containers.
Store at room temperature.
USP REFERENCE STANDARDS (11)
USP Nonoxynol 9 RS
USP Octoxynol 9 RS
sydeibouow 4N
 5466 Octyldodecanol / Official Monographs NF 36

Table 1
Octyldodecanol Hold Time at
Initial Temperature Final Final
Bye NNN Temperature Ramp Temperature | Temperature
©) _(°/min) ©) (min)

f
60 20 180 =
180 10 220 5

HC’ J Carrier gas: Hydrogen

Flow rate: 2.0 mL/min, constant flow mode
Injection volume: 1 wL
Injection type: Split ratio, 100:1
C20H420 298.55
1-Dodecanol, 2-octyl-;
Liner: Single taper, low-pressure drop liner with deac-
tivated wool
2-Octyldodecan-1-ol; Run time: 15 min
2-Octyldodecanol;
2-Octyldodecyl alcohol [5333-42-6]. System suitability
Samples: System suitability solution and Standard
DEFINITION solution
[Not&—See Table 2 for the relative retention times.]
Change to read:
Table 2
Octyldodecanol contains NLT 90.0% and NMT 102.0% of Relative
42-octyldodecan-1-ol (C7oH420),anras the remainder con- Retention
sisting chiefly of related alcohols. It is obtained from Component Time
sources of vegetable, animal, or synthetic origin. 1-Pentadecanol (internal standard) 1.00
Cetyl alcohol 1.08
IDENTIFICATION
Stearyl alcohol 1.25
Oleyl alcohol 1327
Change to read: Aa 2sOctyldodecanol 1.32

e A. CHROMATOGRAPHIC IDENTITY Suitability requirements

Analysis: Proceed as directed in the Assay.
Acceptance criteria: The retention time of the major
peak of the Sample solution, excluding the solvent and
Internal standard peaks, corresponds to the “anr3s octyl-
dodecanol peak of the Standard solution.
Add the following:
Ae B. FATS AND FIXED OlLs (401), Procedures, Hydroxyl
Value: 175-190ane36
ASSAY
Change to read:
Resolution: NLT 10 between the cetyl alcohol and
stearyl alcohol peaks; NLT 2.0 between the stearyl al-
cohol and oleyl alcohol peaks, System suitability
solution
Tailing factor: 0.8-1.8 for the 4anr« octyldodecanol
and 1-pentadecanol peaks, Standard solution
Relative standard deviation: NMT 1%, using the
area ratio of 4anr36 octyldodecanol to 1-pentadecanol,
Standard solution
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of *anrzs octyldodecanol
(C20H420) in the portion of Octyldodecanol taken:
Result = (Ru/Rs) x (Cs/Cu) x 100

e PROCEDURE Ru = peak response ratio of awe octyldodecanol

Internal standard solution: 1 mg/ml of 1-pentadeca-
nol (internal standard) in ethanol
System suitability solution: Prepare 1 mg/mL each of
USP Cetyl Alcohol RS, USP Stearyl Alcohol RS, and USP
Oleyl Alcohol RS in Internal standard solution, and heat
the solution in a sealed container in a 50° water bath
until all fatty alcohols are dissolved. Allow the solution
to cool to room temperature, and mix well.
Standard solution: Prepare 1.0 mg/mL of USP Octyldo-
decanol RS in Internal standard solution.
Sample solution: Prepare 1.0 mg/mL of Octyldodeca-
nol in Internal standard solution.
Chromatographic system
(See Chromatography (621), System Suitability.)
NF Monographs
to the internal standard (peak response of
4,npse OCtyldodecanol/peak response of the
internal standard) from the eae solution
Rs = peak response ratio of Aanrz6 octyldodecanol
to the internal standard (peak response of
Agyris OCtyldodecanol/peak response of the
internal standard) from the Standard solution
Gs = concentration of USP Octyldodecanol RS in
the Standard solution (mg/mL)
Cu = concentration of Octyldodecanol in the
Sample solution (mg/mL)
Acceptance criteria: 90.0%-102.0% 4ane6
IMPURITIES
[Note—On the basis of the manufacturing route, perform

Mode: GC either Organic Impurity Test 1 or eigen Impurity Test 2.]

Detector: Flame ionization e RESIDUE ON IGNITION (281): NMT 0.1%, determined on
Column: 0.25-mm x 30-m fused-silica capillary, coated
with a 0.25-~m layer of phase G7 2g
Temperatures
Injection port: 270°
Detector: 280°
Column: See Table 1.
NF 36
Change to read:
e@ ORGANIC IMPURITY TEST 1: LIMIT OF RELATED FATTY ALCo-
HOLS AND ALKANES
System suitability solution: Prepare 1 mg/mL each of
USP Steary! Alcohol RS, USP Oleyl Alcohol RS, USP Li-
noleyl Alcohol RS, and USP Octyldodecanol RS in etha-
nol, and heat the solution in a sealed container in a 50°
water bath until all fatty alcohols are dissolved. Allow
the solution to cool to room temperature, and mix well.
Dilute the solution with ethanol to obtain a solution
containing 0.05 mg/mL each of USP Stearyl Alcohol RS,
USP Oley! Alcohol RS, USP Linoleyl Alcohol RS, and USP
Octyldodecanol RS.
Sample solution: 1 mg/mL of Octyldodecanol in
ethanol
Chromatographic system: Proceed as directed in the
Assay, except use split injection with a split ratio of 5:1.
System suitability
Samples: System suitability solution and Sample solution
[NotE—See Table 3 for the relative retention times.]
Table 3
Relative
Retention
Component Time
n-Nonadecane? 0.63
9-Methy!l nonadecane? 0.65
2-Octyl-1-decanol or 2-hexyl-1-dodecanol> 0.87
Stearyl alcohols 0.95
Oleyl alcohols 0.96
Linoley!_alcohol« 0.99
Octyldodecanol¢ 1.00
2-Octyl-1-tetradecanol or 2-decyl-1-dodecanol> 1.17
Any other unspecified related fatty alcohol or jn
impurity
@Alkane.
Related branched chain fatty alcohol.
Related linear chain fatty alcohol.
4 Sample.
Suitability requirements
Resolution: NLT 2.0 between the stearyl alcohol and
oleyl alcohol peaks; NLT 2.0 between the linoleyl al-
cohol and 4ans3s octyldodecanol peaks, System suita-
bility solution
Analysis
Samples: System suitability solution and Sample solution
Identify n-nonadecane, 9-methy! nonadecane, and each
of the linear chain fatty alcohols and branched chain
aa alcohols in the Sample solution according to Table
Calculate the percentage of n-nonadecane (9-methyl
nonadecane, each of the linear chain fatty alcohols
and branched chain fatty alcohols, or any other un-
specified related fatty alcohol or impurity) in the por-
tion of Octyldodecanol taken:
Result = (ru/r7) x 100
ru = peak response of n-nonadecane (9-methyl
nonadecane, each of the linear chain fatty
alcohols and branched chain fatty alcohols,
or any other unspecified related fatty alcohol
or impurity) from the Sample solution
tr = sum of all the peak responses, excluding peak
responses due to solvent, from the Sample
solution
Acceptance criteria: Disregard any unspecified peaks
that are less than 0.05%, and any peaks due to solvent.
Sum of unspecified related fatty alcohols and impuri-
ties: NMT 1%
Official Monographs / Octyldodecanol 5467
Sum of n-nonadecane, 9-methyl nonadecane, 2-oc-
tyl-1-decanol, 2-hexyl-1-dodecanol, 2-octyl-1-te-
tradecanol, and 2-decyl-1-dodecanol: NMT 1.5%
© ORGANIC IMPURITY TEST 2: LimiT OF BRANCHED CHAIN FATTY
ALCOHOLS AND BRANCHED CHAIN ALDEHYDE
System suitability solution: Prepare 1 mg/mL each of
USP Stearyl Alcohol RS, USP Oleyl Alcohol RS, USP Li-
noleyl Alcohol RS, and USP Octyldodecanol RS in etha-
nol, and heat the solution in a sealed container in a 50°
water bath until all fatty alcohols are dissolved. Allow
the solution to cool to room temperature, and mix well.
Dilute the solution with ethanol to obtain a solution
containing 0.05 mg/mL each of USP Stearyl Alcohol RS,
USP Oley! Alcohol RS, USP Linoleyl Alcohol RS, and USP
Octyldodecanol RS.
Sample solution: 1 mg/mL of Octyldodecanol in
ethanol
Chromatographic system: Proceed as directed in the
Assay, except use split injection witha split ratio of 5:1
and run time of 30 min.
System suitability
Samples: System suitability solution and Sample solution
{[Note—See Table 4 for the relative retention times for
branched chain fatty alcohols and branched chain
aldehyde.]
Table 4
Relative
Retention
Component Time
2-Octyl-1-decanol or 2-hexyl-1-dodecanol* 0.87
2-Octyldodecanal> 0.93
Stearyl alcohols 0.95
Oleyl alcohols 0.96
Linoleyl alcohol« 0.99
Octyldodecanol? 1.00
2-Octyl-1-tetradecanol or 2-decyl-1-dodecanol 1.17
Any other unspecified fatty alcohol or _
impurity
Related branched chain fatty alcohol.
> Branched aldehyde.
Related linear chain fatty alcohol.
4 Sample.
Suitability requirements
Resolution: NLT 2.0 between the steary! alcohol and
oleyl alcohol peaks; NLT 2.0 between the linoley! al-
cohol and octyldodecanol peaks, System suitability
solution
Analysis
Samples: System suitability solution and Sample
solution
Identify each branched chain fatty alcohol peak and
branched chain aldehyde peak in the Sample solution
according to Table 4.
Calculate the percentage of each branched chain fatty
alcohol (2-octyl-1-decanol, 2-hexyl-1-dodecanol,
2-octyl-1-tetradecanol, or 2-decyl-1-dodecanol),
branched chain aldehyde (2-octyldodecanal), or any
unspecified fatty alcohol or impurity in the portion of
Octyldodecanol taken:
Result = (ru/r7) x 100
sydeibouow 4N
ty = peak response of each branched chain fatty
alcohol (2-octyl-1-decanol, 2-hexyl-
1-dodecanol, 2-octyl-1-tetradecanol, or
2-decyl-1-dodecanol), branched chain
aldehyde (2-octyldodecanal), or any
unspecified fatty alcohol or impurity from
the Sample solution
 5468 Octyldodecanol / Official Monographs NF 36

rr = sum of all the peak responses, excluding peak 9-Octadecenoic acid, (Z)-;
responses due to solvent, from the Sample Oleic acid [112-80-1].
solution
Acceptance criteria: Disregard any unspecified peaks DEFINITION
that are less than 0.05%, and any peaks due to solvent. Oleic Acid is manufactured from fats and oils derived from
Sum of unspecified fatty alcohols and impurities: edible sources, animal or vegetable, and consists chiefly of
NMT 5% (Z)-9-octadecenoic acid [CH3(CH2)7CH:CH(CH2)7,COOH]. It
Branched chain fatty alcohols (2-octyl-1-decanol, contains NLT 65.0% of (Z)-9-octadecenoic acid
2-hexyl-1-dodecanol, 2-octyl-1-tetradecanol, and [CH3(CH2)7CH:CH(CH2);COOH]. It may contain suitable
2-decyl-1-dodecanol): NMT 5% stabilizers.
Srrchied chain aldehyde (2-octyldodecanal):; NMT [Note—Oleic Acid labeled solely for external use is exempt
0 from the requirement that it be prepared from edible
sources.]
SPECIFIC TESTS
e FATS AND FIXED OILS (401), Procedures, Acid Value: NMT IDENTIFICATION
0.5 e A. INFRARED ABSORPTION (197F)
Sample: Undried specimen
Acceptance criteria: Meets the requirements
Delete the following: © B. The retention time of the major peak of the Sample
solution corresponds to that of the Standard solution, as
4e FATS AND FIXED OILS (401), Hydroxy! Value: 175-190anei6 obtained in the Assay.
e FATS AND FIXED OILS (401), Procedures, lodine Value: NMT
8 ASSAY
e FATS AND FIXED OILS (401), Procedures, Peroxide Value: © PROCEDURE
NMT 5.0 Standard solution: 1.7 mg/mL of USP Oleic Acid RS in
e WATER DETERMINATION (921), Method |: NMT 0.5% tetrahydrofuran
Sample solution: 1.7 mg/mL of Oleic Acid in
ADDITIONAL REQUIREMENTS tetrahydrofuran
© PACKAGING AND STORAGE: Preserve in iene containers. Chromatographic system
e LABELING: If a test for Impurities other than Organic Impu- (See Chromatography (621), System Suitability.)
rity Test 1 is used, the labeling states the test with which Mode: GC
the article complies. Label it to indicate whether it is de- Detector: Flame ionization
rived from vegetable, animal, or synthetic sources. Column: 0.53-mm x 30-m capillary; 0.25-11m layer of
e USP REFERENCE STANDARDS (11) phase G25 (or G35)
USP Cetyl Alcohol RS Temperature
USP Linoley! Alcohol RS Detector: 280°
USP Octyldodecanol RS Injection port: 280°
USP Oley! Alcohol RS Column: See Table 1.
USP Stearyl Alcohol RS
Table 1
Hold Time
Initial Temperature Final at Final
Ointment, Hydrophilic—see Hydrophilic Temperature
©
Ramp
(¢/min)
Temperature | Temperature
¢) (min)
Ointment General Monographs 120 = 120 5
120 10 250 20
Carrier gas: Helium
Ointment, White—see White Ointment Flow rate: 7.0 mL/min
General Monographs Injection volume: 1.0 uL
Injection type: Splitless
System suitability
Sample: Standard solution
Ointment, Yellow—see Yellow Ointment ae retention time for oleic acid is about 19.2
min.
General Monographs System suitability requirements
Relative standard deviation: NMT 5.0%
Analysis
Samples: Standard solution and Sample solution
Oleic Acid Calculate the percentage of oleic aci in the portion of
the sample taken:

A
rn nk . pice Result = (ru/rs) x (Cs/Cu) x 100
”
ro) ru = peak response for oleic acid from the Sample
2 solution
i]
- fs = peak response for oleic acid from the Standard
Dp solution
)
iS Cs = concentration of USP Oleic Acid RS in the
) Standard solution (mg/mL)
= Cu = concentration of Oleic Acid in the Sample
J CisH3402 282.46 solution (mg/mL)
Zz
NF 36 Official Monographs / Oleoyl 5469

Acceptance criteria: NLT 65.0% IDENTIFICATION

¢ A. INFRARED ABSORPTION (197F)
IMPURITIES ° B. o_o CHROMATOGRAPHIC IDENTIFICATION TEST
e RESIDUE ON IGNITION (281) (201
Sample: 10 mL Standard solution: 50 mg/mL of USP Oleoyl Polyoxyl-
Acceptance criteria: NMT 1 mg (about 0.01%) glycerides RS in methylene chloride
Sample solution: 50 mg/mL of Oleoyl Polyoxylglycer-
SPECIFIC TESTS ides in methylene chloride
e CONTENT OF FATTY ACIDS Application volume: 10 uL
Oleic Acid exhibits the composition profiles of fatty acids Developing solvent system: Ether and hexanes (70:30)
shown in Table 2 below, as determined in Fats and Fixed Spray reagent: 0.1 mg/mL of rhodamineBin alcohol
Oils (401), Fatty Acid Composition. Analysis
Samples: Standard solution and Sample solution
Table 2 Proceed as directed in the chapter. Then spray the
Carbon-Chain Number of Percentage plate with Spray reagent, and locate the spots on the
Length Double Bonds (%) plate by examination under UV light at a wavelength
of 365 nm.
14 0 $5.0
Acceptance criteria: The R- values of the principal spots
16 0 $16.0 of the Sample solution correspond to those of the Stan-
16 1 8.0 dard solution.
18 0 <6.0 e C. It meets the requirements in Specific Tests (see Table 1)
18 1 265.0 for Fats and Fixed Oils, Fatty Acid Composition (401).
18 2 <18.0 IMPURITIES
18 3 <4.0
20, 222 0 <4.0
Delete the following:
The sum of these fatty acids should be NMT 4.0%.

Test solution: Prepare as directed in the chapter, but °e HEAVY METALS, Method /I (231): NMT 10 Lg/ge ‘omeat-
omitting the initial hydrolysis. Jan-2018)
CONGEALING TEMPERATURE (651): 3°-10° for Oleic Acid © ALKALINE IMPURITIES
derived from animal sources; 10°-16° for Oleic Acid de- Sample: 5.0g
rived from vegetable sources Analysis: To the Sample add 10 mL of alcohol and
FATS AND FIXED OILS, Acid Value (401): 196-204, 2 g be- 0.05 mL of bromophenol blue TS, and mix well. Titrate
ing used with 0.01 N hydrochloric acid VS to change the color
FATS AND FIXED OILS, /odine Value (401): 85-105 to yellow.
FATS AND FIXED OILS, Peroxide Value (401): NMT 10.0 Acceptance criteria: NMT 1.0 mL of 0.01 N hydrochlo-
WATER DETERMINATION, Method | (921): NMT 0.4% ric acid is required.
MINERAL AcIDS © LIMIT OF FREE ETHYLENE OXIDE AND DIOXANE
Sample: 5 mL Analysis: Proceed as directed in Ethylene Oxide and Di-
Analysis: Shake the Sample with an equal volume of oxane (228), Method |.
water at a temperature of about 25° for 2 min, allow Acceptance criteria
the liquids to separate, and pass the water layer Ethylene oxide: NMT 1 ug/g
through a paper filter previously moistened with water. Dioxane: NMT 10 ug/g
Acceptance criteria: The filtrate is not reddened by the e LIMIT OF FREE GLYCEROL
addition of 1 drop of methyl orange TS. Sample: 1.2g
Periodic acetic acid solution: Dissolve 0.446 g of so-
ADDITIONAL REQUIREMENTS dium periodate in 2.5 mL of a 25% (v/v) solution of
© PACKAGING AND STORAGE: Preserve in well-closed contain- sulfuric acid, and dilute with glacial acetic acid to
ers, protected from light. Store at room temperature, 100.0 mL.
and avoid exposure to excessive heat. el iodide solution: 75 mg/mL of potassium
e LABELING: If it is for external use only, the labeling so iodide
indicates. Label it to indicate whether it is derived from Blank: 25 mL of methylene chloride
animal or vegetable sources. Indicate the names and Titrimetric system
quantity of any added stabilizers. (See Titrimetry (541).)
e USP REFERENCE STANDARDS (11) Mode: Residual titration
USP Oleic Acid RS Titrant: 0.1 M sodium thiosulfate VS
Endpoint detection: Visual
Analysis: Dissolve the Sample in 25 mL of methylene
chloride, heating if necessary. Cool, and add 100 mL of
water and 25.0 mL of Periodic acetic acid solution. Shake,
Oleoy! Polyoxylglycerides and allow to stand for 30 min. Add 40 mL of Potassium
iodide solution, and allow to stand for 1 min. Add 1 mL
DEFINITION of starch TS, and titrate the liberated iodine with 0.1 M
Oleoyl Polyoxylglycerides is a mixture of monoesters, dies- sodium thiosulfate VS. Perform a blank determination,
sydesBouow 4N

ters, and triesters of glycerol and monoesters and diesters
of polyethylene glycols. Polyethylene glycols used have a
mean molecular weight between 300 and 400. The article
is produced by partial alcoholysis of unsaturated oils,
mainly containing triglycerides of oleic acid with polyeth-
ylene glycol, by esterification of glycerol and polyethylene
glycol with fatty acids, or as a mixture of glycerol esters
and ethylene oxide condensate with the fatty acids of the
unsaturated oils. It may contain free polyethylene glycols.
and make any necessary correction.
saiae the percentage of glycerol in the sample
taken:
Result = {[(Vs — Vs) x N x F]/W} x 100
Ve = Titrant volume consumed by the Blank (mL)
Vs = Titrant volume consumed by the Sample (mL)
N = actual normality of the Titrant (mEq/mL)
F = equivalency factor, 23.0 mg/mEq
 5470 Oleoyl / Official Monographs NF 36

Ww = Sample weight (mg) IDENTIFICATION

Acceptance criteria: NMT 5.0% ¢ A. CHROMATOGRAPHIC IDENTITY
Analysis: Proceed as directed in the Assay.
SPECIFIC TESTS Acceptance criteria: The retention time of the major
e ARTICLES OF BOTANICAL ORIGIN, Total Ash (561): NMT of the Sample solution, excluding the solvent and
0.1% internal standard peaks, corresponds to the oleyl alco-
e FATS AND FIXED OILS, Acid Value (401) hol peak of the System suitability solution.
Sample: 2.0g
Acceptance criteria: NMT 2.0 ASSAY
© FATS AND FIXED OILS, Fatty Acid Composition (401): Oleoyl © PROCEDURE
Polyoxylglycerides exhibits the composition profile of Internal standard solution: 1 mg/mL of 1-pentadeca-
fatty acids shown in Table 7. nol (internal standard) in ethanol
System suitability solution: Prepare 1 mg/mL each of
Table 1 USP Cetyl Alcohol RS, USP Stearyl Alcohol RS, and USP
Oleyl Alcohol RS in Internal standard solution, and heat
Carbon-Chain Number of Percentage the solution in a sealed container in a 50° water bath
Length Double Bonds (%) until all fatty alcohols are dissolved. Allow the solution
16 0 4.0-9.0 to cool to room temperature, and mix well.
18 oO 36.0 Standard solution: 1.0 mg/mL of USP Oley! Alcohol RS
18 1 58.0-80.0 in Internal standard solution
18 Z. 15.0-35.0
Sample solution: 1.0 mg/mL of Oley! Alcohol in Inter-
nal standard solution
18 3 $2.0 Chromatographic system
20 0 $2.0 (See Chromatography (621), System Suitability.)
20 1 $2.0 Mode: GC
Detector: Flame ionization
© FATS AND FIXED OILS, Hydroxy! Value (401) Column: 0.25-mm x 30-m fused silica capillary; coated
Sample: 1.0g with a 0.25-um layer of phase G7
Acceptance criteria: 45-65 Temperatures
e FATS AND FIXED OLS, /odine Value (401): 75-95 Detector: 280°
FATS AND FIXED OILS, Peroxide Value (401) Injection port: 270°
Sample: 2.0g Column: See Table 1.
Acceptance criteria: NMT 12.0
FATS AND FIXED OILS, Saponification Value (401)
Table 1
Sample: 2.0g
Acceptance criteria: 150-170 Hold Time at
WATER DETERMINATION, Method | (921) Initial Temperature Final Final
Sample: 1.0g Temperature Ramp Temperature | Temperature
Analysis: Instead of using methanol as the solvent, one () (@/min) (°) (min)
of two solvent systems can be used: a mixture of meth- 60 20 180 —
ylene chloride and anhydrous methanol (70:30 v/v), or 180 10 220 5
anhydrous pyridine.
Acceptance criteria: NMT 1.0% Carrier gas: Hydrogen
Flow rate: 2.0 mL/min, constant flow mode
ADDITIONAL REQUIREMENTS Injection volume: 1 wL
¢ PACKAGING AND STORAGE: Preserve in tight containers, Injection type: Split injection; split ratio is 100:1
protected from light and moisture. Store at controlled Liner: Single taper, low pressure drop liner with deac-
room temperature. tivated wool
e LABELING: Label it to indicate thetype and the average Run time: 15 min
nominal molecular weight of polyethylene glycol used as System suitability
part of the official title. Samples: System suitability solution and Standard
e USP REFERENCE STANDARDS (11) solution
USP Oleoyl Polyoxylglycerides RS [Note—See Table 2 for the relative retention times.]

Table 2
Relative
Oleyl Alcohol Retention
Component Time
ian ta oSFes aul 1-Pentadecanol
eS (internal standard)
Cetyl alcohol
1.00
1.08
Stearyl alcohol eZs,
aa
CigH3sO 268.48 Oleyi alcohol 1.27
fe 9-Octadecen-1-ol, (Z)-;
a (Z)-9-Octadecen-1-ol [143-28-2]. Suitability requirements
i]
— Resolution: NLT 30 between the cetyl alcohol and
Dp DEFINITION stearyl alcohol peaks; NLT 2.0 between the stearyl
2} Oleyl Alcohol is a mixture of unsaturated and saturated high and oleyl alcohol peaks, System suitability solution
e molecular weight fatty alcohols consisting of
C) Tailing factor: 0.8-1.8 for the oleyl alcohol and
75.0%-102.0% of oleyl alcohol (CigH36O) and its isomers.
= It is obtained from sources of vegetable, animal, or syn-
1-pentadecanol peaks, Standard solution
— Relative standard deviation: NMT 1%, using the
thetic origin. It may contain suitable stabilizers. area ratio of oleyl alcoho! to 1-pentadecanol, Stan-
Zz
dard solution
 4498 Calcium / Dietary Supplements USP 41

Standard solution: 0.5 mg/mL of USP Calcium DL- Acceptance criteria: 6.0%-17.0%
5-Methyltetrahydrofolate RS in water
Sample solution: 0.5 mg/mL of Calcium L-5-Methylte- ADDITIONAL REQUIREMENTS
trahydrofolate in water e PACKAGING AND STORAGE: Store in a tight container, in a
System suitability solution: Transfer 1.0 mL of Standard cool and dry place.
solution to a 50-mL volumetric flask, and dilute with e USP REFERENCE STANDARDS (11)
Sample solution to volume. USP 4-Aminobenzoylglutamic Acid RS
Chromatographic system N-(4-Aminobenzoyl)-L-glutamic acid.
(See Chromatography (621), System Suitability.) Ci2HiaN20s 266.25
Mode: LC USP Calcium DL-5-Methyltetrahydrofolate RS
Detector: UV 280 nm N[4-[[(2-Amino-1,4,5,6,7,8-hexahydro-5-methyl-4-oxo-
Column: 4.0-mm x 15-cm; 5-um packing L79 6-pteridinyl)methyl]amino]benzoyl]-L-glutamic acid,
Column temperature: 40° calcium salt (1:1).
Flow rate: 1.0 mL/min CooH23CaN7Og — 497.52
Injection volume: 10 uL USP Folic Acid RS
System suitability
Sample: System suitability solution
[Note—The relative retention times of L-5-methylte-
trahydrofolate and D-5-methyltetrahydrofolate are
about 1 and 1.5, respectively.] Calcium L-5-Methyltetrahydrofolate
Suitability requirements Capsules
Resolution: NLT 1.5 between L-5-methyltetrahydrofo-
late and D-5-methyltetrahydrofolate
Analysis DEFINITION
Sample: Sample solution Calcium L-5-Methyltetrahydrofolate Capsules contain NLT
Calculate the percentage of D-5-methyltetrahydrofolate 90.0% and NMT 110.0% of the labeled amount of cal-
in the portion of Calcium L-5-Methyltetrahydrofolate cium L-5-methyltetrahydrofolate (CzoH23CaN7Oo).
taken: IDENTIFICATION
e A. HPLC: The retention time of the major peak of the
Result = [(ro/(ro + 1) x 100] Sample solution corresponds to that of the Standard solu-
tion, as obtained in Strength, and to the L-isomer of the
lp = peak response of D-5-methyltetrahydrofolate
from the Sample solution Standard solution in the test for Enantiomeric Purity.
th = peak response of L-5-methyltetrahydrofolate STRENGTH
from the Sample solution ¢ PROCEDURE
Acceptance criteria: NMT 1.0% of D-5-methyltetra- Antioxidant solution: 1.5% sodium sulfite in water
hydrofolate Buffer: 7.8 g/L of sodium dihydrogen phosphate dihy-
drate in water
SPECIFIC TESTS Solution A: Adjust the Buffer with 32% (w/v) sodium
e CALCIUM
hydroxide solution to a pH of 6.5.
Sample: 250mg Solution B: Methanol and Buffer (35:65). Adjust with
Blank: 150 mL of water, 15 mL of 1 N sodium hydrox- 32% (w/v) sodium hydroxide solution to a pH of 8.0.
ide, and 300 mg of hydroxy naphthol blue Mobile phase: Gradient elution. See Table 7.
Titrimetric system
(See Titrimetry (541).)
Mode: Direct titration Table 1
Titrant: 0.05 M edetate disodium VS Time Solution A Solution B
Endpoint detection: Visual (min) (%) (%)
Analysis: Dissolve the Sample in 150 mL of water, add 0 100 0
15 mL of 1 N sodium hydroxide and 300 mg of hy-
14 45 55
droxy naphthol blue, and titrate with the Titrant until
the solution is deep blue in color, Perform a Blank de- 17 0 100
DS Monographs

termination, and make any necessary correction. 24 0 100

Calculate the percentage of calcium (Ca) in the Sample 24.01 100 0
taken: 33 100 0

Result = [(Vs
— Vs) x M x F/W] x 100 System suitability solution: Transfer 25 mg of USP
Folic Acid RS to a 100-mL volumetric flask. Add about
Vs = volume of Titrant consumed by the Sample 15 mg each of sodium hydrogen carbonate and sodium
(mL) carbonate to the flask, add sufficient water, sonicate to
Vp = volume of Titrant consumed by the Blank (mL) dissolve, and dilute with water to volume. Transfer
M = actual molarity of the Titrant (mmol/mL) 1.0 mL of this solution to a second 100-mL volumetric
F = equivalency factor, 40.08 mg/mmol flask containing 50 mg of USP Calcium D,L-5-Methylte-
Ww = Sample weight (mg) trahydrofolate RS, dissolve, and dilute with water to
Acceptance criteria: 7.0%-8.5% on the anhydrous and volume.
solvent-free basis Standard solution: 0.1 mg/mL of USP Calcium D,L-
e WATER DETERMINATION, Method Ic (921) 5-Methyltetrahydrofolate RS in Antioxidant solution
Sample: Transfer 40 mg of Calcium L-5-Methylte- Sample solution: Remove, as completely as possible,
trahydrofolate to a 20-mL headspace vial, and cap the contents of NLT 30 Capsules, and weigh accurately.
tightly. Heat the vial in a suitable Karl Fischer oven at Transfer a portion of the Capsule contents, nominally
250°. equivalent to 2.5 mg of calcium L-5-methyltetrahydrofo-
Analysis: The released and evaporated water is trans- late, to a 25-mL volumetric flask. Add 20 mL of Antioxi-
ferred into the titration-cell in a stream of dry nitrogen dant solution and sonicate for 20 min with occasional
at a flow rate of about 40 mL/min as directed in Water shaking, cool to room temperature, dilute with Antioxi-
Determination, Method Ic (921). dant solution to volume, mix well, and filter.
NF 36 Official Monographs / Oleyl 5471

Analysis Analysis
Samples: Standard solution and Sample solution Samples: Resolution solution and Sample solution
The peak of elaidyl alcohol, which is an isomer of oleyl Identify each related fatty alcohol peakin the Sample
alcohol, can be resolved from the oleyl alcohol peak solution based on thatin the Resolution solution.
with a resolution of about 1, andwith a relative reten- Calculate the percentage of each related fatty alcohol in
tion time with reference tooleyl alcohol of 0.995. An the portion of Oleyl Alcohol taken:
additional small peak that may be observed on the
peak tail of oleyl alcohol can be assigned to another Result = (ru/rz) x 100
oleyl alcohol isomer. If elaidyl alcohol is observed, a
combination of both peaks of elaidyl alcohol and oleyl ru = peak response of each related fatty alcohol
alcohol is used to determine oley! alcohol content. from the Sample solution
Calculate the percentage of oleyl alcohol (CisH36O) or nr = sum of all the peak responses excluding peak
its isomers in the portion of Oleyl Alcohol taken: responses due to solvent from the Sample
solution
Result = (Ru/Rs) x (Cs/Cu) x 100 Acceptance criteria: See Table 4.Disregard peaks that
are less than 0.05% for any unspecified impurities, and
Ry = peak response ratio of oleyl alcohol (or elaidyl any peaks due to solvent.
alcohol) to the internal standard [peak
response of oleyl alcohol (or elaidyl alcohol)/ Table 4
peak response of the internal standard] from
the Sample solution Acceptance
peakresponse ratio of oleyl alcohol to the Criteria,
2

thera standard(peak response of oley! Component NMT (%)

aoanelipeas response of the internal Cetyl alcohol 8.0
standard) fromthe Standard solution Stearyl alcohol 5.0
Gs = concentration of USP Oley! Alcohol RS in the Linoley! alcohol 7.0
Standard solution (mg/mL) Linolenyl alcohol 1.0
Cu = concentration of Oley! Alcohol in the Sample
solution (mg/mL) Arachidyl alcohol 1.0
Acceptance criteria: = 75.0%-102.0% for oleyl alcohol
and its isomers SPECIFIC TESTS
FATS AND FIXED OILS, Acid Value (401): NMT 1
IMPURITIES FATS AND FIXED OtLs, Hydroxyl Value (401): 205-215
° be ON IGNITION (281): NMT 0.1%, determined on FATS AND FIXED Ous, Peroxide Value(401): NMT 10.0
i} WATER DETERMINATION, Method 1921): NMT 0.5%
e LIMIT OF RELATED FATTY ALCOHOLS
Resolution solution: Prepare 1 mg/mL each of USP ADDITIONAL REQUIREMENTS
Cetyl Alcohol RS, USP Stearyl Alcohol RS, USP Oley! Al- e PACKAGING AND STORAGE: Preserve in well-filled, tight
cohol RS, USP Linoleyl Alcohol RS, USP Linoleny| Alco- containers, and store at controlled room temperature.
hol RS, and USP Arachidyl Alcohol RS in ethanol. Heat e LABELING: Label it to indicate whether it is derived from
the solution in a sealed container in a 50° water bath vegetable, animal, or synthetic sources. Indicate the
until all fatty alcohols are dissolved. Allow the solution names and amounts of any added stabilizers.
to cool to room temperature, and mix well. Dilute the e USP REFERENCE STANDARDS (11)
solution with ethanol to obtain a solution containing USP Arachidyl Alcohol RS
0.05 mg/mL each of USP Cetyl Alcohol RS, USP Stearyl USP Cetyl Alcohol RS
Alcohol RS, USP Oley! Alcohol RS, USP Linoleyl Alcohol USP Linolenyl Alcohol RS
Se USP Linolenyl Alcohol RS, and USP Arachidyl Alcohol USP Linoleyl Alcohol RS
USP Oleyl Alcohol RS
Sample solution: 1 mg/mL of Oleyl Alcohol in ethanol USP Stearyl Alcohol RS
Chromatographic system: Proceed as directed in the
Assay, except use split injection with a split ratio of 5:1.
System suitability
Sample: Resolution solution
[Note—See Table 3 for the relative retention times.] Oleyl Oleate
Table 3
Relative
Retention
Component Time
Cetyl alcohol 0.85
Stearyl alcohol 0.99
Oley! alcohol 1.00
Linoleyl alcohol 1.03
ABET |

Linolenyl alcohol 1.06

Arachidyl alcohol 1.14
EXOCeLBLoLeLeLed

CagHosOr 532.92
Suitability requirements 9-Octadecenoic acid, (Z)-, oleyl ester;
Resolution: NLT 30 between the cetyl alcohol and Oleyl oleate [3687-45-4].
stearyl alcohol peaks; NLT 2.0 between the steary|
and oleyl alcohol peaks; NLT 6.0 between the oleyl DEFINITION
alcohol and linoleyl alcohol peaks Oley! Oleate consists of esters of oleyl alcohol and high mo-
lecular weight fatty acids, principally oleic acid.
 5472 Oleyl / Official Monographs NF 36

IDENTIFICATION e FATS AND FIXED OILS, Peroxide Value (401): NMT 10.0
e A. INFRARED ABSORPTION (197F) e FATS AND FIXED OILS, Fatty Acid Composition (401): Olive
Oil exhibits the composition profile of fatty acids shown
IMPURITIES in Table 1, as determined in the chapter.
e RESIDUE ON IGNITION (281)
Sample: 2g
Acceptance criteria: NMT 0.1% Table 1
e ARSENIC, Method I/ (211): 2 ug/g Carbon-Chain Number of Doub- Percentage
Length le Bonds (%)
Delete the following: <16 0 S0.1
16 0 7.5-20.0
°e HEAVY METALS, Method I! (231): NMT 20 ug/ge ‘otieiat1- 16 1 $3.5
Jan-2018) 18 0 0.5-5.0
18 1 56.0-85.0
SPECIFIC TESTS
© CLARITY OF SOLUTION 18 2 3.5-20.0
Sample solution: 200 mg/mL in ether 18 3 <1.2
Acceptance criteria: The resulting solution is clear. 20 0 <0.7
SPECIFIC GRAVITY (841): 0.860-0.884 at 20° 20 1 <0.4
FATS AND FIXED OILS, Acid Value (401): NMT 3.0 22 0 30.2
FATS AND FIXED OILS, Hydroxy! Value (401): NMT 10 24 oO <0.2
FATS AND FIXED OILS, /odine Value (401): 70-120
FATS AND FIXED OILS, Saponification Value (401): 90-125 ABSENCE OF SESAME OIL
REFRACTIVE INDEX (831): 1.464-1.468 at 20° Sample: 10 mL of Olive Oil
Analysis: Mix the Sample with a mixture of 0.5 mL of a
ADDITIONAL REQUIREMENTS 0.35% (v/v) solution of furfural in acetic anhydride and
¢ PACKAGING AND STORAGE: Preserve in tight containers. No 4.5 mL of acetic anhydride, and shake the mixture for
storage conditions specified. about 1 min. Pass througha filter paper previously wet-
e USP REFERENCE STANDARDS (11) ted with acetic anhydride. Add 0.2 mL of sulfuric acid
USP Oleyl Oleate RS to the filtrate.
Acceptance criteria: No bluish oreen color develops.
e FATS AND FIXED OILS, Unsaponifiable Matter (401): NMT
1.5%. [NoTe—Petroleum ether with a 40°-60° boiling
range can be used to replace ether in the test.]
Olive Oil ULTRAVIOLET ABSORBANCE
Sample solution: Dissolve 1.0 g of Olive Oil in cyclo-
[8001-25-0]. hexane, and dilute with cyclohexane to 100 mL.
DEFINITION Instrumental conditions
Olive Oil is the refined fixed oil obtained from the ripe fruit (See Ultraviolet-Visible Spectroscopy (857).)
of Olea europaea L. (Fam. Oleaceae). It may contain suita- Mode: UV-Vis
ble antioxidants. Wavelength: 270 nm
Path length of the cell: 1m
IDENTIFICATION Analysis: Determine the UV-Vis absorbance using the
e A. IDENTITY BY FATTY ACID COMPOSITION Instrumental conditions described above.
Analysis: Proceed as directed in the test for Fats and Acceptance criteria: The absorbance is NMT 1.20.
Fixed Oils (401), Fatty Acid Composition. WATER DETERMINATION, Method Ic (921): NMT 0.1%
Acceptance criteria: Meets the composition profile of STEROL COMPOSITION
fatty acids in Table 7 2M Alcoholic potassium hydroxide solution: Dissolve
e B. IDENTITY BY TRIGLYCERIDE PROFILE 12 g of potassium hydroxide in 10 mL of water, and
Analysis: Proceed as directed in the test for Identifica- dilute with alcohol (ethanol) to 100 mL.
tion of Fixed Oils by Thin-Layer Chromatography (202). Sample A: Accurately weigh 5 g of Olive Oil into a
Acceptance criteria: Meets the requirements in the 150-mL flask fitted with a reflux condenser. Add 50 mL
chapter of 2M Alcoholic potassium hydroxide solution, and heat
on a water bath for 1 h, shaking frequently. Add 50 mL
IMPURITIES of water through the top of the condenser, shake, and
allow to cool. Transfer the contents of the flask to a
Delete the following: separating funnel. Rinse the flask with several portions
totaling 50 mL of petroleum ether with a 40°-60° boil-
ing range, and add the rinsings to the separating fun-
®e HEAVY METALS, Method II (231): NMT 10 Lg/ge cortical 1- nel. Shake vigorously for 1 min. Allow to separate, and
Jan-2018) transfer the aqueous layer to a second separating fun-
e ALKALINE IMPURITIES nel. If an emulsion forms, add small quantities of alco-
Sample: 10 mL of Olive Oil hol or a concentrated solution of potassium hydroxide.
ms Analysis: Mix 10 mL of freshly opened acetone and Shake the aqueous layer with two 50-mL quantities of
pos 0.3 mL of water, and add 0.05 mL of bromophenol petroleum ether with a 40°-60° boiling range. Combine
. blue TS. Add the Sample, shake, and allow to stand. the petroleum ether layers in a third separating funnel
sS
— Titrate with 0.01 N hydrochloric acid VS to change the and wash with three 50-mL quantities of 50% alcohol.
Dp color of the upper layer to yellow.
° Acceptance criteria: NMT 0.1 mL of 0.01 N hydrochlo-
Transfer thepetroleum ether layer to a tared 250-mL
£ flask. Rinse the separating funnel with small quantities
3 ric acid is required. of petroleum ether with a 40°-60° boiling range, and
2 add to the flask. Evaporate the petroleum ether on a
SPECIFIC TESTS
mn
e FATS AND FIXED OILS, Acid Value (Free Fatty Acids) (401): water bath and oy the residue at 100°-105° for 15
Ps min, keeping the flask horizontal. Allow to cool in a
NMT 0.3. [NoTte—Petroleum ether with a 100°-120° boil-
ing range can be used to replace ether in the test.] desiccator and weigh.
NF 36 Official Monographs / Olive 5473

Reference A: Accurately weigh 5 g of sunflower oil into Temperatures

a 150-mL flask fitted with a reflux condenser. Proceed Injection port: 290°
as directed for Sample A, beginning with “Add 50 mL of Detector: 290°
2MAlcoholic potassium hydroxide solution”. Column: See Table 2.
Separation of the sterol fraction by LC
Mobile phase: Isopropyl alcohol and n-hexane (1:99) Table 2
Sample solution A: Transfer Sample A with three 4-mL
quantities of petroleum ether with a 40°-60° boiling Hold Time
range to a 15-mL test tube. [NoTE—Ether can be used Initial Temperature Final at Final
to replace petroleum ether if Sample A is not well solu- Temperature Ramp Temperature | Temperature
ble in petroleum ether.] Evaporate to dryness under a (°) (¢/min) ©) (min)
stream of nitrogen. Dissolve Sample A in Mobile phase 260 _ 260 38
to obtain a solution with an approximate concentra- 260 5 290 5
tion of 40 mg/mL. Add a few drops of isopropyl alco-
hol to improve the solubility. [NoTE—3 drops are nor- Carrier gas: Helium
mally sufficient to ensure complete solubilization.] Pass Flow rate: 2.6 mL/min
we a membrane filter (nominal 0.45-~m pore Injection volume: 1-3 pL (depending on the ex-
size). pected amount of sterols in the test sample)
Reference solution A: Prepare as directed for Sample Injection type: Split injection; split ratio is 25:1
Solution A, except use Reference A instead of Sample A. System suitability
Chromatographic system Sample: Reference solution B
(See Chromatography (621).) The chromatogram from Reference solution B shows
Mode: LC five principal peaks corresponding to cholesterol,
Detector: UV 210 nm campesterol, stigmasterol, B-sitosterol, and
Columns A7-stigmastenol.
Guard: 4.6-mm x 0.5-cm (or 4.6-mm x 1.0-cm); [Note—The retention times of the sterols with reference
5-um packing L3, with a 6-nm pore size to B-sitosterol are given in Table 3.]
Analytical: 4.6-mm x 25-cm; 5-um packing L3, with
a 6-nm pore size Table 3
Flow rate: 1.0 mL/min
Injection volume: 50 ul Relative
Identification of the peaks due to sterols Retention
Samples: Sample solution A and Reference solution A Identification Time
Sterol identification: The sterol fraction elutes at the Cholesterol 0.65
end of the chromatogram. Locate the fraction to be Brassicasterol 0.71
collected by using the chromatogram from Reference 24-Methylene-cholesterol 0.80
solution A. The chromatogram from Reference solution Campesterol 0.82
A shows two or three principal peaks, which elute at
Campestanol 0.83
approximately 21-35 min depending on the column
used. The chromatogram from Sample solution A may Stiqmasterol 0.87
have one principal peak. A7-Campesterol 0.92
Sterol collection: Collect the fraction at the detector A5,23-Stigmastadienol 0.95
outlet in a 15-mL tube with a screw cap. Evaporate Clerosterol 0.96
the solvent under a stream of nitrogen. [NOTE—If B-Sitosterol 1.00
necessary, to increase the sample amount for later
analysis, make the second injection of 50 uL on the Sitostanol 1.01
HPLC column and collect the fraction at the detector A5-Avenasterol 1.03
outlet in the same 15-mL test tube with a screw cap. A5,24-Stigmastadienol 1.09
Evaporate the solvent under a stream of nitrogen.] A7-Stigmastenol 13)
Determination of sterols by GC A7-Avenasterol 1.17
Sample solution B: Dissolve the residue of the sterol
fraction obtained from Sample solution A in the previ- Suitability requirements
ous LC step in 0.2 mL of anhydrous pyridine and Resolution: NLT 3.0 between the campesterol and
0.2 mL of a mixture of 1 volume of chlorotrimethylsi- stigmasterol peaks
lane and 99 volumes of bis(trimethylsilyl)trifluoro- Analysis
acetamide. Insert the stopper into the test tube tightly, Samples: Sample solution B and Reference solution B
and heat at 80° for 20 min. Allow to cool and use the Use the chromatogram from Reference solution B to
liquid phase. identify the peaks due to cholesterol, campesterol,
Reference solution B: Dissolve 9 parts of the residue stigmasterol, B-sitosterol, and A7-stigmastenol. Iden-
of the sterol fraction obtained from Reference solution A tify the peaks due to the sterols in the chromatogram
in the previous LC step and1 part of cholesterol in from Sample solution B using the chromatograms
0.2 mL of anhydrous pyridine and 0.2 mL of a mixture from Reference solution B and the relative retention
of 1 volume of chlorotrimethylsilane and 99 volumes times with reference to B-sitosterol (main peak) given
of bis(trimethylsilyl)trifluoroacetamide. Insert the stop- in Table 3.
sydesBbouo- 4N

per into the test tube tightly and heat at 80° for 20 Calculate the percentage content of each sterol in the
min. Allow to cool and use the liquid phase. sterol fraction of Olive Oil taken:
Chromatographic system
(See Chromatography (621), System Suitability.) Result = (ru/rr) x 100
Mode: GC
Detector: Flame ionization ty = area of the peak due to the sterol component
Column: 0.25-mm x 30-m fused-silica capillary; 0.25- to be determined
uum layer of phase G27 rr = sum of the areas of the peaks due to the
components indicated in Table 3
Acceptance criteria: Olive Oil exhibits the composition
profiles of sterols shown in Table 4.
 5474 Olive / Official Monographs NF 36

Table 4
Percentage Vehicle for Oral Solution, Sugar Free
Component (%)
Cholesterol $0.5 DEFINITION
Campesterol 4.0 Prepare Vehicle for Oral Solution, Sugar Free as follows (see
A7-Stigmastenol <0.5 Pharmaceutical Compounding—Nonsterile Preparations
Sum of the contents of A5,23-
(795)).
stigmastadienol, clerosterol, B-
sitosterol, sitostanol, AS-avenas- Xanthan Gum 50 mg
terol, and A5,24-stigmastadie- Glycerin 10 mL
nol 293.0 Sorbitol Solution 25 mb
Saccharin Sodium 100 mq
The content of stigmasterol is NMT that of campesterol.
Citric Acid Monohydrate 15q
ADDITIONAL REQUIREMENTS Sodium Citrate 2q
e PACKAGING AND STORAGE: Preserve in tight, light-resistant, Methylparaben 100 mg
well-filled containers, and prevent exposure to excessive Potassium Sorbate 100 mg
heat.
Purified Water, a sufficient quantity to make 100 mL
e LABELING: Label it to indicate the name and quantity of
any suitable antioxidants. Calculate the quantity of each ingredient required for the
total amount to be prepared. Accurately weigh/measure
Delete the following: each ingredient. Heat about 60 mL of Purified Water to
about 70°-75°. Add the Methylparaben, and stir until dis-
°e USP REFERENCE STANDARDS (11) solved. Remove from the heat, and add the Glycerin, Sor-
USP Olive Oil RS bito! Solution, Saccharin Sodium, Citric Acid Monohydrate,
@ (CN 1-May-2018)
Sodium Citrate, Potassium Sorbate, and Xanthan Gum. Add
sufficient Purified Water to volume, and mix well. Adjust
the pH if necessary. Package, and label.
SPECIFIC TESTS
e PH (791): An apparent pH between 4.0 and 5.0
Vehicle for Oral Solution
ADDITIONAL REQUIREMENTS
DEFINITION e PACKAGING AND STORAGE: Package ina tight, light-resis-
Prepare Vehicle for Oral Solution as follows (see Pharmaceuti- tant container, and store at controlled room
cal Compounding—Nonsterile Preparations (795)). temperature.
e LABELING: Label it to indicate that it is for use in com-
Sucrose 80g
pounding sugar-free oral solutions and suspensions.
e BEYOND-USE DATE: NMT 6 months after preparation. A
Glycerin Sg beyond-use date of more than 6 months may be as-
Sorbitol Sq signed if supporting stability data exist. (See Stability Cri-
Sodium Phosphate, Dibasic 120 mg teria and Beyond-Use Dating in Pharmaceutical Compound-
Citric Acid 200 mq ing—Nonsterile Preparations (795).)
Potassium Sorbate 100 mg
Methylparaben 100 mg
Purified Water, a sufficient quantity to make 100 mL

Calculate the quantity of each ingredient required for the Vehicle for Oral Suspension
total amount to be prepared. Accurately weigh/measure
each ingredient. Heat about 30 mL of Purified Water to DEFINITION
70°-75°. Add the Glycerin and Methylparaben, and stir un- Prepare Vehicle for Oral Suspension as follows (see Pharma-
til the Methylparaben is dissolved. Add the Dibasic Sodium ceutical Compounding—Nonsterile Preparations (795)).
Phosphate, Citric Acid, Potassium Sorbate, and Sorbitol, and
mix well. Add the Sucrose, and mix until dissolved; re- Cellulose, Microcrystalline 800 mq
move from the heat, and allow to cool. Add sufficient Xanthan Gum 200 mq
Purified Water to volume, and mix well. Adjust the pH if
Carrageenan 150 mg
necessary. Package, and label.
Carboxymethylcellulose Sodium (High Viscosity) 25 mg
SPECIFIC TESTS Citric Acid 250 mq
e PH (791): An apparent pH between 4.0 and 5.0 Sodium Phosphate, Dibasic 120 mg
ADDITIONAL REQUIREMENTS Simethicone 0.1 mL
e PACKAGING AND STORAGE: Package inatight, light-resis- Potassium Sorbate 100 mg
tant container, and store at controlled room Methylparaben 100 mg
NF Monographs

temperature. Purified Water, a sufficient quantity to make 100 mL

e LABELING: Label it to indicate that it is for use in com-
pounding oral solutions and suspensions.
e BEYOND-USE DATE: NMT 6 months after preparation. A
beyond-use date of more than 6 months may be as-
signed if supporting stability data exist. (See Stability Cri-
teria and Beyond-Use Dating in Pharmaceutical Compound-
ing—Nonsterile Preparations (795).)
Calculate the quantity of each ingredient required for the
total amount to be prepared. Accurately weigh/measure
each ingredient. Heat about 90 mL of the Purified Water to
70°-75°. Dissolve the Methylparaben, followed by the Cit-
ric Acid, Dibasic Sodium Phosphate, and Potassium Sorbate
in the heated water. Remove from the heat. With con-
stant mixing, slowly sprinkle the Microcrystalline Cellulose,
Xanthan Gum, Carrageenan, and Carboxymethylcellulose
NF 36
Sodium into the mixture. Continue to stir until fully hy-
drated, add the Simethicone, and mix well. Add sufficient
Purified Water to volume, and mix well. Adjust the pH if
necessary. Package, and label.
SPECIFIC TESTS
e PH (791): An apparent pH between 4.0 and 5.0
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Package in a tight, light-resis-
tant container, and store at controlled room
temperature.
e LABELING: Label it to indicate that it is for use in com-
pounding oral solutions and suspensions.
e BEYOND-USE DATE: NMT 6 months after preparation. A
beyond-use date of more than 6 months may be as-
signed if supporting stability data exist. (See Stability Cri-
teria and Beyond-Use Dating in Pharmaceutical Compound-
ing—Nonsterile Preparations (795).)
Orange Oil
DEFINITION
Orange Oil is the volatile oil obtained by expression from
the fresh peel of the ripe fruit of Citrus sinensis (L.) Osbeck
(Fam. Rutaceae). The total aldehyde content, calculated as
decanal (CioH200), is NLT 1.2% and NMT 2.5%. It may
contain a suitable antioxidant.
Noe” not use Orange Oil that has a terebinthine
odor.
ASSAY
© TOTAL ALDEHYDE CONTENT
Reagent solution: Dissolve 4.5 g of hydroxylamine hy-
drochloride in 13 mL of water. Add 85 mL of tertiary
a alcohol, mix, and adjust with 0.5 N potassium
hydroxide to a pH of 3.4.
Sample: 5 mL of Orange Oil, accurately weighed
Analysis: Pipet 50 mL of the Reagent solution into a
conical flask containing the Sample. Insert the stopper
in the flask, and allow to stand at room temperature for
30 min, with occasional shaking. Titrate the liberated
hydrochloric acid with 0.5 N alcoholic potassium hy-
droxide VS to a pH of 3.4. Each mL of 0.5 N alcoholic
potassium hydroxide consumed in the titration is equiv-
alent to 78.13 mg of total aldehydes, calculated as dec-
anal (CioH200).
Acceptance criteria: 1.2%-2.5%
IMPURITIES
Delete the following:
°o HEAVY METALS, Method I/ (231): NMT 40 19/ge corciat-
Jan-2018)
SPECIFIC TESTS
© SPECIFIC GRAVITY (841): 0.842-0.846
e a ROTATION, Angular Rotation (781A): +94° to
+99°
e REFRACTIVE INDEX (831): 1.472-1.474 at 20°
¢ ULTRAVIOLET ABSORBANCE
aanpte solution: 250mg of Oil in 100 mL of alcohol
Blank: Alcohol
Instrumental conditions
(See Ultraviolet-Visible Spectroscopy (857).)
Mode: UV-Vis
Wavelength range: 260-400 nm
Analysis: Record the spectrum in 1-cm cell. Determine
the absorbance at the wavelength of maximum absorb-
ance at 330 nm, using the line drawn tangent to the
curves appearing as minima in the spectrum in wave-
Official Monographs / Orange 5475
length regions above and below the maximum wave-
length as the baseline.
Acceptance criteria: The absorbance, calculated on the
basis of a 250-mg specimen, is NLT 0.130 for Califor-
nia-type Orange Oil or NLT 0.240 for Florida-type Or-
ange Oil.
e FOREIGN OILS
Analysis: Place 50 mL of Oil in a four-bulb Ladenburg
flask having the following dimensions: the lower or
main bulb is about 6 cm in diameter, and the smaller
condensing bulbs are about 3.5, 3.0, and 2.5 cm in di-
ameter; the distance from the bottom of the flask to
the side-arm is about 20 cm. Distill Oil at a rate of 1
drop/s until the distillate measures 5 mL.
Acceptance criteria: The angular rotation of the distil-
late does not differ from that of the original Oil by
more than 2°, and the refractive index at 20° is
0.001-0.002 lower than that of the original Oil.
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in well-filled, tight
containers, and avoid exposure to excessive heat.
e LABELING: The label states the Latin binomial and, follow-
ing the official name, the part of the plant source from
which the article was derived. Label it also to indicate
whether it is California-type or Florida-type Orange Oil.
The label indicates that Oil is not to be used if it has a
terebinthine odor.
Compound Orange Spirit
DEFINITION
Compound Orange Spirit contains NLT 25 mL and NMT
30 mL of the mixed oils in 100 mL of Spirit.
Prepare Compound Orange Spirit as follows (see Pharmaceu-
tical Compounding—Nonsterile Preparations (795)).
Orange Oil 200 mL
Lemon Oil 50 mL
Coriander Oil 20 mL
Anise Oil 5 mb
Alcohol, a sufficient quantity to make 1000 mL
Mix the oils with sufficient Alcohol! to make the product
measure 1000 mL.
ASSAY
© PROCEDURE
Sample solution: Transfer 2.0 mL of Compound Or-
ange Spirit and 1.0 mL of kerosene to a Babcock bottle,
graduated to 8%, and mix.
Analysis: To the Sample solution add sufficient saturated
calcium chloride solution, acidified with hydrochloric
acid, to almost fill the bulb of the bottle, Rotate the
bottle vigorously to ensure mixing, then add a sufficient
quantity of the calcium chloride solution to bring the
separated oil into the neck of the bottle. Centrifuge for
5 min at 1500 rpm, and read the volume of oil in the
stem. Subtract five divisions on the volumetric flask for
the kerosene added, and multiply the remaining num-
ber of divisions by 10.5 to obtain the volume, in mL, of =
mixed oils in 100 mL of the Compound Orange Spirit. nm
Acceptance criteria: 25-30 mL Ks
°
OTHER COMPONENTS =]
fo}
e¢ ALCOHOL DETERMINATION, Method | (611): 65.0%-70.0% Ke}
Cf
ADDITIONAL REQUIREMENTS 2
© PACKAGING AND STORAGE: Package in tight containers, mo]
a
protected from light, and store in a cold place. 7
 5476 Orange / Official Monographs NF 36

Acceptance criteria: 0.45%-0.55%

Sweet Orange Peel Tincture OTHER COMPONENTS
e ALCOHOL DETERMINATION, Method | (611): 2.0%-5.0%
DEFINITION
Sweet Orange Peel Tincture is prepared from sweet orange ADDITIONAL REQUIREMENTS
eel, which is the outer rind of the non-artifically colored, @ PACKAGING AND STORAGE: Package in tight containers,
resh, ripe fruit of Citrus sinesis (L.) Osbeck (Fam. and store ina cold place.
Rutaceae). e LABELING: The label states the Latin binomial and, follow-
Prepare Sweet Orange Peel Tincture as follows. ing the official name, the part of the plant source from
which the article was derived. The label indicates that
Sweet Orange Peel 500 g Syrup is not to be used if it has a terebinthine odor or
taste or shows other indications of deterioration.
Alcohol 900 mL.
Alcohol, a sufficient quantity to make 1000 mL

Macerate the Sweet Orange Peel in 900 mL of Alcohol in a

container that can be closed, and put in a warm place.
Agitate it frequently during 3 days or until the soluble Oxyquinoline Sulfate
matter is dissolved. Transfer the mixture toa filter, using
Talc as the filtering medium, and when most of the liquid
has drained away, wash the residue on the filter with a
sufficient quantity of Alcohol, combining the filtrates, to
produce 1000 mL, and mix. [NoTeE—Exclude the inner,
white portion of the rind.]
aa S NS + H,SO,

OTHER COMPONENTS (CoH7NO)2 - H2SO4 388.39

e ALCOHOL DETERMINATION, Method | (611): 62.0%-72.0% 8-Quinolinol sulfate (2:1) (salt) [134-31-6].

ADDITIONAL REQUIREMENTS DEFINITION

e PACKAGING AND STORAGE: Package in tight, light-resistant
containers. Avoid exposure to direct sunlight and exces-
sive heat.
© LABELING: The label states the Latin binomial and the offi-
cial name.
Orange Syrup
DEFINITION
Orange Syrup contains NLT 0.45% and NMT 0.55% of citric
acid (CeHgO7).
Prepare Orange Syrup as follows (see Pharmaceutical Com-
pounding—Nonsterile Preparations (795)).
Sweet Orange Peel Tincture 50 mL
Anhydrous Citric Acid Sq
Talc 15g
Sucrose 820g
Purified Water, a sufficient quantity to make 1000 mL
Triturate Talc with Sweet Orange Peel Tincture and Anhydrous
Citric Acid, and gradually add 400 mL of Purified Water.
Filter, returning the first portions of the filtrate until it
becomes clear, and wash the mortar and the filter with
sufficient Purified Water to make the filtrate measure
450 mL. Dissolve Sucrose in this filtrate by agitation, with-
out heating, and add Purified Water to make the product
measure 1000 mL. Mix, and strain. [NoTE—Do not use
Orange Syrup that has a terebinthine odor or taste or
shows other indications of deterioration.]
ASSAY
NF Monographs
Oxyquinoline Sulfate is 8-hydroxyquinoline sulfate. It con-
tains NLT 97.0% and NMT 101.0% of oxyquinoline sul-
Pe [(CsH7NO)z- H2SO,], calculated on the anhydrous
asis.
IDENTIFICATION
e A. INFRARED ABSORPTION (197M)
Analysis: On the undried specimen
Acceptance criteria: Meets the requirements
e B. IDENTIFICATION TESTS—GENERAL, Sulfate (191)
Analysis: 100 mg/mL
Acceptance criteria: Meets the requirements
ASSAY
© PROCEDURE
Sample: 100mg
Titrimetric system
(See Titrimetry (541).)
Mode: Residual titration
Titrant: 0.1 N bromine VS
Back-titrant: 0.1 N sodium thiosulfate VS
Endpoint detection: Visual
Analysis: Transfer the Sample to an iodine flask, add
30 mL of glacial acetic acid, 25.0 mL of Titrant, 10 mL
of potassium bromide solution (150 mg/mL), and
10 mL of hydrochloric acid. Immediately insert the stop-
ps mix, and allow to stand for 15 min, protected from
ight. Quickly add 10 mL of potassium iodide solution
(100 mg/mL) and 100 mL of water, taking precautions
against the escape of bromine vapor. At once insert the
stopper, and shake the mixture thoroughly. Remove the
stopper, and rinse it and the neck of the flask with a
small quantity of water so that the washing flows into
the flask. Shake the mixture thoroughly. Titrate the lib-
erated iodine with Back-titrant, adding 3 mL of starch
TS as the endpoint is approached. Perform a blank de-
termination. Each mL of Titrant is equivalent to

© Citric AciD 4.855 mg of oxyquinoline sulfate [(CoH7NO)2 - H2SOa].

Sample: 20 mL Ascoptancs criteria: 97.0%-101.0% on the anhydrous
Titrimetric system asis
(See Titrimetry (541).)
Mode: Direct titration
Titrant: 0.1 N sodium hydroxide VS
Endpoint detection: Visual
Analysis: To the Sample add 20 mL of water and add
phenolphthalein TS. Titrate with Titrant. Each mL of Ti-
trant is equivalent to 6.404 mg of citric acid (CsHsO7).
NF 36 Official Monographs / Palm 5477

IMPURITIES Table 1 (Continued)

e RESIDUE ON IGNITION (281): NMT 0.3% Carbon-Chain Number of
Length Double Bonds Percentage
Delete the following: 18 2 7.0-12.0
18 3 $0.5
°e HEAVY METALS, Method |/ (231): NMT 20 11g/ge corin- >20 Qorl <1.0
Jan-2018)
e FATS AND FIXED OILS, Peroxide Value (401): NMT 5.0
SPECIFIC TESTS e FATS AND FIXED OILS, Unsaponifiable Matter (401): NMT
e WATER DETERMINATION, Method | (921): 4.0%-6.0% 1.0%
e@ WATER DETERMINATION, Method Ic (921): NMT 0.1%
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in well-closed ADDITIONAL REQUIREMENTS
containers. e PACKAGING AND STORAGE: Preserve in well-closed contain-
e USP REFERENCE STANDARDS (11) ers. Do not store above 55°.
USP Oxyquinoline Sulfate RS e LABELING: Label it to indicate the name and quantity of
any added antioxidants.

Palm Oil
Palm oil [8002-75-3].
DEFINITION
Palm Oil is the refined fixed oil obtained from the pulp of
the fruit of the oil palm Elaeis guineensis Jacq. (Fam. Are-
caceae). It may contain suitable antioxidants.
IDENTIFICATION
e A. It meets the requirements of the test for Fats and
Fixed Oils, Fatty Acid Composition (401).
e B. It meets the requirements of the test for Melting
Range or Temperature (741).
IMPURITIES
e RESIDUE ON IGNITION (281)
Sample: 5g of Palm Oil
Acceptance criteria: NMT 0.1%
Delete the following:
°e HEAVY METALS, Method /I (231): NMT 10 utg/ge coricia1-
jan-2018)
© ALKALINE IMPURITIES
Sample: 10 mL of Palm Oil
Analysis: Mix 10 mL of acetone and 0.3 mL of water,
and add 0.05 mL of bromophenol blue TS. If necessary,
neutralize the solution to a green color with 0.01 N
hydrochloric acid or 0.01 N sodium hydroxide. Add the
Sample, shake, and allow to stand. Titrate with 0.01 N
hydrosilerc acid VS until the color of the upper layer
changes to yellow.
Acceptance criteria: NMT 0.1 mL of 0.01 N hydrochlo-
ric acid is required.
SPECIFIC TESTS
© MELTING RANGE OR TEMPERATURE (741): 30°-40°
© FATS AND FIXED OILS, Acid Value, Method II (401): NMT
2.0
e FATS AND FIXED OILS, Fatty Acid Composition (401):
Palm Oil exhibits the composition profile of fatty acids as
shown in Table 1.
Hydrogenated Palm Oil
R'COOCH2-CH(OOCR?)-CH200CR?, where R!, R2, and R3
are mainly Cis and Cy7;
Hydrogenated palm oil [68514-74-9].
DEFINITION
Hydrogenated Palm Oil is the pact obtained by refining
and hydrogenating the oil obtained from the pulp of the
fruit of the oil palm Elaeis guineensis Jacq. (Fam. Ara-
caceae). The product consists mainly of triglycerides of
palmitic and stearic acids.
IDENTIFICATION
e A. It meets the requirements of the test for Fats and
Fixed Oils, Fatty Acid Composition (401).
e B. It meets the requirements of the test for Melting
Range or Temperature (741).
IMPURITIES
e RESIDUE ON IGNITION (281)
Sample: 5g
Acceptance criteria: NMT 0.1%
Delete the following:
°e HEAVY METALS, Method f/ (231): NMT 10 29/ge cotiuia-
Jan-2018)
e Limit OF NICKEL
Sample solution: Weigh 5.0 g of Hydrogenated Palm
Oil into a previously tared platinum or silica crucible.
Cautiously heat the substance, and introduce into it a
wick formed from twisted, ashless filter paper. Ignite
the wick. When the substance ignites, stop heating. Af-
ter combustion, ignite in a muffle furnace at about
600°. Continue the incineration until a white ash is ob-
tained. After cooling, with the aid of two 2-mL portions
of diluted hydrochloric acid, transfer the residue to a
25-mL volumetric flask, add 0.3 mL of nitric acid, and
dilute with water to volume.
Nickel standard solution: Immediately before use, di-
lute 10 mL of nickel standard solution TS with water to
sydeiBbouow 4N

Table 1 500 mL. This solution contains the equivalent of 0.2 ig/
mL of nickel.
Carbon-Chain Number of Standard solutions: Into four separate identical 10-mL
Length Double Bonds Percentage volumetric flasks introduce respectively 0, 1.0, 2.0, and
4 0 $2.5 4.0 mL of Nickel standard solution. To each flask add a
14 0 0.5-5.9 2.0-mL portion of the Sample solution, and dilute with
16 0 39.0-47.0 water to volume to obtain four Standard solutions con-
18 oO 2.0-8.0
taining added quantities of 0, 0.2, 0.4, and 0.8 ug of
nickel, respectively.
16 1 $0.5 Instrumental conditions
18 1 36.0-44.0 (See Atomic Absorption Spectroscopy (852).)
 5478 Palm / Official Monographs NF 36

Mode: Atomic absorption spectrophotometer

equipped with a graphite furnace
Analytical wavelength: 232.0 nm Palm Kernel Oil
Lamp: Nickel hollow-cathode Elaeis guineensis seed oil [8023-79-8].
Analysis
Samples: Standard solutions DEFINITION
Concomitantly determine the absorbances of the Stan- Palm Kernel Oil is the refined fixed oil obtained from the
dard solutions at least three times each. Record the kernel of the fruit of the oil palm Elaeis guineensis Jacq.
average of the steady readings for each of the Stan- (Fam. Arecaceae). It may contain suitable antioxidants.
dard solutions. Plot the absorbances of the Standard
solutions versus the added quantity, in ug, of nickel. IDENTIFICATION
Extrapolate the line joining the points on the graph e A. It meets the requirements in Specific Tests for Fats and
until it meets the quantity axis. The distance between Fixed Oils, Fatty Acid Composition (401).
this point and the intersection of the axes represents e B. It meets the requirements in Specific Tests for Melting
the quantity of nickel in the 2-mL portion of the Sam- Range or Temperature (741).
ple solution added to the Standard solutions.
Calculate the content of nickel in the portion of Hy- IMPURITIES
drogenated Palm Oil taken: e Limit oF LEAD
[NoTe—For this test, use reagent-grade chemicals with as
Result = [V x (A/V3)]/W low a lead content as is necicathe, as well as high-
purity water and gases. Before use in this analysis, rinse
Vv = volume of the Sample solution, 25 mL all glassware and plasticware twice with diluted nitric
A = quantity of nickel (us) acid and twice with diluted hydrochloric acid, and then
Va = volume of the Sample solution added to the rinse them thoroughly with Purified Water.]
Standard solutions, 2 mL Hydrogen peroxide-nitric acid solution: 10% hydro-
Ww = weight of Hydrogenated Palm Oil taken to gen peroxide and diluted nitric acid (1:1). [NoTE—Use
prepare the Sample solution (g) caution.]
Acceptance criteria: NMT 1 ug/g Lead nitrate stock solution: Dissolve 159.8 mg of lead
¢ ALKALINE IMPURITIES nitrate in 100 mL of Hydrogen peroxide-nitric acid solu-
Sample: A mixture of 2.0 g of Hydrogenated Palm Oil, tion. Dilute with Hydrogen peroxide-nitric acid solution to
1.5 mL of alcohol, and 3.0 mL of toluene 1000 mL, and mix. Prepare and store this solution in
Analysis: Dissolve the Sample by gentle heating. Add glass containers that are free from lead salts. Each mL
0.05 mL of bromophenol blue TS, and titrate with 0.01 of this solution contains the equivalent of 100 1g of
N hydrochloric acid VS until the mixture turns yellow. lead.
Acceptance criteria: NMT 0.4 mL of 0.01 N hydrochlo- Standard lead solution: On the day of use, dilute
tic acid is required. 10.0 mL of Lead nitrate stock solution with Hydrogen per-
oxide-nitric acid solution to 100.0 mL, and mix. Each mL
SPECIFIC TESTS of Standard lead solution contains the equivalent of
© MELTING RANGE OR TEMPERATURE (741): 58°-62° 10 ug of lead.
e FATS AND FIXED OILS, Acid Value, Method II (401); NMT Butanol-nitric acid solution: Slowly add 50 mL of ni-
2.0 tric acid to approximately 500 mL of butanol in a
e FATS AND FIXED OILS, Fatty Acid Composition (401): Hy- 1000-mL volumetric flask. Dilute with butanol to
drogenated Palm Oil exhibits the composition profile of volume.
fatty acids as shown in Table 7. Standard solutions: Into five separate 100-mL volumet-
ric flasks pipet 0.2, 0.5, 1, 2, and 5 mL, respectively, of
Table 1 Standard lead solution, and dilute with Butanol-nitric
acid solution to volume. The Standard solutions contain
Carbon-Chain Number of 0.02, 0.05, 0.1, 0.2, and 0.5 ug/mL of lead,
Length Double Bonds Percentage respectively.
<12 0 $2.5 Sample solution: [CAuTion—Prepare this solution in a
14 0 0.5-5.9 fume hood, and wear safety glasses Transfer 1.0g of
16 0 32.0-47.0 Oil into a large test tube. Add 1 mL of nitric acid. Place
18 oO 49.0-57.0 the test tube in a rack in a boiling water bath. As soon
as the rusty tint is gone, add 1 mL of 30% hydrogen
20 0 51.0
peroxide dropwise to avoid a vigorous reaction, and
22. 0 $1.0 wait for bubbles to form. Stir with an acid-washed
16 1 $2.5 plastic spatula if necessary. Remove the test tube from
18 1 $2.5 the water bath, and allow it to cool. Transfer the solu-
18 2 <0.5 tion to a 10-mL volumetric flask, and dilute with Buta-
18 3 $0.5 nol-nitric acid solution to volume.
Tungsten solution: Transfer 0.1 g of tungstic acid and
e FATS AND FIXED OILS, Peroxide Value (401): NMT 5.0 5 g of sodium hydroxide pellets to a 50-mL plastic bot-
© FATS AND FIXED OILS, Unsaponifiable Matter (401): NMT tle. Add 5.0 mL of water, and mix. Heat the mixture in
0.8% a hot water bath until complete solution is achieved.
e Loss ON DRYING (731) Cool, and store at room temperature.
NF Monographs

Analysis: Dry a sample at 105° for 4 h. Instrumental conditions

Acceptance criteria: NMT 0.1% (See Atomic Absorption Spectroscopy (852).)
Mode: Graphite furnace atomic absorption
ADDITIONAL REQUIREMENTS ‘ : j spectrophotometry
¢ PACKAGING AND STORAGE: Preserve in tight, light-resistant Analytical wavelength: 283.3 nm lead emission line
containers. No storage requirement specified. Injection size: 20 uL
Lamp: Lead hollow-cathode
Furnace conditioning: Place the graphite tube in the
furnace. Inject the Tungsten solution into the graphite
tube, using an argon flow rate of 300 mL/min. Maintain
NF 36 Official Monographs / Palmitic 5479

the drying temperature at 110° for 20 s, the ashing e MELTING RANGE OR TEMPERATURE (741): 27°-29°
temperature at 700°-900° for 20 s, and with the argon e WATER DETERMINATION, Method | (921): NMT 0.1%,
flow stopped, the atomization temperature at 2700° for 50 mL of chloroform being used instead of 35-40 mL of
10 s; repeat this process once more using a second methanol as the solvent.
20-uL aliquot of the Tungsten solution. Clean the quartz
windows. ADDITIONAL REQUIREMENTS
Analysis © PACKAGING AND STORAGE: Preserve in well-closed contain-
Samples: Standard solutions and Sample solution ers. Do not store above 45°.
[Note—The sample injection technique is the most cru- © LABELING: Label it to indicate the name and quantity of
cial step in controlling the precision of the analysis; the any added antioxidants.
volume of each of the Standard solutions and the Sam-
ple solution must remain constant. Rinse the uL-pipet
tip three times with either the Standard solutions or
the Sample solution before injection. Use a fresh pipet
tip for each injection, and start the atomization pro- Palmitic Acid
cess immediately after injecting the Samples. Between
injections, flush the graphite tube of any residual lead Ci6H3202 256.42
by purging at a high temperature recommended by Hexadecanoic acid [57-10-3].
the manufacturer.]
Concomitantly determine the absorbances of the DEFINITION
Samples. Palmitic Acid is a mixture of solid organic acids obtained
Atomize oc volumes of the Standard solutions and from fats or oils of animal or vegetable origin. It contains
the Sample solution with an argon flow rate of NLT 92.0% of palmitic acid (C;sH3202) and NMT 6.0% of
300 mL/min. stearic acid (CygH36O2).
Maintain the drying temperature of the furnace at
110° for 30 s after a 20-s ramp time and a 10-s hold IDENTIFICATION
time; the ashing temperature at 700° for 42 s after a e A. The retention time of the major peak for palmitic acid
20-s ramp time and a 22-s hold time; and the atomi- of the Sample solution corresponds to that of the Stan-
zation temperature at 2300° for 7 s with the argon dard solution, as obtained in the Assay.
flow stopped.
Plot the absorbance of each of the Standard solutions, ASSAY
© PROCEDURE
compensated for background correction, versus its
content of lead, in g/mL, and draw the best straight Sample solution: Proceed as directed for Test solution
line fitting the five points. From this plot, determine in Fats and Fixed Oils (401), Fatty Acid Composition.
the concentration, C, in g/mL, of lead in the Sample Standard solution: Prepare the Standard solution in the
solution. same manner as the Sample solution, using a mixture of
Calculate the quantity, in ug/g, of lead in the portion 50 mg of USP Palmitic Acid RS and 50 mg of USP Ste-
of Oil taken: aric Acid RS instead of the substance to be examined.
Chromatographic system: Prepare as directed in Fats
Result = (C/W) x V and Fixed Oils (401), Fatty Acid Composition.
Injection size: 1 uL
G. = measured concentration of lead in the Sample System suitability
solution (tug/mL) Sample: Standard solution
Ww = weight of the Oil taken to prepare the Sample [Note—The relative retention times for methyl palmitate
solution (g) and methyl stearate are 0.9 and 1.0, respectively.]
V = final volume of the Sample solution, 10 mL Suitability requirements
Acceptance criteria: NMT 0.1 g/g of lead Resolution: NLT 3.0 between methyl stearate and
methyl palmitate
SPECIFIC TESTS Analysis
e FATS AND FIXED OILS, Acid Value (401): NMT 2.0 Samples: Standard solution and Sample solution
© FATS AND FIXED OILS, Fatty Acid Composition (401): Palm Calculate the percentage of CisH32O2 in the portion of
Kernel Oil exhibits the composition profile of fatty acids Palmitic Acid taken:
shown in Table 1.
Result = (ru/r7) x 100
Table 1
ty = peak response for methyl palmitate from the
Carbon-Chain Number of Double Percentage Sample solution
Length Bonds (%) nr = sum of the responses of all the peaks in the
6 oO $1.5 chromatogram except the solvent peak
8 0 3-5 Similarly, calculate the percentage of CisH36O2 in the
portion of Palmitic Acid taken:
10 0 2.5-6
12 o 40-52 Result = (ru/rz) x 100
14 0 14-18
16 0 7-10 tu = peak response for methyl stearate from the
sydesbouo-= 4N

18 0 1-3 Sample solution

tr = sum of the responses of all the peaks in the
20 0 <1
chromatogram except the solvent peak
16 1 sl Acceptance criteria: NLT 92.0% of palmitic acid
18 1 11-19 (CieH3202) and NMT 6.0% of stearic acid (CigH36O2)
18 2 0.54

e FATS AND FIXED OILS, Peroxide Value (401): NMT 10.0

e FATS AND FIXED OILS, Unsaponifiable Matter (401): NMT
1.5%
 5480 Palmitic / Official Monographs
IMPURITIES
Delete the following:
°e Heavy METALS, Method I] (231): NMT 10 ppme corteiat-
Jan-2018)
SPECIFIC TESTS
© COLOR: Heat a sample of Palmitic Acid to 75°. The result-
ing liquid is not more intensely colored than a solution
prepared by mixing 1.2 mL of ferric chloride CS and
0.3 mL of cobaltous chloride CS with 0.3 N hydrochloric
acid to make 10 mL, and diluting 5 mL of this solution
with 0.3 N hydrochloric acid to make 100 mL. Make the
comparison by viewing the solutions downward in
matched color-comparison tubes against a white surface
(see Color and Achromicity (631)).
CONGEALING TEMPERATURE (651): 60°-66°
FATS AND FIXED OILS, Acid Value (401): 216-220, using
1 Acceptance criteria: The absorbance at any wavelength
Fats AND FIXED OILS, /odine Value (401): NMT 1. Proceed
as directed in Method I, except use 35 mL of chloroform.
MINERAL ACID
Analysis: Shake 5 g of melted Palmitic Acid with an
equal volume of hot water for 2 min. Cool, and filter.
Acceptance criteria: The filtrate is not reddened by the
addition of 1 drop of methyl orange TS.
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in well-closed contain-
ers, and store at room temperature.
e LABELING: Label it to indicate whether it is derived from
animal or vegetable sources.
e USP REFERENCE STANDARDS (11)
USP Palmitic Acid RS
USP Stearic Acid RS
Paraffin
[8002-74-2].
DEFINITION
Paraffin is a purified mixture of solid saturated hydrocarbons
obtained fain petroleum. It may contain suitable
antioxidants.
IDENTIFICATION
e A. INFRARED ABSORPTION (197)
Sample: Use a thin film of melted specimen.
Analysis: Ensure complete melting to avoid doublet
peaks that may be observed at wavenumbers at about
1460 and 730 cm".
Acceptance criteria: Meets the requirements
e B. It meets the requirements in Specific Tests for Con-
gealing Temperature (651).
IMPURITIES
e LIMIT OF SULFUR COMPOUNDS
Sample: 4.0g
Analysis: To the Sample add 2 mL of dehydrated alco-
hol, and add 2 drops of a clear saturated solution of
ww lead(Il) oxide in sodium hydroxide solution (200 mg/
= mL). Heat the mixture at 70° for 10 min with frequent
a time the tube was placed in the bath, the acid (lower
i
—
shaking, and cool.
Dd Acceptance criteria: No dark brown color develops.
° e Limit OF POLYCYCLIC AROMATIC HYDROCARBONS
i<j Dimethyl sulfoxide: Use spectrophotometric grade di-
S the Standard solution when shaken vigorously.
methyl sulfoxide.
= Standard solution: 7.0 ug/mL of USP Naphthalene RS
J
in Dimethy! sulfoxide. Determine the absorbance of this
a solution at 278 nm using Dimethyl sulfoxide as the
blank.
NF 36
Sample: 0.50g
Instrumental conditions
(See Ultraviolet-Visible Spectroscopy (857).)
Mode: UV
Wavelength range: 260-350 nm
Cell: 1cm
Analysis: Dissolve the Sample in 25 mL of n-heptane,
place in a 125-mL separator with unlubricated ground-
glass parts (epee stopcock), and mix. Add 5.0 mL of
Dimethyl! sulfoxide, and shake the mixture vigorously for
1 min. Allow to stand until two clear layers are formed.
Transfer the lower layer to another 125-mL separator,
add 2 mL of n-heptane, and shake the mixture vigor-
ously. Allow to stand until two clear layers are formed.
Separate the lower layer, and determine its absorbance
using as the blank Dimethyl sulfoxide that previously has
been shaken vigorously for 1 min with n-heptane in the
ratio of 5 mL of Dimethy! sulfoxide to 25 mL of n-
heptane.
in the specified range is not greater than one-third o'
the absorbance, at 278 nm, of the Standard solution.
SPECIFIC TESTS
e@ CONGEALING TEMPERATURE (651): 47°-65°
© ACIDITY
Sample: 15g
Analysis: Introduce the Sample into a suitable separator,
add 30 mL of boiling water, and shake vigorously for
about 1 min. Allow to cool, and draw off the separated
water. To 10 mL of the filtrated aqueous layer add
0.1 mL of phenolphthalein TS.
Acceptance criteria: The solution does not produce a
pink color. NMT 1.0 mL of 0.01 M sodium hydroxide is
subsequent required to change the color of the indi-
cator to pink.
© ALKALINITY
Sample: 10 mL of the filtrated aqueous layer obtained
from the test for Acidit}
Analysis: To the Sample add 0.1 mL of methyl red TS2.
Acceptance criteria: The solution produces a yellow
color. NMT 0.5 mL of 0.01 M hydrochloric acid is sub-
sequently required to change the color of the indicator
to red.
e READILY CARBONIZABLE SUBSTANCES (271)
Standard solution: A mix of 3 mL of ferric chloride CS,
1.5 mL of cobaltous chloride CS, and 0.50 mL of cupric
sulfate CS, overlaid with 5 mL of mineral oil
Sample: 5 mL, at a temperature just above the melting
point
Analysis: Use a clean, dry, heat-resistant, glass-stop-
pered test tube, 140 +2 mm in length with an outside
diameter between 14.5 and 15.0 mm, and calibrated at
the 5- and 10-mL liquid levels. The capacity of the tube
with the stopper inserted is between 13.6 and
15.6 mL.! Place the Sample in the test tube, add 5 mL
of sulfuric acid (94.5%-94.9% of H2SO,), and heat in a
water bath at 70° for 10 min. When 5 min have
elapsed, and at each successive min thereafter, remove
the tube from the bath, place a finger over the stopper,
and give the tube three vigorous vertical shakes over an
amplitude of about 12 cm, returning the tube to the
bath within 3 s after the time when it was removed
therefrom.
Acceptance criteria: At the end of 10 min from the
layer) has no more color than the Standard solution. If
the sulfuric acid remains dispersed in the molten paraf-
fin, the color of the emulsion is not darker than that of
1A suitable test tube is available from Kimble Kontes. Item number:
34-19426. Description: Nessler Tube. Contact: phone 800-682-6644, fax
856-692-6644, e-mail customglass@kimkon.com,
USP 41 Dietary Supplements / Calcium 4499

Chromatographic system Analysis

(See Chromatography (621), System Suitability.) Sample: Sample solution
Mode: Calculate the percentage of D-5-methyltetrahydrofolate
Detector: UV 280 nm in the portion of calcium L-5-methyltetrahydrofolate
Column: 4.6-mm x 25-cm; 5-um packing L1 taken:
Column temperature: 32°
Flow rate: 1.1 mL/min Result = [ro/(ro + r)] x 100
Injection volume: 10 pL
System suitability lo = peak response of D-5-methyltetrahydrofolate
Samples: System suitability solution and Standard from the Sample solution
solution nr = peak response of L-5-methyltetrahydrofolate
[Note—For the System suitability solution the relative re- from the Sample solution
tention times of folic acid and L- and D-isomers of Acceptance criteria: NMT 1.0%
5-methyltetrahydrofolate, which co-elute as a single
PERFORMANCE TESTS
peak, are 0.85 and 1.0, respectively.] e DISINTEGRATION AND DISSOLUTION (2040), Disintegration:
Suitability requirements
Resolution: NLT 8 between folic acid and 5-methylte- Meet the requirements
¢ WEIGHT VARIATION OF DIETARY SUPPLEMENTS (2091): Meet
trahydrofolic acid, System suitability solution the requirements
Relative standard deviation: NMT 2.0%, Standard
solution ADDITIONAL REQUIREMENTS
Analysis © PACKAGING AND STORAGE: Store ina tight, light-resistant
Samples: Standard solution and Sample solution container, in a cool and dry place.
Calculate the percentage of the labeled amount of cal- e USP REFERENCE STANDARDS (11)
cium L-5-methyltetrahydrofolate (C2oH23CaN7O<) in the USP Calcium D,L-5-Methyltetrahydrofolate RS
portion of Capsules taken: USP Folic Acid RS
Result = (ru/rs) x (Cs/Cu) x 100
ty = peak response from the Sample solution
Is = peak response from the Standard solution
Cs = concentration of USP Calcium D,L- Calcium L-5-Methyltetrahydrofolate
5-Methyltetrahydrofolate RS in the Standard Tablets
solution (mg/mL)
Cu = nominal concentration of calcium L- DEFINITION
5-methyltetrahydrofolate in the Sample Calcium L-5-Methyltetrahydrofolate Tablets contain NLT
solution (mg/mL) 90.0% and NMT 110.0% of the labeled amount of cal-
Acceptance criteria: 90.0%-110.0% cium L-5-methyltetrahydrofolate (CzoH23CaN7O.).
IMPURITIES IDENTIFICATION
o ENANTIOMERIC PURITY e A. HPLC: The retention time of the major peak of the
Buffer: 4.54 g/L of sodium dihydrogen phosphate dihy- Sample solution corresponds to that of the Standard solu-
drate in water tion, as obtained in Strength, and to the L-isomer of the
Mobile phase: Acetonitrile and Buffer (3:97). Adjust Standard solution in the test for Enantiomeric Purity.
with 32% (w/v) sodium hydroxide to a pH of 6.8.
Standard solution: 0.4 mg/mL of USP Calcium D,L- STRENGTH
5-Methyltetrahydrofolate RS in water ¢ PROCEDURE
Sample solution: Filtered portion, equivalent to Antioxidant solution: 1.5% sodium sulfite in water
0.4 mg/mL of calcium L-5-methyltetrahydrofolate, from Buffer: 7.8 g/L of sodium dihydrogen phosphate dihy-
the contents of NLT 30 Capsules, in water drate in water
System suitability solution: Transfer 0.2 mL of Standard Solution A: Adjust the Buffer with 32% (w/v) sodium
Solution to a 10-mL volumetric flask, and dilute with hydroxide solution to a pH of 6.5.
Sample solution to volume. Solution B: Methanol and Buffer (35:65). Adjust with o
Chromatographic system 32% (w/v) sodium hydroxide solution to a pH of 8.0. “a
(See Chromatography (621), System Suitability.) Mobile phase: Gradient elution. See Table 1. <<
Mode: LC °
Detector: UV 280 nm Table 1 3
Column: 4,.0-mm x 15-cm; 5-um packing L79?
Column temperature: 40° Time Solution A Solution B SI
Flow rate: 1.0 mL/min (min) (%) (%) ES
Injection volume: 10 uL 0 100 0 z
System suitability 14 45 55)
Sample: System suitability solution 7 0 100
[Note—The relative retention times of L-5-methylte- 24 0 100
trahydrofolate and D-5-methyltetrahydrofolate are
about 1 and 1.5, respectively.] 24.01 100 0
Suitability requirements 33: 100 oO
Resolution: NLT 1.5 between L-5-methyltetrahydrofo-
late and D-5-methyltetrahydrofolate System suitability solution: Transfer 25 mg of USP
‘olic Acid RS to a 100-mL volumetric flask. Add about
1A chiral-recognition protein, human serum albumin (HSA), chemically 15 mg each of sodium hydrogen carbonate and sodium
bonded to silica particle, about 5 jum in diameter. For example: Chromtech
Chiral HSA, available at www.chromtech.com.
carbonate to the flask, add sufficient water, sonicate to
dissolve, and dilute with water to volume. Transfer
1.0 mL of this solution to a second 100-mL volumetric
flask containing 50 mg of USP Calcium D,L-5-Methylte-
NF 36 Official Monographs / Peanut 5481

ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in light resistant, well-
closed containers, and avoid exposure to excessive heat.
Peanut Oil
© LABELING: Label it to indicate the name and quantity of [8002-03-7].
any antioxidants.
e USP REFERENCE STANDARDS (11) DEFINITION
USP Naphthalene RS Peanut Oil is the fully-refined (alkali-refined, bleached, and
USP Paraffin RS deodorized at 230°-260°) oil obtained from the seed ker-
nels of one or more of the cultivated varieties of Arachis
hypogaea L. (Fam. Leguminosae). It may contain suitable
antioxidants.

Synthetic Paraffin IDENTIFICATION

e A. IDENTITY BY FATTY ACID COMPOSITION
DEFINITION Analysis: Proceed as directed in the test for Fats and
Synthetic Paraffin is synthesized by the Fischer-Tropsch pro- Fixed Oils (401), Procedures, Fatty Acid Composition.
cess from carbon monoxide and hydrogen, which are cat- Acceptance criteria: Meets the composition profile of
alytically converted to a mixture of paraffin hydrocarbons; fatty acids in Table 7
the lower molecular weight fractions are removed by dis- ¢ B. IDENTITY BY TRIGLYCERIDE PROFILE
tillation, and the residue is hydrogenated and further Analysis: Proceed as directed in Identification of Fixed
treated by percolation through activated charcoal. This Oils by Thin-Layer Chromatography (202), Identification,
mixture may be fractionated into its components by a Method | or Method I.
solvent separation method, using a suitable synthetic Acceptance criteria: Meets the requirements in the
isoparaffinic petroleum hydrocarbon solvent. It may con- chapter
tain NMT 0.005% of a suitable antioxidant. IMPURITIES
IDENTIFICATION
e A. INFRARED ABSORPTION (197): A thin film of it, cast from Delete the following:
a melt onto a cesium bromide plate, exhibits a pair of
very strong IR absorption peaks between 2840 cm-' and °e HEAVY METALS (231), Method Il: NMT 10 ppme (orca 1-
3000 cm, a pair of moderately strong peaks between Jan-2018)
1430 cm-' and 1490 cm, a pair of medium peaks be- e ALKALINE IMPURITIES
tween 720 cm-! and 750 cm-', and only weak peaks at Sample: 10 mL of Peanut Oil
any other wavenumbers. Analysis: Mix 10 mL of freshly opened acetone and
0.3 mL of water, and add 0.05 mL of bromophenol
IMPURITIES blue TS. Add the Sample, shake, and allow to stand.
Titrate with 0.01 N hydrochloric acid VS to change the
Delete the following: color of the upper layer to yellow.
Acceptance criteria: NMT 0.1 mL of 0.01 N hydrochlo-
°e HEAVY METALS, Method {/ (231): NMT 20 ppme coral
1- ric acid is required.
jan-2018)
© LIMIT OF OL CONTENT SPECIFIC TESTS
Analysis: Follow ASTM Method D721-68, “Standard e FATS AND FIXED OILS (401), Procedures, Fatty Acid Compost
Test Method for Oil Content of Petroleum Waxes” tion: Peanut Oil exhibits the composition profile of fatty
(Reapproved 1987).1 acids in Table 1.
Acceptance criteria: NMT 0.5%
Table 1
SPECIFIC TESTS
¢ ABSORPTIVITY Carbon-Chain Number of Percentage
Sample solution: Transfer 50-100 mg to a 100-mL vol- Length Double Bonds (%)
umetric flask. Dissolve in decahydronaphthalene at 88°, <14 0 $0.1
dilute with the same solvent at this temperature to vol- 14 0 <0.2
ume, and mix. 16 0 7.0-16.0
Blank: Decahydronaphthalene 16 1 <1.0
Instrumental conditions 18 oO 1.3-6.5
(See Ultraviolet-Visible Spectroscopy (857).)
18 1 35.0-72.0
Mode: UV
Analytical wavelength: 290 nm 18 2 13.0-43.0
Cell: 10cm (jacketed cells maintained at 88°) 18 3 50.6
Analysis 20 0 0.5—-3.0
Samples: Sample solution and Blank 20 1 0.5-2.1
Determine the absorbance of the Sample solution, and 22 0 1.0-5.0
calculate the absorptivity.
22 1 <0.5
Acceptance criteria: NMT 0.01
sydeiBbouo-W 4N

24 oO 0.53.0
ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Preserve in well-closed e RANCIDITY
containers. Sample: 1 mL of a solution (1 in 10) of Peanut Oil in
e LABELING: The labeling indicates its congealing tempera- ether
ture, viscosity, and needle penetration range under the Analysis: Shake the Sample with 1 mL of hydrochloric
specified conditions. acid, and add 1 mL ofa solution (1 in 1000) of
phloroglucinol in ether.
1 Available from the American Society for Testing and Materials, 1916 Race Acceptance criteria; No red or pink color develops.
St., Philadelphia, PA 19103.
e FATS AND FIXED OILS (401), Procedures, Acid Value: NMT
0.2
 5482 Peanut / Official Monographs
e FATS AND FIXED OILS (401), Procedures, Peroxide Value:
NMT 5.0
e FATS AND FIXED Otis (401), Procedures, Unsaponifiable Mat-
ter: NMT 1.5%
o WATER DETERMINATION (921), Method |, Method Ic: NMT
0.1%
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in tight, light-resistant
containers, and prevent exposure to excessive heat.
© LABELING: Label it to indicate the name and quantity of
any added antioxidant. Where Peanut Oil is intended for
use in the manufacture of injectable dosage forms, it is
so labeled.
© OTHER REQUIREMENTS: For Peanut Oil intended for use in
injectable dosage forms, which is specified in Labeling,
the requirements under Injections and Implanted Drug
Products (1), Product Quality Tests Common to Parenteral
Dosage Forms, Specific Tests, Vehicles and Added Sub-
stances, Nonaqueous Vehicles, must be met.
Pectin—see Pectin General Monographs
Peppermint
DEFINITION
Peppermint consists of the dried leaf and flowering top of
Mentha piperita L. (Fam. Labiatae).
SPECIFIC TESTS
© ARTICLES OF BOTANICAL ORIGIN, Foreign Organic Matter
(561): NMT 2.0% of stems more than 3 mm in diame-
ter and other foreign organic matter
e BOTANIC CHARACTERISTICS
Unground peppermint: Leaves, slender stems, and
flowering tops. The leaves are opposite, usually more or
less crumpled, and frequently detached from the stem.
The petiole is 4-15 mm in length, slightly pubescent;
the blade, when entire, is ovate-oblong to oblong-lan-
ceolate, 1.5—9 cm in length with an acute apex, a nar-
rowed or rounded base, and a sharply serrate margin;
light green to dark green in color; its upper surface is
nearly glabrous, its lower surface has a few hairs on the
veins and many amber-colored glandular hairs. The
stem is quadrangular, 1-3 mm in diameter, glabrous
except for a few scattered deflexed hairs, green to dark
purple. The flowers occur as a compact, oblong or oval
spire of verticillasters, 1-1.5 cm in breadth, rounded at
the summit, and in fruit attaining a length of 3-7 cm.
The bracts are oblong-lanceolate, 4-7 mm in length;
the calyx, tubular-campanulate, equally five-toothed,
pubescent, and glandular-punctate, green to dark pur-
ple; the corolla is glabrous, light purple, tubular-cam-
panulate, four-cleft, 3 mm in length; stamens, 4, short
and equal; style two- or rarely three-cleft at the summit.
The nutlets are ellipsoidal, 500 um in diameter. Pepper-
mint has an aromatic, characteristic odor and a pun-
gent taste, and produces a cooling sensation in the
mouth.
NF Monographs
Histology
Leaf: The lamina is dorsiventral. Both the upper and
the lower epidermis consist of epidermal cells with
wavy, anticlinal walls and stomata, the latter enclosed
by a pair of subsidiary cells with a common wall at
right angles to the guard cells. Many of the epidermal
cells, especially over the veins and midrib, bear non-
glandular and glandular hairs. The nonglandular hairs,
also numerous along the margin, are uniseriate with
NF 36
longitudinally striate and papillose cuticle, up to 8 cells
in length and tapered to a pointed apex. The glandu-
lar hairs occur in two types. The larger of these are
sunken in depressions of the epidermis and consist of
a one- to two-celled stalk and a glandular head of 8
radiating cells beneath the raised cuticle of which vola-
tile oil is secreted. The smaller type of glandular hair
consists of a one- to two-celled stalk and a one-celled
glandular head containing volatile oil. Beneath the up-
per epidermis occurs a single layer of palisade paren-
chyma up to 80 um in length and, directly underneath
it, spongy parenchyma of 3 or 4 layers of chloroplas-
tid-containing cells, through which zone course the fi-
brovascular tissues of the veins.
Stem: The stem is quadrangular. It shows a layer of
epidermis bearing hairs similar to those of the leaf and
possessing cuticularized outer convex walls, a narrow
cortex of chlorenchyma, a clear endodermis of tangen-
tially elongated, thin-walled cells with colorless con-
tents, a narrow phloem, a cambium, and a xylem
broadest in the regions beneath the stem angles and
containing narrow wood-wedges separated by xylem
rays one cell in width. The wood-wedges consist
chiefly of simple pitted and spiral vessels, tracheids,
and wood-fibers. Beneath each of the four angles of
the stem occurs an elliptic to ovate zone of collen-
chyma. A large pith composed of thin-walled paren-
chyma occupies the center.
Powdered peppermint: Green to light olive green.
Shows fragments of leaf epidermis with wavy vertical
walls and, if from the lower surface of the leaf, with
numerous stomata and glandular and nonglandular
hairs, the latter especially numerous along the veins;
glandular hairs with a one- to two-celled stalk and
one- to eight-celled head, usually set in a depression in
the leaf and containing volatile oil and frequently yel-
lowish or brownish css that are birefringent; non-
glandular hairs with thin, papillose walls and fre-
quently with short, longitudinal striations of 1-8 cells
and up to 1.4 mm in length, the terminal cell pointed
or sometimes globular; fragments of chlorenchyma
with vascular tissue, the vessels spiral or with simple
pits and but slightly lignified; fragments of collen-
chyma and of thin-walled, nonlignified fibers associ-
ated with parenchyma. The pollen grains are spheroi-
dal and smooth.
Peppermint Oil
DEFINITION
Peppermint Oil is the volatile oil distilled with steam from
the fresh overground parts of the flowering plant of
Mentha piperita L. (Fam. Labiatae), rectified by distillation
and neitherpartially nor wholly dementholized. It yields
NLT 5.0% of esters, calculated as menthyl acetate
(Ci2H2202), and NLT 50.0% of total menthol (CioH200),
free and as esters.
IDENTIFICATION
oA.
Sample: 6 drops of Oil
Analysis: Place the Sample in a dry test tube and mix
with 5 mL of a 1-in-300 solution of nitric acid in glacial
acetic acid, and place the tube in a beaker of boiling
water,
Acceptance criteria: Within 5 min the liquid develops a
blue color that, on continued heating, deepens and
shows a copper colored fluorescence and then fades,
leaving a golden-yellow solution.
NF 36 Official Monographs / Phenolsulfonphthalein 5483

ASSAY SPECIFIC TESTS

e TOTAL ESTERS SPECIFIC GRAVITY (841): 0.896-0.908
Sample: 10g of Oil OPTICAL ROTATION, Angular Rotation (781A): —18° to -32°
Analysis Place the Samplein a 250-mL conical flask, REFRACTIVE INDEX (831): 1.459-1.465 at 20°
10 mL of neutralized alcohol and 2 drops of phe- SOLUBILITY IN 70% ALCOHOL: One volume dissolves in 3
nolphthalein TS, then add, dropwise, 0.1 N sodium hy- volumes of 70% alcohol, with NMT slight opalescence.
droxide until a faint pink color appears. Add 25.0 mL of
0.5 N alcoholic potassium hydroxide VS, connect the ADDITIONAL REQUIREMENTS
flask to a reflux condenser, and heat on a boiling water e PACKAGING AND STORAGE: Preserve in tight containers,
bath for 1 h. Allow the mixture to cool, add 20 mL of and prevent exposure to excessive heat.
water, and add phenolphthalein TS.
Titrate the excess alkali with 0.5 N hydrochloric acid VS.
Perform a blank determination, disregarding the 0.1 N
sodium hydroxide (see Titrimetry (541), Residual Titra-
tions). Each mL of 0.5 N alcoholic potassium hydrox- Peppermint Spirit—see Peppermint Spirit
ide consumed in the saponification is equivalent to General Monographs
99.15 mg of total esters calculated as C12H2202.
Acceptance criteria: NLT 5.0% of esters, calculated as
Ci2H2202
¢ TOTAL MENTHOL
Sample: 10 mL of Oil Peppermint Water
Analysis: Place the Samplein an acetylation flask of
100-mL capacity, and add 10 mL of acetic anhydride DEFINITION
and 1g of anhydrous sodium acetate. Boil the mixture Peppermint Water is a clear, saturated solution of Pepper-
ently for 1 h, accurately timed, cool, disconnect the mint Oil in Purified Water, prepared by one of the
lask from the condenser,transfer the mixture to a small processes described in Pharmaceutical Dosage Forms
separator, rinsing the acetylation flask with three 5-mL (1151), Solutions, Waters, Aromatic.
portions of warm water, and add the rinsings to the ADDITIONAL REQUIREMENTS
separator. When theliquids have completely separated, © PACKAGING AND STORAGE: Preserve in tight containers.
discard the water layer, and wash the remaining oil
with successive portions of sodium carbonate TS, di-
luted with an equal volume of water, until the last
washing is alkaline to phenolphthalein TS. Dry the re-
sulting oil with anhydrous sodium sulfate, and filter.
Transfer 5 mL of the dry acetylated oil to a tared, Petrolatum—see Petrolatum General
100-mL conical flask, and wean Add 50.0 mL of 0.5 N Monographs
alcoholic potassium hydroxide VS, connect the flask to
a reflux condenser, and boil the mixture on a steam
bath for 1 h,accurately timed. Allow the mixture to
cool, and add 10 drops of phenolphthalein TS. Petrolatum, Hydrophilic—see Petrolatum,
Titrate the excess alkaliwith 0.5 N hydrochloric acid VS.
Perform a blank determination (see yTitrimetry (541), Hydrophilic General Monographs
Residual Titrations).
Calculate the percentage of total menthol in the Oil
tested:
Petrolatum, White—see Petrolatum, White
Result = 7.813 x Ax (1 - 0.0021 x B/W
— 0.021 x A) General Monographs
A = result obtained by subtracting the number of
mL of 0.5 N hydrochloric acid required in
the above titration from the number of mL
of 0.5 N hydrochloric acid required in the Phenol—see Phenol General Monographs
residual titration blank
E = percentage of esters calculated as menthyl
acetate (Ci2H2202)
Ww = weight of acetylated Oil taken (g) Phenolsulfonphthalein
Acceptance criteria: NLT 50.0% of CioH200, free and
as esters 9 20

ao
IMPURITIES

Delete the following:

OH
°e HEAVY METALS, Method II (231): NMT 20 ppme cofficiat 1- HO
sydeibouo-= 4N

Jon-2018)
e Limit OF DIMETHYL SULFIDE CioH14OsS 354.38
Analysis: Distill 1 mL from 25 mL of Oil, and carefully Phenol red;
superimpose the distillate on 5 mL of mercuric chloride Phenol, 4,4’-(3H-2,1-benzoxathiol-3-ylidene)bis-,(5,
5-
TS in a test tube. dioxide);
Acceptance criteria: A white film does not form at the 3,3-Bis(4-hydroxyphenyl)-3H-2, 1-benzoxathiole 1,1-dioxide
zone of contact within 1 min. [143-74-8].
 5484 Phenolsulfonphthalein / Official Monographs
DEFINITION
Phenolsulfonphthalein contains NLT 98.0% and NMT
102.0% of 4,4’-(3H-2,1-benzoxathiol-3-ylidene)bis-,(S, S-di-
oxide) phenol (Ci9H14O05S), calculated on the dried basis.
IDENTIFICATION
eA.
Sample: 5 mg of Phenolsulfonphthalein
Analysis: Transfer the Sample to a 100-mL volumetric
flask, dissolve in and dilute with sodium carbonate solu-
tion (1 in 100) to volume, and mix. Dilute 5.0 mL of
the solution so obtained to 100.0 mL with sodium car-
bonate solution (1 in 100). Examine between 400 and
630 nm.
Acceptance criteria: The solution exhibits an absorp-
tion maximum at 558 nm, and the specific absorbance
at the maximum is between 1900 and 2100.
° B.
Sample: 10mg of Phenolsulfonphthalein
Analysis: Dissolve the Sample in 2 mL of 1 N sodium
hydroxide, and add 8 mL of water. To 5 mL of the solu-
tion so obtained add 1 mL of 0.1 N potassium
bromide—bromate and 1 mL of diluted hydrochloric
acid, shake, and allow to stand for 15 min. Render the
solution alkaline with 1 N sodium hydroxide.
Acceptance criteria: An intense violet-blue color is
produced.
ASSAY
© PROCEDURE
Sample: 0.9g
Titrimetric system
(See Titrimetry (541).)
Mode: Residual titration
Titrant: 0.1 N sodium thiosulfate VS
Endpoint detection: Visual
Analysis: Transfer the Sample to a 250-mL volumetric
flask, dissolve in 15 mL of 1 N sodium hydroxide, dilute
with water to volume, and mix. Transfer 10.0 mL of the
solution so obtained to a glass-stoppered flask. Add
25 mL of glacial acetic acid, 20.0 mL of 0.1 N potas-
sium bromate VS, 5 mL of potassium bromide solution
(1 in 10), and 5 mL of hydrochloric acid, and immedi-
ately insert the stopper into the flask. Allow to stand
protected from light for 15 min. Quickly add 10 mL of
potassium iodide solution (1 in 10), taking care to avoid
the escape of bromine vapor. Immediately insert the
stopper into the flask, and shake vigorously. Rinse the
stopper and the neck of the flask with a small quantity
of water. Titrate the liberated iodine with 0.1 N sodium
thiosulfate VS, using starch TS as the indicator. Perform
a blank determination, and note the difference in
volumes required. Each mL of the difference in volumes
of 0.1 N sodium thiosulfate is equivalent to 4.43 mg of
phenolsulfonphthalein (Ci9H140sS).
Acceptance criteria: 98.0%-102.0% on the dried basis
IMPURITIES
e RESIDUE ON IGNITION (281)
Sample: 0.5g
Acceptance criteria: NMT 0.2%
© CHROMATOGRAPHIC PURITY
Sample solution A: 20 mg/mL of Phenolsulfonphthalein
in 0.1 N sodium hydroxide
Sample solution B: Transfer 0.5 mL of Sample solution A
NF Monographs
to a 100-mL volumetric flask, dilute with 0.1 N sodium
hydroxide to volume, and mix.
Chromatographic system
(See Chromatography (621), Thin-Layer Chromato-
graphy.)
NF 36
Mode: TLC
Adsorbent: 0.25-mm layer of chromatographic silica
gel mixture
Application volume: 10 yl
Developing solvent system: tert-Amyl alcohol, glacial
acetic acid, and water (4:1:1)
Analysis
Samples: Sample solution A and Sample solution B
Allow the plate to air-dry until the solvent has evapo-
rated, and expose the plate to ammonia vapor. Ex-
amine the plate under short-wavelength UV light.
Acceptance criteria: NMT 0.5%; NMT one spot, apart
from the principal spot, appears in the chromatogram
from Sample solution A. This spot is not more intense
panne spot in the chromatogram from Sample solu-
tion B.
© INSOLUBLE SUBSTANCES
Sample: 1 g of finely powdered Phenolsulfonphthalein
Analysis: To the Sample add a solution of 0.5 g of so-
dium bicarbonate in 12 mL of water. Allow to stand for
1h, shaking frequently. Dilute with sufficient water to
make 100 mL, and allow to stand for 15 h. Centrifuge
at 2000-3000 g for 30 min, and decant the superna-
tant. Wash the residue first with 25 mL of sodium bicar-
bonate solution (1 in 100), then with 25 mL of water,
and dry at 105°.
Acceptance criteria: The weight of the insoluble resi-
due does not exceed 0.5% of the weight of the Phenol-
sulfonphthalein taken.
SPECIFIC TESTS
¢ VISUAL TRANSITION INTERVAL
Potassium chloride solution: Dissolve 1.0 g of potas-
sium chloride in 100 mL of water, and adjust with 0.01
N hydrochloric acid or sodium hydroxide to a pH of
6.8.
Sample solution: Dissolve 0.1 g of Phenolsulfonphthal-
ein in 100 mL of alcohol.
Analysis 1: Add 0.15 mL of the Sample solution to the
Potassium chloride solution.
Acceptance criteria 1: The color is yellow with NMT a
faint trace of green color.
Analysis 2: Titrate the solution from Analysis 7 with
0.01 N sodium hydroxide to a pH of 7.0.
Acceptance criteria 2: The color of the solution be-
comes orange.
Analysis 3: Continue the titration of the solution from
Analysis 2 with 0.01 N sodium hydroxide to a pH of
8:25
Acceptance criteria 3: The color of the solution be-
comes red. NMT 0.20 mL of 0.01 N sodium hydroxide
is consumed in the entire titration.
e MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI-
FIED MICROORGANISMS (62): The total aerobic microbial
count does not exceed 103 cfu/g, and the total com-
bined molds and yeasts count does not exceed 10? cfu/
g.
e Loss ON DRYING (731)
Sample: 1g of powdered Phenolsulfonphthalein
Analysis: Dry the Sample at 105° to constant weight.
Acceptance criteria: NMT 1.0%
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in well-closed contain-
ers. No storage requirements specified.
NF 36 Official Monographs / Phenylmercuric 5485

Carrier gas: Helium

Phenoxyethanol Injection volume: 1 uL
Injection type: Split injection mode
Split flow rate: 44 mL/min
co System suitability
Sample: Standard solution
Suitability requirements
Resolution: NLT 10 between the phenol and phenox-
CgH10O2 138.16 yethanol peaks
2-Phenoxyethanol; Relative standard deviation: NMT 2.0% for the phe-
2-Phenoxyethyl alcohol; noxyethanol peak
Ethylene glycol, 2-monopheny! ether [122-99-6]. Analysis
DEFINITION Samples: Standard solution and Sample solution
Phenoxyethanol contains NLT 98.0% and NMT 102.0% of Calculate the percentage of total impurities in the por-
phenoxyethanol (CgHi002). tion of Phenoxyethanol taken:

IDENTIFICATION Result = (ru/rs) x (Cs/Cu) x 100

© A. INFRARED ABSORPTION (197F): On an undried specimen
ru = peak response of all additional peak areas in
ASSAY the Sample solution, excluding the main
© PROCEDURE peak, the solvent peak, and the phenol peak
Phenol solution, Standard solution, and Chromato- rs = peak response of phenoxyethanol from the
graphic system: Prepare as directed in the test for Or- Standard solution
ganic Impurities. Gs = concentration of phenoxyethanol in the
Sample stock solution: 5 mg/mL of Phenoxyethanol in Standard solution (mg/mL)
isopropyl alcohol Cy = concentration of the Sample solution (mg/mL)
Sample solution: Transfer 500 wL of the Sample stock Acceptance criteria: NMT 1.0%
solution to a vial, add 1000 yl of isopropyl alcohol, ¢ LIMIT OF PHENOL
crimp the vial, and mix on a vortex mixer for 15 s. Phenol solution, Standard solution, Sample solution,
Analysis and Chromatographic system: Proceed as directed in
Samples: Standard solution and Sample solution the test for Organic Impurities.
Calculate the percentage of phenoxyethanol (CsH1002) Analysis
in the portion of Phenoxyethanol taken: Samples: Standard solution and Sample solution
Calculate the percentage of phenol in the portion of
Result = (ru/rs) x (Cs/Cu) x 100 Phenoxyethanol taken:
ru = peak response from the Sample solution Result = (ru/rs) x (Cs/Cu) x 100
Is = peak response from the Standard solution
G = concentration of USP Phenoxyethanol RS in ty = peak response of phenol from the Sample
the Standard solution (mg/mL) solution
Cy = concentration of Phenoxyethanol in the rs = peak response of phenol from the Standard
Sample solution (mg/mL) solution
Acceptance criteria: 98.0%-102.0% Gs = concentration of phenol in the Standard
solution (mg/mL)
IMPURITIES Cu = concentration of the Sample solution (mg/mL)
© ORGANIC IMPURITIES Acceptance criteria: NMT 0.1%
Phenol solution: 0.25 mg/mL of phenol in isopropyl
alcohol SPECIFIC TESTS
Standard stock solution: 5 mg/mL of USP Phenoxy- e SPECIFIC GRAVITY (841): 1.105—1.110 at 20°
ethanol RS in the Phenol solution
Standard solution: Transfer 500 ul of the Standard ADDITIONAL REQUIREMENTS
stock solution to a vial, add 1000 ul of isopropyl alco- e PACKAGING AND STORAGE: Preserve in tight containers,
hol, crimp the vial, and mix on a vortex mixer for 15 s. and store in a cool, dry place, protected from light.
Sample solution: Transfer 500 uL of Phenoxyethanol to e USP REFERENCE STANDARDS (11)
a tared vial, and determine the weight of Phenoxyetha- USP Phenoxyethanol RS
nol taken. Add 1000 uL of isopropyl alcohol, crimp the
vial, and mix on a vortex mixer for 15 s.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: GC Phenylethyl Alcohol—see Phenylethy!
Detector: Flame ionization
Column: 0.32-mm x 10-m capillary coated with a
Alcohol General Monographs
5-um film of stationary phase G27
Temperatures
Injection port: 300° Phenylmercuric Acetate
sydesbouo= IN

Detector: 300°

Aang
Column: See Table 1. 9

Table 1
Hold Time at!
Initial Temperature Final Final
Temperature Ramp Temperature | Temperature CsHsHgO2 336.74
@) (¢/min) ©) (min) Mercury, (acetato-O)pheny-;
80 8 260 10 (Acetato)phenylmercury [62-38-4].
 5486 Phenylmercuric / Official Monographs
DEFINITION
Phenylmercuric Acetate contains NLT 98.0% and NMT
100.5% of phenylmercuric acetate (CsHsHgOz).
IDENTIFICATION
oA.
Sample: 0.1g
Analysis: To the Sample add 0.5 mL of nitric acid. Warm
gently until a dark brown color is produced, and dilute
with water to 10 mL.
Acceptance criteria: The characteristic odor of nitro-
benzene is evolved.
° B.
Sample: 0.1g
Analysis: To the Sample add 0.5 mL of sulfuric acid and
1 mL of alcohol, and warm.
Acceptance criteria: The characteristic odor of ethyl
acetate is evolved.
ec.
Sample solution: Saturated solution in water
Analysis: To 5 mL of the Sample solution add a few
drops of sodium sulfide TS.
Acceptance criteria: A white precipitate is formed,
which turns black when the mixture is boiled and then
allowed to stand.
ASSAY
© PROCEDURE
Sample solution: Transfer 500 mg of Phenylmercuric
Acetate to a 100-mL flask. Add 15 mL of water, 5 mL of
formic acid, and 1 g of zinc dust, and reflux for 30 min.
Cool, filter, and wash the filter paper and the amalgam
with water until the washings are no longer acid to
litmus. Dissolve the amalgam in 40 mL of 8N nitric
acid. Heat on a steam bath for 3 min, and then add
500 mg of urea and enough potassium permanganate
TS to produce a permanent pink color. Cool, decolorize
thesolution with hydrogen peroxide TS, and add 1 mL
of ferric ammonium sulfate TS.
Titrimetric system
(See Titrimetry (541).)
Mode: Direct titration
Titrant: 0.1 N ammonium thiocyanate VS
Endpoint detection: Visual
Analysis: Titrate with Titrant, and each mL of 0.1 N
ammonium thiocyanate is equivalent to 16.84 mg of
phenylmercuric acetate (CsHsHgO2).
Acceptance criteria: 98.0%-100.5%
IMPURITIES
e RESIDUE ON IGNITION (281): NMT 0.2%
Delete the following:
°e MERCURIC SALTS AND HEAVY METALS
Sample solution: Heat 100 mg of Phenylmercuric Ace-
tate with 15 mL of water, cool, and filter.
Analysis: To the Sample solution filtrate add a few drops
of sodium sulfide TS.
Acceptance criteria: The resulting precipitate shows no
immediate color. oniciat 1-Jan-2018)
© POLYMERCURATED BENZENE COMPOUNDS
Sample solution: Shake 2.0 g of Phenylmercuric Ace-
“ tate with 100 mL of acetone, and filter.
cm Analysis: Wash the residue with successive portions of
Qa acetone until a total of 50 mL is used, then dry the
ii
es residue at 105° for 1 h. Weigh the residue.
aD Acceptance criteria: NMT 1.5%; the weight of residue
°
i= is NMT 30 mg.
° SPECIFIC TESTS
> © MERCURY IONS
a
C=
NF 36
SPECIFIC TESTS
© MELTING RANGE OR TEMPERATURE (741): 149°-153°
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in tight, light-resistant
containers.
Phenylmercuric Nitrate
Mercury, (nitrato-O)phenyl-;
Nitratophenylmercury [55-68-5].
DEFINITION
Phenylmercuric Nitrate is a mixture of phenylmercuric ni-
trate and phenylmercuric hydroxide containing NLT
87.0% and NMT 87.9% of phenylmercuric ion (CeHsHg*),
and NLT 62.75% and NMT 63.50% of mercury (Hg).
IDENTIFICATION
oA.
Sample: 0.1g
Analysis: To the Sample add 3 mL of sulfuric acid.
Acceptance criteria: The mixture becomes yellow, and
5 the characteristic odor of nitrobenzene is evolved.
eb.
Sample solution: Saturated solution in water
Analysis: To 5 mL of the Sample solution add 1 mL of
3 N hydrochloric acid.
Acceptance criteria: A white precipitate is formed.
eC
Sample solution: Saturated solution in water
Analysis: To 5 mL of the Sample solution add 5 mL of
ammonium sulfide TS.
Acceptance criteria: There is no reaction in the cold,
but upon heating in a boiling water bath for 10 min, a
black precipitate is formed.
ASSAY
e PHENYLMERCURIC [ONS
Sample: 200mg
Analysis: Dissolve the Sample in 90 mL of water and
10 mL of nitric acid. Add 2 mL of ferric ammonium sul-
fate TS. Titrate with 0.05 N ammonium thiocyanate VS.
Each mL of 0.05 N ammonium thiocyanate is equiva-
lent to 13.88 mg of phenylmercuric ion (CsHsHg*).
Acceptance criteria: 87.0%-87.9% of phenylmercuric
ion
o MERCURY
Sample solution: Transfer 400 mg of Phenylmercuric
Nitrate to a 100-mL flask. Add 15 mL of water, 5 mL of
formic acid, and 1g of zinc dust, and reflux for 30 min.
Cool. Filter, and wash the filter paper and the amalgam
with water until the washings are no longer acid to
litmus. Dissolve the amalgam in 40 mL of 8N nitric
acid. Heat on a steam bath for 3 min, then add 0.5 g of
urea and enough potassium permanganate TS to pro-
duce a permanent pink color. Cool. Decolorize the solu-
tion with hydrogen peroxide TS, and add 1 mL of ferric
ammonium sulfate TS.
Analysis: Titrate with 0.1 N ammonium thiocyanate VS.
Each mL of 0.1 N ammonium thiocyanate is equivalent
to 10.03 mg of Hg.
Acceptance criteria: 62.75%-63.50% of mercury
IMPURITIES
e RESIDUE ON IGNITION (281): NMT 0.1%
Sample solution: Saturated solution in water
Analysis: To 5 mL of the Sample solution add 5 mL of
1N sodium hydroxide.
NF 36 Official Monographs / Phosphoric 5487

Acceptance criteria: No yellow precipitate is formed Analysis: To the Sample solution add 1 mL of barium
(mercuric ions), and the solution does not darken (mer- chloride TS.
curous ions). Acceptance criteria: No precipitate is formed
immediately.
ADDITIONAL REQUIREMENTS e ALKALI PHOSPHATES
© PACKAGING AND STORAGE: Preserve in tight, light-resistant Sample: 1 mL
containers. Analysis: Transfer the Sample to a graduated cylinder,
and add 6 mL of ether and 2 mL of alcohol.
Acceptance criteria: No turbidity is produced.
e PHOSPHOROUS OR HYPOPHOSPHOROUS ACID
Sample solution: Dilute 6 mL of Phosphoric Acid with
Phosphoric Acid 14 mL of water.
Analysis: Gently warm 5 mL of the Sample solution, and
H3PQ4 98.00 add 2 mL of silver nitrate TS.
Phosphoric acid [7664-38-2]. Acceptance criteria: The mixture does not become
brownish.
DEFINITION
Phosphoric Acid contains NLT 85.0% and NMT 88.0%, by ADDITIONAL REQUIREMENTS
weight, of H3PO.. e PACKAGING AND STORAGE: Preserve in tight containers.
[CautTion—Avoid contact, because Phosphoric Acid rapidly
destroys tissues.]
IDENTIFICATION
© A. IDENTIFICATION TESTS—GENERAL, Phosphate (191): Diluted Phosphoric Acid
When carefully neutralized with 1 N sodium hydroxide,
phenolphthalein TS being used as the indicator, it meets DEFINITION
the requirements. Diluted Phosphoric Acid contains, in each 100 mL, NLT
ASSAY 9.5 g and NMT 10.5 g of phosphoric acid (HsPOx.).
© PROCEDURE Diluted Phosphoric Acid may be prepared as follows.
samples 1g
Blank: 120 mL of water Phosphoric Acid 69 mL
Titrimetric system Purified Water, a sufficient quantity to make 1000 mL
(See Titrimetry (541).)
Mode: Direct titration Mix the ingredients.
Titrant: 1.N sodium hydroxide VS
Endpoint detection: Visual IDENTIFICATION
Analysis: Place the Sample in a tared, glass-stoppered e A. IDENTIFICATION TESTS—GENERAL, Phosphate (191)
flask, and dilute it with water to 120 mL. Add 0.5 mL of Sample: 100 mL
Hymolphitalein TS. Titrate with 1 N sodium hydroxide Analysis: Carefully neutralize the Sample with 1 N so-
VS to the first appearance of a blue color. Perform a dium hydroxide. Use phenolphthalein TS as the
blank determination. indicator.
Calculate the percentage of phosphoric acid (H3POx) in Acceptance criteria: Meets the requirements
the Sample taken: ASSAY
Result = {[(Vs — Va) x N x F]/W} x 100 ¢ PROCEDURE
Sample: 10 mL
Vs = volume of Titrant consumed by the Sample Blank: 50 mL of water
Titrimetric system
(mL) (See Titrimetry (541).)
Ve = volume of Titrant consumed by the Blank (mL)
N = actual normality of the Titrant (mEq/mL) Mode: Direct titration
E. = equivalency factor, 49.00 mg/mEq Titrant: 1.N sodium hydroxide VS
Ww = weight of the Sample (mg) Endpoint detection: Visual
Acceptance criteria: 85.0%-88.0% by weight Analysis: Dilute the Sample with water to 50 mL. Add
0.5 mL of thymolphthalein TS. Titrate with Titrant to
IMPURITIES the first appearance of a blue color. Perform a blank
determination.
Calculate the amount of phosphoric acid (H3PO,) in the
Delete the following: portion of the sample taken:
°o HEAVY METALS, Method [ (231): NMT 10 ppme comical. Result = (Vs — Vp) x Nx F
Jan-2018)
e LIMIT OF NITRATE Vs = Titrant volume consumed by the Sample (mL)
Sample solution: Dilute 6 mL of Phosphoric Acid with Ve = Titrant volume consumed by the Blank (mL)
14 mL of water. N = actual normality of the Titrant (mEq/mL)
Analysis: Mix 5 mL of the Sample solution with about F = equivalency factor, 4.9 x 10-2 g/mEq
sydeibouo;: 4N

eae of indigo carmine TS, then add 5 mL of sulfuric Acceptance criteria: 9.5-10.5 g per 100 mL
acid.
Acceptance criteria: The blue color is not discharged IMPURITIES
within 1 min.
SPECIFIC TESTS Delete the following:
e CHLORIDE AND SULFATE, Sulfate (221)
Sample solution: Dilute 6 mL of Phosphoric Acid with °e HEAVY METALS, Method | (231)
90 mL of water. Test preparation: Dilute 10 g (9.5 mL) with 10 mL of
water, add 6 mL of 1 N sodium hydroxide, and dilute
 5488 Phosphoric / Official Monographs
with water to 50 mL. Dilute 20 mL of this solution with
water to 25 mL.
Acceptance criteria: NMT 5 ug/ge (oficial 7-Jan-2018)
e LIMIT OF NITRATE
Sample: 100 mL
Analysis: To the Sample add 0.1 mL of indigo carmine
TS, then 5 mL of sulfuric acid.
Acceptance criteria: The blue color is not discharged
within 1 min.
SPECIFIC TESTS
e ALKALI PHOSPHATES
Sample: 20 mL
Analysis: Evaporate the Sample on a steam bath to a
weight of 5 g. Cool, transfer 2 mL to agraduated cylin-
der, and add 6 mL of ether and 2 mL of alcohol.
Acceptance criteria: No turbidity is produced
© PHOSPHOROUS OR HYPOPHOSPHOROUS ACID
Sample: 100 mL
Analysis: Gently warm the Sample, and add 2 mL of
silver nitrate TS.
Acceptance criteria: The mixture does not become
brownish.
e CHLORIDE AND SULFATE, Sulfate (221)
Sample: 100 mL
Analysis: To the Sample add 1 mL of barium chloride
US;
Acceptance criteria: No precipitate is formed
immediately.
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in tight containers.
Polacrilin Potassium
eb Pel,
2-Propenoic acid, 2-methyl-, potassium salt, polymer with
diethenylbenzene;
Potassium methacrylate-divinylbenzene, copolymer
[65405-55-2].
DEFINITION
Polacrilin Potassium is the potassium salt of a unifunctional
low-cross-linked carboxylic cation-exchange resin pre-
pared from methacrylic acid and divinylbenzene. When
previously dried at 105° for 6 h, it contains NLT 20.6%
and NMT 25.1% of potassium (K).
IDENTIFICATION
e A. INFRARED ABSORPTION (197K)
e B. IDENTIFICATION TESTS—GENERAL, Potassium (191)
Sample solution A: Shake 1 g with 10 mL of water.
Sample solution B: Shake 1 g with 10 mL of 0.1 N
hydrochloric acid.
Analysis: Use the aqueous phase from Sample solution A
and Sample solution B.
NF Monographs
Acceptance criteria
Sample solution A: Does not meet the requirements
Sample solution B: Meets the requirements
ASSAY
e CONTENT OF POTASSIUM
Sodium stock solution: 14.612 mg/mL of sodium chlo-
ride, previously dried at 125° for 30 min. This solution
contains 5.76 mg/mL of sodium (Na).
NF 36
Potassium stock solution: 745.5 j1g/mL of potassium
chloride, previously dried at 125° for 30 min. This solu-
tion contains 391 ug/mL of potassium (K).
Surfactant solution: Transfer 5.0 g of a suitable noni-
onic surfactant to a 250-mL beaker, add 200 mL of
water, and stir to dissolve. Transfer this solution to a
500-mL volumetric flask, dilute with water to volume,
and mix. [NoTE—To prevent foaming when using this
solution, gently run the solution down the sides of the
vessel, and use gentle action when mixing.]
Diluted sodium solution: Transfer 50.0 mL of Sodium
stock solution and 10.0 mL of Surfactant solution to a
100-mL volumetric flask, dilute with water to volume,
and mix gently to prevent foaming.
Standard solutions: To three separate 500-mL volumet-
ric flasks transfer, respectively, 3.0-, 4.0-, and 5.0-mL
portions of Potassium stock solution. To each flask add
50.0 mL of Sodium stock solution and 10.0 mL of
Surfactant solution, dilute with water to volume, and
mix gently to prevent foaming. Each mL of these solu-
tions contains 2.346, 3.128, and 3.910 wg of K,
respectively.
Sample solution: Transfer i4g of Polacrilin Potassium,
previously dried, to a 50-mL silica crucible, moisten
with 4 mL of sulfuric acid, heat over a small flame until
the acid has fumed off, moisten the residue with a few
drops of sulfuric acid, and ignite strongly. Allow to cool,
transfer, with the aid of water, to a 1000-mL volumetric
flask, dilute with water to volume, and mix. Transfer
1.00 mL of this solution to a 100-mL volumetric flask,
add 20.0 mL of Diluted sodium solution, dilute with
water to volume, and mix gently to prevent foaming.
Instrumental conditions
Mode: Flame photometry
Analytical wavelength: 766 nm
Analysis
Samples: Standard solutions and Sample solution
Concomitantly determine the emittances of the Stan-
dard solutions and Sample solution, adjusting the in-
strument so that the most concentrated Standard so-
lution gives a reading near 100%.
Prepare a standard curve by plotting the readings from
the Standard solutions versus the square root of the
potassium concentrations. From the curve, determine
the concentration of potassium in the Sample solution.
Calculate the percentage of potassium in the portion
of sample taken:
Result = (Co/Cu) x 100
Co = concentration of the Sample solution
determined from the standard curve (ug/ml)
Cy = concentration of Polacrilin Potassium in the
Sample solution (g/mL)
Acceptance criteria: 20.6%-25.1%
IMPURITIES
Delete the following:
°e HEAVY METALS, Method Ili (231): NMT 20 ug/ge ‘offical 1-
Jan-2018)
e IRON (241)
Sample: 0.10g
Analysis: Transfer the A to a suitable crucible, and
ignite at a low heat until thoroughly ashed. Add to the
carbonized mass 2 mL of nitric acid and 5 drops of sul-
furic acid, and heat cautiously until white fumes are no
longer evolved. Ignite, preferably in a muffle furnace, at
500°-600°, until the carbon is completely burned off.
Cool, add 4 mL of 6 N hydrochloric acid, cover, digest
on a steam bath for 15 min, uncover, and slowly evap-
orate on a steam bath to dryness. Moisten the residue
with 1 drop of hydrochloric acid, add 10 mL of hot
NF 36 Official Monographs / Poloxamer 5489

water, and digest for 2 min. Dilute with water to e Loss ON DRYING (731)
25 mL. Filter, if necessary. Rinse the crucible and the Analysis: Dry at 105° for 6 h.
filter with 10 mL of water, combining the filtrate and Acceptance criteria: NMT 10.0%
rinsing in a 50-mL color-comparison tube, add 2 mL of
hydrochloric acid, dilute with water to 45 mL, and mix. ADDITIONAL REQUIREMENTS
Acceptance criteria: NMT 0.01% © PACKAGING AND STORAGE: Preserve in well-closed
e LIMIT OF SODIUM containers.
Sample solution: Transfer 2 g to a 400-mL borosilicate e USP REFERENCE STANDARDS (11)
beaker, add 20 mL of sulfuric acid, cover with a borosili- USP Polacrilin Potassium RS
cate watch glass, and heat until charring is complete.
While continuing to heat the beaker, add 20 mL of ni-
tric acid dropwise. Continue to heat, and add nitric
acid until all of the organic material has been destroyed
as indicated by the contents of the beaker turning from Poloxamer
brown to a very pale straw-colored or colorless solution.
Continue to evaporate the solution, and if it turns
brown during the evaporation, add nitric acid dropwise
until the brown color disappears. Evaporate just to dry-
ness, cool, and dissolve the residue in 40 mL of water
and 10 mL of 6 N hydrochloric acid. Heat to boiling,
cool, transfer to a 100-mL volumetric flask, dilute with HO(C2H40)a(C3H6O)o(C2H40)2H
water to volume, and mix. Oxirane, methyl-, polymer with oxirane;
Standard solutions: To three separate 100-mL volumet- o-Hydro-w-hydroxypoly(oxyethylene).-poly(oxypropylene))-
tic flasks add, respectively, 1.00, 2.00, and 3.00 mL of a poe lene), block copolymer, in which a and b
solution containing 254.2 mg of sodium chloride in ave the values shown in the following table:
1000 mL of water. Add water to volume, and mix to
obtain sodium chloride solutions having concentrations Poloxamer a b
equivalent to 1, 2, and 3 ug/mL of Na, respectively. 124 12 20
Instrumental conditions
Mode: Flame photometry 188 80 27
Analytical wavelength: 589 nm 237 64 37
Analysis 338 141 44
Samples: Standard solutions and Sample solution 407 101 56
Adjust the instrument so that the emission of the Stan-
dard solution with a concentration of 3 wg/mL reads Polyethylene-polypropylene glycol [9003-11-6].
close to 100% at 589 nm.
Determine the readings of the three Standard solutions DEFINITION
at 589 nm. Readjust the wavelength setting to 580 Poloxamer is a synthetic block copolymer of ethylene oxide
nm, and determine the background emission reading and propylene oxide. It is available in several types, con-
for one of these Standard solutions. forming to the requirements shown in the following table.
Pipet 5 mL of the Sample solution into a 100-mL volu-
metric flask, add water to volume, and mix. Observe Pol- Average Weight
the emission reading of this solution at 589 nm, using Ox- Physical Molecular (% Oxy- Unsaturation
the same instrument settings, then readjust the wave- amer Form Weight ethylene) (mEq/qg)
length setting to 580 nm, and observe the back- 124 Liquid 2090-2360 46.7+1.9 0.020 + 0.008
ground emission reading. 188 Solid 7680-9510 81.8 + 1.9 0.026 + 0.008
Subtract the corresponding background readings from
237 Solid 6840-8830 724419 0.034 + 0.008
the readings of Standard solutions and Sample solution.
Prepare a standard curve by plotting the corrected 12,700-
Standard solution readings versus the square root of 338 Solid 17,400 S37 0.031 + 0.008
the sodium concentration. From this standard curve, 9840-
determine the sodium content in the sample taken. 407 Solid 14,600 TBD EI 0.048 + 0.017
Acceptance criteria: NMT 0.20%
It may contain a suitable antioxidant.
SPECIFIC TESTS
¢ POWDER FINENESS (811) IDENTIFICATION
Sample: 4g e A. INFRARED ABSORPTION (197F): Use a thin film of
Analysis: Transfer the Sample to a No. 100 standard melted specimen if it is a solid. Use USP Poloxamer Liq-
sieve placed on top of a No. 200 standard sieve and uid RS for Poloxamer 124, and use USP Poloxamer Solid
pan. Using a soft 2-cm brush, brush the sample lightly RS for Poloxamer 188, 237, 338, and 407. Because of
across the No. 100 sieve until no more particles pass the differences in the ratios of copeet composition,
through. By brushing and tapping, dust off the particles the intensity of some absorption bands may vary.
on the underside of the No. 100 sieve into the No. 200
sieve. Obtain the weight of the material retained on the ASSAY
e AVERAGE MOLECULAR WEIGHT
No. 100 sieve. Similarly, determine the weight of mate-
sydeibouow 4N

rial retained by the No. 200 sieve.
Acceptance criteria: NMT 1.0% is retained on the No.
100 sieve, and NMT 30.0% is retained on the No. 200
sieve.
Phthalic anhydride-pyridine solution: Dissolve 144 g
of phthalic anhydride in freshly opened or freshly dis-
tilled pyridine containing less than 0.1% of water, and
dilute with pyridine to 1000 mL. Protect from light, and
allow to stand overnight. To verify that the Phthalic
anhydride-pyridine solution has adequate strength, pipet
10 mL into a 250-mL conical flask, add 25 mL of pyri-
dine and 50 mL of water, and after 15 min add 0.5 mL
of a solution of phenolphthalein in pyridine (1 in 100),
then titrate with 0.5 N sodium hydroxide VS: it con-
 5490 Poloxamer / Official Monographs
sumes between 37.6 and 40.0 mL of 0.5 N sodium
hydroxide.
Analysis: Weigh a suitable quantity, not exceeding
15g, of Poloxamer, calculated by multiplying the aver-
age molecular weight by 0.004, into a glass-stoppered,
250-mL boiling flask. Carefully pipet 25 mL of Phthalic
anhydride-pyridine solution into the flask, touching the
tip of the drained pipet to the protrusion in the flask.
Add a few glass beads, and swirl to dissolve the speci-
men. Pipet 25 mL of Phthalic anhydride-pyridine solution
into a second, glass-stoppered, conical flask, add a few
glass beads, and use as the reagent blank. (An addi-
tional 25-mL portion of pyridine may be added to both
the test specimen and reagent blank, before refluxing, if
necessary to ensure fluidity.) Heat both flasks, fitted
with suitable reflux condensers, and allow to reflux for
1h, Allow to cool, and pour two 10-mL portions of
pyridine through each condenser. Remove the flasks
from the condensers, add 10 mL of water to each, in-
sert the stoppers, swirl, and allow to stand for 10 min.
To each flask add 50.0 mL of 0.66 N sodium hydroxide
and 0.5 mL of a solution (1 in 100) of phenolphthalein
in pyridine. Titrate with 0.5 N sodium hydroxide VS to
a light pink endpoint that persists for NLT 15 s.
Calculate the average molecular weight:
Result = 2000 x W/[(Vs — Vs) x NJ
Ww = weight of the sample taken (g)
Ve = volume of 0.5 N sodium hydroxide VS
consumed by the blank (mL)
Vs = volume of 0.5 N sodium hydroxide VS
consumed by the maidtieliacia in the test
solution (mL;
N = actual normality of the 0.5 N sodium
hydroxide VS
Acceptance criteria: See the table in the Definition.
e WEIGHT PERCENT OXYETHYLENE
Solvent: Use deuterated water or deuterochloroform.
NMR reference: Use sodium 2,2-dimethyl-2-si-
lapentane-5-sulfonate (for deuterated water) or tetra-
methylsilane (for deuterochloroform).
Sample solution: Dissolve 0.1-0.2 g of Poloxamer in
deuterated water containing 1% of sodium 2,2-di-
methyl-2-silapentane-5-sulfonate to obtain 1 mL of solu-
tion, or, if the Poloxamer does not dissolve in water,
use deuterochloroform containing 1% of tetramethylsi-
lane as the solvent.
Instrumental conditions
(See Nuclear Magnetic Resonance Spectroscopy (761), Rel-
ative Method of Quantitation.)
Mode: Nuclear magnetic spectrometry
Sample size: 0.5-1.0 mL of the Sample solution
Analysis:
Sample: Sample solution
Transfer the Sample solution to a standard 5-mm NMR
spinning tube, and if deuterochloroform is the solvent,
add 1 drop of deuterated water, and shake the tube.
Scan the region at 0-5 ppm, and use the calculation
formulas specified below. Record as A; the average
area of the doublet appearing at about 1.08 ppm, rep-
resenting the methyl groups of the oxypropylene
units, and record as Az the average area of the com-
posite band at a fangs of 3.2-3.8 ppm, due to the
CH20 groups of both the oxyethylene and oxypropyl-
NF Monographs
ene units and also the CHO groups of the ou
ene units, with reference to the sodium 2,2-dimethyl-
2-silapentane-5-sulfonate or tetramethylsilane singlet at
0 ppm.
Calculate the percentage of oxyethylene, by weight, in
the Poloxamer taken:
= (A2/A1) - 1
NF 36
Az = average area of the composite band at a
range of 3.2-3.8 ppm
Ai = average area of the doublet appearing at
about 1.08 ppm
Result = 3300 x o/(33 x a + 58)
Acceptance criteria: See the table in the Definition.
e UNSATURATION
Solution A: Place303 of mercuric acetate in a
1000-mL volumetric flask, and dissolve with 900 mL of
methanol to which 0.5 mL of glacial acetic acid has
been added. Dilute with methanol to volume, and mix.
Discard the solution if it is yellow. If it is turbid, filter it.
Discard it if it is still turbid. Use fresh reagents if it is
necessary to repeat the preparation of the solution. Pro-
tect the solution from light by storing it in an amber
bottle in the dark.
Sample: 15.0g
Analysis: Transfer the Sample to a 250-mL conical flask.
Pipet 50 mL of Solution A into the flask, and mix on a
magnetic stirrer until solution is complete. Allow to
stand for 30 min with occasional swirling. Add 10 g of
sodium bromide crystals, and stir on a magnetic stirrer
for 2 min. Without delay, add 1 mL of phenolphthalein
TS, and titrate the liberated acetic acid with 0.1 N
methanolic potassium hydroxide VS. Perform a blank
determination. Determine also the initial acidity as fol-
lows. Dissolve 15.0 g of Poloxamer in 75 mL of metha-
nol that has been neutralized with methanolic potas-
sium hydroxide to the phenolphthalein endpoint. Add
1 mL of phenolphthalein TS, and titrate with the same
0.1.N DenARee potassium hydroxide VS under a ni-
trogen sweep.
Calculate the unsaturation, in mEq/g:
Result = (Vy — Vs — Va) x N/15
Vu = volume of 0.1 N methanolic potassium
hydroxide used for titrating the test
specimen (mL)
Vp = volume of 0.1 N methanolic potassium
hydroxide used for titrating the blank (mL)
Va = volume of 0.1 N methanolic potassium
hydroxide used for titrating the initial acidity
N
(mL)
= normality of the titrant
Acceptance criteria: See the table in the Definition.
IMPURITIES
Delete the following:
®o HEAVY METALS, Method | (231): NMT 20 ppme corteia 1.
Jan-2018)
e LIMIT OF FREE ETHYLENE OXIDE, PROPYLENE OXIDE, AND 1,4-
DIOXANE
Stripped poloxamer: Place 500 g of Poloxamer 124
into a suitable 3-neck, round-bottom flask equipped
with a stirrer, a thermometer, a vacuum outlet, and a
heating mantle. Evacuate the flask carefully at room
temperature to a pressure of less than 10 mm of mer-
cury, applying the vacuum slowly to avoid excessive
foaming due to entrapped gases. After any foaming has
subsided, heat the flask to 80° and continue to apply
vacuum for 2 h; then cool to room temperature. Shut
off the vacuum pump, and introduce nitrogen to bring
the flask pressure back to atmospheric pressure. Transfer
the Stripped poloxamer toa suitable nitrogen-filled
container.
Standard solution
[CauTion—Ethylene oxide, propylene oxide, and 1,4-diox-
ane are toxic and flammable. Prepare these solutions in a
well-ventilated fume hood.]
 4500 Calcium / Dietary Supplements USP 41

trahydrofolate RS, dissolve, and dilute with water to Sample solution: Filtered portion, equivalent to
volume. 0.4 mg/m of calcium L-5-methyltetrahydrofolate, from
Standard solution: 0.1 mg/mL of USP Calcium D,L- NLT 3) he ease Tablets, in water
5-Methyltetrahydrofolate RS in Antioxidant solution System suitability solution: Transfer 0.2 mL of Standard
Sample solution: Transfer a portion from NLT 30 finely Folueen to a 10-mL volumetric flask, and dilute with
powdered Tablets, nominally equivalent to 2.5 mg of Sample solution to volume.
calcium L-5-methyltetrahydrofolate, to a 25-mL volu- Chromatographic system
metric flask. Add 20 mL of Antioxidant solution and soni- (See Chromatography (621), System Suitability.)
cate for 20 min with occasional shaking, cool to room Mode: LC
temperature, dilute with Antioxidant solution to volume, Detector: UV 280 nm
mix well, and filter. Column: 4.0-mm x 15-cm; 5-um packing L79"
Chromatographic system Column temperature: 40°
(See Chromatography (621), System Suitability.) Flow rate: 1.0 mL/min
Mode: LC Injection volume: 10 uL
Detector: UV 280 nm System suitability
Column: 4.6-mm x 25-cm; 5-m packing L1 Sample: System suitability solution
Column temperature: 32° {Note—The relative retention times of L-5-methylte-
Flow rate: 1.1 mL/min trahydrofolate and D-5-methyltetrahydrofolate are
Injection volume: 10 uL about 1 and 1.5, respectively.]
System suitability Suitability requirements
Samples: System suitability solution and Standard Resolution: NLT 1.5 between L-5-methyltetrahydrofo-
solution late and D-5-methyltetrahydrofolate
[Note—For the System suitability solution the relative re- Analysis
tention times of folic acid and L- and D-isomers of Sample: Sample solution
5-methyltetrahydrofolate, which co-elute as a single Calculate the percentage of D-5-methyltetrahydrofolate
peak, are 0.85 and 1.0, respectively.] in the portion of calcium L-5-methyltetrahydrofolate
Suitability requirements taken:
Resolution: NLT 8 between folic acid and 5-methylte-
trahydrofolic acid, System suitability solution Result = [ro/(ro + r)] x 100
Relative standard ‘deviation: NMT 2.0%, Standard
solution in) = peak response of D-5-methyltetrahydrofolate
Analysis from the Sample solution
Samples: Standard solution and Sample solution fr = peak response of L-5-methyltetrahydrofolate
Calculate the percentage of the labeled amount of cal- from the Sample solution
cium L-5--ethytetrahydofoate (C20H23CaN7O6¢)in the Acceptance criteria: NMT 1.0%
portion of Tablets taken: PERFORMANCE TESTS
Result = (ru/rs) x (Cs/Cu) x 100 e DISINTEGRATION AND DISSOLUTION (2040), Disintegration:
Meet the requirements
ty =peak response from the Sample solution e@ WEIGHT VARIATION OF DIETARY SUPPLEMENTS (2091): Meet
rs =peak response from the Standard solution the requirements
Cs = concentration of USP Calcium D,L-
5-Methyltetrahydrofolate RS in the Standard ADDITIONAL REQUIREMENTS
solution (mg/mL) © PACKAGING AND STORAGE: Store ina tight, light-resistant
Cu = nominal concentration of calcium L- container, in a cool and dry place.
e USP REFERENCE STANDARDS (11)
5-methyltetrahydrofolate in the Sample
solution (mg/mL) USP Calcium D,L-5-Methyltetrahydrofolate RS
Acceptance criteria: 90.0%-110.0% USP Folic Acid RS

IMPURITIES
e ENANTIOMERIC PURITY
Buffer: 4.54 g/L of sodium dihydrogen phosphate dihy-
DS Monographs

1A chiral-recognition protein, human serum albumin (HSA), chemically

drate in water bonded to silica particle, about 5 um in diameter. For example: Chromtech
Mobile phase: Acetonitrile and Buffer (3:97). Adjust Chiral HSA, available at www.chromtech.com.
with 32% (w/v) sodium hydroxide to a pH of 6.8.
Standard solution: 0.4 mg/mL of USP Calcium D,L-
5-Methyltetrahydrofolate RS in water
NF 36 Official Monographs / Polydecene 5491

To a tared vial that can be sealed add ae of Stripped Relative standard deviation: NMT 15%
poloxamer. Add 60 uL of 1,4-dioxane and 75 uL of pro- Analysis
pylene oxide froma chilled syringe. Add ethylene ox- Samples: Standard solution and Sample solution
ide, using the following special handling procedure. Calculate the concentrations, in ug/g, of ethylene ox-
Ethylene oxide, which is a gas at room temperature, is ide, propylene oxide, and 1,4-dioxane in the portion
usually stored in a lecture-type gas cylinder or a small, of Poloxamer taken:
metal pressure-bomb. Chill the cylinder in a refrigera-
tor before use. Transfer 5 mL of the liquid ethylene Result = (ru/rs) x C
oxide to a 100-mL beaker chilled in wet ice. Using a
gas-tight syringe that has been chilled in a refrigerator, ru = peak response from the Sample solution
transfer 15 uL of the liquid ethylene oxide to the mix- rs = peak response from the Standard solution
ture. Immediately seal the vial, and shake on a vortex G = concentration of ethylene oxide, propylene
mixer for at least 30 s. Transfer 0.20 g of this solution oxide, or 1,4-dioxane in the Standard
to a tared vial that can be sealed, and add Stripped solution (g/g)
poloxamer to obtain a Standard solution having a final Acceptance criteria
weight of 50.0 g. Each g of this Standard solution con- Ethylene oxide: NMT 1 ug/g
tains 1 ug of ethylene oxide, 5 ug of propylene oxide, Propylene oxide: NMT 5 ug/g
and 5 ug of 1,4-dioxane. Transfer 1.00 + 8.01 g of this 1,4-Dioxane: NMT 5 ug/g
solution to a 22-mL pressure headspace vial, and add
about 0.01 g of butylated hydroxytoluene. Seal with a SPECIFIC TESTS
silicone septum with or without a pressure-relief star e PH (791): 5.0-7.5, in a solution (1 in 40)
spring and with a pressure-relief, aluminum, safety ADDITIONAL REQUIREMENTS
sealing-cap, and crimp the cap closed with a cap-seal- e PACKAGING AND STORAGE: Preserve in tight containers. No
ing tool. storage requirements specified.
Sample solution: Transfer 1.00 + 0.01 g of Poloxamer e LABELING: Label it to state, as part of the official title, the
to a 22-mL pressure headspace vial, and add 0.01 g of Poloxamer number. Label it to indicate the name and
butylated hydipaytaluene. Seal, cap, and crimp as di- quantity of any antioxidant.
rected for the Standard solution. e USP REFERENCE STANDARDS (11)
Chromatographic system USP Poloxamer Liquid RS
(See Chromatography (621), System Suitability.) USP Poloxamer Solid RS
Mode: GC (equipped with a balanced-pressure auto-
mated headspace sampler)
Detector: Flame ionization
Column: 0.32-mm x 50-m fused-silica capillary; 5-tum
layer of stationary phase G27 coating
Temperature Hydrogenated Polydecene
Detector: 250°
Injector: 250° CsocneroHane2
Transfer line: 140° 1-Decene, homopolymer, hydrogenated [68037-01-4].
Column: See Table 1.
DEFINITION
Hydrogenated Polydecene is a mixture of saturated, syn-
Table 1 thetic hydrocarbons in the range C3oHe2 through C7oHi42
Hold Time at made from direct oligomerization of 1-decene (Cio alpha
Initial Temperature Final Final olefin). The oligomer mixture may be distilled to fractions
Temperature Ramp Temperature | Temperature of a suitable calculated viscosity and hydrogenated to
«) (¢/min) «@) (min) reach saturation, or it may be hydrogenated to reach sat-
70 = 70 10 uration and then distilled to the desired viscosity. The re-
quirements for specific gravity, viscosity, and content of
70 10 240 10
decene oligomer differ for the various types of Hydrogen-
Carrier gas: Helium ated Polydecene, as set forth in the two tables below.
Flow rate: 1.6 mL/min Hydrogenated Polydecene may contain a suitable
eee size: Separately place the vials containing stabilizer.
the Standard solution and the Sample solution in the
automated sampler, and start the sequence so that the Specific Gravity and Viscosity
vial is heated at a temperature of 110° for 30 min Kinematic
before a suitable portion of its headspace is injected Viscosity
into the chromatograph. Range,
Autosampler Specific Centistokes
Needle-withdrawal time: 0.3 min Type Gravity (mm?/s)
Pressurization time: 1 min
| 0.814-0.819 16.0-20.0
Injection time: 0.08 min
Vial pressure: 22 psig with the vial vent off MW 0.823-0.827 28.0-34.0
System suitability Ml 0.828-0.832 40.0-52.0
Sample: Standard solution v4
al
{Nott—The relative retention times for ethylene oxide,
propylene oxide, and 1,4-dioxane are about 1.0, 1.3, Content of Decene Oligomers ES
and 3.8, respectively.]
Type Csotle2 CaoHs2 Csottio2 CootHi22 Cro ya: Pe
Suitability requirements
Resolution: NLT 2.0 between ethylene oxide and | 70-93 5-25 0-5 0-1 0-1 A
propylene oxide Ul 13-40 35-70 9-25 0-7 0-2 Ry
Ul 3-15 25-55 25-40 13-28 0-10 ‘ce
a
 5492 Polydecene / Official Monographs NF 36

IDENTIFICATION IMPURITIES
e A. The chromatogram of the Sample solution from the e LIMIT OF NICKEL
test for Content of Decene Oligomer exhibits major peaks Nickel stock solution: Immediately before use, dilute
for trimers, tetramers, pentamers, hexamers, and possibly an appropriate quantity of ee standard!
heptamers. The decene oligomer content is within the with kerosene to prepare a solution containing the
range given in the table Content of Decene Oligomers in equivalent of 1.0 j1g/mL of nickel.
the Definition for the labeled type of Hydrogenated Standard solutions: Transfer 0.5, 1.0, 2.0, and 4.0 mL
Polydecene. of Nickel stock solution, respectively, to four identical
10-mL volumetric flasks, dilute the contents of each
ASSAY flask with kerosene to volume, and mix. These Standard
e CONTENT OF DECENE OLIGOMER solutions contain, respectively, 0.05, 0.1, 0.2, and
System suitability solution: 10 mg/mL of hexadecane, 0.40 g/mL of nickel. [NoTE—The calibration range, es-
10 mg/mL of squalane, and 1 mg/mL of tetradecane in pecially the upper limit, can be adjusted for certain in-
pentane struments, provided that instrument validation and cali-
Sample solution: Dissolve 0.1 mL of Hydrogenated bration linearity are achieived.]
Polydecene in 10 mL of pentane. Sample solution: 0.3 g/mL of Hydrogenated
Chromatographic system Polydecene in kerosene. [NoTE—If necessary, dilute with
(See Chromatography (621), System Suitability.) an appropriate quantity of kerosene to obtain a reading
Mode: GC within the calibrated absorbance range.]
Detector: Flame ionization Instrumental conditions
Column: 0.52-mm x 16-m fused-silica capillary; coated (See Atomic Absorption Spectroscopy (852).)
with 0.1-mm stationary phase G2 Mode: Graphite furnace atomic absorption spectro-
Carrier gas: Helium photometer equipped with a deuterium background
Flow rate: 10 mL/min corrector and a pyrolytically coated tube with platform
Injection volume: 2 LL gies wavelength: 232.0 nm (nickel emission
Temperatures ine)
Injection port: 310° Injection volume: 20 pL
Detector: 320° Lamp: Nickel hollow-cathode
Column: See Table 1. Blank: Kerosene
Temperature: See Table 2.
Table 1 [NoTe—The temperature program may be modified to
" obtain optimum furnace temperatures.]
Hold Time
Initial Final at Final
Temperature Ramp Temperature | Temperature Table 2
C) C/min) () (min) Temperature Hold Time
So 5 50 = Step © (s)
50 12 170 = Orying 80 1
170 10 310 18 Drying 120 10

System suitability rene oe =

Sample: System suitability solution saune
[Note—The retention time for squalane is about 18 Ashing 1000 20
min; the relative retention times for tetradecane, hex- Atomization 2500 3
adecane, and squalane are about 0.5, 0.6, and 1.0, Cleaning 2600 5
respectively.]
Suitability requirements Analysis
Resolution: NLT 2.0 between tetradecane and Samples: Standard solutions and Sample solution
hexadecane Place the Standard solutions and the Sample solution in
Relative standard deviation: NMT 2.0% for each an oven, setting the temperature at about 60° during
peak the period of determination, and shake these solu-
Analysis tions vigorously before analysis. Use micropipettor
Sample: Sample solution and pipettor tips to make all injections. [NoTE—Posi-
Record the chromatogram, and measure the areas for tive displacement pipets can be used when viscosity
the major peaks. may become a problem.]
[Note—The tetramer oligomer has a retention time of Pretreat the pipettor tip by pipetting and then discard-
about 23 min. The trimer, pentamer, hexamer, and ing 20 uL of heptane. The tip must be pretreated
heptamer oligomers, if present, have relative retention before each injection. [NoTE—The film of heptane re-
times of about 0.8, 1.1, 1.3, and 1.4, respectively, rel- maining on the wall of the tip facilitates a reproduci-
ative to the tetramer.] ble transfer of the oil sample.]
Calculate the percentage of each oligomer present: Separately inject the Standard solutions and the Sample
solution into a graphite furnace, and concomitantly
Result = (ru/rr) x 100 determine the integrated absorbances of the Standard
solutions and the Sample solution.
tu = response of each oligomer Plot the integrated absorbances of the Standard solu-
NF Monographs

tr = sum of the responses of all the peaks, tions versus concentration, in g/mL, of nickel, and
excluding the solvent peak draw the straight line best fitting the four plotted
Acceptance criteria: The decene oligomer content is points. From the graph so obtained, determine the
within the limits specified in the table Content of Decene concentration of nickel, C, in ug/mL, in the Sample
Oligomers in the Definition. solution.
1 Suitable organometallic standards are available from, e.g., Continental Oil
Co., Ponca City, OK (Conostan, 100 ppm), or Merck, D-6100 Darmstadt,
Germany (metal in standard oil, 1000 ppm).
NF 36
Calculate the content of nickel in the Hydrogenated
Polydecene taken:
Result = C/Cy
Cc = concentration of nickel obtained from the
graph (g/mL) . Polydextrose is a randomly branched polymer prepared by
Cu = concentration of nickel in the Sample solution
(g/mL)
Acceptance criteria: NMT 1 ug/g
e Limit OF SHORT-CHAIN HYDROCARBONS
System suitability solution, Sample solution, Chromat-
ographic system, System suitability, and Analysis:
Proceed as directed in the test for Content of Decene
Oligomer.
Calculate the percentage of each of the short-chain hy-
drocarbons present:
Result = (ru/r7) x 100
tu = peak response of any peak eluting before the
trimer but different from the solvent peak
Ir = sum of the responses of all the peaks in the
chromatogram, excluding the solvent peak
Acceptance criteria: NMT 2.5% of total short-chain hy-
drocarbons is found.
SPECIFIC TESTS
e SPECIFIC GRAVITY (841)
Analysis: Determine at 20°.
Acceptance criteria: Meets the requirements of the
specific gravity range specified in the table Specific Grav-
ity and Viscosity in the Definition for the labeled type
e ViscosiTY—CAPILLARY METHODS (911)
Analysis: Determine using a capil viscometer, in a
liquid bath maintained at 40.0 + 0.7°.
Acceptance criteria: Meets the requirements of the vis-
cosity range specified in the table Specific Gravity and
Viscosity in the Definition for the labeled type.
e READILY CARBONIZABLE SUBSTANCES TEST ort)
Standard solution: 3 mL of ferric chloride CS, 1.5 mL
of cobaltous chloride CS, and 0.5 mL of cupric sulfate
CS in a glass-stoppered test tube previously treated to
remove organic matter (see Cleaning Glass Apparatus
(1051)).
Sample: 5 mL
Analysis: Transfer the Sample to a glass-stoppered test
tube previously treated to remove organic matter (see
Cleaning Glass Apparatus (1051)), add 5 mL of sulfuric
acid, and heat in a boiling water bath for 30 s. Quickly
remove the test tube, and, while holding the stopper in
place, shake three times in a vertically reciprocating cy-
cle with an amplitude of about 13 cm. Repeat this pro-
cedure every 30 s for 10 min. Do not keep the test
tube out of the water bath any longer than 3 s for each
shaking cycle. Remove the test tube from the water
bath, and let it cool for about 20 min to room
temperature.
Acceptance criteria: The oil phase of the Sample may
turn hazy but remains colorless; the interface between
the two layers is free from solids; and the acid layer
does not become darker than the standard color pro-
duced by the Standard solution, the Standard solution
being overlaid with 5 mL of Hydrogenated Polydecene.
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in tight containers. No
storage requirements are specified.
e LABELING: Label it to indicate, as part of the official title,
the Hydrogenated Polydecene type (Type |, Type Il, or
Type Ill), and label it to indicate the name Lrld corkeerr.
tration of any added stabilizer.
Official Monographs / Polydextrose 5493
Polydextrose
[68424-04-4].
DEFINITION
melting and subsequent condensation of the ingredients,
which consist of approximately pales dextrose, 10 parts
sorbitol, and up to 1 part citric acid or 0.1 part phos-
phoric acid. The 1,6-glycosidic linkage predominates in
the polymer but other linkages are present. It contains
NLT 90.0% of dextrose ae units, calculated on the
anhydrous and ash-free basis. It contains small quantities
of free dextrose, sorbitol, and 1,6-anhydro-D-glucose
(levoglucosan), with traces of citric acid or phosphoric
acid.
IDENTIFICATION
e A. To 1 drop of a solution (1 in 10), add 4 drops of 5%
phenol solution, then rapidly add 15 drops of sulfuric
acid TS: a deep yellow to orange color is produced.
e B. With vigorous swirling, add 1 mL of acetone to 1 mL
of a solution (1 in 10): the solution remains clear.
e C. With vigorous swirling, add 2 mL of acetone to the
solution obtained in /dentification test B: a heavy, milky
turbidity develops immediately.
e D. To 71 mL ofa solution (1 in 50), add 4 mL of alkaline
cupric citrate TS. Boil vigorously for 2-4 min. Remove
from heat, and allow the precipitate (if any) to settle: the
supernatant is blue or blue-green.
ASSAY
e PROCEDURE
Mobile phase: 0.001 N sulfuric acid. Pass this solution
througha filter of 0.5-11m pore size, and degas.
Standard solution: 4.0 mg/mL of USP Poly extrose RS,
calculated on the anhydrous and ash-free basis, in Mo-
bile phase
Sample solution: 4.0 mg/mL of Polydextrose, calcu-
salen on the anhydrous and ash-free basis, in Mobile
phase
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: Refractive index
Detector temperature: 35+0.1°
Guard column: 4.6-mm x 3.0-cm, packing L17
Analytical column: 7.8-mm x 30-cm, packing L17
Flow rate: 0.6 mL/min
Injection size: 20 uL
System suitability
Sample: Standard solution
Suitability requirements
Relative standard deviation: NMT 2.0%
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of dextrose polymer units in
the portion of Polydextrose taken:
Result = (ru/rs) x (Cs/Cu) x 100
tu = peak response of dextrose polymer units from
the Sample solution
Ts = peak response of dextrose polymer units from
sydeibouo;- 4N
the Standard solution
Cs = concentration of USP Polydextrose RS in the
Standard solution (mg/mL)
Cu = concentration of Polydextrose in the Sample
solution (mg/mL)
 5494 Polydextrose / Official Monographs NF 36

Acceptance criteria: NLT 90.0% Analysis

IMPURITIES
Inorganic Impurities
e RESIDUE ON IGNITION (281): NMT 0.3%
e Limit OF LEAD
[NoT&—Use reagent-grade chemicals with as low a lead
content as is practicable, as well as high-purity water
and gases. Before use in this analysis, rinse all glassware
and plasticware twice with 10% nitric acid and twice
with 10% hydrochloric acid, and then rinse them thor-
oughly with Purified Water.]
Matrix modifier solution: Prepare a solution in water
containing 100.0 mg of dibasic ammonium phosphate
per 10 mL of solution.
Lead nitrate stock solution: Dissolve 159.8 mg of lead
nitrate in 100 mL of water to which has been added
1 mL of nitric acid, then dilute with water to 1000 mL.
Prepare and store this solution in glass containers free
from soluble lead salts.
Standard lead solution: On the day of use, dilute
10.0 mL of Lead nitrate stock solution with water to
100.0 mL. Each mL of Standard lead solution contains
the equivalent of 10 1g of lead.
Standard solution A: 0.02 g/mL of lead, from Stan-
dard lead solution in water
Standard solution B: 0.05 g/mL of lead, from Stan-
dard lead solution in water
Standard solution C: 0.1 g/mL of lead, from Standard
lead solution in water
Standard solution D: 0.2 ug/mL of lead, from Stan-
dard lead solution in water
Standard solution E: 0.5 g/mL of lead, from Standard
lead solution in water
Sample solution: Transfer 1.0 g of Polydextrose,
weighed and calculated on the anhydrous and ash-free
basis, into a 10-mL volumetric flask, and dissolve in
and dilute with water to volume.
Spikedsans solution: Transfer 1.0 g of Polydex-
trose, weighed and calculated on the anhydrous and
ash-free basis, into a 10-mL volumetric flask, and dis-
solve in water. Add 100 uL of the Standard lead solu-
tion, and dilute with water to volume. This solution
contains 0.1 g/mL of added lead.
Spectrometric conditions
(See Atomic Absorption Spectroscopy (852).)
Mode: Graphite furnace atomic absorption spectro-
Samples: 10 Ll of the Matrix modifier solution was
added into each 10-uL aliquot of the five Standards
solutions, a mixture of 10 uL of the Matrix modifier
solution and 10 ul of the Sample solution, and a mix-
ture of 10 uL of the Matrix modifier solution and 10 wh
of the Spiked sample solution
Concomitantly determine the absorbances of the
Samples using the Spectrometric conditions described
above. Plot the absorbance of each Standard solu-
tion, compensated for background correction, versus
its content of lead, in ug/mL, and draw the best
straight line fitting the five points. From this plot,
determine the concentrations, Cr and Csr, in ug/mL,
of lead in the Sample solution and the Spiked sample
solution, respectively.
Calculate the percentage recovery taken:
Result = [(Csr — Cr)/A] x 100
A = quantity of lead added to the Spiked sample
solution, 0.1 g/mL
Calculate the content, in ug/g, of lead in the portion
of Polydextrose taken:
Result = (C;/W) x V
Ww = weight of Polydextrose taken to prepare the
Sample solution (g)
Vv = volume of the Sample solution, 10 mL
Acceptance criteria: NMT 0.5 g/g. The recovery is
80%-120%.
Organic Impurities
¢ PROCEDURE 1: LIMIT OF 5-HYDROXYMETHYLFURFURAL AND RE-
LATED COMPOUNDS
Sample solution: 1.0g of Polydextrose, weighed and
calculated on the anhydrous and ash-free basis, diluted
with water to 100 mL
Analysis: Determine the absorbance of the Sample solu-
tion in a 1-cm quartz cell at 283 nm, witha suitable
spectrophotometer, using water as the blank.
Calculate the percentage of 5-hydroxymethylfurfural
and related compounds in the Polydextrose taken:
Result = 100 x (V
x M, x A)/(M x L x W)
volume of the Sample solution, 0.1 L
wou

molecular weight of 5-hydroxymethylfurfural,

z> =

photometer, equipped with a pyrolytic tube with a

126 g/mol
platform
Lamp: Alead hollow-cathode lamp, using a slit width absorbance of the Sample solution
ou

molar extinction coefficient of

of 0.7 mm (set low) and a deuterium arc lamp for
background correction 5-hydroxymethylfurfural at a wavelength of
Analytical wavelength: Lead emission line of 283.3 283 nm, 16,830 L/mol cm
a on of the spectrophotometer cell
i

nm
Autosampler cm
Ww = weight of Polydextrose taken to prepare the
Sample volume: 10 pL
Alternative volume: 10 ul of Matrix modifier solution Sample solution (g)
Furnace program: See the temperature program table Acceptance criteria: NMT 0.1%
below. © PROCEDURE 2: LIMIT OF MONOMERS
Mobile phase, Sample solution, and Chromatographic
system: Prepare as directed in the Assay.
Atom- Standard solution: 0.08 mg/mL of each of USP 1,6-
Step Dry Char ize Clean_| Recharge Anhydro-D-glucose RS and USP Sorbitol RS, and
Temperature 130 800 2400 2600 20 0.16 mg/mL of USP Dextrose RS, in Mobile phase
a) (°) System sulnaplllyy
= Sample: Standard solution
rs Ramp time 20 20 0 1 2
[Note—For relative retention times, see Table 1
i] (s)
—
below.]
Dd Hold time 40 40 6 5 20
) (s)
i
iS Argon flow 300 300 50 300 300
= rate
(mL/min)
_
Zz
NF 36 Official Monographs / Polydextrose 5495

Table 1 Check the flow gravimetrically, and adjust it if neces-

Relative
sary. Reduce the flow rate to about 0.1 mL/min when
the system is not in use.]
Retention
Name Time
Injection size: 50 uL
System suitability
Dextrose (glucose) 0.7 Sample: Standard solution
Sorbitol 0.8 [NotE—The retention times for each component deter-
An isomer of 1,6-anhydro-D-glu- mined on replicate injections agree within +2 s.]
cose (D-anhydroglucose fura- Chromatograph five replicate injections of the Stan-
nose form) 0.9 dard solution, allowing 15 min between injections,
1,6-Anhydro-D-glucose (D- and record the retention times of the components of
anhydroglucose pyranose form) 1.0 the Standard solution.
Insert the average retention time along with the mo-
Suitability requirements lecular weight of each component in the Standard
Resolution: NLT 1.0 solution into the calibration table of the molecular
Relative standard derivation: NMT 5.0% weight distribution software. Check the regression re-
Analysis sults for a cubic fit of the calibration points, and ob-
Samples: Standard solution and Sample solution tain a correlation coefficient, R, for the line.
Use peat response of USP 1,6-Anhydro-D-glucose RS Suitability requirements
in the Standard solution for calculation of percentage Resolution: Dextrose and stachyose are baseline re-
of the isomer of 1,6-anhydro-D-glucose in the Sam- solved from one another and from the 5800-MW pul-
ple solution. lulan standard.
Calculate the percentage of each monomer in the [Nott—Prominent negative baseline valleys are usually
portion of Polydextrose taken: observed between the peaks for the 5800-; 23,700-;
and 100,000-MW pullulan standards.]
Result = (ru/ts) x (Cs/Cu) x 100 Correlation coefficient R: NLT 0.9999
Analysis
tu = peak response of the respective monomer Samples: Standard solution and Sample solution
from the Sample solution Use the molecular weight distribution software of the
Is = peak response of the respective monomer data reduction system to generate a molecular
from the Standard solution weight distribution plot of Polydextrose.
Cs = concentration of the neypectine standard Acceptance criteria: No measurable peak above a mo-
monomer in the Standard solution (mg/mL) lecular weight of 22,000 is found.
Cu = concentration of Polydextrose in the Sample e PH (791): 2.5-5.0, in a solution (1 in 10)
solution (mg/mL) e WATER DETERMINATION, Method | (921): NMT 4.0%. Use
Acceptance criteria: NMT 4.0% for 1,6-anhydro-D-glu- a mixture of Hydranal solvent and Hydranal formamide
cose, NMT 4.0% for dextrose, and NMT 2.0% for sor- dry (2:1) as a solvent. Perform the titration at 50° in a
bitol. [NoTE—In the case of 1,6-anhydro-D-glucose, the jacketed beaker.
peak areas for the pyranose and furanose forms are
combined.] ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in tight, light-resistant
SPECIFIC TESTS containers. Store in a cool and dry place.
@ MOLECULAR WEIGHT LIMIT e USP REFERENCE STANDARDS (11)
Mobile phase: Dissolve 35.0 g of sodium nitrate and USP 1,6-Anhydro-D-glucose RS
1.0 g of sodium azide in 100 mL of water. Dilute with USP Dextrose RS
water to 4L. Pass through a filter of 0.45-um pore size, USP Polydextrose RS
and degas byapplying an aspirator vacuum for 30 min. USP Sorbitol RS
The resulting Mobile phase is 0.1 N sodium nitrate con-
taining 0.025% sodium azide.
Standard solution: Transfer 20 mg each of USP Dex-
trose RS, stachyose, and 5800-, 23,700-, and 100,000-
molecular weld tt (MW) pullulan standards into a 10-mL
volumetric flask. Dissolve in and dilute with Mobile Hydrogenated Polydextrose
phase to volume. Pass through a syringe filter of 0.45-
um pore size into a suitable autosampler vial, and seal. DEFINITION
Sample solution: Transfer 50 mg of Polydextrose into a Hydrogenated Polydextrose is obtained by transition metal
10-mL volumetric flask. Dissolve in and dilute with Mo- Dave hydrogenation of Polydextrose in aqueous solu-
bile phase to volume. Pass through a syringe filter of tion. It contains NLT 90.0% of dextrose polymer units,
capm pore size into a suitable autosampler vial, and calculated on the anhydrous and ash-free basis. The poly-
seal. mer chain end groups are mainly sorbitol-terminated.
Chromatographic system IDENTIFICATION
(See Chromatography (621), System Suitability.) e A. To 1 drop of a solution (1 in 10), add 4 drops of 5%
Mode: LC phenol solution, then rapidly add 15 drops of sulfuric
Detector: Refractive index set at a sensitivity of 4 x acid TS: a deep yellow to orange color is produced.
10-6 refractive index units full scale and maintained at
sydesbouow; 4N

B. With vigorous swirling, add 1 mL of acetone to 1 mL

a temperature of 35+0.1°
Column: 7.8-mm x 30-cm; packing L39
Column temperature: 45°
Flow rate: 0.8 mL/min
[Note—After installation of a new column, pump Mobile
phase through the column overnight at a rate of
0.3 mL/min. Before calibration or analysis, increase the
flow slowly over a 1-min period to 0.8 mL/min. Con-
tinue to pump Mobile phase through the column at
this flow rate for at least 1 h before the first injection.
of a solution (1 in 10): the solution remains clear.
C. With vigorous swirling, add 2 mL of acetone to the
solution obtained in /dentification test B: a heavy, milky
turbidity develops immediately.
D. To 1 mL ofa solution (1 in 50), add 4 mL of alkaline
cupric citrate TS. Boil vigorously for 2-4 min. Remove
from heat, and allow the precipitate (if any) to settle: the
supernatant is blue or blue-green.
E. Meets the requirements for dextrose in Procedure 2,
Limit of Monomers
 5496 Polydextrose / Official Monographs NF 36

ASSAY and ash-free basis, into a 10-mL volumetric flask, dis-

e PROCEDURE solve in and dilute with water to volume.
Mobile phase 0.001 N sulfuric acid. Pass through a Spiked sample solution: Transfer 1.0 g of Hydrogen-
filter of 0.5-11m or finer pore size, and degas. ated Polydextrose, weighed and calculated on the an-
Standard solution: 4.0 mg/mL of USP Polydextrose RS, hydrous and ash-free basis, into a 10-mL volumetric
calculated on the anhydrous and ash-free basis, in Mo- flask, and dissolve in water. Add 100 pL of Standard
bile phase lead solution, and dilute with water to volume. This so-
Sample solution: 4.0 mg/mL of Hydrogenated Polydex- lution contains 0.1 j1g/mL of added lead.
trose, calculated on the anhydrous and ash-free basis, in Spectrometric conditions
Mobile phase (See Atomic Absorption Spectroscopy (852).)
Chromatographic system Mode: Graphite furnace atomic absorption spectro-
(See Chromatography (621), System Suitability.) photometer, equipped with a pyrolytic tube with a
Mode: LC platform
Detector: Refractive index Lamp: A lead hollow-cathode lamp, using a slit width
Detector temperature: 35+0.1° of 0.7 mm (set low) and a deuterium arc lamp for
Guard column: 4.6-mm x 3.0-cm; packing L17 background correction
Analytical column: 7.8-mm x 30-cm; packing L17 Analytical wavelength: Lead emission line of 283.3
Flow rate: 0.6 mL/min nm
Injection size: 20 uL Autosampler
System suitability Sample volume: 10 uL
Sample: Standard solution Alternative volume: 10 ul of Matrix modifier solution
Suitability requirements Furnace program: See the temperature program table
Relative standard deviation: NMT 2.0% below.
Analysis
Samples: Standard solution and Sample solution Atom-
Calculate the percentage of dextrose polymer units in Step Dry Char ize Clean | Recharge!
the Hydrogenated Polydextrose taken: Temperature 130 800 2400 2600 20
Result = (ru/ts) x (Cs/Cy) x 100 (°)
Ramp time 20 20 0 1 2
tu = peak response for dextrose polymer units from (s)
the Sample solution Hold time 40 40 6 5 20
Ts = peak response for dextrose polymer units from (s)
the Standard solution Argon flow 300 300 50 300 300
Cs = concentration of USP Polydextrose RS in the rate
Standard solution (mg/mL) (mL/min)
Cu = concentration of Hydrogenated Polydextrose
in the Sample solution (mg/mL) Analysis
Acceptance criteria: NLT 90.0% on the anhydrous and Samples: 10 ul of the Matrix modifier solution was
ash-free basis added into each of the 10-uL aliquots of the five
Standard solutions, a mixture of 10 uL of the Matrix
IMPURITIES modifier solution and 10 wL of the Sample solution, and
Inorganic Impurities a mixture of 10 uL of the Matrix modifier solution and
e RESIDUE ON IGNITION (281): NMT 0.3% 10 uL of the Spiked sample solution
e Limit oF LEAD Concomitantly determine the absorbances of the
[NoTe—Use reagent-grade chemicals with a lead content Samples using the Spectrometric conditions described
of as low as possible, as well as high-purity water and above. Plot the absorbance of each Standard solu-
gases. Before use in this analysis, rinse all glassware and tion, compensated for background correction, versus
plasticware twice with 10% nitric acid and twice with its content of lead, in ug/mL, and draw the best
10% hydrochloric acid, and then rinse them thoroughly straight line fitting the five points. From this plot,
with Purified Water.] determine the concentrations, Cr and Csr, in ug/mL,
Matrix modifier solution: 10.0 mg/mL of dibasic am- of lead in the Sample solution and the Spiked sample
monium phosphate solution, respectively.
Lead nitrate stock solution: Dissolve 159.8 mg of lead Calculate the percentage recovery taken:
nitrate in 100 mL of water to which has been added
1 mL of nitric acid, then dilute with water to 1000 mL. Result = [(Cst — Cr)/A] x 100
Prepare and store this solution in glass containers free
from soluble lead salts. A = quantity of lead added to the Spiked sample
Standard lead solution: On the day of use, dilute solution, 0.1 wg/mL
10.0 mL of Lead nitrate stock solution with water to Calculate the content, in ug/g, of lead in
100.0 mL. Each mL of Standard lead solution contains Hydrogenated Polydextrose taken:
the equivalent of 10 jg of lead.
Standard solution A: 0.02 g/mL of lead, from Stan- Result = (Cr/W) x V
dard lead solution in water
Standard solution B: 0.05 1g/mL of lead, from Stan- Ww = weight of Hydrogenated Polydextrose taken
NF Monographs

dard lead solution in water to prepare the Sample solution (g)

Standard solution C: 0.1 g/mL of lead, from Standard Vv = volume of the Sample solution, 10 mL
lead solution in water Acceptance criteria: NMT 0.5 g/g; recovery is
Standard solution D: 0.2 \1g/mL of lead, from Stan- 80%-120%
dard lead solution in water e Limit OF NICKEL
Standard solution E: 0.5 g/mL of lead, from Standard [Note—All glassware used must be soaked in 1% Nitric
lead solution in water acid for at least 2 h and then rinsed with water.]
Sample solution: Transfer 1.0 g of Hydrogenated Poly- 1% Nitric acid: Cautiously add 10 mL of nitric acid to
dextrose, weighed and calculated on the anhydrous a 1000-mL volumetric flask containing about 500 mL of
water. Mix, and dilute with water to volume.
NF 36
Blank solution: Use 1% Nitric acid.
Nickel stock standard solution: Immediately before
use, dilute an appropriate amount of nickel standard*
with 1% Nitric acid to prepare a solution containing the
equivalent of 10 g/mL of nickel.
Standard solutions: Into four identical 100-mL volu- e PROCEDURE 2: LIMIT OF MONOMERS
metric flasks, introduce respectively 1.0, 2.0, 5.0, and
10.0 mL of Nickel stock standard solution. Dilute with
1% Nitric acid to volume, and mix. These standards
contain 0.1, 0.2, 0.5, and 1.0 ug/mL of nickel.
Sample solution: Weigh 5 g of Hydrogenated Polydex-
trose into a 100-mL volumetric flask. Dissolve in and
dilute with 7% Nitric acid to volume, and mix.
Spectrometric conditions
(See Atomic Absorption Spectroscopy (852).)
Mode: Atomic absorption spectrophotometer
equipped with an air—acetylene flame
Lamp: Nickel hollow-cathode
Analytical wavelength: 352.0 nm
System sty
Sample: Standard solution of 0.2 ug/mL of nickel Sorbitol
Suitability requirements
Relative standard deviation: NMT 20%
Analysis
Samples: Standard solutions and Sample solution
Use the Blank solution to zero the instrument. Con-
comitantly determine the absorbances of the Sam-
ples at least three times each. Record the average of
the Sea readings for each of the Samples. Clear
the nebulizer using the Blank solution, and aspirate
each of the Samples in turn. The standard chosen for
reslope should be run every four to five samples. If
there is a significant change in its response, reslope
and repeat the previous samples.
Plot the absorbances of the Standard solutions versus
concentration, in g/mL, of nickel, and draw the
straight line best fitting the four plotted points.
From the graph so obtained, determine the concen-
tration, C, in ug/mL, of nickel in the Sample solution.
Calculate the quantity, in ug, of nickel in each g of
Hydrogenated Polydextrose taken:
Result = (V x C)/W
Vv = volume of the Sample solution, 100 mL
Ww = weight of Hydrogenated Polydextrose taken to
prepare the Sample solution (g)
Acceptance criteria) NMT 2 ug/g
Organic Impurities
e PROCEDURE 1: LIMIT OF 5-HYDROXYMETHYLFURFURAL AND RE-
LATED COMPOUNDS
Sample solution: 1.0 g of Hydrogenated Polydextrose,
weighed and calculated on the anhydrous and ash-free
basis, diluted with water to 100 mL
Analysis: Determine the absorbance of the Sample solu-
tion in a 1-cm quartz cell at 283 nm, with a suitable SPECIFIC TESTS
spectrophotometer, using water as the blank. © MOLECULAR WEIGHT LIMIT
Calculate the percentage of 5-hydroxymethylfurfural
and related compounds in the Hydrogenated Poly-
dextrose taken:
Result = (V x M, x A)/(€283 x L x W) x 100
Vv = volume of the Sample solution, 0.1 L
M, = molecular weight of 5-hydroxymethylfurfural,
126 g/mol
A = absorbance of the Sample solution
€283 | = molar extinction coefficient of 5-hydroxy-
methylfurfural at a wavelength of 283 nm,
16,830 L- mol. cm"
*Suitable nickel standards are available from e.g., Fisher Scientific, Fair Lawn,
NJ (nickel, reference standard solution, 1000 ppm +1%, certified, application
for atomic absorption) or RICCA Chemical Company, Arlington, TX (nickel
standard, 1000 ppm Ni, for atomic absorption).
Official Monographs / Polydextrose 5497
L = path length of the spectrophotometer cell
(cm)
Ww = weight of Hydrogenated Polydextrose taken
to prepare the Sample solution (g)
Acceptance criteria: NMT 0.1%
Mobile phase, Sample solution, and Chromatographic
system: Prepare as directed in the Assay.
Standard solution: 0.08 mg/mL of each for USP 1,6-
Anhydro-D-glucose RS and USP Sorbitol RS, and
0.04 mg/mL of USP Dextrose RS, in Mobile phase
System suitability
Sample: Standard solution
[Note—See the relative retention times table below.]
Relative
Retention
Name Time
Dextrose (glucose) 0.7
0.8
An isomer of 1,6-anhydro-D-glucose
(b-anhydroglucose furanose form) 0.9
1,6-Anhydro-D-glucose (levoglucosan)
(D-anhydroglucose _pyranose form) 1.0
Suitability requirements
Resolution: NLT 1.0
Relative standard derivation: NMT 5.0%
Analysis
Samples: Standard solution and Sample solution
Use the peak response of USP 1,6-Anhydro-D-glucose
RS in the Standard solution for calculation of the per-
centage of the isomer of 1,6-anhydro-D-glucose in
the Sample solution. Calculate the percentage of
each monomer in the portion of Hydrogenated Poly-
dextrose taken:
Result = (ru/rs) x (Cs/Cu) x 100
tu = peak response for the respective monomer
from the Sample solution
Ts = peak response for the respective monomer
from the Standard solution
Cs = concentration of the respective standard
monomer in the Standard solution (mg/mL)
Cu = concentration of Hydrogenated Polydextrose
in the Sample solution (mg/mL)
Acceptance criteria: NMT 4.0% of 1,6-anhydro-p-glu-
cose, NMT 5.75% for sorbitol and NMT 0.25% for
dextrose
[NoTe—In the case of 1,6-anhydro-D-glucose, the peak
areas for the pyranose and furanose forms are com-
bined.]
Mobile phase: Dissolve 35.0 g of sodium nitrate and
1.0 g of sodium azide in 100 mL of water. Dilute with
water to 4L. Pass throughafilter of 0.45-m or finer
pore size, and degas by applying an aspirator vacuum
for 30 min. The resulting Mobile phase is 0.1 N sodium
nitrate containing 0.025% sodium azide.
Standard solution: Transfer 20 mg each of USP Dex-
trose RS, stachyose, and 5800-, 23,700-, and 100,000-
molecular weight (MW) pullulan standards into a 10-mL
Serele]oLeleLoyAme]
volumetric flask. Dissolve in and dilute with Mobile
phase to volume. Pass througha syringe filter of 0.45-
im or finer pore size into a suitable autosampler vial,
and seal.
Sample solution: Transfer 50 mg of Hydrogenated
Polydextrose into a 10-mL volumetric flask. Dissolve in
and dilute with Mobile phase to volume. Pass through a
syringe filter of 0.45-um or finer pore size into a suita-
ble autosampler vial, and seal.
 5498 Polydextrose / Official Monographs
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: Refractive index set at a sensitivity of 4 x
10-6 refractive index units full scale and maintained at
a temperature of 35+0.1°
Column: 7.8-mm x 30-cm; packing L39
Column temperature: 45°
Flow rate: 0.8 mL/min
[Note—After installation of a new column, pump Mobile
phase through the column overnight at a rate of
0.3 mL/min. Before calibration or analysis, increase the
flow slowly over a 1-min period to 0.8 mL/min. Con-
tinue to pump Mobile phase through the column at
this flow rate for at least 1 h before the first injection.
Check the flow gravimetrically, and adjust it if neces-
sary. Reduce the flow rate to about 0.1 mL/min when
the system is not in use.]
Injection size: 50 pL
System suitability
Sample: Standard solution
Chromatograph five replicate injections of the Stan-
dard solution, allowing 15 min between injections,
and record the retention times of the components of
the Standard solution.
Insert the average retention time along with the mo-
lecular weight of each component in the Standard
solution into the calibration table of the molecular
weight distribution software. Check the regression re-
sults for a cubic fit of the calibration points, and ob-
tain a correlation coefficient, R, for the line.
Suitability requirements
Retention time: The retention times for each compo-
nent determined on replicate injections agree within
+2'S;
Resolution: Dextrose and stachyose are baseline re-
solved from one another and from the 5800-MW pul-
lulan standard.
[Note—Prominent negative baseline valleys are usually
observed between the peaks for the 5800-, 23,700-,
and 100,000-MW pullulan standards.]
Correlation coefficient R: NLT 0.9999
Analysis
Samples: Standard solution and Sample solution
Use the molecular weight distribution software of the
data reduction system to generate a molecular
weight distribution plot of Hydrogenated
Polydextrose.
Acceptance criteria: No measurable peak above a mo-
lecular weight of 22,000 is found.
e PH (791): 5.0-7.0, in a solution (1 in 10)
e WATER DETERMINATION, Method | (921): NMT 4.0%. Use
a mixture of Hydranal Solvent and Hydranal Formamide
dry (2:1) as a solvent. Perform the titration at 50° in a
jacketed beaker.
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in tight, light-resistant
containers. Store in a cool and dry place.
e USP REFERENCE STANDARDS (11)
USP 1,6-Anhydro-D-glucose RS
USP Dextrose RS
USP Polydextrose RS
USP Sorbitol RS
NF Monographs
Polyethylene Glycol
wo 4 (Nominal Value)
° <1000
Poly(oxy-1,2-ethanediyl), o-hydro-w-hydroxy-;
Polyethylene glycol [25322-68-3].
NF 36
DEFINITION
Polyethylene Glycol is an addition polymer of ethylene oxide
and water, represented by the formula H(OCH2CH2),OH,
in which n represents the average number of oxyethylene
groups. The average molecular weight is NLT 95.0% and
NMT 105.0% of the labeled nominal value if the labeled
nominal value is less than 1000; it is NLT 90.0% and
NMT 110.0% of the labeled nominal value if the labeled
nominal value is between 1000 and 7000; and it is NLT
87.5% and NMT 112.5% of the labeled nominal value if
the labeled nominal value is more than 7000. It may con-
tain a suitable antioxidant.
ASSAY
© AVERAGE MOLECULAR WEIGHT
Phthalic anhydride solution: Place 49.0 g of phthalic
anhydride into an amber bottle, and dissolve in 300 mL
of pyridine from a freshly opened bottle or pyridine
that has been freshly distilled over phthalic anhydride.
Shake vigorously until completely dissolved. Add 7 g of
imidazole, swirl carefully to dissolve, and allow to stand
for 16 h before ne
Sample solution for liquid polyethylene glycols: Care-
fully introduce 25.0 mL of the Phthalic oe ‘dride solu-
tion into a dry, heat-resistant pressure bottle. Add an
amount of the specimen equivalent to its expected av-
erage molecular weight divided by 160. Insert the stop-
per in the bottle, and wrap it securely in a cloth bag.
Sample solution for solid polyethylene glycols: Care-
fully introduce 25.0 mL of Phthalic anhydride solution
into a dry, heat-resistant pressure bottle. Add an
amount of the specimen equivalent to its expected mo-
lecularweight divided by 160; however, because of lim-
ited solubility, do not use more than 25 g. Add 25 mL
of pyridine, froma freshly opened bottle or pyridine
that has been freshly distilled over phthalic anhydride.
Swirl to dissolve, insert the stopper in the bottle, and
wep it securely in a clothbag.
Blank: 25.0 mL of Phthalic anhydride solution plus any
additional pyridine added to the bottle
Analysis: Immerse the bottle in a water bath main-
tained at a temperature between 96° and 100°, to the
same depth as that of the mixture in the bottle. Re-
move the bottles from the bath after 5 min and, with-
out unwrapping swirl for 30 s to homogenize. Heat in
the water bath for 30 min (60 min for Be
glycols having molecular weights of 3000 or more),
then remove from the bath, and allow to cool to room
temperature. Uncap the bottle carefully to release any
pressure, remove from the bag, add 10 mL of water,
and swirl thoroughly. Wait 2 min, add 0.5 mL of a solu-
tion of phenolphthalein in pyridine (1 in 100), and ti-
trate with 0.5 N sodium hydroxide VS to the first pink
color that persists for 15 s. Perform a blank
determination.
Calculate the average molecular weight taken:
Result = (2000 x W)/[(Ve — Vs) x N]
Ww = weight of Polyethylene Glycol taken for the
Sample solution (g) | :
Ve = volume of 0.5 N sodium hydroxide consumed
by the Blank (mL) ;
Vs = volume of 0.5 N sodium hydroxide consumed
by the specimen (mL)
N = normality of the sodium hydroxide solution
Acceptance criteria: See Table 1.
Table 1
Label Claim Acceptance Criteria
(%)
95.0-105.0
100"7000 90.0-110.0
>7000 87.5-112.5
NF 36 Official Monographs / Polyethylene 5499

IMPURITIES Immediately seal the vial, and shake. Transfer 1.0 mL of

e RESIDUE ON IGNITION (281) this solution to a 100-mL volumetric flask, and dilute
Sample: 25g with Stripped polyethylene glycol 400 to volume. Transfer
Analysis: Proceed as directed, using a platinum dish, 10.0 mL of this solution to a 100-mL volumetric flask,
moistening the residue with 2 mL of sulfuric acid. and dilute with Stripped polyethylene glycol 400 to vol-
Acceptance criteria: NMT 0.1% ume. Transfer 1.0 mL of the System suitability solution to
a 22-mL pressure headspace vial. Seal, cap, and crimp
as directed for the Standard solution.
Delete the following: Sample solution: Transfer 1.0 g of Polyethylene Glycol
to a 22-mL pressure headspace vial. Seal, cap, and
°e HEAVY METALS (231) crimp as directed in the Standard solution.
Test preparation: 4.0g in 5.0 mL of 0.1 N hydrochloric Chromatographic system
acid. Dilute with water to 25 mL. (See Chromatography (621), System Suitability.)
Acceptance criteria: NMT 5 ppme coficsal 1-Jan-2018) Mode: Headspace GC (balanced pressure automatic
e Limit OF FREE ETHYLENE OXIDE AND 1,4-DIOXANE headspace sampler)
Stripped polyethylene glycol 400: Into a 5000-mL, Detector: Flame ionization
3-neck, round-bottom flask equipped witha stirrer, a Column: 0.32-mm x 50-m fused-silica capillary;
gas dispersion tube, and a vacuum outlet, place 3000 g bonded with 5-um film of phase G27
of a ashe glycol 400. At room temperature, evac- Temperatures
uate the flask carefully to a pressure of less than 1 mm Injection port: 85°
of mercury, applying the vacuum slowly while observ- Detector: 250°
ing for excessive foaming due to entrapped gases. After Column: See Table 2.
any foaming has subsided and while stirring continu-
ously, sparge with nitrogen, allowing the pressure to
rise to 10 mm of mercury. Continue stripping for a min- Table 2
imum of 1 h. Completeness of the stripping procedure Hold Time at}
should be verified by making a headspace injection of Initial Temperature Final Final
the Stripped polyethylene glycol 400. [NoTe—The 10-mm Temperature Ramp Temperature | Temperature
value is a guideline. Deviations from this value affect © (@/min) « (min)
only the total time required to strip polyethylene glycol 70 10 250 =
400.]
Shut off the vacuum pump, and bring the flask pressure Carrier gas: Helium
back to atmospheric pressure while maintaining nitro- Flow rate: 2.9 mL/min
gen sparging: Remove the gas dispersion tube with Injection volume: 1.0 mL of headspace using a 2-mL
the gas still flowing, then turn off the gas flow. Trans- gas syringe preheated in an oven at 90°
fer Stripped polyethylene glycol 400 to a suitable nitro- System suitability
gen-filled container. Sample: System suitability solution
Standard solution: Transfer 4.90 g of Stripped polyethyl- [NotE—The relative retention times for acetaldehyde
ene glycol 400 to a tared 22-mL pressure headspace vial and ethylene oxide are about 0.9 and 1.0,
that can be sealed. Add 48 uL of 1,4-dioxane, equiva- respectively.]
lent to 50 mg of 1,4-dioxane, froma syringe; seal, and Suitability requirements
cap the vial. [CAutlon—Ethylene oxide and 1,4-dioxane Resolution: NLT 1.3 between the acetaldehyde and
are toxic and flammable. Prepare these solutions in a ethylene oxide peaks
well-ventilated fume hood.] Analysis
Using the special handling described in the following, Samples: Standard solution and Sample solution
complete the preparation. Ethylene oxide is a gas at [Note—The relative retention times for ethylene oxide
room temperature. It is usually stored in a lecture-type and 1,4-dioxane are about 1.0 and 3.4, respectively.]
gas cylinder or small metal pressure bomb. Chill the Place the vials containing the Standard solution and the
cylinder in a refrigerator before use. Transfer 5 mL of Sample solution into the automated sampler, and heat
the liquid ethylene oxide to a 100-mL beaker chilled in the vials at a temperature of 80° for 30 min.
wet ice. Using a gas-tight syringe that has been chilled [Note—A headspace apparatus that automatically trans-
in a refrigerator, transfer 57 wL of the liquid ethylene fers the measured amount of headspace may be used
oxide, equivalent to 50 mg of ethylene oxide, to the to perform the injection.]
mixture contained in the headspace vial, and mix. Acceptance criteria: The peak areas for ethylene oxide
With the aid of a syringe, transfer 2 mL of this solution and 1,4-dioxane of the Sample solution are NMT those
to a 5-mL beaker. Transfer 1.0 mL of this solution to a of the corresponding peaks of the Standard solution cor-
100-mL volumetric flask, and dilute with Stripped poly- responding to NMT 10 ug/g of ethylene oxide and
ethylene glycol 400 to volume. Transfer 10 mL of this NMT 10 ug/g of 1,4-dioxane.
solution to a 100-mL volumetric flask, dilute with e Limit OF ETHYLENE GLYCOL AND DIETHYLENE GLYCOL (for
Stripped polyethylene glycol 400 to volume, and mix to Polyethylene Glycol having a nominal molecular weight
obtain a Standard solution having known concentra- less than 450)
tions of 10 ug/g for both ethylene oxide and 1,4-diox- Standard solution: 0.5 mg/mL each of ethylene glycol
ane. Transfer 1.0 mL of the Standard solution to a and diethylene glycol in water
22-mL pressure headspace vial, seal witha silicone Sample solution: 400 mg/mL of Polyethylene Glycol in
septum with or without a pressure relief star spring water im
and a pressure relief safety aluminum sealing cap, and Chromatographic system my
crimp the cap closed with a cap-sealing tool. (See Chromatography (621), System Suitability.) ms
System suitability solution: Transfer 4.90 g of Stripped Mode: GC i}
polyethylene glycol 400 to a 22-mL pressure headspace Detector: Flame ionization =
re)
vial. Pipet 50 uL of acetaldehyde into the vial. Using the
special handling described in the Standard solution,
Column: 3-mm x 1.5-m stainless steel; packing of ro}3
12% G13 on support SINS Ey
transfer 50.0 wl of liquid ethylene oxide into the vial. a}
a
“
 5500 Polyethylene / Official Monographs NF 36

Temperatures Instrumental conditions

Injection port: 250° (See Ultraviolet-Visible Spectroscopy (857).)
Detector: 280° Mode: UV-Vis
Column: 140° Cell: 1cm
Carrier gas: Nitrogen or another suitable inert gas Analytical wavelength: 450 nm
Flow rate: 50 mL/min Analysis
Injection volume: 2.0 LL Samples: Standard solution and Sample solution
Analysis Acceptance criteria: The absorbance of the Sample so-
Samples: Standard solution and Sample solution lution does not exceed that of the Standard solution,
[Note—The elution order is ethylene glycol followed by corresponding to NMT 0.25% of combined ethylene
diethylene glycol.] glycol and diethylene glycol.
Calculate the percentage of ethylene glycol in the por-
tion of Polyethylene Glycol taken: SPECIFIC TESTS
e PH (791)
Result = (rur/rsr) x (Cs1/Cu) x 100 Sample solution: 5.0g of Polyethylene Glycol in
100 mL of carbon dioxide-free water. Add 0.30 mL of
tur = peak height of ethylene glycol from the saturated potassium chloride solution.
Sample solution Acceptance criteria: 4.5-7.5
rst = peak height of ethylene glycol from the e COMPLETENESS AND COLOR OF SOLUTION: A solution of 5
Standard solution of ate: Glycol in 50 mL of water is colorless; it is
Gi = concentration of ethylene glycol in the a or liquid grades and NMT slightly hazy for solid
Standard solution (mg/mL) rades.
Gu = concentration of Polyethylene Glycol in the ° VISCOSITY-=-CAPLLARY METHODS (911): Determine the vis-
Sample solution (mg/mL) cosity Y using a capillary viscometer giving a flow time
Calculate the percentage of diethylene glycol in the of NLT 200 s and usinga liquid bath maintained at 98.9
portion of Polyethylene Glycol taken: + 0.3° (210° F). The viscosity is within the limits specified
in Table 3. For a polyethylene glycol not listed in the
Result = (ru2/tsz) x (Cs2/Cu) x 100 table, calculate the limits by interpolation.
Tu2 = peak height of diethylene glycol from the
Table 3
Sample solution
Ts2 = peak height of diethylene glycol from the Nominal Nominal
Standard solution Average Viscosity Average Viscosity
Csz = concentration of diethylene glycol in the Molecular Range, Molecular Range,
Standard solution (mg/mL) Weight Centistokes Weight Centistokes
Cu = concentration of Polyethylene Glycol in the 200 3.9-4.8 2400 49-65
Sample solution (mg/mL) 300 5.4-6.4 2500 51-70
Acceptance criteria: NMT 0.25% of the sum of ethyl-
400 6.8-8.0 2600 54-74
ene glycol and diethylene glycol
e LIMIT OF ETHYLENE GLYCOL AND DIETHYLENE GLYCOL (for 500 8.3-9.6 2700 57-78
Polyethylene Glycol having a nominal molecular weight 600 9.9-11.3 2800 60-83
of 450 or more but NMT 1000) 700 11,.5-13.0 2900 64-88
Ceric ammonium nitrate solution: 6.25 g of ceric am- 800 12.5-14.5 3000 67-93
monium nitrate in 100 mL of 0.25 N nitric acid. Use 900 15.0-17.0 3250 73-105
within 3 days. 1000 16.0-19.0 = =
Standard stock solution: 2.5 mg/mL of diethylene gly-
col in freshly distilled acetonitrile and water (1:1) 1100 18.0-22.0 3500 87-123
Standard solution: Add 10.0 mL of the Standard stock 1200 20.0-24.5 3750 99-140
solution to 15.0 mL of Ceric ammonium nitrate solution. 1300 22.0-27.5 4000 110-158
Within 2-5 min, determine the absorbance of the Stan- 1400 24-30 4250 123-177
dard solution. 1450 25-32 4500 140-200
Sample stock solution: Dissolve 50.0 g of Polyethylene 1500 26-33 4750 155-228
Glycol in 75 mL of ines ether, previously warmed if
necessary, just to melt the crystals, in a 250-mL distil- 1600 28-36 5000 170-250
ling flask. Slowly distill at a pressure of 1-2 mm of mer- 1700 31-39 5500 206-315
cury into a receiver graduated to 100 mL in 1-mL sub- 1800 33-42 6000 250-390
divisions, until 25 mL of distillate has been collected. 1900 35-45 6500 295-480
Add 20.0 mL of water to the distillate, shake vigorously, 2000 38-49 7000 350-590
and allow the layers to separate. Cool in an ice bath to
2100 40-53 7500 A05~735
solidify the diphenyl ether and facilitate its removal. Fil-
ter the separated aqueous layer, wash the diphenyl 2200 43-56 8000 470-900
ether with 5.0 mL of ice-cold water, pass the washings 2300 46-60 — =
through the filter, and collect the filtrate and washings
in a 25-mL volumetric flask. Warm to room tempera- Note—Polyethlyene Glycol 3350 is listed in the USP official
ture, and dilute with water to volume, if necessary. Mix monographs section to reflect its use as both an active and
NF Monographs

this solution with 25.0 mL of freshly distilled acetonitrile inactive ingredient.]

in a glass-stoppered, 125-mL conical flask.
Sample solution: Add 10.0 mL of the Sample stock solu-
tion to 15.0 mL of Ceric ammonium nitrate solution.
Within 2-5 min, determine the absorbance of the Sam-
ple solution.
Blank: Mixture of 15.0 mL of Ceric ammonium nitrate
solution and 10.0 mL of freshly distilled acetonitrile and
water (1:1)
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in tight containers.
e LABELING: Label it to state, as part of the official title, the
average nominal molecular weight of the Polyethylene
Glycol. Label it to indicate the name and quantity of any
added antioxidant.
USP 41
Calcium Lactate—see Calcium Lactate
General Monographs
Calcium Lactate Tablets—see Calcium
Lactate Tablets General Monographs
Calcium Lactobionate—see Calcium
Lactobionate General Monographs
Calcium Levulinate—see Calcium
Levulinate General Monographs
Calcium Pantothenate—see Calcium
Pantothenate General Monographs
Calcium Pantothenate Tablets—see
Calcium Pantothenate Tablets General
Monographs
Calcium Pantothenate, Racemic —see
Racemic Calcium Pantothenate General
Monographs
Calcium Phosphate, Tribasic—see Tribasic
Calcium Phosphate NF Monographs
Calcium with Vitamin D Tablets
DEFINITION
Calcium with Vitamin D Tablets contain NLT 90.0% and
NMT 125.0% of the labeled amount of calcium (Ca), de-
rived from substances generally recognized as safe, and
NLT 90.0% and NMT 165.0% of the labeled amount of
vitamin D, as cholecalciferol (C27H44O) or ergocalciferol
(C2gH44O). They contain no other vitamins or minerals for
which nutritional value is claimed. They may contain
other labeled added substances or additional ingredients
in amounts that are unobjectionable.
STRENGTH
e CALCIUM
[Note—A commercially available atomic absorption stan-
dard solution for calcium may be used where prepara-
tion of a Calcium standard stock solution is described in
the following assay. Concentrations of the Standard so-
lutions and the Sample stock solution may be modified
to fit the linear or working range of the instrument.]
Lanthanum chloride solution: Dissolve 26.7g of lan-
thanum chloride in 0.125 N hydrochloric acid to make
100 mL.
Calcium standard stock solution: Weigh 1.001 g of
calcium carbonate, previously dried at 300° for 3 h and
cooled in a desiccator for 2 h, and dissolve in 25 mL of
Dietary Supplements / Calcium 4501
1 N hydrochloric acid. Boil to expel carbon dioxide, and
dilute with water to 1000 mL to obtain a solution hav-
ing a concentration of 400 g/mL of calcium.
Standard stock solution: 100 g/mL of calcium from a
volume of Calcium standard stock solution in 0.125 N
hydrochloric acid
Standard solutions: Into separate 100-mL volumetric
flasks, separately pipet 1.0, 1.5, 2.0, 2.5, and 3.0 mL of
the Standard stock solution. To each flask add 1.0 mL of
Lanthanum chloride solution, dilute with water to vol-
ume, and obtain Standard solutions having concentra-
tions of 1.0, 1.5, 2.0, 2.5, and 3.0 pg/mL, of calcium.
Sample stock solution: Weigh and finely powder NLT
20 Tablets. Transfer the equivalent to 500 mg of cal-
cium, in 25 mL of concentrated hydrochloric acid, and
heat for 30 min on a steam bath. Cool, dilute with
water to 1000 mL, and filter.
Sample solution: Quantitatively dilute a volume of
Sample stock solution with 0.125 N hydrochloric acid to
obtain a concentration of 100 g/mL of calcium. Trans-
fer 2.0 mL of this solution to a TO00-mL volumetric flask,
add 1.0 mL of Lanthanum chloride solution, and dilute
with water to volume.
Spectrometric conditions
See Atomic Absorption Spectroscopy (852).)
Mode: Atomic absorption spectrophotometer
Lamp: Calcium hollow-cathode
Flame: Nitrous oxide-acetylene
Analytical wavelength: Calcium emission line, 422.7
nm
Blank: 1 mL of Lanthanum chloride solution per 100 mL
of 0.125 N hydrochloric acid
Analysis
Samples: Standard solutions and Sample solution
Determine the absorbances of the solutions, against the
Blank. Plot the absorbances of the Standard solutions
versus the concentration, in g/mL, of calcium, and
draw the straight line best fitting the five plotted
points. From the graph so obtained, determine the
concentration, C, in ug/mL, of calcium in the Sample
solution.
Calculate the percentage of the labeled amount of cal-
cium (Ca) in the portion of Tablets taken:
Result = (C/Cy) x 100
Cc = measured concentration of calcium in the
Sample solution (ug/mL)
Cy = nominal concentration of calcium in the
Sample solution (g/mL)
Acceptance criteria: 90.0%-125.0% of the labeled
sydeiBouo=: sa
amount of calcium (Ca)
© CHOLECALCIFEROL OR ERGOCALCIFEROL (VITAMIN D)
[Note—Use low-actinic glassware throughout this
procedure.]
Mobile phase: n-Hexane and isopropyl alcohol (99:1)
Standard solution: 2 g/mL of USP Ergocalciferol RS or
USP Cholecalciferol RS in n-hexane
System suitability solution: Heat a volume of Standard
solution at 60° for 1 h to partially isomerize vitamin D
(ergocalciferol or cholecalciferol) to its corresponding
precursor.
Sample solution: Weigh, and grind NLT 20 Tablets.
Transfer the equivalent to 20 ug of cholecalciferol or er-
gocalciferol to a container having a polytef-lined screw
cap. Add 8 mL of dimethyl sulfoxide and 12 mL of n-
hexane, and shake for 45 min on a wrist-action shaker
with tubes in a water bath maintained at 60°. Centri-
fuge for 10 min, withdraw the hexane layer by means
of a pipet, and transfer to an evaporation flask. Add
12 mL of n-hexane to the dimethyl sulfoxide layer, mix
on a vortex mixer for 5 min, and again withdraw the
hexane layer by means of a pipet, and add to the evap-
oration flask. Repeat this extraction with three addi-
tional 12-mL portions of n-hexane, adding the hexane
NF 36
Polyethylene Glycol 3350—see
Polyethylene Glycol 3350 General Monographs
Polyethylene Glycol Ointment
DEFINITION
Prepare Polyethylene Glycol Ointment as follows.
Polyethylene Glycol 3350 400 g
Polyethylene Glycol 400 600 q
To make 1000 g
Heat the two ingredients on a water bath to 65°. Allow to
cool, and stir until congealed. If a firmer preparation is
desired, replace up to 100 g of the Polyethylene Glycol 400
with an equal amount of Polyethylene Glycol 3350.
[Note—If 6%-25% of an aqueous solution is to be incor-
porated in the Ointment, replace 50 g of the Polyethylene
Glycol 3350 with an equal amount of stearyl alcohol.
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Package in well-closed
containers.
Polyethylene Glycol Monomethyl Ether
fealty
Poly(oxy-1,2-ethanediyl), a-methyl-w-hydroxy-;
Methoxy polyethylene glycol [9004-74-4].
DEFINITION
Polyethylene Glycol Monomethyl Ether is an addition poly-
mer of ethylene oxide and methanol, represented as:
CH3(OCH2CH2),0H
in which n represents the average number of oxyethylene
groups. The average molecular weight is NLT 95.0% and
NMT 105.0% of the labeled nominal value if the labeled
nominal value is below 1000; it is NLT 90.0% and NMT
110.0% of the labeled nominal value if the labeled nomi-
nal value is between 1000 and 4750; and it is NLT 87.5%
and NMT 112.5% of the labeled nominal value if the la-
beled nominal value is above 4750.
ASSAY
e AVERAGE MOLECULAR WEIGHT
Phthalic anhydride solution: Place 49.0 g of phthalic
anhydride into an amber bottle, and dissolve in 300 mL
of pyridine, either from a freshly opened bottle or
freshly distilled over phthalic anhydride. Shake vigor-
ously until completely dissolved. Add 7 g of imidazole,
swirl carefully to dissolve, and allow to stand for 16 h
before using.
Sample solution for liquid Polyethylene Glycol Mono-
methyl Ethers: Carefully introduce 25.0 mL of the
Phthalic anhydride solution into a dry, heat-resistant
pressure bottle. Add a weighed amount of the sample,
equivalent to its expected average molecular weight di-
vided by 80. Insert the stopper in the bottle, and wrap
it securely in a cloth bag.
Sample solution for solid Polyethylene Glycol Mono-
methyl Ethers: Carefully introduce 25.0 mL of Phthalic
anhydride solution into a dry, heat-resistant pressure
Official Monographs / Polyethylene 5501
bottle. Add an amount of the sample, equivalent to its
expected molecular weight divided by 80; however, be-
cause of limited solubility, do not use more than 25 g.
Add 25 mL of pyridine, either from a freshly opened
bottle or freshly distilled over phthalic anhydride, swirl
to dissolve, insert the stopper in the bottle, and wrap it
securely in a cloth bag.
Analysis: Immerse the bottle in a water bath main-
tained at 96°-100° to the same depth as that of the
mixture in the bottle. Remove the bottle from the bath
after 5 min, and without unwrapping, swirl for 30 s to
homogenize. Heat in the water bath for 30 min (60
min for Rete iene Glycol Monomethy! Ethers having
molecular weights of 3000 ornigher); then remove
from the bath, and allow to cool to room temperature.
Uncap the bottle carefully to release any pressure, re-
move from the bag, add 10 mL of water, and swirl
thoroughly. Wait for 2 min, add 0.5 mL of a solution of
phenolphthalein in pyridine (1 in 100). Titrate with 0.5
N sodium hydroxide VS to the first pink color that per-
sists for 15 s, recording the volume, in mL, of 0.5 N
sodium hydroxide required as Vs. Perform a blank deter-
mination on 25.0 mL of Phthalic anhydride solution plus
any additional pyridine added to the bottle, and record
the volume, in mL, of 0.5 N sodium hydroxide required
as Vp.
Calculate the average molecular weight:
Result = (1000 x W)/[(Vs — Vs) x NJ
Ww = weight of Palyetiviene Glycol Monomethyl
Ether taken for the Sample solution (g)
Ve = volume of 0.5 N sodium hydroxide consumed
Vs
by the blank (mL)
= volume of 0.5 N sodium hydroxide consumed
by the sample (mL)
N = normality of the sodium hydroxide solution
Acceptance criteria: See Table 7.
Table 1
Label Claim Acceptance Criteria
(nominal value) (%)
<1000 95.0-105.0
1000-4750 90.0-110.0
>4750 87.5-112.5
IMPURITIES
e RESIDUE ON IGNITION (281)
Sample: 25 g of Polyethylene Glycol Monomethyl
Ether, moistened with 2 mL of sulfuric acid in a plati-
num dish
Acceptance criteria: NMT 0.1%
Delete the following:
°o HEAVY METALS (231)
Test preparation: Mix 4.0 g with 5.0 mL of 0.1 N hy-
drochloric acid, and dilute with water to 25 mL.
Acceptance criteria: NMT 5 ppmMe cortical 1-jan-2018)
e LIMIT OF ETHYLENE GLYCOL AND DIETHYLENE GLYCOL (for
ihe a Sah Glycol Monomethy! Ether having a nominal
molecular weight of less than 600)
sydeibouow 4N
Standard solution: 500 g/mL of ethylene glycol and
500 pg/mL of diethylene glycol in water
Sample solution: 400 mg/mL of Polyethylene Glycol
Monomethyl Ether in water
Chromatographic system
(See Chromatography (621), System Suitability.)
 5502 Polyethylene / Official Monographs
Mode: GC
Detector: Flame ionization
Column: 3-mm x 1.0-m; 60- to 80-mesh support $2
Temperatures
Column: 200°
Injection port: 260°
Carrier gas: Nitrogen or another suitable inert gas
Flow rate: 20 mL/min
Injection volume: 1.0 uL
Analysis
Samples: Standard solution and Sample solution
[Note—The elution order is ethylene glycol, diethylene
gel, and polyethylene glycol monomethyl ether.]
Calculate the percentage of ethylene glyco! and diethyl-
ene glycol in the portion of Polyethylene Glycol Mono-
methyl Ether taken:
Result = (ru/rs) x (Cs/Cu) x 100
tu = peak height of ethylene glycol or diethylene
glycol from the Sample solution
ls = peak height of ethylene glycol or diethylene
glycol from the Standard solution
Cs = concentration of ethylene glyco! or diethylene
glycol in the Standard solution
Gy = concentration of Polyethylene Glycol
Monomethyl Ether in the Sample solution
Acceptance criteria: NMT 0.25% of combined ethyl-
ene glycol and diethylene glycol
e Limit OF ETHYLENE GLYCOL AND DIETHYLENE GLYCOL (for
ae Glycol Monomethyl Ether having a nominal
molecular weight of 600-1500)
Solution A: 62.5 mg/mL of ceric ammonium nitrate in
0.25 N nitric acid. Use within 3 days.
Solution B: Freshly distilled acetonitrile and water
(50:50)
Standard solution: 2.5 mg/mL of diethylene glycol in
Solution B
Sample solution: Dissolve no9 of Polyethylene Glycol
Monomethyl Ether in 75 mL of ipneny ether, previ-
ously warmed if necessary, to melt the crystals, in a
250-mL distilling flask. Slowly distill at a pressure of
1-2 mm of mercury into a receiver graduated to
100 mL in 1-mL subdivisions until 25 mL of distillate has
been collected. Add 20.0 mL of water to the distillate,
shake vigorously, and allow the layers to separate. Cool
in an ice bath to solidify the diphenyl ether and facili-
tate its removal. Filter the separated aqueous layer,
wash the diphenyl ether with 5.0 mL of ice-cold water,
ass the washings through the filter, and collect the
Fitrate and washings in a 25-mL volumetric flask. Warm
to room temperature, and dilute with water to volume,
if necessary. Mix this solution with 25.0 mL of freshly
distilled acetonitrile in a glass-stoppered, 125-mL coni-
cal flask.
Instrumental conditions
Mode: Vis
Analytical wavelength: About 450 nm
Cell: 71cm
Blank: Solution A and Solution B (60:40)
Analysis
Samples: Standard solution, Sample solution, and Blank
Transfer 10.0 mL each of the Standard solution and the
Sample solution to separate 50-mL flasks, each con-
taining 15.0 mL of Solution A. Within 2-5 min, deter-
NF Monographs
mine the absorbances of the Samples.
Acceptance criteria: The absorbance of the Sample so-
lution does not exceed that of the Standard solution,
corresponding to NMT 0.25% of combined ethylene
glycol and diethylene glycol.
e FREE ETHYLENE OXIDE AND 1,4-DIOXANE
Stripped MPEG 350: Into a 5000-mL 4-neck, round-
bottom flask, equipped withastirrer, a thermometer, a
gas dispersion tube, a dry ice trap, a vacuum outlet,
and a heating mantle, place 3000 g of Polyethylene
NF 36
Glycol Monomethyl Ether 350. At room temperature,
evacuate the flask carefully to a pressure of less than
1mm of mercury, applying the vacuum slowly while
observing for excessive foaming due to entrapped
gases. After any foaming has subsided, sparge with ni-
trogen, allowing the pressure to rise to 10 mm of mer-
cury. Heat the flask to 130° while increasing the pres-
sure to 60 mm of mercury. Continue stripping for 4 h,
then cool to room temperature. Shut off the vacuum
pump, and bring the flask pressure back to atmospheric
pressure while maintaining nitrogen sparging. Remove
thesparging tube with the gas still flowing, then turn
off the gas flow. Transfer the Stripped MPEG 350 to a
suitable nitrogen-filled container.
Standard solutions: [Caution—Ethylene oxide and 1,4-
dioxane are toxic and flammable. Prepare these solu-
tions in a well-ventilated fume hood.] To a known
weight of Stripped MPEG 350 ina vial that can be
sealed adda suitable quantity of 1,4-dioxane. Deter-
mine the amount added by weight difference. Using
the special handling described in the following, com-
plete the preparation. Ethylene oxide is a gas at room
temperature. It is usually stored in a lecture-type gas
cylinder or small metal pressure bomb. Chill the cylin-
er in a refrigerator before use. Transfer 5 mL of the
liquid ethylene oxide to a 100-mL beaker chilled in wet
ice. Using a gas-tight gas chromatographic syringe that
has been chilled in a refrigerator, transfer a suitable
amount of the liquid ethylene oxide into the mixture.
Immediately seal the vial, and shake. Determine the
amount added by weight difference. By appropriate di-
lution with Stripped MPEG 350, prepare four solutions,
covering a range of 5-20 ppm for the two components
added to the matrix (e.g., 5, 10, 15, and 20 ppm).
Transfer 1.0 mL of each of these solutions to separate
22-mL pressure headspace vials. Seal each withasili-
cone septum, star spring, and pressure relief safety alu-
minum sealing cap. Crimp the cap closed with a cap-
sealing tool.
Sample solution: Transfer 1 +0.01 g of Polyethylene
Glycol Monomethyl Ether to a 22-mL pressure head-
space vial. Seal, cap, and crimp as directed for the Stan-
lard solutions.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: GC (equipped with a balanced pressure auto-
matic headspace sampler)
Detector: Flame ionization
Column: 50-m x 0.32-mm fused silica; bonded phase
G27 in a 5-um film thickness
Temperatures
Detector: 250°
Transfer line: 140°
Column: See Table 2.
Table 2
Hold Time at
Initial Temperature Final Final
Temperature Ramp Temperature | Temperature
«) (¢/min) ©) (min)
70 10 250 —
Carrier gas: Helium
Flow rate: 0.8 mL/min
Calibration: Place the vials containing the Standard so-
lutions in the automated sampler, and start the se-
quence so that each vial is heated at 110° for 30 min
before a suitable portion of its headspace is injected
into the chromatograph. Set the automatic sampler for
a needle withdrawal time of 0.3 min, a pressurization
time of 1 min, an injection time of 0.08 min, and a vial
pressure of 22 psig with the vial vent off. Obtain the
pee areas for ethylene oxide and 1,4-dioxane, which
ave relative retention times of 1.0 and 3.1, respec-
 NF 36 Official Monographs / Polyethylene 5503

tively. Plot the area versus parts per million on linear Acceptance criteria: 4.5-7.5
graph paper, and draw the best straight line Saas © COMPLETENESS AND COLOR OF SOLUTION
the points, On the two Calibration plots, no point di- Sample solution: 5g of Polyethylene Glycol Mono-
gresses from its line by more than 10%. methyl Ether in 50 mL of water
Analysis: Place the vial containing the Sample solution Acceptance criteria: The resulting solution is colorless,
in the automatic sampler, and chromatograph its head- and is clear for liquid grades and NMT slightly hazy for
space as done for the Standard solutions. Obtain the solid grades.
peak areas of each of the components, and read the © ViscosiTY—CAPILLARY METHODS (911): Determine its vis-
concentrations directly from the Calibration plots. cosity, using a capillary viscometer giving a flow time of
Acceptance criteria: NMT 10 ppm of ethylene oxide or NLT 200 s and a liquid bath maintained at 98.9 + 0.3°.
1,4-dioxane The viscosity is within the limits specified in Table 4. For a
@ LIMIT OF 2-IMETHOXYETHANOL Polyethylene Glycol Monomethy! Ether not listed in Table
Stripped MPEG 350 and Sample solution: Prepare as 4, calculate the limits by interpolation.
directed in the test for Free Ethylene Oxide and 1,4-
Dioxane. Table 4
Standard solutions: [CAUTIOoN—2-Methoxyethanol is
toxic and flammable. Prepare these solutions in a well- Nominal Nominal
ventilated fume hood.] To a known weight of Stripped Average Average
MPEG 350 ina vial that can be sealed adda suitable Molecular | Viscosity Range | Molecular | Viscosity Range
quantity of 2-methoxyethanol. Determine the amount Weight (centistokes) Weight (centistokes)
added by weight difference. By appropriate dilution 350 3.5-4.5 2750 50-78
with Stripped MPEG 350, prepare four solutions, cover- 450 4.9-6.0 3000 60-95
ing a range of 5-20 ppm (e.g., 5, 10, 15, and 20 ppm). 550 6 1-7.3 3250 72-113
Transfer 1.0 mL of each of these solutions to separate 650 7.9-9.2 3500 85-133
22-mL pressure headspace vials. Seal each witha sili-
750 9.7-11.1 3750 99-155
cone septum, star spring, and pressure relief safety alu-
minum sealing cap. Crimp the cap closed with a cap- 850 11,.5-13.1 4000 114-178
sealing tool. 950 13.3-15.2 4250 130-204
Chromatographic system 1000 13.3-17.3 4500 148-231
(See Chromatography (621), System Suitability.) 1100 15.0-19.7 4750 167-260
Mode: GC (equipped with a balanced pressure auto- 1200 16.9-22.1 5000 175-305
matic headspace sampler)
1300 18.8-24.6 5500 215-375
Detector: Flame ionization
Column: 15-m x 0.53-mm fused silica capillary; 1400 20.7-27.1 6000 260-455
bonded phase G16 in a 1-um film thickness 1500 23-30 6500 310-545
Temperatures 1600 25-33 7000 365-640
Detector: 275° 1700 27-35 7500 425-745
Transfer line: 140° 1800 29-38 8000 490-860
Column: See Table 3.
1900 31-41 8500 560-980
2000 33-44 9000 640-1110
Table 3
2250 36-54 9500 715-1250
Hold Time at 2500 40-64 10000 775-1475
Initial Temperature Final Final
Temperature Ramp Temperature | Temperature
[seein(E). (¢/min) @) (min) ADDITIONAL REQUIREMENTS
50 = 50 2 © PACKAGING AND STORAGE: Preserve in tight containers.
70 10 250 = © LABELING: Label it to state, as part of the official title, the
average nominal molecular weight of the Polyethylene
Carrier gas: Helium Glycol Monomethyl Ether.
Flow rate: 15 mL/min
Calibration: Place the vials containing the Standard so-
lutions in the automated sampler, and start the se-
quence so that each vial is heated at 100° for 20 min
before a suitable portion of its headspace is injected Polyethylene Oxide
into the chromatograph. Set the automatic sampler for
a needle withdrawal time of 0.3 min, a pressurization
time of 1 min, an injection time of 0.08 min, anda vial “fo
pressure of 22 psig with the vial vent off. Obtain the
peak area for 2-methoxyethanol. Plot the area versus
pm on linear graph paper, and draw the best straight DEFINITION
ine through the points. On the Calibration plot, no Polyethylene Oxide is a nonionic homopolymer of ethylene
point digresses from its line by more than 10%. oxide, represented:
Analysis: Place the vial containing the Sample solution
sydeiGbouow 4N

in the automatic sampler, and chromatograph its head- H(OCH2CH2),0H

space as done for the Standard solutions. Obtain the
peak area, and read the concentration directly from the
Calibration plot.
Acceptance criteria: NMT 10 ppm
SPECIFIC TESTS
© PH (791)
Sample solution: 5.0 g of Polyethylene Glycol Mono-
methyl Ether in 100 mL of carbon dioxide-free water.
Add 0.30 mL of saturated potassium chloride solution.
in which n represents the average number of oxyethylene
groups. The number n varies from about 2000 to
200,000. It is a white to off-white powder obtainable in
several grades, varying in viscosity profile in an aqueous
isopropyl alcohol solution. It may contain a suitable
antioxidant.
 5504 Polyethylene / Official Monographs
IDENTIFICATION
e A. INFRARED ABSORPTION (197K)
Sample: Previously dried in a vacuum at room tempera-
ture to constant weight
¢ B, PROCEDURE
Viscosity
[Note—Based on Labeling information, perform the fol-
lowing test accordingly]
Analysis: Pass Polyethylene Oxide through a 20-mesh
screen. Then transfer Polyethylene Oxide to a 800-mL
low-form beaker the amount which is specified in Ta-
ble 1 to provide the solution concentration.
Table 1
Sample solution
Polyethylene 1% Solution 2% Solution 5% Solution
oxide weight 6g 129 30q
Add 125 mL of dehydrated isopropyl alcohol to the
beaker. Place the stirrer into the Deaker with an ap-
propriate glass cover. Stir the polyethylene oxide-iso-
ropanol mixture at a rate to ensure that a slurry is
ormed. Add the prescribed amount of water to the
polyethylene oxide-isopropanol slurry, refer to Table
2. All solution concentrations are based on the water
content of the aqueous isopropyl alcohol solution. Be
careful to avoid splashing of the water. Adjust the
temperature to near 25° to assist the final solution
coming to temperature.
Table 2
Sample solution
Water weight 1% Solution 2% Solution 5% Solution
594 q 588 g 570 9
Ensure that the stirring is very effective at the begin-
ning for about 1 min. Then allow to gently stir for at
least 3 h. Ensure that Polyethylene Oxide dissolves in
solution, take care to avoid mixing in excess air, and
stop the stirring. [NoTe—Ensure a colloidal dispersion
by stirring for at least 3 h if added antioxidant or
silicon dioxide is not soluble in the system.]
Place a watchglass over the beaker and place in a bath
for at least 30 min. When the solution reaches 25 +
0.1°, determine the viscosity of the Sample solution
using the viscometer, spindle, and speed indicated on
the Labeling. [NOTE—A guard may be required as in-
dicated on the Labeling.] Follow the instrument man-
ufacturer’s directions to measure the apparent
viscosity.
Acceptance criteria: Viscosity falls within the viscosity
range indicated by the Labeling.
IMPURITIES
Inorganic Impurities
Delete the following:
°e HEAVY METALS, Method II (231): NMT 10 ppme coticiai 1-
Jan-2018)
e SILICON DIOXIDE AND NONSILICON DIOXIDE RESIDUE ON
al IGNITION
a Sample: 1g
is Analysis: Weigh the Sample into a previously ignited,
Ss
tared 50-mL platinum crucible. Add 4 drops of sulfuric
a
—
° acid. Heat carefully on a hot plate until the specimen is
< thoroughly charred and fumes no longer are evolved.
SC Ignite the crucible at 700+ 25° (see Residue on Ignition
= (281)) to constant weight. Wet the residue carefully
—— with 1 mL of water, and slowly add 20 drops of hydro-
4 fluoric acid. [Cautlon—Hydrofluoric acid is an extremely
hazardous chemical. When handling it, wear a face
NF 36
shield, arm protection, and rubber gloves, and perform
the operation in a hood.] Evaporate slowly on a hot
plate to dryness, then ignite at 700
+ 25° for 10 min,
cool to room temperature in a desiccator, and weigh.
Repeat the addition of hydrofluoric acid, evaporation,
and ignition, to constant weight.
Calculate the percentage of silicon dioxide residue on
ignition from the difference between the net weights
before and after the hydrofluoric acid treatment.
Calculate the percentage of nonsilicon dioxide residue
on ignition from the final net weight.
Acceptance criteria
Silicon dioxide residue on ignition: NMT 3%
Nonsilicon dioxide residue on ignition: NMT 2%
Organic Impurities
e PROCEDURE: LIMIT OF FREE ETHYLENE OXIDE
Standard stock solution: [CAuTIoN—Ethylene oxide is
toxic and flammable. Prepare solutions of it in a well-
ventilated fume hood.] Prepare the solution using the
special handling described below. Ethylene oxide is a
gas at room temperature. It is usually stored in a lec-
ture-type gas cylinder or small metal pressure bomb.
Chill the cylinder in a refrigerator before use. Transfer
5 mL of the liquid ethylene oxide to a cold, 10-mL se-
rum vial. Seal the vial, and store in a refrigerator. Trans-
fer 40 g of acetone to a tared 50-mL serum vial that is
capable of being tightly sealed with a polytef-lined sep-
tum and a metallic crimp cap. Seal the vial, and weig|
it. Using a gas-tight gas chromatographic syringe that
has been chilled in a refrigerator, transfer 60 wL of the
liquefied ethylene oxide to the same vial. Weigh the
vial, and determine the amount added by weight dif-
ference. The solution contains about 1 ug/L of ethyl-
ene oxide. [NOTE—This solution may bete t for 1
week in the crimp-sealed serum vial, stored in a
freezer.]
[Note—Standard solutions A-D and the Sample solution
should be prepared in vials designed for use in the
headspace sampling system specified in the Chromato-
graphic systema,
Standard solution A: To a tared vial add 1.0 g of Poly-
ethylene Oxide, and seal the vial. Through the septum
add 2.0 uL of Standard stock solution, heat the vial at
100° for 30 min, and cool to room temperature.
Standard solution B: To a tared vial add 1.0 g of Poly-
ethylene Oxide, and seal the vial. Through the septum
add 4.0 uL of Standard stock solution, heat the vial at
100° for 30 min, and cool to room temperature.
Standard solution C: To a tared vial add 1.0g of Poly-
ethylene Oxide, and seal the vial. Through the septum
add 6.0 uL of Standard stock solution, heat the vial at
100° for 30 min, and cool to room temperature.
Standard solution D: Toa tared vial add 1.0 g of Poly-
etivlenie Oxide, and seal the vial. Through the septum
add 8.0 uL of Standard stock solution, heat the vial at
100° for 30 min, and cool to room temperature.
Sample solution: To a tared vial add 1.0g of Polyeth-
ylene Oxide, and seal the vial. Heat the vial at 100° for
30 min, and cool to room temperature.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: GC
Detector: Flame ionization
Column: 0.53-mm x 10-m capillary column bonded
with a 20-um layer of phase G45
Temperature
Injector: 200°
Detector: 250°
Column: See the temperature program table below.
NF 36 Official Monographs / Polyglyceryl 5505

Hold Time at e USP REFERENCE STANDARDS (11)

Initial Temperature Final Final USP Polyethylene Oxide RS
Temperature Ramp Temperature | Temperature
() (°/min) «@) (min)
70 = 70 5
70 10 200 5
Polyglyceryl Dioleate
Flow rate: 15 mL/min
Injection size: 300 uL of headspace gas R-O-(CH2-CH(OR)-CH2-O);-R
Injection type: Split
Carrier gas: Helium R =H, or CO-Ci7H33
[Note—The makeup gas is also helium, with a split 1,2,3-Propanetriol, homotrimer, di[(9Z)-9-octadecenoate];
flow rate of 15 mL/min.] Triglyceril dioleate;
System Sultabllity: Polyglyceryl 3 dioleate [9007-48-1].
Sample: Standard solution C R-O-(CH2-CH(OR)-CH2-O)6-R
Suitability requirements
Relative standard deviation: NMT 5%. [NoTE— R =H, or CO-Cy7H33
Multiple vials are prepared for replicate injections.] 1,2,3-Propanetriol, homohexamer, di[(9Z)-9-octadecenoate];
Analysis Hexaglyceril dioleate;
Samples: Standard solutions A-D and Sample solution Polyglyceryl 6 dioleate [76009-37-5].
[NoTte—A headspace apparatus that automatically DEFINITION
transfers the measured amount of gaseous head- Polyglyceryl Dioleate is a mixture of polyglycery! diesters of
space may be used to perform the injections.] mainly oleic acid, obtained by esterification of poly-
Using a gas-tight syringe, separately inject equal glycerin and oleic acid. The polyglycerin consists mainly
volumes (about 300 lL) of the gaseous headspace of of triglycerin or hexaglycerin.
each of the Standard solutions and the Sample solu-
tion into the gas eee record the chro- IDENTIFICATION
matograms, and measure theareas of the peak re- e A. INFRARED ABSORPTION (197F)
sponses. Determine by a retention time comparison e B. Meets the requirements of the test for Content of Fatty
whether ethylene oxide is detected in the Sample Acids
solution. Plot the responses of the Sample solution eC. Meets the requirements of the test for Fats and Fixed
and the Standard solutions versus the content, in ug, Oils (401), Hydroxy! Value. [NoTe—This test will differenti-
of ethylene oxide in each vial, as furnished by the ate for Polyglyceryl 3 Dioleate and Polyglyceryl 6
Standard stock solution. Draw the straight line best Dioleate.]
fitting the five points, and calculate the correlation
coefficient for the line. ASSAY
[NoTte—The content of ethylene oxide, as furnished © CONTENT OF FATTY ACIDS
by the Standard stock solution, is 0 ug in the Sample 0.5 N methanolic sodium hydroxide solution: Dis-
solution.] solve 20g of sodium hydroxide in 50 mL of water, and
A suitable system is one that yields a line having a mix. Cool to room temperature, and add 950 mL of
correlation coefficient of NLT 0.99. Extrapolate the methanol.
line until it intercepts the content axis on the nega- Boron trifluoride methanol solution: Dissolve 14 g of
tive side. From the intercept, determine the total wor trifluoride in methanol to make 100 mL, and mix
amount, Tu, in Lg, of ethylene oxide in the Sample well.
solution. Saturated sodium chloride solution: Dissolve about
Calculate the percentage of ethylene oxide in the 375g of sodium chloride in water to make 1000 mL.
portion of Polyethylene Oxide taken: Standard solution: Prepare the calibration ester mix-
ture by mixing up each individual ester component (see
Result = (Tu/W) x 100 Table 1 for the component's composition). Dissolve
500 mg of the calibration ester mixture in n-heptane,
Tu = total amount of ethylene oxide in the Sample and dilute with n-heptane to 50 mL. [NoTe—Commer-
solution (ug) cially available mixtures of fatty acid methyl esters may
Ww = weight of Polyethylene Oxide taken to also be used.]
prepare the Sample solution (ug)
Acceptance criteria: NMT 0.001%
Table 1
SPECIFIC TESTS Component in the Composition
e Loss ON DRYING (731): Dry 4g at 105° for 45 min: it Calibration Ester Mixture (%)
loses NMT 1.0% of its weight. USP Methyl Myristate RS (C14:0) 5
ADDITIONAL REQUIREMENTS USP Methy! Palmitate RS (C16:0) 15
e PACKAGING AND STORAGE: Preserve in tight, light-resistant USP Methyl Palmitoleate RS
containers. No storage requirements specified. (16:1) 10
e LABELING: Label it to indicate the viscosity and acceptable USP Methyl Stearate RS (C18:0) 10
limits, giving the viscosity measurement parameters, con- USP Methyl Oleate RS (C18:1) 20 A
centration of the solution, and the type of equipment USP Methyl Linoleate RS (C18:2) 15
used. Label it to indicate the name and quantity of any
USP Methyl Linolenate RS (C18:3) 10 =
added antioxidant.
Methyl arachidate (C20:0) 10 =
Methyl gadoleate (C20:1) 5 a
Sample solution: Introduce 100 mg of Polyglyceryl Di- By
oleate into a 25-mL conical flask fitted with a suitable mo}
1 Boron trifluoride-methanol solution (14% in methanol) is also commercially
a
available from Sigma, B-1252, or equivalent quality.
 5506 Polyglyceryl / Official Monographs NF 36

water-cooled reflux condenser and a magnetic stir bar. A = peak area of each individual fatty acid ester
Add 2 mL of 0.5 N methanolic sodium hydroxide solution, component
mix, and reflux for about 30 min. Add 2 mL of Boron B = sum of the peak areas, excluding the solvent
trifluoride methanol solution through the condenser and peak, of the Sample solution
reflux for about 30 min. Add 4 mL of n-heptane Acceptance criteria: Polyglyceryl Dioleate exhibits the
through the condenser, and reflux for 5 min. Cool, re- following composition profile of fatty acids.
move the condenser, add about 10 mL of Saturated so-
dium chloride solution, shake, add a quantity of Satu- Carbon-Chain Number of
rated sodium chloride solution to bring the upper layer Length Double Bonds Percentage
up to the neck of the flask, and allow the layers to
14 0 $5.0
separate. Collect 2 mL of the n-heptane layer (upper
layer), wash with three quantities, each at 2 mL of 16 0 2.0-16.0
water, and dry the n-heptane phase over anhydrous so- 16 1 $8.0
dium sulfate. 18 oO <6.0
Chromatographic system 18 1 65.0-88.0
(See Chromatography (621), System Suitability.) 18 2 5.0-18.08
Mode: GC
18 3 4.08
Detector: Flame ionization
Column: 0.32-mm x 30-m fused-silica capillary; 0.25- Sum of fatty acids
uum layer of phase G16 with C >18 0 $4.0
Temperature @The content of C18:2 or C18:3 is the content of each fatty acid with its
Detector: 250° respective isomers.
Injection port: 240° IMPURITIES
Column: See temperature program table below. Inorganic Impurities
© RESIDUE ON IGNITION
Hold Time Analysis: Heata silica crucible to redness for 30 min,
Initial Temperature Final at Final allow to cool in a desiccator, and weigh. Evenly dis-
Temperature Ramp Temperature | Temperature tribute about 1.0 g of Polyglyceryl Dioleate in the cru-
@) (¢/min) () (min) cible, and weigh. Dry at 100°-105° for 1 h, and ignite
150 6 250 6 in a muffle furnace at 600 + 25°, until the test sub-
stance is thoroughly charred. Perform the test for Resi-
Carrier gas: Nitrogen due on Ignition (281) on the residue obtained,starting
Flow rate: 1.0-1.2 mL/min with “Moisten the sample with a small amount (usually
Injection size: 1 pL 1 mL) of sulfuric acid.”
eae type: Split injection. The split ratio is about Acceptance criteria: NMT 1%
:80.
System suitability Delete the following:
Sample: Standard solution
[Note—See the relative retention time table below.]
°e HEAVY METALS, Method I! (231): NMT 10 ppMe cotfiiai1-
Jan-2018)
Relative Retention
Component Time SPECIFIC TESTS
Methyl myristate 0.74 © ACID VALUE
Methyl palmitate 1.00 Analysis: Accurately weigh (to within 0.1 mg) 5-10 g of
Methyl palmitoleate 1.03
Folvabce| Dioleate, add 10 mL of alcohol and 3 drops
of phenolphthalein TS, and titrate with 0.1 N potassium
Methyl stearate 1.29 hydroxide VS or 0.1 N sodium hydroxide VS until the
Methyl oleate 1.33 solution remains faintly pink after shaking for 30 s. Pro-
Methyl linoleate 1.378 ceed as directed in Fats and Fixed Oils (401), Acid Value
Methyl linolenate 1.46° to perform the calculation.
Methyl arachidate 1.55 Acceptance criteria
Methyl gadoleate 1.58
Polyglyceryl 3 dioleate: NMT 6
Polyglyceryl 6 dioleate: NMT 6
@ There could be an isomer eluting at a relative retention time of 1.39. ¢ FATS AND FIXED OlLs, Hydroxy! Value (401)
» There could be two isomers eluting at relative retention times of 1.45 Acceptance criteria
and 1.48.
Polyglyceryl 3 dioleate: 195-245, determined on a
Suitability requirements 0.7-g to 1.0-g specimen
Resolution: NLT 1.5 between the peaks due to Polyglyceryl 6 dioleate: 270-320, determined on 0.5-
methyl stearate and methyl oleate g to 0.7-g specimen
Relative standard deviation: NMT 6.0% for the pal- © IODINE VALUE
mitate and stearate peak areas Analysis: Accurately weigh sey of Polyglyceryl Di-
Analysis oleate, transfer to a dry 250-mL flask with a ground-
Samples: Standard solution and Sample solution glass stopper, and add 25 mL of methylene chloride.
”
is; Identify the fatty acid ester peaks of the Sample solu- Add 20 mL of the Wijs’ solution.2 Close the flask, and
a tion by comparing the retention times of these peaks keep it in the dark for 1 h while shaking frequently.
i with those of the Standard solution, and measure the Pertorm the test in Fats and Fixed Oils (401), lodine
i=.)
_
peak areas for all of the fatty acid esters in the Sample Value, starting with “Then add, in the order named,
2) solution. 30 mL of potassium iodide TS and 100 mL of water.”
¢
iS Calculate the percentage of each fatty acid ester com- 2 Wijs’ reagent RPE for analysis from Carlo Erba Reference 491902; Wijs’ solu-
= ponent in the test specimen: tion from www.sigmaaldrich.com, or equivalent quality.
J
Result = (A/B) x 100
2
NF 36 Official Monographs / Polyglyceryl 5507

Acceptance criteria Table 1

Polyglyceryl 3 dioleate: 60-80 Component in the Calibration Composition
Polyglyceryl 6 dioleate: 50-70 Ester Mixture (%)?_
e FATS AND FIXED OILS, Peroxide Value (401): Use 30 mL of
USP Methyl Myristate RS (C14:0) 8
a mixture of glacial acetic acid and methylene chloride
(3:2) to replace 30 mL of a mixture of glacial acetic acid USP Methyl Palmitate RS (C16:0) 70
and chloroform (3:2). USP Methyl Stearate RS (C18:0) 23
Acceptance criteria 2 Composition is proposed according to the relative composition of these
Polyglyceryl 3 dioleate: NMT 12.5 three fatty acid groups in Polyglycery! 3 Diisostearate.
Polyglyceryl 6 dioleate: NMT 12.5
© FATS AND FIXED OILS, Saponification Value (401): Deter- Sample solution: Introduce 100 mg of Polyglyceryl 3
mined on 1-g specimen Diisostearate into a 25-mL conical flask, fitted with a
Acceptance criteria suitable water-cooled reflux condenser and a magnetic
Polyglyceryl 3 dioleate: 135-155 stir bar. Add 2 mL of 0.5 N methanolic sodium hydroxide
Polyglyceryl 6 dioleate: 110-140 Solution, mix, and reflux for about 30 min. Add 2 mL of
© WATER DETERMINATION, Method | (921): NMT 1%, de- Boron trifluoride-methanol solution through the con-
termined on a 2.0-g specimen denser, and reflux for about 30 min. Add 4 mL of n-
heptane through the condenser, and reflux for 5 min.
ADDITIONAL REQUIREMENTS Cool, remove the condenser, add about 10 mL of Satu-
¢ PACKAGING AND STORAGE: Preserve in tight, light-resistant rated sodium chloride solution, shake, add a quantity of
containers, protected from heat and moisture. Saturated sodium chloride solution to bring the upper
e LABELING: Label to indicate whether it is Polyglyceryl 3 layer up to the neck of the flask, and allow the layers to
Dioleate or Polyglyceryl 6 Dioleate. separate. Collect 2 mL of n-heptane layer (upper layer),
e USP REFERENCE STANDARDS (11) wash with three quantities, each of 2 mL of water, and
USP Methyl Linoleate RS aly the n-heptane phase over anhydrous sodium
USP Methyl Linolenate RS sulfate.
USP Methyl Myristate RS Chromatographic system
USP Methy! Ofeate RS (See Chromatography (621), System Suitability.)
USP Methyl Palmitate RS Mode: GC
USP Methyl Palmitoleate RS Detector: Flame ionization
USP Methyl Stearate RS Column: 0.32-mm x 30-m fused-silica capillary col-
USP Polyglyceryl 3 Dioleate RS umn, 0.25-um layer of phase G16
USP Polyglyceryl 6 Dioleate RS Temperature
Detector: 250°
Injection port: 240°
Column: See the temperature program table below.

Polyglyceryl 3 Diisostearate Hold Time at

Initial Temperature Final Final
R-O-(CH>-CH(OR)-CH2-O)s-R Temperature Ramp Temperature | Temperature
(@) (°/min) (a) (min)
R =H, or CO-Cy7H35-iso 150 6 250 6
1,2,3-Propanetriol, homopolymer, diisooctadecanoate;
Triglyceryl diisostearate [63705-03-3]. Carrier gas: Nitrogen
Flow rate: 1.0-1.2 mL/min
DEFINITION Injection size: 1 uL
Polyglyceryl 3 Diisostearate is a mixture of polyglycery! dies- Injection type: Split injection. Split ratio is about 1:80.
ters of mainly isostearic acid, obtained by esterification of System suitability
polyglycerin and isostearic acid. The polyglycerin consists Sample: Standard solution
mainly of triglycerin. [Note—See the relative retention time table below.]
IDENTIFICATION
e A. INFRARED ABSORPTION (197F) Relative
e B. It meets the requirements of the test for Content of Retention
Fatty Acids. Name Time
Methylmyristate 1.0
ASSAY
e CONTENT OF FATTY ACIDS Methyl palmitate 14
0.5 N methanolic sodium hydroxide solution: Dis- Methyl stearate 1.8
solve 20g of sodium hydroxide in 50 mL of water, and
mix. Cool to room temperature, and add 950 mL of Suitability requirements
methanol. Resolution: NLT 10 between the peaks due to methyl
Boron trifluoride-methanol solution: Dissolve 14 g of palmitate and methyl stearate
pon trifluoride in methanol to make 100 mL, and mix
Relative standard deviation: NMT 6.0% for the peak
well. responses for palmitate and stearate
Saturated sodium chloride solution: Dissolve about Analysis ed
Samples: Standard solution and Sample solution Ba
375g of sodium chloride in water to make 1000 mL.
Standard solution: Prepare the calibration ester mix- Identify the fatty acid ester peaks in the chromatogram =
ture by mixing up each individual ester component (see of the Sample solution by comparing the retention °
Table 1), Dissolve 500 mg of the calibration ester mix- times of these peaks with those obtained in the chro- =]
matogramof the Standard solution, and measure the °
ture in n-heptane, and dilute with n-heptane to 50 mL. ©
peak areas for all of the fatty acid ester peaks in the oI
i)
1 Boron trifluoride-methanol solution (14% in methanol) is also commercially chromatogram obtained from the Sample solution. mo]
available from Sigma, B-1252, or equivalent quality.
=
ra
 5508 Polyglyceryl / Official Monographs
Calculate the percentage of each fatty acid ester com-
ponent in the test specimen:
Result = (ru/rr) x 100
tu = peak response for each individual fatty acid
ester component
tr = sum of the peak responses, excluding the
solvent peak, in the chromatogram obtained
from the Sample solution
Acceptance criteria
Sum of the contents of the fatty acids eluting
between palmitic acid and stearic acid (excluding
palmitic acid and stearic acid): NLT 60.0%
Sum of the contents of myristic acid, palmitic acid,
and stearic acid: NMT 11.0%
IMPURITIES
Inorganic Impurities
e RESIDUE ON IGNITION
Analysis: Heat a silica crucible to redness for 30 min,
allow to cool in a desiccator, and weigh. Evenly dis-
tribute about 1.0 g of Polyglyceryl 3 Diisostearate in
the crucible and weigh. Dry at 100°-105° for 1 h, and
ignite in a muffle furnace at 600 + 25°, until the test
substance is thoroughly charred. Perform the test for
Residue on Ignition (281) on the residue obtained, start-
ing with “Moisten the sample with a small amount
(usually 1 mL) of sulfuric acid”.
Acceptance criteria: NMT 0.5%
Delete the following:
°o Heavy Metals, Method I! (231): NMT 10 ppme coficia1-
Jan-2012)
SPECIFIC TESTS
e ACID VALUE
Analysis: Accurately weigh (to within 0.1 mg) 5-10 g of
Polyglyceryl 3 Diiostearate, add 10 mL of alcohol and
3 drops of phenolphthalein TS, and titrate with 0.1 N
potassium hydroxide VS or 0.1 N sodium hydroxide VS
until the solution remains faintly pink after shaking for
30 s. Follow the procedures for Fats and Fixed Oils, Acid
Value (401) to perform the calculation.
Acceptance criteria: NMT 3.0
e FATS AND FIXED OlLs, Hydroxyl Value (401): 180-230, de-
termined on a 0.25-g specimen
e IODINE VALUE
Analysis: Accurately weigh 3 g of Polyglyceryl 3 Diios-
tearate, transfer to a dry 250-mL flask with a ground-
glass stopper, and add 25 mL of methylene chloride.
Add 20 mL of the Wijs’ solution.2 Close the flask, and
keep it in the dark for 1 h while shaking frequently.
Pertorm the test in Fats and Fixed Oils (401) lodine
Value, starting with “Then add, in the order named,
30 mL of potassium iodide TS and 100 mL of water”.
Acceptance criteria: NMT 3.0
FATS AND FIXED OILS, Peroxide Value (401): NMT 6.0. Use
30 mL of a mixture of glacial acetic acid and methylene
chloride (3:2) to replace the 30 mL of a mixture of glacial
acetic acid and chloroform (3:2).
FATS AND FIXED OILS, Saponification Value (401): 128-160
WATER DETERMINATION, Method | (921): NMT 0.5%, de-
NF Monographs
termined on a 2.0-g specimen
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in tight, light-resistant
containers, protected from heat and moisture.
2Wijs’ reagent RPE for analysis from Carlo Erba Reference 491902, Wijs’ solu-
tion from www.sigmaaldrich.com, or equivalent quality.
NF 36
e USP REFERENCE STANDARDS (11)
USP Methyl Myristate RS
USP Methyl Palmitate RS
USP Methyl Stearate RS
USP Polyglyceryl 3 Diisostearate RS
Polyisobutylene
[9003-27-4].
DEFINITION
Polyisobutylene is a synthetic polymer produced by the low-
temperature polymerization of isobutylene in liquid ethyl-
ene, methylene chloride, or hexane, using an aluminum-
chloride or boron-trifluoride catalyst. It may contain a
suitable stabilizer.
IDENTIFICATION
e A. INFRARED ABSORPTION (197F)
Analysis: Prepare the sample by dissolving it in hot tol-
uene and evaporating ona salt plate.
Acceptance criteria: Meets the requirements
IMPURITIES
e LEAD (251)
Sample: 3.3g
Control: 10 mL of Diluted Standard Lead Solution (10 ug
of lead)
Analysis: Transfer the Sample to a porcelain dish, and
heat on a hot plate until completely charred. Then heat
in a muffle furnace at 480° for 8 h, and cool. Cautiously
add 5 mL of nitric acid, evaporate to dryness on a hot
plate, then heat again in the muffle furnace for exactly
15 min, and cool. Extract the ash with two 10-mL por-
tions of water, filtering the extract into a separator.
Leach any insoluble material on the filter with 6 mL of
Ammonium Citrate Solution, 2 mL of Hydroxylamine Hy-
drochloride Solution, and S mL of water, adding the
filtered washings to the separator. To the resulting solu-
tion and Contro! continue as directed in the chapter for
Procedure, beginning with “Add 2 drops of phenol red
TS.
Acceptance criteria: NMT 3 ug/g; the color generated
by the Sample does not exceed that generated by the
Control.
SPECIFIC TESTS
e ViscosiTY—CAPILLARY METHODS (911)
Solvent: Use isooctane.
Sample solution: Prepare a solution of Polyisobutylene
in the Solvent having a known concentration as indi-
cated in Table 1. The solution must be homogenous
before testing. For the Polyisobutylene having a Staud-
inger Index of 100 and higher, add the Solvent to the
weighed material, and allow it to stand in an oven at
80° for 12-24 h. [NoTE—A heated mechanical shaker
may be used to shorten the dissolution time; it is rec-
ommended that a gentle shaker be used to avoid shear-
ing the polymers. Take adequate precautions to prevent
evaporation of the Solvent.]
NF 36 Official Monographs / Polyoxy! 5509

Table 1 calculate the percentage of residue. If it exceeds 0.4%,

Concentration
again heat until constant weight is attained.
Acceptance criteria: NMT 0.4%
Staudinger Index? (g/cm?)
25-60 0.01
60-100 0.005 Delete the following:
100-350 0.002
350-700 0.001
°o HEAVY METALS, Method /! (231): NMT 20 ug/ge (oricia-
Jan-2018)
2The Staudinger Index is equal to 100 times the intrinsic viscosity.
© FREE POLYETHYLENE GLYCOLS
Analysis: Before each measurement, let the solutions be Sample solution: Transfer 12 g to a 500-mL separator
temperature equilibrated for 10 min. Using a suitable containing 50 mL of ethyl acetate. Add 50 mL of so-
Ubbelohde capillary viscometer having dimensions such dium chloride solution (0.29 g/mL), shake vigorously for
that the flow time is NLT 200 s, immersed in a con- 2 min, and allow to separate for 15 min. Drain the
trolled temperature bath, measure the flow of the Sol- lower, aqueous phase into a second 500-mL separator,
vent and of the Sample solution at 20°. Repeat the Anal- and extract the upper layer with a second 50-mL por-
ysis three times, and calculate the average. tion of sodium chloride solution (0.29 g/mL). To the
Calculate the reduced viscosity: combined aqueous layers add 50 mL of ethyl acetate,
shake vigorously for 2 min, and allow to separate as
J = (tte
- 1)/C before. Drain the lower, aqueous phase into a third
500-mL separator, and extract with two 50-mL portions
t = average flow time of the Sample solution (s) of chloroform by shaking for 2 min each time.
to = average flow time of the Solvent (s) Analysis: Evaporate the combined chloroform extracts
Cc = concentration of the Sample solution (g/cm3) in a 150-mL beaker on a steam bath, with the aid of a
Calculate the Staudinger Index: stream of nitrogen, to apparent dryness. Redissolve in
15 mL of chloroform, and transfer toa filter, collecting
Jo = J/[1 + 0.31(t/to — 19] the filtrate in a 150-mL beaker. Rinse the funnel with
several small portions of chloroform, and evaporate the
Acceptance criteria: It is within the limits specified on combined filtrate and rinsings, as described above, until
the label. no odor of chloroform or ethyl acetate is perceptible.
e Loss ON DRYING (731) Cool in a desiccator, and weigh.
Sample: 5g Acceptance criteria: NMT 7.5%
Analysis: Dry for 2 h at 105°. e FREE ETHYLENE OXIDE
Acceptance criteria: NMT 0.3% Internal standard solution: 100 mg/mL of n-butyl
chloride in chlorobenzene. Store in a tightly closed con-
ADDITIONAL REQUIREMENTS tainer. Prepare fresh weekly.
¢ PACKAGING AND STORAGE: Preserve in well-closed contain- Standard stock solution
ers. No storage requirements specified. [CauTion—Ethylene oxide is toxic and flammable. Prepare
¢ LABELING: Label it to indicate the range for intrinsic vis- this solution in a well-ventilated hood, using great care.]
cosity or the range for the Staudinger Index, and the Place 250 mL of chlorobenzene in a glass-stoppered
name and quantity of any added stabilizer. [NoTE—The 500-mL conical flask. Bubble ethylene oxide through
Staudinger Index is equal to 100 times the intrinsic the chlorobenzene at a moderate rate for 30 min, in-
viscosity.] sert the stopper, and store with protection from heat.
e USP REFERENCE STANDARDS (11) Pipet 25 mL of 0.5 N alcoholic hydrochloric acid solu-
USP Polyisobutylene RS tion, prepared by mixing 45 mL of hydrochloric acid
with 1 L of alcohol, into a 500-mL conical flask con-
taining 40 g of magnesium chloride hexahydrate.
Shake the mixture to effect saturation. Pipet 10 mL of
the ethylene oxide solution into the flask, and add
Polyoxyl 10 Oleyl Ether 20 drops of bromocresol green TS. If the solution is
not yellow (acid) at this point, add an additional vol-
Polyoxy-1,2-ethanediyl, o.-(Z)-9-octadecenyl-w-hydroxy-; ume of 0.5 N alcoholic hydrochloric acid to give an
Polyethylene glycol monooleyl ether [9004-98-2]. excess of 10 mL. Record the total volume of 0.5 N
DEFINITION alcoholic hydrochloric acid added. Insert the stopper
Polyoxyl 10 Oleyl Ether is a mixture of the mono-oleyl in the flask, and allow to stand for 30 min. Titrate the
ethers of mixed polyoxyethylene diols, the average poly- excess acid with 0.5 N alcoholic potassium hydroxide
mer length being equivalent to NLT 9.1 and NMT 10. VS. Perform a blank titration, using 10.0 mL of chloro-
oxyethylene units. It may contain suitable stabilizers. benzene instead of ethylene oxide solution, adding the
same total volume of 0.5 N alcoholic hydrochloric
IDENTIFICATION acid, and note the difference in volumes required.
e A. INFRARED ABSORPTION (197F) Each mL of the difference in volumes of 0.5 N alco-
Sample: Use undried specimen. holic potassium hydroxide consumed is equivalent to
Acceptance criteria: Meets the requirements 22.02 mg of ethylene oxide. Calculate the concentra-
tion, in mg/mL, of ethylene oxide in the Standard
IMPURITIES stock solution. Standardize daily.
sydesBouo- 4N

© RESIDUE ON IGNITION (281) Standard solution: Transfer 5 g of Polyoxyl 10 Oleyl

Sample: 25g
Analysis: Weigh the Sample into a tared 40-mL porce-
lain crucible, and heat in contact with air until it ignites
spontaneously or can be ignited with a glowing splint.
Allow the flame to go out, and place the crucible in a
muffle furnace with the door partly open until the car-
bon is consumed. Close the door, and heat at
700+100° for 1 h. Cool in a desiccator, weigh, and
Ether to a suitable glass bottle of 60-mL capacity, and
add 10 mL of chlorobenzene, exactly 50 iL of Internal
standard solution, and a volume of Standard stock solu-
tion containing 0.5 mg of ethylene oxide. Insert a mag-
netic stirring bar, cap the bottle tightly, and stir until
homogeneity is attained.
Sample solution: Transfer 5 g of Polyoxyl 10 Oleyl
Ether to a suitable glass bottle of 60-mL capacity, and
add 10 mL of chlorobenzene and 50 uL of Internal stan-
 5510 Polyoxyl / Official Monographs
dard solution. Add a volume of chlorobenzene equal to
the volume of the Standard stock solution added to pre-
pale the Standard solution. Insert a magnetic stirring
ar, cap the bottle tightly, and stir until homogeneity is
attained.
Interference check solution: Transfer 5 g of Polyoxyl
10 Oleyl Ether to a suitable glass bottle of 60-mL ca-
pacity, and add 10 mL of chlorobenzene. Add a volume
of chlorobenzene equal to the volume of the Standard
stock solution used to prepare the Standard solution. In-
sert a magnetic stirring bar, cap the bottle tightly, and
stir until homogeneity is attained.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: GC
Detector: Flame ionization
Column: 3-mm (OD) x 1.8-m stainless steel packed
with $3
Temperatures
Injection port: 210°
Detector: 230°
Column: 160°
Carrier gas: Helium
Flow rate: 66 mL/min
Injection volume: 2 uL
System suitability
Samples: Chlorobenzene, Internal standard solution,
Standard stock solution, and Interference check solution
Interference check: Inject a suitable volume of chloro-
benzene into the gas chromatograph, and allow the
chromatogram to run until the solvent has eluted.
Similarly inject and chromatograph the Internal stan-
dard solution, the Standard stock solution, and the Inter-
ference check solution. No interfering peaks are
observed.
Analysis
Samples: Standard solution and Sample solution
Calculate the weight of ethylene oxide in the portion
of sample taken:
Wr = (We x Wu x Ru)/[(Wu x Rs) — (Ws x Ru)] x F
W. = weight of ethylene oxide added to the
Standard solution (mg)
Wy = weight of Polyoxyl 10 Oleyl Ether used to
prepare the Sample solution (g)
Ru = peak area ratio of ethylene oxide to the
internal standard for the Sample solution
Rs = peak area ratio of ethylene oxide to the
internal standard for the Standard solution
Ws = weight of Polyoxyl 10 Oleyl Ether used to
prepare the Standard solution (g)
F = unit conversion, mg to g (10-3)
Calculate the percentage of ethylene oxide in the
portion of Polyoxyl 10 Oleyl Ether taken:
Result = (W;/Wy) x 100
W; and Wy are as defined above.
Acceptance criteria: NMT 0.01%
SPECIFIC TESTS
@ WATER DETERMINATION, Method | (921): NMT 3.0%
e FATS AND FIXED OILS, Acid Value (401): NMT 1.0.
e FATS AND FIXED OlLs, Hydroxy! Value (401): 75-95
NF Monographs
e FATS AND FIXED OILS, /odine Value, Method | (401)
Sample: 550mg
Analysis: Proceed as directed in the chapter, with the
reaction time being extended to 60 min.
Acceptance criteria: 23-40
FATS AND FIXED OILS, Saponification Value (401): NMT 3
AVERAGE POLYMER LENGTH
Sample solution: If solid material is present, place the
Polyoxyl 10 Oleyl Ether in a 60° water bath overnight.
Shake vigorously to eliminate any possibility of molecu-
NF 36
lar weight gradients within it. Add 1 mL of the melt to
1 mL of deuterated chloroform in a test tube, and shake
the test tube until dissolution is complete. Transfer
0.5 mL to an NMR tube, and add a small amount of
tetramethylsilane as an internal reference standard. Cap
the tube tightly, and shake thoroughly.
Analysis: Place the tube in an NMR spectrometer that is
capable of performing quantitative analysis, and record
the NMR spectrum (see Nuclear Magnetic Resonance
Spectroscopy (761), Quantitative Applications). Integrate
the areas from 0.4 to 2.35 ppm (A7), and from 2.35 to
4.9 ppm (A2).
Calculate the number of oxyethylene units per molecule
taken:
Result = [(31 x A2/A1) — 3]/4
31 = total number of protons in the molecule not
activated by either oxygen or a double bond
3 = number of oxygen-activated protons not
included in the oxyethylene unit count
4 = number of protons in each oxyethylene unit
Acceptance criteria: 9.1-10.9
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in tight containers,
and store in a cool place.
© LABELING: Label to indicate the names and proportions of
any added stabilizers.
e USP REFERENCE STANDARDS (11)
USP Polyoxyl 10 Oleyl Ether RS
Polyoxyl 15 Hydroxystearate
12-Hydroxyoctadecanoic acid polymer with a-hydro-w-
hydroxypoly(oxy-1,2-ethanediyl);
Polyethylene glycol 15 hydroxystearate [70142-34-6].
DEFINITION
Polyoxyl 15 Hydroxystearate results from the reaction of
about 15 moles of ethylene oxide with 1 mole of
12-hydroxystearic acid. The product consists mainly of
12-hydroxystearic acid polyethoxylated at both the car-
boxyl and the hydroxyl groups with polyethylene glycol.
It contains free polyethylene glycols.
IDENTIFICATION
e A. INFRARED ABSORPTION (197F): If the sample is solid or
too viscous for thin film formation, the sample should be
gently warmed to achieve a mobile liquid, which may
then be used to prepare the thin film.
o B. Onion CHROMATOGRAPHIC IDENTIFICATION TEST
(201
Sample solution: To 1.0g of Polyoxyl 15 Hydroxys-
tearate add 100 mL of a 100-mg/mL solution of potas-
sium hydroxide, and boil under a reflux condenser for
30 min. Acidify the warm solution with 20 mL of hydro-
chloric acid, and cool to room temperature. Shake the
mixture with 50 mL of ether, and allow to stand until a
separation of the layers is visible. Separate the clear up-
per layer, add 5 g of anhydrous sodium sulfate, wait for
30 min, filter, and evaporate to dryness on a water
bath. Dissolve 50 mg of the residue in 25 mL of ether.
Standard solution: 2 mg/mL of USP 12-Hydroxystearic
Acid RS in methylene chloride
Plate: Octadecylsilyl silica gel for chromatography as
the coating substance
Application volume: 2 ul
Developing solvent system: Acetone, methylene chlo-
ride, and glacial acetic acid (50:10:40)
Spray reagent: Prepare a solution of 80 mg/mL of
phosphomolybdic acid in 2-propanol.
NF 36 Official Monographs / Polyoxyl 5511

Analysis: Proceed as directed in the chapter. Develop rst = peak response of polyethylene glycol 1000
over two-thirds of the plate, and dry in a current of from Standard solution A
cold air. Then spray the plate with Spray reagent, heat Ts2 = peak response of polyethylene glycol 1000
the plate at 120° for 1-2 min, and locate the spots on from Standard solution B
the plate. Acceptance criteria: 27.0%-39.0% of free polyethylene
Acceptance criteria: The R- value and color of the prin- glycols
cipal spot of the Sample solution correspond to those of
the Standard solution. IMPURITIES
e C. It meets the requirements of the test for Free Polyethyl- Inorganic Impurities
ene Glycols. e ARTICLES OF BOTANICAL ORIGIN, Total Ash (561): NMT
0.3%, determined on 1.0g
COMPOSITION eo LIMIT OF NICKEL
e FREE POLYETHYLENE GLYCOLS [Caution—When using closed high-pressure digestion ves-
Mobile phase: Methanol and water (8:2) sels and microwave laboratory equipment, the safety pre-
Standard solution A: 1.6 mg/mL of USP Polyethylene cautions and operating instructions given by the manufac-
Glycol 1000 RS in Mobile phase turer must be followed.]
Standard solution B: 0.8 mg/mL of USP Polyethylene [NoTte—lf an alternative apparatus is used, adjustment of
Glycol 1000 RS in Mobile phase, diluted from Standard the instrument parameters may be necessary.]
solution A in Mobile phase Nickel standard stock solution: Dilute nickel standard
Sample solution: 4.8 mg/mL of Polyoxyl 15 Hydroxy- solution TS two-fold with water. This solution contains
stearate in Mobile phase the equivalent of 5 ug/mL of nickel.
Chromatographic system Standard solutions: Transfer 25, 50, 75, and 100 uL of
(See Chromatography (621), System Suitability.) Nickel standard stock solution to four identical 25-mL
Mode: LC volumetric flasks. To each flask add 0.5 mL of a
Detector: Refractive index 10-mg/mL solution of magnesium nitrate, 0.5 mL of a
Columns: 7.8-mm x 30-cm analytical column; 6-14m 100-mg/mL solution of monobasic ammonium phos-
packing L39 and a 12-nm pore size; two 4-mm x phate, and 6.0 mL of nickel-free nitric acid, dilute with
12.5-cm precolumns; 5-14m packing L1 and a 10-nm water to volume, and mix well. [Note—Content of
pore size. nickel in the nickel-free nitric acid is NMT 0.005 ppm.]
Connect both precolumns to the analytical column us- The Standard solutions contain 0.005, 0.01, 0.015, and
ing a 3-way valve, and switch the Mobile phase flow 0.02 g/mL of nickel, respectively.
according to the following program. [NoTE—Shown Sample solution: Transfer about 250 mg of Polyoxyl
in Figure 1, the analysis is started with precolumn 2 15 Hydroxystearate to a suitable high-pressure-resistant
and an analytical column in series. After about 114 s, digestion vessel (fluoropolymer or quartz glass), and
the valves, controlled by the detector program, add 6.0 mL of nickel-free nitric acid and 2.0 mL of 30%
switch over such that the eluent flows past precol- hydrogen peroxide. Place the closed vessel in a labora-
umn 2, and direct to precolumn 1 and the analytical tory microwave oven, and digestusing an appropriate
column. The columns are switched when the compo- program, e.g., 1000 W for 40 min. Allow the digestion
nents to be determined, but not the interfering ma- vessel to cool down before opening. Add 2.0 mL of
trix, are ready to reach the analytical column. Simul- 30% hydrogen peroxide, and repeat the digestion
taneously, precolumn 2 is washed out in the reverse step. Allow the digestion vessel to cool down before
direction by a second pump to remove the unwanted opening. Quantitatively transfer to a 25-mL volumetric
matrix components.] flask, add 0.5 mL of a 10-mg/mL solution of magne-
sium nitrate and 0.5 mL of a 100-mg/mL solution of
Time monobasic ammonium phosphate, dilute with water to
(s) Program volume, and mix well.
0-114 Precolumn 2 and analytical column
Blank solution: Place 6.0 mL of nickel-free nitric acid
and 2.0 mL of 30% hydrogen peroxide in a suitable
115-end Precolumn 1 and analytical column. high-pressure-resistant digestion vessel. Proceed as di-
115-420 Reverse flow of precolumn 2 rected under Sample solution, beginning with “Place
the closed vessel in a laboratory microwave oven, and
Temperature digest using an appropriate program, e.g., 1000 W for
Column: Room temperature 40 min.”
Detector: Room temperature Zero solution: In a 50-mL volumetric flask, introduce
Flow rate: 1.1 mL/min 1.0 mL of a 10-mg/mL solution of magnesium nitrate,
Injection size: 50 uL 1.0 mL of a 100-mg/mL solution of monobasic ammo-
System suitability nium phosphate, and 12.0 mL of nickel-free nitric acid.
Sample: Standard solution A Dilute with water to volume, and mix well.
Suitability requirements Spectrometric conditions
Relative standard deviation: NMT 3.0% (See Atomic Absorption ey (852).)
Analysis Mode: Graphite furnace atomic absorption spectro-
Samples: Standard solution A, Standard solution B, and photometer equipped with a background compensa-
Sample solution tion system, a coated tube resistant to pyrolysis, and
Calculate the percentage of polyethylene glycols in the a nickel hollow-cathode lamp.
portion of Polyoxyl 15 Hydroxystearate taken: Analytical wavelength: Nickel emission line of 232.0
Result = 2 x (Cs/Cu)[ru/(ts1 + 2rs2)] x 100 nm
Temperature: Maintain the drying temperature of
Cs = concentration of USP Polyethylene Glycol the furnace at 120° for 35 s after a 5-s ramp; main-
1000 RS in Standard solution A (mg/mL) tain the ashing temperature at 1100° for 10s after a
Cu = concentration of Polyoxyl 15 Hydroxystearate 30-s ramp; maintain the cooling temperature at 800°
in the Sample solution (mg/mL) for 5 s after a 5-s decrease; and maintain the atomi-
tu = peak response of polyethylene glycol from the zation temperature at 2600° for 7 s. [NoTE—The tem-
Sample solution perature program may be modified to obtain opti-
mum furnace temperatures.]
 4502 Calcium / Dietary Supplements USP 41

extracts to the evaporation flask. Evaporate the com- Tablet, and the salt form of calcium and the chemical
bined hexane extracts in vacuum at room temperature form of vitamin D present in the Tablet.
to dryness. Dissolve in and dilute the residue in a vol- e USP REFERENCE STANDARDS (11)
ume of n-hexane to obtain a concentration of 2 ug/mL. USP Cholecalciferol RS
Chromatographic system USP Ergocalciferol RS
(See Chromatography (621), System Suitability.)
Mode: LC
Column: 4.6-mm x 15-cm; 3-m packing L8
Detector: UV 265 nm
Flow rate: 1 mL/min Calcium and Vitamin D with Minerals
Injection size: 100 uL
System suitability Tablets
Samples: Standard solution and System suitability
solution DEFINITION
Suitability requirements Calcium and Vitamin D with Minerals Tablets contain Vita-
Resolution: NLT 10 between the vitamin D form min D as Ergocalciferol (Vitamin D2) or Cholecalciferol (Vi-
present and its corresponding precursor, System suita- tamin D3), Calcium, and one or more minerals derived
bility solution from substances generally recognized as safe, furnishing
Relative standard deviation: NMT 3.0%, Standard one or more of the following elements in ionizable form:
solution copper, magnesium, manganese, and zinc. Tablets con-
Analysis tain NLT 90.0% and NMT 165.0% of the labeled amount
Samples: Standard solution and Sample solution of vitamin D, as cholecalciferol (C27H14O) or ergocalciferol
Measure the peak areas for vitamin D. (CogH440), and NLT 90.0% and NMT 125.0% of the la-
Calculate the percentage of the labeled amount of cho- beled amounts of calcium (Ca), copper (Cu), magnesium
lecalciferol (C27H440) or ergocalciferol (C2gH44O) in the (Mg), manganese (Mn), and zinc (Zn). They may contain
portion of Tablets taken: other labeled added substances that are acnenie recog-
nized as safe, in amounts that are unobjectionable.
Result = (ru/rs) x (Cs/Cu) x Fx 100
STRENGTH
tu = peak area of cholecalciferol or ergocalciferol e CHOLECALCIFEROL or ERGOCALCIFEROL (VITAMIN D)
from the Sample solution [NoTte—Use low-actinic glassware throughout this
ts = peak area of cholecalciferol or ergocalciferol procedure.]
from the Standard solution Mobile phase: n-Hexane and isopropyl alcohol (99:1)
G = concentration of USP Cholecalciferol RS or Standard solution: 2 g/mL of USP Ergocalciferol RS or
USP Ergocalciferol RS in the Standard solution USP Cholecalciferol RS in n-hexane
System suitability solution: Heat a volume of Standard
(ug/ml) ; f solution at 60° for 1 h to partially isomerize vitamin D
Cu = nominal concentration of cholecalciferol or
ergocalciferol in the Sample solution (g/mL) (ergocalciferol or cholecalciferol) to its corresponding
F = correction factor to account for the average precursor.
amount of previtamin D present in the Sample solution: Weigh NLT 20 Tablets, and grind the
Sample solution, 1.09 Tablets to a fine powder. Transfer the equivalent of
Acceptance criteria: 90.0%-165.0% of the labeled 20 1g of cholecalciferol or ergocalcifero! to a container
having a polytef-lined screw cap. Add 8 mL of dimethyl
amount of vitamin D as cholecalciferol (C27H44O) or er- sulfoxide and 12 mL of n-hexane, and shake for 45 min
gocalciferol (C2sH440)
on a wrist-action shaker with tubes in a water bath
PERFORMANCE TESTS maintained at 60°. Centrifuge for 10 min, withdraw the
e DISINTEGRATION AND DISSOLUTION OF DIETARY SUPPLEMENTS hexane layer by means of a pipet, and transfer to an
(2040): Meet the requirements for Dissolution with re- evaporation flask. Add 12 mL of n-hexane to the di-
spect to calcium methyl sulfoxide layer, mix on a vortex mixer for 5 min,
Medium: 0.1 N hydrochloric acid; 900 mL and again withdraw the hexane layer by means of a
Apparatus 2: 75 rpm pipet, and add to the evaporation flask. Repeat this ex-
Time: 30 min traction with three additional 12-mL portions of n-hex-
DS Monographs

Analysis: Determine the amount of calcium (Ca) dis- ane, adding the hexane extracts to the evaporation
solved, using the procedure in Calcium, making any flask. Evaporate the combined hexane extracts in vac-
necessary volumetric adjustments. uum at room temperature to dryness. Dissolve in and
Tolerances: NLT 75% of the labeled amount of calcium dilute the residue in a volume of n-hexane to obtain a
(Ca) is dissolved. concentration of 2 tug/mL.
e WEIGHT VARIATION OF DIETARY SUPPLEMENTS (2091): Meet Chromatographicsyoterny
the requirements (See Chromatography (621), System Suitability.)
Mode: LC
CONTAMINANTS Detector: UV 265 nm
e MICROBIAL ENUMERATION TESTS (2021): The total aerobic Column: 4.6-mm x 15-cm; 5-um packing L8
microbial count does not exceed 3000 cfu/g, and the Flow rate: 1 mL/min
total combined molds and yeasts count does not exceed Injection size: 100 uL
300 cfu/g. System suitability
e ABSENCE OF SPECIFIED MICROORGANISMS (2022): Meets the Samples: Standard solution and System suitability
requirements of the tests for absence of Salmonella spe- solution
cies and Escherichia coli Suitability requirements
Resolution: NLT 10 between the vitamin D form
ADDITIONAL REQUIREMENTS present and its corresponding precursor, System suita-
© PACKAGING AND STORAGE: Preserve in tight, light-resistant bility solution
containers. Relative standard deviation: NMT 3.0%, Standard
e LABELING: The label states that the product is Calcium solution
with Vitamin D Tablets. The label states also the quanti-
ties of calcium and vitamin D in terms of metric units/
 5512 Polyoxyl / Official Monographs
injection valve
position: inject
valve for col
position: A
GPC column
Detectof
Figure 1. Apparatus
Analysis
Samples: Standard solutions, Sample solution, and
Blank solution
Concomitantly determine the absorbances of the
Samples using the Spectrometric conditions described
above. Use the Zero solution to set the instrument to
zero. Plot the absorbances of the Standard solutions
versus the concentration, in g/mL, of nickel, and
draw the straight line best fitting the plotted points.
From the graph so obtained, determine the concen-
tration, Cr, in g/mL, of nickel in the Sample solu-
tion, and determine the concentration, Cs, in g/mL,
of nickel in the Blank solution. If necessary, dilute
with the Zero solution to obtain a reading within the
calibrated absorbance range.
Calculate the quantity, in ug, of nickel in each g of
Polyoxyl 15 Hydroxystearate taken:
Result = V x (Cy — Cs)/W
NF Monographs
Vv = volume of the Sample solution and the Blank
solution, 25 mL
Ww = weight ofFelon) 15 Hydroxystearate taken
to prepare the Sample solution (g)
Acceptance criteria: NMT 1 g/g of nickel
Organic Impurities
© PROCEDURE: LIMIT OF FREE ETHYLENE OXIDE AND DIOXANE
[CauTion—Ethylene oxide is toxic and flammable. Prepare
these solutions in a well-ventilated fume hood, using
NF 36
injection valve
position: load
GPC column
great care. Protect both hands and face by wearing poly-
ethylene protective gloves and an appropriate face mask.
Store all solutions in hermetic containers, and refrigerate
at a temperature between 4° and 8°.]
[Nott—Before using the polyethylene glycol 200 in this
test, remove any volatile components from it by placing
500 mL of polyethylene glycol 200 in a 1000-mL
round-bottom flask, attaching the flask to a rotary evap-
orator maintained at a temperature of 60° and under a
vacuum of 10-20 mm Hg for 6 h.]
Acetaldehyde solution: 10 g/mL of acetaldehyde.
[Nott—Prepare the Acetaldehyde solution immediately
prior to use.]
Ethylene oxide stock solution: Fill a chilled pressure
bottle with liquid ethylene oxide, and store in a freezer
when not in use. Use a small piece of peer eae film
to protect the liquid from contact with the rubber gas-
ket. Tare a glass-stoppered conical flask, add about
50 mL of polyethylene glycol 200, and reweigh the
flask. Transfer about 5 mL of the liquid ethylene oxide
to a 100-mL beaker chilled in a mixture of sodium
chloride and ice (1:3). Using a gas-tight syringe that
has been previously cooled to —10°, transfer about
300 LL (corresponding to about 250 mg) of liquid eth-
ylene oxide to the polyethylene glycol 200, and swirl
gently to mix. Replace the stopper, reweigh the flask,
and determine the amount of ethylene oxide absorbed
by weight difference. Adjust the weight of the mixture
with polyethylene glycol 200 to 100.0 g, replace the
stopper, and swirl gently to mix. This stock solution
NF 36 Official Monographs / Polyoxyl 5513

contains about 2.5 mg/g of ethylene oxide. [NOTE— System suitabilit

Prepare this stock solution immediately prior to use, Sample: Standard solution A
and store ina refrigerator.] [Note—The relative retention times for acetaldehyde
Ethylene oxide solution: Tare a glass-stoppered coni- and ethylene oxide are 0.94 and 1.0, respectively.]
cal flask, and chill it in a refrigerator. Add about 35 mL Suitability requirements
of polyethylene glycol 200, and reweigh the flask. Us- Resolution: NLT 2.0 between acetaldehyde and eth-
ing agas-tight gas chromatographic syringe that has ylene oxide
been chilled in a refrigerator, transfer about 1 g of the Signal-to-noise ratio: NLT 5, determined from the
chilled Ethylene oxide stock solution, weighed, to the ioxane peak
tared, conical flask. Adjust the weight of the solution Relative standard deviation: NMT 15%
with polyethylene glycol 200 to 50.0 g, replace the Analysis
stopper, and swirl gently to mix. Transfer about 10 g of Samples: Standard solution B and Sample solution
this solution, weighed, to a 50-mL volumetric flask. Using a heated, gas-tight, gas chromatographic sy-
Add 30 mL of water, and mix. Dilute with water to ringe, separately inject equal volumes of the gaseous
volume, and mix to obtain a solution containing about neadspacs of the Samples into the chromatograph,
10 pg/mL of ethylene oxide. [NoTE—Prepare this solu- record the chromatograms, and measure the areas
tion immediately prior to use, and use directly after for the major peaks: the mean areas of the ethylene
preparation.] oxide and dioxane peaks from the Sample solution are
Dioxane solution: 500 g/mL of dioxane not greater than half the mean areas of the corre-
Standard solution A: Transfer 0.1 mL of Ethylene oxide sponding peaks from Standard solution B.
solution to a 10-mL pressure headspace vial. [NOTE— Calculate the content of ethylene oxide, in ppm, in
Other sizes may be used depending on the operating the portion of Polyoxyl 15 Hydroxystearate taken:
conditions, however, the same size must be used for
Standard solution A, Standard solution B, and the Sample Result = (Ae x ru)/[(rs x Wu) — (tu x Ws)]
solution.] Add 0.1 mL of Acetaldehyde solution and
0.1 mL of Dioxane solution, seal the vial, and mix. Ag = quantity of ethylene oxide added to Standard
Standard solution B: Transfer about 1.0g of Polyoxyl solution B Gags
15 Hydroxystearate to another 10-mL pressure head- fu = ethylene oxide peak response from the
space vial, add 0.1 mL of Ethylene oxide solution, 0.1 mL Sample solution
of Dioxane solution, and 1.0 mL of N,N-dimethylaceta- ts = ethylene oxide peak response from Standard
mide. Seal the vial, and mix. solution B
eae solution: Transfer about 1.0 gof Polyoxyl 15 Wu = weight of test substance taken to prepare the
Hydroxystearate to a 10-mL pressure headspace vial, Sample solution (g)
add 1.0 mL of N,N-dimethylacetamide and 0.2 mL of Ws = weight of test substance taken to prepare
water, seal the vial, and mix. Standard solution B (g)
Chromatographic system Calculate the content of dioxane, in ppm, in the
(See Chromatography (621), System Suitability.) portion of Polyoxyl 15 Hydroxystearate taken:
[NoTte—The use of a headspace apparatus that auto-
matically transfers a measured amount of headspace Result = (Ap x ru)/[(rs x Wu) - (ru x Ws)]
is allowed.]
Mode: GC Ao = quantity of dioxane added to Standard
Detector: Flame ionization solution B (ug)
Column: 0.32-mm x 30-m glass or quartz capillary; tu = dioxane peak response from the Sample
1.0-um layer of phase G1 solution
Temperature rs = dioxane peak response from Standard solution
Injector port: 150° B
Detector: 250° Wu = weight of test substance taken to prepare the
Column: See the temperature program table below. Sample solution (g)
Ws = weight of test substance taken to prepare
Standard solution B (g)
Hold Time at| Acceptance criteria
Initial Temperature Final Final Ethylene oxide: NMT 1 ppm
Temperature Ramp Temperature | Temperature Dioxane: NMT 50 ppm
©) (¢/min) ©) (min)
50 a 50 5 SPECIFIC TESTS
50 5 180 = e FATS AND FIXED OILS, Acid Value (401): NMT 1.0, deter-
mined on 2.0g
180 30 230 —
FATS AND FIXED OILS, Hydroxyl Value (401): 90-110
230 a 230 5 FATS AND FIXED OILS, /odine Value, Method | (401): NMT
2.0
Carrier gas: Helium FATS AND FIXED OILS, Peroxide Value (401): NMT 5.0
Linear velocity: 20 cm/s e FATS AND FIXED OILS, Saponification Value (401): 53-63
Injection size: 1 mL (the gaseous headspace) WATER DETERMINATION, Method Ia (921): NMT 1.0%, de-
Injection type: Split ratio 20:1 termined on 2.0g
[Note—If the headspace apparatus is used, then an
injection time of 12 s anda transfer line tempera- ADDITIONAL REQUIREMENTS rs
na,
ture of 150° are recommended.] © PACKAGING AND STORAGE: Preserve in tight containers at a
Headspace sampler: Each vial is heated at a temper- temperature below 25°. =
ature of 90° for 45 min, before a suitable portion of °
=|
its headspace is injected. °
Ko}
2
mo]
s
7
 5514 Polyoxyl / Official Monographs
e USP REFERENCE STANDARDS (11)
USP 12-Hydroxystearic Acid RS
USP Polyethylene Glycol 1000 RS
USP Polyoxyl 15 Hydroxystearate RS
Polyoxyl 20 Cetostearyl Ether
DEFINITION
Polyoxyl 20 Cetostearyl Ether is a mixture of mono-cetostea-
ryl (mixed hexadecyl! andSctadecy)) ethers of mixed poly-
oxyethylene diols, the average polymer length being
equivalent to NLT 17.2 and NMT 25.0 oxyethylene units.
IDENTIFICATION
e A. INFRARED ABSORPTION (197F): Use undried specimen.
IMPURITIES
e RESIDUE ON IGNITION (281)
Sample: 25g
Analysis: Weigh the Sample into a tared 40-mL porce-
lain crucible, and heat in contact with air until it ignites
spontaneously or can be ignited with a glowing splint.
Allow the flame to go out, place the crucible in a muf-
fle furnace with the door partly open until the carbon is
consumed, close the door, and heat at 700 + 100° for 1
h. Cool in a desiccator, weigh, and calculate the per-
centage of residue. If the amount so obtained exceeds
0.4%, heat again until constant weight is attained.
Acceptance criteria: NMT 0.4%
Delete the following:
°e HEAVY METALS, Method fi (231): NMT 20 Ug/ge ctica1-
Jan-2018)
e FREE POLYETHYLENE GLYCOLS
Sample solution: Transfer 12 g to a 500-mL separator
containing 50 mL of ethyl acetate. Add 50 mL of so-
dium chloride solution (0.29 g/mL), shake vigorously for
2 min, and allow to separate for 15 min. Drain the
lower, aqueous phase into a second 500-mL separator,
and extract the upper layer with a second 50-mL por-
tion of sodium chloride solution (0.29 g/mL). To the
combined aqueous layers, add 50 mL of ethyl acetate,
shake vigorously for 2 min, and allow to separate as
before. Drain the lower, aqueous phase intoa third
500-mL separator, and extract with two 50-mL portions
of chloroform by shaking for 2 min each time.
Analysis: Evaporate the combined chloroform extracts
in a 150-mL beaker on a steam bath, with the aid of a
stream of nitrogen, to apparent dryness. Redissolve in
15 mL of chloroform, and transfer toa filter, collecting
the filtrate in a 150-mL beaker. Rinse the funnel with
several small portions of chloroform, and evaporate the
combined filtrate and rinsings, as described above, until
no odor of chloroform or ethyl acetate is perceptible.
Cool in a desiccator, and weigh.
Acceptance criteria: NMT 7.5%
e FREE ETHYLENE OXIDE
Internal standard solution: 100 mg/mL of n-butyl
chloride in chlorobenzene. Store in a tightly closed con-
”“ tainer. Prepare fresh weekly.
pe Standard stock solution
a Calculate the weight of ethylene oxide in the portion
=
is [Caution—Ethylene oxide is toxic and flammable. Prepare
Dp this solution in a well-ventilated hood, using great care.]
S) Place 250 mL of chlorobenzene in a glass-stoppered,
iS 500-mL conical flask. Bubble ethylene oxide through
S the chlorobenzene at a moderate rate for 30 min, in-
Pa sert the stopper, and store with protection from heat.
J
Pipet 25 mL of a 0.5 N alcoholic hydrochloric acid so-
Zz lution, prepared by mixing 45 mL of hydrochloric acid
with 1L of alcohol, into a 500-mL conical flask con-
NF 36
taining 40 g of magnesium chloride hexahydrate.
Shake the mixture to effect saturation. Pipet 10 mL of
the ethylene oxide solution into the flask, and add
20 drops of bromocresol green TS. If the solution is
not yellow (acid) at this point, add an additional vol-
ume of 0.5 N alcoholic hydrochloric acid to give an
excess of 10 mL. Record the total volume of 0.5 N
alcoholic hydrochloric acid added. Insert the stopper
in the flask, and allow to stand for 30 min. Titrate the
excess acid with 0.5 N alcoholic potassium hydroxide
Vs.
Perform a blank titration, using 10.0 mL of chloroben-
zene instead of ethylene oxide solution, adding the
same total volume of 0.5 N alcoholic hydrochloric
acid, and note the difference in volumes required.
Each mL of the difference in volumes of 0.5 N alco-
holic potassium hydroxide consumed is equivalent to
22.02 mg of ethylene oxide. Calculate the concentra-
tion, in mg/mL, of ethylene oxide in the Standard
stock solution. Standardize daily.
Standard solution: Transfer 5 g of Polyoxyl 20 Ceto-
stearyl Ether to a suitable glass bottle of 60-mL capac-
ity. Add 10 mL of chlorobenzene, exactly 50 uL of Inter-
nal standard solution, and a volume of Standard stock
solution containing 0.5 mg of ethylene oxide. Insert a
magnetic stirring bar, cap the bottle tightly, and stir
until homogeneity is attained.
Sample solution: Transfer 5 g of Polyoxyl 20 Cetostea-
ryl Ether to a suitable glass bottle of 60-mL capacity.
Add 10 mL of chlorobenzene and 50 uL of Internal stan-
dard solution. Add a volume of chlorobenzene equal to
the volume of the Standard stock solution added to pre-
pa the Standard solution. Insert a magnetic stirring
ar, cap the bottle tightly, and stir until homogeneity is
attained.
Interference check solution: Transfer 5 g of Polyoxyl
20 Cetostearyl Ether to a suitable glass bottle of 60-mL
capacity, and add 10 mL of chlorobenzene. Add an ad-
ditional volume of chlorobenzene equal to the volume
of the Standard stock solution used to prepare the Stan-
dard solution. Insert a magnetic stirring bar, cap the
bottle tightly, and stir until homogeneity is attained.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: GC
Detector: Flame ionization
Column: 3-mm (OD) x 1.8-m stainless steel packed
with $3
Temperatures
Injection port: 210°
Detector: 230°
Column: 160°
Carrier gas: Helium
Flow rate: 66 mL/min
Injection volume: 2 uL
System suitability
Samples: Chlorobenzene, Internal standard solution,
Standard stock solution, and Interference check solution
Interference check: Inject a suitable volume of chloro-
benzene, and allow the chromatogram to run until the
solvent has eluted. Similarly inject the Internal standard
Solution, Standard stock solution, and Interference check
solution. No interfering peaks are observed.
Analysis
Samples: Standard solution and Sample solution
of the sample taken:
Wr = (We x Wu x Ru)/[(Wu x Rs) — (Ws x Ruy] x F
We = weight of ethylene oxide added to the
Standard solution (mg)
Wy = weight of Polyoxyl 20 Cetostearyl Ether used
to prepare the Sample solution (g)
NF 36 Official Monographs / Polyoxyl 5515

Ru = peak area ratio of ethylene oxide to the DEFINITION

internal standard from the Sample solution Polyoxyl 35 Castor Oil contains mainly the triricinoleate es-
Rs = peak area ratio of ethylene oxide to the ter of ethoxylated glycerol with smaller amounts of poly-
internal standard from the Standard solution ethylene glycol ricinoleate and the corresponding free gly-
Ws = weight of Polyoxyl 20 Cetostearyl Ether used cols. It results from the reaction of glycerol ricinoleate
to prepare the Standard solution (g) with 35 mol of ethylene oxide.
F = unit conversion, mg to g (10-3)
Calculate the percentage of ethylene oxide in the IDENTIFICATION
portion of Polyoxyl 20 Cetostearyl Ether taken: e A. INFRARED ABSORPTION (197F)
© B. CONSTITUTING FATTY ACIDS
Result = (W;/Wy) x 100 Sample: 0.1g
Analysis: Dissolve the Sample in 10 mL of potassium hy-
W; and Wy are as defined above. droxide TS, alcoholic; boil for 3 min; and evaporate to
Acceptance criteria: NMT 0.01% dryness. Mix the residue with 5 mL of water.
Acceptance criteria: The residue dissolves, yielding a
SPECIFIC TESTS clear solution. Add a few drops of glacial acetic acid. A
© FATS AND FIXED OlLs, Acid Value (401): NMT 0.5 white precipitate is formed.
FATS AND FIXED OlLs, Hydroxyl Value (401): 42-60 e C. IDENTITY BY FATTY ACID COMPOSITION
FATS AND FIXED OILS, Saponification Value (401): NMT 2 Diluent: n-Heptane
PH (791) Standard solution 1: 0.2 mg/mL each of methyl! palmi-
Sample solution: 100 mg/mL tate, methyl stearate, methyl oleate, methyl linoleate,
Acceptance criteria: 4.5-7.5 methyl cis-11-eicosenoate, and methyl ricinoleate, from
WATER DETERMINATION, Method | (921): NMT 1.0% USP Methyl Palmitate RS, USP Methyl Stearate RS, USP
AVERAGE POLYMER LENGTH Methyl Oleate RS, USP Methyl Linoleate RS, methyl cis-
Sample solution: Place the Polyoxyl 20 Cetostearyl 11-eicosenoate, and USP Methyl Ricinoleate RS, in
Ether in a 50° water bath overnight to melt it com- Diluent
pletely. Shake vigorously to eliminate any possibility of Standard solution 2: 4 mg/mL each of methyl stearate
molecular weight gradients within it, and transfer and methyl! ricinoleate from USP Methyl Stearate RS
200 pL to a 5- x 180-mm high-resolution NMR sample and USP Methyl Ricinoleate RS in Diluent
tube. Add 200 uL of deuterated chloroform by means Sample solution: Transfer 140 mg of Polyoxy! 35
of a separate microsyringe. Add 5 drops of tetramethyl- Castor Oil to a 10-mL screw-cap test tube, add 3.0 mL
silane as an internal reference standard. Cap the tube of Diluent, and mix well. Add 0.5 mL of 0.5 M sodium
tightly, and shake Hora: methoxide in methanol,’ and mix with the sample. Al-
Analysis: Place the tube in the NMR spectrometer, and low the reaction to proceed at room temperature for 2
record the NMR spectrum at an appropriate RF power h. After 2 h, add 5 mL of water, and mix. Centrifuge
level and a sweep time of 250 s/500 Hz (see Nuclear the test tube at 1000 x g for 10-15 min until a clear
Magnetic Resonance Spectroscopy (761), Qualitative Appli- upper layer forms. Separate the organic layer (the up-
cations ). Adjust the spectrum amplitude so that the per layer), and remove the lower layer. Place an aliquot
signal at 1.1 ppm is at least 80% of full scale. Record of the organic layer into an autosampler vial.
the integral areas, from 0.4 to 2.35 ppm (Aj), and from Chromatographic system
2.35 to 4.9 ppm (Az), at a sweep time of 50 s/500 Hz (See Chromatography (621), System Suitability.)
at an integral power level such that the integra! of the Mode: GC
ethylene oxide peak at 3.5 ppm is at least 80% of full Detector: Flame ionization
chart height. Do not change the power level during the Column: 0.25-mm x 15-m fused silica capillary;
sweep. Record the integral of each peak several times, bonded with a 0.25-um layer of phase G7
and calculate the average integral area. Temperatures
Sicuinte the number of oxyethylene units per molecule Injection port: 240°
taken: Detector: 250°
Column: See Table 1.
Result = [(32 x Ao/A:) - 3]/4
32 = total number of protons in the molecule not Table 1
activated by oxygen, averaged for the cetyl Hold
and stearyl radicals Time at
3. = number of oxygen-activated protons not Initial Tempera- Final Final
included in the oxyethylene unit count Tempera- ture Tempera- | Tempera- Total
4 = number of protons in each oxyethylene unit ture Ramp ture ture Time
Acceptance criteria: 17.2-25.0 (@) (¢/min) ©} (min) (min)
ADDITIONAL REQUIREMENTS 80 0 80 1 1
¢ PACKAGING AND STORAGE: Preserve in tight containers, in 80 30 140 0 3
a cool place. 140 3 150 0 6.3
e USP REFERENCE STANDARDS (11) 150 1 155 0 11.3.
USP Polyoxyl 20 Cetostearyl Ether RS 155 Z 165 0 16.3
165 z 220 10 45
sydesbouo-= 4N

Column mode: See Table 2 for the pressure program.

Polyoxyl 35 Castor Oil
10.5 M sodium methoxide in methanol is available from Sigma-Aldrich
Polyethylene glycol 35 castor oil; (www.sigmaaldrich.com), product #403067. Any other equivalent reagent
Polyoxyethylene 35 castor oil [61791-12-6]. can be used as well.
 5516 Polyoxyl / Official Monographs NF 36

Table 2 tr = sum of all the peak areas, excluding the

Pressure
solvent and methyl ricinoleate peaks and
Pressure Ramp Hold Time Total Time
including the corrected peak area of methyl
(psi) (psi/min) (min) (min)
ricinoleate, from the Sample solution
Acceptance criteria: Polyoxyl 35 Castor Oil exhibits the
10 oO 16 16 composition profile of fatty acids shown in Table 4.
4 5 9 or 08 26.2 or 17.2
3 10 19 or 28 45 Table 4
lf considerable discrimination of late eluting compounds is observed, the
hold time can be adjusted from 9 to 0 min. Thus the total time should be Percentage
17.2 min. Then the next step of the hold time should be 28 min. Component (%)
Carrier gas: Hydrogen Palmitic acid (C16:0) 54.0
Injection volume: 0.5 wL Stearic acid (C18:0) <5.0
Injection type: Split ratio, 60:1 Oleic acid (C18:1) 4.0-10.0
Liner: Single taper, low-pressure drop liner with deac- Linoleic acid (C18:2) $5.0
tivated wool cis-11-Eicosenoic acid (C20:1) S1.0
Run time: 45 min Ricinoleic acid 45.0-75.0
System suitability
Sample: Standard solution 1
[Note—See Table 3 for relative retention times.] IMPURITIES
e RESIDUE ON IGNITION (281): NMT 0.3%
Table 3
Relative Delete the following:
Retention
Component Time °e HEAVY METALS (231), Method li: NMT 10 {tg/gecortical 1-
Methyl palmitate (C16.0) 0.61 Jan-2018)
Methyl stearate (C18:0) 0.98 e ETHYLENE OXIDE AND DIOXANE (228), Method |
Acceptance criteria
Methyl oleate (C18:1) 1.00
Ethylene oxide: NMT 1 ug/g
Methyl linoleate (C18:2) 1.02 Dioxane: NMT 10 ug/g
Methyl cis-11-eicosenoate (C20:1) 1.70 e ETHYLENE GLYCOL, DIETHYLENE GLYCOL, AND TRIETHYLENE
Methyl ricinoleate 2.30 GLYCOL IN ETHOXYLATED SUBSTANCES (469)
Acceptance criteria
Suitability requirements Ethylene glycol: NMT 620 j1g/g
Resolution: NLT 1.5 between methyl stearate and Sum of diethylene glycol and ethylene glycol: NMT
methyl oleate peaks 2500 ug/g
Relative standard deviation: NMT 2.0% for the peak
area ratio of methyl ricinoleate to methyl linoeate SPECIFIC TESTS
Analysis e SPECIFIC GRAVITY (841): 1.05-1.06
Samples: Standard solution 1, Standard solution 2, and © ViscosiTty—CAPILLARY METHODS (911): 600-850 mPa-s
Sample solution at 25°, using a capillary viscometer
The peak of methyl cis-11-octadecenoate, which is an FATS AND FIXED OILS (401), Procedures, Acid Value: NMT
isomer of methyl oleate, can be resolved from the 2.0
methyl oleate peak with a resolution of about 1 and a FATS AND FIXED OILS (401), Procedures, Hydroxy! Value:
relative retention time of 1.01 with respect to methyl 65-80
oleate. FATS AND FIXED OILS (401), Procedures, lodine Value:
Calculate the relative response factor, F, for methyl 25-35
ricinoleate: FATS AND FIXED OlLs (401), Procedures, Saponification
Value: 60-75
F = (ts/ta) X (Ce Cs) e WATER DETERMINATION (921), Method |: NMT 3.0%
rs = peak area of methyl stearate from Standard ADDITIONAL REQUIREMENTS
solution 2 e PACKAGING AND STORAGE: Preserve in tight containers,
tr = peak area of methyl ricinoleate from Standard protected from light and moisture. Store at room tem-
solution 2 perature, and avoid exposure to excessive heat.
Ce = concentration of USP Methyl Ricinoleate RS in e USP REFERENCE STANDARDS (11)
Standard solution 2 (mg/mL) USP Methyl Linoleate RS
Cs = concentration of USP Methyl Stearate RS in USP Methyl Oleate RS
Standard solution 2 (mg/mL) USP Methyl Palmitate RS
Correct the peak area of methyl ricinoleate in the USP Methyl Ricinoleate RS
Sample solution by multiplying by F. USP Methyl Stearate RS
Calculate the percentage of each fatty acid component USP Polyoxyl 35 Castor Oil RS
in the portion of Polyoxyl 35 Castor Oil taken:
NF Monographs

Result = (ru/rr) x 100

Tu = peak area of each individual fatty acid methyl
ester, except for the uncorrected peak area
of methyl ricinoleate (or the corrected peak
area of methyl ricinoleate), from the Sample
solution
Polyoxyl 40 Hydrogenated Castor Oil
Polyethylene glycol 40 hydrogenated castor oil;
Polyoxyethylene 40 hydrogenated castor oil [61788-85-0].
DEFINITION
Polyoxyl 40 Hydrogenated Castor Oil contains mainly the
trihydroxystearate ester of ethoxylated glycerol, with
NF 36 Official Monographs / Polyoxy| 5517

smaller amounts of polyethylene glycol hydroxystearate Table 2 (Continued)

and the corresponding free glycols. It results from the re- Relative
action of glycerol tri-hydroxystearate with 40-45 mol of Retention
ethylene oxide. Component Time
IDENTIFICATION Methyl 12-ketostearate 0.85
e A. IDENTITY BY FATTY ACID COMPOSITION Methyl 12-hydroxystearate 1.00
Diluent: Chloroform
Standard solution 1: 0.4 mg/mL each of methyl palmi- Suitability requirements
tate, methyl arachidate, methyl 12-ketostearate, methyl Resolution: NLT 5 between any two adjacent peaks
stearate, and methyl 12-hydroxystearate, from USP Relative standard deviation: NMT 2.0% for the pak
Methyl Palmitate RS, methyl arachidate, methyl area ratio of methyl 12-hydroxystearate to methy!
12-ketostearate, USP Methyl Stearate RS, and USP 12-ketostearate
Methyl 12-Hydroxystearate RS, in Diluent Analysis
Standard solution 2: 4 mg/mL each of methyl stearate Samples: Standard solution 1, Standard solution 2, and
and methyl 12-hydroxystearate from USP Methyl Stea- Sample solution
rate RS and USP Methyl 12-Hydroxystearate RS in Calculate the relative response factor, F, for methyl
Diluent 12-hydroxystearate:
Sample solution: Transfer 140 mg of Polyoxyl 40ty
drogenated Castor Oil to a 10-mlL screw-cap test tube, F = (s/t) x (Ca!Cs)
add 3.0 mL of Diluent, and mix well. Add 0.5 mL of 0.5
M sodium methoxide in methanol,’ and mix with the rs = peak area of methyl stearate from Standard
sample. Allow the reaction to proceed at room temper- solution 2
ature for 2 h. After 2 h, add 5 mL of water, and mix. Tr = peak area of methyl 12-hydroxystearate from
pauiuge the test tube at 2000 x g for 10 min at 4° Standard solution 2
until a clear organic layer forms. Separate the organic Cr = concentration of USP Methyl
layer, and remove the aqueouslayer. Place an aliquot of 12-Hydroxystearate RS in Standard solution 2
the organic layer into an autosampler vial. (mg/mL)
Chromatographic system Cs = concentration of USP Methyl Stearate RS in
(See Chromatography (621), System Suitability.) Standard solution 2 (mg/mL)
Mode: GC Correct the peak area of methyl 12-hydroxystearate in
Detector: Flame ionization the Sample solution by multiplying by F.
Column: 0.32-mm x 30-m fused silica capillary; Calculate the percentage of each fatty acid component
bonded with a 0.25-1m layer of phase G16 ba portion of Polyoxyl 40 Hydrogenated Castor Oil
Temperatures taken:
Injection port: 240°
Detector: 250° Result = (ru/rz) x 100
Column: See Table 1. ty = peak area of each individual fatty acid methyl
ester except for the uncorrected peak area of
Table 1 methyl 12-hydroxystearate (or the corrected
Hold eak area of methyl 12-hydroxystearate),
Time at tom the Sample solution
Initial Tempera- Final Final rr = sum of all the peak areas, excluding the
Tempera- ture Tempera- | Tempera- Total solvent and methyl! 12-hydroxystearate peaks
ture Ramp ture ture Time and including the corrected peak area of
C) (¢/min) ©) (min) (min) methyl hydroxystearate, from the Sample
solution
80 0 80 1 1
Acceptance criteria: Polyoxyl 40 Hydrogenated Castor
80 30 140 0 3 Oil ee the composition profile of fatty acids shown
140 20 180 5 10 in Table 3.
180 2 250 10 55:
Table 3
Carrier gas: Hydrogen
Flow rate: 5.0 mL/min, constant flow mode Percentage
Injection volume: 1.0 pL Component (%)
Injection type: Split ratio, 120:1 or 60:1 Palmitic acid (C16:0) <4.0
a Single gooseneck liner with deactivated glass Stearic acid (C18:0) 15.0-25.0
woo! Arachidic acid (C20:0) $2.0
Run time: 55 min
12-Ketostearic acid
System suitability
Sample: Standard solution 1 (or 12-oxostearic acid) $5.0
[Note—For relative retention times, see Table 2.] 12-Hydroxystearic acid 50.0-70.0

e B. CONSTITUTING FATTY ACIDS

Table 2 Sample: 0.1g
sydesbouow 4IN

Relative Analysis: Dissolve the Sample in 10 mL of alcoholic po-

Retention tassium hydroxide TS, boil for 3 min, and evaporate to
Component Time dryness. Mix the residue with 5 mL of water.
Methyl palmitate (C16:0) 0.27
Acceptance criteria: The residue dissolves, yielding a
Methyl stearate (C18:0) 0.37
clear solution. Add a few drops of glacial acetic acid. A
white precipitate is formed.
Methyl arachidate (C20:0) 0.54 © C. INDICATION OF SATURATION
10.5 M sodium methoxide in methanol is available from Sigma-Aldrich (www. Analysis: Proceed as directed in Fats and Fixed Oils
sigmaaldrich.com), product #403067. Any other equivalent reagent can be (401), Procedures, lodine Value.
used as well.
 5518 Polyoxyl / Official Monographs
Acceptance criteria: NMT 2.0
IMPURITIES
e RESIDUE ON IGNITION (281): NMT 0.3%
e ETHYLENE OXIDE AND DIOXANE (228), Method |
Acceptance criteria
Ethylene oxide: NMT 1 ug/g
Dioxane: NMT 10 ug/g
Delete the following:
°e HEAVY METALS (231), Method II: NMT 10 pg/ge coral 1-
jan-2018)
e Limit OF NICKEL
[CauTion—When using closed high-pressure digestion ves-
sels and laboratory microwave equipment, the safety pre-
cautions andoperating instructions given by the manufac-
turer must be followed.]
{[Note—lf an alternative apparatus is used, adjustment of
the instrument parameters may be necessary.]
Nickel standard stock solution: Dilute nickel standard
solution TS two-fold with water. This solution contains
the equivalent of 5 g/mL of nickel.
Standard solutions: Transfer 25, 50, 75, and 100 uL of
Nickel standard stock solution to four identical 25-mL
volumetric flasks. To each flask add 0.5 mL of a
10-mg/mL solution of magnesium nitrate, 0.5 mL of a
100-mg/mL solution of monobasic ammonium phos-
phate, and 6.0 mL of nickel-free nitric acid. Dilute with
water to volume, and mix well. [NoTE—Content of
nickel in the nickel-free nitric acid is NMT 0.005 ppm.
The Standard solutions contain 0.005, 0.01, 0.015, and
0.02 g/mL of nickel, respectively.]
Sample solution: Transfer about 250 mg of Polyoxyl 40
Hydrogenated Castor Oil to a suitable high-pressure-re-
sistant digestion vessel (fluoropolymer or quartz glass),
and add 6.0 mL of nickel-free nitric acid and 2.0 mL of
30% hydrogen peroxide. Place the closed vessel in a
laboratory microwave oven, and digest using an appro-
priate program (e.g., 1000 W for 40 min). Allow the
digestion vessel to cool before opening. Add 2.0 mL of
30% hydrogen peroxide, and repeat the digestion step.
Allow the digestion vessel to cool before opening.
Quantitatively transfer to a 25-mL volumetric flask, add
0.5 mL of a 10-mg/mL solution of magnesium nitrate
and 0.5 mL of a 100-mg/mL solution of monobasic am-
monium phosphate. Dilute with water to volume, and
mix well.
Blank solution: Place 6.0 mL of nickel-free nitric acid
and 2.0 mL of 30% hydrogen peroxide in a suitable
high-pressure-resistant digestion vessel. Prepare as di-
rected in the Sample solution, beginning with “Place the
closed vessel in a laboratory microwave oven, and di-
gestusing an appropriate program (e.g., 1000 W for
40 min).”
Zero solution: In a 50-mL volumetric flask, introduce
1.0 mL of a 10-mg/mL solution of magnesium nitrate,
1.0 mL of a 100-mg/mL solution of monobasic ammo-
nium phosphate, and 12.0 mL of nickel-free nitric acid.
Dilute with water to volume, and mix well.
Instrumental conditions
(See Atomic Absorption Spectroscopy (852).)
Mode: Atomic absorption, equipped with a graphite
furnace, a background compensation system, and a
NF Monographs
coated tube resistant to pyrolysis
Analytical wavelength: 232.0 nm
Lamp: Nickel hollow-cathode
Temperature: Maintain the drying temperature of the
furnace at 120° for 35s after a 5-s ramp; maintain the
ashing temperature at 1100° for 10 s after a 30-s
ramp; maintain the cooling temperature at 800° for 5
s after a 5-s decrease; and maintain the atomization
NF 36
temperature at 2600° for 7 s. [NoTE—The temperature
program may be modified to obtain optimum furnace
temperatures.]
Analysis
Samples: Standard solutions, Sample solution, and Blank
solution
Concomitantly determine the absorbances of the Sam-
ples using the Instrumental conditions described above.
Use the Zero solution to set the instrument to zero.
Plot the absorbances of the Standard solutions versus
the concentration, in g/mL, of nickel, and draw the
straight line best fitting the plotted points. From the
graph so obtained, determine the concentration, C;, in
ug/mL, of nickel in the Sample solution, and determine
the concentration, Cs, in g/mL, of nickel in the Blank
solution. If necessary, dilute with the Zero solution to
obtain a reading within the calibrated absorbance
range.
Calculate the quantity, in jig, of nickel in each g of
Polyoxyl 40 Hydrogenated Castor Oil taken:
Result = Vx [(Cr — Cs)/W]
V = volume of the Sample solution and the Blank
solution, 25 mL
G = concentration of nickel in the Sample solution
(ug/ml) oR arts ’
Gs = concentration of nickel in the Blank solution
(g/mL)
w = weight of Polyoxyl 40 Hydrogenated Castor
Oil taken to prepare the Sample solution (g)
Acceptance criteria: NMT 20 1g/g
SPECIFIC TESTS
© CONGEALING TEMPERATURE (651): 16°-26°
e FATS AND FIXED OILS (401), Procedures, Acid Value: NMT
2.0
FATS AND FIXED OILS (401), Procedures, Hydroxy! Value:
57-80
FATS AND FIXED OILS (401), Procedures, Saponification
Value: 45-69
WATER DETERMINATION (921), Method I: NMT 3.0%
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in tight containers,
protected from light and moisture. Store at room tem-
perature, and avoid exposure to excessive heat.
e USP REFERENCE STANDARDS (11)
USP Methyl 12-Hydroxystearate RS
USP Methyl Palmitate RS
USP Methyl Stearate RS
Polyoxyl Lauryl Ether
CH3(CH2)11(OCH2CH2),0H, n = 3-23
Polyethylene glycol monolauryl ether [9002-92-0].
DEFINITION
Polyoxyl Lauryl Ether is a mixture of the monolauryl ethers
of mixed polyethylene glycols, the average polymer
length being equivalent to NLT 3 and NMT 23 oxy-
ethylene units (nominal value). It contains various
amounts of free lauryl alcohol, and it may contain some
free polyethylene glycols.
IDENTIFICATION
e A. INFRARED ABSORPTION (197F)
Sample: Usea thin film of melted Polyoxyl Lauryl Ether
if the material is a solid.
NF 36 Official Monographs / Polyoxyl 5519

Acceptance criteria: Meets the requirements e USP REFERENCE STANDARDS (11)

e B. PROCEDURE USP Polyoxyl! 4 Lauryl Ether RS
Sample: 0.1 g USP Polyoxyl 9 Lauryl Ether RS
Analysis: Dissolve or disperse the Sample in 5 mL of al- USP Polyoxyl 23 Lauryl Ether RS
cohol, and add 10 mL of diluted hydrochloric acid,
5 mL of barium chloride TS, and 10 mL of phosphomo-
lybdic acid solution (1 in 10).
Acceptance criteria: A precipitate is formed.
e C. It meets the requirements of the test for Fats and Fixed Polyoxyl Oleate
Oil, Hydroxyl Values (401).
Polyethylene glycol monooleate [9004-96-0].
IMPURITIES
Organic Impurities DEFINITION
¢ PROCEDURE: LIMIT OF FREE ETHYLENE OXIDE AND DIOXANE Polyoxyl Oleate is a mixture of the monoesters and diesters
Analysis: Proceed as directed in Ethylene Oxide and Di- of oleic acid and mixed polyethylene glycols. It may be
oxane, Method | (228). obtained by ethoxylation of oleic acid or by esterification
Acceptance criteria of polyethylene glycols with oleic acid of animal or vege-
Ethylene oxide: NMT 1 itg/g (ppm) table origin. It may contain free polyethylene glycol. The
Dioxane: NMT 10 ug/g (ppm) average polymer length is equivalent to either 5-6 or 10
oxyethylene units (nominal values). A suitable antioxidant
SPECIFIC TESTS may be added.
e ALKALINITY
Sample: 2.0g of Polyoxyl Lauryl Ether IDENTIFICATION
Analysis: Dissolve the Sample in a hot mixture of 10 mL e A. INFRARED ABSORPTION (197F)
of alcohol and 10 mL of water. Add 0.05 mL of Sample: Undried specimen
bromothymol blue TS, and titrate with 0.1 N hydro- Acceptance criteria: Meets the requirements
chloric acid to a yellow endpoint.
Acceptance criteria: NMT 0.5 mL of 0.1 N hydrochlo- IMPURITIES
ric acid is required. e LIMIT OF FREE ETHYLENE OXIDE AND DIOXANE
¢ APPEARANCE OF SOLUTION: 5.0g of Polyoxy! Lauryl Ether Analysis: Proceed as directed in Ethylene Oxide and Di-
in 50.0 mL of alcohol. The solution is not more intensely oxane (228), Method 1.
colored than a solution prepared immediately before use Acceptance criteria
by mixing 12.0 mL of ferric chloride CS, 5.0 mL of cobal- Ethylene oxide: NMT 1 ug/g
tous chloride CS, and 2.0 mL of cupric sulfate CS with Dioxane: NMT 10 ug/g
dilute hydrochloric acid (10 g/L) to make 50.0 mL, and
SPECIFIC TESTS
diluting 12.5 mL of this solution with dilute hydrochloric e FATS AND FIXED OILS, Acid Value (401)
acid (10 g/L) to make 100.0 mL. Make the comparison
by viewing the substance and the solution downward in Sample: 10.0g
matched color-comparison tubes against a white surface Acceptance criteria: NMT 1.0
(see Color and Achromicity (631)). FATS AND FIXED OILS, Peroxide Value (401): NMT 12.0
eeeo

e ARTICLES OF BOTANICAL ORIGIN, Total Ash (561): NMT FATS AND FIXED OILS, Hydroxyl Value (401): See Table 7.
0.2%, determined on 2.0 g FATS AND FIXED OILS, /odine Value (401): See Table 1.
e FATS AND FIXED OILS, Acid Value (401): NMT 1.0, deter- FATS AND FIXED OILS, Saponification Value (401): See Ta-
mined on 5.0g ble 1.
e FATS AND FIXED OiLs, Hydroxy! Value (401): Within the
ranges specified in the accompanying table Table 1
5-6 Ethylene Oxide 10 Ethylene Oxide
Oxyethylene Units Units
Units/Molecule Hydroxyl value 50-70 65-90
(Nominal Value) Hydroxyl Value lodine value 50-60 27-34
3 165-185 Saponification
4 145-165 value 105-120 68-85
Si 130-140
9 90-100 © FATS AND FIXED OILS, Fatty Acid Composition (401): Poly-
10 85-95
oxy! Oleate exhibits the composition profile of fatty acids
shown in Table 2.
12 73-83
15 64-74
Table 2
20-23 40-60
Number of
e FATS AND FIXED OILS, /odine Value (401): NMT 2.0 Carbon-Chain Double Percentage
© FATS AND FIXED OILS, Saponification Value (401): NMT Length Bonds (%)
3.0, determined on 10.0g 14 0 <5.0
e WATER DETERMINATION, Method | (921): NMT 3.0% 16 0 <16.0 5
ADDITIONAL REQUIREMENTS 18 0 $6.0 n
© PACKAGING AND STORAGE: Preserve in tight containers, 16 1 <8.0 <
and store in a cool, dry place. 18 1 65.0-88.0 =
e LABELING: Label it to indicate the average nominal num- 18 2 <18.0 ray
ber of oxyethylene units. 18 3 <4.0 @
218 = $4.0 aS
a
 5520 Polyoxyl / Official Monographs NF 36

© ALKALINITY for 15 min. If separation is incomplete, carefully insert

Sample solution: 100 mg/mL of Polyoxyl Oleate in the separator into the well of a steam bath for short
alcohol time intervals. Repeat this technique as many times as
Analysis: To 2 mL of the Sample solution add 0.05 mL of necessary to ensure the complete separation of the two
phenol red TS. phases. Cool, and drain the lower, aqueous phase into
Acceptance criteria: The solution is not red. a second 500-mL separator. Extract the upper layer
e ARTICLES OF BOTANICAL ORIGIN, Total Ash (561) with a second 50-mL portion of sodium chloride solu-
Sample: 1.0g tion (29 in 100). Repeat the separation as before, in-
Acceptance criteria: NMT 0.3% cluding the steam bath technique, to facilitate complete
e WATER DETERMINATION, Method | (921): NMT 2.0% separation.
To the combined aqueous layers add 50 mL of ethyl
ADDITIONAL REQUIREMENTS acetate, shake vigorously for 2 min, and allow to sepa-
e PACKAGING AND STORAGE: Preserve in tight containers, rate as before. Drain the lower, aqueous phase into a
and store in a cool, dry place. Protect from moisture. third 500-mL separator, and extract it with two S0-mL
© LABELING: Label it to indicate the number of ethylene ee of chloroform, shaking for 2 min each time.
oxide units per molecule (nominal value), and the name ‘epeat the steam bath technique to ensure complete
and concentration of any added antioxidant. separation.
e USP REFERENCE STANDARDS (11) Evaporate the combined chloroform extracts in a
USP Polyoxyl Oleate RS 150-mL beaker on a steam bath, with the aid of a
stream of nitrogen, to apparent dryness.
Redissolve in about 15 mL of chloroform, and filter, col-
eens filtrate in a weighed 150-mL beaker. Re-
cord the weight of the empty 150-mL beaker, W;, in
Polyoxyl Stearate g. Rinse the funnel with several small portions of chlo-
roform, and evaporate the combined filtrate and rins-
R-CO-(OCH2CH2)n-OH ings, as described above, to remove chloroform or
ethyl acetate.
R-CO-(OCH2CH2)n-OOC-R Dry in vacuum at 60° for 1 h. Cool in a desiccator, and
R = CHs(CHa)is or CH3(CH2)14 weigh. Record the weight, W2, in 9.
Calculate the percentage of free polyethylene glycols in
n = 6-100 Polyoxyl 40 Stearate taken:
Polyethylene glycol stearate;
Polyethylene glycol monostearate; Result = [(W2 — W,)/W] x 100
Poly(oxy-1,2-ethanediyl), a-hydro-w-hydroxyoctadecanoate
[9004-99-3]. Ww = weight of Polyoxyl 40 Stearate (g)
Acceptance criteria: 17%-27% of free polyethylene
DEFINITION glycols for Polyoxyl 40 Stearate only
Polyoxyl Stearate is a mixture of monoesters and diesters of
mainly stearic (octadecanoic) acid and/or palmitic (hex- IMPURITIES
adecanoic) acid and povetrylene glycols. The fatty acids
may be of vegetable, anima , or synthetic origin. Polyoxyl
Stearate Type | or Type II differs in its content of stearic Delete the following:
acid. It may contain free polyethylene glycols. The aver-
age polymer lenge is equivalent to 6-100 ethylene oxide °e HEAVY Metals, Method II (231): NMT 10 ppme coma.
units per molecu ie (nominal value). Jan-2078)
e ARTICLES OF BOTANICAL ORIGIN, Total Ash (561): NMT
IDENTIFICATION 0.3%, determined on 1.0g
e A. INFRARED ABSORPTION (197A) e LIMIT OF ETHYLENE OXIDE AND DIOXANE
Sample: Use an undried specimen. Analysis: Proceed as directed in Ethylene Oxide and Di-
e B. It meets the requirements of the test for Content of oxane (228), Method II.
Stearic Acid and Palmitic Acid. Acceptance criteria
Ethylene oxide: 1 ppm
ASSAY Dioxane: 380 ppm
© CONTENT OF STEARIC ACID AND PALMITIC ACID
Polyoxyl Stearate exhibits the composition profiles of SPECIFIC TESTS
fatty acids shown in Table 1 below, as determined in ¢ ALKALINITY
Fats and Fixed Oils (401), Fatty Acid Composition. Phenol red solution: Dissolve 100 mg of phenolsulfon-
phthalein in a mixture of 2.82 mL of 0.1 M sodium hy-
Table 1 droxide and 20 mL of alcohol, and dilute with water to
100 mL.
Content of Stearic Acid Sample solution: 2.0 g of Polyoxyl Stearate
and Palmitic Acid Analysis: Dissolve the Sample in alcohol and dilute with
Stearic Acid: 40.0%-60.0%; alcohol to 20 mL. To 2 mL of this solution add 0.05 mL
Polyoxyl Stearate sum of Palmitic and Stearic acids: NLT of Phenol red solution.
Type | 90.0% Acceptance criteria: The solution does not turn red.
NMT 6.0
NF Monographs

Stearic Acid: 90.0%-99.0%;
Polyoxyl Stearate sum of Palmitic and Stearic acids: NLT
Type Il 96.0%
e CONTENT OF FREE POLYETHYLENE GLYCOLS
[Note—This test is for Polyoxyl 40 Stearate only.]
Sample: 6g of Polyoxy!l 40 Stearate
Analysis: Transfer the Sample to a 500-mL separator
containing 50 mL of ethyl acetate. Dissolve completely,
then add 50 mL of sodium chloride solution (29 in
100), shake vigorously for 2 min, and allow to separate
FATS AND FIXED OILS, Acid Value (401):
FATS AND FIXED OlLs, Hydroxyl Value (401): Within the
ranges specified in Table 2
FATS AND FIXED OILS, /odine Value (401): NMT 3.0
FATS AND FIXED OILS, Peroxide Value (401): NMT 10.0
FATS AND FIXED OILS, Saponification Value (401): Within
the ranges specified in Table 2
MELTING RANGE OR TEMPERATURE (741)
Sample: 10g
Analysis: Melt the Sample at 80°-90°. Introduce a suffi-
cient amount of the Sample into the tube by capillary
NF 36 Official Monographs / Polyoxyl 5521

action to form a column of the prescribed height in the chloride TS, and 10 mL of phosphomolybdic acid solu-
tube. Allow to stand at 0° for 2 h. tion (1 in 10).
+e criteria; Within the ranges specified in Acceptance criteria: A precipitate is formed.
Table 2 e C. It meets the requirements in the test for Fats and Fixed
Oils, Hydroxyl Value (401).
Table? IMPURITIES
Ethylene Organic Impurities
Oxide Units/ Melting e PROCEDURE: LIMIT OF FREE ETHYLENE OXIDE AND DIOXANE
Molecule Range or Analysis: Proceed as directed in Ethylene Oxide and Di-
(Nominal Temperature | Hydroxyl Saponification oxane (228), Method I.
Value) © Value Value Acceptance criteria
6 26-37 80-110 90-115 Ethylene oxide: NMT 1 ug/g (ppm)
8 26.35 80-105 88-100 Dioxane: NMT 10ug/g (ppm)
32 46-50 20-40 30-45 SPECIFIC TESTS
Measure e FATS AND FIXED OILS, Acid Value (401): NMT 1.0, deter-
Congealing mined on 5.0g
40 Temperature 25-40 25-35 © FATS AND FIXED OILS, Hydroxy! Value (401): Within the
75 53-59 15-35 8-25 ranges specified in the table below
100 48-60 15-30 5-20
Oxyethylene Units/
¢ CONGEALING TEMPERATURE (651): 37°-47° for Polyoxyl 40 Molecule
Stearate only (Nominal Value) Hydroxyl Value
e WATER DETERMINATION, Method | (921): NMT 3.0% 2 150-180
ADDITIONAL REQUIREMENTS 10 75-90
¢ PACKAGING AND STORAGE: Preserve in tight containers, 20 40-60,
and store at room temperature. Protect from light and
moisture. e FATS AND FIXED OILS, Jodine Value (401): NMT 2.0
e LABELING: Label it to indicate the number of ethylene e FATS AND FIXED OILS, Saponification Value (401): NMT
oxide units/molecule (nominal value), and the type of 3.0, determined on 10.0 g
Polyoxyl Stearate. Label it to indicate whether the fatty © ALKALINITY
acids are derived from vegetable, animal, or synthetic Sample: 2.0g of Polyoxyl Stearyl Ether
sources. Analysis: Dissolve the Sample to a hot mixture of 10 mL
e USP REFERENCE STANDARDS (11) of alcohol and 10 mL of water. Add 0.05 mL of
USP Polyoxyl 6 Stearate RS bromothymol blue TS. Titrate with 0.1 N hydrochloric
USP Polyoxyl 8 Stearate RS acid to a yellow endpoint.
USP Polyoxyl 32 Stearate RS Acceptance criteria: NMT 0.5 mL of 0.1 N hydrochlo-
USP Polyoxyl 40 Stearate RS ric acid is required.
USP Polyoxyl 75 Stearate RS e@ WATER DETERMINATION, Method | (921): NMT 3.0%
USP Polyoxyl 100 Stearate RS e APPEARANCE OF SOLUTION: 5.0g of Polyoxy! Stearyl Ether
in 50.0 mL of alcohol. The solution is not more intensely
colored than a solution prepared immediately before use
by mixing 12.0 mL of ferric chloride CS, 5.0 mL of cobal-
tous chloride CS, and 2.0 mL of cupric sulfate CS with
Polyoxyl Stearyl Ether dilute hydrochloric acid (10 g/L) to make 50.0 mL, and
diluting 12.5 mL of this solution with dilute hydrochloric
Polyethylene glycol monosteary| ether acid (10 g/L) to make 100.0 mL. Make the comparison
by viewing the substance and the solution downward in
CH3(CH2z):7(OCH2CH2),0H, n = 2-20 [9005-00-9]. matched color-comparison tubes against a white surface
DEFINITION (see Color and Achromicity (631)).
Polyoxyl Stearyl Ether is a mixture of the monostearyl ethers ADDITIONAL REQUIREMENTS
of mixed polyethylene glycols, the average polymer e PACKAGING AND STORAGE: Preserve in tight containers,
length being equivalent to NLT 2 and NMT 20 oxy- and store in a cool, dry place.
ethylene units (nominal value). It may contain various e LABELING: Label it to indicate the average nominal num-
amounts of free stearyl alcohol and some free polyethyl- ber of oxyethylene units.
ene glycol. e USP REFERENCE STANDARDS (11)
IDENTIFICATION USP Polyoxyl 2 Stearyl Ether RS
e A. INFRARED ABSORPTION (197F): Use a thin film of USP Polyoxyl 10 Stearyl Ether RS
melted Polyoxyl Stearyl Ether. USP Polyoxyl 20 Stearyl Ether RS
e B, PROCEDURE
Sample: 0.1g
Analysis: Dissolve or disperse the Sample in alcohol.
sydeibouo= iN

Add 10 mL of diluted hydrochloric acid, 5 mL of barium

USP 41
Analysis
Samples: Standard solution and Sample solution
Measure the responses for the vitamin D peaks.
Calculate the percentage of the labeled amount of cho-
lecalciferol (C27H44O) or ergocalciferol (C2sH44O) in the
portion of Tablets taken:
Result = (ru/rs) x (Cs/Cu) x F x 100
tu = peak height for cholecalciferol or ergocalciferol
from the Sample solution
rs = peak height for cholecalciferol or ergocalciferol
from the Standard solution
Cs = concentration of USP Ergocalciferol RS or USP
Cholecalciferol RS in the Standard solution
(ug/mL) ‘ Copper standard solution: Dissolve 1.00 g of copper
Cu = nominal concentration of ergocalciferol or
cholecalciferol in the Sample solution (g/mL)
F = correction factor to account for the average
amount of previtamin D present in the
Sample solution, 1.09
Acceptance criteria: 90.0%-165.0% of the labeled
amount of cholecalciferol (C27H44O) or ergocalciferol
(C2gH440)
e CALCIUM, Method 1
[NoTtE—A commercially available atomic absorption stan-
dard solution for calcium may be used where prepara-
tion of a Calcium standard stock solution is described in
the following section. Concentrations of the Standard
solutions and the Sample stock solution may be modified
to fit the linear or working range of the instrument.]
Lanthanum chloride solution: 267 mg/mL of lantha-
ae chloride heptahydrate in 0.125 N hydrochloric
aci
Calcium standard stock solution: 400 g/mL of cal-
cium. Dissolve 1.001 g of calcium carbonate, previous!
dried at 300° for 3 h and cooled in a desiccator for 2
and dissolve in 25 mL of 1 N hydrochloric acid. Boil to
expel carbon dioxide, and dilute with water to
1000 mL.
Standard stock solution: 100 g/mL of calcium from
Calcium standard stock solution diluted with 0.125 N hy-
drochloric acid
Standard solutions: Into separate 100-mL volumetric
flasks pipet 1.0, 1.5, 2.0, 2.5, and 3.0 mL of the Stan-
dard stock solution. To each flask add 1.0 mL of Lantha-
num chloride solution, and dilute with water to volume
to obtain Standard solutions having concentrations of
1.0, 1.5, 2.0, 2.5, and 3.0 g/mL of calcium.
Sample stock solution: Weigh and finely powder NLT
20 Tablets. Mix a portion of the powder equivalent to a
nominal amount of 500 mg of calcium with 25 mL of
concentrated hydrochloric acid, and heat for 30 min on
fe bath. Cool, dilute with water to 1000 mL, and
ilter.
Sample solution: Quantitatively dilute a volume of the
Sample stock solution with 0.125 N hydrochloric acid to
obtain a nominal concentration of 100 g/mL of cal-
cium. Transfer 2.0 mL of this solution to a 100-mL volu-
metric flask, add 1.0 mL of Lanthanum chloride solution,
and dilute with water to volume.
Spectrometric conditions
See Atomic Absorption Spectroscopy (852).)
Mode: Atomic absorption spectrophotometry
Lamp: Calcium hollow-cathode
Flame: Nitrous oxide-acetylene
Analytical wavelength: Calcium emission line, 422.7
nm
Blank: 0.125 N hydrochloric acid containing 1 mL of
Lanthanum chloride solution per 100 mL
Analysis
Samples: Standard solutions and Sample solution
Determine the absorbances of the solutions against the
Blank. Plot the absorbances of the Standard solutions
versus concentration, in g/mL, of calcium, and draw
Dietary Supplements / Calcium 4503
the straight line best fitting the five plotted points.
From the arent determine the concentration, in
ug/mL, of calcium in the Sample solution.
Calculate the percentage of the labeled amount of cal-
cium (Ca) in the portion of Tablets taken:
Result = (C/Cy) x 100
Cc = measured concentration of calcium in the
Sample solution (g/mL)
Cu = nominal concentration of calcium in the
Sample solution (\1g/mL)
Acceptance criteria: 90.0%-125.0% of the labeled
amount of calcium (Ca)
e CoppER, Method 1
foil in a minimum volume of a 50% (v/v) solution of
nitric acid solution, and dilute with a 1% (v/v) solution
of nitric acid to 1000 mL. This solution contains
1000 g/mL of copper.
Standard stock solution: 100 g/mL of copper from
Coppel standard solution diluted with 0.125 N hydro-
chloric acid
Standard solutions: To separate 200-mL volumetric
flasks transfer 1.0, 2.0, 4.0, 6.0, and 8.0 mL of the Stan-
dard stock solution. Dilute with water to volume to ob-
tain concentrations of 0.5, 1.0, 2.0, 3.0, and 4.0 pg/mL
of copper.
Sample solution: Weigh and finely powder NLT 20
Tablets. Transfer the equivalent of 5 mg of copper from
poniseced Tablets to a porcelain crucible. Heat for 6-12
in a muffle furnace maintained at 550°, and cool.
Add 15 mL of hydrochloric acid, and boil gently on a
hot plate or a steam bath for 30 min, intermittently
tinsing the inner surface of the crucible with 6 N hydro-
chloric acid. Cool, and quantitatively transfer the con-
h, tents of the crucible to a 100-mL volumetric flask, rins-
ing the crucible with portions of 6 N hydrochloric acid.
Dilute the contents of the flask with water to volume,
and filter, discarding the first 5 mL of the filtrate. Dilute
the filtrate quantitatively with 0.125 N hydrochloric
acid to obtain a concentration of 2 ug/mL of copper.
Spectrometric conditions
(See Atomic Absorption Spectroscopy (852).)
Mode: Atomic absorption spectrophotometry
Lamp: Copper hollow-cathode
Flame: Air—acetylene
Analytical wavelength: Copper emission line, 324.7
nm
Blank: 0.125 N hydrochloric acid
Analysis
Samples: Standard solutions and Sample solution
sydeibouow Sa
Determine the absorbances of the solutions against the
Blank. Plot the absorbances of the Standard solutions
versus concentration, in g/mL, of copper, and draw
the straight line best fitting the five plotted points.
From the graph, determine the concentration, C, in
ug/mL, of copper in the Sample solution.
Calculate the pce ee of the labeled amount of cop-
per (Cu) in the portion of Tablets taken:
Result = (C/Cy) x 100
Cc = measured concentration of copper in the
Sample solution (ug/mL)
Cu = nominal concentration of copper in the
Sample solution (ug/mL)
Acceptance criteria: 90.0%-125.0% of the labeled
amount of copper (Cu)
e Macnesium, Method 17
Lanthanum chloride solution: 267 mg/mL of lantha-
nm chloride heptahydrate in 0.125 N hydrochloric
aci
Magnesium standard stock solution: Transfer 1.00 g
of magnesium to a 1000-mL volumetric flask, dissolve
 5522 Polysorbate / Official Monographs NF 36

bate 20 must be subjected to further processing durin:

Polysorbate 20 the preparation of injectable dosage forms, the level o
bacterial endotoxins is such that the requirement in the

ie relevant dosage form monograph(s) in which Polysor-

dratky
bate 20 is used can be met.
¢ FATS AND FIXED OILS, Acid Value (401)
Sample: 10.0g
pty tw 42220
4 Analysis: Transfer the Sample to a wide-mouth, 250-mL
conical flask, and add 50 mL of neutralized alcohol.
Heat on a steam bath nearly to boiling, occasionally
R= HO— shaking thoroughly while heating. Invert a beaker over
° the mouth of the flask, cool under running water, and
add 5 drops of phenolphthalein TS. Titrate with 0.1 N
ween en , sodium hydroxide VS. Calculate the acid value as di-
rected in the chapter.
The ratio of the OH group to the (C::H23COO) group is Acceptance criteria: NMT 2.0
mainly 3:1. e FATS AND FIXED OILS, oe! Value (401): 96-108
Polyethylene glycol 20 sorbitan ether monolaurate; e FATS AND FIXED OILS, Peroxide Value (401)
Polyoxyethylene 20 sorbitan monododecanoate; Sample: 10.0g
Polyoxyethylene 20 sorbitan monolaurate [9005-64-5]. Saturated potassium iodide solution: Prepare a satu-
rated solution of potassium iodide in carbon dioxide-
DEFINITION free water. Make sure the solution remains saturated as
Polysorbate 20 is a laurate ester of sorbitol and its anhy- indicated by the presence of undissolved crystals.
rides, copolymerized with approximately 20 moles of Analysis: Introduce the Sample into a 100-mL beaker,
ethylene cee for each mole of sorbitol and sorbitol an- and dissolve with 20 mL of glacial acetic acid. Add 1 mL
hydrides. The fatty acids may be of vegetable, animal, or of Saturated potassium iodide solution, mix, and allow to
synthetic origin. stand for 1 min. Add 50 mL of carbon dioxide-free
water and a magnetic stirring bar. Titrate with 0.01 M
IDENTIFICATION sodium thiosulfate VS, determining the endpoint poten-
© A. INFRARED ABSORPTION (197F) Homeuically (see Titrimetry (541)). Perform a blank titra-
e B. It meets the requirements in the Assay for Composition tion. Calculate the peroxide value as directed in the
of Fatty Acids. chapter.
Acceptance criteria: NMT 10.0
ASSAY For Polysorbate 20 intended for use in the manufac-
© COMPOSITION OF FATTY AcIDs: Polysorbate 20 exhibits the ture of injectable dosage forms: NMT 5.0
composition profiles of fatty acids shown in Table 1, as e FATS AND FIXED OILS, Saponification Value (401): 40-50
determined in Fats and Fixed Oils (401), Fatty Acid e@ WATER DETERMINATION, Method | (921): NMT 3.0%
Composition.
ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Preserve in tight containers,
Table 7 protected from light and moisture. Store at room
Carbon-Chain Number of Percentage temperature.
Length Double Bonds (%) ¢ LABELING: Label it to indicate whether the fatty acids are
6 0 <1.0 derived from animal, vegetable, or synthetic sources.
8 0 <10.0 Where Polysorbate 20 is intended for use in the manufac-
10 0 <10.0 ture of injectable dosage forms, it is so labeled.
12 0 40.0-60.0 ¢ USP REFERENCE STANDARDS (11)
rr USP Polysorbate 20 RS
14 0 14.0-25.0
16 0 7.0-15.0
18 0 511.0
18 1 <11.0
18 2 3.0 Polysorbate 40
IMPURITIES
e RESIDUE ON IGNITION (281): NMT 0.25%
xtytwt2=20
Delete the following:

°e HEAVY METALS, Method II (231): NMT 10 ppme(ortcial1-

Jan-2018)
e Limit ‘OF ETHYLENE OXIDE AND DIOXANE, Method // (228)
Acceptance criteria
NF Monographs

Ethylene oxide: NMT 1 ppm

Dioxane: NMT 10 ppm
SPECIFIC TESTS
e BACTERIAL ENDOTOXINS TEST (85)
For Polysorbate 20 intended for use in the manufac-
ture of injectable dosage forms: The level of bacterial
endotoxins is such that the requirement in the relevant
dosage form monograph(s) in which Polysorbate 20 is
used can be met. Where the label states that Polysor-
The ratio of OH group to C;sH31COO group is mainly 3:1.
Polyethylene glycol 20 sorbitan ether monopalmitate;
Polyoxyethylene 20 sorbitan monohexadecanoate;
Polyoxyethylene 20 sorbitan monopalmitate [9005-66-7].
DEFINITION
Polysorbate 40 is a palmitate ester of sorbitol and its anhy-
tides copolymerized with about 20 moles of ethylene ox-
ide for each mole of sorbitol and sorbitol anhydrides. The
NF 36 Official Monographs / Polysorbate 5523

fatty acids may be of vegetable, animal, or synthetic e FATS AND FIXED OILS, Saponification Value (401): 41-52
origin. e WATER DETERMINATION, Method | (921): NMT 3.0%
IDENTIFICATION ADDITIONAL REQUIREMENTS
¢ A. INFRARED ABSORPTION (197F) e PACKAGING AND STORAGE: Preserve in tight containers,
e B. It meets the requirements in the Assay for Composition protected from light and moisture. Store at room tem-
of Fatty Acids. perature.
e LABELING: Label it to indicate whether the fatty acids are
ASSAY derived from animal, vegetable, or synthetic sources.
e COMPOSITION OF FATTY ACIDS Where Polysorbate 40 is intended for use in the manufac-
Polysorbate 40 exhibits the composition profiles of fatty ture of injectable dosage forms, it is so labeled.
acids shown in Table 1, as determined in Fats and Fixed e USP REFERENCE STANDARDS (11)
Oils (401), Fatty Acid Composition. USP Polysorbate 40 RS

Table 1
Carbon-Chain Number of Percentage
Length Double Bonds (%)
16 0 292.0
Polysorbate 60

dh
tay Ay
IMPURITIES
e RESIDUE ON IGNITION (281): NMT 0.25%

4, PAL
xeyewaz=20
Delete the following:

°e HEAVY METALS, Method II (231): NMT 10 ppme cotta 1-

Jan-2018)
e ETHYLENE OXIDE AND DIOXANE, Method |! (228) ii vad o
Acceptance criteria
Ethylene oxide: NMT 1 ppm pee BA Pe
ji 2
Dioxane: NMT 10 ppm
SPECIFIC TESTS The ratio of OH group to the sum of CisH31COO and
e BACTERIAL ENDOTOXINS TEST (85) Ci7H3sCOO groups is mainly 3:1.
For Polysorbate 40 intended for use in the manufac- Polyethylene glycol 20 sorbitan ether monostearate;
ture of injectable dosage forms: The level of bacterial Polyoxyethylene 20 sorbitan monooctadecanoate;
endotoxins is such that the requirement in the relevant Polyoxyethylene 20 sorbitan monostearate [9005-67-8].
dosage form monograph(s) in which Polysorbate 40 is
used can be met. Where the label states that Polysor- DEFINITION
bate 40 must be subjected to further processing durin Polysorbate 60 is a mixture of stearate and palmitate esters
the preparation of injectable dosage forms, the level o of sorbitol and its anhydrides copolymerized with about
bacterial endotoxins is such that the requirement in the 20 moles of ethylene oxide for each mole of sorbitol and
relevant dosage form monograph(s) in which Polysor- sorbitol anhydrides. The fatty acids may be of vegetable,
bate 40 is used can be met. animal, or synthetic origin.
e FATS AND FIXED OILS, Acid Value (401)
Sample: 10.0g IDENTIFICATION
Analysis: Transfer the Sample to a wide-mouth, 250-mL © A. INFRARED ABSORPTION (197F)
conical flask, and add 50 mL of neutralized alcohol. e B. It meets the requirements in the Assay for Composition
Heat on a steam bath nearly to boiling, shaking thor- of Fatty Acids.
oughly occasionally while heating. Invert a beaker over
the mouth of the flask, cool under running water, and ASSAY
add 5 drops of phenolphthalein TS. Titrate with 0.1 N ¢ COMPOSITION OF FATTY ACIDS
sodium hydroxide VS. Calculate the acid value as di- Polysorbate 60 exhibits the composition profiles of fatty
rected in the chapter. acids shown in Table 1, as determined in Fats and Fixed
Acceptance criteria: NMT 2.0 Oils (401), Fatty Acid Composition.
e FATS AND FIXED O1Ls, Hydroxyl Value (401): 89-105
e FATS AND FIXED OILS, Peroxide Value (401) Table 1
Sample: 10.0g Carbon-Chain Number of Percentage
Saturated potassium iodide solution: Prepare a satu- Length Double Bonds (%)
rated solution of potassium iodide in carbon dioxide-
18 0 40.0-60.0
free water. Make sure the solution remains saturated as
indicated by the presence of undissolved crystals. Sum of stearic acid (C18:0)
Analysis: Introduce the Sample into a 100-mL beaker, and palmitic acid (C16:0) 290.0
and dissolve with 20 mL of glacial acetic acid. Add 1 mL
sydeiBbouow 4N

of Saturated potassium iodide solution, mix, and allow to IMPURITIES

stand for 1 min. Add 50 mL of carbon dioxide-free © RESIDUE ON IGNITION (281): NMT 0.25%
water and a magnetic stirring bar. Titrate with 0.01 M
sodium thiosulfate VS, determining the endpoint poten-
tiometrically (see Titrimetry (541)). Perform a blan Delete the following:
titration.
Calculate the peroxide value as directed in the chapter. °e HEAVY METALS, Method // (231): NMT 10 PPMe ‘official 1-
Acceptance criteria: NMT 10.0 Jan-2018)
For Polysorbate 40 intended for use in the manufac-
ture of injectable dosage forms: NMT 5.0
 5524 Polysorbate / Official Monographs NF 36

e ETHYLENE OXIDE AND DIOXANE, Method // (228) Saturated sodium chloride solution: Sodium chloride
Acceptance criteria and water (1:2). Before use, decant the solution from
Ethylene oxide: NMT 1 ppm any undissolved substance and filter, if necessary.
Dioxane: NMT 10 ppm Reference solution A: Prepare 0.50 g of the mixture of
calibrating substances with the composition described
SPECIFIC TESTS in Table T. Dissolve in heptane, and dilute with heptane
e FATS AND FIXED OlLs, Acid Value (401) to 50.0 mL.
Sample: 10.0 g of Polysorbate 60 Reference solution B: Reference solution A in heptane
Analysis: Transfer the Sample to a wide-mouth, 250-mL (1 in 10)
conical flask, and add 50 mL of neutralized alcohol. Reference solution C: Papas 0.50 g of a mixture of
Heat on a steam bath nearly to boiling, shaking thor- fatty acid methyl esters, which corresponds to the com-
oughly occasionally while heating. Invert a beaker over josition of the substance to be examined. Dissolve in
the mouth of the flask, cool under running water, and eptane, and dilute with heptane to 50.0 mL. [NoTE—
add 5 drops of phenolphthalein TS. Titrate with 0.1 N Commercially available mixtures of fatty acid methyl es-
sodium hydroxide VS. Calculate the acid value as di- ters may also be used.]
rected in the chapter. Sample solution: Dissolve 0.10 g of Polysorbate 80 in
Acceptance criteria: NMT 2.0 2 mL of Diluent in a 25-mL conical flask, and boil under
e FATS AND FIXED OILS, Hydroxy! Value (401): 81-96 a reflux condenser for 30 min. Add 2.0 mL of Boron
e FATS AND FIXED OILS, Peroxide Value (401) trifluoride-methanol solution through the condenser, and
Sample: 10.0g boil for 30 min. Add 4 mL of heptane through the con-
Saturated potassium iodide solution: Prepare a satu- denser, and boil for 5 min. Cool, add 10.0 mL of Satu-
rated solution of potassium iodide in carbon dioxide- rated sodium chloride solution, shake for about 15 s, and
free water. Make sure the solution remains saturated as add a quantity of Saturated sodium chloride solution such
indicated by the presence of undissolved crystals. that the upper phase is brought into the neck of the
Analysis: Introduce the Sample into a 100-mL beaker, flask. Collect 2 mL of the ae phase, wash with three
and dissolve with 20 mL of glacial acetic acid. Add 1 mL uantities, each of 2 mL, of water, and dry over anhy-
of Saturated potassium iodide solution, mix, and allow to rous sodium sulfate.
stand for 1 min. Add 50 mL of carbon dioxide-free
water and a magnetic stirring bar. Titrate with 0.01 M
sodium thiosulfate VS, determining the endpointpolen- Table 1
tiometrically (see Titrimetry (541)). Perform a blan Mixture of the Following Composition
titration. Substances (%)
Calculate the peroxide value as directed in the chapter. Methyl myristate 5
Acceptance criteria: NMT 10.0 Methyl palmitate 10
e FATS AND FIXED OILS, Saponification Value (401): 45-55
Methyl stearate 15
e WATER DETERMINATION, Method | (921): NMT 3.0%
Methyl arachidate 20
ADDITIONAL REQUIREMENTS Methyl oleate 20
e PACKAGING AND STORAGE: Preserve in tight containers, Methyl eicosenoate 10
protected from light and moisture. Store at room tem- Methyl behenate 10
perature.
Methyl lignocerate 10
e LABELING: Label to indicate whether the fatty acids are
derived from animal, vegetable, or synthetic sources. Chromatographic system
e USP REFERENCE STANDARDS (11) (See Chromatography (621), System Suitability.)
USP Polysorbate 60 RS Mode: GC
Detector: Flame ionization
Column: 0.32-mm x 30-m G16 on fused silica; film
thickness 0.5 um
Temperatures
Polysorbate 80 Injection port: 250°
Portions of the monograph text that are national USP text, Detector: 250°
and are not part of the harmonized text, are marked with Column: See Table 2.
symbols (*») to specify this fact.
Sorbitan, mono-9-octadecenoate, poly(oxy-1,2- Table 2
ethanediyl)derivs., (Z)-; Hold Time
Polyoxyethylene 20 sorbitan monooleate [9005-65-6]. Initial Temperature Final at Final
Temperature Ramp Temperature | Temperature
DEFINITION
(@) (C/min) (@) (min)
Polysorbate 80 is a mixture of partial esters of fatty acids,
mainly oleic acid, with sorbitol and its anhydrides ethoxyl- 80 10 220 =
ated with approximately 20 moles of ethylene oxide for 220 — 220 40
each mole of sorbitol and sorbitol anhydrides.
Carrier gas: Helium
IDENTIFICATION Linear velocity: 50 cm/s
Injection volume: 1 uL
NF Monographs

e A. It meets the requirements of the test for Composition

of Fatty Acids. Injection type: Split ratio, 50:1
System suitability
e *B. INFRARED ABSORPTION (197F)* Samples: Reference solution A and Reference solution B
ASSAY Suitability requirements
© COMPOSITION OF FATTY ACIDS Resolution: NLT 1.8 between the peaks due to
Diluent: 20 g/L of sodium hydroxide in methanol methyl oleate and methyl stearate, Reference solution
Boron trifluoride-methanol solution: 140 g/L of bo- A
ron trifluoride in methanol Theoretical plates: NLT 30,000 calculated for the
peak of methyl stearate, Reference solution A
NF 36 Official Monographs / Polysorbate 5525

Signal-to-noise ratio: NLT 5 for the peak of methyl Reference solution: Transfer 2.0 mL of Acetaldehyde
myristate, Reference solution B standard solution and 2.0 mL of Ethylene oxide standard
Analysis solution to a 10-mL headspace vial, and seal the vial
Sample: Sample solution immediately with a Teflon-coated, silicon membrane
Identify the peaks from Reference solution C. and an aluminum cap.
Calculate the percentage of each component in the Chromatographic system
Sample solution: (See Chromatography (621), System Suitability.)
Mode: Headspace GC
Result = (Ac/A7) x 100 Detector: Flame ionization
Column:' 0.53-mm x 50-m G27 on fused silica; film
Ac = peak area for the component of interest thickness 5 um
Ar = total area of all peaks related to fatty acids Temperatures
Acceptance criteria: See Table 3. Injection port: 85°
Detector: 250°
Table 3 Column: See Table 4.
Acceptance Acceptance
Criteria, Criteria, Table 4
Name NMT (%) NLT (%) Hold Time
Myristic acid 5.0 — Initial Temperature Final at Final
Palmitic acid 16.0 =_ Temperature Ramp Temperature | Temperature
Palmitoleic acid 8.0 — «°) (¢/min) ©) (min)
Stearic acid 6.0 — 70 10 250 =
Oleic acid = 58.0 250 = 250 5

eae 18.0 = Split ratio: 3.5:1

Linolenic acid 4.0 = Carrier gas: Helium
Flow rate: 4.0 mL/min
IMPURITIES Injection volume: 1 mL
RESIDUE ON IGNITION System suitability é
Sample: 2.00 Sample: Reference solution
Analysis: Heata silica or platinum crucible to redness [Nott—The relative retention times for ethylene oxide,
for 30 min, allow to cool in a desiccator, and weigh. acetaldehyde, and dioxane are 1.0, 0.9, and 1.9, re-
Evenly distribute the Sample in the crucible. Dry at spectively. The retention time for ethylene oxide is
100°-105° for 1 h and ignite to constant mass in a about 6.5 min.]
muffle furnace at 600 + 25°, allowing the crucible to Suitability requirements
cool in a desiccator after each ignition. Flames should Resolution: NLT 2.0 between the peaks due to acet-
not be produced at any time dure the procedure. If aldehyde and ethylene oxide
after prolonged ignition the ash still contains black par- Analysis
ticles, take up with hot water, pass through an ashless Samples: Sample solution A and Sample solution B
filter paper, and ignite the residue and the filter paper. Calculate the content, in ppm, of ethylene oxide:
Combine the filtrate with the ash, carefully evaporate to
dryness, and ignite to constant mass. Result = (2 x Cro x As)/(As — Aa)
Acceptance criteria: NMT 0.25% Ceo = concentration of ethylene oxide in Sample
solution B (t1g/mL)
Delete the following: As = peak area of ethylene oxide from Sample
solution A
°o *HEAVY METALS, Method I/ (231): NMT 10 ppmee crica Ag = peak area of ethylene oxide from Sample
1-jan-2018) solution B
o ETHYLENE OXIDE AND DIOXANE Calculate the content, in ppm, of dioxane:
Ethylene oxide standard solution: Dilute 0.5 mL of a
commercially available solution of ethylene oxide in Result = (2 x D x Cp x Ay) x 1000/(Ag — Ax)
methylene chloride (50 mg/mL) with water to 50.0 mL.
[Note—The solution is stable for 3 months, if stored in D = density of dioxane, 1.03 g/mL
vials with a Teflon-coated, silicon membrane and Cp = concentration of dioxane in Sample solution B
crimped caps at —20°.] Allow to reach room tempera- (uL/mL)
ture. Dilute 1.0 mL of this solution with water to Ax = peak area of dioxane from Sample solution A
250.0 mL. Ag = peak area of dioxane from Sample solution B
Dioxane standard solution: Dioxane in water (v/v) 1 in Acceptance criteria: NMT 1 ppm for ethylene oxide;
20,000 NMT 10 ppm for dioxane
Acetaldehyde standard solution: 0.01 mg/mL of acet-
aldehyde in water 5 SPECIFIC
*SPECIFICTESTS
GRAVITY (841): 1.06-1.09
Standard solution: Dilute 6.0 mL of Ethylene oxide stan- : oe ee Al (911) or Viscosity—
sydeibouow 4N

dard solution and 2.5 mL of Dioxane standard solution ROTATIONAL METHODS (912): 300-500 centistokes at
with water to 25.0 mL. 25°,
Sample solution A: Transfer 1.0 g of Polysorbate 80 to FATS AND FIXED OILS, Acid Value (401)
a 10-mL headspace vial. Add 2.0 mL of water, and seal Light petroleum: It has the following properties: a
the vial immediately with a Teflon-coated, silicon mem- clear, colorless, flammable liquid without fluorescence;
brane and an aluminum cap. ractically insoluble in water; miscible with alcohol;
Sample solution B: Transfer 1.0 g of Polysorbate 80 to __Piselealy ‘ "
a 10-mL headspace vial. Add 2.0 mL of the Standard 1CP-Sil 8 CB is suitable.
solution, and seal the vial immediately with a Teflon-
coated, silicon membrane and an aluminum cap.
 5526 Polysorbate / Official Monographs NF 36

density at 20° about 0.720; distillation range 100°- e USP REFERENCE STANDARDS (11)
120°; water content NMT 0.03%.2 USP Polysorbate 80 RS»
Sample solution: Dissolve 5.0 g in 50 mL of a mixture
of equal volumes of alcohol and Light petroleum (previ-
ously neutralized with 0.1 N potassium hydroxide or
0.1 N sodium hydroxide), using 0.5 mL of phenol-
phthalein TS as the indicator. If necessary, heat to Polyvinyl Acetate
about 90° to dissolve the substance to be examined.
Analysis: Titrate the Sample solution with 0.1 N potas-
sium hydroxide VS or 0.1 N sodium hydroxide VS until
the ou color persists for at least 15 s. When heating
has been applied to aid dissolution, maintain the tem-
perature at about 90° during the titration.
Acceptance criteria: NMT 2.0
e FATS AND FIXED OILS, Hydroxy! Value (401) (CaH6O2)n
Sample: 2.0g Vinyl acetate homopolymer
Analysis: Transfer the Sample into a 150-mL acetylation Vinyl acetate resin [9003-20-7].
flask fitted with an air condenser. Add 5.0 mL of
Pyridine-Acetic Anhydride Reagent, and attach the air DEFINITION
condenser. Heat the flask in a water bath for 1 h keep- Pobyviny! acetate is a thermoplastic polymer, represented by
ing the level of the water about 2.5 cm above the level the formula:
of the liquid in the flask. Withdraw the flask, and allow
to cool. Add 5 mL of water through the upper end of (CsH6O2)n
the condenser. If a cloudiness appears, add sufficient in which the value of n lies between approximately 100 and
pyridine to clear it, noting the volume added. Shake 17,000.
the flask, and replace in the water bath for 10 min.
Withdraw the flask, and allow to cool. Rinse the con- IDENTIFICATION
denser and the walls of the flask with 5 mL of alcohol, ¢ A, PROCEDURE
previously neutralized with phenolphthalein TS. Titrate Sample: 100 mg of Polyvinyl Acetate
with 0.5 N alcoholic potassium hydroxide VS using Analysis: Dissolve the Sample in 2.5 mL of acetone,
0.2 mL of phenolphthalein TS as the indicator. Carry place two arcs on a potassium bromide plate, and dry
out a blank test under the same conditions. to evaporate the solvent.
Acceptance criteria: 65-80 Acceptance criteria: The IR absorption spectrum of
e FATS AND FIXED OILS, Peroxide Value (401) polyvinyl acetate exhibits maxima corresponding to the
Sample: 10.0g same wavelengths as that of a similar preparation of
Saturated potassium iodide solution: Prepare a satu- USP Polyvinyl Acetate RS, treated in the same manner.
rated solution of potassium iodide in carbon dioxide- e B. PROCEDURE
free water. Make sure the solution remains saturated as Sample: 0.5 g of Polyvinyl Acetate
indicated by the presence of undissolved crystals. Analysis: Saponify the Sample in a mixture of 25.0 mL
Analysis: Transfer the Sample into a 100-mL beaker, of 0.5 N alcoholic potassium hydroxide and 25.0 mL of
and dissolve with 20 mL of glacial acetic acid. Add 1 mL water.
of Saturated potassium iodide solution, and allow to Acceptance criteria: The solution so obtained meets
stand for 1 min. Add 50 mL of carbon dioxide-free the requirements of the tests for /dentification Tests—
water and a magnetic stirring bar. Titrate with 0.01 M General (191), Acetate.
sodium thiosulfate VS, determining the endpoint poten-
tiometrically. Carry out a blank titration. IMPURITIES
Acceptance criteria: NMT 10 Inorganic Impurities
e FATS AND FIXED OILS, Saponification Value (401) e RESIDUE ON IGNITION (281): NMT 0.1%
Sample: 4.0g
Analysis: Transfer the Sample into a 250-mL borosilicate
glass flask fitted with a reflux condenser. Add 30.0 mL Delete the following:
of 0.5 N alcoholic potassium hydroxide VS and a few
Glass beads. Attach the condenser, and heat under re- °e HEAVY METALS, Method /I (231): NMT 10 ppmMe coftiaa1-
ux for 60 min. Add 1 mL of phenolphthalein TS and Jan-2018)
50 mL of dehydrated alcohol, and titrate immediately e RESIDUAL PEROXIDES
with 0.5 N hydrochloric acid VS. Carry out a blank test Sample: 0.85 g of Polyvinyl Acetate
under the same conditions. Analysis: Place the Sample in a borosilicate glass flask
Acceptance criteria: 45-55 with a ground-glass neck. Add 10.0 mL of ethyl acetate,
e WATER DETERMINATION, Method | (921): NMT 3.0%, de- and heat undera reflux condenser with constant agita-
termined on 1.0g tion. Allow to cool. Replace the air in the container with
oxygen-free nitrogen, and add a solution of 1.0 mL of
ADDITIONAL REQUIREMENTS glacial acetic acid and 0.5 g of sodium iodide in
© PACKAGING AND STORAGE: Store in an airtight container, 40.0 mL of water. Shake thoroughly, and allow to stand
protected from light. protected from light for 20 min. Titrate with 0.005 N
sodium thiosulfate VS until the yellow color is dis-
NF Monographs

2Petroleum ether; boiling range 100°-140°; [CAS 64742-49-0] from Fisher

Scientific; catalog number AC23302-0025 is suitable. charged. Perform a blank titration.
Acceptance criteria: The difference between the titra-
tion volumes is not greater than 1.0 mL; and NMT
100 ppm, calculated as hydrogen peroxide, is found.
Organic Impurities
e PROCEDURE: LIMIT OF VINYL ACETATE
Standard stock solution: 1.0 mg/mL of Vinyl Acetate
in toluene
NF 36 Official Monographs / Polyviny! 5527

Standard solutions: 0.1, 0.3, 1, 3, and 10 ug/mL of e Loss ON DRYING (731): Dry 1.5 g at 100° for 2 hina
vinyl acetate in toluene, prepared from Standard stock vacuum: it loses NMT 1.0% of its weight.
solution e AVERAGE MOLECULAR WEIGHT AND MOLECULAR WEIGHT
Sample solution: 0.1 g/mL of Polyvinyl Acetate in DISTRIBUTION
toluene [CautTion—Tetrahydrofuran (THF) is considered to be a car-
Chromatographic system cinogen and embryo-fetal toxic substance. It is also a per-
(See Chromatography (621), System Suitability.) oxide former and is flammable. A safe-handling practice
Mode: GC must be in place in the laboratory. Carefully review ap-
Detector: Hydrogen flame ionization propriate Material Safety Data Sheets before use.]
Column: 0.32-mm x 30-m fused-silica capillary col- Mobile phase: Tetrahydrofuran inhibited with 250 ppm
umn, 5-1um layer of phase G1 butylated hydroxytoluene. Do not sparge or degas.
Temperature Standard solutions: Prepare two sets of mixtures, each
Detector: 250° set containing five narrow polystyrene standards of dif-
Injector port: 150° ferent known molecular weights, totaling 10 narrow
Column: See the temperature program table below. polystyrene standards covering the molecular weight
mnge from about 600 to 3,000,000 g/mol.’ Prepare
Hold Time at each set of five narrow polystyrene standards to have a
Initial Temperature Final Final known concentration at about 0.05% (w/v) for each
Temperature Ramp Temperature | Temperature standard in Mobile phase.
© (¢/min) C) (min) Sample solution: Transfer 0.025g of polyvinyl acetate
100 = 100 8
to a vial, and add 10 mL of Mobile phase. Cap and mix
well, using an appropriate laboratory shaker, for 1 h.
100 20 250 S. Pass the polyvinyl acetate solution through a polytetra-
Carrier gas: Helium fluoroethylene filter having a porosity of 0.45 um, dis-
Flow rate: Adjusted so that the vinyl acetate peak card an appropriate volume of the initial filtrate, and
appears after about 7 min use the rest of the filtered solution for analysis.
Injection size: 1.0 uL Chromatographic system
Injection type: Split ratio is about 8:1. (See Chromatography (621), System Suitability.)
System sultapllity Mode: LC
Detector: Refractive Index (RI)
Sample: Standard solution containing 1 4g/mL of
vinyl acetate in toluene Detector temperature: 35°
Suitability requirements Columns: Two 10-mm x 50-cm analytical columns;
Relative standard deviation: NMT 15% 5-um packing L73, and a 10-mm x 10-cm, 500-A
Analysis guard column; packing L73. [NoTE—The analytical col-
Samples: Standard solution and Sample solution umn is suitable for molecular weight ranges from 100
Plot the peak responses of the vinyl acetate in the to 10,000,000 g/mol.]
Standard solutions versus the concentration, in Flow rate: 1.1 mL/min
ug/mL, of vinyl acetate, and draw the straight line Injection size: 200 ul?
best fitting the five plotted points. From the graph System suitability
so obtained, determine the concentration, C, in
Sample: Standard solutions
tig/mL, of vinyl acetate in the Sample solution. Suitability requirements
Calculate the quantity, in ug, of vinyl acetate in each Resolution: NLT 1.7 between the polystyrene
g of Polyvinyl Acetate taken: standards
Analysis
Result = (C/C,) Samples: Standard solutions and Sample solution
Separately inject equal volumes of the Standard solu-
Cc = as determined above tions and the Sample solution into the chromato-
Cp = concentration of the Polyvinyl Acetate in the graph, record the chromatograms, and determine the
Sample solution (g/mL) elution peak maxima and the corresponding reten-
Acceptance criteria: NMT 5 ug/g (5 ppm) of vinyl tion volumes for the 10 polystyrene standards.
acetate Universal calibration: Analyze each polystyrene stan-
dard, and use a data handling system or a suitable gel
SPECIFIC TESTS permeation chromatography or size exclusion chroma-
e FATS AND FIXED OILS, Acid Value (401) tography (GPC/SEC) software to compute the data
Sample: 10.0g of Polyvinyl Acetate and calibration. Construct the Universal calibration
Analysis: Transfer the Sample to a 250-mL glass-stop- curve as follows, and use it in the section Data analysis
pered conical flask, dissolve in 75 mL of ethylene dichlo- for sample.
tide, add 60 mL of denatured alcoholic TS, and mix. Plot log ({n] x M,) for each polystyrene standard in the
Add 1 mL of phenolphthalein TS, and titrate with 0.02 Standard solutions versus its retention volume, V, in
N alcoholic potassium hydroxide VS until the solution mL, at each standard peak maximum; and construct
remains faintly pink after shaking for 30 s. Perform a the best cubic line fitting the 10 points. In this ex-
blank determination, and make any necessary pression, M, is the molecular weight, in g/mol, of
correction. polystyrene standard; and [n] is the intrinsic viscosity
Acceptance criteria: The acid value is NMT 0.5. ofa polyinier and is related to polymer molecular
e FATS AND FixeD OILS, Ester Value (401) weight (M,), especially viscosity-average molecular Zz
Sample: 0.5 g of Polyvinyl Acetate weight, My, by the following Mark-Houwink equation: Fd
Analysis: Saponify the Sample in a mixture of 25.0 mL
of 0.5 N alcoholic potassium hydroxide VS and 25.0 mL = K x M¢a
[n] = fo)
S
of water. Proceed as directed under Fats and Fixed Oils
(401), Saponification Value, beginning with “Heat the K = constant for a given polymer/solvent system at a
flask on a steam bath”. a specified temperature By
Acceptance criteria: The ester value, calculated from 1 Narrow polystyrene standards are available from polymer laboratories, such me]
the Saponification Value and the Acid Value, is between as EasiCal, or are available as various individual polystyrene standards. =>
ww
615 and 675. 2A sample loop of 400 uL anda syringe of 250 uL were used in the Analysis.
 5528 Polyvinyl / Official Monographs
a = constant for a given polymer/solvent system at
a specified temperature
For pele ene in THF at 25°, K = 0.0128 mL/g, and
a=0.
For polyvinyl acetate in THF at 25°, K = 0.025 mL/g,
and a = 0.63
Based on the Mark-Houwink equation and the fact
that M, can represent the molecular weight (M,), the
following equation is given:
log([n] x M,) = logk + (a + 1)log(M,)
M, can be obtained as M,, a viscosity-average
molecular weight of polystyrene standard.
Data analysis for sample: Analyze the polyvinyl ace-
tate sample by identifying retention volumes V, and
Vp, corresponding to the beginning and end of the
polyvinyl acetate chromatogram. The baseline be-
tween V, and V, is assumed to be linear. [NoTtE—Draw
a straight line between V, and Vp.] Data analysis is
based ona suitable GPC/SEC computer software or a
real-time data acquisition system with either offline or
online data processing that is able to provide a means
of determining chromatographic peak heights or inte-
grated area segments at prescribed intervals under the
SEC chromatogram and a means of handling and re-
porting the data. The following describes the data
processes, which can be computed either by the GPC/
SEC software or by an equivalent data processing sys-
tem. Upon acquisition, handle the data under the pol-
yvinyl acetate elution peak in discrete segments Ai, in-
tegrated area slices, or as digitized chromatogram
heights H,, by recording the vertical displacements be-
tween the chromatogram trace and the baseline at re-
tention volume, Vi, over designated intervals. A mini-
mum of 40 area segments or heights is required.
Obtain the corresponding molecular weight value Mi
for Polyvinyl Acetate at its retention volume, Vj, from
the Universal calibration curve obtained in the section
Universal calibration, since the constants K and a for
Polyvinyl Acetate are known and given above.
Calculate the number-, weight-, and viscosity-average
molecular weights, Mn, Mw, and M,, respectively, in
/mol, of polyvinyl acetate, using the following
ormula:
SA
N
M. =—=!
N A,
>(R]
N range, which is previously neutralized with 0.1 N potas-
M -|=
v N
DA
=1
NF Monographs
If the retention volume internal AV; (for instance, V2 —
V; = V3 — V2, etc.) is constant, parameters Ai and M,
are the chromatographic peak slice area and the Poly-
vinyl Acetate molecular weight associated with the re-
tention volume, Vi; and N is the number of data
points obtained from the chromatogram between V,
and V, (N 2 40). [NoTE—lf N is sufficiently large, the
use of area segments Aj or peak heights H; will yield
equivalent results.]
NF 36
Calculate the molecular weight distribution or polydis-
persity for Polyvinyl Acetate:
Result = Mw/Mn
Acceptance criteria: The values of weight-average
molecular weight and polydispersity are, respectively,
NLT 85% and NMT 115% of their respective values as
stated on the label.
ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Preserve in well-closed contain-
ers. No storage requirement specified.
e LABELING: Label it to indicate its weight-average molecu-
lar weight, Mw, and polydispersity (Mw/Mn). — *
e USP REFERENCE STANDARDS (11)
USP Polyvinyl Acetate RS
Polyvinyl Acetate Dispersion
DEFINITION
Dispersion of Bolin! acetate in water. It contains 25.0%
to 30.0% of polyvinyl acetate. It may contain povidone
and sodium lauryl sulfate as stabilizers.
IDENTIFICATION
e A. FILM FORMATION: Place 1 drop of Dispersion on a glass
plate and allow to dry. A clear and homogeneous film is
formed.
e B. INFRARED ABSORPTION
(See Spectrophotometric Identification Tests (197), Infrared
Absorption.
Analysis: Place 1 drop of the Dispersion on a glass
plate, and cover the test substance with a water-resis-
tant crystal disk (silver chloride or KRS-5).! Gently press
on, and then remove the crystal disk. Dry the crystal
disk in a drying chamber until a homogeneous film is
formed.
Acceptance criteria: The IR absorption spectrum of the
film so formed exhibits maxima corresponding to the
same wavelengths as those of a similar preparation of
USP Polyvinyl Acetate Dispersion RS treated in the same
manner.
ASSAY
© PROCEDURE
Sample 1: 10g of Dispersion
Solvent: 50 mL of a mixture of equal volumes of alco-
hol and petroleum ether with a 100°-120° boiling
sium hydroxide or 0.1 N sodium hydroxide
Analysis 1: Dissolve Sample 1 in the Solvent. Add
0.5 mL of phenolphthalein TS, and titrate with 0.1 N
potassium hydroxide or 0.1 N sodium hydroxide until
the pink color persists for at least 15 s.
Calculate the acid value, 4:
Result = (My x Vx N)/W
Ma = molecular weight of potassium hydroxide,
v
56.11
= volume of 0.1 N potassium hydroxide or 0.1
N sodium hydroxide consumed in the actual
test (mL)
N = exact normality of the potassium hydroxide
solution or sodium hydroxide solution
W = weight of Dispersion taken for the test (g)
1 KRS-5 consists of 42% thallium(!) bromide and 58% thallium(|) iodine by
molecular weight. Suitable disks of silver chloride and of KRS-5 are available
from www.crystals.saint-gobain.com, www.almazoptics.com, and www.in-
ternationalcrystal.net.
NF 36 Official Monographs / Polyvinyl 5529

Sample 2: 1.5 g of Dispersion Table 1

Analysis 2: Transfer Sample 2 to a 250-mL borosilicate Time Solution A Solution B
glass flask fitted with a reflux condenser. Add 25.0 mL (%) (%)
of 0.5 M alcoholic potassium hydroxide and a few glass (min)
oO 100 0
beads. Attach the condenser and heat under reflux for
30 min. Add 1 mL of phenolphthalein TS, and titrate 2 100 0
immediately (while still hot) with 0.5 N hydrochloric 40 85 15
acid VS. Perform a blank determination under the same 42 0 100
conditions (see Titrimetry (541), Residual Titrations.) 48 0 100
Calculate the saponification value, Is:
51 100 0
Result = [My x (Ve — Vr) x N]/W Standard solution: Transfer 50 mg of vinyl acetate to a
100-mL volumetric flask, dissolve in and dilute with
Mn = molecular weight of potassium hydroxide,
56.11 methanol to volume, and mix well. Dilute 5.0 mL of the
Ve = volume of 0.5 N hydrochloric acid consumed solution with Solution A to 100 mL. Dilute 10.0 mL of
in the blank test (mL) this solution with Solution A to 100 mL. The Standard
Vv; = volume of 0.5 N hydrochloric acid consumed solution contains about 2.5 g/mL of vinyl acetate.
in the actual test (mL) [Note—This solution should be analyzed within 1 h
N = exact normality of the hydrochloric acid when stored at room temperature.]
Ww = weight of Dispersion taken for the test (g) System suitability solution: Transfer 50 mg of vinyl
Calculate the percentage content of polyvinyl acetate in acetate and 50 mg of 1-vinylpyrrolidin-2-one to a
the portion of Dispersion taken: 50-mL volumetric flask, add10 mL of methanol, soni-
cate or gently shake the flask to dissolve the materials.
Result = F x {My2 x [(ls — 14)/Mn]} x 100 Dilute with Solution A to volume. Dilute 10 mL of this
solution with Solution A to 100 mL. Dilute 5 mL of this
F = factor converting mg to g, 103 g/mg solution with Solution A to 100 mL. The System suitabil-
M2 = molecular weight of vinyl acetate, 86.09 ity solution contains about 5 g/mL each of vinyl acetate
Is = saponification value and 1-vinylpyrrolidin-2-one.
la = acid value Sample solution: Transfer 250 mg of Dispersion to a
My = molecular weight of potassium hydroxide, 10-mL volumetric flask, add about 4 mL of methanol,
56.11 and sonicate. After cooling to ambient temperature, di-
Acceptance criteria: The content of polyvinyl acetate is lute with water to volume, and mix. Centrifuge at 4000
25.0%-30.0%. x g for 10 min, and pass through a 0.2-um membrane
filter. [NoTE—This solution should be analyzed within 1
OTHER COMPONENTS h when stored at room temperature.]
Stabilizers Chromatographic system
© POVIDONE (See Chromatography (621), System Suitability.)
[NoTe—Perform this test only if the Dispersion contains Mode: LC
povidone.] Detector: UV 205 nm
Sample: 0.25g Columns
Analysis: Perform nitrogen determination by sulfuric Precolumn: 4.0-mm x 3-cm; 5-14m packing L1 may
acid digestion on the Sample as directed in Nitrogen be used if a matrix effect is observed. [NoTE—The
Determination (461), Method Il. matrix effect may result in poor reproducibility of the
Calculate the percentage content of povidone in the retention times and of the peak shapes.]
portion of Dispersion taken: Analytical: 4.0-mm x 25-cm; 5-4m packing L1
Column temperature: 30°
Result = N/Ny Flow rate: 1 mL/min
Injection volume: 10 uL
N = percentage content of nitrogen System suitability
Nv = percentage content, expressed as a decimal Sample: System suitability solution
number, of nitrogen in vinylpyrrolidone, [Nott—The relative retention times for vinyl acetate and
0.126 1-vinylpyrrolidin-2-one are 1.0 and 1.2, respectively.]
Acceptance criteria: The content of povidone is NMT Suitability requirements
4.0%. Resolution: NLT 5.0 between vinyl acetate and
IMPURITIES 1-vinylpyrrolidin-2-one
Relative standard deviation: NMT 5.0% determined
© RESIDUE ON IGNITION (281) from the 1-vinylpyrrolidin-2-one peak
Sample: 1.0g of Dispersion Analysis
Analysis: Heata silica crucible to redness for 30 min, Samples: Standard solution and Sample solution
allow to cool in a desiccator, and weigh. Evenly dis- Acceptance criteria: The response of the vinyl acetate
tribute the Sample in the crucible and weigh. Dry the peak from the Sample solution is NMT that of the vinyl
crucible at 100°-105° for 1 h and ignite in a muffle acetate peak from the Standard solution, corresponding
furnace at 600 + 25°, until the test substance is thor- to NMT 100 ppm of vinyl acetate.
oughly charred. Continue the experiment as directed in e Limit oF Acetic ACID/ACETATE
Residue on Ignition (281) on the residue obtained, be-
Solution A: 5 mM sulfuric acid 2
ginning with “Moisten the sample with a small amount Solution B: Acetonitrile and 5 mM sulfuric acid (1:1) Fd
(usually 1 mL) of sulfuric acid...” Mobile phase: See Table 2. Fey
Acceptance criteria: NMT 0.5%
e Limit OF VINYL ACETATE =]
Solution A: Acetonitrile, methanol, and water (5:5:90) Table 2 eS
Solution B: Acetonitrile, methanol, and water (45:5:50) Time Solution A Solution B By
Mobile phase: See Table 1. (min) (%) (%) S
oO 100 0 7)
10 100 0
 5530 Polyvinyl / Official Monographs NF 36

Table 2 (Continued) with a mesh width of 45 um, and filter the Sample
Time Solution A Solution B
through it. [NoTE—Suitable single-woven wire cloth
(min) (%)
mesh meets the requirements set in ISO 9044.] Wash
(%) the sieve or the cloth with distilled water until a clear
10.5 0 100 filtrate is obtained, and dry the sieve or the cloth to
20 0 100 constant weight at 100°-105°.
20.5 100 0 Acceptance criteria: The weight of the residue is NMT
30 100 0 500 mg (0.5%).

System suitability solution: Transfer 30 mg of glacial ADDITIONAL REQUIREMENTS

acetic acid and 30 mg of malonic acid to a 25-mL volu- e PACKAGING AND STORAGE: Preserve in tight containers at a
metric flask, dilute with methanol to volume, and mix temperature below 25°. Protect from freezing.
well. Transfer 1 mL of the solution to a 25-mL flask, © LABELING: Label it to indicate the amounts ef pavidione:
dilute with water to volume, and mix well. The solution and sodium lauryl sulfate.
aoe en 0.048 mg/mL each of acetic acid and malonic e USP REFERENCE STANDARDS (11)
acid. USP Polyvinyl Acetate Dispersion RS
Standard solution: 0.1 mg/mL of acetic acid in water
Sample solution: Transfer 330 mg of Dispersion to a
50-mL volumetric flask, add about 5 mL of methanol,
and dilute with water to volume, which leads to a pre-
cipitation of sample. Pass the dispersion through a 0.2- Polyvinyl Acetate Phthalate
um regenerated cellulose membrane filter.2 Use the
iltrate. DEFINITION
Chromatographic system Polyvinyl Acetate Phthalate is a reaction product of phthalic
(See Chromatography (621), System Suitability.) anhydride anda partially hydrolyzed polyvinyl acetate. It
Mode: LC contains NLT 55.0% and NMT 62.0% of phthalyl (o-
Detector: UV 205 nm carboxybenzoyl, CsHsO3) groups, calculated on the anhy-
Column: 4.6-mm x 25-cm; 5-um packing L1 drous, acid-free basis.
Column temperature: 25°
Flow rate: 1 mL/min IDENTIFICATION
Injection volume: 20 pL e A. The Sample solution in the Assay exhibits a maximum
Run time: 30 min at 27743 nm.
System suitability ° B.
Samples: System suitability solution and Standard Sample: 10mg
solution Analysis: Place the Sample in a small test tube, add
[Note—The relative retention times for malonic acid 10 mg of resorcinol, and mix. Add 0.5 mL of sulfuric
and acetic acid are 0.9 and 1.0, respectively.] acid, and heat in a liquid bath at 160° for 3 min. Cool,
Suitability requirements and pour the solution into a mixture of 25 mL of 1N
Resolution: NLT 2.0 between malonic acid and acetic sodium hydroxide and 200 mL of water.
acid, System suitability solution Acceptance criteria: The solution shows a vivid green
Relative standard deviation: NMT 5.0% determined fluorescence.
from the acetic acid peak, Standard solution eC.
Analysis Sample solution: 100 mg/mL of Polyvinyl Acetate
Samples: Standard solution and Sample solution Phthalate in methanol
Calculate the percentage of acetic acid in the portion of Analysis: Pour 1 mL of the Sample solution onto a clear
Dispersion taken: glass plate.
Acceptance criteria: A film is deposited as the metha-
Result = (ru/r's) x (Cs/Cu) x 100 nol evaporates.
tu = peak response of acetic acid from the Sample ASSAY
solution e PHTHALYL CONTENT
rs = peak response of acetic acid from the Standard solution: 0.05 mg/mL of phthalic anhydride
Standard solution in alcohol, pepe as follows. Transfer 50 mg of
G = concentration of acetic acid in the Standard phthalic anhydride to a 1000-mL volumetric flask. Dis-
solution (mg/mL) solve with heat in 100 mL of alcohol, dilute with alco-
Cu = concentration of Polyvinyl Acetate Dispersion hol to volume, and mix.
in the Sample solution (mg/mL) Sample solution: 0.1 mg/mL of Polyvinyl Acetate
Acceptance criteria: NMT 1.5% of acetic acid Phthalate in alcohol, prepared as follows. Transfer
100 mg of Polyvinyl Acetate Phthalate to a 1000-mL
SPECIFIC TESTS volumetric flask, dissolve in alcohol, and dilute with al-
© MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI- cohol to volume.
FIED MICROORGANISMS (62): The total aerobic microbial Instrumental conditions
count is NMT 1000 cfu/g, and the total combined molds Mode: UV
and yeasts count is NMT 100 cfu/g. Cell: 1m
e PH (791): 3.0-5.5 Analytical wavelength: 275 nm
NF Monographs

e Loss ON DRYING (731) Analysis

Sample: 1.0g of Dispersion Samples: Standard solution and Sample solution
Analysis: Dry the Sample at 110° for 5 h. Calculate, on the acid-free basis, the percentage of
Acceptance criteria: 68.5%-71.5% phthalyl taken:
e COAGULUM CONTENT
Sample: 100g of Dispersion ; Result = (Au/As) x (Cs/Cu) x 100
Analysis: Accurately weigha stainless steel sieve with
45-l1m openings or a suitable single-woven wire cloth Au = absorbance of the Sample solution
As = absorbance of the Standard solution
2Whatman Spartan HPLC certified syringe filter, Whatman Cat # 10463060 or
equivalent filter.
NF 36 Official Monographs / Potassium 5531

Cs = concentration of phthalic anhydride in the Calculate the percentage of free acid other than
Standard solution (mg/mL) phthalic, as acetic acid, in the portion taken:
Cy = concentration of Polyvinyl Acetate Phthalate in
the Sample solution (mg/mL) Result = [(V
— Vp) x M3 x N]/W
x 100
Acceptance criteria: 55.0%-62.0% of phthalyl (o-
carboxybenzoyl, CsHsO3) groups on the anhydrous, Vv = total volume of 0.1 N sodium hydroxide used
acid-free basis (mL)
Ms = equivalent weight of acetic acid, 60.05 mg/
IMPURITIES mE
e RESIDUE ON IGNITION (281): NMT 1.0% N = actual normality of the Titrant
o FREE PHTHALIC ACID Ww = sample weight on the anhydrous basis (mg)
Standard solution: 0.05 mg/mL of phthalic anhydride Acceptance criteria: NMT 0.6% on the anhydrous
Sample solution: 6 mg/mL of polyvinyl acetate phthal- basis
ate in water prepared as follows. Dissolve 1500 mg of
Polyvinyl Acetate Phthalate in 50 mL of a mixture of SPECIFIC TESTS
methylene chloride and methanol (4:1). Transfer the so- e ViscosiTy—CAPILLARY METHODS (911)
lution to a separator with the aid of 75 mL of water, Sample solution: Dissolve a quantity, equivalent to
and swirl, taking care not to shake. Add 100 mL of hex- 15 g on the anhydrous basis, in 85 g of methanol.
anes, shake, and allow the mixture to stand until it sep- Analysis: Determine the viscosity of the Sample solution,
arates into two layers. Transfer the water layer to a using a capillary viscometer at 25 + 0.2°.
250-mL volumetric flask. Add 100 mL of water to the Acceptance criteria: The apparent viscosity is 7-11
separator, shake, and allow to stand until the layers mPa - s (centipoises).
separate. Transfer the water layer to the same volumet- e WATER DETERMINATION, Method | (921): NMT 5.0%
ric flask, and dilute with water to volume. If the solu-
tion is cloudy, centrifuge a portion until clear. ADDITIONAL REQUIREMENTS
Instrumental conditions © PACKAGING AND STORAGE: Preserve in tight containers.
Mode: UV
Cell: 1.cm
Analytical wavelength: 277 nm
Analysis
Samples: Standard solution and Sample solution Polyvinyl Alcohol—see Polyvinyl Alcohol
Calculate the percentage of free phthalic acid in the General Monographs
portion taken:

Result = (Au/As) x (Cs/Cu) x (Mr/Mj2) x 100

Au = absorbance of the Sample solution Potassium Alginate
As = absorbance of the Standard solution
Cs = concentration of phthalic anhydride in the
Standard solution (mg/mL)
oK
_/ ow

Cu = concentration of Polyvinyl Acetate Phthalate in i of

vtona
a \4o—-
the Sample solution (mg/mL)
Mn = molecular weight of phthalic acid, 166.13
Mz = molecular weight of phthalic anhydride,
148.12 Alginic acid, potassium salt;
go all criteria: NMT 0.6% on the anhydrous Potassium alginate [9005-36-1].
asis
e FREE ACID OTHER THAN PHTHALIC DEFINITION
Sample solution: Proceed as directed for Sample solu- Potassium Alginate is the purified carbohydrate product ex-
tion in Impurities, Free Phthalic Acid, but instead of trans- tracted from various species of brown seaweeds by the
ferring the water extracts to a volumetric flask, transfer use of dilute alkali. It consists chiefly of the potassium salt
them to a 400-mL beaker. of Alginic Acid, a linear glycuronoglycan consisting of B-
Titrimetric system 1,4-linked D-mannuronic acid and |-guluronic acid units in
(See Titrimetry (541).) the pyranose ring form. It yields NLT 16.5% and NMT
Mode: Direct titration 19.5% of carbon dioxide (COz), equivalent to NLT 89.2%
Titrant: 0.1 N sodium hydroxide VS and NMT 105.5% of potassium alginate, calculated on
Endpoint detection: Visual the dried basis.
Analysis: Titrate the Sample solution with Titrant to a
phenolphthalein endpoint. IDENTIFICATION
Calculate the volume, Vp, in mL, of 0.1 N sodium hy- eA.
droxide consumed by the free phthalic acid in the Analysis: To 5 mL of a 1-in-100 solution in 0.1 N so-
sample taken: dium hydroxide add 1 mL of calcium chloride TS.
Acceptance criteria: A voluminous, gelatinous precipi-
Vp = (1/M)
x(1/N)xPx W tate is formed.
° B. 4
M, = equivalent weight of phthalic acid, Analysis: To 10 mL of a 1-in-100 solution in 0.1 N so- a]
83.065 mg/mEq dium hydroxide add 1 mL of 2 N sulfuric acid.
N = actual normality of the Titrant (mEq/mL) Acceptance criteria: A heavy, gelatinous precipitate is
cs
°
P = percentage of free phthalic acid, previously formed. =]
determined, in decimal form °C. °
Ww = sample weight of Polyvinyl! Acetate Phthalate ito}
Analysis: To 5 mg in a test tube add 5 mL of water, a
on the anhydrous basis (mg) 1 ml ofa ines prepared 1-in-100 solution of 1,3- i)
me}
naphthalenediol in alcohol, and 5 mL of hydrochloric =
acid. Heat the mixture to boiling, boil gently for 3 min, vy
then cool to 15°. Transfer the contents of the test tube
 4504 Calcium / Dietary Supplements
in 50 mL of 6 N hydrochloric acid, dilute with water to
volume, and mix to obtain a solution having a known
concentration of 1000 g/mL.
Standard stock solution: 20 ug/mL of magnesium
from Magnesium standard stock solution diluted with
0.125 N hydrochloric acid
Standard solutions: To separate 100-mL volumetric
flasks transfer 1.0, 1.5, 2.0, 2.5, and 3.0 mL of Standard
stock solution. To each flask add 1.0 mL of Lanthanum
chloride solution, and dilute with 0.125 N hydrochloric
acid to volume to obtain concentrations of 0.2, 0.3,
0.4, 0.5, and 0.6 j1g/mL of magnesium.
Sample solution: Finely powder NLT 20 Tablets. Trans-
fer the equivalent of 200 mg of magnesium to a porce-
lain crucible. Heat for 6-12 h in a muffle furnace main-
tained at 550°, and cool. Add 15 mL of hydrochloric
acid, and boil gently on a hot plate or a steam bath for
30 min, intermittently rinsing the inner surface of the
crucible with 6 N hydrochloric acid. Cool, and quantita-
tively transfer the contents of the crucible to a 100-mL
volumetric flask, rinsing the crucible with portions of
6 N hydrochloric acid. Dilute the contents of the flask
with water to volume, and filter, discarding the first
5 mL of the filtrate. Dilute the filtrate quantitatively with
0.125 N hydrochloric acid to obtain a concentration of
0.4 ug/mL of magnesium, adding 1 mL of Lanthanum
chloride solution per 100 mL of the final volume.
Spectrometric conditions
(See Atomic Absorption Spectroscopy (852).)
Mode: Atomic absorption spectrophotometry
Lamp: Magnesium hollow-cathode
Flame: Air-acetylene
Analytical wavelength: Magnesium emission line,
285.2 nm
Blank: 0.125 N hydrochloric acid containing 1 mL of
Lanthanum chloride solution per 100 mL
Analysis
Samples: Standard solutions and Sample solution
Determine the absorbances of the solutions against the
Blank, Plot the absorbances of the Standard solutions
versus concentration, in ug/mL, of magnesium, and
draw the straight line best fitting the five plotted
points. From the graph, determine the concentration,
CG, in ug/mL, of magnesium in the Sample solution.
Calculate the percentage of the labeled amount of
magnesium (Mg) in the portion of Tablets taken:
Result = (C/Cy) x 100
c = measured concentration of magnesium in the
al
Sample solution (g/mL)
Im Cu = nominal concentration of magnesium in the
a Sample solution (g/mL)
i
— Acceptance criteria: 90.0%-125.0% of the labeled
aD amount of magnesium (Mg)
r)
= © MANGANESE, Method 7
C} Manganese standard stock solution: Transfer 1.00 g of
3 manganese, weighed, to a 1000-mL volumetric flask.
va) Dissolve in 20 mL of nitric acid, dilute with 6 N hydro-
Qa chloric acid to volume, and mix to obtain a solution
with a concentration of 1000 ttg/mL of manganese.
Standard stock solution: 50 g/mL of manganese from
Manganese standard stock solution diluted with 0.125 N
hydrochloric acid
Standard solutions: To separate 100-mL volumetric
flasks transfer 1.0, 1.5, 2.0, 3.0, and 4.0 mL of the Stan-
dard stock solution. Dilute the contents of each flask
with 0.125 N hydrochloric acid to volume to obtain
solutions with concentrations of 0.5, 0.75, 1.0, 1.5, and
2.0 g/mL of manganese.
Sample solution: Finely powder NLT 20 Tablets. Trans-
fer the equivalent of 9 mg of manganese from pow-
USP 41
dered Tablets to a porcelain crucible. Heat for 6-12 h in
a muffle furnace maintained at 550°, and cool. Add
15 mL of hydrochloric acid, and boil gently on a hot
plate or a steam bath for 30 min, intermittently rinsing
the inner surface of the crucible with 6 N hydrochloric
acid. Cool, and quantitatively transfer the contents of
the crucible to a 100-mL volumetric flask, rinsing the
crucible with portions of 6 N hydrochloric acid. Dilute
the contents of the flask with water to volume, and
filter, discarding the first 5 mL of the filtrate. Dilute the
filtrate quantitatively with 0.125 N hydrochloric acid to
obtain a concentration of 1 t1g/mL of manganese.
Spectrometric conditions
See Atomic need Spectroscopy (852).)
Mode: Atomic absorption spectrophotometry
Lamp: Manganese hollow-cathode
Flame: Air-acetylene
Analytical wavelength: Manganese emission line,
279.5 nm
Blank: 0.125 N hydrochloric acid
Analysis
Samples: Standard solutions and Sample solution
Determine the absorbances of the solutions against the
Blank. Plot the absorbances of the Standard solutions
versus the concentration, in g/mL, of manganese,
and draw the straight line best fitting the five plotted
points. From the graph so obtained, determine the
concentration, C, in mg/mL, of manganese in the
Sample solution.
Calculate the percentage of the labeled amount of
manganese (Mn) in the portion of Tablets taken:
Result = (C/Cy) x 100
Cc = measured concentration of manganese in the
Sample solution (g/mL)
Cu = nominal concentration of manganese in the
Sample solution (14g/mL)
Acceptance criteria: 90.0%-125.0% of the labeled
amount of manganese
e ZINC, Method 1
Zinc standard stock solution: 1000 g/mL of zinc from
zinc oxide dissolved in 5 M hydrochloric acid (3.89 mg/
mL), and diluted with water to final volume. [NOoTE—
Dissolve in 5 M hydrochloric acid by warming, if neces-
sary, cool, and then dilute to final volume.]
Standard stock solution: 50 g/mL of zinc from Zinc
standard stock solution diluted with 0.125 N hydrochlo-
tic acid
Standard solutions: Transfer 1.0, 2.0, 3.0, 4.0, and
5.0 mL of Standard stock solution to separate 100-mL
volumetric flasks. Dilute the contents of each flask with
0.125 N hydrochloric acid to volume to obtain concen-
trations of 0.5, 1.0, 1.5, 2.0, and 2.5 ug/mL of zinc.
sample solution: Weigh and finely powder NLT 20
Tablets. Transfer the equivalent of 40 mg of zinc from
React Tablets to a porcelain crucible. Heat for 6-12
in a muffle furnace maintained at 550°, and cool.
Add 15 mL of hydrochloric acid, and boil gently on a
hot plate or a steam bath for 30 min, intermittently
rinsing the inner surface of the crucible with 6 N hydro-
chloric acid. Cool, and quantitatively transfer the con-
tents of the crucible to a 100-mL volumetric flask, rins-
ing the crucible with portions of 6 N hydrochloric acid.
Dilute the contents of the flask with water to volume,
and filter, discarding the first 5 mL of the filtrate. Dilute
the filtrate quantitatively with 0.125 N hydrochloric
acid to obtain a concentration of 2 g/mL of zinc.
Spectrometric conditions
(See Atomic Absorption Spectroscopy (852).)
 5532 Potassium / Official Monographs NF 36

to a 30-mL separator with the aid of 5 mL of water, and

extract with 15 mL of isopropyl ether.
Acceptance criteria: The isopropyl ether extract exhib- Potassium Benzoate
its a deeper purplish hue than that from a blank, simi-
larly prepared.
e D. IDENTIFICATION TESTS—GENERAL, Potassium (191) ( = “oOo” K’
Analysis: Ignite completely 0.2 g at as low a tempera- Za
ture as possible.
Acceptance criteria: A solution of the residue meets
the requirements of the tests. CHsKO2 160.21
Benzoic acid, potassium salt;
ASSAY Potassium benzoate [582-25-2].
e ALGINATES ASSAY (311): 16.5%-19.5% of CO2, equiva-
lent to 89.2%-105.5% of potassium alginate on the DEFINITION
dried basis Potassium Benzoate contains NLT 99.0% and NMT 101.0%
of potassium benzoate (C;HsKOz), calculated on the anhy-
IMPURITIES drous basis.
© ARSENIC, Method II (211): NMT 1.5 ppm
e LEAD (251) IDENTIFICATION
Standard solution: 5 mL of Diluted standard lead e A. INFRARED ABSORPTION (197K)
solution Sample: Undried sample
Test Preparation: Add 1.0 g to 20 mL of nitric acid in a Acceptance criteria: Meets the requirements
250-mL conical flask, mix, and heat carefully until the e B. Potassium Benzoate imparts a violet color to a
Potassium Alginate is dissolved. Continue the heating nonluminous flame. Because the presence of small quan-
until the volume is reduced to 7 mL. Cool rapidly to tities of sodium masks the color, screen out the yellow
room temperature, transfer to a 100-mL volumetric color produced by sodium by viewing through a blue
flask, and dilute with water to volume. Use a 50-mL filter that blocks emission at 589 nm (sodium) but is
sample of the Test Preparation. transparent to emission at 404 nm (potassium). [NOTE—
Analysis: Proceed as directed in the chapter using Traditionally, cobalt glass has been used, but other suita-
15 mL of ammonium citrate solution, 3 mL of potas- ble filters are commercially available.]
sium cyanide solution, and 0.5 mL of hydroxylamine hy- e C. The retention time of the major peak of the Sample
drochloride solution being used for the test. After the solution corresponds to that of the Standard solution, as
first dithizone extractions, wash the combined chloro- obtained in the Assay.
form layers with 5 mL of water, discarding the water
ASSAY
layer and continuing in the usual manner by extracting
with 20 mL of 0.2 N nitric acid. © PROCEDURE
Acceptance criteria: 50.0 mL portion of the Test prepa- Solution A: Adjust a 20 mM solution of monobasic po-
ration contains NMT 5 Wg of lead (corresponding to tassium phosphate with phosphoric acid to a pH of 2.5.
NMT 10 ppm of lead) Mobile phase: Solution A ana acetonitrile (70:30)
Diluent: Water and acetonitrile (50:50)
System suitability solution: 0.1 mg/mL of USP Salicylic
Delete the following: Acid RS and 0.1 mg/mL of USP Benzoic Acid RS in
Diluent
°e HEAVY METALS, Method Ii (231) Standard solution: 0.1 mg/mL of USP Benzoic Acid RS
Analysis: Conduct the ignition in a platinum crucible, in Diluent
and use nitric acid in place of sulfuric acid to wet the Sample solution: 0.1 mg/mL of Potassium Benzoate in
sample. Diluent
Acceptance criterias NMT 40 ppme coitcat 1-Jan-2018) Chromatographic system
(See Chromatography (621), System Suitability.)
SPECIFIC TESTS Mode: LC
e ARTICLES OF BOTANICAL ORIGIN, Total Ash (561) Detector: UV 230 nm
Analysis: Proceed as directed for Total Ash under Meth- Column: 4.6-mm x 15-cm; 5-um packing L1
ods of Analysis, carefully igniting 3 g in a tared platinum Column temperature: 25°
dish, until the residue is thoroughly carbonized (5 min), Flow rate: 1.0 mL/min
and then igniting in a muffle furnace at a temperature Injection volume: 10 ul
of 800 + 25° until the carbon is completely burned off System suitability
(approximately 75 min). Samples: System suitability solution and Standard
Acceptance criteria: 24.0%-32.0% of ash is found, cal- Solution
culated on the as-is basis. [Note—The relative retention times for benzoic acid
© MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI- and salicylic acid are approximately 1 and 1.2,
FIED MICROORGANISMS (62): The total aerobic microbial respectively.]
count does not exceed 103 cfu/g, and the total com- Suitability requirements
bined molds and yeasts count does not exceed 10? cfu/ Resolution: NLT 3.0 between benzoic acid and sali-
g. cylic acid, System suitability solution
“
e Loss ON DRYING (731): Dry a sample at 105° for 4 h: it Tailing factor: NMT 2.0 for benzoic acid, Standard
<£ loses NMT 15% of its weight. solution
oe Relative standard deviation: NMT 0.5% for benzoic
scs ADDITIONAL REQUIREMENTS acid, Standard solution
a e PACKAGING AND STORAGE: Preserve in well-closed contain- Analysis
° ers. No storage requirements specified.
re Samples: Standard solution and Sample solution
Co} Calculate the percentage of potassium benzoate
= (C7HsKOz) in the portion of Potassium Benzoate taken:
—
Fs Result = (ru/rs) x (Cs/Cu) x (Mr/M2) x 100
NF 36 Official Monographs / Potassium 5533

tu = peak area of benzoic acid from the Sample IDENTIFICATION

solution e A, IDENTIFICATION TESTS—GENERAL (191), Potassium: A so-
rs = peak area of benzoic acid from the Standard lution (1 in 25) meets the requirements.
solution e B. PH (791)
Cs = concentration of USP Benzoic Acid RS in the Sample solution: 0.1 mg/mL of Potassium Hydroxide
Standard solution, corrected for purity Acceptance criteria: NLT 10.5
(mg/mL)
Cu = concentration of Potassium Benzoate in the ASSAY
Sample solution (mg/mL) e TOTAL ALKALI
M, = molecular weight of potassium benzoate, Sample: 1.5 g of Potassium Hydroxide
160.21 Titrimetric system
M2 = molecular weight of benzoic acid, 122.12 (See Titrimetry (541).)
Acceptance criteria: 99.0%-101.0% on the anhydrous Mode: Direct titration
basis Titrant: 1.N sulfuric acid VS
Endpoint detection: Colorimetric
IMPURITIES Analysis: Dissolve the Sample in 40 mL of carbon diox-
ide-free water. Cool the solution to 15° and add phe-
Delete the following: nolphthalein TS. Titrate with 1 N sulfuric acid VS. At the
discharge of the pink color of the indicator, record the
volume of acid solution required, then add methyl or-
° HEAVY METALS (231) ange TS and continue the titration to a persistent pink
Test preparation: Dilute 4.0 g in 40 mL of water. Add, color. Each milliliter of 1 N sulfuric acid Is equivalent to
dropwise with vigorous stirring, 10 mL of 3 N hydro- 56.11 mg of total alkali, calculated as potassium hy-
chloric acid, and filter. Use 25 mL of the filtrate: droxide (KOH), and each milliliter of acid consumed in
Acceptance criteria: NMT 10 Ug/ge cticia i-jan-2018) the titration with methyl orange is equivalent to
SPECIFIC TESTS 138.2 mg of potassium carbonate (KzCOs3).
e ALKALINITY Acceptance criteria: NLT 85.0% of total alkali, calcu-
Sample: 29 lated as potassium hydroxide (KOH), including NMT
Analysis: Dissolve the Sample in 20 mL of hot water, 3.5% of potassium carbonate (K,CO3)
and add 2 drops of phenolphthalein TS. e¢ CONTENT OF POTASSIUM
Acceptance criteria: If a pink color is produced, it is Diluent: 1% hydrochloric acid solution
disc aged by the addition of 0.20 mL of 0.10 N sulfu- Sodium chloride solution: 0.2 g/mL of sodium chloride
ric acid. Blank solution: Transfer 2.0 mL of the Sodium chloride
e@ WATER DETERMINATION (921), Method I: NMT 1.5% solution to a 100-mL volumetric flask and dilute with
Diluent to volume.
ADDITIONAL REQUIREMENTS Standard stock solution: 57.21 ug/ml of potassium
e PACKAGING AND STORAGE: Preserve in well-closed chloride, previously dried at 105° at 2h, in water. This
containers. solution contains 30 g/mL of potassium.
e USP REFERENCE STANDARDS (11) Standard solutions: Transfer 2.0-, 4.0-, and 6.0-mL
USP Benzoic Acid RS portions of the Standard stock solution to separate
USP Potassium Benzoate RS 100-mL volumetric flasks. To each flask, add 2.0 mL of
USP Salicylic Acid RS the Sodium chloride solution. Dilute the content of each
flask with Diluent to volume and mix to obtain solutions
with known concentrations of 0.6, 1.2, and 1.8 g/mL
of potassium.
cane stock solution: 0.5 mg/mL of Potassium
Hydroxide
Potassium Carbonate—see Potassium Sample solution: Transfer 1.0 mL of the Sample stock
Carbonate General Monographs solution to a 250-mL volumetric flask. Add 5.0 mL of
the Sodium chloride solution and dilute with Diluent to
volume.
Instrumental conditions
Potassium Chloride—see Potassium (See Atomic Absorption Spectroscopy (852).)
Chloride General Monographs Mode: Atomic absorption spectrophotometry
Analytical wavelength: 766.5 nm (potassium emission
line)
Lamp: Potassium hollow-cathode
Flame: Air-acetylene
Potassium Citrate—see Potassium Citrate Blank: Blank solution
General Monographs Standard curve
Samples: Standard solutions
Plot: Absorbance values versus their corresponding
concentrations (g/mL) of potassium. The correlation
Potassium Hydroxide coefficient is NLT 0.999.
Analysis
sydesbouow 4N

KOH 56.11 Sample: Sample solution

Potassium hydroxide [1310-58-3].
DEFINITION
Potassium Hydroxide contains NLT 85.0% of total alkali, cal-
culated as potassium hydroxide (KOH), including NMT
3.5% of potassium carbonate (K,CO3). It also contains
NLT 59.9% of potassium.
[CauTion—Exercise great care in handling Potassium Hy-
droxide because it rapidly destroys tissues.]
From the Standard curve, determine the concentration
of potassium in the Sample solution.
Calculate the percentage of potassium in the portion of
Potassium Hydroxide taken:
Result = (Cs/Cu) x 100
Cs = concentration of potassium in the Sample
solution from the Standard curve (41g/mL)
 5534 Potassium / Official Monographs NF 36

Cu = concentration of Potassium Hydroxide in the DEFINITION

Sample solution (g/mL) Potassium Metabisulfite contains an amount of K2S20s
Acceptance criteria: NLT 59.9% equivalent to NLT 51.8% and NMT 57.6% of SO.
IMPURITIES IDENTIFICATION
e A. IDENTIFICATION TESTS—GENERAL, Potassium (191) and
Sulfite (191): A solution (1 in 20) meets the
Delete the following: requirements.
°e HEAVY METALS (231) ASSAY
Test preparation: 0.67 g of Potassium Hydroxide in a e PROCEDURE
mixture of 5 mL of water and 7 mL of 3 N hydrochloric Sample: 250 mg of Potassium Metabisulfite
acid. Heat to boiling, cool, and dilute with water to Titrimetric system
25 mL. (See Titrimetry (541).)
Acceptance criteria: NMT 30 119/ge cotticiat i-Jan-2019) Mode: Residual titration
e LIMIT OF SODIUM Titrant: 0.1 N iodine VS
Diluent: 1% hydrochloric acid solution Back titrant: 0.1 N sodium thiosulfate VS
Standard stock solution: 12.71 g/mL of sodium chlo- Endpoint detection: Visual
ride,previously dried at 105° for 2 h, in water. This Analysis: Transfer the Sample to a glass-stoppered coni-
solution contains 5 g/mL of sodium. cal flask containing 50.0 mL of 0.1 N iodine VS, and
Standard solutions: Transfer 1.0-, 10.0-, and 15.0-mL swirl to dissolve. Allow to stand for 5 min, protected
portions of the Standard stock solution to separate from light, and then add 1 mL of hydrochloric acid. Ti-
100-mL volumetric flasks. Dilute the content of each trate the excess iodine with 0.1 N sodium thiosulfate
flask with Diluent to volume and mix to obtain solutions VS, adding 3 mL of starch TS as the endpoint is
with known concentrations of 0.05, 0.5, and 0.75 ug/ approached.
mL of sodium. Calculate the percentage of SOz in the portion of Sam-
sane stock solution: 0.5 mg/mL of Potassium ple taken:
Hydroxide
Sample solution: 50 g/mL of Potassium Hydroxide in Result = {[(Vs — Vs) x Nx F]/W} x 100
Diluent, prepared from the Sample stock solution
Instrumental conditions Ve = Back titrant volume consumed by the Blank
(See Atomic Absorption Spectroscopy (852).) (mL)
Mode: Atomic absorption spectrophotometry Vs = Back titrant volume consumed by the Sample
Analytical wavelength: 589.0 nm (ml)
Lamp: Sodium hollow-cathode N = Back titrant actual normality (mEq/mL)
Flame: Air—acetylene F = equivalency factor, 32.03 mg/mEq
Blank: Diluent Ww = weight of the Sample (mg)
Standard curve Acceptance criteria: 51.8%-57.6% of SO2z
Samples: Standard solutions
Plot: Absorbance values versus their corresponding IMPURITIES
concentrations (uug/mL) of sodium. The correlation co- e IRON (241)
efficient is NLT 0.995. Test preparation: Dissolve 1.00g in 14 mL of dilute hy-
Analysis drochloric acid (2 in 7), and evaporate on a steam bat!
Sample: Sample solution to dryness. Dissolve the residue in 7 mL of dilute hydro-
From the Standard curve, determine the concentration chloric acid (2 in 7), and again evaporate to dryness.
of sodium in the Sample solution. Dissolve the resulting residue in a mixture of 2 mL of
Calculate the percentage of sodium in the portion of hydrochloric acid and 20 mL of water. Add 3 drops of
Potassium Hydroxide taken: bromine TS, and boil to expel the bromine. Cool, and
dilute with water to 47 mL.
Result = (Cs/Cu) x 100 Acceptance criteria: NMT 10 ppm

Cs = concentration of sodium in the Sample solution Delete the following:

from the Standard curve (g/mL)
Cu = concentration of Potassium Hydroxide in the
Sample solution (g/mL) °e HEAVY METALS, Method | (231)
Acceptance criteria: NMT 1.0% Test preparation: Dilute 2g in 20 mi of water, add
5 mL of hydrochloric acid, and evaporate on a steam
SPECIFIC TESTS bath to 1 mL. Dissolve the residue in 25 mL of water.
© INSOLUBLE SUBSTANCES Acceptance criteria: NMT 10 ppMee ‘oficial 1jan-2018)
Sample solution: 1g of Potassium Hydroxide in 20 mL
of water ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Preserve in well-fitted, tight
Acceptance criteria: The Sample solution is complete, containers, and avoid exposure to excessive heat.
clear, and colorless.
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in tight containers.
NF Monographs

Potassium Metaphosphate
(KPOs)n
Potassium Metabisulfite Metaphosphoric acid (HPO3), potassium salt;
Potassium metaphosphate;
K2S20s 222.32 Potassium polymetaphosphate [7790-53-6].
Disulfurous acid, dipotassium salt;
Dipotassium pyrosulfite [16731-55-8].
NF 36
DEFINITION
Potassium Metaphosphate is a straight-chain polyphosphate,
having a high degree of polymerization. It contains the
equivalent of NLT 59.0% and NMT 61.0% of phosphorus
pentoxide (P2Os).
IDENTIFICATION
eA.
Sample: 1 g, finely powdered
Analysis: Add the Sample, slowly and with vigorous stir-
ring, to 100 mL of sodium chloride solution (20 mg/
mL).
Acceptance criteria: A gelatinous mass is formed.
© B. IDENTIFICATION TESTS—GENERAL, Potassium (191)
Sample: 0.5
Analysis: Boia mixture of the Sample, 10 mL of nitric
acid, and 50 mL of water for 30 min, and cool.
Acceptance criteria: Meets the requirements
e C. IDENTIFICATION TESTS—GENERAL, Phosphate (191)
Sample: 0.59
Analysis: Boil a mixture of the Sample, 10 mL of nitric
acid, and 50 mL of water for 30 min, and cool.
Acceptance criteria: Meets the requirements
ASSAY
© PROCEDURE
Sample: 200mg
Titrimetric system
(See Titrimetry (541).)
Mode: Residual titration
Titrant: 1.N sodium hydroxide VS
Back-titrant: 1.N sulfuric acid VS
Endpoint detection: Visual
Analysis: Mix the Sample with 15 mL of nitric acid and
30 mL of water. Boil for 30 min, cool, and dilute with
water to 100 mL. Heat to 60°, add an excess of ammo-
nium molybdate TS, and heat at 50° for 30 min. Filter,
and wash the precipitate, first with 0.5 N nitric acid,
and then with 10 mg/mL of potassium nitrate until the
filtrate is no longer acid to litmus. Add 25 mL of water
to the precipitate, dissolve it in 50.0 mL of Titrant, and
add Pisagpeeey TS. Titrate the excess sodium hy-
droxide with Back-titrant. Each mL of Titrant is equiva-
lent to 3.086 mg of phosphorus pentoxide (P20s).
Acceptance criteria: 59.0%-61.0%
IMPURITIES
e LEAD (251)
Sample solution: 1 a Potassium Metaphosphate in
10 mL of 3 N hydrochloric acid
Acceptance criteria: The Sample solution contains NMT
5 ug of lead (corresponding to NMT 5 ppm of lead).
Delete the following:
®e HEAVY METALS, Method | (231)
Sample: Warm 1 g of Potassium Metaphosphate with
10 mL of 3 N hydrochloric acid until no more dissolves.
Add 15 mL of water, mix, and filter.
Acceptance criteria: NMT 20 1g/gecotticial 1-jan-2018)
© LIMIT OF FLUORIDE
Control: Water
Sample solution: Place 5.0
g of Potassium Metaphos-
phate, 25 mL of water, 50 mL of perchloric acid,
5 drops of 500 mg/mL silver nitrate, and a few glass
beads in a 250-mL distilling flask connected with a con-
denser and carrying a thermometer and a capillary
tube, both of “nich extend into the liquid. Connect a
small crepe funnel, filled with water, or a steam
generator to the capillary tube. Support the flask on a
distillation shield with a hole that exposes one-third of
the bottom of the flask to the flame. Distill into a
250-mL volumetric flask until the temperature reaches
135°. Add water from the funnel or introduce steam
Official Monographs / Potassium 5535
through the capillary to maintain the temperature be-
tween 135° and 140°. Continue the distillation until
225-240 mL has been collected, then dilute with water
to volume.
Analysis: Transfer 50.0 mL of the Sample solution to a
100-mL color-comparison tube, and transfer 50.0 mL of
the Control to a similar tube. Add to each tube 0.1 mL
ofa filtered solution of sodium alizarinsulfonate TS and
1 mL of freshly prepared 0.25 mg/mL hydroxylamine
hydrochloride. Add, dropwise and with stirring, 0.05 N
sodium hydroxide to the tube containing the distillate
until its color just matches that of the Control, which is
faintly pink. Then add to each tube 1.0 mL of 0.1 N
hydrochloric acid. From a buret, graduated in 0.05-mL
increments, add slowly to the tube containing the distil-
late, enough 0.25-mg/mL thorium nitrate solution so
that, after mixing, the color of the liquid just changes
to a faint pink. Note the volume of the solution added,
add the same volume to the Control, and mix. Then
add sodium fluoride TS (10 g/mL of fluorine) to the
Control from a buret to make the colors of the two
tubes match after dilution to the same volume. Mix,
and allow all air bubbles to escape before making the
final color comparison. Check the endpoint by adding 1
or 2 drops of sodium fluoride TS to the Control. A dis-
tinct change in color appears.
Acceptance criteria: 10 ug/g; the volume of sodium
fluoride TS required for the Control is NMT 1.0 mL.
SPECIFIC TESTS
© ViscosiTy—CAPILLARY METHODS (911)
Sample solution: Mix 300 mg with 200 mL of 3.5 mg/
mL sodium pyrophosphate, using a magnetic stirrer.
Analysis: Using an Ostwald-Fenske viscometer main-
tained at 25°, determine the viscosity of the clear solu-
tion obtained, or of the liquid phase of the mixture
obtained after 30 min of continuous stirring.
Acceptance criteria: 6.5-15 mPa-s
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in well-closed
containers.
Monobasic Potassium Phosphate
KH2PO4 136.09
Phosphoric acid, monopotassium salt;
Monopotassium phosphate [7778-77-0].
DEFINITION
Monobasic Potassium Phosphate, dried at 105° for 4 h, con-
tains NLT 98.0% and NMT 100.5% of monobasic potas-
sium phosphate (KH2PO,).
IDENTIFICATION
e A. IDENTIFICATION TESTS—GENERAL, Potassium (191)
Sample solution: 50 mg/mL
Acceptance criteria: Meets the requirements
e B. IDENTIFICATION TESTS—GENERAL, Phosphate (191)
Sample solution: 50 mg/mL
Acceptance criteria: Meets the requirements
sydesBbouow 4N
ASSAY
e PROCEDURE
Sample solution: Transfer 5 g of Monobasic Potassium
Phosphate, previously dried, to a 250-mL beaker. Add
100 mL of water and 5.0 mL of 1 N hydrochloric acid
VS, and stir until the assay specimen is completely
dissolved.
Titrimetric system
(See Titrimetry (541).)
 5536 Potassium / Official Monographs NF 36

Mode: Direct titration Calculate the content of fluoride in the portion of

Titrant: 1 .N sodium hydroxide VS
Endpoint detection: Potentiometrically
Analysis: Slowly titrate the excess acid in the Sample
solution, stirring constantly, with Titrant to the inflection
point occurring at about pH 4 (Vs7). Continue the titra-
tion with Titrant until the inflection point occurring at
about pH 8.8 is reached (Vs2).
Calculate the percentage of monobasic potassium phos-
phate (KH2PO,) in the sample taken:
Result = {[(Vsz — Vs1) x N x FI/W} x 100
Titrant volume consumed by the Sample
S&S
Monobasic Potassium Phosphate taken:
Result = (Vx ©/W
Vv = volume of the Sample solution (mL)
Cc = concentration of fluoride ion, determined from
the Standard response line, in the Sample
solution (ug/mL)
w = weight of Monobasic Potassium Phosphate
taken to prepare the Sample solution (g)
Acceptance criteria: NMT 10 g/g
SPECIFIC TESTS

iow

solution to the second inflection point (mL) © INSOLUBLE SUBSTANCES

Titrant volume consumed by the Sample Sample solution: 10g in 100 mL of hot water
Ss
e

solution to the first inflection point (mL) Analysis: Filter the Sample solution throughatared fil-
N = actual normality of the Titrant (mEq/mL) tering crucible, wash the insoluble residue with hot
F = equivalency factor, 0.1361 g/mEq water, and dry at 105° for 2 h.
Ww = weight of monobasic potassium phosphate Acceptance criteria: NMT 20 mg (0.2%)
taken to prepare the Sample solution (g) e Loss ON DRYING (731)
Acceptance criteria: 98.0%-100.5% on the previously Analysis: Dry a sample at 105° for 4 h.
dried basis Acceptance criteria: NMT 1.0%
IMPURITIES ADDITIONAL REQUIREMENTS
© ARSENIC, Method | (211): 3 ug/g ¢ PACKAGING AND STORAGE: Preserve in tight containers.
e LEAD (251) e USP REFERENCE STANDARDS (11)
Test preparation: 1g in 20 mL of water USP Sodium Fluoride RS
Acceptance criteria: NMT 5 ug/g

Delete the following:

Potassium Sorbate
®o HEAVY METALS, Method | (231)
Test preparation: 40 mg/mL
Acceptance criteria: NMT 20 ug/ge coiticiat 14an-2018) MAB
o LIMIT OF FLUORIDE °
[Note—Prepare and store all solutions in plastic
containers.]
Buffer solution: 294 mg/mL of sodium citrate CsH7KO2 150.22
dihydrate 2,4-Hexadienoic acid, (£,6)-, potassium salt;
Standard stock solution: 1.1052 mg/mL of USP So- 2,4-Hexadienoic acid, potassium salt;
dium Fluoride RS Potassium (E,£)-sorbate;
Standard solution: Dilute 20.0 mL of Standard stock so- Potassium sorbate [590-00-1; 24634-61-5].
lution and 50.0 mL of Buffer solution with water to DEFINITION
100 mL. Each mL of this solution contains 100 yg of Potassium Sorbate contains NLT 98.0% and NMT 101.0%
fluoride ion. of CsH7KOz2, calculated on the dried basis.
Sample solution: Transfer 2.0 g of Monobasic Potas-
sium Phosphate to a beaker containing a plastic-coated IDENTIFICATION
stirring bar. Add 20 mL of water and 2.0 mL of hydro- e A. IDENTIFICATION TESTS—GENERAL, Potassium (191)
chloric acid, and stir until dissolved. Add 50.0 mL of Sample: Dissolve 1 g of Potassium Sorbate in 10 mL of
Buffer solution and sufficient water to make 100 mL. water.
Electrode system: Use a fluoride-specific ion-indicatin Acceptance criteria: Meets the requirements
electrode andasilver-silver chloride reference electrode ° B.
connected to a pH meter pave of measuring poten- Sample: 0.2g of Potassium Sorbate
tials with a minimum reproducibility of +0.2 mV (see pH Analysis: Dissolve the Sample in 2 mL of water, and add
(791)). a few drops of bromine TS.
Analysis Acceptance criteria: The color is discharged.
Standard response line: Transfer 50.0 mL of Buffer so-
lution and 2.0 mL of hydrochloric acid to a beaker, and ASSAY
add water to make 100 mL. Add a plastic-coated stir- e¢ PROCEDURE
ring bar, insert the electrodes into the solution, stir for Sample: 300mg of Potassium Sorbate
15 min, and read the potential, in mV. Continue stir- Blank: 40 mL of glacial acetic acid
ting, and at 5-min intervals add 100, 100, 300, and Titrimetric system
”“ 500 uL of Standard solution, reading the potential 5 (See Titrimetry (541).)
a= min after each addition. Plot the logarithms of the cu- Mode: Direct titration
ro
is} mulative fluoride ion concentrations (0.1, 0.2, 0.5, and Titrant: 0.1 N perchloric acid VS
1.0 ug/mL) versus potential, in mV. Endpoint detection: Visual
aD
_

ce} Rinse and dry the electrodes, insert them into the
= Sample solution, stir for 5 min, and read the potential
Sj in mV. From the measured potential and the Stan-
= dard response line determine the concentration, C (in
i g/mL), of fluoride ion in the Sample solution.
rs
Analysis: Dissolve the Sample in 40 mL of glacial acetic
acid, warming, if necessary, to dissolve the solution.
Cool to room temperature, and add 1 drop of crystal
violet TS. Titrate with Titrant to a blue-green endpoint.
Perform a blank determination.
NF 36
Calculate the percentage of potassium sorbate
(CéH7KOz) in the Sample taken:
Result = {[(Vs — Vs) x N x F]/W} x100
Vs = Titrant consumed by the Sample (mL)
Ve = Titrant consumed by the Blank (mL)
N = actual normality of the Titrant (mEq/mL)
F = equivalency factor, 150.2 mg/mEq
Ww = Sample weight (mg)
Acceptance criteria: 98.0%-101.0% on the dried basis
IMPURITIES
Delete the following:
°e HEAVY METALS, Method II (231): NMT 10 ppme coricatt-
Jan-2018)
SPECIFIC TESTS
¢ ACIDITY OR ALKALINITY
Sample solution: 1.1 g of Potassium Sorbate in 20 mL
of water
Analysis: Add phenolphthalein TS to the Sample
solution.
Acceptance criteria: If the solution is colorless, titrate
with 0.10 N sodium hydroxide to a pink color that per-
sists for 15 s: NMT 1.1 mL of 0.10 N sodium hydroxide
is required. If the solution is pink in color, titrate with
0.10 N hydrochloric acid to discharge the pink color:
NMT 0.80 mL of 0.10 N hydrochloric acid is required.
e Loss ON DRYING (731): Dry a sample at 105° for3 h: it
loses NMT 1.0% of its weight.
ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Preserve in tight containers,
rotected from light, and avoid exposure to excessive
eat.
Povidone—see Povidone General
Monographs
Propane
C3He 44.10
[74-98-6].
DEFINITION
Propane contains NLT 98.0% of propane (C3Hs).
[CautTion—Propane is highly flammable and explosive.]
IDENTIFICATION
e A. INFRARED ABSORPTION: Exhibits maxima, amon
others, at the following wavelengths, in um: 3.4 v9, 6.8
(s), and 7.2 (m).
B.
Sample: Use an empty stainless steel cylinder equipped
with a stainless steel valve, having a capacity of NLT
200 mL, and a pressure rating of 240 psi or more. Dry
the cylinder with the valve open at 110° for 2 h, and
evacuate the hot cylinder to less than 1 mm of mercury.
Close the valve, cool, and weigh. Connect one end o
a charging line tightly to the propane container and the
other end loosely to the empty cylinder. Carefully open
the propane container, and allow the propane to flush
out the charging line through the loose connection.
Avoid excessive flushing, which causes moisture to
freeze in the charging line and connections. Tighten the
fitting on the empty cylinder, and open the empty cyl-
Official Monographs / Propane 5537
inder valve, allowing the propane to flow into the evac-
uated cylinder. Continue sampling until the desired
amount of propane is obtained, then close the propane
container valve, and finally close the sample cylinder
valve. [CAUTION—Do not overload the sample cylinder;
hydraulic expansion due to temperature change can
cause overloaded cylinders to explode.] Weigh the
charged sample cylinder, and determine the weight.
Analysis: Determine the vapor pressure of the Sample at
21° by means of a suitable pressure gauge.
Acceptance criteria: 820-875 kPa absolute
(119-127 psia)
ASSAY
© PROCEDURE
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: GC
Detector: Thermal conductivity
Column: 6-m x 3-mm aluminum; acked with 10
weight percent of liquid phase G3 on support $1D
Column temperature: 33°
Carrier gas: Helium
Flow rate: 50 mL/min
Injection volume: 2 uL
System suitability
Sample: Propane
Suitability requirements: The peak responses for Pro-
pane from duplicate determinations agree within 1%.
Analysis: Connect one Propane cylinder to the chro-
matograph through a suitable sampling valve and a
flow control valve downstream from the sampling valve.
Flush the liquid specimen through the sampling valve,
taking care to avoid entrapment of gas or air in the
sampling valve. Calculate the percentage purity by di-
viding 100 times the propane response by the sum of
all of the responses.
Acceptance criteria: NLT 98.0%
SPECIFIC TESTS
e HIGH-BOILING RESIDUES
Sample: Use the Sample from Identification test B.
Analysis: Prepare a cooling coil from copper tubin
(about 6-mm outside diameter x about 6.1-m long) to
fit into a vacuum-jacketed flask. Immerse the cooling
coil in a mixture of dry ice and acetone in a vacuum-
jacketed flask, and connect one end of the tubing to
the Sample. Carefully open the sample cylinder valve,
flush the cooling coil with about 50 mL of the Sample,
and discard this portion of liquefied sample. Continue
delivering liquefied sample from the cooling coil, and
collect it in a previously chilled 1000-mL sedimentation
cone until the cone is filled to the 1000-mL mark. Allow
the sample to evaporate, using a warm water bath
maintained at about 40° to reduce evaporating time.
When all of the liquid has evaporated, rinse the sedi-
mentation cone with two 50-mL portions of pentane,
and combine the rinsings in a tared 150-mL evaporat-
ing dish. Transfer 100 mL of the pentane solvent to a
second tared 150-mL evaporating dish, place both
evaporating dishes on a water bath, evaporate to dry-
ness, and heat the dishes in an oven at 100° for 60
min. Cool the dishes in a desiccator, and weigh. Repeat
the heating for 15-min periods until successive weigh-
ings are within 0.1 mg, and calculate the weight of the ra
residue obtained from the Sample as the difference be- baa
tween the weights of the residues in the two evaporat-
ing dishes.
=
fe)
Acceptance criteria: NMT 5 ug/mL |
© ACIDITY OF RESIDUE °
=}
Sample solution: Add 10 mL of water to the residue =e
obtained in the test for High-Boiling Residues, mix by 2
mo]
swirling for 30 s, add 2 drops of methyl! orange TS, in- >
sert the stopper in the tube, and shake vigorously. 7
 5538 Propane / Official Monographs NF 36

Acceptance criteria: No pink or red color appears in Table 1

the aqueous layer. Hold Time
e Limit OF SULFUR COMPOUNDS Initial Temperature Final at Final
Analysis: Carefully open the container valve to produce Temperature Ramp Temperature | Temperature
a moderate flow of gas. Do not direct the gas stream (¢/min) ©) (min)
(*)
toward the face, but deflect a portion of the stream
50 15, 200 =
toward the nose.
Acceptance criteria: The odor is free from the charac- 200 40 250 IZ
teristic odor of sulfur compounds.
e@ WATER DETERMINATION (921) Carrier gas: Helium
Sample: 100g of the Sample from Identification test B Flow rate: 1.1 mL/min
Analysis: Proceed as directed in the chapter with the Injection volume: 0.2 LL
following modifications. (a) Provide the closed-system Split type: Split ratio of 18:1
titrating vessel with an opening through which passes a System suitability
coarse-porositygas dispersion tube connected to a sam- Sample: System suitability solution
pling cylinder. (b) Dilute the Reagent with anhydrous [NotE—The relative retention times for propylene glycol
methanol to give a water equivalence factor o' and propanediol are 0.7 and 1.0, respectively.]
0.2-1.0 mg/mL; age this diluted solution for NLT 16 h Suitability requirements
before standardization. (c) Introduce the Sample into Resolution: NLT 2.0 between the peaks due to pro-
the titration vessel through the gas dispersion tube at a pylene glycol and propanediol
rate of about 100 mL/min; if necessary, heat the sample Analysis
cylinder gently to maintain this flow rate. Sample: Sample solution
Acceptance criteria: NMT 0.001% Calculate the percentage of propanediol in the portion
of sample taken:
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in tight cylinders, and Result = (ru/rz) x 100
prevent exposure to excessive heat.
tu = peak response for propanediol in the Sample
solution
rr = sum of all peak responses in the Sample
solution
Acceptance criteria: NLT 99.7%
Propanediol
IMPURITIES
Ho er OE e Limit OF RELATED GLYCOL SUBSTANCES
System suitability solution, Sample solution, Chromat-
ographic system, and System suitability: Proceed as
C3HgO2 76.09
directed in the Assay.
1,3-Propanediol; Analysis
1,3-Dihydroxypropane; Sample: Sample solution
Propane, 1-3-diol; Calculate the percentage of each individual impurity in
Trimethylene glycol [504-63-2]. the portion of Propanediol taken:
DEFINITION
Propanediol contains NLT 99.7% of 1,3-propanediol Result = (ru/rr) x 100
(C3HsO2z). It may be of vegetable, other natural source, or ty = peak response of each individual impurity in
synthetic origin. the Sample solution
IDENTIFICATION rr = sum of all peak responses in the Sample
e A. INFRARED ABSORPTION (197F) solution
e B. The retention time of the major peak of the Sample Acceptance criteria
solution corresponds to the 1,3-propanediol peak of the Each individual impurity: NMT 0.1%
System suitability solution, as obtained in the Assay. Total impurities: NMT 0.3%
e LIMIT OF ALDEHYDES
ASSAY Formaldehyde methanol solution:' A solution contain-
© PROCEDURE ing 37%Cited) of formaldehyde and 10%-15% (w/w)
System suitability solution: Mix quantities of USP Pro- of methanol in water
pylene Glycol RS and USP 1,3-Propanediol RS to obtain Phenolphthalein solution: Dissolve 0.1 g of phenol-
a solution containing about 5% propylene glycol and phthalein in 80 mL of alcohol, and dilute with water to
95% propanediol. 100 mL.
Sample solution: Propanediol (neat) Quantification of Formaldehyde methanol solution:
Chromatographic system To 2.0 g of Formaldehyde methanol solution, add 100 mL
(See Chromatography (621), System Suitability.) of a freshly prepared 100 mg/mL solution of sodium
Mode: GC sulfite in carbon-dioxide free water. Add 0.1 mL of Phe-
Detector: Flame ionization nolphthalein solution, and titrate with 0.5 N sulfuric acid
Column: 0.25-mm x 30-m capillary column; bonded until the color changes from pink to colorless. Carry out
a blank titration.
NF Monographs

with a 0.25-1um layer of phase G16

Temperatures Calculate the percentage content of formaldehyde in
Detector: 250° Formaldehyde methanol solution using the following
Injection port: 250° expression:
Column: See Table 1.
Result (Pucno) = {[(Vs — Va) x N x Mw x F]/W} x 100
Vs = volume of 0.5 N sulfuric acid used in the assay
(mL)
1 Formaldehyde TS can be used for Formaldehyde methanol solution.
NF 36 Official Monographs / Propionic 5539

Ve = volume of 0.5 N sulfuric acid used in the SPECIFIC TESTS

blank (mL) e ACIDITY
N = normality of the titrant (mEgq/mL) Sample: 50 mL of Propanediol
My = milliequivalent weight of formaldehyde, Phenolphthalein solution: Dissolve 0.1 g of phenol-
30.03 mg/mEq phthalein in 80 mL of alcohol, and dilute with water to
F = unit conversion factor, 10-3 g/mg 100 mL.
Ww = weight of sample (g) Titrimetric system
Standard stock solution: 1.2 g/mL of Formaldehyde (See Titrimetry (541))
methanol solution in carbon-dioxide free water, pre- Mode: Direct titration
pared from appropriately diluting Formaldehyde metha- Titrant: 0.01 N sodium hydroxide VS
nol solution in carbon-dioxide free water Endpoint detection: Visual
Standard solutions: Introduce into 50-mL volumetric Analysis: To 50 mL of water, add 1 mL of Phenolphthal-
flasks 1.0-, 3.0-, 5.0-, 10.0-, 15.0-, and 25.0-mL of ein solution, then add Titrant until the solution remains
Standard stock solution, respectively. Calculate the con- pink for 30 s. Add the Sample, and titrate with Titrant
tent of formaldehyde, in yg, in the Standard solutions until the color turns back to pink and remains for more
using the following expression. Proceed as directed in than 30 s.
the Analysis below. Calculate the acidity, as acetic acid (CH3COOH):
Result (content of formaldehyde) = V x C x Pucho x 0.01 Result = (Vz x N x Wyeq)/Vs
Vv = volume of the Standard stock solution added Vr Titrantvolume (mL)
oat
into the Standard solution (mL) N Titrant normality (mEq/mL)
G = concentration of Formaldehyde methanol Wwtq = milliequivalent weight of acetic acid,
solution in the Standard stock solution 60.05 mg/mEq
(g/mL) Vs = volume of Propanediol in the Sample (mL)
Pucuo = percentage content of formaldehyde in Acceptance criteria: NMT 0.1 mg/mL, calculated as
Formaldehyde methanol solution, as acetic acid (CH3COOH)
determined above e@ WATER DETERMINATION, Method Ic (921): NMT 0.1%
Sample solution: Introduce 5.0 mL of 0.2 g/mL of
propanediol in carbon-dioxide free water into a 50-mL ADDITIONAL REQUIREMENTS
volumetric flask. Proceed as directed in the Analysis © PACKAGING AND STORAGE: Preserve in well-closed contain-
below. ers. Do not store above 50°. Protect from moisture.
Blank solution: Prepare in the same manner as for the e LABELING: Label it to indicate whether Propanediol is de-
Standard solutions but omitting the Standard stock rived from vegetable, other natural source, or synthetic
solution. origin.
Instrumental conditions ° UsP REFERENCE STANDARDS (11)
(See Ultraviolet-Visible Spectroscopy (857).) USP 1,3-Propanediol RS
Mode: Vis spectrophotometry USP Propylene Glycol RS
Analytical wavelength: 655 nm
Analysis
Samples: Blank solution, Standard solutions, and Sample
solution
To each flask of the Blank solution, Standard solutions, Propionic Acid
and Sample solution add 2 mL of a freshly prepared
5 mg/mL solution of methylbenzothiazolone DEFINITION
hydrazone hydrochloride adjusted with 0.02 N sodium Propionic Acid contains NLT 99.5% and NMT 100.5%, by
hydroxide to a pH of 4.0. Allow the solutions to stand weight, of propionic acid (C3H6Oz).
for 30 min. Add 5 mL of a freshly prepared 7 main,
solution of ferric chloride. Cap anasiid the flasks. Al- ASSAY
low to stand for 5 min. Add methanol to each flask, ¢ PROCEDURE
and dilute with methanol to 50.0 mL. Mix thoroughly, Sample: 1.5 g of Propionic Acid
then allow to stand for 1 min. Titrimetric system
Measure the absorbance of the solutions using the (See Titrimetry (541).)
treated Blank solution as compensation liquid. Mode: Direct titration
Plot the absorbance of the treated Standard solution ver- Titrant: 0.5 N sodium hydroxide VS
sus the content of formaldehyde, in 1g, in the Stan- Endpoint detection: Visual
dard solution. Obtain the content of formaldehyde Analysis: Mix the Sample with 100 mL of recently
Wrcro, in ug, in the treated Sample solution based on boiled and cooled water in a 250-mL conical flask. Add
the calibration curve. phenolphthalein TS to the Sample solution, and titrate
Calculate the content of aldehydes expressed as formal- with Titrant to the first appearance of a faint pink
dehyde (HCHO) in the portion of Propanediol taken: endpoint that persists for NLT 30 s.
Calculate the percentage of propionic acid (C3H¢O2) in
Result = Wucno/(C x V) the sample taken:
Wucro = content of formaldehyde in the treated Sample Result = {[(Vs — Vs) x N x F]/W} x 100
sydesbouo= 4N

Solution, determined from the calibration

curve (ig) volume of Titrant consumed by the sample
S

C = concentration of Propanediol in the Sample (mL)

solution (g/mL) volume of Titrant consumed by the blank (mL)
=U25

V = i of the Sample solution in the analysis actual normality of the Titrant (mEq/mL)
(mL equivalency factor, 74.08 mg/mEq
Acceptance criteria: NMT 20 g/g, expressed as weight of the sample (mg)
HCHO.
 5540 Propionic / Official Monographs NF 36

Acceptance criteria: 99.5%-100.5% by weight ADDITIONAL REQUIREMENTS

e PACKAGING AND STORAGE: Preserve in tight containers.
IMPURITIES
e Limit OF NONVOLATILE RESIDUE: Evaporate 20 g of Propi-
onic Acid in a tared dish, and dry at 105° for 1 h: the
weight of the residue does not exceed 2.0 mg.
Propyl Gallate
Delete the following:

°e HEAVY METALS (231)

Test preparation: To the residue obtained in the test
for Limit of Nonvolatile Residue add 8 mL of 0.1 N hy-
drochloric acid, warm gently until solution is complete,
dilute with water to 100 mL, and use 10 mL of the
solution. CioHi20s 212.20
Acceptance criteria: NMT 10 ppMe cotticial 14an-2018) Benzoic acid, 3,4,5-trihydroxy-, propyl ester;
e LIMIT OF ALDEHYDES Propyl gallate [121-79-9].
Sample solution: Transfer 10.0 mL of Propionic Acid to
a glass-stoppered 250-mL conical flask containing DEFINITION
50 mL of water and 10.0 mL of sodium bisulfite solution Propyl Gallate contains NLT 98.0% and NMT 102.0% of
(1 in 80), insert the stopper, and shake vigorously. Al- propyl gallate (CioH12Os), calculated on the dried basis.
low the mixture to stand for 30 min.
Blank: Add 50 mL of water and 10.0 mL of sodium bi- IDENTIFICATION
sulfite solution (1 in 80) to a glass-stoppered 250-mL e A. INFRARED ABSORPTION (197M)
conical flask, insert the stopper, and Fake vigorously. e B. ULTRAVIOLET ABSORPTION (197U)
Allow the mixture to stand for 30 min. Sample solution: 10 g/mL in methanol
Titrimetric system Acceptance criteria: Meets the requirements
(See Titrimetry (541).) ASSAY
Mode: Residual titration e PROCEDURE
Titrant: 0.1 N iodine VS Standard solution: 10 g/mL of USP Propyl Gallate RS
Endpoint detection: Visual in methanol
Analysis: Titrate the Sample solution and the Blank with Sample solution: 10 g/mL of Propyl Gallate in
Titrant to the same brownish-yellow endpoint. methanol
Acceptance criteria: The difference between the vol- Blank: Methanol
ume of 0.1 N iodine required for the Blank and that Instrumental conditions
required for the Sample solution is NMT 1.75 mL. (See Ultraviolet-Visible Spectroscopy (857).)
SPECIFIC TESTS Mode: UV
e DISTILLING RANGE, Method | (721): 138.5°-142.5° Analytical wavelength: Maximum at about 273 nm
e SPECIFIC GRAVITY (841): 0.988-0.993 Cell: 71cm
e READILY OXIDIZABLE SUBSTANCES Analysis
Sample solution: Dissolve 15 g of sodium hydroxide in Samples: Standard solution and Sample solution
50 mL of water. Cool, add 6 mL of bromine, stirring to Calculate the percentage of propyl gallate (C,oH12Os) in
effect complete solution, and dilute with water to the portion of the sample taken:
2000 mL. Transfer 25.0 mL of this solution to a glass-
stoppered, 250-mL conical flask containing 100 mL of Result = (Au/As) x (Cs/Cu) x 100
water, and add 10 mL of sodium acetate solution (1 in Au = absorbance of the Sample solution
5) and 10.0 mL of Propionic Acid. Allow to stand for 15 As = absorbance of the Standard solution
min, and add 5 mL of potassium iodide solution (1 in Cs = concentration of USP Propyl Gallate RS in the
4) and 10 mL of hydrochloric acid. Standard solution (g/mL)
Blank: Dissolve 15 g of sodium hydroxide in 50 mL of Gu = concentration of the Sample solution (g/mL)
water. Cool, add 6 mL of bromine, stirring to effect Acceptance criteria: 98.0%-102.0% on the dried basis
complete solution, and dilute with water to 2000 mL.
Transfer 25.0 mL of this solution to a glass-stoppered, IMPURITIES
250-mL conical flask containing 100 mL of water, and e RESIDUE ON IGNITION (281); NMT 0.1%
add 10 mL of sodium acetate solution (1 in 5). Allow to
stand for 15 min, and add 5 mL of potassium iodide
solution (1 in 4) and 10 mL of hydrochloric acid. Delete the following:
Titrimetric system
(ce Titrimetry (541).) ®e HEAVY METALS, Method // (231): NMT 10 ppme cfcia 1.
ode: Residual titration jar-2018)
Titrant: 0.1 N sodium thiosulfate VS
Endpoint detection: Visual SPECIFIC TESTS
Analysis: Titrate the Sample solution and the Blank with e MELTING RANGE OR TEMPERATURE (741): 146°-150°
im
”
Titrant just to the disappearance of the brown color. ® Loss ON DRYING (731): Dry a sample at 105° for 4 h: it
ra loses NMT 0.5% of its weight.
3 Acceptance criteria: The difference between the vol-
— ume of 0.1 N sodium thiosulfate required for the Blank
Dp ADDITIONAL REQUIREMENTS
fe) and that required for the Sample solution is NMT © PACKAGING AND STORAGE: Preserve in tight containers,
iS 2.2 mL. protected from light, and avoid contact with metals.
So e USP REFERENCE STANDARDS (11)
= USP Propyl Gallate RS
J
=
NF 36 Official Monographs / Propylene 5541

Propylene Glycol—see Propylene Glycol

Propylene Carbonate General Monographs
2
Soe
HsC’ sg
Propylene Glycol Alginate
C4H6O3 102.09 DEFINITION
4-Methyl-1,3-dioxolan-2-one; Propylene Glycol Alginate is a propylene glycol ester of al-
Cyclic propylene carbonate [108-32-7]. ginic acid. Each gram yields NLT 0.16 and NMT 0.20 g of
carbon dioxide, calculated on the dried basis.
DEFINITION
Propylene Carbonate contains NLT 99.0% and NMT IDENTIFICATION
100.5% of propylene carbonate (C4H.6O3). ° A.
Sample solution: Place 20 mL of the saponified solution
IDENTIFICATION
obtained in the determination of Esterified Carboxyl
A. INFRARED ABSORPTION (197F) Groups in a 250-mL conical flask. Add 50 mL of a solu-
ASSAY tion of periodic acid (1 in 50), swirl, and allow to stand
e PROCEDURE for 30 min. Add 2 g of potassium iodide, titrate with
Barium hydroxide solution: 75 mg/mL of barium hy- sodium thiosulfate TS to a faint yellow color, dilute the
droxide (octahydrate). Filter the solution before use. mixture with water to 200 mL, and mix to obtain the
Sample: 600mg Sample solution for Identification test A and Identification
Titrimetric system test B.
(See Titrimetry (541).) Modified Schiff’s reagent: Dissolve 200 mg of rosani-
Mode: Direct titration line hydrochloride (C2oHaoCIN3) in 120 mL of hot water.
Titrant: 0.5 N hydrochloric acid VS Cool, add 2 g of sodium bisulfite (NaHSOs), followed by
Endpoint detection: Visual 2 mL of hydrochloric acid, and dilute with water to
Analysis: Flush a 250-mL iodine flask with nitrogen to 200 mL. [NoTe—Store this solution in a brown bottle at
expel the air, and insert the stopper in the flask to ex- 15° or lower.]
clude carbon dioxide. Transfer 50.0 mL of Barium hy- Analysis: To 10 mL of the Sample solution add 5 mL of
droxide solution and the Sample to the flask, and loosely hydrochloric acid and 10 mL of Modified Schiff’s reagent.
reinsert the stopper. Moisten the stopper with 3 drops Acceptance criteria: A blue to blue-violet color, due to
of water, and heat the flask on a steam bath for 10 formaldehyde, develops in about 20 min.
min. Remove the flask from the steam bath, add e B.
6 drops of phenolphthalein TS, and titrate while hot Analysis: To 10 mL of the Sample solution prepared in
with Titrant until only a trace of pink color remains. Identification test A add 1 mL of a saturated solution of
Perform a blank determination, using the same Barium piperazine and 0.5 mL of sodium nitroferricyanide TS.
hydroxide solution. Each mL of 0.5 N hydrochloric acid Acceptance criteria: A green color, due to acetalde-
consumed is equivalent to 25.52 mg of propylene car- hyde, develops.
bonate (C4H¢O3). ASSAY
Acceptance criteria: 99.0%-100.5% ¢ CONTENT OF ALGINATE
IMPURITIES Analysis: Proceed as directed for Procedure in Alginates
e RESIDUE ON IGNITION (281): NMT 0.01% Assay (311), without preliminary drying of the Propyl-
ene Glycol Alginate.
SPECIFIC TESTS Acceptance criteria: 0.16-0.20 g of carbon dioxide/g
© SPECIFIC GRAVITY (841): 1.203-1.210 at 20° of Propylene Glycol Alginate, calculated on the dried
PH (791) basis
Sample solution: Mix 10 mL of propylene carbonate
with 0.3 mL of saturated potassium chloride solution in OTHER COMPONENTS
a 100-mL borosilicate volumetric flask. Dilute with car- o FREE CARBOXYL GROUPS
bon dioxide-free water having a pH of 7.0 + 0.5 to Sample: 1g
volume. Titrimetric system
Analysis: Completely purge the solution by vigorous ni- Mode: Direct titration
trogen bubbling, and continue the bubbling during the Titrant: 0.1 N sodium hydroxide VS
pH measurement. Determine the pH potentiometrically Endpoint detection: Potentiometric
when the reading stabilizes. Analysis: Transfer the Sample to a 600-mL beaker. Dis-
Acceptance criteria: 6.0-7.5 solve in 200 mL of water, stirring by mechanical means
for NLT 30 min. Titrate with 0.1 N sodium hydroxide
ADDITIONAL REQUIREMENTS VS to a pH of 7.0.
e PACKAGING AND STORAGE: Preserve in tight containers. Calculate the weight, in g, of free carboxyl groups in the
e USP REFERENCE STANDARDS (11) Sample taken:
USP Propylene Carbonate RS
Result = [(V
x M,
x N)/W] x F vA
Vv = Titrant volume consumed (mL) ay
M, = mEq of CO, 44 mg/mEq =
N = actual normality of the Titrant (mEq/mL) =
Ww = Sample weignt (g) 3}
F = conversion factor, 10-3 g/mg ee)
Acceptance criteria: The weight of free carboxyl ey
groups found, calculated on the dried basis, is NMT me]
35% of the weight of carbon dioxide yielded by an =
“
equal weight of specimen in the Assay.
USP 41
Mode: Atomic absorption spectrophotometry
Lamp: Zinc hollow-cathode
Flame: Air-acetylene
Analytical wavelength: Zinc emission line, 213.8 nm
Blank: 0.125 N hydrochloric acid
Analysis
Samples: Standard solutions and Sample solution
Determine the absorbances of the solutions against the
Blank. Plot the absorbances of the Standard solutions
versus concentration, in g/mL, of zinc, and draw the
straight line best fitting the five plotted points. From
the graph, determine the concentration, C, in ug/mL,
of zinc in the Sample solution.
Calculate the percentage of the labeled amount of zinc
(Zn) in the portion of Tablets taken:
Result = (C/Cy) x 100
c = measured concentration of zinc in the Sample
solution (uug/mL)
Cy — =nominal concentration of zinc in the Sample
solution (g/mL)
Acceptance criteria: 90.0%-125.0% of the labeled
amount of zinc (Zn)
e CALCIUM, COPPER, MAGNESIUM, MANGANESE, and ZINC,
Method 2
Stock aqua regia solution: Prepare a mixture of hydro-
chloric acid and nitric acid (3:1) by adding the nitric
acid to the hydrochloric acid. [NoTE—Periodically vent
the solution in an appropriate fume hood.]
Diluent: Prepare a mixture of Stock aqua regia solution
and water (1:9) by adding one volume of Stock aqua
regia solution to two volumes of water. Dilute with addi-
tional water to volume, and mix well.
System suitability solution: Prepare a mixture of
1000 mg/L of yttrium in 5% (v/v) nitric acid solution
and 1000 mg/L of scandium in 5% (v/v) nitric acid so-
lution with Diluent (1:1:198), and mix.
Standard stock solution (Ca, Cu, Mg, Mn, and Zn):
[Note—It is only necessary to include the minerals of
interest in the solution.] Using commercially available
element standard (single- or multi-element) solutions in
5% (v/v) nitric acid solution, pipet the appropriate
amount of element standard solution into a volumetric
flask, and dilute with 5% (v/v) nitric acid solution to
obtain a solution having final concentrations of about
1000 mg/L of calcium, 100 mg/L of copper, 500 mg/L
of magnesium, 100 mg/L of manganese, and 250 mg/L
of zinc.
Standard solutions: Prepare a mixture of Standard
stock solution in Diluent to prepare a six-point calibra-
tion curve to bracket the concentration range of each
mineral of interest.
Semple solution: Weigh and finely powder NLT 20
Tablets. Transfer a portion equal to the average Tablet
weight to a 250-mL volumetric flask. Slowly add 25 mL
of Stock aqua regia solution in 5-mL increments, fol-
lowed by mixing. [NoTE—If the sample contains a car-
bonate, bubbling will occur. Wait until bubbling ends
to proceed.] Bring the solution to a boil on a hot plate.
Continue to heat gently until fumes cease (about 1 h).
Remove from heat, cool, and dilute with water to vol-
ume, Filter about 30 mL into a centrifuge tube usin
5-uum pore size nylon syringe filter. If necessary, make
any further dilutions using the Diluent.
Spectrometric conditions
(See Plasma Spectrochemistry {730).)
Mode: Inductively coupled plasma spectrometry, using
a spectrometer set to measure the emission of each
mineral of interest at about the corresponding wave-
length. [NoTte—The operating conditions may be de-
veloped and optimized based on the manufacturer’s
recommendation. The wavelengths selected should be
demonstrated experimentally to provide sufficient
Dietary Supplements / Calcium 4505
specificity, sensitivity, linearity, accuracy, and
precision.]
System a ee
[Note—Analyze the System suitability solution and obtain
the response as directed for Analysis.]
Suitability requirements
Relative standard deviation: NMT 2.0%
Analysis
Samples: Standard solutions and Sample solution
Determine the emission of each mineral of interest in
the Standard solutions and Sample solution with an in-
ductively coupled plasma system using the Diluent as
the blank. Plot the emission of the Standard solutions
versus concentration, in mg/L, of the minerals of inter-
est, and draw the straight line best fitting the plotted
points. From the graph, determine the concentration,
CG in mg/L, for each mineral of interest in the Sample
solution.
Calculate the percentage of the labeled amount for
each mineral:
Result = C x (V/W) x F x (W7/L) x 100
€ = measured concentration of the relevant
element in the Sample solution (mg/L)
V volume of the Sample solution (L)
Vout
Ww sample weight (mg)
F dilution factor of the Sample solution
W, T average Tablet weight (mg)
L labeled amount of the relevant element (mg/
Tablet)
Acceptance criteria: NLT 90.0%-125.0% of the labeled
amount of calcium (Ca), copper (Cu), magnesium
(Mg), manganese (Mn), and zinc (Zn).
PERFORMANCE TESTS
¢ DISINTEGRATION AND DISSOLUTION OF DIETARY SUPPLEMENTS
(2040): Meet the requirements for Dissolution with re-
spect to calcium
Medium: 0.1 N hydrochloric acid; 900 mL
Apparatus 2: 75 rom
Time: 30 min
Analysis: Determine the amount of calcium (Ca) dis-
solved, using the procedure in the assay for Calcium,
making any necessary volumetric adjustments.
Tolerances: NLT 75% of the labeled amount of Ca is
dissolved.
e@ WEIGHT VARIATION OF DIETARY SUPPLEMENTS (2091): Meet
the requirements
SPECIFIC TESTS
e MICROBIAL ENUMERATION TESTS (2021): The total aerobic
sydeiBouo-= sa
microbial count does not exceed 3000 cfu/g, and the
total combined molds and yeasts count does not exceed
300 cfu/g. Tablets also meet the requirements of the
tests for absence of Salmonella species, Escherichia coli,
and Staphylococcus aureus.
e ABSENCE OF SPECIFIED MICROORGANISMS (2022): Meet the
requirements of the tests for absence of Salmonella spe-
cies and Escherichia coli
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in tight, light-resistant
containers.
e LABELING: The label states that the product is Calcium
and Vitamin D with Minerals Tablets. The label also states
the quantities of each mineral and vitamin D/dosage
unit, the salt form of the mineral used as the source of
each element present, and the chemical form of vitamin
D present in the dosage unit.
e USP REFERENCE STANDARDS (11)
USP Cholecalciferol RS
USP Ergocalciferol RS
 5542 Propylene / Official Monographs NF 36

© ESTERIFIED CARBOXYL GROUPS Acceptance criteria: NMT 10.0% on the dried basis
Sample solution: The solution obtained in the test for
Free Carboxyl Groups ADDITIONAL REQUIREMENTS
Analysis: Transfer the Sample solution with the aid of e PACKAGING AND STORAGE: Preserve in well-closed
water to a 1000-mL conical flask. Add phenolphthalein containers.
TS and 50.0 mL of 0.1 N sodium hydroxide VS, insert a
stopper in the flask, mix, and allow to stand for 30 min
at ambient temperature. Titrate the excess sodium hy-
droxide with 0.1 N hydrochloric acid VS to a faint pink
endpoint. Transfer the solution with the aid of water to Propylene Glycol Dicaprylate/Dicaprate
a 600-mL beaker, and complete the titration to a pH of
7.0, determining the endpoint potentiometrically. Decanoic acid, mixed diesters with octanoic acid and pro-
Calculate the weight, in g, of esterified carboxyl groups pylene glycol [68583-51-7].
in the weight, W, in g, of the specimen taken:
DEFINITION
Result = [(V
x M, x N)/W] x F Propylene Glycol Dicaprylate/Dicaprate is a mixture of the
propylene glycol mono- and diesters of caprylic acid
Vv = erie of 0.1 N sodium hydroxide consumed (CgHi6O2) and capric acid (CioH20O2), the diesters fraction
mL being predominant.
M, = mEq of COz, 44 mg/mEq
N = actual normality of 0.1 N sodium hydroxide IDENTIFICATION
(mEq/mL) e A. INFRARED ABSORPTION (197F)
e B. THIN-LAYER CHROMATOGRAPHY IDENTIFICATION TEST (201)
w = specimen weight (g)
F = conversion factor, 10-3 g/mg Standard solution: 50 mg/mL of USP Propylene Glycol
Acceptance criteria: The wee of esterified carboxyl Dicaprylate/Dicaprate RS in methylene chloride
groups found, calculated on the dried basis, is Sample solution: 50 mg/mL of Propylene Glycol
40%-85% of the weight of carbon dioxide yielded by Dicaprylate/Dicaprate in methylene chloride
an equal weight of specimen in the Assay. Application volume: 10 uL, as streaks
Developing solvent system: Ether and hexane (7:3)
IMPURITIES Spray reagent: 0.1 mg/mL of rhodamine 6G in alcohol
© ARSENIC, Method II (211): 3 ppm Analysis
Samples: Standard solution and Sample solution
Develop the chromatogram over a path of 15 cm, and
Delete the following: dry the plate in a current of air. Spray the plate with
Spray reagent, and locate the spots on the plate b
°e HEAVY METALS, Method |/ (231): NMT 40 ppm, using a examination under UV light at a wavelength of 365
platinum crucible for the ignition, and nitric acid being nm.
used in place of sulfuric acid to wet the sample speci- Acceptance criteria: The R¢ values of the principal
MENe (Officiat |Jan-2018) spots from the Sample solution correspond to those
e LEAD (251) from the Standard solution.
Standard solution: 5 mL of Diluted Standard Lead e C. It meets the requirements of the test for Fats and Fixed
Solution Oils (401), Fatty Acid Composition.
Test preparation: Add 1.0g to 20 mL of nitric acid in a
250-mL conical flask, mix, and heat carefully until the IMPURITIES
specimen is dissolved. Continue the heating until the Inorganic Impurities
volume is reduced to 7 mL. Cool rapidly to room tem- e ARTICLES OF BOTANICAL ORIGIN, Total Ash (561): NMT
perature, transfer to a 100-mL volumetric flask, dilute 0.1%
with water to volume, and mix. Use a 50-mL portion. e ALKALINE IMPURITIES
Analysis: Proceed as directed in the chapter, using Sample: 2.0g of Propylene Glycol Dicaprylate/
15 mL of ammonium citrate solution, 3 mL of potas- Dicaprate
sium cyanide solution, and 0.5 mL of hydroxylamine hy- Analysis: Dissolve the Sample in a mixture of 1.5 mL of
drochloride solution for the test. After the first dithizone alcohol and 3.0 mL of ether. Add 0.05 mL of bromo-
extractions, wash the combined chloroform layers with phenol blue TS.
5 mL of water, discarding the water layer and continu- Acceptance criteria: NMT 0.15 mL of 0.01 N hydro-
ing in the usual manner by extracting with 20 mL of chloric acid is required in order to change the color of
0.2 N nitric acid. the indicator to yellow.
Acceptance criteria: A 50.0-mL portion of this solution
contains NMT 5 pg of lead (corresponding to NMT SPECIFIC TESTS
10 ppm of Pb). e FATS AND FIXED OILS, Acid Value (401): NMT 0.2
e FATS AND FIXED OILS, Fatty Acid Composition (401): Pro-
SPECIFIC TESTS pylene Glycol Dicaprylate/Dicaprate exhibits the compo-
e MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI- sition profile of fatty acids shown in the following table:
FIED MICROORGANISMS (62): The total bacterial count
does not exceed 200 cfu/g, and the tests for Salmonella Carbon-Chain Number of Percentage
species and Escherichia coli are negative. Length Double Bonds (%)
NF Monographs

e Loss ON DRYING (731): Dry a sample at 105° for 4 h: it 6 0 $2.0

loses NMT 20.0% of its weight.
8 0 50.0-80.0
° ASH
Sample: 3g 10 0 20 0-50.0
Analysis: Weigh the Sample in a tared crucible, and in- 12 0 33.0
cinerate at 650+25° until free from carbon. Cool in a 14 0 <1.0
desiccator, weigh, and determine the weight of the ash.
NF 36 Official Monographs / Propylene 5543

FATS AND FIXED OILS, Hydroxyl Value (401): NMT 10 Temperature

FATS AND FIXED OILS, /odine Value (401): NMT 1.0 Column: 40°
FATS AND FIXED OILS, Peroxide Value (401): NMT 1.0 Detector: 40°
FATS AND FIXED OlLs, Saponification Value (401): 320-340 Flow rate: 1 mL/min
FATS AND FIXED OILS, Unsaponifiable Matter (401): NMT Injection size: 40 uL
0.5%, determined on 5.0g System suitabilit
WATER DETERMINATION, Method Ia (921): NMT 0.1%, us- Sample: Sample solution
ing a mixture of methanol and methylene chloride (1:1) cose order: diesters, monoesters, propylene
in place of methanol in the titration vessel col.
Suitability requirements
ADDITIONAL REQUIREMENTS Relative standard deviation: NMT 1.0% is deter-
e PACKAGING AND STORAGE: Preserve in well-closed contain- mined from the monoester peak.
ers, and protect from moisture. No storage requirements Analysis
specified. Sample: Sample solution
e USP REFERENCE STANDARDS (11) Calculate the percentage of monoesters or diesters in
USP Propylene Glycol Dicaprylate/Dicaprate RS the portion of Propylene Glycol Dilaurate taken:
Result = (ru/rr) x (100 — D)
tu = peak response for monoesters or diesters
Propylene Glycol Dilaurate tr = sum of the responses of the monoester and
diester peaks
D = sum of the percentage content of propylene
lycol and the percentage content of free
fatty acids
Calculate the percentage content of free fatty acids:
Result = (A/561.1) x 200
Co7H5204 A = acid value
Lauric acid, diester with propane-1,2-diol; Acceptance criteria: NLT 70.0% of diesters and NMT
Propane-1,2-diyl didodecanoate 440.70 30.0% of monoesters
22788-19-8].
IMPURITIES
DEFINITION Inorganic Impurities
Propylene Glycol Dilaurate is a mixture of the propylene gly- e ARTICLES OF BOTANICAL ORIGIN, Total Ash (561): NMT
col mono- and diesters of lauric acid. It contains NLT 0.1%
70.0% of diesters and NMT 30.0% of monoesters. Organic Impurities
e PROCEDURE: LIMIT OF PROPYLENE GLYCOL
IDENTIFICATION Mobile phase: Proceed as directed in the Assay.
° * ont CHROMATOGRAPHIC IDENTIFICATION TEST Standard stock solution: 4mg/mL of USP Propylene
20 Glycol RS in tetrahydrofuran
Standard solution: 50 mg/mL of USP Propylene Glycol Standard solutions: Into four 15-mL flasks, introduce
Dilaurate RS in methylene chloride respectively 0.25, 0.5, 1.0, and 2.5 mL of Standard
Sample solution: 50 mg/mL of Propylene Glycol Di- stock solution, and dilute with tetrahydrofuran to
laurate in methylene chloride 5.0 mL. Ina fifth 15-mL flask, introduce 5.0 mL of Stan-
Developing solvent system: Hexane and ether (3:7) dard stock solution.
Spray reagent: 0.1 mg/mL of rhodamine 6G in alcohol Sample solution: Use the Sample solution from the
Analysis: Apply 200 yg of the Standard solution and Assay.
Sample solution, Develop the chromatogram over a path Chromatographic system: Proceed as directed in the
of 15 cm, and dry the plate in a current of air. Spray Assay.
the plate with Spray reagent, and locate the spots on Analysis
the plate by examination under UV light at a wave- Samples: Standard solutions and Sample solution
length of 365 nm. Prepare a standard curve of peak area versus concen-
Acceptance criteria: The R- values of the principal tration, in mg/mL, of propylene glycol in the Stan-
apes of the Sample solution correspond to those of the dard solutions. Obtain the concentration, C, in
tandard solution. mg/mL, of propylene glycol in the Sample solution
e B. FATS AND FIXED OlLS, Fatty Acid Composition (401): from the standard curve.
Meets the requirements Acceptance criteria: NMT 2.0% of propylene glycol is
ASSAY found.
© PROCEDURE SPECIFIC TESTS
Mobile phase: Tetrahydrofuran e FATS AND FIXED OILS, Acid Value (401): NMT 4
Sample solution: 200 mg of Propylene Glycol Dilaurate e FATS AND FIXED OILS, Fatty Acid Composition (401): Pro-
in 5 mL of tetrahydrofuran pylene Glycol Dilaurate exhibits the following composi-
Chromatographic system
sydesbouo-: 4IN

tion profile of fatty acids, determined as directed in the

(See Chromatography (621), System Suitability.) chapter.
Mode: LC
Detector: Refractive index z
Column: 7-mm x 60-cm; 5-m packing L21 (100A) Carbon-
[NotE—Two 7-mm x 30-cm L21 columns may be used Chain Percentage
in place of one 60-cm column, provided system suita- Fatty Acids Length (%)
bility requirements are met.] Caprylic acid C8 NMT 0.5
Capric acid C10 NMT 2.0.
Lauric acid C12 NLT 95.0
 5544 Propylene / Official Monographs NF 36

Carbon- Chromatographic system

Chain Percentage (See Chromatography (621), System Suitability.)
Fatty Acids Length (%) Mode: LC
Myristic acid C14 NMT 3.0 Detector: Refractive index A
Palmitic acid C16 NMT_1.0 Column: 7-mm x 60-cm; 5-1um packing L217 (100 A).
[NoTtt—Two 7-mm x 30-cm L21 columns may be used
e FATS AND FIXED OILS, /odine Value (401): NMT 1 in place of one 60-cm column, provided System suita-
e FATS AND FIXED OILS, Saponification Value (401): 230-250 bility requirements are met.]
e WATER DETERMINATION, Method Ia (921): NMT 1.0%, us- Temperatures
ing a mixture of methanol and methylene chloride (1:1) Column: 40°
in place of methanol in the titration vessel Detector: 40°
Flow rate: 1 mL/min
ADDITIONAL REQUIREMENTS Injection volume: 40 pL
¢ PACKAGING AND STORAGE: Preserve in well-closed contain- System suitabilit
ers, and protect from moisture. No storage requirements Sample: Sample solution
are specified. [Nott—The relative retention times with reference to
e USP REFERENCE STANDARDS (11) propylene glycol for diesters and monoesters are about
USP Propylene Glycol RS 0.85 and 0.90, respectively.]
USP Propylene Glycol Dilaurate RS Suitability requirements
Relative standard deviation: NMT 2.0% for the
monoester peak
Analysis
Sample: Sample solution
Propylene Glycol Monocaprylate Calculate the percentage of monoesters or diesters in
the portion of Propylene Glycol Monocaprylate taken:
Propylene glycol monooctanoate;
Octanoic acid, monoester with 1,2-propane diol; Result = (ru/r7) x (100 — D)
Caprylic acid, monoester with propane-1,2-diol
31565-12-5]. ru = peak response for monoesters or diesters
tr = sum of the peak responses of the monoesters
DEFINITION and diesters
Propylene Glycol Monocaprylate is a mixture of the propyl- D = sum of the percentage content of propylene
ene glycol monoesters and diesters of fatty acids com- glycol and the percentage content of free
posed predominately of caprylic acid. The requirements ‘atty acids
for monoester and diester content differ for the two types Calculate the percentage content of free fatty acids:
of Propylene Glycol Monocaprylate, as shown in the table
below. Result = (A/561.1) x 144
A = acid value
Content of Monoesters Content of Diesters
Acceptance criteria
(%) (%) Type |
Min. Max. Min. Max. Monoesters: 55.0%-80.0%
Type | 55.0 80.0 20.0 45.0 Diesters: 20.0%-45.0%
Type Il 90.0 = = 10.0 Type Il
Monoesters: NLT 90.0%
Diesters: NMT 10.0%
IDENTIFICATION
e A. INFRARED ABSORPTION (197A) IMPURITIES
° B. ee CHROMATOGRAPHIC IDENTIFICATION TEST
(201
Standard solution: 50 mg/mL of USP Propylene Glycol Delete the following:
Monocaprylate Type | RS or USP Propylene Glycol
Monocaprylate Type II RS in methylene chloride. °e HEAVY Metats, Method II (231): NMT 10 pg/Qe cortewii-
Sample solution:” 50 mg/mL in methylene chloride Jan-2018)
Chromatographic system e LIMIT OF PROPYLENE GLYCOL
Application volume: 10 pL Mobile phase, Sample solution, and Chromatographic
Developing solvent system: Ether and hexane (70:30) system: Proceed as directed in the Assay.
Spray reagent: 0.1 mg/mL of rhodamine 6G in Standard stock solution: 4 mg/mL of USP Propylene
alcohol Glycol RS in tetrahydrofuran
Analysis: Develop the chromatogram over a path of Standard solutions: Into four 5-mL volumetric flasks,
15 cm, and dry the plate in a current of air. Spray the introduce respectively 0.25, 0.5, 1.0, and 2.5 mL of
plate with Spray reagent, and locate the spots on the Standard stock solution, and dilute with tetrahydrofuran
plate by examination under UV light at a wavelength of to volume. In a fifth 5-mL volumetric flask, introduce
365 nm. 5.0 mL of Standard stock solution.
Acceptance criteria: The R- values of the principal spots Analysis
NF Monographs

from the Sample solution correspond to those from the Samples: Standard solutions and Sample solution
Standard solution. Prepare a standard curve of peak area versus concentra-
e C. It meets the requirements in Specific Tests for Fats and tion, in mg/mL, of propylene glycol in the Standard
Fixed Oils, Fatty Acid Composition (401). solutions. Obtain the concentration of propylene glycol
in the Sample solution from the standard curve.
ASSAY Calculate the percentage of free propylene ahycal in
e¢ PROCEDURE the portion of Propylene Glycol Monocaprylate taken:
Mobile phase: Tetrahydrofuran
Sample solution: 40 mg/mL of Propylene Glycol Mono- Result = (C/Cy) x 100
caprylate in tetrahydrofuran
NF 36 Official Monographs / Propylene 5545

Cc = concentration of propylene glycol in the IDENTIFICATION

Sample solution trom the standard curve e A. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST
Cu = concentration of the Sample solution (mg/mL) (201)
Acceptance criteria Standard solution: 50 mg/mL of USP Propylene Glycol
Propylene glycol monocaprylate (Type 1): NMT Monolaurate Type | RS (or USP Propylene Glycol Mono-
1.5% laurate Type Il RS) in methylene chloride
Propyiene glycol monocaprylate (Type II): NMT Sample solution: 50 mg/mL in methylene chloride
Chromatographic system
(See ey ategrap Yy (621), Thin-Layer Chromato-
SPECIFIC TESTS grapny.
e FATS AND FIXED OILS, Acid Value (401): NMT 1.5 Developing solvent system: Ether and hexane (70:30)
e FATS AND FIXED OILS, Fatty Acid Composition (401): Pro- Spray reagent: 0.1 mg/mL of rhodamine 6G in
pylene Glycol Monocaprylate exhibits the composition alcohol
profile of fatty acids shown in Table 1. Analysis: Develop the chromatogram over a path of
15 cm, and dry the plate in a current of air. Spray the
Table 1 plate with Spray reagent, and locate the spots on the
plate by examination under UV light at a wavelength of
Carbon-Chain Number of Percentage
365 nm.
Length Double Bonds (%)|
Acceptance criteria: The R; values of the principal spots
8 0 290.0 from the Sample solution correspond to those from the
10 0 $3.0 Standard solution.
12 o 0 e B. It meets the requirements in Specific Tests for Fats and
14 0 0 Fixed Oils, Fatty Acid Composition (401).
16 O 1.0 ASSAY
FATS AND FIXED OILS, lodine Value (401): NMT 1.0 © PROCEDURE
FATS AND FIXED OILS, Saponification Value (401) Mobile phase: Tetrahydrofuran
Propylene glycol monocaprylate (Type I): 285-315 Sample solution: 40 mg/mL of Propylene Glycol Mono-
laurate in tetrahydrofuran
Propylene glycol monocaprylate (Type Il): 270-295
FATS AND FIXED OILS, Peroxide Value (401): NMT 6.0 Chromatographic system
(See Chromatography (621), System Suitability.)
WATER DETERMINATION, Method Ia (921)
Mode: LC
Analysis: Use a mixture of methanol and methylene
Detector: Refractive index .
chloride (1:1) in place of methanol in the titration Column: 7-mm x 60-cm; 5-um packing L21 (100 A)
vessel.
Acceptance criteria: NMT 1.0% [NoTe—Two 7-mm x 30-cm L21 columns may be used
ARTICLES OF BOTANICAL ORIGIN, Total Ash (561): NMT in place of one 60-cm column, provided System suita-
0.1% bility requirements are met.]
Temperatures
ADDITIONAL REQUIREMENTS Column: 40°
© PACKAGING AND STORAGE: Preserve in well-closed contain- Detector: 40°
ers, and protect from moisture. No storage requirements Flow rate: 1 mL/min
specified. Injection volume: 40 uL
© LABELING: Label it to indicate the type (Type | or Type Il). System suitabilit
e USP REFERENCE STANDARDS (11) Sample: Sample solution
USP Propylene Glycol RS Suitability requirements
USP Propylene Glycol Monocaprylate Type | RS [Note—The order of elution is diesters, monoesters, and
USP Propylene Glycol Monocaprylate Type II RS propylene glycol.]
Relative standard deviation: NMT 1.0% for the
monoester peak
Analysis
Sample: Sample solution
Propylene Glycol Monolaurate Calculate the percentage of monoesters or diesters in
the portion of Propylene Glycol Monolaurate taken:
Dodecanoic acid, monoester with 1,2-propanediol;
Lauric acid, monoester with propane-1,2-diol. Result = (ru/rz) x (100 — D)

DEFINITION tu = peak response for monoesters or diesters

Propylene Glycol Monolaurate is a mixture of the propylene i = sum of the peak responses of the monoesters
lycol mono- and diesters of lauric acid. The requirements and diesters
‘or monoester and diester content differ for the two types D = sum of the percentage content of propylene
et Propylene Glycol Monolaurate, as set forth in the table lycol and the percentage content of free
elow. atty acids
Calculate the percentage content of free fatty acids in
the portion taken:
Content of Monoesters Content of Diesters
(%) (%) eA
Result = (A/561.1) x 200 mn
Min. Max. Min. Max.
Type | 45.0 70.0 30.0 55.0 A = acid value os
°
Type II 90.0 = = 10.0 |
=]
=
By
Zz
a
al
 5546 Propylene / Official Monographs NF 36

Acceptance criteria e LABELING: Label it to indicate the type (Type | or Type Il).
Type | e USP REFERENCE STANDARDS (11)
Monoesters: 45.0%-70.0% USP Propylene Glycol RS
Diesters: 30.0%-55.0% USP Propylene Glycol Monolaurate Type | RS
Type Il USP Propylene Glycol Monolaurate Type II RS
Monoesters: NLT 90.0%
Diesters: NMT 10.0%
IMPURITIES
e LIMIT OF PROPYLENE GLYCOL
Mobile phase, Sample solution, and Chromatographic
Propylene Glycol Monostearate
system: Proceed as directed in the Assay. Octadecanoic acid, monoester with 1,2-propanediol;
Standard stock solution: 4 mg/mL of USP Propylene 1,2-Propanediol monostearate [1323-39-3].
Glycol RS in tetrahydrofuran
Standard solutions: Into four 15-mL flasks introduce, DEFINITION
respectively, 0.25, 0.5, 1.0, and 2.5 mL of Standard Propylene Glycol Monostearate is a mixture of the propyl-
stock solution, and dilute with tetrahydrofuran to ene glycol mono- and di-esters of stearic and palmitic
5.0 mL. Ina fifth 15-mL flask, introduce 5.0 mL of Stan- acids. It contains NLT 90.0% of monoesters of saturated
dard stock solution. fatty acids, chiefly propylene glycol monostearate
Analysis (C2iH4203) and propylene glycol monopalmitate
Samples: Standard solutions and Sample solution (CigH38O3).
Prepare a standard curve of peak area versus concen-
tration, in mg/mL, of propylene glycol in the Stan- ASSAY
dard solutions. Obtain the concentration of propylene e@ PROPYLENE GLYCOL MONOESTERS
glycol in the Sample solution from the standard curve. Sample: 25g
Calculate the percentage of free propylene glycol in Analysis: Place the Sample in a 500-mL, round-bottom
the portion of Propylene Glycol Monolaurate taken: flask, and add 250 mL of alcohol and 7.5 g of potas-
sium hydroxide. Connect the flask to a suitable con-
Result = (C/Cy) x 100 denser, reflux the mixture for 2 h, cool, and transfer to
an 800-mL beaker, rinsing the flask with 100 mL of
Cc = concentration of propylene glycol in the water and combining the rinsing with the mixture in
Sample solution from the standard curve the beaker. Heat on a steam bath to evaporate the al-
Cu = concentration of the Sample solution (mg/mL) cohol, adding water occasionally to replace the alcohol,
Acceptance criteria and continue the evaporation until the odor of alcohol
Propylene glycol monolaurate (Type I): NMT 5.0% can no longer be detected. Adjust the volume, with hot
Propylene glycol monolaurate (Type Il): NMT 1.0% water, to 250 mL, neutralize with a mixture of equal
volumes of sulfuric acid and water, noting the volume
SPECIFIC TESTS used, and add a 10% excess of the dilute acid. Heat
e FATS AND FIXED OILS, Acid Value (401): NMT 4 with stirring until the fatty acid layer separates, and
© FATS AND FIXED OILS, Fatty Acid Composition (401): Pro- transfer the fatty acids to a 500-mL separator. Wash the
Pe Glycol Monolaurate exhibits the composition pro- fatty acids with four 200-mL portions of hot water, and
file of fatty acids shown in Table 1. discard the washings. oy the fatty acids at 105° for 1
h, cool, and determine the acid value on a 1-g portion,
Table 1 as directed in Fats and Fixed Oils (401), Acid Value (Free
Fatty Acids).
Carbon-Chain Percentage Calculate the average molecular weight of the monoes-
Fatty Acids Length (%) ee in the portion of Propylene Glycol Monostearate
Caprylic acid C8 NMT 0.5 taken:
Capric acid C10 NMT 2.0
Lauric acid ci2 NLT 95.0 Mravg = (M/A) + Mrz — Ms
Myristic acid C14 NMT 3.0
Ma 1000 times the molecular weight of potassium
Palmitic acid C16 NMT 1.0 hydroxide, 56,110
A = acid value
e FATS AND FIXED OILS, Jodine Value (401): NMT 1 M2 = molecular weight of propylene glycol, 76.10
e FATS AND FIXED OILS, Saponification Value (401) Ms = molecular weight of water, 18.02
Propylene glycol monolaurate (Type !): 210-245
Propylene glycol monolaurate (Type II): 200-230
Calculate F in the portion taken:
© WATER DETERMINATION, Method /a (921)
Analysis: Use a mixture of methanol and methylene F = (Mr x G)/M,s
chloride (1:1) in place of methanol in the titration Mz = 10 times the molecular weight of potassium
vessel. hydroxide, 561.1
Acceptance criteria: NMT 1.0% G = content, in percentage, of aves and
© ARTICLES OF BOTANICAL ORIGIN, Jota! Ash (561): NMT propylene glycol in propylene glycol
0.1% monostearate
NF Monographs

Ms = one-half of the molecular weight of propylene

ADDITIONAL REQUIREMENTS glycol, 38.05
© PACKAGING AND STORAGE: Preserve in well-closed contain- Calculate the percentage of propylene glycol
ers, and protect from moisture. No storage requirements monoesters:
specified.
Result = [CH — PF) X Mravgl/Mra

H = hydroxyl value of propylene glycol

monostearate
NF 36 Official Monographs / Propylparaben 5547

Acceptance criteria: NLT 90.0% of monoesters of satu-

rated fatty acids, chiefly propylene glycol monostearate Propylparaben
and propylene glycol monopalmitate
IMPURITIES O

e RESIDUE ON IGNITION (281): NMT 0.5% . ys oe

© FREE GLYCERIN AND PROPYLENE GLYCOL
Periodic acid solution: Dissolve 5.4 g of periodic acid HO’

in 100 mL of water, and add 1900 mL of glacial acetic

ace Store in a glass-stoppered bottle, protected from
ight. CioH1203 180.20
Chloroform: Use chloroform that meets the following Benzoic acid,senycrony | propyl ester;
additional requirement. To each of three glass-stop- Propyl p-hydroxybenzoate [94-1 3-3].
pered, 500-mL conical flasks add 50.0 mL of Periodic DEFINITION
acid solution, then add 50 mL of chloroform and 10 mL Propylparaben contains NLT 98.0% and NMT 102.0% of
of water to two of the flasks and 50 mL of water to the CyoHi203.
third flask. To each flask add 20 mL of potassium iodide
TS, mix gently, and proceed as directed for Analysis, IDENTIFICATION
beginning with “allow to stand for 1-5 min”. The dif- e A. INFRARED ABSORPTION (197M)
ference between the volumes of 0.1 N sodium thiosul- e B. MELTING RANGE OR TEMPERATURE (741): 96°-99°
fate required in the titrations with and without the
chloroform does not exceed 100 uL. ASSAY
Sample solution: Melt the Propylene Glycol Monostea- ¢ PROCEDURE
rate at a temperature NMT 55°. Transfer a $9 portion Mobile phase, Sample solution, Standard solution B,
to a 100-mL beaker, and dissolve in 25 mL o' and Chromatographic system: Proceed as described
Chloroform. in the procedure for Related Substances.
Analysis: Transfer the Sample solution, with the aid of System suitability
another 25-mL portion of Chloroform, to a separator, Sample: Standard solution B
wash the beaker with 25 mL of water, and add the Suitability requirements
washing to the separator. Insert the stopper, shake vig- Relative standard deviation: NMT 0.85% for 6
orously for 30-60 s, and allow the layers to separate, injections
adding 1-2 mL of glacial acetic acid, if necessary, to Analysis
break any emulsion. Transfer the aqueous layer to a Samples: Sample solution and Standard solution B
lass-stoppered, 500-mL conical flask, wash the chloro- Calculate the percentage of Propylparaben in the Sam-
‘orm layer with two 25-mL portions of water, combin- ple solution:
ing the washings with the aqueous layer, and discard
the chloroform layer. Add, with swirling, 50.0 mL of Pe- Result = P x (ru x Cs)/(r's x Cu)
riodic acid solution to the solution and to another glass-
stoppered, 500-mL conical flask containing 75 mL of P = labeled purity of USP Propylparaben RS
water to provide the blank. Allow to stand for 30-90 expressed as a percentage
min. To each flask add 20 mL of potassium iodide TS, tu = peak area of propylparaben from the Sample
mix gently, and allow to stand for 1-5 min before ti- solution
trating. Add 100 mL of water, and titrate with 0.1 N Cs = concentration of propylparaben in Standard
sodium thiosulfate VS until the brown iodine color fades solution B
to pale yellow, add 3 mL of starch TS, and continue the Ts = peak area of propylparaben from Standard
titration to the disappearance of the blue color. solution B
Calculate the percentage of free glycerin and propylene Cu = concentration of Propylparaben in the Sample
glycol, calculated as propylene glycol, in the portion of solution
Propylene Glycol Monostearate taken: Acceptance criteria: 98.0%-102.0%

Result = [(Vs — Vs) x M, x N]/W IMPURITIES

Inorganic Impurities
Ve = volume of sodium thiosulfate consumed by © RESIDUE ON IGNITION (281): NMT 0.1%, determined on
the blank solution (mL) 1.0
Vs = volume of sodium thiosulfate consumed by Organic Impurities
the Sample solution (mL) © PROCEDURE: RELATED SUBSTANCES
M, = molecular weight of propylene glycol divided Mobile phase: Methanol and a 6.8 g/L solution of po-
by 20, 3.805 tassium dihydrogen phosphate (65:35 v/v)
N = actual normality of the sodium thiosulfate Sample solution: Dissolve 50.0 mg of i lil in
solution 2.5 mL of methanol, and dilute with Mobile phase to
w = weight of Propylene Glycol Monostearate 50.0 mL. Dilute 10.0 mL of this solution with Mobile
taken (g) phase to 100.0 mL.
Acceptance criteria: NMT 1.0% free glycerin and pro- Standard solution A: 5.0 g/mL each of p-hydroxy-
pylene glycol, calculated as propylene glycol benzoic acid, USP Ethylparaben RS, and USP Propylpar-
aben RS in Mobile phase Zz
SPECIFIC TESTS Standard solution B: Dissolve 50.0 mg of USP Propyl- a
© CONGEALING TEMPERATURE (651): NLT 45° paraben RS in 2.5 mL of methanol, and dilute with Mo-
© FATS AND FIXED OILS, Acid Value (Free Fatty Acids) (401): bile phase to 50.0 mL. Dilute 10.0 mL of this solution =
°
NMT 4 with Mobile phase to 100.0 mL. |
FATS AND FIXED OILS, Hydroxy! Value (401): 160-175 Standard solution C: Dilute 1.0 mL of the Sample solu- fo)
FATS AND FIXED OILS, /odine Value (401): NMT 3 tion with Mobile phase to 20.0 mL. Dilute 1.0 mL of this vo}
al
FATS AND FIXED OILS, Saponification Value (401): 155-165 solution with Mobile phase to 10.0 mL. iy
Chromatographic system Tv
ADDITIONAL REQUIREMENTS a
(See Chromatography (621), System Suitability.) 7
© PACKAGING AND STORAGE: Preserve in well-closed
containers.
 5548 Propylparaben / Official Monographs
Mode: LC
Detector: UV 272 nm
Column: 4.6-mm x 15-cm; 5-m packing L1
Flow rate: 1.3 mL/min
Injection size: 10 uL
Run time: About 2.5 times the retention time of
propylparaben
System suitabilit
Sample: Standard solution A
[NotE—The retention time of propylparaben is about
4.5 min; the relative retention times for p-hydroxy-
benzoic acid and ethylparaben are about 0.3 and 0.7,
respectively.]
Suitability requirements
Resolution: NLT 3.0 between the ethylparaben and
ropylparaben peaks
Analysis
Samples: Sample solution and Standard solution C
[Note—Disregard any limit that is 0.2 times the area
of the principal peak in the chromatogram obtained
with Standard solution C (0.1%).]
Acceptance criteria
p-Hydroxybenzoic acid: The peak area in the Sample
solution, multiplied by 1.4 to correct for the calcula-
tion of content, is NMT the area of the principal peak
in Standard solution C (0.5%).
Unspecified impurities: The peak area of each impu-
rity in the Sample solution is NMT the area of the
principal peak in Standard solution C (0.5%).
Total impurities: The total peak area for all impurities
in the Sample solution is NMT twice the area of the
principal peak in Standard solution C (1.0%).
SPECIFIC TESTS
© COLOR OF SOLUTION
Sample solution: 100 mg/mL in alcohol
Comparison solution: Mix 2.4 mL of ferric chloride CS,
1.0 mL of cobaltous chloride CS, and 0.4 mL of cupric
sulfate CS with 0.3 N hydrochloric acid to make 10 mL.
Dilute 5 mL of this solution with 0.3 N hydrochloric
acid to make 100 mL. [NoTE—Prepare and use this solu-
tion immediately.]
Analysis
Samples: Alcohol, Sample solution, and Comparison
solution
Make the comparison by viewing the solutions down-
ward in matched color-comparison tubes against a
white surface (see Color and Achromicity (631)).
Acceptance criteria: The Sample solution is clear and
not more intensely colored than alcohol or the Compari-
son solution.
e ACIDITY
Sample solution: To 2 mL of Sample solution prepared
in the test for Color of Solution, add 3 mL of alcohol,
5 mL of carbon dioxide-free water, and 0.1 mL of
bromocresol green TS.
Analysis: Titrate with 0.10 N sodium hydroxide.
Acceptance criteria: NMT 0.1 mL is required to pro-
duce a blue color.
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in well-closed
containers.
NF Monographs
NF 36
e USP REFERENCE STANDARDS (11)
USP Ethylparaben RS
USP Propylparaben RS
Propylparaben Sodium
‘ om
ia? f Sete
CoH NaO3 202.20
Benzoic acid, te propyl ester, sodium salt;
Propyl p-hydroxybenzoate, sodium salt;
Sodium 4-propoxycarbonylphenolate [35285-69-9].
DEFINITION
Propylparaben Sodium contains NLT 94.0% and NMT
102.0% of propylparaben sodium (CioH1;NaQs), calcu-
lated on the anhydrous basis.
IDENTIFICATION
eo A.
Standard: 0.5 g of USP Propylparaben RS
Sample: 0.59
Analysis: Dissolve the Sample in 5 mL of water. Acidify
with hydrochloric acid, and filter the resulting precipi-
tate. Wash the precipitate with water, and dry over sil-
ica gel for 5 h. Repeat with the Standard.
Acceptance criteria: The IR absorption spectrum of a
mineral oil dispersion of the Sample exhibits maxima
only at the same wavelengths as those of a mineral oil
dispersion of the Standard.
° B.
Sample solution: Ignite 0.3 g of Propylparaben So-
dium, cool, and dissolve the residue in 3 mL of 3 N hy-
drochloric acid.
Acceptance criteria: A platinum wire dipped in the
Sample solution imparts an intense, persistent yellow
color to a nonluminous flame.
ASSAY
© PROCEDURE
Mobile phase: Methanol and a 6.8-g/L solution of po-
tassium dihydrogen phosphate (65:35, v/v)
System suitability solution: 5.0 g/mL each of p-hy-
droxybenzoic acid, USP Ethylparaben RS, and USP Pro-
pylparaben RS in Mobile phase
Standard solution: Dissolve 50.0 mg of USP Propylpar-
aben RS in 2.5 mL of methanol, and dilute with Mobile
phase to 50.0 mL. Dilute 10.0 mL of this solution with
Mobile phase to 100.0 mL.
Sample solution: Dissolve 50.0 mg of Propylparaben
Sodium in 2.5 mL of methanol, and dilute with Mobile
phase to 50.0 mL. Dilute 10.0 mL of this solution with
Mobile phase to 100.0 mL.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 272 nm
Column: 4.6-mm x 15-cm; 5-um packing L1
Flow rate: 1.3 mL/min
Injection volume: 10 wL
Run time: About 2.5 times the retention time of the
propylparaben peak
System suitability
Samples: System suitability solution and Standard
solution
[Note—The retention time of propylparaben is about
4.0 min; the relative retention times for p-hydroxyben-
zoic acid, ethylparaben, and propylparaben are about
0.4, 0.7, and 1.0, respectively.]
NF 36 Official Monographs / Pullulan 5549

Suitability requirements SPECIFIC TESTS

Resolution: NLT 3.0 between the ethylparaben and © PH (791)
propylparaben peaks, System suitability solution Sample solution: 1 mg/mL
Relative standard deviation: NMT 0.85% for six in- Acceptance criteria: 9.5-10.5
jections, Standard solution e@ WATER DETERMINATION, Method | (921): NMT 5.0%
Analysis e COMPLETENESS OF SOLUTION (641)
Samples: Standard solution and Sample solution Sample solution: Dissolve 1 g in water.
Calculate the percentage of propylparaben sodium Acceptance criteria: Meets the requirements
(CioHii1NaQOs) in the portion of Propylparaben Sodium
taken: ADDITIONAL REQUIREMENTS
@ PACKAGING AND STORAGE: Preserve in tight containers.
Result = P x (ry x Cs)/(rs X Cu) X (Ma/M,2) e USP REFERENCE STANDARDS (11)
USP Ethylparaben RS
P = labeled purity of USP Propylparaben RS USP Propylparaben RS
expressed as a percentage
ty = peak area of propylparaben from the Sample
solution
G = concentration of propylparaben in the
Standard solution Protamine Sulfate—see Protamine Sulfate
Is = peak area of propylparaben from the Standard
solution General Monographs
Cy = concentration of Propylparaben Sodium in the
Sample solution
M, — = molecular weight of propylparaben sodium,
202.20 Pullulan
M,z = molecular weight of propylparaben, 180.20
Acceptance criteria: 94.0%-102.0% on the anhydrous
basis . ay a Me pa on
Ho pee Seo} ann c, c,
IMPURITIES
e RELATED COMPOUNDS id WS None ake ting, work. em pox)
Mobile phase, System suitability solution, Sample so- ws on Wo?“on oe on)
lution, and Chromatographic system: Proceed as di-
rected in the Assay.
Standard solution: Dilute 1.0 mL of the Sample solution (C36H60030)
with Mobile phase to 20.0 mL. Dilute 1.0 mL of this so- Poly[6)-c-D-glucopyranosyl-(14)-o.-D-glucopyranosy|-
lution with Mobile phase to 10.0 mL. (1-4)-a-D-glucopyranosyl-(1-] [9057-02-7].
System suitability
Sample: System suitability solution DEFINITION
[Nott—The retention time of propylparaben is about Pullulan is a neutral, simple polysaccharide produced by the
4.0 min; the relative retention times for p-hydroxyben- growth of Aureobasidium pullulans. \t has a chain structure
zoic acid, ethylparaben, and propylparaben are about of repeated a-1,6-bonds of maltotriose composed of three
0.4, 0.7, and 1.0, respectively.] glucoses in o-1,4-bonds. It may contain some maltote-
Suitability requirements traosyl units. It contains NLT 90% of glucan, calculated
Resolution: NLT 3.0 between the ethylparaben and on the dried basis.
propylparaben peaks
Analysis IDENTIFICATION
Samples: Standard solution and Sample solution cA.
Acceptance criteria Sample: 10g
p-Hydroxybenzoic acid: NMT 4.0%; the peak area in maya Dissolve the Sample in 100 mL of water by
the Sample solution, multiplied by 1.4 to correct for adding in small portions with stirring.
the calculation of content, is NMT 8 times the area of Acceptance criteria: A viscous solution is produced.
the principal peak in the Standard solution. ° B.
Unspecified impurities: NMT 0.5%; the peak area of Pullulanase sample solution: 10 units/mL of
each impurity in the Sample solution is NMT the area pullulanase
of the principal peak in the Standard solution. Sample solution: The viscous solution obtained in /den-
Total impurities: NMT 1.0%; the total peak area for tification test A
all unspecified impurities in the Sample solution is NMT Analysis: Mix 10 mL of the Sample solution with 0.1 mL
twice the area of the principal peak in the Standard of Pullulanase sample solution, and allow to incubate at
solution. 25° for 20 min.
e CHLORIDE AND SULFATE, Chloride (221) Acceptance criteria: A substantial loss of viscosity is
Standard solution: 0.10 mL of 0.020 N hydrochloric observed.
aci eC
Sample: 0.29 Sample solution: 20 mg/mL
Analysis: Proceed as directed in the chapter. Analysis: To 10 mL of the Sample solution add 2 mL of
oy |

Acceptance criteria: 0.035%; the Sample shows no polyethylene glycol 600.

ExtleL-MLofeLecANBET

more chloride than the Standard solution. Acceptance criteria: A white precipitate is formed
e CHLORIDE AND SULFATE, Sulfate (221) immediately.
Standard solution: 0.30 mL of 0.020Nsulfuric acid ASSAY
Sample: 0.25g © CONTENT OF MONOSACCHARIDE, DISACCHARIDE, AND
Analysis: Proceed as directed in the chapter. OLIGOSACCHARIDES
Acceptance criteria: 0.12%; the Sample shows no more Sample stock solution: 8 mg/mL, on previously dried
sulfate than the Standard solution. material
Sample solution: To 1.0 mL of the Sample stock solution
add 0.1 mL of saturated potassium chloride solution,
 5550 Pullulan / Official Monographs
and shake vigorously with 3 mL of methyl alcohol. Cen-
trifuge, and use the supernatant.
Standard solution: Dilute 1.0 mL of the Sample stock
solution with water to 50 mL.
Blank: Water
Instrumental conditions
(See Ultraviolet-Visible Spectroscopy (857).)
Mode: Vis
Analytical wavelength: 620 nm
Analysis
Samples: Sample solution, Standard solution, and Blank
Transfer 0.2 mL each of the Standard solution, Sample
solution, and Blank to a test tube containing 5 mL of a
1-in-500 solution of anthrone in 75% (v/v) sulfuric
acid, with the test tube placed in ice water. Mix each
tube immediately, and then heat the test tube at 90°
for 10 min. Remove the tube, and allow it to cool in
cold running water.
Determine the absorbances of the resulting solutions at
the specified wavelength.
Determine the percentage of monosaccharide, disaccha-
tide, and oligosaccharides in the portion of the sample
taken:
Result = (Du/Ds) x (Au — As)/(As — As) x 100
Dy = dilution factor for the Sample solution, 4.1
Ds = dilution factor for the Standard solution, 50
Au = absorbance of the Sample solution
As = absorbance of the Standard solution
Ag = absorbance of the Blank
Acceptance criteria: The total content of monosaccha-
ride, disaccharide, and oligosaccharides is NMT 10.0%,
corresponding to NLT 90% of glucan on the dried
asis.
IMPURITIES
e RESIDUE ON IGNITION (281)
Sample: 2.0g
Acceptance criteria: NMT 0.3%
Delete the following:
°o HEAVY METALS, Method Il (231): NMT 5 1tg/Qe cottciai tan.
2018)
e NITROGEN DETERMINATION, Method II (461)
Sample: 3g, previously dried
Analysis: Proceed as directed in the chapter, replacing
the 7 mL of sulfuric acid with 12 mL of sulfuric acid for
the decomposition and replacing the 30 mL of sodium
hydroxide solution (2 in 5) with 40 mL of a solution of
sodium hydroxide (2 in 5).
Acceptance criteria: NMT 0.05%
SPECIFIC TESTS
© MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI-
FIED MICROORGANISMS (62): The total aerobic microbial
count does not exceed 10? cfu/g, and the total com-
bined molds and yeasts count does not exceed 10? cfu/
° Phi (791)
Sample: 1.0g
Analysis: Dissolve the Sample in 10 mL of freshly boiled
and cooled water.
NF Monographs
Acceptance criteria: 4.5—-6.5
e Loss ON DRrvING (731)
Analysis: Dry at 90° under vacuum for 6 h.
Acceptance criteria: NMT 6.0%
e VISCOSITY—CAPILLARY METHODS (911)
Sample: Exactly 10.0 g, previously dried
Analysis: Dissolve the Sample in water to make exactly
100 9, and perform the test at 30+ 0.1° using a Ub-
belohde-type viscometer.
Acceptance criteria: The kinematic viscosity is
100-180 mm?- s*'.
NF 36
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in well-closed contain-
ers. No storage requirements specified.
e LABELING: Label it to indicate the viscosity, giving the
type of viscosity parameter, concentration of the solution,
and the type of equipment used.
Pumice—see Pumice General Monographs
Racemethionine
CsHiiNO2S 149.21
Methionine, DL-;
DL-2-Amino-4-(methylthio)-butyric acid [59-51-8].
DEFINITION
Racemethionine contains NLT 99.0% and NMT 101.0% of
CsH1i1NO2S, as DL-methionine, calculated on the dried
basis.
IDENTIFICATION
e A. INFRARED ABSORPTION (197K)
Sample: Dry the substances at 105°.
Acceptance criteria: Meets the requirements
e B. The principal spot from Sample solution B is similar in
size, color, and position to the principal spot from Stan-
dard solution A, as obtained in the test for Organic Impuri-
ties, Related Substances.
e C. OPTICAL ROTATION, Angular Rotation (781A)
Sample: 50 mg/mL in 1 M hydrochloric acid
Acceptance criteria: —0.05° to +0.05°
e D. PROCEDURE
Analysis: Dissolve 0.1 g of Racemethionine and 0.1 g of
glycine in 4.5 mL of dilute sodium hydroxide solution
(85 mg/mL). Add 1 mL of sodium nitroferricyanide solu-
tion (25 mg/mL). Heat to 40° for 10 min. Allow to cool,
and add 2 mL of a mixture of hydrochloric acid and
phosphoric acid (90:10).
Acceptance criteria: A deep red color develops.
ASSAY
e PROCEDURE
Sample: 140mg of Racemethionine
Analysis: Dissolve the Sample in a mixture of 3 mL of
formic acid and 50 mL of glacial acetic acid. Titrate with
0.1 N perchloric acid VS, determining the endpoint po-
tentiometrically. Perform a blank determination, and
make any necessary corrections (see Titrimetry (541)).
Each mL of 0.1 N perchloric acid is equivalent to
14.92 mg of CsHi;NO,S.
Acceptance criteria: 99.0%-101.0% on the dried basis
IMPURITIES
Inorganic Impurities
e RESIDUE ON IGNITION (281): NMT 0.1%, determined on
1.0
° CHLOMDE AND SULFATE, Chloride (221): [NoTE—Prepare
the Sample solution and the Standard solution at the
same time.]
Chloride standard solution (5 ppm Cl): 0.824 mg/mL
of NaCl. Just before use, dilute 1 mL of this solution
with water to 100 mL.
Standard solution: To 10 mL of Chloride standard solu-
tion add 10 mL of 0.1 N silver nitrate and 25 mL of
water, and mix.
NF 36
Sample solution: Dissolve 0.25 g in 35 mL of water.
Add 5 mL of dilute nitric acid and 10 mL of 0.1 N silver
nitrate. Allow to stand protected from light for 5 min.
Analysis: Examine the Sample solution and Standard so-
lution laterally against a black background.
Acceptance criteria: Any opalescence in the Sample so-
lution is not more intense than that in the Standard
solution (200 ppm).
¢ CHLORIDE AND SULFATE, Sulfate (221): [NoTE—Prepare the
sempre solution and the Control solution at the same
time.
Barium chloride solution: 250 mg/mL
Sulfate standard solution (10 ppm SO,): 1.81 mg/mL
of potassium sulfate in 30% alcohol (v/v). Just before
use, dilute 1 mL of this solution with 30% alcohol (v/v)
to 100 mL.
Standard solution: Mix 3 mL of the Barium chloride so-
lution and 4.5 mL of the Sulfate standard solution, and
allow to stand for 1 min.
Sample stock solution: 50.0 mg/mL, heated to 60°.
Cool to 10°, and filter.
Sample solution: To 2.5 mL of the Standard solution
add 15 mL of the Sample stock solution and 0.5 mL of
5 N acetic acid.
Control solution: To 2.5 mL of the Standard solution
add 15 mL of the Sulfate standard solution and 0.5 mL
of 5 N acetic acid.
Analysis
Samples: Sample solution and Control! solution
Acceptance criteria: After 5 min, any opalescence in
the Sample solution is not more intense than that in the
Control solution (200 ppm).
Delete the following:
°e HEAVY METALS
Sodium sulfide solution: Dissolve 5 g of sodium sulfide
in 10 mL of water. Add 30 mL of glycerin.
Standard lead solution: Prepare as directed for Special
Reagents in Heavy Metals (231).
Standard solution: Transfer 1.0 mL of the Standard lead
solution to a 10-mL volumetric flask. Add 1 mL of 50%
acetic acid and 2 drops of 25% sodium hydroxide, and
dilute with water to volume.
Sample solution: Dissolve 5 g of Racemethionine b
adding 5 mL of 16% hydrochloric acid and 5 mL o'
water. Adjust with 25% sodium hydroxide to a pH of
3.0-4.0. Dilute with water to 50 mL. Shake for approxi-
mately 15 min, and filter.
Analysis: Add 1 drop of Sodium sulfide solution to 10 mL
of the Sample solution, and add 1 drop of Sodium sulfide
solution to 10 mL of the Standard solution. Let stand for
5 min. View downward over a white surface.
Acceptance criteria: The color of the solution from the
Sample solution is not darker than that of the solution
from the Standard solution (NMT 10 ppm).e cifical14an-
2018)
e LIMIT OF IRON
Standard stock solution (125 ppm): Dissolve 1.727 g
of ferric ammonium sulfate [FeNH4(SO.)2- 12H20] in
water. Add 50 mL of 10% hydrochloric acid, dilute with
water to 1000 mL, and mix. Dilute 1 mL of this solution
with water to 40 mL. Pipet 5 mL of this solution into a
200-mL volumetric flask, dilute with water to volume,
and mix.
Standard solution: Transfer 2 mL of the Standard stock
solution to a 25-mL volumetric flask. Add 5 mL of 16%
hydrochloric acid, 50 mg of ammonium persulfate, and
3 mL of 30% ammonium thiocyanate, and dilute with
water to volume.
Sample solution: Transfer 1 g of Racemethionine to a
25-mL volumetric flask. Add 5 mL of 16% hydrochloric
acid, and dissolve. Add 50 mg of ammonium persulfate
Official Monographs / Racemethionine 5551
and 3 mL of 30% ammonium thiocyanate, and dilute
with water to volume.
Blank: Transfer 5 mL of 16% hydrochloric acid to a
25-mL volumetric flask. Add 50 mg of ammonium per-
sulfate and 3 mL of 30% ammonium thiocyanate, and
dilute with water to volume.
Spectrometric conditions
(See Ultraviolet-Visible Spectroscopy (857).)
Mode: UV-Vis
Analytical wavelength: 475 nm
Cell: 1cm
Analysis
Samples: Standard solution, Sample solution, and Blank
Without delay, concomitantly determine the ab-
sorbances of each sample, correcting for the Blank.
Acceptance criteria: The absorbance of the Sample so-
lution is NMT that of the Standard solution (NMT
10 ppm).
e Limit OF AMMONIUM
Standard solution A: 0.297 mg/mL of USP Ammonium
Chloride RS. This solution contains 0.1 mg/mL or
100 ppm of NH4*.
Standard solution B: 0.297 g/mL of USP Ammonium
Chloride RS. This solution contains 0.1 g/mL or
0.1 ppm of NH4*.
Standard solution C: 2.97 g/mL of USP Ammonium
Sores RS. This solution contains 1.0 j1g/mL or 1 ppm
of NH«+.
Standard solution D: 29.7 g/mL of USP Ammonium
coe RS. This solution contains 10 ug/mL or 10 ppm
of NH4*.
Sample solution: 10 mg/mL of Racemethionine
Electrode system: Use an ammonia-specific,' ion-indi-
cating electrode connected to a pH meter capable of
measuring potentials (see pH (791)).
Analysis
Samples: Standard solution A, Standard solution B,
Standard solution C, Standard solution D, and Sample
solution
Add 100 mL of water to a 150-mL beaker, place the
electrode in the beaker, stir, and measure the poten-
tial. Add 1 mL of 10 N sodium hydroxide. Stir, and
measure the potential after stabilization. [NoTE—It
may take about 5 min.] The potential difference must
be less than 20 mV.
Add 100.0 mL each of Standard solutions A, B, C, and D
to four different 150-mL beakers. To each beaker, add
1 mL of 10 N sodium hydroxide. Place the ammonia
electrode in the beaker, stir, and concomitantly meas-
ure the potential after stabilization. [NoTE—It may take
about 5 min.] Draw acalibration curve of the poten-
tial, in mV, versus, the quantity of ammonium (NH4,*),
in mg.
Add 100.0 mL of the Sample solution to a 150-mL
beaker. Add 1 mL of 10 N sodium hydroxide. Adjust
the pH, if necessary, with 10 N sodium hydroxide to a
pit of NLT 11. Place the ammonia electrode in the
eaker, stir, and measure the potential after stabiliza-
tion. [NoTE—It may take about 5 min.] Obtain the
quantity of NH4*, in mg, in the 100 mL of the Sample
solution based on the calibration curve.
Calculate the percentage of ammonium (NH4*), in the
portion of Racemethionine taken:
Result = (C/W) x F ra
Cc = quantity of ammonium in the Sample solution =
from the standard curve (mg) °
Ww = weight of Racemethionine taken to prepare a
the Sample solution (mg) to)
F = conversion factor to g/g (ppm), 1 x 106 AY
mo]
1 Orion 95-12 is suitable.
a
a)
 4506 Calcium Phosphate, Dibasic / Dietary Supplements USP 41

Anhydrous Dibasic Calcium—see e B. The Sample solution exhibits peaks for speciophylline,
uncarine F, mitraphylline, isomitraphylline, pteropodine,
Anhydrous Dibasic Calcium Phosphate General and isopteropodine at retention times that correspond to
Monographs those in Standard solution A, as obtained in the test for
Content of Pentacyclic Oxindole Alkaloids and Limit of Tetra-
cyclic Oxindoles.

Dibasic Calcium Phosphate Dihydrate— COMPOSITION

¢ CONTENT OF PENTACYCLIC OXINDOLE ALKALOIDS AND LIMIT
see Dibasic Calcium Phosphate Dihydrate OF TETRACYCLIC OXINDOLES
General Monographs Standard solution A: Dissolve an accurately weighed
quantity of USP Powdered Cat’s Claw Extract RS in
methanol, shaking for 1 min. Dilute with methanol to
obtain a solution having a known concentration of
Calcium Phosphate, Dibasic Tablets— about 0.5 mg of the labeled amount of total oxindole
alkaloids per mL. Pass throughafilter of 0.45-14m or
see Dibasic Calcium Phosphate Tablets General finer pore size.
Monographs Standard solution B: 0.1 mg/mL of USP Isopteropodine
RS in methanol. Pass through a nylon filter of 0.45-um
or finer pore size.
Sample solution: Accurately weigh approximately
Cat's Claw 750 mg of ground Cat's Claw, and place in a 10-mL
centrifuge tube. Sonicate with 2.5 mL of methanol for
DEFINITION 10 min. Centrifuge, and transfer this solution to a
Cat’s Claw consists of the inner bark of the stems of Uncaria 10-mL volumetric flask. Repeat the extraction three ad-
tomentosa (Willd.) DC. (Rubiaceae). It contains NLT 0.3% ditional times combining the extracts in the 10-mL vol-
of pentacyclic oxindole alkaloids as isopteropodine, calcu- umetric flask, and dilute with methanol to volume.
lated on the dried basis, as the sum of speciophylline, Transfer about 3 mL of the solution to a test tube con-
uncarine F, mitraphylline, isomitraphylline, pteropodine, taining 300 mg of polyamide powder, and shake for 1
and isopteropodine. min. Pass through a On filter of 0.45-um or finer
pore size, discarding the first part of the filtrate.
IDENTIFICATION Solution A: Prepare a filtered and degassed 10 mM pH
e A. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST 7.0 phosphate buffer by mixing 6 mL of 1 N sodium hy-
(201) droxide, 10 mL of 1 M monobasic potassium phos-
Standard solution: 100mg of USP Powdered Cat's phate, and sufficient water to make 1000 mL. Adjust to
Claw Extract RS in 2 mL of methanol. Sonicate for 5 a pH of 7.0 + 0.1 by adding more of either solution.
min, shaking occasionally, heat in a water bath at 60° Solution B: Acetonitrile
for 15 min, cool, and centrifuge. Solution C: Methanol and glacial acetic acid (99:1)
Sample solution: 5g of powdered Cat’s Claw in 10 mL Mobile phase: See Table 1.
of methanol. Sonicate for 5 min, shaking occasionally.
Heat the mixture in a water bath at 60° for 15 min, Table 1
cool, and filter.
Adsorbent: Chromatographic silica gel mixture with an Time Solution A Solution B Solution C
average particle size of 10-15 um (TLC plates) (min) (%) (%) (%)
Appheate volume: 20 uL, as bands that are 1 cm in 0 65 35 0
lengtl 17 65 35 0
Developing solvent system: Ethyl acetate and hexane 25 50 50 o
(95:5) 30 50 50 0
Derivatization reagent A: Dissolve 0.85 g of basic bis-
muth nitrate in 10 mL of glacial acetic acid and 40 mL 31 0 0 100
of water by heating. Filter if necessary (Solution A). Dis- 36 0 0 100
DS Monographs

solve 8g of potassium iodide in 30 mL of water (Solu- 39 65 35 0

tion B). Mix Solution A and Solution B (1:1) to obtain a 49 65 35 0
stock solution. Dilute 1 mL of the stock solution with
2 mL of glacial acetic acid and 10 mL of water. [NOoTE— Chromatographic system
Use freshly mixed Solution A and Solution B.] (See Chromatography (621), System Suitability.)
Derivatization reagent B: Use a 10% solution of so- Mode: LC
dium nitrite in water. Detector: UV 245 nm
Analysis Column: 4.6-mm x 10-cm; end-capped 3-m packing
Samples: Standard solution and Sample solution u
Develop the chromatogram to a length of NLT 12 cm, Flow rate: 0.75 mL/min
and dry the plate in a current of air. Injection volume: 10 uL
Acceptance criteria: Examine the plate under short- System suitability
wave UV light (254 nm). The Sample solution chromato- Samples: Standard solution A and Standard solution B
gram shows multiple zones that correspond in Ry values Suitability requirements
to those observed in the Standard solution chromato- Chromatogram similarity: The chromatogram ob-
gram. Other zones of varying intensities may be ob- tained using Standard solution A is similar to the refer-
served in the Sample solution. Treat the plate with Der- ence chromatogram provided with the USP Powdered
ivatization reagent A followed by Derivatization reagent Cat's Claw Extract RS.
B, and examine the plate under white light. The Sample Tailing factor: NMT 2.0 for the isopteropodine peak,
solution chromatogram shows multiple orange-brown Standard solution B
zones that correspond in color and Rr values to those Relative standard deviation: NMT 2.0% for the
observed in the Standard solution chromatogram. Other isopteropodine peak in repeated injections, Standard
colored zones of varying intensities may be observed in solution B
the Sample solution.
 5552 Racemethionine / Official Monographs
Acceptance criteria: NMT 200 ppm
Organic Impurities
© PROCEDURE: RELATED SUBSTANCES
Standard solution A: 0.40 mg/mL of USP Raceme-
thionine RS
Standard solution B: 40 1g/mL of USP Racemethion-
ine RS
Sample solution A: 20 mg/mL of Racemethionine
Sample solution B: 0.40 mg/mL of Racemethionine
Chromatographic system
< phromatography (621), Thin-Layer Chromatogra-
phy.
Mode; TLC
Adsorbent: 0.25-mm layer of chromatographic silica
gel mixture
Application volume: 5 wl
Developing solvent system: Butyl alcohol, glacial
acetic acid, and water (3:1:1)
Spray reagent: 2 mg/mL of ninhydrin in a mixture of
butyl alcohol and 2N acetic acid (95:5)
Analysis
Samples: Standard solution A, Standard solution B,
Sample solution A, and Sample solution B
Develop over a path of 10 cm using the Developing
solvent system. After air-drying the plate, spray wit
Spray reagent, and heat between 100° and 105° for
15 min. Examine the plate under white light.
Acceptance criteria: Any spot obtained from Sample
solution A, apart from the panel spot, is not more
intense than the spot obtained from Standard solution B
(NMT 0.2%).
SPECIFIC TESTS
e PH (791): 5.4-6.1, in a 20 mg/mL solution
e Loss ON DRYING (731): Dry a sample at 105° for 3 h: it
loses NMT 0.5% of its weight, determined on 1.000 g.
© TRANSMITTANCE
Sample solution: 10% of Racemethionine in 2 N hy-
drochloric acid, prepared by sonication
Analysis: Determine the transmittance in a 1-cm cell at
430 nm with a suitable spectrophotometer.
Acceptance criteria: Transmittance of NLT 0.98, corre-
sponding to an absorbance of NMT about 0.009
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in well-closed contain-
ers, protected from light.
e USP REFERENCE STANDARDS (11)
USP Ammonium Chloride RS
USP Racemethionine RS
Fully Hydrogenated Rapeseed Oil
Fully hydrogenated rapeseed oil [84681-71-0].
Motes e Result = (C/W) x V
DEFINITION
Fully Hydrogenated Rapeseed Oil is the product obtained by
refining and hydrogenating oil obtained from the seeds of
Brassica napus and Brassica campestris (Fam. Cruciferae).
The product is a mixture of Wobpenac. in which the ‘ fatty
acid composition is a mixture of saturated fatty acids.
re
val
IDENTIFICATION
oy e A. It meets the requirements of the test for Fats and
i]
on Fixed Oils, Fatty Acid Composition (401).
io)
i}
tm IMPURITIES
GC) © ALKALINE IMPURITIES
= Sample solution: Prepare a mixture of 2.0g of Full
J
Hydrogenated Rapeseed Oil, 1.5 mL of alcohol, an
v4 3.0 mL of toluene. Dissolve by gentle heating.
NF 36
Analysis: To the Sample solution add 0.05 mL of bromo-
phenol blue TS, and titrate with 0.01 N hydrochloric
acid to a yellow endpoint.
Acceptance criteria: NMT 0.4 mL of 0.01 N hydrochlo-
ric acid is required.
e RESIDUE ON IGNITION (281): NMT 0.5%, when a 5-g sam-
ple of Fully Hydrogenated Rapeseed Oil is ignited at an
ignition temperature of 800 + 25°
Delete the following:
°e HEAVY METALS, Method Ii (231): NMT 10 ppmMe corficai 1-
fan-2018)
e LIMIT OF NICKEL
Sample solution: Weigh 5.0 g of Fully Hydrogenated
Rapeseed Oil into a previously tared platinum or silica
crucible. Cautiously heat the substance, and introduce
into it a wick formed from twisted ashless filter paper.
Ignite the wick. When the substance ignites, stop heat-
ing. After combustion, ignite in a muffle furnace at
about 600°. Continue the incineration until white ash is
obtained. After cooling, transfer the residue, with the
aid of two 2-mL portions of diluted hydrochloric acid,
to a 25-mL volumetric flask. Add 0.3 mL of nitric acid,
and dilute with water to volume.
Standard stock solution: 0.2 g/mL of nickel prepared
from nickel standard solution TS and water. Prepare im-
mediately before use.
Standard solutions: Into three identical 10-mL volu-
metric flasks introduce 1.0, 2.0, and 4.0 mL of Standard
stock solution, De To each flask add a 2.0-mL
portion of the Sample solution, and dilute with water to
volume.
Instrumental conditions
(See Atomic Absorption Spectroscopy (852).)
Mode: Atomic absorption spectrophotometer
equipped with a graphite furnace
Roccrbance 232.0 an
Lamp: Nickel hollow-cathode
Analysis
Samples: Sample solution and Standard solutions
Concomitantly determine the absorbances of the Sam-
ples at least three times each. Record the average of
the steady readings for each of the Standard solutions
and the Sample solution. Plot the absorbances of the
Standard solutions and the Sample solution versus the
added quantity of nickel. [NoTE—The Sample solution
should be plotted as if it had a content of added
nickel equivalent to 0 g.] Extrapolate the line joining
the points on the graph until it meets the concentra-
tion axis. The distance between this point and the in-
tersection of the axes represents the concentration of
nickel, C, in g/mL, in the Sample solution.
Calculate the content of nickel in the portion of Fully
Hydrogenated Rapeseed Oil taken:
E = concentration of nickel in the Sample solution
(ug/mL)
w = weight of Fully Hydrogenated Rapeseed Oil
(
ee Lani of the Sample solution, 25 mL
Acceptance criteria: NMT ey m
e Limit oF Erucic Acip: NMT 1.0%, as determined under
Specific Tests, in the test Fats and Fixed Oils, Fatty Acid
omposition (401)
SPECIFIC TESTS
e FATS AND FIXED OILS, Fatty Acid Composition (401): Fully
Hydrogenated Rapeseed Oil exhibits the fatty acid com-
position profile shown in Table 1.
NF 36 Official Monographs / Rapeseed 5553

Table 1 to the separator. Close the separator tightly with a

Carbon-Chain Number of Percentage
stopper, shake vigorously for 30-60 s, and allow the
layers to separate.
Length Double Bonds (%) [Note—Add 1-2 mL of glacial acetic acid to break emul-
14 0 <1.0 sions due to the presence of soap.]
16 0 3-5 Collect the aqueous layer in a 500-mL glass-stoppered
18 0 38-42 Erlenmeyer flask, and again extract the chloroform so-
20 0 8-10 lution in the separator with two 25-mL portions of
22 0 42-50 water. Retain the combined aqueous extracts, which
will be used in the test for Limit of Free Glycerin. Trans-
24 0 1.0-2.0
fer the chloroform layer to a 500-mL glass-stoppered
18 1 <1.0 Erlenmeyer flask, and add 50.0 mL of Periodic acid solu-
18 2 <1.0 tion to this flask and to each of two blank flasks con-
20 1 <1.0 taining a mixture of 50 mL of Chloroform and 10 mL of
22 1 1.0 water. Swirl the flasks during the addition of Periodic
@ Erucic acid. acid solution, and allow to stand for at least 30 min,
but no longer than 90 min. To each flask, add 20 mL
FATS AND FIXED OILS, Acid Value (401): NMT 6.0 of potassium iodide TS, and allow to stand at least 1
FATS AND FIXED OILS, lodine Value (401): NMT 4 min, but no longer than 5 min, before titrating. Add
FATS AND FIXED OILS, Peroxide Value (401): NMT 2.0 100 mL of water, and titrate with 0.1 N sodium thio-
FATS AND FIXED OILS, Unsaponifiable Matter (401): NMT sulfate VS, using a magnetic stirrer to keep the solu-
1.5% tion thoroughly mixed, to the disappearance of the
brown iodine color. Add 2 mL of starch TS, and con-
ADDITIONAL REQUIREMENTS tinue the titration to the disappearance of the blue
© PACKAGING AND STORAGE: Preserve in tight, light-resistant color.
containers. No storage requirements specified. Calculate the percentage of 1-monoglycerides in the
portion of Superglycerinated Fully Hydrogenated Rape-
seed Oil taken:
Result = {[M; x (Ve — Vs) x N]/(W x A)} x 100
Superglycerinated Fully Hydrogenated
Rapeseed Oil M, = molecular weight of glyceryl monostearate,
358
Superglycerinated fully hydrogenated rapeseed oil. Ve = volume of sodium thiosulfate VS consumed in
the blank determination (mL)
DEFINITION Vs = volume of sodium thiosulfate VS required in
Superglycerinated Fully Hydrogenated Rapeseed Oil is the the titration of the Superglycerinated Fully
product obtained by refining, hydrogenating, and glycer- Hydrogenated Rapeseed Oil (mL)
inating oil obtained from the seeds of Brassica napus and N = normality of the sodium thiosulfate VS
Brassica campestris (Fam. Cruciferae). The product is a Ww = weight of the Superglycerinated Fully
mixture of mono-, di-, and triglycerides, with triglycerides Hydrogenated Rapeseed Oil taken to prepare
as a minor component. the Sample solution (mg)
[Note—Use compendial grade glycerin as a starting A = factor number, 2
material.] Acceptance criteria: 90.0%-110.0% of that indicated
IDENTIFICATION on the label
e A. It meets the requirements of the test for Fats and IMPURITIES
Fixed Oils, Fatty Acid Composition (401). e RESIDUE ON IGNITION (281): NMT 0.5%, when a 5-g sam-
COMPOSITION ple of Superglycerinated Fully Hydrogenated Rapeseed
© CONTENT OF 1-IMONOGLYCERIDES Oil is ignited at an ignition temperature of 800
+25°
Periodic acid solution: Dissolve 5.4 g of periodic acid
in 100 mL of water, add 1900 mL of glacial acetic acid, Delete the following:
and mix. Preserve in a light-resistant, glass-stoppered
bottle. °e HEAVY METALS, Method I! (231): NMT 10 ppme corsa 1-
Chloroform: Use chloroform that meets the following jan-2018)
test. Add 50.0 mL of Periodic acid solution to each of © Limit OF NICKEL
three 500-mL flasks. Add 50 mL of chloroform and Sample solution: Weigh 5.0 g of Superglycerinated
10 mL of water to two of the flasks, and add 50 mL of Fully Hydrogenated por tinge Oil into a prevoisy tared
water to the third flask. Add 20 mL of potassium iodide platinum or silica crucible. Cautiously heat the sub-
TS to each flask, mix gently, and continue as directed stance, and introduce into it a wick formed from
in the Analysis, beginning with “and allow to stand at twisted ashless filter paper. Ignite the wick. When the
least 1 min, but no longer than 5 min, before titrating”. substance ignites, stop heating. After combustion, ig-
The difference between the volume of 0.1 N sodium nite in a muffle furnace at about 600°. Continue the
thiosulfate VS required in the titrations with and with- incineration until white ash is obtained. After cooling,
out the chloroform is not greater than 0.5 mL.
ExtTe [1 Lelecoy AN BET]

transfer the residue, with the aid of two 2-mL portions

Sample solution: Melt Superglycerinated Fully Hydro-
genated Rapeseed Oil at a temperature not higher than
10° above its melting point, and mix thoroughly. Trans-
fer an accurately weighed quantity of it, equivalent to
about 150 mg of 1-monoglycerides, to a 100-mL
beaker, dissolve in 25 mL of Chloroform, and mix.
Analysis: Transfer the Sample solution, with the aid of
an additional 25 mL ofChloroform, to a separator. Wash
the beaker with 25 mL of water, and add the washing
of diluted hydrochloric acid, to a 25-mL volumetric
flask. Add 0.3 mL of nitric acid, and dilute with water to
volume.
Nickel standard solution: Immediately before use, di-
lute 10 mL of nickel standard solution TS with water to
500 mL. This solution contains the equivalent of 0.2 ug/
mL of nickel.
Standard solutions: Into three identical 10-mL volu-
metric flasks, introduce respectively 1.0, 2.0, and
 5554 Rapeseed / Official Monographs NF 36

4.0 mL of Nickel standard solution. To each flask, add a A = factor number, 4

2.0-mL portion of the Sample solution, and dilute with Acceptance criteria: NMT 1%
water to volume.
Instrumental conditions SPECIFIC TESTS
(See Atomic Absorption Spectroscopy (852).) e FATS AND FIXED OILS, Acid Value (401): NMT 6.0
Mode: Atomic absorption spectrophotometer e FATS AND FIXED Os, Fatty Acid Composition (401): Super-
equipped with a graphite furnace glycerinated Fully Hydrogenated Rapeseed Oil exhibits
Analytical wavelength: 232.0 nm the fatty acid composition profile shown in Table 1.
Lamp: Nickel hollow-cathode
Analysis Table 1
Samples: Sample solution and Standard solutions
Carbon-Chain Number of Percentage
Concomitantly determine the absorbances at least
three times each, at the wavelength of maximum ab- Length Double Bonds (%)
sorbance. Record the average of the steady readings 14 0 <1.0
for each of the Standard solutions and the Sample so- 16 0 3-5
lution. Plot the absorbances of the Sample solution 18 0 38-42
and the Standard solutions versus the added quantity 20 0 8-10
of nickel. 22 0 42-50
[Note—The Sample solution should be plotted as if it
24 oO 1.0-2.0
had a content of added nickel equivalent to 0 yWg.]
Ba the line joining the points on the graph 18 1 $1.0
until it meets the concentration axis. The distance be- 18 2: <1.0
tween this point and the intersection of the axes rep- 20 1 <1.0
resents the concentration of nickel, C, in ug/mL, in 22% 1 1.0
the Sample solution. Erucic acid.
Calculate the content of nickel in the portion of Super-
glycerinated Fully Hydrogenated Rapeseed Oil taken: e FATS AND FIXED OlLs, Hydroxy! Value (401): NLT 90.0%
and NMT 110.0% of that indicated on the label
Result = (Vx O/W e FATS AND FIXED OILS, /odine Value (401): NMT 4
e FATS AND FIXED OILS, Peroxide Value (401): NMT 2.0
Vv = volume of the Sample solution, 25 mL e FATS AND FIXED OlLs, Unsaponifiable Matter (401): NMT
€ = concentration of nickel in the Sample solution 1.5%
(ug/ml)
= weight of Superglycerinated Fully ADDITIONAL REQUIREMENTS
Hydrogenated Rapeseed Oil taken to prepare e PACKAGING AND STORAGE: Preserve in tight, light-resistant
the Sample solution (g) containers. No storage requirements specified.
Acceptance criteria: NMT 1 g/g (ppm) e LABELING: Label it to indicate the hydroxyl value and the
e Limit oF ERucic AciD: NMT 1.0%, as determined in the content of 1-monoglycerides.
test for Fats and Fixed Oils (401), Fatty Acid Composition
© LIMIT OF FREE GLYCERIN
Periodic acid solution and Chloroform: Prepare as di-
rected in the test for Content of 1-Monoglycerides.
Sample solution: Use the combined aqueous extracts Rose Oil
obtained as directed in the test for Content of
1-Monoglycerides. DEFINITION
Analysis: Transfer 50.0 mL of Periodic acid solution to Rose Oil is volatile oil distilled with steam from the fresh
each of two flasks: a 500-mL glass-stoppered flowers of Rosa gallica L, Rosa damascena Miller, Rosa alba
Erlenmeyer flask containing the Sample solution and a L., Rosa centifolia L., and varieties of these species (Fam.
500-mL glass-stoppered Erlenmeyer blank flask contain- Rosaceae).
ing 75 mL of water. Continue as directed for Analysis in
the test for Content of 1-Monoglycerides, beginning with SPECIFIC TESTS
“Swirl the flasks during the addition of Periodic acid so- e SPECIFIC GRAVITY (841)
lution, and allow to stand for at least 30 min, but no Analysis: Measure at 30° compared with water at 15°.
longer than 90 min”. Acceptance criteria: 0.848-0.863
Calculate the percentage of free glycerin in the portion e OPTICAL ROTATION, Angular Rotation (781A): -1° to —4°
of Superglycerinated Fully Hydrogenated Rapeseed Oil e REFRACTIVE INDEX (831): 1.457-1.463 at 30°
taken: ¢ SOLUBILITY TEST
Analysis: 1 mL is miscible with 1 mL of chloroform
Result = {[M; x (Vs — Vs) x N]/(W
x A)} x 100 without turbidity. Add 20 mL of 90% alcohol to this
mixture.
M, = molecular weight of glycerin, 92 Acceptance criteria: The resulting liquid is neutral or
Ve = volume of sodium thiosulfate VS consumed in acid to moistened litmus paper and, upon standing at
the blank determination (mL) 20°, deposits crystals within 5 min.
Vs = volume of sodium thiosulfate VS required in
the titration of the Superglycerinated Fully ADDITIONAL REQUIREMENTS
NF Monographs

Hydrogenated Rapeseed Oil (mL) e PACKAGING AND STORAGE: Preserve in well-filled, tight
N = normality of the sodium thiosulfate VS containers.
Ww = weight of the Superglycerinated Fully
Hydrogenated Rapeseed Oil taken to prepare
the Sample solution as directed in the test for
Content of 1-Monoglycerides (mg)
NF 36
Rose Water Ointment—see Rose Water
Ointment General Monographs
Stronger Rose Water
DEFINITION
Stronger Rose Water is a saturated solution of the odorifer-
ous principals of the flowers of Rosa centifolia L. (Fam.
Rosaceae) prepared by distilling the fresh flowers with
water and separating the excess volatile oil from the clear,
water portion of the distillate. [NoTE—Stronger Rose
Water, diluted with an equal volume of purified water,
may be supplied when “Rose Water” is required.]
IMPURITIES
Delete the following:
®e HEAVY METALS, Method | (231)
Test preparation: Stronger Rose Water, | N acetic acid,
and water (10:2:13)
Acceptance criteria: NMT 2 11g/ge cortiiat 1-jan-2018)
SPECIFIC TESTS
e REACTION: Neutral or acidic to litmus
e RESIDUE ON EVAPORATION
Sample: 100 mL
Analysis: Evaporate the sample on a steam bath, and
dry the residue at 105° for 1 h.
Acceptance criteria: NMT 15 mg (0.015%)
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: The odor of Stronger Rose
Water is best preserved by allowing a limited access of
fresh air to the container.
Saccharin
Portions of this monograph that are national USP text, and
are not part of the harmonized text, are marked with
symbols (%) to specify this fact.
C7HsNO3S
1,2-Benzisothiazol-3(2H)-one, 1,1-dioxide;
1,2-Benzisothiazolin-3-one 1,1-dioxide [81-07-2].
DEFINITION
Saccharin contains NLT 99.0% and NMT 101.0% of
saccharin (C7HsNO3S), calculated on the dried basis.
IDENTIFICATION
¢ A. INFRARED ABSORPTION (197K)
ASSAY
e PROCEDURE
Sample: 500mg
Analysis: Dissolve the Sample in 40 mL of alcohol. Add
40 mL of water and phenolphthalein TS. Titrate with
0.1 N sodium hydroxide. Perform a blank titration, if
necessary, and make the appropriate correction. Each
mL of 0.1 N sodium hydroxide is equivalent to
18.32 mg of saccharin (C7HsNO3S).
Official Monographs / Saccharin 5555
Acceptance criteria: 99.0%-101.0% on the dried basis
IMPURITIES
e RESIDUE ON IGNITION (281): NMT 0.2%, using an ignition
temperature of 600 + 50°
Delete the following:
*e *HEAVY METALS, Method I/ (231): NMT 10 ppmee citical
1.Jan-2018)
© *LIMIT OF TOLUENESULFONAMIDES
Internal standard solution: 0.25 mg/mL of caffeine in
methylene chloride
Standard stock solution: 20.0 g/mL of USP o-Toluene-
sulfonamide RS and 20.0 g/mL of USP p-Toluenesul-
fonamide RS in methylene chloride
Standard solution: Evaporate 5.0 mL of the Standard
stock solution to dryness in a stream of nitrogen. Dis-
solve the residue in 1 mL of the Internal standard
solution.
Sample solution: Suspend 10g of Saccharin in 20 mL
of water, and dissolve using 5-6 mL of 10 N sodium
hydroxide. If necessary, adjust the solution with 1 N so-
dium hydroxide or 1 N hydrochloric acid to a pH of
7-8, and dilute with water to 50 mL. Shake the solution
with four quantities each of 50 mL of methylene chlo-
ride. Combine the lower layers, dry over anhydrous so-
dium sulfate, and filter. Wash the‘titer and the sodium
sulfate with 10 mL of methylene chloride. Combine the
solution and the washings, and evaporate almost to
dryness in a water bath at a temperature not exceeding
40°. Using a small quantity of methylene chloride,
quantitatively transfer the residue into a suitable 10-mL
tube, evaporate to dryness in a stream of nitrogen, and
dissolve the residue in 1.0 mL of the /nternal standard
solution.
Blank solution: Evaporate 200 mL of methylene chlo-
ride to dryness in a water bath at a temperature not
exceeding 40°. Dissolve the residue in 1 mL of methyl-
ene chloride.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: GC
Detector: Flame ionization
Column: 0.53-mm x 10-m fused silica; coated with a
2-uum film of phase G3
Temperatures
Injector: 250°
Detector: 250°
Column: 180°
Carrier gas: Nitrogen
Flow rate: 10 mL/min
Injection volume: 1 wL
183.18 Split ratio: 2:1
System suitability
Samples: Standard solution and Blank solution
[Nott—The substances are eluted in the following or-
der: o-toluenesulfonamide, p-toluenesulfonamide, and
caffeine.]
Suitability requirements: No peaks at the retention
times for the internal standard, o-toluenesulfonamide,
or p-toluenesulfonamide; Blank solution
Resolution: NLT 1.5 between o-toluenesulfonamide
and p-toluenesulfonamide, Standard solution
sydesBouo-= 4N
Analysis
Samples: Standard solution and Sample solution
Acceptance criteria: See Table 1. If any peaks due to o-
toluenesulfonamide and p-toluenesulfonamide appear in
the chromatogram of the Sample solution, the ratio of
their areas to that of the Internal standard solution is
NMT the corresponding ratio in the chromatogram of
the Standard solution.
 5556 Saccharin / Official Monographs
Table 1
Acceptance Criteria
Name (ppm)
o-Toluenesulfonamide 10
p-Toluenesulfonamide 10
ol
e LIMIT OF BENZOATE AND SALICYLATE
Sample solution: 10 mL of a hot, saturated solution of
saccharin
Analysis: Add ferric chloride TS dropwise to the Sample
solution.
Acceptance criteria: No precipitate or violet color ap-
pears in the liquid.
SPECIFIC TESTS
e *IMELTING RANGE OR TEMPERATURE (741): 226°-230°»
e Loss ON DRYING (731)
Analysis: Dry at 105° for 2 h.
Acceptance criteria: NMT 1.0%
© READILY CARBONIZABLE SUBSTANCES TEST (271)
Sample solution: 40 mg/mL in sulfuric acid
[94.5%-95.5% (w/w) of H2SO,]; maintained at 48°-50°
for 10 min
Acceptance criteria: The Sample solution has no more
color than Matching Fluid A, when viewed against a
white background.
© CLARITY OF SOLUTION
[NotE—The Sample solution is to be compared to Refer-
ence suspension A_ in diffused daylight 5 min after
preparation of Reference suspension A.]
Diluent: 200 g/L solution of sodium acetate
Hydrazine solution: 10.0 mg/mL of hydrazine sulfate.
Note—Allow to stand for 4-6 h.]
Methenamine solution: Transfer 2.5 g of methenamine
to a 100-mL glass-stoppered flask, add 25.0 mL of
water, insert the glass stopper, and mix to dissolve.
Primary opalescent suspension: Transfer 25.0 mL of
Hydrazine solution to the Methenamine solution in the
100-mL glass-stoppered flask. Mix, and allow to stand
for 24 h. [NoTte—This suspension is stable for 2 months,
rovided it is stored in a glass container free from sur-
‘ace defects. The suspension must not adhere to the
glass and must be well mixed before use.]
Opalescence standard: Dilute 15.0 mL of the Primary
opalescent suspension with water to 1000 mL. [NoTE—
This suspension should not be used beyond 24 hafter
preparation.]
Reference suspension A: Opalescence standard and
water (1 in 20)
Reference suspension B: Opalescence standard and
water (1 in 10)
Sample solution: 200 mg/mL in Diluent
Analysis
Samples: Diluent, Reference suspension A, Reference sus-
persion B, Sample solution, and water
ransfer a sufficient portion of the Sample solution to a
test tube of colorless, transparent, neutral glass with a
flat base and an internal diameter of 15-25 mm to ob-
tain a depth of 40 mm. Similarly transfer portions of
Reference suspension A, Reference suspension B, water,
and Diluent to separate matching test tubes. Compare
the solutions in diffused daylight, viewing vertically
against a black background (see Nephelometry, Turbi-
NF Monographs
dimetry, and Visual Comparison (855), Visual Compari-
son). [Note—The diffusion of light must be such that
Reference suspension A can readily be distinguished
from water, and that Reference suspension B can readily
be distinguished from Reference suspension A.]
Acceptance criteria: The Sample solution shows the
same clarity as that of water or Diluent, or its opales-
cence is NMT that of Reference suspension A.
NF 36
© COLOR OF SOLUTION
Diluent A: 200-g/L solution of sodium acetate
Diluent B: 10-g/L solution of hydrochloric acid
Standard stock solution: Ferric chloride CS, cobaltous
chloride CS, cupric sulfate CS, and Diluent B
(3.0: 3.0: 2.4: 1.6)
Standard solution: Standard stock solution and Diluent
B (1 in 100). [NotE—Prepare the Standard stock solution
and Standard solution immediately before use.]
Sample solution: Use the Sample solution from the test
for Clarity of Solution.
Analysis
Samples: Diluent A, Standard solution, Sample solution,
water
Transfer a sufficient portion of the Sample solution to a
test tube of colorless, transparent, neutral glass with a
flat base and an internal diameter of 15-25 mm to ob-
tain a depth of 40 mm. Similarly transfer portions of
the Standard solution, Diluent A, and water to separate,
matching test tubes. Compare the solutions in diffused
daylight, viewing vertically against a white background
(see Nephelometry, Turbidimetry, and Visual Comparison
(855), Visual Comparison).
Acceptance criteria: The Sample solution has the ap-
pearance of water or Diluent A, or is not more intensely
colored than the Standard solution.
ADDITIONAL REQUIREMENTS
¢ *PACKAGING AND STORAGE: Preserve in well-closed con-
tainers. Store at room temperature.»
e USP REFERENCE STANDARDS (11)
USP Saccharin RS
USP o-Toluenesulfonamide RS
USP p-Toluenesulfonamide RS
Saccharin Calcium—see Saccharin Calcium
General Monographs
Saccharin Sodium—see Saccharin Sodium
General Monographs
Safflower Oil—see Safflower Oil General
Monographs
Sesame Oil
DEFINITION
Sesame Oil is the refined fixed oil obtained from the seed of
one or more cultivated varieties of Sesamum indicum L.
(Fam. Pedaliaceae). It may contain suitable antioxidants.
IDENTIFICATION
° A. IDENTITY BY TRIGLYCERIDE PROFILE
Analysis: Proceed as directed in the test for Triglyceride
Composition.
Acceptance criteria: The peak responses of the eight
major talycerites thy OLL, PLL, OOL, POL, OOO,
SOL, and POO—elute between 0 and about 40 min, in
the order specified, and at relative retention times of
about 0.55, 0.65, 0.69, 0.77, 0.82, 0.93, 0.97, and 1.0,
respectively, as obtained in the Sample solution in the
test for Triglyceride Composition.
NF 36 Official Monographs / Sesame 5557

ASSAY IMPURITIES
© TRIGLYCERIDE COMPOSITION
[Note—The fatty acid radicals are designated as linoleic Delete the following:
(L), oleic (O), palmitic (P), and stearic (S), and the com-
mon abbreviations for triglycerides used are as follows:
trilinolein (LLL), 1,2-dilinoleoyl-3-oleoyl-rac-glycerol ®e HEAVY METALS, Methed I/ (231): NMT 10 19/ge cotta
1-
(OLL), 1,2-dilinoleoyl-3-palmitoyl-rac-glycerol (PLL), 1,2- Jan-2018)
dicteoy’ > linoleey) rac-glycero) (OOL), 1-palmitoyl- © ALKALINE IMPURITIES
2-oleoyl-3-linoleoyl-rac-glycerol (POL), triolein (OOO), Sample: 10 mL of Sesame Oil
1-linoleoyl-2-oleo' ee eae (SOL), and Analysis: Mix 10 mL of freshly opened acetone and
1,2-dioleoyl-3-palmitoyl-rac-glycero! (POO).] 0.3 mL of water, and add 0.05 mL of bromophenol
Mobile phase: Acetonitrile and methylene chloride blue TS. Add the Sample, shake, and allow to stand.
(60:40) Titrate with 0.01 N hydrochloric acid VS to change the
System suitability solution: 3.0 mg/mL each of USP color of the upper layer to yellow.
Sesame Oil Related Compound A RS and USP Sesame Acceptance criteria: NMT 0.1 mL of 0.01 N hydrochlo-
Oil Related CompoundB RS in Mobile phase. [NoTE— ric acid is required.
USP Sesame Oil Related CompoundARS is OLL, and SPECIFIC TESTS
USP Sesame Oil Related CompoundBRS is PLL.] e SPECIFIC GRAVITY (841): 0.912-0.921
aoe solution: 20 mg/mL of Sesame Oil in Mobile e FATS AND FIXED Olts (401), Acid Value (Free Fatty Acids)
phase Sample: 10g
Chromatographic system Acceptance criteria: NMT 2.0 mL of 0.020 N sodium
(See Chromatography (621), System Suitability.) hydroxide is required for neutralization.
Mode: LC FATS AND FIXED OILS (401), /odine Value: 103-116
Detector: Refractive index FATS AND FIXED OILS (401), Saponification Value: 188-195
Columns: Two 4.6-mm x 25-cm in series; packings L1 FATS AND FIXED OILS (401), Solidification Temperature of
Column temperature: 30° Fatty Acids: 20°-25°
Flow rate: 1.0 mL/min FATS AND FIXED OILS, Peroxide Value (401): NMT 10.0
Injection volume: 20 uL FATS AND FIXED OILS (401), Unsaponifiable Matter: NMT
System suitability 1.5%
Sample: System suitability solution COTTONSEED OIL
[Note—The relative retention times for OLL and PLL are Sample: 5 mL
about 0.93 and 1.0, respectively.] Analysis: Mix the Sample in a test tube with 5 mL of a
Suitability requirements mixture of equal volumes of amyl alcohol and a
Resolution: NLT 1.8 between OLL and PLL 10-mg/mL solution of sulfur in carbon disulfide. Warm
Relative standard deviation: NMT 1.5% determined the mixture carefully until the carbon disulfide is ex-
from peak areas; NMT 2.2% determined from the pelled, and immerse the tube to one-third of its depth
peak area ratio of OLL to PLL in a boiling saturated solution of sodium chloride.
Analysis Acceptance criteria: No reddish color develops within
[Note—The relative retention times for the eight major 15 min.
triglyceride peaks are listed in Table 7.] e WATER DETERMINATION, Method Ic (921): NMT 0.1%
Sample: Sample solution
Calculate the percentage of each of these triglycerides ADDITIONAL REQUIREMENTS
in the portion of the Sample taken: ¢ PACKAGING AND STORAGE: Preserve in tight, light-resistant
containers, and prevent exposure to excessive heat.
Result = (A/B) x 100 © LABELING: Label it to indicate the name and quantity of
any added antioxidant. Where Sesame Oil is intended for
A = peak area of each individual triglyceride use in the manufacture of injectable dosage forms, it is
= sum of the areas of all the peaks, excluding so labeled.
the solvent peak © OTHER REQUIREMENTS: For Sesame Oil intended for use in
injectable dosage forms, which is specified in the label-
Table 1 ing, the requirements must be met for Unsaponifiable
Relative Matter, Acid Value, Peroxide Value, and Water, Method Ic,
Retention Composition under Specific Tests in Injections and Implanted Drug Prod-
Triglyceride Time (%) ucts (1), Vehicles and added substances, Nonaqueous
vehicles.
LLL 0.55 7.0-19.0
e USP REFERENCE STANDARDS (11)
OLL 0.65 13.0-30.0 USP Sesame Oil Related Compound A RS
PLL 0.69 5.0-9.0 USP Sesame Oil Related Compound B RS
OOL 0.77 14.0-25.0
POL 0.82 8.0-16.0
000 0.93 5.0-14.0
SOL 0.97 2.0-8.0
POO 1.0 2.0-8.0
sydesbouow 4N
 5558 Shellac / Official Monographs NF 36

Table 1
Shellac Acid Value Loss on
(on dried Drying Wax
6 i basis) (%) (%)
\ Orange Shellac 68-76 NMT 2.0 NMT 5.5
( (
é

HO
Refined Orange Shellac 68-79 NMT 2.0 NMT 0.2
¥ /
Regular Bleached Shellac 73-89 NMT 6.0 NMT 5.5
)N kr Hoo
¢ \
Refined Bleached Shellac 75-91 NMT 6.0 NMT 0.2
woh |Hcae 2 \ oH Zz
Hom ie } o——_/ , IDENTIFICATION
» é e A. INFRARED ABSORPTION (197K) or(197A): Due to the de-
gree of polymerization, the intensity of some absorption
bands may vai
Use USP Bele Bleached Shellac RS for the following
or y two types:
¢ Orange Shellac
e Regular Bleached Shellac
Use USP Refined Bleached Shellac RS for the following
two types:
e\. ¥Zo I
Ro - © Refined Bleached Shellac
e Refined Orange Shellac
Se
x eh OH
e B. IDENTIFICATION OF ALEURITIC ACID AND SHELLOLIC ACID BY
\
wpeyuy
Can
THIN-LAYER CHROMATOGRAPHY
Standard solution: 6 mg/mL of USP Aleuritic Acid RS in
methanol, heating slightly if necessary.
Sample solution: Weigh and finely powder 500 mg of
HO
k Shellac. Transfer 500 mg of the finely powdered Shellac
to a test tube and heat with 4 mL atas main sodium
Jalane acid
# >on with Aleurtic acid hydroxide solution in a vigorously boiling water bath
‘Sheliofcacd A. ‘on cn with Aleuritieacid for 5 min. Cool, add 10 mL of ethyl acetate, and trans-
fer the content to a separatory funnel. With stirring,
Lakshoiie acid Aron >, with Aleuntic acid add slowly 4 mL of a 120-mg/mL solution ofglacial
acetic acid to the funnel. Shake the solution thoroughly
Lacojalianc acid
-be 4 uy wth Aleuritie acid
and withdraw the lower layer: Transfer the upper layer
Laccishetioicacid A oie withAleurticacid to a small flask, add anhydrous sodium sulfate, and pass
Lacclaksholic acid Peg Le vvathAleumticacid through a membrane disk syringe of 0.45-um pore size.
Collect the filtrate and use it as the sample.
Corresponding epimers ef above acids ‘with Abunte mad
Chromatographic system
(See Chromatography (621), Thin-Layer Chromato-
[9000-59-3]. graphy.)
Mode: TLC
DEFINITION Plate: 10-cm x 20-cm or 20-cm x 20-cm, silica gel 60
Shellac is obtained by the purification of lac, the resinuous
Fasa
secretion of the insect Kerria lacca (Kerr) Lindinger (Lac- Application volume: 10 UL, as 8-mm bands. [NOTE—
cifer lacca Kerr) (Fam. Coccideae). Shellac is a polyester An automated apparatus may be used.]
resin consisting of inter- and intra-esters of polyhydroxyl Developing solvent ae Ethyl acetate, methylene
carboxylic acids formed from certain hydroxyl acids and chloride, methyl alcohol, and acetic acid (60:32:8:1)
sesquiterpenic acids, and also contains variable amounts Spray reagent: Prepare the anisaldehyde solution by
of wax. There are four types of Shellac depending on the mixing in the following order. In 0.5 mL of
nature of the treatment of crude secretion (seedlac). anisaldehyde, add 10 mL of glacial acetic acid, 85 mL
1. Orange Shellac is produced by a process of filtration in of methyl alcohol, and 5 mL of sulfuric acid.
the molten state and/or by a process of solvent extrac- Analysis
tion. Orange Shellac retains most of its wax. Samples: Standard solution and Sample solution
2. Refined (Dewaxed) Orange Shellac is produced by filtra- Development: Apply the Samples in different bands to
tion of the wax in a solvent process. It may also be de- the previously marked starting point on a TLC plate,
colorized by activated carbon. and develop the plate two times over a path of 15 cm.
3. Regular Bleached (white) Shellac is prepared by dissolv- Dry the plate in air.
ing the lac in an alkaline solution and bleaching the solu- Detection: Spray with the Spray reagent. Heat the
tion with sodium hypochlorite. It is precipitated by dilute plate at 100°-105° for 10 min, and examine in
acid and dried. daylight (or white light).
4. Refined Bleached Shellac is prepared by dissolving the lac [Note—The principal band of aleuritic acid shows
in an alkaline solution and bleaching the solution with tong intensity and purple color. The retardation fac-
sodium hypochlorite. During the process, wax is re- tor (Rp) for the principal band of aleuritic acid is 0.41.
NF Monographs

moved by filtration. It is precipitated by dilute acid and
dried.
Shellac conforms to the specifications in Table 1.
A blue-gray band with medium intensity at Rr 0.22
could be assigned to shellolic acid.]
Acceptance criteria: The chromatogram from the Sam-
ple solution shows several colored bands. One of the
colored bands is similar in position and color to the
band in the chromatogram from the Standard solution,
and it is assigned to aleuritic acid. Below the aleuritic
a band, a blue-gray band is assigned to shellolic
acid.
NF 36
IMPURITIES
Delete the following:
®e HEAVY METALS, Method [I (231): NMT 10 ug/ge cotfcial1-
Jar-2018)
o LIMIT OF CHLORIDE
Dilute nitric acid: Dilute 10.5 mL of nitric acid with
water to 100 mL (10%).
Silver nitrate solution: Dissolve 1.75 g of silver nitrate
in water to 100 mL (0.1 mol/L).
Sample: 0.4g
Sample solution: Shake and dissolve the Sample in
5 mL of alcohol while warming. Add 40 mL of water,
and cool. Add 12 mL of Dilute nitric acid and water to
make 100 mL, and filter. Perform the analysis using
50-mL of the filtrate as the Sample solution.
Control solution: 0.8 mL of 0.1 M hydrochloric acid
VS, 2.5 mL of alcohol, 6 mL of Dilute nitric acid, and
water to make 50 mL
Analysis: Add 1 mL of Silver nitrate solution to the Sam-
ple solution and Control solution, mix well, and protect
from light for 5 min. Compare the opalescence devel-
oped in both solutions against a black background by
viewing downward or transversely.
Acceptance criteria: The opalescence of the Sample so-
lution is NMT that of the Contro/ solution, corresponding
to NMT 0.14%.
e TOTAL AsH
Sample: 1g
Analysis: Before sampling, ignite a crucible of platinum,
quartz, or porcelain at 500°-550° for 1 h. Cool and
weigh the crucible. Transfer the Sample to this crucible.
Take off the lid or open slightly if necessary. Heat the
crucible at a low temperature at first, then gradually
heat to 500°-550°. Ignite to incinerate the residue for
more than 4 h until no carbonized substance remains in
the ash. Cool and weigh the ash. Incinerate repeatedly
to constant weight, cool, weigh accurately, and deter-
mine the percentage of total ash. If a carbonized sub-
stance remains and a constant weight cannot be ob-
tained, extract the charred mass with hot water, collect
the insoluble residue on filter paper for assay, and incin-
erate the residue and filter paper until no carbonized
substance remains in the ash. Then add the filtrate,
evaporate to dryness, and incinerate. Cool, weigh accu-
rately, and determine the percentage of the total ash. If
a carbon-free ash still cannot be obtained, moisten the
ash with a small amount of alcohol (ethanol). Break up
the ash with a glass rod, and wash the rod with a small
amount of alcohol. Evaporate carefully, and determine
the mass of the total ash as described above. A desicca-
tor (silica gel) is used for cooling.
Acceptance criteria: NMT 1.0%
© ETHANOL-INSOLUBLE SUBSTANCES
Sample: 5g
Analysis: Dissolve the Sample in 50 mL of alcohol (etha-
nol) in a water bath while shaking. Transfer the ethanol
solution to a tared extraction thimble that was previ-
ously dried at 105° for 2 h in a Soxhlet extractor, and
extract with alcohol for 3 h. Dry the extraction thimble
at 105° for 3 h. Weigh the residue in the thimble.
Acceptance criteria: The mass of the residue is NMT
2.0%.
SPECIFIC TESTS
e Loss ON DRYING (731)
Analysis: Dry at 41 +2° in a well-ventilated oven for 24
h.
Acceptance criteria: See Table 1.
° ACID VALUE
Sample solution: Dissolve 2 g of finely ground Shellac
in 50 mL of alcohol that has been neutralized to phe-
nolphthalein with 0.1 N sodium hydroxide.
Official Monographs / Silica 5559
Analysis: Add additional phenolphthalein TS if neces-
sary and titrate with 0.1 N sodium hydroxide VS to a
pink endpoint, or determine the endpoint potentiomet-
rically. Express the acid value in terms of the number of
mg of potassium hydroxide required per g of dried
Shellac, and calculate the acid value as directed in Fats
and Fixed Oils (401), Acid Value.
[Note—For orange Shellac titrate slowly, stirring vigor-
ously, until a glass rod dipped into the titrated solu-
tion produces a color change when touched to a drop
of thymol blue TS on a spot plate.]
Acceptance criteria: See fable 1.
° WAX
Sample: 10g of finely ground Shellac
Analysis: Transfer the Sample and 2.50 g of sodium car-
bonate to a 200-mL, tall-form beaker. Add 150 mL of
hot water, immerse the beaker in a boiling water bath,
and stir until dissolved. Cover the beaker with a watch
glass, and maintain the heat for more than 3 h without
agitation. Remove the beaker to a cold water bath.
When the wax has floated to the surface, pass the solu-
tion through medium-speed quantitative ashless filter
paper, transferring the wax to the paper, and wash the
filter with water. Pour 5-10 mL of alcohol onto the filter
to facilitate drying. Wrap the paper loosely in a larger
piece of filter paper, bind with a piece of fine wire, and
dry with the aid of gentle heat. Extract with chloroform
in a suitable continuous extraction apparatus for 2 h,
using a weighed flask to receive the extracted wax and
solvent. Evaporate the solvent, and dry the wax at 105°
to constant weight.
Acceptance criteria: See Table 1.
e ROSIN
Sample solution: 200 mg/mL in dehydrated alcohol
Analysis: To 10 mL of Sample solution add slowly, with
shaking, 50 mL of solvent hexane, wash with two suc-
cessive 50-mL portions of water, filter the washed al-
cohol-solvent hexane solution, and evaporate to dry-
ness. To the residue add 2 mL of a mixture of liquefied
phenol, dehydrated alcohol, and solvent hexane
(1: 0.5: 2). Stir, and transfer a portion of the solution to
the cavity of a color-reaction plate. Fill an adjacent cav-
ity with a mixture of bromine and solvent hexane (1:4),
and cover both cavities with an inverted watch glass.
Acceptance criteria: No purple or deep indigo-blue
color is produced in or above the liquid containing the
residue.
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in well-closed contain-
ers. Store in a dry place below 15°. Protect from light.
e LABELING: Label it to indicate the type of Shellac.
e USP REFERENCE STANDARDS (11)
USP Aleuritic Acid RS
USP Refined Bleached Shellac RS
USP Regular Bleached Shellac RS
Dental-Type Silica
DEFINITION
Dental-Type Silica is obtained from sodium silicate solution
sydeibouow 4N
by destabilizing with acid in such a way as to yield very
fine particles. The sum of the Assay values for Silicon Di-
oxide and Sodium Sulfate is NLT 98.0%.
ASSAY
© SILICON DIOXIDE
Sample: 1g
Analysis: Ignite the Sample at 1000° for 1 h, cool in a
desiccator, and weigh. Carefully wet with water, and
add 10 mL of hydrofluoric acid, in small increments.
Evaporate on a steam bath to dryness, and cool. Add
 5560 Silica / Official Monographs
about 10 mL of hydrofluoric acid and about 0.5 mL of
sulfuric acid, and evaporate to dryness. Slowly increase
the temperature until all of the acids have been volatil-
ized, and ignite at 1000°. Cool in a desiccator, and
weigh. The difference between the final weight and the
weight of the initially ignited portion represents the
weight of SiOz.
© SODIUM SULFATE
Sample: 1g
Analysis 1: In a platinum dish, wet the Sample with a
few drops of water, add 15 mL of perchloric acid, and
place the dish on a hot plate. Add 10 mL of hydroflu-
oric acid. Heat until copious fumes are evolved. Add
5 mL of hydrofluoric acid, and again heat to copious
fumes. Add 5 mL of boric acid solution (1 in 25), and
heat to fumes. Cool, and transfer the residue to a
400-mL beaker with the aid of 10 mL of hydrochloric
acid. Adjust the volume with water to about 300 mL,
and bring to boiling on a hot plate. Add 20 mL of hot
barium chloride TS. Keep the beaker on the hot plate
for 2 h, maintaining the volume above 200 mL. After
cooling, transfer the precipitate and solution to a dried,
tared crucible witha filter of 0.8-44m pore size. Wash
the filter and precipitate 8 times with hot water, dry
the crucible at 105° for 1 h, and weigh. The weight,
multiplied by 0.6085, is the sodium sulfate content of
the amount of specimen taken.
Acceptance criteria 1: NMT 4.0%
Analysis 2: Calculate the sum of the Assay values for
the silicon dioxide and the sodium sulfate, and calculate
the percentage in the Dental-Type Silica taken.
Acceptance criteria 2: NLT 98.0%
SPECIFIC TESTS
e PH (791)
Sample solution: 50 pail of slurry
Acceptance criteria: 4.0-8.5
Loss ON DRYING (731)
Analysis: Dry a sample at 105° for 2 h.
Acceptance criteria: It loses NMT the maximum per-
centage of its weight as indicated in the labeling.
Loss ON IGNITION (733)
Sample: 1g, previously dried
Analysis: Ignite the Sample at 1000° for NLT 1 h.
Acceptance criteria: NMT 8.5%
CHLORIDE AND SULFATE, Chloride (221)
Sample solution: Boil 5 g in 50 mL of water under a
reflux condenser for 2 h, cool, and filter.
Control: 1.0 mL of 0.020 N hydrochloric acid
Analysis: Use a 7-mL portion of Sample solution.
Acceptance criteria: 0.1%; the Sample solution shows
no more chloride than the Control.
ARSENIC, Method | (211)
Sample solution: Transfer 4.0 g of Dental-Type Silica to
a patina dish, add 5 mL of nitric acid and 35 mL of
hydrofluoric acid, and evaporate on a steam bath. Cool,
add 5 mL of perchloric acid, 10 mL of hydrofluoric acid,
and 10 mL of sulfuric acid, and evaporate on a hot
plate to the production of heavy fumes. Cool, cau-
tiously transfer to a 100-mL beaker with the aid of a
few mL of hydrochloric acid, and evaporate to dryness.
Cool, add 5 mL of hydrochloric acid, dilute with water
to about 40 mL, and heat to dissolve any residue. Cool,
transfer to a 100-mL volumetric flask, and dilute with
water to volume.
NF Monographs
Analysis: Use a 25.0-mL portion of Sample solution.
Acceptance criteria: NMT 3 ppm
Delete the following:
®e HEAVY Mertats, Method | (231)
Test preparation: 16.7 mL of the solution prepared in
the test for Arsenic
NF 36
Analysis: Transfer the Test preparation to a 100-mL
beaker, and neutralize to litmus paper with ammonium
hydroxide. Adjust with 6 N acetic acid to a pH between
3 and 4. Filter, using medium-speed filter paper, and
ee water until the filtrate and washings measure
mL.
Acceptance criteria: NMT 30 ppMe cinciat j-Jon-z018)
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in tight containers.
e LABELING: Label it to indicate the maximum percentage
of loss on drying.
Hydrophobic Colloidal Silica
[68611-44-9].
DEFINITION
Hydrophobic Colloidal Silica is prepared by partial alkylation
for hydrophobation. It contains NLT 99.0% and NMT
on % of silicon dioxide (SiOz), calculated on the ignited
aSIS.
IDENTIFICATION
co A.
Sample: 25 mg
Analysis: Add the Sample to a platinum crucible, and
ignite at 900° for 2 h. Using a copper wire, mix the
ignited substance with 10 mg of sodium fluoride and a
‘ew drops of sulfuric acid to give a thin slurry. Cover
the crucible with a thin, transparent plate of plastic
under which a drop of water is suspended, and warm
gently.
Acceptance criteria: Within a short time, a white ring
is rapidly formed around the drop of water.
e B. It meets the requirements for Water-Dispersible Sub-
stances in Specific Tests.
ASSAY
¢ PROCEDURE
Sample: The residue obtained in the test for Loss on
Ignition
Analysis: To the Sample add sufficient alcohol to mois-
ten the residue completely, and then add 0.2 mL of sul-
furic acid. Add 6 mL of hydrofluoric acid, and evaporate
to aoe on a hot plate at about 100°, taking care to
avoid loss from sputtering. Wash down the sides of the
platinum crucible with 6 mL of hydrofluoric acid, and
evaporate to dryness. Ignite at 900° for 2 h, cool in a
desiccator, and weigh. The difference between the
weight of the residue obtained in the test for Loss on
Ignition and the weight of the final residue gives the
amount of silicon dioxide (SiOz) in the quantity of the
substance to be examined.
Acceptance criteria: 99.0%-101.0% on the ignited
basis
IMPURITIES
e Loss ON IGNITION (733)
Sample: 0.2g
Analysis: eae the Sample in a platinum crucible at
900° for 2h. Cool in a desiccator before weighing.
[Note—lIt is advisable to place the crucible in a cold
oven and then heat up the oven.]
Acceptance criteria: NMT 6.0%
e Limit OF LEAD
Select reagents having as low a lead content as practica-
ble, and store all solutions in containers of borosilicate
glass. Rinse all glassware thoroughly with warm, dilute
nitric acid (1 in 2) followed by water.
Ammonium acetate buffer solution, pH 3.5: Weigh
25.0 g of ammonium acetate, and dissolve in 25 mL of
water. Add 38.0 mL of dilute hydrochloric acid. Adjust
NF 36
the pH, if necessary, with dilute hydrochloric acid or
weak ammonia solution (containing 460 mL/L of strong
ammonia solution). Dilute with water to 100.0 mL.
Thioacetamide solution: Prepare immediately before
use. To 0.2 mL of thioacetamide TS add 1 mL of a mix-
ture of 5 mL of water, 15 mL of 1 N sodium hydroxide,
and 20 mL of 85% glycerol. Heat in a water bath for 20
Ss
Sample solution: Suspend 2.5 g of Hydrophobic Colloi-
dal Silica in 30 mL of methanol, stir, and add 30 mL of
weak ammonia solution (containing 460 mL/L of strong
ammonia solution). With frequent stirring, evaporate on
a water bath, and dry the residue in an oven at 140°.
When the dried substance is white, break up the mass
with a glass rod. Reduce the residue to a powder, and
add 15 mL of methanol and 25 mL of 1 N hydrochloric
acid. Boil gently for 5 min, stirring frequently with the
glass rod. Centrifuge for 20 min, and pass the superna-
tant through a membrane filter. To the residue in the
centrifuge tube add 3 mL of dilute hydrochloric acid
and 9 mL of water, and bring to a boil. Centrifuge for
20 min, and pass the Supe natant through the same
membrane filter. Wash the residue with small quantities
of water, combine the filtrates and washings, and dilute
with water to 50 mL. To 20 mL of this solution add
50 mg of ascorbic acid and 1 mL of strong ammonia
solution. Neutralize with diluted weak ammonia solu-
tion (containing 160 mL/L of strong ammonia solution).
Dilute with water to 25 mL.
Reference solution: Pipet 10 mL of standard lead solu-
tion TS, and mix with 2 mL of the Sample solution.
Blank solution: A mixture of 10 mL of water and 2 mL
of the Sample solution
Analysis: To the Sample solution, Reference solution, and
Blank solution add 2 mL of Ammonium acetate buffer so-
lution, pH 3.5. Mix, and add 1.2 mL of Thioacetamide
solution. Mix immediately. Examine the solutions after 2
min. The test is invalidit the Reference solution does not
showa slight brown color compared to the Blank
solution.
Acceptance criteria: The brown color in the Sample so-
lution is not more intense than that in the Reference
solution (25 ,1g/g). [NoTE—If the result is difficult to
judge, pass the solutions through a membrane filter of
3-um pore size, and carry out the filtration slowly and
uniformly. Compare the spots on the filters obtained
with the different solutions.]
e LIMIT OF CHLORIDE
Standard solution: Add 10 mL of 0.15 mM sodium
chloride and 5 mL of water. Add 1 mL of dilute nitric
acid, and pour into a test tube containing 1 mL of silver
nitrate TS.
Sample solution: To 1g of Hydrophobic Colloidal Silica
add 30 mL of methanol and 20 mL of dilute nitric acid.
Heat on a water bath for 15 min with frequent stirring.
Cool, dilute with water to 50 mL, and filter. Dilute
10 mL of the filtrate with water to 15 mL. Add 1 mL of
dilute nitric acid, and pour into a test tube containing
1 mL of silver nitrate TS.
Analysis: Examine the tubes laterally against a black
background.
Acceptance criteria: After standing for 5 min protected
from light, any opalescence in the Sample solution is not
more intense than that in the Standard solution
(0.025%).
SPECIFIC TESTS
© WATER-DISPERSIBLE SUBSTANCES
Sample: 0.4g
Analysis: Place the Sample in a 500-mL separating fun-
nel, add 100 mL of water, and shake for 1 min. Allow
to stand for 1 h. Allow 90 mL of the aqueous phase to
run out dropwise without filtration into a suitable dish
dried at 140°, and cool in a desiccator. Evaporate to
Official Monographs / Siliceous 5561
dryness at 140°, starting at a low temperature to avoid
splashing. Cool in a desiccator. :
Acceptance criteria: NMT 3.0%; the weight of the resi-
due does not exceed 12 mg.
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in well-closed contain-
ers. No storage requirements specified.
Purified Siliceous Earth
DEFINITION
Purified Siliceous Earth is a form of silica (SiOz) consisting of
the frustules and fragments of diatoms, purified by
calcining.
IMPURITIES
e Loss ON IGNITION (733)
Sample: 1, previously dried
Analysis: Ignite the Sample at 980+ 25° for 1 hina
tared platinum or porcelain crucible.
Acceptance criteria: NMT 2.0%
e LEACHABLE ARSENIC
Sample solution: To 10.0 g in a 250-mL beaker add
50 mL of 0.5 N hydrochloric acid, cover with a watch
glass, and heat at 70° for 15 min. Cool, and decant
through a Whatman No.3filter paper into a 100-mL
volumetric flask. Wash the slurry with three 10-mL por-
tions of water, preheated to 70°, and dilute with water
to volume.
Analysis: A 3.0-mL portion of the Sample solution meets
the requirements in Arsenic, Method 1 (211).
Acceptance criteria: NMT 10 pg/g
e LEACHABLE LEAD
Sample: A 10.0-mL portion of the Sample solution pre-
pared in the test for Leachable Arsenic
Control: 10 mL of Diluted Standard Lead Solution in
Lead (251)
Anas The Sample meets the requirements in Lead
(251).
Acceptance criteria: NMT 10 ug/g
e LIMIT OF NONSILICEOUS SUBSTANCES
Sample: 200 mg
Analysis: Transfer the Sample to a tared platinum cruci-
ble, add 5 mL of hydrofluoric acid and 2 drops of dilute
sulfuric acid (1 in 2), and evaporate gently to dryness.
Cool, add 5 mL of hydrofluoric acid, evaporate again to
dryness, and ignite to constant weight.
Acceptance criteria: The weight of the residue is NMT
50 mg.
SPECIFIC TESTS
e LOSS ON DRYING (731)
Analysis: Dry a sample at 105° for 2 h.
Acceptance criteria: NMT 0.5%
e ACID-SOLUBLE SUBSTANCES
Sample: 10.0g
Analysis: Digest the Sample with 50 mL of 0.5 N hydro-
chloric acid at 70° for 15 min, and filter. Wash the resi-
due, adding the washings to the filtrate to obtain a
total volume of 100 mL. Evaporate at 110° in a tared
porcelain dish to dryness.
sydesBouow 4N
Acceptance criteria: NMT 2.0% (weight of the dried
residue is NMT 200 mg)
© WATER-SOLUBLE SUBSTANCES
Sample: 12.59
Analysis: Place the Sample in a 500-mL conical flask,
add 250 mL of water, and shake for 2 h at room tem-
perature. Filter with the aid of vacuum, and again filter
if necessary to obtain a clear filtrate. Evaporate in a
tared platinum or porcelain dish, and dry at 110°.
USP 41
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Measure the areas of the analyte peaks. Identify the re-
tention times of the peaks corresponding tospect
ophylline, uncarine F, mitraphylline, isomitraphylline,
rhynchophylline, isorhynchophylline, pteropodine, and
isopteropodine by comparison of the chromatogram
of Standard solution A with the reference chromato-
gram provided with the lot of the USP Powdered Cat's
Claw Extract RS used.
Separately calculate the percentages of speciophylline,
uncarine F, mitraphylline, isomitraphylline, rhyncho-
phylline, isorhynchophylline, pteropodine, and isopter-
opodine, as isopteropodine, in the portion of Cat’s
Claw taken:
Result = (ry/rs) x (Cs/Cu) x 100
tu = peak response for each relevant alkaloid from
the Sample solution
rs = peak response for isopteropodine from
Standard solution B
Cs = concentration of USP Isopteropodine RS in
Standard solution B (mg/mL)
Cu = concentration of Cat’s Claw in the Sample
solution (mg/mL)
Calculate the content of pentacyclic oxindole alkaloids
by adding the percentages of speciophylline, uncarine
F, mitraphylline, isomitraphylline, pteropodine, and
isopteropodine.
Calculate the content of tetracyclic oxindole alkaloids
by adding the individual percentages of
rhynchophylline and isorhynchophylline.
Acceptance criteria
Pentacyclic oxindole alkaloids: NLT 0.3%
Tetracyclic oxindole alkaloids: NMT 0.05%
CONTAMINANTS
© ARTICLES OF BOTANICAL ORIGIN, Limits of Elemental Impuri-
ties (561): Meets the requirements
¢ ARTICLES OF BOTANICAL ORIGIN, Pesticide Residue Analysis
(561): Meets the requirements
e [MICROBIAL ENUMERATION TESTS (2021): The total aerobic
microbial count does not exceed 105/g; the total com-
bined molds and yeasts count does not exceed 103/g;
and the bile-tolerant Gram-negative bacteria do not ex-
ceed 103/g.
e ABSENCE OF SPECIFIED MICROORGANISMS (2022): Meets the
requirements of the tests for absence of Salmonella spe-
cies and Escherichia coli
SPECIFIC TESTS
¢ BOTANICAL CHARACTERISTICS
[Note—The Pharmacopeial article is constituted only by
the stem inner bark of U. tomentosa (Willd.) DC. De-
scriptions of other parts of the plant are given to aid in
the collection of the right species. Compliance should
be determined using the entire monograph and not
only the botanical description.]
Macroscopic: Cat’s Claw is a woody vine with a main
stem up to 20 cm in diameter and up to 30 m long.
The branches are obtusely quadrangular and generally
puberulous. Stipules in the buds are densely tomentose
in the upper side (different from U. guianensis in which
the stipules areglabrous) with the hairs, often with
curved tips, meshed together and with the longer hairs
of the leaf helping to connect the pair of stipules along
the margins, but split when older (different from U. gui-
anensis in which the stipules separate early in the bud
development). Thorns are straight to sickle-shaped, not
spirally twisted (different from U. guianensis), very pun-
gent and woody, from 8 to 20 mm long and from 3 to
6 mm wide. When recently cut, the color of the inner
bark can be whitish gray, yellowish brown, or dark red,
Dietary Supplements / Cat's Claw 4507
with longitudinal fissures and persistent rhytidome. The
internal part has a slightly dusty fibrous and laminar
texture with a characteristic ferruginous dust and an ex-
tremely astringent taste. The terminal branches have a
quadrangular section and yellowish green internal
medulla.
Microscopic: The periderm with cork (phellem) is con-
stituted by 6-8 rows of cells having walls evenly thick-
ened, a compressed phellogen and a phelloderm with
1-7 rows of sclereids. [NOTE—The periderm and phello-
gen should be absent in the Pharmacopeial article.] The
secondary cortex with concentric rings of fibers are sep-
arated by rings of parenchyma; rings of fibers are fre-
quently interrupted by radial rows of parenchyma cells
(predominately 1 cell broad) or narrow medullary rays
(lew cells broad), forming rectangular bundles of fibers
in a regular network. In longitudinal view, the fibers
appear with numerous conspicuous pits; calcium oxa-
late microcrystals (sand-like) are abundant in the paren-
chyma, but usually absent as large polyhedral crystals
or in the form of styloids with bifurcated endings, the
latter forms typically present in the parenchyma of U.
guianensis,; a brown substance is dispersed in paren-
chyma cells; starch is abundant, granules are solitary
(circular in outline, up to 10 um in diameter) or com-
pound (2-3 components up to 15 um in diameter).
e ARTICLES OF BOTANICAL ORIGIN, Foreign Organic Matter
(561): NMT 2.0%
e Loss ON DRYING (731)
Sample: 1.0g of Cat’s Claw, finely powdered
Analysis: Dry the Sample at 105° for 2 h.
Acceptance criteria: NMT 7.0%
e ARTICLES OF BOTANICAL ORIGIN, Total Ash (561): NMT
8.0%
e ARTICLES OF BOTANICAL ORIGIN, Acid-/nsoluble Ash (561):
NMT 2.0%
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in tight, light-resistant
containers, and store at room temperature.
e LABELING: The label states the Latin binomial and, follow-
ing the official name, the parts of the plant contained in
the article.
e USP REFERENCE STANDARDS (11)
USP Isopteropodine RS
USP Powdered Cat’s Claw Extract RS
Powdered Cat’s Claw
sydesbouo;w sq
DEFINITION
Powdered Cat’s Claw is Cat’s Claw reduced to a powder or
very fine powder. It contains NLT 0.3% of pentacyclic ox-
indole alkaloids as isopteropodine, calculated on the dried
basis, as the sum of speciophylline, uncarine F, mitraphy!-
line, isomitraphylline, pteropodine, and isopteropodine.
IDENTIFICATION
° oo CHROMATOGRAPHIC IDENTIFICATION TEST
Standard solution: 100mg of USP Powdered Cat’s
Claw Extract RS in 2 mL of methanol. Sonicate for 5
min, shaking occasionally, heat in a water bath at 60°
for 15 min, cool, and centrifuge.
Sample solution: 5g of Powdered Cat’s Claw in 10 mL
of methanol. Sonicate for 5 min, shaking occasionally.
Heat the mixture in a water bath at 60° for 15 min,
cool, and filter.
Adsorbent: Chromatographic silica gel mixture with an
average particle size of 10-15 jum (TLC plates)
Application volume: 20 LiL, as bands that are 1 cm in
length
 5562 Siliceous / Official Monographs
Acceptance criteria: NMT 0.2% (weight of the residue
is NMT 25 mg)
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in well-closed
containers.
Silicon Dioxide
SiO, + xH20
Anhydrous 60.08
DEFINITION
Silicon Dioxide is obtained by insolubilizing the dissolved
silica in sodium silicate solution. Where obtained by the
addition of sodium silicate to a mineral acid, the product
is termed silica gel. Where obtained by the destabilization
of a solution of sodium silicate in such manner as to yield
very fine particles, the product is termed precipitated sil-
ica. After ignition at 1000° for NLT 1 h, it contains NLT
99.0% of SiOz.
IDENTIFICATION
© PROCEDURE
Sample: 5 mg
Analysis: Transfer the Sample to a platinum crucible,
mix with 200 mg of anhydrous potassium carbonate,
ignite at a red heat over a burner for 10 min, and cool.
Dissolve the melt in 2 mL of recently distilled water,
warming if necessary, and slowly add 2 mL of ammo-
nium molybdate TS.
Acceptance criteria: A deep yellow color is produced.
ASSAY
© PROCEDURE
Sample: 1g
Analysis: Ignite the Sample in a tared platinum dish at
1000° for 1 h, cool in a desiccator, and weigh. Care-
fully wet with water, and add 10 mL of hydrofluoric
acid in small increments. Evaporate on a steam bath to
dryness, and cool. Add 10 mL of hydrofluoric acid and
0.5 mL of sulfuric acid, and evaporate to dryness.
Slowly increase the temperature until all of the acids
have been volatilized, and ignite at 1000°. Cool in a
desiccator, and weigh. The difference between the final
weight and the weight of the initially ignited portion
represents the weight of SiOz.
Acceptance criteria: NLT 99.0% on the previously ig-
nited basis
IMPURITIES
Inorganic Impurities
e LOSS ON IGNITION (733)
Sample: 1g
Analysis: Ignite the Sample, previously dried and
weighed, at 1000° for NLT 1 h.
Acceptance criteria: It loses NMT 8.5% of its weight.
© CHLORIDE AND SULFATE, Chloride (221): Boil 5g in 50 mL
of water under a reflux condenser for 2 h, cool, and
filter. A 7-mL portion of the filtrate shows no more chlo-
ride than corresponds to 1.0 mL of 0.020 N hydrochloric
acid (0.1%).
NF Monographs
© CHLORIDE AND SULFATE, Sulfate (221): A 10-mL portion of
the filtrate from the test for Chloride shows no more sul-
fate than corresponds to 5.0 mL of 0.020 N sulfuric acid
(0.5%).
e ARSENIC, Method | (211)
Sample solution: Transfer 4.0 g to a platinum dish.
Add 5 mL of nitric acid and 35 mL of hydrofluoric acid,
and evaporate on a steam bath. Cool. Add 5 mL of
NF 36
perchloric acid, 10 mL of hydrofluoric acid, and 10 mL
of sulfuric acid, and evaporate on a hot plate to the
production of heavy fumes. Cool. Cautiously transfer to
a 100-mL beaker with the aid of a few mL of hydro-
chloric acid, and evaporate to dryness. Cool. Add 5 mL
of hydrochloric acid, dilute with water to 40 mL, and
heat to dissolve any residue. Cool. Transfer to a
100-mL volumetric flask, and dilute with water to
volume.
Analysis: Use a 25.0-mL portion of the Sample solution.
Acceptance criteria: Meets the requirements of the
test (NMT 3 ppm)
Delete the following:
°e HEAVY METALS, Method | (231)
Sample solution: 16.7 mL of the solution prepared for
the test for Arsenic
Analysis: Transfer the Sample solution to a 100-mL
beaker, and neutralize to Ittmus paper with ammonium
hydroxide. Adjust with 6 N acetic acid to a pH between
3 and 4. Filter, using medium-speed filter paper, and
Woo wies water until the filtrate and washings measure
40 mL.
Acceptance criteria: NMT 30 ppme otticial 1.Jan-2018)
SPECIFIC TESTS
e PH (791): 4-8 ina slurry (1 in 20)
e Loss ON DRYING (731): Dry a sample at 145° for 4 h; it
loses NMT 5.0% of its weight.
ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Preserve in tight containers,
protected from moisture.
e LABELING: Label it to state whether it is silica gel or pre-
cipitated silica.
Colloidal Silicon Dioxide
SiO. 60.08
Silica [7631-86-9].
DEFINITION
Colloidal Silicon Dioxide is a submicroscopic fumed silica
prepared by the vapor-phase hydrolysis ofa silicon com-
pound. When ignited at 1000° for 2 h, it contains NLT
99.0% and NMT 100.5% of SiOz.
IDENTIFICATION
¢ A. PROCEDURE
Analysis: Transfer 5 mg to a platinum crucible, and mix
with 200 mg of anhydrous potassium carbonate. Heat
the crucible to a red color with the aid of a Bunsen
burner for 10 min, and cool. Dissolve the melt in 2 mL
of freshly distilled water, warming if necessary, and
slowly add 2 mL of ammonium molybdate TS to the
solution.
Acceptance criteria: A deep yellow color is produced.
¢ B. PROCEDURE
[Note—Avoid contact with o-tolidine when performing
this test, and conduct the test in a well-ventilated
hood.]
Analysis: Place 1 drop of the yellow silicomolybdate so-
lution from Identification test A ona filter paper, and
evaporate the solvent. Add 1 drop of a saturated solu-
tion of o-tolidine in glacial acetic acid to reduce the
silicomolybdate to molybdenum blue, and place the pa-
per over ammonium hydroxide.
NF 36
Acceptance criteria: A greenish blue spot is produced.
ASSAY
e PROCEDURE
Sample: 500 mg
Analysis: Ignite the Sample in a tared platinum crucible
at 1000 +25° for 2 h, cool in a desiccator, and weigh.
Add 3 drops of sulfuric acid, and add enough alcohol to
just moisten the sample completely. Add 15 mL of hy-
drofluoric acid, and in a well-ventilated hood evaporate
on a hot plate to dryness, using medium heat
(95°-105°) and taking care that the sample does not
spatter as dryness is approached. Heat the crucible to a
red color with the aid of a Bunsen burner. Ignite the
residue at 1000 + 25° for 30 min, cool in a desiccator,
and weigh. If a residue remains, repeat the Analysis, be-
ginning with “Add 15 mL of hydrofluoric acid”. The
weight lost by the assay specimen, previously ignited at
ue + 25°, represents the weight of SiO, in the portion
taken.
Acceptance criteria: 99.0%-100.5% on the previously
ignited basis
IMPURITIES
Inorganic Impurities
e Loss ON IGNITION (733): Ignite the portion of Colloidal
Silicon Dioxide, retained from the test for Loss on Drying,
at 1000 + 25° to constant weight: the previously dried
Colloidal Silicon Dioxide loses NMT 2.0% of its weight.
© ARSENIC, Method 7 (211)
Sample solution: To 2.5 g add 50 mL of 3 N hydro-
chloric acid, and reflux for 30 min using a water con-
denser. Cool, filter with the aid of suction, and transfer
the filtrate to a 100-mL volumetric flask. Wash the filter
and flask with several portions of hot water, and add
the washings to the flask. Cool, and dilute with water
to volume.
Analysis: A 15.0-mL portion of Sample solution, to
which 3 mL of hydrochloric acid has been added,
meets the requirements of the test, the addition of the
7N sulfuric acid being omitted.
Acceptance criteria: NMT 8 ppm
SPECIFIC TESTS
e PH (791): 3.5-5.5, in a (1 in 25) dispersion
e Loss ON DRYING (731): Dry in a tared platinum crucible
at 105° for 2 h: it loses NMT 2.5% of its weight. Retain
the dried specimen in the crucible for the test for Loss on
Ignition.
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in well-closed
containers.
Simethicone—see Simethicone General
Monographs
Simethicone Emulsion—see Simethicone
Emulsion General Monographs
Soda Lime
DEFINITION
Soda Lime is a mixture of Calcium Hydroxide and Sodium
or Potassium Hydroxide or both. It may contain an indica-
tor that is inert toward anesthetic gases such as Ether,
Cyclopropane, and Nitrous Oxide and that changes color
Official Monographs / Soda 5563
when the Soda Lime can no longer absorb Carbon
Dioxide.
IDENTIFICATION
oA.
Analysis: Place a granule on a piece of moistened red
litmus paper.
Acceptance criteria: The paper turns blue immediately.
e B. IDENTIFICATION TESTS—GENERAL, Calcium (191)
Sample solution: A solution in 6 N acetic acid
Acceptance criteria: Meets the requirements. It also
imparts a yellow color to a nonluminous flame that,
when viewed through cobalt glass, may show aviolet
color.
SPECIFIC TESTS
e Loss ON DRYING (731)
Sample: 10g
Analysis: Dry at 105° for 2 h.
Acceptance criteria: 12.0%-19.0%
¢ CARBON DIOXIDE ABSORBENCY
Analysis: Fill the lower transverse section of a U-shaped
drying tube of 15-mm internal diameter and 15-cm
height with loosely packed glass wool. In one arm of
the tube, place 5 g of anhydrous calcium chloride, and
weigh the tube and the contents. In the other arm,
place 9.5-10.5 g of Soda Lime, and again weigh. Insert
stoppers in the open arms of the tube, and connect the
side tube of the arm filled with Soda Lime to a calcium
chloride drying tube, which in turn is connected to a
suitable source of supply of carbon dioxide. Pass the
carbon dioxide through the tube at 75 mL/min for 20
min, accurately timed. Disconnect the tube, cool to
room temperature, remove the stoppers, and welght
Acceptance criteria: NLT 19.0% increase in weight of
the Soda Lime used for the test
e HARDNESS
Sample: 200g
Analysis: Screen the Sample on a mechanical sieve
shaker (see Particle Size Distribution Estimation by Analyti-
cal Sieving (786)) having a frequency of oscillation of
285 + 3 cycles/min, for 3 min, to remove granules both
coarser and finer than the labeled particle size. Weigh
50 g of the granules retained on the screen, and place
them in a hardness pan that has a diameter of 200 mm
and a concave brass bottom 7.9 mm thick at the cir-
cumference and 3.2 mm thick at the center, with an
inside spherical radius of curvature of 109 cm. Add 15
steel balls of 7.9-mm diameter, and shake on a me-
chanical sieve shaker for 30 min. Remove the steel balls,
brush the contents of the hardness pan ontoa sieve of
the fine-mesh size designated on the label, shake for 3
min on the mechanical sieve shaker, and weigh.
Acceptance criteria: NLT 75.0% of Soda Lime is re-
tained on the screen.
e Moisture ABSORPTION
Sample: 10g
Analysis: Place the Sample in a tared 50-mL weighing
bottle having a diameter of 50 mm and a height of
30 mm, and weigh. Then place the bottle, with cover
removed, for 24h in a closed container in which the
atmosphere is maintained at 85% relative humidity by
being in equilibrium with sulfuric acid having a specific
gravity of 1.16. Weigh again.
Acceptance criteria: The weight increase is NMT 7.5%. vA
¢ PARTICLE SIZE DISTRIBUTION ESTIMATION BY ANALYTICAL n
SIEVING, Method | (786)
Sample: 100g
s
°
Analysis: Screen the Sample for 5 min as directed, using =]
a mechanical shaker. °
Xe)
Acceptance criteria: It passes completely through a a
2
No. 2 standard-mesh sieve, and NMT 2.0% passes me}
through a No. 40 standard-mesh sieve. NMT 7.0% is a
retained on the coarse-mesh sieve, and NMT 15.0% a
 5564 Soda / Official Monographs NF 36

asses through the fine-mesh sieve designated on the erature, transfer to a 100-mL volumetric flask, and di-
abel. ute with water to volume.
Analysis: Use 50 mL of the Test preparation, and pro-
ADDITIONAL REQUIREMENTS ceed as directed in the chapter, using 15 mL of ammo-
© PACKAGING AND STORAGE: Preserve in tight containers. nium citrate solution, 3 mL of potassium cyanide solu-
e LABELING: If an indicator has been added, the name and tion, and 0.5 mL of hydroxylamine hydrochloride
color change of such indicator are stated on the con- solution. After the first dithizone extraction, wash the
tainer label. The container label also indicates the mesh combined chloroform layers with 5 mL of water, dis-
size in terms of standard-mesh sieve sizes (see Powder carding the water layer and continuing in the usual
Fineness (811)). manner by extracting with 20 mL of 0.2 N nitric acid.
Acceptance criteria: Contains NMT 5 ug of lead (cor-
responding to NMT 10 ppm)

Delete the following:

Sodium Acetate—see Sodium Acetate
General Monographs °e HEAVY METALS, Method // (231)
[Note—Conduct the ignition in a platinum crucible, and
use nitric acid in place of sulfuric acid to wet the test
specimen.]
Sodium Alginate Acceptance criteria: NMT 40 ppme cortical 1-jan-2018)
SPECIFIC TESTS
e MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI-
FIED MICROORGANISMS (62): The total bacterial count
does not exceed 200 cfu/g, and the tests for Salmonella
species and Escherichia coli are negative.
e Loss ON DRYING (731): Dry a sample at 105° for 4 h: it
loses NMT 15.0% of its weight.
(CgH7NaQo)n e ARTICLES FOR BOTANICAL ORIGIN, Tota! Ash (561): Proceed
Alginic acid, sodium salt; as directed in Methods of Analysis, carefully igniting 3gin
Sodium alginate [9005-38-3]. a tared platinum dish, until the residue is thoroughly car-
bonized (5 min), and then gai in a muffle furnace at
DEFINITION a temperature of 800+25° until the carbon is completely
Sodium Alginate is the purified carbohydrate product ex- burned off (approximately 75 min).
tracted from brown seaweeds by the use of dilute alkali. It Acceptance criteria: 18.0%-27.0% of ash on the dried
consists chiefly of the sodium salt of Alginic Acid, a poly- asis
uronic acid composed of B-D-mannuronic acid residues
linked so that the carboxyl group of each unit is free ADDITIONAL REQUIREMENTS
while the aldehyde group is shielded by a glycosidic link- © PACKAGING AND STORAGE: Preserve in tight containers.
age. It contains NLT 90.8% and NMT 106.0% of sodium
alginate of average equivalent weight 222.00, calculated
on the dried basis.
IDENTIFICATION Sodium Ascorbate—see Sodium Ascorbate
eA. General Monographs
Sample solution: A solution (1 in 100)
Analysis: To 5 mL of Sample solution add 1 mL of cal-
cium chloride TS.
Acceptance criteria: A voluminous, gelatinous precipi-
tate is formed immediately. Sodium Benzoate
° B.
Sample solution: A solution (1 in 100)
Analysis: To 10 mL of Sample solution add 1 mL of 4N
sulfuric acid.
Acceptance criteria: A heavy, gelatinous precipitate is
formed.
C7HsNaO2 144.10
ASSAY Benzoic acid, sodium salt;
© ALGINATES Sodium benzoate [532-32-1].
(See Alginates Assay (311).)
Sample: 250mg DEFINITION
Analysis: Each mL of 0.2500 N sodium hydroxide con- Sodium Benzoate contains NLT 99.0% and NMT 101.0% of
sumed is equivalent to 27.75 mg of sodium alginate. sodium benzoate (C7HsNaOz), calculated on the anhy-
Acceptance criteria: 90.8%-106.0% on the dried basis drous basis.
NF Monographs

IMPURITIES IDENTIFICATION
© ARSENIC, Method I/ (211) NMT 1.5 ppm e A. INFRARED ABSORPTION (197K)
e LEAD (251) Sample: Undried sample
Standard solution: 5 mL of Diluted Standard Lead Acceptance criteria: Meets the requirements
Solution e B. IDENTIFICATION TESTS—GENERAL, Sodium (191): Meets
Test preparation: Add 1.0g to 20 mL of nitric acid in a the requirements
250-mL conical flask, mix, and heat carefully until the eC. The retention time of the major peak of the Sample
Sodium Alginate is dissolved. Continue heating until the solution corresponds to that of the Standard solution, as
volume is reduced to 7 mL. Cool rapidly to room tem- obtained in the Assay.
NF 36 Official Monographs / Sodium 5565

ASSAY ADDITIONAL REQUIREMENTS

© PROCEDURE © PACKAGING AND STORAGE: Preserve in well-closed
Solution A: Adjust a 20-mM solution of monobasic po- containers.
tassium phosphate with phosphoric acid to a pH of 2.5. e USP REFERENCE STANDARDS (11)
Mobile phase: Solution A ana acetonitrile (70:30) USP Benzoic Acid RS
Diluent: Water and acetonitrile (50:50) USP Salicylic Acid RS
System suitability solution: 0.1 mg/mL of USP Salicylic USP Sodium Benzoate RS
Acid RS and 0.1 mg/mL of USP Benzoic Acid RS in
Diluent
Standard solution: 0.1 mg/mL of USP Benzoic Acid RS
in Diluent
Sample solution: 0.1 mg/mL of Sodium Benzoate in Sodium Bicarbonate—see Sodium
Diluent
Chromatographic system Bicarbonate General Monographs
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 230 nm
Column: 4.6-mm x 15-cm; 5-"4m packing L1 Sodium Borate
Column temperature: 25°
Flow rate: 1.0 mL/min NazB4O7 - 10H20 381.37
Injection volume: 10 Ll NazBsO7 201.22
System suitability Borax [1303-96-4].
Samples: System suitability solution and Standard Anhydrous [1330-43-4].
solution
[Note—The relative retention times for benzoic acid DEFINITION
and salicylic acid are approximately 1.0 and 1.2, Sodium Borate contains an amount of Na2B4O7 equivalent
respectively.] to NLT 99.0% and NMT 105.0% of NazB4O;
- 10H20.
Suitability requirements
Resolution: NLT 3.0 between benzoic acid and sali- IDENTIFICATION
cylic acid, System suitability solution © A. IDENTIFICATION TESTS—GENERAL, Sodium (191)
Relative standard deviation: NMT 0.5% for benzoic Sample solution: 1 in 20
acid, Standard solution Acceptance criteria: Meets the requirements
Tailing factor: NMT 2.0 for benzoic acid, Standard o B. IDENTIFICATION TESTS—GENERAL, Borate (191)
solution Sample solution: 1 in 20
Analysis Acceptance criteria: Meets the requirements
Samples: Standard solution and Sample solution
Calculate the percentage of sodium benzoate ASSAY
(C7HsNaOz) in the portion of Sodium Benzoate taken: e PROCEDURE
Sample: 3g of Sodium Borate
Result = (ru/rs) x (Cs/Cu) X (Mn/M,2) X 100 Titrimetric system
(See Titrimetry (541).)
ru = peak area of benzoic acid from the Sample Mode: Direct titration
solution Titrant: 0.5 N hydrochloric acid VS
rs = peak area of benzoic acid from the Standard Blank: 50 mL of water
solution Endpoint detection: Visual
Cs = concentration of benzoic acid in the Standard Analysis: Dissolve the Sample in 50 mL of water, add
solution, corrected for purity (mg/mL) methyl red TS, and titrate with 0.5 N hydrochloric acid
CG = concentration of Sodium Benzoate in the VS. [NoTE—Heating on a steam bath may be required
Sample solution (mg/mL) initially to effect solution.]
Ma = molecular weight of sodium benzoate, 144.10 Calculate the percentage of sodium borate (NazB4O; -
M2 = molecular weight of benzoic acid, 122.12 10H20) in the Sample taken:
Acceptance criteria: 99.0%-101.0% on the anhydrous
basis Result = [(V— B) x N
x F] x 100/W

IMPURITIES = ae of Titrant consumed by the Sample

<

(mL
volume of Titrant consumed by the Blank (mL)
STZ

Delete the following: actual normality of the Titrant (mEg/mL)

ioueoue

equivalency factor, 190.7 mg/mEq

°o HEAVY METALS (231) weight of the Sample (mg)
Test preparation: 4.0 g in 40 mL of water Acceptance criteria: 99.0%-105.0%
Analysis: To the Test preparation add, dropwise with
vigorous stirring, 10 mL of 3 N hydrochloric acid, and IMPURITIES
filter. Use 25 mL of the filtrate.
Acceptance criteria: NMT 10 29/de (offical 1-lan-2018)
Delete the following:
sydesbouo-= 4N

SPECIFIC TESTS
© WATER DETERMINATION, Method | (921): NMT 1.5% °e HEAVY METALS (231)
© ALKALINITY Test pee Dissolve 1g in 16 mL of water and
Sample solution: 2g in 20 mL of hot water 6 mL of 1 N hydrochloric nae: Dilute with water to
Analysis: To the Sample solution add 2 drops of phenol- 25 mL.
phthalein TS. Acceptance criteria: NMT 20 ppme cotticiat 1-4an-2013)
Acceptance criteria: The pink color produced, if any, is © CARBONATE AND BICARBONATE
disc arged by the addition of 0.20 mL of 0.10 N sulfu- Sample solution: To 5 mL of a solution (1 in 20), con-
ric acid. tained in a test tube, add 1 mL of 3 N hydrochloric
acid.
 5566 Sodium / Official Monographs NF 36

Acceptance criteria: No effervescence is observed. Acceptance criteria: NMT 5 g/ge coticiai 1 Jaa-2018)
e CHROMATOGRAPHIC PURITY
ADDITIONAL REQUIREMENTS Standard solution: 1.0 mg/mL of USP Caprylic Acid RS
¢ PACKAGING AND STORAGE: Preserve in tight containers. in ethyl acetate
Sample solution A: Dissolve 116 mg of Sodium Capry-
late in 5 mL of water, add 1 mL of dilute sulfuric acid (1
in 35), and extract with 10 mL of ethyl acetate. Sepa-
rate the organic layer, and dry it over anhydrous so-
Sodium Caprylate dium sulfate.
Sample solution B: Dilute 1.0 mL of Sample solution A
9 with ethyl acetate to 100 mL, transfer 5.0 mL of the
meme ‘ONa solution obtained, and dilute with ethyl acetate to
50 mL.
Chromatographic system
CgHisNaOz 166.19 (See Chromatography (621), System Suitability.)
Sodium octanoate [1984-06-1]. Mode: GC
DEFINITION Detector: Flame ionization
Sodium Caprylate contains NLT 99.0% and NMT 101.0% of Column: 0.25-mm x 30-m fused silica; coated with a
sodium caprylate (CsHisNaOz), calculated on the anhy- 0.25-um layer of phase G25
drous basis. Temperatures
Injection port: 250°
IDENTIFICATION Detector: 250°
e A. The retention time of the major peak of Sample solu- Column: See Table 1.
tion A corresponds to that of the Standard solution, as
obtained in the test for Chromatographic Purity in Table 1
Impurities.
° B. Hold Time
Methoxyphenylacetic reagent: Dissolve 2.7 g of meth- Initial Temperature Final at Final
oxyphenylacetic acid in 6 mL of 10% tetramethylammo- Temperature Ramp Temperature | Temperature
nium hydroxide solution in methanol, and add 20 mL () (</min) @) (min)
of alcohol. Store in a polyethylene container. 100 on 100 1
Sample solution: 20mg 100 5 220 10
Analysis: Dissolve the Sample in 0.5 mL of water, add
1.5 mL of Methoxyphenylacetic reagent, and cool in ice Flow rate: 1.5 mL/min
water for 30 min. A voluminous, white, crystalline pre- Carrier gas: Helium
cipitate is formed. Place in water at 20°, and stir for 5 Injection volume: 1 ul
min. The precipitate does not disappear. Add 1 mL of Injection type: Split ratio, 100:1
ammonia TS. The precipitate dissolves completely. Add System suitabili
1 mL of ammonium carbonate solution (160 mg/mL). Sample: Sample solution B
Acceptance criteria: No precipitate is formed. Suitability requirements
Signal-to-noise ratio: NLT 5
ASSAY Analysis
© PROCEDURE Samples: Standard solution, Sample solution A, and
Sample: 150mg Sample solution B
Blank: Glacial acetic acid Disregard any peaks with an area less than half of the
Titrimetric system area of the principal peak from Sample solution B and
(See Titrimetry (541).) any pak due to the solvent.
Mode: Direct titration Calculate the percentage of each impurity in the por-
Titrant: 0.1 N perchloric acid VS tion of Sodium Caprylate taken:
Endpoint detection: Potentiometric
Analysis: Transfer the Sample to a 125-mL volumetric Result = (ru/r) x 100
flask, and dissolve in 50 mL of glacial acetic acid. Titrate
with Titrant. Perform a blank determination, and make Tu = peak response of the individual impurity
any necessary correction. Each mL of 0.1 N perchloric tr = sum of all the peak responses
acid is equivalent to 16.62 mg of sodium caprylate Acceptance criteria
(CsHisNaQOz). Individual impurities: NMT 0.3%
Acceptance criteria: 99.0%-101.0% on the anhydrous Total impurities: NMT 0.5%
basis
SPECIFIC TESTS
IMPURITIES e APPEARANCE OF SOLUTION
Standard stock solution: Combine 30.0 mL of ferric
chloride CS, 30.0 mL of cobaltous chloride CS, and
Delete the following: 24.0 mL of cupric sulfate CS, and dilute with 1% (w/v)
hydrochloric acid to 100.0 mL.
®e HEAVY METALS, Method !I (231) Standard solution: Dilute 1.0 mL of Standard stock so-
NF Monographs

Test preparation: Dissolve 2.0 g of Sodium Caprylate in
10 mL of glacial acetic acid, and add 10 mL of alcohol.
Standard solution: 1 mL of Standard Lead Solution and
9 mL of a mixture of glacial acetic acid and alcohol
Gay
Analysis: Use 12 mL of the Test preparation, and pro-
ceed as directed in the chapter.
lution with 1% (w/v) hydrochloric acid to 100.0 mL.
Sample solution: Dissolve 2.5 g of Sodium Caprylate in
25.0 mL of freshly boiled and cooled water.
Acceptance criteria: The Sample solution is clear and
colorless, or not more intensely colored than the Stan-
dard solution.
e PH (791)
Sample solution: Use the Sample solution in the test for
Appearance of Solution.
NF 36 Official Monographs / Sodium 5567

Acceptance criteria: 8.0-10.5 Acceptance criteria; NMT 10 ppme (oifciat 1-Jan-2018)

e WATER DETERMINATION, Method | (921):
ADDITIONAL REQUIREMENTS
e USP REFERENCE STANDARDS (11)
USP Caprylic Acid RS
NMT 3.0%
SPECIFIC TESTS
e@ WATER DETERMINATION, Method I! (921)
Sample: 2g
Analysis: Dry the Sample at 105° for 4 h.
Acceptance criteria: The anhydrous form loses NMT
0.5% of its weight, and the hydrous form loses
12.0%-15.0% of its weight.

Sodium Carbonate ADDITIONAL REQUIREMENTS

e PACKAGING AND STORAGE: Preserve in well-closed
containers.
NazCO3 (anhydrous) 105.99 e LABELING: Label it to indicate whether it is anhydrous or
Na2CO3 - H20 124.00 hydrous.
Carbonic acid, disodium salt;
Disodium carbonate [497-19-8].
Monohydrate [5968-11-6].
DEFINITION Sodium Cetostearyl Sulfate
Sodium Carbonate is anhydrous or contains one molecule of
water of hydration. It contains NLT 99.5% and NMT DEFINITION
100.5% of Na»COs, calculated on the anhydrous basis. Sodium Cetostearyl Sulfate is a mixture of sodium cetyl sul-
IDENTIFICATION fate and sodium stearyl sulfate. It contains NLT 40.0% of
e A. IDENTIFICATION TESTS—GENERAL, Sodium (191): Meets sodium cetyl sulfate (CisH33NaSO,), and the sum of the
the requirements sodium cetyl sulfate content and sodium stearyl sulfate
© B. IDENTIFICATION TESTS—GENERAL, Carbonate (191): (CygH37NaSO,) content is NLT 90.0% (both contents cal-
Meets the requirements a on the anhydrous basis). It may contain a suitable
buffer.
ASSAY
¢ PROCEDURE IDENTIFICATION
Sample: 2g of Sodium Carbonate, previously dried, e A. The retention times of the two major peaks of Sample
from the test for Water Determination solution C correspond to those of the System suitability
Blank: 50 mL of water solution, as obtained in the Assay.
Titrimetric system e B. Sodium Cetostearyl Sulfate imparts an intense yellow
(See Titrimetry (541).) color to a nonluminous flame.
Mode: Direct titration eC
Titrant: 1.N sulfuric acid VS Sample solution: 1.0 mg/mL in alcohol
Endpoint detection: Visual Analysis: Heat 10 mL of the Sample solution to boilin:
Analysis: Transfer the Sample to a flask with the aid of on a water bath, shaking frequently. Filter immediately,
50 mL of water. Add methyl red TS, and titrate with and evaporate to dryness. Dissolve the residue in 7 mL
1 N sulfuric acid VS. Add he acid slowly, with constant of water, add 3 mL of diluted hydrochloric acid, and
stirring, until the solution becomes faintly pink. Heat evaporate the solution to half its volume. Allow to cool,
the solution to boiling, cool, and continue the titration. and filter. To the filtrate add 1 mL of barium chloride
Heat again to boiling, and titrate further as necessary solution (60 mg/mL).
until the faint pink color is no longer affected by con- Acceptance criteria: A white crystalline precipitate is
tinued boiling. formed.
Calculate the percentage of sodium carbonate (Na2CO3)
in the Sample taken.
ASSAY
© PROCEDURE
Result = [(V— B) x Nx Fx 100]/W System suitability solution: 5 mg/mL each of USP
Cetyl Alcohol RS and USP Stearyl Alcohol RS in alcohol
Vv = Titrant volume consumed by the Sample (mL) Internal standard solution: 4 mg/mL of 1-heptadeca-
B Titrant volume consumed by the Blank (mL) nol in alcohol
N Titrant actual normality (mEq/mL) Sample solution A: Dissolve 300 mg of Sodium Ceto-
F = equivalency factor, 52.99 mg/mEq stearyl Sulfate in 50 mL of alcohol, and add 2 mL of the
Ww = weight of the Sample (mg) Internal standard solution and 48 mL of water. Extract
Acceptance criteria: 99.5%-100.5% on the anhydrous the solution with four 25-mL portions of pentane, add-
asis ing 10-15 mL of saturated sodium chloride solution, if
necessary, to facilitate the separation of the layers.
IMPURITIES Combine the organic layers, and reserve the
hydroalcoholic layers for the preparation of Sane solu-
tion C and Sample solution D. Wash the organic layer
Delete the following: with two 30-mL portions of water, dry over anhydrous
sodium sulfate, and filter. me
°e HEAVY METALS (231) Sample solution B: Dissolve 300 mg of Sodium Ceto- al
Test preparation: Dissolve 2.0g in 10 mL of water. stearyl Sulfate in 50 mL of alcohol, and add 50 mL of
Analysis: Add 1 drop of phenolphthalein TS to the Test water. Extract the solution with four 25-mL portions of
=
°
preparation, and neutralize the solution with hydrochlo- pentane, adding 10-15 mL of saturated sodium chloride =]
ric acid, added dropwise. Heat the solution to boiling, solution, if necessary, to facilitate the separation of the °
and again neutralize by the dropwise addition of hydro- Ke}
=
layers. Combine the organic layers, wash with two
chloric acid. Cool, and dilute with water to 25 mL. Pro- 30-mL portions of water, dry over anhydrous sodium 2
ceed as directed in the chapter. cS
sulfate, and filter. a
Sample solution C: Transfer 25 mL of the a)

hydroalcoholic solution obtained in the preparation of

 5568 Sodium / Official Monographs NF 36

Sample solution A to a 200-mL flask that can be fitted Inject Sample solution C and Sample solution D into the
with a reflux condenser. Add 20 mL of hydrochloric cpmated taps, record the chromatograms, and
acid and 10 mL of the /nternal standard solution, and measure the areas for the major peaks. Carry out the
boil under reflux for 2 h. Allow to cool. Extract with Correction for interference in the same manner as for
four 20-mL portions of pentane. Wash the combined Sample solution A, and calculate the corrected area of
organic layer with two 20-mL portions of water, dry the peak corresponding to the internal standard of
over anhydrous sodium sulfate, and filter. Sample solution C, Sc¢on.
Sample solution D: Transfer 25 mL of the Samples: System suitability solution, Sample solution C,
hydroalcoholic solution obtained in the preparation of and Sample solution D
Sample solution A to a 200-mL flask that can be fitted [NoTte—The substances are eluted in the following or-
with a reflux condenser. Add 20 mL of hydrochloric der: cetyl alcohol, 1-heptadecanol (internal standard),
acid and 10 mL of alcohol, and boil under reflux for 2 and stearyl alcohol. Identify the cetyl alcohol and
h. Allow to cool. Extract with four 20-mL portions of stearyl alcohol peaks in the chromatograms of the
pentane. Wash the combined organic layer with two Sample solutions by comparison with the System suita-
20-mL portions of water, dry over anhydrous sodium bility solution.]
sulfate, and filter. Calculate the percentage of sodium cetyl sulfate
Chromatographic system (CisH33NaSO,) in the portion of Sodium Cetostearyl
(See Chromatography (621), System Suitability.) Sulfate taken:
Mode: GC
Detector: Flame ionization Result = (rc x Weu)/(Scccom) X Wo) x F x 100
Column: 0.25-mm x 25-m fused silica capillary; phase
G2 Ic = peak response of cetyl alcohol from Sample
Temperatures solution C -
Injection port: 250° Wey = weight of the internal standard added in the
Detector: 250° preparation of Sample solution C (mg)
Column: See Table 1. Scion) = corrected area of aePee corresponding to
the internal standard of Sample solution C
Tablet Wc = weight of Sodium Cetostearyl Sulfate taken to
prepare Sample solution C, calculated on the
Hold Time at anhydrous basis (mg)
Initial Temperature Final Final F = correction factor, 1.421
Temperature Ramp Temperature | Temperature Calculate the percentage of sodium steary! sulfate
©) (¢/min) ©) (min) (CigH37NaSOz) in the portion of Sodium Cetostearyl
Duration of Sulfate taken:
Iysi
150 : fo analysis Result = (Be x Wex)/(Scteon) X We) x FX 100
Carrier gas: Nitrogen
Flow at 1 mlyran Bc = peak response of stearyl alcohol from Sample
Injection volume: 1 ul solution C
fifectlan type: Split atts 100:1 We = weight of the internal standard added in the
System suitability preparation of Sample solution C (mq)
Sample: System suitability solution Sc(con) = Corrected area of the peak corresponding to
Suitability requirements the internal standard of Sample solution C
Resolution: NLT 4.0 between cetyl alcohol and We = weight of Sodium Cetostearyl Sulfate taken to
stearyl alcohol prepare Sample solution C, calculated on the
Relative standard deviation: NMT 1.5% anhydrous basis (mg)
Analysis i 7 alah factor, 1.377
Correction for interference: Inject Sample solution A cceptance criteria
and Sample solution B into the chromatograph, record Sodium cetyl sulfate: NLT 40.0% on the anhydrous
the chromatograms, and measure the areas for the basis . .
j
major peaks. Sum of sodium cetyl sulfate and sodium: stearyl sul-
If Sand le solution B shows a peak at the same retention fate: NLT 90.0% on the anhydrous basis
time as the internal standard peak of Sample solution
IMPURITIES
4; calculate the ratio; « LIMIT OF SODIUM CHLORIDE AND SODIUM SULFATE
Bs Sodium chloride
eta Sample: 5g
5 = peak response of cetyl alcohol from Sample Titrimetric system _
2 solution a if Mode: Direct titration
Si = peak response with the same retention time as Titrant: 0.1 N silver nitrate VS
the internal standard of Sample solution B Endpoint detection: Potentiometric
If R is less than 300, calculate the corrected area, Sacor, in 50 mL of water, and
Analysis: Dissolve the Sample
of the peak corresponding to the internal standard of add diluted nitric acid dropwise until the solution is
Sample solution A: neutral to blue litmus pepee To the resulting solution
aa 1 mL of potassium chromate TS and titrate with
NF Monographs

S = Sua —(Six ScalS itrant.

ie, bce ee Calculate the percentage of sodium chloride (NaCl) in
Sua = peak response of the internal standard from the portion of Sodium Cetosteary! Sulfate taken:
Sample solution A
Si = peak response with the same retention time as Result = (V x N)/W x F
the internal standard of Sample solution B i
Sca = peak response of cetyl alcohol from Sample V. = volume of the Titrant (mL)
solution A N = actual normality of the Titrant
Sce = peak response of cetyl alcohol from Sample W = weight of Sodium Cetostearyl Sulfate (g)
solution B F = equivalence factor for sodium chloride, 5.844
NF 36 Official Monographs / Sodium 5569

Sodium sulfate Sodium Chloride—see Sodium Chloride

Dichloroacetic acid solution: Dilute 67 mL of
dichloroacetic acid with water to 300 mL, and neutral- General Monographs
ize to blue litmus paper using ammonia TS. Cool, add
33 mL of dichloroacetic acid, and dilute with water to
600 mL.
Sample: 0.5g Sodium Chloride injection—see Sodium
Titrimetric system Chloride Injection General Monographs
Mode: Direct titration
Titrant: 0.01 M lead nitrate VS
Endpoint detection: Visual
Analysis: Dissolve the Sample in 20 mL of water, Sodium Chloride Injection,
warming gently if necessary, and add 1 mL ofa solu-
tion containing 0.5 g/L of dithizone in acetone. If the Bacteriostatic —see Bacteriostatic Sodium
solution is red, add 1 N nitric acid dropwise until a Chloride Injection General Monographs
bluish-green color is obtained. To the resulting solution
add 2.0 mL of Dichloroacetic acid solution and 80 mL of
acetone, and titrate with Titrant until a persistent or-
ange-red color is obtained. Sodium Citrate—see Sodium Citrate
Calculate the percentage of sodium sulfate (NazSOx) in
the portion of Sodium Cetosteary! Sulfate taken: General Monographs
Result = (Vx M)/W
x F

Vv = volume of Titrant (mL) Sodium Dehydroacetate

M = actual molarity of Titrant
Ww = weight of Sodium Cetostearyl Sulfate (g) CsH7NaO,y 190.13
F = equivalence factor for sodium sulfate, 14.20 2H-Pyran-2,4(3H)-dione, 3-acetyl-6-methyl-, monosodium
Acceptance criteria: The sum of the percentages of so- salt [4418-26-2].
dium chloride and sodium sulfate is NMT 8.0%.
e LIMIT OF FREE CETOSTEARYL ALCOHOL DEFINITION
Analysis: Examine the chromatogram of Sample solution Sodium Dehydroacetate contains NLT 98.0% and NMT
A, obtained as directed in the Assay. 100.5% of sodium dehydroacetate (CsH7NaOx), calculated
Calculate the percentage of free cetostearyl alcohol in on the anhydrous basis.
the portion of Sodium Cetosteary! Sulfate taken:
IDENTIFICATION
Result = 100(r4 + re) < Wis/(Saccom X W) e A. MELTING RANGE OR TEMPERATURE (741)
Sample solution: 150 mg/mL
vA = peak response of the cetyl alcohol peak from Analysis: To 10 mL of the Sample solution add 5 mL of
Sample solution A 3.N hydrochloric acid, collect the crystals by filtration
ts = peak response of stearyl alcohol from Sample with suction, wash with 10 mL of water, and dry at 80°
solution A for 4 h. Determine the melting point as directed in the
Ws = weight of the internal standard added in the chapter.
preparation of Sample solution A (mg) Acceptance criteria: 109°-111°
Sacon) = Corrected peak area corresponding to the e B. IDENTIFICATION TESTS—GENERAL, Sodium (191)
internal standard of Sample solution A (see Sample solution: 1 in 20
Assay) Acceptance criteria: Meets the requirements
w = weight of Sodium Cetostearyl Sulfate taken to
prepare Sample solution A (mg) ASSAY
Acceptance criteria: NMT 4.0% e PROCEDURE
papier 500 mg
SPECIFIC TESTS Blank: 25 mL of glacial acetic acid containing p-naph-
© ACIDITY OR ALKALINITY tholbenzein TS, which has been previously neutralized
Sample: 500 mg to a blue color
Analysis: Dissolve the Sample by heating in a mixture of Titrimetric system
10 mL of water and 15 mL of 90% alcohol. Add 0.1 mL (See Titrimetry (541).)
of phenolphthalein TS. Mode: Direct titration
Acceptance criteria: The resulting solution is colorless. Titrant: 0.1 N perchloric acid VS
Add 0.1 mL of 0.1 N sodium hydroxide, and the result- Endpoint detection: Visual
ing solution becomes red. Analysis: Transfer the Sample to a 125-mL conical flask,
e@ WATER DETERMINATION (921), Method I: NMT 1.5% and dissolve it in 25 mL of glacial acetic acid containing
p-naphtholbenzein TS,wien has been previously neu-
ADDITIONAL REQUIREMENTS tralized to a blue color. Titrate with 0.1 N perchloric
© PACKAGING AND STORAGE: Preserve in well-closed contain- acid VS to the original blue color. Perform a blank
ers. No storage requirements specified. determination.
e LABELING: Label it to indicate the name and concentra- Calculate the percentage of dehydroacetate (CsH7NaO,) =
tion of any added buffer. in the Sample taken: 4
oe USP REFERENCE STANDARDS (11)
USP Cetyl Alcohol RS Result = {[(Vs — Vs) x N x F]/W} x 100 3
USP Stearyl Alcohol RS °
Vs = volume of the Titrant consumed by the Sample R=)
(mL) Sy
Va = volume of the Titrant consumed by the Blank sy
(ml) 7
N = actual normality of the Titrant (mEq/mL)
F = equivalency factor, 190.1 mg/mEq
 5570 Sodium / Official Monographs NF 36

Ww = weight of the Sample (mg) Acceptance criteria: 45.5%-54.5% of SO2 on the dried
Acceptance criteria: 98.0%-100.5% on the anhydrous basis
basis
IMPURITIES
IMPURITIES © SULFIDE
Analysis: Dissolve 6 g in 14 mL of water in a test tube,
Delete the following: and weta strip of lead acetate test paper with the clear
solution.
Acceptance criteria: No discoloration is evident within
°o HEAVY METALS, Method I! (231): NMT 10 ppme wortcal 1. 5 min.
Jan-2018) e IRON
SPECIFIC TESTS Standard solution: Dissolve 43.2 mg of ferric ammo-
eo WATER DETERMINATION, Method | (921): 8.5%-10.0% nium sulfate in 10 mL of 2.N sulfuric acid, and add
water to make 1000 mL, each mL representing 5 ug of
ADDITIONAL REQUIREMENTS Fe.
e PACKAGING AND STORAGE: Preserve in well-closed Sample solution: Transfer 1.0 g of Sodium Formalde-
containers. hyde Sulfoxylate to a suitable crucible, and carefully ig-
nite, initially at a low temperature until thoroughly
charred, and finally, preferably in a muffle furnace, at
500°-600° until the carbon is all burned off. Cool, dis-
solve the residue in 2 mL of hydrochloric acid, and di-
Sodium Formaldehyde Sulfoxylate lute with water to 50 mL.
Analysis: To 5.0 mL of the Standard solution and 50 mL
Q of the Sample solution add 50 mg of ammonium persul-
Ho. I
p8q 2
fate and 5 mL of ammonium thiocyanate TS, and trans-
“SO” Nat fer each to a separate color comparison tube.
Acceptance criteria: 0.0025%; the color of the Sample
CH3NaQ3S 118.09 is not deeper than that of the Standard solution.
© SODIUM SULFITE
CH3NaO3S - 2H2,O 154.11 Sample solution: 4.0 mL of the solution prepared for
Methanesulfinic acid, hydroxy-, monosodium salt; the Assay in a conical flask containing 100 mL of water
Monosodium hydroxymethanesulfinate [149-44-0]. Titrimetric system
Dihydrate [6035-47-8]. (See Titrimetry (541).)
Mode: Direct titration
DEFINITION Titrant: 0.1 N iodine VS, prepated in the Assay
Sodium Formaldehyde Sulfoxylate contains an amount of Endpoint detection: Visua
sodium formaldehyde sulfoxylate (CH3NaO3S) equivalent Analysis: Add 2 mL of formaldehyde TS to the Sample
to NLT 45.5% and NMT 54.5% of SO2, calculated on the solution, and titrate with the Titrant, adding 3 mL of
dried basis. It may contain a suitable stabilizer, such as starch TS as the endpoint is approached.
sodium carbonate. Calculate the percentage of sodium sulfite (Na2SOs) in
IDENTIFICATION the Sodium Formaldehyde Sulfoxylate taken:
eA.
Result = (V2 — V;) x (N/W) x (F
x 1.25)
Sample solution: Dissolve 4g in 10 mL of water in a
test tube. V2 = volume of 0.1 N iodine VS consumed in the
Analysis: To the Sample solution add 1 mL of silver-am- titration performed in the Assay (mL)
monia-nitrate TS. Vv; = volume of 0.1 N iodine VS consumed in this
Acceptance criteria: Metallic silver is produced, either titration (mL)
as a finely divided, gray precipitate or as a bright metal- N = actual normality of the Titrant (mEq/mL)
lic mirror on the inner surface of the tube. Ww = weight of the Sample in the Assay (g)
° B. F = equivalency weight of sodium sulfite,
Sample solution: Dissolve 40 mg of salicylic acid in 63.02 mg/mEq
5 mL of sulfuric acid, and add 50 mg of Sodium Formal- Acceptance criteria: NMT 5.0% on the dried basis
dehyde Sulfoxylate.
Analysis: Warm very gently. SPECIFIC TESTS
Acceptance criteria: A permanent, deep red color © PH (791)
appears. Sample solution: 20 mg/mL
Acceptance criteria: 9.5-10.5
ASSAY e Loss ON DRYING (731)
e PROCEDURE Analysis: Dry at 105° for 3 h.
Sample: 1g Acceptance criteria: NMT 27.0%
Titrimetric system © ALKALINITY
(See Titrimetry (541).) Sample solution: 1.0 g of Sodium Formaldehyde
Mode: Direct titration Sulfoxylate in 50 mL of water
Titrant: 0.1 N iodine VS. [NoTE—Prepare an adequate Analysis: To the Sample solution add phenolphthalein
NF Monographs

amount for both the Assay and the test for Sodium
Sulfite.]
Endpoint detection: Visual
Analysis: Transfer the Sample to a 50-mL volumetric
flask, dissolve in 25 mL of water, and dilute with water
to volume. Reserve a portion of this solution for the test
for Sodium Sulfite. Transfer 4.0 mL of the remaining so-
lution to a conical flask containing 100 mL of water.
Titrate with Titrant, adding 3 mL of starch TS as the
endpoint is approached. Each mL of 0.1 N iodine is
equivalent to 1.602 mg of SO.
TS, and titrate with 0.10 N sulfuric acid.
Acceptance criteria: NMT 3.5 mL is required for
neutralization.
e CLARITY AND COLOR OF SOLUTION
Sample solution: 1g of Sodium Formaldehyde Sulfoxy-
late in 20 mL of water
Analysis: Transfer 10 mL of the Sample solution to a 20-
x 150-mm test tube. Compare with water in a similar
test tube.
NF 36
Acceptance criteria: The Sample solution and the water
are equally clear and, when viewed transversely by
transmitted light, exhibit no apparent difference in
color.
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in well-closed, light-
resistant containers, and store at controlled room
temperature.
Sodium Hydroxide
NaOH
Sodium hydroxide [1310-73-2].
DEFINITION
Sodium Hydroxide contains NLT 95.0% and NMT 100.5%
of total alkali, calculated as sodium hydroxide (NaOH),
including NMT 3.0% of sodium carbonate (Na2CO3). It
also contains NLT 54.0% and NMT 59.8% of sodium.
[CAuTION—Exercise great care in handling sodium hydrox-
ide, because it rapidly destroys tissues
IDENTIFICATION
¢ A. IDENTIFICATION TESTS—GENERAL (191), Chemical Identifi-
cation Tests, Sodium: A solution (1 in 25) meets the
requirements.
e B. PH (791)
Sample solution: 0.1 mg/mL of Sodium Hydroxide
Acceptance criteria: NLT 11.0
ASSAY
e TOTAL ALKALI
ell 15g
Blank: 40.0 mL of carbon dioxide-free water
Titrimetric system
(See Titrimetry (541).)
Mode: Direct titration
Titrant: 1 .N sulfuric acid VS
Endpoint detection: Visual
Analysis: Dissolve the Sample in 40 mL of carbon diox-
ide-free water. Cool the solution to room temperature,
and add phenolphthalein TS. Titrate with 1 N sulfuric
acid VS. At the discharge of the pink color of the indi-
cator, record the volume of Titrant (Vs:). Add methyl
orange TS, and continue the titration until a persistent
pink color is produced. Record the volume of Titrant
(Vs2). Perform a blank determination, and make any
necessary corrections.
Calculate the percentage of total alkali, calculated as
sodium hydroxide (NaOH), in the Sample taken:
Result = {[(Vs2 — Ve) x N x Fi]/W} x 100
Vs2 = volume of Titrant consumed by the Sample to
the second endpoint (mL)
Ve = volume of Titrant consumed by the Blank (mL)
N = actual normality of the Titrant (mEq/mL)
Fy = equivalency factor, 40.00 mg/mEq
Ww = weight of the Sample (mg)
Calculate the percentage of sodium carbonate (NazCO3)
in the Sample taken:
Result = {[(Vs2 — Vs1) x N x Fo]/W} x 100
Vso = volume of Titrant consumed by the Sample to
the second endpoint (mL)
Vsi = volume of Titrant consumed by the Sample to
the first endpoint (mL)
N = actual normality of the Titrant (mEq/mL)
Official Monographs / Sodium 5571
Fa = equivalency factor, 106.0 mg/mEq
Ww = weight of the Sample (mg)
Acceptance criteria: 95.0%-100.5% of total alkali;
NMT 3.0% of sodium carbonate (NazCO3)
e CONTENT OF SODIUM
Diluent: 1% hydrochloric acid solution
Standard stock solution: 25.41 j1g/mL of sodium chlo-
ride in Diluent. This solution contains 10 g/mL of
sodium.
Standard solutions: Transfer 6.0-, 7.5-, and 9.0-mL
portions of Standard stock solution to separate 100-mL
volumetric flasks. Dilute the content of each flask with
Diluent to volume, and mix to obtain solutions having
known concentrations of 0.6, 0.75, and 0.9 ug/mL of
sodium, respectively.
40.00 Sample stock solution: 1.303 mg/mL of Sodium Hy-
droxide in Diluent
Sample solution: Transfer 0.1 mL of Sample stock solu-
tion to a 100-mL volumetric flask and dilute with Dilu-
ent to volume.
Instrumental conditions
(See Atomic Absorption Spectroscopy (852).)
Mode: Atomic a sorption spectrophotometry
Ae ical wavelength: 589.0 nm (sodium emission
ine
Lamp: Sodium hollow-cathode
Flame: Air-acetylene
Blank: Diluent
Standard curve
Samples: Standard solutions
Plot: Absorbance values versus their corresponding
concentration (ug/mL) of sodium. The correlation co-
efficient is NLT 0.995.
Analysis
Sample: Sample solution
From the Standard curve, determine the concentration
of sodium in the Sample solution.
Calculate the percentage of sodium in the portion of
Sodium Hydroxide taken:
Result = (Cs/Cy) x 100
Cs = concentration of sodium in the Sample solution
from the Standard curve (g/mL)
Cu = concentration of Sodium Hydroxide in the
Sample solution (g/mL)
Acceptance criteria: 54.0%-59.8%
IMPURITIES
© POTASSIUM
Diluent: 1% hydrochloric acid solution
Standard stock solution: 1.907 mg/mL of potassium
chloride, previously dried at 105° for 2 h, in water.
Transfer 5.0 mL of this solution to a 1.0-L volumetric
flask and dilute with Diluent to volume. This solution
contains 5 g/mL of potassium.
Standard solutions: Transfer 2.0-, 5.0-, and 10.0-mL
portions of the Standard stock solution to separate
100-mL volumetric flasks. Dilute the content of each
flask with Diluent to volume, and mix to obtain solu-
tions having known concentrations of 0.10, 0.25, and
0.50 g/mL of potassium, respectively.
appl stock solution: 0.5 mg/mL of Sodium
Hydroxide
Sample solution: 50 g/mL of Sodium Hydroxide in
sydesBbouow 4N
Diluent, prepared from the Sample stock solution
Instrumental conditions
(See Atomic Absorption Spectroscopy {852).)
Mode: Atomic sororptey spectrophotometry
Analytical wavelength: 766.5 nm (potassium emission
line’
 4508 Cat’s Claw / Dietary Supplements USP 41

Deering solvent system: Ethyl acetate and hexane Table 1 (Continued)

5:5
Time Solution A Solution B Solution C
Derivatization reagent A: Dissolve 0.85 g of basic bis- (min) (%) (%) (%)
muth nitrate in 10 mL of glacial acetic acid and 40 mL
of water by heating. Filter if necessary (Solution A). Dis- 25 50 50 0
solve 8g of potassium iodide in 30 mL of water (Solu- 30 50 50 0
tion B). Mix Solution A and Solution B (1:1) to obtain a 31 0 0 100
stock solution. Dilute 1 mL of the stock solution with 36 0 0 100
2 mL of glacial acetic acid and 10 mL of water. [NoTE— 39 65 35 0
Usefreshly mixed Solution A and Solution B.] 49 65 35 0
Derivatization reagent B: Use a 10% solution of so-
dium nitrite in water. Chromatographic system
Analysis (See Chromatography (621), System Suitability.)
Samples: Standard solution and Sample solution Mode: LC
Develop the chromatogram to a length of NLT 12 cm, Detector: UV 245 nm
and dry the plate in a current of air. charm 4.6-mm x 10-cm; end-capped 3-~m packing
Acceptance criteria: Examine the plate under short-
wave UV light (254 nm). The Sample solution chromato- Flow rate: 0.75 mL/min
gram shows multiple zones that correspond in R; values Injection volume: 10 uL
to those observed in the Standard solution chromato- System suitability
gram. Other zones of varying intensities may be ob- Samples: Standard solution A and Standard solution B
served in the Sample solution. Treat the plate with Der- Suitability requirements
ivatization reagent A followed by Derivatization reagent Chromatogram similarity: The chromatogram ob-
B, and examine the plate under white light. The Sample tained using Standard solution A is similar to the refer-
solution chromatogram shows multiple orange-brown ence chromatogram provided with the USP Powdered
zones that correspond in color and R; values to those Cat’s Claw Extract RS.
observed in the Standard solution chromatogram. Other Tailing factor: NMT 2.0 for the isopteropodine peak,
colored zones of varying intensities may be observed in Standard solution B
the Sample solution. Relative standard deviation: NMT 2.0% for the
e B. The Sample solution exhibits peaks for speciophylline, isopteropodine peak in repeated injections, Standard
uncarine F, mitraphylline, isomitraphylline, pteropodine, solution B
and isopteropodine at retention times that correspond to Analysis
those in Standard solution A, as obtained in the test for Samples: Standard solution A, Standard solution B, and
Content of Pentacyclic Oxindole Alkaloids and Limit of Tetra- Sample solution
cyclic Oxindoles. Measure the areas of the analyte peaks. Identify the re-
tention times of the peaks corresponding to speci-
COMPOSITION ophylline, uncarine F, mitraphylline, isomitraphylline,
e CONTENT OF PENTACYCLIC OXINDOLE ALKALOIDS AND LIMIT
rhynchophylline, isorhynchophylline, pteropodine, and
OF TETRACYCLIC OXINDOLES
isopteropodine by comparison of the chromatogram
Standard solution A: Dissolve an accurately weighed of Standard solution A with the reference chromato-
quantity of USP Powdered Cat’s Claw Extract RS in gram provided with the lot of the USP Powdered Cat’s
methanol, shaking for 1 min. Dilute with methanol to
Claw Extract RS used.
obtain a solution having a known concentration of Separately calculate the percentages of speciophylline,
about 0.5 mg of the labeled amount of total oxindole uncarine F, mitraphylline, isomitraphylline, rhyncho-
alkaloids per mL. Pass throughafilter of 0.45-11m or phylline, isorhynchophylline, pteropodine, and isopter-
finer pore size. opodine, as isopteropodine, in the portion of Pow-
Standard solution B: 0.1 mg/mL of USP Isopteropodine dered Cat’s Claw taken:
RS in methanol. Pass through a nylon filter of 0.45-1m
or finer pore size. Result = (ru/rs) x (Cs/Cu) x 100
Sample solution: Accurately weighapprexinatey
750 mg of Powdered Cat’s Claw, and place in a 10-mL ty = peak response for each relevant alkaloid from
DS Monographs

centrifuge tube. Sonicate with 2.5 mL of methanol for
10 min. Centrifuge, and transfer this solution to a
10-mL volumetric flask. Repeat the extraction three ad-
ditional times combining the extracts in the 10-mL vol-
umetric flask, and dilute with methanol to volume.
Transfer about 3 mL of the solution to a test tube con-
taining 300 mg of polyamide powder, and shake for 1
min. Pass through a nylon filter of 0.45-11m or finer
pore size, discarding the first part of the filtrate.
Solution A: Preparea filtered and degassed 10 mM pH
7.0 phosphate buffer by mixing 6 mL of 1 N sodium hy-
droxide, 10 mL of 1 M monobasic potassium phos-
phate, and sufficient water to make 1000 mL. Adjust to
a pH of 7.0 + 0.1 by adding more of either solution.
Solution B: Acetonitrile
Solution C: Methanol and glacial acetic acid (99:1)
Mobile phase: See Table 1.
the Sample solution
rs = peak response for isopteropodine from
Standard solution B
Cs = concentration of USP lsopteropodine RS in
Standard solution B (mg/mL)
Cu = concentration of Powdered Cat’s Claw in the
Sample solution (mg/mL)
Calculate the content of pentacyclic oxindole alkaloids
by adding the percentages of speciophylline, uncarine
F, mitraphylline, isomitraphylline, pteropodine, and
isopteropodine.
Calculate the content of tetracyclic oxindole alkaloids
by adding the individual percentages of
rhynchophylline and isorhynchophylline.
Acceptance criteria
Pentacyclic oxindole alkaloids: NLT 0.3%
Tetracyclic oxindole alkaloids: NMT 0.05%

Table 1 CONTAMINANTS
© ARTICLES OF BOTANICAL ORIGIN, Limits of Elemental Impuri-
Time Solution A Solution B Solution C ties (561): Meets the requirements
(min) (%) (%) (%) © ARTICLES OF BOTANICAL ORIGIN, Pesticide Residue Analysis
0 65 35 0 (561): Meets the requirements
7 65 35 0
 5572 Sodium / Official Monographs
Lamp: Potassium hollow-cathode
Flame: Air-acetylene
Blank: Diluent
Standard curve
Samples: Standard solutions
Plot: Absorbance values versus their corresponding
concentration (g/mL) of potassium. The correlation
coefficient is NLT 0.99.
Analysis
Sample: Sample solution
From the Standard curve, determine the concentration
of potassium in the Sample solution.
Calculate the percentage of potassium in the portion of
Sodium Hydroxide taken:
Result = (Cs/Cu) x 100
Gs = concentration of potassium in the Sample
solution from the Standard curve (\1g/mL)
Cu = concentration of Sodium Hydroxide in the
Sample solution (ug/mL)
Acceptance criteria: NMT 0.5%
Delete the following:
°o HEAVY METALS (231)
Test preparation: Dissolve 0.67g in a mixture of 5 mL
of water and 7 mL of 3 N hydrochloric acid. Heat to
boiling, cool, and dilute with water to 25 mL.
Analysis: Proceed as directed in the chapter.
Acceptance criteria: NMT 30 ppMe cofficisi 140-2018)
SPECIFIC TESTS
© INSOLUBLE SUBSTANCES AND ORGANIC MATTER: Asolution
(1 in 20) is complete, clear, and colorless to slightly
colored.
ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Preserve in tight containers.
Sodium Lauryl Sulfate
Portions of this monograph that are national USP text, and
are not part of the harmonized text, are marked with
symbols (*») to specify this fact.
Sulfuric acid monododecyl ester sodium salt;
Sodium monododecy! sulfate [151-21-3].
DEFINITION
Sodium Laury! Sulfate is a mixture of sodium alkyl sulfates
consisting chiefly of sodium laury! sulfate (Ci2zH2sNaO,S).
It contains NLT 85.0% of sodium alkyl sulfates calculated
as sodium laury! sulfate (Ci2H2sNaO,S).
IDENTIFICATION
A. INFRARED ABSORPTION (197K) or (197A)
e B. SODIUM
Potassium pyroantimonate solution: To 2g of potas-
sium pyroantimonate add 100 mL of water. Boil the so-
lution for about 5 min, cool quickly, and add 10 mL of
a solution of potassium hydroxide (3 in 20). Allow to
stand for 24 h, and filter.
NF Monographs
Sample: 2.5g
Analysis: Place the Sample ina silica or platinum cruci-
ble, and add 2 mL of 10N sulfuric acid. Heat on a
water bath, then cautiously raise the temperature pro-
gressively over an open flame. Ignite, preferably in a
muffle furnace, at 600 + 25°. Continue heating until all
black particles have disappeared. Cool, add a few drops
of 2N sulfuric acid, and heat and ignite as above. Add
a few drops of ammonium carbonate TS, evaporate to
dryness, and ignite as above. Cool, dissolve the residue
NF 36
in 50 mL of water, and mix. To a 2-mL portion of this
solution, add 4 mL of Potassium pyroantimonate solution.
If necessary, rub the inside of the test tube with a glass
rod.
Acceptance criteria: A white, crystalline precipitate is
formed.
© C. SULFATE
Sample: Solution (1 in 10)
Analysis: After acidification with hydrochloric acid and
boiling for 20 min, no precipitate is formed. Add bar-
ium chloride TS; a white precipitate is produced.
Acceptance criteria: Meets the requirements
Sampie: 0.1g
Analysis: Dissolve the Sample in 10 mL of water and
shake.
Acceptance criteria: A copious foam is formed.
Sample: Solution prepared for /dentification D
Analysis: To 0.1 mL of the Sample, add 0.1 mL of a 1 g/
L solution of methylene blue and 2 mL of sulfuric acid,
diluted. Add 2 mL of methylene chloride and shake.
Acceptance criteria: An intense blue color develops in
the methylene chloride layer.
ASSAY
© CONTENT OF SODIUM ALKYL SULFATES
Methylene blue solution: Dissolve 0.003 g of methyl-
ene blue, 5.0 g of anhydrous sodium sulfate, and 1.2 g
of sulfuric acid in 100 mL of water.
[Note—Anhydrous sodium sulfate is the emulsion
breaker.]
Sample: 1.5 g of Sodium Lauryl Sulfate
Titrimetric system
(See Titrimetry (541).)
Mode: Direct titration
Titrant: 0.004 M benzethonium chloride VS
Endpoint detection: Visual
Analysis
Dissolve the Sample in water, warming if necessary, and
dilute with water to exactly 1000.0 mL. To 10.0 mL of
the solution, add 25 mL of Methylene blue solution,
15 mL of methylene chloride, and 20 mL of water. Ti-
trate with Titrant, shaking vigorously and allowing the
layers to Sepatate before each addition, until the two
layers are almost the same blue color. One mL of Ti-
trant is equivalent to 1.154 mg of sodium alkyl
sulfates, calculated as sodium lauryl sulfate
(Ci2H2sNaO,S).
Acceptance criteria: NLT 85.0%, calculated as sodium
lauryl sulfate (C;2H2sNaO.S)
IMPURITIES
Delete the following:
°e HEAVY METALS, Method II (231): 20 ppme (orice '-Jen-2018)
@ SODIUM CHLORIDE| y f
Fluorescein sodium solution: Dissolve 0.2 g of fluores-
cein sodium in water to 100 mL.
Dilute nitric acid: Dilute 105 mL of nitric acid with
water to 1000 mL.
Sample solution: 100 mg/mL of Sodium Lauryl Sulfate
in water
Analysis: Neutralize 50 mL of Sample solution with Di-
lute nitric acid, using litmus paper as the indicator, if
necessary. Add exactly 5.0 mL of 0.1 N sodium chloride
and titrate with 0.1 N silver nitrate VS (indicator:
2 drops of Fluorescein sodium solution) to the first ap-
pearance of turbidity with solution color change from
yellow-green to orange through yellow. Perform a blank
determination, and make any necessary correction. Each
milliliter of 0.1 N silver nitrate is equivalent to 5.844 mg
of sodium chloride.
NF 36
Acceptance criteria: The combined content of sodium
chloride and sodium sulfate is NMT 8.0%.
© SODIUM SULFATE
Sample solution: 100 mg/mL of Sodium Lauryl Sulfate
in water
Analysis: To 10 mL of Sample solution, add 100 mL of
alcohol and heat at a temperature just below the boil-
ing point for 2 h. Pass throughaglass filter (pore size
equivalent to 5-10 jum) while hot, and wash with
100 mL of boiling alcohol. Dissolve the precipitate by
washing with 150 mL of water, collecting the washings
in a beaker. Add 10 mL of hydrochloric acid, diluted,
heat to boiling, add 25 mL of barium chloride TS, and
allow to stand overnight. Collect the precipitate and
wash with water until the last washing shows no opal-
escence with 0.1 N silver nitrate. Dry the precipitate,
ignite to constant mass between 500° and 600° by rais-
ing the temperature gradually, and weigh as barium
sulfate (BaSO4; 233.39).
Amount (mg) of sodium sulfate (NazSO.) = amount (mg)
of barium sulfate (BaSO4) x 0.6086
Acceptance criteria: The combined content of sodium
chloride and sodium sulfate is NMT 8.0%.
SPECIFIC TESTS
@ ALKALINITY
Sample solution: Dissolve 1.0 g in 100 mL of water,
add 0.1 mL of phenol red TS, and titrate with 0.10 N
hydrochloric acid.
Acceptance criteria: NMT 0.5 mL for neutralization
¢ *TOTAL ALCOHOLS: Transfer 5 g to an 800-mL Kjeldahl
flask, and add 150 mL of water, 50 mL of hydrochloric
acid, and a few boiling chips. Attach a reflux condenser
to the Kjeldahl flask, heat carefully to avoid excessive
frothing, and boil for 4 h. Cool the flask, rinse the con-
denser with ether, collecting the ether in the flask, and
transfer the contents to a 500-mL separator, rinsing the
flask twice with ether and adding the washings to the
separator. Extract the solution with two 75-mL portions
of ether, evaporate the combined ether extracts in a
tared beaker on a steam bath, dry the residue at 105° for
30 min, cool, and weigh.
Acceptance criteria: The residue represents the total al-
cohols and is NLT 59.0% of the weight of Sodium
Lauryl| Sulfate taken.*
e UNSULFATED ALCOHOLS
Sample solution: Dissolve 10 g in 100 mL of water,
and add 100 mL of alcohol.
Analysis: Transfer the solution to a separator, and ex-
tract with three 50-mL portions of petroleum ether. If
an emulsion forms, sodium chloride may be added to
poner separation of the two layers. Wash the com-
ined petroleum ether extracts with three 50-mL por-
tions of water, and dry with anhydrous sodium sulfate.
Filter the petroleum ether extract into a tared beaker.
Evaporate on a water bath until the odor of petroleum
ether no longer is perceptible, dry the residue at 105°
for 30 min, cool, and weigh.
Acceptance criteria: The weight of the residue is NMT
4.0% of the weight of Sodium Laury! Sulfate taken.
ADDITIONAL REQUIREMENTS
¢ *PACKAGING AND STORAGE: Preserve in well-closed con-
tainers.*
Official Monographs / Sodium 5573
e USP REFERENCE STANDARDS (11)
USP Sodium Lauryl Sulfate RS
Sodium Metabisulfite
190.11
NazS20s
Disulfurous acid, disodium salt;
Disodium pyrosulfite [7681-57-4].
DEFINITION
Sodium Metabisulfite contains an amount of sodium metabi-
sulfite (Na2S2Os) equivalent to NLT 65.0% and NMT
67.4% of SO2.
IDENTIFICATION
e A. IDENTIFICATION TESTS—GENERAL, Sodium (191) and Sul-
fite (191): A solution (1 in 20) meets the requirements.
ASSAY
@ PROCEDURE
Sample: 200 mg of Sodium Metabisulfite
Blank: 50.0 mL of 0.1 N iodine VS, accurately measured
Titrimetric system
(See Titrimetry (541).)
Mode: Residual titration
Titrant: 0.1 N iodine VS
Back-titrant: 0.1 N sodium thiosulfate VS
Endpoint detection: Visual
Analysis: Add the Sample to 50.0 mL of 0.1 N iodine VS
in a glass-stoppered conical flask, and swirl to dissolve.
Allow to stand for 5 min, protected from light. Add
1 mL of hydrochloric acid, and titrate the excess iodine
with Back-titrant, adding 3 mL of starch TS as the
endpoint is approached. Perform a blank determination.
Calculate the percentage of sodium metabisulfite
(Na2S2Os) in the portion of Sodium Metabisulfite
taken:
Result = {[(Vs — Vs) x N x F]/W} x 100
Ve = Back-titrant volume consumed by the Blank
(mL)
Vs = Back-titrant volume consumed by the Sample
(mL)
N = Back-titrant normality (mEq/mL)
F = equivalency factor, 32.03 mg/mEq
w = Sample weight (mg)
Acceptance criteria: 65.0%-67.4% of SO2
IMPURITIES
Delete the following:
°e HEAVY METALS, Method | (231)
Test preparation: 1g
Analysis: Dissolve the Test preparation in 10 mL water.
Add 5 mL of hydrochloric acid, evaporate on a steam
bath to dryness, and dissolve the residue in 25 mL of
water.
Acceptance criteria: NMT 20 ppme coftciat 1.Jan-2018)
e LIMIT OF CHLORIDE
Standard solution: 0.71 mL of 0.020 N hydrochloric 4
acid in 100 mL of water
Sample solution: 1.0g in 10 mL of water. [NoTE—Pass =
through a small chloride-free filter, if necessary.] Add ms
6 mL of 30% hydrogen peroxide. Add 1 N sodium hy- ro)
droxide until the solution is slightly alkaline to phenol- Ko]
phthalein, and dilute with water to 100 mL. Py
Analysis v
Samples: Standard solution and Sample solution Be
Dilute 2.0 mL of the Samples with water to 20 mL. Add
1 mL of nitric acid and 1 mL of silver nitrate TS. Allow
 5574 Sodium / Official Monographs
to stand for 5 min protected from direct sunlight, and
compare the turbidity from the Samples (see Nephe-
lometry, Turbidimetry, and Visual Comparison (855)).
Acceptance criteria: Any turbidity produced by the
Sample solution does not exceed that of the Standard
solution (0.05%).
e LIMIT OF THIOSULFATE
Standard solution: Mix 0.10 mL of 0.10 N sodium thi-
osulfate with 10 mL of 1 N hydrochloric acid.
Sample solution: Mix 2.2 g with 10 mL of 1 N hydro-
chloric acid.
Analysis
Samples: Standard solution and Sample solution
Gently boil the Samples for 5 min. Cool, then transfer
each solution to a small test tube.
Acceptance criteria: Any turbidity produced by the
Sample solution does not exceed that of the Standard
solution (0.05%).
e IRON (241)
Test preparation: Dissolve 500 mg of Sodium Metabi-
sulfite in 14 mL of dilute hydrochloric acid (2 in 7), and
evaporate on a steam bath to dryness. Dissolve the resi-
due in 7 mL of dilute hydrochloric acid (2 in 7), and
again evaporate to dryness. Dissolve the resulting resi-
due in a mixture of 2 mL of hydrochloric acid and
20 mL of water. Add 3 drops of bromine TS, and boil to
expel the bromine. Cool, then dilute with water to
47 mL.
Analysis: Proceed as directed in the chapter.
Acceptance criteria: NMT 20 ppm
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in well-filled, tight
containers, and avoid exposure to excessive heat.
Sodium Phosphate, Dibasic—see Dibasic
Sodium Phosphate General Monographs
Sodium Phosphate, Monobasic—see
Monobasic Sodium Phosphate General
Monographs
Tribasic Sodium Phosphate
Na3PO, (anhydrous) 163.94
Trisodium phospiiats, monohydrate 181.96
Phosphoric acid, trisodium salt, dodecahydrate;
Trisodium phosphate, dodecahydrate 380.13
{10101-89-0].
Anhydrous [7601-54-9].
DEFINITION
Tribasic Sodium Phosphate is anhydrous or contains one to
twelve molecules of water of hydration. NasPO, (anhy-
drous) and Na3PO, - H2O (monohydrate) contain NLT
97.0% of Na3PO., calculated on the ignited basis.
NF Monographs
Na3POq, - 12H2O (dodecahydrate) contains NLT 92.0% of
Na3PO,, calculated on the ignited basis.
IDENTIFICATION
© A. IDENTIFICATION TESTS—GENERAL, Sodium (191) and
Phosphate (191): A solution (1 in 20) meets the
requirements.
NF 36
ASSAY
¢ PROCEDURE
Sample: 5.5g of Tribasic Sodium Phosphate, on the an-
hydrous basis
Blank: 100.0 mL of 1 N hydrochloric acid, accurately
measured
Titrimetric system
(See Titrimetry (541).)
Mode: Residual titration
Titrant: 1 .N sodium hydroxide VS
Endpoint detection: Potentiometric
Analysis: Transfer the Blank to a 400-mL beaker, and
titrate with the Titrant to the endpoint at a pH of 7.0.
Record as the volume consumed, and designate as A.
Transfer the Sample to a 400-mL beaker, add 100.0 mL
of 1 N hydrochloric acid, and stir until dissolved. Pass a
stream of carbon dioxide-free air, in fine bubbles,
through the solution for 30 min to expel carbon diox-
ide, covering the beaker loosely to prevent any loss by
spraying. Wash the cover and sides of the beaker with a
few mL of water.
Titrate the excess acid potentiometrically with the Ti-
trant to the inflection point at a pH of 4. Record the
buret reading, and designate as B. Protect the solution
from carbon dioxide absorbed from the air, and con-
tinue the titration with 1 N sodium hydroxide VS to
the inflection point at a pH of 8.8. Record the buret
reading, and designate as C.
Calculate the amount of Titrant consumed by the Sam-
ple to the first inflection point, correcting for the Blank
(V, = A — B) and the amount of Titrant consumed by
the Sample between the two inflection points (V2 = C —
B). If V; is equal to or greater than 2V2, calculate the
amount of Na3PO, in the portion of Sample taken:
D=V2x Nx F
Vo = volume of Titrant consumed between the two
inflection points (mL)
N = actual normality of the Titrant (mEq/mL)
F = equivalency factor, 163.9 mg/mEq
If V; is less than 2V2, calculate the amount of Na3PO, in
the portion of Sample taken:
D=(V;-
V2)xNxF
VY; = volume of the Titrant consumed to the first
inflection point, correcting for the Blank (mL)
N = actual normality of the Titrant (mEq/mL)
F = equivalency factor, 163.9 mg/mEq
Calculate the percentage of Na3PO, on the ignited basis
in the portion of Tribasic Sodium Phosphate taken:
Result = [10/(100 — L)] x (D/W)
L = percentage calculated in the test for Loss on
Ignition (733)
D = amount of Na3PO, found (mg)
Ww = weight of the Sample ( )
Acceptance criteria: NLT 57.0% of NasPOx on the ig-
nited basis. Naz3PO4- 12H2O (dodecahydrate) contains
NLT 92.0% of NazPO, on the ignited basis.
IMPURITIES
e Loss ON IGNITION (733)
Sample: 2g
Analysis: Dry the Sample at 110° for 5 h, and then
ignite at 800° for 30 min.
Acceptance criteria: The anhydrous form loses NMT
2.0% of its weight, the monohydrate loses 8.0%-11.0%
of its weight, and the dodecahydrate loses
45.0%-57.0% of its weight.
e ARSENIC, Method | (211)
Test preparation: Dissolve a portion equivalent to 1.0 g
of anhydrous tribasic sodium phosphate in 35 mL of
water.
NF 36 Official Monographs / Sodium 5575

Analysis: Proceed as directed in the chapter. Calculate the percentage of sodium propionate
Acceptance criteria: NMT 3 ppm (C3HsNaOz) in the Sample taken:

Result = [(Vs — Vs) x Nx Fx 100]/W

Delete the following:
Vs = volume of the Titrant consumed by the Sample
°e HEAVY METALS, Method | (231) (ml)
Test preparation: Dissolve a portion equivalent to 2.0 g Ve = volume of the Titrant consumed by the Blank
of anhydrous tribasic sodium phosphate in 25 mL of (mL)
waiter. N = actual normality of the Titrant (mEq/mL)
Analysis: Proceed as directed in the chapter. F = equivalency factor, 96.06 mg/mEq
Acceptance criteria: 10 ppMe cofficia 1-jan-2018) Ww = weight of the Sample (mg)
Acceptance criteria: 99.0%-100.5% on the dried basis
SPECIFIC TESTS
e INSOLUBLE SUBSTANCES IMPURITIES
Sample solution: Dissolve a portion equivalent to
10.0 g of anhydrous tribasic sodium phosphate in
100 mL of hot water. Delete the following:
Analysis: Filter the Sample solution through a tared fil-
tering crucible. [NoTE—Do not use glass.] Wash the in- °e HEAVY METALS, Method | (231)
soluble residue with hot water, and dry at 105° for 2 h. Test prepaner Dissolve 2 g of Sodium Propionate in
Acceptance criteria: The weight of the residue so ob- 1 mL of 1 N acetic acid and sufficient water to make
tained does not exceed 20 mg (0.2%). 25 mL.
Acceptance criteria: NMT 10 ppme ‘official 1-Jen-2018)
ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Preserve in tight containers. No SPECIFIC TESTS
storage requirements specified. e@ WATER DETERMINATION, Method | (921): NMT 1.0%
e LABELING: Label it to indicate whether it is anhydrous, e ALKALINITY
the monohydrate, or the dodecahydrate. Sample solution: 2.0 g of Sodium Propionate in 20 mL
of water
Analysis: Add phenolphthalein TS to the Sample
solution.
Acceptance criteria: If apin color is produced, it is
Sodium Propionate discharged by 0.60 mL of 0.10 N sulfuric acid.
ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Preserve in tight containers.
e USP REFERENCE STANDARDS (11)
USP Sodium Propionate RS
C3HsNaQ> - xH20
C3HsNaO2 96.06
Propanoic acid, sodium salt, hydrate;
Sodium propionate hydrate [6700-17-0]. Sodium Starch Glycolate
Anhydrous [137-40-6]. Portions of this monograph that are national USP-NF text,
and are not part of the harmonized text, are marked with
DEFINITION symbols (*») to specify this fact.
Sodium Propionate, dried at 105° for 2 h, contains NLT
99.0% and NMT 100.5% of sodium propionate Starch carboxymethyl ether, sodium salt.
(CsHsNaQ,). DEFINITION
IDENTIFICATION Sodium Starch Glycolate is the sodium salt of a carboxy-
e A. INFRARED ABSORPTION (197K) methyl ether of starch or of a cross-linked carboxymethyl
Analysis: Perform test on an undried sample. ether of starch. It may contain NMT 7.0% of Sodium
Acceptance criteria: Meets the requirements Chloride. The pH and assay requirements for Type A and
e B. IDENTIFICATION TESTS—GENERAL, Sodium (191) TypeBare set forth in the accompanying table.
Sample solution: 1 in 20
Acceptance criteria: Meets the requirements % Sodium, Combined
as Sodium Starch
ASSAY _pH Glycolate
© PROCEDURE Type Min. Max. Min. Max.
Sample: 200mg of Sodium Propionate, previously
dried at 105° for 2 h A 5.5 7.5 2.8 4.2
Titrimetric system B 3.0 5.0 2.0 3.4
(See Titrimetry (541).)
Mode: Direct titration
sydeibouow- 4N

Titrant: 0.1 N perchloric acid VS
Blank: 50 mL of glacial acetic acid
Endpoint detection: Visual
Analysis: Dissolve the Sample in 50 mL of glacial acetic
acid, and add 1 drop of crystal violet TS. Titrate with
0.1 N perchloric acid VS to a green endpoint. Perform a
blank determination, and make any necessary
correction.
IDENTIFICATION
e *A. INFRARED ABSORPTION (197K)
[Note—Disregard any peaks at about 845, 1285, and
1305 cm, which are attributed to the presence of
citrate.].
e B. An acidified solution of it is colored blue to violet by
the addition of iodine and potassium iodide TS 1.
°C.
Potassium pyroantimonate solution: Dissolve 2 g of
potassium pyroantimonate in 85 mLof hot water. Cool
quickly, and add 10 mL of a solution of potassium hy-
 5576 Sodium / Official Monographs
droxide (3 in 20). Allow to stand for 24 h, filter, and
dilute with water to 100 mL.
Analysis: To a 2-mL portion of the Sample solution pre-
pared for the test for Limit of Iron, add 2 mL of 15%
potassium carbonate, and heat to boiling. No precipi-
tate is formed. Add 4 mL of Potassium pyroantimonate
solution, and heat to boiling. Allow to cool in ice water
and, if necessary, rub the inside of the test tube with a
glass rod.
Acceptance criteria: A dense precipitate is formed.
e *D. Sodium Starch Glycolate imparts an intense yellow
color to a nonluminous flame.
ASSAY
e PROCEDURE
Sample: 1g
Analysis: Transfer the Sample to a conical flask, add
20 mL of 80% alcohol, stir for 10 min, and filter. Repeat
the extraction until the chloride has been completely
extracted, as shown byatest with silver nitrate. Dry the
insoluble portion at 105° to constant weight, and trans-
fer an accurately weighed portion (700 mg) of the dried
80% alcohol-insoluble portion to a suitable flask. Add
80 mL of glacial acetic acid, and heat the mixture under
reflux on a boiling water bath for 2 h. Cool to room
temperature, and titrate with 0.1 N perchloric acid VS,
determining the endpoint potentiometrically.
Calculate the percentage of sodium combined in the
form of sodium starch glycolate:
Result = 100 x 22.99 x Vx (N/W)
Vv = volume of perchloric acid consumed (mL)
N = normality of the perchloric acid
Ww = weight of the dried alcohol-insoluble residue
taken for the Assay (mg)
Acceptance criteria: 2.8%-4.2% for Type A;
2.0%-3.4% for Type B
OTHER COMPONENTS
e LIMIT OF SODIUM CHLORIDE
Sample: 500 mg of Sodium Starch Glycolate
Titrimetric system
(See Titrimetry (541).)
Mode: Direct titration
Titrant: 0.1 N silver nitrate VS
Endpoint detection: Potentiometric
Electrodes
Indicator: Suitable silver-based
Reference: Double junction electrode containing a
10% potassium nitrate filling solution in the outer
jacket, and a standard filling solution in the inner
jacket
Analysis: Transfer the Sample to a beaker, and suspend
in T00 mL of water. Add 1 mL of nitric acid. Titrate with
the Titrant. Each mL of 0.1 Nsilver nitrate is equivalent
to 5.844 mg of sodium chloride.
Acceptance criteria: NMT 7.0%
© LIMIT OF SODIUM GLYCOLATE
{Note—Conduct this test without exposure to daylight.
Use low-actinic glassware.]
Solution A: 0.1 mg/mL of 2,7-dihydroxynaphthalene in
sulfuric acid; allow to stand until decolorized, and use
within 2 days.
Standard solution: Transfer 310 mg of glycolic acid,
NF Monographs
previously dried over phosphorus pentoxide in a desic-
cator at room temperature overnight, to a 500-mL vol-
umetric flask. Dissolve in and dilute with water to vol-
ume. Transfer 5.0 mL of this solution to a 100-mL
beaker, add 4 mL of 6N acetic acid, and allow to stand
for about 30 min. Add 50 mL of acetone and 1 g of
sodium chloride, mix, and pass through fast filter paper
moistened with acetone into a 100-mL volumetric flask.
Rinse the beaker and the filter paper with acetone.
Combine the filtrate and washings, dilute with acetone
NF 36
to volume, and mix. Allow to stand for 24 h without
shaking. Use the clear supernatant as the Standard
solution.
Sample solution: Transfer 200 mg to a 100-mL beaker.
Add 4 mL of 6 N acetic acid and 5 mL of water. Stir
until dissolution is complete (about 10 min). Add 50 mL
of acetone and 1 g of sodium chloride, mix, and pass
through fast filter paper moistened with acetone into a
100-mL volumetric flask. Rinse the beaker and filter pa-
per with acetone. Combine the filtrate and washings,
dilute with acetone to volume, and mix. Allow to stand
for 24 h without shaking. Use the clear supernatant as
the Sample solution.
Analysis: Treat the Sample solution and the Standard so-
lution as follows. Heat 2.0 mL of the solution on a water
bath for 20 min to remove the acetone. Cool to room
temperature. Add 20.0 mL of Solution A to the solution
under test, mix, and heat on a water bath for 20 min.
Cool under running water, and quantitatively transfer to
a 25-mL volumetric flask. Maintain the flask under run-
ning water, and dilute with sulfuric acid to volume.
Within 10 min, determine the absorbance of the solu-
tion at 540 nm with a suitable spectrophotometer, us-
ing water as the blank.
Acceptance criteria: The absorbance of the Sample so-
lution is NMT that of the Standard solution (2.0%).
IMPURITIES
Delete the following:
®e “HEAVY Merais, Method // (231): 20 ppmee cofical an.
2018)
e LIMIT OF IRON
Standard solution: Dissolve 863.4 mg of ferric ammo-
nium sulfate [FeNH4(SO.)2- 12H2O] in water, add 25 mL
of 2.N sulfuric acid, dilute with water to 500.0 mL, and
mix. Pipet 10 mL of this solution into a 100-mL volu-
metric flask, dilute with water to volume, and mix. Pi-
pet 5 mL of this solution into a 100-mL volumetric flask,
dilute with water to volume, and mix. This solution
contains the equivalent of 1.0 g/mL of iron.
Sample solution: Place 2.5g ina silica or platinum cru-
cible, and add 2 mL of 10 N sulfuric acid. Heat on a
water bath, then cautiously raise the temperature pro-
gressively over an open flame. Ignite, preferably in a
muffle furnace, at 600
+25°. Continue heating until all
black particles have disappeared. Cool, add a few drops
of 2N sulfuric acid, and heat and ignite as above. Add
a few drops of 2M ammonium carbonate, evaporate to
dryness, and ignite as above. Cool, dissolve the residue
in 50 mL of water, and mix.
[Note—Reserve a portion of this solution for Identifica-
tion test C.]
Analysis: Treat the Sample solution and the Standard so-
lution as follows. Transfer 10 mL of the solution to a
suitable beaker, add 2 mL of citric acid solution (1 in 5)
and 0.1 mL of thioglycolic acid, and mix. Render the
solution alkaline, using litmus paper as an external indi-
cator, by the addition of ammonium hydroxide. Dilute
with water to 20 mL, and mix. Allow the solutions to
stand for 5 min.
Acceptance criteria: The color of the solution from the
Sample solution is a shade of pink no deeper than that
of the solution from the Standard solution (0.002%).
SPECIFIC TESTS
e MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI-
FIED [MICROORGANISMS (62): It meets the requirements of
he tests for absence of Salmonella species and Escherichia
coli.
e PH (791): Disperse 1g in 30 mL of water. The pH of the
resulting suspension is either 5.5-7.5 for Type A or
3.0-5.0 for Type B.
NF 36 Official Monographs / Sodium 5577

e Loss ON DRYING (731) Temperatures

Analysis: Dry at 130° for 90 min. Injector: 220°
Acceptance criteria: NMT 10.0% Detector: 260°
Column: See Table 1.
ADDITIONAL REQUIREMENTS
© *PACKAGING AND STORAGE: Preserve in well-closed con-
Table 1
tainers, preferably protected from wide variations in tem-
perature and humidity, which may cause caking.+ Hold Time
e *LABELING: Label it to indicate the botanical source of the Initial Temperature Final at Final
starch from which it was derived, the cross-linking agent Temperature Ramp Temperature | Temperature
f used), the pH range, and whether it is Type A or Type ) (¢/min) ¢) (min)
> 70 = 70 2
¢ USP REFERENCE STANDARDS (11) 70 5 240 sh
USP Sodium Starch Glycolate Type A RS
USP Sodium Starch Glycolate Type B RS Carrier gas: Helium, passed through a bed of molecu-
lar sieve for drying, if necessary
Flow rate: 2.4 mL/min
Injection type: Splitless
Injection volume: 1 pL
Sodium Stearate System suitability
Sample: Standard solution
Octadecanoic acid, sodium salt; Suitability requirements
Sodium stearate [822-16-2]. Resolution: NLT 5.0 between the methyl palmitate
and methyl stearate poe [Note—The relative reten-
DEFINITION tion times for methyl palmitate and methyl stearate
Sodium Stearate is a compound of sodium with a mixture of are about 0.9 and 1.0, respectively]
solid organic acids obtained from sources of vegetable or Relative standard deviation: NMT 3.0% for the
animal origin and consists mainly of variable proportions methyl stearate and methyl! palmitate peaks; NMT
of sodium stearate (CigH3sNaOz) and sodium palmitate 1.0% for the ratio of the peak areas of methyl palmi-
(CisHsi1NaOz). The content of stearic acid in the fatty acid tate to the peak areas of methyl stearate, from 6 rep-
fraction is NLT 40.0% of the total content. The sum of licate injections
stearic acid and palmitic acid in the fatty acid fraction is Analysis: Calculate the percentage of stearic acid
NLT 90.0% of the total content. Sodium stearate contains (CigH36Oz) in the fatty acid fraction of the sample taken:
small amounts of the sodium salts of other fatty acids.
IDENTIFICATION Result = (ru/r7) x 100
oA. ru = peak area due to methyl stearate
Analysis: Heat a small quantity of Sodium Stearate in a nr = sum of all the peak areas, excluding the
crucible over a flame until it fuses. Continue heating solvent peak
the sample until it decomposes with emission of flam- Calculate the percentage of palmitic acid (CisH3202) in
mable vapors that should burn when ignited. Moisten the fatty acid fraction of the sample taken:
the residue with water, and test with red litmus paper.
The paper must turn blue. Add a small amount of acid Result = (ru/r7) x 100
to the crucible, and observe the solution effervesce. The
solution must impart an intense yellow color to a tu = peak area due to methyl palmitate
nonluminous flame. tr = sum of all the peak areas, excluding the
Acceptance criteria: Meets the requirements solvent peak
e B. The retention times of the major peaks of the Sample Acceptance criteria
solution correspond to those of the Standard solution, as Stearic acid: NLT 40.0%
obtained in the Assay. Sum of stearic acid and palmitic acid: NLT 90.0%
ASSAY SPECIFIC TESTS
© PROCEDURE e ACIDITY
Boron trifluoride-methanol solution: 140 g/L of bo- Sample solution: Heat 50 mL of alcohol to the same
ron trifluoride in methanol temperature, +5°, as that attained when the pink
Sample solution: Dissolve 100 mg of Sodium Stearate endpoint is reached in the titration of the sample speci-
in a small conical flask, fitted with a suitable reflux at- men. Add 3 drops of phenolphthalein TS and sufficient
tachment, with 5 mL of Boron trifluoride-methanol solu- 0.020 N sodium hydroxide to produce a faint pink
tion. Boil under reflux for 10 min. Add 4.0 mL of n- color. Add 2.00g of Sodium Stearate, and dissolve with
heptane through the condenser, and boil again under the aid of a srr amount of heat. No pink color is
reflux for 10 min. Allow to cool. Add 20 mL of a satu- produced.
rated solution of sodium chloride. Shake, and allow the Analysis: Titrate the solution with 0.020 N sodium hy-
layers to separate. Remove about 2 mL of the organic droxide until a pink color is produced.
layer, and dry it over 0.2 g of anhydrous sodium sulfate. Acceptance criteria: 1.00-4.25 mL of 0.020 N sodium
Dilute 1.0 mL of this solution with n-heptane to hydroxide is required (0.28%-1.2% as stearic acid).
10.0 mL. 74
e Loss ON DRYING (731) n
Standard solution: Prepare as directed in the Sample Sample: Tare a beaker containing 1 g of washed sand,
solution using 50 mg of USP Stearic Acid RS and 50 mg previously dried at 105°, add 500 mg of Sodium Stea- =
of USP Palmitic Acid RS. °
rate, and again weigh. |
Chromatographic system Analysis: To the Sample add 10 mL of alcohol, and fo)
(See Chromatogra, (621), System Suitability.) evaporate at 80° to dryness. Dry at 105° for 4 h. io}
=
Mode: GC aepny ” ¥ Acceptance criteria: NMT 5.0% Ay
Detector: Flame ionization e FATS AND FIXED OILS, Acid Value (401) me]
Column: 30-m x 0.32-mm fused silica; 0.5-4m layer of ma
Sample: 1 g of the fatty acids obtained as follows. Dis- 7
phase G16 solve 25 g of Sodium Stearate in 300 mL of hot water,
 5578 Sodium / Official Monographs NF 36

add 60 mL of 2N sulfuric acid, and heat the solution, OTHER COMPONENTS

with ues stirring, until the separated fatty acid e LIMIT OF SODIUM STEARYL MALEATE AND STEARYL ALCOHOL
layer is clear. Wash the fatty acids with boiling water Solvent: Chloroform and glacial acetic acid (4:1)
until they are free from sulfate, collect in a small beaker, Standard monostearyl maleate stock solution: 1 mg/
and warm on a steam bath until the water has settled mL of USP Monostearyl Maleate RS in Solvent
and the fatty acids are clear. Allow the acids to cool, Standard monostearyl maleate solution: Pipet 5.0 mL
pour off the water layer, then melt the acids, filter into of Standard monosteary! maleate stock solution, and di-
a dry beaker while hot, and dry at 105° for 20 min. lute with chloroform to 50 mL.
Acceptance criteria: 196-211 Standard stearyl alcohol stock solution: 1 mg/mL of
e FATS AND FIXED OILS, /odine Value (401) USP Stearyl Alcohol RS in Solvent
Sample: The fatty acids obtained in Fats and Fixed Oils, Standard stearyl alcohol solution: Pipet 5.0 mL of
Acid Value (401) Standard stearyl alcohol stock solution, and dilute with
Acceptance criteria: NMT 4.0 chloroform to 50 mL.
e ALCOHOL-INSOLUBLE SUBSTANCES Sample solution: Transfer 200 mg of Sodium Stearyl
Sample: 1.0 g of Sodium Stearate Fumarate to a small, glass-stoppered conical flask. Add
Analysis: Reflux the Sample with 25 mL of alcohol until 10.0 mL of Solvent. Dissolve by placing the flask in an
it dissolves completely. ultrasonic bath for 10 min.
Acceptance criteria: The resulting solution is clear or Chromatographic system
NMT slightly opalescent. (See Chromatography (621), Thin-Layer Chromato-
ADDITIONAL REQUIREMENTS
graphy.)
Mode: TLC
© PACKAGING AND STORAGE: Preserve in well-closed, light- Adsorbent: 0.25-mm layer of chromatographic silica
resistant containers. gel (TLC plates)
e LABELING: Label it to indicate the content of stearic acid Spray reagent: Sulfuric acid and alcohol (1:9).
in the fatty acid fraction and to indicate the fatty acids Note—Add cautiously and with stirring.]
used to produce sodium stearate are from sources of Developing solvent system: Hexanes, toluene, and
vegetable or animal origin. glacial acetic acid (5:5:1)
e USP REFERENCE STANDARDS (11) Analysis
USP Palmitic Acid RS Samples: Standard monosteary! maleate solution, Stan-
USP Stearic Acid RS dard stearyl alcohol solution, and Sample solution
Apply 5 ul of Standard monosteary! maleate solution
and 10 pul each of Standard stearyl alcohol solution
and Sample solution to the plate. Immerse the plate in
a tank containing a layer of 10 mm of chloroform on
Sodium Stearyl Fumarate the bottom. Allow the solvent front to reach the up-
per edge of the spots. Withdraw the plate, and dry in
DEFINITION a current of cold air. Repeat the immersion, develop-
Sodium Stearyl Fumarate contains NLT 99.0% and NMT ment, and drying. This results in spots havinga linear
101.5% of sodium stearyl fumarate (C22H3sNaOx), calcu- shape. Develop the chromatograph in a saturated
lated on the anhydrous basis. chamber containing the Developing solvent system un-
til the solvent front has moved 15 cm, and remove
IDENTIFICATION the plate from the chamber. Allow to dry for 10 min,
o INFRARED ABSORPTION (197K) and heat in an oven at 90° for 2 min. Allow to cool
Analysis: Perform test on an undried specimen (1 in to room temperature. Replace the plate in the cham-
300). ber for another 15-cm development, remove the
Acceptance criteria: Meets the requirements plate, and allow to dry at room temperature for 15
min. Spray the plate with Spray eager, Dry the plate
ASSAY in an oven at 150° for 15 min. Dark spots appear on
e PROCEDURE a light background. Allow to cool. Faint spots at an Rr
Sample: 250mg value of 0.9 may result from traces of distearyl
Blank: 10 mL of chloroform maleate and distearyl fumarate.
Titrimetric system Acceptance criteria: The intensity of any spot from
(See Titrimetry (541).) the Sample solution is not greater than that from the
Mode:_ Direct titration corresponding spot from the Standard monosteary!
Titrant: 0.1 N perchloric acid VS maleate solution and Standard stearyl alcohol solution
Endpoint detection: Visual (NMT 0.25% sodium stearyl maleate, NMT 0.5%
Analysis: Transfer the Sample to a 50-mL conical flask, stearyl alcohol).
mix with 10 mL of chloroform, and add 20 mL of gla-
cial acetic acid to dissolve. Add quinaldine red TS, and IMPURITIES
titrate with 0.1 N perchloric acid VS. e LEAD (251): NMT 10 ppm
Calculate the percentage of sodium steary! fumarate
(C22H39NaOz) in the Sample taken:
Delete the following:
Result = {[(Vs — Ve) x N x F]/W} x 100
°e HEAVY METALS, Method |i (231): NMT 20 ppme cowicia1-
NF Monographs

Vs = value of Titrant consumed by the Sample Jan-2018)

m
Vp = volume of Titrant consumed by the Blank (mL) SPECIFIC TESTS
N = actual normality of the Titrant (mEq/mL) e WATER DETERMINATION, Method | (921): NMT 5.0%
F = equivalency factor, 390.5 mg/mEq e FATS AND FIXED OILS, Saponification Value (401)
Ww = weight of the Sample (mg) Sample: 450 mg
Acceptance criteria: 99.0%-101.5% on the anhydrous Ethanolic potassium hydroxide: Dissolve 5.5 g of po-
basis tassium hydroxide in absolute alcohol, heating if neces-
sary to dissolve, and dilute with absolute alcohol to
NF 36 Official Monographs / Sodium 5579

about 1000 mL. Prepare fresh daily, and filter if neces-
sary to remove carbonate.
Titrimetric system
(See Titrimetry (541).)
Mode: Residual titration
Titrant: 0.1 N hydrochloric acid VS
Blank: 50.0 mL of Ethanolic potassium hydroxide
Endpoint detection: Visual
Analysis: Transfer the Sample to a 300-mL conical flask,
and add 50.0 mL of Ethanolic potassium hydroxide, rins-
ing down the inside of the flask during the addition.
Gently reflux the mixture on a steam bath for NLT 2 h,
occasionally swirl gently, but avoid splashing the mix-
ture up into the condenser. Rinse the condenser with
10 mL of 70% alcohol, followed by three 10-mL por-
tions of water, collecting the rinsings in the flask. Cool,
rinse the sides of the flask with two 10-mL portions of
70% alcohol, add phenolphthalein TS, and titrate with
0.1 N hydrochloric acid VS to the disappearance of any
pink color. Perform a blank determination.
Calculate the Saponification Value for Sodium Stearyl
Fumarate in the Sample taken:
Result = [(Vs — Vs) x N x FJ/W
Vp = volume of the Titrant consumed by the Blank
Vs = wee of the Titrant consumed by the Sample
N attach normality of the Titrant (mEq/mL)
F = molecular weight of potassium hydroxide,
Ww = sent of the Sample (g)
Acceptance criteria: 142.2-146.0, calculated on the
anhydrous basis
ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Preserve in well-closed
containers.
e USP REFERENCE STANDARDS (11)
USP Monostearyl Maleate RS
USP Sodium Stearyl Fumarate RS
USP Stearyl Alcohol RS
Sodium Succinate
2Ne* Ho
x=Oor6
NaOOC-CH2CH2-COONa (C4H4Na2Ox) 162.05
Anhydrous disodium 1,4-butanedioate;
Anhydrous butanedioic acid disodium salt [150-90-3].
NaOOC-CH2CH2-COONa - 6H20 (C4H4Na2O04- 6H2O) 270.14
Disodium 1,4-butanedioate hexahydrate;
Butanedioic acid disodium salt hexahydrate [6106-21-4].
DEFINITION
Sodium Succinate, when dried at 120° for 2 h, contains NLT
98.0% and NMT 102.0% of disodium succinate
e B. CHROMATOGRAPHIC IDENTITY
Analysis: Proceed as directed in the Assay.
Acceptance criteria: The retention time of the major
peak of the Sample solution corresponds to that of the
Standard solution.
e C. SoplIuM
Analysis: Proceed as directed in /dentification Tests—
General (191), Sodium.
Acceptance criteria: Meets the requirements
ASSAY
e PROCEDURE
Solution A: Dissolve 6.8 g of monobasic potassium
phosphate in 2 L of water. Adjust with phosphoric acid
to a pH of 2.3. Pass under vacuum through an HNWP
(nylon hydrophilic) membrane filter of 0.45-um pore
size. This is a 25 mM potassium phosphate buffer with
a pH of 2.3.
Mobile phase: Add 100 mL of methanol to 1900 mL of
Solution A and mix well. Sonicate for 30 min and cool
to room temperature.
Diluent: Add 10 mL of phosphoric acid to 1 L of water
and mix well. This is a 1% phosphoric acid solution.
System suitability solution: 3.0 mg/mL of USP Anhy-
drous Sodium Succinate RS and 2.2 t1g/mL of USP Fu-
maric Acid RS in Diluent
Standard solution: 3.0 mg/mL of USP Anhydrous So-
dium Succinate RS in Diluent
Sample solution: 3.0 mg/mL of Anileus Sodium
Succinate or Sodium Succinate Hexahydrate in Diluent.
Dry Anhydrous Sodium Succinate or Sodium Succinate
Hexahydrate at 120° for 2 h before use.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 204 nm
Column: 4.6-mm x 15-cm; 3-um packing L1
Column temperature: 30°
Flow rate: 1.0 mL/min
Injection volume: 10 uL
Run time: 10 min
System suitability
Samples: System suitability solution and Standard
solution
[Note—The relative retention times for succinic acid
and fumaric acid are 1.0 and 1.2, respectively.]
Suitability requirements
Resolution: NLT 2.0 between succinic acid and fu-
maric acid, System suitability solution
Tailing factor: 0.8-2.0, Standard solution
Relative standard deviation: NMT 0.5%, Standard
solution
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of sodium succinate
(C4H4Na2O,) in the portion of sample taken:
Result = (ru/rs) x (Cs/Cu) x 100
Tu = peak response from the Sample solution
ls = peak response from the Standard solution
Cs = concentration of USP Anhydrous Sodium
Succinate RS in the Standard solution
(mg/mL)
Cu = concentration of Anhydrous Sodium Succinate
sydesbouow= IN

(C4H4Na2O,). or Sodium Succinate Hexahydrate in the

Sample solution (mg/mL)
IDENTIFICATION Acceptance criteria: 98.0%-102.0%
e A. INFRARED ABSORPTION (197K) or(197A): Dry the Anhy-
drous Sodium Succinate or Sodium Succinate Hexahy- IMPURITIES
drate sample at 120° for 2 h before use. e LIMIT OF SODIUM ACETATE, SODIUM MALEATE, AND SODIUM
FUMARATE
Solution A, Mobile phase, Diluent, and Chromato-
graphic system: Proceed as directed in the Assay.
 5580 Sodium / Official Monographs NF 36

Acetic acid stock solution: Transfer 37.5 mg of USP Analysis: Proceed as directed in pH (791).
Glacial Acetic Acid RS to a 25-mL volumetric flask that Acceptance criteria: 7.0-9.0
contains 10 mL of Diluent. Dissolve and dilute with Dilu- e Loss ON DRYING (731)
ent to volume. Transfer 1.0 mL of this solution to a Analysis: Proceed as directed in Loss on Drying (731).
10-mL volumetric flask and dilute with Diluent to vol- Dry at 120° for 2 h.
ume. This solution is equivalent to 200 g/mL of so- Acceptance criteria
dium acetate in Diluent. Anhydrous Sodium Succinate: NMT 2.0%
Maleic acid stock solution: Transfer 36.5 mg of USP Sodium Succinate Hexahydrate: 37.0%-41.0%
Maleic Acid RS to a 50-mL volumetric flask. Dissolve
and dilute with Diluent to volume. Transfer 1.0 mL of ADDITIONAL REQUIREMENTS
this solution to a 10-mL volumetric flask and dilute with © PACKAGING AND STORAGE: Preserve in tight containers.
Diluent to volume. This solution is equivalent to 100 ug/ Store at room temperature.
mL of sodium maleate in Diluent. e LABELING: Label it to state, as part of the official title,
Fumaric acid stock solution: Transfer 36.5 mg of USP anhydrous or hexahydrate for sodium succinate.
Fumaric Acid RS to a 50-mL volumetric flask. Dissolve e USP REFERENCE STANDARDS (11)
and dilute with Diluent to volume. Transfer 1.0 mL of USP Anhydrous Sodium Succinate RS
this solution to a 10-mL volumetric flask and dilute with USP Fumaric Acid RS
Diluent to volume. This solution is equivalent to 100 g/ USP Glacial Acetic Acid RS
mL of sodium fumarate in Diluent. USP Maleic Acid RS
System suitability solution: 10 mg/mL of USP Anhy-
drous Sodium Succinate RS, 15 ug/mL of USP Glacial
Acetic Acid RS, and 7.3 g/mL each of USP Maleic Acid
RS and USP Fumaric Acid RS in Diluent
Standard solution: Transfer 1 mL each of Acetic acid Sodium Sulfite
stock solution, Maleic acid stock solution, and Fumaric
acid stock solution to a 10-mL volumetric flask and di- NazSO3 126.04
lute with Diluent to volume. [7757-83-7].
Sample solution: 10 mg/mL of Anhydrous Sodium Suc-
cinate or Sodium Succinate Hexahydrate in Diluent DEFINITION
System suitability Sodium Sulfite contains NLT 95.0% and NMT 100.5% of
Samples: System suitability solution and Standard sodium sulfite (Na2SOs3).
solution
[NoTe—The relative retention times for acetic acid, ma- IDENTIFICATION
leic acid, succinic acid, and fumaric acid are 0.7, 0.8, oA.
1.0, and 1.2, respectively.] Sample solution: 50 mg/mL of Sodium Sulfite. [NoTE—
Suitability requirements Reserve portions of the solution so obtained for use in
Resolution: NLT 1.5 between acetic acid and maleic Identification test B and in the test for Color and Clarity
acid; NLT 2.0 between succinic acid and fumaric acid, of Solution.]
System suitability solution Analysis: Add a drop of phenolphthalein TS.
Relative standard deviation: NMT 5.0%, Standard Acceptance criteria: A pink color is produced.
solution
Analysis Change to read:
Samples: Standard solution and Sample solution
Based on the Standard solution, identify the peaks of o B. IDENTIFICATION TESTS—GENERAL (191), Sulfate
acetic acid, maleic acid, and fumaric acid. Compare Analysis: To 5 mL of the solution from /dentification test
peak areas of acetic acid, maleic acid, and fumaric acid A add 0.5 mL of iodine TS.
in the Standard solution and the Sample solution. Acceptance criteria: The solution is colorless and meets
Acceptance criteria the requirements of °test Ae (cn 1-Mey-2018)
Sodium acetate: The peak area of acetic acid in the
Sample solution is NMT the peak area of acetic acid in
the Standard solution, corresponding to NMT 0.2% of Change to read:
sodium acetate in Sodium Succinate.
Sodium maleate: The peak area of maleic acid in the © C. IDENTIFICATION TESTS—GENERAL (191), Sodium: Meets
Sample solution is NMT the peak area of maleic acid in the requirements of "test Ae cn 41-May-2018}
the Standard solution, corresponding to NMT 0.1% of
sodium maleate in Sodium Succinate. ASSAY
Sodium fumarate: The peak area of fumaric acid in e PROCEDURE
the Sample solution is NMT the peak area of fumaric Sample: 250mg
acid in the Standard solution, corresponding to NMT Titrimetric system
0.1% of sodium fumarate in Sodium Succinate. (See Titrimetry (541).)
o LIMIT OF SULFATE Mode: Residual titration
Standard solution: 0.4 mL of 0.005 mol/L sulfuric acid Titrant: 0.1 N iodine VS
Sample solution: Dissolve 1.0 g of Anhydrous Sodium Back titrant: 0.1 N sodium thiosulfate VS
Succinate or Sodium Succinate Hexahydrate in 30 mL of Blank: 50.0 mL of 0.1 N iodine VS, accurately
measured
NF Monographs

water and neutralize with a diluted hydrochloric acid (1

in 40). Endpoint detection: Colorimetric
Analysis: Proceed as directed in Chloride and Sulfate Analysis: Add the Sample to a 500-mL beaker, add
(221), Sulfate. 50.0 mL of 0.1 N iodine VS, accurately measured, and
Acceptance criteria: NMT 0.019% as SO shake to dissolve. Add 1 mL of starch TS, and titrate
with 0.1 N sodium thiosulfate VS to a clear endpoint.
SPECIFIC TESTS Perform a blank determination, and make any necessary
e ACIDITY AND ALKALINITY correction. Calculate the percentage of sodium sulfite
Sample solution: Dissolve 1.0 g of Anhydrous Sodium (NazSO3) in the Sample taken:
Succinate or Sodium Succinate Hexahydrate in carbon
dioxide-free water and dilute with water to 20 mL. Result = [((B- V)x Nx Fx 100]/W
NF 36 Official Monographs / Sodium 5581

B = 0.1 N sodium thiosulfate VS volume 0.440g of ZnSO,- 7H2O in 100.0 mL of water. [NoTE—
consumed by the Blank pis solution contains the equivalent of 1000 g/mL of
Vv = 0.1 N sodium thiosulfate VS volume Zn.
consumed by the Sample Zinc standard solution: 25 g/mL of zinc from Zinc
N = actual normality of the Back titrant (mEq/mL) standard stock solution
F = equivalency factor, 63.0 mg/mEq Standard solutions: Transfer 1.0-, 2.0-, and 4.0-mL
w = weight of Sample (mg) portions of Zinc standard solution to separate 100-mL
Acceptance criteria: 95.0%-100.5% volumetric flasks. Dilute the contents of each flask with
water to volume, and mix to obtain solutions having
IMPURITIES known concentrations of 0.25, 0.5, and 1.0 g/mL of
zinc.
Delete the following: Sample stock solution: 100 mg/mL of Sodium Sulfite is
prepared as follows. To 10.0 g of Sodium Sulfite add
®e HEAVY METALS, Method | (231) 25 mL of water. Shake until mostly dissolved, and
slowly add 15 mL of hydrochloric acid. Heat to boiling.
Sample solution: To 8.0 9 of Sodium Sulfite add 25 mL
Cool, and dilute with water to 100.0 mL.
of water. Shake until mostly dissolved, and slowly and
carefully add 15 mL of ne rochloric acid. Heat to boil- Sample solution: 20.0 mg/mL of Sodium Sulfite from
ing. Cool, and dilute with water to 100.0 mL. Use a the Sample stock solution
25-mL portion. Instrumental conditions
(See Atomic Absorption Spectroscopy (852).)
Acceptance criteria: NMT 10 ppme coriciat 1-ja7-2018)
e Limit OF IRON Mode: Atomic a sapon spectrophotometry
Standard solution: Immediately before use, dilute 1 Analytical wavelength: Zinc emission line at 213.9
volume of Standard Iron Solution, prepared as directed nm
under /ron (241), to 10 mL with water. [NoTE—This so- Lamp: Zinc hollow-cathode
lution contains the equivalent of 1 ug/mL of iron.] Flame: Air-acetylene
Sample solution: 10.0 g of Sodium Sulfite in 25 mL of Analysis
water, Shake until mostly dissolved, and add 15 mL of Samples: Standard solutions and the Sample solution
hydrochloric acid. Heat to boiling. Cool, and dilute with Plot the absorbances of the Standard solutions versus
water to 100.0 mL. Use a 10-mL portion. concentration of zinc, in fug/mL, and draw the
Analysis: To the Standard solution and the Sample solu- straight line best fitting the plotted points. From the
tion, separately add 2 mL of a citric acid solution graph so obtained, determine the concentration of
(200 g/L), and then add 0.1 mL of thioglycolic acid. zinc, in wg/mL, in the Sample solution.
Make alkaline with stronger ammonia water, and dilute Acceptance criterias NMT 25 ppm
with water to 20 mL. Allow to stand for 5 min. SPECIFIC TESTS
Acceptance criteria: Any pink color in the Sample solu- ¢ COLOR AND CLARITY OF SOLUTION
tion is not more intense than that in the Standard solu- Analysis: Examine the solution prepared for Identifica-
tion (NMT 10 ppm). tion test A.
e LIMIT OF SELENIUM Acceptance criteria: The solution is clear and colorless.
[CauTion—Selenium is toxic; handle with care.]
Selenium standard solution: 100 g/mL of selenium is ADDITIONAL REQUIREMENTS
prepared as follows. Dissolve 0.1 g of metallic selenium © PACKAGING AND STORAGE: Preserve in tight containers.
in 2 mL of nitric acid. Evaporate to dryness, add 2 mL Store at room temperature.
of water, and evaporate to dryness. Repeat the addition
of water and the evaporation to dryness three more
times. Dissolve the residue so obtained in 50 mL of di-
luted hydrochloric acid. Transfer to a 1000-mL volumet-
ric flask, and dilute with diluted hydrochloric acid to Sodium Tartrate
volume.
Standard solution: To 1.0 g of Sodium Sulfite add i OH
0.2 mL of Selenium standard solution and 10 mL of for-
maldehyde TS, and slowly add 2 mL of hydrochloric
acid. Heat in a water bath for 20 min. Ld
Sample solution: To 3.0 g of Sodium Sulfite add 10 mL
of a TS, and slowly add 2 mL of hydrochlo- C4H4Na2O¢ - 2H20 230,08
tic acid. Disodium L-tartrate;
Analysis: Heat the Standard solution and the Sample so- Disodium (+)-2,3-dihydroxybutanedioic acid [868-18-8].
lution in a water bath for 20 min.
Acceptance criteria: Any pink color in the Sample solu- DEFINITION
tion is not more intense than that in the Standard solu- Sodium Tartrate contains NLT 99.0% and NMT 100.5% of
tion (NMT 10 ppm). ant tartrate (C4H4Naz2O¢), calculated on the dried
© LIMIT OF THIOSULFATES asis.
Sample solution: 20 mg/mL of Sodium Sulfite
Analysis: To 100 mL of the Sample solution, add 10 mL IDENTIFICATION
of formaldehyde TS and 10 mL of acetic acid. Allow to e A. IDENTIFICATION TESTS—GENERAL, Sodium (191): Meets
rg
stand for 5 min. Add 0.5 mL of starch TS, and titrate the requirements al
with 0.1 N iodine VS. Perform a blank determination e B. IDENTIFICATION TESTS—GENERAL, TJartrate (191): Meets
(see Titrimetry (541), Residual Titrations), and note the the requirements <<
fo}
difference in volumes required. =]
Acceptance criteria: The difference in volumes is NMT ASSAY fo}
0.15 mL (NMT 0.1%). ¢ PROCEDURE ro}
al
e Limit oF ZINC Sample: 250mg, previously dried at 150° for 3 h i}
Zinc standard stock solution: A solution of 1 mL of Titrimetric system me}
(See Titrimetry (541).) zs
acetic acid and the amount of zinc sulfate equivalent to 7)
USP 41
e MICROBIAL ENUMERATION TESTS (2021): The total aerobic
microbial count does not exceed 105/g; the total com-
bined molds and yeasts count does not exceed 103/g;
and the bile-tolerant Gram-negative bacteria do not ex-
ceed 103/g.
e ABSENCE OF SPECIFIED MICROORGANISMS (2022): Meets the
requirements of the tests for the absence of Salmonella
species and Escherichia coli
SPECIFIC TESTS
e BOTANICAL CHARACTERISTICS: Presence of fragments of
cork and suberized cells, with cell walls evenly thickened;
presence of phelloderm sclereids; fragments of fibers that
are crossed by vascular rays darkened due to the pres-
ence of sand-like calcium oxalate microcrystals; solitary or
two- to three-compound starch grains up to 15 wm in
diameter; absence of styloids, typically present in U.
guianensis
e Loss ON DRYING (731)
Sample: 1.0g of Powdered Cat’s Claw
Analysis: Dry the Sample at 105° for 2 h.
Acceptance criteria: NMT 7.0%
e ARTICLES OF BOTANICAL ORIGIN, Total Ash (561): NMT
8.0%
© ARTICLES OF BOTANICAL ORIGIN, Acid-Insoluble Ash (561):
NMT 2.0%
ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Preserve in tight, light-resistant
containers, and store at room temperature.
e LABELING: The label states the Latin binomial and, follow-
ing the official name, the part of the plant from which
the article was derived.
e USP REFERENCE STANDARDS (11)
USP Isopteropodine RS
USP Powdered Cat’s Claw Extract RS
Powdered Cat's Claw Extract
DEFINITION
Powdered Cat’s Claw Extract is prepared from Cat’s Claw by
extraction with hydroalcoholic mixtures or other suitable
solvents. The ratio of plant material to extract is between
4:1 to 6:1. It contains NLT 90.0% and NMT 110.0% of
the labeled amount of pentacyclic oxindole alkaloids as
isopteropodine, calculated on the dried basis, as the sum
of speciophylline, uncarine F, mitraphylline, isomitraphyl-
line, pteropodine, and isopteropodine. It may contain
suitable added substances.
IDENTIFICATION
¢ A. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST
Standard solution: 100 mg of USP Powdered Cat's
Claw Extract RS in 2 mL of methanol. Sonicate for 5
min, shaking occasionally, heat in a water bath at 60°
for 15 min, cool, and centrifuge.
Sample solution: Shake a quantity of Powdered Ex-
tract, equivalent to about 25 mg of the labeled amount
of pentacyclic oxindole alkaloids, in 20 mL of methanol.
Allow to stand for 15 min before use.
Adsorbent: Chromatographic silica gel mixture with an
average particle size of 10-15 wm (TLC plates)
Rpgal enue volume: 20 uL, as bands that are 1 cm in
lengtl
reeanns solvent system: Ethyl acetate and hexane
Spray reagent A: Dissolve 0.85 g of basic bismuth ni-
trate in 10 mL of glacial acetic acid and 40 mL of water
by heating. Filter if necessary (Solution A). Dissolve 8 g
of potassium iodide in 30 mL of water (Solution B). Mix
Solution A and Solution B (1:1) to obtain a stock solu-
tion. Dilute 1 mL of the stock solution with 2 mL of
Dietary Supplements / Cat's Claw 4509
lacial acetic acid and 10 mL of water. [NoTE—Use
reshly mixed Solution A and Solution B.]
Spray reagent B: Use a 10% solution of sodium nitrite
in water.
Analysis
Samples: Standard solution and Sample solution
Develop the chromatogram to a length of NLT 12 cm,
and dry the plate in a current of air.
Acceptance criteria: Examine the plate under short UV
light. The Sample solution chromatogram shows multi-
ple zones that correspond in Rr values to those ob-
served from the Standard solution chromatogram. Other
zones of varying intensities may be observed in the
Sample solution. Spray the plate with Spray reagent A
followed by Spray reagent B, and examine the plate
under daylight. The Sample solution chromatogram
shows multiple orange-brown zones that correspond in
color and Ry values to those observed in the Standard
solution chromatogram. Other colored zones of varying
intensities may be observed in the Sample solution.
e B. The chromatogram of the Sample solution exhibits
peaks for speciophylline, uncarine F, mitraphylline, isomi-
traphylline, pteropodine, and isopteropodine at retention
times that correspond to those of Standard solution A, as
obtained in the test for Content of Pentacyclic Oxindole
Alkaloids and Limit of Tetracyclic Oxindole Alkaloids. The
sum of the peak areas for the tetracyclic oxindole alka-
loids rh peer lite and isorhynchophylline is less than
25% of the total peak areas detected for pentacyclic ox-
indole alkaloids.
COMPOSITION
© CONTENT OF PENTACYCLIC OXINDOLE ALKALOIDS AND LIMIT
OF TETRACYCLIC OXINDOLES
Standard solution A: Dissolve an accurately weighed
quantity of USP Powdered Cat’s Claw Extract RS in
methanol, shaking for 1 min. Dilute with methanol to
obtain a solution having a known concentration of
about 0.5 mg of the labeled amount of total oxindole
alkaloids per mL. Pass throughafilter of 0.45-um or
finer pore size.
Standard solution B: 0.1 mg/mL of USP Isopteropodine
RS in methanol. Pass ehrough a nylon filter of 0.45-m
or finer pore size.
Sample solution: Transfer an accurately weighed quan-
tity of Powdered Extract, equivalent to about 5 mg of
the labeled content of pentacyclic oxindole alkaloids, to
a 10-mL centrifuge tube. Add 2.5 mL of methanol, and
sonicate for 10 min. Centrifuge, and transfer the super-
natant to a 10-mL volumetric flask. Repeat the extrac-
tion three additional times combining the extracts in
the 10-mL volumetric flask, and dilute with methanol to =]
a)
volume. Transfer about 3 mL of the solution to a test
tube containing 300 mg of polyamide powder, and =
shake for 1 min. Pass through a nylon filter of 0.45-um }
or finer pore size, discarding the first part of the filtrate. =
ro)
Solution A: Prepare a filtered and degassed 10 mM pH Co)2
7.0 phosphate buffer by mixing 6 mL of 1 N sodium hy- i}
droxide, 10 mL of 1 M monobasic potassium phos- so]
phate, and sufficient water to make 1000 mL. Adjust to Pe
a)
a pH of 7.0 + 0.1 by adding more of either solution.
Solution B: Acetonitrile
Solution C: Methanol and glacial acetic acid (99:1)
Mobile phase: See Table 1.
Table 1
Time Solution A Solution B Solution C
(min) (%) (%) (%)
0 65 35 0
17 65 35 0
25 50 50 0
30 50 50 0
31 0 0 100
 5582 Sodium / Official Monographs NF 36

Mode: Direct titration IMPURITIES

Titrant: 0.1 N Perchloric acid VS (in glacial acetic acid) Inorganic Impurities
Endpoint detection: Potentiometric e RESIDUE ON IGNITION (281): NMT 0.2%
Analysis: Dissolve the Sample in 150 mL of glacial acetic
acid by stirring and heating to near the boiling point.
Cool to room temperature. Titrate with 0.1 N perchloric Delete the following:
acid VS (in glacial acetic acid) to a potentiometric
endpoint. Perform a blank determination, and make °e HEAVY METALS, Method H (231): 10 ppMecortical 1-Jan-2018)
any necessary adjustments. Each mL of 0.1 N perchloric SPECIFIC TESTS
acid is equivalent to 9.703 mg of sodium tartrate e MELTING RANGE OR TEMPERATURE (741): 132°-135°
(C4H4Naz0o). e WATER DETERMINATION, Method | (921): NMT 0.5%
Acceptance criteria: 99.0%-100.5% on the dried basis
ADDITIONAL REQUIREMENTS
IMPURITIES © PACKAGING AND STORAGE: Preserve in tight containers,
rotected from light, and avoid exposure to excessive
Delete the following: eat.
e USP REFERENCE STANDARDS (11)
°o HEAVY METALS, Method | (231): NMT 20 ppme cota 1- USP Sorbic Acid RS
Jan-2018)
SPECIFIC TESTS
e PH (791): 7-9 (1 in 10 solution)
e Loss ON DrvING (731): Dry a sample at 150° for 3 h: it Sorbitan Monolaurate
loses 14.0%-17.0% of its weight.
ADDITIONAL REQUIREMENTS Q
OH

© PACKAGING AND STORAGE: Preserve in a tight container. z r NNN

4
No storage requirements specified. Ho" 8
oH

Sorbitan, esters, monododecanoate;

Sorbitan monolaurate [1338-39-2].
Sodium Thiosulfate—see Sodium DEFINITION
Thiosulfate General Monographs Sorbitan Monolaurate is a partial ester of lauric acid with
Sorbitol and its mono- and dianhydrides. It yields, upon
saponification, NLT 55.0% and NMT 63.0% of fatty acids,
and NLT 39.0% and NMT 45.0% of polyols (w/w).
Sorbic Acid IDENTIFICATION
eo A.
PSAP Sample: Residue obtained in the Assay for Fatty Acids
° Acceptance criteria
Fatsaire Fixed Oils, Acid Value (401): 260-280 on 1-g
sample
CeHgO2 112.13 Fats oad Fixed Oils, lodine Value (401): NMT 5
2,4-Hexadienoic acid, (E,£)-; 2,4-Hexadienoic acid; eB.
(E,£)-Sorbic acid; Sorbic acid [110-44-1]. Standard solution: 33 mg/mL of USP Isosorbide RS,
25 mg/mL of USP 1,4-Sorbitan RS, and 25 mg/mL of
DEFINITION sorbitol
Sorbic Acid contains NLT 99.0% and NMT 101.0% of Sample solution: 250 mg/mL of the polyols from the
Ce6HgOz2, calculated on the anhydrous basis. Assay for Polyols
IDENTIFICATION Chromatographic system
e A. INFRARED ABSORPTION (197K)
(See Chromatography (621), Thin-Layer Chromato-
e B. A 1-in-400,000 solution in isopropyl alcohol exhibits graphy.)
an absorbance maximum at 25442 nm. Mode: TLC
Adsorbents 0.25-mm layer of chromatographic silica
ASSAY ge
e PROCEDURE Application volume: 2 uL
Sample solution: Dissolve 250 mg of Sorbic Acid in a Developing solvent system: Acetone and glacial ace-
mixture of 50 mL of methanol and 25 mL of water that tic acid (50:1)
previously has been neutralized with 0.02 N sodium hy- Spray reagent: Sulfuric acid and water (50:50)
droxide. Add phenolphthalein TS. Analysis
Analysis: Titrate with 0.1 N sodium hydroxide VS to the Samples: Standard solution and Sample solution
first pink color that persists for at least 30 s. Each mL of Apply the Standard solution and Sample solution, and
NF Monographs

0.1 N sodium hydroxide is equivalent to 11.21 mg of allow the spots to dry. Develop the chromatogram
CeHgOo. until the solvent front has moved about three-fourths
Acceptance criteria: 99.0%-101.0% on the anhydrous of the length of the plate. Remove the plate from the
basis developing chamber, mark the solvent front, and al-
low the solvent to evaporate. Spray evenly with Spray
reagent until the surface is uniformly wet (do not
overspray), and immediately place the sprayed plate
on a 200° hot plate in a well-ventilated hood. Char
until white fumes of sulfur trioxide cease, and cool.
NF 36
Acceptance criteria: The R; values of the spots of the
Sample solution correspond to those of the spots of the
Standard solution.
ASSAY
e FATTY ACIDS
Sample: 10g
Analysis: Transfer the Sample to a 500-mL conical flask.
Cautiously add 100 mL of alcohol and 3.0g of potas-
sium hydroxide, and a few glass beads. Connecta suit-
able condenser to the flask, reflux the mixture on a hot
plate for 2 h, add 100 mL of water, and heat on a
steam bath to evaporate the alcohol, adding water oc-
casionally to replace the alcohol. Continue the evapora-
tion until the odor of alcohol can no longer be de-
tected, and transfer the saponification mixture, with the
aid of 100 mL of hot water, to a 500-mL separator. Us-
ing extreme caution, neutralize to litmus paper with a
mixture of equal volumes of sulfuric acid and water,
noting the volume used, and add a 10% excess of the
dilute acid. Allow the solution to cool. If salts appear,
add sufficient water to produce a clear solution. Cau-
srt add 100 mL of solvent hexane, shake thor-
oughly, and withdraw the lower layer into a second
500-mL separator. Similarly extract with two more
100-mL portions of solvent hexane. Extract the com-
bined hexane layers with 50-mL portions of water until
neutral to litmus paper. Combine the extracts with the
original aqueous phase to use for the Assay for Polyols.
Evaporate the solvent hexane in a tared beaker on a
steam bath nearly to dryness, dry under vacuum at 60°
for 1h, cool in a desiccator, and weigh the fatty acids.
Acceptance criteria: 55.0%-63.0%
e PoLyoLs
Sample solution: Aqueous solution retained from the
Assay for Fatty Acids
Analysis: Neutralize the Sample solution with potassium
hydroxide solution (1 in 10) to a pH of 7, using a pH
meter. Evaporate on a steam bath to a moist residue,
extract the polyols from the salts with three 150-mL
ortions of dehydrated alcohol, boiling the salt residue
or 3 min, and crushing it, as necessary, with the flat-
tened end of a stirring rod during each extraction; fil-
tering each extract while hot through a medium-poros-
ity sintered-glass funnel provided with a sheet of
retentive filter paper on which alayer of purified sili-
ceous earth has been superimposed; and receiving the
filtrates in a 1-L suction flask. Transfer the clear alcoholic
polyols solution to a tared beaker, evaporate the alco-
ol on a steam bath, dry under vacuum at 60° for 1 h,
cool in a desiccator, and weigh the polyols.
Acceptance criteria: 39.0%-45.0% (w/w)
IMPURITIES
© RESIDUE ON IGNITION (281): NMT 0.5%
Delete the following:
°e HEAVY METALS, Method I (231): NMT 10 U9/ge cortcat i.
Jan-2018)
SPECIFIC TESTS
WATER DETERMINATION, Method | (921): NMT 1.5%
FATS AND FIXED OILS, Acid Value (401): NMT 8
FATS AND FIXED O1Ls, Hydroxy! Value (401): 330-358
FATS AND FIXED OILS, Saponification Value (401): 158-170
ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Preserve in tight containers.
e USP REFERENCE STANDARDS (11)
USP Isosorbide RS
USP 1,4-Sorbitan RS
Ce6Hi20s 164.16
Official Monographs / Sorbitan 5583
Sorbitan Monooleate
CH
2
f
ah
gh fe
ae
OH
a pOgf gg ig
J “
HO" Beg fe)
Sorbitan, esters, mono(Z)-9-octadecenoate;
Sorbitan monooleate [1338-43-8].
DEFINITION
Sorbitan Monooleate is a partial oleate ester of Sorbitol and
its mono- and dianhydrides. It yields, upon saponification,
NLT 72.0% and NMT 78.0% of fatty acids, and NLT
25.0% and NMT 31.0% of polyols (w/w).
IDENTIFICATION
oA.
Sample: Residue obtained in the Assay for Fatty Acids
Acceptance criteria
Fats and Fixed Oils, Acid Value (401): 192-204 on 1-g
sample
Fats and Fixed Oils, lodine Value (401): 75-95
e B.
Standard solution: 33 mg/mL of USP Isosorbide RS,
25 mg/mL of USP 1,4-Sorbitan RS, and 25 mg/mL of
sorbitol
sae solution: 250 mg/mL of the polyols, obtained
in the Assay for Polyols
Chromatographic system
(See Chromatography (621), Thin-Layer Chromato-
graphy.)
Mode: TLC
> inna 0.25-mm layer of chromatographic silica
ge
Application volume: 2 uL
Developing solvent system: Acetone and glacial ace-
tic acid (50:1)
Spray reagent: Sulfuric acid and water (50:50)
Analysis
Samples: Standard solution and Sample solution
Apply the Standard solution and Sample solution, and
allow the spots to dry. Develop the chromatogram
until the solvent front has moved about three-fourths
of the length of the plate. Remove the plate from the
developing chamber, mark the solvent front, and al-
low the solvent to evaporate. Spray evenly with Spray
reagent until the surface is uniformly wet (do not
overspray), and immediately place the sprayed plate
ona 500° hot plate in a well-ventilated hood. Char
until white fumes of sulfur trioxide cease, and cool.
Acceptance criteria: The R- values of the spots from
the Sample solution correspond to those of the spots
from the Standard solution.
ASSAY
e Fatty AcIDsS
Sample: 10g
sydeibouow 4N
Analysis: Transfer the Sample to a 500-mL conical flask.
Cautiously add 100 mL of alcohol and 3.5 g of potas-
sium hydroxide, and a few glass beads. Connect a suit-
able condenser to the flask, reflux the mixture on a hot
plate for 2 h, add 100 mL of water, and heat on a
steam bath to evaporate the alcohol, adding water oc-
casionally to replace the alcohol. Continue the evapora-
tion until the odor of alcohol can no longer be de-
tected, and transfer the saponification mixture, with the
aid of 100 mL of hot water, to a 500-mL separator. Us-
ing extreme caution, neutralize to litmus paper with a
 5584 Sorbitan / Official Monographs
mixture of equal volumes of sulfuric acid and water,
noting the volume used, and add a 10% excess of the
dilute acid. Allow the solution to cool. If salts appear,
add sufficient water to produce a clear solution. Cau-
Say add 100 mL of solvent hexane, shake thor-
oughly, and withdraw the lower layer into a second
500-mL separator. Similarly extract with two more
100-mL portions of solvent hexane. Extract the com-
bined hexane layers with 50-mL portions of water until
neutral to litmus paper. Combine the extracts with the
original aqueous phase to use for the Assay for Polyols.
Evaporate the solvent hexane in a tared beaker on a
steam bath nearly to dryness, dry under vacuum at 60°
for 1 h, cool in a desiccator, and weigh the fatty acids.
Acceptance criteria: 72.0%-78.0%
e POLYOLs
Sample solution: Aqueous solution retained from the
Assay for Fatty Acids
Analysis: Neutralize the Sample solution with a potas-
sium hydroxide solution (1 in 10) to a pH of 7, using a
pH meter. Evaporate on a steam bath to a moist resi-
due, extract the polyols from the salts with three
150-mL portions of dehydrated alcohol, boiling the salt
residue for 3 min, and crushing it, as necessary, with
the flattened end ofa stirring rod during each extrac-
tion; filtering each extract, while hot, through a me-
dium-porosity, sintered-glass funnel provided with a
sheet of retentive filter paper on which a layer of puri-
fied siliceous earth has been superimposed; and receiv-
ing the filtrates in a 1-L suction flask. Transfer the clear
alcoholic polyols solution to a tared beaker, evaporate
the alcohol on a steam bath, dry under vacuum at 60°
for 1h, cool in a desiccator, and weigh the polyols.
Acceptance criteria: 25.0%-31.0% (w/w)
IMPURITIES
e RESIDUE ON IGNITION (281): NMT 0.5%
Delete the following:
°e HEAVY METALS, Method II (231): NMT 10 \g/ge cotta 1-
Jan-2018)
SPECIFIC TESTS
o WATER DETERMINATION, Method | (921): NMT 1.0%
FAT AND FIXED OILS, Acid Value (401): NMT 8&
FAT AND FIXED OILS, Hydroxyl Value (401): 190-215
FAT AND FIXeD OILS, lodine Value (401): 62-76
FAT AND FIXED OILS, Saponification Value (401): 145-160
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in tight containers.
e USP REFERENCE STANDARDS (11)
USP Isosorbide RS
USP 1,4-Sorbitan RS
CeHi20s 164.16
Sorbitan Monopalmitate
rm)
=
Q in a tared beaker on a steam bath nearly to dryness,
i] OH
&
D and weigh the fatty acids.
° Sorbitan, esters, monohexadecanoate;
im Sorbitan monopalmitate [26266-57-9].
fo}
= DEFINITION
re Sorbitan Monopalmitate is a partial ester of palmitic acid
Fa with Sorbitol and its mono- and dianhydrides. It yields,
upon saponification, NLT 63.0% and NMT 71.0% of fatty
acids, and NLT 32.0% and NMT 38.0% of polyols (w/w).
NF 36
IDENTIFICATION
eA.
Sample: Residue obtained in the Assay for Fatty Acids
Acceptance criteria
Fats end Fixed Oils, Acid Value (401): 210-225 on 1-g
sample
Fats and Fixed Oils, lodine Value (401): NMT 4
° v7 wee CHROMATOGRAPHIC IDENTIFICATION TEST
2'
Standard solution: 33 mg/mL of USP Isosorbide RS,
25 mg/mL of USP 1,4-Sorbitan RS, and 25 mg/mL of
sorbitol
bah solution: 250 mg/mL of the polyols, obtained
in the Assay for Polyols
Chromatographic system
(See Chromatography (621), Thin-Layer Chromato-
graphy.)
Mode: TLC
A een 0.25-mm layer of chromatographic silica
e
Application volume: 2 ul
Developing solvent system: Acetone and glacial ace-
tic acid (50:1)
Spray reagent: Sulfuric acid and water (50:50)
Analysis
Samples: Standard solution and Sample solution
Apply the Standard solution and Sample solution, and
allow the spots to dry. Develop the chromatogram
until the solvent front has moved about three-fourths
of the length of the plate. Remove the plate from the
developing chamber, mark the solvent front, and al-
low the solvent to evaporate. Spray evenly with Spray
reagent until the surface is uniformly wet (do not
overspray), and immediately place the sprayed plate
on a 200° hot plate in a well-ventilated hood. Char
until white fumes of sulfur trioxide cease, and cool.
Acceptance criteria: The R; values of the spots of the
Sample solution correspond to those of the spots of the
Standard solution.
ASSAY
e Fatty AcIDs
Sample: 10g
Analysis: Transfer the Sample to a 500-mL conical flask.
Cautiously add 100 mL of alcohol and 3.0g of potas-
sium hydroxide. Connect a suitable condenser to the
flask, reflux the mixture on a hot plate for 2 h, add
100 mL of water, and heat on a steam bath to evapo-
rate the alcohol, adding water occasionally to replace
the alcohol. Continue the evaporation until the odor of
alcohol can no longer be detected, and transfer the sa-
ponification mixture, with the aid of 100 mL of hot
water, to a 500-mL separator. Using extreme caution,
neutralize to litmus paper with a mixture of equal
volumes of sulfuric acid and water, noting the volume
used, and add a 10% excess of the dilute acid. Allow
the solution to cool. If salts appear, add sufficient water
to produce a clear solution. Cautiously add 100 mL of
solvent hexane, shake thoroughly, and withdraw the
lower layer into a second 500-mL separator. Similarly
extract with two more 100-mL portions of solvent hex-
ane. Extract the combined hexane layers with 50-mL
porons of water until neutral to litmus paper. Com-
ine the extracts with the original aqueous phase to use
for the Assay for Polyols. Evaporate the solvent hexane
dry under vacuum at 60° for 1 h, cool in a desiccator,
Acceptance criteria: 63.0%-71.0%
© POLYOLS
Sample solution: Aqueous solution retained from the
Assay for Fatty Acids
Analysis: Neutralize the Sample solution with a potas-
sium hydroxide solution (1 in 10) to a pH of 7, using a
pH meter. Evaporate on a steam bath to a moist resi-
NF 36 Official Monographs / Sorbitan 5585

due, extract the polyols from the salts with three Mode: TLC
150-mL portions of dehydrated alcohol, boiling the salt Adsorbent: 0.25-mm layer of chromatographic silica
residue for 3 min, and crushing it, as necessary, with el
the flattened end of a stirring rod during each extrac- Appliestion volume: 2 LL
tion; filtering each extract while hot through a medium- Developing solvent system: Acetone and glacial ace-
porosity, sintered-glass funnel provided with a sheet of tic acid (50:1)
retentive filter paper on which a layer of purified sili- Spray reagent: Sulfuric acid and water (50:50)
ceous earth has been superimposed; and receiving the Analysis
filtrates in a 1-L suction flask. Transfer the clear alcoholic Samples: Standard solution and Sample solution
papal solution to a tared beaker, evaporate the alco- Apply the Standard solution and Sample solution, and
ol on a steam bath, dry under vacuum at 60° for 1 h, allow the spots to dry. Develop the chromatogram
cool in a desiccator, and weigh the polyols. until the solvent front has moved about three-fourths
Acceptance criteria: 32.0%-38.0% (w/w) of the length of the plate. Remove the plate from the
developing chamber, mark the solvent front, and al-
IMPURITIES low the solvent to evaporate. Spray evenly with Spray
e RESIDUE ON IGNITION (281): NMT 0.5% reagent until the surface is uniformly wet (do not
overspray), and immediately place the sprayed plate
Delete the following: on a 200° hot plate in a well-ventilated hood. Char
until white fumes of sulfur trioxide cease, and cool.
®e HEAVY METALS, Method I/ (231): NMT 10 Ug/ge (orice- Acceptance criteria: The R; values of the spots from
jan-2018) the Sample solution correspond to those of the spots
from the Standard solution.
SPECIFIC TESTS
WATER DETERMINATION, Method | (921): NMT 1.5% ASSAY
FATS AND FIXED OlLs, Acid Value (401): NMT 8 ° Fatty Acips
FATS AND FIXED OILS, Hydroxyl Value (401): 275-305 Sample: 10g
FATS AND FIXED OILS, Saponification Value (401): 140-150 Analysis: Transfer the Sample to a 500-mL conical flask.
Cautiously add 100 mL of alcohol, 3.0 g of potassium
ADDITIONAL REQUIREMENTS hydroxide, and a few glass beads. Connecta suitable
© PACKAGING AND STORAGE: Preserve in well-closed condenser to the flask, reflux the mixture on a hot plate
containers. for 2 h, add 100 mL of water, and heat on a steam
e USP REFERENCE STANDARDS (11) bath to evaporate the alcohol, adding water occasion-
USP Isosorbide RS ally to replace the alcohol. Continue the evaporation
USP 1,4-Sorbitan RS until the odor of alcohol can no longer be detected,
CsHi20s 164.16 and transfer the saponification mixture, with the aid of
100 mL of hot water, to a 500-mL separator. Using ex-
treme caution, neutralize to litmus paper with a mixture
of equal volumes of sulfuric acid and water, noting the
volume used, and add a 10% excess of the dilute acid.
Sorbitan Monostearate Allow the solution to cool. If salts appear, add sufficient
water to produce a clear solution. Cautiously add
100 mL of solvent hexane, shake thoroughly, and with-
OH
0, draw the lower layer into a second 500-mL separator.
Loa LPO IPN Similarly extract with two more 100-mL portions of sol-
Ho% ( 8 vent hexane. Extract the combined hexane layers with
OH 50-mL portions of water until neutral to litmus paper.
Combine the extracts with the original aqueous phase
Sorbitan, esters, monooctadecanoate; to use for the Assay for Polyols. Evaporate the solvent
Sorbitan Monostearate [1338-41-6]. hexane in a tared beaker on a steam bath nearly to
DEFINITION dryness, dry under vacuum at 60° for 1 h, cool in a
Sorbitan Monostearate is a partial ester of Stearic Acid with desiccator, and weigh the fatty acids.
Sorbitol and its mono- and dianhydrides. It yields, upon Acceptance criteria: 68.0%-76.0%
e POLYOLS
saponification, NLT 68.0% and NMT 76.0% of fatty acids,
and NLT 27.0% and NMT 34.0% of polyols (w/w). Sample solution: Aqueous solution retained from the
Assay for Fatty Acids
IDENTIFICATION Analysis: Neutralize the Sample solution with a potas-
cA. sium hydroxide solution (1 in 10) to a pH of 7, using a
Sample: Residue obtained in the Assay for Fatty Acids pH meter. Evaporate on a steam bath to a moist resi-
Acceptance criteria due, extract the polyols from the salts with three
Fats and Fixed Oils, Acid Value (401): 200-215 on 1-g 150-mL portions of dehydrated alcohol, boiling the salt
sample residue for 3 min, and crushing it, as necessary, with
Fats and Fixed Oils, lodine Value (401): NMT 4 the flattened end of a stirring rod, during each extrac-
° B. tion; filtering each extract while hot through a medium-
Standard solution: 33 mg/mL of USP Isosorbide RS, porosity, sintered-glass funnel provided with a sheet of
retentive filter paper on which a layer of purified sili-
sydeibouow- 4N

25 mg/mL of USP 1,4-Sorbitan RS, and 25 mg/mL of

sorbitol ceous earth has been superimposed; and receiving the
santos solution: 250 mg/mL of the polyols, obtained filtrates in a 1-L suction flask. Transfer the clear alcoholic
in the Assay for Polyols pope solution to a tared beaker, evaporate the alco-
Chromatographic system ol on a steam bath, dry under vacuum at 60° for 1 h,
(See Chromatography (621), Thin-Layer Chromato- cool in a desiccator, and weigh the polyols.
graphy.)
 5586 Sorbitan / Official Monographs NF 36

Acceptance criteria: 27.0%-34.0% (w/w) ASSAY

e FATTY AcIDs
IMPURITIES Sample: 10g
© RESIDUE ON IGNITION (281): NMT 0.5% Analysis: Transfer the Sample to a 500-mL conical flask.
Cautiously add 100 mL of alcohol, 3.5 g of potassium
Delete the following: hydroxide, and a few glass beads. Connect a suitable
condenser to the flask, reflux the mixture on a hot plate
°o HEAVY METALS, Method II (231): NMT 10 UQ/Ge circa 1- for 2 h, add 100 mL of water, and heat on a steam
Jan-2018)
bath to evaporate the alcohol, adding water occasion-
ally to replace the alcohol. Continue the evaporation
SPECIFIC TESTS until the odor of alcohol can no longer be detected,
e@ WATER DETERMINATION, Method | (921): NMT 1.5% and transfer the saponification mixture, with the aid of
e FATS AND FIXED OlLs, Acid Value (401): NMT 10 100 mL of hot water, to a 500-mL separator. Using ex-
e FATS AND FIXED O1Ls, Hydroxy! Value (401): 235-260 treme caution, neutralize to litmus paper with a mixture
e FATS AND FIXED OILS, Saponification Value (401): 147-157 of equal volumes of sulfuric acid and water, noting the
volume used, and add a 10% excess of the dilute acid.
ADDITIONAL REQUIREMENTS Allow the solution to cool. If salts appear, add sufficient
© PACKAGING AND STORAGE: Preserve in well-closed water to produceaclear solution. Cautiously add
containers 100 mL of solvent hexane, shake thoroughly, and with-
e USP REFERENCE STANDARDS (11) draw the lower layer into a second 500-mL separator.
USP Isosorbide RS Similarly extract with two more 100-mL portions of sol-
USP 1,4-Sorbitan RS vent hexane. Extract the combined hexane layers with
Co6Hi205 164.16 50-mL portions of water until neutral to litmus paper.
Combine the extracts with the original aqueous phase
to use for the Assay for Polyols. Evaporate the solvent
hexane in a tared beaker on a steam bath nearly to
dryness, dry under vacuum at 60° for 1h, cool in a
Sorbitan Sesquioleate desiccator, and weigh the fatty acids.
Acceptance criteria: 74.0%-80.0%
DEFINITION e POLYOLS
Sorbitan Sesquioleate is a partial oleate ester of Sorbitol and Sample solution: Aqueous solution retained from the
its mono- and dianhydrides. It yields, upon saponification, Assay for Fatty Acids
NLT 74.0% and NMT 80.0% of fatty acids, and NLT Analysis: Neutralize the Sample solution with a potas-
22.0% and NMT 28.0% of polyols (w/w). sium hydroxide solution (1 in 10) to a pH of 7, using a
pH meter. Evaporate on a steam bath to a moist resi-
IDENTIFICATION due, extract the polyols from the salts with three
e A. FATTY ACID COMPOSITION OF CONSTITUTING OLEIC ACID: 150-mL portions of dehydrated alcohol, boiling the salt
It meets the requirements of the test for Fats and Fixed residue for 3 min and crushing it, as necessary, with the
Oils, Fatty Acid Composition (401). flattened end of asting rod during each extraction;
e B. IDENTIFICATION OF CONSTITUTING POLYOLS BY THIN-LAYER filtering each extract while hot through a medium-po-
CHROMATOGRAPHY rosity, sintered-glass funnel provided with a sheet of re-
Standard solution: 33 mg/mL of USP Isosorbide RS, tentive filter paper on which a layer of purified siliceous
25 mg/mL of USP 1,4-Sorbitan RS, and 25 mg/mL of earth has been superimposed; and receiving the filtrates
USP Sorbitol RS in a 1-L suction flask. Transfer the clear alcoholic polyols
Sample solution: 250 mg/mL of polyols, obtained in solution to a tared beaker, evaporate the alcohol on a
the Assay for Polyols steam bath, dry under vacuum at 60° for 1 h, cool ina
Chromatographic system desiccator, and weigh the polyols.
(See Chromatography (621), Thin-Layer Chromato- Acceptance criteria: 22.0%-28.0% (w/w)
graphy.) IMPURITIES
Mode: TLC
Se: 0.25-mm layer of chromatographic silica e RESIDUE ON IGNITION (281): NMT 1.4%
ge
Application volume: 2 uL Delete the following:
Developing solvent system: Acetone and glacial ace-
tic acid (50:1) °o HEAVY METALS, Method // (231): NMT 10 19/ge corals
Spray reagent: Sulfuric acid and water (50:50) Jan-2018)
Analysis
Samples: Standard solution and Sample solution SPECIFIC TESTS
Apply the Standard solution and Sample solution, and © FATS AND FIXED OlLs, Fatty Acid Composition (401): It ex-
allow the spots to dry. Develop the chromatogram hibits the composition profiles of fatty acids shown in
until the solvent front has moved about three-fourths Table 1.
of the length of the plate. Remove the plete from the
developing chamber, mark the solvent front, and al- Table 1
low the solvent to evaporate. Spray evenly with Spray
vr
“
reagent until the surface is uniformly wet (do not Carbon-Chain Number of Double Percentage
is overspray), and immediately place the sprayed plate Length Bonds (%)
i]
— on a 200° hot plate in a well-ventilated hood. Char 14 0 $5.0
a until white fumes of sulfur trioxide cease, and cool.
° 16 0 $16.0
= Acceptance criteria: The R; values of the spots from 16 1 <8.0
5 the Sample solution correspond to those of the spots
18 0 $6.0
= from the Standard solution.
18 1 265.0
J
ra 18 2 318.0
aThe sum of these fatty acids should be NMT 4.0%.
NF 36 Official Monographs / Sorbitan 5587

Table 1 (Continued) ASSAY

Carbon-Chain Number of Double Percentage
° Fatty AciDs
Length Bonds (%)
Sample: 8.6g
Analysis: Transfer the Sample to a 500-mL conical flask.
18 3 34.0 Cautiously add 100 mL of alcohol and 3.5 g of potas-
20, 220 0 $4.0 sium hydroxide, then add a few glass beads, and mix.
*The sum of these fatty acids should be NMT 4.0%. Connect a suitable condenser to the flask, reflux the
mixture on a hot plate for 2 h, add 100 mL of water,
‘WATER DETERMINATION, Method | (921): NMT 1.0% and heat on a steam bath to evaporate the alcohol,
FATS AND FIXED OILS, Acid Value (401): NMT 14 adding water occasionally to replace the alcohol. Con-
FATS AND FIXED OILS, Byam! Value (401): 182-220
tinue the evaporation until the odor of alcohol can no
FATS AND FIXED OILS, Peroxide Value (401): NMT 10.0 longer be detected, and transfer the saponification mix-
FATS AND FIXED OILS, Saponification Value (401): 143-165
ture, with the aid of 100 mL of hot water, to a 500-mL
ADDITIONAL REQUIREMENTS separator. Using extreme caution, neutralize to litmus
¢ PACKAGING AND STORAGE: Preserve in tight containers. paper with a mixture of equal volumes of sulfuric acid
e@ USP REFERENCE STANDARDS (11) and water, noting the volume used, and add a 10%
USP Isosorbide RS excess of the dilute acid. Allow the solution to cool. If
USP 1,4-Sorbitan RS salts appear, add sufficient water to produce a clear so-
CoHi20s 164.16 lution. Cautiously add 100 mL of solvent hexane, shake
USP Sorbitol RS thoroughly, and withdraw the lower layer into a second
500-mL separator. Similarly extract with two more
100-mL portions of solvent hexane. Extract the com-
bined hexane layers with 50-mL portions of water until
neutral to litmus paper. Combine the extracts with the
original aqueous phase to use for the Assay for Polyols.
Sorbitan Trioleate Evaporate the solvent hexane in a tared beaker on a
steam bath nearly to dryness, dry under vacuum at 60°
DEFINITION for 1h, cool in a desiccator, and weigh the fatty acids.
Sorbitan Trioleate is the triester of Oleic Acid and Sorbitol Acceptance criteria: 85.5%-90.0%
and its mono- and dianhydrides. It yields, upon saponifi- © POLYOLs
cation, NLT 85.5% and NMT 90.0% of fatty acids, and Sample solution: Aqueous solution of polyols retained
NLT 13.0% and NMT 19.0% of polyols (w/w). from the Assay for Fatty Acids
IDENTIFICATION Analysis: Neutralize the Sample solution with potassium
eA. hydroxide solution (1 in 10) to a pH of 7, using a pH
Sample: Residue obtained in the Assay for Fatty Acids meter. Evaporate on a steam bath to a moist residue.
Acceptance criteria Extract the polyols from the salts with three 150-mL
Fats and Fixed Oils, Acid Value (401): 192-204 on 1-g en of dehydrated alcohol, boiling the salt residue
sample ‘or 3 min, and crushing it, as necessary, with the flat-
Fats and Fixed Oils, lodine Value (401): 75-95 tened end ofa stirring rod during each extraction. Filter
° einen CHROMATOGRAPHIC IDENTIFICATION TEST each extract while hot through a medium-porosity,
sintered-glass funnel, provided with a sheet of retentive
Standard solution: 33 mg/mL of USP Isosorbide RS, filter paper on whicha layer of purified siliceous earth
25 mg/mL of USP 1,4-Sorbitan RS, and 25 mg/mL of has been superimposed, and receiving the filtrates in a
sorbitol 1-L suction flask. Transfer the clear alcoholic polyols so-
Sample solution: 250 mg/mL of polyols, obtained in lution to a tared beaker, evaporate the alcohol on a
the Assay for Polyols steam bath, dry under vacuum at 60° for 1 h, cool ina
Chromatographic system desiccator, and weigh the polyols.
(See en Vv (621), Thin-Layer Chromato- Acceptance criteria: 13.0%-19.0% (w/w)
raphy. IMPURITIES
Adsorbent 0.25-mm layer of chromatographic silica e RESIDUE ON IGNITION (281): NMT 0.25%
ge
Application volume: 2 pL
Developing solvent system: Acetone and glacial ace- Delete the following:
tic acid (50:1)
Spray reagent: Sulfuric acid and water (50:50) °e HEAVY METALS, Method II (231): NMT 10 Ug/ge corres: 1-
Analysis Jan-2018)
Samples: Standard solution and Sample solution
Apply the Standard solution and the Sample solution, SPECIFIC TESTS
and allow the spots to dry. Develop the chromato- e@ WATER DETERMINATION, Method | (921): NMT 0.7%
gram until the solvent front has moved about three- e FATS AND FIXED OILS, Acid Value (401): NMT 17
fourths of the length of the plate. Remove the plate e FATS AND FIXED OILS, Hydroxyl Value (401): 50-75
from the developing chamber, mark the solvent e FATS AND FIXED OILS, /odine Value (401): 77-85
front, and allow the solvent to evaporate. Spray © FATS AND FIXED OILS, Saponification Value (401): 169-183
evenly with Spray reagent until the surface is uni-
sydeibouo- 4N

formly wet (do not overspray), and immediately place ADDITIONAL REQUIREMENTS
the sprayed plate on a 200° hot plate in a well-venti- ¢ PACKAGING AND STORAGE: Preserve in tight containers.
lated hood. Char until white fumes of sulfur trioxide e USP REFERENCE STANDARDS (11)
cease, and cool. USP Isosorbide RS
Acceptance criteria: The R; values of the spots of the USP 1,4-Sorbitan RS
Sample solution correspond to those of the spots of the CoHi20s 164.16
Standard solution.
 5588 Sorbitol / Official Monographs NF 36

G = concentration of USP Sorbitol RS in the

Standard solution (mg/g)
Sorbitol Cu = concentration of Sorbitor in the Sample
solution (mg/g)
HOH HOH Ww = percentage obtained in the test for Water
na ye 0H
Determination
Hd dH Acceptance criteria: 91.0%-100.5% on the anhydrous
basis
CoHi4O6 182.17 IMPURITIES
D-Glucitol [50-70-4]. e Limit OF NICKEL
Sample solution: Dissolve 20.0 g of Sorbitol in diluted
DEFINITION acetic acid, and dilute with diluted acetic acid to
150 mL.
Change to read: Blank solution: 150 mL of diluted acetic acid
Standard solutions: Prepare three solutions by adding
Sorbitol contains NLT 91.0% and NMT 100.5% of D-sorbi- 0.5, 1.0, and 1.5 mL of nickel standard solution TS to
tol, calculated on the anhydrous basis. The amounts of 20.0 g of Sorbitol dissolved in diluted acetic acid, and
total sugars, other polyhydric alcohols, and any hexitol dilute with the same solvent to 150 mL.
anhydrides, if detected, are not included in the require- Instrumental conditions
ments, nor in the calculated amount under ® General No- (See Atomic Absorption Spectroscopy (852).)
tices, 5.60.10 Other Impurities in USP and NF Articles.e (n1- Mode: Atomic absorption spectrophotometry
May-2018)
Analytical wavelength: 232.0 nm
Lamp: Nickel hollow-cathode
IDENTIFICATION Flame: Air-acetylene
oA. Analysis
Sample solution: 1g of Sorbitol in 75 mL of water Samples: Standard solutions and Sample solution
Analysis: Transfer 3 mL of Sample solution to a 15-cm To each sample add 2.0 mL of a saturated ammonium
test tube, and add 3 mL of freshly prepared catechol pyrrolidinedithiocarbamate solution (containing 10 g/
solution (1 in 10), and mix. Add 6 mL of sulfuric acid, L of ammonium pyrrolidinedithiocarbamate) and
then gently heat the tube in a flame for 30 s. 10.0 mL of methyl isobutyl ketone, and shake for 30
Acceptance criteria: A deep pink or wine-red color s. Protect from bright light. Allow the two layers to
appears. separate, and use the methyl isobutyl ketone layer.
e B. The retention time of the major peak of the Sample Set the instrument to zero using the organic layer
solution corresponds to that from the Standard solution, from the Blank solution. Concomitantly determine the
as obtained in the Assay. absorbances of the organic layer from the Samples at
least three times each. Record the average of the
ASSAY steady readings for each of the Standard solutions and
e PROCEDURE the Sample solution. Between each measurement, as-
Mobile phase: Use degassed water. pirate the organic layer from the Blank solution, and
System suitability solution: Prepare a solution contain- ascertain that the reading returns to zero. Plot the
ing 4.8 mg/g of each USP Sorbitol RS and mannitol absorbances of the Standard solutions and the Sample
Standard solution: 4.8 mg/g of USP Sorbitol RS solution versus the added quantity of nickel. Extrapo-
Sample solution: Dissolve 0.10 g of Sorbitol in water, late the line joining the points on the graph until it
and dilute with water to 20 g. Record the final solution meets the concentration axis. The distance between
weight, and mix thoroughly. this point and the intersection of the axes represents
Chromatographic system the concentration of nickel in the Sample solution.
(See Chromatography (621), System Suitability.) Acceptance criteria: NMT 1 ppm
Mode: LC © RESIDUE ON IGNITION (281): NMT 0.1%, determined on a
Detector: Refractive index 1.5-g portion
Column: 7.8-mm x 10-cm; packing L34
Temperature
Column: 50+ 2° Change to read:
Detector: 35°
Flow rate: 0.7 mL/min e REDUCING SUGARS
Injection size: 10 ul [Note—The amount determined in this test is not in-
System suitability cluded in the calculated amount under ®General No-
Samples: System suitability solution and Standard tices, 5.60.10 Other Impurities in USP and NF Articles.
solution @ (CN 1-May-2018))
[Note—The relative retention times for mannitol and Sample solution: Dissolve 3.3 g of Sorbitol in 3 mL of
sorbitol are about 0.6 and 1.0, respectively.] water with the aid of gentle heat. Cool, and add
Suitability requirements 20.0 mL of cupric citrate TS and a few glass beads.
Resolution: NLT 2.0 between sorbitol and mannitol, Heat so that boiling begins after 4 min, and maintain
System suitability solution boiling for 3 min. Cool rapidly, and add 40 mL of di-
luted acetic acid, 60 mL of water, and 20.0 mL of 0.05
=
al
Relative standard deviation: NMT 2.0%, Standard
rm solution N iodine VS. With continuous shaking, add 25 mL of a
S
sS Analysis mixture of 6 mL of hydrochloric acid and 94 mL of
iy Samples: Standard solution and Sample solution water.
3 Calculate the percentage, on the anhydrous basis, of Analysis: When the precipitate has dissolved, titrate the
= excess of iodine with 0.05 N sodium thiosulfate VS us-
iS D-sorbitol in the portion of Sorbitol taken:
ing 2 mL of starch TS, added toward the end of the
Ps Result = (ru/rs) x (Cs/Cu) x (100/(100 — W)) x 100 titration, as an indicator.
J Acceptance criteria: NLT 12.8 mL of 0.05 N sodium
Fa tu = peak response from the Sample solution thiosulfate VS is required, corresponding to NMT 0.3%
rs = peak response from the Standard solution of reducing sugars, as glucose.
NF 36 Official Monographs / Sorbitol 5589

e CHLORIDE AND SULFATE, Chloride (221) (if labeled for use in e C. LIMIT OF DIETHYLENE GLYCOL AND ETHYLENE GLYCOL
preparing parenteral dosage forms) Diluent: Acetone and water (96:4)
Sample: 1.59 Standard solution: 0.08 mg/mL of USP Diethylene Gly-
Acceptance criteria: The Sample shows no more chlo- col RS and 0.08 mg/mL of USP Ethylene Glycol RS in
ride than corresponds to 0.10 mL of 0.020 N hydro- Diluent.
chloric acid (NMT 0.0050%). Sample solution: Transfer 2.0 g of Noncrystallizing Sor-
e CHLORIDE AND SULFATE, Sulfate (221) (if labeled for use in bitol Solution to a 25-mL volumetric flask. Add 1.0 mL
preparing parenteral dosage forms) of Diluent to the flask, and mix on a vortex mixer for 3
Sample: 1.0g min. Add the remaining Diluent to the flask to volume
Acceptance criteria: The Sample shows no more sulfate in three equal portions. Mix on a vortex mixer for
than corresponds to 0.10 mL of 0.020 N sulfuric acid about 3 min after each addition of Diluent. Pass a por-
(NMT 0.01%). tion of the supernatant layer obtained through a 0.45-
um nylon filter. Discard the first 2 mL of the filtrate, and
SPECIFIC TESTS collect the rest of the filtrate for analysis.
e MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI- [Note—Acetone is used to precipitate sorbitol.]
FIED MICROORGANISMS (62): The total aerobic count us- Chromatographic system
ing the Plate Method is NMT 1000 cfu/g, and the total (See Chromatography (621), System Suitability.)
combined molds and yeasts count is NMT 100 cfu/g. Mode: GC
PH (791): 3.5-7.0, in a 10% (w/w) solution in carbon Detector: Flame ionization
dioxide-free water Column: 0.32-mm x 15-m fused-silica capillary col-
WATER DETERMINATION, Method | (921): NMT 1.5% umn; 0.25-um layer of phase G46
¢ CLARITY AND COLOR OF SOLUTION (if labeled for use in pre- Temperature
paring parenteral dosage forms) Detector: 300°
Sample: 10.0g Injector port: 240°
Analysis: Dissolve the Sample in 100.0 mL of carbon di- Column: See the temperature program table below.
oxide-free water.
Acceptance criteria: The solution is clear and colorless.
Hold Time
BACTERIAL ENDOTOXINS TEST (85) (if labeled for use in pre-
Initial Temperature Final at Final
paring parenteral dosage forms): NMT 4 USP Endotoxin
Temperature Ramp Temperature | Temperature
brits for parenteral dosage forms having a concentra-
tion of less than 100 g/L of sorbitol, and NMT 2.5 USP ©) ¢/min) @) (min)
Endotoxin Units/g for parenteral dosage forms having a 70 = 70 2
concentration of 100 g/L or more of sorbitol 70 50 300 BS

ADDITIONAL REQUIREMENTS Carrier gas: Helium

© PACKAGING AND STORAGE: Preserve in well-closed contain-
ers. No storage requirements are specified.
e LABELING: Sorbitol intended for use in preparing paren-
teral dosage forms is so labeled.
Change to read:
° USP REFERENCE STANDARDS (11)
© (CIN i-May-2018)
USP Sorbitol RS
Noncrystallizing Sorbitol Solution
DEFINITION
Noncrystallizing Sorbitol Solution is an aqueous solution
containing NLT 45.0% of D-sorbitol (CsH140¢) (w/w). The
amounts of total sugars, other powhydne alcohols, and
any hexitol anhydrides, if detected, are not included in
the requirements nor in the calculated amount under
General Notices, 5.60.10. Other Impurities in USP and NF
Articles.
IDENTIFICATION
© A. PROCEDURE
Sample solution: Dissolve 1.4 g of Noncrystallizing Sor-
bitol Solution in 75 mL of water.
Flow rate: 3.0 mL/min
Injection size: 1.0 uL
Injection type: Split injection. The split ratio is about
10:1. [NOTE—A split liner, deactivated with glass wool,
is used.]
System suitability
Sample: Standard solution
[NoTe—Diethylene glycol elutes after ethylene glycol in
the Pvoreargel
Suitability requirements
Resolution: NLT 30 between ethylene glycol and di-
ethylene glycol
Analysis
Samples: Standard solution and Sample solution
Based on the Standard solution, identify the peaks of
ethylene glycol and diethylene Byer Compare the
peak areas of ethylene glycol and diethylene glycol in
the Standard solution and the Sample solution.
Acceptance criteria
Diethylene glycol: The peak area of diethylene glycol
in the Sample solution is NMT the peak area of diethyl-
ene glycol in the Standard solution, corresponding to
NMT 0.10% of diethylene glycol in Noncrystallizing
Sorbitol Solution.
Ethylene glycol: The peak area of ethylene glycol in
the Sample solution is NMT the peak area of ethylene
glycol in the Standard solution, corresponding to NMT
Sue of ethylene glycol in Noncrystallizing Sorbitol
olution.
sydeibouo-: 4IN

Analysis: Transfer 3 mL of Sample solution to a 15-cm ASSAY

test tube. Add 3 mL of freshly prepared catechol solu-
tion (1 in 10), and mix. Add 6 mL of sulfuric acid, mix
again, and gently heat the tube in a flame for 30 s.
Acceptance criteria: A deep pink or wine-red color
appears.
¢ B. The retention time of the major peak from the Sample
solution corresponds to that of the Standard solution, as
obtained in the Assay.
e¢ PROCEDURE
Mobile phase: Use degassed water.
System suitability solution: 4.8 mg/g each of mannitol
and USP Sorbitol RS
Standard solution: 4.8 mg/g of USP Sorbitol RS
Sample solution: Weigh 0.20 g of Noncrystallizing Sor-
bitol Solution, and dissolve in and dilute with water to
20 g. Record the final solution weight.
 5590 Sorbitol / Official Monographs
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: Refractive index
Column: 7.8-mm x 10-cm; packing L34
Temperature
Detector: 35°
Column: 50+ 2°
Flow rate: 0.7 mL/min
Injection size: 10 LL
System suitability
Samples: System suitability solution and Standard
solution
[NoTtt—The relative retention times for mannitol and
sorbitol are about 0.6 and 1.0, respectively, System
suitability solution.]
Suitability requirements
Resolution: NLT 2.0 between sorbitol and mannitol,
System suitability solution
Relative standard deviation: NMT 2.0%, Standard
solution
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of D-sorbitol (CsHi4O¢) in the
portion of Noncrystallizing Sorbitol Solution taken:
Result = (ru/rs) x (Cs/Cu) x 100
tu = peak response from the Sample solution
rs = peak response from the Standard solution
Cs = concentration of USP Sorbitol RS in the
Standard solution (mg/g)
Cu = concentration of Noncrystallizing Sorbitol
Solution in the Sample solution (mg/g)
Acceptance criteria: NLT 45.0%
IMPURITIES
Inorganic Impurities
e RESIDUE ON IGNITION (281): NMT 0.1%, calculated on the
anhydrous basis, determined on a 2-g portion
© Limit OF NICKEL
Solution A: A saturated ammonium pyrrolidine dithio-
carbamate solution (containing 10 mg/mL of ammo-
nium pyrrolidine dithiocarbamate)
Sample solution: Dissolve 20.0 g of Noncrystallizing
Sorbitol Solution in diluted acetic acid, and dilute with
diluted acetic acid to 100.0 mL. Add 2.0 mL of Solution
A and 10.0 mL of methyl isobutyl ketone, and shake for
30 s. Protect from bright light. Allow the two layers to
separate, and use the methyl! isobutyl ketone layer.
Blank solution: Prepare as directed for the Sample solu-
tion, except omit the use of the Noncrystallizing Sorbi-
tol Solution.
Standard solutions: Prepare as directed for the Sample
solution, except prepare three solutions by adding 0.5,
1.0, and 1.5 mL of nickel standard solution TS.
Spectrometric conditions
(See Atomic Absorption Spectroscopy (852).)
Mode: Atomic absorption spectrophotometry
Analytical wavelength: 232.0 nm (maximum
absorbance)
Lamp: Nickel hollow-cathode
Flame: Air—acetylene
Analysis
Samples: Standard solutions, Sample solution, and
NF Monographs
Blank solution
Set the instrument to zero using the Blank solution.
Concomitantly determine the absorbances of the
Standard solutions and the Sample solution at least
three times each. Record the average of the steady
readings for each of the Standard solutions and the
Sample solution. Between each measurement, aspi-
rate the Blank solution, and ascertain that the read-
ing returns to zero. Plot the absorbances of the
Standard solutions and the Sample solution versus the
NF 36
added quantity of nickel. Extrapolate the line joining
the points on the graph until it meets the concen-
tration axis. The distance between this point and the
intersection of the axes represents the concentration
of nickel in the Sample solution.
eee criteria: NMT 1 ug/g on the anhydrous
asis
Organic Impurities
¢ PROCEDURE: REDUCING SUGARS
Sample: An amount of Noncrystallizing Sorbitol Solu-
tion, equivalent to 3.3 g on the anhydrous basis.
Analysis: To the aa add 3 mL of water, 20.0 mL of
cupric citrate TS, and a few glass beads. Heat so that
boiling begins after 4 min, and maintain boiling for 3
min. Cool rapidly, and add 40 mL of diluted acetic
acid, 60 mL of water, and 20.0 mL of 0.05 N iodine
VS. With continuous shaking, add 25 mL of a mixture
of 6 mL of hydrochloric acid and 94 mL of water.
When the precipitate has dissolved, titrate the excess of
iodine with 0.05 N sodium thiosulfate VS using 2 mL of
starch TS, added towards the end of the titration, as an
indicator.
Acceptance criteria: NLT 12.8 mL of 0.05 N sodium
thiosulfate VS is required corresponding to NMT 0.3%
of reducing sugars, on the anhydrous basis, as glucose.
The amount determined in this test is not included in
the calculated amount under General Notices, 5.60.10.
Other Impurities in USP and NF Articles.
SPECIFIC TESTS
© MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI-
FIED MICROORGANISMS (62): The total aerobic microbial
count using the Plate Method is NMT 1000 cfu/mL, and
the total combined molds and yeasts count is NMT 100
cfu/mL.
e PH (791): 5.0-7.5, in a 14% (w/w) solution of Noncrys-
tallizing Sorbitol Solution in carbon dioxide-free water
e@ WATER DETERMINATION, Method | (921): 28.5%-31.5%
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in well-closed contain-
ers. No storage requirements are specified.
e USP REFERENCE STANDARDS (11)
USP Sorbitol RS
USP Diethylene Glycol RS
USP Ethylene Glycol RS
Sorbitol Sorbitan Solution
Former Title: Anhydrized Liquid Sorbitol
DEFINITION
Sorbitol Sorbitan Solution is a water solution containing, on
the anhydrous basis, NLT 25.0% of D-sorbitol (CsH1406)
and NLT 15.0% of 1,4-sorbitan (CsHi20s). The amounts of
total sugars, other polyhydric alcohols, and any other hex-
itol anhydrides, if detected, are not included in the re-
quirements or in the calculated amount under General
Notices, 5.60.10. Other Impurities in USP and NF Articles.
IDENTIFICATION
e A. PROCEDURE
Sample: 1.4g of Sorbitol Sorbitan Solution in 75 mL of
water
Analysis: Transfer 3 mL of Sample to a 15-cm test tube.
Add 3 mL of freshly prepared catechol solution (1 in
10), and mix. Add 6 mL of sulfuric acid, mix again,
then gently heat the tube in a flame for about 30 s.
Acceptance criteria: A deep pink or wine-red color
appears.
e B. The retention times of the Sample solution correspond
to those of the Standard solution, as obtained in the As-
say.
NF 36 Official Monographs / Sorbitol 5591

e C. LIMIT OF DIETHYLENE GLYCOL AND ETHYLENE GLYCOL Record the weight of the final solution, and mix
Diluent: Acetone and water (96:4) thoroughly.
Standard solution: 0.08 mg/mL of USP Diethylene Gly- Chromatographic system
col RS and 0.08 mg/mL of USP Ethylene Glycol RS in (See Chromatography (621), System Suitability.)
Diluent Mode: LC
Sample solution: Transfer 2.0 g of Sorbitol Sorbitan So- Detector: Refractive index
lution to a 25-mL volumetric flask. Add 1.0 mL of Dilu- Column: 7.8-mm x 10-cm; packing L34
ent to the flask, and mix on a vortex mixer for about 3 Temperature
min. Add the remaining Diluent to the flask to volume Detector: 35°
in three equal portions. Mix on a vortex mixer for Column: 50+ 2°
about 3 min after each addition of Diluent. Pass a por- Flow rate: 0.6 mL/min
tion of the supernatant layer obtained through a 0.45- Injection size: 10 pL
uum nylon filter. Discard the first 2 mL of the filtrate, and System suitability
collect the rest of the filtrate for analysis. [NoTE—Ace- Samples: System suitability solution and Standard
tone is used to precipitate sorbitol.] solution
Chromatographic system [Note—The relative retention times for 1,4-sorbitan,
(See Chromatography (621), System Suitability.) isosorbide, mannitol, and sorbitol are about 0.35,
Mode: GC 0.43, 0.7, and 1.0, respectively.]
Detector: Flame ionization Suitability requirements
Column: 0.32-mm x 15-m fused-silica capillary col- Resolution: NLT 2.0 between 1,4-sorbitan and
umn; 0.25-um layer of phase G46 isosorbide, System suitability solution
Temperature Relative standard deviation: NMT 2.0% for each
Detector: 300° analyte, Standard solution
Injector port: 240° Analysis
Column: See the temperature program table below. Samples: Standard solution and Sample solution
aera ae calculate the percentages, on the anhydrous
Hold Time basis, of 1,4-sorbitan and D-sorbitol in the portion of
Initial Temperature Final at Final Sorbitol Sorbitan Solution taken:
Temperature Ramp Temperature | Temperature
Result = (ru/ts) x (Cs/Cu) x [100/(100 - W)] x 100
© (¢/min) @) (min)
70 J 70 2 ty = peak responses of the corresponding analyte
70 50 300 5 from the Sample solution
Ts = peak responses of the corresponding analyte
Carrier gas: Helium from the Standard solution
Flow rate: 3.0 mL/min Cs = concentration of the appropriate USP
Injection size: 1.0 uL Reference Standard in the Standard solution
Injection type: Split injection. The split ratio is about
10:1. [NoTE—A split liner, deactivated with glass wool,
(mg/g) ! ; glcheg
Cu = concentration of Sorbitol Sorbitan Solution in
is used.] the Sample solution (mg/g)
System suitability WwW = percentage from the test for Water
Sample: Standard solution Determination
[Nott—Diethylene glycol elutes after ethylene glycol in Acceptance criteria: NLT 25.0% of CsHi4O¢ and NLT
the chromatogram.] 15.0% of C6Hi205 on the anhydrous basis
Suitability requirements
Resolution: NLT 30 between ethylene glycol and di- IMPURITIES
ethylene glycol Inorganic Impurities
Analysis e RESIDUE ON IGNITION (281): NMT 0.20%, calculated on
Samples: Standard solution and Sample solution the anhydrous basis on a 2-g portion
Based on the Standard solution, identify the peaks of o LIMIT OF NICKEL
ethylene glycol and diethylene glycol. Compare peak Solution A: A saturated ammonium pyrrolidine dithio-
areas of ethylene glycol and diethylene glycol in the carbamate solution (10 mg/mL of ammonium pyr-
Standard solution and the Sample solution. rolidine dithiocarbamate)
Acceptance criteria Sample solution: 200 mg/mL of Sorbitol Sorbitan Solu-
Diethylene glycol: The peak area of diethylene glycol tion in diluted acetic acid. To 100 mL of this solution
in the Sample solution is NMT the peak area of diethyl- add 2.0 mL of Solution A and 10.0 mL of methyl
ene glycol in the Standard solution, corresponding to isobutyl ketone, and shake for 30 s. Protect from bright
NMT 0.10% of diethylene glycol in Sorbitol Sorbitan light. Allow the two layers to separate, and use the
Solution. methyl isobutyl ketone layer.
Ethylene glycol: The peak area of ethylene glycol in Standard solutions: Prepare as directed for the Sample
the Sample solution is NMT the peak area of ethylene solution, except to prepare three solutions by adding
glycol in the Standard solution, corresponding to NMT 0.5, 1.0, and 1.5 mL of nickel standard solution TS.
0.10% of ethylene glycol in Sorbitol Sorbitan Solution. Blank solution: Prepare as directed for the Sample solu-
tion, except to omit the use of Sorbitol Sorbitan Solu-
ASSAY tion. Quantities should be increased five fold to ensure
sydesbouow- 4N

© PROCEDURE that a sufficient volume of Blank solution is available.

Mobile phase: Water
System suitability solution: 10 mg/g of sorbitol, 4 mg/
g of 1,4-sorbitan, 4 mg/g of isosorbide, and 1 mg/g of
mannitol in water
Standard solution: 10 mg/g of USP Sorbitol RS and
4 mg/g of USP 1,4-Sorbitan RS in water
Sample solution: Dissolve 0.40 g of Sorbitol Sorbitan
Solution in water, and dilute with water to about 20 g.
Spectrometric conditions
(See Atomic Absorption Spectroscopy (852).)
Mode: Atomic absorption spectrophotometry
Analytical wavelength: 232.0 nm (maximum
absorbance)
 4510 Cat’s Claw / Dietary Supplements USP 41

Table 1 (Continued) CONTAMINANTS

Time Solution A Solution B Solution C
(min) (%) (%) (%) Delete the following:
36 oO 0 100
39 65. 35 9 °e HEAVY METALS, Method II (231): NMT 10 ppme coiticat1-
49 65 35 0 Jan-2018)
e MICROBIAL ENUMERATION TESTS (2021): Meets the require-
Chromatographic system ments of the tests for absence of Salmonella species and
(See Chromatography (621), System Suitability.) Escherichia coli, The total aerobic microbial count does
Mode: LC not exceed 104/g, and the total combined molds and
Detector: UV 245 nm yeasts count does not exceed 103/g.
Column: 4.6-mm x 10-cm; end-capped 3-4m packing e OTHER REQUIREMENTS: It meets the requirements for Bo-
1 tanical Extracts (565), Residual Solvents and Pesticide
Flow rate: 0.75 mL/min Residues.
Injection size: 10 pL
System suitability SPECIFIC TESTS
Samples: Standard solution A and Standard solution B e Loss ON DRYING (731): Dry 1g at 105° for 2 h: it loses
Suitability requirements NMT 10.0% of its weight.
Chromatogram similarity: The chromatogram ob- ADDITIONAL REQUIREMENTS
tained using Standard solutionA is similar to the Ref- © PACKAGING AND STORAGE: Preserve in tight, light-resistant
erence Chromatogram provided with the USP Pow- containers, and store at room temperature.
dered Cat's Claw Extract RS being used. e LABELING: The label states the Latin binomial and, follow-
Tailing factor: NMT 2.0 for the isopteropodine peak, ing the official name, the part of the plant from which
Standard solution B the article was prepared. The label also indicates the con-
Relative standard deviation: NMT 2.0% for the tent of pentacyclic oxindole alkaloids, the extracting sol-
isopteropodine peak in repeated injections, Standard vent or solvent mixture used for preparation, and the
solution B ratio of the starting crude plant material to Powdered
Analysis Extract. It meets the requirements for Botanical Extracts
Samples: Standard solution A, Standard solution B, and (565), Labeling.
Sample solution e USP REFERENCE STANDARDS (11)
Measure the areas of the analyte peaks. Identify the re- USP Isopteropodine RS
tention times of the peaks corresponding to speci- USP Powdered Cat’s Claw Extract RS
ophylline, uncarine F, mitraphylline, isomitraphylline,
rhynchophylline, isorhynchophylline, pteropodine, and
isopteropodine by comparison of the chromatogram
of Standard solution A with the Reference Chromato-
gram provided with the lot of the USP Powdered Cat's
Claw Extract RS used. Cat's Claw Capsules
Separately calculate the percentages of speciophylline,
uncarine F, mitraphylline, isomitraphylline, rhyncho- DEFINITION
phylline, isorhynchophylline, pteropodine, and isopter- Cat’s Claw Capsules contain Powdered Cat’s Claw Extract.
opodine, as isopteropodine, in the portion of Pow- Capsules contain NLT 90.0% and NMT 110.0% of the
dered Extract taken: labeled amount of Powdered Extract, calculated as penta-
cyclic oxindole alkaloids.
Result = (ru/rs) x (Cs/Cu) x 100
IDENTIFICATION
tu = peak response for each relevant alkaloid from e The Sample solution chromatogram exhibits peaks for
the Sample solution speciophylline, uncarine F, mitraphylline, isomitraphyl-
rs = peak response for isopteropodine from line, pteropodine, and isopteropodine at retention times
Standard solution B that correspond to those in Standard solution A, as ob-
Cs = concentration of USP Isopteropodine RS in tained in the test for Content of Pentacyclic Oxindole Alka-
DS Monographs

Standard solution B (mg/mL)
Cu = concentration of Powdered Extract in the
Sample solution (mg/mL)
Calculate the percentage of the labeled amount of
pentacyclic oxindole alkaloids in the Powdered Extract:
loids and Limit of Tetracyclic Oxindole Alkaloids. The con-
tent of tetracyclic oxindole alkaloids, calculated as the
sum of rhynchophylline and isorhynchophylline, is NMT
25% of the labeled amount of pentacyclic oxindole
alkaloids.

Result = (ZPA/L) x 100 STRENGTH

© CONTENT OF PENTACYCLIC OXINDOLE ALKALOIDS AND LIMIT
=PA = sum of percentages of speciophylline, uncarine OF TETRACYCLIC OXINDOLE ALKALOIDS
F, mitraphylline, isomitraphylline, Solution A: Prepare a 10 mM pH 7.0 phosphate buffer
pteropodine, and isopteropodine (%) by mixing 1 N sodium hydroxide, 1M monobasic po-
L = labeled amount of pentacyclic alkaloids as tassium phosphate, and water (3:5:492), and adjust to
isopteropodine (%) a pH of 7.0 + 0.1 by adding more of either solution.
Calculate the content of tetracyclic oxindole alkaloids Solution B: Acetonitrile
by adding the individual percentages of Solution C: Methanol and glacial acetic acid (99:1)
rhynchophylline and isorhynchophylline. Mobile phase: See the gradient table below.
Acceptance criteria
Pentacyclic oxindole alkaloids: 90.0%-110.0% of the Time Solution A Solution B Solution C
labeled amount on the dried basis (min) (%) (%) (%)
Tetracyclic oxindole alkaloids: NMT 25% of the la- 0 65 35 0
beled amount of pentacyclic oxindole alkaloids on the 7 65 35 oO
dried basis
25. 50 50 0
 5592 Sorbitol / Official Monographs
Lamp: Nickel hollow-cathode
Flame: Air-acetylene
Analysis
Samples: Blank solution, Standard solutions, and Sam-
ple solution
Set the instrument to zero, using the Blank solution.
Concomitantly determine the absorbances of the
Standard solutions and the Sample solution at least
three times each. Record the average of the steady
readings for each of the Standard solutions and the
Sample solution. Between each measurement, aspi-
rate the Blank solution, and ascertain that the read-
ing returns to zero. Plot the absorbances of the
Standard solutions and the Sample solution versus the
added quantity of nickel. Extrapolate the line joining
the points on the graph until it meets the concen-
tration axis. The distance between this point and the
intersection of the axes represents the concentration
of nickel in the Sample solution.
Acceptance criteria: NMT 1 ppm, calculated on the
anhydrous basis
Organic Impurities
© PROCEDURE: REDUCING SUGARS
Sample: An amount of Sorbitol Sorbitan Solution
equivalent to 3.3 g, on the anhydrous basis.
Analysis: To the Sample, add 3 mL of water, 20.0 mL of
cupric citrate TS, and a few glass beads. Heat so that
boiling begins after 4 min, and maintain boiling for 3
min. Cool rapidly, and add 40 mL of diluted acetic
acid, 60 mL of water, and 20.0 mL of 0.05 N iodine
VS. With continuous shaking add 25 mL of a mixture of
6 mL of hydrochloric acid and 94 mL of water. When
the precipitate has dissolved, titrate the excess of io-
dine with 0.05 N sodium thiosulfate VS, using 2 mL of
starch TS, added toward the end of the titration, as an
indicator.
Acceptance criteria: NLT 12.8 mL of 0.05 N sodium
thiosulfate VS is required, corresponding to NMT 0.3%
of reducing sugars, on the anhydrous basis, as glucose.
[NoTte—The amount determined in this test is not in-
cluded in the calculated amount under General Notices,
5.60.10. Other Impurities in USP and NF Articles.]
SPECIFIC TESTS
e MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI-
FIED ORGANISIMS (62): The total aerobic microbial count
using the Plate Method is NMT 1000 cfu/mL. The total
condbined molds and yeasts count is NMT 100 cfu/mL.
e PH (791): 4.0-7.0, in a 14% (w/w) solution of Sorbitol
Sorbitan Solution in carbon dioxide-free water
e WATER DETERMINATION, Method | (921): NMT 31.5%
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in well-closed contain-
ers. No storage pe specified.
e LABELING: The labeling indicates the percentage content,
on the anhydrous basis, of D-sorbitol and 1,4-sorbitan.
e USP REFERENCE STANDARDS (11)
USP Diethylene Glycol RS
USP Ethylene Glycol RS
USP 1,4-Sorbitan RS
Ce6Hi205 164.16
USP Sorbitol RS
fe
”
a section of the axes represents the concentration of
ieS
a
i}
c Sample: 2.0g
5 Analysis: Dissolve the Sample by gently heating in a
= mixture of 1.5 mL of alcohol and 3.0 mL of toluene.
— Add 0.05 mL of bromophenol blue TS, and titrate with
7 0.01 N hydrochloric acid VS to a yellow endpoint.
NF 36
Sorbitol Solution—see Sorbitol Solution
General Monographs
Soybean Oil—see Soybean Oil General
Monographs
Hydrogenated Soybean Oil
[8016-70-4].
DEFINITION
Hydrogenated Soybean Oil is the product obtained by refin-
ing, bleaching, hydrogenation, and deodorization of oil
obtained from seeds of the soya plant Glycine max Merr.
(Fabaceae). The product consists mainly of triglycerides of
palmitic and stearic acids.
IDENTIFICATION
e A. It meets the requirements in Specific Tests for Fats and
Fixed Oils, Fatty Acid Composition (401).
e B. It meets the requirements in Specific Tests for Melting
Range or Temperature, Class Il (741).
IMPURITIES
e LIMIT OF NICKEL
Nickel standard solution: Immediately before use, pre-
pare the equivalent of 0.2 g/g of nickel by diluting
10 mL of nickel standard solution TS with water to
500 mL.
Sample solution: Weigh 5.0 g of Hydrogenated Soy-
bean Oil into a previously tared platinum or silica cruci-
ble. ete heat, and introduce into the substance a
wick formed from twisted ashless filter paper. Ignite the
wick. When the substance ignites, stop heating. After
combustion, ignite in a muffle furnace at 600°. Con-
tinue the incineration until white ash is obtained. After
cooling, transfer the residue, with the aid of two 2-mL
portions of diluted hydrochloric acid, to a 25-mL volu-
metric flask, add 0.3 mL of nitric acid, and dilute with
water to volume.
Standard solutions: Into three identical 10-mL volu-
metric flasks introduce 1.0, 2.0, and 4.0 mL of Nickel
standard solution. To each flask add a 2.0-mL portion of
the Sample solution, and dilute with water to volume.
Instrumental conditions
(See Atomic Absorption Spectroscopy (852).)
Mode: Atomic absorption, equipped with a graphite
furnace
Analytical wavelength: 232.0 nm
Lamp: Nickel hollow-cathode
Analysis
Samples: Sample solution and Standard solutions
Determine the absorbances of the Samples at least
three times each. Record the average of the steady
readings for each of the Samples. Plot the ab-
sorbances of the Standard solutions and the Sample
solution versus the added quantity of nickel, and draw
the straight line best fitting the three plotted points.
Extrapolate the line until it meets the concentration
axis. The distance between this point and the inter-
nickel in the Sample solution.
Acceptance criteria: NMT 1 g/g
© ALKALINE IMPURITIES
NF 36 Official Monographs / Squalane 5593

Acceptance criteria: NMT 0.4 mL of 0.01 N hydrochlo- ASSAY

ric acid VS is required. e PROCEDURE
Diluent: Heptane
SPECIFIC TESTS Standard solution: 4 mg/mL of USP Squalane RS in
e MELTING RANGE OR TEMPERATURE, Class I! (741): 66°-72° Diluent
e FATS AND FIXED OILS, Acid Value (401) System suitability solution: 4 mg/mL of USP Squalane
Sample: 10g of Hydrogenated Soybean Oil RS and 4 uL/mL of methyl erucate in Diluent
Analysis: Dissolve the Sample in 50 mL of a hot mixture Sample solution: 4 mg/mL of Squalane in Diluent
of neutralized alcohol and toluene (1:1). Add 0.5 mL of Chromatographic system
pheseptacs TS, and immediately titrate, while still (See Chromatography (621), System Suitability.)
ot, with 0.1 N potassium hydroxide VS to produce a Mode: GC
permanent, faint pink color. Detector: Flame ionization
Acceptance criteria: NMT 0.5 Column: 0.32-mm x 30-m fused silica capillary col-
e FATS AND FIXED OILS, Fatty Acid Composition (401): Hy- umn bonded with a 1-1m layer of phase G2
drogenated Soybean Oil exhibits the composition profile Temperatures
of fatty acids in Table 1. Detector: 300°
Injection port: 275°
Table 1 Column: See Table 1.
Carbon-Chain Number of Percentage
Length Double Bonds (%) Table 1
<14 0 <0.1 Hold Time at|
14 0 50.5 Initial Temperature Final Final
16 0 9-16 Temperature Ramp Temperature | Temperature
18 0 79-89 ©) (¢/min) Cc) (min)
20 0 <1.0 60 5.9 290 1

22 9 1.0 Carrier gas: Helium

18 1 <4.0 Flow rate: 1.7 mL/min
18 2 <1.0 Injection volume: 1 uL
18 3 <0.2 Injection type: Split injection. The split ratio is about
12:1,
e FATS AND FIXED OlLS, Peroxide Value (401): NMT 5.0 Liner: General purpose split/splitless liner with deacti-
FATS AND FIXED OILS, Unsaponifiable Matter (401) vated wool
Sample: 5.0g System suitability
Acceptance criteria: NMT 1.0% Samples: Standard solution and System suitability
e WATER DETERMINATION, Method | (921): NMT 0.3% solution
[Note—The relative retention times for methyl erucate
ADDITIONAL REQUIREMENTS andpaualane are 0.9 and 1.0, respectively.]
¢ PACKAGING AND STORAGE: Preserve in tight, light-resistant Suitability requirement
containers. No storage requirements are specified. Relative standard deviation: NMT 2%, Standard
solution
Resolution: NLT 5 between the peaks due to methyl
erucate and squalane, System suitability solution
Analysis
Squalane Samples: Standard solution and Sample solution
Calculate the percentage of squalane (C3oH.z) in the
portion of Squalane taken:
HgC. REPRO
Teoetla cas iCH Result = (ru/rs) x (Cs/Cu) x 100

Tu = peak response from the Sample solution

C3oHe2 422.81 Is =peak response from the Standard solution
Tetracosane, 2,6,10,15,19,23-hexamethyl-; Cs = concentration of USP SqualaneRS in the
2,6,10,15,19,23-Hexamethyltetracosane [111-01-3]. Standard solution (m inl)
Cu = concentration of Squalane in the Sample
DEFINITION solution (mg/mL)
Squalaneis a saturated eee obtained by hydrogen- Acceptance criteria: 97.0%-102.0%
ation of squalene,analiphatic triterpene occurring in
some fish oils. It contains NLT 97.0% and NMT 102.0% IMPURITIES
of 2,6,10,15,19,23-hexamethyltetracosane (C3oHea). e RESIDUE ON IGNITION (281): NMT 0.5%

IDENTIFICATION SPECIFIC TESTS

e A. INFRARED ABSORPTION (197F) SPECIFIC GRAVITY (841): 0.807-0.810 at 20°
Sample: Undried specimen FATS AND FIXED OILS, Acid Value (401): NMT 0.2
Eatrel-s orelerolNPETN

Acceptance criteria: Meets the requirements FATS AND FIXED OILS, /odine Value (401): NMT 4
e B, CHROMATOGRAPHIC IDENTITY FATS AND FIXED OILS, Saponification Value (401): NMT 2
Analysis: Proceed as directed in the Assay. REFRACTIVE INDEX (831): 1.4510-1.4525 at 20°
Acceptance criteria: The retention time of the major ADDITIONAL REQUIREMENTS
peak of the Sample solution, excluding the solvent peak, © PACKAGING AND STORAGE: Preserve in tight containers.
corresponds to that of the Standard solution. e USP REFERENCE STANDARDS (11)
USP Squalane RS
 5594 Stannous / Official Monographs
Stannous Chloride
SnClz - 2H20 225.65
Tin chloride (SnClz) dihydrate [10025-69-1].
DEFINITION
Stannous Chloride contains NLT 98.0% and NMT 102.0%
of stannous chloride (SnCl2z- 2H20).
IDENTIFICATION
oA.
Sample solution: To 0.40 g of Stannous Chloride add
1 mL of dilute hydrochloric acid solution (236 mL/L of
hydrochloric acid), and dilute with water to 20 mL.
[Note—Keep a portion for the Limit of Sulfate test.]
Analysis: To 1 mL of the Sample solution add a mixture
of 5 mL of water and 0.05 mL of mercuric chloride TS.
Acceptance criteria: A blackish-gray precipitate forms.
° B.
Sample: 1.0g
Analysis 1: Dissolve the Sample in 3.0 mL of water. Add
0.5 mL of dilute sodium myers solution (85 mg/mL
of sodium hydroxide) to the cloudy solution.
Acceptance criteria 1: A yellowish, flocculent precipi-
tate is formed.
Analysis 2: Add 6.5 mL of water to the solution result-
ing from nee 1. To 1.0 mL of the previously shaken
suspension add 1.0 mL of sodium hydroxide solution
(420 mg/mL of sodium hydroxide).
Acceptance criteria 2: The precipitate dissolves, and
the resulting solution is clear and colorless.
© C. IDENTIFICATION TESTS—GENERAL, Chloride (191)
Sample solution: Dissolve 10 mg of Stannous Chloride
in 2 mL of 20% nitric acid.
Acceptance criteria: Meets the requirements
ASSAY
e PROCEDURE
Sample: 0.1 g of Stannous Chloride
Titrimetric system
(See Titrimetry (541).)
Mode: Direct titration
Titrant: 0.1 N iodine VS
Blank: 50 mL of water
Endpoint detection: Colorimetric
Analysis: Dissolve the Sample in 50 mL of water freed
from oxygen by purging with carbon dioxide or nitro-
gen for 15 min prior to the addition. Add 1.5 mL of 0.8
N hydrochloric acid, 5 g of potassium sodium tartrate,
10 g of sodium bicarbonate, and 1 mL of starch TS. Ti-
trate the resulting solution immediately with Titrant.
Perform a blank determination.
Calculate the percentage of stannous chloride (SnCl2
- solution add 3 mL of sodium hydroxide solution
2H20) in the Sample:
Result = {[(Vs — Vs) x N x F]/W} x 100
Vs = Titrant consumed by the Sample (mL)
Ve = Titrant consumed by the Blank (mL)
N = actual normality of the Titrant (mEq/mL)
F = equivalency factor, 112.8 mg/mEq
Ww = Sample weight (mg)
Acceptance criteria: 98.0%-102.0%
NF Monographs
IMPURITIES
e LIMIT OF SULFATE
Acetic acid solution: Dilute 30 mL of glacial acetic acid
with water to 100 mL.
30% Alcohol: Dilute 30 mL of alcohol with water to
100 mL.
Potassium sulfate solution 1: 1.8 mg/mL of potassium
sulfate in 30% Alcohol. Immediately before use, dilute
with 30% Alcohol to obtain a solution having a known
concentration of about 18 g/mL.
NF 36
Potassium sulfate solution 2: 1.8 mg/mL of potassium
sulfate in water. Immediately before use, dilute with
water to obtain a solution having a known concentra-
tion of about 18 pg/mL.
Standard solution: Mix 3 mL of barium chloride solu-
tion (250 mg/mL) and 4.5 mL of Potassium sulfate solu-
tion 1. Shake, and let stand for 1 min. To 2.5 mL of this
solution add 15 mL of Potassium sulfate solution 2 and
0.5 mL of Acetic acid solution. Allow to stand for 5 min.
Sample solution: Use 15 mL of the solution prepared in
Identification test A.
Analysis: Mix 3 mL of barium chloride solution
(250 mg/mL) and 4.5 mL of Potassium sulfate solution 1.
Shake, and let stand for 1 min. To 2.5 mL of this solu-
tion add the Sample solution and 0.5 mL of Acetic acid
solution. Allow to stand for 5 min.
Acceptance criteria: Any opalescence in the Sample so-
lution is not more intense than that in the Standard so-
lution (500 ppm).
¢ LIMIT OF IRON
Standard iron solution: Prepare as directed in /ron
(241), Special Reagents.
Standard solution: Immediately before use, dilute 1 mL
of Standard iron solution with water to 10 mL. This solu-
tion contains the equivalent of 1 g/mL of iron.
Sample solution: Dilute 5 mL of the Sample solution,
prepared as directed in Substances Not Precipitated by
Thioacetamide, with water to 10 mL.
Analysis: To 10 mL each of the Standard solution and
the Sample solution add 2 mL of dilute citric acid
(200 mg/mL) and 0.1 mL of thioglycolic acid, and mix.
Make alkaline with a dilute ammonia solution (620 mL/
L of ammonium hydroxide). Dilute with water to
20 mL. Allow the treated Standard solution and the
treated Sample solution to stand for 5 min.
Acceptance criteria: Any pink color in the Sample solu-
tion is not more intense than that in the Standard solu-
tion (100 ppm).
o Limit oF LEAD
Dilute thioacetamide solution: [NoTE—Prepare imme-
diately before use.] To 0.2 mL of thioacetamide TS add
1 mL of a mixture of 5 mL of water, 15 mL of 1 N so-
dium hydroxide, and 20 mL of 85% glycerin. Heat in a
water bath for 20 s.
Standard solution: On the day of use, mix 1.0 mL of
standard lead solution TS, 6 mL of water, 3 mL of so-
dium hydroxide solution (420 mg/mL of sodium hy-
droxide), and 0.5 mL of Dilute thioacetamide solution.
Sample solution: Dissolve 1.0 g of Stannous Chloride in
2 mL of a mixture of nitric acid and hydrochloric acid
(1:3). Heat the solution on a water bath until nitrous
vapor is no longer evolved. Dissolve the residue in
water, and dilute with water to 25 mL. To 5 mL of this
(420 mg/mL of sodium hydroxide) and 2 mL of water.
Heat until a clear solution is obtained, and cool. Add
0.5 mL of Dilute thioacetamide solution, and allow to
stand for 2 min.
Analysis: Compare the Standard solution and the Sam-
ple solution.
Acceptance criteria: Any color in the Sample solution is
not more intense than that in the Standard solution
(50 g/g).
SPECIFIC TESTS
© APPEARANCE OF SOLUTION
Standard stock solution: Pipet 30.0 mL of ferric chlo-
ride CS, 30.0 mL of cobaltous chloride CS, and 24.0 mL
of cupric sulfate CS into a 100-mL volumetric flask. Di-
lute with 1% (w/v) hydrochloric acid to volume.
Standard solution: Dilute 1.0 mL of the Standard stock
solution with 1% (w/v) hydrochloric acid to 100 mL.
Sample solution: Dissolve 10.0 g of Stannous Chloride
in dilute hydrochloric acid solution, and dilute with di-
lute hydrochloric acid solution to 20 mL.
NF 36
Acceptance criteria: The Sample solution is clear and
colorless, or if not, not more intensely colored than the
Standard solution.
© SUBSTANCES NOT PRECIPITATED BY THIOACETAMIDE
Sample solution: Dissolve 1.0 g of Stannous Chloride in
dilute hydrochloric acid solution, and dilute with the
same acid to 30 mL. Heat to boiling. Add 30 mL of thi-
oacetamide TS, and boil for 15 min to produce Solution
A. Filter 5 mL of Solution A, and heat the filtrate to boil-
ing. Add 5 mL of thioacetamide TS, and boil for 15
min. If a precipitate is formed, add the remainder of
Solution A to the mixture to produce Solution Al. Add
10 mL of thioacetamide TS, and boil. Repeat the series
of operations from “Filter 5 mL” until a precipitate is no
longer formed on addition of thioacetamide TS to the
filtrate obtained from the 5 mL of Solution A (Solution
Al, Solution A2, and so on, respectively). If no precipi-
tate is formed, or if no more precipitate is formed,
combine the solution obtained with the remainder of
Solution A (Solution A1, Solution A2, and so on, respec-
tively), filter, and wash the precipitate with 10 mL of
water. Heat the filtrate until the resulting vapor no
longer turns a moistened piece of lead acetate test pa-
per blackish-gray. Allow to cool, and dilute with water
to a mL. [NoTeE—Keep a portion for the Limit of Iron
test.
Analysis: Evaporate 25 mL of the Sample solution to
dryness, and ignite at 600°.
Acceptance criteria: The residue weighs NMT 1 mg
(0.2%).
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in well-closed contain-
ers. No storage requirements specified.
Corn Starch
Portions of the monograph text that are national USP text,
and are not part of the harmonized text, are marked with
symbols (%) to specify this fact.
DEFINITION
Corn Starch consists of the starch granules separated from
the mature grain of corn [Zea mays L. (Fam. Gramineae)].
IDENTIFICATION
oA.
Analysis: Examine under a microscope, using a mixture
of glycerin and water (1:1) as a mounting agent.
Acceptance criteria: It aape as either as angular poly-
hedral granules of irregular sizes with diametersranging
from 2-23 um, or as rounded or spheroidal granules o
irregular sizes with diameters ranging from 25-35 um.
The central hilum consists of a distinct cavity or two- to
five-rayed cleft, and there are no concentric striations.
Between orthogonally oriented polarizing plates or
prisms, the starch granules showa distinct black cross
intersecting at the hilum.
° B.
Sample solution: 20 mg/mL in water
Analysis: Boil for 1 min, and cool.
Acceptance criteria: A thin, cloudy mucilage is formed.
eC.
Sample solution: 1 mL of the mucilage obtained in
Identification test B
Analysis: Add 0.05 mL of iodine and potassium iodide
TS 2 to the Sample solution.
Acceptance criteria: An orange-red to dark blue color
is produced, which disappears upon heating.
Official Monographs / Starch 5595
IMPURITIES
© RESIDUE ON IGNITION (281)
Sample: 1.0g
Acceptance criteria: NMT 0.6%
LimIT OF IRON
Standard iron stock solution A: Equivalent to 10 g/
mL of iron prepared as directed in Iron (241)
Standard iron stock solution B: 1 1g/mL of iron from
Standard iron stock solution A in water
[Note—Prepare immediately before use.]
Standard iron solution: Transfer 10 mL of Standard iron
stock solution B to a test tube, and add 2 mL of citric
acid solution (2 in 10) and 0.1 mL of thioglycolic acid.
Add 10 N ammonium hydroxide until the solution is
distinctly alkaline to litmus, and dilute with water to
20 mL.
Sample solution: Shake 1.5 g of Corn Starch with
15 mL of 2.N hydrochloric acid, and filter. Transfer
10 mL of the filtrate to a test tube, add 2 mL of citric
acid solution (2 in 10), and 0.1 mL of thioglycolic acid.
Add 10 N ammonium hydroxide until the solution is
distinctly alkaline to litmus, and dilute with water to
20 mL.
Acceptance criteria: After 5 min, any pink color in the
Sample solution is not more intense than that in the
Standard iron solution, corresponding to a limit of
10 ppm of iron.
e LIMIT OF SULFUR DIOXIDE
Carbon dioxide: Use carbon dioxide, with a flow regu-
lator that will maintain a flow of 100 +10 mL/min.
Bromophenol blue indicator solution: 0.2 mg/mL of
bromophenol blue in dilute alcohol. Filter if necessary.
Hydrogen peroxide solution: Dilute 30% hydrogen
pas with water to obtain a 3% solution. Just
efore use, add 3 drops of Bromophenol blue indicator
solution, and neutralize to a violet-blue endpoint with
0.01 N sodium hydroxide. Do not exceed the endpoint.
Apparatus: See Figure 1.
sydeibouo-= 4N
Figure 1
In this test, the sulfur dioxide is released from the sam-
ple in a boiling acid medium and is removed by a
stream of carbon dioxide. The separated gas is col-
lected in a dilute hydrogen peroxide solution where
the sulfur dioxide is oxidized to sulfuric acid and ti-
trated with standard alkali. The apparatus consists es-
sentially of a 500-mL three-neck, round-bottom boiling
flask, A; a separatory funnel, B, having a capacity of
 5596 Starch / Official Monographs NF 36

100 mL or greater; a gas inlet tube of sufficient length it meets the requirements of the test for the absence of
to permit introduction of the carbon dioxide within Escherichia coli. *Where it is intended for use in preparing
2.5 cm of the bottom of the boiling flask; a reflux con- Absorbable Dusting Powder, it also meets the require-
denser, C, having a jacket length of 200 mm, and a ments of the tests for absence of Staphylococcus aureus
delivery tube, E, connecting the upper end of the re- and Pseudomonas aeruginosa.»
flux condenser to the bottom of a receiving test tube, e Loss ON DRYING (731)
D. Applya thin film of stopcock grease to the sealing Sample: 1
surfaces of all of the joints except the joint between Analysis: Dry the Sample at 130° for 90 min.
the separatory funnel and the boiling flask, and clamp Acceptance criteria: NMT 15.0%
the joints to ensure tightness. e PH (791)
Samale: 25.0 g of Corn Starch Sample solution: Preparea slurry by weighing 5.0g of
Analysis: Add 150 mL of water to the boiling flask. Corn Starch, transferring to a suitable nonmetallic con-
Close the stopcock of the separatory funnel, and begin tainer, and adding 25.0 mL of freshly boiled and cooled
the flow of carbon dioxide at a rate of 100+ 5 mL/min water.
through the Apparatus. Start the condenser coolant Analysis: Apttale continuously at a moderate rate for 1
flow. Add 10 mL of Hydrogen peroxide solution to a re- min. Stop the agitation, and allow to stand for 15 min.
ceiving test tube. After 15 min, without interrupting the Determine the pH to the nearest 0.1 unit.
flow of carbon dioxide, remove the separatory funnel Acceptance criteria: 4.0-7.0
from the boiling flask, and transfer the Sample into the
boiling flask with the aid of 100 mL of water. Apply ADDITIONAL REQUIREMENTS
stopcock grease to the outer joint of the separatory fun- ¢ *PACKAGING AND STORAGE: Preserve in well-closed con-
nel, and replace the separatory funnel in the boiling tainers. No storage requirements specified.
flask. Close the stopcock of the separatory funnel, and e LABELING: Where Corn Starch is intended for use in pre-
add 80 mL of 2 N hydrochloric acid to the separatory paring Absorbable Dusting Powder, it is so labeled, and
funnel. Open the stopcock of the separatory funnel to the label states that it must be subjected to further pro-
pers the hydrochloric acid solution to flow into the cessing during the preparation of Absorbable Dusting
oiling flask, guarding against the escape of sulfur diox- Powder.¢
ide into the separatory funnel by closing the stopcock
before the last few mL of hydrochloric acid drain out.
Boil the mixture for 1 h. Remove the receiving test
tube, and transfer its contents to a 200-mL wide-
necked, conical flask. Rinse the receiving test tube with Hydroxypropyl Corn Starch
a small portion of water, add the rinsing to the 200-mL
conical flask, and mix. Heat on a water bath for 15 Amylose derivative. oH
min, and allow to cool. pr R=Hor “N
Add 0.1 mL of Bromophenol blue indicator solution, and one Hy
titrate the contents with 0.1 N sodium hydroxide VS
until the color changes from yellow to violet-blue. Per- HO
0,
dtOH
sae idee
form a blank determination, and make any necessary ad onl Aner cerivain ( pr
correction (see Titrimetry (541)). fe OH 0
Calculate the content, in ppm, of sulfur dioxide in the o HO.
< ton
ei
ReHor “NO Noy,
Sample taken:
a
Result
= (Vx N x F)/W
x 1000
For the Amylose derivative, m is about 300-1000.
Vv = volume of titrant consumed (mL)
N = normality of the titrant DEFINITION
F = milliequivalent weight of sulfur dioxide, 32.03 Reino Corn Starch is partially substituted 2-hydrox-
w = weight of the Sample (g) ypropylether obtained from corn starch by a chemical
Acceptance criteria: NMT 50 ppm modification of etherification withpeo lene oxide. In ad-
e LIMIT OF OXIDIZING SUBSTANCES dition, this starch may be partially hydrolyzed using acids
Sample solution: Transfer 4.0 g to a glass-stoppered, or enzymes to obtain thinned starch. It contains NLT
125-mL conical flask, and add 50.0 mL of water. Insert 2.0% and NMT 7.0% of hydroxypropyl groups on the
the stopper, and swirl for 5 min. Transfer to a glass- dried basis.
stoppered, 50-mL centrifuge tube, and centrifuge to
clay. Transfer 30.0 mL of the clear supernatant to a IDENTIFICATION
glass-stoppered, 125-mL conical flask. Add 1 mL of gla- ¢ A. PROCEDURE
cial acetic acid and 0.5-1.0 g of potassium iodide. In- Analysis: Examine under a microscope, using NLT 20x
sert the stopper, swirl, and allow to stand for 25-30 magnification and a mixture of glycerin and water (1:1)
min in the dark, Add 1 mL of starch TS. as a mounting agent.
Analysis: Titrate with 0.002 N sodium thiosulfate VS to Acceptance criteria: It presents either as angular pols
the disappearance of the starch-iodine color. Perform a hedral granules of irregular sizes with diameters of 2-23
blank determination, and make any necessary correc- um, or as rounded or spheroidal granules of irregular
tion. Each mL of 0.002 N sodium thiosulfate is equiva- sizes with diameters of 25-35 «um. The central hilum
al
R= lent to 34 wg of oxidant, calculated as hydrogen consists of a distinct cavity or 2- to 5-rayed cleft, and
oe peroxide. there are no concentric striations. Between crossed nicol
Ss
— Acceptance criteria: NMT 1.4 mL of 0.002 N sodium prisms, the Hydroxypropyl Corn Starch granules show a
im) thiosulfate is required (20 ppm, calculated as H2O2). distinct black cross intersecting at the hilum.
} e B. PROCEDURE
c SPECIFIC TESTS Sample solution: Suspend 1 g of Hydroxypropyl Corn
° MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI- Starch in 50 mL of water, boil for 1 min, and cool.
= FIED MICROORGANISMS (62): The total aerobic microbial Acceptance criteria: A translucent or clear mucilage is
I
count does not exceed 103 cfu/g; the total combined formed.
Fa molds and yeasts count does not exceed 10? cfu/g; and
NF 36
e C. PROCEDURE
Analysis: To 1 mL of the Sample solution obtained in
Identification test B add 0.05 mL of iodine and potas-
sium iodide TS 2.
Acceptance criteria: An orange-red to dark blue color
is produced, which disappears upon heating.
e D. PROCEDURE
Ninhydrin solution: Dissolve 3 g of ninhydrin in
100 mL of a 45.5-g/L solution of sodium metabisulfite.
Diluted sulfuric acid: 98 g/L of H2SO4
Sample: 100mg of Hydroxypropyl Corn Starch
Analysis: Transfer the Sample to a 100-mL volumetric
flask, and add 12.5 mL of Diluted sulfuric acid. Place the
flask in a water bath, and heat until the Sample is dis-
solved. Cool, and dilute with water to 100 mL. [Cau-
Tion—When sulfuric acid is miscible with water, it pro-
duces intense heat.]
Pipet 1 mL of this solution to a glass-stoppered, 25-mL
graduated test tube and, with the tube immersed in
cold water, add drop-wise 8 mL of sulfuric acid. Mix
well, and place the tube in a boiling water bath for
exactly 3 min. Immediately transfer the tube to an ice
bath until the solution is chilled. Add 0.6 mL of
Ninhydrin solution, carefully allowing the reagent to
run down the walls of the test tube. Immediately
shake the tube well, and place it in a water bath at
25° for 100 min. Dilute with sulfuric acid to 25 mL
[CauTioN—Use sulfuric acid cautiously.], and mix by
inverting the tube several times. Do not shake.
Acceptance criteria: A violet color develops within 5
min due to the presence of hydroxypropyl groups
(starch ether).
ASSAY
© PROCEDURE FOR HYDROXYPROPYL GROUPS
Deuterium chloride solution: Dilute 1 mL of deuterium
chloride (38% w/w) with 5 mL of deuterium oxide.
Internal standard solution: Disperse 50.0 mg of so-
dium 3-trimethylsilyl-1-propane sulfonate in about 5 g
of deuterium oxide, weighed to the nearest 0.1 mg.
Store in a sealed bottle.
Sample solution: Disperse 20 g of Hydroxypropyl Corn
Starch in 200.0 mL of carbon dlantde ftee water at
room temperature. Agitate for 15 min, and filter. Re-
peat the operation two more times. If poor dispersibility
or slow filtration is observed, use refrigerated carbon
dioxide-free water for the washing operation. Dry the
washed starch for NLT 4 h in vacuum at 30+ 5°. Deter-
mine the moisture content (B) on 5 g of the washed
and dried starch following the Loss on Drying test.
Weigh 12.0 mg of the washed and dried starch in a
S-mm NMR tube. Add 0.75 mL of deuterium oxide and
0.1 mL of Deuterium chloride solution. Cap the tube,
mix, and place it in a boiling water bath until a clear
solution is obtained. [NoTE—This may take 3 min to 1
h.] Whena clear solution is obtained, allow to cool to
room temperature. Dry the exterior of the tube, and
weigh to the nearest 0.1 mg. Add 0.05 mL of Internal
Standard solution, and weigh to the nearest 0.1 mg. De-
termine the mass of the Internal standard solution
added. Mix thoroughly.
Nuclear magnetic resonance spectrometry
(See Nuclear Magnetic Resonance Spectroscopy (761),
Quantitative Applications.)
Apparatus: FT-NMR spectrometer at minimum
300 MHz
Acquisition of 'H NMR spectra: The following param-
eters may be used.
Official Monographs / Starch 5597
Sweep width: 8 ppm (about —1.0 to +7 ppm)
Irradiation frequency offset: None
Time domain: NLT 64 K
Pulse width: 90 degree
Pulse delay: 10s
Dummy scans: 0
Number of scans: 8
Use the CH; signal of the internal standard for shift
referencing. Set the shift of the peak of the singlet to
0 ppm. Record the FID signal.
Analysis
Samples: Internal standard solution and Sample solution
Call the integration sub-routine after phase corrections
and baseline correction between —0.5 and +6 ppm.
Measure the peak areas of the doublet from the methyl!
roups of the hydroxypropyl function at +1.2 ppm
‘A2), and of the methyl groups at 0 ppm of the inter-
nal standard (A:) without 13C-satellites.
Measure the signal coming from the 3 protons of the
methyl group in the hydroxypropyl function.
Calculate the content of hydroxypropyl groups as a per-
centage (w/w, dried basis):
Result = (N x A2/Ai) x (Ci x Wi/W) x (Mi2/Mn) x [100/
(100 — B)} x 100
N = numerical value representing the 3 methyl
groups in the internal standard (sodium
3-trimethylsilyl-1-propane sulfonate), 3
Az = area of the methyl groups of hydroxypropyl in
Hydroxypropyl Corn Starch
A = area of the methyl groups in the internal
standard (sodium 3-trimethylsilyl-1-propane
sulfonate)
G = concentration of the internal standard in the
Internal standard solution (mg/g)
Wi = weight of the Internal standard solution in the
NMR tube (g)
Ww = weight of the washed and dried
icronrpnapy Corn Starch in the NMR tube
mg)
Ma = molecular weight of the internal standard,
218.32 g/mol
M2 = ices mass of hydroxypropyl group, 59.09 g/
mo
B = moisture content of the washed and dried
Hydroxypropyl Corn Starch used in the
Sample solution, as a percentage (w/w)
Acceptance criteria: The content of hydroxypropyl
groups is 2.0%-7.0% on the dried basis.
IMPURITIES
Inorganic Impurities
e RESIDUE ON IGNITION (281): NMT 0.6%, determined on a
1.0-g test specimen
¢ LIMIT OF IRON
Standard iron stock solution: Prepare a solution con-
taining the equivalent of 10 g/mL of iron, as directed
under Iron (241).
Diluted standard iron solution: Immediately before
use, dilute an accurately measured volume of Standard
iron stock solution quantitatively with water to obtain a
solution containing the equivalent of 1 g/mL of iron.
Standard solution: Transfer 10 mL of the Diluted stan-
dard iron solution to a test tube. Add 2 mL of citric acid
sydeibouow 4N
solution (2 in 10) and 0.1 mL of thioglycolic acid, and
mix. Add 10 N ammonium hydroxide until the solution
is distinctly alkaline to litmus, dilute with water to
20 mL, and mix.
Sample solution: Shake 1.0 g of Hydroxypropyl Corn
Starch with 20 mL of 2 N hydrochloric acid, and filter.
Transfer 10 mL of the filtrate to a test tube. Add 2 mL
of citric acid solution (2 in 10) and 0.1 mL of thiogly-
colic acid, and mix. Add 10 N ammonium hydroxide
 5598 Starch / Official Monographs
until the solution is distinctly alkaline to litmus, dilute
with water to 20 mL, and mix.
Acceptance criteria: After 5 min, any pink color in the
Sample solution is not more intense than that in the
soe solution, corresponding toa limit of 20 g/g
of iron.
e Limit OF SULFUR DIoxIDE, Method IV (525): NMT 50 ppm
Organic Impurities
¢ PROCEDURE 1: LIMIT OF OXIDIZING SUBSTANCES
Sample: 4.0 g of Hydroxypropyl Corn Starch
Analysis: Transfer the Sample to a glass-stoppered,
125-mL conical flask, and add 50.0 mL of water. Insert
the stopper, and swirl for 5 min. Transfer to a glass-
stoppered, 50-mL centrifuge tube, and centrifuge to
clarify. Transfer 30.0 mL of the clear supernatant to a
glass-stoppered, 125-mL conical flask. Add 1 mL of gla-
cial acetic acid and 0.5—-1.0 g of potassium iodide. In-
sert the stopper, swirl, and allow to stand for 25-30
min in the dark. Add 1 mL of starch TS, and titrate
with 0.002 N sodium thiosulfate VS to the disappear-
ance of the starch-iodine color. Perform a blank deter-
mination, and make any necessary correction. Each mL
of 0.002 N sodium thiosulfate VS is equivalent to 34 ug
of oxidant, calculated as hydrogen peroxide.
Acceptance criteria: NMT 1.4 mL of 0.002 N sodium
thiosulfate VS is required (20 g/g, calculated as H2O2).
¢ PROCEDURE 2: FOREIGN MATTER
Sample: 50 mg/mL of Hydroxypropyl Corn Starch in a
mixture of glycerin and water (1:1)
Analysis: Examine under a microscope, using NLT 20x
magnification and a mixture of glycerin and water
(1:1) as a mounting agent.
Acceptance criteria: NMT traces of matter other than
Hydroxypropyl Corn Starch granules are present.
SPECIFIC TESTS
e MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI-
FIED MICROORGANISMS (62): The total aerobic microbial
count does not exceed 103 cfu/g, and the total com-
bined molds and yeasts count does not exceed 10? cfu/
g. It meets the requirements of the test for the absence
of Escherichia coli.
e PH (791)
Sample solution: Suspend 5.0 g of Hydroxypropyl Corn
Starch in 25.0 mL of carbon dioxide-free water, and
shake for 60 s. Allow to stand for 15 min.
Acceptance criteria: 4.5-8.0
e Loss ON DRYING (731): Dry about 1 g at 130° for 90 min:
it loses NMT 15.0% of its weight.
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in well-closed contain-
ers. Store at room temperature.
Pregelatinized Hydroxypropyl! Corn
Starch
DEFINITION
Pregelatinized Hydroxypropyl Corn Starch is prepared from
Hydroxypropy! Corn Starch by mechanical processing in
the presence of water, with or without heat, to rupture all
NF Monographs
or some of the starch granules, and is subsequently dried.
It contains NLT 2.0% and NMT 7.0% of hydroxypropyl
groups on the dried basis.
IDENTIFICATION
e A. TEST FOR PREGELATINIZED STATE
Sample: 1g
Analysis: Disperse the Sample in 50 mL of water at a
temperature NMT 25°. Shake vigorously until lumps
completely disperse/solubilize or until lumps disappear.
Allow to stand for 20 min.
NF 36
Acceptance criteria: A translucent or clear mucilage
without precipitate is formed.
o B. TEST FOR STARCH
Analysis: Disperse 0.5 g in 2 mL of water without heat-
ngs and add 0.05 mL of iodine and potassium iodide
Acceptance criteria: A reddish-violet or blue color is
produced.
¢ C. NINHYDRIN TEST
Ninhydrin solution: Dissolve 3 g of ninhydrin in
100 mL of a 45.5-g/L solution of sodium metabisulfite.
Diluted sulfuric acid: 98 g/L of H2SO4
Sample: 100mg
Analysis: Transfer the Sample to a 100-mL volumetric
flask, and add 12.5 mL of Diluted sulfuric acid. Place the
flask in a water bath, and heat until the Sample is dis-
solved. Cool, and dilute with water to 100 mL. [Cau-
TIoN—When sulfuric acid is miscible with water, it pro-
duces intense heat.]
Pipet 1 mL of this solution to a glass-stoppered 25-mL
graduated test tube and, with the tube immersed in
cold water, add dropwise 8 mL of sulfuric acid. Mix
well, and place the tube in a boiling water bath for
exactly 3 min. Immediately transfer the tube to an ice
bath until the solution is chilled. Add 0.6 mL of
Ninhydrin solution, carefully allowing the reagent to
run down the walls of the test tube. Immediately
shake the tube well, and place it in a water bath at
25° for 100 min, Dilute with sulfuric acid to 25 mL.
[CauTion—Use sulfuric acid cautiously.] Mix by in-
verting the tube several times. Do not shake.
Acceptance criteria: A violet color develops within 5
min due to the presence of hydroxypropyl groups
(starch ether).
ASSAY
e ASSAY FOR HYDROXYPROPYL GROUPS
Deuterium chloride solution: Dilute 1 mL of deuterium
chloride (38% w/w) with 5 mL of deuterium oxide.
Internal standard solution: Disperse 50.0 mg of so-
dium 3-trimethylsilyl-1-propane sulfonate in about 5 g
of deuterium oxide, weighed to the nearest 0.1 mg.
Store in a sealed bottle.
Sample solution: Determine the moisture content (8)
of 5 g of Pregelatinized Hydroxypropyl Corn Starch fol-
lowing the Loss on Drying test. Weigh 12.0 mg of Pre-
gelatinized Hydroxypropyl Corn Starch in a 5-mm NMR
tube. Add 0.75 mL of deuterium oxide and 0.1 mL of
Deuterium chloride solution. Cap the tube, mix, and
place it in a boiling water bath until a clear solution is
obtained. [NoteE—This may take from 3 min to 1 h.]
Whena clear solution isobtained, allow it to cool to
room temperature. Dry the exterior of the tube, and
weigh to the nearest 0.1 mg. Add 0.05 mL of Internal
standard solution. Weigh to the nearest 0.1 mg. Deter-
mine the mass of the Internal standard solution added.
Mix thoroughly.
Instrumental conditions
(See Nuclear Magnetic Resonance Spectroscopy (761),
Quantitative Applications.)
Mode: Nuclear magnetic resonance spectrometry
Apparatus: FT-NMR spectrometer at minimum
300 MHz
Acquisition of 'H NMR spectra: The following param-
eters may be used:
Sweep width: 8 ppm (about -1.0 to +7 ppm)
Irradiation frequency offset: None
Time domain: NLT 64 K
Pulse width: 90°
Pulse delay: 10s
Dummy scans: 0
Number of scans: 8
Use the CH3 signal of the internal standard for shift
referencing. Set the shift of the peak of the singlet to
0 ppm. Record the FID signal.
NF 36
Analysis
Samples: Internal standard solution and Sample solution
Call the integration subroutine after phase corrections
and baseline correction between —0.5 and +6 ppm.
Measure the peak areas of the doublet from the methyl
roups of the hydroxypropyl function at +1.2 ppm
tA), and of the methyl groups at 0 ppm of the inter-
nal standard (A;) without 13C-satellites.
Measure the signal originating from the 3 protons of
the methyl group in the hydroxypropyl function.
Calculate the content of hydroxypropyl groups as a per-
centage (w/w, dried basis):
Result = (N x A2/A1) x (Gx Wi/W) x (Mr2/Mr) x [100/
(100 — B)] x 100
N = numerical value representing the 3 methyl
groups in the internal standard (sodium
3-trimethylsilyl-1-propane sulfonate), 3
A2 = area of the methyl groups of hydroxypropyl in
Pregelatinized Hydroxypropyl Corn Starc!
Ai = area of the methyl! groups in the internal
standard (sodium 3-trimethylsilyl-1-propane
sulfonate)
G = concentration of the internal standard in the
Internal standard solution (mg/g)
Ww, = weight of the Internal standard solution in the
NMR tube (g)
Ww = weight of the Pregelatinized Hydroxypropyl
Corn Starch in the NMR tube nig
M2 = i mass of hydroxypropyl groups, 59.09 g/
mol
Mn = molecular weight of the internal standard,
218.32 g/mo
B = moisture content of the Pregelatinized
Hydroxypropy! Corn Starch used in the
Sample solution, as a percentage (w/w)
Acceptance criteria: 2.0%-7.0% of hydroxypropyl
groups on the dried basis
IMPURITIES
© RESIDUE ON IGNITION (281): NMT 0.6%, determined on a
1.0-g test specimen
e LIMIT OF IRON
Standard iron stock solution: Prepare a solution con-
taining the equivalent of 10 ug/mL of iron, as directed
under Iron (241).
Diluted standard iron solution: Immediately before
use, dilute an accurately measured volume of the Stan-
dard iron stock solution quantitatively with water to ob-
tain a solution containing the equivalent of 1 g/mL of
iron.
Sample solution: Shake the residue obtained from the
test for Residue on Ignition with 20 mL of 2 N hydro-
chloric acid, and filter. Transfer 10 mL of the filtrate to a
test tube. Add 2 mL of citric acid solution (2 in 10) and
0.1 mL of thioglycolic acid, and mix. Add 10 N ammo-
nium hydroxide until the solution is distinctly alkaline to
litmus, dilute with water to 20 mL, and mix.
Standard solution: Transfer 10 mL of the Diluted stan-
dard iron solution to a test tube. Add 2 mL of citric acid
solution (2 in 10) and 0.1 mL of thioglycolic acid, and
mix. Add 10 N ammonium hydroxide until the solution
is distinctly alkaline to litmus, dilute with water to
20 mL, and mix.
Acceptance criteria: After 5 min, any pink color in the
Sample solution is not more intense than that in the
Standard solution, corresponding to a limit of 20 ppm of
iron.
e LIMIT OF SULFUR DIOXIDE, Method IV (525): NMT 50 ppm
© LIMIT OF PROPYLENE GLYCOL
Internal standard solution: 0.5 mg/mL of 1,3-
propanediol in anhydrous pyridine
Standard stock solution: 0.5 mg/mL of USP Propylene
Glycol RS in Internal standard solution
Official Monographs / Starch 5599
Standard solution: Transfer 0.1 mL of the Standard
stock solution to a 2-mL vessel with a screw cap fitted
with a septum. Add 0.9 mL of anhydrous pyridine,
0.2 mL of hexamethyldisilazane, and 0.1 mL of trimeth-
ylchlorosilane. Close, and mix. Allow to stand for 15
min before injection.
Sample stock solution: Transfer 200 mg of Pregelati-
nized Hydroxypropyl Corn Starch to a 100-mL volumet-
ric flask. Add 1.0 mL of the Internal standard solution
and 9.0 mL of anhydrous pyridine. Boil under reflux us-
ing a water bath for 20 min. Allow to cool to room
temperature.
Sample solution: Transfer 1.0 mL of the Sample stock
solution to a 2-mL vessel with a screw cap fitted with a
septum. Add 0.2 mL of hexamethyldisilazane and
0.1 mL of trimethylchlorosilane. Close, and mix. Allow
to stand for 15 min before injection.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: GC
Detector: Flame ionization
Column: 0.32-mm x 30-m fused-silica capillary col-
umn; 0.25-uum layer of phase G1
Temperature
Detector: 250°
Injection port: 250°
Column: 70°. [NoTE—The column must be desorbed
regularly. Conditions: Program from 70° to 300° at
7°/min, and maintain 10 min at 300°.]
Carrier gas: Helium
Flow rate: 3 mL/min
Injection type: Split ratio of 1:30
Injection size: 1 uL
System suitability
Sample: Standard solution
[Nott—The relative retention times for the trimethylsyli-
lated derivative of propylene glycol and the trimethyl-
sylilated derivative of 1,3-propanediol are 1.0 and 1.4,
respectively.]
Suitability requirements
Resolution: NLT 2.0 between the peaks due to the
trimethylsylilated derivative of propylene glycol and
the trimethylsylilated derivative of 1,3-propanediol
Analysis
Samples: Standard solution and Sample solution
Calculate theperceniae of propylene glycol in the por-
Hon of Pregelatinized Hydroxypropyl Corn Starch
taken:
Result = (Ru/Rs) x (Cs/Cu) x 100
internal standard ratio (peak response of
éZz
propylene glycol/peak response of 1,3-
propanediol) from the Sample solution
internal standard ratio (peak response of
2
propylene glycol/peak response of 1,3-
propanediol) from the Standard solution
G = concentration of USP Propylene Glycol RS in
the Standard solution (mg/mL)
Cu = concentration of Pregelatinized Hydroxypropyl
Corn Starch in the Sample solution (mg/mL)
Acceptance criteria: NMT 0.1%
© LIMIT OF OXIDIZING SUBSTANCES
Sample: 4.0g
Analysis: Transfer the Sample to a glass-stoppered
sydesbouow 4N
125-mL conical flask, and add 50.0 mL of a mixture of
water and methanol (1:1). Insert the stopper, and swirl
for 5 min. Transfer to a glass-stoppered 50-mL centri-
fuge tube, and centrifuge to clarify. Transfer 30.0 mL of
the clear supernatant to a glass-stoppered 125-mL coni-
cal flask. Add 1 mL of glacial acetic acid and 0.5-1.0g
of potassium iodide. Insert the stopper, swirl, and allow
to stand for 25-30 min in the dark. Add 1 mL of starch
TS, and titrate with 0.002 N sodium thiosulfate VS to
the disappearance of the starch-iodine color. Perform a
 5600 Starch / Official Monographs NF 36

blank determination, and make any necessary correc- Sample solution: Transfer 1.0 g of Hydrogenated Starch
tion. Each mL of 0.002 N sodium thiosulfate VS is Hydrolysate to a 25-mL volumetric flask. Add 1.0 mL of
equivalent to 34 ug of oxidant, calculated as hydrogen water to the flask, and mix on a vortex mixer for 3 min.
peroxide. Add 2.0 mL of the Internal standard stock solution, and
Acceptance criteria: NMT 1.4 mL of 0.002 N sodium add the remaining Diluent to the flask in three equal
thiosulfate VS is required (20 ppm, calculated as H2O2). portions to volume. Mix the contents for about 3 min
after each addition of Diluent. Pass a portion of the su-
SPECIFIC TESTS pernatant layer through a nylon filter of 0.45-1m pore
e MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI- size. Discard the first 2 mL of the filtrate, and collect the
FIED MICROORGANISMS (62): The total aerobic microbial rest of the filtrate for analysis. [NOTE—Acetone is used
count does not exceed 103 cfu/g, the total combined to precipitate sugar alcohols.]
molds and yeasts count does not exceed 10? cfu/g, and Chromatographic system
it meets the requirements of the test for the absence of (See Chromatography (621), System Suitability.)
Escherichia coli. Mode: GC
e PH (791) Detector: Flame ionization
Sample solution: Progressively suspend 3.0 g of Prege- Column: 0.32-mm x 15-m fused-silica capillary; 0.25-
latinized Hydroxypropyl Corn Starch in 100.0 mL of car- um layer of phase G46
bon dioxide-free water, stirring continuously. Determine Temperature
the pH when all the solid is wetted. Detector: 300°
Acceptance criteria: 4.5-8.0 Injection port: 240°
e Loss ON DRYING (731): Dry about 1 g at 130° for 90 min: Column: See Table 7.
it loses NMT 15.0% of its weight.
ADDITIONAL REQUIREMENTS Table 1
© PACKAGING AND STORAGE: Preserve in well-closed contain- Hold Time
ers. Store at room temperature. Initial Temperature Final at Final
e USP REFERENCE STANDARDS (11) Temperature Ramp Temperature | Temperature
USP Propylene Glycol RS cy (¢/min) C) (min)
70 — 70 2
70 50 300 5

Carrier gas: Helium

Hydrogenated Starch Hydrolysate Flow rate: 3.0 mL/min
Injection size: 1.0 uL
Hydrogenated Polysaccharides Injection type: Split injection. The split ratio is about
on oH Pt 10:1. [NOTE—A general-purpose split/splitless, tapered,
glass wool, deactivated liner is used.]
te o ees System suitability
Sample: Standard solution
a on [nh ow if mart [Note—See Table 2. Relative retention times are pro-
vided for information only, and the standards should
no be used to ensure appropriate peak identification.]

Ci2H24O11(CotH005)n Table 2
Polyglucitol; Relative
Polyglycitol syrup [68425-17-2]. Name Retention Time
DEFINITION Ethylene glycol 1.0
Hydrogenated Starch Hydrolysate is a mixture that contains 1,3-Butanediol
NLT 50% of hydrogenated polysaccharides containing (internal standard) 2.0
more than 3 D-glucopyranosyl units terminated with a Diethylene glycol 2:5
D-glucity! unit, calculated on the anhydrous basis. Other
ingredients can include sorbitol, maltitol, and other sugar Suitability requirements
polyols. Resolution: NLT 15 between ethylene glycol and 1,3-
butanediol
IDENTIFICATION Analysis
e A. It meets the requirements of the test for Content of Samples: Standard solution and Sample solution
Maltitol and Sorbitol. Based on the Standard solution, identify the peaks of
e B. It meets the requirements of the test for Content of ethylene glycol, 1,3-butanediol (internal standard), and
Hydrogenated Polysaccharides. diethylene glycol. Compare peak area ratios of ethyl-
e C. Limit OF DIETHYLENE GLYCOL AND ETHYLENE GLYCOL ene glycol to the internal standard and of diethylene
[Note—Perform this test for liquid products of Hydrogen- glycol to the internal standard in the Standard solution
ated Starch Hydrolysate.] and Sample solution, respectively.
Diluent: Acetone and water (96:4) Acceptance criteria
Standard stock solution: 0.5 mg/mL of USP Diethylene
NF Monographs

Diethylene glycol: The peak area ratio of diethylene

Glycol RS and 0.5 mg/mL of USP Ethylene Glycol RS in
Diluent
Internal standard stock solution: 0.5 mg/mL of 1,3-
butanediol (internal standard) in Diluent
Standard solution: 0.04 mg/mL of USP Diethylene Gly-
col RS, 0.04 mgr of USP Ethylene Glycol RS, and
0.04 mg/mL of 1,3-butanediol (internal standard), in
Diluent, prepared from the Standard stock solution and
the Internal standard stock solution
glycol to the internal standard in the Sample solution is
NMT the peak area ratio of diethylene glycol to the
internal standard in the Standard solution, correspond-
ing to NMT 0.10% of diethylene glycol in Hydrogen-
ated Starch Hydrolysate.
Ethylene glycol: The peak area ratio of ethylene glycol
to the internal standard in the Sample solution is NMT
the peak area ratio of ethylene glycol to the internal
standard in the Standard solution, corresponding to
NF 36 Official Monographs / Starch 5601

NMT 0.10% of ethylene glycol in Hydrogenated e CONTENT OF HYDROGENATED POLYSACCHARIDES

Starch Hydrolysate. Mobile phase, Standard solution, Sample solution,
and Chromatographic system: Prepare as directed in
ASSAY the test for Content of Maltitol and Sorbitol.
¢ CONTENT OF MALTITOL AND SORBITOL Analysis
Mobile phase: Degassed water Samples: Standard solution and Sample solution
Standard solution: Dissolve accurately weighed quanti- Inject a volume (about 50 pL) of the Sample solution,
ties of USP Maltose Monohydrate RS, USP Maltitol RS, and measure all the peak areas. The elution pattern
USP Dextrose RS, and USP Sorbitol RS in water to ob- includes the higher-molecular-weight hydrogenated
tain a solution having known concentrations of about polysaccharides containing more than 11 D-gluco-
1.0 mg/mL for each, calculated on the anhydrous basis. pyranosyl units, followed by some individual peaks
Sample solution: Transfer a quantity of Hydrogenated representing hydrogenated polysaccharides containing
Starch Hydrolysate, equivalent to 100 mg on the anhy- NMT 10 D-glucopyranosyl units, if this hydrogenated
drous basis, to a 100-mL volumetric flask. Dilute with species is present. The higher-molecular-weight hydro-
water to volume, and mix. Transfer approximately genated polysaccharides containing more than 11 D-
10 mL of the solution into a separate container, shake glucopyranosyl units can be integrated into one peak;
the solution for 30 s, pass througha filter of 0.45-um the relative retention time is about 0.29 relative to the
or ines pore size into a suitable autosampler vial, and peak of sorbitol. The relative retention times for hydro-
seal. genated polysaccharides containing NMT 10 D-
Chromatographic an glucopyranosyl units are given in Table 4.
(See Chromatography (621), System Suitability.)
Mode: LC Table 4
Detector: Refractive index
Column: 7.7-mm x 30-cm; packing L58 Hydrogenated Species for Relative
Temperature Degree of Polymerization (DP) Retention Time
Detector: 40° “ati Sorbitol (HDP 1) 1.00
Column: 80°, controlled within +2° Maltitol (HDP 2) 0.84
Flow rate: 0.3 mL/min Maltotriitol (HDP 3 0.72
Injection size: 50 uL aoe ne ) :
System suitability Maltotetraitol (HDP 4) 0.64
Sample: Standard solution Maltopentaitol (HDP 5) 0.58
[Note—See Table 3 for relative retention times.] Maltohexaitol (HDP 6) 0.53
Maltoheptaitol (HDP 7) 0.48
Table 3 Maltooctaitol (HDP 8) 0.45
Relative Maltononaito! (HDP 9) 0.42
Name Retention Time Maltodecaitol (HDP 10) 0.40

Maltose 0.81 Calculate the percentage of hydrogenated polysaccha-

Mattitol oes rides containing more than3 D-glucopyranosyl units in
Dextrose 0.94 the solid portion of Hydrogenated Starch Hydrolysate
Sorbitol 1.00 taken:
[NotE—Sorbitol is the last peak to elute.] Result = (ry/r7) x 100
System suitability requirements
Relative standard deviation: NMT 2.0% for the mal- ty = sum of peak areas of hydrogenated
titol peak polysaccharides containing more than 3 D-
Analysis glucopyranosyl units from the Sample
Samples: Standard solution and Sample solution solution
Calculate the percentage of each component, maltose rr = sum of all the peak areas from the Sample
(Pui), Mmaltitol (Pyz), dextrose (Pp), and sorbitol (Ps), in solution
the solid portion of Hydrogenated Starch Hydrolysate Acceptance criteria: NLT 50% of hydrogenated poly-
taken: saccharides containing more than 3 D-glucopyranosy|
units, on the anhydrous basis
Result = V x (ru/rs) x (C/W) x 100
IMPURITIES
v = volume of the Sample solution, 100 mL © RESIDUE ON IGNITION (281): NMT 0.15%, ignition of a
tu = peak response for the respective component quantity of Hydrogenated Starch Hydrolysate equivalent
(maltose, maltitol, dextrose, sorbitol) from to 1.0g of solid, on the anhydrous basis
the Sample solution e LIMIT OF CHLORIDE
rs = peak response for the respective component Sample solution: Transfer a quantity of Hydrogenated
(maltose, maltitol, dextrose, sorbitol) from Starch Hydrolysate, equivalent to 25 g on the anhy-
the Standard solution drous basis, to a beaker, add 100 mL of water, and stir
Cc = concentration of the respective component until the Hydrogenated Starch Hydrolysate is completely
(maltose, maltitol, dextrose, sorbitol) in the dissolved.
Standard solution (mg/mL) Analysis: Add 1.0 mL of potassium chromate indicator r
Ww = weight of Hydrogenated Starch Hydrolysate solution (1 in 20) to the Sample solution. Slowly titrate
that was taken to prepare the Sample with 0.1 N silver nitrate VS until a reddish-orange color <
pluie (mg), calculated on the anhydrous persists. =
asis Calculate the quantity, in 1g, of chloride in each g of 3}
Acceptance criteria: Less than 50% of total maltitol Hydrogenated Starch Hydrolysate taken: ie]
|
and sorbitol and less than 1% of total maltose and dex-
trose, on the anhydrous basis Result = (Fx M,x Nx V)/W |
Ey
F = factor converting mg to jg, 103 ug/mg 5
M, = molar mass of chloride, 35.45
USP 41 Dietary Supplements / Cat's Claw 4511

Time Solution A Solution B Solution C rs = peak response for isopteropodine from

(min) (%) (%) (%) Standard solution B
30 50 50 0 Cs = concentration of USP lsopteropodine RS in
31 9 0 100 Standard solution B (mg/mL)
Vv = final dilution volume of the Sample solution
36 o 0 100
39 65 35 0
(ml)
Calculate the content, in mg, of total pentacyclic
49 65 35. 0 oxindole alkaloids (Cc) in the portion of Capsules
taken by adding the individual contents of
Standard solution A: Dissolve an accurately weighed speciophylline, uncarine F, mitraphylline,
quantity of USP Powdered Cat’s Claw Extract RS in isomitraphylline, pteropodine, and isopteropodine.
methanol, shake for 1 min, and dilute with methanol to Calculate the percentage of Powdered Cat’s Claw
obtain a solution having a known concentration of Extract with respect to the label claim:
about 0.5 mg/mL of the labeled amount of total ox-
indole alkaloids. Pass throughafilter of 0.45-um or Result = Cc x (Awc/W) x (100/Le) x (100/L)
finer pore size.
Standard solution B: 0.1 ate of USP Isopteropodine Cc = content of total pentacyclic oxindole alkaloids
RS in methanol. Pass through a nylon filter of 0.45-m in the portion of Capsules taken (mg)
or finer pore size. Awc = average weight of Capsules contents (mg/
Sample solution: Accurately weigh the contents of not Capsule)
fewer than 20 Capsules and pulverize. Transfer an accu- Ww = weight of the portion of Capsules taken (mg)
rately weighed quantity of the powder, equivalent to Le = content of total pentacyclic oxindole alkaloids,
20 mg of the labeled amount of pentacyclic oxindole mg, in 100 mg of the Extract used to
alkaloids, to a 50-mL centrifuge tube. Sonicate with prepare the Capsules
10 mL of methanol for 10 min. Centrifuge and transfer L: = amount of Extract per Capsule according to
this solution to a 50-mL volumetric flask. Repeat the label claim (mg/Capsule
above extraction three more times, combining the ex- Calculate the percentage of tetracyclic oxindole
tracts in the 50-mL volumetric flask, and dilute with alkaloids with respect to the content of pentacyclic
methanol to volume. Transfer 3 mL of the solution to a oxindole alkaloids in the portion of Capsules taken:
test tube containing 300 mg of polyamide powder, and
shake for 1 min. Pass through a nylon filter of 0.45-m Result = (rr/re) x 100
or finer pore size, and discard the first part of the
filtrate. tr = sum of peak responses for rhynchophylline
Chromatographic system and isorhynchophylline in the chromatogram
(See Chromatography (621), System Suitability.) of the Sample solution
Mode: LC Ip = sum of peak responses for speciophylline,
Detector: UV 245 nm uncarine F, mitraphylline, isomitraphylline,
Selmi 4.6-mm x 10-cm; endcapped 3-14m packing petoropodine and isoperopodine in the
chromatogram of the Sample solution
Flow rate: 0.75 mL/min Acceptance criteria: 90.0%-110.0% of the labeled
Injection size: 10 uL amount of Powdered Extract calculated as pentacyclic
System suitability oxindole alkaloids; and NMT 25% of tetracyclic ox-
Samples: Standard solution A and Standard solution B indole alkaloids with respect to the labeled amount of
Suitability requirements pentacyclic oxindole alkaloids is found.
Chromatogram similarity: The chromatogram ob-
tained using Standard solution A is similar to the Ref- PERFORMANCE TESTS
erence Chromatogram provided with the USP Pow- ¢ DISINTEGRATION AND DISSOLUTION OF DIETARY SUPPLEMENTS
dered Cat’s Claw Extract RS being used. (2040): Meets the requirements for Disintegration
Tailing factor: NMT 2.0 for the isopteropodine peak, © WEIGHT VARIATION OF DIETARY SUPPLEMENTS (2091):
Standard solution B Meets the requirements
Relative standard deviation: NMT 2.0% from the CONTAMINANTS
isopteropodine peak in repeated injections, Standard
sydesbouo- Sa

¢ MICROBIAL ENUMERATION TESTS (2021): The total aerobic

solution B
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Measure the areas of the analyte peaks. Identify the
retention times of the peaks corresponding to speci-
ophylline, uncarine F, mitraphylline, isomitraphylline,
pteropodine, isopteropodine, rhynchophylline, and
isorhynchophylline by comparison of the chromato-
gram of Standard solution A with the Reference Chro-
matogram provided with the lot of the USP Pow-
dered Cat’s Claw Extract RS being used.
Calculate the content, in mg, of speciophylline, un-
carine F, mitraphylline, isomitraphylline, pteropodine,
and isopteropodine, as isopteropodine, in the portion
of Capsules taken:
Result = (ru/rs) x Cs x V
microbial count does not exceed 104 cfu/g, and the total
a molds and yeasts count does not exceed 103
cfu/g.
e ABSENCE OF SPECIFIED MICROORGANISMS (2022): Meets the
requirements of the tests for absence of Salmonella spe-
cies and Escherichia coli.
ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Preserve in tight, light-resistant
containers, and store at room temperature.
e LABELING: The label states the Latin binomial and, follow-
ing the official name, the article from which the Capsules
were prepared. If prepared with Extract, the label also
indicates the quantity, in mg, of Extract per Capsule and
the content, in mg, of pentacyclic oxindole alkaloids per
100 mg of Powdered Extract.

tu = peak response for each relevant alkaloid from

the Sample solution
 5602 Starch / Official Monographs
N = exact normality of the silver nitrate solution
Vv = volume of the silver nitrate solution consumed
in the titration (mL)
Ww = weight of Hydrogenated Starch Hydrolysate
taken to prepare the Sample solution (g)
Acceptance criteria: NMT 50 ug/g(ppm) of chloride
© CHLORIDE AND SULFATE, Sulfate (221): 1.0g of the solid
portion of Hydrogenated Starch Hydrolysate shows no
more sulfate than corresponds to 0.10 mL of 0.020 N
sulfuric acid: NMT 100 ug/g (ppm) of sulfate is found.
e Limit OF NICKEL
[Note—When water is specified as the diluent, use
deionized ultra-filtered water.]
Digester solution (aqua regia): Add 360 mL of hydro-
chloric acid and 240 mL of nitric acid to 1200 mL of
water.
Blank solution: Add 40 mL of nitric acid to a 2-L volu-
metric flask, dilute with water to volume, and mix well.
Internal standard solution: Transfer 2.0 mL of com-
mercially prepared yttrium reference standard solution
(1000 ppm) to a 1-L volumetric flask, dilute with Blank
solution to volume, and mix well. The Internal standard
solution contains 2 g/mL of yttrium.
Standard stock solution: [NoTE—Prepare this solution
fresh every two months.] Quantitatively dilute an accu-
rately measured volume of a commercially prepared
nickel ICP standard (1000 ppm) with Blank solution to
obtain a solution containing 10 ug/mL of nickel (Stan-
dard stock solution 10 ppm).
Standard solutions: [Notr—Prepare these solutions
fresh weekly.] Separately pipet 1.0, 2.0, and 4.0 mL of
Standard stock solution, respectively, into three 200-mL
volumetric flasks. Dilute the content in each flask with
Blank solution to volume, and mix well. These are, re-
spectively, the Standard nickel solution 50ppb, Standard
nickel solution 100 ppb, and Standard nickel solution
200 ppb.
Sample solution: Transfer a quantity of Hydrogenated
Starch Hydrolysate, equivalent to 10.0 g on the anhy-
drous basis, into a 125-mL conical flask. Add 40 mL of
Digester solution, and place on a hot plate. Heat the
solution for about 20 min, being careful to prevent the
solution from boiling over. Pull the sample off the hot
plate just before it turns a dark caramel color. [NOTE—
Do not overburn the sample.] Transfer the flask’s con-
tents into a clean, dry, 50-mL volumetric flask with
washings of Blank solution. Dilute with Blank solution to
volume. Filter the sample into a 15-mL centrifuge tube,
using a 10-mL BD syringe fitted with a syringe filter of
0.45-um pore size.
Instrumental conditions
(See Plasma Spectrochemistry (730).)
Mode: Inductively coupled plasma-optical emission
spectroscopy (ICP-OES) configured in an axial optical
alignment
Detector: Set a UV detector to scan nickel at 232.005
nm and yttrium at 371.029 nm, and set the sample
read time to 10 s minimum and 50 s maximum.
Analysis
Samples: Blank solution, Standard solutions, and Sample
solution
Take three replicate scans with the integration set to
one point per peak. Set forward power from the RF
enerator to 1500 watts. The argon plasma feed gas
NF Monographs
flows at 15 L/min with the auxiliary gas (shear gas) set
to flow at 0.5 L/min. Use a gem cone nebulizer with a
nebulization gas flow rate of 0.55 L/min. Deliver the
sample to the spray chamber with a multichannel peri-
staltic pump set to deliver sample at a rate of
1.00 mL/min. Add the Internal standard solution in-line
via a mixing block between the sample probe and the
spray chamber. Flush the samples through the system
for 30 s at a rate of 4.0 mL/min before analysis. Pro-
gram a 60-s read delay into the sampling routine to
allow for fluid flow equilibration after the high-speed
NF 36
flush, before the first analytical read of the sample. Be-
tween samples, wash the pumping system by flushing
the Blank solution for 30 s at a rate of 4.0 mL/min.
Instrument performance must be verified to conform to
the manufacturer's specifications for resolution and
sensitivity. Before analyzing samples, the instrument
must pass a suitable performance check.
Generate the calibration curve using the Blank solution,
Standard nickel solution 50nan Standard nickel solution
100 ppb, and Standard nickel solution 200 ppb as fol-
lows. Scan the Internal standard solution while running
the Blank solution to measure the intensity of the yt-
trium emission. Hold this value constant throughout
the remainder of the test. Separately scan the Blank
solution, Standard nickel solution 50 ppb, Standard nickel
solution 100 ppb, and Standard nickel solution 200 ppb
for nickel and yttrium. [NoTe—Add the /nternal stan-
dard solution via an in-line mixing chamber.] Normal-
ize the yttrium intensity to the value of the Internal
standard solution. Apply this normalization factor to
the nickel intensity, which is then referred to as the
corrected nickel intensity. Construct a calibration curve
by plotting the corrected nickel intensity versus the
known concentrations, in ng/mL, of the nickel: the lin-
ear regression coefficient is NLT 0.999.
Similarly, analyze the Sample solution on the ICP. Plot
the intensity of the emission of the Sample solution on
the calibration curve. Obtain the concentration of
nickel, C, in ng/mL, in the Sample solution through the
calibration curve.
Calculate the content, in ug/g, of nickel in the solid
portion of Hydrogenated Starch Hydrolysate taken:
Result = Fx Vx (C/W)
F = factor converting ng to ug, 10-3 ug/ng
Vv = volume of the Sample solution, 50 mL
€ = concentration of nickel in the Sample solution
(ng/mL)
Ww = weight of Hydrogenated Starch Hydrolysate
calculated on the anhydrous basis (g)
Acceptance criteria: Nickel content is NMT 1 g/g
(ppm).
e LIMIT OF REDUCING SUGARS
Analysis: Dissolve a quantity of Hydrogenated Starch
Hydrolysate, equivalent to 1.0 g on the anhydrous basis,
in 6 mL of water with the aid of gentle heat, if neces-
sary. Cool, and add 20.0 mL of cupric citrate TS and a
few glass beads. Heat so that boiling begins after 4
min, and maintain boiling for 3 min. Cool rapidly, and
add 40 mL of diluted acetic acid, 60 mL of water, and
20.0 mL of 0.05 N iodine VS. With continuous shaking,
add 25 mL of a mixture of 6 mL of hydrochloric acid
and 94 mL of water. When the precipitate has dis-
solved, titrate the excess of iodine with 0.05 N sodium
thiosulfate VS, using 2 mL of starch TS as an indicator,
added toward the end of the titration.
Acceptance criteria: NLT 12.8 mL of 0.05 N sodium
thiosulfate, corresponding to NMT 1% of reducing
sugars
SPECIFIC TESTS
e MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI-
FIED MICROORGANISMS (62): The total aerobic microbial
count does not exceed 103 cfu/g, and the total com-
bined molds and yeasts count does not exceed 10? cfu/
. It meets the requirements of the tests for absence of
Soimonela species and Escherichia coli,
e PH (791): 3.0-7.0, in a 20% (w/w) solution in freshly
boiled and cooled water
© WATER DETERMINATION, Method | (921)
For dried powder product: NMT 6.0%
For liquid product: Within +1.5 units of the labeled
value
NF 36
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in well-closed contain-
ers. No storage requirements specified for liquid product;
protect from moisture for dried powder product.
e LABELING: Label it to indicate Water content, as the per-
centage of water, for liquid product.
e USP REFERENCE STANDARDS (11)
USP Dextrose RS
USP Diethylene Glycol RS
USP Ethylene Glycol RS
USP Maltitol RS
USP Maltose Monohydrate RS
USP Sorbitol RS
Modified Starch
DEFINITION
Modified Starch is Starch modified by chemical means. Food
Starch may be acid-modified, bleached, oxidized, esteri-
fied, etherified, or treated enzymatically to change its
functional properties (21 CFR 172.892).
IDENTIFICATION
oA.
Corn starch: Polygonal, rounded, or spheroidal gran-
ules up to 35 um in diameter and usually having a
circular or several-rayed central cleft
Tapioca starch: Spherical granules with one truncated
side, typically 5-35 jum in diameter and usually having
a circular or several-rayed central cleft
Potato starch: Irregularly shaped, ovoid, or pear-
shaped granules, usually 30-100 tm in size but occa-
sionally exceeding 100 um; or rounded, 10-35 ym in
size. There are occasional compound granules having
two to four components. The ovoid and pear-shaped
granules have an eccentric hilum, and the rounded
granules have an accentric or slightly eccentric hilum.
All granules show clearly visible concentric striations.
Wheat starch: Large and small granules, usually 10-60
tum in diameter. The central hilum and striations are
visible or barely visible.
eB.
Sodium hydroxide solution: 2% (w/w)
Sample: 0.6g
Analysis: Transfer the Sample to a 25-mL glass vial with
a plastic cap. Add 9.4 g of water, cap, and shake vigor-
ously to evenly disperse the starch. Add 10g of the
Sodium hydroxide solution, cap, and shake vigorously for
1 min to create a smooth mixture. Evaluate within 1
min.
Acceptance criteria: The final solution is translucent to
opaque witha fluid consistency. A yellow tint of the
final solution is acceptable.
e C. A water slurry of the Modified Starch is colored or-
ange-red to deep blue by iodine TS.
IMPURITIES
e RESIDUE ON IGNITION (281)
Sample: 2.0+0.1g
Analysis: Proceed as directed in the chapter.
Acceptance criteria: NMT 1.5%
e IRON (241)
Test preparation: Dissolve the residue obtained in the
test for Residue on Ignition in 8 mL of hydrochloric acid
with the aid of gentle heating. Dilute with water to
100 mL in a volumetric flask, and mix. Dilute 25 mL of
this solution with water to 47+1 mL.
Official Monographs / Starch 5603
Analysis: Proceed as directed in the chapter.
Acceptance criteria: NMT 20 ppm
e Limit OF SULFUR DIOXIDE
Sample: 20.0+0.1g
Analysis: Mix the Sample with 200 mL of 5% alcohol
until a smooth suspension is obtained, and vacuum-fil-
ter through paper (Whatman No.1 or equivalent). To
100 mL of the filtrate add 3 mL of starch TS, and titrate
with 0.01 N iodine VS to the first permanent blue
color.
Acceptance criteria: NMT 1.7 mL of 0.01 N iodine VS
is consumed, which corresponds to NMT 50 ppm of
sulfur dioxide being found.
© OXIDIZING SUBSTANCES
Sample: 4.0g
Titrimetric system
Mode: Direct titration
Titrant: 0.002 N sodium thiosulfate VS
Blank: 30.0 mL of water, accurately measured
Endpoint detection: Visual
Analysis: Transfer the Sample to a glass-stoppered,
125-mL conical flask, and add 50.0 mL of water. Insert
the stopper, and swirl for 5 min. Transfer to a glass-
stoppered, 50-mL ella tube, and centrifuge to
ality Transfer 30.0 mL of the clear supernatant to a
glass-stoppered, 125-mL conical flask. Add 1 mL of gla-
cial acetic acid and 0.5-1.0 g of potassium iodide. In-
sert the stopper, swirl, and allow to stand for 25-30
min in the dark. Add 1 mL of starch TS, and titrate with
Titrant to the disappearance of the starch-iodine color.
Perform a blank determination, and make any necessary
correction. Each mL of 0.002 N sodium thiosulfate is
equivalent to 34 ug of oxidant, calculated as hydrogen
peroxide.
Acceptance criteria: NMT 12.6 mL of 0.002 N sodium
thiosulfate is required (180 ppm, calculated as H2O2),
which corresponds to NMT 0.018% of oxidizing
substances
SPECIFIC TESTS
e MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI-
FIED MICROORGANISMS (62): The total aerobic microbial
count does not exceed 103 cfu/g, and the total com-
bined molds and yeasts count does not exceed 10? cfu/
g. It meets the requirements of the tests for absence of
Salmonella species and Escherichia coli.
© PH (791)
Sample: 20.0+0.1g
Analysis: Transfer the Sample to a suitable nonmetallic
container, and add 100 mL of water to obtain a slurry.
Stir using a magnetic stirrer at a moderate rate for 5
min, and determine the pH to the nearest 0.1 unit.
Acceptance criteria: 3.0-9.0
e Loss ON DRYING (731)
Analysis: Dry a sample at 120° for 4 h.
Acceptance criteria
Corn starch and Wheat starch: NMT 15.0%
Tapioca starch: NMT 18.0%
Potato starch: NMT 21.0%
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in well-closed contain-
ers. No storage requirements specified.
ra
n
E
Pea Starch 2
DEFINITION 8
Pea Starch is obtained from the seeds of Pisum sativum L. Ry
mo]
IDENTIFICATION z
e A. Examined under a microscope using a mixture of —
equal volumes of glycerin and water, it presents a major-
 5604 Starch / Official Monographs NF 36

ity of large elliptical granules, 25-45 um in size, some-

times irregular, or reniform. It also presents a minority of
small rounded granules, 5-8 jum in size. Granules can
pe cracks or irregularities. Sometimes, granules show
arely visible concentric striations. Exceptionally, granules
showaslit along the main axis. Between orthogonally
oriented polarizing plates or prisms, the granules show a
distinct black cross.
e B. Suspend 1 g of it in 50 mL of water, boil for 1 min,
and cool: a thin, cloudy mucilage is formed.
e C. To 1 ml of the mucilage obtained in Identification test
B add 0.05 mL of iodine and potassium iodide TS 2: an
orange-red to dark blue color is produced, which disap-
pears on heating.
IMPURITIES
Inorganic Impurities
@ RESIDUE ON IGNITION (281): NMT 0.6%, determined on a
1.0-g sample
e Limit OF IRON
Standard iron stock solution: Prepare a solution con-
taining the equivalent of 10 ug/mL of iron, as directed
under Iron (241).
Diluted standard iron solution: Immediately before
use, dilute a measured volume of Standard iron stock
solution quantitatively with water to obtain a solution
containing the equivalent of 1 t1g/mL of iron.
Standard solution: Transfer 10 mL of the Diluted stan- Figure 1
dard iron solution to a test tube. Add 2 mL of citric acid
solution (2 in 10) and 0.1 mL of thioglycolic acid, and The apparatus consists of a 500-mL three-neck, round-bot-
mix. Add 10 N ammonium hydroxide until the solution tom boiling flask, A; a separatory funnel, B, having a capac-
is distinctly alkaline to litmus, dilute with water to ity of 100 mL or greater; a gas inlet tube of sufficient length
20 mL, and mix. to permit introduction of the carbon dioxide within 2.5 cm
Sample solution: Shake 1.0g of Pea Starch with 50 mL of the bottom of the boiling flask; a reflux condenser, C,
of 2 N hydrochloric acid, and filter. Transfer 10 mL of having a jacket length of 200 mm; and a delivery tube, £,
the filtrate to a test tube. Add 2 mL of citric acid solu- connecting the upper end of the reflux condenser to the
tion (2 in 10) and 0.1 mL of thioglycolic acid, and mix. bottom of a receiving test tube, D. Apply a thin film of
Add 10 N ammonium hydroxide until the solution is stopcock grease to the sealing surfaces of all the joints ex-
distinctly alkaline to litmus, dilute with water to 20 mL, cept the joint between the separatory funnel and the boiling
and mix. flask, and clamp the joints to ensure tightness.
Acceptance criteria: After 5 min, any pink color in the System suitability test
Sample solution is not more intense than that in the Test A: Using the Potassium metabisulfite solution as
Standard solution, corresponding to a limit of 50 ug/g the standard, proceed as directed in Analysis, except
of iron. for replacing the 25.0 g of Pea Starch with 20 mL of
e LIMIT OF SULFUR DIOXIDE
the Potassium metabisulfite solution.
{[Note—Perform either Test 7 or Test 2.] Calculate the content, in ug/mL, of sulfur dioxide in
Test 1 the Potassium metabisulfite solution taken:
Carbon dioxide: Use carbon dioxide with a flow regu- Result = (F x MW x V x N)/Vp
lator that will maintain a flow of 100410 mL/min.
Hydrogen peroxide solution: Dilute 30% hydrogen F = factor for conversion of mg to Lg, 1000
peroxide with water to obtain a 3% solution. Neutral- MW = milliequivalent weight of sulfur dioxide,
ize the solution with 0.01 N sodium hydroxide to a pH 32.03
of 4.1, determined potentiometrically. Vv = volume of titrant consumed (mL)
Potassium metabisulfite solution: Transfer 0.87 g of N = normality of the titrant
potassium metabisulfite (K2S20s) and 0.2 g of edetate Vp = volume of the Potassium metabisulfite solution
disodium to a 1000-mL volumetric flask. Dilute with taken for the test (mL)
water to volume before mixing. [NoTE—Edetate diso- Test B: In a 100-mL conical flask, add 20 mL of 0.02
dium is used to protect the sulfite ion from oxidation.] N iodine solution and 5 mL of 2 N hydrochloric acid.
Apparat In this test, the sulfur dioxide is released Add 1 mL of starch TS, and titrate with Potassium
rom the sample in a boiling acid medium and is re- metabisulfite solution until the first discoloration is
moved by a stream of carbon dioxide. The separated observed.
gas is collected in a dilute hydrogen peroxide solution Calculate the content, in g/mL, of sulfur dioxide in
where the sulfur dioxide is oxidized to sulfuric acid and Potassium metabisulfite solution:
titrated with standard alkali. A suitable apparatus for
sulfur dioxide determination is shown in the accompa-
NF Monographs

Result = (F x MW x V, x Ni)/Vp
nying diagram (Figure 1).
F = factor for conversion of mg to ug, 1000
MW = milliequivalent weight of sulfur dioxide,
32.03
vi = the volume of the iodine solution used in the
test (mL)
N normality of the iodine solution
Vp volume of the Potassium metabisulfite solution
consumed (mL)
NF 36 Official Monographs / Starch 5605

The difference between the sulfur dioxide contents © PROCEDURE 2: FOREIGN MATTER
obtained from Test A and Test B is NMT 5% of their Analysis: Examine under a microscope, using a mixture
mean value. Test B shall be performed within 15 min of equal volumes of glycerin and water.
after the completion of Test A. [NoTE—This time Acceptance criteria: NMT traces of matter other than
limit avoids potential variation in the sulfur dioxide starch granules are present. No starch grains of any
content of the Potassium bisulfite solution when other origin are present.
stored at room temperature.]
Analysis: Add 150 mL of water to the boiling flask (A) SPECIFIC TESTS
(see Figure 1). Close the stopcock of the separatory fun- e MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI-
nel, and begin the flow of carbon dioxide through the FIED MICROORGANISMS (62): The total aerobic microbial
apparatus at a rate of 100 +5 mL/min. Start the con- count does not exceed 1000 cfu/g, the total combined
denser coolant flow. Place 10 mL of Hydrogen peroxide molds and yeasts count does not exceed 100 cfu/g, and
solution in the receiving test tube (D). After 15 min, it meets the requirements of the test for the absence of
without interrupting the flow of carbon dioxide, re- Escherichia coli.
move the separatory funnel (B) from the boiling flask, PH (791)
and transfer 25.0 g of Pea Starch to the boiling flask Sample solution: Preparea slurry by weighing 5.0 g of
with the aid of 100 mL of water. Apply stopcock grease Pea Starch, transferring to a suitable nonmetallic con-
to the outer joint of the separatory funnel, and replace tainer, and adding 25.0 mL of freshly boiled and cooled
the separatory funnel in the boiling flask. Close the water.
stopcock of the separatory funnel, and add 80 mL of Analysis: Agitate continuously at a moderate rate for 1
2.N hydrochloric acid to the separatory funnel. Open min, Stop the agitation, allow to stand for 15 min, and
the stopcock of the separatory funnel to permit the shake again. Determine the pH to the nearest 0.1 unit:
hydrochloric acid solution to flow into the boiling flask, the pH is determined potentiometrically.
guarding agaltis the escape of sulfur dioxide into the Acceptance criteria: 5.0-8.0
separatory funnel by closing the stopcock before the e Loss ON DRYING (731): Dry about 1 g at 130° for 90 min:
last few mL of hydrochloric acid drain out. Boil the it loses NMT 16.0% of its weight.
mixture for 1 h. Open the stopcock of the funnel, stop
the flow of carbon dioxide, discontinue heating the ADDITIONAL REQUIREMENTS
flask, and turn off the cooling water in the condenser. © PACKAGING AND STORAGE: Preserve in well-closed contain-
Remove the receiving test tube, and transfer its con- ers. Store at room temperature.
tents to a 200-mL wide-necked conical flask. Rinse the
receiving test tube with a small portion of water, add
the rinsing to the 200-mL conical flask, and mix. Heat
on a water bath for 15 min, and allow to cool. Titrate
the contents with 0.1 N sodium hydroxide VS until the Hydroxypropyl Pea Starch
PH reaches 4.1, determined potentiometrically. Perform
a blank determination, and make any necessary correc- Amyloss derivative oy
tion (see Titrimetry (541)). or
s fa
Rater LL
Ny,
Calculate the content, in g/g, of sulfur dioxide in the on
Pea Starch taken: Hom a ston none AK,lb
or’
Result = (F x MW x V x N)/W. ? ‘ ‘Amy lopectin devivat
ro or ” aes ve oH ‘
F = factor for conversion of mg to Ng, 1000 Reno “NS \oy, or HHo-< ton
MW = milliequivalent weight of sulfur dioxide, 32.03
Vv = volume of titrant consumed (mL) ro orl,
N = normality of the titrant
Ww = weight of the Pea Starch taken (g) For the Amylose derivative, m is about 300-1000.
Test 2: Determine the content of sulfur dioxide as di-
rected under Sulfur Dioxide (525), Method I. Use DEFINITION
200 mL of water as a solvent. Then, to 100 mL of the Hydroxypropyl Pea Starch is partially substituted 2-hydroxy-
clear filtrate, add 3 mL of starch TS, and titrate with propylether obtained from pea starch by a chemical mod-
0.01 N iodine VS to the first permanent blue color. ification of etherification with propylene oxide. In addi-
Acceptance criteria: NMT 50 ug/g of sulfur dioxide tion, this starch may be partially hydrolyzed using acids or
Organic Impurities enzymes to obtain thinned starch. It contains NLT 2.0%
© PROCEDURE 1: LIMIT OF OXIDIZING SUBSTANCES an { NMT 7.0% of hydroxypropyl groups, on the dried
Sample: 4.0 g of Pea Starch SIS.
Analysis: Transfer the Sample to a glass-stoppered,
125-mL conical flask, and add 50.0 mL of water. Insert IDENTIFICATION
the stopper, and swirl for 5 min. Transfer to a glass- e A. PROCEDURE
stoppered 50-mL centrifuge tube, and centrifuge to Analysis: Examine under a microscope, using NLT 20x
clarity. Transfer 30.0 mL of the clear supernatant to a magnification and a mixture of glycerin and water (1:1)
glass-stoppered 125-mL conical flask. Add 1 mL of gla- as a mounting agent.
cial acetic acid and 0.51.0 g of potassium iodide. in- Acceptance criteria: It presents a majority of large el-
sert the stopper, swirl, and allow to stand for 25-30 liptical granules 25-45 im in size, sometimes irregular vs
min in the dark. Add 1 mL of starch TS, and titrate or reniform. It also presents a minority of small rounded nn

with 0.002 N sodium thiosulfate VS to the disappear- granules 5-8 im in size. Granules can present cracks or “=
ance of the starch-iodine color. Perform a blank deter- irregularities. Sometimes, granules show barely visible }
mination, and make any necessary correction. Each mL concentric striations. Exceptionally, granules showaslit S
along the main axis. Between orthogonally oriented po- 2}
of 0.002 N sodium thiosulfate VS is equivalent to 34 ug
larizing plates or prisms, the granules showa distinct
ro}=
of oxidant, calculated as hydrogen peroxide. 2
Acceptance criteria: NMT 1.4 mL of 0.002 N sodium black cross. a}
thiosulfate VS is required (20 g/g, calculated as H202). e B. PROCEDURE a
Sample solution: Suspend 1 g of Hydroxypropyl Pea “”

Starch in 50 mL of water, boil for 1 min, and cool.

 5606 Starch / Official Monographs
Acceptance criteria: A translucent or clear mucilage is
formed.
e C. PROCEDURE
Analysis: To 1 mL of the Sample solution obtained in
Identification test B add 0.05 mL of iodine and potas-
sium iodide TS 2.
Acceptance criteria: An orange-red to dark blue color
is produced, which disappears upon heating.
e D. PROCEDURE
Ninhydrin solution: Dissolve 3 g of ninhydrin in
100 mL of a 45.5-g/L solution of sodium metabisulfite.
Diluted sulfuric acid: 98 g/L of H2SO,
Sample: 100mg of Hydroxypropyl Pea Starch
Analysis: Transfer the Sample to a 100-mL volumetric
flask, and add 12.5 mL of Diluted sulfuric acid. Place the
flask in a water bath, and heat until the Sample is dis-
solved. Cool, and dilute with water to 100 mL. [Cau-
TION—When sulfuric acid is miscible with water, it pro-
duces intense heat.]
Pipet 1 mL of this solution to a glass-stoppered, 25-mL
graduated test tube and, with the tube immersed in
cold water, add drop-wise 8 mL of sulfuric acid. Mix
well, and place the tube in a boiling water bath for
exactly 3 min. Immediately transfer the tube to an ice
bath until the solution is chilled. Add 0.6 mL of
Ninhydrin solution, carefully allowing the reagent to
run down the walls of the test tube. Immediately
shake the tube well, and place it in a water bath at
25° for 100 min. Dilute with sulfuric acid to 25 mL
[CauTilon—Use sulfuric acid cautiously.], and mix by
inverting the tube several times. Do not shake.
Acceptance criteria: A violet color develops within 5
min due to the presence of hydroxypropyl groups
(starch ether).
ASSAY
e PROCEDURE FOR HYDROXYPROPYL GROUPS
Deuterium chloride solution: Dilute 1 mL of deuterium
chloride (38% w/w) with 5 mL of deuterium oxide.
Internal standard solution: Dissolve 50.0 mg of so-
dium 3-trimethylsilyl-1-propane sulfonate in about 5 g
of deuterium oxide, weighed to the nearest 0.1 mg.
Store in a sealed bottle.
Sample solution: Disperse 20 g of Hydroxypropyl Pea
Starch in 200.0 mL of carbon dioxide-free water at
room temperature. Agitate for 15 min, and filter. Re-
peat the operation two more times. If poor dispersibility
or slow filtration is observed, use refrigerated carbon
dioxide-free water for the washing operation. Dry the
washed starch for NLT 4 h in vacuum at 30+ 5°. Weigh
12.0 mg of this sample in a 5-mm NMR tube. Add
0.75 mL of deuterium oxide and 0.1 mL of Deuterium
chloride solution. Cap the tube, mix, and place it in a
boiling water bath until a clear solution is obtained.
[Note—This may take 3 min to 1 h.] When a clear solu-
tion is obtained, allow to cool to room temperature.
Dry the exterior of the tube, and weigh to the nearest
0.1 mg. Add 0.05 mL of Internal standard solution, and
weigh to the nearest 0.1 mg. Determine the mass of
the Internal standard solution added. Mix thoroughly.
Nuclear magnetic resonance spectrometry
(See Nuclear Magnetic Resonance Spectroscopy (761),
Quantitative Applications.)
Apparatus: FT-NMR spectrometer at minimum
”“
300 MHz
=
5 Acquisition of 'H NMR spectra: The following param-
i} eters may be used.
im.)
—
° 20 mL, and mix.
iS
S Starch with 50 mL of 2 N hydrochloric acid, and filter.
= Transfer 10 mL of the filtrate to a test tube. Add 2 mL
—
ve
NF 36
Sweep width: 8 ppm (about -1.0 to +7 ppm)
Irradiation frequency offset: None
Time domain: NLT 64 K
Pulse width: 90 degree
Pulse delay: 10s
Dummy scans: 0
Number of scans: 8
Use the CH3 signal of the internal standard for shift
referencing. Set the shift of the peak of the singlet to
0 pem. Record the FID signal.
Analysis
Samples: Internal standard solution and Sample solution
Call the integration sub-routine after phase corrections
and baseline correction between -0.5 and +6 ppm.
Measure the peak areas of the doublet from the methyl
roups of the hydroxypropyl function at +1.2 ppm
Cho), and of the methyl groups at 0 ppm of the inter-
nal standard (A;) without 13C-satellites.
Measure the signal coming from the 3 protons of the
methyl group in the hydroxypropyl function.
Calculate the content of hydroxypropyl groups as a per-
centage (w/w, dried basis):
Result = (N x A2/Ai) x (C, x W,/W) x (My2/Ma) x [100/
(100 — BY] x 100
N = numerical value representing the 3 methyl
groups in the internal standard (sodium
3-trimethylsilyl-1-propane sulfonate), 3
Az = area of the methyl groups of hydroxypropyl in
Hydroxypropyl Pea Starch
A = area of the methyl groups in the internal
standard (sodium 3-trimethylsilyl-1-propane
sulfonate)
G = concentration of the internal standard in the
Internal standard solution (mg/g)
Wi = weight of the Internal standard solution in the
NMR tube (g)
Ww = weight of the washed and dried
Hydroxypropyl Pea Starch in the NMR tube
(mg)
Ma = molecular weight of the internal standard,
218.32 g/mol
Ma = molar mass of hydroxypropyl group, 59.09 g/
mo
B = moisture content of the washed and dried
Hydroxypropyl Pea Starch used in the Sample
solution, as a potest Ge (w/w)
Acceptance criteria: The content of hydroxypropyl
groups is 2.0%-7.0%.
IMPURITIES
Inorganic Impurities
e RESIDUE ON IGNITION (281): NMT 0.6%, determined on a
1.0-g test specimen
© LIMIT OF IRON
Standard iron stock solution: Prepare a solution con-
taining the equivalent of 10 g/mL of iron, as directed
under Iron (241).
Diluted standard iron solution: Immediately before
use, dilute an accurately measured volume of Standard
iron stock solution quantitatively with water to obtain a
solution containing the equivalent of 1 g/mL of iron.
Standard solution: Transfer 10 mL of the Diluted stan-
dard iron solution to a test tube. Add 2 mL of citric acid
solution (2 in 10) and 0.1 mL of thioglycolic acid, and
mix. Add 10 N ammonium hydroxide until the solution
is distinctly alkaline to litmus, dilute with water to
Sample solution: Shake 1.0 g of Hydroxypropyl Pea
of citric acid solution (2 in 10) and 0.1 mL of thiogly-
colic acid, and mix. Add 10 N ammonium hydroxide
NF 36
until the solution is distinctly alkaline to litmus, dilute
with water to 20 mL, and mix.
Acceptance criteria: After 5 min, any pink color in the
Sample solution is not more intense than that in the
SapCOR solution, corresponding to a limit of 50 g/g
of iron.
© Limit OF SULFUR DIOXIDE, Method IV (525): NMT 50 ppm
Organic Impurities
e PROCEDURE 1: LIMIT OF OXIDIZING SUBSTANCES
Sample: 4.0 g of Hydroxypropyl Pea Starch
Analysis: Transfer the Sample to a glass-stoppered,
125-mL conical flask, and add 50.0 mL of water. Insert
the stopper, and swirl for 5 min. Transfer to a glass-
stoppered, 50-mL centrifuge tube, and centrifuge to
char Transfer 30.0 mL of the clear supernatant to a
glass-stoppered, 125-mL conical flask. Add 1 mL of gla-
cial acetic acid and 0.5-1.0 g of potassium iodide. In-
sert the stopper, swirl, and allow to stand for 25-30
min in the dark. Add 1 mL of starch TS, and titrate
with 0.002 N sodium thiosulfate VS to the disappear-
ance of the starch-iodine color. Perform a blank deter-
mination, and make any necessary correction. Each mL
of 0.002 N sodium thiosulfate VS is equivalent to 34 ug
of oxidant, calculated as hydrogen peroxide.
Acceptance criteria: NMT 1.4 mL of 0.002 N sodium
thiosulfate VS is required (20 g/g, calculated as H2O2).
¢ PROCEDURE 2: FOREIGN MATTER
Sample: 50 mg/mL of Hydroxypropyl Pea Starch in a
mixture of glycerin and water (1:1)
Analysis: Examine under a microscope, using NLT 20x
magnification and a mixture of glycerin and water
(1:1) as a mounting agent.
Acceptance criteria: NMT traces of matter other than
Hydroxypropyl Pea Starch granules are present.
SPECIFIC TESTS
e MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI-
FIED MICROORGANISMS (62): The total aerobic microbial
count does not exceed 103 cfu/g, the total combined
molds and yeasts count does not exceed 102 cfu/g, and
it meets the requirements of the test for the absence of
Escherichia coli.
© PH (791)
Sample solution: Suspend 5.0 g of Hydroxypropyl Pea
Starch in 25.0 mL of carbon dioxide-free water, and
shake for 60 s. Allow to stand for 15 min.
Acceptance criteria: 4.5-8.0
e Loss ON DRYING (731): Dry about 1 g at 130° for 90 min:
it loses NMT 15.0% of its weight.
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in well-closed contain-
ers. Store at room temperature.
Pregelatinized Hydroxypropyl Pea
Starch
DEFINITION
Pregelatinized Hydroxypropy! Pea Starch is prepared from
Hydroxypropyl Pea Starch by mechanical processing in
the presence of water, with or without heat, to rupture all
or some of the starch granules, and is subsequently dried.
It contains NLT 2.0% and NMT 7.0% of hydroxypropyl
groups on the dried basis.
IDENTIFICATION
e A. TEST FOR PREGELATINIZED STATE
Sample: 1
Analysis: Disperse the Sample in 50 mL of water at a
temperature NMT 25°. Shake vigorously until lumps
completely disperse/solubilize or until lumps disappear.
Allow to stand for 20 min.
Official Monographs / Starch 5607
Acceptance criteria: A translucent or clear mucilage
without precipitate is formed.
e B. TEST FOR STARCH
Analysis: Disperse 0.5 g in 2 mL of water without heat-
ing, and add 0.05 mL of iodine and potassium iodide
TS2:
Acceptance criteria: A reddish-violet or blue color is
produced.
© C. NINHYDRIN TEST
Ninhydrin solution: Dissolve 3 g of ninhydrin in
100 mL of a 45.5-g/L solution of sodium metabisulfite.
Diluted sulfuric acid: 98 g/L of H2SO.4
Sample: 100mg
Analysis: Transfer the Sample to a 100-mL volumetric
flask and add 12.5 mL of Diluted sulfuric acid. Place the
flask in a water bath, and heat until the Sample is dis-
solved. Cool, and dilute with water to 100 mL. [CAu-
TION—When sulfuric acid is miscible with water, it pro-
duces intense heat.]
Pipet 1 mL of this solution to a glass-stoppered 25-mL
graduated test tube and, with the tube immersed in
cold water, add dropwise 8 mL of sulfuric acid. Mix
well, and place the tube in a boiling water bath for
exactly 3 min. Immediately transfer the tube to an ice
bath until the solution is chilled. Add 0.6 mL of
Ninhydrin solution, carefully allowing the reagent to
run down the walls of the test tube. Immediately
shake the tube well, and place it in a water bath at
25° for 100 min. Dilute with sulfuric acid to 25 mL.
[CauTIoON—Use sulfuric acid cautiously.] Mix by in-
verting the tube several times. Do not shake.
Acceptance criteria: A violet color develops within 5
min due to the presence of hydroxypropyl groups
(starch ether).
ASSAY
e ASSAY FOR HYDROXYPROPYL GROUPS
Deuterium chloride solution: Dilute 1 mL of deuterium
chloride (38% w/w) with 5 mL of deuterium oxide.
Internal standard solution: Disperse 50.0 mg of so-
dium 3-trimethylsilyl-1-propane sulfonate in about 5 g
of deuterium oxide, weighed to the nearest 0.1 mg.
Store in a sealed bottle.
Sample solution: Determine the moisture content (B)
on 5g of Pregelatinized Hydroxypropyl Pea Starch fol-
lowing the Loss on Drying test. Weigh 12.0 mg of the
Pregelatinized Hydroxypropyl Pea Starch in a 5-mm
NMR tube. Add 0.75 mL of deuterium oxide and
0.1 mL of Deuterium chloride solution. Cap the tube,
mix, and place it in a boiling water bath until a clear
solution is obtained. [NoTE—This may take from 3 min
to 1 h.] When a clear solution is obtained, allow to cool
to room temperature. Dry the exterior of the tube, and
weigh to the nearest 0.1 mg. Add 0.05 mL of the /nter-
nal standard solution. Weigh to the nearest 0.1 mg. De-
termine the mass of the /nternal standard solution
added. Mix thoroughly.
Instrumental conditions
(See Nuclear Magnetic Resonance Spectroscopy (761),
Quantitative Applications.)
Mode: Nuclear magnetic resonance spectrometry
Apparatus: FT-NMR spectrometer at minimum
300 MHz
Acquisition of ‘H NMR spectra: The following param-
eters may be used:
N|
Eatrel-B]stoleley ABET
Sweep width: 8 ppm (about -1.0 to +7 ppm)
Irradiation frequency offset: None
Time domain: NLT 64 K
Pulse width: 90°
Pulse delay: 10s
Dummy scans: 0
Number of scans: 8
Use the CH; signal of the internal standard for shift
referencing. Set the shift of the peak of the singlet to
0 ppm. Record the FID signal.
 5608 Starch / Official Monographs
Analysis
Samples: Internal standard solution and Sample solution
Call the integration subroutine after phase corrections
and baseline correction between —0.5 and +6 ppm.
Measure the peak areas of the doublet from the methyl
groups of the hydroxypropyl function at +1.2 ppm
(A2), and of the methyl groups at 0 ppm of the inter-
nal standard (A;) without 13C-satellites.
Measure the signal Shalnetn from the 3 protons of
the methyl group in the hydroxypropyl function.
Calculate the content of hydroxypropyl groups as a per-
centage (w/w, dried basis):
Result = (NX A2/A7) x (CG) x Wi/W) x (Mr2/Mn) x [100/
(100 — B)j x 100
N = numerical value representing the 3 methyl
groups in the internal standard (sodium
3-trimethylsilyl-1-propane sulfonate), 3
A2 = area of the methyl groups of hydroxypropyl in
Pregelatinized Hydroxypropyl Pea Starch
Ai = area of the methyl groups in the internal
standard (sodium 3-trimethylsilyl-1-propane
sulfonate)
G = concentration of the internal standard in the
Internal standard solution (mg/g)
WwW, = weight of the Internal standard solution in the
NMR tube (g)
Ww = weight of the Pregelatinized Hydroxypropyl
Pea Starch in the NMR tube (mg)
M2 = me mass of hydroxypropyl groups, 59.09 g/
mo
Ma = molecular weight of the internal standard,
218.32 g/mol
B = moisture content of the Pregelatinized
Hydroxypropyl Pea Starch used in the Sample
solution, as a percentage (w/w)
Acceptance criteria: 2.0%-7.0% of hydroxypropyl
groups on the dried basis
IMPURITIES
e RESIDUE ON IGNITION (281): NMT 0.6%, determined on a
1.0-g test specimen
e LIMIT OF IRON
Standard iron stock solution: Prepare a solution con-
taining the equivalent of 10 ug/mL of iron, as directed
under /ron (241).
Diluted standard iron solution: Immediately before
use, dilute an accurately measured volume of the Stan-
dard iron stock solution quantitatively with water to ob-
tain a solution containing the equivalent of 1 g/mL of
iron.
Sample solution: Shake the residue obtained from the
test for Residue on Ignition with 50 mL of 2 N hydro-
chloric acid, and filter. Transfer 10 mL of the filtrate to a
test tube. Add 2 mL of citric acid solution (2 in 10) and
0.1 mL of thioglycolic acid, and mix. Add 10 N ammo-
nium hydroxide until the solution is distinctly alkaline to
litmus, dilute with water to 20 mL, and mix.
Standard solution: Transfer 10 mL of the Diluted stan-
dard iron solution to a test tube. Add 2 mL of citric acid
solution (2 in 10) and 0.1 mL of thioglycolic acid, and
mix. Add 10 N ammonium hydroxide until the solution
is distinctly alkaline to litmus, dilute with water to
20 mL, and mix.
NF Monographs
Acceptance criteria: After 5 min, any pink color in the
Sample solution is not more intense than that in the
Standard solution, corresponding to a limit of 50 ug/g
(ppm) of iron.
e LIMIT OF SULFUR DIOXIDE, Method IV (525): NMT 50 ppm
e LIMIT OF PROPYLENE GLYCOL
Internal standard solution: 0.5 mg/mL of 1,3-
propanediol in anhydrous pyridine
Standard stock solution: 0.5 mg/mL of USP Propylene
Glycol RS in Internal standard solution
NF 36
Standard solution: Transfer 0.1 mL of the Standard
stock solution to a 2-mL vessel with a screw cap fitted
with a septum. Add 0.9 mL of anhydrous pyridine,
0.2 mL of hexamethyldisilazane, and 0.1 mL of trimeth-
ylchlorosilane. Close, and mix. Allow to stand for 15
min before injection.
Sample stock solution: Transfer 200 mg of Pregelati-
nized Hydroxypropyl Pea Starch to a 100-mL volumetric
flask. Add 1.0 mL of the Internal standard solution and
9.0 mL of anhydrous pyridine. Boil under reflux using a
water bath for 20 min. Allow to cool to room
temperature.
Sample solution: Transfer 1.0 mL of the Sample stock
solution to a 2-mL vessel with a screw cap fitted with a
septum. Add 0.2 mL of hexamethyldisilazane and
0.1 mL of trimethylchlorosilane. Close, and mix. Allow
to stand for 15 min before injection.
Chromatographic system
(See Chromatography (621), System Suitability).
Mode: GC
Detector: Flame ionization
Column: 0.32-mm x 30-m fused-silica capillary col-
umn; 0.25-um layer of phase G1
Temperature
Detector: 250°
Injection port: 250°
Column: 70°. [NoTeE—The column must be desorbed
regularly. Conditions: Program from 70° to 300° at
7°/min, and maintain 10 min at 300°.]
Carrier gas: Helium
Flow rate: 3 mL/min
Injection type: Split ratio of 1:30
Injection size: 1 ul
System suitability
Sample: Standard solution
[Note—The relative retention times for the trimethylsyli-
lated derivative of propylene glycol and the trimethyl-
sylilated derivative of 1,3-propanediol are 1.0 and 1.4,
respectively.]
Suitability requirements
Resolution: NLT 2.0 between the peaks due to the
trimethylsylilated derivative of propylene glycol and
the trimethylsylilated derivative of 1,3-propanediol
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of propylene glycol in the por-
tion of Pregelatinized Hydroxypropyl Pea Starch taken:
Result = (Ru/Rs) x (Cs/Cu) x 100
Ru = internal standard ratio (peak response of
propylene glycol/peak response of 1,3-
propanediol) from the Sample solution
Rs = internal standard ratio (peak response of
propylene glycol/peak response of 1,3-
propanediol) from the Standard solution
Cs = concentration of USP Propylene Glycol RS in
the Standard solution (mg/mL)
Cu = concentration of Pregelatinized Hydroxypropyl
Pea Starch in the Sample solution (mg/mL)
Acceptance criteria: NMT 0.1%
© LIMIT OF OXIDIZING SUBSTANCES
Sample: 4.0g
Analysis: Transfer the Sample to a glass-stoppered
125-mL conical flask, and add 50.0 mL of a mixture of
water and methanol (1:1). Insert the stopper, and swirl
for 5 min. Transfer to a glass-stoppered 50-mL centri-
fuge tube, and centrifuge to clarify. Transfer 30.0 mL of
the clear supernatant to a glass-stoppered 125-mL coni-
cal flask. Add 1 mL of glacial acetic acid and 0.5-1.0g
of potassium iodide. Insert the stopper, swirl, and allow
to stand for 25-30 min in the dark. Add 1 mL of starch
TS, and titrate with 0.002 N sodium thiosulfate VS to
the disappearance of the starch-iodine color. Perform a
blank determination, and make any necessary correc-
NF 36
tion. Each mL of 0.002 N sodium thiosulfate VS is
equivalent to 34 jg of oxidant, calculated as hydrogen
peroxide.
Acceptance criteria: NMT 1.4 mL of 0.002 N sodium
thiosulfate VS is required (20 ppm, calculated as H202).
SPECIFIC TESTS
e@ MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI-
FIED MICROORGANISMS (62): The total aerobic microbial
count does not exceed 103 cfu/g, the total combined
molds and yeasts count does not exceed 102 cfu/g, and
it meets the requirements of the test for the absence of
Escherichia coli.
© PH (791)
Sample solution: Progressively suspend 3.0 g of Prege-
latinized Hydroxypropyl Pea Starch in 100.0 mL of car-
bon dioxide-free water, stirring continuously. Determine
the pH when all the solid is wetted.
Acceptance criteria: 4.5-8.0
¢ Loss ON DRYING (731): Dry about 1 g at 130° for 90 min:
it loses NMT 15.0% of its weight.
ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Preserve in well-closed contain-
ers. Store at room temperature.
e USP REFERENCE STANDARDS (11)
USP Propylene Glycol RS
Potato Starch
Portions of the monograph text that are national USP text,
and are not part of the harmonized text, are marked with
symbols (*s) to specify this fact.
DEFINITION
Potato Starch is obtained from the tuber of Solanum tuber-
osum L.
IDENTIFICATION
© A. PROCEDURE
Analysis: Examine under a microscope using a mixture
of equal volumes of glycerol and water.
Acceptance criteria: It presents granules, either irregu-
larly shaped, ovoid or pear-shaped, usually 30-100 um
in size but occasionally exceeding 100 um, or rounded,
10-35 um in size. There are occasional compound
granules having 2-4 components. The ovoid and pear-
shaped granules have an eccentric hilum and the
rounded granules acentric or slightly eccentric hilum.
All granules show clearly visible concentric striations.
Between orthogonally oriented polarizing plates or
prisms, the granules showadistinct black cross inter-
secting at the hilum.
e B, PROCEDURE
Sample solution: 20 mg/mL in water
Analysis: Boil for 1 min, and cool.
Acceptance criteria: A thick, opalescent mucilage is
formed,
© C. PROCEDURE
Sample solution: 1 mL of the mucilage obtained in
Identification test B
Analysis: Add 0.05 mL of iodine and potassium iodide
TS2 to the Sample solution.
Acceptance criteria: An orange-red to dark blue color
is produced, which disappears upon heating.
IMPURITIES
e RESIDUE ON IGNITION (281): NMT 0.6%, determined on a
1.0-g sample
e LIMIT OF IRON
Standard iron stock solution A: Equivalent of 10 ug/
mL of iron prepared as directed under Iron (241)
Official Monographs / Starch 5609
Standard iron stock solution B: 1 g/mL of iron from
Standard iron stock solution A in water
[NoTte—Prepare immediately before use.]
Standard iron solution: Transfer 10 mL of Standard iron
stock solution B to a test tube, and add 2 mL of citric
acid solution (2 in 10) and 0.1 mL of thioglycolic acid.
Add 10 N ammonium hydroxide until the solution is
distinctly alkaline to litmus, and dilute with water to
20 mL.
Sample solution: Shake 1.5 g of Potato Starch with
15 mL of 2 N hydrochloric acid, and filter. Transfer
10 mL of the filtrate to a test tube, and add 2 mL of
citric acid solution (2 in 10) and 0.1 mL of thioglycolic
acid. Add 10 N ammonium hydroxide until the solution
i distinctly alkaline to litmus, and dilute with water to
0 mL.
Acceptance criteria: After 5 min, any pink color in the
Sample solution is not more intense than that in the
Standard iron solution, corresponding to a limit of
10 ppm of iron.
e LIMIT OF SULFUR DIOXIDE
Carbon dioxide: Use carbon dioxide, with a flow regu-
lator that will maintain a flow of 100+5 mL/min.
Bromophenol blue indicator solution: 0.2 mg/mL of
bromophenol blue in dilute alcohol. Filter if necessary.
Hydrogen peroxide solution: Dilute 30% hydrogen
ea lge with water to obtain a 3% solution. Just
efore use, add three drops of Bromophenol blue indica-
tor solution, and neutralize to a violet-blue endpoint
with 0.01 N sodium hydroxide. Do not exceed the
endpoint.
Apparatus: Figure 1
Figure 1
In this test, the sulfur dioxide is released from the sam-
ple in a boiling acid medium and is removed by a
stream of carbon dioxide. The separated gas is col-
lected in a dilute hydrogen peroxide solution where Zz
n
the sulfur dioxide is oxidized to sulfuric acid and ti-
trated with standard alkali. The apparatus consists es- =
sentially of a 500-mL three-neck, round-bottom boiling °
|
flask, A; a separatory funnel, B, maving a copay of fo)
100 mL or greater; a gas inlet tube of sufficient length ico}
=
to permit introduction of the carbon dioxide within 2
2.5 cm of the bottom of the boiling flask; a reflux con- a?)
=a
denser, C, having a jacket length of 200 mm; and a 7)
delivery tube, E, connecting the upper end of the re-
flux condenser to the bottom ofa receiving test tube,
 5610 Starch / Official Monographs NF 36

D. Apply a thin film of stopcock grease to the sealing e Loss ON DRYING (731)
surfaces of all of the jane except the joint between Sample: 1g
the separatory funnel and the boiling flask, and clamp Analysis: Dry the Sample at 130° for 90 min.
the joints to ensure tightness. Acceptance criteria: NMT 20.0%
Sample: 25.0 g of Potato Starch e PH (791)
Analysis: Add 150 mL of water to the boiling flask. Sample solution: Prepare a slurry by weighing 5.0 g of
Close the stopcock of the separatory funnel, and begin Potato Starch, transferring to a suitable nonmetallic
the flow of carbon dioxide at a rate of 100+5 mL/min container, and adding 25.0 mL of freshly boiled and
through the Apparatus. Start the condenser coolant cooled water.
flow. Add 10 mL of Hydrogen peroxide solution to a re- Analysis: Aoltate continuously at a moderate rate for 1
ceiving test tube. After 15 min, without interrupting the min. Stop the agitation, and allow to stand for 15 min.
flow of carbon dioxide, remove the separatory funnel Determine the pH to the nearest 0.1 unit.
from the boiling flask, and transfer the Sample into the Acceptance criteria: 5.0-8.0
boiling flask with the aid of 100 mL of water. Apply
stopcock grease to the outer joint of the separatory fun- ADDITIONAL REQUIREMENTS
nel, and replace the separatory funnel in the boiling © *PACKAGING AND STORAGE: Preserve in well-closed con-
flask. Close the stopcock of the separatory funnel, and tainers. No storage requirements specified.»
add 80 mL of 2 N hydrochloric acid to the separatory
funnel. Open the stopcock of the separatory funnel to
perenit the hydrochloric acid solution to flow into the
oiling flask, guarding against the escape of sulfur diox-
ide into the separatory funnel by closing the stopcock Hydroxypropyl! Potato Starch
before the last few mL of hydrochloric acid drain out.
Boil the mixture for 1 h. Remove the receiving test Amyiose derivative oH
tube, and transfer its contents to a 200-mL wide- on iy
ReHor He Noy
necked, conical flask. Rinse the receiving test tube with OH
a small portion of water, add the rinsing to the 200-mL ° Ri=Hor me 3
conical flask, and mix. Heat on a water bath for 15 Hy OH CHy
»— or’
min, and allow to cool. ‘Amylovectin derivative
Add 0.1 mL of Bromophenol blue indicator solution, and rd onl, Y OH 4 °
titrate the contents with 0.1 N sodium hydroxide VS = N
Rano “NNoy or HJ {70H
until the color changes from yellow to violet-blue. Per-
form a blank determination, and make any necessary RoR
correction (see Titrimetry (541)).
Calculate the content, in ppm, of sulfur dioxide in the For the Amylose derivative, m is about 300-1000.
Sample taken:
DEFINITION
Result = 1000 x 32.03 x (VN/W) Hydroxypropyl Potato Starch is partially substituted
2 ROE obtained from potato starch by a
32.03 = milliequivalent weight of sulfur dioxide chemical modification of etherification with Be lene ox-
Vv = volume of titrant consumed (mL) ide. In addition, this starch may be partially drolyzed
N = normality of the titrant using acids or enzymes to obtain thinned starch. It con-
Ww = weight of the Sample (g) tains NLT 2.0% and NMT 7.0% of hydroxypropyl groups,
Acceptance criteria: NMT 50 ppm on the dried basis.
© LIMIT OF OXIDIZING SUBSTANCES
Sample solution: Transfer 4.0 g to a glass-stoppered, IDENTIFICATION
125-mL conical flask, and add 50.0 mL of water. Insert e A. PROCEDURE
the stopper, and swirl for 5 min. Transfer to a glass- Analysis: Examine under a microscope, using NLT 20x
stoppered, 50-mL centrifuge tube, and centrifuge to magnification and a mixture of glycerin and water (1:1)
clarify. Transfer 30.0 mL of the clear supernatant to a as a mounting agent.
glass-stoppered, 125-mL conical flask. Add 1 mL of gla- Acceptance criteria: It presents granules, either irregu-
cial acetic acid and 0.5-1.0 g of potassium iodide. In- larly shaped, ovoid or pear-shaped, usually 30-100 pm
sert the stopper, swirl, and allow to stand for 25-30 in size, but occasionally exceeding 100 um, or rounded
min in the dark. Add 1 mL of starch TS. 10-35 um in size. There are occasional compound
Analysis: Titrate with 0.002 N sodium thiosulfate VS to granules having 2-4 components. The ovoid and pear-
the disappearance of the starch-iodine color. Perform a shaped granules have an eccentric hilum, and the
blank determination, and make any necessary correc- rounded granules have a centric or slightly eccentric hi-
tion. Each mL of 0.002 N sodium thiosulfate is equiva- lum. All granules show clearly visible concentric stria-
lent to 34 yg of oxidant, calculated as hydrogen tions. Between crossed nicol prisms, the Hydroxypropy|
peroxide. Potato Starch granules show a distinct black cross inter-
Acceptance criteria: NMT 1.4 mL of 0.002 N sodium secting at the hilum.
thiosulfate is required (20 ppm, calculated as H2O2). e B. PROCEDURE
Sample solution: Suspend 1 g of Hydroxypropyl Potato
SPECIFIC TESTS Starch in 50 mL of water, boil for 1 min, and cool.
e@ MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI- Acceptance criteria: A translucent or clear mucilage is
NF Monographs

FIED MICROORGANISMS (62): The total aerobic microbial formed.

count does not exceed 103 cfu/g; the total combined
molds and yeasts count does not exceed 10? cfu/g; and
it meets the requirements of the test for the absence of
Escherichia coli.
© C. PROCEDURE
Analysis: To 1 mL of the Sample solution obtained in
Identification test B add 0.05 mL of iodine and potas-
sium iodide TS 2.
Acceptance criteria: An orange-red to dark blue color
is produced, which disappears upon heating.
e D. PROCEDURE
Ninhydrin solution: Dissolve 3 g of ninhydrin in
100 mL of a 45.5-g/L solution of sodium metabisulfite.
NF 36
Diluted sulfuric acid: 98 g/L of H2SO4
Sample: 100mg of Hydroxypropyl Potato Starch
Analysis: Transfer the Sample to a 100-mL volumetric
flask, and add 12.5 mL of Diluted sulfuric acid. Place the
flask in a water bath, and heat until the Sample is dis-
solved. Cool, and dilute with water to 100 mL. [Cau-
TIioN—When sulfuric acid is miscible with water, it pro-
duces intense heat.]
Pipet 1 mL of this solution to a glass-stoppered, 25-mL
graduated test-tube and, with the tube immersed in
cold water, add drop-wise 8 mL of sulfuric acid. Mix
well, and place the tube in a boiling water bath for
exactly 3 min. Immediately transfer the tube to an ice
bath until the solution is chilled. Add 0.6 mL of
Ninhydrin solution, carefully allowing the reagent to
run down the walls of the test tube. Immediately
shake the tube well, and place it in a water bath at
25° for 100 min. Dilute with sulfuric acid to 25 mL
[CauTIon—Use sulfuric acid cautiously.], and mix by
inverting the tube several times. Do not shake.
Acceptance criteria: A violet color develops within 5
min due to the presence of hydroxypropyl groups
(starch ether).
ASSAY
© PROCEDURE FOR HYDROXYPROPYL GROUPS
Deuterium chloride solution: Dilute 1 mL of deuterium
chloride (38% w/w) with 5 mL of deuterium oxide.
Internal standard solution: Dissolve 50.0 mg of so-
dium 3-trimethylsilyl-1-propane sulfonate in about 5g
of deuterium oxide, weighed to the nearest 0.1 mg.
Store in a sealed bottle.
Sample solution: Disperse 20 g of Hydroxypropyl Po-
tato Starch in 200.0 mL of carbon dioxide-free water at
room temperature. Agitate for 15 min, and filter. Re-
peat the operation two more times. If poor dispersibility
or slow filtration is observed, use refrigerated carbon
dioxide-free water for the washing operation. Dry the
washed starch for NLT 4 h in vacuum at 30 + 5°. Deter-
mine the moisture content (B) on 5 g of the washed
and dried starch following the Loss on Drying test.
Weigh 12.0 mg of the washed and dried starch in a
5-mm NMR tube. Add 0.75 mL of deuterium oxide and
0.1 mL of Deuterium chloride solution. Cap the tube,
mix, and place it in a boiling water bath until a clear
solution is obtained. [NoTE—It may take 3 min-1 h.]
Whena clear solution is obtained, allow to cool to
room temperature. Dry the exterior of the tube, and
weigh to the nearest 0.1 mg. Add 0.05 mL of Internal
standard solution, and weigh to the nearest 0.1 mg. De-
termine the mass of the Internal standard solution
added. Mix thoroughly.
Nuclear magnetic resonance spectrometry
(See Nuclear Magnetic Resonance Spectroscopy (761),
Quantitative Applications.)
Apparatus: FI-NMR spectrometer at minimum
300 MHz
Acquisition of 'H NMR spectra: The following param-
eters may be used.
Sweep width: 8 ppm (about —1.0 to +7 ppm)
Irradiation frequency offset: None
Time domain: NLT 64 K
Pulse width: 90 degree
Pulse delay: 10s
Dummy scans: 0
Number of scans: 8
Use the CHs signal of the internal standard for shift
referencing. Set the shift of the peak of the singlet to
0 ppm. Record the FID signal.
Analysis
Samples: Internal standard solution and Sample solution
Call the integration sub-routine after phase corrections
and baseline correction between —-0.5 and +6 ppm.
Measure the peak areas of the doublet from the methyl
groups of the hydroxypropyl function at +1.2 ppm
Official Monographs / Starch 5611
(A2), and of the methyl groups at 0 ppm of the inter-
nal standard (Ai) without 13C-satellites.
Measure the signal coming from the 3 protons of the
ae group in the hydroxypropyl function.
Calculate the content of hydroxypropyl groups as a
percentage (w/w, dried basis):
Result = (N x A2/Ai) x (Ci x Wi/W) x (Mi2/Mn) x [100/
(100 — B)] x 100
N = numerical value representing the 3 methyl
groups in the internal standard (sodium
3-trimethylsilyl-1-propane sulfonate), 3
A2 = area of the methyl groups of hydroxypropyl in
Hydroxypropyl Potato Starch
A = area of the methyl groups in the internal
standard (sodium 3-trimethylsilyl-1-propane
sulfonate)
G = concentration of the internal standard in the
Internal standard solution (mg/g)
Wi = weight of the Internal standard solution in the
NMR tube (g)
WwW = weight of the washed and dried
Hydroxypropyl Potato Starch in the NMR
tube (mg)
Mn = molecular weight of the internal standard,
218.32 g/mo
M2 =ie mass of hydroxypropyl group, 59.09 g/
mo
B = moisture content of the washed and dried
Hydroxypropyl Potato Starch used in the
Sample solution, as a percentage (w/w)
Acceptance criteria: The content of hydroxypropyl
groups is 2.0%-7.0% on the dried basis.
IMPURITIES
Inorganic Impurities
e RESIDUE ON IGNITION (281): NMT 0.6%, determined on a
1.0-g test specimen
e LIMIT OF IRON
Standard iron stock solution: Prepare a solution con-
taining the equivalent of 10 ug/ml of iron, as directed
under Iron (241).
Diluted standard iron solution: Immediately before
use, dilute an accurately measured volume of Standard
iron stock solution quantitatively with water to obtain a
solution containing the equivalent of 1 ug/mL of iron.
Standard solution: Transfer 10 mL of the Diluted stan-
dard iron solution to a test tube. Add 2 mL of citric acid
solution (2 in 10) and 0.1 mL of thioglycolic acid, and
mix. Add 10 N ammonium hydroxide until the solution
is distinctly alkaline to litmus, dilute with water to
20 mL, and mix.
Sample solution: Shake 1.0 g of Hydroxypropyl Potato
Starch with 20 mL of 2 N hydrochloric acid, and filter.
Transfer 10 mL of the filtrate to a test tube. Add 2 mL
of citric acid solution (2 in 10) and 0.1 mL of thiogly-
colic acid, and mix. Add 10 N ammonium hydroxide
until the solution is distinctly alkaline to litmus, dilute
with water to 20 mL, and mix.
Acceptance criteria: After 5 min, any pink color in the
Sample solution is not more intense than that in the
ee solution, corresponding to a limit of 20 ug/g
of iron.
e LIMIT OF SULFUR DIOXIDE, Method !V (525): NMT 50 ppm 74
Organic Impurities ca]
© PROCEDURE 1: LIMIT OF OXIDIZING SUBSTANCES =
Sample: 4.0 g of Hydroxypropy! Potato Starch i}
Analysis: Transfer the Sample to a glass-stoppered, =
S
125-mL conical flask, and add 50.0 mL of water. Insert
the stopper, and swirl for 5 min. Transfer to a glass-
a=
i)
stoppered, 50-mL continue tube, and centrifuge to 2
clarify. Transfer 30.0 mL of the clear supernatant to a 7
glass-stoppered, 125-mL conical flask. Add 1 mL of gla- vw
cial acetic acid and 0.5-1.0 g of potassium iodide. In-
 4512 Cat’s Claw / Dietary Supplements USP 41

e USP REFERENCE STANDARDS (11) Chromatographic system

USP Isopteropodine RS
USP Powdered Cat’s Claw Extract RS
Cat's Claw Tablets
DEFINITION
Cat’s Claw Tablets contain Powdered Cat's Claw Extract.
Tablets contain NLT 90.0% and NMT 110.0% of the la-
beled amount of Powdered Extract, calculated as penta-
cyclic oxindole alkaloids.
IDENTIFICATION
e The Sample solution chromatogram exhibits peaks for
speciophylline, uncarine F, mitraphylline, isomitraphyl-
line, pteropodine, and isopteropodine at retention times
that correspond to those in Standard solution A, as ob-
tained in the test for Content of Pentacyclic Oxindole Alka-
loids and Limit of Tetracyclic Oxindole Alkaloids. The con-
tent oftetmneyelic oxindole alkaloids, calculated as the
sum of rhynchophylline and isorhynchophylline, is NMT
ae a the labeled amount of pentacyclic oxindole
alkaloids.
STRENGTH
© CONTENT OF PENTACYCLIC OXINDOLE ALKALOIDS AND LIMIT
OF TETRACYCLIC OXINDOLE ALKALOIDS
Solution A: Prepare a 10 mM pH 7.0 phosphate buffer
by mixing 1 N sodium hydroxide, 1M monobasic po-
tassium phosphate, and water (3:5:492), and adjust to
a pH of 7.0 + 0.1 by adding more of either solution.
Solution B: Acetonitrile
Solution C: Methanol and glacial acetic acid (99:1)
Mobile phase: See the gradient table below.
(See Chromatogra; (621), System Suitability.
Mode: LC sepny ” ma
Detector: UV 245 nm
eon 4.6-mm x 10-cm; endcapped 3-~m packing
Flow rate: 0.75 mL/min
Injection size: 10 uL
System suitability
Samples: Standard solution A and Standard solution B
Suitability requirements
Chromatogram similarity: The chromatogram ob-
tained using Standard solution A is similar to the Ref-
erence Chromatogram provided with the USP Pow-
dered Cat’s Claw Extract RS being used.
Tailing factor: NMT 2.0 for the isopteropodine peak,
Standard solution B
Relative standard deviation: NMT 2.0% from the
isopteropodine peak in repeated injections, Standard
solution B
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Measure the areas of the analyte peaks. Identify the re-
tention times of the peaks corresponding tosper
ophylline, uncarine F, mitraphylline, isomitraphylline,
pteropodine, isopteropodine, rhynchophylline, and
isorhynchophylline by comparison of the chromato-
gram of Standard solution A with the Reference Chro-
matogram provided with the lot of the USP Powdered
Cat’s Claw Extract RS being used.
Calculate the content, in mg, of specopny lites un-
carine F, mitraphylline, isomitraphylline, pteropodine,
and isopteropodine, as isopteropodine, in the portion
of Tablets taken:
Result = (ru/ts) x Cs x V

Time Solution A Solution B Solution C

tu = peak response for each relevant alkaloid from
the Sample solution
(min) (%) (%) (%) Is = peak response for isopteropodine from
0 65 35 0 Standard solution B
17 65 35 0 Cs = concentration of USP Isopteropodine RS in
25 50 50 0 Standard solution B (mg/mL)
30 50 50 0 Vv = final dilution volume of the Sample solution
31 0 0 100 (mL)
36 0 0 100
Calculate the content, in mg, of total pentacyclic
oxindole alkaloids (Cy) in the portion of Tablets taken
39 65 35 0 by adding the individual contents of Speen nn,
49 65 35. 0 uncarine F, mitraphylline, isomitraphylline,
pteropodine, and isopteropodine.
Standard solution A: Dissolve an accurately weighed
i=
as
Calculate the percentage of Powdered Cat's Claw
ra quantity of USP Powdered Cat’s Claw Extract RS in Extract with respect to the label claim:
i methanol, shake for 1 min, and dilute with methanol to
Dp
=
obtain a solution having a known concentration of Result = Cr x (Awr/W) x (100/Le) x (100/L)
° about 0.5 mg/mL of the labeled amount of total ox-
= indole alkaloids. Pass through afilter of 0.45-14m or
GC} finer pore size.
G = content of total pentacyclic oxindole alkaloids
= in the portion of Tablets taken (mg)
Standard solution B: 0.1 mg/mL of USP Isopteropodine Awr = average weight of Tablets (mg/Tablet)
ww RS in methanol. Passthrough a nylon filter of 0.45-m
a) Ww = weight of the portion of Tablets taken (mg)
or finer pore size. Le = content of total pentacyclic oxindole alkaloids,
Sample solution: Accurately weigh not fewer than 20 mg, in 100 mg of the Extract used to
Tablets and pulverize. Transfer an accurately weighed prepare the Tablets
quantity of the powder, equivalent to 20 mg of the la- L = amount of Extract per Tablet according to
beled amount of pentacyclic oxindole alkaloids, to a label claim (mg/Tablet)
50-mL centrifuge tube. Sonicate with 10 mL of metha- Calculate the percentage of tetracyclic oxindole
nol for 10 min. Centrifuge and transfer this solution to alkaloids with respect to the content of pentacyclic
a 50-mL volumetric flask. Repeat the above extraction oxindole alkaloids in the portion of Tablets taken:
three more times, combining the extracts in the 50-mL
volumetric flask, and dilute with methanol to volume. Result = (r1/rp) x 100
Transfer 3 mL of the solution to a test tube containing
300 mg of polyamide powder, and shake for 1 min. tr = sum of peak responses for rhynchophyiline
Pass through a nylon filter of 0.45-~m or finer pore and isorhynchophylline in the chromatogram
size, and discard the first part of the filtrate. of the Sample solution
 5612 Starch / Official Monographs
sert the stopper, swirl, and allow to stand for 25-30
min in the dark. Add 1 mL of starch TS, and titrate
with 0.002 N sodium thiosulfate VS to the disappear-
ance of the starch-iodine color. Perform a blank deter-
mination, and make any necessary correction. Each mL
of 0.002 N sodium thiosulfate VS is equivalent to 34 ug
of oxidant, calculated as hydrogen peroxide.
Acceptance criteria: NMT 1.4 mL of 0.002 N sodium
thiosulfate VS is required (20 g/g, calculated as H2O2).
© PROCEDURE 2: FOREIGN MATTER
Sample: 50 mg/mL of Hydroxypropy! Potato Starch in
a mixture of glycerin and water (1:1)
Analysis: Examine under a microscope, using NLT 20x
magnification and a mixture of glycerin and water
(1:1) as a mounting agent.
Acceptance criteria: NMT traces of matter other than
Hydroxypropy! Potato Starch granules are present.
SPECIFIC TESTS
e MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI-
FIED MICROORGANISMS (62): The total aerobic microbial
count does not exceed 103 cfu/g, the total combined
molds and yeasts count does not exceed 10? cfu/g, and
it meets the requirements of the test for the absence of
Escherichia coli.
© PH (791)
Sample solution: Suspend 5.0 g of Hydroxypropyl Po-
tato Starch in 25.0 mL of carbon dioxide-free water,
and shake for 60 s. Allow to stand for 15 min.
Acceptance criteria: 4.5-8.0
e Loss ON DRYING (731): Dry about 1 g at 130° for 90 min:
it loses NMT 20.0% of its weight.
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in well-closed contain-
ers. Store at room temperature.
Pregelatinized Hydroxypropyl Potato
Starch
DEFINITION
Pregelatinized Hydroxypropyl Potato Starch is prepared from
Hydroxypropyl Potato Starch by mechanical processing in
the presence of water, with or without heat, to rupture all
or some of the starch granules, and is subsequently dried.
It contains NLT 2.0% and NMT 7.0% of hydroxypropyl
groups on the dried basis.
IDENTIFICATION
© A. TEST FOR PREGELATINIZED STATE
Sample: 1g
Analysis: Disperse the Sample in 50 mL of water at a
temperature NMT 25°. Shake vigorously until lumps
completely disperse/solubilize or until lumps disappear.
Allow to stand for 20 min.
Acceptance criteria: A translucent or clear mucilage
without precipitate is formed.
e B, TEST FOR STARCH
Analysis: Disperse 0.5 g in 2 mL of water without heat-
ing, and add 0.05 mL of iodine and potassium iodide
TS2.
”“
wa Acceptance criteria: A reddish-violet or blue color is
a produced.
i]
— e C. NINHYDRIN TEST
Dp Ninhydrin solution: Dissolve 3g of ninhydrin in
fe) 100 mL of a 45.5-g/L solution of sodium metabisulfite.
= the methyl group in the hy howyprony! function.
2) Diluted sulfuric acid: 98 g/L of H2SO,
= Sample: 100mg
Analysis: Transfer the Sample to a 100-mL volumetric
—
‘a flask, and add 12.5 mL of Diluted sulfuric acid. Place the
NF 36
flask in a water bath, and heat until the Sample is dis-
solved. Cool, and dilute with water to 100 mL. [Cau-
TIioNn—When sulfuric acid is miscible with water, it pro-
duces intense heat.]
Pipet 1 mL of this solution to a glass-stoppered 25-mL
graduated test tube and, with the tube immersed in
cold water, add dropwise 8 mL of sulfuric acid. Mix
well, and place the tube in a boiling water bath for
exactly 3 min. Immediately transfer the tube to an ice
bath until the solution is chilled. Add 0.6 mL of
Ninhydrin solution, carefully allowing the reagent to
run down the walls of the test tube. Immediately
shake the tube well, and place it in a water bath at
25° for 100 min. Dilute with sulfuric acid to 25 mL.
[CauTIoN—Use sulfuric acid cautiously.] Mix by in-
verting the tube several times. Do not shake.
Acceptance criteria: A violet color develops within 5
i due to the
min hi presence of hydroxypropyl groups
(starch ether).
ASSAY
e ASSAY FOR HYDROXYPROPYL GROUPS
Deuterium chloride solution: Dilute 1 mL of deuterium
chloride (38% w/w) with 5 mL of deuterium oxide.
Internal standard solution: Disperse 50.0 mg of so-
dium 3-trimethylsilyl-1-propane sulfonate in about 5 g
of deuterium oxide, weighed to the nearest 0.1 mg.
Store in a sealed bottle.
Sample solution: Determine the moisture content (B)
on 5g of Pregelatinized Hydroxypropy! Potato Starch
following the Loss on Drying test. Weigh 12.0 mg of the
Pregelatinized Hydroxypropyl Potato Starch in a 5-mm
NMR tube. Add 0.75 mL of deuterium oxide and
0.1 mL of Deuterium chloride solution. Cap the tube,
mix, and place it in a boiling water bath until a clear
solution is obtained. [NoTE—This may take from 3 min
to 1 h.] Whena clear solution is obtained, allow it to
cool to room temperature. Dry the exterior of the tube,
and weigh to the nearest 0.1 mg. Add 0.05 mL of the
Internal standard solution. Weigh to the nearest 0.1 mg.
Determine the mass of the Internal standard solution
added. Mix thoroughly.
Instrumental conditions
(See Nuclear Magnetic Resonance Spectroscopy (761),
Quantitative Applications.)
Mode: Nuclear magnetic resonance spectrometry
oe FT-NMR spectrometer at minimum
0 MHz
Acquisition of 'H NMR spectra: The following param-
eters may be used:
Sweep width: 8 ppm (about -1.0 to +7 ppm)
Irradiation frequency offset: None
Time domain: NLT 64 K
Pulse width: 90°
Pulse delay: 10s
Dummy scans: 0
Number of scans: 8
Use the CH3 signal of the internal standard for shift
referencing. Set the shift of the peak of the singlet to
0 ppm. Record the FID signal.
Analysis
Samples: Internal standard solution and Sample solution
Call the integration subroutine after phase corrections
and baseline correction between —0.5 and +6 ppm.
Measure the peak areas of the doublet from the methyl
roups of thebydrexypropy! function at +1.2 ppm
tA), and of the methyl groups at 0 ppm of the inter-
nal standard (A;) without 13C-satellites.
Measure the signal originating from the 3 protons of
Calculate the content of hydroxypropyl groups as a per-
centage (w/w, dried basis):
Result = (N x A2/A1) x (Ci x Wi/W) x (Mr2/Mr) x [100/
(100 — B)] x 100
NF 36
N = numerical value representing the 3 methyl
roups in the internal standard (sodium
-trimethylsilyl-1-propane sulfonate), 3
A2 = area of the methyl groups of hydroxypropyl in
Pregelatinized Hydroxypropyl Potato Starch
Ai = area of the methyl groups in the internal
standard (sodium 3-trimethylsilyl-1-propane
sulfonate)
G = concentration of the internal standard in the
Internal standard solution (mg/g)
Ww, = weight of the Internal standard solution in the
NMR tube (g)
w = weight of the Pregelatinized Hydroxy ropyl
Potato Starch in the NMR tube (ab
M2 = molar mass of hydroxypropyl groups, 59.09 g/
mo
My = molecular weight of the internal standard,
218.32 g/mol
B = moisture content of the Pregelatinized
Hydroxypropyl Potato Starch used in the
Sample solution, as a percentage (w/w)
Acceptance criteria: 2.0%-7.0% of hydroxypropyl
groups on the dried basis
IMPURITIES
© RESIDUE ON IGNITION (281); NMT 0.6%, determined on a
1.0-g test specimen
© LIMIT OF IRON
Standard iron stock solution: Prepare a solution con-
taining the equivalent of 10 ug/ml of iron, as directed
under fron (241).
Diluted standard iron solution: Immediately before
use, dilute an accurately measured volume of the Stan-
dard iron stock solution quantitatively with water to ob-
tain a solution containing the equivalent of 1 g/mL of
iron.
Sample solution: Shake the residue obtained from the
test for Residue on Ignition with 20 mL of 2 N hydro-
chloric acid, and filter. Transfer 10 mL of the filtrate to a
test tube. Add 2 mL of citric acid solution (2 in 10) and
0.1 mL of thioglycolic acid, and mix. Add 10 N ammo-
nium hydroxide until the solution is distinctly alkaline to
litmus, dilute with water to 20 mL, and mix.
Standard solution: Transfer 10 mL of the Diluted stan-
dard iron solution to a test tube. Add 2 mL of citric acid
solution (2 in 10) and 0.1 mL of thioglycolic acid, and
mix. Add 10 N ammonium hydroxide until the solution
is distinctly alkaline to litmus, dilute with water to
20 mL, and mix.
Acceptance criteria: After 5 min, any pink color in the
Sample solution is not more intense than that in the
Standard solution, corresponding to a limit of 20 ppm of
iron.
e Limit OF SULFUR DioxiDE, Method IV (525): NMT 50 ppm
e LIMIT OF PROPYLENE GLYCOL
Internal standard solution: 0.5 mg/mL of 1,3-
propanediol in anhydrous pyridine
Standard stock solution: 0.5 mg/mL of USP Propylene
Glycol RS in Internal standard solution
Standard solution: Transfer 0.1 mL of the Standard
stock solution to a 2-mL vessel with a screw cap fitted
with a septum. Add 0.9 mL of anhydrous pyridine,
0.2 mL of hexamethyldisilazane, and 0.1 mL of trimeth-
ylchlorosilane. Close, and mix. Allow to stand for 15
min before injection.
Sample stock solution: Transfer 200 mg of Pregelati-
nized Hydroxypropyl Potato Starch to a 100-mL volu-
metric flask. Add i mL of the /nternal standard solution
and 9.0 mL of anhydrous pyridine. Boil under reflux us-
ing a water bath for 20 min. Allow to cool to room
temperature.
Sample solution: Transfer 1.0 mL of the Sample stock
solution to a 2-mL vessel with a screw cap fitted with a
septum. Add 0.2 mL of hexamethyldisilazane and
Official Monographs / Starch 5613
0.1 mL of trimethylchlorosilane. Close, and mix. Allow
to stand for 15 min before injection.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: GC
Detector: Flame ionization
Column: 0.32-mm x 30-m fused-silica capillary col-
umn; 0.25-1m layer of phase G1
Temperature
Detector: 250°
Injection port: 250°
Column: 70°. [NoTE—The column must be desorbed
regularly. Conditions: Program from 70° to 300° at
7°/min, and maintain 10 min at 300°.]
Carrier gas: Helium
Flow rate: 3 mL/min
Injection type: Split ratio of 1:30
Injection size: 1 pL
System suitability
Sample: Standard solution
[Not&—The relative retention times for the trimethylsyli-
lated derivative of Pippy ine glycol and the trimethyl-
sylilated derivative of 1,3-propanediol are 1.0 and 1.4,
respectively.]
Suitability requirements
Resolution: NLT 2.0 between the peaks due to the
trimethylsylilated derivative of propylene glycol and
the trimethylsylilated derivative of 1,3-propanediol
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of propylene glycol in the por-
tion of Pregelatinized Hydroxypropy! Potato Starch
taken:
Result = (Ru/Rs) x (Cs/Cu) x 100
Ru = internal standard ratio (peak response of
propylene glycol/peak response of 1,3-
propanediol) from the Sample solution
Rs = internal standard ratio (peak response of
propylene glycol/peak response of 1,3-
propanediol) from the Standard solution
Cs = concentration of USP Propylene Glycol RS in
the Standard solution (mg/mL)
Cu = concentration of Pregelatinized Hydroxypropyl
Potato Starch in the Sample solution (mg/mL)
Acceptance criteria: NMT 0.1%
© LIMIT OF OXIDIZING SUBSTANCES
Sample: 4.0g
Analysis: Transfer the Sample to a glass-stoppered
125-mL conical flask, and add 50.0 mL of a mixture of
water and methanol (1:1). Insert the stopper, and swirl
for 5 min. Transfer to a glass-stoppered 50-mL centri-
fuge tube, and centrifuge to clarify. Transfer 30.0 mL of
the clear supernatant to a glass-stoppered 125-mL coni-
cal flask. Add 1 mL of glacial acetic acid and 0.5-1.0g
of potassium iodide. Insert the stopper, swirl, and allow
to stand for 25-30 min in the dark. Add 1 mL of starch
TS, and titrate with 0.002 N sodium thiosulfate VS to
the disappearance of the starch-iodine color. Perform a
blank determination, and make any necessary correc-
tion. Each mL of 0.002 N sodium thiosulfate VS is
equivalent to 34 wg of oxidant, calculated as hydrogen
peroxide.
Acceptance criteria: NMT 1.4 mL of 0.002 N sodium
sydeubouo-= 4N
thiosulfate VS is required (20 ppm, calculated as H2O2).
SPECIFIC TESTS
e@ MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI-
FIED MICROORGANISMS (62): The total aerobic microbial
count does not exceed 103 cfu/g, the total combined
molds and yeasts count does not exceed 10? cfu/g, and
it meets the requirements of the test for the absence of
Escherichia coli.
 5614 Starch / Official Monographs NF 36

° PH (791) ADDITIONAL REQUIREMENTS

Sample solution: Progressively suspend 3.0g of Prege- ¢ PACKAGING AND STORAGE: Preserve in well-closed contain-
latinized ceeops Potato Starch in 100.0 mL of ers. No storage requirements specified.
carbon dioxide-free water, stirring continuously. Deter- e LABELING: Label it to indicate the botanical source from
mine the pH when all the solid is wetted. which it was derived.
Acceptance criteria: 4.5-8.0
e Loss ON DRYING (731): Dry about 1 g at 130° for 90 min:
it loses NMT 20.0% of its weight.
ADDITIONAL REQUIREMENTS Pregelatinized Modified Starch
© PACKAGING AND STORAGE: Preserve in well-closed contain-
ers. Store at room temperature.
e USP REFERENCE STANDARDS (11) DEFINITION
USP Propylene Glycol RS Pregelatinized Modified Starch is Modified Starch that has
been chemically or cay processed, or both, to
le to produce a product
rupture all or part of the granules
that swells in cold water.
IDENTIFICATION
Pregelatinized Starch eA.
Sample: 0.6g
DEFINITION Analysis: Transfer the Sample to a 25-mL glass vial with
Pregelatinized Starch is Starch that has been chemically and/ a plastic cap. Add 9.4 g of water, cap, and shake vigor-
or mechanically processed to rupture all or part of the ously to evenly disperse the starch. Add 10g of 2% (w/
granules in the presence of water and subsequently dried. w) NaOH solution, cap, and shake vigorously for 1 min
Some types of Pregelatinized Starch may be modified to to create a smooth mixture. Evaluate within 1 min.
render them compressible and flowable in character. Acceptance criteria: The final solution is translucent to
opaque with a fluid consistency. A yellow tint of the
IDENTIFICATION final solution is acceptable.
e A water slurry of it is colored orange-red to deep blue by e B. An aqueous dispersion of Pregelatinized Modified
iodine TS. Starch is colored orange-red to deep blue by iodine TS.
IMPURITIES IMPURITIES
Inorganic Impurities ¢ RESIDUE ON IGNITION (281)
e RESIDUE ON IGNITION (281): NMT 0.5%, determined on a Sample: 2.0+0.1g
2.0-g test specimen Acceptance criteria: NMT 1.5%
e IRON (241): NMT 20 ppm e LIMIT OF SULFUR DIOXIDE
Analysis: Dissolve the residue obtained in the test for Sample solution: Mix 20.0 + 0.1 g of Pregelatinized
Residue on Ignition in 8 mL of hydrochloric acid with Modified Starch with 100 mL of 95% alcohol, and stir
the aid of gentle heating, and dilute with water to for several min to completely wet the starch.
100 mL. Dilute 25 mL of this solution with water to Analysis: Slowly add 100 mL of water to the Sample
47 mL. solution, and stir until a smooth suspension is obtained.
e LIMIT OF SULFUR DIOXIDE Allow the starch mixture to set undisturbed until most
Sample solution: Mix 20g with 200 mL of a 1-in-5 so- of the starch has settled, and filter the aqueous portion
lution of anhydrous sodium sulfate, and filter. through paper (Whatman No. 1 or equivalent). To
Analysis: To 100 mL of the clear filtrate add 3 mL of 100 mL of the clear filtrate add 100 mL of water. Add
starch TS, and titrate with 0.01 N iodine VS to the first 3 mL of starch TS, and titrate with 0.01 N iodine VS to
permanent blue color. the first permanent blue or purple color.
Acceptance criteria: NMT 2.7 mL is consumed Acceptance criteria: NMT 1.7 mL of 0.010 N iodine is
(80 ppm). consumed (NMT 0.005%).
SPECIFIC TESTS SPECIFIC TESTS
e MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI- e PH (791)
FIED MICROORGANISMS (62): It meets the requirements of Sample: 10.040.1g
the tests for absence of Salmonella species and Escherichia Analysis: Wet the Sample with 10 mL of alcohol, then
coli. The total aerobic microbial count does not exceed dilute with water to 300 mL to obtain an aqueous dis-
1000 cfu/g; and the total combined molds and yeasts persion. Stir continuously at a moderate rate for 5 min,
count does not exceed 100 cfu/g. and determine the pH to the nearest 0.1 unit.
© PH (791): 4.5-7.0 Acceptance criteria: 3.0-9.0
Preparea slurrybY weighing 10.0 + 0.1 g in 10 mL of e Loss ON DRYING (731)
alcohol and by diluting with water to 100 mL. Agitate Analysis: Dry at 120° for 4 h.
continuously at a moderate rate for 5 min, then cease Acceptance criteria: NMT 15%
agitation and immediately potentiometrically determine e MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI-
the pH to the nearest 0.1 unit. FIED MICROORGANISMS (62): The total aerobic microbial
e Loss ON DRYING (731): Dry a sample at 120° for 4 h: it count does not exceed 1 x 103 cfu/g, and the total com-
loses NMT 14.0% of its weight. bined molds and yeasts count does not exceed 1 x 102
NF Monographs

© OXIDIZING SUBSTANCES
Sample: 5g
Analysis: To the Sample add 20 mL of a mixture of
equal volumes of methanol and water, then add 1 mL
of 6 N acetic acid, and stir until a homogeneous sus-
pension is obtained. Add 0.5 mL of atreshly prepared,
saturated solution of potassium iodide, and allow to
stand for 5 min.
Acceptance criteria: No distinct blue, brown, or purple
color is observed.
cfu/g. It meets the requirements of the tests for absence
of Salmonella species and Escherichia coli.
e IRON (241)
Sample: The residue obtained in the test for Residue on
Ignition (281)
Analysis: Dissolve the Sample in 8 mL of hydrochloric
acid with the aid of gentle heating. Dilute with water to
100 mL in a volumetric flask. Dilute 25 mL of this solu-
tion with water to 47+1 mL.
NF 36
Acceptance criteria: NMT 20 g/g
© OXIDIZING SUBSTANCES
Sample solution: To 5g of Pregelatinized Modified
er add 20 mL of a mixture of methanol and water
Ct
Analysis: To the Sample solution add 1 mL of 6 N acetic
acid, and stir until a homogeneous suspension is ob-
tained. Add 0.5 mL ofa freshly prepared saturated solu-
tion of potassium iodide, and allow to stand for 5 min.
Acceptance criteria: No distinct blue, brown, or purple
color is observed.
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in well-closed contain-
ers. No storage requirements specified.
Rice Starch
Portions of this monograph that are national USP text, and
are not part of the harmonized text, are marked with
symbols (*») to specify this fact.
DEFINITION
Rice Starch is obtained from the caryopsis of Oryza sativa L.
IDENTIFICATION
e A. PROCEDURE
Analysis: Examine under a microscope, using NLT 20x
magnification and using a mixture of glycerin and
water (1:1) as a mounting agent.
Acceptance criteria: It presents polyhedral, simple
grains 1-10 um, mostly 4-6 um in size. These simple
grains often gather in ellipsoidal, compound grains
50-100 um in diameter. The granules have a poorly
visible central hilum and there are no concentric stria-
tions. Between orthogonally orientated polarising pates
or prisms, the starch granules showadistinct blacl
cross intersecting at the hilum.
e B. PROCEDURE
Sample solution: 20 mg/mL in water
Analysis: Boil for 1 min, and cool.
Acceptance criteria: A thin, cloudy mucilage is formed.
© C. PROCEDURE
Sample solution: 1 mL of the mucilage obtained in
Identification test B
Analysis: Add 0.05 mL of iodine and potassium iodide
TS 2 to the Sample solution.
Acceptance criteria: An orange-red to dark blue color
is produced, which disappears upon heating.
IMPURITIES
Inorganic Impurities
e RESIDUE ON IGNITION (281): NMT 0.6%, determined on a
1.0-g sample
e LIMIT OF IRON
Standard iron stock solution A: Equivalent to 10 ug/
mL of iron prepared as directed in Iron (241)
Standard iron stock solution B: 1 g/mL of iron from
Standard iron stock solution A in water. [NoTE—Prepare
immediately before use.]
Standard iron solution: Transfer 10 mL of Standard
iron stock solution B to a test tube, and add 2 mL of
citric acid solution (2 in 10) and 0.1 mL of thioglycolic
acid. Add 10 N ammonium hydroxide until the solution
is distinctly alkaline to litmus, and dilute with water to
20 mL.
Sample solution: Shake 1.5 g of Rice Starch with
15 mL of 2.N hydrochloric acid, and filter. Transfer
10 mL of the filtrate to a test tube, and add 2 mL of
citric acid solution (2 in 10) and 0.1 mL of thioglycolic
acid. Add 10 N ammonium hydroxide until the solution
is distinctly alkaline to litmus, and dilute with water to
20 mL.
Official Monographs / Starch 5615
Acceptance criteria: After 5 min, ary pink color in the
Sample solution is not more intense than that in the
Standard iron solution, corresponding toa limit of
10 ppm of iron.
e LIMIT OF SULFUR DIOXIDE
Carbon dioxide: Use carbon dioxide, with a flow regu-
lator that will maintain a flow of 100 +5 mL/min.
Hydrogen peroxide solution: Dilute 30% hydrogen
ee with water to obtain a 3% solution. Just
efore use, add 3 drops of bromophenol blue TS, and
neutralize to a violet-blue endpoint with
0.1 N sodium hydroxide. Do not exceed the endpoint.
Apparatus: See Figure 1.
Figure 1
In this test, the sulfur dioxide is released from the sam-
ple in a boiling acid medium and is removed by a
stream of carbon dioxide. The separated gas is col-
lected in a dilute hydrogen peroxide solution where
the sulfur dioxide is oxidized to sulfuric acid and ti-
trated with standard alkali. The apparatus consists es-
sentially of a 500-mL three-neck, round-bottom boil-
ing flask, A; a separatory funnel, B, with a capacity of
100 mL or greater; a gas inlet tube of sufficient
lenge to permit introduction of the carbon dioxide
within 2.5 cm of the bottom of the boiling flask; a
reflux condenser, C, with a jacket length of 200 mm;
and a delivery tube, E, connecting the upper end of
the reflux condenser to the bottom of a receiving test
tube, D. Apply a thin film of stopcock grease to the
sealing surfaces of all of the joints except the joint
between the separatory funnel and the boiling flask,
and clamp the joints to ensure tightness.
Sample: 25.0 g of Rice Starch
Analysis: Add 150 mL of water to the boiling flask.
Close the stopcock of the separatory funnel, and begin
the flow of carbon dioxide at a rate of 100+5 mL/min
sydeibouow- 4N
through the Apparatus. Start the condenser coolant
flow. Add 10 mL of Hydrogen peroxide solution to a re-
ceiving test tube. After 15 min, without interrupung
the flow of carbon dioxide, remove the separatory fun-
nel from the boiling flask, and transfer the Sample into
the boiling flask with the aid of 100 mL of water. Apply
stopcock grease to the outer joint of the separatory
funnel, and replace the separatory funnel in the boiling
flask. Close the sppecsk of the separatory funnel, and
add 80 mL of 2.N hydrochloric acid to the separatory
funnel. Open the stopcock of the separatory funnel to
 5616 Starch / Official Monographs
pam the hydrochloric acid solution to flow into the
oiling flask, guarding against the escape of sulfur di-
oxide into the separatory funnel by closing the stop-
cock before the last few mL of hydrochloric acid drain
out. Boil the mixture for 1 h. Remove the receiving test
tube, and transfer its contents to a 200-mL wide-
necked, conical flask. Rinse the receiving test tube with
a small portion of water, add the rinsing to the 200-mL
conical flask, and mix. Heat on a water bath for 15
min, and allow to cool.
Add 0.1 mL of bromophenol blue TS, and titrate the
contents with 0.1 N sodium hydroxide VS until the
color changes from yellow to violet-blue. Perform a
blank determination, and make any necessary correc-
tion (see Titrimetry (541)).
Calculate the content, in ppm, of sulfur dioxide in the
Sample taken:
Result
= (Vx Nx FA)/W
x 1000
volume of titrant consumed (mL)
=rzS
normality of the titrant
ou
milliequivalent weight of sulfur dioxide, 32.03
weight of the Sample (g)
Acceptance criteria: NMT 50 ppm
¢ LIMIT OF OXIDIZING SUBSTANCES
Sample solution: Transfer 4.0 g to a glass-stoppered,
125-mL conical flask, and add 50.0 mL of water. Insert
the stopper, and swirl for 5 min. Transfer to a glass-
stoppered, 50-mL centrifuge tube, and centrifuge to
a Transfer 30.0 mL of the clear supernatant to a
glass-stoppered, 125-mL conical flask. Add 1 mL of gla-
cial acetic acid and 0.5-1 Og of potassium iodide. In-
sert the stopper, swirl, and allow to stand for 25-30
min in the dark. Add 1 mL of starch TS.
Analysis: Titrate with 0.002 N sodium thiosulfate VS to
the disappearance of the starch-iodine color. Perform a
blank determination, and make any necessary correc-
tion. Each mL of 0.002 N sodium thiosulfate is equiva-
lent to 34 ug of oxidant, calculated as hydrogen
peroxide.
Acceptance criteria: NMT 1.4 mL of 0.002 N sodium
thiosulfate is required [20 ppm, calculated as hydrogen
peroxide (H.0.)1
SPECIFIC TESTS
e MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI-
FIED MICROORGANISMS (62): The total aerobic microbial
count does not exceed 103 cfu/g; the total combined
molds and yeasts count does not exceed 102 cfu/g; and
it meets the requirements of the test for the absence of
Escherichia coli.
e Loss ON DRYING (731)
Sample: 1g
Analysis: Dry the Sample at 130° for 90 min.
Acceptance criteria: NMT 15.0%
e PH (791)
Sample solution: Prepare a slurry by weighing 5.0 g of
Rice Starch, transferring to a suitable nonmetallic con-
tainer, and adding 25.0 mL of freshly boiled and cooled
water.
Analysis: Aaitate continuously at a moderate rate for 1
min. Stop the agitation, and allow to stand for 15 min.
Determine the pH to the nearest 0.1 unit.
Acceptance criteria: 5.0-8.0
NF Monographs
ADDITIONAL REQUIREMENTS
¢ *PACKAGING AND STORAGE: Preserve in well-closed con-
tainers. No storage requirements specified.
NF 36
Tapioca Starch
DEFINITION
Tapioca Starch consists of starch Soars separated from
the tubers of tapioca (cassava) [Manihot utilissima Pohl
(Fam. Euphorbiaceae)].
IDENTIFICATION
cA.
Analysis: Examine Tapioca Starch under a microscope,
using not less than 20x magnification and using glyc-
erin as the mounting agent.
Acceptance criteria: It appears as spherical granules,
each having one truncated side, typically having a 5- to
35-~um diameter, and having circular or several-rayed
central clefts.
° B.
Sample suspension: 1 g of Tapioca Starch in 50 mL of
water
Analysis: Boil the Sample suspension for 1 min, and
cool.
gee criteria: A thin, cloudy mucilage is formed.
° .
Sample: The mucilage obtained in Identification test B
Analysis: To 1 mL of the Sample add 0.05 mL of iodine
and potassium iodide TS 2.
Acceptance criteria: An orange-red to dark blue color
is produced, which disappears on heating.
IMPURITIES
e RESIDUE ON IGNITION (281)
Sample: 1.0g
Acceptance criteria: NMT 0.6%
© IRON (241)
Test preparation: Shake 0.75 g of Tapioca Starch with
15 mL of 0.1. N hydrochloric acid, filter, and use 10 mL.
Acceptance criteria: NMT 20 g/g
© Limit OF OXIDIZING SUBSTANCES
Sample: 4.0g
Blank: 50 mL of water
Titrimetric system
(See Titrimetry (541).)
Mode: Direct titration
Titrant: 0.002 N sodium thiosulfate VS
Endpoint detection: Visual
Analysis: Transfer the Sample to a glass-stoppered,
125-mL conical flask, and add 50.0 mL of water. Insert
the stopper, and swirl for 5 min. Decant into a glass-
stoppered, 50-mL centrifuge tube, and centrifuge to
clarity. Transfer 30.0 mL ofthe clear supernatant to a
glass-stoppered, 125-mL conical flask. Add 1 mL of gla-
cial acetic acid and 0.5-1.0 g of potassium iodide. In-
sert the stopper, swirl, and allow to stand for 25-30
min in the dark. Add 1 mL of starch TS, and titrate with
Titrant to the disappearance of the starch-iodine color.
Perform a blank determination, and make any necessary
correction. Each mL of 0.002 N sodium thiosulfate VS is
equivalent to 34 tg of oxidant, calculated as hydrogen
peroxide.
Acceptance criteria: NMT 1.4 mL of 0.002 N sodium
thiosulfate VS is required (0.002%).
e LIMIT OF SULFUR DIOXIDE
Sample solution: Mix 20g of Tapioca Starch with
200 mL of water until a smooth suspension is obtained,
and filter.
Analysis: To 100 mL of the clear filtrate from the Sam-
ple solution add 3 mL of starch TS, and titrate with 0.01
N iodine solution VS to the first permanent blue color.
Acceptance criteria: NMT 1.7 mL of 0.01 N iodine so-
lution VS is required (0.005%).
SPECIFIC TESTS
e MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI-
FIED MICROORGANISMS (62): The total aerobic microbial
NF 36
count does not exceed 103 cfu/g, and the total com-
bined yeasts and molds count does not exceed 10? cfu/
g. Tapioca Starch meets the requirements of the test for
absence of Escherichia coli.
e PH (791)
Sample: 20.0+0.1g
Analysis: Transfer the Sample to a suitable nonmetallic
container, and add 100 mL of water to obtaina slurry.
Agitate continuously at a moderate rate for 5 min, then
stop agitation, and immediately determine the pH.
Acceptance criteria: 4,.5-7.0
e Loss ON DRYING (731)
Analysis: Dry at 130° for 90 min.
Acceptance criteria: NMT 16.0%
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in well-closed contain-
ers. No storage requirements specified.
e LIMIT OF SULFUR DIOXIDE
Topical Starch—see Topical Starch General
Monographs
Wheat Starch
Portions of the monograph text that are national USP text,
and are not part of the harmonized text, are marked with
symbols (*s) to specify this fact.
DEFINITION
Wheat starch is obtained from the caryopsis of Triticum aes-
tivum L. (T. vulgare Vill.).
IDENTIFICATION
e A, PROCEDURE
Analysis: Examine under a microscope using equal
volumes of glycerol and water.
Acceptance criteria: |t presents large and small gran-
ules, and, very rarely, intermediate sizes. The large
granules, usually 10-60 um in diameter, are discoid or,
more rarely, reniform when seen face-on. The central
hilum and striations are invisible or barely visible, and
the granules sometimes show cracks on the edges. Seen
in profile, the granules are elliptical and fusiform and
the hilum appears as a slit along the main axis. The
small granules, rounded or polyhedral are 2-10 um in
diameter. Between orthogonally oriented polarizing
plates or prisms, the granules showa distinct black
cross intersecting at the hilum.
e B. PROCEDURE
Sample solution: 20 mg/mL in water
Analysis: Boil for 1 min, and cool.
Acceptance criteria: A thin, cloudy mucilage is formed.
© C. PROCEDURE
Sample solution: 1 mL of the mucilage obtained in
Identification test B
Analysis: Add 0.05 mL of iodine and potassium iodide
TS 2 to the Sample solution.
Acceptance criteria: An orange-red to dark blue color
is produced, which disappears upon heating.
IMPURITIES
© RESIDUE ON IGNITION (281): NMT 0.6%, determined on a
1.0-g sample
© LIMIT OF IRON
Standard iron stock solution A: Equivalent to 10 ug/
mL of iron prepared as directed under Iron (241)
Standard iron stock solution B: 1 g/mL of iron from
Standard iron stock solution A in water
Official Monographs / Starch 5617
[Note—Prepare immediately before use.]
Standard iron solution: Transfer 10 mL of Standard iron
stock solution B to a test tube, and add 2 mL of citric
acid solution (2 in 10), and 0.1 mL of thioglycolic acid.
Add 10 N ammonium hydroxide until the solution is
distinctly alkaline to litmus, and dilute with water to
20 mL.
Sample solution: Shake 1.5 g of Wheat Starch with
15 mL of 2.N hydrochloric acid, and filter. Transfer
10 mL of the filtrate to a test tube, and add 2 mL of
citric acid solution (2 in 10) and 0.1 mL of thioglycolic
acid. Add 10 N ammonium hydroxide until the solution
is distinctly alkaline to litmus, and dilute with water to
20 mL.
Acceptance criteria: After 5 min, ate pink color in the
Sample solution is not more intense than that in the
Standard iron solution, corresponding to a limit of
10 ppm of iron.
Carbon dioxide: Use carbon dioxide, with a flow regu-
lator that will maintain a flow of 100+5 mL/min.
Bromophenol blue indicator solution: 0.2 mg/mL of
bromophenol blue in dilute alcohol. Filter if necessary.
Hydrogen peroxide solution: Dilute 30% hydrogen
pseu with water to obtain a 3% solution. Just
efore use, add 3 drops of Bromophenol! blue indicator
solution, and neutralize to a violet-blue endpoint with
0.01 N sodium hydroxide. Do not exceed the endpoint.
Apparatus: Figure 1
Figure 1
In this test, the sulfur dioxide is released from the sam-
ple in a boiling acid medium and is removed by a
stream of carbon dioxide. The separated gas is col-
lected in a dilute hydrogen peroxide solution where
the sulfur dioxide is oxidized to sulfuric acid and ti-
trated with standard alkali. The apparatus consists es-
sydesbouo= 4N
sentially of a 500-mL three-neck, round-bottom boiling
flask, A; a separatory funnel, B, having a capacity of
100 mL or greater; a gas inlet tube of sufficient length
to permit introduction of the carbon dioxide within
2.5 cm of the bottom of the boiling flask; a reflux con-
denser, C, having a jacket length of 200 mm; and a
delivery tube, E, connecting the upper end of the re-
flux condenser to the bottom ofa receiving test tube,
D. Apply a thin film of stopcock grease to the sealing
surfaces of all of the joints except the joint between
 5618 Starch / Official Monographs
the separatory funnel and the boiling flask, and clamp
the joints to ensure tightness.
Sample: 25.0 g of Wheat Starch
Analysis: Add 150 mL of water to the boiling flask.
Close the stopcock of the separatory funnel, and begin
the flow of carbon dioxide at a rate of 100+ 5 mL/min
through the Apparatus. Start the condenser coolant
flow. Add 10 mL of Hydrogen peroxide solution to a re-
ceiving test tube. After 15 min, without interrupting the
flow of carbon dioxide, remove the separatory funnel
from the boiling flask, and transfer the Sample into the
boiling flask with the aid of 100 mL of water. Apply
stopcock grease to the outer joint of the separatory fun-
nel, and replace the separatory funnel in the boiling
flask. Close the ee of the separatory funnel, and
add 80 mL of 2 N hydrochloric acid to the separatory
funnel. Open the stopcock of the separatory funnel to
pematt the hydrochloric acid solution to flow into the
oiling flask, guarding against the escape of sulfur diox-
ide into the separatory funnel by closing the stopcock
before the last few mL of hydrochloric acid drain out.
Boil the mixture for 1 h. Remove the receiving test
tube, and transfer its contents to a 200-mL wide-
necked, conical flask. Rinse the receiving test tube with
a small portion of water, add the rinsing to the 200-mL
conical flask, and mix. Heat on a water bath for 15
min, and allow to cool. Add 0.1 mL of Bromophenol blue
indicator solution, and titrate the contents with 0.1 N
sodium hydroxide VS until the color changes from yel-
low to violet-blue. Perform a blank determination, and
make any necessary correction (see Titrimetry (541)).
Calculate the content, in ppm, of sulfur dioxide in the
Sample taken:
Result = 1000 x 32.03 x (VN/W)
32.03 = milliequivalent weight of sulfur dioxide
Vv = volume of titrant consumed (mL)
N = normality of the titrant
w = weight of the Sample (g)
Acceptance criteria: NMT 50 ppm
e LIMIT OF OXIDIZING SUBSTANCES
Sample solution: Transfer 4.0 g to a glass-stoppered,
125-mL conical flask, and add 50.0 mL of water. Insert
the stopper, and swirl for 5 min. Transfer to a glass-
stoppered, 50-mL centrifuge tube, and centrifuge to
clarify. Transfer 30.0 mL of the clear supernatant to a
glass-stoppered, 125-mL conical flask. Add 1 mL of gla-
cial acetic acid and 0.5-1.0 g of potassium iodide. In-
sert the stopper, swirl, and allow to stand for 25-30
min in the dark. Add 1 mL of starch TS.
Analysis: Titrate with 0.002 N sodium thiosulfate VS to
the disappearance of the starch-iodine color. Perform a
blank determination, and make any necessary correc-
tion. Each mL of 0.002 N sodium thiosulfate is equiva-
lent to 34 ug of oxidant, calculated as hydrogen
peroxide.
Acceptance criteria: NMT 1.4 mL of 0.002 N sodium
thiosulfate is required (20 ppm, calculated as H202).
SPECIFIC TESTS
e MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI-
FIED MICROORGANISMS (62): The total aerobic microbial
count does not exceed 103 cfu/g; the total combined
molds and yeasts count does not exceed 10? cfu/g; and
NF Monographs
it meets the requirements of the test for the absence of
Escherichia coli.
e Loss ON DRYING (731)
Sample: 1g :
Analysis: Dry the Sample at 130° for 90 min.
Acceptance criteria: NMT 15.0%
© PH (791)
Sample solution: Prepare a slurry by weighing 5.0 g of
Wheat Starch, transferring to a suitable nonmetallic
NF 36
container, and adding 25.0 mL of freshly boiled and
cooled water.
Analysis: la continuously at a moderate rate for 1
min. Stop the agitation, and allow to stand for 15 min.
Determine the pH to the nearest 0.1 unit.
Acceptance criteria: 4.5-7.0
© TOTAL PROTEIN
Analysis: Weigh 6.0 g of sample containing 2 mg of ni-
trogen; transfer to a combustion flask; add 4 g of a
owdered mixture consisting of 100 g of potassium sul-
ate, 5g of cupric sulfate, and 2.5 g of selenium; and
add three glass beads. Wash any adhering particles
from the neck into the flask with 5 mL of sulfuric acid,
allowing it to run down the sides of the flask, and mix
the contents by rotation. Close the mouth of the flask
loosely, for example by means of a glass bulb with a
short stem, to avoid excessive loss of sulfuric acid. Heat
gradually at first, then increase the temperature until
there is vigorous boiling with condensation of sulfuric
acid in the neck of the flask; precautions should be
taken to prevent the upper part of the flask from be-
coming overheated. Continue the heating for 30 min,
unless otherwise prescribed. Cool, dissolve the solid ma-
terial by cautiously adding to the mixture 25 mL of
water, cool again, and place in a steam distillation ap-
paratus. Add 30 mL of sodium hydroxide solution (42 in
100), and distill immediately by passing steam through
the mixture. Collect 40 mL of distillate in 20.0 mL of
0.01. N eoeio acid and enough water to cover
the tip of the condenser. Toward the end of the distilla-
tion, lower the receiver so that the tip of the condenser
is above the surface of the acid. Take precautions to
prevent any water on the outer surface of the con-
denser from reaching the contents of the receiver. Ti-
trate the distillate with 0.01 N sodium hydroxide, using
methyl purple TS as the indicator (n; mL of 0.01 N
sodium hydroxide).
Repeat the test using 50 mg of glucose in place of the
substance to be examined (nz mL of 0.01 N sodium
hydroxide).
Content of nitrogen = [0.01401 x (nz — m)]/m
m = amount of test substance weighed g)
Acceptance criteria: NMT 0.3% (corresponding to
0.048% Nz, conversion factor: 6.25)
ADDITIONAL REQUIREMENTS
e *PACKAGING AND STORAGE: Preserve in well-closed con-
tainers. No storage requirements specified.
Stearic Acid
Portions of this monograph that are national USP text, and
are not part of the harmonized text, are marked with
symbols (%) to specify this fact.
Octadecanoic acid;
Stearic acid [57-11-4].
DEFINITION
Mixture consisting of stearic (octadecanoic) acid (CisH36O2;
M,, 284.5) and palmitic (hexadecanoic) acid (Ci6H3202;
M,, 256.4) obtained from fats or oils of vegetable or
animal origin.
NF 36 Official Monographs / Stearic 5619

Content: Chromatographic system

(See Chromatography (621), System Suitability.)
Stearic acid: 40.0%-60.0%. Sum of the Mode: GC
contents of stearic acid and palmitic acids: Detector: Flame ionization
Stearic acid 50 NLT 90.0%. Column: 30-m x 0.32-mm fused silica coated with a
Stearic acid: 60.0%-80.0%. Sum of the
0.5-um layer of stationary phase G16
Temperatures
contents of stearic and palmitic acids: NLT
Stearic acid 70
Injection port: 220°
90.0%.
Detector: 260°
Stearic acid: NLT 90.0%. Sum of the contents Column: See Table 7.
of stearic acid and palmitic acids: NLT
Stearic acid 95 96.0%.
Table 1
*{NoTE—Stearic Acid labeled solely for external use is ex- Hold Time
empt from the requirement that it be prepared from edible Initial Temperature Final at Final
sources.*] Temperature Ramp Temperature | Temperature
IDENTIFICATION (@) (/min) @ (min)
° A. It meets the requirements of the test for Freezing 70 = 70 2
Point. 70 5 240 5
e B. AcID VALUE
Light petroleum: Use a sample that has the following Carrier gas: Helium, passed through a bed of molecu-
properties: a clear, colorless, liquid without fluores- lar sieve for drying, if necessary
cence; practically insoluble in water; miscible with alco- Flow rate: 2.4 mL/min
hol; density at 20° about 0.720; distillation range 100°- Injection volume: 1 wL
120°; water content NMT 0.03%.1 System suitability
Sample solution: Dissolve 0.5 g of Stearic Acid in Sample: Standard solution
50 mL of a mixture of equal volumes of alcohol and Suitability requirements
Lightpetiole previously neutralized with 0.1 N potas- Resolution: NLT 5.0 between the methyl palmitate
sium hydroxide or 0.1 N sodium hydroxide, using and methyl stearate peaks determined on six
0.5 mL of phenolphthalein TS as indicator. If necessary, injections
heat to about 90° to dissolve the substance to be Relative standard deviation: NMT 3.0% for the
examined. methyl stearate and methyl palmitate peaks, from six
Analysis: Titrate the Sample solution with 0.1 N poe replicate injections; NMT 1.0% for the ratio of the
sium hydroxide or 0.1 N sodium hydroxide until the peak areas of methyl palmitate to the peak areas of
ink color persists for at least 15 s. When heating has methyl stearate, from six replicate injections
een applied to aid dissolution, maintain the tempera- Analysis
ture at about 90° during the titration. Sample: Sample solution
Calculate the acid value (/,) of the portion of Stearic Calculate the percentage of stearic acid (CisH36O2) in
Acid taken: the portion of Stearic Acid taken:

Result = (n/m) x N x M, Result = (As/Ar) x 100

n amount of titrant used (mL) As = peak area due to methyl stearate

m amount of Stearic Acid taken to prepare the Ar = sum of the peak areas of all the fatty acid
Sample solution (g) esters
N = normality of the potassium hydroxide solution Calculate the percentage of palmitic acid (CisH3202) in
M, = molecular weight of potassium hydroxide, the portion of Palmitic Acid taken:
56.11
Acceptance criteria: 194-212 Result = (Ap/Ar) x 100
e C. The retention times of the major peaks of the Sample
solution correspond to those of the Standard solution, as Ap = peak area due to methyl palmitate
obtained in the Assay. Ar = sum of the peak areas of all the fatty acid
esters
ASSAY Acceptance criteria
© PROCEDURE For Stearic acid 50: 40.0-60.0% of stearic (octadeca-
Boron trifluoride-methanol solution: 140 g/L of bo- noic) acid (CisH36Q2), and the sum of the stearic acid
ron trifluoride in methanol and palmitic acid is NLT 90.0%
Sample solution: Dissolve 100 mg of Stearic Acid in a For Stearic acid 70: 60.0-80.0% of stearic (octadeca-
small conical flask fitted with a suitable reflux attach- noic) acid (CigH36O2), and the sum of the stearic acid
ment with 5 mL of Boron trifluoride-methanol solution. and palmitic acid is NLT 90.0%
Boil under reflux for 10 min. Add 4.0 mL of heptane For Stearic acid 95: NLT 90.0% of stearic (octadeca-
through the condenser, and boil again under reflux for noic) acid (CigH36O2), and the sum of the stearic acid
10 min. Allow to cool. Add 20 mL of a saturated solu- and palmitic acid is NLT 96.0%
tion of sodium chloride. Shake, and allow the layers to IMPURITIES
separate. Remove about 2 mL of the organic layer, and
sydeibouow 4N

e RESIDUE ON IGNITION (281): NMT 4 mg, determined on a

dry it over 0.2 g of anhydrous sodium sulfate. Dilute 4-g portion (0.1%)>
1.0 mL of this solution with heptane to 10.0 mL.
Standard solution: Prepare as directed in the Sample
solution using 50 mg of USP Stearic Acid RS and 50 mg Delete the following:
of USP Palmitic Acid RS.
1 Petroleum ether; boiling range 100°-140°; CAS 64742-49-0 from Fisher Sci-
°o *HEAVY METALS, Method I! (231): NMT 10 ppmee coral
entific; catalog number AC23302-0025 1s suitable. 1Jan-2018)
 5620 Stearic / Official Monographs
SPECIFIC TESTS
e FATS AND FIXED OILS, /odine Value (401)
Sample: 1g
Analysis: Proceed as directed in Method I, except use
15 mL of chloroform.
Acceptance criteria: See Table 2.
Table 2
Type lodine Value
Stearic acid 50 NMT 4.0
Stearic acid 70 NMT 4.0
Stearic acid 95 NMT 1.5
© *COLOR OF SOLUTION
Standard stock solution Y (yellow): 2.4 mL of ferric
chloride CS, 0.6 mL of cobaltous chloride CS, and
7.0 mL of hydrochloric acid solution (10 g/L)
Standard stock solution BY (brownish-yellow):
2.4 mL of ferric chloride CS, 1.0 mL of cobaltous chlo-
ride CS, 0.4 mL of cupric sulfate CS, and 6.2 mL of hy-
drochloric acid solution (10 g/L)
Standard solution Y: 2.5 mL of Standard stock solution
Y and 97.5 mL of hydrochloric acid solution (10 g/L)
Standard solution BY: 2.5 mL of Standard stock solution
BY and 97.5 mL of hydrochloric acid solution (10 g/L)
Analysis: Heat Stearic Acid to 75°.
Acceptance criteria: The resulting liquid is not more
intensely colored than Standard solution Y or Standard
solution BY.*
e ACIDITY
Sample: rae of Stearic Acid
Analysis: Melt the Sample, shake for 2 min with 10 mL
of hot carbon dioxide-free water, cool slowly, and filter.
To the filtrate add 0.05 mL of methyl orange TS.
Acceptance criteria: No red color develops.
e FREEZING POINT
Apparatus: Consists of a test tube about 25 mm in di-
ameter and 150 mm long placed inside a test tube
about 40 mm in diameter and 160 mm long. The inner
tube is closed by a stopper which carries a thermome-
ter about 175 mm long and graduated in 0.2°, fixed so
that the bulb is about 15 mm above the bottom of the
tube. The stopper has a hole allowing ae ere of
the stem ofa stirrer made from a glass rod or other
suitable material formed at one end into a loop of
about 18 mm overall diameter at right angles to the
rod. The inner tube with its jacket is supported centrally
in a 1-L beaker containing a suitable cooling liquid to
within 20-mm of the top. A thermometer is supported
in the cooling bath. Place in the inner tube sufficient
quantity of the liquid or previously melted substance to
be examined, to cover the thermometer bulb, and de-
termine the approximate freezing point by cooling
rapidly.
Analysis: Place the inner tube in a bath about 5° above
the approximate freezing point until all but the last
traces of crystals are melted. Fill the beaker with water
or a saturated solution of sodium chloride; at a temper-
ature about 5° lower than the expected freezing point,
insert the inner tube into the outer tube, ensuring that
some seed crystals are present, and stir thoroughly until
solidification takes place. Note the highest temperature
observed during solidification.
NF Monographs
Acceptance criteria: See Table 3.
Table 3
Freezing Point
Type ©)
Stearic acid 50 53-59
Stearic acid 70 57-64
Stearic acid 95 64-69
NF 36
ADDITIONAL REQUIREMENTS
¢ *PACKAGING AND STORAGE: Preserve in well-closed con-
tainers.«
e LABELING: lf it is for external use only, the labeling so
indicates.* The label states the type of stearic acid (50,
70, or 95).
e@ USP REFERENCE STANDARDS (11)
USP Palmitic Acid RS
USP Stearic Acid RS
Purified Stearic Acid
(This monograph will be omitted after May 1, 2019 due to
inclusion of the article of commerce in another compendial
standard, Stearic Acid monograph, as Stearic Acid 95.)
(Prior to May 1, 2019, the current practice of labeling the
article of commerce with the name Purified Stearic Acid
may be continued. The use of Stearic Acid 95 will be
mandatory upon May 1, 2019. The 60-month extension
will provide the time needed by manufacturers and users
to make necessary changes.)
DEFINITION
Purified Stearic Acid is a mixture consisting of stearic
(octadecanoic) acid (CisH36O2) and palmitic (hexadeca-
noic) acid (Ci6H3202) obtained from fats or oils of vegeta-
ble or animal origin, which together constitute NLT
96.0% of the total content. The content of stearic acid
(CisH36O2) is NLT 90.0% of the total.
[Note—Purified Stearic Acid labeled solely for external use is
exempt from the requirement that it be prepared from
edible sources.]
IDENTIFICATION
e A. It meets the requirements of the test for Freezing
Point.
o B. It meets the requirements of the test for Acid Value.
e C. The retention times of the major peaks of the Sample
solution correspond to those of the Standard solution, as
obtained in the Assay.
ASSAY
© PROCEDURE
Boron trifluoride-methanol solution: 140 g/L of bo-
ron trifluoride in methanol
Standard solution: Prepare as directed in the Sample
solution using 50 mg of USP Stearic Acid RS and 50 mg
of USP Palmitic Acid RS.
Sample solution: Dissolve 100 mg of Purified Stearic
Acid in a small conical flask fitted with a suitable reflux
attachment with 5 mL of Boron trifluoride-methano! solu-
tion. Boil under reflux for 10 min. Add 4.0 mL of hep-
tane through the condenser, and boil again under re-
flux for 10 min. Allow to cool. Add 20 mL of a
saturated solution of sodium chloride. Shake, and allow
the layers to separate. Remove about 2 mL of the or-
ganic layer, and dry it over 0.2 g of anhydrous sodium
se Dilute 1.0 mL of this solution with heptane to
-OmL.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: GC
Detector: Flame ionization
Column: 30-m x 0.32-mm fused silica coated with a
0.5-um layer of stationary phase G16
Temperatures
Injector: 220°
Detector: 260°
Column: See Table 1.
NF 36 Official Monographs / Stearic 5621

Table 1 Light petroleum previously neutralized with 0.1 N potas-

sium hydroxide or 0.1 N sodium hydroxide, using
Hold Time
0.5 mL of phenolphthalein TS as indicator. If necessary,
Initial Temperature Final at Final
heat to about 90° to dissolve the substance to be
Temperature Ramp Temperature | Temperature
examined.
@°) (°/min) ©) (min) Analysis: Titrate the Sample solution with 0.1 N potas-
70 — 70 Pi sium hydroxide or 0.1 N sodium hydroxide until the
70 5 240 5 ink color persists for at least 15 s. When heating has
been applied to aid dissolution, maintain the tempera-
Carrier gas: Helium, passed through a bed of molecu- ture at about 90° during the titration.
lar sieve for drying, if necessary Calculate the acid value of the portion of Purified Ste-
Flow rate: 2.4 mL/min aric Acid taken:
Injection volume: 1 pL
System suitability Result = V/W
x N x 56.11
Sample: Standard solution
Suitability requirements Vv = volume of the potassium hydroxide VS
Resolution: NLT 5.0 between the methyl palmitate consumed in the titration (mL)
and methyl stearate peaks determined from six Ww sample weight of Purified Stearic Acid taken to

I
injections prepare the Sample solution (g)
[NoTte—The relative retention times for methyl palmitate N = normality of the potassium hydroxide VS
and methyl stearate are about 0.9 and 1.0, 56.11 = molecular weight of potassium hydroxide
respectively.] Acceptance criteria: 194-212
Relative standard deviation: NMT 3.0% for the © FREEZING POINT
methyl stearate and methyl palmitate peaks, from six Apparatus: Consists of a test tube about 25 mm in di-
replicate injections; NMT 1.0% for the ratio of the ameter and 150 mm long placed inside a test tube
peak areas of methyl palmitate to the peak areas of about 40 mm in diameter and 160 mm long. The inner
methyl stearate, from six replicate injections tube is closed by a stopper which carries a thermome-
Analysis ter about 175 mm long and graduated in 0.2°, fixed so
Sample: Sample solution that the bulb is about 15 mm above the bottom of the
Calculate the percentage of stearic acid (CisH36Q2) in tube. The stopper hasa hole allowing the passage of
the portion of the sample taken: the stem of a stirrer made from a glass rod or other
suitable material formed at one end into a loop of
Result = (ru/r7) x 100 about 18 mm overall diameter at right angles to the
rod. The inner tube with its jacket is supported centrally
Tu peak area due to methyl stearate in a 1-L beaker containing a suitable cooling liquid to
as sum of the peak areas of all the fatty acid within 20-mm of the top. A thermometer is supported
esters in the cooling bath. Place in the inner tube sufficient
Similarly, calculate the percentage of palmitic acid quantity of the liquid or previously melted substance to
(Ci6H3202) in the portion of the sample taken: be examined, to cover the thermometer bulb, and de-
Result = (ru/r7) x 100
termine the approximate freezing point by cooling
rapidly.
tu = peak area due to methyl palmitate Analysis: Place the inner tube in a bath about 5° above
tr = sum of the peak areas of all the fatty acid the approximate freezing point until all but the last
esters traces of crystals are melted. Fill the beaker with water
Acceptance criteria: NLT 90.0% of stearic acid or a saturated solution of sodium chloride at a tempera-
(CisH36O2), and the sum of the stearic acid and palmitic ture about 5° lower than the expected freezing point.
acid is NLT 96.0%. Insert the inner tube into the outer tube ensuring that
some seed crystals are present, and stir thoroughly until
IMPURITIES solidification takes place. Note the highest temperature
e RESIDUE ON IGNITION (281) observed during solidification.
Sample: 4g Acceptance criteria: 64°-69°
Acceptance criteria: NMT 4 mg (0.1%) e COLOR OF SOLUTION
Standard stock solution Y (yellow): 2.4 mL of ferric
chloride CS, 0.6 mL of cobaltous chloride CS, and
Delete the following: 7.0 mL of hydrochloric acid solution (10 g/L)
Standard stock solution BY (brownish-yellow):
°e HEAvY METALS, Method // (231): NMT 10 Ug/ge comes. 2.4 mL of ferric chloride CS, 1.0 mL of cobaltous chlo-
Jan-2018) ride CS, 0.4 mL of cupric sulfate CS, and 6.2 mL of hy-
drochloric acid solution (10 g/L)
SPECIFIC TESTS Standard solution Y: 2.5 mL of Standard stock solution
e FATS AND FIXED OILS, lodine Value (401) Y and 97.5 mL of hydrochloric acid solution (10 g/L)
Sample: 1g Standard solution BY: 2.5 mL of Standard stock solution
Analysis: Proceed as directed in Method |, except use BY and 97.5 mL of hydrochloric acid solution (10 g/L)
15 mL of chloroform. Analysis: Heat Purified Stearic Acid to 75°.
Acceptance criteria: NMT 1.5 Acceptance criteria: The resulting liquid is not more
sydesbouo;- 4N

e ACID VALUE intensely colored than Standard solution Y or Standard

Light petroleum: Use a sample that has the following Solution BY.
properties: a clear, colorless, liquid without fluores- e ACIDITY
cence; practically insoluble in water; miscible with alco- Analysis: Melt 5.0 g of Purified Stearic Acid, shake for 2
hol; density at 20° about 0.720; distillation range min with 10 mL of hot carbon dioxide-free water, cool
100°-120°; water content NMT 0.03%. slowly, and filter. To the filtrate add 0.05 mL of methyl
Sample solution: Dissolve 0.5 g of Purified Stearic Acid orange TS.
in 50 mL of a mixture of equal volumes of alcohol and
1 Petroleum ether; boiling range 100°-140°, CAS 64742-49-0 from Fisher Sci-
entific, catalog number AC23302-0025 1s suitable.
USP 41
lp = sum of peak responses for speciophylline,
uncarine F, mitraphylline, isomitraphylline,
petoropodine and isoperopodine in the
chromatogram of the Sample solution
Acceptance criteria: 90.0%-110.0% of the labeled
amount of Powdered Extract calculated as pentacyclic
oxindole alkaloids; and NMT 25% of tetracyclic ox-
indole alkaloids with respect to the labeled amount of
pentacyclic oxindole alkaloids is found.
PERFORMANCE TESTS
© DISINTEGRATION AND DISSOLUTION OF DIETARY SUPPLEMENTS
(2040): Meets the requirements for Disintegration
e@ WEIGHT VARIATION OF DIETARY SUPPLEMENTS (2091):
Meets the requirements
CONTAMINANTS
e MICROBIAL ENUMERATION TESTS (2021): The total aerobic
microbial count does not exceed 104 cfu/g. The total
com placa molds and yeasts count does not exceed 103
u/g.
° ABSENCE OF SPECIFIED MICROORGANISMS (2022): Meets the
requirements of the tests for absence of Salmonella spe-
cies and Escherichia coli.
ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Preserve in tight, light-resistant
containers, and store at room temperature.
© LABELING: The label states the Latin binomial and, follow-
ing the official name, the article from which Tablets were
prepared. The label also indicates the quantity of Pow-
dered Extract per Tablet, in mg. Label Tablets to indicate
the content, in mg, of pentacyclic oxindole alkaloids per
100 mg of Powdered Extract.
e USP REFERENCE STANDARDS (11)
USP Isopteropodine RS
USP Powdered Cat's Claw Extract RS
Centella asiatica
DEFINITION
Centella asiatica consists of the dried aerial parts of Centella
asiatica (L.) Urb. [Syn: Hydrocotyle asiatica L.] (Fam.
Apiaceae). It is also known in commerce as gotu kola. It
contains NLT 2.0% of triterpene derivatives, calculated on
the dried basis.
IDENTIFICATION
e A. Centella asiatica meets the requirements for Specific
Tests, Botanical Characteristics.
¢ B. THIN-LAYER CHROMATOGRAPHY
Standard solution A: 0.5 mg/mL of USP Asiaticoside RS
in methanol
Standard solution B: 10 mg/mL of USP Powdered
Centella asiatica Extract RS in methanol. Sonicate for
about 10 min, centrifuge, and use the supernatant.
Sample solution: About 0.5 g of Centella asiatica, finely
powdered, in 5 mL of methanol. Sonicate for 10 min,
centrifuge, and use the supernatant.
Adsorbent: Chromatographic silica gel with an average
particle size of 10-15 jim (TLC plates) or with an aver-
age particle size of 5 um (HPTLC plates)
Appration volume: 10 ul (TLC plates) or 4 wl (HPTLC
plates
Developing solvent system: Methylene chloride, meth-
anol, and water (14:6:1)
Derivatization reagent: A solution of 10% sulfuric acid
in methanol. [NoTE—Prepare fresh.]
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Dietary Supplements / Centella 4513
Apply the Samples as bands. Use a saturated chamber.
Develop the chromatograms until the solvent front has
moved up about three-fourths of the plate. Remove
the plate from the chamber, dry, treat with Derivatiza-
tion reagent, heat for 3 min at 120°, and examine
under white light.
Acceptance criteria: The Sample solution chromato-
gram exhibits a violet band in the lower third of the
plate due to asiaticoside, corresponding in color and Rr
to that in Standard solution A; a violet band due to
madecassoside at an R; lower than that of asiaticoside;
and two additional violet bands in the upper third of
the plate due to asiatic acid and madecassic acid. Bands
detected in the Sample solution correspond in position
and color to bands in Standard solution B. Other minor
bands may be observed in the Sample solution and
Standard solution B.
e C. HPLC: The Sample solution chromatogram from the
test for Content of Triterpene Derivatives shows a peak at
the retention time corresponding to that of asiaticoside
in Standard solution A. \dentify other triterpene derivative
peaks in the Sample solution by comparison with the
chromatogram of Standard solution B and the reference
chromatogram provided with the lot of USP Powdered
Centella asiatica Extract RS being used. The Sample solu-
tion shows additional peaks corresponding to madecasso-
side and asiaticoside B (these two peaks may co-elute),
madecassic acid, terminolic acid, and asiatic acid.
COMPOSITION
@ CONTENT OF TRITERPENE DERIVATIVES
Solution A: Dilute 3 mL of phosphoric acid with water
to 1000 mL, mix, filter, and degas.
Solution B: Acetonitrile
Mobile phase: See Table 7.
Table 1
Time Solution A Solution B
(min) (%) (%)
0 78 22
65 45 55
66 5 95
75 5 95
76 78 22
85 78 22
Standard solution A: 0.05 mg/mL of USP Asiaticoside
RS in methanol
Standard solution B: Sonicate a portion of USP Pow-
dered Centella asiatica Extract RS in methanol to obtain
sydesbouow= sa
a solution with a concentration of about 5.0 mg/mL.
Before injection, pass through a membrane filter of
0.45-um or finer pore size, discarding the first few mL
of the filtrate.
Sample stock solution: Transfer about 1.0g of Centella
asiatica, finely powdered and accurately weighed, to a
Soxhlet apparatus. Add 100 mL of methanol, extract for
8 h, cool, and dilute with methanol to 100 mL. Pass
through a membrane filter of 0.45-um or finer pore
size, discarding the first few mL of the filtrate. [NoTE—
Use a thimble of a suitable size such that the volume of
methanol used in the Soxhlet extraction is at least twice
the volume of the thimble.]
Sample solution: Dilute 5.0 mL of Sample stock solution
with methanol to 10.0 mL.
Chromatographic system
(See Chromatography (621), System Suitability.)
 5622 Stearic / Official Monographs NF 36

Acceptance criteria: No red color develops. e LIMIT OF FREE ETHYLENE OXIDE AND DIOXANE
Analysis: Proceed as directed in Ethylene Oxide and Di-
ADDITIONAL REQUIREMENTS oxane (228), Method I.
e PACKAGING AND STORAGE: Preserve in well-closed Acceptance criteria
containers. Ethylene oxide: NMT 1 ug/g
e LABELING: If it is for external use only, the labeling so Dioxane: NMT 10 ug/g
indicates. e LIMIT OF FREE GLYCEROL
e@ USP REFERENCE STANDARDS (11) Sample: 1.20g
USP Palmitic Acid RS Periodic acetic acid solution: Dissolve 0.446 g of so-
USP Stearic Acid RS dium periodate in 2.5 mL of a 25% (v/v) solution of
sulfuric acid, diluting with glacial acetic acid to
100.0 mL.
Potassium iodide solution: 75 mg/mL of potassium
iodide
Stearoyl Polyoxylglycerides Analysis: Dissolve the Sample in 25 mL of methylene
chloride, heating if necessary. Cool, and add 100 mL of
DEFINITION water and 25.0 mL of the Periodic acetic acid solution.
Stearoyl Polyoxylglycerides is a mixture of monoesters, dies- Shake, and allow to stand for 30 min. Add 40 mL of the
ters, and triesters of glycerol and monoesters and diesters Potassium iodide solution, and allow to stand for 1 min.
of polyethylene glycols. The pels yicreot cols used Add 1 mL of starch TS, and titrate the liberated iodine
have a mean molecular weight between 300 and 4000. It with 0.1 M sodium thiosulfate VS. Perform a blank de-
is produced by partial alcoholysis of saturated oils, mainly termination, and make any necessary correction (see Ti-
containing triglycerides of stearic acid, with polyethylene trimetry (541)). Calculate the percentage of glycerol in
glycol, by esterification of glycerol and polyethylene gly- the sample taken:
col with fatty acids, or as a mixture of glycerol esters and
ethylene oxide condensate with the fatty acids of the hy- Result = {{(Ve — Vs) x N x FI/W} x 100
drogenated oils. The hydroxyl value is NLT 85% and NMT
115% of the labeled nominal value, and the saponifica- Vg = Titrant volume consumed by the Blank (mL)
tion value is NLT 90% and NMT 110% of the labeled Vs = Titrant volume consumed by the Sample (mL)
nominal value. Stearoyl Polyoxylglycerides may contain N = actual normality of the Titrant (mEq/mL)
free polyethylene glycols. F = equivalency factor, 23.0 mg/mEq
Ww = Sample weight (mg)
IDENTIFICATION Acceptance criteria: NMT 5.0%
e A. INFRARED ABSORPTION (197K)
e B. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST SPECIFIC TESTS
(201) e FATS AND FIXED OILS, Acid Value (401)
Standard solution: 50 Be len of USP Stearoyl Polyoxyl- Sample: 2.0g
glycerides in methylene chloride Acceptance criteria: NMT 2.0
Sample solution: 50 mg/mL of Stearoyl Polyoxylglycer- e FATS AND FIXED OILS, ay Acid Composition (401):
ides in methylene chloride Stearoyl Polyoxylglycerides exhibits the following compo-
Chromatographic system sition profile of fatty acids, as determined in the chapter
Application volume: 10 uL (see Table 1).
Developing solvent system: Ether and hexanes
(70:30) Table 1
Spray reagent: 0.1 mg/mL of rhodamine B in alcohol Carbon-Chain Number of Percentage
Analysis Length Double Bonds (%)
Samples: Standard solution and Sample solution
Proceed as directed in the chapter. Then spray the 12 0 <5.0
plate with Spray reagent, and locate the spots on the 14 0 5.0
plate by examination under UV light at a wavelength 16 0 40.0-50.0
of 365 nm. 18 0 48.0-58.0
Acceptance criteria: The R; values of the principal spots
of the Sample solution correspond to those of the Stan- FATS AND FIXED OlLs, Hydroxy! Value (401)
dard solution. Sample: 1.0g
e C. It meets the requirements in Specific Tests (see Table 1) Acceptance criteria: 25-56, 85%-115% of the labeled
for Fats and Fixed Oils, Fatty Acid Composition (401). nominal value
e FATS AND FIXED OILS, /odine Value (401): NMT 2.0
IMPURITIES e FATS AND FIXED OILS, Peroxide Value (401)
Sample: 2.0g
Delete the following: Acceptance criteria: NMT 6.0
FATS AND FIXED OILS, Saponification Value (401)
°e HEAVY METALS, Method I (231): NMT 10 ug/ge cornciat- Sample: 2.0g
Acceptance criteria: 67-112, 90%-110% of the la-
Jan-2018, beled nominal value
° ARTICLES OF BOTANICAL ORIGIN, Total Ash (561): NMT
WATER DETERMINATION, Method | (921)
NF Monographs

0.2% Sample: 1.0g

© ALKALINE IMPURITIES
Sample: 5.0g
Analysis: Heat the Sample slightly until the test sub-
stance melts, add 10 mL of alcohol and 0.05 mL of bro-
mophenol blue TS, and mix well. While the solution is
still warm, titraté with 0.01 N hydrochloric acid VS to
change the color to yellow.
Acceptance criteria: NMT 1.0 mL of 0.01 N hydrochlo-
ric acid VS is required.
Solvent: Anhydrous pyridine or a mixture of methylene
chloride and anhydrous methanol (70:30)
Acceptance criteria: NMT 1.0%
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in tight containers,
protected from light and moisture. Store at controlled
room temperature.
NF 36 Official Monographs / Stearyl 5623

© LABELING: Label it to indicate the ae and the average Carrier gas: Hydrogen
nominal molecular weight of polyethylene glycols used as Flow rate: 2.0 mL/min, constant flow mode
part of the official title. The label also indicates the hy- Injection volume: 1 uL
droxyl value and the saponification value. Injection ps Split injection; split ratio is 100:1
e USP REFERENCE STANDARDS (11) Liner: Single taper, low pressure drop liner with deac-
USP Stearoyl Polyoxylglycerides RS tivated wool
Run time: 15 min
System suitability
Samples: System suitability solution and Standard
solution
Stearyl Alcohol [Note—See Table 2 for the relative retention times.]

CisH320 270.49
1-Octadecanol;
Octadecan-1-ol [112-92-5].
DEFINITION
Stearyl Alcohol contains NLT 90.0% and NMT 102.0% of
stearyl alcohol (CisH3gQ), the remainder consisting chiefly
of related alcohols. It is obtained from sources of vegeta-
ble, animal, or synthetic origin.
IDENTIFICATION
e A. CHROMATOGRAPHIC IDENTITY
Analysis: Proceed as directed in the Assay.
Acceptance criteria: The retention time of the major
of the Sample solution, excluding the solvent and
internal standards peaks corresponds to the stearyl alco-
hol peak of the System suitability solution.
ASSAY
¢ PROCEDURE
Internal standard solution: 1 mg/mL of 1-pentadeca-
nol (internal standard) in ethanol
System suitability solution: Prepare 1 mg/mL each of
USP Cetyl Alcohol RS, USP Stearyl Alcohol RS, and USP
Oley! Alcohol RS in Internal standard solution. Heat the
solution in a sealed container in a 50° water bath until
all fatty alcohols are dissolved. Allow the solution to
cool to room temperature, and mix well.
Standard solution: Prepare 1.0 mg/mL of USP Stearyl
Alcohol RS in Internal standard solution, and heat the
solution in a sealed container in a 50° water bath until
stearyl alcohol is dissolved. Allow the solution to cool to
room temperature, and mix well.
Sample solution: Prepare 1.0 mg/mL of Stearyl Alcohol
in Internal standard solution. Heat the solution in a
sealed container in a 50° water bath until stearyl alco-
hol is dissolved. Allow the solution to coo! to room
temperature, and mix well.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: GC
Detector: Flame ionization
Column: 0.25-mm x 30-m fused silica capillary; coated
with a 0.25-uum layer of phase G7
Temperatures
Detector: 280°
Injection port: 270°
Column: See Table 7.
Table 1
Table 2
Relative
Retention
Component Time
1-Pentadecanol (internal standard) 1.00
Cetyl alcohol 1.09
Stearyl alcohol 1.25
Oleyl alcohol 1.28
Suitability requirements
Resolution: NLT 30 between the cetyl alcohol and
stearyl alcohol peaks; NLT 2.0 between the steary|
and oleyl alcohol peaks, System suitability solution
Tailing factor: 0.8-1.8 for the stearyl alcohol and
1-pentadecanol peaks, Standard solution
Relative standard deviation: NMT 1%, using the
area ratio of stearyl alcohol to 1-pentadecanol, Stan-
dard solution
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of stearyl alcohol (CigH3sO) in
the portion of Stearyl Alcohol taken:
Result = (Ru/Rs) x (Cs/Cu) x 100
Ru = peakresponse ratio of stearyl alcohol to the
internal standard (peak response of steary|
aera response of the internal
standard) from the Sample solution
Rs = peak jesponse ratio of stearyl alcohol to the
internal standard (peak response of stearyl
alcohol/peak response of the internal
standard) from the Standard solution
Cs = concentration of USP Stearyl Alcohol RS in the
Standard solution (mg/mL)
Cu = concentration of Stearyl Alcohol in the Sample
solution (mg/mL)
Acceptance criteria: 90.0%-102.0%
IMPURITIES
© RESIDUE ON IGNITION (281): NMT 0.1%, determined on
2
° limit OF RELATED FATTY ALCOHOLS
Solution A: 1 mg/mL of 1-pentadecanol in ethanol
Resolution solution: Prepare 1 mg/mL each of USP
Lauryl Alcohol RS, USP Myristyl Alcohol RS, USP Cetyl
Alcohol RS, USP Stearyl Alcohol RS, USP Oley! Alcohol
RS, USP Linoleny| Alcohol RS, and USP Arachidyl Alcohol
RS in Solution A. Heat the solution in a sealed container
in a 50° water bath until all fatty alcohols are dissolved.
Allow the solution to cool to room temperature, and
mix well. Dilute the solution with ethanol to obtain a
sydesbouow 4IN

Hold Time at solution containing 0.05 eat each of USP Lauryl Al-
Initial Temperature Final Final cohol RS, USP Myristyl Alcohol RS, USP Cetyl Alcohol
Temperature Ramp Temperature | Temperature RS, 1-pentadecanol, USP Stearyl Alcohol RS, USP Oleyl
©) (¢/min) ©) (min) Alcohol RS, USP Linolenyl Alcohol RS, and USP Arachidyl
60 20 180 — Alcohol RS.
Sample solution: 1 mg/mL of Stearyl Alcohol in etha-
180 10 220 5
nol. Heat the solution in a sealed container in a 50°
water bath until stearyl alcohol is dissolved. Allow the
solution to cool to room temperature, and mix well.
 5624 Stearyl / Official Monographs NF 36

Chromatographic system: Proceed as directed in the e USP REFERENCE STANDARDS (11)

Assay, except for the split ratio. USP Arachidyl Alcohol RS
Injection type: Split injection; the split ratio is 5:1. USP Cetyl Alcohol RS
System suitability USP Lauryl Alcohol RS
Sample: Resolution solution USP Linolenyl Alcohol RS
[Note—See Table 3 for the relative retention times.] USP Myristyl Alcohol RS
USP Oleyl Alcohol RS
Table 3 USP Stearyl Alcohol RS
Relative
Retention
Component Time
Lauryl alcohol 0.79 Succinic Acid
Myristyl alcohol 0.93
1-Pentadecanol 1.00 °
Cetyl alcohol 1.09 ~ A,
Stearyl alcohol 1:25. 8
Oley! alcohol 1.28
Linolenyl alcohol 1.36 CyHeO4 118.09
Arachidyl alcohol 1.44 Butanedioic acid [110-15-6].

Suitability requirements DEFINITION

Resolution: NLT 15 between myristyl alcohol and Succinic Acid contains NLT 99.0% and NMT 100.5% of suc-
1-pentadecanol peaks; NLT 30 between the cetyl al- cinic acid (C4HeO,).
cohol and stearyl alcohol peaks; NLT 2.0 between the
stearyl and oleyl alcohol peaks IDENTIFICATION
Analysis e A. INFRARED ABSORPTION (197K)
Samples: Resolution solution and Sample solution ¢ B. CHROMATOGRAPHIC IDENTITY
Identify each related fatty alcohol peak in the Sample The retention time of the major peak of the Sample solu-
solution based on that in the Resolution solution. tion pAlb to that of the Standard solution, as ob-
Calculate the percentage of each related fatty alcohol or tained in the Assay.
any unspecified impurity in the portion of Stearyl Alco- ASSAY
hol taken: © PROCEDURE
Result = (ru/rr) x 100 Diluent: Dissolve 6.8 g of potassium phosphate mono-
basic in 2 L of water. Adjust the pH of the solution to
tu = peak response of each related ratty alcohol (or 2.3 with drops of phosphoric acid. This solution is 25
any unspecified impurity) from the Sample mM potassium phosphate buffer, pH 2.3.
solution Mobile phase: Methanol and Diluent (8:92)
rr = sum of all the peak responses excluding peak System suitability solution: 1.0 mg/mL of USP Succinic
responses due to solvent from the Sample Acid RS and 0.003 mg/mL of USP Fumaric Acid RS in
solution Diluent
Acceptance criteria: Disregard peaks that are less than Standard solution: 1.0 mg/mL of USP Succinic Acid RS
0.05% for any unspecified impurities, and any peaks in Diluent
due to solvent. Sample solution: 1.0 mg/mL of Succinic Acid in Diluent
Sum of unspecified impurities: NMT 1% Chromatographic system
Sum of related fatty alcohols and unspecified impuri- (See Chromatography (621), System Suitability.)
ties: NMT 10.0% Mode: LC
Detector: UV 204 nm
SPECIFIC TESTS Column: 4.6-mm x 15-cm; 3-um packing L1
e FATS AND FIXED OiLs, Acid Value (Free Fatty Acids) (401): Flow rate: 1.0 mL/min
NMT 2 Injection volume: 20 LL
e FATS AND FIXED OlLs, Hydroxyl Value (401): 195-220 System suitability
e FATS AND FIXED OILS, lodine Value (401): NMT 2 Sample: System suitability solution
e@ WATER DETERMINATION, Method | (921): NMT 0.5% [NotE—The relative retention times for succinic acid
and fumaric acid are 1.0 and 1.2, respectively.]
ADDITIONAL REQUIREMENTS Suitability requirements
e PACKAGING AND STORAGE: Preserve in well-closed Resolution: NLT 2.0 between succinic acid and fu-
containers. maric acid
e LABELING: Label it to indicate whether it is derived from Relative standard deviation: NMT 0.5% for the suc-
vegetable, animal, or synthetic sources. cinic acid peak
Tailing factor: 0.8-2.0
Analysis
4 Samples: Standard solution and Sample solution
& Calculate the percentage of succinic acid (C4H6Os) in
fo
Ss the portion of the sample taken:
he
DS Result = (ru/rs) x (Cs/Cu) x 100
°
¢
° ty = peak response from the Sample solution
= rs = peak response from the Standard solution
eS Gs = concentration of USP Succinic Acid RS in the
72 Standard solution (mg/mL)
NF 36 Official Monographs / Sucralose 5625

Gu = concentration of Succinic Acid in the Sample Mode: LC

solution (mg/mL) Detector: Refractive index
Acceptance criteria: 99.0%-100.5% Column: 8-mm x 10-cm; packing L1
Flow rate: 1.5 mL/min
IMPURITIES Injection size: 20 wL
e RESIDUE ON IGNITION (281): NMT 0.025% System suitability
Sample: Standard solution
Delete the following: {[Note—The retention time of sucralose is about 9 min.]
Suitability requirements
°e Heavy METALS, Vethod | (231): NMT 20 ug/Ge cotticiat 1- Relative standard deviation: NMT 2.0%
jan-2018)
Analysis : 4
Samples: Standard solution and Sample solution
SPECIFIC TESTS Calculate the percentage of sucralose (C12Hi9Cl3Og) in
e MELTING RANGE OR TEMPERATURE (741): 185.0°-190.0° the portion of Sucralose taken:
ADDITIONAL REQUIREMENTS Result = (ru/rs) x (Cs/Cu) x 100
© PACKAGING AND STORAGE: Preserve in a well-closed con-
tainer. No storage requirements specified. ru = peak response of the Sample solution
e USP REFERENCE STANDARDS (11) rs =peak response of the Standard solution
USP Fumaric Acid RS Cs = concentration of USP Sucralose RSin the
USP Succinic Acid RS Standard solution (mg/mL)
Cu = concentration of Sucralosein the Sample
solution (mg/mL)
Acceptance criteria: 98.0%-102.0% on the anhydrous
basis
Sucralose IMPURITIES
e RESIDUE ON IGNITION (281): NMT 0.7%
8
a
e-
ya No
Delete the following:

HO:
®o HEAVY METALS, Method | (231): 10 ppMe ‘oficial }-1an-2078)
eo Limit OF METHANOL
Internal standard solution: 0.1 wL/mL of n-propyl alco-
Cy2Hi9ClsOg 397.63 hol in pyridine
1,6-Dichloro-1,6-dideoxy-B-D-fructofuranosyl-4-chloro-4-de- Standard solution: 0.2 uL/mL of methanol in Internal
oxy-1-D--galactopyranoside; standard solution
1A, es-Trichlorogalactosucrose [56038-1 3-2]. Sample solution: 0.2 g/mL of Sucralose in Internal stan-
dard solution
DEFINITION Chromatographic system
Sucralose contains NLT 98.0% and NMT 102.0% of (See Chromatography (621), System Suitability.)
Cy2Hi9Cl3Og, calculated on the anhydrous basis. Mode: GC
Detector: Flame ionization
IDENTIFICATION Column: 4-mm x 2-m glass column; packed with 80-
e A. INFRARED ABSORPTION (197K) to 100-mesh silanized support S6
e B. The retention time of the principal peak of the Sample Temperature
solution corresponds to that of the Standard solution, as Column: 150°
obtained in the Assay. Detector: 250°
e C. The R; value of the principal spot of the Sample solu- Injector: 200°
tion corresponds to that of Standard solution A, as ob- Carrier gas: Helium
tained in the test for Related Compounds. Flow rate: 20 mL/min
ASSAY Injection size: 1 ul
© PROCEDURE System suitability
Mobile phase: Acetonitrile and water (3:17) Sample: Standard solution
Standard solution: 1 mg/mL of USP Sucralose RS in
Suitability requirements
Mobile phase Relative standard deviation: NMT 2.0%
Sample solution: 1 mg/mL of Sucralose in Mobile phase Analysis
Chromatographic system Samples: Standard solution and Sample solution
Calculate the percentage of methanol in the portion of
(See Chromatography (621), System Suitability.)
Sucralose taken:
Result = (Ru/Rs) x [(Cs/Cu) x Fi] x Fe x 100
Ru = peak response ratio of methanol to n-propyl
alcohol, from the Sample solution Z
Rs = peak response ratio of methanol to n-propyl
alcohol, from the Standard solution =
Cs = concentration of methanol in the Standard =
solution (uL/mL) 3°
Cu = concentration of Sucralose in the Sample ito)
solution (g/mL) BY
F, = conversion factor from WL to mL mo]
Fy = specific gravity of methanol, 0.79 g/cm3 ie
 5626 Sucralose / Official Monographs
Acceptance criteria: NMT 0.1%
e RELATED COMPOUNDS
Adsorbent: 0.20-mm layer of octadecylsilanized chro-
matographic silica gel. The thin-layer chromatographic
plate also has a preadsorbent zone.
Detection reagent: Sulfuric acid in methanol (3 in 20)
Standard solution A: 10.0 mg/mL of USP Sucralose RS
in methanol
Standard solution B: 0.5 mL Standard solution A diluted
to 10.0 mL with methanol
Sample solution: 100.0 mg/mL of Sucralose in
methanol
Developing solvent system: Acetonitrile and sodium
chloride solution (1 in 20) (3:7)
Application volume: 5 uL
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Proceed as directed under Chromatography (621), Thin-
Layer Chromatography. Spray the plate with Detection
reagent. Heat the plate for 10 min at 125°.
Acceptance criteria: The R; value of the principal spot
from the Sample solution corresponds to that obtained
from Standard solution A, and the color of any other
single spot from the Sample solution is not more intense
than that of the principal spot from Standard solution B
(0.5%).
e LIMIT OF HYDROLYSIS PRODUCTS
[NotE—This test does not require a developing solvent.]
Ape 0.25-mm layer of chromatographic silica
ge
Spray reagent: 12.3 mg/mL of p-anisidine and
16.6 mg/mL of phthalic acid in methanol. Store the so-
lution in the dark and refrigerate to prevent discolora-
tion. Discard if the solution becomes discolored. [Cau-
TION—p-Anisidine is toxic if inhaled or if absorbed
through the skin.]
Standard solution A: 100 mg/mL of mannitol
Standard solution B: 0.4 mg/mL of fructose and 100
mg/mL of mannitol
Sample solution: 250 mg/mL of Sucralose in methanol
Application volume: 5-uL portions separately applied in
1-uL increments, allowing the plate to dry between
applications
Analysis
Samples: Standard solution and Sample solution
Proceed as directed under Chromatography (621), Thin-
Layer Chromatography. Spray the plate with Spray rea-
gent, and heat the plate at 100
+2° for 15 min. If the
ope from Standard solution A has darkened, repeat
the test, heating for a shorter period of time. Imme-
diately after heating, view the plate against a dark
background.
Acceptance criteria: The color of the spot from the
Sample solution is not more intense than that from Stan-
dard solution B (0.1%).
SPECIFIC TESTS
e OPTICAL ROTATION, Specific Rotation (781S): +84.0° to
+87.5° at 20°
Sample solution: 10mg/mL of Sucralose
© WATER DETERMINATION, Method | (921): NMT 2.0%
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in well-closed contain-
NF Monographs
ers, in a cool, dry place, at a temperature not exceeding
21°;
NF 36
e USP REFERENCE STANDARDS (11)
USP Sucralose RS
Sucrose
Portions of the monograph text that are national USP text,
and are not part of the harmonized text, are marked with
symbols (*¢) to specify this fact.
9H
ou
HOw » x) *"
fe Ho” Ma mt
Ci2H2O1n 342.30
a-D-Glucopyranoside, B-D-fructofuranosyl-sucrose [57-50-1].
DEFINITION
Sucrose is a sugar obtained from Saccharum officinarum
Linné (Fam. Gramineae), Beta vulgaris Linné (Fam. Cheno-
podiaceae), and other sources. It contains no added
substances.
IDENTIFICATION
e A. INFRARED ABSORPTION (197K)
IMPURITIES
e SULFITE
Sample solution: 400 mg/mL of Sucrose in freshly pre-
pared distilled water
Sulfite standard solution (80 ppm SO2): 0.1575 mg/
mL of anhydrous sodium sulfite in freshly prepared dis-
tilled water
Reference solution: Dissolve 4.0 g of Sucrose in freshl
prepared distilled water, add 0.5 mL of Sulfite standar
solution (80 ppm SOz), and dilute with freshly prepared
distilled water to 10.0 mL.
Blank: Freshly prepared distilled water
Analysis: Determine the sulfite content by a suitable en-
zymatic method based on the following reactions. Sul-
fite is oxidized = sulfite oxidase to sulfate and hydro-
gen peroxide, which in turn is reduced by
nicotinamide-adenine dinucleotide—peroxidase in the
presence of reduced nicotinamide—adenine dinucleotide
(NADH). The amount of NADH oxidized is proportional
to the amount of sulfite. Separately introduce 2.0 mL
each of the Sample solution, Reference solution, and
Blank in 10-mm cuvettes, and add the reagents as de-
scribed in the kit instructions.! Measure the absorbance
at the maximum at about 340 nm before and at the
end of the reaction time, and subtract the value ob-
tained with the Blank.
Acceptance criteria: The absorbance difference of the
Sample solution is NMT half the absorbance difference
of the Reference solution.
SPECIFIC TESTS
¢ APPEARANCE OF SOLUTION
Sample solution: 500 mg/mL of Sucrose in water.
[Note—Set a portion of this solution aside for the tests
for Dextrins and Reducing Sugars.]
Hydrazine sulfate solution: 10 mg/mL of hydrazine
sulfate in water. Allow to stand for 4-6 h.
Hexamethylenetetramine solution: In a 100-mL
ground glass-stoppered flask dissolve 2.5 g of hexa-
methylenetetramine in 25.0 mL of water.
Primary opalescent suspension: To the Hexamethylene-
tetramine solution in the flask add 25.0 mL of Hydrazine
sulfate solution. Mix, and allow to stand for 24 h. This
1 Test kit for sulfite determination may be ordered from Boehringer Mann-
heim Roche/R-Biopharm, Catalog #10 725 854 035.
NF 36 Official Monographs / Sucrose 5627

suspension is stable for 2 months, provided it is stored Calculate the Color Value using the expression:
in a glass container free from surface defects. The sus-
pension must not adhere to the glass and must be well Result = (A x 1000)/(b x ©)
mixed before use.
Standard of opalescence: Primary opalescent suspension A = absorbance measured at 420 nm
in water (3 in 200). This suspension is freshly prepared b = cell path length (cm)
and may be stored for up to 24 h. c = concentration of the solution (g/mL),
Reference suspension |: Standard of opalescence and calculated from the refractive index of the
water (5:95) solution. Use Table 1, and interpolate the
Acceptance criteria: The clarity of the Sample solution values if necessary.
is the same as that of water or its opalescence is not °Suitability requirements
more pronounced than that of Reference suspension I. Repeatability: The absolute difference between two
e CONDUCTIVITY results is NMT 3.¢ cen i-jun-2017)
Sample solution: 313 mg/mL of Sucrose in freshly
boiled and cooled water Table 1
Apparatus: Use a conductivity meter or resistivity meter
that measures the resistance of the column of liquid 1% ¢ (g/mL)
between the electrodes of the immersed measuring de- 1.4138 0.570
vice. The apparatus is supplied with alternating current 1.4159 0.585
to avoid the effects of electrode polarization. It is 1.4179 0.600
equipped with a temperature compensation device or a 1.4200 0.615
precision thermometer. 1.4221 0.630
Calibration: Choose a conductivity cell that is appropri-
ate for the conductivity of the solution to be examined. 1.4243 0.645
The higher the expected conductivity, the higher the 1.4264 0.661
cell constant that must be chosen so that the value
measured, R, is as large as possible for the apparatus Acceptance criteria: NMT 45 if labeled as parenteral
used. Commonly used conductivity cells have cell con- grade; NMT 75 for nonparenteral grades
stants on the order of 0.1 cm-1, 1 cm-1, and 10 cm-. e DEXTRINS
Use a standard solution of potassium chloride that is [Note—If intended for use in the preparation of large-
appropriate for the measurement. Rinse the cell several volume infusions, it complieswith the test for Dextrins.
times with water that has been previously boiled and Sample solution: Prepare as directed in the test for Ap-
cooled to room temperature and at least twice with the pearance of Solution.
potassium chloride solution used for the determination Analysis: To 2 mL of the Sample solution add 8 mL of
of the cell constant of the conductivity cell. Measure water, 0.05 mL of dilute hydrochloric acid (73 g/L of
the resistance of the conductivity cell using the potas- HCl), and 0.05 mL of 0.05 M iodine.
sium chloride solution at 20+0.1°. Acceptance criteria: The solution remains yellow.
© REDUCING SUGARS
The constant, C (in cm), of the conductivity cell is
given by the expression: Sample solution: Prepare as directed in the test for Ap-
pearance of Solution.
C= Rec X Kcr Analysis: To 5 mL of the Sample solution in a test tube,
about 150-mm long and 16-mm in diameter, add 5 mL
Rea = measured resistance (MQ) of water, 1.0 mL of 1 M sodium hydroxide, and 1.0 mL
Kx = conductivity of the standard solution of of a 1-g/L solution of methylene blue. Mix, and place in
potassium chloride used (uS - cm-') a water bath. After exactly 2 min, take the tube out of
The measured constant, C, of the conductivity cell must the bath, and examine the solution immediately.
be within 5% of the given value. Acceptance criteria: The blue color does not disappear
Analysis completely, ignoring any blue color at the air/solution
Sample: Sample solution interface.
Measure the conductivity of the Sample solution (Cy), e Loss ON DRYING (731)
while gently stirring with a magnetic stirrer, and that Sample: 2.000g
of the water used for preparing the Sample solution Analysis: Dry the Sample at 105° for 3 h.
(C2). The readings must be stable within 1% over a Acceptance criteria: NMT 0.1%
period of 30 s. e BACTERIAL ENDOTOXINS TEST (85): Less than 0.25 IU/mg
Calculate the conductivity of the Sample solution from [Note—ff intended for use in thepreparation of large-
the expression: volume infusions, it complies with t the test for Bacterial
Endotoxins.]
Result = C; — (0.35 x C,)
ADDITIONAL REQUIREMENTS
G = conductivity of the Sample solution © *PACKAGING AND STORAGE: Preserve in well-closed con-
G = water used for preparing the Sample solution tainers.»
Acceptance criteria: NMT 35 uS- cm at 20° e LABELING: The label states, where applicable, that the
¢ OPTICAL ROTATION, Specific Rotation (781S) substance is suitable for use in the manufacture of large-
Sample solution: 260 mg/mL volume parenteral dosage forms.
Acceptance criteria: +66.3° to +67.0° at 20° e USP REFERENCE STANDARDS (11)
sydesbouow- 4N

USP Sucrose RS
Change to read:

© *COLOR VALUE
Sample solution: Dissolve 50.0 g in 50.0 mL of water.
Mix, filter (diameter of pores, 0.45 tum), and degas.
Analysis: Measure the absorbance at 420 nm, using a
cell of at least 4 cm (a cell length of 10 cm or more is
preferred).
 5628 Sucrose / Official Monographs NF 36

e@ WATER DETERMINATION, Method | (921): NMT 1.0%

Sucrose Octaacetate ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in tight containers.
e USP REFERENCE STANDARDS (11)
USP Sucrose Octaacetate RS

Sucrose Palmitate

C2gH32O19 678.59
a-D-Glucopyranoside, 1,3,4,6-tetra-O-acetyl-B-D-fructofura- wa
ong
oly .
nosyl, tetraacetate; Nu a
Octaacetyl sucrose [126-14-7].
DEFINITION 9
Sucrose Octaacetate contains NLT 98.0% and NMT 100.5% R=Hor OANA ANSty
of sucrose octaacetate (C2gH3gO19), calculated on the an-
hydrous basis.
CosHs2012 580.71
IDENTIFICATION
CaaHe2013 819.11
e A. INFRARED ABSORPTION (197K)
CooH 12014 1057.52
ASSAY Sucrose monopalmitate;
e PROCEDURE Sucrose hexadecanoate [26446-38-8].
Semple: 100 mg in a 500-mL conical flask
Blank: 50 mL of 70% alcohol DEFINITION
Titrimetric system Sucrose Palmitate is a mixture of sucrose monoesters, mainly
(See Titrimetry (541).) sucrose monopalmitate, obtained by transesterification of
Mode: Residual titration palmitic acid methyl esters of vegetable origin with su-
Titrant: 0.1 N sodium hydroxide VS crose. The manufacture of the fatty acid methyl esters in-
Back-titrant: 0.1 N sulfuric acid VS cludes a distillation step. It contains variable quantities of
Endpoint detection: Visual mono- and diesters as set forth in the following table:
Analysis: Dissolve the Sample in 50 mL of 70% alcohol.
Neutralize the solution with 0.1 N sodium hydroxide VS Content of Content of Sum of Triesters
using phenolphthalein TS as the indicator. Add 25.0 mL Monoesters Diesters and Polyesters
of 0.1 N sodium hydroxide VS, attach an air condenser (%) (%) _ (%)
to the flask, protect from absorption of carbon dioxide, NLT 55.0 NMT 40.0 NMT 20.0
and reflux on a steam bath for 1 h. Remove from the
steam bath, cool quickly, and titrate the excess alkali
with 0.1 N sulfuric acid VS using phenolphthalein TS as IDENTIFICATION
the indicator. Perform a blank determination. e A. It meets the requirements of the Fatty Acid Composi-
Calculate the percentage of sucrose octaacetate tion test.
(C2gH3gO19) in the portion of Sucrose Octaacetate e B. It meets the requirements of the Content of Monesters,
taken. Each mL of 0.1 N sodium hydroxide is equiva- Diesters, Triesters, and Polyesters.
lent to 8.483 mg of sucrose octaacetate (C2gH3sO19).
Acceptance criteria: 98.0%-100.5% on the anhydrous ASSAY
basis © CONTENT OF MONOESTERS, DIESTERS, TRIESTERS, AND
POLYESTERS
IMPURITIES Mobile phase: Tetrahydrofuran
e RESIDUE ON IGNITION (281): NMT 0.1% Sample solution: 15 mg/mL of Sucrose Palmitate in
tetrahydrofuran
SPECIFIC TESTS Chromatographic system
e MELTING RANGE OR TEMPERATURE (741): NLT 78° (See Chromatography (621), System Suitability.)
° AcIDITY Mode: LC, size-exclusion
Sample: 1g Detector: Differential refractometer .
Analysis: Dissolve the Sample in 20 mL of neutralized Column: 7-mm x 60-cm; packing L21, 100 A
alcohol, and add 2 drops of phenolphthalein TS. [NoTtE—Two 7-mm x 30-cm L21 columns may be used
Acceptance criteria: NMT 2 drops of 0.1 N sodium hy- in place of one 60-cm column, provided system suita-
droxide are required to produce a red color. bility requirements are met.]
Flow rate: 1.2 mL/min
Injection size: 20 uL
NF Monographs

Analysis
Sample: Sample solution
[Note—The relative retention time with reference to the
monoester peak (retention time is approximately 10
min) is about 0.92 for diesters, and about 0.90 for
triesters and polyesters.]
[Note—Disregard solvent peaks and peaks having a sig-
nal-to-noise ratio less than 10.]
NF 36 Official Monographs / Sucrose 5629

Calculate the percentage of monoesters in the portion Sample solution: 50 mg/mL of Sucrose Palmitate in
of Sucrose Palmitate taken: Diluent
Chromatographic system
Result = A x (100 - D - S$ - £)/100 (See Chromatography (621), System Suitability.)
Mode: LC
A = percentage of monoesters determined by peak Detector: Evaporative light-scattering. [NoTE—If the
normalization detector has different setting parameters, adjust the
D = percentage of free fatty acids, using the detector settings so that they comply with the System
following formula: suitability requirements.]
Carrier gas: Nitrogen
Result = AV x 256/561.1 Detector temperature: 45°
Nebulizer temperature: 40°
AV = acid value Column: 4.6-mm x 0.25-m; packing L8
Ss = percentage of free sucrose (see Free Sucrose in Injection size: 20 uL
Organic Impurities) Mobile phase and flow rate: See the gradient table
E = percentage of water (see Water Determination, below.
la in Specific Tests)
Calculate the percentage of diesters in the portion of
Sucrose Palmitate taken: Time Solution A Solution B Flow Rate
(min) (%) (%) (mL/min)
Result = B x (100 — D — S — £)/100 1 100 0 1.0
8 0 100 1.0
= percentage of diesters determined by peak
@
ng

7 0 100 1.0
normalization
= percentage of free fatty acids (above) 0.01 0 100 25.
= percentage of free sucrose (see Free Sucrose in 15:99. 0 100 25
Organic Impurities) 1 100 0 2:5
= percentage of water (see Water Determination,
mm

Bt 100 o 1.0
la in Specific Tests)
Calculate the percentage of triesters and polyesters in System suitability
the portion of Sucrose Palmitate taken: Sample: System suitability solution
[Note—The retention time is about 26 min for su-
Result = C x (100 — D — S — £)/100 crose palmitate.]
Suitability requirements
Cc = percentage of triesters and polyesters Signal-to-noise ratio: 10:1
determined by peak normalization Analysis
D = percentage of free fatty acids (above) Samples: Standard solutions and Sample solution
Ss = percentage of free sucrose (see Free Sucrose in Prepare a standard curve by plotting the peak re-
Organic Impurities) sponse versus concentration of sucrose in the Stan-
E = percentage of water (see Water Determination, lard solutions. Calculate the amount of free sucrose
Ia in Specific Tests) in the Sucrose Palmitate taken.
e FATTY ACID COMPOSITION: Sucrose Palmitate exhibits the Acceptance criteria: NMT 4.0%
following composition profiles of fatty acids, as deter-
aon under Fats and Fixed Oils, Fatty Acid Composition SPECIFIC TESTS
401). e WATER DETERMINATION, Method Ia (921): NMT 4.0% on
a 0.20-g sample
Percentage ¢ TOTAL ASH
Fatty Acid (%) Sample: 1.0g
Lauric acid NMT 3.0
Analysis: Heat a silica or platinum crucible to redness
for 30 min, allow to cool in a desiccator, and weigh.
Myristic acid NMT 3.0 Transfer the Sample into the crucible. Dry at 100°-105°
Palmitic acid 70.0-85.0 for 1 h and ignite to constant weight in a muffle fur-
Stearic acid 10.0-25.0 nace at 600
+ 25°, allowing the crucible to cool in a
Sum of the contents of palmitic acid NLT 90.0 desiccator after each ignition. Flames should not be
and stearic acid produced at any time during the procedure. If after
prolonged ignition the ash still contains black particles,
add hot water, filter through an ashless filter paper, and
IMPURITIES ignite the residue and the filter paper. Combine the
Inorganic Impurities i trate with the ash, carefully evaporate to dryness and
e FATS AND FIXED OILS, Acid Value (401): NMT 6.0%, deter- ignite to constant weight.
mined on a 3-g sample. Use a freshly neutralized mixture Acceptance criteria: NMT 1.5%
of 2-propanol and water (2:1), and gently heat.
Organic Impurities ADDITIONAL REQUIREMENTS
© PROCEDURE: FREE SUCROSE © PACKAGING AND STORAGE: Preserve in a well-closed con-
Solution A: 10 g/mL of ammonium acetate in tainer. Protect from humidity and avoid high
acetonitrile temperatures.
NBT

Solution B: 10 g/mL of ammonium acetate in tetrahy- e USP REFERENCE STANDARDS (11)

ExtrelDLoLeLeroy

drofuran and water (90:10) USP Sucrose RS

Diluent: Tetrahydrofuran and water (87.5:12.5)
System suitability solution: 500 j1g/mL of USP Sucrose
RS in Diluent
Standard solutions: 0.50, 1.0, 2.0, and 2.5 mg/mL of
USP Sucrose RS in Diluent
 5630 Sucrose / Official Monographs NF 36

D = percentage of free fatty acids, obtained by AV

Sucrose Stearate x 284.5/561.1, where AV is the acid value
Ss = percentage of free sucrose (see Free Sucrose in
oR Organic Impurities)
OR E = percentage of water (see Water Determination,
\-,/ie la in Specific Tests)
HO -- O--" ‘ x AV _=acid value
Calculate the percentage of diesters in the portion of
on Ho” Nii Sucrose Stearate taken:
° Result = B x (100 - D — S — £)/100
Rane we I. eS
B = percentage of diesters determined by peak
normalization
CsoHs6O12 608.76 D = percentage of free fatty acids (above)
CasHoO13 875.22 Ss = percentage of free sucrose (see Free Sucrose in
Organic Impurities)
CosHi24O14 1141.68 E = percentage of water (see Water Determination,
Sucrose monostearate;
Ia in Specific Tests)
Sucrose octadecanoate [25168-73-4]. Calculate the percentage of triesters and polyesters in
DEFINITION the portion of Sucrose Stearate taken:
Sucrose Stearate is a mixture of sucrose esters, mainly su-
crose stearate, obtained by transesterification of stearic Result = C x (100 - D — S — £)/100
acid methyl esters derived from vegetable origin with su-
crose. The manufacture of the fatty acid methyl esters in- . me Pees Oo ee ee
cludes a distillation step. The mono- and diesters require- D = percentage of aes fatty acids (above)
Re ee woe res of sucrose stearate as set s = percentage of free sucrose (see Free Sucrose in
9g : Organic Impurities)
E = percentage of water (see Water Determination,
Sum of Tries- la in Specific Tests)
Content of Content of ters e Fatty Actb COMPOSITION: Sucrose Stearate exhibits the
Monoesters Diesters and Polyesters following composition profiles of fatty acids, as deter-
(%) (%) (%) mined in Fats and Fixed Oils (401), Fatty Acid Composition.
Type | NLT 50.0 NMT 40.0 NMT 25.0
Type Il 20.0-45.0 30.0-40.0 NMT 30.0 Percentage
Fatty Acid (%)
IDENTIFICATION Lauric acid NMT 3.0
e A. It meets the requirements of the Fatty Acid Composi- Myristic acid NMT_3.0
tion test. Palmitic acid 25.0-40.0
° B. It meets the requirements of Content of Monoesters, Stearic acid 55.0-75.0
Diesters, Triesters, and Polyesters. Sum of the contents of palmitic acid and stearic NLT 90.0
ASSAY s
e CONTENT OF MONOESTERS, DIESTERS, TRIESTERS, AND
POLYESTERS IMPURITIES
Mobile phase: Tetrahydrofuran Inorganic Impurities
Sample solution: 15 mg/mL of Sucrose Stearate in e FATS AND FIXED OILS, Acid Value (401): NMT 6, deter-
tetrahydrofuran mined on a 3-g sample. Use a freshly neutralized mixture
Chromatographic system of 2-propanol and water (2:1), and gently heat.
(See Chromatography (621), System Suitability.) Organic Impurities
Mode: LC, size-exclusion © PROCEDURE: FREE SUCROSE
Detector: Differential refractometer 5 Solution A: 10 g/mL of ammonium acetate in
Column: 7-mm x 60-cm; packing L21, 100 A. [NoTE— acetonitrile
Two 7-mm x 30-cm L21 columns may be used in Solution B: 10 t1g/mL of ammonium acetate in tetrahy-
place of one 60-cm column, provided system suitabil- drofuran and water (90:10)
ity requirements are met.] Diluent: Tetrahydrofuran and water (87.5:12.5)
Flow rate: 1.2 mL/min System suitability solution: 500 g/mL of USP Sucrose
Injection size: 20 uL RS in Diluent
Analysis Standard solutions: 0.50, 1.0, 2.0, and 2.5 mg/mL of
Sample: Sample solution USP Sucrose RS in Diluent
[Note—The relative retention time with reference to the Sample solution: 50 mg/mL of Sucrose Stearate in
monoester peak (retention time is approximately 10 Diluent
min) is about 0.92 for diesters, and about 0.90 for Chromatographic system
NF Monographs

triesters and polyesters.] (See Chromatography (621), System Suitability.)

[Note—Disregard solvent peaks and peaks havingasig- Mode: LC
nal-to-noise ratio less than 10.] Detector: Evaporative light-scattering. [NoTE—If the
Calculate thepercentage of monoesters in the portion detector has different setting parameters, adjust the
of Sucrose Stearate taken: detector settings so as to comply with the System
Suitability requirements.]
Result = A x (100 - D — $ - £)/100
A = percentage of monoesters determined by peak
normalization
NF 36 Official Monographs / Sugar 5631

Carrier gas: Nitrogen e B. INFRARED ABSORPTION (197K)

Detector temperature: 45°
Nebulizer temperature: 40° ASSAY
Column: 4.6-mm x 0.25-m; packing L8& © CONTENT OF SUCROSE
Injection size: 20 uL Mobile phase: Acetonitrile and water (80:20, v/v)
Mobile phase and flow rate: See the gradient table System suitability solution: Prepare an aqueous solu-
below. tion containing 20 mg/mL of sucrose, 1.0 mg/mL of
dextrose (glucose), 0.6 mg/mL of fructose, 0.6 mg/mL
of maltose, and 0.8 mg/mL of lactose using USP Su-
Time Solution A Solution B Flow Rate crose RS, USP Dextrose RS, USP Fructose RS, USP Malt-
(min) (%) (%) (mL/min)
ose Monohydrate RS, and USP Anhydrous Lactose RS.
1 100 0 1.0 Standard solution: Dissolve USP Sucrose RS in water to
8 0 100. 1.0 obtain a solution having a concentration of about
ie 0 100 1.0 20 mg/mL of sucrose.
0.01 oO 100 25. Sample solution: 20 mg/mL of Compressible Sugar in
15.99 0 100 25. walsh Pass the solution through a 0.2-um nylon syringe
ilter.
1 100 0 2.5
Chromatographic system
3 100 0 1.0 (See Chromatography (621), System Suitability.)
Mode: LC
System suitability Detector: Refractive index
Sample: System suitability solution Column: 4.6-mm x 15-cm; 5-um packing L8&
[Note—The retention time for sucrose stearate is Temperatures
about 26 min.] Column: 45°
Suitability requirements Detector: 40°
Signal-to-noise ratio: 10:1 Flow rate: 2.0 mL/min
Analysis Injection volume: 15 pL
Samples: Standard solutions and Sample solution System suitability
Prepare a standard curve by plotting the peak re- Samples: System suitability solution and Standard
sponse versus concentration of sucrose in the Stan- solution
lard solution. Calculate the quantity of free sucrose [Note—For relative retention times, see Table 1.]
in the Sucrose Stearate taken.
Acceptance criteria: NMT 4.0%
Table 1
SPECIFIC TESTS Relative
e WATER DETERMINATION, Method Ia (921): NMT 4.0%, on Retention
a 0.20-g sample Name Time
© TOTAL ASH
Analysis: Heata silica or platinum crucible to redness Fructose 0.5
for 30 min, allow to cool in a desiccator, and weigh. Dextrose (glucose) 0.6
Transfer a 1.0-g sample into a crucible. Dry at Sucrose 1.0
100°-105° for 1 h and ignite to constant weight in a Maltose 13
muffle furnace at 600 + 25°, allowing the crucible to Lactose 1.5
cool in a desiccator after each ignition. Flames should
not be produced at any time during the procedure. If Suitability requirements
after prolonged ignition the ash still contains black par- Resolution: NLT 1.3 between all adjacent peaks, Sys-
ticles, add hot water, pass through an ashless filter pa- tem suitability solution
per. and ignite the residue and the filter paper. Com- Relative standard deviation: NMT 2.0%, Standard
ine the filtrate with the ash, carefully evaporate to solution
dryness, and ignite to constant weight. Analysis
Acceptance criteria: NMT 1.5% Samples: Standard solution and Sample solution
Calculate, on the dried basis, the percentage of sucrose
ADDITIONAL REQUIREMENTS (Ci2H22011) in the portion of Compressible Sugar
° nee Label to indicate whether it is Type | or Type taken:
© PACKAGING AND STORAGE: Preserve in a well-closed con- Result = (ru/rs) x (Cs/Cu) x 100
tainer. Protect from humidity and avoid high
temperatures. ty = peak response from the Sample solution
e USP REFERENCE STANDARDS (11) rs = peak response from the Standard solution
USP Sucrose RS Gs = concentration of USP Sucrose RS in the
Standard solution (mg/mL)
Cu = concentration of Compressible Sugar in the
Sample solution (mg/mL)
Acceptance criteria: 95,0%-98.0% on the dried basis
Compressible Sugar
IMPURITIES
Bl Leeroy ABET]

DEFINITION e RESIDUE ON IGNITION (281): NMT 0.1%

¢ CHLORIDE AND SULFATE, Chloride (221)
Sirol

Compressible Sugar contains NLT 95.0% and NMT 98.0%

of sucrose (Ci2H22011) on the dried basis. It may contain
starch, maltodextrin, or invert sugar and may contain a
suitable lubricant.
IDENTIFICATION
e A. It meets the requirements of the test for Specific Rota-
tion in Specific Tests.
Standard solution: 0.40 mL of 0.020 N hydrochloric
acid
‘ample solution: Transfer 20 g to a 100-mL volumetric
flask, add 80 mL of water, shake to dissolve the sucrose,
and then add water to volume. sepa the solubilized
sucrose from any insoluble matterby filtration until the
eae is clear, and use the freshly prepared, clear
iltrate.
 4514 Centella / Dietary Supplements USP 41

Mode: LC © ARTICLES OF BOTANICAL ORIGIN, Pesticide Residue Analysis

Detector: UV 200 nm (561): Meets the requirements
Column: 4.6-mm x 25-cm; 5-4um packing L1 e MICROBIAL ENUMERATION TESTS (2021): The total aerobic
Flow rate: 1.0 mL/min bacterial count does not exceed 10° cfu/g, the total com-
Injection volume: 10 uL bined yeasts and molds count does not exceed 103 cfu/
System suitability g, and the bile-tolerant Gram-negative bacteria count
Samples: Standard solution A and Standard solution B does not exceed 103 cfu/g.
Suitability requirements ¢ ABSENCE OF SPECIFIED MICROORGANISMS (2022): Meets the
Chromatogram similarity: The chromatogram from requirements of the tests for absence of Salmonella spe-
Standard solution B is similar to the reference chro- cies and Escherichia coli
matogram provided with the lot of USP Powdered
Centella asiatica Extract RS being used. SPECIFIC TESTS
Tailing factor: Between 0.8 and 2.0 for the asiatico- ¢ BOTANICAL CHARACTERISTICS
side peak, Standard solution A Macroscopic: Stem is slender, yellowish-brown, with
Resolution: NLT 1.5 between the madecassic acid long internodes, rooting at nodes; leaves are grayish-
and terminolic acid peaks, Standard solution B green, simple, alternate or grouped together at the
Relative standard deviation: NMT 2.0% determined nodes, reniform or oblong-elliptic, have palmate vena-
from the asiaticoside peak in replicate injections, tion, usually with 7 veins, apex obtuse, margin crenate,
Standard solution A base cordate, variable in size, 1-4 cm long, 2-4 cm and
Analysis sometimes up to 7 cm wide, young leaves show a few
Samples: Standard solution A, Standard solution B, and trichomes on the lower surface and adult leaves are gla-
Sample solution. [NotE—Standard solution A, Standard brous; petioles are long, grooved, base wider and
solution B, and the Sample solution are stable for 48 h sheathing; the inflorescence, if present, is a single um-
at room temperature.] bel and consists of 3 flowers, rarely 2 or 4; the flowers
Using the chromatograms of Standard solution A, Stan- are very small (about 2 mm), pentamerous, and have
dard solution B, and the reference chromatogrampro- an inferior ovary; the fruit, brownish-gray, orbicular
vided with the lot of USP Powdered Centella asiatica cremocarp, up to 5 mm long, is very flattened laterally
Extract RS being used, identify the retention times of and has 7-9 prominent curved ridges. Pharmacopeial
the peaks corresponding to different triterpene deriva- article is ptsen to yellowish-green masses composed
tives. The approximate relative retention times of the ry of leaves and stem fragments; odor slight; taste
different triterpene derivatives are provided in Table 2. slightly bitter to sweet.
Microscopic
Table 2
Transverse section of stems: Epidermal layer, sub-
rounded or subsquare cells; 2-4 layers of collenchyma
Approximate Relative cells; 6-8 layers of thin-walled parenchyma cells with
Analyte Retention Time intercellular spaces; 6-7 collateral vascular bundles, xy-
Madecassoside 0.71 lem vessels radially arranged, slightly lignified fiber
Asiaticoside B 0.72 groups occurring outside the phloem; pith large, com-
Asiaticoside 1.00
posed of thin-walled parenchyma cells; secretory
canals, composed of 5-7 secretory cells, observed in
Madecassic acid 2.40 cortex and medullary rays
Terminolic acid 2.44 Transverse section of leaves: Upper and lower epider-
Asiatic acid 3:12 mis; mesophyll composed of parenchyma cells, some
contain crystals of calcium oxalate; 2-3 layers of col-
Separately calculate the percentages of the sum of lenchyma present in the midrib region next to both
madecassoside and asiaticoside B (these two peaks epidermal layers; vascular bundles in the center with
may co-elute), asiaticoside, the sum of madecassic acid xylem on the ventral side and phloem on the dorsal
and terminolic acid, and asiatic acid in the portion of side. Transverse section of petioles has a U shape,
Centella asiatica taken: showing an upper and a lower epidermis, followed by
2-3 layers of collenchyma next to both epidermal lay-
Result = (ru/rs) x Cs x (V/W) x Dx Fx 100 ers; a broad parenchymatous zone, some cells contain
a)
crystals of calcium oxalate; 7 vascular bundles forming
os tu = peak areas of the triterpene derivatives from a weeps in the parenchymatous zone, the two pres-
J the Sample solution ent in the projecting arms being less developed.
roy Is = peak area of asiaticoside from Standard ° ARTICLES OF BOTANICAL ORIGIN, Foreign Organic Matter
° solution A (561): NMT 7.0%, of which NMT 5.0% are of under-
B G = concentration of USP Asiaticoside RS in ground organs and NMT 2% are of other foreign matter
Standard solution A (mg/mL)
= Vv = final volume of Sample stock solution (mL)
e Loss ON DRYING (731)
) Sample: 1.0 of finely powdered Centella asiatica
w = weight of Centella asiatica used to prepare Analysis: Dry the Sample at 105° for 2 h.
a Sample stock solution (mg) Acceptance criteria: NMT 12.0%
D = dilution factor to prepare the Sample solution e ARTICLES OF BOTANICAL ORIGIN, Total Ash (561)
from the Sample stock solution Sample: 1.0 of finely powdered Centella asiatica
F = conversion factors for analytes: 1.00 for Acceptance criteria: NMT 12%
asiaticoside, 1.017 for the sum of ¢ ARTICLES OF BOTANICAL ORIGIN, Acid-Insoluble Ash (561):
madecassoside and asiaticoside B, 0.526 for NMT 3.5%
the sum of madecassic acid and terminolic
acid, and 0.509 for asiatic acid ADDITIONAL REQUIREMENTS
Acceptance criteria: Add the percentages of different e PACKAGING AND STORAGE: Preserve in well-closed contain-
triterpene derivatives: NLT 2.0% on the dried basis. ers, protected from light and moisture, and store at
room temperature.
CONTAMINANTS
© ARTICLES OF BOTANICAL ORIGIN, Limits of Elemental Impuri-
ties (561): Meets the requirements
 5632 Sugar / Official Monographs
Acceptance criteria: 0.014%; a 10-mL portion of the
Sample solution shows no more chloride than the Stan-
dard solution.
e CHLORIDE AND SULFATE, Sulfate (221)
Standard solution: 0.50 mL of 0.020N sulfuric acid
Sample solution: 25 mL of the Sample solution from the
test for Chloride and Sulfate (221), Chloride
Acceptance criteria: 0.010%; the Sample solution
shows no more sulfate than the Standard solution.
e Limit oF CaLcium: Proceed as directed in Identification
Tests—General (191), Calcium.
Sample solution: 5 mL of the Sample solution from the
test for Chloride and Sulfate (221), Chloride
Analysis: To the Sample solution add 1 mL of ammo-
nium oxalate TS.
Acceptance criteria: The solution remains clear for NLT
1 min.
Delete the following:
°o HEAVY METALS (231)
Sample solution: 20 mL of the Sample solution from the
test for Chloride and Sulfate (221), Chloride
Analysis: To the Sample solution add 4 mL of water and
1 mL of 0.1 N hydrochloric acid.
Acceptance criteria: NMT 5 Ug/ge (otficia 1-jan-2018)
e LIMIT OF DEXTROSE (GLUCOSE), FRUCTOSE, MALTOSE, AND
LACTOSE
Mobile phase, System suitability solution, Sample so-
lution, and Chromatographic system: Proceed as di-
rected in the test for Content of Sucrose in the Assay.
System suitability
Sample: System suitability solution
[Note—The relative retention times for fructose, dex-
trose (glucose), sucrose, maltose, and lactose are 0.5,
0.6, 1.0, 1.3, and 1.5, respectively.]
Suitability requirements
Resolution: NLT 1.3 between all adjacent peaks
Acceptance criteria: The sum of the peak areas for
dextrose, fructose, maltose, and lactose from the Sam-
ple solution is less than one third of the sum of the peak
areas for dextrose, fructose, maltose, and lactose from
the System suitability solution, corresponding to NMT
5% for the sum of dextrose, fructose, maltose, and
lactose.
SPECIFIC TESTS
© SPECIFIC ROTATION
Sample solution: Transfer 26.0 g of Compressible
Sugar, previously dried, to a 100-mL volumetric flask.
Add 0.3 mL of a saturated aqueous solution of lead ace-
tate, shake with 90 mL of water, and dilute with water
to volume. Distribute evenly on the surface of a sheet
of medium-fast filter paper 8 g of chromatographic sili-
ceous earth suitable Gk column partition chromatogra-
phy (see Reagents, Indicators, and Solutions: Reagents),
and filter the solution, with the aid of vacuum, discard-
ing the first 20 mL of the filtrate.
Instrumental conditions
(See Optical Rotation (781).)
Mode: Specific rotation
Temperature: 20°
Analysis
Uninverted solution: Pipet 25 mL of the Sample solu-
f=
”
a tion into a 50-mL volumetric flask. Cool to 20°, and
dilute with water to volume at 20°. Maintain a tem-
S
S System suitability solution: Prepare an aqueous solu-
a perature of 20° for 30 min.
fe) Acid-inverted solution: Pipet 25 mL of the Sample so-
= lution into a 50-mL volumetric flask. Slowly add 6 mL
5 of dilute hydrochloric acid (1 in 2) while rotating it,
= dilute with water nearly to volume, and mix. Place the
— flask in a water bath maintained at a temperature of
F< 60°, continuously shake the flask in the bath for 3
min, and allow the flask to remain in the bath for a
NF 36
total of 10 min. Immediately cool to 20° by plunging
the flask into a cold bath, and dilute with water to
volume at 20°. Maintain the flask at a temperature of
20° for 30 min.
Determine the specific rotation of the Uninverted solu-
tion and Acid-inverted solution at 20°.
Acceptance criteria
The specific rotation determined from the Uninverted
solution: 62.6°-73.4°
The specific rotation determined from the Acid-in-
verted solution: Levorotatory
e MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI-
FIED MICROORGANISMS (62): The total aerobic microbial
count does not exceed 103 cfu/g, and the total com-
bined molds and yeasts count does not exceed 10? cfu/
g. It meets the requirements of the tests for the absence
of Salmonella species and Escherichia coli.
e Loss ON DRYING (731)
Analysis: Dry at 105° for 4 h.
Acceptance criteria: NMT 1.0%
ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Preserve in well-closed
containers.
© LABELING: Label it to indicate the name and amount of
any added lubricant.
e USP REFERENCE STANDARDS (11)
USP Anhydrous Lactose RS
USP Compressible Sugar RS
USP Dextrose RS
USP Fructose RS
USP Maltose Monohydrate RS
USP Sucrose RS
Confectioner’s Sugar
DEFINITION
Confectioner’s Sugar is Sucrose ground together with corn
starch to a fine powder. It contains NLT 95.0% of sucrose
(Cy2H22011), calculated on the dried basis.
IDENTIFICATION
oA.
Sample solution: Transfer 20 g to a 100-mL volumetric
flask, add 80 mL of water, shake to dissolve the sucrose,
and then add water to volume. Separate the solubilized
sucrose from the insoluble starch component by filtra-
tion until the filtrate is clear. Use the insoluble portion
for the /dentification test, and use the freshly prepared,
clear filtrate in the Impurities tests for Chloride and Sul-
fate (221), Chloride and Chloride and Sulfate (221), Sul-
fate and for Calcium; and in Specific Tests for Optical
Rotation (781), Specific Rotation.
Analysis: To the water slurry of the insoluble portion
add iodine TS.
Acceptance criteria: A reddish-violet to deep blue color
is produced.
e B. It meets the requirements of the test for Optical Rota-
tion (781), Specific Rotation in Specific Tests.
ASSAY
© CONTENT OF SUCROSE
Mobile phase: Acetonitrile and water (80:20, v/v)
tion containing 20 mg/mL of sucrose, 1.0 mg/mL of
dextrose (glucose), 0.6 mg/mL of fructose, 0.6 mg/mL
of maltose, and 0.8 mg/mL of lactose using USP Su-
crose RS, USP Dextrose RS, USP Fructose RS, USP Malt-
ose Monohydrate RS, and USP Anhydrous Lactose RS.
Standard solution: Dissolve USP Sucrose RS in water to
obtain a solution having a concentration of about
20 mg/mL of sucrose.
NF 36 Official Monographs / Sugar 5633

Sample solution: 20 mg/mL of Confectioner’s Sugar in Delete the following:

water. Pass the solution through a 0.2-m nylon syringe
filter. °e HEAVY METALS (231)
Chromatographic system Sample solution: 20 mL of the clear filtrate from /denti-
(See Chromatography (621), System Suitability.) fication test A
Mode: LC Analysis: To the Sample solution add 4 mL of water and
Detector: Refractive index 1 mL of 0.1 N hydrochloric acid.
Column: 4.6-mm x 15-cm; 5-um packing L8 Acceptance criteria: NMT 5 ug/Qe cotticiat 1Jan-2018)
Temperatures
Column: 45° SPECIFIC TESTS
Detector: 40° e MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI-
Flow rate: 2.0 mL/min FIED MICROORGANISMS (62): The total aerobic microbial
Injection volume: 15 pL count does not exceed 103 cfu/g, and the total com-
System suitability bined molds and yeasts count does not exceed 10? cfu/
Samples: System suitability solution and Standard g It meets the requirements of the tests for absence of
solution almonella species and Escherichia coli.
[Note—For relative retention times, see Table 1.] e Loss ON DRYING (731)
Analysis: Dry at 105° for 4 h.
Table 1 Acceptance criteria: NMT 1.0%
e OPTICAL ROTATION, Specific Rotation (781S)
Relative Sample solution: Use the clear filtrate from /dentifica-
Retention tion test A.
Name Time Acceptance criteria: NLT +62.6° corresponding to NLT
Fructose 0.5 ee of sucrose (Ci2H2201)), calculated on the dried
Dextrose (glucose) 0.6 aSIS
Sucrose 1.0
ADDITIONAL REQUIREMENTS
Maltose 133:
e PACKAGING AND STORAGE: Preserve in well-closed
Lactose 1.5 containers.
e USP REFERENCE STANDARDS (11)
Suitability requirements USP Anhydrous Lactose RS
Resolution: NLT 1.3 between all adjacent peaks, Sys- USP Dextrose RS
tem suitability solution USP Fructose RS
Relative standard deviation: NMT 2.0%, Standard USP Maltose Monohydrate RS
solution USP Sucrose RS
Analysis
Samples: Standard solution and Sample solution
Calculate, on the dried basis, the percentage of sucrose
(Ci2H22011) in the portion of Confectioner’s Sugar
taken:
Sugar Spheres
Result = (ru/rs) x (Cs/Cu) x 100
DEFINITION
ty = peak response from the Sample solution Sugar Spheres contain NLT 62.5% and NMT 91.5% of su-
rs = peak response from the Standard solution crose (Cy2H22011), calculated on the dried basis, the re-
G = concentration of USP Sucrose RS in the mainder consisting chiefly of starch. They consist of ap-
Standard solution (mg/mL) proximately spherical particles of a labeled nominal size
Cu = concentration of Confectioner’s Sugar in the range. They may contain color additives permitted by the
Sample solution (mg/mL) FDA for use in drugs.
Acceptance criteria: NLT 95.0% of sucrose on the
dried basis IDENTIFICATION
cA.
IMPURITIES Sample suspension: 1:10
e RESIDUE ON IGNITION (281): NMT 0.08% Analysis: Add iodine TS to the Sample suspension.
e CHLORIDE AND SULFATE, Chloride (221) Acceptance criteria: A violet to deep blue color is
recs solution: 0.40 mL of 0.020 N hydrochloric produced.
aci e B. It meets the requirements of the test for Optical Rota-
Sample solution: 10 mL of the clear filtrate from Identi- tion (7815S), Specific Rotation in Specific Tests.
fication test A
Acceptance criteria: 0.014%; the Sample solution ASSAY
shows no more chloride than the Standard solution. © CONTENT OF SUCROSE
e CHLORIDE AND SULFATE, Sulfate (221) Mobile phase: Acetonitrile and water (80:20, v/v)
Standard solution: 0.30 mL of 0.020Nsulfuric acid System suitability solution: Prepare an aqueous solu-
Sample solution: 25 mL of the clear filtrate from Identi- tion containing 20 mg/mL of sucrose, 1.0 mg/mL of
fication test A dextrose (glucose), 0.6 mg/mL of fructose, 0.6 mg/mL
Acceptance criteria: 0.006%; the Sample solution of maltose, and 0.8 mg/mL of lactose using USP Su-
sydesbouow 4N

shows no more sulfate than the Standard solution.
e CALCIUM
Sample solution: 5 mL of the clear filtrate from Identifi-
cation test A
Analysis: To the Sample solution add 5 mL of water and
1 mL of ammonium oxalate TS.
Acceptance criteria: The Sample solution remains clear
for NLT 1 min.
crose RS, USP Dextrose RS, USP Fructose RS, USP Malt-
ose Monohydrate RS, and USP Anhydrous Lactose RS.
Standard solution: Dissolve USP Sucrose RS in water to
obtain a solution having a concentration of about
20 mg/mL of sucrose.
Sample solution: 20 mg/mL of Sugar Spheres in water.
Pass the solution through a 0.2-14m nylon syringe filter.
Chromatographic system
(See Chromatography (621), System Suitability.)
 5634 Sugar / Official Monographs NF 36

Mode: LC e Loss ON DRYING (731)

Detector: Refractive index Analysis: Dry at 105° for 4 h.
Column: 4.6-mm x 15-cm; 5-um packing L& Acceptance criteria: NMT 4.0%
Temperatures © PARTICLE SIZE DISTRIBUTION ESTIMATION BY ANALYTICAL
Column: 45° SIEVING (786): NLT 90% of it passes the coarser sieve
Detector: 40° size stated in the labeling; all of it passes the next coarser
Flow rate: 2.0 mL/min sieve size listed in Table 7 of the chapter; and NMT 10%
Injection volume: 15 uL passes the finer sieve size stated in the labeling.
System suitability
Samples: System suitability solution and Standard ADDITIONAL REQUIREMENTS
solution e PACKAGING AND STORAGE: Preserve in well-closed
[Note—See Table 7.] containers.
© LABELING: The label states the nominal particle size
Table 1
range. Label it to indicate the name and amount of any
added color additives.
Relative e USP REFERENCE STANDARDS (11)
Retention USP Anhydrous Lactose RS
Name Time USP Dextrose RS
Fructose 0.5 USP Fructose RS
Dextrose (glucose) 0.6 USP Maltose Monohydrate RS
USP Sucrose RS
Sucrose 1.0
Maltose 13
Lactose 15.

Suitability requirements
Resolution: NLT 1.3 between all adjacent peaks, Sys- Sulfur Dioxide
tem suitability solution
Relative standard deviation: NMT 2.0%, Standard SO. 64.06
solution Sulfur dioxide [7446-09-5].
Analysis
DEFINITION
Samples: Standard solution and Sample solution
Calculate the percentage of sucrose in the portion of Sulfur Dioxide contains NLT 97.0%, by volume, of sulfur
Sugar Spheres taken: dioxide (SOz).
[Caution—Sulfur Dioxide is poisonous.]
Result = (ru/rs) x (Cs/Cu) x 100 ASSAY
e PROCEDURE
ru = peak response from the Sample solution
Sample: 100.0 mL of gaseous Sulfur Dioxide
rs = peak response from the Standard solution
Titrimetric system
Cs = concentration of USP Sucrose RS in the
(See Titrimetry (541).)
Standard solution (mg/mL)
Mode:_ Direct titration
Cu = concentration of Sugar Spheres in the Sample
Titrant: 0.1 N iodine VS
solution (mg/mL)
Endpoint detection: Visual
Acceptance criteria: 62.5%-91.5% on the dried basis
Analysis: Collect the Sample over mercury, and note
IMPURITIES the temperature of the Sample and the pressure upon
e RESIDUE ON IGNITION (281) it. Slowly introduce 50.0 mL of 0.1 N sodium hydroxide
Sample: 2.0g into the air space over the mercury, and absorb the
Analysis: Ignite at a temperature of 700 + 25°. Sample in the solution by shaking. When absorption is
Acceptance criteria: NMT 0.25% complete, transfer the solution to a 250-mL conical
flask, add 3 mL of starch TS, and titrate with Titrant
until the solution is pale blue in color.
Delete the following: Calculate the pee ae of sulfur dioxide (SO2) at a
temperature of 0° and a pressure of 760 mm of mer-
°e HEAVY METALS, Method II (231): 5 Ug/Ge cortical :-jan-2018) cury in the portion of Sulfur Dioxide taken. Each mL of
0.1 N iodine is equivalent to 1.094 mL of SO.
SPECIFIC TESTS Acceptance criteria: NLT 97.0%, by volume
e MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI-
FIED MICROORGANISMS (62): The total aerobic microbial IMPURITIES
count does not exceed 10? cfu/g, and the Spheres meet e Limit OF NONVOLATILE RESIDUE
the requirements of the tests for absence of Salmonella Sample: 300g (209 mL)
species, Escherichia coli, Staphylococcus aureus, and Pseu- Analysis: Transfer the Sample to a tared, 250-mL conical
lomonas aeruginosa. flask, and allow the liquid to evaporate spontaneously
© OPTICAL ROTATION, Specific Rotation (781S) in a well-ventilated hood. When evaporation appears
Sample solution: Transfer 20 g to a 200-mL volumetric complete, blow a current of dry, filtered air through the
flask, add 160 mL of water, shake to dissolve the su- flask until the odor of sulfur dioxide is no longer
NF Monographs

crose, add water to volume, and mix. Pass the solubi-
lized sucrose solution by vacuum filtration through fine
filter paper.
Acceptance criteria: +41° to +61°, corresponding to
62.5%-91.5% of sucrose (Ci2H22011), calculated on the
dried basis
apparent.
Acceptance criteria: NMT 7.5 mg (0.0025%)
e SULFURIC ACID
Analysis: To the flask containing the residue obtained in
the test for Limit of Nonvolatile Residue add 25 mL of
water previously neutralized to methyl red TS. Swirl the
flask, and titrate with 0.10 N sodium hydroxide.
Acceptance criteria: NMT 1.3 mL is required (about
0.002%).
NF 36 Official Monographs / Sunflower 5635

SPECIFIC TESTS dense fumes of sulfur trioxide form. Cool, and cau-
e WATER DETERMINATION, Method | (921) tiously wash the solution into an arsine generating flask
Sample: 3g (2.1 mL) with 50 mL of water.
Analysis: Taking precautions to avoid absorption of Analysis: Proceed as directed in the chapter, omitting
moisture, transfer the Sample to a suitable flask, and the addition of the 20 mL of 7N sulfuric acid.
add 20 mL of anhydrous pyridine. Acceptance criteria: NMT 1 ppm
Acceptance criteria: NMT 2.0%
ADDITIONAL REQUIREMENTS Delete the following:
° PACKAGING AND STORAGE: Preserve in cylinders.
[Note—Sulfur Dioxide is used most in the form of a gas °o HEAVY METALS (231)
in pharmaceutical applications, and is described herein Test preparation: Add 2.2 mL (4.0 g) to 10 mg of so-
for such purposes. However, it is usually packaged dium carbonate dissolved in 10 mL of water. Heat until
under pressure, hence the preceding specifications are almost dry. Add 1 mL of nitric acid, and evaporate to
designed for testing it in liquid form.] dryness. Add 2 mL of 1 N acetic acid to the residue,
and dilute with water to 25 mL.
Analysis: Proceed as directed in the chapter.
Acceptance criteria: NMT 5 ppMe coffiaa’ 1.jan-2018)
SPECIFIC TESTS
Sulfuric Acid ¢ REDUCING SUBSTANCES
Sample: 4.4 mL (8.0 g)
H2SO4 98.08 Analysis: Carefully dilute the Sample with 50 mL of ice-
Sulfuric acid [7664-93-9]. cold water, keeping the solution cold during the addi-
tion. Add 0.10 mL of 0.10 N potassium permanganate.
DEFINITION Acceptance criteria: The solution remains pink for 5
Sulfuric Acid contains NLT 95.0% and NMT 98.0%, by min.
weight, of sulfuric acid (H2SO,).
[CauTioN—When sulfuric acid is to be mixed with other liq- ADDITIONAL REQUIREMENTS
uids, always add it to the diluent, and exercise great ¢ PACKAGING AND STORAGE: Preserve in tight containers.
caution.]
IDENTIFICATION
e A. IDENTIFICATION TESTS—GENERAL, Sulfate (191): Meets
the requirements
Sunflower Oil
ASSAY
© PROCEDURE [8001-21-6].
Sample solution: Place 1 mL of Sulfuric Acid in a DEFINITION
weighed, glass-stoppered flask containing 20 mL of Sunflower Oil is a refined fixed oil obtained from the seeds
water, and weigh again to obtain the weight of the of the sunflower plant Helianthus annuus L. (Fam. Astera-
Sulfuric Acid. Dilute with 25 mL of water, and cool. ceae alt. Compositae). It may contain a suitable
Titrimetric system antioxidant.
(See Titrimetry (541).)
Mode: Direct titration IDENTIFICATION
Titrant: 1.N sodium hydroxide VS ¢ A. IDENTITY BY FATTY ACID COMPOSITION
Blank: 45 mL of water Analysis: Proceed as directed in the test for Fats and
Endpoint detection: Visual Fixed Oils, Fatty Acid Composition (401).
Analysis: To the Sample solution add methyl orange TS, Acceptance criteria: It meets the composition profile of
and titrate with 1 N sodium hydroxide VS. Perform a fatty acids in Table 2.
blank determination. Calculate the percentage of sulfu- e B. IDENTITY BY TRIGLYCERIDE PROFILE
ric acid (H2SOx) taken: Analysis: Perform this test for generic oil only. Proceed
as directed in Identification of Fixed Oils by Thin-Layer
Result = {[(Vs — Vs) x N
x FI/W} x 100 Chromatography (202).
Vs = Titrant volume consumed by sulfuric acid in Acceptance criteria: It meets the requirements in the
chapter.
the Sample solution (mL)
Ve = Titrant volume consumed by the Blank (mL) IMPURITIES
N = Titrant actual normality (mEq/mL) © ALKALINE IMPURITIES
F = equivalency factor, 49.04 mg/mEq Sample: 10 mL of Sunflower Oil
Ww = nny of sulfuric acid in the Sample solution Analysis: Mix 10 mL of freshly opened acetone and
(mg
Acceptance criteria: 95.0%-98.0%
0.3 mL of water, and add 0.05 mL of bromophenol
blue TS. Add the Sample, shake, and allow to stand.
Titrate with 0.01 N hydrochloric acid VS to change the
IMPURITIES color of the upper layer to yellow.
© RESIDUE ON IGNITION (281)
Acceptance criteria: NMT 0.1 mL of 0.01 N hydrochlo-
3]oolUC ABET |

Sample: 22 mL (40g)
Analysis: Evaporate the Sample to dryness, and ignite. ric acid is required.
EX¥rel

Acceptance criteria: NMT 2 mg of residue remains

(0.005%). Delete the following:
e CHLORIDE AND SULFATE, Chloride (221): A dilution of
1.1 mL (2.0 g) in water shows no more chloride than °e HEAVY METALS, Method Ii (231): NMT 10 ppme coticiai1-
corresponds to 0.15 mL of 0.020 N hydrochloric acid Jan-2018)
(50 ppm).
e ARSENIC (211)
Test preparation: Add 1.6 mL (3.0 g) to a mixture of
nitric acid and water (3:20). Evaporate the solution until
 5636 Sunflower / Official Monographs NF 36

SPECIFIC TESTS A = peak area of palmitate, stearate, oleate,

© FATS AND FIXED OILS, Acid Value (Free Fatty Acids) (401): linoleate, or linolenate
NMT oe mL of 0.020 N sodium hydroxide is required for B = total area of the five major peaks
neutralization. Acceptance criteria: See Table 2.
© FATS AND FIXED OILS, Fatty Acid Composition (401) e
Standard solution: Prepare an ester mixture containing Table 2
methyl linoleate, methyl oleate, methyl palmitat
methyl stearate, and methyl linolenate (50:35:7:5:3).1 Mid-Oleic High-Oleic
Sample solution: Transfer 100 mg of Sunflower Oil to a Generic Oil oil oil
50-mL conical flask fitted with a suitable water-cooled (%) (%) (%)
reflux condenser and a magnetic stir bar. Add 4 mL of Methyl
0.5 N methanolic sodium hydroxide solution, and reflux palmitate 3-10 2-9 2-9
until fat globules disappear (usually 5-10 min). Add Methyl stearate 2-8 2-8 2-8
5 mL of a solution prepared by dissolving 14 g of boron Methyl oleate 14-39 40-70 70-90
trifluoride in methanol to make 100 mL, swirl to mix, Methyl eel 48-73
and reflux for 2 min. Add 4 mL of chromatographic n- eioy' [oleate = 15-40 S15
heptane through the condenser, and reflux for 1 min. Methyl ‘
cow Pa ue Set a 15 mL of saturated linolenate 0-3 0-3 0-3
sodium chloride solution, shake, and allow the layers to *
separate. Pass the n-heptane layer through 0.1 Gat an- ° Fats AND: FIXED Gis. lodine Value; Method.I<401)
hydrous sodium sulfate (previously washed with chro- < ep! Seok 138 148
matographic n-heptane) into a suitable flask. Transfer Midaleic sil: 98-1 18
1.0 mL of this solution to a 10-mL volumetric flask, di- High-oleic oil 78-98
lite wit ocnremateataphicnenepiane ta Malm’, ard + FATS AND FIXED Outs, Unsaponifiable Matter (401): NMT
. i
Chromatographic system e LIMIT OF PEROXIDE
(See Chromatography (621), System Suitability.) [NoT&—This test must be performed promptly after sam-
Mode: GC pling to avoid oxidation of the test specimen.]
Detector: Flame ionization Solvent A: Chloroform and glacial acetic acid (40:60)
Column: 0.25-mm x 30-m fused-silica capillary col- Potassium iodide solution: Prepare a saturated solution
umn bonded with a 0.25-1m layer of phase GS of potassium iodide in freshly boiled and cooled water,
TemperALES and store protected from light. Discard the solution if it
Detectan 350° becomes colored upon the addition of Solvent A and
iodide-free starch TS.
Column: See Table 7. Sample: 10g of Sunflower Oil
Analysis: Transfer the Sample to a conical flask, add
Table 1 30 mL of Solvent A, and swirl to dissolve. Add 0.5 mL of
Hold Time Potassium iodide solution, swirl for 1.0 min, and add
Initial Temperature Final at Final 30 mL of water. Titrate with 0.01 N sodium thiosulfate
Temperature Ramp Temperature | Temperature VS, with vigorous agitation, to a light yellow color. Add
e) (/min) e) (min) 2.0 mL of iodide-free starch TS, and continue the titra-
2 tion until the blue color has disappeared.
Pp Perform a
120 = 120
blank determination, and make any necessary
120 4 240 5 correction.
c Calculate the amount of peroxide, in mEq/kg, in the
Carrier gas: Timlin
Flow rate: Hydrogen +
portion of Sunflower Oil; taken:sal
Injection volume: 1 pL 7
Injection type: Splitless RESUS LM BSN DIESE
System suitability . Vv = volume of sodium thiosulfate used in the
Sample: Standard solution _ ; titration (mL)
[Note—The relative retention times for methyl palmi- N = normality of sodium thiosulfate VS (mEq/mL)
tate, methyl stearate, and methyl oleate are about Ww = weight of Sunflower Oil taken (g)
0.87, 0.99, and 1.0, respectively.] F = conversion factor, 1000 g/kg
Suitability requirements Acceptance criteria: NMT 10.0 mEq/kg
Resolution: NLT 1.5 between methyl stearate and e WATER DETERMINATION, Method Ic (921): NMT 0.1%
methyl oleate
Relative standard deviation: NMT 6.0% peak areas ADDITIONAL REQUIREMENTS
for the palmitate and stearate peaks for replicate in- ¢ PACKAGING AND STORAGE: Preserve in tight, light-resistant
jections; NMT 1.0% peak area ratio of the palmitate containers. No storage requirement specified.
to stearate peaks from these replicate injections e LABELING: The label states the Latin binomial and, follow-
Analysis ing the official name, the part of the plant source from
Samples: Standard solution and Sample solution which the article was derived. Label it to indicate
”
Measure the areas of the five major peaks for the whether it is Generic Sunflower Oil, Mid-Oleic Sunflower
Py methyl esters of the fatty acids, which elute in the fol- Oil, or High-Oleic Sunflower Oil. The label also indicates
is lowing order: palmitate, stearate, oleate, linoleate, and the name and concentration of any additive.
i}
— linolenate.
Dp Calculate the percentage of palmitate, stearate, oleate,
(2) linoleate, and linolenate in the portion of Sunflower Delete the following:
S
S Oil taken:
°e USP REFERENCE STANDARDS (11)
= USP Sunflower Oil RS
Le
Result = (A/B) x 100
2 —— = (CN i-May-2018 RIE)
@ CES
1 Ester mixtures are available commercially from Nu-Chek-Prep, Inc., P.O. Box
295, Elysian, MN 56028 Typical Nu-Chek-Prep ester mixtures useful in this
test include Nu-Chek 15A. This mixture may contain other components.
NF 36 Official Monographs / Tagatose 5637

Suspension Structured Vehicle Syrup

DEFINITION DEFINITION
Prepare Suspension Structured Vehicle as follows (see Phar- Syrup is a solution of Sucrose in Purified Water. It contains a
maceutical Compounding—Nonsterile Preparations (795)). preservative unless it is used when freshly prepared.

Potassium Sorbate 0.159 Sucrose 850 q

Xanthan Gum 0.15 q Purified Water, a sufficient quantity to make 1000 mL
Citric Acid, Anhydrous 0.15q
Sucrose 20 q
Syrup may be prepared by the use of boiling water or, pref-
erably, without heat, by the following process. Place the
Purified Water, a sufficient quantity to make 100 mL Sucrose in a suitable percolator, the neck of which is
Transfer the Potassium Sorbate to a suitable beaker, and dis-
nearly filled with loosely packed cotton, moistened after
solve in 50 mL of Purified Water. Place the beaker on an packing with a few drops of water. Pour carefully 450 mL
electric hot plate and stirrer, and add the Xanthan Gum
of Purified Water upon the Sucrose, and regulate the out-
flow to a steady drip of percolate. Return the percolate, if
into the vortex while slowly stirring. Apply minimal heat,
necessary, until all of the Sucrose has dissolved. Then wash
and incorporate the Citric Acid and the Sucrose. Add a the inside of the percolator and the cotton with sufficient
sufficient quantity of Purified Water to obtaina final vol- Purified Water to bring the volume of the percolate to
ume of 100 mL, and mix.
1000 mL, and mix.
ADDITIONAL REQUIREMENTS
SPECIFIC TESTS
¢ PACKAGING AND STORAGE: Package in tight, light-resistant
e SPECIFIC GRAVITY (841): NLT 1.30
containers. Store at room temperature, and avoid
freezing. ADDITIONAL REQUIREMENTS
© LABELING: Label it to state that it must be well shaken e PACKAGING AND STORAGE: Package in tight containers,
before use. preferably in a cool place.
© BEYOND-UsE DATE: NMT 30 days after the date on which
it was compounded

Tagatose
Sugar-Free Suspension Structured th
Vehicle wom (on
nh Yn
DEFINITION
Prepare Sugar-Free Suspension Structured Vehicle as follows
(see Pharmaceutical Compounding—Nonsterile Preparations CeHi206 180.16
(795)). D-Tagatose;
D-lyxo-Hexulose [87-81-0].
Xanthan Gum 0.20 q DEFINITION
Saccharin Sodium 0.20 g Tagatose is a ketohexose, an epimer of D-fructose inverted
Potassium Sorbate 0.15 q at C-4. It is obtained from D-galactose by isomerization
Citric Acid 0.10 q under alkaline conditions in the presence of calcium. It
Sabie 204 contains NLT 98.0% of tagatose (CsH120.), calculated on
Manni : the dried basis.
jannitol 20g
Glycerin 2.0 mL IDENTIFICATION
Purified Water, a sufficient quantity to make 100 mL e A. The retention time of the major peak of the Sample
solution corresponds to that of the Standard solution, as
Transfer 30 mL of Purified Water to a beaker, placing it on an obtained in the Assay.
electric hot plate and stirrer. Using moderate heat, stir to e B. It meets the requirements of the test for Optical Rota-
form a vortex, and slowly sprinkle the Xanthan Gum into tion (781), Specific Rotation.
the vortex. In a separate beaker, dissolve the Saccharin e .
Sodium, Potassium Sorbate, and Citric Acid in 50 mL of Pu- Sample solution: 200 mg/mL of Tagatose
rifled Water. Using moderate heat, incorporate the Sorbitol, Analysis: Add 3 mL of the Sample solution to 5 mL of
Mannitol, and Glycerin into this mixture. Add to this mix- hot alkaline cupric tartrate TS.
ture the previously prepared xanthan gum dispersion. Add Acceptance criteria: A copious red precipitate of cu-
a sufficient quantity of Purified Water to obtain a final vol- prous oxide is formed.
ume of 100 mL, and mix.
ASSAY
sydeibouo-: 4N

ADDITIONAL REQUIREMENTS © PROCEDURE

¢ PACKAGING AND STORAGE: Package in tight, light-resistant Mobile phase: 0.05 mg/mL of calcium acetate
containers. Store at room temperature, and avoid Standard solution: 5 mg/mL of USP Tagatose RS. Pass
freezing. throughafilter of 0.2-um pore size.
© LABELING: Label it to state that it must be well shaken Sample solution: 5 mg/mL of Tagatose, previously
before use. dried. Pass through afilter of 0.2-1m pore size.
e BEYOND-Use DATE: NMT 30 days after the date on which Chromatographic system
it was compounded (See Chromatography (621), System Suitability.)
 5638 Tagatose / Official Monographs NF 36

Mode: LC ADDITIONAL REQUIREMENTS

Detector: Refractive index e PACKAGING AND STORAGE: Preserve in well-closed contain-
Column: 7.8-mm x 30-cm; 9-um packing L19 ers, and store at room temperature.
Column temperature: 85° e USP REFERENCE STANDARDS (11)
Flow rate: 0.6 mL/min USP Tagatose RS
Injection size: 20 uL
System suitability
Sample: Standard solution
Suitability requirements
Relative standard deviation: NMT 2.0% of replicate Talc—see Talc General Monographs
injections
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of tagatose (CsHi20¢) in the Tartaric Acid
portion of Tagatose taken:
Result = (ru/rs) x (Cs/Cu) x 100

tu = peak response from the Sample solution

rs = peak response from the Standard solution
Gs = concentration of USP Tagatose RS in the
Standard solution (mg/mL) C4H6Oc 150.09
Cu = concentration of Tagatose in the Sample Butanedioic acid, 2,3-dihydroxy-;
solution (mg/mL) Butanedioicacid,2, 3-dihydrox eee
Acceptance criteria: NLT 98.0% on the dried basis Tartaric acid; L(4)-Tartaricee ies 0; 87-69-4].
DEFINITION
IMPURITIES
e Limit OF LEAD
Tartaric Acid, dried over phosphorus pentoxide for 3 h, con-
Sample solution: 2.5 g of Tagatose dissolved in a mix- tains NLT 99.7% and NMT 100.5% of C4HeOc.
ture of 4 mL of sulfuric acid and 5 mL of hydrochloric IDENTIFICATION
acid. Dilute with water to 50 mL. e A. IDENTIFICATION TESTS—GENERAL, Tartrate (191): It
Standard stock solution A: Dissolve 1.60g of lead ni- meets the requirements.
trate in diluted nitric acid (10 mL of nitric acid diluted e B. INFRARED ABSORPTION (197K)
with 20 mL water, boiled to remove nitrous fumes, and
cooled), and dilute with water to 1000 mL. ASSAY
Standard stock solution B: Standard stock solution A e PROCEDURE
and water (1:50). [NoTtE—This solution contains the Sample: 2g of Tartaric Acid, previously dried, in a coni-
equivalent of 20 ug/mL of lead.] cal flask
Standard solutions: To a series of 100-mL volumetric Analysis: Dissolve the Sample in 40 mL of water, add
flasks pipet 0, 1, 2, 3, 4, and 5 mL of Standard stock phenolphthalein TS, and titrate with 1 N sodium hy-
solution B, and dilute ‘With water to about 50 mL. Add droxide VS. Each mL of 1 N sodium hydroxide is equiv-
8 mL of sulfuric acid and 10 mL of hydrochloric acid to alent to 75.04 mg of C4H6Oc.
each flask, shake to dissolve, and dilute with water to Acceptance criteria: 99.7%-100.5%
volume. [NoTE—These solutions contain 0, 0.2, 0.4, 0.6,
0.8, and 1.0 ug/mL of lead, respectively.] IMPURITIES
Instrumental conditions Inorganic Impurities
(See Atomic Absor oe Spectroscopy (852).) © RESIDUE ON IGNITION (281): NMT 0.1%
Mode: Atomic absorption e CHLORIDE AND SULFATE, Sulfate (221)
Analytical wavelength: 283.3 nm Sample solution: 10 mL of a 1-in-100 solution of Tar-
Analysis taric Acid
Samples: Standard solutions and Sample solution Analysis: Add to the Sample solution 3 drops of hydro-
Concomitantly determine the absorbances of the Stan- chloric acid and 1 mL of barium chloride TS.
dard solutions and the Sample solution. Plot the ab- Acceptance criteria: No turbidity is produced.
sorbances of the Standard solutions versus the con-
centration of lead. Using this graph, determine the Delete the following:
concentration of lead in the Sample solution.
Acceptance criteria: NMT 1 ppm ®e HEAVY METALS, Method |] (231): NMT 10 ppme coriciat 1-
SPECIFIC TESTS Jan-2018)

© MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI-

FIED MICROORGANISMS (62): It meets the requirements of
the tests for absence of Salmonella species and Escherichia
coli. The total aerobic microbial count does not exceed
1000 cfu/g, and the total combined molds and yeasts
count does not exceed 100 cfu/g.
NF Monographs
SPECIFIC TESTS
e OPTICAL ROTATION, Specific Rotation (781S): +12.0° to
+13.0°
Sample solution: 200 mg/mL in water
e Loss ON DRYING (731): Dry over phosphorus pentoxide
for 3 h: it loses NMT 0.5% of its weight.

e MELTING RANGE OR TEMPERATURE, Class | (741):

e LIMIT OF OXALATE
133°-144°
OPTICAL ROTATION, Specific Rotation (781): -4° to -7° Sample solution: 10 mL of a 1-in-10 solution of Tartaric
Sample solution: 10 mg/mL Acid
Loss ON DRYING (731): Dry a sample at 102° for 2 h: it Analysis: Nearly neutralize the Sample solution with 6 N
ammonium hydroxide, and add 10 mL of calcium sul-
loses NMT 0.5% of its weight
NMT fate TS.
ARTICLES OF BOTANICAL ORIGIN, Tota! Ash (561):
0.1%, determined on a 1.0-g specimen
NF 36 Official Monographs / Tocopherols 5639

Acceptance criteria: No turbidity is produced. ADDITIONAL REQUIREMENTS

© PACKAGING AND STORAGE: Preserve in tight, light-resistant
ADDITIONAL REQUIREMENTS containers.
© PACKAGING AND STORAGE: Preserve in well-closed e USP REFERENCE STANDARDS (11)
containers. USP Thymol RS
e USP REFERENCE STANDARDS (11)
USP Tartaric Acid RS

Titanium Dioxide—see Titanium Dioxide

Thimerosal—see Thimerosal General
Monographs
Thymol
CioH14O 150.22
Phenol, 5-methyl-2-(1-methylethyl)-;
Thymol;
p-Cymen-3-ol [89-83-8].
DEFINITION
Thymol contains NLT 99.0% and NMT 101.0% of thymol
(CroH140).
IDENTIFICATION
e A. INFRARED ABSORPTION (197K)
e B. It meets the requirements in Specific Tests for Melting
Range or Temperature (741).
ASSAY
© PROCEDURE
Sample: 100mg
Titrimetric system
(See Titrimetry (541).)
Mode: Direct titration
Titrant: 0.1 N bromine VS
Endpoint detection: Visual
Analysis: Transfer the Sample to a 250-mL iodine flask,
and dissolve in 25 mL of 1 N sodium hydroxide. Add
20 mL of hot dilute hydrochloric acid (1 in 2), and im-
mediately titrate with Titrant to within 1-2 mL of the
calculated endpoint. Warm the solution to between 70°
and 80°, add 2 drops of methyl orange TS, and con-
tinue the titration slowly, swirling vigorously after each
addition. When the color of the methyl orange is
bleached, add 2 drops of Titrant, shake for 10 s, add 1
drop of methyl orange TS, and shake vigorously. If the
solution is red, continue the titration, dropwise and
with shaking, until the color is discharged. Repeat the
alternate addition of the Titrant and methyl orange TS
until the red color is discharged after the addition of
the methyl orange TS. Each mL of Titrant is equivalent
to 3.755 mg of thymol (C1oH140).
Acceptance criteria: 99.0%-101.0%
IMPURITIES
General Monographs
Tocopherols Excipient
DEFINITION
Tocopherols Excipient is a vegetable oil solution containing
NLT 50.0% of total tocopherols, of which NLT 80.0%
consists of varying amounts of beta, gamma, and delta
tocopherols.
IDENTIFICATION
oA.
Sample solution: 50mg of Tocopherols Excipient in
10 mL of dehydrated alcohol
Analysis: To the Sample solution add with swirling 2 mL
of nitric acid, and heat at about 75° for 15 min.
Acceptance criteria: A bright red or orange color
develops.
¢ B. The retention time of the third major peak (i.e., the
peak occurring just before that of the internal standard)
of the Sample solution corresponds to that of the Stan-
dard solution, both relative to that of the internal stan-
dard, as obtained in the Assay.
ASSAY
¢ PROCEDURE
Solution A: Pyridine and propionic anhydride (2:1)
Internal standard solution: 3 mg/mL of hexadecyl hex-
adecanoate in Solution A
Standard solutions: Using low-actinic glassware, add
12-, 25-, 37-, and 50-mg portions of USP Alpha To-
copherol RS to separate 50-mL conical flasks having 19/
38 standard-taper ground-glass necks. Pipet 25 mL of
the Internal standard solution into each flask, and reflux
for 10 min under water-cooled condensers.
Sample solution: Using low-actinic glassware, add
60 mg of Leen Excipient to a 50-mL conical flask
similar to the flasks used in preparing the Standard solu-
tions. Add 10.0 mL of Internal standard solution, and re-
flux for 10 min under a water-cooled condenser.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: GC
Detector: Flame ionization
Column: 4-mm x 2-m borosilicate glass; packed with
2%-5% liquid phase G2 on 80- to 100-mesh support
S1AB using either a glass-lined sample introduction
system or on-column injection
Temperatures
Column: 245°-265°, maintained isothermally
Injection port: 10° higher than the Column
temperature
sydesbouow 4N

e LIMIT OF NONVOLATILE RESIDUE Detector: 10° higher than the Column temperature
Sample: 2g Flow rate: Dry carrier gas is adjusted to obtain a
Analysis: Volatilize the Sample on a steam bath, and dry hexadecyl hexadecanoate peak 30-32 min after sam-
at 105° to constant weight. ple introduction. [NoTE—Cure and condition the col-
Acceptance criteria: NMT 0.05% umn as necessary.]
SPECIFIC TESTS Injection volume: 2-5 ul
e MELTING RANGE OR TEMPERATURE (741): 48°-51° but System suitabilit
when melted, Thymol remains liquid at a considerably Sample: Sample solution
lower temperature. [NoTtt—The relative retention times for delta tocopheryl
propionate, beta plus gamma tocophery! propionate,
 5640 Tocopherols / Official Monographs
and hexadecyl hexadecanoate are about 0.50, 0.63,
and 1.00, respectively.]
Suitability requirements
Resolution: Chromatograph a sufficient number of
injections to ensure that a resolution of NLT 2.5 be-
tween delta tocopheryl propionate and beta plus
gamma tocopheryl propionate relative to hexadecyl
hexadecanoate is met.
Analysis
Samples: Standard solutions and Sample solution
Calibration: Chromatograph each Standard solution,
and calculate the relative response factor, F, for each
concentration of the Standard solution taken:
F = (rs/rp) x (Co/Cs)
rs = peak response of alpha tocopherol in the
Standard solution
tp = peak response of hexadecyl hexadecanoate in
the Standard solution
Cp = concentration of hexadecyl hexadecanoate in
the Standard solution (mg/mL)
Gs = concentration of USP Alpha Tocopherol RS in
the Standard solution (mg/mL)
Chromatograph a sufficient number of injections of
each Standard solution to ensure that F is constant
within a range of 2.0%. Prepare a relative response
factor curve by plotting F versus the alpha tocophery!
propionate peak response.
Inject the Sample solution, and measure the responses
for the four major peaks occurring at relative
retention times of approximately 0.50, 0.63, 0.76,
and 1.00, and record the values as a5, dpy, du, and dp,
corresponding to delta tocopheryl propionate, beta
plus gamma tocopheryl propionates, alpha
tocopheryl propionate, and hexadecyl
hexadecanoate, respectively.
Calculate the quantity of each tocopherol form in the
Tocopherols Excipient taken:
delta tocopherol = (V
x Cp/F) x (as/ap)
beta plus gamma tocopherols = (V x Co/F) x (ap,/a0)
alpha tocopherol = (V
x Cp/F) x (da/ap)
Vv = volume of Internal standard solution used in
the Sample solution (mL)
F = obtained from the relative response factor
curve (see Calibration) for each of the
corresponding responses for the delta, beta
plus gamma, and alpha tocopheryl
propionate peaks produced by the Sample
solution
[NotE—The relative response factor for delta tocopheryl
propionate and for beta Plus gamnira tocopheryl pro-
pionates has been determined empirically to be the
same as for alpha tocopheryl propionate.]
Acceptance criteria: NLT 50.0% of total tocopherols,
of which NLT 80.0% consists of varying amounts of
beta, gamma, and delta tocopherols
SPECIFIC TESTS
e ACIDITY
NF Monographs
Solution A: Alcohol and ether (50%:50%). Neutralize
to phenolphthalein with 0.1 N sodium hydroxide.
Sample solution: Dissolve 1.0 g of Tocopherols Excipi-
ent in 25 mL of Solution A.
Analysis: To the Sample solution add 0.5 mL of phenol-
phthalein TS, and titrate with 0.10 N sodium hydroxide
until the solution remains faintly pink after being
shaken for 30 s.
Acceptance criteria: NMT 1.0 mL of 0.10 N sodium
hydroxide is required.
NF 36
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in tight containers,
protected from light. Protect with a blanket of an inert
gas.
e LABELING: Label it to indicate the content, in mg/g, of
total tocopherols and of the sum of beta, gamma, and
delta tocopherols.
e USP REFERENCE STANDARDS (11)
USP Alpha Tocopherol RS
Tolu Balsam Syrup
DEFINITION
Prepare Tolu Balsam Syrup as follows (see Pharmaceutical
ompounding—Nonsterile Preparations (795)).
Tolu Balsam Tincture 50 mL
Magnesium Carbonate 10q
Sucrose 820 g
Purified Water, a sufficient quantity to make 1000 mL
Add the Tincture all at once to the Magnesium Carbonate
and 60 g of the Sucrose in a mortar, and mix. Gradually
add 430 mL of Purified Water with trituration, and filter.
Dissolve the remainder of the Sucrose in the clear filtrate
with gentle heating, strain the syrup while warm, and add
sufficient Purified Water through the strainer to make the
roduct measure 1000 mL, and mix.
Tolu Balsam Syrup may also be prepared as follows. Place
760g of the Sucrose in a suitable percolator, the neck of
which is nearly filled with loosely packed cotton, moist-
ened after packing with a few drops of water. Pour the
filtrate, obtained as directed in the preceding instructions,
on the Sucrose, and regulate the outflow to a steady drip
of percolate. When all of the liquid has run through, re-
turn portions of the percolate, if necessary, to dissolve all
the Sucrose. Then pass enough Purified Water through the
cotton to make the product measure 1000 mL, and mix.
OTHER COMPONENTS
e ALCOHOL DETERMINATION, Method II (611): 3.0%-5.0%
SPECIFIC TESTS
e FATS AND FIXED OILS, Acid Value (401)
Sample solution: 2% of solution
Analysis: Add phenolphthalein TS, and titrate with 0.5
N alcoholic potassium hydroxide VS.
Acceptance criteria: 112-168
ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Package in tight containers,
and store at controlled room temperature.
e LABELING: The label states the Latin binomial name and,
following the official name, the part of the plant source
from which the article was derived.
Tolu Balsam Tincture
DEFINITION
Tolu Balsam Tincture is prepared from Tolu Balsam obtained
from Myroxylon balsamum (L.) Harms var. balsamum
(Fam. Fabaceae).
Prepare Tolu Balsam Tincture as follows (see Pharmaceutical
Compounding—Nonsterile Preparations (795)).
NF 36 Official Monographs / Trehalose 5641

Tolu Balsam 200 g Acceptance criteria: No pink or red color develops.

Alcohol 750 mL
ADDITIONAL REQUIREMENTS
Alcohol, a sufficient quantity to make 1000 mL. © PACKAGING AND STORAGE: Preserve in well-closed
Macerate the Tolu Balsam with 750 mL of Alcohol in a con- containers.
tainer that can be closed, and put in a warm place. Agi-
tate it frequently during 3 days or until the soluble matter
is dissolved. Transfer the mixture to a filter, and when
most of the liquid has drained away, wash the residue on
the filter with a sufficient quantity of Alcohol, combining Trehalose
the filtrates to produce 1000 mL of Tincture, and mix.
OTHER COMPONENTS
e ALCOHOL DETERMINATION, Method | (611): 77.0%-83.0%
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Package in tight, light-resistant Orsnd o

containers, and store at controlled room temperature.

Avoid exposure to direct sunlight and to excessive heat.
e LABELING: The label states the Latin binomial name and, Ci2H22011 342.30
following the official name, the part of the plant source Ci2H22011 - 2H20 378.33
from which the article was derived. a-D-Glucopyranosyl o-D-glucopyranoside.
Anhydrous [99-20-7].
Dihydrate [6138-23-4].
DEFINITION
Tragacanth Trehalose is a stable, nonreducing disaccharide with two
glucose molecules linked in an o,a-1,1 configuration. It is
DEFINITION obtained through enzymatic conversion of food-grade
Tragacanth is the dried gurimny exudation from Astragalus starch. It contains NLT 97.0% and NMT 102.0% of treha-
ummifer Labill., or other Asiatic species of Astragalus lose (Cy2H22011), calculated on the anhydrous basis.
Fam. Leguminosae).
IDENTIFICATION
IDENTIFICATION e A. INFRARED ABSORPTION (197K)
e A. Add 1g to 50 mL of water: it swells and forms a ° B.
smooth, nearly uniform, stiff, opalescent mucilage free Sample solution: 400 mg/mL of Trehalose
from cellularfragments: Analysis: Add 0.4 mL of a solution containing 1-naph-
thol in 95% alcohol (1 in 20) to 1 mL of the Sample
IMPURITIES solution. Gently add 2 mL of sulfuric acid to the
© LEAD (251): NMT 10 ppm solution.
Acceptance criteria: A violet color develops at the in-
terface between the two solutions.
Delete the following: eC
Glycine solution: 40 mg/mL of glycine
°e Heavy METALS, Method |] (231): NMT 20 pprme cotta 1- Sample solution: 40 mg/mL of Trehalose
Jan-2018) Analysis: Add 1 mL of diluted hydrochloric acid to 2 mL
SPECIFIC TESTS of the Sample solution. Allow to stand for 20 min at
e MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI- room temperature. Add 4 mL of sodium hydroxide TS
FIED MICROORGANISMS (62): It meets the requirements for and 2 mL of Glycine solution to the Sample solution.
absence of Salmonella species and Escherichia coli. Heat the solution for 10 min in boiling water.
¢ BOTANIC CHARACTERISTICS Acceptance criteria: A brown color does not develop.
Tragacanth: It is flattened, lamellated, frequent! ASSAY
curved fragments or straight or spirally twisted linear e PROCEDURE
pieces from 0.5 to 2.5 mm in thickness. It is white to Mobile phase: Water
weak yellow in color, translucent, and horny in texture. Standard solution: 10 mg/mL of USP Trehalose RS, cal-
Its fracture is short. It is rendered more easily pulveriz- culated on the anhydrous basis
able by heating to 50°. It is odorless. Sample solution: 10 mg/mL of Trehalose, calculated on
Histology: Pieces of Tragacanth softened in water and the anhydrous basis
mounted in water or glycerin show numerous lamellae Chromatographic system
and a few starch grains. (See Chromatography (621), System Suitability.)
Powdered tragacanth: It is white to yellowish white. Mode: LC
When examined in water mounts, it shows numerous Detector: Refractive index
angular fragments of mucilage with circular or irregular Column: 8-mm x 30-cm; packing L58
lamellae, and occasional starch grains up to 25 um in Temperatures
sydesbouo-= 4N

diameter, mostly simple, spherical to elliptical, with oc- Detector: 40°

casional two- to four-compound grains, a few of the Column: 80°
grains being swollen and more or less altered. The Flow rate: Adjust so that the retention time of treha-
powder shows few or no fragments of lignified vegeta- lose is about 15 min.
le tissue (Indian gum).
e KARAYA GUM
Sample solution: 1g in 20 mL of water
Analysis: Boil the Sample solution until a mucilage is
formed, add 5 mL of hydrochloric acid, and again boil
the mixture for 5 min.
USP 41
Change to read:
e LABELING: The label states the Latin binomial and, follow-
ing the official name, the parts of the plant contained in
the article. The label states that this article is exempted
from the requirements of °Labeling (7), Labels and Label-
ing for Products and Other Categories, Botanicals,e «cn 1-may-
2018) with respect to the pregnancy and lactation state-
ment. %e (CN }-May-2018)
e¢ USP REFERENCE STANDARDS (11)
USP Asiaticoside RS
USP Powdered Centella asiatica Extract RS
Powdered Centella asiatica
DEFINITION
Powdered Centella asiatica is Centella asiatica reduced to a
powder or very fine powder. It contains NLT 2.0% of
triterpene derivatives, calculated on the dried basis.
IDENTIFICATION
e A. Powdered Centella asiatica meets the requirements for
Specific Tests, Botanical Characteristics.
¢ B. THIN-LAYER CHROMATOGRAPHY
Standard solution A: 0.5 mg/mL of USP Asiaticoside RS
in methanol
Standard solution B: 10 mg/mL of USP Powdered
Centella asiatica Extract RS in methanol. Sonicate for
about 10 min, centrifuge, and use the supernatant.
Sample solution: About 0.5 g of Powdered Centella
asiatica in 5 mL of methanol. Sonicate for 10 min, cen-
trifuge, and use the supernatant.
Adsorbent: Chromatographic silica gel with an average
particle size of 10-15 um (TLC plates) or with an aver-
age particle size of 5 um (HPTLC plates)
_ volume: 10 pl (TLC plates) or 4 wL (HPTLC
plates,
Developing solvent system: Methylene chloride, meth-
anol, and water (14:6:1)
Derivatization reagent: A solution of 10% sulfuric acid
in methanol. [NotE—Prepare fresh.]
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Apply the Samples as bands. Use a saturated chamber.
Develop the chromatograms until the solvent front has
moved up about three-fourths of the plate. Remove
the plate from the chamber, dry, treat with Derivatiza-
tion reagent, heat for 3 min at 120°, and examine
under white light.
Acceptance criteria: The Sample solution chromato-
gram exhibits a violet band in the lower third of the
plate due to asiaticoside, corresponding in color and Rr
to that in Standard solution A; a violet band due to
madecassoside at an R; lower than that of asiaticoside;
and two additional violet bands in the upper third of
the plate due to asiatic acid and madecassic acid. Bands
detected in the Sample solution correspond in position
and color to bands in Standard solution B. Other minor
bands may be observed in the Sample solution and
Standard solution B.
e C. HPLC: The Sample solution chromatogram from the
test for Content of Triterpene Derivatives shows a peak at
the retention time corresponding to that of asiaticoside
in Standard solution A. \dentify other triterpene derivative
peaks in the Sample solution by comparison with the
chromatogram of Standard solution B and the reference
chromatogram provided with the lot of USP Powdered
Centella asiatica Extract RS being used. The Sample solu-
tion shows additional peaks corresponding to madecasso-
Dietary Supplements / Centella 4515
side and asiaticoside B (these two peaks may co-elute),
madecassic acid, terminolic acid, and asiatic acid.
COMPOSITION
e CONTENT OF TRITERPENE DERIVATIVES
Solution A: Dilute 3 mL of phosphoric acid with water
to 1000 mL, mix, filter, and degas.
Solution B: Acetonitrile
Mobile phase: See Table 1.
Table 1
Time Solution A Solution B
(min) (%) (%)
0 78 22.
65 45 55
66 5 95
75 5 95
76 78 22
85 78 22
Standard solution A: 0.05 mg/mL of USP Asiaticoside
RS in methanol
Standard solution B: Sonicate a portion of USP Pow-
dered Centella asiatica Extract RS in methanol to obtain
a solution with a concentration of about 5.0 mg/mL.
Before injection, pass through a membrane filter of
0.45-"um or finer pore size, discarding the first few mL
of the filtrate.
Sample stock solution: Transfer about 1.0 g of Pow-
dered Centella asiatica, accurately weighed, to a Soxhlet
apparatus. Add 100 mL of methanol, extract for 8 h,
cool, and dilute with methanol to 100 mL. Pass through
a membrane filter of 0.45-m or finer pore size, dis-
carding the first few mL of the filtrate. [NoTE—Use a
thimble of a suitable size such that the volume of meth-
anol used in the Soxhlet extraction is at least twice the
volume of the thimble.]
Sample solution: Dilute 5.0 mL of Sample stock solution
with methanol to 10.0 mL.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 200 nm
Column: 4.6-mm x 25-cm; 5-uum packing L1
Flow rate: 1.0 mL/min
Injection volume: 10 pL
System suitability
Samples: Standard solution A and Standard solution B
Suitability requirements
Chromatogram similarity: The chromatogram from
sydeabouo= sa
Standard solution B is similar to the reference chro-
matogram provided with the lot of USP Powdered
Centella asiatica Extract RS being used.
Tailing factor: Between 0.8 and 2.0 for the asiatico-
side peak, Standard solution A
Resolution: NLT 1.5 between the madecassic acid
and terminolic acid peaks, Standard solution B
Relative standard deviation: NMT 2.0% determined
from the asiaticoside peak in replicate injections,
Standard solution A
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution. [Note—Standard solution A, Standard
solution B, and Sample solution are stable for 48 h at
room temperature.
Using the chromatograms of Standard solution A, Stan-
dard solution B, and the reference chromatogrampro-
vided with the lot of USP Powdered Centella asiatica
Extract RS being used, identify the retention times of
the peaks corresponding to different triterpene deriva-
tives. The approximate relative retention times of the
different triterpene derivatives are provided in Table 2.
 5642 Trehalose / Official Monographs
Injection volume: 20 uL
System suitability
Sample: Standard solution
Suitability requirements
Relative standard deviation: NMT 2.0%
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of trehalose (C)2H2201;) in the
portion of Trehalose taken:
Result = (ru/rs) x (Cs/Cu) x 100
ty = peak response from the Sample solution
rs = peak response from the Standard solution
Cs = concentration of USP Trehalose RS in the
Standard solution (mg/mL)
Cu = concentration of Trehalose in the Sample
solution (mg/mL)
Acceptance criteria: 97.0%-102.0% on the anhydrous
basis
IMPURITIES
© RESIDUE ON IGNITION (281): NMT 0.1%, determined on
2.0 g of Trehalose
Delete the following:
°o HEAVY METALS, Method | (231)
Sample: 4.0g
Monitor preparation: Prepare with 2.0 mL of Standard
Lead Solution.
Acceptance criteria: NMT 5 ppMe (ottical 1-Jan-2018)
o RELATED SUBSTANCES
Mobile phase and Chromatographic system: Proceed
as directed in the Assay.
Sample solution: 10 mg/mL of Trehalose
System suitability solution: Transfer 2.5 mL of the
Sample solution, 25 mg of maltotriose, and 25 mg of
glucose to a 10-mL volumetric flask, and dilute with
water to volume.
Standard solution: 0.1 mg/mL of the Sample solution
System suitability
Sample: System suitability solution
[Note—The relative retention times for maltotriose, tre-
halose, and glucose are about 0.9, 1.0, and 1.2,
eapeelyeNeT
Suitability requirements
Resolution: NLT 1.5 between trehalose and
maltotriose
Relative standard deviation: NMT 2.0% for the tre-
halose peak
Analysis
Samples: Sample solution and Standard solution
Determine the peak areas for all peaks.
Acceptance criteria: For the Sample solution, the areas
of any peaks corresponding to maltotriose and other
olysaccharrides and eluting before trehalose are NMT
alt of the area of the peak corresponding to trehalose
in the chromatogram of the Standard solution (0.5%).
The areas of any peaks corresponding to glucose and
eluting after trehalose are NMT half of the area of the
peak corresponding to trehalose in the chromatogram
of the Standard solution (0.5%).
NF Monographs
SPECIFIC TESTS
© COLOR AND CLARITY OF SOLUTION
Sample solution: 33g of Trehalose in 67 g of recently
boiled water
Analysis: Using a suitable spectrophotometer (see Ultra-
violet-Visible Spectroscopy (857)), measure the ab-
sorbances of the Sample solution at 420 and 720 nm in
a 10-cm cuvette. The absorbance of the Sample solution
at 720 nm is NMT 0.050.
NF 36
Determine the absorbance difference:
Result = Aazo — A720
Agzo = absorbance of the Sample solution at 420 nm
Ayzo = absorbance of the Sample solution at 720 nm
Acceptance criteria: The absorbance difference is NMT
0.100.
e OPTICAL ROTATION, Specific Rotation (781S)
Sample solution: 100 mg/mL
Acceptance criteria: +197° to +201° at 20°
¢ MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI-
FIED MICROORGANISMS (62): The total aerobic microbial
count is NMT 100 cfu/g, and the total combined molds
and yeasts count is NMT 100 cfu/g. It meets the require-
ments of the tests for absence of Salmonella species and
Escherichia coli.
© PH (791)
Sample solution: 100 mg/mL
Acceptance criteria: 4.5-6.5
oe WATER DETERMINATION, Method | (921)
Sample: 0.1g
Acceptance criteria
Anhydrous: NMT 1.0%
Dihydrate: 9.0%-11.0%
e BACTERIAL ENDOTOXINS TEST (85): If labeled for use in
prepanng parenteral dosage forms, it also meets the fol-
lowing requirements. The level of bacterial endotoxins is
such that the requirement in the relevant dosage form
monograph(s) in which Trehalose is used can be met.
Where the label states that Trehalose must be subjected
to further processing during the preparation of injectable
dosage forms, the level of bacterial endotoxins is such
that the requirement in the relevant dosage form mono-
graph(s) in which Trehalose is used can be met.
e CHLORIDE AND SULFATE, Chloride (221)
Sample: 2.0g
Acceptance criteria: No more chloride than corre-
sponds to 0.70 mL of 0.01 M hydrochloric acid (NMT
0.0125%)
¢ CHLORIDE AND SULFATE, Sulfate (221)
Sample: 2.0g
Acceptance criteria: No more sulfate than corresponds
to 0.83 mL of 0.005 M sulfuric acid (NMT 0.0200%)
e NITROGEN DETERMINATION, Method |! (461)
Sample: 5.0g
Analysis: Proceed as directed in Method Il, except in-
crease the volume of sulfuric acid for digestion to
30 mL and the volume of the sodium hydroxide solu-
tion (2 in 5) to 45 mL.
Acceptance criteria: NMT 0.005%
© SOLUBLE STARCH
Sample solution: 10% Trehalose (w/v)
Analysis: Add several drops of iodine TS to the Sample
solution.
Acceptance criteria: No blue color develops.
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in tight containers. No
storage requirements specified.
e LABELING: Where Trehalose is intended for use in the
manufacture of injectable dosage forms, it is so labeled.
Where Trehalose must be subjected to further processing
during the preparation of injectable dosage forms to en-
sie accept levels of bacterial endotoxins, it is so
labeled.
Change to read:
° USP REFERENCE STANDARDS (11)
@ (CN 1-May-2018)
USP Trehalose RS
NF 36 Official Monographs / Trichloromonofluoromethane 5643

Triacetin—see Triacetin General Monographs Suitability requirements

Resolution: NLT 1.5 between tributyl citrate and
acetyltributyl citrate
Relative standard deviation: NMT 2.0%, determined
from both the tributyl citrate and acetyltributyl citrate
Tributy! Citrate peaks
Analysis
oh, Sample: Sample solution
Calculate the percentage of tributyl citrate (CisH3207)
2 in the portion of sample taken:
°
om Result = (ru/r) x 100
Ho ) 9
Hy. NO. ir An chy tu = peak area of tributyl citrate from the Sample
8 solution
tr = sum of all peaks excluding the solvent peak
Acceptance criteria: NLT 99.0% on the anhydrous
CisH3207 360.44 basis
DEFINITION IMPURITIES
Tributyl Citrate contains NLT 99.0% of tributyl citrate
(CisH3707), calculated on the anhydrous basis.
Delete the following:
IDENTIFICATION
e A. INFRARED ABSORPTION (197F) °e HEAVY METALS, Method II (231): NMT 10 19/ge wrticia1-
e B. The retention time of the major peak of the Sample Jan-2018)
solution corresponds to that of the System suitability solu-
tion, as obtained in the Assay. SPECIFIC TESTS
© SPECIFIC GRAVITY (841): 1.037-1.045
ASSAY e REFRACTIVE INDEX (831): 1.443-1.445
¢ PROCEDURE ° ACIDITY
System suitability solution: 30 mg/mL each of USP Sample: roa
Tributyl Citrate RS and USP Acetyltributyl Citrate RS in Analysis: Dissolve the Sample in 30 mL of isopropyl al-
toluene cohol, previously neutralized to bromothymol blue. Add
Sample solution: 30 mg/mL of Tributyl Citrate in bromothymol blue TS, and titrate with 0.10 N sodium
toluene hydroxide to a faint blue endpoint.
Chromatographic system Acceptance criteria: NMT 1.0 mL of 0.10 N sodium
(See Chromatography (621), System Suitability.) hydroxide is required.
Mode: GC, nla He with an on-column, tempera- © WATER DETERMINATION, Method | (921): NMT 0.2%
ture-programmable injector
Detector: Flame ionization ADDITIONAL REQUIREMENTS
Column: 0.32-mm x 30-m, bonded with a 0.5-4m e PACKAGING AND STORAGE: Preserve in tight containers.
layer of phase G42 e USP REFERENCE STANDARDS (11)
Temperatures USP Acetyltributyl Citrate RS
Injector: See Table 1. USP Tributyl Citrate RS
Detector: 275°
Column: See Table 2.

Table 1
Hold Time
Trichloromonofluoromethane
Initial Temperature Final at Final
CF
Temperature Ramp Temperature | Temperature VY
Cc) (¢/min) ©) (min) av ~el
85 — 85 0.5
85 20 225: 10 CCI3F 137.37
Methane, trichlorofluoro-;
Trichlorofluoromethane [75-69-4].
Table 2
DEFINITION
Hold Time Trichloromonofluoromethane contains NLT 99.6% and NMT
Initial Temperature Final at Final 100.0% of trichloromonofluoromethane (CCIsF), calcu-
Temperature Ramp Temperature | Temperature lated on the anhydrous basis.
©) (¢/min) C) (min)
80 = 80 0.5 IDENTIFICATION
e A. The IR absorption spectrum, determined in a 10-cm
sydeibouo-= 4N

80 20 220 10
cell with sodium chloride windows, at atmospheric pres-
Carrier gas: Helium sure, exhibits maxima, among others, at the following
Flow rate: 2.3 mL/min wavelengths, in um: 4.67 (m), 5.95 (m), 7.28 (s), 8.06
Injection volume: 1 wL (m), 9.2 (vs), 10.7 (vs), 11.8 (vs), and 13.4 (m). The
System suitability stronger maxima are best obtained at pressures less than
Sample: System suitability solution 10 mm of mercury.
[NoTte—The relative retention times for tributyl citrate
and acetyltributyl citrate are 0.9 and 1.0, respectively.] ASSAY
e PROCEDURE
System suitability solution: Introduce a liquid-phase
mixture of dichlorodifluoromethane, dichlorotetrafluoro-
 5644 Trichloromonofluoromethane / Official Monographs NF 36

ethane, and trichloromonofluoromethane into an evacu- SPECIFIC TESTS

ated headspace vial. © BOILING TEMPERATURE
Sample solution: Introduce the liquid phase of Trichlo- Analysis: Determine as directed in Propellants (602).
epionmnlliekeneteaie into an evacuated headspace Acceptance criteria: Approximately 24°
vial. © WATER
Chromatographic system Analysis: Determine as directed in Propellants (602),
(See Chromatography (621), System Suitability.) and Water Deterimination, Method Ic (921).
Mode: GC Acceptance criteria: NMT 0.001%
Detector: Flame ionization © HIGH-BOILING RESIDUES
Column: 2-mm x 1.8-m stainless steel; 1% phase G25 Analysis: Determine as directed in Propellants (602).
on support $12 Acceptance criteria: NMT 0.01%
Temperatures
Injection port: 110° ADDITIONAL REQUIREMENTS
Detector: 200° e PACKAGING AND STORAGE: Preserve in tight cylinders, and
Column: See Table 7. avoid exposure to excessive heat.

Table 1
Hold Time
Initial Temperature Final at Final Triethyl Citrate
Temperature Ramp Temperature | Temperature
©) (°/min) © (min) HC. 0. 0
70. 10 170 5
Ho, os
ara wa °
Carrier gas: Helium
Flow rate: 20 mL/min
Headspace sampler: The bath temperature is 100°,
the valve/loop temperature is 105°, and the sampling Ci2H2007 276.28
time is 3 s. Make adjustments as necessary to optimize
peak areas to record trace-level impurities. DEFINITION
System sarabilly Triethy! Citrate contains NLT 99.0% and NMT 100.5% of
Sample: Gas phase headspace of the System suitability Ci2H2007, calculated on the anhydrous basis.
solution
[NoTe—The relative retention times for dichlorodifluoro- IDENTIFICATION
methane, dichlorotetrafluoroethane, and trichloro- e A. INFRARED ABSORPTION (197F)
monofluoromethane are 0.5, 0.8, and 1.0, e B. The retention time of the major peak of the Sample
respectively.] solution corresponds to that of a similar preparation of
Suitability requirements USP Triethyl Citrate RS, as obtained in the Assay.
Resolution: NLT 2.0 between dichlorotetrafluoroeth-
ane and trichloromonofluoromethane ASSAY
Analysis © PROCEDURE
System suitability solution: 30 mg/mL each of USP Tri-
Sample: Gas phase headspace of the Sample solution ethyl Citrate RS and USP Acetyltriethyl Citrate RS in
Calculate the percentage of trichloromonofluorome-
thane (CCI3F) in the portion of Trichloromonofluoro-
toluene
methane taken. Sample solution: 30 mg/mL of Triethy! Citrate in
Acceptance criteria: 99.6%-100.0% on the anhydrous toluene
basis
Chromatographic system
(Gee hee alagnap yy (621), System Suitability.)
IMPURITIES Mode:
e INORGANIC CHLORIDES Detector: Flame ionization
Sample: 7g Column: 0.32-mm x 30-m; 0.5-um layer of phase G42
Analysis: Place 5 mL of anhydrous methanol in a test Temperature
tube, add 3 drops of a saturated solution of silver ni- Injector: 225°
trate in anhydrous methanol, shake, and add the Detector: 275°
Sample. Column: See the temperature program table below.
Acceptance criteria: No opalescence or turbidity is
produced. Hold Time
e CHROMATOGRAPHIC PURITY Initial Temperature Final at Final
Analysis: In the chromatogram from the Assay, identi Temperature Ramp Temperature | Temperature
the dichlorodifluoromethane and dichlorotetrafluoroeth- ©) (¢/min) ©) (min)
ane peaks from relative retention times of those peaks 80 = 80 0.5
in the chromatogram of the System suitability solution. 80 20 220 20
Acceptance criteria
Sum of the peak areas for dichlorodifluoromethane Flow rate: 2.3 mL/min
and dichlorotetrafluoroethane: ©NMT 0.2% of the Carrier gas: Helium
NF Monographs

total of all peak areas Injection type: Split, 30:1

Sum of the areas of all peaks other than that for Injection size: 1 uL
trichloromonofluoromethane: NMT 0.4% of the System suitability
total of all peak areas. Sample: System suitability solution
[Note—The relative retention times for triethyl citrate
and acetyltriethyl citrate are 0.9 and 1.0, respectively.]
Suitability requirements
Resolution: NLT 1.5 between triethyl citrate and
acetyltriethyl citrate
NF 36
Relative standard deviation: NMT 2.0% (deter-
mined from both the triethyl citrate and acetyltriethyl
citrate peaks, based on area percentage calculation)
Analysis
Sample: Sample solution
[NoTe—Measure all of the peak areas, excluding the sol-
vent peak.]
Calculate the percentage of Ci2H2007 in the portion of
Triethyl Citrate taken:
Result = (ru/rr) x 100
tu = peak area for triethyl citrate
tr = sum of the area responses of all the peaks
Acceptance criteria: 99.0%-100.5% on the anhydrous
basis
IMPURITIES
Inorganic Impurities
Delete the following:
°e HEAVY METALS, Method I] (231): NMT 10 ppme owicial i-
Jan-2018)
SPECIFIC TESTS
e SPECIFIC GRAVITY (841): 1.135-1.139
e REFRACTIVE INDEX (831): 1.439-1.441
e ACIDITY
Neutralized isopropyl alcohol: To a suitable quantity
of isopropyl alcohol add 2-3 drops of bromothymol
blue TS and just sufficient 0.10 N sodium hydroxide
dropwise to produce a faint blue color. [NoTe—Prepare
Neutralized isopropy! alcohol just prior to use.]
Sample solution: 32.0g of Triethyl Citrate in 30 mL of
Neutralized isopropyl alcohol
Analysis: Add bromothymol blue TS. Titrate with 0.10
N sodium hydroxide to a faint blue endpoint.
Acceptance criteria: NMT 1.0 mL of 0.10 N sodium
hydroxide is required.
© WATER DETERMINATION, Method | (921): NMT 0.25%
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in tight containers.
e USP REFERENCE STANDARDS (11)
USP Acetyltriethy! Citrate RS
USP Triethyl Citrate RS
Medium-Chain Triglycerides
Glycerides, mixed decanoyl and octanoyl;
Caprylic and capric triglycerides.
DEFINITION
Medium-Chain inal cerides consist of a mixture of triglycer-
ides of saturated fatty acids, mainly of caprylic acid
(CgH16O2) and capric acid (CicoH20O2). The fatty acids are
derived from the oil extracted from the hard, dried frac-
tion of the endosperm of Cocos nucifera L. or from the
dried Sucsperm of Elaeis guineensis Jacq. They contain
NLT 95% of saturated fatty acids with 8 and 10 carbon
atoms.
IDENTIFICATION
e A. Meet the requirements in Specific Tests for Fats and
Fixed Oils, Saponification Value (401)
e B. Meet the requirements in Specific Tests for Fats and
Fixed Oils, Fatty Acid Composition (401)
Official Monographs / Triglycerides 5645
IMPURITIES
Delete the following:
®» Heavy METALS, Method I! (231)
{Note—Use this test for Medium-Chain Triglycerides in-
tended for use other than in parenteral nutrition.]
Sample solution: Transfer 2.0 g of Medium-Chain Tri-
glycerides to a quartz crucible. Add 0.5 g of magnesium
oxide, Ignite the crucible to dull redness until a homo-
geneous white or grayish-white mass is obtained. Ignite
at 800° for 1h, cool, and dissolve the residue by add-
ing two 5-ml portions of diluted hydrochloric acid. Add
0.1 mL of phenolphthalein TS and then ammonium hy-
droxide until a pink color is obtained. Cool, add glacial
acetic acid until the solution is decolorized, then add
0.5 mL in excess, and dilute with water to 20.0 mL.
Standard solution: To 0.5 g of magnesium oxide add
2.0 mL of Lead Standard Solution, and evaporate to dry-
ness at 105° for 1 h. Using the same conditions as pre-
scribed for the Sample solution, ignite, dissolve in di-
luted hydrochloric acid, add ammonia and then acetic
acid, and dilute with water to 20.0 mL.
Analysis: To 12 mL of the Sample solution add 2.0 mL of
pH 3.5 Acetate Buffer and 1,2 mL of thioacetamide-
gicere base TS. To 10 mL of the Standard solution add
-O mL of the Sample solution, and add 2.0 mL of pH
3.5 Acetate Buffer and 1.2 mL of thioacetamide-glycerin
base TS. Prepare a blank, using a mixture of 10 mL of
water and 2.0 mL of the Sample solution. Compared to
the blank, the Standard solution shows alight brown
color. Dilute both the Sample solution and the Standard
solution with water to 50 mL, allow to stand for 2 min,
and view downward over a white surface.
Acgeptanes criteria; NMT 10 ug/g; any brown color
of the Sample solution is not darker than that of the
Standard solution. corricial 1-j2n-2018)
e Limit OF CHROMIUM
[Note—Use this test for Medium-Chain Triglycerides in-
tended for use in parenteral nutrition.]
Sample stock solution: 500 mg/mL of Medium-Chain
Triglycerides in diisobutyl ketone
Sample solution: 200 mg/mL of Medium-Chain Triglyc-
erides in diisobutyl ketone, from Sample stock solution
Chromium standard stock solution: 0.283 mg/mL of
potassium dichromate in water, using potassium dichro-
mate previously dried at 105° for 4 h
Chromium standard solution: Immediately before use,
prepare 0.283 g/mL of potassium dichromate in water,
from the Chromium standard stock solution. This solution
contains the equivalent of 0.1 ug/mL of chromium.
Standard solutions: Into each of three 10-mL volumet-
tic flasks, transfer 4.0 mL of Sample stock solution, add
0.5, 1.0, and 2.0 mL, respectively, of Chromium stan-
dard solution, and dilute with diisobutyl ketone to vol-
ume. These solutions contain 0.005, 0.01, and 0.02 ug/
mL of chromium.
Instrumental conditions
(See Atomic Absorption Spectroscopy (852).)
Mode: Atomic absorption spectrophotometer
equipped with a graphite furnace
Analytical wavelength: 357.8 nm
Lamp: Chromium hollow-cathode
Carrier gas: Argon
sydeiBouow 4N
Analysis
Samples: Standard solutions and Sample solution
Determine the abosrbances of the Standard solutions
and the Sample solution in triplicate, and determine
the average of the steady readings for each. Plot the
average absorbances of the Standard solutions and
the Sample solution versus the concentration of added
chromium. Draw the straight line best fitting the
points, and extrapolate the line until it meets the
concentration axis. The distance between this point
 5646 Triglycerides / Official Monographs
and the intersection of the axes represents the con-
centration of chromium in the Sample solution.
Acceptance criteria: NMT 0.05 n9/g
o LIMIT OF COPPER
[Note—Use this test for Medium-Chain Triglycerides in-
tended for use in parenteral nutrition.]
eae es stock solution and Sample solution: Proceed
as directed in the test for Limit of Chromium.
Copper standard stock solution: 0.393 mg/mL of cu-
pric sulfate in water
Copper standard solution: Immediately before use,
prepare 0.393 tg/mL of cupric sulfate in water, from
the Copper standard stock solution. This solution con-
tains the equivalent of 0.1 g/mL of copper.
Standard solutions: Into each of three 10-mL volumet-
ric flasks, transfer 4.0 mL of Sample stock solution. Add
1.0, 2.0, and 4.0 mL, respectively, of Copper standard
solution, and dilute with diisobutyl ketone to volume.
These solutions contain 0.01, 0.02, and 0.04 t1g/mL of
copper.
Instrumental conditions
(See Atomic Absorption Spectroscopy (852).)
Mode: Atomic absorption spectrophotometer
equipped with a graphite furnace
Analytical wavelength: 324.7 nm
Lamp: Copper hollow-cathode
Carrier gas: Argon
Analysis
Samples: Standard solutions and the Sample solution
Record the average of the steady readings for each of
the Standard solutions and the Sample solution in trip-
licate. Plot the absorbances of the Standard solutions
and the Sample solution versus the concentration of
added copper. Draw the straight line best fitting the
points, dnd entiapelne the line until it meets the
concentration axis. The distance between this point
and the intersection of the axes represents the con-
centration of copper in the Sample solution.
Acceptance criteria: NMT 0.1 g/g
o Limit oF LEAD
[Note—Use this test for Medium-Chain Triglycerides in-
tended for use in parenteral nutrition.]
Sample stock solution and Sample solution: Proceed
as directed in the test for Limit of Chromium.
Lead standard stock solution: Dissolve 160 mg of lead
nitrate in 100 mL of water that contains 1 mL of lead-
free nitric acid, and dilute with water to 1000 mL. Pipet
10 mL of this solution into a 100-mL volumetric flask,
and dilute with water to volume.
Lead standard solution: Immediately before use, pre-
pare 0.16 ug/mL of lead nitrate from the Lead standard
stock solution. This solution contains the equivalent of
0.1 g/mL of lead.
Standard solutions: Into each of three 10-mL volumet-
ric flasks, transfer 4.0 mL of Sample stock solution. Add
1.0, 2.0, and 4.0 mL, respectively, of Lead standard solu
tion, and dilute with diisobutyl ketone to volume. These
solutions contain 0.01, 0.02, and 0.04 g/mL of lead.
Instrumental conditions
(See Atomic Absorption Spectroscopy (852).)
Mode: Atomic absorption spectrophotometer
equipped with a graphite furnace coated inside with
palladium carbide
[Note—Calcination is carried out in the presence of ox-
NF Monographs
ygen at a temperature below 800°.]
Analytical wavelength: 283.3 nm
Lamp: Lead hollow-cathode
Carrier gas: Argon
Analysis
Samples: Standard solutions and the Sample solution
Record the average of the steady readings for each of
the Standard solutions and the Sample solution in trip-
licate. Plot the absorbances of the Standard solutions
and the Sample solution versus the concentration of
added lead. Draw the straight line best fitting the
NF 36
points, and extrapolate the line until it meets the
concentration axis. The distance between this point
and the intersection of the axes represents the con-
centration of lead in the Sample solution.
Acceptance criteria: NMT 0.1 ug/g
e Limit OF NICKEL
[Note—Use this test for Medium-Chain Triglycerides in-
tended for use in parenteral nutrition.]
Sample stock solution and Sample solution: Proceed
irected in the test for Limit of Chromium.
Nickel standard solution: Immediately before use, di-
lute 10 mL of nickel standard solution TS with water to
1000 mL. This solution contains the equivalent of
0.1 ug/g of nickel.
Standard solutions: Into each of three 10-mL volumet-
ric flasks, transfer 4.0 mL of Sample stock solution. Add
1.0, 2.0, and 4.0 mL, respectively, of Nickel standard so-
lution, and dilute with diisobutyl ketone to volume.
These solutions contain 0.01, 0.02, and 0.04 j1g/mL of
nickel.
Instrumental conditions
(See Atomic Absorption Spectroscopy (852).)
Mode: Atomic absorption spectrophotometer
equipped with a graphite furnace
Analytical wavelength: 232 nm
Lamp: Nickel hollow cathode
Carrier gas: Argon
Analysis
Samples: Standard solutions and the Sample solution
Record the average of the steady readings for each of
the Standard solutions and the Sample solution in trip-
licate. Plot the absorbances of the Standard solutions
and the Sample solution versus the concentration of
added nickel. Draw the straight line best fitting the
points, and extrapolate the line until it meets the
concentration axis. The distance between this point
and the intersection of the axes represents the con-
centration of nickel in the Sample solution.
Acceptance criteria: NMT 0.1 ug/g
e LIMIT OF TIN
[NoTE—Use this test for Medium-Chain Triglycerides in-
tended for use in parenteral nutrition.]
Sample stock solution and Sample solution: Proceed
irected in the test for Limit of Chromium.
Tin standard stock solution: Dissolve 500 mg of metal-
lic tin (Sn) in a mixture of 5 mL of water and 25 mL of
hydrochloric acid, and dilute with water to 1000 mL.
Tin standard solution: Immediately before use, dilute
10 mL of Tin standard stock solution with dilute hydro-
chloric acid (2.5 in 100) to 1000 mL, and then dilute
10 mL of the solution with water to 500 mL. This solu-
tion contains the equivalent of 0.1 ug/g of tin.
Standard solutions: Into each of three 10-mL volumet-
tic flasks, transfer 4.0 mL of Sample stock solution. Add
1.0, 2.0, and 4.0 mL, respectively, of Tin standard solu-
tion, and dilute with diisobutyl ketone to volume. These
solutions contain 0.01, 0.02, and 0.04 ug/mL of tin.
Instrumental conditions
(See Atomic Absorption Spectroscopy (852).)
Mode: Atomic absorption spectrophotometer
equipped with a graphite furnace coated inside with
palladium carbide
Analytical wavelength: 286.3 nm
Lamp: Tin hollow-cathode
Carrier gas: Argon
Analysis
Samples: Standard solutions and the Sample solution
Record the average of the steady readings for each of
the Standard solutions and the Sample solution in trip-
licate. Plot the absorbances of the Standard solutions
and the Sample solution versus the concentration of
added tin. Draw the straight line best fitting the
points, and extrapolate the line until it meets the
concentration axis. The distance between this point
NF 36 Official Monographs / Vanilla 5647

and the intersection of the axes represents the con-

centration of tin in the Sample solution. Trolamine
Acceptance criteria: NMT 0.1 g/g
¢ ALKALINE IMPURITIES
Sample solution: Dissolve 2.0 g of Medium-Chain Tri- CeHisNO3 149.19
glycerides in a mixture of alcohol and ethyl ether Ethanol, 2,2’,2”-nitrilotris-;
(1.5: 3.0). 2,2’,2”-Nitrilotriethanol [102-71-6].
Analysis: Add 0.05 mL of bromophenol blue TS to the
Sample solution, and titrate with 0.01 N hydrochloric DEFINITION
acid to a yellow endpoint. Trolamine is a mixture of alkanolamines consisting largely of
Acceptance criteria: NMT 0.15 mL of 0.01 N hydro- triethanolamine [N(C2H4OH)3] containing some diethano-
chloric acid is required. lamine [NH(C2HsOH)2] and monoethanolamine
[NH2(C2H,OH)]. It contains NLT 99.0% and NMT 107.4%
SPECIFIC TESTS of alkanolamines, calculated on the anhydrous basis as
e FATS AND FIXED OILS, Unsaponifiable Matter (401) N(C2H4OH)3.
Sample: 5.0g
Acceptance criteria: NMT 0.5% IDENTIFICATION
e SPECIFIC GRAVITY (841): 0.93-0.96 at 20° e A. INFRARED ABSORPTION (197F)
e WATER DETERMINATION, Method | (921): NMT 0.2% ° B.
e APPEARANCE Analysis 1: To 1 mL add 0.1 mL of cupric sulfate TS.
Diluent: Hydrochloric acid and water (2.75%: 97.25%) Acceptance criteria 1: A deep blue color is produced.
Sample: 10 mL Analysis 2: Add 5 mL of 1 N sodium hydroxide, and
Standard solution: Prepare immediately before use by concentrate to one-third of the original volume by
mixing 2.4 mL of ferric chloride CS and 0.6 mL of co- boiling.
baltous chloride CS with Diluent to make 10.0 mL, and Acceptance criteria 2: The blue color remains.
diluting 5.0 mL of the solution with Diluent to make °C
10.0 mL. Analysis: To 1 mL add 0.3 mL of cobaltous chloride TS.
Analysis: Compare the Sample and the Standard solu- Acceptance criteria: A carmine red color is produced.
tion by viewing them downward in matched color-com- ASSAY
parison tubes against a white surface (see Color and © PROCEDURE
Achromicity (631)). Sample: 2g of Trolamine
Acceptance criteria: The Sample is clear and not more Analysis: Transfer the Sample to a 300-mL conical flask.
TE colored than the Standard solution. Add 75 mL of water and 2 drops of methyl red TS, and
e FATS AND p FIXED OILS, Acid Value (Free Fatty Acids) (401): titrate with 1 N hydrochloric acid VS. Each mL of 1N
NMT 0.2 hydrochloric acid is equivalent to 149.2 mg of trietha-
© FATS AND FIXED OILS, Fatty Acid Composition (401): The nolamine, expressed as N(C2H4OH)3.
fatty acid fraction of Medium-Chain Triglycerides exhibits Acceptance criteria: 99.0%-107.4% on the anhydrous
the following composition as seen in Table 1. Disregard basis
any peak with an area less than 0.05% of the total area.
IMPURITIES
Table 1 e RESIDUE ON IGNITION (281): NMT 0.05%
Carbon-Chain Number of SPECIFIC TESTS
Length Double Bonds Percentage (%) e SPECIFIC GRAVITY (841): 1.120-1.128
6 0 82.0 e REFRACTIVE INDEX (831): 1.481-1.486 at 20°
8 0 50.0-80.0 e WATER DETERMINATION, Method | (921): NMT 0.5%, us-
10 0 20.0-50.0 ing a mixture of glacial acetic acid and methanol (1:4) as
the solvent
12, 0 $3.0
14 0 $1.0 ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in tight, light-resistant
FATS AND FIXED OILS, Hydroxy! Value (401): NMT 10 containers.
FATS AND FIXED OILS, /odine Value (401): NMT 1.0 e USP REFERENCE STANDARDS (11)
FATS AND FIXED OILS, Peroxide Value (401): NMT 1.0 USP Trolamine RS
FATS AND FIXED OILS, Saponification Value (401)
Sample: 1.0g
Acceptance criteria: 310-360
e Viscosity—CAPILLARY METHODS (911)
Analysis: Determine at 20 +0.1° with a capillary
viscometer. Tromethamine—see Tromethamine General
Acceptance criteria: 25-33 centipoises Monographs
REFRACTIVE INDEX (831): 1.440-1.452 at 20°
ARTICLES OF BOTANICAL ORIGIN, Total Ash (561)
Sample: 2.0g
Acceptance criteria: NMT 0.1% Tyloxapol—see Tyloxapol General
sydesBbouow 4N

ADDITIONAL REQUIREMENTS Monographs

e PACKAGING AND STORAGE: Preserve in tight containers,
protected from light. Store at temperatures not exceed-
ing 25°.
e LABELING: Where it is intended for use in parenteral nutri- Vanilla
tion, it is so labeled.
DEFINITION
Vanilla is the cured, full-grown, unripe fruit of Vanilla
planifolia Andrews, often known in commerce as Mexican,
Bourbon, or Madagascar vanilla, or of Vanilla tahitensis J.
 5648 Vanilla / Official Monographs
W. Moore, known in commerce as Tahitian vanilla (Fam.
Orchidaceae). Vanilla yields NLT 12.0% of anhydrous, di-
luted alcohol-soluble extractive.
COMPOSITION
e@ CONTENT OF ANHYDROUS, DILUTED ALCOHOL-SOLUBLE
EXTRACTIVE
Sample: 2g of Vanilla
Analysis: Place the Sample, finely cut or coarsely pow-
dered and accurately weighed, in a suitable flask. Add
70 mL of diluted alcohol, shake by mechanical means
for 2 h or for 8 h at 30-min intervals, and allow to
stand overnight. Decant the liquid intoafilter, and
wash the flask and residue with small portions of di-
luted alcohol, passing the washings through the filter
until the filtrate measures 100.0 mL. Mix the filtrate
well, evaporate a 50.0-mL portion in a suitable tared
container on a steam bath to dryness, and dry the resi-
due at 105° for 4 h. The weight obtained represents
the yield of anhydrous, diluted alcohol-soluble extrac-
tive from one-half of the portion of Vanilla taken. Calcu-
late the yield for the entire Sample taken.
Acceptance criteria: NLT 12.0%
CONTAMINANTS
© ARTICLES OF BOTANICAL ORIGIN (561), Pesticide Residue
Analysis: Meets the requirements
SPECIFIC TESTS
e@ BOTANICAL CHARACTERISTICS
Unground Vanilla
Macroscopic: Linear, flattened capsules of 12-35 cm
in length and 5-9 mm in width, with an apex termi-
nating ina flat, circular scar and a gradually tapering
base that is more or less curved or hooked; or in Tahi-
tian vanilla, broad in the middle and tapering toward
either end, the base closely resembling the summit. It
is flexible and tough, nearly black, dusky brown to
moderate brown externally, longitudinally wrinkled,
moist, glossy, and occasionally has efflorescence of
acicular or prismatic crystals of vanillin. Internally it is
unilocular, with a brownish-black pulp and numerous
minute seeds. Occasional capsules are split near the
summit into three parts.
Microscopic: The epidermis has a distinct cuticle and
occasional stomata. The epidermal cells contain red to
brown bodies and occasional prisms of calcium oxalate
or crystals of vanillin. It has a collenchyma layer of one
or two rows of cells, a thick sarcocarp composed of
paler and an interrupted circle of fibrovascular
undles, the latter leptocentric with a few vessels, and
an outer circle of fibers with thin, strongly lignified
walls and numerous transverse simple pits. The vessels
with walls have slit-like pits or spiral thickenings; the
parenchyma cells are usually thin-walled and deeply
undulate, some thick-walled with oblique, slit-like pits
or broad spiral bands, and contain occasional bundles
of acicular crystals of calcium oxalate, up to 400 um in
length, or a thin protoplasmic layer enclosing numer-
ous oil globules. It has an endocarp composed of pla-
cental and interplacental regions; the placental region
consists of six bifid placentas extending into the cavity
of the fruit and bears irregularly trianguloid, black to
reddish, flattened seeds, up to about 250 um in diam-
eter, having a deeply reticulate seed coat; the inter-
NF Monographs
placental regions show long, nearly straight hairs more
or less matted together by their gummy, resinous
secretion.
Powdered Vanilla
Macroscopic: Dusky brown to nearly black
Microscopic: Theprincipal elements of identification
are fragments of parenc' hale of the sarcocarp with
long, oblique, slit-like walls or broad spiral bands, cal-
cium oxalate crystals of acicular outline and up to 400
tum in length, and monoclinic prisms up to 35 wm in
NF 36
length. It has numerous unicellular, nearly straight,
glandular hairs, fragments of the seed coat with poly-
gonal stone cells, and slender crystals of vanillin.
e TEST FOR VANILLIN
Analysis: Place a few of the crystals, occurring as an
efflorescence on the fruit, on a microslide or watch
glass, and add 1 drop of phloroglucinol TS and 1 drop
of hydrochloric acid.
Acceptance criteria: The solution immediately acquires
a red color.
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in tight containers,
and store in a cold place.
e LABELING: The label states the Latin binomial and, follow-
ing the official name, the part(s) of the plant contained
in the article. The commercial variety of Vanilla, whether
Mexican, Bourbon, Madagascar, or Tahitian, is also stated
on the label. The label states that Vanilla that has be-
come brittle is not to be used.
Vanilla Tincture
DEFINITION
Prepare Vanilla Tincture as follows (see Pharmaceutical Com-
pounding—Nonsterile Preparations (795)).
Vanilla, cut into small pieces 100 g
Purified Water 200 mL
Alcohol 207 mL
Sucrose, in coarse granules 200 q
Diluted Alcohol, a sufficient quantity to make 1000 mL
Add Purified Water to the comminuted Vanilla in a suitable
covered container, and macerate for 12 h, preferably in a
warm place. Add Alcohol to the mixture, mix, and macer-
ate for about 3 days. Transfer the mixture to a percolator
containing Sucrose, and drain. Pack the drug firmly, and
percolate slowly, using Diluted Alcohol as the menstruum.
OTHER COMPONENTS
e ALCOHOL DETERMINATION, Method | (611): 38.0%-42.0%
ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Package in tight, light-resistant
containers, and avoid exposure to direct sunlight and ex-
cessive heat.
e LABELING: The label states the Latin binomial name and,
following the official name, the part of the plant source
from which the article was derived.
Vanillin
9
a
HO
CgHgO3 TSZ2;15
Benzaldehyde, 4-hydroxy-3-methoxy-;
Vanillin [121-33-5].
DEFINITION
Vanillin contains NLT 97.0% and NMT 103.0% of vanillin
(CsHgO3), calculated on the dried basis.
NF 36 Official Monographs / Vitamin 5649

IDENTIFICATION SPECIFIC TESTS

¢ A. INFRARED ABSORPTION (197K) e Loss ON DRYING (731)
e B. ULTRAVIOLET ABSORPTION (197U) Analysis: Dry a sample at 105° for 4 h.
Sample solution: 8 g/mL in methanol Acceptance criteria: NMT 0.1%
Acceptance criteria: Meets the requirements e FATS AND FIXED OILS, Acid Value (401)
Sample: 20g in a conical flask
ASSAY Analysis: Melt the Sample on a steam bath, add
¢ PROCEDURE 100 mL of hot alcohol Pievous neutralized with 0.1 N
Standard solution: 8 g/mL of USP Vanillin RS in sodium hydroxide to phenolphthalein TS, swirl, and
methanol add 1 mL of phenolphthalein TS. Titrate with 0.10 N
sample solution: 8 g/mL of Vanillin in methanol sodium hydroxide until the solution remains faintly pink
Blank: Methanol after being shaken for 15 s.
Instrumental conditions Acceptance criteria: NMT 4.0
(See Ultraviolet-Visible Spectroscopy (857).) e FATS AND FIXED OILS, Unsaponifiable Matter (401): NMT
Mode: UV 0.8%
Analytical wavelength: 308 nm
Cell: 1.cm ADDITIONAL REQUIREMENTS
Analysis e PACKAGING AND STORAGE: Preserve in tight containers, in
Samples: Standard solution, Sample solution, and Blank a cool place.
Calculate the percentage of vanillin (CsHgOs) in the por- ¢ LABELING: Label it to state whether it is Type | or Type Il.
tion of Vanillin taken:
Result = (Au/As) x (Cs/Cu) x 100
Au
As
= absorbance of the Sample solution
= absorbance of the Standard solution
Vitamin E Polyethylene Glycol Succinate
Cs = concentration of USP Vanillin RS in the
DEFINITION
Standard solution (\ug/mL)
Cu = concentration of the Sample solution (ug/mL) Vitamin E Polyethylene Glycol Succinate is a mixture formed
Acceptance criteria: 97.0%-103.0% on the dried basis by the esterification of d-alpha tocopheryl acid succinate
and polyethylene glycol. The ester mixture consists prima-
IMPURITIES rily of mono-esterified polyethylene glycol and a small
e RESIDUE ON IGNITION (281): NMT 0.05% amount of di-esterified polyethylene glycol. It contains
NLT 25.0% of d-alpha tocopherol (C29Hs0O2).
SPECIFIC TESTS
e MELTING RANGE OR TEMPERATURE (741): 81°-83° IDENTIFICATION
© Loss ON DRYING (731): Dry the sample over silica gel for © A, GAS CHROMATOGRAPHIC IDENTIFICATION TEST
4h: it loses NMT 1.0% ofis weight. Analysis: Proceed as directed in the test for Content of
Alpha Tocopherol.
ADDITIONAL REQUIREMENTS Acceptance criteria: The retention time of the major
© PACKAGING AND STORAGE: Preserve in tight, light-resistant peak of the Sample solution corresponds to that of the
containers. Standard solution.
© USP REFERENCE STANDARDS (11)
USP Vanillin RS COMPOSITION
© CONTENT OF ALPHA TOCOPHEROL
Solvent: 0.25 mL of phenolphthalein TS in 1 L of
alcohol
Internal standard solution: 12 mg/mL of ethy! arachi-
Hydrogenated Vegetable Oil date in isooctane
Standard solution: Transfer 32.5 mg of USP Alpha To-
copherol RS to a suitable reaction flask. Add 2 mL of
DEFINITION pyridine and 0.5 mL of N,O-bis(trimethylsilyl)trifluor-
oacetamide with 1% trimethylchlorosilane, and heat the
Change to read: flask at 100° for 10 min. Cool the flask, add 5.0 mL of
Internal standard solution followed by 20 mL of isooc-
Hydrogenated Vegetable Oil is a mixture of triglycerides of tane, and shake.
fatty acids. The melting range, heavy metals limit, iodine Sample solution: Transfer a quantity equivalent to
value, and saponification value differ, depending on Type, 0.100-0.160 g of Vitamin E Polyethylene Glycol Succi-
as described in the table below. nate molten at 60° to a culture tube (about 20 cm long
and 2.5 cm in diameter) equipped with a screw cap.
Add 40-50 mg of ascorbic acid and a few boiling chips,
Type! Type Il followed by 20 mL of Solvent. [Note—Reflux the solu-
Melting Range or Temperature (741), tion gently without emission of contents.] Place the
Class Il 57°-85° 20°-50° tube in a heating block set at 100°-150°. When the
0 e e
© (Ome V-fen-2018) © (oMetal 1j00- ©(oMcial 1-4an- sample is fully dissolved, add 0.25g of potassium hy-
28) Zia droxide, and’continue to reflux for 30 min. Remove the
sydeibouow- IN

Fats and Fixed Oils (401), lodine tube from heat, and while contents are still hot, add
Value, Method II 0-5 55-80 1-2 mL of hydrochloric acid dropwise until the pink col-
Fats and Fixed Oils (401), Saponifica- oration as [CauTlon—Exothermic reaction. Al-
tion Value 175-200 175-200 low the acid to trickle down the inside of the tube to
prevent splashing.] Cool the tube, then wash the sides
of the tube with 20 mL of water. Add 5.0 mL of Internal
standard solution, cap, and shake to ensure ros
mixing. Allow the tube to stand until two distinct layers
are formed. Transfer 2.5-3.5 mL of the upper layer into
a suitable reaction flask, and add 2.0 mL of pyridine
 5650 Vitamin / Official Monographs NF 36

followed by 2.5 mL of N,O-bis(trimethylsilyl)trifluoro- tube fitted with a cap, and dissolve in 10.0 mL of alco-
acetamide with 1% trimethylchlorosilane. Heat the flask hol. Place the tube in a heating block set at 100°-105°.
at 100° for 10 min. Cool, and then add 12 mL of [Note—Reflux the solution gently without emission of
isooctane. contents.] When the sample is fully dissolved, add
Chromatographic system 2-3 pellets of sodium hydroxide, and continue to reflux
(See Chromatography (621), System Suitability.) for an additional 30 min. Remove the tube from the
Mode: GC heat, and while contents are still hot, neutralize using
Detector: Flame ionization phenolphthalein as the indicator by slowly adding
Column: 0.25-mm x 15-m fused-silica capillary; coated 10 mL of a mixture of water and hydrochloric acid (1:1)
with a 0.25-m film of phase G27 until the pink color disappears. [CAUTION—Exothermic
Temperature reaction. Allow the acid solution to trickle down the
Injector: 280° inside of the tube to prevent splashing.] Cool the tube,
Detector: 345° cap, and shake until contents are well mixed. Add
Column: See Table 7. 25.0 mL of heptane, cap, and shake for 1 min to ensure
thorough mixing. Allow the tube to stand until two dis-
Table 1 tinct layers are formed. Transfer the top layer to a clean,
dry culture tube, then add 10.0 mL of water to the re-
Hold Time covered solution. Cap, shake, and allow the layers to
Initial Temperature Final at Final eo Transfer the upper layer to a clean, dry tube.
Temperature Ramp Temperature | Temperature Add 10.0 mL of potassium ferricyanide solution, pre-
Gy: (¢/min) () (min) pared by dissolving 2 g of potassium ferricyanide in
260 20 340 1 10.0 mL of 0.2 M sodium hydroxide, and replace the
cap. Shake vigorously for 45 s, and allow the layers to
Carrier gas: Helium separate for 30 min. If the top heptane layer is clear,
Flow rate: 1.5 mL/min proceed with the measurement for specific rotation; if
Injection size: 1 uL not clear, dry over anhydrous sodium sulfate before
Injection type: Split ratio, 200:1 proceeding with the test. [NoTE—Use the results of the
System suitability test for Content of Alpha Tocopherol to calculate the spe-
Sample: Standard solution cific rotation.]
Suitability requirements Acceptance criteria: NLT +24.0°
Falling actor: NMT 2.0 for the alpha tocopherol © SOLUBILITY IN WATER
eal Sample: 20g of melted Vitamin E Polyethylene Glycol
Relative standard deviation: NMT 2.0% for the ratio Succinate
of the alpha tocopherol peak area to the internal Analysis: Place the Sample in a glass container on a
standard peak area magnetic stirrer. Immediately add 80 mL of boiling
Analysis water while stirring. Allow to cool to room temperature
Samples: Standard solution and Sample Solution with constant stirring.
Calculate the percentage of d-alpha tocopherol Acceptance criteria: The solution becomes clear within
(C2sHs0O2) in the portion of Vitamin E Polyethylene 3h.
Glycol Succinate taken: ° ACID VALUE
Sample: 1g of Vitamin E Polyethylene Glycol Succinate
Result = (Ru/Rs) x (Ws/Wu) x 100 Analysis: Dissolve the Sample in 25 mL of a mixture of
alcohol and ether (1:1) that has been neutralized to
Ru = internal standard ratio (peak area of alpha phenolphthalein with 0.1 N sodium hydroxide. Add
tocopherol/peak area of the internal 0.5 mL of phenolphthalein TS, and titrate with 0.10 N
standard) from the Sample solution sodium hydroxide until the solution remains faintly pink
Rs = internal standard ratio (peak area of alpha after shaking for 30 s.
tocopherol/peak area of the internal Acceptance criteria: NMT 0.027 mEq/g, equivalent to
standard) from the Standard solution NMT 0.27 mL of 0.10 N sodium hydroxide
Ws = weight of USP Alpha Tocopherol RS used to
prepare the Standard solution (mg) ADDITIONAL REQUIREMENTS
Wy = weight of Vitamin E Polyethylene Glycol e PACKAGING AND STORAGE: Preserve in tight containers,
Succinate taken to prepare the Sample and store pioicsd from light.
solution (mg) e LABELING: The labeling indicates the d-alpha tocopherol
Acceptance criteria: NLT 25.0% content, expressed in mg/g.
© USP REFERENCE STANDARDS Gi 1)
SPECIFIC TESTS USP Alpha Tocopherol RS
© OPTICAL ROTATION, Specific Rotation (781S)
[Note—This test identifies d-alpha tocopherol after
saponification.]
Sample solution: Transfer 0.9 g of Vitamin E Polyethyl-
ene Glycol Succinate, molten at 60°, to a suitable test
NF Monographs
NF 36
Water for Injection—see Water for
Injection General Monographs
Water for Injection, Sterile—see Sterile
Water for Injection General Monographs
Water for Irrigation, Sterile—see Sterile
Water for Irrigation General Monographs
Water, Purified—see Purified Water General
Monographs
Carnauba Wax
DEFINITION
Carnauba Wax is obtained from the leaves of Copernicia cer-
ifera Mart. (Fam. Palmae).
IDENTIFICATION
e A. INFRARED ABSORPTION (197F) or (197A)
IMPURITIES
e RESIDUE ON IGNITION (281)
Sample: 2g
Analysis: Heat the Sample in an open porcelain or plati-
num dish over a flame; it volatilizes without emitting an
acrid odor. Ignite.
Acceptance criteria: The weight of the residue is NWT
5 mg, corresponding to NMT 0.25%.
Delete the following:
°e HEAVY MeTALs, Method i! (231): 20 ppMecorricia 1-1an-2018)
SPECIFIC TESTS
© MELTING RANGE OR TEMPERATURE (741), Procedures, Proce-
dure for Class Il: 80°-86°
© ACID VALUE
Sample: 3g
Analysis: Weigh the Sample into a 250-mL flask at-
tached to a reflux condenser. Add 50 mL of a mixture
of isopropyl alcohol and toluene (5:4), and boil gently
until the wax is completely dissolved. Remove the flask
from the condenser, add 1 mL of phenolphthalein TS,
and immediately titrate with 0.5 N alcoholic potassium
hydroxide VS to a faint, reddish-yellow color. [NoTE—
Do not allow the solution to cool. Titrate at warm tem-
perature after refluxing.]
Calculate the acid value as the number of milligrams of
potassium hydroxide required to neutralize the free
acids in 1 g of Carnauba Wax.
Acceptance criteria: 2-7
e FATS AND FIXED OILS (401), Saponification Value
Sample: Use the solution from the test for Acid Value.
Analysis: To the Sample add 15.0 mL of 0.5 N alcoholic
potassium hydroxide VS, reflux for 3 h, and titrate the
excess alkali with 0.5 N hydrochloric acid VS to a yel-
low-amber color. Perform a blank determination (see Ti-
trimetry (541), Residual Titrations).
Calculate the ester value. The saponification value is the
sum of the ester value and the acid value.
Official Monographs / Wax 5651
Acceptance criteria: 78-95
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in well-closed
containers.
e USP REFERENCE STANDARDS (11)
USP Carnauba Wax RS
Emulsifying Wax
DEFINITION
Emulsifying Wax is a waxy solid prepated from Cetostearyl
Alcohol containing a polyoxyethylene derivative of a fatty
acid ester of sorbitan.
SPECIFIC TESTS
© MELTING RANGE OR TEMPERATURE (741)
Sample: Emulsifying Wax
Analysis: Melt a quantity of the Sample slowly, while
stirring, until it reaches a temperature of 90°-92°. Re-
move the source of the heat, and allow the molten sub-
stance to cool to a temperature of 8°-10° above the
expected melting point. Chill the bulb of a suitable
thermometer (see General Notices, 6.80.30. Temperature
Reading Devices) to 5°, wipe it dry, and while it is still
cold, dip it into the molten substance so that the bulb
is completely covered. Withdraw it immediately, and
hold it vertically away from the heat until the surface
dulls. Fix the thermometer securely in a test tube so
that the lower point is 15 mm from the bottom of the
test tube. Place the test tube in a water bath at
10°-15°, and allow it to remain at that temperature for
30 min. Raise the temperature of the bath at the rate of
2°/min to 30°, then change toa rate of 1°/min, and
note the temperature at which the first drop of melted
substance leaves the thermometer. Repeat the determi-
nation twice on a freshly melted portion of the sample
substance. If the variation of three determinations is less
than 1°, take the average of the three as the melting
point. Otherwise, make two additional determinations,
and take the average of the five.
Acceptance criteria: 50°-54°
FATS AND FIXED OILS, Hydroxyl Value (401): 178-192
FATS AND FIXED OILS, lodine Value (401): NMT 3.5
FATS AND FIXED OILS, Saponification Value (401): NMT 14
PH (791)
Sample dispersion: Heat 3 Q of Emulsifying Wax in
100 mL of water to 55°, with stirring, followed by cool-
ing to 25°.
Acceptance criteria: 5.5-7.0
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in well-closed
containers.
Microcrystalline Wax
DEFINITION
Microcrystalline Wax is a mixture of straight-chain, Z
branched-chain, and cyclic hydrocarbons, obtained by sol-
vent fractionation of the still bottom fraction of petroleum 4
by suitable dewaxing or deoiling means. <
IMPURITIES Rat
© RESIDUE ON IGNITION (281) mA
Sample: 2g so
Analysis: Heat the Sample in an open porcelain or plati- =>
nw
num dish over a flame.
 4516 Centella / Dietary Supplements
Table 2
Approximate Relative
Analyte Retention Time
Madecassoside 0.71
Asiaticoside B 0.72
Asiaticoside 1.00
Madecassic acid 2.40
Terminolic acid 2.44
Asiatic acid 3.12
Separately calculate the percentages of the sum of
madecassoside and asiaticoside B (these two peaks
may co-elute), asiaticoside, the sum of madecassic acid
and terminolic acid, and asiatic acid in the portion of
Powdered Centella asiatica taken:
Result = (ru/rs) x Cs x (V/W) x D x Fx 100
ty = peak areas of the triterpene derivatives from
the Sample solution
rs = peak area of asiaticoside from Standard
solution A
Gs = concentration of USP Asiaticoside RS in
Standard solution A (mg/mL)
Vv = final volume of Sample stock solution (mL)
w = weight of Powdered Centella asiatica used to
repare the Sample stock solution (mg)
D = dilution factor to prepare the Sample solution
from the Sample stock solution
F = conversion factors for analytes: 1.00 for
asiaticoside, 1.017 for the sum of
madecassoside and asiaticoside B, 0.526 for
the sum of madecassic acid and terminolic
acid, and 0.509 for asiatic acid
Acceptance criteria: Add the percentages of different
triterpene derivatives: NLT 2.0% on the dried basis.
CONTAMINANTS
e ARTICLES OF BOTANICAL ORIGIN, Limits of Elemental Impuri-
ties (561): Meets the requirements
e ARTICLES OF BOTANICAL ORIGIN, Pesticide Residue Analysis
(561): Meets the requirements
e MICROBIAL ENUMERATION TESTS (2021): The total aerobic
bacterial count does not exceed 105 cfu/g, the total com-
bined yeast and mold count does not exceed 103 cfu/g,
and the bile-tolerant Gram-negative bacteria count does
not exceed 103 cfu/g.
e ABSENCE OF SPECIFIED MICROORGANISMS (2022): Meets the
requirements of the tests for absence of Salmonella spe-
cies and Escherichia coli
DS Monographs
SPECIFIC TESTS
e BOTANICAL CHARACTERISTICS: Greenish gray to greenish-
brown in color; odor slight; taste slightly bitter to sweet;
under a microscope, factions of epidermal cells of the
leaves with irregular striated cuticle, showing anisocytic,
some paracytic, and rarely anomocytic stomata; epider-
mal cells of young leaves showing unicellular, occasion-
ally multicellular, non-glandular trichomes; secretory
canals composed of 5-7 secretory cells; parenchyma
cells, some showing prisms or rosettes of calcium oxalate;
bundles of narrow septate fibers from the stem; spiral
vessels; fragments of the fruits, layers of wide cells in a
parquetry arrangement, annular vessels, parenchyma cells
containing simple or compound starch granules
e Loss ON DRYING (731)
Sample: 1.0g of Powdered Centella asiatica
Analysis: Dry the Sample at 105° for 2 h.
Acceptance criteria: NMT 12.0%
e ARTICLES OF BOTANICAL ORIGIN, Total Ash (561)
Sample: 1.0 g of Powdered Centella asiatica
Analysis: NMT 12%
© ARTICLES OF BOTANICAL ORIGIN, Acid-Insoluble Ash (561):
NMT 3.5%
USP 41
ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Preserve in well-closed contain-
ers, protected from light and moisture, and store at
room temperature.
Change to read:
e LABELING: The label states the Latin binomial and, follow-
ing the official name, the parts of the plant contained in
the article. The label states that the article is exempted
from the requirements of °Labeling (7), Labels and Label-
ing for Products and Other Categories, Botanicals, cr 1-may-
2018)with respect to the pregnancy and lactation state-
ment. “e@ cn i-May-2018)
e USP REFERENCE STANDARDS (11)
USP Asiaticoside RS
USP Powdered Centella asiatica Extract RS
Powdered Centella asiatica Extract
DEFINITION
Powdered Centella asiatica Extract is prepared from Centella
asiatica by extraction with alcohol, methanol, acetone, or
a mixture of these solvents. The ratio of plant material to
extract is between 65:1 and 30:1. It contains NLT 90.0%
and NMT 110.0% of the labeled amount of triterpene
derivatives; the labeled amount of triterpene derivatives is
NMT 40%, calculated on the dried basis as the sum of
madecassoside, asiaticoside B, asiaticoside, madecassic
acid, terminolic acid, and asiatic acid. It may contain suit-
able added substances as carriers.
IDENTIFICATION
e A. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST
Standard solution A: 0.5 mg/mL of USP Asiaticoside RS
in methanol
Standard solution B: 10 mg/mL of USP Powdered
Centella asiatica Extract RS in methanol. Sonicate for
about 10 min, centrifuge, and use the supernatant.
Sample solution: Transfer an amount of Powdered
Centella asiatica Extract equivalent to about 5 mg of
triterpene derivatives to a centrifuge tube. Add 5 mL of
methanol, sonicate for 10 min, centrifuge, and use the
supernatant.
Adsorbent: Chromatographic silica gel with an average
particle size of 10-15 tm (TLC plates) or with an aver-
age particle size of 5 um (HPTLC plates)
Application volume: 10 ul (TLC plates) or 4 lL (HPTLC
lates)
Develo ing solvent system: A mixture of methylene
chloride, methanol, and water (14:6:1)
Spray reagent: A solution of 10% sulfuric acid in meth-
anol. [NoTE—Prepare fresh.]
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Apply the samples as bands to a suitable thin-layer
chromatographic plate (see Chromatography {621)).
Use a saturated chamber. Develop the chromato-
grams until the solvent front has moved up about
three-fourths of the plate. Remove the plate from the
chamber, dry, spray with the Spray reagent, heat for
3 min at 120°, and examine under visible light.
Acceptance criteria: The Sample solution chromato-
gram exhibits a violet band in the lower third of the
plate due to asiaticoside, corresponding in color and Rr
to that in Standard solution A; a violet band due to
madecassoside at an R; lower than that of asiaticoside;
and two additional violet bands in the upper third of
the plate due to asiatic acid and madecassic acid. Bands
detected in the Sample solution correspond in position
 5652 Wax / Official Monographs
Acceptance criteria: It volatilizes without emitting an
acrid odor and on ignition yields NMT 0.1%.
SPECIFIC TESTS
e COLOR
Standard solution: Mix 3.8 mL of ferric chloride CS
and 1.2 mL of cobaltous chloride CS in a clear-glass,
16- x 150-mm bacteriological test tube.
Sample solution: Melt 10g of Microcrystalline Wax on
a steam bath, and pour 5 mL of the liquid into a clear-
glass, 16- x 150-mm bacteriological test tube.
Analysis: Visually compare the contents of both tubes
in reflected light against a white background, holding
the tubes directly against the background at such an
angle that there is no fluorescence.
Acceptance criteria: The Sample solution is not darker
than the Standard solution.
© MELTING RANGE OR TEMPERATURE, Class II! (741):
54°-102°
© CONSISTENCY
Sample: Microcrystalline Wax
Apparatus: Determine the consistency of the Sample by
means of a penetrometer fitted with a polished metal
needle weighing 2.5 + 0.05 g and having a truncated
symmetric tapered angle of 9°0’ + 15’. The needle is
tapered, with a length of 25.4 mm, and the shaft at-
tached to the needle is 58 mm in length and 3.17 mm
in diameter. The plunger that fits into the penetrometer
and guides the path of the needle weighs 47.5 +
0.05 g. An additional weight of 50 +0.05 g is added to
the top of the plunger to give a total load of 100g.
Analysis: The Samie is cast in a brass cylinder open at
both ends. The cylinder has an inside diameter of
25.4mm and is 31.8 mm in height. Place the cylinder
on a brass plate wetted with an equal volume mixture
of glycerin and water, and place the plate on two
a Pour the wax, melted at approximately 17°
above its congealing point. into the cylinder. Continue
pouring the wax until a convex meniscus is formed
above the cylinder. Allow the specimen to cool for 1 h
at approximately 24°. Shave excess wax from the top of
the cylinder, and remove the plate. With the smooth
wax surface in the up position, condition the specimen
in a water bath at 25° for 1 h.
Arrange the penetrometer so that the wax specimen is
completely immersed in the water bath while penetra-
tion is run. Lower the needle until the tip just touches
the top surface of the specimen. Release the needle for
5 s, and read the depth of penetration in tenths of
millimeters. Perform four determinations, and calculate
the average value of the four readings.
Acceptance criteria: 3-100 (0.3-10.0 mm)
e ORGANIC ACIDS
Sample solution: 20g of Microcrystalline Wax in
100 mL of a mixture of neutralized alcoho! and water
(1:2). Agitate thoroughly, and heat to ona
Analysis: To the Sample solution add 1 mL of phenol-
phitvatein TS, and titrate rapidly with 0.1 N sodium hy-
droxide VS, with vigorous agitation, to a sharp pink
endpoint in the alcohol-water layer.
Acceptance criteria: NMT 0.4 mL of 0.1 N sodium hy-
droxide is required.
© FIXED OILS, FATS, AND ROSIN
Sample: 10
al
ao Sample solution: Digest the Sample with 50 mL of so-
a dium hydroxide solution (1 in 5) at 100° for 30 min.
HesS Separate the water layer, and acidify it with 2 N sulfuric
D acid.
) Acceptance criteria: No oily or solid matter separates.
Cc
C) © ALKALINITY
= Sample: 35g
a
Analysis: Introduce the Sample into a 250-mL separator,
es add 100 mL of boiling water, and shake vigorously for 5
min. Draw off the separated water into a casserole,
wash further with two 50-mL portions of boiling water,
NF 36
and add the washings to the casserole. To the pooled
washings add 1 drop of phenolphthalein TS, and boil.
Acceptance criteria: The solution does not acquire a
pink color.
e AciDITY
Analysis: f the addition of phenolphthalein TS in the
test for Alkalinity produces no pink color, add 0.1 mL of
methyl orange TS.
Acceptance criteria: No red or pink color is produced.
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in tight containers.
e LABELING: Label it to indicate the name and proportion
of any added stabilizer.
White Wax
DEFINITION
White Wax is the product of bleaching and purifying Yellow
Wax that is obtained from the honeycomb of the bee
[Apis mellifera L. (Fam. Apidae)] and that meets the re-
quirements of the Saponification Cloud Test.
SPECIFIC TESTS
© SAPONIFICATION CLOUD TEST
Sample: 3.00g
Alcoholic potassium hydroxide: Dissolve 40g of potas-
sium hydroxide in about 900 mL of aldehyde-free alco-
hol maintained at a temperature not exceeding 15°,
and then when solution is complete, warm to room
temperature, and add aldehyde-free alcohol to make
1000 mL.
Analysis: Place the Sample in a 100-mL round-bottom
boiling flask fitted with a ground-glass joint. Add 30 mL
of Alcoholic potassium hydroxide. Reflux the mixture gen-
tly for 2 h. At the end of this period, open the flask,
insert a thermometer into the solution, and place the
flask in a container of water at a temperature of 80°.
Rotate the flask in the bath while both the bath and the
solution cool.
Acceptance criteria: The solution shows no cloudiness
or globule formation before the temperature reaches
69°.
© MELTING RANGE OR TEMPERATURE, Class /] (741): 62°-65°
e FATS OR FATTY ACIDS, JAPAN WAX, ROSIN, and SOAP
Sample: 1g
Analysis 1: Boil the Sample for 30 min with 35 mL of
3.5 N sodium hydroxide contained in a 100-mL beaker,
maintaining the volume of solution by the occasional
addition of water, and allow the mixture to cool at
room temperature for about 2 h.
Acceptance criteria 1: The wax separates leaving the
liquid clear, turbid, or translucent, but not opaque.
Analysis 2: Filter the cool mixture obtained in Analysis
1, and acidify the clear filtrate with hydrochloric acid.
Acceptance criteria 2: The liquid remains clear or
shows NMTa slight amount of turbidity or precipitate.
¢ FATS AND FIXED OILS, Acid Value (401)
Sample: 3g
Analysis: Warm the Sample in a 200-mL flask with
25 mL of neutralized dehydrated alcohol until melted,
then shake the mixture. Add 1 mL of phenolphthalein
TS, and titrate the warm liquid with 0.5 N alcoholic
potassium hydroxide VS to produce a permanent, faint
pink color. Calculate the acid value as directed in the
chapter.
Acceptance criteria: 17-24
e FATS AND FIXED OlLs, Ester Value (401)
Sample solution: The solution resulting from the deter-
mination of Acid Value
Analysis: To the Sample solution add 25.0 mL of 0.5 N
alcoholic potassium hydroxide VS and 50 mL of alde-
NF 36
hyde-free alcohol, and reflux the mixture for 4 h. Titrate
the excess alkali with 0.5 N hydrochloric acid VS. Per-
form a blank determination (see Titrimetry (541), Resid-
ual Titrations). Calculate the ester value as directed in
the chapter.
Acceptance criteria: 72-79
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in well-closed
containers.
Yellow Wax
DEFINITION
Yellow Wax is the purified wax from the honeycomb of the
bee [Apis mellifera L. (Fam. Apidae)]. The crude beeswax
used to prepare Yellow Wax conforms to the Saponifica-
tion Cloud Test.
SPECIFIC TESTS
¢ SAPONIFICATION CLOUD TEST
Sample: 3.00g
Alcoholic potassium hydroxide: Dissolve 40g of potas-
sium hydroxide in about 900 mL of aldehyde-free alco-
hol maintained at a temperature not exceeding 15°,
and then when solution is complete, warm to room
temperature, and add aldehyde-free alcohol to make
1000 mL.
Analysis: Place the Sample in a 100-mL round-bottom
boiling flask fitted with a ground-glass joint. Add 30 mL
of Alcoholic potassium hydroxide. Reflux the mixture gen-
tly for 2 h. At the end of this period, open the flask,
insert a thermometer into the solution, and place the
flask in a container of water at a temperature of 80°.
Rotate the flask in the bath while both the bath and the
solution cool.
Acceptance criteria: The solution shows no cloudiness
a globule formation before the temperature reaches
Ds
e [MELTING RANGE OR TEMPERATURE, Class // (741): 62°-65°
© FATS OR FATTY ACIDS, JAPAN WAX, ROSIN, and SOAP
Sample: 1g
Analysis 1: Boil the Sample for 30 min with 35 mL of
3.5 N sodium hydroxide contained in a 100-mL beaker,
maintaining the volume of solution by the occasional
addition of water, and allow the mixture to cool at
room temperature for about 2 h.
Acceptance criteria 1: The wax separates, leaving the
liquid clear, turbid, or translucent,but not opaque.
Analysis 2: Filter the cool mixture obtained in Analysis
1, and acidify the clear filtrate with hydrochloric acid.
Acceptance criteria 2: The liquid remains clear or
shows NMTaslight amount of turbidity or precipitate.
e FATS AND FIXED OILS, Acid Value (Free Fatty Acids) (401)
Sample: 3g
Analysis: Warm the Sample in a 200-mL flask with
25 mL of neutralized dehydrated alcohol until melted,
then shake the mixture. Add 1 mL of phenolphthalein
TS, and titrate the warm liquid with 0.5 N alcoholic
potassium hydroxide VS to produce a permanent, faint
pink color. Calculate the Acid Value as directed in the
chapter.
Acceptance criteria: 17-24
© FATS AND FIXED OILS, Ester Value (401)
Sample solution: The solution resulting from the deter-
mination of Acid Value
Analysis: To the Sample solution add 25.0 mL of 0.5 N
alcoholic potassium hydroxide VS and 50 mL of alde-
hyde-free alcohol, and reflux the mixture for 4 h. Titrate
the excess alkali with 0.5 N hydrochloric acid VS. Per-
form a blank determination (see Titrimetry (541), Resid-
Official Monographs / Xanthan 5653
ual Titrations). Calculate the Ester Value as directed in
the chapter.
Acceptance criteria: 72-79
ADDITIONAL REQUIREMENTS \
e PACKAGING AND STORAGE: Preserve in well-closed
containers.
Xanthan Gum
DEFINITION
Xanthan Gum is a high molecular weight polysaccharide
gum produced by a pure-culture fermentation of a carbo-
hydrate with Xanthomonas campestris, then purified by re-
covery with Isopropyl Alcohol, dried, and milled. It con-
tains D-glucose and D-mannose as the dominant hexose
units, along with D-glucuronic acid, and is prepared as the
sodium, potassium, or calcium salt. It yields NLT 4.2%
and NMT 5.0% of carbon dioxide, calculated on the dried
basis, corresponding to NLT 91.0% and NMT 108.0% of
Xanthan Gum.
IDENTIFICATION
eA.
Sample: Prepare a dry blend of 1.5 g of Xanthan Gum
and 1.5 g of locust bean gum.
Control: 3.0 g of Xanthan Gum
Analysis
Samples: Sample and Control
To two separate 400-mL beakers add 300 mL of water,
and heat to 80°. Stir rapidly by mechanical means.
Add the Sample to one of the beakers and the Control
to the other beaker at the point of maximum agita-
tion. Stir until the mixtures dissolve, and then continue
stirring for 30 min longer. Do not allow the tempera-
ture of the mixtures to drop below 60° during the
stirring. Discontinue a and allow the mixtures to
cool at room temperature for NLT 2 h.
Acceptance criteria: A firm, rubbery gel forms with the
Sample after the temperature drops below 40°, but no
such gel forms with the Control.
ASSAY
© PROCEDURE
Sample: 1.2g
Analysis: Proceed as directed in Alginates Assay (311).
Acceptance criteria: 4.2%-5.0% of carbon dioxide on
the dried basis, corresponding to 91.0%-108.0% of
Xanthan Gum
IMPURITIES
e ARSENIC, Method II (211): NMT 3 ug/g
e LEAD (251)
Sample: Prepare a Test Preparation as directed in the
chapter
Control: Use 5 mL of Diluted Standard Lead Solution
(5 ug of Pb).
Analysis: Proceed as directed in the chapter.
Acceptance criteria: NMT 5 ug/g
Delete the following:
sydeibouo=: 4iN
°e HEAVY METALS, Method I! (231): NMT 30 ug/g. Use a
platinum crucible for the ignition.e (oficial 1Jan-2078)
o LIMIT OF ISOPROPYL ALCOHOL
Internal standard solution: 1 mg/mL of tertiary butyl
alcohol
Standard stock solution: 1 mg/mL of isopropyl alcohol
Standard solution: Pipet 4 mL of the Standard stock so-
lution and 4 mL of the Internal standard solution into a
hal volumetric flask, and dilute with water to
volume.
 5654 Xanthan / Official Monographs NF 36

Sample solution: Disperse 1 mL of a suitable antifoam SPECIFIC TESTS

emulsion in 200 mL of water contained in a 1000-mL, © MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI-
round-bottom distilling flask having a 24/40 standard FIED MICROORGANISMS (62): It meets the requirements of
taper ground joint. Add 5 g of Xanthan Gum, and the tests for Salmonella species and Escherichia coli.
shake for 1 h on a wrist-action mechanical shaker. Con- e Loss ON DRYING (731)
nect the flask to a fractionating column, and distill Analysis: Dry at 105° for 2.5 h.
100 mL, adjusting the heat so that foam does not enter Acceptance criteria: NMT 15.0%
the column. Add by pipet 4 mL of the Internal standard © ASH
solution. Sample: Weigh 3g in a tared crucible.
Chromatographic system Analysis: Incinerate the Sample at 650° until free from
(See Chromatography (621), System Suitability.) carbon. Cool the crucible and its contents in a desicca-
Mode: GC tor, and weigh.
Detector: Flame ionization Acceptance criteria: The weight of the ash is between
Column: 3.2-mm x 1.8-m stainless steel column 6.5%-16.0%, calculated on the dried basis.
packed with 80- to 100-mesh surface-silanized packing © VISCOSITY—ROTATIONAL METHODS (912)
$3, or equivalent Sample: Prepare a dry blend of 3.0 g of Xanthan Gum
Temperatures and 3.0g of potassium chloride.
Column: 165° Instrumental conditions
Detector: 200° Instrument: Rotational viscometer
Injection port: 200° Spindle cylinder dimensions
Carrier gas: Helium Diameter: 1.27 cm
Injection volume: 4-5 uL Height: 0.16cm
Analysis Shaft diameter: 0.32 cm
Samples: Standard solution and Sample solution Distance from top of cylinder to lower lip of shaft:
[NoTe—The retention time of tertiary butyl alcohol is 2.54.cm
1.5 relative to that of isopropyl acakony Immersion depth: 5.00 cm (No. 3 spindle)
Calculate the percentage of isopropy! alcohol in the Spindle rotation speed: 60 rpm
portion of Xanthan Gum taken: Analysis: To a 400-mL beaker add 250 mL of water.
Add the Sampleslew while stirring at 800 rpm, using
Result = (Ru/Rs) x (Cs/Cu) x 100 a low-pitched, propeller-type stirrer. Add 44 mL of
water, rinsing the walls of the beaker. Approximately 10
Ru = peak response ratio of isopropyl alcohol to min after the addition of the Sample to the water, re-
tertiary butyl alcohol from the Sample move the beaker from the propeller-type stirrer, and
solution vigorously stir the solution by hand to ensure that all
Rs = peak response ratio of ropromy! alcohol to the particles around the edge of the beaker are in solu-
tertiary butyl alcohol from the Standard tion. Return the beaker to the stirrer, and agitate at 800
solution rpm for a total mixing time of 2 h. Adjust the tempera-
Gs = concentration of isopropyl alcohol in the ture to 24+1°, and stir by hand in a vertical motion to
Standard solution (mg/mL) eliminate any thixotropic effects or layering. Each hand
Gu = concentration of the Sample solution (mg/mL) mixing should be NMT 15-30 s, and the last hand mix-
Acceptance criteria: NMT 0.075% ing should occur immediately before measuring the vis-
e Pyruvic AcID cosity. With the spindle rotating at 60 rpm, immedi-
Solution A: 5 mg/mL of 2,4-dinitrophenylhydrazine in ately observe and record the scale reading. Convert the
2.N hydrochloric acid scale readings to centipoises by multiplying the read-
Standard stock solution: 90 g/mL of pyruvic acid ings by the constant for the viscometer spindle and
Standard solution: Transfer 10.0 mL of the Standard speed used.
stock solution to a glass-stoppered, 50-mL flask. Add Acceptance criteria: NLT 600 centipoises at 24°
20.0 mL of 1 N hydrochloric acid, weigh the flask, and
reflux for 3 h, taking precautions to prevent loss of va- ADDITIONAL REQUIREMENTS
ors. Cool, and add water to make up for any weight e PACKAGING AND STORAGE: Preserve in well-closed
joss during refluxing. Transfer 2.0 mL of this solution to containers.
a 30-mL separator containing 1.0 mL of Solution A. Mix,
and allow to stand for 5 min. Extract the mixture with
5 mL of ethyl acetate, and discard the aqueous layer.
Extract the hydrazone from the ethyl acetate with three
5-mL portions of sodium carbonate TS, collect the ex- Xanthan Gum Solution
tracts in a 50-mL volumetric flask, and dilute with so-
dium carbonate TS to volume. DEFINITION
Sample stock solution: 6 mg/mL of Xanthan Gum Prepare Xanthan Gum Solution of the designated percent-
Sample solution: Transfer 10.0 mL of the Sample stock age strength as follows (see Pharmaceutical Compound-
solution to a glass-stoppered, 50-mL flask. Proceed as ing—Nonsterile Preparations {795)).
directed in the Standard solution, beginning with “Add
20.0 mL of 1 N hydrochloric acid...”.
Blank: Sodium carbonate TS Xanthan Gum (for a 0.1% solution) 100 mq
Instrumental conditions Xanthan Gum (for 1.0% solution) 10q
NF Monographs

(See Ultraviolet-Visible Spectroscopy (857).) Methylparaben 100 mg

Mode: Spectrophotometry Propylparaben 20 mg
Analytical wavelength: 375 nm Purified Water, a sufficient quantity to
Cell: 1.cm make 100 mL
Analysis
Samples: Standard solution, Sample solution, and Blank Dissolve a weighed quantity of Propylparaben in Purified
Acceptance criteria: The absorbance of the Sample so- Water with heating to 50° and stirring. Cool, and dilute
lution is NLT that of the Standard solution, correspond- quantitatively, and stepwise if necessary, with Purified
ing to NLT 1.5% of pyruvic acid. Water to obtain 90 mL of solution containing 20 mg of
Propylparaben. Heat to 50°, and add the Methylparaben,
NF 36 Official Monographs / Xylitol 5655

with stirring, to dissolve. Cool, stir with a blender, slowly ry = peak response of xylitol from the Sample
sift the Xanthan Gum into the vortex, and continue to solution
blend for 2 min after the Xanthan Gum has been added. r = peak response of xylitol from the Standard
Add 10 mL of Purified Water, and blend for 5 min. Allow solution
to stand for 1 h for excess foam to subside, and remove Cs = concentration of USP Xylitol RS in the
most of the remaining foam by passing the solution Standard solution (mg/mL)
througha strainer. Add Purified Water, if necessary, to Cu = concentration of the Sample solution (mg/mL)
make the final volume 100 mL, and stir. [NoTtE—Depend- Acceptance criteria: 98.5%-101.0% on the anhydrous
ng on the volume needed and the equipment available, basis
adjust the formula proportionately.]
IMPURITIES
ADDITIONAL REQUIREMENTS © RESIDUE ON IGNITION (281): NMT 0.5%
e PACKAGING AND STORAGE: Preserve in tight, light-resistant
containers, and store at controlled room temperature.
e LABELING: Label it to state, as part of the official title, the Delete the following:
percentage content of Xanthan Gum.
e BEYOND-USE DATE: Six weeks after the day on which it °e HEAVY METALS (231): NMT 10 ppm, using 2g of Xylitol
was compounded dissolved in 25 mL of watere (official 1-4an-2018)
e REDUCING SUGARS
Sample: 500mg
Analysis: Dissolve the Sample in 2.0 mL of water in a
10-mL conical flask. Into a similar flask, pipet 2 mL of a
0.5 mg/mL dextrose solution. To each flask add 1 mL of
Xylitol alkaline cupric tartrate TS, heat to boiling, and cool.
Acceptance criteria: Any turbidity in the xylitol flask is
OH
NMT that in the dextrose flask, in which a reddish-
HOS I. 9 OH brown precipitate forms (0.2% reducing sugars, as
OH OH dextrose).
e LIMIT OF OTHER POLYOLS
Cshi 205 152.15 Mobile phase: Acetonitrile and water (20:80)
Xylitol. System suitability solution: 0.5 mg/mL each of USP
L-Arabinitol RS, USP Galactitol RS, USP Mannitol RS, and
DEFINITION USP Sorbitol RS, and 100 mg/mL of USP Xylitol RS in
Xylitol contains NLT 98.5% and NMT 101.0% of CsHi2Os, Mobile phase
calculated on the anhydrous basis. Standard solution: 0.5 mg/mL each of USP L-Arabinitol
RS, USP Galactitol RS, USP Mannitol RS, and USP Sorbi-
IDENTIFICATION tol RS in Mobile phase
e A. INFRARED ABSORPTION (197K) Sample solution: 100 mg/mL of Xylitol in Mobile phase
Sample: Undried Chromatographic system
e B. The retention time of the xylitol peak of the Sample (See Chromatography (621), System Suitability.)
solution corresponds to that of the Standard solution, as Mode: LC
obtained in the Assay. Detector: UV 192 nm
Column: 8.0-mm x 30-cm; 7-m packing L34
ASSAY Column temperature: 80°
© PROCEDURE Flow rate: 0.5 mL/min
Mobile phase: Acetonitrile and water (20:80) Injection size: 25 wL
System suitability solution: 2.5 mg/mL of USP Galac- System suitability
titol RS and 25 mg/mL of USP Xylitol RS in Mobile phase Samples: System suitability solution and Standard
Standard solution: 25 mg/mL of USP Xylitol RS in Mo- solution
bile phase [Note—The relative retention times for L-arabinitol,
Sample solution: 25 mg/mL of Xylitol in Mobile phase mannitol, xylitol, galactitol, and sorbitol are about
Chromatographic system 0.76, 0.81, 1.0, 1.12, and 1.22, respectively.]
(See Chromatography (621), System Suitability.) Suitability requirements
Mode: LC Resolution: NLT 1.5 between all adjacent polyol
Detector: UV 192 nm peaks, System suitability solution
Column: 8.0-mm x 30-cm; 7-11m packing L34 Relative standard deviation: NMT 5.0% for the
Column temperature: 80° galactitol peak, Standard solution
Flow rate: 0.5 mL/min Analysis
Injection size: 25 uL Samples: Standard solution and Sample solution
System suitability Calculate the percentage of each polyol (L-arabinitol,
Sample: System suitability solution and Standard galactitol, mannitol, or sorbitol) in the portion of sam-
solution ple taken:
[Note—The relative retention times for xylitol and
galactitol are about 1.0 and 1.10, respectively.] Result = (ru/rs) x (Cs/Cu) x 100
Suitability requirements
Resolution: NLT 2.0 between galactitol and xylitol, tu = peak response of the individual polyol from Ee
System suitability solution the Sample solution Ed
Relative standard deviation: NMT 2.0%, Standard ig = peak response of the individual polyol from 5)
solution the Standard solution ce}
Analysis Cs = concentration of the individual polyol in the °
Samples: Standard solution and Sample solution Standard solution (mg/mL) ©
Calculate the percentage of xylitol (CsHi2Os) in the por- Cu = concentration of the Sample solution (mg/mL) Ey
tion of sample taken: Acceptance criteria: The sum of the polyols is NMT jt
2.0%, calculated on the anhydrous basis. ry
Result = (ru/rs) x (Cs/Cu) x 100
 5656 Xylitol / Official Monographs NF 36

SPECIFIC TESTS Independent standard working solution: 1 mg/mL of

e WATER DETERMINATION, Method | (921): NMT 0.5% B-lactoglobulin A in water
Independent standard running solution: To the final
ADDITIONAL REQUIREMENTS 1 volume of Independent standard running solution, add
© PACKAGING AND STORAGE: Preserve in well-closed 0.1 volume of Independent standard working solution,
containers. 0.5 volume of Sample loading buffer, 0.1 volume of B-
e USP REFERENCE STANDARDS (11) mercaptoethanol, and sufficient water to obtain
USP L-Arabinitol RS 0.1 mg/mL of B-lactoglobulin A and 10% of B-
USP Galactitol RS mercaptoethanol.
USP Mannitol RS Sample stock solution: 10 mg/mL of Zein in Solvent.
USP Sorbitol RS Mix on a vortex mixer until the sample is fully dis-
USP Xylitol RS solved. Centrifuge at 10,000-12,000 rpm in a
microcentrifuge with a fixed rotor for 10 min to pellet
any undissolved material.
Sample solution: Dilute the Sample stock solution with
the Sample loading buffer (1:1). Incubate the mixture in
Xylose—see Xylose General Monographs a closed microcentrifuge tube for 10 min at 95°. After
incubation, allow the tube to flash-cool on ice. Place
the tube in a microcentrifuge, spin at a top speed for a
few seconds, and allow to stop on its own to collect
Zein any condensation on the sides and top of the tube.
Dilute the solution so obtained with a mixed solution of
[9010-66-6]. Sample loading buffer and water (1:1) to 5 folds.
Electrophoretic system
DEFINITION SDS-PAGE gel and apparatus setup: Following the
Zein is a prolamine derived from corn [Zea mays Linné (Fam. manufacturer’s instructions, assemble and fill a precast
Gramineae)]. 16% Tris-Glycine gels in an appropriate electrophore-
sis module.
IDENTIFICATION Running buffer: Gel running buffer
cA. Voltage: 100 V
Sample solution: Dissolve 0.1 g in 10 mL of 0.1 N so- Run time: 2.5h or until the upper dye front is at the
dium hydroxide. bottom of the gel. [NoTe—The total run time may
Analysis: To the Sample solution add a few drops of need to be altered, depending on the molecular
cupric sulfate TS. Warm in a water bath. weight standards as well on as laboratory equipment
Acceptance criteria: A purple color develops. variability, because the dye front may co-migrate with
° B. or close to the lowest bands of the set.]
Sample solution: In a test tube add 1 mL of nitric acid Analysis
to 25 mg of Zein. Samples: Molecular weight standard solution, Indepen-
Analysis: Agitate the Sample solution vigorously. dent standard running solution, and Sample solution
Acceptance criteria: The solution becomes light yellow. Gel loading: Load 25 uL of the Molecular weight stan-
Further addition of about 10 mL of 6 N ammonium hy- dard solution. Load a volume of the Independent stan-
droxide produces an orange color. dard running solution, equal to 2 ug of B-lactoglobulin
C. IDENTIFICATION OF ALPHA-ZEIN BY SDS-POLYACRYLAMIDE A. Load a volume of the Sample solution, equal to ap-
GEL ELECTROPHORESIS roximately 5 ug of calculated Zein. [NoTE—If the 5 ug
Solvent: 55% Isopropyl! alcohol with 2% beta loading amount is inadequate for the test, the 10 yg
mercaptoethanol loading amount may be used. Loading a lesser quan-
Sample loading buffer:1 0.5 M tris hydrochloride pH tity on the gel results in an overall higher molecular
6.8, 20% glycerin, 4% sodium dodecyl sulfate (SDS), weight determination due to sharper bands. The ac-
and0.008% bromophenol blue tual amount of Zein extracted from the starting mate-
Gel running buffer stock solution:? 0.25 M tris base rial cannot be quantified. Therefore, an estimated
pH 8.6, 1.92 M glycine, and 1.0% SDS amount is derived.]
Gel running buffer: Gel running buffer stock solution Gel staining: After electrophoresis, carefully remove
and water (1:9) the gel from the plates. Rinse the gel three times with
Gel staining solution: A suitable Coomassie blue-based water. Stain the gel by following the manufacturer's
solution? directions for the stain used.
Molecular weight marker: Use a suitable molecular Destaining: After staining as directed by the manufac-
weight marker* containing protein bands at turer, destain as directed by the manufacturer.
10-190 kDa. [NoTeE—A molecular weight marker with Gel scan procedure: Set up a gel scanner accordin
protein bands at 10-100 KDa can also be used.] to the manufacturer’s instructions. Place the gel in the
Molecular weight standard solution: Dilute the Molec- detector, and obtain a single image of all loaded lines
ular weight marker with the Sample loading buffer (1:1). of the gel.
Incubate the mixture in a closed microcentrifuge tube Acceptance criteria: Zein has two major bands for a-
at 95° for 10 min. After incubation, allow the tube to zein at 15-26 kDa.
flash-cool on ice. Place the tube in a microcentrifuge,
spin at a top speed for a few seconds, and allow to IMPURITIES
NF Monographs

stop on its own to collect any condensation on the e RESIDUE ON IGNITION (281): NMT 2.0%, using an ignition
sides and top of the tube. temperature of 800 + 25°
Independent standard stock solution: 10 mg/mL of B-
lactoglobulin A in water
Delete the following:
1 Available from Invitrogen (Life Technologies) as Tris-Glycine SDS Sample
Buffer (2X), catalog number LC2676.
2Available from Invitrogen as Tris-Glycine SDS Running Buffer (10X), catalog °e HEAVY METALS, Method |! (231): NMT 20 119/Ge coreni:-
number LC2675. Jan-2018)
3 Available from Invitrogen as SimplyBlue Stain, catalog number LC6065.
4 Available from Invitrogen as BenchMark Prestained Protein Ladder, catalog 5 Available from Invitrogen, catalog number EC6495. However, these are
number 1074810. readily available from several other manufacturers.
NF 36
© Limit OF HEXANE-SOLUBLE MATTER
Sample: 15g of Zein
Solvent: Alcohol and water (17:3, w/w)
Analysis: Dissolve the Sample in 150 mL of Solvent. Stir
the mixture, using a magnetic stirrer, and heat the solu-
tion to 30°. Once the Sample is dissolved, transfer the
solution to a 500-mL separatory funnel.
Add 60 mL of n-hexane. Shake the mixture, and allow
the phases to separate. Discharge the bottom layer (al-
cohol) to a beaker, and transfer the top layer (hexane)
to a first 500-mL flask. Weigh the first 500-mL flask,
and record the weight. Pour the bottom layer of alco-
hol back into the separatory funnel. Repeat this step
four more times.
After the five 60-mL hexane solutions have been added
to the first 500-mL flask, attach it to a rotary evapora-
tor to distill the hexane. Collect the hexane in a sec-
ond 500-mL flask.
The first 500-mL flask contains a yellow to reddish oil.
Record the weight of the flask containing this oil.
Calculate the percentage of hexane-soluble matter in
the portion of Zein taken:
Result = [(W;
— W)/W] x 100
W, = weight of the flask (g)
W; = weight of the first S00-mL flask (g)
Ww = weight of the Sample (g)
Acceptance criteria
For Zein from normal dent corn: NMT 12.5% for
hexane-soluble matter
Official Monographs / Zinc 5657
For Zein from waxy corn: NMT 16.0% for hexane-
soluble matter
SPECIFIC TESTS
e Loss ON DRYING (731)
Analysis: Dry at 105° for 2 h.
Acceptance criteria: NMT 8.0%
© MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECI-
FIED MICROORGANISMS (62): The total bacterial count
does not exceed 103 cfu/g, and the tests for Salmonella
species and Escherichia coli are negative.
© PROTEIN CONTENT
Analysis: Proceed as directed in Nitrogen Determination
(461), Method |. Calculate the weight percentage of the
protein content in Zein by multiplying the percentage
of nitrogen found by 6.25.
Acceptance criteria: 81.9%-100.0% on the dried basis
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in well-closed contain-
ers, and store at room temperature.
© LABELING: Label it to indicate the corn source from which
it is derived.
Zinc Stearate—see Zinc Stearate General
Monographs
Exerol-B eel UCoyNEE |
Hig fe &
Combined Index to USP 41 and NF 36 Abaca-Acety I-1

Combined Index to USP 41 and

NF 36, Volumes 1-5
Page citations refer to the pages of Volumes 1, 2, 3, 4 and 5 of USP 41-NF 36. This index is repeated in
its entirety in each volume.

1-2302 Volume 1
2303-4414 Volume 2
4415-5658 Volume 3
5659-6698 Volume 4
6699-8228 Volume 5

Numbers in angle brackets such as (421) refer to chapter numbers in the General Chapters section.

A and (salts of) chlorpheniramine,

dextromethorphan, and
Acetate
methyl, 5706
pseudoephedrine, oral powder Acetate buffer, 5676
Abacavir containing at least three of the TS, 5750
oral solution, 19 following, 47 Acetazolamide, 65
sulfate, 23 and (salts of) chlorpheniramine, for injection, 66
tablets, 20 dextromethorphan, and oral suspension, 68
and lamivudine tablets, 21 pseudoephedrine, oral solution tablets, 68
Abiraterone containing at least three of the Acetic acid, 5181, 5664
acetate, 24 following, 49 ammonium acetate buffer TS, 5750
acetate tablets, 26 and (salts of) chlorpheniramine, diluted, 5181, 5664, 5690
Absolute dextromethorphan, and double-normal (2 N), 5762
alcohol, 5666 pseudoephedrine, tablets containing at glacial, 69, 5664, 5697
ether, 5664 least three of the following, 51 glacial, TS, 5750, 5754
Absorbable chlorpheniramine maleate, and and hydrocortisone otic solution, 2062
dusting powder, 1457 dextromethorphan hydrobromide irrigation, 69
gelatin film, 1929 tablets, 53 metaphosphoric, TS, 5756
gelatin sponge, 1929 and codeine phosphate capsules, 55 otic solution, 70
surgical suture, 3901 and codeine phosphate oral solution, 56 strong, TS, 5750
Absorbent and codeine phosphate oral suspension, 1MTS, 5750
cotton, 5664 57 2MTS, 5750
gauze, 1927 and codeine phosphate tablets, 59 Acetic acid in peptides, 6246
Acacia, 5179 dextromethorphan hydrobromide, 0.008 M Acetic acid TS, 5750
syrup, 5179 doxylamine succinate, and 0.3 N Acetic acid TS, 5750
Acarbose, 28 pseudoephedrine hydrochloride oral Acetic anhydride, 5664
Acebutolol hydrochloride, 29 solution, 60 Acetohydroxamic acid, 70
capsules, 30 and diphenhydramine citrate tablets, 61 tablets, 71
Acepromazine maleate, 32 diphenhydramine hydrochloride, and Acetone, 5182, 5664
injection, 33 pseudoephedrine hydrochloride tablets, anhydrous, 5664
tablets, 34 63 buffered, TS, 5664, 5750, 5752
Acesulfame potassium, 5180 and hydrocodone bitartrate tablets, 2053 Acetonitrile, 5664
Acetal, 5664 isometheptene mucate, and spectrophotometric, 5664
Acetaldehyde, 5664 dichloralphenazone capsules, 2251 Acetophenone, 5664
TS, 5750 and oxycodone capsules, 3107 p-Acetotoluidide, 5664
Acetaldehyde ammonia trimer trihydrate, and oxycodone tablets, 3108 Acetylacetone, 5664
5664 and pentazocine tablets, 3221 Acetyl chloride, 5664
Acetaminophen, 34 and pseudoephedrine hydrochloride Acetylcholine chloride, 72, 5665
aspirin and caffeine tablets, 42 tablets, 64 for ophthalmic solution, 73
and aspirin tablets, 41 oral solution, 37 Acetylcysteine, 74
butalbital and caffeine capsules, 596 for effervescent oral solution, 37 Acetylcysteine
butalbital and caffeine tablets, 597 suppositories, 38 compounded solution, 75
and caffeine tablets, 44 oral suspension, 39 and isoproterenol hydrochloride inhalation
capsules, 36 tablets, 39 solution, 76
and (salts of) chlorpheniramine, extended-release tablets, 40 solution, 74
dextromethorphan, and and tramadol hydrochloride oral N-Acetylglucosamine, 4417
pseudoephedrine, capsules containing at suspension, 4157 3-Acetylthio-2-methylpropanoic acid, 5665
least three of the following, 45 Acetanilide, 5664 Acetyltributyl citrate, 5183
|-2. Acety-Alumi Combined Index to USP 41 and NF 36

Acetyltriethy! citrate, 5183 Povidone-iodine topical, 3392 Alkaline

N-Acetyltyrosine, 4418 Terbutaline sulfate inhalation, 3986 borate buffer, 5676
N-Acetyl-L-tyrosine ethyl ester, 5665 Thimerosal topical, 4056 cupric citrate TS, 5750
Acid Tolnaftate topical, 4135 cupric citrate TS 2, 5750
acrylic, 5665 Triamcinolone acetonide topical, 4186 cupric iodide TS, 5750
alpha lipoic, 4740 cupric tartrate TS, 5750
dehydroacetic, 5320 mercuric-potassium iodide TS, 5750
ferric chloride TS, 5750 phosphatase enzyme, 5666
ferrous sulfate TS, 5750 Agar, 5185, 5665 picrate TS, 5750
Folic, compounded oral soultion, 1866 Agarose, 5665 pyrogallol TS, 5758
iminodiacetic, 5701 Air, medical, 91 sodium hydrosulfite TS, 5751
stannous chloride TS, 5750 Air-helium certified standard, 5665 Alkyl (C12-15) benzoate, 5191
stannous chloride TS, stronger, 5750 Alanine, 92 Alkylphenoxypolyethoxyethanol, 5666
Acid-neutralizing capacity (301), 6169 L-Alanyl-L-glutamine, 4420 Allantoin, 120
Acidulated phosphate and sodium fluoride Albendazole, 93 Allopurinol, 121
topical solution, 3791 oral suspension, 93 oral suspension, 123
Acitretin, 77 tablets, 94 tablets, 123
capsules, 78 Albumen TS, 5750 Allyl isothiocyanate, 124
Statistical tools for procedure validation Albumin Almond oil, 5191
(1210), 6702, 7130, 7622 bovine serum, 5665 Almotriptan
Acrylic acid, 5665 human, 95 tablets, 127
Activated rAlbumin human, 5186 Almotriptan malate, 124
alumina, 5665 Albuterol, 95 Aloe, 129
charcoal, 859, 5665 sulfate, 100 Alpha
magnesium silicate, 5665 tablets, 96 lipoic acid, 4740
Acyclovir, 80 extended-release tablets, 97 Alpha-chymotrypsin, 5666
capsules, 80 Alclometasone dipropionate, 100 Alpha cyclodextrin hydrate, 5666
for injection, 81 cream, 102 Alpha-(2-(methylamino)ethyl)benzyl alcohol,
ointment, 82 ointment, 102 5666
oral suspension, 83 Alcohol, 103, 5665 Alphanaphthol, 5666
tablets, 84 70 percent, 80 percent, and 90 percent, Alphazurine 2G, 5745
Adamantane, 5665 5665 Alprazolam, 130
Adapalene, 85 absolute, 5666 oral suspension, 131
gel, 87 aldehyde-free, 5666 tablets, 131
Ademetionine disulfate tosylate, 4419 alpha-(2-(methylamino)ethyl)benzyl, 5666 extended-release tablets, 133
Adenine, 88 amyl, 5666 orally disintegrating tablets, 136
sulfate, 5665 tert-amyl, 5666, 5669, 5702 Alprenolol hydrochloride, 5666
Adenosine, 89 butyl, 5229 Alprostadil, 138
injection, 90 dehydrated, 105, 5666, 5686 injection, 140
Adipic acid, 5184, 5665 dehydrated isopropyl, 5666 Alteplase, 141
Admissions denaturated, 5666 for injection, 144
to NF 36, 5167 denaturated, TS, 5753 Alternative microbiological sampling methods
to USP 41, xxxiii determination (611), 6358 for nonsterile inhaled and nasal products
in dextrose injection, 107 (610), 6356
diluted, 5188, 5666 Altretamine, 146
injection, dehydrated, 107 capsules, 147
isobutyl, 5666 Alum, 5666
Aerosol isopropyl, 5666 ammonium, 148, 5666
Bacitracin and polymyxin B sulfate topical, methyl, 5666 potassium, 148, 5719
439 neutralized, 5666, 5710 Alumina, 5666
Benzocaine, butamben, and tetracaine phenol TS, 5750 activated, 5665, 5666, 5669
hydrochloride topical, 479 n-propyl, 5666 anhydrous, 5666
Benzocaine and menthol topical, 484 rubbing, 108 aspirin, codeine phosphate, and magnesia
Benzocaine topical, 470 secondary butyl, 5666 tablets, 380
Dexamethasone sodium phosphate tertiary butyl, 5666 aspirin, and magnesia tablets, 373
inhalation, 1203 Alcoholic aspirin, and magnesium oxide tablets, 375
Epinephrine bitartrate inhalation, 1533 ammonia TS, 5750 magnesia, and calcium carbonate
Epinephrine inhalation, 1530 mercuric bromide TS, 5750 chewable tablets, 152
Ergotamine tartrate inhalation, 1558 potassium hydroxide TS, 5750 magnesia, calcium carbonate, and
Fluticasone propionate and salmeterol potassium hydroxide TS 2, 5758 simethicone chewable tablets, 153
inhalation, 1847 TS, 5750 magnesia, and calcium carbonate oral
Fluticasone propionate inhalation, 1831 Alcoholometric table, 5861 suspension, 151
Inhalation and nasal drug products: Aldehyde dehydrogenase, 5666 magnesia, and simethicone oral
aerosols, sprays, and powders— Alendronate sodium, 109 suspension, 155
performance quality tests (601), 6327 tablets, 110 magnesia, and simethicone chewable
lsoetharine mesylate inhalation, 2245 Alfadex, 5189 tablets, 157
lsoproterenol hydrochloride inhalation, Alfentanil and magnesia oral suspension, 149
2259 hydrochloride, 112 and magnesia tablets, 150
Isoproterenol hydrochloride and injection, 113 magnesium carbonate, and magnesium
phenylephrine bitartrate inhalation, 2262 Alfuzosin hydrochloride, 114 oxide tablets, 160
Isoproterenol sulfate inhalation, 2264 extended-release tablets, 115 and magnesium carbonate oral suspension,
Lidocaine topical, 2410 Alginates assay (311), 6170 158
Metaproterenol sulfate inhalation, 2607 Alginic acid, 5190 and magnesium carbonate tablets, 159
Polymyxin B sulfate and bacitracin zinc Alizarin complexone, 5666 and magnesium trisilicate oral suspension,
topical, 3350 161
Combined Index to USP 41 and NF 36 Alumi-Ammon 1-3

Alumina (continued) Amiloride hydrochloride, 204 tablets, 249

and magnesium trisilicate tablets, 162 and hydrochlorothiazide tablets, 206 Amlodipine
Aluminon, 5666 tablets, 205 oral suspension, 250
Aluminum, 5666 Amiloxate, 208 and benazepril hydrochloride capsules, 251
acetate topical solution, 163 Aminoacetic acid, 5667 and valsartan tablets, 253
chloride, 163 4-Aminoantipyrine, 5667 valsartan and hydrochlorothiazide tablets,
chlorohydrate, 164 Aminobenzoate 257
chlorohydrate solution, 165 potassium, 209 Amlodipine besylate, 262
chlorohydrex polyethylene glycol, 166 potassium capsules, 211 tablets, 263
chlorohydrex propylene glycol, 167 potassium for oral solution, 212 Ammonia
dichlorohydrate, 167 potassium tablets, 212 alcoholic TS, 5750
dichlorohydrate solution, 168 sodium, 213 detector tube, 5668
dichlorohydrex polyethylene glycol, 169 Aminobenzoic acid, 215 N 13 injection, 2955
dichlorohydrex propylene glycol, 170 gel, 217 nitrate TS, silver, 5759
hydroxide gel, 170 topical solution, 217 solution, diluted, 5668
hydroxide gel, dried, 171 p-Aminobenzoic acid, 5667 solution, strong, 5197
hydroxide gel capsules, dried, 172 2-Aminobenzonitrile, 5667 spirit, aromatic, 264
hydroxide gel tablets, dried, 172 Aminocaproic acid, 218 TS, 5668, 5751
monostearate, 5193 injection, 218 WS 25751
oxide, 5195 oral solution, 219 TS alcoholic, 5751
oxide, acid-washed, 5666, 5667 tablets, 219 TS stronger, 5751
phosphate gel, 172 4-Amino-6-chloro-1,3-benzenedisulfonamide, water, stronger, 5668, 5733, 5751
potassium sulfate, 5667 5667 water, 25 percent, 5668
sesquichlorohydrate, 173 4-Amino-2-chlorobenzoic acid, 5667, 5682 Ammonia-ammonium chloride buffer TS,
sesquichlorohydrate solution, 173 2-Amino-5-chlorobenzophenone, 5667, 5682 5751
sesquichlorohydrex polyethylene glycol, 7-Aminodesacetoxycephalosporanic acid, Ammoniacal potassium ferricyanide TS, 5751
174 5667 Ammonia-cyanide TS, 5751
sesquichlorohydrex propylene glycol, 175 2-Aminoethyl diphenylborinate, 5667 Ammoniated cupric oxide TS, 5751
subacetate topical solution, 175 1-(2-Aminoethyl)piperazine, 5667 Ammonio methacrylate copolymer, 5198
sulfate, 176 Aminoglutethimide, 220 dispersion, 5199
sulfate and calcium acetate tablets for tablets, 221 Ammonium
topical solution, 177 Aminoguanidine bicarbonate, 5667 acetate, 5668
zirconium octachlorohydrate, 177 2-Aminoheptane, 5668 acetate TS, 5751
zirconium octachlorohydrate solution, 179 N-Aminohexamethyleneimine, 5668 alum, 148
zirconium octachlorohydrex gly, 180 Aminohippurate sodium injection, 222 bicarbonate, 5668
zirconium octachlorohydrex gly solution, Aminohippuric acid, 223 bisulfate, 5668
181 4-Amino-3-hydroxy-1 -naphthalenesulfonic bromide, 5668
zirconium pentachlorohydrate, 182 acid, 5668, 5668 carbonate, 5200, 5668
zirconium pentachlorohydrate solution, Aminolevulinic acid carbonate TS, 5751
183 hydrochloride, 223 carbonate TS 2, 5751
zirconium pentachlorohydrex gly, 184 Amino methacrylate copolymer, 5196 chloride, 265, 5668
zirconium pentachlorohydrex gly solution, 1,2,4-Aminonaphtholsulfonic acid, 5668 chloride-ammonium hydroxide TS, 5751
185 Aminonaphtholsulfonic acid TS, 5751 chloride injection, 265
zirconium tetrachlorohydrate, 186 Aminopentamide sulfate, 224 chloride, potassium gluconate, and
zirconium tetrachlorohydrate solution, 187 injection, 225 potassium citrate oral solution, 3380
zirconium tetrachlorohydrex gly, 188 tablets, 225 chloride delayed-release tablets, 265
zirconium tetrachlorohydrex gly solution, 2-Aminophenol, 5668 chloride TS, 5751
189 4-Aminophenol in acetaminophen-containing citrate, dibasic, 5668, 5687
zirconium trichlorohydrate, 190 drug products (227), 6141 citrate, ferric, 266
zirconium trichlorohydrate solution, 191 m-Aminophenol, 5668 citrate for oral solution, ferric, 266
zirconium trichlorohydrex gly, 192 p-Aminophenol, 5668 dihydrogen phosphate, 5668
zirconium trichlorohydrex gly solution, 193 Aminophylline, 226 fluoride, 5668
Aluminum (206), 6107 injection, 228 formate, 5668
Aluminum chloride, 5667 oral solution, 229 Ammonium
Aluminum sulfate rectal solution, 231 glycyrrhizate, 5201
and calcium acetate for topical solution, suppositories, 231 hydroxide, 5668
176 tablets, 232 hydroxide 6 N, 5668
Amantadine hydrochloride, 194 delayed-release tablets, 234 molybdate, 267, 5668
capsules, 195 3-Amino-1-propanol, 5668 molybdate injection, 268
oral solution, 196 3-Aminopropionic acid, 5668 molybdate TS, 5751
Amaranth, 5667 Aminosalicylate sodium, 235 nitrate, 5669
TS, 5751 tablets, 237 nitrate, ceric TS, 5752
Amcinonide, 196 Aminosalicylic acid, 238 nitrate TS, silver, 5759
cream, 197 tablets, 239 oxalate, 5669
ointment, 198 3-Aminosalicylic acid, 5668 oxalate TS, 5751
American ginseng, 4422 Amiodarone persulfate, 5669
capsules, 4426 hydrochloride injection, 243 phosphate, 5202
extract, powdered, 4425 Amiodarone hydrochloride, 240 phosphate, dibasic, 5669, 5687
powdered, 4423 oral suspension, 245 phosphate, dibasic, TS, 5751
tablets, 4428 Amitraz, 245 phosphate, monobasic, 5668, 5669
Amifostine, 198 concentrate for dip, 247 polysulfide TS, 5751
for injection, 199 Amitriptyline hydrochloride, 247 pyrrolidinedithiocarbamate, 5669
Amikacin, 201 and chlordiazepoxide tablets, 872 pyrrolidinedithiocarbamate, saturated, TS,
sulfate, 202 injection, 249 5751
sulfate injection, 203 and perphenazine tablets, 3248 reineckate, 5669
USP 41
and color to bands in Standard solution B. Other minor
bands may be observed in the Sample solution and
Standard solution B.
e B. HPLC IDENTIFICATION TEST: The Sample solution chro-
matogram from the test for Content of Triterpene Deriva-
tives shows a peak at the retention time corresponding to
that of asiaticoside in Standard solution A. Identify other
triterpene derivative peaks in the Sample solution by com-
parison with the chromatogram of Standard solution B
and the reference chromatogram provided with the lot
of USP Powdered Centella asiatica Extract RS being used.
The Sample solution shows additional peaks correspond-
ing to madecassoside and asiaticoside B (these two
sper
may eoslute), madecassic acid, terminolic acid, and
atic acid.
COMPOSITION
© CONTENT OF TRITERPENE DERIVATIVES
Solution A: Dilute 3 mL of phosphoric acid with water
to 1000 mL, mix, filter, and degas.
Solution B: Acetonitrile
Mobile phase: See the gradient table below.
Time Solution A Solution B
(min) (%) (%)
0 78 22
65, 45 55
66 5 95
75 5 95
76 78 22
85 78 22
Standard solution A: 0.1 mg/mL of USP Asiaticoside RS
in methanol
Standard solution B: Sonicate a portion of USP Pow-
dered Centella asiatica Extract RS in methanol to obtain
a solution with a concentration of about 5.0 mg/mL.
Before injection, pass through a membrane filter of
0.45-uum or finer pore size, discarding the first few mL
of the filtrate.
Sample solution: Sonicate a portion of Powdered
Centella asiatica Extract in methanol to obtain a solution
with a concentration of about 5.0 mg/mL. Before injec-
tion, pass through a membrane filter of 0.45-um or
finer pore size, discarding the first few mL of the
filtrate.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 200 nm
Column: 4.6-mm x 25-cm; 5-um packing L1
Flow rate: 1.0 mL/min
Injection size: 10 uL
System suitability
Samples: Standard solution A and Standard solution B
Suitability requirements
Chromatogram similarity: The chromatogram from
Standard solution B is similar to the reference chro-
matogram provided with the lot of USP Powdered
Centella asiatica Extract RS being used.
Tailing factor: Between 0.8 and 2.0 for the asiatico-
side peak, Standard solution A
Resolution: NLT 1.5 between the madecassic acid
and terminolic acid peaks, Standard solution B
Relative standard deviation: NMT 2.0% determined
from the asiaticoside peak in repeated injections,
Standard solution A
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution. [NoTe—Standard solution A, Standard
solution B, and the Sample solution are stable for 48 h
at room temperature.]
Using the chromatograms of Standard solution A, Stan-
dard solution B, and the reference chromatogram pro-
Dietary Supplements / Centella 4517
vided with the lot of USP Powdered Centella asiatica
Extract RS being used, identify the retention times of
the peaks corresponding to different triterpene deriva-
tives. The approximate relative retention times of the
different triterpene derivatives are provided in the fol-
lowing table.
Approximate Relative
Analyte Retention Time
Madecassoside 0.71
Asiaticoside B 0.72
Asiaticoside 1.00
asi- Madecassic acid 2.40
Terminolic acid 2.44
Asiatic acid 3.12
Separately calculate the percentages of the sum of
madecassoside and asiaticoside B (these two peaks
may co-elute), asiaticoside, the sum of madecassic acid
and terminolic acid, and asiatic acid in the portion of
Powdered Centella asiatica Extract taken:
Result = (ru/ts) x (Cs/Cu) x F x 100
Tu = peak response(s) of the triterpene derivative(s)
from the Sample solution
Ts = peak response of asiaticoside from Standard
solution A
Cs = concentration of USP Asiaticoside RS in
Standard solution A (mg/mL)
Cu = concentration of Powdered Centella asiatica
Extract in the Sample solution (mg/mL)
F = conversion factors for analytes: 1.00 for
asiaticoside, 1.017 for the sum of
madecassoside and asiaticoside B, 0.526 for
the sum of madecassic acid and terminolic
acid, and 0.509 for asiatic acid
Acceptance criteria: Add the percentages of different
triterpene derivatives: NLT 90.0% and NMT 110.0% of
the labeled amount of triterpene derivatives; the labeled
amount of triterpene derivatives is NMT 40%, calcu-
lated on the dried basis.
CONTAMINANTS
Delete the following:
°e HEAVY METALS, Method [I! (231): NMT 20 ppme cortica:1-
Jan-2018)
e ARTICLES OF BOTANICAL ORIGIN, General Method for Pesti-
Ure D1XeleCol NIG
cide Residues Analysis (561): Meets the requirements
e MICROBIAL ENUMERATION TESTS (2021): The total aerobic
microbial count does not exceed 10 cfu per g. The total
combined yeast and mold count does not exceed 103 cfu
per g.
© MICROBIOLOGICAL PROCEDURES FOR ABSENCE OF SPECIFIED M-
CROORGANISMS (2022): Meets the requirements of the
tests for absence of Salmonella species and Escherichia coli
SPECIFIC TESTS
© Loss ON DRYING (731): Dry 1.0 g of Powdered Centella
asiatica Extract at 105° for 2 h: it loses NMT 5% of its
weight.
¢ OTHER REQUIREMENTS: Meets the requirements of the test
for Residual Solvents under Botanical Extracts (565)
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in well-closed contain-
ers, protected from light and moisture, and store at con-
trolled room temperature.
e LABELING: The label states the Latin binomial and, follow-
ing the official name, the part of the plant from which
the article was derived. It meets other labeling require-
ments under Botanical Extracts (565).
 1-4. Ammon-Artic Combined Index to USP 41 and NF 36

Ammonium (continued) Analytical procedures for recombinant citrate phosphate dextrose adenine
reineckate TS, 5751 therapeutic monoclonal antibodies (129), solution, 327
sulfamate, 5669 6070 heparin solution, 2025
sulfate, 5202, 5669 Anastrozole, 312 sodium citrate solution, 329
sulfate, cupric TS, 5752 tablets, 313 Anti-D reagent, 5670
sulfate, ferric TS, 5753 Ancillary materials for cell, gene, and tissue- Anti-D (Rho) reagent, 5671
sulfide TS, 5751 engineered products (1043), 6850 Anti-factor Xa and anti-factor Ila assays for
thiocyanate, 5669 Andrographis, 4429 unfractionated and low molecular weight
thiocyanate, tenth-normal (0.1 N), 5762 extract, powdered, 4433 heparins (208), 6113
thiocyanate TS, 5751 powdered, 4431 Antifoam reagent, 5671
vanadate, 5669 Anethole, 5204 Antihuman globulin reagent, 5671
vanadate TS, 5751 (£)-Anethole, 5669 Antimicrobial
Ammonium hydroxide Angustifolia agents—content (341), 6172
1 MTS, 5751 extract, powdered echinacea, 4571 effectiveness testing (51), 5959
2 MTS, 5751 powdered echinacea, 4569 Antimony
Amobarbital sodium, 268 pentachloride, 5671
for injection, 269 potassium tartrate, 329
Amodiaquine, 270 sodium tartrate, 330
hydrochloride, 270
hydrochloride tablets, 271 Anhydrous trichloride, 5671
trichloride TS, 5751
Amoxapine, 272 acetone, 5664 Antipyrine, 330
tablets, 273 alumina, 5669 and benzocaine otic solution, 332
Amoxicillin, 274 barium chloride, 5669 benzocaine, and phenylephrine
boluses, 276 calcium chloride, 5669 hydrochloride otic solution, 333
capsules, 276 calcium phosphate, dibasic, 655 Antithrombin Ill, 5677
and clavulanate potassium for oral citric acid, 968 human, 334
suspension, 284 cupric sulfate, 5669 Apomorphine hydrochloride, 336
and clavulanate potassium tablets, 285 dibasic sodium phosphate, 5669 tablets, 337
and clavulanic acid extended-release magnesium perchlorate, 5669 Apparent intrinsic dissolution—dissolution
tablets, 286 magnesium sulfate, 5669 testing procedures for rotating disk and
for injectable suspension, 279 methanol, 5669 stationary disk (1087), 7155
intramammary infusion, 278 potassium carbonate, 5669 Applications of mass spectrometry (1736),
oral suspension, 279 sodium acetate, 5669 7982
for oral suspension, 280 sodium carbonate, 5669 Applications of nuclear magnetic resonance
tablets, 280 sodium phosphate, monobasic, 5731 spectroscopy (1761), 8004
tablets for oral suspension, 283 sodium sulfate, 5669 Application of water activity determination to
Amphetamine sodium sulfite, 5669 nonsterile pharmaceutical products (1112),
sulfate, 288 7298
sulfate tablets, 290 Apraclonidine
Amphotericin B, 290 hydrochloride, 337
cream, 291 Anileridine, 315 ophthalmic solution, 338
for injection, 291 hydrochloride, 316 Aprepitant, 339
lotion, 292 hydrochloride tablets, 317 capsules, 340
ointment, 292 injection, 316 Aprobarbital, 5671
Ampicillin, 292 Aniline, 5669 Aprotinin, 342
boluses, 298 blue, 5669 injection, 345
sulfate, 5670
capsules, 298 Arcitumomab injection, technetium Tc 99m,
for injectable suspension, 301 Animal drugs for use in animal feeds (1152), 3940
for injection, 300 7450 Argatroban, 346
and probenecid for oral suspension, 303 Anion-exchange resin Arginine, 348
sodium, 304 strong, lightly cross-linked, in the chloride capsules, 4434
soluble powder, 300 form, 5670 hydrochloride, 349
and sulbactam for injection, 305 50- to 100-mesh, styrene-divinylbenzene, hydrochloride injection, 350
for oral suspension, 301 5670, 5734 tablets, 4435
tablets, 302 styrene-divinylbenzene, 5670 Aripiprazole, 351
Amprolium, 306 p-Anisaldehyde, 5670 orally disintegrating tablets, 354
soluble powder, 307 Anise oil, 5205 tablets, 352
oral solution, 307 p-Anisidine, 5670 Aromatic
Amyl Anisole, 5670 castor oil, 741
acetate, 5666, 5669, 5702 Annotations elixir, 5206
alcohol, 5669 to NF 36, 5168 Arsanilic acid, 355
to USP 41, xxxvi Arsenazo Ill acid, 5672
nitrite, 308
nitrite inhalant, 308 Antazoline phosphate, 318 Arsenic
a-Amylase, 5669 Anthracene, 5670 in reagents, 5661
Amylene hydrate, 5203 Anthralin, 319 trioxide, 5672
cream, 320 Arsenic (211), 6124
tert-Amyl alcohol, 5669
ointment, 321 Articaine
Anagrelide
Anthrax vaccine adsorbed, 321 hydrochloride, 356
Index

capsules, 310
hydrochloride, 309 Anthrone, 5670 hydrochloride and epinephrine injection,
Analysis of biological assays (1034), 6818 TS, 5751 358
Analytical data—interpretation and treatment Antibiotics—microbial assays (81), 5991 Articles
(1010), 6706 Anticoagulant appearing in USP 41 that were not
citrate dextrose solution, 324 included in USP 40 including
Analytical instrument qualification (1058),
7005
citrate phosphate dextrose solution, 326 supplements, xxxiv
of Incorporation, xxviii
Combined Index to USP 41 and NF 36 Artic-Balsa 1-5

Articles of botanical origin (561), 6279 insulin (121), 6054 for injection, 434
Ascorbic acid, 359 Assessment of drug product performance— Azure A, 5672
compounded oral solution, 361 bioavailability, bioequivalence, and
injection, 360 dissolution (1090), 7170
oral solution, 361 Assessment of drug product leachables
tablets, 362 associated with pharmaceutical packaging/
10 TS, 5751 delivery systems (1664), 7924
Ascorbyl palmitate, 5206 Assessment of extractables associated with
Ashwagandha root, 4436 pharmaceutical packaging/delivery systems
extract, powdered, 4439 (1663), 7910 Bacillus subtilis subsp. subtilis menaquinone-7
powdered, 4438 Astaxanthin esters, 4446 extract, 4765
Asian ginseng, 4441 Astemizole, 381 Bacitracin, 436
extract, powdered, 4444 tablets, 382 for injection, 437
powdered, 4442 Astragalus root, 4448 methylene disalicylate, soluble, 438
tablets, 4445 dry extract, 4452 methylene disalicylate soluble powder, 439
Asparagine, 5207 powder, 4450 neomycin and polymyxin B sulfates and
L-Asparagine, 5672 Atenolol, 383 hydrocortisone acetate ointment, 2895
Aspart and chlorthalidone tablets, 387 neomycin and polymyxin B sulfates and
insulin, 2164 injection, 384 hydrocortisone acetate ophthalmic
Aspartame, 5208 oral solution, 385 ointment, 2896
acesulfame, 5209 tablets, 384 neomycin and polymyxin B sulfates and
Aspartic acid, 363 Atenolol compounded lidocaine ointment, 2896
L-Aspartic acid, 5672 oral suspension, 386 and neomycin and polymyxin B sulfates
Aspirin, 364 Atenolol compounded, veterinary ointment, 2894
acetaminophen and caffeine tablets, 42 oral suspension, 386 and neomycin and polymyxinB sulfates
and acetaminophen tablets, 41 Atomic absorption spectroscopy (852), 6644 ophthalmic ointment, 2895
alumina and magnesia tablets, 373 Atomic absorption spectroscopy—theory and and neomycin sulfate ointment, 2884
alumina and magnesium oxide tablets, 375 practice (1852), 8109 ointment, 438
boluses, 364 Atomic masses, 5860 ophthalmic ointment, 438
butalbital, and caffeine capsules, 599 Atomic weights, 5859 and polymyxin B sulfate topical aerosol,
butalbital, caffeine, and codeine phosphate Atomoxetine 439
capsules, 601 capsules, 390 zinc, 440
butalbital, and caffeine tablets, 600 Atomoxetine hydrochloride, 388 zinc, neomycin and polymyxinB sulfates,
and butalbital tablets, 597 Atorvastatin calcium, 391 and hydrocortisone ointment, 2898
caffeine, and dihydrocodeine bitartrate Atorvastatin calcium zinc, neomycin and polymyxin B sulfates,
capsules, 378 tablets, 395 and hydrocortisone ophthalmic
capsules, 366 Atovaquone, 399 ointment, 2898
delayed-release capsules, 366 oral suspension, 400 zinc, neomycin and polymyxin B sulfates,
carisoprodol, and codeine phosphate Atracurium besylate, 401 and hydrocortisone acetate ophthalmic
tablets, 718 injection, 403 ointment, 2899
and carisoprodol tablets, 716 Atropine, 404 zinc, neomycin and polymyxin B sulfates,
codeine phosphate, alumina, and sulfate, 405 and lidocaine ointment, 2900
magnesia tablets, 380 sulfate and diphenoxylate hydrochloride zinc and neomycin and polymyxin B
and codeine phosphate tablets, 379 oral solution, 1339 sulfates ointment, 2897
effervescent tablets for oral solution, 371 sulfate and diphenoxylate hydrochloride zinc and neomycin and polymyxin B
orphenadrine citrate and caffeine tablets, tablets, 1340 sulfates ophthalmic ointment, 2897
3052 sulfate injection, 406 zinc and neomycin sulfate ointment, 2884
and oxycodone tablets, 3109 sulfate ophthalmic ointment, 407 zinc ointment, 441
and pentazocine tablets, 3222 sulfate ophthalmic solution, 408 zinc and polymyxinBsulfate topical
suppositories, 367 sulfate tablets, 409 aerosol, 3350
tablets, 368 Attapulgite, activated, 410 zinc and polymyxin B sulfate ointment,
tablets, buffered, 369 colloidal, 410 442
delayed-release tablets, 370 Aurothioglucose, 411 zinc and polymyxin B sulfate ophthalmic
extended-release tablets, 372 injectable suspension, 411 ointment, 442
Assay Auxiliary packaging components (670), 6428 zinc and polymyxin B sulfate topical
alginates (311), 6170 Avobenzone, 412 powder, 3350
antibiotics, iodometric (425), 6205 Azaperone, 412 zinc soluble powder, 442
for citric acid/citrate and phosphate (345), injection, 413 Baclofen, 443
6176 Azatadine maleate, 413 oral suspension, 444
dexpanthenol (115), 6053 tablets, 414 tablets, 445
epinephrine (391), 6183 Azathioprine, 415 Bacopa, 4456
folic acid (411), 6197 oral suspension, 417 extract, powdered, 4459
niacin or niacinamide (441), 6213 sodium for injection, 418 powdered, 4458
riboflavin (481), 6239 tablets, 417 Bacterial
single-steroid (511), 6253 Azelastine hydrochloride, 419 alkaline protease preparation, 5672
for steroids (351), 6177 Azithromycin, 420 endotoxins test (85), 6011
thiamine (531), 6260 capsules, 424 Bacteriostatic
vitamin A (571), 6307 for injection, 425 sodium chloride injection, 3784
vitamin By2 activity (171), 6091 for oral suspension, 428 water for injection, 4346
vitamin D (581), 6315 tablets, 429 Balances (41), 5958
vitamin E (551), 6272 Azo violet, 5745 Balsalazide disodium, 446
Assays Aztec marigold zeaxanthin capsules, 447
antibiotics—microbial (81), 5991 extract, 4454
design and analysis of biological (111), Aztreonam, 432
6049 injection, 433
6 Banab-Bisac Combined Index to USP 41 and NF 36

Banaba leaf, 4461 butamben, and tetracaine hydrochloride sodium phosphate and betamethasone
extract, dry, 4464 topical aerosol, 479 acetate injectable suspension, 512
powder, 4462 butamben, and tetracaine hydrochloride sodium phosphate injection, 512
Bandage gel, 480 oral solution, 503
adhesive, 449 butamben, and tetracaine hydrochloride tablets, 504
gauze, 449 ointment, 482 valerate, 513
Barbital sodium, 5672 butamben, and tetracaine hydrochloride valerate cream, 514
Barbituric acid, 5672 topical solution, 483 valerate and gentamicin sulfate ointment,
Barium cream, 471 1939
acetate, 5672 gel, 473 valerate and gentamicin sulfate otic
chloride, 5672 lozenges, 474 solution, 1940
chloride, anhydrous, 5669, 5672 and menthol topical aerosol, 484 valerate and gentamicin sulfate topical
chloride dihydrate, 5672 ointment, 475 solution, 1941
chloride TS, 5751 otic solution, 476 valerate lotion, 515
hydroxide, 5672 topical solution, 478 valerate ointment, 517
hydroxide lime, 450 Benzoic Betanaphthol, 5673
hydroxide TS, 5751 acid, 486, 5673 Ts, 5751
nitrate, 5672 and salicylic acids ointment, 487 Betaxolol
nitrate TS, 5751 Benzoin, 488 hydrochloride, 518
sulfate, 450 tincture, compound, 488 ophthalmic solution, 519
sulfate for suspension, 453 Benzonatate, 488 tablets, 520
sulfate paste, 451 capsules, 489 Bethanechol chloride, 520
sulfate suspension, 452 Benzophenone, 5673 injection, 522
sulfate tablets, 453 p-Benzoquinone, 5673, 5724 oral solution, 523
0.05 M Barium perchlorate VS, 5762 Benzoyl oral suspension, 523
Basic fuchsin, 5672 chloride, 5673 tablets, 524
BCG live, 454 peroxide and erythromycin topical gel, Beta-lactamase, 5673, 5714
BCG vaccine, 455 1572 Bibenzyl, 5673, 5687
Beclomethasone, 5672 peroxide gel, 491 Bicalutamide, 525
Beclomethasone dipropionate, 455 peroxide, hydrous, 490 tablets, 526
Beclomethasone dipropionate compounded peroxide lotion, 492 Bifidobacterium animalis subsp. lactis, 4469
oral solution, 456 N-Benzoyl-L-arginine ethyl ester Bilberry
Beef extract, 5672 hydrochloride, 5673 extract, powdered, 4472
Behenoyl polyoxylglycerides, 5210 3-Benzoylbenzoic acid, 5673 Bile salts, 5673, 5729
Belladonna Benzoylformic acid, 5673 (S)-Binol, 5674
leaf, 456 Benzphetamine hydrochloride, 5673 Bioburden control of nonsterile drug
extract, 458 Benztropine mesylate, 493 substances and products (1115), 7305
extract tablets, 459 injection, 493 Biocompatibility of materials used in drug
tincture, 460 tablets, 494 containers, medical devices, and implants,
Benazepril hydrochloride, 460 Benzyl the (1031), 6775
and amlodipine hydrochloride capsules, alcohol, 5220 Biological
251 benzoate, 495 assay chapters—overview and glossary
tablets, 462 benzoate lotion, 495 1030), 6764
Benazepril hydrochloride compounded, 2-Benzylaminopyridine, 5673 assay validation (1033), 6803
veterinary 1-Benzylimidazole, 5673 indicators—resistance performance tests
oral suspension, 463 Benzylpenicilloyl polylysine (55), 5962
Bendroflumethiazide, 464 concentrate, 496 indicators for sterilization (1229.5), 7716
and nadolol tablets, 2847 injection, 497 reactivity tests, in vitro (87), 6017
tablets, 465 Benzyltrimethylammonium chloride, 5673 reactivity tests, in vivo (88), 6020
Benoxinate hydrochloride, 465 Beta carotene, 497 Biologics (1041), 6849
and fluorescein sodium ophthalmic capsules, 499 Biotechnology products: stability testing of
solution, 1792 preparation, 4465 biotechnological/biological products,
ophthalmic solution, 466 Betadex, 5222 quality of (1049), 6930
Bentonite, 5211 sulfobutyl ether sodium, 5224 Biotechnology-derived articles
magma, 5214 Beta glucan, 4467 amino acid analysis (1052), 6961
purified, 5212 Betahistine hydrochloride, 500 isoelectric focusing (1054), 6981
Benzaldehyde, 5214, 5672 Betaine hydrochloride, 501 peptide mapping (1055), 6984
elixir, compound, 5215 Beta-lactoglobulin, 5703 polyacrylamide gel electrophoresis (1056),
Benzalkonium chloride, 5215, 5672 Beta-lactoglobulin A, 5703 6991
solution, 5217 Betamethasone, 501 total protein assay (1057), 6998
Benzamidine hydrochloride hydrate, 5672 acetate, 505 Biotechnology products derived from cell
Benzanilide, 5672 acetate and betamethasone sodium lines of human or animal origin, viral safety
Benzene, 5672 phosphate injectable suspension, 512 evaluation of (1050), 6935
Benzenesulfonamide, 5672 acetate and gentamicin sulfate ophthalmic Biotin, 528
Benzenesulfonyl chloride, 5673 solution, 1939 capsules, 529
Benzethonium chloride, 466 benzoate, 506 tablets, 529
concentrate, 467 benzoate gel, 506 Biphenyl, 5674
topical solution, 467 cream, 502 2,2/-Bipyridine, 5674, 5693
tincture, 468 dipropionate, 507 Bis(4-sulfobutyl) ether disodium, 5674
Benzhydrol, 5673 dipropionate and clotrimazole cream, 1046 Bisacodyl, 530
Benzocaine, 469 dipropionate cream, 508 rectal suspension, 532
topical aerosol, 470 dipropionate lotion, 509
and antipyrine otic solution, 332 dipropionate ointment, 510
antipyrine, and phenylephrine sodium phosphate, 511
hydrochloride otic solution, 333
Combined Index to USP 41 and NF 36 Bisac-Butan |-7

Bisacodyl (continued) aspirin, 364

suppositories, 531 dihydrostreptomycin sulfate, 1297
delayed-release tablets, 532
4,4’-Bis(4-amino-naphthylazo)-2,2’-
neomycin, 2882
phenylbutazone, 3274 Buffer
stilbenedisulfonic acid, 5674 tetracycline, 4015 Acetate TS, 5750
Bis(2-ethylhexyl) Borage seed oil, 4488 Acetic acid-ammonium acetate TS, 5750
maleate, 5674 capsules, 4489 Acetone buffered, TS, 5750, 5752
(phosphoric acid), 5674 Boric acid, 5227, 5675 Acid phthalate, 5676
phthalate, 5674 (-)-Bornyl acetate, 5675 Alkaline borate, 5676
sebacate, 5674 Boron trifluoride, 5675 Hydrochloric acid, 5676
Bismuth, 5674 14% Boron trifluoride-methanol, 5675 Neutralized phthalate, 5676
citrate, 533 Boswellia serrata, 4490
iodide TS, potassium, 5758 extract, 4491
milk of, 533 Botanical
nitrate pentahydrate, 5674 extracts (565), 6305 Buffered acetone TS, 5752
nitrate, 0.01 mol/L, 5763 origin, identification of articles of (563), Buffers, 5676
subcarbonate, 534 6293 Buffer solutions, 5676, 5748
subgallate, 535 Bovine collagen, 5675 acetate buffer, 5676
subnitrate, 536, 5674 Bovine serum (1024), 6721 alkaline borate buffer, 5676
subsalicylate, 536 7 percent bovine serum albumin certified hydrochloric acid buffer, 5676
subsalicylate magma, 538 standard, 5675 neutralized phthalate buffer, 5676
subsalicylate oral suspension, 540 Branched polymeric sucrose, 5675 phosphate buffer, 5676
subsalicylate tablets, 540 Bretylium tosylate, 547 Bulk density and tapped density of powders
sulfite, 5745 in dextrose injection, 548 (616), 6360
sulfite agar, 5674 injection, 548 Bulk pharmaceutical excipients—certificate of
Bisoctrizole, 541 Brilliant analysis (1080), 7133
Bisoprolol fumarate, 542 blue G TS, 5752 Bulk powder sampling procedures (1097),
and hydrochlorothiazide tablets, 544 green, 5745 7206
tablets, 543 yellow, 5745 Bumetanide, 562
Bis(trimethylsilyl) Brinzolamide, 549 injection, 563
acetamide, 5674 ophthalmic suspension, 550 tablets, 564
trifluoroacetamide, 5675 Bromelain, 5676 Bupivacaine hydrochloride, 565
trifluoroacetamide with Bromine, 5676 in dextrose injection, 567
trimethylchlorosilane, 5675 sodium acetate TS, 5752 and epinephrine injection, 567
Biuret reagent TS, 5751 tenth-normal (0.1 N), 5763 injection, 566
Black cohosh, 4474 TS, 5752 Buprenorphine
fluidextract, 4480 a-Bromo-2’-acetonaphthone, 5676 hydrochloride, 569
powdered, 4476 p-Bromoaniline, 5676 Buprenorphine and naloxone
powdered extract, 4478 TS, 5752 sublingual tablets, 571
tablets, 4482 Bromocresol Buprenorphine compounded, veterinary
Black pepper, 4483 blue, 5745 buccal solution, 570
powdered, extract, 4487 blue TS, 5752 Bupropion hydrochloride, 573
powdered, 4485 green, 5745 tablets, 575
Bleomycin green-methyl red TS, 5752 extended-release tablets, 576
for injection, 546 green sodium salt, 5745 Buspirone hydrochloride, 586
sulfate, 546 green TS, 5752, 5752 tablets, 588
purple, 5745 Busulfan, 590
purple sodium salt, 5745 tablets, 591
purple TS, 5752 Butabarbital, 591
sodium, 592
Blood Bromocriptine mesylate, 551
capsules, 552 sodium oral solution, 593
Blood, 5675 tablets, 554 sodium tablets, 594
Group A; red blood cells and blood group Bromodiphenhydramine hydrochloride, 555 Butalbital, 595
B red blood cells, 5675 and codeine phosphate oral solution, 556 acetaminophen, and caffeine capsules, 596
Grouping reagent, anti-A, grouping oral solution, 555 acetaminophen, and caffeine tablets, 597
reagent, anti-B, and grouping reagent, Bromofluoromethane, 5676 aspirin, and caffeine capsules, 599
anti-AB, 5675 Bromophenol blue, 5745 aspirin, caffeine, and codeine phosphate
Technetium Tc 99m red blood cells sodium, 5745 capsules, 601
injection, 3954 TS, 5752 aspirin, and caffeine tablets, 600
N-Bromosuccinimide, 5676 and aspirin tablets, 597
Bromothymol blue, 5745 Butamben, 603
TS, 5752 benzocaine, and tetracaine hydrochloride
Blue Brompheniramine maleate, 557 topical aerosol, 479
B, oracet, 5746 injection, 558 benzocaine, and tetracaine hydrochloride
B TS, oracet, 5757 gel, 480
and pseudoephedrine sulfate oral solution,
GTS, brilliant , 5752 559 benzocaine, and tetracaine hydrochloride
tetrazolium, 5675 oral solution, 558 ointment, 482
tetrazolium TS, 5751 tablets, 559 benzocaine, and tetracaine hydrochloride
Board of trustees Brucine sulfate, 5676 topical solution, 483
USP Convention (2015-2020), xi Budesonide, 560 Butane, 5228
Boiling or distilling range for reagents, 5660 Butane-1,2-diol, 5676
Boldine, 5675 Butane-1,4-diol, 5676
Boluses
amoxicillin, 276
ampicillin, 298
|-8 Butan-Capsu Combined Index to USP 41 and NF 36

Butane-2,3-diol, 5676 orphenadrine citrate and aspirin tablets, silicate, 5240

1,3-Butanediol, 5677 3052 stearate, 5242
2,3-Butanedione, 5677, 5686 and sodium benzoate injection, 614 succinate, 658
1-Butanesulfonic acid sodium salt, 5677° Calamine, 615 sulfate, 5243, 5679
1,4-Butane sultone, 5677 topical suspension, phenolated, 616 sulfate TS, 5752
Butanol, 5677 topical suspension, 615 undecylenate, 659
Butoconazole nitrate, 605 Calcifediol, 616 and vitamin D with minerals tablets, 4502
vaginal cream, 605 capsules, 616 with vitamin D tablets, 4501
Butorphanol tartrate, 606 Calcipotriene, 617 Calcium acetate
injection, 607 ointment, 618 and aluminum sulfate for topical solution,
nasal spray, 607 Calcitonin salmon, 620 176
Butyl injection, 623 Calcium glubionate
acetate, normal, 5677 nasal solution, 624 syrup, 641
alcohol, 5229, 5677, 5712 Calcitriol, 625 Calcium L-5-methyltetrahydrofolate, 4496
alcohol, normal, 5677 injection, 626 capsules, 4498
alcohol, secondary, 5666, 5677, 5725 Calcium tablets, 4499
alcohol, tertiary, 5666, 5677, 5735 acetate, 627, 5678 Calconcarboxylic acid, 5679
benzoate, 5677 acetate and aluminum sulfate tablets for triturate, 5679
ether, 5677 topical solution, 177 Calf thymus DNA, 5679
methacrylate, 5677 acetate tablets, 629 dil-Camphene, 5679
palmitostearate, 5230 ascorbate, 630 Camphor, 660
stearate, 5231 carbonate, 630, 5678 spirit, 660
n-Butyl chloride, 5677, 5682 carbonate, alumina, and magnesia d-10-Camphorsulfonic acid, 5680
tert-Butyl methyl ether, 5677 chewable tablets, 152 dl-10-Camphorsulfonic acid, 5680
n-Butylamine, 5677, 5712 carbonate, alumina, magnesia, and Canada balsam, 5680
tert-Butylamine, 5677, 5712 simethicone chewable tablets, 153 Candelilla wax, 5244
4-(Butylamino)benzoic acid, 5678 carbonate, alumina, and magnesia oral Candesartan cilexetil, 661
Butylated suspension, 151 and hydrochlorothiazide tablets, 664
hydroxyanisole, 5232 carbonate, chelometric standard, 5678 tablets, 662
n-Butylboronic acid, 5678 carbonate lozenges, 632 Canola oil, 5244
tert-Butyldimethylchlorosilane in N-methyl-N- carbonate, magnesia, and simethicone Capecitabine, 667
tert-butyldimethylsilyltrifluoroacetamide, (1 chewable tablets, 636 tablets, 668
in 100), 5678 carbonate and magnesia chewable tablets, Capillary electrophoresis (1053), 6973
Butylene glycol, 5234 635 Capreomycin
Butylparaben, 5236, 5678 carbonate oral suspension, 633 for injection, 671
4-tert-Butylphenol, 5678 carbonate tablets, 634 sulfate, 670
t-Butylthiol, 5678 caseinate, 5679 Capric acid, 5680
Butyraldehyde, 5678 chloride, 639, 5679 Caprylic acid, 5245
Butyric acid, 5678 chloride, anhydrous, 5669, 5679 Caprylocaproyl polyoxylglycerides, 5246
Butyrolactone, 5678 chloride injection, 639 Capsaicin, 672
Butyrophenone, 5678 chloride TS, 5752 Capsicum, 673
citrate, 640, 5679 oleoresin, 674
citrate tablets, 4493 tincture, 676
glubionate syrup, 641
gluceptate, 641
Cc gluceptate injection, 642
gluconate, 642
gluconate injection, 645
Capsules
C13 gluconate tablets, 646 Acebutolol hydrochloride, 30
for oral solution, urea, 706 glycerophosphate, 4494 Acetaminophen, 36
urea, 705 hydroxide, 647, 5679 Containing at least three of the
C14 hydroxide topical solution, 647 following—acetaminophen and (salts of)
capsules, urea, 707 hydroxide TS, 5752 chlorpheniramine, dextromethorphan,
Cabergoline, 609 lactate, 648, 5679 and pseudoephedrine, 45
tablets, 610 lactate tablets, 648 Acetaminophen and codeine phosphate,
Cadmium lactobionate, 649 55
acetate, 5678 Leucovorin comounded oral solution, 2361 Acitretin, 78
nitrate, 5678 levulinate, 650 Acyclovir, 80
Caffeine, 611 levulinate injection, 651 Altretamine, 147
acetaminophen and aspirin tablets, 42 and magnesium carbonates oral Aluminum hydroxide gel, dried, 172
and acetaminophen tablets, 44 suspension, 637 Amantadine hydrochloride, 195
aspirin and dihydrocodeine bitartrate and magnesium carbonates tablets, 638 Aminobenzoate potassium, 211
capsules, 378 nitrate, 5679 Amlodipine and benazepril hydrochloride,
butalbital, and acetaminophen capsules, pantothenate, 651 251
596 pantothenate assay (91), 6041 Amoxicillin, 276
butalbital, and acetaminophen tablets, 597 pantothenate, dextro, 5679 Ampicillin, 298
butalbital, and aspirin capsules, 599 pantothenate, racemic, 653 Anagrelide, 310
butalbital, aspirin, and codeine phosphate pantothenate tablets, 652 Aprepitant, 340
capsules, 601 phosphate, anhydrous dibasic, 655 Arginine, 4434
butalbital, and aspirin tablets, 600 phosphate tablets, dibasic, 657 Aspirin, 366
citrate injection, 612 phosphate, tribasic, 5237 Aspirin, caffeine, and dihydrocodeine
citrate oral solution, 613 phosphate dihydrate, dibasic, 654 bitartrate, 378
and ergotamine tartrate suppositories, polycarbophil, 657 Aspirin delayed-release, 366
1561 propionate, 5238 Atomoxetine, 390
and ergotamine tartrate tablets, 1562 saccharate, 658 Azithromycin, 424
Combined Index to USP 41 and NF 36 Capsu-Capsu 1-9

Capsules (continued) Doxycycline extended-release, 1418 Loperamide hydrochloride, 2447

Balsalazide disodium, 447 Doxycycline hyclate, 1429 Loracarbef, 2461
Benzonatate, 489 Doxycycline hyclate delayed-release, 1431 Loxapine, 2487
Beta carotene, 499 Dronabinol, 1440 Lutein, 4744
Biotin, 529 Duloxetine delayed-release, 1450 Magnesium oxide, 2513
Borage seed oil, 4489 Echinacea species, 4595 Meclofenamate sodium, 2546
Bromocriptine mesylate, 552 Echinacea species dry extract, 4590 Mefenamic acid, 2550
Butalbital, acetaminophen, and caffeine, Efavirenz, 1476 Menaquinone-7, 4762
596 Eleuthero root and rhizome dry extract, Mesalamine extended-release, 2596
Butalbital, aspirin, and caffeine, 599 4600 Methacycline hydrochloride, 2626
Butalbital, aspirin, caffeine, and codeine Eleuthero root and rhizome powder, 4604 Methoxsalen, 2655
phosphate, 601 Ephedrine sulfate, 1527 Methsuximide, 2660
Calcifediol, 616 Ergocalciferol, 1548 Methyltestosterone, 2696
Calcium L-5-methyltetrahydrofolate, 4498 Ergoloid mesylates, 1551 Metronidazole, 2722
C 14, urea, 707 Erythromycin delayed-release, 1566 Metyrosine, 2731
Castor oil, 739 Erythromycin estolate, 1573 Mexiletine hydrochloride, 2732
Cat’s claw, 4510 Esomeprazole magnesium delayed-release, Milk thistle, 4775
Cefaclor, 743 1593 Minerals, 4778
Cefadroxil, 750 Ethchlorvynol, 1637 Minocycline hydrochloride, 2753
Cefdinir, 764 Ethosuximide, 1644 Morphine sulfate extended-release, 2810
Cephalexin, 834 Etodolac, 1655 Mycophenolate mofetil, 2829
Cephradine, 843 Etoposide, 1662 Nafcillin sodium, 2849
Chloral hydrate, 860 Evening primrose oil, 4606 Nifedipine, 2937
Chloramphenicol, 863 Fenofibrate, 1697 Nitrofurantoin, 2947
Chlordiazepoxide hydrochloride, 875 Fenoprofen calcium, 1705 Nizatidine, 2962
Chlordiazepoxide hydrochloride and Ferrous gluconate, 1716 Nortriptyline hydrochloride, 2985
clidinium bromide, 877 Fexofenadine hydrochloride, 1727 Oil- and water-soluble vitamins with
Chlorpheniramine maleate extended- Fish oil containing omega-3 acids, 4620 minerals, 5022
release, 901 Fish oil containing omega-3 acids, delayed- Olanzapine and fluoxetine, 3005
Chlorpheniramine maleate and release, 4622 Oleovitamin A and D, 3010
pseudoephedrine hydrochloride Flax seed oil, 4624 Omega-3 ethyl esters, 3019
extended-release, 903 Flucytosine, 1767 Omeprazole delayed-release, 3023
Cholecalciferol, 916 Fluoxetine, 1803 Orlistat, 3045
Clindamycin hydrochloride, 993 Fluoxetine delayed-release, 1804 Oseltamivir phosphate, 3057
Clofazimine, 1012 Flurazepam hydrochloride, 1822 Oxacillin sodium, 3060
Clofibrate, 1014 Flutamide, 1827 Oxazepam, 3078
Clomipramine hydrochloride, 1019 Fluvastatin, 1860 Oxycodone and acetaminophen, 3107
Cloxacillin sodium, 1050 Gabapentin, 1897 Oxytetracycline hydrochloride, 3128
Cod liver oil, 4551 Galantamine extended-release, 1912 Oxytetracycline and nystatin, 3125
Crypthecodinium cohnii oil, 4557 Gemfibrozil, 1934 Pancrelipase, 3149
Curcuminoids, 4561 Ginger, 4656 Pancrelipase delayed-release, 3150
Cyanocobalamin Co 57, 1055 Ginkgo, 4663 Paricalcitol, 3169
Cyanocobalamin Co 58, 1056 Ginseng, American, 4426 Paromomycin sulfate, 3173
Cyclobenzaprine hydrochloride extended- Griseofulvin, 1997 Penicillamine, 3191
release capsules, 1119 Guaifenesin, 2002 Phendimetrazine tartrate, 3254
Cycloserine, 1129 Guaifenesin and pseudoephedrine Phenoxybenzamine hydrochloride, 3266
Cyclosporine, 1130 hydrochloride, 2005 Phensuximide, 3268
Danazol, 1151 Guaifenesin, pseudoephedrine Phentermine hydrochloride, 3269
Dantrolene sodium, 1154 hydrochloride, and dextromethorphan Phenytoin sodium, extended, 3290
Demeclocycline hydrochloride, 1167 hydrobromide, 2006 Piroxicam, 3337
Dextroamphetamine sulfate, 1228 Hydrochlorothiazide, 2049 Potassium chloride extended-release, 3361
Diazepam, 1243 Hydroxyurea, 2090 Potassium perchlorate, 3385
Diazepam extended-release, 1244 Hydroxyzine pamoate, 2097 Prazosin hydrochloride, 3406
Diazoxide, 1247 Imipramine pamoate, 2131 Procainamide hydrochloride, 3440
Dicloxacillin sodium, 1266 Indomethacin, 2151 Procarbazine hydrochloride, 3447
Dicyclomine hydrochloride, 1268 Indomethacin extended-release, 2153 Propranolol hydrochloride extended-
Didanosine delayed-release, 1273 Sodium iodide | 123, 2192 release, 3493
Digitalis, 1288 Sodium iodide | 131, 2196 Pseudoephedrine hydrochloride extended-
Dihydrotachysterol, 1298 lsometheptene mucate, release, 3507
Diltiazem hydrochloride extended-release, dichloralphenazone, and Pygeum, 4809
1305 acetaminophen, 2251 Quinidine sulfate, 3552
Diphenhydramine hydrochloride, 1328 Isosorbide dinitrate extended-release, 2269 Quinine sulfate, 3558
Diphenhydramine hydrochloride and Isotretinoin, 2282 Ramipril, 3574
ibuprofen, 1333 Isradipine, 2288 Rhodiola rosea, 4831
Diphenhydramine and pseudoephedrine, Kanamycin sulfate, 2306 Ribavirin, 3597
1337: Ketoprofen, 2312 Rifabutin, 3606
Disopyramide phosphate, 1351 Ketoprofen extended-release, 2314 Rifampin, 3609
Disopyramide phosphate extended-release, Krill oil, 4721 Rifampin and isoniazid, 3611
1351 Krill oil delayed-release, 4725 Ritonavir, 3648
Divalproex sodium delayed-release, 1354 Lansoprazole delayed-release, 2350 Rivastigmine tartrate, 3660
Docusate calcium, 1376 Levodopa, 2393 Propafenone hydrochloride extended-
Docusate potassium, 1378 Lincomycin hydrochloride, 2422 release, 3477
Docusate sodium, 1379 Alpha lipoic acid, 4741 St. John’s wort flowering top dry extract,
Doxepin hydrochloride, 1406 Lithium carbonate, 2438 4847
Doxycycline, 1416 Lomustine, 2445 Salsalate, 3705
-10 Capsu-Cefme Combined Index to USP 41 and NF 36

Capsules (continued) Carbamazepine, 683 Carmellose, 5270

Saquinavir, 3708 oral suspension, 685 Carmine, 5680
Saw palmetto, 4862 tablets, 686 Carmustine, 720
Schizochytrium oil, 4872 extended-release tablets, 689 for injection, 722
Secobarbital sodium, 3732 Carbamide peroxide, 690 Carprofen, 723
Selegiline hydrochloride, 3735 topical solution, 690 tablets, 724
Simethicone, 3761 Carbazole sulfate, 5680 Carrageenan, 5270
Soy isoflavones, 4879 Carbenicillin Carteolol hydrochloride, 726
Stavudine, 3835 disodium, 691 ophthalmic solution, 727
Sulfinpyrazone, 3884 indanyl sodium, 691 tablets, 728
Tacrine, 3905 indanyl sodium tablets, 692 Carvedilol, 729
Tacrolimus, 3909 for injection, 690 tablets, 731
Tamsulosin hydrochloride, 3925 Carbidopa, 692 (R)-(-)-Carvone, 5680
Temazepam, 3967 and levodopa extended-release tablets, Casanthranol, 733
Temozolomide, 3968 696 Cascara
Terazosin, 3975 and levodopa orally disintegrating tablets, fluidextract, aromatic, 738
Tetracycline hydrochloride, 4017 701 sagrada, 734
Tetracycline hydrochloride and nystatin, and levodopa tablets, 693 sagrada extract, 736
4025 Carbinoxamine maleate, 703 sagrada fluidextract, 738
Thalidomide, 4029 pseudoephedrine hydrochloride, and tablets, 737
Theophylline, 4032 dextromethorphan hydrobromide oral Casein, 5680
Theophylline extended-release, 4033 solution, 3511 hammersten, 5680
Theophylline and guaifenesin, 4042 tablets, 703 Castor oil, 739
Thiothixene, 4069 Carbol-fuchsin topical solution, 704 aromatic, 741
Tienchi ginseng root and rhizome dry Carbomer capsules, 739
extract, 4910 934, 5248 emulsion, 740
Tienchi ginseng root and rhizome powder, 934P, 5249 hydrogenated, 5272
4905 940, 5251 polyoxyl 35, 5515
Tolmetin sodium, 4132 941, 5252 Catechol, 5680
Topiramate, 4141 1342, 5253 Cation-exchange resin, 5680
Triamterene, 4197 copolymer, 5255 carboxylate (sodium form) (50- to 100-
Triamterene and hydrochlorothiazide, 4198 homopolymer, 5257 mesh), 5680
Trientine hydrochloride, 4211 interpolymer, 5260 polystyrene, 5680, 5719
Trihexyphenidy! hydrochloride extended- Carbon styrene-divinylbenzene, 5681
release, 4218 C 13 for oral solution, urea, 706 styrene-divinylbenzene, strongly acidic,
Trimethobenzamide hydrochloride, 4224 C 13, urea, 705 5680
Ubidecarenone, 4920 C 14 capsules, urea, 707 sulfonic acid, 5681, 5735
Ursodiol, 4257 dioxide, 704 Cat’s claw, 4506
Valproic acid, 4273 dioxide detector tube, 5680 capsules, 4510
Vancomycin hydrochloride, 4286 disulfide, chromatographic, 5680 extract, powdered, 4509
Venlafaxine hydrochloride extended- disulfide, CS, 5680 powdered, 4507
release, 4295 monoxide detector tube, 5680 tablets, 4512
Verapamil hydrochloride extended-release, tetrachloride, 5680 Cedar oil, 5681
4302 Carbonates Cefaclor, 741
Vinpocetine, 4933 calcium and magnesium, oral suspension, capsules, 743
Vitamin A, 4328 637 chewable tablets, 746
Vitamin E, 4333 calcium and magnesium, tablets, 638 for oral suspension, 744
Vitamins with minerals, oil-soluble, 4951 Carboplatin, 707 extended-release tablets, 746
Vitamins with minerals, oil- and water- for injection, 709 Cefadroxil, 748
soluble, 5022 Carboprost capsules, 750
Vitamins with minerals, water-soluble, tromethamine, 710 for oral suspension, 752
5109 tromethamine injection, 711 tablets, 753
Vitamins, oil-soluble, 4935 Carboxylate (sodium form) cation-exchange Cefamandole nafate, 754
Vitamins, oil- and water-soluble, 4976 resin (50- to 100-mesh), 5680 for injection, 755
Vitamins, water-soluble, 5086 Carboxymethoxylamine hemihydrochloride, Cefazolin, 756
Zaleplon, 4364 5680 injection, 758
Zidovudine, 4368 Carboxymethylcellulose for injection, 759
Ziprasidone, 4389 calcium, 5262 ophthalmic solution, 760
Zonisamide, 4410 sodium, 712 sodium, 760
sodium 12, 5265 Cefdinir, 763
sodium, low-substituted, 5263 capsules, 764
sodium and microcrystalline cellulose, for oral suspension, 767
Capsules—dissolution testing and related 5278 Cefepime
quality attributes (1094), 7198 sodium paste, 713 hydrochloride, 772
Captopril, 677 sodium tablets, 714 for injection, 770
and hydrochlorothiazide tablets, 680 Carboxymethylcellulose sodium Cefixime, 774
oral solution, 678 enzymatically-hydrolyzed, 5266 for oral suspension, 775
oral suspension, 679 Cardamom tablets, 775
tablets, 679 oil, 5269 Cefmenoxime
Caramel, 5247 seed, 5269 hydrochloride, 777
Caraway, 5247 tincture, compound, 5269 for injection, 776
oil, 5248 Carisoprodol, 714 Cefmetazole, 778
Carbachol, 682 aspirin and codeine phosphate tablets, 718 injection, 778
intraocular solution, 682 and aspirin tablets, 716 for injection, 779
ophthalmic solution, 683 tablets, 715 sodium, 779
Combined Index to USP 41 and NF 36 Cefon-Chlor 1-11

Cefonicid Cephalexin, 833 TS, 5752

for injection, 780 capsules, 834 Chlorambucil, 861
sodium, 781 hydrochloride, 836 tablets, 861
Cefoperazone for oral suspension, 834 Chloramine T, 5682
injection, 781 tablets, 835 Chloramphenicol, 862
for injection, 782 tablets for oral suspension, 836 capsules, 863
sodium, 782 Cephalothin cream, 863
Ceforanide, 783 injection, 837 and hydrocortisone acetate for ophthalmic
for injection, 784 for injection, 838 suspension, 866
Cefotaxime sodium, 838 injection, 863
injection, 785 Cephapirin ophthalmic ointment, 864
for injection, 786 benzathine, 840 ophthalmic solution, 864
sodium, 787 benzathine intramammary infusion, 841 for ophthalmic solution, 865
Cefotetan, 790 for injection, 839 otic solution, 865
disodium, 792 sodium, 842 palmitate, 868
injection, 791 sodium intramammary infusion, 842 palmitate oral suspension, 868
for injection, 791 Cephradine, 843 and polymyxin B sulfate ophthalmic
Cefotiam capsules, 843 ointment, 867
hydrochloride, 793 for injection, 844 sodium succinate, 869
for injection, 794 for oral suspension, 845 sodium succinate for injection, 870
Cefoxitin tablets, 845 oral solution, 865
injection, 796 Ceric tablets, 866
for injection, 797 ammonium nitrate, 5681 Chlordiazepoxide, 870
sodium, 795 ammonium nitrate TS, 5752 and amitriptyline hydrochloride tablets,
Cefpiramide, 798 ammonium nitrate, twentieth-normal (0.05 872
for injection, 799 N), 5763 hydrochloride, 874
Cefpodoxime proxetil, 800 ammonium sulfate, 5681 hydrochloride capsules, 875
for oral suspension, 801 sulfate, 5681 hydrochloride and clidinium bromide
tablets, 802 sulfate, tenth-normal (0.1 N), 5763 capsules, 877
Cefprozil, 802 Cesium chloride, 5682 tablets, 871
for oral suspension, 806 Cetirizine hydrochloride, 846 Chlorhexidine
tablets, 807 and pseudoephedrine hydrochloride acetate, 879
Ceftazidime, 808 extended-release tablets, 853 acetate topical solution, 880
injection, 809 oral solution, 848 gluconate oral rinse, 882
for injection, 810 tablets, 849 gluconate solution, 881
Ceftiofur orally disintegrating tablets, 851 gluconate topical solution, 884
hydrochloride, 812 Cetostearyl alcohol, 5282 hydrochloride, 885
sodium, 814 Cetrimide, 5682 Chlorhexidine gluconate
Ceftizoxime Cetrimonium bromide, 5284 topical gel, 4415
injection, 817 Cetyl Chloride
for injection, 818 alcohol, 5285 cobaltous, TS, 5752
sodium, 816 esters wax, 5286 ferric, TS, 5753
Ceftriaxone palmitate, 5288 ferrous tetrahydrate, 5696
injection, 818 Cetylpyridinium chloride, 857 gold, 5698
for injection, 819 lozenges, 858 gold, TS, 5754
sodium, 821 topical solution, 858 platinic, 5719
Cefuroxime Cetyltrimethylammonium bromide, 5682, platinic, TS, 5758
axetil, 823 5699 in reagents, 5661
axetil for oral suspension, 825 Cetyltrimethylammonium chloride, 25 stannous, 5594
axetil tablets, 826 percent in water, 5682 and sulfate (221), 6139
injection, 822 Chamomile, 4519 Chlorine, 5682
for injection, 823 Characterization of crystalline and partially detector tube, 5682
sodium, 828 crystalline solids by X-ray powder TS, 5752
Celecoxib, 828 diffraction (XRPD) (941), 6692 m-Chloroacetanilide, 5682
Cellaburate, 5275 Characterization of crystalline solids by p-Chloroacetanilide, 5682
Cellacefate, 5276 microcalorimetry and solution calorimetry 1-Chloroadamantane, 5682
Cellular and tissue-based products (1046), (696), 6445 2-Chloro-4-aminobenzoic acid, 5682
6871 Charcoal 5-Chloro-2-aminobenzophenone, 5682
Cellulose activated, 859, 5665, 5682 3-Chloroaniline, 5682
acetate, 5281 Chaste tree, 4521 p-Chloroaniline, 5682
chromatographic, 5681 powdered, 4523 Chlorobenzene, 5682
microcrystalline, 5277, 5681 powdered, extract, 4524 4-Chlorobenzoic acid, 5682
microcrystalline and Chemometrics (1039), 6831 m-Chlorobenzoic acid, 5682
carboxymethylcellulose sodium, 5278 Chenodeoxycholic acid, 5682 4-Chlorobenzophenone, 5682
mixture, chromatographic, 5681 Cherry 1-Chlorobutane, 5682
oxidized, 830 juice, 5289 Chlorobutanol, 5294
oxidized regenerated, 830 syrup, 5289 Chlorocresol, 5295
powdered, 5280 Chia seed 2-Chloroethanol, 5682
silicified microcrystalline, 5279 oil, 4530 2-Chloroethylamine monohydrochloride,
sodium phosphate, 831 Chinese salvia, 4531 5682
sodium phosphate for oral suspension, 832 powdered, 4533 Chloroform, 5683
Centella asiatica, 4513 Chitosan, 5290 alcohol-free, 5683
extract, powdered, 4516 Chloral hydrate, 860 methyl, 5683
powdered, 4515 capsules, 860 Chlorogenic acid, 5683
triterpenes, 4518 oral solution, 860
I-12 Chlor-Clenb Combined Index to USP 41 and NF 36

Chloromethylated polystyrene-divinylbenzene hydrochloride soluble powder, 912 sodium, 931

anion-exchange resin, 5683 hydrochloride tablets, 912 Cilostazol, 932
1-Chloronaphthalene, 5683 and sulfamethazine bisulfates soluble tablets, 934
4-Chloro-1-naphthol, 5683 powder, 911 Cimetidine, 935
2-Chloronicotinic acid, 5683 Chlorthalidone, 912 hydrochloride, 938
2-Chloro-4-nitroaniline, 99%, 5683 and atenolol tablets, 387 injection, 935
Chlorophyllin copper complex sodium, 887 and clonidine hydrochloride tablets, 1027 in sodium chloride injection, 937
Chloroplatinic acid, 5683 tablets, 913 tablets, 936
Chloroprocaine hydrochloride, 889 Chlorzoxazone, 914 Cinchonidine, 5684
injection, 889 tablets, 915 Cinchonine, 5684
Chloroquine, 890 Chocolate, 5295 Cinnamomum cassia
hydrochloride injection, 890 syrup, 5296 twig, 4546
phosphate, 891 Cholecalciferol, 915 twig powder, 4548
phosphate oral suspension, 893 capsules, 916 Ciprofloxacin, 939
phosphate tablets, 893 solution, 917 and dexamethasone otic suspension, 951
5-Chlorosalicylic acid, 5683 Cholestane, 5683 extended-release tablets, 946
Chlorothiazide, 894 Cholestanol, 5683 hydrochloride, 941
and methyldopa tablets, 2669 Cholesterol, 5296, 5683 injection, 942
and reserpine tablets, 3593 Cholesteryl ophthalmic ointment, 944
sodium for injection, 897 benzoate, 5683 ophthalmic solution, 944
oral suspension, 895 n-heptylate, 5683 for oral suspension, 949
tablets, 896 Cholestyramine tablets, 945
1-Chloro-2,2,2- resin, 918 Cisapride, 953
trifluoroethylchlorodifluoromethy! ether, for oral suspension, 919 Cisapride compounded, veterinary
5683 Choline injection, 953
Chlorotrimethylsilane, 5683, 5741 bitartrate, 4535 oral suspension, 954
Chloroxylenol, 898 chloride, 4537, 5683 Cisatracurium besylate, 955
Chlorpheniramine Chondroitin sulfate sodium, 4539 injection, 957
dextromethorphan, pseudoephedrine, and glucosamine tablets, 4667 Cisplatin, 959
(salts of), and acetaminophen, capsules glucosamine, and methylsulfonylmethane for injection, 961
containing at least three of the tablets, 4674 Citalopram
following, 45 shark, 4543 hydrobromide, 966
dextromethorphan, pseudoephedrine (salts tablets, 4541 oral solution, 963
of), and acetaminophen, oral powder Chromate, sodium, Cr 51 injection, 921 tablets, 964
containing at least three of the Chromatographic Citrate
following, 47 columns, 5774 cupric TS, alkaline, 5750, 5752
dextromethorphan, pseudoephedrine (salts fuller’s earth, 5683 cupric TS 2, alkaline, 5750, 5752
of), and acetaminophen, oral solution n-heptane, 5683 Citric acid, 5684
containing at least three of the magnesium oxide, 5683 anhydrous, 968, 5684
following, 49 reagents, 5683 and magnesium carbonate for oral
dextromethorphan, pseudoephedrine (salts silica gel, 5683 solution, 2502
of), and acetaminophen, tablets silica gel mixture, 5684 magnesium carbonate, and potassium
containing at least three of the siliceous earth, 5684 citrate for oral solution, 2503
following, 51 siliceous earth, silanized, 5684 magnesium oxide, and sodium carbonate
maleate, 900 solvent hexane, 5684 irrigation, 972
maleate extended-release capsules, 901 Chromatography (621), 6363 monohydrate, 970
maleate injection, 902 Chromatography, ion (1065), 7075 and potassium citrate oral solution, 3375
maleate, penicillin G procaine, Chromic chloride, 920 and potassium and sodium bicarbonates
dihydrostreptomycin sulfate, and injection, 920 effervescent tablets for oral solution,
dexamethasone injectable suspension, Chromium 3357
3207 Cr 51 edetate injection, 922 and sodium citrate oral solution, 3787
maleate and pseudoephedrine Cr 51 injection, sodium chromate, 921 Citrulline, 4550
hydrochloride extended-release capsules, picolinate, 4545 Cladribine, 973
903 picolinate tablets, 4546 injection, 974
maleate and pseudoephedrine potassium sulfate dodecahydrate, 5684 Clarithromycin, 975
hydrochloride oral solution, 904 trioxide, 5684 for oral suspension, 977
maleate oral solution, 902 Chromogenic tablets, 978
maleate tablets, 903 substrate for amidolytic test, 5684 extended-release tablets, 980
maleate, acetaminophen, and Chromotrope 2R, 5684 Clavulanate
dextromethorphan hydrobromide Chromotropic acid, 5684, 5729 potassium, 983
tablets, 53 disodium salt, 5684 potassium and amoxicillin for oral
Chlorpromazine, 905 TS, 5752 suspension, 284
hydrochloride, 906 Chymotrypsin, 923 potassium and amoxicillin tablets, 285
hydrochloride injection, 907 for ophthalmic solution, 924 Clavulanic acid
hydrochloride oral concentrate, 906 Ciclopirox, 925 and amoxicillin extended-release tablets,
hydrochloride syrup, 908 olamine, 926 286
hydrochloride tablets, 908 olamine cream, 927 Clavulanic acid
suppositories, 906 olamine topical suspension, 928 and ticarcillin injection, 4081
Chlorpropamide, 909 topical solution, 926 and ticarcillin for injection, 4082
tablets, 909 Cidofovir, 928 Cleaning glass apparatus (1051), 6960
Chlortetracycline injection, 930 Clemastine fumarate, 986
bisulfate, 910 Cilastatin tablets, 987
hydrochloride, 911, 5683 and imipenem for injectable suspension, Clenbuterol hydrochloride, 989
hydrochloride ointment, 911 2125
hydrochloride ophthalmic ointment, 912 and imipenem for injection, 2124
Combined Index to USP 41 and NF 36 Clidi-Cotto 1-13

Clidinium bromide, 989 Cloxacillin Colchicine, 1069

and chlordiazepoxide hydrochloride benzathine, 1047 injection, 1070
capsules, 877 benzathine intramammary infusion, 1048 and probenecid tablets, 3437
Clindamycin sodium, 1049 tablets, 1070
hydrochloride, 992 sodium capsules, 1050 Colestipol hydrochloride, 1071
hydrochloride capsules, 993 sodium intramammary infusion, 1051 for oral suspension, 1072
hydrochloride oral solution, 994 sodium for oral solution, 1052 tablets, 1072
injection, 990 Clozapine, 1052 Colistimethate
for injection, 991 tablets, 1054 for injection, 1074
palmitate hydrochloride, 994 Co sodium, 1073
palmitate hydrochloride for oral solution, 57 capsules, cyanocobalamin, 1055 Colistin
995 57 oral solution, cyanocobalamin, 1056 and neomycin sulfates and hydrocortisone
phosphate, 996 58 capsules, cyanocobalamin, 1056 acetate otic suspension, 1075
phosphate gel, 998 Coal tar, 1055 sulfate, 1074
phosphate topical solution, 999 ointment, 1055 sulfate for oral suspension, 1075
phosphate topical suspension, 999 topical solution, 1055 Collagen, 5684
phosphate vaginal cream, 998 Cobalt rat tail, 5684
phosphate vaginal inserts, 1000 chloride, 5684 Collagenase, 5684
Clioquinol, 1001 Co 57 capsules, cyanocobalamin, 1055 Collagenase | (89.1), 6029
cream, 1002 Co 57 oral solution, cyanocobalamin, 1056 Collagenase Il (89.2), 6033
and hydrocortisone cream, 1004 Co 58 capsules, cyanocobalamin, 1056 Collodion, 1076
and hydrocortisone ointment, 1005 nitrate, 5684 flexible, 1076
ointment, 1002 platinum, TS, 5758 Colloidal oatmeal, 1077
topical powder, compound, 1003 uranyl acetate TS, 5752 Color
Clobetasol propionate, 1006 Cobaltous and achromicity (631), 6375
cream, 1007 acetate, 5684 instrumental measurement (1061), 7040
ointment, 1008 chloride, 5684 Colorimetric solutions (CS), 5749
topical solution, 1008 chloride CS, 5749 Compactin, 5684
Clocortolone pivalate, 1009 chloride TS, 5752 Completeness of solution (641), 6376
cream, 1010 Cocaine, 1057 Compound cardamom tincture, 5269
Clofazimine, 1011 hydrochloride, 1058 Compounded topical gel
capsules, 1012 hydrochloride tablets for topical solution, ondansetron, 3038
Clofibrate, 1013 1058 Congealing temperature (651), 6382
capsules, 1014 and tetracaine hydrochlorides and Congo red, 5684, 5745
Clomiphene citrate, 1015 epinephrine topical solution, 1059 TS, 5752
tablets, 1016 Cocoa butter, 5298 Constitution and bylaws, xxix
Clomipramine compounded Coconut Construct human fibroblasts in bilayer
oral suspension, veterinary, 1017 oil, 5299 synthetic scaffold, 1077
Clomipramine hydrochloride, 1018 oil, hydrogenated, 5299 Construct human fibroblasts in polyglactin
capsules, 1019 Codeine, 1062 scaffold, 1082
Clonazepam, 1020 phosphate, 1063 Container content for injections (697), 6449
oral suspension, 1021 phosphate and acetaminophen capsules, Containers
tablets, 1022 55 glass (660), 6390
orally disintegrating tablets, 1023 phosphate and acetaminophen oral performance testing (671), 6436
Clonidine, 1024 solution, 56 Container specifications for capsules and
hydrochloride, 1025 phosphate and acetaminophen oral tablets, 5781
hydrochloride and chlorthalidone tablets, suspension, 57 Coomassie
1027 phosphate and acetaminophen tablets, 59 blue G-250, 5685
hydrochloride tablets, 1026 phosphate, aspirin, alumina, and magnesia brilliant blue R-250, 5685
transdermal system, 1028 tablets, 380 Copovidone, 5300
Clopidogrel phosphate and aspirin tablets, 379 Copper, 5685
bisulfate, 1032 phosphate and bromodiphenhydramine gluconate, 1092
tablets, 1034 hydrochloride oral solution, 556 Copper sulfate pentahydrate, 5685
Clopidogrel compounded phosphate, butalbital, aspirin, and caffeine Coriander oil, 5303
oral suspension, 1033 capsules, 601 Corn
Cloprostenol phosphate, carisoprodol, and aspirin oil, 5303
injection, 1036 tablets, 718 starch, 5595
sodium, 1035 phosphate and guaifenesin oral solution, syrup, 5304
Clorazepate dipotassium, 1037 2004 high fructose syrup, 5307
tablets, 1038 phosphate injection, 1063 syrup solids, 5310
Clorsulon, 1039 phosphate tablets, 1064 Corticotropin
and ivermectin injection, 2297 phosphate and promethazine and injection, 1094
Clotrimazole, 1040 phenylephrine hydrochloride oral for injection, 1095
and betamethasone dipropionate cream, solution, 3473 injection, repository, 1097
1046 phosphate oral solution, 1064 Cortisone, 5685
cream, 1041 sulfate, 1065 acetate, 1099
lotion, 1042 sulfate oral solution, 1066 acetate injectable suspension, 1100
lozenges, 1043 sulfate tablets, 1068 acetate tablets, 1100
topical solution, 1044 and terpin hydrate oral solution, 3999 Cosyntropin, 1101
vaginal inserts, 1045 Cod liver oil, 1060 Cotton
Clove oil, 5298 capsules, 4551 absorbent, 5685
Clover, red, 4811 Coenzyme Q9, 5684 purified, 1103
extract, powdered, 4817 Cohosh Cotton (691), 6443
powdered, 4815 black fluidextract, 4480 Cottonseed oil, 5312
tablets, 4819 hydrogenated, 5313
 4518 Centella / Dietary Supplements USP 41

e USP REFERENCE STANDARDS (11) COMPOSITION

USP Asiaticoside RS e CONTENT OF TRITERPENE DERIVATIVES
USP Powdered Centella asiatica Extract RS Solution A: Dilute 3 mL of phosphoric acid with water
to 1000 mL, mix, filter, and degas.
Solution B: Acetonitrile
Mobile phase: See the gradient table below.

Centella asiatica Triterpenes Time Solution A Solution B

(min) (%) (%)
DEFINITION 0 78 22
Centella asiatica Triterpenes is a fraction enriched in Centella 65 45 55
asiatica triterpenes derivatives. It is prepared from Centella
66 5 95
asiatica Extract using suitable solvents or other means. It
contains NLT 90.0% of triterpene derivatives, calculated 75 , 95
on the anhydrous basis, as the sum of two or more of the 76 78 22,
following: madecassoside, asiaticoside B, asiaticoside, 85 78 22
madecassic acid, terminolic acid, and asiatic acid.
Standard solution A: 0.2 mg/mL of USP Asiaticoside RS
IDENTIFICATION in methanol
© A. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST Standard solution B: Sonicate a portion of USP Pow-
Standard solution A: 0.5 mg/mL of USP Asiaticoside RS dered Centella asiatica Extract RS in methanol to obtain
in methanol a solution with a concentration of about 5.0 mg/mL.
Standard solution B: 10 mg/mL of USP Powdered Before injection, pass through a membrane filter of
Centella asiatica Extract RS in methanol. Sonicate for 0.45-um or finer pore size, discarding the first few mL
about 10 min, centrifuge, and use the supernatant. of the filtrate.
Sample solution: Transfer an amount of Centella asiat- Sample solution: About 1.0 mg/mL of Centella asiatica
ica Triterpenes, equivalent to about 5 mg of triterpene Triterpenes in methanol; sonicate if necessary. Before in-
derivatives, to a centrifuge tube. Add 5 mL of methanol, jection, pass through a membrane filter of 0.45-4um or
sonicate for 10 min, centrifuge, and use the finer pore size, discarding the first few mL of the
supernatant. filtrate.
Adsorbent: Chromatographic silica gel with an average Chromatographic system
particle size of 10-15 jum (TLC plates) or with an aver- (See Chromatography (621), System Suitability.)
age particle size of 5 um (HPTLC plates) Mode: LC
Application volume: 10 ul (TLC plates) or 4 wL (HPTLC Detector: UV 200 nm
plates) Column: 4.6-mm x 25-cm; 5-um packing L1
Developing solvent system: A mixture of methylene Flow rate: 1.0 mL/min
chloride, methanol, and water (14:6:1) Injection size: 10 uL
Spray reagent: A solution of 10% sulfuric acid in meth- System suitability
anol. [NoTe—Prepare fresh.] Samples: Standard solution A and Standard solution B
Analysis Suitability requirements
Samples: Standard solution A, Standard solution B, and Chromatogram similarity: The chromatogram from
Sample solution Standard solution B is similar to the reference chro-
Apply the samples as bands to a suitable thin-layer matogram provided with the lot of USP Powdered
chromatographic plate (see Chromatography (621)). Centella asiatica Extract RS being used.
Use a saturated chamber. Develop the chromato- Tailing factor: Between 0.8 and 2.0 for the asiatico-
grams until the solvent front has moved up about side peak, Standard solution A
three-fourths of the plate. Remove the plate from the Resolution: NLT 1.5 between the madecassic acid
chamber, dry, spray with Spray reagent, heat for 3 and terminolic acid peaks, Standard solution B
min at 120°, and examine under visible light. Relative standard deviation: NMT 2.0% determined
Acceptance criteria: The Sample solution chromato- from the asiaticoside peak in repeated injections,
gram exhibits a violet band in the lower third of the Standard solution A
DS Monographs

plate due to asiaticoside, colrsspontel ng in color and Re Analysis

to that in Standard solution A. The Sample solution Samples: Standard solution A, Standard solution B, and
shows additional bands corresponding to some or all of Sample solution. [NoTe—Standard solution A, Standard
the following triterpene derivatives: a violet band due solution B, and the Sample solution are stable for 48 h
to madecassoside at an Rr lower than that of asiatico- at room temperature.]
side, a violet band in the upper third of the plate due Using the chromatograms of Standard solution A, Stan-
to asiatic acid, and a violet band due to madecassic dard solution B, and the reference chromatogram pro-
acid at an Rr lower than that of asiatic acid. Bands de- vided with the lot of USP Powdered Centella asiatica
tected in the Sample solution correspond in position and Extract RS being used, identify the retention times of
color to bands in Standard solution B. Other minor the peaks corresponding to different triterpene deriv-
bands may be observed in the Sample solution and atives. The approximate relative retention times of
Standard solution B. the different triterpene derivatives are provided in the
e B. HPLC IDENTIFICATION TEST: The Sample solution chro- following table.
matogram from the test for Content of Triterpene Deriva-
tives shows a peak at the retention time corresponding to Approximate Relative
that of asiaticoside in Standard solution A. \dentify other Analyte Retention Time
triterpene derivative peaks in the Sample solution by com-
Madecassoside 0.71
parison with the chromatogram of Standard solution B
and the reference chromatogram provided with the lot Asiaticoside B 0.72
of USP Powdered Centella asiatica Extract RS being used. Asiaticoside 1.00
The Sample solution shows additional peaks correspond- Madecassic acid 2.40
ing to some or all of the following: madecassoside and Terminolic acid 2.44
asiaticoside B (these two peaks may co-elute), madecassic Asiatic acid 3.12
acid, terminolic acid, and asiatic acid.
1-14. Counc-Dacti Combined Index to USP 41 and NF 36

Council of experts Neomycin and polymyxin B sulfates and sulfate, 1111, 5685
(2015-2020), xi pramoxine hydrochloride, 2905 sulfate, anhydrous, 5669, 5685
Cr 51 Neomycin sulfate, 2883 sulfate CS, 5749
edetate injection, chromium, 922 Neomycin sulfate and dexamethasone sulfate injection, 1112
injection, sodium chromate, 921 sodium phosphate, 2884 sulfate test paper, 5747
Cranberry Neomycin sulfate and fluocinolone sulfate TS, 5747, 5753
liquid preparation, 4554 acetonide, 2887 tartrate, alkaline, solution (Fehling’s
Neomycin sulfate and flurandrenolide, solution), 5763
2887 tartrate TS, alkaline, 5750, 5753
Neomycin sulfate and hydrocortisone, Cupriethylenediamine hydroxide solution,
2888 1.0 M, 5685
Cream Neomycin sulfate and hydrocortisone Curcuminoids, 4560
Alclometasone dipropionate, 102 acetate, 2889 capsules, 4561
Amcinonide, 197 Neomycin sulfate and methylprednisolone tablets, 4562
Amphotericin B, 291 acetate, 2893 Cyanoacetic acid, 5685
Anthralin, 320 Neomycin sulfate and triamcinolone Cyanocobalamin, 1113
Benzocaine, 471 acetonide, 2907 Co 57 capsules, 1055
Betamethasone, 502 Nystatin, 2989 Co 57 oral solution, 1056
Betamethasone dipropionate, 508 Nystatin, neomycin sulfate, gramicidin, Co 58 capsules, 1056
Betamethasone valerate, 514 and triamcinolone acetonide, 2991 injection, 1114
Butoconazole nitrate, vaginal, 605 Nystatin, neomycin sulfate, thiostrepton, tablets, 1114
Chloramphenicol, 863 and triamcinolone acetonide, 2992 Cyanogen bromide, 5685
Ciclopirox olamine, 927 Nystatin and triamcinolone acetonide, 4-Cyanophenol, 5685
Clindamycin phosphate, vaginal, 998 2994 4-Cyanopyridine, 5685
Clioquinol, 1002 Piroxicam, 3338 Cyclam, 5685
Clioquinol and hydrocortisone, 1004 Pramoxine hydrochloride, 3398 Cyclandelate, 1115
Clobetasol propionate, 1007 Prednicarbate, 3408 Cyclizine hydrochloride, 1116
Clocortolone pivalate, 1010 Prednisolone, 3412 tablets, 1117
Clotrimazole, 1041 Sulfadiazine, silver, 3863 Cyclobenzaprine hydrochloride, 1118
Clotrimazole and betamethasone Sulfa, vaginal, triple, 3850 extended-release capsules, 1119
dipropionate, 1046 Tetracaine hydrochloride, 4009 tablets, 1121
Crotamiton, 1109 Tolnaftate, 4135 1,1-Cyclobutanedicarboxylic acid, 5685
Desoximetasone, 1190 Tretinoin, 4181 a-Cyclodextrin, 5685
Dexamethasone sodium phosphate, 1203 Triamcinolone acetonide, 4187 B-Cyclodextrin, 5685
Dibucaine, 1249 Cyclohexane, 5685
Diflorasone diacetate, 1283 Cyclohexanol, 5685
Dioxybenzone and oxybenzone, 1322 (1,2-Cyclohexylenedinitrilo)tetraacetic acid,
Estradiol, vaginal, 1601 Creatinine, 5313 5685
Estropipate, vaginal, 1626 Cresol, 5314 Cyclohexylmethanol, 5685
Flumethasone pivalate, 1777 red, 5745 Cyclomethicone, 5319
Fluocinolone acetonide, 1784 red-thymol blue TS, 5752 Cyclopentolate hydrochloride, 1122
Fluocinonide, 1786 red TS, 5752 ophthalmic solution, 1123
Fluorometholone, 1797 m-Cresol purple, 5685 Cyclophosphamide, 1123
Fluorouracil, 1801 TS, 5752 for injection, 1126
Flurandrenolide, 1819 Cromolyn sodium, 1104 tablets, 1126
Fluticasone propionate, 1830 inhalation powder, 1104 Cyclopropane, 1127
Gentamicin sulfate, 1937 inhalation solution, 1105 Cycloserine, 1128
Gentian violet, 1944 nasal solution, 1106 capsules, 1129
Halcinonide, 2015 ophthalmic solution, 1107 Cyclosporine, 1129
Hydrocortisone, 2058 Croscarmellose sodium, 5315 capsules, 1130
Hydrocortisone acetate, 2064 Crospovidone, 5316 injection, 1131
Hydrocortisone butyrate, 2067 Crotamiton, 1109 oral solution, 1133
Hydrocortisone valerate, 2073 cream, 1109 Cyclosporine compounded, veterinary
Hydroquinone, 2082 Cryopreservation of cells (1044), 6858 ophthalmic solution, 1134
Lidocaine and prilocaine, 2418 Crypthecodinium cohnii oil, 4555 Cyproheptadine hydrochloride, 1135
Lindane, 2423 capsules, 4557 oral solution, 1136
Mafenide acetate, 2494 Crystallinity (695), 6445 tablets, 1138
Meclocycline sulfosalicylate, 2545 Crystal violet, 5745 Cyromazine, 1138
Methylprednisolone acetate, 2691 TS, 5752, 5756 Cysteine hydrochloride, 1139
Miconazole nitrate, 2738 Cupric injection, 1140
Mometasone furoate, 2787 acetate, 5685 Cystine, 4564
Monobenzone, 2795 acetate TS, 5752 L-Cystine, 5685
Mupirocin, 2825 acetate TS, stronger, 5752, 5760 Cytarabine, 1140
Naftifine hydrochloride, 2851 ammonium sulfate TS, 5752 for injection, 1142
Neomycin and polymyxin B sulfates, 2893 chloride, 1109, 5685
Neomycin and polymyxin B sulfates and chloride injection, 1111
gramicidin, 2902 citrate, 5685
Neomycin and polymyxin Bsulfates, citrate TS, 5752
gramicidin, and hydrocortisone acetate,
2902
citrate
citrate
TS, alkaline, 5750, 5752
TS 2, alkaline, 5750, 5752 D
Neomycin and polymyxin B sulfates and iodide TS, alkaline, 5750, 5752
hydrocortisone acetate, 2904 nitrate, 5685 Dacarbazine, 1143
Neomycin and polymyxinB sulfates and nitrate hydrate, 5685 for injection, 1143
lidocaine, 2904 nitrate, tenth-normal (0.1 N), 5763 Dactinomycin, 1145
oxide, ammoniated, TS, 5751, 5752, 5759 for injection, 1145
Combined Index to USP 41 and NF 36 Dalfa-Dextr 1-15

Dalfampridine, 1146 Desoxycorticosterone Dextrin, 5321, 5686

Dalteparin pivalate, 1194 Dextro calcium pantothenate, 5686
sodium, 1148 pivalate injectable suspension, 1194 Dextroamphetamine sulfate, 1227
Danazol, 1151 Desoxycorticosterone acetate, 5686 capsules, 1228
capsules, 1151 Detection of irradiated dietary supplements tablets, 1229
Dantrolene sodium, 1152 (2250), 8190 Dextromethorphan, 1230
capsules, 1154 Determination chlorpheniramine, pseudoephedrine (salts
for injection, 1155 methoxy (431), 6212 of), and acetaminophen, capsules
Dapsone, 1156 nitrogen (461), 6219 containing at least three of the
oral suspension, 1157 Deuterated methanol, 5686 following, 45
tablets, 1158 Deuterated water, 5686 chlorpheniramine, pseudoephedrine (salts
Daunorubicin hydrochloride, 1159 Deuterium of), and acetaminophen, oral powder
for injection, 1159 chloride, 5686 containing at least three of the
DEAE-Agarose, 5686 oxide, 5686 following, 47
Decanol, 5686 Deuterochloroform, 5686 chlorpheniramine, pseudoephedrine (salts
Decoquinate, 1160 Devarda’s alloy, 5686 of), and acetaminophen, oral solution
premix, 1160 Dexamethasone, 1195 containing at least three of the
Decyl sodium sulfate, 5686 acetate, 1199 following, 49
Deferoxamine mesylate, 1161 acetate injectable suspension, 1200 chlorpheniramine, pseudoephedrine (salts
for injection, 1162 and ciprofloxacin otic suspension, 951 of), and acetaminophen, tablets
Dehydrated alcohol, 5686 elixir, 1196 containing at least three of the
Dehydroacetic acid, 5320 injection, 1196 following, 51
Dehydrocholic acid, 1163 and neomycin and polymyxin B sulfates hydrobromide, 1231
tablets, 1164 ophthalmic ointment, 2900 hydrobromide, acetaminophen,
Delafield’s hematoxylin TS, 5753 and neomycin and polymyxin B sulfates doxylamine succinate, and
Deliverable volume (698), 6450 ophthalmic suspension, 2901 pseudoephedrine hydrochloride oral
Delta-8-tetrahydrocannabinol, 5736 ophthalmic suspension, 1197 solution, 60
Demecarium bromide, 1164 penicillin G procaine, dihydrostreptomycin hydrobromide, guaifenesin, and
ophthalmic solution, 1165 sulfate, and chlorpheniramine maleate pseudoephedrine hydrochloride
Demeclocycline, 1165 injectable suspension, 3207 capsules, 2006
hydrochloride, 1166 sodium phosphate, 1200 hydrobromide, pseudoephedrine
hydrochloride capsules, 1167 sodium phosphate cream, 1203 hydrochloride, and carbinoxamine
hydrochloride tablets, 1167 sodium phosphate inhalation aerosol, 1203 maleate oral solution, 3511
oral suspension, 1166 sodium phosphate injection, 1204 hydrobromide oral solution, 1232
Denatonium benzoate, 5320 sodium phosphate and neomycin sulfate hydrobromide, acetaminophen, and
Denaturated alcohol TS, 5753 cream, 2884 chlorpheniramine maleate tablets, 53
Denigés’ reagent, 5753 sodium phosphate and neomycin sulfate Dextrose, 1233
Density of solids (699), 6453 ophthalmic ointment, 2885 adenine solution, anticoagulant citrate
Dental paste sodium phosphate and neomycin sulfate phosphate, 327
triamcinolone acetonide, 4188 ophthalmic solution, 2886 anhydrous, 5686
Deoxyadenosine triphosphate, 5686 sodium phosphate ophthalmic ointment, and dopamine hydrochloride injection,
Deoxycytidine triphosphate, 5686 1206 1394
Deoxyguanosine triphosphate, 5686 sodium phosphate ophthalmic solution, excipient, 5322
Deoxyribonucleic acid polymerase, 5686 1207 and half-strength lactated Ringer’s
Deoxythymidine triphosphate, 5686 oral solution, 1198 injection, 3628
Depyrogenation (1228), 7676 tablets, 1198 injection, 1234
Depyrogenation by filtration (1228.3), 7685 and tobramycin ophthalmic ointment, injection, alcohol in, 107
Description and relative solubility of USP and 4117 injection, bretylium tosylate in, 548
NF articles, 5791 and tobramycin ophthalmic suspension, injection, bupivacaine hydrochloride in,
Desflurane, 1170 4119 567
Design, evaluation and characterization of Dexamethasone sodium phosphate injection, dobutamine in, 1369
viral clearance procedures (1050.1), 6950 compounded injection, magnesium sulfate in, 2519
Design and analysis of biological assays injection, 1205 injection, potassium chloride in, 3364
(111), 6049 Dexbrompheniramine maleate, 1208 injection and potassium chloride in
Design and development of biological assays and pseudoephedrine sulfate oral solution, lactated ringer’s, 3367
(1032), 6785 1209 injection and sodium chloride injection,
Desipramine hydrochloride, 1172 Dexchlorpheniramine maleate, 1210 potassium chloride in, 3365
tablets, 1174 oral solution, 1211 injection, tetracaine hydrochloride in, 4013
Deslanoside, 1175 tablets, 1212 injection, theophylline in, 4039
injection, 1176 Dexmedetomidine injection type 1 and multiple electrolytes,
Desloratadine, 1177 injection, 1215 1485
tablets, 1178 Dexmedetomidine hydrochloride, 1213 injection type 2 and multiple electrolytes,
orally disintegrating tablets, 1180 Dexpanthenol, 1216 1488
Desmopressin acetate, 1182 assay (115), 6053 injection type 3 and multiple electrolytes,
injection, 1183 preparation, 1216 1492
nasal spray, 1184 Dextran and lactated Ringer’s injection, 3626
Desogestrel, 1185 1, 1218 and lidocaine hydrochloride injection,
and ethinyl estradiol tablets, 1186 40, 1219 2417
Desonide, 1188 40 in dextrose injection, 1222 and modified lactated Ringer’s injection,
Desoximetasone, 1189 40 in sodium chloride injection, 1223 3631
cream, 1190 70, 1223 and Ringer's injection, 3622
gel, 1190 70 in dextrose injection, 1226 and sodium chloride injection, 1235
ointment, 1191 70 in sodium chloride injection, 1227 solution, anticoagulant citrate, 324
Desoxycholic acid, 1168, 1191 high molecular weight, 5686 solution, anticoagulant citrate phosphate,
Dextrates, 5321 326
1-16 Diace-Dieta Combined Index to USP 41 and NF 36

Diacetyl, 5686 2,6-Dichlorophenylacetic acid, 5688 Calcium L-5-methyltetrahydrofolate

Diacetylated monoglycerides, 5323 2,6-Dichloroquinone-chlorimide, 5688 capsules, 4498
3,3’-Diaminobenzidine hydrochloride, 5686 Dichlorphenamide, 1252 Calcium L-5-methyltetrahydrofolate tablets,
2,3-Diaminonaphthalene, 5687 tablets, 1253 4499
2,6-Diaminopyridine, 5687 Diclazuril, 1253 Calcium and vitamin D with minerals
Diatomaceous earth, 5687 Diclofenac potassium, 1254 tablets, 4502
flux-calcined, 5687 tablets, 1255 Calcium with vitamin D tablets, 4507
silanized, 5687 Diclofenac sodium, 1257 Cat’s claw, 4506
Diatomaceous silica and misoprostol delayed-release tablets, Cat’s claw capsules, 4510
calcined, 5687, 5726 1261 Cat’s claw extract, powdered, 4509
Diatrizoate delayed-release tablets, 1258 Cat’s claw, powdered, 4507
meglumine, 1236 extended-release tablets, 1259 Cat’s claw tablets, 4512
meglumine and diatrizoate sodium Dicloxacillin sodium, 1265 Centella asiatica, 4513
injection, 1237 capsules, 1266 Centella asiatica, powdered, 4515
meglumine and diatrizoate sodium for oral suspension, 1267 Centella asiatica extract, powdered, 4516
solution, 1238 Dicyclohexyl, 5688 Centella asiatica triterpenes, 4518
meglumine injection, 1236 Dicyclohexylamine, 5688 Chamomile, 4519
sodium, 1239 Dicyclohexyl phthalate, 5688 Chaste tree, 4521
sodium and diatrizoate meglumine Dicyclomine hydrochloride, 1268 Chaste tree, powdered, 4523
injection, 1237 capsules, 1268 Chaste tree extract, powdered, 4524
sodium and diatrizoate meglumine injection, 1269 Chia seed oil, 4530
solution, 1238 oral solution, 1270 Chinese salvia, 4531
sodium injection, 1240 tablets, 1271 Chinese salvia, powdered, 4533
sodium solution, 1241 Didanosine, 1272 Choline bitartrate, 4535
Diatrizoic acid, 1241 delayed-release capsules, 1273 Choline chloride, 4537
Diaveridine, 5687 for oral solution, 1275 Chondroitin sulfate sodium, 4539
Diazepam, 1242 tablets for oral suspension, 1275 Chondroitin sulfate sodium, shark, 4543
capsules, 1243 Chondroitin sulfate sodium tablets, 4541
extended-release capsules, 1244 Chromium picolinate, 4545
injection, 1245 Chromium picolinate tablets, 4546
tablets, 1245
Diazobenzenesulfonic acid TS, 5753
Dietary supplements Cinnamomum cassia twig, 4546
Cinnamomum cassia twig powder, 4548
Diazoxide, 1246 N-acetylglucosamine, 4417 Citrulline, 4550
capsules, 1247 Ademetionine disulfate tosylate, 4419 Clover, red, 4811
injection, 1247 L-Alanyl-L-glutamine, 4420 Clover, powdered red, 4815
oral suspension, 1248 Andrographis, 4429 Clover extract, powdered red, 4817
Dibasic Andrographis, powdered, 4431 Clover tablets, red, 4819
ammonium citrate, 5687 Andrographis extract, powdered, 4433 Cod liver oil capsules, 4551
ammonium phosphate, 5687 Arginine capsules, 4434 Cohosh, black, fluidextract, 4480
calcium phosphate, anhydrous, 655 Arginine tablets, 4435 Cranberry liquid preparation, 4554
calcium phosphate dihydrate, 654 Ashwagandha root, 4436 Crypthecodinium cohnii oil, 4555
calcium phosphate tablets, 657 Ashwagandha root extract, powdered, Crypthecodinium cohnii oil capsules, 4557
potassium phosphate, 3386, 5687 4439 Curcuminoids, 4560
sodium phosphate, 3804 Ashwagandha root, powdered, 4438 Curcuminoids capsules, 4561
Dibenzyl, 5687 Astaxanthin esters, 4446 Curcuminoids tablets, 4562
2,6-Dibromoquinone-chlorimide, 5687 Astragalus root, 4448 Diosmin, 4565
Dibucaine, 1249 Astragalus root dry extract, 4452 Echinacea angustifolia, 4566
cream, 1249 Astragalus root powder, 4450 Echinacea angustifolia, powdered, 4569
hydrochloride, 1250 Aztec marigold zeaxanthin extract, 4454 Echinacea angustifolia extract, powdered,
hydrochloride injection, 1251 Bacillus subtilis subsp. subtilis 4571
ointment, 1250 menaquinone-7 extract, 4765 Echinacea pallida, 4574
Dibutyl Bacopa, 4456 Echinacea pallida, powdered, 4576
phthalate, 5323, 5687 Bacopa, powdered, 4458 Echinacea pallida, powdered, extract, 4578
sebacate, 5324 Bacopa extract, powdered, 4459 Echinacea purpurea aerial parts, 4580
Dibutylamine, 5687 Banaba leaf, 4461 Echinacea purpurea, powdered, 4585
Dibutylammonium phosphate, 5687 Banaba leaf dry extract, 4464 Echinacea purpurea, powdered, extract,
1,3-Dicaffeoylquinic acid, 5687 Banaba leaf powder, 4462 4587
Dichloralphenazone, 1251 Beta carotene preparation, 4465 Echinacea purpurea root, 4583
isometheptene mucate and Beta glucan, 4467 Echinacea species dry extract capsules,
acetaminophen capsules, 2251 Bifidobacterium animalis subsp. lactis, 4469 4590
Dichloroacetic acid, 5687 Bilberry extract, powdered, 4472 Echinacea species dry extract tablets, 4592
2,5-Dichloroaniline, 5687 Black cohosh, 4474 Echinacea species powder capsules, 4595
2,6-Dichloroaniline, 5687 Black cohosh, powdered, 4476 Eleuthero, 4597
o-Dichlorobenzene, 5688 Black cohosh extract, powdered, 4478 Eleuthero, powdered, 4602
1,2-Dichloroethane, 5688 Black cohosh tablets, 4482 Eleuthero extract, powdered, 4599
Dichlorofluorescein, 5688 Black pepper, 4483 Eleuthero root and rhizome dry extract
TS, 5753 Powdered black pepper extract, 4487 capsules, 4600
Dichlorofluoromethane, 5688 Powdered black pepper, 4485 Eleuthero root and rhizome dry extract
2,6-Dichloroindophenol sodium, 5688 Borage seed oil, 4488 tablets, 4601
Dichloromethane, 5688 Borage seed oil capsules, 4489 Eleuthero root and rhizome powder
2,4-Dichloro-1-naphthol, 5688 Boswellia serrata, 4490 capsules, 4604
2,6-Dichlorophenol-indophenol sodium, Boswellia serrata extract, 4491 Evening primrose oil, 4605
5688, 5688 Calcium citrate tablets, 4493 Evening primrose oil capsules, 4606
Dichlorophenol-indopheno! solution, Calcium L-5-methyltetrahydrofolate, 4496 Fenugreek seed, 4607
standard, 5764 Fenugreek seed powder, 4609
Combined Index to USP 41 and NF 36
Dietary supplements (continued)
Fenugreek seed powdered extract, 4612
Feverfew, 4615
Feverfew, powdered, 4616
Fish oil containing omega-3 acids, 4617
Fish oil containing omega-3 acids capsules,
4620
Fish oil containing omega-3 acids delayed-
release capsules, 4622
Flax seed oil, 4623
Flax seed oil capsules, 4624
Forskohlii, 4625
Powdered forskohlii, 4627
Powdered forskohlii extract, 4628
Ganoderma lucidum fruiting body, 4629
Ganoderma lucidum fruiting body powder,
4632
Garcinia cambogia, 4635
Garcinia cambogia, powdered, 4637
Garcinia hydroxycitrate extract, powdered,
4638
Garcinia indica, 4639
Garcinia indica, powdered, 4641
Garlic, 4642
Garlic, powdered, 4644
Garlic extract, powdered, 4646
Garlic fluidextract, 4647
Garlic delayed-release tablets, 4648
Ginger, 4650
Ginger, powdered, 4652
Ginger capsules, 4656
Ginger tincture, 4654
Ginkgo, 4657
Ginkgo extract, powdered, 4660
Ginkgo capsules, 4663
Ginkgo tablets, 4665
Ginseng, American, 4422
Ginseng, American, capsules, 4426
Ginseng, American, powdered, 4423
Ginseng, American extract, powdered,
4425
Ginseng, American, tablets, 4428
Ginseng, Asian, 4441
Ginseng, Asian, powdered, 4442
Ginseng, Asian extract, powdered, 4444
Ginseng, Asian, tablets, 4445
Glucosamine and chondroitin sulfate
sodium tablets, 4667
Glucosamine hydrochloride, 4669
Glucosamine tablets, 4669
Glucosamine sulfate potassium chloride,
4670
Glucosamine sulfate sodium chloride, 4671
Glucosamine and methylsulfonylmethane
tablets, 4672
Glucosamine, chondroitin sulfate sodium,
and methylsulfonylmethane tablets,
4674
Glutamic acid, 4676
Glutathione, 4677
Glycyl-L-glutamine, 4678
Glycyl-L-tyrosine, 4679
Goldenseal, 4681
Goldenseal, powdered extract, 4684
Goldenseal, powdered, 4682
Grape seeds oligomeric proanthocyanidins,
4685
Green tea extract, decaffeinated,
powdered, 4687
Guggul, 4689
Guggul extract, native, 4690
Guggul extract, purified, 4691
Guggul tablets, 4692
Gymnema, 4693
Gymnema extract, native, 4696
Gymnema, powdered, 4695
Gymnema extract, purified, 4697
Hawthorn leaf with flower, 4699
Hawthorn leaf with flower, powdered,
4701
Hesperidin, 4703
Holy basil leaf, 4704
Holy basil leaf powdered, 4706
Holy basil leaf extract, powdered, 4708
Horse chestnut, 4526
Horse chestnut, powdered, 4527
Horse chestnut extract, powdered, 4529
5-Hydroxy-L-tryptophan, 4914
Japanese honeysuckle flower, 4709
Japanese honeysuckle flower dry extract,
4712
Japanese honeysuckle flower powder, 4715
Krill oi] capsules, 4721
Krill oil delayed-release capsules, 4725
Lactobacillus acidophilus La-14, 4728
Lactobacillus acidophilus NCFM, 4730
Lactobacillus paracasei LPC-37, 4733
Lactobacillus rhamnosus HNO01, 4732
Licorice, 4735
Licorice, powdered, 4736
Licorice extract, powdered, 4737
Ground limestone, 4738
Linoleic acids-free fatty acids, conjugated,
4739
Lipoic acid, alpha, 4740
Lipoic acid capsules, alpha, 4741
Lipoic acid tablets, alpha, 4742
Lutein, 4743
Lutein capsules, 4744
Lutein preparation, 4745
Lycopene, 4746
Lycopene preparation, 4747
Lysine hydrochloride tablets, 4751
Malabar-nut-tree, leaf, 4752
Malabar-nut-tree, leaf, powdered, 4753
Malabar-nut-tree, leaf extract, powdered,
4754
Maritime pine, 4755
Maritime pine extract, 4757
Melatonin, 4758
Melatonin tablets, 4759
Menaquinone-7, 4761
Menaquinone-7 capsules, 4762
Menaquinone-7 preparation, 4763
Menaquinone-7 tablets, 4764
Methylcobalamin, 4767
Methylcobalamin tablets, 4768
Methylsulfonylmethane, 4769
Methylsulfonyimethane tablets, 4770
Milk thistle, 4770
Milk thistle, powdered, 4772
Milk thistle extract, powdered, 4773
Milk thistle capsules, 4775
Milk thistle tablets, 4776
Minerals capsules, 4778
Minerals tablets, 4785
Northern schisandra fruit, 4865
Northern schisandra fruit dry extract, 4866
Northern schisandra fruit powder, 4868
Olive leaf, 4793
Olive leaf dry extract, 4794
Olive leaf powder, 4796
Omega-3 acids triglycerides, 4797
Phyllanthus amarus, 4800
Phyllanthus amarus, powdered, 4802
Plant stanol esters, 4803
Potassium citrate tablets, 4805
Powdered Rhodiola rosea, 4827
Powdered Rhodiola rosea extract, 4828
Powdered rosemary, 4838
Dieta-Dieta 1-17
Pygeum extract, 4807
Quercetin, 4810
Red clover aerial parts isoflavone aglycones
dry extract, 4814
Rhodiola crenulata root and rhizome, 4821
Rhodiola crenulata root and rhizome dry
extract, 4822
Rhodiola crenulata root and rhizome
powder, 4824
Rhodiola rosea, 4825
Rhodiola rosea capsules, 4831
Rhodiola rosea tablets, 4833
Rhodiola rosea tincture, 4830
Ribose, 4835
Rosemary, 4836
Rosemary leaf dry aqueous extract, 4839
Rutin, 4841
St. John’s wort flowering top, 4842
St. John’s wort flowering top dry extract
capsules, 4847
St. John’s wort flowering top extract, dry,
4845
St. John’s wort flowering top powder,
4844
St. John’s wort flowering top dry extract
tablets, 4849
Salix species bark, 4850
Salix species bark dry extract, 4852
Salix species bark powder, 4854
Saw palmetto, 4856
Saw palmetto, powdered, 4858
Saw palmetto capsules, 4862
Saw palmetto extract, 4860
Schizochytrium oil, 4870
Schizochytrium oil capsules, 4872
Selenomethionine, 4875
Sodium ferrous citrate, 4876
Soy isoflavones capsules, 4879
Soy isoflavones extract, powdered, 4877
Soy isoflavones tablets, 4881
Spirulina, 4882
Spirulina tablets, 4886
Stinging nettle, 4889
Stinging nettle, powdered extract, 4892
Stinging nettle, powdered, 4891
Tangerine peel, 4894
Tangerine peel dry extract, 4896
Tangerine peel powder, 4898
Tienchi ginseng root and rhizome, 4901
Tienchi ginseng root and rhizome dry
extract capsules, 4910
Tienchi ginseng root and rhizome dry
extract, 4909
Tienchi ginseng root and rhizome powder,
4903
Tienchi ginseng root and rhizome powder
capsules, 4905
Tienchi ginseng root and rhizome dry
extract tablets, 4912
Tienchi ginseng root and rhizome powder
tablets, 4907
Tomato extract containing lycopene, 4748
Turmeric, 4915
Turmeric, powdered, 4917
Turmeric extract, powdered, 4918
Ubidecarenone, 4919
Ubidecarenone capsules, 4920
Ubidecarenone tablets, 4921
Ubiquinol, 4922
Ubiquinol capsules, 4923
Valerian, 4924
Valerian, powdered, 4926
Valerian extract, powdered, 4927
Valerian tablets, 4930
Valerian tincture, 4929
1-18 Dieta-Diphe Combined Index to USP 41 and NF 36

Dietary supplements (continued) Digitonin, 5689 Dimercaprol, 1315

Vinpocetine, 4931 Digitoxin, 1288 injection, 1316
Vinpocetine capsules, 4933 injection, 1289 Dimethicone, 5330
Vinpocetine tablets, 4933 tablets, 1290 viscosity 500 centistokes, 5690
Vitamin A oral liquid preparation, 4329 Digoxigenin, 5689 2,5-Dimethoxybenzaldehyde, 5690
Vitamins capsules, oil- and water-soluble, Digoxin, 1291 1,2-Dimethoxyethane, 5690
4976 injection, 1292 Dimethoxymethane, 5690
Vitamins with minerals capsules, oil- and oral solution, 1292 (3,4-Dimethoxyphenyl)-acetonitrile, 5690
water-soluble, 5022 tablets, 1293 Dimethyl
Vitamins with minerals capsules, water- Dihydrocodeine bitartrate, 1294 phthalate, 5690
soluble, 5109 aspirin and caffeine capsules, 378 sulfone, 5690
Vitamins with minerals oral solution, water- Dihydroergotamine mesylate, 1295 sulfoxide, 1316, 5690, 5708
soluble, 5128 injection, 1296 sulfoxide gel, 1317
Vitamins with minerals tablets, oil- and 24,25-Dihydrolanosterol, 5689 sulfoxide irrigation, 1318
water-soluble, 5061 Dihydroquinidine hydrochloride, 5689 sulfoxide topical solution, 1318
Vitamins with minerals tablets, water- Dihydroquinine, 5689 sulfoxide spectrophotometric grade, 5690
soluble, 5137 Dihydrostreptomycin N,N-Dimethylacetamide, 5691
Vitamins tablets, oil- and water-soluble, injection, 1297 p-Dimethylaminoazobenzene, 5691
5004 sulfate, 1296 p-Dimethylaminobenzaldehyde, 5691
Vitamins capsules, oil-soluble, 4935 sulfate boluses, 1297 TS, 5753
Vitamins capsules, water-soluble, 5086 sulfate, penicillin G procaine, p-Dimethylaminocinnamaldehyde, 5691
Vitamins with minerals oral solution, oil- chlorpheniramine maleate, and 2-Dimethylaminoethyl methacrylate, 5691
and water-soluble, 5047 dexamethasone injectable suspension, Dimethylaminophenol, 5691
Oil-soluble vitamins with minerals capsules, 3207 Dimethylaniline (223), 6140
4951 sulfate and penicillin G procaine injectable 2,6-Dimethylaniline, 5691
Oil-soluble vitamins with minerals oral suspension, 3207 N,N-Dimethylaniline, 5691
solution, 4961 sulfate and penicillin G procaine 3,4-Dimethylbenzophenone, 5691
Oil-soluble vitamins with minerals tablets, intramammary infusion, 3206 5,5-Dimethyl-1,3-cyclohexanedione, 5691
4966 sulfate, penicillin G procaine, and N,N-Dimethyldecylamine, 5691
Vitamins oral solution, oil- and water- prednisolone injectable suspension, 3209 1,5-Dimethyl-1,5-diazaundecamethylene
soluble, 4995 Dihydrotachysterol, 1298 polymethobromide, 5691
Oil-soluble vitamins oral solution, 4941 capsules, 1298 N,N-Dimethyldodecylamine-N-oxide, 5691
Vitamins tablets, oil-soluble, 4944 oral solution, 1299 Dimethylethyl(3-hydroxyphenyl)ammonium
Vitamins tablets, water-soluble, 5098 tablets, 1299 chloride, 5691
meso-Zeaxanthin, 5155 Dihydroxyacetone, 1300 Dimethylformamide, 5691
meso-Zeaxanthin preparation, 5157 Dihydroxyaluminum N,N-Dimethylformamide diethyl acetal, 5691
Zinc citrate, 5159 aminoacetate, 1300 1,3-Dimethyl-2-imidazolidinone, 5691
Zinc citrate tablets, 5159 aminoacetate magma, 1301 1,9-Dimethyl-methylene blue, 5691
Zinc and vitamin C lozenges, 5161 sodium carbonate, 1301 N,N-Dimethyl-1-naphthylamine, 5691
sodium carbonate chewable tablets, 1303 N,N-Dimethyloctylamine, 5692
Dihydroxybenzaldehyde, 5689 2,5-Dimethylphenol, 5692
2,5-Dihydroxybenzoic acid, 5689 2,6-Dimethylphenol, 5692
Diethanolamine, 5325 2,7-Dihydroxynaphthalene, 5689 3,5-Dimethylphenol, 5692
Diethylamine, 5688 2,7-Dihydroxynaphthalene TS, 5753 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl
Diethylamine phosphate, 5688 Dihydroxyphenylacetone, 5689 tetrazolium bromide, 5692
N,N-Diethylaniline, 5688 4,5-Dihydroxy-3-(p-sulfophenylazo)-2,7- Dimethyltin dibromide, 5692
Diethylcarbamazine citrate, 1277 napthalenedisulfonic acid, trisodium salt, N,N-Dimethyl-p-phenylenediamine
tablets, 1277 5746 dihydrochloride, 5692
Diethylene glycol, 5689 Diiodofluorescein, 5689 m-Dinitrobenzene, 5692
monoethyl ether, 5326 TS, 5753 3,5-Dinitrobenzoyl chloride, 5692
stearates, 5328 Diisodecyl phthalate, 5690 2,4-Dinitrochlorobenzene, 5692
succinate polyester, 5689 Diisopropanolamine, 5329 2,4-Dinitrofluorobenzene, 5692
Di(ethylene glycol) methyl ether, 5689 Diisopropyl ether, 5690, 5694, 5703 2,4-Dinitrophenylhydrazine, 5692
Diethylenetriamine, 5689 Diisopropylamine, 5690 Dinitrophenylhydrazine TS, 5753
Di(2-ethylhexyl)phthalate, 5689 Diisopropylethylamine, 5690 Dinoprost tromethamine, 1319
Diethyl phthalate, 5325 1,2-Dilinoleoyl-3-oleoyl-rac-glycerol, 5690 injection, 1320
Diethylpropion hydrochloride, 1278 1,2-Dilinoleoy|-3-palmitoyl-rac-glycerol, 5690 Dinoprostone, 1320
tablets, 1279 Diloxanide furoate, 1303 Dioctyl sodium sulfosuccinate, 5692
Diethylpyrocarbonate, 5689 Diltiazem hydrochloride, 1304 Diosmin, 4565
Diethyl sebacate, 5326 extended-release capsules, 1305 Dioxane, 5692
Diethylstilbestrol, 1280 oral solution, 1309 Dioxybenzone, 1322
injection, 1280 oral suspension, 1310 and oxybenzone cream, 1322
tablets, 1281 tablets, 1310 Diphenhydramine
Diethyl sulfone, 5689 Diluted citrate, 1323
Diethyltoluamide, 1281 acetic acid, 5181, 5690 citrate and acetaminophen tablets, 61
topical solution, 1282 alcohol, 5690 citrate and ibuprofen tablets, 1324
Diflorasone diacetate, 1282 hydrochloric acid, 5690 hydrochloride, 1327
cream, 1283 lead subacetate TS, 5753 hydrochloride, acetaminophen, and
ointment, 1284 nitric acid, 5690 pseudoephedrine hydrochloride tablets,
Diflunisal, 1284 sulfuric acid, 5690 63
tablets, 1285 Dimenhydrinate, 1312 hydrochloride capsules, 1328
Digitalis, 1285 injection, 1313 hydrochloride injection, 1330
capsules, 1288 oral solution, 1314 hydrochloride oral solution, 1331
powdered, 1287 tablets, 1314 hydrochloride and ibuprofen capsules,
tablets, 1288 1333
Combined Index to USP 41 and NF 36 Diphe-Elect I-19

Diphenhydramine (continued) sodium tablets, 1381 Duloxetine hydrochloride, 1453

and phenylephrine hydrochloride tablets, 1-Dodecanol, 5693 Dusting powder, absorbable, 1457
1334 Dodecyl Dutasteride, 1454
and pseudoephedrine capsules, 1337 alcohol, 5693 Dyclonine hydrochloride, 1457
Diphenoxylate hydrochloride, 1339 lithium sulfate, 5693 gel, 1458
and atropine sulfate oral solution, 1339 sodium sulfonate, 5693 topical solution, 1458
and atropine sulfate tablets, 1340 3-(Dodecyldimethylammonio) Dydrogesterone, 1459
Diphenyl ether, 5692, 5694, 5717 propanesulfonate, 5693 tablets, 1459
Diphenylamine, 5692 Dodecyltriethylammonium phosphate, 0.5 Dyphylline, 1460
TS, 5753 M, 5693 and guaifenesin oral solution, 1462
Diphenylborinic acid, ethanolamine ester, Dodecyltrimethylammonium bromide, 5693 and guaifenesin tablets, 1462
5667, 5692 Dofetilide, 1382 injection, 1460
Diphenylcarbazide, 5692 Dolasetron mesylate, 1383 oral solution, 1461
Diphenylcarbazone, 5692 oral solution, 1384 tablets, 1461
TS, 5753 oral suspension, 1385
2,2-Diphenylglycine, 5692 Donepezil hydrochloride, 1385
Diphtheria antitoxin potency testing for tablets, 1387
human immune globulins (162), 6088 orally disintegrating tablets, 1391
Dipicolinic acid, 5693
Dipicrylamine, 5693
Dopamine hydrochloride, 1392
and dextrose injection, 1394
E
Dipivefrin hydrochloride, 1341 injection, 1393
ophthalmic solution, 1343 Dorzolamide Earth, chromatographic, silanized, acid-base
Dipropyl phthalate, 5693 Hydrochloride and timolol maleate washed, 5693
Dipyridamole, 1344 ophthalmic solution, 1397 Ecamsule
injection, 1346 Dorzolamide hydrochloride solution, 1464
oral suspension, 1347 ophthalmic solution, 1396 Echinacea
tablets, 1347 Dorzolamide hydrochloride, 1394 angustifolia, 4566
4,4’-Dipyridyl, 5693 Doxapram hydrochloride, 1399 angustifolia, powdered, 4569
a,a’-Dipyridyl, 5693 injection, 1401 angustifolia extract, powdered, 4571
Direct red 80, 5724 Doxazosin mesylate, 1402 pallida, 4574
Dirithromycin, 1348 tablets, 1404 pallida, powdered, 4576
delayed-release tablets, 1349 Doxepin hydrochloride, 1404 pallida extract, powdered, 4578
Disinfectants and antiseptics (1072), 7090 capsules, 1406 purpurea aerial parts, 4580
Disintegration oral solution, 1407 purpurea, powdered, 4585
(701), 6455 Doxercalciferol, 1408 purpurea root, 4583
and dissolution of dietary supplements Doxorubicin hydrochloride, 1409 purpurea extract, powdered, 4587
(2040), 8178 injection, 1411 Echinacea species
Disodium for injection, 1413 dry extract capsules, 4590
chromotropate, 5693 Doxycycline, 1415 dry extract tablets, 4592
ethylenediaminetetraacetate, 5693 calcium oral suspension, 1425 powder capsules, 4595
Disopyramide phosphate, 1350 capsules, 1416 Echothiophate
capsules, 1351 extended-release capsules, 1418 iodide, 1466
extended-release capsules, 1351 hyclate, 1428 iodide for ophthalmic solution, 1467
Dissolution (711), 6459 hyclate capsules, 1429 Econazole nitrate, 1468
The dissolution procedure: development and hyclate delayed-release capsules, 1431 Edetate
validation (1092), 7178 hyclate tablets, 1432 calcium disodium, 1469
Distilling range (721), 6469 hyclate delayed-release tablets, 1434 calcium disodium injection, 1470
Disulfiram, 1352 for injection, 1420 disodium, 1471, 5693
tablets, 1353 for oral suspension, 1421 disodium injection, 1472
5,5’-Dithiobis (2-nitrobenzoic acid), 5693 tablets, 1424 disodium TS, 5753
Dithiothreitol, 5693 Doxycycline compounded, veterinary oral disodium, twentieth-molar (0.05 M), 5764
Dithizone, 5693 suspension, 1427 Edetate disodium
TS, 5753 Doxylamine succinate, 1437 0.01 M TS, 5753
Divalproex sodium, 1353 acetaminophen, dextromethorphan Edetic acid, 5331, 5693
delayed-release capsules, 1354 hydrobromide, and pseudoephedrine Edrophonium
delayed-release tablets, 1357 hydrochloride oral solution, 60 chloride, 1472
extended-release tablets, 1358 oral solution, 1438 chloride injection, 1472
Dobutamine tablets, 1438 Efavirenz, 1473
in dextrose injection, 1369 Drabkin’s reagent, 5693 capsules, 1476
hydrochloride, 1366 Dragendorff’s TS, 5753 Tablets, 1478
injection, 1367 Dried peptone, 5693 Egg phospholipids, 5332
for injection, 1368 Dronabinol, 1438 n-Eicosane, 5693
Docetaxel, 1370 capsules, 1440 Eicosanol, 5693
injection, 1373 Dronedarone Elastomeric closures for injections (381),
Docusate hydrochloride, 1440 6178
calcium, 1375 tablets, 1442 Electrolytes
calcium capsules, 1376 Droperidol, 1443 and dextrose injection type 1, multiple,
potassium, 1377 injection, 1443 1485
potassium capsules, 1378 Drospirenone, 1444 and dextrose injection type 2, multiple,
sodium, 1378 and ethinyl estradiol tablets, 1447 1488
sodium capsules, 1379 Drug release (724), 6471 and dextrose injection type 3, multiple,
sodium and ferrous fumarate extended- Dry heat depyrogenation (1228.1), 7681 1492
release tablets, 1713 Dry heat sterilization (1229.8), 7725 and polyethylene glyco! 3350 for oral
sodium solution, 1380 Duloxetine solution, 3345
sodium syrup, 1380 delayed-release capsules, 1450 injection type 1, multiple, 1480
|-20 Elect-Ethid Combined Index to USP 41 and NF 36

Electrolytes (continued) sulfate oral solution, 1529 ethylsuccinate, sterile, 1577

injection type 2, multiple, 1482 Epiandrosterone, 5693 ethylsuccinate and sulfisoxazole acetyl for
Elemental contaminants in dietary 4-Epianhydrotetracycline (226), 6140 oral suspension, 1580
supplements (2232), 8186 15-Epicarboprost, 5693 ethylsuccinate oral suspension, 1578
Elemental impurities—limits (232), 6147 Epinephrine, 1529 ethylsuccinate for oral suspension, 1578
Elemental impurities—procedures (233), and articaine hydrochloride injection, 358 ethylsuccinate tablets, 1578
6151 assay (391), 6183 topical gel, 1567
Elements bitartrate, 1532 gluceptate, sterile, 1581
injection, trace, 1494 bitartrate inhalation aerosol, 1533 injection, 1568
Eleuthero, 4597 bitartrate ophthalmic solution, 1534 intramammary infusion, 1567
extract, powdered, 4599 bitartrate for ophthalmic solution, 1534 lactobionate for injection, 1581
powdered, 4602 and bupivacaine hydrochloride injection, lactobionate, sterile, 1582
Eleuthero 567 ointment, 1568
root and rhizome dry extract capsules, and cocaine and tetracaine hydrochlorides ophthalmic ointment, 1569
4600 topical solution, 1059 pledgets, 1570
root and rhizome dry extract tablets, 4601 inhalation aerosol, 1530 topical solution, 1571
root and rhizome powder capsules, 4604 inhalation solution, 1531 stearate, 1583
injection, 1530 stearate tablets, 1584
and lidocaine hydrochloride injection, tablets, 1571
2417 delayed-release tablets, 1572
Elixir nasal solution, 1531
ophthalmic solution, 1532
Erythropoietin bioassays (124), 6061
Escin, 5694
Aromatic, 5206 and prilocaine injection, 3429 Escitalopram
Benzaldehyde, compound, 5215 and procaine hydrochloride injection, 3445 oral solution, 1584
Dexamethasone, 1196 Epinephryl borate ophthalmic solution, 1535 Escitalopram oxalate, 1588
Fluphenazine hydrochloride, 1815 Epirubicin hydrochloride, 1536 Escitalopram
Hyoscyamine sulfate, 2103 injection, 1537 tablets, 1587
Epitetracycline hydrochloride, 1538 Esmolol hydrochloride, 1590
Epoetin, 1540 Esomeprazole magnesium, 1591
Eprinomectin, 1543 delayed-release capsules, 1593
Elm, 1496 Eprosartan mesylate, 1544 Esomeprazole strontium, 1596
Emedastine Equilenin, 5694 Estazolam, 1598
difumarate, 1497 Equilin, 1546 tablets, 1599
ophthalmic solution, 1496 Ergocalciferol, 1546 Estradiol, 1600
Emetine hydrochloride, 1498 capsules, 1548 vaginal cream, 1601
injection, 1498 oral solution, 1549 vaginal inserts, 1602
Enalapril maleate, 1499 tablets, 1550 transdermal system, 1604
and hydrochlorothiazide tablets, 1503 a-Ergocryptine, 5694 tablets, 1607
tablets, 1502 Ergoloid mesylates, 1550 benzoate, 1611
Enalaprilat, 1506 capsules, 1551 cypionate, 1612
injection, 1506 oral solution, 1552 cypionate injection, 1614
Enalapril maleate sublingual tablets, 1554 and norethindrone acetate tablets, 1608
oral suspension, 1500 tablets, 1553 valerate, 1614
Enalapril maleate compounded, veterinary Ergonovine maleate, 1554 valerate injection, 1615
oral suspension, 1501 injection, 1555 Estriol, 1616
Endotoxin indicator for depyrogenation, tablets, 1556 Estrogens
1508 Ergotamine tartrate, 1557 conjugated, 1617
Endotoxin indicators for depyrogenation and caffeine suppositories, 1561 esterified, 1622
(1228.5), 7688 and caffeine tablets, 1562 tablets, conjugated, 1619
Enflurane, 1509 inhalation aerosol, 1558 tablets, esterified, 1623
Enoxaparin sodium, 1510 injection, 1559 Estrone, 1624
injection, 1512 sublingual tablets, 1561 injectable suspension, 1625
Enrofloxacin, 1515 tablets, 1560 Estropipate, 1625
Enrofloxacin compounded, veterinary Eriochrome tablets, 1627
oral suspension, 1517 black T, 5746 vaginal cream, 1626
Ensulizole, 1517 black TS, 5753 Eszopiclone, 1628
Entacapone, 1518 black T-sodium chloride indicator, 5694, tablets, 1629
tablets, 1519 5747 Ethacrynate sodium for injection, 1631
Entecavir, 1521 black T trituration, 5746 Ethacrynic acid, 1632
tablets, 1522 cyanine R, 5694 tablets, 1632
Enzacamene, 1524 cyanine TS, 5753 Ethambutol hydrochloride, 1633
Enzymatically-hydrolyzed Erythorbic acid, 5333 rifampin, isoniazid, and pyrazinamide
carboxymethylcellulose sodium, 5266 Erythritol, 5335 tablets, 3614
Enzymes used as ancillary materials in Erythromycin, 1565 compounded oral suspension, 1634
pharmaceutical manufacturing (89), 6025 and benzoyl peroxide topical gel, 1572 tablets, 1635
Eosin Y, 5693, 5746 delayed-release capsules, 1566 Ethanesulfonic acid, 5694
TS, 5753 estolate, 1573 Ethchlorvynol, 1636
Ephedrine, 1525 estolate capsules, 1573 capsules, 1637
hydrochloride, 1525 estolate and sulfisoxazole acetyl oral Ether, 1638, 5694
hydrochloride, theophylline, and suspension, 1575 absolute, 5664
phenobarbital tablets, 4041 estolate oral suspension, 1574 diphenyl, 5694
sulfate, 1526 estolate for oral suspension, 1574 isopropyl, 5694
sulfate capsules, 1527 estolate tablets, 1574 nonyl phenyl polyethylene glycol, 5694
sulfate injection, 1527 ethylsuccinate, 1576 peroxide-free, 5694
sulfate nasal solution, 1528 ethylsuccinate injection, 1577 Ethidium bromide, 5694
Combined Index to USP 41 and NF 36 Ethin-Fenne 1-21

Ethinyl estradiol, 1639 extended-release tablets, 1656 Guggul, native, 4690

and desogestrel tablets, 1186 Etomidate, 1658 Guggul, purified, 4691
and drospirenone tablets, 1447 injection, 1659 Gymnema, native, 4696
and ethynodiol diacetate tablets, 1649 Etoposide, 1660 Gymnema, purified, 4697
and levonorgestrel tablets, 2402 capsules, 1662 Holy basil leaf powdered, 4708
and norethindrone acetate tablets, 2975 injection, 1663 Horse chestnut, powdered, 4529
and norethindrone tablets, 2970 Eucalyptol, 1665 Japanese honeysuckle flower, dry, 4712
and norgestimate tablets, 2981 Eucalyptus oil, 5351 Licorice, powdered, 4737
and norgestrel tablets, 2983 Eugenol, 1665 Licorice fluidextract, 5422
tablets, 1639 Evaluation of plastic packaging systems and Malabar-nut-tree, leaf, powdered, 4754
Ethiodized oil injection, 1641 their materials of construction with respect Maritime pine, 4757
Ethionamide, 1642 to their user safety impact (1661), 7902 Milk thistle, powdered, 4773
tablets, 1642 Evaluation of the inner surface durability of Northern schisandra fruit, dry, 4866
Ethopabate, 1643 glass containers (1660), 7897 Olive leaf dry, 4794
Ethosuximide, 1643 Evening primrose oil, 4605 Powdered Rhodiola rosea, 4828
capsules, 1644 capsules, 4606 Pygeum, 4807
oral solution, 1645 Excipient biological safety evaluation Pyrethrum, 3522
Ethotoin, 1646 guidelines (1074), 7095 Red clover aerial parts isoflavone
tablets, 1647 Excipient performance (1059), 7011 aglycones, dry, 4814
4’-Ethoxyacetophenone, 5694 Excipients Rhodiola crenulata root and rhizome dry,
2-Ethoxyethanol, 5694, 5695 USP and NF, listed by category, 5169 4822
Ethyl Exemestane, 1666 Salix species bark dry, 4852
acetate, 5336, 5694 Exenatide, 1667 Saw palmetto, 4860
acrylate, 5694 Expert committees (2015-2020), xii Senna fluidextract, 3741
acrylate and methacrylic acid copolymer, Food Chemicals Codex, xvii Soy isoflavones, powdered, 4877
5442 National Formulary, xvi Stinging nettle, powdered, 4892
acrylate and methacrylic acid copolymer, United States Pharmacopeia, xii St. John’s wort flowering top, dry, 4845
partially-neutralized, 5446 United States Pharmacopeia and the Dietary Tangerine peel, dry, 4896
acrylate and methyl methacrylate Supplements Compendium, xvi Tienchi ginseng root and rhizome, dry,
copolymer dispersion, 5337 United States Pharmacopeia and USP on 4909
alcohol, 5694 Compounding, xviii Tomato, containing lycopene, 4748
arachidate, 5694 Expert Panels for the Council of Experts Turmeric, powdered, 4918
benzoate, 5694 Executive Committee, xii Valerian, powdered, 4927
chloride, 1648 Extended release tablets Yeast, 5744
cyanoacetate, 5694 nevirapine, 2915
ether, 5694
ether, anhydrous, 5664, 5694
maltol, 5339 Ezetimibe, 1670
oleate, 5339 tablets, 1672
salicylate, 5694
Extract
vanillin, 5340 Andrographis, powdered, 4433
2-Ethylaminopropiophenone hydrochloride, Ashwagandha root, powdered, 4439
5695 Astragalus root, dry, 4452
4-Ethylbenzaldehyde, 5695
Ethylbenzene, 5695
Aztec Marigold Zeaxanthin Extract, 4454
Bacillus subtilis subsp. subtilis
F
Ethylcellulose, 5341 menaquinone-7, 4765
aqueous dispersion, 5342 Bacopa, powdered, 4459 F18
dispersion type b, 5343 Banaba leaf, dry, 4464 injection, fludeoxyglucose, 1794
Ethylene Beef, 5672 injection, sodium fluoride, 1795
dichloride, 5688, 5695 Belladonna, 458 Factor IX complex, 1675
glycol, 5695 Belladonna tablets, 459 Factor Xa (activated factor X) for anti-factor
glycol, diethylene glycol, and triethylene Bilberry, powdered, 4472 X, test, 5695
Famciclovir, 1675
glycol in ethoxylated substances (469), Black cohosh, powdered, 4478
6237 Black pepper, powdered, 4487 Famciclovir compounded
Boswellia serrata, 4491 oral suspension, 1678
glycol monoethyl ether, 5695
glycol stearates, 5348 Cascara fluidextract, aromatic, 738 Famotidine, 1679
glycol and viny! alcohol graft copolymer, Cascara sagrada, 736 injection, 1680
5346 Cascara sagrada fluidextract, 738 for oral suspension, 1682
oxide and dioxane (228), 6142 Cat's claw, powdered, 4509 tablets, 1683
Centella asiatica, powdered, 4516 Fast
oxide in methylene chloride (50 mg/mL),
5695 Chaste tree, powdered, 4524 blue B salt, 5695
Ethylenediamine, 1648, 5695 Clover, red, powdered, 4817 blue BB salt, 5695
N-Ethylmaleimide, 5695 Echinacea angustifolia, powdered, 4571 green FCF, 5696
2-Ethyl-2-methylsuccinic acid, 5695 Echinacea pallida, powdered, 4578 Fat, hard, 5352
Ethylparaben, 5349, 5695 Echinacea purpurea, powdered, 4587 Fats and fixed oils (401), 6184
Ethylparaben sodium, 5350 Eleuthero, powdered, 4599 FD&C blue no. 1, 5696
1-Ethylquinaldinium iodide, 5695 Fenugreek seed, powdered, 4612 Fehling’s solution, 5753
Ethynodiol diacetate, 1648 Garcinia hydroxycitrate, powdered, 4638 Felbamate, 1684
and ethinyl estradiol tablets, 1649 Garlic, powdered, 4646 oral suspension, 1686
and mestranol tablets, 1650 Garlic fluidextract, 4647 tablets, 1688
Etidronate disodium, 1651 Ginkgo, powdered, 4660 Felodipine, 1689
tablets, 1652 Ginseng, American, powdered, 4425 extended-release tablets, 1690
Etodolac, 1654 Ginseng, Asian, powdered, 4444 Fenbendazole, 1694
capsules, 1655 Goldenseal, powdered, 4684 Fennel oil, 5353
tablets, 1655 Green tea, decaffeinated, powdered, 4687
|-22 Fenof-Formo Combined Index to USP 41 and NF 36

Fenofibrate, 1695 Fish oil containing omega-3 acids, 4617 ophthalmic suspension, 1798
capsules, 1697 capsules, 4620 Fluorouracil, 1800
tablets, 1699 delayed-release capsules, 4622 cream, 1801
Fenoldopam mesylate, 1701 Flame photometry for reagents, 5662 injection, 1802
injection, 1703 Flavoxate hydrochloride, 1747 topical solution, 1803
Fenoprofen calcium, 1704 tablets, 1748 Fluoxetine
capsules, 1705 Flax seed oil, 4623 capsules, 1803
tablets, 1706 capsules, 4624 delayed-release capsules, 1804
Fentanyl, 1707 Flecainide acetate, 1749 hydrochloride, 1809
Fentanyl citrate, 1708 oral suspension, 1750 and olanzapine capsules, 3005
injection, 1709 tablets, 1751 oral solution, 1806
Fenugreek seed, 4607 Flow cytometric enumeration of CD34+ cells tablets, 1807
powdered extract, 4612 (127), 6065 Fluoxymesterone, 1810
powder, 4609 Flow cytometry (1027), 6744 tablets, 1811
Ferric Floxuridine, 1752 Fluphenazine
ammonium citrate, 266, 5696 for injection, 1753 decanoate, 1812
ammonium citrate for oral solution, 266 Fluconazole, 1753 decanoate injection, 1812
ammonium sulfate, 5696 in dextrose injection, 1758 enanthate, 1813
ammonium sulfate, tenth-normal (0.1 N), for oral suspension, 1763 enanthate injection, 1814
5764 injection, 1755 hydrochloride, 1814
ammonium sulfate TS, 5753 in sodium chloride injection, 1760 hydrochloride elixir, 1815
chloride, 5696 tablets, 1765 hydrochloride injection, 1816
chloride CS, 5750 Flucytosine, 1766 hydrochloride oral solution, 1817
chloride TS, 5753 capsules, 1767 hydrochloride tablets, 1817
nitrate, 5696 oral suspension, 1767 Flurandrenolide, 1818
oxide, 5353 Fludarabine phosphate, 1768 cream, 1819
subsulfate solution, 1709 injection, 1770 lotion, 1820
sulfate, 1710, 5696 for injection, 1771 and neomycin sulfate cream, 2887
Ferrocyphen, 5696 Fludeoxyglucose F18 injection, 1794 and neomycin sulfate lotion, 2887
Ferroin TS, 5753 Fludrocortisone acetate, 1773 and neomycin sulfate ointment, 2888
Ferrosoferric oxide, 5354 tablets, 1773 ointment, 1820
Ferrous Flumazenil, 1775 tape, 1820
ammonium sulfate, 5696 injection, 1776 Flurazepam hydrochloride, 1821
ammonium sulfate, tenth-normal (0.1 N), Flumethasone pivalate, 1777 capsules, 1822
5764 cream, 1777 Flurbiprofen, 1823
fumarate, 1710 Flunisolide, 1778 sodium, 1825
fumarate and docusate sodium extended- nasal solution, 1779 sodium ophthalmic solution, 1826
release tablets, 1713 Flunixin meglumine, 1780 tablets, 1824
fumarate tablets, 1712 granules, 1781 Flutamide, 1826
gluconate, 1714 injection, 1782 capsules, 1827
gluconate capsules, 1716 paste, 1783 Fluticasone
gluconate oral solution, 1717 Fluocinolone acetonide, 1783 propionate and salmeterol inhalation
gluconate tablets, 1717 cream, 1784 aerosol, 1847
sulfate, 1718, 5696 and neomycin sulfate cream, 2887 propionate and salmeterol inhalation
sulfate, dried, 1721 ointment, 1785 powder, 1852
sulfate oral solution, 1720 topical solution, 1785 Fluticasone propionate, 1828
sulfate syrup, 1720 Fluocinonide, 1786 cream, 1830
sulfate tablets, 1720 cream, 1786 inhalation aerosol, 1831
sulfate TS, 5753 gel, 1787 inhalation powder, 1836
sulfate, acid, TS, 5750, 5753 ointment, 1787 lotion, 1841
0.07 N Ferrous ammonium sulfate, 5764 topical solution, 1788 nasal spray, 1842
Ferulic acid, 5696 Fluorene, 5697 ointment, 1846
Ferumoxides injection, 1722 9-Fluorenylmethy! chloroformate, 5697 Fluvastatin
Ferumoxsil oral suspension, 1724 Fluorescamine, 5697 capsules, 1860
Fetal bovine serum—quality attributes and Fluorescein, 1789 sodium, 1858
functionality tests (90), 6038 injection, 1789 Fluvoxamine maleate, 1862
Feverfew, 4615 sodium, 1790 tablets, 1863
powdered, 4616 sodium and benoxinate hydrochloride Folic acid, 1865
Fexofenadine hydrochloride, 1725 ophthalmic solution, 1792 assay (411), 6197
capsules, 1727 sodium ophthalmic strips, 1791 compounded oral solution, 1866
and pseudoephedrine hydrochloride sodium and proparacaine hydrochloride injection, 1866
extended-release tablets, 1731 ophthalmic solution, 1793 tablets, 1867
tablets, 1729 Fluorescence spectroscopy (853), 6648 Folin-ciocalteu phenol TS, 5753
Fibroblast growth factor-2, 5696 Fluorescence spectroscopy—theory and Fondaparinux sodium, 1868
Fibroblasts practice (1853), 8118 injection, 1872
bilayer synthetic scaffold, construct human, Fluorine Formaldehyde
1077 F 18 injection, fludeoxyglucose, 1794 solution, 1874, 5697, 5754
polyglactin scaffold, construct human, F 18 injection, sodium fluoride, 1795 TS, 5754
1082 4’-Fluoroacetophenone, 5697 Formamide, 5697
Filgrastim, 1739 Fluorometholone, 1796 anhydrous, 5697
Finasteride, 1743 acetate, 1798 Formic acid, 5697
tablets, 1744 acetate and tobramycin ophthalmic 96 percent, 5697
Fingolimod suspension, 4121 anhydrous, 5697
hydrochloride, 1745 cream, 1797 Formoterol fumarate, 1875
and neomycin sulfate ointment, 2887
Combined Index to USP 41 and NF 36 Forsk-Gener 1-23

Forskohlii, 4625 Garlic, 4642 Gelatin, 5360, 5697

extract, powdered, 4628 delayed-release tablets, 4648 film, absorbable, 1929
powdered, 4627 extract, powdered, 4646 sponge, absorbable, 1929
Foscarnet sodium, 1876 fluidextract, 4647 TS, 5754
Fosfomycin tromethamine, 1878 powdered, 4644 Gellan gum, 5362
Fosinopril sodium, 1879 Gaseous sterilization (1229.7), 7722 Gemcitabine
and hydrochlorothiazide tablets, 1882 Gastric fluid, simulated, TS, 5754, 5759 for injection, 1931
tablets, 1881 Gauze hydrochloride, 1930
Fosphenytoin sodium, 1884 absorbent, 1927 Gemfibrozil, 1932
injection, 1885 petrolatum, 1929 capsules, 1934
Fructose, 1887 tablets, 1934
injection, 1887 Gene therapy products (1047), 6900
and sodium chloride injection, 1888
Fuchsin
basic, 1889, 5672, 5697
Gel
Adapalene, 87
pyrogallol TS, 5754
sulfurous acid TS, 5754 Aluminum hydroxide, 170 General chapters
Fuller’s earth, chromatographic, 5683, 5697 Aluminum hydroxide, dried, 171 (1) Injections and implanted drug products
Fulvestrant, 1889 Aluminum hydroxide capsules, dried, 172 (parenterals)—product quality tests,
Fumaric acid, 5357 Aluminum hydroxide tablets, dried, 172 5915
Fuming Aluminum phosphate, 172 (2) Oral drug products—product quality
nitric acid, 5697 Aminobenzoic acid, 217 tests, 5921
sulfuric acid, 5697 Benzocaine, 473 (3) Topical and transdermal drug
Furazolidone, 1891 Benzocaine, butamben, and tetracaine products—product quality tests, 5926
oral suspension, 1891 hydrochloride, 480 (4) Mucosal drug products—product
tablets, 1891 Benzoyl! peroxide, 491 quality tests, 5933
Furfural, 5697 Betamethasone benzoate, 506 (5) Inhalation and nasal drug products
Furosemide, 1892 Chromatographic silica, 5683 general information and product quality
injection, 1893 Chromatographic silica mixture, 5684 tests, 5938
oral solution, 1893 Clindamycin phosphate, 998 (7) Labeling, 5945
tablets, 1894 Desoximetasone, 1190 (11) USP reference standards, 5951
Dimethyl sulfoxide, 1317 (17) Prescription container labeling, 5954
Dyclonine hydrochloride, 1458 (31) Volumetric apparatus, 5957
Erythromycin and benzoyl peroxide, (41) Balances, 5958
topical, 1572 (51) Antimicrobial effectiveness testing,
G Erythromycin, topical, 1567
Fluocinonide, 1787
5959
(55) Biological indicators—resistance
Gelatin, 5360 performance tests, 5962
G designations, 5697 Gelatin film, absorbable, 1929 (61) Microbiological examination of
Ga 67 injection, gallium citrate, 1924 Gelatin sponge, absorbable, 1929 nonsterile products: microbial
Gabapentin, 1896 Gelatin TS, 5754 enumeration tests, 5965
capsules, 1897 Hydrocortisone, 2059 (62) Microbiological examination of
tablets, 1898 Indomethacin, topical, 2155 nonsterile products: tests for specified
Gadodiamide, 1900 Metronidazole, 2723 organisms, 5971
injection, 1902 Naftifine hydrochloride, 2851 (63) Mycoplasma tests, 5978
Gadolinium (Gd III) acetate hydrate, 5697 Phenol topical, camphorated, 3264 (71) Sterility tests, 5984
Gadolinium sulfate, 5697 Salicylic acid, 3696 (81) Antibiotics—microbial assays, 5991
Gadopentetate dimeglumine injection, 1904 Selegiline compounded topical, 3738 (85) Bacterial endotoxins test, 6011
Gadoteridol, 1905 Silica, 5726 (87) Biological reactivity tests, in vitro,
injection, 1908 Silica, binder-free, 5726 6017
Gadoversetamide, 1909 Silica, chromatographic, 5683, 5726 (88) Biological reactivity tests, in vivo,
injection, 1911 Silica, impregnated glass microfiber sheet, 6020
Galactose, 5358 5726 (89.1) Collagenase |, 6029
Galageenan, 5359 Silica mixture, chromatographic, 5684, (89.2) Collagenase II, 6033
Galantamine 5726 (89) Enzymes used as ancillary materials in
extended-release capsules, 1912 Silica mixture, chromatographic, with pharmaceutical manufacturing, 6025
hydrobromide, 1920 chemically bound amino groups, 5726 (90) Fetal bovine serum—dquality attributes
oral solution, 1917 Silica mixture, dimethylsilanized, and functionality tests, 6038
tablets, 1918 chromatographic, 5726 (91) Calcium pantothenate assay, 6041
Gallamine triethiodide, 1923 Silica mixture, octadecylsilanized (92) Growth factors and cytokines used in
injection, 1924 chromatographic, 5726 cell therapy manufacturing, 6045
Gallium citrate Ga 67 injection, 1924 Silica mixture, octylsilanized, (111) Design and analysis of biological
Gamma cyclodextrin, 5317 chromatographic, 5726 assays, 6049
Ganciclovir, 1925 Silica, octadecylsilanized chromatographic, (115) Dexpanthenol assay, 6053
for injection, 1926 5726 (121) Insulin assays, 6054
oral suspension, 1927 Silica, porous, 5726 (121.1) Physicochemical analytical
Ganoderma lucidum fruiting body, 4629 Sodium fluoride and phosphoric acid, procedures for insulins, 6056
Ganoderma lucidum fruiting body powder, 3792 (123) Glucagon bioidentity tests, 6059
4632 Sodium sulfide topical, 3813 (124) Erythropoietin bioassays, 6061
Garcinia cambogia, 4635 Stannous fluoride, 3831 (126) Somatropin bioidentity tests, 6063
powdered, 4637 Tolnaftate, 4135 (127) Flow cytometric enumeration of
Garcinia hydroxycitrate Tretinoin, 4182 CD34+ cells, 6065
extract, powdered, 4638 (129) Analytical procedures for
Garcinia indica, 4639 recombinant therapeutic monoclonal
powdered, 4641 antibodies, 6070
USP 41
Separately calculate the percentages of the triterpene
derivatives in the portion of Centella asiatica
Triterpenes taken:
Result = (ru/ts) x (Cs/Cu) x F x 100
tu = peak response(s) of the triterpene derivative(s)
from the Sample solution
Is = peak response of asiaticoside from Standard
solution A
Cs = concentration of USP Asiaticoside RS in
Standard solution A (mg/mL)
Cu = concentration of Centella asiatica Triterpenes in
the Sample solution (mg/mL)
F = conversion factors for analytes: 1.00 for
asiaticoside, 1.017 for madecassoside, 1.017
for asiaticoside B, 0.526 for madecassic acid,
0.526 for terminolic acid, and 0.509 for
asiatic acid
Acceptance criteria: Add the percentages of different
pies derivatives: NLT 90.0% on the anhydrous
asis.
CONTAMINANTS
Delete the following:
°e HEAVY METALS, Method III (231): NMT 20 ppme corica 1.
Jan-2018)
e ARTICLES OF BOTANICAL ORIGIN, General Method for Pesti-
cide Residues Analysis (S61): Meets the requirements
e MICROBIAL ENUMERATION TESTS (2021): The total aerobic
microbial count does not exceed 103 cfu/g. The total
sa ane yeast and mold count does not exceed 102
cfu/g.
e MICROBIOLOGICAL PROCEDURES FOR ABSENCE OF SPECIFIED Mil-
CROORGANISMS (2022): Meets the requirements of the
tests for absence of Escherichia coli
SPECIFIC TESTS
e@ WATER DETERMINATION, Method | (921): NMT 5%
© OTHER REQUIREMENTS: Meets the requirements of the test
for Residual Solvents under Botanical Extracts (565)
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in well-closed contain-
ers, protected from light and moisture, and store at con-
trolled room temperature.
e LABELING: The label states the Latin binomial and, follow-
ing the official name, the part of the plant from which
the article was derived.
e USP REFERENCE STANDARDS (11)
USP Asiaticoside RS
USP Powdered Centella asiatica Extract RS
COMPOSITION
© CONTENT OF APIGENIN-7-GLUCOSIDE
Chamomile
DEFINITION
Chamomile consists of the dried flower heads of Matricaria
recutita L. (Matricaria chamomilla L., Matricaria chamomilla
L. var. courrantiana, Chamomilla recutita L.) Rauschert
(Fam. Asteraceae alt. Compositae). It contains NLT 0.4%
of blue volatile oil, NLT 0.3% of apigenin-7-glucoside, and
NLT 0.15% of bisabolol derivatives, calculated as
levomenol.
IDENTIFICATION
° A. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST
Standard solution: 1.0 mg/mL of borneol, 2.0 mg/mL
of bornyl acetate, and 0.4 mg/mL of guaiazulene in
toluene
Dietary Supplements / Chamomile 4519
Sample solution: Reduce 1.0 g of Chamomile to a
coarse powder, using a porcelain pestle and mortar.
Transfer to a 1.5-cm x 15-cm chromatographic column,
and tamp lightly with a short length of rubber hose.
Rinse the pestle and mortar twice, each time with
10 mL of methylene chloride. Pour the rinsings into the
column. Collect the percolate in a flask with a long,
narrow neck, and remove the solvent by evaporation
on a water bath. Dissolve the residue in 0.5 mL of
toluene.
Adsorbent: 0.25-mm layer of chromatographic silica
gel
Developing solvent: Chloroform
Spray reagent: Mix anisaldehyde, glacial acetic acid,
and methanol (0.5: 10: 85). Then carefully add 5 mL of
sulfuric acid to this solution.
Application volume: 10 uL, as 3-mm x 20-mm bands
Analysis
Samples: Standard solution and Sample solution
Examine the plate under short-wavelength UV light:
the Sample solution exhibits a number of quenching
areas, the largest of which is due to en-yne-di-
cycloether and has the same R; value as the band due
to bornyl acetate in the Standard solution. There is
also a band due to matricin near the line of applica-
tion. Spray the plate evenly with the Spray reagent.
Examine the plate in daylight while heating at
100°-105° for 5-10 min. The chromatogram ob-
tained from the Standard solution shows in the lower
third a brownish yellow zone that becomes violet-
gray after a few hours and is due to borneol; in the
middle a yellowish brown to gray zone due to bornyl
acetate; and in the upper third a deep red zone witl
a blue edge due to guaiazulene.
Acceptance criteria: The Sample solution exhibits a blue
zone due to matricin near the starting point; several
violet-red zones, one of which is due to bisabolol, at Rr
values between those of borneol and borny! acetate; a
brownish zone, due to en-yne-dicycloether, at an Rr
value corresponding to that of bornyl acetate; red
zones, due to terpenes, at Rr values similar to those of
guaiazulene; and other zones that appear in the middle
and lower parts of the chromatogram.
Analysis: Dissolve 0.25 g of dimethylaminobenzalde-
hyde in a mixture of 5 mL of phosphoric acid, 45 mL of
acetic acid, and 45 mL of water. Transfer 2.5 mL of this
solution and 0.1 mL of the Sample solution, prepared as
directed for Identification test A, to a test tube. Heat on
a water bath for 2 min, and allow to cool. Add 5 mL of
solvent hexane, and shake.
Acceptance criteria: The aqueous layer has a distinct 4
greenish blue or blue color. re
5
ray
Dilute phosphoric acid: Mix 5.0 mL of phosphoric acid [its}
in 50 mL of water. Dilute with water to 100 mL. Py
Solution A: 0.005 M solution of enerasis petosan ne]
phosphate. Adjust with Dilute phosphoric acid to a pH of [ea
2.55 + 0.05.
Solution B: Acetonitrile and methanol (13:7)
Mobile phase: See Table 1.
Table 1
Time Solution A Solution B
(min) (%) (%)
0 74 26
3 74 26
22 15 85
27 74 26
30 74 26
1-24 Gener-Gener
General chapters (continued)
(130) Protein A quality attributes, 6076
(151) Pyrogen test, 6083
(161) Medical devices—bacterial endotoxin
and pyrogen tests, 6085
(162) Diphtheria antitoxin potency testing
for human immune globulins, 6088
(165) Prekallikrein activator, 6090
(171) Vitamin Biz activity assay, 6091
(181) Identification—organic nitrogenous
bases, 6094
(191) Identification tests—general, 6094
(193) Identification—tetracyclines, 6100
(197) Spectrophotometric identification
tests, 6101
(201) Thin-layer chromatographic
identification test, 6102
(202) Identification of fixed oils by thin-
layer chromatography, 6103
(203) High-performance thin-layer
chromatography procedure for
identification of articles of botanical
origin, 6105
(206) Aluminum, 6107
(207) Test for 1,6-anhydro derivative for
enoxaparin sodium, 6108
(208) Anti-factor Xa and anti-factor Ila
assays for unfractionated and low
molecular weight heparins, 6113
(209) Low molecular weight heparin
molecular weight determinations, 6117
(210) Monosaccharide analysis, 6118
(211) Arsenic, 6124
(212) Oligosaccharide analysis, 6125
(221) Chloride and sulfate, 6139
(223) Dimethylaniline, 6140
(226) 4-Epianhydrotetracycline, 6140
(227) 4-Aminophenol in acetaminophen-
containing drug products, 6141
(228) Ethylene oxide and dioxane, 6142
(231) Heavy metals, 6145
(232) Elemental impurities—limits, 6147
(233) Elemental impurities—procedures,
6151
(241) Iron, 6155
(251) Lead, 6155
(261) Mercury, 6157
(267) Porosimetry by mercury intrusion,
6160
(268) Porosity by nitrogen
adsorption-desorption, 6163
(271) Readily carbonizable substances test,
6168
(281) Residue on ignition, 6168
(291) Selenium, 6169
(301) Acid-neutralizing capacity, 6169
(311) Alginates assay, 6170
(341) Antimicrobial agents—content, 6172
(345) Assay for citric acid/citrate and
phosphate, 6176
(351) Assay for steroids, 6177
(381) Elastomeric closures for injections,
6178
(391) Epinephrine assay, 6183
(401) Fats and fixed oils, 6184
(411) Folic acid assay, 6197
(413) Impurities testing in medical gases,
6201
(415) Medical gases assay, 6202
(425) lodometric assay—antibiotics, 6205
(429) Light diffraction measurement of
particle size, 6206
(431) Methoxy determination, 6212
(441) Niacin or niacinamide assay, 6213
(451) Nitrite titration, 6218
(461) Nitrogen determination, 6219
(466) Ordinary impurities, 6220
(467) Residual solvents, 6222
(469) Ethylene glycol, diethylene glycol,
and triethylene glycol in ethoxylated
substances, 6237
(471) Oxygen flask combustion, 6238
(481) Riboflavin assay, 6239
(501) Salts of organic nitrogenous bases,
6245
(503) Acetic acid in peptides, 6246
(503.1) Trifluoroacetic acid (TFA) in
peptides, 6247
(507) Protein determination procedures,
6248
(511) Single-steroid assay, 6253
(525) Sulfur dioxide, 6254
(531) Thiamine assay, 6260
(541) Titrimetry, 6268
(551) Vitamin E assay, 6272
(561) Articles of botanical origin, 6279
(563) Identification of articles of botanical
origin, 6293
(565) Botanical extracts, 6305
(571) Vitamin A assay, 6307
(580) Vitamin C assay, 6313
(581) Vitamin D assay, 6315
(591) Zinc determination, 6325
(601) Inhalation and nasal drug products:
aerosols, sprays, and powders—
performance quality tests, 6327
(602) Propellants, 6353
(603) Topical aerosols, 6354
(604) Leak rate, 6355
(610) Alternative microbiological sampling
methods for nonsterile inhaled and nasal
products, 6356
(611) Alcohol determination, 6358
(616) Bulk density and tapped density of
powders, 6360
(621) Chromatography, 6363
(631) Color and achromicity, 6375
(641) Completeness of solution, 6376
(643) Total organic carbon, 6377
(645) Water conductivity, 6378
(651) Congealing temperature, 6382
(659) Packaging and storage requirements,
6384
(660) Containers—glass, 6390
(661) Plastic packaging systems and their
materials of construction, 6396
(661.1) Plastic materials of construction,
6403
(661.2) Plastic packaging systems for
pharmaceutical use, 6424
(670) Auxiliary packaging components,
6428
(671) Containers—performance testing,
6436
(691) Cotton, 6443
(695) Crystallinity, 6445
(696) Characterization of crystalline solids
by microcalorimetry and solution
calorimetry, 6445
(697) Container content for injections,
6449
(698) Deliverable volume, 6450
(699) Density of solids, 6453
(701) Disintegration, 6455
(705) Quality attributes of tablets labeled
as having a functional score, 6457
(711) Dissolution, 6459
(721) Distilling range, 6469
(724) Drug release, 6471
Combined Index to USP 41 and NF 36
(729) Globule size distribution in lipid
injectable emulsions, 6478
(730) Plasma spectrochemistry, 6482
(731) Loss on drying, 6485
(733) Loss on ignition, 6486
(735) X-ray fluorescence spectrometry,
6486
(736) Mass spectrometry, 6491
(741) Melting range or temperature, 6497
(755) Minimum fill, 6499
(761) Nuclear magnetic resonance
spectroscopy, 6500
(771) Ophthalmic products—quality tests,
6510
(776) Optical microscopy, 6516
(781) Optical rotation, 6519
(785) Osmolality and osmolarity, 6527
(786) Particle size distribution estimation
by analytical sieving, 6530
(787) Subvisible particulate matter in
therapeutic protein injections, 6534
(788) Particulate matter in injections, 6537
(789) Particulate matter in ophthalmic
solutions, 6540
(790) Visible particulates in injections,
6542
(791) pH, 6543
(795) Pharmaceutical compounding—
nonsterile preparations, 6546
(797) Pharmaceutical compounding—
sterile preparations, 6554
(800) Hazardous drugs—handling in
healthcare settings, 6598
(801) Polarography, 6617
(811) Powder fineness, 6621
(821) Radioactivity, 6622
(823) Positron emission tomography drugs
for compounding, investigational, and
research uses, 6629
(831) Refractive index, 6639
(841) Specific gravity, 6639
(846) Specific surface area, 6640
(852) Atomic absorption spectroscopy,
6644
(853) Fluorescence spectroscopy, 6648
(854) Mid-infrared spectroscopy, 6654
(855) Nephelometry, turbidimetry, and
visual comparison, 6658
(857) Ultraviolet-visible spectroscopy, 6660
(861) Sutures—diameter, 6666
(871) Sutures—needle attachment, 6667
(881) Tensile strength, 6668
(891) Thermal analysis, 6669
(905) Uniformity of dosage units, 6673
(911) Viscosity—capillary methods, 6677
(912) Viscosity—rotational methods, 6679
(913) Viscosity—rolling ball method, 6684
(914) Viscosity—pressure driven methods,
6686
(921) Water determination, 6687
(941) Characterization of crystalline and
partially crystalline solids by X-ray
powder diffraction (XRPD), 6692
(1004) Mucosal drug products—
performance tests, 6699
<1005) Acoustic emission, 6702
(1010) Analytical data—interpretation and
treatment, 6706
(1024) Bovine serum, 6721
(1025) Pancreatin, 6734
(1027) Flow cytometry, 6744
(1030) Biological assay chapters—overview
and glossary, 6764
Combined Index to USP 41 and NF 36
General chapters (continued)
{1031) The biocompatibility of materials
used in drug containers, medical
devices, and implants, 6775
(1032) Design and development of
biological assays, 6785
(1033) Biological assay validation, 6803
(1034) Analysis of biological assays, 6818
(1039) Chemometrics, 6831
(1041) Biologics, 6849
(1043) Ancillary materials for cell, gene,
and tissue-engineered products, 6850
(1044) Cryopreservation of cells, 6858
(1046) Cellular and tissue-based products,
6871
(1047) Gene therapy products, 6900
(1048) Quality of biotechnological
products: analysis of the expression
construct in cells used for production of
r-DNA derived protein products, 6928
(1049) Quality of biotechnological
products: stability testing of
biotechnological/biological products,
6930
(1050) Viral safety evaluation of
biotechnology products derived from
cell lines of human or animal origin,
6935
(1050.1) Design, evaluation and
characterization of viral clearance
procedures, 6950
(1051) Cleaning glass apparatus, 6960
(1052) Biotechnology-derived articles—
amino acid analysis, 6961
(1053) Capillary electrophoresis, 6973
(1054) Biotechnology-derived articles—
isoelectric focusing, 6981
(1055) Biotechnology-derived articles—
peptide mapping, 6984
(1056) Biotechnology-derived articles—
polyacrylamide gel electrophoresis, 6991
(1057) Biotechnology-derived articles—
total protein assay, 6998
(1058) Analytical instrument qualification,
7005
(1059) Excipient performance, 7011
(1061) Color—instrumental measurement,
7040
(1062) Tablet compression
characterization, 7042
(1063) Shear cell methodology for powder
flow testing, 7054
(1064) Identification of articles of botanical
origin by high-performance thin-layer
chromatography procedure, 7065
(1065) lon chromatography, 7075
(1066) Physical environments that promote
safe medication use, 7078
(1072) Disinfectants and antiseptics, 7090
(1074) Excipient biological safety
evaluation guidelines, 7095
(1078) Good manufacturing practices for
bulk pharmaceutical excipients, 7100
(1079.1) Storage and transportation of
investigational drug products, 7130
(1079) Good storage and distribution
practices for drug products, 7120
(1080) Bulk pharmaceutical excipients—
certificate of analysis, 7133
(1084) Glycoprotein and glycan analysis—
general considerations, 7141
(1086) Impurities in drug substances and
drug products, 7152
(1087) Apparent intrinsic dissolution—
dissolution testing procedures for
rotating disk and stationary disk, 7155
(1088) In vitro and in vivo evaluation of
dosage forms, 7159
(1090) Assessment of drug product
performance—bioavailability,
bioequivalence, and dissolution, 7170
(1091) Labeling of inactive ingredients,
7178
(1092) The dissolution procedure:
development and validation, 7178
(1094) Capsules—dissolution testing and
related quality attributes, 7198
(1097) Bulk powder sampling procedures,
7206
(1102) Immunological test methods—
general considerations, 7219
(1103) Immunological test methods—
enzyme-linked immunosorbent assay
(ELISA), 7226
(1104) Immunological test methods—
immunoblot analysis, 7237
(1105) Immunological test methods—
surface plasmon resonance, 7248
(1106) Immunogenicity assays—design
and validation of immunoassays to
detect anti-drug antibodies, 7264
(1106.1) Immunogenicity assays—design
and validation of assays to detect anti-
drug neutralizing antibody, 7279
(1111) Microbiological examination of
nonsterile products: acceptance criteria
for pharmaceutical preparations and
substances for pharmaceutical use, 7297
(1112) Application of water activity
determination to nonsterile
pharmaceutical products, 7298
(1113) Microbial characterization,
identification, and strain typing, 7301
(1115) Bioburden control of nonsterile
drug substances and products, 7305
(1116) Microbiological contro! and
monitoring of aseptic processing
environments, 7312
(1117) Microbiological best laboratory
practices, 7325
(1118) Monitoring devices—time,
temperature, and humidity, 7331
(1119) Near-infrared spectroscopy, 7337
(1120) Raman spectroscopy, 7343
(1121) Nomenclature, 7351
(1125) Nucleic acid-based techniques—
general, 7353
(1126) Nucleic acid-based techniques—
extraction, detection, and sequencing,
7359
<1127) Nucleic acid-based techniques—
amplification, 7369
(1128) Nucleic acid-based techniques—
microarray, 7379
(1129) Nucleic acid-based techniques—
genotyping, 7385
(1130) Nucleic acid-based techniques—
approaches for detecting trace nucleic
acids (residual DNA testing), 7389
(1132) Residual host cell protein
measurement in biopharmaceuticals,
7393
(1136) Packaging and repackaging—single
unit containers, 7414
(1151) Pharmaceutical dosage forms, 7425
(1152) Animal drugs for use in animal
feeds, 7450
Gener-Gener 1-25
(1160) Pharmaceutical calculations in
pharmacy practice, 7451
(1163) Quality assurance in pharmaceutical
compounding, 7475
(1174) Powder flow, 7481
(1176) Prescription balances and
volumetric apparatus, 7485
(1177) Good packaging practices, 7492
(1178) Good repackaging practices, 7495
(1180) Human plasma, 7497
(1181) Scanning electron microscopy,
7519
(1184) Sensitization testing, 7529
(1191) Stability considerations in
dispensing practice, 7540
(1195) Significant change guide for bulk
pharmaceutical excipients, 7545
(1197) Good distribution practices for bulk
pharmaceutical excipients, 7556
(1207.1) Package integrity and test
method selection, 7585
(1207.2) Package integrity leak test
technologies, 7597
(1207.3) Package seal quality test
technologies, 7614
(1207) Sterile product packaging—integrity
evaluation, 7578
(1208) Sterility testing—validation of
isolator systems, 7617
(1210) Statistical tools for procedure
validation, 7622
(1211) Sterility assurance, 7633
(1216) Tablet friability, 7634
(1217) Tablet breaking force, 7635
(1222) Terminally sterilized pharmaceutical
products—parametric release, 7638
(1223) Validation of alternative
microbiological methods, 7642
(1223.1) Validation of alternative methods
to antibiotic microbial assays, 7656
(1224) Transfer of analytical procedures,
7663
(1225) Validation of compendial
procedures, 7665
(1226) Verification of compendial
procedures, 7671
(1227) Validation of microbial recovery
from pharmacopeial articles, 7672
(1228) Depyrogenation, 7676
(1228.1) Dry heat depyrogenation, 7681
(1228.3) Depyrogenation by filtration,
7685
(1228.5) Endotoxin indicators for
depyrogenation, 7688
(1229) Sterilization of compendial articles,
7692
(1229.1) Steam sterilization by direct
contact, 7698
(1229.2) Moist heat sterilization of
aqueous liquids, 7701
(1229.3) Monitoring of bioburden, 7706
(1229.4) Sterilizing filtration of liquids,
7709
(1229.5) Biological indicators for
sterilization, 7716
(1229.6) Liquid-phase sterilization, 7719
(1229.7) Gaseous sterilization, 7722
(1229.8) Dry heat sterilization, 7725
(1229.9) Physicochemical integrators and
indicators for sterilization, 7728
(1229.10) Radiation sterilization, 7728
(1229.11) Vapor phase sterilization, 7733
(1229.12) New sterilization methods, 7734
(1229.13) Sterilization-in-place, 7735
1-26 Gener-Gener
General chapters (continued)
(1229.14) Sterilization cycle development,
7737
(1229.15) Sterilization filtration of gases,
7740
(1230) Water for hemodialysis applications,
774)
(1231) Water for pharmaceutical purposes,
7742
(1234) Vaccines for human use—
polysaccharide and glycoconjugate
vaccines, 7778
(1235) Vaccines for human use—general
considerations, 7795
(1237) Virology test methods, 7812
(1238) Vaccines for human use—bacterial
vaccines, 7833
(1240) Virus testing of human plasma for
further manufacture, 7846
(1241) Water-solid interactions in
pharmaceutical systems, 7856
(1251) Weighing on an analytical balance,
7860
(1265) Written prescription drug
information—guidelines, 7866
(1285) Preparation of biological specimens
for histologic and immunohistochemical
analysis, 7868
(1285.1) Hematoxylin and eosin staining of
sectioned tissue for microscopic
examination, 7872
(1601) Products for nebulization—
characterization tests, 7874
(1602) Spacers and valved holding
chambers used with inhalation
aerosols—characterization tests, 7878
(1644) Theory and practice of electrical
conductivity measurements of solutions,
7890
(1660) Evaluation of the inner surface
durability of glass containers, 7897
(1661) Evaluation of plastic packaging
systems and their materials of
construction with respect to their user
safety impact, 7902
(1663) Assessment of extractables
associated with pharmaceutical
packaging/delivery systems, 7910
(1664) Assessment of drug product
leachables associated with
pharmaceutical packaging/delivery
systems, 7924
(1664.1) Orally inhaled and nasal drug
products, 7937
(1724) Semisolid drug products—
performance tests, 7944
(1730) Plasma spectrochemistry—theory
and practice, 7956
(1735) X-ray fluorescence spectrometry—
theory and practice, 7963
(1736) Applications of mass spectrometry,
7982
(1761) Applications of nuclear magnetic
resonance spectroscopy, 8004
(1771) Ophthalmic products—performance
tests, 8024
(1782) Vibrational circular dichroism
spectroscopytheory and practice, 8025
(1787) Measurement of subvisible
particulate matter in therapeutic protein
injections, 8038
(1788) Methods for the determination of
particulate matter in injections and
ophthalmic solutions, 8052
(1790) Visual inspection of injections, 8066
(1821) Radioactivity—theory and practice,
8084
(1823) Positron emission tomography
drugs—information, 8098
(1852) Atomic absorption spectroscopy—
theory and practice, 8109
(1853) Fluorescence spectroscopy—theory
and practice, 8118
(1854) Mid-infrared spectroscopy—theory
and practice, 8127
(1857) Ultraviolet-visible spectroscopy—
theory and practice, 8136
(1911) Rheometry, 8145
(2021) Microbial enumeration tests—
nutritional and dietary supplements,
8153
(2022) Microbiological procedures for
absence of specified microorganisms—
nutritional and dietary supplements,
8158
(2023) Microbiological attributes of
nonsterile nutritional and dietary
supplements, 8164
(2030) Supplemental information for
articles of botanical origin, 8168
(2040) Disintegration and dissolution of
dietary supplements, 8178
(2091) Weight variation of dietary
supplements, 8185
(2232) Elemental contaminants in dietary
supplements, 8186
(2250) Detection of irradiated dietary
supplements, 8190
(2251) Screening for undeclared drugs and
drug analogues, 8193
(2750) Manufacturing practices for dietary
supplements, 8210
(782) Vibrational circular dichroism
spectroscopy, 6520
General chapters
Applications of mass spectrometry (1736),
7982
Acetic acid in peptides (503), 6246
Acid-neutralizing capacity (301), 6169
Acoustic emission (1005), 6702
Alcohol determination (611), 6358
Alginates assay (311), 6170
Alternative microbiological sampling
methods for nonsterile inhaled and nasal
products (610), 6356
Aluminum (206), 6107
4-Aminophenol in acetaminophen-
containing drug products (227), 6141
Analysis of biological assays (1034), 6818
Analytical data—interpretation and
treatment (1010), 6706
Analytical instrument qualification (1058),
7005
Analytical procedures for recombinant
therapeutic monoclonal antibodies
(129), 6070
Ancillary materials for cell, gene, and
tissue-engineered products (1043), 6850
Animal drugs for use in animal feeds
(1152), 7450
Antibiotics—microbial assays (81), 5991
Anti-factor Xa and anti-factor lla assays for
unfractionated and low molecular
weight heparins (208), 6113
Antimicrobial agents—content (341), 6172
Combined Index to USP 41 and NF 36
Antimicrobial effectiveness testing (51),
5959
Apparent intrinsic dissolution—dissolution
testing procedures for rotating disk and
stationary disk (1087), 7155
Applications of nuclear magnetic
resonance spectroscopy (1761), 8004
Application of water activity determination
to nonsterile pharmaceutical products
(1112), 7298
Arsenic (211), 6124
Articles of botanical origin (561), 6279
Assay for citric acid/citrate and phosphate
(345), 6176
Assay for steroids (351), 6177
Assessment of drug product
performance—bioavailability,
bioequivalence, and dissolution (1090),
7170
Assessment of drug product leachables
associated with pharmaceutical
packaging/delivery systems (1664), 7924
Assessment of extractables associated with
pharmaceutical packaging/delivery
systems (1663), 7910
Atomic absorption spectroscopy (852),
6644
Atomic absorption spectroscopy—theory
and practice (1852), 8109
Auxiliary packaging components (670),
6428
Bacterial endotoxins test (85), 6011
Balances (41), 5958
Bioburden control of nonsterile drug
substances and products (1115), 7305
The biocompatibility of materials used in
drug containers, medical devices, and
implants (1031), 6775
Biological assay chapters—overview and
glossary (1030), 6764
Biological assay validation (1033), 6803
Biological indicators—resistance
performance tests (55), 5962
Biological indicators for sterilization
(1229.5), 7716
Biological reactivity tests, in vitro (87),
6017
Biological reactivity tests, in vivo (88),
6020
Biologics (1041), 6849
Biotechnology-derived articles—amino acid
analysis (1052), 6961
Biotechnology-derived articles—isoelectric
focusing (1054), 6981
Biotechnology-derived articles—peptide
mapping (1055), 6984
Biotechnology-derived articles—
polyacrylamide gel electrophoresis
(1056), 6991
Biotechnology-derived articles—total
protein assay (1057), 6998
Botanical extracts (565), 6305
Bovine serum (1024), 6721
Bulk density and tapped density of
powders (616), 6360
Bulk pharmaceutical excipients—certificate
of analysis (1080), 7133
Bulk powder sampling procedures (1097),
7206
Calcium pantothenate assay (91), 6041
Capillary electrophoresis (1053), 6973
Capsules—dissolution testing and related
quality attributes (1094), 7198
Cellular and tissue-based products (1046),
6871
Combined Index to USP 41 and NF 36
General chapters (continued)
Characterization of crystalline and partially
crystalline solids by X-ray powder
diffraction (XRPD) (941), 6692
Characterization of crystalline solids by
microcalorimetry and solution
calorimetry (696), 6445
Chemometrics (1039), 6831
Chloride and sulfate (221), 6139
Chromatography (621), 6363
Cleaning glass apparatus (1051), 6960
Collagenase | (89.1), 6029
Collagenase II (89.2), 6033
Color and achromicity (631), 6375
Color—instrumental measurement (1061),
7040
Completeness of solution (641), 6376
Congealing temperature (651), 6382
Container content for injections (697),
6449
Containers—glass (660), 6390
Containers—performance testing (671),
6436
Cotton (691), 6443
Cryopreservation of cells (1044), 6858
Crystallinity (695), 6445
Deliverable volume (698), 6450
Density of solids (699), 6453
Depyrogenation (1228), 7676
Depyrogenation by filtration (1228.3),
7685
Design, evaluation and characterization of
viral clearance procedures (1050.1),
6950
Design and analysis of biological assays
(111), 6049
Design and development of biological
assays (1032), 6785
Detection of irradiated dietary supplements
(2250), 8190
Dexpanthenol assay (115), 6053
Dimethylaniline (223), 6140
Diphtheria antitoxin potency testing for
human immune globulins (162), 6088
Disinfectants and antiseptics (1072), 7090
Disintegration (701), 6455
Disintegration and dissolution of dietary
supplements (2040), 8178
Dissolution (711), 6459
The dissolution procedure: development
and validation (1092), 7178
Distilling range (721), 6469
Drug release (724), 6471
Dry heat depyrogenation (1228.1), 7681
Dry heat sterilization (1229.8), 7725
Elastomeric closures for injections (381),
6178
Elemental contaminants in dietary
supplements (2232), 8186
Elemental impurities—limits (232), 6147
Elemental impurities—procedures (233),
6151
Endotoxin indicators for depyrogenation
(1228.5), 7688
Enzymes used as ancillary materials in
pharmaceutical manufacturing (89),
6025
4-Epianhydrotetracycline (226), 6140
Epinephrine assay (391), 6183
Erythropoietin bioassays (124), 6061
Ethylene glycol, diethylene glycol, and
triethylene glycol in ethoxylated
substances (469), 6237
Ethylene oxide and dioxane (228), 6142
Evaluation of plastic packaging systems
and their materials of construction with
respect to their user safety impact
(1661), 7902
Evaluation of the inner surface durability of
glass containers (1660), 7897
Excipient biological safety evaluation
guidelines (1074), 7095
Excipient performance (1059), 7011
Fats and fixed oils (401), 6184
Fetal bovine serum—quality attributes and
functionality tests (90), 6038
Flow cytometric enumeration of CD34+
cells (127), 6065
Flow cytometry (1027), 6744
Fluorescence spectroscopy (853), 6648
Fluorescence spectroscopy—theory and
practice (1853), 8118
Folic acid assay (411), 6197
Gaseous sterilization (1229.7), 7722
Gene therapy products (1047), 6900
Globule size distribution in lipid injectable
emulsions (729), 6478
Glucagon bioidentity tests (123), 6059
Glycoprotein and glycan analysis—general
considerations (1084), 7141
Good distribution practices for bulk
pharmaceutical excipients (1197), 7556
Good manufacturing practices for bulk
pharmaceutical excipients (1078), 7100
Good packaging practices (1177), 7492
Good repackaging practices (1178), 7495
Good storage and distribution practices for
drug products (1079), 7120
Growth factors and cytokines used in cell
therapy manufacturing (92), 6045
Hazardous drugs—handling in healthcare
settings (800), 6598
Heavy metals (231), 6145
Hematoxylin and eosin staining of
sectioned tissue for microscopic
examination (1285.1), 7872
High-performance thin-layer
chromatography procedure for
identification of articles of botanical
origin (203), 6105
Human plasma (1180), 7497
Identification of articles of botanical origin
(563), 6293
Identification of articles of botanical origin
by high-performance thin-layer
chromatography procedure (1064),
7065
Identification of fixed oils by thin-layer
chromatography (202), 6103
Identification—organic nitrogenous bases
(181), 6094
Identification tests—general (191), 6094
Identification—tetracyclines (193), 6100
Immunogenicity assays—design and
validation of assays to detect anti-drug
neutralizing antibody (1106.1), 7279
Immunogenicity assays—design and
validation of immunoassays to detect
anti-drug antibodies (1106), 7264
Immunological test methods—surface
plasmon resonance (1105), 7248
Immunological test methods—enzyme-
linked immunosorbent assay (ELISA)
(1103), 7226
Immunological test methods—general
considerations (1102), 7219
Immunological test methods—immunoblot
analysis (1104), 7237
Gener-Gener 1-27
Impurities in drug substances and drug
products (1086), 7152
Impurities testing in medical gases (413),
6201
Inhalation and nasal drug products:
aerosols, sprays, and powders—
performance quality tests (601), 6327
Inhalation and nasal drug products—
general information and product quality
tests (5), 5938
Injections and implanted drug products
(parenterals)—product quality tests (1),
5915
Insulin assays (121), 6054
In vitro and in vivo evaluation of dosage
forms (1088), 7159
lodometric assay—antibiotics (425), 6205
lon chromatography (1065), 7075
Iron (241), 6155
Labeling (7), 5945
Labeling of inactive ingredients (1091),
7178
Lead (251), 6155
Leak rate (604), 6355
Light diffraction measurement of particle
size (429), 6206
Liquid-phase sterilization (1229.6), 7719
Loss on drying (731), 6485
Loss on ignition (733), 6486
Low molecular weight heparin molecular
weight determinations (209), 6117
Manufacturing practices for dietary
supplements (2750), 8210
Mass spectrometry (736), 6491
Measurement of subvisible particulate
matter in therapeutic protein injections
(1787), 8038
Medical devices—bacterial endotoxin and
pyrogen tests (161), 6085
Medical gases assay (415), 6202
Melting range or temperature (741), 6497
Mercury (261), 6157
Methods for the determination of
particulate matter in injections and
ophthalmic solutions (1788), 8052
Methoxy determination (431), 6212
Microbial characterization, identification,
and strain typing (1113), 7301
Microbial enumeration tests—nutritional
and dietary supplements (2021), 8153
Microbiological attributes of nonsterile
nutritional and dietary supplements
(2023), 8164
Microbiological best laboratory practices
(1117), 7325
Microbiological control and monitoring of
aseptic processing environments (1116),
7312
Microbiological examination of nonsterile
products: acceptance criteria for
pharmaceutical preparations and
substances for pharmaceutical use
(1111), 7297
Microbiological examination of nonsterile
products: microbial enumeration tests
(61), 5965
Microbiological examination of nonsterile
products: tests for specified organisms
(62), 5971
Microbiological procedures for absence of
specified microorganisms—nutritional
and dietary supplements (2022), 8158
Mid-infrared spectroscopy (854), 6654
Mid-infrared spectroscopy—theory and
practice (1854), 8127
|-28 | Gener-Gener
General chapters (continued)
Minimum fill (755), 6499
Moist heat sterilization of aqueous liquids
(1229.2), 7701
Monitoring devices—time, temperature,
and humidity (1118), 7331
Monitoring of bioburden (1229.3), 7706
Monosaccharide analysis (210), 6118
Mucosal drug products—performance tests
(1004), 6699
Mucosal drug products—product quality
tests (4), 5933
Mycoplasma tests (63), 5978
Near-infrared spectroscopy (1119), 7337
Nephelometry, turbidimetry, and visual
comparison (855), 6658
New sterilization methods (1229.12), 7734
Niacin or niacinamide assay (441), 6213
Nitrite titration (451), 6218
Nitrogen determination (461), 6219
Nomenclature (1121), 7351
Nuclear magnetic resonance spectroscopy
(761), 6500
Nucleic acid-based techniques—
amplification (1127), 7369
Nucleic acid-based techniques—
approaches for detecting trace nucleic
acids (residual DNA testing) (1130),
7389
Nucleic acid-based techniques—extraction,
detection, and sequencing (1126), 7359
Nucleic acid-based techniques—general
(1125), 7353
Nucleic acid-based techniques—
genotyping (1129), 7385
Nucleic acid-based techniques—microarray
(1128), 7379
Oligosaccharide analysis (212), 6125
Ophthalmic products—performance tests
(1771), 8024
Ophthalmic products—quality tests (771),
6510
Optical microscopy (776), 6516
Optical rotation (781), 6519
Oral drug products—product quality tests
(2), 5921
Orally inhaled and nasal drug products
(1664.1), 7937
Ordinary impurities (466), 6220
Osmolality and osmolarity (785), 6527
Oxygen flask combustion (471), 6238
Package integrity and test method
selection (1207.1), 7585
Package integrity leak test technologies
(1207.2), 7597
Package seal quality test technologies
(1207.3), 7614
Packaging and repackaging—single unit
containers (1136), 7414
Packaging and storage requirements (659),
6384
Pancreatin (1025), 6734
Particle size distribution estimation by
analytical sieving (786), 6530
Particulate matter in injections (788), 6537
Particulate matter in ophthalmic solutions
(789), 6540
pH (791), 6543
Pharmaceutical calculations in pharmacy
practice (1160), 7451
Pharmaceutical compounding—nonsterile
preparations (795), 6546
Pharmaceutical compounding—sterile
preparations (797), 6554
Pharmaceutical dosage forms (1151), 7425
Combined Index to USP 41 and NF 36
Physical environments that promote safe
medication use (1066), 7078
Physicochemical analytical procedures for
insulins (121.1), 6056
Physicochemical integrators and indicators
for sterilization (1229.9), 7728
Plasma spectrochemistry (730), 6482
Plasma spectrochemistry—theory and
practice (1730), 7956
Plastic materials of construction (661.1),
6403
Plastic packaging systems and their
materials of construction (661), 6396
Plastic packaging systems for
pharmaceutical use (661.2), 6424
Polarography (801), 6617
Porosimetry by mercury intrusion (267),
6160
Porosity by nitrogen adsorption—desorption
(268), 6163
Positron emission tomography drugs for
compounding, investigational, and
research uses (823), 6629
Positron emission tomography drugs—
information (1823), 8098
Powder fineness (811), 6621
Powder flow (1174), 7481
Prekallikrein activator (165), 6090
Preparation of biological specimens for
histologic and immunohistochemical
analysis (1285), 7868
Prescription balances and volumetric
apparatus (1176), 7485
Prescription container labeling (17), 5954
Products for nebulization—characterization
tests (1601), 7874
Propellants (602), 6353
Protein A quality attributes (130), 6076
Protein determination procedures (507),
6248
Pyrogen test (151), 6083
Quality assurance in pharmaceutical
compounding (1163), 7475
Quality attributes of tablets labeled as
having a functional score (705), 6457
Quality of biotechnological products:
analysis of the expression construct in
cells used for production of r-DNA
derived protein products (1048), 6928
Quality of biotechnological products:
stability testing of biotechnological/
biological products (1049), 6930
Radiation sterilization (1229.10), 7728
Radioactivity (821), 6622
Radioactivity—theory and practice (1821),
8084
Raman spectroscopy (1120), 7343
Readily carbonizable substances test (271),
6168
Refractive index (831), 6639
Residual host cell protein measurement in
biopharmaceuticals (1132), 7393
Residual solvents (467), 6222
Residue on ignition (281), 6168
Rheometry (1911), 8145
Riboflavin assay (481), 6239
Salts of organic nitrogenous bases (501),
6245
Scanning electron microscopy (1181),
7519
Screening for undeclared drugs and drug
analogues (2251), 8193
Selenium (291), 6169
Semisolid drug products—performance
tests (1724), 7944
Sensitization testing (1184), 7529
Shear cell methodology for powder flow
testing (1063), 7054
Significant change guide for bulk
pharmaceutical excipients (1195), 7545
Single-steroid assay (511), 6253
Somatropin bioidentity tests (126), 6063
Spacers and valved holding chambers used
with inhalation aerosols—
characterization tests (1602), 7878
Specific gravity (841), 6639
Specific surface area (846), 6640
Spectrophotometric identification tests
(197), 6101
Stability considerations in dispensing
practice (1191), 7540
Statistical tools for procedure validation
(1210), 7622
Steam sterilization by direct contact
(1229.1), 7698
Sterile product packaging—integrity
evaluation (1207), 7578
Sterility assurance (1211), 7633
Sterility testing—validation of isolator
systems (1208), 7617
Sterility tests (71), 5984
Sterilization cycle development (1229.14),
7737
Sterilization filtration of gases (1229.15),
7740
Sterilization-in-place (1229.13), 7735
Sterilization of compendial articles (1229),
7692
Sterilizing filtration of liquids (1229.4),
7709
Storage and transportation of
investigational drug products (1079.1),
7130
Subvisible particulate matter in therapeutic
protein injections (787), 6534
Sulfur dioxide (525), 6254
Supplemental information for articles of
botanical origin (2030), 8168
Sutures—diameter (861), 6666
Sutures—needle attachment (871), 6667
Tablet breaking force (1217), 7635
Tablet compression characterization
(1062), 7042
Tablet friability (1216), 7634
Tensile strength (881), 6668
Terminally sterilized pharmaceutical
products—parametric release (1222),
7638
Test for 1,6-anhydro derivative for
enoxaparin sodium (207), 6108
Theory and practice of electrical
conductivity measurements of solutions
(1644), 7890
Thermal analysis (891), 6669
Thiamine assay (531), 6260
Thin-layer chromatographic identification
test (201), 6102
Titrimetry (541), 6268
Topical aerosols (603), 6354
Topical and transdermal drug products—
product quality tests (3), 5926
Total organic carbon (643), 6377
Transfer of analytical procedures (1224),
7663
Trifluoroacetic acid (TFA) in peptides
(503.1), 6247
Ultraviolet-visible spectroscopy (857), 6660
Ultraviolet-visible spectroscopy—theory
and practice (1857), 8136
Uniformity of dosage units (905), 6673
Combined Index to USP 41 and NF 36 Gener-Good 1-29

General chapters (continued) Preservation, packaging, storage, and Gluconolactone, 1958

USP reference standards (11), 5951 labeling, 12 Glucosamine
Vaccines for human use—bacterial vaccines Terms and definitions, 9 and chondroitin sulfate sodium tablets,
(1238), 7833 Test results, 9 4667
Vaccines for human use—general Testing practices and procedures, 7 chondroitin sulfate sodium, and
considerations (1235), 7795 Title and revision, 3 methylsulfonylmethane tablets, 4674
Vaccines for human use—polysaccharide General tests for reagents, 5660 hydrochloride, 4669
and glycoconjugate vaccines (1234), Geneticin, 5697 and methylsulfonylmethane tablets, 4672
7778 Gentamicin sulfate potassium chloride, 4670
Validation of alternative microbiological injection, 1935 sulfate sodium chloride, 4671
methods (1223), 7642 and prednisolone acetate ophthalmic tablets, 4669
Validation of compendial procedures ointment, 1942 Glucose, 5698
(1225), 7665 and prednisolone acetate ophthalmic enzymatic test strip, 1959
Validation of microbial recovery from suspension, 1943 liquid, 5364
pharmacopeial articles (1227), 7672 sulfate, 1936 oxidase-chromogen TS, 5754
Validation of alternative methods to sulfate and betamethasone acetate D-Glucuronolactone, 5698
antibiotic microbial assays (1223.1), ophthalmic solution, 1939 Glutamic acid, 4676, 5698
7656 sulfate and betamethasone valerate L-Glutamic acid, 5698
Vapor phase sterilization (1229.11), 7733 ointment, 1939 hydrochloride, 5365
Verification of compendial procedures sulfate and betamethasone valerate otic Glutamine, 1959
(1226), 7671 solution, 1940 L-Glutamine, 5698
Vibrational circular dichroism sulfate and betamethasone valerate topical Glutaral
spectroscopytheory and practice (1782), solution, 1941 concentrate, 1960
8025 sulfate cream, 1937 disinfectant solution, 5365
Vibrational circular dichroism spectroscopy sulfate ointment, 1938 Glutathione, 4677
(782), 6520 sulfate ophthalmic ointment, 1938 Glyburide, 1960
Viral safety evaluation of biotechnology sulfate ophthalmic solution, 1938 and metformin hydrochloride tablets, 1964
products derived from cell lines of uterine infusion, 1935 tablets, 1961
human or animal origin (1050), 6935 Gentian violet, 1943 Glycerin, 1967, 5698
Virology test methods (1237), 7812 cream, 1944 base TS, 5754
Virus testing of human plasma for further topical solution, 1945 ophthalmic solution, 1968
manufacture (1240), 7846 Ginger, 4650 oral solution, 1969
Viscosity—capillary methods (911), 6677 capsules, 4656 suppositories, 1969
Viscosity—pressure driven methods (914), powdered, 4652 Glyceryl
6686 tincture, 4654 behenate, 5366
Viscosity—rolling ball method (913), 6684 Ginkgo, 4657 dibehenate, 5367
Viscosity—rotational methods (912), 6679 capsules, 4663 distearate, 5369
Visible particulates in injections (790), extract, powdered, 4660 monocaprylate, 5370
6542 tablets, 4665 monocaprylocaprate, 5372
Visual inspection of injections (1790), 8066 Ginseng monolinoleate, 5375
Vitamin A assay (571), 6307 American, 4422 monooleate, 5376
Vitamin By2 activity assay (171), 6091 Asian, 4441 monostearate, 5377
Vitamin C assay (580), 6313 capsules, American, 4426 tristearate, 5378
Vitamin D assay (581), 6315 extract, powdered American, 4425 Glycine, 1969
Vitamin E assay (551), 6272 extract, powdered Asian, 4444 irrigation, 1970
Volumetric apparatus (31), 5957 powdered, American, 4423 Glycolic acid, 5698
Water conductivity (645), 6378 powdered, Asian, 4442 Glycoprotein and glycan analysis—general
Water determination (921), 6687 tablets, American, 4428 considerations (1084), 7141
Water for hemodialysis applications (1230), tablets, Asian, 4445 Glycopyrrolate, 1970
774) Tienchi, root and rhizome, 4901 injection, 1973
Water for pharmaceutical purposes (1231), Tienchi, root and rhizome dry extract, tablets, 1974
7742 4909 Glycyl-L-glutamine, 4678
Water-solid interactions in pharmaceutical Tienchi, root and rhizome powder, 4903 Glycyl-L-tyrosine, 4679
systems (1241), 7856 Girard reagent T, 5697 Gold
Weighing on an analytical balance (1251), Gitoxin, 5697 chloride, 5698
7860 Glacial acetic acid, 69, 5697 chloride TS, 5754
Weight variation of dietary supplements TS, 5754 sodium thiomalate, 1975
(2091), 8185 Glass wool, 5698 sodium thiomalate injection, 1976
Written prescription drug information— Glaze, pharmaceutical, 5363 Goldenseal, 4681
guidelines (1265), 7866 Glimepiride, 1945 extract, powdered, 4684
X-ray fluorescence spectrometry (735), and pioglitazone tablets, 3314 powdered, 4682
6486 tablets, 1947 Gonadorelin
X-ray fluorescence spectrometry—theory Glipizide, 1949 acetate, 1977
and practice (1735), 7963 and metformin hydrochloride tablets, 1953 hydrochloride, 1979
Zinc determination (591), 6325 tablets, 1951 for injection, 1976
Globule size distribution in lipid injectable Gonadotropin
emulsions (729), 6478 chorionic, 1981
Globulin chorionic, for injection, 1982
General notices and requirements, 1 immune, 1955 Good distribution practices for bulk
Conformance to standards, 3 reagent, anti-human, 5671 pharmaceutical excipients (1197), 7556
Monograph components, 5 RH, (D) immune, 1956 Good manufacturing practices for bulk
Monographs and general chapters, 5 Glucagon, 1956 pharmaceutical excipients (1078), 7100
Official status and legal recognition, 3 for injection, 1957 Good packaging practices (1177), 7492
Prescribing and dispensing, 11 Glucagon bioidentity tests (123), 6059 Good repackaging practices (1178), 7495
D-Gluconic acid, 50 percent in water, 5698
I-30 Good -Hydro Combined Index to USP 41 and NF 36

Good storage and distribution practices for Guide to general chapters Hexylene glycol, 5382
drug products (1079), 7120 charts, 5865 Hexylresorcinol, 2034
Goserelin acetate, 1983 table of contents, 13 lozenges, 2035
Goserelin Implants, 1985 Gutta percha, 2013 High-performance thin-layer chromatography
Government liaisons to expert committees Gymnema, 4693 procedure for identification of articles of
and expert panels, xviii extract, native, 4696 botanical origin (203), 6105
Graftskin, 1086 extract, purified, 4697 Histamine
Gramicidin, 1989 powdered, 4695 dihydrochloride, 5699
and neomycin and polymyxin B sulfates phosphate, 2036
cream, 2902 phosphate injection, 2036
and neomycin and polymyxin B sulfates Histidine, 2037
and hydrocortisone acetate cream, 2902 L-Histidine hydrochloride monohydrate, 5699
and neomycin and polymyxin B sulfates Holy basil leaf, 4704
ophthalmic solution, 2902 extract, powdered, 4708
and neomycin sulfate ointment, 2888 powdered, 4706
nystatin, neomycin sulfate, and Halazone, 2014 Homatropine
triamcinolone acetonide cream, 2991 tablets for solution, 2014 hydrobromide, 2038
nystatin, neomycin sulfate, and Halcinonide, 2014 hydrobromide ophthalmic solution, 2039
triamcinolone acetonide ointment, 2992 cream, 2015 methylbromide, 2039
Granisetron, 1989 ointment, 2016 methylbromide and hydrocodone
Granisetron hydrochloride, 1990 topical solution, 2018 bitartrate tablets, 2054
injection, 1992 Halobetasol propionate, 2018 methylbromide tablets, 2040
oral suspension, 1993 Haloperidol, 2019 Homosalate, 2041
tablets, 1994 decanoate, 2022 Honey, purified, 5382
Granules injection, 2020 Horse chestnut, 4526
Flunixin meglumine, 1781 oral solution, 2021 extract, powdered, 4529
Montelukast sodium, oral, 2797 tablets, 2021 powdered, 4527
Grape seeds oligomeric proanthocyanidins, Halothane, 2024 Horseradish peroxidase conjugated to goat
4685 Hawthorn leaf anti-mouse IgG, 5699
Gravity, specific (841), 6639 with flower, 4699 Human plasma (1180), 7497
Green with flower, powdered, 4701 Hyaluronidase
brilliant, 5675, 5705, 5745 Hazardous drugs—handling in healthcare injection, 2042
FCF, fast, 5696 settings (800), 6598 for injection, 2042
soap, 1995 Heavy metals (231), 6145 Hydralazine hydrochloride, 2043
soap tincture, 1996 Heavy metals in reagents, 5662 injection, 2045
Green tea Helium, 2025 oral solution, 2045
extract, decaffeinated, powdered, 4687 oxygen certified standard, 5713 tablets, 2046
Griseofulvin, 1996 Hematein, 5698 Hydrazine
capsules, 1997 Hematoxylin, 5698 dihydrochloride, 5699
oral suspension, 1998 TS, Delafield’s, 5753 hydrate, 85% in water, 5699
tablets, 1999 Hematoxylin and eosin staining of sectioned sulfate, 5700
tablets, ultramicrosize, 2000 tissue for microscopic examination Hydrindantin, 5700
Growth factors and cytokines used in cell (1285.1), 7872 Hydriodic acid, 5700
therapy manufacturing (92), 6045 Hemoglobin, bovine, 5698 Hydrobromic acid, 5700
Guaiacol, 5698 Heparin Hydrochloric acid, 5383, 5700
Guaifenesin, 2001, 5698 lock flush solution, 2025 alcoholic, tenth-molar (0.1 M), 5765
capsules, 2002 sodium, 2026 buffer, 5676
and codeine phosphate oral solution, 2004 sodium injection, 2031 diluted, 5383, 5690, 5700
and dyphylline oral solution, 1462 Hepatitis B half-normal (0.5 N), 5765
and dyphylline tablets, 1462 immune globulin, 2032 half-normal (0.5 N) in methanol, 5765
and pseudoephedrine hydrochloride 1-Heptadecanol, 5699 injection, 2047
capsules, 2005 Heptafluorobutyric acid, 5699 normal (1 N), 5765
pseudoephedrine hydrochloride, and Heptakis-(2,6-di-O-methyl)-B-cyclodextrin, 0.001 N TS, 5754
dextromethorphan hydrobromide 5699 0.01 M TS, 5754
capsules, 2006 n-Heptane, 5699 0.025 N TS, 5754
and theophylline capsules, 4042 chromatographic, 5683, 5699 0.36 N TS, 5754
and theophylline oral solution, 4043 Heptyl p-hydroxybenzoate, 5699 0.05 N TS, 5754
for injection, 2002 Hesperidin, 4703 2N
oral solution, 2003 Hexachlorophene, 2032 3N
tablets, 2003 cleansing emulsion, 2033 5N
Guanabenz acetate, 2007 liquid soap, 2033 6N
tablets, 2008 Hexadecyl hexadecanoate, 5699 0.1 N VS, 5765
Guanethidine monosulfate, 2009 Hexadecyltrimethylammonium bromide, 0.02 N VS, 5765
tablets, 2010 5699 0.08 N hydrochloric acid TS, 5754
Guanfacine Hexadimethrine bromide, 5699 0.125 N hydrochloric acid TS, 5754
hydrochloride, 2011 Hexamethyldisilazane, 5699 Hydrochloride
tablets, 2011 Hexamethyleneimine, 5699 fingolimod, 1745
Guanidine hydrochloride, 5698 Hexamethylenetetramine, 5699 nile blue, 5746
Guanidine isothiocyanate, 5698 n-Hexane, 5699 Hydrochlorothiazide, 2048
Guanine hydrochloride, 5698 Hexane, solvent, 5699, 5716, 5733 and amiloride hydrochloride tablets, 206
Guar gum, 5380 chromatographic, 5684, 5699 amlodipine, valsartan, tablets, 257
Guggul, 4689 Hexanes, 5699 and bisoprolol fumarate tablets, 544
extract, native, 4690 Hexanitrodiphenylamine, 5693, 5699 candesartan cilexetil, tablets, 664
extract, purified, 4691 Hexanophenone, 5699 capsules, 2049
tablets, 4692 Hexylamine, 5699 and captopril tablets, 680
Combined Index to USP 41 and NF 36
Hydrochlorothiazide (continued)
and enalapril maleate tablets, 1503
and fosinopril tablets, 1882
and irbesartan tablets, 2233
and lisinopril tablets, 2434
and losartan potassium tablets, 2480
and methyldopa tablets, 2670
and metoprolol tartrate tablets, 2717
and moexipril hydrochloride and tablets,
2782
and propranolo! hydrochloride tablets,
3497
and quinapril tablets, 3543
and spironolactone oral suspension, 3828
and spironolactone tablets, 3829
tablets, 2051
and telmisartan tablets, 3962
and timolol maleate tablets, 4100
and triamterene capsules, 4198
and triamterene tablets, 4200
and valsartan tablets, 4279
Hydrocodone bitartrate, 2052
and acetaminophen tablets, 2053
and homatropine methylbromide tablets,
2054
tablets, 2052
Hydrocodone diol, 5700
Hydrocortisone, 2057
acetate, 2063
acetate and chloramphenicol for
ophthalmic suspension, 866
acetate and colistin and neomycin sulfates
otic suspension, 1075
acetate cream, 2064
acetate lotion, 2064
acetate, neomycin and polymyxin B
sulfates, and bacitracin ointment, 2895
acetate, neomycin and polymyxin B
sulfates, and bacitracin ophthalmic
ointment, 2896
acetate, neomycin and polymyxin B
sulfates, and bacitracin zinc ophthalmic
ointment, 2899
acetate and neomycin and polymyxin B
sulfates cream, 2904
acetate, neomycin and polymyxin B
sulfates, and gramidicin cream, 2902
acetate and neomycin and polymyxin B
sulfates ophthalmic suspension, 2904
acetate and neomycin sulfate cream, 2889
acetate and neomycin sulfate lotion, 2890
acetate and neomycin sulfate ointment,
2890
acetate and neomycin sulfate ophthalmic
suspension, 2890
acetate ointment, 2065
acetate ophthalmic ointment, 2065
acetate and oxytetracycline hydrochloride
ophthalmic suspension, 3129
acetate, penicillin G, neomycin, polymyxin
B, and hydrocortisone sodium succinate
topical suspension, 3193
acetate, penicillin G procaine, and
neomycin and polymyxin B sulfates
topical suspension, 3210
and acetic acid otic solution, 2062
and clioquinol cream, 1004
and clioquino! ointment, 1005
and neomycin and polymyxinB sulfates
ophthalmic suspension, 2903
and neomycin and polymyxin B sulfates
otic solution, 2903
and neomycin and polymyxin B sulfates
otic suspension, 2903
and neomycin sulfate cream, 2888
and neomycin sulfate ointment, 2889
and neomycin sulfate otic suspension,
2889
and oxytetracycline hydrochloride
ointment, 3130
and polymyxinB sulfate otic solution,
3351
butyrate, 2066
butyrate cream, 2067
cream, 2058
gel, 2059
hemisuccinate, 2067
lotion, 2059
neomycin and polymyxin B sulfates and
bacitracin zinc ointment, 2898
neomycin and polymyxin B sulfates and
bacitracin zinc ophthalmic ointment,
2898
ointment, 2060
rectal suspension, 2060
sodium phosphate, 2068
sodium phosphate injection, 2069
sodium succinate, 2070
sodium succinate for injection, 2071
sodium succinate, penicillin G, neomycin,
polymyxin B, and hydrocortisone acetate
topical suspension, 3193
tablets, 2061
valerate, 2072
valerate cream, 2073
valerate ointment, 2073
Hydroflumethiazide, 2074
tablets, 2074
Hydrofluoric acid, 5700
Hydrogen
peroxide, 10 percent, 5700
peroxide, 30 percent, 5700
peroxide, 30 percent, unstabilized, 5700
peroxide, 50 percent in water, 5700
peroxide concentrate, 2075
peroxide solution, 5700
peroxide topical solution, 2076
peroxide TS, 5754
sulfide, 5700
sulfide detector tube, 5700
sulfide TS, 5754
Hydrogenated lanolin, 5416
Hydrogenated polydextrose, 5495
Hydrogenated vegetable oil, 5649
Hydromorphone hydrochloride, 2077
injection, 2079
oral solution, 2079
tablets, 2081
Hydroquinone, 2082, 5700
cream, 2082
topical solution, 2082
Hydroxocobalamin, 2083
injection, 2084
Hydroxy naphthol blue, 5700
3’-Hydroxyacetophenone, 5700
4’-Hydroxyacetophenone, 5700
Hydroxyamphetamine hydrobromide, 2085
ophthalmic solution, 2085
Hydroxyanisole, butylated, 5232
p-Hydroxybenzoic acid, 5700
4-Hydroxybenzoic acid isopropyl ester, 5700
2-Hydroxybenzyl alcohol, 5700
4-Hydroxybutane-1-sulfonic acid, 5701
4-Hydroxy-2-butanone, 5700
Hydroxychloroquine sulfate, 2086
tablets, 2086
Hydroxyethyl cellulose, 5384
N-(2-Hydroxyethyl)piperazine-N’-(2-
ethanesulfonic acid), 5701
Hydro-I 131 1-31
Hydroxylamine hydrochloride, 5701
TS, 5754
5-Hydroxymethylfurfural, 5701
10B-Hydroxynorandrostenedione, 5701
2’-(4-Hydroxyphenyl)-5-(4-methyl-1-
piperazinyl)-2,5’-bi-1 H-benzimidazole
trihydrochloride pentahydrate, 5701
4-(4-Hydroxyphenyl)-2-butanone, 5701
3-Hydroxyphenyldimethylethyl ammonium
chloride, 5701
D-a.-4-Hydroxyphenylglycine, 5701
4-Hydroxy-4-phenylpiperidine, 5700
Hydroxyprogesterone caproate, 2087
injection, 2087
Hydroxypropyl
betadex, 5385
cellulose, 5387
cellulose, low-substituted, 5389
cellulose ocular system, 2088
corn starch, 5596
pea starch, 5605
potato starch, 5610
Hydroxypropyl-B-cyclodextrin, 5701
Hydroxypropyl cellulose, 5701
8-Hydroxyquinoline, 5701
TS, 5754
Hydroxytoluene
butylated, 5233
butylated, reagent, 5678
Hydroxyurea, 2089
capsules, 2090
Hydroxyzine
hydrochloride, 2090
hydrochloride injection, 2092
hydrochloride oral solution, 2092
hydrochloride tablets, 2094
pamoate, 2095
pamoate capsules, 2097
pamoate oral suspension, 2099
Hymetellose, 5390
Hyoscyamine, 2099
hydrobromide, 2101
sulfate, 2102
sulfate elixir, 2103
sulfate injection, 2103
sulfate oral solution, 2104
sulfate tablets, 2105
tablets, 2100
Hypophosphorous acid, 5391
50 percent, 5701
Hypoxanthine, 5701
Hypromellose, 2105
acetate succinate, 5391
ophthalmic solution, 2107
phthalate, 5394
1123
capsules, sodium iodide, 2192
injection, iobenguane, 2189
injection, iodohippurate sodium, 2191
solution, sodium iodide, 2192
1125
albumin injection, iodinated, 2193
injection, iothalamate sodium, 2194
1131
albumin aggregated injection, iodinated,
2195
albumin injection, iodinated, 2194
capsules, sodium iodide, 2196
1-32 1 131-Injec
1131 (continued)
injection, iobenguane, 2190
injection, iodohippurate sodium, 2195
injection, rose bengal sodium, 2196
solution, sodium iodide, 2197
Ibuprofen, 2109
and diphenhydramine citrate tablets, 1324
diphenhydramine hydrochloride capsules,
1333
and pseudoephedrine hydrochloride
tablets, 2113
oral suspension, 2110
tablets, 2111
Ibutilide fumarate, 2114
Ichthammol, 2115
ointment, 2116
Idarubicin hydrochloride, 2116
injection, 2118
for injection, 2117
Identification
of articles of botanical origin (563), 6293
of articles of botanical origin by high-
performance thin-layer chromatography
procedure (1064), 7065
of fixed oils by thin-layer chromatography
(202), 6103
organic nitrogenous bases (181), 6094
test, thin-layer chromatographic (201),
6102
tests—general (191), 6094
tests, spectrophotometric (197), 6101
tetracyclines (193), 6100
Idoxuridine, 2119
ophthalmic ointment, 2119
ophthalmic solution, 2120
Ifosfamide, 2121
for injection, 2122
IgG-coated red cells, 5701
Imidazole, 5701
Imidurea, 5395
Iminodiacetic acid, 5701
Imipenem, 2123
and cilastatin for injectable suspension,
2125
and cilastatin for injection, 2124
Imipramine pamoate, 2130
Imipramine hydrochloride, 2126
injection, 2127
tablets, 2128
Imipramine pamoate
capsules, 2131
Imiquimod, 2134
cream, 2135
Immunogenicity assays—design and
validation of assays to detect anti-drug
neutralizing antibody (1106.1), 7279
Immunogenicity assays—design and
validation of immunoassays to detect anti-
drug antibodies (1106), 7264
Immunological test methods—surface
plasmon resonance (1105), 7248
Immunological test methods
enzyme-linked immunosorbent assay
(ELISA) (1103), 7226
general considerations (1102), 7219
immunoblot analysis (1104), 7237
Impurities
ordinary (466), 6220
testing in medical gases (413), 6201
Impurities in drug substances and drug
products (1086), 7152
Inamrinone, 2137
injection, 2137
Indapamide, 2139
tablets, 2140
Indene, 5701
Indicator and test papers, 5747
Indicators, 5745
indicator papers, 5747
reagents, and solutions, 5659
test papers, 5747
Indigo carmine, 5701
TS, 5754
Indigotindisulfonate sodium, 2140
injection, 2141
Indinavir sulfate, 2142
Indium In 111
capromab pendetide injection, 2143
chloride solution, 2144
ibritumomab tiuxetan injection, 2145
oxyquinoline solution, 2146
pentetate injection, 2147
pentetreotide injection, 2147
satumomab pendetide injection, 2148
Indocyanine green, 2149
for injection, 2149
Indole, 5701
Indole-3-carboxylic acid, 5701
Indomethacin, 2150
capsules, 2151
extended-release capsules, 2153
for injection, 2155
topical gel, 2155
oral suspension, 2158
sodium, 2159
suppositories, 2156
Indophenol-acetate TS, 5754
Inhalant
amyl nitrite, 308
propylhexedrine, 3500
Inhalation
Acetylcysteine and isoproterenol
hydrochloride solution, 76
Cromolyn sodium powder, 1104
Cromolyn sodium solution, 1105
Dexamethasone sodium phosphate
aerosol, 1203
Epinephrine aerosol, 1530
Epinephrine bitartrate aerosol, 1533
Epinephrine solution, 1531
Ergotamine tartrate aerosol, 1558
Fluticasone propionate aerosol, 1831
Fluticasone propionate powder, 1836
lsoetharine mesylate aerosol, 2245
Isoetharine solution, 2244
Isoproterenol hydrochloride aerosol, 2259
Isoproterenol hydrochloride and
phenylephrine bitartrate aerosol, 2262
Isoproterenol solution, 2258
lsoproterenol sulfate aerosol, 2264
lsoproterenol sulfate solution, 2265
Levalbuterol solution, 2365
Metaproterenol sulfate aerosol, 2607
Metaproterenol sulfate solution, 2608
Racepinephrine solution, 3564
Ribavirin for solution, 3599
Salmeterol powder, 3697
Sodium chloride, solution, 3786
Sterile water for, 4346
Terbutaline sulfate aerosol, 3986
Tobramycin solution, 4114
Combined Index to USP 41 and NF 36
Inhalation and nasal drug products: aerosols,
sprays, and powders—performance quality
tests (601), 6327
Inhalation and nasal drug products general
information and product quality tests (5),
5938
Injection
Acepromazine maleate, 33
Acetazolamide for, 66
Acyclovir for, 81
Adenosine, 90
Alcohol, dehydrated, 107
Alcohol in dextrose, 107
Alfentanil, 113
Alprostadil, 140
Alteplase for, 144
Amifostine for, 199
Amikacin sulfate, 203
Aminocaproic acid, 218
Aminohippurate sodium, 222
Aminopentamide sulfate, 225
Aminophylline, 228
Amiodarone hydrochloride, 243
Amitriptyline hydrochloride, 249
Ammonium chloride, 265
Ammonium molybdate, 268
Amobarbital sodium for, 269
Amphotericin B for, 291
Ampicillin for, 300
Ampicillin and sulbactam for, 305
Anileridine, 316
Aprotinin, 345
Arginine hydrochloride, 350
Articaine hydrochloride and epinephrine,
358
Ascorbic acid, 360
Atenolol, 384
Atracurium besylate, 403
Atropine sulfate, 406
Azaperone, 413
Azathioprine sodium for, 418
Azithromycin for, 425
Aztreonam, 433
Aztreonam for, 434
Bacitracin for, 437
Bacteriostatic sodium chloride, 3784
Bacteriostatic water for, 4346
Benztropine mesylate, 493
Benzylpenicilloyl polylysine, 497
Betamethasone sodium phosphate, 512
Bethanechol chloride, 522
Bleomycin for, 546
Bretylium tosylate, 548
Bretylium tosylate in dextrose, 548
Brompheniramine maleate, 558
Bumetanide, 563
Bupivacaine hydrochloride, 566
Bupivacaine hydrochloride in dextrose, 567
Bupivacaine hydrochloride and
epinephrine, 567
Butorphanol tartrate, 607
Caffeine citrate, 612
Caffeine and sodium benzoate, 614
Calcitonin salmon, 623
Calcitriol, 626
Calcium chloride, 639
Calcium gluceptate, 642
Calcium gluconate, 645
Calcium levulinate, 651
Combined Index to USP 41 and NF 36
Injection (continued)
Capreomycin for, 671
Carbenicillin for, 690
Carboplatin for, 709
Carboprost tromethamine, 711
Carmustine for, 722
Cefamandole nafate for, 755
Cefazolin, 758
Cefazolin for, 759
Cefepime for, 770
Cefmenoxime for, 776
Cefmetazole, 778
Cefmetazole for, 779
Cefonicid for, 780
Cefoperazone, 781
Cefoperazone for, 782
Ceforanide for, 784
Cefotaxime, 785
Cefotaxime for, 786
Cefotetan, 791
Cefotetan for, 791
Cefotiam for, 794
Cefoxitin, 796
Cefoxitin for, 797
Cefpiramide for, 799
Ceftazidime, 809
Ceftazidime for, 810
Ceftizoxime, 817
Ceftizoxime for, 818
Ceftriaxone, 818
Ceftriaxone for, 819
Cefuroxime, 822
Cefuroxime for, 823
Cephalothin, 837
Cephalothin for, 838
Cephapirin for, 839
Cephradine for, 844
Chloramphenicol, 863
Chloramphenicol sodium succinate for,
870
Chloroprocaine hydrochloride, 889
Chloroquine hydrochloride, 890
Chlorothiazide sodium for, 897
Chlorpheniramine maleate, 902
Chlorpromazine hydrochloride, 907
Chorionic gonadotropin for, 1982
Chromic chloride, 920
Chromium Cr 51 edetate, 922
Cimetidine, 935
Cimetidine in sodium chloride, 937
Ciprofloxacin, 942
Cisapride compounded, veterinary, 953
Cisatracurium besylate, 957
Cisplatin for, 961
Cladribine, 974
Clavulanic acid and ticarcillin, 4081
Clindamycin, 990
Clindamycin for, 991
Cloprostenol, 1036
Codeine phosphate, 1063
Colchicine, 1070
Colistimethate for, 1074
Corticotropin, 1094
Corticotropin for, 1095
Corticotropin, repository, 1097
Cr 51, sodium chromate, 921
Cupric chloride, 1111
Cupric sulfate, 1112
Cyanocobalamin, 1114
Cyclophosphamide for, 1126
Cyclosporine, 1131
Cysteine hydrochloride, 1140
Cytarabine for, 1142
Dacarbazine for, 1143
Dactinomycin for, 1145
Dantrolene sodium for, 1155
Daunorubicin hydrochloride for, 1159
Deferoxamine mesylate for, 1162
Dehydrated alcohol, 107
Deslanoside, 1176
Desmopressin acetate, 1183
Dexamethasone, 1196
Dexamethasone sodium phosphate, 1204
Dexamethasone sodium phosphate
compounded, 1205
Dexmedetomidine, 1215
Dextran 40 in dextrose, 1222
Dextran 40 in sodium chloride, 1223
Dextran 70 in dextrose, 1226
Dextran 70 in sodium chloride, 1227
Dextrose, 1234
Dextrose and sodium chloride, 1235
Diatrizoate meglumine, 1236
Diatrizoate meglumine and diatrizoate
sodium, 1237
Diatrizoate sodium, 1240
Diazepam, 1245
Diazoxide, 1247
Dibucaine hydrochloride, 1251
Dicyclomine hydrochloride, 1269
Diethylstilbestro!, 1280
Digitoxin, 1289
Digoxin, 1292
Dihydroergotamine mesylate, 1296
Dihydrostreptomycin, 1297
Dimenhydrinate, 1313
Dimercaprol, 1316
Dinoprost tromethamine, 1320
Diphenhydramine hydrochloride, 1330
Dipyridamole, 1346
Dobutamine, 1367
Dobutamine for, 1368
Dobutamine in dextrose, 1369
Docetaxel, 1373
Dopamine hydrochloride, 1393
Dopamine hydrochloride and dextrose,
1394
Doxapram hydrochloride, 1401
Doxorubicin hydrochloride, 1411
Doxorubicin hydrochloride for, 1413
Doxycycline for, 1420
Droperidol, 1443
Dyphylline, 1460
Edetate calcium disodium, 1470
Edetate disodium, 1472
Edrophonium chloride, 1472
Electrolytes and dextrose type 1, multiple,
1485
Electrolytes and dextrose type 2, multiple,
1488
Electrolytes and dextrose type 3, multiple,
1492
Electrolytes type 1, multiple, 1480
Electrolytes type 2, multiple, 1482
Elements, trace, 1494
Emetine hydrochloride, 1498
Enalaprilat, 1506
Enoxaparin sodium, 1512
Ephedrine sulfate, 1527
Epinephrine, 1530
Epirubicin hydrochloride, 1537
Ergonovine maleate, 1555
Ergotamine tartrate, 1559
Erythromycin, 1568
Erythromycin ethylsuccinate, 1577
Erythromycin lactobionate for, 1581
Estradiol cypionate, 1614
Estradiol valerate, 1615
Ethacrynate sodium for, 1631
Ethiodized oil, 1641
Injec-Injec 1-33
Etomidate, 1659
Etoposide, 1663
Famotidine, 1680
Fenoldopam mesylate, 1703
Fentanyl citrate, 1709
Ferumoxides, 1722
Floxuridine for, 1753
Fluconazole, 1753
Fluconazole in dextrose, 1758
Fluconazole in sodium chloride, 1760
Fludarabine phosphate, 1770
Fludarabine phosphate for, 1771
Fludeoxyglucose F18, 1794
Flumazenil, 1776
Flunixin meglumine, 1782
Fluorescein, 1789
F 18, sodium fluoride, 1795
Fluorouracil, 1802
Fluphenazine decanoate, 1812
Fluphenazine enanthate, 1814
Fluphenazine hydrochloride, 1816
Folic acid, 1866
Fondaparinux sodium, 1872
Fosphenytoin sodium, 1885
Fructose, 1887
Fructose and sodium chloride, 1888
Furosemide, 1893
Gadodiamide, 1902
Gadopentetate dimeglumine, 1904
Gadoteridol, 1908
Gadoversetamide, 1911
Gallamine triethiodide, 1924
Gallium citrate Ga 67, 1924
Ganciclovir for, 1926
Gemcitabine for, 1931
Gentamicin, 1935
Glucagon for, 1957
Glycopyrrolate, 1973
Gold sodium thiomalate, 1976
Gonadorelin for, 1976
Gonadotropin, chorionic for, 1982
Granisetron hydrochloride, 1992
Guaifenesin for, 2002
Haloperidol, 2020
Heparin sodium, 2031
Histamine phosphate, 2036
Hyaluronidase, 2042
Hyaluronidase for, 2042
Hydralazine hydrochloride, 2045
Hydrochloric acid, 2047
Hydrocortisone sodium phosphate, 2069
Hydrocortisone sodium succinate for, 2071
Hydromorphone hydrochloride, 2079
Hydroxocobalamin, 2084
Hydroxyprogesterone caproate, 2087
Hydroxyzine hydrochloride, 2092
Hyoscyamine sulfate, 2103
1123, iobenguane, 2189
1123, iodohippurate sodium, 2191
1125, iothalamate sodium, 2194
1125, albumin, iodinated, 2193
| 131, iobenguane, 2190
1 131, iodohippurate sodium, 2195
1131, rose bengal sodium, 2196
1 131, albumin, iodinated, 2194
| 131, albumin aggregated, iodinated,
2195
Idarubicin hydrochloride, 2118
Idarubicin hydrochloride for, 2117
Ifosfamide for, 2122
Imipenem and cilastatin for, 2124
Imipramine hydrochloride, 2127
Inamrinone, 2137
Indigotindisulfonate sodium, 2141
Indium In 111 capromab pendetide, 2143
 4520 Chamomile / Dietary Supplements USP 41

Standard solution: 25.0 ug/mL of USP Apigenin-7-glu- Mode: GC

coside RS and 10.0 g/mL of 7-methoxycoumarin in Detector: Flame ionization
methanol and water (1:1) Column: 0.32-mm x 30-m fused-silica capillary; coated
Sample solution: Transfer 1.0 g of Chamomile to a suit- with a 0.25-1um film of phase G16
able flask fitted with a reflux condenser anda stirrer. Temperature
Add 80.0 mL of methanol, and reflux the mixture with Column: See Table 2.
pe for 1 h. Cool the flask to room temperature,
ass the extract through a folded filter paper, and col- Table 2
ect the filtrate in a 100-mL volumetric flask. Rinse the
flask with 3 mL of methanol, pour the methanolic rins- Hold Time
ings through the filter paper, and add the filtrate to the Initial Temperature Final at Final
volumetric flask. Dilute with methanol to volume, and Temperature Ramp Temperature | Temperature
mix. Transfer 25.0 mL of the filtered solution to a ©) (¢/min) ©) (min)
round-bottom flask fitted with a reflux condenser and a 70 4 230 10
stirrer; add 5.0 mL of sodium hydroxide solution, pre-
pared by dissolving 0.4 g of sodium hydroxide in Detector: 250°
5.0 mL of water; and reflux the mixture for 25 min. Injection port: 220°
Cool the flask, and adjust the solution with hydrochloric Carrier gas: Helium
acid to a pH of 5.0-6.2. Quantitatively transfer the solu- Flow rate: 1.0 mL/min
tion to a 50-mL volumetric flask, dilute with methanol Injection size: 1 uL
to volume, and filter, discarding the first 10 mL of the System suitability
filtrate. Sample: Standard solution
Chromatographic system Suitability requirements
(See Chromatography (621), System Suitability.) Tailing factor: NMT 1.8 for levomenol
Mode: LC Relative standard deviation: NMT 2.0%
Detector: UV 335 nm Analysis
Column: 4-mm x 12.5-cm; packing L1 Samples: Standard solution and Sample solution
Flow rate: 1 mL/min Measure the peak areas. Identify the peaks due to
[Note—Make adjustments, if necessary, to obtain rela- levomenol, bisabolol oxide B, bisabolol oxide, and
tive retention times of 0.63 for apigenin-7-glucoside bisabolol oxide A in the Sample solution, using the
and 1.0 for demethaxscoumane retention time of levomenol in the Standard solution
Injection size: 15 wL and the approximate relative retention times of 0.89,
System suitability 0.97, and 1.1 for bisabol oxide B, bisabol oxide, and
Sample: Standard solution bisabol oxide A, respectively, with reference to the
[Note—The relative retention times for apigenin-7-glu- levomenol peak.
coside, 7-methoxycoumarin, apigenin, trans-spiroether, Calculate the percentage of bisabolane derivatives in
and cis-spiroether are about 0.63, 1.0, 1.2, 1.6, and the portion of Chamomile taken:
1.8, respectively.]
Suitability requirements Result = (r7/rs) x Cs x (V/W) x 100
Resolution: NLT 3.5 between apigenin-7-glucoside rr = sum of the peak areas for bisabolol oxide B,
and 7-methoxycoumarin bisabolol oxide, levomenol, and bisabolol
Relative standard deviation: NMT 2.0% for oxide A from the Sample solution
apigenin-7-glucoside rs = levomenol peak area from the Standard
Analysis solution
Samples: Standard solution and Sample solution Cs = concentration of USP Levomenol RS in the
[NoTE—Allow the Sample solution to elute for NLT 6 Standard solution (mg/mL)
times the retention time of apigenin-7-glucoside.] Vv = volume of Sample solution (mL)
Calculate the percentage of apigenin-7-glucoside in the w = weight of Chamomile taken to prepare the
portion of Chamomile taken: Sample solution (mg)
Result = (ru/rs) x Cs x (V/W) x 100 Acceptance criteria: NLT 0.15%
al e ARTICLES OF BOTANICAL ORIGIN, Volatile Oi! Determination
no
5 tu = peak response of apigenin-7-glucoside from (561)
s the Sample solution Analysis: Proceed as directed, except use 60 g of
aD rs = peak response of apigenin-7-glucoside from coarsely powdered Chamomile as the test specimen, a
= the Standard solution 2-L round-bottom flask, 300 mL of water as distillation
o Gs = concentration of USP Apigenin-7-glucoside RS re and 0.5 mL of xylene in the graduated tube. Dis-
= in the Standard solution (mg/mL) till for 4 h at a rate of 3-4 mL/min.
Fy Vv = volume of Sample solution (mL) Acceptance criteria: NLT 0.4% of blue volatile oil is
a w = weight of Chamomile taken to prepare the found. [NoTe—Retain the volatile oils for use in the test
Sample solution (mg) for Content of Bisabolane Derivatives.]
Acceptance criteria: NLT 0.3%
CONTAMINANTS
e CONTENT OF BISABOLANE DERIVATIVES e MICROBIAL ENUMERATION TESTS (2021): The total bacterial
Standard solution: 1 mg/mL of USP Levomenol RS in count does not exceed 105 cfu/g, the total combined
cyclohexane molds and yeasts count does not exceed 103 cfu/g, and
Sample solution: Transfer the volatile oils obtained in
the test for Articles of Botanical Origin (561), Volatile Oil the bile-tolerant Gram-negative bacterial count does not
Determination to a 25-mL volumetric flask, rinse the exceed 103 cfu/g.
e ABSENCE OF SPECIFIED MICROORGANISMS (2022): It meets
graduated tube of the apparatus with a small portion of
cyclohexane, transfer the rinsing to the 25-mL volumet- the requirements of the tests for absence of Salmonella
ric flask, add cyclohexane to volume, and mix.
species and Escherichia coli.
e ARTICLES OF BOTANICAL ORIGIN, Pesticide Residues (561):
Chromatographic system
(See Chromatography (621), System Suitability.) Meets the requirements
1-34 Injec-Injec
Injection (continued)
Indium In 111 ibritumomab tiuxetan, 2145
Indium In 111 pentetate, 2147
Indium In 111 pentetreotide, 2147
Indium In 111 satumomab pendetide,
2148
Indocyanine green for, 2149
Indomethacin for, 2155
injection, 930
Insulin, 2163
Insulin aspart, 2166
Insulin glargine, 2169
Insulin human, 2173
Insulin, human, and human insulin
isophane suspension, 2174
Insulin lispro, 2180
Inulin in sodium chloride, 2186
Invert sugar, 3846
lodipamide meglumine, 2198
lodixanol, 2202
lohexol, 2209
lopamidol, 2211
lopromide, 2215
lothalamate meglumine, 2216
lothalamate meglumine and iothalamate
sodium, 2216
loversol, 2219
loxaglate meglumine and ioxaglate
sodium, 2220
loxilan, 2223
Irinotecan hydrochloride, 2237
Iron dextran, 2239
lron sorbitex, 2241
Iron sucrose, 2241
Isoniazid, 2253
Isoproterenol hydrochloride, 2260
Isoxsuprine hydrochloride, 2286
Ivermectin, 2293
Ivermectin and clorsulon, 2297
Kanamycin, 2304
Ketamine hydrochloride, 2308
Ketorolac tromethamine, 2316
Labetalol hydrochloride, 2321
Leucovorin calcium, 2361
Levetiracetam, 2389
Levocarnitine, 2387
Levorphanol tartrate, 2403
Lidocaine hydrochloride, 2413
Lidocaine hydrochloride and dextrose,
2417
Lidocaine hydrochloride and epinephrine,
2417
Lincomycin, 2420
Lorazepam, 2471
Magnesium sulfate, 2519
Magnesium sulfate in dextrose, 2519
Manganese chloride, 2524
Manganese sulfate, 2526
Mannitol, 2529
Mannitol in sodium chloride, 2529
Mechlorethamine hydrochloride for, 2541
Menadiol sodium diphosphate, 2570
Menadione, 2572
Meperidine hydrochloride, 2575
Mepivacaine hydrochloride, 2581
Mepivacaine hydrochloride and
levonordefrin, 2582
Meropenem for, 2592
Mesoridazine besylate, 2602
Metaraminol bitartrate, 2610
Methadone hydrochloride, 2628
Methocarbamol, 2645
Methohexital sodium for, 2648
Methotrexate, 2651
Methotrexate for, 2652
Combined Index to USP 41 and NF 36
Methotrimeprazine, 2653 Potassium chloride in sodium chloride,
Methyldopate hydrochloride, 2672 3370
Methylene blue, 2673 Potassium phosphates, 3387
Methylene blue, veterinary, 2675 Potassium phosphates compounded, 3387
Methylergonovine maleate, 2677 Pralidoxime chloride for, 3395
Methylprednisolone sodium succinate for, Prednisolone sodium phosphate, 3418
2694 Prednisolone sodium succinate for, 3419
Metoclopramide, 2700 Prilocaine and epinephrine, 3429
Metoprolol tartrate, 2713 Prilocaine hydrochloride, 3428
Metronidazole, 2724 Procainamide hydrochloride, 3441
Mezlocillin for, 2734 Procaine hydrochloride, 3444
Miconazole, 2737 Procaine hydrochloride and epinephrine,
Midazolam, 2741 3445
Milrinone lactate, 2748 Procaine and tetracaine hydrochlorides and
Minocycline for, 2751 levonordefrin, 3445
Mitomycin for, 2772 Prochlorperazine edisylate, 3449
Mitoxantrone, 2774 Progesterone, 3455
Morphine sulfate, 2812 Promazine hydrochloride, 3462
Morphine sulfate compounded, 2813 Promethazine hydrochloride, 3465
Morrhuate sodium, 2815 Propofol injectable emulsion, 3489
Mycophenolate mofetil for, 2831 Propoxycaine and procaine hydrochlorides
N 13, ammonia, 2955 and levonordefrin, 3491
Nafcillin, 2847 Propoxycaine and procaine hydrochlorides
Nafcillin for, 2848 and norepinephrine bitartrate, 3492
Nalorphine hydrochloride, 2855 Propranolol hydrochloride, 3495
Naloxone hydrochloride, 2856 Protamine sulfate, 3504
Nandrolone decanoate, 2860 Pyridostigmine bromide, 3523
Neomycin for, 2882 Pyridoxine hydrochloride, 3526
Neostigmine methylsulfate, 2909 Quinidine gluconate, 3548
Netilmicin sulfate, 2910 Ranitidine, 3579
Niacin, 2918 Ranitidine in sodium chloride, 3582
Niacinamide, 2924 Repository corticotropin, 1097
Nicardipine hydrochloride, 2927 Riboflavin, 3603
Nitroglycerin, 2957 Rifampin for, 3610
Norepinephrine bitartrate, 2967 Ringer’s, 3620
Ondansetron, 3031 Ringer’s and dextrose, 3622
Orphenadrine citrate, 3049 Ringer’s and dextrose, half-strength
Oxacillin, 3061 lactated, 3628
Oxacillin for, 3062 Ringer’s and dextrose, lactated, 3626
Oxaliplatin, 3067 Ringer’s and dextrose, modified, lactated,
Oxaliplatin for, 3069 3631
Oxymorphone hydrochloride, 3117 Ringer's, lactated, 3624
Oxytetracycline, 3125 Ritodrine hydrochloride, 3644
Oxytetracycline for, 3128 Ropivacaine hydrochloride, 3679
Oxytocin, 3133 Rose bengal sodium | 131, 2196
Paclitaxel, 3136 Rubidium chloride Rb 82, 3683
Pamidronate disodium for, 3144 Sargramostim for, 3711
Pancuronium bromide, 3152 Scopolamine hydrobromide, 3729
Papaverine hydrochloride, 3163 Selenious acid, 3739
Paricalcitol, 3171 Sisomicin sulfate, 3769
Particulate matter in injections (788), 6537 Sm 153 lexidronam, samarium, 3707
Pemetrexed, 3186 Sodium acetate, 3772
Penicillin G potassium, 3199 Sodium bicarbonate, 3777
Penicillin G potassium for, 3200 Sodium bicarbonate compounded, 3778
Penicillin G sodium for, 3212 Sodium bromide, veterinary, 3780
Pentazocine, 3225 Sodium chloride, 3783
Pentobarbital sodium, 3229 Sodium chloride, bacteriostatic, 3784
Perphenazine, 3246 Sodium chromate Cr 51, 921
Phenobarbital sodium, 3262 Sodium lactate, 3795
Phentolamine mesylate for, 3271 Sodium nitrite, 3799
Phenylbutazone, 3274 Sodium nitroprusside for, 3801
Phenylephrine hydrochloride, 3279 Sodium phosphates, 3805
Phenytoin sodium, 3293 Sodium phosphates compounded, 3806
Physostigmine salicylate, 3296 Sodium sulfate, 3813
Phytonadione injectable emulsion, 3298 Sodium thiosulfate, 3814
Piperacillin for, 3324 Somatropin for, 3816
Piperacillin and tazobactam for, 3325 Strontium chloride Sr 89, 3840
Polymyxin B for, 3349 Streptomycin, 3839
Potassium acetate, 3354 Streptomycin for, 3839
Potassium chloride concentrate for, 3361 Succinylcholine chloride, 3842
Potassium chloride in dextrose, 3364 Sufentanil citrate, 3846
Potassium chloride in dextrose and sodium Sugar, invert, 3846
chloride, 3365 Sulfadiazine sodium, 3864
Potassium chloride in lactated ringer’s and Sulfamethoxazole and trimethoprim, 3874
dextrose, 3367 Sumatriptan, 3893
Combined Index to USP 41 and NF 36 Injec-Iprat I-35

Injection (continued) Vincristine sulfate, 4322 1123 injection, iobenguane, 2189

Technetium Tc 99m albumin, 3936 Vincristine sulfate for, 4323 1 123 injection, iodohippurate sodium,
Technetium Tc 99m albumin aggregated, Vinorelbine, 4326 2191
3937 Warfarin sodium for, 4342 1 123 solution, sodium iodide, 2192
Technetium Tc 99m albumin colloid, 3938 Water for, bacteriostatic, 4346 1125 albumin injection, iodinated, 2193
Technetium Tc 99m apcitide, 3940 Water for, sterile, 4346 1.125 injection, iothalamate sodium, 2194
Technetium Tc 99m arcitumomab, 3940 Water for, 4345 1131 albumin aggregated injection,
Technetium Tc 99m bicisate, 3941 Xenon Xe 133, 4351 iodinated, 2195
Technetium Tc 99m depreotide, 3941 Xylazine, 4354 1131 albumin injection, iodinated, 2194
Technetium Tc 99m disofenin, 3942 Yohimbine, 4358 1131 capsules, sodium iodide, 2196
Technetium Tc 99m etidronate, 3943 Yttrium Y 90 ibritumomab tiuxetan, 4359 | 131 injection, iobenguane, 2190
Technetium Tc 99m exametazime, 3943 Zidovudine, 4369 1 131 injection, iodohippurate sodium,
Technetium Tc 99m fanolesomab, 3944 Zinc chloride, 4378 2195
Technetium Tc 99m gluceptate, 3945 Zinc sulfate, 4386 | 131 injection, rose bengal sodium, 2196
Technetium Tc 99m lidofenin, 3946 Zolazepam and tiletamine for injection, 1 131 solution, sodium iodide, 2197
Technetium Tc 99m mebrofenin, 3947 4093 monobromide, 5702
Technetium Tc 99m medronate, 3948 monochloride, 5702
Technetium Tc 99m mertiatide, 3949 monochloride TS, 5755
Technetium Tc 99m nofetumomab and potassium iodide TS 1, 5755
merpentan, 3950 Injections and implanted drug products and potassium iodide TS 2, 5755
Technetium Tc 99m oxiseonate, 3950 (parenterals)—product quality tests (1), and potassium iodide TS 3, 5755
Technetium Tc 99m pentetate, 3951 5915 solution, strong, 2188
Technetium Tc 99m pertechnetate, Inosine, 5701 topical solution, 2187
sodium, 3951 Inositol, 5395, 5702 tenth-normal (0.1 N), 5755, 5766
Technetium Tc 99m pyrophosphate, 3953 Insoluble matter in reagents, 5663 tincture, 2188
Technetium Tc 99m (pyro- and trimeta-) Insulin, 2161 tincture, strong, 2189
phosphates, 3953 aspart, 2164 TS, 5755
Technetium Tc 99m red blood cells, 3954 assays (121), 6054 twentieth-normal (0.05 N), 5765
Technetium Tc 99m sestamibi, 3955 glargine, 2167 lodipamide, 2198
Technetium Tc 99m succimer, 3956 glargine injection, 2169 meglumine injection, 2198
Technetium Tc 99m sulfur colloid, 3956 human, 2171 lodixanol, 2199
Technetium Tc 99m tetrofosmin, 3957 human injection, 2173 injection, 2202
Temozolomide for injection, 3971 human isophane suspension and human lodobromide TS, 5755
Teniposide, 3974 insulin injection, 2174 lodochloride TS, 5755
Terbutaline sulfate, 3987 human suspension, isophane, 2177 lodoethane, 5702
Teriparatide, 3995 injection, 2163 lodoform, 2204
Testosterone cypionate, 4003 lispro, 2178 lodohippurate sodium
Testosterone enanthate, 4004 lispro injection, 2180 1123 injection, 2191
Testosterone propionate, 4005 suspension, isophane, 2176 1131 injection, 2195
Tetracaine hydrochloride, 4010 zinc suspension, 2181 lodometric assay—antibiotics (425), 6205
Tetracaine hydrochloride for, 4011 zinc suspension, extended, 2182 p-lodonitrotetrazolium violet, 5702
Tetracaine hydrochloride in dextrose, 4013 zinc suspension, prompt, 2184 lodoplatinate TS, 5755
Tetracycline hydrochloride for, 4019 Insulin aspart lodoquinol, 2205
Thallous chloride Tl 201, 4030 injection, 2166 tablets, 2206
Theophylline in dextrose, 4039 Intestinal fluid, simulated, TS, 5755, 5759 lohexol, 2206
Thiamine hydrochloride, 4048 Intramammary infusion injection, 2209
Thiopental sodium for, 4062 amoxicillin, 278 lon chromatography (1065), 7075
Thiotepa for, 4067 cloxacillin benzathine, 1048 lon-exchange resin, 5702
Thiothixene hydrochloride, 4070 Intrauterine contraceptive system lopamidol, 2210
Thiothixene hydrochloride for, 4071 progesterone, 3455 injection, 2211
Ticarcillin and clavulanic acid, 4081 Intrinsic viscosity table, 5863 lopromide, 2212
Ticarcillin and clavulanic acid for, 4082 Inulin, 2185 injection, 2215
Ticarcillin for, 4080 in sodium chloride injection, 2186 lothalamate
Tigecycline for, 4091 Invert sugar, 5397 meglumine injection, 2216
Tiletamine and zolazepam for, 4093 In vitro meglumine and iothalamate sodium
Tilmicosin, 4095 and in vivo evaluation of dosage forms injection, 2216
Tobramycin, 4111 (1088), 7159 sodium | 125 injection, 2194
Tobramycin for, 4112 biological reactivity tests (87), 6017 sodium and iothalamate meglumine
Tolazoline hydrochloride, 4127 In vivo injection, 2216
Tolbutamide for, 4128 biological reactivity tests (88), 6020 lothalamic acid, 2217
Trifluoperazine hydrochloride, 4212 and in vitro evaluation of dosage forms loversol, 2218
Triflupromazine hydrochloride, 4215 (1088), 7159 injection, 2219
Trimethobenzamide hydrochloride, 4224 lobenguane loxaglate
Tripelennamine hydrochloride, 4230 1123 injection, 2189 meglumine and ioxaglate sodium injection,
Tromethamine for, 4238 1131 injection, 2190 2220
Tubocurarine chloride, 4247 sulfate, 5702 sodium and ioxaglate meglumine injection,
Tylosin, 4249 lodic acid, 5702 2220
Urea for, 4256 lodinated loxaglic acid, 2220
Valproate sodium, 4271 1125 albumin injection, 2193 loxilan, 2221
Vancomycin, 4283 1131 albumin aggregated injection, 2195 injection, 2223
Vancomycin hydrochloride for, 4286 1131 albumin injection, 2194 Ipecac, 2224
Vasopressin, 4290 lodine, 2187, 5701 powdered, 2225
Verapamil hydrochloride, 4304 diluted TS, 5755 oral solution, 2226
Verteporfin for, 4312 hundredth-normal (0.01 N), 5765 Ipratropium bromide, 2227
Vinblastine sulfate for, 4319 1123 capsules, sodium iodide, 2192
|-36 Iprat-Lead Combined Index to USP 41 and NF 36

Ipratropium bromide and albuterol sulfate hydrochloride injection, 2260 tablets, 2318
inhalation solution, 2228 hydrochloride and phenylephrine bitartrate Kr 81m
Irbesartan, 2231 inhalation aerosol, 2262 krypton, 2319
and hydrochlorothiazide tablets, 2233 hydrochloride tablets, 2261 Krill oil
tablets, 2232 inhalation solution, 2258 capsules, 4721
Irinotecan hydrochloride, 2235 sulfate, 2264 delayed-release capsules, 4725
Injection, 2237 sulfate inhalation aerosol, 2264 Krypton Kr 81m, 2319
Iron sulfate inhalation solution, 2265
carbonyl, 2238 lsorhamnetin, 5703
dextran injection, 2239 Isosorbide
phenol TS, 5755 concentrate, 2266
powder, 5702
salicylate TS, 5755
dinitrate extended-release capsules, 2269
dinitrate chewable tablets, 2270
L
sorbitex injection, 2241 dinitrate, diluted, 2267
sucrose injection, 2241 dinitrate sublingual tablets, 2272 L designations, 5703
wire, 5702 dinitrate extended-release tablets, 2271 Labeling (7), 5945
Iron (241), 6155 mononitrate, diluted, 2273 Labeling of inactive ingredients (1091), 7178
lsoamyl mononitrate tablets, 2274 Labetalol hydrochloride, 2320
alcohol, 5702 mononitrate extended-release tablets, injection, 2321
Isobutane, 5401 2276 oral suspension, 2321
lsobutyl oral solution, 2267 tablets, 2322
acetate, 5702 Isostearyl isostearate, 5406 alpha-Lactalbumin, 5407
alcohol, 5401, 5666, 5702 Isotretinoin, 2281 Lactase, 2323
4-Isobutylacetophenone, 5702 capsules, 2282 Lactic acid, 2323, 5703
N-lsobutylpiperidone, 5702 Isovaleric acid, 5703 Lactitol, 5411
lsoetharine lsoxsuprine hydrochloride, 2285 Lactobacillus acidophilus
hydrochloride, 2243 injection, 2286 La-14, 4728
inhalation solution, 2244 tablets, 2287 NCFM, 4730
mesylate, 2244 Isradipine, 2287 Lactobacillus paracasei LPC-37, 4733
mesylate inhalation aerosol, 2245 capsules, 2288 Lactobacillus rhamnosus HNO01, 4732
lsoflupredone acetate, 2246, 5702 oral suspension, 2289 Lactobionic acid, 5412
Injectable suspension, 2247 Itraconazole, 2290 Lactose, 5703
neomycin sulfate and tetracaine Ivermectin, 2291 anhydrous, 5413
hydrochloride ointment, 2891 and clorsulon injection, 2297 beta, 5703
neomycin sulfate and tetracaine injection, 2293 monohydrate, 5414
hydrochloride topical powder, 2892 paste, 2294 monohydrate, alpha, 5703
Isoflurane, 2247 and pyrantel pamoate tablets, 2298 Lactulose
Isoleucine, 2249 topical solution, 2296 concentrate, 2324
L-lsoleucine, 5702 tablets, 2295 solution, 2325
lsomalt, 5403 Ixabepilone, 2300 Lamivudine, 2326
lsomaltotriose, 5702 oral solution, 2328
lIsometheptene mucate, 2251 tablets, 2329
dichloralphenazone, and acetaminophen and abacavir tablets, 21
capsules, 2251 and zidovudine tablets, 2331
2-lsoniazid, 2252, 5702 Lamotrigine, 2333
injection, 2253 tablets, 2334
and rifampin capsules, 3611 Lamotrigine
rifampin, pyrazinamide, and ethambutol Japanese honeysuckle flower, 4709 extended-release tablets, 2336
hydrochloride tablets, 3614 dry extract, 4712 tablets for oral suspension, 2340
rifampin and pyrazinamide tablets, 3612 powder, 4715 Lamotrigine compounded
oral solution, 2254 Juniper tar, 2303 oral suspension, 2342
tablets, 2254 Lanolin, 2343
Isonicotinamide, 5702 alcohols, 5415
Isonicotinic acid, 5702 modified, 2345
hydrazide, 5702 Lansoprazole, 2348
Isooctane, 5702
lsopropamide iodide, 2255
K delayed-release capsules, 2350
Lansoprazole compounded
tablets, 2256 oral suspension, 2351
Kaempferol, 5703 Lanthanum
Isopropyl
acetate, 5702 Kanamycin alizarin complexan mixture, 5703
injection, 2304 chloride, 5703
alcohol, 2256, 5666, 5702, 5721
alcohol, azeotropic, 2257
sulfate, 2305 nitrate hexahydrate, 5703
sulfate capsules, 2306 nitrate TS, 5755
alcohol, dehydrated, 5666, 5703
Kaolin, 2307 oxide, 5704
alcohol, rubbing, 2258
Kerosene, 5703 Latanoprost, 2352
ether, 5703
iodide, 5703 Ketamine hydrochloride, 2307 Lauric acid, 5417
injection, 2308 Lauroyl polyoxylglycerides, 5417
myristate, 5404, 5703
Ketoconazole, 2309 Lauryl dimethyl amine oxide, 5704
palmitate, 5405
oral suspension, 2310 Lead
salicylate, 5703
tablets, 2311 acetate, 5704
lsopropylamine, 5703
Ketoprofen, 2311 acetate paper, 5704
Isoproterenol
hydrochloride, 2259
capsules, 2312 acetate test paper, 5747
hydrochloride and acetylcysteine inhalation extended-release capsules, 2314 acetate TS, 5755
Ketorolac tromethamine, 2315 acetate TS, alcoholic, 5755
solution, 76
hydrochloride inhalation aerosol, 2259 injection, 2316 monoxide, 5704
Combined Index to USP 41 and NF 36 Lead-Lotio 1-37

Lead (continued) Licorice, 4735 methoxide, tenth-normal (0.1 N) in

nitrate, 5704 extract, powdered, 4737 methanol, 5766
nitrate, hundredth-molar (0.01 M), 5766 fluidextract, 5422 methoxide, tenth-normal (0.1 N) in
nitrate stock solution TS, 5755 powdered, 4736 toluene, 5767
perchlorate, 5704 Lidocaine, 2409 nitrate, 5704
perchlorate, hundredth-molar (0.01 M), topical aerosol, 2410 perchlorate, 5704
5766 hydrochloride, 2411 oral solution, 2436
perchlorate, tenth-molar (0.1 M), 5766 hydrochloride and dextrose injection, 2417 sulfate, 5704
solution, standard, 5760 hydrochloride and epinephrine injection, Lithocholic acid, 5704
subacetate TS, 5755 2417 Litmus, 5704, 5746
subacetate TS, diluted, 5753, 5755 hydrochloride injection, 2413 paper, blue, 5747
tetraacetate, 5704 hydrochloride jelly, 2413 paper, red, 5747
Lead (251), 6155 hydrochloride oral topical solution, 2414 TS, 5755
Leak rate (604), 6355 hydrochloride topical solution, 2415 Littorine hydrochloride (R)-(-)-, 5704
Lecithin, 5419 neomycin and polymyxin B sulfates and Locke-Ringer’s
Leflunomide, 2353 bacitracin ointment, 2896 solution, 5755
tablets, 2355 neomycin and polymyxin B sulfates and TS, 5755
Lemon bacitracin zinc ointment, 2900 Locust bean gum, 5704
oil, 5421 and neomycin and polymyxinB sulfates Lomustine, 2443
tincture, 5422 cream, 2904 capsules, 2445
Letrozole, 2356 ointment, 2410 Loperamide hydrochloride, 2446
tablets, 2357 and prilocaine cream, 2418 capsules, 2447
Leucine, 2359 oral topical solution, 2411 oral solution, 2448
Leucovorin calcium, 2360 Light diffraction measurement of particle size tablets, 2448
compounded oral suspension, 2361 (429), 6206 Lopinavir, 2450
injection, 2361 Lime, 2420 Lopinavir
tablets, 2362 Limestone and ritonavir oral solution, 2453
Leuprolide acetate, 2363 ground, 4738 and ritonavir tablets, 2457
Levalbuterol (R)-(+)-Limonene, 5704 Loracarbef, 2459
inhalation solution, 2365 Linalool, 5704 capsules, 2461
Levalbuterol hydrochloride, 2367 Lincomycin for oral suspension, 2461
Levamisole hydrochloride, 2369 hydrochloride, 2421 Loratadine, 2462
tablets, 2369 hydrochloride capsules, 2422 chewable tablets, 2466
Levetiracetam, 2370 hydrochloride soluble powder, 2422 oral solution, 2464
extended-release tablets, 2377 injection, 2420 tablets, 2465
injection, 2372 oral solution, 2421 orally disintegrating tablets, 2468
oral solution, 2373 Lindane, 2423 Lorazepam, 2470
tablets, 2375 cream, 2423 injection, 2471
Levmetamfetamine, 2382 lotion, 2424 oral concentrate, 2473
Levobunolol hydrochloride, 2383 shampoo, 2424 tablets, 2474
ophthalmic solution, 2384 Linezolid, 2425 Losartan potassium, 2476
Levocabastine hydrochloride, 2385 Linoleic acid, 5704 and hydrochlorothiazide tablets, 2480
Levocarnitine, 2386 Linoleic acids-free fatty acids tablets, 2477
injection, 2387 conjugated, 4739 Loss on drying (731), 6485
oral solution, 2388 Linolenic acid, 5704 Loss on drying for reagents, 5663
tablets, 2389 Linoleoyl polyoxylglycerides, 5422 Loss on ignition (733), 6486
Levocetirizine dihydrochloride Liothyronine sodium, 2427
tablets, 2391 tablets, 2428
Levodopa, 2392 Liotrix tablets, 2429
Lipid injectable emulsion, 2429
capsules, 2393
Levodopa Lipoic acid Lotion
and carbidopa extended-release tablets, alpha, 4740 Amphotericin B, 292
696 capsules, alpha, 4741 Benzoyl peroxide, 492
and carbidopa orally disintegrating tablets, tablets, alpha, 4742 Benzyl benzoate, 495
701 a-Lipoic acid, 5704 Betamethasone dipropionate, 509
and carbidopa tablets, 693 Liquid petrolatum, 5704 Betamethasone valerate, 515
tablets, 2394 Liquid-phase sterilization (1229.6), 7719 Clotrimazole, 1042
Levofloxacin, 2395 Lisinopril, 2431 Flurandrenolide, 1820
oral solution, 2397 and hydrochlorothiazide tablets, 2434 Fluticasone propionate, 1841
tablets, 2398 oral suspension, 2432 Hydrocortisone, 2059
Levonordefrin, 2401 tablets, 2433 Hydrocortisone acetate, 2064
and mepivacaine hydrochloride injection, Lithium, 5704 Lindane, 2424
2582 carbonate, 2437 Malathion, 2522
and procaine and tetracaine hydrochlorides carbonate capsules, 2438 Methylbenzethonium chloride, 2662
injection, 3445 carbonate tablets, 2438 Neomycin sulfate and flurandrenolide,
and propoxycaine and procaine carbonate extended-release tablets, 2439 2887
hydrochlorides injection, 3491 chloride, 5704 Neomycin sulfate and hydrocortisone
Levonorgestrel, 2401 citrate, 2441 acetate, 2890
and ethinyl estradiol tablets, 2402 hydroxide, 2442, 5704 Nystatin, 2989
Levorphanol tartrate, 2403 metaborate, 5704 Padimate O, 3138
injection, 2403 methoxide, fiftieth-normal (0.02 N) in Triamcinolone acetonide, 4188
tablets, 2404 methanol, 5766
Levothyroxine sodium, 2404 methoxide, tenth-normal (0.1 N) in
oral powder, 2407 chlorobenzene, 5766
tablets, 2407
1-38 Lotio-Menth Combined Index to USP 41 and NF 36

carbonate, alumina, and magnesium oxide Manufacturing practices for dietary

tablets, 160 supplements (2750), 8210
Lovastatin, 2484 carbonate and alumina oral suspension, Maprotiline hydrochloride, 2530
tablets, 2485 158 tablets, 2531
Low molecular weight heparin molecular carbonate and alumina tablets, 159 Marbofloxacin compounded, veterinary
weight determinations (209), 6117 chloride, 2504, 5705 oral suspension, 2532
Loxapine chloride, 0.01 M, 5767 Marfey’s reagent, 5705
capsules, 2487 citrate, 2505 Maritime pine, 4755
succinate, 2486 citrate oral solution, 2506 extract, 4757
Lufenuron, 2488 citrate for oral solution, 2507 Mass spectrometry (736), 6491
Lumefantrine, 2489 gluconate, 2508 Mayer's reagent, 5756
Lutein, 4743 gluconate tablets, 2509 Mazindol, 2533
capsules, 4744 hydroxide, 2510 tablets, 2533
preparation, 4745 hydroxide paste, 2511 Measurement of subvisible particulate matter
Lycopene, 4746 nitrate, 5705 in therapeutic protein injections (1787),
preparation, 4747 oxide, 2512, 5705 8038
tomato extract containing, 4748 oxide, alumina, and magnesium carbonate Mebendazole, 2534
Lysine tablets, 160 oral suspension, 2535
acetate, 2491 oxide, aspirin, and alumina tablets, 375 tablets, 2536
hydrochloride, 2491 oxide capsules, 2513 Mebrofenin, 2537
hydrochloride tablets, 4751 oxide, chromatographic, 5683, 5705 Mecamylamine hydrochloride, 2538
L-Lysine, 5704 oxide, citric acid, and sodium carbonate tablets, 2540
irrigation, 972 Mechlorethamine hydrochloride, 2540
oxide tablets, 2513 for injection, 2541
perchlorate, anhydrous, 5669, 5705 Meclizine hydrochloride, 2541
phosphate, 2514 tablets, 2543
salicylate, 2515 Meclocycline sulfosalicylate, 2544
salicylate tablets, 2516 cream, 2545
silicate, 5429 Meclofenamate sodium, 2545
Mafenide acetate, 2493 silicate, activated, 5665, 5705 capsules, 2546
cream, 2494 silicate, chromatographic, 5705 Medical air, 91
for topical solution, 2494 stearate, 5430 Medical devices—bacterial endotoxin and
Magaldrate, 2496 sulfate, 2518, 5705 pyrogen tests (161), 6085
and simethicone chewable tablets, 2499 sulfate, anhydrous, 5669, 5705 Medical gases assay (415), 6202
and simethicone oral suspension, 2498 sulfate in dextrose injection, 2519 Medium-chain trigylcerides, 5645, 5705
oral suspension, 2497 sulfate injection, 2519 Medroxyprogesterone acetate, 2547
tablets, 2497 sulfate TS, 5756 injectable suspension, 2548
Magnesia trisilicate, 2520 tablets, 2548
alumina and calcium carbonate chewable trisilicate and alumina oral suspension, 161 Mefenamic acid, 2549
tablets, 152 trisilicate and alumina tablets, 162 capsules, 2550
alumina, calcium carbonate, and trisilicate tablets, 2521 Mefloquine hydrochloride, 2551
simethicone chewable tablets, 153 Malabar-nut-tree, leaf, 4752 tablets, 2552
alumina and calcium carbonate oral powdered, 4753 Megestrol acetate, 2553
suspension, 151 extract, powdered, 4754 oral suspension, 2554
alumina and simethicone chewable tablets, Malachite green tablets, 2555
157 G, 5705 Meglumine, 2556
alumina and simethicone oral suspension, oxalate, 5746 Melamine, 5705
155 TS, 5756 Melatonin, 4758
and alumina oral suspension, 149 Malathion, 2521 tablets, 4759
and alumina tablets, 150 lotion, 2522 Melengestrol acetate, 2557
aspirin and alumina tablets, 373 Maleic acid, 5432, 5705 Meloxicam, 2558
aspirin, codeine phosphate, and alumina Malic acid, 5434 oral suspension, 2560
tablets, 380 Mallory’s stain, 5756 tablets, 2562
calcium carbonate and simethicone Malonic acid, 5705 Melphalan, 2563
chewable tablets, 636 Maltitol, 5434 tablets, 2564
and calcium carbonate chewable tablets, solution, 5436 Melting range or temperature (741), 6497
635 Maltodextrin, 5437 Memantine hydrochloride, 2565
milk of, 2500 Maltol, 5439 tablets, 2566
mixture TS, 5755 Maltose, 5440 Members of the United States Pharmacopeial
tablets, 2500 Maltotriose, 5705 Convention, xix
Magnesium, 5705 Mandelic acid, 5441 Menadiol sodium diphosphate, 2569
acetate, 5705 Manganese, 5705 injection, 2570
aluminometasilicate, 5423 chloride, 2523 tablets, 2570
aluminosilicate, 5425 chloride injection, 2524 Menadione, 2571
aluminum silicate, 5426 chloride for oral solution, 2524 injection, 2572
and calcium carbonates oral suspension, dioxide, 5705 Menaquinone-7, 4761
637 dioxide, activated, 5705 capsules, 4762
and calcium carbonates tablets, 638 gluconate, 2525 extract, Bacillus subtilis subsp. subtilis, 4765
carbonate, 2501 sulfate, 2526 preparation, 4763
carbonate and citric acid for oral solution, sulfate injection, 2526 tablets, 4764
2502 Mannitol, 2527 Menthol, 2572
carbonate, citric acid, and potassium injection, 2529 and benzocaine topical aerosol, 484
citrate for oral solution, 2503 In sodium chloride injection, 2529 lozenges, 2573
carbonate and sodium bicarbonate for oral D-Mannitol, 5705 and tetracaine ointment, 4007
suspension, 2504
Combined Index to USP 41 and NF 36 Meper-Methy 1-39

Meperidine hydrochloride, 2574 Metaxalone, 2611 5-Methoxy-2-methyl-3-indoleacetic acid,

injection, 2575 tablets, 2612 5706
oral solution, 2575 Metformin hydrochloride, 2614 Methoxyphenylacetic acid, 5706
tablets, 2576 extended-release tablets, 2616 Methoxyphenylacetic TS, 5756
Mephenytoin, 2576 and glipizide tablets, 1953 Methscopolamine bromide, 2657
tablets, 2577 and glyburide tablets, 1964 tablets, 2658
Mephobarbital, 2579 and pioglitazone tablets, 3317 Methsuximide, 2659
tablets, 2579 tablets, 2615 capsules, 2660
Mepivacaine hydrochloride, 2580 Methacholine chloride, 2624 Methyclothiazide, 2660
injection, 2581 Methacrylic acid, 5706 tablets, 2661
and levonordefrin injection, 2582 and ethyl acrylate copolymer, 5442 Methyl
Meprednisone, 2583 and ethyl acrylate copolymer, partially- acetate, 5706
Meprobamate, 2584 neutralized, 5446 alcohol, 5447
oral suspension, 2585 and methyl methacrylate copolymer, 5444 4-aminobenzoate, 5706
tablets, 2585 Methacycline hydrochloride, 2625 arachidate, 5706
Meradimate, 2586 capsules, 2626 behenate, 5706
2-Mercaptoethanol, 5705 oral suspension, 2626 benzenesulfonate, 5707
Mercaptopurine, 2587 Methadone hydrochloride, 2627 caprate, 5707
tablets, 2589 injection, 2628 caprylate, 5707
Mercuric oral concentrate, 2627 carbamate, 5707
acetate, 5705 oral solution, 2629 chloroform, 5683, 5707, 5739
acetate TS, 5756 tablets, 2629 erucate, 5707
ammonium thiocyanate TS, 5756 tablets for oral suspension, 2630 ethyl ketone, 5707
bromide, 5705 Methamphetamine hydrochloride, 2631 green, 5707
bromide test paper, 5747 tablets, 2631 green-iodomercurate paper, 5747
bromide TS, alcoholic, 5750, 5756 Methanesulfonic acid, 5706 heptadecanoate, 5707
chloride, 5705 Methanol, 5666, 5669, 5706 iodide, 5707
chloride TS, 5756 aldehyde-free, 5706 isobutyl ketone, 5448, 5707
iodide, red, 5705 anhydrous, 5706 laurate, 5707
iodide, TS, 5756 deuterated, 5686 lignocerate, 5707
nitrate, 5705 spectrophotometric, 5706 linoleate, 5707
nitrate, tenth-molar (0.1 M), 5767 Methazolamide, 2632 linolenate, 5707
nitrate TS, 5756 tablets, 2633 methacrylate, 5707
oxide, yellow, 5705, 5744 Methdilazine hydrochloride, 2634 methacrylate and ethyl acrylate copolymer
potassium iodide TS, 5756, 5756 oral solution, 2634 dispersion, 5337
potassium iodide TS, alkaline, 5750, 5756, tablets, 2635 myristate, 5707
5757 Methenamine, 2635, 5699, 5706 oleate, 5708
sulfate, 5705 hippurate, 2637 orange, 5746
sulfate TS, 5753, 5756 hippurate tablets, 2638 orange TS, 5756
thiocyanate, 5705 mandelate, 2638 palmitate, 5708
Mercurous nitrate mandelate for oral solution, 2639 purple TS, 5756
dihydrate, 5705 mandelate oral suspension, 2640 red, 5708, 5746
TS, 5756 mandelate tablets, 2640 red—methylene blue TS, 5756
Mercury, 5705 mandelate delayed-release tablets, 2641 red sodium, 5746
ammoniated, 2590 oral solution, 2636 red TS, 5756
Mercury (261), 6157 tablets, 2637 red TS 2, 5756
Meropenem, 2591 Methimazole, 2641 red TS, methanolic, 5756
for injection, 2592 tablets, 2642 salicylate, 5448
Mesalamine, 2594 Methionine, 2643 stearate, 5708
extended-release capsules, 2596 L-Methionine sulfoxide, 5706 sulfoxide, 5708
rectal suspension, 2597 Methocarbamol, 2644 violet TS, 5756
delayed-release tablets, 2598 injection, 2645 yellow, 5708, 5746
Mesityl oxide, 5705 tablets, 2646 yellow-methylene blue TS, 5756
Mesna, 2600 Methods for the determination of particulate yellow paper, 5747
Mesoridazine besylate, 2601 matter in injections and ophthalmic yellow TS, 5756
injection, 2602 solutions (1788), 8052 3-Methyl-2-benzothiazolinone hydrazone
oral solution, 2602 Methohexital, 2647 hydrochloride TS, 5756
tablets, 2603 sodium for injection, 2648 Methylamine, 40 percent in water, 5708
Mestranol, 2604 Methotrexate, 2649 Methylamine hydrochloride, 5708
and ethynodiol diacetate tablets, 1650 injection, 2651 p-Methylaminophenol sulfate, 5708
and norethindrone tablets, 2971 for injection, 2652 Methylbenzethonium chloride, 2662
Metacresol, 2605 tablets, 2652 lotion, 2662
Metanil Methotrimeprazine, 2653 ointment, 2663
yellow, 5706 injection, 2653 topical powder, 2663
Metaphenylenediamine hydrochloride, 5706 Methoxsalen, 2654 4-Methylbenzophenone, 5708
TS, 5756 capsules, 2655 Methylbenzothiazolone hydrazone
Metaphosphoric-acetic acid TS, 5756 topical solution, 2656 hydrochloride, 5708
Metaphosphoric acid, 5706 5-Methoxy-1H-benzimidazole-2-thiol, 5706 (R)-(+)-alpha-Methylbenzyl isocyanate, 5708
Metaproterenol sulfate, 2606 Methoxyacetophenone p-, 5706 (S)-(-)-a-Methylbenzyl isocyanate, 5708
inhalation aerosol, 2607 7-Methoxycoumarin, 5706 Methylcellulose, 2664
inhalation solution, 2608 Methoxy determination (431), 6212 ophthalmic solution, 2665
oral solution, 2608 Methoxyethanol, 5706 oral solution, 2666
tablets, 2609 2-Methoxyethanol, 5706 tablets, 2666
Metaraminol bitartrate, 2610 Methoxyflurane, 2656 Methylcobalamin, 4767
injection, 2610 tablets, 4768
|-40 Methy-Monit Combined Index to USP 41 and NF 36

Methyldopa, 2666 tartrate injection, 2713 Milrinone lactate

and chlorothiazide tablets, 2669 tartrate oral solution, 2714 injection, 2748
and hydrochlorothiazide tablets, 2670 tartrate oral suspension, 2714 Mineral
oral suspension, 2667 tartrate tablets, 2715 acid, 5709
tablets, 2668 Metrifonate, 2719 oil, 2749
Methyldopate hydrochloride, 2671 Metronidazole, 2720 oil emulsion, 2750
Injection, 2672 benzoate, 2721 oil, light, 5453
Methyl cis-11-eicosenoate, 5707 capsules, 2722 oil, rectal, 2750
Methylene gel, 2723 oil, topical light, 2751
blue, 2672, 5708 injection, 2724 Minerals
blue injection, 2673 tablets, 2726 with calcium and vitamin D tablets, 4502
blue injection, veterinary, 2675 extended-release tablets, 2727 capsules, 4778
blue TS, 5756 Metronidazole benzoate compounded oil- and water-soluble vitamins with,
chloride, 5449, 5688, 5708 oral suspension, 2722 capsules, 5022
5,5’-Methylenedisalicylic acid, 5708 Metyrapone, 2729 oil- and water-soluble vitamins with, oral
Methylergonovine maleate, 2676 tablets, 2730 solution, 5047
injection, 2677 Metyrosine, 2730 oil- and water-soluble vitamins with,
tablets, 2678 capsules, 2731 tablets, 5061
3-O-Methylestrone, 5708 Mexiletine hydrochloride, 2731 tablets, 4785
1-Methylimidazole, 5709 capsules, 2732 water-soluble vitamins with, capsules, 5109
2-Methylimidazole, 5709 Mezlocillin water-soluble vitamins with, oral solution,
Methyl 12-Ketostearate, 5707 for injection, 2734 5128
Methyl methacrylate sodium, 2733 water-soluble vitamins with, tablets, 5137
and methacrylic acid copolymer, 5444 Mibolerone, 2735 Minimum fill (755), 6499
Methylnaltrexone bromide, 2679 oral solution, 2735 Minocycline
2-Methyl-5-nitroimidazole, 5709 Miconazole, 2736 hydrochloride, 2752
N-Methyl-N-nitroso-p-toluenesulfonamide, compounded ophthalmic solution, 2736 hydrochloride capsules, 2753
5709 injection, 2737 periodontal system, 2758
Methylparaben, 5450 nitrate, 2738 hydrochloride oral suspension, 2754
sodium, 5451 nitrate cream, 2738 hydrochloride tablets, 2754
4-Methylpentan-2-ol, 5709 nitrate topical powder, 2739 hydrochloride extended-release tablets,
2-Methylpentane, 5709 nitrate vaginal suppositories, 2740 2755
4-Methyl-2-pentanone, 5707, 5709 Microbial characterization, identification, and for injection, 2751
Methylphenidate hydrochloride, 2681 strain typing (1113), 7301 Minoxidil, 2760
tablets, 2682 Microbial enumeration tests—nutritional and topical solution, 2762
extended-release tablets, 2683 dietary supplements (2021), 8153 tablets, 2761
Methylprednisolone, 2688 Microbiological attributes of nonsterile Mirtazapine, 2763
acetate, 2690 nutritional and dietary supplements tablets, 2765
acetate cream, 2691 (2023), 8164 orally disintegrating tablets, 2766
acetate injectable suspension, 2691 Microbiological best laboratory practices Misoprostol, 2768
acetate and neomycin sulfate cream, 2893 T112)57325 dispersion, 2769
hemisuccinate, 2692 Microbiological control and monitoring of Mission
sodium succinate, 2693 aseptic processing environments (1116), and preface, vii
sodium succinate for injection, 2694 7312 statement, vii
tablets, 2689 Microbiological examination of nonsterile Mitomycin, 2770
2-Methyl-2-propyl-1,3-propanediol, 5709 products: acceptance criteria for for injection, 2772
Methyl p-toluenesulfonate, 5708 pharmaceutical preparations and Mitotane, 2773
N-Methylpyrrolidine, 5709 substances for pharmaceutical use (1111), tablets, 2773
Methylpyrrolidone, 5452 7297 Mitoxantrone
Methylsulfonylmethane, 4769 Microbiological examination of nonsterile hydrochloride, 2774
and glucosamine tablets, 4672 products: microbial enumeration tests (61), injection, 2774
glucosamine, and chondroitin sulfate 5965 Modafinil, 2775
sodium tablets, 4674 Microbiological examination of nonsterile tablets, 2776
tablets, 4770 products: tests for specified Moexipril hydrochloride, 2778
Methyltestosterone, 2695 microorganisms (62), 5971 Moexipril hydrochloride
capsules, 2696 Microbiological procedures for absence of and hydrochlorothiazide tablets, 2782
tablets, 2697 specified microorganisms—nutritional and tablets, 2780
Methylthionine perchlorate TS, 5756 dietary supplements (2022), 8158 Moist heat sterilization of aqueous liquids
Methysergide maleate, 2698 Microscopy, optical (776), 6516 (1229.2), 7701
tablets, 2698 Midazolam, 2740 Molindone hydrochloride, 2784
Metoclopramide injection, 2741 tablets, 2785
hydrochloride, 2699 Mid-infrared spectroscopy (854), 6654 Molybdenum, 5709
injection, 2700 Mid-infrared spectroscopy—theory and Molybdic acid, 5709
oral solution, 2701 practice (1854), 8127 Molybdo-phosphotungstate TS, 5756
tablets, 2702 Midodrine hydrochloride, 2743 Mometasone furoate, 2786
Metolazone, 2704 tablets, 2744 cream, 2787
oral suspension, 2705 Milbemycin oxime, 2745 ointment, 2788
tablets, 2705 Milk thistle, 4770 topical solution, 2790
Metoprolol capsules, 4775 Monensin, 2791
fumarate, 2707 extract, powdered, 4773 granulated, 2792
succinate, 2708 powdered, 4772 premix, 2793
succinate extended-release tablets, 2709 tablets, 4776 sodium, 2794
tartrate, 2712 Millon’s reagent, 5756 Monitoring devices—time, temperature, and
tartrate and hydrochlorothiazide tablets, Milrinone, 2747 humidity (1118), 7331
2717 Monitoring of bioburden (1229.3), 7706
Combined Index to USP 41 and NF 36
Monobasic
potassium phosphate, 5535, 5709
sodium phosphate, 3805, 5709
Monobenzone, 2795
cream, 2795
Monochloroacetic acid, 5709
Mono- and di-glycerides, 5454
Monoethanolamine, 5455, 5709
Monoglyceride citrate, 5455
Monograph and reference material donors
2014 recognition, xxvi
Monosaccharide Analysis, 6118
Monosodium glutamate, 5456, 5709
Monothioglycerol, 5457
Montelukast
sodium oral granules, 2797
sodium tablets, 2800
sodium chewable tablets, 2803
Montelukast sodium, 2795
Montelukast sodium hydrate, 5709
Morantel tartrate, 2805
Moricizine hydrochloride, 2806
tablets, 2808
Morin, 5709
Morphine sulfate, 2809
extended-release capsules, 2810
injection, 2812
suppositories, 2814
Morphine sulfate
compounded injection, 2813
Morpholine, 5709
Morrhuate sodium injection, 2815
Moxidectin, 2815
Moxifloxacin
hydrochloride, 2817
ophthalmic solution, 2819
tablets, 2821
Mucosal drug products—performance tests
(1004), 6699
Mucosal drug products—product quality
tests (4), 5933
Mupirocin, 2823
calcium, 2824
cream, 2825
ointment, 2826
nasal ointment, 2827
Mycophenolate
sodium, 2836
Mycophenolate mofetil, 2828
capsules, 2829
for injection, 2831
for oral suspension, 2832
tablets, 2834
Mycophenolic acid
delayed-release tablets, 2838
Mycoplasma tests (63), 5978
Myristic acid, 5457
Myristyl alcohol, 5458
Myristyltrimethylammonium bromide, 5709
Myrrh, 2841
topical solution, 2841
N 13 injection, ammonia, 2955
Nabumetone, 2843
tablets, 2844
Nadolol, 2844
and bendroflumethiazide tablets, 2847
tablets, 2846
Monob-Neomy 1-41
Nafcillin Oxymetazoline hydrochloride, 3114
injection, 2847 Phenylephrine hydrochloride, 3280
for injection, 2848 Tetrahydrozoline hydrochloride, 4027
sodium, 2849 Xylometazoline hydrochloride, 4355
sodium capsules, 2849
sodium for oral solution, 2850
sodium tablets, 2850
Naftifine hydrochloride, 2850
cream, 2851
Nasal spray
gel, 2851 Butorphanol tartrate, 607, 607
Nalidixic acid, 2852 Desmopressin acetate, 1184
oral suspension, 2853 Fluticasone propionate, 1842
tablets, 2853
Nalorphine hydrochloride, 2854
injection, 2855
Naloxone Natamycin, 2875
hydrochloride, 2855 ophthalmic suspension, 2876
hydrochloride injection, 2856 Nateglinide, 2876
and pentazocine tablets, 3224 tablets, 2878
Naltrexone hydrochloride, 2857 Near-infrared spectroscopy (1119), 7337
Nefazodone hydrochloride, 2879
tablets, 2858
Nandrolone tablets, 2880
decanoate, 2859 Neomycin
decanoate injection, 2860 boluses, 2882
Naphazoline hydrochloride, 2860 and colistin sulfates and hydrocortisone
nasal solution, 2862 acetate otic suspension, 1075
ophthalmic solution, 2862 for injection, 2882
and pheniramine maleate ophthalmic penicillin G, polymyxin B, hydrocortisone
solution, 2862 acetate, and hydrocortisone sodium
Naphthalene, 5709 succinate topical suspension, 3193
1,3-Naphthalenediol, 5709 and polymyxin B sulfates, bacitracin, and
2,7-Naphthalenediol, 5689, 5709 hydrocortisone acetate ointment, 2895
2-Naphthalenesulfonic acid, 5709 and polymyxin B sulfates, bacitracin, and
Naphthol hydrocortisone acetate ophthalmic
dipotassium disulfonate, 5710 ointment, 2896
disodium disulfonate, 5710 and polymyxin B sulfates, bacitracin, and
1-Naphthol, 5666, 5709 lidocaine ointment, 2896
reagent, 5756, 5756 and polymyxin B sulfates and bacitracin
TS, 5756 ointment, 2894
2-Naphthol, 5673, 5709 and polymyxin B sulfates and bacitracin
TS, 5751, 5756 ophthalmic ointment, 2895
p-Naphtholbenzein, 5710, 5746 and polymyxin B sulfates, bacitracin zinc,
TS, 5757 and hydrocortisone ointment, 2898
B-Naphthoquinone-4-sodium sulfonate, 5710 and polymyxin B sulfates, bacitracin zinc,
Naphthoresorcinol, 5710 and hydrocortisone ophthalmic
1-Naphthylamine, 5710 ointment, 2898
1-Naphthylamine hydrochloride, 5710 and polymyxin B sulfates, bacitracin zinc,
2-Naphthy! chloroformate, 5710 and hydrocortisone acetate ophthalmic
N-(1-Naphthyl)ethylenediamine ointment, 2899
dihydrochloride, 5710 and polymyxinB sulfates, bacitracin zinc,
TS, 5757 and lidocaine ointment, 2900
Naproxen, 2864 and polymyxin B sulfates and bacitracin
sodium, 2867 zinc ointment, 2897
sodium tablets, 2868 and polymyxin B sulfates and bacitracin
oral suspension, 2864 zinc ophthalmic ointment, 2897
tablets, 2865 and polymyxin B sulfates cream, 2893
delayed-release tablets, 2866 and polymyxinB sulfates and
Narasin dexamethasone ophthalmic ointment,
granular, 2869 2900
premix, 2871 and polymyxin B sulfates and
Naratriptan dexamethasone ophthalmic suspension,
hydrochloride, 2873 2901
hydrochloride oral suspension, 2875 and polymyxin B sulfates and gramicidin
tablets, 2872 cream, 2902
and polymyxin B sulfates, gramicidin, and
hydrocortisone acetate cream, 2902
and polymyxin B sulfates and gramicidin
ophthalmic solution, 2902
Nasal solution and polymyxin B sulfates and
Calcitonin salmon, 624 hydrocortisone ophthalmic suspension,
Cromolyn sodium, 1106 2903
Ephedrine sulfate, 1528 and polymyxin B sulfates and
Epinephrine, 1531 hydrocortisone otic solution, 2903
Flunisolide, 1779 and polymyxin B sulfates and
Naphazoline hydrochloride, 2862 hydrocortisone otic suspension, 2903
|-42 Neomy-Norge Combined Index to USP 41 and NF 36

Neomycin (continued) Neostigmine 1NTS, 5757

and polymyxin B sulfates and bromide, 2908 2NTS, 5757
hydrocortisone acetate cream, 2904 bromide tablets, 2908 Nitrilotriacetic acid, 5710
and polymyxin B sulfates and methylsulfate, 2909 Nitrite titration (451), 6218
hydrocortisone acetate ophthalmic methylsulfate injection, 2909 4’-Nitroacetophenone, 5711
suspension, 2904 Neotame, 5460 o-Nitroaniline, 5711
and polymyxin B sulfates and lidocaine Nephelometry, turbidimetry, and visual p-Nitroaniline, 5711
cream, 2904 comparison (855), 6658 TS, 5757
and polymyxin B sulfates ophthalmic Nessler’s reagent, 5757 Nitrobenzene, 5711
ointment, 2894 Netilmicin sulfate, 2909 p-Nitrobenzenediazonium tetrafluoroborate,
and polymyxin B sulfates ophthalmic injection, 2910 5711
solution, 2894 Neutralized 4-Nitrobenzoic acid, 5711
and polymyxin B sulfates, penicillin G alcohol, 5710 p-Nitrobenzyl bromide, 5711
procaine, and hydrocortisone acetate phthalate buffer, 5676 4-(p-Nitrobenzyl) pyridine, 5711
topical suspension, 3210 Neutral red, 5746 Nitrofurantoin, 2946
and polymyxin B sulfates and pramoxine TS, 5757 capsules, 2947
hydrochloride cream, 2905 Nevirapine, 2911 oral suspension, 2951
and polymyxin B sulfates and prednisolone Nevirapine tablets, 2952
acetate ophthalmic suspension, 2906 extended release tablets, 2915 Nitrofurazone, 2953
and polymyxin B sulfates solution for oral suspension, 2912 ointment, 2954
irrigation, 2894 tablets, 2914 topical solution, 2955
sulfate, 2882 New sterilization methods (1229.12), 7734 Nitrogen, 5462
sulfate and bacitracin ointment, 2884 Niacin, 2917 97 percent, 5462
sulfate and bacitracin zinc ointment, 2884 extended-release tablets, 2920 certified standard, 5711
sulfate cream, 2883 injection, 2918 compounds in reagents, 5663
sulfate and dexamethasone sodium or niacinamide assay (441), 6213 determination (461), 6219
phosphate cream, 2884 tablets, 2919 N 13 injection, ammonia, 2955
sulfate and dexamethasone sodium Niacinamide, 2924 Nitroglycerin
phosphate ophthalmic ointment, 2885 injection, 2924 diluted, 2956
sulfate and dexamethasone sodium or niacin assay (441), 6213 injection, 2957
phosphate ophthalmic solution, 2886 tablets, 2925 ointment, 2958
sulfate and fluocinolone acetonide cream, Nicardipine hydrochloride, 2925 sublingual tablets, 2958
2887 injection, 2927 Nitromersol, 2959
sulfate and fluorometholone ointment, Nickel-aluminum catalyst, 5710 topical solution, 2960
2887 Nickel, 5710 Nitromethane, 5711
sulfate and flurandrenolide cream, 2887 standard solution TS, 5757 5-Nitro-1,10-phenanthroline, 5710
sulfate and flurandrenolide lotion, 2887 sulfate, 5710 Nitrophenanthroline TS, 5757
sulfate and flurandrenolide ointment, 2888 (ll) sulfate heptahydrate, 5710 1-Nitroso-2-naphthol, 5711
sulfate and gramicidin ointment, 2888 Nickel nitrate hexahydrate, 5710 Nitroso R salt, 5711
sulfate and hydrocortisone cream, 2888 B-Nicotinamide adenine dinucleotide, 5710 Nitrous
sulfate and hydrocortisone ointment, 2889 Nicotinamide adenine dinucleotide oxide, 2960
sulfate and hydrocortisone otic suspension, phosphate-adenosine-5’-triphosphate oxide certified standard, 5711
2889 mixture, 5710 Nizatidine, 2961
sulfate and hydrocortisone acetate cream, Nicotine, 2929 capsules, 2962
2889 polacrilex, 2932 Nomenclature (1121), 7351
sulfate and hydrocortisone acetate lotion, polacrilex gum, 2934 Nonadecane, 5711
2890 transdermal system, 2930 Nonanoic acid, 5711
sulfate and hydrocortisone acetate Nicotinic acid, 5710 Nonionic wetting agent, 5711, 5712
ointment, 2890 Nifedipine, 2935 Nonoxynol 9, 2963, 5712
sulfate and hydrocortisone acetate capsules, 2937 1-Nonyl alcohol, 5712
ophthalmic suspension, 2890 extended-release tablets, 2938 n-Nonylamine, 5712
sulfate, isoflupredone acetate, and Nile blue hydrochloride, 5746 Nonylphenol polyoxyethylene ether, 5712
tetracaine hydrochloride ointment, 2891 Nilutamide, 2944 Nonylphenoxypoly(ethyleneoxy)ethanol,
sulfate, isoflupredone acetate, and Nimodipine, 2945 5712
tetracaine hydrochloride topical powder, Ninhydrin, 5710 Norelgestromin, 2965
2892 TS, 5757 Norepinephrine bitartrate, 2967
sulfate and methylprednisolone acetate Nitrate injection, 2967
cream, 2893 mercurous, dihydrate, 5705 and propoxycaine and procaine
sulfate, nystatin, gramicidin, and mercurous, TS, 5756 hydrochlorides injection, 3492
triamcinolone acetonide cream, 2991 ophthalmic solution, silver, 3759 Norethindrone, 2968
sulfate, nystatin, gramicidin, and in reagents, 5663 acetate, 2972
triamcinolone acetonide ointment, 2992 silver, 3759, 5727 acetate and estradiol tablets, 1608
sulfate, nystatin, thiostrepton, and silver, TS, 5759 acetate and ethinyl estradiol tablets, 2975
triamcinolone acetonide cream, 2992 tenth-normal (0.1 N), silver, 5759, 5770 acetate tablets, 2973
sulfate, nystatin, thiostrepton, and toughened silver, 3759 and ethinyl estradiol tablets, 2970
triamcinolone acetonide ointment, 2993 Nitric and mestranol tablets, 2971
sulfate ointment, 2883 acid, 5461, 5710 tablets, 2969
sulfate ophthalmic ointment, 2883 acid, diluted, 5690, 5710 Norfloxacin, 2976
sulfate and prednisolone acetate acid, fuming, 5697, 5710 ophthalmic solution, 2977
ophthalmic suspension, 2907 acid, lead-free, 5710 tablets, 2978
sulfate oral solution, 2884 oxide-nitrogen dioxide detector tube, Norgestimate, 2978
sulfate tablets, 2884 5710 and ethinyl estradiol tablets, 2981
sulfate and triamcinolone acetonide cream, Nitric acid Norgestrel, 2982
2907 0.01 N TS, 5757 and ethinyl estradiol tablets, 2983
0.2 N TS, 5757 tablets, 2982
Combined Index to USP 41 and NF 36 Norma-Ointm 1-43

Normal Octisalate, 2995 Schizochytrium, capsules, 4872

butyl acetate, 5677 Octocrylene, 2996 Sesame, 5556
butyl alcohol, 5677 Octoxynol 9, 5463, 5712 Soybean, 3822
butylamine, 5712 Octreotide acetate, 2997 Soybean, hydrogenated, 5592
Northern schisandra fruit, 4865 Octyldodecanol, 5466 Sunflower, 5635
dry extract, 4866 (p-tert-Octylphenoxy)nonaethoxyethanol, Vegetable, hydrogenated, 5649
powder, 4868 5694, 5712 Vitamins capsules, oil- and water-soluble,
Nortriptyline hydrochloride, 2984 (p-tert-Octylphenoxy)polyethoxyethanol, 4976
capsules, 2985 5712 Vitamins capsules, oil-soluble, 4935
oral solution, 2986 Octyl sulfate, sodium salt, 5712 Vitamins with minerals capsules, oil- and
Noscapine, 2986 Officers (2015-2020), xi water-soluble, 5022
Novobiocin Ofloxacin, 2998 Vitamins with minerals oral solution, oil-
sodium, 2987 ophthalmic solution, 3000 and water-soluble, 5047
sodium intramammary infusion, 2987 tablets, 3000 Vitamins with minerals tablets, oil- and
sodium and penicillin G procaine water-soluble, 5061
intramammary infusion, 3210 Vitamins oral solution, oil- and water-
sodium, tetracycline hydrochloride, and soluble, 4995
°
prednisolone tablets, 4024
sodium and tetracycline hydrochloride Oil Vitamins tablets, oil- and water-soluble,
5004
tablets, 4024 Almond, 5191 Vitamins tablets, oil-soluble, 4944
Nuclear magnetic resonance spectroscopy Anise, 5205
(761), 6500 Borage seed, 4488
Nucleic acid-based techniques Borage seed, capsules, 4489
amplification (1127), 7369 Canola, 5244 Oil-soluble vitamins
approaches for detecting trace nucleic Caraway, 5248 capsules, 4935
acids (residual DNA testing) (1130), Cardamom, 5269 tablets, 4944
7389 Castor, 739 Oil- and water-soluble vitamins
extraction, detection, and sequencing Castor, aromatic, 741 capsules, 4976
(1126), 7359 Castor, capsules, 739 with minerals capsules, 5022
general (1125), 7353 Castor, emulsion, 740 with minerals oral solution, 5047
genotyping (1129), 7385 Castor, hydrogenated, 5272 with minerals tablets, 5061
microarray (1128), 7379 Cedar, 5681 oral solution, 4995
Nystatin, 2988 Chia seed, 4530 tablets, 5004
cream, 2989 Clove, 5298
lotion, 2989 Coconut, 5299
lozenges, 2989 Coconut, hydrogenated, 5299
neomycin sulfate, gramicidin, and
triamcinolone acetonide cream, 2991
Cod liver, 1060
Cod liver, capsules, 4551
Ointment
neomycin sulfate, gramicidin, and Coriander, 5303 Acyclovir, 82
triamcinolone acetonide ointment, 2992 Corn, 5303 Alclometasone dipropionate, 102
neomycin sulfate, thiostrepton, and Cottonseed, 5312 Amcinonide, 198
triamcinolone acetonide cream, 2992 Cottonseed, hydrogenated, 5313 Amphotericin B, 292
neomycin sulfate, thiostrepton, and Crypthecodinium cohnii, 4555 Anthralin, 321
triamcinolone acetonide ointment, 2993 Crypthecodinium cohnii, capsules, 4557 Atropine sulfate ophthalmic, 407
ointment, 2989 Ethiodized injection, 1641 Bacitracin ophthalmic, 438
and oxytetracycline capsules, 3125 Evening primrose, 4605 Bacitracin zinc, 441
and oxytetracycline for oral suspension, Evening primrose, capsules, 4606 Bacitracin zinc and polymyxinBsulfate,
3126 Fats and fixed oils (401), 6184 442
topical powder, 2989 Fennel, 5353 Bacitracin zinc and polymyxinB sulfate
oral suspension, 2990 Flax seed, 4623 ophthalmic, 442
for oral suspension, 2990 Flax seed, capsules, 4624 Benzocaine, 475
tablets, 2990 Krill, capsules, 4721 Benzocaine, butamben, and tetracaine
and tetracycline hydrochloride capsules, Krill delayed-release capsules, 4725 hydrochloride, 482
4025 Lemon, 5421 Benzoic and salicylic acids, 487
and triamcinolone acetonide cream, 2994 Mineral, 2749 Betamethasone dipropionate, 510
and triamcinolone acetonide ointment, Mineral emulsion, 2750 Betamethasone valerate, 517
2994 Mineral, light, 5453 Bland lubricating ophthalmic, 3038
vaginal inserts, 2991 Mineral, rectal, 2750 Calcipotriene, 618
vaginal suppositories, 2990 Mineral, topical light, 2751 Chloramphenicol and polymyxin B sulfate
Olive, 5472 ophthalmic, 867
Orange, 5475 Chloramphenicol ophthalmic, 864
Palm, 5477 Chlortetracycline hydrochloride, 911
Palm, hydrogenated, 5477 Chlortetracycline hydrochloride
oO Palm kernel, 5478
Peanut, 5481
ophthalmic, 912
Ciprofloxacin ophthalmic, 944
Peppermint, 5482 Clioquinol, 1002
n-Octadecane, 5712 Polyoxyl 35 castor, 5515 Clioquinol and hydrocortisone, 1005
Octadecyl silane, 5712 Polyoxyl 40 hydrogenated castor, 5516 Clobetasol propionate, 1008
Octane-n, 5712 Propyliodone injectable suspension, 3501 Coal tar, 1055
Octanesulfonic acid sodium salt, 5712, 5731 Fully hydrogenated rapeseed, 5552 Desoximetasone, 1191
Octanesulfonic acid sodium salt Superglycerinated fully hydrogenated Dexamethasone sodium phosphate
monohydrate, 5712 rapeseed, 5553 ophthalmic, 1206
1-Octanol, 5712 Rose, 5554 Dibucaine, 1250
Octanophenone, 5712 Safflower, 3692 Diflorasone diacetate, 1284
Octinoxate, 2995 Schizochytrium, 4870 Erythromycin, 1568
USP 41
SPECIFIC TESTS
¢ BOTANIC CHARACTERISTICS
Macroscopic: Flower head is hemispherical, about
6 mm in diameter, composed of a few ray florets and
numerous disk florets (distinction from Matricaria dis-
coidea, which has disk florets only), carried on a recep-
tacle surrounded by an involucre. Involucre is green,
formed of two to three rows of lanceolate, glabrous,
and imbricated bracts with blunt apices and scarious
whitish edges. Ray florets, which usually have fallen off,
have 10-20 pistils; corolla is ligulate, white, but darkens
at a length of 6mm and a width of about 2 mm,
3-toothed, and traversed by four main veins. Disk flo-
rets are yellow, perfect, about 2 mm in length; corolla is
tubular with five teeth; five stamens are epipetalous and
syngenesious. Receptacle is hollow (distinction from
Chrysanthemum and Anthemis species), hemispherical in
the young and conical in the old flower head, 3-10 mm
in width, and lacking paleae. Achene is ovoid, and has
three to five longitudinal ribs.
Microscopic: Separate the capitulum into its parts, and
examine under a microscope. The outer, abaxial epider-
mis of the involucral bracts shows a scarious margin
with a single layer of radially elongated cells and a cen-
tral part made up of chlorophyll tissue covered by elon-
gated epidermal cells with sinuous lateral walls, sto-
mata, and secretory trichomes. The vascular bundles are
surrounded by numerous elongated, pitted sclereids
with fairly large lumens. In surface view, ligulate and
tubular corollas show isodiametric or elongated cells
with more or less wavy walls and a few glandular
trichomes. The outer part of the epidermis of the ligu-
late florets consists of papillary cells with cuticular stria-
tions radiating from their tips. In the mesophyll, very
small clusters of calcium oxalate are sometimes seen.
Four main veins run lengthwise through the entire mes-
ophyll, sometimes accompanied by one or two other
veins, which are shorter and run parallel to the main
veins. Each of the two main median veins split into two
near the tip and, with the lateral veins, anastomose two
by two to form three arcs at the three terminal teeth of
the ligule. The ovaries, oval to spherical, of both kinds
of florets have at their base a sclerous ring consisting of
a single row of cells. The epidermis of the ovary i
made up of elongated cells with sinuous walls between
which secretory trichomes are situated. The ovaries con-
tain numerous, very small clusters of calcium oxalate. In
the tubular florets, the low part of each stamen filament
is surrounded by thick-walled cells. The ends of the two
stigmata have papillose epidermal cells. The pollen
grains have a diameter of about 30 jm and are
rounded and triangular, with three germinal pores and
a spiny exine.
e BROKEN FLOWERS: NMT 25% passes through a No. 25
standard-mesh sieve (see Particle Size Distribution Estima-
tion by Analytical sleving (786)).
© ARTICLES OF BOTANICAL ORIGIN, Foreign Organic Matter
(561): NMT 2.0%
e ARTICLES OF BOTANICAL ORIGIN, Total Ash (561): NMT
13.0%, determined on 1.0 g of powdered Chamomile
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in well-closed contain-
ers, protected from light.
Change to read:
e LABELING: The label states the Latin binomial and, follow-
ing the official name, the part of the plant contained in
the article. This article is exempted from the require-
ments of ° Labeling (7), Labels and Labeling for Products
e «cn TeMay-201g) with respect
and Other Categories, Botanicals,
to the pregnancy and lactation statement. “e ew 1-may-2018)
Dietary Supplements / Chaste Tree 452
e USP REFERENCE STANDARDS (11)
USP Apigenin-7-glucoside RS
USP Levomenol RS
Chaste Tree
DEFINITION
Chaste Tree consists of the dried ripe fruits of Vitex agnus-
castus L. (Verbenaceae). |t contains NLT 0.05% of agnu-
ice and NLT 0.08% of casticin, calculated on the dried
aSsiS.
IDENTIFICATION
e A. THIN-LAYER CHROMATOGRAPHY
Standard solution: 100mg of USP Powdered Chaste
Tree Extract RS in 1 mL of methanol. Heat in a water
bath at 60° for 10 min. Centrifuge, and use the clear
supernatant.
Sample solution: Transfer about 1 g of powdered plant
material to a screw-capped centrifuge tube. Add 10 mL
of methanol, heat in a water bath at 60° for 10-15
min, cool, and filter.
Adsorbent: Chromatographic silica gel with an average
particle size of 10-15 um (TLC plates)
Application volume: 90 uL, Standard solution; 60 uL,
Sample solution; in bands that are 2 cm in length
Developing solvent system: Ethyl acetate, methanol,
and water (77:15:8)
Derivatization reagent: 10 mg/mL of p-dimethylami-
nobenzaldehyde in 1 N hydrochloric acid
Analysis
Samples: Standard solution and Sample solution
Develop to a length of NLT 12 cm, and dry the plate in
a stream of air. Treat the plate with Derivatization rea-
gent, and heat for 10 min at 120°.
Acceptance criteria: The Sample solution shows the fol-
lowing: a blue zone (at an R; value of about 0.21) due
to the presence of aucubin and that corresponds in
color and R; value to a similar zone for the Standard
solution; a blue zone (at an R; value of about 0.44) as a
result of the presence of agnuside that corresponds in
color and R; value to a similar zone for the Standard
solution; and one broad zone, violet in the middle, near
the solvent front and that corresponds in color and Rr
value to a similar zone for the Standard solution. Other
colored zones of varying intensities may be observed
for the Sample solution.
e B. In the test for Content of Casticin, the chromatogram
sydeibouo-: sa
of the Sample solution shows a peak at the retention time
corresponding to the casticin peak in the chromatogram
of the Standard solution.
COMPOSITION
© CONTENT OF CASTICIN
Standard solution: About 0.05 mg/mL of USP Casticin
RS in methanol, with sonication. Pass through acellu-
lose membrane filter of 0.45-m or finer pore size.
Sample solution: Place about 1000 mg of ground plant
material in a container with a stopper. Extract twice
with 40 mL of methanol, using a hand homogenizer at
19,000 rpm for 2 min. Filter each supernatant, and
transfer to a 250-mL round-bottom flask. Rinse the resi-
due with methanol, and filter the resulting solution into
the flask. Evaporate the combined extract to dryness.
Dissolve the residue in methanol, quantitatively transfer
to a 20-mL volumetric flask, and dilute with methanol
to volume. Pass through a cellulose membrane filter of
0.45-um or finer pore size.
-44 Ointm-Ophth
Ointment (continued)
Erythromycin ophthalmic, 1569
Fluocinolone acetonide, 1785
Fluocinonide, 1787
Flurandrenolide, 1820
Fluticasone propionate, 1846
Gentamicin and prednisolone acetate
ophthalmic, 1942
Gentamicin sulfate, 1938
Gentamicin sulfate and betamethasone
valerate, 1939
Gentamicin sulfate ophthalmic, 1938
Halcinonide, 2016
Hydrocortisone, 2060
Hydrocortisone acetate, 2065
Hydrocortisone acetate ophthalmic, 2065
Hydrocortisone valerate, 2073
Hydrophilic, 3002
Ichthammol, 2116
Idoxuridine ophthalmic, 2119
Lidocaine, 2410
Methylbenzethonium chloride, 2663
Mometasone furoate, 2788
Mupirocin, 2826
Mupirocin nasal, 2827
Neomycin and polymyxin B sulfates and
bacitracin, 2894
Neomycin and polymyxin B sulfates,
bacitracin, and hydrocortisone acetate,
2895
Neomycin and polymyxin B sulfates,
bacitracin, and hydrocortisone acetate
ophthalmic, 2896
Neomycin and polymyxin B sulfates,
bacitracin, and lidocaine, 2896
Neomycin and polymyxin B sulfates and
bacitracin ophthalmic, 2895
Neomycin and polymyxin B sulfates and
bacitracin zinc, 2897
Neomycin and polymyxinB sulfates,
bacitracin zinc, and hydrocortisone,
2898
Neomycin and polymyxin B sulfates,
bacitracin zinc, and hydrocortisone
acetate ophthalmic, 2899
Neomycin and polymyxin B sulfates,
bacitracin zinc, and hydrocortisone
ophthalmic, 2898
Neomycin and polymyxin B sulfates,
bacitracin zinc, and lidocaine, 2900
Neomycin and polymyxin B sulfates and
bacitracin zinc ophthalmic, 2897
Neomycin and polymyxin B sulfates and
dexamethasone ophthalmic, 2900
Neomycin and polymyxin B sulfates
ophthalmic, 2894
Neomycin sulfate, 2883
Neomycin sulfate and bacitracin, 2884
Neomycin sulfate and bacitracin zinc, 2884
Neomycin sulfate and dexamethasone
sodium phosphate ophthalmic, 2885
Neomycin sulfate and fluorometholone,
2887
Neomycin sulfate and flurandrenolide,
2888
Neomycin sulfate and gramicidin, 2888
Neomycin sulfate and hydrocortisone,
2889
Neomycin sulfate and hydrocortisone
acetate, 2890
Neomycin sulfate, isoflupredone acetate,
and tetracaine hydrochloride, 2891
Neomycin sulfate ophthalmic, 2883
Nitrofurazone, 2954
Nitroglycerin, 2958
Nystatin, 2989
Nystatin, neomycin sulfate, gramicidin,
and triamcinolone acetonide, 2992
Nystatin, neomycin sulfate, thiostrepton,
and triamcinolone acetonide, 2993
Nystatin and triamcinolone acetonide,
2994
Oxytetracycline hydrochloride and
hydrocortisone, 3130
Oxytetracycline hydrochloride and
polymyxinB sulfate, 3130
Oxytetracycline hydrochloride and
polymyxin B sulfate ophthalmic, 3130
Polyethylene glycol, 5501
Povidone-iodine, 3393
Prednicarbate, 3409
Resorcinol ointment, compound, 3595
Rose water, 3680
Sodium chloride ophthalmic, 3785
Sulfacetamide sodium ophthalmic, 3855
Sulfacetamide sodium and prednisolone
acetate ophthalmic, 3857
Sulfur, 3887
Tetracaine, 4007
Tetracaine and menthol, 4007
Tetracycline hydrochloride, 4020
Tetracycline hydrochloride ophthalmic,
4020
Tobramycin and dexamethasone
ophthalmic, 4117
Tobramycin ophthalmic, 4113
Triamcinolone acetonide, 4188
Undecylenic acid, compound, 4254
White, 3002
Yellow, 3002
Zinc oxide, 4383
Olanzapine, 3002
and fluoxetine capsules, 3005
tablets, 3003
Olanzapine orally disintegrating
tablets, 3007
Olefin detector tube, 5712
Oleic acid, 5468
Oleoresin, capsicum, 674
Oleovitamin A and D, 3009
capsules, 3010
Oleoyl polyoxylglycerides, 5469
Oleyl
alcohol, 5470
oleate, 5471
Oligo-deoxythymidine, 5712
Oligosaccharide analysis (212), 6125
Olive leaf, 4793
dry extract, 4794
powder, 4796
Olive oil, 5472
Olmesartan medoxomil, 3010
tablets, 3012
Olopatadine hydrochloride
ophthalmic solution, 3015
Omega-3
acids triglycerides, 4797
ethyl esters capsules, 3019
ethyl esters, 3016
Omeprazole, 3022
delayed-release capsules, 3023
magnesium, 3026
oral suspension, 3026
Ondansetron, 3028
compounded topical gel, 3038
hydrochloride, 3029
hydrochloride oral suspension, 3033
Combined Index to USP 41 and NF 36
injection, 3031
oral solution, 3032
tablets, 3034
orally disintegrating tablets, 3036
Ophthalmic ointment
Atropine sulfate, 407
Bacitracin, 438
Bacitracin zinc and polymyxin B sulfate,
442
Bland lubricating, 3038
Chloramphenicol, 864
Chloramphenicol and polymyxinB sulfate,
867
Chlortetracycline hydrochloride, 912
Ciprofloxacin, 944
Dexamethasone sodium phosphate, 1206
Erythromycin, 1569
Gentamicin and prednisolone acetate,
1942
Gentamicin sulfate, 1938
Hydrocortisone acetate, 2065
Idoxuridine, 2119
Neomycin and polymyxin B sulfates, 2894
Neomycin and polymyxin B sulfates and
bacitracin, 2895
Neomycin and polymyxinBsulfates,
bacitracin, and hydrocortisone acetate,
2896
Neomycin and polymyxin B sulfates and
bacitracin zinc, 2897
Neomycin and polymyxin B sulfates,
bacitracin zinc, and hydrocortisone,
2898
Neomycin and polymyxin B sulfates,
bacitracin zinc, and hydrocortisone
acetate, 2899
Neomycin and polymyxin B sulfates and
dexamethasone, 2900
Neomycin sulfate, 2883
Neomycin sulfate and dexamethasone
sodium phosphate, 2885
Oxytetracycline hydrochloride and
polymyxin B sulfate, 3130
Sodium chloride, 3785
Sulfacetamide sodium, 3855
Sulfacetamide sodium and prednisolone
acetate, 3857
Tetracycline hydrochloride, 4020
Tobramycin, 4113
Tobramycin and dexamethasone, 4117
Ophthalmic products—performance tests
(1771), 8024
Ophthalmic products—quality tests (771),
6510
Ophthalmic solution
Acetylcholine chloride for, 73
Apraclonidine, 338
Atropine sulfate, 408
Benoxinate hydrochloride, 466
Betaxolol, 519
Carbachol, 683
Carteolol hydrochloride, 727
Cefazolin, 760
Chloramphenicol, 864
Combined Index to USP 41 and NF 36
Ophthalmic solution (continued)
Chloramphenicol for, 865
Chymotrypsin for, 924
Ciprofloxacin, 944
Cromolyn sodium, 1107
Cyclopentolate hydrochloride, 1123
Cyclosporine compounded, veterinary,
1134
Demecarium bromide, 1165
Dexamethasone sodium phosphate, 1207
Dipivefrin hydrochloride, 1343
Dorzolamide hydrochloride, 1396
Dorzolamide hydrochloride and timolol
maleate, 1397
Echothiophate iodide for, 1467
Emedastine, 1496
Epinephrine, 1532
Epinephrine bitartrate, 1534
Epinephrine bitartrate for, 1534
Epinephryl borate, 1535
Fluorescein sodium and benoxinate
hydrochloride, 1792
Fluorescein sodium and proparacaine
hydrochloride, 1793
Flurbiprofen sodium, 1826
Gentamicin sulfate, 1938
Gentamicin sulfate and betamethasone
acetate, 1939
Glycerin, 1968
Homatropine hydrobromide, 2039
Hydroxyamphetamine hydrobromide,
2085
Hypromellose, 2107
Idoxuridine, 2120
Levobunolol hydrochloride, 2384
Methylcellulose, 2665
Miconazole compounded, 2736
Moxifloxacin, 2819
Naphazoline hydrochloride, 2862
Naphazoline hydrochloride and
pheniramine maleate, 2862
Neomycin and polymyxin B sulfates, 2894
Neomycin and polymyxin B sulfates and
gramicidin, 2902
Neomycin sulfate and dexamethasone
sodium phosphate, 2886
Norfloxacin, 2977
Ofloxacin, 3000
Olopatadine hydrochloride, 3015
Oxymetazoline hydrochloride, 3114
Phenylephrine hydrochloride, 3281
Physostigmine salicylate, 3297
Pilocarpine hydrochloride, 3303
Pilocarpine nitrate, 3305
Polymyxin B sulfate and trimethoprim,
3351
Prednisolone sodium phosphate, 3419
Proparacaine hydrochloride, 3486
Scopolamine hydrobromide, 3730
Silver nitrate, 3759
Sodium chloride, 3786
Sulfacetamide sodium, 3856
Suprofen, 3900
Tetracaine hydrochloride, 4012
Tetrahydrozoline hydrochloride, 4027
Timolol maleate, 4098
Tobramycin, 4116
Travoprost, 4174
Tropicamide, 4240
Ophth-Oral 1-45
Voriconazole compounded, veterinary, Containing at least three of the
4338 foliowing—acetaminophen and (salts of)
Zinc sulfate, 4387 chlorpheniramine, dextromethorphan,
and pseudoephedrine, 49
Acetaminophen and codeine phosphate,
56
Acetaminophen, dextromethorphan
Ophthalmic hydrobromide, doxylamine succinate,
suspension and pseudoephedrine hydrochloride, 60
Acetaminophen for effervescent, 37
Brinzolamide, 550 Amantadine hydrochloride, 196
Chloramphenicol and hydrocortisone Aminobenzoate potassium for, 212
acetate for, 866 Aminocaproic acid, 219
Dexamethasone, 1197 Aminophylline, 229
Fluorometholone, 1798 Amprolium, 307
Gentamicin and prednisolone acetate, Aromatic elixir, 5206
1943 Ascorbic acid, 361
Natamycin, 2876 Ascorbic acid compounded, 361
Neomycin and polymyxin B sulfates and Aspirin effervescent tablets for, 371
dexamethasone, 2901 Atenolol, 385
Neomycin and polymyxin B sulfates and Beclomethasone dipropionate
hydrocortisone, 2903 compounded, 456
Neomycin and polymyxin B sulfates and Benzaldehyde elixir, compound, 5215
hydrocortisone acetate, 2904 Betamethasone, 503
Neomycin and polymyxin B sulfates and Bethanechol chloride, 523
prednisolone acetate, 2906 Bromodiphenhydramine hydrochloride,
Neomycin sulfate and hydrocortisone 555
acetate, 2890 Bromodiphenhydramine hydrochloride and
Neomycin sulfate and prednisolone codeine phosphate, 556
acetate, 2907 Brompheniramine maleate, 558
Oxytetracycline hydrochloride and Brompheniramine maleate and
hydrocortisone acetate, 3129 pseudoephedrine sulfate, 559
Prednisolone acetate, 3415 Butabarbital sodium, 593
Rimexolone, 3619 Caffeine citrate, 613
Sulfacetamide sodium and prednisolone Calcium glubionate syrup, 641
acetate, 3858 Captopril, 678
Tetracycline hydrochloride, 4022 C 13 for, urea, 706
Tobramycin and dexamethasone, 4119 Cetirizine hydrochloride, 848
Tobramycin and fluorometholone acetate, Cherry syrup, 5289
4121 Chloral hydrate, 860
Chloramphenicol, 865
eS Chlorpheniramine maleate, 902
Chlorpheniramine maleate and
Opium, 3039
pseudoephedrine hydrochloride, 904
powdered, 3039 Chlorpromazine hydrochloride syrup, 908
tincture, 3039
Chocolate syrup, 5296
Optical Citalopram, 963
microscopy (776), 6516
Clindamycin hydrochloride, 994
rotation (781), 6519
Clindamycin palmitate hydrochloride for,
Oracet blue B, 5746
995
TS, S75Z Cloxacillin sodium for, 1052
Oral drug products—product quality tests
Cyanocobalamin Co 57, 1056
(2), 5921 Codeine phosphate, 1064
Orally inhaled and nasal drug products
Codeine sulfate, 1066
(1664.1), 7937 Cyclosporine, 1133
Cyproheptadine hydrochloride, 1136
Dexamethasone, 1198
Dexamethasone elixir, 1196
Oral powder Dexbrompheniramine maleate and
Containing at least three of the pseudoephedrine sulfate, 1209
following—acetaminophen and (salts of) Dexchlorpheniramine maleate, 1211
chlorpheniramine, dextromethorphan, Dextromethorphan hydrobromide, 1232
and pseudoephedrine, 47 Dicyclomine hydrochloride, 1270
Levothyroxine sodium, 2407 Didanosine for, 1275
Digoxin, 1292
Sodium bicarbonate, 3779
Dihydrotachysterol, 1299
Diltiazem hydrochloride, 1309
Dimenhydrinate, 1314
Diphenhydramine hydrochloride, 1331
Oral solution Diphenoxylate hydrochloride and atropine
Abacavir, 19 sulfate, 1339
Acacia syrup, 5179 Docusate sodium syrup, 1380
Acetaminophen, 37 Dolasetron mesylate, 1384
Doxepin hydrochloride, 1407
Doxylamine succinate, 1438
 |-46 Oral-Oral
Oral solution (continued)
Dyphylline, 1461
Dyphylline and guaifenesin, 1462
Ephedrine sulfate, 1529
Ergocalciferol, 1549
Ergoloid mesylates, 1552
Escitalopram, 1584
Ethosuximide, 1645
Ferric ammonium citrate for, 266
Ferrous gluconate, 1717
Ferrous sulfate, 1720
Ferrous sulfate syrup, 1720
Fluoxetine, 1806
Fluphenazine hydrochloride, 1817
Fluphenazine hydrochloride elixir, 1815
Folic acid, compounded, 1866
Furosemide, 1893
Galantamine, 1917
Glycerin, 1969
Guaifenesin, 2003
Guaifenesin and codeine phosphate, 2004
Haloperidol, 2021
Hydralazine hydrochloride, 2045
Hydromorphone hydrochloride, 2079
Hydroxyzine hydrochloride, 2092
Hyoscyamine sulfate, 2104
Hyoscyamine sulfate elixir, 2103
Ipecac, 2226
Isoniazid, 2254
lsosorbide, 2267
Lamivudine, 2328
Leucovorin calcium compounded, 2361
Levetiracetam, 2373
Levocarnitine, 2388
Levofloxacin, 2397
Lincomycin, 2421
Lithium, 2436
Loperamide hydrochloride, 2448
Lopinavir and ritonavir, 2453
Loratadine, 2464
Magnesium carbonate, citric acid, and
potassium citrate for, 2503
Magnesium carbonate and citric acid for,
2502
Manganese chloride for, 2524
Magnesium citrate, 2506
Magnesium citrate for, 2507
Meperidine hydrochloride, 2575
Mesoridazine besylate, 2602
Metaproterenol sulfate, 2608
Methadone hydrochloride, 2629
Methdilazine hydrochloride, 2634
Methenamine, 2636
Methenamine mandelate for, 2639
Methylcellulose, 2666
Metoclopramide, 2701
Metoprolol tartrate, 2714
Mibolerone, 2735
Nafcillin sodium for, 2850
Neomycin sulfate, 2884
Nortriptyline hydrochloride, 2986
Ondansetron, 3032
Orange syrup, 5476
Oxacillin sodium for, 3062
Oxtriphylline, 3090
Oxybutynin chloride, 3094
Oxycodone hydrochloride, 3103
Index
Paromomycin, 3173
Penicillin G potassium for, 3201
Penicillin V potassium for, 3217
Perphenazine, 3246
Phenobarbital, 3260
Piperazine citrate syrup, 3334
Polyethylene glycol 3350 and electrolytes
for, 3345
Combined Index to USP 41 and NF 36
Potassium bicarbonate effervescent tablets Valproic acid, 4273
for, 3355 Vancomycin hydrochloride for, 4287
Potassium bicarbonate and potassium Vehicle for, 5474
chloride for effervescent, 3356 Vehicle for, sugar free, 5474
Potassium bicarbonate and potassium Verapamil hydrochloride, 4305
chloride effervescent tablets for, 3356 Vigabatrin for, 4315
Potassium bicarbonate, potassium chloride, Vitamins with minerals, oil-soluble, 4961
and potassium citrate effervescent Vitamins with minerals, oil- and water-
tablets for, 3367 soluble, 5047
Potassium bromide, veterinary, 3359 Vitamins with minerals, water-soluble,
Potassium chloride, 3362 5128
Potassium chloride for, 3363 Vitamins, oil-soluble, 4941
Potassium citrate and citric acid, 3375 Vitamins, oil- and water-soluble, 4995
Potassium gluconate, 3377 Zidovudine, 4370
Potassium gluconate and potassium Zinc acetate, 4376
chloride, 3378 Zinc sulfate, 4387
Potassium gluconate and potassium
chloride for, 3379
Potassium gluconate and potassium citrate,
3380
Potassium gluconate, potassium citrate, Oral suspension
and ammonium chloride, 3380 Acetaminophen, 39
Potassium iodide, 3382 Acetaminophen and codeine phosphate,
Potassium and sodium bicarbonates and 57
citric acid effervescent tablets for, 3357 Acetazolamide, 68
Prednisolone, 3412 Acyclovir, 83
Prednisolone sodium phosphate Albendazole, 93
compounded, 3418 Allopurinol, 123
Prednisone, 3422 Alprazolam, 131
Prochlorperazine, 3448 Alumina and magnesia, 149
Promazine hydrochloride, 3462 Alumina, magnesia, and calcium
Promazine hydrochloride syrup, 3463 carbonate, 151
Promethazine and phenylephrine Alumina, magnesia, and simethicone, 155
hydrochloride, 3470 Alumina and magnesium carbonate, 158
Promethazine and phenylephrine Alumina and magnesium trisilicate, 161
hydrochloride and codeine phosphate, Amiodarone hydrochloride, 245
3473 Amlodipine, 250
Promethazine hydrochloride, 3466 Amoxicillin, 279
Pseudoephedrine hydrochloride, 3508 Amoxicillin and clavulanate potassium for,
Pseudoephedrine hydrochloride, 284
carbinoxamine maleate, and Amoxicillin for, 280
dextromethorphan hydrobromide, 3511 Amoxicillin tablets for, 283
Pyridostigmine bromide, 3524 Ampicillin for, 301
Ranitidine, 3580 Ampicillin and probenecid for, 303
Risperidone, 3639 Atenolol compounded, 386
Ritonavir, 3651 Atenolol compounded, veterinary, 386
Saccharin sodium, 3691 Atovaquone, 400
Senna, 3743 Azathioprine, 417
Sertraline hydrochloride, 3749 Azithromycin for, 428
Sodium bromide, veterinary, 3780 Baclofen, 444
Sodium citrate and citric acid, 3787 Benazepril hydrochloride compounded,
Sodium fluoride, 3790 veterinary, 463
Sodium phosphates, 3807 Bethanechol chloride, 523
Stavudine for, 3836 Bismuth subsalicylate, 540
Sulfaquinoxaline, 3880 Calcium carbonate, 633
Syrup, 5637 Calcium and magnesium carbonates, 637
Terpin hydrate, 3998 Captopril, 679
Terpin hydrate and codeine, 3999 Carbamazepine, 685
Theophylline, 4036 Cefaclor for, 744
Theophylline and guaifenesin, 4043 Cefadroxil for, 752
Theophylline sodium glycinate, 4044 Cefdinir for, 767
Thiamine hydrochloride, 4048 Cefixime for, 775
Thiamine mononitrate, 4051 Cefpodoxime proxetil for, 801
Thioridazine hydrochloride, 4064 Cefprozil for, 806
Thiothixene hydrochloride, 4071 Cefuroxime axetil for, 825
Tolu balsam syrup, 5640 Cellulose sodium phosphate for, 832
Triamcinolone diacetate, 4193 Cephalexin for, 834
Cephalexin tablets for, 836
Tricitrates, 4205
Trifluoperazine, 4211 Cephradine for, 845
Trihexyphenidyl hydrochloride, 4219 Chloramphenicol palmitate, 868
Trikates, 4221 Chloroquine phosphate, 893
Trimeprazine, 4222 Chlorothiazide, 895
Triprolidine hydrochloride, 4233 Cholestyramine for, 919
Triprolidine and pseudoephedrine Ciprofloxacin for, 949
hydrochlorides, 4234 Cisapride compounded, veterinary, 954
Combined Index to USP 41 and NF 36 Oral-Oxidi 1-47

Oral suspension (continued) Metronidazole benzoate compounded, Vehicle for, 5474

Clarithromycin for, 977 2722 Verapamil hydrochloride, 4305
Clavulanate potassium and amoxicillin for, Minocycline hydrochloride, 2754 Zonisamide compounded, 4412
284 Mycophenolate mofetil for, 2832
Clomipramine compounded, veterinary, Nalidixic acid, 2853
1017 Naproxen, 2864
Clonazepam, 1021 Naratriptan hydrochloride, 2875 Orange
Clopidogrel compounded, 1033 Nevirapine, 2912 G, 5712
Colestipol hydrochloride for, 1072 Nitrofurantoin, 2951 oil, 5475
Colistin sulfate for, 1075 Nystatin, 2990 peel tincture, sweet, 5476
Dapsone, 1157 Nystatin for, 2990 spirit, compound, 5475
Demeclocycline, 1166 Omeprazole, 3026 syrup, 5476
Diazoxide, 1248 Ondansetron hydrochloride, 3033 Orbifloxacin, 3040
Dicloxacillin sodium for, 1267 Oxcarbazepine, 3082 tablets, 3041
Didanosine tablets for, 1275 Oxfendazole, 3087 Orcinol, 5712
Diltiazem hydrochloride, 1310 Oxytetracycline and nystatin for, 3126 Ordinary impurities (466), 6220
Dipyridamole, 1347 Oxytetracycline calcium, 3127 Organic
Dolasetron mesylate, 1385 Pantoprazole, 3154 nitrogenous bases—identification (181),
Doxycycline for, 1421 Penicillin G benzathine, 3196 6094
Doxycycline calcium, 1425 Penicillin V for, 3214 nitrogenous bases, salts of (501), 6245
Doxycycline compounded, veterinary, Penicillin V benzathine, 3216 Orlistat, 3043
1427 Pentoxifylline, 3231 capsules, 3045
Enalapril maleate, 1500 Pergolide, veterinary, 3238 Orphenadrine citrate, 3047
Enalapril maleate compounded, veterinary, Phenobarbital, 3261 aspirin and caffeine tablets, 3052
1501 Phenoxybenzamine hydrochloride injection, 3049
Enrofloxacin compounded, veterinary, compounded, 3267 extended-release tablets, 3050
1517 Phenytoin, 3286 Orthophenanthroline, 5713
Erythromycin estolate, 1574 Piroxicam compounded, 3338 TS, 5757
Erythromycin estolate for, 1574 Prednisolone compounded, veterinary, Oseltamivir phosphate, 3055
Erythromycin estolate and sulfisoxazole 3415 capsules, 3057
acetyl, 1575 Primidone, 3434 Osmium tetroxide, 5713
Erythromycin ethylsuccinate, 1578 Propylthiouracil, 3501 Osmolality and osmolarity (785), 6527
Erythromycin ethylsuccinate for, 1578 Psyllium hydrophilic mucilloid for, 3516 Otic solution
Erythromycin ethylsuccinate and Pyrantel pamoate, 3518 acetic acid, 70
sulfisoxazole acetyl for, 1580 Pyrazinamide, 3521 antipyrine and benzocaine, 332
Ethambutol hydrochloride compounded, Pyrimethamine, 3530 antipyrine, benzocaine, and phenylephrine
1634 Pyrvinium pamoate, 3532 hydrochloride, 333
Famciclovir compounded, 1678 Quinidine sulfate, 3553 benzocaine, 476
Famotidine for, 1682 Rifabutin, 3607 chloramphenicol, 865
Felbamate, 1686 Rifampin, 3610 gentamicin sulfate and betamethasone
Ferumoxsil, 1724 Sildenafil citrate, 3758 valerate, 1940
Flecainide acetate, 1750 Simethicone, 3762 hydrocortisone and acetic acid, 2062
Fluconazole for, 1763 Sodium phenylbutyrate, 3803 neomycin and polymyxin B sulfates and
Flucytosine, 1767 Sotalol hydrochloride, 3821 hydrocortisone, 2903
Furazolidone, 1891 Spironolactone, 3827 polymyxin B sulfate and hydrocortisone,
Ganciclovir, 1927 Spironolactone and hydrochlorothiazide, 3351
Granisetron hydrochloride, 1993 3828 Otic suspension
Griseofulvin, 1998 Spironolactone compounded, 3826 Ciprofloxacin and dexamethasone, 951
Hydroxyzine pamoate, 2099 Sulfadimethoxine, 3866 Oxacillin
lbuprofen, 2110 Sulfamethizole, 3871 injection, 3061
Indomethacin, 2158 Sulfamethoxazole, 3873 for injection, 3062
Isradipine, 2289 Sulfamethoxazole and trimethoprim, 3875 sodium, 3058
Ketoconazole, 2310 Sulfisoxazole acetyl, 3887 sodium capsules, 3060
Labetalol hydrochloride, 2321 Sumatriptan succinate, 3899 sodium for oral solution, 3062
Lamotrigine compounded, 2342 Tacrolimus, 3914 Oxalic acid, 5713
Lamotrigine tablets, 2340 Tadalafil compounded, 3918 tenth-normal (0.1 N), 5767
Lansoprazole compounded, 2351 Temozolomide, 3972 TS, 5757
Lisinopril, 2432 Terbinafine, 3981 Oxaliplatin, 3063
Loracarbef for, 2461 Terbutaline, 3988 injection, 3067
Magaldrate, 2497 Tetracycline, 4015 for injection, 3069
Magaldrate and simethicone, 2498 Tetracycline hydrochloride, 4022 Oxandrolone, 3072
Magnesium carbonate and sodium Theophylline, 4037 tablets, 3073
bicarbonate for, 2504 Thiabendazole, 4046 Oxaprozin, 3075
Marbofloxacin compounded, veterinary, Thioridazine, 4063 tablets, 3076
2532 Tiagabine hydrochloride, 4077 Oxazepam, 3077
Mebendazole, 2535 Topiramate compounded, 4146 capsules, 3078
Megestrol acetate, 2554 Tramadol hydrochloride, 4150 tablets, 3080
Meloxicam, 2560 Tramadol hydrochloride and Oxcarbazepine, 3080
Meprobamate, 2585 acetaminophen, 4157 oral suspension, 3082
Methacycline hydrochloride, 2626 Tramadol! hydrochloride compounded, tablets, 3084
Methadone hydrochloride tablets for, 2630 veterinary, 4160 Oxfendazole, 3087
Methenamine mandelate, 2640 Triflupromazine, 4214 oral suspension, 3087
Methyldopa, 2667 Trisulfapyrimidines, 4235 Oxidized cellulose, 830
Metolazone, 2705 Ursodiol, 4258 regenerated, 830
Metoprolol tartrate, 2714 Valacyclovir, 4261
1-48 Oxpre-Penta Combined Index to USP 41 and NF 36

Oxprenolol hydrochloride, 3088 Package integrity and test method selection Paroxetine
tablets, 3089 (1207.1), 7585 hydrochloride, 3174
extended-release tablets, 3089 Package integrity leak test technologies tablets, 3177
Oxtriphylline, 3090 (1207.2), 7597 extended-release tablets, 3178
oral solution, 3090 Package seal quality test technologies (1207. Partially-neutralized methacrylic acid and
tablets, 3091 3), 7614 ethyl acrylate copolymer, 5446
extended-release tablets, 3091 Packaging and repackaging—single unit Particle size distribution estimation by
Oxybenzone, 3093 containers (1136), 7414 analytical sieving (786), 6530
and dioxybenzone cream, 1322 Packaging and storage requirements (659), Particulate matter in injections (788), 6537
Oxybutynin chloride, 3093 6384 Particulate matter in ophthalmic solutions
oral solution, 3094 Packings for high-pressure liquid (789), 6540
tablets, 3095 chromatography, 5713 Peanut oil, 5481
tablets, extended-release, 3096 Paclitaxel, 3134 Pea starch, 5603
Oxycodone injection, 3136 Pectate lyase, 5714
and acetaminophen capsules, 3107 Padimate O, 3137 Pectin, 3182
and acetaminophen tablets, 3108 lotion, 3138 Pemetrexed
and aspirin tablets, 3109 Paired ion chromatography reagent, 5713 disodium, 3184
terephthalate, 3110 Paliperidone, 3139 for injection, 3186
Oxycodone hydrochloride, 3100 Palladium Penbutolol sulfate, 3187
oral solution, 3103 catalyst, 5713 tablets, 3188
tablets, 3103 chloride, 5713 Penicillamine, 3189
extended-release tablets, 3104 chloride TS, buffered, 5757 capsules, 3191
3,3’-Oxydipropionitrile, 5713 Palladous chloride, 5713 tablets, 3192
Oxygen, 3112 Pallida Penicillin
21 percent certified standard, 5713 echinacea, 4574 G benzathine, 3194
93 percent, 3112 extract, powdered echinacea, 4578 G benzathine injectable suspension, 3195
93 percent certified standard, 5713 powdered echinacea, 4576 G benzathine and penicillin G procaine
certified standard, 5713 Palm injectable suspension, 3196
flask combustion (471), 6238 oil, 5477 G benzathine oral suspension, 3196
helium certified standard, 5713 oil, hydrogenated, 5477 G benzathine tablets, 3196
Oxymetazoline hydrochloride, 3113 kernel oil, 5478 G, neomycin, polymyxin B, hydrocortisone
nasal solution, 3114 Palmitic acid, 5479 acetate, and hydrocortisone sodium
ophthalmic solution, 3114 Palonosetron succinate topical suspension, 3193
Oxymetholone, 3115 hydrochloride, 3140 G potassium, 3198
tablets, 3115 Pamabrom, 3142 G potassium injection, 3199
Oxymorphone hydrochloride, 3116 Pamidronate disodium, 3143 G potassium for injection, 3200
injection, 3117 for injection, 3144 G potassium for oral solution, 3201
tablets, 3119 Pancreatic digest of casein, 5713, 5742 G potassium tablets, 3202
extended-release tablets, 3121 Pancreatin, 3145, 5713 G procaine, 3203
Oxyquinoline sulfate, 5476 tablets, 3147 G procaine, dihydrostreptomycin sulfate,
Oxytetracycline, 3124 Pancreatin (1025), 6734 chlorpheniramine maleate, and
calcium, 3126 Pancrelipase, 3148 dexamethasone injectable suspension,
calcium oral suspension, 3127 capsules, 3149 3207
for injection, 3128 delayed-release capsules, 3150 G procaine and dihydrostreptomycin
hydrochloride, 3127 tablets, 3150 sulfate injectable suspension, 3207
hydrochloride capsules, 3128 Pancuronium bromide, 3151 G procaine and dihydrostreptomycin
hydrochloride and hydrocortisone acetate injection, 3152 sulfate intramammary infusion, 3206
ophthalmic suspension, 3129 Panthenol, 3153 G procaine, dihydrostreptomycin sulfate,
hydrochloride and hydrocortisone Pantoprazole and prednisolone injectable suspension,
ointment, 3130 oral suspension, 3154 3209
hydrochloride and polymyxinB sulfate Pantoprazole sodium, 3155 G procaine injectable suspension, 3205
ointment, 3130 delayed-release tablets, 3157 G procaine for injectable suspension, 3205
hydrochloride and polymyxinB sulfate Papaic digest of soybean meal, 5713 G procaine intramammary infusion, 3204
ophthalmic ointment, 3130 Papain, 3161 G procaine, neomycin and polymyxin B
hydrochloride and polymyxin B sulfate tablets for topical solution, 3161 sulfates, and hydrocortisone acetate
topical powder, 3131 Papaverine hydrochloride, 3162 topical suspension, 3210
hydrochloride and polymyxin B sulfate injection, 3163 G procaine and novobiocin sodium
vaginal inserts, 3131 tablets, 3163 intramammary infusion, 3210
hydrochloride soluble powder, 3129 Paper G procaine and penicillin G benzathine
injection, 3125 lead acetate, 5704 injectable suspension, 3196
and nystatin capsules, 3125 Para-aminobenzoic acid, 5667, 5713 G sodium, 3211
and nystatin for oral suspension, 3126 Parachlorophenol, 3164 G sodium for injection, 3212
tablets, 3125 camphorated, 3164 V, 3213
Oxytocin, 3132 Paraffin, 5480 V benzathine, 3215
injection, 3133 synthetic, 5481 V benzathine oral suspension, 3216
Paraformaldehyde, 5713 V potassium, 3216
Paraldehyde, 3165 V potassium for oral solution, 3217
Paregoric, 3166 V potassium tablets, 3218
Paricalcitol, 3167 V for oral suspension, 3214
P capsules, 3169
injection, 3171
V tablets, 3214
Penicillinase, 5714
Paromomycin Pentadecane, 5714
P32 oral solution, 3173 1-Pentadecanol, 5714
solution, sodium phosphate, 3295 sulfate, 3173 Pentafluoropropionic acid, 5714
suspension, chromic phosphate, 3295 sulfate capsules, 3173 Pentamidine isethionate, 3219
Combined Index to USP 41 and NF 36 Penta-Physi 1-49

Pentane, 5714 Phenelzine sulfate, 3256 hydrochloride and promethazine oral

1-Pentanesulfonic acid sodium salt, 5714 tablets, 3257 solution, 3470
2-Pentanone, 5714 Pheniramine maleate, 3258 hydrochloride injection, 3279
Pentazocine, 3220 and naphazoline hydrochloride ophthalmic hydrochloride nasal jelly, 3280
and acetaminophen tablets, 3221 solution, 2862 hydrochloride nasal solution, 3280
and aspirin tablets, 3222 Phenmetrazine hydrochloride, 3258 hydrochloride ophthalmic solution, 3281
hydrochloride, 3220 tablets, 3259 hydrochloride tablets, 3281
injection, 3225 Phenobarbital, 3260 Phenylethyl alcohol, 3283
and naloxone tablets, 3224 sodium, 3262 Phenylglycine, 5717
Pentetic acid, 3227 sodium injection, 3262 Phenylhydrazine, 5717
Pentobarbital, 3227 sodium for injection, 3263 acetate TS, 5757
sodium, 3228 oral solution, 3260 hydrochloride, 5717
sodium injection, 3229 oral suspension, 3261 sulfuric acid TS, 5757
Pentoxifylline, 3230 tablets, 3261 Phenylmercuric
oral suspension, 3231 theophylline and ephedrine hydrochloride acetate, 5485
extended-release tablets, 3232 tablets, 4041 nitrate, 5486
People, xi Phenol, 3263, 5717 Phenylmethylsulfony! fluoride, 5717
Peppermint, 5482 alcohol TS, 5750 3-Phenylphenol, 5717
oil, 5482 topical gel, camphorated, 3264 Phenylpropanolamine
spirit, 3234 iron, TS, 5755 hydrochloride, 3283
water, 5483 liquefied, 3265 Phenyltoloxamine citrate, 3284
Pepsin, 5714 ted, 5717, 5746 Phenytoin, 3285
purified, 5716 red, sodium, 5717 chewable tablets, 3287
Peptic digest of animal tissue, 5716 red TS, 5757 sodium, 3289
Peptone, dried, 5693, 5716 red TS, pH 4.7, 5757 sodium capsules, extended, 3290
Perchloric acid, 5716 camphorated, topical solution, 3264 sodium injection, 3293
tenth-normal (0.1 N) in dioxane, 5767 TS, 5757 oral suspension, 3286
tenth-normal (0.1 N) in glacial acetic acid, Phenolated PH indicator paper, short-range, 5747
5767 calamine topical suspension, 616 Phloroglucinol, 5717
TS, 5757 Phenoldisulfonic acid TS, 5757 TS, 5757
Perflubron, 3234 Phenolphthalein, 5746 Phloxine B, 5718
Perflutren protein-type A microspheres paper, 5747 Phosphatase enzyme, alkaline, 5666, 5718
injectable suspension, 3235 Phenolphthalein TS, 5757 Phosphate
Pergolide Phenolsulfonphthalein, 5483, 5717 acidulated, and sodium fluoride topical
mesylate, 3237 Phenoxybenzamine hydrochloride, 3265, solution, 3791
oral suspension veterinary, 3238 5717 buffer, 5676
tablets, 3239 capsules, 3266 diethylamine, 5688
Perindopril Phenoxybenzamine hydrochloride P 32 solution, sodium, 3295
erbumine, 3240 compounded P 32 suspension, chromic, 3295
erbumine tablets, 3243 oral suspension, 3267 in reagents, 5663
Periodic acid, 5716 3-Phenoxybenzoic acid, 5717 Phosphatic enzyme, 5718
Periodontal system 2-Phenoxyethanol, 5717 TS, 5757
minocycline, 2758 Phenoxyethanol, 5485 Phosphomolybdic acid, 5718
Perphenazine, 3245 Phensuximide, 3267 TS; 5757
and amitriptyline hydrochloride tablets, capsules, 3268 Phosphoric acid, 5487, 5718
3248 Phentermine hydrochloride, 3268 diluted, 5487
injection, 3246 capsules, 3269 and sodium fluoride gel, 3792
oral solution, 3246 tablets, 3270 0.01% TS, 5758
syrup, 3247 Phentolamine mesylate, 3271 0.05 M TS, 5757
tablets, 3247 for injection, 3271 0.06 M TS, 5757
Pertussis Phenyl 10 TS, 5758
immune globulin, 3249 ether, 5717 1 NTS, 5757
Petrolatum, 3249 isocyanate, 5717 20% TS, 5758
hydrophilic, 3250 2-Phenylacetamide, 5717 0.01 M TS, 5757
white, 3250 Phenylalanine, 3272, 5717 0.75 M Phosphoric acid TS, 5757
Petroleum benzin, 5716 di-Phenylalanine, 5717 0.02 M Phosphoric acid TS, 5758
pH (791), 6543 Phenylbutazone, 3273 1.5 M Phosphoric acid TS, 5757
Pharmaceutical calculations in pharmacy boluses, 3274 Phosphorous acid, 5718
practice (1160), 7451 injection, 3274 Phosphorus
Pharmaceutical compounding tablets, 3275 pentoxide, 5718
nonsterile preparations (795), 6546 p-Phenylenediamine red, 5718, 5724
sterile preparations (797), 6554 dihydrochloride, 5717 Phosphotungstic acid, 5718
Pharmaceutical dosage forms (1151), 7425 hydrochloride, 5717 TS, 5758
Phases for gas chromatography, 5716 o-Phenylenediamine dihydrochloride, 5717 o-Phthalaldehyde, 5718
Phellandrene Phenylephrine Phthalazine, 5718
(R)-(-)-alpha, 5716 bitartrate, 3275 Phthalic
Phenacetin, 5716 bitartrate and isoproterenol hydrochloride acid, 5718
1,10-Phenanthroline, 5713, 5716 inhalation aerosol, 2262 anhydride, 5718
o-Phenanthroline monohydrochloride Diphenhydramine, hydrochloride tablets, Phthalimide, 5718
monohydrate, 5716 1334 Phyllanthus amarus, 4800
Phenazopyridine hydrochloride, 3251 hydrochloride, 3277 powdered, 4802
tablets, 3252 hydrochloride, antipyrine, and benzocaine Physical environments that promote safe
Phendimetrazine tartrate, 3253 otic solution, 333 medication use (1066), 7078
capsules, 3254 hydrochloride and promethazine and Physicochemical analytical procedures for
tablets, 3255 codeine phosphate oral solution, 3473 insulins (121.1), 6056
50 Physi-Polyv Combined Index to USP 41 and NF 36

Physicochemical integrators and indicators Poloxalene, 3341 and neomycin sulfates and hydrocortisone
for sterilization (1229.9), 7728 Poloxamer, 5489 otic suspension, 2903
Physostigmine Polycarbophil, 3342 and neomycin sulfates and lidocaine
salicylate, 3296 calcium, 657 cream, 2904
salicylate injection, 3296 Polydecene and neomycin sulfates ophthalmic
salicylate ophthalmic solution, 3297 hydrogenated, 5491 ointment, 2894
Phytonadione, 3297 Polydextrose, 5493 and neomycin sulfates ophthalmic
injectable emulsion, 3298 hydrogenated, 5495 solution, 2894
tablets, 3299 Polydimethylsiloxane, viscosity 0.65 and neomycin sulfates, penicillin G
2-Picoline, 5718 centistokes, 5719 procaine, and hydrocortisone acetate
Picrate TS, alkaline, 5750, 5758 Polyethylene topical suspension, 3210
Picric acid, 5718, 5741 glycol, 5498 and neomycin sulfates and pramoxine
TS, 5758 glycol 200, 5719 hydrochloride cream, 2905
Picrolonic acid, 5718 glycol 600, 5719 and neomycin sulfates and prednisolone
Pilocarpine, 3299 glycol 20,000, 5719 acetate ophthalmic suspension, 2906
hydrochloride, 3301 glycol 3350 and electrolytes for oral and neomycin sulfates solution for
hydrochloride ophthalmic solution, 3303 solution, 3345 irrigation, 2894
hydrochloride tablets, 3303 glycol monomethyl ether, 5501 penicillin G, neomycin, hydrocortisone
nitrate, 3305 glycol ointment, 5501 acetate, and hydrocortisone sodium
nitrate ophthalmic solution, 3305 oxide, 5503 succinate topical suspension, 3193
ocular system, 3301 Polyethylene glycol 3350, 3343 sulfate, 3347
Pimobendan, 3305 Polyethylene glycol standards with molecular sulfate and bacitracin topical aerosol, 439
Pimozide, 3306 weights of 1000, 2000, 3000, 4000, and sulfate and bacitracin zinc topical aerosol,
tablets, 3307 6000 daltons (g/mol), 5719 3350
Pindolol, 3309 Polyglyceryl sulfate and bacitracin zinc ointment, 442
tablets, 3309 3 diisostearate, 5507 sulfate and bacitracin zinc ophthalmic
Pinene dioleate, 5505 ointment, 442
(+)-alpha, 5719 Polyisobutylene, 5508 sulfate and bacitracin zinc topical powder,
beta, 5719 Polymyxin B 3350
Pioglitazone for injection, 3349 sulfate and chloramphenicol ophthalmic
and glimepiride tablets, 3314 and neomycin sulfates, bacitracin, and ointment, 867
hydrochloride, 3311 hydrocortisone acetate ointment, 2895 sulfate and hydrocortisone otic solution,
and metformin hydrochloride tablets, 3317 and neomycin sulfates, bacitracin, and 3351
tablets, 3312 hydrocortisone acetate ophthalmic sulfate and oxytetracycline hydrochloride
Pipemidic acid, 5719 ointment, 2896 ointment, 3130
Piperacillin, 3321 and neomycin sulfates, bacitracin, and sulfate and oxytetracycline hydrochloride
for injection, 3324 lidocaine ointment, 2896 ophthalmic ointment, 3130
sodium, 3323 and neomycin sulfates and bacitracin sulfate and oxytetracycline hydrochloride
and tazobactam for injection, 3325 ointment, 2894 topical powder, 3131
Piperazine, 3332, 5719 and neomycin sulfates and bacitracin sulfate and oxytetracycline hydrochloride
adipate, 3333 ophthalmic ointment, 2895 vaginal inserts, 3131
citrate, 3333 and neomycin sulfates, bacitracin zinc, and sulfate and trimethoprim ophthalmic
citrate syrup, 3334 hydrocortisone acetate ophthalmic solution, 3351
citrate tablets, 3334 ointment, 2899 Polyoxyethylene 10 lauryl ether, 5719
dihydrochloride, 3335 and neomycin sulfates, bacitracin zinc, and Polyoxyethylene (20) sorbitan monolaurate,
phosphate, 3335 hydrocortisone ointment, 2898 5719
Piperidine, 5719 and neomycin sulfates, bacitracin zinc, and Polyoxyethylene (23) lauryl ether, 5719
Piroxicam, 3336 hydrocortisone ophthalmic ointment, Polyoxyl
capsules, 3337 2898 10 oleyl ether, 5509
cream, 3338 and neomycin sulfates, bacitracin zinc, and 15 hydroxystearate, 5510
Piroxicam compounded lidocaine ointment, 2900 20 cetostearyl ether, 5514
oral suspension, 3338 and neomycin sulfates and bacitracin zinc 35 castor oil, 5515
Plantago seed, 3339 ointment, 2897 40 hydrogenated castor oil, 5516
Plant Stanol Esters, 4803 and neomycin sulfates and bacitracin zinc lauryl ether, 5518
Plasma protein fraction, 3340 ophthalmic ointment, 2897 oleate, 5519
Plasma spectrochemistry (730), 6482 and neomycin sulfates cream, 2893 stearate, 5520
Plasma spectrochemistry—theory and and neomycin sulfates and dexamethasone stearyl ether, 5521
practice (1730), 7956 ophthalmic ointment, 2900 Polysaccharide molecular weight standards,
Plastic materials of construction (661.1), and neomycin sulfates and dexamethasone 5719
6403 ophthalmic suspension, 2901 Polysorbate
Plastic packaging systems and their materials and neomycin sulfates and gramidicin 20, 5522
of construction (661), 6396 cream, 2902 40, 5522
Plastic packaging systems for pharmaceutical and neomycin sulfates, gramidicin, and 60, 5523
use (661.2), 6424 hydrocortisone acetate cream, 2902 80, 5524
Platinic and neomycin sulfates and gramidicin Polysorbate 80, 5719
chloride, 5719 ophthalmic solution, 2902 Polystyrene
chloride TS, 5758 and neomycin sulfates and hydrocortisone cation-exchange resin, 5719
Platinum acetate cream, 2904 Polytef, 5719
cobalt TS, 5758 and neomycin sulfates and hydrocortisone Polyvinyl
Podophyllum, 3340 acetate ophthalmic suspension, 2904 acetate, 5526
resin, 3341 and neomycin sulfates and hydrocortisone acetate dispersion, 5528
resin topical solution, 3341 ophthalmic suspension, 2903 acetate phthalate, 5530
Polacrilin potassium, 5488 and neomycin sulfates and hydrocortisone alcohol, 3352, 5719
Polarography (801), 6617 otic solution, 2903 alcohol and ethylene glycol graft
Policies, USP, xxix copolymer, 5346
Combined Index to USP 41 and NF 36 Poros-Powde 1-51

Porosimetry by mercury intrusion (267), citrate, magnesium carbonate, and citric phosphate, monobasic, 5535, 5709, 5719,
6160 acid for oral solution, 2503 5721
Porosity by nitrogen adsorption—desorption citrate, potassium chloride, and potassium phosphate, tribasic, 5721
(268), 6163 bicarbonate effervescent tablets for oral phosphates injection, 3387
Positron emission tomography drugs for solution, 3367 pyroantimonate, 5721
compounding, investigational, and citrate, potassium gluconate, and pyroantimonate TS, 5758
research uses (823), 6629 ammonium chloride oral solution, 3380 pyrophosphate, 5721
Positron emission tomography drugs— citrate and potassium gluconate oral pyrosulfate, 5721
information (1823), 8098 solution, 3380 and sodium bicarbonates and citric acid
Potash, sulfurated, 3353 citrate tablets, 4805 effervescent tablets for oral solution,
Potassium citrate extended-release tablets, 3372 3357
acetate, 3353, 5719 cyanide, 5720 sodium tartrate, 3388, 5721
acetate injection, 3354 dichromate, 5720 sorbate, 5536
acetate TS, 5758 dichromate, tenth-normal (0.1 N), 5768 sulfate, 5721
alginate, 5531 dichromate TS, 5758 sulfate TS, 5758
alum, 148, 5719 ferricyanide, 5720 tellurite, 5721
arsenate monobasic, 5719 ferricyanide TS, 5758 thiocyanate, 5721
arsenite, tenth-normal (0.1 N), 5768 ferricyanide, twentieth-molar (0.05 M), thiocyanate, tenth-normal (0.1 N), 5769
benzoate, 5532 5768 thiocyanate TS, 5758
bicarbonate, 3355, 5719 ferrocyanide, 5720 0.025 N Potassium dichromate VS, 5768
bicarbonate effervescent tablets for oral ferrocyanide TS, 5758 Potassium hydroxide
solution, 3355 gluconate, 3376 1.8 N TS, 5758
bicarbonate and potassium chloride for gluconate and potassium chloride oral 45% TS, 5758
effervescent oral solution, 3356 solution, 3378 10 M TS, 5758
bicarbonate and potassium chloride gluconate and potassium chloride for oral 0.1 N VS, 5769
effervescent tablets for oral solution, solution, 3379 Potassium phosphate
3356 gluconate, potassium citrate, and 0.02 M TS, 5758
bicarbonate, potassium chloride, and ammonium chloride oral solution, 3380 0.2 M TS, 5758
potassium citrate effervescent tablets for gluconate and potassium citrate oral Potassium phosphates
oral solution, 3367 solution, 3380 compounded injection, 3387
biphosphate, 5719 gluconate oral solution, 3377 Potato starch, 5609, 5721
biphthalate, 5720 gluconate tablets, 3378 Povidone, 3389
bismuth iodide TS, 5758 guaiacolsulfonate, 3381 Povidone-iodine, 3392
bisulfate, 5720 hyaluronate, 5720 topical aerosol, 3392
bitartrate, 3358 hydrogen sulfate, 5720 cleansing solution, 3393
bromate, 5720 hydroxide, 5533, 5720 ointment, 3393
bromate, tenth-normal (0.1 N), 5768 hydroxide, alcoholic, half-normal (0.5 N), topical solution, 3393
bromide, 3358, 5720 5758, 5768
bromide-bromate, tenth-normal (0.1 N), hydroxide, alcoholic, tenth-molar (0.1 M),
5768 5768
bromide oral solution, veterinary, 3359 hydroxide, methanolic, tenth-normal (0.1
carbonate, 3360, 5720 N), 5769 Powder
carbonate, anhydrous, 5669, 5720 hydroxide, normal (1 N), 5769 Absorbable dusting, 1457
carbonate TS, 5758 hydroxide TS, 5758 Ampicillin soluble, 300
chlorate, 5720 hydroxide 2 N TS, 5758 Amprolium soluble, 307
chloride, 3360, 5720 hydroxide TS, alcoholic, 5758 Astragalus root, 4450
chloride extended-release capsules, 3361 hydroxide TS 2, alcoholic, 5758 Bacitracin methylene disalicylate soluble,
chloride in dextrose injection, 3364 iodate, 5720 439
chloride in dextrose and sodium chloride iodate, twentieth-molar (0.05 M), 5769 Bacitracin zinc soluble, 442
injection, 3365 iodide, 3381, 5720 Banaba leaf, 4462
chloride for injection concentrate, 3361 iodide and iodine TS 1, 5755 Chlortetracycline and sulfamethazine
chloride in lactated ringer’s and dextrose iodide and iodine TS 2, 5755 bisulfates soluble, 911
injection, 3367 iodide and iodine TS 3, 5755 Chlortetracycline hydrochloride soluble,
chloride, potassium bicarbonate, and iodide oral solution, 3382 92
potassium citrate effervescent tablets for iodide and starch TS, 5758 Cinnamomum cassia twig, 4548
oral solution, 3367 iodide tablets, 3382 Compound clioquinol topical, 1003
chloride and potassium bicarbonate for iodide delayed-release tablets, 3382 Cromolyn sodium inhalation, 1104
effervescent oral solution, 3356 iodide TS, 5758 Echinacea species, 4595
chloride and potassium bicarbonate iodide 20% TS, 5758 Eleuthero root and rhizome, capsules,
effervescent tablets for oral solution, iodoplatinate TS, 5758 4604
3356 metabisulfite, 5534, 5720 Fenugreek seed, 4609
chloride and potassium gluconate oral metaphosphate, 5534 Fluticasone propionate and salmeterol,
solution, 3378 nitrate, 3383, 5720 inhalation, 1852
chloride and potassium gluconate for oral nitrate solution, 3384 Fluticasone propionate inhalation, 1836
solution, 3379 nitrite, 5721 Ganoderma lucidum fruiting body, 4632
chloride in sodium chloride injection, 3370 perchlorate, 3384, 5721 Iron, 5702
chloride oral solution, 3362 perchlorate capsules, 3385 Japanese honeysuckle flower, 4715
chloride for oral solution, 3363 periodate, 5721 Levothyroxine sodium oral, 2407
chloride extended-release tablets, 3363 permanganate, 3385, 5721 Lincomycin hydrochloride soluble, 2422
chloroplatinate, 5720 permanganate, tenth-normal (0.1 N), Methylbenzethonium chloride topical,
chromate, 5720 5758, 5769 2663
chromate TS, 5758 permanganate TS, 5758 Miconazole nitrate topical, 2739
citrate, 3371 persulfate, 5721 Neomycin sulfate, isoflupredone acetate,
citrate and citric acid oral solution, 3375 phosphate, dibasic, 3386, 5687, 5721 and tetracaine hydrochloride topical,
phosphate, dibasic, trihydrate, 5721 2892
1-52 Powde-Proma Combined Index to USP 41 and NF 36

Powder (continued) malabar-nut-tree, leaf, 4753 Prednisolone sodium phosphate

Northern schisandra fruit, 4868 milk thistle, 4772 compounded
Nystatin topical, 2989 milk thistle extract, 4773 oral solution, 3418
Olive leaf, 4796 opium, 3039 Prednisone, 3421
Oral, containing at least three of the Phyllanthus amarus, 4802 injectable suspension, 3423
following—acetaminophen and (salts of) rauwolfia serpentina, 3585 oral solution, 3422
chlorpheniramine, dextromethorphan, Rhodiola rosea, 4827 tablets, 3423
and pseudoephedrine, 47 Rhodtola rosea extract, 4828 Preface
Oxytetracycline hydrochloride and rosemary, 4838 and mission, vii
polymyxin B sulfate topical, 3131 saw palmetto, 4858 Pregabalin, 3424
Oxytetracycline hydrochloride soluble, stinging nettle, 4891 Pregnenolone acetate, 5721
3129 stinging nettle extract, 4892 Prekallikrein activator (165), 6090
Polymyxin B sulfate and bacitracin zinc turmeric, 4917 Preparation of biological specimens for
topical, 3350 turmeric extract, 4918 histologic and immunohistochemical
Rhodiola crenulata root and rhizome, 4824 valerian, 4926 analysis (1285), 7868
St. John’s wort flowering top, 4844 valerian extract, 4927 Prescription balances and volumetric
Salix species bark, 4854 zinc chloride, anhydrous, 5744 apparatus (1176), 7485
Salmeterol inhalation, 3697 Prescription container labeling (17), 5954
Sodium bicarbonate oral, 3779 Prilocaine, 3426
Soy isoflavones, powdered extract, 4877 and epinephrine injection, 3429
Sulfadimethoxine soluble, 3865 Powder fineness (811), 6621 hydrochloride, 3427
Tangerine peel, 4898 Powder flow (1174), 7481 hydrochloride injection, 3428
Tetracycline hydrochloride soluble, 4021 Pralidoxime and lidocaine cream, 2418
Tienchi ginseng root and rhizome, 4903 chloride, 3394 Primaquine phosphate, 3430
Tolnaftate topical, 4136 chloride for injection, 3395 tablets, 3431
Zinc oxide, 4384 Pramipexole dihydrochloride, 3395 Primidone, 3432
Pramoxine oral suspension, 3434
hydrochloride, 3397 tablets, 3434
hydrochloride cream, 3398 Probenecid, 3435
hydrochloride jelly, 3398 and ampicillin for oral suspension, 303
Powdered hydrochloride and neomycin and and colchicine tablets, 3437
American ginseng, 4423 polymyxin B sulfates cream, 2905 tablets, 3436
American ginseng extract, 4425 Pravastatin sodium, 3399 Probucol, 3438
andrographis, 4431 tablets, 3401 tablets, 3439
andrographis extract, 4433 Praziquantel, 3403 Procainamide hydrochloride, 3439
ashwagandha root, 4438 tablets, 3404 capsules, 3440
ashwagandha root extract, 4439 Prazosin hydrochloride, 3405 injection, 3441
Asian ginseng, 4442 capsules, 3406 tablets, 3441
Asian ginseng extract, 4444 Prednicarbate, 3407 extended-release tablets, 3442
bilberry extract, 4472 cream, 3408 Procaine
black cohosh, 4476 ointment, 3409 hydrochloride, 3443
black cohosh extract, 4478 Prednisolone, 3411 hydrochloride and epinephrine injection,
black pepper, 4485 acetate, 3413 3445
black pepper extract, 4487 acetate and gentamicin ophthalmic hydrochloride injection, 3444
cat’s claw, 4507 ointment, 1942 and propoxycaine hydrochlorides and
cat’s claw extract, 4509 acetate and gentamicin ophthalmic levonordefrin injection, 3491
cellulose, 5280 suspension, 1943 and propoxycaine hydrochlorides and
Chinese salvia, 4533 acetate injectable suspension, 3414 norepinephrine bitartrate injection, 3492
digitalis, 1287 acetate and neomycin and polymyxin B and tetracaine hydrochlorides and
Echinacea angustifolia, 4569 sulfates ophthalmic suspension, 2906 levonordefrin injection, 3445
Echinacea angustifolia extract, 4571 acetate and neomycin sulfate ophthalmic Procarbazine hydrochloride, 3446
Echinacea pallida, 4576 suspension, 2907 capsules, 3447
Echinacea pallida extract, 4578 acetate ophthalmic suspension, 3415 Prochlorperazine, 3447
Echinacea purpurea, 4585 acetate and sulfacetamide sodium edisylate, 3449
Echinacea purpurea extract, 4587 ophthalmic ointment, 3857 edisylate injection, 3449
eleuthero, 4602 acetate and sulfacetamide sodium maleate, 3450
eleuthero extract, 4599 ophthalmic suspension, 3858 maleate tablets, 3451
fenugreek seed, extract, 4612 cream, 3412 oral solution, 3448
feverfew, 4616 hemisuccinate, 3416 suppositories, 3448
garlic, 4644 penicillin G procaine, and Procyclidine hydrochloride, 3453
garlic extract, 4646 dihydrostreptomycin sulfate injectable tablets, 3453
ginger, 4652 suspension, 3209 Products for nebulization—characterization
ginkgo extract, 4660 sodium phosphate, 3416 tests (1601), 7874
goldenseal, 4682 sodium phosphate injection, 3418 Progesterone, 3454
goldenseal extract, 4684 sodium phosphate ophthalmic solution, injectable suspension, 3456
green tea extract, decaffeinated, 4687 3419 injection, 3455
gymnema, 4695 sodium succinate for injection, 3419 intrauterine contraceptive system, 3455
hawthorn leaf with flower, 4701 oral solution, 3412 vaginal suppositories, 3457
holy basil leaf, 4706 tablets, 3413 Proguanil hydrochloride, 3458
holy basil leaf extract, 4708 tebutate, 3420 Proline, 3460
horse chestnut, 4527 tebutate injectable suspension, 3421 Promazine hydrochloride, 3461
horse chestnut extract, 4529 tetracycline hydrochloride and novobiocin injection, 3462
ipecac, 2225 sodium tablets, 4024 oral solution, 3462
licorice, 4736 Prednisolone compounded oral suspension, syrup, 3463
licorice extract, 4737 veterinary, 3415 tablets, 3463
Combined Index to USP 41 and NF 36 Prome-Queti 1-53

Promethazine Pseudoephedrine root, Echinacea, 4583

and phenylephrine hydrochloride and chlorpheniramine, dextromethorphan (salts Putrescine dihydrochloride, 5723
codeine phosphate oral solution, 3473 of), and acetaminophen, capsules Pygeum, 4806
and phenylephrine hydrochloride oral containing at least three of the capsules, 4809
solution, 3470 following, 45 extract, 4807
Promethazine hydrochloride, 3463 chlorpheniramine, dextromethorphan (salts Pyrantel pamoate, 3517
injection, 3465 of), and acetaminophen, oral powder and ivermectin tablets, 2298
oral solution, 3466 containing at least three of the oral suspension, 3518
suppositories, 3467 following, 47 Pyrantel tartrate, 3519
tablets, 3468 chlorpheniramine, dextromethorphan (salts Pyrazinamide, 3520
Propafenone hydrochloride, 3476 of), and acetaminophen, oral solution rifampin, isoniazid, and ethambutol
extended-release capsules, 3477 containing at least three of the hydrochloride tablets, 3614
tablets, 3481 following, 49 rifampin and isoniazid tablets, 3612
Propane, 5537 chlorpheniramine, dextromethorphan (salts oral suspension, 3521
Propanediol, 5538 of) and acetaminophen, tablets tablets, 3521
Propantheline bromide, 3483 containing at least three of the Pyrazole, 5723
tablets, 3484 following, 51 Pyrene, 5723
Proparacaine hydrochloride, 3485 and diphenhydramine capsules, 1337 Pyrethrum extract, 3522
and fluorescein sodium ophthalmic hydrochloride, 3507 4-(2-Pyridylazo)resorcinol, 5723
solution, 1793 hydrochloride, acetaminophen, Pyridine, 5723
ophthalmic solution, 3486 dextromethorphan hydrobromide, and dried, 5723
Propellants (602), 6353 doxylamine succinate oral solution, 60 Pyridine-pyrazolone TS, 5758
Propionaldehyde, 5721 hydrochloride, acetaminophen, and Pyridostigmine bromide, 3523
Propionic diphenhydramine hydrochloride tablets, injection, 3523
acid, 5539 63 oral solution, 3524
anhydride, 5721 hydrochloride and acetaminophen tablets, tablets, 3524
Propionic acid, 5721 64 Pyridoxal
Propiophenone, 5721 hydrochloride extended-release capsules, hydrochloride, 5723
Propofol, 3486 3507 5-phosphate, 5723
injectable emulsion, 3489 hydrochloride, carbinoxamine maleate, Pyridoxamine dihydrochloride, 5723
Propoxycaine and dextromethorphan hydrobromide Pyridoxine hydrochloride, 3525
hydrochloride, 3490 oral solution, 3511 injection, 3526
and procaine hydrochlorides and hydrochloride and chlorpheniramine tablets, 3527
levonordefrin injection, 3491 maleate extended-release capsules, 903 1-(2-Pyridylazo)-2-naphthol, 5723
and procaine hydrochlorides and hydrochloride and chlorpheniramine 3-(2-Pyridyl)-5,6-di(2-furyl)-1,2,4-triazine-5’,
norepinephrine bitartrate injection, 3492 maleate oral solution, 904 5”-disulfonic acid, disodium salt, 5723
Propranolol hydrochloride, 3493 hydrochloride and guaifenesin capsules, Pyrilamine maleate, 3528
extended-release capsules, 3493 2005 tablets, 3529
and hydrochlorothiazide tablets, 3497 hydrochloride, guaifenesin, and Pyrimethamine, 3529
injection, 3495 dextromethorphan hydrobromide and sulfadoxine tablets, 3868
tablets, 3496 capsules, 2006 oral suspension, 3530
iso-Propyl alcohol, 5721 hydrochloride and ibuprofen tablets, 2113 tablets, 3531
n-Propyl alcohol, 5666, 5721 hydrochloride oral solution, 3508 Pyrogallol, 5723
Propyl gallate, 5540 hydrochloride tablets, 3509 TS, alkaline, 5758
Propylamine hydrochloride, 5721 hydrochloride extended-release tablets, Pyrogen test (151), 6083
Propylene 3510 Pyroxylin, 3531
carbonate, 5541 hydrochloride and cetrizine hydrochloride Pyrrole, 5723
glycol, 3498 extended-release tablets, 853 Pyruvic acid, 5723
glycol alginate, 5541 hydrochloride and fexofenadine Pyrvinium pamoate, 3532
glycol dicaprylate/dicaprate, 5542 hydrochloride extended-release tablets, oral suspension, 3532
glycol dilaurate, 5543 1731 tablets, 3533
glycol monocaprylate, 5544 sulfate, 3512
glycol monolaurate, 5545 sulfate and brompheniramine maleate oral
glycol monostearate, 5546 solution, 559
Propylhexedrine, 3500 sulfate and dexbrompheniramine maleate
inhalant, 3500
Propyliodone, 3500
oral solution, 1209
and triprolidine hydrochlorides oral Q
injectable oil suspension, 3501 solution, 4234
Propylparaben, 5547, 5721 and triprolidine hydrochlorides tablets, Quality assurance in pharmaceutical
sodium, 5548 4235 compounding (1163), 7475
Propylthiouracil, 3501 Psyllium Quality attributes of tablets labeled as having
oral suspension, 3501 hemicellulose, 3513 a functional score (705), 6457
tablets, 3502 husk, 3515 Quality of biotechnological products
Protamine sulfate, 3503 hydrophilic mucilloid for oral suspension, analysis of the expression construct in cells
injection, 3504 3516 used for production of r-DNA derived
Protein Pullulan, 5549 protein products (1048), 6928
molecular weight standard, 5721 Pullulanase, 5721 stability testing of biotechnological/
standard solution (8 g/dL), 5721 5,800, 23,700, and 100,000 molecular biological products (1049), 6930
Protein A quality attributes (130), 6076 weight (MW) pullulan standards, 5709 Quazepam, 3534
Protein determination procedures (507), Pumice, 3516, 5722 tablets, 3534
6248 Pure steam, 4348 Quercetin, 4810
Protocatechuic acid, 5721 Purine, 5723 Quetiapine
Protriptyline hydrochloride, 3505 Purpurea tablets, 3535
tablets, 3506 extract, powdered Echinacea, 4587 extended-release tablets, 3538
powdered Echinacea, 4585 Quetiapine fumarate, 3540
 4522 Chaste Tree / Dietary Supplements USP 41

Solution A: Methanol Solution A: Acetonitrile

Solution B: 5.88 g/L of phosphoric acid in water Solution B: 5.88 g/L of phosphoric acid in water
Mobile phase: See Table 1. Mobile phase: See Table 2.

Table 1 Table 2
Time Solution A Solution B Time Solution A Solution B
(min) (%) (%) (min) (%) (%)
0 50 50 0 Z 93,
oO 50 50 0.6 10 90
13 65 35 5 10 90
18 100 0 Z 14 86
23 50 50 13 15 85

Chromatographic system 13.1 100 o

(See Chromatography (621), System Suitability.) 18 100 0
Mode: LC 18.1 7 93
Detector: UV 348 nm 23: Z 93
Column: 3.1-mm x 12.5-cm; 5-um packing L1
Column temperature: 25° Chromatographic system
Flow rate: 1 mL/min (See Chromatography (621), System Suitability.)
Injection volume: 10 LL Mode: LC
System suitability Detector: UV 258 nm
Sample: Standard solution Column: 3.1-mm x 12.5-cm; 5-um packing L1
Suitabinty requirements Column temperature: 25°
Tailing factor: NMT 2.0 for the casticin peak Flow rate: 1.3 mL/min
Relative standard deviation: NMT 2.0% for the cas- Injection volume: 10 uL
ticin peak, in repeated injections System suitability
Analysis Sample: Standard solution
Samples: Standard solution and Sample solution Suitability requirements
Calculate the percentage of casticin in the portion of Tailing factor: NMT 2.0 for the agnuside peak
Chaste Tree taken: Relative standard deviation: NMT 2.0% for the
agnuside peak, in repeated injections
Result = (ru/rs) x (Cs/Cu) x 100 Analysis
Samples: Standard solution and Sample solution
tu = peak response of casticin from the Sample Calculate the percentage of agnuside in the portion of
solution Chaste Tree taken:
rs = peak response of casticin from the Standard
solution Result = (ru/rs) x (Cs/Cu) x 100
Gs = concentration of USP Casticin RS in the
Standard solution (mg/mL) tu = peak response of agnuside from the Sample
Cu = concentration of Chaste Tree in the Sample solution
solution (mg/mL) rs = peak response of agnuside from the Standard
Acceptance criteria: NLT 0.08% of casticin on the solution
Gs = concentration of USP Agnuside RS in the
dried basis
Standard solution (mg/mL)
e@ CONTENT OF AGNUSIDE
Solvent: Methanol and water (1:19) G = concentration of Chaste Tree in the Sample
solution (mg/mL)
Standard solution: Dissolve a quantity of USP Agnuside
RS in Solvent, with sonication. Dilute with methanol to Acceptance criteria: NLT 0.05% of agnuside on the
obtain a concentration of about 0.125 mg/mL. Pass dried basis
through a cellulose membrane filter of 0.45-u1m or finer CONTAMINANTS
DS Monographs

pore size. e ARTICLES OF BOTANICAL ORIGIN, Limits of Elemental Impuri-

Sample solution: Place about 1000 mg of ground plant
material in a container with a stopper. Extract twice
with 40 mL of methanol, using a hand homogenizer at
19,000 rpm for 2 min. Centrifuge, and transfer each
supernatant to a 250-mL round-bottom flask. Rinse the
residue with methanol, and filter the resulting solution
into the flask. Evaporate the combined extract to dry-
ness, and dissolve the residue in 2 mL of Solvent. Quan-
titatively transfer the solution to a solid-phase extraction
cartridge packed with neutral aluminum oxide previ-
ously conditioned with 5 mL of Solvent. Connect the
cartridge to a vacuum pressure not exceeding 300
mbar, and collect the eluate. Rinse the round-bottom
flask with 2 mL of Solvent, pass this solution through
the cartridge, apply the vacuum, and collect the eluate.
Rinse the cartridge with 4 mL of Solvent, and collect the
eluate. Combine the eluates from the cartridge, transfer
to a 10-mL volumetric flask, and dilute with Solvent to
volume.
ties (561): Meets the requirements
e ARTICLES OF BOTANICAL ORIGIN, Pesticide Residue Analysis
(561): Meets the requirements
e MICROBIAL ENUMERATION TESTS (2021): The total aerobic
microbial count does not exceed 105 cfu/g, the total
combined molds and yeast count does not exceed 103
cfu/g, and the bile-tolerant Gram-negative bacteria count
does not exceed 103 cfu/g.
e ABSENCE OF SPECIFIED MICROORGANISMS (2022): Meets the
requirements of the tests for the absence of Salmonella
species and Escherichia coli
SPECIFIC TESTS
e BOTANICAL CHARACTERISTICS
Macroscopic: Mature chaste tree fruits are spherical to
ovoid, 2-4 mm in diameter, very hard, usually with a
short pedicel. The fruit is reddish brown to black,
slightly rough, and covered with glandular hairs. There
are four grooves perpendicular to one another, and a
slight depression on the apex, more evident on large
fruits. The internal appearance of the fruit is yellowish.
The internal structure of the fruit includes four compart-
1-54 Quina-Relat Combined Index to USP 41 and NF 36

Quinaldine red, 5746
TS, 5759
Quinapril
hydrochloride, 3541
and hydrochlorothiazide tablets, 3543
tablets, 3545
Quinhydrone, 5723
Quinidine gluconate, 3547
Injection, 3548
extended-release tablets, 3549
Quinidine sulfate, 3551
capsules, 3552
oral suspension, 3553
tablets, 3554
extended-release tablets, 3555
Quinine sulfate, 3557
capsules, 3558
tablets, 3560
Quinone, 5724
TS, 5759
Rabeprazole
sodium, 3562
Rabies
immune globulin, 3563
Racemethionine, 5550
Racemic
calcium pantothenate, 653
Racepinephrine, 3564
hydrochloride, 3565
Inhalation solution, 3564
Ractopamine hydrochloride
suspension, 3565
Radiation sterilization (1229.10), 7728
Radioactivity (821), 6622
Radioactivity—theory and practice (1821),
8084
Radiopharmaceuticals
C 13, urea, 705
C 13, urea for oral solution, 706
C 14, urea capsules, 707
Cr 51, sodium chromate injection, 921
Cr 51, chromium edetate injection, 922
Co 57, cyanocobalamin capsules, 1055
Co 57, cyanocobalamin oral solution, 1056
Co 58, cyanocobalamin capsules, 1056
F 18, fludeoxyglucose injection, 1794
F 18, sodium fluoride injection, 1795
Ga 67 injection, gallium citrate, 1924
Indium In 111 capromab pendetide
injection, 2143
Indium In 111 chloride solution, 2144
Indium In 111 ibritumomab tiuxetan
injection, 2145
Indium In 111 oxyquinoline solution, 2146
Indium In 111 pentetate injection, 2147
Indium In 111 pentetreotide injection,
2147
Indium In 111 satumomab pendetide
injection, 2148
1123, iobenguane injection, 2189
1 123, iodohippurate sodium injection,
2191
1123, sodium iodide capsules, 2192
1123, sodium iodide solution, 2192
1125, iodinated albumin injection, 2193 Yttrium Y 90 ibritumomab tiuxetan
1125, iothalamate sodium injection, 2194 injection, 4359
1131, iodinated albumin aggregated
injection, 2195
1131, iodinated albumin injection, 2194
1131, iobenguane injection, 2190 Raloxifene hydrochloride, 3567
| 131, iodohippurate sodium injection, tablets, 3569
2195 Raltegravir potassium, 3571
1131, rose bengal sodium injection, 2196 Raman spectroscopy (1120), 7343
1131, sodium iodide capsules, 2196 Ramipril, 3572
1 131, sodium iodide solution, 2197 capsules, 3574
Krypton Kr 81m, 2319 tablets, 3576
N 13, ammonia injection, 2955 Ranitidine
P 32, chromic phosphate suspension, 3295 hydrochloride, 3578
P 32, sodium phosphate solution, 3295 injection, 3579
Rubidium chloride Rb 82 injection, 3683 in sodium chloride injection, 3582
Samarium Sm 153 lexidronam injection, oral solution, 3580
3707 tablets, 3581
Sr 89 injection, strontium chloride, 3840 Rapeseed oil
Technetium Tc 99m albumin aggregated fully hydrogenated, 5552
injection, 3937 superglycerinated fully hydrogenated,
Technetium Tc 99m albumin colloid 5553
injection, 3938 Rat tail collagen, 5684
Technetium Tc 99m albumin injection, Rauwolfia serpentina, 3583
3936 powdered, 3585
Technetium Tc 99m apcitide injection, tablets, 3585
3940 Rayon, 5724
Technetium Tc 99m arcitumomab purified, 3585
injection, 3940 Rb 82
Technetium Tc 99m bicisate injection, injection, rubidium chloride, 3683
3941 Readily carbonizable substances test (271),
Technetium Tc 99m depreotide injection, 6168
3941 Reagents, 5660
Technetium Tc 99m disofenin injection, arsenic in, 5661
3942 boiling or distilling range for, 5660
Technetium Tc 99m etidronate injection, chloride in, 5661
3943 flame photometry for, 5662
Technetium Tc 99m exametazime general tests for, 5660
injection, 3943 heavy metals in, 5662
Technetium Tc 99m gluceptate injection, indicators and solutions, 5659
3945 insoluble matter in, 5663
Technetium Tc 99m lidofenin injection, loss on drying for, 5663
3946 nitrate in, 5663
Technetium Tc 99m mebrofenin injection, nitrogen compounds in, 5663
3947 phosphate in, 5663
Technetium Tc 99m medronate injection, residue on ignition in, 5663
3948
Technetium Tc 99m mertiatide injection,
sulfate in, 5663
Rectal solution
3949 aminophylline, 231
Technetium Tc 99m nofetumomab sodium phosphates, 3807
merpentan injection, 3950 Red
Technetium Tc 99m oxidronate injection, 80, direct, 5724
3950 phosphorus, 5724
Technetium Tc 99m pentetate injection, Red-cell lysing agent, 5724
3951 Red clover
Technetium Tc 99m pertechnetate aerial parts isoflavone aglycones dry
injection, sodium, 3951 extract, 4814
Technetium Tc 99m pyrophosphate Reference standards
injection, 3953 USP (11), 5951
Technetium Tc 99m (pyro- and trimeta-) Reference tables, 5781
phosphates injection, 3953 Alcoholometric, 5861
Technetium Tc 99m red blood cells Atomic weights, 5859
injection, 3954 Container specifications for capsules and
Technetium Tc 99m sestamibi injection, tablets, 5781
3955 Description and relative solubility of USP
Technetium Tc 99m succimer injection, and NF articles, 5791
3956 Intrinsic viscosity table, 5863
Technetium Tc 99m sulfur colloid injection, Relative atomic masses and half-lives of
3956 selected radionuclides, 5860
Technetium Tc 99m tetrofosmin injection, Solubilities, 5851
3957 Refractive index (831), 6639
Thallous chloride Tl 201 injection, 4030 Rehydration salts, oral, 3585
Xenon Xe 127, 4351 Relative atomic masses and half-lives of
Xenon Xe 133, 4351 selected radionuclides, 5860
Xenon Xe 133 injection, 4351
Combined Index to USP 41 and NF 36 Repag-Saqui I-55

Repaglinide, 3588 Ribavirin, 3596 Ropivacaine hydrochloride, 3677

tablets, 3590 capsules, 3597 injection, 3679
Resazurin (sodium), 5724 for inhalation solution, 3599 Rose
Reserpine, 3591 tablets, 3600 bengal sodium, 5724
and chlorothiazide tablets, 3593 Riboflavin, 3602 bengal sodium | 131 injection, 2196
tablets, 3592 assay (481), 6239 oil, 5554
Residual host cell protein measurement in injection, 3603 water ointment, 3680
biopharmaceuticals (1132), 7393 5’-phosphate sodium, 3604 water, stronger, 5555
Residual solvents (467), 6222 tablets, 3603 Rosiglitazone maleate, 3681
Residue on ignition (281), 6168 Ribonuclease inhibitor, 5724 Roxarsone, 3682
Residue on ignition in reagents, 5663 Ribose, 4835 Rubidium chloride Rb 82 injection, 3683
Rifabutin, 3605 Rufinamide, 3684
capsules, 3606 tablets, 3685
oral suspension, 3607 Rules and procedures, xxix
Rifampin, 3608
Resin capsules, 3609
Ruthenium red, 5725
TS, 5759
Anion-exchange, 50- to 100-mesh, for injection, 3610 Rutin, 4841
styrene-divinylbenzene, 5670, 5734 and isoniazid capsules, 3611
Anion-exchange, chloromethylated isoniazid, pyrazinamide, and ethambutol
polystyrene-divinylbenzene, 5670, 5683 hydrochloride tablets, 3614
Anion-exchange, strong, lightly cross- isoniazid, and pyrazinamide tablets, 3612
linked, in the chloride form, 5670
Anion-exchange, styrene-divinylbenzene,
oral suspension, 3610
Riluzole, 3615
S
5670 tablets, 3616
Capsicum oleoresin, 674 Rimantadine hydrochloride, 3617 Sabinene, 5725
Carboxylate (sodium form) cation- tablets, 3618 Saccharin, 5555
exchange (50- to 100-mesh), 5680 Rimexolone, 3619 calcium, 3688
Cation-exchange, 5680 ophthalmic suspension, 3619 sodium, 3689
Cation-exchange, carboxylate (sodium Ringer’s sodium oral solution, 3691
form) 50- to 100-mesh, 5680, 5680 and dextrose injection, 3622 sodium tablets, 3691
Cation-exchange, polystyrene, 5680, 5719 and dextrose injection, half-strength Saccharose, 5725
Cation-exchange, styrene-divinylbenzene, lactated, 3628 Safflower oil, 3692
5681 and dextrose injection, lactated, 3626 Safranin O, 5725
Cation-exchange, styrene-divinylbenzene, and dextrose injection, modified lactated, St. John’s wort flowering top, 4842
strongly acidic, 5681, 5734 3631 dry extract capsules, 4847
Cation-exchange, sulfonic acid, 5681, injection, 3620 extract, dry, 4845
5735 injection, lactated, 3624 powder, 4844
Chloromethylated polystyrene- irrigation, 3633 dry extract tablets, 4849
divinylbenzene anion-exchange, 5683 lactated, and dextrose injection, potassium Salicylaldazine, 5725
Cholestyramine, 918 chloride in, 3367 Salicylaldehyde, 5725
lon-exchange, 5702 Risedronate sodium, 3633 Salicylamide, 3693
Podophyllum, 3341 tablets, 3636 Salicylic acid, 3694, 5725
Podophyllum topical solution, 3341 Risperidone, 3637 and benzoic acids ointment, 487
Polystyrene cation-exchange, 5719 oral solution, 3639 collodion, 3695
Styrene-divinylbenzene anion-exchange, tablets, 3640 gel, 3696
50- to 100-mesh, 5734 orally disintegrating tablets, 3642 plaster, 3696
Styrene-divinylbenzene cation-exchange, Ritodrine hydrochloride, 3643 topical foam, 3696
strongly acidic, 5734 injection, 3644 and zinc paste, 4384
Sulfonic acid cation-exchange, 5735 tablets, 3645 Saline TS, 5729, 5759
Ritonavir, 3645 pyrogen-free, 5759
capsules, 3648 Salix species bark, 4850
and lopinavir oral solution, 2453 dry extract, 4852
Resorcinol, 3594, 5724 and lopinavir tablets, 2457 powder, 4854
monoacetate, 3596 oral solution, 3651 Salmeterol
ointment, compound, 3595 tablets, 3655 fluticasone propionate, inhalation aerosol,
and sulfur topical suspension, 3595 Rivastigmine, 3657 1847
TS, 5759 Rivastigmine tartrate, 3659 fluticasone propionate, inhalation powder,
6Z-retinoic acid, 5724 capsules, 3660 1852
Retinyl palmitate, 5724 Rizatriptan benzoate, 3662 inhalation powder, 3697
Reverse transcriptase, 5724 Salmeterol xinafoate, 3702
tablets, 3663
Rheometry (1911), 8145 orally disintegrating tablets, 3665 Salsalate, 3703
Rhodamine 6G, 5724 Rocuronium bromide, 3666 capsules, 3705
Rhodamine B, 5724 Root and rhizome tablets, 3706
Rhodiola crenulata dry extract capsules and eleuthero, 4600 Salt
root and rhizome dry extract, 4822 dry extract and Rhodiola crenulata, 4822 octanesulfonic acid sodium, 5712, 5731
root and rhizome powder, 4824 dry extract tablets and eleuthero root, Salts of organic nitrogenous bases (501),
Rhodiola crenulata 4601 6245
root and rhizome, 4821 powder capsules and eleuthero, 4604 Samarium Sm 153 lexidronam injection,
Rhodiola rosea, 4825 powder and Rhodiola crenulata, 4824 3707
capsules, 4831 and Rhodiola crenulata, 4821 Sand
extract, 4828 Ropinirole standard 20- to 30-mesh, 5725, 5733
powdered, 4827 tablets, 3669 washed, 5725, 5743
tablets, 4833 Saquinavir mesylate, 3707
extended-release tablets, 3671
tincture, 4830 capsules, 3708
Ropinirole hydrochloride, 3674
I-56 Sargr-Sodiu Combined Index to USP 41 and NF 36

Sargramostim, 3709 gel, binder-free, 5683, 5726 B-Sttosterol, 5727

for injection, 3711 gel, chromatographic, 5683, 5726 Six-month implementation guideline, ii
Sawdust, purified, 5725 gel-impregnated glass microfiber sheet, Sm 153 lexidronam injection, samarium,
Saw palmetto, 4856 5726 3707
capsules, 4862 gel mixture, chromatographic, 5684, 5726 Soda lime, 5563, 5727
extract, 4860 gel mixture, chromatographic, with Sodium, 5727
powdered, 4858 chemically bound amino groups, 5726 acetate, 3772, 5727
Scaffold gel mixture, dimethylsilanized, acetate, anhydrous, 5669, 5727
bovine dermis, 3714 chromatographic, 5726 acetate injection, 3772
human dermis, 3717 gel mixture, octadecylsilanized acetate solution, 3773
porcine bladder, 3720 chromatographic, 5726 acetate TS, 5759
silk fibroin, 3724 gel mixture, octylsilanized, alendronate, tablets, 110
Scandium oxide, 5725 chromatographic, 5726 alginate, 5564
Scanning electron microscopy (1181), 7519 gel, octadecylsilanized chromatographic, alizarinsulfonate, 5727
Schizochytrium oil, 4870 5726 alizarinsulfonate TS, 5759
capsules, 4872 gel, porous, 5726 aminoacetate TS, 5759
Schweitzer’s reagent, 5759 microspheres, 5726 ammonium phosphate, 5727
Scopolamine hydrobromide, 3728 Siliceous earth arsenate, 5728
injection, 3729 chromatographic, 5684, 5726 arsenite, 5728
ophthalmic solution, 3730 chromatographic, silanized, 5684, 5726 arsenite, twentieth-molar (0.05 M), 5770
tablets, 3730 purified, 5561 ascorbate, 3773
Screening for undeclared drugs and drug Silicic azide, 5728
analogues (2251), 8193 acid, 5726 benzoate, 5564
S designations, 5725 acid—impregnated glass microfilament benzoate and caffeine injection, 614
Secobarbital, 3730 sheets with fluorescent indicator, 5727 bicarbonate, 3774, 5728
sodium, 3731 Silicon bicarbonate injection, 3777
sodium capsules, 3732 carbide, 5727 bicarbonate and magnesium carbonate for
Secondary butyl alcohol, 5725 dioxide, 5562 oral suspension, 2504
Selamectin, 3733 dioxide colloidal, 5562 bicarbonate oral powder, 3779
Selegiline hydrochloride, 3734 Silicone bicarbonate tablets, 3779
capsules, 3735 75 percent phenyl, methyl, 5727 biphenyl, 5728
tablets, 3737 Silicotungstic acid, n-hydrate, 5727 biphosphate, 5728
Selegiline hydrochloride compounded Silicified bisulfite, 5728
topical gel, 3738 microcrystalline cellulose, 5279 bisulfite TS, 5759
Selenious acid, 3738, 5725 Silver bitartrate, 5728
injection, 3739 diethyldithiocarbamate, 5727 bitartrate TS, 5759
Selenium, 5725 diethyldithiocarbamate TS, 5759 borate, 5565, 5729
sulfide, 3739 nitrate, 3759, 5727 borohydride, 5729
sulfide topical suspension, 3740 nitrate ophthalmic solution, 3759 bromide, 3779, 5729
Selenium (291), 6169 nitrate, tenth-normal (0.1 N), 5759, 5770 bromide injection, veterinary, 3780
Selenomethionine, 4875, 5726 nitrate, toughened, 3759 bromide oral solution, veterinary, 3780
Semisolid drug products—performance tests nitrate TS, 5759 butyrate, 3781
(1724), 7944 oxide, 5727 caprylate, 5566
Senna Silver-ammonia-nitrate TS, 5759 carbonate, 5567, 5729
fluidextract, 3741 Silver-ammonium nitrate TS, 5759 carbonate, anhydrous, 5669, 5729
leaf, 3740 Silver nitrate carbonate, citric acid, and magnesium
pods, 3742 0.002 N VS, 5770 oxide irrigation, 972
oral solution, 3743 0.05 N VS, 5770 carbonate, monohydrate, 5729
Sennosides, 3743 Silver sulfate, 5727 carbonate TS, 5759
tablets, 3744 Simethicone, 3760 carboxymethylcellulose, 712
Sensitization testing (1184), 7529 alumina, magnesia, and calcium carbonate carboxymethylcellulose, and
Serine, 3745 chewable tablets, 153 microcrystalline cellulose, 5278
Sertraline alumina and magnesia oral suspension, carboxymethylcellulose, paste, 713
hydrochloride, 3746 155 carboxymethylcellulose, tablets, 714
hydrochloride oral solution, 3749 alumina and magnesia chewable tablets, carboxymethylcellulose, 12, 5265
hydrochloride tablets, 3749 157 cefazolin, 760
Sesame oil, 5556 calcium carbonate and magnesia chewable cefmetazole, 779
Sevoflurane, 3752 tablets, 636 cefoperazone, 782
Shear cell methodology for powder flow capsules, 3761 cefotaxime, 787
testing (1063), 7054 emulsion, 3761 cetosteary! sulfate, 5567
Shellac, 5558 and magaldrate chewable tablets, 2499 chloride, 3781, 5729
Sibutramine hydrochloride, 3754 and magaldrate oral suspension, 2498 chloride and dextrose injection, 1235
Significant change guide for bulk oral suspension, 3762 chloride and fructose injection, 1888
pharmaceutical excipients (1195), 7545 tablets, 3763 chloride inhalation solution, 3786
Sildenafil Simulated gastric fluid TS, 5759 chloride injection, 3783
tablets, 3755 Simulated intestinal fluid TS, 5759 chloride injection, bacteriostatic, 3784
Sildenafil citrate, 3757 Simvastatin, 3763 chloride injection, dextran 40 in, 1223
oral suspension, 3758 tablets, 3764 chloride injection, dextran 70 in, 1227
Silica Single-steroid assay (511), 6253 chloride injection, mannitol in, 2529
calcined diatomaceous, 5726 Sipuleucel-T, 3766 chloride injection, potassium chloride in,
chromatographic, silanized, flux-calcined, Sisomicin sulfate, 3768 3370
acid-washed, 5726 injection, 3769 chloride injection, potassium chloride in
colloidal, hydrophobic, 5560 Sitagliptin dextrose injection and, 3365
dental-type, 5559 phosphate, 3770 chloride injection, ranitidine in, 3582
gel, 5726 tablets, 3769 chloride irrigation, 3785
Combined Index to USP 41 and NF 36
Sodium (continued)
chloride ophthalmic ointment, 3785
chloride ophthalmic solution, 3786
chloride solution, isotonic, 5729
chloride tablets, 3786
chloride tablets for solution, 3786
chloride TS, alkaline, 5759
cholate hydrate, 5729
chromate, 5729
chromate, Cr 51 injection, 921
chromotropate, 5729
cilastatin, 931
citrate, 3786
citrate and citric acid oral solution, 3787
citrate dihydrate, 5729
citrate TS, 5759
citrate TS, alkaline, 5759
cobaltinitrite, 5729
cobaltinitrite TS, 5759
cyanide, 5729
dalteparin, 1148
1-decanesulfonate, 5729
dehydroacetate, 5569
desoxycholate, 5729
dichromate, 5729
diethyldithiocarbamate, 5729
2,2-dimethyl-2-silapentane-5-sulfonate,
5729
dithionite, 5729
dodecyl sulfate, 5729, 5731
ethylparaben, 5350
ferrocyanide, 5729
fluconazole, chloride injection, 1760
fluorescein, 5730
fluoride, 3788, 5730
fluoride and acidulated phosphate topical
solution, 3791
fluoride F18 injection, 1795
fluoride and phosphoric acid gel, 3792
fluoride oral solution, 3790
fluoride tablets, 3790
fluoride TS, 5759
formaldehyde sulfoxylate, 5570
gluconate, 3792
glycocholate, 5730
1-heptanesulfonate, 5730
]-heptanesulfonate, monohydrate, 5730
1-hexanesulfonate, 5730
1-hexanesulfonate, monohydrate, 5730
hydrogen sulfate, 5730
hydrosulfite, 5729, 5730
hydrosulfite TS, alkaline, 5751, $759
hydroxide, 5571, 5730
hydroxide, alcoholic, tenth-normal (0.1 N),
5770
hydroxide, normal (1 N), 5770
hydroxide TS, 5759
hydroxide TS 2, 5759
hydroxide TS 3, 5759
hypobromite TS, 5759
hypochlorite solution, 3793, 5730, 5759
hypochlorite topical solution, 3794
hypochlorite TS, 5759
iodate, 5731
iodide, 3794
iodide | 123 capsules, 2192
iodide | 123 solution, 2192
iodide | 131 capsules, 2196
iodide | 131 solution, 2197
iodohydroxyquinolinesulfonate TS, 5760
lactate injection, 3795
lactate solution, 3795
lauryl sulfate, 5572, 5731
low-substituted carboxymethylcellulose,
5263
Sodiu-Solut |-57
metabisulfite, 5573, 5731 tetraphenylborate, 5733
metaperiodate, 5731 tetraphenylboron, 5733
methoxide, 5731 tetraphenylboron, fiftieth-molar (0.02 M),
methoxide, half-normal (0.5 N) in 5771
methanol, 5771 tetraphenylboron TS, 5760
methoxide, tenth-normal (0.1 N) in thioglycolate, 5733
toluene, 5771 thioglycolate TS, 5760
molybdate, 5731 thiosulfate, 3814, 5733
monofluorophosphate, 3796 thiosulfate injection, 3814
montelukast, oral granules, 2797 thiosulfate, tenth-normal (0.1 N), 5760,
montelukast, tablets, 2800 5771
montelukast, chewable tablets, 2803 thiosulfate TS, 5760
mycophenolate, 2836 L-thyroxine, 5733
nitrate, 5731 3-(trimethylsilyl)-1-propane sulfonate,
nitrite, 3797, 5731 5729, 5733
nitrite injection, 3799 tungstate, 5733
nitrite, tenth-molar (0.1 M), 5771 Sodium bicarbonate
nitroferricyanide, 5731 compounded injection, 3778
nitroferricyanide TS, 5760 Sodium chloride
nitroprusside, 3800 0.5 M TS, 5759
nitroprusside for injection, 3801 Sodium 1-dodecanesulfonate, 5729
1-octanesulfonate, 5731 Sodium ferrous citrate, 4876
oxalate, 5731 Sodium hydroxide
(tri) pentacyanoamino ferrate, 5731 0.0025 N TS, 5759
1-pentanesulfonate, 5714, 5731 0.2 N, TS, 5759
1-pentanesulfonate, anhydrous, 5731 0.02 N TS, 5759
perchlorate, 5731 10 NTS, 5759
peroxide, 5731 2.5 N TS, 5759
pertechnetate Tc 99m injection, 3951 2NTS, 5759
phenylbutyrate, 3801 5 N, TS, 5759
phenylbutyrate oral suspension, 3803 0.1 N VS, 5770
phosphate, dibasic, 3804, 5731 0.01 N VS, 5770
phosphate, dibasic, anhydrous, 5669, 5731 0.5 N VS, 5770
phosphate, dibasic, dihydrate, 5731 Sodium phenylbutyrate, 3801
phosphate, dibasic, dodecahydrate, 5731 Sodium phosphates
phosphate, dibasic, heptahydrate, 5731 compounded injection, 3806
phosphate, dibasic, TS, 5760 Sodium succinate, 5579
phosphate, monobasic, 3805, 5709, 5731 Sodium thiosulfate
phosphate, monobasic, anhydrous, 5731 0.01 M VS, 5772
phosphate, monobasic, dihydrate, 5732 Solubilities, 5851
phosphate P 32 solution, 3295 Soluble starch, 5733
phosphates injection, 3805
phosphates oral solution, 3807
phosphates rectal solution, 3807
phosphate, tribasic, 5574, 5732
phosphite pentahydrate, 5732 Solution
phosphotungstate TS, 5760 Acetaminophen and codeine phosphate
picosulfate, 3807 oral, 56
polystyrene sulfonate, 3809 Acetaminophen, dextromethorphan
polystyrene sulfonate suspension, 3809 hydrobromide, doxylamine succinate,
and potassium bicarbonates and citric acid and pseudoephedrine hydrochloride
effervescent tablets for oral solution, oral, 60
3357 Acetaminophen for effervescent oral, 37
propionate, 5575 Acetaminophen oral, 37
pyrophosphate, 5732 Acetic acid otic, 70
pyruvate, 5732 Acetylcholine chloride for ophthalmic, 73
rabeprazole, 3562 Acetylcysteine, 74
salicylate, 3810, 5732 Acidulated phosphate and sodium fluoride
salicylate tablets, 3811 topical, 3791
selenite, 5732 Aluminum acetate topical, 163
starch glycolate, 5575 Aluminum chlorohydrate, 165
stearate, 5577 Aluminum dichlorohydrate, 168
stearyl fumarate, 5578 Aluminum sesquichlorohydrate, 173
sulfate, 3812, 5732 Aluminum subacetate topical, 175
sulfate, anhydrous, 5669, 5732 Aluminum sulfate and calcium acetate for
sulfate decahydrate, 5732 topical, 176
sulfate injection, 3813 Aluminum sulfate and calcium acetate
sulfide, 3813, 5732 tablets for topical, 177
sulfide topical gel, 3813 Aluminum zirconium octachlorohydrate,
sulfide TS, 5760 179
sulfite, 5580, 5732 Aluminum zirconium octachlorohydrex gly,
sulfite, anhydrous, 5669, 5732 181
p-sulfophenylazochromotropate, 5732 Aluminum zirconium pentachlorohydrate,
tartrate, 5581, 5733 183
tartrate TS, 5760
I-58 Solut-Solut
Solution (continued)
Aluminum zirconium pentachlorohydrex
gly, 185
Aluminum zirconium tetrachlorohydrate,
187
Aluminum zirconium tetrachlorohydrex
gly, 189
Aluminum zirconium trichlorohydrate, 191
Aluminum zirconium trichlorohydrex gly,
193
Amantadine hydrochloride oral, 196
Aminobenzoate potassium for oral, 212
Aminobenzoic acid topical, 217
Aminocaproic acid oral, 219
Aminophylline oral, 229
Aminophylline rectal, 231
Ammonia, diluted, 5668
Ammonia, strong, 5197
Amprolium oral, 307
Anticoagulant citrate dextrose, 324
Anticoagulant citrate phosphate dextrose,
326
Anticoagulant citrate phosphate dextrose
adenine, 327
Anticoagulant heparin, 2025
Anticoagulant sodium citrate, 329
Antipyrine and benzocaine otic, 332
Antipyrine, benzocaine, and phenylephrine
hydrochloride otic, 333
Apraclonidine ophthalmic, 338
Aromatic elixir, 5206
Ascorbic acid oral, 361
Aspirin effervescent tablets for oral, 371
Atenolol oral, 385
Atropine sulfate ophthalmic, 408
Benoxinate hydrochloride ophthalmic, 466
Benzaldehyde elixir, compound, 5215
Benzalkonium chloride, 5217
Benzethonium chloride topical, 467
Benzocaine, butamben, and tetracaine
hydrochloride topical, 483
Benzocaine otic, 476
Benzocaine topical, 478
Betamethasone oral, 503
Betaxolol ophthalmic, 519
Bethanechol chloride oral, 523
Bromodiphenhydramine hydrochloride and
codeine phosphate oral, 556
Bromodiphenhydramine hydrochloride
oral, 555
Brompheniramine maleate and
pseudoephedrine sulfate oral, 559
Brompheniramine maleate oral, 558
Buprenorphine compounded buccal,
veterinary, 570
Butabarbital sodium oral, 593
Caffeine citrate oral, 613
Calcitonin salmon nasal, 624
Calcium hydroxide topical, 647
Captopril oral, 678
Carbachol intraocular, 682
Carbachol ophthalmic, 683
Carbamide peroxide topical, 690
Carbol-fuchsin topical, 704
C 13 for oral, urea, 706
Carteolol hydrochloride ophthalmic, 727
Cefazolin ophthalmic, 760
Cetylpyridinium chloride topical, 858
Chloral hydrate oral, 860
Chloramphenicol for ophthalmic, 865
Chloramphenicol ophthalmic, 864
Chloramphenicol oral, 865
Chloramphenicol otic, 865
Chlorhexidine gluconate, 881
Combined Index to USP 41 and NF 36
Chlorpheniramine maleate and
pseudoephedrine hydrochloride oral,
904
Chlorpheniramine maleate oral, 902
Cholecalciferol, 917
Chymotrypsin for ophthalmic, 924
Ciprofloxacin ophthalmic, 944
Clindamycin hydrochloride oral, 994
Clindamycin palmitate hydrochloride for
oral, 995
Clindamycin phosphate topical, 999
Clobetasol propionate topical, 1008
Clotrimazole topical, 1044
Cloxacillin sodium for oral, 1052
Coal tar topical, 1055
Cyanocobalamin Co 57 oral, 1056
Cocaine hydrochloride tablets for topical,
1058
Cocaine and tetracaine hydrochlorides and
epinephrine topical, 1059
Codeine sulfate oral, 1066
Cromolyn sodium ophthalmic, 1107
Cupriethylenediamine hydroxide, 1.0 M,
5685
Cyclopentolate hydrochloride ophthalmic,
1123
Cyclosporine oral, 1133
Cyproheptadine hydrochloride oral, 1136
Demecarium bromide ophthalmic, 1165
Dexamethasone elixir, 1196
Dexamethasone oral, 1198
Dexamethasone sodium phosphate
ophthalmic, 1207
Dexbrompheniramine maleate and
pseudoephedrine sulfate oral, 1209
Dexchlorpheniramine maleate oral, 1211
Dextromethorphan hydrobromide oral,
1232
Diatrizoate meglumine and diatrizoate
sodium, 1238
Diatrizoate sodium, 1241
Dichlorophenol-indophenol, standard,
5764
Dicyclomine hydrochloride oral, 1270
Diethyltoluamide topical, 1282
Digoxin oral, 1292
Dihydrotachysterol oral, 1299
Diltiazem hydrochloride oral, 1309
Dimenhydrinate oral, 1314
Dimethyl sulfoxide topical, 1318
Diphenhydramine hydrochloride oral, 1331
Diphenoxylate hydrochloride and atropine
sulfate oral, 1339
Dipivefrin hydrochloride ophthalmic, 1343
Docusate sodium, 1380
Dolasetron mesylate oral, 1384
Dorzolamide hydrochloride and timolol
maleate ophthalmic, 1397
Doxepin hydrochloride oral, 1407
Doxylamine succinate oral, 1438
Dyclonine hydrochloride topical, 1458
Dyphylline and guaifenesin oral, 1462
Dyphylline oral, 1461
Ecamsule, 1464
Echothiophate iodide for ophthalmic, 1467
Emedastine ophthalmic, 1496
Ephedrine sulfate oral, 1529
Epinephrine bitartrate for ophthalmic,
1534
Epinephrine bitartrate ophthalmic, 1534
Epinephrine ophthalmic, 1532
Epinephryl borate ophthalmic, 1535
Ergocalciferol oral, 1549
Ergoloid mesylates oral, 1552
Erythromycin topical, 1571
Escitalopram oral, 1584
Ethosuximide oral, 1645
Fehling’s, 5753
Ferric ammonium citrate for oral, 266
Ferric subsulfate, 1709
Ferrous gluconate oral, 1717
Ferrous sulfate oral, 1720
Fluocinolone acetonide topical, 1785
Fluocinonide topical, 1788
Fluorescein sodium and benoxinate
hydrochloride ophthalmic, 1792
Fluorescein sodium and proparacaine
hydrochloride ophthalmic, 1793
Fluorouracil topical, 1803
Fluoxetine oral, 1806
Fluphenazine hydrochloride elixir, 1815
Fluphenazine hydrochloride oral, 1817
Flurbiprofen sodium ophthalmic, 1826
Formaldehyde, 1874, 5697, 5754
Furosemide oral, 1893
Gentamicin sulfate and betamethasone
acetate ophthalmic, 1939
Gentamicin sulfate and betamethasone
valerate otic, 1940
Gentamicin topical, 1941
Gentamicin sulfate ophthalmic, 1938
Gentian violet topical, 1945
Glutaral disinfectant, 5365
Glycerin ophthalmic, 1968
Glycerin oral, 1969
Guaifenesin and codeine phosphate oral,
2004
Guaifenesin oral, 2003
Halazone tablets for, 2014
Halcinonide topical, 2018
Haloperidol oral, 2021
Heparin lock flush, 2025
Homatropine hydrobromide ophthalmic,
2039
Hydralazine hydrochloride oral, 2045
Hydrocortisone and acetic acid otic, 2062
Hydrogen peroxide, 5700
Hydrogen peroxide topical, 2076
Hydroquinone topical, 2082
Hydroxyamphetamine hydrobromide
ophthalmic, 2085
Hydroxyzine hydrochloride oral, 2092
Hyoscyamine sulfate elixir, 2103
Hyoscyamine sulfate oral, 2104
Hypromellose ophthalmic, 2107
Idoxuridine ophthalmic, 2120
Indium In 111 chloride, 2144
Indium In 111 oxyquinoline, 2146
lodine, strong, 2188
Sodium iodide | 123, 2192
Sodium iodide ! 131, 2197
lodine topical, 2187
Ipecac oral, 2226
Ipratropium bromide and albuterol sulfate
inhalation, 2228
Isoniazid oral, 2254
Isosorbide oral, 2267
Ivermectin topical, 2296
Lactulose, 2325
Lead, standard, 5760
Levalbuterol inhalation, 2365
Levobunolol hydrochloride ophthalmic,
2384
Levocarnitine oral, 2388
Levofloxacin oral, 2397
Lidocaine hydrochloride topical, 2415
Lincomycin oral, 2421
Lithium oral, 2436
Locke-Ringer’s, 5755
Loperamide hydrochloride oral, 2448
Combined Index to USP 41 and NF 36
Solution (continued)
Loratadine oral, 2464
Mafenide acetate for topical, 2494
Magnesium carbonate and citric acid for
oral, 2502
Magnesium carbonate, citric acid, and
potassium citrate for oral, 2503
Manganese chloride for oral, 2524
Magnesium citrate for oral, 2507
Magnesium citrate oral, 2506
Maltitol, 5436
Meperidine hydrochloride oral, 2575
Mesoridazine besylate oral, 2602
Metaproterenol sulfate oral, 2608
Methadone hydrochloride oral, 2629
Methdilazine hydrochloride oral, 2634
Methenamine mandelate for oral, 2639
Methenamine oral, 2636
Methoxsalen topical, 2656
Methylcellulose ophthalmic, 2665
Methylcellulose oral, 2666
Metoclopramide oral, 2701
Metoprolol tartrate oral, 2714
Mibolerone oral, 2735
Minoxidil topical, 2762
Mometasone furoate topical, 2790
Moxifloxacin ophthalmic, 2819
Myrrh topical, 2841
Nafcillin sodium for oral, 2850
Naphazoline hydrochloride ophthalmic,
2862
Naphazoline hydrochloride and
pheniramine maleate ophthalmic, 2862
Neomycin and polymyxin B sulfates and
gramicidin ophthalmic, 2902
Neomycin and polymyxin B sulfates and
hydrocortisone otic, 2903
Neomycin and polymyxin B sulfates for
irrigation, 2894
Neomycin and polymyxin B sulfates
ophthalmic, 2894
Neomycin sulfate and dexamethasone
sodium phosphate ophthalmic, 2886
Neomycin sulfate oral, 2884
Nickel standard TS, 5757
Nitrofurazone topical, 2955
Nitromersol topical, 2960
Norfloxacin ophthalmic, 2977
Nortriptyline hydrochloride oral, 2986
Ofloxacin ophthalmic, 3000
Olopatadine hydrochloride ophthalmic,
3015
Ondansetron, oral, 3032
Oral, containing at least three of the
following—acetaminophen and (salts of)
chlorpheniramine, dextromethorphan,
and pseudoephedrine, 49
Oxacillin sodium for oral, 3062
Oxtriphylline oral, 3090
Oxybutynin chloride oral, 3094
Oxycodone hydrochloride oral, 3103
Oxymetazoline hydrochloride ophthalmic,
3114
Papain tablets for topical, 3161
Paromomycin oral, 3173
Penicillin G potassium for oral, 3201
Penicillin V potassium for oral, 3217
Perphenazine oral, 3246
Phenobarbital oral, 3260
Phenol, topical, camphorated, 3264
Phenylephrine hydrochloride ophthalmic,
3281
Phosphate P 32, sodium, 3295
Physostigmine salicylate ophthalmic, 3297
Pilocarpine hydrochloride ophthalmic,
3303
Pilocarpine nitrate ophthalmic, 3305
Podophyllum resin topical, 3341
Polyethylene glycol 3350 and electrolytes
for oral, 3345
Polymyxin B sulfate and hydrocortisone
otic, 3351
Polymyxin B sulfate and trimethoprim
ophthalmic, 3351
Potassium bicarbonate effervescent tablets
for oral, 3355
Potassium bicarbonate and potassium
chloride for effervescent oral, 3356
Potassium bicarbonate and potassium
chloride effervescent tablets for oral,
3356
Potassium bicarbonate, potassium chloride,
and potassium citrate effervescent
tablets for oral, 3367
Potassium bromide oral, veterinary, 3359
Potassium chloride for oral, 3363
Potassium chloride oral, 3362
Potassium citrate and citric acid oral, 3375
Potassium gluconate and potassium
chloride for oral, 3379
Potassium gluconate and potassium
chloride oral, 3378
Potassium gluconate, potassium citrate,
and ammonium chloride oral, 3380
Potassium gluconate and potassium citrate
oral, 3380
Potassium gluconate oral, 3377
Potassium iodide oral, 3382
Potassium nitrate, 3384
Potassium and sodium bicarbonates and
citric acid effervescent tablets for oral,
3357
Povidone-iodine cleansing, 3393
Povidone-iodine topical, 3393
Prednisolone oral, 3412
Prednisolone sodium phosphate
compounded oral, 3418
Prednisolone sodium phosphate
ophthalmic, 3419
Prednisone oral, 3422
Prochlorperazine oral, 3448
Promazine hydrochloride oral, 3462
Promethazine and phenylephrine
hydrochloride and codeine phosphate
oral, 3473
Promethazine and phenylephrine
hydrochloride oral, 3470
Promethazine hydrochloride oral, 3466
Proparacaine hydrochloride ophthalmic,
3486
Protein standard (8 g/dL), 5721
Pseudoephedrine hydrochloride,
carbinoxamine maleate, and
dextromethorphan hydrobromide oral,
3511
Pseudoephedrine hydrochloride oral, 3508
Pyridostigmine bromide oral, 3524
Ranitidine oral, 3580
Risperidone oral, 3639
Saccharin sodium oral, 3691
Scopolamine hydrobromide ophthalmic,
3730
Senna oral, 3743
Silver nitrate ophthalmic, 3759
Sodium acetate, 3773
Sodium bromide oral, veterinary, 3780
Sodium chloride, isotonic, 5729
Sodium chloride ophthalmic, 3786
Sodium chloride tablets for, 3786
Solut-Sorbi 1-59
Sodium citrate and citric acid oral, 3787
Sodium fluoride and acidulated phosphate
topical, 3791
Sodium fluoride oral, 3790
Sodium hypochlorite, 3793, 5730, 5759
Sodium hypochlorite topical, 3794
Sodium lactate, 3795
Sodium phosphate P 32, 3295
Sodium phosphates oral, 3807
Sodium phosphates rectal, 3807
Sorbitol, 3818
Sorbitol noncrystallizing, 5589
Sorbitol sorbitan, 5590
Stavudine for oral, 3836
Sulfacetamide sodium ophthalmic, 3856
Sulfaquinoxaline oral, 3880
Suprofen ophthalmic, 3900
Terpin hydrate and codeine oral, 3999
Terpin hydrate oral, 3998
Tetracaine hydrochloride ophthalmic, 4012
Tetracaine hydrochloride topical, 4012
Tetracycline hydrochloride for topical,
4021
Tetrahydrozoline hydrochloride
ophthalmic, 4027
Tetramethylammonium hydroxide, in
methanol, 5737
Theophylline and guaifenesin oral, 4043
Theophylline oral, 4036
Theophylline sodium glycinate oral, 4044
Thiamine hydrochloride oral, 4048
Thiamine mononitrate oral, 4051
Thimerosal topical, 4057
Thioridazine hydrochloride oral, 4064
Thiothixene hydrochloride oral, 4071
Timolol maleate ophthalmic, 4098
Tobramycin ophthalmic, 4116
Tolnaftate topical, 4136
Travoprost ophthalmic, 4174
Tretinoin topical, 4182
Triamcinolone diacetate oral, 4193
Tricitrates oral, 4205
Trifluoperazine oral, 4211
Trihexyphenidyl hydrochloride oral, 4219
Trikates oral, 4221
Trimeprazine oral, 4222
Triprolidine hydrochloride oral, 4233
Triprolidine and pseudoephedrine
hydrochlorides oral, 4234
Tropicamide ophthalmic, 4240
Valproic acid oral, 4273
Valrubicin intravesical, 4276
Vancomycin hydrochloride for oral, 4287
Vehicle for oral, 5474
Vehicle for oral, sugar free, 5474
Verapamil hydrochloride oral, 4305
Vitamins with minerals, water-soluble oral,
5128
Vitamins with minerals, oil- and water-
soluble oral, 5047
Vitamins, oil- and water-soluble oral, 4995
Xanthan gum, 5654
Zidovudine oral, 4370
Zinc sulfate ophthalmic, 4387
Zinc sulfate oral, 4387
Solutions
reagents, and indicators, 5659
Solvent hexane, 5733
Somatropin, 3815
for injection, 3816
Somatropin bioidentity tests (126), 6063
Sorbic acid, 5582
-60 Sorbi-Sulfa Combined Index to USP 41 and NF 36

Sorbitan sodium, glycolate, 5575 Subvisible particulate matter in therapeutic

monolaurate, 5582 soluble, 5733 protein injections (787), 6534
monooleate, 5583 soluble, purified, 5733 Succinic acid, 5624, 5734
monopalmitate, 5584 tapioca, 5616 Succinylcholine chloride, 3841
monostearate, 5585 topical, 3833 injection, 3842
sesquioleate, 5586 TS, 5760 Sucralfate, 3843
sorbitol, solution, 5590 wheat, 5617 tablets, 3845
trioleate, 5587 Stavudine, 3834 Sucralose, 5625
Sorbitol, 5588, 5733 capsules, 3835 Sucrose, 5626
solution, 3818 for oral solution, 3836 octaacetate, 5628
solution noncrystallizing, 5589 Steam, pure, 4348 palmitate, 5628
sorbitan solution, 5590 Steam sterilization by direct contact (1229. stearate, 5630
Sotalol hydrochloride, 3819 1), 7698 Sudan
oral suspension, 3821 Stearic acid, 5618, 5733 Ill, 5734
tablets, 3821 purified, 5620 lll TS, 5760
Soybean oil, 3822 Stearoyl polyoxylglycerides, 5622 IV, 5734
hydrogenated, 5592 Stearyl alcohol, 5623, 5733 IV TS, 5760
Soy isoflavones Sufentanil citrate, 3845
capsules, 4879 injection, 3846
powdered extract, 4877 Sugar
tablets, 4881
Spacers and valved holding chambers used Sterile compressible, 5631
confectioner’s, 5632
with inhalation aerosols—characterization Erythromycin ethylsuccinate, 1577 free suspension structured vehicle, 5637
tests (1602), 7878 Erythromycin gluceptate, 1581 injection, invert, 3846
Specific gravity (841), 6639 Erythromycin lactobionate, 1582 spheres, 5633
Specific surface area (846), 6640 Pharmaceutical compounding—sterile Sulbactam
Spectinomycin preparations (797), 6554 and ampicillin for injection, 305
hydrochloride, 3824 Sterile product packaging—integrity sodium, 3847
for injectable suspension, 3825 evaluation (1207), 7578 Sulconazole nitrate, 3849
Spectrophotometric identification tests (197), Sterility Assurance (1211), 7633 Sulfa
6101 Sterility testing—validation of isolator vaginal cream, triple, 3850
Spironolactone, 3825 systems (1208), 7617 vaginal inserts, triple, 3851
and hydrochlorothiazide oral suspension, Water, purified, 4348 Sulfabenzamide, 3851
3828 Water for inhalation, 4346 Sulfacetamide, 3852
and hydrochlorothiazide tablets, 3829 Water for injection, 4346 sodium, 3853
tablets, 3827 Water for irrigation, 4347 sodium ophthalmic ointment, 3855
Spironolactone compounded sodium ophthalmic solution, 3856
oral suspension, 3826 sodium and prednisolone acetate
Spironolactone compounded, veterinary ophthalmic ointment, 3857
oral suspension, 3827 Sterile product packaging—integrity sodium and prednisolone acetate
Spirulina, 4882 evaluation (1207), 7578 ophthalmic suspension, 3858
tablets, 4886 Sterility sodium topical suspension, 3856
Squalane, 5593 testing—validation of isolator systems Sulfachlorpyridazine, 3859
Sr 89 injection, strontium chloride, 3840 (1208), 7617 Sulfadiazine, 3860
Stability considerations in dispensing practice tests (71), 5984 cream, silver, 3863
(1191), 7540 Sterility Assurance (1211), 7633 silver, 3861
Stachyose hydrate, 5733 Sterilization cycle development, 7737 sodium, 3864
Standard sand, 20- to 30-mesh, 5733 Sterilization filtration of gases, 7740 sodium injection, 3864
Stannous Sterilization-in-place (1229.13), 7735 tablets, 3861
chloride, 5594, 5733 Sterilization of compendial articles (1229), Sulfadimethoxine, 3865
chloride acid, stronger, TS, 5750, 5760 7692 sodium, 3866
chloride acid TS, 5750, 5760 Sterilizing filtration of liquids (1229.4), 7709 soluble powder, 3865
fluoride, 3830 Stinging nettle, 4889 oral suspension, 3866
extract, powdered, 4892 tablets, 3866
fluoride gel, 3831
powdered, 4891 Sulfadoxine, 3867
Stanozolol, 3832
tablets, 3832 Storax, 3837 and pyrimethamine tablets, 3868
Starch Streptomycin Sulfaguanidine, 5735
corn, 5595 injection, 3839 Sulfamerazine, 5735
corn, pregelatinized hydroxypropyl, 5598 for injection, 3839 Sulfamethazine, 3869
hydrolysate, hydrogenated, 5600 sulfate, 3838 and chlortetracycline bisulfates soluble
hydroxypropyl corn, 5596 Stronger powder, 911
iodate paper, 5747 ammonia water, 5733 granulated, 3869
iodide-free TS, 5760 cupric acetate TS, 5760 Sulfamethizole, 3870
iodide paper, 5747 Strontium oral suspension, 3871
iodide paste TS, 5760 acetate, 5733 tablets, 3871
modified, 5603 chloride Sr 89 injection, 3840 Sulfamethoxazole, 3872
hydroxide, 5734 oral suspension, 3873
pea, 5603
pea, pregelatinized hydroxypropyl, 5607 Strychnine sulfate, 5734 tablets, 3873
potassium iodide TS, 5760 Styrene-divinylbenzene and trimethoprim injection, 3874
potassium iodide and, TS, 5758 anion-exchange resin, 50- to 100-mesh, and trimethoprim oral suspension, 3875
potato, 5609, 5721, 5733 5734 and trimethoprim tablets, 3877
potato, pregelatinized hydroxypropyl, cation-exchange resin, strongly acidic, Sulfamic acid, 5735
5612 5734 Sulfanilamide, 5735
pregelatinized, 5614 copolymer beads, 5734 Sulfanilic
pregelatinized modified, 5614 acid, 5735
Combined Index to USP 41 and NF 36 Sulfa-Suspe 1-61

Sulfanilic (continued) succinate, 3897 Betamethasone sodium phosphate and

acid, diazotized TS, 5760 succinate oral suspension, 3899 betamethasone acetate injectable, 512
acid TS, 5760 tablets, 3895 Bethanechol chloride oral, 523
1-naphthylamine TS, 5760 Sunflower oil, 5635, 5735 Bisacodyl rectal, 532
o-naphthylamine TS, 5760 Supplemental information for articles of Bismuth subsalicylate oral, 540
Sulfapyridine, 3878 botanical origin (2030), 8168 Brinzolamide ophthalmic, 550
tablets, 3879 Supports for gas chromatography, 5735 Calamine topical, 615
Sulfaquinoxaline, 3879 Calamine topical, phenolated, 616
oral solution, 3880 Calcium carbonate oral, 633
Sulfasalazine, 3880 Calcium and magnesium carbonates oral,
tablets, 3881
delayed-release tablets, 3882 Suppositories 637
Captopril oral, 679
Sulfatase enzyme preparation, 5735 Acetaminophen, 38 Carbamazepine oral, 685
Sulfate Aminophylline, 231 Cefaclor for oral, 744
acid, ferrous, TS, 5750, 5753 Aspirin, 367 Cefadroxil for oral, 752
and chloride (221), 6139 Bisacodyl, 531 Cefdinir for oral, 767
ferrous, TS, 5753 Chlorpromazine, 906 Cefixime for oral, 775
magnesium, TS, 5756 Ergotamine tartrate and caffeine, 1561 Cefpodoxime proxetil for oral, 801
mercuric, TS, 5753, 5756 Glycerin, 1969 Cefprozil for oral, 806
potassium, 5721 Indomethacin, 2156 Cefuroxime axetil for oral, 825
potassium, TS, 5758 Miconazole nitrate vaginal, 2740 Cellulose sodium phosphate for oral, 832
in reagents, 5663 Morphine sulfate, 2814 Cephalexin for oral, 834
strychnine, 5734 Nystatin vaginal, 2990 Cephradine for oral, 845
Sulfathiazole, 3883 Prochlorperazine, 3448 Chloramphenicol and hydrocortisone
sodium, 5735 Progesterone vaginal, 3457 acetate for ophthalmic, 866
Sulfinpyrazone, 3883 Promethazine hydrochloride, 3467 Chloramphenicol palmitate oral, 868
capsules, 3884 Thiethylperazine maleate, 4052 Chlorothiazide oral, 895
tablets, 3885 Cholestyramine for oral, 919
Sulfisoxazole, 3885 Chromic phosphate P 32, 3295
acetyl, 3886 Ciclopirox olamine topical, 928
acetyl and erythromycin estolate oral Suprofen, 3900 Ciprofloxacin and dexamethasone otic,
suspension, 1575 ophthalmic solution, 3900 951
acetyl and erythromycin ethylsuccinate for Clarithromycin for oral, 977
oral suspension, 1580 Clavulanate potassium and amoxicillin for
acetyl oral suspension, 3887 oral, 284 .
tablets, 3886
Sulfomolybdic acid TS, 5760
Suspension Clindamycin phosphate topical, 999
Clonazepam oral, 1021
Sulfonic acid cation-exchange resin, 5735 Acetaminophen and codeine phosphate
Clopidogrel compounded oral, 1033
oral, 57
2-(4-Sulfophenylazo)-1,8-dihydroxy-3,6- Colestipol hydrochloride for oral, 1072
Acetaminophen oral, 39
naphthalenedisulfonic acid, trisodium salt, Colistin and neomycin sulfates and
Acetazolamide oral, 68
5746 hydrocortisone acetate otic, 1075
Sulfosalicylic acid, 5735
Acyclovir oral, 83
Colistin sulfate for oral, 1075
Albendazole oral, 93
Sulfur, 5735 Cortisone acetate injectable, 1100
Allopurinol oral, 123
dioxide, 5634 Demeclocycline oral, 1166
dioxide detector tube, 5735 Alprazolam oral, 131
Desoxycorticosterone pivalate injectable,
ointment, 3887 Alumina, magnesia, and calcium carbonate
1194
precipitated, 3887 oral, 151
Dexamethasone acetate injectable, 1200
Alumina and magnesia oral, 149
and resorcinol topical suspension, 3595 Dexamethasone ophthalmic, 1197
sublimed, 3888 Alumina, magnesia, and simethicone oral,
Diazoxide oral, 1248
Sulfur dioxide (525), 6254 155 Dicloxacillin sodium for oral, 1267
Sulfuric acid, 5635, 5735 Alumina and magnesium carbonate oral,
Didanosine tablets for oral, 1275
diluted, 5690, 5735 158 Diltiazem hydrochloride oral, 1310
Alumina and magnesium trisilicate oral,
fluorometric, 5735 Dipyridamole oral, 1347
161
fuming, 5697, 5735 Dolasetron mesylate oral, 1385
half-normal (0.5 N) in alcohol, 5772 Amoxicillin and clavulanate potassium for
Doxycycline calcium oral, 1425
oral, 284
nitrogen free, 5735 Doxycycline compounded oral, veterinary,
Amoxicillin for oral, 280
normal (1 N), 5772 1427
phenylhydrazine, TS, 5757 Amoxicillin for injectable, 279
Doxycycline for oral, 1421
TS, 5760 Amoxicillin oral, 279
Enalapril maleate compounded oral,
0.02 N TS, 5760 Amoxicillin tablets for oral, 283
veterinary, 1501
0.2 N TS, 5760 Ampicillin for injectable, 301
Erythromycin estolate for oral, 1574
Ampicillin for oral, 301
0.5 N TS, 5760 Erythromycin estolate oral, 1574
1 MTS, 5760 Ampicillin and probenecid for oral, 303
Erythromycin estolate and sulfisoxazole
2 NTS, 5760 Atenolol compounded oral, 386
acetyl oral, 1575
Atenolol compounded oral, veterinary, 386
6 NTS, 5761 Erythromycin ethylsuccinate for oral, 1578
Atovaquone oral, 400
7 NTS, 5761 Erythromycin ethylsuccinate oral, 1578
Sulfuric acid—formaldehyde TS, 5761 Aurothioglucose injectable, 411
Erythromycin ethylsuccinate and
0.05 N sulfuric acid VS, 5772 Azathioprine oral, 417
sulfisoxazole acetyl for oral, 1580
Sulfurous acid, 5735 Azithromycin for oral, 428
Estrone injectable, 1625
Sulindac, 3888 Baclofen oral, 444
Famotidine for oral, 1682
tablets, 3889 Barium sulfate, 452
Ferumoxsil oral, 1724
Sulisobenzone, 3891 Barium sulfate for, 453
Flucytosine oral, 1767
Sumatriptan, 3892 Benazepril hydrochloride compounded
Fluorometholone ophthalmic, 1798
injection, 3893 oral, veterinary, 463
Furazolidone oral, 1891
nasal spray, 3894 Ganciclovir oral, 1927
|-62 Suspe-Table
Suspension (continued)
Gentamicin and prednisolone acetate
ophthalmic, 1943
Griseofulvin oral, 1998
Hydrocortisone rectal, 2060
Hydroxyzine pamoate oral, 2099
Ibuprofen oral, 2110
Imipenem and cilastatin for injectable,
2125
Indomethacin oral, 2158
lsophane insulin human, 2177
Human insulin isophane and human insulin
injection, 2174
lsophane insulin, 2176
Insulin zinc, 2181
Insulin zinc, extended, 2182
Insulin zinc, prompt, 2184
lsoflupredone acetate injectable, 2247
Ketoconazole oral, 2310
Labetalol hydrochloride oral, 2321
Lamotrigine compounded oral, 2342
Lansoprazole compounded oral, 2351
Loracarbef for oral, 2461
Magaldrate and simethicone oral, 2498
Magaldrate oral, 2497
Magnesium carbonate and sodium
bicarbonate for oral, 2504
Mebendazole oral, 2535
Medroxyprogesterone acetate injectable,
2548
Megestrol acetate oral, 2554
Meloxicam oral, 2560
Meprobamate oral, 2585
Mesalamine rectal, 2597
Methacycline hydrochloride oral, 2626
Methadone hydrochloride tablets for oral,
2630
Methenamine mandelate oral, 2640
Methyldopa oral, 2667
Methylprednisolone acetate injectable,
2691
Metolazone oral, 2705
Metoprolol tartrate oral, 2714
Metronidazole benzoate compounded oral,
2722
Minocycline hydrochloride oral, 2754
Nalidixic acid oral, 2853
Naproxen oral, 2864
Natamycin ophthalmic, 2876
Neomycin and polymyxin B sulfates and
dexamethasone ophthalmic, 2901
Neomycin and polymyxin B sulfates and
hydrocortisone otic, 2903
Neomycin and polymyxin B sulfates and
hydrocortisone acetate ophthalmic, 2904
Neomycin and polymyxin B sulfates and
hydrocortisone ophthalmic, 2903
Neomycin and polymyxin B sulfates and
prednisolone acetate ophthalmic, 2906
Neomycin sulfate and hydrocortisone otic,
2889
Neomycin sulfate and hydrocortisone
acetate ophthalmic, 2890
Neomycin sulfate and prednisolone acetate
ophthalmic, 2907
Nevirapine oral, 2912
Nitrofurantoin oral, 2951
Nystatin for oral, 2990
Nystatin oral, 2990
Ondansetron hydrochloride oral, 3033
Oxfendazole oral, 3087
Oxytetracycline and nystatin for oral, 3126
Oxytetracycline calcium oral, 3127
Oxytetracycline hydrochloride and
hydrocortisone acetate ophthalmic, 3129
Pantoprazole oral, 3154
Penicillin G benzathine injectable, 3195
Penicillin G benzathine and penicillin G
procaine injectable, 3196
Penicillin G benzathine oral, 3196
Penicillin G, neomycin, polymyxin B,
hydrocortisone acetate, and
hydrocortisone sodium succinate topical,
3193
Penicillin G procaine, dihydrostreptomycin
sulfate, chlorpheniramine maleate, and
dexamethasone injectable, 3207
Penicillin G procaine and
dihydrostreptomycin sulfate injectable,
3207
Penicillin G procaine, dihydrostreptomycin
sulfate, and prednisolone injectable,
3209
Penicillin G procaine, neomycin and
polymyxinB sulfates, and hydrocortisone
acetate topical, 3210
Penicillin G procaine injectable, 3205
Penicillin G procaine for injectable, 3205
Penicillin V benzathine oral, 3216
Penicillin V for oral, 3214
Perflutren protein-type A microspheres
injectable, 3235
Pergolide, oral, veterinary, 3238
Phenoxybenzamine hydrochloride
compounded oral, 3267
Phenytoin oral, 3286
Phosphate P 32, chromic, 3295
Piroxicam compounded oral, 3338
Prednisolone acetate injectable, 3414
Prednisolone acetate ophthalmic, 3415
Prednisolone compounded oral, veterinary,
3415
Prednisone injectable, 3423
Prednisolone tebutate injectable, 3421
Primidone oral, 3434
Progesterone injectable, 3456
Propyliodone injectable oil, 3501
Psyllium hydrophilic mucilloid for oral,
3516
Pyrantel pamoate oral, 3518
Pyrvinium pamoate oral, 3532
Quinidine sulfate oral, 3553
Ractopamine hydrochloride, 3565
Resorcinol and sulfur topical, 3595
Rifampin oral, 3610
Rimexolone ophthalmic, 3619
Selenium sulfide topical, 3740
Simethicone oral, 3762
Sodium polystyrene sulfonate, 3809
Spectinomycin for injectable, 3825
Spironolactone compounded oral, 3826
Structured vehicle, 5637
Structured vehicle, sugar-free, 5637
Sulfacetamide sodium and prednisolone
acetate ophthalmic, 3858
Sulfacetamide sodium topical, 3856
Sulfadimethoxine oral, 3866
Sulfamethizole oral, 3871
Sulfamethoxazole oral, 3873
Sulfamethoxazole and trimethoprim oral,
3875
Sulfisoxazole acetyl oral, 3887
Sumatriptan succinate oral, 3899
Temozolomide oral, 3972
Testosterone injectable, 4002
Tetracycline hydrochloride ophthalmic,
4022
Tetracycline hydrochloride oral, 4022
Tetracycline oral, 4015
Thiabendazole oral, 4046
Combined Index to USP 41 and NF 36
Thioridazine oral, 4063
Tobramycin and dexamethasone
ophthalmic, 4119
Tobramycin and fluorometholone acetate
ophthalmic, 4121
Topiramate compounded oral, 4146
Triamcinolone acetonide injectable, 4192
Triamcinolone diacetate injectable, 4194
Triamcinolone hexacetonide injectable,
4195
Triflupromazine oral, 4214
Trisulfapyrimidines oral, 4235
Vehicle for oral, 5474
Verapamil hydrochloride oral, 4305
Zinc sulfide topical, 4388
Suspension structured vehicle, 5637
sugar-free, 5637
Suture
absorbable surgical, 3901
nonabsorbable surgical, 3903
Sutures
diameter (861), 6666
needle attachment (871), 6667
Syrup
Acacia, 5179
Calcium glubionate, 641
Cherry, 5289
Chlorpromazine hydrochloride, 908
Chocolate, 5296
Corn, 5304
Corn, solids, 5310
High fructose corn, 5307
Docusate sodium, 1380
Ferrous sulfate, 1720
Orange, 5476
Perphenazine, 3247
Piperazine citrate, 3334
Promazine hydrochloride, 3463
Syrup, 5637
Tolu balsam, 5640
T
Tablet breaking force (1217), 7635
Tablet compression characterization, 7042
Tablet friability (1216), 7634
Tablets
Abacavir, 5617
Abiraterone acetate, 26
Acepromazine maleate, 34
Acetaminophen, 39
Containing at least three of the
following—acetaminophen and (salts of)
chlorpheniramine, dextromethorphan,
and pseudoephedrine, 51
Acetaminophen and aspirin, 41
Acetaminophen, aspirin, and caffeine, 42
Acetaminophen and caffeine, 44
Combined Index to USP 41 and NF 36
Tablets (continued)
Acetaminophen, chlorpheniramine
maleate, and dextromethorphan
hydrobromide, 53
Acetaminophen and codeine phosphate,
59
Acetaminophen and diphenhydramine
citrate, 61
Acetaminophen, diphenhydramine
hydrochloride, and pseudoephedrine
hydrochloride, 63
Acetaminophen extended-release, 40
Acetaminophen and hydrocodone
bitartrate, 2053
Acetaminophen and pseudoephedrine
hydrochloride, 64
Acetazolamide, 68
Acetohydroxamic acid, 71
Acyclovir, 84
Albendazole, 94
Albuterol, 96
Albuterol extended-release, 97
Alendronate sodium, 110
Alfuzosin hydrochloride extended-release,
115
Allopurinol, 123
Almotriptan, 127
Alprazolam, 131
Alprazolam extended-release, 133
Alprazolam orally disintegrating, 136
Alumina and magnesia, 150
Alumina, magnesia, and calcium carbonate
chewable, 152
Alumina, magnesia, calcium carbonate,
and simethicone chewable, 153
Alumina, magnesia, and simethicone
chewable, 157
Alumina and magnesium carbonate, 159
Alumina, magnesium carbonate, and
magnesium oxide, 160
Alumina and magnesium trisilicate, 162
Aluminum hydroxide gel, dried, 172
Aluminum sulfate and calcium acetate for
topical solution, 177
Amiloride hydrochloride, 205
Amiloride hydrochloride and
hydrochlorothiazide, 206
Aminobenzoate potassium, 212
Aminocaproic acid, 219
Aminoglutethimide, 221
Aminopentamide sulfate, 225
Aminophylline, 232
Aminophylline delayed-release, 234
Aminosalicylate sodium, 237
Aminosalicylic acid, 239
Amitriptyline hydrochloride, 249
Amlodipine and valsartan, 253
Amlodipine besylate, 263
Amlodipine, valsartan and
hydrochlorothiazide, 257
Ammonium chloride delayed-release, 265
Amodiaquine hydrochloride, 271
Amoxapine, 273
Amoxicillin, 280
Amoxicillin and clavulanic acid extended-
release, 286
Amoxicillin and clavulanate potassium, 285
Amphetamine sulfate, 290
Ampicillin, 302
Anastrozole, 313
Anileridine hydrochloride, 317
Apomorphine hydrochloride, 337
Arginine, 4435
Aripiprazole, 352
Aripiprazole orally disintegrating, 354
Table-Table 1-63
Ascorbic acid, 362 Carbenicillin indany! sodium, 692
Aspirin, 368 Carbidopa and levodopa, 693
Aspirin, alumina, and magnesia, 373 Levodopa and carbidopa extended-release,
Aspirin, alumina, and magnesium oxide, 696
375: Carbidopa and levodopa orally
Aspirin, buffered, 369 disintegrating, 701
Aspirin and codeine phosphate, 379 Carbinoxamine maleate, 703
Aspirin, codeine phosphate, alumina, and Calcium carbonate and magnesia
magnesia, 380 chewable, 635
Aspirin delayed-release, 370 Carboxymethylcellulose sodium, 714
Aspirin effervescent, for oral solution, 371 Carisoprodol, 715
Aspirin extended-release, 372 Carisoprodol, aspirin, and codeine
Astemizole, 382 phosphate, 718
Atenolol, 384 Carisoprodol and aspirin, 716
Atenolol and chlorthalidone, 387 Carprofen, 724
Atorvastatin calcium, 395 Carteolol hydrochloride, 728
Atropine sulfate, 409 Carvedilol, 731
Azatadine maleate, 414 Cascara, 737
Azathioprine, 417 Cat's claw, 4512
Azithromycin, 429 Cefaclor chewable, 746
Baclofen, 445 Cefaclor extended-release, 746
Barium sulfate, 453 Cefadroxil, 753
Belladonna extract, 459 Cefixime, 775
Benazepril hydrochloride, 462 Cefpodoxime proxetil, 802
Bendroflumethiazide, 465 Cefprozil, 807
Benztropine mesylate, 494 Cefuroxime axetil, 826
Betamethasone, 504 Cephalexin, 835
Betaxolol, 520 Cephalexin, for oral suspension, 836
Bethanechol chloride, 524 Cephradine, 845
Bicalutamide, 526 Cetirizine hydrochloride, 849
Biotin, 529 Cetirizine hydrochloride orally
Bisacodyl delayed-release, 532 disintegrating, 851
Bismuth subsalicylate, 540 Cetrizine hydrochloride and
Bisoprolol fumarate, 543 pseudoephedrine hydrochloride
Bisoprolol fumarate and extended-release, 853
hydrochlorothiazide, 544 Chlorambucil, 861
Black cohosh, 4482 Chloramphenicol, 866
Bromocriptine mesylate, 554 Chlordiazepoxide, 871
Brompheniramine maleate, 559 Chlordiazepoxide and amitriptyline
Bumetanide, 564 hydrochloride, 872
Buprenorphine and naloxone sublingual, Chloroquine phosphate, 893
571 Chlorothiazide, 896
Bupropion hydrochloride, 575 Chlorpheniramine maleate, 903
Bupropion hydrochloride extended-release, Chlorpromazine hydrochloride, 908
576 Chlorpropamide, 909
Buspirone hydrochloride, 588 Chlortetracycline hydrochloride, 912
Busulfan, 591 Chlorthalidone, 913
Butabarbital sodium, 594 Chlorzoxazone, 915
Butalbital, acetaminophen, and caffeine, Chondroitin sulfate sodium, 4541
597 Chromium picolinate, 4546
Butalbital and aspirin, 597 Cilostazol, 934
Butalbital, aspirin, and caffeine, 600 Cimetidine, 936
Cabergoline, 610 Ciprofloxacin, 945
Calcium acetate, 629 Ciprofloxacin extended-release, 946
Calcium carbonate, 634 Citalopram, 964
Calcium carbonate, magnesia, and Clarithromycin, 978
simethicone chewable, 636 Clarithromycin extended-release, 980
Calcium citrate, 4493 Clemastine fumarate, 987
Calcium gluconate, 646 Clomiphene citrate, 1016
Calcium L-5-methyltetrahydrofolate, 4499 Clonazepam, 1022
Calcium lactate, 648 Clonazepam orally disintegrating, 1023
Calcium and magnesium carbonates, 638 Clonidine hydrochloride, 1026
Calcium pantothenate, 652 Clonidine hydrochloride and
Calcium phosphate, dibasic, 657 chlorthalidone, 1027
Calcium with vitamin D, 4501 Clopidogrel, 1034
Calcium and vitamin D with minerals, Clorazepate dipotassium, 1038
4502 Clover, red, 4819
Candesartan cilexetil, 662 Clozapine, 1054
Candesartan cilexetil and Cocaine hydrochloride, for topical solution,
hydrochlorothiazide, 664 1058
Capecitabine, 668 Codeine phosphate, 1064
Captopril, 679 Codeine sulfate, 1068
Captopril and hydrochlorothiazide, 680 Colchicine, 1070
Carbamazepine, 686 Colestipol hydrochloride, 1072
Carbamazepine extended-release, 689 Cortisone acetate, 1100
USP 41 Dietary Supplements / Chaste Tree 4523

ments, each containing an oblong seed. A group of up
to six spongy, light tan, immature fruits may also ac-
company mature fruits. The fruit is often covered by a
tubular, greenish-gray, fine tomentose calyx, which is
persistent and has five teeth.
Microscopic: The exocarp is brown and narrow, con-
ns of parenchymatous cells with thin walls and par-
tially lignitied cells with many pitted thickenings on the
inside. In surface view, the exocarp shows an epidermis
of polygonal cells with irregular thickenings and glandu-
lar hairs, each with a short single-celled stalk and a
four-celled head containing essential oil. The outer mes-
ocarp consists of several layers of brown, isodiametric
parenchyma cells. The inner mesocarp consists of finely
pitted sclerenchymatous cells, some with moderately
thickened walls, others consisting of isodiametric stone
cells with small lumen. The endocarp consists of a layer
of small brown sclereid cells. The seeds are small, hav-
ing Ripe cotyledons surrounded by thin-walled, large
parenchymatous cells that have ribbed thickenings. The
nutritive tissue and the cells of the germ contain
aleuron grains and oil globules. Starch is absent. The
outer<p of calyx is composed of aera cells,
covered by abundant unicellular or multicellular curved
trichomes. The inner epidermis of calyx is glabrous and
composed of rectangular, elongated cells with slightly
wavy walls.
e ARTICLES OF BOTANICAL ORIGIN, Foreign Organic Matter
(561): NMT 3.0%
Loss ON DRYING (731)
Sample: 1.0g of Chaste Tree, finely powdered
Analysis: Dry the Sample at 105° for 2 h.
Acceptance criteria: NMT 10.0%
Ano OF BOTANICAL ORIGIN, Total Ash (561): NMT
0%
ARTICLES OF BOTANICAL ORIGIN, Acid-Insoluble Ash (561):
NMT 2.0%
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in well-closed contain-
ers, and store at controlled room temperature.
© LABELING: The label states the Latin binomial and, follow-
ing the official name, the part of the plant contained in
the article.
e USP REFERENCE STANDARDS (11)
USP Agnuside RS
USP Casticin RS
USP Powdered Chaste Tree Extract RS
Derivatization reagent: 10 mg/mL of p-dimethylami-
nobenzaldehyde in 1 N hydrochloric acid
Analysis
Samples: Standard solution and Sample solution
Develop to a length of NLT 12 cm, and dry the plate in
a current of air. Treat the plate with Derivatization rea-
gent, and heat for 10 min at 120°.
Acceptance criteria: The Sample solution shows the fol-
lowing: a blue zone (at an R; value of about 0.21) due
to the presence of aucubin and that corresponds in
color and R; value to a similar zone for the Standard
solution; a blue zone (at an Ry value of about 0.44) as a
result of the presence of agnuside that corresponds in
color and R; value to a similar zone for the Standard
solution; and one broad zone, violet in the middle, near
the solvent front and that corresponds in color and Rr
value to a similar zone for the Standard solution. Other
colored zones of varying intensities may be observed
for the Sample solution.
e B. In the test for Content of Casticin, the chromatogram
of the Sample solution shows a peak at the retention time
corresponding to the casticin peak in the chromatogram
of the Standard solution.
COMPOSITION
e CONTENT OF CASTICIN
Standard solution: About 0.05 mg/mL of USP Casticin
RS in methanol, with sonication. Pass through a cellu-
lose membrane filter of 0.45-1m or finer pore size.
Sample solution: Place about 1000 mg of Powdered
Chaste Tree in a container with a stopper. Extract twice
with 40 mL of methanol, using a hand homogenizer at
19,000 rpm for 2 min. Filter each supernatant, and
transfer to a 250-mL round-bottom flask. Rinse the resi-
due with methanol, and filter the resulting solution into
the flask. Evaporate the combined extract to dryness.
Dissolve the residue in methanol, quantitatively transfer
to a 20-mL volumetric flask, and dilute with methanol
to volume. Pass through a cellulose membrane filter of
0.45-um or finer pore size.
Solution A: Methanol
Solution B: 5.88 g/L of phosphoric acid in water
Mobile phase: See Table 1.
Table 1
Time Solution A Solution B
(min) (%) (%)
0 50 50
0 50 50
3s 65 35
exelVe) Aa

18 100 o
Powdered Chaste Tree 23 50 50
EYiTel-B]

DEFINITION Chromatographic system

Powdered Chaste Tree is Chaste Tree reduced to a powder (See Chromatography (621), System Suitability.)
or a very fine powder. It contains NLT 0.05% of agnuside Mode: LC
and NLT 0.08% of casticin, calculated on the dried basis. Detector: UV 348 nm
Column: 3.1-mm x 12.5-cm; 5-m packing L1
IDENTIFICATION Column temperature: 25°
e A. THIN-LAYER CHROMATOGRAPHY Flow rate: 1 mL/min
Standard solution: 100mg of USP Powdered Chaste Injection volume: 10 uL
Tree Extract RS in 1 mL of methanol. Heat in a water System suitability
bath at 60° for 10 min. Centrifuge, and use the clear Sample: Standard solution
supernatant. Suitability requirements
Sample solution: Transfer about 1 g of Powdered Tailing factor: NMT 2.0 for the casticin peak
Chaste Tree to a screw-capped centrifuge tube. Add Relative standard deviation: NMT 2.0% for the cas-
10 mL of methanol, heat in a water bath at 60° for ticin peak, in repeated injections
10-15 min, cool, and filter. Analysis
Adsorbent: Chromatographic silica gel with an average Samples: Standard solution and Sample solution
particle size of 10-15 tm (TLC plates) Calculate the percentage of casticin in the portion of
Application volume: 90 uL, Standard solution; 60 uL, Powdered Chaste Tree taken:
Sample solution; in bands that are 2 cm in length
Developing solvent system: Ethyl acetate, methanol, Result = (ru/rs) x (Cs/Cu) x 100
and water (77:15:8)
1-64 Table-Table
Tablets (continued)
Curcuminoids, 4562
Cyanocobalamin, 1114
Cyclizine hydrochloride, 1117
Cyclobenzaprine hydrochloride, 1121
Cyclophosphamide, 1126
Cyproheptadine hydrochloride, 1138
Dapsone, 1158
Dehydrocholic acid, 1164
Demeclocycline hydrochloride, 1167
Desipramine hydrochloride, 1174
Desloratadine, 1178
Desloratadine orally disintegrating, 1180
Desogestrel and ethinyl estradiol, 1186
Dexamethasone, 1198
Dexchlorpheniramine maleate, 1212
Dextroamphetamine sulfate, 1229
Diazepam, 1245
Dichlorphenamide, 1253
Diclofenac potassium, 1255
Diclofenac sodium and misoprostol
delayed-release, 1261
Diclofenac sodium delayed-release, 1258
Diclofenac sodium extended-release, 1259
Dicyclomine hydrochloride, 1271
Didanosine, for oral suspension, 1275
Diethylcarbamazine citrate, 1277
Diethylpropion hydrochloride, 1279
Diethylstilbestrol, 1281
Diflunisal, 1285
Digitalis, 1288
Digitoxin, 1290
Digoxin, 1293
Dihydrotachysterol, 1299
Dihydroxyaluminum sodium carbonate
chewable, 1303
Diltiazem hydrochloride, 1310
Dimenhydrinate, 1314
Diphenhydramine citrate and ibuprofen,
1324
Diphenhydramine and phenylephrine
hydrochloride, 1334
Diphenoxylate hydrochloride and atropine
sulfate, 1340
Dipyridamole, 1347
Dirithromycin delayed-release, 1349
Disulfiram, 1353
Divalproex sodium delayed-release, 1357
Divalproex sodium extended-release, 1358
Docusate sodium, 1381
Donepezil hydrochloride, 1387
Donepezil hydrochloride orally
disintegrating, 1391
Doxazosin, 1404
Doxycycline, 1424
Doxycycline hyclate, 1432
Doxycycline hyclate delayed-release, 1434
Doxylamine succinate, 1438
Dronedarone, 1442
Drospirenone and ethinyl estradiol, 1447
Dydrogesterone, 1459
Dyphylline, 1461
Dyphylline and guaifenesin, 1462
Echinacea Species dry extract tablets, 4592
Efavirenz, 1478
Eleuthero root and rhizome dry extract,
4601
Enalapril maleate, 1502
Enalapril maleate and hydrochlorothiazide,
1503
Entacapone, 1519
Entecavir, 1522
Ergocalciferol, 1550
Ergoloid mesylates, 1553
Ergoloid mesylates sublingual, 1554
Combined Index to USP 41 and NF 36
Ergonovine maleate, 1556 Glucosamine and methylsulfonylmethane,
Ergotamine tartrate, 1560 4672
Ergotamine tartrate and caffeine, 1562 Glyburide, 1961
Ergotamine tartrate sublingual, 1561 Glyburide and metformin hydrochloride,
Erythromycin, 1571 1964
Erythromycin delayed-release, 1572 Glycopyrrolate, 1974
Erythromycin estolate, 1574 Granisetron hydrochloride, 1994
Erythromycin ethylsuccinate, 1578 Griseofulvin, 1999
Erythromycin stearate, 1584 Griseofulvin, ultramicrosize, 2000
Escitalopram, 1587 Guaifenesin, 2003
Estazolam, 1599 Guanabenz acetate, 2008
Estradiol, 1607 Guanethidine monosulfate, 2010
Estradiol and norethindrone acetate, 1608 Guanfacine, 2011
Estrogens, conjugated, 1619 Guggul, 4692
Estrogens, esterified, 1623 Halazone for solution, 2014
Estropipate, 1627 Haloperidol, 2021
Eszopiclone, 1629 Homatropine methylbromide, 2040
Ethacrynic acid, 1632 Hydralazine hydrochloride, 2046
Ethambuto! hydrochloride, 1635 Hydrochlorothiazide, 2051
Ethinyl estradiol, 1639 Hydrochlorothiazide and amiloride
Ethionamide, 1642 hydrochloride, 206
Ethotoin, 1647 Hydrocodone bitartrate, 2052
Ethynodiol diacetate and ethinyl estradiol, Hydrocodone bitartrate and
1649 acetaminophen, 2053
Ethynodiol diacetate and mestranol, 1650 Hydrocodone bitartrate and homatropine
Etidronate disodium, 1652 methylbromide, 2054
Etodolac, 1655 Hydrocortisone, 2061
Etodolac extended-release, 1656 Hydroflumethiazide, 2074
Ezetimibe, 1672 Hydromorphone hydrochloride, 2081
Famotidine, 1683 Hydroxychloroquine sulfate, 2086
Felbamate, 1688 Hydroxyzine hydrochloride, 2094
Felodipine extended-release, 1690 Hyoscyamine, 2100
Fenofibrate, 1699 Hyoscyamine sulfate, 2105
Fenoprofen calcium, 1706 Ibuprofen, 2111
Ferrous fumarate, 1712 Ibuprofen and pseudoephedrine
Ferrous fumarate and docusate sodium hydrochloride, 2113
extended-release, 1713 Imipramine hydrochloride, 2128
Ferrous gluconate, 1717 Indapamide, 2140
Ferrous sulfate, 1720 lodoquinol, 2206
Fexofenadine hydrochloride, 1729 Irbesartan, 2232
Fexofenadine hydrochloride and Irbesartan and hydrochlorothiazide, 2233
pseudoephedrine hydrochloride Isoniazid, 2254
extended-release, 1731 lsopropamide iodide, 2256
Finasteride, 1744 Isoproterenol hydrochloride, 2261
Flavoxate hydrochloride, 1748 Isosorbide dinitrate chewable, 2270
Flecainide acetate, 1751 Isosorbide dinitrate extended-release, 2271
Fluconazole, 1765 Isosorbide dinitrate sublingual, 2272
Fludrocortisone acetate, 1773 lsosorbide mononitrate, 2274
Fluoxetine, 1807 Isosorbide mononitrate extended-release,
Fluoxymesterone, 1811 2276
Flurbiprofen, 1824 Isoxsuprine hydrochloride, 2287
Fluvoxamine maleate, 1863 Ivermectin, 2295
Folic acid, 1867 Ivermectin and pyrantel pamoate, 2298
Fosinopril sodium, 1881 Ketoconazole, 2311
Fosinopril sodium and hydrochlorothiazide, Ketorolac tromethamine, 2318
1882 Labetalol hydrochloride, 2322
Furazolidone, 1891 Lamivudine, 2329
Furosemide, 1894 Lamivudine and zidovudine, 2331
Gabapentin, 1898 Lamotrigine, 2334
Galantamine, 1918 Lamotrigine extended-release, 2336
Garlic delayed-release, 4648 Lamotrigine, for oral suspension, 2340
Gemfibrozil, 1934 Leflunomide, 2355
Ginkgo, 4665 Letrozole, 2357
Ginseng, American, 4428 Leucovorin calcium, 2362
Ginseng, Asian, 4445 Levamisole hydrochloride, 2369
Glimepiride, 1947 Levetiracetam, 2375
Glipizide, 1951 Levetiracetam extended-release, 2377
Glipizide and metformin hydrochloride, Levocarnitine, 2389
1953 Levocetirizine dihydrochloride, 2391
Glucosamine, 4669 Levodopa, 2394
Glucosamine and chondroitin sodium Levofloxacin, 2398
sulfate, 4667 Levonorgestrel and ethinyl estradiol, 2402
Glucosamine, chondroitin sulfate sodium, Levorphanol tartrate, 2404
and methylsulfonylmethane, 4674 Levothyroxine sodium, 2407
Liothyronine sodium, 2428
Combined Index to USP 41 and NF 36
Tablets (continued)
Liotrix, 2429
Lipoic acid, alpha, 4742
Lisinopril, 2433
Lisinopril and hydrochlorothiazide, 2434
Lithium carbonate, 2438
Lithium carbonate extended-release, 2439
Loperamide hydrochloride, 2448
Lopinavir and ritonavir, 2457
Loratadine, 2465
Loratadine chewable, 2466
Loratadine orally disintegrating, 2468
Lorazepam, 2474
Losartan potassium, 2477
Losartan potassium and
hydrochlorothiazide, 2480
Lovastatin, 2485
Lysine hydrochloride, 4751
Magaldrate, 2497
Magaldrate and simethicone chewable,
2499
Magnesia, 2500
Magnesium gluconate, 2509
Magnesium oxide, 2513
Magnesium salicylate, 2516
Magnesium trisilicate, 2521
Maprotiline hydrochloride, 2531
Mazindol, 2533
Mebendazole, 2536
Mecamylamine hydrochloride, 2540
Meclizine hydrochloride, 2543
Medroxyprogesterone acetate, 2548
Mefloquine hydrochloride, 2552
Megestrol acetate, 2555
Melatonin, 4759
Meloxicam, 2562
Melphalan, 2564
Memantine hydrochloride, 2566
Menadiol sodium diphosphate, 2570
Menaquinone-7, 4764
Meperidine hydrochloride, 2576
Mephenytoin, 2577
Mephobarbital, 2579
Meprobamate, 2585
Mercaptopurine, 2589
Mesalamine delayed-release, 2598
Mesoridazine besylate, 2603
Metaproterenol sulfate, 2609
Metaxalone, 2612
Metformin hydrochloride, 2615
Metformin hydrochloride extended-release,
2616
Methadone hydrochloride, 2629
Methamphetamine hydrochloride, 2631
Methazolamide, 2633
Methdilazine hydrochloride, 2635
Methenamine, 2637
Methenamine hippurate, 2638
Methenamine mandelate, 2640
Methenamine mandelate delayed-release,
2641
Methimazole, 2642
Methocarbamol, 2646
Methotrexate, 2652
Methscopolamine bromide, 2658
Methyclothiazide, 2661
Methylcellulose, 2666
Methylcobalamin, 4768
Methyldopa, 2668
Methyldopa and chlorothiazide, 2669
Methyldopa and hydrochlorothiazide,
2670
Methylergonovine maleate, 2678
Methylphenidate hydrochloride, 2682
Methylphenidate hydrochloride extended-
release, 2683
Methylprednisolone, 2689
Methylsulfonylmethane, 4770
Methyltestosterone, 2697
Methysergide maleate, 2698
Metoclopramide, 2702
Metolazone, 2705
Metoprolol succinate extended-release,
2709
Metoprolol tartrate, 2715
Metoprolol tartrate and
hydrochlorothiazide, 2717
Metronidazole, 2726
Metronidazole extended-release, 2727
Metyrapone, 2730
Midodrine hydrochloride, 2744
Milk thistle, 4776
Minerals, 4785
Minocycline hydrochloride, 2754
Minocycline hydrochloride extended-
release, 2755
Minoxidil, 2761
Mirtazapine, 2765
Mirtazapine orally disintegrating, 2766
Mitotane, 2773
Modafinil, 2776
Memantine hydrochloride, 2780
Moexipril hydrochloride and
hydrochlorothiazide, 2782
Molindone hydrochloride, 2785
Montelukast sodium, 2800
Montelukast sodium chewable, 2803
Moricizine hydrochloride, 2808
Moxifloxacin, 2821
Mycophenolate mofetil, 2834
Mycophenolic acid delayed-release, 2838
Nabumetone, 2844
Nadolol, 2846
Nadolol and bendroflumethiazide, 2847
Nafcillin sodium, 2850
Nalidixic acid, 2853
Naltrexone hydrochloride, 2858
Naproxen, 2865
Naproxen delayed-release, 2866
Naproxen sodium, 2868
Naratriptan, 2872
Nateglinide, 2878
Nefazodone hydrochloride, 2880
Neomycin sulfate, 2884
Neostigmine bromide, 2908
Nevirapine, 2914
Niacin, 2919
Niacinamide, 2925
Niacin extended-release, 2920
Nifedipine extended-release, 2938
Nitrofurantoin, 2952
Nitroglycerin sublingual, 2958
Norethindrone, 2969
Norethindrone acetate, 2973
Norethindrone acetate and ethinyl
estradiol, 2975
Norethindrone and ethinyl estradiol, 2970
Norethindrone and mestranol, 2971
Norfloxacin, 2978
Norgestimate and ethiny! estradiol, 2981
Norgestrel, 2982
Norgestrel and ethiny! estradiol, 2983
Nystatin, 2990
Ofloxacin, 3000
Olanzapine, 3003
Olanzapine orally disintegrating, 3007
Olmesartan medoxomil, 3012
Ondansetron, 3034
Ondansetron orally disintegrating, 3036
Table-Table 1-65
Orbifloxacin, 3041
Orphenadrine citrate, aspirin, and caffeine,
3052
Orphenadrine citrate extended-release,
3050
Oxandrolone, 3073
Oxaprozin, 3076
Oxazepam, 3080
Oxcarbazepine, 3084
Oxprenolol hydrochloride, 3089
Oxprenolol hydrochloride extended-
release, 3089
Oxtriphylline, 3091
Oxtriphylline extended-release, 3091
Oxybutynin chloride, 3095
Oxybutynin chloride extended-release,
3096
Oxycodone and acetaminophen, 3108
Oxycodone and aspirin, 3109
Oxycodone hydrochloride, 3103
Oxycodone hydrochloride extended-
release, 3104
Oxymetholone, 3115
Oxymorphone hydrochloride, 3119
Oxymorphone hydrochloride extended-
release, 3121
Oxytetracycline, 3125
Pancreatin, 3147
Pancrelipase, 3150
Pantoprazole sodium delayed-release, 3157
Papain, for topical solution, 3161
Papaverine hydrochloride, 3163
Paroxetine, 3177
Paroxetine extended-release, 3178
Penbutolol sulfate, 3188
Penicillamine, 3192
Penicillin G benzathine, 3196
Penicillin G potassium, 3202
Penicillin V, 3214
Penicillin V potassium, 3218
Pentazocine and acetaminophen, 3221
Pentazocine and aspirin, 3222
Pentazocine and naloxone, 3224
Pentoxifylline extended-release, 3232
Pergolide, 3239
Perindopril erbumine, 3243
Perphenazine, 3247
Perphenazine and amitriptyline
hydrochloride, 3248
Phenazopyridine hydrochloride, 3252
Phendimetrazine tartrate, 3255
Phenelizine sulfate, 3257
Phenmetrazine hydrochloride, 3259
Phenobarbital, 3261
Phentermine hydrochloride, 3270
Phenylbutazone, 3275
Phenylephrine hydrochloride, 3281
Phenytoin chewable, 3287
Phytonadione, 3299
Pilocarpine hydrochloride, 3303
Pimozide, 3307
Pindolol, 3309
Pioglitazone, 3312
Pioglitazone and glimepiride, 3314
Pioglitazone and metformin hydrochloride,
3317
Piperazine citrate, 3334
Potassium and sodium bicarbonates and
citric acid effervescent, for oral solution,
3357
Potassium bicarbonate effervescent, for
oral solution, 3355
Potassium bicarbonate and potassium
chloride effervescent, for oral solution,
3356
 1-66 Table-Tacro Combined Index to USP 41 and NF 36

Tablets (continued) Rizatriptan benzoate orally disintegrating, Tizanidine, 4107

Potassium chloride extended-release, 3363 3665 Tocainide hydrochloride, 4124
Potassium chloride, potassium bicarbonate, Ropinirole, 3669 Tolazamide, 4126
and potassium citrate effervescent, for Ropinirole extended-release, 3671 Tolbutamide, 4129
oral solution, 3367 Rufinamide, 3685 Tolcapone, 4130
Potassium citrate, 4805 Saccharin sodium, 3691 Tolmetin sodium, 4133
Potassium citrate extended-release, 3372 St. John’s wort flowering top dry extract, Topiramate, 4143
Potassium gluconate, 3378 4849 Torsemide, 4147
Potassium iodide, 3382 Salsalate, 3706 Tramadol hydrochloride, 4151
Potassium iodide delayed-release, 3382 Scopolamine hydrobromide, 3730 Tramadol hydrochloride and
Pravastatin sodium, 3401 Selegiline hydrochloride, 3737 acetaminophen, 4158
Praziquantel, 3404 Sennosides, 3744 Tramadol hydrochloride extended-release,
Prednisolone, 3413 Sertraline hydrochloride, 3749 4153
Prednisone, 3423 Sildenafil, 3755 Trandolapril, 4161
Primaquine phosphate, 3431 Simethicone, 3763 Trandolapril and verapamil hydrochloride
Primidone, 3434 Simvastatin, 3764 extended-release, 4163
Probenecid, 3436 Sitagliptin, 3769 Tranylcypromine, 4170
Probenecid and colchicine, 3437 Sodium bicarbonate, 3779 Trazodone hydrochloride, 4178
Probucol, 3439 Sodium chloride, 3786 Triamcinolone, 4185
Procainamide hydrochloride, 3441 Sodium chloride, for solution, 3786 Triamterene and hydrochlorothiazide, 4200
Procainamide hydrochloride extended- Sodium fluoride, 3790 Triazolam, 4202
release, 3442 Sodium salicylate, 3811 Trichlormethiazide, 4204
Prochlorperazine maleate, 3451 Sotalol hydrochloride, 3821 Trifluoperazine hydrochloride, 4213
Procyclidine hydrochloride, 3453 Soy isoflavones, 4881 Triflupromazine hydrochloride, 4216
Promazine hydrochloride, 3463 Spironolactone, 3827 Trihexyphenidyl hydrochloride, 4219
Promethazine hydrochloride, 3468 Spironolactone and hydrochlorothiazide, Trimeprazine tartrate, 4223
Propafenone hydrochloride, 3481 3829 Trimethoprim, 4226
Propantheline bromide, 3484 Spirulina, 4886 Trioxsalen, 4229
Propranolol hydrochloride, 3496 Stanozolol, 3832 Tripelennamine hydrochloride, 4231
Propranolol hydrochloride and Sucralfate, 3845 Triprolidine hydrochloride, 4233
hydrochlorothiazide, 3497 Sulfadiazine, 3861 Triprolidine and pseudoephedrine
Propylthiouracil, 3502 Sulfadimethoxine, 3866 hydrochlorides, 4235
Protriptyline hydrochloride, 3506 Sulfadoxine and pyrimethamine, 3868 Trisulfapyrimidines, 4236
Pseudoephedrine hydrochloride, 3509 Sulfamethizole, 3871 Trospium chloride, 4242
Pseudoephedrine hydrochloride extended- Sulfamethoxazole, 3873 Ubidecarenone, 4921
release, 3510 Sulfamethoxazole and trimethoprim, 3877 Ursodiol, 4259
Pyrazinamide, 3521 Sulfapyridine, 3879 Valacyclovir, 4261
Pyridostigmine bromide, 3524 Sulfasalazine, 3881 Valerian, 4930
Pyridoxine hydrochloride, 3527 Sulfasalazine delayed-release, 3882 Valganciclovir, 4266
Pyrilamine maleate, 3529 Sulfinpyrazone, 3885 Valsartan, 4278
Pyrimethamine, 3531 Sulfisoxazole, 3886 Valsartan and hydrochlorothiazide, 4279
Pyrvinium pamoate, 3533 Sulindac, 3889 Venlafaxine, 4292
Quazepam, 3534 Sumatriptan, 3895 Verapamil hydrochloride, 4306
Quetiapine, 3535 Tadalafil, 3917 Verapamil hydrochloride extended-release,
Quetiapine, extended-release, 3538 Tamoxifen citrate, 3922 4307
Quinapril, 3545 Telmisartan, 3959 Vigabatrin, 4316
Quinapril and hydrochlorothiazide, 3543 Telmisartan and amlodipine, 3960 Vinpocetine, 4933
Quinidine gluconate extended-release, Telmisartan and hydrochlorothiazide, 3962 Vitamin A, 4330
3549 Terazosin, 3977 Vitamins with minerals, oil-soluble, 4966
Quinidine sulfate, 3554 Terbinafine, 3982 Vitamins with minerals, oil- and water-
Quinidine sulfate extended-release, 3555 Terbutaline sulfate, 3989 soluble, 5061
Quinine sulfate, 3560 Testolactone, 4001 Vitamins with minerals, water-soluble,
Raloxifene hydrochloride, 3569 Tetracycline hydrochloride, 4023 5137
Ramipril, 3576 Tetracycline hydrochloride and novobiocin Vitamins, oil-soluble, 4944
Ranitidine, 3581 sodium, 4024 Vitamins, oil- and water-soluble, 5004
Rauwolfia serpentina, 3585 Tetracycline hydrochloride, novobiocin Vitamins, water-soluble, 5098
Repaglinide, 3590 sodium, and prednisolone, 4024 Warfarin sodium, 4343
Reserpine, 3592 Theophylline, 4038 Zalcitabine, 4362
Reserpine and chlorothiazide, 3593 Theophylline, ephedrine hydrochloride, Zidovudine, 4372
Rhodiola rosea, 4833 and phenobarbital, 4041 Zinc citrate, 5159
Ribavirin, 3600 Theophylline sodium glycinate, 4044 Zinc gluconate, 4380
Riboflavin, 3603 Thiabendazole chewable, 4046 Zinc sulfate, 4388
Rifampin, isoniazid, and pyrazinamide, Thiamine hydrochloride, 4049 Zolmitriptan, 4399
3612 Thiethylperazine maleate, 4054 Zolmitriptan orally disintegrating, 4400
Rifampin, isoniazid, pyrazinamide, and Thioguanine, 4060 Zolpidem tartrate, 4403
ethambutol hydrochloride, 3614 Thioridazine hydrochloride, 4065 Zolpidem tartrate extended-release, 4405
Riluzole, 3616 Thyroid, 4074
Ticlopidine hydrochloride, 4088
Index

Rimantadine hydrochloride, 3618

Risedronate sodium, 3636 Tienchi ginseng root and rhizome dry
Risperidone, 3640 extract, 4912 Tacrine
Risperidone orally disintegrating, 3642 Tienchi ginseng root and rhizome powder, capsules, 3905
Ritodrine hydrochloride, 3645 4907 hydrochloride, 3906
Ritonavir, 3655 Timolol maleate, 4099 Tacrolimus, 3906
Rizatriptan benzoate, 3663 Timolol maleate and hydrochlorothiazide, capsules, 3909
4100 oral suspension, 3914
Combined Index to USP 41 and NF 36 Tadal-Tetra 1-67

Tadalafil, 3915 Tc 99m oxidronate injection, 3950 Tetrabutylammonium

tablets, 3917 Tc 99m pentetate injection, 3951 bromide, 5735
Tadalafil compounded Tc 99m pertechnetate injection, sodium, hydrogen sulfate, 5735
oral suspension, 3918 3951 hydrogen sulfate ion pairing reagent, 5735
Tagatose, 5637 Tc 99m pyrophosphate injection, 3953 hydroxide, 1.0 M in methanol, 5735
Talc, 3919 Tc 99m (pyro- and trimeta-) phosphates hydroxide, 0.4 M aqueous, 5735
Tamoxifen citrate, 3921 injection, 3953 hydroxide 30-hydrate, 5735
tablets, 3922 Tc 99m red blood cells injection, 3954 hydroxide in methanol/isopropyl alcohol
Tamsulosin hydrochloride, 3923 Tc 99m sestamibi injection, 3955 (0.1 N), 5773
capsules, 3925 Tc 99m succimer injection, 3956 hydroxide, tenth-normal (0.1 N), 5772
Tangerine peel, 4894 Tc 99m sulfur colloid injection, 3956 iodide, 5736
dry extract, 4896 Tc 99m tetrofosmin injection, 3957 phosphate, 5736
powder, 4898 Telmisartan, 3957 Tetrabutylammonium hydrogen sulfate
Tannic acid, 3934, 5735 and hydrochlorothiazide tablets, 3962 0.02 M TS, 5761
TS, 5761 tablets, 3959 Tetrabutylammonium hydroxide, 40 percent
Tape, adhesive, 3934 Telmisartan and amlodipine in water, 5736
Tapioca starch, 5616 tablets, 3960 Tetracaine, 4006
Tartaric acid, 5638, 5735 Temazepam, 3965 and cocaine hydrochlorides and
TS, 5761 capsules, 3967 epinephrine topical solution, 1059
Taurine, 3934 Temozolomide, 3967 hydrochloride, 4008
Tazobactam, 3935 capsules, 3968 hydrochloride, benzocaine, and butamben
and piperacillin for injection, 3325 for injection, 3971 topical aerosol, 479
Tc 99m oral suspension, 3972 hydrochloride, benzocaine, and butamben
albumin aggregated injection, technetium, Temperature gel, 480
3937 congealing (651), 6382 hydrochloride, benzocaine, and butamben
albumin colloid injection, technetium, Teniposide, 3973 ointment, 482
3938 injection, 3974 hydrochloride, benzocaine, and butamben
albumin injection, technetium, 3936 Tensile strength (881), 6668 topical solution, 483
apcitide injection, technetium, 3940 Terazosin hydrochloride cream, 4009
arcitumomab injection, technetium, 3940 capsules, 3975 hydrochloride in dextrose injection, 4013
bicisate injection, technetium, 3941 hydrochloride, 3979 hydrochloride injection, 4010
depreotide injection, technetium, 3941 tablets, 3977 hydrochloride for injection, 4011
disofenin injection, technetium, 3942 Terbinafine hydrochloride, neomycin sulfate, and
etidronate injection, technetium, 3943 hydrochloride, 3984 isoflupredone acetate ointment, 2891
exametazime injection, technetium, 3943 oral suspension, 3981 hydrochloride, neomycin sulfate, and
fanolesomab injection, technetium, 3944 tablets, 3982 isoflupredone acetate topical powder,
gluceptate injection, technetium, 3945 Terbutaline 2892
lidofenin injection, technetium, 3946 sulfate, 3985 hydrochloride ophthalmic solution, 4012
mebrofenin injection, technetium, 3947 sulfate inhalation aerosol, 3986 hydrochloride topical solution, 4012
medronate injection, technetium, 3948 sulfate injection, 3987 and menthol ointment, 4007
mertiatide injection, technetium, 3949 sulfate tablets, 3989 ointment, 4007
nofetumomab merpentan injection, oral suspension, 3988 and procaine hydrochlorides and
technetium, 3950 Terconazole, 3989 levonordefrin injection, 3445
oxidronate injection, technetium, 3950 Teriparatide, 3990 2,3,7,8-Tetrachlorodibenzo-p-dioxin, 3C-
pentetate injection, technetium, 3951 injection, 3995 labeled, 5736
pertechnetate injection, sodium, 3951 Terminally sterilized pharmaceutical 2,3,7,8-Tetrachlorodibenzofuran, '3C-labeled,
(pyro- and trimeta-) phosphates injection, products—parametric release (1222), 7638 5736
technetium, 3953 Terpin hydrate, 3998 1,1,2,2-Tetrachloroethane, 5736
pyrophosphate injection, technetium, 3953 and codeine oral solution, 3999 Tetracosane, 5736
red blood cells injection, technetium, 3954 oral solution, 3998 Tetracycline, 4013
sestamibi injection, technetium, 3955 tert-Butyl hydroperoxide solution, 5735 boluses, 4015
succimer injection, technetium, 3956 Tertiary butyl alcohol, 5666, 5677, 5735 hydrochloride, 4016
sulfur colloid injection, technetium, 3956 Test for 1,6-anhydro derivative for hydrochloride capsules, 4017
tetrofosmin injection, technetium, 3957 enoxaparin sodium (207), 6108 hydrochloride for injection, 4019
T-dodecyl mercaptan ethoxylate, 5693 Testolactone, 4000 hydrochloride, novobiocin sodium, and
Technetium tablets, 4001 prednisolone tablets, 4024
Tc 99m albumin aggregated injection, Testosterone, 4001 hydrochloride and novobiocin sodium
3937 benzoate, 5735 tablets, 4024
Tc 99m albumin colloid injection, 3938 cypionate, 4002 hydrochloride and nystatin capsules, 4025
Tc 99m albumin injection, 3936 cypionate injection, 4003 hydrochloride ointment, 4020
Tc 99m apcitide injection, 3940 enanthate, 4003 hydrochloride ophthalmic ointment, 4020
Te 99m arcitumomab injection, 3940 enanthate injection, 4004 hydrochloride ophthalmic suspension,
Tc 99m bicisate injection, 3941 injectable suspension, 4002 4022
Tc 99m depreotide injection, 3941 propionate, 4005 hydrochloride soluble powder, 4021
Te 99m disofenin injection, 3942 propionate injection, 4005 hydrochloride for topical solution, 4021
Tc 99m etidronate injection, 3943 Test papers hydrochloride oral suspension, 4022
Tc 99m exametazime injection, 3943 and indicator, 5747 hydrochloride tablets, 4023
Tc 99m fanolesomab injection, 3944 indicators and indicator, 5745 oral suspension, 4015
Tc 99m gluceptate injection, 3945 Test solutions, 5750 Tetradecane, 5736
Tc 99m lidofenin injection, 3946 Tetanus Tetradecylammonium bromide, 5736
Tc 99m mebrofenin injection, 3947 immune globulin, 4005 Tetraethylammonium chloride, 5736
Tc 99m medronate injection, 3948 2’A4’,5',7’-Tetrabromofluorescein, 5735 Tetraethylammonium perchlorate, 5736
Tc 99m mertiatide injection, 3949 Tetrabromophenolphthalein ethyl ester, 5735 Tetraethylene glycol, 5736
Tc 99m nofetumomab merpentan TS, 5761 Tetraethylenepentamine, 5736
injection, 3950 Tetraheptylammonium bromide, 5736
|-68 Tetra-Tolme Combined Index to USP 41 and NF 36

Tetrahexylammonium hydrogen sulfate, 5736 2-Thiobarbituric acid, 5738 Timolol maleate

Tetrahydrofuran, 5736 2,2’-Thiodiethanol, 5738 and dorzolamide hydrochloride ophthalmic
peroxide-free, 5736 Thioglycolic acid, 5738 solution, 1397
stabilizer-free, 5736 Thioguanine, 4059 Tin, 5738
Tetrahydro-2-furancarboxylic acid, 5737 tablets, 4060
N-(2-Tetrahydrofuroyl)piperazine, 5737 Thionine acetate, 5738
1,2,3,4-Tetrahydronaphthalene, 5737 Thiopental sodium, 4061
Tetrahydrozoline hydrochloride, 4026
nasal solution, 4027
for injection, 4062
Thioridazine, 4063
Tincture
ophthalmic solution, 4027 hydrochloride, 4064 Belladonna, 460
Tetramethylammonium hydrochloride oral solution, 4064 Benzethonium chloride, 468
bromide, 5737 hydrochloride tablets, 4065 Benzoin, compound, 488
bromide, tenth-molar (0.1 M), 5773 oral suspension, 4063 Capsicum, 676
chloride, 5737 Thiostrepton, 4066 Cardamom, compound, 5269
chloride, tenth-molar (0.1 M), 5773 nystatin, neomycin sulfate, and Ginger, 4654
hydroxide, 5737 triamcinolone acetonide cream, 2992 Green soap, 1996
hydroxide, pentahydrate, 5737 nystatin, neomycin sulfate, and lodine, 2188
hydroxide solution in methanol, 5737 triamcinolone acetonide ointment, 2993 lodine, strong, 2189
hydroxide TS, 5761 Thiotepa, 4066 Lemon, 5422
nitrate, 5737 for injection, 4067 Opium, 3039
Tetramethylbenzidine, 5737 Thiothixene, 4068 Orange peel, sweet, 5476
4,4’-Tetramethyldiaminodiphenylmethane, capsules, 4069 Rhodiola rosea, 4830
5737 hydrochloride, 4070 Thimerosal, 4058
Tetramethylsilane, 5737 hydrochloride injection, 4070 Tolu balsam, 5640
Tetrapropylammonium hydrochloride for injection, 4071 Valerian, 4929
chloride, 5737 hydrochloride oral solution, 4071 Vanilla, 5648
Tetrasodium ethylenediaminetetraacetate, Thiourea, 5738
5737 Thorium nitrate, 5738
Thalidomide, 4028 TS, 5761
capsules, 4029 Threonine, 4072 Tinidazole, 4101
Thallous chloride, 5737 Thrombin human, 5738 Tioconazole, 4102
TI 201 injection, 4030 Thromboplastin, 5738 Tissue human amnion chorion membrane
Theobromine, 5737 Thymidine, 5738 dehydrated, 4104
Theophylline, 4031 Thymol, 5639, 5738 Titanium
capsules, 4032 blue, 5746 dioxide, 4105
extended-release capsules, 4033 blue TS, 5761 tetrachloride, 5738
in dextrose injection, 4039 Thymolphthalein, 5746 trichloride, 5738
ephedrine hydrochloride, and TS, 5761 trichloride-sulfuric acid TS, 5761
phenobarbital tablets, 4041 Thyroglobulin, 5738 trichloride, tenth-normal (0.1 N), 5773
and guaifenesin capsules, 4042 Thyroid, 4073 trichloride TS, 5761
and guaifenesin oral solution, 4043 tablets, 4074 Titration, nitrite (451), 6218
sodium glycinate, 4043 Tiagabine hydrochloride, 4075 Titrimetry (541), 6268
sodium glycinate oral solution, 4044 oral suspension, 4077 Tizanidine
sodium glycinate tablets, 4044 Tiamulin, 4078 hydrochloride, 4106
oral solution, 4036 fumarate, 4079 tablets, 4107
oral suspension, 4037 Ticarcillin TI 201
tablets, 4038 and clavulanic acid injection, 4081 injection, thallous chloride, 4030
Theory and practice of electrical conductivity and clavulanic acid for injection, 4082 Tobramycin, 4109
measurements of solutions (1644), 7890 disodium, 4083 and dexamethasone ophthalmic ointment,
Thermal analysis (891), 6669 for injection, 4080 4117
Thiabendazole, 4045 monosodium, 4085 and dexamethasone ophthalmic
chewable tablets, 4046 Ticlopidine hydrochloride, 4086 suspension, 4119
oral suspension, 4046 tablets, 4088 and fluorometholone acetate ophthalmic
Thiamine Tienchi ginseng root and rhizome, 4901 suspension, 4121
hydrochloride, 4047 dry extract capsules, 4910 inhalation solution, 4114
hydrochloride injection, 4048 powder capsules, 4905 injection, 4111
hydrochloride oral solution, 4048 dry extract, 4909 for injection, 4112
hydrochloride tablets, 4049 powder, 4903 ophthalmic ointment, 4113
mononitrate, 4050 dry extract tablets, 4912 ophthalmic solution, 4116
mononitrate oral solution, 4051 powder tablets, 4907 sulfate, 4122
Thiamine assay (531), 6260 Tigecycline, 4089 Tocainide hydrochloride, 4124
Thiazole yellow, 5737 for injection, 4091 tablets, 4124
paper, 5747 Tiletamine Tocopherols excipient, 5639
Thiethylperazine maleate, 4052 hydrochloride, 4092 Tolazamide, 4125
suppositories, 4052 and zolazepam for injection, 4093 tablets, 4126
tablets, 4054 Tilmicosin, 4094 Tolazoline hydrochloride, 4127
Thimerosal, 4054 injection, 4095 injection, 4127
topical aerosol, 4056 Timolol Tolbutamide, 4128
topical solution, 4057 maleate, 4096 for injection, 4128
maleate and hydrochlorothiazide tablets, tablets, 4129
tincture, 4058
Thin-layer chromatographic identification test 4100 Tolcapone, 4129
maleate ophthalmic solution, 4098 tablets, 4130
(201), 6102
maleate tablets, 4099 o-Tolidine, 5738
Thioacetamide, 5738
Tolmetin sodium, 4132
TS, 5761
capsules, 4132
Thioacetamide-glycerin base TS, 5761
Combined Index to USP 41 and NF 36 Tolme-Triet 1-69

Tolmetin sodium (continued) Mafenide acetate for, 2494 Trazodone hydrochloride, 4176
tablets, 4133 Methoxsalen, 2656 tablets, 4178
Tolnaftate, 4134 Minoxidil, 2762 Trehalose, 5641
topical aerosol, 4135 Mometasone furoate, 2790 Trenbolone acetate, 4179
cream, 4135 Myrrh, 2841 Tretinoin, 4180
gel, 4135 Nitrofurazone, 2955 cream, 4181
topical powder, 4136 Nitromersol, 2960 gel, 4182
topical solution, 4136 Papain tablets for, 3161 topical solution, 4182
Tolterodine tartrate, 4136 Phenol, camphorated, 3264 Triacetin, 4183
Tolualdehyde, 5738 Podophyilum resin, 3341 n-Triacontane, 5739
p-Tolualdehyde, 5738 Povidone-iodine, 3393 Triamcinolone, 4184
Tolu balsam, 4138 Sodium fluoride and acidulated phosphate, acetonide, 4185
syrup, 5640 3791 acetonide cream, 4187
tincture, 5640 Sodium hypochlorite, 3794 acetonide dental paste, 4188
Toluene, 5739 Tetracaine hydrochloride, 4012 acetonide injectable suspension, 4192
p-Toluenesulfonic acid, 5739 Tetracycline hydrochloride for, 4021 acetonide topical aerosol, 4186
TS, 5761 Thimerosal, 4057 acetonide lotion, 4188
p-Toluenesulfonyl-t-arginine methyl ester Tolnaftate, 4136 acetonide and neomycin sulfate cream,
hydrochloride, 5739 Tretinoin, 4182 2907
p-Toluic acid, 5739 acetonide and nystatin cream, 2994
Toluidine acetonide, nystatin, neomycin sulfate, and
blue, 5739 gramicidin cream, 2991
blue O, 5739
o-Toluidine, 5739 Topical suspension acetonide, nystatin, neomycin sulfate, and
gramicidin ointment, 2992
p-Toluidine, 5739 Calamine, 615 acetonide, nystatin, neomycin sulfate and
Tomato extract containing lycopene, 4748 Calamine, phenolated, 616 thiostrepton cream, 2992
Topical aerosols (603), 6354 Ciclopirox olamine, 928 acetonide, nystatin, neomycin sulfate, and
Topical and transdermal drug products— Clindamycin phosphate, 999 thiostrepton ointment, 2993
product quality tests (3), 5926 Penicillin G, neomycin, polymyxin B, acetonide and nystatin ointment, 2994
hydrocortisone acetate, and acetonide ointment, 4188
hydrocortisone sodium succinate, 3193 acetonide nasal spray, 4188
Penicillin G procaine, neomycin and diacetate, 4193
polymyxin B sulfates, and hydrocortisone
Topical solution acetate, 3210
diacetate injectable suspension, 4194
diacetate oral solution, 4193
Aluminum acetate, 163 Resorcinol and sulfur, 3595 hexacetonide, 4194
Aluminum subacetate, 175 Selenium sulfide, 3740 hexacetonide injectable suspension, 4195
Aluminum sulfate and calcium acetate for, Sulfacetamide sodium, 3856 tablets, 4185
176 Zinc sulfide, 4388 2,4,6-Triamino-5-nitrosopyrimidine, 5739
Aluminum sulfate and calcium acetate Triamterene, 4196
tablets for, 177 capsules, 4197
Aminobenzoic acid, 217 and hydrochlorothiazide capsules, 4198
Benzethonium chloride, 467 Topiramate, 4139 and hydrochlorothiazide tablets, 4200
Benzocaine, 478 capsules, 4141 Triazolam, 4201, 5739
Benzocaine, butamben, and tetracaine tablets, 4143 tablets, 4202
hydrochloride, 483 Topiramate compounded Tribasic calcium phosphate, 5237
Calcium hydroxide, 647 oral suspension, 4146 Tribasic sodium phosphate, 5574
Carbamide peroxide, 690 Torsemide, 4146 Tributyl
Carbol-fuchsin, 704 tablets, 4147 citrate, 5643
Cetylpyridinium chloride, 858 Tosylchloramide sodium, 5739 phosphate, 5739
Chlorhexidine acetate, 880 Total organic carbon (643), 6377 Tributylethylammonium hydroxide, 5739
Chlorhexidine gluconate, 884 Tragacanth, 5641 Tributyrin, 5739
Ciclopirox, 926 Tramadol hydrochloride, 4149 Trichlormethiazide, 4203
Clindamycin phosphate, 999 and acetaminophen oral suspension, 4157 tablets, 4204
Clobetasol propionate, 1008 and acetaminophen tablets, 4158 Trichloroacetic acid, 5739
Clotrimazole, 1044 oral suspension, 4150 Trichloroethane, 5739
Coal tar, 1055 tablets, 4151 2,2,2-Trichloroethanol, 5739
Cocaine hydrochloride tablets for, 1058 extended-release tablets, 4153 Trichlorofluoromethane, 5739
Cocaine and tetracaine hydrochlorides and Tramadol hydrochloride compounded, Trichloromonofluoromethane, 5643
epinephrine, 1059 veterinary Trichlorotrifluoroethane, 5739
Diethyltoluamide, 1282 oral suspension, 4160 Tricitrates oral solution, 4205
Dimethyl sulfoxide, 1318 Trandolapril, 4160 Triclocarban, 4206
Dyclonine hydrochloride, 1458 tablets, 4161 Triclosan, 4208
Erythromycin, 1571 Trandolapril and verapamil hydrochloride n-Tricosane, 5739
Fluocinolone acetonide, 1785 extended-release tablets, 4163 Trientine hydrochloride, 4210
Fluocinonide, 1788 Tranexamic acid, 4169 capsules, 4211
Fluorouracil, 1803 Transdermal system Triethanolamine, 5740
Gentamicin sulfate and betamethasone clonidine, 1028 Triethylamine, 5740
valerate, 1941 nicotine, 2930 hydrochloride, 5740
Gentian violet, 1945 Transfer of analytical procedures (1224), phosphate, 5740
Halcinonide, 2018 7663 Triethylammonium acetate
Hydrogen peroxide, 2076 Tranylcypromine 1M, 5740
Hydroquinone, 2082 sulfate, 4171 Triethyl citrate, 5644
lodine, 2187 tablets, 4170 Triethylenediamine, 5740
Ivermectin, 2296 Travoprost, 4173 Triethylene glycol, 5740
Lidocaine hydrochloride, 2415 ophthalmic solution, 4174
|-70 Trifl-Valsa Combined Index to USP 41 and NF 36

Trifluoperazine Triprolidine zinc, TS, 5761

hydrochloride, 4212 hydrochloride, 4231 Urea, 4255, 5742
hydrochloride injection, 4212 hydrochloride oral solution, 4233 C 13, 705
hydrochloride tablets, 4213 hydrochloride tablets, 4233 C 13 for oral solution, 706
oral solution, 4211 and pseudoephedrine hydrochlorides oral C 14 capsules, 707
Trifluoroacetic solution, 4234 for injection, 4256
acid, 5740 and pseudoephedrine hydrochlorides Urethane, 5742
anhydride, 5740 tablets, 4235 Uridine, 5742
Trifluoroacetic acid (TFA) in peptides (503.1), Tris(2-aminoethyl)amine, 5741 Ursodiol, 4256
6247 Tris(hydroxymethyl)aminomethane, 5741 capsules, 4257
0.1% Trifluoroacetic acid TS, 5761 acetate, 5741 oral suspension, 4258
2,2,2-Trifluoroethanol, 5740 hydrochloride, 5741 tablets, 4259
2,2,2-Trifluoroethyldifluoromethy! ether, N-Tris(hydroxymethyl)methylglycine, 5741 USP and NF excipients listed by category,
5740 Trisulfapyrimidines 5169
(m-Trifluoromethylphenyl) oral suspension, 4235 USP policies, xxix
trimethylammonium hydroxide in tablets, 4236 USP reference standards (11), 5951
methanol, 5740 Tritirachium album proteinase K, 5742
5-(Trifluoromethyluracil, 5740 Trolamine, 5647
a,a,0.-Trifluoro-p-cresol, 5740 salicylate, 4236
Trifluorovinyl chloride polymer, 5741 Tromethamine, 4237, 5742
Triflupromazine, 4214
hydrochloride, 4215
carboprost, 710
carboprost, injection, 711
Vv
hydrochloride injection, 4215 for injection, 4238
hydrochloride tablets, 4216 Tropaeolin OO, 5742
oral suspension, 4214 Tropic acid, 5742
Trifluridine, 4216 Tropicamide, 4238
Triglycerides medium-chain, 5645
Trihexyphenidyl hydrochloride, 4217
ophthalmic solution, 4240
Tropine, 5742
Vaccine
extended-release capsules, 4218 Trospium chloride, 4241 Anthrax adsorbed, 321
oral solution, 4219 tablets, 4242 BCG, 455
tablets, 4219 Trypan blue, 5742
Trikates oral solution, 4221 Trypsin, crystallized, 4244
Triketohydrindene hydrate Tryptone, 5742
Vaccines for human use
TS, 5757, 5761 Tryptophan, 4245
bacterial vaccines (1238), 7833
Trimeprazine 5-Hydroxy-L-, 4914
general considerations (1235), 7795
oral solution, 4222 L-Tryptophane, 5742
polysaccharide and glycoconjugate
tartrate, 4221 Tuberculin purified protein derivative
vaccines (1234), 7778
tartrate tablets, 4223 (Tuberculin PPD), 5742
Vaccinia immune globulin, 4261
Trimethobenzamide hydrochloride, 4223 Tubocurarine chloride, 4246, 5742
Valacyclovir
capsules, 4224 injection, 4247
oral suspension, 4261
injection, 4224 Tungstic acid, 5742
tablets, 4261
Trimethoprim, 4225 Turmeric, 4915
Valacyclovir hydrochloride, 4263
and polymyxin B sulfate ophthalmic powdered, 4917
Valerian, 4924
solution, 3351 extract, powdered, 4918
extract, powdered, 4927
and sulfamethoxazole injection, 3874 Turmeric paper, 5747
powdered, 4926
and sulfamethoxazole oral suspension, Tylosin, 4247 tablets, 4930
3875 granulated, 4248
tincture, 4929
and sulfamethoxazole tablets, 3877 injection, 4249
Valeric acid, 5742
sulfate, 4226 tartrate, 4249
Valerophenone, 5742
tablets, 4226 Tyloxapol, 4250
Valganciclovir
Trimethylacethydrazide ammonium chloride, Tyrosine, 4252
hydrochloride, 4267
5697, 5741 L-Tyrosine disodium, 5742
tablets, 4266
Trimethylchlorosilane, 5741 Tyrosol, 5742
Validation
2,2,4-Trimethylpentane, 5702, 5741 Tyrothricin, 4252
of alternative microbiological methods
2,4,6-Trimethylpyridine, 5741
N-(Trimethylsilyl)-imidazole, 5741
(1223), 7642
of compendial procedures (1225), 7665
Trimethyitin bromide, 5741
of microbial recovery from pharmacopeial
Trimipramine maleate, 4227
articles (1227), 7672
2,4,6-Trinitrobenzenesulfonic acid, 5741
Trinitrophenol, 5741
U Validation of alternative methods to
antibiotic microbial assays (1223.1), 7656
TS, 5758, 5761
Ubidecarenone, 4919 Valine, 4270
Trioctylphosphine oxide, 5741
capsules, 4920 Valproate sodium
Trioxsalen, 4228
tablets, 4921 injection, 4271
tablets, 4229
Ubiquinol, 4922 Valproic acid, 4272
Tripelennamine hydrochloride, 4229
capsules, 4923 capsules, 4273
injection, 4230
Ultraviolet-visible spectroscopy (857), 6660 oral solution, 4273
tablets, 4231
Ultraviolet-visible spectroscopy—theory and Valrubicin, 4274
1,3,5-Triphenylbenzene, 5741
practice (1857), 8136 intravesical solution, 4276
Triphenylene, 5741
Undecylenic acid, 4254 Valsartan, 4276
Triphenylmethane, 5741 tablets, 4278
Triphenylmethanol, 5741 ointment, compound, 4254
Uniformity of dosage units (905), 6673 amlodipine, and hydrochlorothiazide
Triphenyltetrazolium tablets, 257
chloride, 5741 Uracil, 5742
Uranyl acetate, 5742 and amlodipine tablets, 253
chloride TS, 5761 and hydrochlorothiazide tablets, 4279
cobalt, TS, 5752
Combined Index to USP 41 and NF 36 Vanad-Water \-71

Vanadium pentoxide, 5742 Vibrational circular dichroism Volumetric

Vanadyl! sulfate, 5743 spectroscopytheory and practice (1782), apparatus (31), 5957
Vancomycin, 4282 8025 solutions, 5761
hydrochloride, 4284 Vigabatrin, 4313 Voriconazole, 4336
hydrochloride capsules, 4286 for oral solution, 4315 Voriconazole compounded, veterinary
hydrochloride for injection, 4286 tablets, 4316 ophthalmic solution, 4338
hydrochloride for oral solution, 4287 Vinblastine sulfate, 4318
injection, 4283 for injection, 4319
Vanilla, 5647 Vincristine sulfate, 4321
tincture, 5648 injection, 4322
Vanillin, 5648
Vapor phase sterilization (1229.11), 7733
for injection, 4323
Vinorelbine
WwW
Varicella-zoster immune globulin, 4289 injection, 4326
Vasopressin, 4289 tartrate, 4325 Warfarin sodium, 4340
injection, 4290 Vinpocetine, 4931 for injection, 4342
Vecuronium bromide, 4290 capsules, 4933 tablets, 4343
Vegetable oil, hydrogenated, 5649 tablets, 4933 Washed sand, 5743
Vehicle Vinyl acetate, 5743
for oral solution, 5474 2-Vinylpyridine, 5743
for oral solution, sugar free, 5474 Vinylpyrrolidinone, 5743
for oral suspension, 5474 Viral safety evaluation of biotechnology Water
suspension structured, 5637 products derived from cell lines of human
or animal origin (1050), 6935 Water, 5743
suspension structured, sugar-free, 5637
Venlafaxine Virology test methods (1237), 7812 ammonia, stronger, 5668, 5733, 5751
hydrochloride, 4293 Virus testing of human plasma for further ammonia, 25 percent, 5668
hydrochloride extended-release capsules, manufacture (1240), 7846 ammonia-free, 5743
4295 Viscosity—capillary methods (911), 6677 carbon dioxide-free, 5743
tablets, 4292 Viscosity—pressure driven methods (914), cetyltrimethylammonium chloride, 25
Verapamil hydrochloride, 4301 6686 percent in, 5682
extended-release capsules, 4302 Viscosity—rolling ball method (913), 6684 conductivity (645), 6378
injection, 4304 Viscosity—rotational methods (912), 6679 deaerated, 5743
oral solution, 4305 Visible particulates in injections (790), 6542 determination (921), 6687
oral suspension, 4305 Visual inspection of injections, 8066 deuterated, 5686
D-Gluconic acid, 50 percent in, 5698
tablets, 4306 Vitamin
extended-release tablets, 4307 A, 4327 for hemodialysis, 4345
Vardenafil A assay (571), 6307 for hemodialysis applications (1230), 7741
hydrochloride, 4287 A capsules, 4328 hydrazine hydrate, 85% in, 5699
for inhalation, sterile, 4346
Verification of compendial procedures A oral liquid preparation, 4329
(1226), 7671 A tablets, 4330 for injection, 4345
Verteporfin, 4311 Biz activity assay (171), 6091 for injection, bacteriostatic, 4346
for injection, 4312 C assay (580), 6313 for injection, sterile, 4346
C and zinc lozenges, 5161 for irrigation, sterile, 4347
D assay (581), 6315 methylamine, 40 percent in, 5708
D and calcium with minerals tablets, 4502 organic-free, 5743
particle-free, 5743
Veterinary D with calcium tablets, 4501
E, 4331 peppermint, 5483
Atenolol compounded oral suspension, E assay (551), 6272 for pharmaceutical purposes (1231), 7742
386 E capsules, 4333 pure steam, 4348
Benazepril hydrochloride compounded oral E polyethylene glycol succinate, 5649 purified, 4347
suspension, 463 E preparation, 4335 purified, sterile, 4348
Buprenorphine compounded buccal Vitamins rose, ointment, 3680
solution, 570 capsules, oil-soluble, 4935 rose, stronger, 5555
Doxycycline compounded oral suspension, capsules, oil- and water-soluble, 4976 solid interactions in pharmaceutical
veterinary, 1427 capsules, water-soluble, 5086 systems (1241), 7856
Enalapril maleate compounded oral with minerals capsules, oil- and water- soluble vitamins capsules, 5086
suspension, 1501 soluble, 5022 soluble vitamins with minerals capsules,
Methylene blue injection, 2675 with minerals capsules, water-soluble, 5109
Pergolide oral suspension, 3238 5109 soluble vitamins with minerals oral
Potassium bromide oral solution, 3359 solution, 5128
with minerals oral solution, oil- and water-
Prednisolone compounded oral suspension, soluble, 5047 soluble vitamins with minerals tablets,
3415 with minerals oral solution, water-soluble, 5137
Sodium bromide injection, 3780 5128 soluble vitamins tablets, 5098
Sodium bromide oral solution, 3780 with minerals tablets, oil- and water- Stronger ammonia, 5733
Spironolactone compounded oral soluble, 5061 vapor detector tube, 5743
suspension, 3827 with minerals tablets, water-soluble, 5137 vitamins capsules, and oil-soluble, 4976
Tramadol hydrochloride compounded oral with minerals capsules, oil-soluble, 4951 vitamins with minerals capsules, and oil-
suspension, 4160 with minerals oral solution, oil-soluble, soluble, 5022
Voriconazole compounded ophthalmic 4961 vitamins with minerals oral solution, and
solution, 4338 with minerals tablets, oil-soluble, 4966 oil-soluble, 5047
oral solution, oil-soluble, 4941 vitamins with minerals tablets, and oil-
oral solution, oil- and water-soluble, 4995 soluble, 5061
tablets, oil-soluble, 4944 vitamins oral solution, and oil-soluble,
Vibrational circular dichroism spectroscopy tablets, oil- and water-soluble, 5004 4995
(782), 6520 tablets, water-soluble, 5098 vitamins tablets, and oil-soluble, 5004
|-72 Water-Zonis Combined Index to USP 41 and NF 36

Zaleplon, 4362
capsules, 4364
Wax Zanamivir, 4366
carnauba, 5651 meso-Zeaxanthin, 5155
emulsifying, 5651 preparation, 5157
microcrystalline, 5651 Zein, 5656
white, 5652 Zidovudine, 4367
yellow, 5653 capsules, 4368
Weighing on an analytical balance (1251), injection, 4369
7860 and lamivudine tablets, 2331
Weight variation of dietary supplements oral solution, 4370
(2091), 8185 tablets, 4372
Wheat Zileuton, 4373
bran, 4348 Zinc, 5744
starch, 5617 acetate, 4375, 5744
Witch hazel, 4349 acetate oral solution, 4376
Wound matrix small intestinal submucosa, activated, 5744
3721 amalgam, 5744
Wright's stain, 5743 carbonate, 4376
Written prescription drug information— chloride, 4377
guidelines (1265), 7866 chloride, anhydrous, powdered, 5744
chloride injection, 4378
citrate, 5159
citrate tablets, 5159
determination (591), 6325
xX gluconate, 4379
gluconate tablets, 4380
oxide, 4381
Xanthan gum, 5653 oxide neutral, 4382
solution, 5654 oxide ointment, 4383
Xanthine, 5743
oxide paste, 4384
Xanthydrol, 5744 oxide and salicylic acid paste, 4384
Xenon Xe 127, 4351 stearate, 4385
Xenon Xe 133, 4351 sulfate, 4385
injection, 4351 sulfate heptahydrate, 5744
X-ray fluorescence spectrometry (735), 6486 sulfate injection, 4386
X-ray fluorescence spectrometry—theory and sulfate ophthalmic solution, 4387
practice (1735), 7963 sulfate oral solution, 4387
Xylazine, 4352 sulfate tablets, 4388
hydrochloride, 4353 sulfate, twentieth-molar (0.05 M), 5773
injection, 4354 sulfide topical suspension, 4388
Xylene, 5744 undecylenate, 4389
m-Xylene, 5744 uranyl acetate TS, 5761
o-Xylene, 5744 and vitamin C lozenges, 5161
p-Xylene, 5744 Zinc oxide
Xylene cyanole FF, 5744 powder, 4384
Xylenol orange, 5746 Zinc sulfate
TS, 5761 0.1 M VS, 5774
Xylitol, 5655 Ziprasidone
Xylometazoline hydrochloride, 4355, 5744 capsules, 4389
nasal solution, 4355 Ziprasidone hydrochloride, 4391
Xylose, 4356, 5744 Zirconyl
nitrate, 5744
Zolazepam
hydrochloride, 4394
and tiletamine for injection, 4093
Y Zolmitriptan, 4395
nasal spray, 4397
tablets, 4399
Yeast extract, 5744
Yellow mercuric oxide, 5744 orally disintegrating tablets, 4400
Yohimbine Zolpidem tartrate, 4402
tablets, 4403
hydrochloride, 4358
injection, 4358 extended-release tablets, 4405
Yttrium Y 90 ibritumomab tiuxetan Zonisamide, 4409
capsules, 4410
injection, 4359
Zonisamide compounded
oral suspension, 4412

Zz
Zalcitabine, 4361
tablets, 4362
 4524 Chaste Tree / Dietary Supplements USP 41

ru = peak response of casticin from the Sample tu = peak response of agnuside from the Sample
solution solution
rs = peak response of casticin from the Standard Is = peak response of agnuside from the Standard
solution solution
Cs = concentration of USP Casticin RS in the Cs = concentration of USP Agnuside RS in the
Standard solution (mg/mL) Standard solution (mg/mL)
Cu = concentration of Powdered Chaste Tree in the Cu = concentration of Powdered Chaste Tree in the
Sample solution (mg/mL) Sample solution (mg/mL)
Acceptance criteria: NLT 0.08% of casticin on the Acceptance criteria: NLT 0.05% of agnuside on the
dried basis dried basis
© CONTENT OF AGNUSIDE
Solvent: Methanol and water (1:19) CONTAMINANTS
Standard solution: Dissolve a quantity of USP Agnuside © ARTICLES OF BOTANICAL ORIGIN, Limits of Elemental Impuri-
RS in Solvent, with sonication. Dilute with methanol to ties (561): Meets the requirements
obtain a concentration of about 0.125 mg/mL. Pass e ARTICLES OF BOTANICAL ORIGIN, Pesticide Residue Analysis
through a cellulose membrane filter of 0.45-1m or finer (561): Meets the requirements
pore size. © MICROBIAL ENUMERATION TESTS (2021): The total aerobic
Sample solution: Place about 1000 mg of Powdered microbial count does not exceed 105 cfu/g, the total
Chaste Tree in a container with a stopper. Extract twice combined molds and yeast count does not exceed 103
with 40 mL of methanol, using a hana homogenizer at cfu/g, and the bile-tolerant Gram-negative bacteria count
19,000 rpm for 2 min. Centrifuge, and transfer each does not exceed 103 cfu/g.
supernatant to a 250-mL round-bottom flask. Rinse the e ABSENCE OF SPECIFIED MICROORGANISMS (2022): Meets the
residue with methanol, and filter the resulting solution requirements of the tests for the absence of Salmonella
into the flask. Evaporate the combined extract to dry- species and Escherichia coli
ness, and dissolve the residue in 2 mL of Solvent. Quan-
titatively transfer the solution to a solid-phase extraction SPECIFIC TESTS
cartridge packed with neutral aluminum oxide previ- ¢ BOTANICAL CHARACTERISTICS: Powdered Chaste Tree is
ously conditioned with 5 mL of Solvent. Connect the dark brown, with a musty, slightly aromatic odor, and a
cartridge to a vacuum pressure not exceeding 300 taste resembling that of sage. The following characteris-
mbar, and collect the eluate. Rinse the round-bottom
tics are present: fragments of the calyx with covering and
flask with 2 mL of Solvent, pass this solution through glandular trichomes on the outer side and rectangular,
the cartridge, apply the vacuum, and collect the eluate. elongated cells with slightly wavy walls on the inner side;
Rinse the cartridge with 4 mL of Solvent, and collect the fragments of exocarp with trichomes and cells with large
eluate. Combine the eluates from the cartridge, transfer pits in the outer wall; thin-walled parenchymatous cells
to a 10-mL volumetric flask, and dilute with Solvent to and globules of fixed oil; stone-pitted cells from the mes-
volume. ocarp; ovoid, lignified cells with bands of reticulate thick-
Solution A: Acetonitrile ening from the testa; and endosperm and cotyledons
Solution B: 5.88 g/L of phosphoric acid in water with fixed oil.
e Loss ON DRYING (731)
Mobile phase: See Table 2.
Sample: 1.0g of Powdered Chaste Tree
Analysis: Dry the Sample at 105° for 2 h.
Table 2 Acceptance criteria: NMT 10.0%
Time Solution A Solution B e ARTICLES OF BOTANICAL ORIGIN, Total Ash (561): NMT
(min) (%) (%) 8.0%
0 Z 93
e ARTICLES OF BOTANICAL ORIGIN, Acid-Insoluble Ash (561):
NMT 2.0%
0.6 10 90
5 10 90 ADDITIONAL REQUIREMENTS
Z 14 86 e PACKAGING AND STORAGE: Preserve in well-closed contain-
13 15 85 ers, and store at controlled room temperature.
e LABELING: The label states the Latin binomial and, follow-
1341 100 0
ing the official name, the part of the plant from which
DS Monographs

18 100 0 the article was derived.

18.1 Z 93 e USP REFERENCE STANDARDS (11)
23 7 93 USP Agnuside RS
USP Casticin RS
Chromatographic system USP Powdered Chaste Tree Extract RS
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 258 nm
Column: 3.1-mm x 12.5-cm; 5-m packing L1
Column temperature: 25 Powdered Chaste Tree Extract
Flow rate: 1.3 mL/min
Injection volume: 10 pL
System suitability DEFINITION
Sample: Standard solution Powdered Chaste Tree Extract is prepared from Chaste Tree
Suitability requirements by extraction with hydroalcoholic mixtures or other suita-
Tailing factor: NMT 2.0 for the agnuside peak ble solvents. It contains NLT 90.0% and NMT 110.0% of
Relative standard deviation: NMT 2.0% for the the labeled amount of casticin and agnuside, calculated
agnuside peak, in repeated injections on the dried basis. It may contain suitable added
substances.
Analysis
Samples: Standard solution and Sample solution IDENTIFICATION
Calculate the percentage of agnuside in the portion of © A. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST
Powdered Chaste Tree taken: Standard solution: 100mg of USP Powdered Chaste
Tree Extract RS in 1 mL of methanol. Heat in a water
Result = (ru/rs) x (Cs/Cu) x 100
USP 41 Dietary Supplements / Chaste Tree 4525

bath at 60° for 10 min. Centrifuge, and use the clear Chromatographic system
supernatant. (See Chromatography (621), System Suitability.)
Sample solution: Shake a quantity of Extract, equiva- Mode: LC
lent to about 10 mg of the labeled amount of agnuside, Detector: UV 348 nm
in 10 mL of methanol. Heat in a water bath at 60°. Column: 3.1-mm x 12.5-cm; 5-um packing L1
Centrifuge or filter before use. Column temperature: 25°
Adsorbent: Chromatographic silica gel with an average Flow rate: 1 mL/min
particle size of 10-15 um (TLC plates) Injection size: 10 wL
Application volume: 90 uL, Standard solution; 60 uL, System suitability
Sample Solution; in bands that are 2 cm in length Sample: Standard solution
Developing solvent system: Ethyl acetate, methanol, Suitability requirements
and water (77:15:8) Tailing factor: NMT 2.0 for the casticin peak
Spray reagent: 10 mg/mL of p-dimethylaminobenzal- Relative standard deviation: NMT 2.0% for the cas-
dehyde in 1 N hydrochloric acid ticin peak, in repeated injections
Analysis Analysis
Samples: Standard solution and Sample solution Samples: Standard solution and Sample solution
Develop the chromatograms to a length of NLT Calculate the percentage of casticin, Pc, in the portion
12cm, and dry the plate in a current of air. Spray the of Extract taken:
plate with Spray reagent, and heat for 10 min at
120°. = (tufts) x (Cs/Cu) x 100
Pc
Acceptance criteria: The Sample solution shows the fol-
lowing: a blue zone (at an Ry value of about 0.21) due tu = peak response of casticin from the Sample
to the presence of aucubin and that corresponds in solution
color and R; value to a similar zone for the Standard rs = peak response of casticin from the Standard
solution; a blue zone (at an Rr value of about 0.44) as a solution
result of the presence of agnuside that corresponds in Cs = concentration of USP Casticin RS in the
color and R; value to a similar zone for the Standard Standard solution (mg/mL)
solution; and one broad zone, violet in the middle, near Cy = concentration of Extract in the Sample solution
the solvent front and that corresponds in color and Rr (mg/ml)
value to a similar zone for the Standard solution. Other Calculate the percentage of the labeled amount of
colored zones of varying intensities may be observed in casticin in the portion of Extract taken:
the Sample solution.
e B. In the test for Content of Casticin, the chromatogram Result = (Pc/L) x 100
of the Sample solution exhibits a peak at the retention
time corresponding to casticin. Pc = content of casticin as calculated above (%)
e C. In the test for Content of Agnuside, the chromatogram L = labeled amount of casticin (%)
of the Sample solution exhibits a peak at the retention Acceptance criteria: 90.0%-110.0% on the dried basis
time corresponding to agnuside. © CONTENT OF AGNUSIDE
Solvent: Methanol and water (1:19)
COMPOSITION Standard solution: Dissolve a quantity of USP Agnuside
e CONTENT OF CASTICIN RS in Solvent, with sonication. Dilute with methanol to
Standard solution: About 0.05 mg/mL of USP Casticin obtain a concentration of about 0.125 mg/mL. Pass
RS in methanol, with sonication. Pass through a cellu- through a cellulose membrane filter of 0.45-l1m or finer
lose membrane filter of 0.45-um or finer pore size. pore size.
Sample solution: Transfer a quantity of Extract, equiva- Sample solution: Transfer an amount of Extract, equiv-
lent to about 2.5 mg of the labeled content of casticin, alent to about 6.25 mg of the labeled content of agnu-
into a 50-mL volumetric flask. Add 25 mL of methanol, side, into a 50-mL volumetric flask. Add 25 mL of Sol-
and sonicate in a bath at 40° for 10 min, shaking to vent, and sonicate in a bath at 40° for 10 min, shaking
disperse the solid. Cool to room temperature, and di- to disperse the solid. Cool to room temperature, and
lute with methanol to volume. Centrifuge or pass dilute with Solvent to volume. Centrifuge or pass
throughafilter of 0.45-11m or finer pore size. throughafilter of 0.45-um or finer pore size.
Solution A: Methanol Solution A: Acetonitrile is}
Solution B: 5.88 g/L of phosphoric acid in water Solution B: 5.88 g/L of phosphoric acid in water nd
Mobile phase: See Table 1. Mobile phase: See Table 2. =
°
Table 1 Table 2 z

Time Solution A Solution B Time Solution A Solution B =

(min) _(%) (%) (min) (%) (%) a
0 50 50 0 Z 93 ws
oO 50 50 0.6 10 90 ie
13 65 35 5 10 90
18 100 0 Z 14 86
23 50 50 13 15 85
13.1 100 0
18 100 0
18.1 Z 93.
23. 7 93
 4526 Chaste Tree / Dietary Supplements USP 41

Chromatographic system e USP REFERENCE STANDARDS (11)

(See Chromatography (621), System Suitability.) USP Agnuside RS
Mode: LC USP Casticin RS
Detector: UV 258 nm USP Powdered Chaste Tree Extract RS
Column: 3.1-mm x 12.5-cm; 5-um packing L1
Column temperature: 25°
Flow rate: 1.3 mL/min
Injection size: 10 pL
System suitability Horse Chestnut
Sample: Standard solution
Suitability requirements DEFINITION
Tailing factor: NMT 2.0 for the agnuside peak Horse Chestnut consists of the dried seeds of Aesculus hippo-
Relative standard deviation: NMT 2.0% for the castanum L. (Fam. Hippocastanaceae) harvested in the fall.
agnuside peak, in repeated injections It contains NLT 3.0% of triterpene glycosides, calculated
Analysis on the dried basis as escin (CssHseOoa).
Samples: Standard solution and Sample solution
Calculate the percentage of agnuside, Pa, in the por- IDENTIFICATION
tion of Extract taken: e A. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST
Standard solution: 5 mg/mL of USP Escin RS in
Pa = (rults) x (Cs/Cu) x 100 methanol
Sample solution: Transfer 1 g of the powdered plant
ty = peak response of agnuside from the Sample material to a screw-capped centrifuge tube, add 10 mL
solution of a mixture of alcohol and water (7:3), and heat on a
rs = peak response of agnuside from the Standard steam bath for 10 min. Centrifuge, and use the clear
solution supernatant.
Cs = concentration of USP Agnuside RS in the Chromatographic system
Standard solution (mg/mL) (See Chromatography (621), Thin-Layer Chromato-
G = concentration of Extract in the Sample solution graphy.)
(mg/mL) Adsorbent: 0.25-mm layer of chromatographic silica
Calculate the percentage of the labeled amount of gel (TLC plates)
agnuside in the portion of Extract taken: Application volume: 10 pL
Developing solvent system: Use the upper phase of a
Result = (Pa/L) x 100 mixture of 1-butanol, glacial acetic acia, and water
Pa = content of agnuside calculated above (%) (5:1:4).
L = labeled amount of agnuside (%) Derivatization reagent: Methanol, glacial acetic acid,
Acceptance criteria: 90.0%-110.0% on the dried basis sulfuric acid, and p-anisaldehyde (85: 10: 5: 0.5)
Analysis
CONTAMINANTS Samples: Standard solution and Sample solution
Develop the chromatograms to a length of NLT 15 cm,
and dry the plate in a stream of air. Spray the plate
Delete the following: with Derivatization reagent, heat at 100° for 5 min,
and examine under white light.
®e HEAVY METALS, Method |] (231): NMT 20 ppme coma 1- Acceptance criteria: The chromatogram of the Sample
jari-201
8) solution shows a blue-violet zone corresponding to es-
° Microstat ENUMERATION TESTS (2021): The total bacterial cin, comparable in position and color to the main zone
count does not exceed 104 cfu/g. The total combined in the chromatogram of the Standard solution. Above
molds and yeasts count does not exceed 103 cfu/g. this zone, the chromatogram of the Sample solution
e ABSENCE OF SPECIFIED MICROORGANISMS (2022): Meets the shows several narrow, brown to brownish-red zones
requirements of the tests for absence of Salmonella spe- that are less intense than the zone corresponding to
cies and Escherichia coli escin.
e OTHER REQUIREMENTS: It meets the requirements for Bo-
DS Monographs

tanical Extracts (565), Residual Solvents and Pesticide COMPOSITION

Residues.
SPECIFIC TESTS
e Loss ON DRYING (731): NMT 6.0%
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in tight containers,
and store in a cool place, protected from light.
e LABELING: The label states the Latin binomial and, follow-
ing the official name, the part of the plant from which
the article was prepared. The label also indicates the con-
tent of casticin and agnuside, the extracting solvent or
solvent mixture used tor preparation, the ratio of the
starting crude plant material to Extract, the percentage of
native extract, and the name and quantity of any added
substances. It meets the requirements for Botanical Ex-
tracts (565), Labeling.
e CONTENT OF TRITERPENE GLYCOSIDES
Solvent A: Methanol and water (13:7)
Solvent B: Use the lower phase of a mixture of chloro-
form, 0.1 N hydrochloric acid, and 1-propanol (5:3:2).
Reagent: Dissolve 75 mg of ferric chloride in 50 mL of
ice-cold glacial acetic acid. Add 50 mL of sulfuric acid,
while swirling on an ice bath. Prepare immediately
before use.
Standard solution A: 0.2 mg/mL of USP Escin RS in
glacial acetic acid, shaken for 1 min
Standard solution B: 0.4 mg/mL of USP Escin RS in
glacial acetic acid, shaken for 1 min
Standard solution C: 0.6 mg/mL of USP Escin RS in
glacial acetic acid, shaken for 1 min
Sample solution: Accurately weigh about 1 g of
ground seeds, and transfer into a 250-mL round-bot-
tom flask. Add exactly 100 mL of Solvent A, and weigh
the filled flask with a precision of +0.1 g. Attach a con-
denser, reflux for 30 min, and allow to cool. Adjust to
the initial weight by adding Solvent A, and filter. Trans-
fer 30.0 mL of the filtrate to a round-bottom flask, and
evaporate under vacuum. Dissolve the residue in 20 mL
of 0.1 N hydrochloric acid, and quantitatively transfer
USP 41
with the aid of two additional 5-mL portions of 0.1 N
hydrochloric acid to a 250-mL separatory funnel. Add
20 mL of 1-propanol and 50 mL of chloroform, and
shake vigorously for 2 min. Collect and retain the lower
chloroform layer, and add 50 mL of Solvent B to the
upper layer remaining in the separatory funnel. Shake
nee for 2 min; collect and retain the lower chlo-
roform layer. Combine the retained chloroform layers in
a round-bottom flask, and evaporate to near-dryness
under vacuum. Evaporate the remaining solvent under
a stream of air. Wash the residue with two 10-mL ali-
quots of ether, filter, wash the filter with 10 mL of
ether, and discard the ether filtrates. After evaporation
of the residual ether, suspend the residue in 10 mL of
glacial acetic acid, and pass through the previously
used dried filter into a 50-mL volumetric flask. Repeat
the addition of glacial acetic acid followed by filtration
two additional times, combining the filtrates in the
50-mL volumetric flask. Wash the round-bottom flask
with small quantities of glacial acetic acid, and filter
into the volumetric flask. Dilute with glacial acetic acid
to volume.
Instrumental conditions
(See Ultraviolet-Visible Spectroscopy (857).)
Mode: Visible
Wavelength: 540 nm
Blank: Glacial acetic acid
Analysis: Accurately transfer 1.0 mL each of Standard
solutions A, B, and C, Sample solution, and Blank into
separate screw-cap test tubes. Add 4.0 mL of Reagent to
each tube, cap the tubes, and keep them on a water
bath at 60° for 25 min, shaking occasionally. Measure
the absorbances of the reacted Sample solution and
Standard solutions A, B, and C, corrected for the Blank.
Plot the absorbances of Standard solutions A, B, and C
against their respective concentrations, and establish
the calibration line by linear regression. From the plot,
determine the concentration, C, in mg/mL, of triterpene
glycosides as escin in the Sample solution.
Calculate the percentage of triterpene glycosides as es-
cin in the portion of Horse Chestnut taken:
Result = (C/W) x (50/3)
¢ = concentration of triterpene glycosides in the
Sample solution as obtained above (mg/mL)
w = weight of Horse Chestnut taken to prepare the
Sample solution (g)
Acceptance criteria: NLT 3.0% of triterpene glycosides,
calculated as escin (CssHgsO24), on the dried basis
CONTAMINANTS
e ARTICLES OF BOTANICAL ORIGIN, Limits of Elemental Impuri-
ties (561): Meets the requirements
e ARTICLES OF BOTANICAL ORIGIN, Pesticide Residue Analysis
(561): Meets the requirements
¢ MICROBIAL ENUMERATION TESTS (2021): The total aerobic
microbial count does not exceed 10 cfu/g, the total
combined molds and yeast count does not exceed 104
cfu/g, and the bile-tolerant Gram-negative bacteria count
is NMT 103 cfu/g.
© ABSENCE OF SPECIFIED MICROORGANISMS (2022): It meets
the requirements of the tests for absence of Salmonella
species and Escherichia coli.
SPECIFIC TESTS
¢ BOTANICAL CHARACTERISTICS
Macroscopic: Horse chestnut seeds are dense and hard,
subspherical to oval, slightly flattened, and from 2 to
4cm in diameter. They have a dark brown seed coat
from 1 to 1.5 mm thick, with a large, round, light
brown spot (hilum). The seed coat is shiny, but only in
fresh condition. The space under the coat is totally filled
with the shiny, massive embryo and its large, pale yel-
low cotyledons lacking endosperm.
Dietary Supplements / Horse Chestnut 4527
Microscopic: The epidermis of the testa in surface view
has yellowish-brown cells of fairly uniform size, with the
majority of cells rounded to polygonal, and a few that
are square to obscurely triangular. The walls of these
cells are considerably but rather unevenly thickened,
and lack pits. In the sectional view, the cells are colum-
nar, eee 3-4 times as high as they are wide,
with the outer periclinal wall markedly thickened, une-
ven, and becoming thinner toward the base; beneath
the epidermis there are a few layers of small collenchy-
matously thickened cells with small intercellular spaces;
the greater part of the testa consists of larger, loosely
packed parenchymatous cells forming a spongy tissue;
the walls are variably and unevenly thickened, with in-
tercellular and large circular spaces well marked, the in-
ner layer of the testa is a narrow zone, with ill-defined
and thinner-walled cells. All the parenchymatous cells of
the testa are darkly pigmented. The embryo has an
outer layer of small colorless cells, almost square in sec-
tional view, with outer and side walls thickened. In the
surface view, only the irregular and more or less poly-
gonal lumens are discernible, giving a reticulate, pitted
appearance. Cotyledons are moderately thickened and
indistinctly pitted, having round to ovoid parenchyma-
tous cells densely filled with starch. Starch granules,
mainly simple, are present in two size ranges: from 15
to 30 um and from 3 to 10 um. The largest granules
vary from circular, ovoid, and bluntly polygonal to pyri-
form, most of them with a well-marked cleft or stellate
hilum, and lacking striations. The smaller starch gran-
ules are less variable, spherical to ovoid, with the hilum
more often a point. Compound starch granules are
found very infrequently.
EXTRACTABLE MATTER
Analysis: Proceed as directed for Articles of Botanical Or-
igin (S61), Alcohol-Soluble Extractives, Method 2, except
use a mixture of methanol and water (8:2) instead of
alcohol.
Acceptance criteria: NLT 18.0%
ARTICLES OF BOTANICAL ORIGIN, Foreign Organic Matter
(561): NMT 2.0%
Loss ON DrYING (731): Dry a sample at 105° for 2 h: it
loses NMT 10.0% of its weight.
ARTICLES OF BOTANICAL ORIGIN, Total Ash (561): NMT
4.0%
ADDITIONAL REQUIREMENTS
PACKAGING AND STORAGE: Preserve in a well-closed, light-
resistant container, protected from moisture.
LABELING: The label states the Latin binomial and, follow-
ing the official name, the part of the plant contained in
sydesbouo=: sa
the article.
USP REFERENCE STANDARDS (11)
USP Escin RS
Powdered Horse Chestnut
DEFINITION
Powdered Horse Chestnut is Horse Chestnut reduced to a
powder or very fine powder. It contains NLT 3.0% of
triterpene glycosides, calculated on the dried basis as es-
cin (CssHscO2a).
IDENTIFICATION
A. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST
Standard solution: 5 mg/mL of USP Escin RS in
methanol
Sample solution: Transfer 1 g of Powdered Horse
Chestnut to a screw-capped centrifuge tube, add 10 mL
of a mixture of alcohol and water (7:3), and heat on a
steam bath for 10 min. Centrifuge, and use the clear
supernatant.
 4528 Horse Chestnut / Dietary Supplements
Chromatographic system
(See Chromatography (621), Thin-Layer Chromato-
graphy.) ; Blank: Glacial acetic acid
Adsorbent: 0.25-mm layer of chromatographic silica
gel (TLC plates)
Application volume: 10 pL
Developing solvent system: Use the upper phase of a
mixture of 1-butanol, glacial acetic acid, and water
(5:1:4).
Derivatization reagent: Methanol, glacial acetic acid,
sulfuric acid, and p-anisaldehyde (85: 10: 5: 0.5)
Analysis
Samples: Standard solution and Sample solution
Develop the chromatograms to a length of NLT 15 cm,
and dry the plate in a current of air. Spray the plate
with Derivatization reagent, heat at 100° for 5 min,
and examine under white light.
Acceptance criteria: The chromatogram of the Sample
solution shows a blue-violet zone corresponding to es-
cin, comparable in position and color to the main zone
in the chromatogram of the Standard solution. Above
this zone, the chromatogram of the Sample solution
shows several narrow, brown to brownish-red zones
that are less intense than the zone corresponding to
escin.
COMPOSITION
@ CONTENT OF TRITERPENE GLYCOSIDES
Solvent A: Methanol and water (13:7)
Solvent B: Use the lower phase of a mixture of chloro-
form, 0.1 N hydrochloric acid, and 1-propanol (5:3:2).
Reagent: Dissolve 75 mg of ferric chloride in 50 mL of
ice-cold glacial acetic acid. Add 50 mL of sulfuric acid,
while swirling on an ice bath. Prepare immediately
before use.
Standard solution A: 0.2 mg/mL of USP Escin RS in
glacial acetic acid, shaken for 1 min
Standard solution B: 0.4 mg/mL of USP Escin RS in
glacial acetic acid, shaken for 1 min
Standard solution C: 0.6 mg/mL of USP Escin RS in
glacial acetic acid, shaken for 1 min
Sample solution: Accurately weigh about 1 g of Pow-
dered Horse Chestnut, and transfer into a 250-mL
round-bottom flask. Add exactly 100 mL of Solvent A,
and weigh the filled flask with a precision of +0.1 g.
Attach a condenser, reflux for 30 min, and allow to
cool. Adjust to the initial veigtt by adding Solvent A,
and filter. Transfer 30.0 mL of the filtrate to a round-
bottom flask, and evaporate under vacuum. Dissolve
the residue in 20 mL of 0.1 N hydrochloric acid, and
quantitatively transfer with the aid of two additional
“ and only a few compounded granules consisting of 2-4
S| 5-mL portions of 0.1 N hydrochloric acid to a 250-mL
5 separatory funnel. Add 20 mL of 1-propanol and 50 mL
s
— of chloroform, and shake vigorously for 2 min. Collect
=) and retain the lower chloroform layer, and add 50 mL
° e EXTRACTABLE MATTER
S of Solvent B to the upper layer remaining in the separa-
iS tory funnel. Shake vigorously for 2 min; collect and re-
= tain the lower chloroform layer. Combine the retained
” chloroform layers in a round-bottom flask, and evapo-
rate to near-dryness under vacuum. Evaporate the re-
a Acceptance criteria: NLT 18.0%
maining solvent under a stream of air. Wash the residue
with two 10-mL aliquots of ether, filter, wash the filter
with 10 mL of ether, and discard the ether filtrates. Af-
ter evaporation of the residual ether, suspend the resi-
due in 10 mL of glacial acetic acid, and pass through
the previously used dried filter into a 50-mL volumetric
flask. Repeat the addition of glacial acetic acid followed
by filtration two additional times, combining the fil-
trates in the 50-mL volumetric flask. Wash the round-
bottom flask with small quantities of glacial acetic acid,
and filter into the volumetric flask. Dilute with glacial
acetic acid to volume.
Instrumental conditions
(See Ultraviolet-Visible Spectroscopy (857).)
USP 41
Wavelength: 540 nm
Mode: Visible
Analysis: Accurately transfer 1.0 mL each of Standard
solutions A, B, and C, the Sample solution, and the Blank
into separate screw-cap test tubes. Add 4.0 mL of Rea-
gent to each tube, cap the tubes, and keep them on a
water bath at 60° for 25 min, shaking occasionally.
Measure the absorbances of the reacted Sample solution
and Standard solutions A, B, and C, corrected for the
Blank. Plot the absorbances of Standard solutions A, B,
and C against their respective concentrations, and es-
tablish the calibration line by linear regression. From
the plot, determine the concentration, C, in mg/mL, of
triterpene glycosides as escin in the Sample solution.
Calculate the percentage of triterpene glycosides as es-
cin in the portion of Powdered Horse Chestnut taken:
Result = (C/W) x (50/3)
€ = concentration of triterpene glycosides in the
Sample solution as obtained above (mg/mL)
w = weight of Powdered Horse Chestnut taken to
prepare the Sample solution (g)
Acceptance criteria: NLT 3.0% of ineKpe ne glycosides,
calculated as escin (CssHgsO24), on the dried basis
CONTAMINANTS
e ARTICLES OF BOTANICAL ORIGIN, Limits of Elemental Impuri-
ties (561): Meets the requirements
e ARTICLES OF BOTANICAL ORIGIN, Pesticide Residue Analysis
(561): Meets the requirements
e MICROBIAL ENUMERATION TESTS (2021): The total aerobic
microbial count does not exceed 106 cfu/g, the total
combined molds and yeast count does not exceed 104
cfu/g, and the bile-tolerant Gram-negative bacteria count
is NMT 103 cfu/g.
e ABSENCE OF SPECIFIED MICROORGANISMS (2022): It meets
the requirements of the tests for absence of Salmonella
species and Escherichia coli.
SPECIFIC TESTS
e BOTANICAL CHARACTERISTICS: Yellowish-brown powder,
odorless, with a somewhat mealy, disagreeably bitter,
and lingering taste. It shows numerous, different-sized
fatty oil droplets that are free or within the thin-walled,
colorless tissue of the cotyledons. Fragments of the testa
consist of thick-walled pitted sclerenchymatous cells. The
following are also present: pyriform, roundish or reniform
larger individual starch granules from 15 to 30 um in
diameter, smaller individual granules from 3 to 10 um,
single grains that form rows up to 45 kum in length.
Many of the starch granules havea bistellate or polystel-
late, but rarely simple, hilum.
Analysis: Proceed as directed for Articles of Botanical Or-
igin (861), Alcohol-Soluble Extractives, Method 2, except
use a mixture of methanol and water (8:2) instead of
alcohol.
e Loss ON DRYING (731): Dry a sample at 105° for 2 h: it
loses NMT 10.0% of its weight.
© ARTICLES OF BOTANICAL ORIGIN, Total Ash (561): NMT
4.0%
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in well-closed, light-
resistant containers, protected from moisture.
e LABELING: The label states the Latin binomial and, follow-
ing the official name, the part of the plant from which
the article was derived.
USP 41
e USP REFERENCE STANDARDS (11)
USP Escin RS
Powdered Horse Chestnut Extract
DEFINITION
Powdered Horse Chestnut Extract is prepared from Horse
Chestnut by extraction with alcohol-water mixtures or
methanol-water mixtures. The ratio of starting plant ma-
terial to extract is between 5:1 and 8:1. It contains NLT
90.0% and NMT 110.0% of the labeled amount of
triterpene glycosides, calculated on the dried basis as es-
cin (CssHgsO24). It may contain suitable added substances.
IDENTIFICATION
© A. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST
Standard solution: 5 mg/mL of USP Escin RS in
methanol
Sample solution: To 10 mL of methanol add a quantity
of Powdered Extract equivalent to 25 mg of the labeled
amount of triterpene glycosides, and shake. Allow to
stand for 15 min before use.
Chromatographic system
(See Chromatography (621), Thin-Layer Chromato-
graphy.) = ee
eeorbent: 0.25-mm layer of chromatographic silica
el
Applitatlot volume: 10 pL
ong solvent system: Use the upper phase of a
mixture of 1-butanol, glacial acetic acid, and water
(5:1:4).
Spray reagent: Methanol, glacial acetic acid, sulfuric
acid, and p-anisaldehyde (85: 10: 5: 0.5)
Analysis
Samples: Standard solution and Sample solution
Develop the chromatograms to a length of NLT 15 cm,
and dry the plate in a current of air. Spray the plate
with Spray reagent, heat the plate at 100° for 5 min,
and examine the plate under daylight.
Acceptance criteria: The chromatogram from the Sam-
ple solution shows a blue-violet zone corresponding to
escin, comparable in position and color to the main
zone in the chromatogram from the Standard solution.
Above this zone, the chromatogram of the Sample solu-
tion shows several narrow, brown to brownish-red
zones that are less intense than the zone corresponding
to escin.
COMPOSITION
¢ CONTENT OF TRITERPENE GLYCOSIDES
Solvent A: Methanol and water (13:7)
Solvent B: Use the lower phase of a mixture of chloro-
form, 0.1 N hydrochloric acid, and 1-propanol (5:3:2).
Reagent: Dissolve 75 mg of ferric chloride in 50 mL of
glacial acetic acid. Add 50 mL of sulfuric acid, while
shaking and cooling. Prepare immediately before use.
Standard solution A: 0.2 mg/mL of USP Escin RS in
glacial acetic acid, shaking for 1 min
Standard solution B: 0.4. mg/mL of USP Escin RS in
glacial acetic acid, shaking for 1 min
Standard solution C: 0.6 mg/mL of USP Escin RS in
glacial acetic acid, shaking for 1 min
Sample solution: Transfer a quantity of Powdered Ex-
tract, equivalent to 50 mg of the labeled content of
triterpene glycosides, into a 50-mL flask. Add 20 mL of
0.1 N hydrochloric acid, and shake for 5 min. Filter into
a 250-mL separatory funnel with the aid of two addi-
tional 5-mL portions of 0.1 N hydrochloric acid. Add
20 mL of 1-propanol and 50 mL of chloroform, and
shake vigorously for 2 min. Separate the chloroform
layer, and add Solvent B to the upper phase remaining
in the separation funnel. Shake vigorously for 2 min,
Dietary Supplements / Horse Chestnut 4529
and separate the chloroform layer. Combine the chloro-
form layers in a round-bottom flask, and evaporate to
dryness under vacuum. Evaporate the remaining sol-
vents with the aid of a current of air. Wash the residue
with two 10-mL portions of ether, filter, wash the filter
with 10 mL of ether, and discard the ether filtrates. Af-
ter evaporation of the residual ether, add to the residue
a 10-mL portion of glacial acetic acid, and pass through
the previously used dried filter into a 100-mL volumet-
ric flask. Repeat the addition of glacial acetic acid fol-
lowed by filtration two additional times, combining the
filtrates in the volumetric flask. Wash the round-bottom
flask with small quantities of glacial acetic acid, and fil-
ter into the volumetric flask. Dilute with glacial acetic
acid to volume.
Instrumental conditions
(See Ultraviolet-Visible Spectroscopy (857).)
Mode: Visible
Wavelength: 540 nm
Blank: Glacial acetic acid
Analysis: Transfer 1 mL each of Standard solutions A, B,
and C, the Sample solution, and the Blank to separate
test tubes with stoppers. Add 4.0 mL of Reagent to each
tube, cap the tubes, and place them in a water bath at
60° for 25 min, shaking occasionally. Measure the ab-
sorbances of the reacted Sample solution and the re-
acted Standard solutions A, B, and C, and correct for the
Blank. Plot the absorbances of the reacted Standard so-
lutions A, B, and C versus concentrations, in mg/mL of
USP Escin RS in the corresponding Standard solution.
From the graphs determine the concentration, C, in
mg/mL, oF tnterpene glycosides as escin (CssHgeO24) in
the Sample solution.
Calculate the percentage of the labeled amount of
triterpene glycosides in the portion of Powdered Ex-
tract taken:
Result = (C/Cy) x 100
¢. = concentration of triterpene glycosides in the
Sample solution as obtained above (mg/mL)
Cu = nominal concentration of triterpene glycosides
in the Sample solution (mg/mL)
Acceptance criteria: 90.0%-110.0% of the labeled
amount of triterpene glycosides as escin (CssHgsO24) on
the dried basis
CONTAMINANTS
Delete the following:
sydesbouo=: Sa
°e HEAVY METALS, Method !/ (231): NMT 20 t1g/ge cotta 1-
n-2018)
° IMicRoBIAL ENUMERATION TESTS (2021): The total aerobic
microbial count does not exceed 10 cfu/g, the total
combined molds and yeasts count does not exceed 10?
cfu/g, and the count for enterobacteria does not exceed
103 cfu/g.
e ABSENCE OF SPECIFIED MICROORGANISMS (2022): It meets
the requirements of the tests for absence of Salmonella
species and Escherichia coli.
SPECIFIC TESTS
© Loss ON DRYING (731): Dry 1g at 105° for 2 h: it loses
NMT 5.0% of its weight.
© OTHER REQUIREMENTS: It meets the requirements in Botan-
ical Extracts (565), General Pharmacopeial Requirements,
for Caeeaieg and Storage, Residual Solvents, and Pesticide
Residues for powdered extracts.
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in tight, light-resistant
containers. Store in a cool place.
e LABELING: The label states the Latin binomial and, follow-
ing the official name, the part of the plant from which
 4530 Horse Chestnut / Dietary Supplements USP 41

the article was prepared. The label also indicates the con- saturated sodium chloride, cap, and mix with a vortex
tent of triterpene glycosides, the extracting solvent or mixer or shake thoroughly for at least 15 s. Let the
solvent mixture used for preparation, the ratio of the solution stand for 5 min, or until the upper layer be-
starting crude plant material to Powdered Extract, and comes clear, and transfer to a separate tube. Shake the
the name and content of any added substance. It meets lower layer once more with 3 mL of n-hexane, and
the requirements for labeling in Botanical Extracts (565). combine the hexane extracts. Evaporate the hexane ex-
e USP REFERENCE STANDARDS (11) tracts with the aid of a nitrogen stream to dryness. Add
USP Escin RS 0.3 mL of pyridine silylated with 1.0 mL of
BSA+TMCS+TMSI mixture (3:2:3)' and let it stand at
room temperature for 15 min. Inject this solution into a
gas chromatograph.
Sample solution: Prepare as directed for the Standard
Chia Seed Oil solution, except replace USP Chia Seed Oil RS with Chia
Seed Oil.
[93384-40-8]. Chromatographic system
(See Chromatography (621), System Suitability.)
DEFINITION Mode: GC
Chia Seed Oil is derived from the seeds of the Chia plant Detector: Flame ionization
(Salvia hispanica L.). The oil is extracted from the seeds by Column: 0.32-mm x 30-m fused silica capillary,
cold pressing. No solvents or external heat are employed bonded with a 0.25-11m film of phase G27
in the extraction process. Tocopherol may be added as an Temperatures
antioxidant. Injection port: 240°
Detector: 325°
IDENTIFICATION Column: See Table 2.
e A. It meets the requirements in Specific Tests for Fats and
Fixed Oils (401), Procedures, Fatty Acid Composition.
© B. IDENTIFICATION OF FIXED OILS BY THIN-LAYER CHROMA- Table 2
TOGRAPHY (202): The R; values of the principal spots of Hold
the Sample solution correspond to those of the Standard Time at
solution. Initial Hold Tempera- Final Final
e C. It meets the requirements in Specific Tests for Sterol Tempera- Time ture Tempera- | Tempera-
Composition. ture at 240° Ramp ture ture
SPECIFIC TESTS C), (min) (¢/min) ©) (min)
e FATS AND FIXED OILS (401), Procedures, Acid Value: NMT 240 3 2 300 7
25 Carrier gas: Helium
FATS AND FIXED OILS (401), Procedures, Peroxide Value: Flow rate: 1.5 mL/min
NMT 10.0 Split ratio: 2:1
FATS AND FIXED OILS (401), Procedures, lodine Value: Injection volume: 1 pL
180-210 System suitability
FATS AND FIXED OILS (401), Procedures, Saponification Sample: Standard solution
Value: 180-230 Suitability requirements
FATS AND FIXED OILS (401), Procedures, Unsaponifiable Mat- Resolution: NLT 1.5 between f-sitosterol and
ter. NMT 1.5 A5-avenasterol
FATS AND FIXED OILS (401), Procedures, Fatty Acid Composi- Relative standard deviation: NMT 2.0% for the ra-
tion: Chia Seed Oil exhibits the composition profile of tios of B-sitosterol to internal standard peak responses
fatty acids in Table 1. from replicate injections
Chromatogram similarity: The chromatogram from
Table 1 the Standard solution is similar to the reference chro-
Area matogram supplied with USP Chia Seed Oil RS. Iden-
Fatty Shorthand Percentage tify the retention times of six relevant sterol methyl
DS Monographs

Acid Notation (%) esters by comparing the chromatogram of the Stan-

dard solution with the reference chromatogram sup-
Palmitic acid 16:0 5.0-9.0
plied with USP Chia Seed Oil RS. The retention times
Stearic acid 18:0 2.0-5.0 of the sterols with reference to B-sitosterol are given
Oleic acid 18:1 4.0-9.0 in Table 3.
Linoleic acid 18:2 17.0-22.0
Alpha-linolenic acid 18:3 (n-3) 57.0-70.0 Table 3
Gammaz-linolenic
Relative
acid 18:3 (n-6) 0.0-0.4
Retention
e STEROL COMPOSITION Identification Time
Internal standard solution: 0.3 mg/mL of USP Choles- Cholestanol (internal standard) 0.73
tanol RS in 2-propanol Campesterol 0.91
Standard solution: Transfer 50 mg of USP Chia Seed Stigmasterol 0.94
Oil RS to a 25-mL screw-cap test tube, add 2.0 mL of B-Sitosterol 1.00
2-propanol, and sonicate to dissolve. Add 3.0 mL of A5-Avenasterol 1.02
1M methanolic potassium hydroxide and 0.8 mL of In-
ternal standard solution to the test tube. Cap the test Cycloartenol 1.06
tube and place in a hot water bath at 80° for 60 min. 24-Methylenecycloartenol 1.11
Remove the test tube from the water bath and cool to 1 BSA+TMCS+TMSI mixture (3:2:3) is available from Sigma-Aldrich, Product
room temperature, then add 3 mL each of water and n- #33030, www.sigmaaldrich.com/catalog/product/supelco/33030.
hexane. Mix the solution with a vortex mixer or shake
vigorously for at least 30 s. Immediately add 5 mL of
USP 41 Dietary Supplements / Chinese Salvia 4531

Analysis Application volume: 5 uL, as 8-mm bands

Sample: Sample solution Developing solvent system A: A mixture of ethyl ace-
Calculate the area percentage of each individual sterol tate, 2B osbforn, toluene, formic acid, and methanol
in the portion of Chia Seed Oil taken: (8:6:4:4:1)
Developing solvent system B: A mixture of solvent
Result = (Ru/R1) x 100 hexane and ethyl acetate (4:1)
Analysis
Ru = peak response ratio of each sterol component Samples: Standard solution A, Standard solution B, and
to the internal standard (internal standard Sample solution
ratio) from the Sample solution Apply the samples as bands to a suitable high perfor-
Rr = sum of six internal standard ratios from the mance thin-layer chromatographic plate. Use a satu-
Sample solution rated chamber, and condition the plate to a relative
Acceptance criteria: Chia Seed Oil exhibits six sterol humidity of about 33% using a suitable device. De-
components, each with the normalized area percentage velop the chromatograms in Developing solvent system
shown in Table 4. A until the solvent front has moved up about 40% of
the plate. Remove the plate, and allow to dry. Develop
Table 4 the chromatograms in a saturated chamber containing
Developing solvent system B until the solvent front has
Normalized
moved up about three-fourths of the plate. Remove
Area
the plate, dry, and examine under visible light and UV
Percentage
light at 254 nm and 365 nm.
Component (%)
Acceptance criteria
Campesterol 11..1-13:2 Under visible light, the chromatogram of the Sample
Stigmasterol 2.5-6.8 solution exhibits three bands similar in positions and
B-Sitosterol 66.2-68.9 colors to bands in the chromatogram of Standard solu-
A5-Avenasterol 6.8-10.4 tion B. These include a pink band in the upper third of
Cycloartenol 3.0-5.6 the chromatogram, similar in Rr and color to the tan-
shinone II, band in the chromatogram of Standard so-
24-Methylenecycloartenol 2 2-3.7 lution A, a yellowish-orange band in the upper third of
e REFRACTIVE INDEX (831): 1.460-1.490 at 20° the chromatogram, and an orange band at about the
middle of the chromatogram due to tanshinone | and
ADDITIONAL REQUIREMENTS cryptotanshinone, respectively.
e PACKAGING AND STORAGE: Preserve in well-closed, tight, Under UV light at 365 nm, the chromatogram of the
light-resistant containers. Sample solution exhibits three blue bands similar in po-
e LABELING: Where Chia Seed Oil is intended for use in the sitions and colors to bands in the chromatogram of
manufacture of dosage forms, it is so labeled. Standard solution B. These include an intense blue
e USP REFERENCE STANDARDS (11) band in the lower third of the chromatogram corre-
USP Chia Seed Oil RS sponding in R; and color to the salvianolic acid B band
USP Cholestanol RS in the chromatogram of Standard solution A, and two
3B-Hydroxy-5a-cholestane. minor blue bands in the lower third of the chromato-
Co7HasO = 3388.67 ram and above the salvianolic acid B band, due to
ithospermic acid and rosmarinic acid.
Under UV light at 254 nm, the chromatogram of the
Sample solution exhibits intense quenching bands at Rr
corresponding to those for tanshinone Il, and salvia-
Chinese Salvia nolic acid B in the chromatogram of Standard solution
A. The chromatogram of the Sample solution also ex-
DEFINITION hibits other quenching bands corresponding in Rr to
Chinese Salvia consists of the dried roots and rhizomes of the bands in the chromatogram of Standard solution B.
Salvia miltiorrhiza Bunge, also known as Danshen (Fam. e C. HPLC
Lamiaceae). It contains NLT 0.1% of tanshinone Il,; NLT Analysis: Proceed as directed in the test for Content of
sydeiBouo- Sa

0.2% of total tanshinones, calculated as the sum of
cryptotanshinone, tanshinone |, and tanshinone Ila; and
NLT 3.0% of salvianolic acid B; all calculated on the dried
basis. It is collected in spring or fall.
IDENTIFICATION
e A. Chinese Salvia meets the requirements for Specific
Tests, Botanic Characteristics.
e B. THIN-LAYER CHROMATOGRAPHY
Standard solution A: A mixture of about 0.5 mg/mL of
USP Tanshinone Il, RS and about 1.5 mg/mL of USP
Salvianolic Acid B RS in alcohol
Standard solution B: About 0.25 g of USP Powdered
Chinese Salvia Extract RS in 5.0 mL of alcohol. Sonicate
for 15 min, conte and use the supernatant.
Sample solution: About 1.0 g of Chinese Salvia, finely
powdered, in 5.0 mL of alcohol. Sonicate for 15 min,
centrifuge, and use the supernatant.
Chromatographic system
(See ea YV (621), Thin-Layer Chromato-
raphy.
dasorncrt: Chromatographic silica gel mixture with
an average particle size of 2-10 um (HPTLC plates)
Tanshinones.
Acceptance criteria: The chromatogram of the Sample
solution exhibits the most intense peak at a retention
time corresponding to that of tanshinone Il, in the
chromatogram of Standard solution A. The iy solu-
tion chromatogram exhibits two additional peaks corre-
sponding to tanshinone | and cryptotanshinone, of less
intensity and accounting for about half of the total tan-
shinones content.
e D. HPLC
Analysis: Proceed as directed in the test for Content of
Salvianolic Acid B.
Acceptance criteria: The chromatogram of the Sample
solution exhibits a peak at a retention time correspond-
ing to that of salvianolic acid B in the chromatogram of
Standard solution A.
COMPOSITION
© CONTENT OF TANSHINONES
Solution A: 0.02% phosphoric acid in water (v/v)
Solution B: Acetonitrile
Mobile phase: See Table 1.
 4532 Chinese Salvia / Dietary Supplements USP 41

Table 1 w = weight of Chinese Salvia taken to prepare the

Time Solution A Solution B Sample solution (mg)
(min) F = conversion factor for analytes (1.18 for
(%) (%)
cryptotanshinone, 1.31 for tanshinone |, and
0 39 61 1.00 for tanshinone Iq)
6 39 61 Add the percentages of cryptotanshinone, tanshinone |,
20 10 90 and tanshinone Ila.
20.5 39 61 Acceptance criteria: NLT 0.1% tanshinone Il, and NLT
25 39 61 0.2% of total tanshinones, calculated on the dried basis
© CONTENT OF SALVIANOLIC ACID B
[NoTe—Proceed under subdued light or use low-actinic Solution A: 0.1% phosphoric acid in water (v/v)
glassware. The Standard solution and Sample solution Mobile phase: Solution A and acetonitrile (78:22)
are stable for 24 h at room temperature.] {[Note—The Standard solution and Sample solution are sta-
Standard solution A: 0.02 mg/mL of USP Tanshinone ble for 12 h at room temperature.]
Il, RS in methanol Solvent: Methanol and water (8:2)
Standard solution B: 2 mg/mL of USP Powdered Chi- Standard solution: 0.1 mg/mL of USP Salvianolic Acid
nese Salvia Extract RS in methanol. Sonicate for 15 min, B RS in Solvent
and pass through a membrane filter having a 0.45-1m Sample stock solution: About 150 mg of Chinese Sal-
pore size. Discard the first few mL of the filtrate. via, finely powdered and accurately weighed, in 40 mL
Sample solution: About 300 mg of Chinese Salvia, of Solvent. Sonicate for 30 min, filter into a 50-mL volu-
fine y powdered and accurately weighed, in 40 mL of metric flask, and wash the residue and the filter paper
methanol. Sonicate for 30 min, filter into a 50-mL volu- with a few mL of Solvent. Adjust with Solvent to vol.
metric flask, and wash the residue and the filter paper ume, mix, and centrifuge a portion.
with a few mL of methanol. Adjust with methanol to Sample solution: Dilute a portion of the supernatant
volume, mix, and pass through a membrane filter hav- from the Sample stock solution (1:2) with Solvent, mix,
ing a 0.45-um pore size. Discard the first few mL of the and pass through a membrane filter having a 0.45-um
filtrate. pore size. Discard the first 2 mL of the filtrate.
Chromatographic system Chromatographic system
(See Chromatography (621), System Suitability.) (See Chromatography (621), System Suitability.)
Mode: LC Mode: LC
Detector: UV 270 nm Detector: UV 286 nm
Column: 4.6-mm x 25-cm; 5-~m packing L1 Column: 4.6-mm x 25-cm; 5-um packing L1
Column temperature: 20° Column temperature: 20+1°
Flow rate: 1.0 mL/min Flow rate: 1.2 mL/min
Injection volume: 10 uL Injection volume: 10 LL
System suitability System suitability
Samples: Standard solution A and Standard solution B Sample: Standard solution
Suitability requirements Suitability requirements
Chromatogram similarity: The chromatogram from Failing actor: NMT 2.0 for the salvianolic acid B
Standard solution B is similar to the reference chro- eal
matogram provided with the lot of USP Powdered Relative standard deviation: NMT 2.0%, determined
Chinese Salvia Extract RS being used. from the salvianolic acid B peak in repeated injections
Tailing factor: NMT 2.0 for the tanshinone Il, peak, Analysis
Standard solution A Samples: Standard solution and Sample solution
Relative standard deviation: NMT 2.0%, determined Using the chromatogram of the Standard solution, iden-
from the tanshinone Il, peak in repeated injections, tify the retention time of the peak corresponding to
Standard solution A salvianolic acid B in the Sample solution.
Resolution: NLT 1.5 between the cryptotanshinone Calculate the percentage of salvianolic acid B in the
and tanshinone | peaks, Standard solution B portion of Chinese Salvia taken:
Analysis
Samples: Standard solution A, Standard solution B, and Result = (ru/rs) x Cs x (V/W) x D x 100
DS Monographs

Sample solution
Using the chromatograms of Standard solution A, Stan- tu = peak area of salvianolic acid B from the
dard solution B, and the reference chromatogram pro- Sample solution
vided with the lot of USP Powdered Chinese Salvia Ex- peak area of salvianolic acid B from the
a

tract RS being used, identify the retention times of the Standard solution
concentration of USP Salvianolic Acid B RS in
2

peaks corresponding to different tanshinones in the

"

Sample solution chromatogram. The approximate rela-
tive retention times of the different peaks for
cryptotanshinone, tanshinone |, and tanshinone Il, are
0.75, 0.79, and 1.00, respectively.
Calculate the percentages of cryptotanshinone, tanshi-
none |, and tanshinone Il, in the portion of Chinese
Salvia taken:
Result = (ru/rs) x Cs x (V/W) x F x 100
ru = peak area of the relevant analyte from the
Sample solution
rs = peak area of tanshinone II, from Standard
solution A
Cs = concentration of USP Tanshinone Il, RS in
Standard solution A (mg/mL)
V = volume of the Sample solution (mL)
the Standard solution (mg/mL)
Vv = volume of the Sample stock solution (mL)
= weight of Chinese Salvia used to prepare the
Sample stock solution (mg)
D = dilution factor to prepare the Sample solution
from the Sample stock solution, 2
Acceptance criteria: NLT 3.0%, calculated on the dried
basis
CONTAMINANTS
e ELEMENTAL IMPURITIES—PROCEDURES (233)
For deionized water: Use deionized water of at least 18
megaohm.
Sample solution: Use Chinese Salvia previously dried
for 2 h at 60°, ground to coarse powder. Accurately
weigh 0.5g of the powder, transfer to a closed micro-
wave vessel, and add 5-10 mL of concentrated nitric
USP 41
acid. [NoTE—In case of a severe reaction, set the vessel
aside until the reaction ceases.] Digest under pressure
following the instrument manufacturer’s recommenda-
tions, cool to below 60°, and remove the vessel. Cool
to room temperature, transfer the contents with the aid
of three 10-mL portions of deionized water to a 250-mL
volumetric flask, dilute with deionized water to volume,
and mix. [NoTte—In case of deposits, centrifuge, and
use the supernatant.]
Acceptance criteria
Arsenic: NMT 2 ug/g
Cadmium: NMT 0.3 ug/g
Lead: NMT 5 ug/g
Mercury: NMT 0.2 ug/g
ARTICLES OF BOTANICAL ORIGIN, General Method for Pesti-
cide Residues Analysis (561): Meets the requirements
MICROBIAL ENUMERATION TESTS (2021): The total aerobic
bacterial count does not exceed 105 cfu/g, the total com-
bined molds and yeasts count does not exceed 103 cfu/
g, and the bile-tolerant Gram-negative bacteria does not
exceed 103 cfu/g.
ABSENCE OF SPECIFIED MICROORGANISMS (2022): Meets the
requirements of the tests for absence of Salmonella spe-
cies and Escherichia coli
ARTICLES OF BOTANICAL ORIGIN, Test for Aflatoxins (561):
Meets the requirements
SPECIFIC TESTS
e@ BOTANIC CHARACTERISTICS
Macroscopic: Rhizomes short and thick, sometimes
with remains of stems at the apex. Roots, long, cylindri-
cal, slightly curved, some branched, with rootlets,
10-20 cm long, 0.3-1.5 cm in diameter. Externally
brownish-red or dark brownish-red, rough, longitudi-
nally wrinkled. The bark of old roots is loose, mostly
purplish-brown, usually scaling off; the bark of young
roots is closely adhering to wood and uneasy to be
scaled off. Texture hard and fragile, fracture loose, with
brownish-red bark and greyish-yellow or purplish-brown
wood, showing bundles of vessels, yellowish-white, ar-
ranged radially.
Microscopic
Transverse section: Cork, 4-8 rows of cells with
brown contents; rhytidome tissues may be present;
cortex broad, parenchyma cells showing reddish-
brown granules; phloem narrow, crescent shape; cam-
bium in a ring; xylem vessels, lignified, mainly scalari-
form and reticulate, numerous near the cambium ring
and fewer near the pith; xylem fibers in bundle, scat-
tered radially; pith in the center.
ARTICLES OF BOTANICAL ORIGIN, Foreign Organic Matter
(561): NMT 2.0%
ARTICLES OF BOTANICAL ORIGIN, Alcohol-Soluble Extractives,
Method 1 (561): NLT 15.0%
ARTICLES OF BOTANICAL ORIGIN, Water-Soluble Extractives,
Method 2 (561): NLT 35.0%
Loss ON DRYING (731)
Sample: 1.0g of finely powdered Chinese Salvia
Analysis: Dry at 105° for 2 h.
Acceptance criteria: NMT 13%
ARTICLES OF BOTANICAL ORIGIN, Total Ash (561)
Sample: 4.0g of finely powdered Chinese Salvia
Acceptance criteria: NMT 10%
ARTICLES OF BOTANICAL ORIGIN, Acid-/nsoluble Ash (561):
NMT 3.0%
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in well-closed contain-
ers, protected from light and moisture, and store at
room temperature.
© LABELING: The label states the Latin binomial and, follow-
ing the official name, the parts of the plant contained in
the article.
Dietary Supplements / Chinese Salvia 4533
e USP REFERENCE STANDARDS (11)
USP Powdered Chinese Salvia Extract RS
USP Salvianolic Acid B RS
USP Tanshinone II, RS
Powdered Chinese Salvia
DEFINITION
Powdered Chinese Salvia is Chinese Salvia reduced to a
powder or very fine powder. It contains NLT 0.1% tanshi-
none Ila; NLT 0.2% of total tanshinones, calculated as the
sum of cryptotanshinone, tanshinone |, and tanshinone
Ila; and NLT 3.0% of salvianolic acid B; all calculated on
the dried basis.
IDENTIFICATION
e A. Powdered Chinese Salvia meets the requirements for
Specific Tests, Botanic Characteristics.
e B. THIN-LAYER CHROMATOGRAPHY
Standard solution A: A mixture of about 0.5 mg/mL of
USP Tanshinone Il, RS and about 1.5 mg/mL of USP
Salvianolic Acid B RS in alcohol
Standard solution B: About 0.25 g of USP Powdered
Chinese Salvia Extract RS in 5.0 mL of alcohol. Sonicate
for 15 min, centrifuge, and use the supernatant.
Sample solution: About 1.0 g of Powdered Chinese Sal-
via in 5.0 mL of alcohol. Sonicate for 15 min, centri-
fuge, and use the supernatant.
Chromatographic system
(See Chromatography (621), Thin-Layer Chromato-
graphy.)
Adsorbent: Chromatographic silica gel mixture with
an average particle size of 2-10 um (HPTLC plates)
Application volume: 5 uL, as 8-mm bands
Developing solvent system A: A mixture of ethyl ace-
tate, chloroform, toluene, formic acid, and methanol
(8:6:4:4:1)
Developing solvent system B: A mixture of solvent
hexane and ethyl acetate (4:1)
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Apply the samples as bands to a suitable high perfor-
mance thin-layer chromatographic plate. Use a satu-
rated chamber, and condition the plate toa relative
humidity of about 33% usinga suitable device. De-
velop the chromatograms in Developing solvent system
A until the solvent front has moved up about 40% of
sydesbouow;w sa
the plate. Remove the plate, and allow to dry. Develop
the chromatograms in a saturated chamber containing
Developing solvent system B until the solvent front has
moved up about three-fourths of the plate. Remove
the plate, dry, and examine under visible light and UV
light at 254 nm and 365 nm.
Acceptance criteria
Under visible light, the chromatogram of the Sample
solution exhibits three bands similar in positions and
colors to bands in the chromatogram of Standard solu-
tion B. These include a pink band in the upper third of
the chromatogram, similar in Rr and color to the tan-
shinone Il, band in the chromatogram of Standard so-
lution A, a yellowish-orange band in the upper third of
the chromatogram, and an orange band at about the
middle of the chromatogram due to tanshinone | and
cryptotanshinone, respectively.
Under UV light at 365 nm, the chromatogram of the
Sample solution exhibits three blue bands similar in po-
sitions and colors to bands in the chromatogram of
Standard solution B. These include an intense blue
band in the lower third of the chromatogram corre-
sponding in R; and color to the salvianolic acid B band
in the chromatogram of Standard solution A, and two
 4534 Chinese Salvia / Dietary Supplements USP 41

minor blue bands in the lower third of the chromato- Mode: LC

gram and above the salvianolic acid B band, due to
lithospermic acid and rosmarinic acid.
Under UV light at 254 nm, the chromatogram of the
Sample solution exhibits intense quenching bands at Rr
corresponding to those for tanshinone II, and salvia-
nolic acid B in the chromatogram of Standard solution
A. The chromatogram of the Sample solution also ex-
hibits other quenching bands corresponding in R; to
the bands in the chromatogram of the Standard solu-
tion B.
e C. HPLC
Analysis: Proceed as directed in the test for Content of
Tanshinones.
Acceptance criteria: The chromatogram of the Sample
solution exhibits the most intense peak at a retention
time corresponding to that of tanshinone Il, in the
chromatogram of Standard solution A. The Sample solu-
tion chromatogram exhibits two additional peaks corre-
sponding to tanshinone | and cryptotanshinone, of less
intensity and accounting for about half of the total tan-
shinones content.
e D. HPLC
Analysis: Proceed as directed in the test for Content of
Salvianolic Acid B.
Acceptance criteria: The chromatogram of the Sample
solution exhibits a peak at a retention time correspond-
ing to that of salvianolic acid B in the chromatogram of
Standard solution A.
COMPOSITION
e CONTENT OF TANSHINONES
Solution A: 0.02% phosphoric acid in water (v/v)
Solution B: Acetonitrile
Mobile phase: See Table 7.
Detector: UV 270 nm
Column: 4.6-mm x 25-cm; 5-um packing L1
Column temperature: 20°
Flow rate: 1.0 mL/min
Injection volume: 10 pL
System suitability
Samples: Standard solution A and Standard solution B
Suitability requirements
Chromatogram similarity: The chromatogram from
Standard solutionB is similar to the reference chro-
matogram provided with the lot of USP Powdered
Chinese Salvia Extract RS being used.
Tailing factor: NMT 2.0 for the tanshinone Il, peak,
Standard solution A
Relative standard deviation: NMT 2.0%, determined
from the tanshinone Il, peak in repeated injections,
Standard solution A
Resolution: NLT 1.5 between the cryptotanshinone
and tanshinone | peaks, Standard solution B
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Using the chromatograms of Standard solution A, Stan-
dard solution B, and the reference chromatogram pro-
vided with the lot of USP Powdered Chinese Salvia Ex-
tract RS being used, identify the retention times of the
peaks corresponding to different tanshinones in the
Sample solution chromatogram. The approximate rela-
tive retention times of the different peaks for
cryptotanshinone, tanshinone |, and tanshinone Ila are
0.75, 0.79, and 1.00, respectively.
Calculate the percentages of cryptotanshinone, tanshi-
none I, and tanshinone Il, in the portion of Powdered
Chinese Salvia taken:

Table 1 Result = (ru/rs) x Cs x (V/W) x F x 100

Time Solution A Solution B ty = peak area of the relevant analyte from the
(min) (%) (%). Sample solution
0 39 61 Is = peak area of tanshinone Il, from the Standard
6 39 61 solution A
20 10 90 Cs = concentration of USP Tanshinone Il, RS in the
20.5 39 61
Standard solution A (mg/mL)
Vv = volume of the Sample solution (mL)
25 39 61 Ww = weight of Powdered Chinese Salvia taken to
[Note—Proceed under subdued light or use low-actinic prepare the Sample solution (mg)
glassware. The Standard solution and Sample solution are = conversion factor for a (1.18 for
stable for 24 h at room temperature.] cryptotanshinone, 1.31 for tanshinone |, and
1.00 for tanshinone II)
Standard solution A: 0.02 mg/mL of USP Tanshinone
al Il4 RS in methanol Add the percentages of cryptotanshinone, tanshinone I,
wo Standard solution B: 2 mg/mL of USP Powdered Chi-
and tanshinone Ila.
% nese Salvia Extract RS in methanol. Sonicate for 15 min, Acceptance criteria: NLT 0.1% tanshinone Il, and NLT
i]
J
and pass through a membrane filter pave a 0.45-um 0.2% of total tanshinones, calculated on the dried basis
=) e CONTENT OF SALVIANOLIC ACID B
i) pore size. Discard the first few mL of the filtrate.
iS Sample solution: About 300 mg of Powdered Chinese Solution A: 0.1% phosphoric acid in water (v/v)
Sj Salvia, accurately weighed, in 40 mL of methanol. Soni- Mobile phase: Solution A and acetonitrile (78:22)
= cate for 30 min, and filter into a 50-mL volumetric flask. [Nott—The Standard solution and Sample solution are sta-
al Wash the residue and the filter pepe with a few mL of ble for 12 h at room temperature.]
fa) Solvent: Methanol and water (8:2)
methanol, adjust to volume with methanol, mix, and
pass through a membrane filter having a 0.45-um pore Standard solution: 0.1 mg/mL of USP Salvianolic acid
size, Discard the first few mL of the filtrate. B RS in Solvent
Chromatographic system Sample stock solution: About 150 mg of Powdered
(See Chromatography (621), System Suitability.) Chinese Salvia, accurately weighed, in 40 mL of Solvent.
Sonicate for 30 min, and filter into a 50-mL volumetric
flask. Wash the residue and the filter paper with a few
mL of Solvent, adjust to volume with Solvent, mix, and
centrifuge a portion.
Sample solution: Dilute a portion of the supernatant
from the Sample stock solution (1:2) with Solvent, mix,
and pass through a membrane filter having a 0.45-um
pore size. Discard the first 2 mL of the filtrate.
Chromatographic system
(See Chromatography (621), System Suitability.)
USP 41 Dietary Supplements / Choline 4535

Mode: LC Microscopic: It shows fragments of cork cells, sub-

Detector: UV 286 nm rectangular or polygonal, containing yellowish-brown
Column: 4.6-mm x 25-cm; 5-um packing L1 pigments, 10-150 yum in diameter; parenchymatous
Column temperature: 20+1° cells of cortex, subsquare or polygonal, containing red-
Flow rate: 1.2 mL/min dish-brown pigments; stone cells, subrounded, subtri-
Injection volume: 10 pL angular, subrectangular or irregular shape, some elon-
System suitability ated, mostly 14-70 um in diameter, up to 270 um in
Sample: Standard solution length; fibers mostly in bundles, long fusiform in shape,
Suitability requirements ends oblique-sharp or blunt-round, with oblique or
Tailing factor: NMT 2.0 for the salvianolic acid B criss-cross striations, 10-60 um in diameter; and reticu-
eak late and pitted vessels, up to 120 um in diameter.
Relative standard deviation: NMT 2.0%, determined e ARTICLES OF BOTANICAL ORIGIN, Alcohol-Soluble Extractives,
from the salvianolic acid B peak in repeated injections Method 1 (561): NLT 15.0%
Analysis e ARTICLES OF BOTANICAL ORIGIN, Water-Soluble Extractives,
Samples: Standard solution and Sample solution Method 2 (561): NLT 35.0%
Using the chromatogram of Standard solution, identify Loss ON DRYING (731)
the retention time of the peak corresponding to salvia- Sample: 1.0 g of Powdered Chinese Salvia
nolic acid B in the Sample solution. Analysis: Dry at 105° for 2 h.
Calculate the percentage of salvianolic acid B in the Acceptance criteria: NMT 13%
portion of Powdered Chinese Salvia taken: ARTICLES OF BOTANICAL ORIGIN, Total Ash (561)
Sample: 4.0 g of Powdered Chinese Salvia
Result = (ru/rs) x Cs x (V/W) x D x 100 Acceptance criteria: NMT 10%
ARTICLES OF BOTANICAL ORIGIN, Acid-/nsoluble Ash (561):
°

tu = peak area of salvianolic acid B from the NMT 3.0%

Sample solution
rs = peak area of salvianolic acid B from the ADDITIONAL REQUIREMENTS
Standard solution © PACKAGING AND STORAGE: Preserve in well-closed contain-
G = concentration of USP Salvianolic Acid B RS in ers, protected from light and moisture, and store at
the Standard solution (mg/mL) room temperature.
Vv = volume of Sample stock solution (mL) e LABELING: The label states the Latin binomial and, follow-
Ww = weight of Powdered Chinese Salvia used to ing the official name, the parts of the plant from which
repare the Sample solution (mg) the article was obtained.
D = dilution factor to prepare the Sample solution e USP REFERENCE STANDARDS (11)
from Sample stock solution, 2 USP Powdered Chinese Salvia Extract RS
Acceptance criteria: NLT 3.0%, calculated on the dried USP Salvianolic Acid B RS
basis USP Tanshinone II, RS
CONTAMINANTS
© ELEMENTAL IMPURITIES—PROCEDURES (233)
For deionized water: Use deionized water of at least 18
megaohm.
ae solution: Use Powdered Chinese Salvia previ- Cholecalciferol—see Cholecalciferol General
ously dried for 2 h at 60°, weigh accurately 0.5 g, Monographs
transfer to a closed microwave vessel, and add 5-10 mL
of concentrated nitric acid. [NoTE—In case of a severe
reaction, set the vessel aside until the reaction ceases.]
Digest under pressure following the instrument manu- Cholecalciferol Capsules—see
facturer’s recommendations, cool to below 60°, remove
the vessel, cool to room temperature, transfer the con-
Cholecalciferol Capsules General Monographs
tent with the aid of three 10-mL portions of deionized
water to a 250-mL volumetric flask, complete to volume
with deionized water, and mix. [NOTE—In case of de-
posits, centrifuge, and use the supernatant.] Cholecalciferol Solution—see v4
Acceptance criteria Cholecalciferol Solution General Monographs =
Arsenic: NMT 2 g/g
Cadmium: NMT 0.3 1g/g F
Lead: NMT 5 ug/g
Mercury: NMT 0.2 g/g Choline Bitartrate
rets
ARTICLES OF BOTANICAL ORIGIN, General Method for Pesti-
cide Residues Analysis (561): Meets the requirements
MICROBIAL ENUMERATION TESTS (2021): The total aerobic
we om Lt z
bacterial count does not exceed 105 cfu/g, the total com- Ho cry is I, r
bined molds and yeasts count does not exceed 103 cfu/
g, and the bile-tolerant Gram-negative bacteria does not
exceed 103 cfu/g. CoHigNO7 253.25
ABSENCE OF SPECIFIED MICROORGANISMS (2022): Meets the 2-Hydroxyethanaminium, -N,N,N-trimethyl-, [R-(R*,R*)]-2,3-
requirements of the tests for absence of Salmonella spe- dihydroxybutanedioate (1:1);
cies and Escherichia coli Oe ee iamanonllnr peta salt (1:1)
ARTICLES OF BOTANICAL ORIGIN, Test for Aflatoxins (561): 87-67-2].
Meets the requirements
DEFINITION
SPECIFIC TESTS Choline Bitartrate contains NLT 99.0% and NMT 100.5% of
e BOTANIC CHARACTERISTICS choline bitartrate (CsHisNO7), calculated on the anhy-
Macroscopic: Yellowish-brown to reddish-brown in drous basis.
color
 4536 Choline / Dietary Supplements USP 41

IDENTIFICATION Delete the following:

© A. INFRARED ABSORPTION (197K)
° B. °e HEAVY MeTALs, Method II (231): NMT 10 ppme coricut1-
Sample: 1g Jan-2018)
Analysis: Dissolve the Sample in 20 mL of water, and © Limit OF TOTAL AMINES
add 2 mL of potassium chloride solution (1 in 4). Standard solution: 500 g/mL of trimethylamine
Acceptance criteria: A white precipitate of potassium hydrochloride
bitartrate is formed. Sample solution: Transfer 10.0 g of Choline Bitartrate
ASSAY to a beaker containing a plastic-coated stirring bar, add
e PROCEDURE 70 mL of sodium hydroxide TS and 130 mL of water,
Sample: 200mg and stir until dissolved.
Titrimetric system System suitability stock solution: 10 pg/mL of trimeth-
(See Titrimetry (541).) ylamine hydrochloride
Mode: Direct titration System suitability solution: Transfer 10.0 mL of System
Titrant: 0.1 N perchloric acid VS Suitability stock solution containing a plastic-coated stir-
Endpoint detection: Potentiometric ring bar, add 160 mL water and 30.0 mL sodium hy-
Blank: 50 mL of glacial acetic acid droxide TS, and stir until dissolved.
Analysis: Dissolve the Sample in 50 mL of glacial acetic Electrode system: Use a gas-sensing, ammonia-specific
acid and titrate with Titrant. indicating electrode with internal reference connected
Calculate the percentage of choline bitartrate to a pH meter capable of measuring potentials with a
(CsHigNO7) in the Sample taken: minimum reproducibility of +0.1 mV (see pH {791)).
Standard response line: Mix 30.0 mL of sodium hy-
Result = [((V— B) x Nx Fx 100]/W droxide TS and 170 mL of water. Add a plastic-coated
stirring bar, insert the electrode into the solution, and
Vv = Sample titrant volume (mL) record the potential, in mV. Continue stirring, and at
B = Blank titrant volume (mL) 5-min intervals, add 0.200, 0.600, 1.00, and 2.00 mL of
N = titrant normality (mEq/mL) Standard solution, and record the potential after each
F = equivalency factor, 253.2 mg/mEq addition. Plot the logarithms of the cumulative trimeth-
Ww = weight of the Sample (mg) ylamine hydrochloride concentrations (0.50, 1.50, 2.50,
fee rane criteria: 99.0%-100.5% on the anhydrous and 5.00 g/mL) versus potential, in mV, and deter-
asis mine the slope (5S) of the Standard response line for the
electrode.
IMPURITIES System suitability
e RESIDUAL SOLVENTS (467): Meet the requirements, except Sample: System suitability solution
that the limit for 1,4-dioxane is 10 ug/g Proceed as directed in Analysis, except to replace the
e RESIDUE ON IGNITION (281): NMT 0.1% Sample solution with the System suitability solution and
e ARSENIC, Method | (211) in the formula below to replace W with V, which
Analysis: Add 30 mL of water and 5 mL of hydrochloric equals 10 mL.
acid to dissolve the sample. Suitability requirements: The total change is NLT 10
Acceptance criteria: NMT 2 ppm mV for a 0.4-mL cumulative addition of the Standard
e LEAD (251) solution; the amount of trimethylamine hydrochloride
[NotE—Use methylene chloride in place of chloroform to found is 8.5-11.5 pg/L.
prepare the Dithizone Extraction Solution and Standard Analysis
Dithizone Solution.] Samples: Standard solution and Sample solution
Solution A: Transfer 8.4 g of sodium hydroxide solution Rinse the electrode, insert it into the Sample solution,
(1 in 2) to a plastic bottle, add 100 mL of ammonium stir, and record the potential, in mV. Add 0.100 mL
hydroxide, and mix. of the Standard solution, and record the potential.
Standard solution: Transfer 1.0 mL of the Diluted Stan- Add another 0.100 mL of the Standard solution, and
dard Lead Solution to a separatory funnel containing record the potential. [NoTe—If the total change after
25.0 mL of water. the second addition of the Standard solution is less
Sample solution: Dissolve 3.00 g of Choline Bitartrate than 10 mV, add a third aliquot of 0.200 mL.]
DS Monographs

in a separatory funnel containing 25.0 mL of water. Calculate the content, inu9/9, of total amines as tri-
Analysis methylamine hydrochloride in the portion of sample
Samples: Standard solution and Sample solution taken:
Separately add 6.0 mL of Ammonium Citrate Solution
and 3.0 mL of Potassium Cyanide Solution to the Stan- Result = (Cs x V,)/[(F
- 1) x W]
dard solution and the Sample solution. Extract each of
the eon solutions three times with 5.0-mL por- Gs = concentration of the Standard solution (g/mL)
tions of Dithizone Extraction Solution, shaking for 60 s Va = total volume of the Standard solution added to
and draining off each extract into another separator. the Sample solution (mL)
Shake the combined dithizone solutions for 30 s with w = weight of Choline Bitartrate taken to prepare
20.0 mL of nitric acid (1 in 100), and discard the the Sample solution (g)
methylene chloride layer. Add 6.0 mL of Ammonia-Cy- F = correction factor, calculated by the formula:
anide Solution, 2 mL of Solution A, and 10 mL of Stan-
dard Dithizone Solution, and shake for 45 s. Allow the F = antilog [(mV — mVo)/S]
phases to separate, and measure the absorbance of
the lower layer at 510 nm with a suitable mV; = final reading after the additions of the
Standard solution (mV)
spectrophotometer.
Acceptance criteria: The absorbance of the Sample so- mVo = initial reading of the Sample solution (mV)
S = slope of the Standard response line for the
lution is NMT the absorbance of the Standard solution
(NMT 0.3 ppm). electrode
Acceptance criteria: NMT 10 g/g
¢ CHROMATOGRAPHIC PURITY
Buffer solution: 7.1 g/L of anhydrous dibasic sodium
phosphate. Adjust with phosphoric acid to a pH of 2.5.
USP 41 Dietary Supplements / Choline 4537

Mobile phase: Buffer solution and acetonitrile (7:3) e USP REFERENCE STANDARDS (11)
Standard solution: Transfer an amount, NMT 100 mg, USP Choline Bitartrate RS
of USP Choline Chloride RS to a 24-mL screw-capped USP Choline Chloride RS
vial, and add 400 mg of 3,5-dinitrobenzoyl chloride and
10 mL of acetonitrile. Cap the vial, heat to 55°, and
continue heating for 2 h. Cool to room temperature,
and add 5 mL of water. Allow to stand for 5 min.
Quantitatively transfer the solution to a 25-mL volumet- Choline Chloride
ric flask and dilute with acetonitrile to volume. Dilute a
volume of this solution with Mobile phase to obtain a
concentration of 2.0 11g/mL of USP Choline Chloride RS.
Sample solution: Transfer 500 mg of Choline Bitartrate
to a centrifuge tube, add 2.0 mL of water, and swirl to
dissolve. Add 0.5 mL of potassium chloride solution (7.5 CsHy4CINO 139.62
in 25), centrifuge, and transfer 1.0 mL of the superna- 2 tesyete ined vianineniunchloride;
tant to a 24-mL Sole cappee vial. Dry at 120° for 2 h. 2-Hydroxy-N,N,N-trimethylethanaminium chloride [67-48-1].
Add 400 mg of 3,5-dinitrobenzoyl chloride and 10 mL
of acetonitrile. Cap the vial, and heat at 55° for 2 h. DEFINITION
Cool to room temperature, add 5 mL of water, and.al- Choline Chloride contains NLT 99.0% and NMT 100.5% of
low to stand for 5 min. Quantitatively transfer this solu- choline chloride (CsHi4CINO), calculated on the anhy-
tion to a 50-mL volumetric flask, and dilute with Mobile drous basis.
phase to volume. Pipet 2.0 mL of the solution to a
25-mL volumetric flask, and dilute with Mobile phase to IDENTIFICATION
volume. e A. INFRARED ABSORPTION (197K)
Chromatographic system © B. IDENTIFICATION TESTS—GENERAL, Chloride (191): A solu-
(See Chromatography (621), System Suitability.) tion (1 in 20) meets the requirements.
Mode: LC ASSAY
Detector: UV 208 nm ¢ PROCEDURE
Column: 4.6-mm x 25-cm; packing L7 Sample: 120mg
Column temperature: 30° Titrimetric system
Flow rate: 1 mL/min (See Titrimetry (541).)
Injection size: 20 ul Mode: Direct titration
System suitability Titrant: 0.1 N silver nitrate VS
Sample: Standard solution Endpoint detection: Potentiometric
Suitability requirements Blank: 35 mL of water. Add 3 drops of acetic acid.
Capacity factor (k’): NLT 2 Analysis: Dissolve the Sample in 35 mL of water and
Relative standard deviation: NMT 5%, determined add 3 drops of acetic acid. Titrate with Titrant.
from the choline derivative peak Calculate the percentage of choline chloride
Analysis (CsHi4CINO) in the Sample taken:
Samples: Standard solution and Sample solution
Calculate the percentage of each impurity in the por- Result = [(V
- B) x Nx F x 100]/W
tion of Choline Bitartrate taken:
Vv = Sample titrant volume (mL)
Result = (ru/rs) x (Cs/Cu) x (Mn/M,2) x 100 B = Blank titrant volume (mL)
N = titrant normality (mEg/mL)
tu = peak response for each impurity, excluding F = equivalency factor, 139.6 mg/mEq
that for the choline derivative and 3,5- Ww = weight of the Sample (mg)
dinitrobenzoic acid from the Sample solution Acceptance criteria: 99.0%-100.5% on the anhydrous
rs = peak response for the choline derivative from basis
the Standard solution
Cs = concentration of USP Choline Chloride RS in IMPURITIES
the Standard solution (mg/mL)
sydesbouo-= Sa

e RESIDUAL SOLVENTS (467): Meets the requirements, ex-

Cu = concentration of Choline Bitartrate in the cept that the limit for 1,4-dioxane is 10 ug/g
Sample solution (mg/mL) © RESIDUE ON IGNITION (281): NMT 0.05%
My = molecular weight of choline bitartrate, 253.25 e ARSENIC, Method | (211)
Mz = molecular weight of choline chloride, 139.62 Analysis: Add 30 mL of water and 5 mL of hydrochloric
Acceptance criteria acid to dissolve the sample.
Individual impurities: NMT 0.3% Acceptance criteria: NMT 2 ppm
Total impurity: NMT 2.0% e LEAD (251)
[Note—Use methylene chloride in place of chloroform to
SPECIFIC TESTS prepare the Dithizone Extraction Solution and Standard
© OPTICAL ROTATION, Specific Rotation (781S) Dithizone Solution.]
Sample solution: 400 mg/mL in water Solution A: Transfer 8.4 g of sodium hydroxide solution
Acceptance criteria: +17.5° to +18.5° (1 in 2) to a plastic bottle, add 100 mL of ammonium
© PH (791): 3.0-4.0, in a solution (1 in 10) hydroxide, and mix.
© WATER DETERMINATION, Method | (921): NMT 0.5% Standard solution: Transfer 1.0 mL of the Diluted Stan-
ADDITIONAL REQUIREMENTS dard Lead Solution to a separatory funnel containing
e PACKAGING AND STORAGE: Preserve in well-closed 25.0 mL of water.
containers. Sample solution: Dissolve 3.00 g of Choline Chloride in
a separatory funnel containing 25.0 mL of water.
Analysis
Samples: Standard solution and Sample solution
Separately add 6.0 mL of Ammonium Citrate Solution
and 3.0 mL of Potassium Cyanide Solution to the Stan-
dard solution and the Sample solution. Extract each of
 4538 Choline / Dietary Supplements USP 41

thepewulting solutions three times with 5.0-mL por-
tions of Dithizone Extraction Solution, shaking for 60 s
and draining off each extract into another separator.
Shake the combined dithizone solutions for 30 s with
20.0 mL of nitric acid (1 in 100), and discard the
methylene chloride layer. Add 6.0 mL of Am-
monia-Cyanide Solution, 2 mL of Solution A, and
10 mL of Standard Dithizone Solution, and shake for
45 s. Allow the phases to separate, and measure the
absorbance of the lower layer at 510 nm with a suita-
ble spectrophotometer.
Acceptance criteria: The absorbance of the Sample so-
lution is NMT the absorbance of the Standard solution
(NMT 0.3 ppm).
© CHROMATOGRAPHIC PURITY
Delete the following:
°e HEAVY METALS, Method Ii (231): NMT 10 ppme cotta t-
Jan-2018)
e LIMIT OF TOTAL AMINES
Standard solution: 500 g/mL of trimethylamine hy-
drochloride in water
Sample solution: Transfer 10.0 g of Choline Chloride to
a beaker containinga plastic-coated stirring bar, add
170 mL of water and 30.0 mL of sodium hydroxide TS,
and stir until dissolved.
System suitability stock solution: 10 g/mL of trimeth-
ylamine hydrochloride in water
System suitability solution: Transfer 10.0 mL of System
suitability stock solution to a beaker containing a plastic-
coated stirring bar, add 160 mL of water and 30.0 mL
of sodium hydroxide TS, and stir until dissolved.
Electrode system: Use a das sort ammonia-specific
indicating electrode with internal reference connected
to a pH meter capable of measuring potentials with a
minimum reproducibility of 0.1 mV (see pH (791)).
Standard response line: Mix 30.0 mL of sodium hy-
droxide TS, and 170 mL of water. Add a plastic-coated
stirring bar, insert the electrode into the solution, and
record the potential, in mV. Continue stirring, and at
5-min intervals add 0.200, 0.600, 1.00, and 2.00 mL of
Standard solution, and record the potential after each
addition. Plot the logarithms of the cumulative trimeth-
ylamine hydrochloride concentrations (0.50, 1.50, 2.50,
and 5.00 pg/mL) versus potential, in mV, and deter-
mine the slope (5S) of the Standard response line for the
electrode.
System suitability
Sample: System suitability solution
Proceed as directed in Analysis, except to replace the
Sample solution with the System suitability solution and
DS Monographs
Cs = concentration of Standard solution (tug/mL)
Va = total volume of the Standard solution added to
the Sample solution (mL)
Ww = weight of Choline Chloride taken to prepare
the Sample solution (g)
F = correction factor, calculated by the formula:
F = antilog [(mV; — mVo)/S]
mV, = final reading after the additions of the
Standard solution (mV)
mVo =initial reading of the Sample solution (mV)
S = slope of the Standard response line for the
electrode
Acceptance criteria: NMT 10 g/g
Buffer solution: 7.1 g/L of anhydrous dibasic sodium
phosphate. Adjust with phosphoric acid to a pH of 2.5.
Mobile phase: Buffer solution and acetonitrile (7:3
Standard solution: Transfer an amount, NMT 100 mg,
of USP Choline Chloride RS to a 24-mL screw-capped
vial, and add 400 mg of 3,5-dinitrobenzoyl! chloride and
10 mL of acetonitrile. Cap the vial, heat to 55°, and
continue heating for 2 h. Cool to room temperature,
and add 5 mL of water. Allow to stand for 5 min.
Quantitatively transfer the solution to a 25-mL volumet-
ric flask, and dilute with acetonitrile to volume. Dilute a
volume of this solution with Mobile phase to obtain a
concentration of 2.0 ug/mL of USP Choline Chloride RS.
Sample solution: Transfer 110 mg of Choline Chloride
to a 24-mL peo anpen vial. Dry at 120° for 2 h. Add
400 mg of 3,5-dinitrobenzoy! chloride and 10 mL of ac-
etonitrile. Cap the vial, heat to 55°, and continue heat-
ing for 2 h. Cool to room temperature, and add 5 mL
of water. Allow to stand for 5 min. Quantitatively trans-
fer the solution to a 50-mL volumetric flask, and dilute
with Mobile phase to volume. Pipet 2.0 mL of the solu-
tion to a 25-mL volumetric flask, and dilute with Mobile
phase to volume.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 208 nm
Column: 4.6-mm x 25-cm; packing L7
Column temperature: 30°
Flow rate: 1.0 mL/min
Injection size: 20 pL
System suitability
Sample: Standard solution
Suitability requirements
Capacity factor (k’): NLT 2

Relative standard deviation: NMT 5%, determined

in the formula below to replace Wwith V, which
equals 10 mL.
Suitability requirements: The total change is NLT 10
mV for a 0.4-mL cumulative addition of the Standard
solution; the amount of trimethylamine hydrochloride
found is 8.5-11.5 tg/L.
Analysis
Samples: Standard solution and Sample solution
Rinse the electrode, insert it into the Sample solution,
stir, and record the potential, in mV. Add 0.100 mL of
the Standard solution, and record the potential. Add
another 0.100 mL of the Standard solution, and record
the potential. [NOTE—If the total change after the sec-
ond addition of the Standard solution is less than 10
mV, add a third aliquot of 0.200 mL.]
Calculate the content, in ug/g, of total amines as tri-
ae hydrochloride in the portion of sample
taken:
Result = (Cs x Va)/[(F- 1) x W]
from the choline derivative peak
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of each impurity in the por-
tion of Choline Chloride taken:
Result = (ru/rs) x (Cs/Cy) x 100
ry = peak response for each impurity, excluding
that for the choline derivative and 3,5-
dinitrobenzoic acid from the Sample solution
rs = peak response for the choline derivative from
the Standard solution
Cs = concentration of USP Choline Chloride RS in
the Standard solution (mg/mL)
Cu = concentration of Choline Chloride in the
Sample solution (mg/mL)
USP 41
Acceptance criteria
Individual impurities: NMT 0.3%
Total impurities: NMT 2.0%
SPECIFIC TESTS
e PH (791): 4.0-7.0, in a solution (1 in 10)
© WATER DETERMINATION, Method | (921): NMT 0.5%
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in well-closed
containers.
e USP REFERENCE STANDARDS (11)
USP Choline Chloride RS
Chondroitin Sulfate Sodium
Chondroitin, hydrogen sulfate, sodium salt [9082-07-9].
DEFINITION
Chondroitin Sulfate Sodium is the sodium salt of the
sulfated linear glycosaminoglycan obtained from bovine,
porcine, or avian cartilages of healthy and domestic ani-
mals used for food by humans. Chondroitin Sulfate So-
dium consists mostly of the sodium salt of the sulfate es-
ter of N-acetylchondrosamine (2-acetamido-2-deoxy-B-D-
galactopyranose) and D-glucuronic acid copolymer. These
hexoses are alternately linked B-1,4 and B-1,3 in the poly-
mer. Chondrosamine moieties in the prevalent glycosami-
noglycan are monosulfated primarily on position 4 and
less so on position 6. It contains NLT 90.0% and NMT
105.0% of chondroitin sulfate sodium, calculated on the
dried basis.
[Note—Chondroitin Sulfate Sodium is extremely hygro-
scopic once dried. Avoid exposure to the atmosphere,
weigh promptly.]
IDENTIFICATION
e A. INFRARED ABSORPTION (197K)
e B. IDENTIFICATION TESTS—GENERAL (191), Sodium
Sample solution: 0.5 g in 10 mL of water
Acceptance criteria: Meets the requirements
e C, DISACCHARIDE COMPOSITION: The chromatogram of the
enzymatically digested Sample solution as obtained in the
test for Limit of Nonspecific Disaccharides shows three
main peaks corresponding to dehydrated glucuronic
acid-[1—3]-chondrosamine-4-sulfated (ADi-4S), dehy-
drated glucuronic acid-[1 >3]-chondrosamine-6-sulfated
(ADi-6S), and nonsulfated dehydrated glucuronic acid-
[1-43]-chondrosamine (ADi-0S) in the enzymatically di-
gested Standard solution. By peak-area response, ADi-4S
is the most abundant, followed by ADi-6S, with ADi-0S
being the least abundant of the three. The ratio of the
peak response of the ADi-4S to the ADi-6S is NLT 1.0.
e D. SPECIFIC ROTATION: Meets the requirements for Optical
Rotation (7815S), Specific Rotation in Specific Tests
COMPOSITION
e CONTENT OF CHONDROITIN SULFATE SODIUM
Standard solutions: 1.5, 1.0, and 0.5 mg/mL of USP
Chondroitin Sulfate Sodium RS in water
Sample solution: Transfer 100 mg of dried Chondroitin
Sulfate Sodium to a 100-mL volumetric flask, dissolve in
30 mL of water, and dilute with water to volume.
Diluent: Weigh about 297 mg of monobasic potassium
phosphate, 492 mg of dibasic potassium phosphate,
and 250 mg of polysorbate 80, and transfer to a 1-L
beaker. Dissolve in 900 mL of water, and adjust with
potassium hydroxide or phosphoric acid to a pH of 7.0
+ 0.2. Dilute with water to 1 L, and mix thoroughly.
Titrimetric system
(See Titrimetry (541).)
Dietary Supplements / Chondroitin 4539
Mode: Photometric titration
Titrant: 1 mg/mL of cetylpyridinium chloride in water.
Degas before use.
Endpoint detection: Turbidimetric with a photo-
electric probe
Analysis
Samples: Standard solutions, Sample solution, and
Diluent
Transfer 5.0 mL each of the Standard solutions and the
Sample solution to separate titration vessels, and add
25 mL of Diluent to each. Stir until a steady reading is
obtained with a phototrode either at 420, 550, or 660
nm. Set the instrument to zero in absorbance mode.
Titrate with Titrant using the phototrode to determine
the endpoint turbidimetrically. From a linear regression
equation, calculated using the volumes of Titrant con-
sumed versus concentrations of the Standard solutions,
determine the concentration of chondroitin sulfate so-
dium in the Sample solution.
Calculate the percentage of chondroitin sulfate sodium
in the portion of Chondroitin Sulfate Sodium taken:
Result = (C/Cy) x 100
Cc = concentration of chondroitin sulfate sodium in
the aliquot of the Sample solution, obtained
from the regression equation (mg/mL)
Cu = concentration of Chondroitin Sulfate Sodium
in the Sample solution (mg/mL)
Acceptance criteria: 90.0%-105.0% on the dried basis
IMPURITIES
° FESDUE ON IGNITION (281): 20.0%-30.0% on the dried
asis
e CHLORIDE AND SULFATE (221), Chloride: NMT 0.50%; a
0.10-g portion shows no more chloride than corresponds
to 0.7 mL of 0.020 N hydrochloric acid.
e CHLORIDE AND SULFATE (221), Sulfate
Sample solution: Dissolve 200 mg in 40 mL of water.
Add 10 mL of a 30-mg/mL solution of cetylpyridinium
chloride, pass through afilter, and use a 25-mL portion
of the filtrate.
Acceptance criteria: NMT 0.24%; the Sample solution
shows no more sulfate than corresponds to 0.25 mL of
0.020 N sulfuric acid.
© ELECTROPHORETIC PURITY
[CauTion—Voltages used in electrophoresis can readily de-
liver a lethal shock. The hazard is increased by the use of
aqueous buffer solutions and the possibility of working in
damp environments. The equipment, with the possible
exception of the power supply, should be enclosed in ei-
ther a grounded metal case or a case made of insulating
sydeibouo-: sa
material. The case should have an interlock that deener-
gizes the power supply when the case is opened, after
which reactivation should be prevented until activation of
a reset switch is carried out. High-voltage cables from the
power supply to the apparaty should preferably be a
type in which a braided metal shield completely encloses
the insulated central conductor, and the shield should be
grounded. The base of the apparatus should be grounded
metal or contain a grounded metal rim that is constructed
in such a way that any leakage of electrolyte will produce
a short that will deenergize the power supply before the
electrolyte can flow beyond the protective enclosure. If
the power supply contains capacitors as part ofa filter
circuit, it should also contain a bleeder resistor to ensure
discharge of the capacitors before the protective case is
opened. A shorting bar that is activated by opening the
case may be considered as an added precaution. Because
of the potential hazard associated with electrophoresis,
laboratory personnel should be completely familiar with
electrophoresis equipment before using it.]
Barium acetate buffer: Dissolve 25.24 g of barium ace-
tate in 900 mL of water. Adjust with acetic acid to a pH
of 5.0, and dilute with water to 1000 mL.
 4540 Chondroitin / Dietary Supplements
Staining reagent: Dissolve 1 g of toluidine blue in
1000 mL of 0.1 M acetic acid.
Standard solution A: 30 mg/mL of USP Chondroitin
Sulfate Sodium RS in water
Standard solution B: Dilute 1 mL of Standard solution A
with water to 50 mL.
Sample solution: 30 mg/mL of Chondroitin Sulfate So-
dium in water
Analysis: Fill the chambers of an electrophoresis appa-
ratus suitable for separations on cellulose acetate mem-
branes? (a small submarine gel chamber or one dedi-
cated to membrane media) with Barium acetate buffer.
Soak a cellulose acetate membrane, 5-6 cm x
12-14 cm, in Barium acetate buffer for 10 min, or until
evenly wetted, then blot dry between two sheets of ab-
sorbent paper. Using an applicator? suitable for electro-
phoresis, apply equal volumes (0.5 pL) of Standard solu-
tion A, Standard solution B, and Sample solution to the
brighter side of the membrane held in position in an
appropriate applicator stand or on a separating bridge
in the chamber. Ensure that both ends of the mem-
brane are dipped at least 0.5—1.0 cm deep into the
buffer chambers. Apply a constant 60 V (6 mA at the
start) for 2 h. [Note Perform the application of solu-
tions and voltage within 5 min because further drying
of the blotted paper reduces sensitivity.]
Place the membrane inaplastic staining tray, and with
the application side down, float or gently immerse in
Staining reagent for 5 min. Then stir the solution gen-
tly for 1 min. Remove the membrane, and destain in
5% acetic acid until the background clears. Compare
the bands. [NoTE—Document the results by taking a
picture within 15 min of completion of destaining.]
Acceptance criteria: The electropherogram from the
Sample solution exhibits a major band that is identical in
osition to the band from Standard solution A. The
and from Standard solutionB is clearly visible at a mo-
bility similar to the band from Standard solution A. Any
secondary band in the electropherogram of the Sample
solution is not more intense than the band from Stan-
dard solution B. NMT 2% of any individual impurity is
found. [NoTE—Document the results by taking a picture
within 15 min of completion of destaining.]
e LIMIT OF PROTEIN
Solution A: 20 mg/mL of sodium tartrate dihydrate
Solution B: 10 mg/mL of cupric sulfate
Solution C: 20 mg/mL of Satveats sodium carbonate
in 0.1 M sodium ydroxide
Dilute Folin-Ciocalteu reagent: Dilute Folin-Ciocalteu
phenol TS with water (1:5). Prepare immediately before
use.
DS Monographs
Alkaline cupric tartaric reagent: Mix 1 mL each of So-
lution A and Solution B, and to the mixture slowly add
100 mL of Solution Cwith stirring. Use within 24 h, and
discard afterward.
Standard solution: 36 g/mL of bovine serum albumin
certified standard in water
Sample solution: Transfer a portion of Chondroitin Sul-
fate Sodium, equivalent to 60 mg of the dried sub-
stance, to a 100-mL volumetric flask, and dissolve in
and dilute with water to volume.
Instrumental conditions
(See Ultraviolet-Visible Spectroscopy (857).)
ae wavelength: 750 nm
Blank: Water
Analysis
Samples: Standard solution, Sample solution, and Blank
Add 2.0 mL of freshly prepared Alkaline cupric tartaric
reagent to test tubes containing 2.0 mL of the Stan-
1 Suitable cellulose acetate membranes for electrophoresis are available from
Malta Chemetron SRL, Milano, Italy; Fluka Chemical Corp., Milwaukee, WI;
and DiaSys Corp., Waterbury, CT (www.diasys.com).
2 Surtable applicators are available from DiaSys Corp., Waterbury, CT (www.
diasys.com) and Helena Laboratories, Beaumont, TX (www.helena com).
USP 41
dard solution, 2.0 mL of the Sample solution, or 2.0 mL
of the Blank. After 10 min, add 1.0 mL of Dilute Folin-
Ciocalteu reagent to each test tube, and mix immedi-
ately and vigorously. After 30 min, measure the ab-
sorbance of the Standard solution and Sample solution
against the Blank.
Acceptance criteria: NMT 6.0% on the dried basis; the
absorbance of the Sample solution is NMT the absorb-
ance of the Standard solution.
CONTAMINANTS
¢ MICROBIAL ENUMERATION TESTS (2021): The total bacterial
count does not exceed 103 cfu/g, and the total com-
bined molds and yeasts count does not exceed 10? cfu/
9.
e ABSENCE OF SPECIFIED MICROORGANISMS (2022): It meets
the requirements of the tests for absence of Salmonella
species and Escherichia coli.
Delete the following:
°e HEAVY MeTALs, Method |i (231): NMT 20 ppme (oie).
Jan-2018)
SPECIFIC TESTS
e LIMIT OF NONSPECIFIC DISSACCHARIDES
Solution A: Water adjusted with 0.1 N hydrochloric
acid to a pH of 3.5
Solution B:_ 1M sodium chloride adjusted with 0.1 N
hydrochloric acid to a pH of 3.5
Mobile phase: See Table 7.
Table:
Time Solution A Solution B
(min) (%) (%)
0.0 100 0
45 100 0
a0 61 36
-
214 100 O
Buffer solution: 50 mM tris(hydroxymethyl)amino-
methane and 60 mM sodium acetate, adjusted with di-
luted hydrochloric acid to a pH of 8.0
Blank: Water
Chondroitinase AC solution: Use appropriate chon-
droitinase AC that is capable of cleaving the N-
acetylhexosaminide linkage in chondroitin 4-sulfate and
chondroitin 6-sulfate, yielding A‘-unsaturated disaccha-
rides (ADi-0S, ADi-4S, and ADi-6S). The working con-
centration of the chondroitinase AC in Buffer solution
must be sufficient for a complete digestion and meet
the enzyme suitability requirement that follows.
[Note—If Chondroitinase AC from Arthrobacter auresens?
is used, 0.2 Units/mL in Buffer solution is a typical
working concentration; if Chondroitinase AC from
Flavobacterium hepariums is used, 3 Units/mL in Buffer
solution is a typical working concentration. The work-
ing enzyme concentration may be increased if a com-
plete digestion could not be achieved. The working
enzyme aliquots should be stored at —20° when not in
use for a period of time to avoid a decrease in the
enzyme activity. A working enzyme solution is typically
stable for 4 days when stored at 4°.]
Enzyme suitability: Dilute the digested Standard solu-
tion and digested Blank (see Analysis section) (1 in 10),
and measure the absorbance at 230 nm in 1-cm path
cells. Make correction with the diluted Blank.
2 Chondroitinase AC from Arthrobacter auresens, Chromadex, part number
ASB-00003613-10.
4 Chondroitinase AC from Flavobacterium heparium, 2200 units/mg protein,
Sigma-Aldrich, catalog number £2039.
USP 41
Calculate the absorptivity of USP Chondroitin Sulfate
Sodium RS:
Result = A/(C x D x d)
A = absorbance of the diluted and digested
Standard solution
G = concentration of USP Chondroitin Sulfate
Sodium RS in the Standard solution (mg/mL)
D = oo factor of digested Standard solution
W/sS
d = dilution factor for the UV measurement (1/10)
Enzyme suitability requirement: The absorptivity of
the digested USP Chondroitin Sulfate Sodium RS is NLT
8 AU-mL-mg"-cm-,
Standard solution: 2.4 mg/mL of dried USP Chondroi-
tin Sulfate Sodium RS in water
Sample solution: Transfer about 250 mg of dried (105°
for 4 h) Chondroitin Sulfate Sodium to a 100-mL volu-
metric flask, and dissolve in and dilute with water to
volume.
System suitability solution: Add 1 volume of Standard
solution to 1 volume of Sample solution.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 230 nm
Column: 4.6-mm x 25-cm; 5-11m packing L14
Flow rate: 1 mL/min
Injection volume: 25 pL
[Note—The Injection volume may be decreased to im-
prove the peak shape of the analytes.]
System suitability
Samples: Standard solution, Sample solution, and Sys-
tem suitability solution (prepared as directed for Sam-
ples in the Analysis)
[Note—The relative retention times for the ADi-OS, ADi-
6S, and ADi-4S peaks are 0.80, 0.97, and 1.0,
respectively.]
Suitability requirements
Chromatogram similarity: The chromatogram of the
Standard solution is similar to that of the reference
chromatogram provided with USP Chondroitin Sul-
fate Sodium RS.
Resolution: NLT 1.0 between the ADi-4S and ADi-6S
peaks, Standard solution
Recovery factor: NLT 95% of the USP Chondroitin
Sulfate Sodium RS added to the Sample solution
[Note—This test is intended to demonstrate the ab-
sence of enzyme inhibition by impurities in the articles
being tested. Performance of this test is required only
for the articles being tested not meeting the Accep-
tance criteria. The Recovery factor can be calculated as
follows:
Result = {[(2 x Zrsy) — Zru]/Ers} x 100
Xrsy = sum of the peak areas of ADi-0S, ADi-4S, and
ADi-6S from the System suitability solution
=u = sum of the peak areas of ADi-0S, ADi-4S, and
ADi-6S from the Sample solution
Zrs = sum of the peak areas of ADi-0S, ADi-4S, and
ADi-6S from the Standard solution}
Analysis
Samples: Blank, Standard solution, Sample solution, and
System suitability solution
In four separate vials, combine 4 volumes (e.g., 800 uL)
of Chondroitinase AC solution with 1 volume (e.g.,
200 uL) each of Standard solution, Sample solution, Sys-
tem suitability solution, and Blank. Mix thoroughly. In-
cubate at 37° for 3 h. [NoTeE—the incubation period
may be increased, if necessary, to complete the diges-
tion.] Allow the solutions to cool before injection.
Dietary Supplements / Chondroitin 4541
Calculate the percentage of specific disaccharides in the
portion of Chondroitin Sulfate Sodium taken:
Result = (Zru/Ers) x (Cs/Cu) x 100
Zr = sum of the peak areas of ADi-0S, ADi-4S, and
ADi-6S from the Sample solution
<rs = sum of the peak areas of ADi-0S, ADi-4S, and
ADi-6S from the Standard solution
Cs = concentration of chondroitin sulfate sodium in
the Standard solution (mg/mL)
Cy = concentration of Chondroitin Sulfate Sodium
in the Sample solution (mg/mL)
Calculate the content of nonspecific disaccharides in the
sample taken:
Result = CSC — SDC
CSC = chondroitin sulfate sodium content from the
oo for Content of Chondroitin Sulfate Sodium
(%
SDC = specific disaccharides content (%)
Acceptance criteria: NMT 10.0%
¢ CLARITY AND COLOR OF SOLUTION
Sample solution: Transfer 2.5 g of Chondroitin Sulfate
Sodium to a 50-mL volumetric flask. Dissolve in and
dilute with carbon dioxide-free water to volume, and
examine immediately.
Instrumental conditions
(See Ultraviolet-Visible Spectroscopy (857).)
Analytical wavelength: 420 nm
Cell: 1.cm
Blank: Carbon dioxide-free water
Analysis: Measure the absorbance of the Sample
solution.
Acceptance criteria: NMT 0.35
© OPTICAL ROTATION (7815), Specific Rotation
Sample solution: 30 mg/mL
Acceptance criteria: -—20.0° to —30.0°
© PH (791): 5.5-7.5, in a solution (1 in 100)
e Loss ON DRYING (731)
[Note—Chondroitin Sulfate Sodium is extremely hygro-
scopic once dried. Avoid exposure to the auriesprers,
and weigh promptly.]
Analysis: Dry at 105° for 4 h.
Acceptance criteria: NMT 12.0%
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in tight containers.
e LABELING: Label it to state the source(s) from which the
article was derived, whether bovine, porcine, avian, or a
mixture of any of them.
sydesbouo=: sa
e USP REFERENCE STANDARDS (11)
USP Chondroitin Sulfate Sodium RS
Chondroitin Sulfate Sodium Tablets
DEFINITION
Chondroitin Sulfate Sodium Tablets contain NLT 90.0% and
NMT 120.0% of the labeled amount of chondroitin sul-
fate sodium.
[Note—Chondroitin Sulfate Sodium is extremely hygro-
scopic once dried. Avoid exposure to the atmosphere,
and weigh promptly.]
IDENTIFICATION
© A, ELECTROPHORESIS
Barium acetate buffer: Dissolve 25.24 g of barium ace-
tate in 900 mL of water. Adjust with acetic acid to a pH
of 5.0, and dilute with water to 1000 mL.
Staining reagent: 0.1% (w/v) toluidine blue in 0.1 M
acetic acid
 4542 Chondroitin / Dietary Supplements
Standard solution: Use the Standard solution of middle
concentration from the Content of Chondroitin Sulfate
Sodium.
Sample solution: Prepare as directed in the Content of
Chondroitin Sulfate Sodium.
Analysis: Fill the chambers of an electrophoresis appa-
ratus suitable for separations on cellulose acetate mem-
branes’ (a small submarine gel chamber or one dedi-
cated to membrane media) with Barium acetate buffer.
Soak a cellulose acetate membrane 5-6 cm x 12-14 cm
in Barium acetate buffer for 10 min, or until evenly wet-
ted, then blot dry between two sheets of absorbent pa-
per. Using an applicator? suitable for electrophoresis,
apply equal volumes (0.5 iL) of the Sample solution and
Standard solution to the brighter side of the membrane
held in position in an appropriate epplentan stand or
on a separating bridge in the chamber. Ensure that
both ends of the membrane are dipped at least
0.5-1.0-cm deep into the buffer chambers. Apply a
constant 60 volts (6 mA at the start) for 2 h. [NOTE—
Perform the application of solutions and voltage within
5 min because further drying of the blotted paper
reduces sensitivity.]
Place the membrane inaplastic staining tray, and with
the application side down, float or gently immerse in
Staining reagent for 5 min. Then stir the solution gen-
tly for 1 min. Remove the membrane, and destain in
5% acetic acid until the background clears.
Acceptance criteria: The principal spot from the Sam-
ple solution has the same migration as the principal spot
from the Standard solution. [Note—Document the re-
sults by taking a picture within 15 min of completion of
destaining.]
STRENGTH
@ CONTENT OF CHONDROITIN SULFATE SODIUM
Standard solutions: 1.5, 1.0, and 0.5 mg/mL of USP
Chondroitin Sulfate Sodium RS in water
Sample solution: Transfer an equivalent to 100 mg of
chondroitin sulfate sodium from NLT 20 Tablets, finely
powdered, to 60 mL of water, and shake to suspend
the powder in solution. Sonicate in a 65° water bath for
20 min. Remove from the bath, stir or shake for 5 min,
dilute with water to 100 mL, and centrifuge or pass
throughasuitable filter.
Diluent: Weigh about 297 mg of monobasic potassium
phosphate, 492 mg of dibasic potassium phosphate,
and 250 mg of polysorbate 80, and transfer into a 1-L
beaker. Dissolve in approximately 900 mL of water, and
adjust with potassium hydroxide or phosphoric acid to
a pH of 7.0 + 0.2. Dilute with water to 1 L, and mix
DS Monographs
thoroughly.
Titrimetric system
(See Titrimetry (541).)
Mode: Photometric titration
Titrant: 1 mg/mL of cetylpyridinium chloride in water
Endpoint detection: Turbidimetric with photoelectric
robe
Analyste Transfer 5.0 mL of each Standard solution and
the Sample solution to separate titration vessels, and add
25 mL of Diluent to each. Stir until a steady reading is
obtained with a photoelectric probe either at 420, 550,
or 660 nm. Setthe instrument to zero in absorbance
mode. Titrate with Titrant using the photoelectric probe
to determine the endpointbee ete eI From a lin-
ear regression equation, calculated using the volumes of
1 Suitable cellulose acetate membranes for electrophoresis are available from
Fluka Chemical Corp., Milwaukee, WI; and DiaSys Corp., Waterbury, CT
(www.diasys com).
2 Suitable applicators are available from DiaSys Corp , Waterbury, CT (www.
diasys.com) and Helena Laboratories, Beaumont, TX (www.helena.com).
USP 41
Titrant consumed versus concentrations of the Standard
solutions, determine the concentration of chondroitin
sulfate sodium in the Sample solution.
Calculate the percentage of the labeled amount of
chondroitin sulfate sodium in the portion of Tablets
taken:
Result = (C/Cu) x 100
Cc = determined concentration of chondroitin
sulfate sodium in the Sample solution
Cu
(mg/ml)
= nominal concentration
of chondroitin sulfate
sodium in the Sample solution (mg/mL)
Acceptance criteria: 90.0%-120.0% of the label claim
PERFORMANCE TESTS
e DISINTEGRATION AND DISSOLUTION OF DIETARY SUPPLEMENTS
(2040): Meet the requirements for Dissolution
Medium: Water; 900 mL
Apparatus 2: 75 rpm
Time: 60 min
Titrant and Diluent: Prepare as directed as in Content
of Chondroitin Sulfate Sodium.
Standard solutions: 1.5, 1.0, and 0.5 mg/mL of USP
Chondroitin Sulfate Sodium RS in water
Sample solution: Combine equal portions of the solu-
tions withdrawn from 6 dissolution vessels and pass
throughasuitable filter; use the pooled sample as the
test specimen.
Analysis: Transfer 5.0 mL of each Standard solution, and
an aliquot of the Sample solution equivalent to about
5 mg of chondroitin sulfate sodium, to separate titration
vessels. Add 25 mL of Diluent to each titration vessel.
Stir until a steady reading is obtained with a photo-
electric probe. Set the instrument to zero in absorbance
mode. Titrate with Titrant using the photoelectric probe
to determine the endpoint turbidimetrically, either at
420, 550, or 660 nm. Froma linear regression equa-
tion, calculated using the volumes of Titrant consumed
versus amount, in mg, of chondroitin sulfate sodium
from each Standard solution, determine the amount, in
mg, of chondroitin sulfate sodium in the aliquot of
Sample solution taken.
Calculate the percentage of the labeled amount of
chondroitin sulfate sodium dissolved:
Result = (Ws/a) x (V/L) x 100
Ws = amount of chondroitin sulfate sodium in the
aliquot of the Sample solution taken (mg)
a = volume of the aliquot of Sample solution taken
Vv = volume of Medium, 900 mL
L = label claim of chondroitin sulfate sodium
(mg/Tablet)
Tolerances: NLT 75% of the labeled amount of chon-
droitin sulfate sodium is dissolved.
e@ WEIGHT VARIATION OF DIETARY SUPPLEMENTS (2091): Meet
the requirements
ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Preserve in tight, light-resistant
containers, and store at room temperature.
© LABELING: Label it to indicate the species of the source
from which the chondroitin used to prepare the Tablets
was derived. Label it to state the source(s) of chondroitin
sulfate sodium, whether bovine, porcine, avian, or a mix-
ture of any of them. The label states on the front panel
the content of chondroitin sulfate sodium on the dried
basis.
USP 41 Dietary Supplements / Chondroitin 4543

e USP REFERENCE STANDARDS (11) cally. From a linear regression equation, calculated us-
USP Chondroitin Sulfate Sodium RS ing the volumes of Titrant consumed versus
concentrations of the Standard solutions, determine the
concentration of chondroitin sulfate sodium in the Sam-
ple solution.
Calculate the percentage of chondroitin sulfate sodium
Chondroitin Sulfate Sodium, Shark inahe portion of Chondroitin Sulfate Sodium, Shark
taken:
Chondroitin, hydrogen sulfate, sodium salt [9007-28-7].
Result = (C/Cy) x 100
DEFINITION
Chondroitin Sulfate Sodium, Shark is the sodium salt of the iC. = concentration of chondroitin sulfate sodium in
sulfated linear glycosaminoglycan obtained from shark the aliquot of the Sample solution, obtained
cartilages used for human foods. Chondroitin Sulfate So- from the regression equation (mg/mL)
dium, Shark consists mostly of the sodium salt of the sul- Cu = concentration of Chondroitin Sulfate Sodium,
fate ester of N-acetylchondrosamine (2-acetamido-2-de- Shark in the Sample solution (mg/mL)
oxy-B-D-galactopyranose) and D-glucuronic acid Acceptance criteria: 90.0%-105.0% on the dried basis
copolymer. These hexoses are alternately linked B-1,4 and e DISACCHARIDE COMPOSITION
B-1,3 in the polymer. Chondrosamine moieties in the Solution A: Water adjusted with 0.1 N hydrochloric
prevalent glycosaminoglycan are monosulfated primarily acid to a pH of 3.5
on position 6 and less so on position 4 with minor disulfa- Solution B: 1M sodium chloride adjusted with 0.1 N
tion on both positions 4 and 6. NLT 8% of the D- hydrochloric acid to a pH of 3.5
glucuronic acid moieties are monosulfated on position 2. Mobile phase: See Table 1.
It contains NLT 90.0% and NMT 105.0% of chondroitin
sulfate sodium, calculated on the dried basis. Table 1
[Nott—Chondroitin Sulfate Sodium, Shark is extremely hy-
groscopic once dried. Avoid exposure to the atmosphere, Time Solution A Solution B
and weigh promptly.] (min) (%) (%)
0.0 100 0
IDENTIFICATION 4.0 100 0
e A. INFRARED ABSORPTION (197K) 45.0 50 50
e B. IDENTIFICATION TESTS—GENERAL (191), Sodium
Sample solution: 0.5 g in 10 mL of water 45.1 100 0
Acceptance criteria: Meets the requirements Buffer solution: 50 mM tris(hydroxymethyl)amino-
© C. SPECIFIC DISACCHARIDES: The chromatogram of the en-
methane and 60 mM sodium acetate, adjusted with di-
zymatically aD Sample solution as obtained in the
test for Disaccharide Composition shows three main peaks luted hydrochloric acid to a pH of 8.0
due to 6-sulfated (ADi-6S), 4-sulfated (ADi-4S), and 2,6- Standard solution: 2.4 mg/mL of dried USP Chondroi-
tin Sulfate Sodium, Shark RS in water
disulfated (ADi-2,6diS) disaccharides, corresponding to Sample solution: Transfer about 250 mg of dried
those of the enzymatically digested Standard solution,
Chondroitin Sulfate Sodium, Shark to a 100-mL volu-
with ADi-6S being the most abundant, followed by ADi-
metric flask, and dissolve and dilute with water to vol-
4S, with NLT 8% corresponding to ADi-2,6diS. Additional
minor peaks corresponding to nonsulfated (ADi-0S) and ume. Filter to obtain a clear solution.
4,6 disulfation may be detected.
Blank: Water
e D. SPECIFIC ROTATION: Meets the requirements in the Spe- Chondroitinase ABC solution: Dissolve 1 unit (U/mg of
cific Tests protein) of chondroitinase ABC! in 1.0 mL of Buffer solu-
tion. Mix thoroughly.
COMPOSITION Chondroitinase ABC solution suitability: Dilute the in-
¢ CONTENT OF CHONDROITIN SULFATE SODIUM cubated Standard solution (1 in 10), and measure its
Standard solutions: 1.5, 1.0, and 0.5 mg/mL of dried absorbance against the incubated Blank at 232 nm. The
USP Chondroitin Sulfate Sodium, Shark RS in water absorptivity is NLT 8 AU» mL- mg". cm".
Sample solution: Transfer 100 mg of dried Chondroitin Chromatographic system
ytrelPleo VG

Sulfate Sodium, Shark into a 100-mL volumetric flask, (See Chromatography (621), System Suitability.)
dissolve in 30 mL of water, and dilute with water to Mode: LC
volume. Detector: UV 232 nm
Diluent: Weigh about 297 mg of monobasic potassium Column: 4.6-mm x 25-cm; 5-4um packing L14
phosphate, 492 mg of dibasic potassium phosphate, Flow rate: 1 mL/min
and 250 mg of polysorbate 80, and transfer to a 1-L Injection volume: 20 uL
beaker. Dissolve in 900 mL of water, and adjust with System suitability
potassium hydroxide or phosphoric acid to a pH of 7.0 Sample: Standard solution (prepared per Analysis
+ 0.2. Dilute with water to 1 L, and mix thoroughly. below)
Titrimetric system [Note—The relative retention times for the ADi-0S, ADi-
(See Titrimetry (541).) 6S, ADi-4S, and ADi-2,6diS peaks are 0.50, 0.75, 0.80,
Mode: Photometric titration and 1.0, respectively.]
Titrant: 1 mg/mL of cetylpyridinium chloride in water. Suitability requirements
Degas before use. Chromatogram similarity: The chromatogram of the
Endpoint detection: Turbidimetric with a photo- Standard solution is similar to the reference chromato-
electric probe gram provided with USP Chondroitin Sulfate Sodium,
Analysis: Transfer 5.0 mL each of the Standard solution Shark RS.
and the Sample solution to separate titration vessels, and Resolution: NLT 2.0 between the ADi-6S and ADi-4S
add 25 mL of Diluent to each. Stir until a steady reading peaks
is obtained with the photoelectric probe set either at Relative standard deviation: NMT 5.0% for the ADi-
420, 550, or 660 nm. Set the instrument to zero in 6S, ADi-4S, or ADi-2,6diS peaks
absorbance mode. Titrate with Titrant using the photo- 1 Chondroitinase ABC from Proteus vulgaris is available from Sigma (www.
electric probe to determine the endpoint turbidimetri- sigmaaldrich.com), Catalog Number C3667.
 4544 Chondroitin / Dietary Supplements
Analysis
Samples: Standard solution, Sample solution, and Blank
In three separate vials, combine 0.8 mL of Buffer solu-
tion, 0.1 mL of Chondroitinase ABC solution, and 0.1 mL
each of the Standard solution, Sample solution, and
Blank. Mix thoroughly. Incubate at 37° for 3 h. Allow
the solution to cool to room temperature, and centri-
fuge prior to injection.
Calculate the percentage of each disaccharide in the
sample taken:
Result = (ru/Zru) x 100
tu = peak area of ADi-0S, ADi-6S, ADi-4S, or ADi-
2,6diS from the Sample solution
Xr = sum of the peak areas of ADi-0S, ADi-6S, ADi-
4S, and ADi-2,6diS from the Sample solution
Acceptance criteria: The area percentage of the ADi-6S
peak is greater than that of the ADi-4S peak, and the
area percentage of the ADi-2,6diS peak is the lowest of
the three. The area percentage of the ADi-2,6diS peak
is NLT 8%.
IMPURITIES
e RESIDUE ON IGNITION (281): 20.0%-30.0% on the dried
basis
© CHLORIDE AND SULFATE (221), Chloride
sn solution: 0.7 mL of 0.020 N hydrochloric
aci
Sample: 0.19
Acceptance criteria: NMT 0.50%
e CHLORIDE AND SULFATE (221), Sulfate
Standard solution: 0.25 mL of 0.020N sulfuric acid
Sample solution: Dissolve 200 mg in 40 mL of water.
Add 10 mL ofa solution of cetylpyridinium chloride
having a concentration of 30 mg/mL, and pass through
a filter. Use a 25-mL portion of the filtrate.
Acceptance criteria: NMT 0.24%; the Sample solution
shows no more sulfate than that of the Standard
solution.
e ELECTROPHORETIC PURITY
[CAUTION—Voltages used in electrophoresis can readily
deliver a lethal shock. The hazard is increased by the
use of aqueous buffer solutions and the possibility of
working in damp environments. The equipment, with
the possible exception of the power supply, should be
enclosed in either a grounded metal case or a case
made of insulating material. The case should have an
interlock that deenergizes the power supply when the
case is opened, after which reactivation should be pre-
vented until activation of a reset switch is carried out.
High voltage cables from the power supply to the appa-
DS Monographs
ratus should preferably be a type in which a braide
metal shield completely encloses the insulated central
conductor, and the shield should be grounded. The
base of the apparatus should be grounded metal or
contain a grounded metal rim which is constructed in
such a way that any leakage of electrolyte will produce
a short which will deenergize the power supply before
the electrolyte can flow beyond the protective enclo-
sure. If the power supply contains capacitors as part of
a filter circuit, it should also contain a bleeder resistor to
ensure discharge of the capacitors before the protective
case is opened. A shorting bar that is activated by
opening the case may be considered as an added pre-
caution. Because of the potential hazard associated with
electrophoresis, laboratory personnel should be com-
pletely familiar with electrophoresis equipment before
using it.]
Barlur acetate buffer: Dissolve 25.24 g of barium ace-
tate in 900 mL of water. Adjust with acetic acid to a pH
of 5.0, and dilute with water to 1000 mL.
Staining reagent: Dissolve 1 g of toluidine blue in
1000 mL of 0.1 M acetic acid.
USP 41
Standard solution A: 30 mg/mL of USP Chondroitin
Sulfate Sodium, Shark RS in water
Standard solution B: Dilute 1 mL of Standard solution A
with water to 50 mL.
Sample solution: 30 mg/mL of Chondroitin Sulfate So-
dium, Shark in water
Analysis: Fill the chambers of an electrophoresis appa-
ratus suitable for separations on cellulose acetate mem-
branes? (a small submarine gel chamber or one dedi-
cated to membrane media) with Barium acetate buffer.
Soak a cellulose acetate membrane, 5-6 cm x
12-14 cm, in Barium acetate buffer for 10 min, or until
evenly wetted, then blot dry between two sheets of ab-
sorbent paper. Using an applicator? suitable for electro-
phoresis, apply equal volumes (0.5 pL) of the Sample
solution, Standard solution A, and Standard solution B to
the brighter side of the membrane held in position in
an appropriate applicator stand or on a separating
bridge in the chamber. Ensure that both ends of the
membrane are dipped at least 0.5-1.0 cm deep into the
buffer chambers. Apply a constant 60 volts (6 mA at
the start) for 2 h. [NoTE—Perform the application of
solutions, and voltage within 5 min because further dry-
ing of the blotted paper reduces sensitivity.]
Place the membrane in a plastic staining tray, and with
the application side down, float or gently immerse in
the Staining reagent for 5 min. Then stir the solution
gently for 1 min. Remove the membrane, and destain
in 5% acetic acid until the background clears. Com-
pare the bands.
[Note—Document the results by taking a picture within
15 min of the completion of destaining.]
Acceptance criteria: The electropherogram from the
Sample solution exhibits a major band that is identical in
osition to the band from Standard solution A. The
and from Standard solutionB is clearly visible at a mo-
bility similar to the band from Standard solution A. Any
secondary band in the electropherogram of the Sample
solution is not more intense than the band from Stan-
dard solution B. NMT 2% of any individual impurity in
Chondroitin Sulfate Sodium, Shark is found.
e LIMIT OF PROTEIN
Solution A: 20 mg/ml of sodium tartrate dihydrate
Solution B: 10 mg/mL of cupric sulfate
Solution C: 20 mg/mL of anhydrous sodium carbonate
in 0.1 M sodium hydroxide
Dilute Folin-Ciocalteu reagent: Dilute Folin-Ciocalteu
phenol TS with water (1:5). Prepare immediately before
use.
Alkaline cupric tartaric reagent: Mix 1 mL each of So-
lution A and Solution B, and to the mixture slowly add
100 mL of Solution C with stirring. Use within 24 h, and
discard afterward.
Standard solution: 36 g/mL of bovine serum albumin
certified standard in water
Sample solution: Transfer a portion of Chondroitin Sul-
fate Sodium, Shark, equivalent to 60 mg of the dried
substance, to a 100-mL volumetric flask, and dissolve in
and dilute with water to volume.
Instrumental conditions
(See Ultraviolet-Visible Spectroscopy (857).)
Analytical wavelength: 750 nm
Blank: Water
Analysis
Samples: Standard solution, Sample solution, and Blank
Add 2.0 mL of freshly prepared Alkaline cupric tartaric
reagent to three test tubes, each containing 2.0 mL of
the Standard solution, 2.0 mL of the Sample solution, or
2.0 mL of the Blank. After 10 min, add 1.0 mL of Dilute
2 Suitable cellulose acetate membranes for electrophoresis are available from
Malta Chemetron SRL, Milano, Italy; Fluka Chemical Corp., Milwaukee, WI;
and Apacor Ltd., Berkshire, England (www.apacor.com/products/electropho-
resis/cellulose-acetate-membranes).
3 Suitable applicators are available from Apacor Ltd., Berkshire, England
(www.apacor.com/PDF/APA092-ElectrophoresisEquipmentSupplies.pdf) and
Helena Laboratories, Beaumont, TX (www.helena.com).
USP 41 Dietary Supplements / Chromium 4545

Folin-Ciocalteu reagent to each test tube, and mix im- DEFINITION

mediately and vigorously. After 30 min, measure the Chromium Picolinate contains NLT 98.0% and NMT
absorbance of the Standard solution and Sample solu- 102.0% of chromium picolinate (CisHi2N306Cn), calcu-
tion against the Blank. lated on the dried basis.
Acceptance criteria: NMT 6.0% on the dried basis; the
absorbance of the Sample solution is NMT the absorb- IDENTIFICATION
ance of the Standard solution. e A. INFRARED ABSORPTION (197M)
eB.
CONTAMINANTS Sample solution: 4mg/mL
e ELEMENTAL IMPURITIES—PROCEDURES (233) Analysis: To 5 mL of the Sample solution add 1 mL of
Acceptance criteria 5.N sodium hydroxide and 10 drops of 30% hydrogen
Arsenic: NMT 2.0 ug/g peroxide, and heat gently for 2 min.
Cadmium: NMT 1.0 ug/g Acceptance criteria: A yellow color develops.
Lead: NMT 1.0 g/g
Mercury: NMT 1.0 ug/g ASSAY
e MICROBIAL ENUMERATION TESTS (2021): The total bacterial © PROCEDURE
count does not exceed 103 cfu/g, and the total com- Standard stock solution: — 100 g/mL of chromium.
bined molds and yeasts count does not exceed 102 cfu/ Transfer 0.283 g of potassium dichromate, previously
g. dried at 120° for 4 h, to a 1000-mL volumetric flask,
e ABSENCE OF SPECIFIED MICROORGANISMS (2022): It meets and dilute with water to volume. Store in a polyethyl-
the requirements of the tests for absence of Salmonella ene bottle.
species and Escherichia coli. Standard solutions: 1.0, 2.0, 3.0, and 4.0 g/mL of
chromium. Separately transfer 1.0 and 2.0 mL of the
SPECIFIC TESTS Standard stock solution to 100-mL volumetric flasks, and
© CLARITY AND COLOR OF SOLUTION transfer 1.5 and 2.0 mL of the Standard stock solution to
Sample solution: Transfer 2.5 g of Chondroitin Sulfate separate 50-mL volumetric flasks. Add 1.0 mL of nitric
Sodium, Shark to a 50-mL volumetric flask. Dissolve in acid to each flask, and dilute the contents of each flask
and dilute with carbon dioxide-free water to volume, with water to volume.
and examine immediately. Sample solution: Transfer 200 mg of Chromium
Instrumental conditions Picolinate to a 100-mL beaker, and add 25 mL of water.
(See Ultraviolet-Visible Spectroscopy (857).) Slowly add 10 mL of nitric acid, and boil for 10 min
Analytical wavelength: 420 nm with constant swirling. Cool the solution, quantitatively
Cell: 1. cm transfer to a 500-mL volumetric flask, and dilute with
Blank: Carbon dioxide-free water water to volume. Filter a portion of the solution, and
Analysis: Measure the absorbance of the Sample transfer 5.0 mL of the filtrate to a 100-mL volumetric
solution. flask. Add 1 mL of nitric acid, and dilute with water to
Acceptance criteria: NMT 0.35 volume.
© OPTICAL ROTATION (7815), Specific Rotation Instrumental conditions
Sample solution: 30 mg/mL in water (See Atomic Absorption Spectroscopy (852).)
Acceptance criteria: —12.0° to —23.0° Mode: Atomic absorption spectrophotometry
e PH (791) Analytical wavelength: 357.9 nm
Sample solution: 10 mg/mL Lamp: Chromium hollow-cathode
Acceptance criteria: 5.5-7.5 Flame: Air-acetylene
e Loss ON DRYING (731) Blank: Diluted nitric acid
Analysis: Dry a sample at 105° for 4 h. [NotE—Chon- Analysis
droitin Sulfate Sodium, Shark is extremely hygroscopic Samples: Standard solutions and Sample solution
once dried. Avoid exposure to the atmosphere, and Determine the absorbances of the Standard solutions
weigh promptly.] and the Sample solution. Plot the absorbances of the
Acceptance criteria: NMT 12.0% Standard solutions versus the chromium concentra-
tion, in g/mL, and draw the straight line best fitting
ADDITIONAL REQUIREMENTS the four plotted points. From the graph so obtained,
© PACKAGING AND STORAGE: Preserve in tight containers. determine the chromium concentration, in g/mL, in 9
e LABELING: Label it to state the source(s) from which the the Sample solution. n“n
article was derived. Calculate the percentage of chromium picolinate =
e USP REFERENCE STANDARDS (11) (CisHi2N3O¢Cr) in the portion of Chromium Picolinate i}
USP Chondroitin Sulfate Sodium, Shark RS taken: |
5
Result = (Co/Cu) x (M/A) x 100
eo}<=
i)
Co = concentration of chromium in the Sample
ae}
7
Chromium Picolinate solution, obtained from the graph (g/mL) al

Cu = concentration of Chromium Picolinate in the

Sample solution (ug/ml)
M, = molecular weight of chromium picolinate,
418.31
Ar = atomic weight of chromium, 51.996
Acceptance criteria: 98.0%-102.0% on the dried basis
IMPURITIES
e CHLORIDE AND SULFATE, Chloride (221)
Sample solution: Dissolve 30 mg of Chromium
Picolinate in 30-40 mL of water, and heat to 70°. Cool
CisHi2N3O06Cr 418.30 overnight, and filter to remove the precipitate.
Chromium Tripicolinate [14639-25-9]. Analysis: Add 1 mL each of nitric acid and silver nitrate
TS, and add sufficient water to make 50 mL. Mix, and
 4546 Chromium / Dietary Supplements
allow to stand for 5 min, protected from direct
sunlight.
Acceptance criteria: Any turbidity formed is NMT that
produced in a similarly treated control solution contain-
ing 0.25 mL of 0.002 N hydrochloric acid (NMT
0.06%).
e CHLORIDE AND SULFATE, Sulfate (221)
Sample solution: Dissolve 100 mg of Chromium
Picolinate in 30-40 mL of water, and heat to 90°. Cool
overnight, and filter to remove the precipitate.
Analysis: Add 1 mL of 3 N hydrochloric acid, 3 mL of
barium chloride TS, and sufficient water to make 50 mL.
Mix, and allow to stand for 10 min.
Acceptance criteria: Any turbidity formed is NMT that
produced in a similarly treated control solution contain-
ing 0.2 mL of 0.02 N sulfuric acid (NMT 0.2%).
SPECIFIC TESTS
e Loss ON DRYING (731): Dry a sample at 105° for 4 h: it
loses NMT 4.0% of its weight.
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in tight containers.
o USP REFERENCE STANDARDS (11)
USP Chromium Picolinate RS
Chromium Picolinate Tablets
DEFINITION
Chromium Picolinate Tablets contain NLT 95.0% and NMT
125.0% of the labeled amount of chromium (Cr).
IDENTIFICATION
e A. The Sample solution prepared as directed in the test
for Strength gives a positive test for chromium, deter-
mined at 357.9 nm using the Instrumental conditions in
the test for Content of Chromium.
STRENGTH
e CONTENT OF CHROMIUM
Standard stock solution A: 1000 g/mL of chromium
from potassium dichromate, previously dried at 120° for
4h, in water. Store in a polyethylene bottle.
Standard stock solution B: Transfer 1.0 mL of Standard
stock solution A to a 100-mL volumetric flask, add
5.0 mL of 6 N hydrochloric acid, and dilute with water
to volume to obtain a solution having a concentration
of 10 ug/mL of chromium.
Standard solutions: Dilute Standard stock solution B
DS Monographs
with 0.125 N hydrochloric acid to obtain concentra-
tions of 1.0, 2.0, 3.0, and 4.0 g/mL of chromium.
Sample solution: Weigh and finely powder NLT 20
Tablets. Transfer a portion of the powder, equivalent to
5 Tablets, to a porcelain crucible, heat the crucible in a
muffle furnace maintained at about 550° for 6-12 h,
and cool. Add 60 mL of hydrochloric acid, and boil
gently on a hot plate or steam bath for 30 min, inter-
mittently rinsing the inner surface of the crucible with
6N hydrochloric acid. Cool, and transfer the contents
of the crucible to a 100-mL volumetric flask. Rinse the
crucible with small portions of 6 N hydrochloric acid,
and add the rinsings to the flask. Dilute with water to
volume, mix, and filter, discarding the first 5 mL of the
filtrate. Dilute this solution with 0.125 N hydrochloric
acid to obtain a solution having a concentration of
2.5 g/mL of chromium.
Instrumental conditions
(See Atomic Absorption Spectroscopy (852).)
USP 41
Mode: Atomic absorption spectrophotometry
Lamp: Chromium hollow-cathode
Flame: Air-acetylene
Analytical wavelength: 357.9 nm (chromium emis-
sion line)
Blank: 0.125 N hydrochloric acid
Analysis
Samples: Standard solutions and Sample solution
Determine the absorbances of the Samples, using the
Blank. From a linear regression equation, calculated
using the absorbances of the Standard solutions versus
the concentration, in g/mL, of chromium, determine
the concentration, C, in ug/mL, of chromium in the
Sample solution.
Calculate the percentage of the labeled amount of
chromium (Cr) in the portion of Tablets taken:
Result = (C/Cy) x 100
€ = determined concentration of chromium in the
Sample solution (ug/mL)
Cu = nominal concentration of chromium in the
Sample solution (\ug/mL)
Acceptance criteria: 95.0%-125.0%
PERFORMANCE TESTS
¢ DISINTEGRATION AND DISSOLUTION OF DIETARY SUPPLEMENTS
(2040): Meet the requirements for Disintegration
e WEIGHT VARIATION OF DIETARY SUPPLEMENTS (2091): Meet
the requirements
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in well-closed contain-
ers, and store at controlled room temperature.
Cinnamomum cassia Twig
DEFINITION
Cinnamomum cassia Twig consists of the dried twigs of Cin-
namomum cassia (L.) J.Presl [syn. Cinnamomum aro-
maticum Nees] (Fam. Lauraceae) collected in spring or
summer. It contains NLT 0.8% of total phenylpropanoids,
calculated as the sum of cinnamyl alcohol, cinnamic acid,
2-methoxycinnamic acid, cinnamaldehyde, and 2-methox-
ycinnamaldehyde on the anhydrous basis.
IDENTIFICATION
e A. HPTLC FOR ARTICLES OF BOTANICAL ORIGIN (203)
Standard solution A: 0.5 mg/mL each of USP Cin-
namaldehyde RS and coumarin in methanol
Standard solution B: USP Cinnamomum cassia Twig
Powder RS in methanol (1 in 5). Sonicate for 10 min,
centrifuge or filter, and evaporate the solvent. Suspend
the residue in a volume of toluene equivalent to one-
fifth the volume of methanol used for the extraction.
Sonicate for about 2 min, centrifuge or filter, and use
the supernatant or the filtrate.
sll solution: 2g of Cinnamomum cassia Twig,
finely powdered, in 10 mL of methanol. Sonicate for 10
min, centrifuge or filter, and transfer the extract to a
round-bottom flask. Evaporate the solution under re-
duced pressure to dryness. Dissolve the residue in 2 mL
of toluene, sonicate for about 2 min, centrifuge or filter,
and use the supernatant or the filtrate.
Chromatographic system
Adsorbent: Chromatographic silica gel F2s4 mixture
with an average particle size of about 5 um
Application volume: 6 uL, as 8-mm bands
Relative humidity: Condition the plate to a relative
humidity of about 33% usinga suitable device.
USP 41
Temperature: About 25°
Developing solvent system: Toluene and ethyl ace-
tate (1 9:19
Developing distance: 6 cm
Derivatization reagent: Methanol, acetic acid, sulfuric
acid, and p-anisaldehyde (170:20:10:1). [NoTE—Pre-
pare fresh. Slowly add sulfuric acid to ice-cold metha-
nol, followed by acetic acid and p-anisaldehyde.]
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Apply the Samples as bands and dry in air. Develop in a
saturated chamber (20 min with filter paper), remove
the plate from the chamber, and dry in air. Examine
under UV light at 254 nm. Then treat the plate with
the Derivatization reagent, heat at 100° for 4 min, and
examine under UV light at 366 nm.
System suitability
Samples: Standard solution A and Standard solution B
Suitability requirements: Prior to derivatization, under
UV light at 254 nm, Standard solution A exhibits a
uenching band due to cinnamaldehyde in the middle
third of the chromatogram, and a quenching band
due to coumarin in the upper part of the lower third.
Under UV light at 254 nm, Standard solution B exhib-
its, in the middle-third section, an intense quenching
band corresponding in R; to the cinnamaldehyde
band in Standard solution A. In the upper part of the
lower-third section, Standard solution B exhibits a
weak quenching band due to coumarin, a quenching
band close to the starting position due to the co-
elution of cinnamic acid and 2-methoxycinnamic
acid, and a quenching band between coumarin and
cinnamic acid due to cinnamyl alcohol. After deriva-
tization, under UV light at 366 nm, Standard solution
B exhibits a band corresponding in Rr and color to
the cinnamaldehyde band in Standard solution A, and
a yellow band immediately below the cin-
namaldehyde band.
Acceptance criteria: Under UV light at 254 nm, the
Sample solution exhibits the most intense quenching
band in the middle-third section, corresponding in Rr to
the cinnamaldehyde band in Standard solution A. The
Sample solution exhibits additional bands corresponding
to similar bands in Standard solution B; these include a
weak quenching band in the upper part of the lower-
third section due to coumarin, a quenching band close
to the SerangPostel due to the co-elution of cin-
namic acid and 2-methoxycinnamic acid, and a
quenching band between the coumarin and cinnamic
acid bands. After derivatization, under UV light at 366
nm, the Sample solution exhibits a band corresponding
in Re and color to the cinnamaldehyde band in Standard
solution A and Standard solution B, and a yellow band
immediately below the cinnamaldehyde band. There is
no red band immediately above the cinnamaldehyde
band (a distinction from Cinnamomum verum).
e B. HPLC
Analysis: Proceed as directed in the test for Content of
Total Phenylpropanoids and Coumarin.
Acceptance criteria: The Sample solution exhibits the
most intense peak at a retention time corresponding to
cinnamaldehyde in Standard solution B; a peak with a
retention time corresponding to cinnamic acid in Stan-
dard solution A; and peaks due to coumarin, cinnamyl
alcohol, 2-methoxycinnamic acid, and 2-methoxycin-
namaldehyde at retention times corresponding to the
same constituents in Standard solution B. The content
ratios of these constituents relative to cinnamic acid are
within the typical ratio ranges listed in Table 2. The
content of cinnamaldehyde is NLT 65% of the content
of total phenylpropanoids.
Dietary Supplements / Cinnamomum cassia 4547
COMPOSITION
© CONTENT OF TOTAL PHENYLPROPANOIDS AND COUMARIN
{NoTe—Protect from light and proceed under low actinic
light. Standard solution A, Standard solution B, and the
Sample solution are stable for 24 h at room
temperature.]
Solution A: 0.05% phosphoric acid in water
Solution B: Acetonitrile
Mobile phase: See Table 7.
Table 1
Time Solution A Solution B
(min) (%) (%)
0 75 25
1 75 25
21 62 38
30 60 40
35 60 40
Solvent: Methanol and water (7:3)
Standard solution A: 0.05 mg/mL of USP Cinnamic
Acid RS in methanol
Standard solution B: Transfer about 250 mg of USP
Cinnamomum cassia Twig Powder RS into a 50-mL
round-bottom centrifuge tube, and add 25 mL of Sol-
vent. Sonicate for 30 min, cool, and centrifuge. Before
injection, pass through a membrane filter of 0.45-um or
finer pore size.
Sample solution: Accurately transfer about 500 mg of
finely powdered Cinnamomum cassia Twig into a 50-mL
round-bottom centrifuge tube, and add 25 mL of Sol-
vent. Sonicate for 30 min (250 W, 33 kHz), cool, centri-
fuge, and transfer the supernatant to a 50-mL volumet-
tic flask. Repeat the extraction one more time by
adding 15 mL of Solvent, and transfer the supernatant
to the same 50-mL volumetric flask, dilute with Solvent
to volume, and mix. Before injection, pass through a
membrane filter of 0.45-11m or finer pore size and dis-
card the first portion of the filtrate.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 265 nm
Column: 4.6-mm x 25-cm; 5-4m packing L1
Column temperature: 25°
Flow rate: 1.0 mL/min
Injection volume: 10 uL
System suitability
Samples: Standard solution A and Standard solution B
Suitability requirements
al
Resolution: NLT 1.5 between the cinnamy! alcohol
and cinnamic acid peaks and between the coumarin
and cinnamyl alcohol peaks, Standard solution B
xiTe -Bloll Uley
Tailing factor: NMT 2.0 for the cinnamic acid peak,
Standard solution A
Relative standard deviation: NMT 2.0% for the cin-
namic acid peak in repeated injections, Standard solu-
tion A
Chromatographic similarity: The chromatogram of
Standard solutionB is similar to the reference chro-
matogram provided with the lot of USP Cinnamomum
cassia Twig Powder RS being used.
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Using the chromatograms of Standard solution A and
Standard solution B and the reference chromatogram
provided with the lot of USP Cinnamomum cassia Twi
Powder RS being used, identify the retention times o'
the peaks corresponding to coumarin, cinnamyl alco-
hol, cinnamic acid, 2-methoxycinnamic acid, cin-
namaldehyde, and 2-methoxycinnamaldehyde in the
 4548 Cinnamomum cassia / Dietary Supplements USP 41

Sample solution. [NotE—see Table 2 for relative reten- walls. Oil cells and stone cells are scattered in the cor-
tion times.] tex. Groups of stone cells in the pericycle are arranged
in an interrupted ring, accompanied by fiber bundles.
Table 2 Secretory cells and fibers are scattered in phloem. Cam-
bium is distinct. Xylem with rays 1-2 cells wide, con-
Approxi- Content taining brown contents; vessels are scattered single or
mate Ratio two to several aggregated; lignified fibers have relativel
Relative Typical Relative thin walls and are difficult to differentiate from lignifie
Reten- Con- Content to parenchymatous cells. In pith, cell walls are slighth
tion version Range Cinnamic thickened and lignified; ray cells contain fine needle
Analyte Time Factor (%) Acid crystals of calcium oxalate.
0.02- © ARTICLES OF BOTANICAL ORIGIN (561), Methods of Analysis,
Coumarin 0.8 2.10 0.15 0.2-1.3 Foreign Organic Matter. NMT 1.0%
Cinnamy! 0.003- © ARTICLES OF BOTANICAL ORIGIN (561), Methods of Analysis,
alcohol 0.9 2.12 0.05 0.02-0.6 Alcohol-Soluble Extractives, Method 1: NLT 6.0%
0.04- ¢@ WATER DETERMINATION (921), Method Il: NMT 15.0%
Cinnamic acid 1.0 1.00 0.16 1.0 © ARTICLES OF BOTANICAL ORIGIN (561), Methods of Analysis,
2-Methoxycin- 0.002- Total Ash: NMT 3.0%
namic acid 11 1.82 0.02 0.04—0.16
¢ ARTICLES OF BOTANICAL ORIGIN (561), Methods of Analysis,
Acid-Insoluble Ash: NMT 1.0%
0.6-
Cinnamaldehyde 1.3 1.47 2.0 6-30 ADDITIONAL REQUIREMENTS
2-Methoxycin- 0.08- ¢ PACKAGING AND STORAGE: Preserve in well-closed contain-
namaldehyde 15 2.69 0.5 0.5-6.0 ers, protected from light and moisture, and store at con-
trolled room temperature.
ey calculate the percentages of coumarin, cin- e LABELING: The label states the Latin binomial following
namyl alcohol, cinnamic acid, 2-methoxycinnamic the official name of the plant from which the article was
acid, cinnamaldehyde, and 2-methoxycinnamaldehyde derived.
in the portion of Cinnamomum cassia Twig taken: e USP REFERENCE STANDARDS (11)
USP Cinnamaldehyde RS
Result = (ru/rs) x Cs x (V/W) x F x 100 USP Cinnamic Acid RS
USP Cinnamomum cassia Twig Powder RS
Ty = peak area of the relevant analyte from the
Sample solution
Is = peak area of cinnamic acid from Standard
solution A
Cs = concentration of USP Cinnamic Acid RS in
Standard solution A (mg/mL) Cinnamomum cassia Twig Powder
volume of the Sample solution (mL)
Ww weight of Cinnamomum cassia Twig taken to DEFINITION
prepare the Sample solution (mg) Cinnamomum cassia Twig Powder consists of the dried twigs
F = conversion factor for the analyte (see Table 2) of Cinnamomum cassia (L.) J.Presl [syn. Cinnamomum aro-
Calculate content of total phenylpropanoids as the sum maticum Nees] (Fam. Lauraceae) collected in spring or
of cinnamyl alcohol, cinnamic acid, 2-methoxycinnamic summer, reduced toa fine or very fine powder. It con-
acid, cinnamaldehyde, and 2-methoxycinnamaldehyde. tains NLT 0.8% of total phenylpropanoids, calculated as
Acceptance criteria: NLT 0.8% of total phenylpropa- the sum of cinnamyl alcohol, cinnamic acid, 2-methox-
noids on the anhydrous basis ycinnamic acid, cinnamaldehyde, and 2-methoxycin-
namaldehyde on the anhydrous basis.
CONTAMINANTS
e ARTICLES OF BOTANICAL ORIGIN (561), Limits of Elemental IDENTIFICATION
Impurities: Meets the requirements e A. HPTLC FOR ARTICLES OF BOTANICAL ORIGIN (203)
e ARTICLES OF BOTANICAL ORIGIN (561), Pesticide Residue Standard solution A: 0.5 mg/mL each of USP Cin-
DS Monographs

Analysis: Meets the requirements namaldehyde RS and coumarin in methanol

e MICROBIAL ENUMERATION TESTS (2021): The total aerobic
bacterial count does not exceed 10° cfu/g, the total com-
bined molds and yeasts count does not exceed 103 cfu/
g, and the bile-tolerant Gram-negative bacteria count
does not exceed 103 cfu/g.
e ABSENCE OF SPECIFIED MICROORGANISMS (2022), Test Proce-
dures, Test for Absence of Salmonella Species and Test for
Absence of Escherichia coli: Meets the requirements
SPECIFIC TESTS
e BOTANICAL CHARACTERISTICS
Macroscopic: Long cylindrical, branched, 30-75 cm
long, 0.3-1 cm in diameter at the thicker end. Exter-
nally brown to reddish brown, with longitudinal ridges,
fine wrinkles, dotted with scars from leaves, branches or
buds, and dotted lenticels. Texture hard and fragile,
easily broken. Slices 2-4 mm thick, cut surface showing
reddish-brown in bark, yellowish white to pale yellow-
brown in wood, pith in subsquare.
Microscopic: In the transverse section, epidermis con-
sists of one layer of cells, unicellular nonglandular hairs
are visible in young branches. Cork consists of 3-5 lay-
ers of cells, the inner-most cells with thickened outer
Standard solution B: USP Cinnamomum cassia Twig
Powder RS in methanol (1 in 5). Sonicate for 10 min,
centrifuge or filter, and evaporate the solvent. Suspend
the ede in a volume of toluene equivalent to one-
fifth the volume of methanol used for the extraction.
Sonicate for about 2 min, centrifuge or filter, and use
the supernatant or the filtrate.
Sample solution: 2g of Powder in 10 mL of methanol.
Sonicate for 10 min, centrifuge or filter, and transfer
the extract to a round-bottom flask. Evaporate the solu-
tion under reduced pressure to dryness. Dissolve the
residue in 2 mL of toluene, sonicate for about 2 min,
centrifuge or filter, and use the supernatant or the
filtrate.
Chromatographic system
Adsorbent: Chromatographic silica gel F2s4 mixture
with an average particle size of about 5 um
Application volume: 6 uL, as 8-mm bands
Relative humidity: Condition the plate to a relative
humidity of about 33% using a suitable device.
Temperature: About 25°
Developing solvent system: Toluene and ethyl ace-
tate (1 o.9
USP 41 Dietary Supplements / Cinnamomum cassia 4549

Developing distance: 6 cm Sample solution are stable for 24 h at room

Derivatization reagent: Methanol, acetic acid, sulfuric temperature.]
acid, and panealdelyde (170:20:10:1). [NoTe—Pre- Solution A: 0.05% phosphoric acid in water
pare fresh. Slowly add sulfuric acid to ice-cold metha- Solution B: Acetonitrile
nol, followed by acetic acid and p-anisaldehyde.] Mobile phase: See Table 1.
Analysis
Samples: Standard solution A, Standard solution B, and Table 1
Sample solution
Apply the Samples as bands and a in air. Develop in a Time Solution A Solution B
saturated chamber (20 min with filter paper), remove (min) (%) (%)
the plate from the chamber, and dry in air. Examine 0 75 25
under UV light at 254 nm. Then treat the plate with 1 75 25
the Derivatization reagent, heat at 100° for 4 min, and 21 62 38
examine under UV light at 366 nm. 30 60 40
System suitability
35 60 40
Samples: Standard solution A and Standard solution B
Suitability requirements: Prior to derivatization, under Solvent: Methanol and water (7:3)
UV light at 254 nm, Standard solution A exhibits a Standard solution A: 0.05 mg/mL of USP Cinnamic
uenching band due to cinnamaldehyde in the middle Acid RS in methanol
third of the chromatogram, and a quenching band Standard solution B: Transfer about 250 mg of USP
due to coumarin in the upper part of the lower third. Cinnamomum cassia Twig Powder RS into a 50-mL
Under UV light at 254 nm, Standard solution B exhib- round-bottom centrifuge tube, and add 25 mL of Sol-
its, in the middle-third section, an intense quenching vent. Sonicate for 30 min, cool, and centrifuge. Before
band corresponding in R; to the cinnamaldehyde injection, pass through a membrane filter of 0.45-um or
band in Standard solution A. In the upper part of the finer pore size.
lower-third section, Standard solution B exhibits a Sample solution: Accurately transfer about 500 mg of
weak quenching band due to coumarin, a quenching Powder into a 50-mL round-bottom centrifuge tube,
band close to the starting position due to the co- and add 25 mL of Solvent. Sonicate for 30 min (250 W,
elution of cinnamic acid and 2-methoxycinnamic 33 kHz), cool, centrifuge, and transfer the supernatant
acid, and a quenching band between coumarin and to a 50-mL volumetric flask. Repeat the extraction one
cinnamic acid due to cinnamylalcohol. After deriva- more time by adding 15 mL of Solvent, and transfer the
tization, under UV light at 366 nm, Standard solution supernatant to the same 50-mL volumetric flask, dilute
B exhibits a band corresponding in R; and color to with Solvent to volume, and mix. Before injection, pass
the cinnamaldehyde band in Standard solution A, and through a membrane filter of 0.45-11m or finer pore size
a yellow band immediately below the cin- and discard the first portion of the filtrate.
namaldehyde band. Chromatographic system
Acceptance criteria: Under UV light at 254 nm, the (See Chromatography (621), System Suitability.)
Sample solution exhibits the most intense quenching Mode: LC
band in the middle-third section, corresponding in R; to Detector: UV 265 nm
the cinnamaldehyde band in Standard solution A. The Column: 4.6-mm x 25-cm; 5-um packing L1
Sample solution exhibits additional bands corresponding Column temperature: 25°
to similar bands in Standard solution B; these include a Flow rate: 1.0 mL/min
weak quenching band in the upper part of the lower- Injection volume: 10 uL
third section due to coumarin, a quenching band close System suitability
to the starting position due to the co-elution of cin- Samples: Standard solution A and Standard solution B
namic acid and 2-methoxycinnamic acid, and a Suitability requirements
quenching band between the coumarin and cinnamic Resolution: NLT 1.5 between the cinnamyl alcohol
acid bands. After derivatization, under UV light at 366 and cinnamic acid peaks and between the coumarin
nm, the Sample solution exhibits a band corresponding and cinnamyl alcohol peaks, Standard solution B
in Re and color to the cinnamaldehyde band in Standard Tailing factor: NMT 2.0 for the cinnamic acid peak,
solution A and Standard solution B, and a yellow band Standard solution A
sydesbouow sa

immediately below the cinnamaldehyde band. There is
no red band immediately above the cinnamaldehyde
band (a distinction from Cinnamomum verum).
e B. HPLC
Analysis: Proceed as directed in the test for Content of
Total Phenylpropanoids and Coumarin.
Acceptance criteria: The Sample solution exhibits the
most intense peak at a retention time corresponding to
cinnamaldehyde in Standard solution B; a peak with a
retention time corresponding to cinnamic acid in Stan-
dard solution A; and peaks due to coumarin, cinnamyl
alcohol, 2-methoxycinnamic acid, and 2-methoxycin-
namaldehyde at retention times corresponding to the
same constituents in Standard solution B. The content
ratios of these constituents relative to cinnamic acid are
within the typical ratio ranges listed in Table 2. The
content of cinnamaldehyde is NLT 65% of the content
of total phenylpropanoids.
COMPOSITION
© CONTENT OF TOTAL PHENYLPROPANOIDS AND COUMARIN
[Note—Protect from light and proceed under low actinic
light. Standard solution A, Standard solution B, and the
Relative standard deviation: NMT 2.0% for the cin-
namic acid peak in repeated injections, Standard solu-
tion A
Chromatographic similarity: The chromatogram of
Standard solution B is similar to the reference chro-
matogram provided with the lot of USP Cinnamomum
cassia Twig Powder RS being used.
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Using the chromatograms of Standard solution A and
Standard solution B and the reference chromatogram
provided with the lot of USP Cinnamomum cassia Twii
Powder RS being used, identify the retention times o
the peaks corresponding to coumarin, cinnamyl alco-
hol, cinnamic acid, 2-methoxycinnamic acid, cin-
namaldehyde, and 2-methoxycinnamaldehyde in the
Sample solution. [Note—See Table 2 for relative reten-
tion times.]
 4550 Cinnamomum cassia / Dietary Supplements USP 41

Table 2 ARTICLES OF BOTANICAL ORIGIN (561), Alcohol-Soluble Ex-

Approxi- Content
tractives, Method 1: NLT 6.0%
mate Ratio
WATER DETERMINATION (921), Method Ii: NMT 15.0%
Relative Typical Relative
ARTICLES OF BOTANICAL ORIGIN (561), Methods of Analysis,
Reten- Con- Content to Total Ash: NMT 3.0%
tion version Range Cinnamic|
ARTICLES OF BOTANICAL ORIGIN (561), Methods of Analysis,
Analyte Time Factor (%) Acid
Acid-Insoluble Ash: NMT 1.0%
0.02- ADDITIONAL REQUIREMENTS
Coumarin 0.8 2.10 0.15 0.2-1.3 e PACKAGING AND STORAGE: Preserve in well-closed contain-
Cinnamyl 0.003- ers, protected from light and moisture, and store at con-
alcohol 0.9 2.12 0.05 0.02-0.6 trolled room temperature.
0.04— e LABELING: The label states the Latin binomial following
Cinnamic acid 1.0 1.00 0.16 1.0 The celal name of the plant from which the article was
2-Methoxycin- 0.002- lerived.
namic acid 11 1.82 0.02 0.04-0.16 e USP REFERENCE STANDARDS (11)
0.6-
USP Cinnamaldehyde RS
USP Cinnamic Acid RS
Cinnamaldehyde 133 1.47 2.0. 6-30
USP Cinnamomum cassia Twig Powder RS
2-Methoxycin- 0.08-
namaldehyde 145 2.69 0.5 0.5-6.0

Separately calculate the percentages of coumarin, cin-

namyl alcohol, cinnamic acid, 2-methoxycinnamic
acid, cinnamaldehyde, and 2-methoxycinnamaldehyde Citrulline
in the portion of Powder taken:
Result = (ru/rs) x Cs x (V/W) x Fx 100
ru = peak area of the relevant analyte from the
Sample solution
rs = peak area of cinnamic acid from Standard Ce6Hi3N303 zl LZ5A9.
solution A (S)-2-Amino-5-ureidopentanoic acid;
Gs = concentration of USP Cinnamic Acid RS in L-Citrulline [372-75-8].
Standard solution A (mg/mL)
DEFINITION
Vv = volume of the Sample solution (mL)
Citrulline contains NLT 98.0% and NMT 102.0% of L-citrul-
Ww = weight of Powder taken to prepare the Sample line (CéH13N303), calculated on the dried basis.
solution (mg)
F = conversion factor for the analyte (see Table 2) IDENTIFICATION
Calculate the content of total phenylpropanoids as the e A. INFRARED ABSORPTION (197K)
sum of cinnamyl alcohol, cinnamic acid, e B. It meets the requirements in Specific Tests for Optical
2-methoxycinnamic acid, cinnamaldehyde, and Rotation (7815S), Procedures, Specific Rotation.
2-methoxycinnamaldehyde. e C. The retention time of the major peak of the Sample
Acceptance criteria: NLT 0.8% of total phenylpropa- solution corresponds to that.of the Standard solution, as
noids on the anhydrous basis obtained in the Assay.
CONTAMINANTS ASSAY
e ARTICLES OF BOTANICAL ORIGIN (561), Limits of Elemental © PROCEDURE
Impurities: Meets the requirements Solution A: 10 mM octanesulfonic acid sodium salt in
e ARTICLES OF BOTANICAL ORIGIN (561), Pesticide Residue water. Adjust with dilute phosphoric acid (1 in 10) to a
Analysis: Meets the requirements pH of 2.5.
e MICROBIAL ENUMERATION TESTS (2021): The total aerobic Solution B: Acetonitrile
DS Monographs

bacterial count does not exceed 10° cfu/g, the total com- Mobile phase: Gradient elution. See Table 1.
bined molds and yeasts count does not exceed 103 cfu/
g, and the bile-tolerant Gram-negative bacteria count
does not exceed 103 cfu/g. Table 1
e ABSENCE OF SPECIFIED MICROORGANISMS (2022), Test Proce- Time Solution A Solution B
dures, Test for Absence of Salmonella Species and Test for (min) (%) (%)
Absence of Escherichia coli: Meets the requirements 0 85 15
4 85 15!
SPECIFIC TESTS
e BOTANICAL CHARACTERISTICS ED) 70 30
Macroscopic: Reddish-brown powder 20 85 15
Microscopic: Stone cells subsquare or subrounded, 25 85 15
30-65 um in diameter with thickened walls, some with
avey thin wall at one side. Phloem fibers mostly in System suitability solution: 0.1 mg/mL of USP L-Citrul-
bundles or scattered singly, colorless or brown, fusi- ine RS and 0.05 mg/mL of USP N-Acetyl-L-leucine RS in
form, some margins serrate, 10-40 um in diameter, water
with heavily thickened walls, lignified, pit canals indis- Standard solution: 0.1 mg/mL of USP L-Citrulline RS in
tinct. Oil cells subrounded or elliptical, 40-105 um in water
diameter. Xylary fibers numerous, usually in bundles, Sample solution: 0.1 mg/mL of Citrulline in water
pits oblique or crossed. Cork cells yellowish brown, pol- Chromatographic system
ygonal in surface view, containing reddish-brown con- (See Chromatography (621), System Suitability.)
tent. Vessels mainly with bordered pits, up to 80 um in
diameter.
USP 41 Dietary Supplements / Cod Liver Oil 4551

Mode: LC Table 2
Detector: UV 200 nm Relative Relative Acceptance
Column: 4.6-mm x 15-cm; 5-um packing L1 Retention Response Criteria,
Flow rate: 1 mL/min Name Time Factor NMT (%)
|
Injection volume: 20 LL
System suitability Citrulline 1.0 = =
Samples: System suitability solution and Standard delta-Acetylornithine? 1.1 2.8 0.5
solution Any unspecified aa
[Note—The relative retention times for L-citrulline and impurity 1.0 0.1
N-acetyl-t-leucine are 1.0 and 2.4, respectively.] Total impurities = = 2.0
Suitability requirements 4 (25)-5-Acetamido-2-aminopentanoic acid.
Resolution: NLT 10.0 between L-citrulline and N-ace-
tyl-L-leucine peaks, System suitability solution SPECIFIC TESTS
Tailing factor: NMT 2.0, Standard solution © OPTICAL ROTATION (7815S), Procedures, Specific Rotation
Relative standard deviation: NMT 2.0%, Standard Sample solution: 80mg/mL in 6 N hydrochloric acid
solution Acceptance criteria: +24.5° to +26.5°
Analysis e LOSS ON DRYING (731)
Samples: Standard solution and Sample solution Analysis: Dry at 105° for 3 h.
Calculate the percentage of L-citrulline (CsHi3N3O3) in Acceptance criteria: NMT 0.2%
the portion of Citrulline taken:
ADDITIONAL REQUIREMENTS
Result = (ru/rs) x (Cs/Cu) x 100 e PACKAGING AND STORAGE: Preserve in well-closed
containers.
ry = peak response from the Sample solution e USP REFERENCE STANDARDS (11)
rs = peak response from the Standard solution USP N-Acetyl-L-leucine RS
Cs = concentration of USP L-Citrulline RS in the Acetyl-L-leucine.
Standard solution (mg/mL) CsHisNO3 173.21
Cu = concentration of Citrulline in the Sample USP L-Citrulline RS
solution (mg/mL)
Acceptance criteria: 98.0%-102.0% on the dried basis
IMPURITIES
e RESIDUE ON IGNITION (281): NMT 0.1% Clover, Red—see Red Clover
© CHLORIDE AND SULFATE (221), Chloride
oer solution: 0.10 mL of 0.020 N hydrochloric
aci
Sample: 0.36 g of Citrulline
Acceptance criteria: NMT 0.02% Cod Liver Oil—see Cod Liver Oil General
e CHLORIDE AND SULFATE (221), Sulfate Monographs
Standard solution: 0.10 mL of 0.020 N sulfuric acid
Sample: 0.48 g of Citrulline
Acceptance criteria: NMT 0.02%
e RELATED COMPOUNDS Cod Liver Oil Capsules
Mobile phase, System suitability solution, Chromato-
graphic system, and System suitability: Proceed as DEFINITION
directed in the Assay. Cod Liver Oil Capsules contain NLT 95.0% and NMT
Standard solution: 2.5 g/mL of USP Citrulline RS in 105.0% of the labeled amount of Cod Liver Oil, where
water Cod Liver Oil is the partially destearinated fixed oil ob-
Sample solution: 0.5 mg/mL of Citrulline in water tained from fresh livers of Gadus morrhua L. and other
Analysis species of Fam. Gadidae. Cod Liver Oil contains, in each
Samples: Standard solution and Sample solution g, NLT 180 ug (600 USP Units) and NMT 750 ug (2500
Calculate the percentage of each impurity in the por-
sydesbouo-: sa

USP Units) of vitamin A and NLT 1.5 jg (60 USP Units)

tion of Citrulline taken: and NMT 6.25 wg (250 USP Units) of vitamin D.
Cod Liver Oil may be flavored by the addition of NMT 1%
Result = (ru/rs) x (Cs/Cu) x (1/F) x 100 of a suitable flavor or a mixture of flavors. A suitable anti-
oxidant may be added.
tu = peak response of each impurity from the
Sample solution IDENTIFICATION
rs = peak response of citrulline from the Standard e A. PRESENCE OF VITAMIN A
solution Sample solution: 25 mg/mL of oil contained in the
Cs = concentration of USP Citrulline RS in the Capsules in chloroform
Standard solution (mg/mL) Analysis: To 1 mL of the Sample solution add 10 mL of
Gu = concentration of Citrulline in the Sample antimony trichloride TS.
solution (mg/mL) Acceptance criteria: A blue color results immediately.
F = relative response factor (see Table 2) e B. FATTY ACID PROFILE
Acceptance criteria: See Table 2. [Note—Depending on Antioxidant solution: 0.05 mg/mL of butylated
the manufacturing process, delta-Acetylornithine impu- hydroxytoluene in hexanes
rity may not be observed in the article being tested. System suitability solution: Prepare a mixture contain-
Disregard any impurity less than 0.1%.] ing equal amounts of methyl palmitate, methyl stearate,
methyl arachidate, and methyl behenate in Antioxidant
solution.
Standard stock solution: 45 mg/mL of USP Cod Liver
Oil RS in Antioxidant solution
 4552 Cod Liver Oil / Dietary Supplements USP 41

Standard solution: Transfer 2.0 mL of the Standard following,in common elution order: 14:0, 15:0, 16:0,
stock solution into a quartz tube, and evaporate with a 16:1 n-7, 16:4 n-1, 18:0, 18:1 n—9, 18:1 n-7, 18:2
gentle stream of nitrogen. Add 1.5 mL of a 2% solution n-6, 18:3 n-3, 18:4 n-3, 20:1 n-11, 20:1 n-9, 20:1
of sodium hydroxide in methanol. Cap tightly with a n-7, 20:2 n-6, 20:4 n—6, 20:3 n-3, 20:4 n-3, 20:5 n-
polytetrafluoroethylene-lined cap, mix, and heat in a 3, 7 n-11, 22:1 n-9, 21:5 n—3, 22:5 n-3, and 22:6
water bath for 7 min. Cool, add 2 mL of a 120 mg/mL n-3
solution of boron trichloride in methanol. Cover with Calculate the area percentage for each fatty acid methyl
nitrogen, cap tightly, and mix. Heatin a water bath for ester in the portion of Capsules taken:
30 min. Cool to 40°-50°. Add 1 mL of isooctane, cap,
and mix in a vortex mixer or shake vigorously for at Result = (ra/rs) x 100
least 30 s. Immediately add 5 mL of saturated sodium
chloride solution. Cover with nitrogen, cap, and mix in in peak area of each individual fatty acid
a vortex mixer or shake thoroughly for at least 15 s. ts total area from all peaks, except the solvent
Allow the upper layer to become clear, and transfer to peak and butylated hydroxytoluene
a separate tube. Shake the methanol layer once more Acceptance criteria: The Sample solution meets the lim-
with 1 mL of isooctane, and combine the isooctane ex- its described in Table 2.
tracts. Wash the combined extracts twice with 1 mL of
water, and dry over anhydrous sodium sulfate.
Sample solution: Proceed as directed in the Standard Jae
solution, except use a weighed quantity of the oil con- Fower, Upper
tained in the Capsules. Shorthand lis Limit
Chromatographic system Fatty Acid - Notation (area %) (area %)
(See Chromatography (621), System Suitability.) Saturated fatty acids
Mode: GC Myristic acid 14:0 2.0 6.0
Detector: Flame ionization Palmitic acid 16:0 7.0 14.0
Column: 0.25-mm x 30-m fused silica capillary col- Stearic acid 18:0 1.0 4.0
Temperature with a 0.25-ym film of G16 Monounsaturated fatty acids
Injector: 250° Palmitoleic acid 16:1 n-7 4.5 11.5
Detector: 280° cis-Vaccenic acid 18:1 n-7 2.0 7.0
Column: See Table 1. Oleic acid 18:1 n-9 12.0 21.0
Gadoleic acid 20:1 n-11 1.0 5.5.
Table 1 Gondoic acid 20:1 n-9 5.0 17.0
Hold Time Erucic acid 22:1-n-9 0 135:
Initial Temperature Final at Final Cetoleic acid 22:1 n=11 5.0 12.0
Temperature Ramp Temperature | Temperature Polyunsaturated fatty acids
(i) (¢/min) ©) (min) Linoleic acid 18:2 n-6 0.5 3.0
170 1 225 20 o-Linolenic acid 18:3 n=3 0 2.0
Carrier gas: Helium Moroctic acid 18:4 n-3 0.5 45
Split flow ratio: 1:200 Eicosapentaenoic acid 20:5 n=3 7.0 16.0
Injection size: 1 wl Docosahexaenoic acid 22:6 n-3 6.0 18.0
System suitability
Samples: System suitability solution and Standard STRENGTH
solution @ VITAMIN A
Suitability requirements Sample: 500 mg to 1g of oil contained in the Capsules
Chromatogram similarity: The chromatogram from Analysis: Proceed as directed under Vitamin A Assay
the Standard solutionis similar to the Reference Chro- (571).
matogram supplied with USP Cod Liver Oil RS. Iden- Acceptance criteria: 180 tg (600 USP Units) to 750 yg
tify the retention times of the relevant fatty acid (2500 USP Units) of vitamin A per g of oil containedin
methyl esters by comparing the chromatogram of the
DS Monographs

Standard solution with the Reference Chromatogram
supplied with USP Cod Liver Oil RS.
Resolution: NLT 1.3 between methyl oleate and
methyl cis-vaccinate, and that between methyl
gadoleate and methyl gondoate is sufficient for pur-
poses of identification and area measurement, Stan-
dard solution
Theoretical area percentages: 24.4 + 1 for methyl
palmitate, 24.8 + 1 for methyl stearate, 25.2 + 1
methyl arachidate, and 25.6 + 1 for methyl behenate,
System suitability solution
Analysis
Samples: Standard solution and Sample solution
Identify the retention times of the relevant fatty acid
methyl esters in the Sample solution by companing the
chromatogram of the Sample solution with that of the
Standard solution.
Determine the number of fatty acid methyl ester peaks
in the Sample solution: The number of fatty acid
methyl ester peaks exceeding 0.05% of the total area
of fatty acid methyl esters is at least 24, and the 24
largest peaks of the methyl esters account for more
than 90% of the total area. (These correspond to the
the Capsules
e VITAMIN D
Solution A: n-Amyl alcohol and dehydrated hexane
(3:997)
Solution B: Acetonitrile, water, and phosphoric acid
(96:3.8:0.2)
Butylatedhydronytoluene solution: 10 mg/mL of bu-
witted hydroxytoluene in chromnarsapanhie: hexane
Aqueous potassium hydroxide solution: 800 mg/mL
of potassium hydroxide in freshly boiled water.Mix,
and cool. [NoTE—Prepare this solution fresh daily.]
Alcoholic potassium hydroxide solution: Dissolve 3 g
of potassium hydroxide in 50 mL of freshly boiled
water. Add 10 mL of alcohol, and dilute with freshly
boiled water to 100 mL. [NoTE—Prepare this solution
fresh daily.]
Ascorbic acid solution: 100 mg/mL of ascorbic acid in
water. [NoTE—Prepare this solution fresh daily.]
Internal standard solution: 5 g/mL of USP Ergocalcif-
erol RS in alcohol
Standard stock solution: 5 tg/mL of USP Cholecalcif-
erol RS in alcohol
USP 41
Standard solution: Transfer 2.0 mL of the Standard
stock solution and 2.0 mL of the Internal standard solu-
tion to a round-bottomed flask. Proceed as directed in
Sample solution 1, beginning with “Add 5 mL of...”.
Sample solution 1: Transfer a quantity equivalent to
4.00 g of oil contained in the Capsules to a round-bot-
tomed flask. Add 5 mL of Ascorbic acid solution, 100 mL
of alcohol, and 10 mL of Aqueous potassium hydroxide
solution, and mix. Reflux the mixture on a steam bath
for 30 min. Add 100 mL of a 10 mg/mL sodium chlo-
ride solution. Cool rapidly under running water, and
transfer the saponified mixture to a 500-mL separator,
rinsing the saponification flask with 75 mL of a 10 mg/
mL sodium chloride solution and then with 150 mL of a
mixture of ether and hexane (1:1). Shake the combined
saponified mixture and rinsings vigorously for 30 s, and
allow to stand until both layers are clear. Discard the
lower layer. Wash the ether-hexane extracts by shaking
vigorously with 50 mL of Alcoholic potassium hydroxide
solution, and then washing with three 50-mL portions
of a 10 mg/mL sodium chloride solution. Pass the upper
layer through 5 g of anhydrous sodium sulfate on a fast
filter paper into a 250-mL flask suitable for a rotary
evaporator. Wash the filter with 10 mL of a mixture of
ether and hexane (1:1), and combine with the extract.
Evaporate the solvent at reduced pressure at a tempera-
ture not exceeding 30°, and fill with nitrogen when the
evaporation is complete. Alternatively evaporate the sol-
vent under a gentle stream of nitrogen at a tempera-
ture not exceeding 30°. Dissolve the residue in 1.5 mL
of Solution A. [NoTe—Gentle heating in an ultrasonic
bath may be required.A large fraction of the white
residue is cholesterol.]
Sample solution 2: To 4.00g of oil contained in the
Capsules add 2.0 mL of Internal standard solution, and
proceed as directed for Sample solution 1, beginning
with “Add 5 mL of...”.
Clean-up chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 265 nm
Mobile phase: Solution A
Column: 25-mm x 4.6-cm stainless steel; packing L10
Injection size: 350 pL
Analysis fclean-up)
Samples: Standard solution, Sample solution 1, and
Sample solution 2
Collect separately the eluates from 2 min before until
2 min after the retention time of cholecalciferol in a
glass tube containing 1 mL of Butylated hydroxytol-
uene solution and fitted with a hermetic closure. Evap-
orate each tube under a stream of nitrogen at a tem-
perature not exceeding 30°. Dissolve each residue in
1.5 mL of acetonitrile.
Analytical chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 265 nm
Mobile phase: Solution B
aa 15-mm x 4.6-cm stainless steel; 5-um pack-
ing
Injection size: 200 uL
System suitability
Sample: Standard solution (after the clean-up)
Suitability requirements
Resolution: NLT 1.4 between cholecalciferol and
ergocalciferol
Relative standard deviation: NMT 2.0% for the cho-
lecalciferol peak from replicate injections
Analysis
Samples: Standard solution, Sample solution 1, and
Sample solution 2 (after the clean-up)
Dietary Supplements / Cod Liver Oil 4553
Calculate the content of vitamin D, in g/g, in the por-
tion of oil taken:
Result = (Ru/Rs) x (Cs/Cu)
Ru = response of cholecalciferol relative to the
corrected internal standard in Sample solution
2, as calculated below
Rs = response of cholecalciferol relative to the
internal standard in the Standard solution
Gs = concentration of USP Cholecalciferol RS in the
Standard solution (ug/ml)
Cu = concentration of oil in Sample solution 2 (g/
mL)
Ru = tual [his2 — Crist X Fu2/tun)]
Tu2 = peak response of cholecalciferol from Sample
solution 2
Nis2 = peak response of the internal standard from
Sample solution 2
Ns1 = peak response of the internal standard from
Sample solution 1
Tur = peak response of cholecalciferol from Sample
solution 1
Acceptance criteria: 1.5 ug (60 USP Units) to 6.25 tg
(250 USP Units) of vitamin D per g of oil contained in
the Capsules
© CONTENT OF COD LIVER OIL
Analysis: Weigh NLT 10 Capsules in a tared weighing
bottle. With a sharp blade, or by other appropriate
means, carefully open the Capsules, without loss of
shell material, and transfer the combined Capsule con-
tents to a 100-mL beaker. Remove any adhering sub-
stance from the emptied Capsules by washing with sev-
eral small portions of isooctane. Discard the washings,
and allow the empty Capsules to dry in a current of dry
air until the isooctane is completely evaporated. Weigh
the eran Capsules in the original tared weighing bot-
tle, and calculate the average net weight per Capsule.
Acceptance criteria: 95.0%-105.0% of the labeled
amount of cod liver oil
PERFORMANCE TESTS
© WEIGHT VARIATION OF DIETARY SUPPLEMENTS (2091): Meet
the requirements
e DISINTEGRATION AND DISSOLUTION OF DIETARY SUPPLEMENTS
(2040): Meet the requirements for Rupture Test for Soft
Shell Capsules
SPECIFIC TESTS
° an AND: FIXED O1Ls, Unsaponifiable Matter (401): NMT
sydeibouo-: sa
-30%
e FATS AND FIXED OILS, Acid Value (401)
Sample solution: Mix 15 mL of alcohol with 15 mL of
ether, add 5 drops of phenolphthalein TS, and neutral-
ize with 0.1 N sodium hydroxide. Dissolve 2.0g of oil
contained in the Capsules in the mixture, and boil the
oil solution gently under a reflux condenser for 10 min.
Analysis: Cool, and titrate the mixture with 0.1 N so-
dium hydroxide VS to the production of a pink color
that persists after shaking for 30 s.
Acceptance criteria: NMT 1.0 mL of 0.1 N sodium hy-
droxide is required.
° FATS AND FIXED OILS, /odine Value (401): 145-180
¢ FATS AND FIXED OlLS, Saponification Value (401): 180-192
[Note—lIf carbon dioxide has been used as a preserva-
tive, expose the oil contained in the Capsules in a shal-
lowdish in a vacuum desiccator for 24 h before weigh-
ing the specimen for determination of the
saponification value.]
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in tight containers at
room temperature. Protect from light.
 4554 Cod Liver Oil / Dietary Supplements USP 41

e LABELING: Vitamin A potency and vitamin D potency, a small chromatographic tube, rinsing the beaker with
when designated on the label, may be expressed in USP water and packing the column evenly. Keep the column
Units per g of oil. The potency may also be expressed in wet until ready for use. Using a volumetric pipet, trans-
metric units, on the basis that 1 USP Vitamin A Unit = fer 1.0 mL of Liquid Preparation to the column, collect
0.3 wg of all-trans retinol and 40 USP Vitamin D Units = the eluate, and discard it. Pipet 4.0 mL of water onto
1 ug. Label them to emphasize the need to avoid freez- the top of the column, collect the eluate in a clean vial,
ing or exposure to excessive humidity or to a tempera- and filter if necessary.
ture above 40°. Where the content of docosahexaenoic Chromatographic system
acid and eicosapentaenoic acid is claimed, state the (See Chromatography (621), System Suitability.)
amount in mg per Capsule on the label. Mode: LC
e@ USP REFERENCE STANDARDS (11) Detector: Refractive index
USP Cholecalciferol RS Columns
USP Cod Liver Oil RS Guard: Packing L19
USP Ergocalciferol RS Analytical: 7.8-mm x 30-cm; packing L19
USP Vitamin A RS Column temperature: 85°
Flow rate: 0.6 mL/min
Injection size: 20 uL
System suitability
Sample: Standard solution
Cohosh, Black—see Black Cohosh [Nott—The approximate relative retention times for
dextrose and fructose are about 0.8 and 1.0,
respectively.]
Suitability requirements
Resolution: NLT 1.8 between the dextrose and fruc-
Copper Gluconate—see Copper Gluconate tose peaks
General Monographs Relative standard deviation: NMT 2.0%
Analysis
Samples: Standard solution and Sample solution
Calculate the percentages of dextrose and fructose in
Co-Q10—see Ubidecarenone in Dietary the volume of Liquid Preparation taken:
Supplements section Result = (ru/rs) x (Cs/V) x 0.5
ru = peak response of each appropriate analyte
from the Sample solution
Cranberry Liquid Preparation Is = peak response of each appropriate analyte
from the Standard solution
DEFINITION Cs = concentration of the appropriate USP
Cranberry Liquid Preparation is a bright red juice derived Reference Standard in the Standard solution
from the fruits of Vaccinium macrocarpon Ait. or Vaccinium
Dayeoccos L. (Fam. Ericaceae). It contains no added
(mg/mL)
= volume of Liquid Preparation taken for the
substances. Sample solution (mL)
Acceptance criteria: NLT 2.4% dextrose and NLT 0.7%
IDENTIFICATION fructose
e A. HPLC The retention times of the quinic acid, malic e CONTENT OF ORGANIC ACIDS
acid, and citric acid peaks of the Sample solution corre- Mobile phase: Transfer 27.2 g of monobasic potassium
spond to those of the Standard solution, as obtained in phosphate to a 1000-mL volumetric flask, and dissolve
the test for Content of Organic Acids. in 950 mL of water. Adjust with phosphoric acid to a
e B. ABSENCE OF ADULTERANTS pH of 2.4, and dilute with water to volume.
Standard solution: 1.0 mg/mL of tartaric acid and Standard solution: 1.0 mg/mL each of USP Citric Acid
0.1 mg/mL of fumaric acid RS, USP Malic Acid RS, and USP Quinic Acid RS
“ Sample solution: Use the Liquid Preparation. Sample solution: Use the filtered Liquid Preparation.
a Mobile phase and Chromatographic system: Proceed Chromatographic system
ici
—
as directed in the test for Content of Organic Acids. (See Chromatography (621), System Suitability.)
iy Analysis Mode: LC
° Samples: Standard solution and Sample solution
iS Injection size: 20 uL
Detector: UV 214 nm
S Acceptance criteria: The retention times of the tartaric
Columns
= acid and fumaric acid peaks of the Standard solution do
Guard: 5-41m; packing L1
” [Note—Before use, condition the column with metha-
not correspond to any of the retention times for peaks nol, then with water, and finally with Mobile phase.]
fa) observed from the Sample solution. Analytical: 4.6-mm x 25-cm analytical; packing L1
Flow rate: 0.6 mL/min
COMPOSITION
© CONTENT OF DEXTROSE AND FRUCTOSE Injection size: 20 uL
Mobile phase: Water System suitability
Standard solution: 6.0 mg/mL of USP Dextrose RS and Sample: Standard solution
2.0 mg/mL of USP Fructose RS in water [NoTtE—The approximate relative retention times for
Sample solution: Transfer 1.0 g of sodium carboxylate guinic acid, malic acid, and citric acid are 0.4, 0.5,
cation-exchange resin to a 50-mL beaker, add 5 mL of and 1.0, respectively.]
water to makea slurry, and transfer the slurry to a poly- Suitability requirements
propylene automatic pipet fitted with a small plug of Resplution: NLT 2.5 between quinic acid and malic
silanized glass wool. Quantitatively transfer the slurry to aci
USP 41 Dietary Supplements / Crypthecodinium 4555

Relative standard deviation: NMT 2.0% e USP REFERENCE STANDARDS (11)

Analysis USP Citric Acid RS
Samples: Standard solution and Sample solution USP Dextrose RS
Measure the peak areas. Calculate the percentages of USP Fructose RS
quinic acid, malic acid, and citric acid in the volume of USP Malic Acid RS
Liquid Preparation taken: USP Quinic Acid RS
USP Sorbitol RS
Result = (ru/rs) x Cs x 0.1 USP Sucrose RS
tu = peak area of each appropriate analyte from
the Sample solution
Is = peak area of each appropriate analyte from
the Standard solution Crypthecodinium cohnii Oil
Cs = concentration of the appropriate USP
Reference Standard in the Standard solution
(mg/mL) DEFINITION
Acceptance criteria: NLT 0.9% each of quinic acid and Crypthecodinium cohnii Oil is obtained from the fermentation
citric acid; NLT 0.7% of malic acid. The ratio of quinic and extraction of algae of the species whee
cohnii and contains NLT 35.0% (w/w) of docosahexaenoic
acid to malic acid is NLT 1.0.
acid (DHA, C22H3202) (C22: 6 n-3), as the only significant
ADULTERANTS polyunsaturated fatty acid present. Suitable antioxidants
¢ LIMIT OF SORBITOL AND SUCROSE in appropriate concentration may be added.
Mobile phase and Sample solution: Prepare as di-
rected in the test for Content of Dextrose and Fructose. IDENTIFICATION
Standard solution: 0.5 mg/mL each of USP Sorbitol RS e A. LONG-CHAIN UNSATURATED FATTY ACID PROFILE: Pro-
ceed as directed in Fats and Fixed Oils (401), Omega-3
and USP Sucrose RS
Chromatographic system: Proceed as directed in the Fatty Acids Determination and Profile, Content of EPA and
test for Content of Dextrose and Fructose. DHA.
System suitability Analysis
Samples: Standard Solution 2a, Standard Solution 2b,
Sample: Standard solution and Test Solution 1
[Note—The relative retention times for sucrose and sor-
bitol are about 0.4 and 1.0, respectively.] Calculate the area percentage for each fatty acid as
Suitability requirements methyl ester in Test Solution 1:
Resolution: NLT 1.8 between the sucrose and sorbi- Result = (ru/rz) x 100
tol peaks
Relative standard deviation: NMT 2.0% ty = peak response of each individual fatty acid as
Analysis methyl ester
Samples: Standard solution and Sample solution tr = sum of all the peak responses, except the
Injection size: 20 uL solvent and butylated hydroxytoluene peaks
Calculate the percentages of sucrose and sorbitol in the Acceptance criteria: The retention times of the peaks
volume of Liquid Preparation taken: for the docosahexaenoic acid methyl ester and the eico-
sapentaenoic acid methyl ester from Test Solution 7 cor-
Result = (ru/rs) x (Cs/V) x 0.5 respond to those from Standard Solution 2a and Stan-
ty = peak response of each appropriate analyte dard Solution 2b, respectively. The area percentage for
from the Sample solution the methyl esters of the fatty acids from Test Solution 7
ls = peak response of each appropriate analyte meets the requirements for each fatty acid indicated in
from the Standard solution Table 1.
Cs = concentration of the appropriate USP
Reference Standard in the Standard solution Table 1
(mg/mL) Relative Lower Upper
V = volume of Liquid Preparation taken for the Reten- Short- Limit | Limit S
Sample solution (mU Fatty tion hand (Area, (Area,
Acceptance criteria: NMT 0.05% each of sorbitol and Acid Time Notation %) %) =
sucrose Linoleic acid 0.52 18:2 n-6 oO 1.0 <
SPECIFIC TESTS Eicosapentaenoic °
e REFRACTIVE INDEX (831): 1.3435-1.3445 acid 0.79 20:5 n-3 0 0.1 =}
e PH (791): 2.5+0.1 Docosapentaenoic a
acid 0.94 22:5 n-6 0 0.1 >
ADDITIONAL REQUIREMENTS Docosahexaenoic 4
© PACKAGING AND STORAGE: Preserve in well-closed contain- acid 1.00 22:6 n-3 35.0 47.0
ers, and store in a refrigerator.

COMPOSITION
Change to read:
e CONTENT OF DHA
Analysis: Proceed as directed in Fats and Fixed Oils
e LABELING: The label states the Latin binomial name and, (401), Omega-3 Fatty Acids Determination and Profile,
following the official name, the parts of the plant source Content of EPA and DHA.
from which the article was derived. The label also states Acceptance criteria: NLT 35.0% (w/w) of docosahexa-
that it is for manufacturing purposes only. This article is enoic acid (DHA)
exempted from the requirements of °Labeling (7), Labels
and Labeling for Products and Other Categories, Botanicals, IMPURITIES
@ (CN i-Niay-201g, With respect to the pregnancy and lactation e LIMIT OF ARSENIC
statement. “e «Nn 1-May-2018) [Note—For the preparation of all aqueous solutions and
for the rinsing of glass, polytef, and plastic vessels
 4556 Crypthecodinium / Dietary Supplements
before use, use water that has been passed through a
strong-acid, strong-base, mixed-bed ion-exchange resin
before use. Select all reagents to have as low a content
of arsenic as practicable, and store all reagent solutions
in containers of borosilicate glass. Cleanse glass, polytef,
and plastic vessels before use by soaking in warm 8N
nitric acid for 30 min and by rinsing with deionized
water.]
Solution A: Transfer 1g of ultrapure palladium metal to
a Teflon beaker. Add 20 mL of water and 10 mL of ni-
tric acid, and warm on a hot plate to dissolve. Allow
the solution to cool to room temperature, transfer it
into a 100-mL volumetric flask, and dilute with deion-
ized water to volume.
Solution B: Transfer 1 g of ultrapure magnesium nitrate
to a Teflon beaker. Add 40 mL of water and 1 mL of
nitric acid, and warm ona hot plate to dissolve the
solids. Allow the solution to cool to room temperature,
transfer it to a 100-mL volumetric flask, and dilute with
deionized water to volume.
Solution C: Solution A, Solution B, and 2% nitric acid
(3:2:5). A volume of 5 LL provides 0.015 mg of palla-
dium and 0.01 mg of magnesium nitrate.
Standard stock solution: Transfer 10.0 mL of Standard
Arsenic Solution, prepared as directed in Arsenic (211),
to a 100-mL volumetric flask. Add 40 mL of water and
5 mL of nitric acid, and dilute with water to volume.
This solution contains 0.10 pg/mL of arsenic.
Blank: Nitric acid and water (1:19)
Standard solutions: Dilute the Standard stock solution
with the Blank to obtain concentrations of 0.002,
0.005, 0.010, 0.025, and 0.050 g/mL of arsenic.
Sample solution: For preparation, use a microwave
oven with a magnetron frequency of 2455 MHz and a
selectable output power of 0-950 watts in 1% incre-
ments, equipped with advanced composite vessels with
100-mL polytef liners. Use rupture membranes to vent
vessels should the pressure exceed 125 psi. The vessels
fit into a turntable, and each vessel can be vented into
an overflow container. Equip the microwave oven with
an exhaust tube to ventilate fumes. [CAUTION—Wear
proper eye protection and protective clothing and
gloves.]
Transfer supreeiniaely 500 mg of Crypthecodinium
cohnii Oil, weighed to the nearest 0.1 mg, into a
Teflon digestion vessel liner. Prepare samples in dupli-
cate. add15 mL of nitric acid, and swirl gently. Cover
the vessels with lids, leaving the vent fitting off. Predi-
gest overnight under a hood. Place the rupture mem-
brane in the vent fitting, and tighten the lid. Place all
vessels on the microwave oven turntable. Connect the
DS Monographs
vent tubes to the vent trap, and connect the pressure-
sensing line to the appropriate vessel. Initiate a two-
stage digestion procedure by peau the microwave
at 15% power for 15 min, followed by 25% power for
45 min. Remove the turntable of vessels from the
oven, and allow the vessels to cool to room tempera-
ture. [NOTE—A cool water bath may be used to speed
the cooling process.] Vent the vessels when they reach
room temperature. Remove the lids, and slowly add
2 mL of 30% hydrogen peroxide to each. Allow the
reactions to subside, and seal the vessels. Return the
vessels on the turntable to the microwave oven, and
heat for an additional 15 min at 30% power. Remove
the vessels from the oven, and allow them to cool to
room temperature. Transfer the cooled digests into
25-mL volumetric flasks, and dilute with water to
volume.
Analysis: Program the graphite furnace as follows. Dry
at 115°, using a 1-s ramp, a 65-s hold, and an argon
flow of 300 mL/min; char the sample at 1000°, using a
1-s ramp, a 20-s hold, and an airflow of 300 mL/min;
cool down, and purge the air from the furnace for 10 s,
using a 20° set temperature and an argon flow of
300 mL/min; atomize at 2400°, using a 0-s ramp and a
USP 41
5-s hold with the argon flow stopped; and clean out at
2600° with a 1-s ramp and a 5-s hold. Separately inject
equal volumes (20 pL) of the Standard solutions, the
Sample solution, and the Blank, followed by an injection
of 5 wL of Solution C for each of the samples, into the
graphite tube of a suitable graphite furnace atomic ab-
sorption spectrophotometer equipped with a hollow-
cathode lamp for arsenic. Determine the peak area at
the arsenic emission line at 193.7 nm, corrected for
background absorption. Plot the corrected peak areas of
the Standard solutions versus their contents of arsenic,
in ug/mL, and calculate the regression line best fitting
the points. Determine the concentration, C, in ug/ml,
of arsenic in each mL of the Sample solution by interpo-
lation from the regression line.
Calculate the content of arsenic in the portion of
Crypthecodinium cohnii Oil taken:
Result = (C/W) x 25
Cc = concentration, as obtained above
Ww = weight of Crypthecodinium cohnii Oil taken to
prepare the Sample solution (g)
Acceptance criteria: NMT 0.1 ug/g
e Limit oF LEAD
[Note—For the preparation of all aqueous solutions and
for the rinsing of glass, polytef, and plastic vessels
before use, use water that has been passed through a
strong-acid, strong-base, mixed-bed ion-exchange resin
before use. Select all reagents to have as low a content
of lead as practicable, and store all reagent solutions in
containers of borosilicate glass. Cleanse glass, polytef,
and plastic vessels before use by soaking in warm 8 N
nitric acid for 30 min and by rinsing with deionized
water.]
Solution A: 10g of ultrapure monobasic ammonium
phosphate in 1 mL of nitric acid and 40 mL of water to
dissolve the phosphate. Dilute with deionized water to
100 mL.
Solution B: Transfer 1 g of ultrapure magnesium nitrate
to a Teflon beaker. Add 40 mL of water and 1 mL of
nitric acid, and warm ona hot plate to dissolve the
solids. Allow the solution to cool to room temperature,
transfer it to a 100-mL volumetric flask, and dilute with
deionized water to volume.
Solution C: Solution A, Solution B, and 2% nitric acid
(2:1:2). A volume of 5 uL provides 0.2 mg of phosphate
and 0.01 mg of magnesium nitrate.
Standard stock solution: Transfer 10.0 mL of lead ni-
trate stock solution TS to a 100-mL volumetric flask.
Add 40 mL of water and 5 mL of nitric acid, and dilute
with water to volume. Transfer 1.0 mL of this solution
to a second 100-mL volumetric flask, add 50 mL of
water and 1 mL of nitric acid, and dilute with water to
volume. This solution contains 0.10 ug/mL of lead.
Blank: Nitric acid and water (1:19)
Standard solutions: Dilute the Standard stock solution
with the Blank to obtain concentrations of 0.002,
0.005, 0.010, 0.025, and 0.050 g/mL of lead.
Sample solution: Prepare as directed for the Sample so-
lution in the test for Limit of Arsenic.
Analysis: Program the graphite furnace as follows. Dry
at 120°, using a 1-s ramp, a 55-s hold, and an argon
flow of 300 mL/min; char the sample at 850°, using a
1-s ramp, a 30-s hold, and an airflow of 300 mL/min;
cool down, and purge the air from the furnace for 10 s,
using a 20° set temperature and an argon flow of
300 mL/min; atomize at 2100°, using a 0-s ramp and a
5-s hold with the argon flow stopped; and clean out at
2600° with a 1-s ramp and a 5-s hold. Separately inject
equal volumes (20 iL) of the Standard solutions, the
Sample solution, and the Blank, followed by an injection
of 5 uL of Solution C for each of the samples, into the
graphite tube of a suitable graphite furnace atomic ab-
sorption spectrophotometer equipped with a hollow-
USP 41
cathode lamp for lead. Determine the peak area at the
lead emission line at 283.3 nm, corrected for back-
ground absorption. Plot the corrected peak areas of the
Standard solutions versus their contents of lead, in
g/mL, and calculate the regression line best fitting the
joints. Determine the concentration, C, in ug/mL, of
ead in each mL of the Sample solution by interpolation
from the regression line.
Calculate the content of lead in the portion of
Crypthecodinium cohnii Oil taken:
Result = (C/W) x 25
G = concentration, as obtained above
w = weight of Crypthecodinium cohnii Oil taken to
prepare the Sample solution (g)
Acceptance criteria: NMT 0.1 ug/g
o Limit OF CADMIUM
[Notr—For the preparation of all aqueous solutions and
for the rinsing of glass, polvtet, and plastic vessels
before use, use water that has been passed through a
strong-acid, strong-base, mixed-bed ion-exchange resin
before use. Select all Penge to have as low a content
of cadmium as practicable, and store all reagent solu-
tions in containers of borosilicate glass. Cleanse glass,
polytef, and plastic vessels before use by soaking in
warm 8N nitric acid for 30 min and by rinsing with
deionized water.]
Solution A: 10g of ultrapure monobasic ammonium
phosphate in 40 mL of water and 1 mL of nitric acid to
dissolve the phosphate. Dilute with deionized water to
100 mL.
Solution B: Transfer 1 g of ultrapure magnesium nitrate
to a Teflon beaker. Add 40 mL of water and 1 mL of
nitric acid, and warm on a hot plate to dissolve the
solids. Allow the solution to cool to room temperature,
transfer it to a 100-mL volumetric flask, and dilute with
deionized water to volume.
Solution C: Solution A, Solution B, and 2% nitric acid to
volume (2:1:2). A volume of 5 uL provides 0.2 mg of
phosphate and 0.01 mg of magnesium nitrate.
Standard stock solution A: 0.1372 mg/mL of cadmium
nitrate
Standard stock solution B: Standard stock solution A,
nitric acid, and water (2:1:97). This solution contains
0.10 ug/mL of cadmium. [NoTtE—Before make-up to fi-
ae dissolve in a portion of water and nitric
acid.
Blank: Nitric acid and water (1:19)
Standard solutions: Dilute Standard stock solution B
with the Blank to obtain concentrations of 0.002,
0.005, 0.010, 0.025, and 0.050 ig/mL of cadmium.
Sample solution: Prepare as directed for the Sample so-
lution in the test for Limit of Arsenic.
Analysis: Program the graphite furnace as follows. Dry
at 120°, using a 1-s ramp, a 55-s hold, and an argon
flow of 300 mL/min; char the sample at 850°, using a
1-s ramp, a 30-s hold, and an airflow of 300 mL/min;
cool down, and purge the air from the furnace for 10 s,
using a 20° set temperature and an argon flow of
300 mL/min; atomize at 2400°, using a 0-s ramp and a
5-s hold with the argon flow stopped; and clean out at
2600° with a 1-s ramp and a 5-s hold. Separately inject
equal volumes (20 LL) of the Standard solutions, the
Sample solution, and the Blank, followed by an injection
of 5 e of Solution C for each of the samples, into the
graphite tube of a suitable graphite furnace atomic ab-
serpiien spectrophotometer equipped with a hollow-
cathode lamp for cadmium, Determine the peak area at
the cadmium emission line at 228.8 nm, corrected for
background absorption. Plot the corrected peak areas of
the Standard solutions versus their contents of cadmium,
in ug/mL, and calculate the regression line best fitting
the points.
Dietary Supplements / Crypthecodinium 4557
Determine the concentration, C, in ug/mL, of cadmium
in each mL of the Sample solution by interpolation
from the regression line.
Calculate the content of cadmium in the portion of
Crypthecodinium cohnii Oil taken:
Result = (C/W) x 25
€ = concentration, as obtained above
Ww = weight of Crypthecodinium cohnii Oil taken to
prepare the Sample solution (g)
Acceptance criteria: NMT 0.1 ug/g
e Limit OF MeRcurY: Proceed as directed in Mercury (261),
Method lla, except use a Standard Mercury Solution hav-
ing the equivalent of 0.1 g/mL of mercury.
Sample solution: Prepare as directed for the Sample so-
lution in the test for Limit of Arsenic, combining the two
duplicate cooled digests into 1.0 mL of Potassium Per-
manganate Solution.
Acceptance criteria: NMT 0.1 ug/g
SPECIFIC TESTS
e FATS AND FIXED OILS (401), Anisidine Value: NMT 20.0
e FATS AND FIXED OILS (401), Acid Value (Free Fatty Acids):
The free fatty acids in 10 g require NMT 1.42 mL of 0.1
N sodium hydroxide for neutralization.
FATS AND FIXED OILS reg Peroxide Value: NMT 5.0
FATS AND FIXED OiLs (401), Tota! Oxidation Value
(TOTOX): NMT 26, calculated as:
Result = (2 x PV) + AV
PV = peroxide value
AV = anisidine value
FATS AND FIXED OILS (401), Unsaponifiable Matter: NMT
3.5%
SPECIFIC GRAVITY (841): 0.91-0.93
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in tight, light-resistant
containers, and avoid exposure to excessive heat.
e LABELING: The label states the content of docosahexae-
noic acid in mg/g. It also states the name and concentra-
tion of any added antioxidant.
Delete the following:
°e USP REFERENCE STANDARDS (11)
USP Docosahexaenoic Acid Ethyl Ester RS
USP Eicosapentaenoic Acid Ethy! Ester RS
USP Methy! Tricosanoate RS
sydesbouow sa
@ (CN 1-May-2018)
Crypthecodinium cohnii Oil Capsules
DEFINITION
ee cohnii Oil Capsules are prepared from
rypthecodinium cohnii Oil and contain NLT 95.0% and
NMT 105.0% of the labeled amount of docosahexaenoic
acid (DHA, C22H3202) (C22:6 n-3).
IDENTIFICATION
@ LONG-CHAIN UNSATURATED FATTY ACID PROFILE: Proceed
as directed in Content of DHA.
Analysis
Samples: Standard Solution 2a, Standard Solution 2b,
and Test solution 7
Calculate the area percentage for each fatty acid as
methy! ester in Test solution 1:
Result = (ru/rz) x 100
 4558 Crypthecodinium / Dietary Supplements USP 41

ty = peak response of each individual fatty acid as of arsenic as practicable, and store all reagent solutions
methyl ester in containers of borosilicate glass. Cleanse glass, polytef,
rr = sum of all the peak responses, except the and plastic vessels before use by soaking in warm 8 N
solvent and butylated hydroxytoluene peaks nitric acid for 30 min and by rinsing with deionized
Acceptance criteria: The retention time of the peaks of water.]
the docosahexaenoic acid methyl ester and the eicosa- Solution A: Transfer 1 g of ultrapure palladium metal
pentaenoic acid methyl ester from Test solution 7 corre- into a Teflon beaker. Add 20 mL of water and 10 mL of
sponds to that from the docosahexaenoic acid methyl nitric acid, and warm on a hot plate to dissolve. Allow
ester and eicosapentanoic acid methyl ester peaks from the solution to cool to room temperature, transfer it
Standard Solution 2a and Standard Solution 2b, respec- into a 100-mL volumetric flask, and dilute with deion-
tively, as obtained in the test for Content of EPA and ized water to volume.
DHA. The area percent for the methyl esters of the fatty Solution B: Transfer 1 g of ultrapure magnesium nitrate
acids from Test solution 1 in the test for Content of EPA into a Teflon beaker. Add 40 mL of water and 1 mL of
and DHA meet the requirements for each fatty acid indi- nitric acid, and warm onahot plate to dissolve the
cated in Table 7. solids. Allow the solution to cool to room temperature,
transfer it into a 100-mL volumetric flask, and dilute
Table 1 with deionized water to volume.
Solution C: Solution A, Solution B, and 2% nitric acid
Lower | Upper (3:2:5). A volume of 5 uL provides 0.015 mg of palla-
Relative Limit Limit dium and 0.01 mg of magnesium nitrate.
Fatty Retention Shorthand | (Area, | (Area, Blank: Nitric acid and water (1:19)
Acid Time Notation %) %) Standard stock solution: Transfer 10.0 mL of Standard
Linoleic acid 0.52 18:2 n-6 0 1.0 Arsenic Solution, prepared as directed in the test for Ar-
Eicosapentanoic senic (211), to a 100-mL volumetric flask. Add 40 mL of
acid 0.79 20:5 n-3 0 0.1 water and 5 mL of nitric acid, and dilute with water to
Docosapentae- volume. This solution contains 0.10 tg/mL of arsenic.
noic acid 0.94 22:5 n-6 0 0.1 Standard solutions: Dilute the Standard stock solution
Docosahexae- with the Blank to obtain concentrations of 0.002,
noic acid 1.00 22°6 n-3 35.0 47.0 0.005, 0.010, 0.025, and 0.050 g/mL of arsenic.
Sample solution: For preparation of the Sample solu-
tion, use a microwave oven with a magnetron fre-
STRENGTH quency of 2455 MHz and a selectable output power of
e CONTENT OF DHA 0-950 watts in 1% increments, equipped with ad-
Test solution 1 and Test solution 2: Weigh NLT 10 vanced composite vessels with 100-mL polytef liners.
Capsules in a tared weighing bottle. With a sharp blade Use rupture membranes to vent vessels should the pres-
or other appropriate means, carefully open the Cap- sure exceed 125 psi. The vessels fit into a turntable, and
sules, without loss of the shell material, and transfer the each vessel can be vented into an overflow container.
combined Capsule contents to a 100-mL beaker. Re- Equip the microwave oven with an exhaust tube to
move any adhering substance from the emptied Cap- ventilate fumes. [CAUTION—Wear proper eye protection
sules by washing with several small portions of isooc- and protective clothing and gloves.]
tane. Discard the washings, and allow the empty Transfer approximately 500 mg of Crypthecondinium
Capsules to dry in a current of dry air until the isooc- cohnii oil from Capsules, weighed to the nearest
tane is completely evaporated. Weigh the empty Cap- 0.1 mg, into a Teflon digestion vessel liner. Prepare
sules in the original tared weighing bottle, and calculate cel ae in duplicate. Add 15 mL of nitric acid, and
the average fill weight (AFW) of Crypthecodinium cohnni swirl gently. Cover the vessels with lids, leaving the
oil/Capsule. Proceed with the content of Capsules as vent fitting off. Predigest overnight under a hood.
directed in the Analysis. Place the rupture membrane in the vent fitting, and
Analysis: Proceed as directed in Fats and Fixed Oils tighten the lid. Place all vessels on the microwave
(401), Omega-3 Fatty Acids Determination and Profile, oven turntable. Connect the vent tubes to the vent
Content of EPA and DHA. trap, and connect the pressure-sensing line to the ap-
Calculate the percentage of the labeled amount of doco- propriate vessel. Initiate a two-stage digestion proce-
DS Monographs

sahexaenoic acid (DHA) in the Capsules taken:
Result = R x AFW/L
R = determined percentage of DHA in the portion
of oil taken from the Capsules (%)
AFW_ = average fill weight of the Capsules taken (mg)
L = labeled amount of DHA (mg/Capsule)
Acceptance criteria: NLT 95.0% and NMT 105.0% of
the labeled amount of DHA
PERFORMANCE TESTS
e DISINTEGRATION AND DISSOLUTION (2040): Meet the re-
quirements for Rupture Test for Soft Shell Capsules
@ WEIGHT VARIATION OF DIETARY SUPPLEMENTS (2091): Meet
the requirements
IMPURITIES
o LIMIT OF ARSENIC
[Note—For the preparation of all aqueous solutions and
for the rinsing of glass,pobtet and plastic vessels
before use, use water that has been passed through a
strong-acid, strong-base, mixed-bed ion-exchange resin
before use. Select all reagents to have as low a content
dure by heating the microwave at 15% power for 15
min, followed by 25% power for 45 min. Remove the
turntable of vessels from the oven, and allow the ves-
sels to cool to room temperature. [NOTE—A cool water
bath may be used to speed the cooling process.] Vent
the vessels when they reach room temperature. Re-
move the lids, and slowly add 2 mL of 30% hydrogen
peroxide to each. Allow the reactions to subside, and
seal the vessels. Return the vessels on the turntable to
the microwave oven, and heat for an additional 15
min at 30% power. Remove the vessels from the oven,
and allow them to cool to room temperature. Transfer
the cooled digests into 25-mL volumetric flasks, and
dilute with water to volume.
Analysis: Program the graphite furnace as follows. Dry
at 115°, using a 1-s ramp, a 65-s hold, and an argon
flow of 300 mL/min; char the sample at 1000°, using a
1-s ramp, a 20-s hold, and an airflow of 300 mL/min;
cool down, and purge the air from the furnace for 10 s,
using a 20° set temperature and an argon flow of
300 mL/min; atomize at 2400°, using a 0-s ramp and a
5-s hold with the argon flow stopped; and clean out at
2600° with a 1-s ramp and a 5-s hold. Separately inject
equal volumes (20 uly of the Standard solutions, the
USP 41
Sample solution, and the Blank, followed by an injection
of 5 ul of Solution C for each of the samples, into the
graphite tube of a suitable graphite furnace atomic ab-
sorption spectrometer equipped with a hollow-cathode
lamp for arsenic. Determine the peak area at the arsenic
emission line at 193.7 nm, corrected for background
absorption. Plot the corrected peak areas of the Stan-
dard solutions versus their contents of arsenic, in g/mL,
and calculate the regression line best fitting the points.
Determine the concentration, C, in yg/mL, of arsenic in
each mL of the Sample solution by interpolation from
the regression line.
Calculate the content of arsenic in the portion of Cap-
sules taken:
Result = (C/W) x 25
G = concentration as obtained above
Ww = weight of Capsules content taken to prepare
the Sample solution (g)
Acceptance criteria: NMT 0.1 ug/g
e Limit oF LEAD
[Note—For the preparation of all aqueous solutions and
for the rinsing of glass, polytef, and plastic vessels
before use, use water that has been passed through a
strong-acid, strong-base, mixed-bed ion-exchange resin
before use. Select all reagents to have as low a content
of lead as practicable, and store all reagent solutions in
containers of borosilicate glass. Cleanse glass, polytef,
and plastic vessels before use by soaking in warm 8 N
nitric acid for 30 min and by rinsing with deionized
water.]
Solution A: 10g of ultrapure monobasic ammonium
phosphate in 1 mL of nitric acid and 40 mL of water to
dissolve the phosphate. Dilute with deionized water to
100 mL.
Solution B: Transfer 1 g of ultrapure magnesium nitrate
to a Teflon beaker. Add 40 mL of water and 1 mL of
nitric acid, and warm on a hot plate to dissolve the
solids. Allow the solution to cool to room temperature,
transfer it to a 100-mL volumetric flask, and dilute with
deionized water to volume.
Solution C: Solution A, Solution B, and 2% nitric acid
(2:1:2). A volume of 5 wL provides 0.2 mg of phosphate
and 0.01 mg of magnesium nitrate.
Blank: Nitric acid and water (1:19)
Standard stock solution: Transfer 10.0 mL of lead ni-
trate stock solution TS to a 100-mL volumetric flask.
Add 40 mL of water and 5 mL of nitric acid, and dilute
with water to volume. Transfer 1.0 mL of this solution
to a second 100-mL volumetric flask, add 50 mL of
water and 1 mL of nitric acid, and dilute with water to
volume. This solution contains 0.10 tg/mL of lead.
Standard solutions: Dilute the Standard stock solution
with the Blank to obtain concentrations of 0.002,
0.005, 0.010, 0.025, and 0.050 jtg/mL of lead.
Sample solution: Prepare as directed for Sample solu-
tion in the test for Limit of Arsenic.
Analysis: Program the graphite furnace as follows. Dry
at 120°, using a 1-s ramp, a 55-s hold, and an argon
flow of 300 mL/min; char the sample at 850°, using a
1-s ramp, a 30-s hold, and an airflow of 300 mL/min;
cool down, and purge the air from the furnace for 10 s,
using a 20° set temperature and an argon flow of
300 mL/min; atomize at 2100°, using a 0-s ramp and a
5-s hold with the argon flow stopped; and clean out at
2600° with a 1-s ramp and a 5-s hold. Separately inject
equal volumes (20 uL) of the Standard solutions, the
Sample solution, and the Blank, followed by an injection
of 5 uL of the Solution C for each of the samples, into
the graphite tube of a suitable graphite furnace atomic
absorption spectrometer equipped with a hollow-cath-
ode lamp for lead. Determine the peak area at the lead
emission line at 283.3 nm, corrected for background
Dietary Supplements / Crypthecodinium 4559
absorption. Plot the corrected peak areas of the Stan-
dard solutions versus their contents of lead, in ug/mL,
and calculate the regression line best fitting the points.
Determine the concentration, C, in ug/mL, of lead in
each mL of the Sample solution by interpolation from
the regression line.
Calculate the content of lead in the portion of Capsules
taken:
Result = (C/W) x 25
Cc = concentration as obtained above
Ww = weight of Capsules content taken to prepare
the Sample solution (g)
Acceptance criteria: NMT 0.1 pg/g
e Limit oF CADMIUM
[Note—For the preparation of all aqueous solutions and
for the rinsing of glass, poles, and plastic vessels
before use, use water that has been passed through a
strong-acid, strong-base, mixed-bed ion-exchange resin
before use. Select all reagents to have as low a content
of cadmium as practicable, and store all reagent solu-
tions in containers of borosilicate glass. Cleanse glass,
polytef, and plastic vessels before use by soaking in
warm 8N nitric acid for 30 min and by rinsing with
deionized water.]
Solution A: 10g of ultrapure monobasic ammonium
phosphate in 40 mL of water and 1 mL of nitric acid to
dissolve the phosphate. Dilute with deionized water to
100 mL.
Solution B: Transfer 1 g of ultrapure magnesium nitrate
to a Teflon beaker. Add 40 mL of water and 1 mL of
nitric acid, and warm ona hot plate to dissolve the
solids. Allow the solution to cool to room temperature,
transfer it to a 100-mL volumetric flask, and dilute with
deionized water to volume.
Solution C: Solution A, Solution B, and 2% nitric acid to
volume (2:1:2). A volume of 5 ul provides 0.2 mg of
phosphate and 0.01 mg of magnesium nitrate.
Blank: Nitric acid and water (1:19)
Standard stock solution A: 0.1372 mg/mL of cadmium
nitrate
Standard stock solution B: Standard stock solution A,
nitric acid, and water (2:1:97). This solution contains
0.10 ng/mL of cadmium. [Note—Before make up to fi-
aa dissolve in a portion of water and nitric
acid.
Standard solutions: Dilute Standard stock solution B
with the Blank to obtain concentrations of 0.002,
0.005, 0.010, 0.025, and 0.050 ttg/mL of cadmium.
Sample solution: Prepare as directed for Sample solu-
sydeibouow-: Sa
tion in the test for Limit of Arsenic.
Analysis: Program the graphite furnace as follows. Dry
at 120°, using a 1-s ramp, a 55-s hold, and an argon
flow of 300 mL/min; char the sample at 850°, using a
1-s ramp, a 30-s hold, and an airflow of 300 mL/min;
cool down, and purge the air from the furnace for 10 s,
using a 20° set temperature and an argon flow of
300 mL/min; atomize at 2400°, using a 0-s ramp and a
5-s hold with the argon flow stopped; and clean out at
2600° with a 1-s ramp and a 5-s hold. Separately inject
equal volumes (20 uL) of the Standard solutions, the
Sample solution, and the Blank, followed by an injection
of 5 uL of the Solution C for each of the samples, into
the graphite tube of a suitable graphite furnace atomic
absorption spectrometer equipped with a hollow-cath-
ode lamp for cadmium. Determine the peak area at the
cadmium emission line at 228.8 nm, corrected for
background absorption. Plot the corrected peak areas of
the Standard solutions versus their contents of cadmium,
in pg/mL, and calculate the regression line best fitting
the points. Determine the concentration, C, in ug/mL,
of cadmium in each mL of the Sample solution by inter-
polation from the regression line.
 4560 Crypthecodinium / Dietary Supplements
Calculate the content of cadmium in the Capsules
taken:
Result = (C/W) x 25
€ = concentration as obtained above
Ww = weight of Capsules content taken to prepare
the Sample solution (g)
Acceptance criteria: NMT 0.7 pg/g
© Limit OF Mercury: Proceed as directed in Mercury (261),
Method Ila, except use a Standard Mercury Solution hav-
ing the equivalent of 0.1 ug/mL of mercury.
Sample solution: Prepare as directed for the Sample so-
lution in the test for Limit of Arsenic, combining the two
duplicate cooled digests into 1.0 mL of Potassium Per-
manganate Solution.
Acceptance criteria: NMT 0.1 pg/g
SPECIFIC TESTS
e FATS AND FIXED OILS, Anisidine Value (401): NMT 20.0,
determined on the contents of the Capsules
FATS AND FIXED OILS, Free Fatty Acids (401): The free fatty
acids in 10 g require for neutralization NMT 1.42 mL of
0.1 N sodium hydroxide.
FATS AND FIXED OILS, Peroxide Value (401): NMT 5.0, de-
termined on the contents of the Capsules
FATS AND FIXED OILS, Total Oxidation Value (TOTOX)
(401): NMT 26 (determined on the contents of the
Capsules), calculated as:
Result = (2 x PV) + AV
PV = peroxide value
AV = anisidine value
FATS AND FIXED OILS, Unsaponifiable Matter (401): NMT
3.5%, determined on the contents of the Capsules
SPECIFIC GRAVITY (841): 0.91-0.93, determined on the
contents of the Capsules
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in tight, light-resistant
containers, and avoid exposure to excessive heat.
e LABELING: The label states the content of docosahexae-
noic acid in mg/Capsule. It also states the name and
concentration of any added antioxidant.
e USP REFERENCE STANDARDS (11)
USP Docosahexaenoic Acid Ethyl Ester RS
USP Eicosapentaenoic Acid Ethyl Ester RS
USP Methyl Tricosanoate RS
DS Monographs
Curcuminoids
DEFINITION
Curcuminoids is a partially purified natural complex of diary!
heptanoid derivatives isolated from Turmeric, Curcuma
longa L. It contains NLT 95.0% of curcuminoids, calcu-
lated on the dried basis, as the sum of curcumin, des-
methoxycurcumin, and bisdesmethoxycurcumin. It con-
tains NLT 70.0% and NMT 80.0% of curcumin, NLT
15.0% and NMT 25.0% of desmethoxycurcumin, and
NLT 2.5% and NMT 6.5% of bisdesmethoxycurcumin.
IDENTIFICATION
e A. HPTLC FOR ARTICLES OF BOTANICAL ORIGIN (203)
Standard solution: 1 mg/mL of USP Curcuminoids RS
in methanol
Sample solution: Suspend about 5 mg of Curcuminoids
in 5 mL of methanol, and sonicate briefly.
USP 41
Chromatographic system
Adsorbent: Chromatographic silica gel with an aver-
age particle size of 5 um (HPTLC plate)!
Application volume: 2 UL each of the Standard solu-
tion and the Sample solution as 8-mm bands
Relative humidity: Condition the plate toa relative
humidity of 33%.
Temperature: Ambient, not to exceed 30°
Developing solvent system: Toluene and glacial ace-
tic acid (4:1)
Developing distance: 6 cm
Derivatization reagent: 85 mL of ice-cold methanol
combined with 10 mL of glacial acetic acid, 5 mL of
sulfuric acid, and 0.5 mL of p-anisaldehyde
Analysis
Samples: Standard solution and Sample solution
Apply the Samples as bands and dry in air. Develop in a
saturated chamber and dry in air. Treat with Deriva-
tization reagent, heat at 100° for 3 min, and examine
inder long-wave UV light (365 nm) and under white
ight.
System suitability: Under long-wave UV light (365
nm), the derivatized chromatogram of the Standard so-
lution exhibits, in its lower half, three bands in the order
of increasing R= an orange band due to bisdesmethoxy-
curcumin, an orange band due to desmethoxycurcu-
min, and the red band due to curcumin. Under white
light, the two lower bands appeat orange, while the
topmost band is reddish-pink.
Acceptance criteria: Under long-wave UV light (365
nm), the derivatized chromatogram of the Sample solu-
tion displays two orange bands and one red band, simi-
lar in position and color to those observed in the Stan-
dard solution. At the bottom part of the upper half of
the plate, two purple bands are seen. Under white
light, two orange bands and a darker red band are seen
coincident with the bands due to bisdesmethoxycurcu-
min, desmethoxycurcumin, and curcumin in the Stan-
dard solution, in the order of increasing Rr. In the upper
half of the plate, the lower of the two bands appears
purple, while the upper band is brown. No bands ap-
pear in the topmost quarter of the plate, which is char-
acteristic of Curcuma zanthorrhiza Roxb. and Curcuma
aromatica Salisb. These confounders, and occasional
adulterants, also lack the lower orange band corre-
sponding to bisdesmethoxycurcumin. Additional weak
bands may be observed in the Sample solution under
either illumination condition.
e B. HPLC
Analysis: Proceed as directed in the test for Content of
Curcuminoids.
Acceptance criteria: The retention times of the peaks
for curcumin, desmethoxycurcumin, and bisdesmeth-
oxycurcumin of the Sample solution correspond to those
of Standard solution A and Standard solution B.
COMPOSITION
© CONTENT OF CURCUMINOIDS
Mobile phase: Tetrahydrofuran and 1 mg/mL of citric
acid in water (4:6)
Standard solution A: 40 g/mL of USP Curcuminoids
RS in Mobile phase
Standard solution B: A composite solution containing
40 g/mL of USP Curcumin RS, 10 ug/mL of USP Des-
methoxycurcumin RS, and 2.0 g/mL of USP Bisdes-
methoxycurcumin RS in Mobile phase. Use sonication if
necessary. Before injection, pass through a filter of
0.45-um pore size, and discard the initial 10 mL of the
filtrate.
Sample stock solution: Transfer about 20 mg of
Curcuminoids, accurately weighed, to a 50-mL volumet-
ric flask, add 30 mL of acetone, and sonicate for 30
1Suitable commercially available plates are HPTLC Silica Gel 60 Fass from EMD
Millipore (e.g., Part No. 1.05642.0001).
USP 41
min. Dilute with acetone to volume, mix, and
centrifuge.
Sample solution: Transfer 5.0 mL of the Sample stock
solution to a 50-mL volumetric flask. Dilute with Mobile
phase to volume, and mix. Before injection, pass
througha filter of 0.45-1um pore size, and discard the
initial 10 mL of the filtrate.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: Vis 420 nm
Column: 4.6-mm x 25-cm; 5-"um packing L1
Flow rate: 1.0 mL/min
Injection volume: 20 pL
System suitability
Samples: Standard solution A and Standard solution B
[Note—The relative retention times for the curcumin,
desmethoxycurcumin, and bisdesmethoxycurcumin
peaks are 1.0, 1.2, and 1.4, eect T ers; protect from light and moisture, and store at room
Suitability requirements
Chromatogram similarity: The chromatogram of
Standard solution A is similar to the reference chro-
matogram provided with USP Curcuminoids RS.
Resolution: NLT 2.0 between curcumin and des-
methoxycurcumin peaks and desmethoxycurcumin
and bisdesmethoxycurcumin peaks, Standard solution
B USP Curcumin RS
Tailing factor: NMT 1.5 for bisdesmethoxycurcumin,
desmethoxycurcumin, and curcumin peaks, Standard
solution B
Relative standard deviation: NMT 2.0% for the des-
methoxycurcumin peak, in replicate injections, Stan-
dard solution B
Analysis
Samples: Standard solution B and Sample solution
Calculate the percentages of curcumin, desmethoxycur-
cumin, and bisdesmethoxycurcumin in the portion of
Curcuminoids taken:
Result = (ru/rs) x Cs x (V/W) x D x 100
ru = peak area of the relevant analyte from the
Sample solution
ts peak area of the relevant analyte from
Standard solution B
Cs = concentration of the relevant analyte in
Standard solution B (mg/mL)
Vv = volume of the Sample stock solution (mL)
Ww = weight of Curcuminoids used to prepare the
Sample stock solution (mg)
D = dilution factor to obtain the Sample solution
from the Sample stock solution, 10
Acceptance criteria: Curcuminoids contains NLT 95.0%
of curcuminoids, calculated on the dried basis, as the
sum of curcumin, desmethoxycurcumin, and bisdes-
methoxycurcumin. It contains 70.0%-80.0% of curcu-
min, 15.0%-25.0% of desmethoxycurcumin, and
2.5%-6.5% of bisdesmethoxycurcumin.
CONTAMINANTS
Delete the following:
°e HEAVY METALs (231), Method Ill: NMT 20 ppme corinult.
Jan-2018)
© ARTICLES OF BOTANICAL ORIGIN (561), Pesticide Residue
Analysis: Meets the requirements
e BOTANICAL EXTRACTS (565), Preparations, General Pharma-
copeial Requirements, Residual Solvents: Meets the
requirements
© ARTICLES OF BOTANICAL ORIGIN (561), Test for Aflatoxins:
Meets the requirements
e MICROBIAL ENUMERATION TESTS (2021): The total aerobic
bacterial count does not exceed 104 cfu/g, and the total
Dietary Supplements / Curcuminoids 4561
combined molds and yeasts count does not exceed 103
cfu/g.
© ABSENCE OF SPECIFIED MICROORGANISMS (2022), Test Proce-
dures, Test for Absence of Salmonella Species and Test for
Absence of Escherichia coli: Meets the requirements
SPECIFIC TESTS
e MELTING RANGE OR TEMPERATURE (741), Procedures, Proce-
dure for Class |, Apparatus Ilz_ 172°-178°
e Loss ON DRYING (731)
Sample: 1.0g of Curcuminoids
Analysis: Dry the Sample at 105° for 2 h.
Acceptance criteria: NMT 2.0%
© ARTICLES OF BOTANICAL ORIGIN (561), Methods of Analysis,
Total Ash: NMT 1.0%
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in well-closed contain-
temperature.
© LABELING: The label states the content of curcuminoids
and the content of the individual curcuminoids, on the
dried basis; the Latin binomial; and the part of the plant
used to prepare the article.
e USP REFERENCE STANDARDS (11)
USP Bisdesmethoxycurcumin RS
USP Curcuminoids RS
USP Desmethoxycurcumin RS
Curcuminoids Capsules
DEFINITION
Curcuminoids Capsules are prepared from Curcuminoids
and contain NLT 90.0% and NMT 110.0% of the labeled
amount of curcuminoids, calculated as the sum of curcu-
min, desmethoxycurcumin, and bisdesmethoxycurcumin.
IDENTIFICATION
e A. THIN-LAYER CHROMATOGRAPHY
Standard solution: 0.2 mg/mL of USP Curcuminoids RS
in acetone
Sample solution: Weigh and finely powder the con-
tents of NLT 20 Capsules. Transfer a portion of the
powder, equivalent to about 10 mg of curcuminoids, to
a suitable container, add 5 mL of acetone, shake for 1
min, and sonicate for 10 min. Allow to stand for 15
min before use.
Adsorbent: Chromatographic silica gel mixture with an
sydeibouow sq
average particle size of 10-15 um (ILC plates)
Application volume: 10 uL, as bands
Developing solvent system: Chloroform, methanol,
and formic acid (96:4:1)
Analysis
Samples: Standard solution and Sample solution
Apply the samples as bands to a suitable thin-layer
chromatographic plate (see Chromatography (621)).
Use a saturated chamber. Develop the chromato-
grams until the solvent front has moved up about
three-fourths of the length of the plate. Remove the
plate from the chamber, dry, and examine under UV
light at 365 nm.
Acceptance criteria: The Sample solution chromato-
gram shows yellowish-brown bands due to bisdesmeth-
Oxycurcumin, desmethoxycurcumin, and curcumin at Rr
values of about 0.4, 0.6, and 0.7, respectively, corre-
sponding in position and color to those obtained from
the Standard solution.
e B. The retention times of the peaks for curcumin, des-
methoxycurcumin, and bisdesmethoxycurcumin of the
Sample solution correspond to those of the Standard solu-
 4562 Curcuminoids / Dietary Supplements
tion for the appropriate USP Reference Standard, as ob-
tained in the test for Content of Curcuminoids.
STRENGTH
© CONTENT OF CURCUMINOIDS
Mobile phase: Tetrahydrofuran and 1 mg/mL of citric
acid in water (4:6)
[Note—Sonication may be necessary to dissolve the RS
in each Standard solution; all solutions should be
passed through a filter with 0.45-1m pore size before
injection. USP Curcumin RS, USP Desmethoxycurcumin
RS, and USP Bisdesmethoxycurcumin RS can also be
prepared in one standard solution containing the final
concentration specified below for each.]
Standard solution A: 40 \tg/mL of USP Curcuminoids
RS in Mobile phase
Standard solution B: 40 g/mL of USP Curcumin RS in
Mobile phase
Standard solution C: 10 g/mL of USP Desmethoxy-
curcumin RS in Mobile phase
Standard solution D: 2 g/mL of USP Bisdesmethoxy-
curcumin RS in Mobile phase
Sample stock solution: Weigh and finely powder the
contents of NLT 20 Capsules. Transfer an accurately
weighed amount of the powder, equivalent to about
20 mg of curcuminoids, to a 50-mL volumetric flask.
Add about 30 mL of acetone, sonicate for 30 min, di-
lute with acetone to volume, mix, and centrifuge.
Sample solution: Dilute a portion of the Sample stock
solution 1 in 10 with Mobile phase, and mix.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV-Vis 420 nm
Column: 4.6-mm x 25-cm; 5-um packing L1
Flow rate: 1 mL/min
Injection size: 20 uL
System suitability
Sample: Standard solution A
[Note—The relative retention times for the curcumin,
desmethoxycurcumin, and bisdesmethoxycurcumin
peaks are about 1.0, 1.2, and 1.4, respectively.]
Suitability requirements
Chromatogram similarity: The chromatogram from
Standard solutionA is similar to the Reference Chro-
matogram provided with the lot of USP Curcumi-
noids RS being used.
Resolution: NLT 2.0 between the curcumin and des-
methoxycurcumin peaks and the desmethoxycurcu-
min and bisdesmethoxycurcumin peaks
Tailing factor: NMT 1.5 for the bisdesmethoxycurcu-
DS Monographs
min, desmethoxycurcumin, and curcumin peaks
Relative standard deviation: NMT 2.0% for des-
methoxycurcumin peak, in repeated injections
Analysis
Samples: Standard solution A, Standard solution B,
Standard solution C, Standard solution D, and Sample
solution
Calculate the quantity, in mg, of curcumin, desmeth-
oxycurcumin, and bisdesmethoxycurcumin in each
Capsule:
Result = (ru/rs) x Cs x D x V x (W/W)
tu = peak area for curcumin, desmethoxycurcumin,
or bisdesmethoxycurcumin from the Sample
solution
Is = peak area for curcumin, desmethoxycurcumin,
or bisdesmethoxycurcumin from the
appropriate Standard solution
Gs = concentration of the appropriate Standard
Solution (mg/mL)
D = dilution factor to prepare the Sample solution
from Sample stock solution
Vv = volume of Sample stock solution (mL)
USP 41
W, = average fill weight of Capsules (mg)
Wu = weight of content of Capsules taken to
prepare the Sample stock solution (mg)
Calculate the percentage of the labeled amount of
curcuminoids in the Capsule:
Result = (ZQ/L) x 100
XQ = =sum of the quantities of curcumin,
desmethoxycurcumin, and
bisdesmethoxycurcumin in the Capsule (mg)
L = labeled amount of curcuminoids (mg/Ca Bae
Acceptance criteria: 90.0%-110.0% of the label claim
PERFORMANCE TESTS
¢ DISINTEGRATION AND DISSOLUTION (2040)
Mode: Dissolution
Medium: Water containing 1% sodium lauryl sulfate;
900 mL
Apparatus 2: 100 rpm
ime: 60 min
Sample solution: Combine 25-mL portions of the solu-
tion under test from each of the six dissolution vessels,
and mix. Transfer 5 mL to a 25-mL volumetric flask, and
dilute with Mobile phase to volume.
Analysis: Determine the amount of curcumin (C2:H20O6¢)
dissolved by using the method used in Strength, making
any necessary modifications.
Tolerances: NLT 75% of the content of curcumin
(C21H200¢) is dissolved.
¢ WEIGHT VARIATION OF DIETARY SUPPLEMENTS (2091): Meet
the requirements
CONTAMINANTS
e MICROBIAL ENUMERATION TESTS (2021): The total aerobic
bacterial count does not exceed 104 cfu/g, and the total
coped molds and yeasts count does not exceed 103
cru/g.
° ABSENCE OF SPECIFIED MICROORGANISMS (2022): Meet the
requirements of the tests for the absence of Salmonella
species and Escherichia coli
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in well-closed contain-
ers, protect from light and moisture, and store at room
temperature.
e LABELING: The label states the content of curcuminoids in
mag Capel.
e USP REFERENCE STANDARDS (11)
USP Bisdesmethoxycurcumin RS
USP Curcumin RS
USP Curcuminoids RS
USP Desmethoxycurcumin RS
Curcuminoids Tablets
DEFINITION
Curcuminoids Tablets are prepared from Curcuminoids and
contain NLT 90.0% and NMT 110.0% of the labeled
amount of curcuminoids, calculated as the sum of curcu-
min, desmethoxycurcumin, and bisdesmethoxycurcumin.
IDENTIFICATION
e A. THIN-LAYER CHROMATOGRAPHY
Standard solution: 0.2 mg/mL of USP Curcuminoids RS
in acetone
mame solution: Weigh and finely powder NLT 20
Tablets. Transfer a portion of the powder, equivalent to
about 10 mg of curcuminoids, to a suitable container,
add 5 mL of acetone, shake for 1 min, and sonicate for
10 min. Allow to stand for 15 min before use.
USP 41
Adsorbent: Chromatographic silica gel mixture with an
average particle size of 10-15 uum (TLC plates)
Application volume: 10 uL, as bands
Developing solvent system: Chloroform, methanol,
and formic acid (96:4:1)
Analysis
Samples: Standard solution and Sample solution
Apply the samples as bands to a suitable thin-layer
chromatographic plate (see Chromatography (621)).
Use a saturated chamber. Develop the chromato-
grams until the solvent front has moved up about
three-fourths of the length of the plate. Remove the
late from the chamber, dry, and examine under UV
ight at 365 nm.
Acceptance criteria: The Sample solution chromato-
gram shows yellowish-brown bands due to bisdesmeth-
area desmethoxycurcumin, and curcumin at Re
values of about 0.4, 0.6, and 0.7, respectively, corre-
sponding in position and color to those obtained from
the Standard solution.
e B. The retention times of the peaks for curcumin, des-
methoxycurcumin, and bisdesmethoxycurcumin of the
Sample solution correspond to those of the Standard solu-
tion for the appropriate USP Reference Standard, as ob-
tained in the test for Content of Curcuminoids.
STRENGTH
e CONTENT OF CURCUMINOIDS
Mobile phase: Tetrahydrofuran and 1 mg/mL of citric
acid in water (4:6)
[NoTe—Sonication may be necessary to dissolve the RS
in each Standard solution; all solutions should be
passed through a filter with 0.45-um pore size before
injection. USP Curcumin RS, USP Desmethoxycurcumin
RS and USP Bisdesmethoxycurcumin RS can also be
prepared in one standard solution containing the final
concentration specified below for each.]
Standard solution A: 40 ug/mL of USP Curcuminoids
RS in Mobile phase
Standard solution B: 40 g/mL of USP Curcumin RS in
Mobile phase
Standard solution C: 10 j1g/mL of USP Desmethoxy-
curcumin RS in Mobile phase
Standard solution D: 2 g/mL of USP Bisdesmethoxy-
curcumin RS in Mobile phase
Sample stock solution: Weigh and finely powder NLT
20 Tablets. Transfer an accurately weighed amount of
the Dowd equivalent to about 20 mg of curcumi-
noids, to a 50-mL volumetric flask. Add about 30 mL of
acetone, sonicate for 30 min, dilute with acetone to
volume, mix, and centrifuge.
Sample solution: Dilute a portion of the Sample stock
solution (1 in 10) with Mobile phase, and mix.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV-Vis 420 nm
Column: 4.6-mm x 25-cm; 5-jum packing L1
Flow rate: 1 mL/min
Injection size: 20 uL
System suitability
Sample: Standard solution A
[Note—The relative retention times for the curcumin,
desmethoxycurcumin, and bisdesmethoxycurcumin
peaks are about 1.0, 1.2, and 1.4, respectively.]
Suitability requirements
Chromatogram similarity: The chromatogram from
Standard solution A is similar to the Reference Chro-
matogram provided with the lot of USP Curcumi-
noids RS being used.
Resolution: NLT 2.0 between the curcumin and des-
methoxycurcumin peaks and the desmethoxycurcu-
min and bisdesmethoxycurcumin peaks
Dietary Supplements / Curcuminoids 4563
Tailing factor: NMT 1.5 for the bisdesmethoxycurcu-
min, desmethoxycurcumin, and curcumin peal
Relative standard deviation: NMT 2.0% for des-
methoxycurcumin peak, in repeated injections
Analysis
Samples: Standard solution A, Standard solution B,
Standard solution C, Standard solution D, and Sample
solution
Calculate the quantity, in mg, of curcumin, desmeth-
oxycurcumin, and bisdesmethoxycurcumin in each
Tablet:
Result = (ru/rs) x Cs x D x Vx (We/ Wu)
tu = peak area for curcumin, desmethoxycurcumin,
or bisdesmethoxycurcumin from the Sample
solution
Is = peak area for curcumin, anime
or bisdesmethoxycurcumin from the
appropriate Standard solution
Cs = concentration of the appropriate Standard
solution (mg/mL)
D = dilution factor to prepare the Sample solution
from the Sample stock solution
V volume of Sample solution (mL)
We average weight of Tablets (mg)
Wu weight of Tablets powder taken to prepare the
Sample stock solution (mg)
Calculate the percentage of the labeled amount of
curcuminoids in the Tablet:
Result = (ZQ/L) x 100
=Q = sum of the quantities of curcumin,
desmethoxycurcumin, and
bisdesmethoxycurcumin in the Tablet (mg)
E = labeled amount of curcuminoids (mg/Tablet)
Acceptance criteria: 90.0%-110.0% of the label claim
PERFORMANCE TESTS
e DISINTEGRATION AND DISSOLUTION (2040)
Mode: Dissolution
Medium: Water containing 1% sodium lauryl sulfate;
900 mL
Apparatus 2: 100 rpm
Time: 60 min
Sample solution: Combine 25-mL portions of the solu-
tion under test from each of the six dissolution vessels,
and mix. Transfer 5 mL to a 25-mL volumetric flask, and
dilute with Mobile phase to volume.
Analysis: Determine the amount of curcumin (C21H2006)
dissolved by using the method used in the Content of
sydesbouo-: sa
Curcuminoids, making any necessary modifications.
Tolerances: NLT 75% of the content of curcumin
(C21H200¢) is dissolved.
e WEIGHT VARIATION OF DIETARY SUPPLEMENTS (2091): Meet
the requirements
CONTAMINANTS
e MICROBIAL ENUMERATION TESTS (2021): The total aerobic
bacterial count does not exceed 10* cfu/g, and the total
ale molds and yeasts count does not exceed 103
u/g.
° Angee OF SPECIFIED MICROORGANISMS (2022): Meet the
requirements of the tests for the absence of Salmonella
species and Escherichia coli
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in well-closed contain-
ers, protect from light and moisture, and store at room
temperature.
© LABELING: The label states the content of curcuminoids in
mg/Tablet.
 4564 Curcuminoids / Dietary Supplements USP 41

e USP REFERENCE STANDARDS (11) Acceptance criteria: 98.5%-101.5% on the dried basis
USP Bisdesmethoxycurcumin RS
USP Curcumin RS IMPURITIES
USP Curcuminoids RS © RESIDUE ON IGNITION (281): NMT 0.1%
USP Desmethoxycurcumin RS ¢ CHLORIDE AND SULFATE, Chloride (221): NMT 200 ppm. A
0.7-g portion shows no more chloride than corresponds
to 0.40 mL of 0.01 N hydrochloric acid.

Delete the following:

Cyanocobalamin—see Cyanocobalamin
General Monographs °e HEAVY METALS, Method | (231): NMT 10 ppme (orteaii-
Jan-2018)
¢ CHLORIDE AND SULFATE, Su/fate (221): NMT 200 ppm. A
1.2-g portion shows no more sulfate than corresponds to
Cysteine Hydrochloride—see Cysteine 0.25 mL of 0.020 N sulfuric acid.
e IRON (241): NMT 10 ppm
Hydrochloride General Monographs © ORGANIC IMPURITIES
System suitability solution: Dissolve quantities of USP
Cystine RS and USP Arginine Hydrochloride RS in 1 N
hydrochloric acid, and dilute with water to obtain a so-
Cystine lution having a known concentration of about 0.4 mg/
mL each.
Standard solution: Dissolve a quantity of USP Cystine
HO. Sy RS in 1 N hydrochloric acid, and dilute with water to
obtain a solution having a known concentration of
about 0.02 mg/mL.
Sample solution: Dissolve a quantity of Cystine in 1 N
CeHi2N204S2 240.30 hydrochloric acid, and dilute with water to obtain a so-
L-Cystine; ingen having a known concentration of about 10 mg/
3,3’-Disulfanediylbis [(2R)-2-aminopropanoic acid] [56-89-3]. mL.
Chromatographic system
DEFINITION (See Chromatography (621), Thin-Layer Chromato-
Cystine contains NLT 98.5% and NMT 101.5% of cystine graphy.)
(CeHi2N204S2), as L-Cystine, calculated on the dried basis. Mode: TLC
IDENTIFICATION Adsorbent: A 0.25-mm layer of chromatographic silica
e A. INFRARED ABSORPTION (197K)
gel mixture
e B. OPTICAL ROTATION, Specific Rotation (781S) Application volume: 5 ul
Sample solution: 20 mg/mL, in 1 N hydrochloric acid. Developing solvent system: A mixture of ammonia
Perform the measurements immediately after and 2-propanol (3:7)
preparation. Spray reagent: Dissolve 0.2 g of ninhydrin in 100 mL
Acceptance criteria: —215 to —-225, determined at 20° of a mixture of butanol and 2 N acetic acid (95:5).
e C. The R; value of the principal spot of the Sample solu- Analysis
tion in the test for Organic Impurities corresponds to that Samples: System suitability solution, Standard solution,
and Sample solution
of the Standard solution.
Proceed as directed inGaromaoarpay (621), Thin-Layer
ASSAY Chromatography. After air-drying the plate, spray with
¢ PROCEDURE Spray reagent, and heat between 100° and 105° for
Sample solution: Transfer about 0.1 g of Cystine to a about 15 min. Examine the plate. The chromatogram
glass-stoppered flask, and dissolve in a mixture of 2 mL from the System suitability solution exhibits two clearly
of dilute sodium hydroxide (1 in 20) and 10 mL of separated spots.
water. Add 10 mL of potassium bromide solution Acceptance criteria: Any secondary spot from the Sam-
DS Monographs

(200 g/L in water), 50.0 mL of 0.1 N potassium bro- ple solution is not larger or more intense than the prin-
mate VS, and 15 mL of dilute hydrochloric acid (17 in cipal spot from the Standard solution.
100). Immediately insert the stopper into the flask, and Individual impurities: NMT 0.2%
cool in an ice water bath. Allow to stand protected Total impurities: NMT 2.0%
from light for 10 min.
Titrimetric system SPECIFIC TESTS
(See Titrimetry (541).) e Loss ON DRYING (731)
Mode: Residual titration Analysis: Dry a sample at 105° for 3 h.
Titrant: 0.1 N potassium bromate VS Acceptance criteria: NMT 0.2%
Back-titrant: 0.1 N sodium thiosulfate VS ADDITIONAL REQUIREMENTS
Endpoint detection: Visual © PACKAGING AND STORAGE: Preserve in well-closed contain-
Equivalency: Each mL of 0.1 N potassium bromate VS ers, and store at a controlled room temperature.
is equivalent to 2.403 mg of cystine (CsHi2N204S2) on e USP REFERENCE STANDARDS (11)
the dried basis. USP Arginine Hydrochloride RS
Analysis: Add 1.5 g of potassium iodide, and after 1 USP Cystine RS
min, titrate with 0.1 N sodium thiosulfate VS, usin:
starch TS as the indicator. Perform a blank determina-
tion, and make any necessary correction.
USP 41 Dietary Supplements / Diosmin 4565

Dexpanthenol—see Dexpanthenol General Analysis

Samples: Standard solution and Sample solution
Monographs Calculate the percentage of diosmin (CzsH32015), in the
portion of Diosmin taken:
Result = (ru/rs) x (Cs/Cu) x 100
Dexpanthenol Preparation—see
Dexpanthenol Preparation General Monographs ru = peak response from the Sample solution
rs = peak response from the Standard solution
Cs = concentration of USP Diosmin RS in the
Standard solution (mg/mL)
Cu = concentration of Diosmin in the Sample
Diosmin solution (mg/mL)
Acceptance criteria: 90.0%-102.0% on the anhydrous

BG SOOCwE LD
basis
LOCH,
IMPURITIES
Ho” ie rte CO ~~ on e RESIDUE ON IGNITION (281)
Ho oH Sample: 1.0g
° Acceptance criteria: NMT 0.2%

CogHs2015 608.54 Delete the following:

5-Hydroxy-2-(3-hydroxy-4-methoxyphenyl)-7-[(25,3R,45,55,
6R)-3,4,5-trihydroxy-6-[[(2R, 3R,4R,5R,65)-3,4,5-trihydroxy- ®e HEAVY METALS, Method iI (231)
6-methyloxan-2-ylJoxymethyl]oxan-2-yl]oxychromen- Sample: 2.0g
4-one; Acceptance criteria: NMT 20 ppme coiticie! 1-jan-2018)
7-[[6-O-(6-Deoxy-a-L-mannopyranosyl)-B-D-glucopyra- e LIMIT OF IODINE
nosylJoxy]-5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-4H- Determine the total content of iodine by potentiometry,
1-benzopyran-4-one [520-27-4]. using an iodide-selective electrode, after oxygen flask
combustion (see Oxygen Flask Combustion (471)).
DEFINITION Sample solution: [CAUTION—Observe rigorously the pre-
Diosmin contains NLT 90.0% and NMT 102.0% of diosmin cautions set forth for Procedure under Oxygen Flask
(C2gH32015), calculated on the anhydrous basis. Combustion (471).] Wrap 0.100 g of Diosmin in a piece
of free-halide filter paper, and place it in the platinum
IDENTIFICATION gauze specimen holder. Introduce into the flask
© A. INFRARED ABSORPTION (197K) 50.0 mL of a 0.2 g/L solution of hydrazine. Flush the
e B. The retention time of the major peak of the Sample flask with oxygen for 10 min. Ignite the filter paper. Stir
solution corresponds to that of the Standard solution, as the contents of the flask immediately after the end of
obtained in the Assay. the combustion to dissolve completely the combustion
ASSAY products. Continue stirring for 1 h.
¢ PROCEDURE Standard solution: 33.2 tig/mL of potassium iodide in
Mobile phase: Methanol, acetonitrile, acetic acid, and water, equivalent to 25.4 t1g/mL of iodine
water (27:2:6:65) Potassium nitrate solution: 200 mg/mL of potassium
Standard solution: 1.0 mg/mL of USP Diosmin RS in nitrate in 0.1 M nitric acid
dimethy! sulfoxide Analysis
System suitability solution: 1 mg/mL of USP Diosmin Samples: Sample solution and Standard solution
‘or System Suitability RS in dimethyl sulfoxide Transfer 30 mL of Potassium nitrate solution to a
Sample solution: 1.0 mg/mL of Diosmin in dimethyl beaker, immerse the electrodes, and stir for 10 min.
sulfoxide The potential (nU;) must remain stable. Measure the
Chromatographic system potential (nU;). Add 1 mL of the Sample solution, and
(See Chromatography (621), System Suitability.) measure the potential (nU2).
sydesbouow; sq

Mode: LC Transfer 30 mL of Potassium nitrate solution to a

Detector: UV 275 nm
Column: 4.6-mm x 10-cm; 3-um packing L1
Column temperature: 40°
Flow rate: 1.2 mL/min
Injection size: 10 pL
System suitability
Samples: System suitability solution and Standard solu-
tion.
[NoTe—Allow the run time about 6 times the diosmin
retention time. The relative retention times for dios-
min, acetoisovanillone, hesperidin, isorhoifolin, linarin,
and diosmetin are 1, 0.5, 0.6, 0.8, 2.6, and 4.5,
respectively.]
Suitability requirements
Chromatogram similarity: The chromatogram from
the System suitability solution is similar to the Refer-
ence Chromatogram provided with the USP Diosmin
for System Suitability RS being used.
Resolution: NLT 2.5 between hesperidin and
isorhoifolin, System suitability solution
Relative standard deviation: NMT 2.0%, Standard
solution
beaker, immerse the electrodes, and stir for 10 min.
The potential (nS;) must remain stable. Measure the
potential (nS;). Add 80 uL of the Standard solution,
and measure the potential (n52).
Acceptance criteria: NMT 0.1%: The absolute value
(nUz) — (nU;)| is not higher than the absolute value
(nSz2) — (nS1)|.
e RELATED COMPOUNDS
Mobile phase, System suitability solution, Sample so-
lution, and Chromatographic system: Proceed as di-
rected in the Assay.
Standard solution: 0.05 mg/mL of USP Diosmin RS in
dimethyl sulfoxide
System suitability
Sample: System suitability solution. [NoTE—Allow the
run time about 6 times that of the diosmin retention
time. The relative retention times for diosmin,
acetoisovanillone, hesperidin, isorhoifolin, linarin, and
diosmetin are 1, 0.5, 0.6, 0.8, 2.6, and 4.5,
respectively.]
 4566 Diosmin / Dietary Supplements USP 41

System suitability requirements e@ USP REFERENCE STANDARDS (11)

Chromatogram similarity: The chromatogram from USP Diosmin RS
the System suitability solution is similar to the Refer- USP Diosmin for System Suitability RS
ence Chromatogram provided with the USP Diosmin
for System Suitability RS being used.
Resolution: NLT 2.5 between hesperidin and
isorhoifolin, System suitability solution
Analysis Echinacea angustifolia
Samples: Standard solution and Sample solution
Calculate the percentage of each impurity in the por- DEFINITION
tion of Diosmin taken: [NoTE—Disregard any impurity Echinacea angustifolia consists of the dried rhizome and
less than 0.1%.] roots of Echinacea angustifolia DC. (Fam. Asteraceae). It is
Result = (ru/rs) x (Cs/Cu) x Fx 100 harvested in the fall after one or more years of growth. It
contains NLT 0.5% of total phenols, calculated on the
tu = peak response for each impurity from the dried basis as the sum of echinacoside (C3sH4¢OQ20), dicaf-
Sample solution feoylquinic acid (C2sH24012), and chlorogenic acid
ls = peak response for diosmin from the Standard (CisHi8O¢). It contains NLT 0.075% of dodecatetraenoic
solution acid isobutylamides (CisH2sNO) on the dried basis.
Cs = concentration of USP Diosmin RS in the IDENTIFICATION
Standard solution (mg/mL) e A. THIN-LAYER CHROMATOGRAPHY
Cy = concentration of Diosmin in the Sample Presence of echinacoside and dicaffeoylquinic acid
solution (mg/mL) (cynarin(e))
F = correction factor for each individual impurity Standard solution A: 0.2 mg/mL of USP Echinacoside
(see Table 71) RS and 0.2 mg/mL of dicaffeoylquinic acid (cynarin) in
Acceptance criteria methanol
Total impurities: NMT 10% Standard solution B: 0.05 mg/mL of USP Caftaric
Individual impurities: See Table 7. Acid RS, 0.1 mg/mL of USP Chlorogenic Acid RS, and
Total other impurities and acetoisovanillone: NMT 0.05 mg/mL of USP Chicoric Acid RS in methanol
1% Standard solution C: 20 mg/mL of USP Powdered
Echinacea angustifolia Extract RS in methanol. Shake to
Table 1 disperse, sonicate for 5 min, and centrifuge. Use the
Relative Correction Acceptance
supernatant.
Retention Factor Criteria,
Sample solution: Transfer ‘9 of finely pulverized Echi-
Name Time (/) NMT (%)
nacea angustifolia to a centrifuge tube, add 10 mL of
methanol, mix well, and sonicate for 10 min. Centri-
Acetoisovanillone= 0.5 0.3 1 fuge, and use the supernatant.
Hesperidin® 0.6 1 5 Chromatographic system
Jsorhoifoline 0.8 1 3 (See ComatOarap Yy (621), Thin-Layer Chromato-
Linarin¢ 2.6 1 3 raphy.)
Diosmetine 4.5 0.5 3 Acareent Chromatographic silica gel mixture with
Any other impuri- = an average particle size of 5 um (HPTLC plates)
ty 1 1
Application volume: 5 jL Standard solution C and
ample solution, and 2 wl Standard solution A and
Total impurities = — 10 Standard solution B as 8-mm bands
2 1-(3-Hydroxy-4-methoxyphenyl)ethanone. Relative humidity: Condition the plate to a relative
(25)
b 7-16;0-(6-Deony-clemaninapyranasy!) B D-glucopyranasylloxy]s: humidity of about 33% using a suitable device.
hydroxy-2-(3-hydroxy-4-methoxyp! enyl)-2,3-dihydro-4H-1 -benzopyran-4-
one. Developing solvent system: A mixture of ethyl ace-
¢ 7-[[6-O-(6-Deoxy-c.-L-mannopyranosyl)-B-D-glucopyranosyl]oxy]-S-hy- tate, methylethyl ketone, water, and formic acid
droxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one. @G:3:181)
4 7-[[6-O-(6-Deoxy-c-L-mannopyranosyl)-B-D-glucopyranosyl]oxy]-5-hy- Developing distance: 6 cm
DS Monographs

droxy-2-(4-methoxyphenyl)-4H-1-benzopyran-4-one. Derivatization reagent: 5 mg/mL of 2-aminoethyl

¢ 5,7-Dihydroxy-2-(3-hydroxy-4-methoxyphenyl)-4H-1 -benzopyran-4-one. diphenylborinate in ethyl acetate
Analysis
SPECIFIC TESTS Samples: Standard solution A, Standard solution B,
e@ WATER DETERMINATION, Method /a (921)
Standard solution C, and Sample solution
Sample: 0.3g Apply the Samples as bands to a suitable thin-layer
Acceptance criteria: NMT 6.0%
chromatographic plate, and dry in air. Develop the
ADDITIONAL REQUIREMENTS chromatograms in a saturated chamber. Remove the
© PACKAGING AND STORAGE: Preserve in well-closed, tight late from the chamber, heat at 100° for 5 min, der-
containers. ivatize the plate while still warm with Derivatization
reagent, dry in air, and examine under UV light at
366 nm.
System suitability: Standard solution A shows two ma-
jor blue bands, one in the lower third of the chromat-
ogram due to echinacoside, and the other band in the
middle section of the chromatogram due to dicaffeoyl-
quinic acid (cynarin). Standard solution B shows two
major blue bands at about the middle of the chromat-
ogram due to caftaric acid (lower Rp) and chlorogenic
acid (higher R,) that are clearly separated, and a blue
band for chicoric acid in the upper third section of the
chromatogram.
Acceptance criteria: The most prominent band in the
Sample solution chromatogram is a blue band in the
USP 41 Dietary Supplements / Echinacea 4567

lower third section of the chromatogram at an R; cor- Re (absent in Echinacea purpurea and Echinacea pallida).
responding to the echinacoside band in the chromato- The yellow band turns reddish pink when the plate is
gram of Standard solution A and Standard solution C heated at 100° for more than 10 min. The Sample
(absent in Echinacea purpurea). The Sample solution solution chromatogram exhibits a minor pink-violet
chromatogram exhibits a prominent greenish blue band at about the middle of the chromatogram (much
band in the middle section of the chromatogram at an more prominent in Echinacea purpurea), a minor pink-
Rr corresponding to the dicaffeoylquinic acid (cynarin) violet band at about two-thirds of the chromatogram
band in the chromatogram of Standard solution A and (much more prominent in Echinacea pallida), an
Standard solution C (absent in Echinacea pallida and broad pink-violet band close to the solvent front.
Echinacea purpurea). The Sample solution chromato- e C. The retention time of the major peak of the Sample
gram does not exhibit, or exhibits very faint blue Solution corresponds to that of the echinacoside peak of
ands at an Rr corresponding to the caftaric acid and Standard solution A and Standard solution B; and the re-
chicoric acid bands in Standard solution B (difference tention time of the peak for 1,3-dicaffeoylquinic acid
from Echinacea pallida and Echinacea purpurea). The from the Sample solution corresponds to that of Standard
Sample solution chromatogram exhibits minor bands solution A, all peaks as obtained in the test for Content of
between the positions of echinacoside and cynarin. Total Phenols.
One of these is due to chlorogenic acid at an Rr corre-
sponding to that of chlorogenic acid in Standard solu- COMPOSITION
tion B. e@ CONTENT OF TOTAL PHENOLS
e B. THIN-LAYER CHROMATOGRAPHY Solution A: Phosphoric acid (0.1 in 100) in water
Presence of alkylamides Solution B: Acetonitrile
Standard solution A: 0.2 mg/mL of USP B-Sitosterol Mobile phase: See Table 7.
RS in methanol
Standard solution B: 100 mg/mL of USP Powdered Table 1
Echinacea angustifolia Extract RS in dichloromethane.
Shake to disperse, sonicate for 5 min, and centrifuge. Time Solution A Solution B
(min) (%) (%)
Use the supernatant.
Sample solution: Transfer 1 g of finely pulverized Echi- 0 90 10
nacea angustifolia to a centrifuge tube, add 10 mL of 3 90 10
dichloromethane, mix well, and sonicate for 10 min. 16 78 22
Centrifuge, and use the supernatant. TZ, 60 40
Chromatographic system 20 60 40
(See ipo Yy (621), Thin-Layer Chromato-
20.5 90 10
raphy.
Adsorbent: Chromatographic silica gel with an aver- 25 90 10
age particle size of 5 um (HPTLC plates)
Application volume: 5 ul Standard solution B and Solvent: Alcohol and water (7:3)
Sample solution, and 2 wL Standard solution A as Standard solution A: Dissolve USP Powdered Echinacea
8-mm bands angustifolia Extract RS in Solvent, shaking and heating in
Relative humidity: Condition the plate to a relative a water bath. Dilute with Solvent to obtain a solution
humidity of about 33% usinga suitable device. having a known concentration of 1 mg/mL. Pass
Developing solvent system: A mixture of toluene, through a membrane filter having a 0.45-um or finer
ethyl acetate, cyclohexane, and formic acid pore size.
(8:22 15053) Standard solution B: 40 j1g/mL of USP Chlorogenic
Developing distance: 6 cm Acid RS in Solvent
Derivatization reagent: Place 85 mL of methanol in Standard solution C: 80 g/mL of USP Echinacoside RS
a 100-mL glass bottle, and cool it down in a in Solvent
water-ice cubes-salt bath or in a freezer. To the ice- Sample solution: Transfer about 125 mg of finely pow-
cold methanol, slowly and carefully add 10 mL of dered Echinacea angustifolia (capable of passing through
acetic acid and 5 mL of sulfuric acid, and mix well. a 40-mesh sieve), accurately weighed, to a round-bot-
Allow the mixture to cool to room temperature, then tom flask equipped with a condenser. Add 25.0 mL of
Solvent, and heat under reflux, while shaking by me-
sydesbouo-: sa

add 0.5 mL of p-anisaldehyde.

Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Apply the Samples as bands to a suitable thin-layer
chromatographic plate, and dry in air. Develop the
chromatograms in a saturated chamber. Remove the
plate from the chamber, dry in air, derivatize with
Derivatization reagent, heat at 100° for 3—5 min, set
aside to cool, and examine under visible light.
System suitability: The B-sitosterol band of the Stan-
dard solution B chromatogram and the two bands be-
low are clearly separated from one another. These two
bands, in decreasing R;, include a major blue violet
band and a yellow band.
Acceptance criteria: The most prominent band of the
Sample solution chromatogram is a blue violet band in
the lower-third section of the chromatogram (much
less prominent in Echinacea purpurea and absent in
Echinacea pallida). This blue violet band is between
two bands: a less prominent blue violet band at a
higher R; corresponding to the B-sitosterol band in the
chromatograms of Standard solution A and Standard
solution B, and a characteristic yellow band at a lower
chanical means for 15 min. Centrifuge, or pass through
a membrane filter having a 0.45-um or finer pore size.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 330 nm
Column: 4.6-mm x 25-cm; 5-um packing L1
Column temperature: 35°
Flow rate: 1.5 mL/min
Injection volume: 5 uL
System suitability
Samples: Standard solution A and Standard solution C
Suitability requirements
Chromatogram similarity: The chromatogram from
Standard solution A is similar to the Reference Chro-
matogram for total phenols provided with the USP
Powdered Echinacea angustifolia Extract RS.
Resolution: NLT 1.0 between the 1,3-dicaffeoylquinic
acid isomer and echinacoside, Standard solution A.
[Not&—Echinacoside peak may be resolved in two
components.]
Capacity factor (k’): NLT 3.0 from Standard solution
 4568 Echinacea / Dietary Supplements
Tailing factor: NMT 2.0 for the echinacoside peak,
Standard solution C
Relative standard deviation: NMT 2.5% for the
echinacoside peaks in repeated injections, Standard
solution C
Analysis
Samples: Standard solution A, Standard solution B,
Standard solution C, and Sample solution
Identify the relevant analytes in the chromatogram
from the Sample solution by comparison with the chro-
matogram from Standard solution A. Measure the areas
for the relevant peaks.
Separately calculate the percentage of chlorogenic acid
(CisHisOs), dicaffeoylquinic acids (C2sH24012), and
echinacoside (C3sH46O20) in the portion of Echinacea
angustifolia taken:
Result = (ru/rs) x Cs x (V/W) x Fx 100
tu = peak response for the relevant analyte from
the Sample solution
fs = peak response for chlorogenic acid or both
components of echinacoside from the
corresponding Standard solution
Cs = concentration of chlorogenic acid or
echinacoside in the corresponding Standard
solution (mg/mL)
V = volume of the Sample solution (mL)
Ww = weight of Echinacea angustifolia taken to
prepare the Sample solution (mg)
F = response factor and is equal to 0.729 for
dicaffeoylquinic acids, relative to chlorogenic
acid, 1.00 for chlorogenic acid, and 1.00 for
echinacoside components
Calculate the percentage of total phenols in the portion
of Echinacea angustifolia taken by adding the individual
percentages calculated.
Acceptance criteria: NLT 0.5% of total phenols on the
dried basis
e CONTENT OF DODECATETRAENOIC ACID ISOBUTYLAMIDES
Mobile phase: Acetonitrile and water (55:45)
Standard solution A: Dissolve, with sonication, USP
Powdered Echinacea angustifolia Extract RS in methanol,
shaking for 10 min, and dilute with methanol to obtain
a solution having a concentration of 5 mg/mL. Pass
through a membrane filter having a 0.45-um or finer
pore size.
Standard solution B: 10 1g/mL of USP 2E,4£-Hexadie-
noic Acid Isobutylamide RS in methanol
Sample solution: Transfer about 2.5 g, aay
weighed, of finely powdered Echinacea angustifolia (ca-
DS Monographs
able of passing through a 40-mesh sieve) to a round-
Bottom flask. Add 80 mL of methanol, and reflux for 30
min. Cool to room temperature, and filter into a
100-mL volumetric flask, using small portions of metha-
nol to rinse the flask and theTilter. Dilute with metha-
nol to volume. Pass through a membrane filter having a
0.45-um or finer pore size.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 254 nm
Column: 4.6-mm x 25-cm; 5-um packing L1
Column temperature: 30°
Flow rate: 1.5 mL/min
Injection volume: 25 uL
System suitability
Samples: Standard solution A and Standard solution B
Suitability requirements
Chromatogram similarity: The chromatogram from
Standard solution A is similar to the reference chro-
matogram for alkamides provided with USP Pow-
dered Echinacea angustifolia Extract RS.
Resolution: NLT 1.0 between dodecatetraenoic acid
isobutylamide peaks, Standard solution A
USP 41
Tailing factor: NMT 2.0 for the 2E,4£-hexadienoic
acid isobutylamide peak, Standard solution B
Relative standard deviation: NMT 2.5% for the 2E,
4E-hexadienoic acid isobutylamide peak in repeated
injections, Standard solution B
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Identify the peaks due to 2£,4£,8Z,10£-dodecatetra-
enoic acid isobutylamide and 2E£,4£,8Z,10Z-dodeca-
tetraenoic acid isobutylamide in the chromatogram
from the Sample solution by comparison with the chro-
matogram from Standard solution A. Measure the areas
for the relevant peaks.
Calculate the percentage of dodecatetraenoic acid iso-
butylamides in the portion of Echinacea angustifolia
taken:
Result = (ru/rs) x Cs x (V/W) x Fx 100
ty = sum of the peak responses of the relevant
analytes from the Sample solution
ts = peak response for 2E,4E-hexadienoic acid
isobutylamide from Standard solution B
Cs = concentration of USP 2F,4E-Hexadienoic Acid
Isobutylamide RS in Standard solution B
(mg/mL)
Vv = volume of the Sample solution (mL)
w = weight of Echinacea angustifolia taken to
prepare the Sample solution (mg)
F = response factor for 2E,4E-hexadienoic acid
isobutylamide, 1.353
Acceptance criteria: NLT 0.075% of dodecatetraenoic
acid isobutylamides (C;sH2sNO) on the dried basis
CONTAMINANTS
e ELEMENTAL IMPURITIES—PROCEDURES (233)
Acceptance criteria
Arsenic: NMT 1.0 ng/g
Cadmium: NMT 0.5 g/g
Lead: NMT 5.0 g/g
Mercury: NMT 1.0 ug/g
e ARTICLES OF BOTANICAL ORIGIN, General Method for Pesti-
cide Residues Analysis (561): Meets the requirements
SPECIFIC TESTS
¢ BOTANIC CHARACTERISTICS
Macroscopic: The outer surface of the rhizome is pale
to yellowish brown, crowned with remains of the aerial
stem, and sometimes showing surface annulations up to
15 mm in diameter. The roots are also pale to yellowish
brown, cylindrical or slightly tapering, sometimes spi-
rally twisted, longitudinally wrinkled and deeply fur-
rowed, up to 4-10 mm in diameter, and passing imper-
ceptibly into rhizome. The fracture is short when dry
and becomes tough and pliable on exposure to air.
Microscopic: The rhizomes and roots in transverse sec-
tion show a thin outer bark separated from a wide xy-
lem by a distinct cambial line. The cork is composed of
several rows of thin-walled cells containing yellowish-
brown pigment. The rhizome has a small circular pith,
occasional small groups of thick-walled, lignified fibers
in the pericycle, and a parenchymatous cortex. The
phloem and xylem are composed of narrow strands of
vascular tissue separated by wide, nonlignified medul-
lary rays. Xylem vessels are lignified, 25-75 um in diam-
eter, usually with reticulate thickening but occasionally
with spiral or annular thickening. Sclereids occur singly
or in small groups, varying considerably in size and
shape from rounded to rectangular to elongated and
fiber-like, up to 300 um long and 20-40 um wide, with
intercellular spaces forming schizogenous oleoresin
canals that are 80-150 um in diameter and contain a
dense black deposit. The canals are present outside of
the central cylinder only (unlike Echinacea pallida, where
USP 41
they are present both inside and outside of the central
cylinder): Spherocrystalline masses of inulin occur
throughout the parenchymatous tissues. Lignified fibers,
300-800 um long, are en in scattered groups, and
are usually surrounded by phytomelanin (unlike fibers in
Echinacea pallida, where they usually occur singly in the
periphery of the cortex and are 100-300 um long, with
phytomelanin often absent).
ARTICLES OF BOTANICAL ORIGIN, Foreign Organic Matter
(561): NMT 3.0%
Loss ON DrYING (731)
e
Analysis: Dry a sample at 105° for 2 h.
Acceptance criteria: NMT 10.0%
ARTICLES OF BOTANICAL ORIGIN, Total Ash (561): NMT
UA
ARTICLES OF BOTANICAL ORIGIN, Acid-Insoluble Ash (561):
NMT 4.0%
ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Store in well-closed, light-resis-
tant containers.
e LABELING: The label states the Latin binomial and, follow-
ing the official name, the parts of the plant contained in
the article.
e USP REFERENCE STANDARDS (11)
USP Caftaric Acid RS
USP Chicoric Acid RS
USP Chlorogenic Acid RS
USP Powdered Echinacea angustifolia Extract RS
USP Echinacoside RS
USP 2E£,4E-Hexadienoic Acid Isobutylamide RS
USP B-Sitosterol RS
Powdered Echinacea angustifolia
DEFINITION
Powdered Echinacea angustifolia consists of the dried rhi-
zome and roots of Echinacea angustifolia DC. (Fam. Aster-
aceae), harvested in the fall after one or more years of
growth, and reduced to powder. It contains NLT 0.5% of
total phenols, calculated on the dried basis as the sum of
echinacoside (C3sH4sO20), dicaffeoylquinic acid (C2sH24012),
and chlorogenic acid (CysHigOs). It contains NLT 0.075%
of dodecatetraenoic acid isobutylamides (CisH2sNO) on
the dried basis.
IDENTIFICATION
¢ A. THIN-LAYER CHROMATOGRAPHY
Presence of echinacoside and dicaffeoylquinic acid
(cynarin(e))
Standard solution A: A mixture of 0.2 mg/mL of USP
Echinacoside RS and 0.2 mg/mL of dicaffeoylquinic
acid (cynarin) in methanol
Standard solution B: 0.05 mg/mL of USP Caftaric
Acid RS, 0.1 mg/mL of USP Chlorogenic Acid RS, and
0.05 mg/mL of USP Chicoric Acid RS in methanol
Standard solution C: 20 mg/mL of USP Powdered
Echinacea angustifolia Extract RS in methanol. Shake to
disperse, sonicate for 5 min, and centrifuge. Use the
supernatant.
Sample solution: Transfer 1 g of Powdered Echinacea
pk gel to a centrifuge tube, add 10 mL of metha-
nol, mix well, and sonicate for 10 min. Centrifuge,
and use the supernatant.
Chromatographic system
(See iymanoarap y (621), Thin-Layer Chromato-
graphy.
Adsorbent: Chromatographic silica gel mixture with
an average particle size of 5 um (HPTLC plates)
Application volume: 5 ul Standard solution C and
ample solution, and 2 wL Standard solution A and
Standard solution B, as 8-mm bands
Dietary Supplements / Echinacea 4569
Relative humidity: Condition the plate to a relative
humidity of about 33% using a suitable device.
Developing solvent system: A mixture of ethyl ace-
tate, methylethyl ketone, water, and formic acid
(5:321:1)
Developing distance: 6 cm
Derivatization reagent: 5 mg/mL of 2-aminoethyl
diphenylborinate in ethyl acetate
Analysis
Samples: Standard solution A, Standard solution B,
Standard solution C, and Sample solution
Apply the Samples as bands to a suitable thin-layer
chromatographic plate, and dry in air. Develop the
chromatograms in a saturated chamber. Remove the
plate from the chamber, heat at 100° for 5 min, der-
ivatize the plate while still warm with Derivatization
reagent, dry in air, and examine under UV light at
366 nm.
System suitability: Standard solution A shows two ma-
jor blue bands, one in the lower third of the chromat-
ogram due to echinacoside, and the other band in the
middle section of the chromatogram due to dicaffeoyl-
quinic acid (cynarin). Standard solution B shows two
major blue bands at about the middle of the chromat-
ogram due to caftaric acid (lower Rp) and chlorogenic
acid (higher R,) that are clearly separated, and a blue
band for chicoric acid in the upper third section of the
chromatogram.
Acceptance criteria: The most prominent band in the
Sample solution chromatogram is a blue band in the
lower third section of the chromatogram at an R; cor-
responding to the echinacoside band in the chromato-
gram of Standard solution A and Standard solution C
(absent in Echinacea purpurea). The Sample solution
chromatogram exhibits a prominent greenish blue
band in the middle section of the chromatogram at an
Re corresponding to the dicaffeoylquinic acid (cynarin)
band in the chromatogram of Standard solution A and
Standard solution C (absent in Echinacea pallida and
Echinacea purpurea). The Sample solution chromato-
ram does not exhibit, or exhibits very faint blue
ands at an Rr corresponding to the caftaric acid and
chicoric acid bands in Standard solution B (difference
from Echinacea pallida and Echinacea purpurea). The
Sample solution chromatogram exhibits minor bands
between the positions of echinacoside and cynarin.
One of these is due to chlorogenic acid at an Rr corre-
sponding to that of chlorogenic acid in Standard solu-
tion B.
e B. THIN-LAYER CHROMATOGRAPHY
Presence of alkylamides
sydesbouo=: sa
Standard solution A: 0.2 mg/mL of USP B-Sitosterol
RS in methanol
Standard solution B: 100 mg/mL of USP Powdered
Echinacea angustifolia Extract RS in dichloromethane.
Shake to disperse, sonicate for 5 min, and centrifuge.
Use the supernatant.
Sample solution: Transfer 1 g of Powdered Echinacea
angustifolia to a centrifuge tube, add 10 mL of dichlo-
romethane, mix well, and sonicate for 10 min. Centri-
fuge, and use the supernatant.
Chromatographic system
(See Chromatography (621), Thin-Layer Chromato-
graphy.) a .
Adsorbent: Chromatographic silica gel with an aver-
age particle size of 5 um (HPTLC plates)
Application volume: 5 yL Standard solution B and
Sample solution, and 2 wL Standard solution A as
8-mm bands
Relative humidity: Condition the plate to a relative
humidity of about 33% using a suitable device.
Developing solvent system: A mixture of toluene,
ethyl acetate, cyclohexane, and formic acid
(8: 2: 1: 0.3)
 4570 Echinacea / Dietary Supplements
Developing distance: 6cm
Derivatization reagent: Place 85 mL of methanol in
a 100-mL glass bottle, and cool it down in a
water-ice cubes-salt bath or in a freezer. To the ice-
cold methanol, slowly and carefully add 10 mL of
acetic acid and 5 mL of sulfuric acid, and mix well.
Allow the mixture to cool to room temperature, then
add 0.5 mL of p-anisaldehyde.
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Apply the Samples as bands to a suitable thin-layer
chromatographic plate, and dry in air. Develop the
chromatograms in a saturated chamber. Remove the
plate from the chamber, dry in air, derivatize with
Derivatization reagent, heat at 100° for 3-5 min, dry
in air, and examine under visible light.
System suitability: The B-sitosterol band of the Stan-
dard solution B chromatogram and the two bands un-
derneath are clearly separated from one another.
These two bands, in decreasing R;, include a major
blue violet band and a yellow band.
Acceptance criteria: The most prominent band of the
Sample solution chromatogram is a blue violet band in
the lower-third section of the chromatogram (much
less prominent in Echinacea purpurea and absent in
Echinacea pallida). This blue violet band is between
two bands: a less prominent blue violet band at a
higher Rr corresponding to the B-sitosterol band in the
chromatograms of Standard solution A and Standard
solution B, and a characteristic yellow band at a lower
Rr (absent in Echinacea purpurea and Echinacea pallida).
The yellow band turns reddish pink when the plate is
heated at 100° for more than 10 min. The Sample
solution chromatogram exhibits a minor pink-violet
band at about the middle of the chromatogram (much
more ee in Echinacea purpurea), a minor pink-
violet band at about two-thirds of the chromatogram
(much more prominent in Echinacea pallida), an
broad pink-violet band close to the solvent front.
e C. The retention time of the major peak of the Sample
solution corresponds to that of the echinacoside peak of
Standard solution A and Standard solution B, and the re-
tention time of the 1,3-dicaffeoylquinic acid peak from
the Sample solution corresponds to that of Standard solu-
tion A, all peaks as obtained in the test for Content of
Total Phenols.
COMPOSITION
e CONTENT OF TOTAL PHENOLS
al
Solution A: Phosphoric acid (0.1 in 100) in water
pra Solution B: Acetonitrile
a Mobile phase: See Table 7.
Ls]
=)
-
re} Table 1
=
6 Time Solution A Solution B
= (min) (%) (%)
a) 0 90 10
[a] 3 90 10
16 78 22
17 60 40
20 60 40
20.5 90 10
25 90 10
Solvent: Alcohol and water (7:3)
Standard solution A: Dissolve USP Powdered Echinacea
angustifolia Extract RS in Solvent, shaking and heating in
a water bath. Dilute with Solvent to obtain a solution
having a known concentration of 1 mg/mL. Pass
through a membrane filter having a 0.45-um or finer
pore size.
USP 41
Standard solution B: 40 g/mL of USP Chlorogenic
Acid RS in Solvent
Standard solution C: 80 g/mL of USP Echinacoside RS
in Solvent
Sample solution: Transfer about 125 mg of Powdered
Echinacea angustifolia Copan of passing through a
40-mesh sieve), accurately weighed, to a round-bottom
flask equipped with a condenser. Add 25.0 mL of Sol-
vent, and heat under reflux while shaking by mechani-
cal means for 15 min. Centrifuge, or pass through a
membrane filter having a 0.45-1m or finer pore size.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 330 nm
Column: 4.6-mm x 25-cm; 5-"um packing L1
Column temperature: 35°
Flow rate: 1.5 mL/min
Injection volume: 5 uL
System suitability
Samples: Standard solution A and Standard solution C
Suitability requirements
Chromatogram similarity: The chromatogram from
Standard solution A is similar to the Reference Chro-
matogram for total phenols provided with the USP
Powdered Echinacea angustifolia Extract RS.
Resolution: NLT 1.0 between the 1,3-dicaffeoylquinic
acid isomer and echinacoside peaks, Standard solution
A. [NoTe—Echinacoside peak may be resolved in two
components.]
Capacity factor (k’): NLT 3.0, Standard solution C
Tailing factor: NMT 2.0 for the echinacoside peak,
Standard solution C
Relative standard deviation: NMT 2.5% for the
echinacoside peaks in repeated injections, Standard
solution C
Analysis
Samples: Standard solution A, Standard solution B,
Standard solution C and Sample solution
Identify the relevant analytes in the chromatogram from
the Sample solution by comparison with the chromato-
gram from Standard solution A. Measure the areas for
the relevant peaks.
Separately calculate the percentage of chlorogenic acid
(CisHisOs), dicaffeoylquinic acids (C2sH24O12), and
echinacoside (C3sH46O20) in the portion of Powdered
Echinacea angustifolia taken:
Result = (ru/rs) x Cs x (V/W) x Fx 100
i = peak response for the relevant analyte from
the Sample solution
rs = peak response for chlorogenic acid or both
components of echinacoside from the
corresponding Standard solution
Cs = concentration of chlorogenic acid or
echinacoside in the corresponding Standard
solution (mg/mL)
Vv = volume of the Sample solution (mL)
w = weight of Powdered Echinacea angustifolia
used to prepare the Sample solution (mg)
F = response factor: 0.729 for dicaffeoylquinic
acids, relative to chlorogenic acid 1.00 for
chlorogenic acid, and 1.00 for echinacoside
components
Calculate the percentage of total phenols in the portion
of Powdered Echinacea angustifolia taken by adding
the individual percentages calculated.
Acceptance criteria: NLT 0.5% of total phenols on the
dried basis
¢ CONTENT OF DODECATETRAENOIC ACID ISOBUTYLAMIDES
Standard solution A: Dissolve, with sonication, USP
Powdered Echinacea angustifolia Extract RS in methanol,
shaking for 10 min, and dilute with methanol to obtain
a solution having a concentration of 5 mg/mL. Pass
USP 41
through a membrane filter having a 0.45-ym or finer
pore size.
Standard solution B: 10 g/mL of USP 2£,4E-Hexadie-
noic Acid Isobutylamide RS in methanol
Sample solution: Transfer about 2.5 g of Powdered
Echinacea angustifolia ope of passing through a
40-mesh sieve), accurately weighed, into a round-bot-
tom flask. Add 80 mL of methanol, and reflux for 30
min. Cool to room temperature, and filter into a
100-mL volumetric flask, using small portions of metha-
nol to rinse the flask and theSitter, Dilute with metha-
nol to volume. Pass through a membrane filter having a
0.45-um or finer pore size.
Mobile phase: Acetonitrile and water (55:45)
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 254 nm
Column: 4.6-mm x 25-cm; 5-um packing L1
Column temperature: 30°
Flow rate: 1.5 mL/min
Injection volume: 25 pL
System suitability
Samples: Standard solution A and Standard solution B
Suitability requirements
Chromatogram similarity: The chromatogram from
Standard solution A is similar to the Reference Chro-
matogram for alkamides provided with USP Pow-
dered Echinacea angustifolia Extract RS.
Resolution: NLT 1.0 between dodecatetraenoic acid
isobutylamide peaks, Standard solution A
Tailing factor: NMT 2.0 for the 2£,4E-hexadienoic
acid isobutylamide peak, Standard solution B
Relative standard deviation: NMT 2.5% for the 2E,
4E-hexadienoic acid isobutylamide peak in repeated
injections, Standard solution B
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Identify the peaks due to 2£,4£,8Z,10£-dodecatetraenoic
acid isobutylamide and 2E,4£,8Z,10Z-dodecatetraenoic
acid isobutylamide in the chromatogram from the
Sample solution by comparison with the chromatogram
from Standard solution A. Measure the areas for the
relevant peaks.
Calculate the percentage of dodecatetraenoic acid iso-
butylamides in the portion of Powdered Echinacea
angustifolia taken:
Result = (ru/rs) x Cs x (V/W) x Fx 100
tu = sum of the peak responses of the relevant
analytes from the Sample solution
Is = peak response of 2£,4E-hexadienoic acid
isobutylamide from Standard solution B
Cs = concentration of USP 2£,4E-Hexadienoic Acid
Isobutylamide RS in Standard solution B
(mg/mL)
= volume of the Sample solution (mL)
Ww = weight of Powdered Echinacea angustifolia
used to prepare the Sample solution (mg)
F = response factor for 2E,4£-hexadienoic acid
isobutylamide, 1.353
Acceptance criteria: NLT 0.075% of dodecatetraenoic
acid isobutylamides (CisH2sNO) on the dried basis
CONTAMINANTS
e@ ELEMENTAL IMPURITIES—PROCEDURES (233)
Acceptance criteria
Arsenic: NMT 1.0 ug/g
Cadmium: NMT 0.5 g/g
Lead: NMT 5.0 ug/g
Mercury: NMT 1.0 ug/g
e ARTICLES OF BOTANICAL ORIGIN, General Method for Pesti-
cide Residues Analysis (561): Meets the requirements
Dietary Supplements / Echinacea 4571
SPECIFIC TESTS
e BOTANIC CHARACTERISTICS: Powdered Echinacea angus-
tifolia is a brown powder witha slight aromatic odor and
a sweet taste that quickly becomes bitter, leaving a tin-
gling sensation on the tongue. Under a microscope, the
following characteristics are observed: thin-walled poly-
gonal cork cells with red-brown contents; lignified reticu-
late vessels; abundant stone cells of various shapes; frag-
ments of oleoresin canals with reddish-brown contents;
and abundant thin-walled parenchyma with spherocrys-
talline masses of inulin.
e Loss ON DRYING (731)
Analysis: Dry a sample at 105° for 2 h.
Acceptance criteria: NMT 10.0%
° Anes OF BOTANICAL ORIGIN, Total Ash (561): NMT
7.0%
e ARTICLES OF BOTANICAL ORIGIN, Acid-Insoluble Ash (561):
NMT 4.0%
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in well-closed, light-
resistant containers.
e LABELING: The label states the Latin binomial and, follow-
ing the official name, the part of the plant from which
the article was derived.
e USP REFERENCE STANDARDS (11)
USP Caftaric Acid RS
USP Chicoric Acid RS
USP Chlorogenic Acid RS
USP Powdered Echinacea angustifolia Extract RS
USP Echinacoside RS
USP 2£,4E-Hexadienoic Acid Isobutylamide RS
USP B-Sitosterol RS
Powdered Echinacea angustifolia Extract
DEFINITION
Powdered Echinacea angustifolia Extract is prepared from
Echinacea angustifolia roots by extraction with
hydroalcoholic mixtures or other suitable solvents. The ra-
tio of the starting crude plant material to Powdered Ex-
tract is 2:1-8:1. Tt contains NLT 4.0% and NMT 5.0% of
total phenols, calculated on the dried basis as the sum of
chlorogenic acid (CisHisOs), dicaffeoylquinic acids
(CosH24012), and echinacoside (C3sH46O20). It contains NLT
0.1% of dodecatetraenoic acid isobutylamides (CisH2sNO)
on the dried basis.
sydeibouo-: sa
IDENTIFICATION
¢ A. THIN-LAYER CHROMATOGRAPHY
Presence of echinacoside and dicaffeoylquinic acid
(cynarin(e))
Standard solution A: A mixture of 0.2 mg/mL of USP
Echinacoside RS and 0.2 mg/mL of dicaffeoylquinic
acid (cynarin) in methanol
Standard solution B: 0.05 mg/mL of USP Caftaric
Acid RS, 0.1 mg/mL of USP Chlorogenic Acid RS, and
0.05 mg/mL of USP Chicoric Acid RS in methanol
Standard solution C: 20 mg/mL of USP Powdered
Echinacea angustifolia Extract RS in methanol. Shake to
disperse, sonicate for 5 min, and centrifuge. Use the
supernatant.
Sample solution: 20 mg/mL of Powdered Echinacea
angustifolia Extract in methanol. Shake to disperse,
sonicate for 5 min, and centrifuge. Use the
supernatant.
Chromatographic system
(See adit y (621), Thin-Layer Chromato-
graphy.
Adsorbent: Chromatographic silica gel mixture with
an average particle size of 5 um (HPTLC plates)
 4572 Echinacea / Dietary Supplements
Application volume: 5 iL Standard solution C and
Sample solution, and 2 wl Standard solution A and
Standard solution B as 8-mm bands
Relative humidity: Condition the plate toa relative
humidity of about 33% using a suitable device.
Developing solvent system: A mixture of ethyl ace-
ag hy deal ketone, water, and formic acid
533312]
Developing distance: 6 cm
Derivatization reagent: 5 mg/mL of 2-aminoethyl
diphenylborinate in ethyl acetate
Analysis
Samples: Standard solution A, Standard solution B,
Standard solution C, and Sample solution
Apply the samples as bands to a suitable thin-layer
chromatographic plate, and dry in air. Develop the
chromatograms in a saturated chamber. Remove the
plate from the chamber, heat at 100° for 5 min, der-
ivatize the plate while still warm with Derivatization
reagent, dry in air, and examine under UV light at
366 nm.
System suitability: Standard solution A shows two ma-
jor blue bands, one in the lower third of the chromat-
ogram due to echinacoside, and the other band in the
middle section of the chromatogram due to dicaffeoyl-
quinic acid (cynarin). Standard solution B shows two
major blue bands at about the middle of the chromat-
ogram due to caftaric acid (lower Rp) and chlorogenic
acid (higher R;) that are clearly separated, and a blue
band for chicoric acid in the upper third of the
chromatogram.
Acceptance criteria: The most prominent band in the
Sample solution chromatogram is a blue band in the
lower third of the chromatogram at an R; correspond-
ing to the echinacoside band in the chromatograms of
Standard solution A and Standard solution C (absent in
Echinacea purpurea). The Sample solution chromato-
gram exhibits a prominent greenish blue band in the
middle section of the chromatogram at an Rr corre-
sponding to the dicaffeoylquinic acid (cynarin) band in
the chromatograms of Standard solution A and Stan-
dard solution C (absent in Echinaceapaliiga and Echina-
cea purpurea). The Sample solution chromatogram does
not exhibit, or exhibits very faint blue bands at an Rr
corresponding to the caftaric acid and chicoric acid
bands in Standard solution B (difference from Echinacea
pallida and Echinacea purpurea). The Sample solution
chromatogram exhibits minor bands between the po-
sitions of echinacoside and cynarin. One of these is
due to chlorogenic acid at an Rr corresponding to that
of chlorogenic acid in Standard solution B.
DS Monographs
e B. THIN-LAYER CHROMATOGRAPHY
Presence of alkylamides
Standard solution A: 0.2 mg/mL of USP B-Sitosterol
RS in methanol
Standard solution B: 100 mg/mL of USP Powdered
Echinacea angustifolia Extract RS in dichloromethane.
Shake to disperse, sonicate for 5 min, and centrifuge.
Use the supernatant.
Sample solution: 100 mg/mL of Powdered Echinacea
angustifolia Extract in dichloromethane. Shake to dis-
perse, sonicate for 5 min, and centrifuge. Use the
supernatant.
Chromatographic system
(See iy oe yy (621), Thin-Layer Chromato-
raphy.
Ausorent: Chromatographic silica gel with an aver-
age particle size of 5 um (HPTLC plates)
Application volume: 5 ul Standard solution B and
Sample solution, and 2 L Standard solution A as
8-mm bands
Relative humidity: Condition the plate toa relative
humidity of about 33% using a suitable device.
USP 41
Developing solvent system: A mixture of toluene,
ethyl acetate, cyclohexane, and formic acid
(8: 2: 1: 0.3)
Developing distance: 6 cm
Derivatization reagent: Place 85 mL of methanol in
a 100-mL glass bottle, and cool it down in a
water-ice cubes-salt bath or in a freezer. To the ice-
cold methanol, slowly and carefully add 10 mL of
acetic acid and 5 mL of sulfuric acid, and mix well.
Allow the mixture to cool to room temperature, then
add 0.5 mL of p-anisaldehyde.
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Apply the samples as bands to a suitable thin-layer
chromatographic plate, and dry in air. Develop the
chromatograms in a saturated chamber. Remove the
plate from the chamber, dry in air, derivatize with
Derivatization reagent, heat at 100° for 3-5 min, dry
in air, and examine under visible light.
System suitability: The B-sitosterol band of the Stan-
dard solution B chromatogram and the two bands un-
derneath are clearly separated from one another.
These two bands, in decreasing R;, include a major
blue violet band and a yellow band.
Acceptance criteria: The most prominent band of the
Sample solution chromatogram is a blue violet band in
the lower-third section of the chromatogram (much
less prominent in Echinacea purpurea and absent in
Echinacea pallida). This blue violet band is between
two bands: a less prominent blue violet band at a
higher R; corresponding to the B-sitosterol band in the
chromatograms of Standard solution A and Standard
solution B, and a characteristic yellow band at a lower
Re (absent in Echinacea purpurea and Echinacea pallida).
The yellow band turns reddish pink when the plate is
heated at 100° for more than 10 min. The Sample
solution chromatogram exhibits a minor pink-violet
band at about the middle of the chromatogram (much
more prominent in Echinacea purpurea), a minor pink-
violet band at about two-thirds of the chromatogram
(much more prominent in Echinacea pallida), and a
broad pink-violet band close to the solvent front.
° C. The retention time of the major peak of the Sample
solution corresponds to that of the echinacoside peak of
Standard solution A and Standard solution B; and the re-
tention time of the peak for 1,3-dicaffeoylquinic acid
from the Sample solution corresponds to that of Standard
solution A, all peaks as obtained in the test for Content of
Total Phenols.
COMPOSITION
© CONTENT OF TOTAL PHENOLS
Solution A: Phosphoric acid (0.1 in 100) in water
Solution B: Acetonitrile
Mobile phase: See Table 1.
Table 1
Time Solution A Solution B
(min) (%) (%)
0 90 10
3 90 10
16 78 22
7 60 40
20 60 40
20.5 90 10
25 90 10
Solvent: Alcohol and water (7:3)
Standard solution A: Dissolve USP Powdered Echinacea
angustifolia Extract RS in Solvent, shaking and heating in
a water bath. Dilute with Solvent to obtain a solution
having a known concentration of 1 mg/mL. Pass
USP 41
through a membrane filter having a 0.45-um or finer
pore size.
Standard solution B: 40 g/mL of USP Chlorogenic
Acid RS in Solvent
Standard solution C: 80 ug/mL of USP Echinacoside RS
in Solvent
Sample solution: Transfer about 60 mg of Powdered
Extract, accurately weighed, to an appropriate round-
bottom flask equipped with a condenser. Add 25.0 mL
of Solvent, and heat under reflux while shaking by me-
chanical means for 15 min. Centrifuge, or pass through
a membrane filter having a 0.45-~m or finer pore size.
Chromatographic ae
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 330 nm
Column: 4.6-mm x 25-cm; 5-um packing L1
Column temperature: 35°
Flow rate: 1.5 mL/min
Injection volume: 5 wl
System suitability
Samples: Standard solution A and Standard solution C
Suitability requirements
Chromatogram similarity: The chromatogram from
Standard solution A is similar to the Reference Chro-
matogram for total phenols provided with the USP
Powdered Echinacea angustifolia Extract RS.
Resolution: NLT 1.0 between the 1,3-dicaffeoylquinic
acid isomer and echinacoside peaks, Standard solution
A. [Note—Echinacoside peak may be resolved in two
components.]
Capacity factor (k’): NLT 3.0, Standard solution C
Tailing factor: NMT 2.0 for the echinacoside peak,
Standard solution C
Relative standard deviation: NMT 2.5% for the
echinacoside peaks, Standard solution C
Analysis
Samples: Standard solution A, Standard solution B,
Standard solution C and Sample solution
Identify the relevant analytes in the chromatogram from
the Sample solution by comparison with the chromato-
gram from Standard solution A. Measure the areas for
the relevant peaks.
Separately calculate the percentage of chlorogenic acid
(Ci6HisOo), dicaffeoylquinic acids (C2sH24012), and
echinacoside (C3sH46O20) in the portion of Powdered
Extract taken:
Result = (ru/rs) x (Cs/Cu) x F x 100
tu = peak response for the relevant analyte from
the Sample solution
rs = peak response for chlorogenic acid or both
components of echinacoside from the
corresponding Standard solution
Gs = concentration of chlorogenic acid or
echinacoside in the corresponding Standard
solution (mg/mL)
Cu = concentration of Powdered Echinacea
angustifolia Extract in the Sample solution
(mg/mL)
F = 0.729 for dicaffeoylquinic acids, 1.00 for
chlorogenic acid, and 1.00 for echinacoside
components
Calculate the percentage of total phenols in the portion
of Powdered Extract taken by adding the individual
percentages.
Acceptance criteria: NLT 4.0% and NMT 5.0% of total
phenols on the dried basis
¢ CONTENT OF DODECATETRAENOIC ACID ISOBUTYLAMIDES
Standard solution A: Dissolve USP Powdered Echinacea
angustifolia Extract RS in methanol, shaking for 1 min,
and dilute with methanol to volume to obtain a solu-
tion having a known concentration of 1 mg/mL. Pass
Dietary Supplements / Echinacea 4573
through a membrane filter having a 0.45-um or finer
pore size.
Standard solution B: 10 g/mL of USP 2£,4F-Hexadie-
noic Acid Isobutylamide RS in methanol
Sample solution: Transfer about 500 mg of Powdered
Extract, accurately weighed, to a 100-mL volumetric
flask. Add 80 mL of methanol, and sonicate for 30 min.
Dilute with methanol to volume, and pass through a
membrane filter having a 0.45-um or finer pore size.
Mobile phase: Acetonitrile and water (55:45)
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 254 nm
Column: 4.6-mm x 25-cm; 5-m packing L1
Column temperature: 30°
Flow rate: 1.5 mL/min
Injection volume: 25 uL
System suitability
Samples: Standard solution A and Standard solution B
Suitability requirements
Chromatogram similarity: The chromatogram from
Standard solution A is similar to the Reference Chro-
matogram for alkamides provided with USP Pow-
dered Echinacea angustifolia Extract RS.
Resolution: NLT 1.0 between dodecatetraenoic acid
isobutylamide peaks, Standard solution A
Tailing factor: NMT 2.0 for the 2E,4E-hexadienoic
acid isobutylamide peak, Standard solution B
Relative standard deviation: NMT 2.5% for the 2E,
4£-hexadienoic acid isobutylamide peak in repeated
injections, Standard solution B
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Identify the peaks due to 2£,4£,8Z,10£-dodecatetraenoic
acid isobutylamide and 2£,4£,8Z,10Z-dodecatetraenoic
acid isobutylamide in the chromatogram from the
Sample solution - comparison with the chromatogram
from Standard solution A. Measure the areas for the
relevant peaks.
Calculate the percentage of dodecatetraenoic acid iso-
butylamides in the portion of Powdered Extract taken:
Result = (ru/rs) x (Cs/Cu) x F x 100
ty = sum of the peak responses of the relevant
analytes from the Sample solution
rs = peak response for 2£,4E-hexadienoic acid
isobutylamide from Standard solution B
Gs = concentration of USP 2£,4£-Hexadienoic Acid
lsobutylamide RS in Standard solution B
sydeibouo-= Sa
(mg/mL)
Cu = concentration of Powdered Echinacea
angustifolia Extract in the Sample solution
(mg/mL)
F = response factor for 2E,4£-hexadienoic acid
isobutylamide, 1.353
Acceptance criteria: NLT 0.1% of dodecatetraenoic
acid isobutylamides (CisH2sNO) on the dried basis
CONTAMINANTS
e ELEMENTAL IMPURITIES—PROCEDURES (233)
Acceptance criteria
Arsenic: NMT 1.0 ug/g
Cadmium: NMT 0.5 ug/g
Lead: NMT 5.0 ug/g
Mercury: NMT 1.0 ug/g
e MICROBIAL ENUMERATION TESTS (2021): The total bacterial
count does not exceed 104 cfu/g and the total combined
molds and yeasts count does not exceed 103 cfu/g.
© ABSENCE OF SPECIFIED MICROORGANISMS (2022): Meets the
requirements of the tests for absence of Salmonella spe-
cies and Escherichia coli.
 4574 Echinacea / Dietary Supplements
e OTHER REQUIREMENTS: It meets the requirements for Bo-
tanical Extracts (565), Residual Solvents and Pesticide
Residues.
SPECIFIC TESTS
e Loss ON DRYING (731)
Sample: 1g
Analysis: Dry the Sample at 105° for 2 h.
Acceptance criteria: NMT 5.0%
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in tight, light-resistant
containers, in a cool place.
e LABELING: The label states the Latin binomial and, follow-
ing the official name, the part of the plant from which
the article was prepared. If standardized by the content
of alkamides, label it to indicate the targeted content of
dodecatraenoic acid isobutylamides. The label bears a
statement indicating that Echinacea angustifolia may
cause rare allergic reactions, rashes, or aggravate asthma.
It meets the requirements for Botanical Extracts (565),
Labeling.
e USP REFERENCE STANDARDS (11)
USP Caftaric Acid RS
USP Chicoric Acid RS
USP Chlorogenic Acid RS
USP Powdered Echinacea angustifolia Extract RS
USP Echinacoside RS
USP 2E,4£-Hexadienoic Acid Isobutylamide RS
USP B-Sitosterol RS
Echinacea pallida
DEFINITION
Echinacea pallida consists of the dried rhizome and roots of
Echinacea pallida (Nutt.) Nutt. (Fam. Asteraceae). It is har-
vested in the fall after three or more years of growth. It
contains NLT 0.5% of total phenols, calculated on the
dried basis as the sum of caftaric acid (C)3Hi20s), chicoric
acid (C22HigO12), chlorogenic acid (CicéHigOs), and
echinacoside (C3sHssO20).
IDENTIFICATION
© A. THIN-LAYER CHROMATOGRAPHY
Presence of echinacoside and absence of dicaffeoyl-
quinic acid (cynarin(e))
Standard solution A: A mixture of 0.2 mg/mL of USP
Echinacoside RS and 0.2 mg/mL of dicaffeoylquinic
DS Monographs
acid (cynarin) in methanol
Standard solution B: 0.05 mg/mL of USP Caftaric
Acid RS, 0.1 mg/mL of USP Chlorogenic Acid RS, and
0.05 mg/mL of USP Chicoric Acid RS in methanol
Standard solution C: 20 mg/mL of USP Powdered
Echinacea pallida Extract RS in methanol. Shake to dis-
perse, sonicate for 5 min, and centrifuge. Use the
supernatant.
Sample solution: Transfer 1 g of finely pulverized Echi-
nacea pallida to a centrifuge tube, add 10 mL of meth-
anol, mix well, and sonicate for 10 min. Centrifuge,
and use the supernatant.
Chromatographic system
(See Chromatography (621), Thin-Layer Chromato-
raphy.)
Alsouberié Chromatographic silica gel mixture with
an average particle size of 5 zm (HPTLC plates)
Application volume: 5 tL Standard solution C and
Sample solution, and 2 wl Standard solution A and
Standard solution B as 8-mm bands
Relative humidity: Condition the plate to a relative
humidity of about 33% using a suitable device.
USP 41
Developing solvent system: A mixture of ethyl ace-
as mw ketone, water, and formic acid
Developing distance: 6 cm
Derivatization reagent: 5 mg/mL of 2-aminoethyl
diphenylborinate in ethyl acetate
Analysis
Samples: Standard solution A, Standard solution B,
Standard solution C, and Sample solution
Apply the Samples as bands to a suitable thin-layer
chromatographic plate, and dry in air. Develop the
chromatograms in a saturated chamber. Remove the
plate from the chamber, heat at 100° for 5 min, der-
ivatize the plate while still warm with Derivatization
reagent, dry in air, and examine under UV light at
366 nm.
System suitability: Standard solution A shows two ma-
jor blue bands, one in the lower third section of the
chromatogram due to echinacoside, and the other
band in the middle section of the chromatogram due
to dicaffeoylquinic acid (cynarin). Standard solution B
shows two major blue bands at about the middle of
the chromatogram due to caftaric acid (lower Rp) and
chioregenig acid (higher R-) that are clearly separated,
and a blue band for chicoric acid in the upper third
section of the chromatogram.
Acceptance criteria: The most prominent band in the
Sample solution chromatogram is a blue band in the
lower third section of the chromatogram at an R; cor-
responding to the echinacoside band in the chromato-
rams of Standard solution A and Standard solution C
absent in Echinacea purpurea). The Sample solution
chromatogram does not exhibit a blue band at an R-
corresponding to the dicaffeoylquinic acid (cynarin)
band in the chromatogram of Standard solution A
(present in Echinacea angustifolia). The Sample solution
chromatogram may exhibit bands of lesser intensity at
the R; of caftaric acid and chicoric acid bands in chro-
matograms of Standard solution B and Standard solu-
tion C (absent in Echinacea angustifolia and much more
prominent in Echinacea purpurea). The Sample solution
chromatogram exhibits minor bands between the po-
sitions of echinacoside and caftaric acid. One of these
is due to chlorogenic acid at an R- corresponding to
that of chlorogenic acid in Standard solution B.
e B. THIN-LAYER CHROMATOGRAPHY
Presence of alkylamides
Standard solution A: 0.2 mg/mL of USP B-Sitosterol
RS in methanol
Standard solution B: 100 mg/mL of USP Powdered
Echinacea pallida Extract RS in dichloromethane. Shake
to disperse, sonicate for 5 min, and centrifuge. Use the
supernatant.
Sample solution: Transfer 1 g of finely pulverized Echi-
nacea pallida to a centrifuge tube, add 10 mL of di-
chloromethane, mix well, and sonicate for 10 min.
Centrifuge, and use the supernatant.
Chromatographic system
(See Chromatography (621), Thin-Layer Chromato-
graphy.) woah
Adsorbent: Chromatographic silica gel with an aver-
age particle size of 5 um (HPTLC plates)
Application volume: 5 wl Standard solution B and
ample solution, and 2 L Standard solution A as
8-mm bands
Relative humidity: Condition the plate to a relative
humidity of about 33% using a suitable device.
Developing solvent system: A mixture of toluene,
ethyl acetate, cyclohexane, and formic acid
(8: 2: 1: 0.3)
Developing distance: 6 cm
Derivatization reagent: Place 85 mL of methanol in
a 100-mL glass bottle, and cool it down in a
water-ice cubes-salt bath or in a freezer. To the ice-
cold methanol, slowly and carefully add 10 mL of
USP 41
acetic acid and 5 mL of sulfuric acid, and mix well.
Allow the mixture to cool to room temperature, then
add 0.5 mL of p-anisaldehyde.
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Apply the Samples as bands to a suitable thin-layer
chromatographic plate, and dry in air. Develop the
chromatograms in a saturated chamber. Remove the
plate from the chamber, dry in air, derivatize with
Derivatization reagent, heat at 100° for 3-5 min, set
aside to cool, and examine under visible light.
System suitability: The Standard solution A chromato-
gram exhibits a violet band corresponding to B-sitos-
terol. The Standard solution B shows the most promi-
nent band as a violet band in the upper third section
of the chromatogram. The Standard solution B chro-
matogram exhibits a less prominent violet band in the
lower third section of the chromatogram and a broad
pink violet band close to the solvent front.
Acceptance criteria: The most prominent band of the
Sample solution chromatogram is a violet band in the
upper third section of the chromatogram, correspond-
ing in Rr to a similar band observed in Standard solu-
tion B (much less prominent in Echinacea angustifolia
and absent in Echinacea purpurea). The Sample solution
chromatogram exhibits a less prominent violet band in
the lower third section of the chromatogram corre-
sponding in R; to a similar band observed in Standard
solution B (much less prominent in Echinacea purpurea
and absent in Echinacea angustifolia). The Sample solu-
tion chromatogram exhibits a minor violet band at an
R- corresponding to the B-sitosterol band in the chro-
matograms of Standard solution A and Standard solu-
tion B and a broad pink violet band close to the sol-
vent front.
e C. The retention time of the major peak in the Sample
solution corresponds to that of the echinacoside peak in
Standard solution A and Standard solution B, as obtained
in the test for Content of Total Phenols. The peak area of
any peak detected in the Sample solution chromatogram
at the locus of 1,3-dicaffeoylquinic acid (Standard solution
9 NMI 1% of the peak area for the echinacoside
peak.
COMPOSITION
e CONTENT OF TOTAL PHENOLS
Solution A: Phosphoric acid (0.1 in 100) in water
Solution B: Acetonitrile
Mobile phase: See Table 7.
Table 1
Time Solution A Solution B
(min) (%) (%)
0 90 10
3 90 10
16 78 22
17 60 40
20 60 40
20.5 90 10
25 90 10
Solvent: Alcohol and water (7:3)
Standard solution A: Dissolve USP Powdered Echinacea
pallida Extract RS in Solvent by shaking and heating in a
water bath. Dilute with Solvent to obtain a solution hav-
ing a known concentration of 1 mg/mL. Pass through a
membrane filter having a 0.45-\1m or finer pore size.
Standard solution B: 40 g/mL of USP Chlorogenic
Acid RS in Solvent
Dietary Supplements / Echinacea 4575
Standard solution C: 80 g/mL of USP Echinacoside RS
in Solvent
Standard solution D: 40 ug/ml of dicaffeoylquinic acid
(cynarin) in Solvent
Sample solution: Transfer 125 mg of finely powdered
Echinacea pallida (capable of passing through a
40-mesh sieve) to a round-bottom flask equipped with
a condenser. Add 25.0 mL of Solvent, and heat under
reflux while shaking by mechanical means for 15 min.
Centrifuge, or pass through a membrane filter having a
0.45-m or finer pore size.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 330 nm
Column: 4.6-mm x 25-cm; 5-um packing L1
Column temperature: 35°
Flow rate: 1.5 mL/min
Injection volume: 5 uL
System suitability
Samples: Standard solution A and Standard solution C
Suitability requirements
Chromatogram similarity: The chromatogram of
Standard solution A is similar to the Reference Chro-
matogram for total phenols provided with USP Pow-
dered Echinacea pallida Extract RS.
Capacity factor (k’): NLT 3.0 for the echinacoside
peak, Standard solution C. [NoTE—Echinacoside peak
may be resolved in two components.]
Tailing factor: NMT 2.0 for the echinacoside peak,
Standard solution C
Relative standard deviation: NMT 2.5% for the
echinacoside peaks in repeated injections, Standard
solution C
Analysis
Samples: Standard solution A, Standard solution B,
Standard solution C, Standard solution D, and Sample
solution
Identify the relevant analytes in the chromatogram
from the Sample solution by comparison with the
chromatogram from Standard solution A. Measure the
areas for the relevant peaks.
Separately calculate the percentage of caftaric acid
(Ci3H120o9), chicoric acid (C22H1gO12), chlorogenic acid
(CisHigOo), and echinacoside (C3sH46O20) in the portion
of Echinacea pallida taken:
Result = (ru/rs) x Cs x (V/W) x Fx 100
ru = peak response for the relevant analyte from
the Sample solution
rs = peak response for chlorogenic acid or both 4
components of echinacoside from the
corresponding Standard solution Ks
Cs = concentration of chlorogenic acid or Sj
echinacoside in the corresponding Standard io)
solution (mg/mL) ie]
Vv = volume of the Sample solution (mL) Py
w = weight of powdered Echinacea pallida used to <
prepare the Sample solution (mg) a]
= response factor: chicoric acid, 0.695; caftaric
acid, 0.881; chlorogenic acid, 1.000; relative
to chlorogenic acid; and echinacoside
components, 1.000
Calculate the percentage of total phenols in the portion
of Echinacea pallida taken by adding the individual
percentages calculated.
Acceptance criteria: NLT 0.5% of total phenols on the
dried basis
 4576 Echinacea / Dietary Supplements
CONTAMINANTS
ELEMENTAL IMPURITIES—PROCEDURES (233)
Acceptance criteria
Arsenic: NMT 1.0 ug/g
Cadmium: NMT 0.5 g/g
Lead: NMT 5.0 ug/g
Mercury: NMT 1.0 ug/g
ARTICLES OF BOTANICAL ORIGIN, General Method for Pesti-
cide Residues Analysis (561): Meets the requirements
SPECIFIC TESTS
BOTANIC CHARACTERISTICS
Macroscopic: The outer surface of the rhizome is pale
to yellowish-brown, crowned with the remains of the
aerial stem, and sometimes shows surface annulations
up to 15 mm in diameter. The roots are pale to yellow-
ish-brown, cylindrical or slightly tapering, sometimes
spirally twisted, longitudinally wrinkled and deeply fur-
rowed, up to 4-10 mm in diameter, and pass impercep-
tibly into rhizome. The short fracture, when dry, be-
comes tough and pliable on exposure to air.
Microscopic: The rhizomes and roots in transverse sec-
tion show a thin outer bark separated from a wide xy-
lem bya distinct cambial line. The cork is composed of
several rows of thin-walled cells containing yellowish-
brown pigment. The rhizome has a small circular pith,
occasional small groups of thick-walled, lignified fibers
in the pericycle, and a parenchymatous cortex. The
phloem and xylem are composed of narrow strands of
vascular tissue separated by wide, nonlignified medul-
lary rays. Xylem vessels are lignified, 25-75 jum in diam-
eter, usually with reticulate thickening but ce Tey
with spiral or annular thickening. Sclereids occur singly
or in small groups, varying considerably in size and
shape from rounded to rectangular to elongated and
fiber-like, are up to 300 um long and 20-40 um wide,
with intercellular spaces forming schizogenous oleoresin
canals that are 80-150 um in diameter and contain a
dense black deposit present both inside and outside of
the central cylinder (unlike Echinacea angustifolia, where
the canals are present only outside of the central cylin-
der). Spherocrystalline masses of inulin occur through-
out the parenchymatous tissues. Lignified fibers, present
in the periphery of the cortex, are 100-300 um long
and occur singly with phytomelanin often absent (un-
like Echinacea angustifolia, where the fibers occur scat-
tered in groups, are 300-800 um long, and are usually
surrounded by phytomelanin).
ARTICLES OF BOTANICAL ORIGIN, Volatile Oi! Determination
(561): 1.0-2.0 mL/100 g
ARTICLES OF BOTANICAL ORIGIN, Foreign Organic Matter
(561): NMT 3.0%
DS Monographs
ARTICLES OF BOTANICAL ORIGIN, Total Ash (561): NMT
7.0%
ARTICLES OF BOTANICAL ORIGIN, Acid-/nsoluble Ash (561):
NMT 4.0%
Loss ON DRYING (731)
Analysis: Dry a sample at 105° for 2 h.
Acceptance criteria: NMT 10.0%
ADDITIONAL REQUIREMENTS
PACKAGING AND STORAGE: Preserve in well-closed, light-
resistant containers.
LABELING: The label states the Latin binomial and, follow-
ing the official name, the parts of the plant contained in
the article.
USP 41
e USP REFERENCE STANDARDS (11)
USP Caftaric Acid RS
USP Chicoric Acid RS
USP Chlorogenic Acid RS
USP Powdered Echinacea pallida Extract RS
USP Echinacoside RS
USP B-Sitosterol RS
Powdered Echinacea pallida
DEFINITION
Powdered Echinacea pallida consists of the dried rhizome
and roots of Echinacea pallida (Nutt.) Nutt. (Fam. Astera-
ceae) harvested in the fall after three or more years of
growth, reduced to powder. It contains NLT 0.5% of total
phenols, calculated on the dried basis as the sum of
caftaric acid (Ci3H120s), chicoric acid (C22Hig012),
chlorogenic acid (CisHigOs), and echinacoside (C3sH4sO20).
IDENTIFICATION
o A, THIN-LAYER CHROMATOGRAPHY
Presence of echinacoside and absence of dicaffeoyl-
quinic acid (cynarin(e))
Standard solution A: A mixture of 0.2 mg/mL of USP
Echinacoside RS and 0.2 mg/mL of dicaffeoylquinic
acid (cynarin) in methanol
Standard solution B: 0.05 mg/mL of USP Caftaric
Acid RS, 0.1 mg/mL of USP Chlorogenic Acid RS, and
0.05 mg/mL of USP Chicoric Acid RS in methanol
Standard solution C: 20 mg/mL of USP Powdered
Echinacea pallida Extract RS in methanol. Shake to dis-
perse, sonicate for 5 min, and centrifuge. Use the
supernatant.
Sample solution: Transfer 1 g of Powdered Echinacea
pallida to a centrifuge tube, add 10 mL of methanol,
mix well, and sonicate for 10 min. Centrifuge, and use
the supernatant.
Chromatographic system
(See Chromatography (621), Thin-Layer Chromato-
graphy.) ila .
Adsorbent: Chromatographic silica gel mixture with
.
an average particle size of 5 um (HPTLC plates)
Application volume: 5 uL Standard solution C and
Sample solution, and 2 wl Standard solution A and
Standard solution B as 8-mm bands
Relative humidity: Condition the plate to a relative
humidity of about 33% using a suitable device.
Developing solvent system: A mixture of ethyl ace-
tate, methylethyl ketone, water, and formic acid
(5:32:11)
Developing distance: 6 cm
Derivatization reagent: 5 mg/mL of 2-aminoethyl
diphenylborinate in ethy! acetate
Analysis
Samples: Standard solution A, Standard solution B,
Standard solution C, and Sample solution
Apply the Samples as bands to a suitable thin-layer
chromatographic plate, and dry in air. Develop the
chromatograms in a saturated chamber. Remove the
late from the chamber, heat at 100° for 5 min, der-
ivatize the plate while still warm with Derivatization
reagent, dry in air, and examine under UV light at
366 nm.
System suitability: Standard solution A shows two ma-
jor blue bands, one in the lower third of the chromat-
ogram, due to echinacoside, and the other band in
the middle section of the chromatogram due to dicaf-
feoylquinic acid (cynarin). Standard solution B shows
two major blue bands at about the middle of the
chromatogram due to caftaric acid (lower Rp) and
chlorogenic acid (higher Rr) that are clearly separated,
USP 41 Dietary Supplements / Echinacea 4577

and a blue band for chicoric acid in the upper third of lution B (much less prominent in Echinacea angustifolia
the chromatogram. and absent in Echinacea purpurea). The Sample solution
Acceptance criteria: The most prominent band in the chromatogram exhibits a less prominent violet band in
Sample solution chromatogram is a blue band in the the lower third section of the chromatogram corre-
lower third section of the chromatogram at an R; cor- sponding in Rr to a similar band observed in Standard
responding to the echinacoside band in the chromato- solution B (much less prominent in Echinacea purpurea
rams of Standard solution A and Standard solution C and absent in Echinacea angustifolia). The Sample solu-
absent in Echinacea purpurea). The Sample solution tion chromatogram exhibits a minor violet band at an
chromatogram does not exhibit a blue band at an Rr Rr corresponding to the B-sitosterol band in the chro-
corresponding to the dicaffeoylquinic acid (cynarin) matograms of Standard solution A and Standard solu-
band in the chromatogram of Standard solution A tion B, and a broad pink violet band close to the sol-
(present in Echinacea angustifolia). The Sample solution vent front.
chromatogram may exhibit bands of lesser intensity at e C. The retention time of the major peak in the Sample
the Rr of caftaric acid and chicoric acid bands in chro- solution corresponds to that of the echinacoside peak in
matograms of Standard solution B and Standard solu- Standard solution A and Standard solution B, as obtained
tion C (absent in Echinacea angustifolia and much more in the test for Content of Total Phenols. Peak area of any
prominent in Echinacea purpurea). The Sample solution peak detected in the Sample solution chromatogram at
chromatogram exhibits minor bands between the po- the locus of 1,3-dicaffeoylquinic acid (Standard solution
sitions of echinacoside and caftaric acid. One of these 9 ee 1% of the peak area for the echinacoside
is due to chlorogenic acid at an R¢ corresponding to peak.
that of chlorogenic acid in Standard solution B.
e B. THIN-LAYER CHROMATOGRAPHY COMPOSITION
Presence of alkylamides e CONTENT OF TOTAL PHENOLS
Standard solution A: 0.2 mg/mL of USP B-Sitosterol Solution A: Phosphoric acid (0.1 in 100) in water
RS in methanol Solution B: Acetonitrile
Standard solution B: 100 mg/mL of USP Powdered Mobile phase: See Table 1.
Echinacea pallida Extract RS in dichloromethane. Shake
to disperse, sonicate for 5 min, and centrifuge. Use the Table 1
supernatant.
Time Solution A Solution B
Sample solution: Transfer 1g of Powdered Echinacea
pallida to a centrifuge tube, add 10 mL of dichloro- (min) (%) (%)
methane, mix well, and sonicate for 10 min. Centri- 0 90 10
fuge, and use the supernatant. 3 90 10
Chromatographic system 16 78 22
(See Su aecane y (621), Thin-Layer Chromato- 17 60 40
graphy.) 20 60 40
Adsorbent: Chromatographic silica gel with an aver-
age particle size of 5 um (HPTLC plates) 20.5 90 10
Application volume: 5 tL Standard solution B and 25 90 10
Sample solution, and 2 wl Standard solution A as
8-mm bands Solvent: Alcohol and water (7:3)
Relative humidity: Condition the plate to a relative Standard solution A: Dissolve USP Powdered Echinacea
humidity of about 33% usinga suitable device. pallida Extract RS in Solvent by shaking and heating in a
Developing solvent system: A mixture of toluene, water bath. Dilute with Solvent to obtain a solution hav-
ethyl acetate, cyclohexane, and formic acid ing a known concentration of about 1 mg/mL. Pass
(8: 2: 1: 0.3) through a membrane filter having a 0.45-um or finer
Developing distance: 6 cm pore size.
Derivatization reagent: Place 85 mL of methanol in Standard solution B: 40 tg/mL of USP Chlorogenic
a 100-mL glass bottle, and cool it down in a Acid RS in Solvent
water-ice cubes-salt bath or in a freezer. To the ice- Standard solution C: 80 j1g/mL of USP Echinacoside RS
cold methanol, slowly and carefully add 10 mL of in Solvent
Standard solution D: 40 ug/mL of dicaffeoylquinic acid
sydesbouo=: sa

acetic acid and 5 mL of sulfuric acid, and mix well.

Allow the mixture to cool to room temperature, then
add 0.5 mL of p-anisaldehyde.
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Apply the Samples as bands to a suitable thin-layer
chromatographic plate, and dry in air. Develop the
chromatograms in a saturated chamber. Remove the
plate from the chamber, dry in air, derivatize with
Derivatization reagent, heat at 100° for 3-5 min, set
aside to cool, and examine under visible light.
System suitability: The Standard solution A chromato-
gram exhibits a violet band corresponding to B-sitos-
terol. Standard solution B shows the most prominent
band asa violet band in the upper third section of the
chromatogram. The Standard solution B chromatogram
exhibits a less prominent violet band in the lower third
section of the chromatogram, and a broad pink violet
band close to the solvent front.
Acceptance criteria: The most prominent band of the
Sample solution chromatogram is a violet band in the
upper third section of the chromatogram, correspond-
ing in Rr to a similar band observed with Standard so-
(cynarin) in Solvent
Sample solution: Transfer 125 mg of Powdered Echina-
cea pallida (capable of passing through a 40-mesh
sieve) to a round-bottom flask equipped with a con-
denser. Add 25.0 mL of Solvent, and heat under reflux
while shaking by mechanical means for 15 min. Centri-
fuge, or pass through a membrane filter having a 0.45-
um or finer pore size.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 330 nm
Column: 4.6-mm x 25-cm; 5-um packing L1
Column temperature: 35°
Flow rate: 1.5 mL/min
Injection volume: 5 pL
System suitability
Samples: Standard solution A and Standard solution C
Suitability requirements
Chromatogram similarity: The chromatogram of
Standard solution A is similar to the Reference Chro-
matogram for total Eee provided with USP Pow-
dered Echinacea pallida Extract RS.
 4578 Echinacea / Dietary Supplements
Capacity factor (k’): NLT 3.0 for the echinacoside
peak, Standard solution C. [NoTE—Echinacoside peak
may be resolved in two components.]
Tailing factor: NMT 2.0 for the echinacoside peak,
Standard solution C
Relative standard deviation: NMT 2.5% for the
echinacoside peaks in repeated injections, Standard
solution C
Analysis
Samples: Standard solution A, Standard solution B,
Standard solution C, Standard solution D, and Sample
solution
Identify the relevant analytes in the chromatogram
from the Sample solution by comparison with the
chromatogram from Standard solution A. Measure the
areas for the relevant peaks.
Separately calculate the percentage of caftaric acid
(Cy3Hi2O¢), chicoric acid (C22HigO12), chlorogenic acid
(CisHigOs), and echinacoside (C3sH4sO20) in the portion
of Powdered Echinacea pallida taken:
Result = (ru/rs) x Cs x (V/W) x F x 100
tu = peak response for the relevant analyte from
the Sample solution
Is = peak response for chlorogenic acid or both
echinacoside components from the
corresponding Standard solution
G& = concentration of chlorogenic acid or
echinacoside in the corresponding Standard
solution (mg/mL)
Vv = final volume of the Sample solution (mL)
Ww = weight of Powdered Echinacea pallida taken to
prepare the Sample solution (mg)
= response factor: chicoric acid, 0.695; caftaric
acid, 0.881; chlorogenic acid, 1.000; relative
to chlorogenic acid; and echinacoside
components, 1.000
Calculate the percentage of total phenols in the portion
of Echinacea pallida taken by alee the individual
percentages calculated.
Acceptance criteria: NLT 0.5% of total phenols on the
dried basis
CONTAMINANTS
eo ELEMENTAL IMPURITIES—PROCEDURES (233)
Acceptance criteria
Arsenic: NMT 1.0 g/g
Cadmium: NMT 0.5 ug/g
Lead: NMT 5.0 ug/g
Mercury: NMT 1.0 ng/g
e ARTICLES OF BOTANICAL ORIGIN, General Method for Pesti-
DS Monographs
cide Residues Analysis (561): Meets the requirements
SPECIFIC TESTS
e BOTANIC CHARACTERISTICS: Powdered Echinacea pallida is a
brown powder with a faint aromatic odor anda slightly
acrid, persistent taste. It turns yellow when mounted in
sodium hydroxide solution. Under a microscope, the fol-
lowing characteristics are observed: groups of secretory
canals with brown contents, surrounded by parenchyma-
tous cells containing cluster crystals of calcium oxalate;
and parenchymatous cells with small starch granules;
thick-walledfignified fibers and fragments of reticulate
and pitted vessels.
e ARTICLES OF BOTANICAL ORIGIN, Volatile Oi! Determination
(561): 1.0-2.0 mL/100 g
e ARTICLES OF BOTANICAL ORIGIN, Total Ash (561): NMT
7.0%
e ARTICLES OF BOTANICAL ORIGIN, Acid-Insoluble Ash (561):
NMT 4.0%
USP 41
e Loss ON DRYING (731)
Analysis: Dry a sample at 105° for 2 h.
Acceptance criteria: NMT 10.0%
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in tight, light-resistant
containers.
e LABELING: The label states the Latin binomial and, follow-
ing the official name, the part of the plant from which
the article was derived.
e USP REFERENCE STANDARDS (11)
USP Caftaric Acid RS
USP Chicoric Acid RS
USP Chlorogenic Acid RS
USP Powdered Echinacea pallida Extract RS
USP Echinacoside RS
USP B-Sitosterol RS
Powdered Echinacea pallida Extract
DEFINITION
Powdered Echinacea pallida Extract is prepared from Echina-
cea pallida roots by extraction with hydroalcoholic mix-
tures or other suitable solvents. The ratio of the starting
crude plant material to Powdered Extract is between 2:1
and 8:1. It contains NLT 4.0% and NMT 5.0% of total
phenols, calculated as the sum of caftaric acid (Ci3H120o),
chicoric acid (C22HisQ12), chlorogenic acid (CisHisO9), and
echinacoside (C3sH46O20), on the dried basis.
IDENTIFICATION
e A. THIN-LAYER CHROMATOGRAPHY
Presence of echinacoside and absence of dicaffeoyl-
quinic acid (cynarin(e))
Standard solution A: A mixture of 0.2 mg/mL of USP
Echinacoside RS and 0.2 mg/mL of dicaffeoylquinic
acid (cynarin) in methanol
Standard solution B: 0.05 mg/mL of USP Caftaric
Acid RS, 0.1 mg/mL of USP Chlorogenic Acid RS, and
0.05 mg/mL of USP Chicoric Acid RS in methanol
Standard solution C: 20 mg/mL of USP Powdered
Echinacea pallida Extract RS in methanol. Shake to dis-
perse, sonicate for 5 min, and centrifuge. Use the
supernatant.
Sample solution: 20 mg/mL of Powdered Extract in
methanol. Shake to disperse, sonicate for 5 min, and
centrifuge. Use the supernatant.
Chromatographic system
(See Chromatography (621), Thin-Layer Chromato-
graphy.)
Adsorbent:
ave ,
Chromatographic silica gel mixture with
.
an average particle size of 5 zm (HPTLC plates)
Application volume: 5 wl Standard solution C and
Sample solution, and 2 wl Standard solution A and
Standard solution B as 8-mm bands
Relative humidity: Condition the plate toa relative
humidity of about 33% using a suitable device.
Developing solvent system: A mixture of ethyl ace-
tate, methylethyl ketone, water, and formic acid
($:3:1 1)
Developing distance: 6cm
Derivatization reagent: 5 mg/mL of 2-aminoethyl
diphenylborinate in ethyl acetate
Analysis
Samples: Standard solution A, Standard solution B,
Standard solution C, and Sample solution
Apply the Samples as bands to a suitable thin-layer
chromatographic plate, and dry in air. Develop the
chromatograms in a saturated chamber. Remove the
plate from the chamber, heat at 100° for 5 min,
derivatize the plate while still warm with Derivatiza-
USP 41
tion reagent, dry in air, and examine under UV light
at 366 nm.
System suitability: Standard solution A shows two ma-
jor blue bands, one in the lower third section of the
chromatogram due to echinacoside, and the other
band in the middle section of the chromatogram due
to dicaffeoylquinic acid (cynarin). Standard solution B
shows two major blue bands at about the middle of
the chromatogram due to caftaric acid (lower Rp) and
pee =O acid (higher Ry) that are clearly separated,
and a blue band for chicoric acid in the upper third
section of the chromatogram.
Acceptance criteria: The most prominent band in the
Sample solution chromatogram is a blue band in the
lower third section of the chromatogram at an R; cor-
responding to the echinacoside band in the chromato-
grams of Standard solution A and Standard solution C
(absent in Echinacea purpurea). The Sample solution
chromatogram does not exhibit a blue band at an Rr
corresponding to the dicaffeoylquinic acid (cynarin)
band in the chromatogram of Standard solution A
(present in Echinacea angustifolia). The Sample solution
chromatogram may exhibit bands of lesser intensity at
the Ry of caftaric acid and chicoric acid bands in the
chromatograms of Standard solution B and Standard
solution C (absent in Echinacea angustifolia and much
more prominent in Echinacea purpurea). The Sample
solution chromatogram exhibits minor bands between
the positions of echinacoside and caftaric acid. One of
these is due to chlorogenic acid at an R- correspond-
ing to that of chlorogenic acid in Standard solution B.
e B. THIN-LAYER CHROMATOGRAPHY
Presence of alkylamides
Standard solution A: 0.2 mg/mL of USP B-Sitosterol
RS in methanol
Standard solution B: 100 mg/mL of USP Powdered
Echinacea pallida Extract RS in dichloromethane. Shake
to disperse, sonicate for 5 min, and centrifuge. Use the
supernatant.
Sample solution: 100 mg/mL of Powdered Extract in
dichloromethane. Shake to disperse, sonicate for 5
min, and centrifuge. Use the supernatant.
Chromatographic system
(See Chromatography (621), Thin-Layer Chromato-
graphy.) : 3
Adsorbent: Chromatographic silica gel with an aver-
age particle size of 5 um (HPTLC plates)
Application volume: 5 ul Standard solution B and
Sample solution, and 2 wL Standard solution A as
8-mm bands
Relative humidity: Condition the plate to a relative
humidity of about 33% using a suitable device.
Developing solvent system: A mixture of toluene,
ethyl acetate, cyclohexane, and formic acid
(822871053)
Developing distance: 6 cm
Derivatization reagent: Place 85 mL of methanol in
a 100-mL glass bottle, and cool it down in a
water-ice cubes-salt bath or in a freezer. To the ice-
cold methanol, slowly and carefully add 10 mL of
acetic acid and 5 mL of sulfuric acid, and mix well.
Allow the mixture to cool to room temperature, then
add 0.5 mL of p-anisaldehyde.
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Apply the Samples as bands toa suitable thin-layer
chromatographic plate, and dry in air. Develop the
chromatograms in a saturated chamber. Remove the
plate from the chamber, dry in air, derivatize with
Derivatization reagent, heat at 100° for 3-5 min, set
aside to cool, and examine under visible light.
Dietary Supplements / Echinacea 4579
System suitability: The Standard solution A chromato-
gram exhibits a violet band corresponding to -sitos-
terol. Standard solution B shows the most prominent
band asa violet band in the upper third section of the
chromatogram. The Standard solution B chromatogram
exhibits a less prominent violet band in the lower third
section of the chromatogram, and a broad pink violet
band close to the solvent front.
Acceptance criteria: The most prominent band of the
Sample solution chromatogram is a violet band in the
upper third section of the chromatogram, correspond-
ing in R; to a similar band observed in Standard solu-
tion B (much less prominent in Echinacea angustifolia
and absent in Echinacea purpurea). The Sample solution
chromatogram exhibits a less prominent violet band in
the lower third section of the chromatogram corre-
sponding in R; to a similar band observed in Standard
solution B (much less prominent in Echinacea purpurea
and absent in Echinacea angustifolia). The Sample solu-
tion chromatogram exhibits a minor violet band at an
R- corresponding to the f-sitosterol band in the chro-
matograms of Standard solution A and Standard solu-
tion B, and a broad pink violet band close to the sol-
vent front.
e C. The retention time of the major peak in the Sample
solution corresponds to that of the echinacoside peak in
Standard solution A and Standard solution B, as obtained
in the test for Content of Total Phenols. The peak area of
any peak detected in the Sample solution chromatogram
at the locus of 1,3-dicaffeoylquinic acid (Standard solution
9 Is NMT 1% of the peak area for the echinacoside
peak.
COMPOSITION
e@ CONTENT OF TOTAL PHENOLS
Solution A: Phosphoric acid (0.1 in 100)
Solution B: Acetonitrile
Mobile phase: See Table 7.
Table 1
Time Solution A Solution B
(min) (%) (%)
0 90 10
90 10
16 78 22
17 60 40
20 60 40
20.5 90 10
25: 90 10
sydesbouo-= sa
Solvent: Alcohol and water (7:3)
Standard solution A: Dissolve USP Powdered Echinacea
pallida Extract RS in Solvent by shaking and heating in a
water bath. Dilute with Solvent to obtain a solution hav-
ing a known concentration of 1 mg/mL. Pass through a
membrane filter having a 0.45-um or finer pore size.
Standard solution B: 40 g/mL of USP Chlorogenic
Acid RS in Solvent
Standard solution C: 80 g/mL of USP Echinacoside RS
in Solvent
Standard solution B: 40 g/mL of dicaffeoylquinic acid
(cynarin) in Solvent
Sample solution: Transfer about 60 mg of Powdered
Extract, accurately weighed, to an appropriate round-
bottom flask equipped with a condenser. Add 25.0 mL
of Solvent, and heat under reflux while shaking by me-
chanical means for 15 min. Centrifuge, or pass through
a membrane filter having a 0.45-um or finer pore size.
Chromatographic system
(See Chromatography (621), System Suitability.)
 4580 Echinacea / Dietary Supplements USP 41

Mode: LC SPECIFIC TESTS

Detector: UV 330 nm
Column: 4.6-mm x 25-cm; 5-um packing L1
Column temperature: 35°
Flow rate: 1.5 mL/min
Injection volume: 5 wL
System suitability
Samples: Standard solution A and Standard solution C.
[Note—Echinacoside peak may be resolved in two
components.]
Suitability requirements
Chromatogram similarity: The chromatogram of
Standard solution A is similar to the Reference Chro-
matogram for total phenols provided with USP Pow-
dered Echinacea pallida Extract RS.
Capacity factor (k’): NLT 3.0 for the echinacoside
peak, Standard solution C
Tailing factor: NMT 2.0 for the echinacoside peak,
Standard solution C
Relative standard deviation: NMT 2.5% for the
echinacoside peaks in repeated injections, Standard
solution C
Analysis
Samples: Sample solution, Standard solution A, Stan-
dard solution B, Standard solution C, and Standard solu-
tion D
Identify the relevant analytes in the chromatogram from
the Sample solution by comparison with the chromato-
gram from Standard solution A. Measure the areas for
the relevant peaks.
Separately calculate the percentage of caftaric acid
(EvaHi20s), chicoric acid (Cz2HisO12), chlorogenic acid
(CisHigOs), and echinacoside (C3sH46O20) in the portion
of Powdered Extract taken:
Result = (ru/rs) x (Cs/Cu) x F x 100
ry = peak response for the relevant analyte from
the Sample solution
rs = peak response for chlorogenic acid or both
echinacoside components from the
corresponding Standard solution
Gs = concentration of chlorogenic acid or
echinacoside in the corresponding Standard
solution (mg/mL)
Cu = concentration of Powdered Extract in the
Sample solution (mg/mL)
F = response factor: chicoric acid, 0.695; caftaric
acid, 0.881; chlorogenic acid, 1.000; relative
to chlorogenic acid; and echinacoside
components, 1.000
Calculate the percentage of total phenols in the portion
DS Monographs
e LOSS ON DRYING (731)
Sample: 1g
Analysis: Dry the Sample at 105° for 2 h.
Acceptance criteria: NMT 5.0%
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in tight, light-resistant
containers, and store in a cool place.
e LABELING: The label states the Latin binomial and, follow-
ing the official name, the parts of the plant from which
the article was prepared. The label bears a statement in-
dicating that Echinacea pallida may cause rare allergic re-
actions, rashes, or aggravate asthma. It meets the re-
quirements for Botanical Extracts (565), Labeling.
e USP REFERENCE STANDARDS (11)
USP Caftaric Acid RS
USP Chicoric Acid RS
USP Chlorogenic Acid RS
USP Powdered Echinacea pallida Extract RS
USP Echinacoside RS
USP B-Sitosterol RS
Echinaceapurpurea Aerial Parts
DEFINITION
Echinacea purpurea Aerial Parts consists of the aerial parts of
Echinacea purpurea (L.) Moench (Fam. Asteraceae). It is
harvested during the flowering stage. It contains NLT
1.0% of the sum of caftaric acid (Ci3Hi20¢) and chicoric
acid (C22HigO12), and NLT 0.01% of dodecatetraenoic acid
isobutylamides (C;sH2sNO), calculated on the dried basis.
IDENTIFICATION
e A. THIN-LAYER CHROMATOGRAPHY
Presence of chicoric acid and absence of echinacoside
Standard solution A: 0.2 mg/mL of USP Echinacoside
RS in methanol
Standard solution B: 0.2 mg/mL of USP Caftaric Acid
RS, 0.1 mg of USP Chlorogenic Acid RS, and 0.2 mg/mL
of USP Chicoric Acid RS in methanol
Standard solution C: 20 mg/mL of USP Powdered Echi-
nacea purpurea Extract RS in methanol. Shake to dis-
perse, sonicate for 5 min, and centrifuge. Use the
supernatant.
Sample solution: Transfer 1 g of finely pulverized Echi-
nacea purpurea Aerial Parts to a centrifuge tube, add
10 mL of methanol, mix well, and sonicate for 10 min.
Centrifuge, and use the supernatant.

of Powdered Extract taken by adding the individual Chromatographic system

percentages calculated. (See Chromatography (621), System Suitability.)
Acceptance criteria: NLT 4.0% and NMT 5.0% of total Adsorbent: Chromatographic silica gel mixture with
phenols on the dried basis an average particle size of 5 um (HPTLC plates)
aie volume: 5 ul Standard solution C and
CONTAMINANTS ample solution, and 2 wL Standard solution A and Stan-
o ELEMENTAL IMPURITIES—PROCEDURES (233) dard solution B as 8-mm bands
Acceptance criteria Relative humidity: Condition the plate to a relative
Arsenic: NMT 1.0 yg/g humidity of about 33% using a suitable device.
Cadmium: NMT 0.5 ug/g Developing solvent system: A mixture of ethyl ace-
Lead: NMT 5.0 ug/g tate, methylethyl ketone, water, and formic acid
Mercury: NMT 1.0 ug/g G:3:121)
e MICROBIAL ENUMERATION TESTS (2021): The total bacterial Developing distance: 6 cm
count does not exceed 104 cfu/g, and the total com- Derivatization reagent: 5 mg/mL of 2-aminoethy!
bined molds and yeasts count does not exceed 103 cfu/ diphenylborinate in ethy! acetate
Analysis
° ABSENCE OF SPECIFIED MICROORGANISMS (2022): Meets the Samples: Standard solution A, Standard solution B,
requirements of the tests for absence of Salmonella spe- Standard solution C, and Sample solution
cies and Escherichia coli Apply the Samples as bands to a suitable thin-layer
¢ OTHER REQUIREMENTS: It meets the requirements for Bo- chromatographic plate, and dry in air. Develop the
one Extracts (565), Residual Solvents and Pesticide chromatograms in a satutated chamber. Remove the
Residues. plate from the chamber, heat at 100° for 5 min, der-
ivatize the plate while still warm with Derivatization re-
USP 41
agent, dry in air, and examine under UV light at 366
nm.
eat suitability: Standard solution A shows one major
lue band in the lower third section of the chromato-
gram due to echinacoside. Standard solution B shows
two major blue bands at about the middle of the chro-
matogram due to caftaric acid (lower Ry) that are clearly
separated, and a blue band for chicoric acid in the up-
per third section of the chromatogram.
Acceptance criteria: The most prominent band in the
Sample solution chromatogram is a blue band in the
upper third section of the chromatogram at an R; corre-
sponding to the chicoric acid band in the chromato-
gram of Standard solution B and Standard solution C.
The second most prominent band in the Sample solu-
tion chromatogram is a blue band at about the middle
of the chromatogram due to caftaric acid, correspond-
ing to a band in the chromatogram of Standard solution
C. The Sore solution chromatogram does not exhibit
a bandat the Rr of echinacoside in Standard solution A
(difference from Echinacea pallida and Echinacea angus-
tifolia). The Sample solution ere ee exhibits mi-
nor blue bands corresponding to similar bands in the
chromatogram of Standard solution C. One of these
bands is due to chlorogenic acid at an R; corresponding
to chlorogenic acid in Standard solution B. The Sample
solution chromatogram exhibits a red band due to chlo-
rophyll close to the solvent front.
e B. The retention time of the major peak in the Sample
solution corresponds to that of the chicoric acid peak in
Standard solution A, and the second most prominent
peak corresponds to that of the caftaric acid peak in
Standard solution B. The Sample solution chromatogram
shows no peak or a very minor peak at the retention
time corresponding to the echinacoside peak in the Stan-
dard solution C chromatogram, all peaks as obtained in
the test for Content of Chicoric Acid and Caftaric Acid.
e C. The retention times for the relevant peaks of the Sam-
ple solution, mainly due to dodecatetraenoic isobutyl am-
ides, correspond to those of Standard solution A, as ob-
tained in the test for Content of Dodecatetraenoic
Isobutylamides.
COMPOSITION
© CONTENT OF CHICORIC ACID AND CAFTARIC ACID
Solution A: Phosphoric acid (0.1 in 100) in water
Solution B: Acetonitrile
Mobile phase: See Table 7.
Table 1
Time Solution A Solution B
(min) (%) (%)
0 90 10
13 78 22
14 60 40
17.5 60 40
18 90 10
30 90 10
Solvent: Alcohol and water (7:3)
Standard solution A: 30 g/mL of USP Chicoric Acid
RS in Solvent
Standard solution B: 20 g/mL of USP Caftaric Acid RS
in Solvent
Standard solution C: 20 g/mL of USP Echinacoside RS
in Solvent
Sample solution: Transfer about 125 mg, accurately
weighed, of finely powdered Echinacea purpurea Aerial
Parts (capable of passing through a 40-mesh sieve) to a
round-bottom flask eau ped with a condenser. Add
25.0 mL of Solvent, and heat under reflux while shaking
by mechanical means for 15 min. Centrifuge, or pass
Dietary Supplements / Echinacea 4581
through a membrane filter of 0.45-1m or finer pore
size.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 330 nm
Column: 4.6-mm x 25-cm; 5-um packing L1
Column temperature: 35°
Flow rate: 1.5 mL/min
Injection size: 5 uL
System suitability
Samples: Standard solution A
Suitability requirements
Relative standard deviation: NMT 2.0% for the
chicoric acid peak in Standard solution A
Analysis
Samples: Standard solution A, Standard solution B,
Standard solution C, and Sample solution
Separately calculate the percentages of caftaric acid
(Ci3Hi2O0s) and chicoric acid (C22HisO12) in the portion
of Echinacea purpurea Aerial Parts taken:
Result = (ru/rs) x Cs x (V/W) x 100
ty = peak area of the relevant analyte from the
Sample solution
rs = peak area of the relevant analyte from the
corresponding Standard solution
Cs = concentration of the relevant analyte in the
corresponding Standard solution (mg/mL)
V = final volume of the Sample solution (mL)
Ww = weight of Echinacea purpurea Aerial Parts taken
to prepare the Sample solution (mg)
Calculate the percentage of the sum of chicoric acid
and caftaric acid in the portion of Echinacea purpurea
Aerial Parts taken by adding the individual percentages
calculated.
Acceptance criteria: NLT 1.0% on the dried basis
¢ CONTENT OF DODECATETRAENOIC ACID ISOBUTYLAMIDES
Mobile phase: Acetonitrile and water (55:45)
Standard solution A: 5 mg/mL of USP Powdered Echi-
nacea purpurea Extract RS in methanol. Dissolve using
sonication and shaking for 10 min. After dilution, pass
through a membrane filter of 0.45-4m or finer pore
size.
Standard solution B: 10 g/mL of USP 2£,4E-Hexadie-
noic Acid lsobutylamide RS in methanol
Sample solution: Transfer about 2.5 g of finely pow-
dered Echinacea purpurea Aerial Parts (capable of pass-
ing through a 40-mesh sieve), accurately weighed, into
a round-bottom flask. Add 80 mL of methanol, and re-
flux for 30 min. Cool to room temperature, and filter
sydesbouo=: sa
into a 100-mL volumetric flask, using small portions of
methanol to rinse the flask and the filter. Dilute with
methanol to volume. Pass through a membrane filter of
0.45-um or finer pore size.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 254 nm
Column: 4.6-mm x 25-cm; 5-um packing L1
Column temperature: 30°
Flow rate: 1.5 mL/min
Injection size: 25 pL
System suitability
Samples: Standard solution A and Standard solution B
Suitability requirements
Chromatogram similarity: The chromatogram from
Standard solution A is similar to the Reference Chro-
matogram for alkamides provided with USP Pow-
dered Echinacea purpurea Extract RS.
Resolution: NLT 1.0 between dodecatetraenoic acid
isobutylamide peaks, Standard solution A
Tailing factor: NMT 2.0 for 2£,4E-hexadienoic acid
isobutylamide, Standard solution B
 4582 Echinacea / Dietary Supplements
Relative standard deviation: NMT 2.5% for the 2E,
4E-hexadienoic acid isobutylamide peak in repeated
injections, Standard solution B
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Identify the peaks of the two isomers of dodecatetra-
enoic acid isobutylamides in the chromatogram from
the Sample solution by comparison with the chromato-
gram from Standard solution A. Measure the areas for
the relevant peaks.
Calculate the percentage of dodecatetraenoic acid iso-
butylamides in the portion of Echinacea purpurea Aerial
Parts taken:
Result = (ru/rs) x Cs x (V/W) x Fx 100
ru = sum of the peak areas of the relevant analytes
from the Sample solution
fs = peak area of 2£,4E-hexadienoic acid
isobutylamide from Standard solution B
Cs = concentration of USP 2£,4£-Hexadienoic Acid
lsobutylamide RS in Standard solution B
(mg/mL)
final volume of the Sample solution (mL)
oil
weight of Echinacea purpurea Aerial Parts taken
to prepare the Sample solution (mg)
response factor to convert 2E,4£-hexadienoic
a
acid isobutylamide into dodecatetraenoic
acid isobutylamides, 1.353
Acceptance criteria: NLT 0.01% of dodecatetraenoic
acid isobutylamides on the dried basis
CONTAMINANTS
o ELEMENTAL IMPURITIES—PROCEDURES (233)
Acceptance criteria
Arsenic: NMT 1.0 pg/g
Cadmium: NMT 0.5 ug/g
Lead: NMT 5.0 g/g
Mercury: NMT 1.0 ug/g
e ARTICLES OF BOTANICAL ORIGIN, General Method for Pesti-
cides Residues Analysis (S61): Meet the requirements
e MICROBIAL ENUMERATION TESTS (2021): The total aerobic
microbial count does not exceed 105 cfu/g, the total
combined molds and yeasts count does not exceed 103
cfu/g, and the enterobacterial count does not exceed 103
cfu/g.
e AnsENeE OF SPECIFIED MICROORGANISMS (2022): It meets
the requirements of the tests for absence of Salmonella
species and Escherichia coli.
SPECIFIC TESTS
DS Monographs
e BOTANIC CHARACTERISTICS
Macroscopic: The herb is an erect, coarse, rough-hairy
perennial, usual up to 90 cm tall, rarely up to 180 cm.
The leaves are alternate and simple; the lowermost
leaves are slender, long, and petioled, ovate to broadly
lanceolate, mostly penta-nerved, acute or acuminated
at the apex, abruptly narrowed or rarely cordate at the
base, usually sharply dentate, and 7-20 cm long and
2.5-7.5 cm wide; the petioles are mostly winged at the
summit. The upper leaves are narrower, often almost
entirely sessile, lanceolate or ovate lanceolate, and usu-
ally with 3 veins.
The flower heads are radiate, up to 15 cm across, soli-
tary or few, and long-peduncled, with 12-20 rays,
purple, crimson, or rarely pale; the bristle disks are of-
ten orange, 3.5-7.5 cm long; the involucre is de-
pressed-hemispheric; the bracts are lanceolate, spread-
USP 41
ing or appressed, imbricated in 2-4 series, and hairy
on the outer surface with ciliate margins; the recepta-
cle is conical, the scales of the receptacle stiff, spines-
cent, and conspicuously longer than the disc flowers;
the chaff is carinate and cuspidate; the achenes are
3-4 mm in length, tetrasided, obypyramidal, and
thick; the pappus has a short, dentate crown.
Microscopic
Leaf: The leaf has a thickness of 200-350 tm, with an
epidermis 9-13 wm thick, largely without chloroplasts;
the stomata are 28-35 um, abundant on the ventral
surface and fewer on the dorsal surface; the mesophyll
is clearly divided into palisade parenchyma and
sponge parenchyma. The palisade parenchyma is one
layer thick, with elongated cells 50-65 wm in length,
oriented at right angles to the leaf surface, containing
numerous chloroplasts. The sponge parenchyma is
150-250 um thick, with cells of irregular shape, and
has multiple cell layers, few chloroplasts, and large in-
tercellular spaces. The phloem bundles of the lateral
veins within the sponge parenchyma are bound by a
one-layer sheath of small parenchymous cells, with
vascular elements of the midrib surrounded by large-
celled parenchyma. The uniseriate trichomes are few in
the ventral surface, numerous on the dorsal surface,
typically tricelled, occasionally tetra- or pentacelled,
250-500 um in length, each arising from an epidermal
cell; the epidermal cell walls appear with moderate
thickening; the vessels are various, scalariform, with va-
triable reticulated width.
Petiole: The parenchyma appear without chloroplasts,
in several layers adjacent to a layer of collenchyma;
5-7 phloem bundles of small- to medium-sized vessels
are weakly lignified and embedded in the parenchyma
in the form of an arc; the wing ribs of the upper sur-
face of the slightly hollowed petiole are marginal.
Inflorescence: The epidermal cells of the ray florets
are square, 50 um, with a transparent, beaded cell
wall; various elements of the Asteraceous exhibit inflo-
rescence; numerous multicellular jointed trichomes of
the involucral bracts are 500-800 jum in length; tan-
gent sections of the paleae with numerous fiber
undles are 10-15 um in diameter and 100-150 pm
in length; cell walls are thin. The epidermis of ray flo-
rets is reddish to violet; the epidermal cells from the
end of the corolla form rounded papillae; a stigma of
papillary cells is present; Asteraceous pollen grains are
20-30 um and spherical with a warty exine.
Calcium oxalate is negative; crystals of inulin and
starch granules are rare.
e ARTICLES OF BOTANICAL ORIGIN, Foreign Organic Matter
(561): NMT 3.0%
Loss ON DRYING (731)
Sample: 1g of the powdered plant material
Analysis: Dry the Sample.
Acceptance criteria: NMT 12%
NMT
ARTICLES OF BOTANICAL ORIGIN, Total Ash (561):
10.0%, determined on 3g
ARTICLES OF BOTANICAL ORIGIN, Acid-/nsoluble Ash (561):
NMT 2.5%
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Store in tight, light-resistant
containers at controlled room temperature.
e LABELING: The label states the Latin binomial and, follow-
ing the official name, the parts of the plant contained in
the article.
USP 41
e@ USP REFERENCE STANDARDS (11)
USP Chlorogenic Acid RS
USP Caftaric Acid RS
USP Chicoric Acid RS
USP Echinacoside RS
USP 2E,4E-Hexadienoic Acid Isobutylamide RS
USP Powdered Echinacea purpurea Extract RS
Echinacea purpurea Root
DEFINITION
Echinacea purpurea Root consists of the dried rhizome and
roots of Echinacea purpurea (L.) Moench (Fam. Astera-
ceae). It is harvested in the fall after three or more years
of growth. It contains NLT 0.5% of total phenols, calcu-
lated on the dried basis as the sum of caftaric acid
(CisHi20s), chicoric acid (C22HisO12), and chlorogenic acid
(CisHigO¢). It contains NLT 0.025% of alkamides calcu-
lated as dodecatetraenoic acid isobutylamides (CisH2sNO).
IDENTIFICATION
¢ A. THIN-LAYER CHROMATOGRAPHY
Presence of chicoric acid and absence of echinacoside
Standard solution A: 0.2 mg/mL of USP Echinacoside
RS in methanol
Standard solution B: 0.2 mg/mL of USP Caftaric Acid
RS, 0.1 mg/mL of USP Chlorogenic Acid RS, and
0.2 mg/mL of USP Chicoric Acid RS in methanol
Standard solution C: 20 mg/mL of USP Powdered
Echinacea purpurea Extract RS in methanol. Shake to
disperse, sonicate for 5 min, and centrifuge. Use the
supernatant.
Sample solution: Transfer 1 g of finely pulverized Echi-
nacea purpurea Root to a centrifuge tube, add 10 mL
of methanol, mix well, and sonicate for 10 min. Cen-
trifuge, and use the supernatant.
Chromatographic system
(See woe yy (621), Thin-Layer Chromato-
raphy.
Adsorbent: Chromatographic silica gel mixture with
an average particle size of 5 um (HPTLC plates)
Application volume: 5 uL Standard solution C and
Sample solution, and 2 ul Standard solution A and
Standard solution B as 8-mm bands
Relative humidity: Condition the plate to a relative
humidity of about 33% using a suitable device.
Developing solvent system: A mixture of ethyl ace-
Ge Teeny ketone, water, and formic acid
Sis
Developing distance: 6 cm
Derivatization reagent: 5 mg/mL of 2-aminoethyl
diphenylborinate in ethyl! acetate
Analysis
Samples: Standard solution A, Standard solution B,
Standard solution C, and Sample solution
Apply the Samples as bands to a suitable thin-layer
chromatographic plate, and dry in air. Develop the
chromatograms in a saturated chamber. Remove the
plate from the chamber, heat at 100° for 5 min, der-
ivatize the plate while still warm with Derivatization
reagent, dry in air, and examine under UV light at
366 nm.
System suitability: Standard solution A shows one ma-
jor blue band in the lower third of the chromatogram
due to echinacoside. Standard solution B shows two
major blue bands at about the middle of the chromat-
ogram due to caftaric acid (lower Rp) and chlorogenic
acid (higher R-) that are clearly separated, and a blue
band for chicoric acid in the upper third section of the
chromatogram.
Acceptance criteria: The most prominent band in the
Sample solution chromatogram is a blue band in the
Dietary Supplements / Echinacea 4583
upper third section of the chromatogram at an R; cor-
responding to the chicoric acid band in the chromato-
grams of Standard solution B and Standard solution C
(less prominent in Echinacea pallida and absent or al-
most absent in Echinacea angustifolia). The second
most prominent band in the Sample solution chromat-
ogram is a blue band at about the middle of the chro-
matogram due to caftaric acid, corresponding to a
band in the chromatogram of Standard solution C (ab-
sent in Echinacea angustifolia and a minor band in Echi-
nacea pallida). The Sample solution chromatogram does
not exhibit a band at the Rr of echinacoside in Stan-
dard solution A (difference from Echinacea pallida and
Echinacea angustifolia). The Sample solution chromato-
gram may exhibit minor blue bands corresponding to
similar bands in the chromatogram of Standard solu-
tion C. One of these is due to chlorogenic acid at an R-
corresponding to chlorogenic acid in the Standard so-
lution B.
e B. THIN-LAYER CHROMATOGRAPHY
Presence of alkylamides
Standard solution A: 0.2 mg/mL of USP B-Sitosterol
RS in methanol
Standard solution B: 100 mg/mL of USP Powdered
Echinacea purpurea Extract RS in dichloromethane.
Shake to disperse, sonicate for 5 min, and centrifuge.
Use the supernatant.
Sample solution: Transfer 1 g of finely pulverized Echi-
nacea purpurea Root to a centrifuge tube, add 10 mL
of dichloromethane, mix well, and sonicate for 10
min. Centrifuge, and use the supernatant.
Chromatographic system
(See 7 eee y (621), Thin-Layer Chromato-
raphy.
Adsarbend Chromatographic silica gel with an aver-
age particle size of 5 um (HPTLC plates)
Application volume: 5 wl Standard solution B and
ample solution, and 2 uL Standard solution A as
8-mm bands
Relative humidity: Condition the plate to a relative
humidity of about 33% using a suitable device.
Developing solvent system: A mixture of toluene,
ethyl acetate, cyclohexane, and formic acid
(8: 2: 1: 0.3)
Developing distance: 6 cm
Derivatization reagent: Place 85 mL of methanol in
a 100-mL glass bottle, and cool it down in a
water-ice cubes-salt bath or in a freezer. To the ice-
cold methanol, slowly and carefully add 10 mL of
acetic acid and 5 mL of sulfuric acid, and mix well.
Allow the mixture to cool to room temperature, then
add 0.5 mL of p-anisaldehyde. Lo]
“
Analysis
Samples: Standard solution A, Standard solution B, and =
Sample solution }
J
Apply the Samples as bands to a suitable thin-layer i)
chromatographic plate, and dry in air. Develop the aBt
chromatograms in a saturated chamber. Remove the i)
plate from the chamber, dry in air, derivatize with ao}
s
Derivatization reagent, heat at 100° for 3-5 min, set al
aside to cool, and examine under visible light.
System suitability: The chromatogram of Standard so-
lution B exhibits the most prominent band as a pinkish
violet band at about the middle of the chromatogram,
and just below this pinkish band, a violet band at a
lower Rr similar in position and color to the B-sitosterol
band in the chromatograms of Standard solution A.
These two bands are clearly separated from each
other. The chromatogram of Standard solution B also
shows a broad pink violet band close to the solvent
front.
Acceptance criteria: The most prominent band of the
Sample solution chromatogram is a pinkish violet band
at about the middle of the chromatogram similar in
position and color to a band in the Standard solution B
 4584 Echinacea / Dietary Supplements USP 41

chromatogram (much less prominent in Echinacea Separately calculate the percentage of caftaric acid
angustifolia and Echinacea pallida), a violet band corre- (Ci3H12O0s), chicoric acid (Cz2HisO12), and chlorogenic
sponding to B-sitosterol band in the chromatograms of acid (CisHigOs) in the portion of Echinacea purpurea
Standard solution A and Standard solution B, and a Root taken:
broad pink violet band close to the solvent front simi-
lar in position and color to the band in the chromato- Result = (ru/rs) x Cs x (V/W) x 100
gram of Standard solution B. The Sample solution chro-
matogram does not exhibit a yellow band below the ry = peak area of the relevant analyte from the
B-sitosterol band (difference from Echinacea angus- Sample solution
tifolia) or a prominent violet band at about two thirds rs = peak area of the relevant analyte from the
of the chromatogram (difference from Echinacea corresponding Standard solution
pallida). Cs = concentration of the relevant analyte in the
e C. The retention time of the major peak in the Sample corresponding Standard solution (mg/mL)
solution corresponds to that of the chicoric acid peak in Vv = volume of the Sample solution (mL)
Standard solution A, and the second most prominent w = weight of Echinacea purpurea taken to prepare
peak corresponds to that of the caftaric acid peak in the Sample solution (mg)
Standard solution B. The Sample solution chromatogram Calculate the percentage of total phenols in the portion
shows no or a very minor peak at the retention time of Echinacea purpurea Root taken by adding the
corresponding to the echinacoside peak in the Standard individual percentages calculated.
solution D chromatogram, all peaks as obtained in the Acceptance criteria: NLT 0.5% of total phenols on the
test for Content of Total Phenols. dried basis
¢ CONTENT OF ALKAMIDES
COMPOSITION Mobile phase: Acetonitrile and water (55:45)
e@ CONTENT OF TOTAL PHENOLS Standard solution A: 5 mg/mL of USP Powdered Echi-
Solution A: Phosphoric acid (0.1 in 100) in water nacea purpurea Extract RS in methanol. Dissolve using
Solution B: Acetonitrile sonication and shaking for 10 min. After dilution, pass
Mobile phase: See Table 1. through a membrane filter having a 0.45-um or finer
pore size.
Table 1 Standard solution B: 10 g/mL of USP 2E,4£-Hexadie-
noic Acid Isobutylamide RS in methanol
Time Solution A Solution B Sample solution: Transfer about 2.5 g of finely pow-
(min) (%) (%) dered Echinacea purpurea Root (capable of passing
0 90 10 through a 40-mesh sieve) into a round-bottom flask.
13 78 22) Add 80 mL of methanol, and reflux for 30 min. Cool to
14 60 40 room temperature, and filter into a 100-mL volumetric
17.5 60 40
flask using small portions of methanol to rinse the flask
and the filter. Dilute with methanol to volume. Pass
18 90 10 through a membrane filter having a 0.45-um or finer
30 90 10 pore size.
Chromatographic system
Solvent: Alcohol and water (7:3) (See Chromatography (621), System Suitability.)
Standard solution A: 30 g/mL of USP Chicoric Acid Mode: LC
RS in Solvent Detector: UV 254 nm
Standard solution B: 20 g/mL of USP Caftaric Acid RS Column: 4.6-mm x 25-cm; 5-um packing L1
in Solvent Column temperature: 30°
Standard solution C: 20 g/mL of USP Chlorogenic Flow rate: 1.5 mL/min
Acid RS in Solvent Injection volume: 25 uL
Standard solution D: 20 g/mL of USP Echinacoside RS System suitability
in Solvent Samples: Standard solution A and Standard solution B
Sample solution: Transfer 125 mg of finely powdered Suitability requirements
Echinacea purpurea Root (capable of passing through a Chromatogram similarity: The chromatogram from
40-mesh sieve) to a round-bottom flask equipped with
DS Monographs

Standard solution A is similar to the Reference Chro-

a condenser. Add 25.0 mL of Solvent, and heat under matogram for alkamides provided with USP Pow-
reflux while shaking by mechanical means for 15 min. dered Echinacea purpurea Extract RS.
Centrifuge, or pass through a membrane filter of 0.45- Resolution: NLT 1.0 between dodecatetraenoic acid
uum or finer pore size. isobutylamide peaks, Standard solution A
Chromatographic system Tailing factor: NMT 2.0 for the 2£,4E-hexadienoic
(See Chromatography (621), System Suitability.) acid isobutylamide peak, Standard solution B
Mode: LC Relative standard deviation: NMT 2.5% for the 2E,
Detector: UV 330 nm 4E-hexadienoic acid isobutylamide peak in repeated
Column: 4.6-mm x 25-cm; 5-m packing L1 injections, Standard solution B
Column temperature: 35° Analysis
Flow rate: 1.5 mL/min Samples: Standard solution A, Standard solution B, and
Injection volume: 5 wl Sample solution
System suitability Identify the peaks of the 10 major alkamides in the
Samples: Standard solution A chromatogra from the Sample solution by compari-
Suitability requirements son with the chromatogram from Standard solution A.
Relative standard deviation: NMT 2% for the Measure the areas for the relevant peaks.
chicoric acid peak in repeated injections, Standard so- Calculate the percentage of alkamides in the portion of
lution A Echinacea purpurea Root taken:
Analysis
Samples: Standard solution A, Standard solution B, Result = (ru/rs) x Cs x (V/W) x Fx 100
Standard solution C, Standard solution D, and Sample
solution ru = sum of the peak areas of the relevant analytes
from the Sample solution
USP 41
Is = peak area of 2E,4E-hexadienoic acid
isobutylamide from Standard solution B
Cs = concentration of USP 2£,4E-Hexadienoic Acid
Isobutylamide RS in Standard solution B
(mg/mL)
Vv = final volume of the Sample solution (mL)
w = weight of Echinacea purpurea root taken to
prepare the Sample solution (mg)
F = response factor to convert 2E,4E-hexadienoic
acid isobutylamide into dodecatetraenoic
acid isobutylamide, 1.353
Acceptance criteria: NLT 0.025% on the dried basis
CONTAMINANTS
e ELEMENTAL IMPURITIES—PROCEDURES (233)
Acceptance criteria
Arsenic: NMT 1.0 ug/g
Cadmium: NMT 0.5 ug/g
Lead: NMT 5.0 ug/g
Mercury: NMT 1.0 ug/g
© ARTICLES OF BOTANICAL ORIGIN, Pesticide Residues (561):
Meets the requirements
SPECIFIC TESTS
¢ BOTANIC CHARACTERISTICS
Macroscopic: The roots are cylindrical and irregularly
branched. The outer surface is dark brown and longitu-
dinally striated; fractures are short and tough. Trans-
verse sections show a thin periderm and yellowish xy-
lem with distinct rays. In older roots, the pith is spongy
with a brownish center surrounded by yellow.
Microscopic: Rhizomes and roots in transverse section
show a thin outer bark separated from a wide xylem by
a brown vascular cambium. The cork is composed of
several rows of thin-walled cells containing brown pig-
ment. Schizogenous resin canals are present in the cor-
tex. The rhizome contains bast fibers and stone cells.
The xylem, with distinct rays, contains tracheary ele-
ments composed of reticulated vessels and tracheids
(about 80 x 30 um) with bordered pits and slanted end
walls. Vessels and tracheids are surrounded by thick-
walled parenchyma and fibers; fibers are elongated with
narrow lumens and funnel-shaped ends (20-40 um
wide). Polygonal sclereids (about 50 um in diameter)
are also present. Xylem fibers have minimal or no
phytomelanin deposits (unlike Echinacea angustifolia and
Echinacea pallida). A melanogenic layer is present be-
tween adjacent xylem parenchyma cell walls. The rhi-
zome, with pith, is composed of pitted parenchyma
cells containing inulin crystals. Starch is minimal to ab-
sent, and calcium oxalate crystals are absent.
ARTICLES OF BOTANICAL ORIGIN, Foreign Organic Matter
(561): NMT 3.0%
Loss ON DRYING (731)
Analysis: Dry a sample at 105° for 2 h.
Acceptance criteria: NMT 10.0%
ane OF BOTANICAL ORIGIN, Total Ash (561): NMT
UP
ARTICLES OF BOTANICAL ORIGIN, Acid-Insoluble Ash (561):
NMT 4.0%
ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Store in well-closed, light-resis-
tant containers.
e LABELING: The label states the Latin binomial and, follow-
ing the official name, the parts of the plant contained in
the article.
Dietary Supplements / Echinacea 4585
e USP REFERENCE STANDARDS (11)
USP Caftaric Acid RS
USP Chicoric Acid RS
USP Chlorogenic Acid RS
USP Powdered Echinacea purpurea Extract RS
USP Echinacoside RS
USP 2E,4E-Hexadienoic Acid Isobutylamide RS
USP B-Sitosterol RS
Powdered Echinacea purpurea
DEFINITION
Powdered Echinacea purpurea consists of the dried rhizome
and roots of Echinacea purpurea (L.) Moench (Fam. Astera-
ceae), harvested in the fall after three or more years of
growth, and reduced to powder. It contains NLT 0.5% of
total phenols, calculated on the dried basis as the sum of
caftaric acid (Ci3H120s), chicoric acid (C22HigO12), and
chlorogenic acid (Ci6HigOs). It contains NLT 0.025% of
alkamides calculated as dodecatetraenoic acid isobutyl-
amides (CisH2sNO).
IDENTIFICATION
e A. THIN-LAYER CHROMATOGRAPHY
Presence of chicoric acid and absence of echinacoside
Standard solution A: 0.2 mg/mL of USP Echinacoside
RS in methanol
Standard solution B: 0.2 mg/mL of USP Caftaric Acid
RS, 0.1 mg/mL of USP Chlorogenic Acid RS, and
0.2 mg/mL of USP Chicoric Acid RS in methanol
Standard solution C: 20 mg/mL of USP Powdered
Echinacea purpurea Extract RS in methanol. Shake to
disperse, sonicate for 5 min, and centrifuge. Use the
supernatant.
Sample solution: Transfer 1 g of Powdered Echinacea
purpurea to a centrifuge tube, add 10 mL of methanol,
mix well, and sonicate for 10 min. Centrifuge, and use
the supernatant.
Chromatographic system
(See ee y (621), Thin-Layer Chromato-
graphy.)
Adsorbent: Chromatographic silica gel mixture with
an average particle size of 5 um (HPTLC plates)
Application volume: 5 uL Standard solution C and
Sample solution, and 2 wl Standard solution A and
Standard solution B as 8-mm bands
Relative humidity: Condition the plate toa relative
humidity of about 33% using a suitable device.
sydesBbouow- sa
Developing solvent system: A mixture of ethyl ace-
tate, methylethyl ketone, water, and formic acid
G33:121)
Developing distance: 6 cm
Derivatization reagent: 5 mg/mL of 2-aminoethyl
diphenylborinate in ethyl acetate
Analysis
Samples: Standard solution A, Standard solution B,
Standard solution C, and Sample solution
Apply the Samples as bands to a suitable thin-layer
chromatographic plate, and dry in air. Develop the
chromatograms in a saturated chamber. Remove the
late from the chamber, heat at 100° for 5 min, der-
ivatize the plate while still warm with Derivatization
reagent, dry in air, and examine under UV light at
366 nm.
System suitability: Standard solution A shows one ma-
jor blue band in the lower third section of the chro-
matogram due to echinacoside. Standard solution B
shows two major blue bands at about the middle of
the chromatogram due to caftaric acid (lower R-) and
enipregetie acid (higher R,) that are clearly separated,
and a blue band for chicoric acid in the upper third
section of the chromatogram.
 4586 Echinacea / Dietary Supplements USP 41

Acceptance criteria: The most prominent band in the Acceptance criteria: The most prominent band of the
Sample solution chromatogram is a blue band in the Sample solution chromatogram is a pinkish violet band
upper third section of the chromatogram at an R; cor- at about the middle of the chromatogram similar in
responding to the chicoric acid band in the chromato- position and color to a band in the Standard solution B
rams of Standard solution B and Standard solution C chromatogram (much less prominent in Echinacea
ee prominent in Echinacea pallida and absent or al- angustifolia and Echinacea pallida), a violet band corre-
most absent in Echinacea angustifolia). The second sponding to B-sitosterol band in the chromatograms of
most prominent band in the Sample solution chromat- Standard solution A and Standard solution B, and a
ogram is a blue band at about the middle of the chro- broad pink violet band close to the solvent front simi-
matogram due to caftaric acid, corresponding to a lar in position and color to the band in the chromato-
band in the chromatogram of Standard solution C (ab- gram of Standard solution B. The Sample solution chro-
sent in Echinacea angustifolia and a minor band in Echi- matogram does not exhibit a yellow band below the
nacea pallida). The Sample solution chromatogram does B-sitosterol band (difference from Echinacea angus-
not exhibit a band at the Rr of echinacoside in Stan- tifolia) or a prominent violet band at about two thirds
dard solution A (difference from Echinacea pallida and of the chromatogram (difference from Echinacea
Echinacea angustifolia). The Sample solution chromato- pallida).
gram may exhibit minor blue bands corresponding to e C. The retention time of the major peak in the Sample
similar bands in the chromatogram of Standard solu- Solution corresponds to that of the chicoric acid peak in
tion C. One of these is due to chlorogenic acid at an Rr Standard solution A, and the second most prominent
corresponding to chlorogenic acid in the Standard so- peak corresponds to that of the caftaric acid peak in
lution B. Standard solution B. The Sample solution chromatogram
e B. THIN-LAYER CHROMATOGRAPHY shows no or a very minor peak at the retention time
Presence of alkylamides corresponding to the echinacoside peak in the Standard
Standard solution A: 0.2 mg/mL of USP B-Sitosterol solution D chromatogram. All peaks as obtained in the
RS in methanol test for Content of Total Phenols.
Standard solution B: 100 mg/mL of USP Powdered
Echinacea purpurea Extract RS in dichloromethane. COMPOSITION
Shake to disperse, sonicate for 5 min, and centrifuge. © CONTENT OF TOTAL PHENOLS
Use the supernatant. Solution A: Phosphoric acid (0.1 in 100) in water
Sample solution: Transfer 1g of Powdered Echinacea Solution B: Acetonitrile
purpurea to a centrifuge tube, add 10 mL of dichloro- Mobile phase: See Table 1.
methane, mix well, and sonicate for 10 min. Centri-
fuge, and use the supernatant. Table 1
Chromatographic system
Time Solution A Solution B
(See Chromatography (621), Thin-Layer Chromato-
raphy.) (min) (%) (%)
Aasonbenis Chromatographic silica gel with an aver- 0 90 10
age particle size of 5 um (HPTLC plates) 13 78 22
Application volume: 5 iL Standard solution B and 14 60 40
ample solution, and 2 wL Standard solution A as 17.5 60 40
8-mm bands 18 90 10
Relative humidity: Condition the plate toa relative
30 90 10
humidity of about 33% usinga suitable device.
Developing solvent system: A mixture of toluene, Solvent: Alcohol and water (7:3)
ethyl acetate, cyclohexane, and formic acid Standard solution A: 30 g/mL of USP Chicoric Acid
(8: 2: 1: 0.3) RS in Solvent
Developing distance: 6 cm Standard solution B: 20 g/mL of USP Caftaric Acid RS
Derivatization reagent: Place 85 mL of methanol in in Solvent
a 100-mL glass bottle and cool it down in a water-ice Standard solution C: 20 g/mL of USP Chlorogenic
cubes-salt bath or in a freezer. To the ice-cold metha- Acid RS in Solvent
nol, slowly and carefully add 10 mL of acetic acid and Standard solution D: 20 pg/mL of USP Echinacoside RS
DS Monographs

5 mL of sulfuric acid, and mix well. Allow the mixture in Solvent

to cool to room temperature, then add 0.5 mL of p-
anisaldehyde.
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Apply the Samples as bands to a suitable thin-layer
chromatographic plate, and dry in air. Condition the
plate to a relative humidity of about 33% using a
suitable device. Develop the chromatograms ina sat-
urated chamber. Remove the plate from the cham-
ber, dry in air, derivatize with Derivatization reagent,
heat at 100° for 3-5 min, set aside to cool, and ex-
amine under visible light.
System suitability: The chromatogram of Standard so-
lution B exhibits the most prominent band as a pinkish
violet band at about the middle of the chromatogram,
and just below this pinkish band, a violet band at a
lower Rr similar in position and color to the B-sitosterol
band in the chromatograms of Standard solution A.
These two bands are clearly separated from each
other. The chromatogram of Standard solution B also
shows a broad pink violet band close to the solvent
front.
Sample solution: Transfer about 125 mg of Powdered
Echinacea purpurea (capable of passing through a
40-mesh sieve), accurately weighed, to a round-bottom
flask equipped with a condenser. Add 25.0 mL of Sol-
vent, and heat under reflux while shaking by mechani-
cal means for 15 min. Centrifuge, or pass through a
membrane filter of 0.45-um or finer pore size.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode:
Detector: UV 330 nm
Column: 4.6-mm x 25-cm; 5-um packing L1
Column temperature: 35°
Flow rate: 1.5 mL/min
Injection volume: 5 uL
System suitability
Samples: Standard solution A and Standard solution B
Suitability requirements
Relative standard deviation: NMT 2% for chicoric
acid peak in repeated injections, Standard solution A
USP 41
Analysis
Samples: Standard solution A, Standard solution B,
Standard solution C, Standard solution D, and Sample
solution
Separately calculate the percentage of caftaric acid
(Ci3Hi20¢), chicoric acid (C22HisO12), and chlorogenic
acid (CisHigOs) in the portion of Powdered Echinacea
purpurea taken:
Result = (ru/rs) x Cs x (V/W) x 100
ru = peak area for the relevant analyte from the
Sample solution
rs = peak area of the relevant analyte from the
corresponding Standard solution
G = concentration of the relevant analyte in the
corresponding Standard solution (mg/mL)
Vv = volume of the Sample solution (mL)
Ww = weight of Powdered Echinacea purpurea used
to prepare the Sample solution (mg)
Calculate the percentage of total phenols in the portion
of Powdered Echinacea purpurea taken by adding the
individual percentages calculated.
Acceptance criteria: NLT 0.5% of total phenols on the
dried basis
© CONTENT OF ALKAMIDES
Mobile phase: Acetonitrile and water (55:45)
Standard solution A: 5 mg/mL of USP Powdered Echi-
nacea purpurea Extract RS in methanol. Dissolve using
sonication and shaking for 10 min. After dilution, pass
through a membrane filter having a 0.45-11m or finer
pore size,
Standard solution B: 10 g/mL of USP 2E,4E-Hexadie-
noic Acid Isobutylamide RS in methanol
Sample solution: Transfer about 2.5 g of Powdered
Echinacea purpurea (capable of passing through a
40-mesh sieve) into a round-bottom flask. Add 80 mL
of methanol, and reflux for 30 min. Cool to room tem-
perature, and filter into a 100-mL volumetric flask using
small portions of methanol to rinse the flask and the
filter. Dilute with methanol to volume. Pass through a
membrane filter having a 0.45-um or finer pore size.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 254 nm
Column: 4.6-mm x 25-cm; 5-11m packing L1
Column temperature: 30°
Flow rate: 1.5 mL/min
Injection volume: 25 pL
System suitability
Samples: Standard solution A and Standard solution B
Suitability requirements
Chromatogram similarity: The chromatogram from
Standard solution A is similar to the Reference Chro-
matogram for alkamides provided with USP Pow-
dered Echinacea purpurea Extract RS.
Resolution: NLT 1.0 between dodecatetraenoic acid
isobutylamide peaks, Standard solution A
Tailing factor: NMT 2.0 for 2E,4£-hexadienoic acid
isobutylamide peak, Standard solution B
Relative standard deviation: NMT 2.5% for the 2E,
4E-hexadienoic acid isobutylamide peak in repeated
injections, Standard solution B
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Identify the peaks of the 10 major alkamides in the
chromatogram from the Sample solution by compari-
son with the chromatogram from Standard solution A.
Calculate the percentage of alkamides in the portion of
Powdered Echinacea purpurea taken:
Result = (ru/rs) x Cs x (V/W) x Fx 100
Dietary Supplements / Echinacea 4587
tu = sum of the peak areas of the relevant analytes
from the Sample solution
ls = peak area of 2£,4£-hexadienoic acid
isobutylamide from Standard solution B
Cs = concentration of USP 2E,4£-Hexadienoic Acid
lsobutylamide RS in Standard solution B
(mg/mL)
Vv = volume of the Sample solution (mL)
w = weight of Powdered Echinacea purpurea used
to prepare the Sample solution (mg)
F = response factor for 2E,4E-hexadienoic acid
isobutylamide, 1.353
Acceptance criteria: NLT 0.025% on the dried basis
CONTAMINANTS
e ELEMENTAL IMPURITIES—PROCEDURES (233)
Acceptance criteria
Arsenic: NMT 1.0 ug/g
Cadmium: NMT 0.5 ug/g
Lead: NMT 5.0 ug/g
Mercury: NMT 1.0 ug/g
© ARTICLES OF BOTANICAL ORIGIN, General Procedure for Pesti-
cide Residues Analysis (561): Meets the requirements
SPECIFIC TESTS
e BOTANIC CHARACTERISTICS: Under a microscope, the fol-
lowing characteristics are observed: vessels (80 x 30 um)
with slanted end walls and spiral or pitted secondary
walls; rectangular cork cells (150 x 60 um) with brown
inclusions; rectangular parenchymatous cells (120 x 30
Lum), some pitted; elongatedfiber cells having a narrow
lumen with funnel-shaped end (20-40 um wide); polygo-
nal sclereids; a melanogenic layer of variable thickness,
interspersed between the cell walls of the parenchyma;
and lignified sclereids, vessels, and fibers. Starch is pres-
ent; calcium oxalate and inulin crystals are absent.
e Loss ON DRYING (731)
Analysis: Dry a sample at 105° for 2 h.
Acceptance criteria: NMT 10.0%
° Amat OF BOTANICAL ORIGIN, Total Ash (561): NMT
Zz
e ARTICLES OF BOTANICAL ORIGIN, Acid-Insoluble Ash (561):
NMT 4.0%
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in well-closed, light-
resistant containers.
e LABELING: The label states the Latin binomial and, follow-
ing the official name, the part of the plant from which
the article was derived.
e USP REFERENCE STANDARDS (11)
USP Caftaric Acid RS
leylU a!
USP Chicoric Acid RS
USP Chlorogenic Acid RS
USP Powdered Echinacea purpurea Extract RS
ExeTel-S]ole
USP Echinacoside RS
USP 2E,4E-Hexadienoic Acid Isobutylamide RS
USP B-Sitosterol RS
Powdered Echinacea purpurea Extract
DEFINITION
Powdered Echinacea purpurea Extract is prepared from dried
Echinacea purpurea Root, Echinacea purpurea Aerial Parts,
or a mixture of them, by extraction with hydroalcoholic
mixtures or other suitable solvents. The ratio of the start-
ing crude plant material to Powdered Extract is between
2:1 and 8:1. It contains NLT 4.0% of total phenols, calcu-
lated as the sum of caftaric acid (C)3H120¢), chicoric acid
(C22HigO12), and chlorogenic acid (Cis6HigOs), on the dried
basis. It contains NLT 0.025% of dodecatetraenoic acid
isobutylamides (CisH2sNO), calculated on the dried basis.
 4588 Echinacea / Dietary Supplements
IDENTIFICATION
e A, THIN-LAYER CHROMATOGRAPHY
Presence of chicoric acid and absence of echinacoside
Standard solution A: 0.2 mg/mL of USP Echinacoside
RS in methanol
Standard solution B: 0.2 mg/mL of USP Caftaric Acid
RS, 0.1 mg/mL of USP Chlorogenic Acid RS, and
0.2 mg/mL of USP Chicoric Acid RS in methanol
Standard solution C: 20 mg/mL of USP Powdered
Echinacea purpurea Extract RS in methanol. Shake to
disperse, sonicate for 5 min, and centrifuge. Use the
supernatant.
Sample solution: 20 mg/mL of Powdered Echinacea
purpurea Extract in methanol. Shake to disperse, soni-
cate for 5 min, and centrifuge. Use the supernatant.
Chromatographic system
(See Somaograp y (621), Thin-Layer Chromato-
raphy.
Aasorbant Chromatographic silica gel mixture with
an average particle size of 5 um (HPTLC plates)
Application volume: 5 ul Standard solution C and
Sample solution, and 2 wL Standard solution A and
Standard solution B as 8-mm bands
Relative humidity: Condition the plate to a relative
humidity of about 33% using a suitable device.
Developing solvent system: A mixture of ethyl ace-
as aa ketone, water, and formic acid
$:3:171
Developing distance: 6 cm
Derivatization reagent: 5 mg/mL of 2-aminoethyl
diphenylborinate in ethyl acetate
Analysis
Samples: Standard solution A, Standard solution B,
Standard solution C, and Sample solution
Apply the Samples as bands to a suitable thin-layer
chromatographic plate, and dry in air. Develop the
chromatograms in a saturated chamber. Remove the
plate from the chamber, heat at 100° for 5 min, der-
ivatize the plate while still warm with Derivatization
reagent, dry in air, and examine under UV light at
366 nm.
System suitability: Standard solution A shows one ma-
jor blue band in the lower third section of the chro-
matogram due to echinacoside. Standard solution B
shows two blue bands at about the middle of the
chromatogram due to caftaric acid (lower Rp) and
chlorogenic acid (higher R-) that are clearly separated,
and a blue band for chicoric acid in the upper third
section of the chromatogram.
Acceptance criteria: The most prominent band in the
Sample solution chromatogram is a blue band in the
DS Monographs
upper third section of the chromatogram at an Rr cor-
responding to the chicoric acid band in the chromato-
grams of Standard solution B and Standard solution C
(less prominent in Echinacea pallida and absent or al-
most absent in Echinacea angustifolia). The second
most prominent band in the Sample solution chromat-
ogram is a blue band at about the middle of the chro-
matogram due to caftaric acid, corresponding to a
band in the chromatograms of Standard solution B and
Standard solution C (absent in Echinacea angustifolia
and a minor band in Echinacea pallida). The Sample
solution chromatogram does not exhibit a band at the
R- of echinacoside in Standard solution A (difference
from Echinacea pallida and Echinacea angustifolia). The
Sample solution chromatogram may exhibit minor blue
bands corresponding to similar bands in the chromat-
ogram of Standard solution C. One of these is due to
chlorogenic acid at an R; corresponding to chlorogenic
acid in Standard solution B.
e B. THIN-LAYER CHROMATOGRAPHY
Presence of alkylamides
Standard solution A: 0.2 mg/mL of USP B-Sitosterol
RS in methanol
USP 41
Standard solution B: 100 mg/mL of USP Powdered
Echinacea purpurea Extract RS in dichloromethane.
Shake to disperse, sonicate for 5 min, and centrifuge.
Use the supernatant.
Sample solution: 100 mg/mL of Powdered Echinacea
purpurea Extract in dichloromethane. Shake to dis-
perse, sonicate for 5 min, and centrifuge. Use the
supernatant.
Chromatographic system
(See ee y (621), Thin-Layer Chromato-
graphy.
Adsorbent: Chromatographic silica gel with an aver-
age particle size of 5 um (HPTLC plates)
Application volume: 5 ul Standard solution B and
Sample solution, and 2 wL Standard solution A, as
8-mm bands
Relative humidity: Condition the plate toa relative
humidity of about 33% usinga suitable device.
Developing solvent system: A mixture of toluene,
ethyl acetate, cyclohexane, and formic acid
(8: 2: 1: 0.3)
Developing distance: 6 cm
Derivatization reagent: Place 85 mL of methanol in
a 100-mL glass bottle, and cool it down in a
water-ice cubes-salt bath or in a freezer. To the ice-
cold methanol, slowly and carefully add 10 mL of
acetic acid and 5 mL of sulfuric acid, and mix well.
Allow the mixture to cool to room temperature, then
add 0.5 mL of p-anisaldehyde.
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Apply the Samples as bands to a suitable thin-layer
chromatographic plate, and dry in air. Develop the
chromatograms in a saturated chamber. Remove the
plate from the chamber, dry in air, derivatize with
Derivatization reagent, heat at 100° for 3-5 min, set
aside to cool, and examine under visible light.
System suitability: The chromatogram of Standard so-
lution B exhibits the most prominent band as a pinkish
violet band at about the middle of the chromatogram,
and just below this pinkish band, a violet band at a
lower Rr similar in position and color to the B-sitosterol
band in the chromatograms of Standard solution A.
These two bands are clearly separated from each
other. The chromatogram of Standard solution B also
shows a broad pink violet band close to the solvent
front. The B-sitosterol band of the Standard solution B
chromatogram and the pinkish violet band underneath
are clearly separated from one another.
Acceptance criteria: The most prominent band of the
Sample solution chromatogram is a pinkish violet band
at about the middle of the chromatogram similar in
position and color to a band in the Standard solution B
chromatogram (much less prominent in Echinacea
angustifolia and Echinacea pallida), a violet band corre-
sponding to B-sitosterol band in the chromatograms of
Standard solution A and Standard solution B, and a
broad pink violet band close to the solvent front simi-
lar in position and color to the band in the chromato-
gram of Standard solution B. The Sample solution chro-
matogram does not exhibit a yellow band below the
B-sitosterol band (difference from Echinacea angus-
tifolia) or a prominent violet band at about two-thirds
of the chromatogram (difference from Echinacea
pallida).
e C. The retention time of the major peak in the Sample
solution corresponds to that of the chicoric acid peak in
Standard solution A, and the second most prominent
peak corresponds to that of the caftaric acid peak in
Standard solution B. The Sample solution chromatogram
shows no or a very minor peak at the retention time
corresponding to the echinacoside peak in the Standard
solution D chromatogram, all peaks as obtained in the
test for Content of Total Phenols.
USP 41 Dietary Supplements / Echinacea 4589

COMPOSITION Standard solution B: 10 t1g/mL of USP 2E,4E-Hexadie-

e CONTENT OF TOTAL PHENOLS noic Acid Isobutylamide RS in methanol
Solvent: Alcohol and water (7:3) Sample solution: Transfer about 500 mg of Powdered
Standard solution A: 30 g/mL of USP Chicoric Acid Echinacea purpurea Extract, accurately weighed, into a
RS in Solvent 100-mL volumetric flask. Add 80 mL of methanol, and
Standard solution B: 20 g/mL of USP Caftaric Acid RS sonicate for 30 min. Cool to room temperature, and
in Solvent dilute with methanol to volume. Pass through a mem-
Standard solution C: 20 g/mL of USP Chlorogenic brane filter of 0.45-t1m or finer pore size.
Acid RS in Solvent Mobile phase: Acetonitrile and water (55:45)
Standard solution D: 20 g/mL of USP Echinacoside RS Chromatographic system
in Solvent (See Chromatography (621), System Suitability.)
Sample solution: Transfer 60 mg of Powdered Echina- Mode: LC
cea purpurea Extract to a round-bottom flask equipped Detector: UV 254 nm
with a condenser. Add 25 mL of Solvent, and heat Column: 4.6-mm x 25-cm; 5-m packing L1
under reflux while shaking by mechanical means for 15 Column temperature: 30°
min. Centrifuge, or pass through a membrane filter of Flow rate: 1.5 mL/min
0.45-uum or finer pore size. Injection volume: 25 pL
Solution A: Phosphoric acid (0.1 in 100) in water System suitability
Solution B: Acetonitrile Samples: Standard solution A and Standard solution B
Mobile phase: See Table 7. Suitability requirements
Chromatogram similarity: The chromatogram from
Table 1 Standard solution A is similar to the Reference Chro-
matogram for alkamides provided with USP Pow-
Time Solution A Solution B dered Echinacea purpurea Extract RS.
(min) (%) (%) Resolution: NLT 1.0 between dodecatetraenoic acid
0 90 10 isobutylamide peaks, Standard solution A
13: 78 22 Tailing factor: NMT 2.0 for the 2£,4E-hexadienoic
14 60 40 acid isobutylamide peak, Standard solution B
17 60 40 Relative standard deviation: NMT 2.5% for the 2E,
4E-hexadienoic acid isobutylamide peak in repeated
AZ, 90 10 injections, Standard solution B
22 90 10 Analysis
Samples: Standard solution A, Standard solution B, and
Chromatographic system Sample solution
(See Chromatography (621), System Suitability.) Identify the peaks due to 2£,4E,8Z,10£-dodecatetraenoic
Mode: LC acid isobutylamide and 2£,4£,8Z,10Z-dodecatetraenoic
Detector: UV 330 nm acid isobutylamide in the chromatogram from the
Column: 4.6-mm x 25-cm; 5-1um packing L1 Sample solution by comparison with the chromatogram
Column temperature: 35° from Standard solution A. Measure the areas for the
Flow rate: 1.5 mL/min relevant peaks.
Injection volume: 5 pL Calculate the percentage of dodecatetraenoic acid iso-
System suitability butylamides in the portion of Powdered Echinacea
Samples: Standard solution A and Standard solution B purpurea Extract taken:
Suitability requirements
Relative standard deviation: NMT 2% for chicoric Result = (ru/rs) x (Cs/Cu) x F x 100
acid peak in repeated injections, Standard solution A
Analysis ty = sum of the peak responses of the relevant
Samples: Standard solution A, Standard solution B, analytes from the Sample solution
Standard solution C, Standard solution D, and Sample rs = peak response from Standard solution B
solution Cs = concentration of USP 2£,4£-Hexadienoic Acid
Separately calculate the percentage of caftaric acid lsobutylamide RS in Standard solution B
(CisH1209), chicoric acid (C22H1s012), and chlorogenic (mg/mL)
Exirel-sloleltlo) Ya

acid (CisHigOs) in the portion of Powdered Echinacea
purpurea Extract taken:
Result = (ru/rs) x (Cs/Cu) x 100
ty = peak response for the relevant analyte from
the Sample solution
fs = peak response of the relevant analyte from the
corresponding Standard solution
Cs = concentration of the relevant analyte in the
corresponding Standard solution (mg/mL)
G = concentration of Powdered Echinacea purpurea
Extract in the Sample solution (mg/mL)
Calculate the percentage of total phenols in the portion
of Powdered Echinacea purpurea Extract taken by
adding the individual percentages calculated.
Acceptance criteria: NLT 4.0% on the dried basis
@ CONTENT OF DODECATETRAENOIC ACID ISOBUTYLAMIDES
Standard solution A: 5 mg/mL of USP Powdered Echi-
nacea purpurea Extract RS in methanol. Dissolve using
sonication and shaking for 10 min. After dilution, pass
through a membrane filter having a 0.45-m or finer
pore size.
Cy = concentration of Powdered Echinacea purpurea
Extract in the Sample solution (mg/mL)
F = response factor to convert 2£,4£-hexadienoic
acid isobutylamide into dodecatetraenoic
acid isobutylamides, 1.353
Acceptance criteria: NLT 0.025% on the dried basis
CONTAMINANTS
e ELEMENTAL IMPURITIES—PROCEDURES (233)
Acceptance criteria
Arsenic: NMT 1.0 g/g
Cadmium: NMT 0.5 ug/g
Lead: NMT 5.0 uG/g
Mercury: NMT 1.0 ug/g
e ARTICLES OF BOTANICAL ORIGIN, General Method for Pesti-
cide Residues Analysis (561): Meets the requirements
e MicROBIAL ENUMERATION TESTS (2021): The total bacterial
count does not exceed 10* cfu/g. The total combined
molds and yeasts count does not exceed 103 cfu/g.
e ABSENCE OF SPECIFIED MICROORGANISMS (2022): It meets
the requirements of the tests for absence of Salmonella
species and Escherichia coli.
 4590 Echinacea / Dietary Supplements
e BOTANICAL ExTRACTS, Residual Solvents (565): Meets the
requirements
SPECIFIC TESTS
e Loss ON DRYING (731)
Sample: 1g
Analysis: Dry the Sample at 105° for 2 h.
Acceptance criteria: NMT 5.0%
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in tight, light-resistant
containers, in a cool place.
e LABELING: The label states the Latin binomial and, follow-
ing the official name, the parts of the plant from which
the article was prepared. If derived from root and aerial
parts, indicate the corresponding percentages. Label it to
indicate the content of total phenols and dodecatetra-
enoic isobutylamides. The label bears a statement indicat-
ing that Echinacea purpurea may cause rare allergic reac-
tions, rashes, or aggravate asthma. It meets the
requirements for Botanical Extracts (565), Labeling.
e USP REFERENCE STANDARDS (11)
USP Caftaric Acid RS
USP Chicoric Acid RS
USP Chlorogenic Acid RS
USP Powdered Echinacea purpurea Extract RS
USP Echinacoside RS
USP 2E,4E-Hexadienoic Acid Isobutylamide RS
USP B-Sitosterol RS
Echinacea Species Dry Extract Capsules
DEFINITION
Echinacea Species Dry Extract Capsules contain one or more
Echinacea Species (Fam. Asteraceae) Dry Extracts prepared
from dried rhizome and roots of Echinacea angustifolia
DC., dried rhizome and roots of Echinacea pallida (Nutt.)
Nutt., dried rhizome and roots of Echinacea purpurea (L.)
Moench, and dried aerial parts of Echinacea purpurea (L.)
Moench. They contain NLT 90% and NMT 110% of the
labeled amount of total phenols, calculated as the sum of
caftaric acid (C13Hi20¢),! chicoric acid (C22H1gO12),?
echinacoside (C3sH4sO20), and cynarin (1,3-di-O-caffeoyl-
quinic acid) (C2sH240y2).3
IDENTIFICATION
e A. HPTLC FOR ARTICLES OF BOTANICAL ORIGIN (203)
For Capsules containing Echinacea angustifolia Dry
Extract
DS Monographs
Standard solution A: 0.2 mg/mL of USP Echinacoside
RS and 0.2 mg/mL of cynarin in methanol
Standard solution B: 0.05 mg/mL of USP Caftaric
Acid RS, 0.1 mg/mL of USP Chlorogenic Acid RS, and
0.05 mg/mL of USP Chicoric Acid RS in methanol
Standard solution C: 20 mg/mL of USP Powdered
Echinacea angustifolia Extract RS in methanol. Shake to
disperse, sonicate for 5 min, and centrifuge. Use the
supernatant.
Sample solution: Transfer a portion of the Capsule
contents, equivalent to 100 mg of Echinacea angus-
tifolia Dry Extract, to a centrifuge tube, add 5 mL of
methanol, shake to disperse, sonicate for 5 min, and
centrifuge. Use the supernatant.
Chromatographic system
Adsorbent: Chromatographic silica gel with an aver-
age particle size of 5 um (HPTLC plates)
1(2R,3R)-2-{[(E)-3-(3,4-Dihydroxyphenyl)acryloylJoxy}-3-hydroxysuccinic acid.
2(2R,3R)-2,3-Bis{[(E)-3-(3,4-dihydroxyphenyl)acryloyljoxy}succinic acid. Other
common names for chicoric acid are cichoric acid and dicaffeoyltartaric acid.
3(1R,3R,45,5R)-1,3-Bis{[(E)-3-(3,4-dihydroxyphenyl)acryloyl]oxy}-4,5-dihydrox-
ycyclohexane-1-carboxylic acid.
USP 41
Application volume: 5 Ll each of Standard solution C
and Sample solution, and 2 wL each of Standard solu-
tion A and Standard solution B as 8-mm bands
Relative humidity: Condition the plate toa relative
humidity of about 33%.
Temperature: Ambient, not to exceed 30°
Developing solvent system: Ethyl acetate, methyl
ethyl ketone, water, and formic acid (5:3:1:1)
Developing distance: 6 cm
Derivatization reagent: 5 mg/mL of 2-aminoethyl
diphenylborinate in ethyl acetate
Analysis
Samples: Standard solution A, Standard solution B,
Standard solution C, and Sample solution
Apply the Samples as bands and dry in air. Develop in
a saturated chamber. Remove the plate from the
chamber, heat at 100° for 5 min, treat while still
warm with Derivatization reagent, dry in air, and ex-
amine under UV light at 366 nm.
System suitability: Standard solution A shows two ma-
jor blue bands, one in the lower third section due to
echinacoside, and the other band in the middle sec-
tion due to dicaffeoylquinic acid (cynarin). Standard
solution B shows two major blue bands at about the
middle section due to caftaric acid (lower Rp) and
chlorogenic acid (higher R,) that are clearly separated,
anda Dive chicoric acid band in the upper third of the
chromatogram.
Acceptance criteria: The Sample solution exhibits the
following: the most prominent blue band in the lower
third section at R- corresponding to that of echinaco-
side in Standard solution A and Standard solution C; a
prominent greenish-blue band in the middle section at
R; corresponding to cynarin in Standard solution A and
Standard solution C; minor bands between the posi-
tions of echinacoside and cynarin. One of these is due
to chlorogenic acid at Rr corresponding to that of
chlorogenic acid in Standard solution B; very faint (or
may be absent) blue bands at Rr corresponding to the
caftaric acid and chicoric acid bands in Standard solu-
tion B.
For Capsules containing Echinacea pallida Dry Ex-
tract: Proceed as directed in For Capsules containin
Echinacea angustifolia Dry Extract. For Standard solution
Cand the Sample solution, substitute USP Powdered
Echinacea angustifolia Extract RS with USP Powdered
Echinacea pallida Extract RS and Echinacea angustifolia
Dry Extract with Echinacea pallida Dry Extract,
respectively.
Acceptance criteria: The Sample solution exhibits the
most prominent blue band in the lower third section
at Rr Corresponding to that of echinacoside in Standard
solution A and Standard solution CG, may exhibit bands
of lesser intensity at the R- corresponding to caftaric
acid and chicoric acid in Standard solution B and Stan-
dard solution C; exhibits minor bands between the po-
sitions of echinacoside and caftaric acid. One of these
is due to chlorogenic acid at an Rr corresponding to
that of chlorogenic acid in Standard solution B. The
Sample solution does not exhibit a blue band in the
middle section at R- corresponding to cynarin in Stan-
dard solution A.
For Capsules containing Echinacea purpurea Root Dry
Extract and Echinacea purpurea Aerial Parts Dry Ex-
tract: Proceed as directed in For Capsules containing
Echinacea angustifolia Dry Extract. For Standard solution
C substitute USP Powdered Echinacea angustifolia Extract
RS with USP Powdered Echinacea purpurea Extract RS.
For the Sample solution, substitute Echinacea angustifolia
Dry Extract with Echinacea purpurea Root Dry Extract
and Echinacea purpurea Aerial Parts Dry Extract.
Acceptance criteria: The Sample solution exhibits the
most prominent blue band in the upper third section
at Rr corresponding to chicoric acid in Standard solu-
tion B and Standard solution C; exhibits the second
USP 41
most prominent blue band at about the middle sec-
tion at R- corresponding to that of caftaric acid in
Standard solution B and Standard solution C; may ex-
hibit minor blue bands corresponding to similar bands
in Standard solution C. One of these is due to
chlorogenic acid at an R; corresponding to chlorogenic
acid in Standard solution B. The Sample solution chro-
matogram does not exhibit a band at the same R; as
echinacoside in Standard solution A.
e B. HPLC For TOTAL PHENOLS
Analysis: Proceed as directed in the test for Content of
Total Phenols.
Acceptance criteria: The chromatogram of the Sample
solution prepared from Capsules labeled to contain ex-
tracts of E. purpurea roots or aerial parts exhibits peaks
at the retention times of those due to caftaric acid,
chlorogenic acid, and chicoric acid in the chromato-
gram of the Standard solution. The chromatogram of
the Sample solution prepared from Capsules labeled to
contain extract of E. angustifolia exhibits peaks at the
retention times of those due to chlorogenic acid,
cynarin, and echinacoside in the chromatogram of the
Standard solution. The chromatogram of the Sample so-
lution prepared from Capsules labeled to contain extract
of E. pallida exhibits peaks at the retention times of
those due to caftaric acid, chlorogenic acid, echinaco-
side, and chicoric acid in the chromatogram of the
Standard solution.
e C. HPLC FOR PRESENCE OF ALKYLAMIDES
Analysis: Proceed as directed in the test for Content of
Dodecatetraenoic Acid Isobutylamides.
Acceptance criteria: The chromatogram of the Sample
solution exhibits peaks at the retention times of those
due to dodecatetraenoic acid isobutylamides in the
chromatogram of Standard solution B or Standard solu-
tion C, and the reference chromatogram provided with
the lot of USP Powdered Echinacea angustifolia Extract
RS or USP Powdered Echinacea purpurea Extract RS be-
ing used.
STRENGTH
© CONTENT OF TOTAL PHENOLS
Solution A: Phosphoric acid in water (0.1 in 100)
Solution B: Acetonitrile
Mobile phase: See Table 1.
Table 1
Time Solution A Solution B
(min) (%) (%)
0 90 10
3 90 10
16 78 22
17 60 40
20 60 40
20.5 90 10
25 90 10
Solvent: Alcohol and water (7:3)
Standard solution: 20 g/mL of USP Caftaric Acid RS,
20 g/mL of USP i ae Acid RS, 20 ng/mL of
cynarin (1,3-di-O-caffeoylquinic acid), 60 ug/mL of USP
Echinacoside RS, and 40 ttg/mL of USP Chicoric Acid RS
in Solvent
Sample solution: Determine the total weight of 20
Capsules. Empty the Capsules, combine, and mix their
contents to obtain a homogenous composite. Weigh
the empty Capsule shells and calculate the average fill
weight per Capsule. Transfer a portion of the Capsule
contents, nominally equivalent to 60 mg of the labeled
Echinacea Species Dry Extract, to a 100-mL round-bot-
tom flask equipped with a condenser. Add 25.0 mL of
Solvent, and heat under reflux for 15 min. Cool to room
Dietary Supplements / Echinacea 4591
temperature and pass throughafilter of 0.45-1m or
finer pore size.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 330 nm
Column: 4.6-mm x 25-cm; 5-lum packing L1
Column temperature: 35°
Flow rate: 1.5 mL/min
Injection volume: 5 uL
System suitability
Sample: Standard solution
Suitability requirements
Resolution: NLT 3.0 between the cynarin and
echinacoside peaks, and NLT 1.0 between the caftaric
acid and chlorogenic acid peaks. [NoTE—Echinacoside
peaks may be resolved in two components. The rela-
tive retention times for caftaric acid, chlorogenic acid,
cynarin, echinacoside, and chicoric acid are 0.7, 0.75,
0.9, 1.0, and 1.4, respectively.]
Relative standard deviation: NMT 2.5% for the sum
of echinacoside peaks
Analysis
Samples: Standard solution and Sample solution
Using the chromatogram of the Standard solution, iden-
tify and measure the areas of the peaks corresponding
to caftaric acid (Ci3H1206¢), cynarin (C2sH24042),
echinacoside (C3sH46O20), and chicoric acid (C22HigOi2)
in the Sample solution chromatogram.
Calculate the percentage of caftaric acid, cynarin,
echinacoside, and chicoric acid in the portion of Cap-
sules taken:
Result = (ru/rs) x Cs x (V/W) x 100
ty = peak area of a relevant analyte from the
Sample solution
Is = peak area of caftaric acid, echinacoside, or
chicoric acid from the Standard solution
Cs = concentration of a relevant analyte in the
Standard solution (mg/mL)
Vv = volume of the solvent taken to prepare the
Sample solution (mL)
Ww = weight of the sample taken to prepare the
Sample solution (mg)
Calculate the percentage of the labeled amount of total
phenols, calculated as the sum of determined caftaric
acid, echinacoside, chicoric acid, and cynarin in the
portion of Capsules taken:
Result = (ZP/L) x 100
sydesbouow Sa
=P, = total combined content of caftaric acid,
echinacoside, chicoric acid, and cynarin as
determined above (%)
L = labeled amount of total phenols (%)
Acceptance criteria: 90%-110%
PERFORMANCE TESTS
© DISINTEGRATION AND DISSOLUTION (2040), Disintegration:
Meet the requirements
e WEIGHT VARIATION (2091): Meet the requirements
SPECIFIC TESTS
© CONTENT OF DODECATETRAENOIC ACID ISOBUTYLAMIDES
[Note—This test is not applicable to Capsules containing
Echinacea pallida extract prepared from dried rhizome
and roots.
Mobile phase: Acetonitrile and water (55:45)
Standard solution A: 10 g/mL of USP 2E,4E-Hexadie-
noic Acid lsobutylamide RS in methanol
Standard solution B: 1 mg/mL of USP Powdered Echi-
nacea angustifolia Extract RS in methanol. Sonicate to
dissolve, and pass throughafilter of 0.45-m or finer
pore size. [NoTe—Prepare when Capsules contain Echi-
nacea angustifolia Dry Extract.]
 4592 Echinacea / Dietary Supplements
Standard solution C: 5 mg/mL of USP Powdered Echi-
nacea purpurea Extract RS in methanol. Sonicate to dis-
solve and pass throughafilter of 0.45-um or finer pore
size. [NoTE—Prepare when Capsules do not contain Ech-
inacea angustifolia Dry Extract.]
Sample solution: Transfer a portion of the Capsule con-
tents, equivalent to 500 mg of Echinacea Species Di
Extract, to a 100-mL volumetric flask. Add 80 mL o'
methanol, and sonicate for 30 min. Dilute with metha-
nol to volume, and pass through a membrane filter of
0.45-um or finer pore size.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 254 nm
Column: 4.6-mm x 25-cm; 5-um packing L1
Column temperature: 30°
Flow rate: 1.5 mL/min
Injection volume: 25 pL
System suitability
Samples: Standard solution A, Standard solution B, or
Standard solution C
Suitability requirements
Resolution: NLT 1.0 between the dodecatetraenoic
acid isobutylamide peaks, Standard solution B or Stan-
dard solution C
Tailing factor: NMT 2.0 for the 2E,4£-hexadienoic
acid isobutylamide peak, Standard solution A
Relative standard deviation: NMT 2.5% for the 2E,
4£-hexadienoic acid isobutylamide peak in replicate
injections, Standard solution A
Chromatogram similarity: The chromatogram of
Standard solution B or Standard solutionC is similar to
the reference chromatogram for alkamides provided
with the USP Powdered Echinacea angustifolia Extract
RS or USP Powdered Echinacea purpurea Extract RS
being used.
Analysis
Samples: Standard solution A, Standard solution B, or
Standard solution C, and Sample solution
Using the chromatogram of Standard solution B or Stan-
dard solution C, and the reference chromatogram pro-
vided with the lot of USP Powdered Echinacea angus-
tifolia Extract RS or USP Powdered Echinacea purpurea
Extract RS being used, identify and measure the areas
of 2E,4E,8Z,10E- and 2£,4£,8Z,10Z-dodecatetraenoic
acid isobutylamide peaks in the Sample solution.
Calculate the percentage of dodecatetraenoic acid iso-
butylamides in the amount of Echinacea Species Dry
Extract taken:
DS Monographs
Result = (rsum/rs) x (Cs x V/W) x (Wov/L) x F x 100
Tum = Sum of the peak areas of the relevant analytes
from the Sample solution
rs = peak area of 2£,4E-hexadienoic acid
isobutylamide from Standard solution A
Cs = concentration of USP 2£,4£-Hexadienoic Acid
Isobutylamide RS in Standard solution A
(mg/mL)
V = volume of the solvent taken to prepare Sample
solution (mL)
Ww = weight of the sample taken to prepare Sample
solution (mg)
W. = average Capsule fill weight (mg/Capsule)
L = labeled amount of Echinacea angustifolia Dry
Extract, Echinacea purpurea Root Dry Extract,
or Echinacea purpurea Aerial Parts Dry Extract
per Capsule (mg)
F = response factor of dodecatetraenoic acid
isobutylamides relative to 2E,4E-hexadienoic
acid isobutylamide, 1.353
Acceptance criteria
For Capsules containing Echinacea angustifolia Dry
Extract: NLT 0.1%
USP 41
For Capsules not containing Echinacea angustifolia
Dry Extract: NLT 0.025%
CONTAMINANTS
e MICROBIAL ENUMERATION TESTS (2021): The total aerobic
bacterial count does not exceed 104 cfu/g, and the total
eae molds and yeasts count does not exceed 103
cfu/g.
° Ansel OF SPECIFIED MICROORGANISMS (2022), Test Proce-
dures, Test for Absence of Salmonella Species and Test for
Absence of Escherichia coli: Meet the requirements
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in well-closed contain-
ers, protected from light and moisture, and store in a
cool place.
e LABELING: The label states the Latin binomial and the offi-
cial name. The label states the amount of total phenols
(as sum of presented caftaric acid, echinacoside, chicoric
acid, and cynarin) and the amount of Echinacea Species
Dry Extract in mg/Capsule.
e USP REFERENCE STANDARDS (11)
USP Caftaric Acid RS
USP Chicoric Acid RS
USP Chlorogenic Acid RS
USP Powdered Echinacea angustifolia Extract RS
USP Powdered Echinacea pallida Extract RS
USP Powdered Echinacea purpurea Extract RS
USP Echinacoside RS
USP 2£,4E-Hexadienoic Acid Isobutylamide RS
Echinacea Species Dry Extract Tablets
DEFINITION
Echinacea Species Dry Extract Tablets contain one or more
Echinacea Species (Fam. Asteraceae) Dry Extracts prepared
from dried rhizome and roots of Echinacea angustifolia
DC., dried rhizome and roots of Echinacea pallida (Nutt.)
Nutt., dried rhizome and roots of Echinacea purpurea (L.)
Moench, and dried aerial parts of Echinacea purpurea (L.)
Moench. They contain NLT 90% and NMT 110% of the
labeled amount of total phenols, calculated as the sum of
caftaric acid (C3H:20¢),! chicoric acid (C22HigO;2),?
echinacoside (C3sH4sO20), and cynarin (1,3-di-O-caffeoyl-
quinic acid) (C2sH24O12).3
IDENTIFICATION
e A. HPTLC For ARTICLES OF BOTANICAL ORIGIN (203)
For Tablets containing Echinacea angustifolia Dry
Extract
Standard solution A: 0.2 mg/mL of USP Echinacoside
RS and 0.2 mg/mL of cynarin in methanol
Standard solution B: 0.05 mg/mL of USP Caftaric
Acid RS, 0.1 mg/mL of USP Chlorogenic Acid RS, and
0.05 mg/mL of USP Chicoric Acid RS in methanol
Standard solution C: 20 mg/mL of USP Powdered
Echinacea angustifolia Extract RS in methanol. Shake to
disperse, sonicate for 5 min, and centrifuge. Use the
supernatant.
Sample solution: Transfer a portion of the powdered
Tablets, equivalent to 100 mg of Echinacea angustifolia
Dry Extract, to a centrifuge tube, add 5 mL of metha-
nol, shake to disperse, sonicate for 5 min, and centri-
fuge. Use the supernatant.
Chromatographic system
Adsorbent: Chromatographic silica gel with an aver-
age particle size of 5 um (HPTLC plates)
1 (2 32103 3, DibydronyphenyDacrHoylloxy:3 Widronysuccuc acid.
2(2R,3R)-2,3-Bis{[(E)-3-(3,4-dihydroxyphenyl)acryloyljoxy}succinic acid. Other
common names for chicoric acid are cichoric acid and dicaffeoyltartaric acid.
3(1R,3R,45,5R)-1 /3-8is((6)-3-G,4-cihydroxypheny!)acryloyloxy -4,5-dihydrox-
ycyclohexane-1-carboxylic acid.
USP 41
Application volume: 5 iL each of Standard solution C
and Sample solution, and 2 uL each of Standard solu-
tion A and Standard solution B as 8-mm bands
Relative humidity: Condition the plate toa relative
humidity of about 33%.
Temperature: Ambient, not to exceed 30°
Developing solvent system: Ethyl acetate, methyl
ethyl ketone, water, and formic acid (5:3:1:1)
Developing distance: 6 cm
Derivatization reagent: 5 mg/mL of 2-aminoethyl
diphenylborinate in ethyl acetate
Analysis
Samples: Standard solution A, Standard solution B,
Standard solution C, and Sample solution
Apply the Samples as bands and dry in air. Develop in
a saturated chamber. Remove the plate from the
chamber, heat at 100° for 5 min, treat while still
warm with Derivatization reagent, dry in air, and ex-
amine under UV light at 366 nm.
System suitability: Standard solution A shows two ma-
jor blue bands, one in the lower third section due to
echinacoside, and the other band in the middle sec-
tion due to dicaffeoylquinic acid (cynarin). Standard
Solution B shows two major blue bands at about the
middle section due to caftaric acid (lower Rp) and
chlorogenic acid (higher R;) that are clearly separated,
and a blue chicoric acid band in the upper third of the
chromatogram.
Acceptance criteria: The Sample solution exhibits the
following: the most prominent blue band in the lower
third section at Ry ree to that of echinaco-
side in Standard solution A and Standard solution CG a
prominent greenish-blue band in the middle section at
Rr corresponding to cynarin in Standard solution A and
Standard solution C; minor bands between the posi-
tions of echinacoside and cynarin. One of these is due
to chlorogenic acid at Rr corresponding to that of
chlorogenic acid in Standard solution B; very faint (or
may be absent) blue bands at Rr corresponding to the
caftaric acid and chicoric acid bands in Standard solu-
tion B.
For Tablets containing Echinacea pallida Dry Extract:
Proceed as directed in For Tablets containing Echinacea
angustifolia Dry Extract. For Standard solution C and the
Sample solution, substitute USP Powdered Echinacea
angustifolia Extract RS with USP Powdered Echinacea
pallida Extract RS and Echinacea angustifolia Dry Extract
with Echinacea pallida Dry Extract, respectively.
Acceptance criteria: The Sample solution exhibits the
most prominent blue band in the lower third section
at Re corresponding to that of echinacoside in Standard
solution A and Standard solution C; may exhibit bands
of lesser intensity at the R- corresponding to caftaric
acid and chicoric acid in Standard solution B and Stan-
dard solution C; exhibits minor bands between the po-
sitions of echinacoside and caftaric acid. One of these
is due to chlorogenic acid at an Rr corresponding to
that of chlorogenic acid in Standard solution B. The
Sample solution does not exhibit a blue band in the
middle section at R- corresponding to cynarin in Stan-
dard solution A.
For Tablets containing Echinacea purpurea Root Dry
Extract and Echinacea purpurea Aerial Parts Dry Ex-
tract: Proceed as directed in For Tablets containing Ech-
inacea angustifolia Dry Extract. For Standard solution C
substitute USP Powdered Echinacea angustifolia Extract
RS with USP Powdered Echinacea purpurea Extract RS.
For the Sample solution, substitute Echinacea angustifolia
Dry Extract with Echinacea purpurea Root Dry Extract
and Echinacea purpurea Aerial Parts Dry Extract.
Acceptance criteria: The Sample solution exhibits the
most prominent blue band in the upper third section
at R- corresponding to chicoric acid in Standard solu-
tion B and Standard solution CG; exhibits the second
Dietary Supplements / Echinacea 4593
most prominent blue band at about the middle sec-
tion at R- corresponding to that of caftaric acid in
Standard solution B and Standard solution C; may ex-
hibit minor blue bands corresponding to similar bands
in Standard solution C. One of these is due to
chlorogenic acid at an R¢ corresponding to chlorogenic
acid in Standard solution B. The Sample solution does
not exhibit a band at the same Rr of echinacoside in
Standard solution A.
e B. HPLC FoR TOTAL PHENOLS
Analysis: Proceed as directed in the test for Content of
Total Phenols.
Acceptance criteria: The chromatogram of the Sample
solution prepared from Tablets labeled to contain ex-
tracts of £. purpurea roots or aerial parts exhibits peaks
at the retention times of those due to caftaric acid,
chlorogenic acid, and chicoric acid in the chromato-
gram of the Standard solution. The chromatogram of
the Sample solution prepared from Tablets labeled to
contain extract of £. angustifolia exhibits peaks at the
retention times of those due to chlorogenic acid,
cynarin, and echinacoside in the chromatogram of the
Standard solution. The chromatogram of the Sample so-
lution prepared from Tablets labeled to contain extract
of E. pallida exhibits peaks at the retention times of
those due to caftaric acid, chlorogenic acid, echinaco-
side, and chicoric acid in the chromatogram of the
Standard solution.
© C. HPLC FOR PRESENCE OF ALKYLAMIDES
Analysis: Proceed as directed in the test for Content of
Dodecatetraenoic Acid Isobutylamides.
Acceptance criteria: The chromatogram of the Sample
solution exhibits peaks at the retention times of those
due to dodecatetraenoic acid isobutylamides in Stan-
dard solution B or Standard solution C, and the reference
chromatogram provided with the lot of USP Powdered
Echinacea angustifolia Extract RS or USP Powdered Echi-
nacea purpurea Extract RS being used.
STRENGTH
e@ CONTENT OF TOTAL PHENOLS
Solution A: Phosphoric acid in water (0.1 in 100)
Solution B: Acetonitrile
Mobile phase: See Table 1.
Table 1
Time Solution A Solution B
(min) (%) (%)
0 90 10
3 90 10
16 78 22 oS
17 60 40
20 60 40 S
20.5 90 10 3
25 90 10 ito)
|
Solvent: Alcohol and water (7:3) a
Standard solution: 20 g/mL of USP Caftaric Acid RS, ra
20 g/mL of USP Chlorogenic Acid RS, 20 g/mL of
cynarin (1,3-di-O-caffeoylquinic acid), 60 g/mL of USP
Echinacoside RS, and 40 g/mL of USP Chicoric Acid RS
in Solvent
Sample solution: Weigh NLT 20 Tablets, determine the
average Tablet weight, and finely powder. Transfer a
portion of finely powdered Tablets, nominally equiva-
lent to 60 mg of the labeled Echinacea Species Dry Ex-
tract, to a 100-mL round-bottom flask equipped with a
condenser. Add 25.0 mL of Solvent, and heat under re-
flux for 15 min. Cool to room temperature, and pass
throughafilter of 0.45-um or finer pore size.
Chromatographic system
(See Chromatography (621), System Suitability.)
 4594 Echinacea / Dietary Supplements
Mode: LC
Detector: UV 330 nm
Column: 4.6-mm x 25-cm; 5-um packing L1
Column temperature: 35°
Flow rate: 1.5 mL/min
Injection volume: 5 ul
System suitability
Sample: Standard solution
Suitability requirements
Resolution: NLT 3.0 between the cynarin and
echinacoside peaks, and NLT 1.0 between the caftaric
acid and chlorogenic acid peaks. [NoTE—Echinacoside
peaks may be resolved in two components. The rela-
tive retention times for caftaric acid, chlorogenic acid,
cynarin, echinacoside, and chicoric acid are 0.7, 0.75,
0.9, 1.0, and 1.4, respectively.]
Relative standard deviation: NMT 2.5% for the sum
of echinacoside peaks
Analysis
Samples: Standard solution and Sample solution
Using the chromatogram of the Standard solution, iden-
tify and measure areas of the peaks corresponding to
caftaric acid (Ci3H120s), cynarin (C2sH24012), echinaco-
side (C3sHasO20), and chicoric acid (C22HigO;2) in the
Sample solution.
Calculate the percentage of caftaric acid, cynarin,
echinacoside, and chicoric acid in the portion of Tab-
lets taken:
Result = (ru/rs) x Cs x (V/W) x 100
tu = peak area of a relevant analyte from the
Sample solution
Is = peak area of caftaric acid, echinacoside, or
chicoric acid from the Standard solution
Gs = concentration of a relevant analyte in the
Standard solution (mg/mL)
Vv = volume of the solvent taken to prepare the
Sample solution (mL)
Ww = weight of the sample taken to prepare the
Sample solution (mg)
Calculate the percentage of the labeled amount of total
phenols, calculated as the sum of determined caftaric
acid, echinacoside, chicoric acid, and cynarin in the
portion of Tablets taken:
Result = (ZP/L) x 100
=P, = total combined content of caftaric acid,
echinacoside, chicoric acid, and cynarin as
determined above (%)
E = labeled amount of total phenols (%)
DS Monographs
Acceptance criteria: 90%-110%
PERFORMANCE TESTS
e DISINTEGRATION AND DISSOLUTION (2040), Disintegration:
Meet the requirements
¢ WEIGHT VARIATION (2091): Meet the requirements
SPECIFIC TESTS
e@ CONTENT OF DODECATETRAENOIC ACID ISOBUTYLAMIDES
[NoTte—This test is not applicable to Tablets containing
Echinacea pallida extract prepared from dried rhizome
and roots.
Mobile phase: Acetonitrile and water (55:45)
Standard solution A: 10 g/mL of USP 2£,4E-Hexadie-
noic Acid Isobutylamide RS in methanol
Standard solution B: 1 mg/mL of USP Powdered Echi-
nacea angustifolia Extract RS in methanol. Sonicate to
dissolve, and pass througha filter of 0.45-1m or finer
pore size. [NoTE—Prepare when Tablets contain Echina-
cea angustifolia Dry Extract.]
Standard solution C: 5 mg/mL of USP Powdered Echi-
nacea purpurea Extract RS in methanol. Sonicate to dis-
solve, and pass through a filter of 0.45-11m or finer pore
USP 41
size. [NoTE—Prepare when Tablets do not contain Echi-
nacea angustifolia Dry Extract.]
Sample solution: Weigh NLT 20 Tablets, determine the
average Tablet weight, and finely powder. Transfer a
portion of finely powdered Tablets, equivalent to
500 mg of Echinacea Species Dry Extract, to a 100-mL
volumetric flask. Add 80 mL of methanol, and sonicate
for 30 min. Dilute with methanol to volume, and pass
through a membrane filter of 0.45-um or finer pore
size.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 254 nm
Column: 4.6-mm x 25-cm; 5-um packing L1
Column temperature: 30°
Flow rate: 1.5 mL/min
Injection volume: 25 wL
System suitability
Samples: Standard solution A, Standard solution B or
Standard solution C
Suitability requirements
Resolution: NLT 1.0 between the dodecatetraenoic
acid isobutylamide peaks, Standard solution B or Stan-
dard solution C
Tailing factor: NMT 2.0 for the 2£,4F-hexadienoic
acid isobutylamide peak, Standard solution A
Relative standard deviation: NMT 2.5% for the 2E,
4E-hexadienoic acid isobutylamide peak in replicate
injections, Standard solution A
Chromatogram similarity: The chromatogram of
Standard solution B or Standard solutionC is similar to
the reference chromatogram for alkamides provided
with the USP Powdered Echinacea angustifolia Extract
RS or USP Powdered Echinacea purpurea Extract RS
being used.
Analysis
Samples: Standard solution A, Standard solution B, or
Standard solution C, and Sample solution
Using the chromatogram of Standard solution B or Stan-
dard solution C, and the reference chromatogram pro-
vided with the lot of USP Powdered Echinacea angus-
tifolia Extract RS or USP Powdered Echinacea purpurea
Extract RS being used, identify and measure the areas
of 2£,4E,8Z,10E- and 2£,4E,8Z,10Z-dodecatetraenoic
acid isobutylamide peaks in the Sample solution
aromateglala
Calculate the percentage of dodecatetraenoic acid iso-
butylamides in the amount of Echinacea Species Dry
Extract taken:
Result = ((eum/Ts) x (Cs x V/W) x (Wov/L) x F x 100
Tum = Sum of the peak areas of the relevant analytes
from the Sample solution
Is = peak area of 2£,4E-hexadienoic acid
isobutylamide from Standard solution A
Cs = concentration of USP 2£,4f-Hexadienoic Acid
lsobutylamide RS in Standard solution A
(mg/mL)
V = volume of the solvent taken to prepare Sample
solution (mL)
w = weight of the sample taken to prepare Sample
solution (mg)
Wa = average Tablet weight (mg/Tablet)
L = labeled amount of Echinacea angustifolia Dry
Extract, Echinacea purpurea Root Dry Extract,
or Echinacea purpurea Aerial Parts Dry Extract
per Tablet (mg)
F = response factor of dodecatetraenoic acid
isobutylamides relative to 2£,4£-hexadienoic
acid isobutylamide, 1.353
Acceptance criteria
For Tablets containing Echinacea angustifolia Dry Ex-
tract: NLT 0.1%
USP 41
For Tablets not containing Echinacea angustifolia Dry
Extract: NLT 0.025%
CONTAMINANTS
© MICROBIAL ENUMERATION TESTS (2021): The total aerobic
bacterial count does not exceed 104 cfu/g, and the total
a es molds and yeasts count does not exceed 103
u/g.
° ABSENCE OF SPECIFIED MICROORGANISMS (2022), Test Proce-
dures, Test for Absence of Salmonella Species and Test for
Absence of Escherichia coli: Meet the requirements
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in well-closed contain-
ers, protected from light and moisture, and store in a
cool place.
e LABELING: The label states the Latin binomial and the offi-
cial name. The label states the amount of total phenols
(as sum of presented caftaric acid, echinacoside, chicoric
acid, and cynarin) and the amount of Echinacea Species
Dry Extract in mg/Tablet.
e USP REFERENCE STANDARDS (11)
USP Caftaric Acid RS
USP Chicoric Acid RS
USP Chlorogenic Acid RS
USP Powdered Echinacea angustifolia Extract RS
USP Powdered Echinacea pallida Extract RS
USP Powdered Echinacea purpurea Extract RS
USP Echinacoside RS
USP 2E,4E-Hexadienoic Acid lsobutylamide RS
Add the following:
«Echinacea Species Powder Capsules
DEFINITION
Echinacea Species Powder Capsules contain one or more of
the following Echinacea Species (Fam. Asteraceae)
powders prepared from aried rhizome and roots of Echi-
nacea angustifolia DC., dried rhizome and roots of Echina-
cea pallida (Nutt.) Nutt., dried rhizome and roots of Echi-
nacea purpurea (L.) Moench, and dried aerial parts of
Echinacea purpurea (L.) Moench. They contain NLT 0.5%
of total phenols, calculated as sum of caftaric acid
(Ci3H120s), chicoric acid (C22HieO12),! echinacoside
(C3sH4sOo9), cynarin (1,3-di-O-caffeoylquinic acid)
(C2sH24O12), and chlorogenic acid (CisHigOo), from the la-
beled amount of Echinacea Species Powder.
IDENTIFICATION
e A. HPTLC FOR ARTICLES OF BOTANICAL ORIGIN (203)
For Capsules containing Echinacea angustifolia powder
prepared from dried rhizome and roots
Standard solution A: 0.2 mg/mL of USP Echinacoside
RS and 0.2 mg/mL of cynarin in methanol
Standard solution B: 0.05 mg/mL of USP Caftaric
Acid RS, 0.1 mg/mL of USP Chlorogenic Acid RS, and
0.05 mg/mL of USP Chicoric Acid RS in methanol
Standard solution C: 20 mg/mL of USP Powdered
Echinacea angustifolia Extract RS in methanol. Shake to
disperse, sonicate for 5 min, and centrifuge. Use the
supernatant.
Sample solution: Transfer a portion of the Capsule
contents, equivalent to 1000 mg of Echinacea angus-
tifolia Powder, to a centrifuge tube, add 10 mL o'
methanol, shake to disperse, sonicate for 20 min, and
centrifuge. Use the supernatant.
| Other common names for chicoric acid are cichoric acid and dicaf-
feoyltartaric acid.
Dietary Supplements / Echinacea 4595
Chromatographic system
Adsorbent: Chromatographic silica gel with an aver-
age particle size of 5 um (HPTLC peso 4
Application volume: 5 tL each of Standard solution C
and the Sample solution, and 2 \tL each of Standard
solution A and Standard solution B as 8-mm bands
Relative humidity: Condition the plate to a relative
humidity of about 33%.
Temperature: Ambient, not to exceed 30°
Developing solvent system: Ethyl! acetate, methy!
ethyl ketone, water, and formic acid (5:3:1:1)
Developing distance: 6 cm
Derivatization reagent: 5S mg/mL of 2-aminoethyl
diphenylborinate in ethyl acetate
Analysis
Samples: Standard solution A, Standard solution B,
Standard solution C, and Sample solution
Apply the Samples as bands and dry in air. Develop in
a saturated chamber. Remove the plate from the
chamber, heat at 100° for 5 min, treat while still
warm with the Derivatization reagent, dry in air, and
examine under UV light at 366 nm.
System suitability: Standard solution A shows two ma-
jor blue bands: one in the lower third section due to
echinacoside, and the other band in the middle sec-
tion due to dicaffeoylquinic acid (cynarin). Standard
solution B shows two major blue bands at about the
middle section due to caftaric acid (lower R,) and
chlorogenic acid (higher R,) that are clearly separated,
andaria chicoric acid band in the upper third of the
chromatogram.
Acceptance criteria: The Sample solution exhibits the
following: the most prominent blue band in the lower
third section at Rr corresponding to that of echinaco-
side in Standard solution A and Standard solution C; a
prominent greenish-blue band in the middle section at
R; corresponding to cynarin in Standard solution A and
Standard solution C; minor bands between the posi-
tions of echinacoside and cynarin. One of these is due
to chlorogenic acid at R- corresponding to that of
chlorogenic acid in Standard solution B, and very faint
(or may be absent) blue bands at R; corresponding to
the caftaric acid and chicoric acid bands in Standard
solution B.
For Capsules containing Echinacea pallida powder pre-
pared from dried rhizome and roots: Proceed as di-
rected For Capsules containing Echinacea angustifolia
powder prepared from dried rhizome and roots. For Stan-
dard solution C and the Sample solution, substitute USP
Powdered Echinacea angustifolia Extract RS with USP
Powdered Echinacea pallida Extract RS and Echinacea
sydesbouow sa
angustifolia powder with Echinacea pallida powder pre-
pared from dried rhizome and roots.
Acceptance criteria: The Sample solution exhibits the
most prominent blue band in the lower third section
at Ry corresponding to that of echinacoside in Standard
solution A and Standard solution C; may exhibit bands
of lesser intensity at the Rr corresponding to caftaric
acid and chicoric acid in Standard solution B and Stan-
dard solution C; exhibits minor bands between the po-
sitions of echinacoside and caftaric acid. One of these
is due to chlorogenic acid at an R: corresponding to
that of chlorogenic acid in Standard solution B. The
Sample solution does not exhibit a blue band in the
middle section at R- corresponding to cynarin in Stan-
dard solution A.
For Capsules containing Echinaceapcan powder
prepared from dried rhizome and roots and Echina-
cea purpurea powder prepared from dried aerial
parts: Proceed as directed For Capsules containing Echi-
nacea angustifolia powder prepared from dried rhizome
and roots. For Standard solution C and the Sample solu-
tion, substitute USP Powdered Echinacea angustifolia Ex-
tract RS with USP Powdered Echinacea purpurea Extract
RS and Echinacea angustifolia powder with Echinacea
 4596 Echinacea / Dietary Supplements
purpurea powder prepared from rhizome and roots or
Echinacea purpurea powder prepared from aerial parts.
Acceptance criteria: The Sample solution exhibits the
following: the most prominent blue band in the upper
third section at R; corresponding to chicoric acid band
in Standard solution B and Standard solution C; the sec-
ond most prominent blue band at about the middle
section at R- corresponding to that of caftaric acid in
Standard solution B and Standard solution CG, may ex-
hibit minor blue bands corresponding to similar bands
in Standard solution C. One of these is due to
chlorogenic acid at an Rr corresponding to chlorogenic
acid in Standard solution B. The Sample solution chro-
matogram does not exhibit a band at the same R; as
echinacoside in Standard solution A.
e B. HPLC FoR TOTAL PHENOLS
Analysis: Proceed as directed in the test for Content of
Total Phenols.
Acceptance criteria: The chromatogram of the Sample
solution prepared from Capsules labeled to contain £.
angustifolia exhibits peaks at the retention times of
those due to chlorogenic acid, cynarin, and echinaco-
side in the chromatogram of the Standard solution. The
chromatogram of the Sample soiution prepared from
Capsules labeled to contain E. pallida exhibits peaks at
the retention times of those due to caftaric acid,
chlorogenic acid, echinacoside, and chicoric acid in the
chromatogram of the Standard solution. The chromato-
gram of the Sample solution prepared from Capsules la-
beled to contain £. purpurea exhibits peaks at the reten-
tion times of those due to caftaric acid, chlorogenic
acid, and chicoric acid in the chromatogram of the
Standard solution.
e C. HPLC FOR PRESENCE OF ALKYLAMIDES
Analysis; Proceed as directed in the test for Content of
Dodecatetraenoic Acid Isobutylamides.
Acceptance criteria: The chromatogram of the Sample
solution exhibits peaks at the retention times of dodeca-
tetraenoic acid isobutylamides in the chromatogram of
Standard solution B or Standard solution C, and the refer-
ence chromatogram provided with the lot of USP Pow-
dered Echinacea angustifolia Extract RS or USP Powdered
Echinacea purpurea Extract RS being used.
STRENGTH
e CONTENT OF TOTAL PHENOLS
Solution A: Phosphoric acid in water (0.1 in 100)
Solution B: Acetonitrile
Mobile phase: See Table 1.
Table 1
DS Monographs
Time Solution A Solution B
(min) (%) (%)
0 90 10
3 90 10
16 738 22
WZ 60 40
20 60 40
20.5 90 10
25 | 90 10
Solvent: Alcoho! and water (7:3)
Standard solution: 20 g/mL of USP Caftaric Acid RS,
20 ug/ml of USP Chlorogenic Acid RS, 20 g/mL of
cynarin (1,3-di-O-caffeoylquinic acid), 60 ug/mL of USP
Evhinacoside RS, and 40 pg/mL. of USP Chicoric Acid RS
in Solvent
Sample solution: Determine the total weight of 20
Capsules. Empty the Capsules, and combine and mix
their contents to obtain a homogenous composite.
Weigh the empty Capsule shells and calculate the aver-
age fill weight per Capsule. Transfer a portion of the
Capsule contents, equivalent to 150 mg of the labeled
USP 41
Echinacea Species Powder, to a round-bottom flask
equleer with a condenser. Add 30.0 mL of Solvent,
and heat under reflux for 20 min. Cool to room tem-
perature and pass througha filter of 0.45-4m or finer
pore size.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 330 nm
Column: 4.6-mm x 25-cm; 5-{um packing L1
Column temperature: 35°
Flow rate: 71.5 mL/min
Injection volume: 5 uL
System suitability
Sample: Standard solution
Suitability requirements
Resolution: NLT 3.0 between the cynarin and
echinacoside peaks, and NLT 1.0 between the caftaric
acid and chlorogenic acid peaks. [NoTr—The
echinacoside peak may be resolved in two compo-
nents. The relative retention times for caftaric acid,
chlorogenic acid, cynarin, echinacoside, and chicoric
acid are 0.7, 0.75, 0.9, 1.0, and 1.4, respectively.]
Relative standard deviation: NMT 2.5% for the sum
of the respective peaks in repeated injections
Analysis
Samples: Standard solution and Sample solution
Identify and measure areas of the peaks corresponding
to caftaric acid (C,3H120s), chlorogenic acid (CisHi3Os),
ae (CosH24012), echinacoside (C3sH4,020), and
chicoric acid (C22H;gO;2) in the Sample solution
chromatogram.
Calculate the quantity, in mg, of caftaric acid,
chlorogenic acid, cynarin, echinacoside, and chicoric
acid in each Capsule taken:
Result = (ru/rs) x Cs x Vx (Wav/W)
hy = peak area of a relevant analyte from the
Sample solution
Is = peak area of caftaric acid, chlorogenic acid,
narin, echinacoside, or chicoric acid from
the Standard solution
Cs = concentration of a relevant analyte in the
Standard solution (mg/mL)
V = volume of the solvent taken to prepare the
Sample solution (mL)
W.y = average Capsule fill weight (mg)
Ww = weight of the sample taken to prepare the
Sample solution (mg)
Calculate the percentage of total phenols, as the sum of
caftaric acid, chlorogenic acid, echinacoside, chicoric
acid, and cynarin in each Capsule taken:
Result = EP, x 100/L
PB = total combined content of caftaric acid,
chlorogenic acid, echinacoside, chicoric acid,
and cynarin as determined above (mq)
L = labeled amount of Echinacea Species Powder
(mg/Capsule)
Acceptance criteria: NLT 0.5%
PERFORMANCE TESTS
¢ DISINTEGRATION AND DISSOLUTION (2040), Disintegration:
Meet the requirements
¢ WEIGHT VARIATION (2091): Meet the requirements
SPECIFIC TESTS
¢ CONTENT OF DODECATETRAENOIC ACID ISOBUTYLAMIDES
[Note—This test is not applicable to Capsules containing
Echinacea pallida powder prepared from dried rhizome
and rake]
Mobile phase: Acetonitrile and water (55:45)
Standard solution A: 10 g/mL of USP 2E,4E-Hexadie-
noic Acid Isobutylamide RS in methanol
USP 41
Standard solution B: 5 ma/mL of USP Powdered Echi-
nacea angustifolia Extract RS in methanol. Sonicate to
dissolve, and pass throughafilter of 0.45-1m or finer
per size. [NoTe—Only ee when Capsules contain
‘chinacea angustifolia powder prepared from dried rhi-
zome and roots.]
Standard solution C: 5 mg/mL of USP Powdered Echi-
nacea purpurea Extract RS in methanol. Sonicate to dis-
solve and pass througha filter of 0.45-um or finer pore
size. [NoTE—Only prepare when Capsules do not con-
tain Echinacea angustifolia powder prepared from dried
rhizome and roots.]
Sample solution: Transfer a portion of the Capsule con-
tents, equivalent to 2500 mg of Echinacea Species Pow-
der, to a round-bottom flask equipped with a con-
denser, Add 80.0 mL of methanol, and heat under
reflux for 30 min. Cool to room temperature and pass
througha filter of 0.45-1m or finer pore size.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 254 nm
Column: 4.6-mm x 25-cm; 5-4im packing L1
Column temperature: 30°
Flow rate: 1.5 mL/min
Injection volume: 25 uL
System suitability
Samples: Standard solution A, Standard solution B, or
Standard solution C
Suitability requirements
Chromatogram similarity: The chromatogram of
Standard solution B or Standard solutionC is similar to
the reference chromatogram for alkamides provided
with the lot of the USP Powdered Echinacea angus-
tifolia Extract RS or USP Powdered Echinacea purpurea
Extract RS being used.
Resolution: NLT 1.0 between the dodecatetraenoic
acid isobutylamide peaks, Standard solution B or Stan-
dard solution C
Tailing factor: NMT 2.0 for the 2£,4£-hexadienoic
acid isobutylamide peak, Standard solution A
Relative standard deviation: NMT 2.5% for the 26,
4£-hexadienoic acid isobutylamide peak in replicate
injections, Standard solution A
Analysis
Samples: Standard solution A, Standard solution B, or
Standard solution C, and Sample solution
Using the chromatogram of Standard solution B or Stan-
dard solution C, and the reference chromatogram pro-
vided with the lot of USP Powdered Echinacea angus-
tifolia Extract RS or USP Powdered Echinacea purpurea
Extract RS being used, identify and measure the areas
of the 2£,4E8Z,10E- and 2F,4E,8Z,102Z-dodecatetra-
enoic acid isobutylamide peaks in the Sample solution
Siomategran.
Calculate the percentage of dodecatetraenoic acid iso-
butylamides in the labeled amount of Echinacea Spe-
cies Powder from the portion of Capsules taken:
Result = (r/rs) x (Cs x V/W) x (Way/L) x Fx 100
tr = sum of the peak areas of the relevant analytes
from the Sample solution
ns = peak area of 2E4E-hexadienoic acid
isobutylamide from Standard solution A
Cs = concentration of USP 2E,4£-Hexadienoic Acid
lsobutylamide RS in Standard solution A
(mg/mL)
V = volume of the solvent taken to prepare the
Sample solution (mL)
Ww = weight of the sample taken to prepare the
Sample solution (mg)
Way = average Capsule fill weight (mg/Capsule)
Dietary Supplements / Eleuthero 4597
L = labeled amount of Echinacea angustifolia
powder or Echinacea purpurea powder, both
prepared from dried rhizome and roots (mg/
Capsule)
F = response factor of dodecatetraenoic acid
isobutylamides relative to 2£,4E-hexadienoic
acid isobutylamide, 1.353
Acceptance criteria
For Capsules containing Echinacea angustifolia
powder prepared from dried rhizome and roots:
NLT 0.075%
For Capsules containing Echinacea‘ee powder
prepared from dried rhizome and roots: NLT
0.025%
For Capsules containing only Echinacea purpurea
pene prepared from dried aerial parts: NLT
0.01
CONTAMINANTS
e [MICROBIAL ENUMERATION TESTS (2021): The total aerobic
bacterial count does not exceed 104 cfu/g, and the total
en molds and yeasts count does not exceed 103
u/g.
edeca OF SPECIFIED MICROORGANISMS (2022), Test Proce-
dures, Test for Absence of Salmonella Species and Test for
Absence of Escherichia coli: Meet the requirements
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in well-closed contain-
ers, protected from light and moisture, and store in a
cool place.
e LABELING: The label states the official name and the
amount of Echinacea Species Powder in mg/Capsule.
e USP REFERENCE STANDARDS (11)
USP Caftaric Acid RS
USP Chicoric Acid RS
USP Chlorogenic Acid RS
USP Powdered Echinacea angustifolia Extract RS
USP Powdered Echinacea pallida Extract RS
USP Powdered Echinacea purpurea Extract RS
USP Echinacoside RS
USP 2£,4£-Hexadienoic Acid Isobutylamide RS
AUSPH?
Eleuthero Root and Rhizome
DEFINITION
Eleuthero Root and Rhizome is the dried rhizome with roots
sydesbouow sa
of Eleutherococcus senticosus (Rupr. & Maxim.) Maxim.
(Fam. Araliaceae) [syn. Acanthopanax senticosus (Rupr. &
Maxim.) Harms]. It contains NLT 0.08% of phenylpropa-
noid glucosides as the sum of eleutheroside B (Ci7H24Os),
also referred to as syringin, and eleutheroside E
(C34H4sO18), also referred to as syringaresinol diglucoside,
calculated on the dried basis.
IDENTIFICATION
e A. HPTLC FoR ARTICLES OF BOTANICAL ORIGIN (203)
Solvent: Alcohol and water (1:1)
Standard solution A: 1 mg/mL of USP Eleutheroside E
RS in methanol
Standard solution B: 1 mg/mL of USP Eleutheroside B
RS in methanol
Standard solution C: 0.1 g of USP Powdered Eleuthero
Extract RS in 5 mL of Solvent. Sonicate for 10 min, cen-
trifuge, and use the supernatant.
Sample solution: Transfer about 1 g of finely powdered
Eleuthero Root and Rhizome to a centrifuge tube, add
5 mL of Solvent, and mix well. Sonicate for 10 min.
Centrifuge or filter the solution, and use the superna-
tant or the filtrate.
 4598 Eleuthero / Dietary Supplements
Adsorbent: Chromatographic silica gel with an average
particle size of 5 um (HPTLC plates)
Application volume: 10 UL, as bands
Relative humidity: Condition the plate to a relative hu-
midity of 33%.
Temperature: Ambient, not to exceed 30°
Developing solvent system: Chloroform, methanol,
and water (35:15:2)
Developing distance: 6 cm
Derivatization reagent: To 18 mL of ice-cold methanol
slowly and carefully add 2 mL of sulfuric acid, and mix
well. Allow the mixture to adjust to room temperature.
Analysis
Samples: Standard solution A, Standard solution B,
Standard solution C, and Sample solution
Apply the Samples as bands and dry in air. Develop in a
saturated chamber and dry in air. Treat the plate with
Derivatization reagent. Heat the plate at 100° for 5
min, and examine under white light and under UV
light (365 nm).
Acceptance criteria: Under white light, the Sample so-
lution exhibits two brown bands due to eleutheroside E
and eleutheroside B at Rr values of about 0.34 and
0.45, corresponding in color and R; to the bands exhib-
ited by Standard solution A and Standard solution B, re-
spectively. The Sample solution also exhibits two addi-
tional brown bands near the application zone,
corresponding in color and R; to the bands exhibited by
Standard solution C. Other bands may be observed in
the Sample solution and Standard solution C chromato-
grams, Under UV light, the Sample solution shows a
rown band due to eleutheroside E corresponding in
color and R; to the band exhibited by Standard solution
A.
e B. HPLC: The Sample solution in the test for Content of
Eleutherosides B and E shows a peak at the retention time
corresponding to that of eleutheroside B in Standard solu-
tion B and a peak at the retention time corresponding to
that of eleutheroside E in Standard solution A.
COMPOSITION
e CONTENT OF ELEUTHEROSIDES B AND E
Solvent: Methanol and water (1:1)
Solution A: Acetonitrile and water (5:95)
Solution B: Acetonitrile and water (60:40)
Mobile phase: See Table 1.
Table 1
Time Solution A Solution B
(min) (%) (%)
DS Monographs
oO 97 3
5 97 3 CONTAMINANTS
30 60 40
31 5 9S.
45 5 95
45.1 97 3 o MICROBIAL ENUMERATION TESTS (2021): The total aerobic
60 97 3:
Standard solution A: 0.1 mg/mL of USP Eleutheroside
E RS in methanol. Transfer 2.0 mL to a 5-mL volumetric
flask, and dilute with Solvent to volume.
Standard solution B: 0.1 mg/mL of USP Eleutheroside
B RS in methanol. Transfer 2.0 mL to a 5-mL volumetric
flask, and dilute with Solvent to volume.
Standard solution C: 5.0 mg/mL of USP Powdered
Eleuthero Extract RS in Solvent. Sonicate for 30 min,
cool to room temperature, decant, and pass through a
nylon filter of 0.45-1m or finer pore size.
Sample solution: Transfer about 5.0 g of finely ground
Eleuthero Root and Rhizome, accurately weighed, to a
round-bottom flask equipped with a condenser. Add
50 mL of Solvent, and heat under reflux for 30 min.
Filter the supernatant through cotton wool into a
USP 41
100-mL volumetric flask. Transfer the cotton wool to
the round-bottom flask, and repeat the extraction
twice, using 22 mL of Solvent for each extraction. Filter
through cotton wool into the volumetric flask, wash the
residue and the cotton wool with Solvent, cool to room
temperature, dilute with Solvent to volume, and mix.
Before injection, pass through a nylon filter of 0.45-um
or finer pore size, discarding the first few milliliters of
the filtrate.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 220 nm
Column: 4.0-mm x 25-cm; 5-{um packing L1
Flow rate: 1 mL/min
Injection volume: 10 uL
System suitability
Samples: Standard solution B and Standard solution C
Suitability requirements
Chromatogram similarity: The chromatogram from
Standard solution C is similar to the reference chro-
matogram provided with the lot of USP Powdered
Eleuthero Extract RS being used.
Relative standard deviation: NMT 2.0% determined
from the eleutheroside B peak in replicate injections,
Standard solution B
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Identify the eleutheroside B and eleutheroside E peaks
in the Sample solution by comparison with the chro-
matograms of Standard solution B and Standard solu-
tion A, respectively.
Separately calculate the percentage of eleutheroside B
and eleutheroside E in the portion of Eleuthero Root
and Rhizome taken:
Result = (ru/rs) x Cs x (V/W) x 100
ty = peak area of the relevant analyte from the
Sample solution
Is = peak area of eleutheroside E or eleutheroside B
from Standard solution A or Standard solution
B, respectively
Gs = concentration of eleutheroside E or
eleutheroside B in Standard solution A or
Standard solution B, respectively (mg/mL)
Vv = volume of the Sample solution (mL)
Ww = weight of Eleuthero Root and Rhizome taken
to prepare the Sample solution (mg)
Acceptance criteria: NLT 0.08% on the dried basis
e ARTICLES OF BOTANICAL ORIGIN (561), Limits of Elemental
Impurities: Meets the requirements
e ARTICLES OF BOTANICAL ORIGIN (561), Pesticide Residue
Analysis: Meets the requirements
bacterial count does not exceed 105 cfu/g, the total com-
bined molds and yeasts count does not exceed 103 cfu/
g, and the bile-tolerant Gram-negative bacteria do not
exceed 103 cfu/g.
e ABSENCE OF SPECIFIED MICROORGANISMS (2022), Test Proce-
dures, Test for Absence of Salmonella Species and Test for
Absence of Escherichia coli: Meets the requirements
SPECIFIC TESTS
e BOTANICAL CHARACTERISTICS
Macroscopic: The rhizome is knotty and of edulis cy-
lindrical shape with a diameter of 15-40 mm. The
heartwood area is light brown, and the connecting
splint wood is pale yellow. The bark is pipon mately
2mm thick and is fonly affixed to the xylem. The sur-
face is gray-brown or black-brown, coarse, and longitu-
dinally valleculate and plicate. A broken rhizome is
USP 41 Dietary Supplements / Eleuthero 4599

coarse and fibrous, particularly inside the xylem. The Standard solution C: 0.1 g of USP Powdered Eleuthero
fractured surface of the bark shows short, thin fibers. Extract RS in 5 mL of Solvent. Sonicate for 10 min, cen-
Numerous roots spring from the underside of the rhi- trifuge, and use the supernatant.
zome. These roots are 35-150 mm long, cylindrical and Sample solution: 0.1 g of Eleuthero Root and Rhizome
knotty, with a diameter of 3-15 mm. The surface of the Dry Extract in 5 mL of Solvent. Sonicate for 10 min,
roots is gray-brown to black-brown, smoother than the centrifuge, and use the supernatant.
rhizome, and has longitudinal stripes. A 0.5-mm thin Adsorbent: Chromatographic silica gel with an average
bark is tightly affixed to the pale yellow xylem. A bro- particle size of 5 um (HPTLC plates)
ken root is sparsely fibrous and appears yellowish-gray Application volume: 10 UL, as bands
where the thin epidermis is flaked off. Relative humidity: Condition the plate to a relative hu-
Microscopic: The roots have five to seven rows of midity of 33%.
brown cork cells. Secretory canals with brown contents Temperature: Ambient, not to exceed 30°
appear in groups of four or five and are NMT 20 um in Developing solvent system: Chloroform, methanol,
diameter. Phloem fibers with thick lignified walls occur and water (35:15:2)
singly or in small groups; there are cluster crystals of Developing distance: 6 cm
calcium oxalate in the phloem parenchyma. Paren- Derivatization reagent: To 18 mL of ice-cold methanol
chymatous cells surround the secretory cells, and med- slowly and carefully add 2 mL of sulfuric acid, and mix
ullary ray cells contain small starch granules. The xylem well. Allow the mixture to adjust to room temperature.
shows reticulately thickened and pitted vessels. The rhi- Analysis
zome is similar to the roots except for larger secretory Samples: Standard solution A, Standard solution B,
canals, up to 25 um in diameter, and the presence of a Standard solution C, and Sample solution
pith with parenchymatous cells containing starch Apply the Samples as bands and dry in air. Develop in a
granules. saturated chamber and dry in air. Treat the plate with
e Loss ON DRYING (731) Derivatization reagent, heat at 100° for 5 min, and ex-
Analysis: Dry at 105° to constant weight. amine under white light and under UV light (365 nm).
Acceptance criteria: NMT 14.0% Acceptance criteria: Under white light, the Sample so-
¢ ARTICLES OF BOTANICAL ORIGIN (561), Methods of Analysis, lution exhibits two brown bands due to eleutheroside E
Total Ash: NMT 8.0% and eleutheroside B at R; values of about 0.34 and
e ARTICLES OF BOTANICAL ORIGIN (561), Methods of Analysis, 0.45, corresponding in color and R; to the bands exhib-
Water-Soluble Extractives, Method 2: NLT 4.0% ited by Standard solution A and Standard solution B, re-
© ARTICLES OF BOTANICAL ORIGIN (561), Methods of Analysis, spectively. The Sample solution also exhibits two addi-
Foreign Organic Matter. NMT 3.0% tional brown bands near the application zone,
corresponding in color and R; to the bands exhibited by
ADDITIONAL REQUIREMENTS Standard solution C. Other bands may be observed in
© PACKAGING AND STORAGE: Preserve in well-closed, light- the Sample solution and Standard solution C chromato-
resistant containers. rams. Under UV light, the Sample solution shows a
© LABELING: The label states the Latin binomial. rown band due to eleutheroside E corresponding in
e USP REFERENCE STANDARDS (11) color and R; to the band exhibited by Standard solution
USP Powdered Eleuthero Extract RS A.
USP Eleutheroside B RS e° B. HPLC: The Sample solution in the test for Content of
B-D-Glucopyranoside, 4-(3-hydroxy-1-propenyl)-2,6- Eleutherosides B and E shows a peak at a retention time
Bion ay corresponding to that of eleutheroside B in Standard solu-
CizH2409 = 372.37 tion B and a peak at a retention time corresponding to
USP Eleutheroside E RS that of eleutheroside E in Standard solution A.
B-D-Glucopyranoside, (tetrahydro-1 H,3H-furo(3,4-
c)furan-1,4-diyl)bis(2,6-dimethoxy-4,1-phenylene)bis-. COMPOSITION
C34H4sO18 = 742.70 © CONTENT OF ELEUTHEROSIDES B AND E
Solvent: Methanol and water (1:1)
Solution A: Acetonitrile and water (5:95)
Solution B: Acetonitrile and water (60:40)
Mobile phase: See Table 7.
Eleuthero Root and Rhizome Dry Oo
wv
Extract Table 1
ms
Time Solution A Solution B °
DEFINITION (min) (%) (%) os
Eleuthero Root and Rhizome Dry Extract is prepared from 0 97 3 a
Eleuthero Root and Rhizome using hydroalcoholic mix-
tures. The ratio of the starting crude plant material to Dry 5 97 3 ey
Extract is between 13:1 and 25:1. It contains NLT 0.8% of 30 60 40 >
phenylpropanoid glucosides as eleutheroside B (Ci7H24Os), 31 5 95 ra
also referred to as syringin, and eleutheroside E 45 5 95
(C34H4sO1), also referred to as syringaresinol diglucoside, 45.1 97 3
calculated on the anhydrous basis. It may contain added 60 97 3
substances.
Standard solution A: 0.1 mg/mL of USP Eleutheroside
IDENTIFICATION E RS in methanol. Transfer 2.0 mL to a 5-mL volumetric
e A. HPTLC For ARTICLES OF BOTANICAL ORIGIN (203) flask, and dilute with Solvent to volume.
Solvent: Alcohol and water (1:1) Standard solution B: 0.1 mg/mL of USP Eleutheroside
Standard solution A: 1 mg/mL of USP Eleutheroside E B RS in methanol. Transfer 2.0 mL to a 5-mL volumetric
RS in methanol flask, and dilute with Solvent to volume.
Standard solution B: 1 mg/mL of USP Eleutheroside B Standard solution C: 5.0 mg/mL of USP Powdered
RS in methanol Eleuthero Extract RS in Solvent. Sonicate for 30 min,
cool to room temperature, and decant. Before injection,
 4600 Eleuthero / Dietary Supplements USP 41

pass through a Ha filter of 0.45-um or finer pore
size, discarding the first few milliliters of the filtrate.
Sample solution: Transfer 500 mg of Eleuthero Root
and Rhizome Dry Extract, accurately weighed, to a
100-mL volumetric flask, add 80 mL of Solvent, and son-
icate for 30 min. Cool to room temperature, dilute with
Solvent to volume, and mix. Before injection, pass
through a nylon filter of 0.45-um or finer pore size,
discarding the first few milliliters of the filtrate.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 220 nm
Column: 4.0-mm x 25-cm; 5-um packing L1
Flow rate: 1 mL/min
Injection volume: 10 uL
System suitability
Samples: Standard solution B and Standard solution C
Suitability requirements
Chromatogram similarity: The chromatogram of
Standard solution C is similar to the reference chro-
matogram provided with the lot of USP Powdered
Eleuthero Extract RS being used.
Relative standard deviation: NMT 2.0% determined
from the eleutheroside B peak in replicate injections,
Standard solution B
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Identify the eleutheroside B and eleutheroside E peaks
in the Sample solution by comparison with the chro-
matograms of Standard solution B and Standard solu-
tion A, respectively, and measure the peak areas.
Separately calculate the percentage of eleutheroside B
and eleutheroside E in the portion of Eleuthero Root
and Rhizome Dry Extract taken:
Result = (ru/rs) x Cs x (V/W) x 100
tu = peak area of eleutheroside E or eleutheroside B
from the Sample solution
Is = peak area of eleutheroside E or eleutheroside B
from Standard solution A or Standard solution
B, respectively
Cs = concentration of eleutheroside E or
eleutheroside B in Standard solution A or
Standard solution B, respectively (mg/mL)
Vv = volume of the Sample solution (mL)
Ww = weight of Eleuthero Root and Rhizome D
Extract used to prepare the Sample solution
(mg)
Acceptance criteria: NLT 0.8% on the anhydrous basis
DS Monographs
© ARTICLES OF BOTANICAL ORIGIN (561), Methods of Analysis,
Total Ash: NMT 10.0%
ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Preserve in tight, light-resistant
containers.
© LABELING: The label states the Latin binomial and, follow-
ing the official name, the content of eleutherosides, the
extracting solvent used for preparation, and the ratio of
the starting crude plant material to Dry Extract. It meets
the requirements in Botanical Extracts (565), Preparations,
General Pharmacopeial Requirements, Labeling.
e USP REFERENCE STANDARDS (11)
USP Powdered Eleuthero Extract RS
USP Eleutheroside B RS
B-D-Glucopyranoside, 4-(3-hydroxy-1-propenyl)-2,6-
dimethoxyphenyl.
CizH24O. =3372.37
USP Eleutheroside E RS
B-D-Glucopyranoside, (tetrahydro-1 H,3H-furo(3,4-
c)furan-1,4-diyl)bis(2,6-dimethoxy-4,1-phenylene)bis-.
C34HacOig = 742.70
Eleuthero Root and Rhizome Dry
Extract Capsules
DEFINITION
Eleuthero Root and Rhizome Dry Extract Capsules contain
Eleuthero Root and Rhizome bry Extract. They contain
NLT 95% of the labeled amount of phenylpropanoid glu-
cosides as the sum of eleutheroside B (C;7H24Os), also re-
ferred to as syringin, and eleutheroside E (C34H46O18), also
referred to as syringaresinol diglucoside. They may con-
tain suitable added substances.
IDENTIFICATION
e° A. HPLC
Analysis: Proceed as directed in the test for Content of
Eleutherosides B and E.
Acceptance criteria: The chromatogram of the Sample
solution exhibits peaks at the retention times corre-
sponding to the peaks due to eleutheroside B and
eleutheroside E in the chromatogram of Standard solu-
tion B.
STRENGTH
e CONTENT OF ELEUTHEROSIDES B AND E
Extraction solvent: Methanol and water (6:4)

Solution A: 0.2% o-phosphoric acid in water

Solution B: Acetonitrile
CONTAMINANTS Mobile phase: See Table 1.

Delete the following: Table 1

°e HEAVY MeTALs (231), Method Il: NMT 20 ppme coricair- Time Solution A Solution B
Jan-2018)
(min) (%)_ (%)
e ARTICLES OF BOTANICAL ORIGIN (561), Pesticide Residue 0 90 10
Analysis: Meets the requirements 2. 90 10
© BOTANICAL EXTRACTS (565), Preparations, General Pharma- 20 70 30
copeial Requirements, Residual Solvents: Meets the 25 70 30
requirements 27 90 10
e MICROBIAL ENUMERATION TESTS (2021): The total aerobic
37 90 10
microbial count does not exceed 104 cfu/g. The total
combined yeasts and molds count does not exceed 103 Standard stock solution A: 0.025 mg/mL of USP
cfu/g. Eleutheroside B RS in methanol. Sonicate for 5 min to
°ABSENCE OF SPECIFIED MICROORGANISMS (2022), Test Proce- dissolve.
dures, Test for Absence of Salmonella Species and Test for Standard stock solution B: 0.1 mg/mL of USP Eleuther-
Absence of Escherichia coli: Meets the requirements oside E RS in methanol. Sonicate for 5 min to dissolve.
SPECIFIC TESTS Standard solution A: Transfer 2.0 mL of Standard stock
e WATER DETERMINATION (921), Method |, Method la: NMT solution A and 1.0 mL of Standard stock solution B to a
5.0%
USP 41 Dietary Supplements / Eleuthero 4601

10-mL volumetric flask, dilute with water to volume, Acceptance criteria: NLT 95%
and mix well.
Standard solution B: _ 1mg/mL of USP Powdered PERFORMANCE TESTS
Eleuthero Extract RS in Extraction solvent. Sonicate for e DISINTEGRATION AND DISSOLUTION (2040), Disintegration:
30 min and cool to room temperature. Dilute with Meet the requirements
water to a final concentration of 0.5 mg/mL. Before in- e WEIGHT VARIATION (2091): Meet the requirements
jection, pass through a PVDF membrane filter of 0.45-
CONTAMINANTS
um or finer pore size.
e MICROBIAL ENUMERATION TESTS (2021): The total aerobic
Sample solution: Determine the total weight of 20
Capsules. Open the Capsules and combine their con- bacterial count does not exceed 104 cfu/g, and the total
coe molds and yeasts count does not exceed 103
tents in an appropriate container. Weigh the empty cru/g.
Capsule shells and calculate the average fill weight per
e ABSENCE OF SPECIFIED MICROORGANISMS (2022), Test Proce-
Capsule. Transfer a portion of the Capsule contents,
equivalent to 0.5 mg of phenylpropanoid glucosides dures, Test for Absence of Salmonella Species and Test for
(sum of eleutheroside B and eleutheroside E) to a Absence of Escherichia coli: Meet the requirements
50-mL volumetric flask. Add 25 mL of Extraction solvent ADDITIONAL REQUIREMENTS
and sonicate for 30 min with occasional shaking. Shake © PACKAGING AND STORAGE: Preserve in well-closed contain-
the flask manually for 1 min, cool to room temperature, ers, protected from light and moisture, and store at
dilute with water to volume, mix well, and pass room temperature.
through a PVDF membrane filter of 0.45-um or finer e LABELING: The label states the Latin binomial and, follow-
pore size. ing the official name, the amount of phenylpropanoid
Chromatographic system glucosides, as the sum of eleutheroside B and eleuthero-
(See Chromatography (621), System Suitability.) side E, and the amount of Eleuthero Root and Rhizome
Mode: LC Dry Extract in mg/Capsule.
Detector: UV 220 nm e USP REFERENCE STANDARDS (11)
Column: 4.6-mm x 25-cm; 5-um packing L1 USP Powdered Eleuthero Extract RS
Column temperature: 35° USP Eleutheroside B RS
Flow rate: 0.8 mL/min USP Eleutheroside E RS
Injection volume: 20 wL
System suitability
Samples: Standard solution A and Standard solution B
Suitability requirements
Chromatogram similarity: The chromatogram from
Standard solution B is similar to the reference chro- Eleuthero Root and Rhizome Dry
matogram provided with the lot of USP Powdered Extract Tablets
Eleuthero Extract RS being used.
Relative standard deviation: NMT 2.0% for the DEFINITION
eleutheroside B and eleutheroside E peaks in replicate Eleuthero Root and Rhizome Dry Extract Tablets contain
injections, Standard solution A Eleuthero Root and Rhizome Dry Extract. They contain
Analysis NLT 95% of the labeled amount of phenylpropanoid glu-
Samples: Standard solution A, Standard solution B, and cosides as the sum of eleutheroside B (Ci7H24Os), also re-
Sample solution ferred to as syringin, and eleutheroside E (C34H4sOi8), also
pert the peaks corresponding to eleutheroside B and referred to as syringaresinol diglucoside. They may con-
eleutheroside E in the Sample solution chromatogram tain added substances.
by comparison with the chromatogram from Standard
solution A and the reference chromatogram provided IDENTIFICATION
with the lot of USP Powdered Eleuthero Extract RS be- e A. HPLC
ing used. Measure the areas of the analyte peaks. Analysis: Proceed as directed in the test for Content of
Calculate the quantity, in mg, of eleutheroside B and Eleutherosides B and E.
eleutheroside E in each Capsule taken: Acceptance criteria: The chromatogram of the Sample
solution exhibits peaks at the retention times corre-
Result = (ru/rs) x Cs x Vx (Wav/W) sponding to the peaks due to eleutheroside B and 9
eleutheroside E in the chromatogram of Standard solu-
ty = peak area of relevant eleutheroside from the tion B. cs
Sample solution o
Is = peak area of corresponding eleutheroside from STRENGTH rm
Standard solution A ¢ CONTENT OF ELEUTHEROSIDES B AND E a
Cs = concentration of relevant eleutheroside in Extraction solvent: Methanol and water (6:4) mn
Standard solution A (mg/mL) Solution A: 0.2% o-phosphoric acid in water ao}
V = volume of the solvent taken for preparation of Solution B: Acetonitrile Pe
the Sample solution (mL) Mobile phase: See Table 7. is
Way = average fill weight per Capsule (mg)
w = weight of the sample taken for preparation of Table 1
the Sample solution (mg)
Calculate the percentage of theTabeled amount of Time Solution A Solution B
phenylpropanoid glucosides, as the sum of (min) (%) (%)
eleutheroside B and eleutheroside E, in each Capsule 0 90 10
taken: 2 90 10
20 70 30
Result = (2Q/L) x 100 25 70 30
xQ; = sum of the quantities of eleutherosides as 27 90 10
determined above (mg) 37 90 10
L = labeled amount of phenylpropanoid
glucosides (mg)
 4602 Eleuthero / Dietary Supplements
Standard stock solution A: 0.025 mg/mL of USP
Eleutheroside B RS in methanol. Sonicate for 5 min to
dissolve.
Standard stock solution B: 0.1 mg/mL of USP Eleuther-
oside E RS in methanol. Sonicate for 5 min to dissolve.
Standard solution A: Transfer 2.0 mL of Standard stock
solution A and 1.0 mL of Standard stock solution B to a
10-mL volumetric flask, dilute with water to volume,
and mix well.
Standard solution B: 1 mg/mL of USP Powdered
Eleuthero Extract RS in Extraction solvent. Sonicate for
30 min and cool to room temperature. Dilute with
water to a final concentration of 0.5 mg/mL. Before in-
jection, pass through a PVDF membrane filter of 0.45-
um or finer pore size.
Sample solution: Weigh NLT 20 Tablets, determine the
average Tablet weight, and finely powder. Transfer a
ortion of Mies popieled Tablets, nominally equiva-
ent to 0.5 mg of phenylpropanoid glucosides (sum of
eleutheroside B and eleutheroside E) to a 50-mL volu-
metric flask. Add 25 mL of Extraction solvent and soni-
cate for 30 min with occasional shaking. Shake the flask
manually for 1 min, cool to room temperature, dilute
with water to volume, mix well, and pass through a
PVDF membrane filter of 0.45-m or finer pore size.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 220 nm
Column: 4.6-mm x 25-cm; 5-um packing L1
Column temperature: 35°
Flow rate: 0.8 mL/min
Injection volume: 20 pL
System suitability
Samples: Standard solution A and Standard solution B
Suitability requirements
Chromatogram similarity: The chromatogram from
Standard solution B is similar to the reference chro-
matogram provided with the lot of USP Powdered
Eleuthero Extract RS being used.
Relative standard deviation: NMT 2.0% for the
eleutheroside B and eleutheroside E peaks in replicate
injections, Standard solution A
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Identify the peaks corresponding to eleutheroside B and
eleutheroside E in the Sample solution chromatogram
by comparison with the chromatogram from Standard
solution A and the reference chromatogram provided
with the lot of USP Powdered Eleuthero Extract RS be-
fn!
“
ing used. Measure the areas of the analyte peaks.
a Calculate the quantity, in mg, of eleutheroside B and
i]
— eleutheroside E in each Tablet taken:
i)
3
= Result = (ru/rs) x Cs x Vx (Wav/W)
3 Sample solution: Transfer about 1 g of Eleuthero Root
= ty = peak area of relevant eleutheroside from the
al Sample solution
peak area of corresponding eleutheroside from
a or filter the solution, and use the supernatant or the
Standard solution A
Cs = concentration of relevant eleutheroside in
Standard solution A (mg/mL)
Vv = volume of the solvent taken for preparation of
the Sample solution (mL)
Wav = average Tablet weight (mg)
Ww = weight of the sample taken for preparation of
the Sample solution (mg)
Calculate the percentage of the labeled amount of
phenylpropanoid glucosides, as the sum of
eleutheroside B and eleutheroside E, in each Tablet
taken:
Result = (£Q/L) x 100
USP 41
=Q =sum of the quantities of eleutherosides as
determined above (mg)
L = labeled amount of phenylpropanoid
glucosides (mg)
Acceptance criteria: NLT 95%
PERFORMANCE TESTS
¢ DISINTEGRATION AND DISSOLUTION (2040), Disintegration:
Meet the requirements
© WEIGHT VARIATION (2091): Meet the requirements
CONTAMINANTS
© MICROBIAL ENUMERATION TESTS (2021): The total aerobic
bacterial count does not exceed 10* cfu/g, and the total
combined molds and yeasts count does not exceed 103
cfu/g.
© ABSENCE OF SPECIFIED MICROORGANISMS (2022), Test Proce-
dures, Test for Absence of Salmonella Species and Test for
Absence of Escherichia coli: Meet the requirements
ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Preserve in well-closed contain-
ers, protected from light and moisture, and store at
room temperature.
© LABELING: The label states the Latin binomial and, follow-
ing the official name, the amount of phenylpropanoid
glucosides, as the sum of eleutheroside B and eleuthero-
side E, and the amount of Eleuthero Root and Rhizome
Po Extract in mg/Tablet.
e USP REFERENCE STANDARDS (11)
USP Powdered Eleuthero Extract RS
USP Eleutheroside B RS
USP Eleutheroside E RS
Eleuthero Root and Rhizome Powder
DEFINITION
Eleuthero Root and Rhizome Powder is Eleuthero Root and
Rhizome reduced to a powder or very fine powder. It
contains NLT 0.08% of phenylpropanoid glucosides as the
sum of eleutheroside B (Ci7H240s), also referred to as syr-
ingin, and eleutheroside E (C34H46O18), also referred to as
syringaresinol diglucoside, calculated on the dried basis.
IDENTIFICATION
e A. HPTLC For ARTICLES OF BOTANICAL ORIGIN (203)
Solvent: Alcohol and water (1:1)
Standard solution A: 1 mg/mL of USP Eleutheroside E
RS in methanol
Standard solution B: 1 mg/mL of USP Eleutheroside B
RS in methanol
Standard solution C: 0.1 g of USP Powdered Eleuthero
Extract RS in 5 mL of Solvent. Sonicate for 10 min, cen-
trifuge, and use the supernatant.
and Rhizome Powder to a centrifuge tube, add 5 mL of
Solvent, and mix well. Sonicate for 10 min. Centrifuge
filtrate.
Adsorbent: Chromatographic silica gel with an average
particle size of 5 um (HPTLC plates)
Application volume: 10 uL, as bands
Relative humidity: Condition the plate toa relative hu-
midity of 33%.
Temperature: Ambient, not to exceed 30°
Developing solvent system: Chloroform, methanol,
and water (35:15:2)
Developing distance: 6 cm
Derivatization reagent To 18 mL of ice-cold methanol
slowly and carefully add 2 mL of sulfuric acid, and mix
well. Allow the mixture to adjust to room temperature.
USP 41
Analysis
Samples: Standard solution A, Standard solution B,
Standard solution C, and Sample solution
Apply the Samples as bands and dry in air. Develop in a
saturated chamber and dry in air. Treat the plate with
Derivatization reagent. Heat the plate at 100° for 5
min, and examine under white light and under UV
light (365 nm).
Acceptance criteria: Under white light, the Sample so-
lution exhibits two brown bands due to eleutheroside E
and eleutheroside B at Rr values of about 0.34 and
0.45, corresponding in color and R- to the bands exhib-
ited by Standard solution A and Standard solution B, re-
spectively. The Sample solution also exhibits two addi-
tional brown bands near the application zone,
corresponding in color and R; to the bands exhibited by
Standard solution C. Other bands may be observed in
the Sample solution and Standard solution C chromato-
grams: Under UV light, the Sample solution shows a
rown band due to eleutheroside E corresponding in
color and R; to the band exhibited by Standard solution
A.
e B. HPLC: The Sample solution in the test for Content of
Eleutherosides B and E shows a peak at the retention time
corresponding to that of eleutheroside B in Standard solu-
tion B and a peak at the retention time corresponding to
that of eleutheroside E in Standard solution A.
COMPOSITION
e CONTENT OF ELEUTHEROSIDES B AND E
Solvent: Methanol and water (1:1)
Solution A: Acetonitrile and water (5:95)
Solution B: Acetonitrile and water (60:40)
Mobile phase: See Table 7.
Table 1
Time Solution A Solution B
(min) (%) (%)
0 97 3 Acceptance criteria: NLT 0.08% on the dried basis
i 97 3
30 60 40
31 5 95
45 5 95
45.1 97 3 Analysis: Meets the requirements
60 97 3 e MICROBIAL ENUMERATION TESTS (2021): The total aerobic
Standard solution A: 0.1 mg/mL of USP Eleutheroside
E RS in methanol. Transfer 2.0 mL to a 5-mL volumetric
flask, and dilute with Solvent to volume.
Standard solution B: 0.1 mg/mL of USP Eleutheroside
B RS in methanol. Transfer 2.0 mL to a 5-mL volumetric
flask, and dilute with Solvent to volume.
Standard solution C: 5.0 mg/mL of USP Powdered
Eleuthero Extract RS in Solvent. Sonicate for 30 min,
cool to room temperature, decant, and pass through a
nylon filter of 0.45-um or finer pore size.
Sample solution: Transfer 5.0 g of Eleuthero Root and
Rhizome Powder, accurately weighed, to a round-bot-
tom flask equipped with a condenser. Add 50 mL of
Solvent, and heat under reflux for 30 min. Filter the
supernatant through cotton wool into a 100-mL volu-
metric flask. Transfer the cotton wool to the round-bot-
tom flask, and ee the extraction twice, using 22 mL
of Solvent for each extraction. Filter through cotton
wool into the volumetric flask, wash the residue and the
cotton wool with Solvent, cool to room temperature,
dilute with Solvent to volume, and mix. Before injection,
pass through a nylon filter of 0.45-um or finer pore
size, discarding the first few milliliters of the filtrate.
Chromatographic system
(See Chromatography (621), System Suitability.)
Dietary Supplements / Eleuthero 4602
Mode: LC
Detector: UV 220 nm
Column: 4.0-mm x 25-cm; 5-um packing L1
Flow rate: 1 mL/min
Injection volume: 10 uL
System suitability
Samples: Standard solution B and Standard solution C
Suitability requirements
Chromatogram similarity: The chromatogram from
Standard solution C is similar to the reference chro-
matogram provided with the lot of USP Powdered
Eleuthero Extract RS being used.
Relative standard deviation: NMT 2.0%, determined
from the eleutheroside B peak in replicate injections,
Standard solution B
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Identify the eleutheroside B and eleutheroside E peaks
in the Sample solution by comparison with the chro-
matograms of Standard solution B and Standard solu-
tion A, respectively.
Separately calculate the percentage of eleutheroside B
and eleutheroside E in the portion of Eleuthero Root
and Rhizome Powder taken:
Result = (ru/rs) x Cs x (V/W) x 100
tu = peak area of the relevant analyte from the
Sample solution
Is = peak area of eleutheroside E or eleutheroside B
from Standard solution A or Standard solution
B, respectively 7
Cs = concentration of eleutheroside E or
eleutheroside B in Standard solution A or
Standard solution B, respectively (mg/mL)
Vv = volume of the Sample solution (mL)
Ww = weight of Eleuthero Root and Rhizome Powder
taken to prepare the Sample solution (mg)
CONTAMINANTS
e ARTICLES OF BOTANICAL ORIGIN (561), Limits of Elemental
Impurities: Meets the requirements
e ARTICLES OF BOTANICAL ORIGIN (561), Pesticide Residue
bacterial count does not exceed 105 cfu/g, the total com-
bined molds and yeasts count does not exceed 103 cfu/
g, and the bile-tolerant Gram-negative bacteria do not
exceed 103 cfu/g.
e ABSENCE OF SPECIFIED MICROORGANISMS (2022), Test Proce-
sydeibouow $a
dures, Test for Absence of Salmonella Species and Test for
Absence of Escherichia coli: Meets the requirements
SPECIFIC TESTS
¢ BOTANICAL CHARACTERISTICS
Macroscopic: The powder is brown witha faint aro-
matic odor andaslightly acrid, persistent taste.
Microscopic: Groups of secretory canals with brown
contents are surrounded by parenchymatous cells con-
taining cluster crystals of calcium oxalate. The paren-
chymatous cells show small starch granules, thick-walled
lignified fibers, and fragments of reticulate and pitted
vessels. It turns bright yellow when mounted in sodium
hydroxide solution.
e Loss ON DRYING (731)
Analysis: Dry at 105° to constant weight.
Acceptance criteria: NMT 14.0%
e ARTICLES OF BOTANICAL ORIGIN (561), Methods of Analysis,
Total Ash: NMT 8.0%
ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Preserve in well-closed, light-
resistant containers.
 4604 Eleuthero / Dietary Supplements
e LABELING: The label states the Latin binomial.
© USP REFERENCE STANDARDS (11)
USP Powdered Eleuthero Extract RS
USP Eleutheroside B RS
B-D-Glucopyranoside, 4-(3-hydroxy-1-propenyl)-2,6-
dimethoxyphenyl.
Ci7zH24O9 372.37
USP Eleutheroside E RS
B-D-Glucopyranoside, (tetrahydro-1 H,3H-furo(3,4-
©)furan-1,4-diyl)bis(2,6-dimethoxy-4, 1-phenylene)bis-.
C3gHacOig = 742.70
Eleuthero Root and Rhizome Powder
Capsules
DEFINITION
Eleuthero Root and Rhizome Powder Capsules contain
Eleuthero Root and Rhizome Powder. They contain NLT
0.08% of phenylpropanoid glucosides as the sum of
eleutheroside B (Ci7H24Os), also referred to as syringin,
and eleutheroside E (C34H4sO1s), also referred to as syrin-
garesinol diglucoside, within the labeled amount of
Eleuthero Root and Rhizome Powder.
IDENTIFICATION
e A. HPTLC FOR ARTICLES OF BOTANICAL ORIGIN (203)
Standard solution A: 1 mg/mL of USP Eleutheroside E
RS in methanol
Standard solution B: 1 mg/mL of USP Eleutheroside B
RS in methanol
Standard solution C: 100 mg of USP Powdered
Eleuthero Extract RS in 5 mL of aqueous ethanol 50%.
Sonicate for 10 min, centrifuge, and use the
supernatant.
Sample solution: Transfer a finely powdered portion of
the contents of the Capsules, equivalent to 1 g of
Eleuthero Root and Rhizome Powder, to a centrifuge
tube. Add 5 mL of aqueous ethanol 50%, and mix well.
Sonicate for 20 min. Centrifuge and use the
supernatant.
Chromatographic system
Adsorbent: Chromatographic silica gel with an aver-
age particle size of 5 um (HPTLC plates)
Application volume: 10 uL, as bands
Relative humidity: Condition the plate to a relative
humidity of 33%.
Temperature: Ambient, not to exceed 30°
Developing solvent system: Chloroform, methanol,
DS Monographs
and water (35:15:2)
Developing distance: 6 cm
Derivatization reagent: To 18 mL of ice-cold metha-
nol, slowly and carefully add 2 mL of sulfuric acid, and
mix well. Allow the mixture to adjust to room
temperature.
Analysis
Samples: Standard solution A, Standard solution B,
Standard solution C, and Sample solution
Apply the Samples as bands and dry in air. Develop in a
saturated chamber, remove the plate from the cham-
ber, and dry in air. Treat the plate with Derivatization
reagent, heat at 100° for 5 min, and examine under
white light and UV light (365 nm).
Acceptance criteria: Under white light, the Sample so-
lution exhibits two brown bands due to eleutheroside E
and eleutheroside B at R; values of about 0.34 and
0.45, corresponding in color and R; to the bands exhib-
ited by Standard solution A and Standard solution B, re-
spectively. The Sample solution also exhibits two addi-
tional brown bands near the application zone,
corresponding in color and R; values to the bands ex-
hibited by Standard solution C. Other bands may be ob-
USP 41
served in the Sample solution and Standard solution C
chromatograms. Under UV light, the Sample solution
shows a brown band due to eleutheroside E corre-
sponding in color and Rr to the band exhibited by Stan-
lard solution A.
e B.HPLC: The chromatogram of the Sample solution ex-
hibits peaks at the retention times corresponding to the
peaks due to eleutheroside B and eleutherosideE in the
chromatogram of Standard solution B.
STRENGTH
e CONTENT OF ELEUTHEROSIDES B AND E
Extraction solvent: Methanol and water (6:4)
Solution A: 0.2% o-phosphoric acid in water
Solution B: Acetonitrile
Mobile phase: See Table 1.
Table 1
Time Solution A Solution B
(min) (%) (%)
0 90 10
2 90 10
20 70 30
25 70 30
27 90 10
37 90 10
Standard stock solution A: 0.025 mg/mL of USP
Eleutheroside B RS in methanol. Sonicate for 5 min to
dissolve.
Standard stock solution B: 0.1 mg/mL of USP Eleuther-
oside E RS in methanol. Sonicate for 5 min to dissolve.
Standard solution A: Transfer 2.0 mL of Standard stock
solution A and 1.0 mL of Standard stock solution B to a
10-mL volumetric flask, dilute with water to volume,
and mix well.
Standard solution B: 1 mg/mL of USP Powdered
Eleuthero Extract RS in Extraction solvent. Sonicate for
30 min and cool to room temperature. Dilute with
water to a final concentration of 0.5 mg/mL. Before in-
jection, pass through a PVDF membrane filter of 0.45-
um or finer pore size.
Sample solution: Determine the total weight of 20
Capsules. Open the Capsules and combine their con-
tents in an appioprate container. Weigh the euipty
Capsule shells and calculate the average fill weight per
Capsule. Transfer a portion of the Capsule contents,
equivalent to 1.5 g of Eleuthero Root and Rhizome
Powder, to a round-bottom flask equipped with a con-
denser. Add 25 mL of Extraction solvent and heat under
reflux for 30 min. Transfer the supernatant to a 100-mL
volumetric flask and repeat the extraction, using 25 mL
of Extraction solvent. Transfer the supernatant to a volu-
metric flask, wash the residue with water, transfer to a
volumetric flask, and cool to room temperature. Dilute
with water to volume, mix well, and pass through a
PVDF membrane filter of 0.45-41m or finer pore size.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 220 nm
Column: 4.6-mm x 25-cm; 5-um packing L1
Column temperature: 35°
Flow rate: 0.8 mL/min
Injection volume: 20 wL
System suitability
Samples: Standard solution A and Standard solution B
Suitability requirements
Relative standard deviation: NMT 2.0% for the
eleutheroside B and eleutheroside E peaks in replicate
injections, Standard solution A
Chromatogram similarity: The chromatogram from
Standard solution B is similar to the reference chro-
USP 41 Dietary Supplements / Evening Primrose 4605

matogram provided with the lot of USP Powdered Elm—see Elm General Monographs
Eleuthero Extract RS being used.
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Using the chromatograms of Standard solution A, Stan-
Ergocalciferol—see Ergocalciferol General
dard solution B, and the reference chromatogram pro- Monographs
vided with the lot of USP Powdered Eleuthero Extract
RS being used, identify the peaks corresponding to
eleutheroside B and eleutheroside E in the Sample solu-
tion. Measure the areas of the analyte peaks. Ergocalciferol Capsules—see Ergocalciferol
Calculate the quantity, in mg, of eleutheroside B and Capsules General Monographs
eleutheroside E in each Capsule taken:
Result = (ru/rs) x Cs x V x (Way/W)
tu = peak area of the relevant eleutheroside from Ergocalciferol Oral Solution—see
the Sample solution Ergocalcifero! Oral Solution General
rs = peak area of the corresponding eleutheroside
from Standard solution A Monographs
Cs = concentration of the relevant eleutheroside in
Standard solution A (mg/mL)
Vv = volume of the solvent taken for preparation of
the Sample solution (mL) Ergocalciferol Tablets—see Ergocalciferol
Way = average Capsule fill weight (mg) Tablets General Monographs
Ww = weight of the sample taken for preparation of
the Sample solution (mg)
Calculate the percentage of phenylpropanoid
glucosides, as the sum of eleutheroside B and Evening Primrose Oil
eleutheroside E, within the labeled amount of
Eleuthero Root and Rhizome Powder in each Capsule: [90028-66-3].
Result = (ZQ/L) x 100 DEFINITION
Evening Primrose Oil is derived from seeds of Oenothera
=Q; =sum of the quantities of eleutherosides as biennis L. The oil is extracted by cold press, where seeds
determined above (mg) are squeezed at very high pressure. It can be extracted
L = labeled amount of Eleuthero Root and using hexane asa solvent. It is then refined. A suitable
Rhizome Powder (mg) antioxidant may be added.
Acceptance criteria: NLT 0.08%
IDENTIFICATION
PERFORMANCE TESTS e A. It meets the requirements in Specific Tests for Fats and
© DISINTEGRATION AND DISSOLUTION (2040), Disintegration: Fixed Oils (401), Fatty Acid Composition.
Meet the requirements B. IDENTIFICATION OF FIXED OILS BY THIN-LAYER CHROMA-
© WEIGHT VARIATION (2091): Meet the requirements TOGRAPHY (202): The R; values of the principal spots of
the Sample solution correspond to those of the Standard
CONTAMINANTS solution.
© MICROBIAL ENUMERATION TESTS (2021): The total aerobic
bacterial count does not exceed 104 cfu/g, and the total IMPURITIES
=oh lle molds and yeasts count does not exceed 103
u/g.
e ABSENCE OF SPECIFIED MICROORGANISMS (2022), Test Proce- Delete the following:
dures, Test for Absence of Salmonella Species and Test for =]
Absence of Escherichia coli: Meet the requirements °e HEAVY METALS, Method I/ (231): NMT 10 Ug/ge comics 1. wn
Jan-2018)
ADDITIONAL REQUIREMENTS =
© PACKAGING AND STORAGE: Preserve in well-closed contain- SPECIFIC TESTS °
FATS AND FIXED OILS, Acid Value (401): NMT 1.0 =]
ers, protected from light and moisture, and store at °
room temperature. FATS AND FIXED OILS, Peroxide Value (401): NMT 5.0 2
FATS AND FIXED OILS, Saponification Value (401): 185-195 =
e LABELING: The label states the Latin binomial and, follow- sy
ing the official name, the amount of Eleuthero Root and Se FIXED OlLs, Unsaponifiable Matter (401): NMT mo]
Rhizome Powder in mg/Capsule. .' O a
7
e USP REFERENCE STANDARDS (11) FATS AND FIXED OILS, Fatty Acid Composition (401): Eve-
USP Eleutheroside B RS ning Primrose Oil exhibits the composition profile of fatty
USP Eleutheroside E RS acids in Table 1.
USP Powdered Eleuthero Extract RS
Table 1
Shorthand Percentage
Fatty Acid Notation (%)
Palmitic acid 16:0 4.0-10.0
Stearic acid 18:0 1.0-4.0
Oleic acid 18:1 5.0-12.0
Gamma-linolenic acid 18:3 7.0-14.0
Linoleic acid 18:2 65.0-85.0
 4606 Evening Primrose / Dietary Supplements USP 41

e REFRACTIVE INDEX (831): 1.477-1.479 at 20° tared 30-mL screw-top glass centrifuge tube and weigh
accurately. Re-tare and accurately weigh about 40 mg
ADDITIONAL REQUIREMENTS of the Internal standard into the same tube. Add 2 mL
© PACKAGING AND STORAGE: Preserve in tight containers, of 0.5 N methanolic sodium hydroxide solution, tightly
preferably under an atmosphere of an inert gas, pro- cap, and transfer to a heating block or another appro-
tected from light. priate heating device. Reflux the solution until fat glob-
e LABELING: Where Evening Primrose Oil is intended for use ules disappear (usually 5-10 min). Add 2 mL of 0.14 g/
in the manufacture of dosage forms, it is so labeled. mL boron trifluoride in methanol, cap, and reflux for 2
min. Add 4 mL of chromatographic n-heptane, cap, and
Delete the following: reflux for 1 min. Cool, add about 8 mL of saturated
sodium chloride solution, shake, and centrifuge to sepa-
°° USP REFERENCE STANDARDS (11) rate the layers. Dilute an aliquot of the upper (heptane)
USP Evening Primrose Oil RS eet with chromatographic n-heptane (1:8) and mix
@ (CN 1-May-2018)
well.
System suitability solution: Using about 80 mg of USP
Evening Primrose Oil RS, proceed as directed for the
Sample solution, beginning with “Transfer 80 mg” but
without the addition of the Internal standard.
Standard solution: Accurately weigh about 20 mg of
Evening Primrose Oil Capsules USP Methyl Oleate RS, 70 mg of USP Methyl Linoleate
RS, and 20 mg of USP Methyl Linolenate RS directly
DEFINITION into a tared 30-mL screw-top less centrifuge tube. Pro-
Evening Primrose Oil Capsules are prepared with Evening ceed as directed for the Sample le solution, beginning with
Primrose Oil and contain NLT 95.0% of the labeled “Re-tare”.
amounts of each, y-linolenic (C18:3 n-6), linoleic (C18:2 Chromatographic system
n-6), and oleic (C18:1 n-9) acids. (See Chromatography (621), System Suitability.)
Mode: GC
IDENTIFICATION Detector: Flame ionization
© A. FATTY ACID PROFILE Column: 0.53-mm x 30-m fused silica capillary; coated
System suitability solution and Chromatographic sys- with a 1.0-uum film of G16
tem: Proceed as directed in Strength. Temperatures
Sample solution: Proceed as directed for the Sample Injection port: 220°
solution in Strength, beginning with “Transfer 80 mg” Detector: 260°
but without the addition of the Internal standard. Column: See Table 2.
Analysis: Identify the specified fatty acid methyl ester
peaks by comparing them to the reference chromato-
gram provided with the lot of USP Evening Primrose Oil Table 2
RS being used. Determine the percentage of each con- Hold Time
stituent relative to the total integrated area. Initial Temperature Final at Final
Acceptance criteria: The Sample solution conforms to Temperature Ramp Temperature| Temperature
the fatty acids composition profile in Table 7. ©) (¢/min) «°) (min)
70 0 70 2
Table 1 70 5 240 5
Shorthand Percentage Carri : Heli
Fatty Acid Notation (%) ATHIEL Jase, Teun
——_ Tél 70.0 Linear velocity: 50 cm/s
Balmnitie acid 6:0 4.0-10. Split mode: Splitless
Stearic acid 18:0 1.0-4.0 Injection volume: 1 pL
Oleic acid 18:1 n-9 5.0-12.0 System suitability
Linoleic acid 18:2 n-6 65.0-85.0 Samples: System suitability solution and Standard
4 yeLinolenic acid 18:3 n-6 7.0-14.0 solution ¢
ot Suitability requirements
ry Chromatogram similarity: The System suitability solu-
nS STRENGTH tion chromatogram is similar to the reference chro-
> matogram provided with the lot of USP Evening
[= . Primrose Oil RS being used.
S Change to read: Resolution: NLT 1.5 between methyl stearate and
e CONTENT OF y-LINOLENIC, LINOLEIC, AND OLEIC ACIDS
methyl
'
oleate, System aero
suitability solution
es [Note—y-Linolenic acid is quantitated against USP Relative standard deviation: NMT 2% for the peak
area ratios of specified analytes to the internal stan-
Methyl Linolenate RS.] dard, Standard solution
0.5 N methanolic sodium hydroxide solution: Dis- Analysis
solve 2 g of sodium hydroxide in 100 mL of methanol.
Samples: Sample solution and Standard solution
Internal standard: Methyl pentadecanoic acid [Nott—The relative retention times for y-methy| linole-
Sample solution: Weigh NLT 10 Capsules. With a sharp nate and a-methyl linolenate are about 0.98 and 1.0,
blade, carefully slice open the Capsules, avoiding loss of respectively.]
shell material. Combine the Capsule contents in a suita- Identify the retention times of the relevant fatty acid
ble container and mix well. Remove any adhering sub- methyl esters by comparing the peaks in the chromat-
stance from the ee Capsules by washing with sev- ogram of the System suitability solution with those in
eral portions of diethyl ether, and discard the washings. the reference chromatogram. Identify the locus for the
Allow the empty Capsule shells to air-dry over a period internal standard peak by comparison of the chromat-
of NMT 30 min, taking precautions to avoid uptake or ograms of the Standard solution and the System suita-
loss of moisture. Weigh the empty Capsule shells and bility solution.
calculate the average fill weight/Capsule (A,). Transfer
80 mg of the combined Capsule contents directly into a
USP 41 Dietary Supplements / Fenugreek 4607

Calculate the content, in mg/g, of y-linolenic, linoleic, e USP REFERENCE STANDARDS (11)
and oleic acids in the portion of Capsule content USP Evening Primrose Oil RS
taken: USP Methyl Linoleate RS
USP Methyl Linolenate RS
Result = (Ru/Rs) x (Au/As) x (ms/W) x (Mr/M,2) USP Methyl Oleate RS
Ru = peak area ratio of the relevant methyl ester
peak to the internal standard peak from the
Sample solution
Rs = peak area ratio of the relevant methyl ester Fenugreek Seed
peak to the internal standard peak from the
Standard solution
Au = weight of the Internal standard in the Sample DEFINITION
solution (mg) Fenugreek Seed consists of the dried ripe seeds of Trigonella
As = weight of the Internal standard in the Standard foenum-graecum L. (Fam. Fabaceae). It contains NLT 0.2%
elution (mg) of 4-hydroxyisoleucine, calculated on the dried basis.
°ms = weight of the relevant USP Methyl Ester RS in IDENTIFICATION
the Standard solution (Mg)e car i-jun-2017) e A. HPTLC FoR ARTICLES OF BOTANICAL ORIGIN (203)—
Ww = sample weight used to prepare the Sample AMINO ACID PROFILE
solution (g Standard solution A: 0.5 mg/mL of USP 4-Hydroxy-
Mn = molecular weight of the relevant fatty acid (g/ isoleucine RS in an ethanol and water (7:3) mixture
mol) Standard solution B: 50 mg/mL of USP Trigonella
Mz = molecular weight of the relevant fatty acid Foenum-graecum Seed Dry Extract RS in an ethanol
methyl ester (g/mol) and water (7:3) mixture. Sonicate for 10 min, centri-
Calculate the percentage of the labeled amounts of fuge, and use the supernatant.
each (y-linolenic, linoleic, and oleic) acid in the portion Sample solution: Suspend about 1 g of Fenugreek
of Capsules taken: Seed, finely powdered, in 5 mL of an ethanol and water
(7:3) mixture, and incubate at 50° for 15 min. Centri-
Result = A x Ar x 100/L
fuge, and use the supernatant.
A = content of the relevant fatty acid in the [NotE—Standard solution B and the Sample solution may
portion of Capsule content taken (mg/g) also be used for Identification test B and for Specific
Ar = average fill weight (g/Capsule) Tests, Presence of Trigonelline.]
L = labeled content of the relevant fatty acid (mg/ Chromatographic system
Capsule) Adsorbent: Chromatographic silica gel with an aver-
Acceptance criteria age particle size of 5 um (HPTLC plate)!
y-Linolenic, linoleic, and oleic acids: NLT 95.0% of Application volume: 2 uL each of Standard solution A
the labeled amount of each and Standard solution B, and 4 uL of the Sample solu-
tion, as 8-mm bands
PERFORMANCE TESTS Relative humidity: Condition the plate toa relative
e DISINTEGRATION AND DISSOLUTION (2040): Meet the re- humidity of 33% using a suitable device.
quirements in Rupture Test for Soft Shell Capsules Temperature: Ambient, not to exceed 30°
e WEIGHT VARIATION (2091): Meet the requirements Developing solvent system: A mixture of n-butanol,
acetic acid, and water (7:2:1)
SPECIFIC TESTS Derivatization reagent: A solution of 0.3% ninhydrin
e FATS AND FIXED OILS, Peroxide Value (401): NMT 10.0 a a pure of isopropanol and glacial acetic acid
19:1
CONTAMINANTS System suitability
e MICROBIAL ENUMERATION TESTS (2021): The total aerobic Samples: Standard solution A and Standard solution B
microbial count does not exceed 1 x 103 cfu/g, and the Suitability requirements: Under white light, the der-
combined molds and yeasts count does not exceed ivatized chromatogram of Standard solution B displays,
3 x 10? cfu/g. in its lower half, five or six brown bands; the darkest
sydesbouo; sa

e ABSENCE OF SPECIFIED MICROORGANISMS (2022): Meet the band corresponding to the 4-hydroxyisoleucine band
requirements of the tests for the absence of Salmonella in the chromatogram of Standard solution A. Under
species and Escherichia coli long-wave UV (365 nm), the derivatized chromato-
ram of Standard solution B exhibits, in its lower half,
ADDITIONAL REQUIREMENTS our or five brown bands; the darkest band corre-
¢ PACKAGING AND STORAGE: Preserve in tightly-closed, light- sponding to the 4-hydroxyisoleucine band in the chro-
resistant containers. matogram of Standard solution A. In the upper half of
e LABELING: The label states the article that the Capsules
were prepared with and the content of y-linolenic, lino- the chromatogram, three diffuse yellow to orange-yel-
low bands are seen, the middle one being the most
leic, and oleic acids in mg/Capsule. intense.
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Apply the samples as bands, and dry in air. Condition
at relative humidity at about 33%. Develop in a satu-
rated chamber, until the solvent front has migrated
over a path of 6 cm. Air-dry, treat with Derivatization
reagent, heat for 3 min at 105°, and immediately ex-
amine under white light and under long-wave UV light
(365 nm).
‘Suitable commercially available plates are HPTLC Silica Gel 60 F2s4 from EMD
Millipore (e.g., Part No. 1.05642.0001).
 4608 Fenugreek / Dietary Supplements USP 41

Acceptance criteria: Under white light, the derivatized diffuse band. In the upper third of the chromatogram,
chromatogram of the Sample solution appears mono- two well-defined bands of medium intensity are ob-
chromatic, with the bands differing in intensity, but not served. Under UV light (365 nm), the lower half of the
the color, which is uniformly reddish-brown. In the chromatogram features three or four deep-blue fluores-
lower third of the plate, two or three thin bands are cent bands of varying intensity interspersed with
seen, proximate to the origin, followed by a very in- greyish-brown zones.
tense band due to 4-hydroxyisoleucine, coincident with
the corresponding bands in Standard solution A and COMPOSITION
Standard solution B, and comparable in intensity to the ¢ CONTENT OF 4-HYDROXYISOLEUCINE
band in the Standard solution B chromatogram. Two Solution A: 0.1% Phosphoric acid in water
lightly colored bands, one just above the 4-hydroxy- Solution B: Acetonitrile
isoleucine band, another further upwards, are seen. The Mobile phase: See Table 1.
upper half of the plate is devoid of discernible features.
Under long-wave UV (365 nm), the derivatized chro- Table 1
matogram of the Sample solution exhibits, in its lower
Time Solution A Solution B
half, two or three light reddish-brown bands followed
by the darker-brown intense band due to 4-hydroxy- (min) (%) (%)
isoleucine, coincident with the corresponding bands in 0 80.0 20.0
Standard solution A and Standard solution B. Above it, 20 40.0 60.0
two lighter-brown and somewhat diffuse bands appear. 21 80.0 20.0
In the upper third of the plate, three yellowish-orange 25 80.0 20.0
bands are seen, corresponding in position and color to
those observed in Standard solution B. Diluent: Methanol and water (1:1)
e B. HPTLC For ARTICLES OF BOTANICAL ORIGIN (203)—STER- Reagent: A mixture of acetonitrile, water, and triethyl-
OIDAL SAPONINS PROFILE amine (10:3:2)
Standard solution: 50 mg/mL of USP Trigonella Standard solution: Transfer about 4.0 mg of USP
Foenum-graecum Seed Dry Extract RS in an ethanol 4-Hydroxyisoleucine RS, accurately weighed, into a
and water (7:3) mixure. Sonicate for 10 min, centri- 50-mL volumetric flask, and dissolve in 5 mL of the Dilu-
fuge, and use the supernatant. ent. Add 10 mL of Reagent and 0.5 mL of phenyl isothi-
Sample solution: Suspend about 1 g of Fenugreek ocyanate, and shake for 5 min. Add 30 mL of methanol,
Seed, finely powdered, in 5 mL of an ethanol and water adjust with water to volume, and mix well.
(7:3) mixture, and incubate at 50° for 15 min. Centri- Sample stock solution: Transfer about 2.0 g of Fenu-
fuge, and use the supernatant. greek Seed, finely powdered and accurately weighed,
[NoTe—The Standard solution and Sample solution may into a centrifuge tube. Add 8 mL of Diluent, place on a
also be used for Identification test A and for Specific water bath at 65° for 5 min, sonicate for 5 min, and
Tests, Presence of Trigonelline.] centtuge, Retain the supernatant, and repeat extrac-
Chromatographic system tion with 8 mL of Diluent two more times. Combine all
Adsorbent: Chromatographic silica gel with an aver- three extracts in the 25-mL volumetric flask, dilute with
age particle size of 5 um (HPTLC plate) Diluent to volume, and mix well.
Application volume: 2 pL, as 8-mm bands Sample solution: Transfer 5.0 mL of Sample stock solu-
Relative humidity: Condition the plate toa relative tion into a 50-mL volumetric flask, add 10 mL of Rea-
humidity of 33% using a suitable device. gent and 0.5 mL of phenyl isothiocyanate, and shake for
Temperature: Ambient, not to exceed 30° 5 min. Add 30 mL of methanol, dilute with water to
Developing solvent system: A mixture of dichloro- volume, and mix well. Pass through a nylon filter hav-
methane, methanol, and water (18:8:1) ing a 0.45-um or finer pore size, discarding the initial
Derivatization reagent: A mixture of methanol, gla- few mL of the filtrate.
cial acetic acid, sulfuric acid, and p-anisaldehyde Chromatographic system
(170:20:10:1). Prepare on an ice bath, and mix well. (See Chromatography (621), System Suitability.)
System suitability Mode: HPLC
Sample: Standard solution Detector: UV 254 nm
Suitability requirements: Under white light, the der- Column: 4.6-mm x 15-cm; 5-4um packing L1
DS Monographs

ivatized chromatogram of the Standard solution exhib- Column temperature: Ambient

its, in its lower half, three or four lightly-shaded bands. Flow rate: 1.5 mL/min
Under long-wave UV (365 nm), the derivatized chro- Injection volume: 20 LL
matogram of the Standard solution exhibits, in its System suitability
lower third, two diffuse bands of blue fluorescence, Sample: Standard solution
and another blue fluorescent band in the middle of Suitability requirements
the plate. Tailing factor: NMT 2.0 for the 4-hydroxyisoleucine
Analysis peak, Standard solution
Samples: Standard solution and Sample solution Relative standard deviation: NMT 2.0% determined
Apply the Samples as bands, and dry in air. Condition for the 4-hydroxyisoleucine peak in replicate injec-
at relative humidity at about 33%. Develop in a satu- tions, Standard solution
rated chamber, until the solvent front has migrated Analysis
approximately 6 cm. Air-dry, treat with Derivatization Samples: Standard solution and Sample solution
reagent, heat for 3 min at 105°, and immediately ex- Using the chromatogram of the Standard solution, iden-
amine under white light and under long-wave UV light tify the retention time of the peak corresponding to
(365 nm). 4-hydroxyisoleucine in the Sample solution
Acceptance criteria: Under white light, the chromato- chromatogram.
gram of the Sample solution, in its lower third, shows Calculate the percentage of 4-hydroxyisoleucine in the
two medium-intensity bands immediately next to the portion of Fenugreek Seed taken:
application line. Further upwards, two closely spaced,
less intense bands are followed by a more prominent Result = (ru/rs) x Cs x (V/W) x D x 100
band, and another pair of closely spaced lighter bands,
which may merge. In the middle third of the chromato- ru = peak area of 4-hydroxyisoleucine from the
gram, a medium-intensity band is followed by a faint Sample solution

peak area of 4-hydroxyisoleucine from the
Standard solution
Cs = concentration of USP 4-Hydroxyisoleucine RS
in the Standard solution (mg/mL)
Vv = volume of the Sample stock solution (mL)
w = weight of Fenugreek Seed taken to prepare
the Sample stock solution (mg)
D = dilution factor to prepare the Sample solution
from the Sample stock solution, 10
Acceptance criteria: NLT 0.2% on the dried basis
CONTAMINANTS
e ELEMENTAL IMPURITIES—PROCEDURES (233)
Acceptance criteria
Arsenic: NMT 2.0 ug/g
Cadmium: NMT 1.0 ng/g
Lead: NMT 10.0 ug/g
Mercury: NMT 1.0 ug/g
© ARTICLES OF BOTANICAL ORIGIN, Pesticide Residue Analysis
(561): Meets the requirements
e MICROBIAL ENUMERATION TESTS (2021): The total aerobic
bacterial count does not exceed 105 cfu/g, the total com-
bined molds and yeasts count does not exceed 103 cfu/
g, and the bile-tolerant Gram-negative bacterial count
does not exceed 103 cfu/g.
© ABSENCE OF SPECIFIED MICROORGANISMS (2022): Meets the
requirements of the tests for absence of Salmonella spe-
cies and Escherichia coli
SPECIFIC TESTS
© HPTLC For ARTICLES OF BOTANICAL ORIGIN (203)—PRES-
ENCE OF TRIGONELLINE
Standard solution A: 1.5 mg/mL of USP Trigonelline RS
in an ethanol and water (7:3) mixture
Standard solution B: 50 mg/mL of USP Trigonella
Foenum-graecum Seed Dry Extract RS in an ethanol
and water (7:3) mixture.sonata for 10 min, centri-
fuge, and use the supernatant.
Sample solution: Suspend about 1 g of Fenugreek
Seed, finely powdered, in 5 mL of an ethanol and water
(7:3) mixture, and incubate at 50° for 15 min. Centri-
fuge, and use the supernatant.
[NoTt—Standard solution B and the Sample solution ma
also By used for Identification test A and for Identification
test B.
Chromatographic system
Adsorbent: Chromatographic silica gel with an aver-
age particle size of 5 um (HPTLC plate)
Application volume: 5 uL, as 8-mm bands
Relative humidity: Condition the plate toa relative
humidity of 33% using a suitable device.
Temperature: Ambient, not to exceed 30°
Developing solvent system: A mixture of isopropyl al-
cohol, methanol, and water (4:1:4)
System suitability
Samples: Standard solution A and Standard solution B
Suitability requirements: Under short-wave UV light
(254 nm), the chromatogram of Standard solution B
displays, in its lower half, a quenching band coincident
with that of the trigonelline band in the chromato-
gram of Standard solution A.
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Apply the Samples as bands, and dry in air. Condition
at relative humidity at about 33%. Develop in a satu-
rated chamber, until the solvent front has migrated
over a path of 6 cm. Air-dry, and examine under short-
wave UV light (254 nm).
Acceptance criteria: Under short-wave UV light, the
chromatogram of the Sample solution displays a
quenching band corresponding to the trigonelline band
in the chromatograms of Standard solution A and Stan-
dard solution B.
Dietary Supplements / Fenugreek 4609
¢ BOTANICAL CHARACTERISTICS
Macroscopic: The seeds are oblong, 3-5 mm long,
2-3 mm wide, 1.5-2.0 mm thick, with rounded corners,
smooth, dull yellowish-brown to reddish-brown. They
are flattened and have a very characteristic rhomboidal
outline. Nearly in the center of one of the long, narrow
sides is a small depression in which both hilum and
micropyle are situated, the former being distinctly visi-
ble as a whitish point. This depression is continued in
the form of a furrow running diagonally across part of
each of the adjoining sides, dividing the seed into two
unequal lobes. A cut made transversely to pass through
both lobes reveals two accumbent cotyledons in the
larger lobe, and the radicle in the smaller lobe. Both are
yellowish in color, and surrounded bya darker, horny,
translucent endosperm, which also separates the radicle
from the cotyledons.
Microscopic: Transverse section shows an epidermis of
alisade cells, one layer, with thick cuticle and thick
amellated walls, and a relatively large lumen at the
lower part. Longitudinal pit-canals fine and close.
Subepidermal layer of basket-like cells, with bar-like
thickening on the radial walls, followed by a paren-
chymatous layer. Endosperm consists of a layer of thick-
walled cells containing aleurone grains, several layers of
polyhedral cells with stratified mucilaginous contents
and thickened walls. Cotyledons of parenchymatous
cells containing fixed oil globules and aleurone grains
up to 15 um in diameter. The paren of the testa
composed of several layers of thin-walled cells which
appear similar in sectional view but in surface view the
ee show structural differences: some are composed
of elongated rectangular cells with slightly thickened
and beaded walls; other layers include thin-walled poly-
gonal cells which may be very irregular in size or may
enclose irregular intercellular spaces.
e ARTICLES OF BOTANICAL ORIGIN, Foreign Organic Matter
(561): NMT 2.0%
e LOSS ON DRYING (731)
Sample: 1g
Analysis: Dry the Sample at 105° for 2 h.
Acceptance criteria: NMT 12.0%
e ARTICLES OF BOTANICAL ORIGIN, Total Ash (561): NMT
5.0%
@ ARTICLES OF BOTANICAL ORIGIN, Alcoho/-Soluble Extractives,
Method 1 (561): NLT 5.0%
e ARTICLES OF BOTANICAL ORIGIN, Water-Soluble Extractives,
Method 1 (561): NLT 9.0%
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in well-closed contain-
ers, protected from light and moisture, and store at
sydeibouo=: sq
room temperature.
e LABELING: The label states the Latin binomial and, follow-
ing the official name, the part of the plant from which
the article was derived.
e USP REFERENCE STANDARDS (11)
USP 4-Hydroxyisoleucine RS
USP Trigonella Foenum-graecum Seed Dry Extract RS
USP Trigonelline RS
Fenugreek Seed Powder
DEFINITION
Fenugreek Seed Powder consists of the dried ripe seeds of
Trigonella foenum-graecum L. (Fam. Fabaceae), reduced to
powder or very fine powder. It contains NLT 0.2% of
4-hydroxyisoleucine, calculated on the dried basis.
 4610 Fenugreek / Dietary Supplements
IDENTIFICATION
e A. HPTLC FOR ARTICLES OF BOTANICAL ORIGIN (203)
Standard solution A: 0.5 mg/mL of USP 4-Hydroxy-
isoleucine RS in an ethanol and water (7:3) mixture
Standard solution B: 50 mg/mL of USP Trigonella
Foenum-graecum Seed Dry Extract RS in an ethanol
and water (7:3) mixture. Sonicate for 10 min, centri-
fuge, and use the supernatant.
Sample solution: Suspend about 1 g of Powder in 5 mL
of an ethanol and water (7:3) mixture, and incubate at
50° for 15 min. Centrifuge, and use the supernatant.
[Not&—Standard solution B and the Sample solution may
also be used for /dentification test B and for Specific
Tests, Presence of Trigonelline.]
Chromatographic system
Adsorbent: Chromatographic silica gel with an aver-
age particle size of 5 um (HPTLC plate)!
Application volume: 2 uL each of Standard solution A
and Standard solution B, and 4 uL of the Sample solu-
tion, as 8-mm bands
Relative humidity: Condition the plate to a relative
humidity of 33% using a suitable device.
Temperature: Ambient, not to exceed 30°
Developing solvent system: A mixture of n-butanol,
acetic acid, and water (7:2:1)
Derivatization reagent: A solution of 0.3% ninhydrin
in a mixture of isopropanol and glacial acetic acid
(19:1)
System suitability
Samples: Standard solution A and Standard solution B
Suitability requirements: Under white light, the der-
ivatized chromatogram of Standard solution B displays,
in its lower half, five or six brown bands; the darkest
band corresponding to the 4-hydroxyisoleucine band
in the chromatogram of Standard solution A. Under
long-wave UV (365 nm), the derivatized chromato-
gram of Standard solution B exhibits, in its lower half,
four or five brown bands; the darkest band corre-
sponding to the 4-hydroxyisoleucine band in the chro-
matogram of Standard solution A. In the upper half of
the chromatogram, three diffuse yellow to orange-yel-
low bands are seen, the middle one being the most
intense.
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Apply the Samples as bands, and dry in air. Condition
at relative humidity at about 33%. Develop in a satu-
rated chamber, until the solvent front has migrated
over a path of 6 cm. Air-dry, treat with Derivatization
reagent, heat for 3 min at 105°, and immediately ex-
DS Monographs
amine under white light and under long-wave UV light
(365 nm).
Acceptance criteria: Under white light, the derivatized
chromatogram of the Sample solution appears mono-
chromatic, with the bands differing in intensity, but not
the color, which is uniformly reddish-brown. In the
lower third of the plate, two or three thin bands are
seen, proximate to the origin, followed by a very in-
tense band due to 4-hydroxyisoleucine, coincident with
the corresponding bands in Standard solution A and
Standard solution B, and comparable in intensity to the
band in the Standard solution B chromatogram. Two
lightly colored bands, one just above the 4-hydroxy-
isoleucine band, another further upwards, are seen. The
upper half of the plate is devoid of discernible features.
Under long-wave UV (365 nm), the derivatized chro-
matogram of the Sample solution exhibits, in its lower
half, two or three light reddish-brown bands followed
by the darker-brown intense band due to 5 eR
isoleucine, coincident with the corresponding bands in
Standard solution A and Standard solution B. Above it,
1Suitable commercially available plates are HPTLC Silica Gel 60 Fas4 from EMD
Millipore (e g., Part No. 1.05642.0001).
USP 41
two lighter brown and somewhat diffuse bands appear.
In the upper third of the plate, three yellowish-orange
bands are seen, corresponding in position and color to
those observed in Standard solution B.
e B. HPTLC For ARTICLES OF BOTANICAL ORIGIN (203)
Standard solution: 50 mg/mL of USP Trigonella
Foenum-graecum Seed Dry Extract RS in an ethanol
and water (7:3) mixture. Sonicate for 10 min, centri-
fuge, and use the supernatant.
Sample solution: Suspend about 1 g of Powder in 5 mL
of an ethanol and water (7:3) mixture, and incubate at
50° for 15 min. Centrifuge, and use the supernatant.
[Note—The Standard solution and Sample solution may
also be used for Identification test A and for Specific
Tests, Presence of Trigonelline.]
Chromatographic system
Adsorbent: Chromatographic silica gel with an aver-
age particle size of 5 um (HPTLC pete)
Application volume: 2 uL each of the Standard solu-
tion and the Sample solution, as 8-mm bands
Relative humidity: Condition the plate toa relative
humidity of 33% using a suitable device.
Temperature: Ambient, not to exceed 30°
Developing solvent system: A mixture of dichloro-
methane, methanol, and water (18:8:1)
Derivatization reagent: A mixture of methanol, gla-
cial acetic acid, sulfuric acid, and p-anisaldehyde
(170:20:10:1). Prepare on an ice bath, and mix well.
System suitability
Sample: Standard solution
Suitability requirements: Under white light, the der-
ivatized chromatogram of the Standard solution exhib-
its, in its lower half, three or four lightly shaded bands.
Under long-wave UV (365 nm), the derivatized chro-
matogram of the Standard solution exhibits, in its
lower third, two diffuse bands of blue fluorescence,
and another blue fluorescent band in the middle of
the plate.
Analysis
Samples: Standard solution and Sample solution
Apply the Samples as bands, and dry in air. Condition
at relative humidity at about 33%. Develop in a satu-
rated chamber, until the solvent front has migrated
over a path of 6 cm. Air-dry, treat with Derivatization
reagent, heat for 3 min at 105°, and immediately ex-
amine under white light and under the long-wave UV
light (365 nm).
Acceptance criteria: Under white light, the chromato-
gram of the Sample solution, in its lower third, shows
two medium-intensity bands immediately next to the
application line. Further upwards, two closely spaced,
less intense, bands are followed by a more prominent
band, and another pair of closely spaced lighter bands,
which may merge. In the middle third of the chromato-
gram, a medium-intensity band is followed by a faint
diffuse band. In the upper third of the chromatogram,
two well-defined bands of medium intensity are ob-
served. Under UV light (365 nm), the lower half of the
chromatogram features three or four deep-blue fluores-
cent bands of varying intensity interspersed with
greyish-brown zones.
COMPOSITION
© CONTENT OF 4-HYDROXYISOLEUCINE
Solution A: 0.1% Phosphoric acid in water
Solution B: Acetonitrile
Mobile phase: See Table 1.
Table 1
Time Solution A Solution B
(min) (%) (%)
0 80.0 20.0
20 40.0 60.0
USP 41 Dietary Supplements / Fenugreek 4611

Table 1 (Continued) D = dilution factor to prepare the Sample solution

Time Solution A Solution B
from the Sample stock solution, 10
(min) (%)
Acceptance criteria: NLT 0.2% on the dried basis
(%)
21 80.0 20.0 CONTAMINANTS
25 80.0 20.0 e ELEMENTAL IMPURITIES—PROCEDURES (233)
Acceptance criteria
Diluent: Methanol and water (1:1) Arsenic: NMT 2.0 ug/g
Reagent: A mixture of acetonitrile, water, and triethyl- Cadmium: NMT 1.0 ug/g
amine (10:3:2) Lead: NMT 10.0 ug/g
Standard solution: Transfer about 4.0 mg of USP Mercury: NMT 1.0 ug/g
4-Hydroxyisoleucine RS, accurately weighed, into a ¢ ARTICLES OF BOTANICAL ORIGIN, Pesticide Residue Analysis
50-mL volumetric flask, and dissolve in 5 mL of the Dilu- (561): Meets the requirements
ent. Add 10 mL of Reagent and 0.5 mL of phenyl isothi- e MICROBIAL ENUMERATION TESTS (2021): The total aerobic
ocyanate, and shake for 5 min. Add 30 mL of methanol, bacterial count does not exceed 105 cfu/g, the total com-
adjust with water to volume, and mix well. bined molds and yeasts count does not exceed 103 cfu/
Sample stock solution: Transfer about 2.0 g of accu- g, and the bile-tolerant Gram-negative bacteria do not
rately weighed Powder into a centrifuge tube. Add exceed 103 cfu/g.
8 mL of Diluent, place on a water bath at 65° for 5 min, e ABSENCE OF SPECIFIED MICROORGANISMS (2022): Meets the
sonicate for 5 min, and centrifuge. Retain the superna- requirements of the tests for absence of Salmonella spe-
tant, and repeat extraction with 8 mL of Diluent two cies and Escherichia coli
more times. Combine all three extracts in the 25-mL
ven flask, dilute with Diluent to volume, and mix SPECIFIC TESTS
well. © PRESENCE OF TRIGONELLINE
Sample solution: Transfer 5.0 mL of Sample stock solu- Standard solution A: 1.5 mg/mL of USP Trigonelline RS
tion into a 50-mL volumetric flask, add 10 mL of Rea- in an ethanol and water (7:3) mixture
gent and 0.5 mL of phenyl isothiocyanate, and shake for Standard solution B: 50 mg/mL of USP Trigonella
5 min. Add 30 mL of methanol, dilute with water to Foenum-graecum Seed Dry Extract RS in an ethanol
volume, and mix well. Pass through a nylon filter hav- and water (7:3) mixture. Sonicate for 10 min, centri-
ing a 0.45-um or finer pore size, discarding the initial fuge, and use the supernatant.
few mL of the filtrate. Sample solution: Suspend about 1 g of Powder in 5 mL
Chromatographic system of an ethanol and water (7:3) mixture, and incubate at
(See Chromatography (621), System Suitability.) 50° for 15 min. Centrifuge, and use the supernatant.
Mode: HPLC [Note—Standard solution B and the Sample solution may
Detector: UV 254 nm also be used for /dentification test A and for Identification
Column: 4.6-mm x 15-cm; 5-4um packing L1 test B.]
Column temperature: Ambient Chromatographic system
Flow rate: 1.5 mL/min (See HPTLC for Articles of Botanical Origin (203).)
Injection volume: 20 uL Adsorbent: Chromatographic silica gel with an aver-
System suitability age particle size of 5 um (HPTLC plate)
Sample: Standard solution Application volume: 5 uL, as 8-mm bands
Sutapility requirements Relative humidity: Condition the plate to a relative
Tailing factor: NMT 2.0 for the 4-hydroxyisoleucine humidity of 33% using a suitable device.
peak, Standard solution Temperature: Ambient, not to exceed 30°
Relative standard deviation: NMT 2.0% determined Developing solvent system: A mixture of isopropyl al-
for the 4-hydroxyisoleucine peak in replicate injec- cohol, methanol, and water (4:1:4)
tions, Standard solution System suitability
Analysis Samples: Standard solution A and Standard solution B
Samples: Standard solution and Sample solution Suitability requirements: Under short-wave UV light
Using the chromatogram of the Standard solution, iden- (254 nm), the chromatogram of Standard solution B
ye the retention time of the peak corresponding to displays, in its lower half, a quenching band coincident
4-hydroxyisoleucine in the Sample solution with that of the trigonelline band in the chromato- 7]
chromatogram. gram of Standard solution A. wn
Calculate the percentage of 4-hydroxyisoleucine in the Analysis E
portion of Powder taken: Samples: Standard solution A, Standard solution B, and 5
Sample solution Ss
Result = (ru/rs) x Cs x (V/W) x D x 100 re)
Apply the Samples as bands, and dry in air. Condition a=
at relative humidity at about 33%. Develop in a satu- i)
Tu = peak area of 4-hydroxyisoleucine from the rated chamber, until the solvent front has migrated 3
Sample solution over a path of 6 cm. Air-dry, and examine under short- a
rs = peak area of 4-hydroxyisoleucine from the waveUVlight (254 nm).
w

Standard solution Acceptance criteria: Under short-wave UV light, the

Gs = concentration of USP 4-Hydroxyisoleucine RS chromatogram of the Sample solution displays a
in the Standard solution (mg/mL) quenching band corresponding to the trigonelline band
= volume of the Sample stock solution (mL) in the chromatograms of Standard solution A and Stan-
z=T

= weight of Powder taken to prepare the Sample dard solution B.

stock solution (mg)
 4612 Fenugreek / Dietary Supplements
BOTANICAL CHARACTERISTICS
Macroscopic: Yellow-brown powder
Microscopic: Fragments of the test in sectional view
with thick cuticle covering lageniform epidermal cells,
with an underlying hypodermis of large cells, narrower
at the upper end and constricted in the middle, with
bar-like thickenings of the radial walls; yellowish-brown
fragments of the epidermis in surface view, composed
of small, polygonal cells with thickened and pitted
walls, frequently associated with the hypodermal cells,
circular in outline with thickened and closely beaded
walls; fragments of the hypodermis viewed from below,
composed of polygonal cells whose bar-like thickenings
extend to the upper and lower walls; parenchyma of
the testa with elongated, rectangular cells with slightly
thickened and beaded walls; fragments of endosperm
with irregularly thickened, sometimes elongated cells,
containing mucilage
ARTICLES OF BOTANICAL ORIGIN, Foreign Organic Matter
(561): NMT 2.0%
Loss ON DRYING (731)
Sample: 1g
Analysis: Dry the Sample at 105° for 2 h.
Acceptance criteria: NMT 12.0%
ARTICLES OF BOTANICAL ORIGIN, Total Ash (561): NMT
5.0%
ARTICLES OF BOTANICAL ORIGIN, Alcohol-Soluble Extractives,
Method 7 (561): NLT 5.0%
ARTICLES OF BOTANICAL ORIGIN, Water-Soluble Extractives,
Method 1 (561): NLT 9.0%
ADDITIONAL REQUIREMENTS
PACKAGING AND STORAGE: Preserve in well-closed contain-
ers, protected from light and moisture, and store at
room temperature.
LABELING: The label states the Latin binomial and, follow-
ing the official name, the part of the plant from which
the article was derived.
USP REFERENCE STANDARDS (11)
USP 4-Hydroxyisoleucine RS
USP Trigonella Foenum-graecum Seed Dry Extract RS
USP Trigonelline RS
Fenugreek Seed Powdered Extract
DEFINITION
Fenugreek Seed Powdered Extract is prepared from the
dried ripe seeds of Trigonella foenum-graecum L. (Fam.
DS Monographs
Fabaceae) by extraction with hydroalcoholic mixtures. It
contains NLT 90.0% and NMT 110.0% of the labeled
amount of 4-hydroxyisoleucine, calculated on the dried
basis.
IDENTIFICATION
A. HPTLC For ARTICLES OF BOTANICAL ORIGIN (203)—
AMINO ACID PROFILE
Standard solution A: 0.5 mg/mL of USP 4-Hydroxy-
isoleucine RS in an ethanol and water (7:3) mixture
Standard solution B: 50 mg/mL of USP Trigonella
Foenum-graecum Seed Dry Extract RS in an ethanol
and water (7:3) mixture. Sonicate for 10 min, centri-
fuge, and use the supernatant.
Sample solution: 50 mg/mL of Powdered Extract in an
ethanol and water (7:3) mixture. Sonicate for 10 min,
centrifuge, and use the supernatant.
[Note—Standard solution B and the Sample solution may
also be used for Identification test B and for Specific
Tests, Presence of Trigonelline.]
USP 41
Chromatographic system
Adsorbent: Chromatographic silica gel with an aver-
age particle size of 5 um (HPTLC plate)"
Application volume: 2 wL, as 8-mm bands
Relative humidity: Condition the plate toa relative
humidity of 33% using a suitable device.
Temperature: Ambient, not to exceed 30°
Developing solvent system: A mixture of n-butanol,
acetic acid, and water (7:2:1)
Derivatization reagent: A solution of 0.3% ninhydrin
fen of isopropanol and glacial acetic acid
‘1)
System suitability
Samples: Standard solution A and Standard solution B
Suitability requirements: Under white light, the der-
ivatized chromatogram of Standard solution B displays,
in its lower half, five or six brown bands; the darkest
band corresponding to the 4-hydroxyisoleucine band
in the chromatogram of Standard solution A. Under
long-wave UV (365 nm), the derivatized chromato-
ram of Standard solution B exhibits, in its lower half,
our or five brown bands; the darkest band corre-
sponding to the 4-hydroxyisoleucine band in the chro-
matogram of Standard solution A. In the upper half of
the chromatogram, three diffuse yellow to orange-yel-
low bands are seen, the middle one being the most
intense.
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Apply the Samples as bands, and dry in air. Condition
at relative humidity at about 33%. Develop in a satu-
rated chamber until the solvent front has migrated
over a path of 6 cm. Air-dry, treat with Derivatization
reagent, heat for 3 min at 105°, and immediately ex-
amine under white light and under the long-wave UV
light (365 nm).
Acceptance criteria: Under white light, and under
long-wave UV light (365 nm), the chromatogram of the
Sample solution displays the bands graelaly similar in
pattern and color to those observed with Standard solu-
tion B, in particular, the prominent band corresponding
to the 4-hydroxyisoleucine band in the chromatograms
of Standard solution A and Standard solution B. However,
depending on the procedures and excipients used in
prepalauet of Powdered Extract, the number, position,
and coloration of the bands in the Sample solution may
differ from those observed in the Standard solution B
chromatogram.
e B. HPTLC FOR ARTICLES OF BOTANICAL ORIGIN (203)—STER-
OIDAL SAPONINS PROFILE
Standard solution: 50 mg/mL of USP Trigonella
Foenum-graecum Seed Dry Extract RS in an ethanol
and water (7:3) mixture. Sonicate for 10 min, centri-
fuge, and use the supernatant.
Sample solution: 50 mg/mL of Powdered Extract in an
ethanol and water (7:3) mixture. Sonicate for 10 min,
centrifuge, and use the supernatant.
[Note—The Standard solution and Sample solution may
also be used for Identification test A and for Specific
Tests, Presence of Trigonelline.]
Chromatographic system
Adsorbent: Chromatographic silica gel with an aver-
age particle size of 5 um (HPTLC plate)
Application volume: 2 uL, as 8-mm bands
Relative humidity: Condition the plate toa relative
humidity of 33% using a suitable device.
Temperature: Ambient, not to exceed 30°
Developing solvent system: A mixture of dichloro-
methane, methanol, and water (18:8:1)
1Suitable commercially available plates are HPTLC Silica Gel 60 Fas« from EMD
Millipore (e.g., Part No. 1.05642.0001).
USP 41 Dietary Supplements / Fenugreek 4613

Derivatization reagent: A mixture of methanol, gla- flask, and dissolve in 5 mL of Diluent. Add 10 mL of
cial acetic acid, sulfuric acid, and p-anisaldehyde Reagent and 0.5 mL of phenyl isothiocyanate, and shake
(170:20:10:1). Prepare on an ice bath, and mix well. for 5 min. Add 30 mL of methanol, adjust with water to
System suitability volume, and mix well.
Sample: Standard solution Chromatographic system
Suitability requirements: Under white light, the der- (See Chromatography (621), System Suitability.)
ivatized chromatogram of the Standard solution exhib- Mode: HPLC
its, in its lower half, three or four lightly shaded bands. Detector: UV 254 nm
Under long-wave UV (365 nm), the derivatized chro- Column: 4.6-mm x 15-cm; 5-um packing L1
matogram of the Standard solution exhibits, in its Column temperature: Ambient
lower third, two diffuse bands of blue fluorescence, Flow rate: 1.5 mL/min
and another blue fluorescent band in the middle of Injection volume: 20 uL
the plate. System suitability
Analysis Sample: Standard solution
Samples: Standard solution and Sample solution Sule requirements
Apply the Samples as bands, and dry in air. Condition Tailing factor: NMT 2.0 for the 4-hydroxyisoleucine
at relative humidity at about 33%. Develop in a satu- peak, Standard solution
rated chamber, until the solvent front has migrated Relative standard deviation: NMT 2.0% determined
over a path of 6 cm. Air-dry, treat with Derivatization for the 4-hydroxyisoleucine peak in replicate injec-
reagent, heat for 3 min at 105°, and immediately ex- tions, Standard solution
amine under white light and under the long-wave UV Analysis
light (365 nm). Samples: Standard solution and Sample solution
Acceptance criteria: Under white light, and under Using the chromatogram of the Standard solution, iden-
long-wave UV light (365 nm), the chromatogram of the iy the retention time of the peak corresponding to
Sample solution displays the bands similar in pattern and 4-hydroxyisoleucine in the Sample solution
color to those observed with the Standard solution. chromatogram.
However, depending on the procedures and excipients Calculate the percentage of 4-hydroxyisoleucine in the
used in preparation of Powdered Extract, the number, portion of Powdered Extract taken:
position, and coloration of the bands in the Sample so-
lution may differ from those observed in the Standard Result = (ru/rs) x Cs x (V/W) x 100
Solution chromatogram.
ty = peak area of 4-hydroxyisoleucine from the
COMPOSITION Sample solution
¢ CONTENT OF 4-HYDROXYISOLEUCINE ls = peak area of 4-hydroxyisoleucine from the
Solution A: 0.1% Phosphoric acid in water Standard solution
Solution B: Acetonitrile Cs = concentration of USP 4-Hydroxyisoleucine RS
Mobile phase: See Table 1. in the Standard solution (mg/mL)
Vv = volume of the Sample solution (mL)
Table 1 Ww = weight of Powdered Extract taken to prepare
the Sample solution (mg)
Time Solution A Solution B Acceptance criteria: 90.0%-110.0% of the labeled
(min) (%) (%) amount of 4-hydroxyisoleucine, on the dried basis
oO 80.0 20.0
20 40.0 60.0 CONTAMINANTS
e ELEMENTAL IMPURITIES—PROCEDURES (233)
21 80.0 20.0
Acceptance criteria
25 80.0 20.0 Arsenic: NMT 2.0 ug/g
Diluent: Methanol and water (1:1) Cadmium: NMT 1.0 ug/g
Reagent: A mixture of acetonitrile, water, and triethyl- Lead: NMT 10.0 ug/g
amine (10:3:2) Mercury: NMT 1,0 ug/g
Standard solution: Transfer about 4.0 mg of USP © ARTICLES OF BOTANICAL ORIGIN, Pesticide Residue Analysis
(561): Meets the requirements
sydeibouow sa

4-Hydroxyisoleucine RS, accurately weighed, into a

50-mL volumetric flask, and dissolve in 5 mL of the Dilu-
ent. Add 10 mL of Reagent and 0.5 mL of phenyl isothi-
ocyanate, and shake for 5 min. Add 30 mL of methanol,
adjust with water to volume, and mix well.
Sample solution: Transfer the amount of accurately
weighed Powdered Extract calculated to contain about
4 mg of 4-hydroxyisoleucine into a 50-mL volumetric
e MICROBIAL ENUMERATION TESTS (2021): The total aerobic
bacterial count does not exceed 104 cfu/g, and the total
combined molds and yeasts count does not exceed 103
cfu/g.
© ABSENCE OF SPECIFIED MICROORGANISMS (2022): Meets the
requirements of the tests for absence of Salmonella spe-
cies and Escherichia coli
 4614 Fenugreek / Dietary Supplements USP 41

SPECIFIC TESTS Ferrous Fumarate—see Ferrous Fumarate

e HPTLC For ARTICLES OF BOTANICAL ORIGIN (203)—PRES-
ENCE OF TRIGONELLINE General Monographs
Standard solution A: 1.5 mg/mL of USP Trigonelline RS
in an ethanol and water (7:3) mixture
Standard solution B: 50 mg/mL of USP Trigonella
Foenum-graecum Seed PD, Extract RS in an ethanol Ferrous Fumarate Tablets—see Ferrous
and water (7:3) mixture. Sonicate for 10 min, centri- Fumarate Tablets General Monographs
fuge, and use the supernatant.
Sample solution: 50 mg/mL of Powdered Extract in
methanol. Sonicate for 10 min, centrifuge, and use the
supernatant. Ferrous Gluconate—-see Ferrous Gluconate
{[NoTe—Standard solution B and the Sample solution may
also a used for Identification test A and for Identification General Monographs
test B.
Chromatographic system
Adsorbent: Chromatographic silica gel with an aver-
age particle size of 5 um (HPTLC plate) Ferrous Gluconate Capsules—see Ferrous
Application volume: 5 uL, as 8-mm bands
Relative humidity: Condition the plate to a relative
Gluconate Capsules General Monographs
humidity of 33% using a suitable device.
Temperature: Ambient, not to exceed 30°
Developing solventsysterns A mixture of isopropyl al-
cohol, methanol, and water (4:1:4) Ferrous Gluconate Oral Solution—see
System suitability Ferrous Gluconate Oral Solution General
Samples: Standard solution A and Standard solution B Monographs
Suitability requirements: Under short-wave UV light
(254 nm), the chromatogram of Standard solution B
displays, in its lower half, a quenching band coincident
with that of the trigonelline band in the chromato- Ferrous Gluconate Tablets—see Ferrous
gram of Standard solution A.
Analysis Gluconate Tablets General Monographs
Samples: Standard solution A, Standard solution B, and
Sample solution
Apply the Samples as bands, and dry in air. Condition
at relative humidity at about 33%. Develop in a satu- Ferrous Sulfate—see Ferrous Sulfate
rated chamber, until the solvent front has migrated
over a path of 6 cm. Air-dry, and examine under short-
General Monographs
wave UV light (254 nm).
Acceptance criteria: Under short-wave UV light, the
chromatogram of the Sample solution displays a
quenching band corresponding to the trigonelline band Ferrous Sulfate Oral Solution—see
in the chromatograms of Standard solution A and Stan- Ferrous Sulfate Oral Solution General
dard solution B. Monographs
e WATER DETERMINATION, Method Ia (921): NMT 9.0%
e RESIDUE ON IGNITION (281): NMT 5.0%
e RESIDUAL SOLVENTS (467): Meets the requirements
ADDITIONAL REQUIREMENTS Ferrous Sulfate Syrup—see Ferrous Sulfate
© PACKAGING AND STORAGE: Preserve in well-closed contain- Syrup General Monographs
ers, protected from light and moisture, and store at
DS Monographs

room temperature.
e LABELING: The label states the Latin binomial and, follow-
ing the official name, the part of the plant from which Ferrous Sulfate Tablets—see Ferrous
the extract was derived. It also meets the requirements
for Labeling in Botanical Extracts (565). Sulfate Tablets General Monographs
e USP REFERENCE STANDARDS (11)
USP 4-Hydroxyisoleucine RS
USP Trigonella Foenum-graecum Seed Dry Extract RS
USP Trigonelline RS Ferrous Sulfate, Dried—see Dried Ferrous
Sulfate General Monographs
USP 41
Feverfew
DEFINITION
Feverfew consists of the dried leaves of Tanacetum
parthenium (L.) Sch. Bip. (Fam. Asteraceae), collected
when the plant is in flower.
IDENTIFICATION
e A. The retention time of the parthenolide peak of the
Sample solution corresponds to that of the Standard solu-
tion, as obtained in the test for Content of Parthenolide.
¢ B. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST
Standard solution: 1.0 mg/mL of USP Parthenolide RS
in methanol
Sample solution: Transfer 1.0 g of finely powdered Fe-
verfew to a suitable flask. Add 20 mL of methanol, heat
the flask over a water bath at 60° for 15 min, cool, and
filter. Evaporate the filtrate under reduced pressure to
dryness, and dissolve the residue in 2.0 mL of
methanol.
Adsorbent: 0.5-mm layer of chromatographic silica gel,
typically 20 cm long (TLC plates)
Application volume: 20 uL
Developing solvent system: Toluene and acetone
(85:15)
Spray reagent: 0.5% Solution of vanillin in a mixture
of sulfuric acid and alcohol (4:1)
Analysis
Samples: Standard solution and Sample solution
Develop the chromatograms until the solvent front has
moved three-fourths of the length of the plate. Re-
move the plate from the chromatographic chamber,
mark the solvent front, and allow it to air-dry. Spray
the plate with Spray reagent. After 5 min, examine the
plate under white light.
Acceptance criteria: A blue spot in the middle portion
of the chromatogram of the Sample solution that corre-
sponds in color and R value to the principal spot ob-
tained in the chromatogram of the Standard solution
indicates the presence of parthenolide. The lower one-
third of the iiverarcerain of the Sample solution may
exhibit two pink spots, and the upper one-third may
exhibit one pink spot.
© C. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST
Standard solution: 0.25 mg/mL of USP Rutin RS in
methanol
Sample solution: To 1 g of finely powdered Feverfew,
add 10 mL of methanol, and heat on a water bath at
60° for 15 min. Cool, and filter.
Adsorbent: 0.25-mm layer of chromatographic silica
gel, typically 20 cm long (TLC plates)
Application volume: 20 uL
Developing solvent system: Ethyl acetate, anhydrous
formic acid, glacial acetic acid, and water
(10: 1.13 1.13 2,7)
Spray reagent A: 1% Solution of 2-aminoethyl
diphenylborinate in methanol
Spray reagent B: 5% (w/v) Solution of polyethylene
glycol 4000 in alcohol
Analysis
Samples: Standard solution and Sample solution
Develop the chromatogram until the solvent front has
moved three-fourths of the length of the plate. Re-
move the plate from the chromatographic chamber,
and allow it to air-dry. Spray the plate with Spray rea-
gent A followed by Spray reagent B, and examine the
plate under UV light at 366 nm.
Acceptance criteria: Relative to the R; value of the prin-
cipal spot of the Standard solution, the chromatogram
of the Sample solution exhibits no blue spot at Rr 1.1
(distinction from Roman chamomile) but exhibits a
green spot at Rr 2.3 (distinction from Matricaria), and
colored spots at the Rr values indicated are as follows:
Dietary Supplements / Feverfew 4615
1.5 (yellowish orange), 1.65 (yellowish green), 2.0
(greenish blue), and 2.25 (whitish blue).
COMPOSITION
e@ CONTENT OF PARTHENOLIDE
Mobile phase: Acetonitrile and water (9:11)
Standard solution: 0.04 mg/mL of USP Parthenolide RS
in methanol
Sample stock solution: Reduce 100 g of Feverfew to a
fine powder. Transfer about Lg of the finely pow-
dered Feverfew, accurately weighed, to a suitable flask.
Add 100 mL of methanol, and feat on a water bath at
60° for 10 min. Remove the flask from the water bath,
cool, and filter. Rinse the flask with three 5-mL portions
of methanol, and filter, adding the rinsings to the fil-
trate. Transfer the residue left within the filter to the
same flask. Add 50 mL of methanol, and continue the
rinse procedure as described above. Evaporate the com-
bined filtrates under reduced pressure to dryness, and
dissolve the residue in 20.0 mL of methanol.
Sample solution: Transfer 10 mL of the Sample stock
Solution to a 25-mL volumetric flask, and dilute with
methanol to volume.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 210 nm
Column: 4.6-mm x 25-cm; packing L1
Flow rate: 2 mL/min
Injection volume: 10 yL
System suitability
Sample: Standard solution
Suitability requirements
Tailing factor: NMT 2.0 for the parthenolide peak
Relative standard deviation: NMT 2.0% for the
parthenolide peak in repeated injections
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of parthenolide in the portion
of Feverfew taken to prepare the Sample solution:
Result = (ru/rs) x Cs x (V/W) x D x 100
tu = peak area of the parthenolide peak in the
Sample solution
rs = peak area of the parthenolide peak in the
Standard solution
Gs = concentration of USP Parthenolide RS in the
Standard solution (mg/mL)
Vv = final volume of the Sample stock solution (mL)
Ww = weight of Feverfew used to prepare the
Sample stock solution (mg)
sydesbouo-: Sa
D = dilution factor to prepare the Sample solution
from the Sample stock solution
Acceptance criteria: NLT 0.2% on the dried basis
CONTAMINANTS
© ARTICLES OF BOTANICAL ORIGIN, Limits of Elemental Impuri-
ties (561): Meets the requirements
e ARTICLES OF BOTANICAL ORIGIN, Pesticide Residue Analysis
(561): Meets the requirements
e MICROBIAL ENUMERATION TESTS (2021): The total bacterial
count does not exceed 104 cfu/g, and the total com-
bined molds and yeasts count does not exceed 10? cfu/
g.
© ABSENCE OF SPECIFIED MICROORGANISMS (2022) It meets
the requirements of the tests for the absence of Salmo-
nella species and Escherichia coli.
SPECIFIC TESTS
¢ BOTANICAL CHARACTERISTICS
Macroscopic: Yellowish green, petiolate, usually 2-5 cm
in length but sometimes up to 10 cm, ovate, deeply
divided into 5 or occasionally 7 segments, each with a
coarsely crenate margin and obtuse apex; both surfaces
downy and the mid-rib prominent on the lower surface
 4616 Feverfew / Dietary Supplements
Microscopic: Upper and lower epidermal cells with
wavy anticlinical walls, striated cuticle and anomocytic
stomata, more frequent on the lower epidermis;
trichomes, more abundant on the lower epidermis, of
two types; covering trichomes uniseriate with up to 6
small isodiametric basal cells and elongated, tapering
apical cells, often at right angles to the axis of the basal
cells; glandular trichomes slightly sunken, composed of
a short, biseriate, 2- or 4-celled stalk and a biseriate
head of 4 cells, around which the cuticle forms a blad-
der-like covering
ARTICLES OF BOTANICAL ORIGIN, Foreign Organic Matter
(561): NMT 10.0%, including the stalk
ARTICLES OF BOTANICAL ORIGIN, Water-Soluble Extractives,
Method 2 (561): NLT 15.0%
Loss ON DRYING (731)
Sample: 1.0gof finely pone Feverfew
Analysis: Dry the Sample at 105° for 1 h.
Acceptance criteria: NMT 10.0%
ARTICLES OF BOTANICAL ORIGIN, Total Ash (561): NMT
12.0%
ARTICLES OF BOTANICAL ORIGIN, Acid-insoluble Ash (561):
NMT 3.0%
ADDITIONAL REQUIREMENTS
PACKAGING AND STORAGE: Preserve in well-closed contain-
ers, and store in a dry place, protected from light.
LABELING: The label states the Latin binomial and, follow-
ing the official name, the part of the plant contained in
the article.
USP REFERENCE STANDARDS (11)
USP Parthenolide RS
USP Rutin RS
Powdered Feverfew
DEFINITION
Powdered Feverfew is Feverfew pulverized toa fine or very
fine powder.
IDENTIFICATION
A. The retention time of the parthenolide peak of the
Sample solution corresponds to that of the Standard solu-
tion, as obtained in the test for Content of Parthenolide.
B. THIN-LAYER CHROMATOGRAPHY
Standard solution: 1.0 mg/mL of USP Parthenolide RS
in methanol
Sample solution: Transfer 1.0 g of Powdered Feverfew
DS Monographs
to a suitable flask. Add 20 mL of methanol, heat the
flask over a water bath at 60° for 15 min, cool, and
filter, Evaporate the filtrate under reduced pressure to
dryness, and dissolve the residue in 2.0 mL of
methanol.
Adsorbent: 0.5-mm layer of chromatographic silica gel,
typically 20 cm long (TLC plates)
Application volume: 20 |
wi solvent system: Toluene and acetone
:15)
Derivatization reagent: 0.5% Solution of vanillin in a
mixture of sulfuric acid and alcohol (4:1)
Analysis
Samples: Standard solution and Sample solution
Develop the chromatograms until the solvent front has
moved three-fourths of the length of the plate. Re-
move the plate from the chromatographic chamber,
mark the solvent front, and allow it to air-dry. Spray
the plate with Derivatization reagent. After 5 min, ex-
amine the plate under white light.
Acceptance criteria: A blue spot in the middle portion
of the chromatogram of the Sample solution that corre-
USP 41
sponds in color and R; value to the principal spot ob-
tained in the chromatogram of the Standard solution
indicates the presence of parthenolide. The lower one-
third of the chromatogram of the Sample solution may
exhibit two pink spots, and the upper one-third may
exhibit one pink spot.
e C. THIN-LAYER CHROMATOGRAPHY
Standard solution: 0.25 mg/mL of USP Rutin RS in
methanol
Sample solution: To 1g of Powdered Feverfew, add
10 mL of methanol, and heat on a water bath at 60°
for 15 min. Cool, and filter.
Adsorbent: 0.25-mm layer of chromatographic silica
gel, typically 20 cm long (TLC plates)
Application volume: 20 uL
Developing solvent system: Ethyl acetate, anhydrous
formic acid, glacial acetic acid, and water
(10:11. 2.7)
Derivatization reagent A: 1% Solution of 2-aminoethyl
diphenylborinate in methanol
Derivatization reagent B: 5% (w/v) Solution of poly-
euyiene glycol 4000 in alcohol
Analysis
Samples: Standard solution and Sample solution
Develop the chromatogram until the solvent front has
moved three-fourths of the length of the plate. Re-
move the plate from the chromatographic chamber,
and allow it to air-dry. Treat the plate with Derivatiza-
tion reagent A followed by Derivatization reagent B, and
examine the plate under UV light at 366 nm.
Acceptance criteria: Relative to the R; value of the prin-
cipal spot of the Standard solution, the chromatogram
of the Sample solution exhibits no blue spot at Re 1.1
(distinction from Roman chamomile) but exhibits a
green spot at R- 2.3 (distinction from Matricaria), and
colored spots at the R- values indicated are as follows:
1.5 (yellowish orange), 1.65 (yellowish green), 2.0
(greenish blue), and 2.25 (whitish blue).
COMPOSITION
e CONTENT OF PARTHENOLIDE
Mobile phase: Acetonitrile and water (9:11)
Standard solution: 0.04 mg/mL of USP Parthenolide RS
in methanol
Sample stock solution: Transfer about 1.0 g of the
Powdered Feverfew, accurately weighed, to a suitable
flask. Add 100 mL of methanol, and heat on a water
bath at 60° for 10 min. Remove the flask from the
water bath, cool, and filter. Rinse the flask with three
5-mL portions of methanol, and filter, adding the rins-
ings to the filtrate. Transfer the residue left within the
filter to the same flask. Add 50 mL of methanol, and
continue the rinse procedure as described above. Evap-
orate the combined filtrates under reduced pressure to
dryness, and dissolve the residue in 20.0 mL of
methanol.
Sample solution: Transfer 10 mL of the Sample stock
solution to a 25-mL volumetric flask, and dilute with
methanol to volume.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 210 nm
Column: 4.6-mm x 25-cm; packing L1
Flow rate: 2 mL/min
Injection volume: 10 uL
System suitability
Sample: Standard solution
Suitability requirements
Tailing factor: NMT 2.0 for the parthenolide peak
Relative standard deviation: NMT 2.0% for the
parthenolide peak in repeated injections
USP 41
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of parthenolide in the portion
of Powdered Feverfew taken to prepare the Sample
solution:
Result = (ru/rs) x Cs x (V/W) x D x 100
tu = area of the parthenolide peak in the Sample
solution
rs = area of the parthenolide peak in the Standard
solution
Cs = concentration of USP Parthenolide RS in the
Standard solution (mg/mL)
Vv = final volume of the Sample stock solution (mL)
Ww = weight of Powdered Feverfew used to prepare
the Sample stock solution (mg)
D = dilution factor to prepare the Sample solution
from the Sample stock solution
Acceptance criteria: NLT 0.2% on the dried basis
CONTAMINANTS
¢ ARTICLES OF BOTANICAL ORIGIN, Limits of Elemental Impuri-
ties (561): Meets the requirements
© ARTICLES OF BOTANICAL ORIGIN, Pesticide Residue Analysis
(561): Meets the requirements
e MICROBIAL ENUMERATION TESTS (2021): The total aerobic
bacterial count does not exceed 104 cfu/g, and the total
carbines molds and yeasts count does not exceed 102
cfu/g.
e ABSENCE OF SPECIFIED MICROORGANISMS (2022): It meets
the requirements of the tests for the absence of Salmo-
nella species and Escherichia coli.
SPECIFIC TESTS
e ARTICLES OF BOTANICAL ORIGIN, Water-Soluble Extractives,
Method
2 (561): NLT 15.0%
e Loss ON DRYING (731)
Sample: 1.0g of Powdered Feverfew
Analysis: Dry the Sample at 105° for 1 h.
Acceptance criteria: NMT 10.0%
© ARTICLES OF BOTANICAL ORIGIN, Total Ash (561): NMT
12.0%
© ARTICLES OF BOTANICAL ORIGIN, Acid-Insoluble Ash (561):
NMT 3.0%
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in well-closed contain-
ers, protected from light and moisture.
e LABELING: The label states the Latin binomial and, follow-
ing the official name, the part of the plant source from
which the article was derived.
© USP REFERENCE STANDARDS (11)
USP Parthenolide RS
USP Rutin RS
Fish Oil Containing Omega-3 Acids
DEFINITION
Fish Oil Containing Omega-3 Acids is the purified, winter-
ized, and deodorized fatty oil obtained fom fish of the
families Engraulidae, Carangidae, Clupeidae, Osmeridae,
Scombroidae, and Ammodytidae. The omega-3 acids are
defined as the following: alpha-linolenic acid (C18:3 n-3),
moroctic acid (C18:4 n—3), eicosatetraenoic acid (C20:4
n—3), eicosapentaenoic acid (EPA) (C20:5 n-3), heneicosa-
pentaenoic acid (C21:5 n—3), docosapentaenoic acid
(C22:5 n-3), and docosahexaenoic acid (DHA) (C22:6 n—
3). It contains NLT 28.0% (w/w) of total omega-3 acids,
expressed as free acids, consisting of NLT 13.0% of EPA
and NLT 9.0% of DHA. Suitable antioxidants in appropri-
ate concentrations may be added.
Dietary Supplements / Fish Oil 4617
IDENTIFICATION ;
e A. The retention times of the docosahexaenoic acid
methyl ester and eicosapentanoic acid methyl ester peaks
from Test solution 1 in Content of EPA and DHA corre-
spond to those of the docosahexaenoic acid methyl ester
and eicosapentanoic acid methyl ester peaks from Stan-
dard solution 2a and Standard solution 2b, respectively, in
Fats and Fixed Oils (401), Content of EPA and DHA. The
sum of the area for EPA and DHA methyl esters is NLT
22% of the total detected area for the methyl esters, and
no other peak has an area higher than 20% of the total
detected area for the methyl esters. In addition to the
EPA and DHA peaks, Test solution 1 exhibits at least 15
more peaks with retention times similar to those of the
Fish oil standard solution, as obtained in the test for Con-
tent of EPA and DHA.
COMPOSITION
e CONTENT OF EPA AND DHA
(See Fats and Fixed Oils (401), Omega-3 Fatty Acids Deter-
mination and Profile.)
Standard solution 1a, Standard solution 1b, Standard
solution 2a, Standard solution 2b, Test solution 1,
Test solution 2, System suitability solution 1, System
suitability solution 2, Chromatographic system, Sys-
tem suitability, and Analysis: Proceed as directed in
Fats and Fixed Oils (401), Content of EPA and DHA for
triglycerides.
Fish oil standard solution: Transfer 300 mg of USP Fish
Oil RS into a 10-mL volumetric flask, and dissolve in
and dilute with Antioxidant Solution to volume. Proceed
as directed for Test Solution 1 (for triglycerides) in Fats
and Fixed Oils (401), Content of EPA and DHA, starting
with “Transfer 2.0 mL”.
Identify the relevant fatty acid methyl esters in the Fish
oil standard solution by comparing their retention times
with those in the reference chromatogram supplied
with the USP Fish Oil RS.
Acceptance criteria: NLT 13.0% (w/w) of EPA and NLT
9.0% (w/w) of DHA
© CONTENT OF TOTAL OMEGA-3 ACIDS
(See Fats and Fixed Oils (401), Omega-3 Fatty Acids Deter-
mination and Profile.)
Analysis: Proceed as directed in Fats and Fixed Oils
(401), Content of Total Omega-3 Acids (for triglycerides).
Acceptance criteria: NLT 28.0% (w/w) of total omega-
3 acids, expressed as free acids
CONTAMINANTS
e LIMIT OF ARSENIC
[Note—For the preparation of all aqueous solutions and
for the rinsing of glass, polytef, and plastic vessels
sydeiBbouow sa
before use, use water that te been passed first through
a strong-acid, strong-base, mixed-bed ion-exchange
resin. Select all reagents to have as low a content of
arsenic as practicable, and store all reagent solutions in
containers of borosilicate glass. Cleanse glass, polytef,
and plastic vessels before use by soaking in warm 8N
nitric acid for 30 min and by rinsing with deionized
water.]
1% Palladium stock solution: Transfer 1 g of ultrapure
palladium metal into a Teflon beaker. Add 20 mL of
water and 10 mL of nitric acid, and warm on a hot
plate to dissolve. Allow the solution to cool to room
temperature, transfer into a 100-mL volumetric flask,
and dilute with deionized water to volume.
1% Magnesium nitrate stock solution: Transfer 1g of
ultrapure magnesium nitrate into a Teflon beaker. Add
40 mL of water and 1 mL of nitric acid, and warm on a
hot plate to dissolve the solids. Allow the solution to
cool to room temperature, transfer into a 100-mL volu-
metric flask, and dilute with deionized water to volume.
Modifier working solution: 1% Palladium stock solution,
1% Magnesium nitrate stock solution, and 2% nitric acid
 4618 Fish Oil / Dietary Supplements
(3:2:5). A volume of 5 UL provides 0.015 mg of palla-
dium and 0.01 mg of magnesium nitrate.
Blank: Nitric acid and water (5 in 100)
Standard stock solution: Transfer 10.0 mL of Standard
Arsenic Solution, prepared as directed in Arsenic (211),
to a 100-mL volumetric flask. Add 40 mL of water and
5 mL of nitric acid, and dilute with water to volume.
This solution contains 0.10 ug/mL of arsenic.
Standard solutions: Dilute the Standard stock solution
with the Blank to obtain concentrations of 0.002,
0.005, 0.010, 0.025, and 0.050 pg/mL of arsenic.
Sample solution: For preparation of the Sample solu-
tion, use a microwave oven with a magnetron fre-
quency of 2455 MHz anda selectable output power of
0-950 watts in 1% increments, equipped with ad-
vanced composite vessels with 100-mL polytef liners.
Use rupture membranes to vent vessels should the pres-
sure exceed 125 psi. The vessels fit into a turntable, and
each vessel can be vented into an overflow container.
Equip the microwave oven with an exhaust tube to
ventilate fumes. [CAUTION—Wear proper eye protection
and protective clothing and gloves.] Transfer approxi-
mately 500 mg of Fish Oil Containing Omega-3 Acids,
weighed to the nearest 0.1 mg, into a Teflon digestion
vessel liner. Prepare samples in duplicate. Add 15 mL of
nitric acid, and swirl gently. Cover the vessels with lids,
leaving the vent fitting off. Predigest overnight under a
hood. Place the rupture membrane in the vent fitting,
and tighten the lid. Place all vessels on the microwave
oven turntable. Connect the vent tubes to the vent
trap, and connect the pressure-sensing line to the ap-
propriate vessel. Initiate a two-stage digestion proce-
dure by heating the microwave at 15% power for 15
min, followed by 25% power for 45 min. Remove the
turntable of vessels from the oven, and allow the vessels
to cool to room temperature. [NOTE—A cool water bath
may be used to speed the cooling process.] Vent the
vessels when they reach room temperature. Remove the
lids, and slowly add 2 mL of 30% hydrogen peroxide to
each. Allow the reactions to subside, and seal the ves-
sels. Return the vessels on the turntable to the micro-
wave oven, and heat for an additional 15 min at 30%
power. Remove the vessels from the oven, and allow
them to cool to room temperature. Transfer the cooled
digests into 25-mL volumetric flasks, and dilute with
water to volume.
Analysis: Program the graphite furnace as follows. Dry
at 115° using a 1-s ramp, a 65-s hold, and an argon
flow of 300 mL/min. Char the sample at 1000° using a
1-s ramp, a 20-s hold, and an airflow of 300 mL/min.
Cool down, and purge the air from the furnace for 10 s
DS Monographs
using a 20° set temperature and an argon flow of
300 mL/min. Atomize at 2400° using a 0-s ramp and a
5-s hold with the argon flow stopped. Clean out at
2600° using a 1-s ramp and a 5-s hold. Separately in-
ject equal volumes (20 L) of the Standard solutions, the
Sample solution, and the Blank, followed by a 5-uL injec-
tion of the Modifier working solution for each of the
samples, into the graphite tube of a suitable graphite
furnace atomic absorption spectrometer equipped with
a hollow-cathode lamp for arsenic. Determine the peak
area at the arsenic emission line at 193.7 nm, corrected
for background absorption. Plot the corrected peak ar-
eas of the Standard solutions versus their contents of
arsenic, in ug/mL, and calculate the regression line best
fitting the points. Determine the concentration, C, in
tug/mL, of arsenic in each mL of the Sample solution by
interpolation from the regression line.
Calculate the content of arsenic in the portion of Fish
Oil Containing Omega-3 Acids taken:
Result = (C/W) x 25
Cc = concentration of arsenic in each mL of the
Sample solution (g/mL)
USP 41
Ww = weight of Fish Oil Senin Omega-3 Acids
taken to prepare the Sample solution (g)
Acceptance criteria: NMT 0.1 g/g
e Limit oF LEAD
[Note—For the preparation of all aqueous solutions and
for the rinsing of glass, polytef, and plastic vessels
before use, use water that has been passed through a
strong-acid, strong-base, mixed-bed ion-exchange resin
before use. Select all reagents to have as low a content
of lead as practicable, and store all reagent solutions in
containers of borosilicate glass. Cleanse glass, polytef,
and plastic vessels before use by soaking in warm 8N
nitric acid for 30 min and by rinsing with deionized
water.]
10% Monobasic ammonium phosphate solution:
10 g of ultrapure monobasic ammonium phosphate in
1 mL of nitric acid and 40 mL of water to dissolve the
phosphate. Dilute with deionized water to 100 mL.
1% Magnesium nitrate solution: Transfer 1 g of ul-
trapure magnesium nitrate to a Teflon beaker. Add
40 mL of water and 1 mL of nitric acid, and warm ona
hot plate to dissolve the solids. Allow the solution to
cool to room temperature, transfer to a 100-mL volu-
metric flask, and dilute with deionized water to volume.
Modifier working solution: 10% Monobasic ammonium
phosphate solution, 1% Magnesium nitrate solution, and
2% nitric acid (2:1:2). A volume of 5 uL provides
0.2 mg of phosphate plus 0.01 mg of magnesium
nitrate.
Blank: Nitric acid and water (5 in 100)
Standard stock solution: Transfer 10.0 mL of lead ni-
trate stock solution TS to a 100-mL volumetric flask.
Add 40 mL of water and 5 mL of nitric acid, and dilute
with water to volume. Transfer 1.0 mL of this solution
to a second 100-mL volumetric flask. Add 50 mL of
water and 1 mL of nitric acid, and dilute with water to
volume. This solution contains 0.10 g/mL of lead.
Standard solutions: Dilute the Standard stock solution
with the Blank to obtain concentrations of 0.002,
0.005, 0.010, 0.025, and 0.050 jig/mL of lead.
Sample solution: Prepare as directed in Limit of Arsenic.
Analysis: Program the graphite furnace as follows. Dry
at 720° using a 1-s ramp, a 55-s hold, and an argon
flow of 300 mL/min. Char the sample at 850° using a
1-s ramp, a 30-s hold, and an airflow of 300 mL/min.
Cool down, and purge the air from the furnace for 10 s
using a 20° set temperature and an argon flow of
300 mL/min. Atomize at 2100° using a 0-s ramp and a
5-s hold with the argon flow stopped. Clean out at
2600° using a 1-s ramp and a 5-s hold. Separately in-
ject equal volumes (20 iL) of the Standard solutions, the
Sample solution, and the Blank, followed by a 5-uL injec-
tion of the Modifier working solution for each of the
samples, into the graphite tube of a suitable graphite
furnace atomic absorption spectrometer equipped with
a hollow-cathode lamp for lead. Determine the peak
area at the lead emission line at 283.3 nm, corrected
for background absorption. Plot the corrected peak ar-
eas of the Standard solutions versus their contents of
lead, in g/mL, and calculate the regression line best
fitting the points. Determine the concentration, C, in
tg/mL, of lead in each mL of the Sample solution by
interpolation from the regression line.
Calculate the content of lead in the portion of Fish Oil
Containing Omega-3 Acids taken:
Result = (C/W) x 25
Cc = concentration of lead in each mL of the
Sample solution (g/mL)
w = weight of Fish Oil Containing Omega-3 Acids
taken to prepare the Sample solution (g)
USP 41
Acceptance criteria: NMT 0.1 ug/g
e Limit oF CADMIUM
[Note—For the preparation of all aqueous solutions and
for the rinsing of glass, polytes, and plastic vessels
before use, use water that has been passed through a
strong-acid, strong-base, mixed-bed ion-exchange resin
before use. Select all reagents to have as low a content
of cadmium as practicable, and store all reagent solu-
tions in containers of borosilicate glass. Cleanse glass,
polytef, and plastic vessels before use by soaking in
warm 8N nitric acid for 30 min and by rinsing with
deionized water.]
10% Monobasic ammonium phosphate solution:
10g of ultrapure monobasic ammonium phosphate in
40 mL of water and 1 mL of nitric acid to dissolve the
phosphate. Dilute with deionized water to 100 mL.
1% Magnesium nitrate solution: Transfer 1 g of ul-
trapure magnesium nitrate to a Teflon beaker. Add
40 mL of water and 1 mL of nitric acid, and warm ona
hot plate to dissolve the solids. Allow the solution to
cool to room temperature, transfer to a 100-mL volu-
metric flask, and dilute with deionized water to volume.
Modifier working solution: 10% Monobasic ammonium
phosphate solution, 1% Magnesium nitrate solution, and
2% nitric acid (2:1:2). A volume of 5 uL provides
0.2 mg of phosphate and 0.01 mg of magnesium
nitrate.
Blank: Nitric acid and water (5 in 100)
Standard stock solution A: 0.1372 mg/mL of cadmium
nitrate in water
Standard stock solution B: Standard stock solution A,
nitric acid, and water (2:1:97). This solution contains
0.10 ug/mL of cadmium. [NoTE—Before diluting to final
volume, dissolve in a portion of water and nitric acid.]
Standard solutions: Dilute Standard stock solution B
with the Blank to obtain concentrations of 0.002,
0.005, 0.010, 0.025, and 0.050 ug/mL of cadmium.
Sample solution: Prepare as directed in Limit of Arsenic.
Analysis: Program the graphite furnace as follows. Dry
at 120° using a 1-s ramp, a 55-s hold, and an argon
flow of 300 mL/min. Char the sample at 850° using a
1-s ramp, a 30-s hold, and an airflow of 300 mL/min.
Cool down, and purge the air from the furnace for 10 s
using a 20° set temperature and an argon flow of
300 mL/min. Atomize at 2400° using a 0-s ramp and a
5-s hold with the argon flow stopped. Clean out at
2600° using a 1-s ramp anda 5-s hold. Separately in-
ject equal volumes (20 uL) of the Standard solutions, the
Sample solution, and the Blank, followed by a 5-uL injec-
tion of the Modifier working solution for each of the
samples, into the graphite tube of a suitable graphite
furnace atomic absorption spectrometer equipped with
a hollow-cathode lamp for cadmium. Determine the
peak area at the cadmium emission line at 228.8 nm,
corrected for background absorption. Plot the corrected
peak areas of the Standard solutions versus their con-
tents of cadmium, in ug/mL, and calculate the regres-
sion line best fitting the points. Determine the concen-
tration, C, in ug/mL, of cadmium in each mL of the
ieee solution by interpolation from the regression
ine.
Calculate the content of cadmium in the Fish Oil Con-
taining Omega-3 Acids taken:
Result = (C/W) x 25
iE = concentration of cadmium in each mL of the
Sample solution (ug/mL)
w = weight of Fish Oil sone Omega-3 Acids
taken to prepare the Sample solution (g)
Acceptance criteria: NMT 0.1 pg/g
e Limit OF MeRcurY: Proceed as directed in Mercury (261),
Method Ila, except use a Standard Mercury Solution hav-
ing the equivalent of 0.1 ug/mL of mercury.
Dietary Supplements / Fish Oil 4619
Sample solution: Prepare as directed for the Sample so-
lution in Limit of Arsenic, combining the two duplicate
cooled digests into 1.0 mL of Potassium Permanganate
Solution.
Acceptance criteria: NMT 0.1 pg/g
e LIMIT OF DIOXINS, FURANS, AND POLYCHLORINATED
BIPHENYLS
Analysis: Determine the content of polychlorinated
dibenzo-para-dioxins (PCDDs) and polychlorinated
dibenzofurans (PCDFs) by Method No. 1613, Revision
B, of the Environmental Protection Agency. Determine
the content of polychlorinated biphenyls (PCBs) by
Method No. 1668, Revision A of the Environmental Pro-
tection Agency.
Acceptance criteria: The sum of PCDDs and PCDFs is
NMT 2.0 pg/g of World Health Organization (WHO)
toxic equivalents. The sum of PCDDs, PCDFs, and di-
oxin-like PCBs (polychlorinated biphenyls, nonortho
IUPAC congeners PCB-77, PCB-81, PCB-126, and PCB-
169, and mono-ortho IUPAC congeners PCB-105, PCB-
114, PCB-118, PCB-123, PCB-156, PCB-157, PCB-167,
and PCB-189) is NMT 10.0 pg/g of WHO toxic
equivalents.
SPECIFIC TESTS
e FATS AND FIXED OILS, Acid Value (401): NMT 3
FATS AND FIXED OILS, Anisidine Value (401): NMT 20.0
FATS AND FIXED OILS, Peroxide Value (401): NMT 5.0
FATS AND FIXED OILS, Total Oxidation Value (TOTOX) (401)
Analysis: Calculate TOTOX as follows:
Result = (2 x PV) + AV
PV = peroxide value
AV = anisidine value
Acceptance criteria: NMT 26
FATS AND FIXED OILS, Unsaponifiable Matter (401): NMT
1.5%
STEARIN
Sample: 10 mL
Analysis: Cool the Sample at 0° for 3 h.
Acceptance criteria: The Sample remains clear.
ABSORBANCE
Sample solution: 0.24 mg/mL in isooctane
Acceptance criteria: The absorbance is NMT 0.70, de-
termined at 233 nm.
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in tight, light-resistant
containers, and store at controlled room temperature. It
may be bottled or otherwise packaged in containers from
which air has been expelled by the production of a vac-
sydesBouo=w sa
uum or by an inert gas.
e LABELING: The label states the average content of DHA
and EPA in mg/g. It also states the name and concentra-
tion of any added antioxidant.
Change to read:
e USP REFERENCE STANDARDS (11)
te (CN 1-May-2018)
YsP Fish Oil RS
@ (CN 1-May-2018)
 4620 Fish Oil / Dietary Supplements
Fish Oil Containing Omega-3 Acids
Capsules
DEFINITION
Fish Oil Containing Omega-3 Acids Capsules contain NLT
95.0% and NMT 105.0% of the labeled amount of Fish
Oil Containing Omega-3 Acids where Fish Oil Containing
Omega-3 Acids is the purified, winterized, and deodorized
fatty oil obtained from fish of the families Engraulidae,
Carangidae, Clupeidae, Osmeridae, Scombroidae, and
Ammodytidae. The omega-3 acids are defined as the fol-
lowing: alpha-linolenic acid (C18:3 n—3), moroctic acid
(C18:4 n-3), eicosatetraenoic acid (C20:4 n-3), eicosa-
pentaenoic acid (EPA) (C20:5 n—3), heneicosapentaenoic
acid (C21:5 n—3), docosapentaenoic acid (C22:5 n—3),
and docosahexaenoic acid (DHA) (C22:6 n-3). It contains
NLT 28.0% (w/w) of total omega-3 acids, expressed as
free acids, consisting of NLT 13.0% of EPA and NLT 9.0%
of DHA. Suitable antioxidants in appropriate concentra-
tions may be added.
IDENTIFICATION
e A. The oil contained in the Capsules meet the require-
ments for the following test. the retention times of the
docosahexaenoic acid methyl ester and eicosapentanoic
acid methyl ester peaks from Test solution 1 in Content of
EPA and DHA correspond to those of the docosahexae-
noic acid methyl ester and eicosapentanoic acid methyl
ester peaks from Standard solution 2a and Standard solu-
tion 2b, respectively, in Fats and Fixed Oils (401), Content
of EPA and DHA. The sum of the areas for EPA and DHA
methyl esters is NLT 22% of the total detected area for
the methyl esters, and no other peak in the chromato-
gram has an area higher than 20% of the total detected
area for the methyl esters. In addition to the EPA and
DHA peaks, Test solution 1 exhibits at least 15 more
peaks with retention times similar to those of the Fish oil
standard solution, as obtained in the test for Content of
EPA and DHA.
STRENGTH
© CONTENT OF FISH OIL
Analysis: Weld NLT 10 Capsules in a tared weighing
bottle; carefully open the Capsules, without loss of shell
material; and transfer the combined Capsule contents
to a 100-mL beaker. Remove any adhering substance
from the emptied Capsules by washing with several
small portions of 2,2,4-trimethylpentane. Discard the
washings, and allow the empty Capsules to dry in a
current of dry air until the 2,2,4-trimethylpentane is
DS Monographs
completely evaporated. egp the empty Capsules in
the original tared weighing bottle, and calculate the av-
erage net weight per Capsule.
Acceptance criteria: 95.0%-105.0% of the labeled
amount
e CONTENT OF EPA AND DHA
(See Fats and Fixed Oils (401), Omega-3 Fatty Acids Deter-
mination and Profile.)
System suitability solution 1, System suitability solu-
tion 2, Standard solution 1a, Standard solution 1b,
Standard solution 2a, Standard solution 2b, Test so-
lution 1, Test solution 2, Chromatographic system,
System suitability, and Analysis: Proceed as directed
in Fats and Fixed Oils (401), Content of EPA and DHA, for
triglycerides.
Fish oil standard solution: Transfer 300 mg of USP Fish
Oil RS into a 10-mL volumetric flask, and dissolve in
and dilute with Antioxidant Solution to volume. Proceed
as directed for Test Solution 1 (for triglycerides) in Fats
and Fixed Oils (401), Content of EPA and DHA, starting
with “Transfer 2.0 mL”.
Identify the relevant fatty acid methyl esters in the Fish
oil standard solution by comparing their retention times
USP 41
with those in the reference chromatogram supplied
with the USP Fish Oil RS.
Calculate the percentage of EPA and DHA in the portion
of fish oil containing omega-3 acids taken from the
Capsules.
Acceptance criteria: NLT 13.0% (w/w) of EPA and NLT
9.0% (w/w) of DHA
e@ CONTENT OF TOTAL OMEGA-3 ACIDS
(See Fats and Fixed Oils (401), Omega-3 Fatty Acids Deter-
mination and Profile.)
Analysis: Proceed as directed in Fats and Fixed Oils
(401), Content of Total Omega-3 Acids (for triglycerides).
Acceptance criteria: NLT 28.0% (w/w) of total omega-
3 acids, expressed as free acids
PERFORMANCE TESTS
e DISINTEGRATION AND DISSOLUTION OF DIETARY SUPPLEMENTS
(2040): Meet the requirements for Rupture Test for Soft
Shell Capsules
e@ WEIGHT VARIATION OF DIETARY SUPPLEMENTS (2091): Meet
the requirements
CONTAMINANTS
e LIMIT OF ARSENIC
[Note—For the preparation of all aqueous solutions and
for the rinsing of glass, polytef, and plastic vessels
before use, use water that has first been passed through
a strong-acid, strong-base, mixed-bed ion-exchange
resin. Select all reagents to have as low a content of
arsenic as practicable, and store all reagent solutions in
containers of borosilicate glass. Cleanse glass, polytef,
and plastic vessels before use by soaking them in warm
8N nitric acid for 30 min and by rinsing them with
deionized water.]
1% Palladium stock solution: Transfer 1 g of ultrapure
palladium metal into a Teflon beaker. Add 20 mL of
water and 10 mL of nitric acid, and warm on a hot
plate to dissolve. Allow the solution to cool to room
temperature, transfer into a 100-mL volumetric flask,
and dilute with deionized water to volume.
1% Magnesium nitrate stock solution: Transfer 1 g of
ultrapure magnesium nitrate to a Teflon beaker. Add
40 mL of water and 1 mL of nitric acid, and warm on a
hot plate to dissolve the solids. Allow the solution to
cool to room temperature, transfer to a 100-mL volu-
metric flask, and dilute with deionized water to volume.
Modifier working solution: 1% Palladium stock solution,
1% Magnesium nitrate stock solution, and 2% nitric acid
(3:2:5). A volume of 5 uL provides 0.015 mg of palla-
dium and 0.01 mg of magnesium nitrate.
Blank: Nitric acid and water (5 in 100)
Standard stock solution: Transfer 10.0 mL of Standard
Arsenic Solution, prepared as directed in the test for Ar-
senic (211), to a 100-mL volumetric flask. Add 40 mL of
water and 5 mL of nitric acid, and dilute with water to
volume. This solution contains 0.10 g/mL of arsenic.
Standard solutions: Dilute the Standard stock solution
with the Blank to obtain concentrations of 0.002,
0.005, 0.010, 0.025, and 0.050 g/mL of arsenic.
Sample solution: For the preparation of the Sample so-
lution, use a microwave oven with a magnetron fre-
quency of 2455 MHz andaselectable qubputpower of
0-950 W in 1% increments, equipped with advanced
composite vessels with 100-mL polytef liners. Use rup-
ture membranes to vent vessels should the pressure ex-
ceed 125 psi. The vessels fit into a turntable, and each
vessel can be vented into an overflow container. Equip
the microwave oven with an exhaust tube to ventilate
fumes. [CAUTION—Wear proper eye protection and pro-
tective clothing and gloves.] Transfer approximate y
500 mg from content of Capsules, weighed to the near-
est 0.1 mg, into a Teflon digestion vessel liner. Prepare
samples in duplicate. Add 15 mL of nitric acid, and swirl
gently. Cover the vessels with lids, leaving the vent fit-
ting off. Predigest overnight under a hood. Place the
USP 41
rupture membrane in the vent fitting, and tighten the
lid. Place all vessels on the microwave oven turntable.
Connect the vent tubes to the vent trap, and connect
the pressure-sensing line to the appropriate vessel. Initi-
ate a two-stage digestion procedure by heating the mi-
crowave at 15% power for 15 min, followed by 25%
power for 45 min. Remove the turntable of vessels from
the oven, and allow the vessels to cool to room tem-
perature. [NOTE—A cool water bath may be used to
speed the cooling process.] Vent the vessels when they
reach room temperature. Remove the lids, and slowly
add 2 mL of 30% hydrogen peroxide to each. Allow the
reactions to subside, and seal the vessels. Return the
vessels on the turntable to the microwave oven, and
heat for an additional 15 min at 30% power. Remove
the vessels from the oven, and allow them to cool to
room temperature. Transfer the cooled digests into
25-mL volumetric flasks, and dilute with water to
volume.
Analysis: Program the graphite furnace as follows. Dry
at 115° using a 1-s ramp, a 65-s hold, and an argon
flow of 300 mL/min. Char the sample at 1000° using a
1-s ramp, a 20-s hold, and an airflow of 300 mL/min.
Cool down, and purge the air from the furnace for 10 s
by using a 20° set temperature and an argon flow of
300 mL/min. Atomize at 2400° using a 0-s ramp and a
5-s hold with the argon flow stopped. Clean out at
2600° using a 1-s ramp and a 5-s hold. Separately in-
ject equal volumes (20 uL) of the Blank, the Standard
solutions, and the Sample solution, followed by a 5-uL
injection of the Modifier working solution for each of the
samples, into the graphite tube of a suitable graphite
furnace atomic absorption spectrometer equipped with
a hollow-cathode lamp for arsenic. Determine the peak
area at the arsenic emission line at 193.7 nm, corrected
for background absorption. Plot the corrected peak ar-
eas of the Standard solutions versus their contents of
arsenic, in g/mL, and calculate the regression line best
fitting the points. Determine the concentration, C, in
tug/mL, of arsenic in each mL of the Sample solution by
interpolation from the regression line.
Calculate the content of arsenic in the portion of Cap-
sules taken:
Result = (C/W) x 25
€ = concentration of arsenic in each mL of the
Sample solution (g/mL)
w = weight of fish oil containing omega-3 acids
taken to prepare the Sample solution (g)
Acceptance criteria: NMT 0.1 ug/g
e Limit OF LEAD
[Note—For the preparation of all aqueous solutions and
for the rinsing of glass, polytef, and plastic vessels
before use, use water that hes first been passed through
a strong-acid, strong-base, mixed-bed ion-exchange
resin. Select all reagents to have as low a content of
lead as practicable, and store all reagent solutions in
containers of borosilicate glass. Cleanse glass, polytef,
and plastic vessels before use by soaking them in warm
8N nitric acid for 30 min andby rinsing them with
deionized water.]
10% Monobasic ammonium phosphate solution:
10 g of ultrapure monobasic ammonium phosphate in
1 mL of nitric acid and 40 mL of water to dissolve the
phosphate. Dilute with deionized water to 100 mL.
1% Magnesium nitrate solution: Transfer 1 g of ul-
trapure magnesium nitrate to a Teflon beaker. Add
40 mL of water and 1 mL of nitric acid, and warm on a
hot plate to dissolve the solids. Allow the solution to
cool to room temperature, transfer to a 100-mL volu-
metric flask, and dilute with deionized water to volume.
Modifier working solution: 10% Monobasic ammonium
phosphate solution, 1% Magnesium nitrate solution, and
2% nitric acid (2:1:2). A volume of 5 uL provides
Dietary Supplements / Fish Oil 4621
0.2 mg of phosphate plus 0.01 mg of magnesium
nitrate.
Blank: Nitric acid and water (5 in 100)
Standard stock solution: Transfer 10.0 mL of lead ni-
trate stock solution TS to a 100-mL volumetric flask.
Add 40 mL of water and 5 mL of nitric acid, and dilute
with water to volume. Transfer 1.0 mL of this solution
to a second 100-mL volumetric flask. Add 50 mL of
water and 1 mL of nitric acid, and dilute with water to
volume. This solution contains 0.10 g/mL of lead.
Standard solutions: Dilute the Standard stock solution
with the Blank to obtain concentrations of 0.002,
0.005, 0.010, 0.025, and 0.050 g/mL of lead.
Sample solution: Prepare as directed in Limit of Arsenic.
Analysis: Program the graphite furnace as follows. Dry
at 120° using a 1-s ramp, a 55-s hold, and an argon
flow of 300 mL/min. Char the sample at 850° using a
1-s ramp, a 30-s hold, and an airflow of 300 mL/min.
Cool down, and purge the air from the furnace for 10 s
by using a 20° set temperature and an argon flow of
300 mL/min. Atomize at 2100° using a 0-s ramp and a
5-s hold with the argon flow stopped. Clean out at
2600° using a 1-s ramp and a 5-s hold. Separately in-
ject equal volumes (20 uL) of the Blank, the Standard
solutions, and the Sample solution, followed by a 5-uL
injection of the Modifier working solution for each of the
samples, into the graphite tube of a suitable graphite
furnace atomic absorption spectrometer equipped with
a hollow-cathode lamp for lead. Determine the peak
area at the lead emission line at 283.3 nm, corrected
for background absorption. Plot the corrected peak ar-
eas of the Standard solutions versus their contents of
lead, in g/mL, and calculate the regression line best
fitting the points. Determine the concentration, C, in
g/mL, of lead in each mL of the Sample solution by
interpolation from the regression line.
eae the content of lead in the portion of Capsules
taken:
Result = (C/W) x 25
Cc = concentration of lead in each mL of the
Sample solution (\ug/mL)
Ww = weight of fish oil containing omega-3 acids
taken to prepare the Sample solution (g)
Acceptance criteria: NMT 0.1 ug/g
e LIMIT FOR CADMIUM
[Note—For the preparation of all aqueous solutions and
for the rinsing of glass, polytef, and plastic vessels
before use, use water that has first been passed through
a strong-acid, strong-base, mixed-bed ion-exchange
resin. Select all reagents to have as low a content of
sydesbouo=:w Sa
cadmium as practicable, and store all reagent solutions
in containers of borosilicate glass. Cleanse glass, polytef,
and plastic vessels before use by soaking them in warm
8N nitric acid for 30 min andbyrinsing them with
deionized water.]
10% Monobasic ammonium phosphate solution:
10 g of ultrapure monobasic ammonium phosphate in
40 mL of water and 1 mL of nitric acid to dissolve the
phosphate. Dilute with deionized water to 100 mL.
1% Magnesium nitrate solution: Transfer 1 g of ul-
trapure magnesium nitrate to a Teflon beaker. Add
40 mL of water and 1 mL of nitric acid, and warm on a
hot plate to dissolve the solids. Allow the solution to
cool to room temperature, transfer to a 100-mL volu-
metric flask, and dilute with deionized water to volume.
Modifier working solution: 10% Monobasic ammonium
phosphate solution, 1% Magnesium nitrate solution, and
2% nitric acid to volume (2:1:2). A volume of 5 UL pro-
vides 0.2 mg of phosphate and 0.01 mg of magnesium
nitrate.
Blank: Nitric acid and water (5 in 100)
Standard stock solution A: 0.1372 mg/mL of cadmium
nitrate in water
 4622 Fish Oil / Dietary Supplements
Standard stock solution B: Standard stock solution A,
nitric acid, and water (2:1:97). This solution contains
0.10 pg/mL of cadmium. [NoTE—Before diluting to final
volume, dissolve in a portion of water and nitric acid.]
Standard solutions: Dilute Standard stock solution B
with the Blank to obtain concentrations of 0.002,
0.005, 0.010, 0.025, and 0.050 g/mL of cadmium.
Sample solution: Prepare as directed in Limit of Arsenic.
Analysis: Program the graphite furnace as follows. Dry
at 120° using a 1-s ramp, a 55-s hold, and an argon
flow of 300 mL/min. Char the sample at 850° using a
1-s ramp, a 30-s hold, and an airflow of 300 mL/min.
Cool down and purge the air from the furnace for 10 s
by using a 20° set temperature and an argon flow of
300 mL/min. Atomize at 2400° using a 0-s ramp and a
5-s hold with the argon flow stopped. Clean out at
2600° using a 1-s ramp and a 5-s hold. Separately in-
ject equal volumes (20 wL) of the Blank, the Standard
solutions, and the Sample solution, followed by a 5-uL
injection of the Modifier working solution for each of the
samples, into the graphite tube of a suitable graphite
furnace atomic absorption spectrometer equipped with
a hollow-cathode lamp for cadmium. Determine the
peak area at the cadmium emission line at 228.8 nm,
corrected for background absorption. Plot the corrected
peak areas of the Standard solutions versus their con-
tents of cadmium, in ug/mL, and calculate the regres-
sion line best fitting the points. Determine the concen-
tration, C, in ug/mL, of cadmium in each mL of the
Sample solution by interpolation from the regression
line.
Calculate the content of cadmium in the portion of
Capsules taken:
Result = (C/W) x 25
Cc = concentration of cadmium in each mL of the
Sample solution (ug/mL)
w = weight of fish oil containing omega-3 acids
taken to prepare the Sample solution (g)
Acceptance criteria: NMT 0.1 pg/g
e Limit OF MERCURY
Sample solution: Prepare as directed in Limit of Arsenic,
combining the two duplicate cooled digests into 1.0 mL
of Potassium Permanganate Solution in Mercury (261),
Method Ila and IIb, Reagents.
Analysis: Proceed as directed for Mercury (261), Method
Ila, except use a Standard Mercury Solution having the
equivalent of 0.1 g/mL of mercury.
Acceptance criteria: NMT 0.1 ug/g
e LIMIT OF DIOXINS, FURANS, AND POLYCHLORINATED
DS Monographs
BIPHENYLS
Analysis: Determine the content of polychlorinated
dibenzo-para-dioxins (PCDDs) and polychlorinated
dibenzofurans (PCDFs) by Method No. 1613, Revision
B, of the Environmental Protection Agency. Determine
the content of polychlorinated biphenyls (PCBs) by
Method No. 1668, Revision A, of the Environmental
Protection Agency.
Acceptance criteria: The sum of PCDDs and PCDFs is
NMT 2.0 pg/g of World Health Organization (WHO)
toxic equivalents. The sum of PCDDs, PCDFs, and di-
oxin-like PCBs (polychlorinated biphenyls, nonortho
IUPAC congeners PCB-77, PCB-81, PCB-126, and PCB-
169, and mono-ortho IUPAC congeners PCB-105, PCB-
114, PCB-118, PCB-123, PCB-156, PCB-157, PCB-167,
and PCB-189) is NMT 10.0 pg/g of WHO toxic
equivalents.
USP 41
SPECIFIC TESTS
© FATS AND FIXED OILS, Acid Value (401): NMT 3
e FATS AND FIXED OILS, Anisidine Value (401): NMT 20.0
© FATS AND FIXED OILS, Peroxide Value (401): NMT 5.0
© FATS AND FIXED OILS, Tota! Oxidation Value (TOTOX)
(401): NMT 26, calculated:
Result = (2 x PV) + AV
PV = peroxide value
AV = anisidine value
FATS AND FIXED OILS, Unsaponifiable Matter (401): NMT
1.5%
STEARIN: 10 mL remains clear after cooling at 0° for 3 h.
ABSORBANCE
Sample solution: 0.24 mg/mL in isooctane
Acceptance criteria: The absorbance is NMT 0.70, de-
termined at 233 nm.
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in tight containers,
and store at room temperature, protected from light.
e LABELING: The label states the amount of docosahexae-
noic acid (DHA) and eicosapentaenoic acid (EPA) in mg
per Capsule.
e USP REFERENCE STANDARDS (11)
USP Docosahexaenoic Acid Ethyl Ester RS
All cis-4,7,10,13,16,19-docosahexaenoic ethyl ester.
CogH3s602 356.55
USP Eicosapentaenoic Acid Ethyl Ester RS
All cis-5,8,11,14,17-eicosapentaenoic ethyl ester.
Co2H3402 330.51
USP Fish Oil RS
USP Methyl Tricosanoate RS
Tricosanoic acid methyl ester.
CosHasO2 3368.64
Fish Oil Containing Omega-3 Acids
Delayed-Release Capsules
DEFINITION
Fish Oil Containing Omega-3 Acids Delayed-Release Cap-
sules are enteric-coated Capsules that contain NLT 95.0%
and NMT 105.0% of the labeled amount of Fish Oil Con-
taining Omega-3 Acids where Fish Oil Containing Omega-
3 Acids is the purified, winterized, and deodorized fatty
oil obtained from fish of the families Engraulidae,
Carangidae, Clupeidae, Osmeridae, Scombroidae, and
Ammodytidae. The omega-3 acids are defined as the fol-
lowing: alpha-linolenic acid (C18:3 n-3), moroctic acid
(C18:4 n-3), eicosatetraenoic acid (C20:4 n-3), eicosa-
pentaenoic acid (EPA) (C20:5 n-3), heneicosapentaenoic
acid (C21:5 n-3), docosapentaenoic acid (C22:5 n-3),
and docosahexaenoic acid (DHA) (C22:6 n-3). It contains
NLT 28.0% (w/w) of total omega-3 acids, expressed as
free acids, consisting of NLT 13.0% of EPA and NLT 9.0%
of DHA. Suitable antioxidants in appropriate concentra-
tions may be added.
IDENTIFICATION
e A. The oil contained in the capes meets the require-
ments for the following test. The retention times of the
docosahexaenoic acid methyl ester and eicosapentanoic
acid methyl ester peaks of Test solution 1 in Content of
EPA and DHA correspond to those of the docosahexae-
noic acid methyl ester and eicosapentanoic acid methyl
ester peaks of Standard solution 2a and Standard solution
2b, respectively, in Fats and Fixed Oils (401), Omega-3
Fatty Acids Determination and Profile, Content of EPA and
DHA. The sum of the area for EPA and DHA methyl esters
is NLT 22% of the total detected area for the methyl
esters, and no other peak has an area higher than 20%
USP 41
of the total detected area for the methyl esters. In addi-
tion to the EPA and DHA peaks, Test solution 1 exhibits at
least 15 more peaks with retention times similar to those
of the Fish oil standard solution, as obtained in Content of
EPA and DHA.
STRENGTH
¢ CONTENT OF FISH OIL
Analysis: Mysiohi NLT 10 Capsules in a tared weighing
bottle, carefully open the Capsules, without loss of shell
material, and transfer the combined Capsule contents
to a 100-mL beaker. Remove any adhering substance
from the emptied Capsulesby washing with several
small portions of 2,2,4-trimethylpentane. Discard the
washings, and allow the empty Capsules to dry in a
current of dry air until the 2,2,4-trimethylpentane is
completely evaporated. welgn the empty Capsules in
the original tared weighing bottle, and calculate the av-
erage net weight per Capsule.
Acceptance criteria: 95.0%-105.0% of the labeled
amount
¢ CONTENT OF EPA AND DHA
(See Fats and Fixed Oils (401), Omega-3 Fatty Acids Deter-
mination and Profile.)
System suitability solution 1, System suitability solu-
tion 2, Standard solution 1a, Standard solution 1b,
Standard solution 2a, Standard solution 2b, Test so-
lution 1, Test solution 2, Chromatographic system,
System suitability, and Analysis: Proceed as directed
in Fats and Fixed Oils (401), Content of EPA and DHA for
triglycerides.
Fish oil standard solution: Transfer 300 mg of USP Fish
Oil RS to a 10-mL volumetric flask, and dissolve in and
dilute with Antioxidant Solution to volume. Proceed as
directed for Test Solution 1 (for triglycerides) in Fats and
Fixed Oils (401), Content of EPA and DHA, starting with
“Transfer 2.0 mL”.
Identify the relevant fatty acid methyl esters in the Fish
oil standard solution by comparing their retention times
with those in the reference chromatogram supplied
with the USP Fish Oil RS.
Calculate the percentage of EPA and DHA in the portion
of fish oil containing omega-3 acids taken from the
Capsules.
Acceptance criteria: NLT 13.0% (w/w) of EPA and NLT
9.0% (w/w) of DHA
© CONTENT OF TOTAL OMEGA-3 ACIDS
(See Fats and Fixed Oils (401), Omega-3 Fatty Acids Deter-
mination and Profile.)
Analysis: Proceed as directed in Fats and Fixed Oils
(401), Content of Total Omega-3-Acids (for triglycerides).
Acceptance criteria: NLT 28.0% (w/w) of total omega-
3 acids, expressed as free acids
PERFORMANCE TESTS
¢ DISINTEGRATION AND DISSOLUTION (2040): Meet the re-
quirements for Disintegration, Delayed-Release (Enteric-
Coated) Soft Shell Capsules
© WEIGHT VARIATION OF DIETARY SUPPLEMENTS (2091): Meet
the requirements
CONTAMINANTS
e FATS AND FIXED OlLs, Trace Metals (401): NMT 0.1 ppm
each of Pb, Cd, As, and Hg
¢ LIMIT OF DIOXINS, FURANS, AND POLYCHLORINATED
BIPHENYLS
Analysis: Determine the content of polychlorinated
dibenzo-para-dioxins (PCDDs) and polychlorinated
dibenzofurans (PCDFs) by Method 1613, Revision B, of
the Environmental Protection Agency. Determine the
content of polychlorinated biphenyls (PCBs) by Method
1668, Revision A, of the Environmental Protection
Agency.
Acceptance criteria: The sum of PCDDs and PCDFs is
NMT 2.0 pg/g of World Health Organization (WHO)
Dietary Supplements / Flax Seed Oil 4623
toxic equivalents. The sum of PCDDs, PCDFs, and di-
oxin-like PCBs (polychlorinated biphenyls, non-ortho
IUPAC congeners PCB-77, PCB-81, PCB-126, and PCB-
169, and mono-ortho IUPAC congeners PCB-105, PCB-
114, PCB-118, PCB-123, PCB-156, PCB-157, PCB-167,
and PCB-189) is NMT 10.0 pg/g of WHO toxic
equivalents.
SPECIFIC TESTS
e FATS AND FIXED OILS, Acid Value (401): NMT 3
FATS AND FIXED OILS, Anisidine Value (401): NMT 20.0
FATS AND FIXED OILS, Peroxide Value (401): NMT 5.0
FATS AND FIXED OILS, Tota! Oxidation Value (TOTOX)
(401): NMT 26, calculated:
Result = (2 x PV) + AV
PV = peroxide value
AV =anisidine value
FATS AND FIXED OILS, Unsaponifiable Matter (401): NMT
1.5%
STEARIN: 10 mL remains clear after cooling at 0° for 3 h.
ABSORBANCE
Sample solution: 0.24 mg/mL in isooctane
Acceptance criteria: NMT 0.70, determined at 233 nm
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in tight containers,
and store at room temperature, protected from light.
e LABELING: The label states the amount of docosahexae-
noic acid (DHA) and eicosapentaenoic acid (EPA) in mg
per Capsule.
e USP REFERENCE STANDARDS (11)
USP Docosahexaenoic Acid Ethyl Ester RS
All cis-4,7,10,13,16,19-docosahexaenoic ethyl ester.
CrsH3602 356.55
USP Eicosapentaenoic Acid Ethyl Ester RS
All cis-5,8,11,14,17-eicosapentaenoic ethyl ester.
CoxH3402 330.51
USP Fish Oil RS
USP Methyl Tricosanoate RS
Flax Seed Oil
[8001-26-1].
DEFINITION
Flax Seed Oil is derived from flaxseed or linseed (Linum
usitatissimum L.). The oil is extracted from the hard, tiny
sydesbouo=: sa
seeds by cold pressing and then refined. No solvents or
external heat are employed in the extraction process. It
contains no added substances.
IDENTIFICATION
e A. It meets the requirements in Specific Tests for Fats and
Fixed Oils (401), Fatty Acid Composition.
¢ B. IDENTIFICATION OF FIXED OILS BY THIN-LAYER CHROMA-
TOGRAPHY (202): The R; values of the principal spots of
the Sample solution correspond to those of the Standard
solution.
SPECIFIC TESTS
FATS AND FIXED OILS, Acid Value (401): NMT 2.0
FATS AND FIXED OILS, Peroxide Value (401): NMT 2.0
FATS AND FIXED OILS, lodine Value (401): 150-165
FATS AND FIXED OILS, Saponification Value (401): 180-190
FATS AND FIXED OILS, Unsaponifiable Matter (401): NMT
1
FATS AND FIXED OILS, Fatty Acid Composition (401): Flax
Seed Oil exhibits the composition profile of fatty acids in
Table 1.
 4624 Flax Seed Oil / Dietary Supplements USP 41

Table 1 Internal standard: Methyl nonadecanoate

Fatty Shorthand Percentage Sample solution: Weigh NLT 10 Capsules. With a sharp
Acid Notation (%) blade, carefully slice open the Capsules, avoiding loss of
shell material. Combine the Capsule contents in a suita-
Palmitic acid 16:0 2,0-7,5 ble container, and mix well. Remove any adhering sub-
Stearic acid 18:0 1.0-6.0 stance from the sated Capsules by washing with sev-
Oleic acid 18:1 12.0-24.0 eral portions of diethyl ether, and discard the washings.
Linoleic acid 18:2 11.0-23.0 Allow the empty Capsule shells to air-dry over a period
Alpha-linolenic acid 18:3 50.0-65.0 of NMT 30 min, rang precautions to avoid uptake or
loss of moisture. Weigh the empty Capsule shells, and
© REFRACTIVE INDEX (831): 1.460-1.490 at 20° calculate the average fill weight/Capsule (A,). Transfer
80 mg of the accurately ae combined Capsule
ADDITIONAL REQUIREMENTS contents directly into a tared 30-mL screw-top glass
© PACKAGING AND STORAGE: Preserve in well-closed, tight, centrifuge tube. Re-tare, and accurately weigh about
light-resistant containers. 50 mg of Internal standard. Add 2 mL of 0.5 N metha-
e LABELING: Where Flax Seed Oil is intended for use in the nolic sodium hydroxide solution, tightly cap, and transfer
manufacture of dosage forms, it is so labeled. to a heating block, or another appropriate heating de-
vice. Reflux the solution until fat globules disappear
Delete the following: (usually 5-10 min). Add 2 mL of 0.14 g/mL boron
trifluoride in methanol, cap, and reflux for 2 min. Add
°e USP REFERENCE STANDARDS (11)
4 mL of chromatographic n-heptane, cap, and reflux for
USP Flax Seed Oil RS 1 min. Cool, add about 8 mL of saturated sodium chlo-
ride solution, shake, and centrifuge to separate layers.
© (CN 1-May-2018) Dilute an aliquot of the upper (heptane) layer 1:8 with
chromatographic n-heptane, and mix well.
System suitability solution: Using about 80 mg of USP
Flax Seed Oil RS, proceed as directed for the Sample
solution, beginning with “Transfer 80 mg” without the
Flax Seed Oil Capsules addition of the Internal standard.
Standard solution: Directly into a tared 30-mL screw-
DEFINITION top glass centrifuge tube accurately weigh about 50 mg
Flax Seed Oil Capsules are prepared with Flax Oil derived of USP Methyl Linolenate RS, 20 mg of USP Methy] Li-
from cold-pressed flax seeds and contain NLT 95.0% of noleate RS, and 20 mg of USP Methyl Oleate RS. Pro-
the labeled amounts of a-linolenic (C18:3 n-3), linoleic ceed as directed for the Sample solution, beginning with
(C18:2 n-6), and oleic (C18:1 n—-9) acids. “Re-tare”.
Chromatographic system
IDENTIFICATION (See Chromatography (621), System Suitability.)
oA. Mode: GC
Sample: A portion of oil, about 2 mL, from NLT 10 Detector: Flame ionization
Capsules Column: 0.53-mm x 30-m fused silica capillary; coated
Analysis: Transfer the Sample to a suitable test tube. with a 1.0-um film of G16
Add 2 mL of glacial acetic acid, and warm the test tube Temperatures
to about 50°, while swirling, for 5 min. Cool, and add 1 Injection port: 220°
drop of sulfuric acid. Detector: 260°
Acceptance criteria: A greenish color develops. Column: See Table 2.
e B. FATTY ACID PROFILE
System suitability solution and Chromatographic sys-
tem: Proceed as directed in Strength. Table 2
Sample solution: Proceed as directed for the Sample Hold Time
solution in Strength, beginning with “Transfer 80 mg” Initial Temperature Final at Final
without the addition of the Internal standard. Temperature Ramp Temperature | Temperature
Analysis: Identify the specified fatty acid methyl ester
DS Monographs

@) (°/min) C) (min)
peaks by comparing them to the reference chromato- 70 0 70 2
gram provided with the lot of USP Flax Seed Oil RS 70 5 240 5
being used. Determine the percentage of each constitu-
ent relative to the total integrated area. Carrier gas: Helium
Acceptance criteria: The Sample solution conforms to Linear velocity: 50 cm/s
the fatty acids composition profile in Table 1. Split mode: Splitless
Injection volume: 1 pL
Table 1 System suitability
Fatty Shorthand Percentage
Samples: System suitability solution and Standard
solution
Acid Notation (%)
Suitability requirements
Palmitic acid 16:0 2.0-7.5 Chromatogram similarity: The System suitability solu-
Stearic acid 18:0 1.0-6.0 tion chromatogram is similar to the reference chro-
Oleic acid 18:1 n-9 12.0-24.0 matogram provided with the lot of USP Flax Seed Oil
Linoleic acid 18:2 n-6 11.0-23.0 RS being used.
a-Linolenic acid 18:3 n-3 50.0-65.0 Resolution: NLT 1.5 between methyl oleate and
methyl stearate, System suitability solution
Relative standard deviation: NMT 2% for the peak
STRENGTH area ratios of analytes to internal standard, Standard
© CONTENT OF c-LINOLENIC, LINOLEIC, AND OLEIC ACIDS solution
0.5 N methanolic sodium hydroxide solution: Dis-
solve 2 g of sodium hydroxide in 100 mL of methanol.
USP 41 Dietary Supplements / Forskohlii 4625

Analysis Folic Acid—see Folic Acid General

Samples: Sample solution and Standard solution
Identify the retention times of the relevant fatty acid
methyl esters by comparing the peaks in the chromat-
ogram of the sys suitability solution with those in
the reference chromatogram. Identify the locus for the
internal standard peak by comparison of the chromat-
ograms of the Standard solution and System suitability
solution.
Calculate the content, in mg/g, of a-linolenic, linoleic,
and oleic acids in the portion of the Capsule contents
taken:
Result = (Ru/Rs) x (Au/As) x (ms/W) x (Mn/Mr2)
Ru = peak area ratio of the relevant methyl ester to
the internal standard in the Sample solution
Rs = peak area ratio of the relevant methyl ester to
the internal standard in the Standard solution
Au = weight of the /nternal standard in the Sample
solution (mg)
As = weight of the Internal standard in the Standard
solution (mg)
Ms = weight of the relevant USP Methyl Ester RS in
the Standard solution (mg)
Ww = wean of sample used to prepare the Sample
solution (g)
Ma = amet weight of the relevant fatty acid (g/
mol
M, = molecular weight of the relevant fatty acid
methyl ester (g/mol)
Calculate the percentage of the labeled amounts of
each individual a-linolenic, linoleic, and oleic acid in
the portion of Capsules taken:
Result = A x Ar x (100/L)
A = content of the relevant fatty acid in the
portion of Capsule contents taken (mg/g)
Ar = average fill weight (9)
L = labeled content of the relevant fatty acid (mg/
Capsule)
Acceptance criteria: 95.0% of the labeled amounts of
a-linolenic, linoleic, and oleic acids
PERFORMANCE TESTS
e DISINTEGRATION AND DISSOLUTION (2040): Meet the re-
quirements for Rupture Test for Soft Shell Capsules
© WEIGHT VARIATION OF DIETARY SUPPLEMENTS (2091): Meet
the requirements
SPECIFIC TESTS
Monographs
Folic Acid Tablets—-see Folic Acid Tablets
General Monographs
Forskohlii
DEFINITION
Forskohlii consists of the dried roots of Plectranthus barbatus
Andrews, also known as Coleus barbatus (Andrews) Benth.
and Coleus forskohlii Briq. (Fam. Lamiaceae). It contains
NLT 0.4% of forskolin, calculated on the dried basis.
IDENTIFICATION
e A. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST
(201)
Standard solution A: 50 g/mL of USP Forskolin RS in
acetonitrile. Sonicate for about 10 min.
Standard solution B: 5 mg/mL of USP Powdered For-
skohlii Extract RS in acetonitrile. Sonicate for about 15
min, centrifuge, and use the supernatant.
Sample stock solution: Use the Sample solution, pre-
pared as directed in the test for Content of Forskolin.
Sample solution: Dilute 10 mL of the Sample stock solu-
tion with acetonitrile to 25 mL.
Adsorbent: Chromatographic silica gel with an average
particle size of 10-15 um (TLC plates)
Application volume: 10 uL, as 4-mm bands
eee solvent system: Toluene and ethyl acetate
(85:15
Spray reagent: 5% vanillin in glacial acetic acid and
10% sulfuric acid in water aly
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Apply the Samples as bands. Use a saturated chamber.
evelop the chromatograms until the solvent front has
moved up about 90% of the plate. Remove the plate
from the chamber, dry, spray with the Spray reagent,
Hae for 5-10 min at 105°, and examine under white
ight.
Acceptance criteria: The chromatogram of the Sample
solution exhibits a violet zone due to forskolin at an R-
value of approximately 0.3, corresponding in color and
Rr to that in the chromatogram of Standard solution A; a
sydesBbouow sa

© FATS AND FIXED OILS, Peroxide Value (401): NMT 10.0 minor violet zone, a pink zone, and a brick-red zone at
Rr values of appreanate 0.1, 0.62, and 0.69, due to
CONTAMINANTS isoforskolin, 1,9-dideoxyforskolin, and crocetindi-
¢ MICROBIAL ENUMERATION TESTS (2021): The total aerobic aldehyde, respectively. Zones detected in the Sample so-
microbial count does not exceed 1 x 103 cfu/g, and the lution chromatogram correspond in position and color
combined molds and yeasts count does not exceed to those in Standard solution B. Other minor zones may
3 x 102 cfu/g. be observed in the Sample solution and Standard solu-
e ABSENCE OF SPECIFIED MICROORGANISMS (2022): Meet the tion B chromatograms.
requirements of the tests for absence of Escherichia coli e B. The chromatogram of the Sample solution from the
ADDITIONAL REQUIREMENTS test for Content of Forskolin shows a main peak at a reten-
¢ PACKAGING AND STORAGE: Preserve in tightly-closed, light- tion time corresponding to that of forskolin in the chro-
resistant containers. matogram of Standard solution A. \dentify other diterpene
e LABELING: The label states the article from which the peaks in the Sample solution chromatogram by compari-
Capsules were prepared and the content of a-linolenic, son with the chromatogram of Standard solution B and
linoleic, and oleic acids in mg/Capsule. the reference chromatogram provided with the lot of
e USP REFERENCE STANDARDS (11) USP Powdered Forskohlii Extract RS. The Sample solution
USP Flax Seed Oil RS chromatogram shows an additional peak corresponding
USP Methyl Linoleate RS to isoforskolin.
USP Methyl Linolenate RS
USP Methyl Oleate RS
 4626 Forskohlii / Dietary Supplements USP 41

COMPOSITION ru = peak area of forskolin from the Sample solution

© CONTENT OF FORSKOLIN ls = peak area of forskolin from Standard solution A
Solution A: Acetonitrile, filtered and degassed Cs = concentration of USP Forskolin RS in Standard
Solution B: Water, filtered and degasse: solution A (mg/mL)
Standard solution A: Sonicate a quantity of USP For- Cu = concentration of Forskohlii in the Sample
skolin RS in acetonitrile to obtain a solution having a solution (mg/mL)
known concentration of about 1.0 mg/mL. Acceptance criteria: NLT 0.4% on the dried basis
Standard solution B: 5 mg/mL of USP Powdered For-
skohlii Extract RS in acetonitrile. Sonicate for about 15 IMPURITIES
min, centrifuge, and use the supernatant. Before injec- © ARTICLES OF BOTANICAL ORIGIN, Acid-Insoluble Ash (561):
tion, pass through a membrane filter of 0.45-11m or NMT 2%
finer pore size. ¢ ARTICLES OF BOTANICAL ORIGIN, Limits of Elemental Impuri-
Sample solution: Transfer about 3.0 g of Forskohlii, ties (561): Meets the requirements
finely powdered and accurately weighed, to a 100-mL e ARTICLES OF BOTANICAL ORIGIN, Foreign Organic Matter
round-bottom flask fitted with a reflux condenser. Add (561): NMT 2.0%
50 mL of acetonitrile, reflux for 20 min, cool to room e ARTICLES OF BOTANICAL ORIGIN, Pesticide Residue Analysis
temperature, and decant the supernatant. Repeat until (561): Meets the requirements
the extract is colorless. Combine the extracts, filter,
concentrate under vacuum, and adjust the volume with SPECIFIC TESTS
acetonitrile to 100 mL. Before injection, pass through a e BOTANICAL CHARACTERISTICS
membrane filter of 0.45-1m or finer pore size, discard- Macroscopic: Fresh root, pale pinkish yellow, cylindrical
ing the first 5 mL of the filtrate. to subcylindrical, with tapering ends, 5—12 in length,
System suitability solution: Standard solution A and 1-2 cm in diameter; surface rough, shows lateral root-
0.01% toluene in acetonitrile (1:1) lets or scars of rootlets and transversely running lenti-
Mobile phase: See Table 1. cels. Pharmacopeial article is dark brown; surface rough,
irregularly cylindrical, longitudinally wrinkled, showing
irregular grooves and prominent ridges; fracture short;
Table 1 cut surface yellowish-white; characteristic and pleasant
Time Solution A Solution B aromatic odor, and slightly bitter to pungent taste.
(min) (%) (%) Microscopic , f -
0 45 55 Transverse section of roots: Irregular circular in out-
3 45 55 line; showing narrow cork, 10-15 rows of tangentially
3 elongated radially arranged cork cells; cortex com-
Zs 95 5 posed of 10-15 rows of thin-walled parenchyma cells
35 95 5 showing sclereids and crystals of calcium oxalate; vas-
36 45 55 cular cambium in the form of a continuous ring; xylem
45 45 55 showing narrow rays of vessels, few lignified fibers are
present in older roots, 3-8 cell-wide medullary rays,
Chromatographic system and perenelyine showing few sclereids, oleoresin
(See Chromatography (621), System Suitability.) canals and simple starch grains; pith composed of pa-
Mode: renchyma cells in young roots and replaced by com-
Detector: UV 220 nm 5 pactly arranged vessels, fibers, and tracheids in mature
Column: 4.6-mm x 25-cm; 5-um, 100 A roots
Column temperature: 30° Loss ON DRYING (731)
Flow rate: 1.8 mL/min Sample: 1.0 of finely perce Forskohlii
Injection volume: 20 uL Analysis: Dry the Sample at 105° for 3 h.
System suitability Acceptance criteria: NMT 12.0%
Samples: Standard solution A, Standard solution B, and ARTICLES OF BOTANICAL ORIGIN, Total Ash (561)
System suitability solution Sample: 1.0g of finely powdered Forskohlii
[Note—The approximate relative retention times for Acceptance criteria: NMT 6%
isoforskolin and forskolin are 0.51 and 1.00, ARTICLES OF BOTANICAL ORIGIN, Alcohol-Soluble Extractives,
respectively.] Method 2 (561): NLT 25.0%
DS Monographs

Suitability requirements: The chromatogram of Stan-
dard solution B is similar to the reference chromato-
gram provided with the lot of USP Powdered Forskohlii
xtract RS being used.
Resolution: NLT 1.5 between the forskolin and tolu-
ene peaks, System suitability solution
Relative standard deviation: NMT 2% determined
from the forskolin peak for replicate injections, Stan-
dard solution A
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Using the chromatogram of Standard solution A, Stan-
dard solution B, and the reference chromatogram pro-
vided with the lot of USP Powdered Forskohlii Extract
RS being used, identify the retention times of the
peaks corresponding to isoforskolin and forskolin.
Calculate the percentage of forskolin in the portion of
Forskohlii taken:
Result = (ru/rs) x (Cs/Cu) x 100
MICROBIAL ENUMERATION TESTS (2021): The total aerobic
bacterial count does not exceed 105 cfu/g, the total com-
bined molds and yeasts count does not exceed 103 cfu/
g, and the bile-tolerant Gram-negative bacterial count
does not exceed 103 cfu/g.
MICROBIOLOGICAL PROCEDURES FOR ABSENCE OF SPECIFIED MI-
CROORGANISMS (2022): Meets the requirements of the
tests for absence of Salmonella species and Escherichia coli
e ARTICLES OF BOTANICAL ORIGIN, Test for Aflatoxins (561):
Meets the requirements
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in well-closed contain-
ers, protected from light and moisture, and store at
room temperature.
e LABELING: The label states the Latin binomial and, follow-
ing the official name, the parts of the plant contained in
the article.
USP 41 Dietary Supplements / Forskohlii 4627

e USP REFERENCE STANDARDS (11) Sample solution: Transfer about 3.0 g of accurately
USP Forskolin RS weighed Powdered Forskohlii to a 100-mL round-bot-
USP Powdered Forskohlii Extract RS tom flask fitted with a reflux condenser. Add 50 mL of
acetonitrile, reflux for 20 min, cool to room tempera-
ture, and decant the supernatant. Repeat until the ex-
tract is colorless. Combine the extracts, filter, concen-
trate under vacuum, and adjust the volume with
Powdered Forskohlii acetonitrile to 100 mL. Before injection, pass through a
membrane filter having a 0.45-um or finer pore size,
DEFINITION discarding the first 5 mL of the filtrate.
Powdered Forskohlii is Forskohlii reduced to a powder or System suitability solution: Standard solution A and
very fine powder. It contains NLT 0.4% of forskolin, calcu- 0.01% toluene in acetonitrile (1:1)
lated on the dried basis. Mobile phase: See Table 7.

IDENTIFICATION Table 1
oA. jueor vs CHROMATOGRAPHIC IDENTIFICATION TEST
(201 Time Solution A Solution B
Standard solution A: 50 j1g/mL of USP Forskolin RS in (min) (%) (%)
acetonitrile. Sonicate for about 10 min. 0 45 55
Standard solution B: 5 mg/mL of USP Powdered For- 25 45 55:
skohlii Extract RS in acetonitrile. Sonicate for about 15 28 95 5
min, centrifuge, and use the supernatant. 35 95 5
Sample stock solution: Use the Sample solution, pre-
36 45 55
pared as directed in the test for Content of Forskolin.
Sample solution: Dilute 10 mL of the Sample stock solu- 45 45 55
tion with acetonitrile to 25 mL.
Adsorbent: Chromatographic silica gel with an average Chromatographic system
particle size of 10-15 um (TLC plates) (See Chromatography (621), System Suitability.)
Application volume: 10 uL, as 4-mm bands Mode: LC
Detector: UV 220 nm ‘
Developing solvent system: Toluene and ethyl acetate Column: 4.6-mm x 25-cm; 5-um, 100 A
(85:15) Column temperature: 30°
Spray reagent: 5% vanillin in lacial acetic acid and
Flow rate: 1.8 mL/min
10% sulfuric acid in water (1:1
Analysis Injection volume: 20 uL
Samples: Standard solution A, Standard solution B, and
System suitability
Sample solution Samples: Standard solution A, Standard solution B, and
Apply the Samples as bands. Use a saturated chamber. System suitability solution
[NoTte—The relative retention times for isoforskolin and
Develop the chromatograms until the solvent front has
moved up about 90% of the plate. Remove the plate forskolin are 0.51 and 1.00, respectively.]
from the chamber, dry, spray with the Spray reagent, Suitability requirements: The chromatogram of Stan-
heat for 5-10 min at 105°, and examine under white dard solutionB is similar to the reference chromato-
ight. gram provided with the lot of USP Powdered Forskohlii
Aen criteria: The chromatogram of the Sample Extract RS being used.
solution exhibits a violet zone due to forskolin at an Rr Relative standard deviation: NMT 2% determined
value of approximately 0.3, corresponding in color and from the forskolin peak in repeated injections, Stan-
R; to that in the chromatogram of Standard solution A; a dard solution A
minor violet zone, a pink zone, and a brick-red zone at Resolution: NLT 1.5 between the forskolin and tolu-
Rr values of approximately 0.1, 0.62, and 0.69, due to ene peaks, System suitability solution
isoforskolin, 1,9-dideoxyforskolin, and crocetindi- Analysis
aldehyde, respectively. Zones detected in the Sample so- Samples: Standard solution A, Standard solution B, and
lution correspond in position and color to those in Stan- Sample solution
Using the chromatogram of Standard solution A, Stan-
sydesbouo-= sa

dard solution B, Other minor zones may be observed in

the Sample solution and Standard solution B.
e B. The Sample solution from the test for Content of For-
skolin shows a main peak at a retention time correspond-
ing to that of forskolin in the chromatogram of Standard
solution A. Identify other diterpene peaks in the Sample
solution by comparison with Standard solution B and the
reference chromatogram provided with the lot of USP
Powdered Forskohlii Extract RS being used. The Sample
solution chromatogram shows an additional peak corre-
sponding to isoforskolin.
COMPOSITION
© CONTENT OF FORSKOLIN
Solution A: Acetonitrile, filtered and degassed
Solution B: Water, filtered and degasse
Standard solution A: Sonicate a quantity of USP For-
skolin RS in acetonitrile to obtain a solution having a
known concentration of about 1.0 mg/mL.
Standard solution B: 5 mg/mL of USP Powdered For-
skohlii Extract RS in acetonitrile. Sonicate for about 15
min, centrifuge, and use the supernatant. Before injec-
tion, pass through a membrane filter having a 0.45-um
or finer pore size.
dard solution B, and the reference chromatogram pro-
vided with the lot of USP Powdered Forskohlii Extract
RS being used, identify the retention times of the
peaks corresponding to isoforskolin and forskolin.
Calculate the percentage of forskolin in the portion of
Powdered Forskohlii taken:
Result = (ru/rs) x (Cs/Cu) x 100
ty = peak area of forskolin from the Sample solution
rs = peak area of forskolin from Standard solution A
Cs = concentration of USP Forskolin RS in Standard
solution A (mg/mL)
Cu = concentration of Powdered Forskohlii in the
Sample solution (mg/mL)
Acceptance criteria: NLT 0.4% on the dried basis
IMPURITIES
© ARTICLES OF BOTANICAL ORIGIN, Acid-Insoluble Ash (561):
NMT 2%
e ARTICLES OF BOTANICAL ORIGIN, Limits of Elemental Impuri-
ties (561): Meets the requirements
 4628 Forskohlii / Dietary Supplements USP 41

© ARTICLES OF BOTANICAL ORIGIN, Pesticide Residue Analysis Analysis

(561): Meets the requirements
SPECIFIC TESTS
e BOTANICAL CHARACTERISTICS: Yellowish brown powder;
characteristic and pleasant aromatic odor; and slightly
bitter to pungent taste. Under a microscope, it shows the
presence of parenchyma cells with oleoresin canals,
starch grains and prisms of calcium oxalate; oil globules;
simple starch grains, cork cells; sclereids; stone cells; pit-
ted vessels; and thin-walled fibers.
e Loss ON DRYING (731)
Sample: 1.0g of Powdered Forskohlii
Analysis: Dry the Sample at 105° for 3 h.
Acceptance criteria: NMT 12.0%
e ARTICLES OF BOTANICAL ORIGIN, Tota! Ash (561)
Sample: 1.0g of Powdered Forskohlii
Acceptance criteria: NMT 6%
e ARTICLES OF BOTANICAL ORIGIN, Alcohol-Soluble Extractives,
Method 2 (561): NLT 25.0%
e MICROBIAL ENUMERATION TESTS (2021): The total aerobic
bacterial count does not exceed 105 cfu/g; the total com-
bined molds and yeasts count does not exceed 103 cfu/
g; and the bile-tolerant Gram-negative bacterial count
does not exceed 103 cfu/g.
© MICROBIOLOGICAL PROCEDURES FOR ABSENCE OF SPECIFIED Mi-
CROORGANISMS (2022): Meets the requirements of the
tests for absence of Salmonella species and Escherichia coli
e ARTICLES OF BOTANICAL ORIGIN, Test for Aflatoxins (561):
Meets the requirements
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in well-closed contain-
ers, protected from light and moisture, and store at
room temperature.
e LABELING: The label states the Latin binomial and, follow-
ing the official name, the parts of the plant contained in
the article.
e USP REFERENCE STANDARDS (11)
USP Forskolin RS
USP Powdered Forskohlii Extract RS
Powdered Forskohlii Extract
DEFINITION
Powdered Forskohlii Extract is prepared from Forskohlii using
suitable solvents such as methanol, ethyl acetate, hexane
or a mixture of these solvents. The ratio of plant material
DS Monographs
Samples: Standard solution A, Standard solution B, and
Sample solution
Apply the samples as bands to a suitable thin-layer
chromatographic plate (see Chromatography (621)).
Use saturated chamber. Develop the chromatograms
until the solvent front has moved up about 90% of
the plate. Remove the plate from the chamber, dry,
spray with the Spray reagent, heat for 5-10 min at
105°, and examine under visible light.
Acceptance criteria: The Sample solution exhibits a vio-
let zone due to forskolin at an Rr value of approximately
0.3, corresponding in color and R; to that from Stan-
dard solution A; and a minor violet zone, a pink zone,
and a brick red zone at R; values of approximately 0.1,
0.62, and 0.69, due to isoforskolin, 1,9-dideoxyfor-
skolin, and crocetindialdehyde, respectively. Zones de-
tected from the Sample solution correspond in position
and color to zones from Standard solution B. Other mi-
nor zones may be observed from the Sample solution
and Standard solution B.
e B. The Sample solution from the test for Content of For-
skolin shows a main peak at a retention time correspond-
ing to that of forskolin from Standard solution A. Identify
other diterpene peaks in the Sample solution by compari-
son with Standard solution B and the reference chromato-
gram provided with the lot of USP Powdered Forskohlii
Extract RS being used. The Sample solution shows an ad-
ditional peak corresponding to isoforskolin.
COMPOSITION
© CONTENT OF FORSKOLIN
Solution A: Use filtered and degassed acetonitrile.
Solution B: Use filtered and degassed water.
Standard solution A: Sonicate a quantity of USP For-
skolin RS in acetonitrile to obtain a solution having a
known concentration of about 1.0 mg/mL.
Standard solution B: 5 mg/mL of USP Powdered For-
skohlii Extract RS in acetonitrile. Sonicate for about 15
min, centrifuge, and use the supernatant. Before injec-
tion, pass through a membrane filter having a 0.45-um
or finer pore size.
Sample solution: Transfer an amount of Powdered For-
skohlii Extract equivalent to about 25 mg of forskolin to
a 25-mL volumetric flask, and add 15 mL of acetonitrile.
Sonicate and heat in a water bath for about 10 min,
cool, dilute with acetonitrile to volume, and mix. Before
injection, filter through a membrane filter having a
0.45-um or finer pore size, discarding the first 5 mL of
the filtrate.
System suitability solution: A mixture of Standard solu-

to extract is between 65:1 and 35:1. It contains NLT tion A and 0.01% toluene in acetonitrile (1:1)
90.0% and NMT 110.0% of the labeled amount of for- Mobile phase: See the gradient table below.
skolin, calculated on the dried basis. It contains suitable
added substances as carriers. Time Solution A Solution B
(min) (%) (%)
IDENTIFICATION
0 45 55
e * oo CHROMATOGRAPHIC IDENTIFICATION TEST
201 25 AS 55
Standard solution A: 50 g/mL of USP Forskolin RS in 28 95 5
acetonitrile. Sonicate for about 10 min. 35 95 5
Standard solution B: 5 mg/mL of USP Powdered For- 36 45 55
skohlii Extract RS in acetonitrile. Sonicate for about 15 AS 45 55
min, centrifuge, and use the supernatant.
Sample solution: 5 mg/mL of Powdered Forskohlii Ex- Chromatographic system
tract in acetonitrile. Sonicate for about 15 min, centri- (See Chromatography (621), System Suitability.)
fuge, and use the supernatant. Mode: LC
Adsorbent: Chromatographic silica gel with an average Detector: UV 220 nm i
particle size of 10-15 um (TLC plates) Column: 4.6-mm x 25-cm; 5-um, 100 A
Application volume: 10 ul, as 4-mm bands Column temperature: 30+ 2°
Developing solvent system: A mixture of toluene and Flow rate: 1.8 mL/min
ethyl acetate (85:15) Injection size: 20 pL
Spray reagent: A mixture of 5% vanillin in glacial ace- System suitability
tic acid and 10% sulfuric acid in water (1:1) Samples: Standard solution A, Standard solution B, and
System suitability solution
USP 41
[Note—The relative retention times for isoforskolin and
forskolin are 0.51 and 1.00, respectively.]
Suitability requirements: The chromatogram from
Standard solutionB is similar to the reference chromat-
ogram provided with the lot of USP Powdered For-
skohlii Extract RS being used.
Relative standard deviation: NMT 2% determined
from the forskolin peak in repeated injections, Stan-
dard solution A
Resolution: NLT 1.5 between the forskolin and tolu-
ene peaks, System suitability solution
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Using the chromatogram of Standard solution A, Stan-
dard solution B, and the reference chromatogram pro-
vided with the lot of USP Powdered Forskohlii Extract
RS being used, identify the retention times of the
peaks corresponding to isoforskolin and forskolin.
Calculate the percentage of forskolin in the portion of
Powdered Forskohlii Extract taken:
Result = (ru/ts) x (Cs/Cu) x 100
tu = peak response of forskolin from the Sample
solution
Is = peak response of forskolin from Standard
solution A
Cs = concentration of USP Forskolin RS in Standard
solution A (mg/mL)
Cu = concentration of Powdered Forskohlii Extract
in the Sample solution (mg/mL)
Acceptance criteria: NLT 90.0% and NMT 110.0% of
the labeled amount of forskolin on the dried basis
IMPURITIES
Inorganic Impurities
Delete the following:
®e HEAVY METALS, Method /I/ (231): NMT 20 ppme cotfical 1-
fan-2018)
Organic Impurities
© PROCEDURE: ARTICLES OF BOTANICAL ORIGIN, General
Method for Pesticide Residues Analysis (561): Meets the
requirements
SPECIFIC TESTS
e Loss ON DRYING (731): Dry 1.0 g of Powdered Forskohlii
Extract at 105° for 3 h: it loses NMT 5.0% of its weight.
e MICROBIAL ENUMERATION TESTS (2021): The total aerobic
microbial count does not exceed 104 cfu/g. The total
ee yeasts and molds count does not exceed 103
cfu/g.
¢ MICROBIOLOGICAL PROCEDURES FOR ABSENCE OF SPECIFIED MI-
CROORGANISMS (2022): It meets the requirements of the
tests for absence of Salmonella species and Escherichia
coli.
e ARTICLES OF BOTANICAL ORIGIN, Test for Aflatoxins (561):
Meets the requirements
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in well-closed contain-
ers, protected from light and moisture, and store at con-
trolled room temperature.
e LABELING: The label states the Latin binomial and, follow-
ing the official name, the part of the plant from which
the article was derived. It meets other labeling require-
ments under Botanical Extracts (565).
© OTHER REQUIREMENTS: It meets the requirements of the
test for Residual Solvents under Botanical Extracts (565).
Dietary Supplements / Ganoderma 4629
e USP REFERENCE STANDARDS (11)
USP Forskolin RS
USP Powdered Forskohlii Extract RS
Ganoderma Lucidum Fruiting Body
DEFINITION
Ganoderma Lucidum Fruiting Body consists of the dried
fruiting body of Ganoderma lucidum (W. Curt.:Fr.) P. Karst.
(Fam. Ganodermataceae). It contains NLT 0.3% of
triterpenoic acids, calculated on the dried basis as a sum
of ganoderic acids A, B, C2, D, F, G, and H and ganoder-
enic acids B, C, and D.
IDENTIFICATION
e A. THIN-LAYER CHROMATOGRAPHY
Standard solution A: 1.0 mg/mL of USP Ganoderic
Acid A RS in alcohol
Standard solution B: 0.3 mg/mL of USP Ergosterol RS
in alcohol
Standard solution C: 50 mg/mL of USP Ganoderma
Lucidum Fruiting Body Powdered Extract RS in alcohol.
Sonicate for about 10 min, centrifuge, and use the
supernatant.
Sample solution: Sonicate about 1 g of Ganoderma
Lucidum Fruiting Body, finely powdered, in 50 mL of
alcohol for 15 min. Centrifuge, withdraw the superna-
tant, and evaporate to dryness under reduced pressure
at 50°. Dissolve the residue in 2.0 mL of alcohol, centri-
fuge, and use the supernatant.
Chromatographic system
(See Chromatography (621), Thin-Layer Chromato-
graphy.)
Mode: HPTLC
Adsorbent: Chromatographic silica gel with an aver-
age particle size of 5-um (HPTLC plate).’ Predevelop
the plate in methanol, and dry at 105° for 30 min.
Application volume: 2 uL each of Standard solution A
and Standard solution B, and 4 uL each of Standard
solution C and the Sample solution as 8-mm bands
Column temperature: Ambient, not to exceed 30°
Developing solvent system: Toluene, ethyl formate,
and formic acid (5: 5: 0.2)
Developing distance: 6 cm
Derivatization reagent: A solution of 10% sulfuric
acid in alcohol. [NoTte—Prepare fresh. Slowly and grad-
oa add sulfuric acid to ice-cold alcohol, and mix
well.]
sydeibouo-: Sa
System suitability
Samples: Standard solution A, Standard solution B, and
Standard solution C
Suitability requirements
Chromatographic pattern: Under long-wave UV
light (365 nm), the chromatogram of Standard solu-
tion C displays, in the bottom third of the plate, the
following bands in the order of increasing Rr. a yel-
lowish or orange band (sometimes, two orange
bands are seen); a bluish-green band correspondin
to the light-blue ganoderic acid A band in Standar
solution A; an intense yellow band corresponding to
ganoderic acid B, ganoderic acid G, ganoderic acid
H, and ganoderenic acid B; and a bluish-green band
coincident with ganoderic acid D and ganoderenic
acid D. In the middle third of the chromatogram, a
variable number of blue-green bands appear. At the
top of the middle third of the Standard solution C
chromatogram, a somewhat diffuse band coincident
with the ergosterol band in Standard solutionB is
seen. In the upper third of the chromatogram, three
1A suitable commercially available plate is the HPTLC Silica Gel 60 Fas4 from
EMD Millipore (e.g., Part No. 1.05642.0001).
 4630 Ganoderma / Dietary Supplements
or four diffuse bands of varying colors appear. Under
white light, Standard solutionC exhibits, in its lower
third, two brownish-red bands, the upper of them
coincident with the ganoderic acid A band in Stan-
dard solution A, followed by a more intense brown
band; and a lighter brown band corresponding to ga-
noderic acid D and ganoderenic acid D. In the mid-
dle third of the chromatogram, five or six light-brown
bands are seen; one of those, deepest in color and
relatively diffuse, corresponds to the ergosterol band
in Standard solution B. Two or three light-brown
bands are seen under white light in the upper third
of the chromatogram of Standard solution C. [NoTE—
The Standard solutions are stable for 72 h at room
temperature.]
Analysis
Samples: Standard solution A, Standard solution B,
Standard solution C, and Sample solution
Apply the samples as bands and dry in air. Develop in a
saturated chamber, remove the plate, air-dry, treat
with Derivatization reagent, and heat at 105°-110° for
5 min. Immediately examine under white light and
under the long-wave UV light (365 nm).
Acceptance criteria: Under long-wave UV light (365
nm) and under white light, the chromatogram of the
Sample solution exhibits bands corresponding in color
and R; to similar bands in the chromatogram of Stan-
dard solution C, at the Rr values listed for System suitabil-
ity. Under white light, the chromatogram of the Sample
solution exhibits an additional violet band above the er-
gosterol band. [NoTe—The Sample solution is stable for
72 h at room temperature.]
e B. HPLC
Analysis: Proceed as directed in the test for Content of
Triterpenoic Acids.
Acceptance criteria: The chromatogram of the Sample
solution exhibits peaks at the retention times corre-
sponding to those of ganoderenic acid C, ganoderic
acid Cz, ganoderic acid G, ganoderenic acid B, ga-
noderic acid B, ganoderic acid A, ganoderic acid H, ga-
noderenic acid D, ganoderic acid D, and ganoderic acid
F in the chromatogram of Standard solution B.
e C. HPLC
Analysis: Proceed as directed in the test for Content of
Water-Soluble Polysaccharides.
Acceptance criteria: The chromatogram of the Sample
solution exhibits peaks at the retention times corre-
sponding to the peaks due to mannose, glucuronic
acid, dextrose, galactose, and L-fucose in the chromato-
gram of the Standard solution.
COMPOSITION
DS Monographs
¢ CONTENT OF TRITERPENOIC ACIDS
Solution A: 0.075% Phosphoric acid in water
Solution B: Acetonitrile
Mobile phase: See Table 1.
Table 1
Time Solution A Solution B
(min) (%) (%)
0 80.0 20.0
3 73.5 26.5
34 73.5 26.5
52 61.5 38.5
53 80.0 20.0
58 80.0 20.0
[NoTte—Maintain the Mobile phase at 73.5% of Solution
A for the period sufficient for complete elution of ga-
noderic acid A.]
Standard solution A: 0.1 mg/mL of USP Ganoderic
Acid A RS in methanol. Sonicate to dissolve if necessary.
USP 41
Standard solution B: Sonicate 40 mg of USP Ga-
noderma Lucidum Fruiting Body Powdered Extract RS in
5 mL of alcohol, and centrifuge. Pass through a nylon
filter of 0.2-m pore size, and discard the initial 1 mL of
the filtrate.
Sample solution: Transfer 2.0 g of Ganoderma Lucidum
Fruiting Body, finely powdered and accurately weighed,
to a 200-mL round-bottom flask, add 75 mL of alcohol,
attach a condenser, reflux for 45 min, cool, and filter.
Rinse the flask with two 10-mL portions of alcohol and
filter, combining the rinsates and the filtrate. Evaporate
to dryness under reduced pressure, and dissolve the res-
idue in about 20 mL of alcohol. Transfer the solution to
a 25-mL volumetric flask, dilute with alcohol to volume,
and mix well. Pass through a nylon filter of 0.2-~m
ore size, and discard the initial 1 mL of the filtrate.
Note—To facilitate the chromatographic column lon-
gevity, the following solid phase extraction procedure
may be employed. Condition the solid phase extraction
column containing about 200 mg of L1 packing with
5 mL of methanol followed by 3 mL of water; do not
allow the column to dry. Transfer 2.0 mL of Ganoderma
Lucidum Fruiting Body solution in alcohol into a 20-mL
volumetric flask, dilute with water to volume, and mix
well. Apply the entire volume onto the column, and
elute at the rate of approximately 1 drop/s, employing
vacuum. Rinse the column with 3 mL of water, and dis-
card the rinsate. Elute with 2.0 mL of methanol and col-
lect the eluate into the 2.0-mL volumetric flask. Adjust
with methanol to volume, and mix well.]
[NoTe—This method may result in coelution of ga-
noderenic acid A and ganoderic acid K.]
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 257 nm
Column: 2.1-mm x 15-cm; 1.8-1um packing L1
Column temperature: 25°
Flow rate: 0.4 mL/min
Injection volume: 5 pL
System suitability
Samples: Standard solution A and Standard solution B
Suitability requirements
Chromatographic similarity: The chromatogram of
Standard solutionB is similar to the reference chro-
matogram provided with the lot of USP Ganoderma
a Fruiting Body Powdered Extract RS being
used.
Resolution: NLT 1.0 between ganoderic acid A and
ganoderic acid H peaks, Standard solution B
Tailing factor: NMT 2.0 for the ganoderic acid A
peak, Standard solution A
Relative standard deviation: NMT 2.0% determined
from the ganoderic acid A peak in replicate injections,
Standard solution A
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
[Note—Standard solution A, Standard solution B, and the
Sample solution are stable for 24 h at room
temperature.]
Using the chromatograms of Standard solution A, Stan-
dard solution B, and the reference chromatogram pro-
vided with the lot of USP Ganoderma Lucidum Fruit-
ing Body Powdered Extract RS being used, identify all
specified ganoderic and ganoderenic acids in the Sam-
ple solution chromatogram. The approximate relative
retention times, with respect to ganoderic acid A, are
provided in Table 2.
USP 41 Dietary Supplements / Ganoderma 4631

Table 2 Internal standard solution: 0.5 mg/mL of D-lyxose in

Relative Relative
water
Retention Response
Standard stock solution: Composite solution contain-
ing 0.20 mg/mL each of USP Mannose RS, USP D-
Analyte Time Factor
Glucuronic Acid RS, and USP Galactose RS; 2.0 mg/mL
Ganoderenic acid C 0.36 0.51 of USP Dextrose RS; and 0.10 mg/mL of USP L-Fucose
Ganoderic acid C2 0.42 1.05 RS in water
Ganoderic acid G 0.56 1.18 Standard solution: Combine 0.125 mL of Standard
Ganoderenic acid B 0.60 0.45 stock solution with 0.125 mL of Internal standard solu-
Ganoderic acid B 0.66 1.10 tion, 0.300 mL of 0.15 M sodium hydroxide solution,
Ganoderic acid A 1.00 1.00
and 0.50 mL of Reagent in a capped reaction vial. Seal
the vial, heat at 70° for 30 min, and cool to room
Ganoderic acid H 1.05 1.54 temperature. Add to the vial 0.300 mL of 0.15 M hy-
Ganoderenic acid D 1.25. 0.51 drochloric acid and 0.65 mL of water, mix well, an
Ganoderic acid D 1.33 1.08 pass through a nylon filter of 0.45-4m or finer pore
Ganoderic acid F 1.54 1.45 size.
[NoTte—The amounts of individual analytes (As) in the
Separately calculate the percentages of each triterpe- 0.125-mL aliquot of the Standard solution submitted to
noic acid in the portion of Ganoderma Lucidum Fruit- derivatization are approximately 0.25 mg for dextrose
ing Body taken: and 0.025 mg for mannose, galactose, and D-
glucuronic acid.]
Result = (ru/rs) x Cs x (V/W) x F x 100 Sample solution: Transfer 2.0 g of Ganoderma Lucidum
Fruiting Body, finely powdered and accurately weighed,
tu = peak area of the relevant analyte from the into a 200-mL round-bottom flask, add 60 mL of water,
Sample solution and allow to stand for 1 h. Attach a condenser, heat
Is = peak area of ganoderic acid A in Standard under reflux for 4 h, and filter immediately. Transfer the
solution A residue and the filter to the same 200-mL round-bot-
Cs = concentration of USP Ganoderic Acid A RS in tom flask. Add 60 mL of water, heat under reflux for 3
Standard solution A (mg/mL) h, and filter immediately. Rinse the flask with three
V = volume of the Sample solution (mL) 5-mL portions of water, and filter. Combine the filtrates
w = weight of Ganoderma Lucidum Fruiting Body and the rinsates in a 250-mL beaker, and evaporate on
taken to prepare the Sample solution (mg) the water bath to dryness. Dissolve the residue in 5 mL
F = relative response factor, with respect to of water, add 75 mL of alcohol, mix well, allow to stand
ganoderic acid A (see Table 2) at 4° for 12 h, and centrifuge at 4000 rpm for 30 min.
Calculate the sum of the percentages of all specified Discard the supernatant, and dry the precipitate on a
triterpenoic acids. water bath. Dissolve the residue in hot water and quan-
Acceptance criteria titatively transfer into a 10-mL volumetric flask. Cool to
om of triterpenoic acids: NLT 0.3% on the dried room temperature, dilute with water to volume, and
asis mix well. Centrifuge at 4000 rpm for 10 min. Accu-
CONTAMINANTS rately transfer 0.250 mL of the supernatant into a reac-
tion vial, and add about 0.25 mL of 4Mtrifluoroacetic
e ELEMENTAL IMPURITIES—PROCEDURES (233)
acid. Seal the vial, heat at 110° for 4 h, cool to room
Acceptance criteria
temperature, add 0.5 mL of methanol, and evaporate to
Arsenic: NMT 2.0 ug/g dryness at 60° under vacuum. Repeat the addition of
Cadmium: NMT 1.0 ug/g
Lead: NMT 5.0 p1g/g 0.5 mL of methanol and subsequent evaporation three
times. Add to the residue 0.125 mL of water, 0.125 mL
Mercury: NMT 1.0 p9/g of the Internal standard solution, 0.300 mL of 0.15 M
© ARTICLES OF BOTANICAL ORIGIN, Pesticide Residue Analysis
(561): Meets the requirements sodium hydroxide solution, and 0.50 mL of the Reagent.
e MICROBIAL ENUMERATION TESTS (2021): The total aerobic Seal the vial, heat at 70° for 30 min, and cool to room
bacterial count does not exceed 10° cfu/g, and the bile- temperature. Add to the vial 0.300 mL of 0.15 M hy-
drochloric acid and 0.65 mL of water, mix well, an
tolerant Gram-negative bacteria count does not exceed
sydesbouow sa

103 cfu/g. pass through a nylon filter of 0.45-11m or finer pore

e ABSENCE OF SPECIFIED MICROORGANISMS (2022): Meets the size.
requirements of the tests for absence of Salmonella spe- Chromatographic system
cies and Escherichia coli (See Chromatography (621), System Suitability.)
Mode: LC
SPECIFIC TESTS Detector: UV 250 nm
e CONTENT OF WATER-SOLUBLE POLYSACCHARIDES Column: 4.6-mm x 25-cm; 5-um packing L1
Solution A: 0.05 M phosphate buffer, pH 6.0 Column temperature: 35°
Solution B: Acetonitrile Flow rate: 1.0 mL/min
Mobile phase: See Table 3. Injection volume: 10 uL
System suitability
Sample: Standard solution
Table 3
Suitability requirements
Time Solution A Solution B Resolution: NLT 1.5 between the D-lyxose peak and
(min) (%) (%) the closest subsequent peak, and NLT 1.5 between
0 84.0 16.0 ig giMcoranis acid peak and the closest preceding
30 82.5 17,5 ea
Thiling factor: NMT 2.0 for the dextrose peak
55 81.0 19.0 Relative standard deviation: NMT 2.0% determined
60 81.0 19.0 for the dextrose peak in replicate injections
61 84.0 16.0 Analysis
Samples: Standard solution and Sample solution
Reagent: 0.1 M solution of 1-phenyl-3-methyl- [Note—Standard solution and Sample solution are stable
5-pyrazolone in methanol for 24 h at room temperature.]
 4632 Ganoderma / Dietary Supplements USP 41

Using the chromatograms of the Standard solution and Analysis: Dry at 105° for 4 h.
the reference chromatogram provided with the lot of Acceptance criteria: NMT 17.0%
USP Ganoderma Lucidum Fruiting Body Powdered Ex- ARTICLES OF BOTANICAL ORIGIN, Jota! Ash (561)
tract RS being used, identify the individual derivatized Sample: 1.0 g of powdered Ganoderma Lucidum Fruit-
monosaccharides at about the following relative reten- ing Body
tion times, with respect to dextrose: 0.48 for man- Acceptance criteria: NMT 4.0%
nose, 0.58 for lyxose, 0.82 for D-glucuronic acid, 1.09 ARTICLES OF BOTANICAL ORIGIN, Alcoho/-Soluble Extractives,
for galactose, and 1.35 for L-fucose. Method 1 (561)
Separately calculate the percentages of derivatized mo- Sample: 2-4 g of powdered Ganoderma Lucidum Fruit-
nosaccharides in the portion of Ganoderma Lucidum ing Body
Fruiting Body taken: Acceptance criteria: NLT 2.0%
ARTICLES OF BOTANICAL ORIGIN, Water-Soluble Extractives,
Result = (Ru/Rs) x As x (F/W) x 100 Method 1 (561)
Sample: 2-4 g of powdered Ganoderma Lucidum Fruit-
Ru = peak response ratio of the relevant analyte to ing Body
the internal standard from the Sample Acceptance criteria: NLT 3.0%
solution
Rs = peak response ratio of the relevant analyte to ADDITIONAL REQUIREMENTS
the internal standard from the Standard PACKAGING AND STORAGE: Preserve in well-closed contain-
solution ers, protected from light and moisture, and store at
As = amount of the relevant analyte in the aliquot room temperature.
of the Standard solution subjected to LABELING: The label states the Latin binomial and, follow-
derivatization (mg) ing the official name, the part of the fungus from which
F = dilution factor to account for the sample the article was derived.
aliquot submitted to derivatization USP REFERENCE STANDARDS (11)
(0.250 mL) relative to the volume of the USP Dextrose RS
Sample solution (10.0 mL), 40 USP Ergosterol RS
w = weight of Ganoderma Lucidum Fruiting Body USP L-Fucose RS
taken to prepare the Sample solution (mg) USP Galactose RS
Calculate the sum of the percentages of mannose, D- USP Ganoderic Acid A RS
glucuronic acid, dextrose, galactose, and L-fucose. USP Ganoderma Lucidum Fruiting Body Powdered E
Acceptance criteria tract RS
Sum of monosaccharides: NLT 0.7% on the dried USP D-Glucuronic Acid RS
basis USP Mannose RS
e BOTANICAL CHARACTERISTICS
Macroscopic: Basidiocarp (fruiting body) morphology is
highly variable. Shape of pileus (cap) ranges from reni-
form to subcircular, convex or concave, 15 cm or more
broad, sings to multiple layers thick (up to 3 cm); mar- Ganoderma Lucidum Fruiting Body
gin generally thick and blunt, sometimes acute. Pileus Powder
surface radially rugose (wrinkled) and concentrically sul-
cate; shiny, yellowish-red to reddish-black. Stipe (stem)
attachment predominantly lateral; stipe length varies DEFINITION
from very short to 10-12 cm long, 1-3 cm thick, cylin- Ganoderma Lucidum Fruiting Body Powder is dried Ga-
drical, reddish to almost black, laccate (lacquered). Hy- noderma Lucidum Fruiting Body reduced to a powder or
menophore (pore surface) yellowish-white to tawny. a very fine powder. It contains NLT 0.3% of triterpenoic
Pores small, circular to irregular, 4-7 per mm, 6-200 acids, calculated on the dried basis as a sum of ganoderic
um in diameter, distance between axes of pores about ae A, B, C2, D, F, G, and H and ganoderenic acids B, C,
260 um. and D.
Microscopic: Hyphal system trimitic with hyaline, thin- IDENTIFICATION
“
walled, clamped, septate generative hyphae, 1-4 um in e A. THIN-LAYER CHROMATOGRAPHY
ie diameter, septa restricted to clamps, scantily branched, Standard solution A: 1.0 mg/mL of USP Ganoderic
¥ abundant at the growth margin of pileus and dissepi- Acid A RS in alcohol
Ss
— ments (partitions). Skeletal hyphae are arboriform, Standard solution B: 0.3 mg/mL of USP Ergosterol RS
Da aseptate, clampless, very long, 3-6 um in diameter,
° scantily branched, branches with limited growth at dis-
in alcohol
a Standard solution C: 50 mg/mL of USP Ganoderma
GS tal end, with thick walls; they compose most of the Lucidum Fruiting Body Powdered Extract RS in alcohol.
= context (flesh) and dissepiments, originating immedi- Sonicate for about 10 min, centrifuge, and use the
” ately behind the growth margin from generative supernatant.
fa) hyphae. Binding hyphae of the “Bovista” type are Sample solution: Sonicate about 1 g of Powder in
aseptate, clampless, profusely branched, generally thin- 50 mL of alcohol for 15 min, centrifuge, withdraw the
ner and lighter than the skeletal, 1-3 um in diameter. supernatant, and evaporate to dryness under reduced
Basidiospores ovoid, double-walled, truncated at apex. paste at 50°. Dissolve the residue in 2.0 mL of alco-
Epispore thin, ovoid, hyaline, 9.0-11.5 x 6.0-8.0 um; ol, centrifuge, and use the supernatant.
endospore thick, ovoid, 6.5-8.5 x 5.0-6.5 1m, bearing Chromatographic system
relatively few long and thick echinules that support the (See Chromatography (621), Thin-Layer Chromato-
epispore, sometimes fused into a short crest.
e ARTICLES OF BOTANICAL ORIGIN, Foreign Organic Matter graphy.)
Mode: HPTLC
(561): NMT 2.0% Adsorbent: Chromatographic silica gel with an aver-
e Loss ON DRYING (731) age particle size of 5 um (HPTLC plate).’ Predevelop
Sample: 1.0 g of powdered Ganoderma Lucidum Fruit- the plate in methanol and dry at 105° for 30 min.
ing Body
1A suitable commercially available plate is the HPTLC Silica Gel 60 Fass from
EMD Millipore (e.g., Part No. 1.05642.0001).
USP 41
Application volume: 2 jl each of Standard solution A
and Standard solution B, and 4 wL each of Standard
solution C and Sample solution as 8-mm bands
Column temperature: Ambient, not to exceed 30°
Developing solvent system: Toluene, ethyl formate,
and formic acid (5: 5: 0.2)
Developing distance: 6 cm
Derivatization reagent: A solution of 10% sulfuric
acid in alcohol. [NoTte—Prepare fresh. Slowly and grad-
oa add sulfuric acid to ice-cold alcohol, and mix
well.]
System suitability
Samples: Standard solution A, Standard solution B, and
Standard solution C
Suitability requirements
Chromatographic pattern: Under long-wave UV
(365 nm), the chromatogram of Standard solution C
displays, in the bottom third of the plate, the follow-
ing bands in the order of increasing Re: a yellowish or
orange band (sometimes, two orange bands are
seen); a bluish-green band corresponding to the
light-blue ganoderic acid A band in Standard solution
A; an intense yellow band corresponding to ga-
noderic acid B, ganoderic acid G, ganoderic acid H,
and ganoderenic acid B; and a bluish-green band co-
incident with ganoderic acid D and ganoderenic acid
D. In themiddle third of the chromatogram, a varia-
ble number of blue-green bands appear. At the top
of the middle third of the Standard solution C chro-
matogram, a somewhat diffuse band coincident with
the ergosterol band in Standard solution B is seen. In
the upper third of the chromatogram, three or four
diffuse bands of varying colors appear. Under white
light, Standard solution C exhibits, in its lower third,
two brownish-red bands, the upper of them coinci-
dent with the ganoderic acid A band in Standard so-
lution A, followed by a more intense brown band;
andalighter brown band corresponding to ga-
noderic acid D and ganoderenic acid D. In the mid-
dle third of the chromatogram, five or six light-brown
bands are seen; one of those, deepest in color and
relatively diffuse, corresponds to the ergosterol band
in Standard solution B. Two or three light-brown
bands are seen under white light in the upper third
of the chromatogram of Standard solution C. [NoTE—
The Standard solutions are stable for 72 h at room
temperature.]
Analysis
Samples: Standard solution A, Standard solution B,
Standard solution C, and Sample solution
Apply the samples as bands and dry in air. Develop in a
saturated chamber, remove the plate. air-dry, treat
with Derivatization reagent, and heat for 5 min at
105°-110°. Immediately examine under white light
and under the long-wave UV light (365 nm).
Acceptance criteria: Under the long-wave UV light
(365 nm) and under white light, the chromatogram of
the Sample solution exhibits the bands corresponding in
color and R; to similar bands in the chromatogram of
Standard solution C. Under white light, the chromato-
gram of the Sample solution exhibits an additional violet
and above the ae band. [NotE—The Sample so-
lution is stable for 72 h at room temperature.]
° B. HPLC
Analysis: Proceed as directed in the test for Content of
Triterpenoic Acids.
Acceptance criteria: The chromatogram of the Sample
solution exhibits peaks at the retention times corre-
sponding to those of ganoderenic acid C, ganoderic
acid C2, ganoderic acid G, ganoderenic acid B, ga-
Dietary Supplements / Ganoderma 4633
noderic acid B, ganoderic acid A, ganoderic acid H, ga-
noderenic acid D, ganoderic acid D, and ganoderic acid
F in the chromatogram of Standard solution B.
e C. HPLC
Analysis: Proceed as directed in the test for Content of
Water-Soluble Polysaccharides.
Acceptance criteria: The chromatogram of the Sample
solution exhibits peaks at the retention times corre-
sponding to the peaks due to mannose, glucuronic
acid, dextrose, galactose, and L-fucose in the chromato-
gram of the Standard solution.
COMPOSITION
e CONTENT OF TRITERPENOIC ACIDS
Solution A: 0.075% Phosphoric acid in water
Solution B: Acetonitrile
Mobile phase: See Table 1.
Table 1
Time Solution A Solution B
(min) (%) (%)
0 80.0 20.0
3 73.5 26.5
34 73.5 26.5
52 61.5 38.5
53 80.0 20.0
58 80.0 20.0
[NotE—Maintain the Mobile phase at 73.5% of Solution
A for the period sufficient for the complete elution of
ganoderic acid A.]
Standard solution A: 0.1 mg/mL of USP Ganoderic
Acid A RS in methanol. Sonicate to dissolve if necessary.
Standard solution B: Sonicate 40 mg of USP Ga-
noderma Lucidum Fruiting Body Powdered Extract RS in
5 mL of alcohol and centrifuge. Pass through a nylon
filter of 0.2-um pore size, and discard the initial 1mL of
the filtrate.
Sample solution: Transfer 2.0 g of Powder, accurately
weighed, to a 200-mL round-bottom flask, and add
75 mL of alcohol. Attach a condenser, reflux for 45
min, cool, and filter. Rinse the flask with two 10-mL
portions of alcohol, and filter, combining the rinsates
and the filtrate. Evaporate to dryness under reduced
pressure, and dissolve the residue in about 20 mL of
alcohol. Transfer the solution to a 25-mL volumetric
flask, dilute with alcohol to volume, and mix well. Pass
through a nylon filter of 0.2-um pore size, and discard
the initial 1 mL of the filtrate. [NoTE—To facilitate the
chromatographic column longevity, the following solid
sydesbouo=: sa
phase extraction procedure may be employed. Condi-
tion the solid phase extraction column containing about
200 mg of L1 packing with 5 mL of methanol followed
by 3 mL of water; do not allow the column to dry.
Transfer 2.0 mL of Powder solution in alcohol to a
20-mL volumetric flask, dilute with water to volume,
and mix well. Apply the entire volume onto the col-
umn, and elute at the rate of approximately 1 drop/s,
employing a vacuum. Rinse the column with 3 mL of
water, and discard the rinsate. Elute with 2.0 mL of
methanol and collect the eluate into the 2.0-mL volu-
wey flask. Adjust with methanol to volume, and mix
well.
[Note—This method may result in coelution of ga-
noderenic acid A and ganoderic acid K.]
Chromatographic system
(See Chromatography (621), System Suitability.)
 4634 Ganoderma / Dietary Supplements USP 41

Mode: LC CONTAMINANTS
Detector: UV 257 nm e ELEMENTAL IMPURITIES—PROCEDURES (233)
Column: 2.1-mm x 15-cm; 1.8-m packing L1 Acceptance criteria
Column temperature: 25° Arsenic: NMT 2.0 g/g
Flow rate: 0.4 mL/min Cadmium: NMT 1.0 ug/g
Injection volume: 5 uL Lead: NMT 5.0 ug/g
System suitability Mercury: NMT 1.0 ug/g
Samples: Standard solution A and Standard solution B e ARTICLES OF BOTANICAL ORIGIN, Pesticide Residue Analysis
Suitability requirements (561): Meets the requirements
Chromatographic similarity: The chromatogram of ¢ MICROBIAL ENUMERATION TESTS (2021): The total aerobic
Standard solution B is similar to the reference chro- bacterial count does not exceed 10° cfu/g, and the bile-
matogram provided with the lot of USP Ganoderma tolerant Gram-negative bacteria count does not exceed
Lucidum Fruiting Body Powdered Extract RS being 103 cfu/g.
used. © ABSENCE OF SPECIFIED MICROORGANISMS (2022): Meets the
Resolution: NLT 1.0 between ganoderic acid A and requirements of the tests for absence of Salmonella spe-
ganoderic acid H peaks, Standard solution B cies and Escherichia coli
Tailing factor: NMT 2.0 for the ganoderic acid A
peak, Standard solution A SPECIFIC TESTS
Relative standard deviation: NMT 2.0% determined ¢ CONTENT OF WATER-SOLUBLE POLYSACCHARIDES
from the ganoderic acid A peak in replicate injections, Solution
1 A: 0.05 M'pphosphate buffer, pH 6.0
Standard solution A Solution B: Acetonitrile
Analysis Mobile phase: See Table 3.
Samples: Standard solution A, Standard solution B, and
Sample solution Table 3
[NotE—Standard solution A, Standard solution B, and the = z
Sample solution are stable for 24 h at room rime Solation:A. Solution B
temperature.] (min) (%) ()
Using the chromatograms of Standard solution A, Stan- o 84.0 16.0
dard solution B, and the reference chromatogram pro- 30 82.5 17.5
vided with the lot of USP Ganoderma Lucidum Fruit- 55 81.0 19.0
ing Body Powdered Extract RS being used, identify all 60 81.0 19.0
specified ganoderic and ganoderenic acids in the Sam- 61 84.0 16.0
ple solution chromatogram. The approximate relative
retention
ided intimes,
Table with
2 respect to ganoderic acid A, are Reagent: 0.1 M solution of 1-phenyl-3-methyl-
provided in fable z. 5-pyrazolone in methanol
Internal standard solution: 0.5 mg/mL of D-lyxose in
Table 2 water . 7 . .
Standard stock solution: Composite solution contain-
Relative Relative ing 0.20 mg/mL each of USP Mannose RS, USP D-
; herengen Response Glucuronic Acid RS, and USP Galactose RS; 2.0 mg/mL
Analyte aime, als of USP Dextrose RS; and 0.10 mg/mL of USP L-Fucose
Ganoderenic acid C 0.36 0.51 RS in water
Ganoderic acid C2 0.42 1.05 Standard solution: Combine 0.125 mL of Standard
Ganoderic acid G 0.56 1.18 stock solution with 0.125 mL of Internal standard solu-
Ganoderenic acid B 0.60 0.45 tion, 0.300 mL of 0.15 M sodium hydroxide solution,
Gansdede-acia'B 0.66 1.10 and 0.50 mL of Reagent in a capped reaction vial. Seal
ic aci 5 si he vial, heat at 70° for 30 min, and cool to room
Ganoderic acid A a06) 1.00
: ‘ 3
temperature. ial 0.300
Add to the vial 0.3 mL of 0.15 M hy-
Ganoderic acid H 1.05 1.54 drochloric acid and 0.65 mL of water, mix well, an
Ganoderenic acid D 1.25 0.51 pass through a nylon filter of 0.45-um or finer pore
Ganoderic acid D 1.33 1.08 size. ; ;
DS Monographs

Ganoderic acid F 1.54 1.45 [NoTtE—The amounts of individual analytes (As) in the
0.125 mL aliquot of the Standard solution submitted to
Separately calculate the percentages of each triterpe- derivatization are approximately 0.25 mg for dextrose
noic acid in the portion of Powder taken: and 0.025 mg for mannose, galactose, and D-
glucuronic acid.]
Result = (ru/rs) x Cs x (V/W) x F x 100 Sample solution: Transfer 2.0 g of Powder, accurately
weighed, to a 200-mL round-bottom flask, add 60 mL
ru = peak area of the relevant analyte from the of water, and allow to stand for 1 h. Attach a con-
Sample solution denser, heat under reflux for 4 h, and filter immedi-
ls = peak area of ganoderic acid A in Standard ately. Transfer the residue and the filter to the same
solution A 200-mL round-bottom flask. Add 60 mL of water, heat
Gs = concentration of USP Ganoderic Acid A RS in under reflux for 3 h, and filter immediately. Rinse the
Standard solution A (mg/mL) flask with three 5-mL portions of water, and filter. Com-
Vv = volume of the Sample solution (mL) bine the filtrates and the rinsates in a 250-mL beaker,
w = weight of Powder taken to prepare the Sample and evaporate on the water bath to dryness. Dissolve
solution (mg) the residue in 5 mL of water, add 75 mL of alcohol, mix
F = relative response factor, with respect to well, allow to stand at 4° for 12 h, and centrifuge at
ganoderic acid A (see Table 2) 4000 rpm for 30 min. Discard the supernatant, and dry
Calculate the sum of the percentages of all specified the precipitate on a water bath. Dissolve the residue in
triterpenoic acids. hot water and quantitatively transfer to a 10-mL volu-
Acceptance criteria metric flask. Cool to room temperature, dilute with
eam of triterpenoic acids: NLT 0.3% on the dried water to volume, and mix well. Centrifuge at 4000 rpm
asis for 10 min. Accurately transfer 0.250 mL of the super-
USP 41
natant to a reaction vial, and add about 0.25 mL of 4M
trifluoroacetic acid. Seal the vial, and heat at 110° for 4
h. Cool to room temperature, add 0.5 mL of methanol,
and evaporate to dryness at 60° under vacuum. Repeat
the addition of 0.5 mL of methanol and subsequent
evaporation three times. Add to the residue 0.125 mL
of water, 0.125 mL of the Internal standard solution,
0.300 mL of 0.15 M sodium hydroxide solution, and
0.50 mL of Reagent. Seal the vial, heat at 70° for 30
min, and cool to room temperature. Add to the vial
0.300 mL of 0.15 M hydrochloric acid and 0.65 mL of
water, mix well, and pass through a nylon filter of 0.45-
um or finer pore size.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 250 nm
Column: 4.6-mm x 25-cm; 5-um packing L1
Column temperature: 35°
Flow rate: 1.0 mL/min
Injection volume: 10 pL
System suitability
Sample: Standard solution
Suitability requirements
Resolution: NLT 1.5 between the D-lyxose peak and
the closest subsequent peak, and NLT 1.5 between
Bis glucuronic acid peak and the closest preceding
ea
Thiling factor: NMT 2.0 for the dextrose peak
Relative standard deviation: NMT 2.0% determined
for the dextrose peak in replicate injections
Analysis
Samples: Standard solution and Sample solution
[Note—The Standard solution and Sample solution are
stable for 24 h at room temperature.
Using the chromatograms of the Standard solution and
the reference chromatogram provided with the lot of
USP Ganoderma Lucidum Fruiting Body Powdered Ex-
tract RS being used, identify the individual derivatized
monosaccharides at about the following relative reten-
tion times, with respect to dextrose: 0.48 for man-
nose, 0.58 for lyxose, 0.82 for D-glucuronic acid, 1.09
for galactose, and 1.35 for L-fucose.
Separately calculate the percentages of derivatized mo-
nosaccharides in the portion of Powder taken:
Result = (Ru/Rs) x As x (F/W) x 100
Ru = peak response ratio of the relevant analyte to
the internal standard from the Sample
solution
Rs = peak response ratio of the relevant analyte to
the internal standard from the Standard
solution
As = amount of the relevant analyte in the aliquot
of the Standard solution subjected to
derivatization (mg)
F = dilution factor to account for the sample
aliquot submitted to derivatization
(0.250 mL) relative to the volume of the
Sample solution (10.0 mL), 40
w = weight of Powder taken to prepare the Sample
solution (mg)
Calculate the sum ofthe percentages of mannose, D-
glucuronic acid, dextrose, galactose, and L-fucose.
Acceptance criteria
a of monosaccharides: NLT 0.7% on the dried
asis
e BOTANICAL CHARACTERISTICS: When milled, the fruiting
body typically grinds into a fibrous mass or fractures into
tiny strips rather than a fine powder. Hyphal system trim-
itic with hyaline, thin-walled, clamped, septate generative
hyphae, 1-4 «um in diameter, septa restricted to clamps,
scantily branched, abundant at the growth margin of pi-
Dietary Supplements / Garcinia 4635
leus and dissepiments (partitions). Skeletal hyphae are ar-
boriform, aseptate, clampless, very long, 3-6 um in di-
ameter, scantily branched, branches with limited growth
at distal end, with thick walls; they compose most of the
context (flesh) and dissepiments, originating immediately
behind the growth margin from generative hyphae.
Binding hyphae of the “Bovista” type are aseptate, clam-
pless, profusely branched, generally thinner and lighter
than the skeletal, 1-3 um in diameter. Basidiospores
ovoid, double-walled, truncated at apex. Epispore thin,
ovoid, hyaline, 9.0-11.5 x 6.0-8.0 um; endospore thick,
ovoid, 6.5-8.5 x 5.0-6.5 tum, bearing relatively few long
and thick echinules that support the epispore, sometimes
fused into a short crest.
e ARTICLES OF BOTANICAL ORIGIN, Foreign Organic Matter
(561): NMT 2.0%
e Loss ON DRYING (731)
Sample: 1.0 g of Powder
Analysis: Dry at 105° for 4 h.
Acceptance criteria: NMT 17.0%
ARTICLES OF BOTANICAL ORIGIN, Tota! Ash (561)
Sample: 1.0g of Powder
Acceptance criteria: NMT 4.0%
ARTICLES OF BOTANICAL ORIGIN, A/cohol-Soluble Extractives,
Method 1 (561)
Sample: 2-4g of Powder
Acceptance criteria: NLT 2.0%
ARTICLES OF BOTANICAL ORIGIN, Water-Soluble Extractives,
Method 7 (561)
Sample: 2-4g of Powder
Acceptance criteria: NLT 3.0%
ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Preserve in well-closed contain-
ers, protected from light and moisture, and store at
room temperature.
e LABELING: The label states the Latin binomial and, follow-
ing the official name, the part of the fungus from which
the article was derived.
e USP REFERENCE STANDARDS (11)
USP Dextrose RS
USP Ergosterol RS
USP L-Fucose RS
USP Galactose RS
USP Ganoderic Acid A RS
USP Ganoderma Lucidum Fruiting Body Powdered Ex-
tract RS
USP D-Glucuronic Acid RS
USP Mannose RS
sydesbouo-; sa
Garcinia cambogia
DEFINITION
Garcinia cambogia consists of the dried pericarp of the fruits
of Garcinia gummi-gutta (L.) N. Robson, also known as
Garcinia cambogia (Gaertn.) Desr. (Fam. Clusiaceae). It
contains NLT 12% of the sum of (-)-hydroxycitric acid
and (-)-hydroxycitric acid lactone, on the dried basis.
IDENTIFICATION
e A. Garcinia cambogia meets the requirements under Spe-
cific Tests, Botanical Characteristics.
e B. HPLC: The Sample solution chromatogram exhibits a
peak for hydroxycitric acid at a retention time corre-
apenas to that of Standard solution A, as obtained in
the test for Content of (—)-Hydroxycitric Acid and
(~)-Hydroxycitric Acid Lactone. The Sample solution also ex-
hibits a peak for hydroxycitric acid lactone. The hydrox-
ycitric acid and the hydroxycitric acid lactone peaks are
the main peaks in the Sample solution chromatogram.
 4636 Garcinia / Dietary Supplements
COMPOSITION
© CONTENT OF (-)-HYDROXYCITRIC ACID AND (—)-HYDROXYCI-
TRIC AcID LACTONE
Solution A: 30% Phosphoric acid in water
Mobile phase: Dissolve 1.36 g of anhydrous potassium
dihydrogen phosphate in 900 mL of water, adjust with
Solution A to a pH of 2.5, dilute to 1000 mL with water,
mix, filter, and degas.
Solvent: A mixture of Solution A and water (1:9)
Standard solution A: A solution of USP Calcium
(-)-Hydroxycitrate RS equivalent to about 2.5 mg/mL of
(-)-hydroxycitric acid in Solvent. Before injection, pass
through a membrane filter of 0.45-um or finer pore
size, discarding the first few mL of the filtrate.
Standard solution B: 5 mg/mL of USP Powdered
Garcinia Hydroxycitrate Extract RS in Solvent. Before in-
jection, pass through a membrane filter of 0.45-11m or
finer pore size.
Sample solution: Transfer about 5 g of Garcinia cambo-
gia, finely powdered and accurately weighed, to a
250-mL round-bottom flask fitted with a reflux con-
denser. Add 50 mL of Solvent, reflux while stirring for
30 min, set aside to settle, and decant the supernatant.
Repeat the extraction using four 50-mL portions of
water, combine all extracts, cool, filter into a 250-mL
volumetric flask, and dilute with water to volume.
Before injection, pass through a membrane filter of
0.45-um or finer pore size, discarding the first few mL
of the filtrate.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 215 nm
Column: 4.6-mm x 25-cm; packing L1
Column temperature: 25°
Flow rate: 1.0 mL/min
Injection volume: 20 uL
System suitability
Samples: Standard solution A, Standard solution B, and
Sample solution
[Notr—The relative retention times for the hydroxycitric
acid lactone and hydroxycitric acid peaks are about
0.9 and 1.0, respectively.]
Suitability requirements
Chromatogram similarity: The chromatogram of
Standard solution B is similar to the reference chro-
matogram provided with the lot of USP Powdered
Garcinia Hydroxycitrate Extract RS being used.
Resolution: NLT 1.0 between hydroxycitric acid lac-
tone and hydroxycitric acid, Sample solution
Tailing factor: NMT 2.0 for the hydroxycitric acid
DS Monographs
peak, Standard solution A
Relative standard deviation: NMT 2.0%, determined
from the hydroxycitric acid peak in repeated injec-
tions, Standard solution A
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
[Note—Standard solution A, Standard solution B, and
Sample solution are stable for 6 h.]
Calculate the percentages of (-)-hydroxycitric acid and
(-)-hydroxycitric acid lactone in the portion of Garcinia
cambogia taken:
Result = (ru/rs) x Cs x (V/W) x Fx 100
Ty = peak area of the relevant analyte from the
Sample solution
rs = peak area of hydroxycitric acid from Standard
solution A
Cs = concentration of (-)-hydroxycitric acid in
Standard solution A (mg/mL)
final volume of the Sample solution (mL)
=<
oil
weight of Garcinia cambogia used to prepare
the Sample solution (mg)
USP 41
F = conversion factor: 2.17 for (—)-hydroxycitric
acid lactone, and 1.00 for (-)-hydroxycitric
acid
Acceptance criteria: The sum of percentages of
(-)-hydroxycitric acid and (~)-hydroxycitric acid lactone:
NLT 12% on the dried basis
IMPURITIES
© ARTICLES OF BOTANICAL ORIGIN, Acid-/nsoluble Ash (561):
NMT 2.0%
ARTICLES OF BOTANICAL ORIGIN, Limits of Elemental Impuri-
ties (561): Meets the requirements
ARTICLES OF BOTANICAL ORIGIN, Foreign Organic Matter
(561): NMT 2.0%
ARTICLES OF BOTANICAL ORIGIN, Pesticide Residue Analysis
(561): Meets the requirements
SPECIFIC TESTS
e BOTANICAL CHARACTERISTICS
Macroscopic: Fresh fruits are spherical to oval in shape,
4-8 cm in height, 3-6 cm in width, yellowish to pale
pinkish when ripe, resemble a miniature pumpkin, with
7-13 deep longitudinal grooves, extending up toa cir-
cular elevated base of stigma with blackish tip, situated
in the depressed end of the fruit, contalning 6-8 seeds
surrounded by a succulent aril. Compendial article con-
sists of dried pieces of pericarp, longitudinal, of variable
size and shape, strongly curved inward; leathery; exter-
nally rough, irregularly wrinkled, longitudinally grooved;
internally smooth, longitudinally faintly striated and
ridged. Dark brown to blackish-brown odor characteris-
tic; taste sour, astringent, and slightly bitter.
Microscopic: Transverse section of thepericalp shows a
layer of epicarp, composed of rectangular to tangen-
tially elongated cells covered with thin cuticle; wide
mesocarp, composed of 100-150 rows of parenchyma
cells of variable size and shape, the outer rows com-
posed of relatively larger cells with wide intercellular
spaces; vascular bundles appear throughout the meso-
carp, more toward the inner zone; dark brown gummy
exudates, simple and compound starch granules and
prisms of calcium oxalate are present in the paren-
chyma cells throughout the mesocarp.
e Limit OF CiTRIc ACID
Solvent and Chromatographic system: Prepare as di-
rected in the test for Content of (—)-Hydroxycitric Acid
and (—)-Hydroxycitric Acid Lactone.
Standard solution: 0.5 mg/mL of USP Citric Acid RS in
Solvent. Before injection, pass through a membrane fil-
ter of 0.45-um or finer pore size, discarding the first
few mL of the filtrate.
Analysis
Sample: Standard solution
Calculate the percentage of citric acid in the portion of
Garcinia comets taken:
Result = (ru/rs) x Cs x (V/W) x 100
ru = peak area of citric acid from the Sample
solution in the test for Content of
(~)-Hydroxycitric Acid and (—)-Hydroxycitric
Acid Lactone (mg/mL)
rs = peak area of citric acid from the Standard
solution
Cs = concentration of USP Citric Acid RS in the
Standard solution (mg/mL)
V = final volume of the Sample solution (mL)
w = weight of Garcinia cambogia used to prepare
the Sample solution in the test for Content of
()-Hydroxycitric Acid and (—)-Hydroxycitric
Acid Lactone (mg)
Acceptance criteria: NMT 2% of citric acid on the
dried basis
USP 41 Dietary Supplements / Garcinia 4637

e Loss ON DRYING (731)
Sample: 2.0g of Garcinia cambogia, finely powdered
Analysis: Dry the Sample at 105° for 3 h.
Acceptance criteria: NMT 12.0%
e ARTICLES OF BOTANICAL ORIGIN, Total Ash (561): Deter-
mined on 1.0g of finely powdered Garcinia cambogia: Chromatographic system
NMT 3.0%; NMT 8.0%if sodium chloride was added as
a preservative during collection of the fruits
e MICROBIAL ENUMERATION TESTS (2021): The total aerobic
bacterial count does not exceed 105 cfu/g, the total com-
bined molds and yeasts count does not exceed 103 cfu/
g, and the bile-tolerant Gram-negative bacteria do not
exceed 103 cfu/g.
© ABSENCE OF SPECIFIED MICROORGANISMS (2022): Meets the
requirements of the tests for absence of Salmonella spe-
cies and Escherichia coli
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in well-closed contain-
ers, protected from light and moisture, and store at
room temperature.
e LABELING: The label states the Latin binomial and, follow-
ing the official name, the part of the plant contained in
the article.
e USP REFERENCE STANDARDS (11)
USP Calcium (—)-Hydroxycitrate RS
USP Citric Acid RS
USP Powdered Garcinia Hydroxycitrate Extract RS
Powdered Garcinia cambogia
DEFINITION
Powdered Garcinia cambogia is Garcinia cambogia reduced
to a powder or very fine powder. It contains NLT 12% of
the sum of (-)-hydroxycitric acid and (—)-hydroxycitric
acid lactone, on the dried basis.
IDENTIFICATION
e A. Powdered Garcinia cambogia meets the requirements
under Specific Tests, Botanical Characteristics. ru
e B. HPLC: The Sample solution chromatogram exhibits a
peak for hydroxycitric acid at a retention time corre- rs
spendin to that of Standard solution A, as obtained in
the test for Content of (—)-Hydroxycitric Acid and Cs
(~)-Hydroxycitric Acid Lactone. The Sample solution also ex-
hibits a peak for hydroxycitric acid lactone. The hydrox-
ycitric acid and the hydroxycitric acid lactone peaks are
the main peaks in the Sample solution chromatogram.
extraction using four 50-mL portions of water, combine
all extracts, Cool, filter into a 250-mL volumetric flask,
and dilute with water to volume. Before eset pass
through a membrane filter of 0.45-11m or finer pore
size, discarding the first few mL of filtrate.
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 215 nm
Column: 4.6-mm x 25-cm; packing L1
Column temperature: 25°
Flow rate: 1.0 mL/min
Injection volume: 20 uL
System suitability
Samples: Standard solution A, Standard solution B, and
Sample solution
[NoTte—The relative retention times for the hydroxycitric
acid lactone and hydroxycitric acid peaks are about
0.9 and 1.0, respectively.]
Suitability requirements
Chromatogram similarity: The chromatogram of
Standard solutionB is similar to the reference chro-
matogram provided with the lot of USP Powdered
Garcinia Hydroxycitrate Extract RS being used.
Resolution: NLT 1.0 between the hydroxycitric acid
lactone and hydroxycitric acid peaks, Sample solution
Tailing factor: NMT 2.0 for the hydroxycitric acid
peak, Standard solution A
Relative standard deviation: NMT 2.0%, determined
from the hydroxycitric acid peak for replicate injec-
tions, Standard solution A
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
[Note—Standard solution A, Standard solution B, and the
Sample solution are stable for 6 h.]
Calculate the percentages of (-)-hydroxycitric acid and
(-)-hydroxycitric acid lactone in the portion of Pow-
dered Garcinia cambogia taken:
Result = (ru/rs) x Cs x (V/W) x Fx 100
= peak area of the relevant analyte from the
Sample solution
= peak area of hydroxycitric acid from Standard
solution A
= concentration of (—)-hydroxycitric acid in
Standard solution A (mg/mL)
V = final volume of the Sample solution (mL)
Ww = weight of Powdered Garcinia cambogia used
to prepare the Sample solution (mg)
= conversion factor: 2.17 for (-)-hydroxycitric
sydesbouow sq

COMPOSITION acd lactone, and 1.00 for (-)-hydroxycitric

© CONTENT OF (—)-HYDROXYCITRIC ACID AND (-)-HYDROXYCI- aci
TRIC AciD LACTONE Acceptance criteria: The sum of percentages of
Solution A: 30% Phosphoric acid in water (-)-hydroxycitric acid and (-)-hydroxycitric acid lactone:
Mobile phase: Dissolve 1.36 g of anhydrous potassium NLT 12% on the dried basis
dihydrogen phosphate in 900 mL of water, adjust with
Solution A to a pH of 2.5, dilute with water to 1000 mL, IMPURITIES
mix, filter, and degas. e ARTICLES OF BOTANICAL ORIGIN, Acid-/nsoluble Ash (561):
Solvent: Solution A and water (1:9) NMT 2.0%
Standard solution A: A solution of USP Calcium © ARTICLES OF BOTANICAL ORIGIN, Limits of Elemental Impuri-
(-)-Hydroxycitrate RS equivalent to about 2.5 mg/mL of ties (561): Meets the requirements
(-)-hydroxycitric acid in Solvent. Before injection, pass e ARTICLES OF BOTANICAL ORIGIN, Pesticide Residue Analysis
through a membrane filter of 0.45-um or finer pore (561): Meets the requirements
size.
Standard solution B: 5 mg/mL of USP Powdered SPECIFIC TESTS
Garcinia Hydroxycitrate Extract RS in Solvent. Before in- e BOTANICAL CHARACTERISTICS: Dark brown ponder odor
jection, pass through a membrane filter of 0.45-m or characteristic; taste sour, astringent and s ightly bitter.
finer pore size, discarding the first few mL of filtrate. Under a microscope, it shows parenchyma cells contain-
Sample solution: Transfer about 5 g of Powdered ing dark reddish-brown gummy exudates, parenchyma
Garcinia cambogia, accurately weighed, to a 250-mL cells containing simple and compound starch granules,
round-bottom flask fitted with a reflux condenser. Add prisms of calcium oxalate, and fragments of spiral and
50 mL of Solvent, reflux while stirring for 30 min, set annular vessels.
aside to settle, and decant the supernatant. Repeat the
 4638 Garcinia / Dietary Supplements
e LIMIT oF Citric AciD
Solvent: Prepare as directed in the test for Content of
(-)-Hydroxycitric Acid and (—)-Hydroxycitric Acid Lactone.
Standard solution: 0.5 mg/mL of USP Citric Acid RS in
Solvent. Before injection, pass through a membrane fil-
ter of 0.45-um or finer pore size, discarding the first
few mL of filtrate.
Analysis
Sample: Standard solution
Calculate the percentage of citric acid in the portion of
Powdered Garcinia cambogia taken:
Result = (ru/rs) x Cs x (V/W) x 100
Ty = peak area of citric acid from the Sample
solution in the test for Content of
(-)-Hydroxycitric Acid and (—)-Hydroxycitric
Acid Lactone
rs = peak area of citric acid from the Standard
solution
Cs = concentration of USP Citric Acid RS in the
Standard solution (mg/mL)
Vv = final volume of the Sample solution (mL)
w = weight of Powdered Garcinia cambogia used
to prepare the Sample solution in the test for
Content of (—)-Hydroxycitric Acid and
(-)-Hydroxycitric Acid Lactone (mg)
Acceptance criteria: NMT 2% of citric acid on the
dried basis
Loss ON DRYING (731)
Sample: 2.0 g of Powdered Garcinia cambogia
Analysis: Dry the Sample at 105° for 3 h.
Acceptance criteria: NMT 12.0%
ARTICLES OF BOTANICAL ORIGIN, Tota! Ash (561): Deter-
mined on 1.0 g of Powdered Garcinia cambogia: NMT
3.0%; NMT 8.0% if sodium chloride was added as a pre-
servative during collection of the fruits
MICROBIAL ENUMERATION TESTS (2021): The total aerobic
bacterial count does not exceed 105 cfu/g, the total com-
bined molds and yeasts count does not exceed 103 cfu/
g, and the bile-tolerant Gram-negative bacteria do not
exceed 103 cfu/g.
ABSENCE OF SPECIFIED MICROORGANISMS (2022): Meets the
requirements of the tests for absence of Salmonella spe-
cies and Escherichia coli
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in well-closed contain-
ers, protected from light and moisture, and store at
room temperature.
e LABELING: The label states the Latin binomial and, follow-
2 ing the official name, the part of the plant contained in
Q the article.
be e USP REFERENCE STANDARDS (11)
Da USP Calcium (-)-Hydroxycitrate RS
= USP Citric Acid RS
io) USP Powdered Garcinia Hydroxycitrate Extract RS
= Result = (ru/ts) x (Cs/Cu) x F x 100
“ Ty
[=]
Powdered Garcinia Hydroxycitrate
Extract
DEFINITION
Powdered Garcinia Hydroxycitrate Extract is prepared from
Garcinia cambogia or Garcinia indica by extraction with
water, alcohol, or mixtures of these solvents, followed by
stabilization of the (-)-hydroxycitric acid content in the
form of a calcium, potassium, magnesium, and/or sodium
salt. The ratio of plant material to extract is about 5:1 to
10:1. It contains NLT 40% of (-)-hydroxycitric acid, calcu-
lated on the dried basis. It may contain suitable added
substances.
USP 41
IDENTIFICATION
e A. HPLC IDENTIFICATION TEST: The Sample solution chro-
matogram exhibits a peak for hydroxycitric acid at a re-
tention time corresponding to that of Standard solution A,
as obtained in the test for Content of (—)-Hydroxycitric Acid
and Limit of (-)-Hydroxycitric Acid Lactone.
COMPOSITION
e CONTENT OF (—)-HYDROXYCITRIC ACID AND LIMIT OF
(-)-Hyproxycitric AcID LACTONE
Solution A: 30% phosphoric acid in water
Mobile phase: Dissolve 1.36 g of anhydrous potassium
dihydrogen phosphate in 900 mL of water, adjust with
Solution A to a pH of 2.5, complete to 1000 mL with
water, mix, filter, and degas.
Solvent: A mixture of Solution A and water (1:9)
Standard solution A: A solution of USP Calcium
(-)-Hydroxycitrate RS equivalent to about 2.5 mg/mL of
(-)-hydroxycitric acid in Solvent. Before injection, pass
through a membrane filter of 0.45-m or finer pore
size.
Standard solution B: 5 mg/mL of USP Powdered
Garcinia Hydroxycitrate Extract RS in Solvent. Before in-
jection, pass through a membrane filter of 0.45-um or
finer pore size.
Sample solution: 5 mg/mL of Powdered Garcinia
Hydroxycitrate Extract in Solvent. Before Injector, pass
through a membrane filter of 0.45-um or finer pore
size.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 215 nm
Column: 4.6-mm x 25-cm; packing L1
Column temperature: 25+1°
Flow rate: 1.0 mL/min
Injection size: 20 pL
System suitability
Samples: Standard solution A and Standard solution B
Suitability requirements
Chromatogram similarity: The chromatogram from
Standard solutionB is similar to the reference chro-
matogram provided with the lot of USP Powdered
Garcinia Hydroxycitrate Extract RS being used.
Tailing factor: NMT 2.0 for the hydroxycitric acid
peak, Standard solution A
Relative standard deviation: NMT 2.0%, determined
from the hydroxycitric acid peak, Standard solution A
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution. [NoTE—Standard solution A, Standard
solution B, and the Sample solution are stable for 6 h.]
Calculate the percentage of (—)-hydroxycitric acid and
the limit of (-)-hydroxycitric acid lactone, if present, in
the portion of Powdered Garcinia Hydroxycitrate Ex-
tract taken:
= peak area for the relevant analyte from the
Sample solution
Is = peak area of hydroxycitric acid from Standard
solution A
Cs = concentration of (—)-hydroxycitric acid in
Standard solution A (mg/mL)
Cu = concentration of Powdered Garcinia
Hydroxycitrate Extract in the Sample solution
(mg/mL)
= conversion factor for each analyte: 2.17 for
(-)-hydroxycitric acid lactone, and 1.00 for
(-)-hydroxycitric acid
Acceptance criteria: NLT 40% of (-)-hydroxycitric acid
and NMT 8% of (-)-hydroxycitric acid lactone on the
dried basis
 USP 41
IMPURITIES
Inorganic Impurities
¢ ARTICLES OF BOTANICAL ORIGIN, Acid-/nsoluble Ash (561):
NMT 3.0%
Delete the following:
°e HEAVY METALS, Method Ii! (231): NMT 20 ppme (orca 1-
Jan-2018)
Organic Impurities
© PROCEDURE: ARTICLES OF BOTANICAL ORIGIN, Pesticide Resi-
dues (561): Meets the requirements
SPECIFIC TESTS
© Limit oF Citric AcID
Solvent: Prepare as directed in the test for Content of
(~)-Hydroxycitric Acid and Limit of (-)-Hydroxycitric Acid
Lactone.
Standard solution: 0.5 mg/mL of USP Citric acid RS in
Solvent. Before injection, pass through a membrane fil-
ter of 0.45-um or finer pore size.
Analysis
Sample: Standard solution
Calculate the percentage of citric acid in the portion of
Powdered Garcinia Hydroxycitrate Extract taken:
Result = (ru/rs) x (Cs/Cu) x 100
tu = peak area of citric acid, using the peak area of
citric acid from the Sample solution in the
test for Content of (—)-Hydroxycitric Acid and
Limit of (—)-Hydroxycitric Acid Lactone
Is = peak area of citric acid from the Standard
solution
Cs = concentration of USP Citric Acid RS in the
Standard solution (mg/mL)
Cu = concentration of Powdered Garcinia
Hydroxycitrate Extract in the Sample solution
in the test for Content of (—)-Hydroxycitric Acid
and Limit of (-)-Hydroxycitric Acid Lactone
(mg/mL)
Acceptance criteria: NMT 5% of citric acid on the
dried basis
IDENTIFICATION TESTS—GENERAL (191): Test for the pres-
ence of calcium, magnesium, potassium, and/or sodium.
Loss ON DRYING (731): Dry 2.0 g of Powdered Extract at
105° for 3 h: Powdered Extract containing calcium
hydroxycitrate loses NMT 5.0% of its weight; Powdered
Extract containing other salts loses NMT 9.0% of its
weight.
MICROBIAL ENUMERATION TESTS (2021): The total aerobic
bacterial count does not exceed 104 cfu/g, and the total
combined molds and yeasts count does not exceed 103
cfu/g.
MICROBIOLOGICAL PROCEDURES FOR ABSENCE OF SPECIFIED Mi-
CROORGANISMS (2022): Meets the requirements of the
tests for absence of Salmonella species and Escherichia
coli.
OTHER REQUIREMENTS: It meets the requirements of the
test for Residual Solvents under Botanical Extracts (565).
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in well-closed contain-
ers, protected from light and moisture, and store at con-
trolled room temperature.
e LABELING: The label states the Latin binomial and, follow-
ing the official name, the part of the plant from which
the article was prepared. It meets other Labeling require-
ments under Botanical Extracts (565).
Dietary Supplements / Garcinia 4639
e USP REFERENCE STANDARDS (11)
USP Calcium (—)-Hydroxycitrate RS
USP Citric Acid RS
USP Powdered Garcinia Hydroxycitrate Extract RS
Garcinia indica
DEFINITION
Garcinia indica consists of the dried pence of the fruits of
Garcinia indica (Thouars) Choisy (Fam. Clusiaceae). It con-
tains NLT 12% of the sum of oe acid and
(-)-hydroxycitric acid lactone, on the dried basis.
IDENTIFICATION
e A. Garcinia indica meets the requirements under Specific
Tests, Botanical Characteristics.
e B. HPTLC FoR ARTICLES OF BOTANICAL ORIGIN (203)
Standard solution: 0.5 mg/mL of garcinol in alcohol
Sample solution: Transfer about 2.0 g of Garcinia in-
dica, finely powdered, to a Soxhlet apparatus, add
100 mL of alcohol, and extract for 6 h. Filter and con-
centrate under vacuum to about 10 mL. [NoTE—Use a
thimble of suitable size such that the volume of alcohol
used in the Soxhlet extraction is at least twice the vol-
ume of the thimble.]
Adsorbent: Chromatographic silica gel with an average
particle size of 5 um (HPTLC plates)
Application volume: 5 wL, as 8-mm bands
Developing solvent system: Toluene, ethyl acetate,
and formic acid (4: 1: 0.5)
Developing distance: 6 cm
Derivatization reagent: A mixture of 1% vanillin in al-
cohol and 10% sulfuric acid in alcohol (1:1)
Analysis
Samples: Standard solution and Sample solution
Apply the Samples as bands. Develop in a saturated
chamber. Remove the plate from the chamber, dry,
treat with Derivatization reagent, heat for 5-10 min at
105°, and examine under white light.
Acceptance criteria: The Sample solution chromato-
gram exhibits a main greenish-gray band due to garci-
nol at an R; value of approximately 0.6, which corre-
sponds in position and color to the main band in the
chromatogram of the Standard solution. The Sample so-
lution exhibits the following additional bands: two pur-
ple bands, two greenish-gray bands, two blue bands
and a poe band at R; values of approximately 0.31,
0.34, 0.37, 0.47, 0.54, 0.83, and 0.93, respectively.
sydesbouo-: sa
Other bands may be observed in the Sample solution.
e C. HPLC: The Sample solution chromatogram exhibits a
peak for hydroxycitric acid at a retention time corre-
spondin to that of Standard solution A, as obtained in
the test for Content of (—)-Hydroxycitric Acid and
(~)-Hydroxycitric Acid Lactone. The Sample solution also ex-
hibits a peak for hydroxycitric acid lactone. The hydrox-
ycitric acid and the hydroxycitric acid lactone peaks are
the main peaks in the Sample solution chromatogram.
COMPOSITION
© CONTENT OF (—)-HYDROXYCITRIC ACID AND (—)-HYDROXYCI-
TRIC ACID LACTONE
Solution A: 30% Phosphoric acid in water
Mobile phase: Dissolve 1.36 g of anhydrous potassium
dihydrogen phosphate in 900 mL of water, adjust with
Solution A to a pH of 2.5, dilute with water to 1000 mL,
mix, filter, and degas.
Solvent: A mixture of Solution A and water (1:9)
Standard solution A: A solution of USP Calcium
(-)-Hydroxycitrate RS equivalent to about 4 mg/mL of
(-)-hydroxycitric acid in Solvent. Before injection, pass
eee a membrane filter of 0.45-11m or finer pore
size, discarding the first few mL of the filtrate.
 4640 Garcinia / Dietary Supplements
Standard solution B: 8 mg/mL of USP Powdered
Garcinia Hydroxycitrate Extract RS in Solvent. Before in-
jection, pass through a membrane filter of 0.45-m or
finer pore size.
Sample solution: Transfer about 5 g of Garcinia indica,
finely powdered and accurately weighed, to a 250-mL
round-bottom flask fitted with a reflux condenser. Add
50 mL of Solvent, reflux while stirring for 30 min, set
aside to settle, and decant the supernatant. Repeat the
extraction using four 50-mL portions of water, combine
all extracts, cool, filter into a 250-mL volumetric flask,
and dilute with water to volume. Before injection, pass
through a membrane filter of 0.45-um or fier pore
size, discarding the first few mL of the filtrate.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 215 nm
Column: 4.6-mm x 25-cm; packing L1
Column temperature: 25°
Flow rate: 1.0 mL/min
Injection volume: 20 uL
System suitability
Samples: Standard solution A, Standard solution B, and
Sample solution
[Note—The relative retention times for the hydroxycitric
acid lactone and hydroxycitric acid peaks are about
0.9 and 1.0, respectively.]
Suitability requirements
Chromatogram similarity: The chromatogram of
Standard solution B is similar to the reference chro-
matogram provided with the lot of USP Powdered
Garcinia Hydroxycitrate Extract RS being used.
Resolution: NLT 1.0 between the hydroxycitric acid
lactone and hydroxycitric acid peaks, Sample solution
Tailing factor: NMT 2.0 for the hydroxycitric acid
peak, Standard solution A
Relative standard deviation: NMT 2.0%, determined
from the hydroxycitric acid peak for replicate injec-
tions, Standard solution A
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution. [Note—Standard solution A, Standard
solution B, and the Sample solution are stable for 6 h.]
Calculate the percentages of (-)-hydroxycitric acid and
(-)-hydroxycitric sek lattone in the portion of Garcinia
indica taken:
Result = (ru/rs) x Cs x (V/W) x F x 100
ru = peak area of the relevant analyte from the
DS Monographs
Sample solution
rs = peak area of hydroxycitric acid from Standard
solution A
Cs = concentration of (—)-hydroxycitric acid in
Standard solution A (mg/mL)
Vv = final volume of the Sample solution (mL)
w = weight of Garcinia indica used to prepare the
Sample solution (mg)
F; = conversion factor: 2.17 for (-)-hydroxycitric
acid lactone, and 1.00 for (-)-hydroxycitric
acid
Acceptance criteria: The sum of percentages of
(-)-hydroxycitric acid and (—)-hydroxycitric acid lactone
is NLT 12% on the dried basis.
IMPURITIES
e ARTICLES OF BOTANICAL ORIGIN, Acid-Insoluble Ash (561):
NMT 0.5%
© ARTICLES OF BOTANICAL ORIGIN, Limits of Elemental Impuri-
ties (561): Meets the requirements
e ARTICLES OF BOTANICAL ORIGIN, Foreign Organic Matter
(561): NMT 2.0%
e ARTICLES OF BOTANICAL ORIGIN, Pesticide Residue Analysis
(561): Meets the requirements
USP 41
SPECIFIC TESTS
e BOTANICAL CHARACTERISTICS
Macroscopic: Fresh fruits are globular in shape, 3-4 cm
in diameter, purplish to pinkish orange when ripe, with
persistant calyx lobes at the base and flattened radiat-
ing sessile stigma at the apex; containing 5-8 seeds sur-
rounded by a succulant aril. Compendial article consists
of dried pieces of pericarp, bluish black, of various size
and shapes, flattened, flexible; remains of pedicels,
calyx, and stigma may be present; odor characteristic;
taste sour.
Microscopic: Transverse section of the pericarp shows a
layer of epicarp, composed of isodiametricGalle with
thin cuticle and stomata; hypodermis consisting of sev-
eral rows of compactly arranged, tangentially elon-
gated, thick-walled cells, containing dark brown con-
tents, showing narrow irregular elongated cavities;
outer mesocarp consisting of loosely arranged, tangen-
tially elongated, parenchyma cells, few are full of starch
grains, traversed by narrow bands of collapsed cells and
oleoresin ducts; inner mesocarp consisting of collapsed
and compactly arranged cells, showing rows of fibrovas-
cular bundles; endocarp is not distinct, consisting of
thin-walled collapsed cells, with dark brown content.
© Limit OF CiTRIC ACID
Solvent and Chromatographic system: Prepare as di-
rected in the test for Content of (—)-Hydroxycitric Acid
and (—)-Hydroxycitric Acid Lactone.
Standard solution: 0.5 mg/mL of USP Citric Acid RS in
Solvent. Before injection, pass through a membrane fil-
ter of 0.45-um or finer pore size, discarding the first
few mL of the filtrate.
Analysis
Sample: Standard solution
Calculate the percentage of citric acid in the portion of
Garcinia indica taken:
Result = (ru/rs) x Cs x (V/W) x 100
tu = peak area of citric acid from the Sample
solution in the test for Content of
(-)-Hydroxycitric Acid and (—)-Hydroxycitric
Acid Lactone
rs = peak area of citric acid from the Standard
solution
Cs = concentration of USP Citric Acid RS in the
Standard solution (mg/mL)
Vv = final volume of the Sample solution (mL)
Ww = weight of Garcinia indica used to prepare the
Sample solution in the test for Content of
(~)-Hydroxycitric Acid and Limit of
(~)-Hydroxycitric Acid Lactone (mg)
Acceptance criteria: NMT 2% of citric acid on the
dried basis
e Loss ON DRYING (731)
Sample: 2.0g of Powdered Garcinia indica
Analysis: Dry the Sample at 105° for 3 h.
Acceptance criteria: NMT 12.0%
e ARTICLES OF BOTANICAL ORIGIN, Total Ash (561): Deter-
mined on 1.0g of finely powdered Garcinia indica: NMT
3.0%; NMT 8.0% if sodium chloride was added as a pre-
servative during collection of the fruits
e MICROBIAL ENUMERATION TESTS (2021): The total aerobic
bacterial count does not exceed 105 cfu/g, the total com-
bined molds and yeasts count does not exceed 103 cfu/
g, and the bile-tolerant Gram-negative bacteria do not
exceed 103 cfu/g.
e ABSENCE OF SPECIFIED MICROORGANISMS (2022): Meets the
requirements of the tests for absence of Salmonella spe-
cies and Escherichia coli
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in well-closed contain-
ers, protected from light and moisture, and store at
room temperature.
USP 41
e LABELING: The label states the Latin binomial and, follow-
ing the official name, the part of the plant contained in
the article.
e USP REFERENCE STANDARDS (11)
USP Calcium (-)-Hydroxycitrate RS
USP Citric Acid RS
USP Powdered Garcinia Hydroxycitrate Extract RS
Powdered Garcinia indica
DEFINITION
Powdered Garcinia indica is Garcinia indica reduced to a fine
or very fine powder. It contains NLT 12% of the sum of
(-)-hydroxycitric acid and (—)-hydroxycitric acid lactone,
on the dried basis.
IDENTIFICATION
e A. Powdered Garcinia indica meets the requirements
under Specific Tests, Botanical Characteristics.
e B. HPTLC FoR ARTICLES OF BOTANICAL ORIGIN (203)
Standard solution: 0.5 mg/mL of garcinol in alcohol
Sample solution: Transfer about 2.0 g of Powdered
Garcinia indica to a Soxhlet apparatus, add 100 mL of
alcohol, and extract for 6 h. Filter and concentrate
under vacuum to about 10 mL. [NoTE—Use a thimble
of a suitable size such that the volume of alcohol used
in the Soxhlet extraction is at least twice the volume of
the thimble.]
Adsorbent: Chromatographic silica gel with an average
particle size of 5 um (HPTLC plates)
Application volume: 5 uL, as 8-mm bands
Developing solvent system: Toluene, ethyl acetate,
and formic acid (4: 1: 0.5)
Developing distance: 6 cm
Derivatization reagent: A mixture of 1% vanillin in al-
cohol and 10% sulfuric acid in alcohol (1:1)
Analysis
Samples: Standard solution and Sample solution
Apply the Samples as bands. Develop in a saturated
chamber. Remove the plate from the chamber, dry,
treat with Derivatization reagent, heat for 5-10 min at
105°, and examine under white light.
Acceptance criteria: The Sample solution chromato-
gram exhibits a main greenish-gray band due to garci-
nol at an R; value of approximately 0.6, which corre-
sponds in position and color to the main band in the
chromatogram of the Standard solution. The Sample so-
lution exhibits the following additional bands: two pur-
ple bands, two greenish-gray bands, two blue bands,
and a purple band at R values of approximately 0.31,
0.34, 0.37, 0.47, 0.54, 0.83, and 0.93, respectively.
Other bands may be observed for the Sample solution.
e C. HPLC: The Sample solution chromatogram exhibits a
peak for hydroxycitric acid at a retention time corre-
sponding to that in the chromatogram of Standard solu-
tion A, as obtained in the test for Content of (—)-Hydrox-
ycitric Acid and (—)-Hydroxycitric Acid Lactone. The Sample
solution also exhibits a peak for hydroxycitric acid lac-
tone. The hydroxycitric acid and the hydroxycitric acid
lactone peaks are the main peaks in the Sample solution
chromatogram.
COMPOSITION
© CONTENT OF (-)-HYDROXYCITRIC ACID AND (-)-HYDROXYCI-
TRIC AcID LACTONE
Solution A: 30% Phosphoric acid in water
Mobile phase: Dissolve 1.36 g of anhydrous potassium
dihydrogen phosphate in 900 mL of water, adjust with
Solution A to a pH of 2.5, dilute with water to 1000 mL,
mix, filter, and degas.
Dietary Supplements / Garcinia 4641
Solvent: A mixture of Solution A and water (1:9)
Standard solution A: A solution of USP Calcium
(-)-Hydroxycitrate RS equivalent to about 4 mg/mL of
(-)-hydroxycitric acid in Solvent. Before injection, pass
through a membrane filter of 0.45-1m or finer pore
size, iscarding the first few mL of the filtrate.
Standard solution B: 8 mg/mL of USP Powdered
Garcinia Hydroxycitrate Extract RS in Solvent. Before in-
jection, pass through a membrane filter of 0.45-m or
finer pore size.
Sample solution: Transfer about 5 g of Powdered
Garcinia indica, accurately weighed, to a 250-mL round-
bottom flask fitted with a reflux condenser. Add 50 mL
of Solvent, reflux while stirring for 30 min, set aside to
settle, and decant the supernatant. Repeat the extrac-
tion using four 50-mL portions of water, combine all
extracts, cool, filter into a 250-mL volumetric flask, and
dilute with water to volume. Before injection, pass
through a membrane filter of 0.45-m or finer pore
size, discarding the first few mL of the filtrate.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 215 nm
Column: 4.6-mm x 25-cm; packing L1
Column temperature: 25°
Flow rate: 1.0 mL/min
Injection volume: 20 pL
System suitability
Samples: Standard solution A, Standard solution B, and
Sample solution
[Note—The relative retention times for the hydroxycitric
acid lactone and hydroxycitric acid peaks are about
0.9 and 1.0, respectively.]
Suitability requirements
Chromatogram similarity: The chromatogram of
Standard solution B is similar to the reference chro-
matogram provided with the lot of USP Powdered
Garcinia Hydroxycitrate Extract RS being used.
Resolution: NLT 1.0 between the hydroxycitric acid
lactone and hydroxycitric acid peaks, Sample solution
Tailing factor: NMT 2.0 for the hydroxycitric acid
peak, Standard solution A
Relative standard deviation: NMT 2.0%, determined
from the hydroxycitric acid peak for replicate injec-
tions, Standard solution A
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution. [NoTe—Standard solution A, Standard
solution B, and the Sample solution are stable for 6 h.]
Calculate the percentages of (-)-hydroxycitric acid and
sydesBouow sa
(-)-hydroxycitric acid lactone in the portion of Pow-
dered Garcinia indica taken:
Result = (ru/rs) x Cs x (V/W) x Fx 100
tu = peak area of the relevant analyte from the
Sample solution
rs = peak area of hydroxycitric acid from Standard
solution A
Cs = concentration of (-)-hydroxycitric acid in
Standard solution A (mg/mL)
V = final volume of the Sample solution (mL)
Ww = weight of Powdered Garcinia indica used to
prepare the Sample solution (mg)
F = conversion factor: 2.17 for (-)-hydroxycitric
acid lactone, and 1.00 for (-)-hydroxycitric
acid
Acceptance criteria: The sum of percentages of
(-)-hydroxycitric acid and (-)-hydroxycitric acid lactone
is NLT 12% on the dried basis.
IMPURITIES
e ARTICLES OF BOTANICAL ORIGIN, Acid-Insoluble Ash (561):
NMT 0.5%
 4642 Garcinia / Dietary Supplements
e ARTICLES OF BOTANICAL ORIGIN, Limits of Elemental Impuri-
ties (561): Meets the requirements
e ARTICLES OF BOTANICAL ORIGIN, Pesticide Residue Analysis
(561): Meets the requirements
SPECIFIC TESTS
© BOTANICAL CHARACTERISTICS
Macroscopic: Dark brown powder; odor characteristic;
taste sour.
Microscopic: It shows cells containing dark brown con-
tent; cells containing yellow content, parenchyma cells
containing simple and compound starch granules; frag-
ments of epicarp cells containing stomata; and frag-
ments of spiral and annular vessels.
e Limit oF Citric AciD
Solvent and Chromatographic system: Prepare as di-
rected in the test for Content of (-)-Hydroxycitric Acid
and (—)-Hydroxycitric Acid Lactone.
Standard solution: 0.5 mg/mL of USP Citric Acid RS in
Solvent. Before injection, pass through a membrane fil-
ter of 0.45-um or finer pore size, discarding the first
few mL of the filtrate.
Analysis
Sample: Standard solution
Calculate the percentage of citric acid in the portion of
Powdered Garcinia indica taken:
Result = (ru/rs) x Cs x (V/W) x 100
ty = peak area of citric acid from the Sample
solution in the test for Content of
(~)-Hydroxycitric Acid and (—)-Hydroxycitric
Acid Lactone
rs = peak area of citric acid from the Standard
solution
Cs = concentration of USP Citric Acid RS in the
Standard solution (mg/mL)
Vv = final volume of the Sample solution (mL)
w = weight of Powdered Garcinia indica used to
prepare the Sample solution in the test for
Content of (-)-Hydroxycitric Acid and
(~)-Hydroxycitric Acid Lactone (mg)
Acceptance criteria: NMT 2% of citric acd on the
dried basis
Loss ON DRYING (731)
Sample: 2.0 g of Powdered Garcinia indica
Analysis: Dry the Sample at 105° for 3 h.
Acceptance criteria: NMT 12.0%
ARTICLES OF BOTANICAL ORIGIN, Tota! Ash (561): Deter-
mined on 1.0 g of Powdered Garcinia indica: NMT 3.0%;
and NMT 8.0% if sodium chloride was added as a pre-
servative during collection of the fruits
DS Monographs
MICROBIAL ENUMERATION TESTS (2021): The total aerobic
bacterial count does not exceed 105 cfu/g, the total com-
bined molds and yeasts count does not exceed 103 cfu/
g, and the bile-tolerant Gram-negative bacteria do not
exceed 103 cfu/g.
ABSENCE OF SPECIFIED MICROORGANISMS (2022): Meets the
requirements of the tests for absence of Salmonella spe-
cies and Escherichia coli
ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Preserve in well-closed contain-
ers, protected from light and moisture, and store at
room temperature.
e LABELING: The label states the Latin binomial and, follow-
ing the official name, the part of the plant contained in
the article.
USP 41
e USP REFERENCE STANDARDS (11)
USP Calcium (-)-Hydroxycitrate RS
USP Citric Acid RS
USP Powdered Garcinia Hydroxycitrate Extract RS
Garlic
DEFINITION
Garlic consists of the fresh or dried compound bulbs of Al-
lium sativum L. (Fam. Liliaceae). It contains NLT 0.5% of
alliin and NLT 0.2% of y-glutamy!-(5)-allyl-L-cysteine, cal-
culated on the dried basis.
IDENTIFICATION
e A. THIN-LAYER CHROMATOGRAPHY
Standard solution A: 0.5 mg/mL of USP L-Methionine
RS in a mixture of methanol and water (1:1)
Standard solution B: 0.5 mg/mL of USP Alliin RS in a
mixture of methanol and water (1:1)
Sample solution: Cut a freeze-dried garlic bulb into
small pieces, transfer 1 g of the cut pieces to an extrac-
tor, and extract with two 20-mL portions of a mixture
of methanol and water (1:1), combining the extracts.
Concentrate to a small volume (about 5 mL), using a
rotary evaporator.
Chromatographic system
Adsorbent: 0,.25-mm layer of chromatographic silica
gel, typically 20-cm long (TLC plates)
Application volume: 20 pL, applied separately as
10-mm bands
Developing solvent system: Butyl alcohol, n-propyl
alcohol, glacial acetic acid, and water (3:1:1:a)
Derivatization reagent: 0.2% Sointiog of ninhydrinin
a mixture of butyl alcohol and 2 N acetic acid (19:1)
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Develop the chromatograms until the solvent front has
moved up about three-fourths of the plate, in a satu-
rated chamber. Remove the plate, and allow the sol-
vent to evaporate. Spray with the Derivatization rea-
gent, heat at 100°-105° for 10 min, and immediately
examine the plate under white light.
Acceptance criteria: The chromatogram of the Sample
solution shows the following zones: a violet zone having
an R value of about 0.89; a pink zone having an Rr
value of about 0.5 and corresponding in color and R;
value to that obtained from the chromatogram of Stan-
dard solution A; a pinkish zone having an R; value of
about 0.43; a strong orange zone having an R; value of
about 0.38; a pinkish violet zone having an R; value of
about 0.3 and corresponding in color and R- value to
that of the ene yan of Standard solution B; and
additional pinkish orange zones situated very close to
each other just below the zone corresponding to alliin
in the chromatogram of Standard solution B.
° B.
Sample: About 10g of garlic bulbs that have been cut
into small pieces
Analysis: Transfer to a suitable flask. Add 10 mL of 1N
sodium hydroxide and 10 mL of water, heat the flask in
boiling water for 10 min, cool, and filter. Add a few
drops of freshly prepared sodium nitroferricyanide TS to
2 mL of the filtrate.
Acceptance criteria: The appearance of a red or or-
ange-red color indicates the presence of sulfur-contain-
ing compounds in the Sample.
° C. The retention time of the major peak in the Sample
solution corresponds to that of one of the alliin diastere-
omer peaks in the Standard solution, as obtained in the
test for Content of Allin.
USP 41
¢ D. THIN-LAYER CHROMATOGRAPHY
Extraction column: 1-cm x 5-cm solid-phase extraction
column containing styrene-divinylbenzene copolymer
packing with a 75- to 150-um diameter and a 400- to
600-A pore size. Condition column before use by wash-
ing with 50 mL of methanol and with 50 mL of a mix-
ture of methanol and water (3:7). [NOTE—Do not allow
the column to dry.]
Standard solution: 0.2 mg/mL each of USP B-
Chlorogenin RS and USP Agigenin RS in methanol
Sample solution: Transfer about 10 g of freshly peeled
garlic cloves to a 37-mL homogenizing cup, and ho-
mogenize with 25 mL of methanol at the highest speed
for 1 min. Centrifuge the mixture, and decant the su-
pernatant to a flask. Add 70 mL of water. Transfer to
the Extraction column, allow to drain, and discard the
eluate. Wash the column with 50 mL of a mixture of
methanol and water (3:7), allow the solvent mixture to
drain, and discard the eluate. Finally, elute the crude
saponin fraction off the column with 20 mL of metha-
nol, collect the eluate, and evaporate todryness. Dis-
solve the residue in 4 mL of a mixture of 8% sulfuric
acid and alcohol (1:1), transfer the solution to a screw-
ope test tube, and heat on a boiling water bath for
5h. Cool the test tube, add 20 mL of water, and trans-
fer the solution to a freshly conditioned Extraction col-
umn, allow to drain, and discard the eluate. Wash the
column with 30 mL of a mixture of methanol and water
(7:3), and discard the eluate. Finally, elute the column
with 50 mL of methanol. Collect the eluate, evaporate it
to dryness, and dissolve the residue in 0.5 mL o
methanol.
Chromatographic system
Adsorbent: 0.25-mm layer of chromatographic silica
gel, typically 20-cm long (TLC plates)
Application volume: 20 uL, as 7-mm bands
Developing solvent system: Methylene chloride and
methanol (15:2)
Derivatization reagent: Dissolve 0.5 mL of 4-methox-
ybenzaldehyde and 0.5 mL of sulfuric acid in sufficient
alcohol to make 10 mL.
Analysis
Samples: Standard solution and Sample solution
Develop the chromatograms until the solvent front has
moved up about three-fourths of the plate, in a satu-
rated chamber. Remove the plate, and allow the sol-
vent to evaporate. Spray the plate with Derivatization
reagent, heat the plate at 100°-105° for 5 min, and
examine the plate under white light.
Acceptance criteria: The chromatogram of the Sample
solution exhibits, among several yellowish and grayish
green spots, a grayish green ae at an R value o
about 0.4, epee to the grayish green spot due
to B-chlorogenin of the Standard solution. The chromat-
ogram of the Sample solution does not exhibit a spot at
an Rr value of about 0.2, corresponding to agigenin of
the Standard solution.
COMPOSITION
e CONTENT OF ALLIIN
Allinase inhibitor solution: Dissolve 109 mg of car-
boxymethoxylamine hemihydrochloride in 100.0 mL of
water.
Solution A: Monobasic sodium phosphate 0.045 M in
waters adjusted with 0.2 M sodium hydroxide to a pH
of 7.1
Buffer: Monobasic sodium phosphate 0.05 M in water,
adjusted with 0.2 M sodium hydroxide to a pH of 9.5
Derivatization reagent: Dissolve 140 mg of o-phthaldi-
aldehyde in 5 mL of methanol, add 100 uL of t-
butylthiol, and dilute with Buffer to 50 mL. [NOoTE—This
reagent may occasionally become opaque during prep-
ey Store at room temperature, and use within 1
week.
Dietary Supplements / Garlic 4643
Mobile phase: Acetonitrile, 1,4-dioxane, tetrahydro-
furan, and Solution A (25: 2.9: 2.2: 69.9)
Standard solution: 0.05 mg/mL of USP Alliin RS in a
mixture of methanol and water (1:1)
Sample stock solution: Transfer about 10.0 g of freshly
eeled garlic cloves, accurately weighed, to a 110-mL
omogenizing cup. Add 70.0 mL of Allinase inhibitor so-
lution, and blend at the highest speed for 30 s. Centri-
fuge, and decant the supernatant into a 100-mL volu-
metric flask. Mix the remaining solids in the cup with
20 mL of Allinase inhibitor solution, centrifuge, and add
the supernatant to the volumetric flask. Dilute the con-
tents of the flask with Allinase inhibitor solution to
volume.
Sample solution: Dilute a portion of the Sample stock
ee 1 in 10 with a mixture of methanol and water
T21)s
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 337 nm
Column: 4-mm x 10-cm; packing L1
Flow rate: 1 mL/min
Injection volume: 10 uL
System suitability
Sample: Standard solution
[NoTe—Alliin exhibits two major peaks representing its
diastereomers.]
Suitability requirements
Relative standard deviation: NMT 2.0% for each of
the major peaks, in repeated injections
Analysis
Samples: Standard solution and Sample solution
Using a syringe, transfer 0.1 mL of the Standard solution
or Sample solution to separate septum-capped vials,
and add 0.5 mL of the Derivatization reagent to each
vial. Allow a reaction time of NLT 2 min before injec-
tion into the chromatograph. Record the chromato-
grams, and measure the areas of the alliin diastere-
omer peaks.
Calculate the percentage of alliin in the portion of Gar-
lic taken:
Result = (ru/rs) x Cs x (V/W) x D x 100
ru = peak area of alliin from the Sample solution
rs = peak areas of alliin diastereomers from the
Standard solution
Gs concentration of USP Alliin RS in the Standard
ul
solution (mg/mL)
V = volume of the Sample stock solution (mL)
w = weight of Garlic used to prepare the Sample
sydesbouow sa
stock solution (mg)
D = dilution factor to prepare the Sample solution
from the Sample stock solution, 10
Acceptance criteria: NLT 0.5% on the dried basis
e CONTENT OF y-GLUTAMYL-(5)-ALLYL-L-CYSTEINE
Solution A: Dissolve 6.80 g of monobasic potassium
phosphate in 900 mL of water, and adjust with phos-
phoric acid to a pH of 2.6. Dilute with water to
1000.0 mL, and mix.
Mobile phase: Methanol and Solution A (3:17)
Standard solution: 0.08 mg/mL of USP y-Glutamyl-(S)-
ail RS in a mixture of methanol and water
el
Sample solution: Transfer about 10 g of freshly peeled
garlic cloves, accurately weighed, to a 110-mLlege
enizing cup. Add 80 mL of a mixture of methanol an
water a1), and homogenize at the highest speed for 1
min. Centrifuge the mixture, and decant the superna-
tant into a 250-mL volumetric flask. Mix the remaining
solids with two 70-mL portions of a mixture of metha-
nol and water (1:1), centrifuge, and transfer the
supernatants to the volumetric flask. Dilute the contents
 4644 Garlic / Dietary Supplements
of the flask with a mixture of methanol and water (1:1)
to volume.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 205 nm
Column: 4.6-mm x 15-cm; packing L1
Flow rate: 0.8 mL/min
Injection volume: 10 uL
System suitability
Sample: Standard solution
Suitability requirements
Relative standard deviation: NMT 2.0% for the y-
glutamyl-(5)-allyl-L-cysteine peak in repeated
injections
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of y-glutamyl-(5)-allyl-L-cys-
teine in the portion of Garlic taken:
Result = (ru/rs) x Cs x (V/W) x 100
ru = peak response of y-glutamyl-(5)-allyl-L-cysteine
from the Sample solution
Is = peak response of y-glutamyl-(5)-allyl-L-cysteine
from the Standard solution
G = concentration of USP y-Glutamyl-(5)-allyl-L-
cysteine RS in the Standard solution (mg/mL)
Vv = volume of the Sample solution (mL)
w = weit of Garlic used to prepare the Sample
solution (mg)
Acceptance criteria: NLT 0.2% on the dried basis
CONTAMINANTS
© ARTICLES OF BOTANICAL ORIGIN, Limits of Elemental Impuri-
ties (561): Meets the requirements
© ARTICLES OF BOTANICAL ORIGIN, Pesticide Residue Analysis
(561): Meets the requirements
SPECIFIC TESTS
e BOTANICAL CHARACTERISTICS
Macroscopic: Subglobular compound bulbs, 3-5 cm in
width, consisting of 8-20 cloves, the whole surrounded
by 2-5 layers of white scale leaves attached toaflat-
tened, circular base; cloves ovoid and 3- to 4-sided,
summit acute, narrowed into a threadlike portion of fi-
ber base, truncate, each clove covered with a white
scale leaf and a pinkish white epidermis, easily sepa-
rated from the solid portion, consisting of two flaky
scale leaves and two yellowish green conduplicate foli-
age leaves
Microscopic: The protective leaf contains an epidermis
DS Monographs
enclosing a mesophyll free from chlorophyll. The outer
epidermis consists of lignified sclereid cells of thick, pit-
ted walls, elongated, covered with thin cuticle, long fi-
bers up to 500 um in length and 30 um in width.
The cortical cells are thick-walled, nonlignified, tending
to collapse on maturity, isodiametric, and contain pur-
ple pigments. The vascular bundles consist of lignitied
spiral and annular vessels. The storage leaves show an
outer epidermis of thin, delicate cells of variable shape,
arranged in somewhat irregular rows, 60 1m in length
and 30 um in width. Stomata are present on the outer
epidermis only at the extreme tip near the base of the
foliage leaves.
The mesophyll consists of swollen storage parenchyma
cells filled with fine granular reserve material; scattered
in the cortex are 20 laticiferous tubes, 500-1000 um
in length. Two series of vascular bundles consisting of
narrow lignified spiral and annular vessels are arranged
in the mesophyll.
e ARTICLES OF BOTANICAL ORIGIN, Water Content (561):
NMT 65.0% for fresh bulbs, and NMT 7.0% for dried
bulbs
USP 41
e ARTICLES OF BOTANICAL ORIGIN, Total Ash (561): NMT
5.0%
© ARTICLES OF BOTANICAL ORIGIN, Acid-/nsoluble Ash (561):
NMT 1.0%
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Store in well-closed containers
in a cool, dry place, protected from light.
e LABELING: The label states the Latin binomial and, follow-
ing the official name, the part of the plant contained in
the article.
© USP REFERENCE STANDARDS (11)
USP Agigenin RS
USP Alliin RS
USP B-Chlorogenin RS
USP y-Glutamyl-(5)-allyl-L-cysteine RS
USP L-Methionine RS
Powdered Garlic
DEFINITION
Powdered Garlic is produced from Garlic that has been cut,
freeze-dried or dried at a temperature not exceeding 65°,
and powdered. It contains NLT 0.3% of alliin and NLT
0.1% of y-glutamyl-(5)-allyl-L-cysteine, calculated on the
dried basis.
IDENTIFICATION
e A. THIN-LAYER CHROMATOGRAPHY
Standard solution A: 0.5 mg/mL of USP L-Methionine
RS in a mixture of methanol and water (1:1)
Standard solution B: 0.5 mg/mL of USP Alliin RS in a
mixture of methanol and water (1:1)
Sample solution: Transfer 1 g of the Powdered Garlic
to an extractor, and extract with two 20-mL portions of
a mixture of methanol and water (1:1), combining the
extracts. Concentrate to a small volume (about 5 mL),
using a rotary evaporator.
Chromatographic system
Adsorbent: 0.25-mm layer of chromatographic silica
gel, typically 20 cm long (TLC plates)
Application volume: 20 uL, applied separately as
10-mm bands
Developing solvent system: Butyl alcohol, n-propyl
alcohol, glacial acetic acid, and water (3:1:1:1
Derivatization reagent: 0.2 in 100 solution of
ninhydrin in a mixture of butyl alcohol and 2N acetic
acid (19:1)
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Develop the chromatograms until the solvent front has
moved up about three-fourths of the plate, in a satu-
tated chamber. Remove the plate, and allow the sol-
vent to evaporate. Spray with Derivatization reagent,
heat at 100°-105° for 10 min, and immediately ex-
amine the plate under white light.
Acceptance criteria: The chromatogram of the Sample
solution shows the following zones: a violet zone having
an Rr value of about 0.89; a pink zone having an Rr
value of about 0.5 and corresponding in color and Ry
value to that obtained from the chromatogram of Stan-
dard solution A; a pinkish zone having an Rr value of
about 0.43; a strong orange zone having an R; value of
about 0.38; a pinkish violet zone having an R; value of
about 0.3 and corresponding in color and Rr value to
that of the chromatogram of Standard solution B; and
additional pinkish orange zones situated very close to
each other just below the zone corresponding to alliin
in the chromatogram of Standard solution B.
USP 41
° B.
Sample: About 10g of Powdered Garlic
Analysis: Transfer to a suitable flask. Add 10 mL of 1N
sodium hydroxide and 10 mL of water, heat the flask in
boiling water for 10 min, cool, and filter. Add a few
drops of freshly prepared sodium nitroferricyanide TS to
2 mL of the filtrate.
Acceptance criteria: The appearance of a red or or-
ange-red color indicates the presence of sulfur-contain-
ing compounds in the Sample.
e C. The retention time of the major peak in the Sample
solution corresponds to that of one of the alliin diastere-
omer peaks in the Standard solution, as obtained in the
test for Content of Allin.
¢ D. THIN-LAYER CHROMATOGRAPHY
Extraction column: 1-cm x 5-cm solid-phase extraction
column containing styrene-divinyloenzene copolymer
packing with a 75- to 150-um diameter and a 400- to
600-A pore size. Condition column before use by wash-
ing with 50 mL of methanol and with 50 mL of a mix-
ture of methanol and water (3:7). [NoTE—Do not allow
the column to dry.]
Standard solution: 0.2 mg/mL each of USP B-
Chlorogenin RS and USP Agigenin RS in methanol
Sample solution: Transfer about 10 g of Powdered Gar-
lic to a 37-mL homogenizing cup, and homogenize
with 25 mL of methanol at the highest speed for 1 min.
Centrifuge the mixture, and decant the supernatant to
a flask. Add 70 mL of water. Transfer to the Extraction
column, allow to drain, and discard the eluate. Wash
the column with 50 mL of a mixture of methanol and
water (3:2), allow the solvent mixture to drain, and dis-
card the eluate. Finally, elute the crude saponin fraction
off the column with 20 mL of methanol, collect the elu-
ate, and evaporate to dryness. Dissolve the residue in
4mL of a mixture of 8% sulfuric acid and alcohol (1:1),
transfer the solution to a eee ered test tube, and
heat ona boiling water bath for 5 h. Cool the test
tube, add 20 mL of water, and transfer the solution to a
freshly conditioned Extraction column, allow to drain,
and discard the eluate. Wash the column with 30 mL of
a mixture of methanol and water (7:3), and discard the
eluate. Finally, elute the column with 50 mL of metha-
nol. Collect the eluate, evaporate it to dryness, and dis-
solve the residue in 0.5 mL of methanol.
Chromatographic system
Adsorbent: 0.25-mm layer of chromatographic silica
gel, typically 20 cm long (TLC plates)
Application volume: 20 UL, as 7-mm bands
Developing solvent system: Methylene chloride and
methanol (15:2)
Derivatization reagent: Dissolve 0.5 mL of 4-methox-
ybenzaldehyde and 0.5 mL of sulfuric acid in sufficient
alcohol to make 10 mL.
Analysis
Samples: Standard solution and Sample solution
Develop the chromatograms until the solvent front has
moved up about three-fourths of the plate, in a satu-
rated chamber. Remove the plate, and allow the sol-
vent to evaporate. Spray the plate with Derivatization
reagent, heat the plate at 100°-105° for 5 min, and
examine the plate under white light.
Acceptance criteria: The chromatogram of the Sample
solution exhibits, among several yellowish and grayis'
green spots, a grayish green a at an R; value o}
about 0.4, conesponuia to the grayish green spot due
to B-chlorogenin of the Standard solution. The chromat-
ogram of the Sample solution does not exhibit a spot at
an R; value of about 0.2, corresponding to agigenin of
the Standard solution.
Dietary Supplements / Garlic 4645
COMPOSITION
@ CONTENT OF ALLIIN
Alliinase inhibitor solution: Dissolve 109 mg of car-
boxymethoxylamine hemihydrochloride in 100.0 mL of
water.
Solution A: 0.045 M monobasic sodium phosphate in
water. Adjust with 0.2 M sodium hydroxide to a pH of
7b:
Buffer: 0.05 M monobasic sodium phosphate in water.
Adjust with 0.2 M sodium hydroxide to a pH of 9.5.
Derivatization reagent: Dissolve 140 mg of o-phthaldi-
aldehyde in 5 mL of methanol, add 100 uL of t-
butylthiol, and dilute with Buffer to 50 mL. [NoTE—This
reagent may occasionally become opaque during prep-
aration. Store at room temperature, and use within 1
week.]
Mobile phase: Acetonitrile, 1,4-dioxane, tetrahydro-
furan, and Solution A (25: 2.9: 2.2: 69.9)
Standard solution: 0.05 mg/mL of USP Alliin RS in a
mixture of methanol and water (1:1)
Sample stock solution: Transfer about 1.0 g of Pow-
dered Garlic, accurately weighed, toa flask. Add
30.0 mL of Alliinase inhibitor solution, and shake vigor-
ously until the powder is fully dispersed. Centrifuge to
obtain a clear solution.
Sample solution: Transfer 5.0 mL of the Sample stock
solution to a 10-mL volumetric flask, and dilute with
Alliinase inhibitor solution to volume.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 337 nm
Column: 4-mm x 10-cm; packing L1
Flow rate: 1 mL/min
Injection volume: 10 uL
System suitability
ample: Standard solution
Suitability requirements
[NoteE—Alliin exhibits two major peaks representing its
diastereomers.
Relative standard deviation: NMT 2.0% for each of
the major peaks, in repeated injections
Analysis
Samples: Standard solution and Sample solution
ve a gngs, transfer 0.1 mL of the Standard solution
or the Sample solution to separate septum-capped vi-
als, add 0.5 mL of the Derivatization reagent to each
vial, and mix. Allow a reaction time of NLT 2 min
before injection into the chromatograph. Record the
chromatograms, and measure the areas of the alliin
diastereomer peaks.
Calculate the percentage of alliin in the portion of Pow- o
dered Garlic taken: Fe
Result = (ru/rs) x Cs x (V/W) x D x 100 =
tu = peak area of alliin from the Sample solution 3
fs = sum of the peak areas of alliin diastereomers By
from the Standard solution mo}
Cs = concentration of USP Alliin RS in the Standard a
solution (mg/mL)
V = volume of the Sample stock solution (mL)
Ww = weight of Powdered Garlic used to prepare
the Sample stock solution (mg)
D = dilution factor to prepare the Sample solution
from the Sample stock solution, 2
Acceptance criteria: NLT 0.3% on the dried basis
© CONTENT OF y-GLUTAMYL-(5)-ALLYL-L-CYSTEINE
Solution A: Dissolve 6.80 g of monobasic potassium
phosphate in 900 mL of water, and adjust with phos-
phoric acid to a pH of 2.6. Dilute with water to
1000.0 mL, and mix.
 4646 Garlic / Dietary Supplements
Mobile phase: Methanol and Solution A (3:17)
Standard solution: 0.08 mg/mL of USP y-Glutamyl-(S)-
one RS in a mixture of methanol and water
qa
sane solution: Transfer about 1.0 g of Powdered
Garlic, accurately weighed, to a 50-mL volumetric flask.
Add 30 mL of methanol and water (1:1), and shake vig-
orously until the powder is fully dispersed. Dilute the
contents of the flask with a mixture of methanol and
water (1:1) to volume. Centrifuge to obtain a clear
solution.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 205 nm
Column: 4.6-mm x 15-cm; packing L1
Flow rate: 0.8 mL/min
Injection volume: 10 uL
System suitability
Sample: Standard solution
Suitability requirements
Relative standard deviation: NMT 2.0% for the y-
glutamyl-(S)-allyl-L-cysteine peak in repeated
injections
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of y-glutamyl-(5)-allyl-L-cys-
teine in the portion of Powdered Garlic taken:
Result = (ru/rs) x Cs x (V/W) x 100
tu = peak response for y-glutamyl-(5)-allyl-l-cysteine
from the Sample solution
ls = peak response for y-glutamyl-(S)-allyl-L-cysteine
from the Standard solution
Cs = concentration of USP y-Glutamyl-(5)-allyl-L-
cysteine RS in the Standard solution (mg/mL)
V = volume of the Sample solution (mL)
Ww = weight of Powdered Garlic used to prepare
the Sample solution (mg)
Acceptance criteria: NLT 0.1% on the dried basis
CONTAMINANTS
e ARTICLES OF BOTANICAL ORIGIN, Limits of Elemental Impuri-
ties (561): Meets the requirements
e ARTICLES OF BOTANICAL ORIGIN, Pesticide Residue Analysis
(561): Meets the requirements
SPECIFIC TESTS
e BOTANICAL CHARACTERISTICS: Under a microscope, Pow-
dered Garlic shows numerous fragments of parenchyma
with large cells containing crystals of calcium oxalate and
DS Monographs
small triangular or quadrangular intercellular spaces at
the corners; spiral vessels accompanied by subquadratic
cells; elongated epidermal cells with thick, pitted walls.
ABSENCE OF STARCH: A water slurry of Powdered Garlic
shows no blue color when iodine TS is added.
Loss ON DRYING (731)
Sample: 1.0g of Powdered Garlic
Analysis: Dry the Sample at 105° for 2 h.
Acceptance criteria: NMT 7.0%
ARTICLES OF BOTANICAL ORIGIN, Total Ash (561): NMT
5.0%
ARTICLES OF BOTANICAL ORIGIN, Acid-Insoluble Ash (561):
NMT 1.0%
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Store in well-closed containers
in a cool, dry place, protected from light.
e LABELING: The label states the Latin binomial and, follow-
ing the official name, the part of the plant from which
the article was derived.
USP 41
e USP REFERENCE STANDARDS (11)
USP Agigenin RS
USP Alliin RS
USP B-Chlorogenin RS
USP y-Glutamyl-(5)-allyl-L-cysteine RS
USP L-Methionine RS
Powdered Garlic Extract
DEFINITION
Powdered Garlic Extract is prepared from fresh Garlic bulbs
by extraction with alcohol. The ratio of the starting crude
plant material to Powdered Extract is 9.5:1-13.5:1. It con-
tains NLT 4.0% of alliin (CsH1;NO3S). It may contain
added Powdered Garlic or other suitable substances.
IDENTIFICATION
¢ A. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST
Standard solution A: 0.5 mg/mL of USP L-Methionine
RS in a mixture of methanol and water (1:1)
Standard solution B: 0.5 mg/ml of USP Alliin RS in a
mixture of methanol and water (1:1)
Sample solution: Transfer a quantity of Powdered Ex-
tract, equivalent to about 5 mg of alliin, to a suitable
container. Add 40 mL of a mixture of methanol and
water (1:1), and shake until the powder is fully dis-
persed. Centrifuge, and decant the supernatant into a
round-bottomed flask. Concentrate to a small volume
(about 5 mL) using a rotary evaporator.
Adsorbent: 0.25-mm layer of chromatographic silica
gel, typically 20 cm long (TLC plates).
Application volume: 20 uL, applied separately as
10-mm bands
Developing solvent system: Buty! alcohol, n-propyl al-
cohol, glacial acetic acid, and water (3:1:1:1)
Spray reagent: 0.2% solution of ninhydrin in a mixture
of butyl alcohol and 2 N acetic acid (19:1)
Analysis
Samples: Standard solutions and Sample solution
Develop the chromatograms until the solvent front has
moved up about three-fourths of the plate, in a satu-
rated chamber. Remove the plate, and allow the sol-
vent to evaporate. Spray with the Spray reagent, heat
at 100°-105° for 10 min, and immediately examine
the plate.
Acceptance criteria: The chromatogram of the Sample
solution shows the following orange and pinkish violet
zones: a violet zone having an Ry value of about 0.89; a
pink zone having an R; value of about 0.5 and corre-
sponding in color and R; value to that of the chromato-
qm of Standard solution A; a pinkish zone having an
Value of about 0.43; a strong orange zone having an
Rr value of about 0.38; a pinkish violet zone having an
Rr value of about 0.3 and corresponding in color and Rr
value to that of the chromatogram of Standard solution
B; and additional pinkish orange zones situated very
close to each other just below the zone attributed to
alliin in the chromatogram of Standard solution B.
e B. The retention time of the alliin peak of the Sample
solution corresponds to that of the Standard solution, as
obtained in the test for Content of Allin.
COMPOSITION
e CONTENT OF ALLIIN
Allinase inhibitor solution: 1.09 mg/mL of carboxy-
methoxylamine hemihydrochloride
Solution A: Monobasic sodium phosphate 0.045 M in
water adjusted with 0.2 M sodium hydroxide to a pH of
7.
Buffer: Monobasic sodium phosphate 0.05 M in water
adjusted with 0.2 M sodium hydroxide to a pH of 9.5
USP 41
Derivatization reagent: Dissolve 140 mg of o-phthaldi-
aldehyde in 5 mL of methanol. Add 100 uL of é-butyl-
thiol, dilute with Buffer to 50 mL, and mix. [NoTE—This
reagent may occasionally become opaque during prep-
aration. Store at room temperature, and use within 1
week.]
Mobile phase: Acetonitrile, 1,4-dioxane, tetrahydro-
furan, and Solution A (25: 2.9: 2.2: 69.9)
Standard solution: 0.05 mg/mL of USP Alliin RS in a
mixture of methanol and water (1:1)
Sample solution: Transfer about 0.10 g of Powdered
Extract, accurately weighed, to a 50-mL volumetric
flask. Add 30 mL of Allinase inhibitor solution, and shake
until the Powdered Extract is fully dispersed. Dilute with
Allinase inhibitor solution to volume. Centrifuge, transfer
5 mL of the clear supernatant to a 10-mL volumetric
flask, and dilute with Allinase inhibitor solution to vol-
ume.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 337 nm
Column: 4-mm x 10-cm; packing L1
Flow rate: 1 mL/min
Injection size: 10 pL
System suitability
Sample: Standard solution
Suitability requirements
[Note—Alliin exhibits two major peaks representing its
diastereomers.]
Relative standard deviation: NMT 2.0% for each of
the major peaks
Analysis
Samples: Standard solution and Sample solution
Using a volumetric syringe, transfer 0.1 mL of the Sam-
ple solution or the Standard solution to separate sep-
tum-capped vials, and add 0.5 mL of the Derivatization
reagent to each vial. Allow a reaction time of NLT 2
min before injection into the chromatograph. Record
the chromatograms, and measure the areas of the al-
liin diastereomer peaks.
Calculate the percentage of alliin in the portion of Pow-
dered Extract taken:
Result = (ru/rs) x Cs x (V/W) x 100
tu = peak area of alliin from the Sample solution
rs = peak areas of alliin diastereomers from the
Standard solution
Gs = concentration of USP Alliin RS in the Standard
Solution (mg/mL)
V = volume of the Sample solution (mL)
w = weight of Powdered Garlic Extract used to
prepare the Sample solution (mg)
Acceptance criteria: NLT 4.0% on the dried basis
CONTAMINANTS
Delete the following:
°e HEAVY METALS, Method | (231): NMT 10 ppme criear1-
Jan-2018)
© MICROBIAL ENUMERATION TESTS (2021): The total aerobic
bacterial count does not exceed 104 cfu/g, and the total
combined molds and yeasts count does not exceed 103
cfu/g.
e ABSENCE OF SPECIFIED MICROORGANISMS (2022): Meets the
requirements of the tests for absence of Salmonella spe-
cies and Escherichia coli
SPECIFIC TESTS
© ALLIINASE ACTIVITY
Allinase inhibitor solution, Solution A, Buffer, Deriva-
tization reagent, Mobile phase, Standard solution,
Dietary Supplements / Garlic 4647
Chromatographicsystem, and Analysis: Proceed as
directed in the test for Content of Allin.
Sample solution: Incubate 200 mg of Powdered Extract
with 20 mL of water at room temperature for 5 min.
Immediately after incubation, add 80.0 mL of Allinase
inhibitor solution, mix, and centrifuge.
Acceptance criteria: The area of the alliin peak of the
Sample solution is NMT 1% of the area of thealliin peak
of the Standard solution.
e ALCOHOL DETERMINATION, Method I! (611): NMT 0.5%
e ARTICLES OF BOTANICAL ORIGIN, Water Content (561):
NMT 5.0%
¢ OTHER REQUIREMENTS: Meets the requirements under Bo-
tanical Extracts (565), Packaging and Storage and Pesticide
Residues
ADDITIONAL REQUIREMENTS
© PACKAGING AND STORAGE: Preserve in tight containers, in
a cool place, protected from light.
e LABELING: The label states the Latin binomial and, follow-
ing the official name, the part of the plant from which
the article was prepared. The label also indicates the con-
tent of alliin, the extracting solvent or solvent mixture
used for preparation, and the ratio of the starting crude
plant material to Powdered Extract. It meets the require-
ments under Botanical Extracts (565), Labeling.
© USP REFERENCE STANDARDS (11)
USP Alliin RS
USP L-Methionine RS
Garlic Fluidextract
DEFINITION
Garlic Fluidextract is prepared as follows. Soak 1000g of
Garlic, whole or sliced, in a volume of a mixture of water
and alcohol (between 80:20 and 50:50) sufficient to cover
the cloves. Store in a suitable container for a length of
time sufficient to extract the constituents, avoiding any
contamination, and then filter. Concentrate the filtrate, if
necessary, at the lowest possible temperature, and add
sufficient water or alcohol to make the product measure
aol: [Note—Complete extraction may require 30
ays.
IDENTIFICATION
© A. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST
Extraction column: Use a solid-phase extraction col-
umn that contains benzenesulfonylpropyl bonded to sil-
Sire BLalelere val
ica gel in the hydrogen form having a sorbent mass-to-
column volume ratio of 1g per 6 mL, or equivalent.
Condition the column before use by washing with
10 mL of methanol and 10 mL of water. [NOTE—Do not
allow the column to dry.]
Standard solution: 0.5 mg/mL of USP S-Allyl-L-cysteine
RS in a mixture of methanol and water (1:1)
Sample solution: Mix 1 mL of Fluidextract and 5 mL of
water, and transfer to the Extraction column. Allow to
drain, and discard the eluate. Wash the column with
10 mL of water and 10 mL of methanol, discarding the
eluates. Elute the amino acid fraction with 3 mL of am-
monium hydroxide solution in methanol (7 in 100),
and collect the eluate.
Adsorbent: 0.25-mm layer of chromatographic silica
gel, typically 20 cm long (TLC plates)
Application volume: 5 uL
Developing solvent system: Ethyl acetate, methanol,
acetone, glacial acetic acid, and water (10:4:3:1:3)
Spray reagent: lodoplatinate TS
Analysis
Samples: Standard solution and Sample solution
Develop the chromatograms until the solvent front has
moved up about three-fourths of the plate, in a satu-
 4648 Garlic / Dietary Supplements
rated chamber. Remove the plate, and allow the sol-
vent to evaporate. Spray with the Spray reagent, and
examine the plate.
Acceptance criteria: The chromatogram of the Sample
solution exhibits, among several yellow spots on the
purple plate, a yellow spot at an R; value of about 0.4
corresponding to that of the yellow spot obtained in
the chromatogram of the Standard solution (presence of
S-allyl-L-cysteine).
COMPOSITION
© CONTENT OF S-ALLYL-L-CYSTEINE
Mobile phase: Transfer 15.8 g of sodium citrate dihy-
drate to 250 mL of water, and carefully add 10.5 mL of
hydrochloric acid. Using a pH meter, adjust with 6 N
sodium hydroxide to a pH of 4.0. Dilute with water to
1000 mL, and mix.
Derivatizing reagent: Dissolve 0.8 g of o-phthalal-
dehyde in 2 mL of 2-mercaptoethanol. Add to a solu-
tion containing 24.70 g of boric acid and 22.35 g of
potassium hydroxide in 1000 mL of water, and mix.
Reactivating solution: 0.2 N sodium hydroxide. Pre-
pare by dissolving 0.8 g of sodium hydroxide in 100 mL
of water.
Standard solution: 0.01 mg/mL of USP S-Allyl-L-cys-
teine RS in water
Sample solution: Transfer about 2.0 g of Fluidextract,
accurately weighed, to a 100-mL volumetric flask, dilute
with trichloroacetic acid solution (5 in 100) to volume,
and mix. Centrifuge for 5 min, and filter the
supernatant.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: Fluorometric detector; excitation wave-
length of 340 nm and emission wavelength of 455 nm
Column: 4.6-mm x 12-cm; packing L17
Column temperature: 40°
Injection size: 10 pL
[NotE—The Mobile phase and the Reactivating solution
are pumped separately, each at the rate of 0.4 mL/
min, by pumps connected to the opposing arms of a
tee. The outlet of the tee is connected to the injector
and the chromatographic column. The outlet of the
column isattached to a tee, the opposing arm of
which is attached to a tube from which the Derivati-
zing reagent is constantly pumped through the system
at a rate of 0.6 mL/min. The outlet of the tee is con-
nected to a 0.5-mm x 2.0-m postcolumn polytef reac-
tion coil maintained at 40°. The outlet of the reaction
coil is connected to the detector. The system is pro-
DS Monographs
grammed to deliver the Mobile phase for 10 min, the
Reactivating solution for the next 6 min, and the Mobile
phase forthe 24 min remaining before the next
injection.]
System suitability
Sample: Standard solution
Suitability requirements
Capacity factor (k’): 2.5-4.5
Tailing factor: NMT 2.0 for the S-allyl-L-cysteine peak
Relative standard deviation: NMT 2.0% for the S-
allyl-L-cysteine peak in repeated injections
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of S-allyl-L-cysteine (CséH11SN)
in the portion of Fluidextract taken:
Result = (ru/rs) x Cs x (V/W) x 100
tu = peak height of S-allyl-L-cysteine from the
Sample solution
rs = peak height of S-allyl-L-cysteine from the
Standard solution
Cs = concentration of the USP S-Allyl-L-cysteine RS
in the Standard solution (mg/mL)
USP 41
Vv = volume of the Sample solution (mL)
Ww = weight of Fluidextract used to prepare the
Sample solution (mg)
Acceptance criteria: NLT 0.05% on the dried basis
CONTAMINANTS
Delete the following:
°e HEAVY METALS, Method Il 231): NMT 10 ppme (orca.
jan-2018)
e MICROBIAL ENUMERATION TESTS (2021): The total aerobic
bacterial count does not exceed 103 cfu/mL, and the to-
tal combined molds and yeasts count does not exceed
102 cfu/mL.
© ABSENCE OF SPECIFIED MICROORGANISMS (2022): Meets the
requirements of the tests for absence of Salmonella spe-
cies and Escherichia coli
SPECIFIC TESTS
e RESIDUE ON EVAPORATION: Proceed as directed under Bo-
tanical Extracts (565): NLT 20% of the Fluidextract por-
tion taken remains as residue.
a OF BOTANICAL ORIGIN, Tota! Ash (561): NMT
0%
ARTICLES OF BOTANICAL ORIGIN, Acid-insoluble Ash (561)
NMT 0.2%
PH (791): 4.5-6.5
OTHER REQUIREMENTS: Meets the requirements under Bo-
tanical Extracts (565), General Pharmacopeial Regie
ments, sections for Packaging and Storage, Labeling, Pesti-
cide Residues, and Alcohol Content for Fluidextracts
ADDITIONAL REQUIREMENTS
e USP REFERENCE STANDARDS (11)
USP. S-Allyl-L-cysteine RS
Garlic Delayed-Release Tablets
DEFINITION
Garlic Delayed-Release Tablets are prepared from Powdered
Garlic or Powdered Garlic Extract and contain NLT 90.0%
and NMT 140.0% of the labeled amount of alliin
(C6Hi;NO3S) and NLT 90.0% and NMT 140.0% of the
labeled amount of potential allicin (CsH100Sz).
IDENTIFICATION
e A. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST
Standard solution A: 0.5 mg/mL of USP L-Methionine
R:
Standard solution B: 0.5 mg/mL of USP Alliin RS, in a
mixture of methanol and water (1:1)
Sample solution: Transfer an amount of pulverized Tab-
lets, equivalent to 30 mg of alliin, to a 100-mL volumet-
ric flask. Add 70 mL of a mixture of methanol and
water (1:1), shake, and centrifuge. Concentrate to a
small volume (about 5 mL) using a rotary evaporator.
Chromatographic system
(See re Yy (621), Thin-Layer Chromato-
graphy.
Application volume: 20 uL, applied separately as
10-mm bands
Developing solvent system: Butyl alcohol, n-propyl
alcohol, glacial acetic acid, and water (3:1:1:1
Apia reagent: 2 mg/mL of ninhydrin, in a mixture of
uty! alcohol and 2 N acetic acid (19:1)
Analysis
Samples: Standard solutions and Sample solution
Proceed as directed in the chapter. Spray with the
Spray reagent, heat at 100°-105° for 10 min, and im-
mediately examine the plate.
USP 41
Acceptance criteria: The chromatogram of the Sample
solution shows the following orange and pinkish violet
zones: a violet zone raving an R; value of 0.89; a pink
zone having an R; value of 0.5 and corresponding in
color and R; value to that of the chromatogram of Stan-
dard solution A; a pinkish zone having an R; value of
0.43; a strong orange zone having an R; value of 0.38;
a pinkish violet zone having an R; value of 0.3 and cor-
responding in color and Ry value to that of the chro-
matogram of Standard solution B; and additional pinkish
orange zones situated very close to each other, just be-
low the zone attributed to alliin in the chromatogram
of Standard solution B.
e B. HPLC IDENTIFICATION TEST
iAanysts: Proceed as directed in the test for Content of
Allin.
Acceptance criteria: The Sample solution exibits a peak
for alliin corresponding to one of the diasteroisomer
pairs of peaks in the Standard solution.
STRENGTH
© CONTENT OF ALLIIN
Alliinase inhibitor solution: Dissolve 109 mg of car-
boxymethoxylamine hemihydrochloride in 100.0 mL of
water.
Solution A: Dissolve 1.24 g of monobasic sodium phos-
phate in 100 mL of water, adjust with 0.2 M sodium
ydroxide to a pH of 7.1, and dilute with water to
200.0 mL.
Buffer: Dissolve 1.38 g of monobasic sodium phosphate
in 100 mL of water, adjust with 0.2 M sodium hydrox-
ide to a pH of 9.5, and dilute with water to 200.0 mL.
Derivatization reagent: Dissolve 140 mg of o-phthaldi-
aldehyde in 5 mL of methanol, add 100 uL of ¢
butylthiol, and dilute with Buffer to 50 mL. [NoTE—This
reagent may occasionally become opaque during prep-
aration. Store at room temperature, and use within 1
week.]
Mobile phase: Acetonitrile, 1,4-dioxane, tetrahydro-
furan, and Solution A (25: 2.9: 2.2: 69.9)
Standard solution: 0.05 mg/mL of USP Alliin RS in a
mixture of methanol and water (1:1). Use a syringe to
transfer 0.1 mL of this solution to a septum-capped vial,
and add 0.5 mL of the Derivatization reagent. Allow a
reaction time of NLT 2 min before injection into the
chromatograph.
Sample solution: Pulverize a counted number of Tab-
lets, equivalent to 50 mg of alliin, with a mortar and
pestle. Transfer a quantity of powder equivalent to
5 mg of alliin to a 100-mL volumetric flask. Add 70 mL
of Alliinase inhibitor solution, and shake for 1 min. Dilute
with Alliinase inhibitor solution to volume. Use a volu-
metric syringe to transfer 0.1 mL of this solution to a
septum-capped vial, and add 0.5 mL of the Derivatiza-
tion reagent. Allow a reaction time of NLT 2 min before
injection into the chromatograph.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 337 nm
Column: 4-mm x 10-cm; packing L1
Flow rate: 1 mL/min
[NoTe—Alliin exhibits two major peaks, representing its
diastereomers.]
Injection size: 10 uL
System suitability
Sample: Standard solution
Suitability requirements
Relative standard deviation: NMT 2.0% for each of
the major peaks
Analysis
Samples: Standard solution and Sample solution
[Note—Record the chromatograms, and measure the
areas of the responses of the alliin diastereomer peaks.]
Dietary Supplements / Garlic 4649
Calculate the percentage of alliin in the portion of Tab-
lets taken:
Result = (ru/rs) x (Cs/Cu) x 100
tu = peak area for alliin from the Sample solution
rs = sum of the peak area for alliin diastereomers
from the Standard solution
Cs = concentration of USP Alliin RS in the Standard
solution (g/mL)
Cu = nominal concentration of alliin in the Sample
solution (tug/mL)
Acceptance criteria: 90%-140.0%
e CONTENT OF POTENTIAL ALLICIN
Alliinase inhibitor solution: Dissolve 109 mg of car-
boxymethoxylamine hemihydrochloride in 100.0 mL of
water.
Crude alliinase solution: Homogenize 5 g of raw garlic
cloves with 25 mL of water. Filter, and extract three
times with 50 mL of tert-butyl methyl ether. Discard the
organic phase, and remove the residual solvent from
the aqueous phase by rotary evaporation in vacuum for
5 min. Filter, and store frozen in small vials. [NoTeE—This
solution is stable for 6 months when stored as directed.
Thaw at room temperature just before use.]
Mobile phase: Methanol and water (3:2)
Standard stock solution: 50 g/mL of USP Alliin RS
Standard solution: Transfer 1.0 mL of the Standard
stock solution to a 5-mL volumetric flask containing
100 uL of Crude alliinase solution, and allow to stand for
5 min at room temperature. Dilute with water to vol-
ume, and pass througha filter having a 0.45-1m or
finer pore size.
Sample solution: Transfer an equivalent to 5 mg of po-
tential allicin, from finely powdered Tablets (NLT 20), to
a 200-mL volumetric flask, and add 25 mL of water.
Incubate at room temperature for exactly 30 min. Stop
the enzymatic reaction by diluting with Alliinase inhibi-
tor solution to volume. Centrifuge a portion of this solu-
tion, transfer 1.0 mL of the supernatant to a 5-mL volu-
metric flask, and dilute with water to volume.
Blank solution: 100 ul of Crude alliinase solution diluted
with water to 1 mL
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 240 nm
Column: 4.6-mm x 25-cm; packing L1
Flow rate: 1 mL/min
Injection size: 100 uL
System suitability
Samples: Standard solution, Sample solution, and Blank
sydesbouow sa
solution
[Note—The allicin peak is identified by comparing the
chromatograms of the Blank solution and the Standard
solution.]
Suitability requirements
Resolution: NLT 2.0 between the allicin peak and the
preceding peak at a relative retention time of 0.80
(allyl methyl thiosulfinates), Sample solution
Relative standard deviation: NMT 2.0%
Analysis
Samples: Standard solution, Sample solution, and Blank
solution
Calculate the percentage of potential allicin in the por-
tion of Tablets taken:
Result = (ru/rs) x (Cs/Cu) x (Mu/Mr2) x 100
tu = peak area of allicin, corrected by the response
of the Blank solution, from the Sample
solution
rs = peak area of allicin, corrected by the response
of the Blank solution, from the Standard
solution
 4650 Garlic / Dietary Supplements
Cs = concentration of USP Alliin RS in the Standard
solution (g/mL)
Cu = nominal concentration of potential allicin in
the Sample solution (g/mL)
Ma = molecular weight of allicin, 162.26
Mz = twice the molecular weight of alliin, 354.42
Acceptance criteria: 90.0%-140.0%
PERFORMANCE TESTS
e ALLICIN RELEASE: Proceed as directed in Dissolution (711)
for Method A in Apparatus 1 and Apparatus 2, Delayed-
Release Dosage Forms. Place a number of Tablets, equiva-
lent to 5 mg of potential allicin, in each vessel.
Apparatus 2: 100 rpm
Time: 60 min for the Buffer stage
Crude alliinase solution, Mobile phase, Blank solution,
and Chromatographic system: Proceed as directed in
the test for Content of Potential Allicin.
Standard stock solution: 50 g/mL of USP Alliin RS
Standard solution: Transfer 1.0 mL of the Standard
stock solution to a S-mL volumetric flask containing
100 uL of Crude alliinase solution, and allow to stand for
5 min at room temperature. Dilute with water to vol-
ume, and pass through a membrane filter having a
0.45-um or finer pore size.
Sample solution: Transfer 1.0 mL of the solution under
test to a test tube containing 50 pL of 0.21 M carboxy-
methoxylamine hemihydrochloride solution.
[NoTte—The solution must be transferred immediately
upon removal from the dissolution vessel to inhibit the
alliinase enzyme.]
Injection size: 100 pL
Analysis
[NotE—Do not perform the allicin determination in the
Acid stage.]
Samples: Standard solution and Sample solution
Calculate the percentage of potential allicin released in
the Buffer stage:
Result = (ru/rs) x (Cs x D x V/L) X (Mi/M2) x 100
ty = peak area of allicin from the Sample solution
rs = peak area of allicin from the Standard solution
Cs = concentration of USP Alliin RS in the Standard
solution (g/mL)
D = dilution factor for the Sample solution, 1.050
(1 mL of the Sample solution + 50 wL of 0.21
M carboxymethoxylamine hemihydrochloride
solution)
Vv = volume of final medium, 1000 mL
L = labeled amount of potential allicin (ug/Tablet)
Ma = molecular weight of allicin, 162.26
DS Monographs
Mz = twice the molecular weight of alliin, 354.42
Tolerances: It meets the requirements of Acceptance Ta-
ble 4 in Dissolution (711). [NOTE—Q is the percentage of
the labeled amount of potential allicin released only in
the Buffer stage.]
@ WEIGHT VARIATION OF DIETARY SUPPLEMENTS (2091): Meet
the requirements
SPECIFIC TESTS
© ALLIINASE ACTIVITY
Alliinase inhibitor solution, Solution A, Buffer, Deriva-
tization reagent, Mobile phase, Standard solution,
and Chromatographic system: Proceed as directed in
the test for Content of Allin.
Sample solution: Transfer an equivalent to 5 mg of al-
liin, from finely powdered Tablets (NLT 20), to a
100-mL volumetric flask, and add 25 mL of water. Incu-
bate at room temperature for exactly 5 min. Stop the
enzymatic reaction by diluting with Alliinase inhibitor so-
lution to volume. Centrifuge a portion of this solution,
and use a volumetric syringe to transfer 0.1 mL of the
supernatant to a septum-capped vial. Add 0.5 mL of the
USP 41
Derivatization reagent, and allow a reaction time of NLT
2 min before injection into the chromatograph.
Analysis
Samples: Standard solution and Sample solution
Proceed as directed in the test for Content of Allin.
Acceptance criteria: The area of the alliin peak from
the Sample solution is NMT 1% of the area of the alliin
peak from the Standard solution.
ADDITIONAL REQUIREMENTS
¢ PACKAGING AND STORAGE: Preserve in tight containers.
e LABELING: The label states the Latin binomial and, follow-
ing the official name, the article from which the Tablets
were prepared. Label it to indicate the amount of total
alliin, in ug/Tablet, and the amount of potential allicin, in
ug/Tablet.
e USP REFERENCE STANDARDS (11)
USP Alliin RS
USP L-Methionine RS
Ginger
DEFINITION
Ginger is the dried rhizome of Zingiber officinale Roscoe
(Fam. Zingiberaceae), scraped, partially scraped, or un-
scraped. It is known in commerce as unbleached ginger.
IDENTIFICATION
oA.
Analysis: Pulverize 5 g of Ginger. To 1 g of the pulver-
ized Ginger add 5 mL of dilute acetic acid, prepared by
diluting 1 part of glacial acetic acid with 1 part of
water, and shake for 15 min. Filter, and add a few
drops of ammonium oxalate TS to the filtrate.
Acceptance criteria: NMT a slight turbidity is
produced.
e B.
Sample: 50mg of the residue obtained in the test for
Articles of Botanical Origin, Alcohol-Soluble Extractives,
Method 2
Analysis: Dissolve the Sample in 25 mL of water, and
extract this solution with two 15-mL portions of ether.
Combine the ether extracts, and evaporate in a porce-
lain dish. To the residue add 5 mL of sulfuric acid solu-
tion (7.5 in 10.0) and 5 mg of vanillin. Allow to stand
for 15 min, and add an equal volume of water.
Acceptance criteria: The solution turns azure blue.
e C. HPTLC FoR ARTICLES OF BOTANICAL ORIGIN (203)
Standard solution A: 0.2 mg/mL of USP Ginger Con-
stituent Mixture RS in methanol
Standard solution B: 100 mg/mL of USP Powdered
Ginger RS in methanol. Sonicate for 10 min, and centri-
fuge or filter. Use supernatant or filtrate.
Sample solution: Pulverize 5 g of Ginger. Prepare a
100-mg/mL on of Ginger in methanol. Sonicate
for 10 min, and centrifuge or filter. Use supernatant or
filtrate.
Adsorbent: Chromatographic silica gel with an average
particle size of 5 um (HPTLC plate)!
Application volume: 6 wL of Standard solution A and
2 ul each of Standard solution B and Sample solution as
8-mm bands
Temperature: Ambient, not to exceed 30°
Developing solvent system: Toluene and ethyl acetate
(3:1)
Derivatization reagent: To 170 mL of ice-cold metha-
nol add 20 mL of glacial acetic acid, 10 mL of sulfuric
acid, and 1 mL of anisaldehyde. Mix well.
1 Suitable commercially available plates are HPTLC Silica Gel 60 Fasa from
EMD Millipore (e.g., Part No. 1.05642.0001).
USP 41 Dietary Supplements / Ginger 4651

System suitability Table 1

Samples: Standard solution A and Standard solution B Time Solution A Solution B
Apply the Savi and dry in air. Condition at relative (min) (%) (%)
humidity of about 33%. Develop in a saturated cham-
0 100 0
ber until the solvent front has migrated over a path of
6m. Dry in a current of cold air, and immerse into 2 0 100
Derivatization reagent for 1 s. Heat for 3 min at 100°, 12 0 100
and examine under white light and under long-wave 14 100 0
UV (365 nm). 29 100 0
Suitability requirements
Chromatographic pattern: Under white light, the Standard solution: 0.1 mg/mL of USP Capsaicin RS in
derivatized chromatogram of Standard solution A dis- methanol
plays two prominent bands: the lower due to System suitability solution: Reconstitute the content of
panel, the upper due to 6-shogaol. Under white one vial of USP Ginger Constituent Mixture RS in 1 mL
light, the derivatized chromatogram of Standard solu- of the Standard solution.
tion B shows a succession of dark-violet bands be- Sample solution: Use the filtrate retained from the test
tween the origin and the intense dark-brown band for Articles of Botanical Origin, Alcohol-Soluble Extractives,
corresponding to that of the 6-gingerol in Standard Method 2.
solution A. Immediately proximate to the 6-gingerol Chromatographic system
band in the Standard solution B chromatogram, less (See Chromatography (621), System Suitability.)
intense bands due to 8-gingerol and 10-gingerol are Mode: LC
observed. A variable number of low-intensity dark- Detector: UV 282 nm
gray bands appear between 10-gingerol and the sec- Column: 4.6-mm x 25-cm; packing L1
ond prominent band corresponding to 6-shogaol in Flow rate: 1 mL/min
Standard solution A. |In the distal part of the chromat- Injection volume: 25 uL
ogram, a dark-purple somewhat diffuse band is ob- System suitability
served. Under long-wave UV (365 nm), the chromat- Samples: Standard solution and System suitability
ograms of the Standard solutions exhibit patterns Solution
similar to those observed under white light. The [Note—The relative retention times for 6-gingerol, cap-
bands due to gingerols and shogaols are bright or- saicin, and 6-shogaol are about 0.8, 1.0, and 1.9, re-
ange; the bands between the origin and the spectively, System suitability solution.]
6-gingerol band are dark-red to brown, somewhat Suitability requirements
less prominent than when observed in white light. Resolution: NLT 3.0 between the 6-gingerol and cap-
The bands between 10-gingerol and 6-shogaol are saicin peaks; NLT 10.0 between the capsaicin and
variably colored; frequently, a light-gray band ap- 6-shogaol peaks, System suitability solution
pears halfway between them, with a light-purple dif- Tailing factor: NMT 2.0 for the 6-gingerol, capsaicin,
fuse band between it and the orange band due to and 6-shogaol peaks, System suitability solution
6-shogaol. The distal diffuse band assumes a purple- Relative standard deviation: NMT 2.5%, Standard
pink hue. solution
Analysis Analysis
Samples: Standard solution A, Standard solution B, and Samples: Standard solution, System suitability solution,
Sample solution and Sample solution
Treat and examine the Samples as described under Sys- Calculate the sum of the peak responses due to ginger-
tem suitability. ols and gingerdiones occurring at about the following
Acceptance criteria: Under white light and under long- retention times, relative to 1.0 for capsaicin: 0.8 for
wave UV (365 nm), the chromatogram of the Sample 6-gingerol, 1.5 for 8-gingerol A, 2.2 for 8-gingerol B,
solution exhibits the band pattern similar to that ob- 2.5 for 6-gingerdiol, 2.6 for 6-gingerdione, 3.4 for
served with Standard solution B. The band in the distal 10-gingerol, and 5.2 for 8-gingerdione.
part of the chromatogram, however, has no diagnostic Calculate the percentage of gingerols and gingerdiones
significance. Its color may range from purple-pink to in the portion of Ginger taken:
muddy yellow, or the band may be altogether absent.
sydesbouow sq

Potential adulterants lack the band pattern characteristic Result = (rr/rs) x (Cs/W) x 10
of the gingerol-shogaol succession. Kaempferia galanga
L. rhizome shows no diagnostic bands under UV, but rr = sum of the peak responses for gingerols and
under white light a purple band is seen at about two- gingerdiones from the Sample solution
thirds from the application line. Lesser galangal (Alpinia ls = peak response of capsaicin from the Standard
officinarum Hance) rhizome presents a yellow band at solution
an R; just below the 6-gingerol band, followed by a Cs = concentration of USP Capsaicin RS in the
continuous broad blue smudge, and a distinct tandem Standard solution (mg/mL)
of light-orange and yellow bands close to the middle of Ww = weight of Ginger used in the test for Articles of
the plate. Botanical Origin, Alcohol-Soluble Extractives,
Method 2 (g)
COMPOSITION Acceptance criteria: NLT 0.8%
© CONTENT OF GINGEROLS AND GINGERDIONES
Solution A: Acetonitrile, dilute phosphoric acid (1 in CONTAMINANTS
1000), and methanol (55:44:1) ¢ ARTICLES OF BOTANICAL ORIGIN, Limits of Elemental Impuri-
Solution B: Acetonitrile ties (561): Meets the requirements
Mobile phase: Use Solution A for NLT 7 times the re- e ARTICLES OF BOTANICAL ORIGIN, Pesticide Residue Analysis
tention time of capsaicin. (561): Meets the requirements
Column washing: After each chromatographic run, e MICROBIAL ENUMERATION TESTS (2021): The total bacterial
wash the column, using Table 1. count does not exceed 10° cfu/g, the total combined
molds and yeasts count does not exceed 103 cfu/g, and
the bile-tolerant Gram-negative bacteria count does not
exceed 103 cfu/g.
 4652 Ginger / Dietary Supplements
e ABSENCE OF SPECIFIED MICROORGANISMS (2022): It meets
the requirements of the tests for absence of Salmonella
species and Escherichia coli.
SPECIFIC TESTS
e BOTANICAL CHARACTERISTICS
Macroscopic: Ginger occurs in horizontal, laterally flat-
tened, sympodially branching pieces. Whole rhizomes
are 5-15 cm long, 1.5-6 cm wide, and up to 2cm
thick, sometimes split longitudinally, pale yellowish buff
or light brown externally, longitudinally striated, some-
what fibrous; branches are flattish, obovate, short,
about 2 cm long, each ending with a depressed stem
scar; fracture is short with projecting fibers, or some-
times resinous; internally it is yellowish brown, showing
a yellow endodermis separating the narrow cortex from
the wide stele, and numerous yellowish points, secre-
tion cells and numerous bigger grayish points, and vas-
cular bundles are scattered on the whole surface. The
unscraped rhizome shows in addition an outer layer of
dark-brown cork. Morphological characteristics of differ-
ent varieties and forms of Ginger from different geo-
graphical areas are listed in Table 1 of Supplemental In-
formation for Articles of Botanical Origin (2030).
Microscopic: The scraped rhizome in transverse section
shows a cortex composed of multiple layers of paren-
chyma ce
USP 41
Acceptance criteria: NLT 4.5% residue
© ARTICLES OF BOTANICAL ORIGIN, Starch Content, Method 7
(561): NLT 42%, Method 1A of the General Procedure
being used
e ARTICLES OF BOTANICAL ORIGIN, Foreign Organic Matter
(561): NMT 1.0%
°vatcet OF BOTANICAL ORIGIN, Tota! Ash (561): NMT
U7
© ARTICLES OF BOTANICAL ORIGIN, Acid-/nsoluble Ash (561):
NMT 2.0%
e ARTICLES OF BOTANICAL ORIGIN, Volatile Oil Determination
(561): NLT 1.8 mL/100g
e ARTICLES OF BOTANICAL ORIGIN, Water-Soluble Ash (561):
NLT 1.9%
e ARTICLES OF BOTANICAL ORIGIN, Water-Soluble Extractives,
Method
2 (561): NLT 10.0%
© WATER DETERMINATION, Method Ia (921): NMT 10%
ADDITIONAL REQUIREMENTS
e PACKAGING AND STORAGE: Preserve in well-closed contain-
ers, protected from light and moisture, and store in a
cool area.
© LABELING: The label states the Latin binomial and, follow-
ing the official name, the part of the plant contained in
the article. This article is exempted from the require-
ments of Labeling (7), Labels and Labeling for Products and