Analytical Profiles of Substances

Volume 8
Edited by

Klaus Florey
The Squibb Institute for Medical Research New Brunswick, New Jersey

Contributing Editors

Norman W. Atwater Lester Chafetz Rafik Bishara Boen T. Kho Glenn A. Brewer, Jr. Hans-Georg Leemann Bruce C. Rudy
Compiled under the auspices of the Pharmaceutical Analysis and Control Section Academy of Pharmaceutical Sciences

Academic Press New York San Francisco London 1979

A Subsidiary of Harcourt Brace Jovanovich. Publishers

EDITORIAL BOARD
Norman W. Atwater Rafik Bishara Jerome I. Bodin Glenn A. Brewer, Jr. Lester Chafetz Edward M. Cohen Klaus Florey Salvatore A. Fusari Derek W. Houghton Erik H. Jensen Boen T. Kho Hans-Georg Leemann Arthur F. Michaelis Gerald J. Papariello Bruce C. Rudy Bernard Z. Senkowski

Academic Press Rapid Manuscript Reproduction

COPYRIGHT @ 1979, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR A N Y INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

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79 80 81 82

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AFFILIATIONS OF EDITORS, CONTRIBUTORS, AND REVIEWERS

H . Y. Aboul-Enein, Riyadh University, Riyadh, Saudi Arabia A . A . Al-Badr, Riyadh University, Riyadh, Saudi Arabia T. Alexander, Food and Drug Administration, Washington, D.C. N. W. Atwater, E. R. Squibb and Sons, Princeton, New Jersey S . A . Benezra, Burroughs Wellcome Company, Research Triangle Park, North Carolina R. Bishara, Eli Lilly and Company, Indianapolis, Indiana J. I . Bodin, Carter Wallace, Inc., Cranbury, New Jersey G . A . Brewer, The Squibb Institute for Medical Research, New Brunswick, New Jersey L. Chafetz, Warner-Lambert Research Institute, Morris Plains, New Jersey Z. L. Chang, Abbott Laboratories, North Chicago, Illinois A. P . K. Cheung, SRI International, Menlo Park, California E. M . Cohen, Merck Sharp & Dohme, West Point, Pennsylvania R . D . Daley, Ayerst Laboratories, Rouses Point, New York E. Debesis, Hoffmann-LaRoche, Inc., Nutley, New Jersey K . Florey, The Squibb Institute for Medical Research, New Brunswick, New Jersey K . Furnkranz, Food and Drug Administration, Washington, D.C. S. A . Fusari, Parke-Davis, Inc., Detroit, Michigan D. A . Giron-Forest, Sandoz Limited, Basel, Switzerland P . E. Crubb, Sterling- Winthrop Research Institute, Rensselaer, New York W. F. Heyes, The Squibb Institute for Medical Research, Moreton, Wirral, England D . W . Houghton, G . D. Searle and Company Ltd., High Wycombe, Bucks, England E. Jensen, The Upjohn Company, Kalamazoo, Michigan B. T. Kho, Ayerst Laboratories, Rouses Point, New York J . Kirschbaum, T h e Squibb Institute for Medical R e s e a r c h , N e w Brunswick, New Jersey
vii

viii

AFFILIATIONS OF EDITORS, CONTRIBUTORS, AND REVIEWERS

W . L . Koch, Eli Lilly and Company, Indianapolis, Indiana H. G. Leemann, Sandoz Limited, Basel, Switzerland P . Lim, SRI International, Menlo Park, California H. B. Long, Eli Lilly and Company, Indianapolis, Indiana J. W . McRae, Burroughs Wellcome Company, Research Triangle Park, North Carolina A. F. Michaelis, KV Pharmaceutical Company, St. Louis, Missouri N. G. Nash, Ayerst Laboratories, Rouses Point, New York C. E. Orzech, Ayerst Laboratories, Rouses Point, New York G. Pupariello, Wyeth Laboratories, Philadelphia, Pennsylvania L . 0. Pont, SRI International, Menlo Park, California B. Rudy, Burroughs Wellcome Company, Greenville, North Carolina W . D . SchBnleber, Sandoz Limited, Basel, Switzerland G. Schwartzman, United States Pharmacopeia, Rockville, Maryland G. Selzer, Food and Drug Administration, Washington, D.C. B. Senkowski, Alcon Laboratories, Fort Worth, Texas E. R . Townley, Schering Plough Corporation, Bloomfield, New Jersey L . Wuyland, Food and Drug Administration, Washington, D.C. C-H. Yung, Burroughs Wellcome Company, Research Triangle Park, North Carolina

PREFACE
Although the official compendia list tests and limits for drug substances related to identity, purity, and strength, they normally do not provide other physical or chemical data, nor do they list methods of synthesis or pathways of physical or biological degradation and metabolism. For drug substances important enough to be accorded monographs in the official compendia, such supplemental information should also be made readily available. To this end the Pharmaceutical Analysis and Control Section, Academy of Pharmaceutical Sciences, has undertaken a cooperative venture to compile and publish Analytical Profiles of Drug Substances in a series of volumes of which this is the seventh. The concept of analytical profiles is taking hold not only for compendia1 drugs but, increasingly, in the industrial research laboratories. Analytical profiles are being prepared and periodically updated to provide physicochemical and analytical information of new drug substances during the consecutive stages of research and development. Hopefully, then, in the not too distant future, the publication of an analytical profile will require a minimum of effort whenever a new drug substance is selected for compendial status. The cooperative spirit of our contributors has made this venture possible. All those who have found the profiles useful are requested to contribute a monograph of their own. The editors stand ready to receive such contributions. Thanks to the dedicated efforts of Dr. Morton E. Goldberg, a long cherished dream has come to fruition with the publication of Pharmacological and Biochemical Properties of Drug Substances, M. E. Goldberg, editor, published by APhA Academy of Pharmaceutical Sciences. This new series supplements the comprehensive description of the physical, chemical, and analytical characteristics of drug substances covered in Analytical Profiles of Drug Substances with the equally important description of pharmacological and biochemical properties.
ix

PREFACE

Drug substances appearing in the new series will be cross-referenced in the cumulative index. The goal to cover all drug substances with comprehensive monographs is still a distant one. It is up to our perseverance to make it a reality. Klaus Florey

Analytical Profiles of Drug Substances, 8

ASPIRIN
Klaus Florey
I . Introduction 1 . 1 Foreword 1.2 History 2. Description 2. I Name, Formula, Molecular Weight 2.2 Appearance, Color, Odor 3. Synthesis 4. Physical Properties 4.1 Spectra 4.11 Infrared 4.12 Ultraviolet 4.13 Fluorescence-Phosphorescence 4.14 Raman 4.15 Nuclear Magnetic Resonance 4.16 Mass 4.2 Solid Properties 4.2 I Melting Range 4.22 Differential Thermal Analysis 4.23 Thermogravimetric Analysis 4.24 Crystal Properties 4.3 Solution Properties 4.3 I Solubility 4.32 Dissociation Constant (pKa) 4.33 Partition Coefficients 4.34 Dielectric Constant, Dipole Moment 4.35 Radiation Absorption 5. Methods of Analysis 5.1 Historical Synopsis 5.2 Identity and Color Tests 5.3 Quantitative Analysis 5.31 Elemental 5.32 Colorimetric 5.33 Ultraviolet 5.34 Infrared 5.35 Fluorescence-Phosphorescence 5.36 Titrimetric-Electrochemical 5.37 Miscellaneous (NMR) 5.4 Chromatographic Methods 5.41 Paper 5.42 Thin-Layer 5.43 Column
I
Copyright @ 1979 by Academic Press, inc. All rights of reproduction in any form reserved. ISBN 0-12-260808-9

KLAUS FLOREY 5.44 High Pressure Liquid 5.45 Gas-Liquid 5.5 Electrophoretic Methods 5.6 Determination of Impurities 5.61 Salicylic Acid 5.62 Acetic Acid 5.63 Acetylsalicylic Anhydride and Acetylsalicylsalicylic Acid 6. Stability-Degradation 7. Pharmacokinetics-Drug Metabolism Products 8. Bioavailability-Dissolution 9. Determination in Biological Fluids and Tissues 10. Determination in Pharmaceutical Preparations 1 1 . Acknowledgments 12. References

ASPIRIN

Introduction 1.1 Foreword The writing of an analytical profile of aspirin, this drug of drugs, poses two major dilemmas. The first is in the name itself. There are many countries where Aspirin is still a tradename of the German firm Bayer AG, and Acetylsalicylic Acid is used as the generic. Yet, I decided to use the former because it is the U . S . P . and B.P., the better known worldwide and the more elegant name. Aspirin has now been available for close to 80 years, and its usefulness and popularity are undiminished. Consequently, the literature is voluminous and also undiminished. A complete coverage would be beyond the scope of an analytical profile. I have endeavored to cover the newer literature as comprehensibly as possible and have included only those older references which I found of historical interest. To all those who have labored in the vineyard of aspirin and who go unreferenced in this profile, I tender my sincere apologies.
1.

Bistor d u m e n t e d facts of the discovery of aspirin are quickly told. It was synthesized-by the German chemist Felix Hoffmann (1868-1946) in the laboratories of Farbenfabriken Bayer, Elberfeld, Germany in 1897 (Fig. 1). The compound was tested pharmacologically by H. Dreser' and clinically among others by Wohlgemuth and Witthauer3 who documented the antirheumatic, antipyretic and analgesic properties free of the undesirable side effects of salicylic acid. Apparently, there was some initial reluctance at Bayer to market the new compound since it was thought that the field was already crowded with new drugs. But opposition faded when the new drug got the support of Carl Duisberg, then the general manager of Bayer. Duisberg, of course, was the great chemist and industrialist who built Bayer into the chemical giant of world renown. After the inspired trade name Aspirin, a contraction of acetyl and "spirssure" (salicylic acid), was coined in the offices of Bayer - Euspirin was also considered and fortunately discarded4 -it was marketed in tablet form in 1899 and conquered the world.

1.2

KLAUS FLOREY

44

-. .. .

&...
.

. ,.,... . .

..

'

..I .

F i g u r e 1.

L a b o r a t o r y notebook e n t r y of F e l i x Hoffmann, d e s c r i b i n g h i s f i r s t p r e p a r a t i o n of a s p i r i n . The i n i t i a l s CD o n t h e p a g e are t h o s e of C a r l D u i s b e r g . ( C o u r t e s y of Bayer A . G . , L e v e r k u s e n , Germany)

ASPIRIN

What motivated Hoffmann to undertake this momentous synthesis? Legend has it that he wanted to help his father who was suffering from rheumatism and who was no longer able to tolerate sodium salicylate, then widely used in rheumatic and arthritic diseases. Salicylic acid occurs naturally in several plants. The analgesic and antipyretic properties of willow bark were already known in antiquity to Hippocrates and the blossoms of spiraea ulmaria (meadow sweet) were used in the middle ages. Salicylic acid was crystallized from willow bark extracts in the early years of the last century, and Kolbe, in 1859, was able to synthesize it from sodium phenolate and carbon dioxide. His student von Heyden worked out a commercially feasible process and started a factory to produce salicylic acid which made possible its widespread use in rheumatic diseases. However, its bad taste, stomach irritation and other side effects were a strong incentive to search for derivatives which retained its efficacy without its disadvantages. Acetylation of the hydroxyl group was one of the logical modifications. Acetylated salicylic acid had already been described three times in the literature (see Section 3 ) . Von Heyden and possibly also Merck, Darmstadt,are reputed to have experimented with aspirin without being able to produce the pure drug. At the time when Felix Hoffmann prepared pure aspirin successfully in the Bayer laboratories, one of his colleagues was Arthur Eichengrun, who had been hired by Carl Duisberg in 1896, while Hoffmann had been hired in 1894. Eichengrun,as an old man, had to undergo the horrors of the infamous Nazi concentration camp in Theresienstadt which he survived. In 1949, Eichengrun published his memoirs relating to the invention of aspirin5 which was then a half-century old. He claimed that it was he who told Hoffmann to prepare acetylsalicylic acid. Acetylation certainly was on Eichengrh's mind, since he had also experimented successfully with the acetylation of cellulose about the same time. He went cn to fame as the inventor and developer of rayon and safety film. Eichengrcn also claimed that anothercolleague of Bayer, the pharmacologist Dreser, opposed clinical trials. However, the memory of the 82 year-old Eichengrun must have been faulty when he

KLAUS FLOREY

wrote these rather bitter reminiscences concerning Hoffmann's, Dreser's and his own role in the discovery of aspirin because, in 1913, Eichengrun wrote a chapter on "The Pharmaceutical Research Laboratory" in the book History and Development of Farbenfabriken Bayer, V o r m . Friedr. Bayer & Co., Elber?eld by F. Fischer, 1913'+, where he laid no paternity claim to aspirin and described Dreser's role correctly. The pertinent passage (p. 412) translates as follows: "Acetylsalicylic acid, prepared by Felix rested unnoticed for 1+ years Hoffmann among thepreparations rejected by the pharmacological laboratory until in 1898,during unrelated investigations, Dreser's attention was again drawn to it. On account of the observation that the acetyl compound was increasing cardiac activity in contrast to salicylic acid itself, he recommended a clinical trial of the product . . . . I '

........

Felix Hoffmann did not publish his version of the discovery, nor did he obtain a German patent, since the synthesis had been previously described. Farbenfabriken Bayer did obtain a U.S. Patent6 in 1900 which named him as the inventor. Chemical Abstracts reveal no subsequent publications by him, nor is there any record that he was publiclyhonored for his contribution. However, in 1899, he was appointed director of the pharmaceutical research and marketing division of Bayer. He retired in 19287. In many ways, the story of the discovery of aspirin is typical for the way in which new drugs are invented and developed in pharmaceutical research laboratories, where many individuals have to make a contribution and where it is often difficult to fathom completely what thought processes, suggestions and interactions lead to a successful new drug. Description 2.1 Name, Formula, Molecular Weight Aspirin is acetylsalicylic acid, also salicylic acid acetate and 2-(acetyloxy)-benzoic acid (50-78-2). The last name is currently popular in Chemical Abstracts.
2.

ASPIRIN

H g84
2.2

M.W.

180.16

Appearance, Color, Odor Aspirin is a white, crystalline powder. It is odorless but might have a faint odor of acetic acid.
S nthesis hsynthesis of aspirin is credited to Gerhardt* in 1853. Gerhardt was investigating

3.

mixed organic acid anhydrides and, among others, reacted acetylchloride with sodium salicylate. He obtained a solid product, undoubtedly impure acetylsalicylic acid,which immediately and without further characterization he hydrolyzed with aqueous sodium carbonate to salicylic and acetic acids. Next it was prepared by reaction of salicylic acid with acetylchloride by H. von Gilmg in 1859, who described a crystalline product. In 1869, K.Krautlo had a student, A . Prinzhorn, prepare acetylsalicylic acid by the methods of Gerhardt and von Gilm and obtained an identical product by both methods with a reported melting point of 118.5O. Kraut also correctly observed that the product is not an acid anhydride as assumed by Gerhardt but rather a phenolic ester. Felix Hoffmann6 used acetic anhydride for its preparation. CH 3 ,COC1

(gooH +
or OH CH CO 3 \ CH3CO or CH2=C0 catalyst

KLAUS FLOREY

Essentially, all methods of synthesis are variations of the reaction of acetylchloride, acetic anhydride or ketenell with salicylic acid using a variety of catalysts such as pyridine12 or sulfuric acid13 and reaction conditions (c.f. 14). The preparation of aspirin labeled with a 14C-labeled acetyl group has also been reported.15 Efforts to improve the commercial processes continue to the present day. Physical Properties 4.1 Spectra 4.11 Infrared The assignment of the KBr infrared spectrum (Figure 2) of aspirin (U.S.P. reference standard #0675-F-4) is summarized in Table 1.16 It agrees essentially with a spectrum published A previ~usly?~ reflection spectrum has also been presented. 4.

TABLE 1 Infrared Spectrum Interpretation Wavelength (m' c-) 2300-2500 1760 1690
1490 1220 1 1190 760

Assignment carboxyl OH vinyl ester C=O aromatic acid C=O aromatic C=C stretch =C-0 (acid and ester) ortho subst. phenyl C-H bending

Ultraviolet Aspirin in 0.1N sulfuric acidlg and in dilute1trichloroacetic acid20 exhibits maxima at 229 nm (El 484) and 276 nm (E 65.5) .1 In chloroform a maximum was found at 2 7 nm (El 68).21 Fluorescence - Phosphorescence The native fluorescence of aspirin, in contrast to salicylic acid, is a weak one and has been studied only recently.22 Excitation wavelength maximum is at 280 nm and emission maximum is at 335 nm. Maxima for salicylic acid are at 308 and 450 nm respectively. 4.13

4.12

a E
rd

id

10

KLAUS FLOREY

The phosphorescence emission maximum was found at 410 nm.23 Raman. Raman spectra are described and discussed in the following references: 24, 25 Nuclear Magnetic Resonance The 60 and 100 MHz proton macrnetic resonance spectra of aspirin have been published as part of a n a l y t i ~ a l ~ ~ - ~ ~ and biochemical studie~.~O?~l The 100 MHz pmr spectrum of a deuter ochloroform solution containing tetramethylsilane as an internal reference was obtained on a Varian Associates XL-100-15 spectrometer equipped with a Nicolet pulsed Fourier accessory. 3 2 (Figure 3 ) The rms error for the experimental and calculated spectra shown in Figure 4 is 0.2. The proton assignment is shown below. 6 J = 8.05Hz H1 12.04 3?4 = 1.34Hz J H* 2.34(3H) 3?5 J3,6 = 0.3Hz H3 7.13 J = 7.80Hz H4 7.61 4?5 = 1.74Hz J H5 7.33 4?6 = 7.96Hz J H6 8.11 5?6 4.15 4.14

The differences in the pmr spectrum previously reported in aqueous media30 are attributed to the solvent used. The proton-proton couplings of the aryl protons are virtually identical, however. The fully decoupled I3C-NMR spectrum of aspirin in CD30D (200 mg/ml) is shown in Figure 5. The spectrum was obtained on a Varian XL-100-15 NMR spectrometer equipped with a Transform Technology TT-100 FT data system. The data represent the transformation of a 400 pulse FID obtained using 4096 data points with a 15 second delay time between accumulations.33 Peaks - a-i (Figure 5) arise from the nine carbons of aspirin. The seven peak multiplet centered at 6=49.0 ppm

12.0

1. 10

10.0

9.o

8 .O

70 .

6.0

5.0

4 .O

3.0

2.0

I.o

0.o

PPM

Figure 3.

100 MHz PMR Spectrum of Aspirin.

Instrument:

Varian XL-100-15

- -

=z
-4

-4

2
rCI

0 Id

)-I
+J

u
a,
v)

a
p :

a, 4J
rl

Id

I
rl

Id

a
c
Id

4J
rl

a,

Id

-4

w
a,

k 3

.rl

m
k l

12

6,

.-

13

14

KLAUSFLOREY

arises from the solvent. Assignment of each of the peaks can be made on the basis of their chemical shifts and the coupling information summarized in Peak a is the sole peak in the aliphatic Table 2 . region of the spectrum and is, therefore, assigned to methyl carbon (C-9). As expected, the fully coupled spectrum exhibits four peaks in this region --JH with a ' C of 130 + 2 hz. Peaks b,d,e and f can be assigned to the Four singly protonated aromatic carbons Each is strongly coupled to one proton ~ ~ with a i J of '65 + 2 hz. Weak long range coup--ling can also be observed. Peak b,d,e and f can be assigned to carbons 3,5,6 and 4, respectivery by comparison of their chemical shifts to values predicted on the basis of substituent effects observed in model systems. Peaks c,g,h and i must arise from non-protonated carbons since they do not exhibit appreciable splitting in the fully coupled spectrum. Chemical shift predictions based on substituent effects enable assignment of peaks c and 2 to the C-1 and C-2 aromatic ring carbons, respectively. The remaining peaks h and i must arise from the carbonyl carbons. Comparrson 07 their respective chemical shifts to those of model compounds suggest their assignment to the carboxyl (C-7) and a-cetoxy carbonyls (C-81, respectively. This assignment is confirmed by the observation of a long range coupling of peak i to the three protons of the methyl group. TABLE 2 l3C-NMR Data for Aspirin Assignment Jcab Multiplicityb Peak G(ppm)a Carbon #
21.6 124.6 124.9 126.9 132.6 134.7 151.9 167.4 171.2
9 3 1 5 6 4 2 7 8

a) ppm from TMS external via the relationship 6 (CD30D) =49.0 ppm. b) obtained from the fully coupled spectrum.

ASPIRIN

c) none observed. m) weakly coupled multiplets due to long range effects. spectrum was obtained on an AEI MS-902 double-focussing mass spectrometer equipped with a frequency-modulated analog tape recorder at a source temperature of looo C. above ambient (approximately 1300 C.). 32 By adjusting the sample flow and the electron multiplier gain, the maximum sensitivity was obtained without clipping the most intense peak. The recorded analog spectrum was processed on a PDP-11.34 Except for differences in the intensity of the molecular ion (M), the intensities shown in Figure 6 are virtually identical to the previously published spectrum.35 Differences could be ascribed to instrument design, source temperatures or even source design. The assignment of a number of fragment ions of the mass spectrum is shown in Figure 7 . Figure 7. 4.16 Mass - resolution mass The low

120

+H 138

m* 104 3 m/z 138 -H20 m/z 64

63

m/z 120

92

HCO -/ mz

The metastable ion at m/z 104.3 supports the loss of the elements of water from the m/z 138 rearrangement ion.

16

KLAUS FLOREY

5292 R S P I R I N
03-RUG-78 100 MF16761 30

90
>I H

80 70 60 50 40
30

W : Z
I -

25
20

0
H

cn

a
Z

Z W I Z
H

kl
H

0
H

W
l -

>

15 a I cj

_I

I -

I W rY

10

I -

Z
W

20 10

5
0

a:

I N T E N S I T Y SUM = 4 0 1 4 0

BRSE PERK X = 3 3 . 0 7

Figure 6.

Mass Spectrum of Aspirin. Instrument: AEI MS 902

ASPIRIN

17

The mass spectrum of aspirin has been used as an aid in the rapid identification of toxic materials isolated from urine, blood or gastric aspirates of drug abuse patients.36r37 Solid Properties 4.21 Melting Range The melting point of aspirin is nothing very definitive having variously been given between 118-14438 and a good deal of work has been done to get the best methods for compendia1 Early commercial preparation melted around 135.44 The European P h a r m a ~ o p e i agives a ~~ melting point of 141 to 144O as determined by the instantaneous method. For further discussion, see Polymorphism (Section 4.242) 4.2

Differential Thermal Analysis When aspirin (USP reference standard) was heated at a rate of 15O/min. in air, a single endotherm was observed with a T onset=1340 and T peak = 139.46 DTA and TGA patterns of aspirin have also been previously studied4&nd used for forensic drug identification.48 Thermogravimetric Analysis When aspirin (USP reference standard) was heated at 20/min. and a N2 flow of 20 cc/min.,no weight loss was observed at less than 1300. Crystal Properties 4.241 Single Crystal X-Ray Diffraction The crystal structure of aspirin was determined by Wheatley.49 The monoclinic crystals have a space group of P21/C. The dimension of the unit cell are: a=11.446A; b=6.596A; c=11.388 A; ~ = 9 5 033'; n=4. Powder X-Ray Diffraction The powder x-ray diffraction pattern of aspirin is presented in Table 3 and Figure 8. 5 0 Polymorphism Tn 1968, Tawashi5' claimed that aspirin exists in several polymorphic forms. 4.243 4.242 4.24 4.23

4.22

a,

8 k
W

aJ

PI

4
W

aJ

ASPIRIN

19

TABLE 3

P o w d e r X-Ray D i f f r a c t i o n P a t t e r n o f A s p i r i n (U.S.P. Reference S t a n d a r d )

20 (Deg . I 7.80 14.09 15.63 16.78 18.19 20.63 20.95 21.53 22.56 23.20 25.00 27.05

(b)

R e l at i v e

11.3 6.25 5.68 5.28 4.88 4.30 4.22 4.12 3.93 3.83 3.55 3.30

Intensity 0.539 0.033 1,000 0.087 0.038 0.079 0.035 0.030 0.268 0.210 0.033 0.427

20 (Deg . I 27.56 28.85 29.62 30.26 31.54 32.57 33.85 34.50 36.04 36.55 37.45 39.37

R el ati v e
. .

3.23 3.08 3.02 2.96 2.84 2.74 2.67 2.60 2.50 2.46 2.40 2.29

Intensity 0.054 0.068 0.040 0.039 0.120 0.110 0.051 0.037 0.049 0.048 0.034 0.038

This started a f l u r r y of a c t i v i t y . DeBis~chop~~ claimed t o have o b t a i n e d t h r e e d i f f e r e n t c r y s t a l forms b u t s t r e s s e d t h a t t h e o n l y s t a b l e one i s t h e monoclinic one m e l t i n g a t 142O C . However, t h e c l a i m f o r t r u e polymorphism of a s p i r i n w a s u e s t i o n e d o r ref u t e d i n several l a b ~ r a t o r i e s ~ ~ - ~ c a n be b e s t and % summarized i n t h e words o f G . Schwartzman:57
" I t i s g e n e r a l l y a c c e p t e d t h a t t r u e polymorphism r e s u l t s i n d i s t i n c t o p t i c a l and s p e c t r a l p r o p e r t i e s . The data p r e s e n t e d a r e e n t i r e l y n e g a t i v e i n t h e s e r e s p e c t s . The e v i d e n c e accumulated a g r e e s w i t h t h e f i n d i n g s o f P f e i f f e r 5 5 and q u e s t i o n s t h e f o r m a t i o n of a s p i r i n polymorphs. W e believe t h a t the d i f f e r e n t crystal habits w e r e c a u s e d by t h e s o l v e n t s used f o r c r y s t a l l i z a t i o n . The d i s s i m i l a r m e l t i n g p o i n t s a r e probabl y due t o t h e poor t r a n s f e r of h e a t caused by the larger crystal s i z e o r t o possible crystal d e f e c t s . I'

Optical Constants A s p i r i n h a s been d e s c r i b 8=95O ed a s m o n o c l i n i c , a:b:c = 1.7322:1:1.7322, 8=1.6424 4.25'. I n d i c e s f o r 576 p p a r e : d=1.5042;

4.244

20

KLAUS FLOREY

and 2=1.6554; 2V=15' 46'. The optical plane is normal to (010) and lies in obtuse angle f3.58 Very similar constants are presented in reference 41. Polarized Crystal Absorption Spectrum The polarized absorption spectrum of a single crystal of aspirin was measured which indicated that aspirin may be spectroscopically treated as perturbed benzoic acid.59
4.245

Calorimetry The heat of combustion at constant volume was determined as 859.3 kcal/Mol.60 Aspirin tablets make good samples for use in oxygen bomb calorimetry.61
4.25 4.3

Solution Properties 4.31 S ~ l u b i l i t v ~ ~ ' ~ ~

g/ml 5 ' Water at 2 0.0033 Water at 37O 0.01 Water at 10 0' 0.03 0.2 - 0.4 Ethanol Chloroform 0.025 - 0.06 Carbon tetrachloride 0.0004 0.1 - 0.2 Ether Abs. ether sparingly soluble Benzene 0.0033 Petroleum ether insoluble The solubility in polyethylene glycol 400 and in aqueous solution of other polyethylene glycols has been described.65-66 The effect of selected surfactants above and below the critical micelle concentration (CMC) on aspirin solubility6 was studied. Dissociation Constant (pKa) In 1913, Springer and determined the dissociation in-aqueous solution at various temperatures. At 25O they determined the (pKa 3.55). The dissociation constant as 2.8 x Merck Index62 gives a value of 3.27 x (pKa 3.49) at 25O. When the apparent pKa was de4.32

ASPIRIN

21

termined in DMF, using quaternary butyl ammonium hydroxide as the titrant, the pKa observed depended on the solvent of crystallization. From ethanol (m.p. 140-142O) , a value of 8.99; from hexane (m.p. 121-124O), a value of 9.19 was obtained. The latter higher value was ascribed to internal hydrogen bonding of the carbonyl to the hydroxy group.69 This apparent pKa should not be confused with the true pKa of 3.5 (see above). Partition Coefficients When aspirin was partitioned between buffers pH 1-7 and octyl alcoho1,partition coefficients ranging from k=17.7 (pH 1) to k=0.025 (pH 7) were obtained.70 Earlier, coefficients of 0.32 in to1uene:water and 1.81 in ch1oroform:water were determined.71
4.33 4.34

Dielectric Constant, Dipole Moment Dipole Moment: 2.09 Ref. 72 73 74

Dielectric constant: fb 2.35 5 to 7

5.65 calc.; 4.36 observed 0.93 (acc. to 75 Onsager by immersion)

Radiation Absorption The absorption coerricient of a collimated beam of 6oCo z-radiation was determined for aspirin. See reference 76 for details.
4.35

Methods of Analysis 5.1 Historical Synopsis As the following pages of this section will show, there is hardly a new method of analysis which is not immediately tried for the determination of aspirin as such, or in formulations and biological fluids. The analysis of aspirin is intricately interwoven with that of salicylic acid, its precursor and degradation product. From the very first,residual salicylic acid was determinedby the convenient reaction with ferric salts --typical for phenols -- which give a violet complex with salicylic acid. In spite of the plethora of methods, the
5.

22

KLAUS FLOREY

compendia1 approach to determine purity and strength of aspirin has been very conservative. A monograph for aspirin was introduced into U.S.P. not earlier than Volume X (1926) and has not been changed in its essentials for the last fifty years; Aside from such niceties as ash, carbonizable substances, chloride, sulfate and heavy metals, the mainstays then (U.S.P. X, 1926) and now (U.S.P. XIX, 1975) are: a) identification by a color test with ferric chloride after heating, saponification to salicylic acid, identified as a white precipitate and an odor test (ethyl acetate), b) residual salicylates as determined by a color matching test with ferric ammonium sulfate with a 0.1% limit and c) an assay involving saponification to salicylic and acetic acids and back titration of excess alkali with a purity specification of 99.5 to 100.5%. The European P h a r m a c ~ p e i auses essential~~ ly the same analytical methods as U.S.P. In contrast to aspirin itself, the U.S.P. monograph for aspirin tablets has undergone considerable changes. For some reason, U.S.P. does not use the ferric salt test for free salicylic acid, as does the British Pharmacopeia of 1973. Apparently, certain excipients such as citric and tartaric acid interfere with this reaction.77 Already in 1913, a double titration method was developed78 which was made an official method in 1926.79 This method was used as the assay method when the aspirin tablets monograph was introduced into U.S.P. XI1 in 1942. For identification, the same two tests as for aspirin itself were prescribed then (U.S.P. XII) and now (U.S.P. XIX) ; however, due to the pioneering work of Higuchi, Banes, Smith and Levine in the ' ~ O ' S , ~ ~ a test for non-aspirin salicylates was introduced using a siliceous earth column for separation from excipients and aspirin, and spectrophotometric finish at 306 nm. A limit of 0.3% is specified. The same column method with different eluting solvents and a spectrophotometric finish at 280 nm is used for the assay with limits of 95 tb 105 percent. 5.2 Identity and Color Tests Aspirin can be identified by the following name tests:

ASPIRIN

23

Test Color Ref. Trinder's reagent Purple after hydrolysis 63 McNally's test Red 63 Mandelin's reagent Green with blue 81 (Ammonium vanadate ) tint; changes to red-violet Feigl Saponif. to salicylic 82 acid. Reaction with KOH at 130: Violet fluorescence Kulberg Saponif. to salicylic 83 acid. Addition of FeC13 in HC1: red-violet coloration Vitali Morin Orange to red 84 Kofler Microscopic Identifi85 cation

For identification by infrared, see Section 4.11. Identification in combination products by mass spectrometry has been described.86
Quantitative Analysis 5.31 Elemental The percent of carbon, hydrogen and oxygen is as follows:62 % (theoretical) 60.00 c9 4.48 H8 35.53 O4 Colorimetric The use of basic organic dyes for ion pair extraction-photometric determination has been described.87 After ammonia treatment,,an orange-red color with CuSoq and H202 (Deniges) can be quantitated.88 A water insoluble violet complex ( X max. 620 nm) with 2-picoline-Cu(II) has also been reported.89 Ultraviolet The maximum at 277 nm has been used to determine aspirin in tablets after chromatography (see Section 5.43). It also has been used to determine aspirin in mixtures with other drugs (cf. 21). For simultaneous determination of 5.33 5.32 5.3

24

KLAUS FLOREY

aspirin and salicylic acid, see Section 5.61. Infrared The infrared spectrum has been used to determine aspirin in combination products. Accuracy of 1-2% has been claimed (cf. 90,911. For aspirin, the absorption maximum at 1765 cm-1 has been used.92 Fluorescence - Phosphorescence Although fluorescence of aspirin, as contrasted to that of salicylic acid, is weak (see Section 4.13), it has been used for the determination in tablets.2 2 Phosphorimetry (see Section 4.13) has been described as useful in the determination of aspirin in blood serum and plasma.23 Since the phosphorescence of salicylic acid at the maximum of 410 nm is about 500 times weaker, it does not interfere. 5.35 Titrimetric - Electrochemical Although theoretically aspirin could be titrated directly with alkali, this tends to give inaccurate results due to its instability in alkali and, therefore, the compendia1 methods back titrate after saponification (cf. 7 9 ) . However, non-aqueous titration is possible and desirable, particularly for determination in combination products. Sodium methoxide in benzene-methanol is used as the titrant and methylisobutyl ketone as the solvent. The end point is determined potentiometrically.93 Alternately, tetrabutyl ammonium hydroxide and DMF as titration solvent have also been used.94,95 Titration in ethylene diamine has also been described.96 Potentiometric measurements of ion-pair association and selective acid stren th in ethylene diamine and water has been reported.7 7 Potentiometric titration in aqueous medium has also been described9* as has colorimetric.99 5.36 The direct current and alternating current polarographic response of aspirin in an a aprotic organic solvent system (acetonitrile - 0.1M tetrabut 1 ammonium perchlorate) has been studied.Y0 The following values were obtained: 1. dc half-wave potential: E%= -1.64 2. ac fundamental harmonic peak potential: Ep = -1.76 5.34

ASPIRIN

25

3. ac second harmonic minimum potential: E min = 1.87 n values calculated from the three modes are: 0.44; 0.45 and 0.40. Approximate detection limits (moles/liter) for the three modes are: 5 x 10-5; 1 x 10-4; 1 x 10-4. On a rotating disk electrode, aspirin was reduced to the aldehyde.lo1 Miscellaneous (NMR) Nuclear magnetic resonance spectrometry has been used to quantitate aspirin in a combination product with a coefficient of variation of 1.1.12 For quantitation, the shift at 2.3 ppm representing the ester methyl group was used. Chromatographic Methods 5.41 Paper Paper chromatographic systems have been tabulated in Table 4 . TABLE 4 Solvent systems: Pet-ether, Methanol, Benzene and Water (25:20:20:0.05) Iso-propyl alcohol, Water, Ammonia (15:85: 10) Methanol, Water, Ammonia (10:90:10) Butanol, 25% Ammonia (4:1) 0.75% Nitric Acid *RfA
=

5.37

5.4

RfA* - RfS* -

Detection FeC13 FeC13 FeC13

Ref. 103 104 104

0.05

0.45
0.8

0.6

U.V. 105 Fluorimetry 106

aspirin; RfS

salicylic acid.

Cellulose anion-exchange paper chromatography with 5 % acetic acid-n-propanol (5:l) gave good separation,detected by U.V. light, of aspirin ( R f . 0.07) from salicylic acid ( R f . 0.51) . l o 7

26

KLAUS FLOREY

Thin Layer TLC has been used to identifv and quantify aspirin in pharmaceutical preparations and body fluids. Data have been summarized in Table 5. Readout by d e n s i t o m e t e r ~ ~ ~ ~ TLC ~ ~ and , ~ separation as student experimentsllO have also been described. Column mentioned in the historical synopsis (Section 5.1) , Levine perfected the compendia1 partition column procedure in which aspirin in chloroform is first trapped in an immobile phase of sodium bicarbonate on a column of siliceous earth (celite) then eluted with a solution of acetic acid in chloroform and measured spectrophotometrically. This has been also used for separation in combination products.80 For the determination of salicylic acid in presence of aspirin by this method, see Section 5.61. Ion exchange columns filled with strongly or weakly basic anion exchange resin in the acetate or chloride cycle have also been used for se aration of aspirin ,724 This has also in combination products. 2 2 I been adapted for a student experiment.125 A Sephadex-G25 column has been used for the separation of aspirin from salicylic acid.
As

5.42

5.43

High Pressure Liquid This newest of chromatographic techniques has already been used quite-extensively for the determination of aspirin in pharmaceutical products. Separation on columns filled with anionexchange resin127 or cation-exchange resin with and without counter ions128r129were investigated in detail. Silica surface columns have also been used130 as have been reverse phase (octadecyl) ones!31-134 U . V . detection was used throughout. Gas-liquid Determination of aspirin by qaschromato raphy was first reported by Hoffkan and Mitchellq35 in 1963 who separated it from other tablet ingredients by direct chromatography on tetrafluoroethylene polymer coated with Dow-Corning silicone, using a flame ionization detector and an integrator. A glass-bead column, coated with carbowax and isophthalic acid has also been used.136 Most other investigators made the methyl- or trimethylsilyl-derivative prior to chromatography. A s 5.45

5.44

TABLE 5 Thin Layer Chromatography of Aspirin Support Silica Silica Gel G Silica Po1yamide Polyamide Polyamide Polyamide Silica Gel G Polyamide Solvent System MeOH-HOAc-Et2O-CgH6 (1:18: 60/120) Et20-AcOEt (1 4) : C6H6-HOAC-MeOH-CHC13Pet-ether CH3C13-C6H6-90% HC02H (5:l:O.l) iso-PrOH-H20-90% HC02H' (1: 6: 01) 5: CH3C13-90% HC03H (20:0.1) Cyclohexane-CHC13-HOAc (4:5:1) Cyclohexane-CHC13-HOAc (50:40:10) CHC13-Cyclohexane-HOAcDioxane (40:60:1:10) CHC13-Cyclohexane-HOAc (40:60:1) EtOH-IsoProOH-XyleneCHC13 (12.5:12.5:25:50) Rf.Asp.
QJo.9

Rf.Sa1.

Detection

Ref.

0.26 0.42 0.44 0.32 0.47 0.61 0.36 0.48 0.37

'

0.44 0.24 0.08 0.24 0.41

w
W

111 112
113 114 114 114 114 115 116 116

w
W

w
K4(Fe(CNa))3

0.14 0.12

w
Iodine

Silica Gel G

0.04

117

TABLE 5 (continued) Support Silica Silica Gel GF

S i l i c a Gel GF

Solvent System CHC13-(CH3)2C0 (9:1) MeOH NH3(100:1.5)

Rf.Asp. 0.075 0.75 0.25 0.92 0.10

Rf.Sa1.

Detection NH4 Vanadate NH4 Vanadate

Ref. 118 118

EtOAc-t-PrOH-NH3 (40:30:3) E~OH-HOAC-H~O (60:30:10) CgHg C6Hg-Dioxane-HOAc ( 6 0 : 20: 2)

W W

Silica Gel GF Silica Gel 6 0

w
Fluorimetry

119 119 119

120

ASPIRIN

29

meth lating agents, diazomethane in ether alcohollY7 or tetrahydrofurane, 3 8 methanol with boron t r i f l ~ o r i d e land methyl iodide with potassium ~~ carbonate140 were used. For trimethylsilylation hexamethyl disilazane,141-143 N-0-bis(trimethy1silyl) acetamide and other trimethylsilyl BSTFA145-146and MSTFA147 have been tried. It was noted that derivatization with BSTFA or MSTFA resulted in slight hydrolysis of aspirin.147 Electrophoretic Methods Aspirin can be separated from salicylic Separation acid by ionophoresis at a pH of 4-5.14* of aspirin in combination products has been achieved with paper strip electrophoresis, ysinq4$uffers at pH 2-8 and a 200 V. applied potential. Aspirin was separated from metabolites by paper electrophoresis in a phthalate buffer of pH 3.2 and an ionic strength of 0.0125-0.0500.150
5.5

Determination of Impurities 5.61 Salicylic Acid As has alreadv been pointed out, the determination of salicylic acid is intricately interwoven with that of aspirin itself. There is the convenient color reaction with ferric chloride which was already used by Dreserl to determine free salicylic acid in his own urine after the ingestion of aspirin. However, this reaction is not too specific and considerable work has gone in the development of interference free methods.
5.6

My task has been made easy since there is an excellent review by C.A. Kelly151 on the determination of salicylic acid in aspirin and aspirin products. A more recent review has been compiled by S.L. Ali.152

I , therefore, have restricted my tabulation of methods to those references which discuss determination of salicylic acid in biological specimens, which were not covered by Kelly, to important references which have been published since the Kelly review and references which are pertinent to other sections of this profile. References for the determination of salicylic acid: 1. Colorimetric (iron complex): 77,78,153,154 (Folin-Ciocalteu reagent): 218,219

30

KLAUS FLOREY

Nonaqueous titration: 104 Iodometric: 131 Spectrophotometric: 20, 156-158 Fluorometric: 22,159,160 Infrared: 92 Column: 57,137,138,103 PC (for Rf values and solvent systems see Section 5.41): 106,107 9 . TLC (for Rf values and solvent systems see Section 5.42): 114,116,119 10. VPC: 106,107,140,141,144,145,146,147 11. HPLC: 152,164 12. Automated: 165-167 2 . 3. 4 . 5. 6. 7. 8. Acetic Acid It is easily forgotten that aspirin degrades to acetic as well as salicylic acid. And, indeed, any smell aspirin might have is due to acetic acid. However, the volatility of acetic acid does not make the determination of acetic acid a reliable tool to measure stability or degradation. A.N. Smith in 192016* devised a method to determine acetic acid in aspirin by bubbling dry air through a thin layer of powdered aspirin, trapping the acetic acid in water and backtitrating it with alkali. Gas chr~matographyl~~ has also been used. Acetylsalicylic Anhydride and Acetylsalicylsalicylic Acid In addition to salicylic and acetic acids, very small quantities of acetylsalicylanhydride (0.0012 to 0.024%) and acetylsalicylsalicylic acid (0.03 to 0.1%) have been found in aspirin preparations. The former has been determined by gas chromatography, TLC1 and spectro hotometry , the latter by gas chromatography. tT4 A controversy is still ongoing (cf. 152) whether the occasionally observed hypersensitivity against aspirinis caused by these two impurities and whether the basis of the adverse reaction is immunological. Stability - Deqradation That aspirin is sensitive to moisture is wellknown and most of us at one time or another have observed the growth of salicylic acid whiskers on aspirin tablets left f o r too long in a humid bath6. 5.63 5.62

ASPIRIN

31

room wall cabinet. The hydrolysis of aspirin to salicylic acid was already of concern to the pharmacologist, Dreser' (see Section 1.21, who in 1899 prepared what nowadays one would call a preformulation profile. He tested the hydrolysis of aspirin in acid and alkaline solutions and,by perusing textbooks of Ostwald and Nernst to devise the proper equations, determined hydrolysis constants of 3.3 x at "body temperature" in acid medium and 2.5 x at room temperature in alkaline medium. He also observed that aspirin was easily split in weakly alkaline medium which had consequences for the metabolism (see Section 7 ) . One could say that he established the first profile for the pH dependence of hydrolysis of aspirin. Of course, the definition of pH was unknown at the time. The next systematic study of the hydrolysis of aspirin in water, albeit at 1000 C., was undertaken by Rath172 who determined a hydrolysis constant of about 0.17 depending on experimental conditions. Much work has been done since, and it is quite evident that this seemingly simple hydrolysis to acetic and salicylic acids is both complex and controversial. I am fortunate that I can refer the reader to the excellent and detailed review by Clark A . KellylS1 already mentioned in Section 5.61. The most complete and thorough kinetic studies of the factors involved in the hydro1 sis of aspirin are undoubtedly those by Edwards 73, which were further elaborated by Garrett.174 Stability and decomposition kinetics of aspirin both as a solid and in solution continue to be studied. The topochemical decomposition pattern of aspirin tablets has been explored.175 The degradation of aspirin in the presence of sodium carbonate and hi h humidity was studied by x-ray diffraction. l q 6 The activation energy of decomposition by water vapor in the solid state was found to be 30 kcal/mo1.177 The effect of common tablet excipients on aspirin in aqueous suspension was also studied.178 An exhaustive study of the stability of aspirin in polyethylene glycols (substituted, unsubstituted and esterified), as well as other polyhydric alcohols, was undertaken by Whithworth and collaborator^!^^-^^^

32

KLAUS FLOREY

It was found that decomposition is in part due to transesterification and that substitution increases stability. The effect of gamma radiation on aspirin has been described.lE4 The pH stability profile of aspirin according to Edwards has been made the subject of a student experiment.lE5 Pharmacokinetics - Drug Metabolic Products When Dreser' investigated the pharmacological properties of aspirin in 1897 (see Section 1.21, he postulated that, based on the easy hydrolysis of aspirin in weakly alkaline medium, aspirin also should be converted to salicylic acid in vivo. And, indeed, he found that only 22 minutes Z t a n gestion of aspirin, his own urine gave a positive reaction for salicylic acid with ferric chloride. After 12 hours, he could no longer detect salicylic acid in his urine. He found no evidence for aspirin itself in urine, nor did he find a combination of aspirin with glycine analogous to the formation of hippuric acid already known at the time. He had evidence for formation of another nitrogen containing derivative. This early investigation should give food for thought to those who believe that pro-drugs and pharmacokinetics are recent discoveries.

7.

In 1911, NeuberglE6 detected small amounts of gentisic acid (2,5-dihydroxybenzoic acid) in the urine of dogs dosed with aspirin. The metabolism of aspirin is intertwined with that of salicylic acid, but I was unable to ascertain who first reported the metabolic formation of salicyluric acid, the major metabolite of both salicylic acid and aspirin, specifically after administration of aspirin. Reviews by PuetterlE7 and by Levy,188-190taken together present a comprehensive picture of the pharmacokinetics and metabolism of aspirin. Once absorbed, aspirin is rapidly converted to salicylic acid. After i.v. administration, the half life of aspirin in the human organism was found to be only 15 minuteslgl by Rowland and Riegelman who also estimated that only 2 0 % of the -in vivo hydrolysis takes place in blood.192 Apparently, all tissues investigated possess

ASPIRIN

33

esterases which can split aspirin; however, most of it seems to be hydrolyzed in the liver.187 While the major pathway of hydrolysis leads to free acetic acid, it is noteworthy that a significant portion of the acetic acid is not set free but used for transacetylation of certain factors which pla a role in the inhibition of platelet aggregation1J 3 only shown by aspirin but not by salicylic acid. The kinetics of this important pathway still need exploration.187 Aspirin serum esterase activity is species dependent.Ig4 In man it is sex linked since it was found to be higher in men than women.lg5 It also seems to be age dependent.lg6 Once aspirin is hydrolyzed to salicylic acid, it follows the metabolic pathway of the latter. The metabolic products of salicylic acid are presented in Figure 9 . Over the last decade, the pharmacokinetics of salicylates in the healthy and diseased organism have been explored in great detail by Levy.188 Salicylic acid (11) is eliminated by five parallel and competing pathways (Fig. 9 ) leading to renal excretion. These are: excretion unchanged (II), conjugation with glycine to form salicyluric acid (111) -- the major pathway -conjugation of the carboxyl or phenolic hydroxyl group to form glucuronides IV and V and hydroxylation to gentisic acid (VI) To a minor degree, salicyluric acid can be further conjugated with glucuronic and sulfuric acids.187 The major metabolic pathways (salicyluric and salicyl phenol glucuronide) are easily saturated in the usual dosage range for treatment of inflammation.lg7 For the clinical implications of these non-linear pharmacokinetic characteristics, I refer to the reviews by Levy.188'190

To properly treat bovine "sufferers of headaches" (inflammation) the pharmacokinetics of aspirin in cattle have also been explored.1g8 Bioavailability - Dissolution In the last two decades, the concept of bioavailability has gained prominence and-with it dissolution as a possible in vitro model for drug absorption. In 1960, S w i n t o s k y d BlytheIg9
8.

I
0

E
I

W Ocn

E o '
u

/
I
X

rcl

8 o' u

ASPIRIN

35

compared the relative availability of aspirin from entexic coated and compressed tablets by measuring the excretion of salicylate. About the same time, Levy200,201started to compare dissolution and absorption rates of different commercial aspirin tablets, found good correlation and proposed that the U.S.P. tablet disintegration test be replaced by a dissolution test; a suggestion which as of this writing has not been heeded. These studies were extended and further perfected by Gibaldi.163 Many other papers on this subject have appeared. Particularly,the influence of the crystal habitat (see Section 4.243) on absorption has been studied (cf. 202). Significant intraindividua1,but not i n t e r i n d i v i d u a 1 , v a r i a t i o n s in blood plasma aspirin levels were found in young male subjects after administration of aspirin as tablets or solutions. The plasma level curves obtained for tablets were more variable than those obtained for solutions.203 Levy204 found that using urinary excretion measurements for evaluation of aspirin dosage forms,with different absorption rates in man, requires that such measurements be made during the first hour after drug administration. The use of a pHstat for testing dissolution of various aspirin dosage forms has been described.205 Determination in Biological Fluids and Tissues All the advances in pharmacokinetics and drug metabolism described in Sections 7 and 8 would not have been possible without the availability of the proper analytical methods. The following is a tabulation of publications in this field, most of which have already been discussed in Section 5. It should be mentioned that a few publications talk about aspirin blood levels, but really mean salicylate levels. The following tabulation covers only those papers where aspirin was differentiated from other salicylates by chromatography or other means. It seems that the "workhorse" for serum salicylate levels is still the colorimetric (ferricnitrate) method of Brodie, Udenfriend and C ~ b u r n ' ~ ~ published in 1944, or modifications thereof. Simplified versions (cf. 206) may lead to erroneous results under certain conditions.207 The method is also applicable for urinary metabolites after proper hydrolysis (cf. 2 0 8 ) . For other methods restricted to salicylic acid, see Section 5.61. The first gas chromatographic separation of aspirin
9.

36

KLAUS FLOREY

and salicylic acid in plasma was described by Rowland and Riegelman, 4 1 who presented a brief review of earlier methods. Methods for aspirin: Colorimetric (with differential extraction):218,219 Spectrophotometric (differential): 20, 209 Fluorometric (as salicylic acid): 160, 210 Phosphorimetric: 23 VPC: 137,141,143,145,146 PC: 106,107,119 TLC: 118,211 Mass Spectrometric: 37 Radioisotopic: 212-214 Determination in Pharmaceutical Preparations The following tabulation of references highlights those methods (see Section 5 ) useful in pharmaceutical analysis.
10.

1.

Determination in tablets: Differential U.V.: 157,158 Automation: 166 22 Fluorometric : 121 Column : TLC : 120 GC : 136,139,144 Determination in buffered tablets: Fluorometric: 159 161,162 Column : Determination in combination products: a. General Identity tests: 83,86,103,104,105, 112 b. Aspirin, caffeine, acetophenetidine (and more components) w: 21,215 IR: 91-93 NMR: 102 Electrophoresis: 149 110 ,111 TLC : Column: 80 Ion-exchange column: 123 HPLC : 127,130,131 135 ,142 VPC : Pot. titration: 216

2.

3.

ASPIRIN

37

c.

Aspirin, acetaminophen, caffeine: Nonaqueous titration: 94,217 Potentiometric: 102 Aspirin-barbiturate combinations: Nonaqueous titration: 93 Cough-cold mixtures: HPLC: 132

d. e. 11.

Acknowledgments

I would like to express my gratitude to the Archives of Bayer AG, Leverkusen, Germany for historical information, to G. Levy, School of Pharmacy, S.U.N.Y., Buffalo for a critical review of the section on pharmacokinetics, to A . I. Cohen, M. Porubcan and B. Toeplitz for providing spectral information and interpretations, and to M. Bruno for her expert secretarial assistance.

38

KLAUS FLOREY

12.
1 .

References

H. Dreser, Pflug. Arch. Gen. Physiol., 76, 306 (1899). 2. J. Wohlgemuth, Ther. Mh., 13, 276 (1899). 3 3. K. Witthauer, Ther. Mh., 1 ,330 (1899). 4. Bayer A.G. Grchives 5. A. Eichengrun, Pharmazie, 4, 582 (1949). 6. F. Hoffmann, U.S. Patent c44077 of February 27, 1900. 7. Neue Deutsche Biographien, p. 415. 8. C. Gerhardt, Annalen der Chemie, 67, 149(1853). 9. H. von Gilm, Annalen, 112, 181 (1859). 15 10. K. Kraut, Annalen, -0 3 (1869). 11. D.A. Nightingale, U.S. Patent 1,604,472; C.A. 21, 157 (1927). 12. BayerT Co., DRP 386679 (1924); Beilstein 111, p . 41. 21, 13. Fernandez, Torres, An.Soc. espaz., - 23 (1923); Beilstein 111, p. 41. 14. Norwich Pharmacal.Co., Brit. Pat. 893,967 (1962); C.A. 57, 3572f (1962). lpio J.P. Hummel, W.D. Paul and 15. J.W. C a p s : J. I. Routh, J. Am. Pharm. ASSOC., 40, 532 (1951). 16. B. Toeplitz, The Squibb Institute for Medical Research, Personal Communication. 17. O.R. Sammul, W.L. Brannon and A.L. Hayden, J. Assoc. Offic. Agr. Chemists, 47, 918 (1964). 18. S.G. Il'yasov, V.L. Dragun, C.A.82, 91861p (1975). 19. Isolation and Identification of Drugs, E.C.G. Clarke, Editor, The Pharmaceutical Press, London (1969). 6, 20. A. Garner, J.K. Sugden, Anal. Lett. - 275 (1973). 21. M. Jones and R.L. Thatcher, Anal. Chem., 23, 957 (1951). 22. C.I. Miles and G.H. Schenk, Anal. Chem., 42, 656 (1970). 23. J.D. Winefordner and H.W. Latz, Anal. Chem., - 1517 (1963). 35, 74, 24. L. Kahovec and K.W.F. Kohlrausch, Monotsh. 333 (1943); C.A. 38, 51475 (1944). 25. G.V.L.N. Murty andT.R. Seshadri, Proc.Indian Acad. Sci., - 17 (1944); C.A. - 6197-8 19A, 38, (1944).

ASPIRIN

39

26. 27. 28. 29. 30. 31. 32. 33. 34.

35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50.

E.G. Brame, Encycl. Ind. Chem. Anal., - 707 2, (1966). G.A. Olah, P.W. Westerman, J. Org. Chem., 2 1307 (1974). B.L. Shapiro, L.E. Mohrmann, J. Phys. Chem. Ref. Data, 6 919 (1977). K.N. Scott,-J. Magn. Resonance, 5, 55 (1972). B.D. Sykes, W.E. Hull, Ann. N.Y. Acad. Sci., - 60 (1973); C.A. 80, 74282s (1974). 226, A.J. Marcus, S.E. Lasker, Haernostasis, - 92 2, (1973); C.A. 82, 8 0 3 6 0 ~(1975). A.I. Cohen, The Squibb Institute for Medical Research, Personal Communication. M. Porubcan, The Squibb Institute for Medical Research, Personal Communication. P.J. Black, A.I. Cohen, presented at the 20th Annual Conference on Mass Spectrometry and Allied Topics, Dallas, Texas, June 4-9, 1972. H.M. Fales, G.W.A. Milne, N.C. Law, Arch. Mass Spectral Data, 2, 628 (1971). N.C. Law, V. Aandahl; H.M. Fales, G.W.A. Milne, Clin. Chim. Acta, 32, 221 (1971). W. Boerner, S. Abbzt, J.C. Eidson, C.E. C.E. Becker, H.T. Horio, K. Loeffler, Clin. Chim. Acta, 49, 445 (1973). L. Kofler, Pharm. Zentralhalle, - 223 (1950); 89, C.A. 45, 1301a (1951). G.D. =a1 and C.R. Szalkowski, J. Am. Pharm. ASSOC., 22, 36 (1933). T.S. Carswell, J. Am. Pharm. ASSOC., 16,306 (1927). J.L. Hayman, L.R. Wagener and E.F. Holden, J. Am. Pharm. ASSOC., 14, 388 (1925). H.W. Johnson and C.W. Ballard, Pharm. J., - 88 (1946). 157, M. deBisschop, J. Pharm. Belg., - 330 (1970). 25, Beilstein X, p. 67 (1927). Europeian Pharmacopeia I, 236 (1969). T. Prusik, The Squibb Institute for Medical Research, Personal Communication. W.W. Wendlandt, J. Hoiberg, Anal. Chim. Acta, 29, 539 (1963). K W . Wendlandt, L.W. Collins, Anal. Chim. Acta, - 411 (1974). 71, P.J. Wheatley, J. Chem. S O C . Suppl., 1964,6039. Q. Ochs, The Squibb Institute for Medical Research, Personal Communication.

40

KLAUSFLOREY

51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74.

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199.

Analytical Profiles of Drug Substances, 8

BROMOCRIPTINE METHANESULPHONATE
Danielle A . Giron-Forest and W . Dieter Schonleber
Introduction 1 . 1 History I .2 Name, Formula, Molecular Weight 1.3 Appearance, Color, Odor Ph ysicochemical Properties 2. I Elemental Analysis 2.2 Spectra 2.21 Infrared 2.22 Ultraviolet 2.23 Fluorescence 2.24 Proton Nuclear Magnetic Resonance 2.25 Carbon-13 Nuclear Magnetic Resonance 2.26 Mass 2.3 Crystal Properties 2.3 I Melting Characteristics 2.32 Polymorphism 2.33 X-Ray Diffraction 2.34 Differential Scanning Calorimetry 2.35 Thermogravimetry 2.4 Solubility 2.5 Dissociation Constant 2.6 Partition Coefficients 2.7 Optical Rotation Synthesis Stability and Degradation Processes 4.1 InBulk 4 . 2 In Solution 4.3 In the Dosage Form Biopharmaceutics 5. I Pharmacokinetics 5.2 Metabolism Toxicology Analytical Methods 7. I Application of General Tests 7.2 Titration 7.3 Spectroscopic Methods 7.31 Infrared 7.32 Ultraviolet 7.33 Colorimetry 7.34 Nuclear Magnetic Resonance 7 . 4 Chromatography 7.41 Paper 7.42 Thin-Layer 7.43 Gas-Liquid 7.44 High Performance Liquid 7.5 Differential Scanning Calorimetry 7.6 Phase Solubility 7.7 Analysis of the Dosage Form 7.8 Determination in Body Fluids and Tissues References All 41

3. 4.

5.

6. 7.

8.

Copyright @ 1979 by Academic Press, Inc. rights of reproduction in any form reserved. ISBN 0 12-260808-9

48

DANIELLE A. GIRON-FOREST AND W. DIETER SCHONLEBER

1.

Introduction
1.1 History

The most valuable pharmacological p r o p e r t i e s of the pept i d e type e r g o t a l c a l o i d s induced a v a r i e t y of attempts of chemical d e r i v a t i s a t i o n of t h e p a r e n t compounds ( 1 , 2 + lit. quoted t h e r e i n ) . I n one of these the bromination of a-ergoc r y p t i n e l e d t o a product of h i g h l y i n t e r e s t i n g pharmacology, namely bromocriptine.
I t became known t o suppress p r o l a c t i n e s e c r e t i o n , and i t i s t h e r e f o r e a u s e f u l t o o l i n t h e treatment of p r o l a c t i n e dependent d i s o r d e r s , such a s g a l a c t o r r h e a a s s o c i a t e d with hyperprolactinemia and postpartum, a s w e l l as c e r t a i n kinds of s t e r i l i t y ( 3 - 9 ) . I n more e l e v a t e d doses, t h e drug i s a p o t e n t antiparkinsonicum. I n a d d i t i o n , t h e r e i s r e c e n t evidence of bromocriptine playing an important r o l e i n the trace heavy metals balance of t h e b r a i n ( 1 0 ) .

1.2

Name, Formula, Molecular Weight

Bromocriptine mesilate i s 2-bromo-a-ergocryptine methanesulphonate o r 2-bromo-12'-hydroxy-2'-(l-methylethyl)-5'-(2methylpropyl-5'a-ergotaman-3',6',18-trione methanesulphonate o r Bromocriptinum ( I N N ) . I t i s the a c t i v e i n g r e d i e n t i n ParlodelB dosage forms.

/..

CH3

x CH3S03H

BROMOCRIPTINE METHANESULPHONATE

49

Chemical A b s t r a c t s R e g i s t r y Numbers :
25614-03-3 (26409-15-4) ( 4783 0- 26- 2 )

Average Molecular Weights : base : mesilate: Average Formulas : base :

C

654.61 750.71

32 4 0

BrN 0 5 5

mesilate: C

H BrN 0 S 33 44 5 8

1 . 3 . Appearance, Colour, Odour Grey tinged white o r l i g h t yellow, f i n e l y c r y s t a l l i n e powder, odourless o r of weak, c h a r a c t e r i s t i c odour.
2.

Physicochemical P r o p e r t i e s
2.1

Elemental Analysis

The elemental a n a l y s i s of bromocriptine mesilate y i e l d e d t h e following r e s u l t s (11) : element

C
%

calculated
52.8 5.9 10.6 9.3 17.0 4.3

found
53.2 6.0 10.5 9.2 16.8 4.4

H Br N 0
S

2.2

Spectra

2.21 I n f r a r e d

The i n f r a r e d spectrum of bromocriptine m e s i l a t e i n a

KBr p e l l e t i s given i n f i g . 1. I t was recorded on a Perkin

Elmer 257 i n f r a r e d spectrophotometer. The main c h a r a c t e r i s t i c bands are t h e following

%Absorption

cr
rl .d

a,

ffl

BROMOCRIPTINE METHANESULPHONATE wave number ( c m 3200 1728

51

-1

assignment C-H-stretching v i b r a t i o n s C=O-stretching o f 5-membered r i n g lactam ( c y c l o l ) C=O-stretching o f 6-membered r i n g l a c t a m (amide I ) amide-11 band and C=C- st r e t c h i n g

2900

1642, 1672 1560

The s p e c t r u m of t h e b a s e h a s been r e p o r t e d by P.A. S t a d l e r and co-workers (11). 2.22 U l t r a v i o l e t The u l t r a v i o l e t spectrum o f b r o m o c r i p t i n e mesilate was r e c o r d e d on a P h i l i p s 1700 uv s p e c t r o p h o t o m e t e r i n 0 . 1 M met h a n o l i c methanesulphonic a c i d s o l u t i o n . I t i s g i v e n i n f i g . 2. A maximum o c c u r s a t a b o u t 308 n w i t h a l o g molar absorpm t i v i t y o f 4.0. I n 1:l dichloromethane/methanol s o l u t i o n t h e a b s o r p t i o n maximum was r e p o r t e d as 306 nm l o g = 3.988 (11). 2.23 F l u o r e s c e n c e Bromocriptine m e s i l a t e e x h i b i t s fluorescence l i k e the o t h e r l y s e r g i c and i s o l y s e r g i c a c i d type a l c a l o i d s . I n 2 p e r c e n t e t h a n o l i c t a r t a r i c a c i d s o l u t i o n , t h e e m i s s i o n maximum a p p e a r s a t 402 m ( e x c i t a t i o n a t 325 nm) See f i g . 3 .

2 . 2 4 P r o t o n Nuclear Magnetic Resonance The P R spectrum of b r o m o c r i p t i n e mesilate i n d e u t e r a t e d M d i m e t h y l s u l p h o x i d e a s o b t a i n e d on a Bruker HX-90 NMR s p e c t r o meter i s p r e s e n t e d i n f i g . 4. T S s e r v e d a s i n t e r n a l s t a n d a r d . M The c h a r a c t e r i s t i c s of t h e spectrum a r e g i v e n i n the f o l l o w i n g t a b l e (see a l s o 1 2 , 1 3 and lit. q u o t e d t h e r e i n ) : PMR-spectrum and Assignment Intensity Multiplicity Assignment
1 H 1 H 1 H

Chemical S h i f t downf i e l d (ppm) 11.85 10.45 9.55 7

Singlet Singlet Singlet

indole-Ng
I

NH CNOH

(18-19)

7.3

4H and

OH

52

DANIELLE A. GIRON-FOREST AND

w. DIETER SCHONLEBER

230

270

30 1

350

390

nm

Figure 2.

Ultraviolet Spectrum of Bromocriptine Mesilate in 0.1 M Methanolic Methanesulphonic Acid. CA = 0.05 mg/ml, C = 0.012 m g / m l . B Instrument: Philips SP 1700

B LOMI3CRIPTINE METHANESULPHONATE R

53

nm

Figure 3.

Fluorescence Spectrum of Bromocriptine Mesilate in 2 % Ethanolic Tartaric Acid. C = 65,ug/ml. Instrument: Perkin Elmer MPF-3

Figure 4.

PMR Spectrum of Bromocriptine Mesilate in (CD ) SO, 3 2 Instrument: Bruker HX-90 E

BROMOCRIPTINE METHANESULPHONATE

55

6.47 4.3

1H

Singlet

H-C

4.5 3.2
16 H

- 4.2

{
{

Triplet singlet Multiplet

- 5' 6-CH ((2-17) -3 g-C C ; -! ;H-C4; 8' 11' H-C5 ; F-C * H-C 7 ' 8 CH C0CK2CH3 ; H20 3 (recryst. solvent)
CH -SO H

H-C

2.37

2.1

2*1 1.5

3 H
8-9 H

Singlet Singlet Multiplet

-3

CH COCH CH

-3 2 3 5'-C12 ; 5'-Cg ; 2 ' - C g

; g-C 1 0 '

H-Cg,

0.75-1.15

12 H

Multiplet

2'CH

-3

5'-CH -3

and

CH3COCH CH

2 -3

2.25 Carbon-13 Magnetic Resonance Spectrum The spectrum o f b r o m o c r i p t i n e m e s i l a t e h a s been r e c o r d e d i n d i m e t h y l s u l p h o x i d e u s i n g a Bruker HX-90 NMR s p e c t r o p h o t o meter ( f i g . 5 ) . The a s s i g n m e n t o f t h e i n d i v i d u a l s i g n a l s i s g i v e n i n f i g . 6. 2.26 Mass Spectrum

The low r e s o l u t i o n e l e c t r o n impact mass s p e c t r a l p a t t e r n of bromocriptine m e s i l a t e ( f i g . 7 ) corresponds q u i t e n i c e l y t o t h o s e o f t h e o t h e r non- and d i h y d r o g e n a t e d e r g o t a l c a l o i d s ( 1 2 , 1 3 and lit. quoted h e r e i n ) . The m o l e c u l a r peak M of t h e b a s e shows up a t 653 and 655, r e s p e c t i v e l y , r e f l e c t i n g t h e n a t u r a l abundance o f t h e bromine i s o t o p e s , and M - 1 8 a t 635/637 mass u n i t s . A s i n t h e p a r e n t compounds t h e n e x t s m a l l e r f r a g m e n t a p p e a r s a t M-228, i n d i c a t i c g a s u b s t a n t i a l loss of t h e p e p t i d e s e c t i o n c o n s t i t u e n t s w i t h t h e t e n t a t i v e fragment s t r u c t u r e F1. From t h i s s p e c i e s t h e i s o p r o p y l group s p l i t s o f f e a s i l y t o y i e l d t h e l i n e p a i r a t 382/384 m a s s u n i t s . L o s s o f t h e e n t i r e p e p t i d e moiety l e a d s t o t h e f o r m a t i o n o f 2-bromo-lysergamide F2 a t 345/347 m.u., t h e mass s p e c t r a l b e h a v i o u r of which c o r r e s p o n d s t o t h a t o f t h e bromine-free compound. Thus, i t g i v e s r i s e t o peak p a i r s a t 300/302 and 299/301, r e s p e c t i v e l y , presumably by l o s s of formamide o r by t h e r u p t u r e of t h e t e t r a - h y d r o - p y r i d i n e r i n g ,

6000

4003

2000

3000

2000

>m
xa

'%a

rm

t
T
T
lb

4a c

y1

I t m

L
I
0

lbo

Figure 5 .

13-C-NMR Spectrum of Bromocriptine Mesilate in (CD ) SO, 3 2 Instrument: Bruker HX-90 E

57

100 90 80

'O

70

60
50
YO

30

20
10

36

I__
582
'25

635 653

1 " " " " ' 1 " " " ' " l ' " ' ' " " " " " ' ' ~

M/Z

100

200

300

900

500

600

700

Figure 7 .

Low Resolution Electron Impact Mass Spectrum of Bromocriptine Mesilate Instrument: CEC 21-llOB; Energy 70 eV, Ion Source 0 Temperature 160 - 200 C

BROMOCRIPTINE METHANESULPHONATE

59

a decay mode t h a t w a s proposed e a r l i e r (14,15) for l y s e r g i c a c i d d e r i v a t i v e s . Peak d o u b l e t s a t 285/287 and 274/276 m.u. i n d i c a t e fragments of i l l i c i t s t r u c t u r e s t i l l c o n t a i n i n g bromine.

425/427 m.u.
The p e p t i d e moiety i t s e l f (308 rn.u.1 obviously does n o t appear i n t h e spectrum, whereas i t s fragmentation p a t t e r n with peaks a t m/e 195 (F3), 167 (F3-CO), 155 - 153 (F4), 125 (FS), 86 (F6), and base peak 70 (F7) mass u n i t s , i s c l e a r l y understood (14,15) The m e s i l a t e shows a s i g n a l a t m/e 96.

I
195 m.u.

F3

F4
1 5 4 m.u

60

DANIELLE A. GIRON-FOREST AND W. DIETER SCHONLEBER

D0 ' q
F5
125 m.u. 86 m.u. 2.3 Crystal Properties 2.31 M e l t i n g C h a r a c t e r i s t i c s 2.32 Polymorphism

F7
70 m.u.

Bromocriptine m e s i l a t e decomposes between 180 and 200 O C , t h u s a m e l t i n g p o i n t o r m e l t i n g r a n g e c a n n o t be g i v e n .

I n v e s t i g a t i o n s f o r t h e o c c u r r e n c e o f polymorphism have been u n d e r t a k e n by i r s p e c t r o s c o p y , d i f f e r e n t i a l s c a n n i n g c a l o r i m e t r y and x-ray powder d i f f r a c t i o n ( G u i n i e r - d e W o l f f ) . N o polymorphism h a s been observed so f a r . An amorphous form may be p r e p a r e d a r t i f i c i a l l y by r a p i d e v a p o r a t i o n o f a methanolic s o l u t i o n of t h e drug substance. 2.33 X-Ray D i f f r a c t i o n X-ray s t r u c t u r a l a n a l y s i s o f b r o m o c r i p t i n e , c r y s t a l l i z e d a s t h e b a s e from dichloromethane/diethylether, h a s been carr i e d o u t on a CAD 4 - d i f f r a c t o m e t e r w i t h CuKa-radiation ( 1 6 ) . 3371 r e f l e x i o n s were w i t h i n s i n @/A<0.62 8-l. Assessment o f t h e s t r u c t u r e was a c h i e v e d by computation t o a r e f i n e m e n t o f R = 0.033 f o r t h e a b s o l u t e c o n f i g u r a t i o n . The c r y s t a l d a t a w e r e : Space g r o u p P 2 a = 10.681, 1 ' b = 13.454, c = 11.049, @ = 9 9 - 7 5 " , Z = 2. I n f i g . 8 t h e r e s u l t a n t conformation of bromocriptine base i s depicted. 2.34 D i f f e r e n t i a l Scanning C a l o r i m e t r y The D C thermogram o f b r o m o c r i p t i n e m e s i l a t e , o b t a i n e d S w i t h a P e r k i n E l m e r DSC-2 i n s t r u m e n t a t a h e a t i n g r a t e o f 20 'C/min. and i n a n i t r o g e n atmosphere, i s shown i n f i g . 9.

2a

Figure 8.

Conformation of Broaocriptine Base established on the Basis of the X-ray Structural Analysis.
=

Nitrogen, Hydrogen

= oxygen,

= Carbon,

62

DANIELLE A. GIRON-FOREST AND W. DIETER SCHONLEBER

TG

\
T

40

60

80

100

120

140

160

180

200

O C

Figure 9.

Differential Scanning Calorimetry and. Thermogravimetry Curves of Bromocriptine Mesilate, each at a heating rate of 200C/min. and a Nitrogen Flow of 15 ml/min. Instruments: Perkin Elmer DSC-2 Perkin Elmer TGS-1

BROMOCRIF'TINE METHANESULPHONATE

63

The m e l t i n g endotherm i s f o l l o w e d immediately by a s t r o n g exothermic d e g r a d a t i o n . S i n c e b r o m o c r i p t i n e mesilate decomposes under m e l t i n g , the t r a n s i t i o n t e m p e r a t u r e i s s t r o n g l y dependent on the h e a t i n g r a t e . A b r o a d b u t weak endotherm between 40 and 100 "C i n d i c a t e s t h e v o l a t i l i z a t i o n o f s o r b e d r e c r y s t a l l i z a t i o n s o l v e n t ( u s u a l l y butanone-2, see s e c t i o n 3 ) . 2.35 Thermogravimetry The thermogram o f b r o m o c r i p t i n e m e s i l a t e , c a r r i e d o u t on a P e r k i n E l m e r TGS-1 thermobalance, i s g i v e n i n f i g . 9. The sample t e m p e r a t u r e w a s r a i s e d a t a r a t e o f 20"C/minute m a i n t a i n i n g a n i t r o g e n atmosphere. A c o n s i d e r a b l e loss o f weight, a t t r i b u t e d t o a loss of sorbed s o l v e n t ( s . above), i s observed below 130 'C. Sample decomposition o b v i o u s l y s t a r t s a f t e r melting. 2.4. Solubility

The s o l u b i l i t y of b r o m o c r i p t i n e mesilate w a s d e t e r m i n e d i n a v a r i e t y of s o l v e n t s e q u i l i b r a t e d by v i b r a t i o n d u r i n g 2 4 h o u r s a t 25 OC. They are a s f o l l o w s : Solvent S o l u b i l i t y i n mg/g

0.8

S o l u b i l i t y i n g/100 m l
0.08 72 1.8 0.1 0.12 0.015 0.015 0.06 <0.02

water m e t h a no 1 ethanol 2-propanol a c e t o n i tr i l e acetone ethyl acetate chloroform benzene hexane

910
23.0 1.2 1.6 0.2 0.2 0.45

<0.1 <<0.1

<<0.01

A t 22 ?r 2 OC b r o m o c r i p t i n e m e s i l a t e d i s s o l v e s r e a d i l y ( > 2 % ) i n p r o p y l e n e g l y c o l , 50 % e t h a n o l , 95 % e t h a n o l and n - o c t a n o l , whereas i t i s p o o r l y s o l u b l e (<0.1 % ) i n s i m u l a t e d g a s t r i c and i n t e s t i n a l f l u i d s a t 37 ? 2 "C.

2.5

Dissociation Constant

Due t o t h e low s o l u b i l i t y o f b r o m o c r i p t i n e m e s i l a t e i n water, t h e p K v a l u e had t o be determined i n methyl c e l l o a s o l v e / w a t e r 8: 2 (w/w) T i t r a t i o n a t ambient t e m p e r a t u r e y i e l d 0.05 f o r a 0.0078 M s o l u t i o n . e d p K a a s 4.90

64

DANIELLE A . GIRON-FOREST AND

w. DIETER SCHONLEBER

2.6

P a r ti t i o n C o e f f i c i e n t s

The p a r t i t i o n c o e f f i c i e n t s of b r o m o c r i p t i n e mesilate between water of pH 1 . 2 and n - o c t a n o l o n t h e o n e hand, and water of pH 7.5 and n - o c t a n o l o n t h e o t h e r , h a v e b e e n determined a t 37.0 ? 0 . 5 "C.

water p H 1 . 2 / n - o c t a n o l water pH 7.5/n-octanol

2.7

1 : 90 1 : 235

Optical R o t a t i o n

T h e o r e t i c a l l y , w i t h 6 c h i r a l c e n t e r s w i t h i n the m o l e c u l e ( a t 6 , 9 , 2 ' , 5 ' , 11' and 1 2 ' p o s i t i o n s ) 64 d i a s t e r e o m e r i c forms are p o s s i b l e . However, b r o m o c r i p t i n e i s s t e r i c a l l y w e l l d e f i n e d a t a l l o f t h e s e p o s i t i o n s , a s it i s d e r i v e d from the n a t u r a l l y occurring a-ergocryptine. The s p e c i f i c o p t i c a l r o t a t i o n of b r o m o c r i p t i n e mesilate i n d i f f e r e n t s o l v e n t s i s g i v e n below f o r 20 C corrected f o r l o s s on d r y i n g . A P e r k i n E l m e r p o l a r i m e t e r 241 w a s u s e d , the a c t u a l c o n c e n t r a t i o n b e i n g 1 0 mg/ml. wavelength (nm) solvent 589 578 546 436 365

s p e c i f i c o p t i c a l r o t a t i o n i n degrees dichloromethane/ methanol 1:l ethanol d i m e t h y l formamide 101.0 100.6 127.4 107.5 107.0 135.2 130.1 129.6 162.0 327.8 328.7 388 .O 329.4 331.3 392.7 and - i n i n e

F o r t h e s p e c i f i c r o t a t i o n of 2-bromo-a-ergocryptine bases see (11). 3. Synthesis

B r o m o c r i p t i n e b a s e i s manufactured by b r o m i n a t i o n of a - e r g o c r y p t i n e w i t h N-bromosuccinimide i n d i o x a n e s o l u t i o n . The mesilate i s t h e n formed by a d d i t i o n of methanesulphonic a c i d . The s a l t i s r e c r y s t a l l i z e d from butanone-2 ( s . f i g . 1 0 ) (2,3111)

BROMOCRIPTINE METHANESULPHONATE

65

a-Ergocryptine Base

N-Bromo-succinimide

Bromocriptine Base

Bromocriptine Methanesulphonate

Methanesulphonic Acid

Figure 10.

Route of Synthesis

66

DANIELLE A. GIRON-FOREST AND W. DIETER SCHONLEBER

S t a b i l i t y and Degradation Processes

4 .

P o s s i b l e degradation pathways of e r g o t a l c a l o i d s a t t h e example of ergotamine t a r t r a t e have been c l e a r l y summarized by H. Bethke e t a l . ( 1 7 ) and B. Kreilgaard (13).
A s a nonhydrogenated e r g o t a l c a l o i d , bromocriptine i s r e l a t i v e l y s e n s i t i v e t o autoxidation both i n s o l i d and i n d i s solved states, b u t degradation products have not y e t been e l u c i d a t e d . S i m i l a r i t y t o t h e o x i d a t i v e transformation of t h e p a r e n t compounds i s t o be s t r o n g l y a n t i c i p a t e d ( 1 2 ) .

I n hydroxyl-containing s o l v e n t s , nonhydrogenated e r g o t p e p t i d e a l c a l o i d s a r e r e a d i l y epimerized a t C-8 t o an e q u i l i b r a t e d mixture of the l y s e r g i c and i s o - l y s e r g i c a c i d series , c a l l e d t h e -ine/-inine forms (-ine = 88, - i n i n e = 8 a ) ( 1 2 , 1 8 and lit. quoted t h e r e i n ) . The h y d r o l y s i s of t h e l y s e r g i c a c i d amide bond i s of minor importance f o r t h e beginning degradat i o n , b u t , of course, p r e v a l e n t under more d r a s t i c h y d r o l y s i s conditions. Under t h e same conditions, b u t a t e l e v a t e d temperatures, t h e C-2I-center i s very l i k e l y t o be i n v e r t e d ( a c i - i n v e r s i o n ) y i e l d i n g a more a c i d i c isomeric compound.

The light-induced a d d i t i o n of water t o t h e 9,lO-double bond of bromocriptine y i e l d i n g t h e so-called lumi-products, i s of high p r o b a b i l i t y ( 1 2 , 1 8 ) . They have, however, n o t y e t been i s o l a t e d o r c h a r a c t e r i z e d . The corresponding l0a-methoxylumi-derivative could be prepared by t h e photo-catalyzed a d d i t i o n of methanol (19) under s l i g h t l y a c i d i c conditions. 4.1

i n Bulk

Although bromocriptine m e s i l a t e i s s e n s i t i v e t o h e a t and l i g h t , i t i s s t a b l e f o r up t o 3 y e a r s a t ambient temperat u r e ( 2 0 ) when s t o r e d i n s e a l e d polyethylene bags contained i n twist-off amber g l a s s b o t t l e s . I n warm and t r o p i c a l climat e (30 "C/75 p e r c e n t r e l a t i v e humidity) and under i d e n t i c a l package c o n d i t i o n s , it i s s t a b l e f o r 1 y e a r , however o n l y f o r 3 months a t 50 "C. I f not properly p r o t e c t e d , bromocriptine adsorbs up t o 6 p e r c e n t by weight of water i n t r o p i c a l climate.

BROMOCRIPTINE METHANESULPHONATE

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4.2

i n Solution

Bromocriptine follows t h e behaviour sketched above and i s t h u s r a t h e r l a b i l e i n aqueous o r aqueous/alcoholic s o l u t i o n s , p a r t i c u l a r l y i n t h e presence of a c i d , y i e l d i n g mainly t h e e q u i l i b r a t e d mixture with i t s 8-epimer (1) and t o a smaller e x t e n t , i t s h y d r o l y s i s products 2-bromo-lysergamide and 2-bromo-lysergic a c i d and t h e i r 8a-isomers, r e s p e c t i v e l y . 4.3 i n t h e Dosage Form

Bromocriptine m e s i l a t e i s marketed a s ParlodelB c a p s u l e s ( 1 0 m f o r t h e treatment of Parkinson's d i s e a s e ) and t a b l e t s g ( 2 . 5 m a s l a c t a t i o n s u p p r e s s o r ) . Both forms have proved g s t a b l e a t l e a s t f o r 4 y e a r s when s t o r e d a t ambient temperature i n amber g l a s s b o t t l e s ( 2 2 ) . 5. Biopharmaceutical Aspects (23) 5.1. Pharmacokinetics

The d i s p o s i t i o n of bromocriptine has been s t u d i e d i n s e v e r a l animal s p e c i e s and man following s i n g l e o r a l and intravenous a d m i n i s t r a t i o n of t h e drug l a b e l l e d with e i t h e r t r i t i u m o r carbon-14. The e n t e r a l absorption of bromocriptine from an aqueous s o l u t i o n amounts t o 30 - 40 8 as determined from t h e sum of t h e cumulative b i l i a r y and u r i n a r y e x c r e t i o n of r a d i o a c t i v i t y ( p a r e n t drug + m e t a b o l i t e s ) i n b i l e d u c t cannulated animals. The blood l e v e l s following o r a l and intravenous doses a r e very low i n a l l animal s p e c i e s . This, most l i k e l y , i s due t o t h e marked a f f i n i t y of t h e drug f o r v a r i o u s t i s s u e s and t h e r a p i d h e p a t i c e x t r a c t i o n of t h e absorbed f r a c t i o n . The main r o u t e of e x c r e t i o n i s t h e b i l e . L e s s than 5 % of t h e dose a r e recovered i n t h e u r i n e of i n t a c t animals a f t e r o r a l o r i n t r a venous a d m i n i s t r a t i o n . I n man, t h e e x t e n t of e n t e r a l absorption i s estimated t o be a t l e a s t of t h e same order of magnitude than determined f o r animals. Absorption i s r a p i d with an approximate r a t e c o n s t a n t of 1 . 4 h-' ( t '/2 = 30 min)

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DANIELLE A. GIRON-FOREST AND W. DIETER SCHONLEBER

Peak plasma l e v e l s are reached about 1 . 5 h a f t e r o r a l i n g e s t i o n , t h e maximum c o n c e n t r a t i o n s being i n t h e o r d e r of 2 - 3 ng equivalents/ml ( p a r e n t drug + m e t a b o l i t e s ) f o r an g o r a l 1 m dose. The e l i m i n a t i o n from t h e plasma i s b i p h a s i c and proceeds with mean h a l f - l i v e s of 6 h (a-phase) and 50 h (6-phase). S i m i l a r e l i m i n a t i o n h a l f - l i v e s are obtained from t h e u r i n a r y e x c r e t i o n . The cumulative r e n a l e x c r e t i o n i s p r a c t i c a l l y t h e same a f t e r o r a l and intravenous a d m i n i s t r a t i o n 7 % of t h e r a d i o a c t i v i t y dosed. The main and amounts t o 6 p o r t i o n of t h e dose, e i t h e r o r a l or intravenous, i s e l i m i n a t e d by t h e b i l i a r y r o u t e i n t o t h e faeces. The k i n e t i c s of bromoc r i p t i n e has been demonstrated t o be l i n e a r i n t h e o r a l dose range from 2.5 t o 7 . 5 mg.

P a r e n t drug i s bound t o bovine and human plasma p r o t e i n s t o an e x t e n t of 89 - 96 % ( i n v i t r o c o n c e n t r a t i o n range 0.2 80 pg/ml) and e x e r t s a pronounced a f f i n i t y to v a r i o u s t i s s u e s . 5.2 Metabolism

Bromocriptine i s r a p i d l y and completely metabolised i n animals and man. The major components of t h e u r i n a r y metabol i t e s have been i d e n t i f i e d as 2-bromo-lysergic a c i d and 2-bromo-isolysergic a c i d . Apart from t h e h y d r o l y t i c cleavage of t h e amine bond and t h e isomerization a t p o s i t i o n 8 of t h e l y s e r g i c a c i d moiety, a t h i r d p r i n c i p a l biotransformation pathway cons i s t s i n t h e o x i d a t i v e a t t a c k of t h e molecule a t t h e p r o l i n e fragment of t h e peptide p a r t , predominantly a t p o s i t i o n 8 ' , giving r i s e t o t h e formation of a number of hydroxylated and f u r t h e r oxidized d e r i v a t i v e s of bromocriptine, and i n a d d i t i o n of conjugated d e r i v a t i v e s t h e r e o f . 6. Toxicology

The a c u t e t o x i c i t y of bromocriptine mesilate h a s been determined i n t h e mouse a s 230 and 2620 mg/kg i . v . and P.o., r e s p e c t i v e l y . I n t h e r a b b i t , t h e corresponding values w e r e 1 2 and >lo00 mg/kg ( 2 ) . Thus, bromocriptine proved less t o x i c than t h e nonhydrogenated e r g o t a l c a l o i d s by one o r d e r of magnitude, resembling t h e behaviour of t h e dihydrogenated derivatives. Chronic t o x i c i t y s t u d i e s were c a r r i e d o u t with r a t s , dogs, and rhesus monkeys ( 2 ) . I n n e a r l y a l l c a s e s , the p r i n c i p a l e f f e c t produced w a s ischemia of some p a r t of t h e body. The well-known e m e t i c e f f e c t of e r g o t a l c a l o i d s i n dogs w a s p a r t i c u l a r l y pronounced with bromocriptine, even with o r a l doses of a s low a s 0 . 1 mg/kg.

BROMOCRIPTINE METHANESULPHONATE

69

7.

A n a l y t i c a l Methods 7.1 Application of General T e s t s

Compared with t h e u n s u b s t i t u t e d e r g o t a l c a l o i d s (1,181 t h e i n t r o d u c t i o n of bromine i n t o t h e 2-position of t h e indole nucleus diminishes t h e r e a c t i v i t y of t h e molecule i n r e s p e c t t o general a l c a l o i d t e s t s . Nevertheless, they s t i l l remain applicable. 1. KeLlgrLs-tgsL. Bromocriptine m e s i l a t e i s d i s s o l v e d i n g l a c i a l a c e t i c a c i d t o which have been added t r a c e s of f e r r i c c h l o r i d e . A f t e r c a r e f u l l y l a y e r i n g with concentrated s u l f u r i c a c i d , a green colour i s produced a t t h e i n t e r f a c e . U s u a l l y an i n t e n s e b l u e - v i o l e t colour occurs with 2-H-substituted compounds. 2 . I a n grk's gegcLion, ( 2 4 ) Bromocriptine m e s i l a t e i s dissolved i n methanol. After a d d i t i o n of van U r k ' s r e a g e n t and vigorous shaking a blue colour develops slowly, which i s s u b s t a n t i a l l y weaker than with 2-H-ergot a l c a l o i d s .

3. Meyeg's t e z t L Bromocriptine m e s i l a t e i s d i s s o l v e d i n methanol/water 3:lO. After a d d i t i o n of one drop of d i l u t e d hydrochloric a c i d and one drop of Meyer's reagent (mercuric potassium iodide) and s h a k b g , a white p r e c i p i t a t e i s produced.
7.2
Titration

Bromocriptine m e s i l a t e may be assayed i n g l a c i a l a c e t i c a c i d / a c e t i c anhydride 1 : 7 by t i t r a t i o n with 0 . 1 N p e r c h l o r i c a c i d . The endpoint may be determined p o t e n t i o m e t r i c a l l y using a glass/calomel e l e c t r o d e system. The methanesulphonic a c i d c o n t e n t of bromocriptine m e s i l a t e i s u s u a l l y determined by t i t r a t i o n with 0 . 1 N methanolic potassium hydroxide. The endpoint may be d e t e c t e d p o t e n t i o m e t r i c a l l y using a glass/calomel e l e c t r o d e system. Residual N-bromosuccinimide from t h e manufacturing process may be i d e n t i f i e d and/or q u a n t i f i e d by making use of i t s o x i d a t i o n p o t e n t i a l by t i t r a t i o n of l i b e r a t e d i o d i n e a f t e r a d d i t i o n of potassium iodide i n a c e t i c a c i d ( 2 5 ) .

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DANIELLE A. GIRON-FOREST AND W. DIETER SCHONLEBER

7.3

Spectroscopic Methods

7.31 Infrared
I n f r a r e d spectroscopy i s u t i l i z e d f o r i d e n t i f i c a t i o n purposes during t h e a n a l y s i s of t h e drug substance. (see 2 . 2 1 ) 7.32 U l t r a v i o l e t Spectrophotometric a n a l y s i s of bromocriptine m e s i l a t e i s c a r r i e d o u t d i r e c t l y using t h e uv maximum a t about 305 run i n methanolic methanesulphonic a c i d . However, t h e method i s n o t very s p e c i f i c .
I t was p r e f e r r e d t o f i r s t s e p a r a t e t h e i m p u r i t i e s from bromocriptine by t h i n l a y e r chromatography and then t o i s o l a t e t h e substance by e l u t i o n from t h e s i l i c a g e l of t h e p l a t e with methanol. The i n t a c t a c t i v e i n g r e d i e n t i s measured i n 0.01 M methanolic methanesulphonic a c i d ( 2 6 ) .

7 . 3 3 Colorimetry
I n moderately a c i d i c s o l u t i o n s bromocriptine m e s i l a t e r e a d i l y forms i o n p a i r s with a n i o n i c dyes such a s p i c r i c a c i d , bromothymol blue, methyl orange, which a r e e x t r a c t a b l e with an organic s o l v e n t . A procedure has been developed both f o r d i r e c t assay and f o r assay following chromatographic separat i o n from t h e i m p u r i t i e s . Therein bromocriptine m e s i l a t e i s allowed t o r e a c t with bromothymol blue a t p 2.5. The r e s u l H t i n g i o n p a i r i s then e x t r a c t e d with benzene and i t s concent r a t i o n determined a t 410 nm ( 2 5 ) . 7.34 Proton Magnetic Resonance
PMR spectroscopy may be used f o r i d e n t i f i c a t i o n of t h e drug substance. ( s e e 2 . 2 4 )

7.4

Chromatography

7 . 4 1 Paper
Paper chromatography was applied formerly t o t h e d e t e r mination of bromocriptine. conditions: system 1: s t a t i o n a r y phase: 25 % formamide mobile phase : carbon t e t r a c h l o r i d e / d i e t h y l e t h e r 1:l

BROMOCRIPTINE METHANESULPHONATE

71

system 2 : s t a t i o n a r y phase: 10 % dimethylphthalate mobile phase : dimethylformamide/ 0 . 5 N hydrochloric a c i d 15:85 The drug substance i s v i s u a l i z e d by r e a c t i o n with i o d i n e vapour

Rf values of bromocriptine and precursor system 1 Bromocriptine a- ergocr yp t i n e (precursor) 7 . 4 2 Thin Layer The r e l a t i v e i n s t a b i l i t y of bromocriptine makes it very d i f f i c u l t t o avoid a r t i f a c t formation i n t e s t s o l u t i o n s o r during s p o t t i n g on t h e p l a t e . Therefore, bromocriptine mesil a t e , u s u a l l y d i s s o l v e d i n chloroform/methanol 1:1, has t o be s p o t t e d on t h e p l a t e very r a p i d l y and with t h e exclusion of l i g h t . The s e p a r a t i o n being terminated, t h e mobile phase i s removed by means of high vacuum f o r 30 minutes. A g r e a t number of t l c systems have been i n v e s t i g a t e d f o r t h e s e p a r a t i o n of by-products, degradation products, metabol i t e s , and e x c i p i e n t s . Also a v a r i e t y of spraying r e a g e n t s have been t e s t e d ( s e e t a b l e ) . The most advantageous one w a s Dragendorff's r e a g e n t with consecutive spraying by 30 % hydrogen peroxide. methods: s t a t i o n a r y phase: s i l i c a g e l 60 F254, Merck t l c p l a t e s , no.5715 mobile phase 1
:

system 2

0.88 0.70

0.09

0.31

(27)

dichloromethane/dioxane/96 p e r c e n t
ethanol/conc.ammonia (v/v/v/v) 180:15:5:0.1

mobile phase 2 mobile phase 3

chloroform/methanol/formic a c i d 7 8 :20 : 2 (v/v/v)

chloroform/96 p e r c e n t ethanol/conc. ammonia 192:8:0.35 (v/v/v)

I n the procedure with mobile phase 1 and 2 , t h e visual i z a t i o n i s accomplished by screening under uv l i g h t (254 and 366 nm) and i n a d d i t i o n by spraying with Dragendorff's reagent, modified by Munier and Deboeuf, followed by 30 perc e n t hydrogen peroxide.

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DANIELLE A. GIRON-FOREST AND W. DIETER SCHONLEBER

values st mobile phase 1 mobile phase 2
R

compound bromocr i p t i n e

2-bromo-a-ergocryptinine

1 . 0 (Rf 0.5) 1.5

1 . 0 (Rf 0.7) 1.1

1.0 0.5 0.25 0.35 0.15 0.35

a-ergocryptinine (isomer of precursor) a-ergocryptine (precursor) 2-bromo-lysergic a c i d 2-bromo-isolysergic a c i d 2-bromo-lysergamide 2-bromo-isolysergamide

1.4 0.6 0.0

0.0
0.5 0.5

Mobile phase 3 may be used f o r t h e d e t e c t i o n and s e m i q u a n t i t a t i v e determination of r e s i d u a l N-bromosuccinimide. Thereby, t h e p l a t e i s sprayed with water and then placed i n a c h l o r i n e atmosphere f o r 10 minutes. Excess c h l o r i n e i s removed by p l a c i n g t h e p l a t e i n a stream of w a r m a i r f o r another 1 0 minutes. A f t e r spraying with o - t o l u i d i n e reagent, the eval u a t i o n i s made a g a i n s t a d i l u t i o n of N-bromosuccinimide. i s 0.3 with r e s p e c t t o The d e t e c t i o n l i m i t i s 0 . 1 pg, t h e R st bromocriptine R f value.
A high performance t l c system has been developped f o r t h e in-process-control (28) using Merck s i l i c a g e l HPTLC p l a t e s , no. 5628, a s t h e s t a t i o n a r y phase and tetrahydrofuran/ chloroform/n-heptane/methanol/conc. ammonia 20:20:57:7:1 p e r m volume a s t h e mobile phase. The chromatography ( 6 c ascending) i s c a r r i e d o u t without preceding chamber s a t u r a t i o n . The compounds separated a r e v i s u a l i z e d by uv a t 254 and 366 nm, r e s p e c t i v e l y , and by i o d i n e vapour. Rf of bromocriptine i s 0.27. The p o t e n t i a l by-products y i e l d s p o t s a t R 1.5 s (2-bromo-a-ergocryptinine) , and 0.55 (a-ergocryptiney 2-Bromo-lysergic a c i d remains a t the s t a r t i n g p o i n t .

Beside t h e modified Dragendorff's reagent/hydrogen peroxide a l r e a d y mentioned, a v a r i e t y of spraying reagents has been used f o r t h e v i s u a l i z a t i o n of bromocriptine. They a r e compiled i n t h e following l i s t .

BROMOCRIPTINE METHANESULPHONATE

reagent

spraying r e a g e n t s f o r bromocriptine detection l i m i t colour on s i l i c a g e l Merck no. 5715

Dragendorff (Munier,Deboeuf) + 30 p e r c e n t H 2 0 2 0.05 p e r c e n t aqueous permangana t e van U r k ' s r e a g e n t cinnamic aldehyde formaldehyde/hydrochloric acid formaldehyde/sulphuric a c i d chlorine/o-toluidine folin/ammonia ninhydrin/uv d e t . 360 iodine/potassium i o d i d e E h r l i c h ' s reagent

brown yellow grey yellow grey violet violet grey red brown orange

The i d e n t i t y of methanesulphonic a c i d may be determined by t l c on c e l l u l o s e p l a t e s , Merck no. 5728, with ethanol/ water/conc. ammonia 80:16:4 (v/v/v) a s a mobile phase. Detection i s achieved by spraying with acid-base i n d i c a t o r s , e.g. bromocresol green o r s i m i l a r s p e c i e s . Rf of methanesulphonic a c i d i s 0.5 ( t h a t of bromocriptine base = 0.9). 7.43 G a s Chromatography
GC cannot be a p p l i e d t o t h e a n a l y s i s of bromocriptine m e s i l a t e due t o i t s low v o l a t i l i t y and i t s thermal i n s t a b i l i t y . A procedure according t o 29 o r 30, which claims e x c e l l e n t i d e n t i f i c a t i o n and q u a n t i t a t i o n on t h e b a s i s of well-defined p e p t i d e s e c t i o n p y r o l y s i s products, h a s n o t y e t been attempted. However, GC i s very u s e f u l determining t h e r e s i d u a l r e c r y s t a l l i z a t i o n s o l v e n t butanone-2. The c o n d i t i o n s a r e t h e following:

Column : Porapak Q 80/100 mesh, i n 1.8 m x 2mm g l a s s Temperatures: i n j e c t o r 240 OC, column 130 O C flame i o n i z a t i o n d e t e c t o r , 240 OC. 7.44 High Performance Liquid
A s e r i e s of HPLC systems have been u t i l i z e d both f o r assay and p u r i t y of bromocriptine m e s i l a t e . S a t i s f a c t o r y procedures have been accomplished both on s t r a i g h t phase ( s i l i c a g e l ) and on reversed phase ( o c t a d e c y l s i l a n i s e d s i l i c a g e l ) columns.

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DANIELLE A. GIRON-FOREST AND W. DIETER SCHONLEBER

HPLC-conditions s rai h -t - -g- t-phase: -s t a t i o n a r y phase: s i l i c a g e l , 5 pm, i n s t a i n l e s s s t e e l 25 c x 3 mm i.d. m mobile phase: water-saturated dichloromethane/methanol 100:2 (v/v) uv d e t e c t i o n a t 254 nm. F i g . 1 shows a chromatogram of bromocriptine mesilate spiked 1 w i t h dodecylbenzene, p o t e n t i a l by-products and t h e p r e c u r s o r . The flow was 1.7 ml/minute ( 3 1 ) . Key: 1 = dodecylbenzene, 2 = bromocriptinine, 3 = a-ergocrypt i n i n e , 4 = bromocriptine, 5 = a-ergocryptine ( p r e c u r s o r ) .

- - - - -phase, reversed - -

z sel I yt?_:

s t a t i o n a r y phase: Merck F@-18, 1 0 pm i n s t a i n l e s s s t e e l 25 c m x 4.6 mm i . d . mobile phase: g r a d i e n t : 35 t o 60 p e r c e n t B w i t h i n 60 min. A = water, 0 . 2 p e r c e n t t r i e t h y l a m i n e added B = a c e t o n i t r i l e , 0.2 p e r c e n t t r i e t h y l a m i n e added. uv d e t e c t i o n a t 280 nm. This system was found optimal f o r t h e p u r i t y t e s t . Fig. 1 2 shows a chromatogram of t h e drug substance spiked with p o t e n t i a l by-products and the precursor. The flow was s e t a t 4.0 ml/minute. Key: I = 2-bromo-a-ergocryptinine, I1 = a-ergocryptinine, 1 1 = a-ergocryptine ( p r e c u r s o r ) , I V = bromocriptine, 1 V = 2-bromo-lysergamide, V I = 2-bromo-lysergic a c i d , V I I = 2-bromo-isolysergamide.

---reversed-p&azeL ZyEtgm-II-

(26) :

s t a t i o n a r y phase: Merck RP-18, 10 pm i n s t a i n l e s s s t e e l , 25 c x 4.6 mm m mobile phase: 0.01 M ammonium carbonate o r 0.05 M ammonium hydrogen carbonate s o l u t i o n / a c e t o n i t r i l e 35:65 i s o c r a t i c , flow 1 . 5 ml/min.

BROMOCRIPTINE METHANESULPHONATE

75

I
Figure 1 . 1

High Performance Liquid Chromatogram of Bromocriptine Mesilate, spiked with Dodecylbenzene, Precursor and p o t e n t i a l By-products. Adsorption Mode, i s o c r a t i c , Uv-detection a t 254 nm

76

DANIELLE A. GIRON-FOREST AND W. DIETER SCHONLEBER

1
c

c
2
4

L
14

L
16 18

10

12

20 min

Figure 1 2 .

High Performance Liquid Chromatogram of Bromocriptine M e s i l a t e , spiked w i t h Precursor and p o t e n t i a l By-products. Reversed-phase Mode, Solvent Gradient, Uv-detection a t 280 nm.

BROMOCRIF'TINE METHANESULPHONATE

71

7.5

D i f f e r e n t i a l Scanning Calorimetry

D i f f e r e n t i a l scanning c a l o r i m e t r y cannot be a p p l i e d t o q u a n t i f y t h e p u r i t y of t h e drug substance f o r t h e reasons mentioned i n 2.31 and 2.34. However, i t may be v a l u a b l e i n q u a l i t a t i v e comparisons from sample t o sample o r batch t o batch on t h e b a s i s of t h e i r corresponding d i f f e r e n t i a l scanning c a l o r i m e t r y p a t t e r n s . 7.6 Phase S o l u b i l i t y Analysis

For phase s o l u b i l i t y a n a l y s i s , a c e t o n i t r i l e appears t o be t h e most s u i t a b l e s o l v e n t . A t y p i c a l p l o t i s given i n f i g u r e 13 along w i t h t h e experimental conditions. 7.7 Analysis of t h e Dosage Form

The i d e n t i f i c a t i o n o f bromocriptine m e s i l a t e i n t h e dosage form c a n be c a r r i e d o u t by t h i n l a y e r chromatography using Merck p l a t e s with dichloromethane/methanol/formic a c i d 78:20:2 (v/v/v) and subsequent u v - v i s u a l i z a t i o n a t 254 and 360 run. Using t h i s method, i t i s important t o only a i r - d r y t h e s p o t a f t e r a p p l i c a t i o n t o t h e p l a t e , s i n c e more vigorous evaporation of t h e s o l v e n t w i l l g i v e r i s e t o a r t i f a c t s ( 3 2 ) . Bromocriptine can a l s o be i d e n t i f i e d a s t h e base by i r spectroscopy a f t e r e x t r a c t i o n from t h e dosage form with ethanol and removal of t h e s o l v e n t , both i n s o l u t i o n and i n a KBr p e l l e t ( 3 3 ) . Bromocriptine m e s i l a t e i n P a r l o d e l a t a b l e t s may be assayed i n a non-specific way by d i r e c t uv-spectrophotometry following e x t r a c t i o n with methanol ( 3 2 )

Fluorimetry i n 0 . 1 N hydrochloric a c i d has been a p p l i e d during t h e measurement of d i s s o l u t i o n r a t e of t h e dosage forms with e x c i t a t i o n a t 335 and emission a t 425 nm, r e s p e c t i v e l y (26)

A s p e c i f i c a s s a y of bromocriptine m e s i l a t e i n t h e dosage form may be c a r r i e d o u t by t l c followed by uv-spectrophotometry ( 2 6 ) (The system can a l s o s e r v e f o r i d e n t i f i c a t i o n p u r p o s e s ) . The drug substance i s e x t r a c t e d with methanol i n t h e absence of l i g h t , t h e chromatographic c o n d i t i o n s a r e : Merck p l a t e s F 254, mobile phase: dichloromethane/dioxane/ ethanol abs./conc. ammonia 180:15:5:0.1 per volume. The s p o t corresponding t o bromocriptine i s e x t r a c t e d with m methanol, and t h e c o n c e n t r a t i o n i s determined a t about 300 n by spec tropho tometry.

78

DANIELLE A. GIRON-FOREST AND

w. DIETER SCHONLEBER

SYSTEfl COMPOSITION: MG OF SAMPLE PER

G OF SOLVENl

Figure 13.

Phase Solubility Analysis Plot of Bromocriptine Mesilate, dried in High Vacuum for 15 Hours. Solvent: dry Acetonitrile, Vibration for 24 hrs. in the Absence of Light. Slope 1.41~0.05 %.

BROMOCRIF'TINE METHANESULPHONATE

79

A f u r t h e r s p e c i f i c assay i s t h e HPLC-determination of bromocriptine m e s i l a t e following e x t r a c t i o n with methanol from t h e dosage form using RP-18 a s t h e s t a t i o n a r y and aceton i t r i l e / O . O l M ammonium carbonate s o l u t i o n 65:35 a s t h e mobile phase. Uv-detection wavelength i s s e t a t 300 nm ( 2 6 ) .

For t h e d e t e c t i o n and e s t i m a t i o n of degradation products

i n dosage forms, a s o l v e n t g r a d i e n t , c o n t a i n i n g t h e components mentioned above, i s u t i l i z e d with advantage.

7.8
Determination i n Body F l u i d s and T i s s u e s ( 2 3 )

Due t o i t s p o t e n t e f f i c a c y i n t h e treatment of hyperprolactinemia and acromegaly, bromocriptine i s administered i n low doses leading t o minute c o n c e n t r a t i o n s i n body f l u i d s and t i s s u e s . Therefore, none b u t t h e most s e n s i t i v e a n a l y t i c a l methods can be used t o measure i t s c o n c e n t r a t i o n i n b i o l o g i c a l specimens. The only method so f a r a p p l i c a b l e f o r pharmacok i n e t i c s t u d i e s with bromocriptine i s t h e u s e of t h e r a d i o a c t i v e l y l a b e l l e d drug, measurement of t o t a l r a d i o a c t i v i t y and i t s f r a c t i o n a t i o n by chromatographic s e p a r a t i o n techniques f o r t h e assay of p a r e n t drug and major metabolites. Recently, a radioimmunoassay k i t f o r t h e a n a l y s i s of picogram q u a n t i t i e s of unchanged bromocriptine i n body f l u i d s has become a v a i l a b l e . G a s chromatography, mass fragmentography and l i q u i d chromatography a l s o appear t o be s u i t a b l e f o r determining bromocriptine i n plasma from p a t i e n t s with P a r k i n s o n ' s d i s e a s e which a r e on treatment a t high dose l e v e l s . These c u r r e n t l y developped procedures permit q u a n t i t a t i v e determinations down t o c o n c e n t r a t i o n s of 0.5 (GC) , 1 . 0 (MF), and 1 0 ng/ml (LC), respectively ( 3 4 ) . The p a t t e r n of m e t a b o l i t e s i n b i l e (animals o n l y ) and i n u r i n e have been i n v e s t i g a t e d using column chromatography (Amberlite XAD 2 and Sephadex D E A E ) , t l c and reversed phase HPLC i n combination with r a d i o a c t i v i t y monitoring. The p r i n c i p a l metabolites have been i s o l a t e d from t h e b i l e of r a t s t r e a t e d with high doses of bromocriptine. The s t r u c t u r e of t h e i s o l a t e d m e t a b o l i t e s was e l u c i d a t e d by means of spectroscopic techniques.

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w. DIETER SCHONLEBER

8.

References F.Troxler and A.Hofmann, Helv.Chim.Acta 2, 2160 (1957) Handbook of Experimental Pharmacology, Vol. 49:"Ergot Alcaloids and Related Compounds", B.Berde and H.O.Schild, Ed., Springer-Verlag, Berlin, Heidelberg, New York, 1978 E.Fluckiger, F.Troxler, and A.Hofmann, German Offenlegungsschrift no. 1 926 045 (1969) E. Kovacs and E.Fluckiger, Experientia 30, 1172 (1974) J.K.Murdoch, Can.J.Hosp.Pharm. (1977) 149 H.Gjonnoes, Tidsskr.Nor.Laegeforen 97, 1405 (1977) A.Saarinen, V.Myllila, M.Reunanen, and E-Hokkanen, Duodecim 93, 1219 (1977) Triangle, Sandoz Journal of Medical Science, g(1) (1978) Symposium on Bromocriptine, Special Supplement, The Medical Journal of Australia, Vol - no. 3 (1978) 2, W.J.Weiner, P.A.Nausieda, and H.I.Klawans, Neurology 28, 734 (1978) H.R.Schneider, P.A.Stadler, P.Stutz, F.Troxler,and J.Seres, Experientia 33, 1412 (1977) W.D.Schonleber , A.L. Jacobs, and G. A.Brewer, jr. :"Dihydroergotoxine mesilate" in Analytical Profiles of Drug Substances 1, 81 B. Kreilgaard, "Ergotamine tartrate" in Analytical Profiles of Drug Substances 6, 113 T.Inone, Y.Nakahara, and T.Niwaguchi, Chem.Pharm.Bul1. 20, 409 (1972) D.Voigt, S.Johne, and D.Groger, Pharmazie - 697 (1974) 29, H.P.Weber, Sandoz Ltd., personal communication H-Bethke, B.Delz, and K.Stich, J.Chromatog. 123, 193 (1976) A.Hofmann: Die Mutterkornalkaloide, F.Enke Verlag, Stuttgart, GFR, (1964) P. Gull and P.A.Stadler, Sandoz Ltd., personal communication Sandoz stability report for drug substance Sandoz stability reports for Parlodel capsules and tablets This part was kindly contributed by E.Schreier, Sandoz Ltd. A.Ehmann, J-Chromatog. 132, 267 (1977) V.Creutzburg-Biermanova, Sandoz Ltd., personal communication
L

1. 2.
3.

4.
5. 6. 7.
8. 9. 10. 11.

12.

13.

14.
15. 16. 17.
18.

19.

20. 22. 23. 24. 25.

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26. 27. 28. 29.

30.

31.
32.

33. 34.

Control procedure for dosage form, Sandoz Ltd. Control procedure for drug substance, Sandoz Ltd. R.Stampfli, Sandoz Ltd., personal communication F.J.W.Van Mansvelt, J.E.Greving, and R.A.De Zeeuw, J.Chromatog. 151, 113 (1978) T.A.Plomp, J.G.Leferink, R.A.A.Maes, J.Chromatog. 151, 121 (1978) H.P.Keller, Sandoz Ltd., personal communication W-Ableidinger,Sandoz Ltd., personal communication M.Devaud, Sandoz Ltd., personal communication N.-E.Larsen, R.Oehman, M.Larsson, E.F.Hvidberg, to be published in Clin. Chem. Acta.

Acknowledgments The authors are indebted to many colleagues at SandozWander Ltd. for their most valuable help, in particular to Messrs. H.-R. Loosli, H.P. Weber and E. Schreier. The technical assistance for the proper presentation of the mass spectrum by Prof. W. Simon, at the Swiss Federal Institute of Technology in Zurich, is gratefully acknowledged. Furthermore, the authors wish to express special thanks to Miss Moret for her secretarial assistance in preparing this manuscript.

Analytical Profiles of Drug Substances, 8

CALCITROL
Eileen Debesis
I.
2.

3. 4. 5. 6.

7. 8.

Description I . I Name, Formula, Molecular Weight I .2 Appearance, Color, Odor Physical Properties 2.I Infrared Spectrum 2.2 Nuclear Magnetic Resonance Spectrum 2.3 Ultraviolet Spectrum 2.4 Mass Spectrum 2.5 Melting Range 2.6- Differential Scanning Calorimetry 2.7 Thermogravimetric Analysis 2.8 Hot Stage Microscopy 2.9 Solubility 2.10 X-Ray Powder Diffraction 2.1 I Optical Rotation Synthesis Stability and Degradation Drug Metabolic Products Methods of Analysis 6. I Elemental Analysis 6.2 Chromatographic Methods 6.2 I Thin-Layer Chromatography 6.22 High Performance Liquid Chromatography 6.23 Gas Chromatography-Mass Spectrometry 6.3 Biological Methods 6.3 I Radioimmunoassay 6.32 Protein Binding Assay 6.33 Bioassay 6.4 Polarography Acknowledgments References

83

Copyright @ 1979 by Academic Press. Inc. All rights of reproduction in any form reserved.

ISBN 0-12-260808-9

84

EILEEN DEBESIS

1.

Description 1.1 Name, Formula, Molecular Weight Calcitriol is 1 a, 2 5 - d i h y d r o x y c h o l e c a l c i f e r o l .

MW = 416.65 274' "43

1.2 Appearance, Color, Odor Calcitriol is an odorless white crystalline powder. Physical Properties 2.1 Infrared Spectrum The infrared spectrum of calcitriol is shown in Figure 1 ( ) 1 . The spectrum was recorded on a Perkin-Elmer Model 283 Grating Infrared Spectrophotometer and was measured in a KBr pellet which contained 1 mg of calcitriol in 300 mg of KBr. Figure 1:
a.

2.

The following absorptions have been assigned for OH stretching (bonded): 3391 cm-l Aliphatic CH stretching: 2943,12872 cm-l CH deformation: 1468, 1318 cmC-0 stretching: 1056 cm-

b. c. d.

2.2 Nuclear Magnetic Resonance Spectrum (NMR) The NMR spectrum of calcitriol, recorded on a Varian XL-lOO/Nicolet TT-100 pulsed Fourier Transform NMR spectrometer, with internal deuterium lock, is shown in Figure 2 (2). The spectrum was recorded using a solution of 0.84 mg of sample dissolved in 50 microliters of CD30D (100%D) containing 1% v/v tetramethylsilane in a 1.7 mm capillary tube. The spectral assignments are given in Table I.

WAVELENGTH (MICRONS)

2.5
100 -

30 .
I

4.0
I

5.0
I

6.0
I

8.0
I

1 0
I

1 20 25 6
1 I I

Figure 1 Infrared Spectrum oE Calcitriol

CALCITRIOL

87

Table I NMR Spectral Assignments for Calcitriol Proton -CH3(C18) -CH3(C21> -CH3(C26,27 X H 2 , ;CH HO. ;H C
~

Chemical Shift (a) 0.57 0.95

Multiplicity Sing1et Doublet Singlet Complex Comp 1ex Triplet Complex AB Quartet JAB = 11 Hz

-1.16 -1.38-3 4.08 4.32 4.90, 5.28 6.07, 6.32

/ -

Ho> I CH -

- CH2

HDO + 3 x OH (exchangeable protons) 4.78

88

EILEEN DEBESIS

2.3 Ultraviolet Spectrum The ultraviolet spectrum of calcitriol (1 mg of Calcitrio1/100 ml of absolute ethanol) in the region of 220 to 400 nm exhibits one maximum at 264 nm (E = 1.9 x 10 ) and one minimum at 226 nm. The spectrum is shown in Figure 3 (3). Mass Spectrum The low resolution mass spectrum of calcitriol is shown in Figure 4 (4). The spectrum was obtained using a Varian MAT CH5 spectrometer, which was interfaced with a Varian data system 620 I. The data system accepts the output of the spectrometer, calculates the masses, compares the intensities to the base peak, and plots this information as a series of lines whose heights are proportional to the intensities,
2.4
'

The molecular ion was measured at m/e = 416. Other characteristic masses were observed at m/e = 398, 380 and 362, corresponding to the l o s s of one, two and three molecules of water, respectively, from the molecular ion; m/e = 383, corresponding to the loss of water and CH3 from the parent peak; and m/e = 365, corresponding to the l o s s of two water molecules and one CH from the molecular ion. The base peak is 3 observed at m/e = 134, and corresponds to the

..

moiety
H

2.5

Melting Range Calcitriol melts at 111-115"C ( ) 3.

2.6 Differential Scanning Calorimetry Melting was accompanied by decomposition (5). Thermogravimetric Analysis Calcitriol was subjected to thermogravimetric analysis on a Perkin-Elmer Model TGS-1 Thermogravimetric Analyzer. The sample exhibited two overlapping weight losses. The initial weight loss, of 0.4%, began at 55C and ended at 105"C, and was due to surface moisture or solvent. The second weight l o s s , due to decomposition, amounted to 2.5% at 205"C, 7.5% at 255"C, 33% at 305"C, and 93% at 355C ( ) 5.
2.7

2.8 Hot Stage Microscopy As observed on a Mettler Hot Stage FP 52 with FP5 controller, the sample appeared as birefringent needle-like crystals which melted from 115-117C. The sample did not re5. crystallize from the melt ( )

!$fH+

CALCITRIOL

89

0.6

0.5

0.4
W

z
a
U

3 0.3
m

8
0.2

0. I

0
250

300
NANOMETERS

350

Figure 3 Ultraviolet Spectrum of Calcitriol

Q)

K n

CALCITRIOL

91

2.9 Solubility Extensive solubility data is not readily obtainable due to the scarcity of calcitriol. The material is slightly soluble in methanol, ethanol, ethyl acetate and tetrahydrofuran ( 3 ) . 2.10 X-Ray Powder Diffraction The X-ray powder diffraction data for calcitriol are presented in Table I11 ( 3 ) ; instrumental conditions are given below. The diffraction pattern is shown in Figure 5. Instrument and Operating Conditions Instrument Generator Detector Sample Densitometer" Optics Detector Optical density Beam exit slit Guinier-De Wolff Camera GE XRD-6 50 KV, 12.5 mA Film
As is

Gelman DCD-16 Tungsten Lamp 575 nm Visible mode Silicon phototransistor

0.25
0.53 mm

*The diffraction pattern was recorded on film; the density of the individual lines in the pattern was determined by densitometry.

id a

a
c .

CALCITRIOL

93

TABLE I11

Calcitriol Powder Diffraction Data 20 26.52 13.33 22.41 20.89 18.01 28.97 27.33 23.70 33.55 30.54 31.77 17.19 24.95 21.54 28.04 32.66 19.46 20.35 16.07 11.37 8.03 34.18 12.07
*d
**I/Io
--

I/Io**

4.993 9.863 5.892 6.316 7.314 4.578 4.847 5.577 3.968 4.348 4.183 7.662 5.302 6.129 4.727 4.072 6.776 6.480 8.192 11.559 16.343 3.896 10.889
A

100 90
88

82 70 66 49 44 36 34 33
33

26 26 15 12 12 12
2

2 2
2

2 Sin 0

(interplanar distance)

= percent relative intensity (based on maximum

intensity of 1.00)

2.11 Optical Rotation The specific rotation of calcitriol in absolute methanol, measured at 589 nm and 2SoC, was +48' ( 7 ) .

94

EILEEN DEBESIS

Synthesis Several procedures for the synthesis of calcitriol have et a1.(8) and Norman been reported in the literature. Parren - -- (9) described the preparation of calcitriol from et al. 25-hydroxyvitamin D3. Uskokovic and others (10-18) have described lengthier syntheses utilizing more readily available starting materials. A typical reaction scheme, utilizing 1 a, 25-diacetoxy-7-dehydrocholesterol as the starting material, is shown in Figure 6.
3.

Stability and Degradation Calcitriol must be protected from air and light. The drug substance exhibits good stability when stored at - 5 C 1' to -5C in an argon atmosphere. The material is stable 2' at room temperature when dissolved in a vegetable oil derivative, containing antioxidants, such as is used in calcitriol soft gelatin capsules (19).
4.

Drug Metabolic Products Calcitriol may be absorbed directly into the intestine or bone, or may be hydroxylated to form 1 a, 24, 25-trihydroxycholecalciferol prior to intestinal absorption (20-24).
6.

5.

Methods of Analysis 6.1 Elemental Analysis A typical elemental analysis of a sample of calci7; triol is presented in Table IV ( ) TABLE IV Elemental Analysis of Calcitriol E 1ement
C H

% Theory

% Found

77.84 10.64 11.52

77.53 10.75 11.92 (by difference)

2 o 2
v

I
0
r(

x3R
0 ,
0

g
1

y z
1

rn

8
.rl *+-I

O--

c,

r r

.rl
L V I .rl

0 I

'.

96

EILEEN DEBESIS

6.2 Chromatographic Methods 6.21 Thin-Layer Chromatography (TLC) The following TLC procedure is useful for determining the purity of calcitriol. It separates the previtamin, 1 a, 25-dihydroxyprecholecalciferol. A silica gel GF plate is activated by heating for one hour at 105OC and is then cooled in a desiccator. A low actinic all glass chromatographic chamber is equilibrated with the developing solvent, and 0.8 mg of calcitriol is applied to the plate from ethyl acetate. The plate is developed in an ascending mode in ethyl acetate:spectroquality heptane:methanol (100:10:2) for 15 cm. After air drying, the plate is viewed under shortwave ultraviolet radiation, then sprayed with a 15% w/v solution of phosphomolybdic acid in ethanol, followed by heating at 105OC for 10 minutes to develop the colors. The approximate R f values are summarized in Table V ( 3 ) . TABLE V Summary of TLC Data Compound 1 a, 25-Dihydroxyprecholecalciferol Calcitriol Approximate R 0.4
0.5

6.22 High Performance Liquid Chromatography High performance liquid chromatography is used to determine the purity of calcitriol, and to separate it from related compounds. Using a 10 micron silica column of 25 cm length, and a mobile phase of spectroquality heptane: ethyl acetate:methanol (50:SO:l) at a flow rate of 1.7 ml/ minute, separation and quantitation are achieved. p-Dimethylaminobenzaldehyde may be used as an internal standard to compensate for variations in injection technique and instrumental conditions. With a 254 nm ultraviolet absorbance detector, 0.01 ug of calcitriol may be detected ( 3 ) . This procedure, with a mobile phase of spectroquality heptane:ethyl acetate:methanol (70:25:5) is also useful for analyzing calcitriol in soft gelatin capsules. The capsule fill solution may be injected directly. The amount of calcitriol in the capsule is determined by comparison to a calcitriol reference standard, prepared in a medium similar to the capsule fill ( 3 ) .

CALCITRIOL

97

6.23 Gas Chromatography - Mass Spectrometry Halket and Lisboa (25) examined several Vitamin D derivatives by capillary gas chromatography coupled with mass spectrometry. This technique offered the advantages of great sensitivity and separating power. Retention times and fragmentation patterns for ergocalciferol, cholecalciferol and calcitriol were reported. 6.3 Biological Methods 6.31 Radioimmunoassav Several workers (26-32) have reported on the use of radioimmunoassay for measuring calcitriol at very low (pg) levels in serum. 6.32 Protein Binding Assays Various protein binding techniques are reported (32-42) for the determination of calcitriol in plasma. Generally, a preliminary purification step is required to avoid interference from other plasma components. 6.33 Bioassays Stern. - al. (43.44) reDorted a bioassav et technique based on fetal rat bone absorption of calcitriol. Parkes and Reynolds (45) developed an in-vitro bioassay using duodenal tissue from chicken embryos.
. . <

6.4 Polarography Calcitriol drug substance may be analyzed polarographically, using a glassy carbon working electrode. The limiting current of the observed oxidation wave (E 0.96 v> is linear with concentration in the 0.01 tol6203 m~ region ( 6 . 4)
Acknowledgments The author wishes to thank the staffs of the Scientific Literature Department and Research Records Office of HoffmannLa Roche Inc., Nutley, NJ. 7.

98

EILEEN DEBESIS

8.

References 1. 2. 3.
4.
5.

6.

7.
8. 9.

10.

11. 12.

13. 14. 15. 16. 17. 18. 19.

20.

Ng, S., Hoffmann-La Roche, Inc. Personal Communication. Grant, Anne, Hoffmann-La Roche, Inc. Personal Communication. Rubia, Linda, Hoffmann-La Roche, Inc. Personal Communication. Benz, W . , Hoffmann-La Roche, Inc. Personal Communication. Ramsland, Arnold, Hoffmann-La Roche, Inc. Personal Communication. Macri, Frank, Hoffmann-La Roche, Inc. Personal Communication. Scheidl, F . , Hoffmann-La Roche, Inc. Personal Communication. Paaren, H. E., Hamer, D. E., Schnoes, H. K., and DeLuca, H. F . , Proc. Nat2. Acad. S c i . USA, 75, 2080 (1978). Norman, A. W., Myrtle, J. F., Midgett, R. J., and Nowicki, H. G., U.S. 3 , 772, 150 (November 13, 1973); CA 80:105146g (1974). Uskokovic, M. Baggiolini, E., Mahgoub, A., Narwid, T., and Partridge, J. J. , Vitamin D and ProbZems Related t o Uremic Bone Disease, Walter de Gruyter, Berlin, New York, 279 (1975). Okamura, U. H., Am. Chem. Soc. Abstr. Pap. 275th ACS Natl. Meet., Anaheim, California, Mar. 13-17, 1978, MEDI #32 (1978). Matsunaga, I., Ochi, K., Nagano, H., Shindo, M., Ishikawa, M., Kaneko, C., and DeLuca, F . H., to Chugai Pharmaceutical Co., Ltd. Japan Kokai 76 100,056 (September 3, 1976); CA 8639014313 (1977). Suda, T. , Horiuchi, N., and O g a t c E. , Tampakushitsu Kakusan Koso, 21, 844 (1956). DeLuca, H. F., TetrahehTn Lee&, 1972, 4147. Cohen, Z., Keinan, E . , Mazur, Y., and Ulman, A., J . Org. Chern., - 2651 (1976). 41, Mielczarek, I., Wiad. Chem., 28, 813 (1974). 33, Kaneko, C. , Yuki Gosei KagakuTyokai Shi, 75 (1975). Barton, D. H. R., Hesse, R. H., Pechet, M. M., and Rizzardo, E., J . Chem. Soc., Chem. Commun., 1974, 203. Johnson, J. B., Hoffmann-La Roche, Inc. Personal Communication. Napoli, J. L., Vitamin D and Its Metabolites, Annu. Rep. Med. Chem., - 295 (1975). 10,

r,

CALCITRIOL

99

21. 22. 23. 24. 25. 26. 27. 28. 29.

30.

31. 32. 33.

34.

35.
36.

Kumar, R., and Deluca, H. F., Biochm. Biophys. Res. Commun., 69, 197 (1976). Kumar, R., Harnden, D., and DeLuca, H. F., Biochemistry, 15, 2420 (1976). Harnden, D., Kzar, R., Holick, M. F., and DeLuca, H. F., Science, 193,493 (1976). Castillo, L., DeLuca, H. F., and Ikekawa, N., J . BioZ. Chem., 252, 1421 (1977). Halket, J. M., and Lisboa, B. P., Acta. Endocrinol., (Copenhagen), - (Suppl. 215), 120 (1978). 87, Clemens, T. L., Hendy, G. N., Graham, R. F., Baggiolini, E. G., Uskokovic, M. R., and O'Riordan, J. L. H., Endocrinology, 102 (Suppl.), 490 (1978). Schaefer, P. C., LifschitrM. D., Fadem, S. Z. and Goldsmith, R. S., Endocrinology, 102, (Suppl.), 320, (1978). Clemens, T. L., Hendy, G. N., Graham, R. F., Baggiolini, E. G., Uskokovic, M. R., and O'Riordan, J. L. H., J . Endocrinol. 77, 49P (1978). Clemens, T. L., Hendy, G.N., Graham, R. F . , Baggiolini, E. G . , Uskokovic, M. R., and O'Riordan, J. L. H., Clin. Sci. Mol. Med. Med., 54, 329 (1978). Fairney, A . , Turner, C., Baggiolini, E . G., Uskokovic, M. R., in Vitamin D BiochLmioal, : Chemical, and Clinical Aspects Related t o Calciwn MetaboZism, A. W. Norman, K. Schaefer, J. W. Coburn, H. F. DeLuca, D. Fraser, H. G. Grigoleit, D. von Herrath [eds.], Walter de Gruy-ter, Berlin, New York, 459 (1977). Eisman, J. A . , Hanistra, A . J., Kream, B. E., DeLuca, H. F., Arch. Biochem. Biophys., - 235 176, 1976). Etsuko, A . , Suda, T., Nakano, H., Igaku No A y w n i , - 509 (1977). 102, Brumbaugh, P. F . , Haussler, D. H., Bressler, R., and Haussler, M. R., Science, 1 3 1089 (1974). 8, Caldas, A . E., Gray, R. W., Lemann, J., Jr., J . Lab. CZin. Med., 91, 840 (1978). Shimura, F . , and TamGa,.M., Vitamins, - 173 52, (1978). Horst, P. L., Merchant, C . , Shephard, R., Hamstra, A . Jorgensen, N. A . , DeLuca, H. F . , J . Dairy S c i . , - (Suppl. l), 123 (1977). 60,

100

EILEEN DEBESIS

37.

38.

39.
40.

41. 42. 43. 44.

45.

46.

Kream, B. E., Eisman, J. A., DeLuca, H. F., in Vitamin D Biochemical, Chemical and Clinical : Aspects Related t o Calcium Metabolism, A. W. Norman, K. Schaefer, J . W. Coburn, H. F. DeLuca, D. Fraser, H. C. Grigoleit, and D. von Herrath, [eds.], Walter de Gruyter, Berlin, New York, 501 (1977). Haussler, M. R., Hughes, M. R., Pike, J. W., McCain, T. A,, in Vitamin D Biochemical, Chemical : and Clinical Aspects Related t o Calcium Metabolism, A. W. Norman, K. Schaefer, J . W. Coburn, H. F. DeLuca, D. Fraser, H. C. Grigoleit, and D. von Herrath, [eds.], Walter de Gruyter, Berlin, New York, 473 (1977). Norman, A. W., Henry, H., Bishop, J. E., Coburn, J. W., CZin. Res., 26, 423A (1978). Eisman, J. A., Hamstra, A. J., Kream, B. E., DeLuca, H. F., Science, 193, 1021 (1976). Hughes, M. R., Baylink, D. J., Jones, P. G., Haussler, M. .R., J. CZin. Inuest. , - 61 (1976). 58, Brumbaugh, P. F., Haussler, D. H., Bursac, K. M., Haussler, M. R., Biochemistry, 13, 4091 (1974). Stern, P. H., Hamstra, A. J., Dxuca, H . F., Bell, N. H., J. Clin. Edocrinol. Metab., 46, 891 (1978). Stern, P. H., Phillips, T. E., Lucas, S. V., Hamstra, A. J., DeLuca, H. F., Bell, N. H., in Vitamin D Biochemical, Chemical and Clinical : Aspects Related t o Calciwn Metabolism, A. W. Norman, K. Schaefer, J. W. Coburn, H. F. DeLuca, D. Fraser, H. G. Grigoleit, D. von Herrath, [eds.], Walter de Gruyter, Berlin, New York, 459 (1977). Parkes, C. O., Reynolds, J . J . , Moz. Cell. Endocrinol., - 25 (1977). 7, Rucki, R. J., Hoffmann-La Roche, Inc. Personal Communication.

Analytical Profiles of Drug Substances, 8

CHLORTETRACYCLINE HYDROCHLORIDE
George Schwartzman I ;Lola Wayland, Thomas Alexander Kenneth Furnkranz, George Selzer, and the USASRG *
Description I . I Drug Properties I .2 Physical Description and Optical Crystallography 1.3 Chemical Properties 1.4 Structure I .5 The FDA Chlortetracycline Standard 2. Physical Properties 2. I Thermal Properties (DTA, TGA) 2.2 X-Ray Powder Diffraction 2.3 Solubility 2.4 Acid-Base Properties 2.5 Polymorphism3. Spectral Properties (Optical) 3.1 Ultraviolet 3.2-Infrared 3.3 Spectropolarimetry 3.4 Fluorescence 4. Spectral Properties (Other) 4. I Proton NMR 4.2 ' T - N M R 4.3 Mass Spectrometry 5 . Chromatography 5 . I Paper and Thin-Layer 5.2 Gas-Liquid 6. Manufacture 6. I Fermentation 6.2 Isolation 7. Stability 8. Analytical Methods 8. I Microbiological 8.2 Chemical 9. References
*The US.Antibiotics Standards Research Group (USASRG) is an ad hor collaboration of antibiotics researchers at the U S . Food and Drug Administration. Contributors to this monograph from the Bureau of Drugs include: T. Alexander. R. Barron, V. Folen. K. Furnkranz. G . Mack (Baltimore District), M. Maientbal. G. Mazzola (Bureau of Foods). G . Schwartzman. G. Selzer. E. Sheinin. J. Taylor, L.Wayland. and J. Weber. The USASRG was formed at the request of P. Weiss. National Center for Antibiotics Analysis. Individual contributions are referenced where possible. 'Retired. Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. 101 ISBN 0-12-260808-9

I.

I02

GEORGE SCHWARTZMAN ET A L .

1.

DESCRIPTION

1.1 Drug P r o p e r t i e s C h l o r t e t r a c y c l i n e h y d r o c h l o r i d e (CTC-HCL) i s the h y d r o c h l o r i d e s a l t of a n a n t i b i o t i c s u b s t a n c e produced by t h e growth of Streptomyces a u r e o f a c i e n s (Fam. Streptomycetaceae) I t was d i s c o v e r e d i n 1948 by Duggar (1).

CTC-HC1 is a broad-spectrum a n t i b i o t i c and a n t i protozoan a c t i v e a g a i n s t many Gram-positive b a c t e r i a , some Gram-negative b a c t e r i a , s p i r o c h e t e s , amebae, and c e r t a i n l a r g e v i r u s e s . S t a p h y l o c o c c i have become g e n e r a l l y r e s i s t a n t to t h e drug, and d r u g - r e s i s t a n t s t r a i n s may b e found among o t h e r b a c t e r i a l genera and s p e c i e s g e n e r a l l y s e n s i t i v e t o i t . C r o s s - r e s i s t a n c e t o o t h e r a n t i b i o t i c s of t h e t e t r a c y c l i n e family is a u t o m a t i c ( 2 ) . Organisms may be c o n s i d e r e d s u s c e p t i b l e i f t h e Minimum I n h i b i t o r y Concentration (MIC) is n o t more t h a n 4.0 pg/ml and i n t e r m e d i a t e i f t h e M I C is 4.0-12.5 pg/ml ( s e e Table 1). T e t r a c y c l i n e s are r e a d i l y absorbed and are bound t o plasma p r o t e i n s i n v a r y i n g d e g r e e s . They a r e c o n c e n t r a t e d by t h e l i v e r i n t h e b i l e and e x c r e t e d i n t h e u r i n e and f e c e s a t high c o n c e n t r a t i o n s and i n a b i o l o g i c a l l y a c t i v e form. The mode of a c t i o n a g a i n s t microorganisms i n v o l v e s t h e i n h i b i t i o n of phosphorylation p r o c e s s e s i n b a c t e r i a l c e l l s ( 3 ) .
TABLE 1 (5) Antimicrobial Spectrum of Chlortetracycline Minimum Inhibitory Microorganism Concentration (Vg/ml) Micrococcus pyrogenes aureus 209P Micrococcus pyrogenes v z . aureus 1248A Streptococcus pyrogenes Streptococcus Q Streptococcus faecalis Micrococcus flavus Diplococcus pneumoniae Sarcina lutea ~Bacillus subtilis Escherichia Haemophilus influenzae Klebsiella pneumoniae Neisseria catarrhalis Aerobacter aerogenes Proteus vulgaris Pseudomonas aeruginosa Salmonella schottmuelleri Salmonella typhi Shigella dysenteriae Brucella bronchiseptica Mycobacterium tuberculosis Mycobacterium friedmanii Mycobacterium smegmatis Mycobacterium s p . Candida albicans Clostridium butyricum Pasteurella multocida Vibrio percolans

E.

0.292 0.39 0.29 0.147 0.39 0.292 0.098 0.147 0.195 1.45 0.312 0.29 0.098 1.17 4.6 25 3.12 1.17 3.12 0.292 0.147 0.122 0.073 0.58 100 0.078 0.049 0.098

CHLORTETRACYCLINE HYDROCHLORIDE

103

Physical Description and Optical Crystallography CTC-HC1 is a yellow, odorless powder composed mainly of crystals in the shape of small hexagons, and has the following optical crystallographic characteristics ( ) 4:

1.2

a: 1.635; optic sign negative; 2V = 59" (calc.);

8:

1.706; orthorhombic; extinction parallel; and

y: 1.730; symmetrical
6. It is stable in air, but is slowly affected by light ()
Chemical Properties CTC-HC1 is the HC1 salt of amphoteric CTC; it is multifunctional with two chromophores. It is a parachlorophenol with an aYf3-unsaturatedketone in conjugation. The second chromophore involves another a,B-unsaturated ketone that is in conjugation with an anomalously behaving amide ( ) 7. The tertiary amine is responsible for the basic character and the phenolic group is acidic. CTC is fluorescent and can be assayed polarographically ( ) 8. A particularly intriguing aspect of the chemistry of the compounds of the tetracycline family is their ability to form metallic complexes. CTC shares in this intensively studied capability, which is very likely related to the therapeutic activity ( ) 9 . This property is also used in the purification (10) and analysis (11) of CTC. 1.3

1.4

Structure C22H23ClN208.HCl Empirical Formula 515.35 Molecular Weight

7-Chloro-4-(dirnethylamino)-1,4,4a,5,5a,6,11,12aoctahydro-3,6,10,12,12a-pentahydroxy-6-methyl-l,ll-dioxo-2naphthacenecarboxamide monohydrochloride (CAS-64-72-2) (6,12-14) (Figure 1 . )

The FDA Chlortetracycline Standard The current official FDA Chlortetracycline Working Standard is chlortetracycline hydrochloride, Lot W501-632B95-1 (9/29/53), obtained from Lederle, which markets the antibiotic under the proprietary name Aureomycin. The current working standard has an assigned potency o 1 0 pg/mg (the f 00 term pg applied to chlortetracycline means the chlortetracycline activity (potency) contained in l u g of the FDA Chlortetracycline Master Standard (Lot #990-107-141-1),which is also chlortetracycline hydrochloride).

1.5

CHLORTETRACYCLINE HYDROCHLORIDE

I05

The Working S t a n d a r d is s t o r e d i n l o t s of 250 mg a t -2O"C, p r o t e c t e d from l i g h t and m o i s t u r e , a t t h e h a t i o n a l C e n t e r f o r A n t i b i o t i c s A n a l y s i s , Washington, DC. PHYSICAL PROPERTIES 2.1 Thermal P r o p e r t i e s D i f f e r e n t i a l Thermal A n a l y s i s (DTA) and Thermal G r a v i m e t r i c A n a l y s i s (TGA).CTC-HC1 i s s t a b l e u n t i l i t b e g i n s t o decompose e x o t h e r m i c a l l y a t a p p r o x i m a t e l y 230C ( F i g u r e s 2 and 3 ) ( 1 5 ) . The compound d o e s n o t l o s e any m a s s u n t i l t h e f i n a l d e c o m p o s i t i o n t a k e s p l a c e . No polymorphs have b e e n s e e n i n t h e samples examined. X-Ray Powder D i f f r a c t i o n The X-ray d i f f r a c t i o n p a t t e r n of t h e FDA Working S t a n d a r d h a s been d e t e r m i n e d . It d e m o n s t r a t e s c r y s t a l l i n e s t r u c t u r e ; t h e d a t a are l i s t e d i n T a b l e 2 ( 1 6 ) . TABLE 2 X-Ray D i f f r a c t i o n Data d 2.2

2.

(1)

1/11

d (i) 3.34 3.27 3.23 (3.17 D(3.12 3.095 2.982 2.948 2.910 (2.878 D(2.854 2.788 2.710 D ( 2 . 653 (2.632 2.560 2.482 2.440 2.421 2.390 2.343 2.290 2.235 2.210 2.170 2.142 2.093

1/11

d 2.060B D(1.985 (1.962 1.897 1.880 1.868 1.856 1.827 1.805 1.784 ~(1.748 (1.737 1.705 1.682 1.638 1.631 1.608 1.595 1.583

1/11

9.10 8.48 (7.75a D(7.43 6.65 6.38 (5.72 D(5.61 5.28 5.16 D(4.96 (4.88 4.68 4.54 4.42 4.29 4.24 (4.15 D(4.09 3.88 3.80 3.71 3.64 3.57 3.52 3.37

1 1 12 56 44 13 43 81 42 61 8 8 15 3 40 100 70 25 52 78 37 31 59 33 16 25 23

17 37 16 73 74 38 8 9 47 25 31 35 36 14 10 30 22 13 27 12 24 7 14 37 13 23 21

13 1 1 13 12 10 13 13 8 9 6 8 1 1 9 6 5 4 5 4 4

Doublet.

106

GEORGE SCHWARTZMAN ET AL.

120'

140"

160'

180" 200'

220'

240"

Figure 2.

Differential thermogram of CTC-HC1.

120

160'

200"

240

Figure 3.

Thermal gravimetric analysis curve of CTC-HC1.

CHLORTETRACYCLINE HYDROCHLORIDE

I07

Solubility The s o l u b i l i t y of a n t i b i o t i c s , i n c l u d i n g CTC-HC1, w a s r e p o r t e d by Andrew and Weiss (17). CTC-HC1 i s a n amphoteric s u b s t a n c e and consequently i t is solu'ble i n aqueous a c i d and b a s e . However, i t can r a p i d l y degrade i n t h e s e s o l v e n t s . Its s o l u b i l i t y i n w a t e r is about 8 mg/ml and i n methanol about 1 7 mg/ml. I n h i g h e r m o l e c u l a r weight a l c o h o l s , t h e s o l u b i l i t y of CTC-HC1 i s c o n s i d e r a b l y le ss t h a n i n methanol. For p r a c t i c a l purposes, i t i s i n s o l u b l e i n many common s o l v e n t s such a s t h e a l i p h a t i c hydrocarbons, benzene, I t i s r e a d i l y s o l u b l e i n p y r i d i n e and e t h e r , and chloroform. t o t h e e x t e n t of about 5 mg/ml i n formamide. P y r i d i n e is an u n d e s i r a b l e s o l v e n t because of i t s b a s i c i t y , and formamide i s n o t d e s i r a b l e because of t h e d i f f i c u l t y i n o b t a i n i n g and maintaining i t a s a s t a b l e solvent.

2.3

2.4

Acid-Base P r o p e r t i e s

CTC e x h i b i t s t h r e e a c i d i c d i s s o c i a t i o n c o n s t a n t s when t i t r a t e d i n aqueous s o l u t i o n s ( 1 8 ) . Stephens e t a l . (19)

i d e n t i f i e d t h e t h r e e a c i d i c groups ( F i g u r e 4 ) , and r e p o r t e d thermodynamic pKa v a l u e s of 3.30, 7 . 4 4 , and 9.27. Leeson e t a l . (20) a s s i g n e d pKa v a l u e s t o t h e f o l l o w i n g a c i d i c groups:

pKa
3.30 7.44 9.27

Assignment Tricarbonylmethane System (A) P h e n o l i c Diketone System (B) Dimethylamino System (C)

Kalnins and B e l e n ' s k i i (21) v e r i f i e d t h e assignments by i n f r a r e d (IR) spectroscopy. The pH o f a 10 mg/ml aqueous s o l u t i o n , as d e s c r i bed i n t h e Code of F e d e r a l R e g u l a t i o n s monograph f o r CTC-HC1 (22) should l i e between 2.3 and 3.3. Polymorphism I n r e c e n t y e a r s , w i t h growing concern about t h e r e l a t i v e b i o a v a i l a b i l i t i e s of d i f f e r e n t samples of t h e same drug s u b s t a n c e , polymorphism h a s become of p r i m e i n t e r e s t . Miyazaki and co-workers (23) have r e p o r t e d t h e e x i s t e n c e of The X-ray powder d i f f r a c two c r y s t a l l i n e forms of CTC-HC1. t i o n p a t t e r n s , I R s p e c t r a , d i s s o l u t i o n b e h a v i o r s , and h y g r o s c o p i c i t i e s t h a t t h e y r e p o r t e d were d i s t i n c t l y d i f f e r e n t and t h e r e w e r e d i s c r e p a n c i e s i n t h e b i o a v a i l a b i l i t i e s . I n l a t e r work on two forms of CTC b a s e p r e p a r e d by c r y s t a l l i z i n g from water o r methanol, t h e X-ray p a t t e r n s and 2.5

CHLORTETRACYCLINE HYDROCHLORIDE

I09

water c o n t e n t s were d i f f e r e n t , b u t o t h e r p h y s i c a l p r o p e r t i e s were i n d i s t i n g u i s h a b l e . T h e r e a p p e a r e d t o be no d i f f e r e n c e i n b i o a v a i l a b i l i t y ( 2 4 ) . No polymorphs were found i n t h e FDA CTC s t a n d a r d .

3. SPECTRAL PROPERTIES (OPTICAL) 3.1 Ultraviolet Medium Maxima (nm) 368, 340sh, 322sh, 265, 229 345, 283, 253, 222 372, 342sh, 322sh, 2 6 2 s h , 253, 233

0 . 1 N HC1 0 . 1 N NaOH Methanol

See F i g u r e 5 ( 2 5 ) .

Infrared The I R a b s o r p t i o n s p e c t r a of CTC-HC1 a r e shown i n F i g u r e s 6 and 7 . S p e c t r a were r u n on a Perkin-Elmer Model 467 g r a t i n g s p e c t r o p h o t o m e t e r as a K C 1 p e l l e t ( 1 mg/200 mg K C I ) , The s p e c t r a are e s s e n and as a N u j o l m u l l ( 2 0 mg/3 d r o p s ) . t i a l l y i d e n t i c a l . The CTC-HC1 s p e c t r u m may a l s o b e f o u n d i n a c o m p i l a t i o n of I R s p e c t r a of Drug R e f e r e n c e S t a n d a r d s by Hayden e t a l . ( 2 6 ) . Major a b s o r p t i o n f r e q u e n c i e s h a v e b e e n compiled (27) and a s s i g n m e n t s h a v e b e e n made on inany of t h e bands(28-31). Some of t h e m a j o r f r e q u e n c i e s and band a s s i g n ments are:

3.2

&
3360 3315 3200-3450 2800-2400

x
2.98 3.02 2.90-3.12 3.57-4.17
vNH of NH2 (asym.) vNH of NH2 (sym.)

vOH ( a l c o h o l i c , masks NH)

t e r t i a r y amine h a l i d e s a l t , g r o u p of b r o a d bands

1675 1582 1450 1368 1042

5.97 6.32 6.90

7.31

vc-0
6NH2 6CH ( b e n d i n g of CH3)
VCN

9.60

OH ( d e f o r m a t i o n , a l c o h o l s )

110

GEORGE SCHU ,RTZMAN ET AL.

m
v)

0
XI 00

2 0

Figure 5 .

WAVELENGTH (nm) Ultraviolet absorption spectra of CTC-HC1 i n a H a , 0.1N HC1; b , 0.1N N O ; and c , methanol.

c,
rl
rl

a,

-ai

rl

v)

(d

W 0

B 2
a

I800 1600 1400 WAVELENGTH (CM")

1200

10 0

Figure 7 .

Infrared absorption spectrum of CTC-HC1 a s Nujol mull.

CHLORTETRACYCLINE HYDROCHLORIDE

113

Spectropolarimetry Because c o n f i g u r a t i o n a l i n f o r m a t i o n can be d e r i v e d from o p t i c a l r o t a t o r y d i s p e r s i o n and c i r c u l a r d i c h r o i s m s c a n s , c o n s i d e r a b l e work h a s been conducted u s i n g t h e s e t e c h n i q u e s t o s t u d y t h e t e t r a c y c l i n e s ( 3 2 ) . The a b s o l u t e c o n f i g u r a t i o n of CTC w a s determined u s i n g o p t i c a l r o t a t o r y d i s p e r s i o n d a t a (33) S p e c t r a l c u r v e s are p r e s e n t e d i n F i g u r e s 8 and 9. The c i r c u l a r d i c h r o i s m spectrum is similar t o t h a t p r e s e n t e d by Mitscher e t a l . ( 3 4 ) , e x c e p t t h a t t h e v a l u e s d i f f e r by a f a c t o r of about 1.5. I n Table 3, d a t a o b t a i n e d by M i t s c h e r and i n F A l a b o r a t o r i e s are compared. D Fluorescence The CTC-HC1 F A Working Standard g i v e s a y e l l o w D f l u o r e s c e n c e under 1or.pwave u l t r a v i o l e t (W) l i g h t (375 m) (35). The e x c i t a t i o n (324 and 357 nm) and emission (443 nm) s p e c t r a of t h i s s t a n d a r d d i s s o l v e d i n 0.05N NaOH (11.4 mg/50 ml) a r e p r e s e n t e d i n F i g u r e 10.

3.3

3.4

TABLE 3 Molecular E l l i p t i c i t i e s of CTC Wavelength (nm) M i t s c h e r ' s Valuea F A Value D (X 103) (X 103)
236 254 288 318 355 ( s h ) +10 -31 +4 8 -24 +2 3 -48 +88 -32 -12

Ratio

-14 aThese v a l u e s w e r e o b t a i n e d from t h e g r a p h i n R e f e r e n c e

0.43 0.64 0.54 0.73 1.09 34

SPECTRAL PROPERTIES (OTHER) P r o t o n NMR The 60 MHz NMR s p e c t r a of CTC-HC1 i n DMSO-d6 and i n methanol-d4 are shown i n F i g u r e s 1 and 1 2 , r e s p e c t i v e l y . 1 The s h a r p s i g n a l s due t o t h e methyl groups are r e a d i l y a s s i g n a b l e ; t h e i r chemical s h i f t s and i n t e n s i t i e s are c o n s i s t e n t w i t h t h e s t r u c t u r e . The 2 methylene p r o t o n s a t C-5 are n o t e q u i v a l e n t and g i v e two sets of m u l t i p l e t s . Only one m u l t i p l e t is a s s i g n a b l e i n t h e methanol spectrum: t h e s i g n a l c e n t e r e d a t 2.20 ppm. I n DMSO-db t h e s e p r o t o n s appear between 1 . 5 and 2 . 3 ppm. The o n l y methine p r o t o n a c t u a l l y a s s i g n e d i s t h e one a t C-4. The c l o s e p r o x i m i t y t o t h e p o s i t i v e l y charged N c a u s e s a downfield s h i f t away from t h e o t h e r s i g n a l s . When D20 i s added t o t h e DMSO-db s o l u t i o n , t h e H-4 s i g n a l remains c o n s t a n t However, t h e two amide p r o t o n s , o r i g i n a l l y a t 9.15 and 9.55 ppm, exchange w i t h t h e deuterium and a r e no l o n g e r observed. These s i g n a l s were n o t observed i n methanol-d4, a g a i n , due t o

4.

4.1

~-

NOIWMSIO W O l V l O Y lWl1dO

I I4

<

115

AlISN3lNI

I I6

u X

V
I 3 u

CI V

a,

I I7

CHLORTETRACYCLINE HYDROCHLORIDE

119

deuterium exchange. Data from t h e l i t e r a t u r e ( 3 6 ) , based on a spectrum o b t a i n e d i n DMSO-db, a r e i n c l u d e d i n T a b l e 4. As c a n be s e e n from t h e t a b l e , t h e s e v a l u e s are g e n e r a l l y u p f i e l d from o u r r e s u l t s . Although t h e s e d i f f e r e n c e s were unexpected, no e x p l a n a t i o n w i l l be a t t e m p t e d h e r e (37). TABLE 4 Chemical S h i f tsa Proton L i t e r a t u r e (36) 1.7 DMSO-dh Solvent Methanol-dq 1.98 ( s ) 3.05 ( s ) 7.55 ( d ) 6.93 ( d ) 4.10 ( d ) 2.20 (m)b n o t observedC not assignedd

1.88 ( s )
2.93 ( s )

2.7
6.4 6.4

4.0

7.0

7.58 (d) 6.98 ( d ) 4.36 ( b r . s )

- 7.0

1.7

2.7 2.7

2.1

1.5

2 . 3 (m,s)

8.4, 8 . 9

9.15, 9.55 not assignedd not assignedd

n o t assignedd a S p e c t r a were o b t a i n e d on a Perkin-Elmer R-12B equipped w i t h a N i c o l e t TT-7 F o u r i e r t r a n s f o r m a c c e s s o r y . Chemical s h i f t s are r e p o r t e d i n ppm downfield from i n t e r n a l TMS. M u l t i p l i c i t i e s a r e i n d i c a t e d as s = s i n g l e t , d = d o u b l e t , and m = m u l t i p l e t . bThis m u l t i p l e t r e p r e s e n t s one of t h e H-5 p r o t o n s . The chemical s h i f t of t h e o t h e r one w a s n o t a s s i g n e d . CThese p r o t o n s exchange w i t h t h e deuterium of t h e s o l v e n t and t h u s are n o t observed. dThe chemical s h i f t s of t h e s e p r o t o n s are n o t a s s i g n e d . They a r e obscured by t h e -N(CH3)2 a n d / o r t h e s o l v e n t .

4.2

I3C NMR

Proton-noise decoupled and s i n g l e - f r e q u e n c y o f f resonance decoupled carbon-13 NMR s p e c t r a were determined f o r t h e CTC Working Standard ( F i g u r e 1 3 ) . The observed chemical s h i f t s of t h e 22 carbon atoms compare c l o s e l y w i t h t h o s e r e p o r t e d by Frank (38). The o n l y s i g n i f i c a n t d i f f e r e n c e observed w a s t h a t of t h e dimethylamino carbons which were found a t 48.5 ppm r a t h e r t h a n a t F r a n k ' s r e p o r t e d v a l u e of 4 1 ppm. This d i f f e r e n c e i n chemical s h i f t s can probably be a s c r i b e d t o d i f f e r i n g amounts of w a t e r p r e s e n t i n t h e DMSO-db s o l v e n t s and hence i n t h e p of t h e s e r e s p e c H tive solutions.

rl

V H V
Ict

0 k V a,

4J

4 ! z 8
m

V
4

m
rl

a, k

-4

m
h

CHLORTETRACYCLINE HYDROCHLORIDE

121

S p e c t r a were d e t e r m i n e d u s i n g a p u l s e w i d t h of 4 p s e c o n d s , which c o r r e s p o n d s t o a f l i p a n g l e of 18" and a 1 second p u l s e d e l a y t i m e . The 4000 Hz spectrum w a s d e s c r i b e d u s i n g 8192 d a t a p o i n t s . The o b s e r v e d r e s o n a n c e l i n e s of CTC-HC1 t h e i r a s s i g n m e n t s are shown i n T a b l e 5 . (39) and

'CABLE 5 Observed Resonance L i n e s o f CTC-HC1 and T h e i r Assignments Carbon Assignment Chemical S h i f ta

CcH3

C-4a C-5a N (CH-3)2 c-4 C-6 C-12a c- 2 C-lla C-lOa c-9 c-7 C-8 C-6a c-10 corn2 c-12 c- 3

c-1 c-11

25.1 27.0 34.9 42.0 48.5 68.3 70.4 73.3 95.5 106.1 117.0 118.9 121.3 139.7 143.6 160.8 172.0 175.3 187.1 192.1 193.3

q t d d q d

d d

DMSO-d6, p a r t s p e r m i l l i o n from T S (0.00 ppm). M quadruplet, t = t r i p l e t , d = doublet. 4.3

q =

Mass S p e c t r o m e t r y Mass s p e c t r a l s t u d i e s , i n c l u d i n g low v o l t a g e t e c h n i ques and a c c u r a t e m a s s measurements, have been r e p o r t e d by Hoffman (40) on t e t r a c y c l i n e and e i g h t r e l a t e d compounds. A s a r e s u l t of good s p e c t r a l c o r r e l a t i o n s among t h e s e compounds, t h e m a j o r f r a g m e n t a t i o n p r o c e s s e s are d i s c u s s e d i n terms of two compounds i n t h e series: 50,,6-anhydrotetracycline and dedimethylaminotetracycline, w i t h o n l y a s e l e c t e d number of i o n s r e p o r t e d f o r CTC. F u r t h e r s t u d i e s on t h e s e a n t i b i o t i c s , i n c l u d i n g CTC, w e r e conducted by M o r r i s and C a i r n s ( 4 1 ) . However, l i t t l e i n f o r m a t i o n on t h e i r r e s u l t s is i n c l u d e d i n t h e extended a b s t r a c t of t h e m e e t i n g , and a f u l l r e p o r t h a s n o t

122

GEORGE SCHWARTZMAN ET A L .

been p u b l i s h e d . P e r s o n a l communications w i t h t h e a u t h o r s i n d i c a t e t h a t t h e r e s u l t s of t h e i r work a r e i n c l o s e agreement with d a t a reported here. The f r a g m e n t a t i o n p a t t e r n of CTC can be d i s c u s s e d i n terms of t h r e e d i f f e r e n t mechanisms. These are: a. P r o c e s s e s r e s u l t i n g from t h e c a p a c i t y of t h e dimethylamino group t o d i r e c t l o s s e s of small fragments t o g e t h e r w i t h i t s a b i l i t y t o r e t a i n and s t a b i l i z e t h e p o s i t i v e charge. b. Fragmentation of t h e D r i n g system w i t h c h a r g e r e t e n t i o n on t h e f u s e d r i n g p o r t i o n of t h e molecule. c. Fragmentation of t h e D r i n g system w i t h c h a r g e r e t e n t i o n elsewhere t h a n on t h e f u s e d r i n g moiety. The mass spectrum of CTC, shown i n F i g u r e 1 4 ( 4 2 ) , is c h a r a c t e r i z e d by a r e a s o n a b l y i n t e n s e r ; o l e c u l a r i o n a t m / e 4 7 8 w i t h t h e concomitant i s o t o p e peak a t P+2 r e p r e s e n t i n g one c h l o r i n e atom i n t h e r i n g system. Although i t h a s been suggested t h a t t h i s c h l o r i n e atom b e employed as a t r a c e r v i a t h e 3 7 ~ 1 s o t o p e r a t i o f o r d e t e c t i o n of s p e c i e s c o n t a i n i n g i t h e A r i n g and a d j u n c t r i n g systems, many of t h e s e i o n s are of such low r e l a t i v e i n t e n s i t y t h a t t h i s would have o n l y l i m i t e d u s e f u l n e s s toward t h a t end, e x c e p t perhaps a t h i g h e r mass values. The dominance of t h e f r a g m e n t a t i o n p r o c e s s e s by t h e p r e s e n c e of t h e 4-dimethylamino group i v e s r i s e t o t h e i n t e n s e i o n s a t m / e 4 4 , m / e 58 (CH3)2-i=CH2, m / e 7 1 (CH3)2ftCH=CH2, and m / e 84 (CH3)2-$=CH-CH=CH2. The i o n a t m / e 98 (C+8NO+) a p p e a r s t o i n v o l v e a c y c l i z a t i o n of t h e dimethylamino group w i t h e l e m e n t s of t h e D r i n g . Of p a r t i c u l a r n o t e i s t h e l o s s of 43 atomic mass u n i t s from t h e m o l e c u l a r i o n t o g i v e t h e i n t e n s e i o n a t m / e 435 and from t h e i o n a t m / e 443 t o y i e l d m / e 400 ( F i g u r e 1 5 ) . The p r e s e n c e of t h e amide f u n c t i o n a l group i s i n d i c a t e d by o b s e r v a t i o n s of s u c c e s s i v e l o s s e s of NH3 and OH ( a s w a t e r ) from t h e m o l e c u l a r i o n t o y i e l d u l t i m a t e l y t h e i o n a t m / e 443. The l o s s of water a t m/e 460 a p p e a r s t o be s l i g h t l y f a v o r e d over t h e competing l o s s of NH3 a t m / e 462 from t h e abundances of t h e r e s p e c t i v e i o n s . Fragmentation of t h e D r i n g can p r o v i d e t h e d u a l purpose of d e t e r m i n i n g t h e s u b s t i t u e n t a t t h e 4 p o s i t i o n (dimethylamino i n t h e case of CTC) a n d / o r h e l p t o l o c a t e f u n c t i o n a l i t i e s e l s e w h e r e i n t h e r i n g system. This,however, r e q u i r e s a d e t a i l e d examination of a n a l o g s such a s i n t h e work of Hoffman ( 4 0 ) . The i o n a t m / e 365 w i l l l o c a t e t h e s u b s t i t u e n t s a t t h e 2 and 3 p o s i t i o n s of t h e D r i n g by d e t e r m i n a t i o n of t h e mass of t h e l o s t s u b s t i t u e n t and a l s o g i v e e v i d e n c e c o n c e r n i n g t h e e x t e n t of s u b s t i t u t i o n on t h e A , B, and C r i n g s . T h i s

SPEC.

NO.

2 0 0 3 9 LS CHLORTETRRCYCLINE HCL

- '"1 * * c
z
L

100-

In
U
U

44

v)

w IW

75-

; so1 a
W 2

25

MASS T O CHRRCE RRIIO

w
433
170

M '

Figure 14. Electron impact mass spectrum of CTC-HC1. Instrument: Varian MAT 311; source temperature sufficient to produce vaporization. (Chemical ionization using ammonia as reagent gas gave MH+ as base peak. 1

I24

GEORGE SCHWARTZMAN ET A L .

365 435

461

460

I70

400 I

275 -

370

3--'O

Figure 15.

Major fragmentation pathways of CTC involving molecular ion and other significant high mass ions. Underlined m/e values indicate confirmation by accurate mass measurements.

CHLORTETRACYCLINE HYDROCHLORIDE

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knowledge p l u s t h e observed s h i f t of t h e i o n a t m / e 170 s h o u l d s u f f i c e t o a l l o w p o s t u l a t i o n of t h e s u b s t i t u t i o n ( F i g u r e 1 6 ) .

CHROMATOGRAPHY 5.1 Paper and Thin Layer Some of t h e e a r l y r e p o r t s on t h e chromatography of t h e t e t r a c y c l i n e a n t i b i o t i c s p r i o r t o 1957 are of l i m i t e d v a l u e . Fischbach and Levine (43) d e s c r i b e d a c o n t i n u o u s ascending t e c h n i q u e and B e r t i and C i m a (44) r e p o r t e d an ascending method u s i n g aqueous sodium a r s e n i t e as t h e mobile s o l v e n t . Other a u t h o r s (45,46) r e p o r t e d descending t e c h n i q u e s and b i o a u t o g r a p h i c means f o r l o c a t i n g t h e zones of a c t i v i t y . A l l of t h e s e methods f a i l t o show t h e p r e s e n c e of t h e e p i m e r i c form of t h e t e t r a c y c l i n e s and i n most i n s t a n c e s s t r e a k i n g of t h e s p o t s i s a problem. A b a s i c improvement i n t h e paper chromatography of t h e s e a n t i b i o t i c s w a s a c h i e v e d by S e l z e r and Wright (47) and K e l l y and Bryske ( 4 8 ) when t h e y r e p o r t e d methods f o r t h e p r e t r e a t m e n t of t h e paper w i t h comp l e x i n g a g e n t s t o bind t h e m e t a l l i c i o n s which may be p r e s e n t . The t e t r a c y c l i n e s are w e l l known f o r t h e i r a b i l i t y t o form complexes w i t h p o l y v a l e n t c a t i o n s . T h i s p r o p e r t y changes t h e i r s o l u b i l i t y c h a r a c t e r i s t i c s i n t h e m o b i l e s o l v e n t s and o f t e n r e s u l t s i n troublesome s t r e a k i n g . To overcome t h i s d i f f i c u l t y , S e l z e r and Wright used paper dipped i n M c I l v a i n e ' s b u f f e r (pH 3.5) which c o n t a i n s c i t r a t e i o n s c a p a b l e of b i n d i n g t h e metallic i o n s . The chromatograms were developed w i t h a m i x t u r e of n i t r o m e t h a n e , chloroform, and p y r i d i n e (20:10:3) on paper s t i l l damp from t h e t r e a t m e n t w i t h the buffer solution. For t h e same purpose, K e l l y and Bryske used p a p e r impregnated w i t h 0.1N disodium ethylenediaminetetraacetate (EDTA) and two mobile s o l v e n t s : t h e o r g a n i c phase from a m i x t u r e of E-butanol, ammonia, water (4:1:5) and t h e o r g a n i c phase from a m i x t u r e of g - b u t a n o l , a c e t i c a c i d , water ( 4 : l : 5 ) . Disodium EDTA (0.1N) works as w e l l as M c I l v a i n e ' s b u f f e r when i t is used t o t r e a t t h e paper i n t h e method of Walton e t a l . ( 4 9 ) . A c i r c u l a r paper chromatographic method a l s o u s i n g paper dampened w i t h M c I l v a i n e ' s b u f f e r (pH 4.5) w a s r e p o r t e d by Urx e t a l . ( 5 0 ) . They used a m i x t u r e of chloroform and c - b u t a n o l ( 4 : l ) as t h e mobile s o l v e n t . Most of t h e methods i n which t h e paper i s t r e a t e d with a c h e l a t i n g a g e n t are c a p a b l e of showing a s e p a r a t i o n of some of t h e t e t r a c y c l i n e d r u g s from e a c h o t h e r and from t h e i r r e s p e c t i v e e p i m e r i c forms. They a r e a l s o c a p a b l e of r e v e a l i n g t h e p r e s e n c e of common d e g r a d a t i o n compounds of t h e s e d r u g s . The u s u a l method of d e t e c t i n g chromatographed t e t r a c y c l i n e a n t i b i o t i c s i n v o l v e s fuming t h e paper w i t h ammonia vapor and o b s e r v i n g t h e yellow f l u o r e s c e n c e under UV l i g h t . A s l i t t l e as 0.2-0.5 pg can b e v i s u a l i z e d by t h i s technique.

5.

2 ; :&f (

" CH30H

,N$ CH3 CH3

W e 365

-6
iJ? ' \
N+ / \
II

0 0 6-NH2 \ / C

II

/'\OH Ch CH, CH3 CH3 CH3

Mf
Figure 16.

W e 170

Proposed structures for ions at m/e 365 and m/e 170 for use in determination of ring substituents.

CHLORTETRACYCLINE HYDROCHLORIDE

I27

Some of t h e f e a t u r e s t h a t are s u c c e s s f u l f o r t h e chromatography of t h e t e t r a c y c l i n e s o n paper have been adapted t o t h i n l a y e r chromatography (TLC). Complexing a g e n t s are almost always used i n t h e p r e p a r a t i o n of t h e p l a t e s a n d , i n a d d i t i o n , s e v e r a l methods f o r h y d r a t i n g t h e p l a t e s by t h e a d d i t i o n of g l y c e r i n a n d / o r p o l y e t h y l e n e g l y c o l have been reported. Several i n v e s t i g a t o r s f i n d i t d e s i r a b l e t o acidwash t h e i n o r g a n i c s u p p o r t i n o r d e r t o remove m e t a l l i c c a t i o n s . These TLC methods are much more time-consuming t h a n most of t h e paper chromatographic p r o c e d u r e s because of t h e s p e c i a l care o f t e n r e q u i r e d t o p r e p a r e and s t o r e t h e TL,C d a t e s . For t h i s r e a s o n , where a p p l i c a b l e , t h e p a p e r chromaHowever, f o r q u a n t i t a t i v e t o g r a p h i c methods .are p r e f e r a b l e . a n a l y s i s of t h e t e t r a c y c l i n e epimers and d e g r a d a t i o n p r o d u c t s , TLC i s u s u a l l y c o n s i d e r e d t o b e b e t t e r t h a n p a p e r chromatography. I n 1964, Somanini and Anker (51) d e s c r i b e d a method u s i n g K i e s e l g u h r l a y e r s impregnated w i t h a b u f f e r s o l u t i o n c o n t a i n i n g g l y c e r i n . Nishimoto e t a l . (52) r e p o r t e d t h e u s e of s i l i c a g e l l a y e r s t r e a t e d w i t h EDTA. I n 1967, Ascione e t a l . (53) p u b l i s h e d a method u s i n g l a y e r s made from acid-washed diatomaceous e a r t h . They p r e p a r e d p l a t e s c o n t a i n i n g 0.1N EDTA, g l y c e r i n , and p o l y e t h y l e n e g l y c o l 400. Other i n v e s t i g a t o r s (54,55) have d e s c r i b e d methods which are m o d i f i c a t i o n s of improvements of t h e methods p r e v i o u s l y p u b l i s h e d . G a s and L i q u i d Chromatography I n v e s t i g a t o r s have found i t q u i t e d i f f i c u l t t o chromatograph t h e t e t r a c y c l i n e s by g a s - l i q u i d t e c h n i q u e s . Often, o n l y fragments o f t h e o r i g i n a l sample are o b t a i n e d (56). T s u j i and Robertson (57) d i d manage t o chromatograph s i l y l a t e d CTC u s i n g 3% methyl s i l i c o n e on Gas-Chrom Q and o t h e r s t a t i o n a r y phases. However, w i t h t h e a d v e n t of r e f i n e d h i g h p r e s s u r e l i q u i d chromatographic (HPLC) t e c h n i q u e s , i n t e r e s t i n g a s chromatographic methods f o r t h e t e t r a c y c l i n e s has diminished. A number of p a p e r s have appeared r e p o r t i n g t h e HPLC s e p a r a t i o n of CTC from i t s isomers a n d / o r o t h e r t e t r a c y c l i n e s . There i s n o t a consensus of o p i n i o n as t o t h e most s a t i s f a c t o r y approach; t h u s , i t a p p e a r s t h a t a t t h i s t i m e one must s t i l l v e r i f y t h e o p t i m a l system f o r a p a r t i c u l a r i n s t r u m e n t . Methods found i n t h e l i t e r a t u r e f o r CTC a r e d e s c r i b e d i n Table 6. EDTA is added t o p r e v e n t t h e f o r m a t i o n of complexes of t h e t e t r a c y c l i n e s w i t h m e t a l l i c s u r f a c e s . Other chromatographic t e c h n i q u e s t h a t have been a p p l i e d t o t h e t e t r a c y c l i n e s , i n c l u d i n g CTC, i n v o l v e low p r e s s u r e column chromatography. Ascione e t a l . (64) developed a semiautomated system whereby sample s o l u t i o n s are a u t o m a t i c a l l y i n j e c t e d o n t o a column of diatomaceous e a r t h mixed w i t h

5.2

TABLE 6 High P r e s s u r e L i q u i d Chromatography of CTC

Mobile Phase 20% Methanol, 80% 0.05M ammonium c a r b o n a t e , 0.02M EDTA Phosphate b u f f e r i n 13% methanol, 0.85 ml/min., pH 2.5 Aqueous p e r c h l o r a t e - c i t r a t e b u f f e r mixed w i t h CH3CN
EDTA, N a C l i n 30% methanol, pH 9.9 EDTA, PO4, isopropanol-water, pH 7.6, 2 ml/min.

S t a t i o n a r y Phase C8/Lichrosorb 1 0 pm, 25 c m x 3 . 2 mm Zipax-hydrocarbon polymer, 2 . 1 x 1000 mm Sil-X, 13 Urn, 5 x 125 mm Ion-X-SA, a n i o n exchanger

Migration T i m e (min. ) 34.5

Ref. 58

59 60

1.3

NH3,

3
11.6

61

UBondapak c18, 300 x 4 mm Zipak, 1.8 x 1000 mm

62

EDTA, NO3, 9.6% e t h a n o l , pH 9 , 1 rnllmin.

22

63

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a s o l u t i o n of EDTA and p o l y e t h y l e n e g l y c o l . CTC i s e l u t e d w i t h t h e o r g a n i c phase of a m i x t u r e of chloroform, benzene, aqueous s o l u t i o n of EDTA, and p o l y e t h y l e n e g l y c o l . Ragazzi and Veronese (65) s e p a r a t e d CTC from o t h e r t e t r a c y c l i n e s by means of g e l permeation chromatography.
MANUFACTURE 6.1 Fermentation A medium ( c o n t a i n i n g c o r n s t e e p l i q u o r ; c a l c i u m c a r b o n a t e : s u c r o s e ; ammonium, f e r r o u s , manganese, and z i n c s u l f a t e s ; and ammonium, c o b a l t , and magnesium c h l o r i d e s ) i s s t e r i l i z e d and d i l u t e d w i t h water t o t h e d e s i r e d c o n c e n t r a t i o n . It is i n o c u l a t e d w i t h Streptomyces a u r e o f a c i e n s , k e p t a t 27"C, and a e r a t e d and a g i t a t e d f o r %60 h o u r s , w i t h l a r d o i l added t o c o n t r o l foaming ( 6 6 ) . Isolation The mash from t h e Streptomyces a u r e o f a c i e n s ferment a t i o n b r o t h i s a c i d i f i e d and f i l t e r e d . The f i l t r a t e i s a d j u s t e d t o t h e d e s i r e d pH, u s u a l l y 7-8.5, and v a r i o u s f l o c c u l a t i n g o r c h e l a t i n g a g e n t s may be added ( e . g . , v i n y l acetatem a l e i c a n h y d r i d e copolymer, sodium EDTA, ammonium o x a l a t e , Arquad). The p r e c i p i t a t e i s ( 1 ) s t i r r e d w i t h f i l t e r a i d , f i l t e r e d , s t i r r e d w i t h HCI., r e f i l t e r e d , mixed w i t h 2e t h o x y e t h a n o l , f i l t e r e d , washed, and t h e f i l t r a t e s are combined, a c i d i f i e d w i t h HC1, N a C l i s added, and t h e c r y s t a l s a r e c o l l e c t e d , washed w i t h 2-ethoxyethanol, water, and e t h a n o l , and d r i e d ( 6 7 ) , o r (2) e x t r a c t e d i n t o methyl i s o b u t y l k e t o n e , t h e e x t r a c t s are combined, f i l t e r e d , and a c i d i f i e d w i t h H C 1 , and t h e c r y s t a l s are c o l l e c t e d and washed w i t h w a t e r , 2-ethoxyethanol, and i s o p r o p a n o l , and vacuumd r i e d . I f t h e c r y s t a l s are g r e e n i s h , t h e y a r e t r e a t e d w i t h sodium h y d r o s u l f i t e a t p 1 . 8 , f i l t e r e d , washed, and d r i e d as H i n ( 1 ) above (68). STABILITY CTC-HC1, a s a d r y powder, i s a s t a b l e y e l l o w c r y s t a l l i n e material. The s i t u a t i o n i n aqueous s o l u t i o n , however, i s q u i t e d i f f e r e n t . I n sodium hydroxide s o l u t i o n s , CTC is c o n v e r t e d t o iso-CTC on s t a n d i n g ( 6 9 ) . The s o l u t i o n becomes c o l o r l e s s and e x h i b i t s a s t r o n g b l u e f l u o r e s c e n c e under UV l i g h t . D i l u t e s o l u t i o n s of CTC, i n pH 7.5 b u f f e r , make t h e same c o n v e r s i o n a t 100C. I n a c i d s o l u t i o n s , CTC i s c o n v e r t e d t o anhydro-CTC ( 7 0 ) . T h i s change is g r e a t l y a c c e l e r a t e d by h e a t i n g and r e s u l t s i n a yellow p r o d u c t t h a t h a s a maximum absorbance a t 445 nm. I n a d d i t i o n , CTC undergoes r e v e r s i b l e e p i m e r i z a t i o n of t h e 4-dimethylamino group ( 7 1 ) . T h i s o c c u r s s l o w l y i n w a t e r o r methanol b u t i s h a s t e n e d i n b u f f e r s o l u t i o n s i n t h e r a n g e 6.2

6.

7.

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GEORGE SCHWARTZMAN ET AL.

of pH 2-6. The rate of e p i m e r i z a t i o n i s u n d e t e c t a b l e i n s o l u t i o n s more a c i d i c t h a n pH 2 ( 7 2 ) . The a n t i m i c r o b i a l a c t i v i t y of t h e epimer is probably z e r o . The s l i g h t a c t i v i t y found f o r t h i s material i s probably due t o t h e reemergence o f CTC under t h e t e s t c o n d i t i o n s . I n a f a s h i o n analogous t o t h a t of CTC, i t s epimer can be converted t o epianhydro-CTC i n a c i d s o l u t i o n .
ANALYTICAL METHODS 8.1 Microbiological The m i c r o b i o l o g i c a l methods used f o r t h e determinat i o n of CTC potency i n body t i s s u e s and f l u i d s , b u l k p r o d u c t s , and p h a r m a c e u t i c a l f o r m u l a t i o n s can b e s e p a r a t e d i n t o two (1) a g a r d i f f u s i o n p l a t e method t e s t i n g procedures: ( c y l i n d e r - p l a t e ) and ( 2 ) t u r b i d i m e t r i c method. 1. Agar d i f f u s i o n p l a t e method ( c y l i n d e r - p l a t e ) : T h i s method is employed f o r d e t e r m i n i n g t h e potency of CTC i n human and animal p h a r m a c e u t i c a l formulat i o n s , b u l k p r o d u c t s , serum, t i s s u e s , u r i n e , d a i r y p r o d u c t s , and animal f e e d s . The c y l i n d e r - p l a t e procedure i s d e s c r i b e d by Grove and Randall (73) and t h e Code of F e d e r a l R e g u l a t i o n s (74). A d d i t i o n a l methods u s i n g t h i s a s s a y are d e s c r i b e d by K r a m e r e t a l . (75). The o f f i c i a l f i n a l a c t i o n method f o r CTC a s s a y i n animal f e e d s i s d e s c r i b e d i n t h e A s s o c i a t i o n of O f f i c i a l A n a l y t i c a l Chemists' O f f i c i a l Methods of A n a l y s i s

8.

(76).
method: T h i s method i s used i n l i e u of t h e d i f f u s i o n p l a t e method f o r human and animal p h a r m a c e u t i c a l f o r m u l a t i o n s and b u l k p r o d u c t s . The t u r b i d i m e t r i c method i s d e s c r i b e d i n t h e Code of F e d e r a l R e g u l a t i o n s (77). Chemical The p h y s i c a l s t r u c t u r e o f CTC h a s provided a good s o u r c e of c h a r a c t e r i s t i c s u s e f u l f o r t h e a n a l y s i s of t b i s a n t i b i o t i c . Although t i t r i m e t r i c (78) and p o l a r o g r a p h i c (79) methods have been r e p o r t e d , t h e most u s e f u l p r o c e d u r e s have been based on t h e s p e c t r o s c o p i c p r o p e r t i e s of CTC and i t s d e r i v a t i v e s (80). I n 1949, Levine e t a l . (81) p u b l i s h e d two p r o c e d u r e s f o r t h e a s s a y of CTC. One method w a s based on t h e c o n v e r s i o n of CTC by h e a t i n g i n a c i d t o t h e more i n t e n s e l y yellow anhydro-CTC d e r i v a t i v e . The o t h e r method w a s based on measuri n g t h e b l u e f l u o r e s c e n c e of iso-CTC, which w a s p r e p a r e d by h e a t i n g CTC i n pH 7.5 phosphate b u f f e r . O t h e r s have d e s c r i b e d m o d i f i c a t i o n s of t h e s e methods f o r v a r i o u s purposes. Hiscox (82) s u g g e s t e d t h e d i r e c t spect r o p h o t o m e t r i c a s s a y of CTC i n e i t h e r a c i d o r a l k a l i n e s o l u t i o n a t v a r i o u s UV wavelengths. The p o s s i b l e c o n t a m i n a t i o n of CTC w i t h o t h e r t e t r a c y c l i n e d r u g s w a s a d d r e s s e d by C h i c e a r e l l i e t 8.2

2.

Tur -b i d i m e t r i c -

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a l . ( 8 3 ) . They c o r r e c t e d f o r t h e p o s s i b l e p r e s e n c e of t e t r a c y c l i n e i n CTC u s i n g t h e f a c t t h a t t h e former i s unchanged i n d i l u t e a l k a l i w h i l e CTC is converted t o t h e c o l o r l e s s isoCTC The t e t r a c y c l i n e which may be p r e s e n t i s t h e n converted t o a n anhydro d e r i v a t i v e by h e a t i n g i n a c i d and i s measured s p e c t r o p h o t o m e t r i c a l l y . Feldman e t a l . ( 8 4 ) developed t h e a l k a l i n e d e g r a d a t i o n method t o measure CTC i n f e r m e n t a t i o n mash and Spock and Katz (85) used t h i s method t o determine CTC i n animal f e e d premixes. The n a t u r a l f l u o r e s c e n c e of CTC and i t s d e r i v a t i v e s has been used e x t e n s i v e l y t o determine s m a l l amounts of CTC i n b i o l o g i c a l m a t e r i a l s . Kohn (86) showed t h a t t h e f l u o r e s c e n t complex formed by CTC w i t h calcium i o n s and b a r b i t a l could be e x t r a c t e d from animal t i s s u e s i n t o an o r g a n i c s o l v e n t and t h e n measured spectrofluorometrically. The i n t e n s e f l u o r e s c e n c e of anhydro-CTC was used by Hayes and DuBuy (87) t o determine CTC i n animal t i s s u e s , t i s s u e c u l t u r e c e l l s , and b a c t e r i a . P o i g e r and S c h l a t t e r (88) e x t r a c t e d CTC from biol o g i c a l material i n t o e t h y l acetate as t h e CTC-calcium t r i c h l o r o a c e t a t e i o n p a i r . The f l u o r e s c e n c e of t h e a n t i b i o t i c w a s t h e n enhanced by t h e a d d i t i o n of magnesium i o n s and a base.

132

GEORGE SCHWARTZMAN ET REFERENCES

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2. Remington's Practice of Pharmacy, 12th Ed., Mack Publishing Co., Easton, PA, p. 1172 (1961). 3. Physicians' Desk Reference, 31st Ed., Medical Economics Inc., Oradell, NJ, p. 890 (1977).

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12. C. Stephens, L. Conover, R. Pasternack, F. Hochstein, W. Moreland, P. Regna, F. Pilgrim, K. Brunings, and R. Woodward, J. her. Chem. SOC. 76, 3568-3575 (1954). 13. V. Dobryin, A. Gurevich, M. Karapetyan, M. Kolosov, and M. Shemyakin, Tetrahedron Lett. 901 (1962). 14. J. Donohue, J. Dunitz, K. Trueblood, and M. Webster, J. Amex. Chem. SOC. 85, 851-856 (1963). -

15. M. Maienthal, FDA, Washington, DC, unpublished data (1977).

16. V. Folen, FDA, Washington, DC, unpublished data (1977).

17. M. Andrew and P. Weiss, Antibiot. Chemother. 2, 277-279 (1959).

CHLORTETRACYCLINE HYDROCHLORIDE

133

18.

T. P a r k e and W. Davis, Anal. Chem.

2 , 642-645

(1954).

19. C . S t e p h e n s , K. Murai, K. B r u n i n g s , and R. Woodward, J . A m e r . Chem. SOC. 78, 4155 (1956). 20. L. Leeson, J. Krueger, and R. Nash, T e t r a h e d r o n L e t t . 1155-1160 (1963).

18,

21. K. K a l n i n s and B.G. B e l e n ' s k i i , Dokl. Akad. Nauk. SSSR, 1 5 7 ( 3 ) , 619-621 (1964); CA 61, 9381f (1964). 22. Code of F e d e r a l R e g u l a t i o n s , T i t l e 21, p a r t 446.10a ( a ) (1)( v i i ) (1977).

23. S. Miyazaki, M. Nakano, and T. A r i t a , Chem. Pharm. B u l l . 81, Tokyo 22, 638 ( 1 9 7 4 ) ; CA - 413312 (1974). 24. S. Miyazaki, M. Nakano, and T . A r i t a , Chem. Pharm. B u l l . Tokyo 23, 552-558 (1975). 25. K. F u r n k r a n z , FDA, Washington, D.C.,unpublished d a t a (1977). 26. A. Hayden, 0. Sammul, G . S e l z e r , and J . C a r o l , J. Assoc. Off. A g r i c . Chem. 4 5 , 797 (1962).

27. A t l a s of S p e c t r a l Data and P h y s i c a l C o n s t a n t s f o r Organic Compounds, J . G . G r a s s e l l i , Ed., CRC P r e s s , C l e v e l a n d , OH, B-189 (1974). 28. J . L a c h e r , J. B i t n e r , and J . P a r k , J . Phys. Chem. 50, 610-614 (1955).
29. M. Avram and G . Matescu, I n f r a r e d S p e c t r o s c o p y , WileyI n t e r s c i e n c e , New York, p. 323 (1972). 30. K. N a k a n i s h j I n f r a r e d A b s o r p t i o n S p e c t r o s c o p y , Holden-Day I n c . , San F r a n c i s c o , CA, p. 4 1 and 1 9 8 (1962). 31. R . Conley, I n f r a r e d S p e c t r o s c o p y , 2nd Ed., A l l y n & Bacon, I n c . , Boston, p. 1 4 8 (1972). 32. Methods i n Enzymolopy, Vol. 43, J . H . P r e s s , New York, p. 363 (1975). Hash, Ed., Academic

33. V . Dobryin, A. Gurevich, M. K a r a p e t y a n , M. Kolosov, and M. Shemyakin, T e t r a h e d r o n L e t t . 20, 901-904 (1962). 34. L.A. M i t s c h e r , A.C. Bonacci,and T.D. S o k o l o s k i , Antimicrob. Agents Chemother. 78-86 (1968).

134

GEORGE SCHWARTZMAN ET A L .

35. V. Folen, FDA, Washington, DC, unpublished data (1977).

- 2397-2405 (1975). 97,

38.

36.

D. Williamson and G. Everett, Jr., J. Amer. Chem. SOC.

37. E Sheinin, FDA, Washington, DC, unpublished data (1978). . C. Frank, J. Amer. Chem. SOC. 97, 6246 (1975).

39. E. Mazzola, FDA, Washington, DC, unpublished data (1978).

40. D. Hoffman, J. Org. Chem.

2,792-796

(1966).

41. W. Morris and T. Cairns, 25th Annu. Conf. on Mass Spectrosc. and Allied Topi., paper Tp-20, Washington, DC, p. 601 (1977).
42. R. Barron, FDA, Washington, DC, unpublished data (1977). 43. H. Fischbach and J. Levine, Antibiot. Chemother. (1955)

2, 640

44. T. Berti and L. Cima, Z l . 1st Sieroter. Milan.. 3 3 , 643

(1954). 45. H. Bird and C. Pugh, Antibiot. Chemother. 4, 750 (1954).

46. R. Hickey and W. Phillips, Anal. Chem.

6, 1640

(1954).

47. G. Selzer and W. Wright, Antibiot. Chemother. (1957).

I, 292

48. R. Kelly and D. Bryske, Antibiot. Chemother. (90. 16)

10, 604

49. V. Walton, M. Howlett, and G. Selzer, J. Pharm. Sci. 59, 1160 (1970).
50. M. Urx, L. Vondrackova, L. Kovarike, 0 Hersky, and . M. Herold, LChromatogr. l , (1963). l 62 51. D. Somanini and L. Anker, Pharm. Acta Helv. (1964).

2,518

-- 87, 516 Zasshi

52. Y. Nishimoto, E. Tsuchida, and S. Toyoshima, Yakugaku (1967).

53. P. Ascione, J. Zager, and G. Chrekian, J. Pharm. ScL. 56, 1393 (1967).

CHLORTETRACYCLINE HYDROCHLORIDE

135

- 59, Sci.
55.

54.

N. Gyanchandani, I. McGilivery, and D. Hughes, J. Pharm. 224 (1970). I. Dijkhuis and M. Brommet, J. Pharm. Sci. 59, 558 (1970).

56. M. Margosis, FDA, Washington, DC, private communication (1977). 57. K. Tsuji and J. Robertson, Anal. Chem. - 2136-2140 45, (1973). 58. E. White, M. Carroll, and J. Zarembo, J. Antibiot. 30(10) , 811-818 (1977). Tokyo 59. K. Tsuji, J. Robertson, and W. Beyer, Anal. Chem. 539-543 (1974).

9,

60. J. Knox and J. Jurand, J. Chromatogr. 110, 103-115 (1975).

61. J. Sharma, G. Koritz, E. Perkins, and R. Bevill, L. Pharm. Sci. 66, 1819-1822 (1977).

62. G. Mack, FDA, Baltimore, MD, unpublished data (1977). 63. A. Butterfield, D. Hughes, N. Pound, and W. Wilson, Antimicrob. Agents Chemother. k, 11.-15 (1973).

- 377-386 65,

64. P. Ascione, J. Zager, and G. Chrekian, J. Chromatogr. (1972).

65. E. Ragazzi and G. Veronese, J. Chromatogr. (1977).

134, 223-229

66. American Cyanamid, U.S. Patent 2,709,672 (May 31, 1955); CA 49,11966a (1955). 67. American Cyanamid, British Patent 808,362 (Feb. 4, 1959); CA 53,11769~(1959).
68. American Cyanamid, U.S. Patent 2,875,247 (Feb. 24, 1959); CA !i3,11770a (1959). 69. C. Waller, B. Hutchings, C. Wolf, A Goldman, . 74, R. Broschard, and J. Willi&s, J. Amer. Chem. SOC. 4981 (1952).

70. C. Waller, B. Hutchings, R. Broschard; A. Goldman, W. Stein, C. Wolf, and J. Williams, J. Amer. Chem. SOC. 4981 (1952).

74,

136

GEORGE SCHWARTZMAN ET A L .
A. Doerschuk, B. B i t l e r , and J . McCormack, J . Amer. 4687 (1955).

71.

SOC.

z,

=.

72. J. McCormack, S. Fox, L. Smith, B. B i t l e r , J . R e i c h e n t h a l , V. O r i g o n i , W. M u l l e r , R. Winterbottom, and A. Doerschuk, J. Amer. Chem. SOC. 2, 2849 (1957). 73. D. Grove and W. R a n d a l l , Assay Methods of A n t i b i o t i c s : A L a b o r a t o r y Manual, Medical E n c y c l o p e d i a , I n c . , New York (1955). 74. Code of F e d e r a l R e g u l a t i o n s , T i t l e 21, C h a p t e r 1, p a r t 446.10a ( b ) ( i - v i i ) (1977). 75. J. Kramer, G. Carter, B. Arret, J. W i l n e r , W. W r i g h t , and A. Kirshbaum, A n t i b i o t i c R e s i d u e s i n M i l k , D a i r y P r o d u c t s , and Animal T i s s u e s : M e t h o d s , R e p o r t s , and P r o t o c o l s , N a t i o n a l C e n t e r f o r A n t i b i o t i c s and I n s u l i n A n a l y s i s , FDA, Washington, DC (1968). 76. O f f i c i a l Methods of A n a l y s i s , 1 2 t h Ed., s e c s . 42.20742.210, A s s o c i a t i o n of O f f i c i a l A n a l y t i c a l Chemists, Washington, DC (1975).

77. Code o f F e d e r a l R e g u l a t i o n s , T i t l e 21, C h a p t e r 1. p a r t 446.10a ( b ) ( v i i i ) (a-d) (1977).

42,

78.

C . S i d e r i and A. Osol, J. Amer. Pharm. Assoc. S c i . Ed. 688 (1953).

79.

C. O l l i f f and L. C h a t t e r , . J .

Pharm. S c i .

66,

1564 (1977). 63-67

80. D. Labza and B. J a n i k , Acta Pol. P h a r m . , X ( l ) , (1977).

81. J . L e v i n e , E. Garlock, and H. F i s c h b a c h , J . A m e r . Pharm. Assoc. S c i . Ed. 38, 473 (1949). 82. D. Hiscox, J. h e r . Pharm. A s s o c . . S c i . (1951). Ed. 40, 237

83. S. C h i c e a r e l l i , M. Wodford, and M. Avery, J. Amer. Pharm. Assoc. S c i . Ed. 48, 263 (1959). 84. D. Feldman, H. Kelsey, and J. Cauagnol, Anal. Chem. 1607 (1957). 85. J . Spock and S. Katz, J.ssoc. 434 (1963). O f f i c . e i c . Chem.

2,

46,

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K . Kohn, Anal. Chem.

137

3 , 862

(1961).

J . Hayes and H . DuBuy, Anal. Biochem.

7,

322 ( 1 9 6 4 ) . (1976).

H . Poi ger and C . S c h l a t t e r , Analyst

101, 808

Analytical Profiles of D u Substances, 8 rg

DOBUTAMINE HYDROCHLORIDE
RaJk H . Bishara and Harlan B. Long
I.

2.

3. 4. 5.

6.

7.

Description I . I Nomenclature I . I . 1 Chemical Name I . I .2 Nonproprietary Name I . I . 3 Proprietary Name 1.2 Formula I .2.1 Empirical 1.2.2 Structural I . 3 Molecular Weight I .4 Appearance, Color, Odor, and Taste Physical Properties 2. I Melting Range 2.2 Simple Solubility Profile 2.3 pHRange 2.4 Dissociation Constant (pKa) 2.5 Thermal Analyses 2.5. I Differential Thermal Analysis 2.5.2 Thermogravimetric Analysis 2.6 Crystallinity 2.6. I Crystalline Habit 2.6.2 X-Ray Powder Diffraction 2.7 Ultraviolet Spectrum 2.8 Infrared Spectrum 2.9 Nuclear Magnetic Resonance Spectrum 2.10 Mass Spectrum Synthesis Stability-Degradation Absorption, Metabolism, and Excretion 5.1 In Dog 5.2 In Man Methods of Analysis 6. I Elemental Analysis 6.2 Chloride Identity 6.3 Nonaqueous Titration 6.4 Chloride Determination 6.5 Chromatography 6.5.I Thin-Layer Chromatography 6.5.2 Gas Chromatography 6.5.3 High Performance Liquid Chromatography Analysis of Biological Samples 7. I Enzymatic Assay 7.2 Chromatographic Assays 7.2.1 Thin-Layer Chromatography 7.2.2 Gas Chromatography 7.2.3 High Performance Liquid Chromatography 7.3 Mass Spectrometry (GUMS)
139
Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-260808-9

140

RAFIK H. BISHARA AND HARLAN B. LONG

8. Analysis of Pharmaceutical Formulations 8. I Chromatographic Assays 8. I . 1 Thin-Layer Chromatography 8.1.2 Gas Chromatography 8.1.3 High Performance Liquid Chromatography 8.2 Spectrophotometric (UV) 9. Acknowledgments 11. References

1. Description 1.1 Nomenclature 1.1.1 Chemical Name (f)-4-[2-[ [3-(4-Hydroxyphenyl)-lmethylpropyl]amino]ethyl]-1,2-benzenediol, hydrochloride 1.1.2 NonDrowietarv Name Dobutamine hydrochloride 1.1.3 Proprietary Name

DOBUTREF
1.2 Formula 1.2.1 Empirical C, 8H23N03 * HC 1 1.2.2 Structural

HO HO&)CH2-CH2-NH-LH-CH HCI

CH3 2 C H 2 0 0 H

DOBUTAMINE HYDROCHLORIDE

141

1 . 3 M o l e c u l a r Weight
337.85 1 . 4 Appearance, C o l o r , Odor, and Taste White t o o f f - w h i t e , s l i g h t l y b i t t e r taste. 2. P h y s i c a l P r o p e r t i e s 2 . 1 M e l t i n g Range 189 o d o r l e s s powder w i t h a

191C

2 . 2 Simple S o l u b i l i t y P r o f i l e The sample is s o n i c a t e d f o r one m i n u t e a t ambient t e m p e r a t u r e . Solvent mg/ m 1 >3.33 >2.50 >3.33 >3.33 >5.00 > l o . 00 >5.00 <O .50 <O . 5 0 <O. 50 <O .50 <O. 50 <O .50

Water p 1 . 2 (USP X I X ) H p 4 . 5 (USP X I X ) H pH 7 . 0 (USP X I X ) Ethanol Methanol Pyr i d i n e Oc t a n o l D i e t h y l ether Ethyl acetate Chloroform Benzene Cyc lohexane
2 . 3 DH Ranne

T h e pH of a 5% w/v s o l u t i o n i n a water/ e t h a n o l (1: 1) s o l u t i o n is a b o u t 4 . 9 .

2 . 4 Dissociation Constant The pKa i n dimethylformamide/water is 9 . 4 5 . (66:34)

142

RAFIK H. BISHARA AND HARLAN B. LONG

2.5 Thermal Analyses

2.5.1 Differential Thermal Analysis

A DTA thermogram of dobutamine hydrochloride, at a heating rate of 5C per min. in a nitrogen atmosphere of 40 cc per min., shows (figure 1) an endotherm at 196C which appears to indicate a melt,
2.5.2. Thermogravimetric Analysis

A TGA thermogram of dobutamine hydrochloride, run simultaneously with the DTA, shows (figure 1) no weight loss until 233C which results from decomposition.
2.6 Crystallinity

2.6.1 Crystalline Habit Dobutamine hydrochloride generally crystallizes in a random manner usually from an oil (1). This results in a nondescript crystalline formation. In only few cases does the drug exhibit any crystalline habit of interest. Upon careful and patient crptallization small thin plates and/ or small needles are formed. 2.6.2 X-Rav Powder Diffraction The following data describe the pattern for dobutamine hydrochloride, where d is equal to the integplanar spacing measured in terms of Angstroms (A). The ratio I/I, is the intensity of the X-ray maxima based upon a value of 100 for the strongest line. A b indicates a broad line resulting from failure to-resolve two closely spaced diffraction maxima.

Figure 1.

Thermogravimetric Analysis and Differential Thermal Analysis Thermograms of Dobutamine Hydrochloride

144

RAFIK H. BISHARA AND HARLAN B. LONG

CU-Ni- X 1.5418 A

d
9.07 6.87 6.37 5.27 4.98 4.50 4.13 4.04 3.78 3.60 3.44 3.18 3.11 3.01 2.86

1/11

d 2.74 2.63 2.51 2.40 2.35 2.27 2.22 2.14 2.11 2.06 2.01 1.99 1.95

1/11

7 5 7 18b 18 100 16 61 45 45 16b 18 7 14 7

14 9 16 5 2

7 7 7

2 . 7 U l t r a v i o l e t SDectrum The u l t r a v i o l e t s p e c t r u m o f d o b u t a m i n e h y d r o c h l o r i d e i n m e t h a n o l is g i v e n i n f i g u r e 2 . The s p e c t r u m e x h i b i t s maxima a t 2 8 1 and 223 nm w i t h molar a b s o r p t i v i t i e s of 4 , 7 6 8 and 1 4 , 4 0 0 , r e s p e c t i v e l y . When a q u e o u s p o t a s s i u m h y d r o x i d e is a d d e d t o t h e m e t h a n o l i c s o l u t i o n o f d o b u t a m i n e hydroc h l o r i d e t h e maxima a t 2 8 1 a n d 223 s h i f t t o 293 ( e = 6 , 1 0 0 ) and 240 nm, r e s p e c t i v e l y . T h e s e s h i f t s are r e v e r s i b l e by a d d i t i o n of h y d r o c h l o r i c a c i d . When t h e a b s o r p t i o n s p e c t r u m o f t h e d r u g is r e c o r d e d i n water r a t h e r t h a n m e t h a n o l , s l i g h t s h i f t s i n peak p o s i t i o n s and i n t e n s i t i e s are observed : x max = 278 nm ( e = 4 , 1 1 2 ) X max = 220 nm ( E = 1 3 , 5 0 0 ) . 2 . 8 I n f r a r e d Spectrum The i n f r a r e d s p e c t r u m of d o b u t a m i n e hydroc h l o r i d e i n a p o t a s s i u m bromide d i s k is g i v e n i n f i g u r e 3. Major band a s s i g n m e n t s are a s f o l l o w s :

DOBUTAMINE HYDROCHLORIDE

145

10

WAVELENGTH, nm

Figure 2 .

U l t r a v i o l e t Spectrum of Dobutamine Hydrochloride

.Ooo

c
Infrared Spectrum of Dobutamine Hydrochloride

Figure 3.

DOBUTAMINE HYDROCHLORIDE

147

Band P o s i t i o n , cm-' 3400, 3300 and 3140 2960 and 2840 2700 and 2450 (weak bands) 1610, 1530, 1520 and 1450 1440, 1390 and 1380 1360, 1280-1190 ( s e v e r a l bands) 1150 and lower

Assignment phenolic 0-H s t r e t c h i n g C-H s t r e t c h i n g (overl a p i n g 0 - H bands 1 Mainly NH2, N s t r e t c h i n g H Aromatic r i n g s t r e t c h i n g C H 2 , C H 3 , C-H bending phenolic C-0 s t r e t c h i n g Mainly s k e l e t a l and a r o matic C-H bending

2 . 9 N u c l e a r Magnetic Resonance Spectrum

T h e 60 M z p r o t o n NMR Spectrum of dobutamine H h y d r o c h l o r i d e i n d e u t e r a t e d d i m e t h y l s u l f o x i d e is g i v e n i n f i g u r e 4. Assignments of t h e bands are as follows:

CH2-CH2-N+CI -

I I I
I

H2

c
I

a
.l4 rl Jz

a,

Lc 0

a
X
a,
.rl

Lc

c,

cd

n
w
0

7 0

E 1 Lc

DOBUTAMINE HYDROCHLORIDE

149

Chemical Shift (ppm) Multiplicity Area Assignment 1.3 doublet 3 a 1.9 unresolved 2 b mu 1t ip 1et 2.5 unresolved 2 C (overlapping solvent) 3.0 unresolved 5 d,e,f triplets 6.8 overlapping 7 aromatic protons mu 1t ip lets 8.9 very broad 5 g,h,i,j singlet 2.10 Mass Spectrum The mass spectrum of dobutamine hydrochloride given in figure 5 shows the molecular ion of the free base at m/e 301. The major fragmentation consists of a cleavage beta to the nitrogen to yield peaks at m/e 123 and 178 with relative intensities, to the base peak, of 15.2 and 97.6%, respectively. The base peak is at m/e 107.

3. Synthesis

A mixture of crotonic acid, thionyl chloride, and a catalytic amount of dimethylformamide is stirred in a solvent such as benzene to give 2butenoyl chloride ( I ) . The Friedel-Crafts reaction of methoxybenzene ( 1 1 ) with ( I ) using AlC1, in carbon disulf ide yields 1-( 4-methoxyphenyl)-2buten-1-one ( 1 1 1 ) . 3,4-Dimethoxybenzeneethanamine ( I V ) is then condensed with ( 1 1 1 ) to give 3-"2(3,4-dimethoxyphenyl)ethyl]amino]-l-(4-methoxypheny1)-1-butanone ( V ) . This ketone is reduced with hydrogen over Pd/C to give N-[2-(3,4dimethoxyphenyl)ethyl]-4-methoxy-a-methylbenzenepropanamine ( V I ) . An alternate .synthesis f o r compound ( V I ) involves the reduction of 4-(4methoxyphenyl)-3-buten-2-one ( V I I ) with hydrogen over Raney nickel to yield the corresponding butanone ( V I I I ) , which is then condensed with ( I V ) to produce the imine ( I X ) . Compound ( 1 x 1 is then reduced again with hydrogen over Pd/C to give ( V I ) .

150

RAFIK H. BISHARA A N D HARLAN B. LONG

Figure 5 .

Mass Spectrum of Dobutamine Hydr och l o r ide

DOBUTAMINE HYDROCHLORIDE

151

A second alternate synthesis of compound (VI) in-

volves the reaction of 4-methoxy-a-methylbenzenepropanamine (X) with 3,4-dimethoxybenzeneacetic acid (XI) at 200C to yield 3,4-dimethoxy-N-[3(4-methoxyphenyl)-l-methylpropyl]benzeneacetamide (XII), which is then reduced with borane in THF to produce (VI). The trimethoxy secondary amine (VI) is demethylated by refluxing its solution in glacial acetic acid and HBr to yield dobutamine hydrobromide (XIII). Compound (XIII) is added to aqueous methanol then small amounts of hydrochloric acid are added to produce dobutamine hydrochloride (XIV). The flow diagram of the synthesis presented above (2) is shown in figure 6 .

4. Stability-Degradation
Dobutamine hydrochloride is quite stable to refluxing in acid and to heating in air for 20 hours at 115C (2). However, the drug is very rapidly oxidized to the corresponding aminochrome at pH 11-13. Approximate kinetic measurements suggest a half life of 30-45 minutes. This is similar to catecholamines which produce aminochromes that undergo further rapid and complex oxidations and/or condensations. These reactions yield products of unknown structure which finally are converted to dark colored polymers related to the melanins. Photolysis of an aqueous solution of the drug at 40-50C for 5 days using a 325 w mercury lamp in the presence of oxygen also produced the aminochrome as the photooxidation product. 5. Absorption, Metabolism, and Excretion 5.1 In Dog The short plasma half-life of dobutamine (1-2 minutes) was found by Murphy et al. ( 4 ) to be due to the rapid redistribution of the drug from the plasma to the tissue. However, plasma halflife of radioactivity following the administration of 14C-dobutamine was 1.9 hours. The major circulating metabolite is the glucuronide conjugate of 3-0-methyldobutamine. During a continuous intravenous infusion of dobutamine, the plasma level of the parent drug reach a maximum within 8 to 10 minutes, while those of the metabolites peak be-

152

RAFIK H . BISHARA A N D HARLAN B. LONG

Figure 6.

Synthesis of Dobutamine Hydrochloride

DOBUTAMINE HYDROCHLORIDE

I53

tween 3 and 4 hours. Dobutamine and/or its metabolites are eliminated via the urine and bile in both the dog and rat (5). After 4 8 hours from administering 14C-dobutamine to dogs, 67% of the radioactivity was excreted in urine and 20% in feces. Dogs with canulated bile ducts excreted 30 to 35% of the administered drug in the bile. The major urinary metabolites are the glucuronide conjugates of both dobutamine and 3-0-methyldobutamine. At very high doses of the drug, small amounts of hydroxylated dobutamine and hydroxylated 3-0-methyldobutamine were observed in the urine. The exact position of the extra hydroxyl group was not determined. The metabolites were not observed at therapeutic dosages. 5 . 2 In Man Serum levels of dobutamine reached a maximum of 20 ng/ml during a 15-minute infusion of dobutamine at a rate of 2 pg/Kg/min. and declined to 3 ng/ml within 5 minutes after the infusion. Rapid clearance from the plasma is indicated by its short half-life of approximately 2 minutes. Dobutamine is rapidly metabolized by methylation and conjugation to 3-0-methyldobutamine and conjugates of dobutamine. The major portion of the metabolites are excreted in the urine within the first 2 hours following infusion and the remainder within 6 hours. Metabolism and excretion in man are similar to the processes described above in the dog. F o r the detailed pharmacological and biochemical properties of dobutamine, the reader should consult the profile by Weber and Tuttle ( 5 ) .
6 . Methods of Analysis

6 . 1 Elemental Analysis ( A s C18H23N03-HC1) Element Theory ( % >

63.99 7.16 4.15 14.21 10.49

C H

N 0 c1

I54

RAFIK H. BISHARA A N D HARLAN B. LONG

6 . 2 Chloride I d e n t i t y About 5 m l of d o b u t a m i n e h y d r o c h l o r i d e s o l u t i o n i n water, 1 mg/ml, is a c i d i f i e d w i t h t w o d r o p s of c o n c e n t r a t e d n i t r i c a c i d . When a d r o p of 0 . 1 N s i l v e r n i t r a t e is a d d e d t o t h i s s o l u t i o n a w h i t e p r e c i p i t a t e is formed which is r e a d i l y s o l u b i l i z e d by t h e a d d i t i o n of 3 d r o p s of ammonium h y d r o x i d e . T h i s i n d i c a t e s t h e p r e s e n c e of c h l o r i d e i o n . 6 . 3 Non-Aaueous Titration

The s e c o n d a r y amine f u n c t i o n of d o b u t a m i n e h y d r o c h l o r i d e may b e d e t e r m i n e d by p o t e n t i o m e t r i c t i t r a t i o n w i t h p e r c h l o r i c a c i d using g l a c i a l acetic a c i d as a nonaqueous s o l v e n t . M e r c u r i c a c e t a t e is used t o t i e up t h e c h l o r i d e i o n . 6 . 4 Chloride Determination

A s a m p l e o f d o b u t a m i n e h y d r o c h l o r i d e cont a i n i n g a t l e a s t 2 m o f c h l o r i n e is i g n i t e d i n a g S c h o n i g e r f l a s k c o n t a i n i n g 20 m l o f water. T h r e e d r o p s o f d i p h e n y l c a r b a z o n e ( 5 mg/ml) i n m e t h a n o l a r e added t o t h e s o l u t i o n of t h e c o m p l e t e l y b u r n e d s a m p l e . M e r c u r i c n i t r a t e , 0 . 5 N, is t h e n u s e d t o t i t r a t e t h i s s o l u t i o n t o t h e f i r s t s i g n of rose c o l o r , u s i n g a 1 m l microburette:

percent chlorine = m l m e r c u r i c n i t r a t e X n o r m a l i t y X 35.5 X 1 0 0 mg s a m p l e p e r c e n t p u r i t y of d o b u t a m i n e h y d r o c h l o r i d e = p e r c e n t c h l o r i n e f o u n d X 100 percent chlorine theory

6 . 5 Chromatographv

6 . 5 . 1 T h i n L a y e r Chromatography The Rf v a l u e f o r d o b u t a m i n e hydroc h l o r i d e when c h r o m a t o g r a p h e d on a s i l i c a g e l 6 0 F254 t h i n l a y e r p l a t e d e v e l o p e d by e t h y l a c e t a t e / n p r o p a n o l / w a t e r / a c e t i c a c i d (100/40/ 15/5 v / v / v / v ) i n an u n s a t u r a t e d chamber is a b o u t 0 . 6 7 . The s p o t of t h e d r u g may b e v i s u a l i z e d u n d e r s h o r t w a v e l e n g t h UV l i g h t ( 2 5 4 nm), or u n d e r w h i t e l i g h t a f t e r e x p o s u r e t o iodine vapors.

DOBUTAMINE HYDROCHLORIDE

155

6.5.2 Gas ChromatograDhv Silylated dobutamine hydrochloride (by reaction with N-trimethylsilylimidazole) may be chromatographed on a 3 foot glass column packed with 3% OV-225 on Chromosorb G AW-DMCS (100/120 mesh). The column is operated at 230C using helium as a carrier gas at the rate of 60 ml/min. The retention time of the drug is approximately 4 minutes. A flame ionization detector is used. n-Triacontane is used as an internal standard. 6.5.3 High Performance Liquid Chromatography Dobutamine hydrochloride may be analyzed on a C , reversed-phase column eluted with 75% , 0.05M KH2P04, pH 4.4, and 25% methanol at 2 ml/minUte. The compound is detected at 280 nm. The retention time of the drug is approximately 6 minutes.
7. Analvsis of Biological Samples
7.1 Enzymatic Assay

Plasma levels of dobutamine hydrochloride are determined by reaction of the drug with 3Hme thyl-S-adenosy lmethionine in the presence of catechol 0-methyl-transferase. The radioactivity of the labeled methyl derivative is determined by a liquid scintillation counter using an external standard. The final recovery of added dobutamine as 3H-CH3-dobutamine is 24.9 f 1.3% in the range of 2 to 170 ng/ml ( 4 ) . When 14C-dobutamine is administered the samples are counted by a double isotope method.

7.2 Chromatographic Assays

7.2.1 Thin Layer Chromatography A Silica gel G plate is developed with a 15% aqueous solution of NaHSO,. The Rf Values in this system for dobutamine and 3-0-methyldobutamine are 0.50 and 0.35, respectively ( 4 ) .

156

RAFIK H. BISHARA AND HARLAN B . LONG

7.2.2Gas Chromatography Plasma and urine levels of the drug are determined by chromatographing the trimethylsilyl derivative of dobutamine on a 6-foot column packed with 3.0% UC-W98 silicon gum rubber (methylvinyl) on Diatoport S operated at 260C. The hydrogen flame detector is maintained at 280C. Helium flow rate is 60 ml/min. The retention time of dobutamine derivative (TMS) under these conditions is 3.8 minutes. This method measures plasma levels as low as 1 bg/ml (4). The levels of free and conjugated 3-0-methyldobutamine in plasma and urine are determined using electron capture detection of the pentafluoropropionate derivative of the metabolite. A 4-fOOt coiled column is packed with SP-2100 and maintained at 240C. The temperature of the 63Ni electron capture detector is 250C. The retention time of the pentafluoropropionate derivative is 1.6 minutes. Plasma levels as low as 50 ng/ml are readily measured using this method. 3-Hydroxy-N3-(4-hydroxyphenyl)-l-methyl-N-propyl)phenethylamine hydrobromide is used as an internal standard. 7.2.3. High Performance Liquid Chromatography Dobutamine hydrochloride may be determined in plasma levels, after extraction, on a C , reversed-phase column eluted with 22% aceto, nitrile-78% 0.1 M phosphate buffer (pH 2.0) at 2 ml/minute. The drug and its metabolite are detected by a fluorescent detector with an excitation wavelength of 195 nm and a 330 nm emission cut off filter. The retention times of dobutamine and the 3-methoxy metabolite are 5.2 and 7.9 min., respectively. The lower limit of sensitivity is 10 ng/ml. Reproducibility is f 5% over a 25-300 ng/ml range. Nylidrin is used as an internal standard (6). 7.3 Mass Soectrometrv (GC/MS) The biological samples are analyzed with an LKB 900 GC mass spectrometer containing a 4-foot coiled glass column packed with 1% UC-W98 on Gas Chrom 9 . The column is maintained at 240C and the

DOBUTAMINE HYDROCHLORIDE

157

flow rate of helium is 60 ml/min. The ion source voltage is 70 eV. The retention times of the trimethy Is ily 1 dobutamine and trimethy Is ilyl-3-0methyldobutamine are 3.8 and 3 . 6 min., respectively. The mass spectrum of the dobutamine-TMS shows a molecular ion at 517 and fragments at 250 and 2 6 7 . The spectrum of the derivatized metabolite has a molecular ion at 459 with major fragments at 250 and 2 0 9 . This fragmentation pattern confirms the presence of the methyl group on the catechol moiety.
8. Analysis of Pharmaceutical Formulations
8 . 1 Chromatographic Assays

8.1.1 Thin Layer Chromatography

Samples are dissolved in methanol. The insoluble excipients are removed by centrifugation. The solution is applied on a silica gel plate using the same conditions as listed previously in section 6 . 5 . 1 . Additional detection sensitivity may be obtained by spraying with a 6% solution of ferric chloride followed by a 2% solution of potassium ferricyanide.
8 . 1 . 2 Gas Chromatography

Samples are extracted into ethyl acetate from pH 9.0 buffer.. After evaporation of the solvent, n-triacontane, the internal standard, in pyridine/chloroform is added to the residue. The trimethylsilyl derivative is formed and chromatographed according to the details mentioned in section 6 . 5 . 2 .
8 . 1 . 3 High Performance Liquid Chromatography

Samples are dissolved in water to a concentration of approximately 0.5 mg/ml and injected directly into the liquid chromatograph without additional preparation. The conditions of section 6 . 5 . 3 apply also to the analysis of formu lat ions.

158

RAFIK H. BISHARA A N D HARLAN B. LONG

8.2 Spectrophotometric (UV)

Dobutamine hydrochloride may be determined spectrophotometrically in 0.5 M hydrochloric acid at the maximum of 278 nm. If excipients interfere, the drug may be extracted into ethyl acetate from pH 9 buffer followed by extraction into 0.5 M hydrochloric acid for the UV measurement. 9. Acknowledgments The authors are thankful to Dr. A. Hunt, Mr. H. W. Smith and Dr. A . D. Kossoy for their help in generating and permission to use the UV, crystallinity and stability degradation data.

References 1. H.W. Smith, personal communication, Eli Lilly and Company, Indianapolis, Indiana 46206.

2. R.R. Tuttle and J. Mills, French Patent No. 2,182,947 (1973); Ger. Offen. No. 2,317,710 (1973) and U.S. Patent No. 3, 987,200 (1976).
3. A.D. KOSSOY, personal communication, Eli Lilly and Company, Indianapolis, Indiana 46206.

4. P.J. Murphy, T.L.Williams and D.L.K. Kau, J. Pharmacol. Exp. Ther., 199,423 (1976).
5 . R. Weber and R.R. Tuttle, in M.E. Goldberg

(Editor), Pharmacological and Biochemical Properties of Drug Substances, American Pharmaceutical Association, Academy of Pharmaceutical Sciences, Washington, D . C . (1977) p. 109.

6. D.W. McKennon and R.E. Kates, J. Pharm. Sci., - 1756 (1978). 67,

The literature is surveyed through January, 1979.

Analytical Profiles of Drug Substances, 8

ERYTHROMYCIN
William L . Koch
1.

2.

3.

4. 5. 6.

Description I , I Name, Formula, Structure, and Molecular Weight I . 2 Appearance. Color, and Odor Physical Properties 2. I Solubilities 2.2 Infrared Spectrum 2.3 Ultraviolet Spectrum 2.4 Thermal Gravimetrk Analysis 2.5 Differential Thermal Analysis 2.6 X-Ray Diffraction 2.7 Nuclear Magnetic Resonance 2.8 pKa Methods of Analysis 3.1 Ultraviolet Assay 3.2 Gas Chromatographic Analysis 3.3 Colorimetric Analysis 3.4 Thin-Layer Chromatography 3.5 Microbiological Analysis 3.6 High Performance Liquid Chromatography Stability Bioavailability References

159

Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reSeNed. ISBN 0-12-260808-9

I60

WILLIAM L. KOCH

1.

Description
1.1

Name, Formula, S t r u c t u r e , and Molecular Weight Erythromycin, erythromycin A

CH3 CH3

\/

Erythromycin A
c3 7H6 7 N 0 1 3

Mol. w t . :

733.92

Erythromycin is a m a c r o l i d e a n t i b i o t i c c o n s i s t i n g of t h e a g l y c o n e , e r y t h r o n o l i d e A; t h e aminosugar, desosamine; and t h e n e u t r a l s u g a r , c l a d i n o s e . Using NNIR and CD s t u d i e s , 1 * 2 t h e conformation f e a t u r e s were deduced ( F i g . 1 ) .

162

WILLIAM L. KOCH

1.2

Appearance, Color, and Odor

The compound is a w h i t e c r y s t a l l i n e powder, p r a c t i c a l l y odorless, and h a s a b i t t e r taste. 2. P h y s i c a l Propert ies 2.1 Solubilities

Webs - - reported t h e s o l u b i l i t y e t al.3 of e r y t h r o m y c i n summarized i n T a b l e 1.

2.2

I n f r a r e d Spectrum

The i n f r a r e d s p e c t r u m of e r y t h r o m y c i n is commonly u s e d f o r its i d e n t i f i c a t i o n . F i g u r e 2 shows t h e s p e c t r u m of a 75 mg./ml. chlo_soform s o l u t i o n . The bands a t 1685 and 1730 c m are due t o t h e k e t o n e c a r b o n y l and t h e l a c t o n e c a r b o n y l , r e s p e c t i v e l y . The a b s o r p t i o n p e a k s a r e d u e t o t h e ethers between 1000 and 1200 cm' and m i n e f u n c t i o n s . T h e CH2 b e n d i n g is e v i d e n c e d by peaks between 1340 and 1460 cm' , and a l k a n e s r e t c h i n g p e a k s a p p e a r between 2780 Hydrogen bonded OH and wa er and 3020 cm' a p p e a r a s bands between 3400 and 3700 cm'

t.

1.

2.3

U l t r a v i o l e t Spectrum

The u l t r a v i o l e t s p e c t r u m of e r y t h r o mycin in m e t h a n o l e x h i b i t s Xmax a t 288 MI. The c v a l u e of 31.1 and t h e Xmax a r e c o n s i s t e n t w i t h t h e n +n* t r a n s i t i o n of C = 0 . 4

ERYTHROMYCIN

I63

TABLE 1 S o l u b i l i t i e s of Erythromycin Solvent mg. / m l 0.477 4.69 0.2*

5.05
7 7 7 7 7 7 7

Isooct ane Petroleum e t h e r Cyclohexane Carbon d i s u l f i d e Carbon t e t r ach l o r i d e Toluene Benzene Diethyl ether Chloro f o m Ethylene c h l o r i d e Methyl e t h y l k e t o n e Acetone 1,4-Dioxane Isoamyl a c e t a t e Ethyl a c e t a t e Isoamyl a l c o h o l Pyr i d i n e Formam i d e Benzyl a l c o h o l Isopropanol Ethanol M e t hano 1 Ethylene g l y c o l Water

20 20
20 20

20 20 20 > 20 7 20 7 20 > 20 9.65

> 20 > 20
7 7 7 7

>

20 20 20 20 20 2.1

Weiss reported t h i s v a l u e a s > 20. found 0 . 2 i n our l a b o r a t o r i e s .

W e

10

3600

3200

2800

2400

2000 1800 1600 WAVENUMBER CM-'

UO O

1200

1000

800

Fig. 2.

Infrared abeorption spectrum of erythromycin

ERYTHROMYCIN

I65

2.4

Thermal Gavimetric A n a l y s i s

A TGA o f erythromycin h y d r a t e i n d i c a t e s a loss o f v o l a t i l e s o f about 4 2 , t h e n decomposit i o n s t a r t i n g a t 195OC.S

2.5

D i f f e r e n t i a l Thermal A n a l y s i s

A D A o f erythromycin h y d r a t e shows T an endotherm a t a b o u t 128'C., i n d i c a t i n g simulT t a n e o u s loss o f v o l a t i l e s and m e l t i n g . A D A o f erythromycin a n h y d r a t e i n d i c a t e s m e l t i n g s t a r t i n g a t 193'C., t h e n decomposition.6

2.6

X-Ray D i f f r a c t i o n

The x-ray powder d i f f r a c t i o n p a t t e r n s o f erythromycin d i h y d r a t e and a n h y d r a t e are shown i n T a b l e 11. R a d i a t i o n : C r / V , X 2.2896.7 2.7 Nuclear Magnetic Resonance

The spectrum shown i n F i g u r e 3 was o b t a i n e d a s a CDsOD s o l u t i o n u s i n g a V a r i a n T-60A 60mHz i n s t r u m e n t , General band a s s i g n ments a r e l i s t e d i n T a b l e I11 a c c o r d i n g t o Underbr ink.
2.8

pKa

The pKa f o r erythromycin i n 66% DMF/34X w a t e r is 8.6.

166

WILLIAM L. KOCH

TABLE I1 X-Ray Eowder Diffract ion Data Erythromycin

Anhydr at e

D ihydr at 8

ZLLa
19.02 15.63 13.40 11.14 10.11 9.74 8.43 7.79 7.24 6.79 6.43 6.31 6.02 5.71 5.46 5.20 5.01 4.85 4.78 4.57 4.51 4.43 3.91 3.75 3.58 3.35 2.98 2.23 2.06 50 10 70 60 60 80 100 60 70 15 60 60 15 70 20 70 10 60 50 20 30 40 20 10 05 02 02 02 02 11.84 9.01 8.57 7.26 6.73 6.40 6.12 5.43 5.05 4.99 4.60 4.42 4.25 4.12 4.00 3.90 3.78 3.39 3.30 3.16 2.99 2.90 10 50 100 10 100 20 40 30 40 30 30 20 20 20 10 10 10 10 10 10 10 10

168

WILLIAM L. KOCH

TABLE I11

NMR S p e c t r a l Assignments of m y t h r o m y c i n
Solvent : Instrument:
0 3 OD

V a r i a n T-60A 6 0 mHz

Proton ( 8 )
c!!3a2

Resonance P o s i t i o n (PPM)
.9

Peak Type Unresolved triplet Overlapping s i n g l e t s and doublets Singlet S i n g l e t (overlapping solvent mu 1 i p l e t ) t

A l l methyls not l i s t e d above

1-1.5

o r below
2.35 3.35

O t h e r p r o t o n s (methyne and m e t h y l e n e ) g i v e u n r e s o l v e d and o v e r l a p p i n g m u l t i p l e t s i n t h e 60 mHz s p e c t r u m of t h e CD30D s o l u t i o n .

ERYTHROMYCIN

169

3.

Methods of Analysis

3.1

U l t r a v i o l e t Assay

The u l t r a v i o l e t chemical a s s a y f o r erythromycin remains l a r g e l y unchanged from t h a t described by Kuzel e t a l . 9 i n 1954. T h i s procedure is e s s e n t i a l v 8.8 follows. The reference s t a n d a r d , a l k a l i reagent, and b u f f e r s o l u t i o n s a r e prepared p r i o r t o t h e assay.

Phosphate b u f f e r pH 7 . 0 is prepared by d i s s o l v i n g 13.6 g. KH2FO4 (anhydrous) and 27.2 g. KzHFOl (anhydrous) i n s u f f i c i e n t p u r i f i e d water t o make 5 liters.
The r e f e r e n c e s t a n d a r d s o l u t i o n i s prepared by d i s s o l v i n g about 35 mg. a c c u r a t e l y weighed erythromycin s t a n d a r d i n 100 m l . methanol in a 250 m l . v o l u m e t r i c f l a s k . T h i s is d i l u t e d w i t h phosphate b u f f e r pH 7 . 0 t o 250 m l . , mixed, and allowed t o cool t o room t e m p e r a t u r e , t h e n a g a i n d i l u t e d t o t h e mark and mixed w e l l .
The a l k a l i reagent is prepared by s l u r r y i n g 42 g. Na3F04*12Hz0 i n about 125 m l . 0 . 5 N NaOH i n a 250 m l . v o l u m e t r i c f l a s k . An add i t i o n a l 100 m l . p u r i f i e d water is added and t h e s l u r r y heated on t h e steam b a t h t o aid i n s o l u s t i o n . The s o l u t i o n i cooled s l o w l y t o room temperature and d i l u t e d t o 250 m l . w i t h p u r i f i e d w a t e r , t h e n filtered p r i o r t o use.

Four 10 m l . a l i q u o t s of t h e s t a n d a r d s o l u t i o n are p i p e t t e d i n t o separate 25 m l . v o l u m e t r i c f l a s k s , two are labelled s t a n d a r d and t h e others blank. One m l . of 0 . 5 N HzSOd is added t o t h e blank f l a s k s and they are allowed t o s t a n d a f t e r mixing a t room temperature for 60 minutes f 5 minutes. Two ml. of p u r i f i e d water a r e added t o t h e s t a n d a r d f l a s k s . A t t h e
end of t h i s time, 1.0 m l . of 0 . U NaOH is added t o t h e blank f l a s k s and t h e i r c o n t e n t s s w i r l e d

I70

WILLIAM L. KOCH

t o m i x . Then, 2 . 0 m l . of a l k a l i r e a g e n t s a r e added t o a l l f o u r f l a s k s , t h e y a r e s w i r l e d t o m i x and p l a c e d i n a 60.C. water b a t h f o r 15.0 m i n u t e s . The f l a s k s a r e t h e n cooled r a p i d l y i n a n ice b a t h , b r o u g h t t o room t e m p e r a t u r e , t h e n d i l u t e d t o 2 5 . 0 m l . w i t h p u r i f i e d w a t e r . The W a b s o r b a n c e is r e a d a t 236 nm. v e r s u s p u r i f i e d w a t e r i n 1.0 cm. s i l i c a c e l l s . The b l a n k v a l u e s a r e s u b t r a c t e d from t h e s t a n d a r d v a l u e s and t h e a v e r a g e n e t a b s o r b a n c e u s e d f o r c a l c u l a t ion.
Bulk e r y t h r o m y c i n raw m a t e r i a l is t r e a t e d t h e same a s t h e s t a n d a r d . F o r m u l a t i o n s a r e made up t o t h e same c o n c e n t r a t i o n a s t h e s t a n d a r d i n m e t h a n o l and b u f f e r and 10 m l . a l i q u o t s u s e d for chromophore development.

The s u l f u r i c a c i d t r e a t e d a l i q u o t r e p r e s e n t i n g t h e b l a n k forms a c y c l i c ether anhydroerythromycin.10 The a l k a l i n e t r e a t m e n t c a u s e s t h e f o r m a t i o n of a n n,R u n s a t u r a t e d k e t o n e (9-keto-10-ene) h a v i n g its a b s o r b a n c e max inum a s a s h o u l d e r a t 236 nm. ( c 6000) . I 1 9 1 2 T h u s , any o t h e r W a b s o r b i n g s p e c i e s a r e measured w i t h t h e b l a n k and s u b t r a c t e d from t h e a b s o r b a n c e before c a l c u l a t i o n of t h e e r y t h r o m y c i n c o n c e n t r a t i o n . A t y p i c a l s p e c t r u m is shown i n F i g u r e 4 .
3.2

Gas Chromatographic Assay

T s u j i and Robertson13 r e p o r t e d a g a s chromatographic procedure for erythromycin u s i n g a n OV-225 column o r a PPE-20 column. T h e proc e d u r e i n v o l v e s s i l y l a t i n g 10 mg. e r y t h r o m y c i n w i t h a m i x t u r e of t r i m e t h y l c h l o r o s i l a n e , N , O - b i s t rime t h y 1si l y l a c e t am i d e , and N - t r imethy 1 i l y l s Ten i m i d a z o l e i n p y r i d i n e f o r 24 h o u r s a t 75'C. micrograms a r e i n j e c t e d o n t o t h e column (3 mm. x 1850 mm., 3% OV-225 o n GCQlOO-120 mesh o r 3 mm. x 1850 mm. 3% PPE-20 on S u p e l c o p o r t a t 275OC.) of a n F and M model 400 gas c h r o m a t o g r a p h e q u i p p e d w i t h a f l a m e i o n i z a t i o n detector. They r e p o r t b e i n g a b l e to s e p a r a t e e r y t h r o m y c i n s A , B, C ,

ERYTHROMYCIN

171

1.0

v)

0.5

0.0

220 240 260 280 300

nm
Fig. 4 .
W s p e c t r a of sample and blank a f t e r

chrornophore development

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WILLIAM L. KOCH

anhydroerythromycin A, and erythralosamine. Good agreement w i t h t h e microbiological a s s a y is shown. However, t h e biggest drawbacks appear t o be in s i l y l a t i o n time and t h e i n s t a b i l i t y of t h e GC column, 3 weeks a t 275C.
These authors1 4 l a t e r report u s i n g t h e GC method for e n t e r i c coated t a b l e t s of e r y t h r o mycin, g i v i n g a recovery of 99.8% and a coeff i c i e n t of v a r i a t i o n of 2.3% based o n placebo

tablets s p i k e d w i t h erythromycin.
3.3

Colorimetric Assays
,

Two procedures are worthy of note here. The first, published i n 1967 by Kuzel and C o f f e y l s i s based on t h e i o n p a i r dye complex of bromcresol p u r p l e (5' ,5"-dibromo-oc r e s o l - s u l f o n p h t h a l e i n ) and t h e desosamine moiety of erythromycin i n pH 1.2 b u f f e r . The method l a c k s s p e c i f i c i t y for erythromycin, measuring a l l t e r t i a r y amines; however, it is q u i t e s e n s i t i v e and precise, being r o u t i n e l y used for concentrations of 250 mcg. erythromycin/ml. i n t a b l e t s i n f e r m e n t a t i o n broth. A and 20-100 mcg./ml. more r e c e n t method by Sanghavi and Chandramohan16 is a l s o based on a complex of t h e desosamine moiety, but t h e y u s e p-dimethyl amino benzaldehyde a s t h e coupling a g e n t , The procedure is non-spec i f ic , but s e n s it i v e and l i n e a r over a c o n c e n t r a t i o n range of 10-35 mcg./ml.
3.4

Thin Layer Chromatography

Egon S t a h l l 7 described four TLC systems. Table IV summarizes the s o l v e n t s and R f ' s o n s i l i c a gel G.

ERYTHROMYCIN

I73

TABLE IV

Solvent Methanol Chlorof orm-me t hano 1

95

EL

Detect i o n Brownish-green color after spraying w i t h 10%s u l f u r i c acid and h e a t i n g 5-10 minutes a t
8OoC.

.16

5
50

.03
.29 .39

Chloroform-methanol
50

Butanol-acet ic acid-water 6 0 + 20 + 20

Spraying w i t h 10%molybdophosphoric acid i n alcohol, followed by h e a t i n g produces a b l u e s p o t on a yellow background. The s p o t d i s a p p e a r s i n 2 hours. Vilim e t a l . l * d e v i s e d a TLC i d e n t i f i c a t i o n s y s t e m f o r erythromycin b a s e , s t e a r a t e , e s t o l a t e and e t h y l s u c c i n a t e . T h i s combination cannot d i f f e r e n t i a t e between t h e e s t o l a t e and ethylsuccinate.
Our l a b o r a t o r i e s l 9 have developed a system s e p a r a t i n g erythromycin e s t o l a t e , e r y t h r o mycin base, and anhydroerythromycin on a s i l i c a g e l 6 0 F-254 p l a t e u t i l i z i n g e t h a n o l , methanol, t r i e t h y l a m i n e , 170:30:1. V i s u a l i z a t i o n is made by s p r a y i n g w i t h 0.15% x a n t h y d r o l and 7 . 5 % a c e t i c acid i n water. T a b l e V summarizes t h e R f ' s .

--

TABLE V_

Component Erythromycin Base Anhydroerythromgc i n Er y t hromyc i n Es t ol a t e

Rf 0.30
0.43
0.60

Color Violet Violet Violet

I74

WILLIAM L. KOCH

3.5

M i c r o b i o l o g i c a l Analysis

Kavanagh and Dennen2 r e p o r t microb i o l o g i c a l t u r b i d i m e t r i c and p l a t e a s s a y s f o r erythromycin base i n A n a l y t i c a l Microbiology, vol. 1. Staphylococcus a u r e u s (ATCC 9144) is used for t h e t u r b i d i m e t r m c e d u r e . The bulk raw m a t e r i a l o f f i c i a l a s s a y is found i n 2lCFR 452.10 and t h e o f f i c i a l t a b l e t a s s a y is found i n The sample is d i l u t e d from 0.3 t o 21CFR452.110. 2.0 tig./ml. i n pH 7 . 0 b u f f e r and comparison is made t o a s t a n d a r d curve of 0 , 0 . 3 , 0 . 4 , 0 . 6 , 0 . 8 , 1 . 2 , 1 . 6 , and 2.0 !ig./ml. Sarcina lutea (ATCC 9341) is used f o r t h e p l a t e a s s a y . A i n e a r response i n t h e r a n g e of 0.5 2.0 ug./ml, is o b t a i n e d when p 8 . 0 b u f f e r is used for sample H and s t a n d a r d . I n b o t h methods, a s m a l l amount of methanol is used t o s o l u b i l i z e t h e erythromycin p r i o r t o b u f f e r i n g a t pH 7 . 0 or 8 . 0 .

3.6

High Performance Liquid Chromatography

Omura e t a1.21 used a r e v e r s e phase high performance l i q u m Z r o m a t o g r a p h i c column, JASCO PACK SV-02-500@, f o r macrolide a n t i b i o t i c s w i t h methanol, W15 a c e t a t e b u f f e r p 4 . 9 , and a c e t o H n i t r i l e (35:60:5) a s s o l v e n t . A v a r i a b l e wavel e n g t h W detector u s i n g t h e a b s o r p t i o n of t h e i n d i v i d u a l compounds gave t h e r e q u i r e d s e n s i t i v i t y . A l t e r a t i o n s of b u f f e r pH and t h e composition r a t i o of t h e mobile phase gave s e l e c t i v i t y f o r s e p a r a t i o n of i n d i v i d u a l macrolide a n t i b i o t i c s .
2 . . I Hash22 r e p o r t e d chromatographic c o n d i t i o n s f o r s e p a r a t i n g anhydroerythromycin from erythromycin u s i n g a normal phase C o r a s i l II@ s i l i c a g e l column, w i t h chloroform a s mobile phase and r e f r a c t i v e index detect ion.

White e t a l . 2 3 d e v i s e d a r e v e r s e phase high performance-riquid chromatographic procedure f o r erythromycin. R e f r a c t i v e index d e t e c t i o n was used s i n c e t h e compound absorbs weakly i n t h e W. A 10 inn C1,/Lichrosorb r e v e r s e phase column was used w i t h 80% methanol, 19.9% w a t e r , 0.1% ammonium hydrochloride a s t h e developing s o l v e n t .

ERYTHROMYCIN

175

T s u j i and Goetz2' d e v e l o p e d a q u a n t i t a t i v e high performance l i q u i d chromatographic method f o r s e p a r a t i n g and m e a s u r i n g e r y t h r o m y c i n s A , B, and C , t h e i r e p i m e r s and d e g r a d a t i o n produ c t s . T h i s method u s e s a p o n d a p a k @ C1 r e v e r s e column w i t h a c e t o n i t r ile-met hanol-0.2M ammonium a c e t a t e - w a t e r (45 :10 :10 :25) a s s o l v e n t . The pH and c o m p o s i t i o n of t h e mobile p h a s e may be adj u s t e d t o o p t i m i z e r e s o l u t i o n and e l u t i o n volume. The a u t h o r s u t i l i z e d t h e p r o c e d u r e o n USP r e f e r e n c e s t a n d a r d and r e p o r t a r e l a t i v e s t a n d a r d d e v i a t i o n of f 0.64%.
4.

Stability

Erythromycin is u n s t a b l e i n a c i d i c o r a l k a l i n e s o l u t i o n s and shows its maximum s t a b i l i t y between pH 6 . 0 and 9.525. Its a q u e o u s , a l c o h o l i c 8 . 0 is s t a b l e f o r s o l u t i o n b u f f e r e d a t pH 7 . 0 a b o u t o n e week u n d e r r e f r i g e r a t i o n .

5.

Bioavailability

Maximum l e v e l s of e r y t h r o m y c i n i n serum a r e o b t a i n e d i n 1 t o 2 h o u r s a f t e r a s i n g l e dose. T h e U. S D i s p e n s a t o r y 2 ' reports maximum serum . l e v e l s of 0 . 2 i j g . / m l . 1 hour a f t e r a d m i n i s t r a t i o n of a 250 mg. d o s e , 0 6 !ig,/ml. 2 h o u r s a f t e r a . 500 mg. dose, and 1 . 2 t g . / m l . 2 hours a f t e r a 1 g. dose. Higher blood l e v e l s a r e a c h i e v e d o n a m u l t i p l e d o s a g e s c h e d u l e . S i n c e it is a c i d l a b i l e , a r e s i s t a n t c o a t i n g is u s e d i n t a b l e t f o r m u l a t i o n s t o overcome t h e d e l e t e r i o u s e f f e c t of g a s t r i c f l u i d on e r y t h r o m y c i n b a s e ; o r t h e s t e a r a t e s a l t is p r e p a r e d w h i c h does n o t d i s s o l v e r e a d i l y i n t h e stomach, The B i o a v a i l a b i l i t y Monograph f o r E r y t h r o mycin27 p r o v i d e s d a t a f o r c o m p a r i s o n of s e v e r a l m a n u f a c t u r e r s * t a b l e t s of e r y t h r o m y c i n , T h e c r i t e r i a f o r b i o a v a i l a b i l i t y tests a r e d i s c u s s e d .

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WILLIAM L. KOCH

6.

References

1. K. Gerzon, p e r s o n a l communication, L i l l y Research L a b o r a t o r i e s . 2. R. S. Egan, T. J. Perun, J. R. Martin, and L. A. M i t s c h e r , T e t r a h e d r o n 2 9 , 25252538 (1973). 3. P. J. Weiss, 1. L. Andrew, and W. W. Wright, A n t i b i o t i c s and Chemotherapy 7 ( 7 ) , 374-377 (1957) a 4. R. M. S i l v e r s t e i n and G. C. Bassler, S p e c t r o m e t r i c I d e n t i f i c a t i o n o f Organic Compounds, 2nd Ed., John Wiley and Sons, New York, p. 151. 5 . T. E. Cole, p e r s o n a l communication, L i l l y Research L a b o r a t o r i e s . . 6. T. E Cole, p e r s o n a l communication, L i l l y Research L a b o r a t o r i e s 7. H A. R o s e , A n a l y t i c a l Chemistry - 938 . 26 (1954). 8. C , D. Under b r i n k , persona 1 communi c a t i o n , L i l l y Research L a b o r a t o r i e s . 9. N. R. Kuzel, J. M. Woodside, J. P Comer, . and E. E. Kennedy, A n t i b i o t i c s and Chemot h e r a p y 6 , 1234-1241 (1954). 10. P. Kuratii, P H, Jones, R. S. Egan, and . T. J. PBrun, l k p e r e n t i a 27 ( 4 ) , 362 (1971). 1 . E. H Flynn, M. V. S i g a l , J r . , P F. 1 . . W i l e y , K Gerzon, J o u r n a l o f t h e American . Chemical S o c i e t y 76, 3121-3131 (1954). 12. T. J. Perun, J o u r n a l o f Organic Chemistry 3 2 , 2324-2330 (1967). 13. K T s u j i and J. Robertson, A n a l y t i c a l . I . Chemistry 43 ( 7 ) , 818-821 (1971). 14. J. H. Robertson and K. T s u j i , J o u r n a l o f Pharmaceutical S c i e n c e s 6 1 (lo), 16331635 (1972). 15. N. R. Kuzel and H F. Coffey, Technicon . Symposium (1966), V o l . 1, Automation in A n a l y t i c a l Chemistry, Medical, Inc. , White P l a i n s , N.Y., 1967, pp. 235-239.. 16. N. M. Sanghavi and H. S. Chandramohan, Canadian J o u r n a l o f Pharmaceutical S c i e n c e s - (2), 59-61 (1975). 10

ERYTHROMYCIN

1I1

17. E. S t a h l , Ed., Thin Layer Chromatography, A Laboratory Handbook, 2nd Ed., S p r i n g e r V e r l a g , N . Y . , 1969, pp. 572. 18. A. V i l i m , M. J. I s B e l l e , W. L. Wilson, and K. C. Graham, J o u r n a l of Chromatography 133, 239-244 (1977). 19. H. F. Hugar, p e r s o n a l communication, L i l l y Research L a b o r a t o r i e s . 20. D. W. Dennen, i n F. Kavanagh ( E d i t o r ) , A n a l y t i c a l Microbiology, V o l . 1, Academic Press (1963), pp. 209-294. . 2 1. S. Omura, Y S u z u k i , A. Nakagawa, and T. Hata, The J o u r n a l of A n t i b i o t i c s V o l . XXVI, NO. 12, 794-795 (1973). 22. Z. H. Hash, Methods i n Enzymology, V o l . LXIII, p. 3 0 8 , Academic mess, New York, 1975. 23. E. R. White, M. A. C a r r o l l , and J. E. Zarembro, The J o u r n a l of A n t i b i o t i c s , V O l . XXX, NO. 1 0 , 811-818 (1977). 24. K. T s u j i , J. F. Goetz, J o u r n a l of Chromatography, 147, 359-367 (1978). 25. F. Kavanagh, A n a l y t i c a l Microbiology, V o l . 1, Academic Press (1963), pp. 209294. 26. U. S. D i s p e n s a t o r y , 2 7 t h Ed., A. -01, R. P r a t t , and A. R. Gennaro E d i t o r s , J. B. L i p p i n c o t t , P h i l a d e l p h i a (1973), pp. 487. 27. C. H. N i g h t i n g a l e , J o u r n a l of t h e American Pharmaceutical A s s o c i a t i o n NS 16, ( 4 ) , 203-206 (1976).

Analytical Profiles of D u Substances. 8 rg

GRAMICIDIN
Glenn A . Brewer
I . Introduction 2. Chemistry 3. Description 3. I Composition, Formula, Molecular Weight 3.1 I GramicidinA(l1029-61-1) 3.12 Grarnicidin B ( I 1041-38-6) 3.13 Gramicidin C (9062-61-7) 3.14 Gramicidin D (1405-97-6) 3.2 Appearance, Color, Odor 4. Physical Properties 4.1 Spectra 4. I I Infrared Spectra 4. I2 Nuclear Magnetic Resonance Spectra 4.13 Ultraviolet Absorption Spectra 4.14 Fluorescence Spectra 4.15 Rarnan Spectra 4.2 Crystal Properties 4.2 I Crystalline Modifications 4.22 X-Ray Powder Diffraction 4.23 Crystal Density 4.24 Differential Thermal Analysis 4.3 Solubility 4.31 Solubility in Pure Solvents 4.32 Solubility in Solutions of Quaternary Ammonium Compounds 4.33 Solubility in Solutions of Other Surface Active Agents 4.4 Physical Properties of Solutions 4.41 Diffusion 4.42 Surface Tension 4.43 Specific Volume 4.4 Conformation 5. Production 5. I Fermentation 5.2 Isolation 5.3 Derivatives 6. Stability 6. I Stability in Solution 6.2 Effect of Light 6.3 Stability of Formulations 7. Analytical Methods. 7. I Identity Tests 7.2 Microbiological Assays

I79

Copyright @ 1979 by Academic Press. Inc. All rights of reproduction in any form reserved. ISBN 0-12-260808-9

I80
7.21 Dilution Methods 7.22 Dye Reduction Methods 7.23 Turbidimetric Assays 7.24 Agar Diffusion Assays 7.25 Potentiation of Microbiological Assays 7.3 Chemical Assays 7.31 Colorimetric Assays 7.32 Spectrophotometric Methods 7.33 Miscellaneous Methods 7.4 Electrochemical Methods 7.5 Hemolytic Methods 7 . 6 Enzymatic and Other Biochemical Methods 7.7 Chromatographic Methods 7.71 Counter Current Distribution 7.72 Paper Chromatography 7.73 Thin-Layer Chromatography 7.74 Electrophoresis 7.75 Column Chromatography 7.76 High Performance Liquid Chromatography 8. Reviews References

GLENN A. BREWER

GRAMICIDIN

181

1.

Introduction Gramiddin is a peptide antibiotic first isolated by

peptide antibiotic known as tyrocidin. The mixture of the two antibiotics was called tyrothricin. Although the mixture was discovered about ten years after penicillin2, tyrothricin was the first antibiotic utilized in clinical practice. It was soon found that tyrothricin causes severe hemolysis when administered parenterally and was destroyed when given orally. The antibiotic complex or individual components are effective topically and are used in various cream, ointment, lotion and solution preparations alone or in combination with other antibiotics or topical steroids3. The aspect of gramicidin which is of most interest to the analytical chemist is the continued.study of the structure of the antibiotic,from its discovery to the present day. As more and more sophisticated separation and structural elucidation techniques have been deueloped, various scientists have applied these to the problem of understanding the complete structure of the gramicidin complex. Thus, we can trace the development of our understanding of the structure from the crude extract prepared by Dubosl to our current knowledge of not only the structure of the various components of gramicidin, but the conformation of the major fraction in s~lution.~ is It indeed interesting that so many scientists have applied their knowledge and skill to solve this difficult structural problem,considering the relative minor role of this material product in modern medicine.
2.

D u b o s in 1939l as a crude complex together with a second

Chemistry

Tyrothricin was obtained by acidification of the fermentation broth of Bacillus brevis to precipitate the antibiotic activity along with various proteins and then dissolving the antibiotic complex in alcohol. The alcohol was removed under vacuum, the residue was washed with ether, then redissolved in alcohol and finally reprecipitated with 1% sodium chloride5 6 . It was soon recognized that tyrothricin was not a pure compound but could be separated into a neutral fraction called gramicidin and a basic fraction called tyroci-

182

GLENN A. BREWER

dine by extraction with acetone and ether mixtures. The individual fractions were thought to be homogeneous because they were crystalline and had constant physical properties on recrystalli~ation~1 1' 1 1 2. At the time, these constant properties were considered to be sufficient proof to indicate chemical purity.
A n empirical formula of C74H106N14014 was proposed for gramicidin based on elemental analysis and a Rast molecular The application of various bioweight determination'. chemical methods led the investigators to the conclusion that gramicidin was a peptide containing ten a-amino acid residues and a saturated aliphatic fatty acid containing 14 to 16 cabon atoms. It was established that a hydrolysate of gramicidin contained tryptophane and that histidine, arginine, tyrosine and ammonia were absent. The authors further noted that about half of the amino acid residues have the D-(unnatura1)configuration. This was shown by oxidation with d-amino acid oxidase' 1 2.

Other workers began to study the structure of gramicidin. Christensen and coworkers isolated crystalline tryptophane and leucine from a hydrolysate. They found no evidence for a fatty acid component and established that phenylalanine, proline and hydroxyproline were absent from a hydrolysate. These workers isolated alanine dioxpyridate from a hydrolysate and also established that gramicidin contained a compound with vicinal hydroxy and amino groups. They speculated that this compound might be serine or isoserine and proposed that gramicidin contains two tryptophane, 2 leucine, 2 or 3 alanine and 1 hydroxyamino residues or a multiple of this composition. Hotchkissl isolated optically and analytically pure d-leucine from the hydrolysate. This was the first nonenzymatic proof that d-amino acids actually occurred in gramicidin. He also noted the presence of an amino-hydroxy compound, but indicated that it was not isoserine. Gordon, Martin and Synge15 utilized their new and elegant technique, chromatography, to establish the amino acid composition of gramicidin. They proposed a 24 unit cyclic peptide consisting of six moles each of leucine, and tryptophane, 5 moles of valine, 3 moles of alanine and 2 moles each of glycine and unknown hydroxyamino compound. They confirmed that the leucine was the d-form while the tryptophane and alanine had the L-configuration. The

GRAMICIDIN

183

valine appeared to be racemic. Christensenl isolated the dipeptide valylvaline from completely hydrolyzed gramicidin. This worker later showed that he had isolated a racemic mixture of D(-)-valylD(-)-valine and I,(+)-valyl-L(t)-valine rather than dipeptides containing one d and one L-residue. l 7 Synge, using starch columns,confirmed the presence of valylvaline and identified L-valylglycine in partial hydrolysates. This unexpected finding triggered a good deal of work on the kinetics of peptide hydrolysis in an attempt to develop a rational explanation. Synge2 isolated the elusive hydroxyamino compound by azeotropic distillation and identified it as 2-aminoethanol. He proposed a structure of 6 L-tryptophane, 6 D-leucine, 5 D and L-valine, 3 L-alanine, 2 glycine and 2 aminoethanol residues. There was some evidence that the preparations of gramicidin used in this structural work were not completely homogeneous15, 22 but the evidence was not strong until Gregory and Craig2 separated crystalline gramicidin into three major fractions by counter current distribution. Fraction B contained only 55% as much tryptophane as Fraction A based on an ultraviolet analysis. The third fraction was called gramicidin C. Still using heterogeneous gramicidin,S ~ n g e ~ ~ isolated the D-leucylglycine, L-alanyl-D-valine and L-alanyl-Dleucine from partial hydrolysates of gramicidin. He also had less conclusive evidence for the tripeptides alanylvalylleucine or alanylleucylvaline. In a review paper, Dr. S ~ n g e ~ ~ recapitulated the early structural work on gramicidin and indicated that x-ray diffraction was incapable of distinguishing a gramicidin fraction purified by counter current analysis from the heterogeneous starting material. Hinman, Caron and ChristensenZ6 corrected the earlier report by Christensen16. They reported that the dipeptides found on the hydrolysis of gramicidin were D-valyl-L-valine and L-valyl- D-valine.

I84

GLENN A. BREWER

James and S ~ n g e ~ ~ speculated on the nature of the nonpeptide bonds in gramicidin. Hodgkin2' examined crystals of gramicidins A and B by x-ray diffraction. She estimated that the molecular weight of gramicidin A was approximately 3800, Previous estimates by chemical methods were approximately 7000. In 1953, Cowan and Hodgkin2' published a second report on gramicidin B.
A provisional structure for gramicidin was proposed by Gavrilov and Akimova30.

Okuda and coworkers31 determined the amino acid compoestabsition of gramicidins A, B and C. Ishii and W i t k ~ p ~ ~ lished the complete optical assignment of the amino acids in gramicidin A using enzymatic degradation and quantitative gas chromatography. The composition established was 4 moles of L-tryptophane, 4 moles of D-leucine, 2 moles of D-valine, 2 moles of L-valine", 2 moles of L-alanine, 1 mole of glycine and 1 mole of aminoethanol. (*The authors actually found 1 6 moles of L-valine and 0 6 moles of L-isoleucine. . . This indicated the possibility of the non-homogeneity of gramicidin A . ) The evidence that gramicidin A was actually heterogeneous was obtained by Ramachandran3 using counter current distribution with more than 1000 transfers. The new gramicidin which contained isoleucine was called gramicidin D. This was an unfortunate choice of terminology since the crude mixture of gramicidin fractions had been previously called gramicidin D (gramicidin Dubos) to distinguish it from other gramicidins (S and J). Sarges and established the amino acid sequence of gramicidins A and D. The same authors also established the amino acid sequences of gramicidins B36 and C37. In addition, they synthesized gramicidins A and D. 3 ' Gross and Witkop3' subjected commercial gramicidin to counter current distribution. They found that gramicidins A , B and C all consisted of a pair of congeners containing primarily valine-gramicidins (00-95%) with some isoleucine-gramicidins (5-20%) as minor components. In addition, they isolated a more hydrophilic, strongly antibiotic group of antibiotics which they called gramicidin D.

GRAMlCIDlN

185

Urry and coworkers4 41 proposed a left-handed helical structure for gramicidin A. This conformation can undergo ion induced relaxations which provides a mechanism for the movement of the ion along the channel. These workers confirmed this proposed structure by nuclear magnetic resonance spe~trometry~~. Using circular dichroism and nuclear magnetic resonance spectrometry, Veatch and coworkers4 established that four conformational species of gramicidin A exist in solution. Two were postulated to be helixes of opposite handedness.
3.

Description 3.1 Composition, Formula, Molecular Weight

The gramicidin of commerce is a complex of at least four compounds. The identified fractions are called gramicidins A, B, C and D. The major component of the mixture is gramicidin A. (See Section 2.) The mixture of gramicidins is called gramicidin The latter name is used to distinguish the gramicidin discovered by Dubos from gramicidins S and J.
[1405-97-61 or gramicidin D (Dubos) [1393-88-01.

As discussed in Section 2, the chemical structure of the various fractions has now been elucidated. The general formula for gramicidin is shown below, where R and R ' are different amino acid residues depending on the particular type of gramicidin.
HCO-R-Glycine-L-Alanine-D-Leucine-L-Alanine-D-ValineL-Valine-D-Valine-L-Tryptophane-D-Leucine-R'-D-LeucineL-Tryptophane-D-Leucine-L-Tryptophane-NHCH CH2OH 2

3.11

Gramicidin A [11029-61-11

Gramicidin A consists of a pair of congeners containing primarily L-valine gramicidin A (8095%) but also containing L-isoleucine gramicidin A (5-20%) 9 8 3 34.

I86

GLENN A. BREWER

3.111

L-Valine Gramicidin A [4419-81-21

R = L-valine R = L-tryptophane N O 9gH140 20 17 Molecular Weight 1882.349

3.112 L-Isoleucine Gramicidin A [553603-81

L-isoleucine

R = L-tryptophane

N O 100H142 20 17 Molecular Weight 1896.376 3.12 Gramicidin B [11041-38-61

Gramicidin B consists of a pair of congeners containing primarily L-valine ramicidin B but also containing L-isoleucine gramicidin B 3%

3.121

L-Valine Gramicidin B [4422-52-01

R = L-valine R= L-phenylalanine N O C97H139 19 17 Molecular Weight 1843.312

3.122

L-Isoleucine Gramicidin B
R = L-isoleucine R = L-phenylalanine N O C98H141 19 17 Molecular Weight 1857.339

3.13

Gramicidin

[9062-61-71

Gramicidin C also consists of a pair of congeners containing primarily L-valine gramicidin C but also small amounts of L-isoleucine gramicidin C are present37. 3.131 L-Valine Gramkidin C [58442-65-21 R = L-valine R = L-tyrosine C97H139N19018 Molecular weight 1859.312

GRAMICIDIN

I87

3.132

L-Isoleucine Gramicidin C R
=

L-isoleucine N O

R= L-tyrosine

9BH141 19 18

Molecular Weight 1873.339

3.14

Gramicidin D [1405-97-61

As previously discussed, the term gramicidin D has been used to designate the entire gramicidin complex of gramicidins A, B, C, D or the isoleucine component of gramicidin A33. Gross and W i t k ~ p ~ ~ used it have to name a minor and still undefined, polar fraction of the gramicidin complex. In the rest of this Analytical Profile, the physical and chemical properties which will be described will be of the complex mixture of gramicidins A, B, C and D unless otherwise noted.
3.2

Appearance, Color, Odor

Gramicidin is described as a crystalline powder, which is white or nearly white and is odorless43.
4.

Physical Properties
4.1

Spectra 4.11 Infrared Spectra

The infrared spectrum of gramicidin has been reported by Hayden, et al.44. The infrared spectra of the U.S.P. gramicidin standard are presented in Figures 1 and 245.
4.12

Nuclear Magnetic Resonance Spectra

Nuclear Magnetic Resonance has been used to establish the conformation of the pure fractions of the gramicidin complex42. The proton resonance spectrum of the U.S.P. gramicidin standard is shown in Figure 346.

189

GRAM IC ID1N

191

4.13

Ultraviolet Absorption Spectra

The tryptophane content of the gramicidin complex was measured by the ultraviolet absorbanceY7. Cann has demonstrated the shift to longer wavelengths when acetic acid complexes with gramicidin4'. The ultraviolet spectrum of the gramicidin complex has been reported by Hayden and coworker^^^. Figure 4 presents the ultraviolet spectrum of the U.S.P. Reference standard taken in 95% ethanol".

4.14

Fluorescence Spectra

Sommermeyer and coworkers50 reported that solutions of gramicidin exhibit fluorescence when irradiated with soft x-rays. Gramicidin exhibits strong fluorescence in 95% ethanol. The excitation maximum is at 286 nm and the emission maximum is at 337 nm. The fluorescence intensity was linear with respect to concentration in solution5'. 4.15 Raman Spectra

Rothchild and Stanley studied the con. formation of Gramicidin A using Raman s p e c t r o ~ c o p y ~ ~Two types of conformation were found depending on the solvent used. 4.2 Crystal Properties 4.21 Crystalline Modifications

Dr. Synge noted that when gramicidin complex was crystallized from acetone, he obtained small crystals that were not suitable for x-ray diffraction25. When the sample was allowed to crystallize from alcoholic solution by slow evaporation, large chunky crystals were obtained. These gave very good x-ray diffraction patterns. Olesen and Szabo obtained crystals from ethanol and acetone53. They found the crystals to have different solubility, melting point and x-ray diffraction patterns. Since acetone is retained in the crystalline lattice, it was indicated that the forms are pseudopolymorphs.

GRAMICIDIN
4.22

I93

X-ray Powder Diffraction

A s was mentioned in Section 2, x-ray diffraction was used in an effort to establish the structure of the gramicidin complex25'2 8 ' 29. These studies were frustrated by the fact that the gramicidin was not chemically pure but was a mixture of components.

Belavtseva found that crystalline gramicidin was converted to amorphous gramicidin by the action of x-rays54. The powder x-ray diffraction pattern of the gramicidin U.S.P. reference standard is shown in Figure 5 5 5 . The relative peak intensities are presented in Table 1. TABLE 1 Relative Peak Intensities of U.S.P. Gramicidin Reference Standard as Measured by Powder X-Ray Diffraction
20 D (DEG.) (ANGSTROMS) 6.64 7.40 9.44 13.69 14.46 16.75 16.92 17.86 19.39 20.07 21.26 21.68 22.28 23.55 23.89 13.31 11.95 9.37 6.47 6.13 5.29 5.24 4.97 4.58 4.42 4.18 4.10 3.99 3.78 3.72

PEAK
143.5 104.1 28.4 31.1 28.6 46.5 45.3 48.7 63.8 50.7 38.8 39.5 37.2 36.2 34.6

REL. PEAK
1.000 0.725 0.198 0.217 0.199 0.324 0.316 0.339 0.445 0.353 0.270 0.275 0.259 0.252 0.241

AREA
850.0 791.4 301.2 327.6 246.0 349.9 274.6 473.6 716.2 414.9 288.3 250.9 275.6 312.1 306.0

REL. AREA
1.000
0.931 0.354 0.385 0.289 0.412 0.323 0.557 0.843 0.488 0.339 0.295 0.324 0.367 0.360

1 94

.GLENN A. BREWER

4.23

Crystal Density

Low and Richards used a density gradient column to study the density of crystals of "mixed gramicidir. fractions"56. 4.24 Differential Thermal Analysis

The U.S.P. gramicidin standard shows a small endothem at 161OC and a larger endotherm at 244OC. Both endotherms are broad57. 4.3 solubility 4.31 Solubility in Pure Solvents

The solubility of the gramicidin complex in a variety of solvents has been determined by Weiss and coworkers5*. TABLE 2 Solubility of Gramicidin Complex in Various Solvents SOLUBILITY mg/ml
0.140 > 20 > 20 > 20 14.10 0.02
0.19 0.04

SOLVENT

SOLVENT

SOLUBILITY mg/ml
11.90 > 20 18.10 18.80 10.7 2.15 >20 >20 0.100 > 20 > 20
> 20

Water Methanol Ethanol Isopropanol Isoamyl Alcohol Cyclohexane Benzene Toluene Petroleum Ether Isooctane Carbon Tetrachloride Ethylene Glycol

0.007 0.005 0.047 > 20

Ethyl Acetate Isoamyl Acetate Methyl Ethyl Ketone Acetone Diethyl Ether Ethylene Chloride 1,4-Dioxane Chloroform Carbon Disulfide Pyridine Formamide Benzyl Alcohol

GRAMlCIDlN

195

4.32

Solubility in Solutions of Quaternary Ammonium Compounds

A number of reports have indicated that tyrothricin is more soluble in solutions of quaternary ammonium compounds , O.

When it was realized that tyrothricin was a mixture of antibiotics,the same principle was applied to gramicidin6 I 6 2 64 6 5 . The addition of quaternary pounds to gramicidin formulations to increase the solubility of the antibiotic in aqueous solution has been utilized66,67. 4.33 Solubility in Solutions of Other Surface Active Agents

A number of other materials have been shown to increase the solubility of gramicidin in aqueous solution. Alcohols6, ~ a n t h o c i l l i n ~ ~ , fatty acid amidesO, fatty acid alcohols71, aliphatic and polyvinylpyrrolidone73 # 74.

4.4

Physical Properties of Solutions 4.41 Diffusion

The diffusion constants for the gramicidin complex were determined in acetic acid and ethanol solut i ~ n ~ The molecular weight range of gramicidin was ~ . calculated as 2800-5000. Polson using a similar method obtained a molecular weight of 300076. 4.42 Surface Tension

Gramicidin decreases the surface tension of aqueous solution7. The bactericidal and hemolytic properties of gramicidin were destroyed by heat but the surface tension depression was not changed. Kemp and coworkers found that when they partitioned gramicidin between water and heptane, it migrated to the walls of the vessel7.

I96

GLENN A. BREWER

4.43

Specific Volume

Derechin and coworkers noted the anomalous behavior of gramicidin A in absolute ethanolirg. The apparent partial specific volume increased with decreasing concentration of gramicidin. The same was not true for aqueousethanol solutions.
4.44

Conformation

The conformation of gramicidin in aqueous solution has been extensively studied. A lipophilic lefthanded helical structure has been proposed for gramicidin A401 41. It was proposed that the mode of action of gramicidin is due to the formation of ion transport channels across biological membranes. Bamberg and coworkers have studied the single channel conductance of gramicidins A, B and C8 Significant differences between gramicidin A and B were found
I

Cabon 13 NMR has been used to confirm the presence of a double helical dimer modela2. The conformation of gramicidin in various 84 I 86. organic solvents has also been established"I
I I

Circular dichroism at high pressures has been used to study the conformation of a derivative of gramicidin A in trifluoroethanol solutionsa7. Kyogoku and Kawano have prepared an extensive review of the use of NMR techniques to study the conformation of gramicidin and other antibiotics in solutionaa. Lotz and coworkers have used poly (y-benzylD-L-glutamate) as a stereochemical model to study the conformation of gramicidin Aa9. 5. Production
5.1

Fermentation

The gramicidin complex was originally isolated by Dubos as a component of the antibiotic mixture called tyrothricin formed by an aerobic sporulating bacillus'.

GRAMICIDIN

197

Dubos and Hotchkiss found that a number of species of aerobic sporulating bacilli produced gramicidin
I

Stokes and Woodward established that Bacillus brevis produces the antibiotic in stationary cultures of both complex natural and synthetic mediag0. They noted that production of the antibiotic occurred in an aerated synthetic medium but not in an aerated complex nitrogenous medium. Konikova and coworkers compared the productivity of two strains of bacilli in the production of gramicidingl. Lewis and coworkers studied the production of the antibiotic complex by Bacillus brevis in both natural and complex mediag2. They noted requirements for calcium, magnesium and manganese ions. Stokes patented a submerged culture fermentation production procedure utilizing a synthetic medium6. Appleby and coworkers studied the addition of vitamins to synthetic mediumg3. Konikova and Dobbert studied the effect of the addition of amino acids to a synthetic medium on the production of tyrothricing4. They found that these additions had no effect on antibiotic production although growth was stimulated. Udalova and Fedorova have studied the effect of different carbon sources on the yield of antibioticg5. A number of workers have described the production of tyrothricin in a synthetic medium supplemented with organic nitrogen compounds96r97r98r99r100. Several investigators have attempted to establish the biosynthetic pathways for the production of tyrothric~n101,102,103,104~ Akers, Lee and Lipmann have isolated two enzymes from Bacillus brevis that are responsible for the synthesis of the initial portion of the g r a m i ~ i d i n s ~ ~ ~ .
5.2

Isolation

Hotchkiss and Dubos have utilized solvent extraction to separate gramicidin and tyrocidine 5. Several patents have been issued on procedures for isolating grami: cidin from fermentation brothlo6 ,lo7,lo8.

198

GLENN A. BREWER

5.3

Derivatives

Shepel and coworkers have reported the properties of analogs of gramicidin A with shorter peptide chainslog. It has been found that the treatment of gramicidin with formaldehyde results in the formation of a compound with the same antibiotic activity but with much less hemolytic activity110 1 1 1 112,113,114. Various esters of gramicidin have also been prepared 5, 6 , 7. Although these compounds have reduced hemolytic activity, they also possess less antibiotic activity.

6.

Stability
61 .

Stability in Solution

Nitti and Nislo have shown that gramicidin is stable to autoclaving in aqueous solutionll8. Certain aqueous solutions were patented because they produced stable gramicidin solutions

Ishii and Witkop have found that treatment of gramicidin A with 1.5N_hydrochloric acid in absolute methanol for one hour at room temperature selectively cleaved one peptide bond120.
6.2

Effect of Light

The ultraviolet inactivation spectrum for gramicidin has been published by Setlow and Doyle. Sugimoto and coworkers have presented infrared, ultraviolet, visible and ESR spectra for gramicidin solutions irradiated with various amounts of ultraviolet light122.
6.3

Stability of Formulations

Buckwalter has indicated that gramicidin solutions in propylene glycol and carbowaxes are stable to aut~claving~. He also showed that the antibiotic can be solubilized in water with the aid of non-ionic wetting agents.

GRAMICIDIN

I99

7.

Analytical Methods 7.1 Identity Tests

Brustier and coworkers utilized the AdamkiewiczHopkins-Cole reaction as an identity test for gra~nicidinl~~. The test detects the indole ring structure of the tryptophane residue. Fischbach and Levine described the use of Ehrlichs reagent as an identity test for grami~idinl~~. They also utilized a modified biuret reaction125. Ramachandran reported on the reaction of hydrazine with gramicidin to yield formic hydrazide126. This product was detected by a color reaction. 7.2 Microbiological Assays

Microbiological assays are the primary assay method for antibiotics. They provide sensitive but nonselective methods. A variety of microbiological methods have been described for the assay of gramicidin. The official method in the United States is the turbidimetric method described in the Code of Federal regulation^^^^^ 128. 7.21 Dilution Methods

Dilution assays are generally utilized as early microbiological methods before well defined standards are available. They have the advantage that one can compare the activity of one preparation to another without having a standard. Reedy and Wolfson have described a tube dilution assay for gramicidin12.
A n agar dilution assay using Micrococcus lysodeikticus has been reported30.

7.22

Dye Reduction Methods

The addition of a dye that is reduced by the growing microorganisms gives a microbiological assay a sharper end point.

200

GLENN A. BREWER

Prevot reported on Janus green reduction methods with a number of different organisms 1 32.

DeFelip and coworkers use sodium resazurin as a dye to give a rapid assay in three to six hours133.
7.23

Turbidimetric Assays

Ceriotti has reported a turbidimetric assay using Micrococcus lysodeikticus34. Berridge and Barrett have reported an assay with Streptococcus agalactiae which can be read after 4 hour i n c u b x l - 3 - 5 7 Kaiser and Camboni reported a turbidimetric assay utilizing Staphylococcus aureusl 3 6 . Kramer and Kirschbaum have reported an assay with Streptococcus faecalis37. Leclercq has described a nephelometric assay for gramicidin138,139. Pain and coworkers have reported a turbidimetric assay utilizing lactic acid bacteria140. Kreuzig has described a turbidimetric assay utilizing Streptococcus faecalisl41.
7.24

Agar Diffusion Assays

Since gramicidin is not very soluble in aqueous solution, relatively few agar diffusion assays have been reported. Ceriotti has reported an agar diffusion assay for gramicidin utilizing Micrococcus lysodeikticus34. Miller, Matt and Ciminera reported an agar diffusion assay for grami~idinl~~. Raitio and Bonn used this method to assay pharmaceutical preparations 43. The Swiss Pharmacopeia utilizes an agar diffusion assay with Sarcina l ~ t e a l ~ ~ .

GRAMICIDIN

20 1

Viola and Canestrini reported on an agar well technique with Sarcina Kreuzig has studied the agar diffusion assay for tyrocidine in detail using gel chromatography as an analytical technique146. 7.25 Potentiation of Microbioloaical Assavs

Nisonger reported that the addition of hexadecylpyridinium chloride to gramicidin potentiates the activity found by microbiological assay4 7 . Forni found that traces of cobalt chloride enhances the activity of gramicidin toward Escherichia & and Staphlococcus aureus4 8 . Gillissen indicated that while cationic surfactants like cetylpyridinium chloride have a synergistic effect, Tween 80 inhibits the activity of gramicidin14. This effect was confirmed by Barr and Ticel5O. Casilli and Ragni assayed gramicidin in the presence of cetrimide without interference using a cetrimide resistant strain of Staphlococcus aureus 51. 7.3 Chemical Assays 7.31 Colorimetric Assavs

Rittenberg and coworkers used a colorimetric assay for tryptophane to determine tyrothricin in fermentation broth 5 2 . Kreuzig described a semi-automated colorimetric assay for gramicidin utilizing the reaction of tryptophane with perchloric acid-butanol and ferric chloride
141,153.

Pate1 and Naravane have published an assay utilizing p-dimethylaminobenzaldehyde and nitrite 54. Ivashkiv has used this reaction to assay gramicidin and tyrocidine in fermentation broth 5 5 . The antibiotics are separated by a simple solvent partition.
7.32

Spectrophotometric Methods

Oberzill has described a spectrophotometric assay for gramicidinl 56. Thombs and coworkers have used the

202

GLENN A. BREWER

absorbance of the peptide bond at 210 nm to measure gramicidin157.

7.33

Miscellaneous Methods

White and Secor have measured gramicidin by measuring the Kjeldahl nitrogen58.
7.4

Electrochemical Methods

Kramarczyk and Berg have described an indirect polarographic assay for gramicidinl597.5

Hemolytic Methods

As has been mentioned previously, gramicidin has limited utility as an antibiotic because of its hemolytic activity (Section 1.0). Various workers have utilized the hemolytic property of the antibiotic as an assay tool. The hemolytic activity of gramicidin is probably due to its ability to form ion conducting channels in the membranes of red blood cells. The loss of isotonicity causes the cells to rupture.

Heilman and Herrell reported the first use of the hemolytic assay to the assay of gramicidin60. Mann and coworkers compared the hemolytic activity of gramicidin and tyrocidine161. They reported that the addition of serum inhibits the hemolytic activity of gramicidin. Dimick reported that the hemolytic assay can be used to measure the concentration of antibiotic in fermentation broth162. Other authors have reported on modified hemolytic r n e t h o d ~ ~ ~ ~ , ~ ~ , ~ ~ .
7.6

Enzymatic and Other Biochemical Methods

Many antibiotics have been found to have inhibitory activity on enzyme systems. This inhibition can be utilized as the basis of an assay system. Creaser reported that Staphlococcus aureus forms an inducible 8-galactosidasel66, The production of this enzyme is inhibited by the addition of gramicidin. Strictly speaking, gramicidin does not inhibit the enzyme directly but this method could be used as the basis of a sensitive assay method

GRAMICIDIN

203

c167,168,169,170,171,172,173,174~175~

Gramicidin was found to uncouple the phosphorylation of ADP from the enzymatic reduction of ferricytochrome

Hinkson reported that gramicidin inhibited the photoreduction of NAD by photosynthetic bacterial 76. 7.7 Chromatographic Methods 7.71 Counter Current Distribution

Gramicidin was first shown to be heterogeneous by counter current distribution23 . A water-methanolchloroform-benzene (7:23:15:15) system was used to show the presence of two components. Craig later showed that gramicidin contained three crystalline components177. Stamm discussed the application of counter current distribution to the separation of gramicidins 78. Ramachandran showed that there were at least four components in gramicidin and gave the fourth component the name gramicidin D33.
Goss and Witkop separated each of gramicidins A,B and C into a pair of congeners and identified the major congener as valine- ramicidin and the minor component as isoleucine-gramicidin3 7

Okamoto and coworkers showed that gramicidin could be separated into three fractions (A, B and C) using droplet counter current distribution79.
7.72 Paper Chromatography

Snell, Ijichi and Lewis published a series of paper chromatographic systems capable of separating various antibiotics including gramicidinl Detection was by bioautography. Forni and Cavalli used the following systems on Whatman No. 1 paper to distinguish between bacterial peptide antibioticslS1. t-Butanol-Acetic Acid-Water (74:3:25) n-Butanol-Acetic Acid-Water (79:6:15) Acetone-Water (70:30) + Ammonia t-Butanol-Water (80:20)

Ammonia

204

GLFNN A. BREWER

Cunha and Baptista found that a butanolacetic acid-water (50:25:25) system using Schleicher and Schull 2043a paper buffered to pH 3.0 gave the best separation of peptide antibiotics182. The same authors utilized salting out chromatography to separate gramicidin and tyrothri~inl~~. Singh utilized anionic dyes to detect gramicidin on paper chr~matogramsl~~. Paris and Theallet described three paper chromatographic systems for gramicidin185. Mtschel and Lercher described two solvent systems for antibiotics186. The solvent systems were butanolpyridine-acetic acid-water (15:10:3:12) and water saturated butanol-water saturated ethyl ether-acetic acid (5:l:l) on Schleicher and Schull 204333 paper. The antibiotics were visualized by ninhydrin. DeFranca and coworkers described a system of 25 ml 9:l acetone-water, 5 ml chloroform, 2 ml ethylene glycol and 1 ml of ~ y r i d i n e l ~ ~ . claim the method is as They accurate as the microbiological assay. 7.73 Thin Layer Chromatography

Nussbaumer utilized a solvent system of butanol-acetic acid-water (10:1:3 ) on acid Silica gel G1 Pitton described the following five thin layer systems for several antibiotics including gramicidinl

*.

Water-Methanol-Butanol-Butyl Acetate-Acetic Acid (12:2.5:7.5:40:20)

Water-Butanol-Pyridine-Acetic Acid (14:30:20:6)
Water-Butanol-Pyridine-Acetic Acid (16:40:8:16)

Water-Butanol-Acetic Acid (39:55:6) Aqueous phase of Methanol-Ammonia-Chloroform (10:10:20) Guven and Ozsari reported some thin layer systems to use as identity tests for antibiotics including gramicidinl O . McGilveray and Stickland described two thin layer chromatographic systems to use to identify several antibiotics including gramicidinl l. Nekola published a thin layer chromatographic assay for gramicidin in fermentation brothlg2. The

GRAMICIDIN

205

broth was adjusted to pH 4.5 with HC1 and the antibiotics precipitated. The residue was dissolved in methanol and purified on an alumina column. The eluate was chromatographed on silica gel using water-methanol-butanol-butyl acetate-acetic acid (24:5:15:80). The absorbance of the gramicidin spot was then determined. Kreuzig reported a high performance TLC method for gramicidin in fermentation broth1 94. A Kieselgel 60 plate is developed with acetic acid-butyl acetate-butanolmethanol-water (40:80:15:5:12). The gramicidin spot was visualized by spraying with ethanolic 4-dimethylaminobenzaldehyde-HC1 and quantitated by scanning at 570 nm. 7.74 Electrophoresis

Cunha and Baptista reported an electrophoresis method to determine gramicidin in pharmaceutical preparations 9 5 . Paris and Theallet have described two Cunha and paper electrophoresis systems for gramicidin18. Gomes have published paper electrophoresis methods for various peptide antibiotics196. Lightbown and DeRossi utilized agar electrophoresis to identify various antibiotics including gramicidinl97. 7.75 Column Chromatography

Moses has patented a process for the separaThe 80% aqueous tion of gramicidin from tyrothricin1 methanol solution is passed through a cation ion exchange resin in the hydrogen cycle followed by an anion resin in the hydroxyl cycle. Bartley and coworkers separated gramicidin from tyrocidine using Sephadex W 2 O 1 99. 7.76 High Performance Liquid Chromatography

Axelsen and Vogelsang have reported the separation of gramicidins A, B and C by HPLC using Zorbax ODS (5 pm) eluted at 6OoC with methanol-5 mM ammonium sulfate (37:13)200.

206

GLENN A. BREWER

8.

Reviews

A lar e number of general reviews have been written on 1 gramicidin2?1, 202,20 3,204,205,206,207, 2 08 209 21 0,2 1 1 1 21212 3

214,215,216,217,218,219,220,221,222~

These reviews cover the chemical properties, biological properties and medical use of gramicidin.

A review on the analytical methods for gramicidin and other antibiotics has been prepared by Brewer and Platt224.

GRAMICIDIN

207

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209

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(e.

C e r i o t t i , G.; S t u d i a g h i s l e r i a n a 5 6078g ( 1 9 5 4 ) ) .

(G.
( A = .

1 189-96 ,

(1951)

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Analytical Profiles of Drug Substances, 8

GRISEOFULVIN
Ecl~1at-d . Tou1nley R
1.

? -.

3. 4. 5.
6.

7.

8 9 I0

Description I . I Name, Formula. Molecular Weight I .2 Appearance, Color. Odor I . 3 Compendia1 References. Other Physical Properties 2.01 Circular Dichroisni 2.02 Nuclear Magnetic Resonance 2.03 Mass Spectrum 2.04 Ultraviolet Spectrum 2.05 Infrared Spectrum 2.06 X-Ray Diffraction 2.07 Fluorescence and Luminescence 2.08 Photolysis 2.09 Optical Rotation 2.10 Melting Range 2.11 Differential Scanning Calorimetry 2.12 Thermogravimetry 2.13 Electrophoretic Properties 2. I4 Solubilitv Production and Synthesis Impurities Stahility Drug Metabolic Products Methods of Analysis 7.01 Identification 7.02 Elemental Analysis 7.03 Spectrophotometric Analysis 7.04 Spectrofluorometric Analysis 7.05 Colorimetric Analysis 7.06 lodometric Analysis 7.07 Turbidimetric Analysis 7.08 Polarographic Analysis. 7.09 Chromatographic Analysis 7.091 Partition Column Chromatography 7.092 Paper Chromatography 7.093 Thin-Layer Chromatography 7.094 Gas chromatography 7.095 High Performance Liquid Chromatography 7.10 Biological Methods of Analysis Identification and Determination in Body Fluids Analysis of Dosage Forms Acknowledgments
219

Copyright 0 1979 by Academic Press. Inc. All rigtits of reproduction in any form reserved. ISBN 0-12-260808-9

220

EDWARD R. TOWNLEY

1.

Description 1.1 Name, Formula, Molecular Weight

Chemical Names (2S-trans)-7-Chloro-2',4,6-trimethoxy-6'-methylspiro [benxofuran-2(3H), 1 - 2 ' ( ) cyclohexene]3,4'-dione

7-chloro-4,6-dimethoxycoumaran-3-one-2-spir0-1'-(2~me thoxy-6'-me thylcyclohex-2'-en-4 '-one)

Generic Names Griseofulvin Trade Names Fulcin; Fulvicin UfF, Fulvicin PIG, Grifulvin; Grisactin; Grisovin, Gris-PEG, Grysio, Lamoryl, Likuden, NeoFulcin, Poncyl; Spirofulvin; Sporostatin. Formula and Molecular Weight

OCH

CH 0
3

c1

CH

1 7H17 ' ' 1 6

1.2 der.

Molecular Weight

352.77

Appearance, Color, Odor Griseofulvin is a white, odorless, crystalline pow-

Cornpendial References, Other Griseofulvin is listed in the following compendia: The United States Pharmacopia (1) The British Pharmacoepia (2) The Europeon Pharmacoepia ( 3 ) and the Merck Index ( ) 4. A previously published review (5) is a good source for physical and chemical data, production, use, occurrence and biological information.

1.3

GRISEOFULVIN

22 I

Physical Properties P h y s i c a l measurements by S c h e r i n g C o r p o r a t i o n are provided f o r Batch Number UGFP-1961. T h i s b a t c h i s chromatographic a l l y p u r e w i t h t h e e x c e p t i o n of 1.1% d e c h l o r o g r i s e o f u l v i n .
2.

C i r c u l a r Dichroism S p e c t r a The c i r c u l a r d i c h r o i s m s p e c t r a ( F i g u r e 1) w a s obt a i n e d on a 0.0155 mg/ml s o l u t i o n i n methanol w i t h a Cary Model 61 C i r c u l a r Dichroism Spectrophotometer. The f o l l o w i n g molar e l l i p t i c i t y v a l u e s were o b t a i n e d : Table I Molar E l l i pt i c it y

2.01

Wavelength n m 345 326 314 294 236 218 2.02 shoulder shoulder shoulder peak peak peak

PI

+ + + +

7,520 19,100 23,200 43,700

+110,000

- 97,500

Nuclear Magnetic Resonance S p e c t r a The p r o t o n NMR spectrum of g r i s e o f u l v i n ( F i g u r e 2) w a s o b t a i n e d i n DMSO-d s o l u t i o n (conc. w/v = 10 mg/0.40 m l ) c o n t a i n i n g TMS as k t e r n a l r e f e r e n c e u t i l i z i n g t h e V a r i a n A s s o c i a t e s CFT-20 S p e c t r o m e t e r o p e r a t i n g a t a frequency of 79.5 MHz. The chemical s h i f t s ( 6 , ppm) are w i t h r e f e r e n c e t o TMS. The e x p e r i m e n t a l c o n d i t i o n s a r e : Sweep w i d t h Pulse width Acquisition t i m e Data t a b l e
=
= = =

800 Hz 6.0 1.1 sec ( 25'tip) 5.1 s e c 8K

222

E D W A R D R. T O W N L E Y

200

li
250
WAVELENGTH [nm]

300

350

400

FIGURE 1 :

Circular Dichroism Spectra of Griseofulvin

224

EDWARD R. T O W N L E Y

T a b l e I1 Proton 6 '-CH3 5'-CH 6 '-CH 21 2'-OCH3 Chemical S h i f t s ( 6 , ppm) 0.82 2.3-2.9 3.63 Aromatic 3.96 4.04 d ( J x 6 . 5 Hz) (3H) (3H) (3H)

4-0CH3 } 6-OCH3 3 '-H 5-I

8
S

d= d o u b l e t , s= s i n g l e t , m- m u l t i p l e t . The carbon-13 NMR s p e c t r u m of g r i s e o f u l v i n ( F i g u r e 3 ) w a s o b t a i n e d a t am bi ent t e m p e r a t u r e i n DMSO-d 6 c o n t a i n i n g TMS a s i n t e r n a l r e f e r e n c e u t i l i z i n g V a r i a n A ssoci a t e s XL-100-15 s p e c t r o m e t e r equi pped w i t h F o u r i e r a c c e s s o ries. The s y s t e m was l o c k e d t o t h e d e u t e r i u m r e s o n a n c e f r e quency o f t h e s o l v e n t , and o p e r a t e d a t a f r e q u e n c y of 25.2 MHz f o r carbon-13. The chem i cal s h i f t s are r e p o r t e d ( c , ppm.) from t h e i n t e r n a l s t a n d a r d TMS. Sweep w i d t h Pulse width Acquisition t i m e Acquisition delay

= 5500 Hz

15 P sec (66'

tip)

= 1.6 sec = 0.20 sec

T a b l e 111 Carbon 1' 2' 3' Chemical S h i f t (6,) 90.11 170.22 104.60 195.45 39.49 35.52 13.77 57.54 56.97 Carbon 6 OCH3 3 3a 4 5 6 7 7a Chemical S h i f t ( 56.52 191.12 104 04 157.59 91.27 164.44 95.24 168.57
6,)

4 '
5' 6' 6' CH 4' ocd, 2' OCH3

I I

I
1

I
1

m
I
I

I
I

I
I

I
I
#

*,

m
I I

1,

I "

' I '

FIGURE 3:

Carbon 1 3 , Nuclear Magnetic Resonance Spectra of Griseofulvin i n DMSO d6

226

EDWARD R. T O W N L E Y

The spectrum is in substantial agreement with the data reported by Wenkert et. al, ( ) 6 . However, on the basis the proton c coupled carbon 13 N M R spectra, the assignments for 6 and 7i are reversed [Brambilla (16)] from those previously reported. The new assignments are based on long range H-C-0-C couplings. Mass Spectrum The medium resolution, electron-impact mass spectrum of griseofulvin (Figure 4) was run on a Varian-Mat CH-5 Mass Spectrometer. Instrumental conditions were; Electron Energy 70eV; Source Temperature 25OoC; Sample Probe Temperature 14OoC. The fragmentation ions, given below, are consistent with the griseofulvin structure. Table IV Mass (amu) 352 337 321 310 284 Ions Losses 2.03

CH30 CH3 CH CO Me6=CHCO

OCH

l+

215

138

I+

al

-t

vl

[) I
[) I

i-

..

8 s
F

228

EDWARD R. TOWNLEY

I+
123

U l t r a v i o l e t Spectrum The u l t r a v i o l e t spegtrum of g r i s e o f u l v i n i n anhyd r o u s methanol s o l u t i o n a t 25 C gave t h e f o l l o w i n g absorpt i v i t y values Wavelength maximum i s a t 324 nm; a = 15.5 Wavelength maximum i s a t 291 nm; a = 68.3 Wavelength maximum i s a t 235 nm; a = 64.0 The u l t r a v i o l e t spectrum i s shown i n F i g u r e 5. I n f r a r e d Spectrum The i n f r a r e d spectrum of g r i s e o f u l v i n , o b t a i n e d as a m i n e r a l o i l m u l l , was r u n on a Perkin-Elmer Model 180 g r a t i n g Important a b s o r p t i o n assignments are I R spectrophotometer. given i n Table V. The spectrum i s g i v e n i n F i g u r e 6. Table V Wavenumber (cm

2.04

2.05

-1

Assignment
C=O s t r e t c h ; benzofuranone r i n g carbonyl C-0 s t r e t c h ; cyclohexenone c a r b o n y l C=C s t r e t c h , a r o m a t i c and c y c l i c unsaturation C=C s t r e t c h , a r o m a t i c C-0 s t r e t c h , a r y l methoxyl

1703 ( s ) 1658 ( s ) 1615, 1597, 1580 ( s ) 1501 (m) 1220, 1210 ( 8 )

I nt e n s i t y
s

- strong
medium

X-ray D i f f r a c t i o n The X-ray d i f f r a c t i o n p a t t e r n of g r i s e o f u l v i n w a s o b t a i n e d on a P h i l l i p s ADP-3500 X-ray D i f f r a c t o m e t e r u s i n g Cu K r a d i a t i o n (1.5405A0) and N i f i l t e r . The d a t a is g i v e n i n , Table VI.

2.06

GRISEOFULVIN

229

2.06

X-Ray Powder D i f f r a c t i o n P a t t e r n of G r i s e o f u l v i n Table V I

20 4 009 4.117 10.679 13.123 13.844 14.497 16.422 17.669 19.194 19.625 20.184 21.562 21.986 22.462 23.765 24.067 24.295 25.780 26.567 28.418 29.835 31.104 31.185 31.385 32.624 32.674 34.852 34.914 35.896 36.142 36 202 36.290 37.077 37.270 38.500 38.569 38.659

d(Ao)*
22.038 21.460 8.284 6.746 6.397 6.110 5.398 5.020 4.624 4.523 4.399 4.121 4.043 3.958 3.744 3.698 3.663 3.456 3.355 3.141 2.995 2.875 2.868 2.850 2.745 2.741 2.574 2.570 2.502 2.485 2.481 2.475 2.425 2.413 2.338 2.334 2.329

II* /'*
18 14 46 48 8 59 100 7 30 16 24 34 23 45 72 10 13 26 87 57 29 20 19 24 14 14 12 11 18 14 15 14 14 13 18 18 19

**Ill'

*d ( i n t e r p l a n a r d i s t a n c e ) = n / 2 s i n 0 = r e l a t i v e i n t e n s i t y (based on t h e h i g h e s t i n t e n s i t y of 100)

230

EDWARD R. T O W N L E Y

WAVELENGTH [nm]
FIGURE 5: U l t r a v i o l e t Spectrum of G r i s e o f u l v i n Obtained i n Anhydrous Methanol Solvent

2.5

WAVELENGTH 5 6

MICRONS 8 9 1 0

12 14

18 22

3550

4600

3500

3000

2500

2000

1700 1400 FREQUENCY (CM)

1100

800

500

205

FIGURE 6:

Infrared Spectrum of Griseofulvin Obtained a s a Mineral O i l Mull.

232

EDWARD R. TOWNLEY

Fluorescence and Luminescence Griseof u l v i n e x h i b i t s b o t h f l u o r e s c e n c e and luminescence. A r e p o r t by Neely e t al., ( 7 ) g i v e s c o r r e c t e d f l u o r e s c e n c e e x c i t a t i o n (max. 295 nm) and e m i s s i o n (max. 420 nm) s p e c t r a , v a l u e s f o r quantum e f f i c i e n c y of f l u o r e s c e n c e (0.108) c a l c u l a t e d f l u o r e s c e n c e l i f e t i m e (0.663 n s e c ) and phosphorescence decay t i m e (0.11 s e c . ) . The f l u o r e s c e n c e e x c i t a t i o n and emission s p e c t r a a r e g i v e n i n F i g u r e 7. Photolysis There i s no change i n t h e t h i n l a y e r chromatogram (single spot) o r i n the fluorescence o r u l t r a v i o l e t spectra a f t e r i r r a d i a t i o n i n methanol w i t h a xenon lamp f o r 20 hours. It is t h e r e f o r e concluded t h a t t h e r e is no s i g n i f i c a n t photod e g r a d a t i o n of g r i s e o f u l v i n under r e a s o n a b l e c o n d i t i o n s of l i g h t exposure ( 7 ) . 2.09
O p t i c a l Rotation

2.07

2.08

Griseofulvin e x h i b i t s the following o p t i c a l r o t a t i o n when d i s s o l v e d i n t h e s e s o l v e n t s . Table V I I S a t u r a t e d chloroform Acetone Dimethylformamide Dioxane 2.10 [a]:60= [a]:60= +358 +302 17 [a]

k7'=

+370

17

M e l t i n g Range The D i f f e r e n t i a l Thermal Gravimetry c u r v e ( F i g u r e 9 ) demonstrates t h a t t h e g r i s e o f u l v i n m e l t i n g p o i n t t a k e s p l a c e with decomposition. The m e l t i n g range of g r i s e o f u l v i n from s e v e r a l s o u r c e s i s g i v e n i n Table VIII. Table V I I I

Me1t i n g Range 0 C

Reference 3 4 17 51

218 t o 224 2 20 218 217-224

GRISEOFULVIN

233

8C
7c

60

50
Ik

>

w 40

z -

30

20
1 0

0 200
FIGURE 7:

300

400 WAVELENGTH

500

Corrected Fluorescence and Emission Spectra of Griseofulvin: la, Excitation Spectrum with Emission at 420 nm; l b , Emission Spectrum with Excitation at 295 nm.

234

EDWARD R. TOWNLEY

D i f f e r e n t i a l Scanning C a l o r i m e t r y F i g u r e 8 shows t h e DSC thermogram of g r i s e o f u l v i n o b t a i n e d w i t h a DuPont Model 900 Thermal Analyzer. A single s h a r p m e l t i n g endokherm o c c u r s f o r t h i s s u b s t a n c e w i t h o n s e t t e m p e r a t u r e a t 216 C. Thermogravimetry F i g u r e 9 shows t h e TG thermogram of g r i s e o f u l v i n o b t a i n e d w i t h a DuPont Model 950 Thermogravimetric Analyzer. The thermogram shows no weight l o s s from ambient t o about 2OO0C followed by weight loss due t o s u b l i m a t i o n . Electrophoretic Properties Zeta p o t e n t i a l s of d i s p e r s e d g r i s e o f u l v i n have been s t u d i e d b o t h a l o n e , and i n t h e p r e s e n c e of s u r f a c e - a c t i v e a g e n t s , t h e l a t t e r a t a c o n t r o l l e d pH ( 8 ) . A M o b i l i t y / Z e t a P o t e n t i a l pH p l o t of = +25mV g r i s e o f u l v i n , shows a p o s i t i v e c h a r g e a t pH 1 . 5 which r a p i d l y d e c r e a s e s t o z e r o a t p 2 . 4 . H There i s t h e n r e v e r s a l of c h a r g e followed by a n i n c r e a s e o v e r t h e pH r a n g e 2 . 4 t o 7 . 0 . The z e t a p o t e n t i a l a t t h e l a t t e r pH i s -45 mV. The p o t e n t i a l t h e n s t a y s c o n s t a n t o v e r t h e pH range 7 t o 10. Solubility The f o l l g w i n g d a t a are g i v e n f o r t h e s o l u b i l i t y of g r i s e o f u l v i n a t 25 C ; a c e t o n e 30 g/L, carbon t e t r a c h l o r i d e 2 g/L, d i c h l o r o e t h a n e 80 g/L, dimethylacetamide, 40 g/L, dioxane 30 g/L, e t h y l e t h e r 0 . 7 g/L, h e p t a n e 0.3 g/L, methanol 0 . 4 g/L; m i n e r a l o i l (0.1 g/L; propylene g l y c o l 2 g/L; Span 80 0.2 g/L, Tween 80, 7 g/L water 0 . 2 g/L ( 1 7 ) . P r o d u c t i o n and S y n t h e s i s Griseof u l v i n is b i o s y n t h e t i c a l l y manufactured by e l a b o r a t i o n w i t h P e n i c i l l i u m griseofulvum and r e l a t e d s t r a i n s of Penicillia. The b i o s y n t h e s i s h a s been t h e s u b j e c t of numerous chemical and b i o l o g i c a l s t u d i e s , t h e l a t e s t of which is g i v e n by Harris, e t . a l . ( 9 ) F i g u r e 10. Other proposed b i o s y n t h e t i c pathways a r e d i s c u s s e d . G r i s e o f u l v i n was f i r s t i s o l a t e d i n 1938 by Oxford ( 1 0 ) (1939); i t s t o t a l s y n t h e s i s was accomplished i n 1960 and f o l l o w i n g y e a r s i n s e v e r a l l a b o r a t o r i e s ( B r o s s i e t a l . , 1960 ( 1 1 ) Grove, 1963; ( 1 2 ) Mutant s t r a i n s of P. patulum are used f o r t h e commercial p r o d u c t i o n of t h e a n t i b i o t i c by f e r mentation (9). 2.13

2.11

2.12

2.14

3.

et. al.,

Impurities Some f e r m e n t e r b r o t h i m p u r i t i e s have been l i s t e d by Holbrook, B a i l e y and B a i l e y (13) and r e p e a t e d i n a d e s c r i p t i o n

4.

FIGURE 8:

Differential Scanning Calorimetry Curve of Griseofulvin

20

\
50 100 T. 'C

200 250 300 350 150 (CORRECTED FOR CHROMEL ALUMEL THERMOCOUPLES)

400

450

500

FIGURE 9 :

Thermogravimetry Curve of Griseofulvin

GRISEOFULVIN

237

acetate

OH

CHJO

OH H,C

I
CHJ

CHJ

FIGURE 10:

A Biosynthetic Route f o r G r i s e o f ulvin.

238

EDWARD R. TOWNLEY

of e f f i c i e n t l i q u i d chromatographic s e p a r a t i o n systems by B a i l e y and B r i t t a i n ( 1 4 ) and i n a g a s chromatographic s e p a r a t i o n system by Margosis ( 1 5 ) . S t r u c t u r e s are i n F i g u r e 11. The common i m p u r i t y found i n commercial b a t c h e s of g r i s e o f u l v i n is d e c h l o r o g r i s e o f u l v i n ( 1 4 , 1 5 ) which a p p e a r s t o b e i n t h e range of 0.5 t o 3 . 5 % . Stability G r i s e o f u l v i n is a s t a b l e drug s u b s t a n c e . After 1 2 y e a r s s t o r a g e a t room t e m p e r a t u r e no decomposition was d e t e c t e d by d i f f e r e n t i a t i n g LC methods (16). There is no p h o t o d e g r a d a t i o n under r e a s o n a b l e c o n d i t i o n s of l i g h t exposure ( 7 ) . G r i s e o f u l v i n is c o n v e r t e d t o g r i s e o f u l v i c a c i d under a c i d i c c o n d i t i o n s . Drug Metabolic P r o d u c t s The major human m e t a b o l i t e of g r i s e o f u l v i n i s 6-demethylg r i s e o f u l v i n and i t s g l u c u r o n i d e ( 1 7 , l B ) which a c c o u n t f o r about 65% of t h e i n t r a v e n o u s d o s e ( 1 9 ) and 3 5 t o 65% of t h e o r a l dose ( 2 0 , 2 1 ) . The 6 - d e m e t h y l g r i s e o f u l v i n is a l s o t h e major m e t a b o l i t e i n dogs ( 2 2 ) and r a b b i t s ( 2 3 ) w h i l e b o t h 4d e m e t h y l g r i s e o f u l v i n and 6 - d e m e t h y l g r i s e o f u l v i n are major m e t a b o l i t e s i n r a t s ( 2 4 ) and mice ( 2 5 ) . These m e t a b o l i t e s can b e determined by g a s l i q u i d chromatography v i a i s o p r o poxyl d e r i v a t i v e s ( 1 8 ) o r t r i m e t h y l s i l y l e t h e r d e r i v a t i v e s ( 2 6 , 2 7 ) . The 6-demethylgriseofulvin h a s been measured i n u r i n e by h i g h performance l i q u i d chromatography ( 2 8 ) and u l t r a v i o l e t spectrophotometry ( 1 9 ) . Only t r a c e amounts of g r i s e o f u l v i n are found i n t h e u r i n e ( 2 8 ) . 6.

5.

7.

Methods of A n a l y s i s

Identification A wine r e d c o l o r is produced when about 5 mg of g r i s e o f u l v i n are d i s s o l v e d i n 1 m l of s u l f u r i c a c i d w i t h about 5 m of powdered potassium dichromate ( 2 9 ) . g Elemental A n a l y s i s A n a l y s i s of g r i s e o f u l v i n , w a s determined f o r c a r b o n , hydrogen, and c h l o r i n e . The carbon, and hydrogen a n a l y s i s w a s performed on a P e r k i n . E l m e r Model 240 i n s t r u m e n t . A n a l y s i s f o r c h l o r i n e w a s performed by combustion of t h e sample and c o u l o m e t r i c t i t r a t i o n u s i n g a n American I n s t r u m e n t Co. Chloride T i t r a t o r .
The r e s u l t s from t h e e l e m e n t a l a n a l y s i s are l i s t e d i n

7.01

7.02

Table I X .

GRISEOFULVIN

239

0
I1

0
CH03
0 CH 3
0

Dechlorgr i seo fulvin

-0 cii 3 0

@i0
\
CHO
0

c1

CII 3

CH 3

=c

Dihydrogr iseofulvin

c1

Dehydrogriseofulvin

C H 30

c1

CH 3

Tetrahydrogriseofulvin
CH30

C1

CtI 3

OCR 3

Griseofulvic Acid

c1
Isogriseofulvin FIGURE 11: Impurities Found in the Fermenter Broth

240

EDWARD R. TOWNLEY

Table I X Elemental A n a l y s i s of G r i s e o f u l v i n : Element

C H
% Theory

Batch UGFP-1961
% Found

c1
7.03

57.88 4.86 10.05

57.97 4.84 9.92

Spectrophotometric Analysis Q u a n t i t a t i v e u l t r a v i o l e t a n a l y s i s of g r i s e o f u l v i n may b e performed by comparison t o a R e f e r e n c e S t a n d a r d . The u l t r a v i o l e t absorbance i s d e s c r i b e d i n S e c t i o n 2.04 and F i g u r e 5. Spectrofluorometric Analysis Griseof u l v i n e x h i b i t s f l u o r e s c e n t p r o p e r t i e s which have been u t i l i z e d f o r h i g h l y s e n s i t i v e a n a l y s e s i n blood and serum (30-33) s k i n and sweat ( 3 4 ) . Riegelman (32) h a s combined TLC s e p a r a t i o n w i t h a f l u o r i m e t r i c d e n s i t o m e t e r r e a d o u t t o g i v e a h i g h l y s p e c i f i c and s e n s i t i v e g r i s e o f u l v i n d e t e r mination i n plasma. Other a n a l y s e s are commonly performed i n e i t h e r 1%aqueous e t h a n o l ( 3 0 ) , a c t i v a t i o n maxima 295 and 335 m nm, f l u o r e s c e n c e maxima a t 450 n o r anhydrous methanol (31) a c t i v a t i o n maxima unchanged a t 295 and 335 nm, f l u o r e s c e n c e maxima 420 nm. Values are u n c o r r e c t e d . Other a p p l i c a t i o n s t o t h e a n a l y s i s of b u l k d r u g s , dosage forms o r as a d e t e c t i o n method f o r high performance l i q u i d chromatography are f e a s i ble. Colorimetric Analysis A c o l o r i m e t r i c a s s a y of g r i s e o f u l v i n , based on t h e yellow-orange c o l o r (Xmax=420 nm) which d e v e l o p s when g r i s e o f u l v i n is h e a t e d w i t h i s o n i c o t i n i c a c i d h y d r a z i d e i n a l k a l i n e medium h a s been d e s c r i b e d by Unterman (35) and t h e mechanism i n v e s t i g a t e d by Unterman and Duca (36). Iodometric Analysis Iodometric a n a l y s i s has been a p p l i e d t o t h e d e t e r mination of g r i s e o f u l v i n i n s t a g e s of t h e manufacturing proc e s s (37). The mycelium i s e x t r a c t e d w i t h chloroform and t h e a n a l y s i s c a r r i e d out i n a l c o h o l i c solution. The 0.01N i o d i n e s o l u t i o n is s t a n d a r d i z e d w i t h g r i s e o f u l v i c a c i d . Turbidimetric Analysis A t u r b i d i m e t r i c a s s a y f o r potency e v a l u a t i o n of g r i s e o f u l v i n h a s been r e p o r t e d (38). The drug i s d i s s o l v e d i n 7.07 7.06 7.05 7.04

GRISEOFULVIN

24 1

e t h y l e n e g l y c o l monomethyl e t h e r (niethyl c e l l o s o l v e ) . Polym e r i z a t i o n i s induced w i t h g l y c e r o l and guanosine-5'-triphosp h a t e (GTP). Polarographic Analysis A s t u d y d i r e c t e d toward a comparison of t h e reduct i o n p o t e n t i a l s f o r g r i s e o f u l v i n homologs and a n a l o g s s u g g e s t s t h a t polarography i s a method of g r i s e o f u l v i n i d e n t i f i c a t i o n ( 3 9 ) . In e t h a n o l i c s o l u t i o n w i t h 0 . 2 M K C 1 s u p p o r t i n g elect r o l y t e , g r i s e o f u l v i n shows two p o l a r o g r a p h i c waves w i t h h a l f wave p o t e n t i a l s a t about -1.58 V and -1.84 V. T h i s system h a s been a p p l i e d w i t h good r e s u l t s t o t h e d e t e r m i n a t i o n of f i n i s h ed p r o d u c t s i n c l u d i n g t a b l e t s . The a c c u r a c y , p r e c i s i o n and s e l e c t i v i t y of t h e method w a s compared w i t h t h e i o d o m e t r i c method ( 4 0 ) . I s o g r i s e o f u l v i n and g r i s e o f u l v i c a c i d do n o t interfere. (41,42) 7.09 Chromatographic Analyses 7.091 7.08

P a r t i t i o n Column Chromatography G r i s e o f u l v i n maybe s e p a r a t e d from o b s e r v e d s t r u c t u r a l l y s i m i l a r i m p u r i t i e s i n t h e f e r m e n t e r b r o t h by means of p a r t i t i o n column chromatography ( 1 3 ) . A C e l i t e c o l umn packing and s o l v e n t system c o n s i s t i n g of methano1:water: hexane:chloroform ( 8 : 2 : 9 : 1 ) was used. Tetrahydrogriseofulvin, dihydrogriseofulvin, i s o g r i s e o f u l v i n , dechlorogriseofulvin are s e p a r a t e d from g r i s e o f u l v i n . The s t r u c t u r e s f o r t h e s e compounds have been g i v e n i n S e c t i o n 5. 7.092 T a b l e X. (43) Table X S o l v e n t System Benzene :Cyclohexane; Methanol :Water (5:5:6:4) Glacial a c e t i c a c i d , 0.5%, w a s added t o t h e o r g a n i c phase of t h e s o l v e n t after equilibration. 7.093 i n T a b l e XI.
Paper P a p e r Chromatography A p a p e r chromatography system i s g i v e n i n

Detection

Reference 43

Whatman No. 1

uv

Thin Layer Chromatography Thin l a y e r chromatographic systems are g i v e n The d e t e c t i o n method w a s U.V.

242

EDWARD R. T O W N L E Y

Table XI Solvent System Methanol :n-bu tanol; 95% ethano1:conc. ammonium hydroxide (4:1:2:1, by ~01.) Ch1oroform:isopropanol (3:1, by vole) Adsorbent Silica Gel

Tr
0.64

Reference 44

Silica Gel

0.86 0.86 0.64

44 44 44

n-butano1:formic acid: Silica Gel water (77:10:13 by vol.) n-Butanol:95% ethanol conc. ammonium hydroxide:water (4:1:2:1 by vole) Ch1oroform:acetone (93:7 by vole) Ch1oroform:methanol (lot1 by vole) Silica Gel

Silica Gel Silica Gel

0.65

45 46 47 48 17

0.82

Ch1oroform:acetic acid Silica Gel diethyl ether (17:1:3) Methanol :benzene (2:98 by vol.) Ethyl acetate 7.094 Silica Gel Silica Gel

0.50 0.50

Gas Chromatopraphy Successful griseofulvin analyses by gas chromatography are reported for simulated samples (49) fermentation extracts (45) and pharmaceutical bulk and dosage forms (50). Margosis (15) describes the application of gas chromatography to the purity determination of griseofulvin. Separation from the related compounds; dehydrogriseofulvin, isogriseofulvin and dechlorogriseofulvin was demonstrated. In a subsequent collaborative study (50) the accuracy and precision of the method was established. Although griseofulvin is thermally stable, the high GLC temperatures require precautions similar to those taken for steroids when preparing columns, column supports and associated equipment (45).

GRISEOFULVIN

243

The s p e c i f i c and s e n s i t i v e GLC d e t e r m i n a t i o n of g r i s e o f u l v i n i n body f l u i d s and t i s s u e s such as s k i n , sweat, u r i n e and plasma w i t h e l e c t r o n c a p t u r e d e t e c t i o n h a s been used by s e v e r a l i n v e s t i g a t o r s (34,51,52). Gas chromatographic c o n d i t i o n s are g i v e n in Table XII. Table XI1

Column

Carrier Gas

Column Temp.

Internal Standard diphenylphthalate

Reference

150 cm x 4 mm I.D.; Ushaped s t a i n l e s s s t e e l t u b i n g , 1.5% QF-1 on Anakrom ABS 3 f t x 4 mm I.D.; coiled g l a s s tubing, 3% OV-101 on Gas Chrom Q

N2

20 3'

45,49

He2

25 4'

tetraphenylcyclopentadienone

15

5 f t x 4 mm I.D.; g l a s s 10% Methane column, 3% OV-17 on 90% Argon Chromosorb W. o r Gas or Chrom Q. N2
3 f t x 4 mm I.D.; glass coiled. % OV-17 on Gas Chrom. Q
He 2

27OoC

diazepam

34,50, 51,52

25 2'

tetraphenyl15 cyc l o p e n t adienone

High Performance L i q u i d Chromatography G r i s e o f u l v i n can b e r e a d i l y chromatographed on e i t h e r normal o r r e v e r s e p h a s e columns. S e p a r a t i o n of dec h l o r o g r i s e o f u l v i n , t h e most common s y n t h e t i c i m p u r i t y i s accomplished on C / 1 8 , CN (17) o r ETH columns. (15) I s o g r i s e o f u l v i n , i s s e p a r a t e d on e i t h e r CN (17) o r ETH columns (15). L i q u i d chromatography c o n d i t i o n s are g i v e n i n T a b l e XIII.

7.095

244

EDWARD R. T O W N L E Y

Table XI11 Mobile Phase Reverse phase

II

Column PBondapak C/18 ( o c t y l d e c y l c h e m i c a l l y bonded to silica) mondapak C/18 ( o c t y l d e c y l c h e m i c a l l y bonded to silica)

Internal Standard n-butyl p-hydroxy benzoate

ence
16

Refer-

Methanol: water 3:2

45% a c e t o - d i a z e p a n nitrile in 4 m KH PO4 5 M pn = 3.6

53

Zorbax CN (cyanop r o p y l c h e m i c a l l y bonded to silica)

11

Methanol: water 3:2

m-phenyl phenol

16

Permaphase ETH Normal (C-7 e t h e r c h e m i c a l l y phase bonded t o p e l l i c u l a r 30-50 mesh g l a s s packing) 7.10

5% c h l o r o form i n hexane

14

B i o l o g i c a l Methods o r A n a l y s i s M i c r o b i o l o g i c a l p r o c e d u r e s have been developed f o r a s s a y of g r i s e o f u l v i n and a p p l i e d t o t h e a n a l y s i s of b u l k d r u g s and dosage forms ( 1 ) . The c y l i n d e r p l a t e a g a r d i f f u s i o n method is t h e o f f i c i a l m i c r o b i o l o g i c a l method of d e t e r m i n a t i o n ( 5 5 ) . Microsporum gypseum (ATCC 14683) i s t h e t e s t organism. I d e n t i f i c a t i o n and D e t e r m i n a t i o n i n Body F l u i d s and T i s s u e G r i s e o f u l v i n h a s u s u a l l y been determined i n body f l u i d s and t i s s u e s by s p e c t r o f l u o r i m e t r i c (30-34) o r g a s chromatograp h i c methods (45,46,48). More r e c e n t l y g r i s e o f u l v i n h a s been determined i n plasma by h i g h performance l i q u i d chromatography (53,541. A n a l y s i s of Dosage forms Usual dosage forms of g r i s e o f u l v i n are c a p s u l e s , t a b l e t s and b o l u s e s . These may b e p r e p a r e d f o r a n a l y s i s by s i m p l e l i q u i d s o l i d e x t r a c t i o n of drug s u b s t a n c e . Margosis (15,50) used chloroform as an e x t r a c t i n g s o l v e n t w i t h g e n t l e h e a t . A compendia procedure d e s c r i b e s t h e e x t r a c t i o n of g r i s e o f u l v i n from t a b l e t s w i t h b o i l i n g a l c o h o l . The a n a l y s i s f o r e x t r a c t e d drug s u b s t a n c e has been performed by s e v e r a l methods. Most common is a simple u l t r a v i o l e t a n a l y s i s ( 2 , 3 , 5 5 ) . Polarography u t i l i z i n g t h e system g i v e n i n S e c t i o n 7.08 h a s been used (39). Thin-layer, g a s and l i q u i d chromagraphy may a l s o be 9. 8.

G R I S EO F U LV I N

245

used u t i l i z i n g systems d e s c r i b e d i n S e c t i o n s 7.093, 7.094, and 7.095 r e s p e c t i v e l y . These l a t t e r methods are v a l u a b l e because of t h e i r s p e c i f i c i t y . I n t h e United S t a t e s , g r i s e o f u l v i n drug s u b s t a n c e and dosage forms must conform t o t h e r e g u l a t i o n s of t h e F e d e r a l Food and Drug A d m i n i s t r a t i o n concerning a n t i b i o t i c d r u g s (55, 5 6 ) . M i c r o b i o l o g i c a l a s s a y r e s u l t s o b t a i n e d by a n a l y t i c a l methods d e s c r i b e d i n t h e s e compendia a r e c o n c l u s i v e . Acknowledgements The a u t h o r wishes t o thank D r . M. D. Yudis and D r . H. Suprenant f o r t h e i r encouragement f o r t h i s work and t o acknowledge t h e v a l u a b l e a s s i s t a n c e of members of t h e S c h e r i n g C o r p o r a t i o n , P h y s i c a l Organic Research S e c t i o n : D r . R. B r a m b i l l a , M r . P. B a r t n e r , M r . C. Eckhart and M r . R. F o e s t e r f o r t h e a c q u i s i t i o n and i n t e r p r e t a t i o n of t h e p h y s i c a l d a t a , and t o t h e S c h e r i n g l i b r a r y s t a f f p a r t i c u l a r l y M s . J . Nocka, f o r t h e l i t e r a t u r e s e a r c h e s and S c h e r i n g General O f f i c e S t a f f f o r t h e c a r e f u l t y p i n g of t h i s monograph. 10.

246

EDWARD R. T O W N L E Y

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56.

Analytical Profiles of Drug Substances, 8

HALCINONIDE
Joel Kirschbaum
I. History, Description, Precautions, Synthesis 1 . 1 History I . 2 Name, Formula, Molecular Weight I . 3 Appearance, Color. Odor I .4 Precautions I .5 Synthesis Physical Properties 2.01 Single Crystal X-RayDiffraction 2.02 Mass Spectrometry 2.03 Nuclear Magnetic Resonance Spectrometry (NMR) 2.031 'H-NMR 2.032 IT-NMR 2.04 Infrared Spectrometry 2.05 X-Ray Powder Diffraction 2.06 Ultraviolet Spectrometry 2.07 Optical Rotation 2.08 Fluorescence Spectrometry 2.09 Melting Range 2.10 Differential Thermal Analysis 2. I I Thermal Gravimetric Analysis 2.12 Microscopy and Crystal Size 2. I3 Particle Size 2.14 Surface Area 2. I5 Polymorphism 2.16 Hydration Solution Properties 3 . 1 Intrinsic Dissolution Rate 3 . 2 Solubilities in Aqueous and Nonaqueous Solvents 3.3 Partition Coefficients Methods of Analysis 4. I Elemental and Inorganic Analyses 4.2 Identification, Ultraviolet and Colorimetric Analyses 4.3 Chromatographic Analyses 4.3 I High Pressure Liquid Chromatography (HPLC) 4.32 Thin-Layer Chromatography (TLC) 4.33 Column Chromatography 4.34 Paper Chromatography 4.4 Polarographic Analysis 4.5 Halcinonide in Tissues and Body Fluids Stability Acknowledgments References
25 I
Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

2.

3.

4.

5.
6.

7.

ISBN 0-12-260808-9

252

JOEL KIRSCHBAUM

1 History, Description, Precautions and Synthesis

1.1 History Halcinonide is a topical antiinflammatory agent. 192 Introduction of a 9a-fluoro group3 to the hydrocortisone molecule resulted in an eight-fold increase in antiinflammatory activity. Such other groups as 16a-hydroxy, 16a-methyl, 168methyl, 16,17-acetonide and 6a-methyl moieties have been found to diminish or eliminate mineralocorticoid activity resulting from the 9a-fluoro substituent, while retaining he enhanced antiinflammatory activity of the halo derivative Halcinonide lipophilicity is increased by masking the 16,17-hydroxyl groups as the acetonide, which increases the availability of the steroid at the site of action in the skin. The 21-chloro group also increased antiinflammatory properties. Halcinonide has systemic3 as well as topical activity.

E.

1.2 Names, Formula and Molecular Weight Halcinonide is the United States adopted name5 (USAN). The preferred chemical names6 are 21-chloro-9-fluoro-11Bhydroxy-l6a, 17-[(l-methylethylidene)bis(oxy)]pregn-4-ene-3, 20-dione, and 21-chloro-9a-fluoro-ll~-l6a,l7-trihydroxypregn-4 -ene-3,20-dione cyclic 16,17-acetal with acetone. Other chemical names are 21-chloro-9-fluoro-ll~-hydroxy-l6~,l7a-isopropylidenedioxy-4-pregnene-3,2O-dione, 9a-fluoro-21-chloro-llf3, 16a, 17a-trihydroxypregn-4-ene-3, 20-dione 16,17 acetonide and 21chloro-9-fluoro-118,16~,l7-trihydroxypregn-4-ene-3,2O-dione cyclic 16,17-acetal with acetone.
2 1

CHZCI I

in

1 2

I* I

I7

0 .O

>tt(,,
24

2qH 32 5 454.97 Daltons

14

Halcinonide has also been called Halog and Squibb SQ 18,566. The Chemical Abstracts systematic number is CAS-3093-35-4.

HALCINONIDE

253

1.3

Appearance, Color and Odor

Halcinonide is a white or practically white, odorless powder consisting of free flowing crystals.
1.4

Precautions

Since halcinonide is a corticosteroid, large doses of unformulated steroid are needed systemically before the unwanted side effects appear. Halcinonide is usually formulated at concentrations of 0 . 1 % , or less. Normal handling precautions are adequate.

1.5

Synthesis

The of halcinonide is summarized in Figure 1, starting with 16a-hydroxy-9a-fluorohydrocortisone (A4-pregnene-9a-f luoro-11@, 16a,1 7 a , 21-tetrol-3,20-dione; dihydrotriamcinolone, I , which is available c ~ m m e r c i a l l y . l ~ - ~ ~ ) This tetrahydroxy steroid is slurried in acetone, and then 70% perchloric acid is added slowly. The acetonide, I1 (9a-fluoro11~,16a,l7,21-tetrahydroxypregn-4-ene-3,2O-dioney cyclic 16,17acetal with acetone; d i h y d r o t r i a m c i n o l o n e - a c e t o n i d e ) precipitates spontaneously from solution. Mesyl chloride is added to the acetonide in pyridine to give the 21-mesylate derivative (dihydrotriamcinolone acetonide-21-mesylate, 111). Compound I11 is dissolved in dimethylformamide, lithium chloride is added and the mixture is refluxed to produce halcinonide (IV), which is recrystallized from a solution of n-propanol in water. 2. Physical Properties

2.01 Single Crystal X-Ray Diffraction

The three dimensional structure was obtained by means of single crystal X-ray diffraction. CuKa radiation, a graphite monochromator, and a photomultiplier tube were used to collect 1825 total reflections on an automated diffractometer. Of these, 1162 were used for the analysis. Figure 2 shows a computer generated drawing of halcinonide. The position of the chlorine atom was not clear from the Patterson map, but the direct method program "MLJLTAN" gave its position. Least squares refinement of coordinates together with anisotropic temperature factors, in the final stages, gave an R factor of 0.11.

14

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JOEL KIRSCHBAUM

2.02 Mass S p e c t r o m e t r y F i g u r e . 3 i s t h e p l o t t e d low r e s o l u t i o n mass s p e c t r u m of h a l c i n o n i d e found u s i n g a n A E I S c i e n t i f i c Apparat u s L t d , Model 902 mass s p e c t r o m e t e r . S p e c t r a were c o l l e c t ed on f r e q u e n c y modulated a n a l o g t a p e and p r o c e s s e d on a D i g i t a l Equipment C o r p . , P D P - l l . The t a b l e 1 5 below summarizes h i g h r e s o l u t i o n d a t a from m/z 39 t o t h e p r o m i n e n t m o l e c u l a r i o n a t 454.1922, which c o r r e s p o n d s t o C24H3205FC1. Each f r a g m e n t i o n c o n t a i n i n g c h l o r i n e a p p e a r s as a d o u b l e t p e a k b e c a u s e c h l o r i n e is a m i x t u r e of two i s o t o p e s ( 3 5 C l and 3 7 C l > . T h e s e p e a k s a r e two mass u n i t s a p a r t w i t h a n i n t e n s i t y r a t i o of 3 t o 1. Fragmentation proceeds a l o n g s e v e r a l pathways,
a s shown below.

439 419 396?)m/z

361

377

m/z 2 6 4

m/z

278

These s t r u c t u r e s do n o t n e c e s s a r i l y r e p r e s e n t the a c t u a l i o n i c s t r u c t u r e b u t o n l y t h e o r i g i n of t h e f r a g m e n t . The weak i o n s a t m/z 121, 122 and 1 2 3 (CgHgO, 0) a r e c h a r a c t e r i s t i c of A4-3-one s t e r o i d s . 8 11

C H

8 10

0 and C H

Figure 3
Low Resolution Mass Spectrum of Halcinonide. See text for details. 5433 S Q 1 8 , 5 6 6 BFI NN009NFl 175 DEG

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High-Resolution Massa Spectrum of H a l c i n o n i d e Found Mass Calc. Mass Unsat.

bO/EC
0 E E 0 E E E 0 0 0 E E 0 E E E

c H
24 23 24 21 22 21 19 17 16 16 9 8 8
8

- 0

F 1 1 1 1 1 1 1 1 1 1
0 0 0 0 0 0

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454.1925 439.1680 419.2252 396.1501 377.2147 361.1838 319.1665 278.1695 264.1524 246.1422 135.0808 123.0769 122.0729 121.0683 76.9790 43.0184
a

454.1922 439.1687 419.2233 396.1503 37 7.2128 361.1815 319.1709 278.1682 264.1525 246.1420 135.0810 123.0810 122.0732 121.0653 76.9794 43.0184

8. 0 8. 5 8.5 8. 0 7.5 8.5 7.5 6.0 6.0 7.0 4.5 3.5 4.0 4.5 1.5 1.5

32 29 32 26 30 26 24 23 21 19 1 1 1 1 10
9

2 2

2
3

5 5 5 4 4 4 3 2 2 1 1 1 1 1 1 1

1 0 0 0 0 0 0 0 0 0 0

1
0

Only t h o s e peaks c o n s i d e r e d t o b e s i g n i f i c a n t t o t h e d i s c u s s i o n are l i s t e d . element map c a n b e o b t a i n e d on r e q u e s t .

A complete

bNumber of double bonds and r i n g s .

C

0-odd e l e c t r o n i o n ; E-even e l e c t r o n i o n .

HALCINONIDE 2 . 0 3 Nuclear Magnetic Resonance Spectrometry

259

(NMR)

2.031

'H-NMR

Figure 4 is the 100 MHz NMR Spectrum of halcinonide in deuterochloroform containing tetramethylsilane as internal reference at 0 Hz. The instrument used is a Varian Associates, Inc., Model XL-100 NMR spectrometer. Below is an interpretation of the various resonances.
'H-NMR

Assignments Peak Appearance S = singlet D = doublet B = broad

S

Proton at Carbon

Chemical Shift 5.78 4.58 5.05 0.87 1.51 4.19 4.57 1.15

c-4
c-11
C-16
C-18

D J15,16= 4 . 0 Hz
S

c-19 c-21 8-Acetonide, methyl a-Acetonide, methyl

AB quartet

1.44

These data agree with the published results of the 21hydroxy-A' analog, triamcinolone acetonide.l 7

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2.032

13C-NMR

The 13C-NMR spectrum of halcinonide, Figure 5, was obtained18 using a Jeol FX-60Q NMR spectrometer operating in the Fourier transform mode at 15 MHz. Numbers on the vario u s peaks refer to the assignments in the table below. These assignments were made by comparison of chemical shifts and 3C-19F coupling constants to those assigned to such related steroids as 9a-fluorocortisol (9a-fluoro-llf3,17,21-trihydroxypregn-4-ene-3,20-dione). Four pairs of closely related peaks show chemical shift differences small enough to lead to possible reversals o f their tentative assignments. These pairs are the peaks assigned to the acetonide methyl groups (6 = 28.6 and 6 = 25.1 ppm), the two carbonyl peaks (6 = 202.1 and 199.1 ppm), the methylene carbons assigned to C-1 and C-7 (6 = 26.2 and 28.5 ppm), and the methylene carbons assigned to C-2 and C-15 (6 = 33.2 and 33.7 ppm). These assignments must be verified by additional experimentation. 'C-NMR Assignments Carbon Chemical Shift 6 (ppm>a 26.2 33.7 199.1 124.7 169.0 30.7 28.5 33.1 99.0 43.7 70.2 37.3 (28.5) (33.2) (202.1) C-F Coupling Constant (Hz) 3

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

1 2 3

(26.2)

3 19 175 21

43.4 33.2 81.9 98.1 17.0 21.9 202.1 47.1 111.5 28.6 25.1

44.8

2 (33.7)

6 (199.1) (25.1) (28.6)

a Referenced from center peak of deuterochloroform = 77.0 pprn from tetramethyl silane. Numbers in parentheses refer to alternative assignments (see text for details).

I-(
(d

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4

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r J

HALCINONIDE

263

2.04

I n f r a r e d Spectrometry

F i g u r e 6 shows t h e i n f r a r e d s p e c t r u m of h a l c i n o n i d e r u n as a p o t a s s i u m bromide p e l l e t , u s i n g a Perkin-Elmer Model 6 2 1 i n f r a r e d s p e c t r o m e t e r . Below a r e t h e i n t e r p r e t a t i o n s of v a r i o u s a b s o r b a n c e s . l 9

Frequency (cm )

Interpretation
-OH S t r e t c h

3450 2950 17 3 0 1660 1620 2.05

X-ray Powder D i f f r a c t i o n

-OH S t r e t c h
C z 0 Keto

C3
A4

Keto C=C

F i g u r e 7 is t h e powder X-ray d i f f r a c t i o n p a t t e r n of h a l c i n o n i d e a s o b t a i n e d on a P h i l i p s gowder Using a d i f f r a c t i o n u n i t e m i t t i n g CuKa r a d i a t i o n a t 1.54A. s c i n t i l l a t i o n c o u n t e r d e t e c t o r , t h e sample w a s scanned and r e c o r d e d from a p p r o x i m a t e 1 2 t o 4 0 d e g r e e s ( 2 0 ) . The t a b l e below i s t h e s o r t e d d a t a . 21;

20 (Degrees) 14.80 12.53 18.49 17.94 19.82 29.31 18.10 11.67 32.14 39.59 25.86 21.47 26.80 25.24 15.67 40.37 22.33 33.08 25.47 34.02
'Interplanar

' d ' (Angstrom) 1

5.99 7.06 4.80 4.94 4.48 3.05 4.90 7.58 2.78 2.28 3.45 4.14 3.33 3.53 5.66 2.23 3.98 2.71 3.50 2.64
distance

Relative Area

1.000 0.866 0.738 0.526 0.422 0.266 0.246 0.225 0.195 0.171 0.165 0.142 0.135 0.135 0.131 0.124 0.117 0.114 0.053 0.052

2 R e l a t i v e a r e a , o r i n t e n s i t y , i s based on h i g h e s t i n t e n s i t y
of 1.00 u s i n g CuKa r a d i a t i o n .

Figure 6 I n f r a r e d Spectrum of H a l c i n o n i d e , Potassium Bromide P e l l e t . See t e x t o r d e t a i l s .

25

WAVELENGTH (MICRONS) 8 6 7

1 0

12

1 5

20

30 4050

4ooo

3500

3ooo

2500

2ooo

1800

FREQUENCY (CM)

1600

1400

1200

lo00

800

600

400

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2.06

U l t r a v i o l e t Spectrometry

U l t r a v i o l e t s p e c t r a 2 ' of h a l c i n o n i d e w e r e r e c o r d e d on a Beckman Acta C I I I s p e c t r o p h o t o m e t e r . The s p e c t r a i n m e t h a n o l , 0.M m e t h a n o l i c h y d r o c h l o r i c a c i d and 0 . W m e t h a n o l i c sodium h y d r o x i d e show p e a k maxima a t 238 nm, and r e m a i n unchanged a f t e r 24 h o u r s . Absorbances are t a b u l cm l a t e d below u s i n g t r a d i t i o n a l E 1% n o t a t i o n .

1% Elcm
So l v e n t

Initial 379 370

3 Hours 374 372

24 Hours 373 370

M thano1 e

0.M Methanolic
H y d r o c h l o r i c Acid
0. I Methanolic M

380

382

380

Sodium Hydroxide The s p e c t r u m i n 95% e t h a n o l a l s o h a s a peak maximum a t 238 nm and a p p e a r s s t a b l e f o r a t l e a s t 3 h o u r s . I n a c e t o n i t r i l e and e t h y l e t h e r t h e maximum i s s h i f t e d t o 234 nm and 229 nm, r e s p e c t i v e l y . 2.07 Optical Rotation

The s p e c i f i c r o t a t i o n s i n d e t e r m i n e d 2 3 t o b e as f o l l o w s ,

were

Wavelength (nm) 589 578 546 436 305 2.08

Specific Rotation +156 +164

+188
+348 +436

F l u o r e s c e n c e Spectrometry

Using a Perkin-Elme54Model 204 f l u o r e s c e n c e s p e c t r o p h o t o m e t e r , no f l u o r e s c e n c e w a s found a t e i t h e r 1 o r 1000 ug h a l c i n o n i d e p e r mL o f m e t h a n o l . No f l u o r e s c e n c e w a s induced i n 0 . U h y d r o c h l o r i c a c i d o r 0 . U sodium h y d r o x i d e .

HALCINONIDE

267

2.09

Melting Range

Following the USP procedure25 for class lA compounds, the melting range.of halcinonide is 270-272" (reference 21). This is in excellent agreement with the results of differential thermal analysis, below, as well as hot stage microscopy, section 2.12. 2.10 Differential Thermal Analysis

A duPont Model 900 Differential Thermal Analyzer shows halcinonide to have one endothermZ6 at 269". Decomposition on melting precludes differential scanning colorimetry studies for purity.

2.11

Thermal Gravimetric Analysis

Using a Perkin-Elmer Model TGS-2 Thermogravimetric Analyzer, halcinonide was found26 to lose 0.3% total volatile material at 70". No further l o s s was found up to 150". The heating rate was 20"/minute under a nitrogen atmosphere. 2.12 the method to 4, 5 to three lots (acicular) Microscopy and Crystal Type

Microscopically, the crystal type depends on of preparation of halcinonide. Slabs, either 2 10, or 10 to 15 microns square, were found in of halcinonide, intermingled with needle-like crystals 5 to 10microns 10ng.27

14 Single crystal x-ray diffraction studies showed that the crystals of halcinonide recrystallized from n-propyl alcohol-water azeotrope (79:22) are orthorhombic and belong to the gpace group P2 2 2 , with unit sell constants 1 of a = 10.007 A , b = 11.875 an& c = 19.460 A. Density is 1.330 gm/cm3, as measured by flotation in a hexane-carbon tetrachloride gradient. The molecular weight calculated from the unit cell volume and density is 461 daltons (theoretical is 455 daltons).

Hot stage microscopy26 was performed using a Mettler FP52 temperature controller at a rate o f 3"/minute. Melting began at 268.2", and by 271.4" all crystals were melted. The yellow melt slowly turned orange-brown. Cooling showed no recrystallization at ambient temperature.

268

JOEL KIRSCHBAUM

2.13

Particle

Size

By l i g h t s c a t t e r i n g u s i n g a Royco i n s t r u m e n t , a l l p a r t i c l e s are below 20 microns.27 C o u l t e r Counter ana l y s i s of ground h a l c i n o n i d e suspended i n a sodium c h l o r i d e s o l u t i o n shows 100% t o be <10.2p (p = m i c r o n ) , 98.2% <6.4p, 95.3% <5.1p, 52.0% <2.6p and 19.9% <1.6p.

2.14

S u r f a c e Area

A s measured by g a s a d ~ o r p t i o n , h e~ s u r f a c e t~ areas of t h r e e l o t s of ground h a l c i n o n i d e a r e 2.61, 3.85 and 4.08 m2/g. 2.15 Polymorphism

There i s no evidence o r polymorphism from i n f r a r e d s p e c t r o s c o p y and d i f f e r e n t i a l t h e r m a l a n a l y s i s , and o n l y i n c o n c l u s i v e d a t a from x-ray d i f f r a c t i o n and microscopy studies

2.16

Hydration

The c r y s t a l s of h a l c i n o n i d e are n o t s o l v a t e d w i t h water, based on a t o t a l v o l a t i l e c o n t e n t of 0.3% o b t a i n ed by t h e r m a l g r a v i m e t r i c a n a l y s i s , a c o r r e c t e l e m e n t a l a n a l y s i s , and a loss-on-drying v a l u e of 0.6% (cf. s e c t i o n 4 . 1 , Elemental and I n o r g a n i c A n a l y s e s ) . 3. Solution Properties 3.1 I n t r i n s i c D i s s o l u t i o n Rate

The i n t r i n s i c d i s s o l u t i o n r a t e w a s determined a f t e r compressing powder under 2000 p . s . i . g . p r e s s u r e u s i n g 318" d i a m e t e r disc-shaped d i e s . I n one l i t e r of 0 . M hydroc h l o r i c a c i d a t 37", a g i t a t e d a t a r a t e of 50 r.p.m., t h e g i n t r i n s i c d i s s o l u t i o n rate of h a l c i n o n i d e i s 8.33 x loW3 m min.-l cm-' computed from d a t a o b t a i n e d by u l t r a v i o l e t s p e c t r o m e t r y . 28 3.2 S o l u b i l i t i e s i n Aqueous and Nonaqueous S o l v e n t s

S o l u b i l i t i e s of h a l c i n o n i d e were determined i n v a r i o u s s o l v e n t s . 2 9 R e s u l t s a r e r e p o r t e d u s i n g t h e U.S.P. d e f i n i t i o n s . 30

HALCINONIDE

269

Solvent

Solubility

Water
H y d r o c h l o r i c a c i d , 0.W Sodium h y d r o x i d e , 0.N Acetone Ace t o n i t r i l e Acetonitrile-water Benzene Chloroform Castor o i l Dimethylsulf o x i d e Ethanol Ethyl e t h e r G l y c e r y l monooleate Hexanes Isopropyl myristate Methanol n-Oc t a n o l P o l y e t h y l e n e g l y c o l 200 P o l y e t h y l e n e g l y c o l 400 Polypropylene g l y c o l Propylene g l y c o l

Insoluble Insoluble Insoluble Soluble Sparingly soluble Slightly soluble Slightly soluble Freely soluble Slightly soluble Freely soluble Slightly soluble Slightly soluble Slightly soluble I n so l u b l e Slightly soluble Slightly soluble Slightly soluble Slightly soluble Slightly soluble Slightly soluble Very s l i g h t l y s o l u b l e

270

JOEL K I R S C H B A U M

3.3

Partition Coefficients

H a l c i n o n i d e was p a r t i t i o n e d 3 ' between h e x a n e s and m e t h a n o l , and between h e x a n e s and a q u e o u s a c e t o n i t r i l e a t a p p a r e n t pH v a l u e s of 2 , 4 , 6 ( u n a d j u s t e d ) , and 10. A f t e r one h o u r of m i x i n g , t h e s t e r o i d c o n t e n t w a s d e t e r m i n e d by u l t r a v i o l e t s p e c t r o m e t r y of b o t h p h a s e s . I n a l l cases, a b s o r b a n c e a t t h e p e a k maximum of 239 nm was d e t e c t e d o n l y i n t h e a c e t o n i t r i l e - w a t e r o r m e t h a n o l l a y e r . The aqueous a c e t o n i t r i l e (pH 6) r e s u l t was v e r i f i e d 3 2 u s i n g 1 4 C - h a l c i n o n i d e l a b e l e d a t t h e 2- c a r b o n of t h e a c e t o n i d e m o i e t y . Thus, t h e halcinonide i s completely r e t a i n e d i n e i t h e r the a c e t o n i t r i l e water o r m e t h a n o l l a y e r s , i n d i c a t i n g t h e u t i l i t y of t h e s e s o l v e n t s y s t e m s f o r e x t r a c t i n g t h e s t e r o i d from f o r m u l a t i o n s . Another s t u d y 3 3 i n v o l v e d i s o p r o p y l m y r i s t a t e and aqueous p r o p y l e n e g l y c o l . A f t e r e u i l i b r a t i o n f o r two weeks a t 3 7 " , t h e l a y e r s were s e p a r a t e d 3 2 and t h e aqueous g l y c o l a s s a y e d f o r s t e r o i d u s i n g h i g h - p r e s s u r e l i q u i d chromatog r a p h y (cf. s e c t i o n 4 . 3 1 ) . The i s o p r o p y l m y r i s t a t e / a q u e o u s propylene g l y c o l p a r t i t i o n c o e f f i c i e n t s are as follows: p r o p y l e n e g l y c o l - w a t e r (4:l); 1 4 4 ; p r o p y l e n e g l y c o l - w a t e r ( 2 : 3 ) , 44.4; p r o p y l e n e g l y c o l - w a t e r ( 3 : 2 ) , 8.14, and propyl e n e g l y c o l - w a t e r ( 9 : 1 ) , 0.82. 4. Methods of A n a l y s i s

4.1

E l e m e n t a l and I n o r g a n i c A n a l y s e s

The e l e m e n t a l a n a l y s i s 3 5 of h a l c i n o n i d e i s c a r b o n 63.38% (63.35%, t h e o r e t i c a l ) ; hydrogen 7.39% ( 7 . 1 0 % , t h e o r e t i c a l ) ; c h l o r i n e , 7.89 ( 7 . 7 9 % , t h e o r e t i c a l ) , and f l u o r i n e , 4.30% ( 4 . 1 7 % , t h e o r e t i c a l ) . Emission s p e c t r o c h e m i c a l a n a l y s i s f o r metals w a s performed u s i n g a Spec I n d u s t r i e s c a r b o n a r c A . C . u n i t w i t h a Bausch and Lomb d u a l g r a t i n g s p e c t r o g r a p h . Data were r e c o r d e d on g l a s s p l a t e s and i n t e r p r e t e d by means of a m i c r o p h o t o m e t e r . H a l c i n o n i d e c o n t a i n e d t r a c e amounts (% 60 i o pg/g t o t a l ) of t h e f o l l o w i n g m e t a l l i c i m p ~ r i t i e s ~ r~ ;n , manganese, c o p p e r , n i c k e l , l e a d , z i n c , aluminum, sodium, c a l c i u m , and magnesium. R e s i d ~ e - o n - i g n i t i o n ~ ~ t h e same of l o t 3 8 i s less t h a n 0.1%. Heavy metals c o n t e n t ( r e f e r e n c e 39, method 11) i s less t h a n 0.003%.38 A f t e r 3 h o u r s a t 100" i n vacuum, t h e l o ~ s - o n - d r y i n g ~ ~ l u e 3 8 i s 0 . 6 % . va Four l o t s of h a l c i n o n i d e were a n a l y z e d f o r r e s i d u a l s o l v e n t s by d i s s o l v i n g p o r t i o n s i n p y r i d i n e . A f t e r r e t e n t i o n on a precolumn, water c o n t e n t w a s d e t e r m i n e d by

H ALCINON I DE
v a p o r p h a s e ( g a s ) chromatography u s i n g a n a u t h e n t i c s t a n d a r d b e f o r e and a f t e r e a c h i n j e c t i o n . Water c o n t e n t s of 0 . 6 % , o r l e s s , were found by c o m p a r i s o n w i t h a u t h e n t i c s t a n d a r d s . n-Propanol h a s o c c a s i o n a l l y b e e n found t o b e present. 4.2 I d e n t i f i c a t i o n , U l t r a v i o l e t and C o l o r i m e t r i c Analyses P r o p o s e d compendia1 i d e n t i f i c a t i o n tests f o r t h e

27 I

United States Pharmacopeia i n v o l v e comparing e i t h e r t h e i n f r a r e d o r u l t r a v i o l e t a b s o r p t i o n s p e c t r u m of s a m p l e h a l c i n n o n i d e w i t h t h a t o f a n a u t h e n t i c sample4'. To t n i s a u t h o r , a c h r o m a t o g r a p h i c method i s s u p e r i o r s i n c e e l u t i o n t i m e u s u a l l y d e p e n d s on much o f t h e m o l e c u l e i n t e r a c t i n g w i t h t h e s o l i d and m o b i l e p h a s e s v i a weak bonding f o r c e s . U l t r a v i o l e t a b s o r p t i o n d e p e n d s p r i n c i p a l l y on t h e 3-one-4-eneY A and B r i n g region being i n t a c t . H a l c i n o n i d e h a s b e e n q u a n t i t a t e d i n v a r i o u s formul a t i o n s o r a s b u l k powder by a d i f f e r e n t i a l u l t r a v i o l e t , T b o r o h y d r i d e r e d u c t i o n a s ~ a y . ~ 2 h i s d i f f e r e n t i a l a s s a y inv o l v e s m e a s u r i n g t h e u l t r a v i o l e t a b s o r b a n c e of a n a l i q u o t of m e t h a n o l i c s t e r o i d s o l u t i o n c o n t a i n i n g sodium b o r o h y d r i d e decomposed p r i o r t o t h e a d d i t i o n of s t e r o i d . Its absorbance i s d e t e r m i n e d a g a i n s t a m e t h a n o l i c r e f e r e n c e s o l u t i o n of s t e r o i d r e d u c e d by sodium b o r o h y d r i d e t o d e s t r o y t h e 3-one4-ene chromophore. The u t i l i t y of t h i s p r o c e d u r e i s t h a t many i n t e r f e r e n c e s from e x c i p i e n t s and o t h e r , u n c o n j u g a t e d , s t e r o i d s can b e e l i m i n a t e d i n t h e a s s a y of a f o r m u l a t i o n . The a d d i t i o n of h a l c i n o n i d e t o v a r i o u s c o l o r i m e t r i c r e a g e n t s i v e s r e s u l t s t y p i c a l of s t e r o i d s w i t h i t s s u b s t i t ~ e n t s . ~ q - 4 5 A s t e s t e d i n o u r labor at or^,^^ h a l c i n o 47 , nide r e a c t s with a c i d i c ethanolic 4-nitrophenylhydrazine a f t e r h e a t i n g , c o o l i n g and t h e a d d i t i o n of sodium h y d r o x i d e , t o g i v e a r i l l i a n t p u r p l e "plum" c o l o r . With m e t h a n o l i c isoniazid4', halcinonide g i v e s a b r i g h t yellow c o l o r , w i t h no n o t i c e a b l e f a d i n g a f t e r two h o u r s . H a l c i n o n i d e added t o 4 - a m i n o a n t i ~ y r i n e ~n methanolic hydrochloric a c i d g i v e s a i~ p a l e g r e e n c o l o r . H a l c i n o n i d e added t o e t h a n o l i c t e t r a methylammonium hydroxide5', and h e a t e d , g i v e s a c l o u d y amber If c o l o r . T h i s c a n b e t h e b a s i s of a q u a n t i t a t i v e a say.51 added t o e t h a n o l i c tetramethylammonium hydroxide5' and p i c r i c a c i d , a n orange-red ( t e a c o l o r e d ) s o l u t i o n r e s u l t s with halcinonide. I n concentrated s u l f u r i c acid,53 halcinon i d e g i v e s a d e e p y e l l o w c o l o r . Adding water s l o w l y c a u s e s a v i o l e t c o l o r t o a p p e a r a t t h e i n t e r f a c e . Halc' o n i d e g i v e s a r o y a l p u r p l e c o l o r w i t h b l u e t e t r a z o l i u m 3 ' and a yellow-brown c o l o r w i t h aluminum c h l o r i d e i n n i t r o m e t h a n e .

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4.3

Chromatographic Analyses 4.31 High-pressure Liquid Chromatography (HPLC)

Reverse phase high-pressure liquid chromatography is used to separate and quantitate bulk and formulated h a l ~ i n o n i d e . ~ ~ Commercially available, prepacked octadecylsilane columns, 10 pm in particle size, United S t a t e s Phamacopeia designation L-1 (such as Partisil, MicroPak or UBondapak), are used. The mobile phase is aqueous acetonitrile (1:l to 1:3) with a flow rate of 0.3 to 1.0 mL/min. Detection is at 254 nm. Figure 7 shows the elution of halcinonide and progesterone internal standard. Using a precision loop injector, repetitive injections gave a relative standard deviation of 0.6%. Without the progesterone internal standard, the relative standard deviation is 0.8%. Formulated halcinonide, in concentrations of 1 to 0.25 mg steroidlg, is either diluted with mobile phase, extracted into methanol, or partitioned into aqueous acetonitrile from h e ~ a n e 9s ~ ~ Halcinonide can be separated from kenalogl 56. (triamcinolone acetonide) by HPLC.57 Intermediates in the synthesis of halcinonide (cf. section 1.5) have the following relative retention times; Dihydrotriamcinolone, RRT = 0.18;dihydrotriamcinolone acetonide, RRT = 0.32, and dihydrotriamcinolone acetonide-21~ . mesylate, RRT = 0.72 (Halcinonide, RRT = ~ 0 0 ) ~The purity of dihydrotriamcinolone can be determined by HPLC using the same reverse phase column and a mobile phase consisting of 0.8% ammonium nitrate, 0.23% monobasic ammonium phosphate, 32.5% methanol, 8% tetrahydrofuran and water.58 Cortisone acetate is used as an internal standard.59 Impurities are determined at a higher concentration.

HALCINONIDE

273

Halcinonide

Progesterone

+r
Figure 8 R e v e r s e P h a s e H i g h - p r e s s u r e L i q u i d Chromatography of H a l c i n o n i d e . P r o g e s t e r o n e i s u s e d as i n t e r n a l s t a n d a r d . See t e x t f o r d e t a i l s .

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4.32

Thin Layer Chromatography (TLC)

TLC, using silica gel GF 254 plates, can separate halcinonide from its synthetic precursors (cf. section 1 5 . .) Using a developing solvent of chloroform60 ethyl acetate (5:1), the following Rf values were found : Dihydrotriamcinolone, Rf = 0.0; dihydrotriamcinolone acetonide, Rf = 0.08; dihydrotriamcinolone acetonide-21-mesylate, Rf = 0.18 and halcinonide, Rf = 0.36. Continuous TLC development gives respective relative Rf values of 0.02, 0.02, 0.48 and 1.0. With benzene (CAUTION)-acetone-water (70:30:07) the respective Rf values are 0.12, 0.42, 0.66 and 0.81. Traces of these intermediates have been found in some lots of halcinonide, as has dihydrotriamcinolone-2l-acetate, an impurity occasionally found in some preparations of dihydrotriamcinolone. Elution from the TLC plate with ethanol permits quantitation of bulk halcinonide and halcinonide extracted from formulations. C-Halcinonide (labelled at the acetonide carbon) was examined for impurities using silica gel TLC plates and chloroform-ethyl acetate (5 :1)61 mobile phase. The Rf value of halcinonide is 0.5. 4.33 Column Chromatography

A diatomaceous earth column was used to separate halcinonide from excipients62 The formulation is mixed with column packing material and then is transferred to the top of the column. Steroidal material is eluted with benzene (CAUTION) under a well-ventilated hood and then quantitated using thin layer chromatography60 or tetramethylammonium hydroxide51 colorimetry

4.34 Paper Chromatography Paper chromatography using Whatman No. 1 paper was once used to determine the homogeneity of halcinonide.60 Twenty percent formamide in methanol comprises one stationary phase and methylisobutyl ketone-formamide ( 2 0 : l ) is the mobile phase. A second solvent system uses 25% propylene glycol-chloroform as the stationary phase and toluene saturated with propylene glycol as the mobile phase.

4.4 Polarographic Analysis

In dimethylformamide, halcinonide is reduced in two steps.63 The 2la-chloroketo group exhibits a halfwave reduction potential of -1.17 volts U S Hg. This is easily distinguished from the half-wave potential of -1.62 volts 2)s Hg of the A4-3-keto group. Thus, halcinonide can be

HALCINONIDE

275

a s s a y e d i n t h e p r e s e n c e o f o t h e r 4-ene-3-keto steroids that l a c k t h e 21-chloro group. This response r e q u i r e s a concentrat i o n between 0.5 and 2.0 d of s t e r o i d p e r l i t e r . The more s e n s i t v e t e c h n i q u e of d i f f e r e n t i a l p u l s e p ~ l a r o g r a p h y ~ ~ should a l s o b e a p p l i c a b l e t o halcinonide. 4.5 Halcinonide

& Tissues I

and Body F l u i d s

The a n t i i n f l a m m a t o r y p r o p e r t i e s of s u c h t o p i c a a g e n t s a s h a l c i n o n i d e are u s u a l l y d e t e r m i n e d by a vasoconstrictor assay. Topically applied corticosteroids c a u s e a b l a n c h i n g a t t h e s i t e o f a p p l i c a t i o n , which c a n b e t h e forearm65 o r t h e u p p e r back of h e a l t h y a d u l t s where s t r a t u m corneum i s removed w i t h c e l l o p h a n e t a p e . 6 6 The t e s t a r e a s , c o n t a i n i n g v a r i o u s c o n c e n t r a t i o n s of h a l c i n o n i d e , are o c c l u d e d w i t h p l a s t i c wrap and are e v a l u a t e d on a n a l l - o r P e r c u t a n e o u s a b s o r p t i o n s t u d i e s 6 7 w i t h 0.1% none b a s i s . 1 4 C - h a l c i n o n i d e cream, 1 g cream p e r dog o r r a b b i t , showed t h a t t h e s t e r o i d i s absorbed through intact o r abraded s k i n . I n d o g s , 0 . 4 t o 0.5% i s e s t i m a t e d t o b e a b s o r b e d t h r o u g h 0 In rabbits, 6 t o i n t a c t and 4 t o 1 %t h r o u g h a b r a d e d s k i n . 16% of t h e 4 C - h a l c i n o n i d e w a s a b s o r b e d t h r o u g h i n t a c t and 1 4 t o 23% t h r o u g h a b r a d e d s k i n . Metabolism68 w a s s t u d i e d w i t h h a l c i n o n i d e l a b e l l e d w i t h carbon-14 i n t h e 2 - p o s i t i o n of t h e a c e t o n i d e It w a s a d m i n i s t e r e d i n t r a v e n o u s l y t o dogs a t a d o s e group. The m a j o r p o r t i o n o f t h e r a d i o a c t i v i t y w a s o f 5 mg/kg. excreted i n b i l e . Radio-autography of b i l e showed a t l e a s t 10 d i s t i n c t m e t a b o l i t e s t o be p r e s e n t . Four of t h e metabol i t e s were i d e n t i f i e d . The two most a b u n d a n t m e t a b o l i t e s , t h a t were i d e n t i f i e d , a c c o u n t e d f o r 43% ( F i g u r e 8 , M1) and 30%(M2) o f t h e r a d i o a c t i v i t y . The two minor m e t a b o l i t e s (M3 and M4) a c c o u n t e d f o r 2% e a c h . I n dog u r i n e , t h e s e f o u r m e t a b o l i t e s (Ml-4) a c c o u n t e d f o r 1 0 , 1 5 , 5 and 18% o f t h e radioactivity, respectively. I n dog b l o o d , unchanged h a l c i n o n i d e and m e t a b o l i t e s M3 and M4 e a c h a c c o u n t e d f o r a b o u t M 1 and M w e r e n o t d e t e c t e d . 2 15% of t h e r a d i o a c t i v i t y . These f o u r m e t a b o l i t e s , none of which c o n t a i n e d c h l o r i n e , were i d e n t i f i e d by comparing t h e i r u l t r a v i o l e t , n u c l e a r m a g n e t i c r e s o n a n c e o r mass s p e c t r a , and t h e i r t h i n - l a y e r c h r o m a t o g r a p h i c b e h a v i o r ( e i t h e r u n d e r i v a t i z e d , o r as a s u i t a b l e d e r i v a t i v e ) w i t h similar d a t a obtained f o r authent i c r e f e r e n c e s a m p l e s . M 1 w a s i d e n t i f i e d as t h e 21-carboxy d e r i v a t i v e of h a l c i n o n i d e (-CH2C1 + -C02H), M i s t h e 6B2 hydroxy d e r i v a t i v e , M i s t h e 21-hydroxy d e r i v a t i v e (-CH2C1 3 -CH20H) and M i s t h e 66-hydroxy d e r i v a t i v e of M3. 4 Figure 9 shows t h e p r o p o s e d pathway. M 3 i s t h e a c e t o n i d e d e r i v a f

Figure 9 Proposed M e t a b o l i c Pathways f o r H a l c i n o n i d e i n t h e Dog.

HALCINONIDE

277

t i v e i n t h e s y n t h e s i s of h a l c i n o n i d e (cf. F i g . 2) and i t i s a l s o found a s a d e g r a d a t i o n p r o d u c t i n in vitro s t u d i e s summarized below. T h i s compound, d i h y d r o t r i a m c i n o l o n e a c e t o n i d e , p o s s e s s e s t o p i c a l and s y s t e m i c a n t i i n f l a m m a t o r y a c t i v i t y . 69

5.

Stability:

Halcinonide d i s s o l v e d i n e i t h e r methanol, deuterom e t h a n o l , a q u e o u s ammonia-methanol, o r d e u t e r i u m o x i d e d e u t e r a t e d ammonia-methanol s o l v e n t s a p p e a r s s t a b l e a f t e r s t o r a g e f o r s i x d a y s a t 50", u s i n g n u c l e a r m a g n e t i c r e s o n a n c e and mass s p e c t r o m e t r y . 70 Using t h i n l a y e r chromatography (cf. s e c t i o n 4 . 3 2 , f i r s t s y s t e m ) 6 0 no change w a s f o u n d i n two l o t s of h a l c i n o n i d e a f t e r But t h e s t o r a g e a t 50" f o r s i x months i n brown g l a s s b o t t l e s . d i r e c t e x p o s u r e t o 900 f o o t - c a n d l e s of l i g h t f o r one month c a u s e d a n i n c r e a s e i n i m p u r i t i e s of two l o t s from 0.5% t o 3.9% o r 4.9%. P h o t o l y t i c d e g r a d a t i o n of t h e A-ring i s e x p e c t e d s i n c e h y d r o c o r t i s o n e and p r e d n i s o l o n e u n d e r g o r e a r r a n g e m e n t s when s o l u t i o n s of t h e s e s t e r o i d s i n a l c o h o l a r e exposed t o u l t r a v i o l e t r a d i a t i o n o r o r d i n a r y f l u o r e s c e n t l i g h t i n g . 71-73 F o r m u l a t e d h a l c i n o n i d e , a t a c o n c e n t r a t i o n of 0.1% i n e i t h e r a cream b a s e o r p o l y e t h y l e n e g l y c o l - w a t e r l o t i o n , a f t e r s t o r a g e a t a p p r o x i m a t e l y 23" f o r 3 y e a r s , showed no l o s s o n h a l c i n o n i d e c o n t e n t , u s i n g h i g h p r e s s u r e l i q u i d chromatography. 4 6 The c o n t e n t s remained unchanged w i t h i n 2% of l a b e l e d concentration. One t r i a l f o r m u l a t i o n , s t o r e d a t 60" f o r 7 months showed d e g r a d a t i o n . 7 4 The one i m p u r i t y t h a t w a s i s o l a t e d and i d e n t i f i e d ( w i t h t h e a i d of mass s p e c t r o m e t r y ) i s d i h y d r o t r i a m c i n o l o n e a c e t o n i d e ( F i g u r e l), a n i n t e r m e d i a t e i n t h e s y n t h e s i s of halcinonide. T h i s compound a l s o p o s s e s s e s t o p i c a l a n t i i n f l a m matory a c t i v i t y . 69 6. Acknowledgements

The a u t h o r g r a t e f u l l y acknowledges t h e c o u r t e o u s assist a n c e of t h e many c o n t r i b u t o r s c i t e d a s " p e r s o n a l communication." Some of t h e i n f o r m a t i o n c i t e d i n t h i s manner w a s e s p e c i a l l y obtained f o r t h i s analytical profile. Unless o t h e r w i s e i d e n t i f i e d , t h e work w a s done a t E. R . S q u i b b & S o n s , I n c . Much c r e d i t f o r t h i s p r o f i l e b e l o n g s t o D r . K l a u s F l o r e y , who wrote t h e o r i g i n a l one f o r u s e a t Squibb. S p e c i a l t h a n k s go to R . P o e t , G . B r e w e r , K . F l o r e y and S . Perlman f o r t h e i r c r i t i c a l r e a d i n g of t h i s MS, t o C . Pope f o r t y p i n g i t and t o my i a m i l y , f o r t h e i r p a t i e n c e d u r i n g t h e c o m p i l i n g of t h i s a n a l y tical profile.

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64. 65. 66. 67. 68. 69.

70.

R.N. Yadav and F.W. Teare, J . Pharm. Sci. 67, 436 (1978).

A. McKenzie and R. Stoughton, A r c h . Derm., 86, 608

(1962).
G . Wells,

Brit. J . Derm., 6 9 , 11 (1957).

A. Heald and C. Ita, Personal communication. K. Kripalani, A. El-Abdin, A . Dean and A. Cohen, Pharmacologist 29, 168 (1977) [Abs. No. 2401. L. Lerner, Personal communication. P. Funke and M. Puar, Personal communication. D. Barton and W. Taylor, J . C h e m . Soc. 2958, 2500.

71. 72.
73.

Ibid. J . Arner. C h e m . Soc., 8 0 , 244 1958.

W. Hamlin, T. Chulski, P. Johnson and J . Wagner, J . h e r . Pharm. Assoc. ( S c i . Ed.! 4 9 , 253 (1963).
K. Bush and G. Petrie, Personal communication. Literature reviewed to January 1, 1979.

74.

Analytical Profiles of Drug Substances, 8

HYDRALAZINE HYDROCHLORIDE
Chester E . Orzech, Norris G . Nash, and Raymond D. Daley
Description 1 . 1 Name, Formula, Molecular Weight I .2 Appearance, Color, Odor 1.3 History Physical Properties 2.1 Infrared Spectra 2 . 2 Ultraviolet Spectra 2.3 Mass Spectrum 2.4 Nuclear Magnetic Resonance Spectra 2.5 Differential Thermal Analysis (DTA) 2 . 6 Crystal Properties 2.7 Microchemical Characterization 2.8 Solubility 2 . 9 Dissociation Constants 2.10 Melting Points Synthesis Stability-Degradation Metabolism Methods of Analysis 6. I Identification Tests 6.2 Elemental Analysis 6 . 3 Spectrophotometric Methods 6 . 4 Fluorescence 6.5 Titration Methods 6.6 Gasometric Methods 6 . 7 Polarography 6.8 Paper chromatography 6 . 9 Thin-Layer Chromatography 6.10 High Pressure Liquid Chromatography 6.1 I Gas Chromatography Acknowledgments References

3. 4. 5. 6.

7. 8.

283

Copyright @ I979 by Academic Press. Inc. All rights of reproduction in any form reserved.

ISBN 0-12-260808-9

284

CHESTER E. ORZECH ET AL.

1.

DESCRIPTION 1.1

N a m e , Formula, Molecular Weight The Chemical A b s t r a c t s name f o r h y d r a l a z i n e hydroc h l o r i d e i s 1( 2H)-phthalazinone hydrazone monohydrochloride, s t a r t i n g w i t h volume 80; p r e v i o u s l y t h e name l-hydrazinoA p h t h a l a z i n e monohydrochloride w a s used. The C S R e g i s t r y No. i s [304-20-1] f o r t h e h y d r o c h l o r i d e s a l t , [86-54-4] f o r t h e base. NHNH2

C8H8N4OHC1
1.2

Molecular Weight:

196.64

Appearance, C o l o r , Odor Hydralazine h y d r o c h l o r i d e i s a white t o o f f - w h i t e o d o r l e s s c r y s t a l l i n e powder. History The a b i l i t y of h y d r a l a z i n e and similar hydrazino compounds t o reduce blood p r e s s u r e w a s r e p o r t e d by Gross e t a1 ( 1 ) i n 1950. Methods of s y n t h e s i s were p u b l i s h e d by Druey and R i n g i e r ( 2 ) i n 1951, along w i t h t y p i c a l r e a c t i o n s d i s p l a y e d by t h e compound , i n c l u d i n g s e v e r a l r e a c t i o n s which l a t e r became t h e b a s e s f o r a n a l y s i s of h y d r a l a z i n e and i t s m e t a b o l i t e s . The drug h a s been widely used f o r t r e a t m e n t of h y p e r t e n s i o n , and p a p e r s on i t s p r o p e r t i e s and methods f o r i t s d e t e r m i n a t i o n have been p u b l i s h e d i n many languages. Hydralazine i s u s u a l l y used i n combination w i t h o t h e r drugs. In r e c e n t y e a r s , it h a s been found t o b e e s p e c i a l l y u s e f u l when used with b e t a - a d r e n e r g i c b l o c k i n g a g e n t s and d i u r e t i c s (3,4). 2. PHYSICAL PROPERTIES 2.1 Infrared Spectra 1.3

The i n f r a r e d spectrum of h y d r a l a z i n e h y d r o c h l o r i d e (Figure I > w a s o b t a i n e d w i t h a Beckman IR-12 spectrophotometer. A mineral o i l d i s p e r s i o n between potassium bromide p l a t e s w a s scanned from 423 t o 4000 cm-1, and a t h i c k e r l a y e r of t h e d i s p e r s i o n , supported on p o l y e t h y l e n e f i l m ,

HYDRALAZINE HYDROCHLORIDE

285

was scanned from 200 t o 530 cm-. bands can b e a s s i g n e d as f o l l o w s :

Frequency, c m -1

Some of t h e a b s o r p t i o n

Assignment ( 5 )

3220
9. 25
2800-3000

N-H s t r e t c h
Aromatic C-H s t r e t c h Mineral o i l C-H stretch C=C s t r e t c h Mineral o i l C-H band

1590- 1600 1460

785

H atoms on an aromatic r i n g The m i n e r a l o i l a b s o r p t i o n a t 2800 t o 3000 cm-I and at 1460 cm- o b s c u r e s a b s o r p t i o n bands of h y d r a l a z i n e h y d r o c h l o r i d e at 2810, 2320, and 2970 cm-1 (N-H s t r e t c h ) and a w e a k s h a r p band a t 1470 cm-l; t h e s e bands can be observed i n potassium bromide d i s p e r s i o n s p e c t r a . The bands a t 1070 and 1082 cm-1 tend t o merge i n t o a s i n g l e band i n potassium bromide dispersion spectra. The i n f r a r e d spectrum of h y d r a l a z i n e h y d r o c h l o r i d e b a s e i n a potassium bromide d i s p e r s i o n ( F i g u r e 2 ) w a s 0 recorded from 4 0 t o 4 0 cm-1 , and t h e 2 0 t o 550 cm-1 0 00 region w a s o b t a i n e d from a m i n e r a l o i l d i s p e r s i o n s u p p o r t e d on p o l y e t h y l e n e f i l m . The s p e c t r a o f potassium bromide d i s p e r s i o n s of t h e b a s e a r e q u a l i t a t i v e l y i d e n t i c a l t o t h o s e of m i n e r a l o i l d i s p e r s i o n s . The assignment of a b s o r p t i o n bands i n t h e spectrum of t h e b a s e i s s i m i l a r + t o t h a t of t h e h y d r o c h l o r i d e except o r t h e p r e s e n c e of N-H stretch a b s o r p t i o n i n t h e l a t t e r . A spectrum of t h e b a s e h a s been published ( 6 ) . Ultraviolet Spectra F i g u r e 3 is t h e u l t r a v i o l e t a b s o r p t i o n spectrum of h y d r a l a z i n e h y d r o c h l o r i d e i n water s o l u t i o n , run on a Cary Model 14 spectrophotometer. The s o l u t i o n contained 9.9 m g of h y d r a l a z i n e h y d r o c h l o r i d e p e r l i t e r , and w a s run a g a i n s t water ( 1 c c e l l s ) . The d i s c o n t i n u i t y i n t h e spectrum a t m 219 nm i s a change of a b s o r p t i o n s c a l e ; t h e absorbance s c a l e range i s 0.0 t o 1.0 f o r t h e wavelength range 350 t o 2.2

4 adjacent

Out of p l a n e bending,

rl rl

*rl

a ,

0 0
0

v)

0 0

....
.

.
,

.~ . . .

t
!

287

288

CHESTER E. ORZECH ET A L .

219 nm, and 1.0 t o 2.0 f o r t h e wavelength range 219 t o 200 nm. A scan of water a g a i n s t water i s a l s o recorded on t h e c h a r t . The spectrum e x h i b i t s m a x i m a a t 315, 303, 260, 239, and 211 nm, w i t h r e s p e c t i v e a b s o r p t i v i t i e s as f o l l o w s : 4,200; 5,200; 10,600; 11,000; 33,800 l./mole cm. Druey and Tripod ( 3 ) , Kuhnert-Brandstbtter e t al (71, Sharkey e t al ( 8 ) , and Solomonova e t al ( 9 ) r e p o r t e d similar m a x i m a and a b s o r p t i v i t i e s , except t h a t t h e y d i d Clarke (6) g i v e s d a t a not r e p o r t t h e 211 n m a x i m u m . m similar t o t h a t o f t h e above i n v e s t i g a t o r s . Sunshine ( 1 0 ) g i v e s t h e spectrum i n 0.1N h y d r o c h l o r i c a c i d , w i t h m a x i m a at 312, 302, 259, and 233 nm, and t h e spectrum i n 0.1N sodium hydroxide, w i t h m a x i m a at 304, 27.1, and 262 nm. Naik e t al (11) gave p a r t i a l u l t r a v i o l e t s p e c t r a of t h e b a s e , t h e monohydrochloride, and t h e d i h y d r o c h l o r i d e : ( a ) t h e b a s e e x h i b i t e d m a x i m a a t 305 and 273 nm, w i t h a t h i r d maximum n e a r 262 nm; ( b ) t h e monohydrochloride e x h i b i t e d m a x i m a a t 315, 302, 2'30, and 260 nm; ( c ) t h e d i h y d r o c h l o r i d e e x h i b i t e d a m a x i m u m a t 318 nm w i t h a broad The Merck Index (12) l i s t s s h o u l d e r a t about 300 nm. m a x i m a at 315, 304, 260, 240, and 211 nm f o r t h e aqueous s o l u t i o n of t h e hydrochloride.
Mass Spectrum F i g u r e 4 shows t h e low r e s o l u t i o n mass spectrum of h y d r a l a z i n e hydrochloride. The d a t a were o b t a i n e d with an LKB 9000s mass s p e c t r o m e t e r , w i t h an i o n i z a t i o n voltage of 70 eV, s o u r c e temperature z O o C . Some of t h e peaks m a y b e a s s i g n e d as f o l l o w s ( 1 3 ) :

2.3

Mass Charge R a t i o
160

Assignment

M+

129 103

+-

.NH-NH,

76
51

C6H4

C4H3
HC1'

36/38

. . . . . .
I
I

. .

! -I f ::.I--:
1 .
.

,
.

. .

.
1 -

.
,
a * . . .

..
.
A

a
T . . ?

. . .
. .

.
..........
. .-

. . . . . . _ ~

. . . . . . .

..

. ._ . . . . . .
~

__

. . . . .

_ - --./ . _

# -;
~
. .

. . . . . . .
.
&

.......... . . .
J

. . . . . .

.rl

a,
0

k a

I
I

A l I S N 3 1 N I 3A11Vl31J

HYDRALAZIN E HYDROCHLORIDE

29 I

Nuclear Magnetic Resonance S p e c t r a The NMR spectrum shown i n F i g u r e 5 w a s o b t a i n e d by d i s s o l v i n g h y d r a l a z i n e h y d r o c h l o r i d e i n deuterium oxide c o n t a i n i n g 3- ( t r i m e t h y l s i l y 1 ) - I-propane-sulfonic a c i d sodium s a l t ( E X ) . The s e r i e s of p e a k s a t 0 , 0.6, 1.8, and 3 ppm are all due t o t h e WS. The peak a t 4.8 pprn i s due t o HDO which forms on exchange w i t h t h e s o l v e n t and t h e p e a k s at 8.01 and 8.61 ppm a r e due t o t h e a r o m a t i c p r o t o n s . The N M R spectrum of t h e b a s e ( F i g u r e 6 ) w a s o b t a i n e d i n a 1 :1 mixture of dirnethylsulfoxide-d :deuterochloroform. The p e a k s a t 2.53 ppm a r e due t o t 8 e s o l v e n t . The N-H p r o t o n s a r e a t 4.1 pprn and t h e aromatic p r o t o n s a r e a t 7.6 and 8 ppm. The s p e c t r a were produced u s i n g a Varian EM-360 NMR s p e c t r o m e t e r . D i f f e r e n t i a l Thermal A n a l y s i s (IYTA) The DTA c u r v e s i n F i g u r e s 7 and 8 were o b t a i n e d with a W o n t Model 900 instrument. F i g u r e 8 shows t h a t t h e b a s e m e l t s s h a r p l y and F i g u r e 7 shows t h a t t h e hydroc h l o r i d e melts w i t h decomposition. V i s u a l l y t h e hydroc h l o r i d e melts at 274"C, b u t decomposition t a k e s p l a c e o v e r a l a r g e t e m p e r a t u r e range ( F i g u r e 7). Crystal Properties Chojnacki e t a1 ( 1 4 ) i n v e s t i g a t e d t h e c r y s t a l p r o p e r t i e s of h y d r a l a z i n e hydrochloride. Recrystallized from water, t h e c r y s t a l s were found t o be monoglinic, w i t h t h e u n i t c e l l a = 9.408, b = 14.529, c = 6.643A, p = 103.59", Z = 4. The c a l c u l a t e d d e n s i t y w a s 1.486, t h e measured d e n s i t y 1.479 g p e r cm3. The s p a c e group w a s P21/c. Powder d i f f r a c t i o n d a t a , g e n e r a l l y similar t o t h a t of Table 1, i s a l s o given. X-ray powder d i f f r a c t i o n d a t a f o r h y d r a l a z i n e h y d r o c h l o r i d e and h y d r a l a z i n e b a s e are g i v e n i n Table 1. These d a t a were o b t a i n e d with a Norelco d i f f r a c t o m e t e r u s i n g n i c k e l - f i l t e r e d copper Ka r a d i a t i o n . Microchemical C h a r a c t e r i z a t i o n S a n d r i (15) r e p o r t e d t h e formation of c h a r a c t e r i s t i c c r y s t a l s w i t h cadmium bromide-hydrobromic a c i d , bismuth iodide-potassium i o d i d e , and bismuth bromide-hydrobrornic a c i d , with compositions CdBr -HBr*C H N - 4 H 2 0 , 8 8 2 ~ i I q - c 8 H ~ N 4 , HEiiBr - C H N r e s p e c t i v e l y . 4 and u h n e r t - B r a n d s4 s t e r e al ( 7 ) r e p o r t e d t h e microt* s c o p i c h o t - s t a g e c h a r a c t e r i z a t i o n of h y d r a l a z i n e hydroc h l o r i d e . Above 21OoC, small r o d s , g r a n u l e s , and p r i s m s sublime. The m e l t i n g p o i n t i s 268-a5"C. G a s bubbles are evolved as m e l t i n g b e g i n s , and m e l t i n g p r o c e e d s w i t h 2.7

2.4

2.5

2.6

9 41

292

CHESTER E. ORZECH ET A L .

TABLE 1
X-RAY POWDER DIFFRACTION PATTERNS OF HYDRALAZINE HYDROCHLORIDE AND HYDRALAZINE BASE Hydrochloride Base
0 0 -

d(A)

d(A)

7.74 7- 5 2 5-92 5-54 48 .2 46 .2 43 .7 39 .9 38 .7 3-63 33 .9 3-38 3-32

3 - 29

10 0

93 9 1 8 47 1 5
2 2

54 9
11

12

32 .2 3- 5 1 3-05 2-99 2-95 2-93 28 .5 2-77 27 .2 26 .4 25 .8 24 .4 24 .1 2-39 23 .4 22 .4

22 .2

3 1 65 92 7 9 4

22 2 2

2 2

73 .7 5-87 5.62 5.04 48 .6 44 .8 39 .8 37 .4 36 .6 34 .7 32 .8 32 .4 3-12 29 .6 28 .0 27 1 . 26 .7 26 .0 24 .9 24 .4 23 .8

2-30

10 0

49
11

11

1 7 1 8

1 0

2 2

10 0

1 5

1 7 7

11 2 1 2 2 2 2 1 2 1 2 1
2

4 7

22 .8 22 .5 21 .8

1 6

2 1

21 .2

21 .8 21 .6
21 .0

20 .6
20 .2

3 4 4

2 2 2 2 2 1 2 2

19 .9 1-97 I. 4 9 18 .9

2-07 20 .4 1-99 19 .5 18 .7 I. 5 8 18 .3 1-79 I. 8 7 17 .6 17 .2 16 .7 16 .2

3
1

1 2 2 1 1 1 1 1 1

HYDRALAZINE HYDROCHLORIDE

293

1 0

5 4 PPY (61

Figure

5.

Nuclear Magnetic Resonance Spectrum of Hydralazine

Hydrochloride.

1 0

5
PPY 16)

Figure

6.

Nuclear Magnetic Resonance Spectrum of Hydralazine Base.

h)

Figure 7.

D i f f e r e n t i a l Thermal Analysis Curve of Hydralazine Hydrochloride.

Figure 8.

D i f f e r e n t i a l Thermal Analysis Curve of Hydralazine Base.

296

CHESTER E. ORZECH ET A L .

t h e separation of high melting (over 300C) needles and bushlike aggregates. The e u t e c t i c temperature with p-acetamidophenyl s a l i c y l a t e was 181 "C, with dicyandiami.de 162C. The p i c r a t e salt forms from aqueous s o l u t i o n as long needles and columns t h a t melt a t 210 t o 212C with decomposition. The styphnate forms needles and r o s e t t e s with a melting point of 200 t o 203C. Sunshine (10) and Clarke ( 6 ) give two microc r y s t a l t e s t s , both from aqueous s o l u t i o n : ( a ) l e a d iodide-potassium a c e t a t e reagent forms dense r o s e t t e s ; (b) iodine-potassium i o d i d e ( 1 :5O) reagent forms c l u s t e r s or masses of needles. Solubility The Merck Index (12) g i v e s t h e s o l u b i l i t y i n water as 9 . 1 m p e r m l at 15"C, and 44.2 m p e r m l a t 25C. g g In 95% ethanol, t h e s o l u b i l i t y i s given as 2 m p e r ml. These g a r e consistent with t h e following approximate s o l u b i l i t y d a t a , determined at room temperature: Solvent Water Methanol Ethanol (95%) 2-Propanol Chloroform Ethyl e t h e r (anhydrous) Ethyl a c e t a t e Acet oni t r i l e 2.9 Approximate S o l u b i l i t y , mg/ml

2.8

39

6 -7
1-9

0.1

< 0.1

< 0.1
< 0.1 < 0.1

Dissociation Constants Evstratova and Ivanova (16) reported a pKa of 7.1. Evstratova e t a1 (17) reported a p K of 7.1 i n water, a a p K of 4.7 i n aqueous 90 percent acetone, and a p\ of 15.6 inathe l a t t e r solvent. N a i k e t a1 (11) reported a pK of 6.9 for t h e d i s s o c i a t i o n of t h e monohydrochloride anda0.5 f o r d i s s o c i a t i o n of t h e dihydrochloride, determined absorptiometrically. Artamanov et al reported a pKa of 7.2 (18).

HYDRALAZINE HYDROCHLORIDE

297

2.10 Melting P o i n t s The m e l t i n g p o i n t of h y d r a l a z i n e h y d r o c h l o r i d e i s n e a r n3"C, t h a t of t h e b a s e i s n e a r 173C. Melting p o i n t s r e p o r t e d i n t h e l i t e r a t u r e a r e as f o l l o w s : Melting P o i n t of Hydrochloride ( a ) ,

OC

Reference (3,191
( 2 , s 21 ,22,23 )

no 273
271

280

272

(24)

265

(25)

273

274

(26)
Reference (2,21,22) (24)

( a ) The h y d r o c h l o r i d e m e l t s w i t h decomposition. Melting P o i n t of Base, 172

OC

173

165 - 172 172 173

(25)

175

(27)

3.

SYNTHESIS

Hydralazine has been p r e p a r e d by v a r i o u s p r o c e d u r e s from I - c h l o r o p h t h a l a z i n e ( 2 , 2 1 , 2 2 , 2 4 , 2 8 ) , p h t h a l a z i n e - l t h i o n e (20,23,25), 1 , 4 - d i c h l o r o p h t h a l a z i n e (29,30,31), I-(methylsulfony1)-phthalazine (271, o r 1-cyano-2-benzal a c e t a t e (26). Phthalazone, used t o p r e p a r e l-chlorop h t h a l a z i n e , h a s been p r e p a r e d from naphthalene (3,281 o r from p h t h a l i c anhydride ( 3 ) . A comprehensive survey on h y d r a z i n o p h t h a l a z i n e s and r e l a t e d compounds h a s been p u b l i s h e d by Druey and Tripod ( 3 ) . Some of t h e s e p r o c e d u r e s a r e o u t l i n e d i n F i g u r e s 9 and 10.

4.

STABILITY

DEGRADATION

Hydralazine h y d r o c h l o r i d e i s q u i t e s t a b l e as t h e c r y s t a l l i n e s o l i d . A t room t e m p e r a t u r e , it i s s t a b l e i n In aqueous d i s t i l l e d water s o l u t i o n f o r weeks (32).

ri V

&o J
E XN

-1
0
V

0 X

O7 &

(H

298

aCN
H( OAC 1

HNNH

ci

Figure 10.

c1 Several Syntheses f o r Hydralazine.

300

CHESTER E. ORZECH E7 A L .

s o l u t i o n w i t h a pH g r e a t e r t h a n 7 , h y d r a l a z i n e decomposes, forming p h t h a l a z i n e ; t h e r a t e of decomposition depends on pH, t e m p e r a t u r e , and t h e k i n d and c o n c e n t r a t i o n of anion p r e s e n t (33). I n b i o l o g i c a l samples t h e drug d i s a p p e a r s r a p i d l y a t room temperature, a p p a r e n t l y by enzymatic r e a c t i o n s . The hydrazino group i s h i g h l y r e a c t i v e , forming It i s a l s o a hydrazones w i t h aldehydes and ketones. reducing a g e n t , and i t forms complexes w i t h many metal i o n s (34). A t 275 t o 280C i t decomposes t o h y d r a z i n e , ammonia, n i t r o g e n , and 1 , k d i h y d r o - l , 1 ' - b i p h t h a l a z i n e (35).

5.

METABOLISM

Hydralazine h y d r o c h l o r i d e i s r a p i d l y metabolized and excreted. Experiments w i t h carbon-14 l a b e l e d drug i n humans i n d i c a t e d t h a t l e s s t h a n 10 p e r c e n t of t h e i n t a c t drug was e x c r e t e d (36). Within 5 days a f t e r a dose, 83 t o 89 p e r c e n t was e x c r e t e d i n t h e u r i n e and 9 t o 12 p e r c e n t i n t h e f e c e s . O t h e m a t e r i a l e x c r e t e d i n t h e u r i n e , 96 f p e r c e n t w a s recovered i n t h e first 24 hours. Individuals who are slow a c e t y l a t o r s e x h i b i t h i g h e r h y d r a l a z i n e blood l e v e l s t h a n f a s t a c e t y l a t o r s , f o r t h e same dose (37). Reported metabolic p r o d u c t s of h y d r a l a z i n e i n v a r i o u s s p e c i e s are as follows: Metabolite Species Man Reference (36,37,38,39, 40,41,42,43,44) (3,39,43,45,

I.

3-Methyl-s-triazolo !3,t d p h t h a l a z i n e I

Rat
Guinea p i g Rabbit Pigeon

46 1

(45) (38 1

(45)
(36,38,47,85 ) (38,46) (38)

1. 1

N- ( I-Phthalaziny1)hydrazone of p y r u v i c acid
N- ( 1-Phthalaziny1)hydrazone of acetone N- ( 1-Phthalaziny1)hydrazone of aketoglutaric acid

Man
Rat Rabb i t

111.
IV.

Man
Rat Man

(47) (44,461
(47)

HYDRALAZINE HYDROCHLORIDE

30 I

V.

s-Triazolo[3,4al phthalazine 1(2H)-Phthalazinone

Man
Rat

VI.

Man Rat

VII.
VIII.

Phthalazine

Man
Rat

(37)
(46)

4- ( 2-Acetylhydrazino )phthalazinone Man

9-Hydroxy-3-methyl-striazol0[3,4a! phthalazine Man 3-Hydroxymethyl-striazolot3,4a! phthalazine

(36)

IX.

(36)

X.

Man
Rat

In c a s e s where t h e m e t a b o l i t e s above p o s s e s s s u i t a b l e f u n c t i o n a l g r o u p s , t h e y a r e u s u a l l y e x c r e t e d as g l u c u r o n i d e s or s u l f a t e s . F i g u r e 11 i s a scheme i l l u s t r a t i n g t h e metabolism of h y d r a l a z i n e , based p r i m a r i l y on t h o s e proposed by Haegele e t al ( 4 6 ) and Wagner e t a1 (36).

6.

METHODS OF ANALYSIS

Identification Tests Hydralazine h y d r o c h l o r i d e can be e a s i l y i d e n t i f i e d by t h e p h y s i c a l p r o p e r t i e s d e s c r i b e d i n S e c t i o n 2 above. Where i d e n t i f i c a t i o n o f h y d r a l a z i n e i n f o r m u l a t i o n s i s n e c e s s a r y , t h e f o l l o w i n g t e s t s may b e u s e f u l . Cooper ( 4 9 ) used s p o t t e s t s on p a p e r , r e a c t i n g f e r r i c i o n or dirnethylarninobenzaldehyde w i t h t h e unknown. The i r o n r e a g e n t y i e l d s a b l u e c o l o r , and dimethylaminobenzaldehyde an orange c o l o r . The r e s u l t s o b t a i n e d w i t h 95 o t h e r d r u g s a r e given. Belikov e t a1 (50)d e s c r i b e d t h r e e r e a c t i o n s t o i d e n t i f y hydralazine. Sodium n i t r o p r u s s i . d e r e a c t s w i t h an a l k a l i n e aqueous s o l u t i o n of h y d r a l a z i n e t o y i e l d a r e d c o l o r ; when t h e m i x t u r e c o n t a i n s m i n e r a l a c i d s , a g r e e n p r e c i p i t a t e i s formed, b u t w i t h a c e t i c a c i d , a r e d p r e c i p i t a t e i s formed. Cinnamaldehyde r e a c t s w i t h a h y d r o c h l o r i c a c i d s o l u t i o n of h y d r a l a z i n e t o g i v e a y e l l o w p r e c i p i t a t e , m.p. 197-200"C. p-Nitrobenzaldehyde r e a c t s w i t h a n

6.1

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CHESTER E. ORZECH ET AL.

aqueous s o l u t i o n of h y d r a l a z i n e t o g i v e an orange p r e c i p i t a t e , m.p. 260-26I0C. Modras ( 5 1 ) r e p o r t e d s p o t t e s t r e a c t i o n s t o d i f f e r e n t i a t e h y d r a l a z i n e from c l o s e l y r e l a t e d drugs. Reagents used were aqueous copper (I) c h l o r i d e , aqueous ammonium molybdate, i o d i n e i n potassium i o d i d e s o l u t i o n , aqueous c o b a l t (11) n i t r a t e , a l c o h o l i c n i n h y d r i n , and a l c o h o l i c bromophenol b l u e . The t e s t s were performed on paper o r on S i l i c a G e l G. Roshchenko e t al ( 5 2 ) r e a c t e d h y d r a l a z i n e w i t h p ,p'-dichlorobenzene sulfonamidate sodium i n water at 80 t o 90C t o produce a c r y s t a l l i n e p r e c i p i t a t e C H 0 N S C 1 20.17452 2 with a m e l t i n g p o i n t of 196-197"C. I n t h e USP ( 1 9 ) method f o r i d e n t i f y i n g h y d r a l a z i n e i n t a b l e t s and i n j e c t i o n s , o-nitrobenzaldehyde i s added t o form an orange p r e c i p i t a t e . S t o h s and S c r a t c h l e y ( 5 3 ) used t h i n l a y e r chromatography t o i d e n t i f y h y d r a l a z i n e i n dosage forms and b i o l o g i c a l f l u i d s ( s e e S e c t i o n 6.8). Elemental Analyses The elemental compositions of h y d r a l a z i n e hydroc h l o r i d e and h y d r a l a z i n e b a s e are as f o l l o w s : Hydrochloride Calculated Found(2) 6.2

Element Carbon Hydrogen Nitrogen Chlorine

Base Calculated Found( 2)

48.87
4.61

48 -99
4.83
28-39
18.17

59.99

----35 -09

5 -03
34.98

28.49
18.03

---

---

Spe ct rop ho t omet r i c Methods Solomonova e t al ( 9 ) determined h y d r a l a z i n e i n t a b l e t s by a d i r e c t u l t r a v i o l e t a b s o r p t i o n method. Although t h e compound can b e e x t r a c t e d from f o r m u l a t i o n s with a l c o h o l o r w a t e r , o t h e r components of t h e m i x t u r e may i n t e r f e r e w i t h d i r e c t u l t r a v i o l e t a b s o r p t i o n measurement by c o n t r i b u t i n g t o t h e observed a b s o r p t i o n . The s o l u t i o n s u s u a l l y cannot be cleaned up by e x t r a c t i o n t e c h n i q u e s because h y d r a l a z i n e decomposes i n a l k a l i n e s o l u t i o n . However, t h e r e a r e many r e a c t i o n s t h a t g i v e r i s e t o n e a r u l t r a v i o l e t o r v i s i b l e a b s o r p t i o n bands s u i t a b l e f o r quant i t a t i on.

6.3

HYDRALAZINE HYDROCHLORIDE

305

A f r e q u e n t l y r e p o r t ed s p e ct ropho t omet r i c t e c h i que f o r t h e d e t e r m i n a t i o n of h y d r a l a z i n e i s based on r e a c t i o n s with a r o m a t i c aldehydes t o form hydrazones w i t h a b s o r p t i o n i n t h e v i s i b l e region. Luk'yanchikova e t al ( 5 4 ) used p-nitrobenzaldehyde; Wesley-Hadzi j a and Abaffy (55) and Ruggieri (56 ) used p-dimethylaminobenzaldehyde; Luk'yanchikova (57,581 used cinnamaldehyde; S c h u l e r t (33) used p-hydroxybenzaldehyde; and Zak e t al ( 5 9 ) used p-methoqbenzaldehyde, a f t e r t e s t i n g cinnamaldehyde, s a l i c y l a l d e h y d e , 3,4,5- t r i m e t h o q b e n z a l d e h y d e , and I-napht haldehyde P e r r y (32), and Grabowicz and B r u l i n s k a (60)used n i n h y d r i n t o o b t a i n a s o l u t i o n w i t h an a b s o r p t i o n maximum n e a r 450 nm. E l l e r t and Modras (61 ) t r e a t e d h y d r a l a z i n e w i t h f e r r o u s i o n i n a l k a l i n e s o l u t i o n , and measured t h e c o l o r produced a t 540 nm. Ruggieri (56) r e p o r t e d a c o l o r i m e t r i c t e s t u s i n g 2-naphthoquinonesulfonate sodium t o form a r o s e - v i o l e t color. Urbanyi and O'Connell (62,63)developed an automated c o l o r i m e t r i c method u s i n g b l u e t e t r a z o l i u m f o r t h e a n a l y s i s of h y d r a l a z i n e i n t h e p r e s e n c e of r e s e r p i n e and hydrochlorothiazide.

Fluorescence N a i k e t al (11) e x t r a c t e d h y d r a l a z i n e hydroc h l o r i d e from t a b l e t s w i t h 50 p e r c e n t aqueous methanol and mixed a p o r t i o n of t h e e x t r a c t w i t h 99 volumes of concent r a t e d s u l f u r i c a c i d t o o b t a i n f l u o r e s c e n c e a t 353 n w i t h m e x c i t a t i o n a t 323 nm. The f l u o r e s c e n c e i n t e n s i t y v a r i e d l i n e a r l y w i t h c o n c e n t r a t i o n i n t h e range 2 t o 8 pg hydralazine hydrochloride p e r m l . I n j e c t i o n s were analyzed similarly. T i t r a t i o n Methods Ruggieri (56) r e p o r t e d t i t r a t i n g h y d r a l a z i n e w i t h p e r c h l or i c a c i d Ruzhentseva e t a1 ( 6 4 ) converted h y d r a l a z i n e t o ammonia by h e a t i n g with z i n c i n s u l f u r i c a c i d , forming 3 moles of ammonia p e r mole of h y d r a l a z i n e . The m i x t u r e w a s made a l k a l i n e and t h e ammonia w a s d i s t i l l e d i n t o b o r i c a c i d s o l u t i o n , which w a s t h e n t i t r a t e d . S a n d r i ( 1 5 ) t e s t e d t h r e e t i t r a t i o n methods w i t h good r e s u l t s . One was t i t r a t i o n w i t h potassium bromate i n t h e p r e s e n c e of potassium bromide and h y d r o c h l o r i c a c i d , u s i n g a s t a r c h - i o d i n e end-point. Another w a s a d d i t i o n of e x c e s s p e r i o d i c acid-potassium i o d i d e with sodium

6.4

6.5

306

CHESTER E. ORZECH ET AL.

thiosulfate back-titration. The t h i r d w a s a d d i t i o n of sodium hydroxide and a c o n c e n t r a t e d s o l u t i o n of potassium f e r r o c y a n i d e , then a c i d i f y i n g and t i t r a t i n g with potassium permanganat e s o l u t i o n . P e r e l 'man and Evst rat ova (65 ) t i t r a t e d h y d r a l a z i n e h y d r o c h l o r i d e i n dimethylformamide s o l u t i o n t o a p o t e n t i o m e t r i c end-point w i t h sodium methoxide s o l u t i o n . Artamanov e t a1 (18) determined h y d r a l a z i n e h y d r o c h l o r i d e by conductometric t i t r a t i o n with sodium hydroxide s o l u t i o n . Goryacheva and Prikhodkina ( 6 6 ) t i t r a t e d hydralaz i n e from t a b l e t s w i t h N-bromosuccinimide s o l u t i o n . One mole of h y d r a l a z i n e h y d r o c h l o r i d e w a s e q u i v a l e n t t o 2 moles of N-bromosuccinimide. Methyl r e d was used as t h e indicator. Soliman and B e l a l i n v e s t i g a t e d a r g e n t i m e t r i c (67,681 and m e r c u r i m e t r i c (69) methods. Hydralazine p r e c i p i t a t e s s i l v e r from ammoniacal s i l v e r n i t r a t e s o l u t i o n . The s i l v e r i s d i s s o l v e d w i t h h o t n i t r i c a c i d and t i t r a t e d w i t h ammonium t h i o c y a n a t e s o l u t i o n . A l t e r n a t i v e l y , mercury i s p r e c i p i t a t e d from a l k a l i n e potassium mercuric i o d i d e s o l u t i o n . The p r e c i p i t a t e d mercury i s d i s s o l v e d by adding e x c e s s s t a n d a r d i o d i n e s o l u t i o n . The excess i o d i n e i s b a c k - t i t r a t e d w i t h sodium t h i o s u l f a t e solution a f t e r acidifying with a c e t i c acid. USP X I X d i r e c t s t h a t h y d r a l a z i n e b e determined i n t h e raw material, t a b l e t s , and i n j e c t i o n s by potassium i o d a t e t i t r a t i o n i n s t r o n g l y a c i d s o l u t i o n , u s i n g chloroform t o d e t e c t t h e p r e s e n c e of i o d i n e (19). Gasometric Methods McKennis and Yard (70) s t u d i e d t h e n i t r o g e n e v o l u t i o n from a s e r i e s of hydrazino compounds when t r e a t e d w i t h 0.0% K I O i n 0z H 04 i n a Warburg a p p a r a t u s at . 37OC. I n an aqueous s o l u i o n , h y d r a l a z i n e r e l e a s e d 102 3 p e r c e n t of t h e t h e o r e t i c a l amount of n i t r o g e n i n I5 minutes. Viala (71) determined h y d r a l a z i n e i n s o l u t i o n s o r t a b l e t s by measuring t h e n i t r o g e n f r e e d from t h e hydrazine group, u s i n g potassium permanganate i n s u l f u r i c a c i d , o r i o d i n e i n sodium b i c a r b o n a t e s o l u t i o n .

6.6

Polarographx P o l a r o g r a p h i c s t u d i e s o f h y d r a l a z i n e and r e l a t e d compounds were r e p o r t e d by Giovanoli-Jakubezak e t a1 (72) and by Modras ( 7 3 ) . The r e d u c t i o n p r o c e e d s i n two 2-electron s t a g e s w i t h t h e formation of t h e t e t r a h y d r o d e r i v a t i v e . Hydralazine could b e determined i n t h e

6.7

HYDRALAZINE HYDROCHLORIDE

307

p r e s e n c e of decomposition p r o d u c t s , at a c o n c e n t r a t i o n of about 0.0OlM i n 1 h y d r o c h l o r i c a c i d c o n t a i n i n g a s m a l l 2 amount of g e l a t i n . The half-wave p o t e n t i a l s of t h e two waves were -0.7 and -0.95 V a g a i n s t a s a t u r a t e d calomel electrode

Paper Chromatography Ruggieri (56) and McIsaac and Kanda (38) d e s c r i b e chromatography on Whatman No. 1 p a p e r w i t h b u t a n o l / a c e t i c acid/water (4:1:5) as t h e s o l v e n t . Detection w a s by quenching of t h e background f l u o r e s c e n c e of t h e p a p e r under u l t r a v i o l e t l i g h t or by ammoniacal s i l v e r n i t r a t e . The Rf value f o r hydralazine w a s 0 9 . Several in v e s tig a to rs .0 have used a l k a l i n e systems f o r p a p e r chromatog(41,45,74) raphy of h y d r a l a z i n e and i t s m e t a b o l i t e s , b u t Lesser e t al (75) suggest t h a t a l k a l i n e chromatography systems may n o t be s u i t a b l e f o r h y d r a l a z i n e i t s e l f . Thin Layer Chromatography S t o h s and S c r a t c h l e y (53) i n v e s t i g a t e d s e v e r a l t h i n l a y e r chromatography systems f o r thiazi.de d i u r e t i c s and a n t i h y p e r t e n s i v e d r u g s , u s i n g s i l i c a g e l G and a v a r i e t y of d e t e c t i o n r e a g e n t s . Solvent System Methyl e t h y l ketone/n-hexane Methyl e t h y l ketone/n-hexane Methyl e t h y l ketone/n-hexane
( 1 :1) ( 2 : 1)

68 .

69 .

Hydralazine R

0.72 0.62
0.oo

(3: ) 1

Chl oro fo rm/ac e t on e/t r i et hanol amin e

(50:50: . ) 15

06 .8

Reagents f o r d e t e c t i n g h y d r a l a z i n e were D r a g e n d o r f f ' s r e a g e n t , a l k a l i n e dimethylaminobenzaldehyde, a n i s a l d e h y d e , and Bratton-Marshall r e a g e n t . Z a k e t a1 (59) used 3g h y d r o c h l o r i c acid/methanol a s c o r b i c a c i d (44:6:1) on s i l i c a g e l and found an R v a l u e f o f 0.53 f o r h y d r a l a z i n e . Lesser e t a1 (75) used c e l l u l o s e s h e e t s w i t h f l u o r e s c e n t i n d i c a t o r , a c e t i c acid/0.0 1% aqueous disodium .8 e d e t a t e (3:97), and observed an Rf v a l u e of 0 7 for hydralazine. On s i l i c a g e l w i t h f l u o r e s c e n t i n d i c a t o r , t h e Rf v a l u e w a s l e s s t h a n 0 0 w i t h ( a ) chloroform/n-heptane/ .5 a c e t i c a c i d (6:4: I), and ( b ) cyclohexane/chloroform/ a c e t o n i t r i l e ( I :2:I ).

308

CHESTER E. ORZECH ET A L

High P r e s s u r e Liquid Chromatography Smith e t al (76,771 analyzed h y d r a l a z i n e i n a drug mixture c o n t a i n i n g h y d r a l a z i n e h y d r o c h l o r i d e , hydrochlorot h i a z i d e , and an i m p u r i t y d e r i v e d from t h e l a t t e r . The column w a s 1 m x 2.1 mm (ID) s t a i n l e s s s t e e l , packed w i t h a s t r o n g anion exchanger on 3 pin Zipax@. The mobile phase was pH 9.2 b o r a t e b u f f e r c o n t a i n i n g 0.005z sodium s u l f a t e (5%methanol), a t 1.7 m l p e r minute. Detection w a s by u l t r a v i o l e t a b s o r p t i o n a t 254 nm. Honigberg e t a1 ( 7 8 ) t e s t e d reversed-phase chromatography f o r s e p a r a t i o n of a number of d r u g s , i n c l u d i n g hydralazine. The columns contained e i t h e r octadecyltrichlorosilane o r d i p h e n y l d i c h l o r o s i l a n e , bonded Of t h e numerous t o 37 t o 50 p n p e l l i c u l a r s i l i c a packing. mobile p h a s e s t e s t e d , t h e b e s t f o r s e p a r a t i n g h y d r a l a z i n e , h y d r o c h l o r o t h i a z i d e , and r e s e r p i n e w a s a c e t o n i t r i l e / O . 1% ammonium a c e t a t e (20:80), pH 7.35. The columns were 1.22 m x 2.3 mm (ID) and t h e flow rate w a s 1.4 m l p e r minute. Detection w a s by u l t r a v i o l e t a b s o r p t i o n at 254 nm. 6.11 G a s Chromatography Jack e t al ( 7 9 ) determined h y d r a l a z i n e i n plasma. The sample w a s t r e a t e d w i t h n i t r o u s a c i d , which r e a c t s with h y d r a l a z i n e t o form t e t r a z o l o [ 1,5a]phthalazine (2). The d e r i v a t i v e was e x t r a c t e d with benzene and determined by g a s chromatography. I-Hydrazino-4-methylphthalzine w a s used as an i n t e r n a l s t a n d a r d . The same p r o c e d u r e , or m o d i f i c a t i o n s o f i t , was used by Zak e t a1 (801, T a l s e t h ( 4 2 , 8 2 , 8 2 , 8 3 ) , and Haegele e t a1 ( 4 6 ) f o r m e t a b o l i c s t u d i e s . Zak e t a1 (80) p o i n t o u t t h a t h y d r o l y s i s of c o n j u g a t e s of t h e drug m a y cause a n a l y t i c a l r e s u l t s on b i o l o g i c a l samples t o be v a r i a b l e , depending on t h e a c i d c o n c e n t r a t i o n d u r i n g d e r i v a t i z a t i o n , and t h a t s e l e c t i v e a n a l y s i s f o r unchanged h y d r a l a z i n e and a c i d - l a b i l e m e t a b o l i t e s can be c a r r i e d o u t by s u i t a b l e adjustment of t h e a c i d c o n c e n t r a t i o n . Smith e t a1 (84) determined h y d r a l a z i n e i n t a b l e t s . An aqueous e x t r a c t of t h e t a b l e t s w a s t r e a t e d with 2,4-pentanedione, forming 1- (3,5-dimethylpyrazole) phthalazine. The method w a s a p p l i e d t o s t a b i l i t y s t u d i e s of t a b l e t s s u b j e c t e d t o e l e v a t e d t e m p e r a t u r e s , where t a b l e t s could not b e analyzed by t h e USP method.

6.10

7.

ACKNOWLFEMENTS

The w r i t e r s wish t o thank D r . B. T. Kho f o r h i s review of t h e manuscript, Dr. G. S c h i l l i n g of Ayerst Research L a b o r a t o r i e s f o r h i s mass s p e c t r a l d a t a and

Table 2 G S C R M T G A H SYSTEMS USED FOR DERIVATIZED HYDRALAZINE DETERMINATIONS A HO AO R P Y Reference Number Column
Carrier G a s

Column Temp., 220 O C

OC

Detector Pulsed electron capture (Ni-63, 10 mCi ) Electron capture

(79)

15 m x 2 mm I D , g l a s s , . packed w i t h 3% OV-225 on 80-100 mesh m Chromosorb W

1.2 m x 2 mm I D , glass, packed w i t h 3% OV-225 on Gas-Chrom Q 1 m x 4 mm I D , g l a s s , packed with 3% OV-I7 on 80-100 mesh Chromosorb W 1.8 m x 4 mm I D , g l a s s , packed w i t h 7 % S E - 3 0 on 80-100 mesh G a s Chrom Q

N2' ml/min.

(80)

220 O C

(46)

He 7

40 ml/min.

Programmed, a t 4"/min., 170- 240 O C

Mass spectrometer

(84)

55

2' ml/min.

200 O C

Flame ionization

3 10

CHESTER E. ORZECH ET AL.

i n t e r p r e t a t i o n , t h e l i b r a r y s t a f f (Ms. J. Mcbnough and M s . G. Smith) f o r t h e l i t e r a t u r e s e a r c h , and Mrs. S. W i l l e t t e f o r t y p i n g t h e p r o f i l e .

8.

RFFERENCES F. Gross, J. Druey, and R. Meier, E x p e r e n t i a 6, 19 (1950). 2. J. Druey and B. H. R i n g i e r , Helv. Chim. Acta 195-210 (1951); C.A. 10248f. 3. J. Druey and J. Tripod, Med. Chem. (New York) 2, 223-62 (1967); C.A. 70, 77829e. 4. J. Koch-Weser, N. Engl. J. Med. 295, 320-3 (1976); C.A. 85, 103541rn. 5. L. J . B e l l a m y , "The I n f r a - r e d S p e c t r a of Complex Molecules", 3 r d . ed., John Wiley & Sons, New York, N.Y., 1975. 6. E. G. C. C l a r k e , e d i t o r , " I s o l a t i o n and I d e n t i f i c a t i o n of Drugs", The Pharmaceutical P r e s s , London, England, 1969. 7. M. Kuhnert-BrandstBtter, A. K o f l e r , R. Hoffmann, and H.-C. Rhi, S c i . Pharrn. 33, 205-30 (1965); C.A. 64, 7966g. 8. M. F . S h a r k e y , C. N. Andres, S. W. Snow, A. Major, T. K r a m , V. Warner, and T. G. Alexander, J. A s s . Offic. Anal. Chem. 2, 1124-54 (1968). 9. S. G. Solomonova, N. M. Turkevich, and N. V. Kurinnaya, Farm. Zh. (Kiev) - 42-4 (1973); 28, C.A. 78, 140445j. 10. I. S u n h i n e , e d i t o r , ffHandbook of A n a l y t i c a l Toxicology", The Chemical Rubber Co., Cleveland, Ohio, 1969. 11. D. V. Naik, B. R. Davis, K. M. Minnet, and S. G. Schulman, J. Pharm. S c i . - 8 4 - 6 (1976); C.A. 65, 126826b. 12. "The Merck Index", 9 t h ed., Merck & Co., Inc., Rahway, N.J., 1976. 13- G. S c h i l l i n g , Ayerst Research L a b o r a t o r i e s , P e r s o n a l Communication. 14. J. Chojnacki, L. Lebioda, and K. S t a d n i c k a , Rocz. Chem. 49, 1163-6 (1975); C.A. &, 106677e. G. C. L d r i , B o l l . chim. farm. 431-6 (1957); 15 C.A. 52, 4109g. 16. K. I. E v s t r a t o v a and A. I. Ivanova, Farmatsiya (Moscow) 1 7 ( 2 ) , 41-5 ( 1 9 6 8 ) ; C.A. 46128a. K. I. E v s G a t o v a , N. A. Goncharova, and V. Ya. I7 Solomko, Farmatsiya (Moscow) -( 4 ) , 33-6 ( 1 9 6 8 ) ; 17 C.A. 99338a. 1.

5,

3,

84,

96,

69,

69,

HYDRALAZINE HYDROCHLORIDE

31 I

18.

19

20.
21. 22. 23 24.

25.

26.
28.

n.

33.

34

3. 5

37.
38
39
9

40.
41.

B. P. Artamanov, T. Ya. Konenkova, and S. L. M a i o f i s , Med. Prom. SSSR -( 9 ) , 57-9 (1965); 19 C.A. 63, 17800f. " U n i t z S t a t e s Pharmacopeia XIX" , Mack P u b l i s h i n g CO., Easton, Pa., 1975. K. F u j i i and S. S a t o , Ann. Rept. G. Tanabe Co. L t d . -, 1-3 (1956); C.A. 51, 6650~. 1 U.S. P a t e n t 2,484,029; C.A. 44, 4046b. B r i t . P a t e n t 629,177; C.A. 4 q 4516i. X Japan. P a t e n t 5526 ( ' 5 4 ) ; C . 49, 1598221. S B i n i e c k i and B. Gutkowska, Axa Pol. Pharm. 3, . 3 - 3 0 ( 1 9 5 5 ) ; C.A. 50, 1206211. B r i t . P a t e n t 735,899; C.A. 50, 1080ii. B r i t . P a t e n t 719,183; C.A. 3, 15982i. E. O i s h i , Yakugaku Zasshi 959-71 ( 1969 1; C.A. 7 2 , 3 4 5 0 ~ . T. P . S y c h e v a , T. P. Kuz'micheva, A. T. Chernyaeva, T. Kh. Trupp, and M. N. Shchukina, Med. Prom. SSSR - ( 2 ) , 13-17 ( 1 9 6 0 ) ; C.A. 54, 22669g. 14 Fr. P a t e n t 2,193,824; C.A.-82, 73014k. Ger. P a t e n t 2,311,510; C.A.30, 1 0 8 5 6 0 ~ . Can. P a t e n t 992,082; C.A. 86743728m. H. M. P e r r y , J. Lab. Clin.Med. - 566-73 ( 1 9 5 3 ) ; 41, C.A. 47, 1060ii. A. R . S c h u l e r t , Arch. i n t e r n . pharmacodynamie B, 1-15 (1961); C.A. 55, 2 2 4 7 1 ~ . S. F a l l a b , Helv. C z m . Acta 45, 1957-65 ( 1 9 6 2 ) ; C.A. 5242~. S. B i n i e c k i , M. M o l l , K. Niewiadomski, a n d M. Rzewuski, Acta P o l . Pharm. 33, 425-7 ( 1 9 7 6 ) ; C.A. - 161223r. 86, J. Wagner, J. W. F a i g l e , P. Imhof, and G. L i e h r , Arzneim.-Forsch. -, 2388-95 (1977); C.A. 8 88, 13066111. M. M. Reidenberg, D. Drayer, A. L. DeMarco, and C. T. B e l l o , C l i n . Pharmacol. Ther. 970-7 (1973); C.A. 449. W. M. McIsaac and M. Kanda, J. Pharmacol. Exptl. Therap. 7-13 (1964); C.A. &, 11233bH. Zimmer, J: McManus, T. Novinson, E. V. Hess, and A. H. L i t w i n , Arzneim.-Forsch. 20, 1586-7 ( 1 9 7 0 ) ; C.A. 40761h. H. Zimmer, J. Kokosa, and D. A. G a r t e i z , Arzneim.Forsch. 23, 1028-9 ( 1 9 7 3 ) ; C.A. @, 22460d. A. L i t w i n , L. E. Adams, E. V. Hess, J. McManus, and H. Zimmer, A r t h r i t i s Rheum. 5, 217-23 ( 1 9 7 3 ) ; C.A. - 43862b. 80,

&,

2,

x,

9,
2,

2,

312

CHESTER E. ORZECH ET A L .

42.

43.

44. 45
46. 47

48.
49
50
5152.
9

T. T a l s e t h , Eur. J. Clin. Pharmacol. 2, 395-401 (1976); C.A. 100742n. H. Zimmer, R. Glaser, J. Kokosa, D. A. G a r t e i z , E. V. Hess, and A. L i t w i n , J. Med. Chem. 18, 188169~. 1031-3 (1975); C.A. A. J. McLean, K. D. Haegele, P. DuSouich, and J. L = McNay , Eur. J. Drug Metab. Pharmacokinet 1 1, 17-20 (1977); C.A. 9911011. C. D. Douglass and R. Hogan, Proc. SOC. Exptl. 100, Biol. Med. - 446-8 (1959); C.A. 53, 14319f. K. Haegele, H. B. S k r d l a n t , N. W. R x i e , D. Lalka, and J. L. McNay, J. Chromatogr. 517-34 (1976); C.A. 86, 25798e. K. D.Haegele, A. J. McLean, P. DuSouich, H. B. S k r d l a n t , B. Werckle, and J. L. McNay, Clin. Res. 25, 46OA (1977). S. B. Zak, T. G. G i l l e r a n , J. K a r l i n e r , G. Lukas, J. Med. Chem. 17, 381-2 ( 1 9 7 4 ) ; C.A. 8 1 , 45171~. P. Cooper, P h a x . J. 177, 495-6 ( 1 9 5 6 7 C.A. 2, 6945g V. G. Belikov, G. I. Luk'yanchikova, V. N. Bern12, s h t e i n , and I. Ya. Kul, Aptechn. Delo - 60-2 ( 1 9 6 3 ) ; C.A. 9357b. Z. Modras, Farm. Pol. 3, 347-8 (1969); C.A. 71,

86,

5,

88,

. z(

126,

61,

84594~.
A. I. Roshchenko, E. G. Rezchik, and T. V. Gorenko , Farmat s i y a (Moscow) &(5 ) , 76-8 ( 1975 ) ; C.A. 84, 35406~. S. J . S t o h s and G. A. S c r a t c h l e y , J. Chromatogr. - 329-33 ( 1 9 7 5 ) ; C.A. 114, 65305g. G. I. Luk'yanchikova, E. N. Vergeichik, A. N. Baranova, A. S. Davydenko, E. N. Pelekhova, 0. I. Turubarova, G. V. Alfimova, and S. G. T i r a s p o l l skaya, Aktual. Vop. Farm. 71-5; C.A. 76, 90123k. B. Wesley-Hadzija and F. Abaffy, Croat. Chem. Acta 30, 15-19 (1958); C.A. 54, 2661i. R. Ruggieri, Farmaco ( P G i a ) Ed. p r a t . 2, 571-6 (1956); C.A. 53, 14421a. U.S.S.R. P a t e n t 148,956; C.A. 58, 1311h. G. I. Luk'yanchikova, Med. P r o z SSSR -( 9 ) , 46-8 16 (1962); C.A. 5454h. S . B. Z a k , M. F. B a r t l e t t , W. E. Wagner, T. G. G i l l e r a n , and G. Lukas, J. Pharm. S c i . 225-9 (1974); C.A. 140963t. W. Grabowicz and J. B r u l i n s k a , Farm. Pol. 29, 805-7 (1973); C.A. 112718~.

53

54

84,

m,

55
56

57 58.
59
60.

58,

80,

a,

80,

HY DRALAZINE HYDROCHLORIDE

313

61. 62.

6364.

65

66.
67

68.
69
70.

7172-

73.

74.
75

76

H. E l l e r t a n d Z. Modras, Farm. Pol. 511-15 ( 1 9 7 1 ) ; C.A. 6766v. T. Urbanyi and A. O'Connell, Anal. Cheni. 44, 565-70 ( 1 9 7 2 ) ; C.A. - 90100d. 76, T. Urbanyi and A. W. O'Connell, Adv. Autorn. Anal., Technicon I n t . Congr. 2, 15-21 (1972); C.A. 82, 116 154a. A. K. Ruzhentseva, I. S. Tubina, and L. N. Bragina, Med. Prom. SSSR -( 1 1 ) , 34-6 ( 1 9 6 0 ) ; C.A. 55, 14 9789g. Ya. M. Perel'man and K. I. E v s t r a t o v a , Aptechn. Delo 1 2 ( 1 ) , 45-9 (1963); C.A. 6 1 , 9360h. N. S.Goryacheva and L. N. P r f l o d k i n a , Farmatsiya 61575n. (Moscow) 17(3), 69-72 (1968); C.A. R. Solirnan and S. A. B e l a l , Pharmazie 3, 204 ( 1 9 7 4 ) ; C.A. 8 1 , 111504j. R. Solirnan a n T S . A. B e l a l , J. Drug Res. 7-11 (1974); C.A. 84918e. S. B e l a l and R. Soliman, Pharmazie 59-60 (1975); C.A. &, 1 5 7 1 2 ~ . H. McKennis and A. S. Yard, U S Dept. Corn., .. O f f i c e Tech. Serv., PB Rept. 143,914 ( 1 9 5 7 ) ; C.A. 55, 17375i. A. Viala, Trav. soc. pharm. M o n t p e l l i e r - 96-100 18, (1958); C.A. 53, 8534g. T. Giovanoli-Jakubezak, J. Chodkowski, and D. Gralewska, Rocz. Chem. - 1315-28 (1971); C.A. 45, 76, 30113a. Z. Modras, Chern. Anal. (Warsaw) 1349-53 (1973); C.A. 78, 128457g. H. ZiKer, R. G l a s e r , J. Kokosa, D. A. G a r t e i z , E. V. Hess, and A. L i t w i n , J. Med. Chem. 18, 1031-33 ( 1 9 7 5 ) ; C.A. 188169~. J. M. L e s s e r , Z. H. Israili, D. C. Davis, and P. G. Dayton, Drug Metab. Dispos. 2, 351-60 (1974); C.A. 8 2 , 64s. J. B . S m i t h , J. A. M o l l i c a , H. K. Govan, and I. M. Nunes, Amer. Lab. -( 5 ) , 13-17, 19 (1972); C.A. 4

76,

a,

69,

83,

6,

2,

17,

83,

77.

J. B. Smith, J. A.

78
79

M o l l i c a , H. K. Govan, and I. M. Nunes, I n t . Lab. 1972 (July-Aug.), 15-16, 19-20, 22-23; C.A. 83, 4 m g . I. L. Honigberg, J. T. S t e w a r t , A. P. Smith, and D. W. H e s t e r , J. Pharm. S c i . - 1201-4 ( 1 9 7 5 ) ; 64, C.A. Q, 103357s. D. B. J a c k , S. Brechb'ihler, P. H. Degen, P. Zbinden, and W. R i e s s , J. Chrornatogr. 115,87-92 ( 1 9 7 5 ) ; C.A. 38500k.

39 28%.

77,

84,

3 14

CHESTER E. ORZECH ET AL.

80.
81.
82.

83. 84.

85.

S. B. Zak, G. L u k a s , and T G. G i l l e r a n , Drug . Metab. Dispos. 2, 116-21 (1977); C.A. 95267a. T. T a l s e t h , Eur. J. Clin. P h a m a c o l . 2, 183-7 (1976); C.A. 1864599. T. T a l s e t h , Clin. Pharmacol. Ther. 3, 715-23 (1977); Index Medicus (Sept. 1, 362-3. T. T a l s e t h , Clin. Pharmacokinet. 2, 317-19 (1977); Index Medicus (Feb.), 363. K. M. Smith, R. N. Johnson, and B. T. Kho, J. Chromatogr. 137,431-7 (1977); C.A. 73417~. P. A. Reece, P. E. S t a n l e y , and R. Z a c e s t , J. Pharm. S c i . 1150-3 (1978).

87,

5,

17 97

1978

87,

67,

L i t e r a t u r e surveyed through 1977.

Analytical Profiles of Drug Substances, 8

CALCIUM LEUCOVORIN
Leslie 0.Pont, Andrew P . K . Cheung, and Peter Lirn
I.
Description 1 . 1 Name, Structure, Empirical Formula, Molecular Weight 1.2 Appearance, Odor, Color 1.3 Isomeric Forms Physical Properties 2. I Infrared Spectrum 2.2 Proton Magnetic Resonance Spectrum 2.3 Carbon- 13 Magnetic Resonance Spectrum 2.4 Ultraviolet Spectrum 2.5 Mass Spectrum 2.6 X-Ray Crystallographic Data 2.7 Optical Rotation 2.8 Circular Dichroism 2.9 Dissociation Constants 2.10 Solubility Synthesis Stability 4.1 Bulk 4 . 2 Solution Metabolism Methods of Analysis 6.1 Elemental Analysis 6 . 2 Equivalent Weight Determination 6.3 Biological Assay 6.3.I Microbiological Assay 6.3.2 Enzyme Assay 6.4 Polarographic Assay 6.5 Spectrophotometric Analysis 6.5. I Fluorimetric Analysis 6 . 5 . 2 Ult ravioletlvisible Analysis 6 . 6 Chromatography 6.6. I Paper Chromatography 6.6.2 Thin-Layer Chromatography 6.6.3 Column Chromatography 6.6.4 High Pressure Liquid Chromatography 6.6.5 Affinity Chromatography 6 . 7 Radioassay Acknowledgments References

2.

3. 4.

5.
6.

7. 8.

3 15

Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-260808-9

316

LESLIE 0 . PONT ET A L .

1.

Description

1.1 Name, Structure, Empirical Formula, and Molecular Weight

Citrivorum factor (CF), folinic acid, and leucovorin are all N-[4[[(2-amino-5-formyl-l,4,5,6,7,8-hexahydro4-oxo-6-pteridinyl)methyl]-amino]benzoyl]glutamic acid, I, the Leuconostoc citrovorum 8081* growth factor first reported by Sauberlich and Baumann. Leucovorin refers to the chemically synthesized material that contains both dL and 1L diastereomers; "citrovorum factor" and "folinic acid" generally apply to the biologically synthesized 1L isomer. Much of the data reported here was obtained on calcium leucovorin, the stable, isolatable, biologically compatible salt.
__ Name

Empirical Formula C20Hz3N707 CZ0H2,N707Ca

Molecular Weight

Leucovorin Calcium leucovorin

473.449
511.513

Calcium leucovorin is also identified by the National Cancer Institute number: NSC 3590.

I
Citrovorum f a c t o r CF, f o l i n i c a c i d , l e u c o v o r i n , 5fHbF

CALCIUM LEUCOVORIN

317

R,
C-NH-CH CH 2 I CH z I COOH I1 IIa IIb IIc

R 1 = OH R1
=

R2

Folic acid, FA Methotrexate, amethopterin, MTX

10 formylfolic acid, lOfFA

R 1 = NH2 R 2 = CH3
OH

R 2 = CHO

COOH I11 IIIa IIIb IIIc R1

R1

Rz = H
Rz = H

Tetrahydrofolic acid, H4F 5-methyltetrahydrofolic acid, 5mH F 10-formyltetrahydrofolic acid, 10fH4F

= CH3

R1 = H

R2 = CHO

+
c1-

IV
N 5 , N 1O-methenyltetrahydrofolic acid , anhydroleucovorin, 5,lO-methenylH4F

3 18

LESLIE 0. PONT ET AL.

CH z
CH z I COOH
I

v
N',N'o-methylenetetrahydrofolic

acid , 5 ,lo-methylene H4F

1.2

Appearance, Odor, C o l o r

Calcium l e u c o v o r i n o c c u r s a s a n amorphous, odorless powder t h a t i s o f f - w h i t e t o l i g h t b e i g e i n c o l o r . The s a l t is n o t i s o l a t e d i n a n anhydrous form, b u t g e n e r a l l y c o n t a i n s 10-15% w a t e r (3-5 m o l e c u l e s o f h y d r a t i o n ) .

1.3

I s o m e r i c Forms

Two a s y m m e t r i c c a r b o n a t o m s , t h e a c a r b o n i n t h e g l u t a m i c a c i d p o r t i o n o f t h e m o l e c u l e and t h e C 6 c a r b o n i n the tetrahydropteridine r i n g , allow four p o s s i b l e isomers. S i n c e s y n t h e t i c p r o c e d u r e s would u n d o u b t e d l y s t a r t w i t h L-glutamic a c i d , t h e i s o m e r i c p o s s i b i l i t i e s a r e r e d u c e d t o t h e dL a n d 1 L d i a s t e r e o m e r s . Of t h e s e , t h e b i o l o g i c a l l y more a c t i v e form i s t h e 1 L ; s e p a r a t i o n o f t h e d i a s t e r e o m e r s i s e f f e c t e d by s o l u b i l i t y d i f f e r e n c e s o f t h e c a l c i u m s a l t s . '
x

R e c l a s s i f i e d as P e d i c o c c u s c e r e v i s i a e .

CALCIUM LEUCOVORIN

3 19

Although a p u r e dL sample had n o t been o b t a i n e d , a sample p o s s e s s i n g a h i g h p o s i t i v e r o t a t i o n and lower b i o l o g i c a l a c t i v i t y had been i s o l a t e d . '

2.

Physical Properties 2.1 I n f r a r e d Spectrum

F i g u r e 1 i s t h e i n f r a r e d spectrum t a k e n from a m i n e r a l o i l s u s p e n s i o n of a r e p r e s e n t a t i v e sample o f calcium l e u c o v o r i n . A common f e a t u r e of f o l a t e d e r i v a t i v e i r s p e c t r a o b t a i n e d i n t h i s l a b o r a t o r y i s t h e a b s e n c e of s h a r p a b s o r p t i o n b a n d s , which i s u s u a l l y a t t r i b u t e d t o l a c k of c r y s t a l l i n i t y . Below a r e l i s t e d t h e major a b s o r p t i o n assignments:

Ir a b s o r p t i o n ( p )
'L

Assignment
H Z O , N-H(2)

3.0 6.0-6.7

H'O, amide I , a r y l s y s t e m s , CO,-,

amide I1

'L

7.1

c0'-

1.1.9

(tentative)

2.2

P r o t o n Magnetic Resonance Spectrum Due t o t h e i o n i c n a t u r e of c a l c i u m l e u c o v o r i n , a n

' nmr s p e c t r u m c o u l d n o t b e o b t a i n e d i n a n a p r o t i c s o l v e n t ; H
t h e r e f o r e , t h e s p e c t r u m was o b t a i n e d from a d e u t e r a t e d aqueous s o l u t i o n . The f o l l o w i n g a s s i g n m e n t s h a v e b e e n made r e l a t i v e t o TMS = 0 . 0 0 ppm from a 100-MHz V a r i a n XL100 s p e c trum ( F i g u r e 2 ) : Assignment Chemical S h i f t (ppm) 1.70-2.60 2.95-3.80 4.00-4.60 ( o b s c u r e d by HOD) Multiplicity
m m
q

J (Hz)

H 8, Y
H 7, 9 H a

z
0
LT

8 3
w
I

>

2
u
U

I
LT I -

13

n
0

n
w LT

2
U

LT 13

c7

r"

0 0 0 0 0
i-9

t"??

g
0 0 0 0
7

3 3 ~ w a osaw tl
320

8
7

vl i

32 I

322

LESLIE 0. PONT ET A L .

Assignment H 3 ' , 5' H 2'c 6'

0
II

Chemical S h i f t (ppm) 6.64 7.60

Multiplicity d d

J (Hz)

H-C-,

8.59 7.89
+

unknown H, 6

4.8

m (broad)

The above a s s i g n m e n t s a r e i n g e n e r a l a g r e e m e n t w i t h t h o s e of Pastore3 for folates. The C, p r o t o n i s o b s c u r e d by t h e l a r g e HOD p e a k , and i t s l o c a t i o n was c o n f i r m e d by s h i f t i n g t h e HOD a b s o r b a n c e a t 8 0 " C. The p e a k a t + 7.9 ppm d u e t o a n unknown s p e c i e s h a s b e e n p r e s e n t i n a l l c a l c i u m l e u c o v o r i n samples. The i d e n t i t y o f t h e s p e c i e s r e s p o n s i b l e f o r t h i s s i n g l e t r e m a i n s unknown, b u t a t t h e p r e s e n t t i m e t h e s e a u t h o r s h y p o t h e s i z e t h a t t h e dL d i a s t e r e o m e r ' s f o r m y l g r o u p c o n t r i b u t e s t h i s peak. Since chemically prepared calcium l e u c o v o r i n i s a s s a y e d a t t h i s l a b o r a t o r y , a n 'H nmr s p e c trum h a s n o t been o b t a i n e d on a b i o l o g i c a l l y o r enzymatic a l l y p r e p a r e d m a t e r i a l . E i t h e r s h o u l d b e f o l i n i c a c i d and t h e r e f o r e have t h e 1 L c o n f i g u r a t i o n . Investigations t o r e s o l v e t h i s problem a r e c o n t i n u i n g . 2.3 Carbon-13 Magnetic Resonance Spectrum

The 1 3 C nmr s p e c t r u m r e p r o d u c e d i n F i g u r e 3 w a s o b t a i n e d from a D,O/NaOD s o l u t i o n of c a l c i u m l e u c o v o r i n on a V a r i a n X L l O O S p e c t r o m e t e r . The a s s i g n m e n t s on t h e T S M s c a l e a n d r e f e r e n c e d t o d i o x a n e are l i s t e d below: Assignment Chemical S h i f t (ppm) 93.7 102.0
0.0

c- 2 c- 4
C- 6

c- 7
0

23.3 98.2

- C-H

I 1

c- 9

- 25.0

D IOXAN E

c
PPm
FIGURE 3 13C NMR OF CALCIUM LEUCOVORIN
LSJ-57

324

LESLIE 0 . PONT ET A L .

A s s i gnmen t
C-4a o r C-8a

Chemical S h i f t (ppm) 25.2 86.5 54.7

C-8a o r C-4a

c-1'
C-2
I ,

C-6' c-5'

62.4 45.7 84.8

C-3',

c-4
c-7
C-Ci

'
-

102.8 10.8 37.9 32.3 112.6 115.6

C- B C-Y

a- cooy-coo-

The above a s s i g n m e n t s a r e i n r e a s o n a b l e agreement w i t h t h o s e previously reported f o r p t e r i d i n e s . 2.4 U l t r a v i o l e t Spectrum

F i g u r e 4 r e p r e s e n t s t h e uv s p e c t r u m of c a l c i u m l e u c o v o r i n i n pH 7.0 p h o s p h a t e b u f f e r (0.10 M ) , A ,, = 286 nm, Amin = 243 nm, and i n 0.10N NaOH (pH 1 3 ) , A ,, = 282 nm, A m i n = 242 nm. These s p e c t r a l f e a t u r e s a r e s i m i l a r t o t h o s e r e p o r t e d i n t h e l i t e r a t u r e . 5-12 The most commonly r e p o r t e d a b s o r p t i v i t i e s f o r l e u c o v o r i n o r f o l i n i c a c i d were o b t a i n e d from s o l u t i o n s prepa:ed i n 0.1N NaOH; t h e s e v a l u e s v a r y from 2.7 x lo4 M-' cm-' t o 3 . 2 6 x 10' M-' crn-l'l. Robinson" r e p o r t s E~~~ a p p r o x i m a t e l y 5% lower ( a t pH 8 . 4 ) t h a n t h o s e i n b a s i c s o l u t i o n . The s i n g l e a b s o r p t i o n i s due t o c o n t r i b u t i o n s from p-amino-benzoylglutamate and t h e u n s a t u r a t e d p o r t i o n of t h e p t e r i d i n e r i n g . Loss of a b s o r b a n c e a t s h o r t e r and l o n g e r w a v e l e n g t h s i n b a s i c s o l u t i o n (256 nm and 368 nm) i s c h a r a c t e r i s t i c o f r e d u c e d f o l a t e s . Spectral d a t a o b t a i n e d from s o l u t i o n s p r e p a r e d i n a c i d s do n o t r e f l e c t l e u c o v o r i n a b s o r b a n c e s b e c a u s e t h e compound i s n o t s t a b l e . Leucovorin d e h y d r a t e s under a c i d i c c o n d i t i o n s t o produce anhydroleucovorin, N5 ,N"-methenyltetrahydrofolic acid, IV. T h i s compound h a s been i s o l a t e d i n more t h a n o n e form, d e p e n d i n g on pH. l 3
Q

CALCIUM LEUCOVORIN

325

1.0

0.9
0.8

0.7
w

z a

0.6 0.5
0.4

0.3

0.2
0.1

0 200

250

nm

300

350
LSJ-58

FIGURE 4

ULTRAVIOLET SPECTRUM OF CALCIUM LEUCOVORIN 0.1N NaOH pH 7 Phosphate buffer -~

326

LESLIE 0. PONT ET A L .

2.5

Mass Spectrum-

An e l e c t r o n impact mass s p e c t r u m of c a l c i u m l e u c o v o r i n h a s n o t b e e n o b t a i n e d b e c a u s e t h e compound i s n o t s u f ficiently volatile. I t would b e d i f f i c u l t t o i s o l a t e t h e f r e e a c i d w i t h o u t f i r s t d e h y d r a t i n g t h e compound. Due t o i t s i o n i c n a t u r e , calcium leucovorin w i l l n o t d i s s o l v e i n common s i l y l a t i n g r e a g e n t s . F i e l d d e s o r p t i o n , a n o t h e r mass s p e c t r a l t e c h n i q u e , g e n e r a l l y l e n d s i t s e l f more t o compounds l i k e leucovorin. I n d e e d , t h i s t e c h n i q u e h a s been a p p l i e d s u c c e s s f u l l y t o m e t h o t r e x a t e and o t h e r f o l i c a c i d a n a l o g s . l 4

2.6

X-ray C r y s t a l l o g r a p h i c Data

C r y s t a l l o g r a p h i c d a t a have n o t been p u b l i s h e d f o r c a l c i u m l e u c o v o r i n , b u t l i t e r a t u r e v a l u e s do e x i s t f o r t h e D i f f r a c t i o n p a t t e r n s could n o t be obtained barium s a l t . ' on a l l s a m p l e s a l t h o u g h t h e y had b e e n r e c r y s t a l l i z e d s e v eral t i m e s . I n t e r p l a n a r s p a c i n g s a r e l i s t e d , b u t t h e s e numb e r s a p p e a r t o change w i t h t h e amount o f m o i s t u r e p r e s e n t i n t h e sample. 2.7 Optical Rotation
21
0

a]
5 8 9

=
21
0

14.3 k 0.4"

(c1, N/10
( 1, N/10 c
(C 1, H20)

NaOH)

a]

546 21
0

1 7 . 9 k 0.4'

NaOH)

0.3
a]

=
5 8 9

+
+

14.9 ? 0.4"

21

=
5 4 6

18.8 t 0.4"

(c1, H z O )

The above v a l u e s were o b t a i n e d on a n a v e r a g e l o t o f c a l c i u m l e u c o v o r i n examined in t h i s l a b o r a t o r y . C o r r e c t i o n s were made f o r water c o n t e n t . D i f f e r e n c e s i n r o t a t i o n v a l u e s may p a r t l y r e f l e c t s o l u b i l i t y i n t h e two s o l v e n t s ; t h e compound w a s more d i f f i c u l t t o d i s s o l v e i n N / 1 0 NaOH. The H 2 0 r o t a tion is c l o s e t o t h e l i t e r a t u r e value: (rID = 1 4 . 2 6 " 3.42 H,O).'

(c,

CALCIUM LEUCOVORIN

327

2.8

C i r c u l a r Dichroism

The f o l l o w i n g d a t a were o b t a i n e d on an a v e r a g e sample of c a l c i u m l e u c o v o r i n : Solvent 0.1N NaOH

H20

&

(nm)

[ e l (deg
3.89

M-lcm-')

282 287

103

3.47 x lo3

The molar e l l i p t i c i t i e s a r e c a l c u l a t e d f o r l e u c o v o r i n f r e e a c i d and have been c o r r e c t e d f o r water c o n t e n t . These v a l u e s may b e q u i t e d i f f e r e n t from t h o s e of f o l i n i c a c i d , Although a s i n g l e v a l u e i s r e p o r t e d f o r each s o l v e n t , b o t h s p e c t r a c o n t a i n more t h a n one a b s o r p t i o n band. Whether t h e e x t r a n e o u s a b s o r p t i o n s a r e due t o i m p u r i t i e s o r a r e a c t u a l l y due t o l e u c o v o r i n ' s a b s o r p t i o n b e h a v i o r i s n o t known at this time.

2.9

Dissociation Constants

Three pKa v a l u e s have been r e p o r t e d f o r l e u c o v o r i n ( f r e e a c i d ) ; t h e y a r e 3 . 1 , 4 . 8 , and 1 0 . 4 , as d e t e r mined by e l e c t r o m e t r i c t i t r a t i o n . 6 The f i r s t two v a l u e s a r e a t t r i b u t e d t o t h e g l u t a m y l c a r b o x y l s , and 10.4 is a s s i g n e d t o t h e h y d r o x y l group a t t h e 4 p o s i t i o n , ' by comparison t o model compounds.

2.10 S o l u b i l i t y
The i o n i c n a t u r e o f c a l c i u m l e u c o v o r i n s e v e r e l y r e s t r i c t s i t s s o l u b i l i t y i n common o r g a n i c s o l v e n t s ( s o l I n water, t h e s o l u b i l i t y is u b i l i t y i n D S << 1 mg/ml). MO l a r g e (% 100 mg/ml), b u t i n 0.1N NaOH i t d r o p s s i g n i f i c a n t l y ( < 20 mg/ml). Acid s o l u b i l i t y i s d i f f i c u l t t o i n t e r p r e t s i n c e t h e s o l u t e may no l o n g e r b e l e u c o v o r i n .

3.

Synthesis

With s l i g h t m o d i f i c a t i o n s i n p r o c e d u r e , one b a s i c synt h e s i s of l e u c o v o r i n and t h e s u b s e q u e n t i s o l a t i o n of i t s s a l t s have remained unchanged f o r n e a r l y t h i r t y y e a r s . The method i n v o l v e s h y d r o g e n a t i o n of f o l i c a c i d ( p t e r o y l g l u tamic a c i d ) i n t h e p r e s e n c e of a p l a t i n u m o r p a l l a d i u m c a t a l y s t , as f i r s t d e s c r i b e d by O ' D e l l e t a l . 1 5 T h i s

328

LESLIE 0. PONT ET A L .

r e d u c t i o n s t e p i s c a r r i e d on c o n c u r r e n t l y . o r a f t e r 1 6 - 1 9 i n i t i a l f o r m y l a t i o n of f o l i c a c i d i n f o r m i c a c i d . The pH of t h e r e a c t i o n mixture i s a d j u s t e d w i t h base i n t h e presence of a n a n t i o x i d a n t and h e a t e d ( i n c u b a t e d 50-150" C P u r i f i c a t i o n by column w i t h o r w i t h o u t reduced p r e s s u r e ) . chromatography i s f o l l o w e d by p r e c i p i t a t i o n o f t h e d e s i r e d s a l t by a d d i t i o n of t h e a p p r o p r i a t e c a t i o n and o r g a n i c s o l vent. The c a l c i u m s a l t i s p r e c i p i t a t e d from a n aqueous s o l u t i o n by a d d i t i o n o f C a C l z f o l l o w e d by e t h a n o l . * '

IV

CALCIUM LEUCOVORIN

329

NaHCO, or NaOH or both (antioxidant)

IIIC

c m

CaC1. or Ca(OH), Ethanol

Calcium lcucovorio

Early syntheses' ' ' employed ascorbic acid as the antioxidant to protect the newly formed tetrahydro ring. But Roth et ala9 found that ascorbic acid had no effect on leucovorin yield (40-50%) as long as incubation was performed under anaerobic conditions. More recent syntheses have used Z-mer~aptoethanol~~ a stream of nitrogenz2 to or to protect the tetrahydro system. Isolation of the active component is carried out chromatographically. Roth et al. used a magnesol (magnesium silicate) column to absorb colored impurities, followed by a Darco G 6 0 activated carbon column to eliminate sodium formate and inorganics. Elution with an alcohol/ammonia solvent was followed by rechromatographing in magnesol.

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LESLIE 0. PONT ET AL.

Flynn et a1.6 used a potato starch column, elution with

n-butanol/water/ethanolfglacial acetic acid, 100/50/40/ 0 . 4 , followed by a Florisil column, treated with pH 5.5

acetate buffer, CaC12 and ascorbic acid and elution with distilled water. Subsequent recrystallizations were made from water. Other authors prefer ion-exchange cellulose chromatography. Zakrzewski and Sanson' use DEAE cellulose in the O H form, eluting with a 2-mercaptoethanol/ ammonia gradient. Beavon and Blair ' recommend 1-mm cellulose plates developed in n-propanol/NH40H/Hz0, 200/1/99. The chemical synthetic methods of obtaining leucovorin or its salts are discussed above, but of course the originally identified material came from biological synthesis of citrovorum factor in animal liver.' Early isolation techniques included electrolysis of liver concentrates in the presence of acetic acid.23 Folinic acid synthesis has been reported in a variety of biological systems, including rat liver," avian liver homogenates,25"6 microorganisms, (i. e. , bacteria' " '' and virus3') and plants. 3 1 Factors affecting synthesis (e.g. , temperature, pH, antioxidants) are discussed in several of these papers. In living systems, folinic acid can be synthesized ultimately from folic acid by reduction to tetrahydrofolic acid followed by addition of a 1-carbon fragment to the molecule (N5,N1 O-methylenetetrahydrofolate, V) After a 2-step oxidation, the formyl group resides either at the N5 or NIO position or as an equilibrium mixture. The essential reactions are summarized below: 3 2

H Folic acid + dihydrofolate

H dihydrofolate' reductase ND' AP

t et rahydrofolate

N5 ,N1O-methenyltetrahydrofolateN 5-formyltetrahydrofolate

N 1 O - formyltet rahydrofo late

lt

N 5 , N -methylenetetrahydrofolate + glycine NADPH "active formaldehyde''

serine

NADH NAD+ N5-methyltetrahydrofolate

lr

CALCIUM LEUCOVORIN

33 I

R e c e n t l y , t h e enzymatic f o r m a t i o n of r o l i n i c a c i d h a s been u t i l i z e d t o s y n t h e s i z e r a d i o a c t i v e l y l a b e l e d prod u c t s . 3 4 The p r e p a r a t i o n of 5-formyl t e t r a h y d r o f o l a t o , 9 , 3 ' ,5'-3H and 5 - f 0 m y l - ~ ~ C - t e t r a h y d r o f o l a t e s t a r t s w i t h t r i t i a t e d f o l i c a c i d , which is reduced t o d i h y d r o f o l a t e , i n c u b a t e d i n t h e p r e s e n c e of formaldehyde, d i h y d r o f o l a t e r e d u c t a s e , and NADPH, and f i n a l l y i n c u b a t e d w i t h 5,lOmethylenetetrahydrofolate d i h y d r o g e n a s e . The p r o d u c t , N 5 ,N1'-methenyltetrahydrofolate (with ascorbic a c i d ) w a s a d j u s t e d t o n e u t r a l pH, a u t o c l a v e d , and s t o r e d a t -20" C p r i o r t o column p u r i f i c a t i o n on DEAE and G-15 Sephadex. These l a b e l e d p r o d u c t s are t h e b i o l o g i c a l l y a c t i v e d i a s t e r e o m e r s , and t h e y a r e used t o s t u d y t h e metabolism of f o l i n i c a c i d i n c e l l s , t i s s u e s , and a n i m a l s .

4.

Stability

4.1

Bulk

Calcium l e u c o v o r i n i s s t a b l e i n b u l k form a f t e r f o u r weeks' s t o r a g e a t 6 0 " C. The s t a b i l i t y w a s m o n i t o r e d by HPLC i n t h i s l a b o r a t o r y , u s i n g t h e c o n d i t i o n s d i s cussed i n S e c t i o n 6.6.4.

4.2

Solution

Citrovorum f a c t o r h a s been r e p o r t e d t o b e s t a b l e under s e v e r e a l k a l i n e c o n d i t i o n s by B r o q u i s t e t a1.35 and e a r l i e r by Lyman and P r e s c o t t . 3 6 Steam-heating f o r 30 m i n u t e s i n 0.2" NaOH d i d n o t decompose t h e compound. A l k a l i n e s t a b i l i t y i s n o t s u r p r i s i n g when one c o n s i d e r s t h a t many o f t h e s y n t h e t i c p r o c e d u r e s r e q u i r e a u t o c l a v i n g a t h i g h pH as a f i n a l s t e p i n c o n v e r t i n g anhydroleucovorin. I n v e s t i g a t i o n s conducted i n t h i s l a b o r a t o r y i n 0.1N N a O H (pH % 1 3 ) and i n d e i o n i z e d w a t e r (pH % 6 show l e u c o v o r i n t o b e s t a b l e a t room t e m p e r a t u r e and under l a b o r a t o r y i l l u m i n a t i o n o r a t l e a s t 24 h o u r s ( c o n c e n t r a t i o n 1 mg/ml). S t a b i l i t y w a s m o n i t o r e d by h i g h - p r e s s u r e l i q u i d chromatography u s i n g c o n d i t i o n s found i n S e c t i o n 6.6.4. As s t a t e d previously, leucovorin is not s t a b l e under a c i d i c c o n d i t i o n s . l 3 Depending on t h e pH, d i f f e r e n t forms o f a n h y d r o l e u c o v o r i n have been i s o l a t e d : pH 5 1 . 3 i s o l e u c o v o r i n c h l o r i d e ; pH = 2 a n h y d r o l e u c o v o r i n A; p H = 4 ( h o t ) a n h y d r o l e u c o v o r i n B. A l l t h r e e forms c o n v e r t back t o l e u c o v o r i n when d i s s o l v e d i n NaOH under a n a e r o b i c c o n d i t i o n s

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LESLIE 0 . PONT ET AL.

5.

Metabolism

Citrovorum f a c t o r f u n c t i o n s ( p r i m a r i l y ) as a 1-carbon donor i n t h e s y n t h e s i s of s e r i n e from g l y c i n e . 3 7 - 4 0 The N S formyl group i s i n c o r p o r a t e d from t h e 5 , l O methylene comSilverman e t a 1 . 4 1 have shown t h a t t h e c i t r o pound. 3 3 y vorum f a c t o r , when i n t h e p r e s e n c e of a l a r g e e x c e s s of g l u t a m i c a c i d and a hog l i v e r enzyme, l o s e s i t s formyl group t o form t h e N-formylglutamic a c i d and t e t r a h y d r o f o l i c a c i d . These a u t h o r s d i s m i s s t h e p o s s i b l e f o r m a t i o n of 10fH4F o r 5,lO-methenyl H4F i n t h e i r r e a c t i o n s . Howe v e r , Peters and Greenberg4 r e p o r t e d s e p a r a t i n g a n enzyme from s h e e p l i v e r , c i t r o v o r u m f a c t o r c y c l o h y d r a s e , which c o n v e r t e d 5fH4F t o a compound much l i k e , b u t n o t i d e n t i c a l t o , 5,lO-methenyl H4F. Anhydroleucovorin, t h e 5,lOmethenyl H4F compound, e x i s t s i n e q u i l i b r i u m w i t h t h e 5 and 1 0 formyl compounds ( s e e b i o s y n t h e t i c scheme). The l a t t e r , 10fH4F, h a s been r e p o r t e d t o d o n a t e i t s formyl group t o m e t h i o n i n e , which i s a s s o c i a t e d w i t h a n e s t e r i f i e d s p e c i e s A t r a n s f e r RNA, which i n t u r n c o n t i n u e s i n t h e s y n t h e s i s of p r o t e i n s . 4 3 I n t h e p r e s e n c e of appropr i a t e enzymes, f o r m y l t e t r a h y d r o f o l a t e s d o n a t e formyl groups f o r p u r i n e s y n t h e s i s 4 4 :

CALCIUM LEUCOVORIN

333

E q u a t i o n 1 i s c a t a l y z e d b y g l y c i n a m i d e r i b o t i d e (GAR) t r a n s f o r m y l a s e and E q u a t i o n 2 i s c a t a l y z e d b y a m i n o i m i d a z o l e carboxamide r i b o t i d e (AICAR) t r a n s f o r m y l a s e .

I n humans and r a t s , e a r l y i n v e s t i g a t i o n s showed t h a t l a r g e doses of f o l i c a c i d r e s u l t e d i n increased e x c r e t i o n of a s u b s t a n c e t h a t s t i m u l a t e d t h e growth o f P. c e r e v i s i a e and t h a t w a s presumed t o b e c i t r o v o r u m This r e s p o n s e i s now a l s o a s s o c i a t e d w i t h o t h e r r e d u c e d f o l a t e s .
R e c e n t l y a g r e a t d e a l of e f f o r t h a s b e e n s p e n t i n s t u d y i n g t h e metabolism of leucovorin i n vivo. This emphasis was prompted by t h e c h e m i c a l s t a b i l i t y of t h i s f o l a t e and by t h e o b s e r v a t i o n o f a r e d u c t i o n i n t o x i c i t y o f m e t h o t r e x a t e when i t w a s g i v e n i n c o n j u n c t i o n w i t h l e u c o v o r i n . Foli n i c a c i d i s found i n human l i v e r , b u t i t i s n o t t h e m a j o r c i r c u l a t i n g f o l a t e , which i s 5-methyltetrahydrofolate. Most of t h e f o l a t e s t h a t o c c u r n a t u r a l l y i n man a r e polyglutamates, a l t h o u g h t h e y a r e t r a n s p o r t e d as monoglutamates. S e v e r a l g r o u p s i n v e s t i g a t i n g human m e t a b o l i s m o f f o l i n i c a c i d have used a d i f f e r e n t i a l m i c r o b i o l o g i c a l method f o r d e t e r m i n i n g f o l a t e s i n b i l e , serum, and u r i n e . 4 7 4 9 Using t h r e e m i c r o o r g a n i s m s t h a t r e s p o n d t o d i f f e r e n t f o l a t e s , t h e y deduced t h e i d e n t i t y o f some metab o l i c products. Generally, c a s e i r e s p o n d s t o a l l monoglutamates, f a e c a l i s a l s o m e a s u r e s monoglutamates e x c e p t 5mH4F, and p. c e r e v i s i a e growth q u a n t i t a t e s t e t r a h y d r o f o l a t e s , a g a i n e x c l u d i n g 5mH4F. P r a t t and Cooper4 casei r e s p o n s e and a l e s s showed a l a r g e i n c r e a s e i n pronounced i n c r e a s e i n 2. f a e c a l i s growth i n b i l e and plasma a f t e r o r a l a d m i n i s t r a t i o n of l e u c o v o r i n t o p a t i e n t s . L i t t l e , i f a n y , i n c r e a s e w a s n o t i c e d i n p. c e r e v i s i a e . From t h e s e o b s e r v a t i o n s t h e y c o n c l u d e d t h a t 5fH,F i s r a p i d l y m e t a b o l i z e d t o 5mH4F, a l t h o u g h a v e r y s m a l l amount may b e a b s o r b e d as t h e i n t a c t 5-formyl compound. P e r r y and C h a n a r i n 4 found s i m i l a r r e s u l t s i n u r i n e s a m p l e s . With t h e s y n t h e s i s o f t h e doubly l a b e l e d r a d i o a c t i v e compound, 5-formyl- 4 C - t e t r a h y d r o f o l a t e - 3 H , i n v e s t i g a t o r s have been a b l e t o t r a c e t h e m e t a b o l i c p r o d u c t s when t h e drug is administered o r a l l y o r i n t r a v e n o u s l y .

s.

L.

L.

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LESLIE 0.PONT ET AL.

Nixon and B e r t i n o have done a n e x t e n s i v e s t u d y o f f o l i n i c a c i d metabolism i n human s u b j e c t s g i v e n t h e d r u g b o t h ways. When g i v e n o r a l l y , a f t e r 75 m i n u t e s t h e majori t y (90%) of 3H i n serum w a s found i n 5mH4F, w i t h 20% o f 1 4 C a s s o c i a t e d w i t h t h e same compound; 8 t o 9% of e a c h l a b e l w a s found a s 10fH4F o r 5,lO-methenyl H4F. Due t o t h e l a b i l e n a t u r e of t h e formyl group, 70% of t h e 1 4 C was n o t absorbed on Sephadex as f o l a t e under t h e chromatog r a p h i c c o n d i t i o n s u s e d , and t h e a u t h o r s presumed t h a t r a d i o a c t i v i t y had been i n c o r p o r a t e d i n t o amino a c i d s . V i r t u a l l y no r a d i o a c t i v e 5fH4F was found. However, t h e r a d i o a c t i v i t y a s s o c i a t e d w i t h u r i n e (0-1 hour a f t e r admini s t r a t i o n ) was % 40% 3H-labeled 5fH4F. Less t h a n 20% of t h e remaining 3H w a s i d e n t i f i e d as 5mH4F o r p-aminobenz o y l g l u t a m a t e , less t h a n 40% as 10fH4F o r 5,lO-methenyl H4F. The 1 4 C w a s e x c r e t e d as a m i x t u r e of t h e t h r e e f o l a t e s . A t longer t i m e i n t e r v a l s , t h e s e proportions v a r i e d . Ninety minutes a f t e r i n j e c t i o n , 60% 3H w a s found as 5mH4F, 40% of r a d i o a c t i v i t y as 5fH4F, and 40% 1 4 C w a s no l o n g e r a s s o c i a t e d w i t h serum f o l a t e . U r i n e showed most r a d i o a c t i v i t y a s 5fH4F i n i t i a l l y , and s e v e r a l h o u r s l a t e r a r i s e w a s s e e n i n l a b e l e d 10fH4F a n d / o r 5,10-methenyl H4F. From t h i s s e r i e s of s t u d i e s , t h e a u t h o r s concluded t h a t most 1 4 C w a s removed from c i r c u l a t i n g f o l a t e s , most of t h e 3H w a s a s s o c i a t e d w i t h 5mH4F, most of t h e drug w a s a b s o r b e d by t i s s u e s , and t h e m a j o r i t y of 5fH4F w a s c o n v e r t e d t o 5mH4F. E q u a l l y i m p o r t a n t w a s t h e s i t e o f metabolism. S i n c e t h e 5f -+ 5m c o n v e r s i o n w a s more r a p i d i n p a t i e n t s r e c e i v i n g o r a l admini s t r a t i o n , t h e authors f e l t t h a t t h e r e a c t i o n took place The i n t h e i n t e s t i n e , a view s u p p o r t e d by o t h e r s . l voided f o l a t e s seemed t o b e 10fH4F o r 5,lO-methenyl H4F. Another i n t e r e s t i n g s u g g e s t i o n was t h a t 5mH4F w a s cons e r v e d o v e r 5fH4F by t h e k i d n e y s . Although l e u c o v o r i n i s r a p i d l y a b s o r b e d and metabol i z e d i n t h e i n t e s t i n e , i t s m e t a b o l i c p r o d u c t , 5mH4F, i s c o n c e n t r a t e d i n c e r e b r o s p i n a l f l u i d (CSF). I n a s t u d y w i t h dogs, L e v i t t e t a1. found t h a t i n j e c t e d l a b e l e d f o l a t e s d i s a p p e a r e d from serum and a p p e a r e d as 5mHbF i n CSF f o l l o w i n g f i r s t - o r d e r k i n e t i c s . With doubly l a b e l e d 5fH4F, t h e a u t h o r s were a b l e t o trace t h e 1 4 C formyl moiety t o s e r i n e o r m e t h i o n i n e and t h e 3H p o r t i o n t o 5mH4F. The u p t a k e o f d r u g o c c u r r e d i n two s t e p s : a ) e q u i l i b r a t i o n i n e x t r a c e l l u l a r f l u i d with nonlabeled

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compound,53 followed by b ) c e l l u l a r uptake and metabolism. E a r l y p a p e r s r e p o r t e d a n a t u r a l c o n c e n t r a t i o n of f o l a t e i n CSF t h a t w a s t h r e e t i m e s t h e e q u i l i b r i u m c o n c e n t r a t i o n i n serum. T h i s c o u l d i n d i c a t e t h e importance of f o l a t e s i n n e u r a l metabolism, and t h e r e have been some r e p o r t s regardi n g t h e t h e r a p e u t i c t r e a t m e n t of n e u r o p s y c h i a t r i c d i s o r d e r s with f o l a t e s . The r e c e n t i n t e r e s t i n l e u c o v o r i n i s due t o i t s a b i l i t y t o r e d u c e m e t h o t r e x a t e (MTX) t o x i c i t y when b o t h a r e admini s t e r e d t o c a n c e r p a t i e n t s . E a r l y s t u d i e s s 4 i n mice showed t h a t most b e n e f i t o c c u r r e d when l e u c o v o r i n w a s g i v e n 12-24 h o u r s a f t e r MTX i n f u s i o n . The d e l a y i n a d m i n i s t e r i n g l e u c o v o r i n reduced t h e t o x i c e f f e c t s of MTX w i t h o u t r e d u c i n g the l a t t e r s antitumor a c t i v i t y . T h e r e f o r e , an enhancement of MTX e f f e c t can b e produced by l a r g e r dosages witho u t t h e t o x i c i t y a s s o c i a t e d w i t h t h e s e amounts. When g i v e n c o n c u r r e n t l y , l e u c o v o r i n reduced MTX t o x i c i t y a t t h e expense of tumor a c t i v i t y . However, r e c e n t work5 h a s shown some advantage i n c o n c u r r e n t a d m i n i s t r a t i o n . I n t h e case of MTX-sensitive c e l l s , f o l i n i c a c i d p r o t e c t i o n i n c r e a s e s t h e c e l l s u r v i v a l percentage. Rescue p r o c e d u r e , o r leucov o r i n given a f t e r a d e l a y p e r i o d , was most e f f e c t i v e (meas u r e d i n p e r c e n t a g e of c e l l s u r v i v a l ) i n t r e a t i n g MTXr e s i s t a n t c e l l s . The t h e r a p e u t i c e f f e c t of l e u c o v o r i n l i e s i n an e a r l i e r resumption of DNA s y n t h e s i s t h a n i f MTX were a d m i n i s t e r e d a l o n e . 5 6 Nahas e t a l . attribute this resumption t o s e v e r a l p o s s i b l e a c t i o n s of l e u c o v o r i n ( a s s t u d i e d i n L1210 leukemia c e l l s ) : a ) e f f l u x of MTX i s i n c r e a s e d i n t h e p r e s e n c e of 5fH4F; b) 5fH4F could deblock d i h y d r o f o l a t e r e d u c t a s e bound by MTX by b e i n g a p o t e n t i a l supply of d i h y d r o f o l a t e ; c ) t h e i n t a c t drug could p o s s i b l y d i s p l a c e some of t h e MTX; and d) 5fH4F competes w i t h MTX f o r c e l l u l a r uptake. C e l l u l a r uptake seems t o b e enhanced by methyl o r formyl groups a t N 5 on reduced f o l a t e s , by amino s u b s t i t u t i o n s on C 4 o f o x i d i z e d f o l a t e s , and by t e r minal glut am ate^.^' Thus 5fH4F, 5mH4F, and MTX appear t o u s e t h e s a m e c a r r i e r - m e d i a t e d t r a n s p o r t i n t o c e l l s , a system used by f o l i n i c a c i d t o a lesser e x t e n t . Another a u t h o r d e s c r i b e s t h e t r a n s p o r t of f o l a t e s a c r o s s memb r a n e s a s an exchange phenomenon because a l t h o u g h leucov o r i n competes w i t h MTX f o r c e l l u p t a k e , i t can a l s o s t i m u l a t e i n f l u x of t h e l a t t e r i f t h e c e l l s a r e a l r e a d y

336

LESLIE 0. PONT ET A L .

preloaded w i t h f o l i n i c a c i d . The i n f l u x i s n o t e d even i n c a s e s where d i h y d r o f o l a t e r e d u c t a s e h a s been i n a c t i v a t e d by p r e v i o u s MTX dosage.

6.

Methods of A n a l y s i s

6.1

Elemental A n a l y s i s

Below a r e l i s t e d e l e m e n t a l a n a l y s i s r e s u l t s o b t a i n e d from a t y p i c a l sample of calcium l e u c o v o r i n : Element C

H

% Theoretical

% Found*

46.96 4.14 19.17 7.84

46.87 4.11 19.07 6 . 9 4 t , 7.71$

Ca
f o r 10.4% HzO. +Found by a t o m i c a b s o r p t i o n . ?Found by s u l f a t e a s h .

*C o r r e c t e d

The h i g h e r calcium v a l u e o b t a i n e d by t h e a s h method i s no doubt due t o t h e p r e s e n c e of sodium ( t h e c o u n t e r i o n f o r a c e t a t e commonly found i n some l o t s ) .

6.2

E q u i v a l e n t Weight Determination

Because s y n t h e t i c p r o d u c t s a r e i s o l a t e d a s t h e barium o r , more f r e q u e n t l y , t h e c a l c i u m s a l t of l e u c o v o r i n , common a c i d - b a s e t i t r a t i o n s are n o t r e p o r t e d . I f t h i s t y p e of t i t r a t i o n o r one i n which t h e c a t i o n i s exchanged were f e a s i b l e , t h e r e s u l t s would r e q u i r e c a r e f u l i n t e r p r e t a t i o n because i m p u r i t i e s c o n t a i n i n g t h e g l u t a m i c a c i d moiety would respond s i m i l a r l y t o l e u c o v o r i n when t h e c a r b o x y l groups are b e i n g a n a l y z e d .

6.3

B i o l o g i c a l Assay

6.3.1

M i c r o b i o l o g i c a l Assay

Several microbiological procedures f o r a s s a y i n g CF have been d e s c r i b e d i n t h e l i t e r a t u r e . In g e n e r a l , t h e s e methods a r e designed t o measure CF c o n t e n t i n a b i o l o g i c a l f l u i d o r specimen. They a l l make u s e of t h e growth promotion of L. c i t r o v o r u m 8 0 8 1 , o r p. E v i s i a e , as i t h a s been renamed. Before CF w a s i s o l a t e d

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o r l e u c o v o r i n w a s s y n t h e s i z e d , S a u b e r l i c h and Baumann m e a s u r e d e n h a n c e d growth o f t h e m i c r o o r g a n i s m i n t h e p r e s e n c e o f v a r i o u s b i o l o g i c a l e x t r a c t s , e s p e c i a l l y l i v e r conc e n t r a t e s . Q u a n t i t a t i o n came from t u r b i d o m e t r i c r e a d i n g s a n d f o r N a O H t i t r a t i o n of a c i d p r o d u c e d . T h e i r r e s u l t s are r e p o r t e d i n t e r m s of c i t r o v o r u m u n i t s t h a t c o r r e s ponded t o t h e h a l f - o p t i m a l g r o w t h i n a s t a n d a r d r e t i c u l o g e n s a m p l e . The a u t h o r s r e a l i z e d t h e l i m i t s o f t h e i r a s s a y method s i n c e f o l i c a c i d a d d i t i o n s c a u s e d e n h a n c e d growth a f t e r a n i n i t i a l l a g . W i n s t e n a n d E i g e n t r i e d t o improve t h e method by c h r o m a t o g r a p h i n g t h e s a m p l e s on p a p e r b e f o r e t e s t i n g f o r p. c e r e v i s i a e g r o w t h , b u t t h e y , The b a s i s t o o , found more t h a n a s i n g l e a c t i v e component. Coopero f t h i s p a r t i c u l a r technique is s t i l l being used. man6 r e f i n e d t h e p r o c e d u r e somewhat, b u t c o n t i n u e d t o measure t u r b i d i t y . F o l i c a c i d i n t e r f e r e n c e is minimized by i n c u b a t i o n f o r s h o r t p e r i o d s . Quantitation is in t e r m s o f mg/ml, u s i n g commercial s a m p l e s o f l e u c o v o r i n as s t a n In o n e r e c e n t l y p u b l i s h e d method, p a p e r d i s c s dards. a r e i m p r e g n a t e d w i t h t h e t e s t s o l u t i o n s and CF c o n t e n t i s found by m a n u a l l y m e a s u r i n g L. c i t r o v o r u m ATCC 8 0 8 1 growth on agar p l a t e s . The a d v a n t a g e o f t h i s method i s t h a t CF c a n b e q u a n t i t a t e d i n s o l u t i o n w i t h a l a r g e excess o f amethopterin because t h e microorganisms are resistant t o S t a t i s t i c a l v a r i a t i o n on t h i s method i s 10%. the latter.

6.3.2

Enzyme Assay

S i l v e r m a n e t a 1 . 4 1 p u r i f i e d a hog l i v e r enzyme t h a t c a t a l y z e d t h e t r a n s f e r o f t h e f o r m y l g r o u p o f CF t o g l u t a m i c a c i d . I f n o t p r o t e c t e d from o x i d a t i o n , t h e t e t r a h y d r o f o l i c a c i d formed would s p o n t a n e o u s l y decompose t o p - a m i n o b e n z o y l g l u t a m a t e and p t e r i d i n e . A r y l a m i n e f o r m a t i o n c o u l d be m o n i t o r e d b y t h e B r a t t o n - M a r s h a l l method. T h i s method g i v e s a b e t t e r i n d i c a t i o n o f enzyme a c t i v i t y t h a n CF p u r i t y . The t e c h n i q u e d e v e l o p e d by P e t e r s a n d G r e e n b e r g , 4 z l a t e r r e f i n e d by G r e e n b e r g , 6 2 i s p e r h a p s more straightforward. The t e s t s o l u t i o n s a r e mixed w i t h ATP, MgSO,, a n d enzyme i s o l a t e d from s h e e p l i v e r i n a b u f f e r e d aqueous s o l v e n t . Absorbance a t 343 nm i s r e a d i n a specThe 5,lO-methenyltetrahydrofolate t r o p h o t o m e t e r a t 30 C . b e i n g formed s h o u l d g i v e a d i r e c t m e a s u r e o f t h e CF o r leucovorin present.

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The above b i o l o g i c a l a s s a y s a r e i n t e n d e d t o measure CF i n b i o l o g i c a l s o u r c e s r a t h e r t h a n t o d i r e c t l y measure l e u c o v o r i n o r CF p u r i t y . I n f a c t , commercial samples o f l e u c o v o r i n a r e u s u a l l y used t o d e t e r m i n e s t a n d a r d c u r v e s . 6.4 P o l a r o g r a p h i c Assay

Leucovorin, s i n c e i t i s t o t a l l y r e d u c e d , i s p o l a r o g r a p h i c a l l y i n e r t i n a pH 9 b u f f e r e d s o l u t i o n . 6 3 A f t e r a c i d t r e a t m e n t , t h r e e p o l a r o g r a p h i c waves a r e g e n e r a t e d , c o r r e s p o n d i n g t o a n a n o d i c o x i d a t i o n of a t e t r a h y d r o compound and two c a t h o d i c r e d u c t i o n s of unreduced p t e r i a i n e s ; presumably a t l e a s t one of t h e s e t h r e e i s a dihydro s p e c i e s . Polarography i s u s e f u l as a technique i n s t r u c t u r a l e l u c i d a t i o n , b u t a n a l y t i c a l d a t a would b e d i f f i c u l t t o o b t a i n from an a c i d - t r e a t e d s o l u t i o n c o n t a i n i n g several s p e c i e s , each w i t h i t s own p o l a r o g r a p h i c b e h a v i o r .

6.5

Spectrophotometric Analysis 6.5.1 Fluorometric Analysis

I n 1957, Duggan e t a1.64 r e p o r t e d t h a t maximum n a t u r a l f l u o r e s c e n c e o f f o l i n i c a c i d o c c u r r e d a t pH 7 , w i t h e x c i t a t i o n a t 370 nm and e m i s s i o n a t 460 nm. A c o n c e n t r a t i o n o f 0.15 vg/ml gave a f l u o r e s c e n c e of 10% f u l l - s c a l e d e f l e c t i o n a t maximum i n s t r u m e n t a l s e n s i t i v i t y . These a u t h o r s e x p l o r e d a n a l y z i n g f o l i n i c a c i d i n t h e p r e s e n c e of f o l i c a c i d and found t h a t e x c i t a t i o n a t 290 nm e f f e c t i v e l y s h i f t e d t h e e m i s s i o n band of t h e compound o f i n t e r e s t t o 370 nm, t h u s e n a b l i n g a n a l y s i s of a m i x t u r e . A l a t e r p a p e r s 5 r e p o r t e d a f l u o r e s c e n c e maximum f o r l e u c o v o r i n a t 365 n when e x c i t e d a t 314 nm i n a pH 7 s o l u t i o n ; m t h e c o n c e n t r a t i o n w a s 5 x lo-* M. V a r i a t i o n between t h e s e d a t a and o t h e r v a l u e s w a s a t t r i b u t e d t o sample i m p u r i t y , pH of s o l u t i o n , and quenching. The a u t h o r s made an a t t e m p t t o c o r r e l a t e s t r u c t u r e and f l u o r e s c e n c e of reduced f o l a t e s . S i m i l a r i t y between t e s t e d compounds and p-aminobenzoylg l u t a m a t e l e a d them t o c o n c l u d e t h a t t h i s p o r t i o n of t h e molecule i s r e s p o n s i b l e f o r maxima a t 360-425 n when m e x c i t e d a t 300-320 nm. They s u g g e s t e d t h a t i n t e n s i t y d i f f e r e n c e s may a r i s e from v a r i o u s s u b s t i t u t i o n s on t h e t e t r a h y d r o p t e r i d i n e moiety.

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I n 1964, N e t r a w a l i e t a 1 . 6 6 d e s c r i b e d f l u o r e s c e n c e measurements of c a l c i u m l e u c o v o r i n i n NaHC03 s o l u t i o n a f t e r p a p e r chromatography and r e a c t i o n w i t h a c i d i c o r c i n o l . The gray-blue f l u o r e s c e n t s p e c i e s formed on t h e developed papergram w a s v e r y s e n s i t i v e f o r l e u c o v o r i n , showing a d e t e c t i o n l i m i t of 0 . 1 ug. I n t h e e l u t i n g s o l u t i o n , however, t h e r e q u i r e d c o n c e n t r a t i o n i n c r e a s e d t o 0 . 5 ug. Recovery by t h i s method w a s 100 k 15%, and r e s u l t s o b t a i n e d from f l u o r e s c e n c e were lower by 10 t o 20% t h a n t h o s e o b t a i n e d by m i c r o b i o l o g i c a l methods. 6.5.2 U l t r a v i o l e t / V i s i b l e Analysis

U l t r a v i o l e t s p e c t r o s c o p y does n o t l e n d i t s e l f t o l e u c o v o r i n a n a l y s i s f o r two r e a s o n s . F i r s t , b e c a u s e commercial samples a r e f r e q u e n t l y c o n t a m i n a t e d w i t h uv-absorbing i m p u r i t i e s , a r e l i a b l e molar a b s o r p t i v i t y h a s n o t been d e t e r m i n e d f o r l e u c o v o r i n . R e c e n t l y , i n t h i s l a b o r a t o r y a v a l u e of 3.09 x l o 4 I T 1 cm-' w a s d e r i v e d from thorough a n a l y s i s o f a r e l a t i v e l y p u r e sample. T h i s v a l u e i s i n r e a s o n a b l e agreement w i t h t h a t o f Zakrzewski and Sanson. ' Second, l e u c o v o r i n i s known t o d e h y d r a t e under a c i d i c c o n d i t i o n s t o form a n h y d r o l e u c o v o r i n , 5,10-methenyl H4F, which a b s o r b s a t 352-353 nm. I n t h e a b s e n c e of i n t e r f e r i n g s p e c i e s , * l e u c o v o r i n may b e a n a l y z e d by a c i d i f i c a t i o n w i t h 0 . 1 N H C 1 f o l l o w e d by u v measurement a f t e r 1.52 . 0 h o u r s . P u r i t y may b e d e t e r m i n e d r e l a t i v e t o a sample of known p u r i t y o r r e l a t i v e t o l i t e r a t u r e v a l u e s : E~~~ = 2.39-2.41 x l o 4 M-' cm-1.''

A b s o r p t i o n i n t e r f e r e n c e s would a r i s e from compounds o t h e r than le uc ovori n t h a t convert t o anhydroleucovorin a t a c i d i c pH. An example of such a compound i s 10fH4F, although i t i s not expected t o c o n t r i b u t e t o ab s o r p tio n due t o i t s i n s t a b i l i t y . However, 10-formyl-7,8d i h y d r o f o l a t e i s a p o s s i b l e contaminant. Ad d itio n al a b s o r p t i o n c o u l d a l s o r e s u l t from u n s a t u r a t e d p t e r i d i n e s .

340

LESLIE 0. PONT ET A L .

6.6

Chromatography 6.6.1 P a p e r Chromatography

Both p a p e r and t h i n - l a y e r chromatography s e r v e as s e p a r a t i o n t e c h n i q u e s b e f o r e q u a n t i t a t i o n o f a c t i v e components. P a p e r chromatography h a s been u s e d by many e a r l y i n v e s t i g a t o r s t o s e p a r a t e b i o l o g i c a l s a m p l e s b e f o r e d e t e c t i n g by m i c r o b i o l o g i c a l a s s a y , t h e b i o a u t o g r a p h i c method. Winsten and E i g e n S 9 used 2 , 4 , 6 t r i m e t h y l p y r i d i n e t o d e v e l o p papergrams on Whatman No. 1 o r No. 4 . C o l 6 n Z 9 d e v e l o p e d h i s chromatograms i n i s o a m y l a l c o h o l : 5 % d i b a s i c sodium p h o s p h a t e , 1 : 2 . Netrawali e t a1.66 used ethanol:n-butanol:water:25% NH3, 50:15:35:5, on Whatman No. 1 and d e t e c t e d l e u c o v o r i n by forming a f l u o r e s c e n t o r c i n o l d e r i v a t i v e . The m o b i l e p h a s e s i n t h e s e s y s t e m s a r e n e u t r a l t o b a s i c due t o t h e a c i d i n s t a b i l i t y of leucovorin. 6.6.2 T h i n - l a y e r Chromatography

T h i n - l a y e r chromatography on c e l l u l o s e h a s been used a s a n i n t e r m e d i a t e clean-up p r o c e d u r e and f i n a l i s o l a t i o n t e c h n i q u e i n t h e s y n t h e s i s of l e u c o v o r i n . 2 2 A c a t i o n e x c h a n g e / c e l l u l o s e s u p p o r t w a s p r e p a r e d by Copenh a v e r and O ' B ~ i e n , ~and t h e m o b i l e p h a s e w a s 15% Na,HPO,* ' 12H20 (pH 8 . 5 ) c o n t a i n i n g 0 . 1 M m e r c a p t o e t h a n o l . Unfort u n a t e l y , t h i s s y s t e m d i d n o t s e p a r a t e 5f a n d 10fH4F from e a c h o t h e r o r from 5,10-methenyl H4F. S e p a r a t i o n o f t h e former two from t h e * l a t t e r w a s a c h i e v e d w i t h 1 N a 2 H P 0 4 * 1 1 2 H 2 0 . Leucovorin w a s d e t e c t e d w i t h a 6N HC1/ZnClz/sodium c i t r a t e spray. Brown e t a 1 . 6 8 h a v e developed a c e l l u l o s e p l a t e w i t h a fluorescent indicator. Compounds a r e d e v e l o p e d i n 3.0% (w/v) N H 4 C 1 and d e t e c t e d by f l u o r e s c e n c e q u e n c h i n g . These a u t h o r s a l s o u s e 0.5% m e r c a p t o e t h a n o l i n t h e i r m o b i l e p h a s e , b u t t h i s i s o n l y t o p r e v e n t o x i d a t i o n of t h e l a b i l e r e d u c e d p t e r i d i n e s , which a r e n o t a d e q u a t e l y p r o t e c t e d by s u b s t i tution a t the N5 position. Since n e u t r a l o r a l k a l i n e solut i o n s of l e u c o v o r i n a r e r e l a t i v e l y s t a b l e i n a i r , t h i s p r e c a u t i o n may n o t b e r e q u i r e d f o r r o u t i n e a s s a y .

CALCIUM LEUCOVORIN

34 I

6.6.3

Column Chromatography

L e u c o v o r i n h a s been chromatographed on columns of d i f f e r e n t p a c k i n g m a t e r i a l s s i n c e i t w a s f i r s t synthesized. E a r l y i n v e s t i g a t o r s used magnesium s i l i c a t e and a c t i v a t e d c h a r c o a l , g s t a r c h , 6 and--more r e c e n t l y - DEAE c e l l u l o s e ' ' i n t h e i r p u r i f i c a t i o n and i s o l a t i o n of leucovorin. Column t e c h n i q u e s h a v e b e e n c o u p l e d w i t h v a r i o u s d e t e c t i o n methods t o i d e n t i f y and q u a n t i t a t e CF and o t h e r f o l a t e s i n n a t u r a l l y o c c u r r i n g p r o d u c t s . Noronha and S i l ~ e r m a nused &. c a s e i , P . c e r e v i s i a e , a n d ~~ -. f a e c a l i s t o d e t e c t f o l a t e s i n c h i c k e n l i v e r e x t r a c t s S chromatographed on DEAE c e l l u l o s e . A s i m i l a r method w a s u s e d by R o h r i n g e r e t a 1 . 7 0 t o m o n i t o r f o l a t e s i n h e a l t h y An e a r l i e r p u b l i c a t i o n " and i n f e c t e d wheat l e a v e s . r e p o r t e d anion-exchange chromatography on Dowex-lc h l o r i d e u s i n g uv a b s o r p t i o n o f c o l l e c t e d f r a c t i o n s f o r d e t e c t i o n . Due t o i t s i n s t a b i l i t y i n a c i d , l e u c o v o r i n w a s e l u t e d w i t h N a C l r a t h e r t h a n H C 1 , which i s commonly u s e d f o r o t h e r f o l a t e s . T h i s method w a s l i m i t e d t o synt h e t i c products r a t h e r than b i o l o g i c a l mixtures. The a u t h o r s f e l t t h a t s e n s i t i v i t y c o u l d be g a i n e d by microbiological detection. More r e c e n t l y , a t t e n t i o n h a s been p l a c e d on g e l p e r meation te c h n iq ues . Sephadex G-15 and G-25 columns c o u p l e d w i t h uv d e t e c t o r s w e r e u s e d t o s e p a r a t e f o l a t e s u~ found i n r a t k i d n e y e x t r a c t s . 7 2 K B s and C e r 1 - 1 9 ~s e d Sephadex G-10 t o s e p a r a t e f o l a t e s i n cow's m i l k , u t i l i z i n g s p e c t r o s c o p i c and m i c r o b i o l o g i c a l d e t e c t i o n . Nixon and B e r t i n ~ ' used f l u o r e s c e n c e r a t i o s and m a r k e r s on ~ DEAE Sephadex A-25 t o s e p a r a t e f o l a t e coenzymes. I n s e p a r a t i n g f o l a t e s from b i o l o g i c a l s o u r c e s , s e v e r a l a u t h o r s t r e a t e d t h e i r s a m p l e s w i t h t h e a p p r o p r i a t e conjugase t o hydrolyze polyglutamates. R a d i o a c t i v i t y measurement h a s been used t o d e t e c t doubly l a b e l e d 5fH,F and its metabolic products. 5 0

6.6.4

H i g h - p r e s s u r e L i q u i d Chromatography

A n a t u r a l e x t e n s i o n o f column chromatogr a p h y i s h i g h - p r e s s u r e l i q u i d c h r o m a t o g r a p h y , which comb i n e s s e p a r a t i o n , d e t e c t i o n , and q u a n t i t a t i o n (and i s o l a Perhaps t h e s i n g l e t i o n i n t h e c a s e of p r e p a r a t o r y w o r k ) .

CALC IU h LEUCOVOR IF 8

FOLIC ACID (INTERNAL STANDARD)

I NJ ECT I ON

9 12 15 18 21 24 MINUTES LSJ-59

FIGURE 5

HIGH PRESSURE LIQUID CHROMATOGRAM OF CALCIUM LEUCOVORIN

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343

most a t t r a c t i v e f e a t u r e o f t h i s t e c h n i q u e , i s s p e e d , o n c e t h e chromatographic c o n d i t i o n s have been determined. V a r i o u s modes a r e a v a i l a b l e , b u t f o l a t e s h a v e b e e n r e s t r i c t e d t o i o n exchange o r p a r t i t i o n i n g d u e t o l i m i t e d s o l u b i l i t y i n o r g a n i c s o l v e n t s . D e t e c t i o n i s u s u a l l y by uv a b s o r p t i o n , a l t h o u g h i n t h e case of l e u c o v o r i n , s e n s i t i v i t y i s i n c r e a s e d s e v e r a l o r d e r s of m a g n i t u d e by e l e c t r o c h e m i c a l d e t e c t i o n . HPLC h a s b e e n u s e d r o u t i n e l y i n t h i s l a b o r a t o r y s i n c e 1973, and among t h e compounds a n a l y z e d a r e f o l a t e s . 7 5 The most v e r s a t i l e s y s t e m t h a t t h e s e a u t h o r s h a v e found i s a c h e m i c a l l y bonded C I S column w i t h 0 . 1 M KHIPOL (pH a d j u s t e d t o 4 . 0 ) c o n t a i n i n g v a r y i n g amounts of m e t h a n o l ( s e e F i g u r e 5 ) . An e a r l i e r s y s t e m u s i n g t h e same reverse p h a s e C I S column w i t h t r i s b u f f e r , 2-amino-2-(hydroxymethyl)-lY3-propanediol a t pH 6 . 7 , w a s abandoned i n f a v o r of t h e above p h o s p h a t e e l u e n t which b e t t e r r e s o l v e s c o n t a m i n a n t s from t h e m a j o r component. A s i m i l a r s e t of c o n d i t i o n s w a s u t i l i z e d by R e i f e t a 1 . 7 6 t o a n a l y z e f o l i c a c i d , u s i n g l e u c o v o r i n as a n i n t e r n a l s t a n d a r d . Other a u t h o r s 7 7 have used similar c o n d i t i o n s t o s t u d y e x t e n s i v e l y t h e i d e n t i t y of p o s s i b l e l e u c o v o r i n contaminants. In a d d l t i o n t o t h e s e p a r t i t i o n ' / i o n i z a t i o n s u r p r e s s i o n m e t h o d s , a n i o n exchange h p l c h a s b e e n u s e d t o s e p a r a t e CF from o t h e r n a t u r a l l y o c c u r r i n g f o l a t e s , 7 e and some c o r r e l a t i o n h a s been made between t h e number of glutamyl r e s i d u e s and r e t e n t i o n t i m e . 6.6.5 A f f i n i t y Chromatography

Although a f f i n i t y chromatography h a s n o t b e e n u s e d d i r e c t l y as a n a n a l y t i c a l method, i t may b e modified i n t h e f u t u r e t o produce a v i a b l e technique. L e u c o v o r i n h a s been used a s a n e f f e c t i v e s p a c e r i n o b t a i n i n g a c t i v e samples o f d i h y d r o f o l a t e r e d u c t a s e . 79 I f t h e enzyme c o u l d b e i m m o b i l i z e d w i t h o u t l o s i n g i t s a c t i v i t y , p e r h a p s i t c o u l d b e used t o s e p a r a t e f o l a t e s .

6.7

Radioassay

R e c e n t l y , r a d i o a s s a y methods h a v e been r e f i n e d t o measure f o l a t e s in b i o l o g i c a l s a m p l e s . These t e c h n i q u e s u s e r a d i o a c t i v e l y l a b e l e d f o l a t e s and c o m p e t i t i v e p r o t e i n binding. Johnson e t a l . compare t h i s method w i t h t r a d i t i o n a l m i c r o b i o l o g i c a l a s s a y w i t h L. c a s e i .

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Although t h i s method w a s d e s i g n e d f o r f o l a t e s i n g e n e r a l , d i f f e r e n t i a l microbiological t e s t i n g could give an indic a t i o n of r e d u c e d f o l a t e c o n c e n t r a t i o n . 7. Acknowledgments

S u p p o r t e d by C o n t r a c t N01-CM-33723 from t h e D i v i s i o n of Cancer T r e a t m e n t , N a t i o n a l Cancer I n s t i t u t e , N a t i o n a l I n s t i t u t e s o f H e a l t h , Department o f H e a l t h , E d u c a t i o n , and W e l f a r e . The o p i n i o n s e x p r e s s e d a r e t h o s e of t h e a u t h o r s and n o t n e c e s s a r i l y t h o s e o f t h e N a t i o n a l Cancer Institute. The a u t h o r s wish t o t h a n k Michael C u l c a s i f o r t h e HPLC development, B a r b a r a S e n u t a , F l o r e n c e Yoshikawa, M a r t i n S t r u b l e , and J o h n Jee f o r t h e t e c h n i c a l a s s i s t a n c e and A r d a t h Trine f o r s e c r e t a r i a l support.

8.

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A. G o l d i n , M. Manatel, S. W. Greenhouse, J . M. V e d i t t i , and S. R . Humphreys, Cancer Res. 43

14,

(1954). 55. 56.

B. T. H i l l , L. A. P r i c e , S . I . H a r r i s o n , and J . H . G o l d i e , Europ. J . Cancer. g, 6 1 ( 1 9 7 7 ) . 8
F. M. S i r o t n a k , R. C . Donsbach, D. M. Eloccio, and D. M. Dorick, Cancer R e s . 4679 ( 1 9 7 6 ) .

2,

57. 58.

A. Nahas, P. F. Nixon, and J . R. B e r t i n o , Cancer R e s .

- 1416 (1972). 32,

D. Goldman, The c h a r a c t e r i s t i c s of t h e membrane t r a n s p o r t o f a m e t h o p t e r i n and n a t u r a l l y o c c u r r i n g f o l a t e s . I n F o l a t e A n t a g o n i s t s as Chemotherapeutic Agents, Ann. N . Y . Acad. S c i . - 400 ( 1 9 7 1 ) . 186,
W. A. Winsten and E . E i g e n , J . B i o l . Chem. - 1 5 5 184,

59. 60.

(1950).
J . M. Cooperman, M i c r o b i o l o g i c a l a s s a y of f o l i c a c i d a c t i v i t y i n serum and whole b l o o d . I n Methods i n Enzymology, X V I I I , P a r t B ( e d s . D . G. McCormick and L . D. W r i g h t ) , Academic P r e s s , New York, 1 9 7 1 , p . 629.

61. 62.

B . M. Mehta and D. J . H u t c h i s o n , Cancer Chemother. Rep., P a r t 1, - 935 ( 1 9 7 5 ) . 59, D. Greenberg, NS-Formyltetrahydrofolic a c i d cyclohyd r a s e . I n Methods i n Enzymology, X V I I I , P a r t B ( e d s . D. B. McCormick and L . D. W r i g h t ) , Academic P r e s s , New York, 1 9 7 1 , p . 7 8 6 .

CALCIUM LEUCOVORIN

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63. 64.

W.

A l l e n , R. L. P a s t e r n a k , and W. SOC. 74, 3264 ( 1 9 5 2 ) .

Seaman, J . Am. Chem.

D . E. Duggan, R. L. Bowman, B. B. B r o d i e , and S . Udenf r i e n d , Arch. Biochem. Biophys. 68, 1 ( 1 9 5 7 ) . K. Uyeda and J . C . Rabinowitz, Anal. Biochem.

65. 66. 67. 68. 69. 70.

(1963)

5, 100

M. S . N e t r a w a l i , R. Radhakrishnamurty, and A. Sreeni v a s a n , Anal. Biochem. -, 1 4 3 ( 1 9 6 4 ) . 8

J. H . Copenhaver and K . L. O ' B r i e n , - 454 ( 1 9 6 9 ) . 31,

Anal. Biochem.

J. P. Brown, G . E. Davidson, and J . M. S c o t t , J . Chromatog. 79,1 9 5 ( 1 9 7 3 ) .

J . M. Noronha and M. S i l v e r m a n , J . B i o l . Chem. 3299 ( 1 9 6 2 ) .

237,

R. R o h r i n g e r , W . K. K i m , and D . J. Samborski, Can. J . Biochem. -, 1 1 6 1 ( 1 9 6 9 ) . 47

M. R. H e i n r i c h , V. C . Dewey, and C. W. K i d d e r , J . Chromatog. 2, 296 ( 1 9 5 9 ) . Y. S . S h i n , K. U . Buehring, and E.L.R. B i o l . Chem. 2 4 7 , 7266 ( 1 9 7 2 ) .
~

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124,53

(1976).

P. F. Nixon and J . R. B e r t i n o , S e p a r a t i o n and i d e n t i f i c a t i o n of f o l a t e coenzymes on DEAE Sephadex. I n Methods i n Enzymology, X V I I , P a r t B ( e d s . D. B. McCormick and L. D. W r i g h t ) , Academic P r e s s , N e w York, 1 9 7 1 , p. 6 6 1 f f .
A . Cheung, L. P o n t , and P . Lim, A p p l i c a t i o n s of h i g h s p e e d l i q u i d chromatography t o some f o l i c a c i d a n a l o g s . I n N u c l e i c Acid Chemistry Improved and New S y n t h e t i c P r o c e d u r e s , Methods and T e c h n i q u e s , P a r t Two, ( e d s . L. B. Townsend and R . S . T i p s o n ) , John Wiley and Sons, I n c . , New York, 1 9 7 8 , p . 1 0 4 5 f f .

75.

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LESLIE 0. PONT ET A L .

76.

V. D. R e i f , J. T. R e a m e r , and L. T. Grady, J . Pharm. S c i . - 1 1 1 2 (1977). 66, J . A. Montgomery, T. P. J o h n s t o n , H. J. Thomas, J. R. P i p e r , and C. Temple, J r . , The u s e of m i c r o p a r t i c u l a t e reversed-phase packing i n h i g h p r e s s u r e l i q u i d chromatography of compounds of b i o l o g i c a l i n t e r e s t . I n Advances i n Chromatography ( e d . J. C a l v i n G i d d i n g s ) , Dekker, New York, -, 169. 15

77.

78.

R. W. S t o u t , A. R. Cashmore, J. K. Coward, C . G. Horv a t h , and J . R. B e r t i n o , Anal. Biochem. 71, 119 (1976).

79.

J. G. Dann, N.G.L. Harding, P.C.H. Newbold, and J. M. W h i t e l e y , Biochem. J. - 28P (1972). 127,
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80. 81.

I. Johnson, H. G u i l f o r d , and M. Rose, J. C l i n . P a t h o l .

- 645 (1977). 30,

Analytical Profiles of Drug Substances, 8

METHIMAZOLE
Hassan Y. Aboul-Enein and A . A . Al-Badr
I,
Description I . I Nomenclature 1. I I Chemical Names I . 12 Generic Names I . 13 Trade Names 1 . 1 Formulae 1.2 I Empirical 1.22 Structural 1.3 Molecular Weight I .4 Elemental Composition 1.5 Appearance. Color, Odor Physical Properties 2. I Crystal Properties 2. I I Crystallinity 2. I2 Melting Point 2.2 Dipole Moments 2.3 Solubility 2.3 Identification 2.5 Spectral Properties 2.51 Ultraviolet Spectrum 2.52 Infrared Spectrum 2.53 Nuclear Magnetic Resonance Spectrum 2.54 Mass Spectrum and Fragmentometry Synthesis Stability, Decomposition Products. and Metal Complexes Metabolism Methods of Analysis 6. I Titrimetric Analysis 6.11 Aqueous 6.12 Nonaqueous 6.2 Spectrophotometric Methods 6.21 Infrared Spectrophotometric 6.22 Nuclear Magnetic Resonance Spectrometric 6.23 Ultraviolet Spectrophotometric 6.3 Chromatographic Analysis 6.3 I Thin-Layer Chromatography 6.32 Gas-Liquid Chromatography 6.33 Paper chromatography 6.0 Colorimetric Analysis 6.5 Potentiometric Analysis 6.6 Coulometric Analysis References 35 I

2.

3. 4. 5. 6.

7.

Copyright @ 1979 by Academic Press. Inc. All rights of reproduction in any form reserved. ISBN 0-12.260808-9

352

HASSAN Y . ABOUL-ENEIN AND A. A. AL-BADR

METHIMAZOLE

1.

Description

1.1

Nomenclature

1.11

Chemical Names 1,3-dihydro-l-methyl, 2-H-Imidazole-2-thione. 1-methylimidazole-2-thiol

1-methyl-2-thioimidazole 1-methy1-2-mercap to imidazo1e

1.12 Generic Names 1.13

Methimazole, thiamazole

Trade Names Thiamethazole, Bazolan, Dananti201, Faristan, Frentorox, Mercazole, Metazole, Tapazole, Thacapazol, Thycapazol, Strumazol, Meto thyr ine

1.2

Formulae 1.21 Empirical 1.22 Structural

qHgN2 (iH3

FH3

1.3

Molecular weight

114.17
S 28.09%

1.4
1.5

Elemental composition C 42.08%, H 5.30%, N 24.54%,

Appearance, color, odor White to pale buff, crystalline powder, having a faint characteristic odor. Its solution is practically neutral to litmus. properties Crystallinity Crystal properties 2.11
No detailed studies on the crystal structure

2. Physical 2.1

of methimazole is reported in the literature. Methimazole can be microscopically identified using Koflers method, the occurrence of polymorphous modification is indicated in methimazole.

METHIMAZOLE

353

2.12

Melting point USP X I X (1) s p e c i f i e s a m e l t i n g r a n g e f o r methimazole between 144 and 147O. L e a f l e t s from a l c o h o l m.p. 146-148O(2), b.p. 280' (some d e c o m p o s i t i o n ) .

2.2

Dipole Moments The d i p o l e moment of methimazole w a s determined i n benzene and 1 , 4 d i o x a n e s o l u t i o n a t 25O ( 3 ) and r e p o r t e d t o be 4 . 7 4 D and 5.53 r e s p e c t i v e l y . The r e s u l t s were d i s c u s s e d i n terms of tautomerism and m o l e c u l a r conformations. Solubility F r e e l y s o l u b l e i n water, i n a l c o h o l and i n c h l o r o form, s l i g h t l y s o l u b l e i n e t h e r , petroleum e t h e r and benzene. Identification The f o l l o w i n g t e s t s a r e c i t e d from USP X I X (1) :The i n f r a r e d a b s o r p t i o n spectrum of a potassium bromide d i s p e r s i o n of i t exhibitsmaxima o n l y a t t h e same wavelength a s t h a t of a s i m i l a r p r e p a r a t i o n of USP methimazole Reference S t a n d a r d . M e r c u r i c c h l o r i d e TS produces i n a s o l u t i o n (1 i n 200)a w h i t e p r i c i p i t a t e , b u t no p r e c i p i t a t i o n i s produced by t r i n i t r o p h e n o l TS. The s o l u t i o n i s c o l o u r e d i n t e n s e l y b l u e by molybdo-phosphotungst a t e TS. Methimazole c a n be i d e n t i f i e d by forming c r y s t a l s of w i t h gold bromide/HCl s o l u t i o n . P l a t e s , o f t e n i n c r o s s e s ( s e n s i t i v i t y : 1 i n 1000); potassium t r i - i o d i d e s o l u t i o n - bunches of r o d s o r n e e d l e s ( s e n s i t i v i t y 1 i n 1000) F i g . 1.

2.3

2.4

2.5

Spectral Properties 2.51 U l t r a v i o l e t spectrum Methimazole i n 0.1N s u l f u r i c a c i d shows maxima a t 211 n ( E 1 % , 1 cm 593) and 251.5 n m m ( E 1%, 1 c m 1 5 2 8 ) . I n n e u t r a l aqueous s o l u t i o n , methimazole a b s o r b s u l t r a v i o l e t r a d i a t i o n a t 251.5 nm ( F i g . 2 ) . Hayden _ - ( 5 ) p u b l i s h e d e t a1 a r e p o r t on t h e r e l a t i o n between t h e s p e c t r a and s t r u c t u r e of methimazole and some r e l a t e d compounds. The replacement of an oxygen atom a t C2 by s u l f u r c a u s e s a b i g s h i f t t o l o n g e r wavelength w i t h i n c r e a s e d a b s o r p t i o n ( 6 ) .

3 54

HASSAN Y . ABOUL-ENEIN AND A. A. AL-BADR

Fig. 1 -

Methimazole

- KI/I2

crystals.

Fig. 2

- Ultraviolet spectrum of Methimazole in methanol.

METHIMAZOLE

355

2.52

I n f r a r e d spectrum The i n f r a r e d spectrum of methimazole i s shown i n Fig. 3. The spectrum w a s o b t a i n e d from Nuj 0 m u l l . 1 The s t r u c t u r a l assignments have been c o r r e l a t ed w i t h t h e f o l l o w i n g band f r e q u e n c i e s : Frequency (Cm-l) 2500 1580 Assignment

- 2450 broad weak

SH

C = N aromatic

Other f i n g e r p r i n t bands c h a r a c t e r i s t i c t o methimazole are a t 1466, 1570 and 1271 Cm-l a s shown i n F i g u r e 4 . Further information with regards t o the inf r a r e d s p e c t r a of methimazole i s g i v e n i n several r e f e r e n c e s ( 4 , 5 , 7 ) . 2.53 Nuclear Magnetic Resonance Spectrum A t y p i c a l N M R spectrum of methimazole i s shown i n F i g . 5. The sample w a s d i s s o l v e d i n CDC13. The spectrum w a s determined on ir Varian T-60 A NMR s p e c t r o m e t e r w i t h TMS a s t h e i n t e r n a l s t a n d a r d . The f o l l o w i n g s t r u c t u r a l a s s i g n ments have been made f o r Fig. 5. Chemical S h i f t ( 6 ) S i n g l e t a t 3.63 S i n g l e t a t 6.70 Assignment 3 a r o m a t i c H and 4 H5 of t h e imidaz o l e r i n g system.
N-CH

Further information concerning t h e NMR spectrum of methimazole c a n be o b t a i n e d from S a d t l e r NMR c a t a l o g (8) and a l s o from CRC A t l a s of s p e c t r a l d a t a (7) and A l d r i c h NMR catalog (9). 2.54
Mass Spectrum and Fragmentometry The mass spectrumof rnethimazole o b t a i n e d by e l e c t r o n impact i o n i z a t i o n shows a pronounced m o l e c u l a r i o n d a t m / e 114 ( F i g . 6 ) .

Bowie e t a 1 (10) s t u d i e d t h e mass s p e c t r a of s e v e r a l i m i d a z o l e s and methimazole w a s i n c l u d e d . They d i s c u s s e d t h e f r a g m e n t a t i o n

a
w C
al k cd k

P
356

357

z
2
I

358

359

360

HASSAN Y . ABOUL-ENEIN AND A. A. AL-BADR

patterns and modes which is substantiated by deuterium labelling, exact mass measurements and appropriate metastable ions. Skeletal rearrangements are rare in the imidazole ring system. 3. Synthesis The method of preparation of methimazole illustrates the general synthesis of 2-mercaptoimidazoles by ring closure from a -amino-aldehydes or ketones and alkylisothiocyanates. This reaction gives 1-alkyl 2-imidazolethiols, and the simpler compounds unsubstituted in the 1-position can be made by the use of simple metallic thiocyanate in place of the alkyl compound. In the original synthesis of Wohl & Marckwald (Ber., 22, 1354, 1889) of which a number of variations have since been introduced. Methimazole is made by condensing methyl isothiocyanate with amino-acetal, NH2-CH2CH(0 Et)2 (a convenient substitute for aminoacetaldehyade) and cyclizing the product by heating with acid. The 2-mercaptoimidazols can be alkyl-

"H-CH

ated or acylated on the sulphur or the second nitrogen atom or the whole SH group can be removed by oxidation with HNO to give a simple imidazole. 3 Methimazole is also prepared (12,13) by the reaction of CH -NHCH2-CH(0-Et) with KSCN in the presence of dil. HC1. Hydrochloric acid t300 ml., 2N) was added gradually to a mixture of 72.5 gm of CH -NHCH2-CH(0 Et) and 56.4 gm of KSCN, the mixture kept 13 hours, evaporaged to dryness, the residue refluxed 1 hour with 200 ml. of anhydrous acetone. The mixture filtered, the precipitate washed with 50 ml acetone and the solution evaporated to give 63-8% of methimazole m.p. 147-8O Yield (75-80%). CH3-NH-CH2-CH(0CH CH ) + KSCN dil HC1 2 3 2

4. Stability, Decomposition product and metal complexes

Methimazole is a relatively stable compound at room temperature. However, it is recommended that it should

METHIMAZOLE

36 I

be kept i n a well-closed andinadry place.

container

p r o t e c t e d from l i g h t

Methimazvje forms metal complexes w i t h heavy metals e . g . C U + ~ , A 1 , Fe+3 i o n s . Foye and Lo (14) s t u d i e d t h e c h e l a t i n g p r o p e r t i e s of some i m i d a z o l e s and o t h e r r e l a t e d h e t e r o c y c l i c t h i o n e s , and r e p o r t e d a r e l a t i o n between t h e metal b i n d i n g s t r e n g t h s and t h e i r a n t i m i c r o b i a l a c t i v i t y . 5. Metabolism When 20 mg/kg of methimazole w a s a d m i n i s t e r e d i . p . o r o r a l l y t o r a t s , u r i n a r y methimazole g l u c u r o n i d e s a c c o u n t e d f o r 36-48% of t h e d o s e i n 24 h o u r s . The o n l y o t h e r u r i n a r y m e t a b o l i t e accounted f o r 10-20% and w a s n o t c h a r a c t e r i z e d . An a d d i t i o n a l 14-20% of methimazole w a s e x c r e t e d unchanged i n 24 hour u r i n e . The b i l e c o n t a i n e d methimazole g l u c u r o n i d e and two u n i d e n t i f i e d m e t a b o l i t e s . One of which w a s t h e same a s t h e u n i d e n t i f i e d u r i n a r y m e t a b o l i t e s . Plasma p r o t e i n s bound 5% of methimazole which had no a f f i n i t y f o r a n y s p e c i f i c t i s s u e . Methimazole had a much g r e a t e r CHC13/H20 p a r t i t i o n c o e f f i c i e n t and H 2 0 s o l u b i l i t y t h a n d i d p r o p y l t h i o u r a c i l . Between 7 7 and 95% of t h e m e t h i mazole w a s e x c r e t e d i n t h e u r i n e and a p p r o x i m a t e l y 10% i n the bile. Since f e c a l excretion w a s neglegible; an e n t e r o h e p a t i c c i r c u l a t i o n w a s p r e s e n t . The h a l f l i f e of u r i n a r y e x c r e t i o n w a s 5-7 h o u r s r e g a r d l e s s of t h e r o u t e o f administration (15).

35S - l a b e l l e d methimazole g i v e n i . p . t o r a t s accumulated i n t h e t h y r o i d g l a n d where i t w a s m a i n l y o x i d i z e d t o methimazole s u l p h a t e ( 1 6 ) . Most of t h e l a b e l w a s e x c r e t e d i n t h e u r i n e . The same r e s u l t s were found i n man t o o ( 1 7 ) . P i t t m a n e t a1 (18) showed t h a t b o t h t h e t h y r o i d and a d r e n a l g l a n d s had t h e h i g h e s t o r g a n t o plasma r a t i o s of t h e d r u g a f t e r f o u r d a y s of i . v . a d m i n i s t r a t i o n i n r a t s . I t w a s r e p o r t e d t h a t methimazole i n man, i s more s l o w l y a b s o r b e d and e x c r e t e d t h a n p r o p y l t h i o u r a c i l . The plasma h a l f - l i f e i n h o u r s o f methimazole 35S w a s 2 o r 5 t i m e s t h a t of l a b e l l e d p r o p y l t h i o u r a c i l . The blood r a d i o a c t i v i t c u r v e a f t e r t h e o r a l a d m i n i s t r a t i o n of c a r b i m a z o l e - 3 5 S was v e r y s i m i l a r t o t h a t of methimazole. I t w a s s u g g e s t e d by Alexander - - t h a t t h e r e n a l f u n c t i o n may have a more et a1 i m p o r t a n t i n f l u e n c e on t h e b i o l o g i c a l h a l f - l i f e of t h e d r u g t h a n t h e t h y r o i d s t a t u s ( 1 9 ) . However, t h e i d e n t i f i c a t i o n of methimazole m e t a b o l i t e s s t i l l need more i n v e s t i g a t ion.

362

HASSAN Y . ABOUL-ENEIN AND A. A. AL-BADR

6, Methods of analysis

6.1. Titrimetric methods

Aqueous Several titremetric methods were developed for the analysis of methimazole Silver nitrate method (1) for the standard methimazole Dissolve about 250 mg of methimazole, accurately weighed, in 75 ml of water. Add from a buret 15 ml of 0 1 N NaOH, mix and add with . agitation, about 30 ml of 0.1 N AgN03. Add 1 ml of bromothymol blue TS, and continue the titration with the 0 1 N NaOH until a perma. nent, blue green color is produced. Each ml. of 0.1N NaOH is equivalent to 11.42 mg of C4H6N2S. For methimazole tablets Weigh and finely powder not less than 20 methimazole tablets. Weigh accurately a portion of the powder equivalent to about 120 mg of methimazole and place in 100 ml volumetric flask. Add about 80 ml of water, insert the stopper and shake by mechanical means or occasionally by hand during 30 minutes, dilute with water to volume and mix. Filter and transfer 50.0 ml of the filtrate to a 125 ml conical flask. Add from a burett3.5 ml of 0.1N NaOH, mix, and add with agitation about 7 ml of 0.1N AgN03. Add 1 ml of bromothymol blue T.S. and continue the titration with 0.1N NaOH until a permanent, blue green color is produced. Each ml of 0.1N NaOH is equivalent to 11.42 mg of C H N S. 4 6 2 Iodometric methods Kossakowski _ _ (20) developed the followet a1 ing method for determination of methimazole (thiamazole) in substances and tablets. solution containing 10-60 ug thiamazole was buffered at pH 5.6, treated with NaN3 and then with 0.02 M iodine solution, left 10 minutes and titrated for unchanged iodine with 0.02 M Na3As03. The method was useful in determining methimazole in compound drugs provided they
A

METHIMAZOLE

363

c o n t a i n no s-'

ions.

b) By t h e method Blazek e t a1 (21) The d e t e r m i n a t i o n i s done by o x i d a t i o n of t h e -SH group w i t h 0.1N i o d i n e s o l u t i o n i n NaHC03 solution v i s u a l l y with respect t o strength o r potentiometrically ( t h e equivalence point i s d i f f i c u l t t o r e c o g n i z e ) o r i n NaOH s o l u t i o n p o t e n t i o m e t r i c a l l y , which i s more f a v o r a b l e . The e r r o r of t h e method f o r a weight of lOmg amounts is a p p r o x i m a t e l y 0.5%. iii) I o d i n e complex method ( 2 2 ) . The p o t e n t i a l c o l o r i m e t r i c u s e of t h e i o d i n e complexes of methimazole i n CHC13 and CCl4 i s s t u d i e d . The a b s o r p t i o n maximum of methimaz o l e - i o d i n e complex a t 269 n could b e used m analytically. Bromometry method The method was d e s c r i b e d by Varga e t a1 ( 2 3 ) . To a 2-5 m sample i n a q u e o u s s o l u t i o n , 10 m l g of w a t e r , 10 m l of 0.1N KBr03, and e x a c t l y 0.5 gm. K B r a r e a c i d i f i e d w i t h 1 . 0 m l 50% H2S04. A f t e r 15 min. 5 ml 20% K I i s added, w i t h s t a r c h i n d i c a t o r and t h e i o d i n e t i t r a t e d w i t h 0.1N NazS203. 1 m l 0.1 KBr03 = 0.0009517 methimazole. The bromometric d e t e r m i n a t i o n of methimazole and o t h e r s i m i l a r compounds i s markedly i n f l u e n c e d by t h e p r e s e n c e of e x c e s s bromide. I n g e n e r a l , t h e g r e a t e r t h e amount of bromide p r e s e n t t h e lower t h e p e r c e n t a g e of t h e compound found. The d e v i a t i o n approa c h e s a l i m i t which i s d i f f e r e n t f o r each compound (24)

iv)

v)

Cerimetry method The method w a s developed by Varga e t a 1 (23) 0.03 - 0.10 g sample i n 5 m l water i s cooled m t o Oo and t i t r a t e d w i t h 0.1N C e (SO!) ,using t 2 p-ethoxychrysoiodine o r ( n o t r e v e r s i b l e ) methyl orange, methyl r e d , o r thymol b l u e a s indicator. 1 m l 0.1N C e ( S 0 4 ) ~= 0.011417 g m methimazole. The e r r o r i s l e s s t h a n + 1% V e h i c l e s of t a b l e t s do n o t i n t e r f e r e .

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HASSAN Y . ABOUL-ENEIN AND A. A. AL-BADR

6.12. Non-aqueous t r t r a t i o n A non-aqueous t i t r a t i o n method w a s developed (25,26) f o r t h e d e t e r m i n a t i o n of b a s i c compounds w i t h thio(-S-) and mercapto (-SH-) groups. The r e a c t i o n of t h e S group w i t h H (OAc)2 i n a c e t i c a c i d makes t h i s p o s s i b l e . g Methimazole and o t h e r compounds were a l l t i t rated with HC104 i n a c e t i c acid using gentian v i o l e t as indicator. 6.2 S p e c t r o p h o t m e t r i c methods 6.21 I n f r a r e d s p e c t r o p h o t o m e t r i c method A method recommended f o r a d o p t i o n a s o f f i c i a l , f i r s t a c t i o n (27). The method i n which methimazole i s s e p a r a t e d from t a b l e t e x c i p i e n t s by column chromatography on C e l i t e 545 w i t h chloroform as t h e e l u e n t and t h e n q u a n t i t a t i v e l y measured and i d e n t i f i e d by I R spectrophotometry. T h i s method w a s s t u d i e d c o l l a b o r a t i v e l y by 10 a n a l y s t s ; a v e r a g e r e c o v e r i e s from two s i m u l a t e d and two t a b l e t s m i x t u r e s ranged from 96.6% -t 1 . 0 t o 101.1% - 0.9 ( 2 8 ) .

6.22

Nuclear magnetic resonance s p e c t r o m e t r i c A NMR procedure i s d e s c r i b e d by which methin mazole i s determined i n p u r e and t a b l e t f o r m u l a t i o n . The method i s r a p i d , a c c u r a t e , p r e c i s e (S.d. 0.95%) and a l s o p r o v i d e s a s p e c i f i c i d e n t i f i c a t i o n of methimazole. The spectrum was r u n i n 10% methylene c h l o r i d e i n carbon t e t r a c h l o r i d e w i t h u s e of b e n z o i c a c i d a s a n i n t e r n a l s t a n d a r d , u s i n g t h e N-CH3 p r o t o n s of methimazole a t 3.566 and t h e a r o matic p r o t o n s of benzoic a c i d a t 7 . 5 and 8.136 as c r i t e r i a f o r a n a l y s i s ( 2 9 ) .

6.23

U l t r a v i o l e t S p e c t r o p h o t o m e t r i c method I n t h i s method, a b s o r p t i o n s p e c t r a have been determined f o r methimazole (mercazolyl) i n aqeuous a c i d and a l k a l i n e s o l u t i o n s . Aqeuous s o l u t i o n s of methimazole have an a b s o r p t i o n band i n t h e medium have r e g i o n w i t h a max a t 250 nm. I n s t r o n g a c i d and a l k a l i n e media hypsochromic and hypochromiceffects a r e o b s e r ved ( 3 0 ) .

METHIMAZOLE

365

6.3

Chromatographic A n a l y s i s

6.31

Thin Layer Chromatography Methimazole w a s determined i n chloroform e x t r a c t s of r a t u r i n e by t h i n l a y e r chromatography on s i l i c a g e l G. ( c o n t a i n i n g 1% Zn S i l i c a t e phosphor) p l a t e s w i t h CHC13/CH30H/H20 (32 :8 :5) as d e v e l o p i n g s o l v e n t and 2,6-dichloroquinone c h l o r i m i d e a s d e t e c t i o n r e a g e n t by d e n s i t o m e t r i c scanning ( 3 1 ) . T h i s method i s s u i t a b l e f o r routine a n a l y s i s ( b e t t e r than GLC because of t h e i n s t a b i l i t y of t h e S . methylmethimazole d e r i v a t i v e ) .
Gas Liquid Chromatography T h i s method i s used f o r S-methyl methimazole on a 10% Apiezon L 5% KOH/Chromosob W column a t looo w i t h N c a r r i e r g a s . The method i s more s e n s i t i v e and more p r e c i s e t h a n TLC method, b u t d u p l i c a t e measurements must b e made on t h e same day due t o i n s t a b i l i t y of Smethylmethimazole (32). C l a r k e ( 4 ) r e p o r t e d r e t e n t i o n t i m e of 0.43 r e l a t i v e t o diphenhydramine under c o n d i t i o n of methimazole d e s c r i b e d i n t h e monograph.

6.32

6.33

Paper Chromatography C l a r k e (4) d e s c r i b e d s e v e r a l s o l v e n t s systems used f o r paper chromatography of methimazole a s shown i n T a b l e 1. Table 1.

S o l v e n t System
Acetate B u f f e r (pH 4.58)

V i s u a l i s i n g Agent ultraviolet i o d o p l a t i n a t e spray (white)

Rf 0.27

Ref. 33

Phosphate B u f f e r (PH 7.4)

ultraviolet 0.00 i o d o p l a t i n a t e spray (white) ultraviolet iodoplatinate 0.72

34,35

C i t r i c acid: H20: n-butanol ( 4 . 8 g : 130 m l : 870 m l ) .

36,37

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HASSAN Y. ABOUL-ENEIN AND A. A . AL-BADR

6.4

Colorimetric A n a l y s i s I n t h i s method t h e r e a c t i o n of d i p h e n y l p i c r y l h y d r a aim w i t h methimazole ( t h i a m a z o l e ) w a s s u f f i c i e n t l y rapid f o r its use i n a spectrophotometric assay. -5 When t h e c o n c e n t r a t i o n of t h e r e a g e n t w a s 4 x 1 0 M and t h e molar r a t i o of methimazole t o t h e r e a g e n t w a s 2 : 1 0 , d e c o l o r a t i o n stopped a f t e r 90 m i n u t e s a t 26'. The r e a c t i o n m i x t u r e t h e n c o n t a i n e d b i s (l-methylimidazol-2-yl) disulphide, t h e reagent, diphenylpicrylhydrazine. A f t e r s e v e r a l h o u r s t h e decoloration s t a r t e d again giving rise t o a multip l i c i t y of c o l o r e d compounds some of which were a l s o formed i n a m e t h a n o l i c s o l u t i o n of t h e r e a g e n t . R e a c t i o n mechanisms were proposed f o r equimolar amounts of t h e r e a c t a n t s as w e l l as f o r t h e r e a c t i o n of methimazole w i t h e x c e s s of t h e r e a g e n t and f o r t h e r e a c t i o n of t h e r e a g e n t w i t h t h e d i s u l p h i d e of methimazole (38). Szabo e t a1 (39) d e s c r i b e d method f o r i d e n t i f i c a t i o n and d e t e r m i n a t i o n of methimazole by c o n v e r s i o n i n t o i t s coloured 1 : l C U + ~ complex which h a s an a b s o r p t i o n max. a t 614 nm. The complex i s n o t s t a b l e , b u t on t r e a t m e n t w i t h H C 1 , a 2 : l methimazole CuCl complex i s p r e c i p i t a t e d a s white c r y s t a l s .

6.5

P o t e n t i m e t r i c Analysis Methimazole w a s a n a l y s e d i n p h a r m a c e u t i c a l p r e p a r a t i o n s p o t e n t i m e t r i c a l l y u s i n g 0.1N chloramine. The method can d e t e c t amounts of up t o 5 m of t h e g drug w i t h an a c c u r a c y of 98-99.5% ( 3 9 ) .

_ _ e t a1

A a l t e r n a t i v e method w a s d r s c r i b e d by P i n z a u t i n ( 4 0 ) f o r d e t e r m i n a t i o n of s e v e r a l a n t i t h y r o i d d r u g s p o t e n t i m e t r i c a l l y w i t h 0.01 M m e r c u r i c acet a t e w i t h u s e of a m e r c u r i c s u l f a t e r e f e r e n c e e l e c t r o d e and an amalgamated gold o r a s i l v e r i n d i cator-electrode. The method i s r a p i d and t h e res u l t s a r e reproducible; t h e e r r o r s are a l l within + 0.36%. -

6.6

Coulometric d e t e r m i n a t i o n C e r t a i n t h i o l s , i n c l u d i n g methimazole, were d e t e r mined c o u l o m e t r i c a l l by d i r e c t t i t r a t i o n w i t h e l e c t r o - g e n e r a t e d HgT2 ( 4 2 ) . The c o u l o m e t r i c a s s a y i s c a r r i e d o u t i n a c e l l having t h r e e compartments s e p a r a t e d from each o t h e r by s i n t e r e d - g l a s s d i s c s . One compartment c o n t a i n s t h e mercury f o r t h e e l e c t r o -

METHIMAZOLE

367

g e n e r a t i o n of Hg+2, t h e mercury i n d i c a t o r e l e c t r o d e and t h e S . C . E . ( e n c l o s e d i n a P e r l e y t u b e c o n t a i n i n g 4 M-sodium n i t r a t e t o a v o i d i n t e r f e r e n c e w i t h C S ) ; t h e second i s f i l l e d w i t h s u p p o r t i n g e l e c t r o l y t e (an aqueous s o h . 0.05 M i n Na2B407 and 0.5M H KNO3 and of p 9 . 3 ) ; and t h e t h i r d a c t s as a n a u x i l l i a r y compartment. The c o u n t e r - e l e c t r o d e i s of p o l i s h e d p l a t inium

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I n c . , Rahaway,

3. C.W.N. Camper, G.D. P i r k e r i n g , J. Chem. SOC., P e r k i n s T r a n s . , 2, 2045 (1972). 4. E . G . C . C l a r k e , " I s o l a t i o n and I d e n t i f i c a t i o n of Drugs", The P h a r m a c e u t i c a l P r e s s , London, 1969, p. 413. 5. A . L . Hayden and M. M a i e n t h a l , J . Assoc. O f f i c e . Agr. Chemists, 48, 596 (1965); t h r o u g h Chem., A b s t r . , 6793 ( 1 9 6 5 c 6. H . Y . Aboul-Enein, unpublished r e s u l t s

63,

7 . "CRC Atlas of s p e c t r a l d a t a and P h y s i c a l C o n s t a n t s of Organic Compounds'' e d i t e d by J . G . G r a s s e l l i , CRC Press, C l e v e l a n d , Ohio, 1973.

8 . S a d t l e r NMR C a t a l o g , S a d t l e r R e s e a r c h L a b o r a t o r i e s , I n c . , P h i l a d e l p h i a , Pa. 1970. 9. A l d r i c h L i b r a r y of NMR S p e c t r a , by C . J . P o u c h e r t and John R. Campbell, Vol. V I I I , s p e c t r u m 30A.

10. J . H . Bowie, R.G. Cooks, S . O . Lawesson and G. S c h r o l l , Aust. J . Chem., 20, 1613 (1967).
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J o n e s , E.C. K o r n f e l d , K.C. McLaughlin, and R.C. Anderson, J . h e r . Chem. S O C . , 71, 4000 ( 1 9 4 9 ) .

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12. I . B .

Simon and 1.1. Kovtunovskaya - Levshina, T r . Ukr., I n s t . Eksperim, E n d o r k r i n o l , 18,345 (1961), t h r o u g h Chem. A b s t r . , 50, 7 9 2 1 (1963), a l s o see Chem. A b s t r . , - 3 4 1 6 h (1960). 50, Simon and 1.1. Kovtunovskaya-Levshina, T i o l o y e Soedinen. Med., Ukrain, Nauch, - I s s l e d o v a t e . S a n i t Khim I n s t . , Trudy Nauch Konf., 1957, 406 (Pub. 1959), through Chem. A b s t r . - 24760 (1960). 54,

13. I . B .

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Foye, J. Lo, J . Pharm. S c i . ,

61,

1209 (1972).

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W.D.

16. B .

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M a r c h a n t , W . D . A l e x a n d e r , J. C l i n . E n d o c r i n o l . Metab., 34, 847 (1972). P i t t m a n , R . J . Beschi, T . C . Smitherman, J. C l i n . E n d r o c r i n o l . Metab. - 182 (1971). 33, Alexander, V. Evans, A. MacAulay, T.F. G a l l a g h e r , Jr. and J. Londons,Brit. Med. J . , 290 (1969).

18. J . A .

19. W.D.

2,

Act. 20. J . Kossakowski; J. Klopocki; T. Kuryl; B. Zbikowski, P o l . Pharm. - 465 (1972). 29,
21. J. Blazek; J . Kracemar and Z . S t e j s k a l ; Ceskoslov. f a r m . , - 4 4 1 (1957); through _ _ _ _t_ 6, Chem. A b s r . 54, 9204 (1960). 22. E . Z o e l l n e r , G . Vastagh, Acta. Pharm. Hung., through Chem. A b s t r . 73, 69923c (1970).

5, 29,

(1970)

23. E. Varga and E. Zb'llner, - - Hung. 25, 1 5 0 (1955), Acta. Pharm. through Chem. Abstr. 52, 12327 (1958).

Acta. Chem. Acad. S c i . 24. E. Z o e l l n e r and E. Varga, - - - -Hung., 12, Chem. A b s t r 52, - 1 (1957), through - - . , - 520SF (1958).
25. G. Posgay, Pharm. Z e n t r a l h a l l e 100, 65 (1961); Chem. A b s t r . 55, 14819 a (1961).

through

26. I . Bayer and G. Posgay, Acta. Pharm. Hung. 2, Suppl. 43 (1961), through Chem. A b s t r . 56, 7 4 3 1 (1962).

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2 7 . A.L. Hyyden and L. Brannon, J. A s s . O f f i c . Anal. Chem.,

- 674 ( 1 9 6 7 ) . 50,

28. O f f i c i a l methods of A n a l y s i s of t h e A s s o c i a t i o n of O f f i c i a l A n a l y t i c a l Chemist., p u b l i s h e d by OAAC, Washington DC 2 0 t h Ed., William Horwitz ( e d i t o r ) 1 1 t h ed. (1970) p . 685.
29. H. Y . Aboul-E n e i n , press). 30. T s . I . Shakh; A.E. 31. J . B . S t e n l a k e ; W.D.

2. Pharm.

Pharmacol. 31, 1979, ( i n

Verzina, - Farm. Zh. (Kiev.) - 28(1968) 23,

W i l l i a m s ; G.G.

S k e l l e r n , J . Chromatog.

- 285, (1970) and r e f e r e n c e s were c i t e d T h e r e i n . 53,

32. E. Niomiya; H . U s a m i ; H . Yamada, A. Morikawa, K . Matsuyama, K . K a t o , I r y o 23, 273 (1969) t h r o u g h Chem. A b s t r . , 71, 536124. (1969). 33. H . V .
Street, (1962).

&.
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Pharmacal. T o x i c a l . ,

19,312

and 325

34. H.V. 35. H . V . 36. A . S .

S t r e e t , - F o r e n s i c S c i . SOC. J. Street,

2,

118 (1962).

Pharm. Pharmacol.

Curry and H. P o w e l l , N a t u r e ,

14,56 (1962) 2, ( 1 9 5 4 ) . 1143

37. E . G . C . C l a r k e , Methods of F o r e n s i c S c i e n c e , ed. Frank L u n d g u i s t , New York, I n t e r s c i e n c e P u b l i s h e r s , Vol. 1, p. 31, 1962. 38. H.B. 39. A . E . Berg, A c t a , - S u e c i c a - Pharm.

S,

431 ( 1 9 7 1 ) .

Szabe, G . S t a j e r , and E . V i n k l e r , Pharmazie, (1974).

2, 615

40. S.G. Avakyants, A.M. Murtazaev, DoL1. Akad. Nauk Uzb. S R . , - 35 ( 1 9 6 9 ) ; t h r o u g h -A- 72,103778a ( 1 9 7 0 ) . 26 Chem. bstr., 41. S . P i n z a u t i , V . D a l P i a z and E. L a P o r t a , Farmaco., 3. - 28, 396 ( 1 9 7 3 ) ; t h r o u g h - - . , 26, 1220 Prat. Anal. A b s t r 42. C . A . Mairesse Patriarche,

- Ducarmois,
2.

J . L . Vandenbalck, and G . J . Pharm. B e l g . , 28, 300 ( 1 9 7 3 ) .

370

HASSAN Y . ABOUL-ENEIN AND A. A. AL-BADR

ACKNOWLEDGEMENT

The authors would like to thank Mr. Dennis Charkowski, Department of Pharmacology, University of Iowa, Iowa City, Iowa 5 2 2 4 2 , U.S.A., for determining mass spectrum

of methimazole, Mr. Essam A. Lotfi and Mr. Muhammad Naeem

A. Akhtar, Faculty of Pharmacy, University of Riyad, Riyad

for the ultraviolet determination and typing the manuscript respectively.

Analytical Profiles of Drug Substances, 8

NALIDIXIC ACID
Patricia E . Grubb
I.

2.

3. 4. 5.

6.

7.
8.

9.

Description 1 . I Name, Formula, Molecular Weight 1.2 Appearance, Color, Odor Physical Properties 2. I Spectral Properties 2.1 I Mass Spectrum 2.12 Infrared 2.13 NMR 2.14 Ultraviolet Spectrum 2.15 Fluorescence 2.2 Crystal Properties 2.21 Crystallinity and X-Ray Diffraction 2.22 Solubility 2.23 pKa 2.24 Melting Range 2.25 Differential Scanning Calorimetry Synthesis stability and Degradation Drug Metabolic Products and Pharmacokinetics 5 . I Metabolic Products 5.2 Pharmacokinetics Methods of Analysis 6 . I Elemental Analysis 6 . 2 Nonaqueous Titration 6.3 Spectrophotometric 6 . 4 Colorimetric 6.5 Polarographic 6.6 Chromatographic 6.61 Thin-Layer and Paper Chromatography 6.62 Liquid Chromatography 6.63 Gas Chromatography 6.7 Spectrofluorimetric Identification and Determination in Biological Fluids Identification and Determination in Biological Fluids References

37 I

Copyright @ 1979 by Academic Press. Inc. All rights of reproduction in any form reserved. ISBN 0-12-260808-9

312

PATRICIA E. GRUBB

1.

Description 1.1 Name, Formula, Molecular Fjeight

Nalidixic acid is l-ethyl-1,4-dihydro-7-methyl-4oxo-1,8-naphthyridine-3-carboxylic acid.

Molecular Formula: Molecular Weight: 1.2

12 12 2 3

H N 0

232.24'l)

Appearance, Color, Odor

Nalidixic acid is a white to slightly yellow, odor(2) less crystalline powder. 2. Physical Properties 2.1. Spectral Properties 2.11 Mass Spectrum The low resolution mass spectrum of nalidixic is presented in Figure 1. It was obtained on a Joel JMS OlSC Mass Spectrometer at an ionization potential of 75 eV. The fragmentation pattern is presented in Figure 2. The molecular ion (m/e=232) is present at an intensity of 18%; the loss of CO gives a fragment of mass 188 which is the most agundant ion. 2.12 Infrared The infrared spectrum of nalidixic acid in a
K B r pellet is presented in Figure 3 . The spectrum was obtained on a Perkin-Elmer Infrared Spectro-

photometer Model 21. It agrees with the spectrum presented by Salim and Shupe. ( 2 )

0 0
7

0
Lo

374

PATRICIA E. CRUBB

Figure 2

Fragmentation Pattern

of Nalidixic Acid

376

PATRICIA E. GRUBB

The c a r b o x y l i c a c i d OH b a n d s i n t h e r e g i o n o f 3300-2500 cm-1 a r e weak and b r o a d , i n d i c a t i n g t h a t hydrogen bonding w i t h t h e c a r b o n y l may b e present.(3) The i n t e n s e p e a k a t a b o u t 1 7 1 5 cm-1 i s p r o b a b l y d u e t o t h e C=O s t r e t c h i n g of t h e c a r b o x y l i c a c i d . The peak a t 1620 c m - 1 may b e d u e t o t h e C=O s t r e t c h of t h e c a r b o n y l a t p o s i t i o n 4 o r t h e C=C s t r e t c h of C-2 and C-3, c o n j u g a t e d w i t h t h e c a r b o n y l , o r a c o m b i n a t i o n of t h e s e two vibrations. 2.13

NMR

The NMR s p e c t r u m of n a l i d i x i c a c i d i s p r e s e n t e d i n F i g u r e 4. I t w a s o b t a i n e d on a V a r i a n A60 s p e c t r o m e t e r . T h i s s p e c t r u m i s i n agreement w i t h t h e s e c t r u m p r e s e n t e d by Hamilton and cow o r k e r s . ( l ) The f o l l o w i n g a s s i g n m e n t s h a v e b e e n made.

ppm
1.75 2.98 5.12 7.85 8.85 9.47

I protons /
3(triplet) 3(singlet) 2(quartet) l(doub1et)
1( d o u b l e t )

description
-CH CH

2 -3 -CH ( a r o m a t i c ) -3 N-CH -CH3 -2 C(6)H

C(5)H C(2)H

l(sing1et)

2.14 U l t r a v i o l e t Spectrum F i g u r e 5 shows t h e u l t r a v i o l e t s p e c t r a of n a l i d i x i c a c i d a t a b o u t 7 . 5 mcg/ml i n 0 . 1 N NaOH, m e t h a n o l , and c h l o r o f o r m , o b t a i n e d on a P e r k i n E l m e r 323 r e c o r d i n g s p e c t r o p h o t o m e t e r . The i n t e n s i t y , p o s i t i o n , and f i n e s t r u c t u r e p r e s e n t i n each spectrum i s r e l a t e d t o t h e s o l v e n t p o l a r i t y . These s p e c t r a are i n a g r e e m e n t w i t h t h e s p e c t r a p u b l i s h e d by S a l i m and Shupe, (2) Zubenko and Shcherba a l s o r e p o r t t h a t t h e r e a r e two bands i n methanol and 0 . 1 N NaOH, a t 258 n and 324 o r m 332 nm, r e s p e c t i v e l y T ( 5 ) G a f a r i ( 6 ) h a s r e p o r t e d t h r e e bands i n m e t h a n o l : 213-216 nm ( 1 L a > , 255 nm (1Lb) and 320-322 nm ( m ) . G a f a r i a l s o r e p o r t s a n a b s o r b a n c e i n 0 . 1 N NaOH a t 279-281 nm and 325 nm. These w a v e l e n g t h s a r e a b o u t 3-10 nm l o w e r t h a n t h o s e r e p o r t e d by o t h e r s o u r c e s . (2) ( 5 ) ( 7 )

0-0

0
c

--n

--(?

: :

0 w

--h

-0

NALIDIXIC ACID

379

2.15 Fluorescence Nalidixic acid exhibits strong fluorescence in acidic solutions. McChesney and co-workers used 0.1 N H SO ( 8 ) with an excitation wavelength - 2 4 o f 330 nm, measuring emission at 375 nm. Browning(9) used 21.5 H SO with excitation at 4 325 nm and emission at 406 nm. Figure 6 shows the fluorescence excitation and emission spectra of nalidixic acid in 0.5 N H SO determined on an 2 4 Aminco Bowman Spectrophotofluorimeter

2.2

Crystal Properties 2.21 Crystallinity and X-ray Diffraction Prismatic crystals of nalidixic acid elongated along the c axis were grown in ethanollwater solution by Achari and Neidle.(lO) The crystals were found to be monoclinic and of the space group P21/C. The x-ray diffraction pattern of the single crystal was solved for the structure of the molecule. The bond lengths and angles of all nonhydrogen atoms were determined. The values found compare favorably with those reported for 1,8naphthyridine and 3-ethoxycarbonyl-4-oxo-6-methyl homopyrimidazole. Nalidixic acid is calculated to be slightly non-planar; each ring is planar but the ring fusion has induced a slight buckling of the ten-membered ring. The above-mentioned compounds also have similar buckling. An intramolecular hydrogen bond is found between the carbonyl oxygen and the hydrogen of the carboxylic acid. The parameters of the monoclinic crystal are a

a b =13.133 a
=

8.913 9.31

2
2

=99.75

The measured density is 1.41, the calculated density is 1.425 g/cm3 for Z=4. The refractive indices of crystals of nalidixic acid have been reported as a=1.510, 8=1.800, and 6=1.880. (4)(11)

380

PATRICIA E. GRUBB

Figure 6

FLUORESC ENCE SPECT RUM

of Nalidixic Acid

Excitation maximum 330 nm Emission maximum 365 nm (uncorr e ded spectrum)

200

25 0

300

350

400

450

500

nm

NALlDlXlC ACID

38 I

2.22 Solubility The solubilities of nalidixic acid in various solvents at 23" are listed below. Solvent chloroform toluene ethyl acetate methanol ethanol isopropanol water (distilled) ethyl ether Solubility (mg/ml)
35

1.6
.8

1.3

.9 .4
.I

.1

The partition coefficients between water and various organic solvents have been reported by Sulkowska and Staroscik. ( 1 2 ) The pH-solubility profile has been studied by these workers and also by Takasugi and coworkers. (13) 2.23 pKa The pKa of the protonation of the nitrogen in position 8 has been reported as 6.02 and the pKa for the carboxylate anion formulation has been reported as -0.94. These were determined by Staroscik and Sulkowska by a spectrophotometric method.(l4) Further study by the same workers on the partition equilibria of nalidixic acid between water and various organic solvents led to calculations of the pKa values of 5.99 + 0.03 for Nprotonation and -0.86 + 0.07 for-carboxylate anion formation. (12) Takasugi and co-workers reported the apparent pKa of nalidixic acid to be 5.9 at 28" by a spectrophotometric method. ( 1 3 ) 2.24 Melting Range The melting range of nalidixic acid is reported to be 225-231", determined as a class 1 compound. (1) (2)

382

PATRICIA E. GRUBB

2.25 Differential Scanning Calorimetry

A DSC scan for nalidixic acid was performed The instrument used was a by Houghtaling.(g) Perkin-Elmer DSC-1B at a scan rate of l"/min. The curve is presented in Figure 7. A sharp peak at 229-230.5" (corr) represents the sample melting.

3.

Synthesis

The synthesis of nalidixic acid was reported by Lesher and Gruett. (15) (16) It may be prepared by the procedure shown in Figure 8.
4.

Stability and Degradation

Nalidixic acid is stable up to five years under reasonable conditions of temperature and humidity. Pawelczyk and Plotkowiakowa(l7) subjected sodium nalidixate solutions to accelerated aging, but were unable to identify decomposition products. Detzer and Huber(l8) studied the photolysis and thermolysis of nalidixic acid in the presence of oxygen. Photolysis produced de-carboxylated nalidixic acid, structure A, and a diketone product, structure B y as well as carbon dioxide and ethylamine.

Structure A

Structure B

Thermolysis also produced the decarbonylation product plus a dimer, structure C.

Structure C

NALIDIXIC ACID

383

Figure 7
.-.

-I

1 -

---ti-t
I

--t-- ---I

p / n LI n

T t

+ t.
---

-.---r-

__

. ,

_.

t
-

-- -

t----

t
._

. _.

._ +_

ii
- - r .I ' C

it9

I 1
I

1
i

---?---

4--

n
CI

8
0
v

r
\ I

"p"
I
N

I "

+
I c
I
v)
n

Z *

(/ J \=
I"'

fiZ
Q l

om I
I

NALIDIXIC ACID

385

5.

Drug M e t a b o l i c P r o d u c t s and P h a r m a c o k i n e t i c s 5.1 Metabolic Products

N a l i d i x i c a c i d i s a s y n t h e t i c a n t i b a c t e r i a l compound t h a t i s used i n t h e t r e a t m e n t of u r i n a r y t r a c t i n f e c tions. It i s more a c t i v e a g a i n s t gram-negative t h a n g r a m - p o s i t i v e organisms. ( 4 ) The compound i s r a p i d l y a b s o r b e d as t h e f r e e a c i d ; i t i s e x c r e t e d i n t h e u r i n e by man i n s e v e r a l forms. ( 8 ) A s m a l l amount is e x c r e t e d unchanged, b u t a much l a r g e r f r a c t i o n is c o n j u g a t e d as t h e monoglucuronide, The most i m p o r t a n t m e t a b o l i t e i s

l-ethyl-1,4-dihydro-7(hydroxymethyl)-4-oxo-l,8naphthyridine-3-carboxylic a c i d , r e f e r r e d t o as hydrox y n a l i d i x i c a c i d . T h i s m e t a b o l i t e h a s shown t h e same o r d e r o f m i c r o b i o l o g i c a l a c t i v i t y i n v i t r o as n a l i d i x i c a c i d . The monoglucuronide c o n j u g a t e of 7-hydroxynalid i x i c a c i d h a s a l s o been found, a s w e l l as t h e 3,7No a n t i b a c t e r i a l a c t i v i t y d i c a r b o x y l i c a c i d product. h a s been found f o r t h e g l u c u r o n i d e s o r f o r t h e 3,7d i c a r b o x y l i c a c i d . I n a d d i t i o n , no g l u c u r o n i d e of t h e 3 , 7 - d i c a r b o x y l i c a c i d h a s been r e p o r t e d .
S i m i l a r m e t a b o l i t e s were found produced by man(l9) (20) ( 2 1 ) , monkeys ( 8 ) , dogs ( 8 ) , c h i c k e n s ( 2 2 ) , c a l v e s ( 2 3 ) and m i c r o o r g a n i s m s ( 4 ) . The r a t i o s of t h e s e m e t a b o l i t e s , however, were found t o v a r y w i t h t h e i n d i v i d u a l . The o v e r a l l c o n v e r s i o n of n a l i d i x i c a c i d t o h y d r o x y n a l i d i x i c a c i d had been r e p o r t e d by McChesney(8) t o b e n o r m a l l y a b o u t 32%; b i c a r b o n a t e supp l e m e n t a t i o n i n c r e a s e d t h i s t o a b o u t 40%. B i c a r b o n a t e s u p p l e m e n t a t i o n a l s o i n c r e a s e d t h e amount of t o t a l n a p h t h y r i d i n e e x c r e t e d i n t h e b i o l o g i c a l l y a c t i v e form.

Two s e p a r a t e s t u d i e s have r e p o r t e d on t h e r a t i o s of t h e various metabolic products excreted i n t h e u r i n e by man. I n a d d i t i o n , Portmann and co-workers c a l c u l a t e d t h e o r e t i c a l amounts based on t h e i r r a t e equations. ( 2 4 )

386

PATRICIA E. GRUBB

Compound Nalidixic acid Hydroxynalid ixic acid

Calc.amts. Found (24) Found (21) (mg per lg dose) (mg per lg dose) % 9 105 8 + 3 129 + 8 537 + 49 229 + 32 43 + 6 -

0.5-5 % 2.5-6 %

NA-glucuronide 1-HNA-glucuronide 3,7-dicarboxylic acid

517 221 74

24-80% 11-12% not reported

In glucuronides were not found as metabolic products by Hamilton(4) in studies of various fungi. The major metabolite of the microorganisms was hydroxynalidixic acid. The dicarboxylic acid was also found as a product in a number of microorganisms.

-E. coli. was found to be inhibition of DNA synthesis.(25)

No selective effect on purines or pyrimidines was found and no inhibition of initiator synthesis was demonstrated. In addition the nalidixic acid could be removed from cultures after exposures up to 75 minutes by rinsings and cells would recover from the block. 5.2 Pharmacokinetics

The mechanism of action of nalidixic acid against

The pharmacokinetics of nalidixic and hydroxynalidixic acids have been studied by several different groups. Takasugi et a1 studied in-situ and in-vitro absorption of nalidixic acid from the gastrointestinal tracts of rats as a function of pH. They reported that the absorption of non-ionized nalidixic acid was faster than the ionized form, with the maximum absorption rate constant found when the drug was administered from a pH=3 buffer solution. The absorption in-situ was found to be ten times the rate in-vitro, but this was dependent on several factors. (13) Moore and co-workers(19) described a simplified model for nalidixic acid metabolism by combining the

NALlDIXlC ACID

387

two m i c r o b i o l o g i c a l l y a c t i v e f o r m s , n a l i d i x i c and h y d r o x y n a l i d i x i c a c i d , and t h e two c o n j u g a t e d p r o d u c t s of t h e a c t i v e forms. T h i s model i s shown below i n F i g u r e 9. Figure 9 S i m p l i f i e d Model f o r K i n e t i c pathways of N a l i d i x i c a c i d i n Man

Nalidixic acid

lag time

> A kA,B

A =Nalidixic acid in GI tract B =totalactivedrug in body CB=conjugated drug in body U =total active drug excreted CU= conjugated drug excreted

.I

cu

The r a t e c o n s t a n t s f o r metabolism and e x c r e t i o n were f u r t h e r combined t o a r a t e c o n s t a n t f o r t h e d i s a p p e a r a n c e of t h e d r u g , kd. Four d o s a g e forms were s t u d i e d and i t w a s found t h a t k was d c o n s t a n t f o r a l l forms e x c e p t f o r a c o a r s e , s l o w l y d i s s o l v i n g powder. Portmann and co-workers t h e n s t u d i e d t h e k i n e t i c pathways i n man f o r h y d r o x y n a l i d i x i c a c i d , t h e a c t i v e p r i m a r y m e t a b o l i t e . ( 2 6 ) The r a t e constants f o r glucuronide formation, oxidation t o t h e d i c a r b o x y l i c a c i d and e x c r e t i o n of hydroxyn a l i d i x i c a c i d were c a l c u l a t e d . E s s e n t i a l l y t o t a l a b s o r p t i o n of h y d r o x y n a l i d i x i c a c i d w a s found i n e v e r y c a s e . Good agreement between e x p e r i m e n t a l and t h e o r e t i c a l plasma l e v e l s , based on t h e f i r s t o r d e r r a t e a p p r o x i m a t i o n s used f o r t h e model, w a s found. Again, t h e d i s a p p e a r a n c e r a t e c o n s t a n t , kd2, w a s found t o b e v e r y s i m i l a r f o r e a c h s u b j e c t , a l t h o u g h t h e i n d i v i d u a l e x c r e t i o n and m e t a b o l i c The d i s a p p e a r a n c e r a t e c o n s t a n t s v a r i e d widely. r a t e c o n s t a n t , k d 2 , w a s d e f i n e d as t h e sum of t h e e x c r e t i o n r a t e c o n s t a n t , kE2, and t h e m e t a b o l i c r a t e c o n s t a n t s t o t h e g l u c u r o n i d e and d i c a r b o x y l i c acid, k M and kM4, r e s p e c t i v e l y . 3

388

PATRICIA E. GRUBB

This data was then used in another study by Portmann and co-workers(24) of nalidixic acid metabolism in man in which a more elaborate model was developed and various rate constants were reported (Figure 1 ) This model was based on the 0. oral administration of 1 g of nalidixic acid. Theoretical curves for plasma levels of nalidixic and hydroxynalidixic acid vs. time agreed with experimental values. McChesney and co-workers(27) then studied the effect of repeated oral dosage of nalidixic acid and found some carry-over of nalidixic acid from day to day but reported no important change in the metabolism due to multiple dosings. The half-life of nalidixic acid in plasma in man was found to be between 85 and 100 minutes by McChesney and co-workers(8) and was reported as about 100 minutes by Bruehl and co-workers(27). Another study by McChesney and co-workers on the metabolism of nalidixic acid in the immature calf (23) demonstrated a pattern of metabolism very different than in man. The half-life of nalidixic acid was found to be about 24 hours and a large amount was excreted into the feces. The immature calves were also unable to excrete nalidixic acid into the urine at concentrations greater than found in the plasma and conjugated drug was present at low levels only. Calves seven months old had metabolic patterns much closer to man; the plasma half-life was about 1.5 hours, the concentration of excretion into the urine was at least ten times that in plasma and the extent of conjugation was increased. The inability to metabolize nalidixic acid by the immature calf was considered to be due to incomplete development of its metabolic system. A similar effect was seen in human infants. (29)

6.

Methods of Analysis

6.1 Elemental Analysis

The molecular formula is C H N 0 12 12 2 3'

NALIDIXIC ACID

389

F i g u r e 10

METABOLIC PATHWAYS of Nalidixic Acid in Man

Nalidixic Acid inGI tract

kA Nalidixic Acid in body

Nalidixic Acid
in

urine

lkMl
4
kMz

Nalidixic Acid GIuc ur onide

:*r

Hydroxynalidixic Acid in urine Hydroxynalidixic Acid Glucuronide in body

y l k ~ 3

Hydroxynalidixic Acid in body

Nalidixic Acid Glucuronide in urine Dicarboxylic Acid in body

Hydroxynalidixic Acid Dicarboxylic Acid Glucuronide in urine In urine kMl=4.5 3 xIO- min kA=1.8x10-2 mill- kEl=1.0x min -I kM2= 5.77xIO- min-l kM3= 5xlO-mn- 4.0
k,,=

I.

k, 2=1 3X 10 -3 min-1 .9

1.37~10-~i d m

390

PATRICIA E. GRUBB

Element C H N
6.2

Theory 62.07% 5.21% 12.07%

Found for standards(30) 62.11 5.21 12.00

61.98 5.20 12.32

Non-aqueous Titration

The titration of nalidixic acid in DMF with lithium methoxide has been reported(1) (2) with thymolphthalein as the indicator. It has also been titrated with sodium methoxide in ethylenediamine or DMF:methanol 1:2 with thymol blue indicator.(31) An error for this titration was reported as + 0.7%. A titration with sodium borohydride Followed potentiometrically or with thymol blue indicator has also been reported by Bachrata and co-workers. The standard deviation was reported as + 0.60%. (32) 6.3 Spectrophotometric

The ultraviolet absorption spectrum of nalidixic acid in methanol or chloroform has an absorption maximum at about 258 nm and a broad double peak at 324 to 333 nm. In 0.1 N NaOH the band at 324 nm is shifted to a single peak at about 332 nm. The a of the band at about 258 nm is approximately 110 but varies with the solvent. ( 2 ) (5) (6)(7)

6.4 Colorimetric
Nalidixic acid and sodium nalidixate form a strong colored complex with iron 111. The maximum of absorbance of the complex is 410 nm and Beer's Law is obeyed from 10 to 250 ug of nalidixic acid per m1(33), and from 0.43 to 17.05 mg iron I11 per ml. (34) Nalidixic acid complexes through the 0x0-group on C-4 and the carboxylic acid group on C-3. Three moles o f nalidixic acid complex with one mole of iron 111. The instability constant of the complex was calculated to be 2.11 x 10-8 by Dick and Murgu.(34)

NALlDIXlC ACID

391

6.5 Polarographic The polarographic behavior of nalidixic acid has been studied by Staroscik and co-workers. (35) The pH range of -2.9 to 11 in 20% DMF was investigated in the concentration range of 5 x 1 - 1 1 and 041 three stages of reduction were found. The potentials were found to vary linearly with pH for the first two reduction stages, while the third was constant and appeared at pH >8. The carbonyl on C-4 was shown to be reduced to the 4-hydroxy product. Nalidixic acid was reduced with sodium borohydride and the product was demonstrated to be the same as that in the polarographic reduction by TLC. 6.6 Chromatographic

6.61 Thin-layer and paper chromatography

Several thin-layer and paper chromatographic systems for nalidixic acid are listed below. Chromatographic Systems for Nalidixic Acid Ad sorbent paper Mobile Phase toluene:dioxane(9:1) descending butano1:acetic acid: H 2 0 (4:l:l) propano1:ethanol:water (6:1:3) paper impreg. with 0.1 M Na and HPZ4 silica gel silica gel silica gel silica gel
iso-butano1:ethanol:ethyl acetate:water:acetone (8.2: 1:5 : 2 ) ascending

Rf
.6

Ref. -

8
36 36 17

--

-.73

methano1:water:ammonia (100:12:16)
ch1oroform:methanol:formic

.88

35 37 37 23

acid (90:7:3) ethyl acetate:methanol: isopropylamine (76:20:4) benzene in ethano1:acetic acid (80: 10) 10:

.54

.18
.5

392

PATRICIA E. GRUBB

6.62 Liquid chromatography

A high-performance liquid chromatographic method for nalidixic acid on a strong anionexchange resin column has been reported, using a mobile phase of 0.01 sodium tetraborate at pH 9.2 and 0.003 sodium sulfate. The relative retention time for nalidixic acid in the system reported by Sondach and Koch was 0.86 with sulfanilic acid as the standard at

1 . 0 0 , (38) 6 . 6 3 Gas chromatography

A gas chromatographic procedure for nalidixic acid after esterification has been reported for biological samples. (39)

6.7

Spectrofluorimetric

Nalidixic acid has a strong fluorescence spectrum which has been used for its determination in biological fluids. ( 8 ) ( 9 ) (24) (26) ( 4 0 )

7.

Identification and Determination in Biological Fluids

The first spectrofluorimetric methods reported for the determination of nalidixic acid and its metabolites in biological fluids did not differentiate between nalidixic acid and hydroxynalidixic acid. The determination of free nalidixic acid and the hydroxy-metabolite in human urine plasma and feces was performed by extraction by toluene from acidified biological fh i d and subsequent fluorimetric measurement at 3251375 nm of sample re-extracted into aqueous solution. (8) Conjugated nalidixic and hydroxynalidixic acids were determined by acid hydrolysis and then toluene extraction for fluorimetric measurement of the total drug. The conjugated nalidixic acid was then determined by difference.
A refined method involving extractfon of two aliquots of biological material, buffered at two different p H ' s , by toluene, and re-extraction into aqueous solution allowed the simultaneous determination of nalidixic and hydroxynalidixic acid(24) ( 2 6 ) by their differential extractabilities and fluorescent intensities. This method was extended to the differential determination of the conjugated forms of nalidixic and hydroxynalidixic acid.

NALIDIXIC ACID

393

The 3,7-dicarboxylic acid metabolite was also measured fluorimetrically by extraction with ethyl acetate:chloroform 1:l at pH=l and re-extraction into aqueous solution. The fluorescence was determined at 3501435 nm; there was no cross interference with the nalidixic acid determination. ( 8 ) Extraction of nalidixic acid with chloroform from urine has also been reported. ( 4 0 ) Another fluorimetric method for chicken liver and muscle containing not less than 100 ppb nalidixic acid was reported by Browning(9) using an ethylacetate extraction and alumina column to retain the nalidixic acid. The fluorescence was measured at 3251408 nm. Spectrophotometric measurements of nalidixic acid have been reported. Gafari -et al(41) used an extraction with ultraviolet measurement at 255 or 327 nm for blood and tissue samples. Takasugi and co-workers extracted buffered tissue homogenate with chloroform and measured the optical density at 334 nm.(l3) Several chromatographic procedures have been used for nalidixic acid and metabolites in biological fluids. Urine samples were extracted with chloroform and chr.omatographed on filter paper using the system toluene dioxane (9:l) descending chromatography. The known spots were eluted off the paper with methanol and quantitatively measured spectrophotometrically. ( 8 ) Other chromatographic systems reported include butano1:acetic acid:water (4:l:l) and propanol: ethano1:water (6:1:3) for paper chromatography. The glucuronides were also separated without prior hydrolysis using isopropano1:ethyl acetate:water (6:1:3) or propano1:ethyl acetate:acetic acid:water (5:5:1:3). (36)
A high performance liquid chromatographic method was developed by Shargel and co-workers for the assay of nalidixic and hydroxynalidixic acids in human plasma and urine. (42) Extraction into chloroform and re-extraction into aqueous solution was used as the sample treatment. The column used was a 0.5 M stainless steel (2.1 mm i.d.) packed with Zipax SAX strong anion exchange resin. The column pressure was 600 psi with a flow rate of 0.8 ml/min. A modification of this procedure, using a 1 m column with a column pressure of 900 psi and a flow rate of 0.7 ml/min. and an internal standard of l-ethyl-1,4-dihydro-4-oxo-7-(4-pyridiyl)3-quinolinecarboxylic acid, was used by Goehl and coworkers (43) or improved separation. A separate liquid chromato raphic procedure for the dicarboxylic acid was used by Lee( 4) - - The column used was a 25 cm x 4.6 mm i.d. et al.

3 94

PATRICIA E. GRUBB

Partisil PXS 10125 PAC pre-packed Magnum 9 column with a mobile phase of methano1:pH 3 0.1 citrate buffer 85:15 with a flow rate of 1.6 mlfmin. The retention time of the dicarboxylic acid was 12 minutes. A thin-layer gas chromatographic system was devised by Pittman and Shekosky(39) for chicken tissue and feces. A TLC system of benzene:methanol:acetic acid (9:l:l) was used for prior separation; the spots were then removed and esterified in 14% BF3 in methanol. A 4 ft. 3% OV-17 column at 240" was used. Retention times of about 7 minutes for nalidixic acid, 10 minutes for hydroxynalidixic acid and 17 minutes for the dicarboxylic acid were reported. A pulse polarographic system for detection of the dicarboxylic acid in the presence of nalidixic and h droxynalidixic acids was devised by Koss and Warner. (457 The reduction potential in the system used was -.54V vs. SCE. Microbiological assay procedures for nalidixic acid have also been used for biological samples. Since nalidixic and hydroxynalidixic acids have the same order of antibacterial activity in-vitro, then cannot be determined separately. Organism P. boviseptica Reference
8

B. pumilus
E. coli

46
25,47, 48,49

8.

Identification and Determination in Dosage Forms

Nalidixic acid has been determined spectrophotometrically in tablets after chloroform extraction at a wavelength maximum of 258 nm.(1)(2) Another source reported 259 nm as the maximum for chloroform and 258 nm for 0.1 N NaOH. (7) The infrared spectrum has also been used to identify nalidixic acid in tablets.(7) Non-aqueous titration of tablet extracts with sodium methoxide(27) or lithium methoxide(1) ( 2 ) has been reported. A polarographic determination of nalidixic acid in tablets at -0.6V v s . SCE in 20% aq. DMF with 0.1 - HC1 has also been N used. (35)

NALIDIXIC ACID

3 95

High performance liquid chromatography was used by Sondack and K ~ c h ( ~to ) ~ assay nalidixic acid in aluminum hydroxide gel suspension.

A microbiological detection has been described for use for pharmaceutical preparations of nalidixic acid. A diffusimetric determination using E. coli (Bruxelles) was reported by Monciu. ( 4 8 )

396

PATRICIA E. G R U B B

References

1. National Formulary, =:477. 2. Salim,E.; Shupe,I.; J.Pharm.Sci. (1966) 55 - (11) :1289-90. 3. Parikh,V.M., Absorption Spectroscopy of Organic Molecules, Addison-Wesley Publishing Co., (1974) :244. 4. Hamilton,P.B.: et al; Appl.Microbio1. (1969) - (2) ~237-41. 17 5. Zubenko,V.; Shcherba,I.; Farm.Zh, (1975) - (3):28-33 through CA 83:152437Z. 30 6. Gafari,A.; Farm.Zh., (1977) 1:53-6 through - 86:179821u. CA 7. da Silva,M.; Noqueira,M.; Rev.Port.Farm. (1965) 15 (3):290-4 through CA 64:9513h. 8. McChesnG,E.; et al; Toxicolxnd Appl.Pharmacol (1964) 6:292-309. (1970) 9. J.A.o.A.c. :723-4. 10. Achari,A.; Neidle,S.; Acta Crysta1logr.Sect.B (1976) 32 (2) ~600-2. 11. HoughtaFng,W. ; Sterling-Wintrhop Research Institute; unpublished data. 12. Sulkowska,J.; Staroscik,R; Pharmazie (1975) 30:405-6. 13. Takasugi,N.: et al; Chem.Pharm.Bul1. (1968) 16 - (1) :13-16. 14. Staroscik,R.; Sulkowska,J.; Acta Pol.Pharm. (1971) - (6):601-6 through CA 76:158322k. 28 15. Lesher,G. ; et al; J.Med.PharEChem. (1962) 5~1063-4. 16. Lesher,G.; et al; US Patent //3,149,104. 17. Pawelczyk,E.; Plotkowiakowa,Z.; Acta Pol. Pharm.(1970) 27 (2):105-11 through CA 73:91300u. 18. Detzer,N.; Hucr,B.; Tetrahedron (1975) 31:1937-41 1 :36-41. 19. Moore,W. ; et al; J.Pharm.Sci. (1965) 54 ( ) 20. Pereny1,T. ; et al; Acta Microbiol.AcaxSci. Hung. (1975) 22 (4):433-45 through CA 84385914h. 21. Graber,H.; etTl; Gyogyszereink (19E) 25 (3):97-106 through= 84:173528r. 22. Browning,R.; Sterling-Winthrop Research Institute, unpublished report. 23. McChesney ,E. , et al; Toxicol Appl Pharmacol , (1969) 14 (1)~138-50. 55 24. PortmanKG.; et al; J.Pharm.Sci. (1966) - (1):72-8.

NALIDlXlC ACID

397

25. Goss,W.; et al; J.Bacterio1, (1965) 89:1068-74. 26. Portmann,G.; J.Pharm.Sci. (1966) - (1):59-62. 55 27. McChesney,E.; et al; J.Pharm.Sci. (1967) 56 (5) :594-9. 28. Brueh1,P.; et al; Arzneim.-Forsch (1973) 23 CA (9):1311-13 through - 80:33692g. 29. Rohwedder,H.; et al; Z.Kinderheilk (1970) 109 (2):124-34 through 974:97455r. 30. Auerbach,M., Sterling Wintrhop Research Institute, unpublished data. 31. Ignat,V.; Bera1,H.; Rev.Chim.(Bucharest) (1966) - (1):50 through - 64:17360h. 17 CA 32. Bachrata,M.; et al; Pharmazie (1977) 32 7) : 398-401. 33. Murgu,N.; Pharmazie (1964) 2 (11):724-5 34. Dick,I.; Murgu,N.; Rev.Chim(Bucharest) ( 964) - (12):757-8 through CA 62:15600h. 15 6: 35. Staroscik,R.; et al; Pharmazie (1974) 2 ( ) 387-90. 36. Perenyi,T.; Acta Pharm.Hung. (1975) 45 ( 5 ) : 196-206 through CA 84:27577q. 37. Crain,A.V.R. ; Sterling Winthrop Research Institute, unpublished data. 38. Sondack,D., Koch,W.; J.Chromatog. (1977) 132:322-5. 39. Pittman,K.; Shekosky,J. ; Sterling W i n t h r o p Research Institute; unpublished data. 40. Staroscik,R.; Sulkowska,J.; Acta Pol.Pharm. (1973) 30 (5):459-66 through CA 81:33082d. 41. Gafari,X; et al; Farm.Zh. (1F7) 2:92-3 through CA 87:33391c. 62 9 : 42. Sharge1,c; et al; J.Pharm.Sci. (1973) - ( ) 1452-4. 43. Goeh1,T; et al; Sterling Winthrop Research Institute; unpublished report. 44. Lee,F.; et al; Sterling Winthrop Research Institute; unpublished report. 45. Koss,R.; Warner,C.; Sterling Winthrop Research Institute, unpublished report. 46 Ciuro,C.; Cienc.Ind.Farm. (1973) 5 (2):54-7 through CA 82:152437z. 47. Bauernfezd,A. ; Gruemmer,G. ; Z.Med.Mikrobio1. Immunol., (1968) 154 (1):35-9 through CA 69:33474k. 48. Monciu,D. ; et al; Farmacia(Bucharest) (1972), - (8):459-66 through CA 17:168693s. 20 49. Goss,W.; Deitz,W.; Bacteriol.Proc. (1963):93. The literature was reviewed to June 19, 1978.

Analytical Profiles of D u Substances, 8 rg

NEOMYCIN
William F. Heyes
I. Description 1.1 Composition, Name, Formula, Molecular Weight 1.2 Appearance, Color, Odor, Taste I .3 Definition of International Standard Physical Properties 2.1 Spectra 2. I 1 Infrared Spectrum 2.12 Ultraviolet Spectrum 2.13 NMR Spectrum 2.14 Mass Spcetrum 2.2 Physical Properties of the Solid 2.21 Hygroscopic Nature 2.22 Solubility 2.23 Specific Surface Area 2.2 Physical Properties of Solutions 2.31 Density 2.32 Viscosity 2.33 Surface Tension 2.34 Adsorption by Clays, Soils, and Minerals 2.35 Adsorption by Ion Exchange Resins Salts, Derivatives, and Complexes 3.1 Salts 3.2 Derivatives 3.3 Complexes Synthesis and Production 4.1 Commercial Biosynthesis 4.2 Synthesis of Radio-Labelled Material Stability and Degradation 5.1 Hydrolytic Degradation 5.2 Stability of Bulk Material 5.3 Stability in Aqueous Solution 5.4 Stability in Pharmaceutical Formulations 5 . 5 Compatibility with Excipient Materials 5.6 Compatibility with Other Actives Methods of Analysis 6. I Identification 6.2 Chemical Procedures 6.21 Titrimetry 6.22 Polarography 6.23 Polarimetry 6.24 Radio-Chemical Procedures 6.25 Fluorimetry 6.26 Spectrophotometry 6.3 Chromatographic Procedures 6.3 1 Counter-Current Distinction 6.32 Electrophoresis 6.33 Column Chromatography 399
Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-260808-9

2.

3.

4.
5.

6.

400 6.34 Paper and Thin-Layer Chromatography 6.35 Gas-Liquid Chromatography Microbiological Procedures 6.41 Turbidimetric Assay 6.42 Agar-Diffusion Assay Automated Procedures Use as an Analytical Reagent Determination in Body Fluids and Tissues

WILLIAM F . HEYES

6.4

7 6.5 6.6 6.7

1.

Description.
1.1. C o m p o s i t i o n , N a m e , Formula, M o l e c u l a r Weight

Composition Commercial neomycin i s a complex mixt u r e of a m i n o g l y c o s i d e a n t i b i o t i c s o r i g i n a l l y i s o l a t e d from a c u l t u r e of S t k c p X o m y c e b d k a d i a e by Waksman and h i s c o - w o r k e r s i n 1 9 4 9 . The p r i n c i p l e components of t h e m i x t u r e are neomycin B ( 1 ) and neomycin C ( I 1 ) t o g e t h e r w i t h a s m a l l q u a n t i t y of n e a m i n e l , a d e g r a d a t i o n p r o d u c t of neomycin f o r m e r T a b l e 1 shows t h e c o n t e n t l y known as neomycin A. v a r i a b i l i t y o f neomycin B and C and neamine i n commercial s a m p l e s of neomycin a s r e p o r t e d i n t h e literature. Neomycins LP-A, LP-B and LP-C which c h e m i c a l l y a r e t h e mono N - a c e t y l d e r i v a t i v e s o f neomycins A , B and C2 may a l s o b e p r e s e n t i n s m a l l amounts. S e v e r a l o t h e r minor components h a v e rec e n t l y been i d e n t i f i e d as paromamine, paromomycin I and paromomycin I13. ( A l s o known as neomycins D , E and F r e s p e c t i v e l y ) . Table 1 C o n t e n t V a r i a b i l i t y of Neomycin Source Canada Reference Neomycin B Neomycin C Neamine C o n t e n t % C o n t e n t % C o n t e n t P,
69.2

to 97.5 U.K. 80.5 to 92.0 63.7 to

91.6

30.8 to 4.0 17.5 to 8.0

40.0

N i l

to 2.5
N i l

U.S. S. R.

7,8

to 3.4

NEOMYCIN

40 1

.............

neamine..

. .:
H 2 6N5 q

...........

R l = H

R 2 = CH2NH2; R2 = H
;

neomycin B neomycin C

I1

R1 = CH2NH2

F i g u r e 1.

S t r u c t u r e of neomycin and neamine

402

WILLIAM F. HEYES

Names The following are alternative names for the neomycin complex, all of which have been used by Chemical Abstracts:Colimycin, Colistin(not to be confused with Polymixin) , Dextromycin,Flavomycin,Fradiomycin, Framycetin, Mycerin, Mycifradin, Neomix, Soframycin, Streptothricin BI, BII. 4 Chemical Names Neomycin B
0-2,6-diamin0-2,6-3dideoxy-aD-glucopyranosyl-(1-+4) [0-"2, -06-diamino-2,6-dideoxy-@-L-a idopyranosyl- (1+3)- B-D-ribofuranosyl-s(1+5)] -2-deoxy-Dstreptarnine. 0-2,6-diamino-2,6-3dideox -aD-glucopyranosyl- (1+4)-0-f0-=2, 6-diamino-2,6-dideoxy-a-D-c) glucopyranosyl-(1+3)-@-D-ribofuranosyl-3(1+5)] -2-deoxy-Dstreptamine

Neomycin C

Ne amine ( formerly Neomycin A) Neomycin B Neomycin C Neamine Formula

2-deoxy-4-0- (2,6-Cdiamino 2,6-dideoxy-a-D-glucopyranosyl) -D-streptamine

Chemical Abstracts Registry No. 119-04-0 66-86-4 3947-65-7 C23H46N6013 C12H26N406 614.67 322.36

Neomycin B,C Ne amine Molecular Weight Neomycin B,C Ne amine

NEOMYCIN

403

1.2.

A p p e a r a n c e , C o l o u r , Odour, Taste

The s u l p h a t e s a l t o f neomycin ( t h e u s u a l commercial f o r m ) i s an amorphous, w h i t e o d o u r l e s s , powder which is p r a c t i c a l l y t a s t e l e s s . 1.3. Definition of I n t e r n a t i o n a l Standard

The I n t e r n a t i o n a l R e f e r e n c e S t a n d a r d f o r neomycin9 i s d e f i n e d as c o n t a i n i n g 6 8 0 units o f a c t i v i t y p e r mg o r a l t e r n a t i v e l y 1 u n i t of a c t i v i t y i s c o n t a i n e d i n 0.0014 7mg of s t a n d a r d material. Similarly, the International R e f e r e n c e S t a n d a r d f o r neomycin B i s d e f i n e d a s containing 670 u n i t s per mg, or a l t e r n a t i v e l y 1 u n i t o f a c t i v i t y i s c o n t a i n e d i n 0.001492 m g of s t a n d a r d material.
2.

P h y s i c a l Properties 2.1. Spectra 2 . 1 1 . Infra-Red Spectrum

The s o l i d s t a t e i n f r a r e d s p e c t r a o f neomycins B & C s u l p h a t e h a v e been r e c o r d e d as a d i s p e r s i o n i n p o t a s s i u m bromide and are i l l u s t r a t e d i n F i g . 2 and 3 . The i n f r a r e d s p e c t r u m of neamine s u l p h a t e ( f o r m e r l y c a l l e d neomycin A ) a l s o as a p o t a s s i u m bromide d i s p e r s i o n i s i l l u s t r a t e d i n A l l s p e c t r a are o f a u t h e n t i c m a t e r i a l Fig. 4 . s u p p l i e d by The Upjohn Company, Kalamazoo. Sammul e t a l l o h a v e p u b l i s h e d t h e i n f r a r e d s p e c t r u m o f neomycin u n d e c y l e n a t e .
2.12.

U l t r a v i o l e t Spectrum

N o a b s o r p t i o n maxima i n t h e u l t r a v i o l e t r e g i o n 200-340nm i s o b s e r v e d w i t h s o l u t i o n s o f neomycin.

2.13.

NMR S p e c t r u m

The p r o t o n NMR s p e c t r u m of neomycins B and C h a s been u s e d by R i n e h a r t and c o - w o r k e r s 1 t o e l u c i d a t e t h e s t r u c t u r e and

2.5 0.0

3
1
00

5
I

I ,

WAVELENGTH (MICRONS) 8 6 7. . . . , I I . , . , II ,, ... I J ... . I

I

9
1

10
I . . .

12
'

15

20

03

'

,
.

30 4Ou) an ..

- .

~- ' 1

3 m

2500

2ooo

la00

1600
FREQUENCY

(CM')

1400

1200

lo00

800

wo

400

200

Figure 2.

I n f r a r e d Spectrum of Neomycin B

2.5

W V L N T (MICRONS) AEE GH 7 8 6

10

12

15

20

30 4050

4Ooo

3500

3000

250

2000

1800

1600 1400 F E U N Y (CM) RQ E C

1200

1000

800

600

A00

200

Figure 3.

Infrared Spectrum of Neomycin C

u-l

..

33NVWOSW
31dWVS

N EOMY CIN

407

stereochemical configuration of these a n t i b i o t i c s . T r u i t t has extensively studied t h e 1 3 C NMR o f t h e neomvcins and d e m o n s t r a t e d t h e pH d e p e n d a n c e o f t h e s p e c t r u m o f neomycins B and C . The s p e c t r a o f t h e h e x a N - a c e t y l d e r i v a t i v e s however, w e r e shown t o be i n d e p e n d a n t o f pH t h u s allowing assignment of t h e observed chemical s h i f t s as g i v e n i n T a b l e 2 .
2.14.

Mass Spectrum

Neomycin i s i n s u f f i c i e n t l y v o l a t i l e f o r d i r e c t mass s p e c t r o m e t r i c a n a l y s i s . T o overcome t h i s p r o b l e m I n o u y e 1 4 p r e p a r e d t h e v o l a t i l e N - s a l i c y l i d e n e S c h i f f s b a s e , t h e M.S. o f w h i c h , however, d i d n o t e x h i b i t a peak f o r t h e molecular i o n . T o o b s e r v e t h e m o l e c u l a r i o n i t w a s n e c e s s a r y t o use t h e o - t r i m e t h y l s i l y l e t h e r o f the N-salicylidene S c h i f f s base. The s p e c t r u m o f N - s a l i c y l i d e n e neomycin w a s f o u n d t o b e d e p e n d a n t on t h e ion-chamber t e m p e r a t u r e i n d i c a t i n g t h a t thermal decomposition plays a s i g n i f i c a n t p a r t i n the fragmentation process. The M.S. e x a m i n a t i o n o f neomycin t r i m e t h y l s i l y l e t h e r f o l l o w i n g GLC h a s been rep o r t e d by Murata e t a l 1 5 . The f r a g m e n t a t i o n p a t t e r n o b t a i n e d by t h e s e w o r k e r s i s somewhat d i f f e r e n t t o t h a t o b s e r v e d by I n o u y e 1 4 .
It is interesting t o note t h a t the m o l e c u l a r i o n c o r r e s p o n d i n g t o neamine ( a major p r o d u c t o f neomycin h y d r o l y s i s ) w a s n o t o b s e r v e d i n t h e mass s p e c t r u m o f e i t h e r t h e N - s a l i c y l i d e n e o r t h e T.M.S. d e r i v a t i v e .

I n an a t t e m p t t o a v o i d t h e n e e d f o r d e r i v a t i s a t i o n R i n e h a r t and Cook16 a p p l i e d t h e t e c h n i q u e o f F i e l d D e s o r p t i o n Mass S p e c t r o m e t r y t o neomycin a n d s u c c e s s f u l l y o b s e r v e d an i n t e n s e r e s p o n s e f o r t h e m o l e c u l a r i o n a t m / e 615(M + H ) t o g e t h e r w i t h v e r y s m a l l p e a k s a t m / e 455, 307 and 2 0 6 r e p r e s e n t i n g t h e loss o f s u g a r f r a g m e n t s .

408

WILLIAM F. HEYES

Table 2 ~13C NMR Absorptions of hexa-N-acetyl-neomycin12 Carbon atom (see Fig.1) A1 A2 A3 A4 A5 A6 D1 D2 D3 D4 D5 D6 R1 R2 R3 R4 R5 B2 B3 B4 B5 B6

CH 3
B1

6 ppm (from TMS)

Hex a-N- acetyl neomycin B 97.0 54.2 71.4 71.4 71.4 40.8 50.4 33.2 48.9 76.7 86 .O 74.5 109.3 74.5 77.2 82.4 62.2 98.8 51.8 70.3 68.5 74 .O 40.8 23.0(x 3) 22.9(x 3) 175.2 174.9 174.8 174.7 174.5 174.1

Hexa-N-acetyl neomycin C 96.9 54.1 71.4 71.4 71.4 40.8 50.4 33.1 48.9 76.8 86.0 74.5 110.0 73.5 75.2 81.8 62.9 97.3 54.3 71.7 72.3 71.4 40.6 23.2 22.8(x 5) 175.2 175.1 174.9(x 2) 174.6 174.1

co

NEOMYCIN

409

2.2 .Physical Properties of the Solid

2.21.

Hygroscopic N a t u r e

H i r t z e t a l l 7 demonstrated t h e h y g r o s c o p i c n a t u r e of neomycin s u l p h a t e u s i n g a thermogravimetric technique. I n a more r e c e n t s t u d y Russian w o r k e r s 1 8 determined t h e a d s o r p t i o n o f wate8 by neomycin s u l p h a t e a t t e m p e r a t u r e s of 23O, 9 0 & l O O z C . Table 3 g i v e s t h e r e s u l t s o b t a i n e d a t 23 C .

Tab -l e 3 A d s o r p t i o n o f M o i s t u r e by Neomycin S u l p h ate

0, 0

Humidity
0

Moisture adsorbed % (350 hours storage)

0

10

4.15

15 25 40
60

5.25
6.90

10.20
16.70

80
2.22.

38.90

Solubility

The s o l u b i l i t i e s of e l e v e n s a l t s o f neomycfg ,30,3pme t w e n t y s i x s o l v e n t s h a v e been reported. The v a l u e s f o r t h e most commonly encountered s a l t s a r e l i s t e d i n Table 4. 2.23. S p e c i f i c S u r f a ce Area

Using a method b a s e d on t h e f i l t r a t i o n of a i r t h r q y g h a c o m p r e s s e d l a y e r o f determined t h e v i s i b l e powder E z e r s k i i e t a 1 s p e c i f i c s u r f a c e a r e a o f neomycin s u l p h a t e t o b e 2 . 6 rn2g-l.

rq
d

O V V N
N

d c n W N
W

0
d d

r-cnr-rl
N

~ c n m m ....

c,

-4

c
u4J

h l a
N

w
m O
0 0 0 0
Q)

m
d d
A A A A
A A

0 0
I n

r(

Q ) dO A

0
N

* * *
0 0 0 0
NNNN
A A A A

+I

*
N O O N 0

d m W O m

~ In mn * I mrN

4J

m o

ddddd
N O
d

. . 99 g
0 0

z
N

*
w d m w m
v )

InW

0
*v)

m
0 0

0 0 0 0
+I

C o W O N W

m o o o m

.....

;Id d d

;
d

ia

3
.rl

u
k
d

d d

4
0

0
410

T a b l e 4 (Contd.. ) S o l u b i l i t y of Neomycin S a l t s ( m g / m l , c o r r e c t e d Solvent Petroleum e t h e r Is o o c t a n e E t h y l acetate Isoamyl acetate Acetone Methyl e t h y l ketone Diethyl ether Ethylene chloride Chloroform Carbon tetrachloride 1 , 4 dioxane Carbon d i s u l p h i d e Pyridine Form a m i d e Dimethylsulphoxide Neomycin B hydrochl o r e
0.0
N e omy c i n N e omy c i n

for solvent blank)

N e omy c i n

su l p h a t e
0.005

undecylenate 2.62 0.342 4.70" 7.36* 16.00* 5.795" 7.989 0.77* >20* 7.938 6.485 9.40* >20*
>20

oleate
>20 15.852 6.885 14.080 9.260 6.578

Neomycin stearate 0.188

0.0

0.03 0.03 0.06 0.04 0.058 1.485* 0.005 0.06

0.14"

0.028 0.06 0.15 C. 005

0.07
0.0

1.030 7.014 2.083 1.742 7.328 0.217 9.463 1.046 4.500 3.918 >20 0.2 1 7 3.862

0.075
0.14"

11.288 3.257 >20 >20 13.038 >20 >20 1.365 10.580

0.323* 0.45"

0.923* 0.35" 0.025mg e x c e p t

A l l values

+ -

+ * -

0.05mg.

412

WILLIAM F . HEYES

2.3 .Physical P r o p e r t i e s of S o l u t i o n s 2.31. Density

K i g e l e t a l l 8 have r e p o r t e d t h e d e n s i t y o f 6 & 1 8 % a q u e o u s neomycin s u l p h a t g s o l u t i o n s o v e r t h e t e m p e r a t u r e r a n g e 20 t o 8 0 C . The p u b l i s h e d v a l u e s a r e g i v e n i n T a b l e 5 . T a b-l e 5 D e n s i t i e s o f Neomycin S u l p h a t e S o l u t i o n s

N e omy c i n

Density ooc 1.025 1.086

4 OC
L

Concen t r a t i on
%

6 OC 1.014 1.076

8 OC

6 18

1.021 1.081

1.007
1.068

2.32. V i s c o s i t y T a b l e 6 shows t h e r e p o r t e d 1 8 v i s c o s i t i e s f o r 6 and 1 8 % a q u e o u s n e o m y c i n o s u l p b a t e s o l u t i o n s o v e r t h e t e m p e r a t u r e r a n g e 2 0 C-80 C . Table 6

---i s c o s i t y o f Neomycin S u l p h a t e S o l u t i o n s V
Neomycin Concentration
9:

Viscosity (cps)
2 ooc
4OoC

6 OC
0.64

8OoC 0.623
0.74

1.25
1.99

0.86 1.30

18 2.33.

0.95

S u r f a c e Tensiofl

Kigel e t a l l 8 h a v e r e p o r t e d t h e s u r f a c e t e n s i o n v a l u e s of 6 & 1 8 % aqueous neomycin sulphate s o l u t i o n s (Table 7 ) .

NEOMYCIN

413

Table 7 S u r f a c e T e n s i o n V a l u e s f o r Neomycin _Sulphate Solutions

N e omy c i n

S u r f ace T e n s i o n (dynes/cm)
2 ooc

Concentration

-%
6

4OoC
66.00

6 OC

8OC

69.81
72.01

63.30

60.30 62.36

18
2.34.

68.12

65.45

A d s o r p t i o n by C l a y s , S o i l s & Minerals -

The a d s o r p t i o n of a n t i b i o t i c s from a q u e o u s s o l u t i o n by c l a y s and m i n e r a l s h a s r e p o r t e d by a number of a u t h o r s . P i n c h e t concluded t h a t , f o r s t r o n g l y b a s i c a n t i b i o t i c s s u c h a s neomycin, a d s o r p t i o n by c l a y s s u c h as montmorillonite d i s t o r t e d the c r y s t a l l a t t i c e o f t h e c l a y 4.42, c o r r e s p o n d i n g t o m o n o l a y e r a d s o r p t i o n . The same a u t h o r s also s u g g e s t e d t h e bonding i n t h e monolayers t o be s t r o n g l y e l e c t r o s t a t i c . A t t e m p t s t o r e l e a s e t h e a n t i b i o t i c by the addition of b u f f e r s has en demonstrated t o be o n l y p a r t i a l l y successful". Phosphate b u f f e r s r e l e a s e d a p p r o x . 50% o f t h e a d s o r b e d a n t i b i o t i c w h e r e a s c i t r a t e , g l y c i n e and u n i v e r s a l b u f f e r s were c o m p l e t e l y i f f e c t i v e . In a recent study h a v e shown d i v a l e n t magnesium McGinity a n d H i l l " i o n s t o be more e f f i c i e n t t h a n m o n o v a l e n t sodium i o n s i n d i s p l a c i n g neomycin from t h e n e g a t i v e l y c h a r g e d c l a y s a t t a p u l g i t e and m o n t m o r i l l o n i t e .

!??'

From t h e s e s t u d i e s t h e l i m i t i n g a d s o r p t i v e c a p c i t i e s f o r t h r e e c l a y s w e r e calc u l a t e d u s i n g t h e Langmuir e q u a t i o n . Wayman e t a126 s t u d i e d t h e b i n d i n g of neomycin t o t h e f i l t r a t i o n m a t e r i a l s c e l i t e , c e l l u l o s e powder and S e i t z f i l t e r s . Neomycin w a s f o u n d t o b e a d s o r b e d on a l l t h r e e materials. Acid-washing t h e c e l l u l o s e powder f a i l e d t o d e s o r b a l l o f t h e neomycin.

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WILLIAM F. HEYES

2 . 3 5 . A d s o r p t i o n by I o n Exchange R e s i n s During a s t u d y o f t h e physicoc h e m i c a l p r o p e r t i s o f some a m i n o g l y c o s i d e a n t i b i o t i c s Kuzyaeval' d e t e r m i n e d t h e s t a t i c and dynamic e x c h a n g e c a p a c i t i e s o f neomycin on t h e c a t i o n e x c h a n g e r e s i n KB-4P-2 ( a p h e n o x y a c e t i c acid-formaldehyde r e s i n ) u s i n g t h e r e s i n i n t h e Na form.
K i l l f i n e t a127 h a v e r e p o r t e d v a l u e s f o r t h e e x c h a n g e e n t h a l p y o f neomycin o r t h r e e i o n - e x c h a n g e r e s i n s . The v a l u e s of A H w e r e c a l c u l a t e d by a p p l i c a t i o n o f t h e Gibbs-Helmholtz e q u a t i o n ; t h e p u b l i s h e d r e s u l t s are t a b u l a t e d below:-

T a-e -b l-8 Some Exchange E n t h a l p i e s f o r Neomycin Ion exchange resin

KFU,Na

Type methacrylic acidd iv i n y 1 ben z e n e methacrylic acidhexame t h y l e n e d i m e t h y 1 a a cr y 1 m id e phenoxyacetic acidformaldehyde

AH -1 cal e q u i v

+ form

-40

KMDM6 ,Na+form

+34 0

KB4P2 ,Na+form

+5 7 0

Ig8a f u r t h e r p u b l i c a t i o n reported investigations i n t o t h e v a r i a t i o n of the S e l e c t i v i t y Coefficient ( K ) w i t h t h e amount o f neomycin a d s o r b e d f o r f o u r t e e n d i f f e r e n t ion-exchange r e s i n s . In a l l cases t h e s e l e c t i v i t y c o e f f i c i e n t i n c r e a s e d w i t h t h e amount o f a d s o r b e d neomycin ( e x p r e s s e d as t h e % surface-coverage o f t h e i o n exchange r e s i n ) , t h o u g h d i f f e r e n t t y p e s of i o n e x c h a n g e r s r e s u l t e d i n d i f f e r e n t r e s p o n s e s . T h e d e g r e e o f crossl i n k i n g of t h e i o n e x c h a n g e r e s i n was a l s o r e p o r t e d t o a f f e c t e d t h e v a l u e of t h e s e l e c t i v i t y coefficient. The a u t h o r s c o n c l u d e d t h a t p o l y c o n d e n s a t i o n - t y p e i o n exchange r e s i n s a d s o r b
K i l ' f i n and Samsonov

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neomycin more s e l e c t i v e l y t h a n t h e p o l y m e r i s a t i o n t y p e r e s i n s and t h a t v a r i a t i o n of t h e components of e i t h e r t y p e of r e s i n had l i t t l e e f f e c t on t h e a d s o r p t i o n of neomycin. Klyueva and G e l p e r i n 2 have compared t h e e q u i l i b r i u m d i s t r i b u t i o n c u r v e s f o r t h e a d s o r p t i o n o f neomycin on i o n - e x c h a n g e r e s i n s from b o t h p u r e s o l u t i o n s and t y p i c a l f e r m e n t a t i o n broths.
3.

S a l t s , Derivatives
3.1.

&

Complexes

Salts

Many a t t e m p t s t o a l t e r t h e p h y s i c a l p r o p e r t i e s o f neomycin by t h e f o r m a t i o n o f v a r i o u s s a l t s have b e e n d e s c r i b e d . Thus t h e neomycin s a l t s of t h e h i g h e r f a t t y a c i d s , s u c h a s t h e s t e a r a t e , p a l m i t a t e and m y r i s t a t e 3 0 r 31 were p r e p a r e d and b e i n g w a t e r - i n s o l u b l e w e r e f o r m u l a t e d i n ointment bases. Similarly, t h e undecylenate s a l t h a s been re a r e d and p r o c e s s e s f o r i t s p r o d u c t i o n p a t e n t e d 3 5 39, 3 4 * The u n d e c y l e n a t e and c a p r y l a t e ~ s a l t s h a v e been d e s c r i b e d as b e i n p a r t i c u l a r l y s u i t a b l e as a n t i m i t o t i c compounds 85

V a r i o u s c a r b o x y l i c a c i d s a l t s have a l s o gal lard^^^ p r o d u c e d t h e m a l e a t e been r e p o r t e d . s a l t o f neomycin w h i c h , i t was c l a i m e d , improved t h e a q u e o u s s t a b i l i t y of t h e a n t i b i o t i c . A p r a c t i c a l l y t a s t e l e s s compound t h e c i t r a t e s a l t , h a s b e e n d e s c r i b e d by S z y s ~ k a ~ Neomycin m a n d e l a t e ~ . h a s been claimed t o be p a r t i c u l a r 1 u s e f u l i n t h e t r e a t m e n t of u r o g e n i t a l i n f e c t i o n s s g w h i l e t h e d i hydroxy-d i n a p h t h l m e t h a n e - d i c a r b o x y l a t e 40 and t h e pamoate s a l t s 4 1 , g 7 1 6 8 h a v e a l o w i n t e s t i n a l abs o r p t i o n and a r e t h u s e f f e c t i v e t r e a t m e n t s f o r intestinal infections.
F i s h e r and H a l l r e p o r t e d t h e p r o p i o n a t e s a l t t o be u n s u i t a b l e f o r u s e i n o p h t h a l m i c preparation^^^. O t h e r s a l t s d e s c r i b e d i n t h e l i t e r a t u r e a r e neom c i n g l u c u r o n a t e 4 2 , s u c c i ~, n a t e 4 3 I 4 4 , l a c t a t e 42 a ~ c o r b a t e ~ h t h a l a t e ~ ~ a n d t h e s u l p h o s ~ c c i n a t e4~ ~ r 8 which h a s t h e p r o p e r t y of improved s k i n p e n e t r a t i o n .

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t o p r e p a r e neomycin s a l t s , t h e p - t o l u e n e s u l p h o n a t e h a s been r e p o r t e d and i n c o r p o r a t e d i n an

Amongst t h e o r g a n i c s u l p h o a c i d s u s e d

a e r o s o l s p r a y f o r m u l a t i o n 4 9 . S a l t s o f neomycin w i t h h a l o g e n s u b s t i t u t e d 8 - h y d r o x y q u i n o l i n e 5s u l p h o n i c a c i d s h a v e been d e s c r i b e d a s p o s s e s s i n g l o w t o x i c i t y , a n t i s e p t i c and a n t i a m e b i c b e s i d e s a n t i m i c r o b i a l pro erties50f51. Stankovics e t a 1 5 2 and V e ~ i a n a r e ~ i p i t a t e d a complex s a l t of p~ c neomycin, by m i x i n g a q u e o u s s o l u t i o n s of neomycin s u l p h a t e and sodium l a u r y l s u l p h a t e , which had improved a c t i v i t y a g a i n s t Staphylocaccub a u k e u d . I n a more r e c e n t i n v e s t i g a t i o n o f t h i s r e a c t i o n , found t h a t t h e p r e c i p i t a t e d Leucuta e t complex d i s s o l v e d on a d d i t i o n o f e x c e s s sodium I.R. s t u d i e s of t h e complex lauryl sulphate. have i n d i c a t e d t h a t amide g r o u p s , c o u p l i n g rea c t i o n s and H-bonding a r e i n v o l v e d i n t h e format i o n o f t h e compound. M i c h e l e and V a l e t t e h a v e rep o r t e d an enhanced a n t i b i o t i c a c t i v i t y and a reduced t o x i c i t y f o r t h e e u c a l y p t 0 1 ~ u l p h o n a t e ~ ~ . P r e p a r a t i o n of t h e d o d e c l b e n z e n e - s u l p h o n a t e 5 5 and t h e cyclohexylsulphonatex6has a l s o been d e s c r i b e d . The p a n t o t h e n a t e s a l t of neomycin , f i r s t d e s c r i b e d by K e l l e r e t a 1 5 7 , a l s o e x h i b i t s t h e p r o p e r t y of a lower t o x i c i t y t h a n t h e p a r e n t a n t i b i o t i c . A f u r t h e r p a p e r by t h e same a u t h o r s 5 8 a t t r i b u t e d t h e lower t o x i c i t y o f t h e p a n t o t h e n a t e t o be due t o a r e d u c t i o n i n t h e c a l c i u m - b i n d i n g a b i l i t y of t h e a n t i b i o t i c when i n t h e form of t h e p a n t o t h e n a t e s a l t . A s i m i l a r e f f e c t h a s been n o t e d by W e i t n a u e r e t a159 w i t h a n e q u i m o l a r m i x t u r e of neomycin and c a l c i u m g l u c o n a t e . Numerous p r o cedures f o r the preparation o t e pantothenate res a l t have been p a t e n t e d 6 0 , 6 1 , 2 r k 3 . A b b ~ u ~ ~ p o r t e d t h e p r e p a r a t i o n of a complex s a l t of neomycin w i t h a m i x t u r e of g l u t a m i c and p a n t o t h e n i c a c i d s and d e s c r i b e d t h e compound a s p o s s e s s i n g b e t t e r o r g a n o l e p t i c p r o p e r t i e s t h a n o t h e r neomycin salts. S a l t s w i t h t h e amino a c i d s N-methyls t e a r o y l g l y c i n e 6 5 and l u t a m i c a c i d 6 6 , and t h e v i t a m i n s a s c o r b i c a c i d 2 3 and n i c o t i n i c a c i d 6 3 have a l s o been e v a l u a t e d .

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C h l o r o p h e n o l s a r e of a s u f f i c i e n t l y a c i d i c n a t u r e t o be a b l e t o form s a l t s w i t h neomycin and h a v e been r e p o r t e d t o p o s s e s s h t h a n t i b a c t e r i a l and m y c o c i d a l proper tie^^^ ,7 0 r 7p. Shaw70 h a s d e s c r i b e d t h e t o p i c a l u s e of s u c h a compound, neomycin h e x a k i s p e n t a c h l o r o p h e n a t e , i n t h e t r e a t m e n t o f d e n t a l r o o t i n f e c t i o n and p e r i o dontic cavities.
A s a l t formed by r e a c t i n g neomycin w i t h p-amino b e n z e n e s u l h o a c e t a m i d e was d e s c r i b e d by t w o g r o u p s of w o r k e r s T 2 I 7 3 . The s a l t combined t h e c h e m o t h e r a p e u t i c p r o p e r t i e s of t h e components. A n o t h e r u n u s u a l s a l t of neomycin d e s c r i b e d i n t h e l i t e r a t u r e i s t h a t w i t h m- u l p h o n y l b e n z a l d e h y d e i s o n i c o t i n o y l h y d r a z o n e787. The compound i s rep u t e d t o be l e s s t o x i c t h a n neomycin and t o be more a c t i v e a g a i n s t t u b e r c u l e b a c i l l i t h a n t h e individual constituents.

S a l t s of neomycin w i t h t h e i n o r g a n i c The s u l p h a t e s a l t a c i d s have a l s o b e e n d e s c r i b e d . i s t h e u s u a l c o m m e r c i a l form i n u s e t o d a y b u t neomycin b o r a t e h a s been u s e d i n o p h t h a l m i c p r e p a r a t ions46. Not a l l neomycin s a l t s , however, p o s s e s s a d v a n t a g e s s u c h as a r e d u c e d t o x i c i t y . I n f a c t , t h e orotic acid (1,2,3,6-tetrahydro-2 ,6d i o x o - 4 - p y r i m i d i n e c a r b o x y l i c a c i d ) s a l t w a s rep o r t e d t o h a v e a g r e a t e r t o x i c i t y t h a n neomycin i t ~ e l f ~ ~ u n c e r ~a i .n t y of t h i s f a c t i s Some ~ ~ t a p p a r e n t a s o t h e r a u t h o r s have r e p o r t e d t h e two compounds t o b e o f a s i m i l a r t o x i c i t y 7 6 . The s a l t of neomycin w i t h u s n i c a c i d ( a n a n t i b a c t e r i a l s u b s t a n c e f o u n d i n l i c h e n s ) h a s been r e p ~ r t e d ~ ~ t o h a v e l e s s a n t i b a c t e r i a l a c t i v i t y t h a n t h e neomycin used i n t h e p r e p a r a t i o n of t h e s a l t . 3.2. Derivatives The neomycin m o l e c u l e c o n t a i n s b o t h f r e e amino and f r e e .hydroxy g r o u p s . Many w o r k e r s have e x p l o i t e d t h e p o s s i b i l i t y o f c h e m i c a l d e rivatisation a t these positions in attempts t o r e d u c e t h e t o x i c i t y of t h e p a r e n t a n t i b i o t i c . S u b s t i t u t i o n of methane s u l p h o n a t e g r o u p s a t t h e amino n i t r o g e n h a s been r e p o r t e d by U m e z a w a e t a183184 and by B o i s s i e r e t a185. Both g r o u p s o f w o r k e r s d e s c r i b e d t h e d e r i v a t i v e t o b e less t o x i c

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than t h e p a r e n t a n t i b i o t i c . Boissier claimed t h a t t h e ' i n v i t r o ' and ' i n v i v o ' a n t i b a c t e r i a l a c t i v i t y o f t h e neomycin i s p r e s e r v e d f o l l o w i n g derivatisation. However, Umezawa, w o r k i n g w i t h f r a d i o m y c i n , d e s c r i b e d t h e N-methyl s u l p h o n a t e d e r i v a t i v e a s having h a l f t h e a c t i v i t y of f r a d i o mycin when r e a s u r e d a g a i n s t gram-pos i t i v e b a c t e r i a and o n l y one t e n t h t h e a c t i v i t y when measured again s t gram-negative b a c t e r i a . Russian workers89 have r e p o r t e d t h e r e d u c t i o n i n a n t i b a c t e r i a l a c t i v i t y t o b e p r o p o r t i o n a l t o t h e d e g r e e o f Ns u b s t i t u t i o n . Alternative procedures f o r t h e p r e p a r a t i o n o f N-methyl s u l p h o n y l neomycin h a v e been r e p o r t e d by a number o f a u t h o r s 8 6 1 8 7 1 8 8 and t h e pharmacology o f t h e compound h a s been e x t e n s i v e l y i n v e s t i g a t e d by D i Marco and B e r t a z z o i g o . T h e p r e p a r a t i o n of t h e f o l l o w i n g s u l p h o d e r i v a t i v e s h a s a l s o been d e s c r i b e d :
N-me t hy l s u l p h i no n e omyc i n
I

N - p h e n y l s u l p h o n y l neomycin93
N- a c e t amidop hen y Is u l phony 1 neomyc i n

N-p-aminophenylsulphonyl neomycing3 N- t r i c h lorome t h y l t h i o neomycin9

N - e t h y l t h i o c a r b o n y l neomycing3 and neomycin N - s u l p h o n a t e g 4 Vanderhaeghe8O h a s s t u d i e d t h e a l k y l a t i o n of neomycin. A r e d u c t i o n i n t o x i c i t y was n o t e d f o l l o w i n g a l k y l a t i o n b u t t h i s w a s c o u p l e d w i t h a c o m p l e t e loss o f a n t i b a c t e r i a l properties. The r e s u l t i n g compound, however, w a s found t o p o s s e s s good h y p o c h o l e s t e r e m i c a c t i v i t y . The e f f e c t of N - a l k y l a t i o n , 0 - a l k y l a t i o n , Na c y l a t i o n and O - a c y l a t i o n h a s been i n v e s t i g a t e d by Magyar e t a181. These w o r k e r s r e p o r t e d t h e l o s s o f b i o l o g i c a l a c t i v i t y t o be p r o p o r t i o n a l t o t h e number o f g r o u p s s u b s t i t u t e d . F u r t h e r i n v e s t i g a t i o n s by t h e s e w o r k e r s d e m o n s t r a t e d t h a t conv e r s i o n o f amino g r o u p s i n t o q u a t e r n a r y ammonium g r o u p s r e s u l t e d i n t h e p r o d u c t i o n of compounds having a mild t u b e r c u l o s t a t i c e f f e c t . Penasse e t a182 have d e s c r i b e d an a l t e r n a t i v e p r o c e d u r e f o r t h e p r e p a r a t i o n of N-alkyl d e r i v a t i v e s .

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3 . 3 . Complexes

The f o r m a t i o n o f a zinc-neomycin complex h a s been r e p o r t e d by Chornock95 and by K e l l e r and C o s a r g 6 . E l e c t r o p h o r e s i s w a s u s e d t o d e m o n s t r a t e t h e f o r m a t i o n o f a complex which was d e s c r i b e d as p o s s e s s i n g a s i m i l a r b i o l o g i c a l a c t i v i t y t o neomycin b u t a d e c r e a s e d t o x i c i t y . More r e c e n t l y Agrawal, H a r m a l k e r and V i j a y a w a r g i y a g 7 have d e s c r i b e d t h e f o r m a t i o n o f a copper-neomycin complex. With a s t o i c h i o m e t r y o f 1:1, t h e b l u e c o l o u r e d complex h a s been made t h e b a s i s o f a s p e c t r o p h o t o m e t r i c a s s a y method f o r neomycin ( S e e S e c t i o n 6 . 2 6 ) . The c o m p l e x a t i o n o f neomycin w i t h a number of b i o c h e m i c a l l y i m p o r t a n t compounds h a s been r e p o r t e d . I n t e r a c t i o n of t h e a n t i b i o t i c with h e p a r i n w a s f i r s t i n v e s t i g a t e d by Higginbotham and D o u g h e r t y 9 8 . U s i n g a s p e c t r o p h o t o m e t r i c p r o c e d u r e , which measured t h e amount o f dye r e l e a s e d f r o m a t o l u i d i n e b l u e - h e p a r i n complex on a d d i t i o n of neomycin s o l u t i o n , t h e s e a u t h o r s s t u d i e d a number of a n t i b i o t i c s and t h e i r r e a c t i o r . w i t h heparin. G u b e r n i e v a and S i l a e v g 9 employed elect r o p h o r e s i s t o i n v e s t i g a t e t h e n a t u r e of t h e n e o m y c i n - h e p a r i n complex and s u g g e s t e d t h e compound may b e more t h a n a s i m p l e c a t i o n - a n i o n i n t e r a c t i o n . The p o s s i b i l i t y o f a d d i t i o n a l H-bonding f o l l o w i n g t h e i n i t i a l i o n i c i n t e r a c t i o n was p o s t u l a t e d . A more d e t a i l e d s t u d y o f t h e c o m p l e x a t i o n o f n e o mycin w i t h h e p a r i n , DNA,RNA and p o l y h l o r e t i n p h o s p h a t e h a s been d e s c r i b e d by HeinyOO. The c o m p l e x e s w e r e p r e p a r e d by m i x i n g a q u e o u s s o l u t i o n s of neomycin s u l p h a t e and t h e c o m p l e x i n g a g e n t u n d e r i n v e s t i g a t i o n . The p r e c i p i t a t e which formed r e d i s s o l v e d on a d d i t i o n o f sodium h y d r o x i d e s o l u t i o n o r aqueous s o l u t i o n s of s i l v e r n i t r a t e , manganese s u l p h a t e and c o b a l t c h l o r i d e . S u b j e c t i o n o f t h e p r e c i p i t a t e d complex t o an a g a r d i f f u s i o n e x p e r i m e n t d e m o n s t r a t e d t h e compound t o b e b i o logically active. U s i n g an a g a r o s e g e l s y s t e m Kunin and Tupasi.101 o b s e r v e d t h e f o r m a t i o n o f a p r e c i p i t a t i o n band between z o n e s of d e x t r a n s u l p h a t e and neomycin. The a u t h o r s a t t r i b u t e d t h e p r e c i p i t a t i o n t o be a r e s u l t o f complex f o r m a t i o n

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and n o t t h e r e s u l t of an a n t i g e n - a n t i b o d y i n t e r a c t i o n which t h e s y s t e m i s u s u a l l y employed t o detect.

H a r r i s l o 2 h a s s t u d i e d t h e complexat i o n of neomycin w i t h p e c t i n and d e m o n s t r a t e d t h e i n h i b i t i o n of complex f o r m a t i o n i n t h e p r e s e n c e of an e l e c t r o l y t e . P o t e n t i o m e t r i c measurements i n d i c a t e t h e mechanism o f t h e r e a c t i o n t o be a cation-anion i n t e r a c t i o n . H-bonding between t h e hydroxy g r o u p s o f p e c t i n and s u g a r m o i e t i e s o f neomycin h a s been s u g g e s t e d and would f u r t h e r s t a b i l i s e t h e compound.

The c o m p l e x a t i o n of neomycin w i t h a n i o n i c d y e s s u c h a s a m a r a n t h ( F . D . & C . Red No.2) i s w e l l known100,102 and h a s been made t h e b a s i s of a q u a n t i t a t i v e a s s a y f o r n e o m y ~ i n ~ ~ These 5 . ~ 1 ~ complexes a r e a g a i n of t h e c a t i o n / a n i o n t y p e and t h e i r f o r m a t i o n i s d e p e n d a n t on t h e i o n i c s t r e n g t h of t h e s o l u t i o n . I n o r g a n i c c o n d e n s e d p h o s p h a t e s have been r e p o r t e d t o complex w i t h neomycin and i n a s t u d y of t h e s e compounds I S i n g h l o 3 d e m o n s t r a t e d t h e s t r e n g t h o f t h e complex t o be d e p e n d a n t on t h e d e g r e e of p o l y m e r i s a t i o n of t h e p h o s p h a t e . By i n c r e a s i n g t h e number of p h o s p h a t e u n i t s i n t h e polymer o v e r t h e r a n g e 1 t o 1 6 u n i t s an i n c r e a s i n g s t r e n g t h of c o m p l e x a t i o n w a s o b s e r v e d . Beyond t h i s u p p e r l i m i t , however, t h e s t r e n g t h of t h e complex remained c o n s t a n t . Relative complex-strengths w e r e a s s e s s e d by t i t r i m e t r y w i t h p o t a s s i u m c h l o r i d e solution.
A number of w o r k e r s h a v e u t i l i s e d e l e c t r o p h o r e s i s t o e s t a b l i s h t h e f o r m a t i o n of a complex between neom c i n and t h e r o t e i n s i n b l o o d Serum and plasma104 ,Yo511 0 6 1 1 0 7 , ,881 1 0 9 , 1 1 0 , 1 1 1 . However, t h e n a t u r e of t h e complex i s s t i l l unknown. Geitman h a s e x t e n s i v e l y s t u d i e d t h e phenomena of p r o t e i n - b i n d i n g o f a n t i b i o t i c s l l 1 t 1 l 2 . I n a p a p e r p u b l i s h e d j o i n t l y w i t h Kivman1l2, Geitman r e p o r t e d t h a t neomycin d i d n o t b i n d t o any of t h e p r o t e i n f r a c t i o n s when added t o serum b u t bound t o t h e i s o l a t e d albumin and g l o b u l i n f r a c t i o n s . An e a r l i e r p a p e r l l l d e s c r i b e d t h e g l o b u l i n - n e o m y c i n complex t o b e o f g r e a t e r s t a b i l -

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i t y t h a n t h e albumin-neomycin complex and e s t a b l i s h e d lmg o f serum p r o t e i n t o b i n d 0 . 0 6 u n i t s o f neomycin

4.

S y n t h e s i s and P r o d u c t i o n
4.1.

Commercial B i o s y n t h e s i s -

Waksman e t a1286, i n 1 9 4 9 , f i r s t rep o r t e d t h e p r o d u c t i o n of neomycin by f e r m e n t a t i o n of a c u l t u r e o f S . d h a d i a ~ ( 3 5 3 5 ) .The same o r g a n i s m s u b s e q u e n t l y formed t h e b a s i s o f an i n d u s t r i a l f e r m e n t a t i o n rocess f o r t h e b i o s y n t h e s i s o f neomycin28712i8. I s o l a t i o n o f t h e a n t i b i o t i c from t h e f e r m e n t a t i o n media i s a c c o m p l i s h e d b y u s e of i o n - e x c h a n e r e s i n s , s u c h a s Amberlite I R C 50225 1 9 5 0 , 2 5 1 .

F o l l o w i n g t h e o r i g i n a l r e p o r t by Waksman, a number o f o t h e r a u t h o r s have d e s c r i b e d S . Q a a d i a e t o y i e l d a n t i b i o t i c s on f e r m e n t a t i o n . The r e s u l t i n g s u b s t a n c e s w e r e named s t r e p t o t h r i c i n B 1 2 8 9 , m y c e r i n 2 9 0 and c 0 l i m y c i n 2 9 1 ~b u t h a v e s i n c e b e e n shown t o be i d e n t i c a l t o t h e neomycin complex.

4.2.

S y n t h e s i s o f R a d i o - L a b e l l e d Neomycin

The b i o s y n t h e t i c p r e p a r a t i o n o f l 4 C l a b e l l e d neomycin w a s f i r s t d e s c r i b e d by Sebek267 who s t u d i e d a number o f s u b s t r a t e s f o r t h i s p u r p o s e b u t c o n c l u d e d 14C'labelled glucose was t h e most s a t i s f a c t o r y . A l t h o u g h 6 8 % o f t h e r a d i o n u c l i d e w a s l o s t as r e s p i r a t o r y I 4 C O , 1 9 % o f t h e t o t a l a c t i v i t y added p r i o r t o f e r m e n $ a t i o n w a s i n c o r p o r a t e d i n t o t h e neomycin s u l p h a t e . T h i s method o f p r e p a r i n g 1 4 C - l a b e l l e d neomycin h a s b e e n e x t e n s i v e l y u s e d t o s t u d y t h e mechanism o f neomycin b i o s y n t h e s i s . Stroshane13 has described t h e s y n t h e s i s o f 13C-neomycin and 15N-neomycin, a g a i n a s p a r t o f an i n v e s t i g a t i o n o f neomycin b i o s n t h e s i s . 13C-glucose s e r v e d a s p r e c u r s o r f o r y3C-neomycin and g l u c o s a m i n e l a b e l l e d w i t h I 5 N w a s u t i l i s e d f o r t h e p r e p a r a t i o n o f 15Nneomycin.
A p r o c e d u r e f o r 1abe l l i n g neomycin w i t h t r i t i u m h a s been d e s c r i b e d by J a c k s o n e t

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a t 2 9 2 and i n v o l v e s p a s s i n g an e l e c t r i c d i s c h a r g e across a v e s s e l c o n t a i n i n g t r i t i u m and neomycin sulphate. L a b i l e t r i t i u m was removed by w a s h i n g w i t h a p o l a r s o l v e n t and t h e t r i t i u m - l a b e l l e d neomycin p u r i f i e d by m u l t i p l e r e c r y s t a l l i s a t i o n .

5. S t a b i l i t y and D e g r a d a t i o n 5.1. Hydrolytic Degradation

The d e g r a d a t i o n of neomycin h a s b e e n e G t e n s i v e l y s t u d i e d by R i n e h a r t l a s a means o f e s t a b l i s h i n g t h e s t r u c t u r e s o f t h e neomycin compon e n t s . F i g . 5 i l l u s t r a t e s t h e r o u t e by which comp l e t e d e g r a d a t i o n of t h e a n t i b i o t i c w a s a c h i e v e d . Extremely vigorous c o n d i t i o n s are n e c e s s a r y f o r t h e h y d r o l y s i s o f n e m i n e a s t h i s compound i s r e s i s t a n t t o acid-hydrolysis. However, u n d e r t h e s e c o n d i t i o n s t h e c h e m i c a l e n t i t i e s neosamine C ( f r o m n e a m i n e ) and r i b o s e are n o t o b s e r v e d b e c a u s e v i g o r o u s h y d r o l y s i s i m m e d i a t e l y decomposes t h e compounds f u r t h e r . By d e r i v a t i s i n g t h e amino g r o u p s o f t h e neamine and n e o b i o s a m i n i d e m o l e c u l e s R i n e h a r t w a s a b l e t o lower t h e r e s i s t a n c e of t h e s e compounds t o h y d r o l y s i s and t h u s u s e mild h y d r o l y t i c c o n d i t i o n s t o s u c c e s s f u l l y i s o l a t e neosamine C and r i b o s e :
(CHJCO)

a ) neamine

CH30H

*,5 o C

t e t r a N - a c e t y l neamine

blmethyl neob i o s a m i n i d e

PhCOCl N aOH H SO

deoxystreptamine

3N HC1

lOhr @ 9 5 O C

neosamine

methyl N,N'-dibenzoyl neob i o s a m i n i d e

1.6N HC1

0.1N

ribose

d 4 H6 O(OH)3 CH OC 3 5

Rus s i a n w o r k e r s attempted t o prep a r e t h e d e g r a d a t i o n p r o d u c t s i n a p u r e f o r m by c a r r y i n g o u t t h e h y d r o l y s i s i n t h e p r e s e n c e of an i o n e x c h a n g e r e s i n which a d s o r b e d t h e i n t e r m e d i a t e

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n eomy c i n
I

F i g u r e 5.

H y d r o l y t i c d e g r a d a t i o n of neomycin C

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WILLIAM F. HEYES

hydrolysis products,thereby preventing f u r t h e r dec o m p o s i t i o n . The d e g r a d a t i o n p r o d u c t s w e r e rel e a s e d from t h e r e s i n by g r a d i e n t - e l u t i o n . 5.2. S t a b i l i t y o f Bulk M a t e r i g

J a p a n e s e w o r k e r s l5 h a v e d e s c r i b e d t h e s t a b i l i t y of b u l k f r a d i o m y c i n (neomycin B l s u l p h a t e which was shown t o h a v e r e t a i n e d 9 9 % o f i t s microb i o l o g i c a l p o t e n c y a f t e r s t o r a g e f o r 2 4 months. Neomycin s u l p h a t e powder h a s been r e o r t e d t o b e s t a b l e f o r a t l e a s t 3 y e a r s a t 2OoC 5 9 8 ,305. Neomycin s u l p h a t e may a l s o b e h e a t e d a t llOC f o r 10 hours, during dry-heat sterilisat i o n , w i t h o u t l o s s o f p o t e n c y , t h o u g h some d e g r e e of yellowing i s apparent298. 5.3. S t a b i l i t y i n Aqueous S o l u t i o n

Swart e t a 1 h a v e d e m o n s t r a t e d t h e s t a b i l i t y of neomycin h y d r o c h l o r i d e i n a q u e o u s Further ins o l u t i o n o v e r t h e pH r a n g e 2-9293. v e s t i g a t i o n s by Simone and P o p i n o 2 9 8 c o n f i r m e d t h e a q u e o u s s t a b i l i t y a t 23OC b u t a t 45OC p o t e n c y l o s s e s o f u p t o 9 4 % w e r e n o t e d o v e r a p e r i o d of 2 y e a r s w i t h s o l u t i o n s i n t h e pH r a n g e 4-8. Leach e t a 1 2 9 4 d e s c r i b e d a p u r i f i e d neomycin t o be s t a b l e t o t h e a c t i o n o f a l k a l i b u t Refluxing t h e a n t i b i o t i c w i t h barium not t o acids. h y d r o x i d e f o r a p e r i o d of e i g h t e e n h o u r s f a i l e d t o show a l o s s i n m i c r o b i o l o g i c a l p o t e n c y . Japanese w o r k e r s have d e s c r i b e d a stable a q u e o u s s o l u t i o n of neomycin w i t h t h e i n c o r p o r a t i o n o f a b o r a t e b u f f e r (pH 6 ) and E . D . T . A . 2 9 5 The p r e s e n c e of 1-10% o f g l y c e r o l , p r o p y l e n e g l y c o l o r m a n n i t o l h a s b e e n c l a i m e d t o improve t h e s o l u t i o n a p p e a r a n c e by p r e v e n t i n g d i s c o l ~ r a t i o n ~ The . ~~ p r e s e n c e of p o l y o l s a l s o p r e v e n t e d a d e c r e a s e i n pH v a l u e o f t h e s o l u t i o n . D i s c o l o r a t i o n may a l s o b e p r e v e n t e d by a d d i t i o n of 0.1% sodium m e t a b i H s u l p h i t e a t a s o l u t i o n p of 6 . 6 t o 6 . 8 2 9 7 .

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5.4.

S t a b i l i t y i n Pharmaceutical Formulations

Numerous r e p o r t s c o n c e r n i n g t h e s t a b i l i t y of neomycin i n v a r i o u s d o s a e forms have been p u b l i s h e d . Simone and Popino j 9 8 s t u d i e d t h e s t a b i l i t y o f neomycin i n l i q u i d dosage forms s u c h a s n a s a l d r o p s , mouth washes and t i n c t u r e s . The a n t i b i o t i c w a s s t a b l e i n a l l t h e formulations t e s t e d , e x c e p t D o b e l l s s o l u t i o n ( a mouth w a s h ) , f o r a t l e a s t 6 months a t 2 0 O C . Some f o r m u l a t i o n s were s t a b l e f o r considerably longer. Heyd2 h a s s t u d i e d t h e s t a b i l i t y o f neomycin i n aqueous g e l s and f o r m u l a t e d a s a t i s f a c t o r y p r o d u c t by a d s o r b i n g t h e a n t i b i o t i c on an i o n exchange r e s i n , Amberlite IRP-69M, p r i o r t o incorporation i n the gel. R e c o n s t i t u t e d aqueous s u s p e n s i o n s o f neomycin s u l p h a t e , c o n t a i n i n g t r a g a c a n t h and s u g a r have been shown t o b e s t a b l e f o r 10 d a y s when s t o r e d a t 4OC b u t some loss of p o t e n c y w a s o b s e r v e d when t h e s u s p e n s i o n was exposed t o d a y l i g h t 3 . Non-aqueous s u s p e n s i o n s c o n t a i n i n g p e a n u t o i l and l a n o l i n have been r e p o r t e d 2 9 8 which are s t a b l e f o r 1 year a t 200C. Simone and pop in^^'^ have c o n s i d e r e d t h e s t a b i l i t y of neomycin i n b o t h h y d r o p h o b i c and hydrophilic ointment bases. No l o s s of p o t e n c y o v e r a p e r i o d of 1 y e a r a t 2OoC w a s r e p o r t e d f o r formulations containing carboxymethylcellulose, polyethylene glycol(P.E.G.) o r white-soft paraffin. However, f o r m u l a t i o n s c o n t a i n i n g h y d r o u s A l l materlanolin w e r e reported t o be unstable. i a l s u s e d i n t h e f o r m u l a t i o n s were o b t a i n e d from U.S. sources. C o a t e s e t a1301 i n v e s t i g a t e d t h e u s e o f P . E . G . from B r i t i s h s o u r c e s and d e s c r i b e d neomycin a s b e i n g i n c o m p a t i b l e w i t h t h e m a t e r i a l s tested. S o l i d d o s a g e forms of neomycin as t a b l e t s and t r o c h e s have been p r e p a r e d 2 9 8 and a r e r e p o r t e d t o b e s t a b l e a t 2OoC and a t 56OC. I n t h e a r e a of animal h e a l t h n u t r i t i o n a feed-premix c o n t a i n i n g neomycin and oxyt e t r a c y c l i n e h a s been d e s c r i b e d . With neomycin

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a t a l e v e l of 20g/lb t h e p r o d u c t is s t a b l e f o r 1 y e a r a t 20C, though an 8 % loss o f p o t e n c y was obs e r v e d on s t o r a g e f o r 6 months a t 380C305. A medi c a t e d d r i n k i n g - w a t e r c o n t a i n i n g 100 ppm o f neomycin and p r e p a r e d w i t h t a p - w a t e r , showed n o s i g n i f i c a n t l o s s of p o t e n c y a f t e r 48 h o u r s s t o r a g e a t 38OC i n e i t h e r g l a s s c o n t a i n e r s o r g a l v a n i s e d troughs305. 5 . 5 . C o m p a t i b i l i t y w i t h E x c i p i e n t Materials The r e a c t i o n of neomycin w i t h many compounds h a s been d e s c r i b e d i n S e c t i o n 3 , h e n c e numerous r e p o r t s o f neomycin i n c o m p a t i b i l i t y may b e e x p e c t e d . D a l e and Rundman304 h a v e e x t e n s i v e l y reviewed t h e c o m p a t i b i l i t y o f neomycin w i t h s u b s t a n c e s t h a t may b e e n c o u n t e r e d by t h e f o r m u l a t i o n pharmac i s t . K u d a l k e r e t a1303 have d e s c r i b e d t h e incomp a t i b i l i t y o f t h e a n t i b i o t i c w i t h r a n c i d o i l s , and the i n c o m p a t i b i l i t y with b e n t o n i t e , a montomorillo n i t e c l a y , h a s been r e p o r t e d by D a n t i and Guth306. The i n c o m p a t i b i l i t y w i t h l a c t o s e , c a u s i n g a d i s c o l o r a t i o n o f t h e m i x t u r e h a s b e e n s t u d i e d by Hammouda and Sa1akawy3O7. The amount of browning produced w a s shown t o be d e p e n d a n t on t h e i n i t i a l pH of t h e s o l u t i o n . The r a t e of d i s c o l o r a t i o n of t h e l a c t o s e / n e o m y c i n powder w a s d i r e c t l y r e l a t e d t o t h e t e m p e r a t u r e o f s t o r a g e and t h e r e l a t i v e h u m i d i t y of t h e a t m o s p h e r e . D i s c o l o r a t i o n w a s o v e r come by a d d i t i o n of sodium b i s u l p h i t e . F l o r e s t a n o e t a l 308 compared t h e rel e a s e of neomycin from P . E . G . d i e s t e r o i n t m e n t b a s e s and g r e a s e - t y p e b a s e s and c o n c l u d e d t h e former t o b e p r e f e r r e d though Coates e t a1301 h a v e d e s c r i b e d P . E . G . from B r i t i s h s o u r c e s t o b e incomp a t i b l e w i t h neomycin. 5.6. Compatibility with Other Actives

Neomycin forms an i n s o l u b l e complex when added t o aqueous s o l u t i o n s of c o r t i c o s t e r o i d phosp h a t e s . T o overcome t h i s i n c o m p a t i b i l i t y , McGinty and Brown304 i n c o r p o r a t e d d i s o d i u m h y d r o g e n phosp h a t e i n t h e o p t h a l m i c f o r m u l a t i o n . The mechanism by which t h e c o m p l e x a t i o n i s p r e v e n t e d h a s n o t y e t been f u l l y d e t e r m i n e d .

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6 . Methods of A n a l y s i s 6.1.

I d e n t i f i c a t i on

T a b l e 9 l i s t s a number of c o l o r i m e t r i c t e s t s which h a v e b e e n u s e d a s i d e n t i t y t e s t s f o r neomycin. N o one t e s t , h o w e v e r , h a s been demons t r a t e d t o b e s p e c i f i c f o r neomycin and i t i s t h u s a l s o a d v i s a b l e t o e s t a b l i s h t h e a b s e n c e of o t h e r c h e m i c a l l y s i m i l a r a n t i b i o t i c s by s u i t a b l e means. Table 9 Colorimetric Identity T e s t s f o r N eomy c i n
Test

Chemical g r o u p ing u t i l i s e d i n re a c t i o n g l y c o si d e pentose amino ribose ribose

Colour obtained

Reference

G l u cos amine Molisch Ninhydrin Fur f u r a 1 Ph lorog l u c i n o1

cherry purple purple pink-red pink-red

114 115 115,116 116 117

Both t h i n - l a y e r and p a p e r chromatog r a p h y w i l l p r o v i d e s p e c i f i c i d e n t i f i c a t i o n of neomycin. Numerous s y s t e m s f o r t h i s p u r p o s e w i l l b e found l i s t e d i n s e c t i o n 6 . 3 4 . I n a d d i t i o n t o t h e above c h e m i c a l t e s t s a number o f m i c r o b i o l o g i c a l p r o c e d u r e s have b e e n have d e s c r i b e d a reported. Heinmann e t simple t e s t t o d i f f e r e n t i a t e neomycin-like m o l e c u l e s from t e t r a c y c l i n e s and c h l o r a m p h e n i c o l . When added t o a column of i n n o c u l a t e d a g a r o n l y t h e n e o m y c i n - l i k e m o l e c u l e s show a n a r e a of g r o w t h i n h i b i t i o n a t t h e t o p o f t h e column.
A more s p e c i f i c m i c r o b i o l o g i c a l p r o c e d u r e h a s b e e n r e p o r t e d by F r e r e s and B u l l a n d 1 l g . By u s i n g f o u r t e s t OrganiSmS(8aCillUb c e k e u n , BacilCub b u b t i l i b , Sahcina C u t e a and M i c k u c o c c u b 6Cavub)patterns of i n h i b i t i o n w e r e determined f o r e a c h of t h e 1 2 a n t i b i o t i c s e x t r a c t e d i n t o t h r e e solvents. Comparing t h e i n h i b i t i o n p a t t e r n o f a n

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unknown a n t i b i o t i c w i t h t h e s t a n d a r d p a t t e r n s , t h e a u t h o r s were a b l e t o i d e n t i f y t h e p r e s e n c e o f i n d i v i d u a l s u b s t a n c e s i n animal t i s s u e . A s i m i l a r t e s t h a s a l s o b e e n d e s c r i b e d by T i e c c o e t

6.2.

Chemical P r o c e d u r e s
6.21.

T i t r i m e t r i c Assay

The d e t e r m i n a t i o n of neomycin by non-a u e o u s t i t r a t i o n h a s been d e s c r i b e d by Penau e t a l q 2 1 . Neomycin b a s e i s a l l o w e d t o r e a c t w i t h standardised perchloric acid; the excess acid i s t h e n b a c k - t i t r a t e d w i t h p o t a s s i u m hydrogen p h t h a l a t e u s i n g c r y s t a l v i o l e t a s i n d i c a t o r . To d e t e r mine t h e neomycin c o n t e n t of t h e s u l p h a t e s a l t t h e same a u t h o r s p r e c i p i t a t e d t h e s u l p h a t e w i t h b e n z i d i n e b e f o r e r e a c t i n g t h e neomycin w i t h p e r c h l o r i c a c i d . The amount o f b e n z i d i n e r e q u i r e d t o p r e c i p i t a t e t h e s u l p h a t e i s c a l c u l a t e d from t h e s u l p h a t e c o n t e n t which i s i t s e l f d e t e r m i n e d by t i t r a t i o n w i t h sodium h y d r o x i d e .
An a l t e r n a t i v e method f o r d e t e r m i n i n g t h e s u l p h a t e c o n t e n t o f neomycin s u l h a t e h a s been r e p o r t e d by Roets and Vanderhaeghey22. The p r o c e d u r e i n v o l v e s a t i t r a t i o n w i t h b a r i u m c h l o r i d e and m u s t be c a r r i e d o u t i n a 30-40% s o l u t i o n of a l c o h o l i n o r d e r t o o b t a i n a s h a r p endA s the antibiotic is insoluble i n alcohol point. it i s n e c e s s a r y t o remove t h e neomycin p a r t o f t h e This i s accomplished molecule p r i o r t o t i t r a t i o n . u s i n g a c a t i o n e x c h a n g e r e s i n s u c h a s Dowex 5 0 - X 8 .

D u r i n g an i n v e s t i g a t i o n of t h e c o m p l e x a t i o n p r o p e r t i e s of neomycin, H a r r i s 1 O 2 r e p o r t e d t h e ti t r a t i o n o f neomycin conduc t o m e t r i c a l l y w i t h a m a r a n t h ( F . D . and C Red N o . 2 ) . The e n d - p o i n t o f t h e t i t r a t i o n i s s h a r p and h a s good reproducibility.

6.22.

Polarography

The u s e of d i f f e r e n t i a l p u l s e p o l a r o g r a p h y t o d e t e r m i n e neomycin s u l p h a t e i n aqueous s o l u t i o n s h a s been d e s c r i b e d by S i e g e r m a n et F o l l o w i n g a c i d h y d r o l y s i s of neomycin, H t h e s o l u t i o n was a d j u s t e d t o p 4 and 0 . 1 M a c e t a t e

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b u f f e r ( p H 4 ) added a s s u p p o r t i n g e l e c t r o l y t e . R e d u c t i o n p o t e n t i a l s of -0.19 and -0.36V w e r e obtained. The above a u t h o r s a l s o d e s c r i b e d t h e a p p l i c a t i o n of t h i s t e c h n i q u e t o t h e e x a m i n a t i o n o f s a m p l e s of s k i n p r e v i o u s l y t r e a t e d w i t h neomycin-containing formulations. 6.23. Polarimetry Neomycins B and C h a v e b e e n shown t o d i f f e r i n t h e i r s p e c i f i c r o t a t i o n v a l u e s , neomycin B h a v i n g a s p e c i f i c r o t a t i o n of + 80 and neomycin c a s e c i f i c r o t a t i o n o f + 1 2 0 0 1 2 4 , 1 2 7 . Brooks e t a 1 12g made t h i s f a c t t h e b a s i s f o r a number o f methods t o d e t e r m i n e t h e B & C c o n t e n t o f commercial neomycin. The s p e c i f i c r o t a t i o n o f t h e t e s t s o l u t i o n i s d e t e r m i n e d a t 25OC and t o t a l neomycin d e t e r m i n e d e i t h e r t i t r i m e t r i c a l l y o r s p e c t r o p h o t o m e t r i c a l l y . By s u b s t i t u t i o n of t h e s e v a l u e s i n a s u i t a b l e e q u a t i o n t h e c o n c e n t r a t i o n o f neoI n a s e c o n d method mycins B and C a r e c a l c u l a t e d . t h e same a u t h o r s d e t e r m i n e d t h e s p e c i f i c r o t a t i o n of t h e t e s t - s o l u t i o n a t t e m p e r a t u r e s o f 25O and 7 5 O C . The c h a n g e i n t h e v a l u e of s p e c i f i c r o t a t i o n on i n c r e a s i n g t h e t e m p e r a t u r e from 25Oto 75OC c a n t h e n b e u s e d t o c a l c u l a t e t h e amounts of neomycin B and C i n t h e s a m p l e . The u s e o f an a u t o m a t i c p o l a r i m e t e r w i t h a f l o w - c e l l h a s been r e p o r t e d by d e Rosri126 , t o m o n i t o r t h e e l u a t e from an i o n e x c h a n g e column (Bio-Rad A G l - X 2 ) t h r o u g h which a s o l u t i o n of neomycin w a s p a s s e d . The d e t e c t i o n o f an o p t i c a l l y a c t i v e s u b s t a n c e was r e c o r d e d e l e c t r o n i c a l l y w i t h a s u i t a b l e pen r e c o r d e r . By d e t e r m i n i n g t h e a r e a s of t h e p e a k s r e c o r d e d , t h e amounts of neomycir,s B and C and neamine i n a number of c o m m e r c i a l s a m p l e s have b e e n d e t e r m i n e d .
6.24.

R a d i o - c h e m i c a l Assay

To d e t e r m i n e t h e r e l a t i v e amounts o f neomycins B C and neamine by r a d i o chemical method, K a i s e r i 2 8 s e p a r a t e d t h e C - l a b e l l e d Na c e t y l d e r i v a t i v e s by p a p e r c h r o m a t o g r a p h y and q u a n t i t a t e d t h e chromatograms by l i q u i d s c i n t i l l a t i o n c o u n t i n g . A c o e f f i c i e n t of v a r i a t i o n of 3 . 6 % w a s obtained.

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Seaman and Stewart1 have described a radio-chemical assay f o r determining neomycin on c o t t o n - f a b r i c . Neomycin i s r e a c t e d with carbon d i s u l p h i d e forming a d i t h i o c a r b o n a t e silver which i s t h e n decomposed w i t h [lloAg] n i t r a t e . The p r e c i p i t a t e d [ll0Ag] s i l v e r s u l p h i d e , which i s d i r e c t l y r e l a t e d t o t h e amount o f neomycin p r e s e n t , i s e s t i m a t e d by c o u n t i n g . Recently an i s o t o p e d i l u t i o n method h a s been r e p o r t e d 1 3 0 f o r a s s a y i n g neomycin s u l p h a t e . However, it i s f i r s t n e c e s s a r y t o p r e p a r e I 4 C - l a b e l l e d neomycin s u l p h a t e T h i s i s a c c o m p l i s h e d by a d d i n g 1 4 C - l a b e l l e d g l u c o s e t o a small-scale fer m entation of S . dtadiae.. 14C-labelled neomycin c a n t h e n be e x t r a c t e d by s o l v e n t - e x t r a c t i o n o r by ion-exchange c h r o m a t o g r a p h y .

6.25.

Uorimetric Assay

The neomycin m o l e c u l e d o e s n o t e x h i b i t fluor escence b u t following s u i t a b l e der i v a t i s a t i o n two s p e c t r o f l u o r i m e t r i c d e t e r m i n a t i o n s reported the have been d e s c r i b e d . Maeda e t c o m p l e x a t i o n of s i m p l e h e x o s a m i n e s w i t h p y r i d o x a l and z i n c i o n s t o r e s u l t i n a f l u o r e s c e n t d e r i v a l y i n g t h i s p r o c e d u r e t o neomycin, tive. demonstrated a l i n e a r response over t h e Simpson c o n c e n t r a t i o n r a n g e 0-50 uq/ml o f neomycin s u l p h a t e .

33

The r e a c t i o n of neomycin w i t h f l u o r e s c a m i n e t o form a f l u o r e s c e n t complex h a s been r e p o r t e d by K u s n i r & Barna133. The f l u o r e s c e n c e i n t e n s i t y v a r i e s w i t h b o t h t h e p of t h e H s o l u t i o n (optimum pH r a n g e 7 . 5 - 9 . 5 ) and t h e amount of f l u o r e s c a m i n e added. T h i s p r o c e d u r e may b e u s e d t o d e t e r m i n e neomycin a t v e r y low l e v e l s , t h e minimum c o n c e n t r a t i o n d e t e r m i n a b l e b e i n g 45 ng/ml.
6.26.

S p e c t r o p h o t o m e t r i c Assay

Spectrophotometric a s s a y s of neomycin may be c o n v e n i e n t l y d i v i d e d i n t o t w o groups: -

(a)
(b)

Direct methods, i n v o l v i n g t h e i n t a c t neomycin m o l e c u l e . I n d i r e c t methods , i n v o l v i n g h y d r o l y s i s of neomycin p r i o r t o a s s a y .

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( a )D i r e c t Methods
0 ' Keefe and R u ~ s o - A l e s i l ~ r sft r e p o r t e d i~ t h e a p p l i c a t i o n of n i n h y d r i n t o t h e a s s a y o f n e o mycin, t h e p r o c e d u r e b e i n g an a d a p t a t i o n of t h a t d e s c r i b e d by Moore and S t e i n 1 3 5 f o r t h e a s s a y o f amino a c i d s and i n v o l v i n g measurement a t 570nm o f t h e p u r p l e - c o l o u r e d complex formed. Maehr and S h a f f n e r 1 3 6 have a p p l i e d t h i s p r o c e d u r e t o t h e d e t e r m i n a t i o n o f neomycin i n c h r o m a t o r a p h y column e l u a t e s . Gerosa and M e l a n d r i attempted t o a p p l y t h e Moore and S t e i n method t o t h e q u a n t i t a t i v e d e t e r m i n a t i o n of neomycin i n p h a r m a c e u t i c a l preparations b u t reported a l i n e a r response only o v e r t h e s h o r t c o n c e n t r a t i o n r a n g e o f 400-600pg/ml o f neomycin. B o r ~ w i e c k a l ~ ~ Thorburn-Burns e t and a1139 however, r e p o r t e d no s u c h l i m i t a t i o n and s u c c e s s f u l l y a p p l i e d t h e method t o t h e a s s a y o f neomycin i n p h a r m a c e u t i c a l f o r m u l a t i o n s . P r e g n a l a t t o and S a b i n o 2 0 0 i n c o r p o r a t e d g l y c e r i n e i n t o t h e n i n h y d r i n s o l u t i o n and r e p o r t e d an i m proved s e n s i t i v i t y and r e p r o d u c i b i l i t y e n a b l i n g neomycin c o n c e n t r a t i o n s a s l o w a s 4 pg/ml t o be d e t e r m i n e d . An a u t o m a t e d n i n h y d r i n a s s a y h a s b e e n d e s c r i b e d by K a p t i o n a k , B i e r n a c k a and P a z d e r a l 4 0 b a s e d on a m o d i f i e d Moore and S t e i n m e t h o d l 4 1 . An a l t e r n a t i v e r e a g e n t which a l s o reacts w i t h t h e p r i m a r y amino g r o u p s of neomycin h a s r e c e n t l y been d e s c r i b e d l 7 2 . The r e a g e n t , d i c l o n e ( 2 , 3 - d i c h l o r o - l , 4 - n a p h t h o q u i n o n e ) , r e a c t s w i t h neomycin b a s e t o form an o r a n g e - c o l o u r e d p r o d u c t when t h e s o l u t i o n i s a d j u s t e d t o pH 4 .

C o m p l e x a t i o n of neomycin w i t h v a r i o u s d y e s was r e p o r t e d by H e i n l o o and t h e a q u e o u s i n s o l u b i l i t y o f t h e complex w i t h a m a r a n t h ( F . D . & C Red N o . 2 ) h a s been u t i l i s e d by H i l l 2 5 and by B u f t o n and S a d d l e r 1 4 2 t o a s s a y neomycin i n a q u e o u s s o l u t i o n . Amaranth may a l s o be u s e d t o d e t e r m i n e neomycin i n p r o d u c t i o n s a m p l e s from f e r m e n t a t i o n r e c o v e r 1 6 8 , The d y e , Orange 11, h a s b e e n s i m i l a r l y 1 4 3 d e s c r i b e d ( 1 max. = 484 nm) Complex f o r m a t i o n between neomycin and t h e dye i s v e r y d e p e n d a n t on t h e i o n i c s t r e n g t h o f t h e s o l u t i o n , thus n e c e s s i t a t i n g c a r e f u l c o n t r o l of reaction c o n d i t i o n s t o e n s u r e c o m p l e t e p r e c i p i t a t i o n of t h e complex d u r i n g t h e a s s a y p r o c e d u r e . R e a c t i o n of sodium l 1 2 - n a p h t h o - q u i n o n e - 4-su l p h o n a t e w i t h

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n e 0 m y c i n l 4 ~ p r o d u c e san o r a n g e / y e l l o w product,Xmax 460nm i n a c e t i c a c i d , which h a s been u t i l i s e d t o d e t e r m i n e t h e neomycin c o n t e n t o f o p h t h a l m i c s o l u t i o n s g i v i n g r e s u l t s i n good a g r e e m e n t w i t h microbiological assays. S t r o n g l y b a s i c a n t i b i o t i c s may be p r e c i p i t a t e d by f o r m a t i o n of t h e c o l o u r e d r e i n e c k a t e s a l t which may t h e n be d e t e r m i n e d s p e c t r o p h o t o m e t r i ~ a l l y l ~B~ c . f o r d l 6 6 d i s s o l v e d t h e p r e c i p i t a t e d i k neomycin r e i n e c k a t e i n a c e t o n e and h a s s u c c e s s f u l l y u s e d t h i s p r o c e d u r e t o a s s a y neomycin e x t r a c t e d from t o p i c a l f o r m u l a t i o n s . Roushdi e t a1173 p r e f e r r e d t o oxidise the p r e c i p i t a t e with potassium p e r m a n g a n a t e and t h e n c o l o r i m e t r i c a l l y e s t i m a t e t h e chromate produced w i t h d i p h e n y l c a r b a z i d e . A r o m a t i c a l d e h y d e s r e a c t w i t h p r i m a r y amines f o r m i n g S c h i f f ' s b a s e s which a r e o f t e n c o l o u r e d . The c o l o u r may t h e n be u s e d t o q u a n t i t a t e t h e amine. Using t h i s p r i n c i p l e Kocy167 h a s shown neomycin t o form a y e l l o w - c o l o u r e d S c h i f f ' s b a s e w i t h s a l i c y l a l d e h y d e t h o u g h t h e q u a n t i t a t i v e a s p e c t of t h i s p r o c e d u r e i s , as y e t , i n c o m p l e t e . The c o m p l e x a t i o n o f neomycin w i t h c o p p e r r e s u l t s i n t h e f o r m a t i o n of a b l u e - c o l o u r e d compound which h a s a l s o been made t h e b a s i s o f a c o l o r i m e t r i c d e t e r m i n a t i o n o f neomycin i n pharmac e u t i c a l f o r m u l a t i o n s g 7 . Maximum c o l o u r i n t e n s i t y was o b s e r v e d a t a s o l u t i o n pH of 10. F u r t h e r i n v e s t i g a t i o n showed t h e s t o i c h i o m e t r y of t h e comp l e x t o be 1:l i n a l k a l i n e s o l u t i o n . ( b) I n d i r e c t Methods H y d r o l y s i s p r o d u c t s o f neomycin may b e an a m i n o - s u g a r , a p e n t o s e o r f u r f u r a l d e p e n d i n g on t h e r e a c t i o n c o n d i t i o n s c h o s e n . Each o f t h e s e e n t i t i e s h a s been u t i l i s e d f o r i n d i r e c t s p e c t r o p h o t o m e t r i c d e t e r m i n a t i o n o f neomycin. The p r e s e n c e o f a s u g a r m o i e t y i n neomycin was d e m o n s t r a t e d by Hamre e t a1145 who u s e d c a r b a z o l e and a n t h r o n e t o q u a n t i t a t e neomycins B and C i n commercial neomycin. The p r o c e d u r e s employed were t h o s e p r e v i o u s l y a p p l i e d t o manosido~ t r e p t o m y c i n,147. Penau e t l~~ r e p o r t e d poor

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a g r e e m e n t b e t w e e n r e s u l t s o b t a i n e d by t h e a n t h r o n e a s s a y and m i c r o b i o l o g i c a l p r o c e d u r e s and s u g g e s t e d t h e p r e s e n c e of c a r b o h y d r a t e i m p u r i t i e s i n neomycin t o be t h e r e a s o n . O t h e r r e a g e n t s which f o r m c o l u d comp l e x e s w i t h s u g a r s s u c h a s o r c i n o l 1 4 % 1 5 ? 9 ,1 5 0 ( 3,5d i h y d r o x y t o l u e n e ) and p h l o r o g l ~ c i n o (1 3 ,~ l ~ ~ 5, t r i h y d r o x y b e n z e n e ) have b e e n r e p o r t e d f o r t h e s p e c t r o p h o t o m e t r i c a s s a y o f neomycin. Hoodless c l a i m e d t h e p h l o r o g l u c i n o l p r o c e d u r e t o be less time-consuming t h a n o t h e r methods117 and a p p l i e d t h i s p r o c e d u r e t o neomycin s u l p h a t e raw m a t e r i a l . D ~ u l a k a s l dopted a s i m i l a r procedure t o d e t e r a~ ~ mine t h e neomycin s u l p h a t e c o n t e n t o f o p h t h a l m i c ointments. I n t e r f e r i n g substances co-extracted from t h e o i n t m e n t were s e p a r a t e d from neomycin by TLC b e f o r e r e a c t i n g t h e a n t i b i o t i c w i t h p h l o r o glucinol. The o r c i n o l p r o c e d u r e h a s been u s e d t o a s s a y t h e neomycin c o n t e n t of f e r m e n t e r b r o t h s 1 4 8 I 1 4 9 I 150 though i t i s n e c e s s a r y t o f i r s t s e p a r a t e t h e a n t i b i o t i c by i o n - e x c h a n g e c h r o m a t o g r a p h y . Korchegin15O r e p o r t e d good a g r e e m e n t between t h e o r c i n o l a s s a y f production samand t h e m i c r o b i o l o g i c a l a s s a ples. Khromov-and S t a r u n o v ay 7 ' and K o r c h a g i n e t compared t h e o r c i n o l and m i c r o b i o l o g i c a l a s s a y s f o r d e t e r m i n i n g t h e neomycin c o n t e n t of p o l y m e r - f i l m s a n d , a g a i n , good a g r e e m e n t b e t w e e n t h e two p r o c e d u r e s was n o t e d . A f t e r m i l d h y d r o l y s i s , neomycin h a s been shown t o g i v e a p o s i t i v e Elson-Morgan r e a c t i o n 1 5 1 , a r e a c t i o n c h a r a c t e r i s t i c o f amino-sugars152. A method i n v o l v i n g t h i s r e a c t i o n h a s been made t h e b a s i s of a q u a n t i t a t i v e a s s a y f o r n e ~ m y c i n l ~ ~ which h a s been u s e d t o d e t e r m i n e t h e neomycin cont e n t of f e r m e n t a t i o n b r o t h s 1 5 4 . V i g o r o u s h y d r o l y s i s o f neomycin r e s u l t s i n be q u a n t i t a t e d i n a number of d i f f e r e n t ways15xf156 . D u t c h e r e t a1157 u s e d a UV s p e c t r o p h o t o m e t r i c p r o c e d u r e , m e a s u r i n g t h e a b s o r b a n c e of t h e s o l u t i o n a t 280nm. A s i m i l a r p r o c e d u r e combined w i t h measurement of t h e o p t i c a l r o t a t i o n h a s b e e n u s e d by Brooks e t a l l 5 8 t o d e t e r m i n e t h e neomycin B and C c o n t e n t
t h e f o r m a t i o n o f f ~ r f u r a l which ma l ~ ~

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of t h e a n t i b i o t i c . Colorimetrically, the furfural o b t a i n e d from neomycin may be a s s a y e d w i t h an a n i l i n e by m e a s u r i n g t h e amount o f p i n k / r e d complex formed. Morgan e t a1159 u s e d 4 - b r o m o a n i l i n e i n t h i s c o n t e x t , t h e 4-bromo d e r i v a t i v e b e i n g more resist a n t t o t h e formation of i n t e r f e r i n g coloured prod u c t s t h a n a n i l i n e i t s e l f l 6 0 . T h i s method h a s been a p p l i e d t o v a r i o u s p h a r m a c e u t i c a l f o r m u l a t i o n s and r e s u l t s show good a g r e e m e n t w i t h m i c r o b i o l o g i c a l a s s a y s . A n i l i n e h a s been u s e d t o d e t e r m i n e t h e neomycin c o n t e n t o f f e r m e n t e r b r o t h s and o t h e r p r o d u c t i o n s a m p l e s l 6 1 , neomycin u n d e c y l e n a t e and neomycin c a p r y l a t e l 6 2 . Much work h a s been c a r r i e d o u t t o e s t a b l i s h optimum c o n d i t i o n s f o r a r e p r o d u c i b l e c o n v e r s i o n of neomycin t o f u r f u r a l , q u a n t i t a t i v e c o n v e r s i o n of t h e p e n t o s e m o i e t y t o f u r f u r a l b e i n g d i f f i c u l t and The a c i d s t r e n g t h t h e procedures tedious163t164. f o r optimum p r o d u c t i o n o f f u r f u r a l must b e s u c h t h a t i t i s s t r o n g enough t o h y d r o l y s e t h e s u g a r m o l e c u l e , y e t i n s u f f i c i e n t l y s t r o n g t o decompose t h e f u r f u r a l formed. A r e v i e w o f t h e l i t e r a t u r e by I v a s h k i v 1 7 0 showed 1 2 % h y d r o c h l o r i c a c i d t o b e a c c e p t e d a s optimum f o r c o n v e r s i o n o f p e n t o s e s t o f u r f u r a l . However, a s neomycin must f i r s t b e decomposed t o t h e pentose b e f o r e h y d r o l y s i s of t h e s u g a r can t a k e p l a c e , more v i o r o u s c o n d i t i o n s a r e r e q u i r e d . Dutcher e t a l l Z 4 used 4 0 % s u l p h u r i c a c i d t o produce an a c i d c o n c e n t r a t i o n o f 37% i n t h e s o l u t i o n f o r h y d r o l y s i s and o b t a i n e d maximum f o r m a t i o n o f f u r f u r a l a f t e r 1% h r s r e f l u x . Morgan e t a1159 however, u s e d 7 0 % s u l p h u r i c a c i d t o p r o d u c e a 25% a c i d conc e n t r a t i o n i n t h e h y d r o l y s i s s o l u t i o n and o b t a i n e d maximum f o r m a t i o n o f f u r f u r a l a f t e r one h o u r . I v a s h k i v 1 7 0 h a s e v a l u a t e d t h e u s e of b o t h hydroc h l o r i c and s u l p h u r i c a c i d s and found up t o f o u r t i m e s more f u r f u r a l t o be p r o d u c e d by s u l p h u r i c acid, the y i e l d being extremely s e n s i t i v e t o the c o n c e n t r a t i o n of a c i d employed. C o n t i n u i n g f u r t h e r work w i t h s u l p h u r i c a c i d , t h e f o r m a t i o n o f f u r f u r a l a s a f u n c t i o n o f neomycin c o n c e n t r a t i o n was e x amined and t h e c o n c l u s i o n r e a c h e d t h a t b e t t e r y i e l d s of f u r f u r a l a r e o b t a i n e d a t low c o n c e n t r a t i o n s of t h e a n t i b i o t i c . Heyes171 h a s shown a cons t a n t amount of f u r f u r a l t o be p r o d u c e d on hydrol y s i s w i t h 27-28% s u l p h u r i c a c i d , r e f l u x i n g f o r 1 h o u r . T h i s makes i t u n n e c e s s a r y t o a c c u r a t e l y

NEOMYCIN

435

p i p e t t e concentrated s o l u t i o n s of sulphuric acid. From t h e above d i s c u s s i o n i t would, t h e r e f o r e a p p e a r n e c e s s a r y t o e s t a b l i s h optimum c o n d i t i o n s f o r f u r f u r a l p r o d u c t i o n i n e a c h i n d i v i d u a l case. 6.3. Chromatographic Procedures 6.31. Counter-Current D i s t r i b u t i o n S w a r t , L e c h e v a l i e r and Waksman 1 7 6 have examined neomycin by t h e method o f c o u n t e r c u r r e n t d i s t r i b u t i o n u s i n g a p a r t i t i o n - s y s t e m of b o r a t e b u f f e r ( p H 7 . 6 ) / p e n t a s o l ( s y n t h e t i c amyl a l cohol)/stearic acid. T h r e e m a j o r components w i t h v a r y i n g amounts of neamine were found i n s a m p l e s of neomycin B from v a r i o u s s o u r c e s . D e t e r m i n a t i o n o f t h e amount of a n t i b i o t i c r e m a i n i n g i n e a c h t u b e f o l l o w i n g p a r t i t i o n was c a r r i e d o u t m i c r o b i o l o g i cally. I n an e x t e n s i o n o f t h i s s t u d y t o t h e whole g r o u p of n e o m y c i n - l i k e m o l e c u l e s S c h a f f n e r 1 7 7 however, h a s been u n a b l e t o s e p a r a t e neomycin B i n t o t h e s e p a r a t e components r e p o r t e d by S w a r t e t u s i n g e i t h e r o f f o u r p a r t i t i o n s y s t e m s . The s y s t e m s employed w e r e : Systems 1-3) 0 . 5 M b o r a t e b u f f e r a t pH 7 . 3 , 7.6 o r 7 . 8 / p e n t a s o l / s t e a r i c acid.
4 ) 0 . 5 % b i c a r b o n a t e buf f e r / p e n t a s o l / stearic acid.

Both c h e m i c a l and m i c r o b i o l o g i c a l assays w e r e u t i l i z e d t o determine the d i s t r i b u t i o n of a n t i b i o t i c i n t h e t u b e s . In a l l experiments t h e m i c r o b i o l o g i c a l a s s a y r e s u l t s w e r e more e r r a t i c t h a n t h e c o r r e s p o n d i n g c h e m i c a l a s s a y s and on t h i s b a s i s S c h a f f n e r e x p l a i n e d t h e d i f f e r e n c e between h i s r e s u l t s and t h o s e of S w a r t e t who u s e d o n l y m i c r o b i o l o g i c a l d e t e r m i n a t i o n s . I n t h i s same work S c h a f f n e r w a s a b l e t o d e m o n s t r a t e t h e same d i s t r i b u t i o n p a t t e r n f o r b o t h f r a m y c e t i n and neomycins B and C and t h u s t e n t a t i v e l y i d e n t i Similarly, but using f i e d f r a m y c e t i n a s neomycin. a p a r t i t i o n s y s t e m o f 3% p - t o l u e n e s u l p h o n i c a c i d i n 1N a c e t a t e b u f f e r (pH 3 . 8 ) / b u t a n o l B a i k i n a e t have i d e n t i f i e d m c e r i n and c o l i m y c i n a s neomycin a n d S a n n i k o v 1 7 5 h a s i d e n t i f i e d f r a m i c i n w i t h neomycin.

436

WILLIAM F. HEYES

From a p r e p a r a t i v e a s p e c t , Peck e t a l l 8 ' have employed t h e c o u n t e r - c u r r e n t d i s t r i b u t i o n t e c h n i q u e w i t h a p a r t i t i o n s y s t e m of water/ b u t a n o l / p - t o l u e n e s u l p h o n i c a c i d t o s e p a r a t e and p u r i f y t h e neamine (neomycin A ) p r e s e n t i n commerc i a l neomycin.
6.32 Electrophoresis

Neomycin i s a r e a d i l y i o n i s a b l e m o l e c u l e and s h o u l d t h u s b e s e p a r a b l e from o t h e r a n t i b i o t i c s by a p p l i c a t i o n of an e l e c t r i c f i e l d ( z o n e e l e c t r o p h o r e s i s ) . V a r i o u s w o r k e r s have s u c c e s s f u l l y a p p l i e d t h i s t e c h n i q u e t o neomycin and T a b l e 10 summarises t h e c o n d i t i o n s r e p o r t e d i n t h e l i t e r a t u r e . A number o f a u t h o r s d e s c r i b e d t h e q u a l i t a t i v e s e p a r a t i o n o f neomycin from o t h e r c h e m i c a l - t p e s of a n t i b i o t i c s u s i n g p a p e r - e l e c t r o p h o r e s i s l 8 y t 1 8 5 , 1 8 7 w h i l e Ochab189 d e s c r i b e d s y s r e m s s p e c i f i c a l l y d e s i g n e d t o s e p a r a t e compounds within the i n o g l y c o s i d e group of a n t i b i o t i c s . C a r r e t all' have r e p o r t e d t h e q u a n t i t a t i v e d e t e r m i n a t i o n o f neomycin i n t h e p r e s e n c e o f p o l y m i x i n and b a c i t r a c i n . Using p a p e r e l e c t r o p h o r e s i s q u a n t i t a t i o n of neomycin w a s a c c o m p l i s h e d c o l o r i metrically with ninhydrin. Lightbown and DeRossi 188 s u b s t i t u t e d an a g a r - g e l s u p p o r t f o r t h e more u s u a l c h r o m a t o g r a p h y - p a p e r and s u c c e s s f u l l y s e p a r a t e d neomycin from a v a r i e t y o f a n t i b i o t i c s b u t n o t from paromomycin. However, ' q u a n t i t a t i o n o f t h i s p r o c e d u r e w a s n o t p o s s i b l e d u e t o t h e e l o n g a t e d zone of i n h i b i t i o n produced by neomycin on i n c u b a t i o n o f the agar-gel. The same a u t h o r s a l s o r e p o r t e d some anomalous b e h a v i o u r of t h e neomycin i o n i n t h e p r e s e n c e of c e r t a i n t y p e s of a g a r . Neomycin i s a b a s i c m o l e c u l e and u n d e r n o r m a l c i r c u m s t a n c e s would be e x p e c t e d t o b e h a v e a s a c a t i o n and m i g r a t e t o t h e anode. However, w i t h c e r t a i n t y p e s of a g a r t h e neomycin w a s o b s e r v e d t o m i g r a t e t o w a r d s t h e c a t h o d e . T h i s phenomena w a s r e p e a t e d l y c o n f i r m e d though n o t e x p l a i n e d . A s y s t e m s u i t a b l e f o r t h e q u a n t i t a t i v e e s t i m a t i o n of neomycin u s i n an a g a r g e l s u p p o r t h a s been d e s c r i b e d by Grynney83. T o o b t a i n a good c l e a r zone f o r neomycin i t w a s n e c e s s a r y t o change t h e b u f f e r e d e l e c t r o l y t e / a g a r - g e l t o a p h o s p h a t e s y s t e m a t pH 6 . 5 . D e t e c t i o n of t h e

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Table lO(Contd..

.)
D e t e c t i n g Agent (See a l s o S ectio n 6.34) Reference

E l e c t r o p h o r e s i s of Neomycin Electrolyte Medium ~Conditions

5. Trishydroxymethylaminomethane/maleic a c i d / sodium h y d r o x i d e ( 0 . 9 0 7 % :0.870%:0 . 0 0 2 % ) p 5.6 H

P

agar-gel buffered p 5.6 H

2 ooov 2 0 0 mA

B .n u b L L L L A
NCTC 8 2 4 1

188

6.

m w

2 M f o r m i c acid/O.lM toluene sulphonate/ p r o p ano l / w a C e r ( 5 0 : 50:5 :40)pH 1 . 8

paper

3 40v

B . A u bLLk5A
ATCC 6633

189

(40:5:5:50)

pH 1 . 9 paper 340v

1 89

7.

2 M f o r m i c acid/O.lM

73. Aub&.k%A
ATCC 6633

189

toluene sulphonate/ water ( 4 0 : 5 :55)pH 1 . 9

paper 340v

8 . 1 M ammonium h y d r o x i d e / 1 M sodium h y d r o x i d e / 0 . 1 M toluene sulphonate/ p r o p a n ol / w at e r ( 2 0 : O . 5 : 10: 1 0 : 6 9 . 5 ) p 10.8 H

B . AubLL&iA
ATCC 6633

189

T a b l e 10 (Contd.

.. )
Detect i n g Agent R e f e r en c e (See a l s o S e c t i o n 6.343

E l e c t r o p h o r e s i s o f Neomycin Electrolyte Medium Conditions

9.

1M ammonium h y d r o x i d e / 1M sodium h y d r o x i d e / w a t e r
(20:0.25:79.75)pH 11.5

paper

3 40v

B.bubXilib
ATCC 6633

189

10. i M sodium h y d r o x i d e / H w a t e r ( 5:95) p 1 2 - 2

paper a cr y 1 amide ge 1 a g a r -ge 1 buffered a t pH 6 . 5

3 40v

B.aubtiLib
ATCC 6633

189
190

11. Acrylamide g e l
'd P

p 4.6 H

ic

lOOV, 2mA
310 t o 360v

Naphthalene Black
Staphalococcub

1 2 . pH 6.5 p h o s p h a t e b u f f e r (KHPO4 22g/l; KH2P04,28g/l)diluted 1 : 4 0 with d i s t i l l e d water.

c p i d etr mid i b

183

(current 5 50mA)

ATCC 1 2 2 2 8

440

WILLIAM F. HEYES

a n t i b i o t i c was a c h i e v e d b i o a u t o g r a p h i c a l l y . The o r g a n i s m S f a p h a l o c o c c u a e p i d e h m i d i a ATCC 12228 w a s found t o be t h e m o s t s e n s i t i v e f o r t h i s p u r p o s e . (The d e s c r i p t i o n o f a l t e r n a t i v e b i o a u t o g r a p h i c a l s y s t e m s w i l l be found i n s e c t i o n 6 . 3 4 . ) Using an a c r y l a m i d e - g e l as s u p p o r t , CoombelgO h a s d e m o n s t r a t e d t h e q u a n t i t a t i v e a s s a y of neomycin w i t h a r e c o v e r y o f 9 6 % i n t h e p r e s e n c e of b a c i t r a c i n and p o l y m i x i n . I n t h i s case q u a n t i t a t i o n w a s a c h i e v e d by d e n s i t o m e t r y a f t e r s t a i n i n g the g e l with naphthalene black. E l e c t r o p h o r e s i s h a s a l s o been employed t o s e p a r a t e neomycin f r o m a n a l y t i c a l l y i n t e r f e r i n g s u b s t a n c e s s u c h a s p r o t e i n s . Hence B r a m m e r and Hemson182 have d e t e r m i n e d t h e neomycin c o n t e n t of b l o o d serum. Neomycin was s e p a r a t e d from t h e serum p r o t e i n s by e l e c t r o p h o r e s i s on c e l l u l o s e a c e t a t e and a s s a y e d c o l o r i m e t r i c a l l y f o l l o w i n g e l u t i o n from t h e s u p p o r t .
A modified technique c o n s i s t i n g of an e l e c t r i c f i e l d a p p l i e d p e r p e n d i c u l a r t o a f l o w i n g b u f f e r s o l u t i o n and u s i n g c h r o m a t o g r a p h y p a p e r a s support(cross-electrophoresis) h a s been e x p l o i t e d t o s t u d y t h e r e a c t i o n of neomycin w i t h h e ~ a r i n ~ ~ E v i~ e~ c e f o r t h e f o r m a t i o n o f a t l d n. neomycin-heparin complex was o b t a i n e d by t h i s means.

6.33.

Column Chromatoqraphy

The column c h r o m a t o g r a p h i c s e p a r a t i o n of neomycin B , neomycin C and neamine on an ion-exchan e column h a s been d e s c r i b e d by Maehr and S c h a f f n e r ?3 6 Dowex 1 X 2 ( O H f o r m ) was t h e i o n e x c h a n g e r e s i n and w a t e r t h e e l u t i n g s o l v e n t . The column e l u a t e was m o n i t o r e d by r e a c t i o n w i t h n i n h y d r i n . Using a slower e l u t i o n r a t e and a s m a l l e r p a r t i c l e s i z e of t h e same i o n - e x c h a n g e r e s i n Inouye and Ogawa19 r e p o r t e d improved r e s o l u t i o n of t h e p e a k s . The a p p l i c a t i o n o f t h i s p r o c e d u r e t o t h e q u a n t i t a t i v e d e t e r m i n a t i o n of neomycin C and n e a mine i n commercial s a m p l e s of raw m a t e r i a l h a s a l s o b e e n d e s c r i b e d 6 . When compared w i t h TLC res u l t s on t h e same s a m p l e s , t h e column chromatog r a p h i c method gave c o n s i s t e n t l y h i g h e r r e s u l t s f o r t h e neamine c o n t e n t . TLC e x a m i n a t i o n o f t h e

NEOMYCIN

44 I

f r a c t i o n of e l u a t e c o n t a i n i n g neamine , d e m o n s t r a t e d t h e p r e s e n c e o f t h r e e o t h e r u n i d e n t i f i e d compounds which gave a p o s i t i v e n i n h y d r i n r e a c t i o n t h u s exp l a i n i n g t h e o b s e r v e d d i f f e r e n c e between t h e t w o procedures.

R o e t s and Vanderhaeghe l9 h a v e a l s o examined neomycin by c h r o m a t o g r a p h y on Dowex 1x2 b u t t h e s e a u t h o r s m o n i t o r e d t h e column e l u a t e c o n d u c t o m e t r i c a l l y . G i l l e t e t a l l g 4 appl i e d a s i m i l a r p r o c e d u r e t o t h e e x a m i n a t i o n of v a r i o u s s a m p l e s of neomycin t o a s c e r t a i n t h e r a t i o of neomycin B:C.
A number of minor components p r e s e n t i n commercial neomycin h a v e been s e p a r a t e d by column c h r o m a t o g r a p h y on a c a r b o x y l i c c a t i o n exchange r e s i n I A m b e r l i t e ~ ~ - 5 The ~ ~ ~ . 0 components were e l u t e d from t h e r e s i n w i t h ammonium h y d r o x i d e s o l u t i o n . T . L . C . o f t h e e l u e n t f r a c t i o n s showed t h e p r e s e n c e of two p r e v i o u s l y u n r e p o r t e d i m p u r i t i e s which were t h e n i s o l a t e d on Dowex 1 x 2 and t e n t a t i v e l y i d e n t i f i e d u s i n g NMR and mass s p e c t r o metry.

When i n v e s t i g a t i n g t h e a c i d h y d r o l y s i s o f neomycin I B r a z h n i k o v a and K u d i n o v a l g 8 added a s u l p h o c a t i on i c - t y p e i o n -exchange re s i n t o t h e a c i d s o l u t i o n of a n t i b i o t i c t o a d s o r b i n t e r m e d i a t e h y d r o l y s i s p r o d u c t s and t h u s p r e v e n t f u r t h e r decomposition d u r i n g h e a t i n g . Following e l u t i o n o f t h e r e s i n . i n a column t h e p r o d u c t s o f neomycin h y d r o l y s i s w e r e i d e n t i f i e d and shown t o be d e p e n d a n t on t h e t i m e o f h y d r o l y s i s .
An i o n - e x c h a n g e c h r o m a t o g r a p h i c p r o c e d u r e u t i l i s i n g Dowex 1x2 h a s been made t h e b a s i s of an i n d u s t r i a l process f o r manufacturing neomycin B which i s f r e e from neomycin C l g 5 .

The i s o l a t i o n o f neamine on A m b e r l i t e I R C 5 0 ( N a f o r m ) f o l l o w i n g t h e a c i d hydrol y s i s of neomycin h a s been d e s c r i b e d l g 6 . Neamine i s e l u t e d from t h e i o n - e x c h a n g e column w i t h h d r o chloric acid. T h i s p r o c e s s had been p a t e n t e d 7 9 7 a s a means of m a n u f a c t u r i n g neamine.

442

WILLIAM F. HEYES

Many column c h r o m a t o g r a p h i c p r o c e d u r e s have been d e s c r i b e d f o r t h e commercial s e p a r a t i o n and p u r i f i c a t i o n of neomycin f o l l o w i n g fermentation. 6.34.

Paper and Thin-Layer Chromatograph

The e x a m i n a t i o n of neomycin by p a p e r and t h i n - l a y e r c h r o m a t o g r a p h y h a s b e e n f o r t w o main p u r p o s e s : a ) t o s e p a r a t e and i d e n t i f y neomycin i n t h e p r e s e n c e of o t h e r a n t i b i o t i c s , and b ) t o s e p a r a t e i n d i v i d u a l components and d e g r a d a t i o n p r o d u c t s o f neomycin. The s o l v e n t s y s t e m s employed f o r t h e s e p u r p o s e s a r e summarised i n T a b l e s 1 2 and 1 3 ( p a p e r c h r o m a t o g r a p h y ) and T a b l e s 1 4 and 1 5 (thin-layer procedures)

V i s u a l i s a t i o n of t h e chromatograms h a s been r e p o r t e d u s i n g b o t h c h e m i c a l and m i c r o b i o l o g i c a l means. Chemical s p r a y r e a g e n t s which h a v e been d e s c r i b e d a r e l i s t e d i n T a b l e 1 6 . The v a r i o u s o r g a n i s m s t h a t have b e e n employed f o r b i o a u t o g r a p h y a r e g i v e n i n T a b l e 11. T a b l e 11

- s u a l i s a t i o n o f Chromatograms V iBioautographically Organism Reference 201,202,204,228 234,235

B putniluh
a c h e t r i c h i a c o l i

22 4

205,206
2 0 7 , 2 2 0 , 2 3 1 , 2 32

S t a p h a l o c o c c u h aukeuh
ATCC 6538 s t r a i n

Table 12 P a p e r C h r o m a t o g r a p h i c S y s t e m s S e p a r a t i n g Neomycin from o t h e r A n t i b i o t i c s Paper Whatman N o . 1 Whatman No.1 Whatman N o . 1 Direction

A s c e n d i n9

S o l v e n t Sys t e m Water - s a t u r a t e d butanol

Rf V a l u e 0.84
)

Use

Reference
202

1
0.85
202

Ascend i n g Ascending

n-Butano l/ace t i c a c i d / w a t e r ( 2 :1:1)

n-Butanol/pyridine/ w a t e r (1:0 . 6 :1) 3 % Aqueous ammonium chloride

1
0.94
0.00
) Identifi-

202
202 202

Whatman N o . 1 A s c e n d i n g Whatman No.1 Ascending

Benzene/acetic acid/ w a t e r ( 2 : 1:1), o r g a n i c 1 yer a

n -Bu t a n 0 1 s a t u r a t e d -

0.89

) c a t i o n of Ineomycin i n ) t h e presence )of o t h e r )antibiotics

1
) )

Whatman No.1

A s cend i n g

0.07

202
203

water
Ascending Toyo No. 5 0 Descending for 9 hrs. Methanol/3 % a q u e o u s sodium c h l o r i d e ( 5 :1) n-Butanol/pyridine/ acetic acid/water ( 1 5 : 10: 3 :1 2 ) 0.03 ) S e p a r a t i o n of neomycin from )other I streptomyces )antibiotics

204

Table 1 2 (Contd.. 1 P a p e r C h r o m a t o g r a p h i c Systems S e p a r a t i n g Neomycin from o t h e r A n t i b i o t i c s Paper Toyo N o . 5 0

D i r e c tion

S o l v e n t System
2 % p-toluene sulphoni c acid + 2% pyridine i n wet n - b u t a n o l

Rf V a l u e

Use

Reference
204

Descending for 9 hrs.

0 . 4 8 - 0 . 5 4 ) S e p a r a t i o n of Ineomycin f r o m ) other 1s t r e p t o m y c e s )antibiotics.

0.06
0.98
Oe0

Toyo No. 5 0 Whatman No.1

P

Ascending Ascending Ascending

t-Butanol/acetic w a t e r ( 5 5 :6 :39)

acid/

1
)

20 4

Water
Water-saturated n -bu t a n 0 1 Water-saturated e t h y l acetate W a t e r - s a t u r a t e d benzene Methanol/water ( 4 0 : 6 0 ) n-Propanol/water ( 4 0 : 6 0 ) Methanol/3% a q u e o u s ammonium c h l o r i d e ( 7 0 : 3 0 ) Methyl e t h y l k e t o n e / n b u t a n o l / w a t e r ( 3 0 : 5 :6 5 )

205 Systematic
2 05 2 05

Whatman N o . 1

!i d e n t i f i c a t i o n
'of the antibiotics

Whatman N o . 1 Ascending Whatman N o . 1 Ascending Whatman No.1 Whatman N o . 1 Whatman No.1 Ascending Ascending Ascending Whatman N o . 1 Ascending

1
0.0
)

2 05
205,206 205,206 205,206 205,206

0.21 )
0.20 ) 0.11 ) 1

1
0.27
)

Table 1 2 (Contd..

.)
__ Use

P a p e r C h r o m a t o g r a p h i c Systems S e p a r a t i n g Neomycin from o t h e r A n t i b i o t i c s Paper Whatman No.1 Direction Ascending S o l v e n t System

6 6 . 6 % aqueous propanol/benzene/ethylene glycol/acetic a c i d ( 5 :5 :1 . 5 :1)

Rf V a l u e
0.03 )
)

R e f e re n c e

207

1
0.27
O.O0

Whatman No.1 Whatman N o . 1

Ascending Ascending

5 0 % aqueous p r o p a n o l / a c e t i c a c i d ( 2 5 : 1) Water-saturated butanol/acetic acid/potassium c y a n i d e ( 100: 1:0: 0 5 9 ) n-Butanol/water/acetic acid(30:13:8) n-Butanol/acetic acid/ pyridine/water/ethanol ( 6 0 : 15: 6 :5 :5) n-Butanol/water/acetic acid/pyridine/sodium c h l o r i d e ( 3 0 : 1 2 : 7 :2: 0.1)

' S e p a r a t i o n of neomycin from ch l o r a m p h e n i c o l , tetracycline ) and p e n i c i l l i n

1
)

207

207

Whatman No.3 Whatman No.3

Ascending Ascending

Oso2

0.04

' s e p a r a t i o n of neomycin f r o m 'polymixin B and b a c i t r a c i n

186,208 186,208

!
1

Whatman No. 3

Ascending

1 0.05 )

186,208

Table 13

P a p e r C h r o m a t o g r a p h i c S y s t e m s S e p a r a t i n g Neomycin Components and D e g r a d a t i o n P r o d u c t s Paper Direction S o l v e n t Sy s t e m Rf Value Neomycin Neamine

B C

Use

Reference

Water /bu t a n o 1 / m e t h a n ol / m e t h y 1 orange . ( 2 : 4 : 1:1:5 ) Butanol/pyridine/ w a t e r ( 6 0 : 30: 4 0 )

N o t available

Stability testing fradiomycin Separation o f B and C as a c e t y l derivatives Separation of B and C as a c e t y l derivatives

) ) )
)

209

P P m

Whatman No.1

Descending

0.29

0.19

2 10

Butanol/pyridine/ w a t e r ( 3 :2: 1)

N o t available

211

Toyo No.50

Descending for 9 hrs

n-Propanol/ pyridine/acetic acid/water (15:10:3:12)

0.13

0.15

0.28)

Separation of neamine f r o m neomycins B and C

204

Table 13 (Contd.. ) P a p e r C h r o m a t o g r a p h i c Systems S e p a r a t i n g Neomycin Components and D e g r a d a t i o n P r o d u c t s Paper Direction S o l v e n t System Rf Value Neomycin Neamine
B C

Use

Reference

Toyo N o . 50 Toyo

Descending for 9 hrs Ascending Ascending

2 % p-toluene sulphonic acid in w a t e r s a t u r a t e d n-butanol

1 . 5 % sodium c h l o r i d e i n 80% m e t h a n o l
Butanol/acetic acid/wat er (4:l: 5) n-Butanol/water/ piperidine (84:16:2)

0.11-

0.20

0.23

)of neamine
) from neo)mycins B )and C

)Separation

20 4

No. 5 0
P 4 P

0.08-

0.11

0.22

1
Not a v a i l a b l e Separation of B and C as acetyl derivatives 212

Grade M

Whatman NO. 4

Descending f o r 24-3g h r s , 28 C

0.70 0.35 1-00 Q u a n t i t a t i v e (values r e l a t i v e spectrophotot o neamine) m e t r i c determ i n a t i o n of components

213

Table 1 3 ( C o n t d . .

.)
Use

P a p e r C h r o m a t o g r a p h i c Systems S e p a r a t i n g Neomycin Components and D e g r a d a t i o n P r o d u c t s Paper Direction S o l v e n t System

B

Rf V a l u e Neomycin
C

Reference

Neamine

Whatman No. 1

Descending f o r 2 4- 36 h r s , 2527OC

Methyl e t h y l ke t o n e / t -bu t ano 1/ methanol/6.5N ammonium h y d r o x i d e (16:3:1:6)

0.54 0.30 1.00 (values r e l a t i v e t o neamine)

Separation a s f r e e bases Q u a n ti t a t i on by s p e c t r o ph ot o m e t r y

214

m P

Whatman No. 1

Descending f o r 8-400 hrs , 24 C Radial

Methyl e t h y l k e t o n e / 0 . 5 7 0.34 1.00 isopropanol/6.5N (values r e l a t i v e ammonium h y d r o x i d e t o neamine) (80:20:30) But an o l / p y r i d i n e / a c e t i c acid/water (15: 10: 3 :1 2 )
N o t available

2 15

Separation of degradation products S e p a r a t i o n of neamine from neomycin B and C

216

Whatman D e s c e n d i n g No.limf o r 18 h r s pregnated with P 8 H buffer

Butanol/water(l:2) 0.5 containing 4% t o l u e n e s u l p h o n ic a c i d and 2 % p i p e r i d i n e hydrochloride

0.6

228

Table 1 4 T h i n L a y e r C h r o m a t o g r a p h i c Systems S e p a r a t i n q Neomvcin from O t h e r A n t i b i o t i c s Ads o r b e n t

S i l i c a ge 1 G/

- l v e n t Sys tern So
n-Propanol/ethylacetate/ w a t e r / 1 3 . 4 M ammonium h y d r o x i d e ( 5 0 : 10: 30: 10) n-Propanol/pyridine/ a c e t i c acid/water ( 1 5 :10: 3 :1 2 1

Rf Value 0.52

Use
S e p a r a t i o n of neomycin from o t h e r aminoglycoside antibiotics S e p a r a t i o n of neomycin from other w a t e r soluble basic a n t i b i o t ic s

Reference
217

aluminium o x i d e (1:1) Cellulose MN

0.10

2 18

P \c

Kieselgel G

Wate r / m e t hano l / b u t an o l / butyl acetate/acetic a c i d ( 1 2 :2.5 :7 . 5 :4 0 :2 0 ) Water/butanol/pyridine/ a c e t i c a c i d . ( 1 4 :30: 2 0 : 6 ) ( 1 6 :4 0 : 8: 1 6 ) Water/butanol/acetic a c i d (39 :55 :6 )

0.0

219

1 S e p a r a t i o n of 1 neomycin from 1 bacitracin,

) p o l y m i x i n and

Kieselgel G

0.12

tyrothricin

219

0.00 )

Kieselgel G

1 1
0.00
)

219

T a b l e 1 4 (Contd..

.)
Use ) Sep aratio n of ) neomycin from )bacitracin, ) p o l y m i x i n and )tyrothricin

Thin L a y e r C h r o m a t o g r a p h i c Systems S e p a r a t i n g Neomycin from O t h e r A n t i b i o t i c s A s or b e n t d Kieselgel G S o l v e n t System Methanol/l7% aqueous ammonia c h l o r o f o r m , a q u e o u s l a y e r (10:10:20) Rf V a l u e Reference

0.31

219

Se ph adex G-15
VI P

pH6 p h o s p h a t e b u f f e r (0.025M) + 0.5M sodium chloride Ben z e n e / a c e t one/ ace t i c a c i d ( 4 :4 :2 ) But a n o l / a c e t i c a c i d / pyridine/water (30: 2 2 : 6 : 3 8 ) Butanol/water/acetic acid/ethanol/pyridine ( 6 0 : 10:15: 5: 6 )

1 1
0.75
) Identification

220

)of antibiotics Silica gel G Kieselgel G 0.35

1 1

221

? Separation
0.14
0.05

Kieselgel G

of ? nebmycinc i fromi n zin 1 and cpb lay mtirxai cn o

186 208

186 208

T a b l e 14 (Contd..

.)
Use
)

Thin Layer Chromatographic Systems S e p a r a t i n g Neomycin from Other A n t i b i o t i c s A so r ben t d Kieselgel G Kieselgel G + Kieselguhr
G(1:l)

S o l v e n t System Propanol/ethyl acetate/water/ 2 5 % ammonium hydroxide (100:20:60:20).

Rf Value 0.08

Reference
222 222

1
0.14
)

Kieselgel G + Kieselguhr G(1:2)

C e1 ulose l MN-300

1
0.22
)

222

0.32 0.12

) S e p a r a t i o n of 'glycosidic

222 222

Kieselgel G + aluminium oxide G type E ( 1 : l ) Kieselgel G impregnated w i t h sodium acetate

1
)

antibiotics

0.33

222

T a b l e 2 (Contd.. ) Thin Layer Chromatographic Systems S e p a r a t i n g Neomycin from Other A n t i b i o t i c s Ads o r b e n t Kieselgel G Kieselgel G + Kieselguhr G ( 1 : 1) Kieselgel G + Kieselguhr G(1:2)
VI P
h ,

S o l v e n t System Prop a n o l / e t hy 1 ace t a t e / w a t e r / 2 5 % ammonium hydroxide/ p y r i d i n e / 3 . 8 5 % aqueous ammonium a c e t a t e ( 100:2 0 :6 0 :2 0 :10:2 00)
II

Rf

Value
0.11
0.13

Use

Reference
222
222

1 1

0.16

222

Kieselgel G impregnated w i t h 3.85% aqueous ammonium acetate Kieselgel G Kieselgel G + Kieselguhr G(1:l) Kieselgel G + Kieselguhr G(1:2)

0.08

1 1
1
S e p a r a t i o n of glycosidic antibiotics

222

1
Ethanol/ethylacetate/water/ 25% ammonium hydroxide/ p y r i d i n e / 3 . 8 5 % aqueous ammonium a c e t a t e
(100:20:60:20:10:200)
It

0.08

222

1
0.11

222

1
0.12 222

1 1

Table 1 4 (Contd. ) Thin Layer Chromatographic Systems S e p a r a t i n q Neomycin from Other A n t i b i o t i c s Adsorbent Kieselgel G + aluminium oxide G ( t y p e E ) (1:1) Kieselgel G
VI P

S o l v e n t System

Rf Value 0.08
)

Use
1
1

Reference
222

Ethanol/ethylacetate/water/ 2 5 % ammonium hydroxide/ p y r i d i n e / 3 . 8 5 % aqueous ammonium a c e t a t e (100: 2 0 : 6 0 : 2 0 : 10: 2 0 0 )

Methanol/ethyl a c e t a t e / water/25 % ammonium hydroxi d e / p y r i d i n e / 3 . 8 5 % aqueous ammonium a c e t a t e (100:20:60:20:10:200)
11

1
0.06
) )

222

Kieselgel G + Kieselguhr G (1:1) Kieselgel G + Kieselguhr

G(1:2)

0.17

222

0.14
0.11

)
1 1
)

S e p a r a t i o n of glycosidic antibiotics

222

Kieselgel G + aluminium oxide G , type E ( 1 : l )

Methanol/ethyl a c e t a t e / water/25% ammonium hydroxi d e / p y r i d i n e / 3 . 8 5 % aqueous ammonium acetate ( 100 :2 0 :6 0 :2 0 : 10: 2 0 0 )

222

1 1

- 1 4 (Contd. . I Table Thin Layer Chromatographic Systems S e p a r a t i n g Neomycin from Other A n t i b i o t i c s Ads o r b e n t Kieselgel G impregnated w i t h 3.85% aqueous ammonium acetate
VI

So lven t System

Rf v a l u e 0.08

Reference 222

Methanol/ethyl acetate/ water/25% ammonium hydroxi d e / p y r i d i n e / 3 . 8 5 % aqueous ammonium a c e t a t e (100: 20: 60: 2 0 : 10: 2 0 0 ) Butanol/methanol/ethyl a c e t a t e / w a t e r / 2 5 % ammonium

1 1 1 1 1

Kieselgel G Kieselgel G + Kieselguhr G ( 1 : 1) Kieselgel G + Kieselguhr G(1:2) Kieselgel G + aluminium oxide G , type E ( 1 : l ) Kieselgel G + Kieselguhr G(1:2)

0.06

222

fr

hydroxide/pyridine/3.85% aqueous ammonium acetate

(100: 100: 20: 60: 20: 10: 2 0 0 )

1
1
) S e p a r a t i o n of ) glycosidic ) antibiotics

0.07

222

0.13 0.05

222
222

1
)

1
25 % ammonium hydroxide/ water/acetone (16: 144: 40) 0.36

222

T a b l e 1 4 (Contd.. 1 Thin Layer Chromatographic Systems S e p a r a t i n g Neomycin from Other A n t i b i o t i c s Adsorbent Kieselgel G + Kieselguhr G(1:2)impregnated with 2 % sodium a c e t a t e Kieselgel G + aluminium oxide G , t y p e E (1:1) Kieselgel G + Kieselguhr G ( 1 : 1) impregnated with 2 % sodium a c e t a t e Kieselgel G S o l v e n t System
2 5 % ammonium hydroxide/ w a t e r/ a c e t on e (16:144 :40)

Rf Value 0.34

Use -

Reference
222

1 1 1 1

If

0.48

) S e p a r a t i o n of glycosidic antibiotics

222

1
11

0.43

222

1 1 1 1
Water/sodium c i t r a t e / c i t r i c acid (100:20:5)
0.95

Classification of a n t i b i o t i c s

223

Table 15 Thin Layer Chromatographic Systems S e p a r a t i n g Neomycin Components and Degradation P r o d u c t s Adsorbent S o l v e n t System Neomycin
B

Rf Value Neomycin
C

Use
Neamine
0.60 0.61
0.00

Reference

Acidified carbon Acidified carbon

VI P

Water 0.5N s u l p h u r i c acid Water 0.5N s u l p h u r i c acid

0.10

0.10

0.21
0.00

0.43
0.00

)Assay of ) neomycin )components ) i n commer) c i a 1 form)ulations

)

22 4 224 224

Untreated carbon Untreated carbon

0.24 0.37-0.45

0.45 0.53-0.74

0.54 0.63-0.92

1 1
)

224
204

0.5N hydroCarbon chloric acid treated w i t h hydrochloric acid

I I

1
) Separation ) of neomycins

)B and C and

0.5N hydroc h l o r i c acid/ methanol ( 4 :1)

0.44-0.72

0.58-0.81

0.64-0.91

) neamine )

20 4

1 1

Tab -l e 1 5 ( C o n t d . . -

.)
Use -

Thin L a y e r C h r o m a t o q r a p h i c Systems S e p a r a t i n g Neomycin Components and D e g r a d a t i o n P r o d u c t s

Ad s or b e n t

S o l v e n t System Neomycin

Rf V a l u e Neomycin

Reference

Neamine
0.62
)

B
Carbon treated with w a t e r 0.5N hydrochloric acid 0.5N hydrochloric acid/ m e t h a n o l ( 4 : 1) Carbon/ gypsum treated with sulphuric acid 0.5N s u l p h u r i c acid 0.41

C
0.57
20 4

0.48

0.58

0.68

1
)

204

1
0.20

0.59

0.80 ) Sep aratio n of neomycins ) B and C and ) neamine

20 4

1
1

1
0.5N s u l p h u r i c a c i d / m e t h a n o l ( 4 : 1) 0.5N hydrochloric acid 0.64
0.16

0.82
0.28

0.85 )

204
20 4

1
0.30

Carbon/ gypsum treated with water

Table 15 (Contd.. ) Thin Layer Chromatographic Systems S e p a r a t i n g Neomycin Components and Degradation P r o d u c t s Adsorbent S o l v e n t System Neomycin
B

R f Value

Use

R e f er e n c e

Neomycin C
0.40

Neamine

Carbon/ gypsum treated with water

W v ,

0.5N s u l p h u r i c acid

0.24

1 1

20 4

I1

0.5N hydrochloric acid/ methanol (1 1) :

0.36

0.36

0.46

)Separation ) o f neomycin )B and C and ) neamine

20 4

11

0.5N hydroc h l o r i c acid/ p r o p a n o l ( 1 : 1)

3.85% aqueous ammonium acetate
3 . 4 % ammonium hydroxide

0.48

0.48

0.52

1 1
Assay of neamine Assay of neomycin C

20 4

Kieselgel H Kieselgel H

0.13 0.37

0.13
0.44

0.36

196,9

0.47

196,9

Table 15 (Contd.. ) Thin Layer Chromatographic Systems S e p a r a t i n g Neomycin Components and Degradation P r o d u c t s Ads orben t S o l v e n t System Neomycin
B

R f Value -

Use -

R e fe r e n ce

Neomycin
C

Neamine 0.31 Separation components

2 15

C e 1l u l o s e MN

Methyl e t h y l
ketone/iso-

0.21

0.12

propanol/6.5N ammonium hydroxide (80:20:30)

of neomycin

2 \o

Cellulose MN-300

Methyl e t h y l k e tone/me t h a n o 1/ isopropanol/7.9N ammonium hydro x i d e (10: 8.5 :3 :7 ) Propanol/pyridine/ a c e t i c acid/water ( 100 : 6 :2 0 :80) 6
11

0.52

0.24

0.61

236

Cellulose MN-300 Silica gel G

single 0.25 double 0.33

0.57

development 0.20 0.47 development 0.25 0.60

0.66

)Assay of )neomycins B 226 land C and nea)mine i n pharm) a c e u t i c a l form0.73 ) u l a t i o n s 226

Table 15 (Contd.. ) Thin Layer Chromatographic Systems S e p a r a t i n g Neomycin Components and Deqradation P r o d u c t s A s o r ben t d S o l v e n t System Neomycin
B

Rf Value Neomycin C 0.46 Neamine 0.73 )

Use

Reference

Cellulose MN-300/ Kieselguhr G(1:l)

m

Propanol/pyridine/ acetic acid/water (100: 66 :20: 80) Prop an o l /e t hy 1 acetate/water/25% ammonium hydroxide (100:20:60:20)

0.53

226

1 1
0.23 0.23 0.42
)

Silica gel G

226

1 1
0.17
)Assay of )neomycins B 0.43 ) a n d C and 226 )nearnine i n )pharmaceutical ) formulations

Cellulose MN-300/ Kieselguhr G(1:l)

Silica gel G/ Kieselguhr G(1:2)

Methanol/3% aqueous 0.36 sodium c h l o r i d e ( 2 :1)

1
3.85 % aqueous ammonium a c e t a t e

0.22

0.22

'0.39 )

226

1 1 1

Table 1 5 (Contd.. ) Thin Layer Chromatographic Systems S e p a r a t i n g Neomycin Components and Degradation P r o d u c t s Adsorbent S o l v e n t System Neomycin
B

Rf Value Neomycin
C

Use -

Reference

Neamine

Silica gel G

Methyl e t h y l ketone/ t b u t ano l / m e t han o1 / 6.5N ammonium hydroxide ( 1 6 0 : 30: 10: 6 0 )

II

0.14

0.11

0.22

226

Kieselguhr
G

0.46

0.68

0.84

purified Silica gel G Propanol/e than0.18 ol/ethyl acetate/ water/25% ammonium hydroxide/pyridine/ 3.85%ammonium a c e t a t e (100: 100: 2 0 : 6 0 : 20: 10: 2 0 0 )
I I

0.15

)Assay of )neomycins B ) a n d C and lneamine i n )pharmaceutical 0 - 38 f o r m u l a t i o n s

226

Silica g e l G/ Kieselguhr G(1:2)

0.18

0.11

0.32 )

1
226

Table 1 5 (Contd.. ) Thin Layer Chromatographic Systems S e p a r a t i n g Neomycin Components and Degradation P r o d u c t s Adsorbent S o l v e n t System Neomycin B
R f Value

Use -

Reference

Neomycin
C

Neamine 0.28 S e p a r a t i o n of neamine from neomycins B and C Quantitative a n a l y s i s of n e omy c i n sulphate S t a b i l i t y of neomycin a f t e r e t h y l e n e oxide st e r i l i s a t i o n

Dowex 50x8

25SA)

sodium form(1onex

+ 58.49 sodium chloride/ t - b u t a n o l ( 1 0 : 1)

3% Ammonium hydroxide / a c e t o n e ( 1 6 0 : 40) 3% Ammonium hydroxide / a c e t o n e ( 1 6 0 : 90) 0.33 0.33

1.5M Sodium a c e t a t e , p 8.5 H

0.15

0.15

227

Silica ge 1

229

Kieselgel G

N o t available

233

N EOMY CIN

463

T a b l e 16

V i s u a l i s a t i o n of Chromatograms by C h e m i c a l Means Spray Reagent Col -o u r o f Neomycin zone Dark b l u e

R e f e r e n ce
210,211

1. E t h a n o l i c sodium h ypoch 1 r it e o f o l l o w e d by s t a r c h / potassium iodide solution 2 . Ch l o r i n e /e t h a n o 1 f o l l o w e d by s t a r c h / potassium iodide solution 3. C h l o r i n e / c a r b o n tetrachloride f o l l o w e d by s t a r c h / p o t as s ium i o d i d e / pyridine 4 . t - b u t y l hypochlor i t e / d i ch 1 oromethane/acetic acid f o l l o w e d by s t a r c h / potassium iodide solution 5. N i n h y d r i n

Dark b l u e

212

Dark b l u e

213

Dark b l u e

196

Purple

186,203,208 2 1 4 , 2 1 5 ,2 1 6 217,218,219 229,231 227 223 226

6.

Ninhydrin/cadmium acetate 7. p - d i m e t h y l aminoben z a l d e h y d e Ninhydrin/pd i m e t h y l aminobenzaldehyde 9 . Copper s u l p h a t e / ammonium h y d r oxide

8.

Purple/p ink Pink o r purple

Blue

221

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WILLIAM F. HEYES

Q u a n t i t a t i v e a n a l y s i s o f neomycin a f t e r c h r o m a t o g r a p h y u t i l i s i n g some o f t h e 1 v i s u a l i s a t i o n t e c h n i q u e s g i v e n i n T a b l e s 1 and 1 6 have been r e p o r t e d . Majumder a n d Majumder213 s e p a r a t e d neomycins B a n d C a s t h e a c e t y l d e r i v a t i v e s on p a p e r t h e n , f o l l o w i n g c o n v e r s i o n t o t h e c h l o r o d e r i v a t i v e s , t h e c o l o u r formed by r e a c t i o n w i t h t h e starch/iodine/hydrochloric a c i d r e a g e n t w a s measure d a t 570nm. A l a t e r p a p e r by t h e same a u t h o r s 2 1 4 d e s c r i b e d t h e s e p a r a t i o n o f neomycins B and C as t h e f r e e b a s e s . The r e s u l t i n g chromatograms w e r e ded e v e l o p e d w i t h n i n h y d r i n t h e n t h e c o l o u r e d complex e l u t e d i n t o m e t h a n o l and t h e a b s o r b a n c e o f t h e s o l u t i o n measured a t 570nm. Thin- l a y e r c h r o m a t o g r a p h y h a s been u s e d b y F o p p i a n o and Brown229 t o a s s a y t h e t o t a l neomycin B and C c o n t e n t of neomycin s u l p h a t e . The neomycin zone w a s s c r a p e d o f f t h e p l a t e and rea c t e d w i t h o r c i n o l / f e r r i c c h l o r i d e r e a g e n t , t h e abs o r b a n c e o f t h e r e s u l t i n g c o l o u r b e i n g measured a t 665nm. By t h i s p r o c e d u r e a p r e c i s i o n o f 2% was achieved. Ninhydrin h a s also been used t o quantitate neomycin c o l o r i m e t r i c a l l y f o l l o w i n g t h i n - l a e r c h r o m a t o g r a p h y . P r e g n o l a t t o and S a b i n 0 2 5 0 r e p o r t e d t h e a d d i t i o n of g l y c e r i n e t o t h e n i n h y d r i n s o l u t i o n t o improve t h e s e n s i t i v i t y and An a l t e r n a t i v e r e p r o d u c i b i l i t y of t h e p r o c e d u r e . c o l o r i m e t r i c p r o c e d u r e f o r t h e q u a n t i t a t i o n of neomycin a f t e r c h r o m a t o g r a p h y h a s been d e s c r i b e d by D o u l a k a s l 7 4 . Neomycin i s e l u t e d f r o m t h e s i l i c a g e l with p 12.5 phosphate b u f f e r th en o x i d i s e d H w i t h sodium h y p o b r o m i t e . The r e s u l t i n g a l d e h y d e i s complexed w i t h p h l o r o g l u c i n o l y i e l d i n g a p i n k c o l o u r e d p r o d u c t which i s measured s p e c t r o p h o t o metrically. The u s e o f b i o a u t o g r a p h y f o r t h e q u a n t i t a t i o n of chromatograms h a s b e e n d e s c r i b e d by many w o r k e r s . E m i l i a n o w i c z - C z e r s k a and Herman228 s e p a r a t e d neamine f r o m neomycins B and C t h e n q u a n t i t a t i v e l y d e t e r m i n e d t o t a l neomycin B and C by b i o a u t o g r a p h y w i t h B.hubtiCih. A l i n e a r res p o n s e f o r c o n c e n t r a t i o n s between 0 . 2 5 and 1 O p g o f neomycin w a s o b t a i n e d . With a s i m i l a r t e c h n i q u e b u t using a mathematical a n a l y s i s of the bioauto-

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g r a m s , Simon230 r e p o r t e d a q u a n t i t a t i v e d e t e r m i n a t i o n o f neomycin s u i t a b l e f o r u s e w i t h i r r e g u l a r Brodasky224 employed b i o a u t o g r a p h y shaped zones. w i t h BaciLLuh pumiluh f o r t h e a s s a y o f neomycin and neamine a f t e r s e p a r a t i o n by T.L.C. on a l a y e r of c a r b o n . The q u a n t i t a t i o n o f s m a l l amounts of n e o mycin C however, p r o v e d d i f f i c u l t , t h e d i f f i c u l t ies b e i n g a s s o c i a t e d w i t h t h e enhanced growth of t h e o r g a n i s m i n t h e p r e s e n c e o f c a r b o n ( f r o m t h e T.L.C. p l a t e ) and ammonium i o n s ( f r o m t h e c h r o m a t o g r a p h i c procedure). Similar d i f f i c u l t i e s w e r e experienced by S o k o l s k i and L ~ m m i s 2 3 ~ who a t t r i b u t e d p o o r neomycin z o n e s t o be t h e r e s u l t o f a n t a g o n i s m by t h e sodium c h l o r i d e p r e s e n t i n t h e b i o a u t o g r a p h i c s y s tem. An e x t e n s i v e s t u d y by L a n g n e r 2 0 1 d e m o n s t r a t e d maximum s e n s i t i v i t y f o r t h e d e t e r m i n a t i o n of neomycin t o b e a p p a r e n t u s i n g t h e o r g a n i s m B.dubtiLih (ATCC 6633) t o g e t h e r w i t h pH 8 T r i s a g a r . The d e t e c t i o n l i m i t f o l l o w i n g chromatography w a s r e p o r t e d t o be 0.05pg. 6.35. G a s - L i q u i d Chromatography

T s u j i and R o b e r t s o n l g 2 a c h i e v e d t h e s e p a r a t i o n of neomycin B , neomycin C and neamine as t h e t r i m e t h y l s i l y l e t h e r s on a 6 f t . column of 0 . 7 5 % OV-1 on G a s Chrom Q a t a t e m p e r a t u r e of 290OC. The same c o n d i t i o n s have a l s o b e e n shown t o s e p a r a t e n e o b i o s a m i n e B , neosamine and d e o x y s t r e p t a m i n e from neomycin and neamine. Hence t h e method c o u l d b e u s e d t o s t u d y t h e s t a b i l i t y o f neomycin o r t o m o n i t o r t h e b i o s y n t h e t i c p r o d u c t i o n U s e of t h e p r o c e d u r e t o assess t h e s t a b process. i l i t y o f neomycin i n p h a r m a c e u t i c a l f o r m u l a t i o n s h a s b e e n d e m o n s t r a t e d by Van G i e s s e n and T s u j i 2 3 7 w i t h t r i l a u r i n as i n t e r n a l s t a n d a r d . H o w e v e r , t h e s e a u t h o r s recommended a 2 f t . column packed w i t h 3 % OV-1 on G a s Chrom Q as l o n g e r columns r e q u i r e d a h i g h e r t e m p e r a t u r e t o c h r o m a t o g r a p h neomycin and c o n s e q u e n t l y h a d a r e d u c e d column l i f e . A concent r a t i o n o f 3 % OV-1 w a s p r e f e r r e d , a s 2 % o r less r e s u l t e d i n i n c r e a s e d column a d s o r p t i o n o f neomycin. A f t e r a f u r t h e r s t u d y of t h e p r o c e d u r e Margosis and T s u j i 2 3 8 recommended a number of improvements t o o p t i m i s e t h e a n a l y s i s o f neomycin by G . L . C . The improvements t o t h e a s s a y i n c l u d e d t h e modif icat i o n o f t h e i n j e c t i o n p o r t t o p r e v e n t s a m p l e dec o m p o s i t i o n by c o n t a c t o f i n j e c t e d m a t e r i a l w i t h

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WILLIAM F. HEYES

m e t a l o r Teflon and t h e a d d i t i o n of an a c c u r a t e volume of s i l y l a t i n g r e a g e n t t o each sample and s t a n d a r d t o overcome i n c o n s i s t e n t d e r i v a t i s a t i o n .

A comparison of t h e G.L.C. and microbiological(agar-diffusion) a s s a y s h a s been rep o r t e d by T s u j i e t a1239 who showed t h e neomycin c o n t e n t of neomycin s u l p h a t e powders determined by t h e G.L.C. method, t o c o r r e l a t e w e l l w i t h v a l u e s o b t a i n e d by t h e m i c r o b i o l o g i c a l a s s a y when it i s assumed neomycin C h a s 35% o f t h e b i o l o g i c a l act i v i t y of neomycin B.

Neomycin h a s been s e p a r a t e d from m i x t u r e s of o t h e r aminoglycoside a n t i b i o t i c s cont a i n i n g t h e 2-deoxystreptamine moiety a s b o t h t h e p e r t r i m e t h y l s i l y l d e r i v a t i v e and t h e N - t r i f l u o r o a c e t y l p e r t r i m e t h y l s i l y l d e r i v a t i v e u s i n g a column of 0.75% OV-1 on Gas Chrom Q 2 4 0 . The p r o c e d u r e may be used t o e s t i m a t e t h e number of s u g a r moieti e s bound i n t h e a n t i b i o t i c as a close r e l a t i o n s h i p e x i s t s between t h e number of r i n g s and t h e retention t i m e . Examination of neomycin B by a combined G.C.-M.S. procedure h a s been r e p o r t e d by Murata e t a l l 5 . G.C. w a s accomplished w i t h t h e t r i m e t h y l s i l y l d e r i v a t i v e on a l m column of 1% OV-1 on Chromosorb W , by which means neomycin was s e p a r a t ed from kanamycin. The r e s u l t i n g mass s p e c t r a of t h e neomycin d e r i v a t i v e e x h i b i t e d a minute molec u l a r i o n peak a t m / e 1550 i n d i c a t i n g t h a t a l l a c t i v e hydrogens of b o t h hydroxy and amine groups were completely s i l y l a t e d . 6.4.Microbiological Procedures 6 . 4 1 . T u r b i d i m e t r i c Assay Various organisms have 'been used t o a s s a y neomycin t u r b i d i m e t r i c a l l y . These are summarised i n T a b l e 1 7 w i t h t h e working concent r a t i o n f o r t h e a n t i b i o t i c as r e p o r t e d by t h e respective authors.
O f t h e organisms t a b u l a t e d overl e a f K.pneumaniae i s g e n e r a l l y used f o r r o u t i n e assays.

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Table 1 7 C o n d i t i o n s of T u r b i d i m e t r i c Assay Organism

- -

S.auheub NRRLB314 I I II 209P

11
11

C o n c e n t r a t i o n Range of Assay ( T o t a l pg neomycin base required i n s o l u t i o n t o be assayed) 2.5 5.0

Reference

2 41 242 243 2 48 244 245 I 246 I 268 247 253 2 48 248 248 248 249

100-300

F1

0.016

0.04

s t h epzo cu cc U b

ATCC 10031
II

daecalib ATCC 1054 K l e bb i e l l a pneumaniae PC1602 Klebbiella pneumo n i a e

120-200 6-15 6-14 1- 3 1-20 3-7 20-60 2-6 4-6

0-6

E.coli ATCC 10536

II

ATCC 11105

Lacto b a c i l l u b ahabino zua La c t o ba c i l l ub cab ei Lacto b a c i l l u a dehmenti L a c t u ba c i l l u n L eic hm a n n i Aeho b a c t e h aehogeneb

I P L 22K5118

H e i n 2 4 3 n o t e d an a p p a r e n t d e c r e a s e i n t h e p o t e n c y of neomycin when a s s a y e d t u r b i d i m e t r i c a l l y i f t h e i n n o c u l u m c o n t a i n e d e i t h e r sodium c h l o r i d e o r p o t a s s i u m phos h a t e . With S.auheub a s o r g a n i s m l S o k o l s k i e t a 12! d e m o n s t r a t e d t h e p r e 5? s e n c e of K C 1 t o a n t a g o n i s e t h e a c t i v i t y of neomycin C more t h a n t h a t of neomycin B.

WILLIAM F. HEYES

A c o m p a r a t i v e s t u d y o f t h e responce o f neomycin B and neomycin C i n t h e t u r b i d i metric a s s a y u s i n g K . pneumakziae (ATCC 10031) h a s been r e p o r t e d 2 3 9 . The r e s p o n s e - r a t i o neomycin B: neomycin C w a s 100:38.9.
An a u t o m a t e d t u r b i d i m e t r i c p r o c e d u r e u s i n g a T e c h n i c o n A u t o a n a l y z e r h a s a l s o been d e s c r i b e d (See S e c t i o n 6.5)

6.42.Agar-Diffusion

Assay

T a b l e 18 l i s t s t h e o r g a n i s m s t h a t have been recommended f o r d e t e r m i n i n g t h e microb i o l o g i c a l p o t e n c y o f neomycin. Tab -l e 1 8 Recommended Organ i s m Working Concentration (P g h l )
4-2 1 4-20
0.64

Reference

8. p u m i l i n
NCTC 8241

9 245

S aua eun ATCC 6538

S e p i d ekmidics ATCC 12228

1.56

245,268

I n a l l t h e above r e f e r e n c e s , t h e c y l i n d e r - p l a t e a s s a y p r o c e d u r e i s t h e one d e s c r i b e d though a l t e r n a t i v e p r o c e d u r e s have been r e p o r t e d i n t h e l i t e r a t u r e . Thus Davis and P a r k e 2 4 5 r e p o r t e d a l i n e a r - d i f f u s i o n s y s t e m i n which a s o l u t i o n o f t h e a n t i b i o t i c i s allowed t o d i f f u s e i n t o a g a r - f i l l e d The s t a n d a r d d e v i a t i o n s c a l glass capilliaries c u l a t e d f o r s e v e n e x p e r i m e n t s w e r e between 5 and 10&(100 r e s u l t s ) f o r t h e a s s a y o f neomycin. The p r o c e d u r e i s more economic i n t e r m s o f a g a r and n u t r i e n t s t h a n o t h e r d i f f u s i o n methods. K u z e l and Coffey255 m o d i f i e d t h e c o n v e n t i o n a l c y l i n d e r p l a t e p r o c e d u r e by u s i n g a c y l i n d e r s e a l e d a t one e n d , f o r m i n g a c u p . The c u p s are f i l l e d w i t h t h e a n t i b i o t i c s o l u t i o n s t o b e a s s a y e d , t h e n a p l a t e of i n o c u l a t e d a g a r i s i n v e r t e d and p l a c e d over t h e t o p of t h e c u p s . With t h i s method a s i g n i f i c a n t r e d u c t i o n i n t h e s t a n d a r d d e v i a t i o n f o r t h e a s s a y o f neomycin

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w a s c l a i m e d . The r e p l a c e m e n t o f c l i n d e r s by a e r 47Y d i s c s h a s a l s o been r e p o r t e d 2561275,278. ~~~~i claimed a standard d e v i a t i o n of 2% f o r the assay of neomycin by t h i s means and recommended t h e p a p e r d i s c p r o c e d u r e i n p r e f e r e n c e t o t h e c y l i n d e r method. The t y p e of p a p e r u s e d h a s been shown t o a f f e c t t h e ~~~ d i a m e t e r of t h e i n h i b i t i ~ n - z o n eWhatman s e e d t e s t p a p e r p r o d u c i n g t h e s m a l l e s t z o n e s . When d e t e r m i n i n g neomycin l e v e l s i n m i l k , K o s i k ~ w s k i ~ ~ ~ improved t h e s e n s i t i v i t y o f t h e p r o c e d u r e by s u b s t i t u t i n g d r i e d milk t a b l e t s f o r paper d i s c s .
S e v e r a l s t u d i e s of t h e f a c t o r s a f f e c t i n g t h e i n h i b i t i o n - z o n e s i z e i n t h e neomycin a g a r d i f f u s i o n a s s a y have been r e p o r t e d . P i n z e l i k e t a1257 d e m o n s t r a t e d t h a t v a r i o u s d e l a y s d u r i n g t h e a n a l y t i c a l procedure a f f e c t e d t h e s i z e of t h e i n h i b i t i o n zone. Refrigeration of t h e agar-plates p r i o r t o i n c u b a t i o n c a u s e d an i n c r e a s e i n zones i z e when compared w i t h s i m i l a r p l a t e s h e l d a t 2OoC. T h i s p r i n c i p l e h a s been u t i l i z e d by S i d d i q u e e t a1264 t o improve s e n s i t i v i t y when d e t e r m i n i n g t h e neomycin c o n t e n t o f m i l k . The p r e s e n c e o f K C l , N a C l , C a C 1 2 h a s been shown t o m a r k e d l y i n c r e a s e neomycin d i f f u s i o n i n a g a r 2 5 8 , 2 6 5 194. C o n v e r s e l y t h e p r e f p h o s p h a t e s 2 5 8 and of g l u c o s e / s t a r c h w i t h NaCl d e c r e a s e s t h e s i z e of t h e i n h i b i t i o n z o n e . I n c o r p o r a t i o n of non- f at milk i n t h e a g a r s i g n i f i c a n t l y r e d u c e s t h e s i z e o f t h e i n h i b i t i o n z o n e bec a u s e o f t h e i n t e r a c t i o n o f neom c i n w i t h t h e f r e e c a r b o x 1 g r o u p of m i l k p r o t e i n 2 6 3 F e d o r k o and K a t z 2 6 3 d i l u t e d neomycin s o l u t i o n s w i t h b l o o d serum and r e p o r t e d an i n c r e a s e i n t h e s i z e o f t h e i n h i b i t i o n zone when compared w i t h s i m i l a r s o l u t i o n s d i l u t e d w i t h pH 8 phos h a t e b u f f e r . S o k o l s k i e t a1252 and Yousef e t a1559 d e m o n s t r a t e d t h e p h y s i c a l b i n d i n g o f neomycin t o a g a r , a p r o c e s s which may be r e v e r s e d by a d d i t i o n o f o t a s s i u m o r sodium c h l o r i d e . F u r t h e r s t u d i e s 2 6 0 t 5 6 1 , 2 5 2 have r e p o r t e d t h e b i n d i n g t o be a f u n c t i o n o f b o t h t h e p H o f t h e a g a r and t h e m a n u f a c t u r e r . I n c o r p o r a t i o n o f 0.1M t r i s b u f f e r w i t h t h e a g a r (pH 8 ) h a s b e e n recommende d 2 6 0 , 261 i n o r d e r t o a c h i e v e t h e h i g h e s t s e n s i tivity. T r i s buffer results in a greater sensitivi t y t h a n p h o s p h a t e b u f f e r 2 6 1 and d o e s n o t a f f e c t t h e s i z e o f t h e i n h i b i t i o n zone w h e r e a s t h e p r e sence o f p h o s p h a t e r e d u c e s zone s i z e 2 5 2 . G i l l e t e t , 1 1 9 4 c o n c l u d e d t h a t t h e q u a l i t y of t h e a g a r a f f e c -

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t e d t h e i n h i b i t i o n zone s i z e . A comparison of 3 a g a r s showed t h e b e s t response f o r a g i v e n concent r a t i o n of neomycin w a s o b t a i n e d w i t h agarose.Comb i n i n g a g a r o s e w i t h recommendations d e s c r i b e d by o t h e r a u t h o r s , such a s t h e a d d i t i o n of C a C l and t h e use of t r i s b u f f e r , f u r t h e r i n c r e a s e d tge s i z e of t h e i n h i b i t i o n zone. The l i t e r a t u r e d e s c r i b e d above r e f e r s t o t h e a s s a y of t h e t o t a l neomycin complex i . e . neomycins B and C and neamine. To a s s a y neomycin B i n t h e p r e s e n c e of neomycin C and neamine by a m i c r o b i a l method S o k o l s k i and Carpenter265 adopted t h e f o l l o w i n g p r o c e d u r e . These a u t h o r s e m ployed a neomycin C - r e s i s t a n t organism (8.b u b i i l i b UC 564) and added K C 1 t o t h e a g a r t o t o t a l l y dep r e s s d i f f u s i o n of neamine and i n c r e a s e d i f f u s i o n of neomycin B . Neomycin B and C d i f f e r i n t h e i r b i o l o g i c a l a c t i v i t i e s though t h i s d i f f e r e n c e may v a r y w i t h t h e c o n d i t i o n s of t h e a s s a y . T y p i c a l l y neomycin C h a s an a c t i v i t y of 30-50% of t h a t of The p r e s e n c e of potassium c h l o neomycin B 2 6 6 r 2 3 9 . r i d e o r phosphate r e d u c e s t h e d i f f u s i o n of neomycin C more t h a n t h a t o f neomycin B252. Furthermore, t h i s a n t a g o n i s t i c e f f e c t of t h e s a l t s h a s been shown t o be more pronounced when u s i n g S . U U t r e U b a s organism than when u s i n g B.bubiilib. By a l t e r i n g t h e i o n i c s t r e n g t h o f t h e medium a system h a s been developed i n which t h e r e s p o n s e s of neomycins B and C a r e i d e n t i c a l 2 6 6 . The m i c r o b i a l assay of neomycin i n t h e p r e s e n c e of o t h e r a n t i b i o t i c s can p r e s e n t d i f f i c u l t i e s i f b o t h a n t i b i o t i c s are a c t i v e a g a i n s t t h e same t e s t organisms. The a s s a y o f neomycin i n t h e p r e s e n c e of d i h y d r o s t r e p t o m y c i n , t o which t h e t e s t organism S . U U t r e U b i s a l s o s e n s i t i v e , h a s been Levine e t a 1 2 6 9 r e n d e r e d t h e d i h y d r o reported. s t r e p t o m y c i n i n a c t i v e by h y d r o l y s i s w i t h barium hydroxide p r i o r t o t e r m i n i n g neomycin w i t h S. UUfLeUb DeNuzio 2% however, chose t o overcome t h e same problem by c u l t i v a t i n g a s t r a i n of S.uutreub which was r e s i s t a n t t o d i h y d r o s t r e p t o mycin. When a s s a y i n g neomycin i n a m i x t u r e cont a i n i n g t e t r a c y c l i n e , e n i c i l l i n and d i h y d r o s t r e p t omycin, Tanguay e t a12T1 found i t n e c e s s a r y t o

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s e p a r a t e t e t r a c y c l i n e and p e n i c i l l i n by s o l v e n t ext r a c t i o n b e f o r e d e t e r m i n i n g neomycin by t h e method of DeNuzio. The d e t e r m i n a t i o n of neomycin i n animal f e e d s h a s r e c e i v e d much a t t e n t i o n . B a r b i e r s and Neff272 recommended t h e u s e of a t r i s b u f f e r a t p 8 t o p r e p a r e neomycin s o l u t i o n s and t h e a d d i t i o n H of magnesium o r calcium c h l o r i d e t o t h e a g a r f o r enhancement of t h e i n h i b i t i o n zones. E x t r a c t i o n of neomycin from t h e f e e d r e q u i r e d t h e u s e of sodium c h l o r i d e s o l u t i o n f o r 100%r e c o v e r y . A c o l l a b o r a t i v e s t u d y of t h i s procedure by e i g h t e e n l a b o r a t o r i e s r e s u l t e d i n an average r e c o v e r y of 100.3% w i t h a c o e f f i c i e n t of v a r i a t i o n of 15.2%279. Modif i c a t i o n of t h e p r e v i o u s method h a s been d e s c r i b e d by Williams and Wornick273 who i n c o r p o r a t e d t h e t r i s b u f f e r i n t h e a g a r and d e l e t e d t h e a d d i t i o n of calcium c h l o r i d e . A t h r e e - f o l d i n c r e a s e i n s e n s i t i v i t y was r e p o r t e d . However, a c o l l a b o r a t i v e s t u d y of t h e modified procedure r e s u l t e d i n an ave r a g e r e c o v e r of 1 1 5 % w i t h a c o e f f i c i e n t of v a r i a t i o n of 20.4% 7 4 3

An i n v e s t i g a t i o n of t h e a g a r d i f f u s i o n a s s a y of neomycin a t v e r y low l e v e l s ( 5 0 n g 1000ng) i n meat p r o d u c t s h a s been r e p o r t e d 2 8 0 . The p o i n t s on t h e dose-response graph c o v e r i n g t h e above c o n c e n t r a t i o n range w e r e s c a t t e r e d about t h e a v e r age s t r a i g h t l i n e . However, by j o i n i n g a l l t h e p o i n t s t o g e t h e r a complex c u r v e r e s u l t e d , t h e equat i o n of which was d e r i v e d .

6.5.

Automated Procedures

An a t t e m p t t o automate t h e t u r b i d i metric method f o r t h e d e t e r m i n a t i o n of neomycin w i t h K.pneurnaniae has been r e p o r t e d by Gerke e t a1281 who o b t a i n e d s a t i s f a c t o r y a s s a y s w i t h s o l An u t i o n s c o n t a i n i n g 150-1200 pg/ml of neomycin. automated r e s p i r o m e t r i c method, which measured t h e amount of C 0 2 produced by i n t e r a c t i o n of a n t i b i o t i c and t h e b a c t e r i a E.cali w a s a l s o r e p o r t e d w i t h a similar sensitivity. I n b o t h methods Auto Analyzer systems were employed.

The m o d i f i c a t i o n of t h e above r e s p i r o m e t r i c procedure t o i n c l u d e c o n t i n u o u s c u l t u r e of

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t h e t e s t o r g a n i s m E . c o L i h a s been d e s c r i b e d 2 8 2 . With t h e o r g a n i s m grown i n t h i s manner t h e s e n s i t i v i t y o f t h e a s s a y i s improved. G r e e l y e t a1283 have a p p l i e d t h e automated r e s p i r o m e t r i c method t o t h e d e t e r m i n a t i o n o f neomycin i n p h a r m a c e u t i c a l p r o d u c t s and compared t h e s e a s s a y s w i t h t h e r e s u l t s o b t a i n e d by t h e c y l i n d e r - p l a t e p r o c e d u r e on t h e same s a m p l e s . Good c o r r e l a t i o n between t h e t w o p r o c e d u r e s was d e m o n s t r a t e d .
A n automated c o l o r i m e t r i c a s s a y f o r t h e q u a n t i t a t i o n of t h e s e p a r a t e components o f neom c i n ( B , C and neamine) h a s a l s o been r e p o r t The method u t i l i z e s a s e p a r a t i o n o f t h e ed148. components on a column o f c a r b o n and K i e s l g u h r G (4:l). The column e l u a t e i s reacted w i t h n i n h y d r i n t o d e t e r m i n e t h e amounts o f neomycin B , C and neamine.

6.6.

U s e as a n A n a l y t i c a l Reagent

Turbidimetric procedures f o r d e t e r mining r i b o n u c lease2 and d e o x y r i b o n u c l e a s e 85 u s i n g neomycin as t h e p r e c i p i t a n t h a v e been d e s c r i bed. The t u r b i d i t y w a s shown t o b e d e p e n d a n t on t h e r e l a t i v e c o n c e n t r a t i o n s o f r e a c t a n t s , t h e molec u l a r w e i g h t o f t h e RNA o r DNA and t h e i o n i c s t r e n g t h of t h e s o l u t i o n t h u s n e c e s s i t a t i n g t h e c a r e f u l c o n t r o l of assay conditions. Extraneous p r o t e i n s do n o t i n t e r f e r e e n a b l i n g t h e p r o c e d u r e t o be a p p l i e d d i r e c t l y t o blood serum.
6.7.

D e t e r m i n a t i o n i n Body F l u i d s and Tissues

An a g a r d i f f u s i o n a s s a y , based upon t h e method d e s c r i b e d by Groves and Randa11246, h a s been u t i l i s e d by t h e m a j o r i t y of w o r k e r s f o r det e r m i n i n g neomycin l e v e l s i n c l i n i c a l samples. T o compensate f o r t h e p r o t e i n - b i n d i n g e f f e c t e x h i b i t ed by neomycin, Kivman and Geitman309 recommended t h e a d d i t i o n of serum and 3 % KC1 t o t h e neomycin standard solution. D a n i e l ~ v a ~ ~ ~ , however, p r e f e r r e d t o release t h e bound a n t i b i o t i c by enzymat i c h y d r o l y s i s p r i o r t o m i c r o b i o l o g i c a l a s s a y . An a g a r d i f f u s i o n method r e q u i r i n g o n l y 2 0 ~ 1 f serum o sample h a s been d e s c r i b e d by A x l i n e e t a1311.

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The a p p l i c a t i o n of a t u r b i d i m e t r i c method t o t h e e x a m i n a t i o n o f s e r u m s a m p l e s h a s been r e p o r t e d by Fe l s e n f e l d 3 l 2 . More r e c e n t l y a laser l i g h t s c a t t e r i n g b i o a s s a y f o r d e t e r m i n i n g t h e neomycin c o n t e n t of m i l k , s e r u m , u r i n e and b i l e h a s been r e p o r t e d 3 1 3 . The p r o c e d u r e u s e s a helium-neon l a s e r l i g h t s o u r c e and h a s a l i n e a r d o s e - r e s p o n s e g r a p h over t h e r a n g e 0.1 t o 1 0 u g / m l f o r serum.
A n enzymic a s s a y i n v o l v i n g t h e r e a c t i o n of neomycin w i t h a m i n o g l y c o s i d e 4 ' a d e n y l t r a n s f e r a s e h a s b e e n d e s c r i b e d 3 1 4 . A l i n e a r r e s p o n s e f o r neomycin w a s o b s e r v e d over t h e r a n g e 2.5 t o 2Oug/ml serum.

The c o n c e n t r a t i o n of neomycin i n u r i n e h a s been d e t e r m i n e d w i t h a f l u o r i m e t r i c method315. I n t e r f e r i n g amines w e r e s e p a r a t e d by c h r o m a t o g r a p h y on SE Sephadex C-25 b e f o r e r e a c t i n g t h e i s o l a t e d neomycin w i t h f l u o r e s c a m i n e

B r a m m e r and Hemson182 employed a n e l e c t r o p h o r e t i c p r o c e d u r e t o s e p a r a t e neomycin from b l o o d - p r o t e i n s b e f o r e d e t e r m i n i n g t h e neomycin c o n c e n t r a t i o n c o l o r i m e t r i c a1l y

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189-92 ( 1 9 5 7 ) . P a i n S.K., B o s e B.K. a n d D u t t a B . N . , J . P r o c . I n s t . C h e m i s t s ( I n d i a ) , 36 (2), 82-6 ( 1 9 6 4 ) . Yavordios D . , C a r r a z M . , and K o e b e r l e J . , Ann.Pharm. F r a n c . , 28 (6) ,437-41 ( 1 9 7 0 ) . W a c h t e l J . L . and B r y a n W.L., U . S . P a t e n t 2,793,978(1975). D e n k e w a l t e r R.G. and G i l l i n J . , G e r . P a t e n t 1,062,891 (1959). S o k o l s k i W.T., C h i d e s t e r C.G. and K a i s e r D.G., J . P h a r m . S c i . , 53 ( 7 1 , 7 2 6 - 9 (1964). Rappe A . , Ann. Pharm. F r . , 31 ( 6 1 , 4 3 5 - 4 0 (1973). Davis W.W. a n d P a r k e T . V . , J.Am.Pharm. ASSOC., 2 , 3 2 7 - 3 3 ( 1 9 5 0 ) . K u z e l N . a n d C o f f e y H.F., U . S . P a t e n t 3,401,087 (1968). Abbey A . , A n t i b i o t i c s 8, C h e m o t h e r a p y , 3, 528-31 (1952). P i n z e l i k J . , Nisonger L.L. a n d Murray F . J . , A p p l . M i c r o b i o l . , 1, 2 9 3 - 6 ( 1 9 5 3 ) . K o c h e t k o v a G.V., An-tibiotiki, - ( 4 1 , 52-56 2 (1957). Yousef R . T . , El-Nakeeb M.A. a n d E l m a s r i M.H., A c t a Pharm.Suecica, 4(4),253-60 (1967). El-Nakeeb M.A. and Yousef R . T . , A r z n e i m Forsch., (1)1 0 3 - 7 ( 1 9 7 0 ) , El-Nakeeb, M.A. and Yousef R . T . , Indian J. Pharm., 32 (1) ,6-9 (1970). M a r s h a l l R . T . , A l - S h a i k h l i J . B . and Edmondson J . E . , Am. J . V e t . R e s . , 1044-7 (1964). F e d o r k o J. a n d K a t z S . , J . B a c t e r i o 1 , 89 (2), 548 (1963). Siddique I . H . , Loken K . I . and Hoyt H . H . , A p p l . M i c r o b i o l . , 2 ( 5 ) ,635-7 (1965). S o k o l s k i W.T. and C a r p e n t e r O . S . , A n t i b i o t i c s Ann. 1 9 5 5 / 6 , 3 8 3 - 9 0 . S o k o l s k i FI.T., C h i d e s t e r C.G. a n d Carpenter O.S., J.Pharm.Sci., 53 ( 7 1 , 826-8 ( 1 9 6 4 ) . Sebek O . K . ,Arch.Biochem.and B i o p h y s . 71-9 ( 1 9 5 5 ) .

7,

W i n t e r m e r e D . M . , E i s e n b e r g W.H. and K i r s h b a u m A. , A n t i b i o t i c s and C h e m o t h e r a p y ,

20

25,

,=,

486

WILLIAM F. HEYES

268. 269. 270. 271. 272. 273. 274. 275. 276. 277. 278. 279. 280.
281.

U n i t e d S t a t e s Pharmacopiea XlX, 1975

p. 598.

282. 283. 284. 285. 286. 287.

Levine J . , Fischbach H. and A r r e t B . , A n t i b i o t i c s and C h e m o t h e r a p y , ? , (3), 266-269 (1954). DeNuzio J.C., B o w m a n F.W. and K i r s h b a u m A . , Antibiotics and C h e m o t h e r a p y , 4, 300-3 (1954). T a n g u a y A.E., B o g e r t A.B. and Wehner D . C . , A n t i b i o t i c s and C h e m o t h e r a p y , 5 , 232-7 (1955). B a r b i e r s R. and N e f f A.W., J.Ass.Offic. Anal.Chem., 50 (2), 462-7 (1967). W i l l i a m s B . J . and Wornick R.C. , J . A s s . O f f i c . Anal.Chem., 54 (11, 121-4 (1971). W i l l i a m s B. J. , J . A s s . O f f i c . A n a l . C h e m . , 56 (5)I 1154-60(1973). R o s s i L., Farmaco ( P a v i a ) , E d . p r a t . , 16, 203-6 (1961). Kramer J. and K i r s h b a u m A . , A p p l . M i c r o b i o l . , - 334-6 (1961). 9, K o s i k o w s k i F.V. , 14th 1 n t e r n . D a i r y C o n g r . , R o m e , 1956; 2 P t . 2, 203-9. G a r r e t t E . R . and Savage G.M. , A n t i b i o t i c s and C h e m o t h e r a p y , 5 (51, 273-80 (1955). N e f f A.W., M i l l e r C . C . and B a r b i e r s A.R., J.Ass.Offic.Anal.Chem., 53 (1), 60-68 (1970). Langner H. J . , Weiss H. and T e u f e l U . , Z e n t r a l b l . V e t e r i n a e r m e d . , R e i k e B. , 20 (6)I 435-63 (1973). G e r k e J.R., H a n e y T.A., Pagano J . F . and F e r r a r i A . , A n n . N . Y . A c a d . S c i . , 87, 782-91 (1960). Shaw W.H.C. and D u n c o m b e R . E . , A n a l y s t , - (1050), 694-701 (1963). 8 G r e e l y V. J . , H o l l W.W., Michaels T . P . and S i n o t t e L.P. , A n n . N . Y . A c a d . S c i . , 130 (2), 657-63 (1965). A l t e s c u E.J., Anal.Biochem., 8 (31,373-92 (1964). A l t e s c u E.J. , Enzymol.Biol.Clin., 6 (41, 305-23 (1966). Waksman S . A . and Lechevalier H.A., Science, 2 ,305-7 (1949). B r i t i s h P a t e n t 683,632 (1952).

NEOMYCIN

487

288. 289. 290. 291. 292. 293. 294. 295. 296. 297. 298. 299. 300. 301. 302. 303. 304. 305. 306. 307. 308. 309.

Waksman S.A. a n d L e c h e v a l i e r H . A . , U.S. P a t e n t 2,799,620 (1957). Umezawa H . , T a k e u c h i T. a n d Yamagiwa S . , J a p a n . Med. J . , 3 , 25-30 ( 1 9 5 0 ) . A r a i T. a n d A i i s o K . , J a p a n e s e P a t e n t 5450 (1953). Game G.F., K o c h e t k o v a G . V . , P r o b r a z e n s k a y a T.P. a n d P e v z n e r N.S., A n t i b i o t i k i , - ( 5 1 , 1 4-8 ( 1 9 5 6 ) . J a c k s o n F.L., K i t t i n g e r G.W. and K r a u s e F.P. , N u c l e o n i c s , 1 ( 8 ) ,102-5 ( 1 9 6 0 ) . 8 S w a r t E.A. , H u t c h i n s o n D . a n d Waksman S.A. , A r c h . Biochem., 24, 92-103 ( 1 9 4 9 ) . L e a c h B.E., D e V r i e s W . H . , N e l s o n H.A., J a c k s o n W.G. a n d E v a n s J . S . , J.Am.Chem.Soc., -I 2797-2800 ( 1 9 5 0 ) 73 Makino S. , H a y a s h i E . a n d Okamura K . , J a p a n . P a t e n t 6247 ( 1 9 6 0 ) . T a k e n a g a M. a n d Okada Am, G e r . P a t e n t 2,165,373 (1972). T a n a k a I . , Yoshizawa H., O t a n i S., N a k a y a s h i k i J. a n d Hara E . ,Y a k u z a i g a k u , -, 1 4 7 - 5 0 ( 1 9 6 2 ) . 22 Simone R.M. a n d P o p i n o R.P., J.Am.Pharm. A S ~ O C .I 44, 275-80 ( 1 9 5 5 ) . Beyd A . , J . P h a r m . S c i . , 60 ( 9 ) ,1343-5 (1971). Gecgil S . , I s t a n b u l U n i v . E c z a c i l i k F a k . Mecmuasi , 2 (1) 56-57 ( 1 9 6 8 ) , Coates L . V T , P a s h l e y M.M. a n d T a t t e r s a l l K . , J.Pharm.Pharmaco1. , 13, 6 2 0 - 4 ( 1 9 6 1 ) . D a l e J . K . a n d Rundman S. J . , J.Am.Pharm. ASSOC., P r a c t . Pharm. E d . , 1 8 , 4 2 1 - 5 ( 1 9 5 7 ) . K u d a l k a r V.G., H a l l N . A . a n d R i s i n g L.W. , Am.J.Pharm., 1 3 1 , 166-73 ( 1 9 5 9 ) . M c G i n i t y J . W . a n d Brown R.L., J . P h a r m . S c i . , 64 ( 9 ) ,1528-30 ( 1 9 7 5 ) . E r n i c k R.C., P r o c . , Annu. P f i z e r R e s . C o n f . , 1 5 t h , 54-90 1 1 9 6 7 ) . Danti A . G . a n d G u t h E . P . , J.Am.Pharm.Assoc., S c i . E d . , 4 6 , 249-52 ( 1 9 5 7 ) . Hammouda Y T a n d S a l a k a w y S.A., P h a r m a z i e , - (10),636-40 ( 1 9 7 1 ) . 26 F l o r e s t a n o H . J . , B a h l e r M.E. a n d J e f f r i e s S . F . , J.Am.Pharm.Assoc. , 4 5 , 538-41 ( 1 9 5 6 ) . Kivman G. Y a . a n d G e i t m a n I. Y a . , Antibiotiki, 1 6 ( 4 ) ,331-5 ( 1 9 7 1 ) .

488

WILLIAM F. HEYES

310. 311. 312. 313. 314. 315.

D a n i e l o v a L . T . , A n t i b i o t i k i , 1 9 (111, 1032-8 ( 1 9 7 4 ) . Axline S.G., Yaffe S . J . a n d Simon H . J . , P e d i a t r i c s , 39 ( 1 ) , 9 7 - 1 0 7 ( 1 9 6 7 ) . F e l s e n f e l d O . , V o l i n i I . F . , K a d i s o n E.R., Zimmermann E . a n d I s h i h a r a S . J . , Am.J. Clin.Path., 3 , 670-1 (1950). Wyatt P . J . , P h i l l i p s D . T . and A l l e n E . H . , J . A g r i c . F o o d Chem., 24 ( 5 ) ,984-8 ( 1 9 7 6 ) . Santanam P . a n d K a y s e r F.H., A n t i m i c r o b . A g e n t s C h e m o t h e r . , lo ( 4 ) ,664-7 ( 1 9 7 6 ) . K u s n i r J . a n d B a r n a K . , Cesk. Farm., 24 ( 6 ) ,253-5 ( 1 9 7 6 )

This p r o f i l e attempts t o cover t h e published l i t e r a t u r e on neomycin u p t o a n d i n c l u d i n g C h e m i c a l Abstracts, Volume 8 5 .

Analytical Profiles of Drug Substances, 8

PSEUDOEPHEDRINE HYDROCHLORIDE
Steven A . Benezra and John W . McRae
1.

2.

3. 4. 5. 6.

Description 1.1 Name, Formula, Molecular Weight 1.2 Appearance, Color, Odor Physical Properties 2. I Infrared Spectrum 2.2 Nuclear Magnetic Resonance Spectrum 2.3 Ultraviolet Spectrum 2.4 Mass Spectrum 2.5 Melting Point 2.6 Specific Rotation 2.7 Solubility 2.8 Partition Coefficient 2.9 Differential Scanning Calorimetry 2.10 Crystal Structure 2.1 I Dissociation Constant Synthesis Stability Metabolism and Pharmacokinetics Methods of Analysis 6.1 Elemental Analysis 6.2 Nonaqueous Titration 6.3 Ultraviolet Spectrophotometric Analysis 6.4 Colorimetric Analysis 6.5 Chromatography 6.51 High Performance Liquid Chromatography 6.52 Thin-Layer Chromatography 6.53 Paper Chromatography 6.54 Gas Chromatography

489

Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-260808-9

490

STEVEN A . BENEZRA AND

JOHN W. MCRAE

1.

Description

1.1 Name, Formula, Molecular Weight d-Pseudoephedrine hydrochloride is (+)-threo-a-(1(methy1amino)ethyl)benzyl alcohol hydrochloride. Throughout this analytical profile, d-pseudoephedrine will be referred to as pseudoephedrine.

* o

H
I

HCI

C10H15NO*HC1

CH3

201.72

1.2 Appearance, Color, Odor Pseudoephedrine hydrochloride occurs as fine white t o off-white crystals o r as a powder having a faint odor1

2.

Physical Properties

2.1 Infrared Spectrum The infrared spectrum of pseudoephedrine hydrochloride is shown in Figure 1. It was obtained as a 0.2% dispersion of pseudoephedrine hydrochloride in KBr with a Nicolet Model 7199 FT-IR spectrophotometer.2 Table I gives the infrared assignments consistent with the structure of pseudoephedrine hydrochloride. Table I Infrared Spectral Assignments for Pseudoephedrine Hydrochloride Frequency (cm-l) Assignment OH stretch 3270 3010 Asym. C-H stretch

0 0

0 0

3 3

0 d

0
( u

492

STEVEN A. BENEZRA AND

JOHNW. MCRAE

2930 2700 1587, 1490 1430 762, 702 2.2

+NH

Sym. C-H stretch stretch

C=C aromatic stretch OH bend, secondary alcohol C-H bend, monosubst. benzene

Nuclear Magnetic Resonance (NMR) Spectrum The NMR spectrum o f pseudoephedrine hydrochloride The spectrum was obtained with a

is shown in Figure 2.

H Varian model CFT-20 80 M z NMR spectrometer. Deuterated DMSO was used as the solvent with tetramethylsilane as an internal standard. Table I1 gives the NMR assignments consistent with the structure of pseudoephedrine hydrochloride.

8 H H HN-

G9

OH H
Table I1

@@

NMR Assignments for Pseudoephedrine Hydrochloride

Proton a b
C

No. of Protons 3 3
1

Shift (ppm) 0.96 2.55 3.25 4.54 6.32 7.34 8.90

Multiplicity doublet singlet quartet (partially obscured by H20) doublet of doublets doublet singlet broad singlet

d e f
8

1
5

PSEUDOEPHEDRINE HYDROCHLORIDE

493

1
I

1
I S

5
PPm

'

I'

Figure 2

I.o

NMR Spectrum of Pseudoephedrine Hydrochloride

ae

0.6
U

4
[L

v 0 )

a
0.4

0.2

0.9

Figure 3

nrn

Ultraviolet Spectrum of Pseudoephedrine Hydrochloride

494

STEVEN A. BENEZRA A N D

JOHN W. MCRAE

2.3 Ultraviolet Spectrum The ultraviolet spectrum of pseudoephedrine hydrochloride in ethanol was obtained with a Beckman ACTA CIII ultraviolet spectrophotometer and is shown in Figure 3. Pseudoephedrine hydrochloride exhibits absorption maxima at 208, 251, 257, and 264 nm with extinction coefficients of 8300, 161, 201, and 161, respectively. 2.4. Mass Spectrum The low resolution mass spectrum of pseudoephedrine hydrochloride is shown in Figure 4.5 It was obtained with a 0 ' Varian MAT CH5-DF mass spectrometer. Direct probe at 8C into the ion source was used to obtain the mass spectrum. The electron energy was 70 eV. The assignments of the major ions formed in the mass spectrometer are shown below. molecular ion is not observed. The

H +HC /NzCH3 'CHq 3

m/e 58 (100%) 2.5 and 15C 8'.

0
+

HCI

HNCH3

m/e 77 (9%)

m/e 36 (10%)

m/e 30 (12%)

Melting Point Pseudoephedrine hydrochloride melts between 1 2 8'

2.5 Specific Rotation The specific rotation, of d-pseudoephedrine hydrochloride in water is between +61.0' and + 2 5 . 6.'l

[cy]iO,

2.7

Solubility The solubility of pseudoephedrine hydrochloride in

PSEUDOEPHEDRINE HYDROCHLORIDE

495

1001

I00

I50

200

m /e

Figure 4

Mass Spectrum of Pseudoephedrine Hydrochloride

1 I

178

179

180

11 8

182

13 8

1 1

184
OC

15 8

1 1

1
1

186

187

TEMPERATURE

Figure 5

DSC Curve of Pseudoephedrine Hydrochloride

496

STEVEN A. BENEZRA AND JOHN W. MCRAE

various solvents at 25OC is given in Table 111.' Table I11 Solubility of Pseudoephedrine Hydrochloride at 25OC Solvent Water Chloroform
E thano1

Solubility (gm/ml) 2.0 0.011 0.278 1.4

Ether

2.8 Partition Coefficient

The partition coefficients of pseudoephedrine hydrochloride at 25OC in n-octanol/aq. pH 1.2 and n-octanol/ aq. pH 6.0 are 0.010 and 0.049 respectively.6 2.9 Differential Scanning Calorimetry (DSC) The DSC curve of pseudoephedrine hydrochloride obtained with a Perkin Elmer DSC-1B differential scanning calorimeter is shown in Figure 5 . ? point (uncorrected) is 184OC. 2.10 Crystal Structure The crystal properties of pseudoephedrine hydrochloride were determined with a GE model XRD-6 x-ray diffractometer using Zr filtered MoKa radiation on a crystal grown from water. Pseudoephedrine hydrochloride has an orthorhombic crystal system belonging to the P2 2 2 space group. 0 l a 1 0 The cell dimensions are a=25.358 A , bz6.428 A , ~ ~ 6 . 9 0 1 A with each cell containing four molecules. 2.11 Dissociation Constant The pKa of pseudoephedrine hydrochloride determined The heating rate was 5OC/min. The heat of fusion is 6.4 Kcal/mol. The melting

PSEUDOEPHEDRINE HYDROCHLORIDE

491

titrimetrically in 80% aqueous methylcellosolve is 9.22.

3.

Synthesis Pseudoephedrine hydrochloride is prepared by a

Welsh rearrangementl o of R-ephedrine hydrochloride with acetic anhydride followed by deacetylation with hydrochloric acid. l 1 2-Ephedrine can be resolved from dR-ephedrine with R-Ephedrine occurs naturally in certain 2-mandelic acid. l2

plants of the Ma Huang species. 4. Stability Pseudoephedrine hydrochloride can be considered a stable compound in bulk and in formulations. After 4-weeks under fluorescent lights (2400 ft. candles) and ultraviolet light (190 pw/cm2) no discoloration or chemical degradation was observed. The bulk drug was stable for at least 6 months at 37OC and 3 months at 5OoC. Tablet and syrup formulations stored at 15-30C for 5 years showed no appreciable degradation. l 3

5.

Metabolism and Pharmacokinetics The major biotransformations of pseudoephedrine

hydrochloride are parahydroxylation, N-demethylation, and oxidative deamination. l4 The proposed pathways for the metabolism of pseudoephedrine are shown in Figure 6 . In a study with human subjects, whose urine pH was controlled with sodium bicarbonate and ammonium chloride, it was found that 10-25% of the administered pseudoephedrine hydrochloride was metabolized to norpseudoephedrine and the elimination of pseudoephedrine and norpseudoephedrine was related to urine pH. ed.15 As the urine pH increased, the serum half-life o f pseudoephedrine and norpseudoephedrine increas-

In another similar study it was found that a decrease

H
p-&-+ HQ +

CH3

OH NH
I

CH3 Pseudoephedr ine

o( +/
OH
CH3
I/

\ I

OH NH,
Nor pseudoephedrine

O W H ,
OH OH

I-Hydroxy-l-phenyl-2-proponone

I -Phenyl-l,Z-propanediol

OH Benzoic acid
Figure 6

Metabolism of Pseudoephedrine Hydrochloride

PSEUDOEPHEDRINE HYDROCHLORIDE

499

in urine pH caused a decrease in plasma half-life of pseudoephedrine. l6 Plasma half-lives measured in normal human subjects were 5 . 2 - 8 . 0 hours.l6 In a rat study using 14C-labelled dQ-ephedrine,
85% of the i.p.-administered drug was eliminated in the first 40 hours.

Two major metabolic pathways were postulated after

analysis of the metabolites. The major metabolic pathway was ring para-hydroxylation forming para-hydroxyephedrine and
para-hydroxynorephedrine.

The minor metabolic pathway was

oxidative deamination, giving acidic metabolites such as hippuric and benzoic acids. l7 The relative tissue distribution in mice 15 minutes after i.v.-administered l4C2ephedrine was kidney > lung, adrenal, spleen, liver > intestines, stomach > brain, heart > plasma. l7 The LDs0 in mice o f pseudoephedrine administered i.p. is 1.0 mmole/kg.18
6.

Methods of Analysis
6.1

Elemental Analysis

The results of the elemental analysis o f pseudoephedrine hydrochloride are given in Table IV.6 The analysis was performed on a NF Reference Standard. Table IV Elemental Analysis of Pseudoephedrine Hydrochloride Element
C

Theory (%)
59.55

Found ( X )
59.54 8.11 6.81

8.00 6.95

500

STEVEN A. BENEZRA AND

JOHN W.M C W E

6.2

Nonaqueous Titration Pseudoephedrine hydrochloride is dissolved in a

mixture of glacial acetic acid and mercuric acetate test solution. A standardized solution of 0 . 1 N perchloric acid is used to titrate the solution to a blue-green end point with crystal violet indicator. Each ml of 0.1N perchloric acid is equivalent to 0.1 mmole of pseudoephedrine hydrochloride.
6.3

Ultraviolet Spectrophotometric Analysis An ultraviolet spectrophotometric analysis is used

to assay pseudoephedrine hydrochloride in tablets. A portion of finely powdered tablets equivalent to approximately 30 mg of pseudoephedrine hydrochloride is placed in a distilling flask which is part of a micro-steam distillation apparatus. Sodium chloride, water, and concentrated sodium hydroxide are added. A minimum of 30 ml of distillate is collected in a volumetric flask containing dilute hydrochloric acid. The flask is made to volume with distilled water and the absorbance of the solution is determined at 257 nm in 1 cm cells and compared to a solution of known concentration of NF Pseudoephedrine Hydrochloride Reference Standard. An ultraviolet spectrophotometric method based on the absorbance of a periodate oxidation product o f pseudoephedrine hydrochloride will be the official method of analysis in the USP XX.19,20 A portion of tablets or syrup in water is placed in a separatory funne.1. Sodium bicarbonate and sodium metaperiodate are added. After standing for 15 minutes, 1 N HC1 is added. The solution is extracted with hexane. The hexane extract is filtered and its absorbance determined at 242 nm in 1 cm cells. The amount of the oxidation product of pseudoephedrine hydrochloride is determined by comparison of the sample absorbance against the absorbance of a Pseudoephedrine Hydrochloride Reference Standard treated in the same manner.

PSEUDOEPHEDRINE HYDROCHLORIDE

50 1

6.4 Colorimetric Analysis

Pseudoephedrine hydrochloride in syrup formulations has been analyzed by colorimetry. Pseudoephedrine forms a stable blue-colored chelate with cupric sulfate at pH 12.5. The complex has a maximum absorbance at 500 nm. The complex is extracted from an aqueous layer with 1-pentanol. Interfering substances such as glycerine and sugars normally found in syrup formulations, which form complexes with cupric aulfate, are not extracted into l-pentanol.*l
6.5

Chromatography 6.51 High Performance Liquid Chromatography (HPLC) High performance liquid chromatography has been

used to analyze pseudoephedrine hydrochloride and dosage

forms containing pseudoephedrine hydrochloride.

Table V

gives the HPLC conditions used for separations. Table V HPLC Conditions or Pseudoephedrine Hydrochloride Column Corasil@/Phenyl Corasil@/C18 Mobile Phase Rention Time (min) 1 8 (phenyl) . acetonitrile: 0.1% ammonium carbonate
(9:l) pH 8.9

Reference 22

1.9 (C18)

Corasil@/Phenyl Corasil@/C18

acetonitrile:
1% ammonium

22

carbonate (6:4) pH 7.4

502

STEVEN A. BENEZRA AND JOHN W.M C M E

Zipax@/SCX

0.02 M dibasic ammonium phosphate:dioxane (64:36) 7 23

Nucleosil@/ silica gel

methanol:0.5M sodium dihydrogen phosphate: phosphoric acid (195:50:2)

24

Spherisorb@/ silica gel

ethanol:0.4% ammonium acetate (85: 15)

25

6.52 Thin Layer Chromatography (TLC) Table VI lists the various TLC systems used for pseudoephedrine hydrochloride.
Table VI TLC Systems for Pseudoephedrine Hydrochloride Mobile Phase ethyl acetate: cyclohexane: methano1:conc. M140H(70:15:10:5) n-butano1:ethanol: water:acetic acid (60:30:10:0.2) silica gel 27 0.18 (free base) Adsorbent silica gel

Rf
0.25

Reference

26

0.46

PSEUDOEPHEDRINE HYDROCHLORIDE

503

chlorof o ri: n methanol : acetone (7:3:5) ethyl ether: benzene (1:l)

silica gel

0.33

28

0.10 (free base) silica gel 0.35 (as 4-chloro7-nitrobenzo-2,1,3oxadiazole derivative)

29

chloroform : water:acetic acid (20:75:20:1) (lower phase)

alumina

0.70 (as acetylated product)

16

6.53 Paper Chromatography Paper chromatography has been used to separate and detect pseudoephedrine hydrochloride from other pharmacologically active amines. Whatman No. 1 paper developed in n-butanol:water:95% acetic acid (4:5:1), n-butano1:toluene: water:95% acetic acid (10:10:5:5), ethyl acetate:water:95% acetic acid (3:3:1), o r chloroform:water:95% acetic acid (10:5:4) gave Rf values of 0.73, 0.35, 0.57, and 0.52 for pseudoephedrine hydrochloride respectively. Visualization of pseudoephedrine hydrochloride was done by spraying the chromatogram with 0.5% bromcresol green in methanol or 0.2% ninhydrin in acetic acid:butanol 5 :95.30 6.54 Gas Chromatography Pseudoephedrine hydrochloride has been separated from other arnines by gas chromatography. The oxazolidine

504

STEVEN A. BENEZRA AND JOHN

w.MCRAE

derivative has been prepared by the reaction of pseudoephedrine with anhydrous acetone. On a 1.15% SE-30, glass column, 2.4 m x 3 mm i.d. at 104OC the oxazolidine derivative has a retention time of 16.4 minutes.31

On a 15% PEG 6000, glass column 2 m x 4 mm i.d. at 175OC the oxazolidine derivative had a retention time of 10.1 minutes.32 The N-trifluroacetyl-L-prolylchloride derivative of pseudoephedrine has retention time of 105 minutes on a 3% SE-30, stainless steel column, 2 m x 3 mm i.d. at 170C.33 An on-column acetic anhydride derivatization technique has been described for pseudoephedrine hydrochloride. Immediately after injection of a solution of pseudoephedrine onto a 20% SE-30, 1.8 m x 7 mm i.d. glass column at 125OC, an injection of acetic anhydride was made. The pseudoephedrine derivative formed on column has a retention time of 55.5 minutes as compared to a retention time of 8.7 minutes for underivatized pseudoephedrine.34 A variety of methods have been used to determine pseudoephedrine hydrochloride levels in plasma and urine by extracted basefied gas chromatography. Bye and c o - w o r k e r ~ ~ ~ plasma or urine with diethyl ether. The ether extract concentrate was chromatographed on a 1.2 m x 2mm i.d. glass column packed with 2% Carbowax 20 M +5% KOH. The column was maintained at 187OC for plasma samples and 15OoC for urine samples. The heptafluorobutyric anhydride derivative of pseudoephedrine and electron capture detector have been used to enhance the sensitivity of the gas chromatographic method. Lin and c o - ~ o r k e r s ~ ~ Cummins and Fourier37 extracted and basefied urine or serum with benzene. Heptafluorobutyric anhydride is added to the benzene extract. The heptafluoroibutyric anhydride derivative extracted was chromatographed

PSEUDOEPHEDRINE HYDROCHLORIDE

505

on a 3% OV-17, 1.82 m x 2 mm i.d. glass column at 150C36 or a 5% ethylene glycol succinate, 1.82 m x 2 mm i.d. stainless steel column at 140C. 37 Pseudoephedrine in urine was analyzed by gas chromatography using a 2% polyethylene glycol 600 + 5% KOH,

2 m x 2 mm i.d. stainless steel column at 165OC. The urine was extracted with diethyl ether and then made basic with 5N NaOH. The pseudoephedrine was extracted with diethyl ether, concentrated, and injected directly into the gas chromatograph, or derivatized with acetone and then chromatographed.38 Pseudoephedrine was determined after acidification, precipitation, and acetic anhydride derivatization. The ester derivative was injected onto a 2.5% SE-30, 1.8 m x 4 mm i.d. column at 190C.39 References
1.

N . F . XIV, Mack Printing Co., 1975

2. W. Martin, Burroughs Wellcome Co., personal communication 3 . R . Crouch, Burroughs Wellcome Co., personal communication 4.
J.W. McRae, unpublished data 5. D. Brent, Burroughs Wellcome Co., personal communication

6. B.S. Hurlbert, Burroughs Wellcome Co., personal communication 7. J. Ebron, Burroughs Wellcome, personal communication 8. M. Mathew, G.J. Palenik, Acta. Cryst., E, 1016 (1977)

9. V. Prelog, P. Haflinger, Helv. Chim. Acta., 33, 2021 (1950) 10. L.H. Welsh, J. h e r . Chem. SOC., 3500 (1949) 11. R. Seaman, Burroughs Wellcome Co., personal communication

c,

506

STEVEN A. BENEZRA AND JOHN W.MCRAE

12.

R.H. Manski, T.B. Johnson, J. h e r . Chem. SOC., 51,

(1929)

1906

13. 14.

T. Morgan, Burroughs Wellcome Co., personal communication

D.R. Feller, P. Basu, W. Mellon, J. Curott, L. Malspeis,

Arch. Int. Pharmacodyn.,

203,

187 (1973)

15. 16.

D.C. Brater, L.Z. Benet, E. Lin, R.C. Morris, Jr.,

17. 18.

K.L. Melmon, Clin. Res., 24, 252A (1976) R.G. Kuntzman, I. Tsai, L. Brand, L. Mark, Clin. Pharmacol. Ther., - 62 (1971) 12, J. Bralet, Y. Cohen, G . Valette, Biochem. Pharm.,
2319 (1968)

1 1

M.D. Fairchild, G.A. Alles, J. Pharmacol. Exp. Ther.

19. 20. 21.

- 135 (1967) 158, J.E. Wallace, J. Pharm. S c i . , g ,1489 (1969) L. Chafetz, J. Pharm. Sci., 60, 291 (1971)
J.A. McCrerie, Wellcome Foundation Ltd., personal communication

22.

I.L. Honigberg, J.T. Stewart, A.P. Smith, J. Pharm.

Sci.,

3, 766

(1974)

23. 24.

T.L. Spriek, J. Pharm. Sci., - 5 9 1 (1974) 64, G.T. Hill, Wellcome Foundation Ltd., personal communication M.L. Blackmon, Burroughs Wellcome Co., personal communication

25. 26. 27. 28

K.K. Kaistha, R. Tadrus, R. Janda, J. Chromatog.,

359 (1975)

107,

29. 30.

Y. Hashimoto, Y. Ikeshiro, T. Higashiyama, T. Ando, M. Endo, Yakugaku Zasshi, 97, 1594 (1977) L.N. Mikhailova, M.N. Preobrazhenskaya, G.M. Kadatskii, S.D. Sokolov, Khim.-Farm. Zh., 2, 49 (1975) J.C. Hudson, W.P. Rice, J. Chromatog., 117, 449 (1976) A. WickstrZim, B. Salvesen, J. Pharm. Pharmacol., 4, 631
(1952)

PSEUDOEPHEDRINE HYDROCHLORIDE

507

31.

E.B.-Hanssen, A.B. Svendsen, J. Pharm. Sci.,

(1962)

51,

938

32. 33.

K. Yamasaki, K. Fujia, M. Sakamoto, M. Okeda, M.

Yoshida, 0 Tanaka, Chem. Pharm. Bull., .
(1973)

22,

2898 (1974)

A.H. Beckett, B. Testa, J. Pharm. Pharmacol., 9, 382

34. 35.

M.W. Anders, G.J. Mannering, Anal. Chem., - 730 (1962) 34, C. Bye, H.M. Hill, D.T.D. Hughes, A.W. Peck, Eur. J.
Clin. Pharmacol., 8, 47 (1974)

36.

E.T. Lin, D.C. Brater, L.Z. Benet, J. Chromatog., 140,

273 (1977)

37. 38. 39.

L.M. Cummins, M.J. Fourier, Anal. Lett., Suppl. 1 7 , 104s (1965) P. Leblish, B . S . Finkle, J . W . Chem.,

2,

403 (1969)

A.H. Beckett, G.R. Wilkinson, J. Pharm. Pharmacol., Brackett, Jr., Clin.

16,195

(1970)

Analytical Profiles of Drug Substances, 8

TRIPROLIDINE HYDROCHLORIDE
Steven A . Benezra and Chen-Hwa Yang
I.
2.

3. 4. 5. 6.

Description I. I Name, Formula, Molecular Weight I .2 Appearance, Color, Odor Physical Properties 2. I Infrared Spectrum 2.2 Nuclear Magnetic Resonance Spectrum 2.3 Ultraviolet Spectrum 2.4 Mass Spectrum 2.5 Fluorescence Spectrum 2.6 Crystal and Molecular Structure 2:7 Melting Point 2.8 Solubility 2.9 Partition Coefficient 2.10 Dissociation Constant Synthesis Stability Drug Metabolic Products and Pharmacokinetics Methods of Analysis 6. I Elemental Analysis 6.2 Nonaqueous Titration 6.3 Ultraviolet Spectrophotometric Analysis 6.4 Quantitative Infrared Analysis 6.5 Chromatography 6.5 I High Performance Liquid Chromatography 6.52 Thin-Layer Chromatography 6.53 Quantitative Thin-Layer Chromatography 6.54 Gas Chromatography 6.6 Fluorimetric Analysis

509

Copy-t @ 1979 by Academic Fress, Inc. AU rights of reproduction i any form reserved. n

ISBN 0-12-2608089

5 10

STEVEN A. BENEZRA AND CHEN-HWA YANG

1.

Description
1.1

Name, Formula, Molecular Weight Triprolidine hydrochloride is E-2-[3-(l-pyrrolimonohydrochloride; monohydro-

diny1)-1-p-tolylpropenyllpyridine,

E-l-(2-pyridyl)-3-pyrrolidino-l-p-tolylprop-l-ene,

chloride.
J

M.W. 314.85
1.2 Appearance, Color, Odor Triprolidine hydrochloride occurs as a white, crystalline powder, with no more than a slight unpleasant odor.

2.

Phvsical ProDerties 2.1 Infrared Spectrum (IR) The infrared spectrum of triprolidine hydrochloride The IR spectrum was recorded with a Table I gives

is shown in Figure 1.

Nicolet model 7199 FT-IR spectrophotometer as a 0.2% dispersion of triprolidine hydrochloride in KBr.2 of triprolidine hydrochloride. the infrared assignments consistent with the structure

TRIPROLIDINE HYDROCHLORIDE

511

Table I Infrared Spectral Assignments or Triprolidine Band (m' e-) Hydrochloride Monohydrate Assignment

3480 2958 2690 1630 1582 1562 1462 1386 1358 846 824 776

OH stretch (hydrate) CH stretch (-CH3) N stretch H ' C=C stretch (conj. diene) C=C stretch (aromatic) C=C stretch (pyridine) -CH a s p . bend 3 -CH3 sym. bend C-N stretch (tert. amine) =C-H rock
para subst. benzene 1-subst. pyridine

2.2 Nuclear Magnetic Resonance (NMR) Spectrum The NMR spectrum of triprolidine hydrochloride is shown in Figure 2. It was obtained with a Varian XL-100 100 MHz NMR spectrometer. Deuterated DMSO was used as the
solvent with tetramethylsilane as an internal standard. The interpretation of the NMR spectrum is given in Table 11.

H3cQ

c I'

?=====-

c rP

0
P

33NVlllWSNVU

1
I

5 I4

STEVEN A. BENEZRA AND CHEN-HWA YANG

Table I1

NMR Assignments for Triprolidine Hydrochloride Chemical Shift (ppm) No. of Protons Multiplicity Assignments 1 1 11.55 j a 1 multiplet 8.63 b 6 1 7.74 multiplet C 1 7.35 d multiplet 7.00 1 4 4 benzene ring 7.12-7.38 e 1 3 6.95 f 2 2 3.78 h 1 (broad) 2 3.49 h 1 (broad) 2 2.94 1 i 2.37 3 mu1tiplet 1.88 4 l z =8, Jc,d=1.2, Jaf=7 J =4.7, Ja,d=0.9, Ja,b=2.0, J
a,c b,c

2.3 Ultraviolet (W) Spectrum The ultraviolet spectrum of triprolidine hydrochloride in 0.1N HC1 was taken with a Beckman ACTA CIII W spectrophotometer and is shown in Figure 3.4 Table I11 gives the W data for triprolidine hydrochloride in various solvents. Table I11 W Spectral Data for Triprolidine Hydrochloride Solvent h a x (NO)

EmaX

0.1N NaOH

0.1N HC1
Methanol

234 275 232 290 235 282

17000 8000 13000 9900 15000 7200

TRIPROLIDINE HYDROCHLORIDE

5 15

0.6
0 Z
W

I-

a m c r
a

0 cn

220
Figure 3

260

300 340 nm

380

- Ultraviolet

Spectrum of Triprolidine Hydrochloride

250
m /e

300

Figure 4

Mass Spectrum of Triprolidine Hydrochloride

516

STEVEN A. BENEZRA AND CHEN-HWA YANG

2.4 Mass Spectrum The mass spectrum of triprolidine hydrochloride is shown in Figure 4. It was obtained with a Varian MAT CH5-DF mass spectrometer. The electron energy was 70 eV and the sample was introduced via direct probe at 120C.5 The characteristic fragments are in agreement with those found by Kuntzman and co-workers.6 The major fragments characteristic of triprolidine are shown in Figure 5.
2.5 Fluorescence Spectrum Triprolidine hydrochloride in 0.1M sulfuric acid has an excitation maximum at 305 nm and an emission maximum at 445 nm. The fluorescence is much diminished in hydrochloric acid and almost non-existent in distilled water. The addition of halide ions to 0.1M sulfuric acid solutions of triprolidine hydrochloride has a quenching effect on the fluorescent intensity. The degree of quenching is I- > BrZC1 > F-. A concentration of 10-3M C1- has little effect on the fluorescent intensity, while 0.1M C1- reduces the intensity by a proximately 75% of the chloride-free sulfuric acid solution.

2.6 Crystal and Molecular Structure James and Williams have determined that triprolidine hydrochloride monohydrate crystals belong to the P2 /c 1 space group.8 There are 4 molecules per unit cell. The cell parameters are a = 14.777 b = 9.5785 c = 13.099 0 A, and f? = 90.48O. The 2-pyridyl ring and p-tolyl group make dihedral angles of 29.7O and 55.3O respectively with the double bond plane. The inter-aryl dihedral angle is 106.5O. The data was obtained on crystals grown from a solution of the compound in anisole. A Picker FACS-1 diffractometer with CuK01 radiation was used for the measurements.

i,

i,

R2, RI

r c=c
H

R l?
3

R2 \

, c=c
H

C; H R,

c=c

+CH2 / H

RI

m/e 278 ( 33%)

m/e 209 (90%)

m/e 208 (100%)

R2,

+c=c

/-R3

R2,

c=c+

R2\+

CH

+CHz-R,

m/e 200 ( 1 % 0 )

R, H m/e 194 ( 1 %) 8

Rl m/e 182 (7%)

m/e 84 (15 %)

R2=QCH

R3=-N

Figure 5

- Major

Ions in Mass Spectrum of Triprolidine Hydrochloride

518

STEVEN A. BENEZRA AND CHEN-HWA YANG

2.7 Melting Point Triprolidine hydrochloride melts at 115-12OoC in a sealed capillary tube.

2.8 Solubility The solubility of triprolidine hydrochloride, as the monohydrate, in various solvents at 25OC is given in Table IV.9
Table IV Solubility of Triprolidine Hydrochloride at 25OC Solvent Water 0.1N HC1 95% Ethanol n-Octanol Chloroform Propylene Glycol Diethyl Ether 2.9 Partition Coefficients The partition coefficients of triprolidine hydrochloride in n-octanol/aqueous pH 1.2 and n-octanol/aqueous pH 7.4 are 0.041 and 7.0 respectively.9 2.10 Dissociation Constant The pKal and pKa2 of triprolidine are 3.6 and 9.3 respectively.9 3. Synthesis The synthesis of triprolidine hydrochloride is The 4-methyl-w-pyrrolidinopropiophenone used in the first step is prepared by the Mannich shown in Figure 6.l o Solubility (mg/ml) 316 319 374 75

480 237 <1

c-3 z
i

+
I T

AI

+
0

m I

ma z
I
/

520

STEVEN A. BENEZRA AND CHEN-HWA YANG

reaction of 4-methylacetophenone and pyrrolidine. With proper dehydration conditions of the carbinol, a product almost pure in the desired E-conformation is obtained. 4. Stability Ultraviolet light will convert the E-form of triprolidine hydrochloride to the Z-form. After 2 years at
37OC, triprolidine hydrochloride in a syrup formulation does

not decompose more than 10%. After 3 years at 37OC triprolidine hydrochloride in tablet formulations does not decompose more than 10%. The formulations are kept in light resistant containers since triprolidine hydrochloride discolors on exposure to light.' Drug Metabolic Products and Pharmacokinetics Figure 7 shows the major metabolic pathway of triprolidine hydrochloride as determined in a study of 14C labelled triprolidine hydrochloride in guinea pigs. Triprolidine is converted to metabolite I by liver microsomes. Metabolite I is converted to metabolite I1 which is the major metabolite found in guinea pig urine. The plasma levels of triprolidine hydrochloride were determined in 16 normal male subjects.12 When administered orally at a concentration of 3.75 mg triprolidine hydrochloride in 15 ml of syrup, peak plasma levels of 8 . 2 ng/ml were achieved in 2 hours with a drug half-life of 5 hours. The low plasma levels found indicate a large volume of tissue distribution which was consistent with data obtained from rat studies.
5. 6.

Methods of Analysis 6.1 Elemental Analysis The results of the elemental analysis of triprolidine hydrochloride are given in Table V.3 The theoretical

H
k
0
I
W

e
W
Q

0=0
I
&

I
01

I z I
H

k
0
J

$1
I

k W

0
VJ

aJ

I z I

522

STEVEN A. BENEZRA AND CHEN-HWA YANG

figures are based on the monohydrate. Table V Elemental Analysis of Triprolidine Hydrochloride Element Theory (%) Found (%>*
C

58.50

58.59 7.58 8.39

H 7.51 N 8.41 *N.F. Reference Standard

6.2 Nonaoueous Titration

Triprolidine hydrochlor-Je -s dissolved, w th warming if necessary, in glacial acetic acid. Mercuric acetate test solution is added and the solution titrated with 0.1N perchloric acid, the end-point being determined potentiometrically. Each ml. of 0.1N perchloric acid is equivalent to 0.05 m o l e of triprolidine hydrochloride.
6.3 Ultraviolet Spectrophotometric Analysis

An ultraviolet spectrophotometric analysis is

used to determine content uniformity of triprolidine hydrochloride in tablet formulations. The triprolidine hydrochloride is extracted from finely powdered tablets with dilute hydrochloric acid. The solution is filtered and diluted to a concentration of approximately 10 pg triprolidine hydrochloride per ml. The absorbance at 290 nm of the extracted triprolidine hydrochloride solution in 1 cm cells is compared against N Triprolidine Hydrochloride F Reference Standard prepared in dilute hydrochloric acid at the 10 pg/ml level.
6.4 Quantitative Infrared Analysis

Quantitative infrared analysis is used as an assay

TRIPROLIDINE HYDROCHLORIDE

523

procedure for triprolidine hydrochloride in syrup and tablets.

A portion of syrup or tablets equivalent to 20 mg

and 12.5 m g triprolidine hydrochloride, respectively, is placed in a separatory funnel or glass-stoppered test tube. Distilled water is added to dilute the sample followed by concentrated NaOH solution to liberate triprolidine free base. The triprolidine free base is extracted into cyclo-1

hexane. The infrared absorbance of the cyclohexane extract is determined at 824 cm in 1 mm thick cells. A base line The absorbance of is drawn between 840 cm- and 806 cm-.

the sample is compared against the absorbance of a NF Triprolidine Hydrochloride Reference Standard treated in the same manner as the sample. 6.5 Chromatography

6.51 High Performance Liquid Chromatography (HPLC) Separation of the E and 2 isomers of triprolidine hydrochloride has been performed with HPLC. A DuPont SAX column (lm x 2 mm i.d.) with a mobile phase of 0.04M NaN03 and 0.01M Na HP04 at a pressure of 1500 psi gave retention 2 times of 4 minutes and 9 minutes for the E and 2 isomers
A re~pective1y.l~ 0.45m x 4 mm i.d.

Partisil@ silica gel

column with a mobile phase of acetonitri1e:concentrated

NH40H:H20 (1000:5:45) at 2 ml/min gave retention times of 15 and 22 minutes for E and 2 isomers respectively.* A mixture
of triprolidine hydrochlaride with other drugs normally found

in combination with triprolidine hydrochloride was separated with a 1.22m x 2.3 mm i.d. column containing a chemically bonded diphenyldichlorosilane pellicular packing. A mobile phase consisting of 6 0 : 4 0 acetonitrile:l% aqueous ammonium acetate (pH 7.4) with a flow rate of 1.4 ml/min gave a retention time of 310 seconds for triprolidine.15 Syrup and

524

STEVEN A. BENEZRA AND CHEN-HWA YANG

tablet formulations of triprolidine hydrochloride can be analyzed by HPLC using a SpherisorbG3 lop C18 reversed phase column eluted with ethanol:0.5% aqueous ammonium acetate (4:l) at 2 ml/min. In this system triprolidine hydrochloride has a retention time of 22 minutes. l6 6.52 Thin Layer Chromatography (TLC) Various TLC systems for triprolidine hydrochloride are given in Table VI. Table VI Thin Layer Chromatography Systems for Triprolidine Hydrochloride Mobile Phase ch1oroform:methanol:ammonia 80:20:1 ch1oroform:diethylamine 95:5 2-butanone:dimethylformamide 1:1 n-buty1acetate:acetone: n-butano1:methanol:lOX aq. ammonia 4:2:2:1:1 2-butanone:toluene: methano1:diethylamine 60:4 :7:3 0 cyc1ohexane:benzene: diethylamine 75:15:10 methano1 Adsorbant Silica gel F Silica gel F Silica gel F Silica gel F Rf Reference 0.60 17

0.44
0.60 0.60

17 17 17

Silica gel F

0.40

17

Silica gel G

0.41

18

Silica gel G 0.40 pre-coated with

18

TRIPROLIDINE HYDROCHLORIDE

525

0.1M NaOH

acetone

Silica gel G pre-coated with 0.1M NaOH

0.13

19

methano1

Silica gel G pre-coated with 0.1M KHS04

0.17

18

95% ethanol

Silica gel G pre-coated with 0.1M KHS04

0.02

19

benzene:dioxane:amonia 60:35: 35 ethano1:acetic acid:water 5:3:2 methano1:n-butanol 6:4 n-butano1:n-butyl ether: acetic acid 4:8:1

Silica gel G Silica gel G Silica gel G Alumina

0.40
0.48

20

20
20

0.26
0.81

20

6.53 Quantitative Thin Layer Chromatography Quantitative TLC has been used to determine triprolidine hydrochloride in human plasma. l 2 Plasma was extracted with dichloroethane at physiological pH (7.4). The organic phase was evaporated and the residue dissolved in chloroform. A portion of the chloroform solution was spotted on silica gel plates which were then developed in
ch1oroform:methanol:anunonia

(89:lO:l). After development,

the plate was air dried and sprayed with a 2M aqueous solution of ammonium bisulfate. After the plate was air dried for
1 hour, the fluorescent spot representing triprolidine was

quantitated with a spectrodensitometer in the reflectance mode with 300 nm excitation and emission above 405 nm. With

526

STEVEN A. BENEZRA AND CHEN-HWA YANG

this technique it was possible to quantitate as little as

0.4 ng triprolidine spotted on the plate.

6.54 Gas Chromatography Triprolidine hydrochloride has been separated from other antihistamines with a 1.52111x 2 . 4 m m i.d. column packed with 2% carbowax 20M + 10% KOH on 60180 mesh Chromosorb W. Retention time of 32 minutes was obtained with a column temperature of 190OC. Triprolidine hydrochloride was probably eluted as the free base.21

6.6 Fluorimetric Analysis

Triprolidine hydrochloride in syrups and tablets can be analyzed by fluorimetry. A portion of the tablets or syrup is made basic with 1N NaOH and extracted with ethylene chloride. The organic phase is then extracted with 0.1N H2S04. The fluorescence of the acid extract is determined with a fluorometer using a UGll filter for excitation and a Wratten 2A filter for emission. The fluorescence of the sample preparation is compared against a Reference Standard prepared in the same manner22.

TRIPROLIDINE HYDROCHLORIDE

527

References
1.

N.F. XIV, Mack Printing Co., communication

1975

2.

W. Martin, Burroughs Wellcome Co., personal B.S. Hurlbert, Burroughs Wellcome Co., personal communication C-H. Yang, Burroughs Wellcome Co., unpublished data

3.
4. 5. 6.

D. Brent, Burroughs Wellcome

communication

Co.,

personal

R. Kuntzman, E. Sernatinger, I. Tsai, A. Klutch,

Importance Fundam. Princ. Drug Eval. Proc. Symp., 87
(1968)

7.
8.

J. McCrerie, Wellcome Foundation Ltd., personal communication M.N.G. James, G.J.B. Williams, Can. J. Chem.,
(1974)

52,

1880

9.

T.A. Morgan, Burroughs Wellcome Co., personal

communication

U.S. Patent 2,712,023 11. D. Adamson, J. Billinghurst, J. Chem. SOC., 1039 (1950) 12. R.L. DeAngelis, M.F. Kearney, R.M. Welch, J. Pharm. Sci., - 841 (1977) 66, 13. K. Holmes, Burroughs Wellcome Co., personal communication 14. G.T. Hill, Wellcome Foundation Ltd., personal communication 15. I.L. Honigberg, J.T. Stewart, A.P. Smith, J. Pharm. Sci., 63, 766 (1974) 16. R. Jones, Wellcome Foundation Ltd., personal communication 17. A.C. Caws, Wellcome Foundation Ltd., personal communication 18. W.W. Fike, I. Sunshine, Anal. Chem., 37, 127 (1965)
10.

528

STEVEN A. BENEZRA AND CHEN-HWA YANG

19. W.W. Fike, Anal Chem., (1963)

38, 1697

(1966)

20. J. Cochin, J.W. Daly, J. Pharm. Exptl. Therap.,

139, 160
35, 591

21. C.R. Fontan, W.C. Smith, P.L. Kirk, Anal. Chem.,

(1963)
22. C. Skinner, Burroughs Wellcome Co., personal

communication

Analytical Profiles of Drug Substances, 8

SODIUM VALPROATE AND VALPROIC ACID

Zui L . Chang
I.

2.

3. 4. 5. 6.

7. 8. 9.

Description 1. I Nomenclature 1.1 I Chemical Names I . 12 Trade Names 1.2 Formulas and Molecular Weights 1.3 Appearance, Color, Odor Physical Properties 2.1 Infrared Spectra 2.2 Nuclear Magnetic Resonance Spectra 2.3 Mass Spectrum 2.4 Raman Spectra 2.5 Ultraviolet Spectrum 2.6 Solubility 2.7 Crystal Properties 2.8 Dissociation Constants 2.9 Hygroscopic Behavior 2.10 Sublimation 2. I 1 Melting Range 2.12 Boiling Point 2.13 Differential Thermal Analysis 2.14 Specific Gravity 2.15 Refractive Index Synthesis Stability-Degradation Drug Metabolism and Pharmacokinetics Methods of Analysis 6. I Identification 6.2 Elemental Analysis 6.3 Chromatographic Analysis 6.3 1 Thin-Layer Chromatography 6.32 Gas-Liquid Chromatography 6.4 Titrimetry Determination of Valproic Acid and Its Metabolites in Biological Fluids References Acknowledgments

529

Copyright @ 1979 by Academic Rcss, Inc. All rights of reproduction in any form reserved.

ISBN 0-12-2!48W-9

530

ZUI L. CHANG

1. Description 1.1 Nomenclature

1.11 Chemical Names Sodium valproate is sodium 2-propylpentanoate. It is also known as sodium dipropylacetic acid, sodium 2-propylvalerate, sodium dipropylacetate, sodium di-n-propylacetate, and by many slight variations of the particular nomenclature.
Valproic acid is also known propylvaleric variations of 1.12 Trade Names DepakeneB, Depakinea, EurekeneB, Epilim, ErgenylB, and LabazeneB. 1.2 Formulas and,MolecularWeights CH3CH2CH2, CH3CH2CH2/ C8H1502Na CH3CH2CH2,
~

is 2-propylpentanoic acid. It as n-dipropylacetic acid, 2acid, DPA, and by many slight the particular nomenclature.

CHCOONa M.W. 166.19

CH3CH2CH2 C8H1602

CHCOOH
M.W.

144.21

1.3 Appearance, Color, Odor Sodium valproate is a white crystalline powder with a very slight characteristic odor of valproic acid. Valproic acid is a colorless slightly viscous liquid, and it has a characteristic odor of valproic acid.

SODIUM VALPROATE A N D VALPROIC ACID

531

2.

Physical Properties 2.1 Infrared Spectra The infrared spectrum of sodium valproate is presented in Figure 1. The spectrum was measured in the solid state as a potassium bromide dispersion. The following bands (cm-1) have been assigned for 1. Figure 1 ( ) a.
3000-2800 cm-l

Complex of strong bands due to the overlapping C-H stretching vibrations of the various methyl and methylene groups. Strong bands due to the antisymmetrical and symmetrical stretching vibrations of the COO- grouping.

b.

1560 and 1412 cm-1

The infrared spectrum of valproic acid is presented in Figure 2. The spectrum was measured using the capillary method. The following bands (cm-1) have been assigned for Figure 2 ( ) 1. a.
3400-2300 cm-l

Broad and diffuse absorption due to the OH stretching vibration of the carboxylic acid. Complex of strong bands due to the overlapping C-H stretching vibrations of the various methyl and methylene groups. Strong band due to the C=O stretching vibration of the carboxylic acid. Broad band due to the OH bending vibration of the carboxylic acid.

b.

3000-2800 cm-1

c. 1700 cm'

d.

930 cm-l

2.2

The nuclear magnetic resonance spectrum of sodium valproate as shown in Figure 3 was obtained on a Varian Associates T-60 N M R Spectrometer in deuterium oxide containing tetramethylsilane as the internal standard. The spectral peak assignments (2) are presented in Table I.

FIGURE 1

- INFRARED SPECTRUM O F SODIUM VALPROATE

4000

3500

3000

2500

2000

1500

1000

700

FREQUENCY ( C M - l )

FIGURE 2
2.5

- INFRARED SPECTRUM OF VALPROIC ACID

WAVELENGTH (MICRONS)
4

9 10

12

15

VI

w W

4000

3500

3000

2 500

2000

1500

1000

700

FREQUENCY (CM-')

FIGURE 3
I
I

NUCLEAR MAGNETIC RESONANCE SPECTRUM OF SODIUM VALPROATE

I I I
1 I I

ii
VI W P

SODIUM VALPROATE A N D VALPROIC ACID

535

Table I

NMR Spectral Assignments for Sodium Valproate

Proton Assignment
\

Chemical Shift (ppm) 1.9-2.5 1.1-1.8

Mu1tiplicity Mu1tiplet Complex

CH-CO

CH3-

0.5-1.1

Complex

The nuclear magnetic resonance spectrum of valproic acid as shown in Figure 4 was obtained on a Varian Associates T-60 NMR Spectrometer as a 10% w/v solution in a solvent of deuterated chloroform. The spectral peak assignments ( 2 ) are presented in Table 11. Table I1

NMR Spectral Assignments for Valproic Acid

Proton Assignment C02H CHCO -CH2CH2 Chemical Shift (ppm) 11.3-11.6 1.9-2.5 1.1-1.8 Multiplicity Singlet Multiplet Complex

CH32.3 Mass Spectrum

0.5-1.1

Complex

Sodium valproate was not sufficiently volatile for mass spectral analysis. The mass spectrum of valproic acid as shown in Figure 5 was obtained using an Associated Electrical Industries Model MS-902 Mass Spectrometer with the ionization electron beam energy at 70 eV. High resolution data were compiled and tabulated with the aid of an on-line PDP11 Computer.

536

ZUI L. CHANG

Valproic acid was quite volatile and vaporized as soon as it was admitted to the source of the mass spectrometer. Only a very weak ion was detectable in the molecular ion region at m/e 145. This would correspond to (M+H)+, but exact mass measurement was not possible because of the peak's small size and the short lifetime of the sample in the mass spectrometer. The mass spectrum assignments of the prominent ions and subsequent fragments are shown in Table I11 and 3. Figure 6 ( ) Table I11 High Resolution Mass Spectrum of Valproic Acid Measured Mass (m/e) 126.1044 102.0690 73.0295 2.4 Raman Spectra The Raman spectrum of sodium valproate as shown in Figure 7 was obtained in the solid state on a Cary Model 83 Spectrometer. The following bands (cm-l) 1. have been assigned for Figure 7 ( ) a. 3000-2800 cm' Complex of strong bands due to the overlapping C-H stretching vibrations of the various methyl and methylene groups. Due to the superimposing C-H bending vibrations of the various methyl and methylene groups. Calculated Mass 126.1045 102.0681 73.0290 Formula

C8H140
CgH1002 C3H502

b.

1450 cm-l

The Raman spectrum of valproic acid as shown in Figure 8, was obtained in the undiluted liquid state on a Cary Model 83 Spectrometer. The following bands (cm-1) have been assigned for Figure 8 () 1.

FIGURE 4

- NUCLEAR MAGNETIC RESONANCE SPECTRUM

OF VALPROIC ACID
I 1

FIGURE 5

MASS SPECTRUM O F VALPROIC ACID

E m
z c z Lu
Y

>

100

130

539

0 0

e l

0 0 d

2
0
Y
0
0 a0

0 0
9

4 a > E
0

2
0
0

2 Z ' r
U

g
0

cn

5
L
v)

P
0

e
g f
2 5
o

cn

a E a

5 0 0 0
N

z 3 E

0 0
a0
(Y

0 0 m
(Y

0
0

0
9

O P

0
N

0 0 0 t

AlISN31NI

540

I
1

I
I I

0 0
(Y

0 0 d

0 0 9 0

2
P
2
0 0 0 0

8
5

B
0

0
0

0 N

8
0 0

>

aD

0 0

5
I I I

0
(Y

3
AllSN31NI

S4 I

542

ZUI L. CHANG

FIGURE 9 ULTRAVIOLET SPECTRUM O F VALPROIC ACID

0.6

0.5

0.4

2 SI
9

e z

0.3

0.2

0.1

0 200

2 50

300
WAVELENGTH (nm)

350

SODIUM VALPROATE AND VALPROIC ACID

543

a.

3000-2800 cm-1

Complex of strong bands due to the overlapping C-H stretching vibrations of the various methyl and methylene groups. Weak band due to C=O stretching vibration of the carboxylic acid. Due to the superimposing C-H bending vibrations of the various methyl and methylene groups.

b.

1660 cm-l

c.

1450 cm-l

2.5

Ultraviolet Spectrum (UV) Sodium valproate in methanol solution has no W maximum between 400 and 205 nm. When the UV spectrum of 0.1% solution of valproic acid in methanol solution was scanned from 400,to205 nm, one maximum at 213 nm ( E = 86) was observed (Figure 9 ) . The spectrum was obtained with a Beckman Acta V Spectrophotometer.

2.6

Solubility Approximate solubility data have been determined for sodium valproate at room temperature. One gram of sodium valproate is soluble in 0 . 4 ml of water and also in 1.5 ml of ethanol. It is freely soluble in methanol (1 in 5 ) . It is practically insoluble in common organic solvents such as ether, chloroform, benzene, n-heptane, etc. The following solubility data have been determined for valproic acid at room temperature: n-Heptane Chloroform Ethyl Acetate Methanol 100% Ethanol Acetone Diethyl Ether Benzene 1 N Aqueous NaOH 0.1 N Aqueous HCl Water Greater than Greater than Greater than Greater than Greater than Greater than Greater than Greater than Greater than 1.15 mg/ml 1 . 2 7 mg/ml

v/v v/v v/v v/v lo%, v/v lo%, v/v lo%, v/v
lo%, lo%, lo%, lo%,

lo%, v/v lo%, v/v

544

ZUI L. CHANG

2.7

Crystal Properties The X-ray powder diffraction pattern of sodium valproate was determined by visual observation of a film obtained with a 143.2 mm Debye-Scherrer Powder V . An Enraf-Nonius Difractis 6 0 1 Camera (Table I ) Generator; 38 KV and 1 8 MA with nikel filtered copper radiation; A = 1.5418, was employed ( 4 ) . Table I V X-Ray Powder Diffraction Pattern d-Spacings and Intensities dA 16.0 13.4 7.7 6.7 5.8 5.25 4.9 4.77 4.42 4.21 4.09 3.95 3.85 3.64 3.40 3.20 3.13 3.02 2.87 2.84

111 1
60 100 50 3 5 20 5 2 5 30 35 5 1 15 20 1 3 4 5 5

dA
2.80 2.66 2.61 2.57 2.46 2.42 2.37 2.23 2.20 2.06 2.01 1.97 1.93 1.90 1.86 1.81 1.75 1.69 1.66

1/11
5 2

1
1

1 1
2 2 2 2

1 1
2

1 1
5 1 1 1

2.8

Dissociation Constants Sodium valproate exhibits basic properties. Titration of an aqueous solution of sodium valproate with aqueous hydrochloric acid gave a pKa value of 4.8 (proton gained). Titration of valproic acid with aqueous sodium hydroxide using acetone-water as the sample solvent and extrapolated to pure water gave a pKa value of 4.6 (proton lost).

SODIUM VALPROATE AND VALPROIC ACID

545

2.9

Hygroscopic Behavior Sodium valproate is hygroscopic. The rate of moisture absorption was tested in a humidity chamber using a Cahn Electro Balance and the results are 5. shown in Table V ( ) Table V Rate of Moisture Absorption of Sodium Valproate

Relative Time, min. 10 20 30 40 50 60 Humidity - - - - - - Overnight 12, 22 33, 43% 53% 1.76 3.17 4.39
No gain

5.61

6.78 7.80

42.9%*

*Sample was completely liquified. Expressed as precent weight gain. 2.10 Sublimation Sodium valproate did not sublime when it was stored at 105C for 10 days. 2.11 Melting Range Sodium valproate does not melt, decompose, or physically change form in the normal working range of the Thomas-Hoover Capillary Melting Point apparatus. 2.12 Boiling Point The following boiling points have been reported for valproic acid: bpi4 120-121C (6), bp20 128-13OoC (6) and bp760 221-222"C ( ) 7. 2.13 Differential Thermal Analysis Differential thermal analysis of sodium valproate shows a large endotherm beginning at 100C and ending at 118C which is possible due to the loss of water. A sharp endothermic peak at 450C is indicative of the melting point of sodium valproate. Differential thermal analysis of valproic acid shows. a sharp endothermic response at 225C indicative of the boiling point of valproic acid.

FIGURE 1 0
I

- DIFFERENTIAL THERMAL ANALYSIS CURVE OF SODIUM VALPROATE

I
I I I I
I
I

I
I

50 T,

100
O C

300 350 400 (CORRECTED FOR CHROME1 ALUMEL THERMOCOUPLES)

200

150

250

450

500

FIGURE 11

- DIFFERENTIAL THERMAL ANALYSIS CURVE

OF VALPROIC ACID

50

100

150

200

250

300

350

400

450

500

1, O C (CORRECTED FOR CHROME1 ALUMEL THERMOCOUPLES)

548

ZUI L. CHANG

2.14

Specific Gravity The specific gravity of valproic acid was determined It has a in a calibrated 25 ml pycnometer at 2 ' . 5C 8. value of 0.904 g/ml ( )

2.15

Refractive Index Valproic acid has a refractive index of N D ~ ~ - ~ 1.425 ( ) 6.

3.

Synthesis The synthesis of valproic acid was first described in the literature by Oberreit (9) in 1896. The sodium valproate is preferably formed from valproic acid by the interaction of sodium hydroxide in an aqueous solution. The synthetic pathways are shown in Figure 12.

4. Stability-Degradation
Sodium valproate was found to be extremely stable when refluxed in water, 1.0 N hydrochloric acid, or 1.0 sodium hydroxide for 3 Kours. Also, it was very stable when it was subjected to heat at llOC for 10 days and to natural sunlight for 30 days in the dry state. Valproic acid is a very stable compound. No degradation has been observed by the action of heat, light, and strong aqueous alkali, or acid. 5. Drug Metabolism and Pharmacokinetics In 1971, Eymard et. al. (10) studied the distribution and the absorption of carbon-14 labeled sodium valproate in rats by oral administration. The metabolites and metabolic pathway of a new anticonvulsant drug, sodium valproate, in rats were investigated using carbon-14 labeled sodium valproate. Most of the metabolites in urine and bile were a glucuronide conjugate of ValprOic acid. Free sodium valproate was as little as one-seventh of the total metabolites. In feces, only free sodium valproate was detected, and the possibility of enterohepatic circulation of sodium valproate was presumed. A part of dosed sodium valproate was excreted in expired air in the form of C02. This degradative reaction took place in liter mitochondria and required CoA and oxygen. It was stimulated by ATP

FIGURE 12

- SYNTHETIC PATHWAYS OF VALPROIC ACID

AND SODIUM VALPROATE
CH2= CHCH2 * C CH2= CHCH2

/
CH2

C02C2H5

\ C02C2H5

2CI-CH2CH

= CH2

(or NaOCzHS)
(or C,H,OH)

c02r

CHjOH

/ \ C02R

Diethyl Malonate

Ally1 Chloride

Esters of Diallyl Malonic Acid

R=CHs and/or C2H5

Pd/C H
-c02

CH3CH2CH2

\ / CH3CH2CH2/ c \

Co2R

1) KOH, H 2 0
c

CH3CH2CH2 C CH3CH2CH2

C02H

CO2R

2) HCI

/ \ C02H

Esters of Dipropyl Malonic Acid

Dipropyl Malonic Acid

CH3CH2CH2

CHsCH2CH2

N aOH H2O

CH3CH2C H2

/ CH-C02H

CH3CH2CH2

/ CH-c02No

2-Propylpentonoic Acid (Valproic Acid)

Sodium 2-propylpentanoate (Sodium Valproate)

550

ZUI L. CHANG

and EDTA, and inhibited by various enzyme reaction inhibitors such as malonate, Antimycin-A, chloropromazine, p-chloromercuribenzoate (PCMB) and 2,4-dinitrophenol. Therefore, this degradation is not a one-step reaction, decarboxylation, but must be @-oxidation of a fatty acid. Thin-layer chromatography and gas-liquid chromatography were used for assay of metabolites ( 1 . 1) The pharmacokinetics of distribution and elimination of sodium valproate in mice and dogs has been reported by Schobben and van der Kleijn ( 2 . 1) The omega-oxidation of sodium valproate in rats has been reported by Kuhara, et. al. ( 3 . 1) The absorption, excretion, and biotransformation of val1) proic acid were studied by Kukino and Matsumoto ( 4 . A preliminary pharmacokinetic profile of sodium valproate in monkey has been written by Levig, et. al. ( 5 . 1) The pharmacokinetics of sodium valproate have been studied in 7 patients by Schobben, et. al. ( 6 . The plas1) ma concentrations were determined by gas-liquid chromatography during and following subchronic treatment. Elimination of the drug appeared to follow a monophasic exponential course; biological half lives were 8 to 15 hours. The drug appeared to have a relatively restricted distribution: calculated relative distribution volumes ranged from 0.15 to 0.40 llkg. There were large interindividual differences in clearance rate. The therapeutic range was considered to be between 50 and 100 mg/l of plasma. In 1976, Matsumoto, et. al. (17, 18) discovered several new metabolites of sodium valproate in rat urine, which support the hypothesis that the drug is also metabolized by a B-oxidation mechanism. One of the metabolites, 2n-propyl 3-0x0-pentanoic acid, was recently found by Gompertz, et. al. (19) and Kochen, et. al. (20) to be a major constituent in urine of children who were receiving sodium valproate. The urinary 3-0x0 derivative of valproate was reported to account or 20% of the administered dose.
6.

Methods of Analysis

6.1 Identification The presence of sodium cation may be identified by

SODIUM VALPROATE AND VALPROIC ACID

55 1

a flame test. A positive test for sodium is produced in a non-luminous flame imparting a yellow color. The presence of carboxylic anion may be ider!.tified by the following two tests:

A.
B.
6.2

A 5% aqueous solution gives a violet precipitate with a 5% aqueous solution of cobalt nitrate. A 5% aqueous solution gives a violet precipitate with potassium iodobismuthite.

Elemental Analysis
A typical elemental analysis of a sample of sodium valproate is present in Table VI (21).

Table VI Elemental Analysis of Sodium Valproate Element

% Theory
57.82 9.10 19.25 13.83
% Found

H
0

Na

-----* -----*

57.82 9.38

*The oxygen value cannot be determined due to presence of sodium.

A typical elemental analysis of a sample of valproic acid is present in Table VII (22).
Table VII Elemental Analysis of Valproic Acid Element
C

% Theory

% Found

H
0

66.63 11.18 22.19

66.39 11.32 22.43

6.3

Chromatographic Analysis
6.31

Thin-Layer Chromatography A number of thin-layer chromatographic systems

552

ZUI L. CHANG

on silica gel have been found to induce the migration of the compound. However, no system has been found which gives resolution equivalent to the gas-liquid chromatographic system detailed in section 6.32. Thin-layer chromatography is not the preferred method for determining the impurities. The following systems have been studied :
1. Ch1oroform:Methanol:Glacial Acetic Acid ) (17:2 :1
2.
3.

Ethyl Acetate:Formic Acid:Water (5:l:l)

Benzene:Methanol:Acetone:Ammonium Hydrox-

ide (2:2:5:1)

4. Ch1oroform:Methanol:Ammonium Hydroxide
(20:15 :3)
5. n-Butanol Saturated with 10% Ammonium Hydroxide Solution (Aqueous) 6.32 Gas-Liquid Chromatography The author has found the following GLC procedure to be suitable to determine the purity of sodium valproate and valproic acid. Preparatory to chromatography, the sodium valproate was acidified with a strong aqueous hydrochloric acid solution, and the valproic acid which is practically insoluble in water was separated. The separated free acid was then analyzed. The following is the typical chromatographic condition for the GLC determination of valproic acid: Column: 10% DEGS-PS on Supelcoport 80/100 mesh, Two 6 ft x 114 in 0.d. stainless steel. Detection: Thermal Conductivity Detector. Temperature: Inj. Port Column Detector Flow Rate: 225OC 160C 300C -20 ml/min helium

SODIUM VALPROATE AND VALPROIC ACID

553

Attenuation: 2 x 4 Slope Sensitivity: 0.03 or adjust for proper sensitivity Sample Size:
2 11 .1

Impurities as low as 0.01% could be detected using the above conditions.

6.4

Titrimetry Sodium valproate exhibits basic properties. It can be titrated with 0.1 - hydrochloric acid. N In addition, sodium valproate can be potentiometrically titrated with standardized 0 1 perchloric . acid using a modified glass-calomel electrode system, in which 0.1 N lithium perchlorate in acetic acid has been substituted for potassium chloride, and employing glacial acetic acid as the sample solvent

Valproic acid can be potentiometrically titrated with standardized 0.1 N tetra-n-butylammonium hydroxide in chlorobenzene using a modified glasscalomel electrode system, in which 1 0 aqueous . tetra-n-butylammonium chloride has been substituted for potassium chloride, and employing acetone as the sample solvent.
7.

Determination of Valproic Acid and Its Metabolites in Biological Fluids Many gas-liquid chromatographic methods for determination of valproic acid in biological fluids have been reported. Early methods required derivatization of valproic acid
( 2 3 , 24, 25, 2 6 ) .

Although Meijer and Hessing-Brand in 1973 (27) developed a micro diffusion method without derivatization, it required special equipment. Recently, most of the methods which have been used for the analysis of valproic acid in plasma, serum, cerebral spinal fluid, saliva, breast milk, and urine involve acidification of the biological sample, extraction into an organic solvent, and direct injection onto a gasliquid chromatographic column (28, 29, 16, 30, 31, 3 2 ,

554

ZUI L. CHANG

33, 34, 35, 36, 37, 38, 3 ) 9. Most of the methods employ an internal standard, and all are reported as accurate and reproducible. However, Schmidt, et. al. (40) recently reported great interlaboratory variation in the analysis of such biological samples.
8 References . 1. W. Washburn, Abbott Laboratories, Personal Communication.

2. M. Cirovic and R. Egan, Abbott Laboratories, Personal Communication. 3. S. Mueller, Abbott Laboratories, Personal Communication.
4.

J. Quick, Abbott Laboratories, Personal Communication.

5. M. Yunker, Abbott Laboratories, Personal Communication. 6. The Merck Index, 9th Edition, 9574, Merck ti Co., Inc., Rahway, N.J., 1976. 7. Dictionary of Organic Compounds, Vol. 5, p. 2799, Oxford University Press, New York, (1965).

8. J. Fornnarino, Abbott Laboratories, Perwnal Communication.

9. E. Oberreit, Berichte der Deutschen Chemischen 1998 (1896). Gesellschaft, 3,

10. P. Eymad, et. al., J. Pharmacol. (Paris), 2 ( ) 251 2, (1971)

11. K. Kukino, K. Mineura, T. Deguchi, A. Ishii and H. Takahira. Yaku-Gaku Zasshi, 92 (71, 896 (1972).
12. F. Schobben and E. van der Kleijn, Pharm. Weekbl., 109 (2), 33 (1974). 13. T. Kuhara, et. al., Biomed. Mass Spectrom., 291 (1974).

1 (4), .

SODIUM VALPROATE AND VALPROIC ACID

555

14. K. Kukino and I. Matsumoto, J. of Kurukome Med. ASSOC., 34 (41, 369 (1975).
15. R.H. Levig, et. al., Epilepsia - (l), 202 (1975). 1 6 16. 17. F. Schobben, E. van der Kleijn and F.J. M. Gabreels, Europ. J. Clin. Pharmacol., 8, 98 (1975).
I. Matsumoto, T. Kuhara and M. Yoshino, Advances in Mass Spectrometry in Biochemistry and Medicine, 1, 17-26, (1976).

18. I. Matsumoto, T. Kuhara and M. Yoshino, Biomedical Mass Spectrometry, 3 (5), 235-240, (1976).
19. D. Gompertz, P. Tippett, K. Bartlett, and T. Baillie, Clinica Chimica Acta, 74, 153-160, (1977).
20. W. Kochen, H. Imbeck and C. Jakobs, ArzneimittelI Forschung, 27 ( ) No. 5, 1090-1099, (1977). 21. Galbraith Laboratories, Inc., Knoxville, TN.

22. J. Hood, Abbott Laboratories, Personal Communication. 23. J. Alary, D. Cantin, A. Coeur and G. Carraz, Trav. SOC. Pharm. Lyon., 1 (2), 53 (1972). 6

u .

24. B. Ferrands, P. Eymard and C. Gautier, Ann. Pharm. 5, (4), 279 (1973). 31 25. J.M.H.G. Cremers and Ing. P.E. Verheesen, Pharm. Weekbl. , 1 9 (22), 522 (1974). 0 26. F.N. Ijdenberg, Pharm, Weekbl.,

1 0 (2), 1

21 (1975).

27. J.W.A. Meijer and L. Hessing-Brand, Clin. Chim. , . Acta - ( 2 ) , 215 (1973). 43 28. I.C. Dijkhuis and E. Vervloet, Pharm. Weekbl., ( ) 42 (1974). 2,

109

29. F. Schobben, E. van der Kleijn and Neth. Nijmegen, Pharm. Weekbl, 1 9 ( ) 30 (1974). 0 2,
30. C.R. Chard, N.J. Legg, Ed., Clinical and Pharmacological Aspects of Sodium Valproate in the Treatment of Epilepsy, pp. 89-91 (1975).

556

ZUI L. CHANG

31. P.N. Patsalos, V.C. Goldberg and P.T. Lascelles, Proc. Analyt. Div. Chem. SOC., 12 (lo), 270 (1975). 32. M.C. Pfaff and G. Mahuzier, Ann. Pharm. Fr., (1975).

33, 355

. . 33. T.B. Vree, E van der Kleijn and H.J. Knop, J Chromatography, 1 1 ( ) 150 (1976). 2 l, 34. N.H. Wood, D.C. Sampson and W.J. Hensley, Clinica Chimica Acta, 77, 343 (1977). 35. L.J. Dusci and L.P. Hackett, J. of Chromatography, 132, 145 (1977). 36. L.J. Dusci and L.P. Hackett, J. Chromatography, 132 ( ) 145 (1977). l, 37. C.J. Jensen and R. Gugler, J. Chromatography, 137, 188 (1977).

8 2, 38. W. Loescher, Epilepsia 1 ( ) 225 (1977). 3 39. A. Sengupta and M.A. Peat, J. Chromatography, 1 7 206 (1977).
40.

w,

D. Schmidt, B. Ferrandes, D. Grandjean, R. Gugler, C. Jakobs, S . Johannessen, U. Klotz, W. Kochen, H.J. Kupferberg, J.M.A. Meijer, A. Richens, H. Schaefer, H.U. Schulz and.A. Windorfer, Arzneim-ForschIDrug 27 (l), 1078 (1977).

9. Acknowledgements The author wishes to thank Mr. V.E. Papendick and Mr. J. B. Martin for their review of the manuscript, and Miss Diane Penza for typing the manuscript.

CUMULATIVE INDEX
Italic numerals refer to volume numbers Acetaminophen, 3, I Acetohexamide, I , 1 ;2,573 Allopurinal, 7, 1 Alpha-tocopheryl acetate, 3, 11 1 Amitriptyline hydrochloride, 3, 127 Amoxicillin, 7, 19 AmphotericinB,6, 1;7,502 Ampicillin,2, I ; # , 517 Aspirin, 8, 1 Bendroflumethiazide, 5 , I ;6,597 Betamethasone dipropionate, 6,43 Bromocriptine methanesulfonate, 8 , 4 7 Calcitriol, 8,83 Cefazolin*; 4, I Cephalexin, 4,21 Cephalothin sodium, I , 319 Cephradine*, 5 , 2 1 Chloral hydrate, 2,85 Chloramphenicol, 4,47,517 Chlordiazepoxide, I , I5 Chlordiazepoxide hydrochloride, I , 39; 4,517 Chloroquine phosphate, J , 6 1 Chlorpheniramine maleate, 7,43 Chloroprothixene, 2,63 Chlortetracycline hydrochloride, 8; 101 Clidinium bromide, 2, 145 Clonazepam, 6 , 6 1 Clorazepate dipotassium, 4,91 Cloxacillin sodium, 4,113 Cyclizine, 6,83; 7, 502 Cycloserine, I , 53 Cyclothiazide, I , 66 Dapsone, 5 , 8 7 Dexamethasone, 2, 163; 4,518 Diatrizoic acid, 4,137; 5,556 Diazepam,1,79;4,517 Digitoxin, 3, 149 Dihydroergotoxine methane sulfonate, 7,8 1 Dioctyl sodium sulfosuccinate, 2, 199 Diperodon, 6.99 Diphenhydramine hydrochloride, 3, 173 Diphenoxylate hydrochloride, 7, 149 Disulfram, 4,168 Dobutamine hydrochloride, 8 , 139 Droperidol, 7,171 Echothiophate iodide, 3,233 Epinephrine, 7, 193 Ergotamine tartrate, 6, 113 Erythromycin, 8 , 139 Erythromycin estolate, 1, 101; 2, 573 Estradiol valerate, 4,192 Ethambutol hydrochloride, 7,231 Ethynodiol diacetate, 3,253 Fenoprofencalcium*, 6, 161 Flucytosine, 5 , 115 Fludrocortisone acetate, 3,281 Fluorouracil, 2,221 Fluoxymesterone, 7,251 Fluphenazine enanthate, 2,245; 4,523 Fluphenazine hydrochloride, 2,263; 4,518 Gluthethimide,S, 139 Gramicidin, 8 , 179 Griseofulvin, 8,219 Halcinonide, 8,251 Halothane, I , 119;2,573 Hexetidine, 7,277 Hydralazine hydrochloride, 8,283 Hydroflumethiazide, 7,297 Hydroxyprogesterone caproate, 4,209 Hydroxyzine dihydrochloride, 7,319 Iodipamide, 3,333 Isocarboxazid, 2,295

*Monographs in Pharmacological and Biochemical Properties of Drug Substances: M. E. Goldberg, D. Sc., Editor American Pharmaceutical Association.

558

CUMULATIVE INDEX

Isoniazide, 6,183 Isopropamide, 2,315 Isosorbide dinitrate, 4,225; 5,556 Kanamycin sulfate, 6,259 Ketamine, 6,297 Leucovonn Calcium, 8,3 I5 Levarterenol bitartrate, I , 49;2,573 Levallorphan tartrate, 2,339 Levodopa, 5,189 Levothyroxine sodium, 5,225 Meperidine hydrochloride, I, 175 Meprobamate, I, 209;4,519 &Mercaptopurine, 7,343 Methadone hydrochloride, 3,365; 4,519 Methaqualone, 4,245,519 Methimazole, 8,351 Methotrexate, 5,283 Methyclothiazide, 5,307 Methyprylon, 2,363 Metronidazole, 5,327 Minocycline, 6,323 Nalidixic Acid, 8.371 Neomycin, 8,399 Nitrohrantoin, 5 , 345 Norethindrone, 4,268 Norgestrel, 4,294 Nortriptyline hydrochloride, I, 233;2,573 Nystatin, 6,341 Oxazepam, 3,441 Phenazopyridine hydrochloride, 3,465 Phenelzine sulfate, 2,383 Phenformin hydrochloride, 4,319; 5,429 Phenobarbital, 7,359 Phenoxymethyl penicillin potassium, I, 249 Phenylephrine hydrochloride, 3,483 Piperazine estrone sulfate, 5,375 Primidone, 2,409 Procainamide hydrochloride, 4,333 Procarbazine hydrochloride, 5,403 Prornethazine hydrochloride, 5,429

Proparacaine hydrochloride, 6,423 Propiomazine hydrochloride, 2,439 Propoxyphene hydrochloride, I, 301 ;4 , 5 19; 6,598 Propylthiouracil, 6,457 Pseudoephedrine hydrochloride, 8,489 Reserpine, 4,384;5,557 Rifampin, 5,467 Secobarbital sodium, I, 343 Spironolactone, 4,431 Sodium nitroprusside, 6,487 Sulphamerazine, 6,515 Sulfamethazine, 7,401 Sulfamethoxazole, 2,467; 4,520 Sulfasalazine,5,515 Sulfisoxazole, 2,487 Testolactone,S, 533 Testosterone enanthate, 4,452 Theophylline, 4,466 Thiostrepton, 7,423 Tolbutamide,3,513;5,557 Triamcinolone, 1,367; 2,571; 4,520,523 Triamcinolone acetonide, I, 397,416; 2,571; 4,520; 7 Triamcinolone diacetate, I , 423 Triamcinolone hexacetonide, 6,579 Triclobisonium chloride, 2,507 Triflupromazine hydrochloride, 2,523; 4,520; 5,557 Trimethaphan camsylate, 3,545 Trimethobenzamide hydrochloride, 2,551 Trimethoprim, 7,445 Triprolidine hydrochloride, 8,509 Tropicamide, 3,565 Tubocurarine chloride, 7,477 Tybamate, 4,494 Valproate Sodium and valproic acid*, 8, 529 Vinblastine sulfate, I, 443 Vincristine sulfate, I , 463