Profiles of Drug Substances Vol 08

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 Chafe tz 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

Norman W. Atwater Rafik Bishara Jerome I. Bodin Glenn A. Brewer, Jr. Lester Chafetz Edward M. Cohen Klaus Florey Salvatore A. Fusari

EDITORIAL BOARD

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 ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

ACADEMIC PRESS, INC. 11 1 Fifth Avenue, New York, New York 10003

United Kingdom Edition published by ACADEMIC PRESS, INC. (LONDON) LTD. 24/28 Oval Road, London NW1 7DX

ISBN 0-12-260808-9

PRINTED IN THE UNITED STATES OF AMERICA

79 80 81 82 9 8 7 6 5 4 3 2 1

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,

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,

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,

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,

D. W . Houghton, G . D. Searle and Company Ltd., High Wycombe, Bucks,

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 Research , New

North Carolina

New Jersey

New Jersey

England

England

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,

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

North Carolina

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 compen- dial 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

X PREFACE

Drug substances appearing in the new series will be cross-referenced in

The goal to cover all drug substances with comprehensive monographs is the cumulative index.

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 2.2 Appearance, Color, Odor

Name, Formula, Molecular Weight

3. Synthesis 4. Physical Properties

4.11 Infrared 4.12 Ultraviolet 4.13 Fluorescence-Phosphorescence 4.14 Raman 4.15 Nuclear Magnetic Resonance 4.16 Mass

4.1 Spectra

4.2 Solid Properties 4.2 I Melting Range 4.22 Differential Thermal Analysis 4.23 Thermogravimetric Analysis 4.24 Crystal Properties

4.3 I Solubility 4.32 Dissociation Constant (pKa) 4.33 Partition Coefficients 4.34 Dielectric Constant, Dipole Moment 4.35 Radiation Absorption

4.3 Solution Properties

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)

5.41 Paper 5.42 Thin-Layer 5.43 Column

5.4 Chromatographic Methods

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

ISBN 0-12-260808-9 I

2 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 3

1. 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 trade- name of the German firm Bayer AG, and Acetylsali- cylic 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.

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.

Aspirin has now been available for close

1.2 Bistor d u m e n t e d facts of the discovery of

aspirin are quickly told. 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.

It was synthesized-by the

Apparently, there was some initial reluc- tance 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.

KLAUS FLOREY

44

-. .. .

&... . ,., ... . .

. , .

' ..I . .. .

F i g u r e 1. Labora tory 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 irst p repa ra - t i o n of a s p i r i n . The i n i t i a l s CD on t h e page are t h o s e of C a r l Duisberg. (Cour tesy of Bayer A . G . , Leverkusen, Germany)

ASPIRIN 5

What motivated Hoffmann to undertake this momentous synthesis? Legend has it that he wanted to help his father who was suffering from rheuma- tism 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 sur- vived. 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

6 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 Farben- fabriken Bayer, V o r m . Friedr. Bayer & Co., Elber- ?eld by F. Fischer, 191 3'+, 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 Hoffmann ........ rested unnoticed for 1+ years among thepreparations rejected by the pharmacological laboratory until in 1898,during unrelated in- vestigations, 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.

2 . 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.

ASPIRIN

‘gH8’4 M . W . 180.16

2 . 2 Appearance, Color, Odor Aspirin is a white, crystalline powder. It

is odorless but might have a faint odor of acetic acid.

3. S nthesis

Gerhardt* in 1853. Gerhardt was investigating 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. 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.

h synthesis of aspirin is credited to

Kraut also

CH ,COC1 (gooH + OH

3

or

CH CO 3 \

CH3CO”

catalyst - or

CH2=C0

8 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.

4. Physical Properties

4.11 Infrared 4.1 Spectra

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 previ~usly?~ presented. * A reflection spectrum has also been

TABLE 1

Infrared Spectrum Interpretation

Wavelength (cm-') - Assignment

2300-2500 carboxyl OH 1760 vinyl ester C=O 1690 aromatic acid C=O

aromatic C=C stretch

=C-0 (acid and ester) 1490 1220 1 1190 760 ortho subst. phenyl C-H bending

4.12 Ultraviolet

1

Aspirin in 0.1N sulfuric acidlg and in dilute trichloroacetic acid20 exhibits maxima at

form a maximum was found at 2 7 nm 229 nm (El 484) and 276 nm (E 65.5) .1 In chloro-

(El 68).21

4.13 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.

id a E

rd

10 KLAUS FLOREY

The phosphorescence emission maximum was found at 410 nm.23

4.14 Raman. Raman spectra are described and dis-

cussed in the following references: 24, 25

4.15 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. 32 (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

H1 12.04

H* 2.34(3H)

H3 7.13

H4 7.61

H5 7.33

H6 8.11

= 8.05Hz

= 1.34Hz 3?4

3?5

4?5

4?6

5?6

J

J

J3,6 = 0.3Hz

J

J

J

= 7.80Hz

= 1.74Hz

= 7.96Hz

The differences in the pmr spectrum previously reported in aqueous media30 are attrib- uted 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 Trans- form 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) The seven peak multiplet centered at 6=49.0 ppm

- - arise from the nine carbons of aspirin.

12.0 11.0 10.0 9 .o 8 .O 7.0 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

c

--

=z

d 2

-4

k

-4 a

rCI 0

Id )-I +J

u

a, a

v)

p:

E a a,

4J Id rl I

u

rl

Id u

a c Id

a a,

4J Id rl

-4

m

2 w a, k 3

m

.rl kl

12

6, 5

.-

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 Table 2 . Peak a is the sole peak in the aliphatic 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 with a 'JCH 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 coupling 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 pre- dicted 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

b Assignment Peak G(ppm)a Carbon # Jcab Multiplicity

21.6 9 124.6 3 124.9 1 126.9 5 132.6 6 134.7 4 151.9 2 167.4 7 171.2 8

a) ppm from TMS external via the relationship

b) obtained from the fully coupled spectrum. 6 (CD30D) =49.0 ppm.

ASPIRIN

c) none observed. m) weakly coupled multiplets due to long range

effects.

4.16 Mass - The low resolution mass 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

By adjusting the sample flow and the electron multi- plier 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.

ment ions of the mass spectrum is shown in Figure 7.

looo C. above ambient (approximately 1300 C.). 32

The assignment of a number of frag-

Figure 7.

120 +H 138

m/z

92

m* 104 3 138 -H20

m/z 64

-m/z 63 ‘HCO

m/z 120

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

>- I-

cn Z W I- Z

H

H

W > l-

-I W rY

H

a

100

90

80

70

60

50

40

30

20

10

5292 R S P I R I N

03-RUG-78 MF16761

30

25

20

15

10

5

0

W: Z 0

I-

kl

Z 0

H

a H

H

_I

I- cj I-

I- Z W 0 a: W a

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

4.2 Solid Properties 4.21 Melting Range

The melting point of aspirin is nothing very definitive having variously been given between 118-144°38 and a good deal of work has been done to get the best methods for compendia1

around 135°.44 The European Pharma~opeia~~ gives a melting point of 141° to 144O as determined by the instantaneous method. For further discussion, see Polymorphism (Section 4.242) .

Early commercial preparation melted

4.22 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 aspirin have also been previously studied4&nd used for forensic drug identification.48

DTA and TGA patterns of

4.23 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.

4.24 Crystal Properties

Diffraction The crystal structure of

4.241 Single Crystal X-Ray

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; ~=950 33'; n=4.

4.242 Powder X-Ray Diffraction The powder x-ray diffrac-

tion pattern of aspirin is presented in Table 3 and Figure 8. 5 0

4.243 Polymorphism Tn 1968, Tawashi5' claim-

ed that aspirin exists in several polymorphic forms.

a, 0

k

aJ W

8 2 PI . 4 4

W

0 aJ

k

ASPIRIN

TABLE 3

Powder X-Ray D i f f r a c t i o n P a t t e r n of Asp i r in (U.S.P. Reference Standard)

19

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

D (b) 11.3 6.25 5.68 5.28 4.88 4.30 4.22 4.12 3.93 3.83 3.55 3.30

R e l a t i v e I n t e n s i t y

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

. .

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

R e l a t i v e I n t e n s i t y

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 s tar ted a f l u r r y of a c t i v i t y . D e B i s ~ c h o p ~ ~ claimed t o have obtained 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 only s t a b l e one i s t h e monoclinic one melt ing a t 142O C.

However, t h e claim f o r t r u e polymorphism of a s p i r i n w a s ues t ioned o r re- fu t ed i n several l a b ~ r a t o r i e s ~ ~ - ~ % and can be b e s t summarized i n t h e words of G. Schwartzman:57

"It is gene ra l ly accepted 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 proper- t i es . The data presented a r e e n t i r e l y nega t ive i n these r e s p e c t s . The evidence accumulated agrees wi th t h e f ind ings of P f e i f f e r 5 5 and ques t ions t h e formation of a s p i r i n polymorphs. W e be l i eve t h a t t h e d i f f e r e n t c r y s t a l h a b i t s w e r e caused by t h e s o l v e n t s used for 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 mel t ing p o i n t s are probab- l y due t o t h e poor t r a n s f e r of h e a t caused by t h e l a r g e r c r y s t a l s i z e o r t o p o s s i b l e c r y s t a l d e f e c t s . I'

4.244 Opt ica l Constants Aspi r in has been desc r ib -

ed a s monoclinic, a:b:c = 1.7322:1:1.7322, 8=95O 4.25'. Ind ices f o r 576 p p a re : d=1.5042; 8=1.6424

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.

4.245 Polarized Crystal Absorption Spectrum The polarized absorption

spectrum of a single crystal of aspirin was measured which indicated that aspirin may be spectroscop- ically treated as perturbed benzoic acid.59

4.25 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

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

Water at 25' Water at 37O Water at 100' Ethanol

g/ml 0.0033 0.01 0.03 0.2 - 0.4

Chloroform 0.025 - 0.06 Carbon tetrachloride 0.0004 Ether 0.1 - 0.2 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 effect of selected surfactants above and below the critical micelle concentration (CMC) on aspirin solubility6 was studied.

The

4.32 Dissociation Constant (pKa) In 1913, Springer and

determined the dissociation in- aqueous solution at various temperatures. dissociation constant as 2.8 x (pKa 3.55). The Merck Index62 gives a value of 3.27 x (pKa 3.49) at 25O.

At 25O they determined the

When the apparent pKa was de-

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).

4.33 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

4.34 Dielectric Constant, Dipole Moment

Dielectric constant: Dipole Moment: Ref. fb 2.35 2.09 72

5 to 7 - 73 5.65 calc.; 74

0.93 (acc. to 75 4.36 observed

Onsager by immersion)

4.35 Radiation Absorption The absorption coerricient of a

collimated beam of 6oCo z-radiation was determined for aspirin. See reference 76 for details.

5 . Methods of Analysis

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. very first,residual salicylic acid was determinedby the convenient reaction with ferric salts --typical for phenols -- which give a violet complex with salicylic acid.

5.1 Historical Synopsis As the following pages of this section

From the

In spite of the plethora of methods, the

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%.

ly the same analytical methods as 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.

ing solvents and a spectrophotometric finish at 280 nm is used for the assay with limits of 95 tb 105 percent.

The European Pharmac~peia~~ uses essential-

In contrast to aspirin itself, the U.S.P.

The same column method with different elut-

5.2 Identity and Color Tests Aspirin can be identified by the following

name tests:

ASPIRIN 23

Test

Trinder's reagent McNally's test Mandelin's reagent (Ammonium vanadate )

Feigl

Kulberg

Vitali Morin Kofler

Color Ref.

Purple after hydrolysis 63 Red 63

Green with blue 81 tint; changes to red-violet

Saponif. to salicylic 82 acid. Reaction with KOH at 130°: Violet fluorescence Saponif. to salicylic 83 acid. Addition of FeC13 in HC1: red-violet coloration

Orange to red 84 Microscopic Identifi- 85 cation

For identification by infrared, see Section 4.11. Identification in combination products by mass spectrometry has been described.86

5.3 Quantitative Analysis 5.31 Elemental

The percent of carbon, hydrogen and oxygen is as follows:62

% (theoretical) - 60.00

4.48

35.53

c9

O4

H8

5.32 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 ( X max. 620 nm) with 2-picoline-Cu(II) has also been reported.89

A water insoluble violet complex

5.33 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

24 KLAUS FLOREY

aspirin and salicylic acid, see Section 5.61.

5.34 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

5.35 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.

5.36 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 also been described.96 Potentiometric measurements of ion-pair association and selective acid stren th

Potentiometric titration in aqueous medium has also been described9* as has colorimetric.99

Titration in ethylene diamine has

in ethylene diamine and water has been reported. 7 7

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

ASPIRIN

3. ac second harmonic minimum potential: E min = 1.87

25

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

5.37 Miscellaneous (NMR) Nuclear magnetic resonance spectro-

metry has been used to quantitate aspirin in a combination product with a coefficient of variation of 1.1.1°2 For quantitation, the shift at 2.3 ppm representing the ester methyl group was used.

5.4 Chromatographic Methods 5.41 Paper

Paper chromatographic systems have been tabulated in Table 4 .

TABLE 4

RfA* RfS* Detection Ref. - - Solvent systems:

Pet-ether, Methanol, Benzene and Water (25:20:20:0.05) 0.05 - FeC13 103

Iso-propyl alcohol, Water, Ammonia (15 : 85: 10) - - FeC13 104

Methanol, Water, Ammonia (10:90:10) - - FeC13 104

Butanol, 25% Ammonia (4: 1) 0.45 - U.V. 105

0.75% Nitric Acid 0.8 0.6 Fluorimetry 106

*RfA = aspirin; RfS = salicylic acid.

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

Cellulose anion-exchange paper

26 KLAUS FLOREY

5.42 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 ~ ~ ~ ~ , ~ ~ ~ and TLC separation as student experimentsllO have also been described.

5.43 Column As 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 im- mobile 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 in combination products. 22 I ,724 This has also been adapted for a student experiment.125 A Sepha- dex-G25 column has been used for the separation of aspirin from salicylic acid.

5.44 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 anion- exchange resin127 or cation-exchange resin with and without counter ions128r129 were 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.

5.45 Gas-liquid Determination of aspirin by qas-

chromato 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. As

TABLE 5

Thin Layer Chromatography of Aspirin

Solvent System Rf.Asp. Rf.Sa1. Detection Ref.

MeOH-HOAc-Et2O-CgH6 (1: 18: 60/120) QJo.9 - W 111

Et20-AcOEt (1 : 4) 0.26 - - 112

C6H6-HOAC-MeOH-CHC13- Pet-ether 0.42 0.44 w 113

CH3C13-C6H6-90% HC02H (5:l:O.l) 0.44 0.24 W 114

iso-PrOH-H20-90% HC02H' (1: 5: 6: 01) 0.32 0.08 w 114

CH3C13-90% HC03H (20: 0.1) 0.47 0.24 W 114

Cyclohexane-CHC13-HOAc (4:5:1) 0.61 0.41 w 114 Cyclohexane-CHC13-HOAc (50:40:10) 0.36 - K4(Fe(CNa))3 115

CHC13-Cyclohexane-HOAc- Dioxane (40:60:1:10) 0.48 0.14 W 116 CHC13-Cyclohexane-HOAc (40: 60: 1) 0.37 0.12 w 116 EtOH-IsoProOH-Xylene- CHC13 (12.5:12.5:25:50) ' 0.04 - Iodine 117

Support

Silica

Silica Gel G

Silica

Po 1 y ami de

Polyamide

Polyamide

Po lyamide

Silica Gel G

Polyamide

Silica Gel G

TABLE 5 (continued)

Support Solvent System Rf.Asp. Rf.Sa1. Detection Ref.

Silica CHC13-(CH3)2C0 (9:1) 0.075 - NH4 Vanadate 118

MeOH - NH3(100:1.5) 0.75 - NH4 Vanadate 118

Silica Gel GF EtOAc-t-PrOH-NH3 (40:30:3) 0.25 - W 119

S i l i c a Gel GF E~OH-HOAC-H~O (60:30:10) 0.92 - W 119

Silica Gel GF CgHg 0.10 - w 119

Silica Gel 60 C6Hg-Dioxane-HOAc (60 : 20: 2) - - Fluorimetry 120

ASPIRIN 29

meth lating agents, diazomethane in ether alco- hollY7 or tetrahydrofurane, 3 8 methanol with boron trifl~oridel~~ and methyl iodide with potassium carbonate140 were used. For trimethylsilylation hexamethyl disilazane,141-143 N-0-bis(trimethy1- silyl) acetamide and other trimethylsilyl BSTFA145-146 and MSTFA147 have been tried. It was noted that derivatization with BSTFA or MSTFA resulted in slight hydrolysis of aspirin.147

5.5 Electrophoretic Methods Aspirin can be separated from salicylic

acid by ionophoresis at a pH of 4-5.14* Separation 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

5.6 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.

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

2. 3. 4. 5. 6. 7. 8.

9.

10. 11. 12.

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

TLC (for Rf values and solvent systems see Section 5.42): 114,116,119

VPC: 106,107,140,141,144,145,146,147 HPLC: 152,164 Automated: 165-167

Section 5.41): 106,107

5.62 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.

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 back- titrating it with alkali. Gas chr~matographyl~~ has also been used.

A.N. Smith in 192016* devised a

5.63 Acetylsalicylic Anhydride and Acetyl- salicylsalicylic Acid In addition to salicylic and acetic

acids, very small quantities of acetylsalicylanhy- dride (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. t T 4 A contro- versy 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.

6. Stability - Deqradation That aspirin is sensitive to moisture is well-

known 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 bath-

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 preformula- tion profile. He tested the hydrolysis of aspirin in acid and alkaline solutions and,by perusing text- books 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 depend- ence 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

7. Pharmacokinetics - Drug Metabolic Products 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 discover- ies.

When Dreser' investigated the pharmacological

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,188-190 taken 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 20% 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 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

only shown by aspirin but not by salicylic acid. The kinetics of this important pathway still need exploration.187

dependent.Ig4 In man it is sex linked since it was found to be higher in men than women.lg5 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 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

While

a role in the inhibition of platelet aggregation 1J 3

Aspirin serum esterase activity is species

It also

The major

To properly treat bovine "sufferers of head- aches" (inflammation) the pharmacokinetics of aspirin in cattle have also been explored.1g8

8. Bioavailability - Dissolution In the last two decades, the concept of bio-

availability has gained prominence and-with it dissolution as a possible in vitro model for drug absorption. In 1960, Swintoskyd BlytheIg9

I I 0

W

Ocn

U /

E'

u

o

I 8 8

'

uo

E I 0

X

rcl 0

ASPIRIN 35

compared the relative availability of aspirin from entexic coated and compressed tablets by measuring the excretion of salicylate. About the same time, Levy200,201 started 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 ind iv idua1 ,va r i a t ions 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 pH- stat for testing dissolution of various aspirin dosage forms has been described.205

9. Determination in Biological Fluids and Tissues

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 (ferric- nitrate) 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

All the advances in pharmacokinetics and drug

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

10. Determination in Pharmaceutical Preparations The following tabulation of references high-

lights those methods (see Section 5 ) useful in pharmaceutical analysis.

1. Determination in tablets: Differential U.V.: 157,158 Automation: 166 Fluorometric : 22 Column : 121 TLC : 120 GC : 136,139,144

2. Determination in buffered tablets: Fluorometric: 159 Column : 161 , 162

3. Determination in combination products: a. General Identity tests: 83,86,103,104,105,

b. Aspirin, caffeine, acetophenetidine 11 2

(and more components) w: 21,215 IR: 91-93 NMR: 102 Electrophoresis: 149 TLC : 110 , 111 Column: 80 Ion-exchange column: 123 HPLC : 127,130,131 VPC : 135 , 142 Pot. titration: 216

ASPIRIN 37

c. Aspirin, acetaminophen, caffeine: Nonaqueous titration: 94,217 Potentiometric: 102

d. Aspirin-barbiturate combinations: Nonaqueous titration: 93

e. Cough-cold mixtures: HPLC: 132

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. References

1.

2. 3. 4. 5. 6.

7. 8. 9.

10. 11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

H. Dreser, Pflug. Arch. Gen. Physiol., 76, 306 (1899). J. Wohlgemuth, Ther. Mh., 13, 276 (1899). K. Witthauer, Ther. Mh., 13, 330 (1899). Bayer A.G. Grchives A. Eichengrun, Pharmazie, 4, 582 (1949). F. Hoffmann, U.S. Patent c44077 of February 27, 1900. Neue Deutsche Biographien, p. 415. C. Gerhardt, Annalen der Chemie, 67, 149(1853). H. von Gilm, Annalen, 112, 181 (1859). K. Kraut, Annalen, - 1 5 0 3 (1869). D.A. Nightingale, U.S. Patent 1,604,472; C.A. 21, 157 (1927). BayerT Co., DRP 386679 (1924); Beilstein 111, p . 41. Fernandez, Torres, An.Soc. espaz., - 21, 23 (1923); Beilstein 111, p. 41. Norwich Pharmacal.Co., Brit. Pat. 893,967 (1962); C.A. 57, 3572f (1962). J.W. Clappiso: J.P. Hummel, W.D. Paul and J. I. Routh, J. Am. Pharm. ASSOC., 40, 532 (1951). B. Toeplitz, The Squibb Institute for Medical Research, Personal Communication. O.R. Sammul, W.L. Brannon and A.L. Hayden, J. Assoc. Offic. Agr. Chemists, 47, 918 (1964). S.G. Il'yasov, V.L. Dragun, C.A.82, - 91861p (1975). Isolation and Identification of Drugs, E.C.G. Clarke, Editor, The Pharmaceutical Press, London (1969). A. Garner, J.K. Sugden, Anal. Lett. - 6, 275 (1973). M. Jones and R.L. Thatcher, Anal. Chem., 23, 957 (1951). C.I. Miles and G.H. Schenk, Anal. Chem., 42, 656 (1970). J.D. Winefordner and H.W. Latz, Anal. Chem., - 35, 1517 (1963). L. Kahovec and K.W.F. Kohlrausch, Monotsh. - 74, 333 (1943); C.A. 38, 51475 (1944). G.V.L.N. Murty andT.R. Seshadri, Proc.Indian Acad. Sci., - 19A, 17 (1944); C.A. - 38, 6197-8 (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., - 2, 707 (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., - 226, 60 (1973); C.A. 80, 74282s (1974). A.J. Marcus, S.E. Lasker, Haernostasis, - 2, 92 (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, - 89, 223 (1950); 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., - 157, 88 (1946). M. deBisschop, J. Pharm. Belg., - 25, 330 (1970). 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, - 71, 411 (1974). 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.

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ASPIRIN 41

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199. J.V. Swintosky and R.H. Blythe, Drug

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- - -

-

-

-

- The literature has been covered systematically through Chemical Abstracts,Volume 88 (1978), with a few stray references beyond.


BROMOCRIPTINE METHANESULPHONATE

3. 4 .

5 .

6. 7 .

8.

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 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

Application of General Tests

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 Copyright @ 1979 by Academic Press, Inc.

All rights of reproduction in any form reserved. ISBN 0 12-260808-9

41

48 DANIELLE A. GIRON-FOREST AND W. DIETER SCHONLEBER

1. Introduction

1.1 History

The most valuable pharmacological p rope r t i e s of the pep- t i d e type e rgo t a l ca lo ids induced a va r i e ty of attempts of chemical de r iva t i s a t ion of the parent compounds ( 1 , 2 + lit. quoted the re in ) . In one of these the bromination of a-ergocryptine l ed t o a product of highly i n t e r e s t i n g pharmacology, namely bromocriptine.

I t became known to suppress p ro lac t ine secret ion, and it i s therefore a useful t o o l i n the treatment of prolact ine dependent disorders , such as galactorrhea associated with hyperprolactinemia and postpartum, as well as c e r t a i n kinds of s t e r i l i t y ( 3 - 9 ) . In more elevated doses, the drug i s a potent antiparkinsonicum. In addi t ion, t he re i s recent evidence of bromocriptine playing an important r o l e i n the trace heavy metals balance of the b ra in (10 ) .

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'-(2- methylpropyl-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 ingredient i n ParlodelB dosage forms.

/.. CH 3

x CH3S03H

BROMOCRIPTINE METHANESULPHONATE 49

Chemical Abstracts Registry Numbers :

25614-03-3 (26409-15-4) ( 4783 0- 26- 2 )

Average Molecular Weights :

base : 654.61

mesilate: 750.71

Average Formulas :

base : C H B r N 0

mesilate: C H B r N 0 S

32 40 5 5

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 Propert ies

2.1 Elemental Analysis

The elemental ana lys i s of bromocriptine mesilate yielded the following r e s u l t s (11) :

element % calculated % found

C 52.8 53.2 H 5.9 6.0 B r 10.6 10.5 N 9.3 9.2 0 17.0 16.8 S 4.3 4.4

2.2 Spectra

2.21 Infrared

The in f r a red spectrum of bromocriptine mesilate 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 red spectrophotometer. The main c h a r a c t e r i s t i c bands are the following :

%Absorption

a, cr m rl

.d

ffl

P


-1 wave number ( c m ) assignment

3200 - 2900 C-H-stretching v i b r a t i o n s 1728 C=O-stretching of 5-membered

1642, 1672 C=O-stretching of 6-membered

1560 amide-11 band and

r i n g lactam (cyc lo l )

r i n g l a c t a m (amide I)

C=C- s t r e tch ing

The spectrum of t h e base has been r epor t ed 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 of bromocript ine mesilate was recorded on a P h i l i p s 1700 uv spectrophotometer i n 0 .1 M me- t hano l i c methanesulphonic a c i d so lu t ion . I t is given i n f i g . 2. A maximum occurs a t about 308 nm wi th a log molar absorp- t i v i t y of 4.0. I n 1:l dichloromethane/methanol s o l u t i o n t h e absorp t ion maximum was r epor t ed as 306 nm log = 3.988 (11).

2.23 Fluorescence

Bromocriptine mes i l a t e e x h i b i t s f luorescence 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 ac id type a l c a l o i d s . I n 2 per- c e n t e thano l i c tartaric a c i d s o l u t i o n , t he emission maximum appears 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 Proton Nuclear Magnetic Resonance

The PMR spectrum of bromocript ine mesilate i n deu te ra t ed dimethyl sulphoxide a s ob ta ined on a Bruker HX-90 NMR spec t rometer is presented i n f i g . 4. TMS served a s i n t e r n a l s tandard . The c h a r a c t e r i s t i c s of t h e spectrum are given i n the fol lowing t a b l e (see a l s o 12 , 13 and lit. quoted t h e r e i n ) :

PMR-spectrum and Assignment Chemical S h i f t Intensity downf i e l d (ppm)

M u l t i p l i c i t y Assignment

11.85 1 H S i n g l e t indole-Ng I

NH 10.45 1 H S i n g l e t -

9.55 1 H S i n g l e t CONH- (18-19)

7 - 7.3 4 H

and OH

52 DANIELLE A. GIRON-FOREST AND w. DIETER SCHONLEBER

230 270 310 350 390 nm

Figure 2. Ultraviolet Spectrum of Bromocriptine Mesilate in 0.1 M Methanolic Methanesulphonic Acid. CA = 0.05 mg/ml, C Instrument: Philips SP 1700

= 0.012 m g / m l . B

BR LOMI 3CRIPTINE METHANESULPHONATE

nm

53

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


1 H S i n g l e t H-C

T r i p l e t H-C

s i n g l e t 6-CH ((2-17)

- 9

- 5 '

6.47

4.3 - 4.5

3.2 16 H { -3 Mul t ip l e t g-C ;!-C ;H-C4;

8 ' 11' - H-C5 ; F-C * H-C - 7 ' - 8 CH C0CK2CH3 ; H20

( r e c r y s t . so lven t )

-3 3

3 - 4 . 2

3

3 H S i n g l e t CH -SO H 2.37

S i n g l e t CH COCH CH

Mul t ip l e t -3 2 3

5'-C€12 ; 5'-Cg ; 2 ' - C g ; 2 * 1 1 8-9 H {

2 . 1 - 1.5

- H-Cg, ; g-C 10 ' 0.75-1.15 1 2 H M u l t i p l e t 2 ' C H - 5'-CH and

-3 -3 CH3COCH CH

2 -3

2.25 Carbon-13 Magnetic Resonance Spectrum

The spectrum of bromocript ine mes i l a t e has been recorded i n dimethyl sulphoxide us ing a Bruker HX-90 NMR spectrophotometer ( f i g . 5 ) . The assignment of t h e ind iv idua l s i g n a l s is given 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 bromocript ine mes i l a t e ( f i g . 7 ) corresponds q u i t e n i c e l y t o those o f t he o t h e r non- and dihydrogenated e r g o t a l c a l o i d s ( 1 2 , 13 and lit. quoted h e r e i n ) .

The molecular peak M of t h e base 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 the n a t u r a l abundance of t he bromine i so topes , 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 nex t smal le r fragment appears a t M-228, ind ica- t i c g a s u b s t a n t i a l loss of the pep t ide s e c t i o n c o n s t i t u e n t s wi th the 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 spec ie s the i sopropyl 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 . Loss of t he e n t i r e pep t ide moiety l eads t o t h e formation of 2-bromo-lysergamide F2 a t 345/347 m.u., t he mass s p e c t r a l behaviour of which corresponds t o t h a t of t h e bromine-free compound. Thus, i t g ives 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 loss of formamide o r by t h e rup tu re of t he te t ra-hydro-pyridine r i n g ,

6000

3000

'%a

4 0 0 3

2000

rm

2000

>m xa

I 4ca

t I tm y1 - L

I T T T T I lb lbo 0

Figure 5 . 13-C-NMR Spectrum of Bromocriptine Mesilate in (CD ) SO, 3 2

Instrument: Bruker HX-90 E

57

I 100

I ' O

90 1 I 80

70

60

50

YO

30

20

10

I__ 36

I

M/Z 582 '25 635 653 1 " " " " ' 1 " " " ' " l ' " ' ' " " " " " ' ' ~ f

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


a decay mode t h a t w a s proposed earlier (14,15) for l y se rg ic acid der ivat ives . Peak doublets a t 285/287 and 274/276 m.u. i nd ica 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 containing bromine.

425/427 m.u.

The peptide moiety i t s e l f (308 rn.u.1 obviously does not appear i n the 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 , is c l e a r l y under- stood (14,15) - The mesilate shows a s igna l a t m/e 96.

1

F3 I F4

195 m.u. 154 m.u


H D ' q0 F5

125 m.u.

F7

86 m.u. 70 m.u.

2.3 Crys t a l P rope r t i e s

2.31 Melting C h a r a c t e r i s t i c s

Bromocriptine mes i la te decomposes between 180 and 200 O C ,

t hus a mel t ing p o i n t o r mel t ing range cannot be given.

2.32 Polymorphism

Inves t iga t ions f o r t h e occurrence of polymorphism have been undertaken by ir spectroscopy, d i f f e r e n t i a l scanning ca lor imet ry and x-ray powder d i f f r a c t i o n (Guinier-de Wolf f ) . N o polymorphism has been observed so f a r . An amorphous form may be prepared a r t i f i c i a l l y by r a p i d evapora t ion of a methanolic s o l u t i o n of t h e drug substance.

2.33 X-Ray Di f f r ac t ion

X-ray s t r u c t u r a l a n a l y s i s of bromocript ine, c r y s t a l l i z e d a s t he base from dichloromethane/diethylether, has been car- r i e d o u t on a CAD 4-diffractometer wi th CuKa-radiation ( 1 6 ) . 3371 r e f l e x i o n s were wi th in s i n @/A<0.62 8-l . Assessment of t h e s t r u c t u r e was achieved by computation t o a ref inement of R = 0.033 f o r t h e abso lu te conf igura t ion . The c r y s t a l d a t a w e r e : Space group P 2 a = 10.681, b = 13.454, c = 11.049, @ = 99-75", Z = 2. I n f i g . 8 t he r e s u l t a n t conformation of bromocript ine base i s depic ted .

1'

2.34 D i f f e r e n t i a l Scanning Calor imetry

The DSC thermogram of bromocript ine mes i l a t e , ob ta ined wi th a Perkin E l m e r DSC-2 instrument a t a hea t ing ra te of 20 'C/min. and i n a n i t rogen 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.

= Carbon, 0 = oxygen, 0 = Nitrogen,

0 = Hydrogen


TG

\

T

40 60 80 100 120 140 160 180 200 OC

Figure 9. Differential Scanning Calorimetry and. Thermogravimetry Curves of Bromocriptine Mesilate, each at a heating rate of 20 C/min. and a Nitrogen Flow of 15 ml/min. Instruments: Perkin Elmer DSC-2

Perkin Elmer TGS-1

0

BROMOCRIF'TINE METHANESULPHONATE 63

The melt ing endotherm i s followed immediately by a s t rong exothermic degradat ion. Since bromocript ine mesilate decomposes under mel t ing , the t r a n s i t i o n temperature i s s t rong ly dependent on the hea t ing r a t e . A broad b u t weak endotherm between 40 and 100 "C i n d i c a t e s t he v o l a t i l i z a t i o n of sorbed r e c r y s t a l l i z a t i o n so lven t (usua l ly butanone-2, see s e c t i o n 3 ) .

2.35 Thermogravimetry

The thermogram of bromocript ine mesilate, c a r r i e d o u t on a Perk in E l m e r TGS-1 thermobalance, i s given i n f i g . 9. The sample temperature w a s r a i s e d a t a r a t e of 20"C/minute maintaining a n i t rogen atmosphere. A cons iderable loss of 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 obviously s t a r t s a f t e r mel t ing.

2.4. S o l u b i l i t y

The s o l u b i l i t y of bromocript ine mesilate w a s determined i n a v a r i e t y of so lven t s e q u i l i b r a t e d by v i b r a t i o n dur ing 2 4 hours a t 25 OC. They are a s fol lows:

Solvent S o l u b i l i t y i n mg/g S o l u b i l i t y i n g/100 m l

water m e t h a no 1 e thanol 2-propanol ace ton i tr i l e acetone e t h y l a c e t a t e chloroform benzene hexane

0.8

23.0 1 . 2 1.6 0.2 0.2 0.45 <0.1 <<0.1

910 0.08

1.8 0 .1 0.12 0.015 0.015 0.06

<0.02 <<0.01

72

A t 22 ?r 2 OC bromocript ine mes i l a t e d i s s o l v e s r e a d i l y ( > 2 % ) i n propylene g lyco l , 50 % e thano l , 95 % e thanol and n-octanol, whereas i t i s poor ly so lub le (<0.1 % ) i n s imulated 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 Dissoc ia t ion Constant

Due t o the low s o l u b i l i t y of bromocript ine mesilate i n water , t h e pK va lue had t o be determined i n methyl c e l l o - solve/water 8: 2 (w/w) . T i t r a t i o n a t ambient temperature y i e ld - ed pKa a s 4.90 * 0.05 f o r a 0.0078 M so lu t ion .

a


2.6 Par 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 bromocr ip t ine mesilate between water of pH 1.2 and n-octanol on t h e one hand, and water of pH 7.5 and n-octanol on t h e other, have been determined a t 37.0 ? 0.5 "C.

water p H 1 .2/n-octanol 1 : 90 water pH 7.5/n-octanol 1 : 235

2.7 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 molecule ( 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 romocr ip t ine 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 of 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 o c c u r r i n g a -ergocrypt ine .

The specific optical r o t a t i o n of bromocr ip t ine mesilate i n d i f f e r e n t s o l v e n t s i s g iven below for 20° C corrected for loss on dry ing . 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 used, 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 10 mg/ml.

wavelength (nm)

s o l v e n t 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 d e g r e e s

dichloromethane/ methanol 1:l 101.0 107.5 130.1 327.8 329.4 e t h a n o l 100.6 107.0 129.6 328.7 331.3 d imethyl formamide 127.4 135.2 162.0 388 .O 392.7

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

3. S y n t h e s i s

Bromocript ine base i s manufactured by brominat ion of a -ergocrypt ine w i t h N-bromosuccinimide i n d ioxane 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 is r e c r y s t a l l i z e d from butanone-2 (s. f i g . 10 ) (2 ,3111) -


a-Ergocryptine Base

N-Bromo-succinimide

Bromocriptine Base Bromocriptine Base

Methanesulphonic Acid

* Bromocriptine Methanesulphonate

Figure 10. Route of Synthesis


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

Possible degradation pathways of e rgo t a l ca lo ids a t the 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 . (17) and B. Kreilgaard (13).

A s a nonhydrogenated ergot a l ca lo id , bromocriptine i s r e l a t i v e l y sens i t i ve t o autoxidation both i n s o l i d and i n d i s - solved states, but degradation products have not y e t been e l u - cidated. S imi l a r i t y t o the oxidative transformation of the parent compounds i s t o be strongly an t i c ipa t ed ( 1 2 ) .

I n hydroxyl-containing solvents, nonhydrogenated e rgo t peptide a l ca lo ids a r e r ead i ly epimerized a t C-8 t o an equ i l i - brated mixture of the lysergic and iso-lysergic acid series , ca l l ed the -ine/-inine forms (-ine = 88, - inine = 8a)(12,18 and lit. quoted the re in ) . The hydrolysis of the lysergic acid amide bond i s of minor importance f o r the beginning degrada- t i on , but, of course, prevalent under more d r a s t i c hydrolysis conditions.

Under the same conditions, but a t elevated temperatures, the C-2I-center is very l i k e l y t o be inverted (aci-inversion) yielding a more ac id i c isomeric compound.

The light-induced addi t ion of water t o the 9,lO-double bond of bromocriptine yielding the so-called lumi-products, i s of high probabi l i ty (12,18). They have, however, not y e t been i so l a t ed o r characterized. The corresponding l0a-methoxy- lumi-derivative could be prepared by the photo-catalyzed addition 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 mesilate is s e n s i t i v e t o hea t and l i g h t , it is s t a b l e f o r up t o 3 years a t ambient tempera- t u r e (20 ) when s tored i n sealed polyethylene bags contained i n twist-off amber g l a s s bo t t l e s . In warm and t rop ica l clima- t e (30 "C/75 percent r e l a t i v e humidity) and under i d e n t i c a l package conditions, it i s s t a b l e fo r 1 year, however only f o r 3 months a t 50 "C.

I f not properly protected, bromocriptine adsorbs up t o 6 percent by weight of water i n t rop ica l climate.


4.2 i n Solution

Bromocriptine follows the behaviour sketched above and is thus r a the r l a b i l e i n aqueous o r aqueous/alcoholic so lu t ions , p a r t i c u l a r l y i n the presence of acid, yielding mainly the equi l ibrated mixture with i t s 8-epimer (1) and t o a smaller extent , i t s hydrolysis products 2-bromo-lysergamide and 2-bromo-lysergic ac id and t h e i r 8a-isomers, respect ively.

4.3 i n the Dosage Form

Bromocriptine mesilate is marketed a s ParlodelB capsules (10 mg f o r the treatment of Parkinson's disease) and t a b l e t s (2 .5 mg a s l a c t a t i o n suppressor). Both forms have proved s t a b l e a t l e a s t f o r 4 years when s tored 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 spos i t i on of bromocriptine has been studied i n several animal species and man following s ing le o r a l and intravenous administration of the drug label led 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 solut ion amounts t o 30 - 40 8 as determined from the sum of the cumulative b i l i a r y and ur inary excretion of r ad ioac t iv i ty (parent drug + metabolites) i n b i l e duct 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 species. This, most l i k e l y , i s due t o the marked a f f i n i t y of the drug f o r various t i s s u e s and the rapid hepatic ex t r ac t ion of t he absorbed f r ac t ion . The main route of excretion is the b i l e . L e s s than 5 % of the dose a r e recovered i n the ur ine 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 administration.

In man, the extent of en te ra l absorption i s estimated t o be a t l e a s t of the same order of magnitude than determined f o r animals. Absorption is rapid with an approximate r a t e constant of 1 . 4 h-' ( t '/2 = 30 min) .


Peak plasma l eve l s are reached about 1 . 5 h a f t e r o r a l ingestion, the maximum concentrations being i n the order of 2 - 3 ng equivalents/ml (parent drug + metabolites) f o r an o r a l 1 mg dose. The elimination from the plasma i s biphasic and proceeds with mean ha l f - l i ves of 6 h (a-phase) and 50 h (6-phase). Similar elimination ha l f - l i ves are obtained from the urinary excretion. The cumulative r ena l excretion is p rac t i ca l ly the same a f t e r o r a l and intravenous administration and amounts t o 6 - 7 % of the r ad ioac t iv i ty dosed. The main portion of the dose, e i t h e r o r a l or intravenous, is eliminated by the b i l i a r y route i n t o the faeces. The k i n e t i c s of bromo- c r i p t i n e has been demonstrated t o be l i n e a r i n the o r a l dose range from 2.5 t o 7 . 5 mg.

Parent drug is bound t o bovine and human plasma p ro te ins t o an extent of 89 - 96 % ( i n v i t r o concentration range 0.2 - 80 pg/ml) and exe r t s a pronounced a f f i n i t y to various t i s sues .

5.2 Metabolism

Bromocriptine is rapidly and completely metabolised i n animals and man. The major components of the urinary metabo- l i t e s have been iden t i f i ed as 2-bromo-lysergic acid and 2-bromo-isolysergic acid. Apart from the hydrolyt ic cleavage of the amine bond and the isomerization a t pos i t i on 8 of the ly se rg ic acid moiety, a t h i r d p r inc ipa l biotransformation pathway consists i n the oxidative a t t ack of the molecule a t the p ro l ine fragment of the peptide p a r t , predominantly a t posi t ion 8 ' , giving r i s e t o the formation of a number of hydroxylated and fu r the r oxidized der ivat ives of bromocriptine, and i n addi t ion of conjugated der ivat ives thereof.

6. Toxicology

The acute tox ic i ty of bromocriptine mesilate has been determined i n the mouse a s 230 and 2620 mg/kg i .v . and P.o., respectively. In the r abb i t , the corresponding values w e r e 1 2 and >lo00 mg/kg ( 2 ) . Thus, bromocriptine proved less toxic than the nonhydrogenated ergot a l ca lo ids by one order of magnitude, resembling the behaviour of the dihydrogenated der ivat ives .

Chronic tox ic i ty s tud ie s were ca r r i ed o u t with r a t s , dogs, and rhesus monkeys ( 2 ) . In nearly a l l cases, the p r inc i - p a l e f f e c t produced w a s ischemia of some p a r t of the body. The well-known e m e t i c e f f e c t of ergot a l ca lo ids 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.


7 . Analytical Methods

7 . 1 Application of General Tests

Compared with the unsubsti tuted ergot a l ca lo ids (1,181 the introduction of bromine i n t o the 2-position of the indole nucleus diminishes the r e a c t i v i t y of the molecule i n r e spec t t o general a l ca lo id t e s t s . Nevertheless, they s t i l l remain applicable.

1. - KeLlgrLs-tgsL. Bromocriptine mesilate i s dissolved i n g l a c i a l a c e t i c acid t o which have been added t r aces of f e r r i c chloride. After ca re fu l ly layering with concentrated s u l f u r i c acid, a green colour i s produced a t the in t e r f ace . Usua l ly an intense blue-violet colour occurs with 2-H-substituted compounds.

2 . I a n grk's gegcLion, ( 2 4 ) Bromocriptine mesilate i s dissolved i n methanol. After addi t ion of van U r k ' s reagent and vigorous shaking a blue colour develops slowly, which is subs t an t i a l ly weaker than with 2-H-ergot a lcaloids .

3. Meyeg's t e z t L Bromocriptine mesilate i s dissolved i n methanol/water 3:lO. After addi t ion of one drop of d i lu t ed hydrochloric acid 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 is produced.

7 . 2 T i t r a t i o n

Bromocriptine mesilate may be assayed i n g l a c i a l a c e t i c acid/acet ic anhydride 1 :7 by t i t r a t i o n with 0.1 N perchlor ic acid. The endpoint may be determined potentiometrically using a glass/calomel e lectrode system.

The methanesulphonic acid content of bromocriptine mesilate i s usually determined by t i t r a t i o n with 0 .1 N

methanolic potassium hydroxide. The endpoint may be detected potentiometrically using a glass/calomel e lectrode system.

Residual N-bromosuccinimide from the manufacturing process may be iden t i f i ed and/or quant i f ied by making use of i t s oxidation po ten t i a l by t i t r a t i o n of l i be ra t ed iodine a f t e r addi t ion of potassium iodide i n a c e t i c acid ( 2 5 ) .


7.3 Spectroscopic Methods

7 .31 Infrared

Infrared spectroscopy is 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 the analysis of the drug substance. (see 2 .21)

7.32 Ul t r av io l e t

Spectrophotometric ana lys i s of bromocriptine mesilate i s carr ied ou t d i r e c t l y using the uv maximum a t about 305 run i n methanolic methanesulphonic acid. However, the method i s not very spec i f i c .

I t was preferred t o f i r s t separate the impuri t ies from bromocriptine by thin layer chromatography and then t o i s o l a t e the substance by e lu t ion from the s i l i c a ge l of the p l a t e with methanol. The i n t a c t a c t i v e ingredient is measured i n 0.01 M methanolic methanesulphonic acid (26 ) .

7 .33 Colorimetry

In moderately ac id i c solut ions bromocriptine mesilate readi ly forms ion p a i r s with anionic dyes such a s p i c r i c ac id , bromothymol blue, methyl orange, which a r e ex t r ac t ab le with an organic solvent. A procedure has been developed both f o r d i r e c t assay and fo r assay following chromatographic separa- t i o n from the impurit ies. Therein bromocriptine mesilate i s allowed t o r e a c t with bromothymol blue a t pH 2.5. The resul- t i ng ion p a i r is then extracted with benzene and i ts concen- t r a t i o n determined a t 410 nm ( 2 5 ) .

7.34 Proton Magnetic Resonance

PMR spectroscopy may be used for i d e n t i f i c a t i o n of the drug substance. (see 2 . 2 4 )

7.4 Chromatography

7 .41 Paper

Paper chromatography was applied formerly t o the determination of bromocriptine.

conditions: system 1: s t a t iona ry phase: 25 % formamide mobile phase : carbon te t rachlor ide/

d i e thy l e ther 1:l


system 2 : s t a t iona ry phase: 10 % dimethylphthalate mobile phase : dimethylformamide/

0.5 N hydrochloric acid 15:85

The drug substance is visual ized by react ion with iodine vapour .

Rf values of bromocriptine and precursor

system 1 system 2

Bromocriptine 0.88 0.09 a- ergocr yp t i n e

(precursor) 0.70 0.31

7 .42 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 solut ions o r during spo t t ing on the p l a t e . Therefore, bromocriptine mesi- l a t e , usually dissolved i n chloroform/methanol 1:1, has t o be spotted on the p l a t e very rapidly and with the exclusion of l i g h t . The separation being terminated, the mobile phase is 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 invest igated f o r the separat ion of by-products, degradation products, metabo- l i t e s , and excipients . Also a v a r i e t y of spraying reagents have been t e s t ed (see t a b l e ) . The most advantageous one w a s Dragendorff 's reagent with consecutive spraying by 30 %

hydrogen peroxide.

methods: s t a t iona ry phase: s i l i c a g e l 60 F254, ( 2 7 ) Merck t l c p l a t e s , no.5715

mobile phase 1 : dichloromethane/dioxane/96 percent ethanol/conc.ammonia 180:15:5:0.1

(v/v/v/v)

mobile phase 2 : chloroform/methanol/formic ac id 78 : 20 : 2 (v/v/v)

mobile phase 3 : chloroform/96 percent ethanol/conc. ammonia 192:8:0.35 (v/v/v)

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


R values s t

compound mobile phase 1 mobile phase 2

bromocr i p t i n e 1.0 (Rf 0.5) 1.0 (Rf 0.7)

a-ergocryptinine

a-ergocryptine (precursor) 0.6 0.5 2-bromo-lysergic acid 0.0 0.25 2-bromo-isolysergic acid 0.0 0.35 2-bromo-lysergamide 0.5 0.15 2-bromo-isolysergamide 0.5 0.35

2-bromo-a-ergocryptinine 1 .5 1.1

(isomer of precursor) 1 .4 1.0

Mobile phase 3 may be used f o r the detect ion and s e m i - quan t i t a t ive determination of res idual N-bromosuccinimide. Thereby, the p l a t e i s sprayed with water and then placed i n a chlorine atmosphere f o r 10 minutes. Excess chlorine i s removed by placing the p l a t e i n a stream of w a r m a i r f o r another 10 minutes. After spraying with o-toluidine reagent, the eva- luat ion i s made against a d i l u t i o n of N-bromosuccinimide. The detect ion l i m i t is 0 .1 pg, the R is 0.3 with r e spec t t o bromocriptine R f value.

s t

A high performance t l c system has been developped f o r the in-process-control (28) using Merck s i l i c a gel HPTLC pla- t e s , no. 5628, as the s t a t iona ry phase and tetrahydrofuran/ chloroform/n-heptane/methanol/conc. ammonia 20:20:57:7:1 per volume a s the mobile phase. The chromatography (6 cm ascending) i s carr ied ou t without preceding chamber saturat ion. The compounds separated a r e visualized by uv a t 254 and 366 nm, respect ively, and by iodine vapour. Rf of bromocriptine i s 0.27. The po ten t i a l by-products y i e ld spots a t R 1.5 (2-bromo-a-ergocryptinine) , and 0.55 (a-ergocryptiney . 2-Bromo-lysergic acid remains a t the s t a r t i n g point.

s

Beside the modified Dragendorff's reagent/hydrogen peroxide already mentioned, a va r i e ty of spraying reagents has been used fo r the visual izat ion of bromocriptine. They a r e compiled i n the following list.

BROMOCRIPTINE METHANESULPHONATE

spraying reagents f o r bromocriptine detect ion l i m i t

Merck no. 5715 reagent colour on s i l i c a ge l

Dragendorff (Munier,Deboeuf) + 30 percent H 0

0.05 percent aqueous permangana te

van U r k ' s reagent cinnamic aldehyde formaldehyde/hydrochloric acid formaldehyde/sulphuric acid chlorine/o-toluidine folin/ammonia ninhydrin/uv det . 360 iodine/potassium iodide Ehr l i ch ' s reagent

2 2 brown

yellow

grey yellow

grey v i o l e t v i o l e t

grey red brown orange

The i d e n t i t y of methanesulphonic acid may be determined by t l c on ce l lu lose p l a t e s , Merck no. 5728, with ethanol/ water/conc. ammonia 80:16:4 (v/v/v) as a mobile phase. Detection is achieved by spraying with acid-base ind ica to r s , e.g. bromocresol green o r similar species. Rf of methanesulphonic acid is 0.5 ( t h a t of bromocriptine base = 0.9).

7.43 G a s Chromatography

GC cannot be applied t o the ana lys i s of bromocriptine mesilate 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 exce l l en t i d e n t i f i c a t i o n and quan t i t a t ion on the bas i s of well-defined peptide sect ion pyrolysis products, has not y e t been attempted. However, GC i s very useful determining the r e s idua l r e c r y s t a l l i z a t i o n solvent butanone-2. The conditions a r e the following:

Column : Temperatures: i n j e c t o r 240 OC, column 130 O C

Porapak Q 80/100 mesh, i n 1.8 m x 2mm g l a s s

flame ionizat ion de tec to 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 fo r assay and pu r i ty of bromocriptine mesilate. Sa t i s f ac to ry 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 (octadecylsilanised s i l i c a ge l ) columns.


HPLC-conditions

s t r a i g h t phase:

s t a t iona ry phase: s i l i c a g e l , 5 pm, i n s t a i n l e s s steel

mobile phase: water-saturated dichloromethane/methanol

uv detect ion a t 254 nm.

Fig. 11 shows a chromatogram of bromocriptine mesilate spiked w i t h dodecylbenzene, po ten t i a l by-products and the precursor. The flow was 1.7 ml/minute (31 ) .

Key: 1 = dodecylbenzene, 2 = bromocriptinine, 3 = a-ergocryp- t i n i n e , 4 = bromocriptine, 5 = a-ergocryptine (precursor) .

- - - - - - -

25 cm x 3 mm i.d.

100:2 (v/v)

- reversed - - - - phase, - - zyste?l_I:

s t a t iona ry phase: Merck F@-18, 1 0 pm i n s t a i n l e s s steel 25 c m x 4.6 mm i . d .

mobile phase: gradient: 35 t o 60 percent B within 60 min. A = water, 0 . 2 percent tr iethylamine added B = a c e t o n i t r i l e , 0.2 percent tr iethylamine

added. uv detect ion a t 280 nm.

This system was found optimal f o r the p u r i t y t e s t .

Fig. 1 2 shows a chromatogram of the drug substance spiked with po ten t i a l by-products and the precursor. The flow was set a t 4.0 ml/minute.

Key: I = 2-bromo-a-ergocryptinine, I1 = a-ergocryptinine, 111 = a-ergocryptine (precursor) , I V = bromocriptine, V = 2-bromo-lysergamide, V I = 2-bromo-lysergic acid, V I I = 2-bromo-isolysergamide.

- - - - reversed-p&azeL ZyEtgm-II- (26) :

s t a t iona ry phase: Merck RP-18, 10 pm i n s t a i n l e s s s t e e l ,

mobile phase: 0.01 M ammonium carbonate o r 0.05 M

25 cm x 4.6 mm

ammonium hydrogen carbonate solution/ 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.


1

3

I I

5

Figure 11. High Performance Liquid Chromatogram of Bromocriptine Mesilate, spiked with Dodecylbenzene, Precursor and po ten t i a l By-products. Adsorption Mode, i s o c r a t i c , Uv-detection a t 254 nm

76

5

1

c

DANIELLE A. GIRON-FOREST AND W. DIETER SCHONLEBER

c L L 0 2 4 6 8 10 12 14 16 18 20 min

Figure 1 2 . High Performance Liquid Chromatogram of Bromocriptine Mesilate, spiked w i t h Precursor and po ten t i a l By-products. Reversed-phase Mode, Solvent Gradient, Uv-detection a t 280 nm.


7.5 Di f f e ren t i a l Scanning Calorimetry

Di f f e ren t i a l scanning calorimetry cannot be applied t o quantify the pu r i ty of the drug substance f o r the reasons mentioned i n 2.31 and 2.34. However, i t may be valuable 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 the bas i s of t h e i r corresponding d i f f e r e n t i a l scanning calorimetry pa t t e rns .

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 analysis , a c e t o n i t r i l e appears t o be the most s u i t a b l e solvent. A t yp ica l p l o t i s given i n f i g u r e 13 along w i t h the experimental conditions.

7.7 Analysis of the Dosage Form

The i d e n t i f i c a t i o n of bromocriptine mesilate i n the dosage form c a n be ca r r i ed o u t by t h i n layer chromatography using Merck p l a t e s with dichloromethane/methanol/formic ac id 78:20:2 (v/v/v) and subsequent uv-visualization a t 254 and 360 run. Using t h i s method, i t is important t o only air-dry the spot a f t e r appl icat ion t o the p l a t e , s ince more vigorous evaporation of the solvent w i l l give r i s e t o a r t i f a c t s (32) .

Bromocriptine can a l s o be i d e n t i f i e d a s the base by ir spectroscopy a f t e r ex t r ac t ion from the dosage form with ethanol and removal of the solvent, both i n so lu t ion and i n a KBr p e l l e t (33 ) .

Bromocriptine mesilate i n Parlodela 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 ex t r ac t ion with methanol ( 3 2 ) .

Fluorimetry i n 0 .1 N hydrochloric acid has been applied during the measurement of d i s so lu t ion r a t e of the dosage forms with exc i t a t ion a t 335 and emission a t 425 nm, respect ively

(26) - A spec i f i c assay of bromocriptine mesilate i n the

dosage form may be ca r r i ed o u t by t l c followed by uv-spectrophotometry ( 2 6 ) (The system can a l s o serve f o r i d e n t i f i c a t i o n purposes). The drug substance i s extracted with methanol i n the absence of l i g h t , the chromatographic conditions a re : 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 spot corresponding t o bromocriptine i s extracted with methanol, and the concentration i s determined a t about 300 nm by spec tropho tometry.


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 %.


A fu r the r s p e c i f i c assay is the HPLC-determination of bromocriptine mesilate following extract ion with methanol from the dosage form using RP-18 a s the s t a t iona ry and aceto- nitr i le/O.Ol M ammonium carbonate solut ion 65:35 a s the mobile phase. Uv-detection wavelength i s s e t a t 300 nm (26 ) .

For the detect ion and estimation of degradation products i n dosage forms, a solvent gradient , containing the components mentioned above, is u t i l i z e d with advantage.

7 .8 Determination i n Body Fluids and T i s s u e s (23)

Due t o i t s potent eff icacy i n the treatment of hyperprolactinemia and acromegaly, bromocriptine is administered i n low doses leading t o minute concentrations i n body f l u i d s and t i s sues . Therefore, none but the most s ens i t i ve a n a l y t i c a l methods can be used t o measure i t s concentration i n b io log ica l specimens. The only method so f a r appl icable f o r pharmaco- k i n e t i c s tud ie s with bromocriptine is the u s e of the radio- ac t ive ly l abe l l ed drug, measurement of t o t a l r ad ioac t iv i ty and i ts f r ac t iona t ion by chromatographic separation techniques f o r the assay of parent drug and major metabolites. Recently, a radioimmunoassay k i t f o r the ana lys 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 avai la- ble . 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 Parkinson's disease which a r e on treatment a t high dose l eve l s . These cu r ren t ly developped procedures permit quan t i t a t ive determinations down t o concentrations of 0.5 (GC) , 1 . 0 (MF) , and 10 ng/ml (LC) , respect ively ( 3 4 ) .

The pa t t e rn of metabolites i n b i l e (animals only) and i n urine have been invest igated using column chromatography (Amberlite XAD 2 and Sephadex DEAE), t l c and reversed phase

HPLC i n combination with r ad ioac t iv i ty monitoring. The p r inc ipa l metabolites have been i so l a t ed from the b i l e of r a t s t r ea t ed with high doses of bromocriptine. The s t r u c t u r e of the i so l a t ed metabolites was elucidated by means of spectroscopic techniques.


8.

1. 2.

3.

4. 5. 6. 7.

8. 9.

10.

11.

12.

13.

14.

15. 16. 17.

18.

19.

20. 22.

23.

24. 25.

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 Offen- legungsschrift 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 - 2, no. 3 (1978) 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. : "Dihydro- ergotoxine 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 29, 697 (1974) 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

-


26. 27. 28. 29.

3 0 .

31. 3 2 . 3 3 . 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 Sandoz- Wander 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.


CALCITROL

Eileen Debesis

I . Description I . I I .2 Appearance, Color, Odor

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

3 . Synthesis 4. Stability and Degradation 5 . Drug Metabolic Products 6. 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 7. Acknowledgments 8. References

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

ISBN 0-12-260808-9 83

84 EILEEN DEBESIS

1. Description 1.1 Name, Formula, Molecular Weight

Calcitriol is 1 a, 25-dihydroxycholecalciferol .

MW = 416.65

2 7"44'3

1.2 Appearance, Color, Odor Calcitriol is an odorless white crystalline

powder.

2. Physical Properties 2.1 Infrared Spectrum

The infrared spectrum of calcitriol is shown in Figure 1 (1). Model 283 Grating Infrared Spectrophotometer and was measured in a KBr pellet which contained 1 mg of calcitriol in 300 mg of KBr.

The spectrum was recorded on a Perkin-Elmer

The following absorptions have been assigned for Figure 1:

a. OH stretching (bonded): 3391 cm-l b. Aliphatic CH stretching: 2943, 12872 cm-l c. CH deformation: 1468, 1318 cm- d. C-0 stretching: 1056 cm-

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). 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.

The spectrum was recorded using a solution of

WAVELENGTH (MICRONS) 2.5 3.0 4.0 5.0 6.0 8.0 10 16 20 25 !

I I I I I I 1 I I

100 -

0 I I I I I I I I I I I I

Figure 1

Infrared Spectrum oE Calcitriol

CALCITRIOL

H

Table I

87

NMR Spectral Assignments for Calcitriol

Proton Chemical Shift (a) Multiplicity

-CH3(C18) 0.57 Sing1 et

-CH3(C21> 0.95 Doublet

-CH3(C26,27 1 -1.16 Singlet

XH2, ;CH -1.38-3 Complex \ \

HO. ~

;CH

Ho> I - CH

/ -

- - CH2

4.08 C omp 1 ex

4.32 Triplet

4.90, 5.28 Complex

6.07, 6.32 AB Quartet JAB = 11 Hz

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).

2.4 Mass Spectrum The low resolution mass spectrum of calcitriol is

shown in Figure 4 (4). Varian MAT CH5 spectrometer, which was interfaced with a Varian data system 620 I. 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,

The spectrum was obtained using a

The data system accepts the output

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 observed at m/e = 134, and corresponds to the 3

!$fH+ moiety

2 .5

2.6

2 . 7

. . H

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

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 55°C 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 355°C (5).

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 115-117°C. crystallize from the melt (5).

The sample did not re-

CALCITRIOL 89

0.6

0.5

0.4

W 0 z a 3 0.3 m 8 U

0.2

0. I

0 250 300 350

NANOMETERS

Figure 3

Ultraviolet Spectrum of Calcitriol

Q) a

Kn

CALCITRIOL 91

2.9 Solubility Extensive solubility data is not readily obtain-

able 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 Guinier-De Wolff Camera

Generator GE XRD-6 50 KV, 12.5 mA

Detector Film

Sample As is

Densitometer" Gelman DCD-16

Optics Tungsten Lamp 575 nm Visible mode

Detector Silicon phototransistor

Optical density 0.25

Beam exit slit 0.53 mm

*The diffraction pattern was recorded on film; the density of the individual lines in the pattern was determined by dens i tome try.

mu

id

oa

a

c.

CALCITRIOL 93

TABLE I11

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

Calcitriol Powder Diffraction Data

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

I/Io**

100 90 88 82 70 66 49 44 36 34 33 33 26 26 15 12 12 12

2 2 2 2 1

- - - A (interplanar distance) 2 Sin 0

*d

**I/Io = 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 ) .

EILEEN DEBESIS 94

3 . Synthesis Several procedures for the synthesis of calcitriol have

been reported in the literature. -- et al. (9) described the preparation of calcitriol from 25-hydroxyvitamin D3. described lengthier syntheses utilizing more readily available starting materials. ing 1 a, 25-diacetoxy-7-dehydrocholesterol as the starting material, is shown in Figure 6.

Parren -- et a1.(8) and Norman

Uskokovic and others (10-18) have

A typical reaction scheme, utiliz-

4. Stability and Degradation Calcitriol must be protected from air and light. The

drug substance exhibits good stability when stored at -15'C to -25'C in an argon atmosphere. at room temperature when dissolved in a vegetable oil derivative, containing antioxidants, such as is used in calcitriol soft gelatin capsules (19).

The material is stable

5. Drug Metabolic Products

or bone, or may be hydroxylated to form 1 a, 24, 25-trihydro- xycholecalciferol prior to intestinal absorption (20-24).

Calcitriol may be absorbed directly into the intestine

6. Methods of Analysis 6.1 Elemental Analysis

A typical elemental analysis of a sample of calcitriol is presented in Table IV (7);

TABLE IV

Elemental Analysis of Calcitriol

E 1 ement % Theory % Found

C H 0

77.84 77.53 10.64 10.75 11.52 11.92

(by difference)

22

v

o

yz

I

x3R 0 g 0, O

--

0

r - I 8

rn 1

1

0 r

m

'.

r(

0

.rl k

c,

*+-I

.rl

LV

I

.rl

0 I

8

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 pre- vitamin, 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 values are summarized in Table V ( 3 ) . f

TABLE V

Summary of TLC Data

Compound Approximate R -

1 a, 25-Dihydroxyprecholecalciferol 0.4 Calcitriol 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-Dimethyl- aminobenzaldehyde 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. and fragmentation patterns for ergocalciferol, cholecalciferol and calcitriol were reported.

Retention times

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. et al. (43.44) reDorted a bioassav . . < A --

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 polaro-

graphically, 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 (46).

-

7. Acknowledgments

Literature Department and Research Records Office of Hoffmann- La Roche Inc., Nutley, NJ.

The author wishes to thank the staffs of the Scientific

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. r, 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 Ogatc E. , Tampa- kushitsu Kakusan Koso, 21, 844 (1956). DeLuca, H. F., TetrahehTn Lee&, 1972, 4147. Cohen, Z., Keinan, E . , Mazur, Y., and Ulman, A., J . Org. Chern., - 41, 2651 (1976). Mielczarek, I., Wiad. Chem., 28, 813 (1974). Kaneko, C. , Yuki Gosei KagakuTyokai Shi, - 33, 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., 10, 295 (1975). -

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), - 87, (Suppl. 215), 120 (1978). 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., - 176, 235 1976). Etsuko, A . , Suda, T., Nakano, H., Igaku No Aywni , - 102, 509 (1977). Brumbaugh, P. F . , Haussler, D. H., Bressler, R., and Haussler, M. R., Science, 183, 1089 (1974). Caldas, A . E., Gray, R. W., Lemann, J., Jr., J . Lab. CZin. Med., 91, 840 (1978). Shimura, F . , and TamGa,.M., Vitamins, - 52, 173 (1978). Horst, P. L., Merchant, C . , Shephard, R., Hamstra, A . Jorgensen, N. A . , DeLuca, H. F . , J . Dairy Sci. , - 60, (Suppl. l), 123 (1977).

100 EILEEN DEBESIS

37.

38.

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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 to 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. , - 58, 61 (1976). 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., - 7, 25 (1977). Rucki, R. J., Hoffmann-La Roche, Inc. Personal Communication.


CHLORTETRACYCLINE HYDROCHLORIDE

George Schwartzman I ;Lola Wayland, Thomas Alexander Kenneth Furnkranz, George Selzer, and the

USASRG *

I . Description I . I Drug Properties I .2 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 Polymorphism-

3.1 Ultraviolet 3.2-Infrared 3.3 Spectropolarimetry 3.4 Fluorescence

4. I Proton NMR

4.3 Mass Spectrometry 5 . Chromatography

5 . I Paper and Thin-Layer 5.2 Gas-Liquid

6. I Fermentation 6.2 Isolation

7. Stability 8. Analytical Methods

8. I Microbiological 8.2 Chemical

Physical Description and Optical Crystallography

3. Spectral Properties (Optical)

4. Spectral Properties (Other)

4.2 'T-NMR

6. Manufacture

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.

contributions are referenced where possible. The USASRG was formed at the request of P. Weiss. National Center for Antibiotics Analysis. Individual

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

ISBN 0-12-260808-9 101

I02 GEORGE SCHWARTZMAN ET A L .

1. DESCRIPTION 1.1 Drug P r o p e r t i e s

Ch lo r t e t r acyc l ine hydrochlor ide (CTC-HCL) i s the hydrochlor ide s a l t of an a n t i b i o t i c substance produced by t h e growth of Streptomyces aureofac iens (Fam. Streptomycetaceae) . It was discovered i n 1948 by Duggar (1).

protozoan a c t i v e aga ins t many Gram-positive b a c t e r i a , some Gram-negative b a c t e r i a , sp i roche te s , amebae, and c e r t a i n l a r g e v i r u s e s . Staphylococci have become gene ra l ly r e s i s t a n t to t he drug, and d rug- re s i s t an t s t r a i n s may b e found among o the r b a c t e r i a l genera and s p e c i e s gene ra l ly s e n s i t i v e t o i t . Cross-resis tance 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 automatic ( 2 ) .

Organisms may be considered 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 not more than 4.0 pg/ml and in te rmedia te i f t h e M I C is 4.0-12.5 pg/ml ( see Table 1). Te t r acyc l ines 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 varying degrees . by the l i v e r i n t h e b i l e and excre ted i n t h e u r i n e and f e c e s a t high concent ra t ions 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 aga ins t microorganisms involves t h e i n h i b i t i o n of phosphorylation processes i n b a c t e r i a l cells ( 3 ) .

TABLE 1 (5) Antimicrobial Spectrum of Chlortetracycline

Microorganism Concentration (Vg/ml)

Micrococcus pyrogenes E. aureus 209P 0.292

Streptococcus pyrogenes 0.29 Streptococcus Q 0.147 Streptococcus faecalis 0.39 Micrococcus flavus 0.292 Diplococcus pneumoniae 0.098 Sarcina lutea 0.147 Bacillus subtilis 0.195 Escherichia 1.45 Haemophilus influenzae 0.312 Klebsiella pneumoniae 0.29 Neisseria catarrhalis 0.098 Aerobacter aerogenes 1.17 Proteus vulgaris 4.6 Pseudomonas aeruginosa 25 Salmonella schottmuelleri 3.12 Salmonella typhi 1.17 Shigella dysenteriae 3.12 Brucella bronchiseptica 0.292 Mycobacterium tuberculosis 0.147 Mycobacterium friedmanii 0.122 Mycobacterium smegmatis 0.073 Mycobacterium s p . 0.58 Candida albicans 100 Clostridium butyricum 0.078 Pasteurella multocida 0.049

CTC-HC1 is a broad-spectrum a n t i b i o t i c and a n t i -

They a r e concentrated

Minimum Inhibitory

Micrococcus pyrogenes v z . aureus 1248A 0.39

~-

Vibrio percolans 0.098

CHLORTETRACYCLINE HYDROCHLORIDE 103

1.2 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):

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

8: 1.706; orthorhombic; extinction parallel; and

y: 1.730; symmetrical

It is stable in air, but is slowly affected by light (6).

1.3 Chemical Properties CTC-HC1 is the HC1 salt of amphoteric CTC; it is

multifunctional with two chromophores. It is a para- chlorophenol with an aYf3-unsaturated ketone 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.

1.4 Structure C22H23ClN208.HCl Empirical Formula

515.35 Molecular Weight

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

1.5 The FDA Chlortetracycline Standard The current official FDA Chlortetracycline Working

Standard is chlortetracycline hydrochloride, Lot W501-632B- 95-1 (9/29/53), obtained from Lederle, which markets the antibiotic under the proprietary name Aureomycin. working standard has an assigned potency of 1000 pg/mg (the 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).

The current

CHLORTETRACYCLINE HYDROCHLORIDE I05

The Working Standard 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 Center f o r A n t i b i o t i c s A n a l y s i s , Washington, DC.

2 . 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 Analys is (DTA) and Thermal Gravimet r ic 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 exothermica l ly a t approximately 230°C ( F i g u r e s 2 and 3) (15) . f i n a l decomposi t ion t a k e s p l a c e . i n t h e samples examined.

The compound does n o t l o s e any m a s s u n t i l t h e No polymorphs have been s e e n

2.2 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

Standard has been determined. 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 Table 2 (16) .

It demonst ra tes c r y s t a l l i n e

TABLE 2 X-Ray D i f f r a c t i o n Data

d (1) 1/11 d (i) 1/11 d 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

11 1 2 56 44 1 3 43 81 42 6 1

8 8

1 5 3

40 100

70 25 52 78 37 31 59 33 1 6 25 23

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 7 37 16 7 3 74 38 8 9

47 25 31 35 36 14 10 30 22 13 27 1 2 24

7 14 37 1 3 2 3 2 1

2.060B D(1.985

(1.962 1.897 1.880 1.868 1.856 1.827 1.805 1.784

(1.737 1.705 1.682 1.638 1.631 1.608 1.595 1 .583

~ ( 1 . 7 4 8

1 3 11 1 3 1 2 10 1 3 1 3 8 9 6 8

11 9 6 5 4 5 4 4

3 = 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.


2.3 S o l u b i l i t y The s o l u b i l i t y of a n t i b i o t i c s , inc luding CTC-HC1,

w a s repor ted by Andrew and Weiss (17). CTC-HC1 is an amphoteric subs tance and consequently i t is solu'ble i n aqueous ac id and base. However, i t can r a p i d l y degrade i n these so lven t s . Its s o l u b i l i t y i n water is about 8 mg/ml and i n methanol about 1 7 mg/ml. t he s o l u b i l i t y of CTC-HC1 i s cons iderably less than i n methanol. For p r a c t i c a l purposes, i t i s in so lub le i n many common so lven t s such a s t h e a l i p h a t i c hydrocarbons, benzene, e t h e r , and chloroform. It i s r e a d i l y so lub le i n py r id ine and t o t h e ex ten t of about 5 mg/ml i n formamide. Py r id ine is an undes i rab le so lven t because of i t s b a s i c i t y , and formamide is not 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 ob ta in ing and maintaining i t a s a s t a b l e so lven t .

I n h igher molecular weight a l coho l s ,

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 cons t an t s

when t i t r a t e d i n aqueous s o l u t i o n s (18) . 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 (Figure 4 ) , and r epor t ed thermodynamic pKa va lues of 3.30, 7 . 4 4 , and 9.27. Leeson e t a l . (20) ass igned pKa values t o t h e fol lowing a c i d i c groups:

pKa Assignment

3.30 Tricarbonylmethane System (A)

7.44 Phenol ic Diketone System (B)

9.27 Dimethylamino System (C)

Kalnins and Be len ' sk i i (21) v e r i f i e d t h e assignments by i n f r a r e d ( I R ) spectroscopy.

The pH of a 10 mg/ml aqueous s o l u t i o n , as desc r i - bed i n the Code of Federal Regulat ions monograph f o r CTC-HC1 (22) should l i e between 2.3 and 3.3.

2.5 Polymorphism I n r ecen t yea r s , w i th 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 subs tance , polymorphism has become of p r i m e i n t e r e s t . Miyazaki and co-workers (23) have repor ted t h e ex i s t ence of 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 behaviors , and hygroscop ic i t i e s t h a t they r epor t ed 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 sc repanc ie 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 base prepared 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

The X-ray powder d i f f r a c -


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 . The re appea red 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 (24 ) . 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 U l t r a v i o l e t

Medium Maxima (nm)

0 . 1 N H C 1 368, 340sh, 322sh, 265, 229 0 . 1 N NaOH 345, 283, 253, 222 Methanol 372, 342sh, 322sh, 262sh, 253, 233

See F i g u r e 5 (25 ) .

3 .2 I n f r a r e d The I R a b s o r p t i o n s p e c t r a of CTC-HC1 are shown i n

S p e c t r a were run on a Perkin-Elmer Model 467 F i g u r e s 6 and 7 . g r a t i n g spec t ropho tomete r as a K C 1 p e l l e t ( 1 mg/200 mg K C I ) , and as a Nujo l m u l l (20 mg/3 d r o p s ) . The s p e c t r a are es sen - t i a l l y i d e n t i c a l . 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 . (26) . Major a b s o r p t i o n f r e q u e n c i e s have been compiled (27) and a s s ignmen t s have been made on inany of t h e bands(28-31). Some of t h e ma jo r f r e q u e n c i e s and band a s s i g n -

The CTC-HC1 spec t rum may a l s o b e found i n a

ments are:

& - x

3360 2.98

3315 3.02

3200-3450 2.90-3.12

2800-2400 3.57-4.17

1675 5.97

1582 6.32

1450 6.90

1368 7 . 3 1

1042 9.60

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 , group of b road bands

vc-0

6NH2

6CH (bending of CH3)

VCN

OH (de fo rma t ion , a l c o h o l s )

110

b m v) 0 X I 00 B 2 0 m

GEORGE SCHU

a

WAVELENGTH (nm)

,RTZMAN ET AL.

0

Figure 5 . Ultraviolet absorption spectra of CTC-HC1 in a, 0.1N HC1; b, 0.1N NaOH; and c , methanol.

c, a, rl

rl

-ai a

rl V

M v)

(d

W 0 t B 2 8

a

I800 1600 1400 1200 100 WAVELENGTH (CM")

Figure 7. Infrared absorption spectrum of CTC-HC1 as Nujol mull.


3.3 Spec t ropolar imet ry Because conf igu ra t iona l in format ion can be der ived

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 dichroism scans , cons iderable work has been conducted us ing t h e s e techniques t o s tudy t h e t e t r a c y c l i n e s (32) . The abso lu te conf igu ra t ion of CTC w a s determined using 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 curves are presented i n F igures 8 and 9. The c i r cu - l a r dichroism spectrum is similar t o t h a t p resented by Mitscher e t a l . (34) , except t h a t t h e va lues 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 obta ined by Mitscher and i n FDA l a b o r a t o r i e s are compared.

3 .4 Fluorescence The CTC-HC1 FDA Working Standard g ives a yel low

f luorescence 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 tandard d i s so lved i n 0.05N NaOH (11.4 mg/50 ml) a r e presented i n F igure 10.

TABLE 3 Molecular E l l i p t i c i t i e s of CTC

Wavelength (nm) Mi tscher ' s Valuea FDA Value Ra t io (X 103) (X 103)

236 254 288 318 355 (sh)

+10 -31 +4 8 -24 -14

+2 3 0.43 -48 0.64 +88 0.54 -32 0.73 -12 1.09

These va lues w e r e obtained from the graph i n Reference 34 . a

4 . SPECTRAL PROPERTIES (OTHER) Proton 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 igures 11 and 12 , r e spec t ive ly . The sharp 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 ignab le ;

4 .1 ~-

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 th t h e s t r u c t u r e . The 2 methylene pro tons a t C-5 are n o t equ iva len 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 ignab le i n the methanol spectrum: t h e s i g n a l cen tered a t 2.20 ppm. I n DMSO-db t h e s e pro tons appear between 1 . 5 and 2.3 ppm. The only methine proton a c t u a l l y ass igned i s t h e one a t C-4. The c l o s e proximity t o t h e p o s i t i v e l y charged N causes 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 cons tan t However, t he two amide protons, o r i g i n a l l y a t 9.15 and 9.55 ppm, exchange wi th t h e deuterium and are no longe r observed. These s i g n a l s were n o t observed i n methanol-d4, aga in , due t o

NO

IWM

SIO

WO

lVlO

Y lW

l1dO

I I4

<

115

AlIS

N3lN

I I I6

d

u

X I V

I3 u 5 8 k

CI V a,

c b

2

I I7

w 0

b


deuterium exchange. Data from t h e l i t e r a t u r e (36) , based on a spectrum obta ined i n DMSO-db, are inc luded i n Table 4. As can be seen from the t a b l e , t h e s e va lues are g e n e r a l l y u p f i e l d from our 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 explana t ion w i l l be a t tempted he re (37).

TABLE 4 Chemical Shif tsa

Solvent Proton L i t e r a t u r e (36) DMSO-dh Methanol-dq

1.7 1.88 (s) 1.98 ( s )

2 .7 2.93 (s)

6.4 - 7.0 7.58 (d)

6.4 - 7.0 6.98 (d)

4.0 4.36 (br . s)

1 . 7 - 2 . 1 1 . 5 - 2.3 (m,s)

8.4, 8 .9 9.15, 9.55

d 2.7 not ass igned

2.7 no t ass ignedd

3.05 (s)

7.55 (d )

6.93 (d)

4.10 (d)

2.20 (m)b

n o t observedC

d n o t ass igned

n o t ass ignedd

aSpectra were obta ined on a Perkin-Elmer R-12B equipped wi th a Nicole t TT-7 Four i e r t ransform accessory. repor ted 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 are ind ica t ed as s = s i n g l e t , d = doub le t , and m = m u l t i p l e t .

bThis m u l t i p l e t r ep resen t s one of t h e H-5 protons. chemical s h i f t of t h e o t h e r one w a s n o t ass igned.

CThese protons exchange wi th t h e deuterium of t h e s o l v e n t and thus are n o t observed.

dThe chemical s h i f t s of t h e s e protons are no t ass igned. They are obscured by t h e -N(CH3)2 and/or t h e so lven t .

Chemical s h i f t s are

The

4.2 I3C NMR Proton-noise decoupled and s ingle-frequency o f f -

resonance decoupled carbon-13 NMR s p e c t r a were determined f o r t he CTC Working Standard (Figure 13 ) .

compare c l o s e l y wi th those r epor t ed by Frank (38). The only 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 than a t Frank 's repor ted va lue 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 sc r ibed t o d i f f e r i n g amounts of w a t e r p r e sen t i n the DMSO-db s o l v e n t s and hence i n t h e pH of t h e s e respec- t i v e s o l u t i o n s .

The observed chemical s h i f t s of t h e 22 carbon atoms

t

rl V

V

H

V

Ict 0

T 5 4

8 k

4J V

a,

!z V

m

4 m

rl

a, k

3

m

-4

h


S p e c t r a were determined u s i n g a p u l s e wid th of 4 pseconds, which cor responds 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 observed resonance l i n e s of CTC-HC1 (39) and t h e i r ass ignments are shown i n Table 5 .

'CABLE 5 Observed Resonance Lines of CTC-HC1 and T h e i r Assignments

Carbon Assignment Chemical Shi f ta

25.1 q C- 27.0 t

34.9 d C-4a C-5a 42.0 d N (CH-3) 2 48.5 q c-4 68 .3 d C-6 70.4 C-12a 73.3 c- 2 95.5 C - l l a 106.1 C - l O a 117 .0 c-9 118.9 d c-7 121.3 C-8 139.7 d C-6a 143.6 c-10 160.8

c-12 175.3 c- 3 187 .1 c-1 192.1 c-11 193.3

cH3

corn2 172.0

DMSO-d6, p a r t s p e r m i l l i o n from TMS (0.00 ppm). q = q u a d r u p l e t , t = t r i p l e t , d = double t .

4 . 3 Mass Spectrometry 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 major f ragmenta t ion 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 only 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 Morr i s and C a i r n s (41) . However, l i t t l e informat ion on t h e i r r e s u l t s is inc luded i n t h e extended a b s t r a c t of t h e meet ing, 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 publ ished. 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 are i n c lose agreement with d a t a r epor t ed here .

i n terms of t h r e e d i f f e r e n t mechanisms. These are:

dimethylamino group t o d i r e c t l o s s e s of small fragments toge ther w i th i ts 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.

r e t e n t i o n on t h e fused r i n g po r t ion of t h e molecule.

r e t e n t i o n elsewhere than on t h e fused r i n g moiety.

Personal communications wi th t h e au tho r s

The fragmentat ion p a t t e r n of CTC can be d iscussed

a. Processes r e s u l t i n g from t h e capac i ty of t h e

b. Fragmentation of t h e D r i n g system wi th charge

c. Fragmentation of t h e D r i n g system wi th charge

The mass spectrum of CTC, shown i n F igure 14 (42) , is cha rac t e r i zed by a reasonably i n t e n s e r ;olecular i o n a t m / e 478 wi th t h e concomitant i so tope peak a t P+2 r ep resen t ing one c h l o r i n e atom i n t h e r i n g system. suggested t h a t t h i s c h l o r i n e atom be employed as a t r a c e r v i a t h e 3 7 ~ 1 i so tope 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 conta in ing t h e A r i n g and ad junc t r i n g systems, many of t hese ions 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 only l i m i t e d usefu lness toward t h a t end, except perhaps at h igher mass va lues .

Although i t has been

The dominance of t h e fragmentat ion processes by t h e presence of t h e 4-dimethylamino group i v e s r ise t o t h e i n t e n s e ions at m / e 44 , m / e 58 (CH3)2-i=CH2, m / e 7 1 (CH3)2ft- CH=CH2, and m / e 84 (CH3)2-$=CH-CH=CH2. (C+8NO+) appears t o involve a c y c l i z a t i o n of t h e dimethylamino group wi th elements 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 molecular i o n t o g ive the i n t e n s e ion a t m / e 435 and from t h e ion a t m / e 443 t o y i e l d m / e 400 (Figure 15 ) .

i nd ica t ed by observa t ions of success ive l o s s e s of NH3 and OH ( a s water) from t h e molecular i on t o y i e l d u l t i m a t e l y t h e ion a t m / e 443. s l i g h t l y favored over t h e competing l o s s of NH3 a t m / e 462 from the abundances of t h e r e s p e c t i v e ions .

purpose of determining 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) and/or 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 lsewhere i n t h e r i n g system. r equ i r e s a d e t a i l e d examination of analogs such as i n t h e work of Hoffman (40) .

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 de termina t ion 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 ive evidence concerning the ex ten 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 .

The i o n a t m / e 98

The presence of t h e amide f u n c t i o n a l group i s

The l o s s of water a t m/e 460 appears t o be

Fragmentation of t h e D r i n g can provide t h e dua l

This,however,

The ion a t m / e 365 w i l l l o c a t e the s u b s t i t u e n t s a t

This

SPEC. NO. 20039 LS CHLORTETRRCYCLINE HCL

100- * c v)

I-

- w 7 5 - z

W

- ;1 so- a

a 2 W

25 -

44

U

U

w

* L '"1 In

MASS T O CHRRCE RRIIO

- v)

I- w 7 5 - z

W

- ;1 so- a

a 2 W

25 -

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

(Chemical ionization using ammonia

U

U

w 433 M'

170 433 M'

170

I24 GEORGE SCHWARTZMAN ET AL.

365 435 461 460 I70

2 75 - 400 I

3--'O

370

Figure 15. Major fragmentation pathways of CTC involving molecular ion and other significant high mass ions. Underlined m/e values indicate confirma- tion by accurate mass measurements.


knowledge p lus t h e observed s h i f t of t he ion a t m / e 170 should s u f f i c e t o a l low 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 (Figure 1 6 ) .

5. 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 va lue . Fischbach and Levine (43) descr ibed a cont inuous ascending technique and B e r t i and C i m a (44) r epor t ed an ascending method us ing aqueous sodium a r s e n i t e as t h e mobile so lven t . Other authors (45,46) r epor t ed descending techniques and b ioautographic 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 hese methods f a i l t o show t h e presence of t he epimeric 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 he s p o t s is a problem. A b a s i c improvement i n the paper chromatography of t h e s e a n t i b i o t i c s w a s achieved by Se lze r and Wright (47) and Kel ly and Bryske ( 4 8 ) when they repor ted methods f o r t h e pre t rea tment of t h e paper wi th complexing agents t o bind t h e metal l ic i o n s which may be p re sen t .

l i t y t o form complexes wi th polyvalen t c a t i o n s . This proper ty 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 mobile s o l - vents and o f t e n r e s u l t s i n troublesome s t r eak ing . To overcome t h i s d i f f i c u l t y , Se l ze r and Wright used paper dipped i n McIlvaine 's bu f fe r (pH 3.5) which con ta ins c i t r a t e ions capable of binding the metallic ions . The chromatograms were developed wi th a mixture of ni t romethane, chloroform, and py r id ine (20:10:3) on paper s t i l l damp from t h e t rea tment w i th the b u f f e r s o l u t i o n .

impregnated wi th 0.1N disodium ethylenediaminetetraacetate (EDTA) and two mobile so lven t s : t h e o rgan ic phase from a mixture of E-butanol, ammonia, water (4:1:5) and t h e o rgan ic phase from a mixture of g-butanol , a c e t i c a c i d , water (4: l : 5 ) . b u f f e r when i t is used t o t rea t the paper i n t h e method of Walton e t a l . (49) . A c i r c u l a r paper chromatographic method a l s o us ing paper dampened wi th McI lva ine ' s b u f f e r (pH 4.5) w a s r epor t ed by Urx e t a l . (50) . They used a mixture of chloroform and c-butanol ( 4 : l ) as t h e mobile s o l v e n t .

with a che la t ing agent are capable 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 drugs from each o t h e r and from t h e i r r e spec t ive epimeric forms. They a r e a l s o capable of r evea l ing the presence of common degrada t ion compounds of t h e s e drugs .

t e t r a c y c l i n e a n t i b i o t i c s involves fuming t h e paper w i th ammonia vapor and observing t h e yellow f luorescence under UV l i g h t . A s l i t t l e as 0.2-0.5 pg can be v i s u a l i z e d by t h i s technique.

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 ab i -

For the same purpose, Kel ly and Bryske used paper

Disodium EDTA (0.1N) works as w e l l as McIlvaine 's

Most of t h e methods i n which t h e paper is t r e a t e d

The usua l method of d e t e c t i n g chromatographed

(f&:;2

I

" CH30H ,N$ CH3 CH3

W e 365

iJ? ' \

CH, CH3

M f

0 0 -6 6-NH2

C II

C h

\ /

/'\OH

II N+

CH3 CH3 / \

W e 170

Figure 16. Proposed structures for ions at m/e 365 and m/e 170 for use in determination of ring substituents.


Some of t h e f e a t u r e s t h a t are success fu 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 on paper have been adapted t o t h i n l a y e r chromatography (TLC). Complexing agen t s are almost always used i n t h e p repa ra t ion of t he p l a t e s and , in a d d i t i o n , s e v e r a l methods f o r hydra t ing 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 and/or po lye thylene g lyco l have been repor ted . Seve ra l 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 ac id- wash t h e inorganic suppor t i n o rde r t o remove m e t a l l i c ca t ions . These TLC methods are much more time-consuming than most of t h e paper chromatographic procedures because of t h e s p e c i a l care o f t e n r equ i r ed t o prepare and s t o r e t h e TL,C d a t e s . For t h i s reason, where app l i cab le , t he paper chromatographic methods .are p re fe rab le . However, f o r q u a n t i t a t i v 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 degrada t ion products , TLC i s u s u a l l y considered t o be b e t t e r than paper chromatography.

using Kieselguhr l a y e r s impregnated wi th a b u f f e r s o l u t i o n conta in ing g lyce r in . Nishimoto e t a l . (52) r epor t ed t h e use of s i l i c a g e l l a y e r s t r e a t e d wi th EDTA. I n 1967, Ascione e t al . (53) publ ished a method us ing l a y e r s made from acid-washed diatomaceous e a r t h . They prepared p l a t e s conta in ing 0.1N EDTA, g l y c e r i n , and polyethylene g lyco l 400. Other i n v e s t i - g a t o r s (54,55) have descr ibed methods which are mod i f i ca t ions of improvements of t h e methods previous ly publ i shed .

I n 1964, Somanini and Anker (51) descr ibed a method

5.2 G a s and Liquid Chromatography I n v e s t i g a t o r s have found it 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 gas- l iqu id techniques. Often, only fragments o f t he o r i g i n a l sample are obta ined (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 us ing 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 th t h e advent of r e f i n e d h igh p res su re l i q u i d chromatographic (HPLC) techniques, in te res t i n gas 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 papers have appeared r epor t ing t h e HPLC sepa ra t ion of CTC from i ts isomers and/or 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 opinion as t o t h e most satis- f ac to ry approach; t h u s , i t appears 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 the opt imal system f o r a p a r t i c u l a r ins t rument . Methods found i n t h e l i t e r a t u r e f o r CTC a r e descr ibed i n Table 6. EDTA is added t o prevent t he formation of complexes of t h e t e t r a c y c l i n e s wi th metall ic s u r f a c e s .

appl ied t o t h e t e t r a c y c l i n e s , i nc lud ing CTC, involve low pressure column chromatography. Ascione e t a l . (64) developed a semiautomated system whereby sample s o l u t i o n s are automati- c a l l y i n j e c t e d onto a column of diatomaceous e a r t h mixed wi th

Other chromatographic techniques t h a t have been

TABLE 6 High Pressure Liquid Chromatography of CTC

Migrat ion T i m e Mobile Phase S ta t iona ry Phase (min. ) Ref.

20% Methanol, 80% 0.05M ammonium C8/Lichrosorb 1 0 pm, carbonate , 0.02M EDTA 25 c m x 3 . 2 mm

Phosphate bu f fe r i n 13% methanol, Zipax-hydrocarbon polymer, 0.85 ml/min., pH 2.5 2.1 x 1000 mm

Aqueous pe rch lo ra t e -c i t r a t e bu f fe r Sil-X, 13 Urn, mixed with CH3CN 5 x 125 mm

EDTA, N a C l i n 30% methanol, NH3, Ion-X-SA, anion exchanger pH 9.9

EDTA, PO4, isopropanol-water, UBondapak c18, pH 7.6, 2 ml/min. 300 x 4 mm

EDTA, NO3, 9.6% e thanol , pH 9 , Zipak, 1.8 x 1000 mm 1 rnllmin.

34.5 58

6 59

1 .3

3

11.6

22

60

61

62

63


a s o l u t i o n of EDTA and polye thylene g lyco l . CTC i s e l u t e d wi th t h e o rgan ic phase of a mixture of chloroform, benzene, aqueous s o l u t i o n of EDTA, and polye thylene g lyco l . Ragazzi and Veronese (65) sepa ra t ed 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.

6 . MANUFACTURE 6.1 Fermentat ion

A medium (con ta in ing corn s t e e p l i q u o r ; calcium carbonate: suc rose ; 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 ) is s t e r i l i z e d and d i l u t e d wi th water t o t h e d e s i r e d concentra- t i o n . It is inocula ted wi th Streptomyces aureofac iens , kep 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 hours , w i th l a r d o i l added t o c o n t r o l foaming (66) .

6.2 I s o l a t i o n The mash from t h e Streptomyces au reo fac i ens fermen-

t a t i o n b r o t h is 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 is ad jus t ed 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 loccu- l a t i n g o r c h e l a t i n g agen t s may be added (e .g . , v i n y l acetate- maleic anhydride 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 wi th f i l t e r a i d , f i l t e r e d , s t i r r e d wi th HCI., r e f i l t e r e d , mixed wi th 2- e thoxyethanol , f i l t e r e d , washed, and the f i l t r a t e s are combined, a c i d i f i e d wi th HC1, N a C l is added, and t h e c r y s t a l s are c o l l e c t e d , washed wi th 2-ethoxyethanol, water, and e thano l , and d r i e d (67) , 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 ketone, 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 wi th 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 wi th water, 2-ethoxyethanol, and i sopropanol , and vacuum- d r i e d . I f t h e c r y s t a l s are g reen i sh , they are t r e a t e d wi th sodium h y d r o s u l f i t e a t pH 1 . 8 , f i l t e r e d , washed, and d r i e d as i n (1) above (68).

7. STABILITY

material. The s i t u a t i o n i n aqueous s o l u t i o n , however, is 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 converted t o iso-CTC on s t and ing (69) . 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 lue f luo rescence 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 conversion a t 100°C.

This change is g r e a t l y a c c e l e r a t e d by hea t ing and r e s u l t s i n a yellow product t h a t has a maximum absorbance a t 445 nm.

the 4-dimethylamino group (71) . This occurs s lowly i n w a t e r o r methanol but is hastened i n b u f f e r s o l u t i o n s i n t h e range

CTC-HC1, a s a d ry powder, is a s t a b l e yel low c r y s t a l l i n e

I n a c i d s o l u t i o n s , CTC i s converted t o anhydro-CTC (70) .

I n a d d i t i o n , CTC undergoes r e v e r s i b l e ep imer i za t ion of

I30 GEORGE SCHWARTZMAN ET AL.

of pH 2-6. The rate of ep imer iza t ion is undetec tab le i n s o l u t i o n s more a c i d i c than pH 2 (72) . The an t imic rob ia l a c t i v i t y of t h e epimer is probably zero. The s l i g h t a c t i v i t y found f o r t h i s material is probably due t o t h e reemergence o f CTC under t h e test condi t ions .

I n a f a sh ion analogous t o converted t o epianhydro-CTC i n

8. ANALYTICAL METHODS 8.1 Microbio logica l

The microbio logica l

t h a t of CTC, i t s epimer can be a c i d s o l u t i o n .

methods used f o r t h e determina- t i o n of CTC potency i n body t i s s u e s and f l u i d s , bu lk products , and pharmaceut ical formula t ions can be sepa ra t ed i n t o two t e s t i n g procedures: (cy l inder -p la te ) 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

1. Agar d i f f u s i o n p l a t e method ( cy l inde r -p l a t e ) : Th i s method is employed f o r determining t h e

potency of CTC i n human and animal pharmaceut ical formula- t i o n s , bu lk products , serum, t i s s u e s , u r i n e , d a i r y products , and animal feeds . The cy l inde r -p l a t e procedure is descr ibed by Grove and Randall (73) and t h e Code of Federa l Regula t ions (74). 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 assay i n animal f eeds is descr ibed i n t h e Assoc ia t ion of O f f i c i a l Ana ly t i ca l Chemists ' O f f i c i a l Methods of Analys is (76).

Addi t iona l methods us ing t h i s assay are descr ibed by

2. Turb id imet r ic method: -- This 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 pharmaceut ical formula t ions and bulk products . The t u r b i d i m e t r i c method is descr ibed i n the Code of Federal Regulat ions (77).

8.2 Chemical The phys ica l s t r u c t u r e of CTC has provided a good

source 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 polarographic (79) methods have been r epor t ed , t he most u s e f u l procedures have been based on t h e spec t roscop ic 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) .

f o r t h e assay of CTC. One method w a s based on t h e conversion of CTC by hea t ing i n a c i d t o t h e more i n t e n s e l y yellow anhydro-CTC de r iva t ive . i ng t h e b lue f luorescence of iso-CTC, which w a s prepared by hea t ing CTC i n pH 7.5 phosphate bu f fe r .

Others have descr ibed modi f ica t ions of t h e s e methods f o r var ious purposes. Hiscox (82) suggested t h e d i r e c t spec- t rophotometr ic assay 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 va r ious UV wavelengths. The p o s s i b l e contaminat ion of CTC wi th o t h e r t e t r a c y c l i n e drugs w a s addressed by C h i c e a r e l l i e t

I n 1949, Levine e t al . (81) publ i shed two procedures

The o t h e r method w a s based on measur-


al. (83) . They co r rec t ed f o r t h e poss ib l e presence of t e t r a - cyc l ine i n CTC using t h e f a c t t h a t t h e former is unchanged i n d i l u t e a l k a l i whi le CTC is converted t o t h e c o l o r l e s s iso- CTC . converted t o an anhydro d e r i v a t i v e by hea t ing i n ac id and is measured spec t rophotometr ica l ly . Feldman e t a l . ( 8 4 ) developed t h e a l k a l i n e degradat ion method t o measure CTC i n fermentat ion mash and Spock and Katz (85) used t h i s method t o determine CTC i n animal feed premixes.

has been used ex tens ive ly t o determine small amounts of CTC i n b i o l o g i c a l ma te r i a l s . Kohn (86) showed t h a t t h e f luo res - cent complex formed by CTC wi th calcium ions and b a r b i t a l could be ex t r ac t ed from animal t i s s u e s i n t o an organic so lven t and then measured spectrofluorometrically. The i n t e n s e f luorescence 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 . Poiger and S c h l a t t e r (88) ex t r ac t ed CTC from bio- l 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 ion p a i r . The f luorescence of t h e a n t i b i o t i c w a s then enhanced by the a d d i t i o n of magnesium ions and a base.

The t e t r a c y c l i n e which may be p re sen t is then

The n a t u r a l f luorescence of CTC and its d e r i v a t i v e s


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137 CHLORTETRACYCLINE HYDROCHLORIDE

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DOBUTAMINE HYDROCHLORIDE

RaJk H . Bishara and Harlan B. Long

I . Description I . I Nomenclature

I . I . 1 Chemical Name I . I .2 Nonproprietary Name I . I . 3 Proprietary Name

I .2.1 Empirical 1.2.2 Structural

I . 3 Molecular Weight I .4 Appearance, Color, Odor, and Taste

2. I Melting Range 2.2 Simple Solubility Profile 2.3 pHRange 2.4 Dissociation Constant (pKa) 2.5 Thermal Analyses

1.2 Formula


2.5. I Differential Thermal Analysis 2.5.2 Thermogravimetric Analysis

2.6. I Crystalline Habit 2.6.2 X-Ray Powder Diffraction

2.6 Crystallinity

2.7 Ultraviolet Spectrum 2.8 Infrared Spectrum 2.9 Nuclear Magnetic Resonance Spectrum 2.10 Mass Spectrum

3. Synthesis 4. Stability-Degradation 5 . Absorption, Metabolism, and Excretion

5.1 In Dog 5.2 In Man

6. I Elemental Analysis 6.2 Chloride Identity 6.3 Nonaqueous Titration 6.4 Chloride Determination 6.5 Chromatography

6. Methods of Analysis

6.5. I Thin-Layer Chromatography 6.5.2 Gas Chromatography 6.5.3 High Performance Liquid Chromatography

7. 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) Copyright 0 1979 by Academic Press, Inc.

All rights of reproduction in any form reserved. ISBN 0-12-260808-9

139

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)-l- methylpropyl]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

CH3 HO

HO&)CH2-CH2-NH-LH-CH 2 C H 2 0 0 H

HCI

DOBUTAMINE HYDROCHLORIDE 141

1.3 Molecular Weight

337 .85

1 . 4 Appearance, Co lo r , Odor, and Taste

White t o o f f -wh i t e , o d o r l e s s powder wi th a s l i g h t l y b i t t e r t as te .

2. P h y s i c a l P r o p e r t i e s

2 . 1 Mel t ing Range

189 - 191°C

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 minute a t

So lven t mg/ m 1

ambient t empera tu re .

Water pH 1 . 2 (USP X I X ) pH 4 . 5 (USP X I X ) pH 7 . 0 (USP X I X ) E thanol Methanol Pyr i d i n e Oc t a n o l D i e t h y l ether E t h y l acetate Chloroform Benzene Cyc lohexane

2 . 3 DH Ranne

>3.33 >2.50 >3 .33 >3.33 >5.00

> l o . 00 >5.00 <O .50 <O .50 <O. 50 <O .50 <O. 50 <O .50

The 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 about 4 . 9 .

2 . 4 D i s s o c i a t i o n Cons tan t

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


2.5 Thermal Analyses

2.5.1 Differential Thermal Analysis

A DTA thermogram of dobutamine hydrochloride, at a heating rate of 5°C per min. in a nitrogen atmosphere of 40 cc per min., shows (figure 1) an endotherm at 196°C 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 233°C 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


0

CU-Ni - X 1.5418 A

d - d 1/11 -

9 .07 7 2 .74 6 .87 5 2 . 6 3 6 . 3 7 7 2 . 5 1 5 . 2 7 18b 2 .40 4 . 9 8 18 2 . 3 5

4.50 100 2.27 4 .13 16 2 .22 4 .04 6 1 2 . 1 4 3 . 7 8 45 2 . 1 1 3 .60 45 2.06

3 . 4 4 16b 2 . 0 1 3.18 18 1 . 9 9 3.11 7 1 . 9 5 3 . 0 1 14 2.86 7

2 . 7 U l t r a v i o l e t SDectrum

1/11

14 9

16 5 2

7 7 7

The u l t r a v i o l e t spec t rum of dobutamine h y d r o c h l o r i d e i n methanol is g i v e n i n f i g u r e 2 . The spec t rum e x h i b i t s maxima a t 281 and 223 nm w i t h molar a b s o r p t i v i t i e s of 4 ,768 and 14 ,400 , r e spec - t i v e l y . When aqueous potass ium hydroxide is added t o t h e methanol ic s o l u t i o n of dobutamine hydro- c h l o r i d e t h e maxima a t 281 and 223 s h i f t t o 293 (e=6,100) and 240 nm, r e s p e c t i v e l y . These 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 spec t rum of t h e d rug is reco rded i n water r a t h e r t han methanol , 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 = 4,112) 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 spec t rum of dobutamine hydro- c h l o r i d e i n a potass ium bromide d i s k is g i v e n i n f i g u r e 3. Major band ass ignments are as f o l l o w s :


10

5

WAVELENGTH, nm

Figure 2 . U l trav io le t Spectrum of Dobutamine Hydrochloride

.Ooo c

Figure 3. Infrared Spectrum of Dobutamine Hydrochloride


c I

Band P o s i t i o n , cm-' Assignment 3400, 3300 and 3140 pheno l i c 0 - H s t r e t c h i n g 2960 and 2840 C-H s t r e t c h i n g (over-

2700 and 2450 (weak Mainly NH2, NH s t r e t c h i n g bands) 1610, 1530, 1520 and 1450 Aromatic r i n g s t r e t c h i n g 1440, 1390 and 1380 C H 2 , C H 3 , C-H bending 1360, 1280-1190 ( s e v e r a l pheno l i c C - 0 s t r e t c h i n g bands) 1150 and lower Mainly ske l e t a l and aro-

l a p i n g 0 - H bands 1

matic C-H bending

2 . 9 Nuclear Magnetic Resonance Spectrum

The 60 MHz pro ton NMR Spectrum of dobutamine hydroch lo r ide i n d e u t e r a t e d d ime thy l su l fox ide is g iven i n f i g u r e 4. Assignments of t h e bands are as f o l l o w s :

d c i

I I I I I H2

, CH2-CH2-N+- CI -

- I

a, a

.l4 Lc 0

rl

Jz 0 0

Lc a h

X

a, d

.rl E

cd c, 7

P 0

w 0

E 1

Lc

n


Chemical Shift (ppm) Multiplicity Area Assignment 1.3 doublet 3 a 1.9 unresolved 2 b

mu 1 t i p 1 e t 2.5 unresolved 2 C

(over lapping solvent)

3.0 unresolved 5 d,e,f triplets

mu 1 t i p le t s

singlet

6.8 overlapping 7 aromatic protons

8.9 very broad 5 g,h, i,j

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 2- butenoyl chloride ( I ) . The Friedel-Crafts reaction of methoxybenzene ( 1 1 ) with ( I ) using AlC1, in carbon disulf ide yields 1-( 4-methoxyphenyl)-2- buten-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-methoxy- pheny1)-1-butanone ( V ) . This ketone is reduced with hydrogen over Pd/C to give N-[2-(3,4- dimethoxyphenyl)ethyl]-4-methoxy-a-methylbenzene- propanamine ( V I ) . An alternate .synthesis f o r compound ( V I ) involves the reduction of 4-(4- methoxyphenyl)-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 ) .


Figure 5 . Mass Spectrum of Dobutamine Hydr oc h l o r i de


A second alternate synthesis of compound (VI) involves the reaction of 4-methoxy-a-methylbenzene- propanamine (X) with 3,4-dimethoxybenzeneacetic acid (XI) at 200°C 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 115°C (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 40-50°C 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 half- life of radioactivity following the administration of 14C-dobutamine was 1.9 hours. The major circu- lating 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 withi’n 8 to 10 minutes, while those of the metabolites peak be-

152 RAFIK H . BISHARA A N D HARLAN B. LONG

I

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 48 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. Dobu- tamine 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. For the detailed pharmacological and biochemical properties of dobutamine, the reader should consult the profile by Weber and Tuttle ( 5 ) .


6.1 Elemental Analysis ( A s C18H23N03-HC1)

Element

C H N 0 c1

Theory ( % >

63.99 7.16 4.15 14.21 10.49

I54 RAFIK H. BISHARA A N D HARLAN B. LONG

6 . 2 C h l o r i d e I d e n t i t y

About 5 m l of dobutamine 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 added 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 hydrox ide . 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 T i t r a t i o n

The secondary amine f u n c t i o n of dobutamine h y d r o c h l o r i d e may be de t e rmined 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 u s i n g g l a c i a l acet ic a c i d as a nonaqueous s o l v e n t . Mercu r i c acetate is used t o t i e u p t h e c h l o r i d e i o n .

6 . 4 C h l o r i d e D e t e r m i n a t i o n

A sample of dobutamine h y d r o c h l o r i d e con- t a i n i n g a t least 2 mg of c h l o r i n e is i g n i t e d i n a Schon ige 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 of d i p h e n y l c a r b a z o n e ( 5 mg/ml) i n methanol are 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 burned sample . Mercur ic 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:

p e r c e n t c h l o r i n e = 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 100

mg sample

p e r c e n t p u r i t y of dobutamine 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 found X 100 p e r c e n t c h l o r i n e t h e o r y

6 . 5 Chromatographv

6 . 5 . 1 Thin Layer Chromatography

The Rf v a l u e f o r dobutamine hydro- c h l o r i d e when chromatographed 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 deve loped 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 abou t 0 . 6 7 . The s p o t of t h e d r u g may be v i s u a l i z e d unde r s h o r t wavelength UV l i g h t (254 nm), or under 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 i o d i n e v a p o r s .


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 230°C 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/min- Ute. 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 3H- me 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 ) .


7.2.2 Gas 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 (methyl- vinyl) on Diatoport S operated at 260°C. The hydrogen flame detector is maintained at 280°C. 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 240°C. The temperature of the 63Ni electron capture detector is 250°C. 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-N- 3-(4-hydroxyphenyl)-l-methyl-N-propyl)phenethyl- amine 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% acetonitrile-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 240°C and the


flow rate of helium is 60 ml/min. The ion source voltage is 70 eV. The retention times of the trimethy Is i ly 1 dobutamine and tr imethy Is i lyl-3-0- methyldobutamine 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 267. The spectrum of the derivatized metabolite has a molecular ion at 459 with major fragments at 250 and 209. 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 centrifug- ation. 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 form- u 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., - 67, 1756 (1978).

The literature is surveyed through January, 1979.


ERYTHROMYCIN

William L. Koch

1 . Description I , I I . 2

2. 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

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

Name, Formula, Structure, and Molecular Weight Appearance. Color, and Odor


4. Stability 5. Bioavailability 6 . References

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

ISBN 0-12-260808-9 159

I60 WILLIAM L. KOCH

1. Descr ip t ion

1 .1 Name, Formula, S truc ture , 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 macrol ide a n t i b i o t i c c o n s i s t i n g of t h e aglycone , ery throno l ide A; t h e aminosugar, desosamine; and t h e neutra l sugar , 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 (Fig. 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 bitter tas te .

2. P h y s i c a l Propert ies

2 . 1 S o l u b i l i t i e s

Webs -- et al.3 reported t h e s o l u b i l i t y of e ry thromycin 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 spec t rum of ery thromycin is commonly used for 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 spec t rum 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 ke tone c a r b o n y l and the l a c t o n e ca rbony l , r e s p e c t i v e l y . The a b s o r p t i o n peaks between 1000 and 1200 cm' a r e due t o the ethers and m i n e f u n c t i o n s . The CH2 bending is evidenced by peaks between 1340 and 1460 cm' , and a l k a n e s r e t c h i n g peaks appea r between 2780

appear as bands between 3400 and 3700 cm' . 1 and 3020 cm' t . Hydrogen bonded OH and wa er

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 spec t rum of e r y t h r o - mycin in methanol 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

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

* Weiss reported t h i s va lue a s

mg. / m l

0.477 4.69 0.2* 5 . 0 5

7 20 7 20 7 20 7 20 7 20 7 20 7 20 > 20 7 20 7 20 > 20

> 20 > 20 7 20 7 20 7 20 7 20 > 20

9.65

2 . 1

> 20. We found 0.2 i n our l a b o r a t o r i e s .

I I I I I 1 I I I I 1 10 3600 3200 2800 2400 2000 1800 1600 UOO 1200 1000 800

WAVENUMBER CM-'

Fig. 2. Infrared abeorption spectrum of erythromycin

ERYTHROMYCIN I65

2.4 Thermal Gavimetric Analysis

A TGA of erythromycin hydrate i n d i c a t e s a loss of v o l a t i l e s of about 42, then decomposi- t 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 Analysis

A DTA of erythromycin hydrate shows an endotherm a t about 128'C., i nd ica t ing simultaneous loss of v o l a t i l e s and melting. A DTA of erythromycin anhydrate i n d i c a t e s mel t ing s t a r t i n g a t 193'C., then 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 of erythromycin d ihydra t e and anhydrate are shown i n Table 11. Radiat ion: C r / V , X 2.2896.7

2.7 Nuclear Magnetic Resonance

The spectrum shown i n Figure 3 was obtained a s a CDsOD s o l u t i o n us ing a Varian T-60A 60mHz instrument, General band assignments a r e l i s t e d i n Table I11 according t o Underbr ink. *

2 .8 pKa

The pKa f o r erythromycin i n 66% DMF/34X water is 8.6.

166 WILLIAM L. KOCH

TABLE I1

X-Ray Eowder Diffract ion Data

Erythromycin

An hy dr at e D ihydr a t 8

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

ZLLa 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 mythromycin

So lven t : 0 3 OD

Ins t rument : V a r i a n T-60A 60 mHz

Resonance Pro ton ( 8 ) P o s i t i o n (PPM) Peak Type

c!!3a2 - . 9 Unresolved t r i p l e t

A l l methyls no t 1 -1 .5 Over lapping l is ted above s i n g l e t s and or below d o u b l e t s

2.35 S i n g l e t

3.35 S i n g l e t (over- l a p p i n g s o l v e n t mu 1 t i p l e t )

O t h e r p r o t o n s (methyne and methylene) g i v e un reso lved and o v e r l a p p i n g mul t i p l e t s i n t h e 60 mHz spec t rum of t h e CD30D s o l u t i o n .

ERYTHROMYCIN 169


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 assay for erythromycin remains l a r g e l y unchanged from t h a t described by Kuzel e t al . 9 i n 1954. T h i s procedure is e s s e n t i a l v 8.8 follows. ence s tandard , a l k a l i reagent, and buf fer so lu- t i o n s a r e prepared p r i o r t o the assay.

Phosphate buf fer 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 purif ied water t o make 5 liters.

The refer-

The r e fe rence s t anda rd s o l u t i o n is prepared by d i s s o l v i n g about 35 mg. accu ra t e ly weighed erythromycin s tandard i n 100 m l . methanol in a 250 m l . volumetr ic f lask. This is d i l u t e d w i t h phosphate buf fer pH 7 . 0 t o 250 m l . , mixed, and allowed to cool t o room temperature , then aga in d i l u t e d to t h e mark and mixed w e l l .

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 . volumetr ic f lask . An ad- d i t i o n a l 100 m l . p u r i f i e d water is added and the s l u r r y heated on t h e steam bath t o aid i n so lu- t i o n . The s o l u t i o n is cooled slowly t o room temperature and d i l u t e d to 250 m l . w i t h p u r i f i e d water , t hen filtered prior t o use.

The a l k a l i reagent is prepared by

Four 10 m l . a l i q u o t s of t h e s t anda rd 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 . volumetr ic f lasks, two are labelled s t anda rd and t h e others blank. One m l . of 0 . 5 N HzSOd is added to t h e blank flasks and they are allowed t o s t a n d after 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 the s tandard f l a sks . A t t h e end of t h i s time, 1.0 m l . of 0.U NaOH is added to the blank f lasks and t h e i r con ten t s swi r l ed

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 fou r f l a s k s , t h e y are swir led t o m i x and p l aced i n a 60.C. water bath for 15.0 minutes . The f l a s k s a r e t h e n cooled r a p i d l y i n an ice b a t h , brought 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 25 .0 m l . w i t h p u r i f i e d wa te r . The W absorbance is r e a d a t 236 nm. v e r s u s p u r i - f i e d wa te r i n 1.0 cm. s i l i c a cells . The b l ank 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 ave rage n e t absorbance used for c a l c u l a - t ion.

Bulk e ry thromycin raw m a t e r i a l is treated t h e same a s t h e s t a n d a r d . Formula t ions a r e made up to 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 methanol and b u f f e r and 10 m l . a l i q u o t s used 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 the b lank 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 fo rma t ion of an n,R u n s a t u r a t e d ke tone (9-keto-10-ene) hav ing its absorbance max inum a s a s h o u l d e r a t 236 nm. ( c 6000) .I1 9 1 2 Thus, any other W abso rb ing s p e c i e s a r e measured w i t h t h e b l ank and s u b t r a c t e d from t h e absorbance before c a l c u l a t i o n of t h e e ry thromycin c o n c e n t r a t ion. A t y p i c a l spec t rum 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 fo r e ry thromycin u s i n g an OV-225 column or a PPE-20 column. The pro- c 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 ry thromycin 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 1s i l y l a c e t am ide , and N - t r imethy 1s i l y l - imidazole i n p y r i d i n e for 24 hours a t 75'C. Ten 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 on GCQlOO-120 mesh or 3 mm. x 1850 mm. 3% PPE-20 on Supe lcopor t a t 275OC.) of a n F and M model 400 gas chromatograph equipped w i t h a f lame i o n i z a t i o n detector. They r e p o r t be ing a b l e to s e p a r a t e e ry th romyc ins A , B, C ,

ERYTHROMYCIN

1.0

v)

0.5

0.0 I I

220 240 260 280 300 nm

Fig . 4 . W spectra of sample and blank a f t e r chrornophore development

171

172 WILLIAM L. KOCH

anhydroerythromycin A, and erythralosamine. Good agreement w i t h t he microbiological assay is shown. However, t h e biggest drawbacks appear t o be in s i l y l a t i o n t ime and the i n s t a b i l i t y of the GC column, 3 weeks a t 275°C.

These authors1 4 la ter report us ing t h e GC method for e n t e r i c coated tab le t s of e ry thromycin, g iv ing a recovery of 99.8% and a coef- f i c i e n t of v a r i a t i o n of 2.3% based on placebo tablets spiked 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 Coffeyls i s based on t h e ion p a i r dye complex of bromcresol purp le (5' ,5"-dibromo-o- cresol-sulfonphthalein) and t h e desosamine moiety of erythromycin i n pH 1.2 buffer . The method lacks 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 and 20-100 mcg . /ml . i n fermentat ion broth. A more r ecen t method by Sanghavi and Chandramo- han16 is a l s o based on a complex of the desosamine moiety, but they use p-dimethyl amino benzaldehyde a s the coupling agent , The procedure is non-spec i f ic , but s ens it ive and l i n e a r over a concent ra t ion range of 10-35 mcg. /ml .

3.4 Thin Layer Chromatography

Egon S tah l l7 described four TLC systems. Table IV summarizes the s o l v e n t s and R f ' s on s i l i ca gel G.

ERYTHROMYCIN I73

TABLE IV

Solvent EL

Methanol .16

Chlorof orm-me t hano 1 .03 95 + 5

Chloroform-methanol .29 50 + 50

Detect ion

Brownish-green color after spraying w i t h 10% s u l f u r i c acid and hea t ing 5-10 minutes a t 8OoC.

Butanol-acet ic acid-water .39 60 + 20 + 20

Spraying wi th 10% molybdophosphoric acid i n alcohol, followed by hea t ing produces a b lue s p o t on a yellow background. The s p o t d i sappears i n 2 hours.

Vilim e t a l . l * devised 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 base, s t e a r a t e , e s t o l a t e and e thylsucc ina te . This 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 e thy l succ ina te .

system s e p a r a t i n g erythromycin es tolate , e r y t h r o - mycin base, and anhydroerythromycin on a s i l i c a g e l 60 F-254 p l a t e u t i l i z i n g ethanol , methanol, t r i e thy lamine , 170:30:1. V i sua l i za t ion is made by sp ray ing w i t h 0.15% xanthydrol and 7.5% a c e t i c acid i n water. Table V summarizes the R f ' s .

Our l a b o r a t o r i e s l 9 have developed a

Component

Erythromycin Base

TABLE V_

Rf Color

0.30 Violet -

Anhydroerythromgc i n 0.43 Vio le t

Er y t hromyc i n Es t o l a te 0.60 Violet

I74 WILLIAM L. KOCH

3.5 Microbiological Analysis

Kavanagh and Dennen2 r e p o r t micro- b i o l o g i c a l tu rb id imet r ic and p l a t e assays f o r erythromycin base i n Ana ly t i ca l Microbiology, vol. 1. Staphylococcus aureus (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 assay 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 assay is found i n 21CFR452.110. The sample is d i l u t e d from 0.3 t o 2.0 tig./ml. i n pH 7 . 0 buffer and comparison is made t o a s tandard curve of 0 , 0.3 , 0.4, 0.6, 0 . 8 , 1.2 , 1 .6 , and 2.0 !ig./ml. Sarc ina l u t e a (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 range of 0.5 - 2.0 u g . / m l , is obtained when pH 8 . 0 buf fe r is used for sample and s tandard . In both methods, a sma 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 bu f fe r ing a t pH 7 . 0 or 8 .0 .

3.6 High Performance Liquid Chromatography

performance l i q u m Zromatographic 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 bu f fe r pH 4.9, and aceto- n i t r i l e (35:60:5) a s so lvent . A v a r i a b l e wavelength W detector us ing t h e absorp t ion of t h e ind iv idua l compounds gave t h e r equ i r ed s e n s i t i v i t y . A l t e r a t i o n s of buf fer 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 separa- t i o n of i nd iv idua l macrolide a n t i b i o t i c s .

Omura e t a1.21 used a r e v e r s e phase high

2. €I. Hash22 r epor t ed chromatographic cond i t ions f o r s e p a r a t i n g anhydroerythromycin from erythromycin us ing 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.

high performance-riquid chromatographic procedure for erythromycin. Ref rac t ive index d e t e c t i o n was used s i n c e the compound absorbs weakly i n t h e W. A 10 inn C1,/Lichrosorb” r eve r se phase column was used w i t h 80% methanol, 19.9% wate r , 0.1% ammonium hydrochloride a s t h e developing so lven t .

White et a l . 23 devised a r e v e r s e phase

ERYTHROMYCIN 175

T s u j i and Goetz2' deve loped a q u a n t i - t a t i v e h igh performance l i q u i d chromatographic method f o r s e p a r a t i n g and measur ing e ry th romyc ins A , B, and C , t h e i r ep imers and d e g r a d a t i o n prod- u 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 tr 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 compos i t ion of t h e mobile phase may be ad- j 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 rocedure on 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 . S t a b i l i t y

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 aqueous, a l c o h o l i c s o l u t i o n b u f f e r e d a t pH 7 . 0 - 8 . 0 is s t a b l e for about one week under r e f r i g e r a t i o n .

5. B i o a v a i l a b i l i t y

o b t a i n e d i n 1 t o 2 hour s a f t e r a s i n g l e dose. The U. S. Dispensa tory2 ' 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. dose , 0.6 !ig,/ml. 2 hour s a f t e r a 500 mg. dose, and 1.2 t g . / m l . 2 hour s a f t e r a 1 g. dose. Higher blood l e v e l s a r e ach ieved o n a m u l t i p l e dosage 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 used i n t a b l e t f o r m u l a t i o n s to 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 ry th romyc in base ; or t h e s t e a r a t e s a l t is p r e p a r e d which does n o t d i s s o l v e r e a d i l y i n t h e s tomach,

Maximum l e v e l s of e ry th romyc in i n serum a r e

The B i o a v a i l a b i l i t y Monograph for Ery thro- mycin27 p r o v i d e s d a t a for comparison of s e v e r a l manufac tu re r s * t a b l e t s of e ry th romyc in , The c r i t e r i a for 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 .

176 WILLIAM L. KOCH

6. References

1. K. Gerzon, personal communication, L i l l y

2. R. S. Egan, T. J. Perun, J. R. Martin,

3. P. J. Weiss, 1. L. Andrew, and W. W.

4. R. M. S i l v e r s t e i n and G. C. Bassler,

Research Laboratories.

and L. A. Mitscher, Tetrahedron 29, - 2525- 2538 (1973).

Wright, A n t i b i o t i c s and Chemotherapy - 7

Spectrometr ic I d e n t i f i c a t i o n of Organic Compounds, 2nd Ed., John Wiley and Sons, New York, p. 151.

5 . T. E. Cole, personal communication, L i l l y Research Laborator ies .

6. T. E. Cole, personal communication, L i l l y Research Laborator ies .

7. H. A. R o s e , Analy t ica l Chemistry 26 -’ 938 (1954).

8. C , D. Under br ink, persona 1 commun i c a t ion , L i l l y Research Laboratories.

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 Chemo- therapy 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).

11. E. H. Flynn, M. V. S i g a l , Jr. , P. F. Wiley, K. Gerzon, Jou rna l of t h e American Chemical Soc ie ty 76, 3121-3131 (1954).

12. T. J. Perun, Journa l of Organic Chemistry

13. K. T s u j i and J. €I. Robertson, Ana ly t i ca l Chemistry 43 (7) , 818-821 (1971).

14. J. H. Rober’tson and K. T s u j i , Jou rna l o f Pharmaceutical Sciences 6 1 - ( lo) , 1633- 1635 (1972).

15. N. R. Kuzel and H. F. Coffey, Technicon Symposium (1966), V o l . 1, Automation in Analy t ica l Chemistry, Medical, Inc. , White P la ins , N.Y., 1967, pp. 235-239..

16. N. M. Sanghavi and H. S. Chandramohan, Canadian Journa l o f Pharmaceutical Sciences - 10 (2), 59-61 (1975).

(7 ) , 374-377 (1957) a

32 , 2324-2330 (1967).

ERYTHROMYCIN 1 I1

17.

18.

19.

20.

2 1.

22.

23.

24.

25.

26.

27.

E. S t a h l , Ed., Thin Layer Chromatography, A Laboratory Handbook, 2nd Ed., Spr inger - Ver lag , N . Y . , 1969, pp. 572. 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 Chromato- graphy 133, 239-244 (1977). H. F. Hugar, personal communication, L i l l y Research Labora tor ies . D. W. Dennen, i n F. Kavanagh ( E d i t o r ) , Ana ly t i ca l Microbiology, V o l . 1, Academic Press (1963), pp. 209-294. S. Omura, Y. Suzuki , A. Nakagawa, and T. Hata, The Journa l of A n t i b i o t i c s

Z. H. Hash, Methods i n Enzymology, V o l . LXIII, p. 308, Academic mess, New York, 1975. 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 ,

K. T s u j i , J. F. Goetz, J o u r n a l of Chromato- graphy, 147, 359-367 (1978). F. Kavanagh, Ana ly t i ca l Microbiology, V o l . 1, Academic Press (1963), pp. 209- 294. U. S. Dispensatory, 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.

-

V o l . XXVI, NO. 12, 794-795 (1973).

V O l . XXX, NO. 10 , 811-818 (1977).

B. L ippincot t , Phi lade lphia (1973), pp. 487. C. H. Night inga le , Journa l of t h e American Pharmaceutical Associat ion NS 16, (4 ) , 203-206 (1976).

Analytical Profiles of Drug Substances. 8

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

4.1 Spectra 4. Physical Properties

4. I I Infrared Spectra 4. I2 4.13 Ultraviolet Absorption Spectra 4.14 Fluorescence Spectra 4.15 Rarnan Spectra

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 4.33

4.41 Diffusion 4.42 Surface Tension 4.43 Specific Volume 4.4 Conformation 5. Production

Nuclear Magnetic Resonance Spectra


Solubility in Solutions of Quaternary Ammonium Compounds Solubility in Solutions of Other Surface Active Agents

4.4 Physical Properties of Solutions

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


ISBN 0-12-260808-9 I79

I80 GLENN A. BREWER

7.21 Dilution Methods 7.22 Dye Reduction Methods 7.23 Turbidimetric Assays 7.24 Agar Diffusion Assays 7.25 Potentiation of Microbiological Assays

7.31 Colorimetric Assays 7.32 Spectrophotometric Methods 7.33 Miscellaneous Methods

7.3 Chemical Assays

7.4 Electrochemical Methods 7.5 Hemolytic Methods 7.6 7.7 Chromatographic Methods

Enzymatic and Other Biochemical 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

GRAMICIDIN 181

1. Introduction

D u b o s in 1939l as a crude complex together with a second peptide antibiotic known as tyrocidin. two antibiotics was called tyrothricin. ture was discovered about ten years after penicillin2 , tyrothricin was the first antibiotic utilized in clinical practice.

Gramiddin is a peptide antibiotic first isolated by

The mixture of the Although the mix-

It was soon found that tyrothricin causes severe hemo- lysis when administered parenterally and was destroyed when given orally. nents 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 antibiotic complex or individual compo-

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.~ It is 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. Chemistry

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 .

Tyrothricin was obtained by acidification of the

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. 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.

The

An empirical formula of C74H106N14014 was proposed for

The application of various bio- gramicidin based on elemental analysis and a Rast molecular weight 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 diox- pyridate 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. compound, but indicated that it was not isoserine.

He also noted the presence of an amino-hydroxy

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(-)-valyl- D(-)-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. work on the kinetics of peptide hydrolysis in an attempt to develop a rational explanation.

This unexpected finding triggered a good deal of

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 Gregory and Craig2 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.

but the evidence was not strong until separated crystalline gramicidin into

Still using heterogeneous gramicidin, S ~ n g e ~ ~ isolated the D-leucylglycine, L-alanyl-D-valine and L-alanyl-D- leucine from partial hydrolysates of gramicidin. He also had less conclusive evidence for the tripeptides alanyl- valylleucine 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.

GLENN A. BREWER I84

James and S ~ n g e ~ ~ speculated on the nature of the non- peptide bonds in gramicidin.

Hodgkin2' examined crystals of gramicidins A and B by x-ray diffraction. 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.

She estimated that the molecular

A provisional structure for gramicidin was proposed by Gavrilov and Akimova30.

Okuda and coworkers31 determined the amino acid composition of gramicidins A, B and C. Ishii and W i t k ~ p ~ ~ established 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 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).

using counter current

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. D3'.

In addition, they synthesized gramicidins A and

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. ness.

Two were postulated to be helixes of opposite handed-

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 [1405-97-61 or gramicidin D (Dubos) [1393-88-01. The latter name is used to distinguish the gramicidin discovered by Dubos from gramicidins S and J.

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-Valine- L-Valine-D-Valine-L-Tryptophane-D-Leucine-R'-D-Leucine- L-Tryptophane-D-Leucine-L-Tryptophane-NHCH CH OH 2 2

3.11 Gramicidin A [11029-61-11

Gramicidin A consists of a pair of congeners containing primarily L-valine gramicidin A (80- 95%) but also containing L-isoleucine gramicidin A (5-20%) 398 34.

I86 GLENN A. BREWER

3.111

3.112

L-Valine Gramicidin A [4419-81-21

R = L-valine R ’ = L-tryptophane

N O ‘9gH140 20 17 Molecular Weight 1882.349

L-Isoleucine Gramicidin A [5536- 03-81

R = L-isoleucine R ’ = L-tryptophane

‘100H142 20 17 N O

Molecular Weight 1896.376

3.12 Gramicidin B [11041-38-61

Gramicidin B consists of a pair of con- ramicidin B but also geners containing primarily L-valine

containing L-isoleucine gramicidin B 3% . 3.121

3.122

L-Valine Gramicidin B [4422-52-01

R = L-valine R’= L-phenylalanine

N O C97H139 19 17 Molecular Weight 1843.312

L-Isoleucine Gramicidin B

R = L-isoleucine R ’ = L-phenylalanine

N O C98H141 19 17 Molecular Weight 1857.339

3.13 Gramicidin C [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 R’= L-tyrosine

‘9BH141 19 18 Molecular Weight 1873.339

N O

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 ~ ~ have used it 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.


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


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.

t9

d

d

189

GRAM IC ID1 N 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 conformation of Gramicidin A using Raman spectro~copy~~. Two types of conformation were found depending on the solvent used.


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 I93

4.22 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' 28 ' 29. frustrated by the fact that the gramicidin was not chemically pure but was a mixture of components.

These studies were

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 555. 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) PEAK REL. PEAK AREA REL. AREA

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

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

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

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

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

4.23 Crystal Density

.GLENN A. BREWER

Low and Richards used a density gradient column to study the density of crystals of "mixed grami- cidir. 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 SOLVENT mg/ml

Water Methanol Ethanol Isopropanol Isoamyl Alcohol Cyclohexane Benzene Toluene Petroleum Ether I sooc t ane Carbon Tetrachloride Ethylene Glycol

0.140 > 20 > 20 > 20

14.10 0.02 0.19 0.04 0.007 0.005 0.047

> 20

SOLUBILITY SOLVENT mg/ml

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

11.90 > 20

18.10 18.80 10.7 2.15 >20 >20

0.100 > 20 > 20 > 20

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 65. 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. fatty acid alcohols71, aliphatic pyrrolidone73 # 74.

Alcohols6’, ~anthocillin~~, fatty acid amides’O, and polyvinyl-

4.4 Physical Properties of Solutions

4.41 Diffusion

The diffusion constants for the gramicidin complex were determined in acetic acid and ethanol solu- t i ~ n ~ ~ . calculated as 2800-5000.

The molecular weight range of gramicidin was

Polson using a similar method obtained a molecular weight of 300076.

4.42 Surface Tension

Gramicidin decreases the surface tension of aqueous solution7’. properties of gramicidin were destroyed by heat but the surface tension depression was not changed.

The bactericidal and hemolytic

Kemp and coworkers found that when they partitioned gramicidin between water and heptane, it migrated to the walls of the vessel7’.

I96

4.43 Specific Volume

GLENN A. BREWER

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

The apparent

4.44 Conformation

The conformation of gramicidin in aqueous solution has been extensively studied. A lipophilic left- handed helical structure has been proposed for gramicidin A401 41.

cidin is due to the formation of ion transport channels across biological membranes.

It was proposed that the mode of action of grami-

Bamberg and coworkers have studied the single channel conductance of gramicidins A, B and C8 I

Significant differences between gramicidin A and B were found .

.

Cabon 13 NMR has been used to confirm the presence of a double helical dimer modela2.

The conformation of gramicidin in various organic solvents has also been established" I I 8 4 I I 86.

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-benzyl- D-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. effect of the addition of amino acids to a synthetic medium on the production of tyrothricing4. 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 anti- bioticg5.

Konikova and Dobbert studied the

They found that these

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 tyrothri- c~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 grami~idins~~~.

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 11 112, 113,114.

Various esters of gramicidin have also been prepared’ ’ 5, ’ ’ 6 , ’ 7. hemolytic activity, they also possess less antibiotic activity.

Although these compounds have reduced

6. Stability

6.1 Stability in Solution

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

Certain

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’’’. and coworkers have presented infrared, ultraviolet, visible and ESR spectra for gramicidin solutions irradiated with various amounts of ultraviolet light122.

Sugimoto

6 . 3 Stability of Formulations

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

He also showed that the antibiotic can

GRAMICIDIN I99

7. Analytical Methods

7.1 Identity Tests

Brustier and coworkers utilized the Adamkiewicz- Hopkins-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 Ehrlich’s 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. product was detected by a color reaction.

This

7.2 Microbiological Assays

Microbiological assays are the primary assay method for antibiotics. They provide sensitive but non- selective 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 reported’ 30.

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 lysodeikticus’ 34.

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 faecalis’ 37.

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 lysodeikticus’ 34.

Miller, Matt and Ciminera reported an agar diffusion assay for grami~idinl~~. used this method to assay pharmaceutical preparations 43. The Swiss Pharmacopeia utilizes an agar diffusion assay with Sarcina l ~ t e a l ~ ~ .

Raitio and Bonn

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 assay’ 47.

Forni found that traces of cobalt chloride enhances the activity of gramicidin toward Escherichia & and Staphlococcus aureus’ 48.

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 . colorimetric assay for gramicidin utilizing the reaction of

Kreuzig described a semi-automated

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 gramici- din157.

7.33 Miscellaneous Methods

White and Secor have measured gramicidin by measuring the Kjeldahl nitrogen’ 58.

7.4 Electrochemical Methods

Kramarczyk and Berg have described an indirect polarographic assay for gramicidinl 59-

7.5 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 gramicidin’60. 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. lytic r n e t h o d ~ ~ ~ ~ , ’ ~ ~ , ’ ~ ~ .

Other authors have reported on modified hemo-

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

Strictly an inducible 8-galactosidasel 66, enzyme is inhibited by the addition of gramicidin. speaking, gramicidin does not inhibit the enzyme directly but this method could be used as the basis of a sensitive assay method .

The production of this

GRAMICIDIN 203

Gramicidin was found to uncouple the phosphorylation of ADP from the enzymatic reduction of ferricytochrome c167,168,169,170,171,172,173,174~175~

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 distribution2 3 . chloroform-benzene (7:23:15:15) system was used to show the presence of two components. Craig later showed that gramicidin contained three crystalline components177. cussed the application of counter current distribution to the separation of gramicidins 78. there were at least four components in gramicidin and gave the fourth component the name gramicidin D33.

A water-methanol-

Stamm dis-

Ramachandran showed that

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-gramicidin 3 7 .

Okamoto and coworkers showed that gramicidin could be separated into three fractions (A, B and C) using droplet counter current distribution’ 79.

7.72 Paper Chromatography

Snell, Ijichi and Lewis published a series of paper chromatographic systems capable of separating various antibiotics including gramicidinl by bioautography. Forni and Cavalli used the following systems on Whatman No. 1 paper to distinguish between bacterial peptide antibioticslS1.

Detection was

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 butanol- acetic acid-water (50:25:25) system using Schleicher and Schull 2043a paper buffered to pH 3.0 gave the best separation of peptide antibiotics182. salting out chromatography to separate gramicidin and tyrothri~inl~~.

The same authors utilized

Singh utilized anionic dyes to detect gramicidin on paper chr~matogramsl~~. described three paper chromatographic systems for gramici- din185. Mtschel and Lercher described two solvent systems for antibiotics186. pyridine-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.

Paris and Theallet

The solvent systems were butanol-

DeFranca and coworkers described a system of 25 ml 9:l acetone-water, 5 ml chloroform, 2 ml ethylene glycol and 1 ml of ~yridinel~~. accurate as the microbiological assay.

They claim the method is as

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

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

(12:2.5:7.5:40:20)

(10:10:20)

Guven and Ozsari reported some thin layer

McGilveray and Stickland described two thin systems to use as identity tests for antibiotics including gramicidinl ’ O . 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-butanol- methanol-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 95 . Paris and Theallet have described two paper electrophoresis systems for gramicidin 18’. Gomes have published paper electrophoresis methods for various peptide antibiotics1 96. utilized agar electrophoresis to identify various antibiotics including gramicidinl 97.

Cunha and

Lightbown and DeRossi

7.75 Column Chromatography

Moses has patented a process for the separation 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.

The 80% aqueous

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 gramicidin2?1, 202,20 3,204,20 5,206,207, 2 08 209 21 0,2 1112 1212 13

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

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-

-

Biochim. Biophys. Acta 419, 223-8 (1976)-(=. 84 70598 j (1976) ) . Bamberg, E. and Laeuger, P.; Biochim. Biophys. Acta - 367, 127-33 (1974) (G. 81 169801~ (1974)). Fossel, E. T.; Veatch, W. R.; Ovchinnikov, Yu. A., and Blout, E. R.; Biochemistry 13, 5264-75 (1974) (C.. - 82 69439k (1975)).

GRAMICIDIN 21 1

83.

84.

85.

86.

87.

88.

89.

90.

91.

92.

93.

94.

95.

96.

97.

98.

99.

100.

Isbell, B. E.; Rice-Evans, C. and Beaven, G. H.; Fed. Eur. Biochem. SOC. Lett. 25, 192-6 (1972) (G. 77 161266~ (1972)). Grell, E.; Egqers, F. and Funck, Th.; Chimia 26, 632-7 (1972) (C. A. 78 84803k (1973)).

-

- - Grell, E. and Funck, Th.; J. Supramolecular Struct. 1, 307-35 (1973) (C. A. 80 79316r (1974)). - - Veatch, W. R. and Blout, E. R.; Biochemistry 2, 5257- 64 (1974) (G. 82 69438j (1975)). Harris, R. D.; Jacobs, M.; Long, M. M. and Urry, D. W.; Anal. Biochem. 73, 363-8 (1976) (C. A. 85 43280p (1976) ) . - - -

Kyogoku, Y. and Kawano, K.; Tampakushitsu Kakusan Koso, Bessatsu 76, 280-96 (1976) (C. A. 85 118022e (1976)). - - Lotz, B.; Colonna-Cesari, F.; Heitz, F. and Spach, G.; J. Mol. Biol. 106, 915-42 (1976) (C. A. 86 90231d (1977) ) . Stokes, J. L. and Woodward, C. R. Jr.; J. Bact. 46, 83- 88 (1943) (C. - - A. 37 5997(9) (1943)). Konikova, A. S.; Azarkh, R. M.; Blinnikova, E. I. and Dobbert, N. N.; Microbiology U.S.S.R. 13, 171-179 (1944) (G. 2 4111 (4) (1945)). Lewis, J. C.; Dimick, K. P. and Feustel, I. C.; E. Eng. Chem. - 37, 996-1004 (1945) (e. 40 921(2) (1946)). Appleby, J. C.; Knowles, E.; McAllister, R. C. A.; Pearson, J. and White, T.; J. Gen. Microbiol. L, 145-157 (1947) (C. A. - 41 63709 (1947)).

Konikova, A. S. and Dobbert, N. N.; Biokhimiya 13, 115-23 (1948) (C. A. 42 7832a (1948)). Udalova, T. P. and Fedorova, R. I.; Mikrobiologiya 34, 631-635 (1965) (C. A. 63 18988f (1965)).

Mitchell, W. R.; U. S. Patent 2,465,338 March 29, 1949 (G. 43 4342f (1949) ) . Mitchell, W. R.; U. S. Patent 2,602,043 July 1, 1952 (u. 46 92679 (1952)).

- -

- -

- - -

Mercere, N. M. I.; Rev. fac. cienc. quim. 25, 29-41 (1950) (C. A. 48 46303. (1954)). - - Tudesca, M. V.; Anales Fac. Quim. Farm., Univ. Chile - 14, 105-13 (1962) (U. 60 4741f (1964)). Fraile, E. R.; Prechel, W.; Lugones, 2. M.; D'Aquino, M. and Valdez, I.; SAFYBI 9, 296-303 (1979) (C. A. 73 43874n (1970) ) . - -

212 GLENN A. BREWER

101.

102.

103.

104.

105.

106.

107.

108.

109.

110.

111.

112.

113.

114.

115.

116.

117.

118.

Okuda, K.; Edwards, G. C. and Winnick, T.; J. Bacteriol. 85, 329-38 (1963) (C. - - A. 58 7149e (1963)).

Bodley, J. W. Dissertation Abstr. 25, 2738-9 (1964) (C. A. 62 5465f (1965)). -- Jerez Guevara, C.; An. Fac. Quim. Farm., Univ. Chile - 20, 65-8 (1968) (G. 2 42548 x (1970)). Jerezguevara, C.; Caviedes, R. and Basilio, C.; H. Biol. Med. Exp. 6, 1-9 (1969)(C. A. 73 1065272 (1970)).

Hotchkiss, R. D. and Dubos, R. J.; J. Biol. Chem. 136, 803-804 (1940) (C. - A. 35 1083(4) (1941)). Olcott, H. S. and Fraenkel-Conrat, H. L.; U. S. Pat. 2,453,534 Nov. 9, 1946 (C. - - A. 43 1534a (1949)).

Baron, A. L.; U. S. Pat. 2,534,541 December 19, 1950 (C. A. 45 2158b (1951)). Anon., Brit. Pat. 966,540 August 12, 1964 (C. A. 61 13133g (1964) 1. Shepel, E.N.; Iordanov, S.; Sychev, S. V.; Miroshnikov, A. I.; Ivanov, V. T.; and Ovchinnikov, Yu.A.;

- - -

- -

Tezisy Dok1.-Vses. Simp. Khim. Pept. Belkov. 3rd - 171 (1974) (G. 86 44012k (1977) ) . Lewis, J. C.; Dimick, K. P.; Feustel, I. C.; Fevold, H. L.; Olcott, H. S. and Fraenkel-Conrat, H. L.; Science 102, 274-5 (1945) (C. A. 39 5282 (1945)).

Fraenkel-Conrat, H.; Brandon, B. A. and Olcott, H. S.; J. Biol. Chem. 168, 99-118 (1947) (G. 41 4182e (1947) ) . Leurquin, J.; Experientia 2, 198-9 (1947) (C. - - A. 41 6989~ (1947) ) . Fraenkel-Conrat, H. L.; Humfeld, H.; Lewis, J. C.; Dimick, K. P. and Olcott, H. S.; U. S. Pat. 2,438,209 March 23, 1948 (G. 42 3914a (1948)). Bustois, A. C. and Cotte, J.; French Patent 1,007,485 May 6, 1952 (G. 51 lOOlOa (1957)). Olcott, H. S.; Lewis, J. C . ; Dimick, K. P.; Fevold, H. L. and Fraenkel-Conrat, H. L.; Arch. Biochem. l0,

Moreau, H.; Chovin, P. and Rivoal, G.; Bull. SOC. chim. biol. 31, 1062-1069 (1949) (G. 44 3678a (1950) ) . Rambhav, S. and Ramachandran, L. K.; Indian J. Biochem. Biophys. 9, 225-229 (1972) (u. 78 143631~ (1973)).

- --

553-555 (1946) (G. 40 7386(3) (1946)).

Nitti, G. and Nislo, V.; Boll. SOC. ital. biol. sper. 28, 1807-8 (1953) (C. A. 47 7735a (1953)). - - -

GRAMICIDIN 2 13

119.

120.

1 2 1 .

1 2 2 .

123.

124.

125.

126.

127.

128.

129.

130.

131.

132.

133.

134.

135.

136.

137.

138.

Anon.; German P a t . 820,949 November 15, 1951 (C. A. - 49 5788c ( 1 9 5 5 ) ) .

I s h i i , S . and Witkop, B.; J. Am. Chem. SOC. - 86, 1848-53 (1964) (G. 61 717h ( 1 9 6 4 ) ) .

Set low, R. B. and Doyle, B.; Biochim. e t Biophys. A c t a 24, 27-41 (1957) (C. A. 51 11423g (1957)) .

Sugimoto, S. ; Ohnishi , S. and N i t t a , I.; Nippon Genshiryoku Kenkyusho Nempo, JAERI 5027, 143-6 (1971) (C. A. 76 106364f ( 1 9 7 2 ) ) .

Buckwalter, F. H . ; J. Am. Pharm. Assoc., P r a c t . Pharm. - Ed. 15, 694-700 (1954) (G. 49 3469f (1955)) .

B r u s t i e r , V.; Bourbon, P. and Vignes, R.; B u l l . SOC.

chim. France 113-14 (1950) (C. A. 44 6579h ( 1 9 5 0 ) ) .

Fischbach, H. and Levine, J.; Antibiotics & Chemo- t h e r a p y 2, 1159-69 (1953) (C. A. 48 9621g ( 1 9 5 4 ) ) .

- -

- - -

- -

- -

- - Ramachandran, L. K . ; I n d i a n J. Biochem. 4, 137-141 (1967) (Anal. Abst. - 1 5 7532 ( 1 9 6 8 ) ) .

- Anon.; 1, 1978.

- Anon.; F e d e r a l R e g i s t e r 29, 7625-57 June 1 3 , 1964 (C. A. 61 6857d ( 1 9 6 4 ) ) .

Reedy, R. J. and Wolfson, S. W . ; J. Am. Pharm. Assoc. - 39, 1-3 (1950) (e. 44 3 2 1 1 ~ (1950)) .

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 1, 189-96 (1951) (G. 5 6078g ( 1 9 5 4 ) ) .

Code of F e d e r a l Regula t ions P a r t 448.25 A p r i l

- -

Prgvot , A. R. ; Compt. rend . SOC. b iol . 141, 264-5 (1947) (=A. 42 246b ( 1 9 4 8 ) ) .

Pr6vot , A. R.; Rept. Proc. 4 t h I n t e r n . Congr. Microbiol. 106-7 (1947) (G. 43 4339g ( 1 9 4 9 ) ) .

DeFel ip , G. ; A l b e r t i , S. and von Lorch, L.; Rend. ist. super . s a n i t a 2 2 , 534-42 (1959) (C. A. 54 8 2 1 f ( 1 9 6 0 ) ) . C e r i o t t i , G. ; B o l l . SOC. i t a l . biol . sper. 24, 1234-7 (1948) (C. A. 43 8614a ( 1 9 4 9 ) ) .

- - -

Berr idge , N. J. and Barre t t , J.; J. Gen. Microbiol. 6, 14-20 (1952) (G. 46 10295e ( 1 9 5 2 ) ) .

K a i s e r , E. and Camboni, V.; B o l l . ist. sieroterap. mi lan 32, 298-302 (1953) (C. A. 48 13822a ( 1 9 5 4 ) ) .

Kramer, J. and Kirschbak, A . ; Antibiotics & Chemo- t h e r a p y 5, 561-5 (1955) (C. A. 50 52383. ( 1 9 5 6 ) ) .

- -

Leclercq , S.; J. pharm. Belg. 11, 33-44 (1956) (G. - 50 16958e (1956) ) .

214 GLENN A. BREWER

139. Leclercq, S.; J. pharm. Bel. 23, 83-105 (1968) (C. A. - - 69 30126n (1968)).

140. Pain, S. K.; Bose, B. K. and Dutta, B. N.; J. Proc. Inst. Chemists 36, 82-6 (1964) (=. 61 936033 (1964)).

141. Kreuzig, F.; Allg. Prakt. Chem. 23, 145-6 (1972)(C. - A. - 77 135003~ (1972)).

142. Miller, A. K.; Matt, C. and Ciminera, J. L.; J. Am. Pharm. Assoc. 41, 23-6 (1952) (C. - - A. 46 4175c (1952)). -

143.

144.

145.

146.

147.

148.

149.

150.

151.

152.

153.

154.

155.

156.

157.

158.

Raitio, A. and Bonn, K. E.; J. pharm. Belg. 10, 358-60 (1955) (C. A. 50 1732533 (1956)). - - Vuilleumier, M. and Anker, L.; Pharm. Acta Helv. 33, 621-33 (1958) (C. A. 53 10661~ (1959)).

Viola, M. R. and Canestrini, C.; Boll. Chim. Farm. 105, - -

688-94 (1966) (G. 66 222532 (1967)). Kresuzig, F.; Pharm. Acta Helv. - 47, 288-94 (1972) (C. A. 77 92895m (19731). . . - - Nisonger, L. L.; J. Am. Pharm. Assoc. 43, 716-18 (1954) (C. A. 49 3477a (1955)).

Forni, P. V.; Boll. SOC. ital. patol. - 3, 183-4 (1953) (x. 50 12172~ (1956) ) . Gillissen, G.; Arzneimittel-Forsch. 2, 460-3 (1955) (C. A. 49 16065f (1955)). -- Barr, M. and Tice, L. F.; Am. J. Pharm. - 127, 260-9 (1955) (C. A. 50 7395e (1956)).

Casilli, I. and Ragni, G.; Boll. Chim. Farm. 104, 432-7 (1965) (x. 67 14878a (1967)).

- -

Rittenberg, S. C.; Sternberg, H. E. and Bywater, W. C.; J. Biol. Chem. 168, 183-9 (1947) (G. 41 4609h (1947)). Kreuzig, F.; Allg. Prakt. Chem. 20, 224-5 (1969) (C. A. 71 1165803 (1969)). - - Patel, S. Z. and Naravane, J. S.; Indian J. Pharm. - 19, 67-9 (1957) ( L A . 52 10256f (1958)). Ivashkiv, E.; Biotechnol. Bioeng. 15, 821-5 (1973) (G. 79 76903y (1973) ) . Oberzill, W.; Sci. Pharm. 25, 148-63 (1957) (=. 52 3267e (1958) ) . Tombs, M. P.; Cooker K. B.; Souter, F. and MacLagan,

Bruges, Belg. 37-4 (1959) (Q. 54 15497f (1960)). White, L. M. and Secor, G. E.; Ind. Eng. Chem., Anal. - Ed. 18, 457-8 (1946) (G. 40 5357(6) (1946)).

N. F.; prot ides Biol. Fluids. Proc. 7th Colloa . I

GRAMICIDIN 215

159.

160.

161.

162.

163.

164.

165.

166.

167.

168.

169.

170.

171.

172.

173.

174.

175.

176.

177.

Kramarczyk, K. and Berg, H.; Abhandl. Deut. Akad. Wiss. Berlin, K1. Chem., Geol. Biol. 23-36 (1964) (C. A. 62 5141f (1965)). - - Herrell, W. E.; Proc. S O ~ . Exptl. Biol. Med. 46, 182-4 (1941) (C. A. 35 2217(7) (1941)). -_ Mann, F. C.; Heilman, D. H. and Herrell, W. E.; e. SOC. Exptl. Biol. Med. 52, 31-3 (1943) (C. A. 37 1763 (7) (1943) ) . Dimick, K. P.; J. Biol. Chem. - 149, 387-93 (1943) (C. A. 37 6295(7) (1943)).

Villela, G. G. and Cury, A.; Rev. brasil. biol. - 5, 361-6 (1945) (C. A. 40 2175(3) (1946)).

Friedrich, W.; Mikrochemie ver. Mikrochim-Acta 36/37,

Dimick, K. P.; Proc. S O ~ . Exptl. Biol. Med. - 78, 782-4 (1951) (g. 46 3157g (1952)). Creaser, E. H.; J. Gen. Microbiol. 12, 288-97 (1955) (C. A. 49 9739d (1955)).

BorgstrGm, B.; Sudduth, H. C. and Lehninger, A. L.; J. Biol. Chern. 215, 571-7 (1955) (G. 49 14063d (1955)).

- - -

--

- -

894-901 (1951) (C. A. 45 574717 (1951)). - -

- -

Cooper, C. and Lehninger, A. L.; J. Biol. Chem. 219, 489-506 (1956) (C. _ _ - A. 50 7911d (1956)).

Devlin, T. M. and Lehninger; J. Biol. Chem. 219, 507- 18 (1956) (C. A. 50 7911g (1956)). - - Cooper, C. and Lehninger, A. L.; J. Biol. Chem. 219, 519-529 (1956) (C. A. 50 7912a (1956)). - - Brodie, A. F. and Gray, C. T.; J. Biol. Chem. 219, 853-62 (1956) (C. A. 50 10188d (1956)). - - Cooper, C. and Lehninger, A. L.; J. Biol. Chem. 224 547-60 (1957) (C. A. 51 8848e (1957)). - - Hiltibran, R. C.; Trans. Ill. State Acad. Sci. 50, 176-82 (1965) (G. 64 6968a (1966)). Hiltibran, R. C.; Trans. I l l . State Acad. Sci. 59, 249-53 (1966) (C. - A. 65 18904~ (1966)).

Hotchkiss, R. D.; Arch. Biochem. and Biophys. 65, 302-18 (1956) (e. 2 3739g (1957)). Hinkson, J. W.; Arch. Biochem. Biophys. 112, 478-87 (1965) (G. 64 2299e (1966)). Craig, L. C.; Harvey Lectures 45, 64-86 (1949-1950) (C. A. 46 11295a (1952)). - -

216 GLENN A. BREWER

178.

179.

180.

181.

182.

183.

184.

185.

186.

187.

188.

189.

190.

191.

192.

193.

194.

195.

Stamm, W.; Pharm. Ind. 15, 120-4 (1953) (C. A . 47 8814d (1953) 1 .

- -

Okamoto, K.; Yonezawa, H. and Izumiya, H . ; J. Chroma-

Snell, N.; Ijichi, K. and Lewis, J. C.; Appl. Micro- biol. 4, 13-17 (1956) (C. - - A. 50 5086d (1956)).

togr. 92, 147-56 (1974) (=. 81 25929~ (1974)).

- - Forni, P. V . and Cavalli, F.; Minerva med. 4545 (1958) (C. A. 53 7306g (1959)). - - daCunha, A. P. and Baptista, M. L. D. M.; Bol. escola farm.,Univ. Coimbra 19-20, 225-30 (1959-60) (G. - 55 19136e (1961)).

daCunha, A. P. and Baptista, M. L. D. M.; Bol. escola farm., Univ. Coimbra 19-20, 217-24 (1959-60) (G. - 55 21481g (1961)).

Singh, C.; Cesk. Farm. 12, 294-7 (1963) (C. - - A . 61 2906b (1961) ) . Paris, R. R. and Theallet, J. P.; Ann. Pharm. Franc. - 20, 436-42 (1962) (G. 57 16746i (1962) ) . Ritschel, W. A . and Lercher, H.; Pharm. Ztg. Ver. Apotheker-Ztg.106, 120-2 (1961) (C. A. 62 397h (1965)).

DeFranca, F. P.; Rosemberg, J. A. and DeJesus, A. M.; Rev. Brasil. Farm. 50, 343-6 (1969) (C. - - A. 73 28952t (1970) 1 . Nussbaumer, P. A.; Pharm. Acta Helv. 2, 647-52 (1964) (C. A. 62 3886e (1965)).

Pitton, J. S.; Antibiot., Advan. Res., Prod. Clin. Use, Proc. Congr., Prague 490-5 (1964) (C. - - A. 66 40743p (1967)).

- -

Guven, K. C. and Ozsari, G.; Eczacilik BuL. - 9, 19-29 (1967) (C. A. 68 71008c (1968)). - - McGilveray, I. J. and Strickland, R. D.; J. Pharm. Sci. -- 56, 77-79 (1967) (G. 66 40746s (1967)). Nekola, M.; 2. analyt. Chem. 268, 272-274 (1974) (C. A. 81 103174m (19741). . . - - Kreuzig, F.; Fresenius' Z. anal. Chem. 282, 447-449 (1976) (e. 86 60589j (1977)). Kreuzig, F.; J. Chromatogr. - 142, 441-447 (1977) (G. 88 11966h (1978) ) . - daCunha, A. P. and Baptista, M. L. D. M.; Bol. escola. farm., Univ. Coimbra 19-20, 331-40 (1959-60) (C. A. 55 19136i (1961)).

- -

GRAMICIDIN 2 I7

196.

197.

198.

199 *

200.

201.

202.

203.

204.

205 -

206.

207.

daCunha, 0. R. P. and Gomes, M. E. B.; Bol. Escola Farm., Univ. Coimbra Ed. Cient. 22, 129-36 (1962) (C. A. 61 1711d (1964)).

Lightbown, J. W. and DeRossi, P.; Analyst 3, 89-98 (1965) (C. A. 62 11630~ (1965)).

Moses, W.; U. S. Pat. 2,992,164 July 11, 1961 (C. - A. 55 22721a (1961)).

Bartley, I. M.; Hodqson, B.; Walker, J. S.; Holme, G.; Biochem. J. 127, 489-502 (1972) (C. A. 77 58417x (1972) ) . Axelsen, K. S. and Vogelsang, S. H.; J. Chromatogr. - 140, 174-78 (1977) (Anal. Abst. - 34 5E31 (1978)).

- -

-

- - -

Dubos, R. J.; J. Pediatrics 2, 588-95 (1941) (G. 40 376(5) (1946)). - Lions, F.; Australian J. Sci. - 6, 46-8 (1943) (C. A. 38 21658 (1944)).

- - Gauze, G. F.; Advances in Modern Biol. 16, 215-19 (1943) (C. A. 38 138(5) (1944)).

Hotchkiss, R. D.; Advances in Enzymology A, 153-99 (1944) (G. 38 4623(6) (1944)).

Petersen, H. K. J. and Vermehren, E.; Arch. Pharm. Chem. - 51, 109-28, 161-94 (1944) (G. 41 6306b (1947) ) . Hoogerheide, J. C.; Botan. Rev. - 10, 599-638 (1944) (e. 2 4913 (5) (1945) ) . Bergol'ts, M. K h . ; Farmatsiya z, 35-8 (1944) (C. A. 43 5551c (1949)).

- -

- 208. Broch, P. J. F. and Jacob, F.; Farmatsiya I, 139-40

209. Filho, A. R.; Rev. brasil. farm. 27, 335-61 (1946) (1944) (C. A. 43 5826e (1949)).

(C. A. 41 4193d (1947)).

--

- - 210. Massey, F. C.; Penna. Med. J. 49, 1090-4 (1946) (C. - A.

211. Rivoal, G.; Rev. sci. 86, 369-76 (1948) (e. 43

212. Postovskil, I. Ya. and Bednyaqina, N. P.; Uspekhi Khim.

213. Macheboeuf, M.; Actualit& pharmacol. 2, 111-22 (1950)

214. Werner, G.; Scientia Pharm. 19, 95-103 (1951) (G.

- 43 3061a (1949)).

3881d (1949) ) .

16, 3-28 (1947) (C. A. 41 5578a (1947)).

(C. A. 47 5026d (1953)).

- 45 10510a (1951)).

- - -

- -

218 GLENN A. BREWER

215.

216.

217.

218.

219.

220.

221.

222.

223.

224.

Macheboeuf, M. and Gros, F.; Expos& ann. biochim. m6d. 12, 141-60 (1951) (C. - - A. 46 8716e (1952)). Tr6foue1, J.; Cheymol, J.; Nau, A.; Paul, R.; Pgnau, H.; Hagemann; Romain, R.; Ziegl6, M.; Vignalou, J. and Quevauviller, A.; The'rapie 2, 961-1029 (1956) (C. A. 52 20665d (1958)).

Brunner, R.; Osterr. Apotheker Ztg. 11, 455-60, 477-9 (1957) (u. 52 57489 (1958)). Baker, W. B.; Drug & Cosmetic Ind. 87, 172-3, 258-62 (1960) (s. 54 25568h (1960)). Schumacher, E.; Schweiz. Arch. Tierheilk. 104, 350-60 (1962) (C. A. 57 11307b (1962)).

DeLogu, I.; Corriere Farm. 21, 419-20 (1966) (C. A. - 66 102608j (1967)).

- -

- -

- -

Bogentoft, C.; Farm. Revy 67, 749-51 (1968) (C.. 70 80793u (1969) 1 .

Hladky, S. B.; Drugs Transp. Processes Symp. 193-210 (1973) (u. 82 149063~ (1975) ) . AkerS, H. A.; Lee, S. G. and Lipmann, F.; Biochemistry - 16, 5722-9 (1977) (G. 88 17964p (1978) ) . Brewer, G. A. and Platt, T. B.;Encyclopedia of Indus- trial Chemical Analysis Volume 5 pages 460-633 published by John Wiley & Sons, N.Y. 1967.


1 .

? -.

3 . 4 . 5 . 6 . 7.

8 9

I0

GRISEOFULVIN

Ecl~1at-d R . Tou1nley

Description I . I I .2 Appearance, Color. Odor I . 3 Compendia1 References. Other Physical Properties

Name, Formula. Molecular Weight

2.01 2.02 2.03 2.04 2.05 2.06 2.07 2.08 2.09 2.10 2.11 2.12 2.13 2. I4

Circular Dichroisni Nuclear Magnetic Resonance Mass Spectrum Ultraviolet Spectrum Infrared Spectrum X-Ray Diffraction Fluorescence and Luminescence Photolysis Optical Rotation Melting Range Differential Scanning Calorimetry Thermogravimetry Electrophoretic Properties 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

Copyright 0 1979 by Academic Press. Inc. All rigtits of reproduction in any form reserved.

ISBN 0-12-260808-9 219

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 thylcyc lohex-2 '-en-4 '-one)

Generic Names Griseof ulvin

Trade Names Fulcin; Fulvicin UfF, Fulvicin PIG, Grifulvin; Gris-

actin; Grisovin, Gris-PEG, Grysio, Lamoryl, Likuden, Neo- Fulcin, Poncyl; Spirofulvin; Sporostatin.

Formula and Molecular Weight

OCH

CH 0 3

c 1 CH 3

Molecular Weight 352.77 '1 7H1 7'1°6

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

der.

1.3 Cornpendial References, Other Griseof ulvin 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.

GRISEOFULVIN 22 I

2 . Phys i ca l P r o p e r t i e s Phys ica l measurements by Schering Corporat ion are provid-

ed f o r Batch Number UGFP-1961. This ba t ch is chromatographi- c a l l y pure wi th t h e except ion of 1.1% dech lo rogr i seo fu lv in .

2.01 C i r c u l a r Dichroism Spect ra The c i r c u l a r dichroism s p e c t r a (F igure 1) w a s ob-

t a ined on a 0.0155 mg/ml s o l u t i o n i n methanol wi th a Cary Model 61 C i r c u l a r Dichroism Spectrophotometer. The fo l lowing molar e l l i p t i c i t y va lues were obta ined:

Table I

Wavelength nm

345 shoulder 326 shoulder 314 shoulder 294 peak 236 peak 218 peak

Molar E l l i p t i c i t y

P I

+ 7,520 + 19,100 + 23,200 + 43,700 +110,000 - 97,500

2.02 Nuclear Magnetic Resonance Spec t ra The pro ton NMR spectrum of g r i s e o f u l v i n (F igu re 2)

w a s ob ta ined i n DMSO-d s o l u t i o n (conc. w/v = 10 mg/0.40 m l ) conta in ing TMS as k t e r n a l r e fe rence u t i l i z i n g t h e Varian Assoc ia tes CFT-20 Spectrometer ope ra t ing a t a frequency of 79.5 MHz. The chemical s h i f t s ( 6 , ppm) are wi th r e f e r e n c e t o TMS. The experimental cond i t ions a re :

Sweep width = 800 Hz Pulse width = 6.0 1.1 sec ( 25'tip) Acqu i s i t i on t i m e = 5.1 s e c Data t a b l e = 8 K

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

1 li 200 250 300 350 400

WAVELENGTH [nm]

FIGURE 1 : Circular Dichroism Spectra of Griseofulvin


T a b l e I1

Pro ton Chemical S h i f t s ( 6 , ppm)

6 '-CH3 0.82 d ( Jx6 .5 Hz) (3H)

21 5'-CH 6 '-CH

2.3-2.9 m (3H)

2'-OCH3 3.63 8 (3H)

4-0CH3 } Aromatic 6-OCH3

3 '-H 5 -€I

3.96 8

4.04 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 spec t rum of g r i s e o f u l v i n

6 ( F i g u r e 3 ) w a s o b t a i n e d a t ambient t e m p e r a t u r e i n DMSO-d c o n t a i n i n g TMS as 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 Var i an Associ- a tes XL-100-15 s p e c t r o m e t e r equipped w i t h F o u r i e r accesso - ries. The sys t em was locked 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 equency of 25.2 MHz f o r carbon-13. The chemical 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 wid th = 5500 Hz P u l s e wid th A c q u i s i t i o n t i m e = 1.6 sec A c q u i s i t i o n d e l a y = 0.20 sec

- 15 P sec (66' t i p )

Tab le 111

6,) Carbon Chemical S h i f t (6,) Carbon Chemical S h i f t (

1' 2' 3' 4' 5' 6' 6' CH

2' OCH3 4' ocd,

90.11 170.22 104.60 195.45

39.49 35.52 13.77 57.54 56.97

6 OCH3 3 3a 4 5 6 7 7a

56.52 191.12

157.59 91.27

104 04

164.44 95.24

168.57

I A

m I I I 1 I I I I I I , I I I I I 1 I I I I I I # I

1 1 I I I I ~

% *, ,

m

I I I I I I I 1 , 1 I " ' I ' I I I I I , I I I I

FIGURE 3: Carbon 13, Nuclear Magnetic Resonance Spectra of Griseofulvin i n DMSO d6


The spectrum is in substantial agreement with the data reported by Wenkert et. al, (6). However, on the basis the proton coupled carbon 13 NMR spectra, the assignments for 6 and 7ci are reversed [Brambilla (16)] from those previously reported. The new assignments are based on long range H-C-0-C couplings.

2.03 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) Ions Losses

35 2 337 32 1 310 284

O C H l+

CH3 CH30

CH CO Me6=CHCO

215

138 I+

-t k i-

U

al a

vl

[I) [I)

9 .. U 8 s F


123

I +

2.04 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 anhy-

drous methanol s o l u t i o n a t 25 C gave t h e fol lowing absorp- t i v i t y va lues

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 igu re 5.

2.05 I n f r a r e d Spectrum The in f r a red spectrum of g r i s e o f u l v i n , ob ta ined as a

mineral o i l mul l , was run on a Perkin-Elmer Model 180 g r a t i n g I R spectrophotometer. given i n Table V.

Important absorp t ion assignments are The spectrum i s g iven i n F igure 6.

Table V

Assignment -1 Wavenumber (cm 1

1703 ( s ) C=O s t r e t c h ; benzofuranone r i n g

1658 (s ) C-0 s t r e t c h ; cyclohexenone carbonyl carbonyl

1615, 1597, 1580 ( s ) C=C s t r e t c h , aromatic and c y c l i c unsa tu ra t ion

1501 (m) C=C s t r e t c h , aromatic 1220, 1210 ( 8 ) C-0 s t r e t c h , a r y l methoxyl

I n t e n s i t y

s - s t rong m - medium

2.06 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

obtained on a P h i l l i p s ADP-3500 X-ray Dif f rac tometer us ing Cu K, r a d i a t i o n (1.5405A0) and N i f i l t e r . Table VI.

The d a t a is given i n

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 Gr i seo fu lv in

Table V I

20 d(Ao)* I/I'**

4 009 4.117

10.679

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

32.624 32.674 34.852

35.896 36.142

36.290 37.077 37.270 38.500

38.659

13.123

31.385

34.914

36 202

38.569

22.038 21.460

6.746 6.397 6.110

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

8.284

5.398

2.850 2.745 2.741

2.570 2.502 2.485

2.475

2.413 2.338 2.334 2.329

2.574

2.481

2.425

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 1 2 11 18 14 15 14 14 13 18 18 19

*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 **Ill' = r e l a t i v e i n t e n s i t y (based on the h ighes t

i n t e n s i t y of 100)


WAVELENGTH [nm]

FIGURE 5: U l t r a v i o l e t Spectrum of Gr i seo fu lv in Obtained i n Anhydrous Methanol Solvent

WAVELENGTH MICRONS 2.5 3 4 5 6 7 8 9 10 12 14 18 22 3550

4600 3500 3000 2500 2000 1700 1400 1100 800 500 205 FREQUENCY (C M’)

FIGURE 6: Infrared Spectrum of Griseofulvin Obtained as a Mineral O i l Mull.


2.07 Fluorescence and Luminescence Griseof u lv in e x h i b i t s both f luorescence and lumines-

cence. A r e p o r t by Neely e t al., ( 7 ) g ives co r rec t ed f luorescence e x c i t a t i o n (max. 295 nm) and emission (max. 420 nm) spec t r a , va lues f o r quantum e f f i c i e n c y of f luorescence (0.108) ca l cu la t ed f luorescence l i f e t i m e (0.663 nsec) and phosphorescence decay t i m e (0.11 sec.) . The f luorescence e x c i t a t i o n and emission spec t r a a r e g iven i n F igure 7.

2.08 Photo lys i s There i s no change i n the t h i n l a y e r chromatogram

( s i n g l e spo t ) o r i n the f luorescence o r u l t r a v i o l e t s p e c t r a a f t e r i r r a d i a t i o n i n methanol with a xenon lamp f o r 20 hours. It is the re fo re concluded t h a t t h e r e is no s i g n i f i c a n t photodegradat ion of g r i s e o f u l v i n under reasonable condi t ions of l i g h t exposure (7 ) .

2.09 O p t i c a l Rota t ion Gr iseofu lv in e x h i b i t s t he fol lowing o p t i c a l r o t a t i o n

when d isso lved i n these so lvents .

Table V I I

Sa tura ted chloroform [a] k7'= +370 4

Acetone

Dimethylformamide [a]:60= +358 17

Dioxane [a]:60= +302 1 7

2.10 Melt ing Range The D i f f e r e n t i a l Thermal Gravimetry curve (F igure 9 )

demonstrates t h a t t he g r i s e o f u l v i n mel t ing poin t t akes p l ace with decomposition. The melt ing range of g r i s e o f u l v i n from seve ra l sources i s given i n Table VIII.

Table V I I I

Me1 t ing Range

C 0 - Reference

218 t o 224 2 20 218 217-224

3 4

17 51

GRISEOFULVIN 233

8C

7c

60

50 > I-

w k z

- 40

- 30

20

10

0 200 300 400 500

WAVELENGTH

FIGURE 7: Corrected Fluorescence and Emission Spectra of Griseofulvin: la, Excitation Spectrum with Emis- sion at 420 nm; lb , Emission Spectrum with Excita- tion at 295 nm.


2.11 D i f f e r e n t i a l Scanning Calor imetry F igure 8 shows t h e DSC thermogram of g r i s e o f u l v i n

obtained wi th a DuPont Model 900 Thermal Analyzer. A s i n g l e sharp mel t ing endokherm occurs f o r t h i s subs tance wi th onse t temperature a t 216 C.

2.12 Thermogravimetry F igure 9 shows t h e TG thermogram of g r i s e o f u l v i n

obtained wi th 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 subl imat ion.

2.13 E lec t rophore t i c P r o p e r t i e s Zeta p o t e n t i a l s of d i spersed g r i s e o f u l v i n have been

s tud ied both a lone , and i n t h e presence of su r face -ac t ive agents , t h e l a t te r a t a con t ro l l ed pH (8 ) . A Mobi l i ty /Zeta 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 charge a t pH 1 .5 which r a p i d l y decreases t o zero a t pH 2 . 4 . There i s then r e v e r s a l of charge followed by an inc rease over t he pH range 2 . 4 t o 7 . 0 . The z e t a p o t e n t i a l a t t he l a t t e r pH i s -45 mV. range 7 t o 10.

The p o t e n t i a l then s t a y s cons t an t over t he pH

2 .14 S o l u b i l i t y The fol lgwing d a t a are g iven f o r t he 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 ; acetone 30 g/L, carbon t e t r a c h l o r i d e 2 g/L, d ich loroe thane 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, heptane 0.3 g/L, methanol 0 . 4 g/L; minera l o i l (0.1 g/L; propylene g lyco l 2 g/L; Span 80 0.2 g/L, Tween 80, 7 g/L water 0 . 2 g/L ( 1 7 ) .

3. Product ion and Syn thes i s

t i o n wi th Pen ic i l l i um griseofulvum and r e l a t e d s t r a i n s of P e n i c i l l i a . The b iosyn thes i s has been t h e sub jec 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 latest of which is g iven by Harris, e t . a l . (9) F igure 10. Other proposed biosynthe- t i c pathways are discussed.

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 labora-

Gr i seo fu lv in was f i r s t i s o l a t e d i n 1938 by Oxford ( 1 0 ) et. a l . , (1939); i t s t o t a l s y n t h e s i s was accomplished i n 1960 and fol lowing yea r s i n s e v e r a l l a b o r a t o r i e s (Bross i e t a l . , 1960 (11 ) Grove, 1963; (12 ) Mutant s t r a ins of P. patulum are used f o r t h e commercial p roduct ion of t h e a n t i b i o t i c by f e r - mentat ion (9) .

4. Impur i t i e s

Holbrook, Bai ley and Bai ley (13) and repeated i n a d e s c r i p t i o n Some fermenter b ro th impur i t i e s have been l i s t e d by

FIGURE 8: Differential Scanning Calorimetry Curve of Griseofulvin

FIGURE 9:

20

\

200 250 3 0 0 3 5 0 400 4 5 0 5 0 0 0 5 0 100 150

T. 'C (CORRECTED FOR CHROMEL ALUMEL THERMOCOUPLES)

Thermogravimetry Curve of Griseofulvin

GRISEOFULVIN 237

0 0 0 0 0 0 0

OH acetate -

- - CHJO OH H,C

a I CHJ

a I CHJ

FIGURE 10: A Biosynthetic Route f o r Griseof ulvin.


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 Bai ley and B r i t t a i n (14 ) and i n a gas chromatographic separa- t i o n system by Margosis ( 1 5 ) . S t r u c t u r e s are i n F igu re 11.

The common impuri ty found i n commercial ba tches of g r i s e o f u l - v i n is dech lo rogr i seo fu lv in (14 ,15 ) which appears t o be i n the range of 0.5 t o 3 .5%.

5 . S t a b i l i t y Gr i seo fu lv in is a s t a b l e drug substance. After 12 y e a r s

s to rage a t room temperature no decomposition was de tec t ed by d i f f e r e n t i a t i n g LC methods (16). There is no photodegradat ion under reasonable condi t ions of l i g h t exposure ( 7 ) . Griseofu l - v i n is converted t o g r i s e o f u l v i c ac id under a c i d i c cond i t ions .

6. Drug Metabolic Products

g r i s e o f u l v i n and i ts g lucuronide ( 1 7 , l B ) which account f o r about 65% of the in t ravenous dose (19 ) and 35 t o 65% of the o r a l dose ( 2 0 , 2 1 ) . The 6-demethylgr iseofulvin is a l s o t h e major me tabo l i t e i n dogs (22 ) and r a b b i t s (23 ) whi le both 4- demethylgr iseofulvin and 6-demethylgr iseofulvin are major metabol i tes i n rats (24 ) and mice ( 2 5 ) . These me tabo l i t e s can be determined by gas l i q u i d chromatography v i a isopro- poxyl d e r i v a t i v e s (18 ) 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 has been measured i n u r ine by h igh performance l i q u i d chromatography (28 ) and u l t r a v i o l e t spectrophotometry ( 1 9 ) . Only trace amounts of g r i s e o f u l v i n are found i n the u r i n e ( 2 8 ) .

The major human me tabo l i t e of g r i s e o f u l v i n is 6-demethyl-


7.01 I d e n t i f i c a t i o n A wine red co lo r is produced when about 5 mg of

g r i s e o f u l v i n are d isso lved i n 1 m l of s u l f u r i c ac id wi th about 5 mg of powdered potassium dichromate ( 2 9 ) .

7 .02 Elemental Analysis Analysis of g r i s e o f u l v i n , w a s determined f o r carbon,

hydrogen, and ch lor ine . The carbon, and hydrogen a n a l y s i s w a s performed on a Perkin.Elmer Model 240 inst rument . Analysis f o r ch lo r ine w a s performed by combustion of t he sample and coulometr ic t i t r a t i o n us ing a n American Instrument Co. Chlor ide T i t r a t o r .

The r e s u l t s from t h e elemental a n a l y s i s are l i s t e d i n Table I X .

GRISEOFULVIN 239

C H 0 3

0 I1 C

0

0 0 CH 3

Dechlorgr i seo f ulvin - - 0

cii 3 0 @i0 c1 C I I 3

Dihydrogr iseofulvin = c

- 0 \

C H O c 1 CH 3

Dehydrogriseofulvin

CH 30

C H 3 0

C 1 C t I 3

Griseofulvic Acid

c 1 C H 3

Tetrahydrogriseofulvin

O C R 3

c1

Isogriseofulvin

FIGURE 11: Impurities Found i n the Fermenter Broth


Table I X

Elemental Analysis of Gr i seo fu lv in : Batch UGFP-1961

Element % Theory % Found

C H c1

57.88 4.86

10.05

57.97 4.84 9.92

7.03 Spectrophotometr ic Analysis Quan t i t a t ive 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 be performed by comparison to a Reference Standard. The u l t r a v i o l e t absorbance i s descr ibed i n Sec t ion 2.04 and Figure 5.

7.04 Spec t rof luorometr ic 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 ighly s e n s i t i v e ana lyses i n blood and serum (30-33) s k i n and sweat (34) . Riegelman (32) has combined TLC sepa ra t ion wi th a f l u o r i m e t r i c dens i tometer readout t o g ive a h ighly 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 de t e r - mination i n plasma. Other ana lyses are commonly performed i n e i t h e r 1% aqueous e thano l (30) , a c t i v a t i o n maxima 295 and 335 nm, f luorescence maxima a t 450 nm o r anhydrous methanol (31) a c t i v a t i o n maxima unchanged a t 295 and 335 nm, f luo rescence maxima 420 nm. Values are uncorrected. Other a p p l i c a t i o n s t o the a n a l y s i s of bu lk drugs , 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 - b le .

7.05 Color imet r ic Analys is A co lo r ime t r i c assay of g r i s e o f u l v i n , based on t he

yellow-orange co lo r (Xmax=420 nm) which develops when gr i seo- f u l v i n is heated with i s o n i c o t i n i c ac id hydraz ide i n a l k a l i n e medium has been descr ibed by Unterman (35) and t h e mechanism inves t iga t ed by Unterman and Duca (36).

7.06 Iodometr ic Analys is Iodometric a n a l y s i s has been appl ied t o the de 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 he manufacturing process (37). The mycelium is ex t r ac t ed wi th 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 so lu t ion . The 0.01N iod ine s o l u t i o n is s tandard ized wi th g r i s e o f u l v i c ac id .

7.07 Turbid imet r ic Analys is A t u r b i d i m e t r i c assay 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 has been repor ted (38). The drug i s d i s so lved i n

GRISEOFULVIN 24 1

e thylene g l y c o l monomethyl e t h e r (niethyl c e l l o s o l v e ) . Poly- mer iza t ion is induced wi th g l y c e r o l and guanosine-5'-triphos- phate (GTP).

7.08 Polarographic Analys is A s tudy d i r e c t e d toward a comparison of t h e reduc-

t 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 ana logs sugges 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 (39) . In e t h a n o l i c s o l u t i o n wi th 0 . 2 M K C 1 suppor t ing elec- t r o l y t e , g r i s e o f u l v i n shows two polarographic waves with 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. This system has been appl ied wi th good r e s u l t s t o the de te rmina t ion of f i n i s h - ed products i nc lud ing t a b l e t s . The accuracy, p r e c i s i o n and s e l e c t i v i t y of t he method w a s compared with t h e iodometr ic method (40) . 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 ac id do no t in- t e r f e r e . (41,42)

7.09 Chromatographic Analyses

7.091 P a r t i t i o n Column Chromatography Gr i seo fu lv in maybe separa ted from observed

s t r u c t u r a l l y s imilar i m p u r i t i e s i n the fermenter b r o t h by means of p a r t i t i o n column chromatography (13) . A Celite column packing and so lven t system c o n s i s t i n g of methano1:water: hexane:chloroform (8:2:9:1) was used. Te t r ahydrogr i seo fu lv in , d ihydrogr i seo fu lv in , i s o g r i s e o f u l v i n , dech lo rogr i seo fu lv in are sepa ra t ed from g r i seo fu lv in . The s t r u c t u r e s f o r t hese compounds have been g iven i n Sec t ion 5.

7.092 Pape r Chromatography A paper chromatography system i s g iven i n

Table X. (43)

Table X

Solvent System Pape r

Benzene : Cyclohexane; Whatman Methanol :Water No. 1 (5:5:6:4) Glacial a c e t i c a c i d , 0.5%, w a s added t o the organic phase of t he so lven t a f t e r e q u i l i b r a t i o n .

Detec t ion Reference

uv 43

7.093 Thin Layer Chromatography Thin l a y e r chromatographic systems are g iven

i n Table XI. The d e t e c t i o n method w a s U.V.


Table XI

Solvent System Adsorbent

Methanol : n-bu tanol ; 95% ethano1:conc. ammonium hydroxide

Silica Gel

(4:1:2:1, by ~01.)

Ch1oroform:isopropanol Silica Gel (3:1, by vole)

n-butano1:formic acid: Silica Gel water (77:10:13 by vol.)

n-Butanol:95% ethanol Silica Gel conc. ammonium hydroxide:water (4:1:2:1 by vole)

- Tr Reference

0.64 44

0.86

0.86

0.64

44

44

44

Ch1oroform:acetone Silica Gel 0.65 45 (93:7 by vole)

Ch1oroform:methanol Silica Gel 0.82 46 (lot1 by vole)

Ch1oroform:acetic acid Silica Gel - diethyl ether (17:1:3)

47

Methanol :benzene Silica Gel 0.50 48 (2:98 by vol.)

Ethyl acetate Silica Gel 0.50 17

7.094 Gas Chromatopraphy Successful griseofulvin analyses by gas chro-

matography 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. Separa- tion from the related compounds; dehydrogriseofulvin, isogriseofulvin and dechlorogriseofulvin was demonstrated. subsequent collaborative study (50) the accuracy and precision of the method was established. Although griseofulvin is ther- mally stable, the high GLC temperatures require precautions similar to those taken for steroids when preparing columns, column supports and associated equipment (45).

In a

GRISEOFULVIN 243

The s p e c i f i c and s e n s i t i v e GLC de te rmina t ion 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 wi th e l e c t r o n cap tu re d e t e c t i o n has 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 cond i t ions are g iven in Table XII.

Table XI1

Column Carrier Column I n t e r n a l

Gas Temp. Standard Reference

230' diphenyl- 45,49 p h t h a l a t e

N2 150 cm x 4 mm I.D.; U- shaped s t a i n l e s s steel tub ing , 1.5% QF-1 on Anakrom ABS

3 f t x 4 mm I.D.; c o i l e d g l a s s tub ing , 3% OV-101 on Gas dienone Chrom Q

5 f t x 4 mm I.D.; g l a s s 10% Methane 27OoC diazepam 34,50, column, 3% OV-17 on 90% Argon 51,52 Chromosorb W. o r Gas o r Chrom Q.

3 f t x 4 mm I.D.; g l a s s co i l ed . % OV-17 on Gas Chrom. Q dienone

245' t e t raphenyl - 15 cyclopenta-

He2

N2

He 2 225' t e t raphenyl - 15 cyc lopen t a-

7.095 High Performance Liquid Chromatography Gr i seo fu lv in can be r e a d i l y chromatographed on

e i t h e r normal o r r eve r se phase columns. Separa t ion of dech lo rogr i seo fu lv in , t h e most common s y n t h e t i c impuri ty is accomplished on C / 1 8 , CN (17) o r ETH columns. (15) I sogr i seo- f u l v i n , i s separa ted on e i t h e r CN (17) o r ETH columns (15).

Liquid chromatography cond i t ions are g iven i n Table XIII.

244 EDWARD R. T O W N L E Y

Table XI11

Column

PBondapak C/18 (oc ty l - decyl chemical ly bonded t o s i l i c a )

mondapak C/18 (oc ty l - decyl chemical ly bonded t o s i l i ca )

Zorbax CN (cyano- propyl chemical ly bonded t o s i l i c a )

Permaphase ETH (C-7 e t h e r chemical ly bonded t o p e l l i c u l a r 30-50 mesh g l a s s packing)

Mobile I n t e r n a l Refer- Phase Standard ence

Reverse Methanol: n-butyl 16 phase water 3:2 p-hydroxy

benzoate

I I 45% aceto- diazepan 53

n i t r i l e i n 45mM KH PO4 pn = 3.6

11 Methanol: m-phenyl 16 water 3:2 phenol

Normal 5% chloro- phase form i n

hexane

14

7.10 B io log ica l Methods o r Analys is Microbio logica l procedures have been developed f o r

assay of g r i s e o f u l v i n and appl ied t o the a n a l y s i s of bu lk drugs and dosage forms (1) . The c y l i n d e r p l a t e aga r d i f f u s i o n method is t h e o f f i c i a l microbio logica l method of de te rmina t ion (55) . Microsporum gypseum (ATCC 14683) i s t h e test organism.

8. I d e n t i f i c a t i o n and Determinat ion i n Body F lu ids and T i s sue Gr i seo fu lv in has usua l ly 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 gas chromatographic methods (45,46,48). More r ecen t ly g r i s e o f u l v i n has been determined i n plasma by high performance l i q u i d chromatography (53,541.

9. Analys is of Dosage forms Usual dosage forms of g r i s e o f u l v i n are capsu le s , t a b l e t s

and boluses . l i q u i d s o l i d e x t r a c t i o n of drug substance. Margosis (15,50) used chloroform as an e x t r a c t i n g s o l v e n t wi th g e n t l e hea t . A compendia procedure d e s c r i b e s the 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 wi th b o i l i n g a lcohol . The a n a l y s i s f o r e x t r a c t e d drug subs tance has been performed by s e v e r a l methods. 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 ,55) . Polarogra- phy u t i l i z i n g t h e system given i n Sec t ion 7.08 has been used (39). Thin-layer, gas and l i q u i d chromagraphy may a l s o be

These may be prepared f o r a n a l y s i s by s imple

Most

G R I S EO F U LV I N 245

used u t i l i z i n g systems descr ibed i n Sec t ions 7.093, 7.094, and 7.095 r e spec t ive ly . These l a t te r methods are va luab le because of t h e i r s p e c i f i c i t y .

I n the United States , g r i s e o f u l v i n drug subs tance and dosage forms must conform t o the r egu la t ions of t h e Federa l Food and Drug Adminis t ra t ion concerning a n t i b i o t i c drugs (55, 5 6 ) . Microbio logica l assay r e s u l t s obtained by a n a l y t i c a l methods descr ibed i n these compendia a r e conclus ive .

10. Acknowledgements

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 the va luab le a s s i s t a n c e of members of t h e Schering Corporat ion, Phys ica l Organic Research Sec t ion : D r . R. Brambil la , M r . P. Bar tne r , M r . C. Eckhart and M r . R. Foes te r f o r t he 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 phys ica l d a t a , and t o the Schering 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 he l i t e r a t u r e searches and Schering General Of f i ce S t a f f f o r t h e c a r e f u l typ ing of t h i s monograph.

The au thor wishes t o thank D r . M. D. Yudis and


1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14

15.

16.

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British Pharmacopeia Commission (1973) British Pharmacopeia, London, HMSO, p. 221.

Council of Europe (1971) Europeon Pharmacopoeia, Europeon Treaty Series, 50, 2, Paris, Maisonnmeuve, p. 234.

Stecher, P.G., ed (1976). The Merck Index, 9th ed., Rahway, N.J., Merck & CO., 4392.

International Agency for Research on Cancer, 10, 153 (1976). WHO; Geneva, Switzerland.

S.G. Levine, R.E. Hicks, H.E. Gottlieb and E. Wenkert, J. Org. Chem., 40, 2540 (1975).

W.C. Neely and J.R. McDuffie, J. Assoc. Off. Anal. Chem., - 55, 1300 (1972).

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GRISEOFULVIN 247

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P.A. Harris and S . Riegelman, J. Pharm. Sci., 58, 93, 1969

S. Symchowicz, M. S . Staub and K. K. Wong, Biochem. Pharmacol., l6, 2405 (1967).

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C. Lin, R. Chang, J. Magat and S . Symchowicz, J. Pharm. and Pharmacol., 24, 9 1 1 (1972).

A. Zlatkis, "Advances in Gas Cromatography 1967," Pro- ceedings of the Fourth International Symposium held in New York, N.Y., April 3-6, 1967. Preston Technical Abstracts Co., Evanston, Ill. 1967, p. 129.

P. Kabasakalian, M. Katz, B. Rosenkrantz and E. Townley, J. Pharm. Sci., 59, 595 (1970).

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GRISEOFULVIN 249

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HALCINONIDE

Joel Kirschbaum

I . History, Description, Precautions, Synthesis 1 . 1 History I . 2 I . 3 Appearance, Color. Odor I .4 Precautions I .5 Synthesis

2. 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

3. Solution Properties 3 . 1 Intrinsic Dissolution Rate 3 . 2 3 .3 Partition Coefficients

4. I 4.2 4.3 Chromatographic Analyses

Solubilities in Aqueous and Nonaqueous Solvents

4. Methods of Analysis Elemental and Inorganic Analyses Identification, Ultraviolet and Colorimetric Analyses

4.3 I 4.32 Thin-Layer Chromatography (TLC) 4.33 Column Chromatography 4.34 Paper Chromatography

Halcinonide in Tissues and Body Fluids

High Pressure Liquid Chromatography (HPLC)

4.4 Polarographic Analysis 4.5

5. Stability 6. Acknowledgments 7. References

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

ISBN 0-12-260808-9 25 I

252 JOEL KIRSCHBAUM

1 History, Description, Precautions and Synthesis

1.1 History

192 Halcinonide is a topical antiinflammatory agent. Introduction of a 9a-f luoro group3 to the hydrocortisone molecule resulted in an eight-fold increase in antiinflammatory activity. Such other groups as 16a-hydroxy, 16a-methyl, 168- methyl, 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 . Halcino- nide 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. has systemic3 as well as topical activity.

E

Halcinonide

1.2 Names, Formula and Molecular Weight

Halcinonide is the United States adopted name5 (USAN) . The preferred chemical names6 are 21-chloro-9-f luoro-11B- hydroxy-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-isopropyl- idenedioxy-4-pregnene-3,2O-dione, 9a-fluoro-21-chloro-llf3, 16a, 17a-trihydroxypregn-4-ene-3, 20-dione 16,17 acetonide and 21- chloro-9-fluoro-118,16~,l7-trihydroxypregn-4-ene-3,2O-dione cyclic 16,17-acetal with acetone.

21 CHZCI I

in 12 I* I I7 I

14 2

4 6 W

0 .O >tt(,,

24

2 qH 3 2 5 454.97 Daltons

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 un- wanted 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~mmercially.l~-~~ This tetrahydroxy steroid is slurried in acetone, and then 70% perchloric acid is added slowly. The acetonide, I1 (9a-fluoro- 11~,16a,l7,21-tetrahydroxypregn-4-ene-3,2O-dioney cyclic 16,17- acetal with acetone; dihydro t r iamcinolone-ace tonide) precipi- tates 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.


2.01 Single Crystal X-Ray Diffraction

14 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. tion 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 aniso- tropic temperature factors, in the final stages, gave an R factor of 0.11.

The posi-

rcl

0

m

w

w

w

>

w

w

w

254

255

256

2.02 Mass Spec t romet ry

JOEL KIRSCHBAUM

F i g u r e . 3 is t h e p l o t t e d low r e s o l u t i o n mass spectrum 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 Appara- t 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 equency modulated a n a l o g t a p e and p rocessed on a D i - g i t a l Equipment Corp. , 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 prominent 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. 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 peak because 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 > . 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.

Each fragment i o n c o n t a i n -

These peaks are two

Fragmen ta t ion p roceeds a l o n g s e v e r a l pathways, as shown below.

439

419 396?)m/z 361

377

m/z 264 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 bu t o n l y t h e o r i g i n of t h e f r agmen t .

The weak i o n s a t m/z 121, 122 and 123 (CgHgO, C H 0 and C H 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 10 8 11

> I-

v) Z W I- Z

- H

W > I- U

a W w

100

90

80

70

60

50

40

30

20

10

0

Figure 3

Low Resolution Mass Spectrum of Halcinonide. See text for details.

5433 S Q 1 8 , 5 6 6 BFI NN009NFl 175 DEG

MF17080 2 5 - S E P - 7 8

r u 3 u l ~ 8 c u f O a 3 8 c u f f . G ~ 8 c u 3 f . G - - - + cu cu cu (U cu 0 rn Q c1-l

MFISSICHRRGE INTENSITY SUM =65775 BQSE PERK % =6.46

G z 0

I-

N

z 0

H

a U

H

-J

I- 0 t-

t- z w 0

w a

a

Found Mass

454.1925 439.1680 419.2252 396.1501 3 77.2147 361.1838 319.1665 278.1695 264.1524 246.1422 135.0808 123.0769 122.0729 121.0683

76.9790 43.0184

High-Resolution Massa Spectrum of Halcinonide

Calc. Mass Unsat. b O / E C c - H - 0

454.1922 8.0 0 24 32 5 439.1687 8.5 E 23 29 5 419.2233 8 . 5 E 24 32 5 396.1503 8.0 0 2 1 26 4 37 7.2128 7 .5 E 22 30 4 361.1815 8 .5 E 2 1 26 4 319.1709 7.5 E 1 9 24 3 278.1682 6 .0 0 17 23 2 264.1525 6.0 0 1 6 2 1 2 246.1420 7.0 0 1 6 1 9 1 135.0810 4.5 E 9 11 1 123.0810 3 . 5 E 8 11 1 122.0732 4.0 0 8 10 1 121.0653 4.5 E 8 9 1

76.9794 1 . 5 E 2 2 1 43.0184 1 . 5 E 2 3 1

F

1 1 1 1 1 1 1 1 1 1 0 0 0 0 0 0

- c1 1 1 0 1 0 0 0 0 0 0 0 0 0 0 1 0

-

a Only those peaks considered t o be s i g n i f i c a n t t o t h e d i scuss ion are l i s t e d . A complete element map can be obtained on r eques t .

bNumber of double bonds and r i n g s .

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

HALCINONIDE 259

2.03 Nuclear Magnetic Resonance Spectrometry (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

Proton at Carbon

c-4

c-11

C-16

C-18

c-19

c-21

8-Acetonide, methyl

a-Acetonide, methyl

Chemical Shift

5.78

4.58

5.05

0.87

1.51

4.19 4.57

1.15

1.44

Peak Appearance S = singlet D = doublet B = broad

S

B

D J15,16= 4 .0 Hz

S

S

AB quartet

These data agree with the published results of the 21- hydroxy-A' analog, triamcinolone acetonide. l7

w 0

k

ra a,

HALClNONlDE

2.032 13C-NMR

26 1

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 vari- o 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 rever- sals of 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

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Chemical Shift C-F Coupling 6 (ppm>a Constant (Hz)

26.2 (28.5) 33.7 (33.2) 199.1 (202.1) 124.7 169.0 30.7 28.5 (26.2) 33.1 99.0 43.7 70.2 37.3 44.8 43.4 33.2 (33.7) 81.9 98.1 17.0 21.9

47.1 111.5 28.6 (25.1) 25.1 (28.6)

202.1 (199.1)

3

3 19

175 21

2

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

a, 4

rJ 1 z I

V

m

4

3 z

HALCINONIDE 263

2 . 0 4 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 spectrum of h a l c i n o n i d e r u n as a potassium 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 spec t rometer . 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 absorbances. l9

1 Frequency (cm )

3450

2950

17 3 0

1 6 6 0

1 6 2 0

I n t e r p r e t a t i o n

-OH S t r e t c h

-OH S t r e t c h

C z 0 Keto

C 3 Keto

A 4 C=C

2 .05 X-ray Powder D i f f r a c t i o n

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 ob ta ined on a P h i l i p s gowder 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. Using a 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 recorded from approximate1 2 t o 40 degrees ( 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) 1 ' d ' (Angstrom)

2 Relative Area

1 4 . 8 0 5 . 9 9 1.000 12.53 7 .06 0 .866 18 .49 4.80 0 . 7 3 8 17 .94 4 . 9 4 0.526 19.82 4 . 4 8 0 .422 2 9 . 3 1 3 . 0 5 0.266 18.10 4.90 0 .246 11.67 7 . 5 8 0 .225 32 .14 2.78 0 . 1 9 5 39.59 2.28 0 . 1 7 1 25.86 3.45 0 . 1 6 5 21.47 4 . 1 4 0 . 1 4 2 26.80 3.33 0 .135 25.24 3.53 0.135 15.67 5.66 0 . 1 3 1 40.37 2.23 0.124 22.33 3.98 0 .117 33 .08 2 .71 0 .114 25.47 3.50 0 . 0 5 3 34 .02 2.64 0 . 0 5 2

' I n t e r p l a n a r d i s t a n c e

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 us ing CuKa r a d i a t i o n .

Figure 6 I n f r a r e d Spectrum of Halcinonide, Potassium Bromide P e l l e t .

See t e x t €o r d e t a i l s .

WAVELENGTH (MICRONS) 8 9 10 12 15 20 30 4050 2 5 3 4 5 6 7

4ooo 3500 3ooo 2500 2ooo 1800 1600 1400 1200 lo00 800 600 400 FREQUENCY (CM’)

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266 JOEL KIRSCHBAUM

2.06 U l t r a v i o l e t Spec t romet ry

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 eco rded on a Beckman Acta C I I I spec t ropho tomete r . The s p e c t r a i n methanol , 0.M methano 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 hydrox ide show peak maxima a t 238 nm, and remain unchanged a f t e r 24 hour s . l a t e d below u s i n g t r a d i t i o n a l E l %

Absorbances are tabu- n o t a t i o n .

1 cm

1% Elcm

So l v e n t I n i t i a l 3 Hours 24 Hours

Me thano 1 379 374 373

0 .M Methano l i c 370 372 370

Hydroch lo r i c Acid

0 . IM Methano l i c 380 382 380

Sodium Hydroxide

238 nm and a p p e a r s s t a b l e f o r a t least 3 hour 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 .

The spec t rum i n 95% e t h a n o l a l s o h a s a peak maximum a t

2.07 O p t i c a l R o t a t i o n

The s p e c i f i c r o t a t i o n s i n were de te rmined23 t o be as f o l l o w s ,

Wavelength (nm) S p e c i f i c R o t a t i o n

589

578

546

436

305

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

+156

+164

+188

+348

+436

Using a Perkin-Elme54Model 204 f l u o r e s c e n c e spec t ropho tomete 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 methanol . 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 hydrox ide .

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.


A duPont Model 900 Differential Thermal Ana- lyzer 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 Thermogravi- metric 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 Microscopy and Crystal Type

Microscopically, the crystal type depends on the method of preparation of halcinonide. Slabs, either 2 to 4, 5 to 10, or 10 to 15 microns square, were found in three lots of halcinonide, intermingled with needle-like (acicular) crystals 5 to 10 microns 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 A 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 of 3"/minute. began at 268.2", and by 271.4" all crystals were melted. yellow melt slowly turned orange-brown. recrystallization at ambient temperature.

Melting The

Cooling showed no

268

2.13 P a r t i c l e Size

JOEL KIRSCHBAUM

By l i g h t s c a t t e r i n g us ing a Royco ins t rument , a l l p a r t i c l e s are below 20 microns.27 a l y s i s of ground ha lc inonide 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 = micron) , 98.2% <6.4p, 95.3% <5.1p, 52.0% <2.6p and 19.9% <1.6p.

Coul te r Counter an-

2.14 Surface Area

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

2.15 Polymorphism

There is no evidence €o r polymorphism from i n f r a r e d spectroscopy and d i f f e r e n t i a l thermal a n a l y s i s , and only inconclus ive d a t a from x-ray d i f f r a c t i o n and microscopy s t u d i e s .

2.16 Hydration

The c r y s t a l s of ha lc inonide are no t so lva ted wi th water, based on a t o t a l v o l a t i l e conten t of 0.3% obta ined by thermal g rav ime t r i c a n a l y s i s , a c o r r e c t e lemental a n a l y s i s , and a loss-on-drying va lue of 0.6% (cf. s e c t i o n 4 . 1 , Elemental and Inorganic Analyses) .

3. So lu t ion P r o p e r t i e s

3 . 1 I n t r i n s i c D i s so lu t ion Rate

The i n t r i n s i c d i s s o l u t i o n ra te w a s determined

I n one l i t e r of 0.M hydro- a f t e r compressing powder under 2000 p . s . i . g . p re s su re us ing 318" diameter disc-shaped d i e s . c h l o r i c a c i d a t 37", a g i t a t e d a t a rate of 50 r.p.m., t h e i n t r i n s i c d i s s o l u t i o n rate of ha lc inonide i s 8.33 x loW3 mg min.-l cm-' computed from d a t a obtained by u l t r a v i o l e t spectrometry. 28

3.2 S o l u b i l i t i e s i n Aqueous and Nonaqueous Solvents

S o l u b i l i t i e s of ha lc inonide were determined Resu l t s are r epor t ed using t h e U.S.P. i n va r ious so lvents .29

d e f i n i t i o n s . 30

HALCINONIDE 269

S o l v e n t

Water Hydroch lo r i c a c i d , 0.W Sodium hydrox ide , 0.N Acetone Ace t o n i t r i l e A c e t o n i t r i l e - w a t e r Benzene Chloroform C a s t o r o i l Dimethylsulf o x i d e E thano l E t h y l e t h e r G lyce ry l monooleate Hexanes I s o p r o p y l m y r i s t a t e 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

S o l u b i l i t y

I n s o l u b l e I n s o l u b l e I n s o l u b l e S o l u b l e S p a r i n g l y s o l u b l e S l i g h t l y s o l u b l e S l i g h t l y s o l u b l e F r e e l y s o l u b l e S l i g h t l y s o l u b l e F r e e l y s o l u b l e S l i g h t l y s o l u b l e S l i g h t l y s o l u b l e S l i g h t l y s o l u b l e I n so l u b l e S l i g h t l y s o l u b l e S l i g h t l y s o l u b l e S l i g h t l y s o l u b l e S l i g h t l y s o l u b l e S l i g h t l y s o l u b l e S l i g h t l y s o l u b l e Very s l i g h t l y s o l u b l e

270

3 . 3 P a r t i t i o n C o e f f i c i e n t s

JOEL KIRSCHBAUM

Halc inon ide was pa r t i t i o n e d 3 ' between hexanes and methanol , and between hexanes and aqueous 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 hour of mixing, t h e s t e r o i d c o n t e n t w a s de t e rmined 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 phases . I n a l l cases, abso rbance a t t h e peak 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 me thano 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 14C-halcinonide l a b e l e d a t t h e 2- carbon of t h e a c e t o n i d e moiety. Thus, t h e h a l c i n o n i d e i s comple t e ly r e t a i n e d i n e i t h e r t h e a c e t o n i t r i l e - water o r methanol 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 sys t ems 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 tudy33 invo lved i s o p r o p y l m y r i s t a t e and aqueous p ropy lene 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 sayed 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 chromatography (cf. s e c t i o n 4 .31 ) . 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 p ropy lene 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 f o l l o w s : p ropy lene g lyco l -wa te r (4:l); 1 4 4 ; p r o p y l e n e g lyco l -wa te r ( 2 : 3 ) , 44.4; p ropy lene g lyco l -wa te r ( 3 : 2 ) , 8.14, and propy- l e n e g lyco l -wa te r ( 9 : 1 ) , 0.82.

4 . Methods of A n a l y s i s

4 . 1 E lemen ta l and I n o r g a n i c Ana lyses

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 ca rbon 63.38% (63.35%, t h e o r e t i c a l ) ; hydrogen 7.39% (7.10%, t h e o r e - t i c a l ) ; c h l o r i n e , 7.89 (7 .79%, t h e o r e t i c a l ) , and f l u o r i n e , 4.30% (4.17%, 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 ca rbon a rc 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 reco rded 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 microphotometer . H a l c i n o n i d e c o n t a i n e d trace amounts (% 60 pg/g t o t a l ) of t h e f o l l o w i n g metal l ic i m p ~ r i t i e s ~ ~ ; i r o n , manganese, coppe r , n i c k e l , l e a d , z i n c , aluminum, sodium, ca l c ium, and magnesium. l o t 3 8 i s less than 0.1%. 39, method 11) i s less t h a n 0.003%.38 i n vacuum, t h e

R e s i d ~ e - o n - i g n i t i o n ~ ~ of t h e same Heavy metals c o n t e n t ( r e f e r e n c e

A f t e r 3 h o u r s a t 100" l o ~ s - o n - d r y i n g ~ ~ va lue38 i s 0.6%.

Four l o t s of h a l c i n o n i d e were ana lyzed f o r resi- 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 de t e rmined by

H ALCINON I DE 27 I

vapor phase ( 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 comparison 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 been found t o b e p r e s e n t .

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 Ana lyses

Proposed compendia1 i d e n t i f i c a t i o n tests f o r t h e United States Pharmacopeia i n v o l v e comparing e i t h e r t h e in - 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 spec t rum of sample h a l c i n - non ide w i t h t h a t o f an a u t h e n t i c sample4'. To t n i s a u t h o r , a chromatographic method is 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 depends on much o f t h e molecu le i n t e r a c t i n g w i t h t h e s o l i d and mob i l e phases 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 depends 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 r e g i o n b e i n g i n t a c t .

H a l c i n o n i d e h a s been q u a n t i t a t e d i n v a r i o u s formu- l a t i o n s o r as 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 , bo rohydr ide r e d u c t i o n a s ~ a y . ~ 2 v o l v e s measuring t h e u l t r a v i o l e t abso rbance of an a l i q u o t of me thano 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 abso rbance i s de te rmined 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 reduced by sodium borohydr ide t o d e s t r o y t h e 3-one- 4-ene chromophore. The u t i l i t y of t h i s p rocedure 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 , uncon juga ted , s t e r o i d s can be 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 .

T h i s d i f f e r e n t i a l a s s a y in-

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 color ime- 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 ou r labor at or^,^^ h a l c i n o - n i d e r e a c t s w i t h a c i d i c e t h a n o l i c 4 - n i t r o p h e n y l h y d r a z i n e , 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 hydrox ide , 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 i son iaz id4 ' , h a l c i n o n i d e g i v e s a b r i g h t y e l l o w 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 hour s . Ha lc inon ide added t o 4 - a m i n o a n t i ~ y r i n e ~ ~ i n 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 g i v e s a 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 te t ra- methylammonium hydroxide5', and h e a t e d , g i v e s a c loudy amber 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 I f 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 w i t h h a l c i n o n i d e . n i d e g i v e s a deep 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 te t razol ium3' 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 .

47

I n c o n c e n t r a t e d s u l f u r i c a c i d , 5 3 h a l c i n o -

272 JOEL KIRSCHBAUM

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 hal~inonide.~~ Commercially available, prepacked octa- decylsilane columns, 10 pm in particle size, United S ta tes 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 s ~ ~ 9 56. (triamcinolone acetonide) by HPLC. 57

Halcinonide can be separated from kenalogl’

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 ) ~ ~ . 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.

The purity

HALCINONIDE

+ r

273

Halcinonide

Progesterone

F i g u r e 8

Reverse Phase High-p res su re L iqu id Chromatography of Halcino- n i d e . See t e x t f o r d e t a i l s .

P r o g e s t e r o n e i s used as i n t e r n a l s t a n d a r d .

274 JOEL KIRSCHBAUM

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 chloroform- 60 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 excipients 62 . 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 tetra- met hy lammonium hydroxide51 colorimetry .

The formulation is


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. propylene glycol-chloroform as the stationary phase and toluene saturated with propylene glycol as the mobile phase.

A second solvent system uses 25%

4.4 Polarographic Analysis

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

This is

Thus, halcinonide can be

HALCINONIDE 275

s e n s i t shou ld

t o p i c a

a s sayed i n t h e p r e s e n c e o f o t h e r 4-ene-3-keto s t e r o i d s t h a t l a c k t h e 21-chloro group. T h i s r e s p o n s e r e q u i r e s a c o n c e n t r a - t 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

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 ~ ~ a l s o b e a p p l i c a b l e t o h a l c i n o n i d e .

4 . 5 H a l c i n o n i d e &-I T i s s u e s 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 a g e n t s as h a l c i n o n i d e are u s u a l l y de t e rmined by a

v a s o c o n s t r i c t o r a s s a y . T o p i c a l l y a p p l i e d c o r t i c o s t e r o i d s 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 can b e t h e forearm65 o r t h e upper back of h e a l t h y a d u l t s where s t r a t u m corneum is removed w i t h c e l l o p h a n e t ape .66 The test areas, 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 occluded w i t h p l a s t i c wrap and are e v a l u a t e d on an a l l - o r - none b a s i s . 14C-ha lc inon ide 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 th rough in tac t o r ab raded s k i n . I n dogs , 0 .4 t o 0.5% i s e s t i m a t e d t o b e absorbed th rough i n t a c t and 4 t o 10% t h r o u g h ab raded s k i n . I n r a b b i t s , 6 t o 16% of t h e 1 4 t o 23% t h r o u g h ab raded s k i n .

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%

4C-halcinonide w a s abso rbed th rough i n t a c t and

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 group. 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 o f 5 mg/kg. The major 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 e x c r e t e d i n b i l e . Radio-autography of b i l e showed a t least 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 metabo- l i t e s were i d e n t i f i e d . The two most abundant 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 , accounted 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) accoun ted f o r 2% each. 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) accoun ted f o r 10, 15 , 5 and 18% of t h e r a d i o a c t i v i t y , r e s p e c t i v e l y . I n dog b lood , 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 accounted f o r a b o u t 15% of t h e r a d i o a c t i v i t y . M 1 and M2 w e r e n o t d e t e c t e d . 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 magne t i c r e sonance 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 ch romatograph ic 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 s imilar d a t a o b t a i n e d f o r au then- t i c r e f e r e n c e samples . 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), M2 i s t h e 6B- hydroxy d e r i v a t i v e , M3 i s t h e 21-hydroxy d e r i v a t i v e (-CH2C1 -f -CH20H) and M4 is t h e 66-hydroxy d e r i v a t i v e of M3. 9 shows t h e proposed pathway. M 3 i s t h e a c e t o n i d e d e r i v a -

F i g u r e

F i g u r e 9 Proposed Metabol ic Pathways f o r Halcinonide 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 as 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 . S t a b i l i t y :

H a l c i n o n i d e d i s s o l v e d i n e i t h e r me thano l , d e u t e r o - me thano l , aqueous ammonia-methanol, o r deu te r ium 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 days a t 50", u s i n g n u c l e a r magne 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 4.32, f i r s t system)60 no change w a s found i n two l o t s of h a l c i n o n i d e a f t e r 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 . But t h e d i r e c t exposure 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 caused 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 is 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 undergo r ea r r angemen 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 re 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

Formulated 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 lyco l -wa te r l o t i o n , a f t e r s t o r a g e a t approx ima te ly 23" f o r 3 y e a r s , showed no l o s s on 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. 46 c e n t r a t i o n .

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 con-

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 h a l c i n o n i d e . 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 assis- t 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 as " p e r s o n a l communica- t i o n . " 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 espe- c i a l l y o b t a i n e d f o r t h i s a n a l y t i c a l p r o f i l e . 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 . Squibb & Sons, I n c . Much c r e d i t f o r t h i s p r o f i l e be longs t o D r . Klaus F l o r e y , who wro te 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 compi l ing of t h i s a n a l y - t i c a l p r o f i l e .

278

7 .

1.

2 .

3 .

4 .

5 .

6 .

7 .

8 .

9 .

10 .

11.

1 2 .

1 3 .

1 4 .

1 5 .

JOEL KIRSCHBAUM

References

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J. Fried and E. Sabo, J . Amer. Chem Soc., 75, 2273 ( 1 9 5 3 ) .

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M.E.

S. -S. Lee and Y. Rapp, Int. J . DermatoZ., 14, 412 ( 1 9 7 5 ) .

"USAN and the USP Dictionary of Drug Names," American Pharmaceutical Association, Washington, D.C., 1 9 7 8 , p . 1 5 1 .

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Martindale, "The Extra Pharmacopoeia," A. Wade, editor, 2 7 t h edition, The Pharmaceutical Press, London, 1 9 7 7 , p. 389.

U.S. Patent 3 , 0 4 8 , 5 8 1 , August 7 , 1 9 6 2 , to E. R. Squibb.

U.S. Patent 3 , 8 9 2 , 8 5 7 , July 1, 1 9 7 5 , to E. R. Squibb.

U.S. Patent 3 , 0 3 2 , 4 7 5 , May 1, 1 9 6 2 , to Chas. Pfizer & co.

U.S. Patent, 3 , 1 1 6 , 2 1 9 , December 3 1 , 1 9 6 3 , to American Cyanamid.

U.S. Patents, 2 , 7 7 3 , 0 5 8 and 2 , 7 7 3 , 0 8 0 , to American Cyanamid.

S. Bernstein, R. Lenhard, W. Allen, M. Heller, R. Littell, S. Stolar, L. Feldman, and R. Blank, J . h e r . Chem. SOC, 78, 5693 ( 1 9 5 6 ) .

J . Z . Gougoutas and B. Toeplitz, Personal communication.

P.T. Funke and A.I. Cohen, Personal Communication.

1 6 . M. Puar and A . I . Cohen, Personal Communication.

279 HALCINONIDE

1 7 . K. F l o r e y , Ann. ProfiZes, 2, 397 (1972) .

18. M. Porubcan, Personal communication.

19. B. Toeplitz, Personal communication.

20. Q. Ochs and T. Prusik, Personal communication.

21. D. Whigan, Personal communication.

22. United S t a t e s Pharmacopeia, 18, 936 (1970) .

23. P . Grabowich, Personal communication.

24 . P. Valatin, Personal communication.

25. united S t a t e s Pharmacopeia, 19, 6 5 1 (1975) .

26. T. Prusik, Personal communication.

27. H. Jacobson and V. Valenti, Personal communication.

28. A. Dhruv, Personal communication.

29. M. Augustine, J.M. Battaglia, C.G. Hughes and S. Perl- man, Personal communications.

30 . United S t a t e s Pharmacopeia, 29, 6 (1975) .

3 1 .

32.

33 .

3 4 .

35.

36.

37.

38.

39.

4 0 .

S. Perlman, Personal communication.

R. Poet, Personal communication.

M. Augustine and J. Battaglia, Personal communications.

V. Valenti, Personal communication.

J . Alicino, Personal communication.

N. Trandifir, Personal communication.

United S t a t e s Pharmacopeia, 29, 620 (1975) .

G . Brewer, Personal communication.

United S t a t e s Pharmacopeia, 19, 619 (1975) .

A . Niedermayer, Personal communication.

280 JOEL KIRSCHBAUM

4 1 .

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4 3 .

44 .

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46 .

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49 .

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5 1 .

5 2 .

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5 4 .

H. Lerner, Personal communication.

J. Kirschbaum, J . Pharm. Sei . , 67, 275 ( 1 9 7 8 ) .

J. Bartos and M. Pesez, i n "Colorimetric and Fluori- metric Analysis of Steroids," York, 1 9 7 6 .

Academic Press, New

M. Pesez and J. Bartos i n "Colorimetric and Fluori- metric Analysis of Organic Compounds and Drugs," Marcel Dekker, New York, 1 9 7 4 .

A.A. Forist and J.L. Johnson in "Pharmaceutical Analy- sis," T. Higuchi and E. Brochmann-Hanssen, Editors, Interscience Publishers (J. Wiley), New York, 1 9 6 1 , p. 7 1 .

L. Kerr and J. Kirschbaum, Personal communication.

M. Kimura, T. Nishina, and T. Sakamoto, Chem. Pharm. B u l l . (Tokyo), l 5 , 454 ( 1 9 6 2 ) .

E. Cingolani, G. Cavina, and V. Amormino, Farmaco (Pavia) Ed. Prat. , 15 , 3 0 1 ( 1 9 6 0 ) .

E.P. Schulz, M. Diaz, G. Lopez, L. Guerrero, H. Barrera, A Pereda, and A. Aquilera, A n a l . Chem., 36, 1 6 2 4 ( 1 9 6 4 ) .

J. Cross, H . Eisen and R. Kedersha, A n a l . Chem., 24, 1 0 4 9 ( 1 9 5 2 ) .

E. Ivashkiv, Personal Communication.

M. Pesez and J. Bartos in "Colorimetric and Fluori- metric Analysis of Organic Compounds and Drugs," Marcel Dekker, New York, 1 9 7 4 , p. 4 8 1 .

S . Berstein and R. Lenhard, J . Org . Chem., Z8, 1 1 4 6 ( 1 9 5 3 ) .

S. Gijr'cig and Gy. Szdsz in "Analysis of Steroid Hormone Drugs," Akadgmiai Kiadb, Budapest, 1 9 7 8 .

5 5 . J. Kirschbaum, R. Poet, K. Bush and G. Petrie, Personal communication.

HALCINON IDE 28 1

56.

57.

58.

59.

60.

61.

62.

6 3 .

64.

65.

66.

67.

68.

69.

70 .

71.

72.

73 .

74.

J.E. Fairbrother i n "Assay of Drugs and Other Trace Compounds in Biological Fluids, Ed. by E. Reid, "Meth- odological Developments in Biochemistry, Vol. 5, Longman Group, London, 1976, p. 141.

G. Gordon and P.R. Wood. A n a l . Div. C h e m . SOC. 14, 30 (1977) .

W. Beyer, The Upjohn Company, Personal communication.

J . Kirschbaum, Personal communication.

H. Roberts, Personal communication.

P. Egli, Personal communication.

E. Ivashkiv and R. Poet, Personal communication.

0. Kocy and C. Smith, Personal communication.

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., 69, 11 (1957).

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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.

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.! 49 , 253 (1963) .

K. Bush and G. Petrie, Personal communication.

Literature reviewed to January 1, 1979.


3 . 4 . 5 . 6 .

7 . 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

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

ISBN 0-12-260808-9 283

284 CHESTER E. ORZECH ET AL.

1. DESCRIPTION

1.1 N a m e , Formula, Molecular Weight The Chemical Abs t rac ts name f o r hydralazine hydro-

ch lor ide i s 1 ( 2H)-phthalazinone hydrazone monohydrochloride, s t a r t i n g with volume 80; previous ly t h e name l-hydrazino- phtha laz ine monohydrochloride w a s used. The CAS Regis t ry No. i s [304-20-1] f o r t h e hydrochlor ide sa l t , [86-54-4] f o r t he base.

NHNH2 I

C8H8N4OHC1 Molecular Weight: 196.64

1.2 Appearance, Color , Odor Hydralazine hydrochlor ide is a white t o off-white

odor less c r y s t a l l i n e powder.

1.3 History The a b i l i t y of hydralazine and similar hydrazino

compounds t o reduce blood pressure w a s repor ted by Gross e t a1 (1) i n 1950. Druey and Ringier ( 2 ) i n 1951, along with t y p i c a l r e a c t i o n s displayed by t h e compound , inc luding seve ra l r e a c t i o n s which la te r became t h e bases f o r a n a l y s i s of hydralazine and i t s metabol i tes . t reatment of hypertension, and papers on i t s p r o p e r t i e s and methods f o r i t s determinat ion have been publ ished i n many languages.

o the r drugs. e spec ia l ly use fu l when used with beta-adrenergic blocking agents and d i u r e t i c s (3,4).

2. PHYSICAL PROPERTIES

Methods of syn thes i s were publ ished by

The drug has been widely used f o r

Hydralazine i s usua l ly used i n combination wi th In recent yea r s , it has been found t o be

2.1 In f r a red Spec t ra

The i n f r a r e d spectrum of hydralazine hydrochlor ide (Figure I > w a s obtained with a Beckman IR-12 spectrophotometer. A mineral o i l d i spers ion 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 ispers ion , supported on polyethylene f i lm ,

HYDRALAZINE HYDROCHLORIDE 285

was scanned from 200 t o 530 cm-’. bands can be assigned as fol lows:

Frequency, cm Assignment (5 )

Some of t h e absorp t ion

-1

3 220 N-H s t r e t c h

92.5 Aromatic C-H s t r e t c h

2800-3000 Mineral o i l C-H s t r e t c h

1590- 1600 C=C s t r e t c h

1460 Mineral o i l C-H band

785 Out of p lane bending, 4 adjacent H atoms on an aromatic r i n g

The mineral o i l absorp t ion at 2800 t o 3000 cm-I and at 1460 cm- obscures absorpt ion bands of hydralazine hydrochlor ide at 2810, 2320, and 2970 cm-1 (N-H’ s t r e t c h ) and a w e a k sharp band a t 1470 cm-l; t hese bands can be observed i n potassium bromide d ispers ion spec t ra . The bands at 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 d ispers ion spec t ra .

base i n a potassium bromide d ispers ion (Figure 2) w a s recorded from 400 t o 4000 cm-1 , and t h e 200 t o 550 cm-1 region w a s obtained from a mineral o i l d i spers ion supported on polyethylene f i lm. The s p e c t r a o f potassium bromide d ispers ions of t he base 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 those of mineral o i l d i spers ions . The assignment of absorp t ion bands i n t h e spectrum of t h e base is s imi l a r+ to t h a t of t h e hydrochlor ide except €o r t h e presence of N-H s t r e t c h absorpt ion i n the l a t t e r . publ ished ( 6 ) .

The i n f r a r e d spectrum of hydralazine hydrochlor ide

A spectrum of the base has been

2.2 U l t r a v i o l e t Spec t ra Figure 3 is the u l t r a v i o l e t absorpt ion spectrum of

hydralazine hydrochlor ide i n water so lu t ion , run on a Cary Model 14 spectrophotometer. The so lu t ion contained 9.9 mg of hydralazine hydrochlor ide p e r l i t e r , and w a s run aga ins t water ( 1 cm c e l l s ) . The d i scon t inu i ty i n t h e spectrum at 219 nm is a change of absorpt ion sca l e ; t he 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

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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 aga ins t water i s a l s o recorded on t h e char t . The spectrum e x h i b i t s m a x i m a at 315, 303, 260, 239, and 211 nm, wi th r e spec t ive a b s o r p t i v i t i e s as fol lows: 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 ) repor ted 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 they d id not r epor t t h e 211 nm m a x i m u 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 . g ives t h e spectrum i n 0.1N hydrochlor ic a c i d , with m a x i m a at 312, 302, 259, and 233 nm, and t h e spectrum i n 0.1N sodium hydroxide, with 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 base , t h e monohydrochloride, and t h e dihydrochlor ide: ( a ) t h e base exhib i ted m a x i m a at 305 and 273 nm, wi th a t h i r d maximum near 262 nm; ( b ) t h e monohydrochloride exhib i ted m a x i m a at 315, 302, 2'30, and 260 nm; ( c ) t h e dihydrochlor ide exhib i ted a m a x i m u m a t 318 nm wi th a broad shoulder at about 300 nm. The Merck Index (12) l ists m a x i m a at 315, 304, 260, 240, and 211 nm f o r t h e aqueous so lu t ion of t h e hydrochloride.

Clarke (6) g i v e s d a t a Sunshine (10)

2.3 Mass Spectrum Figure 4 shows t h e low r e so lu t ion mass spectrum

of hydralazine hydrochloride. The d a t a were obtained with an LKB 9000s mass spectrometer , with an i o n i z a t i o n voltage of 70 eV, source temperature z O o C . Some of t h e peaks m a y be ass igned as fol lows (13) :

Mass Charge Rat io Assignment

160 M+

+ 129 M - .NH-NH,

103

76

51

36/38

t

C6H4 +

C4H3

HC1'

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HYDRALAZIN E HYDROCHLORIDE 29 I

2.4 Nuclear Magnetic Resonance S p e c t r a The NMR spectrum shown i n Figure 5 w a s obtained by

d i s so lv ing hydralazine hydrochlor ide i n deuterium oxide containing 3- ( t r ime thy l s i ly1 ) - I-propane-sulfonic a c i d sodium salt ( E X ) . The s e r i e s of peaks a t 0 , 0.6, 1.8, and 3 ppm are all due t o t h e WS. t o HDO which forms on exchange with t h e so lvent and t h e peaks at 8.01 and 8.61 ppm a r e due t o t h e aromatic protons. The NMR spectrum of t h e base (Figure 6 ) w a s obtained i n a 1 : 1 mixture of dirnethylsulfoxide-d :deuterochloroform. The peaks a t 2.53 ppm a r e due t o t 8 e so lvent . The N-H p ro tons a r e at 4.1 pprn and t h e aromatic pro tons are a t 7.6 and 8 ppm. NMR spectrometer .

The peak at 4.8 pprn i s due

The s p e c t r a were produced using a Varian EM-360

2.5 D i f f e r e n t i a l Thermal Analysis (IYTA) The DTA curves i n F igures 7 and 8 were obtained

with a W o n t Model 900 instrument. Figure 8 shows t h a t t h e base melts sharp ly and Figure 7 shows t h a t t h e hydroch lo r ide melts wi th decomposition. Visua l ly t h e hydroch lo r ide melts at 2 7 4 " C , but decomposition t akes p l ace over a l a r g e temperature range (F igure 7) .

2.6 Crys t a l P r o p e r t i e s Chojnacki e t a1 (14) inves t iga t ed the c r y s t a l

p r o p e r t i e s of hydra laz ine hydrochloride. Rec rys t a l l i zed from water, t h e c r y s t a l s were found t o be monoglinic, with 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 ca l cu la t ed dens i ty w a s 1.486, t h e measured dens i ty 1.479 g p e r cm3. Powder d i f f r a c t i o n da ta , gene ra l ly 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 hydralazine hydrochlor ide and hydralazine base are given i n Table 1. These da t a were obtained with a Norelco d i f f r ac tomete r using n i c k e l - f i l t e r e d copper Ka r ad ia t ion .

The space group w a s P21/c.

2.7 Microchemical Charac te r iza t ion Sandr i (15) repor ted t h e formation of charac te r -

i s t i c c r y s t a l s wi th cadmium bromide-hydrobromic a c i d , bismuth iodide-potassium i o d i d e , and bismuth bromide-hydro- brornic a c i d , with compositions CdBr -HBr*C H N - 4 H 2 0 , ~ i I q - c 8 H ~ N 4 , and HEiiBr - C H N respec t ive ly .

uhnert-Brandsts t 4 * 9 e r 41 e al ( 7 ) repor ted t h e microscopic hot-s tage cha rac t e r i za t ion of hydralazine hydrochlor ide. Above 21OoC, small rods , g ranules , and pr isms sublime. The melt ing po in t i s 268-a5"C. G a s bubbles are evolved as melt ing begins , and mel t ing proceeds wi th

2 8 8 4


TABLE 1

X-RAY POWDER DIFFRACTION PATTERNS OF HYDRALAZINE HYDROCHLORIDE AND HYDRALAZINE BASE

H y d r o c h l o r i d e 0

d(A)

Base 0 -

d ( A )

7.74 7- 25 5-92 5 -54 4.82 4.62 4.37 3.99 3.87 3 -63 3.39 3-38 3-32 3- 29 3.22 3- 15 3-05 2-99 2-95 2-93

2-77 2.85

2.72 2.64

2.44 2.41

2.34 2.24 2.22 2.18 2.16 2.10 2.06 2.02 1.99 1-97 I .94

2.58

2-39

1.89

100 93 9 2 2 18 47 15 12 54 9 11 31 65 92 7 9 4 2 2 6 22 2 2 4 7 2 1 16 2 2 2 2 3 2 1 2 4 4 2

7.37 5 -87 5.62 5.04 4.86 4.48 3.98 3.74 3.66 3.47 3.28 3.24

2.96 2.80

2.67

3-12

2.7 1

2.60 2.49 2.44 2.38 2-30 2.28 2.25 2.18 2.12 2-07 2.04 1-99 1.95 1.87 I .85 1.83

I .78 1-79

1.76

1.67 1.62

1.72

100 11 49 11 17 18 2 10 2 15 100 17 11 2 7 1 2 2 2 2 1 2 1 2 1 3 2 3 1 2 2 1 1 1 1 1 3 1 1


1 1 I 1 1 1 1 1 1

10 9 8 7 6 5 4 3 2 I

PPY (61

Figure 5. Nuclear Magnetic Resonance Spectrum of Hydralazine

Hydrochloride.

1 1 1 1 I I I 1 I 1 1

10 9 0 7 6 5 4 3 2 I 0 PPY 16)

Figure 6. Nuclear Magnetic Resonance Spectrum of Hydralazine Base.

h)

P

Figure 7. Differen t ia l Thermal Analysis Curve of Hydralazine Hydrochloride.

Figure 8. Different ia l Thermal Analysis Curve of Hydralazine Base.


t he separation of high melting (over 300°C) needles and bushlike aggregates. p-acetamidophenyl s a l i c y l a t e was 181 "C, with dicyandiami.de 162°C. The p i c r a t e salt forms from aqueous solut ion as long needles and columns t h a t melt at 210 t o 212°C with decomposition. with a melting point of 200 t o 203°C.

c r y s t a l tes ts , both from aqueous solut ion: iodide-potassium ace ta t e reagent forms dense r o s e t t e s ; (b) iodine-potassium iodide ( 1 :5O) reagent forms c l u s t e r s or masses of needles.

The eu tec t i c temperature with

The styphnate forms needles and r o s e t t e s

Sunshine (10) and Clarke (6) give two micro- ( a ) l e a d

2.8 So lub i l i t y The Merck Index (12) gives the s o l u b i l i t y i n water

as 9.1 mg pe r m l at 15"C, and 44.2 mg pe r m l at 25°C. I n 95% ethanol, t he s o l u b i l i t y i s given as 2 mg pe r ml. a r e consistent with the following approximate s o l u b i l i t y data , determined at room temperature:

These

Solvent Approximate So lub i l i t y , mg/ml

Water 39

Methanol 6 -7

Ethanol (95%) 1-9

2-Propanol 0.1

Chloroform < 0.1

Ethyl e the r (anhydrous) < 0.1

Ethyl ace t a t e < 0.1

Acet oni t r i l e < 0.1

2.9 Dissociation Constants

Evstratova e t a1 (17) reported a pK pK inathe l a t t e r solvent. of 6.9 for t he dissociat ion of t he monohydrochloride anda0.5 f o r dissociat ion of t he dihydrochloride, determined absorptiometrically. 7.2 (18).

Evstratova and Ivanova (16) reported a pKa of 7.1.

of 4.7 i n aqueous 90 percent acetone, and a p\ of 15.6 of 7.1 i n water, a

a

N a i k e t a1 (11) reported a pK

Artamanov et al reported a pKa of


2.10 Melting P o i n t s The mel t ing po in t of hydra laz ine hydrochlor ide i s

nea r n3"C, t h a t of t h e base is nea r 173°C. repor ted i n t h e l i t e r a t u r e a r e as fol lows:

Melting p o i n t s

Melting Poin t of Hydrochloride ( a ) , O C

no - 280

Reference

(3,191

273

271 - 272 (24)

( 2 , s , 21 ,22,23 )

265 ( 2 5 )

273 - 274 (26)

( a ) The hydrochlor ide me l t s wi th decomposition.

Melting Poin t of Base, O C Reference

172 - 173 (2,21,22)

165 - 172 (24)

172 ( 2 5 )

173 - 175 (27)

3. SYNTHESIS

Hydralazine has been prepared by va r ious procedures from I-chlorophthalazine (2,21,22,24,28) , phthalazine- l - th ione (20,23,25), 1 ,4-dichlorophthalazine (29,30,31), I-(methylsulfony1)-phthalazine (271, o r 1-cyano-2-benzal a c e t a t e (26). phtha laz ine , has been prepared from naphthalene (3,281 o r from p h t h a l i c anhydride ( 3 ) . hydrazinophthalazines and r e l a t e d compounds h a s been publ ished by Druey and Tripod (3 ) . a r e ou t l ined i n F igures 9 and 10.

Phthalazone, used t o prepare l-chloro-

A comprehensive survey on

Some of t h e s e procedures

4. STABILITY - DEGRADATION

Hydralazine hydrochlor ide 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 so l id . A t room temperature , it i s s t a b l e i n d i s t i l l e d water s o l u t i o n f o r weeks (32). In aqueous

ri

V

&Jo

EN

X

-1 O&7

0

V

0

X

(H 0

298

aCN H( OAC 1

I

H N N H -

c1 ci Figure 10. Several Syntheses f o r Hydralazine.

300 CHESTER E. ORZECH E7 A L .

so lu t ion with a pH g r e a t e r than 7 , hydralazine decomposes, forming phtha laz ine ; t h e r a t e of decomposition depends on pH, temperature , and t h e k ind and concentrat ion of anion present (33).

at room temperature, apparent ly by enzymatic reac t ions .

hydrazones wi th aldehydes and ketones. It i s a l s o a reducing agent , and it forms complexes wi th many metal i ons (34).

ammonia, n i t rogen , and 1 , k d i h y d r o - l , 1 ' -b iphtha laz ine (35).

In b i o l o g i c a l samples the drug disappears r ap id ly

The hydrazino group is highly r e a c t i v e , forming

A t 275 t o 280°C i t decomposes t o hydrazine,

5. METABOLISM

Hydralazine hydrochlor ide i s rap id ly metabolized Experiments with carbon-14 l abe led drug i n and excreted.

humans ind ica t ed t h a t l e s s than 10 percent of t h e i n t a c t drug was excreted (36). 89 percent was excreted i n t h e u r ine and 9 t o 12 percent i n the feces . Of the ma te r i a l excreted i n t h e u r ine , 96 percent w a s recovered i n t h e first 24 hours. Ind iv idua ls who are slow a c e t y l a t o r s exh ib i t h igher hydralazine blood l e v e l s than f a s t a c e t y l a t o r s , f o r t h e same dose (37).

var ious spec ie s are as follows:

Within 5 days a f te r a dose, 83 t o

Reported metabolic products of hydralazine i n

Metabolite Spec ies Reference

I. 3-Methyl-s-triazolo Man (36,37,38,39, !3, Itdphtha laz ine 40,41,42,43,44)

R a t ( 3 , 3 9 , 4 3 , 4 5 ,

Guinea p i g (45) Rabbit (38 1 Pigeon (45)

46 1

11. N- ( I-Phthalaziny1)- M a n (36,38,47,85 ) hydrazone of pyruvic R a t (38,46) ac id Rabb it (38)

111. N- ( 1-Phthalaziny1)- M a n (47) hydrazone of acetone R a t (44,461

I V . N- ( 1-Phthalaziny1)- Man hydrazone of a- k e t o g l u t a r i c ac id

(47)

HYDRALAZINE HYDROCHLORIDE 30 I

V. s-Triazolo[3,4al Man ph tha laz ine R a t

V I . 1(2H)-Phthalazinone M a n R a t

V I I . Ph tha laz ine Man R a t

V I I I . 4- ( 2-Acetylhydrazino )- ph t halazinone M a n

I X . 9-Hydroxy-3-methyl-s- t r iazol0[3,4a! ph t ha l az ine Man

X. 3-Hydroxymethyl-s- Man t r i azo lo t3 ,4a ! R a t p h t ha l az ine

( 3 7 ) (46)

(36)

( 3 6 )

In cases where t h e me tabo l i t e s above posses s s u i t a b l e func t iona l groups, they a r e usua l ly excre ted as g lucuronides or s u l f a t e s .

of hydra laz ine , based p r imar i ly on those proposed by Haegele e t al ( 4 6 ) and Wagner e t a1 (36).

Figure 11 is a scheme i l l u s t r a t i n g t h e metabolism

6. METHODS OF ANALYSIS

6.1 I d e n t i f i c a t i o n T e s t s Hydralazine hydrochlor ide can be e a s i l y i d e n t i f i e d

by t h e phys ica l p r o p e r t i e s descr ibed i n Sec t ion 2 above.

i s necessary , t h e fol lowing t e s t s may b e use fu l .

f e r r i c i on or dirnethylarninobenzaldehyde with t h e unknown. The i r o n reagent y i e l d s a b lue c o l o r , and dimethylaminobenzaldehyde an orange co lor . The r e s u l t s ob ta ined wi th 95 o t h e r drugs a r e given.

Belikov e t a1 (50) descr ibed 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. a l k a l i n e aqueous s o l u t i o n of hydra laz ine t o y i e l d a red co lor ; when t h e mixture con ta ins mineral a c i d s , a green p r e c i p i t a t e i s formed, bu t with a c e t i c a c i d , a red p rec ip i - t a t e is formed. Cinnamaldehyde r e a c t s with a hydrochlor ic a c i d s o l u t i o n of hydra laz ine t o g ive a yel low p r e c i p i t a t e , m.p. 197-200"C. p-Nitrobenzaldehyde r e a c t s wi th an

Where i d e n t i f i c a t i o n of hydra laz ine i n formula t ions

Cooper (49) used spot t e s t s on paper , r e a c t i n g

Sodium ni t ropruss i .de r e a c t s wi th an

/

z-2 EN'

K)

X

vb-=

&L w 1: V X 2

-24 );i 8 w 3

'H

0

302

2-2

m

R-R

?

Ot

1

H

H

gg P 303


aqueous so lu t ion of hydralazine t o g ive an orange p rec ip i - t a te , m.p. 260-26I0C.

d i f f e r e n t i a t e hydralazine from c lose ly r e l a t e d drugs. Reagents used were aqueous copper (I) ch lo r ide , aqueous ammonium molybdate, i od ine i n potassium iod ide s o l u t i o n , aqueous cobal t (11) n i t r a t e , a l coho l i c ninhydrin, and a l coho l i c bromophenol blue. 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 (52) reac ted hydralazine with p ,p'-dichlorobenzene sulfonamidate sodium i n water at 80 t o 90°C 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 with a melt ing poin t of 196-197"C.

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 .

chromatography t o i d e n t i f y hydralazine i n dosage forms and b i o l o g i c a l f l u i d s ( see Sec t ion 6.8).

Modras (51) repor ted spot t e s t r e a c t i o n s t o

H 0 N S C 1 2 0 . 1 7 4 5 2 2

I n t h e USP (19) method f o r i d e n t i f y i n g hydralazine

S tohs and Scra tch ley (53) used t h i n l a y e r

6.2 Elemental Analyses The elemental compositions of hydralazine hydro-

ch lor ide and hydralazine base are as fol lows:

Hydrochloride Base Element Calculated Found(2) Calculated Found( 2)

- - - Carbon 48.87 48 -99 59.99

- - - Hydrogen 4.61 4.83 5 -03

Nitrogen 28.49 28-39 34.98 35 -09

Chlorine 18.03 18.17 - - - - - -

6.3 Sp e c t rop ho t ome t ri c Methods Solomonova e t al ( 9 ) determined hydralazine 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 absorp t ion method.

formulat ions with a lcohol o r water , o t h e r components of t h e mixture may i n t e r f e r e with d i r e c t u l t r a v i o l e t absorp t ion measurement by cont r ibu t ing t o t h e observed absorpt ion. The s o l u t i o n s usua l ly cannot be cleaned up by e x t r a c t i o n techniques because hydralazine decomposes i n a l k a l i n e so lu t ion . However, t h e r e a r e many r e a c t i o n s t h a t g ive r i se t o near u l t r a v i o l e t o r v i s i b l e absorpt ion bands s u i t a b l e f o r quant i tat i on.

Although t h e compound can be ex t r ac t ed from


A f r equen t ly r ep o r t ed spec t ropho t ome t r i c t e c h i que f o r t h e determinat ion of hydra laz ine i s based on r e a c t i o n s with aromatic aldehydes t o form hydrazones wi th absorp t ion i n t h e v i s i b l e region. Luk'yanchikova e t al (54) used p-nitrobenzaldehyde; Wesley-Hadzi ja and Abaffy (55) and Ruggieri (56 ) used p-dimethylaminobenzaldehyde; Luk'yanchikova (57,581 used cinnamaldehyde; Schu le r t (33) used p-hydroxybenzaldehyde; and Zak e t al (59) used p-methoq- benzaldehyde, a f t e r t e s t i n g cinnamaldehyde, s a l i cy la ldehyde , 3,4,5- t r ime t hoqbenzaldehyde , and I-napht haldehyde . ninhydrin t o ob ta in a s o l u t i o n wi th an absorp t ion maximum near 450 nm.

E l l e r t and Modras (61 ) t r e a t e d hydra laz ine wi th f e r r o u s ion i n a l k a l i n e s o l u t i o n , and measured t h e co lo r produced at 540 nm.

2-naphthoquinonesulfonate sodium t o form a rose -v io l e t color .

mated co lo r ime t r i c method us ing b lue te t razol ium f o r t h e a n a l y s i s of hydra laz ine i n t h e presence of r e se rp ine and hydrochlorothiazide.

Per ry (32), and Grabowicz and Brul inska (60) used

Ruggieri (56) r epor t ed a co lo r ime t r i c t e s t us ing

Urbanyi and O'Connell (62,63) developed an auto-

6.4 Fluorescence N a i k e t al (11) ex t r ac t ed hydra laz ine hydro-

ch lo r ide from t a b l e t s wi th 50 percent aqueous methanol and mixed a po r t ion of t h e e x t r a c t wi th 99 volumes of concen- t r a t e d s u l f u r i c a c i d t o ob ta in f luorescence at 353 nm wi th e x c i t a t i o n at 323 nm. The f luorescence i n t e n s i t y va r i ed l i n e a r l y wi th concent ra t ion i n t h e range 2 t o 8 pg hydra laz ine hydrochlor ide p e r m l . s imi l a r ly .

I n j e c t i o n s were analyzed

6.5 T i t r a t i o n Methods Ruggieri (56) r epor t ed t i t r a t i n g hydra laz ine wi th

p e r chlo r i c a c i d . Ruzhentseva e t a1 (64) converted hydra laz ine t o

ammonia by hea t ing with z inc i n s u l f u r i c a c i d , forming 3 moles of ammonia p e r mole of hydralazine. 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 ac id s o l u t i o n , which w a s then t i t r a t e d .

good r e s u l t s . i n t h e presence of potassium bromide and hydrochlor ic a c i d , using a s t a rch - iod ine end-point. Another w a s add i t ion of excess p e r i o d i c acid-potassium iodide with sodium

The mixture w a s

Sandr i (15) t e s t e d t h r e e t i t r a t i o n methods with One was t i t r a t i o n with potassium bromate


t h i o s u l f a t e back- t i t r a t ion . The t h i r d w a s add i t ion of sodium hydroxide and a concentrated s o l u t i o n of potassium fer rocyanide , then ac id i fy ing and t i t r a t i n g with potassium permanganat e so lu t ion .

hydrochlor ide i n dimethylformamide s o l u t i o n t o a poten t io- met r ic end-point with sodium methoxide so lu t ion .

hydrochlor ide by conductometric t i t r a t i o n with sodium hydroxide so lu t ion .

z ine from t a b l e t s with N-bromosuccinimide so lu t ion . One mole of hydralazine hydrochlor ide w a s equivalent t o 2 moles of N-bromosuccinimide. i nd ica to r .

(67,681 and mercurimetr ic (69) methods. 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 so lu t ion . The s i l v e r i s d isso lved with hot n i t r i c ac id and t i t r a t e d with ammonium th iocyanate so lu t ion . Al te 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 iod ide so lu t ion . The p r e c i p i t a t e d mercury i s d isso lved by adding excess s tandard iod ine so lu t ion . The excess iod ine is back- t i t r a t ed wi th sodium t h i o s u l f a t e so lu t ion a f t e r ac id i fy ing wi th a c e t i c ac id .

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 ioda te t i t r a t i o n i n s t rong ly a c i d s o l u t i o n , using chloroform t o de t ec t t he presence of i od ine (19).

P e r e l 'man and Evst rat ova (65 ) t i t r a t e d hydralazine

Artamanov e t a1 (18) determined hydralazine

Goryacheva and Prikhodkina (66) t i t r a t e d hydrala-

Methyl red was used as t h e

Soliman and B e l a l i nves t iga t ed a rgen t ime t r i c Hydralazine

USP X I X d i r e c t s t h a t hydralazine be determined i n

6.6 Gasometric Methods McKennis and Yard (70) s tud ied t h e n i t rogen

evolut ion from a s e r i e s of hydrazino compounds when t r e a t e d with 0.0% K I O

percent of t h e t h e o r e t i c a l amount of n i t rogen i n I5 minutes. Viala (71) determined hydralazine i n s o l u t i o n s o r t a b l e t s by measuring the n i t rogen f r eed from t h e hydrazine group, using potassium permanganate i n s u l f u r i c a c i d , o r i od ine i n sodium bicarbonate so lu t ion .

i n 0.z H 04 i n a Warburg appara tus at 3 7 O C . In an aqueous 3 s o l u B i on , hydra laz ine r e l eased 102

6.7 Polarographx Polarographic s t u d i e s of hydra laz ine and r e l a t e d

compounds were repor ted by Giovanoli-Jakubezak e t a1 (72) and by Modras (73 ) . 2-electron s t a g e s with t h e formation of t h e te t rahydro der iva t ive . Hydralazine could be determined i n t h e

The reduct ion proceeds i n two


presence of decomposition products , at a concent ra t ion of about 0.0OlM i n 12 hydrochlor ic a c i d containing a s m a l l 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 aga ins t a s a t u r a t e d calomel e l ec t rode .


chromatography on Whatman No. 1 paper with butanol /ace t ic acid/water (4:1:5) as t h e so lvent . Detection w a s by quenching of t h e background f luorescence of t h e paper 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 0.90. (41,45,74) have used a l k a l i n e systems f o r paper chromatography of hydra laz ine and i t s me tabo l i t e s , bu t Lesser e t al (75) suggest t h a t a l k a l i n e chromatography systems may not be s u i t a b l e f o r hydralazine i t s e l f .

Ruggieri (56) and McIsaac and Kanda (38) descr ibe

Severa l i n v e s t i g a t o r s

6.9 Thin Layer Chromatography Stohs and Scra tch ley (53) i nves t iga t ed seve ra 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 an t ihype r t ens ive drugs, us ing s i l i c a g e l G and a v a r i e t y of de t ec t ion reagents .

f Solvent System Hydralazine R

Methyl e t h y l ketone/n-hexane ( 1 : 1) 0.72

Methyl e t h y l ketone/n-hexane ( 2 : 1 ) 0.62

Methyl e t h y l ketone/n-hexane (3: 1) 0 .oo

Chl or o f o rm/ac e t on e/t ri e t hanol amin e (50:50: 1.5) 0.68

Reagents f o r de t ec t ing hydra laz ine were Dragendorff ' s reagent , a l k a l i n e dimethylaminobenzaldehyde , anisaldehyde, and Bratton-Marshall reagent .

Zak e t a1 (59) used 3g hydrochlor ic acid/methanol ascorb ic a c i d (44:6:1) on s i l i c a g e l and found an R value o f 0.53 f o r hydralazine.

f luorescent i n d i c a t o r , a c e t i c acid/0.0 1% aqueous disodium ede ta t e (3:97), and observed an Rf value of 0.78 for hydralazine. On s i l i c a g e l with f luo rescen t i n d i c a t o r , t h e Rf value w a s l ess than 0.05 with ( a ) chloroform/n-heptane/ a c e t i c ac id (6:4: I), and ( b ) cyclohexane/chloroform/ a c e t o n i t r i l e ( I : 2: I ).

f

Lesser e t a1 (75) used c e l l u l o s e s h e e t s wi th

308 CHESTER E. ORZECH ET A L

6.10 High Pressure Liquid Chromatography Smith e t al (76,771 analyzed hydra laz ine i n a drug

mixture conta in ing hydralazine hydrochlor ide, hydrochloro- t h i a z i d e , and an impuri ty der ived 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 ee l , packed with a s t rong anion exchanger on 3 pin Zipax@. The mobile phase was pH 9.2 bo ra t e b u f f e r containing 0.005z sodium s u l f a t e (5% methanol), at 1.7 m l p e r minute. Detection w a s by u l t r a v i o l e t absorpt ion at 254 nm.

Honigberg e t a1 (78) t e s t e d reversed-phase chromatography f o r separa t ion of a number of drugs , inc luding hydralazine. The columns contained e i t h e r octadecyltrichlorosilane o r d iphenyld ich loros i lane , bonded 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. Of t h e numerous mobile phases t e s t e d , t h e b e s t f o r s epa ra t ing hydra laz ine , hydrochlorothiazide, and r e se rp ine w a s ace toni t r i le /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 absorp t ion at 254 nm.

6.11 G a s Chromatography Jack et al (79) determined hydralazine i n plasma.

The sample w a s t r e a t e d with n i t r o u s a c i d , which r e a c t s with hydralazine t o form t e t r azo lo [ 1,5a]phthalazine (2). The d e r i v a t i v e was ex t r ac t ed with benzene and determined by gas chromatography. I-Hydrazino-4-methylphthalzine w a s used as an i n t e r n a l s tandard.

used by Zak e t a1 (801, Ta lse th (42,82,82,83) , and Haegele e t a1 (46) f o r metabol ic s tud ie s . t h a t hydro lys i s of conjugates 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 the a c i d concent ra t ion during 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 hydra laz ine and ac id - l ab i l e me tabo l i t e s can be c a r r i e d out by s u i t a b l e adjustment of t h e ac id concentrat ion.

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. of t a b l e t s subjec ted t o e leva ted temperatures , where t a b l e t s could not be analyzed by t h e USP method.

The same procedure, or modif ica t ions of i t , was

Zak e t a1 (80) po in t ou t

Smith e t a1 (84) determined hydralazine i n

The method w a s appl ied t o s t a b i l i t y s t u d i e s

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 Labora tor ies f o r h i s mass s p e c t r a l d a t a and

Table 2

GAS CHROMATOGRAPHY SYSTEMS USED FOR DERIVATIZED HYDRALAZINE DETERMINATIONS

Reference Number Column

(79) 1.5 m x 2 mm I D , g l a s s , packed with 3% OV-225 on 80-100 mesh Chromosorb W-m

(80) 1.2 m x 2 mm I D , glass, packed with 3% OV-225 on Gas-Chrom Q

(46) 1 m x 4 mm I D , g l a s s , packed with 3% OV-I7 on 80-100 mesh Chromosorb W

(84) 1.8 m x 4 mm I D , g l a s s , packed with 70% S E - 3 on 80-100 mesh G a s - Chrom Q

Carrier G a s Column Temp., O C

220 O C N2'

ml/min.

220 O C

He 7 Programmed, at 40 ml/min. 4"/min.,

170- 240 O C

200 O C

2' N

55 ml/min.

Detector

Pulsed elec- t r o n capture (Ni-63, 10 m C i )

Elec t ron capture

Mass spectrometer

Flame i o n i z a t i o n

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 search , and Mrs. S. W i l l e t t e f o r typing t h e p r o f i l e .

8. RFFERENCES

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13-

14.

15

16.

I7 -

F. Gross, J. Druey, and R. Meier, Experent ia 6, 19 (1950). J. Druey and B. H. Ringier , Helv. Chim. Acta 3, 195-210 (1951); C.A. 5, 10248f. J. Druey and J. Tripod, Med. Chem. (New York) 2, 223-62 (1967); C.A. 70, 77829e. J. Koch-Weser, N. Engl. J. Med. 295, 320-3 (1976); C.A. 85, 103541rn. L. J .Bel lamy, "The Infra-red Spec t ra of Complex Molecules", 3 rd . ed., John Wiley & Sons, New York,

E. G. C. Clarke, e d i t o r , " I so l a t ion and Iden t i f i ca - t i o n of Drugs", The Pharmaceutical P res s , London, England, 1969. M. Kuhnert-BrandstBtter, A. Kofler , R. Hoffmann, and H.-C. Rhi, Sc i . Pharrn. 33, 205-30 (1965); C.A. 64, 7966g. M. F .Sharkey, 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). S. G. Solomonova, N. M. Turkevich, and N. V. Kurinnaya, Farm. Zh. (Kiev) - 28, 42-4 (1973); C.A. 78, 140445j. I. S u n h i n e , e d i t o r , ffHandbook of Analy t ica l Toxicology", The Chemical Rubber Co., Cleveland, Ohio, 1969. D. V. Naik, B. R. Davis, K. M. Minnet, and S. G. Schulman, J. Pharm. Sci . - 65, 8 4 - 6 (1976); C.A. 84, "The Merck Index", 9 t h ed., Merck & Co., Inc., Rahway, N.J., 1976. G. S c h i l l i n g , Ayerst Research Labora tor ies , Personal Communication. J. Chojnacki, L. Lebioda, and K. Stadnicka, Rocz. Chem. 49, 1163-6 (1975); C.A. &, 106677e. G. C. L d r i , B o l l . chim. farm. 96, 431-6 (1957); C.A. 52, 4109g. K. I. Evstratova and A. I. Ivanova, Farmatsiya (Moscow) 17 (2 ) , 41-5 (1968); C.A. 69, 46128a. K. I. EvsGatova, N. A. Goncharova, and V. Ya. Solomko, Farmatsiya (Moscow) - 17(4 ) , 33-6 (1968); C.A. 69, 99338a.

N.Y., 1975.

126826b.

HYDRALAZINE HYDROCHLORIDE 31 I

18.

19

20.

21. 22. 23 24.

25. 26. n. 28.

33.

34

35.

37.

38

39 9

40.

41.

B. P. Artamanov, T. Ya. Konenkova, and S. L. Maiofis , Med. Prom. SSSR - 19(9) , 57-9 (1965); C.A. 63, 17800f. " U n i t z S t a t e s Pharmacopeia XIX" , Mack Publ ishing CO., Easton, Pa., 1975. K. F u j i i and S. Sa to , Ann. Rept. G. Tanabe Co. L td .

- 1, U.S. Pa ten t 2,484,029; C.A. 44, 4046b. B r i t . Pa ten t 629,177; C.A. 4 q 4516i. Japan. Pa ten t 5526 ( ' 5 4 ) ; CX. 49, 1598221. S. Biniecki and B. Gutkowska, Axa Pol. Pharm. 3, 3 - 3 0 (1955); C.A. 50, 1206211. B r i t . Pa ten t 735,899; C.A. 50, 1080ii. B r i t . Pa ten t 719,183; C.A. 3, 15982i. E. Oish i , Yakugaku Zasshi &, 959-71 ( 1969 1; C.A. 72, 3 4 5 0 ~ . T. P .Sycheva, T. P. Kuz'micheva, A. T. Chernyaeva, T. Kh. Trupp, and M. N. Shchukina, Med. Prom. SSSR

Fr. Pa ten t 2,193,824; C.A.-82, 73014k. Ger. Pa ten t 2,311,510; C.A.30, 108560~. Can. Pa ten t 992,082; C.A. 86743728m. H. M. Pe r ry , J. Lab. Clin.Med. - 41, 566-73 (1953); C.A. 47, 1060ii. A. R . S c h u l e r t , Arch. i n t e r n . pharmacodynamie B,

S. Fa l l ab , Helv. C z m . Acta - 45, 1957-65 (1962); C.A. 2, 5 2 4 2 ~ . S. B in ieck i , M. Moll , K. Niewiadomski, and M. Rzewuski, Acta Pol . Pharm. 33, 425-7 (1976); C.A. - 86, 161223r. J. Wagner, J. W. Fa ig l e , P. Imhof, and G. L iehr , Arzneim.-Forsch. - 8, 2388-95 (1977); C.A. - 88,

M. M. Reidenberg, D. Drayer, A. L. DeMarco, and C. T. Be l lo , Cl in . Pharmacol. Ther. 2, 970-7 (1973); C.A. 9, 449. W. M. McIsaac and M. Kanda, J. Pharmacol. Exptl. Therap. x, 7-13 (1964); C.A. &, 11233b- H. Zimmer, J: McManus, T. Novinson, E. V. Hess, and A. H. Li twin, Arzneim.-Forsch. 20, 1586-7 (1970); C.A. 2, 40761h. H. Zimmer, J. Kokosa, and D. A. Ga r t e i z , Arzneim.- Forsch. 23, 1028-9 (1973); 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 (1973); C.A. 80, 43862b.

1-3 (1956); C.A. 51, 6650~.

- 14(2 ) , 13-17 (1960); C.A. 54, 22669g.

1-15 (1961); C.A. 55, 22471~.

-

13066111.

-


42.

43.

44.

45

46.

47

48.

49 9

50

51-

52.

53

54 9

55

56

57 58.

59

60.

T. Ta lse th , Eur. J. Clin. Pharmacol. 2, 395-401 (1976); C.A. 86, 100742n. H. Zimmer, R. Glaser, J. Kokosa, D. A. Ga r t e i z , E. V. Hess, and A. Li twin, J. Med. Chem. - 18, 1031-3 (1975); C.A. 5, 188169~. A. J. McLean, K. D. Haegele, P. DuSouich, and J. L = McNay , Eur. J. Drug Metab. Pharmacokinet . z( 1 1, 17-20 (1977); C.A. 88, 9911011. C. D. Douglass and R. Hogan, Proc. SOC. Exptl. Biol. Med. - 100, 446-8 (1959); C.A. 53, 14319f. K. Haegele, H. B. Skrd lan t , N. W. R x i e , D. Lalka, and J. L. McNay, J. Chromatogr. 126, 517-34 (1976); C.A. 86, 25798e. K. D.Haegele, A. J. McLean, P. DuSouich, H. B. Skrd lan t , B. Werckle, and J. L. McNay, Clin. Res. - 25, 4 6 O A (1977). S. B. Zak, T. G. G i l l e r an , J. Kar l iner , G. Lukas, J. Med. Chem. 17, 381-2 (1974); C.A. 81 , 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. Bern- s h t e i n , and I. Ya . Kul, Aptechn. Delo - 12, 60-2 (1963); C.A. 61, 9357b. Z. Modras, Farm. Pol. 3, 347-8 (1969); C.A. 71, 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. Scra tch ley , J. Chromatogr.

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 raspol l - skaya, Aktual. Vop. Farm. m, 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 ra t . 2, 571-6 (1956); C.A. 53, 14421a. U.S.S.R. Pa ten t 148,956; C.A. 58, 1311h. G. I. Luk'yanchikova, Med. P r o z SSSR - 16(9) , 46-8 (1962); C.A. 58, 5454h. S. B. Z a k , M. F. Bar t le t t , W. E. Wagner, T. G. G i l l e r an , and G. Lukas, J. Pharm. Sci . a, 225-9 (1974); C.A. 80, 140963t. W. Grabowicz and J. Brul inska, Farm. Pol. 29, 805-7 (1973); C.A. 80, 112718~.

- 114, 329-33 (1975); C.A. 84, 65305g.

HY DRALAZINE HYDROCHLORIDE 313

61.

62.

63-

64.

65 - 66.

67

68.

69

70.

71-

72-

73.

74.

75

76 - 77.

78 - 79

H. E l l e r t and Z. Modras, Farm. Pol. a, 511-15 (1971); C.A. 76, 6766v. T. Urbanyi and A. O'Connell, Anal. Cheni. - 44, 565-70 (1972); C.A. - 76, 90100d. 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 - 14(11) , 34-6 (1960); C.A. 55, 9789g. Ya. M. Perel'man and K. I. Evs t ra tova , Aptechn. Delo 12(1) , 45-9 (1963); C.A. 61, 9360h. N. S.Goryacheva and L. N. P r f l o d k i n a , Farmatsiya (Moscow) 17(3), 69-72 (1968); C.A. 69, 61575n. R. Solirnan and S. A. Be la l , Pharmazie 3, 204 (1974); C.A. 81 , 111504j. R. Solirnan anTS. A. Belal , J. Drug Res. 6, 7-11 (1974); C.A. 83, 84918e. S. Bela l and R. Soliman, Pharmazie 2, 59-60 (1975); C.A. &, 15712~ . H. McKennis and A. S. Yard, U.S. Dept. Corn., Off ice Tech. Serv., PB Rept. 143,914 (1957); C.A. 55, 17375i. A. Viala, Trav. soc. pharm. Montpel l ier - 18, 96-100 (1958); C.A. 53, 8534g. T. Giovanoli-Jakubezak, J. Chodkowski, and D. Gralewska, Rocz. Chem. - 45, 1315-28 (1971); C.A. - 76, 30113a. Z. Modras, Chern. Anal. (Warsaw) 17, 1349-53 (1973); C.A. 78, 128457g. H. ZiKer, R. G lase r , J. Kokosa, D. A. Ga r t e i z , E. V. Hess, and A. Li twin, J. Med. Chem. - 18, 1031-33 (1975); C.A. 83, 188169~. J. M. Lesser , Z. H. Israili, D. C. Davis, and P. G. Dayton, Drug Metab. Dispos. 2, 351-60 (1974); C.A. 82 , 64s. J. B.Smi th , J. A. Moll ica , H. K. Govan, and I. M. Nunes, Amer. Lab. - 4 ( 5 ) , 13-17, 19 (1972); C.A. 77, 39 28%. J. B. Smith, J. A. Moll ica , 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 tewar t , A. P. Smith, and D. W. Hes te r , J. Pharm. Sci . - 64, 1201-4 (1975); C.A. Q, 103357s. D. B. Jack , S. Brechb'ihler, P. H. Degen, P. Zbinden, and W. R iess , J. Chrornatogr. 115, 87-92 (1975); C.A. 84, 38500k.

3 14 CHESTER E. ORZECH ET AL.

80. S. B. Zak, G. Lukas , and T. G. G i l l e r an , Drug

81.

82.

83.

84.

85.

Metab. Dispos. 2, 116-21 (1977); C.A. 87, 95267a. T. Ta lse th , Eur. J. Clin. Phamacol . 2, 183-7 (1976); C.A. 5, 1864599. T. Ta lse th , Clin. Pharmacol. Ther. 3, 715-23 (1977); Index Medicus 1977 (Sept. 1, 362-3. T. Ta lse th , Clin. Pharmacokinet. 2, 317-19 (1977); Index Medicus 1978 (Feb.), 363. K. M. Smith, R. N. Johnson, and B. T. Kho, J. Chromatogr. 137, 431-7 (1977); C.A. 87, 7 3 4 1 7 ~ . P. A. Reece, P. E. S t an ley , and R. Zaces t , J. Pharm. Sc i . 67, 1150-3 (1978).

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


CALCIUM LEUCOVORIN

Leslie 0. Pont, Andrew P . K . Cheung, and Peter Lirn

I . Description 1 . 1 1.2 Appearance, Odor, Color 1.3 Isomeric Forms

2. I Infrared Spectrum 2.2 Proton Magnetic Resonance Spectrum 2.3 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

Name, Structure, Empirical Formula, Molecular Weight


Carbon- 13 Magnetic Resonance Spectrum

3. Synthesis 4. Stability

4.1 Bulk 4.2 Solution

5. Metabolism 6. 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. 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 . 6 Chromatography

6.7 Radioassay 7. Acknowledgments 8. References


ISBN 0-12-260808-9 3 15

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-hexahydro- 4-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 Molecular Weight

Leucovorin C20Hz3N707 473.449

Calcium leucovorin CZ0H2 ,N707Ca 511.513

Calcium leucovorin is also identified by the National Can- cer Institute number: NSC 3590.

H

I

Citrovorum f a c t o r CF, f o l i n i c a c i d , l eucovor in , 5fHbF

CALCIUM LEUCOVORIN 317

R,

C-NH-CH I CH 2 I CH z I COOH

I1

IIa R 1 = OH R2 = H Folic acid, FA

IIb R1 = NH2 R2 = CH3 Methotrexate, amethopterin, MTX

IIc R 1 = OH R2 = CHO 10 formylfolic acid, lOfFA

COOH

I11

IIIa R 1 = H R z = H Tetrahydrofolic acid, H4F

IIIb R1 = CH3 R z = H 5-methyltetrahydrofolic acid,

IIIc R1 = H R2 = CHO 10-formyltetrahydrofolic acid, 5mH F

10fH4F

+ c1-

IV

N5 ,N1 O-methenyltetrahydrofolic acid , anhydroleucovorin, 5,lO-methenylH4F

3 18 LESLIE 0. PONT ET AL.

I

CH z I CH z I COOH

v N',N'o-methylenetetrahydrofolic acid , 5 ,lo-methylene H4F

1 . 2 Appearance, Odor, Co lo r

Calcium l e u c o v o r i n o c c u r s as 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 molecu le s of h y d r a t i o n ) .

1 . 3 I s o m e r i c Forms

Two asymmetr ic ca rbon a toms, t h e a ca rbon 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 molecu le and t h e C 6 ca rbon i n t h e t e t r a h y d r o p t e r i d i n e r i n g , a l l o w f o u r p o s s i b l e i s o m e r s . S i n c e s y n t h e t i c p rocedures would undoub ted ly 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 are reduced t o t h e dL and 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 1L; s e p a r a t i o n of 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 of t h e ca l c ium s a l t s . ' x

R e c l a s s i f i e d as Ped icoccus 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 . P h y s i c a l P r o p e r t i e s

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 of 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 is t h e absence of s h a r p a b s o r p t i o n bands, which is 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 are l i s t e d t h e major a b s o r p t i o n a s s ignmen t s :

Ir a b s o r p t i o n ( p ) Assignment

'L 3.0 H Z O , N-H(2)

6.0-6.7

'L 7 . 1

1.1.9

H'O, amide I , a r y l sys t ems ,

c0'-

CO,-, amide I1

( t e n t a t i v e )

2 .2 P ro ton Magnetic Resonance Spectrum

Due t o t h e i o n i c n a t u r e of calcium l e u c o v o r i n , a n 'H nmr spectrum cou ld 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 ; t h e r e f o r e , t h e spectrum 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 ignmen t s have been made r e l a t i v e t o TMS = 0.00 ppm from a 100-MHz Varian XL100 spectrum (F igure 2 ) :

Assignment Chemical S h i f t (ppm) M u l t i p l i c i t y J (Hz)

H 8 , Y 1.70-2.60 m

H 7 , 9 2.95-3.80 m

H a 4.00-4.60 (obscured by HOD) q

0 00000 0

g z

r" i-9

t"??

7

0

0 0 0000

33

~w

atl osaw

320

m

m

z LT 0

>

3

w

-I

8

5

2

2 a u U 0

I

13 LT I- n

n

$ 0

w

LT

2 U z - W

LT 13 c7 U

8 7

vl i

E

Q

P

32 I

322 LESLIE 0. PONT ET AL.

Assignment Chemical S h i f t (ppm) M u l t i p l i c i t y J (Hz)

H 3 ' , 5 ' 6 .64 d 8

H 2 ' c 6 ' 7.60 d 8

0 II

H-C-, 5 8.59

unknown 7.89

H , 6 + 4 . 8

S

S

m (b road)

The above a s s ignmen t s are i n g e n e r a l agreement w i t h t h o s e of P a s t o r e 3 f o r f o l a t e s . l a r g e HOD peak, and i t s l o c a t i o n was confirmed by s h i f t i n g t h e HOD abso rbance a t 80" C. The peak a t + 7.9 ppm due t o a n unknown s p e c i e s h a s been p r e s e n t i n a l l ca l c ium leuco- 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 remains 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 formyl group c o n t r i b u t e s t h i s peak. S i n c e chemica l ly p r e p a r e d ca l c ium l e u c o v o r i n i s a s sayed a t t h i s l a b o r a t o r y , a n 'H nmr spectrum 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 enzymati- c a l l y p repa red m a t e r i a l . E i t h e r shou ld 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 . I n v e s t i g a t i o n s t o r e s o l v e t h i s problem are c o n t i n u i n g .

The C, p r o t o n i s obscured by t h e

2.3 Carbon-13 Magnetic Resonance Spectrum

The 1 3 C nmr spec t rum reproduced 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 Varian X L l O O Spec t romete r . The a s s ignmen t s on t h e TMS scale and r e f e r e n c e d t o d ioxane are l i s t e d below:

Assignment Chemical S h i f t (ppm)

c- 2 93 .7

c- 4 102 .0

C- 6 0.0

c- 7 - 2 3 . 3

0 I1

- C-H 98.2

c- 9 - 25.0

I I I I 1 I 1 I I 1 1 1

D IOXAN E

c

I I I I I I I

PPm

FIGURE 3 13C NMR OF CALCIUM LEUCOVORIN

LSJ-57


A s s i gnmen t

C-4a o r C-8a

C-8a o r C-4a

c-1' C-2 I , C-6'

C-3', c-5'

c-4

c-7 ' C-Ci

C- B

C-Y

a- coo- y-coo-

Chemical S h i f t (ppm)

25.2

86.5

54 .7

62 .4

45.7

84 .8

102.8

- 1 0 . 8

- 37.9

- 32 .3

112.6

1 1 5 . 6

The above a s s ignmen t s are i n r e a s o n a b l e agreement w i t h t h o s e p r e v i o u s l y r e p o r t e d 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 spec t rum of ca l c ium l e u c o v o r i n i n pH 7.0 phospha te 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 are s imi 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 - 1 2 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 Q 2.7 x lo4 M-' cm-' t o 3 .26 x 10' M-' crn-l'l. Robinson" r e p o r t s E~~~ app rox ima te ly 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 abso rbance a t s h o r t e r and l o n g e r wave leng ths 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 educed f o l a t e s . S p e c t r a l 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 abso rbances because 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 anhydro leucovor in , N 5 ,N"-methenyl te t rahydrofol ic a c i d , I V . T h i s compound h a s been i s o l a t e d i n more t h a n one form, depending on pH. l 3


1.0

0.9

0.8

0.7

w 0.6 0 z a

0.5

2 0.4

0.3

0.2

0.1

0 200 250 300 350

nm LSJ-58

FIGURE 4 ULTRAVIOLET SPECTRUM OF CALCIUM LEUCOVORIN 0.1N NaOH ~

pH 7 Phosphate buffer - -

326

2 . 5 Mass Spectrum-

LESLIE 0. PONT ET A L .

An e l e c t r o n impact mass spec t rum of ca l c ium l euco- v o r i n h a s n o t been o b t a i n e d because t h e compound i s n o t s u f - f i c i e n t l y v o l a t i l e . 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 , ca l c ium l e u c o v o r i n 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 l e u c o v o r i n . Indeed, 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 calcium 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 barium s a l t . ' D i f f r a c t i o n p a t t e r n s cou ld n o t b e o b t a i n e d on a l l samples a l t h o u g h t h e y had been r e c r y s t a l l i z e d sev- e r a l 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 re l i s t e d , b u t t h e s e num- b e r s appea 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 O p t i c a l R o t a t i o n 0

2 1

a ] = + 1 4 . 3 k 0 .4" (c 1, N/10 NaOH) 5 8 9

0 2 1

a ] = 17.9 k 0.4' (c 1, N/10 NaOH) 5 4 6

0 2 1

0.3 = + 14.9 ? 0 .4" (C 1, H20) 5 8 9

2 1

a ] = + 18.8 t 0.4" (c 1, H z O ) 5 4 6

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 ca l c ium 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 - t i o n i s c l o s e t o t h e l i t e r a t u r e v a l u e : 3.42 H,O).'

( r I D = + 1 4 . 2 6 " (c,

CALCIUM LEUCOVORIN

2.8 C i r c u l a r Dichroism

327

The fo l lowing d a t a were o b t a i n e d on an average sample of calcium leucovor in :

Solvent & (nm) [ e l (deg M-lcm-')

0.1N NaOH 282 3.89 103

H 2 0 287 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 is 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 than one a b s o r p t i o n band. Whether t h e ex t raneous a b s o r p t i o n s are due t o i m p u r i t i e s o r are ac tu- 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 behavior i s n o t known a t t h i s t i m e .

2.9 D i s s o c i a t i o n Cons tan ts

Three pKa v a l u e s have been r e p o r t e d f o r leuco-

The f i r s t two v a l u e s v o r i n ( f r e e a c i d ) ; they a r e 3.1, 4 . 8 , and 10 .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 a r e a t t r i b u t e d t o t h e glutamyl 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 hydroxyl 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 of ca lc ium l e u c o v o r i n s e v e r e l y restricts 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 ( so l - u b i l i t y i n DMSO << 1 mg/ml). I n water, t h e s o l u b i l i t y is l a r g e (% 100 mg/ml), b u t i n 0.1N NaOH i t drops 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 longer b e l e u c o v o r i n .

3 . S y n t h e s i s

With s l i g h t m o d i f i c a t i o n s i n procedure , one b a s i c syn- t h e s i s of l e u c o v o r i n and t h e subsequent i s o l a t i o n of i t s sa 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 hydrogenat ion 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 presence of a p la t inum o r pa l lad ium 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 .15 This


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 fo rmyla t ion of f o l i c a c i d i n fo rmic a c i d . of t h e r e a c t i o n mix tu re i s a d j u s t e d w i t h b a s e i n t h e p re s - ence 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 w i th o r w i t h o u t reduced p r e s s u r e ) . P u r i f i c a t i o n by column chromatography i s fol lowed 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 so l - ven t . s o l u t i o n by a d d i t i o n of C a C l z fol lowed by e t h a n o l . * '

The pH

The calcium s a l t i s p r e c i p i t a t e d from a n aqueous

IV

CALCIUM LEUCOVORIN

NaHCO, or NaOH or both (antioxidant)

329

cm IIIC

CaC1. or Ca(OH), Ethanol I

Calcium lcucovorio

Early syntheses' ' ' oxidant 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~~ or a stream of nitrogenz2 to to protect the tetrahydro system.

employed ascorbic acid as the anti-

Isolation of the active component is carried out chro- matographically. 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.


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 concen- trates 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 ulti- mately 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. essential reactions are summarized below: 3 2

Folic acid + dihydrofolate

The

t et rahydrof olate H H

+ serine I dihydrofolate' reduc tase

NADP' N 5 , N N5 ,N1 O-methenyltetrahydrofolate- hydrof olate + glycine

NADPH "active formaldehyde''

-me thy lene t e tra-

NADH lr NAD+ M lt N 5-f ormyltetra- - N1 O- f ormyl- hydrofolate te t rahydro f o la te

N5-methyl tetrahydrof olate

CALCIUM LEUCOVORIN 33 I

R e c e n t l y , t h e enzymatic format ion 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 pro- d u c t s . 3 4

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 starts 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 , incubated i n t h e presence 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 incubated w i t h 5,lO- methylenetetrahydrofolate dihydrogenase. The p r o d u c t , N 5 ,N1'-methenyltetrahydrofolate (wi th a s c o r b i c 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 products 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 are 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 ce l l s , t i s s u e s , and animals .

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 ,

4 . S t a b i l i t y

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 60" C. The s t a b i l i t y w a s monitored 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 S o l u t i o n

Citrovorum f a c t o r has 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 Broquis 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 minutes 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 procedures 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 anhydroleuco- v o r i n . 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 % 13) and i n de ionized water (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 tempera ture 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 hours (concent ra - t i o n 1 mg/ml). S t a b i l i t y w a s monitored by h igh-pressure 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 . A s s t a t e d p r e v i o u s l y , l e u c o v o r i n i s n o t 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 of anhydroleucovorin 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 anhydroleucovorin A; p H = 4 ( h o t ) anhydroleucovorin B. A l l t h r e e forms conver 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 anaerobic c o n d i t i o n s

Steam-heating f o r 30


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 formyl group i s i n c o r p o r a t e d from t h e 5 , lO methylene compound. 3 3 y vorum f a c t o r , when i n t h e presence of a l a r g e excess of glutamic 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 ismiss t h e p o s s i b l e format ion of 10fH4F o r 5,lO-methenyl H4F i n t h e i r r e a c t i o n s . How- e v e r , Peters and Greenberg4’ r e p o r t e d s e p a r a t i n g an enzyme from sheep l i v e r , c i t rovorum f a c t o r cyc lohydrase , which converted 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,lO- methenyl 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, has been r e p o r t e d t o donate i t s formyl group t o methionine, which i s a s s o c i a t e d w i t h an esteri- 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 cont inues 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 presence of approp- r 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 donate formyl groups f o r p u r i n e s y n t h e s i s 4 4 :

The N S

Silverman e t a1.41 have shown t h a t t h e c i t r o -


Equa t ion 1 i s c a t a l y z e d by g lyc inamide r i b o t i d e (GAR) t r a n s - fo rmylase and Equa t ion 2 i s c a t a l y z e d by aminoimidazole- 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 rats, 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 o f f o l i c a c i d r e s u l t e d i n i n c r e a s e d 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. cerevisiae and t h a t w a s presumed t o b e c i t rovorum T h i s r e sponse 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 reduced 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 been s p e n t i n s t u d y i n g t h e metabol ism o f l e u c o v o r i n i n v i v o . This emphasis was prompted by t h e chemica 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 of methotrex- a t e when i t w a s g iven 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 . Fol- i 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 ma jo 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 are poly- g l u t a m a t e s , a l t h o u g h t h e y are t r a n s p o r t e d as monoglutamates.

S e v e r a l groups i n v e s t i g a t i n g human metabol ism 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

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 meta- b o l i c p r o d u c t s . Gene ra l ly , L. casei r e sponds t o a l l mono- g l u t a m a t e s , s. f a e c a l i s a l s o measures monoglutamates excep 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 ra - 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’ showed a l a r g e i n c r e a s e i n L. casei r e s p o n s e and a l e s s 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 any , 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 concluded t h a t 5fH,F i s r a p i d l y me tabo l i zed t o 5mH4F, a l t h o u g h a v e r y small amount may b e absorbed as t h e i n t a c t 5-formyl compound. P e r r y and Chanarin4’ found s i m i l a r r e s u l t s i n u r i n e samples . 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-’ 4C- t e t r ahydro fo la t e -3H, i n v e s t i g a t o r s have been a b l e t o t race t h e m e t a b o l i c p r o d u c t s when t h e d rug is a d m i n i s t e r e d o r a l l y o r i n t r a v e n o u s l y .

Using t h r e e microorganisms t h a t respond t o


Nixon and B e r t i n o have done an e x t e n s i v e s tudy of f o l i n i c a c i d metabolism i n human s u b j e c t s g iven t h e drug both ways. When given o r a l l y , a f t e r 75 minutes t h e major- i t y (90%) of 3H i n serum w a s found i n 5mH4F, w i t h 20% of

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 each l a b e l w a s found as 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 chromatographic c o n d i t i o n s used, 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 admin- i s t r a t i o n ) was % 40% 3H-labeled 5fH4F. Less than 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-aminoben- zoylg lu tamate , 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 mixture 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 p r o p o r t i o n s 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 assoc- i a t e d w i t h serum f o l a t e . Urine showed most r a d i o a c t i v i t y as 5fH4F i n i t i a l l y , and s e v e r a l hours l a t e r a rise w a s seen i n l a b e l e d 10fH4F and/or 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 assoc- i a t e d w i t h 5mH4F, most of t h e drug w a s absorbed 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 conver ted t o 5mH4F. Equal ly important w a s t h e s i t e of metabolism. S ince t h e 5f -+ 5m convers ion 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 admin- i s t r a t i o n , t h e a u t h o r s f e l t t h a t t h e r e a c t i o n took p l a c e i n t h e i n t e s t i n e , a view suppor ted by o t h e r s . ’ l The voided f o l a t e s seemed t o be 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 con- served over 5fH4F by t h e kidneys.

1 4

Although leucovor in is r a p i d l y absorbed and metabo- l i z e d i n t h e i n t e s t i n e , i t s metabol ic product , 5mH4F, is concent ra ted 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 sappeared from serum and appeared as 5mHbF i n CSF fo l lowing 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 methionine and t h e 3H p o r t i o n t o 5mH4F. The uptake of drug occurred 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 w i t h nonlabe led


compound,53 followed by b) c e l l u l a r uptake and metabolism. Early papers repor ted a n a t u r a l concent ra t ion 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 equ i l ib r ium concent ra t ion i n serum. This could i n d i c a t e the importance of f o l a t e s i n neu ra l metabolism, and t h e r e have been some r e p o r t s regard- ing t h e the rapeu t i c t reatment of neu ropsych ia t r i c d i s o r d e r s with f o l a t e s .

The r ecen t i n t e r e s t i n leucovor in i s due t o i t s a b i l i t y t o reduce methotrexate (MTX) t o x i c i t y when both are admin- i s t e r e d t o cancer p a t i e n t s . t h a t most b e n e f i t occurred when leucovor in w a s given 12-24 hours a f t e r MTX in fus ion . The de lay i n admin i s t e r ing leucovorin reduced t h e tox ic e f f e c t s of MTX without reducing the l a t t e r ’ s ant i tumor a c t i v i t y . Therefore , an enhancement of MTX e f f e c t can be produced by l a r g e r dosages with- ou t t h e t o x i c i t y a s s o c i a t e d wi th these amounts. When given concurren t ly , l eucovor in reduced MTX t o x i c i t y a t t h e ex- pense of tumor a c t i v i t y . However, r ecen t work5’ has shown some advantage i n concurren t admin i s t r a t ion . I n t h e case of MTX-sensitive c e l l s , “ f o l i n i c ac id p ro tec t ion” 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 procedure, o r leuco- vo r in given a f t e r a delay per iod , was most e f f e c t i v e (measured i n percentage of c e l l s u r v i v a l ) i n t r e a t i n g MTX- r e s i s t a n t c e l l s . The the rapeu t i c e f f e c t of leucovor in l i e s i n an ear l ie r resumption of DNA s y n t h e s i s than i f MTX were adminis tered a lone . 5 6 Nahas e t a l . a t t r i b u t e t h i s resumption t o s e v e r a l poss ib l e a c t i o n s of leucovor in (as 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 is increased i n t h e presence of 5fH4F; b) 5fH4F could deblock d ihydro fo la t e reductase bound by MTX by be ing a p o t e n t i a l supply of d ihydro fo la t e ; c ) t h e i n t a c t drug could poss ib ly d i s p l a c e some of t h e MTX; and d) 5fH4F competes wi th 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 be 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 of ox id ized f o l a t e s , and by ter- minal glut am ate^.^' Thus 5fH4F, 5mH4F, and MTX appear t o use the s a m e car r ie r -media ted 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 ex ten t . Another au thor” desc r ibes 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 membranes as an exchange phenomenon because al though leuco- vo r in competes wi th MTX f o r c e l l uptake, 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 are a l r eady

Ear ly s t u d i e s s 4 i n mice showed


preloaded wi th f o l i n i c ac id . The i n f l u x i s noted even i n cases where d ihydro fo la t e reductase has been i n a c t i v a t e d by previous MTX dosage.


6 . 1 Elemental Analysis

Below are l i s t e d elemental a n a l y s i s r e s u l t s ob ta ined from a t y p i c a l sample of calcium leucovorin:

Element % Theore t i ca l % Found*

C 46.96 46.87

H 4.14 4 . 1 1

N

Ca

19.17

7.84

19.07

6 . 9 4 t , 7 .71$

* Corrected f o r 10.4% HzO.

+Found by atomic absorp t ion . ?Found by s u l f a t e ash .

The h ighe r calcium va lue obtained by t h e ash method i s no doubt due t o t h e presence of sodium ( t h e counter i on f o r a c e t a t e commonly found i n some l o t s ) .

6.2 Equivalent Weight Determination

Because s y n t h e t i c products are i s o l a t e d as t h e barium o r , more f r equen t ly , t h e calcium s a l t of leucovor in , common acid-base t i t r a t i o n s are not r epor t ed . I f t h i s type 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 impur i t i e s conta in ing the glutamic a c i d moiety would respond s i m i l a r l y t o leucovor in when the carboxyl groups are be ing analyzed.

6.3 B io log ica l Assay

6 .3 .1 Microbio logica l Assay

Severa l microbio logica l procedures f o r assaying CF have been descr ibed i n t h e l i t e r a t u r e . I n genera l , t hese methods are designed t o measure CF content i n a b i o l o g i c a l f l u i d o r specimen. t he growth promotion of L. c i t rovorum 8081 , o r p. E- visiae , as i t has been renamed. Before CF w a s i s o l a t e d

They a l l make use of


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’ measured enhanced growth o f t h e mic roorgan i sm i n t h e p r e s - ence 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 con- c 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 roduced . 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 o f 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 growth i n a s t a n d a r d r e t i c - u logen sample . The a u t h o r s r e a l i z e d the 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 caused enhanced growth a f t e r a n i n i t i a l l a g . Wins t en and Eigen” t r i e d t o improve t h e method by ch romatograph ing t h e samples 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 rowth , b u t t h e y , t o o , found more t h a n a s i n g l e ac t ive component. The b a s i s o f t h i s p a r t i c u l a r t e c h n i q u e i s s t i l l b e i n g u s e d . Cooper- 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 i s 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 . Q u a n t i t a t i o n i s i n t e r m s o f mg/ml, u s i n g commercial s amples o f l e u c o v o r i n as s t a n - d a r d s . In one 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 are impregna ted w i t h t h e test s o l u t i o n s and CF c o n t e n t i s found by manua l ly m e a s u r i n g L. c i t r o v o r u m ATCC 8081 growth o n a g a r 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 be 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 a m e t h o p t e r i n b e c a u s e t h e mic roorgan i sms a re res i s tan t t o t h e l a t t e r . 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 is 1 0 % .

6 .3 .2 Enzyme Assay

S i lve rman e t a1.41 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 the fo rmyl group 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 -aminobenzoy lg lu t ama te and p t e r i d i n e . Arylamine f o r - mat ion c o u l d be mon i to red by t h e Bra t ton -Marsha 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 of enzyme a c t i v i t y t h a n C F 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 and 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 s t r a i g h t f o r w a r d . The t e s t s o l u t i o n s a re mixed w i t h ATP, MgSO,, and 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 n m i s r e a d i n a spec - t r o p h o t o m e t e r a t 30” C . The 5,lO-methenyltetrahydrofolate b e i n g formed s h o u l d g i v e a d i r e c t measure o f t h e CF o r l e u c o v o r i n p r e s e n t .


The above b i o l o g i c a l a s s a y s are i n t e n d e d t o measure CF i n b i o l o g i c a l sources r a t h e r than t o d i r e c t l y measure leucovor in o r CF p u r i t y . I n f a c t , commercial samples of l e u c o v o r i n are u s u a l l y used t o de te rmine s t a n d a r d c u r v e s .

6.4 Polarographic Assay

Leucovorin, s i n c e i t i s t o t a l l y reduced, is 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 are genera ted , cor responding t o a n anodic o x i d a t i o n of a t e t - rahydro 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 least one of t h e s e t h r e e is 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 behavior .

6 . 5 Spec t rophotometr ic Analys is

6 . 5 . 1 F luorometr ic Analys is

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 of 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 emiss ion a t 460 nm. A c o n c e n t r a t i o n of 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 explored 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 pres - ence 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 emission band of t h e compound of 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 mixture . A l a t e r papers5 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 leuco- v o r i n a t 365 nm 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 ; 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-aminobenzoyl- g lu tamate l e a d them t o conclude t h a t t h i s p o r t i o n of t h e molecule is r e s p o n s i b l e f o r maxima a t 360-425 nm when e x c i t e d a t 300-320 nm. They sugges ted 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.


I n 1964, Net rawal i e t a1.66 d e s c r i b e d f l u o r e s c e n c e measurements of ca lc ium 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 paper 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 paper- gram w a s very 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 ob ta ined from f l u o r - escence were lower by 10 t o 20% t h a n t h o s e obta ined 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 Analys is

U l t r a v i o l e t spec t roscopy 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 , because commercial samples are f r e q u e n t l y contaminated 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 determined f o r l e u c o v o r i n . Recent ly , 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 pure 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 of Zakrzewski and Sanson. '

Second, l e u c o v o r i n i s known t o dehydra te under a c i d i c c o n d i t i o n s t o form anhydroleucovor in , 5,10-methenyl H4F, which absorbs a t 352-353 nm. I n t h e absence 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 ana lyzed by a c i d i f i c a - t i o n w i t h 0 . 1 N H C 1 fol lowed by uv measurement a f t e r 1.5- 2 . 0 hours . P u r i t y may be determined 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.''

* Absorpt ion i n t e r f e r e n c e s would arise from compounds o t h e r than l e u c o v o r i n t h a t conver t t o anhydroleucovorin a t a c i d i c pH. An example of such a compound i s 10fH4F, a l though i t i s n o t expected t o c o n t r i b u t e t o a b s o r p t i o n due t o i t s i n s t a b i l i t y . However, 10-formyl-7,8- d i h y d r o f o l a t e i s a p o s s i b l e contaminant . A d d i t i o n a l a b s o r p t i o n could 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

6.6 Chromatography

6 . 6 . 1 Paper Chromatography

LESLIE 0. PONT ET A L .

Both paper 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. Paper chromatography h a s been used 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 samples 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 ioau to - g r a p h i c method. t r i m e t h y l p y r i d i n e t o deve lop papergrams on Whatman No. 1 o r No. 4 . Col6nZ9 developed h i s chromatograms i n isoamyl a l coho l :5% d i b a s i c sodium phospha te , 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 mob i l e phases i n t h e s e systems are 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 o f l e u c o v o r i n .

Winsten and E igenS9 used 2,4,6-

6 .6 .2 Th in - l aye r Chromatography

Thin- layer chromatography on c e l l u l o s e h a s been used as a n i n t e r m e d i a t e clean-up p rocedure 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 Copen- haver and O ' B ~ i e n , ~ ' and t h e mobile phase w a s 15% Na,HPO,* 12H20 (pH 8 .5 ) c o n t a i n i n g 0 .1 M mercap toe thano l . Unfor- t u n a t e l y , t h i s system d i d n o t s e p a r a t e 5f and 10fH4F from each 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 * la t te r w a s ach ieved w i t h 11 N a 2 H P 0 4 * 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 s p r a y .

Brown e t a1 .68 have developed a c e l l u l o s e p l a t e w i t h a f l u o r e s c e n t i n d i c a t o r . Compounds a re developed 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 quenching. These a u t h o r s a l s o u s e 0.5% mercap toe thano l i n t h e i r mobile phase , bu 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 reduced p t e r i d i n e s , which a re 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 - t u t i o n a t t h e N 5 p o s i t i o n . S i n c e n e u t r a l o r a l k a l i n e s o l u - t i o n s of l e u c o v o r i n are 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

6 . 6 . 3 Column Chromatography

34 I

Leucovorin h a s been chromatographed on columns of d i f f e r e n t pack ing mater ia l s s i n c e i t w a s f i r s t s y n t h e s i z e d . 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 l e u c o v o r i n . Column t e c h n i q u e s have been coupled 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 . Nor- onha and S i l ~ e r m a n ~ ~ used &. c a s e i , P . c e r e v i s i a e , and - S . 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 ch icken l i v e r e x t r a c t 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 used by Rohringer e t a 1 . 7 0 t o mon i to r f o l a t e s i n h e a l t h y 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-l- c 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 used 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 syn- t h e t i c p r o d u c t s r a t h e r t h a n b i o l o g i c a l m i x t u r e s . 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 gained by micro- b i o l o g i c a l d e t e c t i o n .

An ea r l i e r p u b l i c a t i o n "

More r e c e n t l y , a t t e n t i o n h a s been p l aced on g e l per- meat ion t e c h n i q u e s . Sephadex G-15 and G-25 columns coupled w i t h uv d e t e c t o r s w e r e used t o s e p a r a t e f o l a t e s found i n r a t kidney e x t r a c t s . 7 2 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 markers 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 samples w i t h t h e a p p r o p r i a t e con- j u g a s e t o h y d r o l y z e p o l y g l u t a m a t e s . R a d i o a c t i v i t y measurement has been used t o d e t e c t doubly l a b e l e d 5fH,F and i t s m e t a b o l i c p r o d u c t s . 5 0

K B s and Cer1-19~~ used

6 . 6 . 4 High-p res su re L iqu id Chromatography

A n a t u r a l e x t e n s i o n o f column chromatography is h i g h - p r e s s u r e l i q u i d chromatography, which com- b 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 - 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 work) . Pe rhaps t h e s i n g l e

9

8

7

6

5

4

3

2

1

0

1 I I 1 1 1 1 I

CALC IU h LEUCOVOR IF

-

-

I N J ECT I ON

FOLIC ACID (INTERNAL STANDARD)

0 3 6 9 12 15 18 21 24

LSJ-59 MINUTES

FIGURE 5 HIGH PRESSURE LIQUID CHROMATOGRAM OF CALCIUM LEUCOVORIN


most a t t r a c t i v e f e a t u r e o f t h i s technique, i s s p e e d , once t h e chromatographic c o n d i t i o n s have been de te rmined . Various modes a re a v a i l a b l e , b u t f o l a t e s have been 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 due 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 is 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 magni tude by e l e c t r o - chemica l d e t e c t i o n . HPLC h a s been used 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 ana lyzed are f o l a t e s . 7 5 The most versa t i le system t h a t t h e s e a u t h o r s have 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 methanol ( s e e F i g u r e 5 ) . An e a r l i e r sys t em u s i n g t h e same reverse phase 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 phospha te e l u e n t which b e t t e r r e s o l v e s con taminan t s from t h e major component. A similar 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 Re i f e t a1 .76 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 . O the r a u t h o r s 7 7 have used s imilar 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 con taminan t s . 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 methods, a n i o n exchange h p l c h a s been used 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 g lu t amyl 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 been used 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 mod i f i ed i n t h e f u t u r e t o p roduce a v i a b l e t e c h n i q u e . Leucovorin h a s been used as 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 immobil ized w i t h o u t l o s i n g i t s a c t i v i t y , pe rhaps i t c o u l d be 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 have 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 samples . These t ech - 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 b i n d i n g . 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. casei.


Although t h i s method w a s des igned 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 m i c r o b i o l o g i c a l t e s t i n g c o u l d g i v e a n i n d i - c a t i o n of reduced f o l a t e c o n c e n t r a t i o n .

7. Acknowledgments

Supported 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 Treatment , 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 , Educa t ion , and Welfare . The o p i n i o n s e x p r e s s e d are 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 of t h e N a t i o n a l Cancer I n s t i t u t e .

The a u t h o r s wish t o thank Michael C u l c a s i f o r t h e HPLC development, Barbara 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 John 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 Ardath T r i n e f o r s e c r e t a r i a l s u p p o r t .

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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

1.2 I Empirical 1.22 Structural

1 . 1 Formulae

1.3 Molecular Weight I .4 Elemental Composition 1.5 Appearance. Color, Odor

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

3. Synthesis 4. 5 . Metabolism 6. Methods of Analysis

6. I Titrimetric Analysis 6.11 Aqueous 6.12 Nonaqueous

6.21 Infrared Spectrophotometric 6.22 Nuclear Magnetic Resonance Spectrometric 6.23 Ultraviolet Spectrophotometric

6.3 I Thin-Layer Chromatography 6.32 Gas-Liquid Chromatography 6.33 Paper chromatography

Stability, Decomposition Products. and Metal Complexes

6.2 Spectrophotometric Methods

6.3 Chromatographic Analysis

6.0 Colorimetric Analysis 6.5 Potentiometric Analysis 6.6 Coulometric Analysis

7. References

35 I 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

1.2

1.3

1.4

1.5

Nomenclature

1.11 Chemical Names 1,3-dihydro-l-methyl, 2-H-Imidazole-2-thione. 1-methylimidazole-2-thiol 1-methyl-2-thioimidazole 1 -met h y 1 - 2 -me r c a p t o im id a z o 1 e

1.12 Generic Names Methimazole, thiamazole

1.13 Trade Names Thiamethazole, Bazolan, Dananti- 201, Faristan, Frentorox, Mercazole, Metazole, Tapazole, Thacapazol, Thycapazol, Strumazol, Meto t hyr ine .

Formulae 1.21 Empirical

1.22 Structural ‘qHgN2’

FH3 (iH3

Molecular weight 114.17

Elemental composition C 42.08%, H 5.30%, N 24.54%, S 28.09%

Appearance, color, odor White to pale buff, crystalline powder, having a faint characteristic odor. Its solution is practically neutral to litmus.

2. Physical properties

2.1 Crystal properties

2.11 Crystallinity No detailed studies on the crystal structure of methimazole is reported in the literature. Methimazole can be microscopically identified using Kofler’s method, the occurrence of poly- morphous modification is indicated in methimazole.

METHIMAZOLE 353

2.2

2.3

2 . 4

2 . 5

2.12 Melt ing po in t USP X I X (1) s p e c i f i e s a me l t ing range f o r methimazole between 144 and 147O.

L e a f l e t s from a l coho l m.p. 146-148O(2), b.p. 280' (some decomposi t ion) .

Dipole Moments The d i p o l e moment of methimazole w a s determined i n benzene and 1 , 4 dioxane s o l u t i o n a t 25O (3) and repor ted 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 iscussed i n terms of tautomerism and molecular conformations.

S o l u b i l i t y F ree ly s o l u b l e i n water, i n a l coho l and i n ch loro- 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.

I d e n t i f i c a t i o n The fol lowing tests a r e c i t e d from USP X I X (1) :-

The i n f r a r e d abso rp t ion spectrum of a potassium bromide d i s p e r s i o n of i t exhibitsmaxima only a t the same wavelength as t h a t of a similar pre- p a r a t i o n of USP methimazole Reference Standard.

Mercur i cch lo r ide TS produces i n a s o l u t i o n (1 i n 200)a white p r i c i p i t a t e , bu 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 is coloured i n t e n s e l y b lue by molybdo-phosphotungs- t a t e TS.

Methimazole can be i d e n t i f i e d by forming c r y s t a l s of w i th gold bromide/HCl so lu t ion . P l a t e s , o f t e n i n c ros ses ( 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 rods o r need le s ( s e n s i t i v i t y 1 i n 1000) F ig . 1.

S p e c t r a l P r o p e r t i e s

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 ac id shows maxima a t 211 nm (E 1%, 1 cm 593) and 251.5 nm ( E 1%, 1 c m 1528) . I n n e u t r a l aqueous so lu t ion , methimazole absorbs 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 ig . 2 ) . Hayden _ - e t a1 (5) publ ished 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. atom a t C2 by s u l f u r causes a b ig s h i f t t o longer wavelength wi th increased absorp t ion(6) .

The replacement of an oxygen

3 54 HASSAN Y. ABOUL-ENEIN AND A. A. AL-BADR

Fig. 1 - Methimazole - KI/I c r y s t a l s . 2

F i g . 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 obtained from Nuj 01 mull .

The s t r u c t u r a l assignments have been co r re l a t - ed wi th t h e fol lowing band f requencies :

Frequency (Cm-l) Assignment

2500 - 2450 broad weak - SH

1580 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 as shown i n F igure 4 .

Fur ther in format ion wi th r ega rds t o t h e in - f r a r e d s p e c t r a of methimazole is given i n several r e fe rences (4 ,5 ,7 ) .

A t y p i c a l NMR spectrum of methimazole is shown i n Fig. 5. The sample w a s d i s so lved i n CDC13. The spectrum w a s determined on ir Varian T-60 A NMR spectrometer wi th TMS as t h e i n t e r n a l s tandard . The fo l lowing s t r u c t u r a l ass ignments have been made f o r Fig. 5.

2.53 Nuclear Magnetic Resonance Spectrum

Chemical S h i f t ( 6 ) Assignment

3 S i n g l e t a t 3.63 N-CH

aromatic H and H5 of t h e imida- z o l e r i n g system.

4 S i n g l e t a t 6.70

Fur ther in format ion concerning t h e NMR spectrum of methimazole can be obta ined from S a d t l e r NMR c a t a l o g (8) and a l s o from CRC Atlas of s p e c t r a l d a t a (7) and Aldr ich NMR c a t a l o g ( 9 ) .

2.54 Mass Spectrum and Fragmentometry The mass spectrumof rnethimazole obtained by e l e c t r o n impact i o n i z a t i o n shows a pronounced molecular ion d a t m / e 114 (Fig. 6 ) .

Bowie e t a1 (10) s tud ied t h e mass s p e c t r a of s e v e r a l imidazoles and methimazole w a s included. They d iscussed t h e fragmentat ion

P

a

al k

cd k

w

C H

356

357

m

z I 2 m

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 alkylisothiocyan- ates. 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. Methi- mazole 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-

3 "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) 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%).

with KSCN in the presence of dil. HC1.

dil HC1 CH3-NH-CH2-CH(0CH CH ) + KSCN 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 wel l -c losed c o n t a i n e r p r o t e c t e d from l i g h t a n d i n a d r y p l a c e .

Methimazvje forms metal complexes w i t h heavy metals e . g . C U + ~ , A 1 c h e l a t i n g p r o p e r t i e s of some imidazoles 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 inding 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 .

, Fe+3 ions . Foye and Lo (14) s t u d i e d t h e

5. Metabolism When 20 mg/kg of methimazole w a s adminis te red i . p . o r o r a l l y t o rats, u r i n a r y methimazole g l u c u r o n i d e s accounted f o r 36-48% of t h e d o s e i n 24 hours . The only 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 conta ined 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 as 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 any 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 than d i d p r o p y l t h i o u r a c i l . Between 77 and 95% of t h e methimazole w a s e x c r e t e d i n t h e u r i n e and approximately 10% i n t h e b i l e . S i n c e f e c a l e x c r e t i o n w a s n e g l e g i b l e ; 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 hours r e g a r d l e s s of t h e r o u t e of a d m i n i s t r a t i o n (15) .

35S - l a b e l l e d methimazole g iven i . p . t o ra t s accumulated i n t h e t h y r o i d gland where i t w a s mainly oxid ized 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 tman 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 organ t o plasma r a t i o s of t h e drug a f t e r f o u r days of i .v . a d m i n i s t r a t i o n i n rats. 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 lowly absorbed 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 hours of 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 carb imazole - S was v e r y s imilar t o t h a t of methimazole. I t w a s sugges ted by Alexander e t a1 t h a t t h e r e n a l f u n c t i o n may have a more impor tan 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 drug t h a n t h e t h y r o i d s t a t u s (19) . 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 inves- t i g a t ion .

35

--


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. 1 ml of bromothymol blue TS, and continue the titration with the 0.1 N NaOH until a permanent, 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.

Iodometric methods

Kossakowski _ _ et a1 (20) developed the following method for determination of methimazole (thiamazole) in substances and tablets.

A 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. methimazole in compound drugs provided they

Add

4 6 2

The method was useful in determining

METHIMAZOLE

con ta in no s-' i ons .

363

b) By t h e method Blazek e t a1 (21) The de termina t ion i s done by ox ida t ion of t h e -SH group wi th 0.1N iod ine s o l u t i o n i n NaHC03 s o l u t i o n v i s u a l l y wi th r e s p e c t t o s t r e n g t h o r p o t e n t i o m e t r i c a l l y ( t h e equiva lence po in t i s d i f f i c u l t t o recognize) 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 is more f avorab le . The e r r o r of t h e method f o r a weight of lOmg amounts is approximately 0.5%.

The p o t e n t i a l c o l o r i m e t r i c u se of t h e iod ine complexes of methimazole i n CHC13 and CCl4 is s tud ied . The abso rp t ion maximum of methima- zo le- iodine complex a t 269 nm could be used a n a l y t i c a l l y .

i i i ) Iod ine complex method (22) .

i v ) Bromometry method The method was desc r ibed by Varga e t a1 ( 2 3 ) . To a 2-5 mg sample i n a q u e o u s s o l u t i o n , 10 m l of water , 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 wi th 1 .0 m l 50% H2S04. Af t e r 15 min. 5 ml 20% K I i s added, w i th s t a r c h i n d i c a t o r and t h e iod ine t i t r a t e d wi th 0.1N NazS203. 1 m l 0.1 KBr03 = 0.0009517 methimazole. The bromometric de te rmina t ion of methimazole and o t h e r s imi l a r compounds i s markedly inf luenced by t h e presence of excess bromide. I n gene ra l , t h e g r e a t e r t h e amount of bromide p resen t t he lower t h e percentage of t h e compound found. The d e v i a t i o n appro- aches a l i m i t which is d i f f e r e n t f o r each compound (24) .

v) Cerimetry method The method w a s developed by Varga e t a1 (23) 0.03 - 0.10 gm sample i n 5 m l water is cooled t o Oo and t i t r a t e d wi th 0.1N C e (SO!) ,us ing p-ethoxychrysoiodine o r (not r e v e r s i b l e ) methyl orange, methyl r e d , o r thymol b lue a s i n d i c a t o r . 1 m l 0.1N C e ( S 0 4 ) ~ = 0.011417 gm methimazole. Vehicles of t a b l e t s do not i n t e r f e r e .

t 2

The e r r o r i s less than + - 1%


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 de te rmina t ion of b a s i c compounds wi th thio(-S-) and mercapto (-SH-) groups. The r e a c t i o n of t h e S group wi th Hg (OAc)2 i n a c e t i c a c i d makes t h i s poss ib l e . Methimazole and o t h e r compounds were a l l t it- ra t ed wi th H C 1 0 4 i n a c e t i c ac id us ing gen t i an v i o l e t as i n d i c a t o r .

6.2 S p e c t r o p h o t m e t r i c methods

6.21 In f r a red spectrophotometr ic method A method recommended f o r adopt ion 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 separa ted 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 wi th chloroform as the e l u e n t and then 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. This method w a s s tud ied 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 ; average r ecove r i e s from two s imulated and two t a b l e t s mix tures ranged from 96.6% -t 1 .0 t o 101.1% + - 0.9 (28) .

6.22 Nuclear magnetic resonance spec t romet r ic

-

An NMR procedure is descr ibed by which methimazole is determined i n pure and t a b l e t f o r - mulat ion. The method is r a p i d , accu ra t e , p r e c i s e (S.d. 0.95%) and a l s o provides 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 run 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 wi th use of benzoic ac id as an i n t e r n a l s t anda rd , us ing t h e N-CH3 protons of methimazole a t 3.566 and t h e a ro- matic pro tons 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 (29) .

6.23 U l t r a v i o l e t Spectrophotometr ic method In t h i s method, abso rp t ion 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 abso rp t ion band i n t h e medium have reg ion wi th a max a t 250 nm. I n s t rong a c i d and a l k a l i n e media hypsochromic and hypochromiceffects are observed (30) .

METHIMAZOLE 365

6.3 Chromatographic Analys is

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 ca g e l G. ( conta in ing 1% Zn S i l i c a t e phosphor) p l a t e s w i th CHC13/CH30H/H20 (32 : 8 : 5) as developing so lvent and 2,6-dichlo- roquinone ch lor imide as d e t e c t i o n reagent by dens i tome t r i c scanning (31) . This method is s u i t a b l e f o r r o u t i n e 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 ) .

6.32 Gas Liquid Chromatography This method i s used f o r S-methyl methimazole on a 10% Apiezon L + 5% KOH/Chromosob W column a t looo wi th N c a r r i e r gas . The method i s more s e n s i t i v e and more p r e c i s e than TLC method, bu t d u p l i c a t e measurements must be made on t h e same day due t o i n s t a b i l i t y of S- methylmethimazole (32). Clarke ( 4 ) r epor t ed 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 cond i t ion of methimazole descr ibed i n t h e monograph.

6.33 Paper Chromatography Clarke (4) descr ibed s e v e r a l so lven t s systems used f o r paper chromatography of methimazole as shown i n Table 1.

Table 1.

Ref. Rf Solvent System Visua l i s ing Agent

Acetate Buffer u l t r a v i o l e t 0.27 33 (pH 4.58) i o d o p l a t i n a t e spray

Phosphate Buffer u l t r a v i o l e t 0.00 34,35

(white)

(PH 7.4) i o d o p l a t i n a t e spray (white)

C i t r i c ac id : H 2 0 : u l t r a v i o l e t 0.72 36,37 n-butanol (4 .8g: i o d o p l a t i n a t e 130 m l : 870 ml) .

366 HASSAN Y. ABOUL-ENEIN AND A. A . AL-BADR

6 . 4 Color imet r ic 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 iphenylp icry lhydra- aim wi th methimazole ( thiamazole) w a s s u f f i c i e n t l y r ap id f o r i ts use i n a spectrophotometr ic a s say . -5 When t h e concen t r a t ion of t h e reagent w a s 4 x 10 M and the molar r a t i o of methimazole t o t h e reagent w a s 2 : 10, deco lo ra t ion stopped a f t e r 90 minutes a t 26'. The r e a c t i o n mixture then contained b i s ( l-methylimidazol-2-yl) d i s u l p h i d e , t h e r eagen t , diphenylpicrylhydrazine. After s e v e r a l hours t h e deco lo ra t ion s t a r t e d aga in g iv ing r ise t o a mul t i - p l i c i t y of colored compounds some of which were a l s o formed i n a methanol ic s o l u t i o n of t h e reagent . React ion 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 wi th excess of t h e reagent and f o r t h e r e a c t i o n of t h e reagent w i th t h e d i s u l p h i d e of methimazole (38).

Szabo e t a1 (39) descr ibed method f o r i d e n t i f i c a t - i on and de termina t ion of methimazole by conversion i n t o i ts coloured 1:l C U + ~ complex which has an absorp t ion max. a t 614 nm. The complex is no t s t a b l e , bu t on t rea tment wi th H C 1 , a 2 : l methimazole CuCl complex is p r e c i p i t a t e d as whi te 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 analysed i n pharmaceut ical prepara- t i o n s p o t e n t i m e t r i c a l l y us ing 0.1N chloramine. The method can d e t e c t amounts of up t o 5 mg of t h e drug wi th an accuracy of 98-99.5% (39) .

An a l t e r n a t i v e method w a s d r sc r ibed by P i n z a u t i _ _ e t a1 ( 4 0 ) f o r de te rmina t ion of s e v e r a l a n t i t h y r o i d drugs p o t e n t i m e t r i c a l l y wi th 0.01 M mercuric ace- t a t e wi th use of a mercuric 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 nd i - ca tor -e lec t rode . The method is rap id and t h e re- s u l t s a r e reproducib le ; t h e e r r o r s are a l l w i th in + 0.36%. -

6.6 Coulometric de te rmina t ion Cer t a in t h i o l s , inc luding methimazole, were d e t e r - mined cou lomet r i ca l l by d i r e c t t i t r a t i o n wi th e lec t ro-genera ted HgT2 (42) . is c a r r i e d out i n a c e l l having t h r e e compartments separa ted from each o t h e r by s in t e red -g la s s d i s c s . One compartment con ta ins t h e mercury f o r t h e electro-

The coulometr ic a s say

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. (enclosed 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 avoid 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 KNO3 and of pH 9 .3) ; and t h e t h i r d a c t s as an auxi- l l i a r y compartment. The c o u n t e r - e l e c t r o d e is of p o l i s h e d p l a t inium .

REFERENCES

1. The United States Pharmacopeia X I X , United S t a t e s Phar- macopeial Convention, I n c . , R o c k v i l l e , Md., 20852, p. 313-314.

2 . Merck Index, Nin th e d i t i o n , Merck & Co., I n c . , Rahaway, N . J . , U . S . A . , p. 78, 5841.

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 Trans . , 2, 2045 (1972).

4. E . G . C . C larke , " I s o l a t i o n and The Pharmaceut ica l P r e s s ,

5. A.L. Hayden and M. Maientha l , Chemists, 48, 596 (1965); 6793 ( 1 9 6 5 c

6. H . Y . Aboul-Enein, unpubl ished

I d e n t i f i c a t i o n of Drugs", London, 1969, p. 413.

J. Assoc. O f f i c e . Agr. through Chem., Abs t r . , 63,

r e s u l t s . 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 Cons tan ts of

Organic Compounds'' e d i t e d by J . G . Grassell i , CRC Press, Cleveland, Ohio, 1973.

8 . S a d t l e r NMR Cata log , S a d t l e r Research 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. Aldr ich L i b r a r y of NMR S p e c t r a , by C . J . Pouchert and John R. Campbell, Vol. V I I I , spectrum 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).

11. R.G. Jones , E.C. Kornfe ld , K.C . McLaughlin, and R.C. Anderson, J. h e r . Chem. SOC. , 71, 4000 (1949) .


12. I . B . Simon and 1.1. Kovtunovskaya - Levshina, T r . Ukr., I n s t . Eksperim, Endorkrinol , 18, 345 (1961), through Chem. Abs t r . , 50, 7921 (1963), a l s o see Chem. Abs t r . , - 50, 3416h (1960).

13. I . B . Simon and 1.1. Kovtunovskaya-Levshina, Tioloye Soedinen. Med., Ukrain, Nauch, - I s s l edova te . S a n i t - Khim I n s t . , Trudy Nauch Konf., w., 1957, 406 (Pub. 1959), through Chem. Abstr . - 54, 24760 (1960).

14 . W.O. Foye, J. Lo, J. Pharm. S c i . , 61, 1209 (1972).

15. D.S., S i t a r , D.P., Thornh i l l , J . Pharmacol, Exp. Ther . , 184, 432 (1973).

16 . B . M a r c h a n t , W.D. Alexander, Endocrinology, 91, 747 (1972).

17. B . M a r c h a n t , W . D . A l e x a n d e r , J. Cl in . Endocrinol . Metab., 34, 847 (1972).

18. J . A . Pi t tman, R . J . Beschi, T . C . Smitherman, J. C l in . Endrocrinol . Metab. - 33, 182 (1971).

19. W.D. Alexander, V. Evans, A. MacAulay, T.F. Gal lagher , Jr. and J. Londons,Brit . Med. J . , 2, 290 (1969).

20. J. Kossakowski; J. Klopocki; T. Kuryl; B. Zbikowski, - Act. Pol . Pharm. - 29, 465 (1972).

21. J. Blazek; J. Kracemar and Z . S t e j s k a l ; Ceskoslov. farm. , - 6, 4 4 1 (1957); through _____- Chem. Abs t r . 54, 9204 (1960).

22. E . Zoel lner , G . Vastagh, Acta. Pharm. Hung., 5, 29, (1970) through Chem. Abstr . 73, 69923c (1970).

23. E. Varga and E. Zb'llner, Acta. Pharm. Hung. 25, 150 (1955), -- through Chem. Abstr . 52, 12327 (1958).

24. E. Zoel lner and E. Varga, Acta. Chem. Acad. S c i . Hung., ---- 12, 1 (1957), through Chem. Abs t r . , 52, 520SF (1958). -- - -

25. G. Posgay, Pharm. Z e n t r a l h a l l e 100, 65 (1961); through Chem. Abstr . 55, 14819 a (1961).

26. I. Bayer and G. Posgay, Acta. Pharm. Hung. 2, Suppl. 43 (1961), through Chem. Abstr . 56, 7431 (1962).

METHIMAZOLE 369

2 7 . A.L. Hyyden and L. Brannon, J. A s s . O f f i c . Anal. Chem., - 50, 674 (1967) .

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 . , publ i shed by OAAC, Washington DC 20th Ed., Will iam Horwitz ( e d i t o r ) 1 1 t h ed. (1970) p . 685.

29. H. Y . Aboul - Enein, 2. Pharm. Pharmacol. 31, 1979, ( i n p r e s s ) .

30. T s . I . Shakh; A.E. Verzina, - - Farm. Zh. (Kiev.) - 2 3 , 28(1968)

31. J . B . S t e n l a k e ; W . D . W i l l i a m s ; G.G. S k e l l e r n , J . Chromatog. - 53, 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 .

32. E. Niomiya; H . U s a m i ; H . Yamada, A. Morikawa, K . Matsuyama, K . Kato, I r y o 23, 273 (1969) through Chem. A b s t r . , 71, 536124. (1969).

33. H . V . S t ree t , &. Pharmacal. T o x i c a l . , 19, 312 and 325 (1962).

34. H.V. S t ree t , - J. F o r e n s i c S c i . SOC. 2, 118 (1962).

35. H . V . S t reet , 2. Pharm. Pharmacol. 14, 56 (1962)

36. A . S . Curry and H. Powell , Nature , 2, 1143 (1954) .

37. E . G . C . C larke , Methods of F o r e n s i c Sc ience , ed. Frank Lundguist , 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 . Berg, Acta , Pharm. Suec ica S , 431 (1971) .

39. A . E . Szabe, G . S ta je r , and E . V i n k l e r , Pharmazie, 2, 615

--

(1974).

40. S.G. Avakyants, A.M. Murtazaev, DoL1. Akad. Nauk Uzb. S R . , - 26 35 (1969); through -- Chem. A b s t r . , 72, 103778a (1970) .

41. S . P i n z a u t i , V . D a l P i a z and E. LaPor ta , Farmaco., 3. - P r a t . 28, 396 (1973); through -- Anal. A b s t r . , 26, 1220

42. C . A . Mairesse - Ducarmois, J . L . Vandenbalck, and G . J . P a t r i a r c h e , 2. Pharm. Belg . , 28, 300 (1973) .


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.


I .

2.

3 . 4 . 5 .

6.

7 . 8. 9.

NALIDIXIC ACID

Patricia E . Grubb

Description 1 . I 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

2.21 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


ISBN 0-12-260808-9 37 I

312 PATRICIA E. GRUBB

1. Description

1.1 Name, Formula, Molecular Fjeight

Nalidixic acid is l-ethyl-1,4-dihydro-7-methyl-4- oxo-1,8-naphthyridine-3-carboxylic acid.

Molecular Formula: C H N 0

Molecular Weight: 232.24'l)

12 12 2 3


Nalidixic acid is a white to slightly yellow, odorless crystalline powder. (2)


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


The c a r b o x y l i c a c i d OH bands i n t h e r e g i o n o f 3300-2500 cm-1 are weak and b road , 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 be p r e s e n t . ( 3 ) The i n t e n s e peak a t abou t 1715 cm-1 i s p robab ly due 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 . 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, con juga ted w i t h t h e c a r b o n y l , o r a combina t ion of t h e s e two v i b r a t i o n s .

The peak a t 1620 c m - 1 may be due

2.13 NMR

The NMR spec t rum of n a l i d i x i c a c i d i s p re - 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 Var i an A60 s p e c t r o m e t e r . T h i s spec t rum is i n agreement w i t h t h e s ectrum p r e s e n t e d by Hamilton and co-

made.

ppm I/ p r o t o n s d e s c r i p t i o n

w o r k e r s . ( l t ) The f o l l o w i n g a s s ignmen t s have been

-CH CH

-3

2 -3 1 . 7 5 3 ( t r i p l e t )

2.98

-2 5 .12 2 ( q u a r t e t )

7.85 l ( d o u b 1 e t ) C(6)H

8 .85 1 ( d o u b l e t ) C(5)H

9.47 l ( s i n g 1 e t ) C(2)H

3 ( s i n g l e t ) -CH ( a r o m a t i c )

N-CH -CH3

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 abou t 7 .5 mcg/ml i n 0 .1 N NaOH, methanol , and ch lo ro fo rm, o b t a i n e d on a Pe rk in - E l m e r 323 r e c o r d i n g spec t ropho tomete 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 spec t rum 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 agreement w i t h t h e s p e c t r a p u b l i s h e d by Sa l im 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 are two bands i n methanol and 0 . 1 N NaOH, a t 258 nm and 324 o r 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 methanol : 213-216 nm (1La> , 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 abso rbance i n 0 .1 N NaOH a t 279-281 nm and 325 nm. 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 )

These wave leng ths are abou t 3-10 nm lower

0-0

0

0 c --n

--(?

0

:: 0

0

w --h

-0

n

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 of 330 nm, measuring emission at 375 nm. Browning(9) used 21.5 H SO with excitation at 325 nm and emission at 406 nm. fluorescence excitation and emission spectra of nalidixic acid in 0.5 N H SO determined on an Aminco Bowman Spectrophotofluorimeter .

- 2 4

4 Figure 6 shows the

2 4


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) found to be monoclinic and of the space group P21/C. crystal was solved for the structure of the molecule. The bond lengths and angles of all non- hydrogen atoms were determined. The values found compare favorably with those reported for 1,8- naphthyridine 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 crystals were

The x-ray diffraction pattern of the single

The parameters of the monoclinic crystal are

a = 8.913 a b =13.133 a c = 9.31 2 a =99.75 2

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)


F i g u r e 6

F L UO RE SC E NC E SPECT RUM

of Nalidixic Acid

Excitation maximum 330 nm

Emission maximum 365 nm

(uncor red ed 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) the partition equilibria of nalidixic acid between water and various organic solvents led to calcula- tions of the pKa values of 5.99 + 0.03 for N- protonation and -0.86 + 0.07 for-carboxylate anion formation. (12) the apparent pKa of nalidixic acid to be 5.9 at 28" by a spectrophotometric method. ( 1 3 )

Further study by the same workers on

Takasugi and co-workers reported

2.24 Melting Range

The melting range of nalidixic acid is reported to be 225-231", determined as a class 1 compound. (1) (2)


2.25 Differential Scanning Calorimetry

A DSC scan for nalidixic acid was performed 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.

The instrument used was a

3. Synthesis

The synthesis of nalidixic acid was reported by Lesher It may be prepared by the procedure and Gruett. (15) (16)

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 , .-. , .

I I

1 1-

I p / n L n I

--t-- ---- I T t

---ti-- + t

- t . - - -

-.---r- I

i _ _ . , _.

I

--t I

- t---- I - -- -

ii it9 I - - r . 'C 1

---?--- I

i

. ~ *

-t

- ._ . _. I .+__

I 1

I

4--

n

a

CI

0

Y

I

c

Z

* v)

N

m

N

- r 0

0

V

8

v

I"

+ m

m

n

I

+ N

I

(=J

\/

I"'

r

fiZ

Ql

I "p" \I om I

NALIDIXIC ACID 385

5 . Drug Metabol ic Products and Pharmacokinet ics

5 .1 Metabol ic 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 - t i o n s . It is more a c t i v e a g a i n s t gram-negative than gram-posi t ive organisms. (4) The compound i s r a p i d l y absorbed 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 conjugated as t h e monoglucuronide, The most impor tan t m e t a b o l i t e is l-ethyl-1,4-dihydro-7(hydroxymethyl)-4-oxo-l,8- naphthyridine-3-carboxylic a c i d , r e f e r r e d t o as hydro- x y n a l i d i x i c a c i d . This m e t a b o l i t e has shown t h e same o r d e r of 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 conjugate of 7-hydroxynali- d i x i c a c i d h a s a l s o been found, as w e l l as t h e 3,7- 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 lucuronides o r f o r t h e 3,7- d 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 lucuronide of t h e 3 ,7-d icarboxyl ic a c i d has been r e p o r t e d .

No a n t i b a c t e r i a l a c t i v i t y

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) (21) , monkeys (8) , dogs (8) , chickens ( 2 2 ) , c a l v e s ( 2 3 ) and microorganisms(4) . 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 convers ion of n a l i d i x i c a c i d t o hydroxynal id ix ic a c i d had been r e p o r t e d by McChesney(8) t o be normally about 32%; b i c a r b o n a t e sup- p lementa t ion i n c r e a s e d t h i s t o about 40%. Bicarbonate supplementa t ion 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 excre ted i n t h e b i o l o g i c a l l y a c t i v e form.

The r a t i o s of t h e s e

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 o f t h e v a r i o u s metabol ic products excre ted 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 ra te e q u a t i o n s . ( 2 4 )


Calc.amts. Found (24) Found (21) Compound (mg per lg dose) (mg per lg dose) %

acid Nal id ix ic 9 8 + 3 0.5-5 % -

Hydroxy- 105 129 + 8 2.5-6 % - nalid ixic acid

NA-glucuronide 517 537 + - 49 24-80%

1-HNA-glucur- 221 229 + 32 11-12% - onide

3,7-dicarbo- 74 xylic acid

4 3 + 6 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.

The mechanism of action of nalidixic acid against -- 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 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 forms, 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 conjugated p r o d u c t s of t h e a c t i v e forms. T h i s model is shown below i n F igure 9.

F i g u r e 9

N a l i d i x i c a c i d i n Man S i m p l i f i e d Model f o r K i n e t i c pathways of

Nalidixic acid lag time > A k A , 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 excre- t i o n were f u r t h e r combined t o a rate c o n s t a n t f o r t h e d isappearance of t h e drug, kd. Four dosage 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 except f o r a c o a r s e , s lowly d i s s o l v i n g powder.

Portmann and co-workers then 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 pr imary m e t a b o l i t e . ( 2 6 ) c o n s t a n t s f o r g lucuronide format ion , o x i d a t i o n 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 hydroxy- n 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 every case . Good agreement between exper imenta 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 ra te approximations used f o r t h e model, w a s found. Again, t h e d isappearance ra te c o n s t a n t , kd2, w a s found t o be v e r y s imilar f o r each 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 r a t e c o n s t a n t s v a r i e d widely. ra te c o n s t a n t , kd2, 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 ra te 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 lucuronide and d i c a r b o x y l i c a c i d , kM and kM4, r e s p e c t i v e l y .

The r a t e

The d isappearance

3


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 10). This model was based on the 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)



The molecular formula is C H N 0 12 12 2 3'

NALIDIXIC ACID

F i g u r e 10

389

METABOLIC PATHWAYS of Nalidixic Acid in Man

Nalidixic Acid inGI tract

I kA

Nalidixic Acid Nalidixic Acid kMz ~

GI uc ur on ide

4 in body

Nalidixic Acid lkMl :*r in urine

Hydroxynalidixic Acid Nalidixic Acid in body Glucuronide

in urine

Dicarboxylic Acid in body

‘y l k ~ 3 Hydroxynalidix i c Acid

in urine Hydroxynalidixic Acid Glucuronide in body

Dicarboxylic Acid In urine

kMl= 4.5 3 xIO-’ min

kEl= 1.0 x min - I kM2= 5.77 xIO-’ min-l

k, 2=1 .9 3 X 1 0 -3 min-1 kM3= 4.0 5 xlO-’mn-’

k,,= 1.37~10-~ m i d

I“.’ Hydroxynalidixic Acid Glucuronide in urine

kA=1.8x10-2 mill-’


Element Theory Found for standards(30)

C 62.07% 62.11 61.98 H 5.21% 5.21 5.20 N 12.07% 12.00 12.32

6 . 2 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 ethylene- diamine 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 of 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 10-4111 and three stages of reduction were found. The poten- tials 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 sorb en t

paper

paper impreg. with 0.1 M Na and HPZ4

silica gel

silica gel

silica gel

silica gel

Mobile Phase

toluene:dioxane(9:1) descending

butano1:acetic acid: H 2 0 (4:l:l)

propano1:ethanol:water (6:1:3)

iso-butano1:ethanol:ethyl acetate:water:acetone (8.2: 1 : 5 : 2 ) ascending

methano1:water:ammonia (100:12:16)

ch1oroform:methanol:formic acid (90:7:3)

ethyl acetate:methanol: isopropylamine (76:20:4)

benzene in ethano1:acetic acid (80: 10: 10)

- Rf

.6

--

--

.73

.88

.54

.18

.5

Ref.

8

-

36

36

17

35

37

37

23


6.62 Liquid chromatography

A high-performance liquid chromatographic method for nalidixic acid on a strong anion- exchange 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 1.00, (38)

6 .63 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 f h 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) (26 ) 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 Another fluorimetric method for has also been reported. ( 4 0 )

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. 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)

Gafari -- et al(41) used an extraction with

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. fication 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. mato raphic procedure for the dicarboxylic acid was used by

A modi-

A separate liquid chro-

-- Lee( 8 4) et al. The column used was a 25 cm x 4.6 mm i.d.

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 dicarboxylic acid was 12 minutes.

The retention time of

A thin-layer gas chromatographic system was devised by Pittman and Shekosky(39) for chicken tissue and feces. TLC system of benzene:methanol:acetic acid (9:l:l) was used for prior separation; the spots were then removed and esterified in 14% BF in methanol. A 4 ft. 3% OV-17 column 3 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

A pulse polarographic system for detection of the dicarboxylic acid in the presence of nalidixic and h droxy- nalidixic 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 anti- bacterial activity in-vitro, then cannot be determined sepa- rately.

Organism Reference

P. boviseptica 8

B. pumilus 46

E. coli 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) the maximum for chloroform and 258 nm for 0.1 N NaOH. (7) infrared spectrum has also been used to identify nalidixic acid in tablets. (7)

Another source reported 259 nm as The

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 vs. SCE in 20% aq. DMF with 0.1 - N HC1 has also been 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

References

PATRICIA E. GRUBB

1. 2.

3.

4.

5.

6.

7.

8.

9. 10.

11.

12.

13.

14.

15.

16. 17.

18. 19. 20.

21.

22.

23.

24.

National Formulary, =:477. Salim,E.; Shupe,I.; J.Pharm.Sci. (1966) 55 (11) :1289-90. Parikh,V.M., Absorption Spectroscopy of Organic Molecules, Addison-Wesley Publishing Co., (1974) : 244. Hamilton,P.B.: et al; Appl.Microbio1. (1969)

Zubenko,V.; Shcherba,I.; Farm.Zh, (1975) - 30 (3):28-33 through CA 83:152437Z. Gafari,A.; Farm.Zh., (1977) - 1:53-6 through - CA 86:179821u. da Silva,M.; Noqueira,M.; Rev.Port.Farm. (1965) 15 (3):290-4 through CA 64:9513h. McChesnG,E.; et al; Toxicolxnd Appl.Pharm- acol (1964) 6:292-309.

-

- 17 (2) ~237-41.

J.A.o.A.c. (1970) :723-4. Achari,A.; Neidle,S.; Acta Crysta1logr.Sect.B

HoughtaFng,W. ; Sterling-Wintrhop Research (1976) 32 (2) ~600-2.

Institute; unpublished data. Sulkowska,J.; Staroscik,R; Pharmazie (1975)

Takasugi,N.: et al; Chem.Pharm.Bul1. (1968)

Staroscik,R.; Sulkowska,J.; Acta Pol.Pharm. (1971) - 28 (6):601-6 through CA 76:158322k. Lesher,G. ; et al; J.Med.PharEChem. (1962)

Lesher,G.; et al; US Patent //3,149,104. Pawelczyk,E.; Plotkowiakowa,Z.; Acta Pol. Pharm.(1970) 27 (2):105-11 through CA 73:91300u. Detzer,N.; Hucr,B.; Tetrahedron (1975) 31:1937-41 Moore,W. ; et al; J.Pharm.Sci. (1965) 54 (1) :36-41. Pereny1,T. ; et al; Acta Microbiol.AcaxSci. Hung. (1975) 22 (4):433-45 through CA 84385914h. Graber,H.; etTl; Gyogyszereink (19E) 25 (3):97-106 through= 84:173528r. Browning,R.; Sterling-Winthrop Research Institute, unpublished report.

- 30:405-6.

16 (1) :13-16. -

- 5~1063-4.

McChesney , E. , et al; Toxicol . Appl . Pharmacol , PortmanKG.; et al; J.Pharm.Sci. (1966) 55 (1):72-8. (1969) 14 (1) ~138-50.

-

NALIDlXlC ACID 397

25. Goss,W.; et al; J.Bacterio1, (1965) 89:1068-74. 26. Portmann,G.; J.Pharm.Sci. (1966) - 55 (1):59-62. 27. McChesney,E.; et al; J.Pharm.Sci. (1967) 56

28. Brueh1,P.; et al; Arzneim.-Forsch (1973) 23

29. Rohwedder,H.; et al; Z.Kinderheilk (1970) 109

(5) :594-9.

(9):1311-13 through - CA 80:33692g.

(2):124-34 through 9 74:97455r. 30. Auerbach,M., Sterling Wintrhop Research

Institute, unpublished data. 31. Ignat,V.; Bera1,H.; Rev.Chim.(Bucharest) - 17 (1):50 through - CA 64:17360h.

32. Bachrata,M.; et al; Pharmazie (1977) 32

33. Murgu,N.; Pharmazie (1964) 2 (11):724-5 34. Dick,I.; Murgu,N.; Rev.Chim(Bucharest) (

398-401.

- 15 (12):757-8 through CA 62:15600h.

(1966)

7) :

964)

35.

36.

37.

38. 39.

40.

41.

42.

43.

44.

45.

46

47.

48.

49.

- Staroscik,R.; et al; Pharmazie (1974) 2 (6):

Perenyi,T.; Acta Pharm.Hung. (1975) 45 ( 5 ) : 196-206 through CA 84:27577q. Crain,A.V.R. ; Sterling Winthrop Research Institute, unpublished data. Sondack,D., Koch,W.; J.Chromatog. (1977) 132:322-5. Pittman,K. ; Shekosky,J. ; Sterling Winthrop Research Institute; unpublished data. Staroscik,R.; Sulkowska,J.; Acta Pol.Pharm. (1973) 30 (5):459-66 through CA 81:33082d. Gafari,X; et al; Farm.Zh. (1F7) 2:92-3 through CA 87:33391c. Sharge1,c; et al; J.Pharm.Sci. (1973) - 62 (9):

Goeh1,T; et al; Sterling Winthrop Research Institute; unpublished report. Lee,F.; et al; Sterling Winthrop Research Institute; unpublished report. Koss,R.; Warner,C.; Sterling Winthrop Research Institute, unpublished report. Ciuro,C.; Cienc.Ind.Farm. (1973) 5 (2):54-7 through CA 82:152437z. Bauernfezd,A. ; Gruemmer,G. ; Z.Med.Mikrobio1. Immunol., (1968) 154 (1):35-9 through CA 69:33474k. Monciu,D. ; et al; Farmacia(Bucharest) (1972), - 20 (8):459-66 through CA 17:168693s. Goss,W.; Deitz,W.; Bacteriol.Proc. (1963):93.

The literature was reviewed to June 19, 1978.

387-90.

1452-4.


NEOMYCIN

William F. Heyes

I . Description 1.1 1.2 Appearance, Color, Odor, Taste I .3

2.1 Spectra

Composition, Name, Formula, Molecular Weight

Definition of International Standard 2. Physical Properties

2. I 1 Infrared Spectrum 2.12 Ultraviolet Spectrum 2.13 NMR Spectrum 2.14 Mass Spcetrum 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

3.1 Salts 3.2 Derivatives 3.3 Complexes

4.1 Commercial Biosynthesis 4.2 Synthesis of Radio-Labelled Material

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

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

2.2

3. Salts, Derivatives, and Complexes

4. Synthesis and Production

5 . Stability and Degradation



ISBN 0-12-260808-9 399

400

6.34 Paper and Thin-Layer Chromatography 6.35 Gas-Liquid Chromatography

6.4 Microbiological Procedures 6.41 Turbidimetric Assay 6.42 Agar-Diffusion Assay

6.6 Use as an Analytical Reagent 6.7 Determination in Body Fluids and Tissues

7 6.5 Automated Procedures

WILLIAM F. HEYES

1. D e s c r i p t i o n .

1.1. Composition, N a m e , Formula, -- Molecular Weight

Composition

Commercial neomycin i s a complex mix- t u r e of aminoglycoside a n t i b i o t i c s o r i g i n a l l y iso- 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 co-workers i n 1 9 4 9 . The p r i n c i p l e components of t h e mix tu re 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 neaminel , a d e g r a d a t i o n p roduc t of neomycin former- l y known as neomycin A. Table 1 shows t h e c o n t e n t v a r i a b i l i t y of neomycin B and C and neamine i n commercial samples of neomycin a s r e p o r t e d i n t h e l i t e r a t u r e . Neomycins LP-A, LP-B and LP-C which chemica l ly a r e t h e mono N-acetyl d e r i v a t i v e s of neomycins A , B and C2 may a l s o be 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 have re- c 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

Content V a r i a b i l i t y of Neomycin

S o u r c e

Canada

U.K.

U.S. S. R.

Neomycin B Neomycin C Neamine Reference Content % Content % Content P,

6 9 . 2 30.8 N i l 5 t o t o

9 7 . 5 4 . 0

80.5 17.5 0 6 t o t o t o

9 2 . 0 8 .0 2.5

63.7 4 0 . 0 N i l 7 , 8 t o t o

9 1 . 6 3.4

NEOMYCIN

............. 40 1

neamine . . . .:

H 2 N q 6 5

...........

I R l = H R 2 = CH2NH2; neomycin B

I1 R1 = CH2NH2 R 2 = H ; neomycin C

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, Sofra- mycin, Streptothricin BI, BII.

4 Chemical Names

Neomycin B 0-2,6-diamin0-2,6-3dideoxy-a- D-glucopyranosyl- (1-+4) -0- [0-"2, 6-diamino-2,6-dideoxy-@-L-a idopyranosyl- (1+3) - B-D-ribo- furanosyl-s (1+5)] -2-deoxy-D- streptarnine.

Neomycin C 0-2,6-diamino-2,6-3dideox -a- D-glucopyranosyl- (1+4) -0-f0-=2, 6-diamino-2,6-dideoxy-a-D-c) glucopyranosyl-(1+3)-@-D-ribo- furanosyl-3(1+5)] -2-deoxy-D- s treptamine .

Ne amine 2-deoxy-4-0- (2,6-Cdiamino ( formerly 2,6-dideoxy-a-D-glucopyranosyl) Neomycin A) -D-streptamine .

Chemical Abstracts Registry No.

Neomycin B 119-04 -0 Neomycin C 66-86-4 Neamine 3947-65-7

Formula

Neomycin B,C C23H46N6013

Ne amine C12H26N406

Molecular Weight

Neomycin B,C 614.67 Ne ami ne 322.36

NEOMYCIN 403

1 . 2 . Appearance, Co lour , Odour, Taste

u s u a l commercial form) 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 tas te less .

1 .3 . D e f i n i t i o n o f I n t e r n a t i o n a l S t anda rd

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 m a t e r i a l .

Reference S tanda rd f o r neomycin B i s d e f i n e d as c o n t a i n i n g 6 7 0 u n i t s p e r m g , 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 0.001492 mg of s t a n d a r d mater ia l .

The s u l p h a t e s a l t o f neomycin ( t h e

The I n t e r n a t i o n a l Reference S tanda rd

S i m i l a r l y , t h e I n t e r n a t i o n a l

2 . P h y s i c a l Properties

2 . 1 . S p e c t r a 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 of neomycins B & C s u l p h a t e have been r eco rded as a d i s p e r s i o n i n po ta s s ium bromide and are i l l u s - t r a t e d i n F ig . 2 and 3 .

The i n f r a r e d spec t rum of neamine s u l p h a t e ( fo rmer ly c a l l e d neomycin A ) a l so as a potass ium 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 F ig . 4 . A l l s p e c t r a are o f a u t h e n t i c mater ia l s u p p l i e d by The Upjohn Company, Kalamazoo.

Sammul e t a l l o have p u b l i s h e d t h e i n f r a r e d spec t rum of neomycin undecy lena te .

2 . 1 2 . U l t r av io 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 observed w i t h s o l u t i o n s o f neomycin.

2.13. NMR Spectrum

The p ro ton NMR spec t rum of neomycins B and C has been used by R i n e h a r t and co-workers1 t o e l u c i d a t e t h e s t r u c t u r e and

WAVELENGTH (MICRONS) 8 9 10 12 15 20 30 4Ou) 2.5 3 4 5 6 7

I , . I , I I . , . , I , . . . I...JI....,I I . . . I 0.0 1 00 03 I ' . an ..

I 1 1 , '

. * 1 ' - . ~-

L

3 m 2500 2ooo la00 1600 1400 1200 lo00 800 wo 400 200 FREQUENCY (CM')

Figure 2. I n f r a r e d Spectrum of Neomycin B

WAVELENGTH (MICRONS) 7 8 9 10 12 15 20 30 4050 2.5 3 4 5 6

4Ooo 3500 3000 2 5 0 2000 1800 1600 1400 1200 1000 800 600 A00 200 FREQUENCY (CM’)

Figure 3. Infrared Spectrum of Neomycin C

33NV

WO

SW

31dW

VS

u-l 0

.. c H

N EOMY CIN 407

s t e r e o c h e m i c a l c o n f i g u r a t i o n

T r u i t t ” h a s t h e 1 3 C NMR of t h e neomvcins

o f t h e s e a n t i b i o t i c s .

e x t e n s i v e l y s t u d i e d and demonst ra ted t h e

pH dependance o f t h e spec t rum o f neomycins B and C. The s p e c t r a o f t h e hexa N-acetyl d e r i v a t i v e s however, w e r e shown t o be independant o f pH t h u s a l l o w i n g ass ignment o f t h e observed chemica l s h i f t s as g iven i n Table 2 .

2 . 1 4 . Mass Spectrum

Neomycin is i n s u f f i c i e n t l y vo la - t i l e f o r d i r e c t mass spectrometric a n a l y s i s . T o overcome t h i s problem Inouye14 p repa red t h e vo la - 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 which, 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 obse rve t h e molecu la r i o n it 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 t h e N - s a l i c y l i d e n e S c h i f f ’ s base . The spec t rum of N - s a l i c y l i d e n e neomycin w a s found t o be dependant 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 t he rma l decomposi t ion p l a y s a s i g n i f i c a n t p a r t i n t h e f r a g m e n t a t i o n p r o c e s s .

The M.S. examinat ion of 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 has been re- p 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 observed by Inouye14.

I t i s i n t e r e s t i n g t o n o t e t h a t t h e molecular i o n co r re spond ing t o neamine (a major p roduc t o f neomycin h y d r o l y s i s ) w a s n o t observed i n t h e mass spec t rum 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 avoid t h e need 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 echn ique of F i e l d Desorp t ion Mass Spec t rometry t o neomycin and s u c c e s s f u l l y observed an i n t e n s e response f o r t h e molecu la 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 peaks 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 agmen t s .


Table 2 ~- 12 13C NMR Absorptions of hexa-N-acetyl-neomycin

Carbon atom (see Fig.1)

A1 A2 A3 A4 A5 A6

D1 D2 D3 D4 D5 D6

R1 R2 R3 R4 R5

B 1 B2 B3 B4 B5 B6

CH 3

co

6 ppm (from TMS)

Hex a-N- ace tyl Hexa-N-acetyl neomycin B - neomycin C

97.0 96.9 54.2 54.1 71.4 71.4 71.4 71.4 71.4 71.4 40.8 40.8

50.4 50.4 33.2 33.1 48.9 48.9 76.7 76.8 86 .O 86.0 74.5 74.5

109.3 110.0 74.5 73.5 77.2 75.2 82.4 81.8 62.2 62.9

98.8 97.3 51.8 54.3 70.3 71.7 68.5 72.3 74 .O 71.4 40.8 40.6

23.0(x 3) 23.2 22.9(x 3) 22.8(x 5)

175.2 175.2 174.9 175.1 174.8 174.9(x 2) 174.7 174.6 174.5 174.1 174.1

NEOMYCIN 409

2 . 2 . P h y s i c a l P r o p e r t i e s o f t h e S o l i d

2 . 2 1 . Hygroscopic N a t u r e

H i r t z e t a l l 7 demonst ra ted t h e hygroscop ic n a t u r e of neomycin s u l p h a t e u s i n g a the rmograv ime t r i c t echn ique . 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 de te rmined t h e a d s o r p t i o n of wate8 by neomycin s u l p h a t e a t t empera tu res 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.

Table 3

Adsorpt ion of M o i s t u r e by Neomycin Su lpha te

Humidity Mois ture adsorbed %

--

-

0, -- 0 (350 hour s s torage)

0

10

15

25

4 0

6 0

0

4.15

5.25

6 . 9 0

1 0 . 2 0

1 6 . 7 0

80 38 .90

2 . 2 2 . S o l u b i l i t y

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 of neomycfg ,30,3pme twenty s i x s o l v e n t s have been r e p o r t e d . The v a l u e s f o r t h e most commonly encoun te red 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 c e Area --

Using a method based on t h e f i l t r a t i o n of a i r thrqygh a compressed l a y e r of powder E z e r s k i i e t a1 de termined t h e v i s i b l e s p e c i f i c s u r f a c e area of neomycin s u l p h a t e t o b e 2 . 6 rn2g-l.

W

r- q

0

W

OV

VN

d

cn

WN

~

cn

mm

....

r-cn

r-rl d

d

N

d

N

m

-4

c, hal

w

0

N

c u

4J

d

0

In

mO

m

0

00

0

Q)

r(

Q)d

OA

d

A

AA

A

m

d

u .rl k 0

d

0

**

*

00

00

N

NN

N

AA

AA

m~

~m

d

mW

Om

N

OO

N0

dd

dd

d

* *

v)

In

W

wd

mw

m

Co

WO

NW

mo

oo

m

.....

NO

V

W

mm

d

d

00

N

N

AA

*In

m

r- m

o

.. N

O

d m

*v)

00

;Id

+I

* 4J a

In

N

99

0

0

g z m

N

0

m

0

0

00

+

I

dd

;

7

d

ia 3

d

d 4

410

S o l v e n t

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 f o r s o l v e n t b l a n k )

P e t r o l e u m e t h e r I s o o c t a n e E t h y l ace ta te I s o a m y l acetate Acetone Methyl e t h y l

k e t o n e - D i e t h y l e t h e r - E t h y l e n e c h l o r i d e

C h l o r o f o r m Carbon

t e t r a c h l o r i d e 1 , 4 d i o x a n e Carbon d i s u l p h i d e P y r i d i n e Form a m i d e D i m e t h y l s u l p h o x i d e

Neomycin B h y d r o c h l o r e

0.0 0 .03 0 .03 0 .06 0 . 0 4 0 .058

1.485* 0.005 0 .06

0 . 1 4 "

0.323* 0.45"

N e omy c i n s u l p h a t e

0 .005

0.028 0.06 0 .15 C. 005

0 . 0 7

0.0 0.075

0 . 1 4 "

0 .923* 0.35"

N e omy c i n u n d e c y l e n a t e

2 .62 0.342 4.70" 7.36*

16 .00* 5 .795"

7 .989 0 .77* > 2 0 * 7 . 9 3 8

6.485 9 .40* >20* > 2 0

N e omy c i n oleate

> 2 0 15 .852

6.885 1 4 . 0 8 0

9 .260 6 . 5 7 8

1 1 . 2 8 8 3.257 > 2 0 > 2 0

13 .038 > 2 0 > 2 0 1 . 3 6 5

10 .580

Neomycin s tearate

0 .188 0.0 1 . 0 3 0 7.014 2.083 1 .742

7.328 0.217 9.463 1 . 0 4 6

4.500 3 .918 > 2 0 0 . 2 1 7 3.862

+ + A l l v a l u e s - 0.025mg except * - 0.05mg.

412 WILLIAM F . HEYES

2.3 . P h y s i c a l P r o p e r t i e s o f S o l u t i o n s

2.31. Dens i ty

Kige 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 of 6 & 18% aqueous neomycin s u l p h a t g so l - u t i o n s o v e r t h e t empera tu re range 20° t o 80 C. The p u b l i s h e d v a l u e s are g iven i n Table 5 .

Table 5 -- D e n s i t i e s o f Neomycin Su lpha te S o l u t i o n s

N e omy c i n Dens i ty Concen t r a t i on 4 O°C 6 O°C 8 O°C

6 1.025 1 . 0 2 1 1 . 0 1 4 1 . 0 0 7

18 1.086 1 .081 1 . 0 7 6 1 .068

ooc L - %

2.32. V i s c o s i t y

Table 6 shows t h e r e p o r t e d 1 8 v i s - cosit ies f o r 6 and 18% aqueous neomycinosulpbate s o l u t i o n s ove r t h e t empera tu re range 2 0 C-80 C .

Table 6

--- V 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

Neomycin V i s c o s i t y ( c p s ) Concen t r a t ion 2 ooc 4OoC 6 O°C 8OoC

9:

6 1 . 2 5 0.86 0 . 6 4 0.623

18 1 . 9 9 1 .30 0.95 0 . 7 4

2 .33. Su r face Tensiofl

Kigel e t a l l 8 have 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 & 18% aqueous neomycin s u l p h a t e s o l u t i o n s (Table 7 ) .

NEOMYCIN 413

Table 7

S u r f a c e Tension Values f o r Neomycin _- S u l p h a t e S o l u t i o n s

N e omy c i n Surf ace Tension (dynes/cm) Concen t r a t ion

2 ooc 4OoC 6 O°C 8 O°C % I -- - - 6 69 .81 6 6 . 0 0 63.30 60.30

1 8 7 2 . 0 1 68.12 65.45 62.36

2.34. Adsorp t ion by C lays , S o i l s & Minera ls -

The a d s o r p t i o n of a n t i b i o t i c s from aqueous 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 inch 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 such a s neomycin, a d s o r p t i o n by c l a y s such a s m o n t m o r i l l o n i t e d i s t o r t e d t h e c r y s t a l l a t t i c e of t h e c l a y 4.42, cor re spond ing t o monolayer 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 lec t ro- s t a t i c . A t t e m p t s t o release t h e a n t i b i o t i c by t h e a d d i t i o n o f b u f f e r s h a s en demonst ra ted t o be o n l y p a r t i a l l y successful" . r e l e a s e d approx. 50% of t h e adsorbed a n t i b i o t i c whereas 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 comple te ly i f f e c t i v e . I n a r e c e n t s t u d y McGinity and H i l l " have shown d i v a l e n t magnesium i o n s t o be more e f f i c i e n t than monovalent 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 charged 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 .

Phosphate b u f f e r s

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 cal- c 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 ind- i n g of neomycin t o t h e f i l t r a t i o n mater ia 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 found t o be adso rbed on a l l t h r e e m a t e r i a l s . Acid-washing t h e c e l l u l o s e powder f a i l e d t o deso rb a l l of t h e neomycin.


2.35. Adsorp t ion by Ion Exchange Res ins

During a s t u d y o f t h e phys ico- chemical p r o p e r t i s of some aminoglycoside a n t i - b i o t i c s Kuzyaeval' de te rmined t h e s t a t i c and dynamic exchange c a p a c i t i e s of neomycin on t h e c a t i o n exchange r e s i n KB-4P-2 ( a phenoxyace 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 N a form.

K i l l f i n e t a127 have r e p o r t e d v a l u e s f o r t h e exchange e n t h a l p y o f neomycin €or t h r e e ion-exchange 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:-

Table 8 --- Some Exchange E n t h a l p i e s f o r Neomycin

-1 r e s i n c a l . e q u i v Ion exchange Type A H

- 4 0 +

K F U , N a form m e t h a c r y l i c a c i d - d i v i n y 1 ben z ene

KMDM6 ,Na+form m e t h a c r y l i c a c i d - +3 4 0 hexame thylenedime t h y 1 ac r y 1 a m i de

formaldehyde KB4P2 ,Na+form phenoxyace t ic a c i d - +5 7 0

Ig8a f u r t h e r p u b l i c a t i o n K i l ' f i n and Samsonov r e p o r t e d i n v e s t i g a t i o n s i n t o t h e v a r i a t i o n o f 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 ( K ) w i t h t h e amount o f neomycin adsorbed 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 . I n 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 adsorbed neomycin ( expres sed as t h e % sur face-coverage o f t h e ion exchange r e s i n ) , though d i f f e r e n t t y p e s of i o n exchangers r e s u l t e d i n d i f f e r e n t r e sponses . The deg ree of cross- l i n k i n g of t h e i o n exchange 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 c o e f f i c i e n t . The a u t h o r s concluded t h a t poly- condensa t ion- type i o n exchange r e s i n s adso rb

NEOMYCIN 415

neomycin more s e l e c t i v e l y than t h e p o l y m e r i s a t i o n - type 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 ype 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 Ge l ’pe r in2 ’ 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 ion-exchange r e s i n s from bo th pure 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 b r o t h s .

3 . S a l t s , D e r i v a t i v e s & Complexes

3 . 1 . S a l t s

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 of neomycin by t h e format ion o f v a r i o u s s a l t s have been 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 , such 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 - pared and b e i n g w a t e r - i n s o l u b l e w e r e fo rmula t ed i n o in tmen t b a s e s . S i m i l a r l y , t h e undecy lena te s a l t has 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, 34* The undecy lena te and c a p r y l a t e s a l t s have 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 .

Various c a r b o x y l i c a c i d s a l t s have a l so gal lard^^^ produced t h e m a l e a t e been r e p o r t e d .

s a l t of neomycin which, i t was c la imed, improved t h e aqueous 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 rac - t i c a l l y t as te less compound t h e c i t r a t e s a l t , h a s been d e s c r i b e d by S z y s ~ k a ~ ~ . Neomycin mandela te has been c la imed 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 wh i l e t h e d i - hydroxy-d i n a p h t h l m e t hane -d ica rboxy la t e 40 and t h e pamoate s a l t s 4 1 , g 7 1 6 8 have a l o w i n t e s t i n a l ab- s 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 i n t e s t i n a l i n f e c t i o n s .

F i s h e r and H a l l r e p o r t e d t h e propion- a t e s a l t t o be u n s u i t a b l e f o r u se i n oph tha lmic preparation^^^. Other 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 lucurona te42 , s u c c i - n a t e 4 3 I 4 4 , l a c t a t e 42 t h e s u l p h o s ~ c c i n a t e ~ ~ r 4 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 .

a ~ c o r b a t e ~ ~ , ~ h t h a l a t e ~ ~ a n d


Amongst t h e o r g a n i c su lpho a c i d s used t o p repa re neomycin s a l t s , t h e p- to luene sulphon- a te 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 a e r o s o l sp ray formula t ion49. S a l t s of neomycin w i t h halogen s u b s t i t u t e d 8-hydroxyquinol ine 5- su lphon ic a c i d s have been d e s c r i b e d as 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 an t i ameb ic b e s i d e s a n t i m i c r o b i a l p r o ert ies50f51. S tankov ics e t a152 and V e ~ i a n a ~ ~ p r e c i p i t a t e d a complex s a l t of neomycin, by mixing aqueous 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 . In a more r e c e n t i n v e s t i g a t i o n of t h i s r e a c t i o n , Leucuta e t found t h a t t h e p r e c i p i t a t e d complex d i s s o l v e d on a d d i t i o n of e x c e s s sodium l a u r y l s u l p h a t e . I.R. s t u d i e s of t h e complex have i n d i c a t e d t h a t amide groups , coup l ing re- a c t i o n s and H-bonding are involved i n t h e forma- t i o n o f t h e compound. Michele and V a l e t t e have re- p 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 dodec lbenzene-sulphonate55 and t h e cyclohexylsulphonatex6has a l s o been d e s c r i b e d .

f i r s t d e s c r i b e d by Keller e t a157, 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 paper by t h e same au thor s58 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 ca lc ium-binding 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 pan to thena te s a l t . A s i m i l a r e f f e c t has been not - e d by Weitnauer e t a159 w i t h an equimolar mix tu re of neomycin and c a l c i u m g lucona te . Numerous procedures f o r t h e p r e p a r a t i o n o t e p a n t o t h e n a t e s a l t have been pa ten ted60,61 , ‘ 2 r k 3 . A b b ~ u ~ ~ re- 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 wi th a mix tu re of g lu t amic 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 as 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 han o t h e r neomycin s a l t s .

The p a n t o t h e n a t e s a l t of neomycin ,

S a l t s w i th t h e amino a c i d s N-methyl- s t e a r o y l g l y c i n e 6 5 and lu t amic ac id66 , and t h e v i t a m i n s a s c o r b i c ac id23 and n i c o t i n i c ac id63 have a l so been e v a l u a t e d .

N EOMY CIN 417

Chlorophenols 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 have 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 mycocidal 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 use of such a compound, neomycin hexak i spen tach lo rophena te , i n t h e t r e a t m e n t of 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 - d o n t i c c a v i t i e s .

w i t h p-amino benzene s u l hoacetamide was d e s c r i b e d by t w o groups of workersT2 I 73. The s a l t combined t h e chemothe rapeu t i c p r o p e r t i e s of t h e components. Another unusua 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 lphonylbenzaldehyde i s o n i c o t i n o y l h y d r a z o n e 78’7’. The compound i s reputed t o be less t o x i c than 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 than t h e i n d i v i d u a l c o n s t i t u e n t s .

a c i d s have a l so been d e s c r i b e d . The s u l p h a t e s a l t i s t h e u s u a l commercial form i n u s e today b u t neomycin b o r a t e has been used i n ophtha lmic pre- p a r a t ions46 .

Not a l l neomycin sa l t s , however, p o s s e s s advantages such as a reduced t o x i c i t y . I n f a c t , t h e o ro t ic a c i d (1 ,2 ,3 ,6 - t e t r ahydro -2 , 6 - d ioxo-4-pyrimidine c a r b o x y l i c a c i d ) s a l t w a s re- p o r t e d t o have 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 ~ ~ ~ ~ ~ . 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 be of a similar t o x i c i t y 7 6 . of neomycin w i t h u s n i c a c i d (an a n t i b a c t e r i a l sub- s t a n c e found i n l i c h e n s ) has been r e p ~ r t e d ~ ~ t o

A s a l t formed by r e a c t i n g neomycin

S a l t s of neomycin w i t h t h e i n o r g a n i c

Some u n c e r t a i n t y of t h i s f a c t i s

The s a l t

have used

f r e e have

less a n t i b a c t e r i a l a c t i v i t y than t h e neomycin 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 . D e r i v a t i v e s

The neomycin molecule c o n t a i n s b o t h amino and f r e e .hydroxy groups . Many w o r k e r s e x p l o i t e d t h e p o s s i b i l i t y of chemica l de-

r i v a t i s a t i o n a t t h e s e p o s i t i o n s i n a t t e m p t s t o reduce 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 groups 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 Boiss ie r e t a185. Both groups 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 be less t o x i c


than t h e p a r e n t a n t i b i o t i c . Boiss ie r c la imed 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 of t h e neomycin i s p r e s e r v e d f o l l o w i n g d e r i v a t i s a t i o n . However, Umezawa, working w i t h f rad iomycin , 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 su red a g a i n s t gram-pos i t i v e b a c t e r i a and on ly one t e n t h t h e a c t i v i t y when measured aga in 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 be p r o p o r t i o n a l t o t h e deg ree o f N- s u b s t i t u t i o n . A l t e r n a t i v e p rocedures f o r t h e p r e p a r a t i o n of N-methyl su lphony l neomycin have been r e p o r t e d by a number of au thors86187188 and t h e pharmacology of 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 Ber t azzo igo . The p r e p a r a t i o n of t h e fo l lowing su lpho d e r i v a - t i v e s has 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 ne omyc i n I

N-phenylsulphonyl neomycin93

N- a ce 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 thy l th ioca rbony l neomycing3

and neomycin N-sulphonateg4

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 coupled w i t h a complete loss of a n t i b a c t e r i a l p r o p e r t i e s . 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 hypocholes te remic a c t i v i t y . The e f f e c t of N-a lky la t ion , 0 - a l k y l a t i o n , N- a c y l a t i o n and O-acyla t ion has been i n v e s t i g a t e d by Magyar e t a181. l o s s of 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 groups 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 workers demonst ra ted t h a t con- v e r s i o n of amino groups i n t o q u a t e r n a r y ammonium groups r e s u l t e d i n t h e p roduc t ion of compounds having a mi ld 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 procedure 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 .

These w o r k e r s r e p o r t e d t h e

NEOMYCIN

3 . 3 . Complexes

419

The fo rma t ion of a zinc-neomycin complex h a s been r e p o r t e d by Chornock95 and by Kel ler and Cosa rg6 . E l e c t r o p h o r e s i s w a s used t o demons t r a t e t h e format ion 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 dec reased t o x i c i t y . More r e c e n t l y Agrawal, Harmalker and V i j ayawargiyag7 have d e s c r i b e d t h e fo rma t ion o f a copper-neomycin complex. With a s t o i c h i o m e t r y of 1:1, t h e b l u e c o l o u r e d complex has been made t h e b a s i s of 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 (See S e c t i o n 6 . 2 6 ) .

The complexa t ion o f neomycin w i t h a number of b iochemica l ly i m p o r t a n t compounds has 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 w i t h 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 Dougherty98. Using a s p e c t r o p h o t o m e t r i c pro- 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 lue -hepa r in 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 eac t io r . w i t h h e p a r i n . Gubernieva and S i l a e v g 9 employed elec- t 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 neomycin-heparin complex and sugges t ed t h e compound may be more t h a n a s imple ca t ion -an ion i n t e r a c t i o n . The p o s s i b i l i t y of 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 complexat ion o f neomycin w i t h h e p a r i n , DNA,RNA and po ly h l o r e t i n phosphate 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 repa red by mixing aqueous s o l u t i o n s of neomycin s u l p h a t e and t h e complexing a g e n t under 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 hydroxide so 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 of 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 exper iment demonst ra ted t h e compound t o be b i o - l o g i c a l l y a c t i v e .

Using an aga rose g e l sys tem Kunin and Tupasi.101 observed t h e format ion o f a pre- c i p i t a t i o n band between zones 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 of complex fo rma t ion


and n o t t h e r e s u l t of an an t igen -an t ibody i n t e r - a c t i o n which t h e system i s u s u a l l y employed t o d e t e c t .

Harr is lo2 has s t u d i e d t h e complexa- t i o n of neomycin w i t h p e c t i n and demonst ra ted t h e i n h i b i t i o n of complex format ion i n t h e p re sence 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 of t h e r e a c t i o n t o be a ca t ion -an ion i n t e r a c t i o n . H-bonding between t h e hydroxy groups o f p e c t i n and s u g a r moieties of neomycin has been sugges t ed and would f u r t h e r s t a b i l i s e t h e compound.

The complexat ion of neomycin wi th a n i o n i c dyes such as amaranth ( 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 ~ ~ ~ 1 ~ 5 . These complexes are aga in 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 format ion i s dependant 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 condensed phosphates have been r e p o r t e d t o complex w i t h neomycin and i n a s tudy of t h e s e compounds I Singhlo3 demonst ra ted t h e s t r e n g t h of t h e complex t o be dependant on t h e degree of p o l y m e r i s a t i o n of t h e phosphate . By i n - c r e a s i n g t h e number of phosphate u n i t s i n t h e polymer ove r t h e range 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 complexat ion w a s observed . Beyond t h i s upper 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 . R e l a t i v e complex-s t rengths w e r e a s s e s s e d by t i t r i m e t r y wi th potass ium c h l o r i d e s o l u t i o n .

A number of workers have 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 fo rma t ion of a complex between neom c i n and t h e r o t e i n s i n blood-

However, t h e n a t u r e of t h e complex i s s t i l l unknown. Geitman has 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 . In a pape r pub l i shed 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 ind 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 . globulin-neomycin complex t o be of g r e a t e r s t a b i l -

Serum and plasma104 ,Yo51 1 0 6 1 1 0 7 , ,881 1 0 9 , 1 1 0 , 1 1 1 .

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

NEOMYCIN 42 1

i t y t han t h e albumin-neomycin complex and estab- l i s h e d lmg of serum p r o t e i n t o b ind 0 . 0 6 u n i t s of neomycin . 4 . S y n t h e s i s and P roduc t ion

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 re- p o r t e d t h e p roduc t ion 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 organism subsequen t ly 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. t h e f e r m e n t a t i o n media is accomplished by use of ion-exchan e r e s i n s , such as Amberlite I R C 50225 1950,251.

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

Fol lowing 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.Qaadiae 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 , mycerin290 and c 0 l i m y c i n 2 9 1 ~ b u t have s i n c e been shown t o be i d e n t i c a l t o t h e neomycin com- p l e x .

4 . 2 . S y n t h e s i s o f Radio-Label led 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 purpose b u t concluded 14C'labelled g lucose w a s t h e most s a t i s f a c t o r y . Although 68% of 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 % of t h e t o t a l a c t i v i t y added p r i o r t o fermen$at ion 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 repa r ing14C- labe l l ed neomycin h a s been e x t e n s i v e l y used 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 . S t roshane13 has d e s c r i b e d t h e s y n t h e s i s o f 13C-neomycin and 15N-neomycin, aga in as p a r t of 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 as p r e c u r s o r f o r y3C-neomycin and glucosamine labelled wi th 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 of 15N- neomycin.

A procedure f o r 1 abe 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 Jackson e t


a t 2 9 2 and i n v o l v e s p a s s i n g an e l ec 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 s u l p h a t e . L a b i l e t r i t i u m was removed by washing wi th 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 Degrada t ion

5.1. Hydro ly t i c Degrada t ion

The d e g r a d a t i o n of neomycin h a s been 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 of 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 of t h e neomycin compo- n e n t s . F ig . 5 i l l u s t r a t e s t h e r o u t e by which com- p 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 ach ieved . Extremely v igo rous 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 of n e m i n e a s t h i s compound is r e s i s t a n t t o a c i d - h y d r o l y s i s . However, under t h e s e c o n d i t i o n s t h e chemica l e n t i t i e s neosamine C(f rom neamine) and r i b o s e are n o t observed because v i g o r o u s h y d r o l y s i s immediately 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 groups of t h e neamine and neobiosaminide molecules R ineha 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 :

( C H J C O ) 0 a ) neamine *, t e t r a N-ace ty l neamine

C H 3 0 H 5 o C 3 N H C 1

lOhr @ 9 5 O C J deoxys t rep tamine + neosamine

P h C O C l

N a O H - methyl N,N'-dibenzoyl

neob iosamin i d e b lmethyl

neob io s aminide

1 . 6 N H C 1 0 . 1 N H S O

r i b o s e d4 CH OC H O(OH)3 3 5 6

Rus s i a n w o r k e r s a t t empted t o p re - pa re t h e d e g r a d a t i o n p roduc t s i n a pu re form 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 re sence of an i o n exchange r e s i n which adsorbed t h e i n t e r m e d i a t e

NEOMYCIN

n eomy c i n I

Figure 5. Hydro ly t i c d e g r a d a t i o n of neomycin C

423


h y d r o l y s i s p r o d u c t s , t h e r e b y p r e v e n t i n g f u r t h e r decomposi t ion . The d e g r a d a t i o n p r o d u c t s w e r e re- l 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 of Bulk M a t e r i g

Japanese w o r k e r s l5 have d e s c r i b e d t h e s t a b i l i t y of bu lk f rad iomycin (neomycin B l s u l p h a t e which was shown t o have r e t a i n e d 9 9 % o f i t s micro- b i o l o g i c a l potency 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 has been re 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 598 , 305.

a t l l O ° C f o r 10 h o u r s , d u r i n g d ry -hea t s te r i l i sa - t i o n , w i t h o u t loss of po tency , though some d e g r e e of ye l lowing i s apparent298.

Neomycin s u l p h a t e may a l s o be h e a t e d

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 a1 have demonst ra ted 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 aqueous s o l u t i o n o v e r t h e pH range 2-9293. v e s t i g a t i o n s by Simone and Popino298 confirmed t h e aqueous s t a b i l i t y a t 23OC b u t a t 45OC potency l o s s e s of up t o 9 4 % w e r e no ted 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 range 4-8.

Leach e t a1294 d e s c r i b e d a p u r i f i e d

F u r t h e r i n -

neomycin t o be s t a b l e t o t h e a c t i o n of a l k a l i b u t n o t t o a c i d s . Re f lux ing t h e a n t i b i o t i c w i t h bar ium hydroxide f o r a p e r i o d of e i g h t e e n hour s f a i l e d t o show a loss i n m i c r o b i o l o g i c a l po tency .

Japanese w o r k e r s have d e s c r i b e d a s table aqueous 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 of 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 resence of 1-10% of g l y c e r o l , p ropylene g l y c o l o r mann i to l h a s been claimed t o improve t h e s o l u t i o n appearance 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 resence of p o l y o l s a l s o p reven ted a d e c r e a s e i n pH v a l u e of 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 be p reven ted by a d d i t i o n of 0.1% sodium metabi- s u l p h i t e a t a s o l u t i o n pH of 6 . 6 t o 6 .8297 .

NEOMYCIN 425

5.4. S t a b i l i t y i n Pharmaceut ica l Formula t ions

Numerous r e p o r t s concern ing t h e sta- b i l i t y of neomycin i n v a r i o u s dosa e forms have been pub l i shed . 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 of neomycin i n l i q u i d dosage forms such 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 f o r m u l a t i o n s t e s t e d , e x c e p t Dobe l l s s o l u t i o n (a mouth wash) , f o r a t least 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 c o n s i d e r a b l y longe r .

Heyd2” has 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 formula ted a s a t i s - f a c t o r y p roduc t by adso rb ing 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 i n c o r p o r a t i o n i n t h e g e l .

R e c o n s t i t u t e d aqueous suspens ions 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 be s t a b l e f o r 10 days when s t o r e d a t 4OC b u t some loss of potency w a s observed when t h e suspens ion was exposed t o day l igh t3” . Non-aqueous suspens ions c o n t a i n i n g peanu t o i l and l a n o l i n have been repor ted298 which are s t a b l e f o r 1 y e a r a t 2 0 0 C .

Simone and pop in^^'^ have cons ide red t h e s t a b i l i t y of neomycin i n both hydrophobic and h y d r o p h i l i c o in tment bases . No l o s s of po tency over 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 fo r fo rmula t ions c o n t a i n i n g ca rboxymethy lce l lu lose , po lye thy lene g lycol (P .E .G. ) o r w h i t e - s o f t para- f f i n . However, f o r m u l a t i o n s c o n t a i n i n g hydrous l a n o l i n w e r e r e p o r t e d t o be u n s t a b l e . A l l mater- i a l s used 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 . sou rces . Coates e t a1301 i n v e s t i g a t e d t h e use of 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 as be ing incompa t ib l e w i t h t h e ma te r i a l s t e s t e d .

S o l i d dosage forms of neomycin as t a b l e t s and t r o c h e s have been prepared298 and are r e p o r t e d t o be 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 oxy- t e t r a c y c l i n e has been d e s c r i b e d . With neomycin


a t a l e v e l of 20g/lb t h e p roduc t is s t a b l e f o r 1 y e a r a t 20°C, though an 8 % loss of potency was observed on s t o r a g e f o r 6 months a t 380C305. i c a t e d d r ink ing -wa te r c o n t a i n i n g 100 ppm of neomycin and prepared w i t h t ap -wa te r , showed no s i g n i - f i c a n t loss of po tency a f t e r 48 hour 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 t roughs305.

A med-

5 .5 .Compa t ib 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 , hence numerous r e p o r t s of neomycin i n c o m p a t i b i l i t y may be expec ted . D a l e and Rundman304 have 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 of neomycin w i t h s u b s t a n c e s t h a t may b e encountered by t h e f o r m u l a t i o n pharma- c i s t . Kudalker e t a1303 have d e s c r i b e d t h e incom- p 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 t h e i n c o m p a t i b i l i t y w i th b e n t o n i t e , a montomori l l - o n i t e c l a y , h a s been r e p o r t e d by Dan t i and Guth306. The i n c o m p a t i b i l i t y w i t h lactose, caus ing a d i s - c o l o r a t i o n of t h e mix tu re h a s been s t u d i e d by Hammouda and Sa1akawy3O7. The amount of browning produced w a s shown t o be dependant 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 lactose/neomycin 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 empera tu re of s t o r a g e and t h e r e l a t i v e humidi ty of t h e atmosphere. D i s c o l o r a t i o n w a s overcome 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 re- l e a s e of neomycin from P .E .G . d i e s t e r o in tment bases and g rease - type b a s e s and concluded t h e former t o be p r e f e r r e d though Coates e t a1301 have 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 be incom- p a t i b l e w i th neomycin.

5 .6 . C o m p a t i b i l i t y w i t h Other Ac t ives

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 phos- p 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 disodium hydrogen phospha te i n t h e optha lmic fo rmula t ion . The mechanism by which t h e complexat ion i s p reven ted has n o t y e t been f u l l y de te rmined .

NEOMYCIN 427

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 o n -

Table 9 l i s t s a number of c o l o r i m e t r i c tes ts which have been used a s i d e n t i t y t es t s f o r neomycin. N o one t es t , however, has been demon- s t r a t e d t o be s p e c i f i c f o r neomycin and it is 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 absence of o t h e r chemica l ly 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

N eomy c i n

T e s t Chemical group- Colour Reference i n g u t i l i s e d i n o b t a i n e d

C o l o r i m e t r i c I d e n t i t y T e s t s f o r

re a c t i o n

G l u cos amine g l y c o s i d e c h e r r y 1 1 4 Molisch p e n t o s e p u r p l e 115 Ninhydrin amino p u r p l e 115,116 Fur f u r a 1 r i b o s e p ink- red 1 1 6 Ph loro- r i b o s e p ink- red 1 1 7

g l u c i n o 1

Both t h i n - l a y e r and pape r chromatography w i l l p rov ide 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 systems f o r t h i s purpose w i l l be 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 chemica l tests a number of m i c r o b i o l o g i c a l p rocedures have been r e p o r t e d . Heinmann e t have d e s c r i b e d a s imple t es t t o d i f f e r e n t i a t e neomycin-l ike m o l e - c u l e s from t e t r a c y c l i n e s and ch loramphenicol . 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 neomycin-like molecules show an a r e a of growth- i n h i b i t i o n a t t h e t o p of t h e column.

cedure h a s been 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 test 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)pa t t e rns of i n h i b i t i o n w e r e de te rmined f o r each 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 s o l v e n t s . Comparing t h e i n h i b i t i o n p a t t e r n of an

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 pro-


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 re sence of 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 tes t has a l s o been d e s c r i b e d by T iecco e t

6 . 2 . Chemical Procedures

6 . 2 1 . 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 ueous t i t r a t i o n has been d e s c r i b e d by Penau e t alq21. Neomycin base i s al lowed t o react w i t h s t a n d a r d i s e d p e r c h l o r i c a c i d ; t h e e x c e s s a c i d i s then b a c k - t i t r a t e d w i t h potass ium hydrogen phtha- 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 benz 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 of benz id ine r e q u i r e d t o pre- 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 - pha te c o n t e n t which i s i t s e l f de te rmined by t i t r a - t i o n w i t h sodium hydrox ide .

An a l t e r n a t i v e method f o r d e t e r - mining t h e s u l p h a t e c o n t e n t of neomycin s u l h a t e has been r e p o r t e d by Roets and Vanderhaeghey22. The procedure i n v o l v e s a t i t r a t i o n w i t h bar ium 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 end- p o i n t . A s t h e a n t i b i o t i c i s i n s o l u b l e i n a l c o h o l it i s n e c e s s a r y t o remove t h e neomycin p a r t of t h e molecule p r i o r t o t i t r a t i o n . Th i s i s accomplished us ing a c a t i o n exchange r e s i n such a s Dowex 5 0 - X 8 .

During an i n v e s t i g a t i o n of t h e complexat ion 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 of neomycin conduc t o m e t r i - c a l l y w i t h amaranth ( F . D . and C Red N o . 2 ) . The end-poin t of t h e t i t r a t i o n i s s h a r p and h a s good r e p r o d u c i b i l i t y .

6 . 2 2 . Po larography

The u s e of d i f f e r e n t i a l p u l s e polarography t o de t e rmine 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 Siegerman e t Fol lowing a c i d h y d r o l y s i s of neomycin, t h e s o l u t i o n was a d j u s t e d t o pH 4 and 0 . 1 M acetate

N EOMYCIN 429

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 . Reduct ion p o t e n t i a l s of -0.19 and -0.36V w e r e ob- t a i n e d . 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 examinat ion of samples 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-conta in ing f o r m u l a t i o n s .

6 .23 . P o l a r i m e t r y

Neomycins B and C have been 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 having 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 of methods t o de te rmine 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 of t h e t e s t s o l u t i o n i s de te rmined a t 25OC and t o t a l neomycin de t e rmined 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 of neomycins B and C a r e c a l c u l a t e d . I n a second method t h e same a u t h o r s de te rmined 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 of 25O and 7 5 O C . The change 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 empera tu re from 25Oto 75OC can t h e n b e used 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 sample.

The u s e of an au tomat 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 has been r e p o r t e d by d e Rosri126 , t o moni tor t h e e l u a t e from an ion - exchange column (Bio-Rad A G l - X 2 ) th rough which a s o l u t i o n of neomycin w a s passed . The d e t e c t i o n of 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 eco rded e lectro- 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 de te rmin - i n g t h e a r e a s of t h e peaks 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 commercial samples have been de te rmined .

6 . 2 4 . Radio-chemical Assay

To de te rmine t h e r e l a t i v e amounts of neomycins B C and neamine by r a d i o chemica l method, Kaiser i28 s e p a r a t e d t h e “C - l a b e l l e d N- a c e t y l d e r i v a t i v e s by paper chromatography 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 coun t ing . 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 o b t a i n e d .


Seaman and Stewart1’’ have de- s c r i b e d a rad io-chemica l a s s a y f o r de t e rmin ing neomycin on c o t t o n - f a b r i c . Neomycin i s r e a c t e d wi th 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 which i s then decomposed wi th [lloAg] s i l v e r 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 coun t ing .

Recent ly an i s o t o p e d i l u t i o n method has been r epor t ed130 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 . This i s accomplished by adding 14C- labe l l ed g lucose t o a s m a l l - s c a l e f e r m e n t a t i o n of S . d t a d i a e . . 14C-labe l led neomycin can then 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 chromatography.

6.25. U o r i m e t r i c A s s a y

The neomycin molecule does n o t e x h i b i t f l u o r e s c e n c e b u t fo l lowing s u i t a b l e de- r 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 have been d e s c r i b e d . Maeda e t r e p o r t e d t h e complexat ion of s imple hexosamines 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 procedure t o neomycin, Simpson tive. 3 3 demonst ra ted a l i n e a r r e sponse o v e r t h e c o n c e n t r a t i o n range 0-50 uq/ml of neomycin s u l p h a t e .

The r e a c t i o n of neomycin w i t h f luo rescamine t o form a f l u o r e s c e n t complex has been r e p o r t e d by Kusni r & Barna133. The f l u o r e - scence 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 pH of t h e s o l u t i o n (optimum pH range 7.5-9.5) and t h e amount of f luo rescamine added. Th i s p rocedure may be used t o de te rmine neomycin a t ve ry low l e v e l s , t h e minimum c o n c e n t r a t i o n de te rminab le be ing 45 ng/ml.

6 . 2 6 . Spec t ropho tomet r i c Assay

Spec t ropho tomet r i c 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 ) Direct methods, i n v o l v i n g t h e i n t a c t neomycin molecule .

( b ) 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 .

NEOMYCIN 43 1

( a ) D i r e c t Methods

0 ' Keefe and R u ~ s o - A l e s i l ~ ~ f i rst r e p o r t e d 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 of neomycin, t h e procedure be ing 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 te in135 f o r t h e a s s a y of amino a c i d s and i n v o l v i n g measurement a t 570nm of t h e purp le-co loured complex formed. Maehr and Shaffner136 have a p p l i e d t h i s procedure t o t h e d e t e r m i n a t i o n of neomycin i n chromato raphy - column e l u a t e s . Gerosa and Melandri a t t empted t o app ly 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 pha rmaceu t i ca l p r e p a r a t i o n s b u t r e p o r t e d a l i n e a r r e sponse o n l y over t h e s h o r t c o n c e n t r a t i o n range of 400-600pg/ml of neomycin. B o r ~ w i e c k a l ~ ~ and Thorburn-Burns e t a1139 however, r e p o r t e d no such 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 pha rmaceu t i ca 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 Sabino200 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 inhydr in 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 as 4 pg/ml t o be de te rmined . An automated n inhydr in a s s a y has been d e s c r i b e d by Kapt ionak, Biernacka and Pazde ra l40 based on a modi f ied Moore and S t e i n methodl41. 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 pr imary amino groups of neomycin has 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-naphthoquinone) , r e a c t s w i t h neomycin base t o form an orange-coloured p roduc 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 .

Complexation of neomycin w i t h v a r i o u s dyes was r e p o r t e d by Hein loo and t h e aqueous i n - s o l u b i l i t y of t h e complex wi th amaranth ( F . D . & C Red N o . 2 ) ha s been u t i l i s e d by H i l l 2 5 and by Bufton and Saddler142 t o a s s a y neomycin i n aqueous s o l u t i o n . Amaranth may a l s o be used t o de te rmine neomycin i n p roduc t ion samples from f e r m e n t a t i o n - r ecove r 168, The dye , Orange 11, h a s been 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 - mat ion between neomycin and t h e dye i s v e r y dependant 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 , t h u s 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 o f r e a c t i o n c o n d i t i o n s t o e n s u r e complete 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 rocedure . Reac t ion of sodium l12-naphtho-quinone- 4-su lphona te w i t h


n e 0 m y c i n l 4 ~ p r o d u c e s an orange/ye l low product,Xmax 460nm i n a c e t i c a c i d , which has been u t i l i s e d t o de te rmine t h e neomycin c o n t e n t of ophtha lmic 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 agreement 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 .

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 format ion of t h e co lou red r e i n e c k a t e s a l t which may t h e n be de te rmined spec t ropho tomet r i - ~ a l l y l ~ ~ . Bickford l66 d i s s o l v e d t h e p r e c i p i t a t e d neomycin r e i n e c k a t e i n ace tone and h a s s u c c e s s f u l l y used t h i s procedure t o a s say 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 re - f e r r e d t o o x i d i s e t h e p r e c i p i t a t e w i t h po ta s s ium permanganate and then c o l o r i m e t r i c a l l y es t imate 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 .

Aromatic a ldehydes r e a c t w i t h pr imary amines forming S c h i f f ' s b a s e s which a r e o f t e n co lou red . The c o l o u r may t h e n be used 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 has shown neomycin t o form a ye l low-coloured Sch i f f ' s base w i t h s a l i c y l a l d e h y d e though 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 procedure i s , as y e t , incomple te .

The complexat ion of neomycin wi th copper r e s u l t s i n t h e format ion of a b lue -co lou red compound which has a l so 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 of neomycin i n pharma- c 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 observed 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 com- p 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

Hydrolys is p r o d u c t s of neomycin may be an amino-sugar, a pen tose o r f u r f u r a l depending on t h e r e a c t i o n c o n d i t i o n s chosen . Each of these 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 resence o f a s u g a r moiety i n neomycin was demonst ra ted by Hamre e t a1145 who used c a r - bazo le and an th rone t o q u a n t i t a t e neomycins B and C i n commercial neomycin. The p rocedures 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 l ~ ~ ,147. Penau e t r e p o r t e d poor

N E O M Y C I N 433

agreement between r e s u l t s o b t a i n e d by t h e an th rone a s s a y and m i c r o b i o l o g i c a l p rocedures and sugges t ed t h e p re sence of ca rbohydra 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 .

Other r e a g e n t s which form col u d com- p l e x e s w i t h s u g a r s such a s o r c i n o l 1 4 % 1 5 ? 9 , 1 5 0 ( 3 ,5- d ihydroxy to luene ) and p h l o r o g l ~ c i n o l ~ ~ ~ (1 , 3 , 5- t r ihydroxybenzene) have been 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 say of neomycin. Hoodless c la imed t h e p h l o r o g l u c i n o l procedure 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 procedure t o neomycin s u l p h a t e raw mater ia l . D ~ u l a k a s l ~ ~ adopted a s i m i l a r p rocedure t o d e t e r - mine t h e neomycin s u l p h a t e c o n t e n t of ophtha lmic o in tmen t s . I n t e r f e r i n g s u b s t a n c e s c o - e x t r a c t e d from t h e o in tmen 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 ph lo ro - g l u c i n o l .

The o r c i n o l procedure has been used t o a s s a y t h e neomycin c o n t e n t of f e rmen te r b ro ths148 I 1 4 9 I 150 though it 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 ion-exchange chromatography. Korchegin15O r e p o r t e d good agreement between t h e o r c i n o l a s s a y and t h e m i c r o b i o l o g i c a l a s s a f p roduc t ion s a m - p l e s . Khromov-and Starunova y7' and Korchagin e t

compared the 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 de t e rmin ing t h e neomycin c o n t e n t of polymer-f i lms and , a g a i n , good agreement between t h e two p rocedures was no ted .

A f t e r mi ld h y d r o l y s i s , neomycin has been shown t o g i v e a p o s i t i v e Elson-Morgan reaction151, a r e a c t i o n c h a r a c t e r i s t i c of 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 has 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 has been used t o de t e rmine t h e neomycin con- t e n t of f e r m e n t a t i o n broths154.

Vigorous h y d r o l y s i s of neomycin r e s u l t s i n t h e format ion o f f ~ r f u r a l l ~ ~ which ma 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 .Dutcher e t a1157 used a UV s p e c t r o p h o t o m e t r i c p rocedure , measuring t h e absorbance of t h e s o l u t i o n a t 280nm. A s i m i l a r procedure 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 has been used by Brooks e t a l l 5 8 t o de te rmine t h e neomycin B and C c o n t e n t


of t h e a n t i b i o t i c . C o l o r i m e t r i c a l l y , t h e f u r f u r a l ob ta ined from neomycin may be a s sayed w i t h an a n i l - i n e by measuring t h e amount of p ink / r ed complex formed. Morgan e t a1159 used 4-bromoanil ine 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 resis- t a n t t o t h e format ion o f i n t e r f e r i n g co lou red pro- d u c t s than 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 pha rmaceu t i ca l f o r m u l a t i o n s and r e s u l t s show good agreement 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 used t o de te rmine t h e neomycin c o n t e n t of f e rmen te r b r o t h s and o t h e r pro- d u c t i o n samples l61 , neomycin undecy lena te 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 conve r s ion of neomycin t o f u r f u r a l , q u a n t i t a t i v e conve r s ion of t h e p e n t o s e moiety t o f u r f u r a l be ing d i f f i c u l t and t h e p rocedures ted ious163t164. f o r optimum produc t ion of f u r f u r a l must b e such t h a t i t i s s t r o n g enough t o hydro lyse t h e s u g a r molecule, 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 review of t h e l i t e r a t u r e by Ivashkiv170 showed 1 2 % h y d r o c h l o r i c a c i d t o be accep ted a s optimum f o r conve r s ion of 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 be 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 rous 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 of 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 fo rma t ion 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, used 7 0 % s u l p h u r i c a c i d t o produce a 25% a c i d con- c 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 format ion o f f u r f u r a l a f t e r one hour . Ivashkiv170 has e v a l u a t e d t h e u s e of bo th hydro- c 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 produced by s u l p h u r i c a c i d , t h e y i e l d b e i n g ex t r eme ly s e n s i t i v e t o t h e c o n c e n t r a t i o n of a c i d employed. Con t inu ing 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 fo rma t ion o f f u r f u r a l a s a f u n c t i o n of neomycin c o n c e n t r a t i o n was examined and t h e conc lus ion reached 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 concen t r a - t i o n s of t h e a n t i b i o t i c . Heyes171 has shown a con- s t a n t amount of f u r f u r a l t o be produced on hydro- l y s i s w i th 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 hour . Th i s makes i t unnecessary t o a c c u r a t e l y

The a c i d s t r e n g t h

NEOMYCIN 435

p i p e t t e c o n c e n t r a t e d s o l u t i o n s o f s u l p h u r i c a c i d .

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 appear 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 each i n d i v i d u a l case.

6 .3 . Chromatographic P rocedures

6 .31. Counter-Current D i s t r i b u t i o n

1 7 6 Swart , L e c h e v a l i e r and Waksman have examined neomycin by t h e method o f coun te 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 - c o h o l ) / s t e a r i c a c i d . Three major components w i t h v a r y i n g amounts of neamine were found i n samples of neomycin B from v a r i o u s s o u r c e s . Determina t ion o f t h e amount of a n t i b i o t i c remain ing i n each t u b e fo l lowing 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 - c a l l y . In an e x t e n s i o n of t h i s s t u d y t o t h e whole group of neomycin-l ike molecules Schaf fner177 however, h a s been unab le 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 Swart e t

u s i n g e i t h e r of f o u r p a r t i t i o n systems. The systems 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 or 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 / s t e a r i c a c i d .

Both chemica l and m i c r o b i o l o g i c a l a s s a y s w e r e u t i l i z e d t o de te rmine t h e 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 . I n a l l expe r imen t s 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 erra- t i c t h a n t h e co r re spond ing chemica l a s s a y s and on t h i s b a s i s Scha f fne 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 Swart e t who used 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 demons 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 - f i e d f r amyce t in as neomycin. S i m i l a r l y , b u t u s i n g a p a r t i t i o n s y s t e m of 3% p- to luene 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 Ba ik ina e t

neomycin and Sannikov175 has i d e n t i f i e d f r amic in w i t h neomycin.

have i d e n t i f i e d m c e r i n and co l imycin a s


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 echn ique w i t h a p a r t i t i o n system of water/ bu tanol /p- to luene 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 commer- c i a l neomycin.

6 . 3 2 E l e c t r o p h o r e s i s

Neomycin i s a r e a d i l y i o n i s a b l e molecule and should t h u s be 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 ec t r i c f i e l d (zone e l e c t r o p h o r e s i s ) . Various workers 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 echn ique t o neomycin and Table 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 of 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 chemica 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 185,187 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 des igned t o s e p a r a t e compounds w i t h i n t h e inog lycos ide 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 - mina t ion of neomycin i n t h e p re sence of polymixin and b a c i t r a c i n . Using paper 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 accomplished c o l o r i - m e t r i c a l l y wi th n i n h y d r i n .

Lightbown and DeRossi 188 s u b s t i - t u t e d an aga r -ge l s u p p o r t f o r t h e more u s u a l chromatography-paper 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 procedure w a s n o t p o s s i b l e due t o t h e e longa ted 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 of t h e aga r -ge l . The same a u t h o r s a l so r e p o r t e d some anomalous behaviour of t h e neomycin i o n i n t h e presence 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 molecule and under normal c i r cums tances would be expec ted t o behave as 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 observed t o mig ra t e towards t h e ca thode . Th i s phenomena w a s r e p e a t e d l y confirmed though n o t e x p l a i n e d . A system 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 aga r - g e l s u p p o r t has been d e s c r i b e d by Grynney83. To o b t a i n a good c l e a r zone f o r neomycin it w a s neces- 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 phosphate system a t pH 6 . 5 . De tec t ion of t h e

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7

30

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7

30

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7

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Table lO(Contd. . . ) E l e c t r o p h o r e s i s of Neomycin

E l e c t r o l y t e Medium ~-

5. Trishydroxymethylamino- aga r -ge l methane/maleic a c i d / b u f f e r e d I

sodium hydroxide pH 5.6 ( 0 . 9 0 7 % : 0.870% : 0 . 0 0 2 % ) pH 5 . 6

P 6 . 2 M formic acid/O.lM paper w m t o l u e n e s u lphona te /

p rop ano l / w a C e r (50: 50:5 :40)pH 1 .8

(40:5:5:50) pH 1 . 9

t o l u e n e su lphona te / water (40:5 :55)pH 1 . 9

1 M sodium hydroxide/ 0 . 1 M t o l u e n e su lphona te / prop ano l / w a t e r ( 2 0 : O . 5 : 10: 10:69.5) pH 10.8

7 . 2 M formic acid/O.lM paper

8 . 1 M ammonium hydroxide/ paper

Cond i t ions D e t e c t i n g Agent Reference (See a l s o S e c t i o n

6 .34)

2 ooov 2 0 0 mA NCTC 8241

B . n u b L L L L A

3 40v B . A u bLLk5A ATCC 6633

188

189

18 9

340v 73. Aub&.k%A 189 ATCC 6633

340v B . AubLL&iA ATCC 6633

189

Tab le 10 (Contd. . . ) E l e c t r o p h o r e s i s of Neomycin

Medium E l e c t r o l y t e --

9 . 1M ammonium hydroxide / pape r 1M sodium hydroxide /water (20:0.25:79.75)pH 11 .5

w a t e r ( 5 :95) pH 1 2 - 2 10. i M sodium hydroxide/ paper

11. Acrylamide g e l pH 4 . 6 ac r y 1 amide

1 2 . pH 6.5 phosphate b u f f e r a g a r -ge 1 ( K H P O 4 22g/ l ; b u f f e r e d KH2P04,28g/ l )di luted a t pH 6.5 1 : 4 0 w i th d i s t i l l e d w a t e r .

P 'd

ic ge 1

Cond i t ions

3 40v

3 40v

lOOV, 2mA

310 t o 360v

( c u r r e n t 5 50mA)

Detect i n g Agent Refer en c e (See a l s o S e c t i o n

B . b u b X i l i b 189 ATCC 6633

6.343

B.aubtiLib 189 ATCC 6633

Naphthalene 1 9 0 B l a c k

S t a p h a l o c o c c u b 183 c p i d etr mid i b ATCC 1 2 2 2 8


a n t i b i o t i c was achieved b i o a u t o g r a p h i c a l l y . The organism Sfaphalococcua e p i d e h m i d i a ATCC 12228 w a s found t o be t h e most s e n s i t i v e f o r t h i s purpose. (The d e s c r i p t i o n of 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 systems w i l l be found i n s e c t i o n 6.34. )

Using an ac ry lamide -ge l as s u p p o r t , CoombelgO h a s demonst ra ted t h e q u a n t i t a - t i v e a s s a y of neomycin wi th a r ecove ry of 96% i n t h e p re sence of b a c i t r a c i n and polymixin. In t h i s case q u a n t i t a t i o n w a s ach ieved by dens i tome t ry a f t e r s t a i n i n g t h e g e l w i th naph tha lene b l a c k .

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 from 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 such a s p r o t e i n s . Hence B r a m m e r and Hemson182 have de termined t h e neomycin c o n t e n t of blood 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 assayed c o l o r i m e t r i c a l l y fo l lowing e l u t i o n from t h e s u p p o r t .

A modi f ied t echn ique 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 lowing b u f f e r s o l u t i o n and u s i n g chromatography paper 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 wi th h e ~ a r i n ~ ~ t l ~ ~ . neomycin-heparin complex was o b t a i n e d by t h i s means.

Evidence f o r t h e format ion of a

6.33. Column Chromatoqraphy

The column chromatographic sep- a r a t i o n of neomycin B , neomycin C and neamine on an ion-exchan e column has been d e s c r i b e d by Maehr and Scha f fne r ?3 6 . Dowex 1 X 2 ( O H form) was t h e ion- exchange 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 monitored by r e a c t i o n w i t h n i n - hydr in . Using a slower e l u t i o n r a t e and a smal le r p a r t i c l e s i z e of t h e same ion-exchange r e s i n Inouye and Ogawa19 peaks. The a p p l i c a t i o n o f t h i s procedure t o t h e q u a n t i t a t i v e de t e rmina t ion of neomycin C and neamine i n commercial samples of raw m a t e r i a l h a s a l s o been desc r ibed6 . When compared wi th TLC re- s u l t s on t h e same samples , t h e column chromato- g 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 examinat ion of t h e

r e p o r t e d improved r e s o l u t i o n of 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 ,demonst ra ted t h e p re sence of 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 ex- p l a i n i n g t h e observed d i f f e r e n c e between t h e t w o p rocedures .

R o e t s and Vanderhaeghe l9 have a l s o examined neomycin by chromatography on Dowex 1x2 b u t t h e s e a u t h o r s monitored 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 app- l i e d a s i m i l a r procedure t o t h e examinat ion of v a r i o u s samples 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 have been s e p a r a t e d by column chromatography 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 Amber l i te ~ ~ - 5 0 ~ ~ ~ . The components were e l u t e d from t h e r e s i n w i t h ammonium hydroxide s o l u t i o n . T .L .C . of t h e e l u e n t f r a c t i o n s showed t h e p re sence of two p r e v i o u s l y u n r e p o r t e d impur i - t i e s which were then i s o l a t e d on Dowex 1x2 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 Brazhnikova and Kudinovalg8 added a s u lph oc a t i on ic- t y p e ion -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 adsorb i n t e r - mediate 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 decomposi t ion d u r i n g h e a t i n g . Fol lowing e l u t i o n o f t h e r e s i n . in a column t h e p r o d u c t s of 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 dependant on t h e t i m e of h y d r o l y s i s .

An ion-exchange chromatographic procedure u t i l i s i n g Dowex 1x2 h a s been made t h e b a s i s o f an i n d u s t r i a l p rocess f o r manufac tu r ing 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 of neamine on Amber l i te I R C 50(Na form) f o l l o w i n g t h e a c i d hydro- l y s i s of neomycin has 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 ion-exchange column w i t h h dro- c h l o r i c a c i d . This p r o c e s s had been pa ten ted797 a s a means of manufac tur ing neamine.


Many column chromatographic p rocedures 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 fo l lowing f e rmen ta t ion .

6.34. Paper and Thin-Layer Chromatograph

The examinat ion of neomycin by paper and t h i n - l a y e r chromatography has been f o r t w o main purposes : -

a ) t o s e p a r a t e and i d e n t i f y neomycin

b ) t o s e p a r a t e i n d i v i d u a l components and

i n t h e p re sence of o t h e r a n t i b i o t i c s , 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 systems employed f o r t h e s e purposes a r e summarised i n Tab les 1 2 and 1 3 ( p a p e r chromatography) and Tab les 1 4 and 15 ( t h i n - l a y e r p rocedures ) .

V i s u a l i s a t i o n of t h e chromatograms has been r e p o r t e d u s i n g b o t h chemical 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 have been d e s c r i b e d are l i s t e d i n Table 1 6 . The v a r i o u s organisms t h a t have been employed f o r b ioautography a r e g iven i n Tab le 11.

Table 11

-- V i s u a l i s a t i o n of Chromatograms B i o a u t o g r a p h i c a l l y

Organism Reference

201,202,204,228 234,235

B . putn i luh 2 2 4

€a chetr ich ia c o l i 205,206

S tapha lococcuh aukeuh 2 0 7 , 2 2 0 , 2 3 1 , 2 3 2 ATCC 6538 s t r a i n

Table 1 2

Paper

Whatman N o . 1

Whatman No.1

Whatman N o . 1

Whatman N o . 1

Whatman No.1

Whatman No.1

Toyo No. 50

Paper Chromatographic 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

D i r e c t i o n So lven t Sys t e m Rf Value - U s e Reference

A s cendin 9 Water - s a t u r a t ed 0.84 ) b u t a n o l 1

Ascend i n g n-Butano l/ace t i c 0.85

1 ac id /wa te r ( 2 : 1 : 1)

Ascending n-Butanol /pyr id ine / w a t e r (1: 0 . 6 : 1)

Ascending 3% Aqueous ammonium c h l o r i d e

Ascending Benzene /ace t i c a c i d / w a t e r ( 2 : 1: 1) , o r g a n i c 1 a y e r

n -Bu t a n 0 1 - s a t u r a t e d water

A s cend i n g

2 0 2

2 0 2

0 . 9 4 ) I d e n t i f i - 2 0 2 ) c a t i o n of Ineomycin i n

0.00 ) t h e p re sence 2 0 2 )of o t h e r

2 0 2 ) a n t i b i o t i c s 1 0.89

) 0.07 ) 2 0 2

Ascending Methanol/3 % aqueous 0.03 ) S e p a r a t i o n of 203

2 0 4 Descending n-Butanol /pyr id ine / f o r 9 h r s . acet ic ac id /wa te r

sodium c h l o r i d e (5 : 1) neomycin from ) o t h e r I s t r ep tomyces ) a n t i b i o t i c s (15: 10: 3 : 1 2 )

Table 1 2 (Contd. . 1

Paper Chromatographic Systems S e p a r a t i n g Neomycin

Paper D i r e c t i o n

Toyo N o . 50 Descending f o r 9 h r s .

Toyo No. 5 0 Ascending

Whatman No.1 Ascending

Whatman N o . 1 Ascending P P

Whatman N o . 1 Ascending






from o t h e r A n t i b i o t i c s

S o l v e n t System Rf Value - U s e Reference

2 % p - to luene sulphon- 0.48-0.54) S e p a r a t i o n of 2 0 4 i c a c i d + 2 % p y r i d i n e i n wet n-butanol

t - B u t a n o l / a c e t i c a c i d / w a t e r (55 : 6 :39)

Water

Wate r - sa tu ra t ed n -bu t a n 0 1

Wate r - sa tu ra t ed e t h y l a c e t a t e

Wate r - sa tu ra t ed benzene

Methanol/water ( 4 0 : 6 0 )

n-Propanol /water ( 4 0 : 6 0 )

Methanol/3% aqueous ammonium c h l o r i d e (70 : 30)

Methyl e t h y l ke tone /n- bu tano l /wa te r ( 3 0 : 5 : 6 5 )

Ineomycin from ) o t h e r 1 s t r ep tomyces ) a n t i b i o t i c s . 1 2 0 4 0 . 0 6

0 .98 ) 205

2 05 S y s t e m a t i c O e 0 ! i d e n t i f i c a t i o n

' o f t h e a n t i b i o t i c s 2 05

1 0.0 ) 2 05

0 . 2 1 ) 205,206

0 . 2 0 ) 205,206

0.11 ) 1 205,206

1 0 . 2 7 )

1 205,206

Table 1 2 (Contd. . . )

from o t h e r A n t i b i o t i c s Paper Chromatographic Systems S e p a r a t i n g Neomycin

Paper D i r e c t i o n






Whatman No. 3 Ascending

So lven t System Rf Value __ U s e

6 6 . 6 % aqueous pro- 0.03 ) panol /benzene/ethy- ) l e n e g l y c o l / a c e t i c 1 a c i d ( 5 : 5 : 1 .5 : 1) 1

R e f e re n c e

2 0 7

50% aqueous p ropano l / 0 . 2 7 ' S e p a r a t i o n of 2 0 7 acet ic a c i d (25: 1) 1 neomycin from

ch loramphenicol , 207 O.O0 t e t r a c y c l i n e Wate r - sa tu ra t ed but -

a n o l / a c e t i c ac id /po t - assium cyan ide )

) and p e n i c i l l i n

( 100: 1 : 0: 059)

186,208 O s o 2 ' s e p a r a t i o n of ! neomycin from

n -Bu tano l /wa te r / ace t i c ac id (30 :13 :8 )

n -Bu tano l / ace t i c a c i d / 0 . 0 4 ' po lymixin B 186,208 p y r i d i n e / w a t e r / e t h a n o l ( 6 0 : 15: 6 :5 : 5 )

and b a c i t r a c i n 1 1

n-Bu tano l /wa te r / ace t i c 0.05 ) ac id /pyr id ine /sodium 1 c h l o r i d e (30: 1 2 : 7 : 2: 0.1) 1

186,208

Table 13

Paper

Whatman P No.1 P m

Toyo No.50

Paper Chromatographic Systems S e p a r a t i n g Neomycin Components and Degrada t ion P roduc t s

So lven t Sy s t e m Rf Value U s e Reference D i r e c t i o n - Neomycin Neamine B C

Water /bu t ano 1/ N o t a v a i l a b l e S t a b i l i t y 2 09 m e t han o l / m e t h y 1 t e s t i n g orange . f r a d i o - ( 2 : 4 : 1: 1: 5 ) mycin

-

Descending Bu tano l /py r id ine / 0 . 2 9 0 . 1 9 S e p a r a t i o n 2 10 w a t e r ( 6 0 : 30: 4 0 ) o f B and C

as a c e t y l d e r i v a t i v e s

- Butano l /py r id ine / N o t a v a i l a b l e S e p a r a t i o n 2 1 1 wa te r ( 3 :2: 1) of B and C

as a c e t y l d e r i v a t i v e s

Descending n-Propanol/ 0.13 - 0.15 0.28) S e p a r a t i o n 2 0 4 f o r 9 h r s p y r i d i n e / a c e t i c ) of neamine

ac id /wa te r ) from neo- (15:10:3:12) ) mycins B

) and C

Tab le 13 (Contd. . )

Paper Chromatographic Systems S e p a r a t i n g Neomycin

Paper D i r e c t i o n S o l v e n t System Rf Value - U s e Reference

Components and Degrada t ion P roduc t s

Toyo N o . 50

Toyo No. 5 0

Grade M

P P 4

Descending 2 % p - to luene su lph- f o r 9 h r s o n i c a c i d i n w a t e r

s a t u r a t e d n-butanol

Ascending 1 . 5 % sodium c h l o r - i d e i n 80% methanol

Ascending B u t a n o l / a c e t i c ac id /wat e r ( 4 : l : 5 )

Neomycin Neamine B C

0.11- 0 . 2 0 0.23 ) S e p a r a t i o n 2 0 4 )of neamine ) from neo- )mycins B

1 0.08- 0.11 0 . 2 2 ) a n d C

Not a v a i l a b l e S e p a r a t i o n 2 1 2 of B and C a s a c e t y l d e r i v a t i v e s

Whatman Descending n-Butanol /water / 0 . 7 0 0.35 1-00 Q u a n t i t a t i v e 213 NO. 4 f o r 24-3g p i p e r i d i n e ( v a l u e s r e l a t i v e spec t ropho to -

h r s , 28 C (84:16:2) t o neamine) m e t r i c d e t e r - mina t ion of components

Table 1 3 (Contd. . . ) Paper Chromatographic Systems S e p a r a t i n g Neomycin

Components and Degrada t ion P roduc t s

Paper D i r e c t i o n So lven t System Rf Value - U s e Reference

Neomycin Neamine B C

Whatman Descending Methyl e t h y l 0.54 0.30 1.00 S e p a r a t i o n 2 1 4 No. 1 f o r 2 4- 3 6 ke tone / t -bu t ano 1/ ( v a l u e s r e l a t i v e a s f r e e b a s e s

h r s , 25- methanol/6.5N t o neamine) Quant i t a t i on 27OC ammonium hydroxide by s p e c t r o -

P (16:3:1:6) ph o t o m e t r y P m

Whatman Descending Methyl e t h y l ke tone / 0.57 0.34 1.00 N o . 1 f o r 8-400 isopropanol /6 .5N ( v a l u e s r e l a t i v e

h r s , 2 4 C ammonium hydroxide t o neamine) (80:20:30)

2 15

- R a d i a l But an o l / p y r i d i n e / N o t a v a i l a b l e S e p a r a t i o n o f 2 1 6 a c e t i c ac id /wa te r d e g r a d a t i o n (15: 10: 3 : 1 2 ) p r o d u c t s

Whatman Descending B u t a n o l / w a t e r ( l : 2 ) 0 .5 0 . 6 0 S e p a r a t i o n of 228 N o . l i m - f o r 18 h r s c o n t a i n i n g 4 % neamine from pregnat - t o l u e n e s u l p h o n i c a c i d neomycin B ed wi th and 2 % p i p e r i d i n e and C PH 8 hydroch lo r ide b u f f e r

Table 1 4

Thin Layer Chromatographic Systems S e p a r a t i n q Neomvcin from Other A n t i b i o t i c s

Ads o r b e n t - S o l v e n t Sys tern

S i l i c a ge 1 G/ n-Propanol/ethylacetate/ aluminium ox ide water /13.4 M ammonium (1: 1) hydroxide (50: 10: 30: 10)

C e l l u l o s e M N n-Propanol /pyr id ine / a c e t i c a c i d / w a t e r (15 : 10: 3 : 1 2 1

P P \c

K i e s e l g e l G Wate r / m e t hano l /bu t an o l / b u t y l a c e t a t e / a c e t i c a c i d ( 1 2 : 2.5 : 7 . 5 : 4 0 : 2 0)

K i e s e l g e l G Wate r /bu tano l /py r id ine / acet ic a c i d . ( 1 4 : 30: 2 0 : 6 ) ( 1 6 : 4 0 : 8: 1 6 )

K i e s e l g e l G Wate r /bu tano l / ace t i c a c i d (39 : 55 : 6 )

Rf Value

0.52

0.10

0.0 ) 1 1 1 )

0 . 1 2 ) 0.00 )

1 1

0.00 )

- U s e Reference

S e p a r a t i o n of 2 1 7 neomycin from o t h e r aminoglyco- s i d e a n t i b i o t i c s

S e p a r a t i o n of 2 18 neomycin from o t h e r w a t e r s o l u - b l e b a s i c a n t i b i o t i c s

2 1 9 S e p a r a t i o n of neomycin from b a c i t r a c i n , polymixin and t y r o t h r i c i n 2 1 9

2 1 9

Tab le 1 4 (Contd. . . )

Ad so rben t

K i e s e l g e l G

Se ph adex G-15

P VI 0

S i l i c a g e l G

K i e s e l g e l G

K i e s e l g e l G

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

So lven t System Rf Value U s e Reference -

Methanol / l7% aqueous ammonia chloroform,aqueous l a y e r (10:10:20) 0 .31

pH6 phosphate b u f f e r (0.025M) + 0.5M sodium c h l o r i d e 0.75

Ben z ene/ace t one/ ace t i c a c i d ( 4 : 4 : 2 ) 0.35

But anol /ace t i c a c i d / py r id ine /wa te r (30: 22: 6 : 38)

Bu tano l /wa te r / ace t i c a c i d / e t h a n o l / p y r i d i n e ( 6 0 : 10:15: 5: 6 )

0 . 1 4

0.05

) S e p a r a t i o n of ) neomycin from ) b a c i t r a c i n , 2 1 9 ) polymixin and ) t y r o t h r i c i n

1 1 ) I d e n t i f i c a t i o n 2 2 0 ) o f a n t i b i o t i c s 1

2 2 1 1

? S e p a r a t i o n of

186 208 ’ nebmycin from ? z i n c b a c i t r a c i n ’ and polymixin 1 1 186 208

Table 14 (Contd.. . )

Ad s o r ben t

Kiese lge l G

Kiese lge l G + Kieselguhr G ( 1 : l )

Kiese lge l G + Kieselguhr G ( 1 : 2 )

C e 1 l u l o s e MN-300

Kiese lge l G + aluminium oxide G type E ( 1 : l )

Kiese lge l G impregnated with sodium a c e t a t e

Thin Layer Chromatographic Systems Sepa ra t ing Neomycin from Other A n t i b i o t i c s

Solvent System Rf Value - U s e Reference

Propanol/ethyl ace ta te /water / 0.08 ) 2 5 % ammonium hydroxide 1 (100 :20 :60 :20) . 0 . 1 4 )

1

222

222

1 0 . 2 2 ) 222

222 )Separa t ion of

0.32 'g lycos id ic

a n t i b i o t i c s 1

0 . 1 2 ) 1

0 . 3 3 )

1 1

222

222

Ads or bent

Kiese lge l G

Kiese lge l G + Kieselguhr G ( 1 : 1) Kiese lge l G + Kieselguhr G ( 1 : 2 )

P VI

h, Kiese lge l G impregnated with 3.85% aqueous ammonium a c e t a t e

Kiese lge l G

Kiese lge l G + Kieselguhr G ( 1 : l ) Kiese lge l G + Kieselguhr G ( 1 : 2 )

Table 2 (Contd.. )

Thin Layer Chromatographic Systems Separa t ing Neomycin from Other A n t i b i o t i c s

Solvent System Rf Value U s e

Prop anol/e t hy 1 ace t a t e / 0.11 w a t e r / 2 5 % ammonium hydroxide/ pyridine/3.85 % aqueous 0.13 ammonium a c e t a t e ( 100 : 2 0 : 6 0 : 2 0 : 10 : 2 00)

II 0.16

0 .08

Ethanol/ethylacetate/water/ 0.08 25% ammonium hydroxide/

ammonium a c e t a t e (100:20:60:20:10:200)

pyridine/3.85% aqueous 0.11

0 .12 I t

1 1

1 1 1

Separa t ion of g l y c o s i d i c a n t i b i o t i c s

1

1

1

1 1

Reference

222

2 2 2

2 2 2

2 2 2

2 2 2

2 2 2

222

Table 1 4 (Contd. . )

Adsorbent

Kiese lge l G + aluminium oxide G ( type E ) (1:1)

--

Kieselgel G

VI P Kieselgel G + Kieselguhr G (1: 1) Kiese lge l G + Kieselguhr G ( 1 : 2 )

Kiese lge l G + aluminium oxide G , type E ( 1 : l )

Thin Layer Chromatographic Systems Separa t inq Neomycin from Other A n t i b i o t i c s

Solvent System Rf Value - U s e

Ethanol/ethylacetate/water/ 0.08 ) 2 5 % ammonium hydroxide/ 1 pyridine/3.85% aqueous ammonium a c e t a t e 1 (100: 2 0 : 60 : 2 0 : 10: 2 0 0 )

1

Reference

2 2 2

Methanol/ethyl a c e t a t e / 0 . 0 6 ) 222

ide/pyridine/3.85% aqueous 0.17 ) 222 water/25 % ammonium hydrox-

ammonium a c e t a t e (100:20:60:20:10:200)

Separat ion of g lycos id i c

11 2 2 2 ’ a n t i b i o t i c s 0 . 1 4 ) 1 1

Methanol/ethyl a c e t a t e / 0.11 ) water/25% ammonium hydrox- 1 ide/pyridine/3.85% aqueous 1 ammonium acetate ( 100 : 2 0 : 6 0 : 20 : 10: 200) 1

2 2 2

-- Table 1 4 (Contd. . I Thin Layer Chromatographic Systems Separa t ing

Neomycin from Other A n t i b i o t i c s

Ads o r bent

Kieselgel G impregnated w i t h 3.85% aqueous ammonium a c e t a t e

Kieselgel G

Kieselgel G + Kieselguhr G ( 1 : 1) Kiese lge l G + Kieselguhr G ( 1 : 2 )

Kiese lge l G + aluminium oxide G , type E ( 1 : l )

Kieselgel G + Kieselguhr G ( 1 : 2 )

VI fr

So lven t System Rf value

Methanol/ethyl acetate/ 0.08 water/25% ammonium hydrox- 1 ide/pyridine/3.85% aqueous 1 ammonium a c e t a t e 1 (100: 20: 60: 20: 10: 2 0 0 )

1 1

Butanol/methanol/ethyl 0.06

hydroxide/pyridine/3.85% 0.07 acetate/water/25 % ammonium 1 -

aqueous ammonium acetate (100: 100: 20: 60: 20: 10: 2 0 0 )

1 ) Separat ion of ) g lycos id i c

1

1

0.13 ) a n t i b i o t i c s

0.05 )

1 25 % ammonium hydroxide/ 0.36 water/acetone (16 : 1 4 4 : 4 0 )

Reference

222

222

222

2 2 2

222

2 2 2

Table 1 4 (Contd.. 1 Thin Layer Chromatographic Systems Separa t ing

Neomycin from Other A n t i b i o t i c s

U s e Solvent System Rf Value - 25% ammonium hydroxide/ 0.34 1 w a t e r/ ace t on e 1 (16 : 144 : 40)

1

Adsorbent

Kiese lge l G + Kieselguhr G(1:2)impregnated wi th 2 % sodium acetate

Kiese lge l G + aluminium

type E (1: 1) % oxide G ,

Kiese lge l G + Kieselguhr G ( 1 : 1) impregnated with 2 % sodium a c e t a t e

Kieselgel G

If

11

Water/sodium c i t r a t e / c i t r i c ac id (100:20:5)

Reference

2 2 2

1 0.48 )Separa t ion of 222

g l y c o s i d i c a n t i b i o t i c s

1

1 1 1 1

0.43 ) 2 2 2

0.95 C l a s s i f i c a t i o n 223 of a n t i b i o t i c s

Adsorbent

Acid i f ied carbon

Acidif ied carbon

Untreated carbon

P VI

Untreated carbon

Carbon t r e a t e d

Table 15

Thin Layer Chromatographic Systems Separa t ing Neomycin Components and Degradation Products

Solvent System

Water

0.5N su lphur i c ac id

Water

0.5N su lphur ic ac id

0.5N hydro- c h l o r i c ac id

with hydro- c h l o r i c ac id

II 0.5N hydro- c h l o r i c acid/ methanol ( 4 : 1)

Rf Value - U s e Reference

Neomycin Neomycin Neamine B C

0.10 0.10 0 . 6 0 )Assay of 22 4 ) neomycin )components

) c i a 1 form- ) u l a t i o n s

1 1

0 . 2 1 0.43 0 . 6 1 ) i n commer- 2 2 4

0.00 0.00 0.00 ) 2 2 4

0 . 2 4 0.45 0.54 ) 2 2 4

0.37-0.45 0.53-0.74 0.63-0.92 ) 2 0 4 1 ) Separa t ion ) of neomycins )B and C and ) neamine

1 1

0 . 4 4 - 0 . 7 2 0.58-0.81 0 .64-0 .91 ) 2 0 4

Tab le 15 (Contd. . . ) -- Thin Layer Chromatoqraphic Systems S e p a r a t i n g Neomycin Components and Degrada t ion P roduc t s

So lven t System Rf Value U s e Reference - Ad so r b e n t

Carbon t r e a t e d wi th w a t e r

Carbon/ gypsum t r e a t e d w i t h s u l - p h u r i c a c i d

Carbon/ gypsum t r e a t e d wi th wa te r


0.5N hydro- 0 . 4 1 0.57 0 . 6 2 c h l o r i c a c i d )

0.5N hydro- 0.48 0.58 0.68 1 c h l o r i c a c i d / ) methanol ( 4 : 1) 1

2 0 4

2 0 4

0.5N s u l p h u r i c 0 . 2 0 0.59 0.80 ) S e p a r a t i o n 2 0 4 a c i d of neomycins

) B and C and ) neamine 1 1 1

0.5N s u l p h u r i c 0 . 6 4 0 .82 0.85 ) ac id /me thano l (4 : 1) 1 0.5N hydro- 0 . 1 6 0.28 0.30 c h l o r i c a c i d )

1

2 0 4

2 0 4

Table 15 (Contd.. )


Adsorbent

Carbon/ gypsum t r e a t e d with water

P v, W

I1

11

Kieselgel H

Kiese lge l H

Solvent System

0.5N su lphur i c ac id

0.5N hydro- c h l o r i c ac id / methanol (1 : 1)

0.5N hydro- c h l o r i c acid/ propanol(1: 1)

3.85% aqueous ammonium acetate

3 . 4 % ammonium hydroxide

R f Value

Neomycin Neomycin Neamine -- B C

- 0 . 2 4 0 . 4 0

0.36

0 . 4 8

0.13

0.37

0.36

0.48

0.13

0 . 4 4

0 .46

0 . 5 2

0.36

0 .47

U s e

1 1 )Separa t ion )of neomycin )B and C and ) neamine

1 1

Assay of neamine

Assay of neomycin C

R e f e rence

2 0 4

2 0 4

2 0 4

1 9 6 , 9

1 9 6 , 9

Table 15 (Contd.. )

Ads orben t

C e 1 lu lose MN

2 Cel lu lose \o

MN-300

Cel lu lose MN-300

S i l i c a g e l G

Thin Layer Chromatographic Systems Sepa ra t ing Neomycin Components and Degradation Products

Solvent System

Methyl e t h y l ke tone/ isopropanol/6.5N ammonium hydroxide (80:20:30)

Methyl e t h y l ke tone/me thano 1/ isopropanol/7.9N ammonium hydroxide (10: 8.5 : 3 : 7 )

R e f e ren ce U s e - R f Value -- Neomycin Neomycin Neamine

B C

0 . 2 1 0 . 1 2 0.31 Separa t ion 2 15 of neomycin components

0.52 0 . 2 4 0 . 6 1 236

s i n g l e development )Assay of Propanol/pyridine/ 0.25 0 . 2 0 0 . 4 7 )neomycins B 226 a c e t i c acid/water double development land C and nea- ( 100 : 6 6 : 2 0 : 80) 0.33 0.25 0 . 6 0 )mine i n pharm-

) a c e u t i c a l form- 11

0.57 0 .66 0.73 ) u l a t i o n s 226

Table 15 (Contd.. )

Thin Layer Chromatographic Systems Separa t ing Neomycin Components and Deqradation Products

Solvent System Rf Value - U s e Reference Ad sorb en t

Cel lu lose MN-300/ Kieselguhr G ( 1 : l )

S i l i c a m 0 g e l G

C e l l u l o s e MN-300/ Kieselguhr G ( 1 : l )

S i l i c a g e l

Kieselguhr G ( 1 : 2 )

G/


Propanol/pyridine/ 0.53 acetic acid/water (100: 66 : 20: 80)

Prop an o l /e t hy 1 acetate/water/25% ammonium hydroxide (100:20:60:20)

Methanol/3% aqueous 0.36 sodium ch lo r ide ( 2 : 1)

0.23

3.85 % aqueous 0 .22 ammonium acetate

0 . 4 6 0.73 ) 226 1 1

0.23 0 . 4 2 ) 226 1 1 )Assay of )neomycins B

0.43 )and C and 226 )nearnine i n )pharmaceut ical ) formulat ions 1

'0.39 ) 226 1 1 1

0.17

0 .22

Table 1 5 (Contd.. )


Adsorbent

S i l i c a g e l G

Kieselguhr G p u r i f i e d

S i l i c a g e l G

S i l i c a

Kieselguhr G ( 1 : 2 )

g e l G/

Solvent System

Methyl e t h y l ketone/ t - but ano l / m e t han o 1 / 6.5N ammonium hydroxide (160 : 30: 10: 6 0 )

II

Propanol/e than- o l / e t h y l a c e t a t e /

U s e Rf Value - Neomycin Neomycin Neamine

B C

0 . 1 4 0.11 0 . 2 2 )

0 . 4 6

0.18

water/25% ammonium hydroxide/pyridine/ 3.85%ammonium a c e t a t e (100: 100: 2 0 : 60: 20: 10: 2 0 0 )

II 0.18

0.68

0.15

0.11

Reference

226

)Assay of

)and C and lneamine i n )pharmaceut ical

0 . 8 4 )neomycins B 226

0 - 38 formulat ions

1 0.32 ) 226

Table 15 (Contd.. )


Adsorbent Solvent System R f Value

Neomycin Neomycin B C

Dowex 50x8 1.5M Sodium 0.15 sodium acetate, pH 8.5 form(1onex + 58.49 sodium 25SA) ch lo r ide /

t -butanol(10: 1)

Si l ica 3% Ammonium 0.33 ge 1 hydroxide /acetone

(160: 40)

Kiese lge l 3% Ammonium G hydroxide /acetone

(160: 90)

0.15

0.33

N o t a v a i l a b l e

U s e R e f e rence - Neamine

0.28 Separa t ion of 227 neamine from neomycins B and C

- Q u a n t i t a t i v e 2 2 9 a n a l y s i s of ne omy c i n su lpha te

S t a b i l i t y of 233 neomycin a f t e r e thy lene oxide s te r i l i s a t i o n

N EOMY CIN

T a b l e 16

V i s u a l i s a t i o n of Chromatograms by Chemical Means

463

1.

2.

3.

4 .

5.

6.

7.

8.

9 .

Spray Reagent Colour of R e f e r en ce - Neomycin zone

E t h a n o l i c sodium Dark b l u e 2 1 0 , 2 1 1 h ypoch 1 or i t e fo l lowed by s t a r c h / po ta s s ium i o d i d e s o l u t i o n

Ch l o r i n e /e thano 1 Dark b l u e fo l lowed by s t a r c h / po ta s s ium i o d i d e s o l u t i o n

Chlor ine /carbon Dark b l u e t e t r a c h l o r i d e fo l lowed by s t a r c h / p o t as s ium i o d i d e / p y r i d i n e

t - b u t y l hypochlo- Dark b l u e r i t e / d i c h 1 oro- me thane /ace t i c a c i d fo l lowed by s t a r c h / po ta s s ium i o d i d e s o l u t i o n

Ninhydrin P u r p l e

2 1 2

2 1 3

1 9 6

186 ,203 ,208 214,215 , 216 217,218,219 229 ,231

Ninhydrin/cad- Purp le /p i n k 2 2 7 m i u m a c e t a t e

p-dimethyl amino- P ink o r ben zaldehyde p u r p l e

Ninhydrin/p- d ime thy l aminobenzaldehyde

Copper s u l p h a t e / Blue ammonium hydr- o x i d e

223

226

2 2 1


Q u a n t i t a t i v e a n a l y s i s of neomycin a f t e r chromatography u t i l i s i n g some of t h e v i s u a l i s a t i o n t e c h n i q u e s g iven i n Tab les 11 and 1 6 have been r e p o r t e d . Majumder and Majumder213 sep- a r a t e d neomycins B and C as t h e a c e t y l d e r i v a t i v e s on paper t h e n , f o l l o w i n g conve r s ion 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 measur- e d a t 570nm. d e s c r i b e d t h e s e p a r a t i o n of 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 de- developed 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 methanol and t h e absorbance of t h e s o l u t i o n measured a t 570nm.

A l a t e r pape r by t h e same au thor s214

Thin- l a y e r chromatography has been used by Foppiano 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 raped o f f t h e p l a t e and re- a 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 absorbance o f t h e r e s u l t i n g c o l o u r be ing measured a t 665nm. By t h i s p rocedure a p r e c i s i o n of 2 % w a s ach ieved .

Ninhydrin h a s a lso been used t o q u a n t i t a t e 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 er chromatography. P r e g n o l a t t o and Sabin0250 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 inhydr in 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 r e p r o d u c i b i l i t y of t h e procedure . An a l t e r n a t i v e color imetr ic procedure 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 chromatography h a s been d e s c r i b e d by Doulakasl74. Neomycin i s e l u t e d from t h e s i l i c a g e l w i t h pH 12.5 phosphate b u f f e r t h e n o x i d i s e d w i t h sodium hypobromite. The r e s u l t i n g a ldehyde 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 pink- co lou red p roduc t which i s measured spec t ropho to - m e t r i c a l l y .

The use of b ioautography f o r t h e q u a n t i t a t i o n of chromatograms h a s been d e s c r i b - ed by many w o r k e r s . Emilianowicz-Czerska and Herman228 s e p a r a t e d neamine f r o m neomycins B and C then q u a n t i t a t i v e l y de te rmined t o t a l neomycin B and C by b ioautography w i t h B.hubtiCih. A l i n e a r response f o r c o n c e n t r a t i o n s between 0.25 and 1 O p g o f neomycin w a s ob ta ined . With a s i m i l a r t e c h n i q u e b u t u s i n g a mathemat ica l a n a l y s i s o f t h e b i o a u t o -

NEOMYCIN 465

grams, Simon230 r e p o r t e d a q u a n t i t a t i v e de te rmina- t i o n of neomycin s u i t a b l e f o r use w i t h i r r e g u l a r - shaped zones . Brodasky224 employed b ioau tography 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 carbon. The q u a n t i t a t i o n o f s m a l l amounts of neomycin C however, proved d i f f i c u l t , t h e d i f f i c u l t - ies be ing a s s o c i a t e d w i t h t h e enhanced growth of t h e organism i n t h e p re sence of ca rbon( f rom t h e T.L.C. p l a t e ) and ammonium i o n s (from t h e chromatographic p r o c e d u r e ) . S i m i l a r d i f f i c u l t i e s w e r e e x p e r i e n c e d by Soko l sk i and L ~ m m i s 2 3 ~ who a t t r i b u t e d poor neomycin zones t o be t h e r e s u l t of antagonism 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 sys- t e m . 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 be a p p a r e n t u s i n g t h e organism 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 de- 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.

An e x t e n s i v e s t u d y by Langner201 demonst ra ted

6.35. Gas-Liquid Chromatography

T s u j i and Rober t son lg2ach iev - ed 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 0.75% OV-1 on G a s Chrom Q a t a tempera- t u r e of 2 9 0 O C . The same c o n d i t i o n s have a l s o been shown t o s e p a r a t e neobiosamine B, neosamine and deoxys t rep tamine from neomycin and neamine. Hence t h e method could be used t o s t u d y t h e s t a b i l i t y of neomycin o r t o moni tor 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 p r o c e s s . U s e of t h e p rocedure t o assess t h e s t a b - i l i t y of neomycin i n pha rmaceu t i ca l f o r m u l a t i o n s has been demonst ra ted by Van Giessen 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 . However , these 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 empera tu re t o chromatograph neomycin and consequen t ly had a reduced column l i f e . A concen- t r a t i o n of 3% OV-1 w a s p r e f e r r e d , as 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 rocedure 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 of 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 ica- t i o n of 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 sample decomposi t ion by c o n t a c t of i n j e c t e d mater ia l w i t h


metal o r Teflon and the add i t ion of an accu ra t e volume of s i l y l a t i n g reagent t o each sample and s tandard 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) assays has been reported by T s u j i e t a1239 who showed t h e neomycin content of neomycin su lpha te 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 wi th va lues obtained by the microbio logica l assay when it is assumed neomycin C has 35% of the b i o l o g i c a l ac- t i v i t y of neomycin B.

mixtures of o t h e r aminoglycoside a n t i b i o t i c s con- t a i n i n g t h e 2-deoxystreptamine moiety a s both 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 luoro- 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 using a column of 0.75% OV-1 on Gas Chrom Q240. may be used t o e s t ima te t h e number of sugar moieties 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 r e t e n t i o n t i m e .

Neomycin has been separa ted from

The procedure

Examination of neomycin B by a combined G.C.-M.S. procedure has been repor ted by Murata e t a l l 5 . G.C. w a s accomplished with t h e tr i- me thy l s i ly 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 separa t - ed from kanamycin. The r e s u l t i n g mass s p e c t r a of the neomycin d e r i v a t i v e exh ib i t ed a minute mole- c u l a r ion 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 both hydroxy and amine groups were completely s i l y l a t e d .

6.4.Microbiological Procedures

6.41.Turbidimetric Assay

Various organisms have 'been used t o assay neomycin t u r b i d i m e t r i c a l l y . These are summarised i n Table 1 7 with t h e working concen- t r a t i o n f o r t h e a n t i b i o t i c as repor ted by t h e r e spec t ive au thors .

O f t h e organisms t abu la t ed over- l e a f K.pneumaniae is gene ra l ly used f o r r o u t i n e assays.

NEOMYCIN 467

Tab le 1 7

Cond i t ions of T u r b i d i m e t r i c Assay

Organism Concen t ra t ion Range Reference of Assay

(Tota l pg neomycin b a s e r e q u i r e d i n s o l u t i o n t o be a s sayed )

---

S.auheub NRRLB314 2.5 - 5.0 2 41

209P 100- 300 242

F1 0.016 - 0.04 243

II II

11 11

s th epzo cu cc Ub daecalib ATCC 1054 12 0-200

K l e bb i e l l a pneumaniae PC1602 6-15

K l e b b i e l l a pneumo niae ATCC 10031

6-14

2 48

244

245 I 246 I 268

E.coli ATCC 10536 1- 3 247

ATCC 11105 1-20 253 II II

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 ac tu 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

3-7 2 48

20-60 248

2-6 248

4-6 248

0- 6 249

Hein243 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 potency of neomycin when a s sayed t u r b i d i - m e t r i c a l l y i f t h e innoculum 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 po ta s s ium phos h a t e . With S.auheub as organisml S o k o l s k i e t a1 25!? demonst ra ted t h e p r e - s ence 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 than t h a t of neomycin B.

WILLIAM F. HEYES

A comparat ive s t u d y of t h e res- ponce of 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 repor ted239. neomycin C w a s 100:38.9.

The r e s p o n s e - r a t i o neomycin B:

An automated t u r b i d i m e t r i c procedure u s i n g a Technicon Autoanalyzer h a s a l so 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

Table 18 l i s ts t h e organisms t h a t have been recommended f o r de t e rmin ing t h e micro- b i o l o g i c a l potency o f neomycin.

Table 18 -- Recommended Working Reference

Organ i s m Concen t r a t ion ( P g h l )

8 . p u m i l i n NCTC 8241

S . aua eun ATCC 6538

4-2 1 9

4 - 2 0 245

S . e p i d ekmidics 0 . 6 4 - 1.56 245,268 ATCC 12228

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 procedure 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 rocedures 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 Parke245 r e p o r t e d a l i n e a r - d i f f u s i o n sys tem i n which a s o l u t i o n of t h e a n t i b i o t i c i s al lowed t o d i f f u s e i n t o a g a r - f i l l e d g l a s s c a p i l l i a r i e s . The s t a n d a r d d e v i a t i o n s cal- c u l a t e d f o r seven exper iments 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 of neomycin. The procedure i s more economic i n t e r m s of a g a r and n u t r i e n t s than o t h e r d i f f u s i o n methods. Kuzel and Coffey255 modi f ied 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 procedure by u s i n g a c y l i n d e r s e a l e d a t one end , forming a cup. The cups 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 be a s sayed , t hen a p l a t e of in - 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 cups . 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

NEOMYCIN 469

w a s c la imed. The rep lacement o f c l i n d e r s by a er

c la imed a s t a n d a r d d e v i a t i o n o f 2 % f o r t h e a s s a y of neomycin by t h i s means and recommended t h e pape r d i s c procedure 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 used has 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 e ~ ~ ~ Whatman s e e d t e s t pape r producing t h e sma l l e s t zones. When de- 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 rocedure by sub- s t i t u t i n g d r i e d mi lk t a b l e t s f o r pape r d i s c s .

d i s c s h a s also been r e p o r t e d 2561275,278. ~~~~i 47Y

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 demonst ra ted 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 p rocedure 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. R e f r i g e r a t i o n o f t h e a g a r - p l a t e s p r i o r t o i n c u b a t i o n caused an i n c r e a s e i n zone- s 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 idd ique 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 mi lk . The p r e s e n c e o f K C l , N a C l , C a C 1 2 has been shown t o markedly i n c r e a s e neomycin d i f f u s i o n i n aga r258 , 265 194. Converse ly t h e p r e -

f phosphates258 and of g l u c o s e / s t a r c h w i t h N a C l 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 zone. I n c o r p o r a t i o n of n o n - f a t mi lk i n t h e a g a r s i g n i f i - c a n t l y r educes t h e s i z e of t h e i n h i b i t i o n zone because of 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 carbox 1 group of mi lk p r o t e i n 2 6 3 . Fedorko 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 lood 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 of 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 demonst ra ted 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 of 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 tud ie s260 t561 ,252 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 of bo th t h e p H o f t h e a g a r and t h e manufac tu re r . I n c o r p o r a t i o n of 0.1M tr is b u f f e r w i th t h e a g a r (pH 8 ) h a s been recommend- ed260, 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 - t i v i t y . T r i s b u f f e r r e s u l t s i n a g r e a t e r s e n s i t i v - i t y t han phosphate b u f f e r 2 6 1 and does n o t a f f e c t t h e s i z e of t h e i n h i b i t i o n zone whereas t h e p re - sence of phosphate r educes zone s i z e 2 5 2 . G i l l e t e t ,1194 concluded 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 -


t ed t h e i n h i b i t i o n zone s i z e . A comparison of 3 agars showed t h e b e s t response f o r a given concen- t r a t i o n of neomycin w a s obtained with agarose.Com- bining agarose wi th recommendations descr ibed by o t h e r au tho r s , such as t h e add i t ion of C a C l and the use of t r is b u f f e r , f u r t h e r increased tge s i z e of t he i n h i b i t i o n zone.

The l i t e r a t u r e descr ibed above r e f e r s t o the assay of t h e t o t a l neomycin complex i . e . neomycins B and C and neamine. To assay neomycin B i n t h e presence of neomycin C and neamine by a microbia l method Sokolski and Carpenter265 adopted t h e fol lowing procedure. These au thors 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 the agar t o t o t a l l y de- p re s s d i f f u s i o n of neamine and inc rease 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 vary wi th t h e condi t ions of t he assay.Typical ly neomycin C has an a c t i v i t y of 30-50% of t h a t of neomycin B 2 6 6 r 239 . The presence of potassium chlo- r i d e o r phosphate reduces t h e d i f f u s i o n of neomycin C more than t h a t of neomycin B252. 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 has been shown t o be more pronounced when us ing S . U U t r e U b a s organism than when us ing 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 of t h e medium a system has been developed i n which t h e responses of neomycins B and C a r e ident ica l266 .

Furthermore,

The microbia l assay of neomycin i n t h e presence of o t h e r a n t i b i o t i c s can p resen t d i f f i c u l t i e s i f both 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 tes t organisms. The assay of neomycin i n the presence of dihydrostreptomycin, 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 , has been repor ted . streptomycin i n a c t i v e by hydro lys is with barium hydroxide p r i o r t o termining neomycin with S. UUfLeUb . 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 ihydros t rep to- mycin. When assaying neomycin i n a mixture con- t 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 ihydros t r ep t - omycin, Tanguay e t a12T1 found it necessary t o

Levine e t a1269 rendered t h e dihydro-

DeNuzio 2% however, chose t o overcome

NEOMYCIN 47 1

sepa ra 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 so lven t ex- t r a c t i o n before determining neomycin by t h e method of DeNuzio.

The determinat ion of neomycin i n animal feeds has received much a t t e n t i o n . Barb iers and Neff272 recommended the use of a tris b u f f e r a t pH 8 t o prepare neomycin s o l u t i o n s and t h e add i t ion of magnesium o r calcium ch lo r ide t o t h e agar f o r enhancement of t h e i n h i b i t i o n zones. Ex t r ac t ion of neomycin from t h e feed requi red t h e u s e of sodium ch lo r ide s o l u t i o n f o r 100% recovery. A co l labora- t i v e s tudy of t h i s procedure by e ighteen labora- t o r i e s r e s u l t e d i n an average recovery of 100.3% with 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. Modi- f i c a t i o n of the previous method has been descr ibed by Williams and Wornick273 who incorporated t h e t r is b u f f e r i n t h e agar and de le t ed t h e add i t ion of calcium ch lo r ide . A th ree- fo ld increase i n sens i - t i v i t y was repor ted . However, a c o l l a b o r a t i v e s tudy of t h e modified procedure r e s u l t e d i n an average recover of 115% with 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% 37 4 .

An i n v e s t i g a t i o n of t h e agar d i f - fus ion assay of neomycin a t very low levels(50ng- 1000ng) i n meat products has been reported280. The p o i n t s on t h e dose-response graph covering the above concent ra t ion range w e r e s c a t t e r e d about t h e average s t r a i g h t l i n e . However, by jo in ing a l l t he p o i n t s t oge the r a complex curve r e s u l t e d , t h e equa- t i o n of which was der ived .

6.5. Automated Procedures

An at tempt t o automate t h e t u r b i d i - metric method f o r t h e determinat ion of neomycin with K.pneurnaniae has been repor ted by Gerke e t a1281 who obtained s a t i s f a c t o r y assays with so l - u t i o n s conta in ing 150-1200 pg/ml of neomycin. An automated r e sp i romet 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 the b a c t e r i a E.cali w a s a l s o repor ted wi th a s imi la r s e n s i t i v i t y . In both methods Auto Analyzer systems were employed.

The modi f ica t ion of t h e above r e sp i ro - m e t r i c procedure t o inc lude continuous c u l t u r e of


t h e tes t organism E . c o L i has been desc r ibed282 . With t h e organism grown i n t h i s manner t h e s e n s i - t i v i t y of t h e a s s a y i s improved. Gree ly 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 pha rmaceu t i ca 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 procedure on t h e same samples . Good c o r r e l a t i o n between t h e t w o p rocedures was demonst ra ted .

A n automated colorimetric 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 of neom c i n ( B , C and neamine) h a s a l so been r e p o r t - ed148. components on a column o f carbon and Kies lguhr G ( 4 : l ) . The column e l u a t e i s reacted w i t h n i n -

hydr in t o de te rmine t h e amounts o f neomycin B , C and neamine.

The method u t i l i z e s a s e p a r a t i o n of t h e

6 . 6 . U s e as an A n a l y t i c a l Reagent

T u r b i d i m e t r i c p rocedures f o r d e t e r - mining r ibonuc lease2 u s i n g neomycin as t h e p r e c i p i t a n t have been d e s c r i - bed. The t u r b i d i t y w a s shown t o be dependant 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 mole- c u l a r weight of 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 o f a s s a y c o n d i t i o n s . Ext raneous 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 rocedure t o be a p p l i e d d i r e c t l y t o blood serum.

and deoxyr ibonuc lease 85

6 . 7 . Determina t ion i n Body F l u i d s and T i s s u e s

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 workers f o r de- t e rmin ing neomycin levels i n c l i n i c a l samples. To 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 s t a n d a r d s o l u t i o n . D a n i e l ~ v a ~ ~ ~ , however, p re- f e r r e d t o release t h e bound a n t i b i o t i c by enzyma- t 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 o f serum sample h a s been d e s c r i b e d by Axl ine e t a1311.

NEOMYCIN 473

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 examinat ion o f serum samples has 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 , serum, u r i n e and b i l e has been r epor t ed313 . The p rocedure u s e s a helium-neon laser l i g h t - s o u r c e and h a s a l i n e a r dose- response g raph over t h e range 0.1 t o 1 0 u g / m l f o r serum.

An 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 aminoglycoside 4 ' a d e n y l t r a n s f e r a s e h a s been d e s c r i b e d 3 1 4 . mycin w a s observed over t h e range 2.5 t o 2Oug/ml serum.

A l i n e a r r e sponse f o r neo-

The c o n c e n t r a t i o n of neomycin i n u r i n e has been de termined 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 chromatography 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 luorescamine .

B r a m m e r and Hemson182 employed an e l e c t r o p h o r e t i c procedure t o separate neomycin from b lood-p ro te ins b e f o r e de t e rmin ing t h e neomycin c o n c e n t r a t i o n colorimetric a1 l y .

414

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63. B r i t . P a t e n t 852 ,334 ( 1 9 6 0 ) . 64. Abbou R . , F r . P a t e n t M5,989 ( 1 9 6 8 ) . 65. Miura T., S a i t o K . , Mayama T. a n d Umezawa H . ,

Japan P a t e n t 69 27837 ( 1 9 6 9 ) . 66. B r i t . P a t e n t 857 ,875 ( 1 9 6 1 ) . 67. Coppi G. , M a r a z z i - U b e r t i E. and Maselli A. ,

B o l l . Chim. Farm. , 103 (lo), 736-47 ( 1 9 6 4 ) . 68. Casadio S., B r i t . P a t e n t 1 , 0 2 5 , 4 3 1 ( 1 9 6 6 ) . 69. Shaw C.G. , B r i t . P a t e n t 902,725 ( 1 9 6 2 ) . 70. Shaw W.B. , F r . P a t e n t 1 , 3 3 2 , 5 2 2 ( 1 9 6 3 ) . 71 . Carras M. and B o u c h e r i e A . , F r . P a t e n t

72. L a t t W.A., B e r n s t e i n J. and Dolliver M.A.,

73. Abbou R. , G e r . P a t e n t 2 ,017 ,554 ( 1 9 7 0 ) .

954,874 ( 1 9 5 6 ) .

1 , 0 1 1 , 8 0 0 ( 1 9 5 7 ) .

M3445 ( 1 9 6 5 ) .

U.S. P a t e n t 2 , 5 0 9 , 1 9 1 ( 1 9 5 0 ) .

NEOMYCIN 477

74 .

75 .

76.

7 7 .

78 . 79 .

80.

81 .

82.

83.

84.

85.

86.

87. 88.

89.

90.

91.

92.

Sobek V. and K r a l Z. ,Arzneim.Forsch. ,

R e i n i s S . , Suva J. and K r i s k a M . , A c t a His tochem. , 27 (21, 248-56 ( 1 9 6 7 ) . Sobek V . , Kral Z . , P a d e v e r t M. and Kargerova A . , Ant ibiot . , Advan.Res. ,Prod. C l i n . U s e , P r o c . Congr . , Prague , 196 4 , 2 8 5 - 9 1 ( p u b l i s h e d 1 9 6 5 ) . V i r t a n e n O.E . , Suomen K e m i s t i l e h t i B , - 27, 6 7 - 7 0 ( 1 9 5 4 ) . B r i t . P a t e n t 789,683 ( 1 9 5 8 ) . Penau H. and Hagemann G. , U . S . P a t e n t 3 ,042 ,581 ( 1 9 6 2 ) . Vanderhaeghe H. , Belg . P a t e n t 630,462 ( 1 9 6 3 ) . Magyar K . , S t v e r t e c z n y G . , Toth Sarudy E . and Hollosi M . , Magy. K e m . Lapja, 25 ( 7 ) , 332-4 ( 1 9 7 0 ) . P e n a s s e L . , Bar the lemy P . and Nomine G . , Bu l l .Soc . Chim.Fr. , ( 7 ) , 2391-4 ( 1 9 6 9 ) . U m e z a w a S., I t o Y . and F a k a t s u S . ,

- 1 7 ( 6 ) , 711-14 ( 1 9 6 7 ) .

J . A n t i b i o t . ( J a p a n ) , Ser.A. , 12, 114-15 ( 1 9 5 9 ) .

Umezawa H . , Takeuchi T . , Yamazaki S . , N i t t a K . and Osata T . , J. A n t i b i o t . ( J a p a n ) , Ser .A, 12, 117-25 ( 1 9 5 9 ) . Boiss ie r J . R . , P h i l i p p e J . , Zuckerkandl Fa , O r e s B . , T e i l l o n J . , Dumont C . and B o i l o t T . , C o m p t . r e n d . , 249, 1415-17 ( 1 9 5 9 ) . Grinev A . N . , Mezentsev A.S. and S i b i r y a k o v a D.V. , A n t i b i o t i k i , 5, 894-7 ( 1 9 6 1 ) . B r i t . P a t e n t 874 ,028 ( 1 9 5 9 ) . Hermansky M. , S b . P r a c i 10 ( i . e . D e s e t i n ) Vyroc i Za lozeni Us tavu , 45-52 ( 1 9 6 2 ) . S h o r i n V.A. , Goldberg L . E . , Muraveiskaya V.S. , Pevzner N.S. , Shapovalova S.P. , Kunra t I . A . , Belova I . P . , K r e m e r V.E. and F i l i p p o s ' y a n S.T. , A n t i b i o t i k i , 5, 897-904 I

( 1 9 6 1 ) . D i Marco A. and B e r t a z z o i i C . , A n t i b i o t . Chemotherapia , - 11, 2 - 2 0 ( 1 9 6 3 ) . Zuckerkandl F. , O r e s B. and P h i l i p p e J. , F r . P a t e n t CAM 34 ( 1 9 6 3 ) . (Chemical Abstracts, 60, 3 0 8 2 a ) . Umezawa S. , J a p a n P a t e n t 15,682 ( 1 9 6 4 ) .


93. Magyar K., Stverteczky J. and Toth-Sarudy E., Antibiot., Advan.Res., Prod. Clin.Use, Proc.Congr., Prague 1964, 435-7(published 1965).

Fr.Patent 1,360,154 (1964). 94. Nomine G., Penasse L. and Barthelemy P.,

95. Chornock F.W., U.S. Patent 2,811,481(1957). 96. Kellar H. and Cosar C., Ger.Patent

97. Agrawal J.K., Harmalkar S.G. and 1,002,336 (1957).

Vijayavargiya R., Microchem.J., 21, 202-8 (1976).

Proc.Soc.Exptl.Biol.Med., 96, 466-70(1957).

29 (5), 831-6 (1964). 100. Hein H., Zentr. Bakteriol.Parasitenk.,

Abt. I, Orig., 185 (4), 416-25 (1962). 101. Kunin C.M. and Tupasi T., Proc.Soc.Exp.Bio1.

Med., 133 (31, 858-61 (1970). 102. Harris W.A., Australas. J. Pharm., 52 (621),

S69-S71 (1971).

98. Higginbotham R.D. and Dougherty T.F.,

99. Gubernieva S. and Silaev A.B., Biokhimiya,

103.

104.

105.

106.

107.

108.

109.

110.

111.

112.

113.

Singh C.; Indian J.Chem., 2 (21, 67-71 (1964). Santarato R. I Arch. Intern. Pharmacodynamie, - 98, 394-8 (1954). Namiki S., Tokyo Iji Shinski, 75, 401-4 (1958). Kharchenko G.I., Vop. Med. Khim., 1, 468-9 (1961). Kharchenko G.I., Mikrobiol.Zh., Akad. Nauk. Ukr. RSR, 24 (31, 28-31 (1962). Kharchenko G.I., Lab.Delo, 8 (41, 23-26 (1962). Gubernieva L.M. and Silaev A.B.,Biokhimiya, - 28 (3), 572-4 (1963). Kharchenko G . I . , Zentr.Bakteriol., Parasitenk.Abt. I, Orig. 189 (11, 85-101 (1963). Geitman I. Ya., Vop.Med.Khim., 15 (41, 369-73 (1969). Kivman G. Ya and Geitman I. Ya., Vop. Med. Khim., 16 (41, 405-9 (1970). Leucuta S., Schwartz I. and Popovici A., -

Farmacia (Bucharest), 24 (1) , 47-54 (1976).

NEOMYCIN 479

1 1 4 .

115.

116.

117 . 118.

119.

1 2 0 .

1 2 1 .

1 2 2 .

123 .

124.

125 .

126 . 127. 1 2 8 . 129.

130.

131.

132 . 133.

134.

135.

136 .

F i s c h b a c h H. a n d L e v i n e J . , Antibiotics and Chemotherapy , 3 , 1159-69 ( 1 9 5 3 ) . H a y a s h i T . , MiyashTta T . , K o j i m a C. a n d I s h i i T . , J. F e r m e n t a t i o n T e c h n o l . ( J a p a n ) , - 33, 170-2, ( 1 9 5 5 ) . L a r b r e S . and Dorche J . , B u l l . T r a v . SOC. Pharm. Lyon, 2, 61-2 ( 1 9 6 2 ) . H o o d l e s s R.A. , A n a l y s t , 91, 333-4 ( 1 9 6 6 ) . Heinmann B . , Hunt G.A. and M i s i e k M . , Ant ib io t ics Ann. 1955/56, 391-5. F r e r e s D. a n d B u l l a n d F . , B u l l A c a d . V e t . F r . , 42 ( 8 ) , 835-45 ( 1 9 6 9 ) . T i e c c o G . , G h i s o l i M.P., V i t i A. a n d Ronchi P . , Arch. V e t . I t a l . , 2 ( 6 ) , 385-95 ( 1 9 7 0 ) . Penau H , S a i a s E . a n d F e r d e t J. , Ann. Pharm. F r a n c . , 11, 740-4 ( 1 9 5 3 ) . Roets E . , and Vanderhaeghe H . , J. Pharm. P h a r m a c o l . , 24 ( l o ) , 795-7 ( 1 9 7 2 ) . S i e g e r m a n H . D . , F l a t o J . B . and O'Dorn G.W., Autom. Microbiol. Immunol. (Pap . Symp.) , 1 9 7 3 ( P u b l i s h e d 1 9 7 5 ) , 305-34, W i l e y , N e w York. F o r d J . H . , Bergy M.E., Brooks A . A . , G a r r e t t E . R . , A l b e r t i J. , Dyer J . R . and Carter H.E., J. Am. Chem. SOC., 77, 5311-14 ( 1 9 5 5 ) . Brooks A.A. , A r l i n g t o n A.F. a n d L o e h r B.F., Anal.Chem., 28, 1788-90 ( 1 9 5 6 ) . D e R o s s i P . , A n a l y s t , 100, 25-28 ( 1 9 7 5 ) . Merck I n d e x , 8 t h e d . , 1968.MercklRahway,USA. Kaiser D . G . , Anal. Chem., 35, 552-4 ( 1 9 6 3 ) . Seaman W. and S t e w a r t D . , Am. D y e s t u f f R e p t r . , 54 ( 4 1 , 29-30 ( 1 9 6 5 ) . S z a b o - F e j e s G . , S z a b o V. and E r d e i - H o r v a t h A. , A c t a - P h y s . Chem. D e b r e c i n a , 18, 217-24 ( 1 9 7 3 ) . Maeda M. , K i n o s h i t a T . a n d T s u j i A. , Anal. Biochem., 38, 121-129 ( 1 9 7 0 ) . Simpson K . , S q u i b b P r i v a t e Communicat ion. K u s n i r J. and B a r n a K , F r e s e n i u s Z . A n a 1 . Chem., 271, 288 ( 1 9 7 4 ) . O ' K e e f e A.E. a n d R u s s o - A l e s i F.M. , Am.Chem. SOC. A b s t r . 1 1 6 t h M e e t i n g (19491, 63c-64c. Moore S . and S t e i n W . H . , J .Bio l .Chem. , 176, 367-388 ( 1 9 4 8 ) . Maehr H . a n d S c h a f f n e r C.P., Anal.Chem., - 36, 104-108 ( 1 9 6 4 ) .


137.

138.

139.

1 4 0 .

1 4 1 .

1 4 2 .

143. 1 4 4 .

145.

1 4 6 .

147.

148.

1 4 9 . 150.

151.

152.

153.

154.

155.

156.

157.

Gerosa V. and Melandr i M . , Farmaco Ed .P ra t . , - 1 2 , 13-17 (1956) . Borowiecka B. , D i s s e r t a t i o n s Pharm. , 17, 313-318 (1965) . Thorburn-Burns D . , Lloyd G.H. and Watson- Walker J . , L a b . P r a c t . , =, 448-449 (1968) . Kapt ionak A.E . , B ie rnacka E. and Pazde ra H . J . , Technicon Symp., 2nd, N . Y . London, 1965, 27-33. Moore S. and S t e i n W.H. , J.Biol.Chem. , - 2 1 1 , 907-913 (1954) . Bufton R . J . L . and S a d d l e r N . G . , Squibb P r i v a t e communication. Kohn M . , Squibb P r i v a t e Communication. Kakemi K . and Ohashi S . , Yakugaku Z a s s h i , - 80, 183-186 ( 1 9 6 0 ) . Hamre D . M . , Pansy F.E., Lapedes D.N. , Perlman D . , Bayan A.P. and Donovick R. , A n t i b i o t i c s and Chemotherapy, 2, 135-141 (1952) . Emery W.B. and Walker A.D. , A n a l y s t , 2, 455-457 ( 1 9 4 9 ) . Perlman D . , J .Biol.Chem., 179, 1147-1154 ( 1 9 4 9 ) . Lavrova M.F., A n t i b i o t i k K o l i m i t s i n i ego Premenenie u K l i n i k e , Akad. Med. Nauk S.S.S.R. , 1959, 19-23. Lavrova M.F., A n t i b i o t i k i , 1, 55-59 ( 1 9 5 6 ) . Korchagin V.B. , Korob i t skaya A.A. , Druzh in ina E.N. and Semenov S.M., A n t i b i o t i k i , 1, 124-128 ( 1 9 6 2 ) . Penau H . , S a i a s E . , A n d r e e t t i C. and F e r d e t M . J . , Ann.Pharm.Franc., 11, 431-438 (1953) . E lson L.A. and Morgan W.T.J . , Biochem. J . , - 27, 1824-1828 (1933) . Penau H . , S a i a s E . , A n d r e e t t i C. and F e r d e t M . J . , Ann.Pharm.Franc., 12, 49-53 (1954) . Dutcher J . D . , Hosansky N., Donin M.N. and W i n t e r s t e i n e r 0. , J.Am.Chem.Soc. , 73, 1384-5 (1951) . S t i l l i n g s R.A. and Browning B.L. , Ind. and Eng. Chem. , Anal.Ed. , 12, 499 ( 1 9 4 0 ) . Dunstan S. and G i l l a m A.E. , J.Chem.Soc., 1 4 0 ( 1 9 4 9 ) . Dutcher J . D . , Hosansky N. and Sherman J . H . , A n t i b i o t i c s and Chemotherapy,2,534-536(1953).

NEOMYCIN 48 1

158.

159.

160.

161. 162. 163.

164.

165.

166. 167. 168. 169.

170. 171. 172.

173.

174.

175.

176.

177.

178.

179.

180.

181.

Brooks A.A., Forist A.A., and Loehr B.F., Anal.Chem. , 28, 1788-1790 (1956). Morgan J.W., Honig A.B., Warren A.T. and Levine S., J.Pharm.Sci., 50, 668-671 (1961). Roe J.H. and Rice E.W., J.Biol.Chem., - 173, 507 (1948). Ivashkiv E., Squibb Private Communication. Ivashkiv E., Squibb Private Communication. Hughes E.E. and Acree S.F., J.Research Natl. Bur. Standards, 3, 327 (1938). Reeves L.E. and Munro J., Ind. Eng. Chem., Anal. Ed., 12, 551 (1940). Basu K. and Dutta B.N., J.Proc.Inst. Chemists (India), 34, 142 (1962). Bickford, Squibb Private Communication. Kocy O.R., Squibb Private Communication. Kohn M.B., Squibb Private Communication. Korchagin V.B., Satarova D., Kochetkova E.V. and Vakulenko N.A., Antibiotiki (MOSCOW), 20 (2), 116-119 (1975). Ivashkiv E., Squibb Private Communication. Heyes W.F., Unpublished work. Abou-0uf A.A., Taha A.M. and Saidhom M.B., J.Pharm.Sci., 62 (10) , 1700-2 (1973). Roushdi I.M., Ibrahim A. and Issa A. , Egypt. J.Pharm.Sci., 15 (1) , 87-90 (1974). Doulakas J., Pharm.Acta Helv. , 5.1 (121, 391-2 (1976). Khromov G.L., and Starunova L.N., Khim- Farm. Zh., 3 (71, 53-5 (1974). Swart E.A., Lechevalier H.A. and Waksman S.A., J.Am.Chem.Soc. , 73, 3253 (1951). Schaffner C.P., Antibiotics Ann., 2, 153 (1954-5). Baikina V.M., Mamiofe S.M., Rozanova T.N., Sinitsyna Z.T., Slugina M.D. and Dzegilenko N.B., Antibiotiki, 8, 1059 (1963). Sannikov V.A., Materialy 2-oi (Vtoroi)Konf. Molodykk Uchenykk Leningr.Inst.Antibiotikov, Leningrad, Sb., 1964, 76-80. Peck R.L., Hoffhine C.E., Gale P. and Folkers K., J. Am.Chem.Soc., 71, 2590 (1949). Paris R.R. and Theallet J.P., Ann.Pharm. Franc., 20, 436-42 (1962).


182.

183 .

184.

185.

186.

187 .

188.

189.

190 .

191.

192.

193.

194.

195 . 196 .

197 . 198.

199.

200 .

201 .

202.

203.

B r a m m e r K.W. and Hemson L . J . , J . C h r o m a t o g r . , - 1 9 ( 2 ) , 456-8 ( 1 9 6 5 ) . Grynne B . , A c t a . P a t h o l . M i c r o b i o l . S c a n d . , S e c t i o n B . , 81 ( 5 ) , 583-8 ( 1 9 7 3 ) . Govorovich E.A. and Bogomolova N.S., A n t i b i o t i k i , 14 ( 2 ) , 129-32 ( 1 9 6 9 ) . Apreotesei C . and T e o d o s i u M . , F a r m a c i a , - 10, 321-30 ( 1 9 6 2 ) . Carr J . P . , S t r e t t o n R . J . and Watson-Walker J . , Loughborough Univ. Technol.Dep.Chem. Sum.Fina1 Year S t u d . P r o j . T h e s e s , lo, 17-18 ( 1 9 6 9 ) . Caste1 P . , Mus R.and S t o r c k J . , Ann.Pharm. F r a n c . , 17, 63-71 ( 1 9 5 9 ) . Lightbown J . W . a n d D e R o s s i P . , A n a l y s t , - 9 0 , 89-98 ( 1 9 6 5 ) . Ochab S. , P o l . J. Pharmacol .Pharm., 25, 105-8 ( 1 9 7 3 ) . Coombe R . G . , A u s t . J . P h a r m . S c i . , I, 6-8 ( 1 9 7 2 ) . Inouye S. and O g a w a H . , J . C h r o m a t o g r . , - 1 3 ( 2 ) , 536-41 ( 1 9 6 4 ) . T s u j i K . a n d R o b e r t s o n J . H . , Anal.Chem., - 41, 1332-5 ( 1 9 6 9 ) . Roets E . and Vanderhaeghe H . , Pharm. T i j d s c h r . B e l g . , 44 ( 4 ) , 57-64, ( 1 9 6 7 ) . G i l l e t A . , Vanderhaeghe H., Bogaerts R . , Boudru I . , B r o u c k a e r t A . , Coucke G . , Dony G . , Drion P . , Dumont P . , H a e m e r s A . , P i j c k J. and Van Kerchove C . , J . P h a r m . B e l g . , 27 ( 4 ) , 381-401 ( 1 9 7 2 ) . P i n e s S.H., U . S . P a t e n t 3 , 3 2 9 , 5 6 6 ( 1 9 6 7 ) . Leach B.E. a n d Teeters C.M., J.Am.Chem. SOC., 73, 2794-7 ( 1 9 5 1 ) . Leach B . E . , U.S. P a t e n t 2 , 6 7 9 , 5 3 2 ( 1 9 5 4 ) . B r a z h n i k o v a M.G. and Kudinova M.K.,Nature, - 200 ( 4 9 0 2 ) , 167-8 ( 1 9 6 3 ) . Claes P. J . , Compernol le F. and Vanderhaeghe H . , J .Ant ibiot . , 3 (121 , 931-42 ( 1 9 7 4 ) . P r e g n o l a t t o W . a n d S a b i n o M . , Rev. I n s t . Adolfo L u t z , 34, 41-6 ( 1 9 7 4 ) . Langner H . J . a n d T e u f e l U . , Chem.Mikrobio l . , T e c h n o l . Lebensm., 2 ( 3 ) , 71-78 ( 1 9 7 4 ) . Ammann A . a n d G o t t l i e b D . , Appl.Microbiol., - 3 , 181-6 ( 1 9 5 5 ) . Rangaswami G . , S c h a f f n e r C.P. and Waksman S.A., Antibiot ics and Chemotherapy , 6, 675-683 ( 1 9 5 6 ) .

NEOMYCIN 483

2 0 4 .

205.

206.

207.

208.

2 0 9 .

2 1 0 .

211.

2 1 2 .

213.

214.

2 1 5 .

2 1 6 .

2 1 7 .

218.

2 1 9 .

220.

221.

222.

2 2 3 .

Kondo S. , Sezaki M.and Shimaru M., J.Antibiot. (Tokyo) Ser.B., - 1 7 , 1-6 ( 1 9 6 4 ) . Betina V. , J.Chromatogr. , 15, 379-92 ( 1 9 6 4 ) . Betina V., Sb.Prac.Chem.Fak.S.V.S.T., 1 9 6 4 , 33-40. De Franca F.P. and De Oliveira Dias G., Rev. Brasil.Farm., 47, 6 7 - 7 1 ( 1 9 6 6 ) . Stretton R.J., Carr J.P. and Watson- Walker J. , J-Chromatogr. , 45, 1 5 5 - 8 ( 1 9 6 9 ) . Hayashi T. , Takao M., Kojima C. and Ishii T., J. Fermentation Technol. (Japan) , - 33, 1 7 0 - 2 ( 1 9 5 5 ) . Pan S.C. and Dutcher J.D., Anal.Chem., - 28, 836-8 ( 1 9 5 6 ) . Sugano Y. , Katsumi M. and Wakazawa T., Meiji Seika Kenkyu Nempo, 1 9 6 0 , 1 - 3 . Sannikov V.A., Materialy 2-oi (Vtoroi) Konf. Molodykk Uchenykk Leningr. Inst. Antibiotikov, Leningrad, Sb, 1 9 6 4 , 7 4 - 7 6 . Majumdar M.K. and Majumdar S.K., Anal. Chem. , 2, 2 1 5 - 7 ( 1 9 6 7 ) . Majumdar M.K. and Majumdar S.K., Appl. Microbiol., 17, 763-4 ( 1 9 6 9 ) . Deshmukh P.V., Mehta S.I. and Vaidya Madhukar G., Hindustan Antibiot. Bull.,

Kuzyaeva V.A., Antibiotiki, 9 , 7 8 4 - 8 ( 1 9 6 4 ) . Huettenrauch R. and Schulze J., Pharmazie, - 19, 334-5 ( 1 9 6 4 ) . Ito Y., Namba M., Nagahama N.,Yamaguchi T. and Okuda T. , J. Antibiot. (Tokyo) , Ser.A, - 1 7 , 218-9 ( 1 9 6 4 ) . Pritton J.S., Antibiot.,Advan.Res.Prod. Clin.Use, Proc.Congr.,Prague 1 9 6 4 , 490-5 (Published 1 9 6 5 ) . Zuidweg M.H.J., Oostendorp J.G. and B o s C.J.K., J.Chromatogr., 42, 5 5 2 - 4 ( 1 9 6 9 ) . Guven K.C. and Ozsari G., Eczacilik Bu1.I - 9, 1 9 - 2 9 ( 1 9 6 7 ) . Borowiecka B., Diss.Pharm.Pharmacol., 22, 3 4 5 - 5 0 ( 1 9 7 0 ) . Schmitt J.P. and Mathis C . , Ann.Pharm.Fr., 23 , 2 0 5 - 1 0 ( 1 9 7 0 ) .

- 1 2 , 6 8 - 7 0 ( 1 9 6 9 ) .

-


224.

225. 226.

227. 228.

229.

230. 231.

232.

233.

234.

235.

236.

237.

238.

239.

240.

241.

242.

243.

244.

245.

246.

Brodasky T.F., Anal.Chem., 35, 343-5 (1963). Nager U.F., U.S. Patent 2,667,441(1954). Borowiecka B., Diss.Pharm.Pharmacol., - 24, 209-15 (1972). Pauncz J.K., J.Antibiot.,z, 677-8 (1972). Emilianowicz-Czerska W. and Herman H., Med. Doswiadezalna Mikrobiol., 2, 183-7 (1961). Foppiano R. and Brown B.B., J.Pharm.Sci., - 54, 206-8 (1965). Simon A., Mikrobiologiya, 37, 552-7 (1968). Yakobsen, L.M., Snezhnova L.P., Astanina L.N. and Skiryaeva V.L., Antibiotiki, - 16, 983-6 (1971). Snezhnova L.P. and Astanina L.N. , Antibiotiki, 17, 263-6 (1972). Klaushofer H. and Nekola M. , Pharm.Ind. , - 34, 359-63 (1972). Sokolski W.T. and Lummis N.E.,Antibiotics & Chemotherapy, 11, 271-5 (1961). Peterson D.H. and Reineke L.M., J.Am.Chem. SOC., 72, 3598-3603 (1950). Chatterjee N.R., Indian J.Chem., 13 (121, 1282-4 (1975). Van Giessen B. and Tsuji K., J.Pharm.Sci., - 60, 1068-70 (1971). Margosis M. and Tsuj i K., J. Pharm. Sci . , - 62, 1836-8 (1973). Tsuji K., Robertson J.H., Baas R. and McInnis D.J., Appl. Microbiol., 18, 396-8 (1969). Omoto S., Inouye S. and Niida T., J.Antibiot., 24, 430-4 (1971). Philippe J., Benoist D. and Patte F., Ann. Pharm. Fr., 12, 339-50 (1954). Dony J. and Conter J., J.Pharm.Belg., 9, 403-7 (1954). Hein H., Zentralbl. Bakeriol.Parasitenk. Infectionskr. Hyg. Abt. 1:Orig. 214 (2), 259-61 (1970). Dony J. and Conter J., J.Pharm.Belg., 10, 104-8 (1955).

- -

Analytical Microbiology, Vol. 2 , Editor Kavanagh Fa, Academic Press, 1972. Assay Methods of Antibiotics, Grove D.C. and Randell W.A.,Med.Encyclopedia, 1955.

NEOMYCIN 485

247.

248.

249.

250.

251.

252.

253.

254.

255.

256.

257.

258.

259.

260.

261.

262.

263.

264.

265.

266.

267.

Wintermere D . M . , E i s e n b e r g W.H. and Kirshbaum A. , Ant ib io t ics and Chemotherapy, - 7, 189-92 ( 1 9 5 7 ) . P a i n S.K., B o s e B.K. and 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 Koeber l e J . , Ann.Pharm. F r a n c . , 28 ( 6 ) , 437 -41 ( 1 9 7 0 ) . Wachtel J . L . and Bryan W.L., U.S.Patent 2 ,793 ,978(1975) . Denkewal te r R.G. and G i l l i n J. , G e r . P a t e n t 1,062,891 ( 1 9 5 9 ) . S o k o l s k i W.T., C h i d e s t e r C.G. and Kaiser D . G . , J .Pha rm.Sc i . , 53 (71,726-9 ( 1 9 6 4 ) . Rappe A . , Ann. Pharm. F r . , 31 (61 ,435-40 ( 1 9 7 3 ) . Davis W.W. and Pa rke T . V . , J.Am.Pharm. ASSOC., 2, 327-33 ( 1 9 5 0 ) . K u z e l N . and Cof fey H.F., U.S .Pa ten t 3,401,087 ( 1 9 6 8 ) . Abbey A . , A n t i b i o t i c s 8, Chemotherapy, - 3, 5 2 8 - 3 1 ( 1 9 5 2 ) . P i n z e l i k J . , Nisonger L.L. and Murray F . J . , Appl. M i c r o b i o l . , 1, 293-6 ( 1 9 5 3 ) . Kochetkova G.V., An-t ibiot iki , - 2 ( 4 1 , 52-56 ( 1 9 5 7 ) . Yousef R .T . , El-Nakeeb M.A. and E l m a s r i M . H . , A c t a Pharm.Suecica , 4 ( 4 ) , 2 5 3 - 6 0 ( 1 9 6 7 ) . El-Nakeeb M.A. and Yousef R . T . , A r z n e i m - F o r s c h . , 20 (1) ,103-7 ( 1 9 7 0 ) . El-Nakeeb, M.A. and Yousef R . T . , Ind ian J. Pharm., 32 (1) ,6-9 ( 1 9 7 0 ) . 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 . , 25, 1044-7 ( 1 9 6 4 ) . Fedorko J. and K a t z S . , J .Bacter io1, 89 ( 2 ) , 5 4 8 ( 1 9 6 3 ) . S i d d i q u e I . H . , Loken K . I . and Hoyt H . H . , Appl. M i c r o b i o l . , 2 ( 5 ) ,635-7 ( 1 9 6 5 ) . 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. 1955/6 , 383-90. S o k o l s k i FI.T., C h i d e s t e r C.G. and C a r p e n t e r O . S . , J .Pharm.Sci . , 53 (71 , 826-8 ( 1 9 6 4 ) . Sebek O . K . ,Arch.Biochem.and Biophys. ,=, 71-9 ( 1 9 5 5 ) .


268.

269.

270.

271.

272.

273.

274.

275.

276.

277.

278.

279.

280.

281.

282.

283.

284.

285.

286.

287.

U n i t e d S t a t e s Pharmacopiea XlX, 1975 p. 598. Levine J . , Fischbach H. and A r r e t B. , Ant ibiot ics and C h e m o t h e r a p y , ? , (3) , 266-269 (1954). D e N u z i o J . C . , Bowman 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. , Ant ibiot ics 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 . A s s . O f f i c . A n a l . C h e m . , 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 . A n a l . C h e m . , 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 . , - 9, 334-6 (1961). 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. ,Ant ib io t ics and C h e m o t h e r a p y , 5 (51, 273-80 (1955). N e f f A.W., Miller C . C . and B a r b i e r s A.R. , J . A s s . O f f i c . A n a l . C h e m . , 53 (1) , 60-68 (1970). Langner H. J. , Weiss H. and Teufe l U . , Zen t r a lb 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 Ferrari 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 . , Analys t , - 8 (1050), 694-701 (1963). 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 . , A n a l . B i o c h e m . , 8 (31,373-92 (1964).

A l t e s c u E . J . , E n z y m o l . B i o l . C l i n . , 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 , 7 9 9 , 6 2 0 ( 1 9 5 7 ) . Umezawa H . , T a k e u c h i T. a n d Yamagiwa S . , Japan . Med. J . , 3 , 25-30 ( 1 9 5 0 ) . A r a i T. and A i i s o K . , J a p a n e s e P a t e n t 5 4 5 0 ( 1 9 5 3 ) . Game G.F., Kochetkova G.V. ,Probrazenskaya 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 , - 1 ( 5 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. , Nucleonics , 18 ( 8 ) ,102-5 ( 1 9 6 0 ) . S w a r t E.A. , H u t c h i n s o n D . and Waksman S.A. , Arch. Biochem., 24, 92-103 ( 1 9 4 9 ) . Leach 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. and Evans J . S . , J.Am.Chem.Soc., - 73 I 2797-2800 ( 1 9 5 0 ) . Makino S. , H a y a s h i E . a n d Okamura K . , Japan. P a t e n t 6247 ( 1 9 6 0 ) . Takenaga M. a n d Okada Am, G e r . P a t e n t 2 , 1 6 5 , 3 7 3 ( 1 9 7 2 ) . Tanaka I . , Yoshizawa H., Otani S., N a k a y a s h i k i J. a n d Hara E . , Yakuzaigaku , - 2 2 , 147-50 ( 1 9 6 2 ) . 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 .Pharm. Sc i . , 60 ( 9 ) ,1343-5 ( 1 9 7 1 ) . Gecgil S . , I s t a n b u l Univ. E c z a c i l i k Fak . Mecmuasi , 2 (1) , 56-57 ( 1 9 6 8 ) . Coates L.VT, 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 . and 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 . and R i s i n g L.W. , Am.J.Pharm., 1 3 1 , 166-73 ( 1 9 5 9 ) . McGini ty J . W . and 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 Res .Conf . , 1 5 t h , 54-90 1 1 9 6 7 ) . Danti A.G. and Guth E . P . , J .Am.Pharm.Assoc., S c i . E d . , 46, 249-52 ( 1 9 5 7 ) . Hammouda YTand Salakawy S.A., P h a r m a z i e , - 26 (10) , 636-40 ( 1 9 7 1 ) . 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. , 45, 538-41 ( 1 9 5 6 ) . Kivman G. Y a . a n d Gei tman I. Y a . , A n t i b i o t i k i , 16 ( 4 ) ,331-5 ( 1 9 7 1 ) .


310. Danie lova L .T . , A n t i b i o t i k i , 19 (111, 1032-8 (1974) .

311. Ax l ine S . G . , Yaffe S . J . and Simon H . J . , P e d i a t r i c s , 39 (1) ,97-107 (1967) .

312. F e l s e n f e l d O . , V o l i n i I .F . , Kadison E.R. , Zimmermann E. and I s h i h a r a S . J . , Am.J. C l i n . P a t h . , 3, 670-1 (1950) .

313. Wyatt P . J . , P h i l l i p s D.T. and A l l e n E.H. , J .Agr ic .Food Chem., 24 ( 5 ) ,984-8 (1976) .

314. Santanam P. and Kayser F.H., Ant imicrob . Agents Chemother. , lo ( 4 ) ,664-7 (1976) .

315. Kusn i r J. and Barna K . , Cesk. Farm., 24 ( 6 ) , 253-5 (1976)

T h i s p r o f i l e a t t e m p t s t o c o v e r t h e p u b l i s h e d l i t e r a t u r e on neomycin up t o and i n c l u d i n g Chemical Abstracts, Volume 85 .


PSEUDOEPHEDRINE HYDROCHLORIDE

Steven A . Benezra and John W . McRae

1. Description 1.1 Name, Formula, Molecular Weight 1.2 Appearance, Color, Odor

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


3. Synthesis 4. Stability 5. Metabolism and Pharmacokinetics 6. 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


ISBN 0-12-260808-9 489

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 0 HCI

I

201.72 CH3

C10H15NO*HC1


Pseudoephedrine hydrochloride occurs as fine white

t o off-white crystals o r as a powder having a faint odor1


2.1 Infrared Spectrum

The infrared spectrum of pseudoephedrine hydro-

chloride 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

the infrared assignments consistent with the structure of

pseudoephedrine hydrochloride.

Table I gives

Table I Infrared Spectral Assignments for Pseudoephedrine

Hydrochloride

Frequency (cm-l) Assignment

3270 OH stretch

3010 Asym. C-H stretch

1

1

1 1

I

I

I 1 0

(u

0

8 3

d

0

0

0

(u

0

8 3

d

0

0

492 STEVEN A. BENEZRA AND JOHN W. MCRAE

2930 Sym. C-H stretch

2700 NH stretch

1587, 1490 C=C aromatic stretch

1430 OH bend, secondary alcohol

762, 702 C-H bend, monosubst. benzene

+

2.2 Nuclear Magnetic Resonance (NMR) Spectrum

The NMR spectrum o f pseudoephedrine hydrochloride

is shown in Figure 2. The spectrum was obtained with a

Varian model CFT-20 80 MHz 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.

G9 8 H H HN-

OH H @ @

Table I1

NMR Assignments for Pseudoephedrine Hydrochloride

Proton No. of Protons Shift (ppm) Multiplicity

a 3 0.96 doublet

b 3 2.55 singlet

C 1 3.25 quartet (partially

obscured by H20)

d 1 4.54 doublet of doublets

e 1 6.32 doublet

f 5 7.34 singlet

8 2 8.90 broad singlet

PSEUDOEPHEDRINE HYDROCHLORIDE 493

J

1 1

I S I ' I ! 1 I 1 I'

9 8 7 6 5 4 3 2 I 0

Figure 2 - NMR Spectrum of Pseudoephedrine Hydrochloride PPm

I .o

ae

0.6 U

4

[L

0 v)

a 0.4

0.2

0.9

nrn

Figure 3 - Ultraviolet Spectrum of Pseudoephedrine Hydrochloride

494 STEVEN A. BENEZRA A N D JOHN W. MCRAE

2.3 Ultraviolet Spectrum

The ultraviolet spectrum of pseudoephedrine hydro-

chloride 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

Varian MAT CH5-DF mass spectrometer.

into the ion source was used to obtain the mass spectrum.

The electron energy was 70 eV.

ions formed in the mass spectrometer are shown below. The

molecular ion is not observed.

It was obtained with a

Direct probe at 80'C

The assignments of the major

+ H

'CHq

+HC /NzCH3 0 3

m/e 58 (100%) m/e 77 (9%)

HCI

m/e 36 (10%)

+ HNCH3

m/e 30 (12%)

2.5 Melting Point

Pseudoephedrine hydrochloride melts between 182'

and 185'C.

2.5 Specific Rotation

The specific rotation, [cy]iO, of d-pseudoephedrine

hydrochloride in water is between +61.0' and +62.5'.l

2.7 Solubility

The solubility of pseudoephedrine hydrochloride in

PSEUDOEPHEDRINE HYDROCHLORIDE

1001

495

I00 I50 200

m /e

Figure 4 - Mass Spectrum of Pseudoephedrine Hydrochloride

1 1 1 I 1 I 1 I 1 I I 1 i 1 1

1 1 1

178 179 180 181 182 183 184 185 186 187

TEMPERATURE O C

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 Solubility (gm/ml)

Water 2.0

Chloroform 0.011

E thano 1 0.278

Ether 1.4

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 . ?

5OC/min. The heat of fusion is 6.4 Kcal/mol. The melting

point (uncorrected) is 184OC.

The heating rate was

2.10 Crystal Structure

The crystal properties of pseudoephedrine hydro-

chloride were determined with a GE model XRD-6 x-ray diffrac-

tometer using Zr filtered MoKa radiation on a crystal grown

f rom water. Pseudoephedrine hydrochloride has an ortho-

rhombic crystal system belonging to the P2 2 2

The cell dimensions are a=25.358 A , bz6.428 A , ~ ~ 6 . 9 0 1 A

with each cell containing four molecules.

space group. 0 l a 1 0

2.11 Dissociation Constant

The pKa of pseudoephedrine hydrochloride determined


titrimetrically in 80% aqueous methylcellosolve is 9.22.

3. Synthesis

Pseudoephedrine hydrochloride is prepared by a

Welsh rearrangement lo of R-ephedrine hydrochloride with ace-

tic anhydride followed by deacetylation with hydrochloric

acid. l 1

2-mandelic acid. l2

plants of the Ma Huang species.

2-Ephedrine can be resolved from dR-ephedrine with

R-Ephedrine occurs naturally in certain

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.

at 37OC and 3 months at 5OoC.

stored at 15-30°C for 5 years showed no appreciable degra-

dation. l3

The bulk drug was stable for at least 6 months

Tablet and syrup formulations

5. Metabolism and Pharmacokinetics

The major biotransformations of pseudoephedrine

hydrochloride are parahydroxylation, N-demethylation, and

oxidative deamination. l4

metabolism of pseudoephedrine are shown in Figure 6 .

The proposed pathways for the

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. As the urine pH increased, the serum

half-life o f pseudoephedrine and norpseudoephedrine increas-

ed.15 In another similar study it was found that a decrease

H CH3

Q-++H-&-p

\ I OH NH OH NH,

Nor pseudoephedrine

I

CH3 Ps eu doe p hed r in e

/ o+(- OH CH3

OW,H

OH OH

I / I -Hydroxy-l-phenyl-2-proponone I -Phenyl-l,Z-propanediol

OH

Benzoic acid

Figure 6 - Metabolism of Pseudoephedrine Hydrochloride


in urine pH caused a decrease in plasma half-life of pseudo-

ephedrine. l6

subjects were 5 . 2 - 8 . 0 hours. l6

Plasma half-lives measured in normal human

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.

oxidative deamination, giving acidic metabolites such as

hippuric and benzoic acids. l7

The minor metabolic pathway was

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 of pseudoephedrine administered

i.p. is 1.0 mmole/kg.18


6 . 1 Elemental Analysis

The results of the elemental analysis o f pseudo-

ephedrine hydrochloride are given in Table IV.6

was performed on a NF Reference Standard.

The analysis

Table IV

Elemental Analysis of Pseudoephedrine Hydrochloride

Element Theory (%) Found ( X ) C 59.55 5 9 . 5 4

H 8 . 0 0 8 . 1 1

N 6 . 9 5 6 . 8 1

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 0.1N 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

A portion to assay pseudoephedrine hydrochloride in tablets.

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 absorb-

ance 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 of pseudo-

ephedrine hydrochloride will be the official method of analy-

sis in the USP XX.19,20

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 oxi-

dation 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.

A portion of tablets or syrup in

PSEUDOEPHEDRINE HYDROCHLORIDE 50 1

6.4 Colorimetric Analysis

Pseudoephedrine hydrochloride in syrup formulations

has been analyzed by colorimetry.

stable blue-colored chelate with cupric sulfate at pH 12.5.

The complex has a maximum absorbance at 500 nm.

is extracted from an aqueous layer with 1-pentanol. Inter-

fering substances such as glycerine and sugars normally found

in syrup formulations, which form complexes with cupric aul-

fate, are not extracted into l-pentanol.*l

Pseudoephedrine forms a

The complex

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 Mobile Phase Rention Time (min) Reference

Corasil@/Phenyl acetonitrile:

Corasil@/C18 0.1% ammonium

carbonate

(9:l) pH 8.9

Corasil@/Phenyl acetonitrile:

Corasil@/C18 1% ammonium

carbonate (6:4)

pH 7.4

1.8 (phenyl) 22

1.9 (C18)

22

502 STEVEN A. BENEZRA AND JOHN W. M C M E

Zipax@/SCX 0.02 M dibasic

ammonium 7 23

phosphate:dioxane

(64:36)

Nucleosil@/ methanol:0.5M

silica gel sodium dihydrogen 6

phosphate:

phosphoric acid

(195:50:2)

Sphe risorb@/ ethanol:0.4%

silica gel ammonium 8

acetate (85: 15)

24

25


Table VI lists the various TLC systems used for

pseudoephedrine hydrochloride.

Table VI

TLC Systems for Pseudoephedrine Hydrochloride

Mobile Phase Adsorbent Rf - Reference

ethyl acetate: silica gel 0.25 26

cyclohexane:

methano1:conc.

M140H(70:15:10:5)

n-butano1:ethanol: silica gel

water:acetic acid

(60:30:10:0.2)

0.46 27

0.18 (free base)


chlo ro f o rni :

methanol :

acetone (7:3:5)

ethyl ether:

benzene (1:l)

chloroform :

water:acetic

acid (20:75:20:1)

(lower phase)

silica gel

silica gel

alumina

0.33 28

0.10 (free base)

0.35 (as 29

4-chloro-

7-nit ro-

benzo-2,1,3-

oxadiazole

derivative )

0.70 (as 16

acetylated

product)


Paper chromatography has been used to separate and

detect pseudoephedrine hydrochloride from other pharmacologi-

cally 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 chro-

matogram with 0.5% bromcresol green in methanol or 0.2% nin-

hydrin 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 pseudo-

ephedrine with anhydrous acetone. On a 1.15% SE-30,

glass column, 2.4 m x 3 mm i.d. at 104OC the oxazoli-

dine 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 170°C.33

An on-column acetic anhydride derivatization tech-

nique 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.

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

The pseudoephedrine

A variety of methods have been used to determine

pseudoephedrine hydrochloride levels in plasma and urine by

gas chromatography.

plasma or urine with diethyl ether.

centrate 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.

Bye and co-worker~~~ extracted basefied

The ether extract con-

The heptafluorobutyric anhydride derivative of

pseudoephedrine and electron capture detector have been used

to enhance the sensitivity of the gas chromatographic method.

Lin and co-~orkers~~ and Cummins and Fourier37 extracted

basefied urine or serum with benzene.

anhydride is added to the benzene extract.

ibutyric anhydride derivative extracted was chromatographed

Heptafluorobutyric

The heptafluoro-


on a 3% OV-17, 1.82 m x 2 mm i.d. glass column at 150°C36 or

a 5% ethylene glycol succinate, 1.82 m x 2 mm i.d. stainless

steel column at 140°C. 37

Pseudoephedrine in urine was analyzed by gas chro-

matography 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 chromato-

graph, or derivatized with acetone and then chromato-

graphed. 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 190°C.39

References

1.

2.

3 .

4 .

5.

6.

7.

8.

9.

10.

11.

N . F . XIV, Mack Printing Co., 1975

W. Martin, Burroughs Wellcome Co., personal communi-

cation

R . Crouch, Burroughs Wellcome Co., personal communi-

cation

J.W. McRae, unpublished data

D. Brent, Burroughs Wellcome Co., personal communication

B.S. Hurlbert, Burroughs Wellcome Co., personal communi-

cation

J. Ebron, Burroughs Wellcome, personal communication

M. Mathew, G.J. Palenik, Acta. Cryst., E, 1016 (1977) V. Prelog, P. Haflinger, Helv. Chim. Acta., 33, 2021 (1950)

L.H. Welsh, J. her. Chem. SOC., c, 3500 (1949) R. Seaman, Burroughs Wellcome Co., personal communi-

cation

STEVEN A. BENEZRA AND JOHN W. MCRAE 506

12.

13.

14.

15.

16.

17 .

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28

29.

30.

R.H. Manski, T.B. Johnson, J. her. Chem. SOC., 51, 1906

(1929)

T. Morgan, Burroughs Wellcome Co., personal communi-

cation

D.R. Feller, P. Basu, W. Mellon, J. Curott, L. Malspeis,

Arch. Int. Pharmacodyn., 203, 187 (1973)

D.C. Brater, L.Z. Benet, E. Lin, R.C. Morris, Jr.,

K.L. Melmon, Clin. Res., 24, 252A (1976)

R.G. Kuntzman, I. Tsai, L. Brand, L. Mark, Clin.

Pharmacol. Ther., - 12, 62 (1971)

J. Bralet, Y. Cohen, G . Valette, Biochem. Pharm., 11 2319 (1968)

M.D. Fairchild, G.A. Alles, J. Pharmacol. Exp. Ther.

- 158, 135 (1967)

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

I.L. Honigberg, J.T. Stewart, A.P. Smith, J. Pharm.

Sci., 3, 766 (1974)

T.L. Spriek, J. Pharm. Sci., - 64, 591 (1974)

G.T. Hill, Wellcome Foundation Ltd., personal com-

munication

M.L. Blackmon, Burroughs Wellcome Co., personal communi-

cation

K.K. Kaistha, R. Tadrus, R. Janda, J. Chromatog., 107, 359 (1975)

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)


31.

32.

33.

34.

35.

36.

37.

38.

39.

E.B.-Hanssen, A.B. Svendsen, J. Pharm. Sci., 51, 938

(1962)

K. Yamasaki, K. Fujia, M. Sakamoto, M. Okeda, M.

Yoshida, 0. Tanaka, Chem. Pharm. Bull., 22, 2898 (1974)

A.H. Beckett, B. Testa, J. Pharm. Pharmacol., 9, 382

(1973)

M.W. Anders, G.J. Mannering, Anal. Chem., - 34, 730 (1962)

C. Bye, H.M. Hill, D.T.D. Hughes, A.W. Peck, Eur. J.

Clin. Pharmacol., 8, 47 (1974)

E.T. Lin, D.C. Brater, L.Z. Benet, J. Chromatog., 140, 273 (1977)

L.M. Cummins, M.J. Fourier, Anal. Lett., 2, 403 (1969)

A.H. Beckett, G.R. Wilkinson, J. Pharm. Pharmacol.,

Suppl. 1 7 , 104s (1965)

P. Leblish, B . S . Finkle, J . W . Brackett, Jr., Clin.

Chem., 16, 195 (1970)


TRIPROLIDINE HYDROCHLORIDE

Steven A . Benezra and Chen-Hwa Yang

I . Description I . I I .2 Appearance, Color, Odor

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



3. Synthesis 4. Stability 5. 6. Methods of Analysis

Drug Metabolic Products and Pharmacokinetics

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

Copy-t @ 1979 by Academic F’ress, Inc. AU rights of reproduction in any form reserved.

ISBN 0-12-2608089 509

5 10 STEVEN A. BENEZRA AND CHEN-HWA YANG

1. Description

1.1 Name, Formula, Molecular Weight

Triprolidine hydrochloride is E-2-[3-(l-pyrroli-

diny1)-1-p-tolylpropenyllpyridine, monohydrochloride;

E-l-(2-pyridyl)-3-pyrrolidino-l-p-tolylprop-l-ene, monohydro-

chloride.

J

M.W. 314.85


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

is shown in Figure 1. The IR spectrum was recorded with a

Nicolet model 7199 FT-IR spectrophotometer as a 0.2% disper-

sion of triprolidine hydrochloride in KBr.2

the infrared assignments consistent with the structure

of triprolidine hydrochloride.

Table I gives

TRIPROLIDINE HYDROCHLORIDE 511

Table I

Infrared Spectral Assignments €or Triprolidine

Hydrochloride Monohydrate

Band (em-') Assignment

3480 OH stretch (hydrate)

2958 CH stretch (-CH3)

2690 NH' stretch

1630 C=C stretch (conj. diene)

1582 C=C stretch (aromatic)

1562 C=C stretch (pyridine)

1462

1386 -CH3 sym. bend

1358 C-N stretch (tert. amine)

-CH asp. bend 3

846 =C-H rock

824 para subst. benzene

776 1-subst. pyridine

2.2 Nuclear Magnetic Resonance (NMR) Spectrum

The NMR spectrum of triprolidine hydrochloride is

shown in Figure 2.

100 MHz NMR spectrometer.

solvent with tetramethylsilane as an internal standard.

The interpretation of the NMR spectrum is given in Table 11.

It was obtained with a Varian XL-100

Deuterated DMSO was used as the

i

H3cQ 9 c I'

b

e

?=====-

c- P

r-

{ 1

1

1

1 I

I I

I 1

0

8 8

8 P

33

NV

lllWS

NV

U

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

11.55

8.63

7.74

7.35

7.00

7.12-7.38

6.95

3.78

3.49

2.94

2.37

1.88

1

1

1

1

1

4

1

2

2

2

3

4

1

multiplet

6

multiplet

multiplet

4

3

2

1 (broad)

1 (broad)

1

mu1 t iplet

j

a

b

C

d

benzene ring

e

f

h

h

i

lz a,b=2.0, J =8, Jc,d=1.2, Jaf=7 J =4.7, Ja,d=0.9, J

b,c a,c

2.3 Ultraviolet (W) Spectrum

The ultraviolet spectrum of triprolidine hydro-

chloride in 0.1N HC1 was taken with a Beckman ACTA CIII W

spectrophotometer and is shown in Figure 3.4

gives the W data for triprolidine hydrochloride in various

solvents.

Table I11

Table I11

W Spectral Data for Triprolidine Hydrochloride

Solvent h a x (NO)

0.1N NaOH 234 17000

275 8000

EmaX

0.1N HC1

Methanol

232

290

235

13000

9900

15000

282 7200

TRIPROLIDINE HYDROCHLORIDE

0.6 I- W 0 Z

cr 0 cn

a m

m a

5 15

220 260 300 340 380 nm

Figure 3 - Ultraviolet Spectrum of Triprolidine Hydrochloride

Figure 4 - Mass Spectrum of Triprolidine

250 300 m /e

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 120°C.5

characteristic fragments are in agreement with those found

by Kuntzman and co-workers.6

istic of triprolidine are shown in Figure 5.

The

The major fragments character-

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.

chloric 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- > Br- ZC1 > F-. on the fluorescent intensity, while 0.1M C1- reduces the

intensity by a proximately 75% of the chloride-free sulfuric

acid solution.

The fluorescence is much diminished in hydro-

A concentration of 10-3M C1- has little effect

s

2.6 Crystal and Molecular Structure

James and Williams have determined that triproli-

dine hydrochloride monohydrate crystals belong to the P2 /c

space group.8 There are 4 molecules per unit cell. The

cell parameters are a = 14.777 i, b = 9.5785 i, c = 13.099 A, and f? = 90.48O. make dihedral angles of 29.7O and 55.3O respectively with

the double bond plane.

106.5O.

solution of the compound in anisole. A Picker FACS-1 dif-

fractometer with CuK radiation was used for the measurements.

1

0

The 2-pyridyl ring and p-tolyl group

The inter-aryl dihedral angle is

The data was obtained on crystals grown from a

01

+CH2 , CH;’ / c = c R2 \ R l?

R2, r 3 c = c c = c

RI’ ‘H RI’ ‘H R, ’ ‘H

m/e 278 ( 33%) m/e 209 (90%) m/e 208 (100%)

R 2 \ + CH +CHz-R,

R2, /-R3 R2, c = c + /

+ c = c

m/e 200 ( 10%) ‘H R,’ ‘H R l

m/e 182 (7%) m/e 84 (15 %) m/e 194 ( 18 %)

R2=QCH R 3 = - N 3 Figure 5 - Major Ions in Mass Spectrum of Triprolidine Hydrochloride


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 Solubility (mg/ml)

Water 316

0.1N HC1 319

95% Ethanol 374

n-Octanol 75

Chloroform 480

Propylene Glycol 237

Diethyl Ether <1

2.9 Partition Coefficients

The partition coefficients of triprolidine hydro-

chloride 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

shown in Figure 6. lo

phenone used in the first step is prepared by the Mannich

The 4-methyl-w-pyrrolidinopropio-

I

c-3 z

+

Ob

i

0

XI

A

+ + m

I

0

IT

ma

z

I

/

I


reaction of 4-methylacetophenone and pyrrolidine.

proper dehydration conditions of the carbinol, a product

almost pure in the desired E-conformation is obtained.

4. Stability

With

Ultraviolet light will convert the E-form of

triprolidine hydrochloride to the Z-form.

37OC, triprolidine hydrochloride in a syrup formulation does

not decompose more than 10%. After 3 years at 37OC tripro-

lidine hydrochloride in tablet formulations does not decom-

pose more than 10%. The formulations are kept in light re-

sistant containers since triprolidine hydrochloride dis-

colors on exposure to light.'

After 2 years at

5. 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. Tri-

prolidine 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

istered 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.

When admin-



The results of the elemental analysis of tripro-

lidine hydrochloride are given in Table V.3 The theoretical

0=

0

I II z

$1 I II z

H

W

k

-I 0

m

I

e W

H

W

k

J

0

m

a

k

W

z

01 Q

4

& 0

W 0

VJ aJ


figures are based on the monohydrate.

Table V

Elemental Analysis of Triprolidine Hydrochloride

Found (%>* Element Theory (%)

C 58.50

H 7.51

N 8.41

*N.F. Reference Standard

6.2 Nonaoueous Titration

58.59

7.58

8.39

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 mole of triprolidine hydrochloride.

6.3 Ultraviolet Spectrophotometric Analysis

An ultraviolet spectrophotometric analysis is

used to determine content uniformity of triprolidine hydr-

ochloride in tablet formulations. The triprolidine hydro-

chloride is extracted from finely powdered tablets with

dilute hydrochloric acid. The solution is filtered and

diluted to a concentration of approximately 10 pg tripro-

lidine hydrochloride per ml. The absorbance at 290 nm of

the extracted triprolidine hydrochloride solution in 1 cm

cells is compared against NF Triprolidine Hydrochloride

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


procedure for triprolidine hydrochloride in syrup and

tablets. ’ and 12.5 mg 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.

hexane. The infrared absorbance of the cyclohexane extract

is determined at 824 cm in 1 mm thick cells. A base line

is drawn between 840 cm-’ and 806 cm-’. The absorbance of

the sample is compared against the absorbance of a NF Tri-

prolidine Hydrochloride Reference Standard treated in the

same manner as the sample.

A portion of syrup or tablets equivalent to 20 mg

The triprolidine free base is extracted into cyclo-

-1

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.

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

times of 4 minutes and 9 minutes for the E and 2 isomers

re~pective1y.l~ A 0.45m x 4 mm i.d. Partisil@ silica gel

column with a mobile phase of acetonitri1e:concentrated

NH40H:H 0 (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.

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

A DuPont SAX

2

2

A mobile

Syrup and


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.

has a retention time of 22 minutes. l6

In this system triprolidine hydrochloride


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 : 40 : 7 : 3

cyc1ohexane:benzene:

diethylamine

75: 15: 10

me thano 1

Adsorbant

Silica gel F

Silica gel F

Silica gel F

Silica gel F

Silica gel F

Silica gel G

Silica gel G

pre-coated with

Rf

0.60

0.44

0.60

0.60

0.40

0.41

0.40

Reference

17

17

17

17

17

18

18


acetone

met hano 1

95% ethanol

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

0.1M NaOH

Silica gel G 0.13

pre-coated with

0.1M NaOH

Silica gel G 0.17

pre-coated

with 0.1M KHS04

Silica gel G 0.02

pre-coated with

0.1M KHS04

Silica gel G 0.40

Silica gel G 0.48

Silica gel G 0.26

Alumina 0.81

19

18

19

20

20

20

20

6.53 Quantitative Thin Layer Chromatography

Quantitative TLC has been used to determine tri-

prolidine hydrochloride in human plasma. l2

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 solu-

tion of ammonium bisulfate.

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

Plasma was

After the plate was air dried for


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 1.52111 x 2 .4 mm 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.

ly eluted as the free base.21

Triprolidine hydrochloride was probab-

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.

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.

The fluorescence of the acid extract is determined


1.

2.

3 .

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

References

N.F. XIV, Mack Printing Co., 1975

W. Martin, Burroughs Wellcome Co., personal

communication

B.S. Hurlbert, Burroughs Wellcome Co., personal

communication

C-H. Yang, Burroughs Wellcome Co., unpublished data

D. Brent, Burroughs Wellcome Co., personal

communication

R. Kuntzman, E. Sernatinger, I. Tsai, A. Klutch,

Importance Fundam. Princ. Drug Eval. Proc. Symp., 87

(1968)

J. McCrerie, Wellcome Foundation Ltd., personal

communication

M.N.G. James, G.J.B. Williams, Can. J. Chem., 52, 1880

(1974)

T.A. Morgan, Burroughs Wellcome Co., personal

communication

U.S. Patent 2,712,023

D. Adamson, J. Billinghurst, J. Chem. SOC., 1039 (1950)

R.L. DeAngelis, M.F. Kearney, R.M. Welch, J. Pharm.

Sci., - 66, 841 (1977)

K. Holmes, Burroughs Wellcome Co., personal

communication

G.T. Hill, Wellcome Foundation Ltd., personal

communication

I.L. Honigberg, J.T. Stewart, A.P. Smith, J. Pharm.

Sci., 63, 766 (1974)

R. Jones, Wellcome Foundation Ltd., personal

communication

A.C. Caws, Wellcome Foundation Ltd., personal

communication

W.W. Fike, I. Sunshine, Anal. Chem., 37, 127 (1965)


19.

20. J. Cochin, J.W. Daly, J. Pharm. Exptl. Therap., 139, 160 W.W. Fike, Anal Chem., 38, 1697 (1966)

(1963)

21. C.R. Fontan, W.C. Smith, P.L. Kirk, Anal. Chem., 35, 591 (1963)

22. C. Skinner, Burroughs Wellcome Co., personal

communication


SODIUM VALPROATE AND VALPROIC ACID

Zui L . Chang

I . Description 1. I Nomenclature

1 .1 I Chemical Names I . 12 Trade Names

1.2 Formulas and Molecular Weights 1.3 Appearance, Color, Odor

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

3. Synthesis 4. Stability-Degradation 5. Drug Metabolism and Pharmacokinetics 6. 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 7.

8. References 9. Acknowledgments

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

ISBN 0-12-2€!48W-9 529

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 dipro- pylacetate, sodium di-n-propylacetate, and by many slight variations of the particular nomenclature.

Valproic acid is 2-propylpentanoic acid. It is also known as n-dipropylacetic acid, 2- propylvaleric acid, DPA, and by many slight variations of the particular nomenclature.

1.12 Trade Names

DepakeneB, Depakinea, EurekeneB, Epilim, ErgenylB, and LabazeneB.

1.2 Formulas and,Molecular Weights

CH3CH2CH2,

CH3CH2CH2/ CHCOONa

C8H1502Na M.W. 166.19

CH3CH2CH2, ~ CHCOOH

CH3CH2CH2

C8H1602 M.W. 144.21


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 53 1

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 Figure 1 (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.

b. 1560 and Strong bands due to the anti- 1412 cm-1 symmetrical and symmetrical

stretching vibrations of the COO- grouping.

The infrared spectrum of valproic acid is presented in Figure 2. capillary method. 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.

b. 3000-2800 cm-1 Complex of strong bands due to the overlapping C-H stretching vibrations of the various methyl and methylene groups.

stretching vibration of the carboxylic acid.

ing vibration of the carboxylic acid.

The spectrum was measured using the The following bands (cm-1) have

c. 1700 cm-' Strong band due to the C=O

d. 930 cm-l Broad band due to the OH bend-

2.2

The nuclear magnetic resonance spectrum of sodium valproate as shown in Figure 3 was obtained on a Varian Associates T-60 NMR 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 (CM- l )

2.5

FIGURE 2 - INFRARED SPECTRUM OF VALPROIC ACID

3

WAVELENGTH (MICRONS)

5 6 7 8 9 10 12 15 4

VI W w

4000 3500 3000 2 500 2000 1500 1000 700

FREQUENCY (CM-')

VI W P

FIGURE 3 - NUCLEAR MAGNETIC RESONANCE SPECTRUM OF SODIUM VALPROATE

I I I I I 1 I I

I I I I I I I I

8 7 6 5 4 3 2 1 0

ii

SODIUM VALPROATE A N D VALPROIC ACID 535

Table I

NMR Spectral Assignments for Sodium Valproate

Proton Chemical Assignment Shift (ppm) Mu1 t iplicity

1.9-2.5 Mu1 t iple t \ . CH-CO 1.1-1.8 Complex

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 Ta- ble 11.

Table I1

NMR Spectral Assignments for Valproic Acid

Proton Chemical Assignment Shift (ppm) Multiplicity

C02H 11.3-11.6 Singlet

CHCO 1.9-2.5 Multiplet

-CH2CH2 1.1-1.8 Complex

CH3- 0.5-1.1 Complex

2.3 Mass Spectrum

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 PDP- 11 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. in the molecular ion region at m/e 145. 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.

Only a very weak ion was detectable This would

The mass spectrum assignments of the prominent ions and subsequent fragments are shown in Table I11 and Figure 6 (3).

Table I11

High Resolution Mass Spectrum of Valproic Acid

Measured Mass (m/e) Calculated Mass Formula

126.1044 126.1045 C8H140

102.0690 102.0681 CgH1002

73.0295 73.0290 C3H502

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) have been assigned for Figure 7 (1).

a. 3000-2800 cm-' Complex of strong bands due to the overlapping C-H stretching vibrations of the various methyl and methylene groups.

b. 1450 cm-l Due to the superimposing C-H bending vibrations of the various methyl and methylene groups.

The Raman spectrum of valproic acid as shown in Figure 8, was obtained in the undiluted liquid state on a Cary Model 83 Spectrometer. lowing bands (cm-1) have been assigned for Figure

The fol-

8 (1).

FIGURE 4 - NUCLEAR MAGNETIC RESONANCE SPECTRUM OF VALPROIC ACID

I I I I 1 I I I I I

FIGURE 5 = MASS SPECTRUM O F VALPROIC ACID

E m

Lu c z z

> - Y

0 100 130

539

m

0

2 4 a

E

2

Y

>

g 5 e cn

L 3

P

cn z a

E

a

z Y

h

3

I

E

0

ON

0

9

P

AlIS

N31N

I

0

0

0

el 0

0

d

0 0

9

0

0

a0

0 0

2

0

0-

Z'

r

- PE

25

5

U

0-

0

1

v)

gf

U

ow

- 0

0

0

N

0

0

P

N

0

0

a0 (Y

0

0

(Y

m

0

0

9

m

0

0

0

t

540

I 1

I

I

I

~ >

I

I

I

I I

0

0

0

(Y

w

0

0

m

9

AllS

N31N

I

0

0

aD N

0

0

0

(Y

0

0

d

0

0

9

0

2 0

0

P

0

0

2

8

5

0

B 0 0

z 0

0

8

0

N

0

0

0

5 0 0

3

S4 I

542 ZUI L. CHANG

FIGURE 9 - ULTRAVIOLET SPECTRUM OF VALPROIC ACID

0.6

0.5

0.4

e 2 SI 9

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.

ing vibration of the carboxylic acid.

b. 1660 cm-l Weak band due to C=O stretch-

c. 1450 cm-l Due to the superimposing C-H bending vibrations of the various methyl and methylene groups.

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,to 205 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-Hep tane Chloroform Ethyl Acetate Methanol 100% Ethanol Ace tone Diethyl Ether Benzene 1 N Aqueous NaOH 0.1 N Aqueous HCl Water

Greater than lo%, v/v Greater than l o%, v/v Greater than lo%, v/v Greater than lo%, v/v Greater than lo%, v/v Greater than lo%, v/v Greater than l o%, v/v Greater than lo%, v/v Greater than lo%, v/v 1.15 mg/ml 1 .27 mg/ml

544 ZUI L. CHANG


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 Camera (Table IV). An Enraf-Nonius Difractis 601 Generator; 38 KV and 1 8 MA with nikel filtered copper radiation; A = 1.5418, was employed ( 4 ) .

Table IV

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

- -1 111 dA - 1/11

60 100

50 3 5

20 5 2 5

30 35

5 1

15 20 1 3 4 5 5

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 . 8 1 1.75 1.69 1.66

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. tion 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).

Titra-


2.9 Hygroscopic Behavior

Sodium valproate is hygroscopic. ture absorption was tested in a humidity chamber using a Cahn Electro Balance and the results are shown in Table V (5).

The rate of mois-

Table V

Rate of Moisture Absorption of Sodium Valproate

Relative Time, min. Humidity 10 20 30 40 50 60 Overnight ------

12, 22 33, 43%

No gain

53% 1.76 3.17 4.39 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 105°C for 10 days.

2.11 Melting Range

Sodium valproate does not melt, decompose, or physi- cally 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 120-121°C (6), bp20 128-13OoC (6) and bp760 221-222"C (7).


Differential thermal analysis of sodium valproate shows a large endotherm beginning at 100°C and end- ing at 118°C which is possible due to the loss of water. A sharp endothermic peak at 450°C is indicative of the melting point of sodium valproate.

Differential thermal analysis of valproic acid shows. a sharp endothermic response at 225°C indicative of the boiling point of valproic acid.

FIGURE 10 - DIFFERENTIAL THERMAL ANALYSIS CURVE OF SODIUM VALPROATE

I I I I I I I I I

- -

I I I I I I I I I

0 50 100 150 200 250 300 350 400 450 500

T, O C (CORRECTED FOR CHROME1 ALUMEL THERMOCOUPLES)

FIGURE 11 - DIFFERENTIAL THERMAL ANALYSIS CURVE OF VALPROIC ACID

0 50 100 150 200 250 300 350 400 450 500

1, O C (CORRECTED FOR CHROME1 ALUMEL THERMOCOUPLES)

ZUI L. CHANG 548

3 .

4.

5.

2.14

2.15

Specific Gravity

The specific gravity of valproic acid was determined in a calibrated 25 ml pycnometer at 25'C. value of 0.904 g/ml (8).

It has a

Refractive Index

Valproic acid has a refractive index of N D ~ ~ - ~ 1.425 (6).

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.

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. when it was subjected to heat at llO°C for 10 days and to natural sunlight for 30 days in the dry state.

Also, it was very stable

Valproic acid is a very stable compound. No degradation has been observed by the action of heat, light, and strong aqueous alkali, or acid.

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 anticon- vulsant 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 c02r * ‘C’

/ C02C2H5 (or NaOCzHS) + 2CI-CH2CH = CH2

C 0 2 R CHjOH / \

C02C2H5 (or C,H,OH) CH2= CHCH2 \

CH2

Diethyl Malonate Ally1 Chloride Esters of Diallyl Malonic Acid R=CHs and/or C2H5

CH3CH2CH2 C02H

Pd/C \ / c ‘C’ Co2R 1) KOH, H 2 0 CH3CH2CH2 -

C02H 2) HCI / \

/ c \ CO2R CH3CH2CH2 CH3CH2CH2 H

Esters of Dipropyl Malonic Acid Dipropyl Malonic Acid

CH3CH2CH2 CHsCH2CH2 -c02 \ NaOH \

c

/ CH-c02No -

CH3CH2CH2 / CH-C02H H2O CH3CH2C H2

2-Propylpentonoic Acid (Valproic Acid)

Sodium 2-propylpentanoate (Sodium Valproate)

550 ZUI L. CHANG

and EDTA, and inhibited by various enzyme reaction inhi- bitors 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 (11).

The pharmacokinetics of distribution and elimination of sodium valproate in mice and dogs has been reported by Schobben and van der Kleijn (12).

The omega-oxidation of sodium valproate in rats has been reported by Kuhara, et. al. (13).

The absorption, excretion, and biotransformation of valproic acid were studied by Kukino and Matsumoto (14).

A preliminary pharmacokinetic profile of sodium valproate in monkey has been written by Levig, et. al. (15).

The pharmacokinetics of sodium valproate have been studied in 7 patients by Schobben, et. al. (16). The plasma concentrations were determined by gas-liquid chromatography during and following subchronic treatment. Elimi- nation of the drug appeared to follow a monophasic expo- nential course; biological half lives were 8 to 15 hours. The drug appeared to have a relatively restricted distribution: 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.

calculated relative distribution volumes ranged

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, 2- n-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.


6.1 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. A 5% aqueous solution gives a violet precipitate with a 5% aqueous solution of cobalt nitrate.

B. A 5% aqueous solution gives a violet precipitate with potassium iodobismuthite.


A typical elemental analysis of a sample of sodium valproate is present in Table VI (21).

Table VI

Elemental Analysis of Sodium Valproate


C 57.82 57.82 H 9.10 9.38 0 19.25 -----* Na 13.83 -----*

*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


C 66.63 66.39 H 11.18 11.32 0 22.19 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.

2 .

3 .

4.

5.

Ch1oroform:Methanol:Glacial Acetic Acid (17 : 2 : 1)

Ethyl Acetate:Formic Acid:Water (5:l:l)

Benzene:Methanol:Acetone:Ammonium Hydrox- ide (2:2:5:1)

Ch1oroform:Methanol:Ammonium Hydroxide (20 : 15 : 3)

n-Butanol Saturated with 10% Ammonium Hy- droxide 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. Pre- paratory 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 225OC Column 160°C Detector 300°C

Flow Rate: -20 ml/min helium


Attenuation: 2 x 4

Slope Sensitivity: 0.03 or adjust for proper

Sample Size: 2 1.11

sensitivity

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 - N hydrochloric acid.

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 glass- calomel 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 , 2 4 , 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 gas- liquid chromatographic column (28, 29, 16, 30, 31, 3 2 ,

554 ZUI L. CHANG

33, 34, 35, 36, 37, 38, 39).

Most of the methods employ an internal standard, and all are reported as accurate and reproducible. However, Schmidt, et. al. (40) recently reported great interlabo- ratory variation in the analysis of such biological samples.

8. References

1. W. Washburn, Abbott Laboratories, Personal Communica- t ion.

2. M. Cirovic and R. Egan, Abbott Laboratories, Personal Communication.

3. S. Mueller, Abbott Laboratories, Personal Communica- t ion.

4 . J. Quick, Abbott Laboratories, Personal Communica- tion.

5. M. Yunker, Abbott Laboratories, Personal Communica- tion.

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 Communi- cation.

9. E. Oberreit, Berichte der Deutschen Chemischen Gesellschaft, 3, 1998 (1896).

10. P. Eymad, et. al., J. Pharmacol. (Paris), 2 (2), 251 (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., 1. ( 4 ) , 291 (1974).


14. K. Kukino and I. Matsumoto, J. of Kurukome Med. ASSOC., 34 (41, 369 (1975).

15. R.H. Levig, et. al., Epilepsia - 16 (l), 202 (1975).

16. F. Schobben, E. van der Kleijn and F.J. M. Gabreels, Europ. J. Clin. Pharmacol., 8, 98 (1975).

17. 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, Arzneimittel- Forschung, 27 (I) 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, u. Trav. SOC. Pharm. Lyon., 16 (2), 53 (1972).

24. B. Ferrands, P. Eymard and C. Gautier, Ann. Pharm. 5, 31 (4), 279 (1973).

25. J.M.H.G. Cremers and Ing. P.E. Verheesen, Pharm. Weekbl. , 109 (22), 522 (1974).

26. F.N. Ijdenberg, Pharm, Weekbl., 110 (2), 21 (1975).

27. J.W.A. Meijer and L. Hessing-Brand, Clin. Chim. ., Acta - 43 ( 2 ) , 215 (1973).

28. I.C. Dijkhuis and E. Vervloet, Pharm. Weekbl., 109 (2), 42 (1974).

29. F. Schobben, E. van der Kleijn and Neth. Nijmegen, Pharm. Weekbl, 109 (2), 30 (1974).

30. C.R. Chard, N.J. Legg, Ed., Clinical and Pharmacolo- gical Aspects of Sodium Valproate in the Treatment of Epilepsy, pp. 89-91 (1975).

556 ZUI L. CHANG

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

P.N. Patsalos, V.C. Goldberg and P.T. Lascelles, Proc. Analyt. Div. Chem. SOC., 12 (lo), 270 (1975).

M.C. Pfaff and G. Mahuzier, Ann. Pharm. Fr., 33, 355 (1975).

T.B. Vree, E. van der Kleijn and H.J. Knop, J. Chromatography, 121 (l), 150 (1976).

N.H. Wood, D.C. Sampson and W.J. Hensley, Clinica Chimica Acta, 77, 343 (1977).

L.J. Dusci and L.P. Hackett, J. of Chromatography, 132, 145 (1977).

L.J. Dusci and L.P. Hackett, J. Chromatography, 132 (l), 145 (1977).

C.J. Jensen and R. Gugler, J. Chromatography, 137, 188 (1977).

W. Loescher, Epilepsia 18 (2), 225 (1977).

A. Sengupta and M.A. Peat, J. Chromatography, 137 206 (1977).

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 w, 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 ,47 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 ,87 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 ,25 1 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.

5 5 8 CUMULATIVE INDEX

Isoniazide, 6, 183 Isopropamide, 2,3 15 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, 8.37 1 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;

Propylthiouracil, 6,457 Pseudoephedrine hydrochloride, 8,489 Reserpine, 4,384;5,557 Rifampin, 5,467 Secobarbital sodium, I , 343 Spironolactone, 4,43 1 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;

Triamcinolone diacetate, I , 423 Triamcinolone hexacetonide, 6,579 Triclobisonium chloride, 2,507 Triflupromazine hydrochloride, 2,523; 4,520;

Trimethaphan camsylate, 3,545 Trimethobenzamide hydrochloride, 2,55 1 Trimethoprim, 7,445 Triprolidine hydrochloride, 8,509 Tropicamide, 3,565 Tubocurarine chloride, 7,477 Tybamate, 4,494 Valproate Sodium and valproic acid*, 8,

Vinblastine sulfate, I , 443 Vincristine sulfate, I , 463

6,598

4,520; 7

5,557

529

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