Special Methods used in

Pharmaceutical Analyses
 crude drugs and products derived from them
 PURPOSES:
1. to establish purity
2. to determine the amount of therapeutically
active constituent present
 CLASSIFICATION:
1. Chemical Methods
2. Biological Methods – measure the effects
of drugs upon microbes, animals or
animal tissues
 Ash Determination

Ash Content
 the residue remaining after incineration
 represents the inorganic salts naturally occurring in the drug
and adhering to it
 may also include inorganic matter added for the purposes of
adulteration

Ash Determination
 furnishes a basis for judging the identity and cleanliness of a
drug
 gives information relative to its adulteration with inorganic
matter
 GRAVIMETRIC METHOD OF ANALYSIS
 careful control of temperature is the most important analytical
factor to regulate
 Ash Determination

Temperature Equivalents – Electric Furnace

VERY DULL-RED HEAT 500 – 550oC

DULL-RED HEAT 550 – 700oC
BRIGHT-RED HEAT 800 – 1000oC
YELLOW-RED HEAT 1000 – 1200oC
WHITE HEAT 1200 – 1600oC
 Ash Determination

Total Ash
 residue remaining after incineration
% Total Ash = ( wt TA / wt sx ) x 100

Acid-Insoluble Ash
 part of the total ash which is insoluble in diluted
HCl
 consists almost entirely of silica derived from the
soil adhering to the drug
% Acid-Insoluble Ash = ( wt AIA / wt sx ) x 100
 Substances with Ash Limits

Substances Total Ash, % Acid-Insoluble Ash, %

USP
Acacia 4.0 0.5
Benzoin (Sumatra) … 1.0
Benzoin (Siam) … 0.5
Cocoa 8.0 0.4
NF
Digitalis 5.0 …
Plantago Seed 4.0 1.0
Senna … 3.0
Sodium Alginate 18-24 …
Source: Jenkins’ Quantitative Pharmaceutical Chemistry – 7th Edition
 Ash Determination

Residue on Ignition
 expensive chemicals
 determined by ignition to dull redness
 yields negligible amount of ash
 quantity not exceeding 500 µg
 residue on ignition limits
 acetazolamide - upper residue limit of 0.1%
 aspirin – upper residue limit of 0.05%
 cocaine HCl – residue which remains must not exceed
500 µg
 Ash Determination

Loss on Ignition
 technique which provides a means of determining the
percentage of test material which is volatilized and driven
off under the conditions specified
 definite limitations on the amounts of volatile matter they
will lose when heated
 permitted loss in percent weight
 USP calamine (2.0), magnesium sulfate (40.0 to 52.0)
 NF calcium phosphate tribasic (8.0), kaolin (15.0)
 Water Determination

 drugs official in the USP and NF contain varying

quantities of water
 water of crystallization
 water in the adsorbed form

 to ensure uniformity in the official drugs

Computation:

% H2O = (wt H2O / wt drug) x 100

 Methods of Water Determination

1. Gravimetric Method A
 for drugs containing no constituents, other than
water, volatile at 105oC

2. Gravimetric Method B
 for drugs containing ether-soluble constituents
volatile at 105oC

3. Azeotropic Method (USP)

Xylene Method (US Forestry Service)
Moisture Method by Toluene Distillation (NF)
 for vegetable drugs containing 2% or more of
moisture
 disadvantage – requires a comparatively large
amount of drug
 Methods of Water Determination
4. Karl Fischer Electrometric Titration Method
 employs the use of Na2C4H4O6 . 2 H2O as primary
standard or water-methanol solution of known
concentration as secondary standard
 end point of titration – increase current
 % H2O = [ ( S x F ) / wt drug ] x 100
 Water Equivalence Factor (F) – mg H2O / mL KFR
F = 0.1566 x W/V

5. Dew Point Method

 drugs with low concentration of water

6. Electrolytic Hygrometric Method

 drugs with very low concentration of water
 Sample Problems

1. Calculate the water equivalence factor of Karl

Fischer reagent if a 180-mg sample of
Na2C4H4O6 . 2 H2O required 15.00-mL of Karl
Fischer reagent.

2. Calculate the percent moisture in

aminosalicylic acid if 9.00-mL of Karl Fischer
reagent, having a water equivalence factor 0f
4.10, was consumed by a 5.100-g sample.
 Extractive

 an approximate measure of the amount of a certain

constituent or group of related constituents
present in a drug

 amount of a drug soluble in a given solvent is an

index of its purity

 GRAVIMETRIC METHOD OF ANALYSIS

 Soxhlet Extraction Method

 continuous extraction
 uses the same portion
of solvent repeatedly
 separation of the
solvent and solute after
the extraction
 Solvents for Extraction
Solvents Drugs containing Extractive
1. Absolute Ether Volatile Oils Total Ether –Soluble
Volatile Extrractives
(TESVE)

active constituents Non-Volatile Ether-

associated with Soluble Extractives
Volatile Matter (NVESE)

2. Alcohol Resinous Matter Alcohol Soluble

Extractives (ASE)
3. Diluted Alcohol Dilute Alcohol-Soluble
* Intermittent Agitation Extractives (DASE)

4. Water Water Soluble Extractives

* Intermittent Agitation (WSE)

5. Hexane Fats & Fatty Oils Hexane Soluble

Extractives (HSE)
 Exercise 12.1
Determination of the Non-Volatile Ether-Soluble Content of Cocoa

1.
Extract about 10-g of Cocoa, accurately weighed, with absolute ether in a
continuous extraction apparatus for 8 hours.
2.
Allow the ether solution to evaporate spontaneously in a suitable tared
container, dry at 105oC for 1 hour, and weigh the NVESE.
3.
The ether-insoluble residue is dried at 105oC for 1 hours and tests for total ash,
crude fiber, etc. may be made.

USP Official Requirement:

Cocoa must yield not less than 10% and not more than 22% of NVESE.
 Exercise 12.1
Determination of the Non-Volatile Ether-Soluble Content of Cocoa

Notes:
 8 hours extraction – ensure complete removal of the ether-soluble constituents
from the powdered cocoa
 ordinary ether contains water which dissolves some tannin, sugar, etc.
 ether boils 35oC
 Residue = Ether-Soluble Extractive (ESE) consists of fixed oil and resin
 residue heated at 105oC , volatile substances are volatilized
 Non-Volatile Ether Soluble Extractive (NVESE) consists of resin, coloring
matter and fixed oil
 Ether-Insoluble Residue (EIR)
 drying to constant weight:
drying and weighing at 1-hour intervals until the loss is not more than
0.25% in 1 hour of drying
 Crude Fiber Content

 the residue, consisting chiefly of cellulose, that

remains undissolved after successive treatment
with boiling acid and alkali
 important for detection of adulterants

 limitations on the amount of substance that is

insoluble in a given solvent serve to check the
purity and identity of the drug
 GRAVIMETRIC METHOD OF ANALYSIS

% Crude Fiber
= [ ( wt residue – wt ash ) / wt drug ] x 100
 Constants of
Fats, Fatty Oils, Waxes, Balsams, Resins, etc.

 methods of analysis consist of the determination of

physical and chemical properties or values
commonly known as constants
 which when taken in conjunction with color, odor,
taste and special identity tests for the given
substance and for common adulterants are the
basis upon which the purity and quality of these
substances are judged
 turbidity of oil sample is due to the separation of
stearin
 Acid Value
[ Acid Number / Acidity Index ]

 number of milligrams of KOH necessary

to neutralize the free acids in 1 g of sample
 number of milliliters of 0.1-N NaOH
required to neutralize the free acid in 10 g
of sample
 DIRECT ALKALIMETRIC METHOD
 presence of free acids due to the
hydrolysis of esters and caused by
chemical treatment, by bacterial action or
by the catalytic action of light and heat
 SAPONIFICATION VALUE
[ Saponification Number / Koettsdorfer Number ]

 number of milligrams of KOH required to

neutralize the free acids and saponify the esters
contained in 1 gram sample
 serves to aid in the detection of the presence of
the glycerides of acids containing less than 16 or
more than 18 carbon atoms
 indicate adulteration with unsaponifiable matter

 inversely proportional to the mean molecular

weights of the acids present
 ALKALIMETRIC METHOD USING BACK-
TITRATION (w/ Blank Test)
 ESTER VALUE [ Ester Number ]

 number of milligrams of KOH required to

saponify the esters in 1-g of sample

 important in the analysis of yellow and

white wax – it serves to indicate the
presence of adulterants ( e.g. paraffin)

 ALKALIMETRIC METHOD USING BACK-

TITRATION (w/ Blank Test)

 SV = AV + EV
 UNSAPONIFIABLE MATTER

 substances present in oils or fats that are

not saponified by alkali hydroxides but are
soluble in ordinary fat solvents
 PHYTOSTEROL – vegetable origin
 CHOLESTEROL - animal origin

 indicative of the quality and purity of the oil

 GRAVIMETRIC METHOD OF ANALYSIS

IODINE VALUE [ Iodine Number ]

 number of grams of iodine absorbed under specified

conditions by 100-g sample
 quantitative measure of the proportion of unsaturated
fatty acids present
 serves to characterize fats and oils and to indicate
whether they are pure or admixtures
 serves as an aid to indicate in a definite manner the class
to which an unknown fat or oil belongs
 when considered in conjuncture with saponification
value, it serves as a means to of detecting adulteration
 IODOMETRIC METHOD (w/ Blank Test)
IODINE VALUE [ Iodine Number ]

Classification of Oils
a. DRYING OIL
 very high iodine value
 usually above 120
b. SEMI-DRYING OIL
 intermediate iodine value
 between 100 and 120
c. NON-DRYING OIL
 relatively low iodine value
 below 100
N.B.
In case of animal fats, iodine value is not very high, usually
being less than 90.
IODINE VALUE [ Iodine Number ]

Methods for Determination of Iodine Value

1. Hubl Method
2. Hanus Method
3. Wijs Method

Reasons for Blank Test

1. corrects for the presence of impurities in the reagents

2. corrects changes in volume at different temperature

3. makes it unnecessary to know the normality of the

iodochloride solution
 HYDROXYL VALUE
[ Hydroxyl Number ]

 number of milligrams of KOH equivalent to the

hydroxyl content of 1-g of the sample

 gives an indication of the identity and purity of

fatty substances possessing alcoholic hydroxyl
groups

 inversely proportional to the molecular weight

 INDIRECT ALKALIMETRIC METHOD (w/

Blank Test)
 ACETYL VALUE of Fatty Acids
 number of milligrams of KOH required to neutralize
the acetic acid obtained by the saponiifcation of 1-g of
acetylated fatty acids
 corresponds closely to the hydroxyl value of fatty
alcohols and two constants (SV & AV) have much the
same significance with respect to identity and purity
of substances
 Computation:
A = ( S – F ) / ( 1 – 0.00075S )
A = acetyl value of free fatty acids
S = saponification value of acetylated fatty acids
F = acid value of original fatty acids
0.00075 = number of grams of acetyl group that
corresponds to 1-mg of KOH
 WATER & SEDIMENTS in Fatty Oils

 moisture and non-fatty tissue residues in fatty oils

of animal origin

 determination carried out in a pear-shaped

graduated centrifuge tubes

 centrifuge that has a diameter of swing of 38 to 43

cm is operated at 1500 r/min

 Computation:
% by volume = mL CF 1 + mL CF 2
 ASSAY OF VOLATILE OILS
[ ETHEREAL OILS ; ESSENTIAL OILS ; ESSENCES ]

 complex products composed of mixtures of

compounds of widely variant chemical
characteristics

 important chemical components of official

volatile oils
 Hydrocarbons
 Alcohols
 Aldehydes
 Ketones
 Phenols
 Acids
 Sulfur Compounds
 ASSAY OF VOLATILE OILS
[ ETHEREAL OILS ; ESSENTIAL OILS ; ESSENCES ]

 analysis of volatile oils for the purpose of

determining their purity and value is based on:
 the measurement of certain physical characteristics
 the quantitative estimation of certain components
 the qualitative tests for the various substances commonly
employed as adulterants
 ASSAY FOR ESTER CONTENT

 are mostly the acetates of alcohols

 determination of the total esters when taken in
conjunction with the official tests for purity serves to
detect adulteration and to establish the quality of oils
valued for their ester content
 ALKALIMETRIC METHOD USING BACK TITRATION
(w/ Blank Test)
 Each milliliter of 0.5-N alcoholic KOH consumed in the
saponification is equivalent to 99.15-mg of total esters
calculated as menthyl acetate (C12H23O2)
ASSAY FOR ALCOHOL CONTENT

 alcohols present in volatile oils occur both free and

combined as esters
 establish the purity and value of an oil with respect
to its content of alcoholic constituents
 determined by transforming the free alcohols into
the corresponding acetates by boiling the oil with
acetic anhydride in an acetylization flask and then
determining the saponification value of the
acetylized product
 ACETYLIZATION FLASK
ASSAY FOR ALCOHOL CONTENT

 computation of % total menthol

= A x 7.813
_____________ x [ 1 – (E x 0.0021) ]
B – (A x 0.021)
A = ( mL BT – mL AT ) volume of 0.5-N HCl
B = wt acetylated oil taken
E = % esters as menthyl acetate
ASSAY FOR ALDEHYDE CONTENT

1. Bisulfite Method
 form addition products with certain reagents
 bisulfite addition product dissolve in water
 non-aldehyde constituents as a water insoluble layer
(residual layer)
 CASSIA FLASK

2. Hydroxylamine Method
 very small amounts of aldehydes
 contain other constituents that form water-soluble
addition products
 INDIRECT ALKALIMETRIC METHOD (w/ Blank
Test)
 ASSAY FOR KETONE CONTENT

 only caraway oil and spearmint oil are assayed for

their ketone (CARVONE) content
1. Bisulfite Method
 bisulfite addition product dissolve in water
 non-ketone constituents as a water insoluble layer
(residual layer)
 CASSIA FLASK

2. Hydroxylamine Method
 INDIRECT ALKALIMETRIC METHOD (w/ Blank
Test)
 ASSAY FOR PHENOL CONTENT

 volatile oils that contain phenols when shaken with

solutions of NaOH diminish in volume because of
the ready solubility of the phenol constituents in the
alkali

 the non-phenolic portion of the oils remains

undissolved (residual layer)

 CASSIA FLASK
 DETERMINATION OF VOLATILE OIL CONTENT
OF CRUDE DRUGS AND OLEORESINS

 crude drugs and oleoresins, used as medicinal or

flavoring agents owe their virtues primarily to
volatile oil constituents

 separation of the oil from other components by

means of steam distillation

 accurate measurement of the volume of oil is

obtained
 ASSAY OF VOLATILE OIL IN SPIRITS

 based upon the separation of the volatile oil by

means of an immiscible solvent (e.g. kerosene)

 salting-out effect – CaCl2 TS

 BABCOCK BOTTLE
 1 division = 0.2-mL

 correction factor ( 0.21 ) due to the contraction of

liquid
ASSAY OF ALKALOIDS AND AMINE DRUGS

 chemical substances which are obtained from

plant, animal or synthetic sources, contain
organic nitrogen within their chemical structure
and usually possess physiological activity
 alkaloidal drugs and preparations derived from
them constitute a relatively important group of
the official substances employed in modern
therapy
 as a class of medicinal agents, alkaloids are
characterized by their high potency
 are performed for purposes of standardization,
proof of purity, commercial evaluation or
pharmacolegal purposes
ASSAY OF ALKALOIDS AND AMINE DRUGS

 methods of quantitative estimation

 Gravimetric Method
 Titrimetric Method – Volumetric
 Spectrometric Method
 Electrometric Method
 Physiological Method

 amount of alkaloids in crude drugs vary due to:

 age of the plant when collected
 season of the year when drug is harvested
 soil and climate in which the drug is grown
 conditions under which the drug is collected, dried
and stored
ASSAY OF ALKALOIDS AND AMINE DRUGS

 amount alkaloids present in galenical

preparations vary due to:
 quality of drug employed
 menstrum used in the extraction
 amount of decomposition of the alkaloid during the
process of extraction and of storage

 properties of alkaloids
 free alkaloids – sparingly soluble in water ; readily
soluble in immiscible solvents
 alkaloidal salts – readily soluble in water ; sparingly
soluble in immiscible solvents
 combine with acids to form salts
 liberated from aqueous solutions of their salts by alkali
 form highly insoluble precipitates with a number of reagents
ASSAY OF ALKALOIDS AND AMINE DRUGS

 methyl red solution is the indicator of choice for

alkaloidal titrations
 Alkaloidal Test Solutions
 Valser’s Reagent – HgI2 TS ; white ppt.
 Wagner’s Reagent – I2 TS ; reddish or red-brown ppt.
 Mayer’s Reagent – K2HgI4 TS ; white or slightly yellow ppt.

 Steps in Alkaloidal Assay

 Collection & Separation
 Analysis

 Types of Alkaloidal Assays

 Proximate Assay – total of a class of plant principles
 Ultimate Assay – single chemical species
 Assay of Crude Drugs and Galenicals

 Assay of Belladona Leaf / Tincture

 Acidimetric Method using Back-Titration Method
 Each mL of 0.02-N acid is equivalent to 5.788-mg of the
alkaloids of belladonna leaf, calculated as hyoscyamine or
atropine.

 Assay of Ipecac for Ether-Soluble

Alkaloids
 Acidimetric Method using Back-Titration Method
 Each mL o f 0.1-N H2SO4 is equivalent to 24.0-mg of the total
ether-soluble alkaloids of ipecac calculated as emetine.
 Assay of Pharmaceutical Dosage Forms for
Alkaloidal Content

 Assay of Ephedrine Sulfate Injection

 Acidimetric Method using Direct Titration (w/ Blank Test)
 Each mL of 0.1-N HClO4 is equivalent to 21.43 mg of
(C10H15NO)2 . H2SO4.

 Assay of Aminophylline Tablets

 Argentometric Method using Back-Titration Method
 Each mL of 0.1-N AgNO3 is equivalent to 21.02-mg of
C16H24N10O4.
 Computation for Alkaloidal Assays

 equivalent weight from given titer

 concentration of alkaloid/s present
 percentage
 mg/tablet
 mg/mL
 percentage labelled amount/claim
= ( computed conc. / potency ) x 100