Analytical Profiles of Drug Substances

Volume 9
Edited by

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

Contributing Editors

Jerome I. Bodin Hans-Georg Leemann Rafik Bishara Gerald J . Papariello Glenn A. Brewer, Jr. Bruce C. Rudy Milton D. Yudis
Compiled under the auspices of the Pharmaceutical Analysis and Control Section f Academy o Pharmaceuticul Sciences

A Subsidiary of Harcourt Brace Jovanovich, Publishers

ACADEMIC PRESS London Sydney

1980

New York

Toronto San Francisco

EDITORIAL BOARD
Norman W. Atwater Rafii Bishara Jerome I. Bodin Glenn A. Brewer, Jr. Lester Chafetz Edward M. Cohen John E. Fairbrother Klaus Florey
Salvatore A. Fusari Boen T. Kho Hans-Georg Leemann Gerald J. Papariello Bruce C. Rudy Bernard Z. Senkowski Milton D. Yudis

Academic Press Rapid Manuscript Reproduction

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Library of CongressCataloging in PublicationData

Main entry under title: (Revised) Analytical profiles of drug substances. Compiled under the auspices of the Pharmaceutical Analysis and Control Section, Academy of Pharmaceutical Sciences. Includes bibliographical references. 1. Drugs-Analysis-Collected works. 2. Chemistry, Pharmaceutical-Collected works. I. Florey, Klaus, ed. 11. Brewer, Glenn A. 111. Academy of Pharmaceutical Sciences. Pharmaceutical Analysis and Control Section. [DNLM: 1. Drugs-AnalysisYearbooks. QV740 AAl A551 RS189.AS8 615l.1 70- 187259 ISBN 0-12-260809-7 (V.9)
PRINTED I N THE UNITED STATES OF AMERICA 80 81 82 83

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AFFILIATIONS OF EDITORS, CONTRIBUTORS, AND REVIEWERS

H . Y . Aboul-Enein, Riyadh University, Riyadh, Saudi Arabia E. A . Abourubl, Faculty of Pharmacy, Cairo University, Cairo, Egypt A . A . Al-Budr, Riyadh University, Riyadh, Saudi Arabia A . H . Amann, American Critical Care, McGraw Park, Illinois N. W. Afwurer, E. R. Squibb and Sons, Princeton, New Jersey D. M . Baaske, American Critical Care, McGraw Park, Illinois S . A . Benezru, Burroughs Wellcome Company, Research Triangle Park, North Carolina R . Bisharu, Eli Lilly and Company, Indianapolis, Indiana J. I. Bodin, Carter Wallace, Inc., Cranbury, New Jersey G. A . Brewer, The Squibb Institute for Medical Research, New Brunswick, New Jersey J . E . Carrer, American Critical Care, McGraw Park, Illinois L. Chuferz, Warner-Lambert Research Institute, Moms Plains, New Jersey G . Clarke, The Squibb Institute for Medical Research, Moreton, Wirral, England E. M . Cohen, Merck Sharp & Dohme, West Point, Pennsylvania A . Egli, Sandoz Limited, Basel, Switzerland J. Fairbrorher, Department of Pharmacy, University of Nottingham, Nottingham, England K . Florey, The Squibb Institute for Medical Research, New Brunswick, New Jersey P. R. B . Foss, Burroughs Wellcome Company, Research Triangle Park, North Carolina H . L. Fung, School of Pharmacy, S.U.N.Y. at Buffalo, Amherst, New York S . A . Fusari, Parke-Davis, Inc., Detroit, Michigan J. R. Greco, Schering Corporation, Bloomfield, New Jersey M . M . A . Hassun, Riyadh University, Riyadh, Saudi Arabia J. G. Hoogerheide, Schering Corporation, Bloomfield, New Jersey A. I. Judo, Riyadh University, Riyadh, Saudi Arabia
vii

viii

AFFILIATIONS OF EDITORS, CONTRIBUTORS, AND REVIEWERS

C . A . Janicki, McNeil Laboratories, Fort Washington, Pennsylvania B. T. Kho, Ayerst Laboratories, Rouses Point, New York C. Y. KO, McNeil Laboratories, Fort Washington, Pennsylvania H. G. Leemunn, Sandoz Limited, Basel, Switzerland L . J . Lorenz, Eli Lilly and Company, Indianapolis, Indiana M, A, Lou&, Riyadh University, Riyadh, Saudi Arabia E. F. McNif, School of Pharmacy, S.U.N.Y. at Buffalo, Amherst, New York W . R . Michaefis, Sandoz Limited, Basel, Switzerland E. M . Oden, Schering Corporation, Bloomfield, New Jersey G. Paparieflo, Wyeth Laboratories, Philadelphia, Pennsylvania A. Posr, Smith Kline & French Laboratories, Philadelphia, Pennsylvania E. C. Rickard, Eli Lilly and Company, Indianapolis, Indiana B. E. Rosenkranrz, Schering Corporation, Bloomfield, New Jersey B. Rudy, Burroughs Wellcome Company, Greenville, North Carolina I. G. Rutgers, Wyeth Laboratories, Philadelphia, Pennsylvania B. Senkowski, Alcon Laboratories, Forth Worth, Texas C. M . Shearer, Wyeth Laboratories, Philadelphia, Pennsylvania L . Sfusarek, Eastman-Kodak, Rochester, New York A. Vigevani, Pharmitalia-Carlo Erba SPA, Milan, Italy R . J. Warren, Smith Mine & French Laboratories, Philadelphia, Pennsylvania M . J. Williamson, Adria Laboratories, Columbus, Ohio P . S . K. Yap, School of Pharmacy, S.U.N.Y. at Buffalo, Amherst, New York M . D . Yudis, Schering Corporation, Bloomfield, New Jersey J. E. Zarernbo, Smith Kline & French Laboratories, Philadelphia, Pennsylvania M . U. Zubair, Riyadh University, Riyadh, Saudi Arabia

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 ninth. The concept of analytical profiles is taking hold not only for compendial drugs but, increasingly, in the industrial research laboratories. Analytical profiles are being prepared and periodically updated to provide physicochemical and analytical information of new drug substances during the consecutive stages of research and development. Hopefully, then, in the not too distant future, the publication of an analytical profile will require a minimum of effort whenever a new drug substance is selected for compendial status. The cooperative spirit of our contributors has made this venture possible. It is gratifying to note that increasingly profiles are being written not only in industrial laboratories but also academic institutions worldwide. 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. The goal to cover all drug substances with comprehensive monographs is still a distant one. It is up to our perseverance to make it a reality. Klaus Florey

ix

BACITRACIN
Glenn A . Brewer
1.

2. 3. 4.

5. 6.

7.

8. 9.
10.

Introduction Chemistry 2.1 structure 2.2 Biosynthesis Description 3.1 Composition, Formula, Molecular Weight Physical Properties 4.1 Spectra 4.2 Crystal Properties 4.3 Solubility 4.4 Physical Properties of Solutions Production 5.1 Microbiological 5.2 Isolation Stability 6.1 Stability of Solid 6.2 Stabliiy of Solutions 6.3 Light Stability 6 . 4 Formulation Stability 6.5 Stability of Metal Salts Analytical Methods 7 . 1 Identity Tests 7.2 Microbiological Assays 7.3 Chemical Methods 7.4 Chromatographic Methods Mode of Action Derivatives of Bacitracin Reviews References

2 4 4 8
10

10 12 12 16 18 19 20 20 24 25 25 26 27 27 21 28 28 30 34 35 42 43 44 45

Analytical Pmfiles of Drug Substances, 9

Copyright @ 1980 by Academic Press. Inc.

All rights of reproduction i any form reserved. n

ISBN: 0-12-260809-7

GLENN A . BREWER

Introduction The organism which produces bacitracin was isolated by Miss B.A. Johnson in June 1943 from the debrided tissue removed from a compound fracture of the tibia of a seven year old girl named Margaret Traceyl. Miss Johnson was working on a project directed by Dr. Frank L. Meleney. These workers thought that it might be possible to isolate an antibiotic producing organism from the mixed bacterial flora present in a severe wound. A crude concentrate was soon produced, and in October 1943 the first human clinical trial was The process for the manufacture of started2 bacitracin was scaled up and the first large scale clinical studies were reported in 19473. Bacitracin was approved as a certifiable antibiotic in July 1949.

1.

In 1944, Magargo and co-workers isolated a strain of Bacillus subtilis which had in vitro activity toward Mycobacterium tuberculosis4. The culture was studied in England and it was found that the culture no lonqer showed activity aqainst Mycobacterium tube;culosis. Subsequentiy , a strain of Bacillus licheniformis was isolated from the culture and this isolate was found-to produce an antibiotic which was called Ayfivin5. When the composition of bacitracin was better understood, it was realized that it and Ayfivin were probably identical, and the latter name was no longer used6. Although bacitracin was known to be active primarily against Gram positive organisms, it was widely used in all types of infections. It was administered topically, by intramuscular injection, as lozenge for infections of the mouth and throat, intervaginally and as an ophthalmic preparation. Apparently, as more potent preparations of bacitracin were produced, the material also increased in nephrotoxicity7. In a review on bacitracin published in 1952, the author states8: "The side effects resulting from the administration of any therapeutic agent are of secondary importance in assessing the clinical value of the drug. They assume importance only if they limit either the dosage or duration of treatment because

BACITRACIN

of harmful effects on any organ or tissue of the body or any body function." This statement is interesting in the present era in which the importance of side effects practically eclipses the therapeutic activity and a potent therapeutic agent may be discarded because of relatively minor side effects. Today, the U.S.P. recognizes bacitracin ointments for topical and ophthalmic use and sterile bacitracin for intramuscular injection9. In addition, the C.F.R. provides for the certification of bacitracin oral dosage forms and bacitracin combination products with other antibiotics and steroids for ophthalmic and topical uselo. It is probable that the veterinary use of bacitracin is more economically important than the clinical use,although volume figures are not readily available. The C.F.R. provides certification for bacitracin powder, the manganese and zinc salts and unrefined feed grade zinc bacitracin powder. In addition, bacitracin methylene disalicylate oral dosage forms, combination oral products with streptomycin sulfate, implantation pellets and a large variety of ophthalmic and topical dosage forms are monographed. It is interesting to note that the number of publications on bacitracin chemistry and production have not waned in the thirty four years since the discovery of the antibiotic. It is rare to find a year in which a patent was not issued on the production of bacitracin, apd the literature on the chemistry of the antibiotic continues to grow.
2.

Chemistrv 2.1 Structure

The key to the establishment of the structure of a natural product is the isolation of the pure substance. Counter-current distribution analysis was used by Craig and co-workers to demonstrate that at least three components were present in commercial bacitracin12. The major component was hydrolyzed and the following dipeptides were found: phenylalanine and leucine phenylalanine and ornithine. In addition, phenylalanine, leucine, isoleucine,

GLENN A. BREWER

glutamic acid, aspartic acid, lysine, histidine, cystine and ammonia were found by amino acid analysis using starch column chromatography. It was recognized by Craig and co-workers that some of the amino acids probably had the D-configuration, as racemic mixtures were isolated in some cases. Newton and Abraham also used countercurrent distribution to study the purity of the antibiotic ayfivin6. They demonstrated that there were at least seven components in the mixture with the three major components being present in the ratio 4:1:4. Two'components were shown to be identical to components in bacitracin and the name ayfivin was dropped (see section 1). The same workers showed that at least ten components were present in crude bacitracinl3. They were designated bacitracins E , D, B, A l l A, C, G I F1 and F2. Bacitracins E, D, B and A showed a broad absorption band in the U.V. at 253 nm. Components C and G showed a sharper band at 250 nm while the three F components had a broad maximum at 288 nm. They established that all the components contained cysteine, ornithine, lysine, histidine, aspartic acid, glutamic acid, phenylalanine and leucine (or isoleucine). Bacitracin C also contained a component which was not separable from glycine in the chromatographic system used, while the bacitracins B, D and E yielded valine. Bacitracins D and E apparently do not contain amide groupings while A, B, C, G and the F components do. Newton and Abraham continued their examination of the structure of acitracin A, the major They established that component of the complex19 the antibiotic had three basic centers, one amide and had a unit molecular weight of 1500.

In addition, they established that each unit contained two carboxyl, one a-amino, one 6-amino and one histidine glyoxaline as ionizable groups. Bacitracin A did not contain a disulfide linkage, but a thiol group was liberated on acid hydrolysis. An amide group was also liberated and the ultraviolet absorbance at 254 nm disappeared on acid hydrolysis. On hydrogenation with Raney nickel, the group which contained the cystine residue was converted to an alanine residue. The authors postulated that

BACITRACIN

bacitracin A contains a thiazoline ring. Craig and co-workers used their newly developed ion exchange amino acid analyzer to establish the amino acid composition of bacitracin A 15. The same group established a molecular weight of 1470 for bacitracin A using a partial substitution methodI6. They also proposed a cyclic structure for the molecule. Ingram reported that bacitracin A contained no free amino end group based on methylation studiesl7. Porath, using partial acid hydrolysis, established the amino acid sequence for the ring as glutamic acid, cysteine, isoleucine, ornithine, histidine and 2 moles of aspartic acidlg. He postulated that the sulfur of cysteine was involved in a hetero cyclic ring between lysine and glutamic acid. An unidentified ninhydrin-negative compound is attached to lysine. Lockhart, Newton and Abraham performed acid hydrolysis at 37OCl9. They found the amino acid sequences:

isoleucine-cysteine-leucine-glutamic acid and ornithine-phenylalanine-isoleucine.

The latter peptide appeared to be an N-terminal peptide. Lockhart and Abraham postulated the following partial structure for bacitracin A 2 0

Aspartate Aspartated-a

.) Phenylalanine Lysine 9 -Isoleucine . % Ornithine-Isoleucine Cys ine lsoleucine

Histidine

tL

+\ Glutamate-

Leucine

GLENN A. BREWER

They also indicated that the sequence lysine-ornithine-valine-phenylalanine occurs in bacitracin B. Craig and co-workers confirmed the presence of three isoleucine residues in bacitracin A and, on this basiq postulated the emperical formula C66H168 014N 17S for the antibiotic2l. The same workers proposed the following structure for bacitracin A based on the products obtained after partial hydrolysis22,23,24,

Isoleucine-cysteine-leucine-glutamic acid-isoleucine-lysine
Aspartic acid-aspartic acid-histidine-phenylalanineisoleucine-ornithine It should be noted that this structure differs significantly from the one proposed by Abraham's group20, and does not explain their earlier findingsl4. Craig and co-workers proposed that bacitracin A contains a thiazole ring formed by the condensation of cysteine and isoleucine25. They began a study of the chemistry of bacitracin F. Further studies by Abraham and co-workers confirmed the fact that there were three isoleucine residues in bacitracin A26,27. This had been previously indicated by Craig and co-workers21. Lockhart and Abraham concluded that the lysine residue in bacitracin A is linked to isoleucine through the a-amino group and to aspartic acid through the -amino group28. This aspartic acid residue has the L-configuration while the other aspartic acid in bacitracin A has the D-configuration. Wrinch proposed a structure for bacitracin A based on the published information29. Several reviews of the chemistry of bacitracin A have been published30131132133,34. Craig and Konigsberg established that bacitracin F was a degradation product of bacitracin A35. The conversion was accompanied by the loss of

BACITRACIN

ammonia. Swallow and Abraham found that the glutamic acid residue was connected via the a-carboxyl group and that the y-carboxyl group is free36. One of the aspartic acid residues was present as an amide. Stoffel and Craig synthesized a number of cysteine peptides modeled on the N-terminal portion of bacitracin A37. They hoped to establish the substitution that would give stable thiazoline rings. Craig and co-workers studied the acid isomerization of bacitracin A38. The transformation involves the epimerization of the N-terminal isoleucine residue. Theodoropoulos established that both lysine residues in bacitracin A are a-isoleucyl-(E-aspartyl)-1ysine39. Kaneko and co-workers published a series of papers on the synthesis of peptide intermediates to be used in the total synthesis of bacitracin A40r41, 42,43,44,45,46. Ratti and co-workers established the optical configuration of the aspartyl and asparaginyl residues of bacitracin A as D and L r e ~ p e c t i v e l y ~ ~ . Cornell and Guiney established that the coordination sites for zinc in bacitracin were the thiazolene ring and histidine residue48. Manning developed a method to establish the amount of racemization that occurred during acid hydrolysis 4 9 I50. On the basis of NMR studies, a space-filling model of bacitracin A was proposed51. The presently accepted structures for the bacitracins can be found in Section 3.
2.2

Biosynthesis

The cell-free enzymatic synthesis of bacitracin A has been extensively studied by a number of workers.

GLENN A. BREWER

Bernlohr and Sievert noted the similarity of the amino acid composition of bacitracin and Bacillus licheniformis spore coats52. This suggested that the antibiotic was a precursor of a structural entity of the bacterial cell. Bernlohr and Novelli indicated that bacitracin was produced by postlogarithmic cells of Bacillus lichenifnrmis which are in the process of producing spores53. The amino acids were not incorporated into bacitracin by a normal mechanism. Shimura and co-workers found that the amount of bacitracin produced by B. licheniformis was governed by the amount of cysteine present in the medium54. Cornell published a thesis synthesis of b a ~ i t r a c i n ~ ~ .
OR

the bio-

The cell-free synthesis of bacitracin was They first achieved by Shimura and c o - ~ o r k e r s ~ ~ . utilized lysed protoplasts of B. licheniformis. The incorporation of L-histidine was inhibited when various D-amino acids were added. The biosynthesis was not inhibited by ribonuclease, chloramphenicol or puromycin so it was concluded that the biosynthetic pathway was different from that involved in protein biosynthesis. Pfaender also reported the bios nthesis of bacitracin with a cell-free preparation5 7 . He found that leaving out one of the required amino acids or the substitution of a D-amino acid for an L-amino acid stopped the synthesis. Pfaender and co-workers fractionated the enzyme system and found two fractions with molecular weights of 200,000 and 350,000,which dissociated to 50,000 units on storage for one day in the cold5*. Froyshov and Laland purified bacitracin synthetase about ll-fold59. They showed that two fractions were present,both of which were required for the synthesis of bacitracin. The amino acids required for the pyrophosphate-ATP exchange reactions were determined for each fraction. Froyshov reported that he had resolved

BACITRACIN

bacitracin synthetase nto three fractions by affinity chromatography66 . Ishihara and Shimura purified bacitracin synthetase 25-fold6I. Froyshov continued his work and found that fraction A was responsible for the chain lengthening of bacitracin A62. Ishihara published a review on the biosynthesis of bacitracin A with cell-free enzyme preparations63. Froyshov also reviewed rogress in cellfree biosynthesis of bacitracin A 6% Wang and c o - ~ o r k e r sand Umezawa and co~~ workers66 have published reports on the practical cell-free synthesis of bacitracin A. 3. DescriDtion
3.1

Composition, Formula, Molecular Weight

The bacitracin of commerce is a mixture of components. The major component is bacitracin A. The mixture of bacitracin components [1405-87-41 will be referred to in this monograph as bacitracin. Certain salts and derivatives of the bacitracin complex have been utilized in feeds or formulations Zinc bacitracin 11405-89-61 Manganese bacitracin [1405-99-81 Sodium bacitracin 139436-06-11 Methylenebis [2-hydroxybenzoate]-[55852-84-11 3.11 Bacitracin A r22601-59-81

The structure of bacitracin A was elucidated after almost twenty years of work by a It is no number of different groups (Section 2 ) . wonder then that there is disagreement in the literature on which group established the definitive structure. Ressler and Kashelikar, using a dehydration-reduction technique established the final position of the amino acids in the seven membered

10

GLENN A . BREWER

ring67. Craig and co-workers established the conformation of bacitracin A6*. The structure has been confirmed by total chemical synthesis69.

CO+Leu+

Glu

Ile -P Lvs+

Asn 4- Asp 4- His

Orn+

Ile ---c Phe

C66H103N170 16s Molecular Weight 3.12 1422.73

Bacitracin B r1402-99-91

The structure of bacitracin B is very similar to that for bacitracin A except that valine replaces one of the isoleucine residues. The exact residue is not certain but evidence suggests the isoleucine in the seven membered ring is replaced by valine. C65H101N17O 16S Molecular Weight 3.13 1408.70

Bacitracin F [22601-63-41

Bacitracin F is a degradation product of bacitracin A (see section 6).

o=c- C k CH3

fC$ I
C-

' 2HC'

CH3

H (L) (D) (L) (L) (D) (L) (D) CO+Leu+Glu+Ile+L s+Orn+Ile+Phe LYAsn+D-AspeL-HiX

BACITRACIN

11

Molecular Weight 3.14

1406.66

Other Bacitracin Components

A number of other minor components have been identified in the bacitracin complex. The structures of these components are not known at the present time.
Bacitracin Bacitracin Bacitracin Bacitracin Bacitracin Bacitracin Bacitracin Bacitracin B1 B2 C D E F1
G

F2

(57762-79-5) (57762-78-4) (1403-00-5) (1403-01-6) (1403-07-7) (1403-04-9) (1403-05-0) (1403-03-8)

Unless otherwise specified, in the remainder of this profile when we use the name bacitracin we refer to the bacitracin complex. 4. Physical Properties 4.1 Spectra 4.11 Infrared SDectrum

The infrared spectrum of bacitracin has been published by Hayden and co-workers70. The infrared curves of bacitracin and zinc bacitracin taken as mineral oil mulls and as KBr pellets are shown in Figures 1-471. 4.12 Nuclear Magnetic Resonance Spectrum Chapman and Golden used NMR to exchange in bacitracin A51. This the tritium exchange studies conand co-workers6* extablished the bacitracin A in aqueous solution.

study deuterium work along with ducted by Craig conformation of

Coates and co-workers used 270 MHz NMR to measure pro9qn spin lattice relaxation times for bacitracin A .

d rl

4J a,

PC

a,

. %
-ti

4J

Id k

a
u-4

Id

0 k

4J

a,

zo

a a
a k a
u-4
H

33NVBMOSBV 0

0 7
12

k c1

al

rn

a a
w
k rd k

al

33NVBIOSBV

13

c,
rl rl

a,

PI

al

.ti

m z I c u

.ti

c,

rd k

rd

u
. d
N

5
c,
k

a,

a a
k rd k
a,

14

WAVELENGTH (MICRONS)

2.5

10

12

15

20

30 4050

Figure 4.

Infrared Spectrum of Zinc Bacitracin-Mineral Oil Mull

16

GLENN A. BREWER

Reynolds and co-workers used C 1 3 magnetic resonance spectroscopy to establish the tautomeric equilibrium of the histidine ring in bacitracin A73. The NMR spectrum of bacitracin in D20 is shown in Figure 5 7 4 .
4.13

Ultraviolet Absorlstion Slsectrum

The ultraviolet absorption spectrum of bacitracin was reported by Hayden, gt The ultraviolet spectrum of bacitracin was determined in water, methanol, dilute acid and dilute alkali75. In all solvents, a small peak with an E ( 1 $ , 1 cm) of about 2 0 was exhibited at about 2 5 0 nm. There was no significant shift in wavelength or decrease in absorbance on standing in dilute acid or alkali for periods up to 2 4 hours at room temperature.
4.14

Fluorescence Spectrum

Bacitracin ex ibits a very weak fluorescence in aqueous solution9 6 In both acid and alkaline solutions the excitation wavelength is at about 2 9 2 nm and the emission occurs at about 3 2 5 nm.

4.15

Acoustic Absorption Spectrum

Slutsky, Madsen and White determined the acoustic bsorption spectrum of bacitracin and other peptides 7 9

4.2

Crystal Properties
4.21

X-Ray Powder Diffraction

Samples of U.S.P. Reference standard of bacitracin and zinc bacitracin were examined by powder x-ray diffraction. Both substances were found to be amorphous as indicated y the absence of any peaks in the x-ray pattern7B

4.22

Hygroscopicity Hayashi and co-workers determined

Figure 5.

N M R Spectrum of Bacitracin in D20

18

GLENN A. BREWER

the hygroscopicity of bacitracin at 63% relative humidity82.

loo%,

93% and

Lannung also reported on the hygroscopicity of b a ~ i t r a c i n ~ ~ . 4.3 Solubility 4.31 Solubility in Pure Solvents

Weiss, Andrew and Wright published data on the solubility of bacitracin and zinc bacitracin in a number of solvents79. Solvent Solubility in mg/ml Bacitracin water acetone 1,4-dioxane ethanol ethylene glycol formamide isopropyl alcohol me thano1 pyridine benzene benzyl alcohol carbon disulfide carbon tetrachloride chloroform cyclohexane ethyl acetate diethyl ether ethylene chloride isoamyl alcohol isooctane methyl ethyl ketone petroleum ether toluene isoamyl acetate >20 0.75 0.70 9.1 220 19.9 1.85 >20 9.15 0.025
>20

Zinc Bacitracin 5.1 1.0 0.49 2.0 7.95 >20 0.16 6.55 4.05 0.065 10.35 0.30 0.12 0.01 0.06 1.3 0.02 1.1 2.6 0.015 0.85 0.025 0.02 0.45

0.30 0.18 0.0 0.075 0.047 0.065 0.025 1.65 0.55 0.20 0.35 0.15 0.09

Gross noted that bacitracin is more sol ble in aqueous solution in the pH range 6.5 to 7.5 j 0

BACITRACIN

19

4.32

Distribution Coefficient

Carpenter and co-workers determined the distribut n coefficient of bacitracin in 2-butanol-O.1N acid -

An .

4.33

Formulation Release

Nesbit and co-workers determined the release of bacitracin from ointment bases84. 4.4 Physical Properties of Solutions 4.41 Metal Bindincr

Selzer noted that while mostantibiotics contain less than 30 p.p.m. of heavy metals, bacitracin, by virtue of salt formation, maf;5 contain more than three times this concentration

Garbutt, Morehouse and Harisen established the following order for complex formation of metal salts and bacitracin: Cu>Ni>Co=Zn>Mn86
A l l the metals, except manganese,complexed the baci-

tracin group which titrates between 5.5 and 7.5. Using titration data and the U.V. spectra of the complexes, these workers postulated the involvement of the imidazole group of histidine in the complex. Using NMR and ORD measurements, Cornell and co-workers found that zinc comp xes between the thiazoline and histidine residues

&.

Weinberg measured the stability constants of the binding of copper, nickel, cobalt, zinc and manganese to b a ~ i t r a c i n ~ ~ . Storm and Strominger established the association constants for bacitracins A and F with magnesium88. Bacitracin F has a lower association constant. 4-42 Optical Rotary Dis2ersion

Konigsberg and Craig reported that bacitracin undergoes a change in rotation

20

GLENN A. BREWER

below p H 4 due tggthe epimerization of the terminal isoleucine group

of bacitracin Ago.

Craig reported on O.R.D. studies

Cornell and co-workers used O.R.D. to study the attachment of zinc to bacitracin A48. Craig and co-workers studied the conformation of bacitracin A in aqueous solutiong1.
4.43

Isoelectric Point

Messing patented a method to determine the isoelectric point of proteinsg2. The method was used to establish the isoelectric point of bacitracin as 8.8. This value agrees well with a determination of 8.5 using electrophoresis.
4.44

Dialysis

Craig and co-workers developed the technique of thin-film dialysis to stud conformation of large molecules in solutionjiOfhegacitracin A was one of the model compounds studied. Klein and co-workers used bacitracin as a model compound to establish the properties of four cellulosic membranes93. Craig and co-workers reported additional studies on the dialysis o bacitracin Ag4. Krogerus used dialysis to study the release rate of bacitracin from various ointment bases95. Several reviews have been published on the ph sical and chemical properties of the bacitracinsg: I 9 7 I 98. 5. Production

5.1

Microbiological

Meleney and co-workers described the production of bacitracin on L-glutamic acid

BACITRACIN

21

synthetic and soybean digest media99. Hendlin studied the formation of bacitracin by Bacillus subtilis and evaluated the effect of the addition of various ions, organic acids, amino acids and carbohydrateslOO. Inskeep and co-workers described a new plant built for the production of bacitracinlOl
I

Darker patented the addition of various salts to so bean medium to stimulate production of bacitracin1 ; . 42 Keko, Bennett and Arzberger patented a soybean meal-starch medium for the production of bacitracinlo3. Su and Lu noted the increased production of bacitracin in a peanut oil meal-starch medium when calcium lactate and potassium phosphate were addedlo4. Cohen patented a soybean meal-dextrin medium for the production of bacitracin105. Wilk specified the pH ranges for the growth and antibiotic production phases of a bacitracin-producing culture106 . Freaney and Allen patented a fermentation medium capable of su orting a yield of about 320 units/ml in 24 hours

187.

Ziffer patented a soybean-sucrose medium for bacitracin productionl08. Ripoli published a report on the production of bacitracin in five-liter flaskslog. Siquiroff found that the production of bacitracin was higher in surface culture than in shaken flasks1l0 Zorn patented a fermentation medium containing a water-soluble salt of cobaltlll- He proposed that cobalt complexes of bacitracin were formed which stabilized the bacitracin for use in animal feed supplements.

22

GLENN A. BREWER

Aida and Ito describe the formation of bacitracin A and bacitracin X com lex from bacterial protoplasts (see Section 2.2) 112 ,P13 ,114,115 ,116. Bacitracin X complex has a similar amino acid composition to bacitracins A and B but can be separated by paper chromatography. Cornell and Snoke showed by adding various antibiotics and D-phenylalanine that the biosynthesis of protein and bacitracin by Bacillus licheniformis was accomplished by different metabolic pathwaysll7. The same workers showed that B. licheniformis is inhibited by bacitracin in the early stages of growth118. Brand1 and co-workers studied oxygen transfer in the bacitracin fermentation119. Weinberg and Tonnis showed that although inhibitors of nucleic acid metabolism, messenger RNA synthesis and protein synthesis inhibited the production of bacitracin, the inhibition could be overcome by the addition of a manganese saltl20. Weinberg postulated the function of the bacitracin peptide and other peptide antibiotics for Bacillus species121. Styczynska and co-workers noted that the production of bacitracin by Bacillus subtilis was stimulated when fermentation was conducted as a mixed culture process with a Pseudomonas strain122. Lubinski patented a process using a strain of Bacillus subtilis ada ted to iron and grown on a soy-fish meal medium538 . Feuer and co-workers obtained a patent on an antifoam composition which was useful in the bacitracin fermentation123 . Chigaleichik and co-workers defined a synthetic medium for bacitracin production by Bacillus polymyxa124 . Simlot, Pfaender and Specht noted that changes in the fermentation medium did not alter the quantity of bacitracin synthesized but did change the type produced125.

BACITRACIN

23

Haavik suggested that glucose inhibited the formation of bacitracin primarily by lowering the pH of the fermentation,and not by catabolite repression controlla6 # 127. The same worker found that phosphate only has an adverse effect when it alters the optimum pH of the fermentation128. Haavik postulated that bacitracin may participate in manganese-ion transport throu h the , 90 cell membrane of Bacillus l i c h e n i f ~ r m i s l1~ ~ 132. Kurima and co-workers atented a process for the production of bacitracin 133 Pass and Raczynska-Bojanowska found that high bacitracin-producing strains of Bacillus subtilis lack ornithine 6-transaminaselj4,ljb If ornithine is added to low producing strains, their productivity is increased.

Vitkovic and Sadoff found that bacitracin is a constituent of vegetative cell proteinl36. Makukhina and co-workers described the production of bacitracinl37. Tyc and co-workers have patented a process or the production of bacitracin utilizing a non-sporulating strainl38. Lipavska and associates used acriflavine to prevent infection of Bacillus licheniformis with bacteriophage BLE139. They found that acriflavine did not inhibit the production of bacitracin. Tyc and Kadzikiewicz described their method of producing ultraviolet mutants of Bacillus licheniformis,and evaluating selected isolates for bacitracin production140. Increases of 5 0 to 75% were obtained with four isolates. Haavik studied the metabolism of a high yielding mutant strain of B. licheniformis and found that the addition of L-leucine stimulated bacitracin productionl41. Raczynska-Bojanowska and co-workers patented a process for the simultaneous production of

24

GLENN A . BREWER

bacitracin and p r ~ t e a s e s l ~ ~ . 5.2 Isolation

Anker and co-workers used butanol extraction to isolate the bacitracin from the fermentation brothg9. Gorley used ammonium sulfate salt fractionation to purify crude bacitracinl43. Johnson and Meleney patented a process for the production and recovery of bacitracinl44. There are four common ways in which bacitracin is isolated from fermentation broth. A number of patents and papers have been published on these. 5.21 Precipitation From Broth Various workers have used salts to precipitate bacitracin from the fermentation broth. After the bacitracin salt mixture is filtered off,the pH is adjusted and the antibiotic is extracted into a solvent145,146i147i148,149,150, 151,152,153,154,155,15611571158,1591160. 5.22 Ion Exchange of Bacitracin

A number of patents have been issued for processes which involve the removal of bacitracin from broth b means of an ion exchan e ~~~~~~161i162~163i164~1~5~166,167,168.169~17 172.

5.23

Solvent Extraction of Bacitracin

Solvent extraction has been used less extensive1 than the first two methods based on Apparently, the depatents issued 5gi173,174i175. velopment of this isolation procedure has been carried out pri-marily by one company. 5.24 Metal Salts of Bacitracin

The metal salts of bacitracin are used extensively as animal feed supplements (See Section 1). These insoluble salts can be formed directly in the fermentation broth and isolated as a

BACITRACIN

25

crude concentrate for animal feed use177117811791 180,181,182,183. 5.25 Miscellaneous Methods

Namiki has published a report on a method used to isolate high potency bacitracinl84. Monroe and Ward have patented a process to precipitate bacitracin on diatomaceous earth185. The dried solid can be used as an animal feed supplement. Ores and Rauber have used the non-ionic resin XAD-2 to isolate bacitracinl86. Kindraka and Gallagher have used ultrafiltration to remove bacitracin from fermentation broth187. Malitskii and Mikhel'son have noted that dry bacitracin has tendency to undergo spontaneous combustionl88. Brecka and co-workers inoculated a bacitracin fermentor with Rhodotorula flava after the antibiotic was produced189. The fermentor contained both bacitracin and y-carotene at harvest. The use of the second fermentation was to remove fermentation by-products. Stepanov and Rudenskaya have used immobilized bacitracin to purify proteolytic enzymes 190.
6.

Stability
6.1

Stabilitv of Solid

Bond, Himelick and MacDonald reported that bacitracin was stable at temperatures up to 370C191. Craig and co-workers also indicated that bacitracin is relatively stable as a solid192. Gross studied the stability of bacitracin powder at temperatures up to 60C193. He indicated that after a minor initial drop,the preparations were relatively stable. There was no difference in stability between high and low potency preparations.

26

GLENN A. BREWER

Babin , Coustou and Brisou showed that bacitracin in a mixture with papain enzyme powder maintained its potency for a six month periodl94. Gupta, Vyas and Sekhon showed that 15 Mrads of neutron and y-radiation did not change the activity of bacitracin powderl95. Tsuji and Robertson also showed that 6oCo radiation did not cause potency loss of bacitracin powder196. Ethylene oxide treatment caused 46% reduction in potency, but did not cause the formation of bacitracin F. Dry heat sterilization caused a 35% decrease in potency with a corresponding increase in bacitracin F.
6.2

Stabilitv of Solutions

Anker and co-workers reported that solutions of bacitracin were stable for 8 to 12 months at 50Cg9. Hayashi and co-workers found that a solution of bacitracin in pH 7 phosphate buffer lost 2 5 % of the initial potency after 6 days at room temperature82. Vasilescu and Molss found that solutions of bacitracin were most stable at pH 4.498. Craig and Konigsberg showed that bacitracin B was inactivated more rapidly than bacitracin A35. In both cases,bacitracin F was a major decomposition product. The same workers showed that below pH 4.0 bacitracin undergoes an epimerization of the terminal isoleucine residue89r38. Pirila, Saukkonen and Santaoja separated the degradation products of bacitracin in solutionl97. Herrmann, Woodward and Pulaski postulated that the inactivation of bacitracin on passage through the gastrointestinal tract of rats is due to degradationl98. Pirila, Salo and Pirila found that the complex of bacitracin with sodium dodecyl sulfate was stable in solution, although the complex showed

BACITRACIN

27

diminished skin penetrationlgg. Makinen found that bacitracin inhibits the activity oz0gapain, subtilisin and leucine . aminopeptidase 6.3 Light Stability

Wurtzen found that exposure to sunlight and temperature variations between 2OoC and 35OC caused 20-35% l o s s of activity in 6 days201. 6.4 Formulation Stability

Bond and co-workers reported that anhydrous grease based ointments were stable while water miscible ointments were notlgl. A numb r f other investigators agree with these findings82,902. Hegarty and Verwey atented formulations for bacitracin that were stable5 0 3 . Plaxco and Husa established the stability of bacitracin in a number of ointment bases204. Other authors evaluated various other formulation
excipients205,206,207,208,2091239~

Gordon patented aerosol compositions of bacitracin2I0. Snyder patented a stable formulation of bacitracin in animal feed211. The bacitracin was coated with oil and the droplets absorbed on diatomaceous earth to form a free-flowing powder. Saito, Kawano and Ichijima patented a bacitracin feed additive stabilized with 2-0x0-4methyl-6-ureidohexahydropyrimidine212. 6.5 Stability of Metal Salts

Gross, Johnson and Lafferty showed that zinc bacitracin was more stable than bacitracin in troches, ointments and tablets2l3. Other additives have confirmed the increased stabilit Of bacitracin with zinc and other metals 215,216, 217,91.

Crisler and Weinberg indicated that

28

GLENN A. BREWER

while zinc salt of bacitracin was not more stable than bacitracin to autoclaving, the salt enhances the antibiotic activity of bacitracin ll-fold2l8 219.
f

Tanaka, Seki and Ito patented the use of mineral salts of bacitracin as animal feed supplements220, These salts were reported to have enhanced stability. Other patents have been issued on the use of metal salts221t222. 7. Analytical Methods 7.1 Identitv Tests 7.11 Physical Methods

Landgren differentiated antibiotics by measuring the refractive index of the crystals using liquids of known refractive index223. Zief and co-workers prepared the tetraphenylboron derivatives of several antibiotics 224. The melting points of these derivatives were used to identify them. Matta and co-workers also utilized the tetraphenyl borate derivative for antibiotic i dentifi~at i o n 2 ~ ~ . 7.12 Colorimetric Tests

Fischbach and Levine utilized the ninhydrin reaction as an identity test226. Hayashi and co-workers reported that bacitracin gave positive biuret, Adamkewitz, Millon and Molisch reactions82. Wornick and Kuhn indicated that bacitracin produces a violet color with ninhydrin spray on paper227. 7.13 Chromatographic Methods

Almost any chromatographic system for bacitracin could be used as an identity test for the antibiotic. In this section we are listing those systems specifically indicated as identity tests, other chromatographic methods can be found in Section 7.4.

c
m
N N N O

m
N N

m - 3

c
.d

c,

c,

a,

a
c, r d c,
h

a,

a,
0

N
v

..
rl

2
c
h

a,
0

z
c,
I

h
51
h

-3

c,
rl

z
N -3
v

$
0

a
z
-J

m
PI

2
3
v

.. ..
rd
0 k

I rl

c, z w ow
N

Lrl
I +

c
k
I
rl

a
CI
a, I n

.. m .. ..

Lrl

z ! UJ

c,
k
0

a
m
?

4
B

4 B

4 B

30

GLENN A . BREWER

7.14 ElectroDhoresis Methods Peptides are commonly separated by electrophoretic methods. A few methods specifically designated as identity tests are listed here. Other electrophoretic systems may be found in section 7.42. Lightbrown and DeRossi utilized Using this basic methagar gel e l e c t r o p h ~ r e s i s ~ ~ ~ . od, Bozzi and Valdebouze developed a bioautogra hic system for 14 antibiotics including bacitracin 276 . Grynne developed a paper electrophoresis-bioautographic system for a number of antibiotics including bacitracin237. 7.2 Microbiolosical Assavs 7.21 Tube Dilution Assay

Patrick, Craig and Bachman correlated the results of serial dilution assa s with those obtained by agar diffusion assays248 . 7.22 Turbidimetric Assay

Although the agar diffusion assay technique is the primary method for bacitracin, a number of turbidimetric methods have been reported. Method Notes Staphylococcus aureus Na resazurin indicator Streptococcus faecalis Autoanalyzer nethod Na resazurin indicator Escherichia Coli Zinc bacitracin in feeds Authors Darker, g

g.

Reference 241 242 243 244 245 246 247

De Felip, et al. Pain, Bose, Dutta Platt, Gentile and George Ruffo and Socci Rappe , Mauquoy and Bauer Ragheb, Black and Graham

BACITRACIN

31

Kirschbaum, Arret and Harrison published statistical procedures for determining the dose-response curve or turbidimetric assays248. 7.23 Agar Diffusion Assays

The majority of microbiological assays or bacitracin involve the use of agar diffusion methods. Some highlights of these methods are presented in tabular form. Method Notes Staphlococcus aureus, prediffusion Development of diagnostic discs Effect of medium composition Corynebacterium xerosis Assay of bacitracin in Galenical products Micrococcus flavus Organisms resistant to other antibiotics Assay of emulsion formulation Sarcina lutea Diagnostic discs Disc plate assay Bacitracin in feed Disc plate assay Sensitivity of method Authors Darker,- et al. Patrick, Craig, Bachman Neter , Murdock , Kunz Porath Trolle-Lassen Pinzelik, Nisonger Murrcly Friedman, Kirschbaum Varma, Hall, Rising Vuilleumier, Anker Kirschbaum, Kramer, Arret Rossi Craig Bauer Pitton Reference 241 240 249 250 251 252 253 254 255 256 257 258 259 260

32

GLENN A. BREWER

Method Notes Antibiotic mixtures Bacitracin in feed Bacitracin in tissue Diffusion characteristics Bacitracin in milk Bacillus stearothermophilus Bacitracin in feedsgel filtration Sensitivity tablets Bacitracin in milk, tissue Bacitracin in fermentation broth Use of tetrazolium dyes Bacitracin in tissue Bacitracin in tissue Bacitracin in feedsmolecular sieve Interference Bacitracin in animal feeds Frozen inoculum Bacitracin in antibiotic mixtures

Authors et YonezawaI - al Craig

Reference

_.

261 262 263 264 265 266 267 268 269 128 270 271

Freres, Valdebouze Cluzel, Cluzel, Michel, Sirot Read, Bradshaw, Swartzentruber Kabay Skodova, et al. Casals, Gylling, Pedersen Ryb inska Haavik Picmanova,- et al. Smither

Kr st a-Skonieczna, 2 7 2 Rygrnzta Skodova, Skarka 273 Liskova , Kohoutkova Pacini , Meneghini Hadfield DeCarneri
274 275 276 277

BACITRACIN

33

Method Notes Vertical agar diffusion

7.24

Authors Lameris,et Feed Assays

Reference

s.

278

Several of the microbiological assays already mentioned can be used to assay bacitracin or zinc bacitracin in feeds. The following papers detail extraction methods which can be used to extract bacitracin from complex animal feeds. Author Randall Randall and Burton Wright and Burton Craig Grynne and Hoff Grynne Grynne, Hoff, Silsand and Vaaje Fassbender and Katz
7.25

Reference
279 280 281 282 283 284

285
286

Miscellaneous Assavs

The microbiological assay of bacitracin in antibiotic mixtures, soils and body fluids has been discussed in some of the papers specified The following papers in sections 7 . 2 1 through 7 . 2 4 . are of special interest in the assay of these samples: Assay Notes Bacitracin and neomycin Bacitracin and neomycin Bacitracin in soil Authors Lingnau and Machek Balliu and Boteanu Soulides Reference
287 288 289

34

GLENN A . BREWER

Assay Notes Blood level assay Stool assay

Authors Eagle, et al. Wilson, Ing, Metcalfe-Gibson and Wrong Kline and Rathmacher Kirschbaum, Arret and Kramer Dennin Hinks, DaneoMoore and Braverman Morris and Jennings

Reference 290 291

Animal tissue Bacitracin standard Review of methods Temperature of incubation Electrical polarization 7.3

292 293 294 295

296

Chemical Methods

Although microbiological methods appear to be preferred f o r bacitracin,several types of chemical and biochemical assays have been proposed for the antibiotic.
7.31

Gravimetric and Colorimetric

Maturana, Dannier and Brieva have proposed a gravimetric phosphotungstic acid method for b a ~ i t r a c i n ~ ~ ~ . Doulakas has published a colorimetric assay involving the reaction with phloroglucinol after the oxidation of the antibiotic with hyp~bromite~~~. 7.32 Electrochemical Assays

Caplis, Ragheb and Schall have proposed an alternating current polarographic assay for bacitracin2 9. Skarka and Sestakova have reported a oscillopolarographic method as well as a

BACITRACIN

35

sensitive colorimetric method300. Jacobsen, Pederstad and Oeystese have utilized differential pulse polarography to assay bacitracin and zinc bacitracin301. The degradation product,bacitracin F, is reduced at a less negative potential. 7.33 Determination of Zinc in Zinc Bacitracin

Charles and Weiss utilized an EDTA titration to measure the concentration of zinc in zinc bacitracin302. More recently, atomic absor tion s ectroscopy has been utilized for this assay503 , 394. 7.34 Biochemical Assays

As is the case with other antibiotics, investigators have established that certain enzyme systems are inhibited by the presence of bacitracin. In general, these methods have not been shown to be as useful as microbiological assays but we have included a few references which may be of general interest. Enzyme System D-Amino acid oxidase Arginine diaminase Proteolytic enzymes Pancreatic lipase Human spermatozoa 7.4 Author Hayashi Mikolajcik Coppi and Bonardi Coppi and Bonardi Schirren Reference 305 306 307 308 309

Chromatographic Methods

7.41

Countercurrent Distribution

At the time when bacitracin was discovered, countercurrent distribution was probably the most popular separation technique. Although it has been supplanted by various types of

36

GLENN A. BREWER

chromatography on solid supports it is still useful for the separation of large molecules such as the bacitracins. System Notes Separation of bacitracin in one major and two minor fractions Separation into more than one component Amy1 alcohol-butanolpH 7.0 buffer Isolated pure A, B and C Separation into 1 major and 4 minor components Separation into 10 components CHC13-methanol-water (2:2:l) Authors Barry, Gregory and Craig Craig Newton and Abraham Newton and co-workers Craig and co-workers Newton and Abraham Konigsberg and Craig Reference 12

310 6 311 312 13 313 314 315

PJ~~OH-H~O-C~H~-CHC~~ Ramachandrar? (23:7:15: 15) Separation of commercial bacitracin into 10 components Hausmann, Weisiger and Craig Craig a i rd Konigsberg Craig, Hausmann and Weisiger Craig, King and Konigsberg Konigsberg, Hill and Craig

A number of systems utilized

Countercurrent dist. of DNP derivative Separation of bacitracin A into two isomers BuOH-C H N-AcOH-H~O (20:5 :$ :30) separation of degradation products

35 316 317 38

BACITRACIN

31

System Notes 30% Ethyl acetate-70% 1 butanol-pH 5.43 buffer 7.42

Authors Craig and co-workers

Reference 91

Electrophoresis

Electrophoresis on a variety of substances has been utilized frequently in the separation of large molecular weight molecules posessing an ionic charge. Method Notes Starch column Cellulose column Paper electrophoresis Paper electrophoresis Paper electrophoresis Paper electrophoresis Paper electrophoresis Agar gel Paper electrophoresis Authors Flodin and Porath Porath Proenca da Cunha and Baptista Paris and Theallet Apreotesei and Teodosiu Proenca Ca Cunha and Gomes Pirila, Saukkonen and Santaoja Swank and Munkres Maeda, Y a g i , Naganawa, Kondo and Umezawa Swank and Munkres Dubost and Pascal Coombe

Re erence
318 319 320 321 322 323 197 325 324

Polyacrylamide gel Agar gel Polyacrylamide gel

325 326 327

38

GLENN A. BREWER

Method Notes Low voltage Electrophoresis of feed and foods Gelatin gel Identification test Isoelectric focusing in gel 7.43

Authors Langner and co-workers Langner Bozzi and Valdebouze Grynne Froeyshov

Reference 328 329 236 237 330

Column Chromatography

Column chromatography utilizing a variety of support materials has been used to perform crude separations of bacitracin fractions. Method Notes Charcoal-celite column Charcoal-celite (1: 3) 0.1u acetic acid Carboxymethylcellulose Carboxymethylcellulose Carboxymethylcellulose 7.44 Authors Porath Porath Konigsberg and Craig Konigsberg, Hill and Craig Storm and Strominger Reference

250
319 89 38 331

Gel Filtration

Gel filtration has been extensively used to separate macro molecules on the basis of molecular size. Bacitracin has been utilized as a standard in several systems since it is well characterized.

BACITRACIN

39

Method Notes Sephadex G-25 (Propanol-acetic acidwater) Sephadex G-10 (acetic acid-NaC1) Sephadex G-100 Sephadex LH-20

Authors P.R. Carnegie

Reference 332

Eaker and Porath Reickert and co-workers Gregerman, Weaver and Kowatch Bryce and Crichton Randau, Bayer and Schnell Catsimpoolas and Kenny Stewart Skarka, Skodova and Skoda

333 334 335

Agarose Polyethyleneglycol dimethacrylate gel Sephadex G-25, G - 5 0 Polyacrylamide gel Bio-Gel P-2 (tissues) 7.45

336 337 338 339 340

Paper Chromatography

Paper chromatography is frequently used for the separation of antibiotics because the components can conveniently be located by bioautography

Method Notes Bioautography of various antibiotics Ninhydrin pyridineacetic acid Butanol-acetic acidwater (50:25:25)

Authors Snell , Ij ichi and Lewis Castel, Mus and Storck daCunha and Baptista

Reference 228 341 342

40

GLENN A . BREWER

Method Notes "Salting out" chromatography Three solvent systems Dyes as detection reagents Hydrophobic system Det. of Bacitracin in fodder Separation of 42 antibiotics 7.46

Authors daCunha and Baptista Paris and Theallet Singh Ritschel and Lercher Louis Schmitt and Mathis

Reference 343 321 344 345 346 347

Thin Layer Chromatography

Thin layer chromatography is also widely used for the chromatography of antibiotics because of its rapidity. Method Notes Silica gel and kieselgel Silica gel ethanol, NH40H-H20 (8:1:1) Silica gel ethanolwater (4:l) Butanol-acetic acidwater (3:l:l) Separates Bacitracins A and F Separates various antibiotics Authors Paris and Theallet Umezawa and coworkers Akita and Ikekawa Umezawa and coworkers Nussbaumer Pitton McGilveray and Strickland CuSO4 color reaction Guven and Ozsari Reference
321

348 349 350 351 352

353

229

BACITRACIN

41

Method Notes Identification of sensitivity discs Bioautography

Authors Wayland and Weiss Aszalos, Davis and Frost Fooks, McGilveray and Strickland Reimers

Reference 230 354 355 356 357 358 359 232 231 328 329 233 360 234

5 Solvent systems Butanol-H20-pyridineAcOH-ethanol Dowex-50 plates Resin coated plates Detection of antibiotics in meat Bioautography Cellulose plates Determination in feed Determination in tissue Determination i n milk 7.47

Stretton, Carr, Watson-Walker Carr, Stretton and Watson-Walker Pauncz Pauncz Langner and Tuefel Langner and Teufel Langner and Tuefel Freres and Va 1deb0uz e Baldini and co-workers Bossuyt and co-workers

High Pressure Liquid Chromatography

High pressure liquid chromatography is one of the newest chromatographic methods. The technique combines a high resolution column with a detector, so the method is generally not only selective but precise. Spechter has utilized a silica

42

G L E N N A. BREWER

column coated with Carbowax 2OM361. Tsuji, Robertson and Bach used Bondapak C18/Corasil with gradient elution to separate the components of b a ~ i t r a c i n ~ ~ ~ . Tsuji and Robertson improved on the previous method by using a micro-Bondapak c18 columnl96. Dr. Yeh adopted the general method of Tsuji and Robertson196 for the examination of some samples of commercial bacitracin obtained by our laboratory407. (Samples of bacitracin and zinc bacitracin were generously supplied by International Minerals and Chemicals Corporation and by A/S Dumex Ltd. In addition, the U.S.P. Standard of zinc bacitracin was chromatographed). Although the column and solvent system employed by Dr. Yeh were the same as those reported by Tsuji and Robertson, he was unable to reproduce the exact exponential gradient they utilized because of equipment limitations. As a result, the peaks were not as sharp and he was unable to obtain separation of some of the components. The major component in all the samples appeared to be bacitracin A. A component eluting just before bacitracin A was probably bacitracin B1 or B2. The other components appeared to be present in much smaller concentrations. In all samples, eight to ten components could be seen. Dr. Yeh experienced some base line drift because of the change in gradient composition. We were gratified with the separation that Yeh was able to achieve with the limited amount of time he was able to devote to the project. 8. Mode of Action

Gale found that,like other antibiotics bacitracin interfered with protein biosynthesis 365. Gale and Folkes studied the inhibition of incorporation of amino acids into proteins using a cell homogenate364. Schechter, Momose and Rudney found that

BACITRACIN

43

bacitracin interfered with biosynthetic athways which involved polyprenylpyrophosphates 365. Storm and Strominger found that bacitracin interacted with C55 isoprenylpyrophosphate in the cell membrane366. This altered the permeability of the bacterial cell.
9.

Derivatives of Bacitracin

A number of bacitracin derivatives have been produced. Some of these have been suggested for use in animal feeds. Siminoff, Price and Bywater suggested that the methylene disalicylic acid complex of bacitracin was useful as a feed additive for swine and poultry 367. Radomski, Hagan, Nelson and Welch established the toxicity and safety of this derivative368 This complex was approved as a feed a d d i t i ~ e ~ 6 ~ . Man anese bacitracin is an approved feed additive3 7 3 . A Japanese patent was issued for the sodium methanesulfate derivative of b a ~ i t r a c i n ~ ~ ~ .
A U.S. patent was issued to Lewis, Ninger and Pattison for the synthesis of the sodium methanesulfonate derivative of bacitracin which they suggested was suitable or parenteral administration 37 2 Baldwin patented sodium, potassium, calcium, zinc and manganese salts of bacitracin methanesulfonate373.

SPOFA United Pharmaceutical Works reacted bacitracin with a number of aldehydes and then isolated the corresponding zinc salts374. Vondracek, Toscaniova and Hoffman have patented a furfural derivative of b a ~ i t r a c i n ~ ~ ~ . Kalina, Ulbert and Masita patented the diisobutylnaphthalenesulfonate derivative of bacitracin 376. Atassi and Rosenthal reduced bacitracin with Shipchandler was issued a patent on dib~rane~~~. derivatives of bacitracin reduced with sodium boroh~dride~~*.

44

GLENN A. BREWER

Mancino, Tigelaar and Ovary compared the antigenic properties of the three monodinitrophenyl derivatives of bacitracin with that of the tri-dinitrophenyl derivative379.
A Japanese patent was issued in which bacitracin was reacted with polyamine ion exchange resins by means of an aldehyde380. The resulting product was insoluble. In the same way, a dimer of bacitracin was produced by reacting the antibiotic with g l y ~ x a l ~ ~ l .
10.

Reviews

Two reviews have been published on the assay of bacitracinlg3I 382.

A number of reviews have been ublished on bacitracin383 I384 ,385,386 I 387 I 388 g8 I 389 390 I 391.
Many more reviews have included bacitracin alon with other antibiotics 392 I 393 I 394 I 395 I 396 I 397,~98,399,400,401,402,403, 404,405,406,

BACITRACIN

45

References

1.
2. 3. 4. 5. 6. 7. 8. 9.
10. 11.

12. 13. 14. 15. 16. 17.

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BACITRACIN

63

(C.A. 87 1 7 8 3 1 5 r ( 1 9 7 7 ) 1 . 296. M 0 r r i s F V . J . and J e n n i n g s , B . R . ; Biochim. B i o h y s . A c t a . 497 253-9 ( 1 9 7 7 ) . 7 C . A . 86 1 6 5 8 1 9 n ( 1 9 7 7, ).) . 297. M a t u r a G , M.H.; D a n n i e r , C.A. a n d Brieva, A . J . ; R e v . R e a l Acad. C i e n c . E x a c t . , F i s . N a t . Madrid 56 365-82 ( 1 9 6 2 ) . (C.A. 5 7 1 3 8 8 8 b ( 1 9 6 2 )) 298. D o u l a k z , J . ; J . P h a r m . S c i . - 307-10 ( 1 9 7 5 ) . 64 (C.A. 8 2 1 2 9 3 1 7 f ( 1 9 7 5 ) ) . 2 9 9 . C a p 1 i s T M . E . ; R a g h e b , H.S. and S c h a l l , E . D . ; J . Pharm. S c i . 54 694-8 ( 1 9 6 5 ) . (C.A. 6 3 1 6 5 6 e n 9 6 5 ) ) . 300. S k a r k a T P . and S e s t a k o v a , I . ; B i o l . Chem. Vyz. Z v i r a t . 1 2 167-74 ( 1 9 7 6 ) . (C.A. 8 5 1 7 5 4 8 2 r (1976)) 301. Jacobson, E . ; P e d e r s t a d , J . H . a n d O e y s t e s e , B . ; A n a l . Chim. A c t a 9 1 1 2 1 - 8 ( 1 9 7 7 ) . (C.A. 87 90769f ( 1 9 7 7 1 ) . 302. C h a r l e s , J . L . a n d W e i s s , P . J . ; A n t i b i o t i c s and C h e m o t h e r a p y 8 496-9 ( 1 9 5 8 ) . ( C . A . 5 3 1 2 5 8 3 h ( 1 9 5 9 ). ) . . 303. S a l v e s = , B . a n d 'Aaro, B. , Medd. N o r . F a r m . S e l s k . 34 9 - 1 3 ( 1 9 7 2 ) . (C.A. - 1 1 2 7 1 9 ~( 1 9 7 4 ) 1 . 80 304. Anon.; F e d . R e g i s t . 4 0 ' 1 5 0 8 8 A p r i l 4 , 1 9 7 5 . (C.A. 8 3 33121g ( 1 9 7 5 ) ) . 3 0 5 . H a y a s h c H . ; S e i k a g a k u - 45-52 ( 1 9 6 0 ) . 32 ( C . A . 60 4 4 0 0 d ( 1 9 6 4 ) ) . 3 0 6 . M i k o l a j c i k , E . M . ; J . D a i r y S c i . 48 1 4 4 5 - 9 (1965). (C.A. - 5437g ( 1 9 6 6 ) ) . 64 3 0 7 . C o p p i , G . and B o n a r d i , G . ; - 185-7 ( 1 9 6 5 ) . 4 (C.A. - 18232d ( 1 9 6 6 ) ) . 64 308. Coppi, G . a n d B o n a r d i , G . ; B i o c h i m . B i o l . S p e r . - 191-3 ( 1 9 6 5 ) . 4 ( C . A . 64 1 8 2 3 2 f ( 1 9 6 6 ) ) 3 0 9 . S c h i r r e n , C . ; A r c h . G y n a e k o l . 1 9 8 253-60 ( 1 9 6 3 ) . ( C . A . - 1204512 ( 1 9 6 3 ) ) . 59 3 1 0 . C r a i g , L . C . ; H a r v e y L e c t u r e s 45 64-86 ( 1 9 4 9 ) ( C . A . 46 1 1 2 9 5 a ( 1 9 5 2 ) ) . 311. N e w t o n , G.G.F.; Abraham, E . P . , F l o r e y , H . W . ; S m i t h , N . and ROSS, J . ; B r i t . J . P h a r m a c o l . 417-29 ( 1 9 5 1 ) . ( C . A . 46 2 2 0 i ( 1 9 5 2 ) ) . 312. C r a i g , x . C . ; W e i s i g e r , J . R . ; Hausmann, W. a n d H a r f e n i s t , E . J . ; J . B i o l . Chem. - 259-66 199 (1952).
I ,

.-

64

GLENN A. BREWER

(C.A. 47 1337e (1953)). 313. Konigssrg, W. and Craig, L.C.; J. Org. Chem. - 934 (1962). 27 (C.A. 57 1388033 (1962)). 2 314. Ramachzdran, L.K.; Biochemistry - 1138-42 (1963). (C.A. 59 10363h (1963)). 315. Hausma=, W,; Weisiger, J.R. and Craig, L.C.; J. Amer. Chem. SOC. 77 723-31 (1955). 49 15. 9) (C.A. - 5582i (5) 316. Craig, L.C., Hausmann, W. and Weisiger, J.R.; J. Amer. Chem. SOC. 76 2839-40 (1954). 49 15. 9) (C.A. - 5583a (5) 317. Craig, L.C.; King, T.P. and Konigsberg, W.H.; Ann. N.Y. Acad. Sci. 88 571-85 (1960). (C.A. 55 23637h (6) 11. 9) 318. FlodinTP. and Porath, J.; Biochim. et Biophys. Acta 13 175-82 (1954). (C.A. 48 6498b (1954)). 319. PorathTJ.; Acta Chem. Scand. - 1813-26 (1954). 8 (C.A. 49 10579g (1955)). 320. Proencada Cunha, A. and Baptista, M.L.D.M.; Bol. escola. farm, Univ. Coimbra 19-20 231 (1959-60). (C.A. - 19136i (1961)). 55 321. Paris, R.R. and Theallet, J.P.; Ann. Pharm. Franc. 20 436-42 (1962). (C.A. 5716746i (1962)). 322. ApreosGsei, C. and Teodosiu, M.; Farmacia 10 321-30 (1962). (C.A. 58 437513 (1963). 323. Proencada Cunha, O.R. and Gomes, M.E.B.; Bol. Escola Farm. Univ. Coimbra, Ed. Cient. - 12922 36 (1962). 61 (C.A. - 1711d (1964)). 324. Maeda, K.; Yagi, A.; Naganawa, H.; Kondo, S. and Umezawa, H.; J. Antibiot. Tokyo - 635-636 22 (1969). (Anal. Abst. 20 500 (1971)) 325. Swank, R.T. and Munkres, K.D.; Anal. Biochem. - 462-77 (1971). 39 (C.A. 74 72609f (1971)). 326. DubostTM. and Pascal, C.; Ann. Fals. Expert. Chim. 63 189-202 (1970). (C.A. 75 1 3 9 4 5 6 ~(1971)1 . .. 327. Coombe7R.G.; Aust. J. Pharm. Sci. - 6-8 (1972). 1 (C.A. 77 168680k (1972)). 328. LangnerH.J.; Teufel, v . - Sie ert M. and Frommhdld, M: FleischwirtschaPt 55 243-6 (1973).

BACITRACIN

65

(C.A. 78 134569m ( 1 9 7 3 ) ) . 329. L a n g n e 7 H . J . a n d T e u f e l , U . ; Chem., M i k r o b i o l . , T e c h n o l . Lebensm. 2 . 71-8 ( 1 9 7 3 ) . ( C . A . 80 35901y ( 1 9 7 4 ) ) . 330. F r o e y s h o v , 0 . ; A n a l . Chim. A c t a - 137-9 ( 1 9 7 8 ) 98 (C.A. - 3 0 8 3 8 j ( 1 9 7 8 ) ) . 89 331. S t o r m , D . R . a n d S t r o m i n g e r , J . L . ; J . B i o l . Chem. 248 3940-45 ( 1 9 7 3 ) . ( C . A . F 1 2 2 3 1 3 f ( 1 9 7 3 ).) . . 332. C a r n e g z , P.R.; Biochem. J . - 9 p ( 1 9 6 5 ) . 95 (C.A. - 3196c ( 1 9 6 5 ) ) . 63 333. E a k e r , D. a n d P o r a t h , J . ; S e p a r . S c i . - 518 2 (1967). ( C . A . 68 7 2 6 7 0 v ( 1 9 6 8 ) ) . 334. R e i c h e z , L . E . ; Rasco, M . A . ; Ward, D . N . ; N i s w e n d e r , G . D . a n d Midgley, A . R . ; J . B i o l . Chem. 244 5110-5117 ( 1 9 6 9 ) . (C.A. 7 1 9 8 6 1 2 9 ( 1 9 6 9 ) ) . 335. G r e g e r m a n , R . I . ; Weaver, T. a n d Kowatch, M.A.; J . C h r o m a t o g r . 47 369-375 ( 1 9 7 0 ) . (C.A. 73 821a ( n 7 0 ) ) . 336. B r y c e , C.F.A. a n d C r i c h t o n , R . R . ; J . C h r o m a t o g r . 6 3 267-280 ( 1 9 7 1 ) . [C.A. 75 5869 1r(19%1) 1 . 337. K a n d a u T D . ; B a y e r , H . a n d S c n e l l , W . ; J . C h r o m a t o g r . 57 77-82 ( 1 9 7 1 ) . ( C . A . 7 4 150928a-(1971) ) 338. C a t s i m p o o l a s , N . a n d Kenney, J . ; J . C h r o m a t o g r . - 77-83 ( 1 9 7 2 ) . 64 ( C . A . 76 69619v ( 1 9 7 2 ) ) . 339. S t e w a r c J . A . ; Biochem. B i o p h y s . R e s . Commun. - 1405-1410 ( 1 9 7 2 ) . 46 ( C . A . 76 1 2 3 0 9 3 j ( 1 9 7 2 ) ) . 340. S k a r k a F P . ; S k o d o v a , H . a n d S k o d a , J . ; A g r i c . B i o l . Chem. 4 1 1303-4 ( 1 9 7 7 ) . ( C . A . 87 1 2 6 8 5 8 a ( 1 9 7 7 ) 1 . 341. C a s t e l T P . ; MUS, R . a n d S t o r c k , J . ; Ann. p h a r m . f r a n c . 1 7 63-71 (19591. (c.A. 5 3 1 7 4 2 8 i (1959j 342. d a C u n h z A.P.M.A. a n d B a p t i s t a , M.L.D.M. B o l . e s c o l a a r m . , U n i v . C o i m b r a 19-20 225-30 ( 1 9 5 9 60). (C.A. 55 19136e ( 1 9 6 1 ) ) . 343. d a C u n h z A.P. a n d B a p t i s t a , M . L . D . M . ; escola f a r m . U n i v . C o i m b r a 1 9 / 2 0 217-24 ( 1 9 5 9 / 60). (C:A. - 21481h ( 1 9 6 1 ) ) 55 344. S i n g h , C . ; C e s k . Farm. 1 2 294-7 ( 1 9 6 3 ) . ( C . A . - 290633 ( 1 9 6 4 ) ) .61

G.

66

GLENN A. BREWER

345. Ritschel, W.A. and Lercher, H.; Pharm. Ztg., Ver. Apotheker - Ztg. 106 120-2 (1961). (C.A. - 397h (1965)). 62 346. Louis, R.; Mitt. Gebiete Lebensm. Hyg. 56 244-9 (1965). (C.A. 64 17357d (1966)). 347. Schmitc J.P. and Mathis, C.; Ann. pharm. fr. - 205-210 (1970). 28 (C.A. 73 113005f (1970)). 348. Ikekawr T.; Iwami, F.; Akita, E. and Umezawa, 16 J.; J. Antibiot. Ser A - 56 1963). (C.A. - 1539c (1964)). 60 12 349. Akita, E. and Ikekawa; T. ; J. Chromatogr. 250 (1963). (C.A. 60 368d (1964)). 350. I k e k a w z T.; Iwami, F.; Akita, E. and Umezawa, H.; Penishirin Sono Ta Koseibusshitsu Ser A - 56-7 (1963). 16 (C.A. 60 1539c (1964)). 40 351. Nussbaumer, P.A.; Pharm. Acta. Helv. - 210-18 (1965). (C.A. 63 437g (1965)). 352. Pitton7J.S.; Antibiot.; Advan. Res. Prod. Clin. Use, Proc. Congr., Prague, 1964 490-495 (1965). (C.A. 66 40743p (1967)). 353. McGilvZay, I.J. and Strickland, R.D.; J. Pharm. Sci. 56 77-79 (1967). (C.A. 66 4 0 7 4 6 ~ 7 1 9 6 7 ) . ) 354. Aszalo c A.; Davis, S. and Frost, D.; J. Chromtogr. 37 487-498 (1968). 70 (C.A. - 14444e(1969) ) 355. Fooks, J.R.; McGilveray, I.J. and Strickland, R.D.; J. Pharm. Sci. 57 314-317 (1968). (C.A. 68 62732c (8 1) 9) 6. 356. Reimerc F.; Arch. Pharm. Chemi. - 1064-68 75 (1968). (C.A. 70 60851h (1969)). 357. Stretton, R.J.; Carr. J.P. and Watson-Walker, J.; J. Chromatosr. 45 155-8 (1969). 72 (C.A. - 35815h (19%)). 358. Carr, J.P.; Stretton, R.J. and Watson-Walker, J.; Loughbrough Univ. Technol., Dep. Chem., 10 Sum. Final Year Stud. Pro]. Theses - 17-18 (1969). (C.A. 7 3 28954v (1970)). 25 359. Pauncz7J.K.; J. Antibiot. - 677-78 (1972). (C.A. 78 54975a (1973). 360. Baldin17 P.; Frati, G.; Pezzani, G. and

BACITRACIN

61

361. 362. 363. 364. 365. 366. 367.

368.

369. 370. 371. 372. 373. 374. 375. 376. 377.

Ambanelli, G.; Ind. Conserve - 135-9 (1973). 48 (C.A. 80 131710f (1974)). 58 Shechter, I.; Anal. Biochem. - 30-38 (1974). 80 (C.A. - 1 0 5 4 8 9 ~(1974)). Tsuji, K.; Robertson, J.H. and Bach, J.A.; J. Chromatogr. 99 597-608 (1974). (C.A. 82 64576t71975)). .. Gale , F F . ; 2nd Consr. intern. biochim., Chim. biol. VI 5-20 (1952). (C.A. - 4955c (1953)). 47 .. Gale, E.F. and Folkes, J.P., Biochem. J. 59 661-75 (1955). (C.A. 49 10416d (1955). Schechter, N.; Momose, K. and Rudney, H.; 48 Biochem. Biophys. Res. Commun. - 833-9 (1972). 77 (C.A. - 147922a (1972)). Storm, D.R. and Strominger, J.L., J. Biol. Chem. 249 1823-7 (1974). (C.A. E 3 1 2 a (1974)). Siminoff, P.; Price, R.W. and Bywater, W.G.; Antibiotics Ann. 1953-54, Proc. Symposium Antibiotics 395-400 (1953). (C.A. 48 7207e (1954)). Radomski, J.L.; Hagan, E.C.; Nelson, A.A. and 4 Welch, H.; Antibiotics & Chemotherapy - 304-7 (1954). 48 (C.A. - 13075e (1954). Anon., Fed. Regist. 34 200469 (1969). 71 (C.A. - 69415x (1969)). Anon. Fed. Regist. 27 8072-3 (1962). (C.A. 57 12963g (1962)). KoyamaTY.; Kurosawa, A. and Sato, H.; Japanese Patent 1296, April 30, 1962. (C.A. 58 4648g (1963)). Lewis, A.D.; Ninger, F.C. and Pattison, I.; U.S. Patent 3,205,137, Sept. 7, 1965. (C.A. 63 129831.1 (1965)). Baldwin, R.S.; French Patent 1,463,679, Dec. 23, 1966. (C.A. 67 91116g (1967)). Anon.; Neth. Appl. 6,512,824, April 7, 1966. (C.A. 65 10434c (1966)). Vondracek, M.; Toscaniova, E. and Hoffman, J.; Czech. Patent 123,980, August 15, 1967. (C.A. 68 103878a (1968)). KalinaTV. ; Ulbert , S. and Masita, A. ; Czech. Patent 118,498, May 15, 1966. (C.A. 66 74945w (1967)). Atassi7M.Z. and Rosenthal, A.F.; Biochem. J.
~~

68

GLENN A . BREWER

378. 379. 380.

381.

382.

co. 47 (C.A. - 39309 ( 1 9 5 3 ) ) . 385. Anon.; Arch. Pharm. Chemi.; 61 1-7 ( 1 9 5 4 ) . (C.A. 48 4 1 8 1 i ( 1 9 5 4 ) ) . 95 386. Paylos, R.N. and Seijo, E.; Rev. farm. - 1 4 6 &

(1967). 383. Meleney, F.L. and Johnson , B.A. ; Conn. State Med. J. 1 4 305-7 ( 1 9 5 0 ) . (c.A. -1 0 7 8 5 a (1350) j . 44 384. Anon.; Bacitracin 1 2 7 pages ( 1 9 5 2 ) S . B . Penick

(C.A. 70 1 0 6 8 6 7 ~( 1 9 6 9 ) ) . Shipchandler, M.T.; U . S . Patent 3 , 9 6 6 , 6 9 9 , June 2 9 , 1 9 7 6 . (C.A. 8 5 143521m ( 1 9 7 6 ) ) . Mancinc D.; Tigelaar, R.E. and Ovary, 2 . ; Immunology 1 8 739-47 ( 1 9 7 0 ) . (C.A. - 6 4 X l t ( 1 9 7 0 ) ) 73 Sato, K.; Kurosawa, A.; Koyama, Y.; Nagai, Y.; Abe, M.; Ouchi, M. and Shimizu, S. Japan Kokai 7 3 6 7 , 2 5 1 , September 1 3 , 1973(C.A. - 6 9 6 5 s ( 1 9 7 4 ) ) . 80 Sato, H.; Kurosawa, A.; Koyama, Y.; Nagai, H.; Ohuchi, M. and Shimizu, M.; Japan Kokai 77 4 1 2 0 1 , November 2 2 , 1 9 7 7 . (C.A. 88 1 5 2 9 4 1 ( 1 9 7 8 ) ) . Brewer, G.A. and Platt, T.B.; Encyclopedia of Industrial Chemical Analysis Vol. 5 533-549

- 593-601 1 1 1

(1969).

387. Aubertin, E . ; J. Med. Bordeaux 1 3 0 1257-63 (1953). (C.A. 48 6652d ( 1 9 5 4 ) ) . 388. MeleneE F . L . and Johnson, B.A.; Semana med. 495-508 ( 1 9 5 4 ) . (C.A. 49 1215d ( 1 9 5 5 ) ) . 389. JawetzTE.; Pediat. Clin. N. Am. 329-44 ( 1 9 5 6 ) . (C.A. 5 1 6 8 7 4 a ( 1 9 5 7.) 1 . . 390. HickeyTR. J . ; Progr. Ind. Microbiol. - 93-150 4 (1964). (C.A. 62 584313 ( 1 9 6 5 ) 1 . 391. Weinberg, E.D. ; 'Antibiotics - 90-101 ( 1 9 6 7 ) . 1 (C.A. 68 479621 ( 1 9 6 8 ) ) . 392. Goldstein, A.; New Engl. J. Med. 240 9 8 - 1 0 7 , 1 3 7 - 4 7 , 180-8 ( 1 9 4 9 ) . (C.A. 4 3 388433 ( 1 9 4 9 ) ) . 71 3 9 3 . Brainerd, H.D.; Calif. Med. - 9-14 ( 1 9 4 9 ) . (C.A. 4 3 8 5 5 0 c ( 1 9 5 9 ) ) . 394. Hoe,R; opr.. Science Counselor - 1 2 8 - 9 , 148-9 12 (1949).

56 ( 1 9 5 3 ) . (C.A. 48 47749 ( 1 9 5 4 ) ) .

BACITRACIN

69

(C.A. - 1613f (1950)). 44 395. Anon.; Chem. Eng. News 29 1190-5 (1951). (C.A. 45 4001f (1951)). 19 396. WernerTG.; Scientia Pharm. - 95-103 (1951). (C.A. - 105101a (1951)). 45 6 397. Baker, W.B.; J. SOC. Cosmetic Chemists - 31323 (1955). (C.A. 50 7404e (1956)). 398. JawetzTE.; Polymyxin, Neomycin, Bacitracin Med. Encyclopedia, N.Y. (1956). (C.A. 50 14875c (1956)). 399. Trefoux, J.; Cheymol, J.; Nau, A.; Paul, R.; Penau, H.; Hagemann; Romain, R.; Ziegle, M.; 11 Vignalou, J. and Quevauviller, A.; Therapie 961-1029 (1956). (C.A. 52 20665e (1958)). 400. Brunnec R.; Osterr. Apotheker Ztg. - 455-60 11 477-9 (1957). (C.A. 52 5748g (1958)). 401. JawetzTE.; Antibiotics Monographs - 1-95 5 (1956). 53 (C.A. - 22520g (1959)). 402. Baker, W.B.; Drug t Cosmetic Ind. - 172-3, i 87 258-62, 264-7 (1960). (C.A. 54 25568i (1960)). 403. Schumacher, E.; Schweiz. Arch. Tierheilk. 104 350-60 (1962). (C.A. 57 11307b (1962)). 404. Bogentoft, C.; Farm. Revy 67 749-51 (1968). (C.A. 70 80793u (1969)). 405. JawetzTE.; Antimicrob. Ther. 91-101 (1970). (C.A. - 30462p (1972)). 76 406. Sakai, K.; Sogo Rinsho 21 2849-58 (1972). (C.A. 78 923282 ( 9 3 ) 17): 407. Yeh, P.; Personal Communication, 1980.

Literature search complete through 1978.

BRETYLIUM TOSYLATE
James E. Carter, Anton H . Amann, and David M . Baaske
1.

2.

3. 4.

5. 6. 7. 8. 9.

Description I . 1 Chemical and Proprietary Names 1.2 Empirical Formula, Molecular Weight, and Structure 1.3 Appearance, Color, Odor, and Taste Physical Properties 2.1 Melting Range 2.2 Solubility Profile 2.3 Infrared Spectrum 2.4 Ultraviolet Spectrum 2.5 Proton Magnetic Resonance Spectrum 2.6 Mass Spectrum 2.7 Differential Scanning Colorimetry 2.8 Crystal Properties Synthesis Analysis 4.1 Elemental Analysis 4.2 Nonaqueous Titration 4 . 3 High Performance Liquid Chromatography (HPLC) 4.4 Gas- Liquid Chromatography (GLC) 4.5 Thin- Layer Chromatography (TLC) Stability Analysis of Biological Samples by Gas- Liquid Chromatography. Absorption, Metabolism, and Excretion Acknowledgment References

72 72 72 72 72 72 73 73 73 73 78 78 80 80 80 80 81 81 82 84 84 84 85 85 86

Analytical Profiles of Drug Substances, 9

71

Copyright 0 1980 by Academic Ress, Inc. All rights of reproductioni any form reserved. n ISBN: 0.12-260809-7

12

JAMES E. CARTER eta1

1.

Description

1.1 Chemical and Proprietary Names

Bretylium tosylate is the non-proprietary name for o-bromobenzylethyldimethylammonium p-toluenesulfonate. It has been marketed as an antihypertensive agent but is no longer used for this indication in the United States. Proprietary names listed by the Merck Index are Bretylan, Bretylate, Darenthin and Ornid. The drug is now marketed as an antiarrhythmic agent with the proprietary name Bretylol. 1.2 Empirical Formula C18H24BrN03S Molecular Weight 414.36 The Merck Index (1) lists the molecular weight as 414.39. Based upon atomic weights defined in 1973 by the International Union of Pure and Applied Chemistry 414.36 is correct. Structure

CH2-N-C2H,

+ I

CH3

so;
I

Br@

LH3

CH,
1.3 Appearance, Color, Odor and Taste

Bretylium tosylate is a white to off-white free flowing, fine, odorless powder. It has an extremely bitter taste. 2. Physical Properties
2.1

Melting Range 96OC - 99OC

BRETYLIUM TOSYLATE

73

2.2

Solubility Profile

Bretylium tosylate is freely soluble in water, methanol and ethanol. It is commonly recrystallized from hot acetone. Chloroform and methylene chloride are the best extraction solvents. Bretylium tosylate is essentially insoluble in ether, ethylacetate and hexane. 2.3 Infrared Spectrum

The KBr pellet infrared spectrum of 0.5% bretylium tosylate obtained with a Perkin-Elmer 283 Infrared Spectrophotometer is contained in Figure 1. Bretylium tosylate is very hygroscopic. Unless the spectrum is obtained on dried material the broad 0-H stretching band centered at 3460 cm-I will be present. The aromatic (3100 - 3000 cm-l) and aliphatic (3000 - 2900 cm-l) C-H stretching bands are present but not as strong as might be anticipated. The strong broad peak centered at 1200 cm-l is the S - 0 stretching band. The molecule contains both a para substituted aromatic (strong C-H bending at 815 cm-l) and an ortho substituted aromatic (strong C-H bending at 772 cm-l) ring. For routine identification purposes a liquid infrared spectrum is generally more reproducible. The spectrum of a 2% solution in dry chloroform is shown in Figure 2.
2.4

Ultraviolet Swectrum

Bretylium tosylate absorbs strongly in the ultraviolet region of the spectrum with three distinct maxima between 230 nm and 300 nm. The spectrum (Figure 3) was obtained with a Beckman Acta I11 double beam spectrophotometer. The wavelength maxima and molar absorptivities are:

278 271 264 257 (shoulder) 2.5

671 885 886

---

Proton Magnetic Resonance Spectrum

H The 60 M z proton magnetic resonance spectrum was obtained with a Varian Associates T-60A spectrometer. The spectrum in CDC13 with tetramethylsilane (TMS) as internal reference is contained in Figure 4 . The integration and

Figure 1.

KBr Infrared Spectrum of Bretylium Tosylate.

rl

0 k 0

-4

4J

5 k
u
a,

Cn

a
a
a, k
rd

u c

a,

L l

b -4 h

16

JAMES E. CARTER et al.

Figure 3 .

Ultraviolet Spectrum of Bretylium Tosylate.

Figure 4.

Proton Magnetic Resonance Spectrum of Bretylium Tosylate in CDC13.

78

JAMES E. CARTER er al.

multiplicities are consistent with the proton assignments. Chemical shifts (6) in ppm relative to TMS are:

Proton Assignment

2.6

Mass Spectrum

The direct probe electron impact mass spectrum of bretylium tosylate i s shown in Figure 5. The spectrum was obtained with a Dupont Dimaspec GC/MS Model 321 (2). No parent ion is seen because bretylium tosylate is a salt and will not travel through the spectrometer intact. Principal fragment ions in the spectrum are identifiable. The base peak at m/z 91 is the tropylium ion (C7H7+) probably formed by loss of SO3- from tosylate. The tropylium ion is also possible from fragmentation of the bretylium ion. The m/z 58 is C H N 3 8 ' formed by loss of C2H5 (which is possible by a number of different paths) from the bretylium quaternary ammonium side chain. The two isotopes of bromine of mass 79 and 81 make the peaks at m/z 169, 171 and m/z 185, 187 readily identifiable as ~ 7 Br+ and C7HgN Br+ respectively. ~ 6 2.7 Differential Scanning Calorimetry

Bretylium tosylate was heated at a rate of 20/min in a Perkin-Elmer Model DSC-2 differential scanning calori-

N+-*

Br

ii
CH

: : t d

7H3 -CH -2
A 4 3

i i
Multiplicity triplet singlet singlet quartet singlet mu1tiplet multiplet

# of Protons
3

Chemica1 Shift ( 6 ) 1.35 2.27 3.07 3.65 4.73 7.17 7.67

3 6 2 2

4
4

19

Figure 5.

Electron Impact Mass Spectrum of Bretylium Tosylate.

80

JAMES E. CARTER c t a l .

meter. A single endotherm was observed with an onset temperature of 97.5OC with the endotherm maximum at 102.5OC. The onset temperature corresponds to the melting point. The heat of transition ( H) calculated in relation to an indium standard is 16.8 cal/g. 2.8 Crvstal Prowerties

Bretylium tosylate crystals examined with a polarizing microscope were found to be tetragonal prisms, elongated parallel to the c crystallographic axis (3). X-ray diffraction patterns (Table I) were also determined (3). Table I. Powder x-ray diffraction pattern of Bretylium Tosylate Re1ative Intensity 50 50 10 25 30 10 5 5 100 5 15 50 75 10 100 80

20 7.65 11.05 12.65 14.10 15.25 16.60 18.00 18.65 19.35 20.10 21.30 22.15 23.10 24.35 24.75 26.45 3. Synthesis

&

11.6 8.00 6.99 6.28 5.81 5.34 4.92 4.75 4.58 4.41 4.17 4.01 3.85 3.65 3.60 3.37

The Bretylium Unites States patent contains examples for the synthesis of numerous bretylium salts (4).

4.

Analysis

4.1 Elemental Analysis

Elemental analysis of a typical bretylium tosylate

BRETYLlUM TOSYLATE

81

sample is as follows: Element C H Br N

0
%

Theoretical* 52.18 5.84 19.28 3.38 11.58 7.74

Found** 52.40 5.76 19.56 3.28 11.69

----

*Calculated for C18H24BrN03S **Determined on a dried sample 4.2 Non-aaueous Titration

Bretylium tosylate may be measured by non-aqueous titration with 0.025 N perchloric acid in dioxane. The end point is visually detected by a change from violet to bluegreen using crystal violet as the indicator. 4.3 High Performance Liquid Chromatography (HPLC)

Two reversed phase HPLC methods have been developed for the quantitation of bretylium tosylate. In the first method (5) bretylium and tosylate ions are determined simultaneously with benzenesulfonic acid as an internal standard. Chromatography is carried out on a 10 um octadecylsilane column with an isocratic mobile phase consisting of 30% methanol in water (pH 5.0) containing a paired ion reagent, tetrabutylammonium phosphate. Flow rate through the column was 2.0 ml/min and the variable wavelength UV detector was set at 220 nm. Standards containing from 0.1 to 0.5 mg bretylium tosylate per ml of solution were employed. The method is applicable for raw drug evaluations, analysis of intravenous solutions and compatibility studies with other drugs. It is not amenable to determination of bretylium or tosylate in biological fluids. Total analysis time is less than 12 minutes. The second reversed phase HPLC method was employed for the quantitation of bretylium ion (6). Bretylium tosylate standard concentrations ranged from 10 to 400 ug/ml; the internal standard was the 2,4-dichloro congener of bretylium tosylate. The 30 cm by 3.9 mm column was packed with 10 um alkylnitrile bonded silica. The compounds were eluted with a mobile phase consisting of acetonitrile and 0.005 M sodium phosphate monbasic in purified water (30:70) at a flow rate of 2.0 ml/min. A fixed wavelength UV detector at 254 nm was

82

JAMES E. CARTER et al.

used to monitor the column effluent. As with the first HPLC method this method is only applicable to evaluation of the raw material and dosage forms. Total analysis time by this method is approximately 1 5 minutes. Both methods are specific, accurate, rapid and precise.
4.4

Gas-Liquid Chromatography (GLC)

A quantitative, stability indicating GLC assay for bretylium tosylate is also applicable to dosage forms ( 7 ) .
The method is based upon a published assay for estimating plasma and urine levels of the drug (8). p-Chlorobenzylethyldimethylammonium p-toluene sulfonate (bretylium is the o-bromo congener) was synthesized and used as the internal standard. The method has been used to evaluate the stability of raw materia1,tablets and injections. The identity of the peaks appearing in the chromatogram has been confirmed by GLC-mass spectrometry. The method involves reaction of bretylium and internal standard with sodium thiophenolate at 70 for 15 minutes. The resultant halogenated benzylthioethers are quantitated by GLC. The reaction is specific for quaternary amines. Chromatography was performed with a suitable gas chromatograph equipped with a flame ionization detector. The 1 8 m . by 4 mm id glass column was packed with 3% OV 225 on 100-120 mesh Chromosorb W-HP. The column and inlet temperatures were maintained at 210 while the detector temperature was 275O. The carrier gas was helium at a flow rate of 5 0 ml/min. The reaction scheme for bretylium and internal standard with sodium thiophenolate is shown in Figure 6. Three peaks appear in the chromatogram following the solvent front. These were identified by GLC-mass spectrometry ( M S ) with a DuPont DP-1 system in the electron impact ( E I ) mode ( 9 ) . Diphenyldithiol appears at 3.0 min; EIMS, 218 ( M ' ) . pChlorobenzylphenylthioether appears at 4.0 min; EIMS, 234 (M+) while o-bromobenzylphenylthioether appears at 4.8 min; EIMS 278 (M') and 280 (M'). All EIMS spectra showed the base peak at 110 corresponding to the C H S ' o . ggHin The 15 minute reaction time and 6 minute analysis time is much more practical than the 1 hour reaction time and 2 0 minute analysis time reported previously (8).

c,
a,

31 k

c,
cl
R1

i? k

a,

t
0

s
0 4
0

a,

*a
a,

0
pc

m
+ +
d

a, Q

d
.0 3

Jz

u >.

a,

84

JAMES E. CARTER e t a l .

4.5

Thin-layer Chromatography (TLC)

Purity and stability of the raw drug have also been assessed by thin-layer chromatography. Bretylium tosylate has an Rf of 0.50 when chromatographed on Alumina-G with 1butanol saturated with water as solvent. o-Bromobenzyldimethyl amine is the most likely contaminant and degradation product. When the plate is sprayed with modified Dragendorff's reagent it appears as a pink spot with an Rf of 0.85. 5. Stability

Bretylium tosylate is a very stable molecule. Solutions hydrochloric acid, of bretylium tosylate at 50 mg/ml in 1 1 N sodium hydroxide and 10% hydrogen peroxide were heated for one hour at 90C. The solutions were analyzed by the stability indicating gas chromatographic (section 4.4) and thin-layer chromatographic (section 4.5) methods. The ultraviolet absorbance at 271 nm was also monitored. The results (Table 11) indicate the potency of the solutions did not change with this drastic treatment. Table 11. Subjection of bretylium tosylatc to drastic acid, alkaline and oxidative conditions. Initial Assay
100.0%

Solution

Assay* GLC TLC

Final Assay 100.5% one spot 0.832 100.5% one spot 0.838 102.9% one spot 0.821

Change From Initial

HC1

1 - NaOH N
10% H202

uv uv

GLC TLC

GLC TLC

uv

one spot** 0.824 101.5% one spot 0.825 101.5% one spot 0.835

+O. 5% no change +l.0%

-1.0%

no change +1.6% +1.4% no change -1.7%

*GLC - gas-liquid chromatography TLC - thin-layer chromatogrpahy UV - ultraviolet absorbance at 271 nm. **one spot indicates one spot with an Rf matching bretylium tosylate standarii.
6.

Analysis of Biological Samples by Gas-Liquid Chromatography

BRETYLIUM TOSYLATE

85

A quantitative method for the analysis of low concentrations of bretylium in plasma and urine has only recently been developed (10). The method is based upon derivatization as are the previously described GLC procedures ( 7 , 8 ) . To enhance the sensitivity of the assay an electron capture detector was employed and 2 , 4 , 5 trichloro sodium thiophenolate was substituted for sodium thiophenolate., Internal standards employed were p-bromobenzylethyldimethylammonium p-toluenesulfonate or o-methoxybenzylethyldimethylammonium ptoluene sulfonate. Bretylium tosylate was quantitatively extracted with methylene chloride after deproteinization with acetonitrile.

The sensitivity of the method is 5 ng/ml. Analysis was performed with a gas chromatograph and a 63Ni electron capture detector. The 1.8 m by 4 mm id glass column was packed with 3% OV 2 2 5 on 100/120 Supelcoport. Injection port, column and detector temperatures were maintained isothermally at 270, 250 and 300, respectively. Argon/ methane ( 9 5 / 5 ) was the carrier gas at a flow rate of 50 ml/min (30 ml/min through the column and 20 ml/min directly t o the detector as a scavenger gas). The retention times of the 2 , 4 , 5 trichlorophenylthioether derivatives of the o-methoxybretylium congener, bretylium and the p-bromobretylium congener were 6.1, 7.4 and 9.4 min respectively.
7.

Absorption, Metabolism and Excretion

Bretylium is not absorbed from the stomach and is poorly absorbed from the gastrointestinal tract (11). Radioactive tracer studies indicate that the drug is not metabolized and is excreted primarily in the urine ( 8 , 1 2 ) . After an intramuscular administration of I 4 C bretylium 63% of the dose was recovered in the urine and 31% was in the feces in the following 4 days (8). Bretylium was found in high concentration in the bile which suggests bile as the source of bretylium found in the feces. The pharmacological and biochemical properties of bretylium have been reviewed (13).
8.

Acknowledgement

The manuscript was expertly typed by Ms. Deborah Canfield.

86

JAMES E. CARTER et al.

References
1.

2. 3.
4. 5.
6.

7.

8 . 9.

10. 11. 12. 13.

The Merck Index, 9th Edition, 1376, Merck & Co. Inc., Rahway, NJ, 1976. V. Diaz, Shilstone Engineering testing laboratory Inc., New Orleans, LA, 70112. Personal communication. S. Palenik, Walter C. McCrone Associates, Inc., Chicago, IL 60616. Personal communication. - Unites States Patent 3,038,004, June 5, 1962. Anon., Y.C. Lee, D.M. Baaske, A.H. Amann and J.E. Carter, Chromatography Newsletter, 8, 9 (1980). C.M. Lair Z.M. Look, P.K. Lai and A. Yacobi, J. Liquid Chromatography, 3, 93 (1980). J.E. Carter, H. Kesler, L.R. Klein, D.P. Carney, A.H. Amann and L.A. Gardella, presented in part, American Pharmaceutical Association 126 annual meeting, Anaheim, CA, Apr., 1979. R. Kuntzman, I. Tsai, R. Chang and A.H. Conney, Clin. Pharmacol. and Therap. , 1 ,829 (1970). 1 E. Chait, EI DuPont DeNemours and Co. Inc., Instrument Products, Wilmington, DE 19898. Personal communication. C.M. Lair B.L. Kamath, J.E. Carter, P. Erhardt, Z.M. Look and A. Yacobi, J . Pharm. Sci., accepted for publication. A.L.A. Boura and A. McCoubrey, 2. Pharm. Pharmacol., 14, 647 (1962). W.G. Duncombe and A. McCoubrey, J . Pharmacol., 15, 260 (1960). Pharmacological and Biochemical Properties of Drug Substances, Vol 2, M.E. Goldberg, Ed., American Pharmaceutical Association, Academy of Pharmaceutical Sciences, Washington, D.C., p. 148.

&.

CARBAMAZEPINE
Hassan Y. Aboul-Enein and A. A . Al-Badr

I.

2.

3. 4. 5. 6.

Description 1 . 1 Nomenclature 1.2 Formulae 1.3 Molecular Weight I . 4 Elemental Composition 1.5 Appearance Physical Properties 2.1 Melting Point 2.2 Solubility 2.3 Identification 2.4 Spectral Properties Synthesis Stability, Decomposition Products Metabolism, Pharmacokinetics, and Absorption Methods of Analysis 6.1 Spectrophotometric Methods 6.2 Chromatographic Methods Acknowledgments References

88 88 83 88 88 88 88 88 89 89 89 94 96 96 99 99
100

103 104

Analytical h f i l e s of Drug Substances, 9

87

Copyright 0 1980 by Academic Ress. Inc. All rights of reproductioni any form ~ S C N C ~ . n
ISBN: 0-12-260809-7

88

HASSAN Y . ABOUL-ENEIN AND A. A. AL-BADR

CARBAMAZEPINE 1. Description

1.1 Nomenclature

1.11 Chemical names

5H-Dibenz [b,f] azepine-5-carboxamide 5-Carbamoyl-5H-dibenz [ b , f] azepine 2,3 : 6,7-Dibenzazepine-l-carboxylic acid, amide 1.12 Generic Name Carbamazepine 1.3 Trade names Finlepsin, Tegretol, Tegretal 1.2 Formulae 1.21 Empirical C15H12N20 1.22 Structural

a?o
cow2
236.26 5.12%, N 11.86%,
0 6.77%

1.23 Wiswcsser Line Notation : TC 676 BNJ BVZ (1) 1.3 Molecular weight

1.4 Elemental composition

C

76.25%,

1.5 Apearance White to off-white powder. 2. Physical properties 2.1 Melting point Melts within a range of 3O between 187 and 193' ( ) 2.

C ARB AMAZE PINE

89

2 . 2 Solubilig

Practically insoluble in water; soluble in alcohol, acetone and propylene glycol ( 3 ) .

2 . 3 Identification 2 . 3 1 Infrared Spectroscopic test

USP XIX ( 4 ) cites the use of infrared absorption spectrum of carbamazepine in methylene chloride as a mean of identification comparing some characteristic absorption bands of the drug. This will be discussed in the infrared spectral properties of the drug.
2 . 3 2 Color test

Carbamazepine can be identified ( 5 ) by color test with ammonium molybdate. A faint to blue color .8. is produced (sensitivity 1.0 11) BP 1 9 7 3 ( 6 ) describes a color test in which 0.1 g of the drug is treated with 2 ml nitric acid in a water-bath for three minutes where an orange color is produced.
2.33 Crystal test

Carbamazepine can be identified by forming crystals with lead iodide solution where needles are formed ( 5 ) .
2 . 4 Spectral properties 2 . 4 1 Ultraviolet spectrum

Carbamazepine in neutral methanol solution shows maxima at 212 nm, an inflection at 2 3 6 nm, 283 nm; and a minimum at 256 nm. (Fig. 1 . ) Carbamazepine ( 5 ) in ethanol shows a maxima at 215 nm and at 285 nm, minimum at about 257 nm. In 0 . 1 N sulphuric acid, the drug shows maxima at 283 nm (E 1%,1 cm 1 4 7 ) and an inflection at about 255 nm (E 1%,1 cm 2 7 4 ) .

90

HASSAN Y. ABOUL-ENEIN AND A. A. AL-BADR

400 380 360 340 320 300 280 260 240 220'200

Fig. 1 - Ultraviolet spectrum of carbamazepine in methanol.

C ARB A MAZE PINE

91

The ultraviolet absorption spectrum of the drug is used as a mean of identification of carbamazepine in BP 1973 (6). A 2 cm layer of 0.001 w/v solution in alcohol (95%) exhibits a maximum only at 285 nm; extinction at 285 nm, about 0.98. The drug also exhibits an intense blue florescence in the ultraviolet light at 366 nm.

2.42 Infrared spectrum

The infrared spectrum of carbamazepine is shown (Fig. 2. The spectrum was obtained from nujol ) mull. The structural assignments have been correlated with the following band frequencies:Frequency (Cm-l) Assignments

3470 1680 1600 shoulder and 1590

Aromatic C = C

c=o

NH2

Clarke (5) cited the following bands as characteristic principal peaks for carbamazepine when determined in potassium bromide; 1678, 1388 and

1594 Cm-l. 2.43 Nuclear Magnetic Resonance Spectrum

A typical NMR spectrum of carbamazepine is shown in (Fig. 3). The sample was dissolved in CDCl 3 The Spectrum was determined on a Varian T-60A, NMR spectrometer with TMS as the internal standard. The following structural assignments have been made for (Fig. 3). Chemical Shift (6) Broad singlet at 4.83 Singlet at 6.87 Multiplet centered at 7.33 Assignments

CH = CH at CloCll
Eight aromatic protons on the two phenyl groups.

NH2

2.44 Mass spectrum and fragmentometry

The mass spectrum of carbamazepine obtained by

C
C
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W
1

a
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0 N 1

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0 0

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c

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N
r

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0 r

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PPM(6) 4.0

1 . . . . 1 . . , , 1 . . . . 1 . . . .
3 .O
2.0

1 .o

Fig. 3 - NMR spectrum of carbamazepine in CDCl containing TMS as internal standard. 3

94

HASSAN Y . ABOUL-ENEIN AND A. A. AL-BADR

e ectron impact ionization shows a molecular ion $1 at m/e 236 (relative intensity 9.1%) Fig. 4 , and a base peak at m/e 193.

Frigerio et. a1 (7,8) had published the mass spectrometric properties of carbamazepine and its metabolites, carbamazepine - 1 0 , U epoxide and 10,11-dihydro-10,11-dihydroxy-5H-dibenz [b,f] azepine-5-carboxamide. The fragmentation pattern are shown in Scheme 1. Frigerio et.al., discussed the fragmentation pattern of carbamazepine and its epoxide in details (7,8).

CONH 2

H
mie 193 m/e 192

m/e 236

3. Synthesis

a
H
1

coc12

Me

Toluene

@&
I

(PhC02)

COCl

Br Pressure COCl

coNH2

a)

Carbamazepine can be synthesized as follows:Iminodibenzyl in toluene was treated with C0Cl2 to give 95% of 5-chlorocarbonyl iminodibenzyl which in turn was dissolved in CC1 and was treated with 1,3-dibromo-5, 4 5-dimethyl hydantoin and (PhCO ) to give 90% 5-chlorocarbonyl-10-bromoimino-dibenzyj. The latter was dissolved in xylene and heated at about 10 in an auto0' clave with gaseous NH to give 85% o f 5-carbamoyl-5H3 dibenzo [b,f] azepine (9).

11846 SCAN 50 CQRBWAXPXNE

S I G M A 4 RT-0

1 1 B ~ C K = 1 7 0 ~ X 1 0 100'/.= 0

444000

Fig. 4

- Mass spectrum of carbamazepine (EI) determined

by direct probe insertion.

96

HASSAN Y. ABOUL-ENEIN AND A. A . AL-BADR

cow2
b) 5H-Dibenzo [b,f] azepine, which may be prepared by thermal decomposition of 2-(0-aminostyry1)-aniline hydrochloride, is condensed with carbamoyl chloride by refluxing in an inert solvent in the presence of sodamide (10).

4 . Stability, Decomposition products

Carbamazepine is relatively stable drug at room temperature. However, it is recommended that it should be kept and stored in a well closed container, protected from light and in dry place.

BP 1973 (6) had described a test for identification of

foreign substances namely iminodibenzyl using tlc for this purpose

5. Metabolism, Pharmacokinetics and absorption

Meinardi (11) had published a review on carbamazepine in 1972 in which he discussed the determination, metabolism and pharmacology of the drug. Carbamazepine is readily absorbed from the gastrointestinal tract. Peak concentration in serum have been reported at about 2% h after a dose. It is believed to have a halflife between 14-29 h. (12). Studies on the plasma kinetics of carbamazepine suggested that it induced its own metabolism (13). Frigerio et. al., ( 7 ) had isolated carbamazepine-lO-11epoxide as a urinary metabolite from humans following oral

CA RB A M AZE PINE

91

administration. The epoxide formation was confirmed by the in vitro studies of the activity of the liver microsomal -monooxygenases. SKF 525A inhibited the formation of carbamazepine oxide by 80X (14). Goenechea and HeckeSeibicke (15) had detected seven metabolites in human urine in addition to unchanged drug by tlc. l0,ll-Dihydro10-11-dihydroxy-5H-dibenzo [b,f] azepine-5-carboxamide was identified on the basis of UV, IR, mass and NMR Spectra. Iminostilbene was also isolated as a minor urinary metabolite from rats (16). The N-glucuronide of carbamazepine was identified in the bile of isolated perfused rat lever by the mean of permethylation GC/MS (17). The pharmacokinetic of carbamazepine was studied in several species :A)

Humans Gerardin et. al., (18) had discussed the pharmacokinetics of the drug in normal humans after single and repeated doses. It was reported that the plasma concentration of the drug following single dose (100, 200, 600 mg) to normal healthy humans were fitted by a one-compartment open model. The elimination halflife after a single dose was 37.7h; it decreased during chronic treatment to a calculated value around 21h. The steady-state plasma concentration, lowers than expected from the single dose study, was adequately predicted from the single-dose data when a correction was made for the increased elimination rate constant. These findings contrast with the apparantly unpredictable plasma levels reported during carbamazpine therapy. Palmer et. al., (19) reported that following oral administration of the drug (200 mg) to two healthy fasting subjects, peak plasma concentration occured after 6-8 h . and remained constant for 24 h before declining over the subsequent 6 days. The plasma halflife was about 36 h.

B)

Rhesus monkey The pharmacokinetics of carbamazepine (20) after a 20 mg/kg dose was administered by I.V. (5. min) infusion and orally. A l l semilogarethmic plasma concen-

&
I
corn2
OH

Km 0.34nM

0.41 nmo1/
corn2

minlmg protein

(igb
OH

O K

I
I
I

\
OH

"I
I

Glucuronide

Glucuronide

CONJQ

cowz

Oil

Glucuronide Identified Metabolites of Carbamazepine.

CARBAMAZEPINE

99

tration-time curve after I . V . administration exhibited an irregular decay behavior in the first 3-hr period, followed by a linear disappearance phase (T% =, 1.02.4 h ) r . Urinary extraction measurements confirmed the short elimination half-time and showed that < 1% of the dose was excreted unchanged. Oral studies also yielded a short elimination half-life (1.0-1.60 hr), which was confirmed by urinary excretion measurements. The fraction of the oral dose reaching the systemic circulation ranged between 58 and 87%. Measurable (but insignificant) amounts of drug were found in the feces after I.V. and oral administrations.
C)

Adult male, female and pregnant rats After treatment with single and repeated doses of carbamazepine, male rats eliminated the drug faster than females; the total body clearance (TBC) was 1 6 ml/min/ kg and 9.4 ml/min/kg. respectively. Two dose levels (25 and 50 mg/kg) had the same pharmacokinetic properties in young rats. Pregnant rats cleared the drug to a lesser extent than controls. Carbamazepine accelerated its own elimination after repeated administration in both adult and young rats as revealed by the shortening of its half-life and an increase of 50% in clearance. Moreover the protection against electrosock was significantly reduced after repeated administration, compared with a single-dose administration,
(21)
9

The mean amount of carbamazepine not bound in vitro to plasma protein from 24 healthy subjects was 18.2%; the mean amount not bound in plasma from 5 4 patients taking the drug was 26.9% (range 7 . 9 to 60%). There was no significant difference in binding capacity between plasma from patients with renal disease and that from healthy subjects but the plasma from patients with level disease bound a slightly lower percentage o f carbamazepine than did normal plasma ( 2 . 2)
6. Methods of Analysis 6.1 Spectrophotometric methods 6 . 1 1 Ultraviolet spectrophotometric methods

a) Both BP 1973 and USP XIX ( 4 ) describe an analytical procedure for carbamazepine and its tablet formulation depending on measuring

HASSAN Y . ABOUL-ENEIN AND A. A. AL-BADR

the absorbance of the solution prepared at 285 nm. The solvent used in BP 1973 is alcohol 95% while USP XIX uses dehydrated alcoholmethanol (95:5) as a solvent system in the ultraviolet determination of carbamazepine.

b) Fellenberg eta (23,24) reported a method for the determination of carbamazepine in blood. The method has a detection threshold of <O.l mg/ 100 ml of blood. The method is based on the catalytic rearrangement of carbamazepine to 9-methylacridine. After extraction of the drug from blood with methylene chloride, which is then washed with alkali and acid. An aliquot of the extract is evaporated to dryness and the residue heated briefly with HC1 at Following removal of nonspecific inter150'. ference with n-heptane, the absorbance of 9-methylacridine is determined at 258 nm. The method is reported to be rapid, sensitive and specific and is suitable for routine clinical use.
6.12 Nuclear magnetic resonance spectrometry Carbamazepine has been assayed (25) in its tablet form by the application of PMR spectrometry. The method involves comparing the integral of the aromatic and olifinic protons of the drug, in the range of 6.60-7.60 ppm, to that of the singlet of known amount of hexamethylcyclotrisilazine (at 0.00 ppm) used as internal standard. The method is simple, rapid and accurate. The average percent recovery of carbamazepine from its tablets is 96.88 5 0.93. 6.2 Chromatographic methods 6.21 High pressure liquid chromatography Several methods have been published for the quantitative determination of carbamazepine and its epoxide metabolite in biological fluids (plasma, serum, urine) by HPLC. Kitazawa and Komuro (26) had described a method for determination of carbamazepine and other anti-convulsant drugs in human blood plasma. The method involved two step extraction procedures with chloroform and use of 2x50 cm long stainless steel column packed with an

CARBAMAZEPINE

101

a n i o n exchange r e s i n . The m o b i l e p h a s e w a s 4mH ammonium p h o s p h a t e b u f f e r s o l u t i o n of pH 6.2 a t a f l o w r a t e o f 0 . 4 0 ml/min. The r e s u l t s p r e s e n t e d showed l i n e a r c a l i b r a t i o n c u r v e s and 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 as low a s 1 . 0 v g I 0 . 5 m l plasma. The method w a s e f f i c i e n t t o d e t e c t t h e d r u g i n plasma a f t e r t h e r a p e u t i c c l i n i c a l doses. Eichelbaum and B e r t i l s s o n ( 2 7 ) d e s c r i b e d a method u s i n g HPLC-mass s p e c t r o m e t r y f o r s i m u l t a n e o u s d e t e r m i n a t i o n of carbamazepine and i t s a c t i v e 1 0 , 11-epoxide m e t a b o l i t e i n plasma. The method r e q u i r e d no d e r i v a t i z a t i o n and had a l o w e r l i m i t of s i n s i t i v i t y of 4 ng f o r carbamazepine and 4 ng f o r i t s m e t a b o l i t e . The method i s v e r y s p e c i f i c and had a p r e c i s i o n s of 2.2% f o r t h e d r u g and 4.2% o r i t s m e t a b o l i t e s . Karba Marton (28) p u b l i s h e d a method f o r d e t e r m i n a t i o n of carbamazepine i n t h e whole blood by HPLC. The d r u g w a s w e l l s e p a r a t e d from normal blood c o n s t i t u e n t s i n less t h a n 8 m i n u t e s . The s e n s i t i v i t y o f t h i s method i s 0.25 mg of t h e d r u g 1 1 i n a 2 m l s a m p l e , and t h e l o w e r l i m i t of d e t e c t i o n i s 100 ng. Another method (29) w a s r e p o r t e d f o r d e t e r m i n a t i o n of carbamazepine i n plasma i s d e s c r i b e d i n which t h e drug w a s e x t r a c t e d with e t h e r , i s o l a t e d with L i c h r o s o r b RP8 and d e t e c t e d by UV s p e c t r o s c o p y a t 280 nm. The r e c o v e r y of t h e d r u g w a s a t c o n c e n T h e r e w a s no i n t e r f e r e n c e by t r a t i o n 1-2 ug/ml. t h e d r u g m e t a b o l i t e o r endogenous plasma component s

6.22 P a p e r chromatography C l a r k e ( 5 ) d e s c r i b e d s e v e r a l s o l v e n t s y s t e m s used f o r p a p e r c h r o m a t o g r a p h i c d e t e c t i o n of carbamazep i n e as shown i n T a b l e 1. 6 . 2 3 Thin Layer Chromatography Carbamazepine WAS d e t e r m i n e d among o t h e r a n t i c o n v u l s a n t a g e n t s i n serum by t l c . The serum w a s e x t r a c t e d w i t h toluene and t h e d r i e d e x t r a c t w a s d i s s o l v e d i n c h l o r o f o r m and s p o t t e d on t o a t l c

102

HASSAN Y . ABOUL-ENEIN AND A. A. AL-BADR

Table 1 Solvent System Citric acid : H 0 : n-butanol ( 4 . 8 gm : 1 3 0 m12: 870 ml) Acetate Buffer
(pH 4 . 5 8 )

Visualizing agent Ultraviolet blue fluorescence Ultraviolet, blue fluorescence Ultraviolet, blue fluorescence.

Rf
0.34

0.29
0.30

Phosphate Buffer (PH 7 . 4 )

plate. After development, the plate was scanned at 215 nm without staining. Most of the interfering substances that occur naturally in serum were soluble in and eliminated by the liq. front.
(30).

Clarke (5) reported the use of a solvent system consisting of strong ammonia solution :methanol (1.5 : loo), the solvent system is recommended to be changed after two runs. Carbamazepine gives an R value o f 0 . 7 3 . The chromatogram (Silicagel G) f is visualized by potassium permangnate spray.
6.24 Gas-Liquid Chromatography

Gas-Liquid chromatography is considered to be the main procedure for the quantitation and analysis of carbamazepine specially in biological fluids and tissues. The drug has been determined by several authors ( 3 1 , 32, 3 3 , 3 4 ) through derivatization to its methylated derivative. The methods reported show lower limit of detection of about 0.5 mgflitre. Carbamazepine had beer, determined by GLC without derivatization on several stationary phases as shown in Table 2. Toseland et al., ( 3 8 ) described the determination of carbamazepine among other anticonvulsants and barbiturates in plasma and tissue using the nitrogen flame detector.

C ARB A M AZEPl NE

103

Table 2 Stationary phase

3% OV - 17 2% SP - 1000

Detector used Flame ionization. Isothermal

Reference
(35) (36)

Cab-0-Sil deactivated with benzyltriphenyl phosphonium chloride and OV - 225

Isothermal

(37)

Marozzis. &., (39) had reported the gas chromatographic retention indexes (I ) of 232 compounds of toxicological interest whi% were determined isothermally at 10 8' on SE 30, OV.1, OV-17, among which was carbamazepine. Clarke (5) reported the retention time of carbamazepine to be 0.81 relative to codeine and 3.76 relative to diphenhydramine using 2.5% SE-30 on 80-100 chromosorb W AWHMDS and the conditions specified in the monograph. ACKNOWLEDGEMENTS

The authors would like to thank Mr. Dennis Charkowski, Department o f Pharmacology, Unitersity of Iowa, Iowa City, Iowa 5 2 2 4 2 , U.S.A., for determining the mass spectrum of carbamamazepine, Mr. Said E. Ibrahim, for his help in the Library search, Mr. Essam A. Lotfi and Mr. Khalid N.K. Lodhi, for their technical assistance in the ultraviolet and nmr determination, and Mr. Altaf Hussain Naqvi for typing the manuscript.

104

HASSAN Y . ABOUL-ENEIN AND A. A. AL-BADR

REFERENCES
1.

"Atlas of Spectral data and physical constants of organic compounds'', edited by J.G. Grasselli and W.M. Ritchey. Volume 3, CRC Press 1975, page 178. Remington's Pharmaceutical Sciences, 15th edition, Mack publishing Co., Easton, Pa., 18042, 1975, page 1014. Merck Index, Ninth edition, Merck 6 Co., Inc., Rahaway, N . J . , U.S.A., 1976, page 226. The United States Pharmacopeia XIX, United States Pharcopeial Convention, Inc., Rockville, Md., 20852, page 69. E.C.G. Clarke, "Isolation and Identification of Drugs". The Pharmaceutical Press, London 1969, page 238. British Pharmacopoeia 1973, London Her Majesty's Stationery Office 1973, page 80. A. Frigerio, R. Fanelli, P. Biandrate, G. Passerini, P.L. Morselli and S. Garattini, J. Pharm. Sci., 61, 1144 (1972) A. Frigerio, K.M. Baker and P.L. Morselli, Mass Spectrum. Biochem. Med. Symp., 65-82 (1973); through Chem. Abstr., 82, 38760 d (1975).
A. Rudnicki, D. Krementowska, A. Osowski, H. Rozentalski

2. 3. 4.

5.
6. 7. 8.

9.

and T. Szszepkowska, Starogardzkie Zaklady Farmaceutyczne Chem. Abstr., 77, "Polfa" (1972); through - - - 101406 g (1972)

10. Schindler, U . S . Pat. 2, 948, 718 (1960, Geigy).

11. H. Meinardi, Antiepileptic Drugs, edited by D.M. Woodbury and M. Dixon, 487 (1972). Raven Press New York, N.Y., - - 77,134933 e (1972). Chem. Abstr.,
12. Martindale, The Extra Pharmacopoeia, 27th edition, The

Pharmaceutical Press, London, edited by Ainley Wade,

1236 (1977).

13. M. Eichelbaum, Eur. J . Clin. Pharmac., 14. J. Pachecka, M. Salmona,

6,

8,

337 (1975).

L. Cantoin, E. Mussini, C. Pantarotto, A. Frigerio and G. Belvedere, Xanobiotica,

593 (1976).

CARBAMAZEPINE

105

1 5 . S. Goenechea and E. Hecke-Seibicke, Z. K l i n . Chem. K l i n Biochem. 10,1 1 2 ( 1 9 7 2 ) ; t h r o u g h Chem. A b s t r . , 13852 u (1972).

77,

16. J. C s e t e n y i , K . M. Baker, A. F r i g e r i o , and P.L. -J. Pharm. Pharmacal., 25, 340 ( 1 9 7 3 ) .

Morselli,

17. J . E . Bauer, N . G e r b e r , R.K. Lynn, R.G. Smith and R.M. Thompson, E x p e r i e n t i a 32, 1032 (1976).

18. A.P. G e r a r d i n , F.V. Abadie, J . A . C a m p e s t r i n i and W. Theobald, J. Pharmacokint, Biopharm, 4, 521 ( 1 9 7 6 ) .

19. L. Palmer, e t a l , C l i n . Pharmac. T h e r a p . ,

14,827

(1973).

20. R.H. Levy, J . S . Lockard, J . R . Green, P. F r i e l and L. Martis, J . Pharm. S c i . , 64,302 (1975). 21. H.M. F a r g h a l l i , B.M. Assael, L. B o s s i , S. G a r a t t i n i , M. Gerna, R. Gomeni and P.L. M o r s e l l i , Arch.. I n t . Pharmacodyn. T h e r . , 220, 1 2 5 ( 1 9 7 6 ) .

22. W.D.

Hooper, e t a l . ,

m.Pharmac.

T h e r a p . , 1 7 , 4 3 3 (1975).

23. A . J . F e l l e n b e r g and A.C. 423 (1976). 24. A . J . F e l l e n b e r g and A.C. 429 (1976).

P o l l a r d , C l i n . Chim. Acta,

3,

P o l l a r d , C l i n Chim A c t a , 69, and E.A. Lotfi,

25. H.M. E l - F a t a t r y , H.Y. Aboul-Enein, Letters, 951 (1979).

12,

&.

26. S. Kitazawa and T. Komuro,ClinChim A a , (1975). 28. P.M. Kabra and L.J. - 1070 ( 1 9 7 6 ) . 22,

73,

31 (1976).

27. M. Eichelbaum and R. B e r t i l s s o n , J . Chromatogr.,

1 3 135 0,
N.C)

Marton, C l i n Chem (Winston-Salem,

29. I . M . House and D . J . B e r r y , High P r e s s u r e L i q . Chromatogr. C l i n . Chem., p r o c . Symp; 1 5 5 (1976) e d i t e d by P.F. Dixon, C.H. Gray and C.K. Lim, Academic, London, England. 30. N . Wad, E. H a n i f l and H. Rosenmund, J. Chromatogr. 89, (1977).

143,

106

HASSAN Y . ABOUL-ENEIN AND A. A. AL-BADR

31. C.V. Abraham, and H.D. (1976). 32. C.V. Abraham and H.D. N.C) 22 769, (1976).

J o s l i n , J. Chromatogr.

128,281

J o s l i n , C l i n Chem (Winston-Salem,

33. 0. Drummer, P. M o r r i s and F. Vajda, C l i n . Exp. Pharmacol. P h y s i o l . 2, 497 (1976). 34. R . J . P e r c h a l s k i and B . J . W i l d e r , C l i n Chem.

20,

492 (1974)

35. W. Beyer, W. Truppe and W. Mlekusch, 2. Med. L a b o r t e c k . -, 267 ( 1 9 7 6 ) ; t h r o u g h Chem. A b s t r . , 86, 65258 k (1977). 17
36. P.A. T o s e l a n d , J. Grove and D . J . - 321 (1972). 38, B e r r y , C l i n . Chim. Acta

37. C.A. Gramers, E.A. Vermeer, L.G. Van Kuik, J . A . Hulsnan and C.A. Meijers, C l i n . Chem. Acta, 73, 97 (1976). 38. P.A. T o s e l a n d , M. A l b a n i and F.D. Gauchel, C l i n Chem., (Winston-Salem, N.C.) 21, 98 (1975). 39. E. Marozzi, V. Gambaro, F.Lodi and A. P a r i a l i , Farmaco Ed. P r a t . 2, 180 (1976); t h r o u g h Chem. A b s t r . , 85, 14793 c (1976).

CEFACLOR
Leslie J . Lorenz
1.

2.

3. 4.

5. 6.

7. 8. 9.

Description 1.1 Name 1.2 Structure, Formula, and Molecular Weight 1.3 Appearance Physical Properties 2.1 Infrared Spectrum 2.2 Nuclear Magnetic Resonance Spectrum 2.3 Mass Spectrum 2.4 Ultraviolet Spectrum 2.5 Optical Rotation 2.6 Differential Thermal Analysis 2.7 Thermogravimetric Analysis 2.8 Dissociation Constants (pKa) 2.9 Solubility Properties 2.10 Crystal Properties Chemical Synthesis Stability 4.1 Bulk Stability 4.2 Solution Stability Drug Metabolism Method of Analysis 6.1 Identification tests 6.2 Quantitative tests 6.3 Impurity Tests Determination in Body Fluids Acknowledgments References

108 108 108 108

108

108 109 112

112

112 112 114 1 I4 114 1 I4 115 117 1 I7 117 117 118 118 118 119 122 122 123

Analytlcal Rofiles of Drug Substances, 9

107

Copyright 0 1980 by Academic Ress. Inc. All rights of reproduction in any form reserved. ISBN: 0-12-260809-7

108

LESLIE J . LOREN2

1.

Description 1.1. Name

C e f a c l o r i s 3-chloro-7-d- (2-phenylglycinamido) 3-cephem-4-carboxylic a c i d , monohydrate.

1.2.

S t r u c t u r e , formula and m o l e c u l a r weight

mC

COOH

molecular weight 385.82 1.3. Appearance

C e f a c l o r is a w h i t e t o cream c o l o r e d c r y s t a l l i n e powder. The m a t e r i a l i s o d o r l e s s going t o s l i g h t l y sulphurous.

2.

Physical properties
2.1.

I n f r a r e d spectrum The i n f r a r e d spectrum o f c e f a c l o r monohydrate

A i n t e r p r e t a t i o n o f t h e spectrum i s given i n t a b l e 1. n

i n a potassium bromide p e l l e t i s p r e s e n t e d i n f i g u r e 1.

-Q
-8
(D

0 -0
O o
d d

+
a ,

0 -0

z
a
a ,

0 -0

a ,

k rd k

-I3 c9

0
a ,

I
I

-8
' p

110

LESLIE J. LOREN2

Table 1

Wave1ength (cm-' ) 3680-3000 ( s e r i e s o f broad bands) 2580 (broad) 1775 (strong) 1693 (strong) 1600 (strong) 1560 (weak) 1500 (medium) 1365 (strong) 697 (sharp)
2.2.

Ass ignment
OH from H20 and amide NH s t r e t c h

C0.j 8-lactam C=O s t r e t c h

R -0 C

amide C=O s t r e t c h carboxylate s t r e t c h i n g

aromatic C=C aromatic C=C

co2 (sym) c-c1

Nuclear magnetic resonance spectrum

Figure 2 shows t h e proton magnetic resonance spectrum of cefaclor. The spectrum was recorded on a 60 MHz instrument. A i n t e r p r e t a t i o n of t h e spectrum i s n presented i n t a b l e 2 .

Table 2 Proton Magnetic Resonance Spectrum

COOD

8.00

7.00

I 6.00

5.00 PPM

4.00

30 .0

2.00

I 1.oo

.oo

Figure 2

The NMR Spectrum of Cefaclor i n D20+DC1

112

LESLIE J . LORENZ

Peak Assignments

PP!
3.66 5.17 5.35 5.78 7.60 2.3.

Mu1t ip l i c it y q u a r t e t AB J=19Hz doublet singlet d o u b l e t J=5HZ singlet Mass spectrum

Assignment CH2 (2)

H (6) @-CH- COI ND3

phenyl

C e f a c l o r i s amenable t o f i e l d d e s o r p t i o n t e c h n i q u e s or o b t a i n i n g a meaningful mass spectrum. Using t h i s t e c h n i q u e , a small quasi-molecular i o n i s s e e n or c e f a c l o r a t m/e o f 369 which corresponds t o t h e molecular weight o f c e f a c l o r a n h y d r a t e . The major ion i n t h e mass spectrum appears a t m/e o f 331. T h i s i s caused by t h e l o s s o f HC1 from t h e molecule. The oiily o t h e r s i g n i f i c a n t i o n i n t h e spectrum a p p e a r s a t m/e o f 287. T h i s i o n corresponds t o a molecule which has l o s t HC1 and decarboxyolated. 2.4. U l t r a v i o l e t spectrum

F i g u r e 3 shows t h e u l t r a v i o l e t spectrum f o r c e f a c l o r . The chromophores i n c e f a c l o r a r e 3-cephem, phenyl and amide. O f t h e s e , o n l y t h e 3-cephem group c o n t r i b u t e s s i g n i f i c a n t l y about 2 2 0 nm. T h e w r * 3-cephem t r a n s i t i o n has ~ ( 2 6 5 nm) 2 8400. There i s a n + r * transition at about 230 nm due t o t h e 3-cephem group. The phenyl group has a weak a b s o r p t i o n a t 260 nm with E N 200. 2.5. Optical 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 o r c e f a c l o r determined on a one perceng s o l u t i o n o f c e f a c l o r i n 0.1 M h y d r o c h l o r i c a c i d a t Na2' i s +105.6'0n an anhydrous b a s i s .

D

2.6.

D i f f e r e n t i a l thermal a n a l y s i s

The thermogram o f c e f a c l o r g e n e r a l l y shows a small broad endotherm between 40OC and 12OoC corresponding t o t h e l o s s o f water and o t h e r v o l a t i l e s from t h e sample. The major endotherm i n t h e DTA curve f o r c e f a c l o r i s o b s e r v e d around 22OoC where t h e m a t e r i a l decomposes.

cu

I14

LESLIE J . LORENZ

2.7.

Thermogravimetric a n a l y s i s

Cefaclor gives a reasonable thermogravimetric curve and shows a l o s s of w a t e r and o t h e r v o l a t i l e s from about 4 0 O C t o 12OoC. A t about 18OoC c e f a c l o r samples begin t o l o s e weight i n d i c a t i n g t h e b e g i n n i n g o f decomposit i o n o f t h e sample. 2.8. D i s s o c i a t i o n c o n s t a n t pKa

The f o l l o w i n g d i s s o c i a t i o n c o n s t a n t s have been determined f o r c e f a c l o r : Solvent Carboxvl 1.5*0.2 4.33 PKa Amino 7.17 7.34

H2
66% DMF

2.9.

Solubility properties

The s o l u b i l i t y p r o p e r t i e s o f c e f a c l o r are d e s c r i b e d i n t a b l e 3. Table 3

So 1v e n t

S o l u b i l i t y mg/ml
>5 b u t <10 >5 b u t <10 co.5 <0.5 <0.5 <0.5 C0.5 <0.5 <0.5 <0.5

Water pH 1 . 2 (USP XIX) pH 4 . 5 (USP XIX) pH 7.0 (USP XIX) Met hano 1 Octanol Is opropano 1 Diethyl e t h e r Ethyl a c e t a t e Ch 1or0 form Benzene Cyclohexane
2.10.

10.0

Crystal properties

Polymorphs o f c e f a c l o r are p o s s i b l e . Such polymorphs are a f u n c t i o n o f t h e s o l v e n t from which c e f a c l o r i s c r y s t a l l i z e d . The o n l y polymorph o f g e n e r a l importance i s c e f a c l o r monohydrate. The X-ray powder d i f f r a c t i o n d a t a f o r c e f a c l o r monohydrate are given i n table 4.

CEFAC LO R

Table 4 X-ray Powder D i f f r a c t i o n Data C e f a c l o r Monohydrate x=l.5418 "d" Value (A) 12.90 10.05 6.58 6.08 5.42 5.01 4.75 4.06 3.86 3.69 3.53 3.41 3.29 3.23 3.13 2.99 2.81 2.67 2.52 2.48 2.35 2.26 2.15 2.07 1.99 1.94 3. Chemical s y n t h e s i s I n t ens i t i e s ( 1/ I ) , 0.75 0.17 0.13 0.13 0.96 1.oo 0.04 0.54 0.04 0.29 0.58 0.04 0.17 0.13 0.04 0.21 0.25 0.08 0.08 0.04 0.17 0.17 0.04 0.08 0.21 0.08

F i g u r e 4 p r o v i d e s a flow s h e e t o f t h e chemical s y n t h e s i s f o r c e f a c l o r . In t h i s procedure, p e n i c i l l i n V (1) i s e s t e r i f i e d with p - n i t r o b e n z y l bromide (PNB-Br) and oxidized with peracetic acid t o give p e n i c i l l i n sulfoxide e s t e r ( 2 ) . Ring expansion and o z o n o l y s i s p r o v i d e t h e Sul f o x i d e r e d u c t i o n 3-hydroxy- 3- cephem s u l f o x i d e ( 4 ) and e n o l c h l o r i n a t i o n o c c u r s w i t h phosphorous t r i c h l o r i d e i n N , N-dimethyl formamide. S i d e c h a i n c l e a v a g e i s accomplished w i t h phosphorous p e n t a c h l o r i d e and p y r i d i n e followed by a l c o h o l y s i s w i t h i s o b u t y l a l c o h o l . The r e s u l t i n g n u c l e u s h y d r o c h l o r i d e (5) i s n e u t r a l i z e d w i t h

116

LESLIE J. LORENZ

?
1 ,)

?
PNB-Br \2)CH3CO3H
2

COzK
1

COzPNB

SnC14 Toluene, 2)

, CaO, 8 1) NCP,
A

0
II

1) PCl3, DMF

2) PC15, Py, CHpClz

COzPNB

OH 3)i-BuOH COzPNB
4

HclHzNz/cl

CH3CN:HzO TEA

0
5

COzPNB

HzNu,
D

CI C02PNB

coz 7

coz -

Cefaclor Monhydrate

Figure 4

The Chemical Synthesis o f Cefaclor

CEFACLOR

117

t r i e t h y l a m i n e and a c y l a t e d w i t h a N-protected D-phenylglycine t o give c e f a c l o r (7). This is then hydrated i n w a t e r t o y i e l d t h e d e s i r e d monohydrate ( 8 ) . 4.1. Bulk s t a b i l i t y

C e f a c l o r i s a r e a s o n a b l y s t a b l e molecule i n t h e dry s t a t e . When c e f a c l o r i s p r e s e n t i n t h e monohydrate c r y s t a l l i n e form i n t h e dry powder, two y e a r s t a b i l i t y can b e e a s i l y o b t a i n e d . The powder becomes l i g h t l y yellow upon aging, however, l i t t l e d e c r e a s e i n t h e potency of c e f a c l o r i s observed. On d e g r a d a t i o n , c e f a c l o r appears t o l o s e HC1 q u i t e e a s i l y . F u r t h e r d e g r a d a t i o n s t e p s seem t o b e q u i t e r a p i d and no o t h e r compounds have been i s o l a t e d . In an a t t e m p t t o g e n e r a t e such compounds, some s t u d i e s have been c a r r i e d o u t on t h e p - n i t r o b e n z y l e s t e r o f c e f a c l o r . This s t u d y showed t h a t c e f a c l o r can undergo i n t r a m o l e c u l a r n u c l e o p h i l i c a t t a c k by t h e s i d e c h a i n amine group t o produce a d i k e t o p i p e r a z i n e with t h e f o l l o w i n g s t r u c t u r e (1) :

4.2.

Solution s t a b i l i t y

..

C e f a c l o r i s s t a b l e i n s o l u t i o n s o f pH n o t h i g h e r t h a n 4.5. S o l u t i o n s p r e p a r e d i n pH 2 . 5 and 4.5 b u f f e r s c o n t a i n a t l e a s t 90 p e r c e n t o f t h e i r i n i t i a l a c t i v i t y a f t e r 72 hours a t 4OC ( 2 ) . In 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 , c e f a c l o r undergoes a r a p i d l o s s o f a c t i v i t y . When h e l d i n Mueller-Hinton b r o t h a t 37OC o v e r n i g h t , 30 t o 60 p e r c e n t o f t h e i n i t i a l a c t i v i t y o f t h e s o l u t i o n is l o s t ( 3 , 4 ) . 5. Drug metabolism

When c e f a c l o r was a d m i n i s t e r e d t o normal v o l u n t e e r s , peak serum c o n c e n t r a t i o n s o f c e f a c l o r o c c u r r e d about one hour g a f t e r a d m i n i s t r a t i o n . A 250 m dose gave an approximate g peak l e v e l of 7 mcg/ml. A 500 m dose gave an approximate peak level o f 13 mcg/ml, and a 1 gram dose gave a n approximate peak l e v e l o f 23 mcg/ml ( 5 , 6 ) . The mean serum

118

LESLIE J. LORENZ

h a l f l i f e o f c e f a c l o r i n normal a d u l t v o l u n t e e r s a s d e t e r mined by s e v e r a l i n v e s t i g a t o r s ranges from 29 t o 60 minutes (5-10). C e f a c l o r i s r a p i d l y e x c r e t e d i n t h e u r i n e . In s e v e r a l s t u d i e s , 38 t o 54 p e r c e n t o f t h e drug was d e t e c t e d i n t h e u r i n e i n t h e f i r s t two hours a f t e r a d m i n i s t r a t i o n ( 7 ) . After e i g h t hours 43 t o 79 p e r c e n t o f t h e drug was found i n t h e urine (6,lO).

These s t u d i e s would t e n d t o i n d i c a t e t h a t about 85 p e r c e n t of t h e drug is e x c r e t e d i n t o t h e u r i n e a s t h e unchanged drug. When c e f a c l o r i s metabolized i n t h e body o r n a t u r a l d e g r a d a t i o n o c c u r s , t h e e f f e c t on t h e molecule i s s e v e r e and no r e c o g n i z a b l e p r o d u c t s have been found.
6. Methods o f a n a l y s i s 6.1. Identification tests 6.1.1. Infrared

The i n f r a r e d spectrum o f a sample i n a potassium bromide p e l l e t may be used f o r i d e n t i t y . In such c a s e s , t h e i n f r a r e d spectrum compares f a v o r a b l y with t h e c e f a c l o r r e f e r e n c e spectrum o v e r t h e range o f 2 . 5 t o 16 microns when recorded i n a s i m i l a r manner. 6.1.2. Nuclear magnetic resonance The NMR spectrum of a c e f a c l o r sample d i s s o l v e d i n deuterium oxide with deuterium c h l o r i d e present, over t h e r a n g e of 0 t o 10 ppm has chemical s h i f t s and i n t e g r a t i o n s which compare f a v o r a b l y t o a c e f a c l o r r e f e r ence m a t e r i a l handled i n l i k e manner. 6.2 Quantitative t e s t s 6.2.1. Microbiological

For b u l k and formulated p r o d u c t s , e i t h e r an a g a r d i f f u s i o n a s s a y w i t h B a c i l l u s s u b t i l i s (ATCC 6633) ( 2 ) o r an automated t u r b i d i m e t r i c a s s a y (AUTOTURW ) with Staphylococcus a u r e u s (ATCC 9144) (2) may b e used f o r a s s a y of c e f a c l o r . Agar d i f f u s i o n a s s a y s w i t h e i t h e r B . s u b t i l i s (2,8,10,11,12,13) o r S a r c i n a l u t e a (ATCC 9341) (2,13) are used t o a s s a y t h e a n t i b i o t i c i n t i s s u e s and b i o l o g i c a l f l u i d s . The g r e a t e s t s e n s i t i v i t y i s o b t a i n e d w i t h - -u t e a S. l a s c o n c e n t r a t i o n s o f c e f a c l o r a s low as 0.025 mcg p e r m g may be determined ( 2 ) .

CEFAC LOR

119

6.2.2.

High performance l i q u i d chromatography

HPLC i s t h e t e c h n i q u e o f c h o i c e f o r determining t h e p u r i t y o f c e f a c l o r i n raw m a t e r i a l , f o r m u l a t e d p r o d u c t s and i n body f l u i d s . C e f a c l o r i s run i n an a c i d i c medium on a r e v e r s e phase column. A Waters Microbondapaks C18 o r o t h e r a l t e r n a t i v e c o l m w i t h similar r e t e n t i o n c h a r a c t e r i s t i c s i s u s e d t o determine t h e p u r i t y . The approximate s o l v e n t system c o n s i s t s o f 2 p a r t s g l a c i a l a c e t i c a c i d , 1 2 p a r t s a c e t o n i t r i l e and 86 p a r t s w a t e r . The s u b s t a n c e i s u s u a l l y monitored a t 254 nm; however, s l i g h t l y improved s e n s i t i v i t y can be o b t a i n e d a t about 265 nm. The samples a r e d i s s o l v e d i n an a c i d i c media s u c h a s a pH 4.6 b u f f e r o r i n d i l u t e f o r m i c o r a c e t i c a c i d . 6.2.3. Iodometric t i t r a t i o n

An i o d o m e t r i c t i t r a t i o n procedure similar t o t h a t used f o r c e p h a l e x i n (14) has been adapted f o r c e f a c l o r . In t h i s procedure a s w i t h o t h e r c e p h a l o s p o r i n s , t h e i n t a c t a n t i b i o t i c does n o t consume i o d i n e , w h i l e t h e a l k a l i - h y d r o l y s i s product o f c e f a c l o r does. The a l k a l i n e h y d r o l y s i s o f c e f a c l o r r e s u l t s i n t h e cleavage o f t h e 6-lactam r i n g . This product t h e n reacts w i t h i o d i n e t o give a q u a n t i t a t i v e t i t r a t i o n procedure f o r cefaclor. This t e s t can be run i n a manual mode a s well a s i n an automated mode with b e h a v i o r s i m i l a r t o t h a t o f c e p h a l e x i n (15). T h i s t e s t i s n o t n e c e s s a r i l y a s t a b i l i t y i n d i c a t i n g t e s t f o r c e f a c l o r s i n c e any molecule w i t h an i n t a c t 6lactam moiety w i l l g i v e a t e s t i n t h i s procedure. 6.2.4. 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 with hydroxylamine

The r e a c t i o n o f hydroxylamine w i t h c e f a c l o r has been used t o determine t h e drug (16). The method i s based on t h e f a c t t h a t hydroxylamine c l e a v e s t h e Plactam r i n g (pH 7 . 0 ) t o form a hydroxamic a c i d . T h i s hydroxamic a c i d forms a c o l o r e d complex w i t h f e r r i c i o n . Again any e n t i t y having t h e B-lactam r i n g i n t a c t w i l l g i v e a t e s t w i t h t h i s p r o c e d u r e . Thus, t h i s t e s t may n o t b e a s t a b i l i t y indicating t e s t f o r cefaclor. 6.3. I m p u r i t i e s 6.3.1. Colorimetric determination o f 3-chloro nuc 1e u s

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LESLIE J . LORENZ

The 3-chloro n u c l e u s o f c e f a c l o r

COOH
has a f r e e a-amino group a d j a c e n t t o a 6-lactam. T h i s t y p e o f moiety i s s e n s i t i v e t o a t e s t w i t h n i n h y d r i n t o form a c o l o r e d r e a c t i o n p r o d u c t . The r e a c t i o n i s c a r r i e d out i n a c i t r a t e b u f f e r and a pH o f about 3.0. A f t e r 30 minutes f o r c o l o r development, t h e absorbance i s r e a d a t 560 nm. 6.3.2. Phenyl g l y c i n e

Phenylglycine c o n t e n t o f c e f a c l o r can b e determined by a high performance l i q u i d chromatographic procedure. I n t h i s procedure, p h e n y l g l y c i n e i s determined u s i n g a Waters Microbondapaks C18 column o r o t h e r s i m i l a r l y s u i t a b l e r e v e r s e phase column. The e l u t i n g s o l v e n t c o n s i s t s o f 0.01 M potassium dihydrogen phosphate t i t r a t e d t o a pH o f 2.7 with phosphoric a c i d and 1 p e r c e n t by volume o f a c e t o n i t r i l e . The column e l u e n t i s monitored a t 2 2 0 nm f o r d e t e r m i n a t i o n o f p h e n y l g l y c i n e . Phenylglycine i s t h u s determined v e r s u s t h e r e s p o n s e o f a p h e n y l g l y c i n e s t a n d a r d handled i n l i k e manner.
A s might be expected, c e f a c l o r e l u t e s very l a t e i n such a system and may be removed from t h e column i n a quick g r a d i e n t o r s t e p g r a d i e n t procedure where t h e a c e t o n i t r i l e composition o f t h e mobile s o l v e n t i s i n c r e a s e d u n t i l t h e e l u t i o n o f t h e c e f a c l o r has been completed.

6.3.3.

Cephalexin

Cephalexin might be a p o t e n t i a l i m p u r i t y a r i s i n g from t h e rearrangement o f t h e 3-exomethylene 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 o f c e f a c l o r . Trace l e v e l s o f c e p h a l e x i n may b e determined by high performance l i q u i d chromatograph t e c h n i q u e s . To determine t h e c e p h a l e x i n c o n t e n t o f c e f a c l o r , a r e v e r s e phase system i s employed u s i n g a Waters Microbondapaks C18 o r o t h e r s u i t a b l y s i m i l a r HPLC column. The e l u t i n g s o l v e n t c o n s i s t s o f 9 1 p a r t s water, 4 p a r t s a c e t o n i t r i l e and 5 p a r t s g l a c i a l a c e t i c a c i d . The column e l u e n t i s monitored a t 265 n f o r m d e t e r m i n a t i o n o f c e p h a l e x i n . The r e s p o n s e o b t a i n e d a t t h e

CEFACLOR

121

e l u t i o n volume o f c e p h a l e x i n i s compared t o t h e r e s p o n s e of a sample c o n t a i n i n g c e p h a l e x i n a t a known composition t o d e t e r m i n e t h e c o n t e n t of c e p h a l e x i n i n t h e c e f a c l o r sample. 6.3.4. 3-Exomethylene a n a l o g o f c e f a c l o r The 3 -e xomethylene a n a l o g o f c e f a c l o r

COOH
i s a p o t e n t i a l i m p u r i t y which may c a r r y through t h e s y n t h e sis i f t h e ozonolysis o f t h e corresponding intermediate s h o u l d be incomplete. T h i s compound i s determined by h i g h performance l i q u i d chromatography. To determine t h i s compound, a Waters Microbondapaks C 1 8 o r o t h e r s u i t a b l y s i m i l a r r e v e r s e phase column i s employed. The e l u t i n g solvent f o r t h i s determination i s 1 p a r t g l a c i a l a c e t i c a c i d , 7 . 5 p a r t s methanol, and 91.5 p a r t s water. The column e l u e n t i s monitored a t 225 nm. The 3-exomethylene a n a l o g i s determined by measuring t h e r e s p o n s e o f t h e 3-exomethylene peak i n t h e sample chromatogram and comparing i t t o t h e r e s p o n s e o f a sample with a known c o n t e n t of t h e 3-exomethylene a n a l o g .
6.3.5. Other i m p u r i t i e s

G r a d i e n t HPLC p r o c e d u r e s can be u t i l i z e d f o r t h e determination o f o t h e r unidentified impurities i n c e f a c l o r . I n t h i s p r o c e d u r e , a Waters Microbondapap C18 column, o r a Dupont Zorbaxs T S column o r o t h e r M s u i t a b l y s i m i l a r HPLC r e v e r s e phase column i s employed. A l i n e a r g r a d i e n t i s run from 2 p e r c e n t g l a c i a l a c e t i c a c i d i n water t o 2 percent g l a c i a l a c e t i c a c i d i n aceton i t r i l e . Most compounds o f i n t e r e s t e l u t e e a r l y i n t h e system s o a 2 p e r c e n t change p e r minute i s used f o r 25 minutes f o l l o w e d by a f a s t e r s l o p e such as 5 p e r c e n t change p e r minute f o r t h e remainder o f t h e chromatogram. Samples a r e p r e p a r e d a t about 25 m p e r m l i n f o r m i c a c i d and a b o u t g 2 0 u l o f such a s o l u t i o n i s c h r o m a t o g r a p h i c a l l y examined. The column e l u e n t i s monitored a t 254 nm. The peak a r e a s of a l l u n i d e n t i f i e d peaks a r e i n t e g r a t e d and compared t o t h e r e s p o n s e o f a c e f a c l o r s t a n d a r d a t about one p e r c e n t o f t h e c o n c e n t r a t e d s o l u t i o n . The assumption i s made t h a t

122

LESLIE J . LOREN2

a l l o t h e r i m p u r i t i e s have s i m i l a r s p e c t r a l p r o p e r t i e s a s c e f a c l o r and an approximation of t h e i r l e v e l s i n t h e sample can t h e n be made.

7.

Determination i n body f l u i d s

M i c r o b i o l o g i c a l and h i g h performance l i q u i d chromatog r a p h i c procedures have been employed f o r t h e d e t e r m i n a t i o n of c e f a c l o r i n b i o l o g i c a l f l u i d s . Generally, p r o t e i n has been p r e c i p i t a t e d from t h e samples by c l a s s i c a l means and t h e f l u i d s are t h e n examined by one o f t h e s e t e c h n i q u e s . When h a n d l i n g b i o l o g i c a l f l u i d s which c o n t a i n c e f a c l o r , care must b e t a k e n so t h a t t h e s o l u t i o n s a r e kept c o l d i n an i c e b a t h o r f r o z e n from t h e time of sampling t o t h e t i m e o f a s s a y . Also, i f p o s s i b l e , i t i s a d v i s a b l e t o a c i d i f y t h e samples t o p r e v e n t l o s s of c e f a c l o r due t o i t s i n s t a b i l i t y a t h i g h e r pH's.
8.

Acknowledgements The a u t h o r wishes t o e x p r e s s h i s s i n c e r e t h a n k s t o t h e f o l l o w i n g people who have provided t h e necessary information f o r s p e c i f i c portions of t h i s chapter:
D. E. Dorman or t h e NMR i n t e r p r e t a t i o n . L. D . H a t f i e l d f o r t h e s y n t h e t i c p r e p a r a t i o n o f ce f a c l o r J . L . Occolowitz f o r t h e mass s p e c t r a l i n t e r p r e t a t ion. H. W. Smith f o r t h e X-ray c r y s t a l i n t e r p r e t a t i o n . L. G. Tensmeyer f o r t h e i n f r a r e d assignments. T. C. T r o x e l l f o r t h e u l t r a v i o l e t s p e c t r a l interpretation. P. G . Wassel f o r t h e c e p h a l e x i n t e s t and t h e 3-chloronucleus t e s t . C. L . Winely f o r t h e m i c r o b i o l o g i c a l a s s a y port ions.

CEFACLOR
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References
1. 2. J . M. I n d e l i c a t o , A. Dinner, L . R. P e t e r s , and W. L. Wilham, J . Med. Chem., 20, 961 (1977). M. A. Fogelsong, J . W. Lamb, a n d J . V. D i e t z , Antimicrob. Agents Chemother. , 13, 49 (1978). C . C. Sanders, Antimicrob. Agents Chemother., -, 490 (1977). 12 D. A . P r e s t o n , Postgrad. Med. J . , - (Supplement 55, No. 4 ) , 2 2 (1979). G . R. Hodges, C. Liu, D . R. Hinthorn, J . L. Harms, and D. L . Dworzack, Antimicrob. Agents Chemother., - 454 (1978). 14, B. R. Meyers, S . Z . Hirschman, G . Wormser, G . Gartenberg, and E. S r u l e v i t c h , J . C l i n . Pharmacol., - 274 (1978). 18, 0. M. Korzeniowski, W. M. Scheld, M. A. Sande, Antimicrobl. Agents Chemother. , 1 2 , 157 (1977). J . Santoro, B. N. Agarwal, R. M a z i n e l l i , N . Wenger, and M. E. Levison, Antimicrob. Agents Chemother., 13, 951 (1978). D. A. SpykerTB. L. Thomas, M. A. Sande, and W. K. Bolton, Antimicrobl. Agents Chemother., - 1 7 2 (1978). 14, R. Bloch, J . J . Szwed, R. S. Sloan, and F. C . L u f t , Antimicrob. Agents Chemother. , 1 2 , 730 (1977). S. J . Berman, W. H. Broughton, G. Sugihara, E. G . C . Wong, M. M. Sato, and A. W . Siemsen, Antimicrob. Agents Chemother., 1 4 , 281 (1978). C. Simon, and U. Gatzemeier, Postgrad. Med. J . , - (Supplement No. 4) , 30 (1979). 55 G . H . McCracken J r . , C. M. Ginsburg, J . C . Clahsen, and M. L. Thomas, J . Antimicrob. Chemother., -, 515 (1978). 4 Federal R e g i s t e r , 21CFR 141, 506. C . E. Stevenson, and L . D. Bechtol, Private Communication. Federal R e g i s t e r , 21CFR 442, 40(b) (1) ( i i )

3. 4. 5. 6.
7.

8. 9. 10. 11.

12.
13.
14. 15.

16.

CEFAMANDOLE NAFATE
Rafik H . Bishara and Eugene C . Rickard
1.

2.

3. 4.

5.

6.

7.

8. 9.
10.

i26 Introduction 126 Description 126 1. I Nomenclature 127 1.2 Formula 127 1.3 Molecular Weight 127 1.4 Appearance, Color, Odor, and Taste 127 Physical Properties 127 2.1 Melting Range 127 2.2 Simple Solubility Profile 128 2.3 Specific Rotation 128 2.4 pH Range 128 2.5 Dissociation Constant (pKJ 128 2.6 Thermal Analysis 128 2.7 Crystallinity 131 2.8 Ultraviolet Spectrum 131 2.9 Circular Dichroism Spectrum 132 2. I 0 Infrared Spectrum 2. 11 Nuclear Magnetic Resonance 134 Spectrum 136 Synthesis 138 Stability-Degradation 140 Pharmacology, Bacteriology, Pharmacokinetics, and Metabolism 140 5.1 Pharmacological Action 140 5.2 Antibacterial Activity 142 5.3 Protein Binding 142 5.4 Pharmacokinetics 143 5.5 Metabolism 144 Method of Analysis 144 6.1 Elemental Analysis 144 6 . 2 Microbiological Assay 145 6.3 Iodometric Assay 145 6.4 Hydroxylamine Assay 146 6.5 Electrochemical Assay 147 6.6 Chromatography 148 6.7 Analysis of Related Materials 148 Analysis of Biological Samples 148 7.1 Microbiological Assay 148 7.2 Liquid Scintillation Assay 149 7.3 Chromatographic Assay 149 Analysis of Pharmaceutical Formulations 150 Acknowledgments 151 References Copyright 0 1980 by Academic Press. Inc.
125
All rights of reproduction i any form resewed. n ISBN: 0-12-260809-7

Analytical Profiles of D u Substances. 9 rg

126

RAFIK H. BISHARA AND EUGENE C. RICKARD

Introduction Cefamandole nafate is a semisynthetic broad-spectrum cephalosporin antibiotic for parenteral administration. The dosage form of cefamandole nafate also contains 6 3 mg of sodium carbonate per gram of cefamandole free acid activity (0.275 moles of sodium carbonate per mole of cefamandole free acid activity). After addition of diluent, cefamandole nafate rapidly hydrolyzes to cefamandole, and both compounds have microbiologic activity in vivo.

1. Description
1.1 Nomenclature
1.1.1 Chemical Name

7-D-Mandelamido-3-<<(l-methyl-lH-tetrazo1-5yl)thio>methyl>-3-cephem-4-carboxylic acid, formate (ester), sodium salt 7 4 D - <(Formyloxy)phenylacetyl>amino>-3-< < ( 1methyl-1H-tetrazol-5-yl)thio~methyl~-3-cephem-4-carboxylic acid, sodium salt 7-D-Mandelamido-3- [ [ ( 1-methy1-lH-tetrazol-5yl)thio] methyl] -8-0x0-5-thia-1-azabicyclo [4.2.0] -oct-2-ene2-carboxylate formate(ester)

1.1.2 1.1.3

Nonproprietary Name Cefamandole nafate Proprietary Name Mandol @ Mandokef ,

CEFAMANDOLE NAFATE

127

1.2

Formula
1.2.1

Empirical
C19H17N606S2-Na

Salt

1.2.2

Structural

H
1.3

Molecular Weiaht 512.49

1.4

Appearance, Color, Odor, and Taste

White t o off-white, o d o r l e s s powder with a s l i g h t l y bitter taste.

2 . Physical P r o p e r t i e s 2.1

Meltina Ranae

Cefamandole n a f a t e s t a r t s t o d i s c o l o r with e v o l u t i o n of gas a t about 190C under U P c o n d i t i o n s f o r C l a s s I S substances (1).

2.2

Simple S o l u b i l i t y P r o f i l e

The sample i s s o n i c a t e d f o r one minute a t ambient temperature. Solvent Water p 1 . 2 (USP X I X ) H p 4.5 (USP X I X ) H p 7 . 0 (USP X I X ) H Met hano 1 Octanol I sopropanol D i e thy l e t h e r Ethylacetate Chloroform Benzene Cyclohexane mg/ml
) 3 3 3 - <lo00

c0.5
3 3 3 3 - <lo00

>,333-<1000 310-<33.3 <O. 5

< 0.5
<0.5 < 0.5

<0.5 <0.5 < 0.5

128

RAFIK H. BISHARA A N D EUGENE C. RICKARD

2.3

Specific Rotation

R o t a t i o n measured a t sodium D l i n e (approximately 589nm) o f a 10% s o l u t i o n of cefamandole n a f a t e i n pH 5.0 a c e t a t e b u f f e r (1.04M) is -38 f 5 c a l c u l a t e d on an anhydrous b a s i s . I t i s t o be noted t h a t t h e s p e c i f i c r o t a t i o n i s a f u n c t i o n of c o n c e n t r a t i o n . I n unbuffered s o l u t i o n s o r s o l u t i o n s b u f f e r e d a t p H 6.0-7.5, t h e conversion o f cefamandole n a f a t e t o cefamandole c a u s e s a d r i f t , w i t h t i m e , of t h e measured r o t a t i o n . There i s minimal d r i f t and pH dependence i n t h e range o f pH 4.5-5.5.
2.4

pH Range The pH o f a 10% aqueous s o l u t i o n i s between 3.5 and

7.0. 2.5 D i s s o c i a t i o n Constant The c a r b o x y l a t e PKa o f cefamandale n a f a t e i s about 2.6-2.9 as determined by aqueous t i t r a t i o n o r 3.0 a s determined by spectrophotometry ( 2 ) . 2.6 Thermal Analysis 2.6.1 D i f f e r e n t i a l Thermal A n a l y s i s

A DTA thermogram of cefamandole n a f a t e , a t a h e a t i n g r a t e o f 5 C p e r minute i n a n i t r o g e n atmosphere o f O 40cc p e r minute, shows ( f i g u r e 1) an exotherm a t 207'C i n d i c a t i n g decomposition.

2.6.2

Thermogravimetric A n a l y s i s

A TGA thermogram of cefamandole n a f a t e , run simultaneously with t h e above DTA, shows ( f i g u r e 1) a weight l o s s beginning a t 63OC r e s u l t i n g i n a 0 . 2 % loss a t 137OC. A t 163'C a n o t h e r loss b e g i n s r e s u l t i n g i n a continuous loss through decomposition.

2.7

Crystallinity
2.7.1

C r y s t a l l i n e Habit

The anhydrate form ( y ) o f cefamandole n a f a t e g e n e r a l l y c r y s t a l l i z e s a s small n e e d l e s . 2.7.2 X-Ray Powder D i f f r a c t i o n The following d a t a d e s c r i b e t h e p a t t e r n f o r t h e anhydrate form ( y ) o f cefamandole n a f a t e , where d i s equal t o t h e i n t e r p l a n a r s p a c i n g measured i n terms of Angstroms The r a t i o 1/11 i s t h e i n t e n s i t y of t h e X-ray maxima based upon a v a l u e o f 100 f o r t h e s t r o n g e s t l i n e .

(x).

CEFAMANDOLE NAFATE

129

Cu-Ni-A 1.5405
-

I/I 1

17.80 11.76 9.39 7.49 7.18 6.20 5.52

30
30
10

3.72 3.51 3.32 3.06 2.91 2.83 2.75 2.56 2.36 2.17 2.11

100

5
2

70 20 15 40 40 20
80

10 15 15
10

5.00
4.74 4.54 4.20 3.98
TGA

10
10

50
10 63OC 137OC

10

163OC

100%

X D T A

i
K

90%

80%

70%

60 '10

Figure 1.

Thermogravimetric Analysis and Differential Thermal Analysis Thermograms of Cefamandole Nafate

PH UNBUFFERED CONDITIONS 0.5 NM RESOLUTION 1 CM PATHLENGTH

MTE
2-eW

CMPD LOT#
CONC - 7

CEFAMANDOLE NAFATE REFERENCE STANDARD

MCG./ML

IN WATER

Figure 2.

Ultraviolet Spectrum of Cefamandole Nafate

CEFAMANDOLE NAFATE

131

2.8

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

The u l t r a v i o l e t spectrum o f cefamandole n a f a t e i n w a t e r i s g i v e n i n f i g u r e 2 . The spectrum e x h i b i t s a maximum a t 269nm w i t h a molar a b s o r p t i v i t y of 10,800 ( E 1 - c m / l % a b o u t 211). The chromophores i n cefamandole n a f a t e a r e 3-cephem, t h i o t e t r a z o l e , p h e n y l , amide, and e s t e r . Of t h e s e , o n l y t h e 3-cephem and t h i o t e t r a z o l e make s i g n i f i c a n t c o n t r i b u t i o n s above a b o u t 225nm. For t h e 3-cephem group i n H 2 0 , ~( 2 6 1 nm) = 9200 i s e x p e c t e d from a TI + IT* t r a n s i t i o n ; t h e r e i s p r o b a b l y a TI TI* t r a n s i t i o n a t a b o u t 230 nm. a l s o . Thiotetrazole m peak w i t h E a b o u t 1 2 , 8 0 0 . On s u b s t i t u t i o n i n t o h a s a 245 n t h e a n t i b i o t i c , t h e peak a p p a r e n t l y r e d s h i f t s t o around 275 n m w i t h a d r a m a t i c i n t e n s i t y d e c r e a s e t o E a b o u t 4000. T h i s can be s e e n by comparison o f -OH v e r s u s t h i o t e t r a z o l e substitution.
-f

2.9

C i r c u l a r Dichroism Spectrum

The c i r c u l a r d i c h r o i s m (CD) spectrum o f cefamandole n a f a t e i n w a t e r i s g i v e n i n f i g u r e 3. The A E maxima and z e r o s are: A ,nm AE 264 7.11 0 246.7 -19.81 228.5 -12.70 209 0 195
lo

AE

- 10

- 20
195 220 245 270 295

Wavelength, nm

F i g u r e 3.

C i r c u l a r Dichroism Spectrum o f Cefamandole N a f a t e

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RAFIK H. BISHARA A N D EUGENE C . RICKARD

The CD of cefamandole above 220 n m appears t o be The 228.5 nm negative t h a t t y p i c a l t o t h e 3-cephem group. CD comes from t h e n -+ T* 3-cephem t r a n s i t i o n . The p o s i t i v e CD peak a t 264 n m probably l o c a t e s t h e p o s i t i o n of t h e T 7 ~ * 3-cephem t r a n s i t i o n . The f a c t t h a t t h e absorption m r a t h e r than 264 i s probably due t o t h e peak i s a t 269 n t h i o t e t r a z o l e a b s o r p t i o n , from which CD i s e i t h e r weak o r absent. S i m i l a r l y , t h e t h i o t e t r a z o l e group i s responsible f o r t h e absorption above 295 nm, where t h e CD i s zero. The lack of o p t i c a l a c t i v i t y (CD) i n t h e t h i o t e t r a z o l e 275 nm t r a n s i t i o n may r e s u l t from i t s conformational m o b i l i t y , a s implied by molecular models ( 3 ) .
-f

2.10

I n f r a r e d Spectrum

The i n f r a r e d spectrum of cefamandole n a f a t e i n a potassium bromide p e l l e t i s given i n f i g u r e 4. Major band assignments a r e a s follows: I n f r a r e d Absorption, cm- 1 Group Responsible
3 5 0 0 , very broad

H-bonded H20 H-bonded t r a n s N-H secondary amide in

3260, sharp 3040-3020 2940 2880 1755

1712

CH i n mono-substituted phenyl r i n g
CH s t r e t c h i n N-CH3

and

S-CH2 carbonyl i n @-lactam carbonyl i n e s t e r

1668 1620 1610 1600 1530 1490 1450 1385 o r 1357

carbonyl i n amide (termed Amide I )

.carboxylate s a l t C=C, conjugated t o a c i d group C=C i n phenyl r i n g Amide I1 aromatic C=C

N-CH3

carboxylate s a l t
C=O i n e s t e r

1174
1100

t e r t i a r y n i t r o g e n , -NCH p a t t e r n f o r mono-

745 692

s u b s t i t u t e d phenyl r i n g

PATH PELLET ISM# 16 MINUTE SCAN TIME CONDITIONS 3 WAVENUWBER RESOLUTION

CMPD LOT#

CEFAMANDOLE NAFATE REFERENCE STANDARD

IN KBr

CONC DATE 4-20-79 Figure 4. Infrared Spectrum of Cefamandole Nafate

0 8 MG. .7

134

RAHK H. BISHARA AND EUGENE C. RICKARD

2.11

Nuclear Magnetic Resonance Spectrum 2.11.1 Proton NMR

The 60 MHz proton NMR spectrum of cefamandole nafate in deuterated dimethylsulforide is given in figure 5. Assignment of the resonances are as follows:

o=c

a Description of Resonance:-

4
(J = 8.5)

C02Na
NH CHO -

Assignment:

d/9.38 p.p.m. s/8.37 p.p.m. m/7.45 p.p.m. s/6.13 p.p.m.

(5H)

aromatic protons -CH-CO

I--

/O

dd/5.53 p.p.m. d/4.87 p.p.m. s/3.90 p.p.m.

(J = 8.5, 5)
(J = 5)

H-7 H-6 CH2(3) NCH3 CH2 (2) solvent

AB-system/4.32 p.p.m. (3H)

b
2

(2H) (JAB = 12)

18)

m/E. 3.4 (2H)- (J AB m/2.53

Unless otherwise specified, each resonance represents a single proton. Coupling constants (J) are in Hz. This resonance is superimposed by that due to moisture in the solid sample. 2.11.2 3C-NMR

The fully decoupled 13C-NMR spectrum of cefamandole nafate in deuterium oxide is given in Figure 6. The spectrum was obtained on a Varian FT80-A instrument at

3. 75

2.50

1.25

TIME 250 SWEEP WIDTH 1000 SPIN RATE 45

SWEEP

CMPD LOT#
CONC

CEFAMANDOLE NAFATE REFERENCE STANDARD

MG./ML

108.2

IN DMSB-DB

DATE

2/12/80

F i g u r e 5.

P r o t o n NMR Spectrum o f Cefamandole N a f a t e

136

RAFIK H. BISHARA A N D EUGENE C. RICKARD

ambient temperature and using a 5mm sample tube. The data consist of 15,000 acquisitions of 16,384 data points over a 5000 Hz spectral width. The reference line is at 1348 Hz (67.46). Assignment of the resonances are as follows: a Assignment Description 27.4 34.8 37.1 58.3
59.5

CH2 ( 2 ) N-CH3 CH2 (3) C (6)

75.6
119.0

c (7) cgH5 -cH c (3)

C (c),C (e) -aromatic -aromatic C (b),C (f) C (d)-aromatic

128.4 129.9 130.6 131.7 134.5 154.6 162.8 164.6 168.3 171.8 a

c (4)
C (a) -aromatic -tetrazole C (1) CHO C (8)

c (4)
CO-NH -

Chemical shift, 6 , in ppm from TMS (0.0 ppm). Each resonance represents a single carbon unless otherwise stated. Synthesis

3.

D ( - ) Mandelic acid (I) is formylated to produce 0-formylmandelic acid (II), which is then treated with excess thionylchloride to form D (-1 0-formylmandeloyl chloride (111). Formylation of 7-aminocephalosporanic acid (IV) produces 7-formamidocephalosporanic acid (V), which is then treated with l-methyl-1H-tetrazole-5-thi01, sodium salt (VI) to afford 7-formamido-3-(l-methyl)-1H-tetrazol-5-ylthiomethyl) -3-cephem-4-carboxylic acid (VII). Deformylation of (VII) yields 7-amino-3-(l-methyl-lH-tetrazol-5-ylthiomethyl)-3cephem-4-carboxylic acid (VIII) The nucleus (VIII) is silylated with monosilylacetamide (MSA) and is then acylated

zw

,80

150

isu

110

170

110

i w
cwmlhl

'IU

an

70

1x1

50

1
40

10

20

to

Figure 6.

I3C-NMR Spectrum of Cefamandole Nafate

138

RAFIK H . BISHARA AND EUGENE C. IUCKARD

with 0-formylmandeloyl chloride (111) to provide 7- (D-2fonnyloxy-2-phenylacetamido)-3-(l-methyl-lH-tetrazol-5ylthiomethyl)-3-cephem-4-carboxylic acid (IX). Alternatively, (IV) is acylated with (111) to produce 7- (D-2-formyloxy-2phenylacetamido)-3-cephem-4-carboxylic acid (X). Addition of 1-methyl-5-thio-1, 2, 3, 4-tetrazole (XI) to (X) produces (IX). The sodium salt (XII) is produced by treating (1x1 with sodium 2-ethylhexanoate in acetone. The flow diagram of the synthesis presented above (4-7) is shown in figure 7.

4. Stability-Degradation
The ester function of cefamandole nafate is quite labile to nuclcophilic attack by water or hydroxide ion in slightly acidic to slightly alkaline aqueous solutions in vitro (81, giving cefamandole (11) as the product (figure 8). Indelicato et.al..found that the formyl moiety of cefamandole nafate hydrolyzes with a half-life at 37OC which ranges from about 290 minutes at pH 5.5 to about 7 minutes at p H 8. In unbuffered solutions, the addition of bases such as sodium carbonate, ethanolamine and tromethainine produce rapid hydrolysis. For sodium carbonate, the fraction of cefamandole nafate which hydrolyzes is approximately equal to the number of equivalents of carbonate added per mole of cefamandole nafate and the hydrolysis reaches steady state in about 30 minutes or less for 0.28, 0.60 and 0.90 mole equivalents of carbonate. Ester hydrolysis is essentially complete within a few minutes when one mole of amine is added per mole of cefamandole nafate. Retention of chirality in the 7-D-mandelamido sidechain is observed for carbonate hydrolysis, which indicates cleavage of the acyl-oxygen bond. The hydrolysis of cefamandole nafate also occurs very rapidly in vivo with half-lives of 6-7 minutes and 10-17 minutes for dogs and humans respectively (9). In artificially accelerated degradation studies (2, 10, ll), ester hydrolysis is observed in unbuffered solutions stored at 25, 37 or 6OoC for 24 hours, in 0.1N hydrochloric acid at 25OC for 24 hours, in aqueous solutions (46OC) exposed to a high intensity UV light source for 64 hours, and in the reconstituted formulation. Hydrolysis of the solid sample varies with water content (11); no hydrolysis is observed for a dry (<0.1% water) sample after 2 months at 5OoC (11) 24 hours at 100C or 22 days at 6OoC (10) but , , hydrolysis is observed in a wet sample heated for 3 weeks at 5OoC (11). The 1-methyl-5-thio-1, 2, 3, 4-tetrazole (111) product is formed under the same conditions which produce ester hydrolysis. In addition to these

a,

a,

O="

140

RAFIK H. BISHARA AND EUGENE C. RICKARD

reactions, other products can be formed. In neutral or unbuffered solutions, the nucleus which remains after loss of I11 may form the hydroxymethyl compound (IV) or, in slightly acidic solutions, dehydrate to the a,@-unsaturated lactone (V). In extremely acidic conditions, the mandelic acid moiety (VI) is cleaved and the 8-lactam system is destroyed. Strongly basic conditions produce mandelic acid and destruction of the @-lactam. This information is summarized in figure 8 ; the protonation of compounds I-IV and VI will depend upon pH.
5.

Pharmacology, Bacteriology, Pharmacokinetics and Metabolism

5.1

Pharmacological Action

Cefamandole nafate is a parenteral cephalosporin antibiotic. In vitro, it is rapidly converted to cefamandole by hydrolysis of the formyl ester after dissolution ( 8 ) . Because of rapid in vivo conversion of cefamandole nafate to cefamandole, the latter is the predominant circulating moiety after administration of cefamandole nafate to laboratory animals and humans (9). The calculated rate constant for hydrolysis of cefamandole nafate is higher in dogs than in humans, yielding t+ values of 6-7 minutes and 10-17 minutes respectively. Disappearance of cefamandole nafate from the plasma of dogs (due to hydrolysis and elimination) is slightly faster than from humans with a half-life (t+) of 4-6 minutes and 6-9 minutes, respectively.
5.2

Antibacterial Activity
5.2.1

In Vitro

In many conventional laboratory evaluation procedures, the in vitro antibacterial activities of cefamandole and cefamandole nafate appear virtually identical due to the hydrolysis of cefamandole nafate (2). Cefamandole is active in vitro against a variety of gram-pos,itive and gram-negative microorganisms. In addition, it is active against a number of gram-negative aerobic organisms and both gram-positive and gram-negative anaerobes that have not traditionally been in the spectrum of the cephalosporins. Extensive in vitro studies have documented this expanded spectrum for cefamandole ( 1 3 - 2 6 ) .
5.2.2

In Vivo

The efficacy of cefamandole is identical to that of cefamandole nafate in treating experimental animal infections, indicating that rapid conversion of cefamandole nafate to cefamandole occurs in vivo ( 1 2 ) .

HO

H003- t k l ( 0 )

IA

@NO03

A1

CH3
I

eN003

I1

CH3 eN003 I " I S - ' H 3 f z N

0
HN-03-H3

H 3=0

io

I 1

N-

142

RAFIK H. BISHARA A N D EUGENE C. RICKARD

5.3

Protein Binding

The protein binding of cefamandole is 74 percent when determined by an ultrafiltration method (27) and 67 percent with a range of 56-78 percent when measured by equilibrium dialysis (28). However, this data is obtained from an initial antibiotic concentration of 25mcg/ml and 20mcg/ml, respectively. Cefamandole appears to be rapidly dissociated from the serum proteins as indicated by its relatively short half-life and its rapid appearance in the urine (18). 5.4 Pharmacokinetics 5.4.1 Serum Concentrations

After intramuscular administration of a 500mg dose of cefamandole to normal volunteers, the mean peak serum concentration is 13mcg/ml (18, 26, 29, 30). After lg doses, the mean peak level is 25 mcg/ml (18, 26, 27, 30, 31). These peaks occur at 30-120 minutes. The decline of antibiotic concentration in the serum is biphasic with a rapid fall in the first two hours. Thereafter, levels decrease more slowly. Detectable concentrations are present for six to eight hours (18). Multiple-dose studies in patients given cefamandole intramuscularly show no evidence of accumulation (30, 32). Following intravenous doses of 1, 2 and 39, serum concentrations are 139, 240 and 533mcg/ml at 10 minutes, respectively (18, 27, 29, 33). These concentrations decline to 0.8, 2.2 and 2.9mcg/ml at four hours. Intravenous administration of 49 doses every 6 hours produce no evidence of accumulation in the serum. 5.4.2 Half-Life The half-life after an intravenous dose is 32 minutes: after intramuscular administration, the half-life is 60 minutes (27, 28, 33, 34). 5.4.3 Serum Clearance and Apparent Volume of Distribution (AVD)

A mean serum clearance of 230 ? 99/ml/min./ 1 73m2 is found (27, 28) following intravenous administration . of cefamandole. The AVD ranges from 10-67 percent of body weight. Following intramuscular administration of cefamandole the mean AVD is 17.11 liters, or 24 percent of the body weight (29), which is similar to the findings in the intravenous studies described above.

CEFAMANDOLE NAFATE

143

5.4.4

Urine Concentrations, Excretion, and Renal Clearance

Cefamandole, in addition to being excreted by glomerular filtration, is also secreted by the renal tubules (32). Sixty-five to eighty-five percent of cefamandole is excreted by the kidneys after intramuscular injection of the drug, in the first 8 hours (18). The mean eight-hour urine concentrations are 254mcg/ml after 500mg doses and 1357 mcg/ml after lg doses. Similar results are obtained by other investigators (27, 30). After intravenous administration of cefamandole, 75 to 100 percent is excreted in the urine in the first six to eight hours, and concentrations exceed 1000 mcg/ml with 500mg doses (27, 29). Probenecid slows tubular excretion and doubles the peak serum level and the duration of measurable serum concentrations. The renal clearance of cefamandole before and after administration of probenecid is 302 f 60 and 80 ? 14ml/min./l. 73m2 , respectively (32, 34) In the presence of renal impairment, urinary excretion of cefamandole is slowed (32).

5.4.5.

Body Fluid and Tissue Concentrations

Distribution of cefamandole in body fluids and tissues following therapeutic doses of the antibiotic has been determined in bones and joints (351, gallbladder (361, interstitial fluid (37) and uterine tissue (38, 39). Tissue analysis gives primarily qualitative rather than meaningful quantitative data as to the presence or absence of an antibiotic in a particular body fluid or tissue. Therapeutic efficacy cannot be predicted by the level attained in a specific body fluid or tissue. 5.5 Metabolism A study of the metabolic fate of 14C-cefamandole in rats and dogs shows that after rapid in vivo hydrolysis of cefamandole nafate to cefamandole, the antibiotic is very resistant to metabolic degradation in both species (40). In dogs, cefamandole escapes metabolism and is eliminated as unaltered antibiotic almost exclusively by renal excretion. In rats, cefamandole is somewhat labile to metabolism. However , a major portion of the administered antibiotic is eliminated unchanged principally by renal excretion. Essentially all of the administered radiocarbonnoteliminated by renal excretion is eliminated via biliary excretion. The fraction of radiocarbon dose remaining in the body of the rats after 24 hours amounts to less than 2%. The half-life of cefamandole in the blood of both species ranges from 30 to 42 minutes. Tissue level studies reveal no abnormal deposition of the antibiotic or metabolite in any tissue,

144

RAFIK H. BISHARA A N D EUGENE C. RICKARD

although all tissues examined contained concentrations of the antibiotic. The only tissues possessing significantly higher levels than that found in the blood are the kidney in both species and liver in dogs.
6.

Method of Analysis 6.1 Elemental Analysis Element C H N

0
S

Theory ( % ) _____ Sodium Salt

44.53 3.34 16.40 18.73 12.51 4.49

Free Acid
46.53 3.70 17.13 19.57 13.07

Na
6.2

Microbiological Assay

Cefamandole nafate is rapidly hydrolyzed to cefamandole in vivo ( 9 ) or in aqueous solutions of pH 5.5-8 (8). The in vitro activity of cefamandole nafate relative to that of cefamandole is different for some organisms when the assay conditions do not produce hydrolysis of cefamandole nafate (12, 4 1 ) . Thus, a chemical hydrolysis is required prior to the microbiological assay in order to obtain results which are valid measurements of the bioactivity, and to avoid experimental difficulties due to partial hydrolysis during the assay. The microbiological assay is described for turbidimetric and agar diffusion methods. These assays are not specific for cefamandole nafate in the presence of impurities and/or degradation products. However, most contaminants will tend to have lower specific activity than cefamandole nafate so that a certain degree of selectivity is achieved.
6.2.1

Turbidimetric Method

The turbidimetric assay is performed after hydrolysis to cefamandole, e.g., 1 5 minutes at room temperature with 0.87 moles of sodium carbonate per mole of cefamandole nafate followed by dilution with 0.1M phosphate buffer, pH6. The sample is further diluted to the reference concentration with O.lM, pH6 phosphate buffer and added to medium # 3 ( 4 2 ) inoculated with Staphylococcus aureus (ATCC 9 1 4 4 ) . Dose response concentrations are 0.02 to l.0mcg cefamandole per ml of inoculated medium. The precision of the assay is about 2.5% as measured by the relative standard

CEFAMANDOLE NAFATE

145

d e v i a t i o n ( R S D ) of t h e assay ( 4 3 ) . 6.2.2 Agar Diffusion Method For p e n i c y c l i n d e r agar d i f f u s i o n a s s a y s of r a w m a t e r i a l s o r f i n a l dosage forms of cefamandole n a f a t e , e i t h e r S aureus (ATCC 6538P) o r B a c i l l u s s u b t i l i s (ATCC 6633) may -. be used. With an a g a r p l a t e system c o n s i s t i n g of 1 0 m l of agar medium N o . 2 ( 4 2 ) as base l a y e r and 5 m l of a g a r medium No. 1 ( 4 2 ) a s seed l a y e r , dose response c o n c e n t r a t i o n s of 0.5 t o 2 . 0 mcg of cefamandole n a f a t e p e r m l are a p p r o p r i a t e f o r both organisms. Sample p r e p a r a t i o n , h y d r o l y s i s followed by d i l u t i o n i n 0 . 1 M phosphate b u f f e r (pH 6.0), i s c a r r i e d o u t i n t h e same manner a s f o r t h e t u r b i d i m e t r i c assay. For assay of b i o l o g i c a l f l u i d s , t h e B. s u b t i l i s assay i s used. When s e n s i t i v i t y g r e a t e r than 0.s mcg p e r m l i s r e q u i r e d , a 5 m l s i n g l e l a y e r p l a t e , medium No. 1, with dose response concentrations of 0 . 1 t o 1 . 0 rncg p e r m l may be employed. Samples a r e n o t hydrolyzed s i n c e cefamandole n a f a t e i s conv e r t e d t o cefamandole i n vivo ( 9 , 1 2 ) , b u t standard m a t e r i a l m u s t be hydrolyzed t o cefamandole f o r p r e p a r a t i o n of s t a n d a r d curves. 6.3 I o d a n e t r i c Assay

Cefamandole n a f a t e can be determined b y a n i o d o m e t r i c t i t r a t i o n procedure s i m i l a r t o t h a t a p p l i e d t o o t h e r cephalosporins ( 4 5 ) . The 6-lactam r i n g i s hydrolyzed f o r about 1 0 minutes with a l k a l i (0.2N N a O H ) , a c i d i f i e d (0.5N HC1) and allowed t o r e a c t with i o d i n e f o r about 5 minutes. A l l r e a c t i o n s are thermostated t o 37'C. The d i f f e r e n c e i n i o d i n e uptake i s measured between a blank (no sodium hydroxide added) and t h e sample. The RSD of t h i s assay i n an automated mode i s about 1-2% ( 2 ) . The measurement of i o d i n e uptake i s not s p e c i f i c f o r cefamandole n a f a t e , e s p e c i a l l y i n t h e presence of i m p u r i t i e s and/or degradation products which contain t h e i n t a c t 6-lactam system. 6.4 Hydroxylamine Assay The hydroxylamine assay i s an a l t e r n a t e chemical assay procedure f o r cefamandole n a f a t e ( 4 4 ) . The method i s based upon cleavage of t h e 6-lactam by hydroxylamine t o form a hydroxamic a c i d which i s then r e a c t e d with a c i d i f i e d f e r r i c ion t o give a colored complex t h a t can be monitored a t 480nm. A blank c o r r e c t i o n or i n t e r f e r i n g non-6-lactam chemical s p e c i e s which r e a c t with hydroxylamine i s incorporated by adding t h e hydroxylamine t o an a c i d i c s o l u t i o n of t h e sample ( t h e a c i d d e s t r o y s a l l 6-lactam e n t i t i e s ) . However, i t i s not p o s s i b l e t o c o r r e c t f o r i n t e r f e r e n c e s due t o i m p u r i t i e s and/or degradation products which contain an i n t a c t 6-lactam.

146

RAFlK H. BISHARA AND EUGENE C. RICKARD

6.5

Electrochemical Assay

The electrochemical assays r e l y on t h e r e d u c t i v e cleavage of t h e t h i o e t h e r linkage a t t h e 3' p o s i t i o n of t h e cephalosporin ( 2 ) . This reduction occurs a t t h e dropping mercury e l e c t r o d e (DME) with a half-wave p o t e n t i a l of about -0.75V VS. SCE ( s a t u r a t e d calomel e l e c t r o d e ) f o r a 0 . 4 m sample concentration (0.2mg/ml) i n a p 2 . 4 McIlvaine b u f f e r , H and v a r i e s with concentration and p ( 2 ) . The electrochemiH c a l methods include c o n t r o l l e d p o t e n t i a l coulometry, DC o r sampled DC polarography and d i f f e r e n t i a l p u l s e polarography. 6.5.1 Controlled P o t e n t i a l Coulometry The c o n t r o l l e d p o t e n t i a l coulometric d e t e r mination has been p r e v i o u s l y described ( 2 ) . Coulometry i s an absolute method i n which t h e t o t a l charge consumed i s measured during an exhaustive e l e c t r o l y s i s . That i s , no comparison t o a standard i s r e q u i r e d ( 4 6 ) . Thus, it i s an important a n a l y t i c a l t o o l f o r e v a l u a t i o n of p u r i t y o f s t a n d a r d m a t e r i a l s , b u t i t i s not normally used f o r r o u t i n e a s s a y s . For cefamandole n a f a t e , a p o t e n t i a l of -0.875V vs. SCE i s used and t h e r e d u c t i o n i s performed a t a s t i r r e d mercury pool e l e c t r o d e . The p r e c i s i o n of t h i s method applied t o cefamanS dole n a f a t e i s approximately 0.95% as measured by t h e R D ( 4 6 ) . This assay measures a l l compounds which have functiona l groups t h a t a r e reduced a s e a s i l y o r more e a s i l y than cefamandole n a f a t e . However, t h e most common i m p u r i t i e s and degradation products do not i n t e r f e r e ( 2 ) . 6.5.2
DC and Sampled DC Polarography
DC and sampled DC ( T a s t ) polarography can be applied t o cefamandole n a f a t e ( 2 ) . These methods e x h i b i t a p r e c i s i o n of about 1.4-1.5% f o r t h e measurement of a sample versus a standard ( 2 , 4 7 ) . Their s e l e c t i v i t y depends upon t h e d i f f e r e n c e between half-wave p o t e n t i a l s of t h e v a r i o u s s p e c i e s . Since t h e s e methods can d i s c r i m i n a t e a g a i n s t more e a s i l y r e d u c i b l e a s w e l l as l e s s e a s i l y r e d u c i b l e s p e c i e s , both a r e more s e l e c t i v e than c o n t r o l l e d p o t e n t i a l coulometry ( 2 , 48). These methods a r e more s e l e c t i v e than microb i o l o g i c a l , iodometric o r hydroxylamine a s s a y s , b u t n o t as s e l e c t i v e a s t h e high performance l i q u i d chromotographic assay. A automated, microprocessor c o n t r o l l e d polarographic n system has r e c e n t l y been described f o r t h i s assay (49) and t h e l i n e a r i t y range i n v e s t i g a t e d ( 4 7 ) .

6.5.3

D i f f e r e n t i a l P u l s e 'Polarography

The d i f f e r e n t i a l p u l s e polarographic assay i s t h e o f f i c i a l chemical assay i n t h e Code of Federal Regulat i o n s ( 4 5 ) . I t r e l i e s upon t h e same electrochemical

CEFAMANDOLE NAFATE

I47

reduction process and achieves s i m i l a r ( o r s l i g h t l y g r e a t e r ) s e l e c t i v i t y than DC polaroqraphy. The p r e c i s i o n f o r t h i s assay i s about 1 . 2 % f o r a sample measured versus a s t a n d a r d using t h e automated, microprocessor c o n t r o l l e d system. The l i n e a r i t y and o t h e r c h a r a c t e r i s t i c s of t h i s assay have been r e c e n t l y described ( 4 7 ) . 6.6 Chromatography 6.6.1 Paper Chromatography

Cefamandole n a f a t e may be chromatographed on u n t r e a t e d Whatman N o . 4 paper using a methyethyl ketone/water (92:8 v/v) developing s o l v e n t ( 4 0 , 5 0 ) . When about 30 ml of developing s o l v e n t i s used and development t i m e i s 6-7 h o u r s , t h e s o l v e n t f r o n t w i l l run o f f t h e paper and t h e cefamandole n a f a t e zone w i l l move about 0.75 of t h e d i s t a n c e t o t h e end of t h e paper. g . s u b t i l u s innoculated i n an agar medium (6g peptone, 39 y e a s t e s t r a c t , 1.59 beef e x t r a c t , 209 a g a r d i s s o l v e d i n 1 l i t e r of water and p a d j u s t e d t o 7 . 2 ) can be H used f o r d e t e c t i o n when approximately 1 mcg of sample i s applied. 6.6.2 Thin Layer Chromatography

The R value i s about 0.52 f o r cefamandole f n a f a t e when chromatographed on a s i l i c a g e l 60 F254 t h i n l a y e r p l a t e developed by ethylacetate/acetone/glacial a c e t i c acid/water (5:2:1:1 v/v/v/v) i n a s a t u r a t e d chamber ( 2 ) . The sample may be dissolved i n water o r i n t h e developing s o l vent. The sample i s v i s u a l i z e d under s h o r t wavelength UV l i g h t (254nm) o r under white l i g h t a f t e r exposure t o i o d i n e describes a vapors. The Code of Federal Regulations 45 continuous flow t h i n l a y e r chromatographic (TLC) system f o r i d e n t i f i c a t i o n which employs a s i l i c a g e l G t h i n l a y e r p l a t e developed by n-butanol/glacial a c e t i c acid/water ( 4 : l : l v / v / v ) . This t e s t u s e s a s t a r c h i o d i d e / g l a c i a l a c e t i c acid/O.lN i o d i n e spray reagent f o r v i s u a l i z a t i o n . Other TLC systems and v i s u a l i z a t i o n procedures f o r cephalosporins are d e s c r i b e d by M a r r e l l i ( 4 4 ) . 6.6.3 High Performance Liquid Chromatography

A reverse-phase high performance l i q u i d chromatographic (HPLC) assay may be performed using a C8 column with an i s o c r a t i c e l u t i n g s o l v e n t ( 2 % g l a c i a l a c e t i c a c i d ) , 20% a c e t o n i t r i l e and 78% water, by volume) a t a f l o w r a t e of 2 m l / m i n u t e ( 5 1 ) . Samples are prepared i n g l a c i a l a c e t i c a c i d , d e t e c t i o n i s by W absorbance a t 254 nm. and t h e r e t e n t i o n t i m e f o r cefamandole n a f a t e i s about 9.5 minutes. This method e x h i b i t s a p r e c i s i o n of about 2-3% (RSD). Cefamandole n a f a t e may a l s o be determined by ion-pair

148

RAFIK H. BISHARA AND EUGENE C. RICKARD

chromatography using the C8 column with an eluting solvent of 25% acetonitrile, 2% acetic acid and 73% water containing 2% glacial acetic acid and 500 mg/ml of tetrabutylammonium dihydrogenphosphate (52). With a flow rate of 2.5 ml/minute, the retention time is about 15 minutes. Sample preparation and detection are identical to that described above. 6.7 Analysis of Related Materials Water may be determined by the KarlFischertitration procedure. Other solvents, such as methanol, may be determined by a gas chromatographic (GC) assay. In the GC assay, the sample is dissolved into water and an internal standard solution such as ethanol is added. A 6 foot glass column packed with Porapak Q (60/80 mesh) and operated at 15OoC with a helium carrier gas flow rate of 80 ml/minute gives retention times of approximately 1.1 and 2.4 minutes for methanol and ethanol, respectively. A Chromosorb 104 column may be used also. The flame ionization detector is used. The hydrolysis product cefamandole and a potential impurity, 7-(D-2-formyloxy-2-phenylacetamido)-3-cephem-4cephalosporanic acid (Compound X, figure 7 ) may be observed by TLC (R values of 0.46 and 0.58 respectively) (2) or determine5 using either of the HPLC methods described in section 6.5.3 (retention times of about 4.5 and 8.75 minutes for the first method and 6 . 2 5 and 13.5 minutes for the second method, respectively). Another possible impurity and degradation product 1-methyl-5-mercepto-1, 2, 3, 4-tetrazoleI can be measured polarographically (2). It is oxidized at the DME in a pH 5.8 acetate buffer. The concentration of this species is proportional to the sum of the currents for the absorption prewave and the main wave (half-wave potentials about -0.20V and -0.05V vs. SCE, respectively). 7. Analysis of Biological Samples

71 .

Microbiological Assay

- subtilis as the microorganism (18, 30, 40, 53-59). B.

Serum and urine are assayed microbiologically using The microbiological assay is also used for determining the cefamandole levels in aqueous humor (591, occular tissues (60-611, interstitial fluids, bile (55), pulmonary and subcutaneous tissue (58). A klebsiella strain is used as the indicator organism for assaying urine samples by an Autoturb (18). 7.2 Liquid Scintillation Assay 14C-cefamandole is assayed by liquid scintillation procedure in metabolic and tissue distribution studies (40,

CEFAMANDOLE NAFATE

149

60, 61).

7.3

Chromatographic Assay 7.3.1 Paper Chromatography

Cefamandole is assayed in biological samples on Whatman No. 4 paper developed with methylethylketone/water (92:8) for 7 hours to give a mobility of about 15cm from the point of application (40). 7.3.2 Thin Layer Chromatography Separation of cefamandole, cefamandole nafate and two metabolites is accomplished on silica Gel F plates developed with ethylacetate/acetone/glacial acetic acid/water (5:2:2:1) solvent. The R values are 0 . 5 5 , 0.63 and 0.81, E respectively (40). 7.3.3 High Performance Liquid Chromatography In studying the hydrolysis of cefamandole nafate to cefamandole, the biological samples are chromatographed on a column packed with Vydac reverse phase (30/44um) and eluted with 20-25% acetonitrile in 0.1% aqueous acetic acid. At a flow rate of 2.0 ml/minute and monitoring the absorption at 254 nm, the retention time for cefamandole nafate and cefamandole are 4.4 and 7.2 minutes, respectively (9). Other HPLC work is performed on VBondapak C18 column eluted with 30% methanol-0.01M sodium acetate, pH 5.2 at a flow rate of 2 ml/minute and monitoring at 270nm. The accuracy of the latter system is ? 3% and the reproducibility measurements yield a coefficient of variation of 4.6% (62).
8. Analysis of Pharmaceutical Formulations

The pharmaceutical formulation contains a buffering agent such that the reconstituted material has a pH between 6.0 and 80 . . Thus, when the material is reconstituted in water, it will be a mixture of cefamandole nafate and cefamandole (the hydrolysis product). The hydrolysis can be minimized by dissolving the sample in acetic acid or other acidic solvents. Somewhat different chromatographic, spectroscopic and physical characteristics will be observed on partially hydrolyzed samples compared to the corresponding data for the raw material. The microbiological, iodometric, hydroxylamine, and polarographic (63) assays described in section 6 for the raw material are applicable to the formulation. The chromatographic methods will generally yield two zones (or peaks) corresponding to cefamandole nafate and cefamandole. Specifically, cefamandole gives a zone which is about 0.50 of the

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RAFIK H. BISHARA A N D EUGENE C . RICKARD

d i s t a n c e t o t h e end o f t h e paper i n t h e paper chromatographic system, a R v a l u e o f 0.45 i n t h e TLC system ( 2 ) and i s f observed i n t h e HPLC systems as d e s c r i b e d i n s e c t i o n 6.6.

9.

Acknowledgements

The a u t h o r s a r e t h a n k f u l t o M r . H. W . Smith, D r . T . T r o x e l l , D r . L . G . Tensmeyer, and D r . D. Dorman f o r t h e i r h e l p w i t h t h e d a t a and i n t e r p r e t a t i o n f o r c r y s t a l l i n i t y ; UV and CD; I R ; and NMR s p e c t r o s c o p y , r e s p e c t i v e l y .

CEFAMANDOLE NAFATE

151

10. References
1. The United States Pharmacopeia, XX, The National Formulary XV, p. 961. 2. E. C. Rickard and G. G. Cooke, J. Pharm. Sci., 66, 379 (1977). 3. R. Nagarajan and D. 0. Spry, J. Amer. Chem. SOC., 93, 2310 (1971). 4. L. G. Tensmeyer, U.S. Patent No. 3,947,414 (1976). 5. L. G. Tensmeyer, U.S. Patent No. 3,947,415 (1976). 6. J. M. Greene and J. M. Indelicato, U.S. Patent No. 3,928,592 (1975). 7. L. D. Hatfield, Proc. R. SOC. London, Ser. 3, in press (1980). 8. J. M. Indelicato, W. L. Wilham & B. J. Cerimele, J. Pharm. Sci., 65, 1175 (1976). 9. J. S. Wold, R. R. Joost, H.R. Black and R.S.Griffith, J. Infect. Dis., 1 7 (Suppl.), S17 (1978). 3 10. A. Dinner, R. J. Templeton and A. D. KOSSOY, Eli Lilly and Co., Indianapolis, IN 46206,personal communication. 11. M. J. Pikal, A. L. Lukes and J. E. Lang, J. Pharm. Sci., 66, 1312 (1977). 12. J. R. Turner, D. A. Preston and J. S. Wold, Antimicrob.Agents, Chemother. , 12, 67 (1977). 13. G. P. Bodey and S. Weaver, Antimicrob. Agents Chemother., 9, 452 (1976). 14. G. Darland and J. Birnbaum, Antimicrob. Agents Chemother., 1 ,725 (1977). 1 15. E. C. Ernst, S. Berger, M. Barza, N. V. Jacobus and F. P. Tally, Antimicrob. Agents Chemother., 9, 852 (1976). 16. S. Eykyn, C. Jenkins, A. King and I. Phillips, Antimicrob. Agents Chemother., 3, 657 (1973). 17. S. Eykyn, C. Jenkins, A. King and I. Phillips, Antimicrob. Agents Chemother., 9, 690 (1976). 18. R. S. Griffith, H. R. Black, G. L. Brier and J. D. Wolny, Antimicrob. Agents Chemother., 10,814 (1976). 19. B. R. Meyers, B. Leng and S. Z. Hirschman, Antimicrob. Agents Chemother., S,737 (1975). 20. B. R. Meyers and S. 2 . Hirschman, J. Infect. Dis., 137 (Suppl.), S25 (1978). 21. T C . Neu, Antimicrob. Agents Chemother., 6, 177 (1974). 22. V. L. Sutter and S. M. Finegold, Antimicrob. Agents Chemother. , 10, 736 (1976). 23. J. A. Washington, Mayo Clin. Proc., 51, 237 (1976). 24. A. E. Weinrich and V. E. Del Bene, Antimicrob. Agents Chemother., 10,106 (1976).

152

RAFIK H. BISHARA AND EUGENE C. RICKARD

25. W. E. Wick and D. A. Preston, Antimicrob. Agents Chemother. , 221 (1972). 26. Data on file, Lilly Research Laboratories, Indianapolis, IN 46206. 27. I. W. Fong, E. D. Ralph, E. R. Engelking and W. M. M. Kirby, Antimicrob. Agents Chemother., 2, 65 ( 1 9 7 6 ) . 28. M. Barza, S. Melethil, S. Berger and E. C. Ernst, 421 (1976). Antimicrob. Agents Chemother., 29. B. R. Meyers, B. Ribner, S. Yancovitz and S. Z. Hirschman, Antimicrob. Agents Chemother., 9, 1 4 0 (1976). 30. N. K. Shemonsky, J. Carrizosa and M. E. Levison, Antimicrob. Agents Chemother. , 679 (1975). 31. H. D. Short, L. 0 Gentry and S. Sessoms, J. Antimicrob. Chemother., 2, 345 ( 1 9 7 6 ) . 32. H. E. Mellin, P. G. Welling and P. 0. Madsen, Antimicrob. Agents Chemother., 262 ( 1 9 7 7 ) . 33. W. E. Grose, G. P. Bodey and D. Stewart, Clin. Pharmacol. Ther., 20, 579 ( 1 9 7 6 ) . 34. R. S. Griffith, H. R. Black, G. L. Brier and J. D. Wolny, Antimicrob. Agents Chemother., 809 (1977). 35. D. J. Schurman, data on file, Lilly Research Laboratories, Indianapolis, IN 46206. 36. E. L. Quinn, T. Madhaven, R. Wixson, E. Guise,

I,

10,

8,

11,

1, 1

37. 38. 39. 40.

73 ( 1 9 7 7 ) . 41. C. L. Winely, J. C. Spears and J. K. Scott, Antimicrob. Agents Chemother., 424 ( 1 9 7 9 ) . 42. U.S. Code of Federal Regulations (1974, admended March 15, 19771, Food and Drugs, Title 21, part 436.102 (a), Washington, D.C. , U . S . Government

N. Levin, M. Block, K. Burch, E. Fisher, A. Suarez and R. del Busto, proceedings of the 10th International Congress of Chemotherapy, Zurich, September 18-23, 1977, p. 803. Washington, D.C.: American Society for Microbiology, 1978. J. S. Tan and S. J. Salstrom, Antimicrob. Agents Chemother. , 1 ,698 ( 1 9 7 7 ) . 1 R. Carter, data on file, Lilly Research Laboratories, Indianapolis IN 46206. S. Gall, data on file, Lilly Research Laboratories, Indianapolis, IN 46206. H. R. Sullivan, S. L. Due, D. L. Kau, J. F. Quay and W. M. Miller, Antimicrob. Agents Chemother., 12,

1, 6

printing office.

43. C. L. Winely, Lilly Research Laboratories, Indianapolis, IN 46206, personal communication. 44. L. P . Marrelli, "Analytical Procedures for Cephalos-

porins," in Cephalosporins and Penicillins, E. H. Flynn, ed., Academic Press, New York NY, 1972, chapter 14.

CEFAMANDOLE NAFATE

153

45. U . S . Code of Federal Regulations (1974, admended April 6, 1979), Food and Drugs, Title 21, part 442.8, Washington, D.C., U.S. Government printing office. 46. E. C Rickard , "Coulometry," in Modern Methods of Pharmaceutical Analysis, R. E. Schirmer,ed., CRC Press, West Palm Beach, FL, in press (1980), Chapter V. 47. T. Getek (Food and Drug Administration, Washington, D.C. 20204) and E. C. Rickard (Lilly Research Laboratories, Indianapolis, IN 46206), manuscript in preparation. 48. E. C Rickard , "Polarography," in Modern Methods of Pharmaceutical Analysis, R. E. Schirmer, ed., CRC Press, West Palm Beach, FL, in press (19801, Chapter VI. 49. R. E. Cooley, C. E. Stevenson and E. C. Rickard, J. Automated Chem., in press (1980). 50. N. E. Davis, Lilly Research Laboratories, Indianapolis, IN 46206, personal communication. 51. L. J. Lorenz, Eli Lilly and Co., Indianapolis, IN 46206, personal communication. 52. J. Kennedy, Eli Lilly and Co., Indianapolis, IN 46206, personal communication. 3 53. H. C. Neu, J. Infect. Dis., 1 7 (Suppl.), S80 (1978). 54. G. B. Appel, H. C. Neu, M. F. Parry, M. J. Goldberger and G. B. Jacob, Antimicrob. Agents Chemother., 10, - 623 (1976). 55. N. G. Waterman, H. U. Eickenberg and L. Scharfenberger, Antimicrob. Agents Chemother., 10,733 (1976). 56. M. M. Agbayani, A. J. Khan, P. Kemawikasit, W. Rosenfeld, D. Salazar, K. Kumar, L. Glass and H. E. Evans, Antimicrob. Agents Chemother., 15,674 (1979). 57. P.H. Azimi, Antimicrob. Agents Chemother., 2, 955 (1978). 58. F. Daschner, E. Blume, H. Langmaack and W. Wolfart, J. Antimicrob. Chemother., 2, 474 (1979). 59. J. L. Axelrod and R. S. Kochman, Amer. J. Ophthalmol., 85, 342 (1978). Barza, A. Kane and J. Baum, Amer. J. Ophthal., 86, 60. 121 (1978). 61. P. Young, M. Barza, A. Kane and J. Baum, Arch. Ophthalmol., 97, 717 (1979). 62. N. S. Aziz, J. G. Gambertoglio, E. T. Lin, H. Grausz and L. 2 . Benet, J. Pharmacokinet. Biopharm., 6, - 153 (1978).

154

RAFIK H. BISHARA A N D EUGENE C . RICKARD

63. U . S . Code of Federal Regulations (1974, Admended April 6, 1 9 7 9 ) , Food and Drugs, Title 21, part 442.208, Washington, D.C., U . S . Government printing office. The literature is surveyed through January 1980.

CYPROHEPTADINE
Hassan Y. Aboul-Enein and A . A. Al-Badr

1.

2.

3. 4. 5.

6.

Description 1.1 Nomenclature 1.2 Formulae 1 .3 Molecular Weight 1.4 Elemental Composition 1.5 Appearance, Odor, Color, and Stability Physical Properties 2.1 Melting Point 2.2 Solubility 2.3 Identification 2.4 Spectral Properties Synthesis Metabolism Methods of Analysis 5.1 Spectrophotornetric Methods 5.2 Titrimetric Method 5.3 Chromatographic Methods Acknowledgments References

156 156 156 157 157 157 157 157 157 157 158 166 167 170 170 171 172 177 178

Analytical Pmfiks of Drug Substances, 9

155

Copyright @ 1980 by Academic Rcss. Inc. All rights of reproduction i any form reserved. n ISBN: 0-12-2608097

156

HASSAN Y. ABOUL-ENEIN AND A. A. AL-BADR

CYPROHEPTADINE

1. Description
1.1 Nomenclature

1.11 Chemical names

4-(5H-Dibenzo [a,d] cyclohepten-5-ylidene)-1-methyl piperidine; l-methyl-4-( 5H-dibenzo- [a,d] cycloheptenylidene ) piperidine; 5-( 1-methyl piperidylidene-4)-5H-dibenzo [a,d] cycloheptene; l-methyl4-(5-dibenzo [a,e] cycloheptatrienylidene) piperidine; 4-(1,2: 5,6-dibenzocycloheptatrienylidene) -1-methyl piperidine.
1.12 Generic name
Cyproheptadine. 1.13 Trade names Anarexol, Antegan, Nuran, Periactin, Vimicon, Cipractin, Peritol, Dronactine, Periactinol. 1.2 Formulae 1.21 Empirical
1.22 Structural
-1

c21 H21

1.23 Wiswesser line notation

L C676 BYJ BUDT6N DYTJ A (1)

CY PRO HE PTADINE

157

1.3 Molecular weight

Anhydr0u.s base 287.39, Anhydrous HC1 323.86 Sesquihydrate HC1 350.89.

1.4 Elemental composition

C 87.76%, H 7.37%, N 4.87%.
1.5 Appearance, color, odor and stability White to slightly yellow, crystalline powder that is odorless or practically odorless and has a slightly bitter taste; relatively stable in light, stable at room temperature and nonhygroscopic; the sequihydrate is stable in air (2 ).
2. Physical properties

2.1 Melting point The anhydrous form melts at agout 2' 5. 0 The sesquihydrate melts about about 162 (2). Crystals from absolute ethanol + ether, dec. 252.6 - 253.606 Hydrochloride monohydrate crystal melts at 214-216 ( 3 ). Crystals frgm dilute ethanol (the base ) melts at 112.3-113.3 ( 3 ) . 2.2 Solubility
1 Gm in about 1.5 m l methanol, about 16 m l chloroform, about 35 m l alcohol and about 275 ml H20; practically insoluble in ether (2).

2.3 Identification
The following tests are cited from the BP 1973 (4).
a) The infrared absorption spectrum exhibits maxima which are compared to the authentic cy-proheptadine hydrochloride. The light absorption, ir. the range of 230-350 nm, of a 2 cm layer of 0.0016% w/v solution in ethanol 95%, exhibits a maximum only at 286 nm, and extinction about one.

b)

c)

A saturated solution yield the characteristic test

for chloride.

158

HASSAN Y. ABOUL-ENEIN AND A. A. AL-BADR

The following are certain color tests ( 5 ) which are useful in the identification of cyproheptadine in micro amount :Reagent. Sulphuric acid/formaldehyde. Ammonium molybdate. Ammonium vanadate Color Gray green Blue green to green. Purple-brown. Sensitivity
0.5 Pg

0.1 Pg
0.5 Pg

Furthermore, crystal tests can be used for the identification of the drug ( 5 ) , f o r example, with ammonium thiocyanate solution, it gives branching needles (sensitivity 1 : 1000). With potassium iodide solution, dense roset and fans of rods (sensitivity 1 : 1000). Yalcindag and Onur ( 6 ) had published a report which described the identification for some drug containing basic nitrogen including cyproheptadine through the microscopic appearance of crystals formed with a number of reagents and by some color tests.
2 . 4 Spectral properties
2 . 4 1 Infrared spectrum

The infrared spectrum of cyproheptadine base (Nujol mull) is given in Fig. 1, major brand assignments are as follows :-1 Frequency, cm Assignment.
3380, 2 4 0
1590 1640

N-CH3 Aromatic phenyl stretch. C=C at Cl0 - cll

Other finger print bands has been assigned by Clarke ( 5), these peaks are :all of which are seen in the spectrum which is shown in Fig. 1. Further information with regard to the infrared spectrum of cyproheptadine is given in reference (1).
749, 797, 765 and 776cm-,

Fig. 1

- Infrared spectrum of cyproheptadine in

N u j o l mull.

HASSAN Y . ABOUL-ENEIN AND A. A. AL-BADR

2.42 Ultraviolet spectrum

Cyproheptadine in methanol solution exhibits maxima at 224 nm, an inflextion at 240 m and at 283 nm as shown in Fig. 2 . Clarke ( 5 ) reported the "ultraviolet absorption spectrum for cyproheptadine in 0.1N H Sc! to show maxima at 224 nm (El% 1 en 1656) and 2 at4 285 nm r (El% 1 em 3 5 5 ) , and an inflextion at 240 nm.
2.43 Nuclear Magnetic Resonance Spectrum:

NMR spectrum of cyproheptadine is preFig. 3 . It was obtained on a Varian Spectrometer with TMS as the internal The sample was dissolved in CDCL 3' The following structural assignments have been made for Fig. 3 :
A typical sented in T-60A NMR standard. Chemical shift (8) Assignment . N-CH 3 8 protons of the piperidine ring system. CH = CH bridge at Cl0 and C . 8 protons'$or the aromatic phenyl. rings.

- Singlet at 2.1 - Broad complex multiplets between 2.27 and 3.47

Singlet at 6.87. Multiplet centered at 7 . 2 3 .

These data are in agreement with the data published by Englehardt - - ( 7 ) . et al.
2.44 Mass spectrum and Fragmentometry

The low resolution mass spectrum of cyproheptadine is shown in Fig. 4 . It was obtained on a Finnigan 1015 L quadrupole mass spectrometer at an ionization potential of 70eV. The spectrum shown is obtained by direct insertion of cyproheptadine base. It shows a molecular ion I at m/e 287 d (Relative intensity 5 . 2 % ) , a promenant peak at m/e 96. The most important prominant ions are shown in Table 1.

CYPROHEPTADINE

161

Fig. 2

U l t r a v i o l e t spectrum of cyprohept a d i n e i n methanol.

I . I . . . i . . . . i .1 . . . i . . . . i I . . . . . ' i . . . . I i .
8.0

7.0

6.0

S.O(PPM)s 4.0

3.0

2.0

1.0

Fig. 3 - NMR spectrum of cyproheptadine i n C D C l 3 as i n t e r n a l standard.

with TMs

96

cn
W

215

50

100

150

200

250

308

Fig.

4-

Mass spectrum of cyproheptadine ( E I ) determined by d i r e c t probe i n s e r t i o n .

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HASSAN Y . ABOUL-ENEIN AND A. A. AL-BADR

T+*
Fragment
Table 1 m/e

Relative dntensity
5.1

287

I CH3

CI,

l+
4 H3

Frigerio - - ( 8 , 9 ) had discussed the fragmentaet a1 tion of some of the metabolites of cy-proheptadine mainly cyprohepladine-lO,ll-epoxide, desmethyl cyproheptadine, desmethyl cyproheptadine-10, 11epoxide. Frigerio - - (8)suggested a fraget a1 mentation pathway of these epoxide metabolites as shown on Scheme 1.

-+

' 0

'+

&
CH3 M+ - CH

272

1.3

215

17.4

202

4.5

96

80.2

70

39.1

CY PROHEPTADINE

165

I H
I

m / e 289 ( R = H) m / e 303 ( R = CH3)

- CHO'

II
R
m / e 260 (R = H)

cH2
m / e 203

S c h e m e 1.

CH

166

HASSAN Y . ABOUL-ENEIN AND A. A. AL-BADR

3. Synthesis
a) Phthalic anhydride ( I ) is reacted with phenylacetic acid (11) to form 3-benzylidenephthalide (111) which on isomerization and hydrogenation, gives 2-phenylbenzoic acid (IV). This is converted to its acid chloride which then undergoes condensation to close the 7-nembered ring and give 10, 11-dehydro-5H-dibenzo [a,b] cyclohepten-5-one (V). Bromination at the 1- pesition followed by dehydrobromination introduces the 10, 11double-bond. Grignardization of this ketone with 4chloro-1-methyl piperidine followed by dehydration of the resulting carbinol yields cy-proheptadine (base) which on reaction with equimolar quantity of hydrogen chloride, forms the hydrochloride salt ( 10) .

I1

I11

IV

V
1 ) N-methgl-4piperidyl
~

-HBr

magnesium chloride 2 ) Hydrolysis.

9-" "4i
CH3 I H \ / CH3

CY PROHEPTADINE

I67

b)

Converting 5-cyanodibenzo [a,dl cycloheptadine ( I ) to its corresponding pipertdine derivative I1 which on treatment with sodium hydride, anhydrous dimethylsulphoxide at 170-180 then refluxed with 10% HC1, cyproheptadine was obtained in 87% yield (11).

11

CH3

CH

c)

Dibenzo [a,d] cycloheptene I11 was treated with phenyl lithuim in tetrahydrofuran and ether, the anion generated was reacted with l-methyl-4-piperidone to give IV which was dehydrated by refluxing with acetic acid in concentrated HC1 ( 11).

I11

IV

dH3

CH3

4. Metabolism
The metabolism of cyproheptadine has been extensively studied in several species including humans. Hucker - - (12) had published a report on the physiologiet a1 cal disposition and urinary metabolite in the dog, rat and

168

HASSAN Y . ABOUL-ENEIN AND A. A. AL-BADR

CH3

9
0

Rat

Dog, c a t

I -CH3

Dog,cat

Scheme 2.

CH3

Urinary metabolites of cy-proheptadine identified i n dog, r a t and c a t . Underlining i n d i c a t e s t h a t t h e s t r u c t u r e was a major metabolite i n t h a t species.

CY PROHEPTADINE

169

cat. It was found that, cyproheptadine was well absorbed and excreted almost equally in the urine and feces of these species. However, the plasma levels of the radioactivity were considerably higher in the dog than in the rat. About 17% or the dose was excreted in the dog bile in 6 hours. The urinary metabolites identified in the dog, rat and cat as, reported by Hucker -- (12), are shown et a1 in Scheme 2. Rats excreted the drug almost entirely as the desmethyl cyproheptadine-10, 11-epoxide. This statement was substantiated by the study reported by Hintze - - (13). et a1 Hintze -- (13) reported that after intzoducing a dose of et a1 labelled cyproheptadine hydrochloride, the major meta14c bolite in the rat urine was unconjugated, but the majority of the radioactive materials found in mouse and human urine were conjugated with glucuronic acid. The rat metabolite (the desmethyl cy-proheptadine-10, 11-epoxide) accounted for 25% of 45 mg dose of the drug per kg. None of this epoxide was found in human. The dihydrcdiclswhich could arise from the 10, 11 epoxy metabolite were not found in the urine of the rat, mice and humans. The epoxide found in the rat urine reported to be et a1 unusually stable in the in vivo hydrolysis. Hintze - ( 1 3 ) suggested possible implications of these results in the species-selected pancreatotoxicity of cyproheptadine in the rat. A detailed study on the B-sell toxicity of cyproheptadine is published by Rickert (14) and by Wold and et a1 Fischer (15). Frigerio - - (8)had identified cyproheptadine-10, 11-epoxide, desmethylcyproheptadine-10, 11epoxide, and desmethylcyproheptadine in rat urine after administration of 40 mg/kg I.P. of the drug by mass spectrometry and confirmed their structure Furthermore, et a1 Frigerio - - (9)in another report had identified the presence of the desmethylcyproheptadine-10, 11-epoxide in et a1 the urine of human volunteers. Porter - - (15) had reported a full study on the metabolism of cyproheptadine in humans. The metabolites identified are shown in scheme 3 . Aromatic ring hydroxylation followed by glucuronide conjugation, N-demethylation and heterocyclic ring oxidation were shown to occur in man. The principal metabolite, however, was identifed as a quarternary ammonium glucuronide-like conjugate of cyproheptadine. They reported (16) Cll bridge no evidence for any metabolic changes at C 10

170

HASSAN Y . ABOUL-ENEIN AND A. A. AL-BADR

N
O HO # =

\CH3

Glucuronide

Scheme 3. of the drug in humans. All metabolites seen in scheme 3, were identified by GLC, Ms, NMR and IR spectrometric techniques.
5. Methods of Analysis%
5.1 Spectrophotometric methods

5.11 Colorimetric methods

Cyproheptadine had been determined in pharmaceutical formulation colorimetrically by Adamski (18). The ground tablets were extzacted with chloroform, the extract was shaken with phosphate buffer and bromocresol green. The chloroform was re-extracted with 0.N NaOH measuring the extinction at 615 nm refering the results to a calibrating graph.
~ ~~ ~ ~~

Shapoval - - (17)published a report which include et a1 the physical, chemical and biological properties of the drug in tablets and the metho&of its evaluation.

CY PROHEPTADINE

171

The error was reported to be > 1.2%. Beltagy - - (19)reported a method for the deet a1 termination of 18 drugs in the free form and in various formulations colorimatrically using tro; peolin 000. Cyproheptadine was included in that method of assay in which the drug was treated with tropeolin 000 in a pH 1.09 buffer. The complex formed was extracted with methylene chloride, the dye liberated by the acid addition was measured at 485 nm. The method gave results comparable to those obtained by the method of the B.P. 1973 ( 4 ) . 5.12 Ultraviolet Spectrophotometric method This method has been adopted by the B.P. 1973 ( 4 ) for the assay of cy-proheptadine tablets. The method cited in the B.P. 1973 depends on the measurement of the extinction of 1-cm layer of the alcoholic (95%) solution at a maximum at about 286 nm. The content of cyproheptadine hydrochloride is calculated taking 355 as value of E 1% 1-cm. Demir and h a 1 (20) had published a similar procedure. 5.2 Titrimetric methods 5.21 Nonaqueous titration The B.P. 1973 (4) analyses cy-proheptadine hydrochloride, free drug, by the non-aqueous titration using 0.1 N perchloric acid as a titration after the addition of mercuric acetate solution using crystal violet solution as indicator. 5.3 Chromatographic methods 5.31 Counter-Current Distribution Hintze - - (13) had isolated cyproheptadine and et a1 its epoxide metabolite from rat urine by the counter current distribution method. The pooled urine was adjusted to pH 8 and extracted several times with methylene chloride, the organic layer was concentrated -in vaccu to 2 m l . After addition of

172

HASSAN Y . ABOUL-ENEIN AND A. A . AL-BADR

10 ml of 0.05M of phosphate b u f f e r (pH 7 . 5 ) , t h e remainder of t h e organic phase was evaporated. The buffer s o l u t i o n was placed i n a 100 tube-counter current d i s t r i b u t i o n apparatus and d i s t r i b u t e d b e t ween 0.05M phosphate buffer (pH 7.5 ) and benzene. After a 100 cycles, the solvents were decanted i n t o n glass-receiving tubes a ? a l i q u o t s of t h e benzene l a y e r were removed f o r determination of radioa c t i v i t y . The benzene l a g e r s of tubes 75-90 were combined and dried out over sodium s u l f a t e . Mass spectrometry, and TLC were u t i l i z e d t o d e t e r mine t h e p u r i t y of t h e metabolite and t h e unchanged drug t h a t was i s o l a t e d from r a t urine.

Another method reported by P o r t e r -- ( 1 6 ) f o r et a1 t h e counter current d i s t r i b u t i o n . A gum i s o l a t e d from human u r i n e iiigesting 1 cy-proheptadine ( 5 , % 10,11-11, 4mg, 16 JJ C i per s u b j e c t ) a f t e r passing C t h e urine through XAD-2 r e s i n columns. The gum was subjected t o f r a c t i o n between water and butanol/ benzene (1:l v/v). Cyproheptadine and o t h e r metab o l i t e s were separated by t h i s system and i d e n t i f i e d by TLC and GC.

5.32 Paper chromatography

Clarke ( 5 ) described a s e v e r a l solvent systems which a r e used f o r t h e i d e n t i f i c a t i o n of cy-proheptadine a s shown i n Table 2. Table 2

Solvent system C i t r i c acid : water : n-but anol 4.8g

:

I
:

Visualizing agent U l t r a v i o l e t , Iodoplatinate. (Weak reaction),Bromocresol green (weak reaction). U l t r a v i o l e t Iodoplatinate. U l t r a v i o l e t Iodoplatinate

1 Rf
0.77

103 ml
870 ml

Acetate Buffer (PH 4 . 5 8 ) Phosphate Buffer (PH 7 . 4 )

0.22

0.00

CYPROHEPTADINE

173

5.33 Thin k y e r Chromatography

Several reports had appeared in the literature concerning the tlc of cyproheptadine and its metabolites describing the separation and identification of cyproheptadine and its metabolites ( 5 , 8 , 12, 1 3 , 21, 22). The systems are given in Table 3. Ultraviolet light at 254 nm was used to detect the drug and its metabolites unless otherwise stated.

Hucker - - (12), Hintze - - ( 1 3 ) an$ Porter et a1 et a1 et - (16)had published the Rf values of cyprohepa1 tadine metabolites in several solvent systems which can be useful i~nclinical identification of the drug and its metabolites in biological fluids. Furthermore,Virgnoli -- - (21) published a report et a1 on the identification of cyproheptadine among other drugs using Silica-gel as an absorbsnt in the following solvent systems :-

A) Diethyl ether : acetone 90 19

: diethylamine

1
: diethylamine

B) Benzene
400

: dioxane

95

The chromatograms were sprayed by iodoplatinate followed by dilute H2S0 or 1% potassium permangnate in 5% H2S04 and iohoplatinate reagent.

5.34 Gas Liquid Chromatography

During the metabolic study of cyproheptadine in humans and other species, several gas chromatographic analyses were reported f o r the determination, identification and quantitation o f cyproheptadine and its metabolites. The drug was chromatographed without derivatization. The gas chromatographic conditions are given in Table 4 .

r:
ni

t . 0
0 0

m
D
0
d

m
W

0 0
0

2
w

. .. . ..
M
d

..
.. ..

a,
k \ kIn Ln

0
d

In

..
0
d

Ln
M

03

Ln
r(

..

Ln W

0
d

In
0

I74

ci \

ri
r-

ri

m
M

m r-

..

r-

0 0

..

..

I75

176

HASSAN Y. ABOUL-ENEIN AND A. A . AL-BADR

Table 4 Column. Carrier Gas. Column Temp. C h-ogramned from L50-250' a t a :ate of 5% per ninute. 250 lefer:nces.

6 f e e t column packed w i t h 3% OV-17 on acid-washed and s i l a n i z e d Gas-Chrom F

Glass tubing ( 1m long a n d

16

4 n i . d . )packed with m

100-120 mesh Gas-Chrom Q and coated with OV-17.

6 f e e t x &-inch g l a s s column packed w i t h 1.5% OV-17/Gas-Chrom Q.

5 f e e t x &-inch 0 . d . g l a s s column, 3% OV-225 on Supelcoport ( 80-100 mesh).

238

12

225

13

5 f e e t x 4 mm i . d . g l a s s column, packed w i t h 2.5% SE 30 on 80-100 mesh Chromosob W AWHMDS.

225

5.35 P a r t i t i o n Column Chromatography

e t a1 Porter -- ( 1 6 ) have separated cy-proheptadine metabolites by f r a c t i o n a t i o n on columns packed H ' ) resin with Cellex SE (H') and on Bio-Rex 63 ( column.

CYPROHEPTADINE

177

ACKNOWLEDGEMENTS The authors would like to thank M r . Dennis Charkowski, Dept. of Pharmacology, the University of Iowa, Iowa City, Iowa 52242, U.S.A., for determining the mass spectrum of cyproheptadine; M r . Said E. Ibrahim, for his help in the library search, M r . Essam A. Lotfi and M r . Khalid N.K.Lodhi for determining the ultraviolet and nmr spectra, and M r . Altaf Hussain Naqvi for typing the manuscript.
D r . E.L. Engelhardt of Merck Sharp and Dohme Research labo-

A sample of cyproheptadine HC1 was kindly donated by

ratories, West Point, Pa. 19486, U.S.A.

178

HASSAN Y . ABOUL-ENEIN AND A. A. AL-BADR

REFERENCES
1. IfAtlasof Spectral data and physical constants of Organic compounds", edited by J.G. Grasselli and W.M. Ritchey. Vol 3, CRC Press 1975, p. 160.

2. Remington's Pharmaceutical Sciences, 15th edition, Mack Publishing Co., Easton, Pa 18042, 1975, Page 1066.

3 , Merck Index, Ninth edition, Merck & Co. Inc., Rahaway, N.J., U.S.A., 1976, Page 2765.

4. British Pharmacopoeia 1973, London Her Majesty's Stationary Office 1973, Page 139. 5. E.C.G. Clarke, "Isolation and Identification of Drugs", The Pharmaceutical Press, London, 1969, Page 278. 6. O.N. Yalcindag and E. Onur, Turk. Hij terc. - Biyol. Derg., 33 25 (1971)Through - - - 22, Abstract No.2649 Anal. Abst. (1972 ) .

7. E.L. Engelhardt, E.C. Zell, W.S. Saari, M.E. Christy and Med. Chem., 8, C.D. Colton, J. -- - 829 (1965), and references were cited therein.

8 A. Frigerio, N. Sossi, G. Belvedere, C. Pantarotto and . S. Garattini, J. Pharm. Sci, 63, 1536 (1974). 9. A . Frigerio, N. Sossi, G. Belvedere, C. Pantarotto and S. Garattini, Adv. Mass Spectrum. Biochem. Med., - 1 9 1, 0 ( 1976) .
10. L.M. Atherden, "Bentley and Driver's Textbook of Pharmaceutical Chemistry", Eight edition, "London, Oxford University Press, 1969, Page 591.

11. L. Vargha, E. Kasztreiner, E. Meszaros and G. Szidagyi, 72, Ger. Offen. 1, 921, 934; through Chem. Abstr. - 31625~

(1970r

12. H.B. Hucker, A.J. Balleto, S.C. Stauffer, A.G. Zacchei and B.H., Arison, Drug.Metab. Dispos. - 406 (1974). 2,

13. K.L. Hintze, J.S. Wold and L.J. Fisher, Drug Metab.
Dispos.,

2,

1, (1975).

CY PROHEPTADINE

179

1 4 . D.E. Rickert, - - - B, - 4079 (1975). Diss. Abstr. I n t . 35,

1 5 . J . S . Wold and L . J . Fischer, J. Pharmacol. E X ~ .Ther., 183, 188 (1972 ).
16. C . C . P o r t e r , B.H. b i s o n , V.F. Gruber, D.C. T i t u s and W.J.A. Vandenheuvel, Drug Metab. Dispos., - 189 (1975). 3, 17. E.E.S. Schapoval, M.M. M a r t i n e l l i , L.C. Chiaaini and E . J . C . De Castro, Rev. B r a z i l . Farm., 53, 1 % (1972) through - -A- t r . 7 9 m O a m 7 3 ) . Chem. bs

18. S. Adamski, Acta Pol. Pharm., 311 (1965 ) through Anal. Abstr. - - 13, Abstract No. 6503 (1966).

19. Y.A. Beltagy, A. I s s a and S.M. R i d a , Pharmazie, - 484, 31,

( 1976 )

20. S. Demir and H. Amal, I s t a n b u l Univ. E c z a c i l i k Fak. Mecm., - 1 4 , ( 1970) ; through Chem. Abstr. 73, 1 1 3 0 2 4 - 9 v 6, 21. L. Virgnoli, B. C r i s t a n , F. Gouezo and J.M. Vassalo, Bull. Tran. SOC. Pharm. u o n . , 9, 277 (1965); through Anal.Abstr. - Abstract No. 3731. (1.5176). 14, 22. F. Schmidt, Dtsch. Apoth. Ztg., 114, 1593 (1974); through Chem. Abstr. 82, -- - 64614d (1975).

DIBENZEPIN HYDROCHLORIDE
Alfred Egli and Werner R. Michaelis
I.
Introduction 1.1 History 1.2 Name, Formula, Molecular Weight 1.3 Appearance, Colour, Odour Physicochemical Properties 2.1 Elemental Analysis 2.2 Spectra 2.3 Crystal Properties 2.4 Solubility 2.5 Dissociation Constant 2.6 Partition Coefficients Synthesis Stability 4.1 Stability in Bulk 4 . 2 Stability in Solution 4.3 Stability in Dosage Forms Biophannaceutical Aspects 5.1 Pharmacokinetics 5 . 2 Metabolism Acute Toxicities Analytical Methods 7.1 Titration 7.2 Spectroscopic Methods 7.3 Chromatography 7.4 Analysis of the Dosage Forms 7.5 Determination in Body Fluids References

2.

3. 4.

5.
6. 7.

8.

182 182 182 182 182 182 183 191 191 193 193 193 194 194 195 195 195 195 196 197 198 198 198 199 20 1 204 205

Analytical Profiles of Drug Substances. 9

181

Copyright @ 1980 by Academic R s s . Inc. All rights of reproductionin any form reserved. ISBN: 0-12-260809-7

182

ALFRED EGLI A N D WERNER R. MICHAELIS

1.
1.1

Introduction History

In 1959 and 1962, patent applications were filed for dibenzepin hydrochloride [l]. The drug substance shows remarkable histaminolytic and anti-anaphylactic effects [21. According to clinical trials this antidepressant can be classified among the thymoleptic drugs between Imipramine and Amitryptiline [ 3 , 41. Dibenzepin hydrochloride is the active ingredient of the NOVERIL@ dosage forms. 1.2 Name, Formula, Molecular Weiqht

Dibenzepin hydrochloride is 10-[2-(dimethylamino)ethyl]-5,10-dihydro-5-methyl-11H-dibenzo[b,e][1,4]diazepin-11-one, monohydrochloride CH34jCH3

N

TI@ &iD
0 II
1

- HCI
La
L

Molecular Formula: C18H22C1N30 Molecular Weight: 331.85

5a

CH3
Chemical Abstracts Registry Number: 315-80-0 1.3 Appearance, Colour, Odour

Finely crystalline to crystalline, white or buff white powder; odourless o r of weak, characteristic odour. 2.
2.1

Physicochemical Properties Elemental Analysis Element C H c1

%

Calculated 65.2 6.7 10.7 12.7

4.8

Found 65.3 6.5 10.6 12.6 5.0

DIBENZEPIN HYDROCHLORIDE

183

2.2

Spectra

2.21 I n f r a r e d The I R spectrum i n a KBr p e l l e t as obtained on a PERKIN-ELMER 283 i n f r a r e d spectrophotometer i s presented i n fig. 1 . The main c h a r a c t e r i s t i c bands a r e t h e f o l l o w i n g : Wave number (cm-') 3100 2400 Assignment

- 2800 - 2560

1630
1600 775

C-H N-H+ C=O C=O C-H

stretching vibrations stretching vibrations stretching vibration i n - p l a n e deformation v i b r a t i o n out-of-plane deformation vibration (1,2 disubstitution)

2.22 U l t r a v i o l e t The UV spectrum i n 0.1 N h y d r o c h l o r i c a c i d as o b t a i n e d on a Z E I S S DM4 spectrophotometer i s presented i n f i g . 2. A maximum occurs a t about 204 nm w i t h a l o g molar a b s o r p t i v i t y m o f 4.530, another maximum a t about 220 n w i t h a l o g molar a b s o r p t i v i t y o f 4.458 and a shoulder a t about 285 n w i t h a m l o g molar a b s o r p t i v i t y o f 3.421. 2.23 Fluorescence I n 0.1 N h y d r o c h l o r i c a c i d t h e drug substance shows no fluorescence ( e x c i t a t i o n from 220 t o 400 nm). 2.24 P r o t o n Nuclear Magnetic Resonance The PMR spectrum i n d e u t e r a t e d d i m e t h y l sulphoxide as o b t a i n e d on a BRUKER HX-90-E spectrometer i s presented i n f i g . 3. TMS served as i n t e r n a l standard. The c h a r a c t e r i s t i c s o f t h e spectrum a r e g i v e n i n t h e f o l l o w i n g t a b l e :

4 a,
rl 4

a
m
Y k

a,

m
.rl -0 .rl
rl

c
a,

k 0

r
o
k -0

I
.rl

c a .
NOJ C N

a,M

ncc w
.rl

a,

DIBENZEPIN HYDROCHLORIDE

185

F i q u r e 2 : U l t r a v i o l e t Spectrum o f D i b e n z e p i n H y d r o c h l o r i d e i n 0.1 N H y d r o c h l o r i c Acid.

CA

0.0505 m g / m l ;

CB

0.0101 m g / m l .

I n s t r u m e n t : Z E I S S DM4.

[ PPm 1
F i g u r e 3: P r o t o n Nuclear Magnetic Resonance Spectrum o f Dibenzepin H y d r o c h l o r i d e i n (CD3) SO. Instrument: BRUKER HX-90-E.

DIBENZEPIN HYDROCHLORIDE

187

Chemical S h i f t [PPml 11.5 7.6 7.4-7.5 7.3

Intensity

Multiplicity singlet (broad) doublet o f doublet multiplet doublet o f doublet mu1t i p l e t triplet
m 1 i p l et u t

Assignment 3'-H+

1 H 1 H
2 H

H-Cl
H-C3, H-C6 H-C9

1 H
3 H

7.15-7.3 7.1 4.6 4.2 3.35 3.3 2.8

ti-C4, H-C2
H-C1

H-C7,

H-C8

1 H 1 H 1 H
2 H 3 H 6 H

mu1t i p l e t

'
'

triplet singlet singlet

H-C2

5-CH3 3 ' -CH3

2.25 Carbon-13 N u c l e a r Maqnetic Resonance The C-13 NMR spectrum i n d e u t e r a t e d d i m e t h y l s u l p h o x i d e as o b t a i n e d on a BRUKER HX-90-E s p e c t r o m e t e r i s p r e s e n t e d i n f i g . 4. TMS s e r v e d a s i n t e r n a l standard. The assignment o f the i n d i v i d u a l signals i s given i n the following table: Carbon Chemical S h i f t PPm 1 131.2 122.6 132.4 116.4 153.6 36.6 148.3 119.1 126.3
Carbon

Chemical S h i f t [PPml 124.6 123.5 134.8 168.0 126.5 44.6 53.5 42.0

c-4 C- 4a 5-CH3 C-5a C-6 c- 7

c- 1 c- 2 c- 3

C-8 c-9 C-9a

C-lla

c-11

c-1'

c-2' 3 ' -CH3

.tlz

low0

8 WO 4wo

6wO

4wo

2wo
,000
5w

tiz
HZ

5000
25M

3O W

2wo
1

22.53 MHz-C"

2 ow

1500

000

m
50

l W

1 W

w
50 '

50 25

2b0

1'50

1'00

[ PPm I
F i g u r e 4: C-13 Nuclear Magnetic Resonance Spectrum o f Dibenzepin H y d r o c h l o r i d e i n (CD3)gS0. Instrument: BRUKER HX-90-E.

DIBENZEPIN HYDROCHLORIDE

189

2.26 Mass

The low r e s o l u t i o n e l e c t r o n impact mass spectrum ( 7 0 eV) as o b t a i n e d on a AEI MS 30 mass spectrometer u s i n g d i r e c t i n s e r t i o n probes a t 80 O C i s presented i n f i g . 5. The f r a g m e n t a t i o n pathways a r e as f o l l o w s :

- CH3,
CH3'

CH3
N- CH = CH2

H I

0 II

.q

- C H O -29

r3
-+

&b i
CHI 0
I

-cHi-14
m/e=237

CH3

CH3

100-

F i q u r e 5: Low R e s o l u t i o n E l e c t r o n Impact Mass Spectrum o f Dibenzepin H y d r o c h l o r i d e . I n s t r u m e n t : AEI MS 30 (Energy: 70 eV, Ion S o u r c e Temperature: 80
OC).

90 -

80-

70 -

60.

50-

40-

3020 -

100
11
I

I,

,1 2 :

, , ,

,
350

, , , ,

100

150

200

250

300

400

DIBENZEPIN HYDROCHLORIDE

191

2.3

Crystal Properties

2.31 M e l t i n g P o i n t 238 O C ; t h e d e t e r m i n a t i o n was c a r r i e d o u t on a METTLER FP 1 ( s t a r t i n g t e m p e r a t u r e 230 O C , h e a t i n g r a t e 2 O C / m i n > . 2.32 Polymorphism

So f a r no polymorphism has been observed by I R s p e c t r o s copy and d i f f e r e n t i a l scanning c a l o r i m e t r y .

2.33 D i f f e r e n t i a l Scanninq C a l o r i m e t r y The DSC thermogram, o b t a i n e d w i t h a PERKIN-ELMER DSC-2 i n s t r u m e n t a t a h e a t i n g r a t e o f 10 O C / m i n and i n a n i t r o g e n atmosphere, i s shown i n f i g . 6. The DSC c u r v e shows o n l y a s h a r p m e l t i n g endotherm accompanied by decomposition o r s u b l i m a t i o n . 2.34 Thermoqravimetry The t h e r m o g r a v i m e t r i c curve, c a r r i e d o u t on a PERKINELMER TGS-1 thermobalance, i s g i v e n i n f i g . 6. The sample t e m p e r a t u r e was r a i s e d a t a r a t e o f 1 0 O C / m i n m a i n t a i n i n g a n i t r o g e n atmosphere.
No l o s s o f w e i g h t i s observed u n t i l m e l t i n g . A s t r o n g l o s s o f w e i g h t i s observed d u r i n g t h e m e l t i n g process.

2.4

Solubility

The s o l u b i l i t y was determined i n a v a r i e t y o f s o l v e n t s e q u i l i b r a t e d by v i b r a t i o n d u r i n g 24 h o u r s a t 25 O C . Solvent water methanol ethanol 2-propanol acetonitrile acetone e t h y l acetate chloroform benzene hexane Solubility i n mg/g more t h a n 200 more t h a n 200 86 9.2 14.4 2.2 0.5 13.1 Solubility i n g/1OO m l more t h a n 20 more t h a n 20 7.0 0.69

1.7 0.7

1.1 0.17 0.04 19.3 0.15 0.04

192

ALFRED EGLI AND WERNER R. MICHAELIS

50

100

150

200

"C

F i q u r e 6: D i f f e r e n t i a l Scanning C a l o r i m e t r y and Thermog r a v i m e t r y Curves o f Dibenzepin Hydrochloride. Instruments: PERKIN-ELMER DSC-2 PERKIN-ELMER TGS-1 (Heating r a t e s 10 T/min>.

DIBENZEPIN HYDROCHLORIDE

193

A t 22 2 O C dibenzepin h y d r o c h l o r i d e d i s s o l v e s more t h a n 2 % (w/v) i n propylene g l y c o l and e t h a n o l 95 per cent, and more t h a n 20 % (w/v) i n e t h a n o l 50 per cent; i t i s p o o r l y s o l u b l e (0.056 76 (w/v>) i n n-octanol. 2.5 D i s s o c i a t i o n Constant

T i t r a t i o n o f a 0.003 M s o l u t i o n i n water a t 20 y i e l d e d as pKa 5.25 0.05 f o r t h e 3'-Nitrogen.

- 22

OC

2.6

Partition Coefficients

The p a r t i t i o n c o e f f i c i e n t s between s i m u l a t e d g a s t r i c f l u i d pH 1.2 ( w i t h o u t enzyme) and n-octanol on one hand, and s i m u l a t e d i n t e s t i n a l f l u i d pH 6.8 ( w i t h o u t enzyme) and n - o c t a n o l on t h e o t h e r , have been determined a t 37.0 0.5 O C

g a s t r i c f l u i d pH 1.2/n-octanol: i n t e s t i n a l f l u i d pH 6.8/n-octanol:
3.

1 : 0.27 1 : 18.3

Synthesis

C a t a l y t i c hydrogenation o f 2-[methyl(2-nitrophenyl) aminolbenzoic a c i d m e t h y l e s t e r l e a d s t o t h e corresponding aminoester, 2-[(2-arninophenyl)methylamino]benzoic a c i d m e t h y l e s t e r , which i s t h e n converted by c y c l i z a t i o n w i t h a s t r o n g base (e.g. sodium amide) t o t h e lactam 5,10-dihydro-5-methyl11H-dibenzo[b,e][l,4]diazepin-ll-one. Alkylation with 2-chloro-N,N-dimethylethanamine y i e l d s dibenzepin base, whose h y d r o c h l o r i d e i s formed by r e a c t i o n w i t h gaseous h y d r o c h l o r i c a c i d i n e t h a n o l i c s o l u t i o n . F i n a l l y t h e product i s r e c r y s t a l l i z e d f r o m e t h a n o l [2l. The s y n t h e s i s o f C-14-labelled drug substance i s d e s c r i b e d i n [51.

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ALFRED EGLI A N D WERNER R. MICHAELIS

NaNHz
H

1
I

CI-CHz -CHz - N ( C H 3 4 CH3, ,CH3 CY,

H CI

K% ;
I

CH3

* K;D
N

,CH3

1
I

CH3

5:
CH3

4.

Stability

Dibenzepin h y d r o c h l o r i d e i s a v e r y s t a b l e s u b s t a n c e ; a d e g r a d a t i o n c o u l d o n l y b e o b s e r v e d i n a c i d s o l u t i o n under d r a s t i c conditions.

4.1

S t a b i l i t y i n Bulk

Samples s t o r e d i n g l a s s b o t t l e s f o r 1 5 y e a r s a t 2 1 O C and f o r 8 y e a r s a t 35 O C were i n v e s t i g a t e d by TLC ( 3 s y s t e m s ) : no d e g r a d a t i o n p r o d u c t c o u l d b e d e t e c t e d ( d e t e c t i o n l i m i t 0.05 ?A).

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4.2

S t a b i l i t y i n Solution

Dibenzepin h y d r o c h l o r i d e i s a l s o very s t a b l e i n s o l u t i o n : a f t e r r e f l u x i n g a 1 0 p e r c e n t aqueous s o l u t i o n (pH 3.6) f o r 10 days o n l y t h e a c t i v e i n g r e d i e n t and no d e g r a d a t i o n p r o d u c t c o u l d be d e t e c t e d by TLC ( 3 systems, d e t e c t i o n l i m i t 0.05 %).
To degrade t h e a c t i v e i n g r e d i e n t v e r y d r a s t i c c o n d i t i o n s a r e necessary: a f t e r r e f l u x i n g a 10 p e r c e n t aqueous s o l u t i o n o f pH 1 f o r 15 days about 10 E (w/w) o f t h e f o l l o w i n g degradation p r o d u c t c o u l d be i s o l a t e d and i d e n t i f i e d :

CH3,

KNB
I
(3-43

HC'

N-(Z-(dimethylamino)ethyl)-N'-methyl-N'-phenyl-1,Z-benzenediamine h y d r o c h l o r i d e

No o t h e r d e g r a d a t i o n p r o d u c t c o u l d be detected.

4.3

S t a b i l i t y i n Dosaqe Forms

Dibenzepin h y d r o c h l o r i d e i s marketed as NOVERIL@ t a b l e t s , sugar-coated t a b l e t s , i n j e c t i o n and c o n c e n t r a t e i n t e n d e d f o r i n j e c t i o n by i n t r a v e n o u s i n f u s i o n . Since t h e a c t i v e i n g r e d i e n t i s s t a b l e i n these dosage forms too, t h e s h e l f - l i v e s i n a temperate c l i m a t e and i n a h o t c l i m a t e o f a l l these p r e p a r a t i o n s a r e a t l e a s t 5 years [61. 5. Biopharmaceutical Aspects Pharmacokinetics

5.1

71

The a b s o r p t i o n , d i s t r i b u t i o n and e x c r e t i o n o f t h e C-14l a b e l l e d drug substance was i n v e s t i g a t e d i n t h e mouse a f t e r o r a l and i . v . a d m i n i s t r a t i o n o f s i n g l e doses and a l s o a f t e r S.C. a d m i n i s t r a t i o n t o t h e r a b b i t . I n a d d i t i o n , radiochromatog r a p h i c examinations o f t h e b r a i n e x t r a c t s o f mice, r a t s and r a b b i t s were made.

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I n t h e mouse, o r a l l y a d m i n i s t e r e d dibenzepin h y d r o c h l o r i d e was promptly and completely absorbed. A f t e r i.v. a p p l i c a t i o n , t h e r a d i o a c t i v i t y disappeared r a p i d l y from t h e b l o o d because t h e substance i s taken up r a p i d l y by t h e organs. The d i s t r i b u t i o n o f t h e a c t i v i t y i n t h e v a r i o u s organs i s independent o f t h e mode o f a d m i n i s t r a t i o n . The l a r g e s t c o n c e n t r a t i o n s were found i n t h e l i v e r , kidneys, g a l l bladder, and t h e lungs. The drug substance i s r a p i d l y excreted. H a l f o f t h e administ e r e d a c t i v i t y had a l r e a d y been e x c r e t e d 5 h a f t e r o r a l admin i s t r a t i o n and 100 min a f t e r i . v . a p p l i c a t i o n . A f t e r e i t h e r o r a l o r i . v . a d m i n i s t r a t i o n 80 76 were e x c r e t e d i n t h e u r i n e and 20 76 i n t h e feces. The a c t i v i t y p a t t e r n i n t h e r a b b i t was s i m i l a r t o t h a t i n t h e mouse: r a p i d and complete a b s o r p t i o n and a c t i v i t y concent r a t i o n i n l i v e r , kidneys, g a l l bladder, and lungs. 5 min a f t e r i . v . a p p l i c a t i o n , 2.6 76 o f t h e dose were found i n t h e b r a i n o f t h e mouse and 1.6 % i n t h e b r a i n o f t h e r a t . 30 min a f t e r S.C. a d m i n i s t r a t i o n t o t h e r a b b i t , 0.3 76 were found i n t h e b r a i n . Between 1/2 and 4 h a f t e r a d m i n i s t r a t i o n t o t h e r a b b i t t h e s p e c i f i c a c t i v i t y found i n t h e b u l b i o l f a c t . was lower and t h a t i n t h e caudate nucleus was somewhat h i g h e r than i n the r e s t o f the brain. Radiochromatographic examination showed t h a t t h e a c t i v i t y found i n t h e b r a i n o f mice, r a t s , and r a b b i t s c o n s i s t e d m o s t l y o f unchanged dibenzepin. Besides t h i s t h e r e were found t h e m e t a b o l i t e I11 ( c f . 5.2) and two minor b a s i c components o f unknown s t r u c t u r e , which t o g e t h e r amounted t o no more t h a n

10
5.2

x.

Metabolism

[El
Compound

R1
CH3

R2

R3

R1,

I
I1 111 IV
V

CH3
Y 3 CH3 CH3

CH3
CH3 H

H CH3 H
H

H
CH3 H

R3

VI

H H

DIBENZEPIN HYDROCHLORIDE

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The m e t a b o l i t e s o f o r a l l y a d m i n i s t e r e d d i b e n z e p i n h y d r o c h l o r i d e e x c r e t e d i n t h e u r i n e o f man, dog and r a b b i t have been s t u d i e d . The compound i s n o t r e t a i n e d i n t h e body, b u t i s r a p i d l y metab o l i z e d and e x c r e t e d i n t h e u r i n e . I n a l l o f t h e s p e c i e s , none o f t h e m e t a b o l i t e s was more t o x i c t h a n t h e p a r e n t compound. Man and dog e x c r e t e d t h e unchanged compound and 5 d e m e t h y l a t e d R a b b i t s e x c r e t e d t h e unchanged compound derivatives (11-VI) and t h e compounds I1 and 111. I n a l l t h r e e s p e c i e s , metab o l i t e s c o n t a i n i n g p h e n o l i c h y d r o x y g roups were e x c r e t e d . F o r t h e most p a r t , t h e s e appeared i n t h e u r i n e a s g l u c u r o n i d e s . The dog e x c r e t e d a b o u t 1 6 76 o f t h e a d m i n i s t e r e d doses as f r e e b a s i c m e t a b o l i t e s . About 8 76 were c o n j u g a t e d w i t h g l u c u r o n i c a c i d . 48 h a f t e r t h e l a s t dose, n o e x c r e t o r y p r o d u c t s r e l a t e d t o t h e d r u g s ub s ta n c e were f o u n d i n t h e u r i n e .

Man e x c r e t e d 20 - 30 76 o f t h e a d m i n i s t e r e d dose a s f r e e b a s i c m e t a b o l i t e s . The amounts p r e s e n t a s t h e g l u c u r o n i d e s were r e l a t e d t o t h e dose. The f o r m a t i o n o f g l u c u r o n i d e s was depend e n t o n t h e dosage s c h e d u l e ; d i v i d e d doses gave l a r g e r amounts t h a n a s i n g l e dose. The r a b b i t e x c r e t e d t h e d r u g p r i n c i p a l l y a s c o n j u g a t e s o f t h e metabolites.

6.

A c ut e t o x i c i t i e s

The a c u t e t o x i c i t i e s ( LD50) o f d i b e n z e p i n h y d r o c h l o r i d e were f ound t o be: i n t h e mouse, 22 mg/kg i . v . and 225 mg/kg p.0.; i n t h e r a t , 22.2 mg/kg i . v . and 220 mg/kg p.0.; and i n t h e g u i n e a - p i g , 110 mg/kg p.0. [61.

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ALFRED EGLI AND WERNER R. MICHAELIS

7.

A n a l y t i c a l Methods Titration

7.1

Dibenzepin h y d r o c h l o r i d e may be assayed i n g l a c i a l a c e t i c a c i d / a c e t i c anhydride 1:l ( v / v ) by t i t r a t i o n w i t h 0.1 N perc h l o r i c acid. The end p o i n t i s determined p o t e n t i o m e t r i c a l l y u s i n g a glass/calomel e l e c t r o d e system. The h y d r o c h l o r i c a c i d c o n t e n t o f t h e dibenzepin h y d r o c h l o r i d e i s u s u a l l y determined by t i t r a t i o n w i t h 0.1 N s i l v e r n i t r a t e . The end p o i n t i s detected p o t e n t i o m e t r i c a l l y u s i n g a s i l v e r / potassium s u l f a t e e l e c t r o d e system. 7.2 Spectroscopic Methods

7.21 I n f r a r e d I n f r a r e d spectroscopy i s u t i l i z e d f o r i d e n t i f i c a t i o n purposes d u r i n g t h e a n a l y s i s o f t h e drug substance (see 2.21). 7.22 U l t r a v i o l e t The drug substance can be assayed d i r e c t l y by measurement o f t h e e x t i n c t i o n a t about 221 n (maximum) or a t about 280 n m m (shoulder) i n 0.1 N h y d r o c h l o r i c acid. The method i s n o t s p e c i f i c , because by-products w i t h t h e same chromophore a r e determined simultaneously. F o r t h e s p e c i f i c assay o f t h e a c t i v e i n g r e d i e n t i t i s necessary f i r s t t o separate t h e byproducts by t h i n l a y e r chromatography and t h e n t o i s o l a t e t h e substance by e l u t i o n from t h e s i l i c a g e l o f t h e p l a t e w i t h 0.1 N h y d r o c h l o r i c acid. The a c t i v e i n g r e d i e n t i s determined i n t h e f i l t e r e d 0.1 N h y d r o c h l o r i c acid. 7.23 C o l o r i m e t r y

I n moderately a c i d i c s o l u t i o n s dibenzepin h y d r o c h l o r i d e r e a d i l y forms i o n p a i r s with m e t h y l orange, which a r e e x t r a c t a b l e w i t h chloroform. A procedure has been developed f o r assay w i t h AUTO ANALYZER. Therein dibenzepin h y d r o c h l o r i d e i s allowed t o r e a c t w i t h m e t h y l orange a t pH 4.0. The r e s u l t i n g i o n p a i r i s e x t r a c t e d w i t h c h l o r o f o r m and i t s c o n c e n t r a t i o n determined a t 425 nm.
7.24 P r o t o n Magnetic Resonance

PMR spectroscopy may be used f o r i d e n t i f i c a t i o n o f t h e drug substance (see. 2.24).

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7.3

Chromatoqraphy

7.31 T h i n Layer Chromatoqraphy The f o l l o w i n g systems can be used f o r t h e s e p a r a t i o n o f by-products, degradation products, m e t a b o l i t e s and e x c i p i e n t s : System 1 S t a t i o n a r y Phase s i l i c a g e l 60 F 254 (MERCK t l c p l a t e s , no 5 7 1 5 ) s i l i c a g e l 60 F 254 (MERCK t l c p l a t e s , no 5 7 1 5 ) s i l i c a g e l 60 F 254 (MERCK t l c p l a t e s , no 5 7 1 5 ) s i l i c a g e l 60 F 254 (MERCK t l c p l a t e s , no 5 7 1 5 ) Mobile Phase chloroform/methanol 1:l (v/v) e t h y l acetate/ g l a c i a l acetic acid/ water 5 : 2 : 2 (v/v/v) chloroform/cyclohexane/diethy lamine 5 : 4 : 1 (v/v/v) c h l o r o f o rm/cy c l o hexane/diethylamine 1 : E : l (v/v/v), t w i c e developed n-heptane/chloroform/ e t h a n o l ( 9 5 per c e n t ) Y : 9 : 2 (v/v/v)

aluminium o x i d e F 254 (MERCK t l c p l a t e s , no 5 7 1 3 )

m V i s u a l i s a t i o n i s accomplished under UV l i g h t 254 n and by s p r a y i n g w i t h D r a g e n d o r f f ' s reagent. The RSt values are:

RSt Substance dibenzepin hydrochloride degradation product" System 1 System 2

Value System 4 System 5

System 3

1.0 1.0 1.0 ( R f 0 . 4 0 ) ( R f 0 . 4 5 ) ( R f 0.50)

1.0
( R f 0.42)

1.0
( R f 0.64)

0. Y O

1.15

1.20

1.75

1.15

N- ( 2 - (dimethylamino) e t h y l ) - N ' -methyl-" -phenyl-1, 2-benzenediamine h y d r o c h l o r i d e ( f o r f o r m u l a see 4 . 2 ) .

System 4 i s most s u i t a b l e f o r t h e d e t e c t i o n and semiquantitat i v e d e t e r m i n a t i o n o f t h e d e g r a d a t i o n product.

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ALFRED EGLI AND WERNER R. MICHAELIS

The f o l l o w i n g reagents can be used f o r t h e v i s u a l i s a t i o n o f dibenzepin h y d r o c h l o r i d e : Systems 1 - 4 Reagent Colour Dragendorff I s reagent(1) i o d i n e vapor 2,6-dichloro-p-benzoquinone-4-chlorimine(modified Gibbs r e a g e n t ) ( 2 ) Folin-Ciocalteus reagent ( 3 sodium n i t r o prusside/ acetaldehyde potassium j o d i d e / hexachlorop l a t i n i c acid potassium d i c hr omat e/ sulfuric acid (40 per c e n t ) brown brown grey t o g r ey b r own Detection Limit [pgl System 5 Colour brown brown grey t o green Detection L i m i t [pgl

0.02-0.05 0.2

0.05 0.5 0.2

1.2

blue white t o light violet pinkbrown to violet blue

0.1

blue pink

0.05

0.5

0.5

0.5

pink

0.2-0.5

brown

(1) Dragendorff s reagent with consecutive s p r a y i n g with a m i x t u r e o f 20 r n l hydrogen peroxide ( 3 0 per c e n t ) and 10 m l o f e t h a n o l (95 per c e n t ) .

(2) 90

- 110 mg 2,6-dichloro-p-benzoquinone-4-chlorimine a r e d i s s o l v e d i n a m i x t u r e o f 25 m l chloroform, 25 m l e t h a n o l 95 per c e n t and 3 m l dimethylforrnamide.

( 3 ) Sprayed w i t h F o l i n - C i o c a l t e u s reagent, MERCK no 9001 d i l u t e d with water 1:3 ( v / v > and a f t e r w a r d s t r e a t e d w i t h ammonia gas.

The d e t e c t i o n l i m i t s under UV l i g h t 254 nm a r e 0.1

- 0.2

pg.

DIBENZEPIN HYDROCHLORIDE

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7.32 Gas L i q u i d Chromatoqraphy The f r e e base o f dibenzepin h y d r o c h l o r i d e can be d e t e r mined b y GC due t o i t s v o l a t i l i t y and i t s t h e r m a l s t a b i l i t y . The c o n d i t i o n s a r e t h e f o l l o w i n g : Column: glass; l e n g t h 2 m; i n t e r n a l diameter 2 mm AW-DMCS S t a t i o n a r y phase: D e x s i l @ 300, 1 X on Chromosorb@ W, ( 8 0 - 100 mesh) M o b i l e phase: n i t r o g e n , flow r a t e 35 m l / m i n Temperatures: i n j e c t o r : 250 O C d e t e c t o r : 300 O C column: 200 O C f o r 2 min; temperature g r a d i e n t : 8 OC/min; f i n a l temperature: 300

OC.

F i g . 7 shows a gas chromatogram o f a dichloromethane s o l u t i o n o f dibenzepin s p i k e d w i t h t h e degradation product and octacosane as an i n t e r n a l standard.

7.33 H i g h Performance L i q u i d Chromatoqraphy

A HPLC system has been developed on reversed phase ( o c t y l s i l a n i s e d s i l i c a g e l column) f o r assay and p u r i t y t e s t i n g o f dibenzepin h y d r o c h l o r i d e . HPLC-conditions S t a t i o n a r y phase: LiChrosorb@ RP-8 (MERCK), 1 0 pm i n s t a i n l e s s s t e e l , 25 cm x 4.6 mm i.d. Mobile phase: i s o c r a t i c : m e t h a n o l / l p e r c e n t ammonium carbonate s o l u t i o n 65:35 ( v / v ) UV d e t e c t i o n : a t 221 n m F i g . 8 shows a chromatogram o f t h e drug substance s p i k e d w i t h t h e d e g r a d a t i o n product. The f l o w was s e t a t 2.0 m l / m i n . 7.4 A n a l y s i s o f t h e Dosaqe Forms

7.41 I d e n t i f i c a t i o n The i d e n t i f i c a t i o n o f dibenzepin h y d r o c h l o r i d e i n t h e dosage forms can be c a r r i e d o u t b y t h i n l a y e r chromatography u s i n g s i l i c a g e l p l a t e s w i t h chloroform/cyclohexane/diethylamine 1:8:1 (v/v/v) and subsequent UV v i s u a l i s a t i o n a t 254 nm. The most advantageous s p r a y i n g reagents i s D r a g e n d o r f f ' s reagent w i t h consecutive s p r a y i n g by a m i x t u r e o f 20 m l hydrogen p e r o x i d e 30 p e r c e n t and 1 0 m l o f e t h a n o l ( c f . 7.31).

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ALFRED EGLI A N D WERNER R. MICHAELIS

i ' -! - ! ' - !I

_c

min 18 16 14 12 10

8.

4 2

F i q u r e 7: Gaschromatogram o f Dibenzepin s p i k e d w i t h t h e degradation product and octacosane ( i n t e r n a l standard). Instrument: PERKIN-ELMER 900. Key: 1 =dichlormethane ( s o l v e n t ) 2 = N-( 2-(dimethylamino)ethyl)-N' -methyl-N' -phenyl-1,2-benzenediarnine h y d r o c h l o r i d e ( d e g r a d a t i o n product 1 3 = dibenzepin 4 = octacosane ( i n t e r n a l standard)

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min 15

12

Fiqure 8: High Performance Liquid Chromatogram o f Dibenzepin Hydrochloride spiked with the degradation product.
UV detection at 2 2 1 nrn.

Reversed-phase Mode, isocratic,

Key: 1 dibenzepin hydrochloride 2 N-( 2-(dimethylamino)ethyl)-N'-methyl-N'-phenyl-1,2-benzenediamine hydrochloride (degradation product)

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ALFRED EGLI AND WERNER R. MICHAELIS

Dibenzepin can a l s o be i d e n t i f i e d by I R spectroscopy a f t e r e x t r a c t i o n from t h e dosage form with chloroform. 7.42 Assay Dibenzepin h y d r o c h l o r i d e i n N o v e r i l @ coated t a b l e t s and t a b l e t s may by assayed i n a n o n - s p e c i f i c way by d i r e c t UV spectrophotometry a f t e r e x t r a c t i o n with 0.1 N h y d r o c h l o r i c a c i d o r , i n case o f s o l u t i o n s ( i n j e c t i o n o r concentrate intended f o r i n j e c t i o n by i n t r a v e n o u s i n f u s i o n ) a f t e r d i l u t i o n w i t h 0.1 N h y d r o c h l o r i c acid. A s p e c i f i c assay o f dibenzepin h y d r o c h l o r i d e i n t h e dosage form may be c a r r i e d o u t by t l c f o l l o w e d b y UV spectrophotometry ( t h e system can a l s o be used f o r i d e n t i f i c a t i o n purposes). The a c t i v e i n g r e d i e n t i s e x t r a c t e d w i t h methanol. The chromatographic c o n d i t i o n s are: s i l i c a g e l , mobile phase: chloroform/cyclohexane/diethylamin 1 8: 1 (v/v/v) : The spot corresponding t o dibenzepin i s e x t r a c t e d with 0.1 N hydrochlor i c acid, and t h e c o n c e n t r a t i o n i s determined a t about 285 n m (shoulder) by spectrophotometry. A f u r t h e r s p e c i f i c assay i s t h e HPLC d e t e r m i n a t i o n o f dibenzep i n h y d r o c h l o r i d e a f t e r e x t r a c t i o n with methanol/water 8:2 (v/v) from t h e dosage form u s i n g LiChrosorb@ RP-8 as s t a t i o nary phase and a c e t o n i t r i l e / l per c e n t ammonium carbonate s o l u t i o n 65:35 (v/v) as t h e m o b i l e phase. UV d e t e c t i o n wavel e n g t h i s s e t a t 221 nm.

7.5

Determination i n Body F l u i d s

The i s o l a t i o n , s e p a r a t i o n and i d e n t i f i c a t i o n o f dibenzep i n h y d r o c h l o r i d e and i t s m e t a b o l i t e s from u r i n e o f man, r a b b i t s and dogs i s described i n [81. The methods used a r e e x t r a c t i o n , t h i n l a y e r and gas chromatography; 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 s were made by UV spectroscopy. Gas chromatog r a p h i c procedures f o r t h e d e t e r m i n a t i o n o f t h e drug and i t s b a s i c m e t a b o l i t e s v i a a c e t y l a t i o n o f t h e demethylated compounds a r e described i n [ 9 131.

The i s o l a t i o n o f dibenzepin h y d r o c h l o r i d e from plasma and o t h e r body f l u i d s by e x t r a c t i o n or column chromatography and i t s i d e n t i f i c a t i o n by gas chromatography a r e g i v e n i n [14,151. A gas chromatographic d e t e r m i n a t i o n o f t h e a c t i v e i n g r e d i e n t and i t s b a s i c m e t a b o l i t e s i n b i o l o g i c a l m a t e r i a l a f t e r trif l u o r a c e t y l a t i o n i s described i n C161. I n t h a t paper a s i m p l e procedure i s g i v e n f o r t h e s e p a r a t i o n o f dibenzepin hydrochlor i d e and i t s demethylated m e t a b o l i t e s by i o n - p a i r e x t r a c t i o n .

DIBENZEPIN HYDROCHLORIDE

205

HPLC may a l s o be used f o r t h e s e p a r a t i o n and d e t e r m i n a t i o n o f t h e a c t i v e i n g r e d i e n t [17, 181. Acknowledqements The a u t h o r s a r e i n d e b t e d t o many c o l l e a g u e s f o r t h e i r most v a l u a b l e h e l p , i n p a r t i c u l a r t o Mrs. D.A. Giron-Forest and Messrs. H.-R. L o o s l i , Ch. Quiquerez and W.D. Schoenleber o f SANDOZ L t d . Furthermore, t h e a u t h o r s wish t o express s p e c i a l thanks t o Miss I. Andre f o r h e r s e c r e t a r i a l a s s i s t a n c e i n p r e p a r i n g t h i s manuscript.

8. 1. 2. 3. 4. 5.
6.

References

F. Hunziker and J. Schmutz ( t o WANDER L t d . ) , Chem. Abstr. - 13331 (1964) and - 64455 (1967) 61, 67,
F. Hunziker, H. Lauener and J. Schmutz, Arzneim.-Forsch. (Drug Res.) 2, 324 (1963) D. Bente, M.P. Engelmeier, K. H e i n r i c h , H. H i p p i u s and W. Schrnitt, Arzneim.-Forsch. (Drug Res.) 14,538 (1964)
G. S t i l l e , H. Lauener and E. Wschr. - 366 (1965) 95,

Eichenberger, Schweiz. med.

F. Hunziker and 0. S c h i n d l e r , Helv.chirn.acta (1965)

WANDER L t d . unpublished r e s u l t s
W. M i c h a e l i s , Arzneim.-Forsch. (Drug Res.)

48,

1590

7.
8.

17,181

(1967)

H. Lehner, R. Gauch and W. M i c h a e l i s , Arzneim.-Forsch. 185 (1967) (Drug Res.)

17,

9.
10
a

R. Brochon, H. Lehner, R. Gauch and 0. Rudin, Arch. T o x i c o l . - 249 (1969) 24,

R.

Bonnichsen and B. Schubert, Z.

Rechtsmed.

(1971) 11.
E. Klug, Z. Rechtsmed. W.

68,

253

71, 27

(1972)

12.
13. 14.

K i s s e r , Wien Med. Wochenschr.

123, 747

(1973)

A. De Leenher and A. Heyndrickx, J. Pharm. Sci. (1973)

62,

31

39,

H.P.

Gelbke, T.H. 2 1 1 (1978)

G r e l l and G.

Schmidt, Arch. T o x i c o l .

206

ALFRED EGLI AND WERNER R. MICHAELIS

15.

R. Pentz and A.

Schutt, Arch.

Toxicol.

16. 17.

H.J. S c h l i c h t and H.P. (1978)

Gelbke, J.

2, ( 1 9 7 8 ) 225 Chromatogr. 166,599

Sci. Ed. ,I

D.R.A. Uges and P. Bouma, Pharm. Weekbl., (1979)

Gerontol. [Lect.]

417

18.

J. Husser and C. Hesse, 5 t h E u r . Symp. Basic Res.

1976 (Pub. 19771, 739

DIGOXIN
Penelope R . B. Foss and Steven A . Benezra
1. Description 1.1 Names 1.2 Formula, Structure, Molecular Weight 1.3 Appearance, Color, Odor 2. Physical Properties 2.1 Infrared Spectrum 2.2 Nuclear Magnetic Resonance Spectrum 2.3 Ultraviolet Spectrum 2.4 Mass Spectrum 2.5 Optical Rotation 2.6 Melting Point 2.7 Solubility 3. Synthesis 4. Stability 5 . Pharmacokinetics, Metabolism, and Protein Binding 5.1 Pharmacokinetics and Metabolism 5.2 Protein Binding 6. Methods of Analysis 6.1 Elemental Analyses 6.2 Identification Tests 6.3 Fluorometric Analysis 6.4 Chromatography 6.5 Polarography 6.6 Colorimetry 7. Methods of Analysis-Biochemical Applications 7.1 Chromatography 7.2 Polarography 7.3 Radioimmunoassay 8. References 208 208 208 208 209 209 209 214 214 214 214 217 217 217 217 217 219 220 220 220 220 22 1 225 230 230 230 239 239 240

Analytical Profiles of Drug Substances, 9

207

Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN: 0-12-260809-7

208

PENELOPE R. B . FOSS A N D STEVEN A . BENEZRA

1.

Description

Digoxin is a cardiotonic glycoside obtained from the leaves of Digitalis lanata Ehrhart (Fam. Scrophulariaceae). 1.1 Names

3~-[(O-2,6-Dideoxy-~-D-~-hexopyranosyl-(l~4)-0-2,6dideoxy-~-D-~-hexopyranosyl-(l~4)-2,6-dideoxy-~-D-ribohexopyranosyl)oxy] - 1 2 ~ , 1 4 - d i h y d r o x y - 5 ~ - c a r d - 2 0 ( 2 2 )-enolide2
Cordioxil, Davoxin, Digacin, Dilanacin, Dixina, Lanocardin, Lanicor , Lanoxin, Rougoxin, Vanoxin2 1.2 Formula, Structure, Molecular Weight 41H6414
780.96

HO

1.3

Appearance, Color, Odor

Digoxiii is an odorless, white crystalline powder.

DIGOXIN

209

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

The i n f r a r e d spectrum of d i g o x i n i s shown i n F i g u r e 1.3 I t was t a k e n a s a 0.2% d i s p e r s i o n of d i g o x i n i n KBr w i t h a N i c o l e t Model 7199 FT-IR. Table I g i v e s t h e i n f r a r e d assignments c o n s i s t e n t w i t h t h e s t r u c t u r e of d i g o x i n . Table I I n f r a r e d S p e c t r a l Assignments f o r Digoxin

3445 2930 1725

Band (cm-l)

0-H s t r e t c h

Assignment

C-H s t r e t c h of CH3-, -CH -

1625 1445, 1405, 1375, 1320, 1270 1163, 1150, 1080, 1020 865

i s t i c of 01, $ u n s a t urated y lactone C=C s t r e t c h C-H bending v i b r a t i o n s of -CH , and -CH2C-0 s t r e ? c h f o r a l c o h o l s and e t h e r s C-H bend of t r i s u b s t i t u t e d C=C

C=O s g r e t c h c h a r a c t e r -

2.2 Nuclear Magnetic Resonance (NMR) S p e c t r a H The ' and 13C NMR of d i g o x i n a r e shown i n F i g u r e s 2 and 3.5 T e t r a m e t h y l s i l a n e i s t h e i n t e r n a l s t a n d a r d i n t h e s o l v e n t s used f o r t h e p r o t o n and carbon NMR.
The ' NMR was o b t a i n e d w i t h a Varian XL-100A a t H 100 M z w i t h d e u t e r a t e d chloroform a s t h e s o l v e n t . The lH H NMR i s v e r y complex and n o t a l l p r o t o n s can be a s s i g n e d . P r o t o n s 18-CH3 and 19-CH , p l u s t h e HOD s i g n a l a r e between 0.82-0.95 ppm. The 4',%', and 4"' p r o t o n s a r e from 3.16-3.30 ppm. P r o t o n s 5', 5", 5"' appear between 3.70-3.98 ppm, and p r o t o n s 3', 3", 3"' and 3 a r e between 3.98-4.34 ppm. P r o t o n s l', 1" and 1"' a r e between 4.80-5.02 ppm.6 The 13C NMR was o b t a i n e d w i t h a Varian CFT-20 i n s t r u ment a t 20 MHz. D e u t e r a t e d d i m e t h y l s u l f o x i d e was t h e s o l v e n t . Table I1 g i v e s t h e carbon assignments f o r t h e 13C NMR.6

5:

0 0 0
7

0 0

m
7

u
U

a C n
V

aJ

0 0

cu

0 0 m N

0 0
@Y

0 0
Cr)

0
0 0

0 0 ~ Q )

0 0 c o b

0 0 c D m

0 0 - r c

0 c

0 u -

-r

33NVlll WSNVtll

210

21 1

F i g u r e 2 - 'H Nuclear Magnetic Resonance Spectrum of Digoxin

Figure 3

13C

Nuclear Magnetic Resonance Spectrum of Digoxin

DIGOXIN

213

Table I1 13C NMR Assignments for Digoxin Carbon No. 7

10 13 14 17 18 19 20 22 23

Chemical Shift (ppm)

21.31 34.59 55.64 84.30 45.16 9.34 23.58 176.69 115.84 173.82 98.91 98.91 95.30

1 1 1

HO

214

PENELOPE R . B. FOSS AND STEVEN A. BENEZRA

2.3

Ultraviolet (W) Spectrum

The W spectrum of digoxin in ethanol was taken with a Beckman ACTA CIII W spectrophotometer and is shown in Figure 4.4 Digoxin has o2e maximum in the W spectrum at 220 nm with E = 1.28 x 10 . 2.4 Mass Spectrum The mass spectrum of digoxin as shown in Figure 5 was obtained with a Varian MAT CH5-DF mass spectrometer.7 The direct probe temperature was 290', and the electron energy was 70 eV. The major fragmentation pattern characteristic of the aglycone portion of digoxin is outlined below.a
0

HO

- -H20
C23H3304

Ci?3H3103

M/E 390'
C23H3406

M/E

373

M/E

355

2.5 Optical Rotation

The optical rotation of di.goxin has been determined under different conditions.
25 ['I Hg + 13.6' to 14.2' (C=lO in pyridine)' [a]:'

+ 18.9'
+ 30.4'

(C=l in pyridine)2 (C=1.2 in alcohol)2

2.6 Melting point Digoxin melts and decomposes between 23Oo-265'C.

0 0

m
.I4

0 Lo

.I4

0 M

cv

n
W

0 0
7

I
I

co

CD

w-

cv

AlISN31NI 3A11Vl3tl

DIGOXIN

217

2.7 Solubility
Digoxin is freely soluble in pyridine, slightly soluble in 1 : l ethanol:water, chloroform, and practically insoluble in water and in ether.1'2
3.

Synthesis

No successful synthesis of digoxin has been reported. Digoxin is obtained commercially from the ethanolic extraction of Digitalis lanata leaves followed by chromatographic purification.

4.

Stabilityg

Digoxin is stable indefinitely when kept in the dark in well closed containers. No degradation is noted in tablets after five years when stored in tightly closed containers. Solutions of digoxin hydrolyze in the presence of acids yielding digoxigenin bis-digitoxoside, digoxigenin monodigitoxoside and digoxigenin. The latter degrades further to anhydrodigoxigenin under anhydrous acid conditions. Neutral solutions of digoxin in ethanol and propylene glycol are stable up to five years. Digoxin solutions are relatively stable to light except when stored under intense light for long periods o f time. Degradation is by apparent opening of the lactone ring and can be detected by a lowering of the ultraviolet absorbance and by HPLC assays. Only chromatographic procedures can be used to determine digoxin in the presence of all its breakdown products.
5.

Pharmacokinetics, Metabolism, and Protein Binding

5.1

Pharmacokinetics and Metabolism

In man digoxin is 60-80% absorbed and has a biologic half life of 1.5 to 2.0 days.1 In the anuric patient the half-life is prolonged to four to six days. To determine which dosage form has the best bioavailability digoxin was given by intravenous infusion, intramuscular injection, oral The fate of the elixir, and tablet to human subjects." glycoside is similar regardless of the dosage method used. l2 The bioavailability of the dosage forms was compared by

218

PENELOPE R . B. FOSS AND STEVEN A. BENEZRA

serum concentration levels and cumulative urinary excretion.l1 The dosage forms, in order of highest to lowest resulting serum concentration levels, and excretion, were intravenous infusion, intramuscular injection, oral elixir, and tablet. An improved digoxin tablet with a more rapid dissolution rate, showed twice the absorption rate of the previous tablet and a forty percent increase in urinary excretion.l3 A new encapsulated liquid digoxin (a soft gelatin capsule containing the glycoside in a dissolved form) was superior in bioavailability to the rapid dissolution tablet and the The capsule's absorption approaches that solution.14' of the intravenous dosage forms.

''

In postmortem examinations of patients with normal renal function, the highest concentration of digoxin was in the kidney, followed by the heart and liver.16 The lowest concentration of digoxin was in the brain. Studies in anephric patients and those with renal failure show the highest concentration of digoxin to be in the heart followed by the liver, and the kidney. When digoxin content was measured in samples of left ventricular papillary muscle,l7 skeletal muscle, and plasma of human patients during heart surgery, the papillary muscle digoxin concentration averaged 77 ng/g, the skeletal muscle, 1 1 . 3 ng/g and plasma, 1-2 ng/mL. A significant amount of total body digoxin is stored in the skeletal muscle since skeletal muscle represents 43% of the body weight. A relatively wide range of digoxin concentration in atrial heart tissue is commensurate with satisfactory digitalization. Myocardial tissue samples taken two hours after the intravenous administration of tritiated digoxin revealed a significant variation in digoxin concentration in and around the infarcted zone. The infarcted tsu' ise' demonstrated a tissue to serum ratio of 12:l. The therapeutic activity of digitalis is likely to depend on the concentration at the active sites in the tissues rather than in the plasma. The quantity of digoxin excreted each day is a function of the amount present in the body. Excretion during the first twenty-four hours has been determined to be between 20-50% of the dose.l g y 2 O Digoxin undergoes appreciable biliary excretion after intravenous dosing in man, however, total fecal recovery is low, with figures ranging from 6-20% of the dose of digoxin.lg''O'l' Doherty - et a1.21 determined that only 6-8% of the given dose of digoxin is recycled through the bowel. Digoxin is excreted predominantly through the kidney. In dogs, it was found that approximately sixty percent2*

DIGOXIN

219

of the metabolism of digoxin takes place in sites other than the liver. The heart muscle was found to have a negligible role in digoxin metabolism. A significant amount of digoxin is excreted unmetabolized. The following digoxin metabolites are present in the lipid-extractable fraction of urine or plasma : dihydrodigoxin, digoxigenin bis-digitoxoside, digoxigenin mono-digitoxoside, dihydrodigoxigenin monodigitoxide, and digoxigenin. 23 Dihydrodigoxin is the major metabolite. In various animals the activity of dihydrodigoxin has been measured to be 1 / 7 to 1/20 the activity of digoxin.24 All glycolytic reduced and nonreduced metabolites were found except for dihydr~digoxigenin~~ which is often detectable only in patients using very high doses of digoxin. Metabolic conversion of digoxin includes the stepwise hydrolysis of the sugar units, conjugation to form water soluble metabolites, epimerization at C-3, and reduction of the lactone ring which destroys the activity of digoxin.23
5.2

Protein Binding

Digitalis protein binding is important because tissue uptake is related to free drug and not to total drug concentration. The variations reported in protein binding of digoxin may result from differences in methodology and may a l s o occur when using the same method, but in different laboratories. Significant species differences in binding have been reported the range to be reported for digoxin. B a g g ~ t ~ ~ 17-40% binding. Storstein26 reported the range to be 5-60%. Most investigators found digoxin binding to be about 20%. Storstein26 used equilibrium dialysis and ultrafiltration for measuring the protein binding of digoxin. With equilibrium dialysis 21-24% of digoxin was found to be protein bound. The glycoside concentration was within therapeutic range, and the dialysis was performed at room temperature. Serum or human albumin was used for the equilibrium dialysis. Storstein reported that the ultrafiltration results were not accurate. Doherty and Hall27 reported that the lack of affinity for serum protein binding for digoxin appears to be a function of its polarity. The polar structure of digoxin tends to render chemical protein binding of the drug less likely to occur. Protein binding of digoxin was found to be normal in

220

PENELOPE R. B. FOSS AND STEVEN A. BENEZRA

uremic patients, but decreases during hernodialysis.26

6.

Methods of Analvsis
6.1

Elemental Analysis
C41H64014

Elemental analysis2 of digoxin as C 63.06% H 8.26%

0
6.2 28.69%

Identification Tests'

Digoxin is dissolved and diluted with hot ethanol and an aliquot is evaporated to dryness. Acid-ferric chloride TS is added to the residue. A green color develops that slowly changes to a deep green-blue. Digoxin is dissolved and diluted with hot ethanol. An aliquot of the solution is evaporated to dryness then dissolved in a solution of methanol and chloroform (1:Z). The sample is spotted onto Whatman No. 1 filter paper that has been impregnated with a solution of formamide and acetone ( 3 : 7 ) . The sample is developed with chloroform saturated with formamide. After development the paper is heated for fifteen minutes at 90C then sprayed with trichloroacetic acid in chloroform and hydrogen peroxide and reheated to 90C for ten minutes. The sample is viewed under W light and compared to the standard.
6.3

Fluorometric Analvsis

Fluorometry has been used to simultaneously determine digitoxin and digoxin in leaves, tincture, tablets, and drug.28 An Aminco Bowman spectrofluorometer was used for the determination of the excitation and emission spectra, and a Turner model 110 was the fluorometer used for the analysis. The reagent was a mixture of acetic anhydride, acetyl chloride, and trifluoroacetic acid. Digoxin has two excitation peaks, one at 470 nm, the same as digitoxin, and a second at 350 nm. The fluorescence peaks for both digitoxin and digoxin occur at 500 nm. With a 47B + 2A-12 filter combination the reading was found to be a sum o f the fluorescence of digoxin and digitoxin. To correct for digoxin fluorescence the 7-60 + 2A-2ND filter combination was used because it allows the determination o f the emission of digoxin alone. The results were linear over the concentration range of 0.5 to 6 pg/mL. The accuracy, based on 2 pg/mL was 99.2% of theory.

DIGOXIN

22 1

Fluorinietric analysis was also used for the determination of digoxin in tablets.29 A Technicon automatic analyzer was used for the analysis. The reagents and solutions were, 70% SD3A alcohol in water, hydrochloric acid, ascorbic acid, hydrogen peroxide, and standard digoxin. Three standards of appropriate levels and samples of the intact or powdered tablets were used. Excitation and emission wavelength maxima for digoxin were 350 nm and 490 nm respectively. Spectral measurements were made on a Farrand Spectrofluorometer. The procedure was stability indicating, and a linear relationship existed between fluorescent intensity and digoxin concentration. The relative standard deviation of a 0 . 2 5 mg digoxin sample was 21.2%. None of the tablet excipients interfered with the procedure. The following fluorimetric assay procedure has also been used for the analysis of digoxin in tablets.30 Ten mL of 80% alcohol was added to a tablet, in a volumetric flask, warmed on a steam bath until the tablet was dispersed, and the alcohol boiled. The mixture was cooled, swirled, and diluted to 20 mL with 80% alcohol. After standing for 15 minutes 5 mL of the supernatant was pipetted into a 20 mL volumetric flask and diluted to volume with 80% alcohol. Three mL of 0.1% solution of ascorbic acid in methanol, 0 . 2 mL of .009 M hydrogen peroxide in water were added to a 1 mL aliquot of the sample solution. The solution was diluted to ten mL with hydrochloric acid and allowed to stand for two hours in the dark. The standard was prepared in a similar manner. For the fluorescence measurement the excitation maximum was at 355 m and the emission maximum was at 490 nm.
6.4

Chromatography
6.41

Paper Chromatography

Paper c h r ~ m a t o g r a p h yhas been used to separate the ~~ M components of a digitalis tincture. Whatman 3 M paper impregnated with formamide and developed in chloroform gave an R f = 0 . 3 3 for digoxin. a variety of spray reagents were used to detect digoxin.
6.42

Thin Layer Chromatography

Table I11 gives various thin layer chromatography systems which have been used for the separation of digoxin.

Table I11 Thin Layer Chromatography for Digoxin Adsorbent Silica Gel G Comment, R f or Relative Order of Elution Digoxin appears as a blue spot under 385 nm W light

Mobile Phase Cyclohexane-acetoneacetic acid ( 4 9 : 4 9 : 2 ) Ethyl acetate-watermethanol ( 8 0 : 5 : 5 )

or

50% aq sulfuric acid or 30% aq soln chloramine and 25% alcoholic soln (1:4)

Spray Reagent

Ref.

32

trichloracetic acid

Silica Gel F

Silica Gel (non-fluorescent)

Ethyl acetate-methanol- 6 g of trichloroacetic Digoxin 0 . 3 33 acid in 25 mL chlorowater ( 8 0 : 5 : 5 ) form and 0.5 mL 30% w/v hydrogen peroxide Chloroform-acetone 50% methanolic sulfuric Digoxin, digoxigenin 34 acid bis-digitoxoside, (1:l) digoxigenin monodigitoxoside, a-anhydrodigoxigenin, 8-anhydrodigoxigenin Comment: 2-25 hrs continuous development Dichloromethanemethanol ( 9 :1 ) Digoxin, gitoxigenin, digitoxigenin

Table I11 (continued) Comment, R or f Relative Order of Elution Ref. Mobile Phase Spray Reagent Ethylacetate-dichloro- 20% v/v orthophosphoric Gitoxigenin, 35 methane-methanol-water acid digoxigenin, f3-acetyl (60:36:3.5:2) digoxin, digoxigenin mono-digitoxoside, or-acetyl digoxin, digoxigenin bis-digitoxoside, gitoxin, digoxin, digitoxin, digitoxigenin Chloroform-pyridine
(60: 1 0 )

Adso rbent Silica Gel 60 F

254

N N W

Digitoxin, digoxin Digitoxin, digoxin

20% v/v orthophosphoric Digoxigenin, dig-

Dichloromethanemethanol (9O:lO) Silica Gel F

254

Chloroform-acetone
(1:l)

acid

oxigenin monodigitoxoside, digoxigeninbis-digitoxoside, digoxin All cardenolides are referenced relative t o digoxin.

36

Kieselgel 60 DC-Fertigplatten

Ethylacetate-dichloromethane-methanolwater ( 1 2 0 : 7 2 : 7 : 4 )

37

Table I11 [continued) Comment, R or f Relative Order of Elution

Ad so rben t

Mobile Phase and Dichloromethanemethanol (9 :1)

Spray Reagent

Ref. -

Comment : Mobile phase 1: continuous development 3 hrs, mobile phase 2: continuous development 2 hrs. The plate is turned 90 after development by mobile phase 1.

DIGOXIN

225

6.43

Gas Chromatography

Digoxin, as a tablet or as the powdered drug,32 was converted to digoxigenin for analysis by gas chromatography. The separation was achieved by using three columns, (A) a two meter glass U tube packed with 2.5% OV-1 on 80-100 mesh Chromosorb A , (B) a 0.5 meter copper U tube with 2.5% OV-1 on 80-100 mesh Chromosorb A , and (C) 3% OV-17 on 80-100 mesh Chromosorb A . Cholesterol was used as the internal standard. The oven temperature was 285OC. The injection port and the flame ionization detector temperature were 330OC. All injections were made with a 5 pL syringe. The detection range was 0.05-0.2 mg. There was little difference in the retention times of the silylated drug and standard versus those of the unsilylated drug and standard. Retention times (min) Cholesterol unsilylated s ilylated Digoxigenin unsilylated silylated
6.44

Column A 2.0 2.0 15.0 15.0

B 0.67 0.67

C 0.5 0.5

5.33 5.0

3.67 3.67

High Performance Liquid Chromatography

Table IV gives various HPLC systems used for digoxin.

6.5

Polarography

Polarography has been used for the assay of digoxin tablets.4 7 The working electrode was a dropping mercury electrode with a one second drop time, and the reference electrode was a saturated calomel electrode (SCE) with a platinum wire as an auxillary electrode. The linear potential sweeps were constant at 5 mV/sec, and the pulse modulation was 25 mV. A 2-mL aliquot of the extraction of the ground tablets plus 0.2 mL of 0.2 M TBAI, tetrabutylammonium iodate, or of 0 . 2 M TBAH, tetrabutylammonium hydroxide, the supporting electrolytes, was added to 2 mL of isopropanol. Before each experiment the solution was deaerated with isopropanol saturated nitrogen, which was also passed over the solution during the assay. The potential was scanned cathodically from -1.8 volt. The peak potential of digoxin was -2.285 xolts. The usef 1 analytical range of the assay -8 was 5 x 10- M to 2.5 x 1 0 M of digoxin.

Table IV HPLC Systems for Digoxin Flow (mL/min) or Pressure

Column Li Chrosorb SI60 (25 cm x 3 mm id)

Mobile Phase n-Butanolacetonitrile-heptanewater (230:100:700:10) t-Butanol-acetonitrileheptane-water

Retention Time (min)

Detection

Ref. 38

1.3

3.5

225 nm

2.2

10

(220:70:800:10)

(204:93:712:10.4)
n-Pentanol-acetonitrile-iso-octanewater (270:93:660:9.3)

3.6 1.3 3.8

(230:100:700:10) (170:60:620:10) (175:60:620:6)

1.4 1.3

5.2 10.4 8.2

Table IV continued Flow (mL/min) or Pressure

Column Merckosorb S160 5 Ir .m (15 cm x 3 mm id)

Mobile Phase

Retention Time (min)

Detection Ref. -~ __ 254 nm or 260 nm 39

Comment: The digitalis glycosides are derivatized with 4-nitrobenzoyl chloride (4N BC1) n-Hexane-methylene chloride-acetonitrile (10:3 :3 ) n-Hexane-chloroformacetonitrile (30:10:9) 1.5 5.6

1.5
2.0

5.9 1.3 230

40

Li Chrosorb S160 5 I.rm (15 cm x 3 mm id) Nucleosil CI8 (30 cm x 3.5 mm id)

8 Methanol in methy% lene chloride saturated with water

37% Acetonitrile in water

1.4 1.3

4
5.4

220

40

40% Solution of 1:l acetonitriledioxane in water

Table IV continued Flow (mL/min) or Pressure

3.0

Column Whatman ODs-1 (25 cm x 4.2 mm id)

Mobile Phase
540 mL of acetonitrile diluted to two liters with water

Retention Time (min)

5.6 (tablets) 7.0 (injection) 5.4 (pediatric injection) 12.4 (elixir)

Detection
220 nm

Ref.
41

450 mL of acetonitrile diluted to two liters with water

3.0

Li Chrosorb S160 (25 cm x 4 mm id) Whatman ODs-1 (30 cm x 4.2 mm id) Perisorb RP ( 1 x 2 mm id)

Cyclohexane-absolute ethanol-acetic acid (60:9: 1) 780 mL Acetonitrile diluted to three liters with water Water with 25% acetonitrile

265,234 nm

42

2.25

220 n m

43

0.75

4.5

254 nm

44

Table IV continued Flow (mL/min) or Pressure 1500 p s i

Column

Mobile Phase

Retention Time (min)

4.5

Detection
254 nm

Ref.
45

Zo rbax-S IL (25 cm x 2 . 1 mm id)

6% Methanol + 0.15%
acetic acid in methylene chloride

3% Methanol + 0.1%
acetic acid in methylene chloride
N

0.5 1200 p s i

10

or

254 nm, 235 nm

46

W N

230

PENELOPE R. B. FOSS AND STEVEN A. BENEZRA

6.6 Colorimetry
An alkaline d i n i t r ~ b e n z e n ereagent has been used for ~~ a colorimetric assay of crystalline powder, tablets, injections, and elixirs containing digoxin. The dinitrobenzene reagent was added to standard and sample preparations that have been evaporated to dryness. The mixture was allowed to stand for five minutes, with frequent stirring, at a room temperature not exceeding 3OOC. The absorbance o f the resulting blue color was measured at 620 nm versus the reagent blank and the USP digoxin standard. The following method of assaying dosage samples used a color reagent of glacial acetic acid49 containing ferric chloride and sulfuric acid. After an initial extraction procedure tailored to the sample type, the sample was dissolved or diluted in a chloroform-methanol solution (65:35) then diluted with glacial acetic acid. An aliquot of digoxin solution was diluted with color reagent and allowed to stand for two hours. The absorbance of the sample was measured at 590 nm versus that of a digoxin standard. The following two colorimetric procedures have been used for the assay of tablet samples. The first employed an alkaline sodium picrate reagent. A crushed digoxin tablet was placed in a 10-mL volumetric flask and diluted with 6-mL of absolute alcohol. The flask was heated to 4OoC and shaken for two hours. The solution was diluted to volume with alcohol, then centrifuged. A 3-mL aliquot of the reagent was added to a 5-mL aliquot of the sample. The solution was stored in darkness for 30 min. The absorbance was measured at 490 nm versus a reagent blank. The Xanthydrolso method was another tablet assay procedure. In a 50-mL volumetric flask a tablet was crushed in a solution of three mL of hot chloroform/methanol (65:35) and two mL of glacial acetic acid. Twenty mL of xanthydrol reagent was added to the mixture. The flask was heated for five min in a 7 5 O C water bath then cooled for five min in an ice bath. The standard and blank were prepared in a similar manner. The absorbance was measured at 540 nm.

7. Methods of Analysis
7.1

Biochemical Applications

Chromatography
7.11 Paper Chromatography

Table V gives various paper chromatography systems

Table V Paper Chromatography for Digoxin and Metabolites Comment, Rf, or Relative Order of Elution dihydrodigoxin,

Adsorbent Whatman No. 1 filter paper impregnated with formamide (30% in acetone) Whatman No. 3 filter paper soaked with formamide-acetone
(1:3)

Mobile Phase Chloroform saturated with formamide

Spray Reagent
25% trichloroacetic

Ref. 51

acid solution in chloroform with four drops of hydrogen peroxide/50 mL m-dinitrobenzene

Chlorofo rm-methanol (1:l)

digoxin 0.50

52

232

PENELOPE R. B. FOSS AND STEVEN A. BENEZRA

which have been used for the separation of digoxin and its metabolites.

7.12

High Performance Liquid Chromatography

Digoxin and its metabolites have been separated and assayed by reverse phase high performance liquid chromatography.53 The column was a pBondapak C 1 8 (30 cm x 4 mm id). The sample solvent was 95% ethanol, and the injection size was 50-75 pL. The detector was set to 220 nm. Listed below are four mobile phases that achieved the desired separation. Isocratic systems: The flow rate was 3 mL/min 1. 25% acetonitrile in water R of digoxin was 13 min. t 2. 33% acetonitrile in water Rt of digoxin was 23 min. Gradient systems: The flow rate was 2.2 mL/min 1. 25% acetonitrile in water to 40% acetonitrile in water at 5%/min. Rt of digoxin was 10 min. 2. 100% water to 30% acetonitrile in water at 6.67%/ min. R of digoxin was 23 min.
t

7.13

Column Chromatography

Column chromatographys4 has been used for the separation of digoxin and dihydrodigoxin extracted from urine samples. The adsorbent was diethylaminoethoxypropylated Sephadex LH-20 (DEAE-Sephadex LH-20). The mobile phase was chloroform-methanol (85:15). Samples were applied in 0.2-0.5 mL volumes of eluting solvent. The flow rate for a 40 x 1.0 cm column was 0.25 mL/min. Dihydrodigoxin Ve/Vt = 0.25 Ve/Vt = 0.34 Digoxin The flow rate for a 36 x 2.5 cm column was 0.20 mL/min. Ve/Vt = 0.43 Dihydrodigoxin Ve/Vt = 0.48 Digoxin

DIGOXIN

233

7.14 Thin Layer Chromatography Table VI gives various thin layer chromatography systems which have been used for the separation of digoxin and its metabolites extracted from biological samples. 7.15 Gas Chromatography
A single column gas chromatographic determination62 of digoxin and its metabolites has been achieved with either isothermal or temperature programming. Digoxin and its metabolites were converted to trimethylsilyl (TMS) derivatives prior to analysis. The column (U shaped, 1 ft x 4 mm id) was packed with 1.6% SE 30 on 80-100 mesh Gas Chrom Q. The sample injection volume was 1 pL. The instrument used 0 was a Barber-Colman 5000 gas chromatograph equipped with a hydrogen flame ionization detector. Under isothermal conditions the column temperature was set to 3OOOC and the detector temperature was set to 320OC. The injection block temperature was maintained at column temperature. The nitrogen flow was 125 mL/min. The retention time o f digoxin was approximately eighteen minutes. With temperature programming from 23OOC to 33OOC at boC/rnin, one minute initial delay, the retention time of digoxin was approximately twenty-four minutes. The detector temperature was 3 4 O O C and the nitrogen flow was 60 mL/min.

Digoxin and its metabolites, derivatized with heptafluorobutyric anhydride,6 0 5 6 have been resolved on a gas chromatographic column packed with 3% OV-1 on Gas Chrom Q. Digoxin and its metabolites were extracted from urine, plasma, biological tissue, and fecal samples. The compounds were initially separated by paper and/or thin layer chromatography. Before the extraction 3H-digoxin can be added as an internal standard. The gas chromatograph used was a Tracer MT-220 with a 63Ni electron capture detector. With a U-shaped column ( 4 ft x 2 mm id) at 25OOC and a detector at 35OoC the retention time for digoxigenin HFB was nine minutes. With a U-shaped column ( ft x 2 mm id) at 250C and detector 6 at 325OC the digoxigenin HFB retention time was eight minutes.
A Varian CH-7 GC-MS combinations6 was used for an determination of digoxin and its metabolites. For the GC a column packed with 3% OV-1 on Chromosorb W AW DMCS 100/120 ( ft x 2 mm id) was used. The column temperature was 6 25OoC, injector temperature, 26OoC, and the molecular separator, 260OC. For the mass spectrometer, electron energy was 20 eV, the ion source temperature, 25OoC, and the trap current, 300 PA. The accelerating voltage was 3KV and the

Table VI Thin Layer Chromatography for Digoxin Comment, Rf or Relative Order of Elution Digoxin: 0.21 Comment: Plates are developed six times to a height of 15 cm. Digoxin: 0.77 Comment: Plates are developed once to 10 cm Digoxin: 0.32
20% sulfuric acid soln 56

Adsorbent Silica Gel G

Mobile Phase Cyclohexane-acetoneacetic acid (65:33:2)

Spray Reagent Lieberman-Burchard (acetic anhydridesulfuric acid-ethanol

(5:5 :50)

Ref. 55

Chloroform-ethanol
E P
(2: 1)

Silica Gel GF Silica Gel H

Chloroform-acetone
(13:7)

Cyclohexane-acetoneacetic acid (65:33:2)

or Anisaldehyde reagent (0.5 mL anisaldehyde, 1.0 mL sulfuric acid, 50 mL acetic acid)

Digoxin: 0.09 Comment: plates developed four times

57

Table VI (continued) Comment, Rf o r Relative Order of Elution

Adsorb en t Silica Gel F

254

Mobile Phase

Spray Reagent Solution of conc sulfuric acid in ethanol ( 1 : 4 )

Ref.
~

58

Cyclohexane-acetoneacetic acid
( 4 9 :49 :2) ( 4 9 :49 :2 ) (45:45:10) (16:80:4 ) (64:6)

Digoxin: 0 . 1 6 (lined tank)

0.33 0 . 3 4 (lined tank) 0.59 0.13

Chloroform-pyridine

% formamide in
acetone for impregnation
10% 10 10 15 20 10 15 (64:6)

2-Butanone-xylene-formamide
( 5 0 : 5 0 :0) (50:50:4 ) (50:50:4) (50:50:4 ) ( 7 0 :3 0 : 0) (50:50)

0.38 0.12
0.09 0.10 0.09

0.36
0.16

2-Butanone-xylene

Table VI (continuedl Comment, Rf or Relative Order of Elution Digoxin 0.15 one development 0 . 2 8 two developments Digoxin: 0.29

Adsorbent Silica Gel G

Or

Mobile Phase Ethyl acetate-chloroform-acetic acid

( 9 0 : 5 :5 )

Spray Reagent

Ref. 59

G254

Cyclohexane-acetoneacetic acid ( 4 9 : 4 9 : 2 ) Cyclohexane-acetoneacetic acid ( 6 5 : 3 3 : 2 and Cyclohexane-acetoneacetic acid ( 4 9 : 4 9 : 2 )

Cyclohexane-acetone-acetic acid ( 6 5 : 3 3 : 2 ) and Ethyl acetate-chloroform

0.36 one development

in each mobile phase

0.12 one development

(9:1 )

in each mobile phase

Chloroform-isopropanol-acetone
(80:5: 15)

0 . 1 8 two developments

Table VI (con tinued) Comment, Rf or Relative Order of Elution

Adsorbent Silica Gel

F254

Mobile Phase Chloroform-methanol (saturated with AgN03)-ammonia ( 9 : l : l )

Spray Reagent

Ref. 51

Chose one of following: 0.33 two developments ( 1 ) 25% trichoroacetic acid soln in chloroform with four drops of hydrogen peroxide per 50 mL. ( 2 ) acetic anhydridesulfuric acid-abs ethanol
( 5 : 5 : 100)

( 3 ) 0 . 0 5 mL p-anisaldehyde, 0.2 mL conc sulfuric acid, 10 mL acetic acid

( 4 ) 20 mg ascorbic acid, 19 mL methanol, 30 mL conc hydrochloric acid, 2.1 pL 30% hydrogen-peroxide. ( 5 ) 10 mL of 3% aq soln chloramine T, 40 mL 25% trichloroacetic acid in ethanol

Table VI (continued) Comment, Rf or Relative Order of Elution 0.33

Adsorbent

Mobile Phase

Spray Reagent Reagent 5 above

Ref. 51

Cellulose Chloroform saturated (MN-300) with formamide predipped with formamide in acetone Mallinckrodt Chromar 7GF Isopropyl ethermethanol (9:l)

0.09 developed five

times 0.26 developed four times in mobile phase 1, developed one time in mobile phase 2 Digoxin: 0 . 2 4

Isopropyl ethermethanol (9:1) and 2-Butanone-chloroform (3:l) Chloroform-methanolacetone-water Fertigplatten (64:6:28:2) Kieselgel 60

DC

61

Any of the following techniques can be used for enhanced detection: (1) Chloramine-trichloroacetic acid spray (2) HC1 vapor ( 3 ) Coating the plate with a thin film of parafin

DIGOXIN

239

spectrum was scanned every six seconds. 7.2 Polarography The polarographic analysis63 of digoxin has been used for assaying the drug as well as blood samples containing the drug. The study of the polarographic characteristics of digoxin in a 50% alcohol solution containing tetraethyl ammonium hydroxide showed a half wave potential of -1.965 volts for an alcoholic solution of the drug and -1.958 volts for the drug extracted from blood samples. At concentrations of 0.1-0.4 pg of digoxin in the blood, the error o f the method was 20.02 pg. 7.3 Radioimmunoassay Employing 3H digoxin tracer and antiserum solutions available in a commercial kit, optimum conditions were determined for the radioimmunoassay of digoxin in plasma, serum, and urine.64 A summary of the procedure is given below. Phosphate buffered saline solution, plasma, and 30% ethanol water were added to each tube and vortexed. The antiserum was added, vortexed, and preincubated, then the tracer solution was added, vortexed, and incubated. The charcoal suspension was added, vortexed, and centrifuged. The supernatant was decanted into 15 mL of liquid scintillation fluid and counted. The range of the assay was 0.05 pg/mL to 5 ng/mL of digoxin. Tritiated digoxin has also been used or the determination of digoxin in liver tissue.65 A liquid-liquid extraction was used to obtain the glycoside. The radioactivity was determined with a liquid scintillation counter. The solvent was 5 mL of 95% ethanol plus 15 mL of toluene. The total counting volume contained 4 g/L of 2,5 diphenyloxazole (PPO) and 50 mg/L of 1,4-bis-2(5-phenyloxazole)benzene (POPOP). The average recovery rate for the procedure was 95.6% and the sample size was 1 mg. The amount of digoxin in human plasma has been assayed by radioimmunoassay with an iodinated tracer.66 The reagents for the assay were digoxin, labelled digoxin (3-0-succinyl digoxigenin [1251]tyrosine derivative), a dilute phosphate buffer containing sodium chloride, bovine albumin powder, sodium azide, anti-digoxin serum, dextran coated charcoal, and normal digoxin free human serum. All standards and specimens were set up in assay tubes and had cold dextran-

240

PENELOPE R. B. FOSS AND STEVEN A. BENEZRA

coated charcoal suspension added. The tubes were centrifuged and the supernatant placed in a separate assay tube. The supernatant fluid and charcoal were both counted for one minute. Digoxin values of pg/L of plasma were calculated from a standard curve of the percent tracer bound versus pg of digoxin per liter. The useful working range of the assay is 0.2 to 8 pg/L of plasma. Fifty pL of plasma was used. The amount of 1251-labelled digoxin has been determined in a 10-pL sample of serum with a modification of a Curtis Digoxin RIA kit assay procedure. The following is a description of the micro-radioimmunoassay procedure. Each polymer tablet was dissolved in the sodium chloride solution, and 100-pL aliquots of the solution were pipetted into the test tubes. Ten pL aliquots of each standard (0-4.8 pL of digoxinlliter) and patients sera were pipetted into a polymer slurry, mixed, and let stand for ten minutes. Ten pL of 1251-labelled digoxin was pipetted into each test tube, mixed and let stand for 30 min. Twice, saline was added to each test tube, centrifuged, and decanted. The contents of the test tubes were counted for ten minutes for radioactivity.
8. References

1. The United States Pharmacopeia, XIX, (1975). 2. Merck Index, Ninth Edition, (1976). 3 . W. Martin, Burroughs Wellcome Co., private communications, (1979). 4. P.R. Booze FOSS, Burroughs Wellcome Co., unpublished data. 5. R. Crouch, Burroughs Wellcome Co., private communication, (1979). 6. S . Hurlbert, Burroughs Wellcome Co., private communication, (1979). 7. R.L. Johnson, Burroughs Wellcome Co., private communication, (1978). 8 . P. Chandrasurin, Burroughs Wellcome Co., private communication, (1979). 9. J . E . Murphy, Burroughs Wellcome Co., private communication, (1979). 10. A.F. Lyon, C.C. DeGraff, American Heart Journal, 7216, 838 (1966). 11. D.J. Greenblatt, D.W. Duhme, J. Koch-Weser, T.W. Smith, The New England Journal of Medicine, - 651 (1973). 289, 12. J.E. Doherty, W.H. Perkins, The American Journal of Cardiology, - 170 (1965). 15,

D1GO XI N

24 1

13. 14. 15. 16.

17.

18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35.

D. Falch, A. Teien, C.J. Bjerklund, British Medical Journal, 1, 695 (1973). J. LindenEaum, Clinical Pharmacology and Therapeutics, 21, 278 (1977). G.I. Mallis, D X . Schmidt, J. Lindenbaum, Clinical Pharmacology and Therapeutics, 18, 761 (1975). J.E. Doherty, W.H. Perkins, W.J. Flanigan, Annals o f Internal Medicine, 66, 116 (1967). J. Coltart, M. Howard,x. Chamberlain, British Medical Journal, 2, 318 (1972). A.J. Thompson, J. Hargis, M.L. Murphy, J.E. Doherty, American Heart Journal, 88, 319 (1974). J.E. Doherty, W.H. Perkins, American Heart Journal,
Burkhalter, C. Cuccia, J. Pavlovich, G.G. Hapedia, Circulation, 34, 864 (1966). J.E. Doherty, W.H. Hall, M.L. Murphy, O.W. Beard, - - 59, 433 (1971). Chest, R.M. Abel, R.J. Luchi, G.W. Perkin, H.L. Corn, Jr., L.D. Miller, The Journal of Pharmacology and Experimental Therapeutics.,L O , 463 (1965). U. Peters, L.C. Falk, S.M. Kalman, Archives o f Internal Medicine, 138, 1074 (1978). D.R. Cork, S.M. K a l G , Drug Metabolism and 2, Disposition, - 148 (1974). J.D. Baggot, L.E. Davis, Res. Vet. Sci., 15, 8 1 L. Storstein, Clinical Pharmacology and Therapeutics, 20, 6 (1976). J.E. Doherty, C H . Hall, J. Sherwood, D. Gerkin, J. Gammill, The American Journal o f Cardiology, I.M. Jakovljavic, Analytical Chemistry,
1513 (1963). (1973).

E .Marcus, L. I

63, 528 (1962).

- 326 (1971). 28,

35,

L.F. Cullen, D.L. Packman, G.J. Papariello, Journal o f Pharmaceutics Sciences, 3, 697 (1970). A. Waghorn, J.A. McCrerie, The Wellcome Foundation Ltd., private communication. H. Brindle, G . Rigby, S.N. Sharma, Journal of Pharmacy and Pharmacology, 1,942 (1955). A.H. Kibbe, O.E. Aracys, Journal of Pharmaceutical Sciences, 62, 1703 (1973). L. Hicks, The Wellcome Foundation Ltd., private communication. J.D. Mills, The Wellcome Foundation Ltd., private communication. C.J. Clarke, P.H. Cobb, The Wellcome Foundation, private communication.

242

PENELOPE R. B. FOSS AND STEVEN A. BENEZRA

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

C.J. Clarke, The Wellcome Foundation; private communication. C.J. Clarke, P.H. Cobb, Journal of Chromatography, K L i n d e r , R.W. Frei, Journal of Chromatography, TNachtmann, H. Spitzy, R.W. Frei, Journal of Chromatography, 122, 293 ( 1 9 7 6 ) . F. Erni, R.W. Frei, Journal of Chromatography, C.H. Powell, Jr., Burroughs Wellcome Co., private communication. P.H. Cobb, Analyst, 1 0 1 , 768 ( 1 9 7 6 ) . J.L. Ebron, Burroughsellcome Co., private communication. Hewlett Packard, Application Sheet E-6, ACS Short Course: "Solving Problems with Liquid Chromatography. D.R. Baker, Dupont Instruments, private communication. D.R. Baker, Dupont Instruments, Application Sheet E - 7 , 7A-C, ACS Short Course: "Solving Problems with Liquid Chromatography." K.M. Kadish, V.R. Spiehlor, Analytical Chemistry,
-, 1714 ( 1 9 7 5 ) . 47

1 6 8 , 541 ( 1 9 7 9 ) . 117, 81 ( 1 9 7 6 ) .

1 3 0 , 169 ( 1 9 7 7 ) .

51. 52. 53. 54. 55. 56. 57. 58.

A.C. Caws, The Wellcome foundation Ltd., private communication. A Waghorn, J.A. McCrerie, The Wellcome Foundation Ltd., private communication. J.J. Sabatka, D.A. Brent, J. Murphy, J. Charles, J. Vance, M.H. Gault, Journal of Chromatography, T W a t s o n , P. Tramell, S.M. Kalman, Journal of Chromatography, 69, 157 ( 1 9 7 2 ) . M.C. Castle, Journal of Chromatography, 115,437
1 2 5 , 523 ( 1 9 7 6 ) .

A.E.H. Hoak, T.G. Alexander, D. Banes, Journal of the American Pharmaceutical ASSOC., 48, 217 ( 1 9 5 9 ) .

D. Sugden, M. Ahmed, M.H. Gault, Journal of

(1975).

Chromatography, 1 2 1 , 401 ( 1 9 7 0 ) . W.E. Wilson, S .AxJohnson, W.A. Perkins, J.E. Ripley, Analytical Chemistry, 2 , 40 ( 1 9 6 7 ) . E. Watson, D.R. Clark, S.M. Kalman, Journal of Pharmacology and Experimental Therapeutics, 184, W.H. Bulger, R.E. Talcott, S.J. Stohs, Journal o f Chromatography, 7 0 , 187 ( 1 9 7 2 ) . L. Storstein, Journal of Chromatography, 117,
87 ( 1 9 7 6 ) . 424 ( 1 9 7 3 ) .

DIGOXIN

243

59. 60. 61.

62.

M.L. Carvalhas, M.A. Figueira, Journal of Chromatography, 86, 254 (1973). E. Watson, P. Tramell, S.M. Kalman, Journal o f Chromatography, 69, 157 (1972). D.B. Faber, A . d z o k , U.A.T. Brinkmann, Journal o f Chromatography, 143, 95 (1977). W.E. Wilson, S . A . Johnson, W.H. Perkins, J.E. Ripley, Analytical Chemistry, 39, 40 J.H. Hilton, Science, 110, 526 (1949). J.G. Wagner, M.R. Hallmark, E. Sakmar, J.W. Cryres, 29, Steroids, - 787 (1977). K.C. Wang, L.J. Spratt, Biochemical Pharmacology,
(1967).

63. 64. 65. 66. 67.

K D . Horgan, W.J. Riley, Clinical Chemistry, 19,

B . Calesnick, A. Dinan, Clinical Chemistry,
903 (1976). 187 (1973).

12, 577 (1963).

22,

DOXORUBICIN
Aristide Vigevani and Martin J . Williamson
I.
Description 1 . 1 History 1.2 Name, Formula, Molecular Weight 1.3 Appearance, Color Physical Properties 2.1 Infrared Spectrum 2.2 Nuclear Magnetic Resonance Spectra 2.3 Mass Spectra 2.4 Ultraviolet and Visible Spectrum 2.5 Fluorescence Spectra 2.6 Circular Dichroism 2.7 Optical Rotation 2.8 Melting Point 2.9 X-ray Diffraction 2.10 Differential Scanning Calorimetry 2.11 Solubility 2.12 Ionization Constant 2. I3 Polarography Synthesis 3.1 Microbiological 3.2 Chemical Stability Metabolism Methods of Analysis 6.1 Elemental Analysis 6.2 Spectrophotometric Analysis 6.3 Electrochemical Analysis 6.4 Paper Chromatography 6.5 Thin Layer Chromatography 6.6 Liquid Chromatography Determination of Doxorubicin in Biological Fluids Analysis of Pharmaceutical Formulations Miscellaneous Acknowledgments References

2.

3.

4. 5.
6.

7. 8. 9. 10.
1 1.

246 246 246 247 241 241 247 25 1 253 255 255 255 255 255 260 260 260 260 260 260 263 263 265 261 267 267 267 267 268 268 270 270 270 270 270

Analytical h f i l c s of Drug Substances, 9

245

CopytighI @ 1980 by Academic Ress, Inc. AIIrighrs Of n - ~ u c t i o nin any form resewed.

ISBN: &12-260809-7

246

ARISTIDE VIGEVANI A N D MARTIN J . WILLIAMSON

Description 1 1 History . Doxorubicin is an antineoplastic antibiotic isolated from a culture of Streptomyces peucetius var. caesius or by chemical synthesis from daunorubicin. The injectable dosage form is supplied as the hydrochloride salt in combination with lactose as a freeze-dried powder.
1 .

Name, Formula, Molecular Weight Doxorubicin is chemically named (8S-lOS)-lO(3-amino-2,3,6-trideoxy-a. -L-e-hexopyranosy1)oxy7,8,9 ,l0-tetrahydro-6,8,1l-trihydroxy-8-hydroxyacetyl-lmethoxy-5,12-naphthacenedione. (CAS-23214-92-8) Originally named (7S:9S)-9-hydroxyacetyl-4-methoxy-7~8,9,lO-tetrahydro6,7,9,11-tetrahydroxy-7-~-(2,3,6-trideoxy-3-amino-cl - L - w hexopyranosyl)-5,12-naphthacenedione.

1.2

NHq

27H29N01 1 Hydrochloride salt (CAS-25316-40-9) C27H2gNOll.HC1

Mw

543.5

Mw

580.0

DOXORUBICIN A p p e a r a n c e , Color The h y d r o c h l o r i d e s a l t i s a r e d , f r e e - f l o w i n g c r y s t a l l i n e powder, a n d t h e f r e e z e - d r i e d f o r m u l a t i o n c o n t a i n i n g lactose i s a r e d cake. 1.3

247

Physical Properties 2.1 I n f r a r e d Spectrum A review of the c a r b o n y l a b s o r p t i o n s o f a n t i n e o p l a s t i c ( a n t i tumor) a n t h r a c y c l i n e s h a s b e e n The i n f r a r e d s p e c t r u m o f d o x o r u b i c i n published1. h y d r o c h l o r i d e recorded from a KBr p e l l e t ( 0 . 4 ) % o n a Perkin-Elmer model 457 g r a t i n g s p e c t r o p h o t o m e t e r is shown i n F i g u r e 1. The i n t e r p r e t a t i o n o f t h e m a i n a b s o r p t i o n b a n d s i s g i v e n i n T a b l e 1. TABLE 1 I n f r a r e d spectrum o f doxorubicin hydrochloride
~

I R A b s o r p t i o n Band, c m - l

A s s i g n men ts

3560-3160 3160-2300 1724 1 6 1 3 and 1580 1282 1115 1071 1008 2.2

0-H s t r e t c h ( h y d r o g e n bonded) NH3+ s t r e t c h and OH s t r e t c h (hydrogen bonded) C=O (ketone) C=O s t r e t c h ( i n t r a hydrogen bonded q u i n o n e ) C-0-C s t r e t c h ( e t h e r ) C-0 ( t e r t i a r y alcohol) C-0 (secondary a l c o h o l ) C-0 (primary a l c o h o l )

N u c l e a r M a g n e t i c Resonance Spectra Proton magnetic resonance spectrometry h a s been e x t e n s i v e l y u s e d a s a f u n d a m e n t a l tool for t h e d e t e r m i n a t i o n o f t h e s t r u c t u r e o f daunorubicin and r e l a t e d compounds2r3r4. The lH-NMR s p e c t r u m o f a d r i a m y c i n o n e p e n t a a c e t a t e i n CDC13 h a s b e e n d e s c r i b e d and t e n t a t i v e l y assigned5. The lH-NMR s p e c t r u m o f d o x o r u b i c i n h y d r o c h l o r i d e i n DMSO-d6 s o l u t i o n r e c o r d e d a t 1 0 0 MHz on a V a r i a n HA-100 s p e c t r o m e t e r a t 8 O o C ( f o r b e t t e r r e s o l u t i o n ) i s shown i n F i g u r e 2. The i n t e r p r e t a t i o n o f t h e s p e c t r u m is g i v e n i n T a b l e 2.

I
(v

0
L n

-s
0 -0

0 W

.d

<-0 z3
0

-s

0-

-0 N 0

Icl
U

bl

DO XO R UBICIN

249

TABLE 2
lH-NMR d a t a of d o x o r u b i c i n h y d r o c h l o r i d e i n D M s 0 - d ~ s o l u t i o n a t 80C (TMS as i n t e r n a l r e f e r e n c e ) .

Proton CH3-5 H2-2 H2-9 H2-7 H-3 I H-4 I CH3O H-5 H2-14 OH-4 H-10
H-1

M 1 i p l i c it y a u t
d

J or WH

(Hz)

m
m
s

m
bs
S

dq
S

1.15 1.77 2.15 2.92 3.31 3.62 3.94 4.14 4.57 4.46 4.90 5.25 7.53 7.79 7.96 13. 08b and 13. 85b

6.5

6.0 and

bs bs
bs

H-3
H-4 H-2

NH3

t
d bs

10.0 7.0 7.0 7.0

OH-11

two s

a)
b)

s - S i n g l e t ; d = Doublet; t = Triplet; m = M u l t i p l e t ; b s = Broad s i g n a l ; dq = Double quartet. A t room t e m p e r a t u r e .

.d

u
0

DOXORUBICIN

25 I

The 13C NMR spectra of doxorubicin hydrochloride, daunorubicin hydrochloride, the corresponding aglycones and of a-methyl daunosaminide in DMSO-d6 solutions and the interpretation has been reported6. Figure 3 shows the FT 13C NMR proton noise-decoupled spectrum of doxorubicin hydrochloride in D20 solution, recorded at 80 MHz on a Varian CFT-20 NMR spectrometer. Dioxane, which is not shown, was used as the internal standard. The interpretation of the spectrum is given in Table 3 .
TABLE 3

13C-NMR data of doxorubicin hydrochloride in D20 solution values (ppm) are referred to 734s. Carbon
1 2 3 4 5 6 7 8 9 10 11 12 13 14 6

Carbon 4a 5a 6a 10a lla 12a CH3O 1'

2'

161.0 (119.2) 137.3 (120.2) (185.9) 154.6 32.8 76.6 36.0 69.0 156.2 (186.1) 213.9 65.3

3' 4' 5' CH3-5 '

(134.5) 111.0 (134.0) (134.5) 111.0 (120.0) 57.2 99.4 28.5 47.7 (67.9) (67.0) 16.6

Assignments with similar shift values given in parentheses may be interchanged.

M s Spectra as The mass spectrum of doxorubicin hydrochloride itself cannot be obtained by electron-impact ionization, but this technique can be used to obtain the spectra of A study of adriamycinone5 and daunosamine'. N-acetyldaunosamine derivatives has been published8. The mass spectra of some E-acylated daunorubicin derivatives have been published9 and also the GC-MS of persilylated aglycone derivatives of doxorubic in and daunorubic inlo.

23 .

E a
P
-m 0
.r(

w 0

-0

m
r-l

u
-r
0

m aJ
U
-4

cn
E

-8 N

DOXORUBICIN

253

The i n t a c t molecule can be examined by f i e l d desorption i o n i z a t i o n mass s p e c t r o m e t r y l l . Figure 4 shows t h e spectrum obtained on a Varian MAT31lA spectrometer, equipped w i t h a combined FD/FI/EI source ( e m i t t e r heating c u r r e n t 2 O m A ) l 2 . Table 4 g i v e s t h e assignments of t h e major fragmentation peaks.
TABLE 4

F i e l d desorption mass spectrum of doxorubicin hydrochloride

m/e
544 543 414

assignment
M+1

Molecular ion a d r iamycinone

l +

CH30

bH

7,
0
OH

-b isanhyLi0a-L iamycinone

336

U l t r a v i o l e t and V i s i b l e Spectrum The u l t r a v i o l e t and v i s i b l e spectrum of doxorubicin hydrochloride i n methanol ( c = l % ) shown i n Figure 513. is The molecular a b s o r p t i v i t i e s a r e given i n Table 5.
2.4

=
II

=.t

co

u a J a

m I
V

a,
U
( D

m m

m
h

DOXORUBICIN

255

TABLE 5

U l t r a v i o l e t a n d V i s i b l e Molecular Absorptivities of
Doxorubicin Hydrochloride i n Methanol Wavelength 233 253 290 471 495 530 2.5 E 38150 255 00 8400 13050 13000 7200

Fluorescence Spectra T h e f l u o r e s c e n c e spectra of d o x o r u b i c i n h y d r o c h l o r i d e i n water a n d e t h a n o l a t a p p r o x i m a t e l y 5 ppm, d e t e r m i n e d u s i n g a P e r k i n - E l m e r MPF-2A s p e c t r o f l u o r i m e t e r , a r e shown i n F i g u r e 6.13 C i r c u l a r Dichroism T h e c h i r a l c e n t e r s a t C-8 a n d C-10 are r e s p o n s i b l e f o r t h e C o t t o n e f f e c t s a t 3 4 5 a n d 2 8 5 nm. T h e c i r c u l a r d i c h r o i s m c u r v e s of m e t h a n o l s o l u t i o n s of d o x o r u b i c i n a n d d a u n o r u b i c i n h y d r o c h l o r i d e s a n d of a d r i a m y c i n o n e a n d daunomycinone i n d i o x a n e , d e t e r m i n e d u s i n g a Roussel-Jouan D i c h r o g r a p h 11, a r e shown i n F i g u r e s 7 a n d 8. T h i s t e c h n i q u e h a s been used i n t h e deduction o f stereochemical r e l a t i o n s h i p s i n t h e f i e l d of a n t h r a c y ~ l i n o n e s ~ ~ . Optical R o t a t i o n The o p t i c a l r o t a t i o n C C ] ~ ~ of d o x o r u b i c i n h y d r o c h l o r i d e i n m e t h a n o l ( 0 . 1 ) was d e t e r m i n e d a t 5 8 9 nm C u s i n g a P e r k i n - E l m e r Model 2 4 1 M polarimeter t o b e +255O. Melting Point D o x o r u b i c i n h y d r o c h l o r i d e melts a t 2 0 5 T w i t h decomposition. 2.9 d i f f r a c t i o n s t u d i e s have been r e p o r t e d o n d o x o r u b i c i n h y d r o c h l o r i d e or i t s d e r i v a t i v e s .
A t t h i s t i m e n o X-ray

2.6

2.7

2.8

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

N-bromoacetyldaunorubicin s o l v a t e

S i n g l e c r y s t a l X-ray d i f f r a c t i o n of with acetone15 confirmed t h e s t r u c t u r e and a b s o l u t e c o n f i g u r a t i o n o f daunorubicin, which h a d p r e v i o u s l y been d e t e r m i n e d b y c h e m i c a l s t u d i e s 2 , 3,4. More r e c e n t l y t h e s e r e s u l t s were c o n f i r m e d b y a n X-ray a n a l y s i s of d a u n o r u b i c i n , a s t h e h y d r o c h l o r i d e

0
4

ln
0

aD

2 m

ln
I n
.r(

4J

0 d

d
.d

a J

o c
n 'l I n k

0 0 I n
0 a ,

ln
d 0 0

d
0
( u

w 0

0 0

d
0 a ,

ln

c)

5
0 0
0 0
( u

c
4J
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Q)
.r(

0
0 a ,
( u

0 3

m
0

rl

4J

3
ln
( u

In
0
d n l

256

70
> 60 h

70
60
50 40 30 20
10

u l
I -

5 50
z

z 40 0

2 30
5
W

g 20 -

!z

10

&

0
480 520 560 600 640 680

0
r 1 1 1 1 1

WAVELENGTH (nm)

480 520 560 600 640 680

L
1 1 1 ~

F i g u r e 6.

F l u o r e s c e n c e S p e c t r a of D o x o r u b i c i n H y d r o c h l o r i d e i n Water ( l e f t ) and E t h a n o l ( r i g h t ) C o n c e n t r a t i o n s a p p r o x i m a t e l y 5 mg/l.

258

ARISTIDE VIGEVANI AND MARTIN J . WILLIAMSON

3t

At2

D AU NORUB ICI N

At2 3r

h(nrn1

2
1
- ) - . - c - - c- - .

-1

DOXORUBICI N

-2
-3
-4 4

260 2i O 360 3O ;

3iO 3 i O 3iO 400 460 A(nmi

Figure 7.

Circular Dichroism Curves of Doxorubicin and Daunorubicin Hydrochlorides in Methanol

DOXORUBICIN

259

27

A&

-2 1 260 O 2 ;

360 3O ;

3iO ?dnm 1

3kO

360 460

-2 260

260 360 3iO 3 i O A (nml

3kO

360 460

Figure 8.

Circular Dichroism Curves of Adriamycinone and Daunomycinone in Dioxane Solution

260

ARISTIDE VIGEVANI A N D MARTIN J . WILLIAMSON

monohydrate p y r i d i n e s a l t 1 6 . Some c o n f o r m a t i o n a l d i f f e r e n c e s were o b s e r v e d w i t h respect t o t h e N-bromoacetyl derivative. 2.10 D i f f e r e n t i a l Scanning Calorimetry The h e a t i n g c u r v e of d o x o r u b i c i n h y d r o c h l o r i d e o b t a i n e d w i t h a Perkin-Elmer Model DSC-1B s c a n n i n g calorimeter a t a t e m p e r a t u r e g r a d i e n t o f 8OC/min. i s shown i n I t shows a n endotherm, c o r r e s p o n d i n g t o t h e F i g u r e 917. s o l i d - l i q u i d t r a n s i t i o n a t 202-2O5OCI p a r t i a l l y superimposed by a n endotherm due t o d e c o m p o s i t i o n which i s a t a maximum a t 26OOC and c o n t i n u e s t o h i g h e r t e m p e r a t u r e s .
2.11 S o l u b i l i t y Doxorubicin h y d r o c h l o r i d e i s r e a d i l y s o l u b l e i n water, normal s a l i n e , methanol, a c e t o n i t r i l e and t e t r a h y d r o f u r a n , b u t o n l y s l i g h t l y s o l u b l e or i n s o l u b l e i n less polar o r g a n i c s o l v e n t s . The a p p a r e n t p a r t i t i o n c o e f f i c i e n t (Papp) between 1 - o c t a n o l and T r i s b u f f e r a t pH 7.0 w i t h c o n s t a n t i o n i c s t r e n g t h ( I = 0.1) is 0.52 a t room t e m p e r a t u r e (22-24OC) a f t e r s h a k i n g f o r 1 5 hours18.

2.12 I o n i z a t i o n C o n s t a n t A pKa o f 8.22 was d e t e r m i n e d f o r t h e h y d r o c h l o r i d e w i t h N/20 sodium hydroxide. Solutions of doxorubicin h y d r o c h l o r i d e show i n d i c a t o r - 1 i k e p r o p e r t i e s I t u r n i n g from orange-red t o b l u e - v i o l e t a b o u t pH = 913. V a l u e s o f -5.9, 8.2, 10.2, and 13.2 f o r pK1, pK2, pK3 and pK4, d e t e r m i n e d by s p e c t r o p h o t o m e t r i c methods, have been r e p o r t e d 1 9 . 2.13 m l a r o g r a p h y Due t o i t s q u i n o i d a l system, d o x o r u b i c i n g i v e s c h a r a c t e r i s t i c p o l a r o g r a m s a t d i f f e r e n t pH v a l u e s . These c u r v e s , d e t e r m i n e d u s i n g a Leeds-Northrup Electro-Chemograf t y p e E p o l a r o g r a p h , are shown i n F i g u r e Synthesis 3.1 M i c r o b i o l o g i c a l Doxorubicin c a n be o b t a i n e d by a e r o b i c f e r m e n t a t i o n o f S t r e p t o m y c e s peucetius v a r . c a e s i u s f o l l o w e d by e x t r a c t i o n w i t h a c i d i c a c e t o n e and p u r i f i c a t i o n by p a r t i t i o n chromatography o n a column o f cellulose b u f f e r e d a t pH 5.4. The a n t i b i o t i c is r e c o v e r e d from t h e e l u a t e s i n 1 - b u t a n o l s a t u r a t e d w i t h pH 5.4 p h o s p h a t e b u f f e r by back e x t r a c t i o n w i t h d i l u t e a c i d p 3 , f o l l o w e d by r e - e x t r a c t i o n i n t o H c h l o r o f o r m a t pH 8.6. The c h l o r o f o r m s o l u t i o n i s c o n c e n t r a t e d and d o x o r u b i c i n c r y s t a l l i z e d a s t h e h y d r o c h l o r i d e on a d d i t i o n o f a n e q u i v a l e n t o f m e t h a n o l i c 3.

F
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262

ARISTIDE VIGEVANI AND MARTIN J . WILLIAMSON

0.3 0.4 0.5 0.6 0,7 0.8 0.9

4 1 1.1 1.2 1.3Volt

Figure 10.

Polarograms of Doxorubicin i n S o l u t i o n s of D i f f e r e n t pH Values

DOXORUBICIN

263

hydrogen chloride. Final purification is performed by crystallization from ethanol or from a methanol/l-propanol mixture20. Chemical Doxorubicin can be obtained2l by reacting daunorubicin hydrochloride in a methanol/dioxane solvent mixture with a chloroform solution of bromine, forming 14-bromodaunorubicin. This is then hydrolyzed with an aqueous methanolic solution of sodium hydroxide under a nitrogen atmosphere. After dilution with water, the solution is extracted with chloroform and the organic extracts dried over anhydrous sodium sulfate, concentrated, treated with hydrogen chloride in anhydrous methanol, and then diluted with ethyl ether. The precipitate formed is doxorubicin hydrochloride, which is purified by crystallization from a mixture of methanol and 1-propanol. The above reaction pathway can be summarized as shown in Figure ll, in which the anthraquinone moiety is not shown. Stability Doxorubicin hydrochloride is very stable in the solid state. It has been stored for years at room temperature without any loss in potency or indications of degradation. The lyophilized powder of doxorubicin hydrochloride with lactose is also stable, if dry and stored in well closed containers at room temperature13. The active drug substance has also been found to be stable for three months at 60C, and for three months in light of 500 ft. candles of illumination at room temperature. The lyophilized formulation is stable mder similar lighting conditions, and at 6OoC if the moisture content in the sealed vial is less than 1.0%. The effect of pH values and buffer concentrations on the stability of aqueous solutions of doxorubicin hydrochloride has been determined by spectrophotometric and chromatographic methods. Doxorubicin is stable in acidic solutions in the pH range 3.0 to 6.5, but decomposes at increasing rates as the pH is increased from 6.5 to 12. Decomposition in aqueous solution gives complex mixtures of pigmented compounds with a wide range of chromatographic polarities. Apart from the isolation of adriamycinone from dilute acid solutions13 the identification of the components of these mixtures has not been accomplished. 4. 3.2

Figure 1 . 1

S y n t h e t i c Pathway for Doxorubicin

DOXORUBICIN

265

Metabolism The two major metabolic transformations of doxorubicin in laboratory animals and in man are:
5.

a) b)

The reduction of the side chain carbonyl group to a secondary alcohol, giving 13-dihydrodoxorubicin (adriamycinol) The reductive cleavage of the daunosamine moiety with the formation of 10-deoxyadriamycinone.

The first reaction is catalyzed by an enzyme named "daunorubicin reductase," an aldo-keto reductase of a very ubiquitous nature. The reductive splitting of the benzylic glycosidic bond is, on the contrary, rather unique as no other examples of enzyme catalysis of this otherwise chemically very facile reaction have been described1. The aglycone-like compounds thus formed are then further metabolized by other typical reactions such as 2-methylation and conjugat ion22* 23. Doxorubicin and its metabolites extracted from the urine of patients treated with the drug were separated by chromatography on columns of silicic acid. The following compounds were isolated (in order of increasing polarity) (see Figure 1 2 ) : 13-dihydroadriamycinone (3), lO-deoxy-13dihydroadriamycinone(4), l-demethyl-lO-deoxy-13dihydroadriamycinone(5), doxorubicin(1) , 13-dihydrodoxorubicin(2), l-demethyl-lO-deoxy-13d ihydr oadr iamyc inone-1-2- u1fate( 6 ) , 1-demethy1-10-deoxy-13 s dihydroadriamycinone-l-~-R-D-glucuronide(7). A total of 60% of the fluorescence in the urine was due to metabolites and the remainder was unchanged drug. As the recovery of doxorubicin fluorescence in bile and urine from a patient was about 60% of the administered dose, the authors pointed out the possibility of the presence of non-fluorescent metabolite^^^. The above mentioned metabolites were also detected in the plasma of patients under doxorubicin treatment25. Protein binding studies using the ultracentrifugation method suggested that doxorubicin is bound to rabbit and human plasma proteins to an extent of 50%26, but a re-examination of the original Scatchard plot data changed this value to 90%27. Other studies using equilibrium dialysis have suggested complex binding relationships that need further investigation2*.

I
0 I

= I
0

0
00

0 00 X

I
N

I I

0 -

0 I

I
0

&8p
I 0
0

It

0\

I
0

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I

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IOU2

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DOXORUBICIN

261

The main biochemical effects of doxorubicin are concerned with nucleic acid synthesis. The binding of this drug to DNA is considered responsible for the interference with template DNA function29. The DNA-doxorubicin binding constant has been determined to be approximately 2 x lo6 M-l (30).
6 .

Methods of Analysis 6 1 Elemental Analysis . The elemental analysis of doxorubicin hydrochloride (Farmitalia reference standard batch GDA 1) is as follows:
%

Theory 55.91 5.22 2.41 6.11

Found 56.08 5.33 2.16 5.85

C H N c1 6.2

Spectrophotometric Analysis

The visible absorption maximum at 495 nm (El% = 223) lcm can be used for the quantitation of doxorubicin in dosage forms. The fluorescence properties of doxorubicin can be used for the determination of total anthracycline at low concentrations31. Electrochemical Analysis ChronopotentiometricJ1, cyclic voltammetr ic32 and p~larographic~~r 33 assays of doxorubicin hydrochloride have been reported. These techniques determine the total anthracycline content. Paper Chromatography Doxorubicin can be separated from the aglycone, adriamycinone, by paper chromatography using either of the following two systemsl3.
64 .

6.3

A) B)

1-butanol saturated with pH 5.4 M/15 phosphate buffer. 1-propanol/ethyl acetate/water, 7/1/2 by volume.

The Rf values for doxorubicin and adriamycinone are 0.1 and 0.3, and 0.25 and 0.65 for systems A and B respectively

268

ARISTIDE VIGEVANI AND MARTIN J. WILLIAMSON

Thin Layer Chromatography Thin l a y e r c h r o m a t o g r a p h i c s y s t e m s f o r d o x o r u b i c i n are g i v e n i n T a b l e 6.

6.5

TABLE 6 Thin l a y e r c h r o m a t o g r a p h i c s y s t e m s f o r d o x o r u b i c i n . Adsorbent Silica gel Silica g e l Silica gel S o l v e n t System Methylene c h l o r i d e / me t h a n o l / w a t e r (100/20/2) 1-butanol/acetic (4/1/5) acid/water

Rf
0.17 0.33

Reference

13 13

Chloroform/95% e t h a n o l / trifluoroacetic acid (75/20/5) Chloroform/me t h a n o l / a c e t i c acid (93/5/2, p l a t e d r i e d , t h e n 76/20/4) Ch loroform/me thanol/wa ter (140/60/10)

0.23

34

Silica gel

0.2

35

Silica gel sprayed with phosphate buffer (pH = 7.0) Polyamide/ cellulose

0.3 l-Butanol/2-propanol/isopropyl e t h e r / a c e t i c acid/wa ter (35/6/6/9/44) 0.3

36

37

L i q u i d Chromatography L i q u i d c h r o m a t o g r a p h i c s y s t e m s for d o x o r u b i c i n h y d r o c h l o r i d e a r e g i v e n i n T a b l e 7.

6.6

DOXORUBICIN

269

TABLE 7 Liquid chromatographic systems for doxorubicin Stationary Phase Silica (5 micron) Silica (5 micron) Silica (5 micron) Mobile Phase Approximate Doxorubicin Capacity Factor (k)

Ref.

2-pr opanol/i sopropyl e ther/0 .125 M acetate buffer pH 4.5 (65/30/5) 2-propanol/0.5 sodium acetate buffer pH 4.5 (9 6.2/3.8) Methylene chloride/ methanol/25% ammonia/ water (90/9/0.1/0.8)

10

38

39

40

Cyanopropylsilica Chloroform/methanol/ (10 micron) acetic acid/water (79.8/14.1/4.7/14 ) Cyanopropylsilica Ch lor oform/me thanol/ water (96/5/1) (10 micron)
Corasi1-pheny 1 (37-75 micron)

41 42

Linear gradient, 16% acetonitrile/in water to 20% acetonitrile/ 80% pH 4 formate buffer Linear gradient 0 to 40% acetonitrile in pH 4.0 ammonium formate buffer
Me thanol/aqueous solution of PIC B-7 (heptanesulfonic acid) (50/50)

10

43

Coras i1-pheny1 (37-50 micron) Octadecyl-silica (10 micron)

10

44

14

45

Oc tadecyl-s ilica Acetonitrile/aqueous phosphoric acid pH 2, (10 micron) (31/69) Octadecyl-silica (10 micron) Octyl-silica (10 micron)
Me thanol/water/acetic

3 1.4

46

acid (66/33.2/0.8) Acetonitrile/102 M. aq. phosphoric acid (40/60)

47

48

210

ARISTIDE VIGEVANI AND MARTIN J . WILLIAMSON

Determination of Doxorubicin in Biological Fluids Total anthracycline compounds in biological fluids can be determined by fluorimetric method^^^,^^. Radioimmunoassay procedures have also been reported50. The emphasis is now on the separate determination of metabolites and intact drug in biological fluids. One such method coupled liquid chromatography followed by RIA^^ but it was rather TLC followed by fluorescence scanning has time-consuming. been reported35 and used for disposition prediction^^^. The most recently published methods have used ~ rever sed-phase 1 iquid ~ h r o m a t o g r a p h y 5s2 or normal phase liquid ~ h r o m a t o g r a p h y ~ ~ , ~ ~ with fluorescence detection. These methods have been applied to tissue distribution studies5l. Similar methodss4 used for daunorubicin and metabolites should also be applicable. Analysis of Pharmaceutical Formulations The identification and/or determination of doxorubicin hydrochloride in Adriamycin involves the use of visible spectrophotometry, thin layer chromatography followed by spectrophotometry or microbiological agar diffusion55. However, the recently published liquid chromatographic procedure46 is replacing the above physical methods. 9. Miscellaneous Ph a rmace u t ica 1 preparations of doxor u b i c in hy dr och lor ide , trade-marked Adriamycin, have been patented20. 10. Acknowledgments Acknowledgment is made to Drs. F. Arcamone and S Penco . of Farmitalia-Carlo Erba SPA. and Drs. G Davis, W. Hausmann . and J. Short of Adria Inc., for their useful advise during the preparation of the manuscript.
11. References - Literature covered until March, 1979. F. Arcamone, in "Topics in Antibiotics Chemistry," P. Sammes Ed., Vol. 2, Ellis Horwood Publ., Chichester, 1978, pp. 102-239, and references therein.

7.

8.

F. Arcamone, G. Franceschi, P. Orezzi, S. Penco, and R. Mondelli, Tetrahedron Letters, 3349 (1968). . F. Arcamone, G. Cassinelli, G Franceschi, P. Orezzi, and R. Mndelli, Tetrahedron Letters, 3353 (1968). F. Arcamone, G Cassinelli, G Franceschi, . . R. Mondelli, P. Orezzi, and S. Penco, Gazz. Chim. Ital., 100, 949 (1970), and references therein.

DOXORUBICIN

27 1

F. Arcamone, G. F r a n c e s c h i , S. Penco, and A. S e l v a , T e t r a h e d r o n Letters, 1007 (1969).

A. Arnone, G. Fronza, R.

M o n d e l l i , and A. V i g e v a n i , T e t r a h e d r o n Letters, 3349 (1976).

B. G i o i a , F a r m i t a l i a Research L a b o r a t o r i e s , P r i v a t e Communication (1972).

A V i g e v a n i , B.

as.,

32, 321

Gioia, and G. C a s s i n e l l i , C a r b o h y d r a t e (1974).

P. P. Roller, M.

Mass Spectrom.,

3,

S u t p h i n , and A. A. A s z a l o s , Biomed. 166 (1976). Sci.,

K. K. Chan, and E. Watson, J. Pharm. (1978).

67,1748

K. H. Maurer, U. Rapp, K. Chan, and W. Sadee, Communication p r e s e n t e d a t t h e "Mario Negri 2nd I n t e r n a t i o n a l Symposium on M s S p e c t r o m e t r y i n as B i o c h e m i s t r y and Medicine", M i l a n , 24-26 J u n e , 1974. B. Gioia, F a r m i t a l i a Research L a b o r a t o r i e s , P r i v a t e Communication (1978).

F. Arcamone, G. C a s s i n e l l i , G. F r a n c e s c h i , S. Penco, C. P o l , S. R e d a e l l i , a n d A. S e l v a , " I n t e r n a t i o n a l Symposium o n Adriamycin," S. K . C a r t e r , A. D i Marco, M. Ghione, I. H. K r a k o f f , and G. Mathe Eds., S p r i n g e r - V e r l a g , B e r l i n , 1972, pp. 1-22.
H. Brockmann, H Brockmann Jr., and J. Niemeyer, . T e t r a h e d r o n Letters, 4719 ( 1 9 6 8 ) . R. A n g i u l i , E. F o r e s t i , L. Riva d i S a n s e r v e r i n o , W. Isaacs, 0. Kennard, W. D. S. Motherwell, D. L. Wampler, and F. Arcamone, Nature. New Biology, 234, 78 (1978)
S. N e i d l e and G.

N.

479,

T a y l o r , Biochim. Biophys. Acta.,

450 (1977).

E. P e l l a , Carlo Erba Research Laboratories, P r i v a t e Communication (1978). G. G o n f a l o n i e r i and G. Vasconi, Carlo Erba R e s e a r c h L a b o r a t o r i e s , P r i v a t e Communication, (1975).

212

ARISTIDE VIGEVANI AND MARTIN J . WILLIAMSON

19) 20)

- 958 66,

R. J S t u r g e o n , and S. S. Schulman, J. Pharm. .

Sci.,

(1977).

F. Arcamone, G. C a s s i n e l l i , G. F a n t i n i , A. G r e i n , P. O r e z z i , C. p o l , and C. Spalla, B i o t e c h n o l . Bioeng., &, 1 1 0 1 (1969). F. Arcamone, G. F r a n c e s c h i , and S. Penco, U S .. 3,803,124 (Apr. 9, 1974). Patent

21)

22) M. A. A s b e l l , R. Schwartzbach, F. J. B u l l o c k , and D. W. Yesair, J. Pharmacol. Exp. Ther., 182, 63 (1972). 23) 24) 25) 26) 27)
F. J. B u l l o c k , R. J. B r u n i , M. A. A s b e l l , J. Pharmacol. Exp. Ther., 182, 70 (1972).
S Takanashi and N. R. Bachur, Drug Metabolism and . D i s p o s i t i o n , Q, 79 (1976).
R. S. Benjamin, C. E. Riggs, Jr., Cancer Res., 37, 1416 (1977).

and N.

R.

Bachur,

59,

P. A. H a r r i s and J. F. Gross, Cancer Chemother, Rep., 819 (1975).

K. K. Chan, J. L. Cohen, J. F. Gross, K. J. H i m e l s t e i n , J. R. Bateman, Y. Tsu-Lee, and A. S. Marlis, Cancer Treatment Reports, 62, 1161

(1978).

28)

M.

Menozzi, F a r m i t a l i a Research L a b o r a t o r i e s , P r i v a t e Communication (1978).

29) A. DiMarco and F. Arcamone, Arzneim.-Fbrsch. (Drug R e s e a r c h ) , 25, 368 (1975) and r e f e r e n c e s c i t e d
t h e r ein.

30) 31) 32) 33)

S. R.

Byrn and G. D. Dolch, J. Pharm. S c i . ,

(1978) and r e f e r e n c e s c i t e d .

67, 688
Res.

L Dusonchet, N. Gebbia, and F. G e r b a s s i , Pharm. . Commun., 2, 55 (1971).

G. M. Rao, J. W. LOwn, and J. A. Plambeck, J. Electrochem. Soc., 125, 534 (1978).

L A. S t e r n s o n and G. Thomas, A n a l . Letters, . (1977).

10, 99

DO XO RUBICIN
G. W.

273

Communication, (1979).

C l a r k , Adria Laboratories , P r i v a t e

60,

E. Watson and K.

K.

Chan, Cancer Treatment R e p o r t s , A p r i l 2, 1976 S e c t i o n

1611 (1976)

Federal Register

436.315.
Federal Register

4 , 14184, 1

436.314.

4, 1

14184, A p r i l 2, 1976 S e c t i o n

F a r m i t a l i a SpA., J u n e 1975, P r i v a t e Communication.

H . G B a r t h and A. .

Z. COnner, J. Chromatogr.,

375 (1977).
R. Hulhoven and J. P. Desager, J. Chromatogr.,

131,

369 (1976). 131 (1976).

V. P. M a r s h a l l , E. J. A n t i b i o t i c s ,

125,
2,

P. A. Harris, Proc. Amer. A s s o c . Cancer R e s e a r c h , R e i s e n d e r , and P. F. W i l l e y .

29, 966

A.

(1976).
Bachur, Biochem.

J. J. Langone, H v a n Vunakis, and N.

Med.,

2,283

(1975).

M. I s r a e l , W. J. Pegg, P. M. W i l k i n s o n and M. B. G a r n i c k , " B i o c h e m i c a l / B i o l o g i c a l A p p l i c a t i o n s of L i q u i d Chromatography," G. L. Hawk, e d i t o r , Marcel D e k k e r Inc., N.Y. 1978, Chap. 22.

Waters P h a r m a c e u t i c a l A p p l i c a t i o n Note #312.

Federal Register S e c t i o n 436.322.

43, 44836,

September 29, 1978,

L. M a l s p e i s , Ohio S t a t e U n i v e r s i t y , P e r s o n a l Communication, (1978). S. Eksborg, J. Chromatogr.,

N.

149, 225 (1978).

R. Bachur, A. L. Moore, J. G. B e r n s t e i n a n d A. L i u , Cancer Chemotherap. Rep., 54, 89 (1970).

H.van Vunakis, J. J. Langone, L. J. R i c e b e r g and L. Levine, Cancer R e s e a r c h , 34, 2546 (1976).

274

ARISTIDE VIGEVANI A N D MARTIN J . WILLIAMSON

51) 52) 53) 54) 55)

M. Israel, W. J. Pegg., P M. Wilkinson and M. B . . Garnick, J. Liquid Chromatogr., 1,795 (1978).

. R. Baurain, D Deprez-DeCampeneere and A. Trouet, Analytical Biochemistry, 99, 112 (1979).

J. H. Peters and J. F Murray Jr., J. Liquid . , Chromatogr., 2 45 (1979)

S. Eksborg, H Ehrsson, B. Anderson and M. Beran, J. . Chromatogr., 153, 211 (1978).

Federal Register 450.24.

4 , 14184, April 2, 1976, Section 1

FLUPHENAZINE DECANOATE.
Geofiey Clarke
1.

2.

3. 4. 5. 6.

7. 8.

Description I . 1 Name, Formula, Molecular Weight 1.2 Appearance, Color, Odor Physical Properties 2.1 lnfrared Spectrum 2.2 Ultraviolet Spectrum 2.3 Nuclear Magnetic Resonance Spectrum 2.4 Fluorescence Spectrum 2.5 Mass Spectrum 2.6 Melting Range 2.7 Refractive Index 2.8 Solubility 2.9 pKa 2.10 Differential Thermal Analysis Synthesis Stability Drug Metabolism Methods of Analysis 6.1 Elemental Analysis 6.2 Non-aqueous Titration 6.3 Spectrophotometric Analysis 6.4 Colorimetric Analysis 6.5 Fluorometric Analysis 6.6 Chromatographic Analysis Body Fluid and Tissue Analysis References

276 276 276 216 279 219 219 28 1 28 1 28 1 28 1 284 284 284 284 284 286 286 286 286 286 287 287 29 1 293

Analytical Profiles of D u Substances. 9 rg

215

Copyright 01980 by Academic Press. Inc All rights ofreproduction in any form reserved. ISBN: 012-260809-7

216

GEOFFREY CLARKE

1 .

Description

11 Name, Formula, Molecular Weight. ..

Fluphenazine decanoate is 4-[3-[2-(trifluoromethyl)phenothiaz i n -1O-ylI propyl] -1 piperazine ethanol decanoate ester; P r o l i x i n decanoate; S Q 10,733

(CH2)3 N

n(CH2)2 N

9
c

(CH2)8 CH3

Molecular weight 591.7

1.2. Appearance, Odor, Color.

Fluphenazine decanoate is a pale yellow to yellow orange viscous liquid w i t h a characteristic odor. A t r o o m temperature the l i q u i d w i l l slowly crystallise.

2.

Physical properties 2.1 Infrared spectrum The infrared spectrum o f fluphenazine decanoate(1ot 117102, p u r i t y 99%) in the liquid phase (as a thin f i l m ) is given in figure 1 The following W j g n m e n t s have been made f o r the . most characteristic bands

Frequency(cm-')

Ass ignment A r o m a t i c C-H stretching vibrations Ester carbonyl stretching vibrations aromatic r i n g skeletal vibrations C-H out o f plane bending vibrations

2 940
1740 1605 1575 930,870 820,750

"t

I I

II

F i g u r e 1. I n f r a r e d s p e c t r u m o f F l u p h e n a z i n e d e c a n o a t e as a t h i n f i l m . I n s t r u m e n t : Unicam SP 1000.

Figure 2.

U l t r a v i o i f t s p e c t r u m of F l u p h e n a z i n e d e c a n o a t e i n m e t h a n o l (15ug m l ) . I n s t r u m e n t : P e r k i n E l m e r 137.
0
SLH
ffl

a,d

FLUPHENAZINE DECANOATE

279

The four bands between 750 and 930cm-1 are reported t o be characteristic o f 2, 1 0 disubstitu ed phenothiazines and t h e t w o bands a t 1605 d 1575cm c h a r a c t e r i s t i c f o r phenothiazines in general (397

-!

2.2. U l t r a v i o l e t spectrum The u l t r a v i o l e t spectrum o f fluphenazine decanoate(1ot 117102, p u r i t y 9%) in methanol is given in figure 2. Similar spectra are obtained i n ethanol and c h l o r o f o r m although t h e shoulder a t 240nm i s obscured in c h l o r o f o r m due t o absorption o f t h e solvent. The spectrum given in figure 2 is characteristic o f a phenothiazine and t h e location o f the m o s t intense peak a t i s consistent w i t h a halogen substituent in t h e 2-position

4cm 240nm 261nm 551 562 586 315nm 74.3 65.9 68.4

1Yo

Chlor0 f o r m
Methanol Ethanol(95%)

220 221

2.3 Fluorescence spectrum Fluphenazine decanoate does n o t e x h i b i t any s i g n i f i c a n t l y measurable fluorescence in ethanolic solution. Fluorescence measurement can be made however a f t e r p r i o r oxidation to t h e sulphoxide. (See section 6.5).

2.4 Nuclear magnetic resonance spectrum.

The 100 M H z spectrum o f fluphenazine decanoate in DMSOd&internal reference TMS) i s given i n figure 3.

F i g u r e 3 . N u c l e a r m a g n e t i c resonance s p e c t r u m of F l u p h e n a z i n e d e c a n o a t e i n DMSO-d6. I n s t r u m e n t : Thompson P a c k a r d .

FLUPHENAZINE D E C A N O A T E

281

The c h y g i c a l s h i f t s produced can be assigned to the following protons d e a b c n f g h i k

proton
a

chemical shi fts(dppm). 3.97 1.77 2.22 4.07 2.22 1.20 0.82 6.97 7.11 7.22 triplet(6.5 Hz) multiplet multiplet triplet(6.5 Hz) multiplet singlet triplet(7.0 Hz)

b c,d,e,d 9 h j

Y ,f e

Aromatic

2.5 Mass Spectrum The low resolution mass spectrum i s given in figure 4. The molecular ion a t rn/e 591 and t h e fragmentation p a t t e r n depicted i n figure 5 are consistent w i t h the structure given f o r fluphenazine decanoate. The m / e 4ffi may be due t o either fragment a t i o n or t o fluphenazine base

2.6 M e l t i n g range
The m e l t i n g range o f crystallised fluphenazine decanoate has been determined as 30-3ZC.(Lot 117102,purity 99%). 2.7 R e f r a c t i v e index The r e f r a c t i v e index has been determined as 1.5395 a t 25OC on a sample o f m a t e r i a l o f 99% purity.(Lot 117102).

2.8 Solubility
Fluphenazine decanoate is inso uble in water ( d O u g mlml) b u t extremely soluble(>lOOOrng m l ) in chloroform, d i e t h y l ether, It is also extremely soluble cyclohexane, methanol an$ythanol. i n coconut and sesame oil.

-1

282

RELATIVE INTENSITY

a
2a

; 8 g : % 8 8 S f 3 $ E

40

60
80 100
120

140 160 180 200 220 240 260

'? 300
7,

280

' 0

rtm w m
. .

5 320
U

340
I Y

360

380 400 420 440 460 480 500 520 540 560 580

600
620 0
h )

U l

PERCENT TOTAL IONIZATION Z 39

I----110

O t-

hl S

I1 s a,
-4
N

I
0

e l

I SI -rll I I

c,
rd
0

a,

E
c
rd

f
c
rd

a"
a,

r:

. c

r---

a
b4
4-1

I
I
1 I I

3
N +

283

284

GEOFFREY CLARKE

2.9 pKa
The p K a and p K a values f o r fluphenazine decanoate have n o t been reported. dowever they would be expected t o be very similar t o those reported f o r fluphenazine enanthate o f about 3.4 and 8.0.(see analytical profiles of drug substances, volume 2).

2.10 D i f f e r e n t i a l thermal analysis

Beoween 15OC and 200C only the endotherm due t o m e l t i n g a t 30 C is observed f o r the crystalline material. Thg c(iidrochloride salt gives an endotherm due t o m e l t i n g a t 180 C.

3.

Synthesis Fluphenazine decanoate(1) can be prepared from fluphenazine(I1) by refluxing a chloroform solution o f (11) w i t h decanoyl chloride. (See Figure 6). The fluphenazine decanoate is e x t r a c t e d as t h e hydrochloride salt and recrystallised f r o m a m i x t u r e o f anhydrous acetone and ether. A f t e r reconversion t o the base w i t h aqueous sodium carbonate t h e fluphenazine decanoa extract ed into ether, dried and concentrated b y evaporation

t 8 , h.

4. Stability
Fluphenazine dpjanoate(1) w i l l hydrolyse i n alkaline medium t o fluphenazine(I1) In the presence o f peroxides, oxidation o f t h e piperazine nitrogen(506afj N -oxide(III) occurs probably by a f r e e radical mechanism. Fluphen ine decanoate w i l l undergo photolysis t o f o r m a sulphoxide(IV{?(See Figure 6).

5.

Drug metabolism Studies w i t h 14C labelled fluphenazine decanoate i n t h e dog have been reported. The fluphenazine decanoate i s hydrolysed t o fluphenazine by plasma esterases and excreted in the urine. The metabolism o f fluphenazine decanoate, in the dog, is therefore similar to(@at o f fluphenazine dihydrochloride and fluphenazine enanthate .(See analytical profiles o f drug substances, volume 2). Traces o f residual fluphenazine decanoate and/or i t s meta~ liver, kidney, skin and h e a r t of , bolites were found in t h e l the dog b u t none i n the brain

light

(CH2)3.N- . N(

c H2)2 0

Figure 6.

Chemistry of Fluphenazine decanoate.

286

GEOFFREY CLARKE

6. Methods o f analysis 6.1 Elemental analysis The following analysis has been made(10)
Ca1c u1a t ed

Found 65.19 7.68

H N
6.2 Non-aqueous t i t r a t i o n

64.94 7.42 7.09

7.2Y

Fluphenazine decanoate can be t i t r a t e d w i t h perchloric a c i d in glacial acetic acid using c r y s t a l violet as the indicator. The neutralisation equivalent is 295.75. End point detection either visually v f h malachite green or potentiometrically has also been reported

6.3 Spectrophotometric analysis The U V absorbance a t 261nm o f fluphenazine decanoate in methanol can be used as a quantitative assay. UV spectrophotom e t r y however w i l l only d i f f e r e n t i a t e between fluphenazine decanoate and its sulphoxide, therefore a chromatographic sepa r a t i o n o f fluphenazine decanoate f r o m other related substances usually precedes UV measurement(See section 6.6). 6.4 C o l o r i m e t r i c analysis Fluphenazine decanoate can be extracted as an ion-pair w i t h bromothymol blue into toluene f r o m a p H 2.55 g l y c i n e / q I buffer. The absorbance o f the solution i s measured a t 400nm.

(7

A solution of fluphenazine decanoate in e t h y l acetate when shaken w i t h a pH2 b u f f e r e d solution o f palladium chloride w i l l f o r m a complex. This complex is s o h@e,jtj, the e t h y l acetate Palladium comphase and can be measured a t 440nm. plexes o f any other esters o f fluphenazine present as trace impurities, would also be formed i n the e t h y l acetate phase. However, the major hydrolysis product fluphenazine forms an aqueous soluble complex and would r e m a i n in the b u f f e r phase. Therefore, spectrophotometric measurement o f the aqueous b u f f e r phase coulq& used as an assay f o r fluphenazine i n fluphenazine decanoate

FLU PH EN A ZI N E DEC A N 0ATE

287

6.5 F l u o r i m e t r i c analysis

A spectrofluorimetric procedure has been reported i n which a solution of fluphenazine decanoate in methanol/sulphuric acid (80:20),after oxidation w i t h c e r i c ioTy@ t h e sulphoxide, fluoresces a t 400nm when activated a t 343nm.

6.6 Chromatographic analysis 6.6.1 Column chromatography Fluphenazine decanoate and r e l a t e d substances can be adsorbed onto a column o f silica gel f r o m a chloroform solution. The fluphenazine decanoate can be selectively eluted f r o m t h e column w i t h a solvent m i x t u r e o f cyclohexane/methanol/methylacetate (67.2:35.6:97.2). A f t e r removing t h e solvent by evaporation t h e fluphenazine decanoate can be quantified b y f i y o l v i n g i n methanol and measuring the U V absorbance a t 261nm The major degradation products, fluphenazine and fluphenazine decanoate N-oxide are n o t eluted f r o m t h e silica gel column.

6.6.2

Paper chromatography

The following systems have been reported, although no R values are quoted. Benzene/acetic/water(Z:Z:l) descending o n Wfiatman No.1 paper and sodium formate(1 molar) ascending on Whatman 3 M M paper for the separation o f fluphenazine decanoate, fluphenazine and fluphenazine sulphoxide. Methanol/water(85:15) descending on Whatman No.1 paper impregnated w i t h castor oi1(2% in ether) f o r t h e separation o f fluphenazine decanoate, fluphenyfae, fluphenazine octanoate and fluphenazine dodeLocation i s by U V l i g h t and quantitation by elution canoate w i t h 95% ethanol and measuring the absorbance a t 261nm.

6.6.3 Thin layer chromatography A summary o f t h e solvent systems and separations reported is given in table 1 The adsorbent used in a l l systems is silica gel G . w i t h a fluorescent indicator. Location o f separated compounds i s made b y fluorescence quenching of UV light(366 or 254nm) or c o l o r i m e t r i c a l l y b y spraying w i t h 50% sulphuric a c i d t o produce r e d zones. The solvent systems r e f e r r e d t o in table 1 a r e as follows

Rf values
Solvent System Fluphenazine Fluphenazine sulphoxide Fluphenazine dec anoat e N-oxides Fluphenazine dec anoat e Fluphenazine dec anoate sulphoxide

I II III

06 .0
0.73

0.25

0 0 , 0.16 .0
0.00, 0.20" 0.08, 0.25

0. ao 0.84
0.50

0.73

00 . 00 .

0.80 0.48
0.75

0.10

00 .
0.0

IV V

01 0 .
0. ao

0. ao
0.90

*All 4 zones have n o t been positively identified as N-oxides. Table 1 Thin layer chromatography. .

FLUPHENAZINE DECANOATE

289

I II
111
IV

Cyclohexane/acetone/ammonia(30:80:5) (17)

Chloroform(saturated w i t h ammonia)/methanol(80:2) (18) Cyclohexane/acetone/ammonia(36:60:0.6)

(17)

Methanol/ethyl acetate/cyclohexane/chloroform (19)

(9: :17 :3 8) 25
V

Chloroform/methanol/ammonia(9:10:0.5) (20)

Solvent system (111) has been used as t h e basis f o r a quantitative assay, the separated zones being ed w i t h methanol and the UV absorbance measured a t 261nm

@if

6.6.4 Gas l i q u i d chromatography Fluphenazine decanoate has been separated f r o m fluphenazine by chromatographing the silylated omixture on a 5' x 4'' column o f 3% JXR* on Gas C h r o m Q a t 280 C. The carrier gas was nitrogen and detection was b y f l a m e ionisation. Perphenazine was used as an i n t T S y 1 standard and t h e following retention times were report.

Fluphenazine Perphenazine Fluphenazi ne decanoa t e

4 minutes 7 minutes 24 minutes

The silylation procedure was necessary in t h e above method t o satisfactorily chromatograph the fluphenazine. However, fluphenazine esters do n o t require s i l y l a t i n g and t w o other procedures have been reported f o r the separation o f fluphenazine decanoate f r o m o t h e r fluphenazine esters. W i t h the exception o f t h e temperature and the absence o f silylation the conditions were as above. The following separations were reported. 305C(23) Fluphenazine octanoate Fluphenazine decanoate Fluphenazine dodecanoate Fluphenazine stearate/ oleate/linoleate 5.2 minutes 7.4 minutes 11.2 minutes 3.4 minutes 330C(24)

1m i n u t e

*JXR-Applied Science Laboratories 1nc.State College P.A.U.S.A.

290

GEOFFREY CLARKE

6.6.5 High performance liquid chromatography A reversed phase HPLC system f o r t h e separation o f fluphenazine f r o m fluphenafB7 decanoate and other fluphenazine esters The e f f e c t s o f t h e p H o f t h e mobile has been reported. phase and the chain length o f the stationary phase were studied and the following separation reported.

Column Mobile phase

Partisil-TMS*(trimethylsilane)
200 x 4.6mm ID
Methanol/acetonitrile/ 1% ammonium carbonate (1:l:O. 3)

Flow r a t e D e t e c t ion

: :

-1 2ml min

U V a t 260nm Fluphenazine Fluphenazine decanoate Fluphenazine m y r i s t a t e Fluphenazine palmi t a t e Fluphenazine stearate

Retention times :

2.6 minutes
3.3 minutes
4.2 minutes 4.8 minutes 5.8 minutes

Slight variation in the ammonium carbonate concentration had l i t t l e e f f e c t over the range 0.1-1%. However, changes in the r a t i o o f t o t a l organic p m t o aqueous phase has a marked e f f e c t on r e t v $ p n times Hence t h e following separation has been reported

Column Mobile phase

: :

Partisil-TMS* 250 x 4.6mm ID. Methanol/acetonitrile/ 0.45% ammonium carbonate

Flow r a t e Detection

: :

(1:l:l) -1
2 m l min U V a t 260nm. Fluphenazine Fluphenazine decanoate N-oxi des Fluphenazine decanoate 4.5minutes a) 9 minutes b) 1 minutes 1 14.5 minutes

Retention times :

FLUPHENAZINE DECANOATE

29 1

Using a similar mobile phase t o the above a separation o f the octanoate and d o q y y n o a t e impurities i n fluphenazine decanoate has been reported

Column Mobile phase

: :

Bondapak C18** 300 x 3.9mm ID Methanol/acetonitrile/ 0.4% ammonium carbonate (1:1:0.5). 2ml min-l 254nm Fluphenazine Fluphenazine octanoate Fluphena z i ne de canoa t e Fluphenazine dodecanoate 2 minutes

Flow rate Detection

: :

Retention times :

3 minutes
4.5 minutes 6.5 minutes

*Partisil, Reeve Ange1,Clifton NJ. US.A. **Bond a pa k, Waters Ass oci a t es

7.

Body f l u i d and tissue analysis

i t s hydrolysis product fluphenazine. Whilst traces o f the hy dr o I ys edc2 f $ ~hena z ine d e c ano a t e ha v e been d e t ec t ed by p

The metabolism o f fluphenazine decanoate is m a i n l y t h a t o f

C tracing, most o f the reported work has been directed towards the detection and estimation o f fluphenazine and i t s metabolites. In man, p p o d plasm 1 o f fluphenazine have C tracing a f t e r i n i t i a l extracbeen determined by t i o n of the alkaline plasma w i t h n-heptane. Further partitioninq34yeparated various metabolites in the urine and faeces U r i n e and plasma e x t r a c t s have also been analysed b y GLC(34) using an alkali-bead nitrogen sensitive detector and a column o f 3% OV-17" o n Chromosorb W a t 215OC. A similar p r o c e q y e using a f l a m e ionisation detector has also Another G L C procedure has been repo been reported f o r determining fluphenazine and i t s metabolites in urine The urine was extracted by adsorption onto a n A m b e r l i t e X A D - 2 column, washing w i t h pH8.5 ammonium chloride b u f f e r and elution w i t h methanol. The extracted metabolites were chromatographed as t h e i r t r i r n e t h y l s i l y l derivatives on 2% SE30 on Gas Chrom Q a t 225OC. D e t e c t i o n was b y f l a m e ion is at ion.

y8

" i, f'

fW.

292

GEOFFREY CLARKE

A f l u o r i m e t r i c procedure f o r blood plasma has been reported in which the plasma is e x t r a c t e d w i t h hepiary&yamyl alcoho1(98.3:1.7) a f t e r alkaline hydrolysis a t 100 C The metabolites are back e x t r a c t e d i n t o 0.1M phosphate b u f f e r and oxidised w i t h hydrogen peroxide. Fluorescence measurement was a t 405nm, e x c i t i n g a t 350nm. A n HPLC procedure has been reported f o r hexane e x t r a c t e d serum a glassy carbon electrode as an electrochemical detector The mobile phase was methanol/O.O5M phosphate buffer, p H 6.9(53:47) and t h e separation was achieved on a Lichrosorb S1(60)column(Merck, Darmstadt, GFR), t r e a t e d w i t h dichlorodirnethylsilane.

UTW

( 3 2 16Zl. ::)

A TLC r a t i o n of metabolites i n animal tissues has been reportedy5? The tissues were extracted w i t h dichloromethane and chromatographed on silica gel i n chloroform/isopropyl alcohol(l0:l) and isopropyl alcohol/chloroform/arnmonia/water

*OV-17, Applied Science Laboratories Inc., State College P A U.S.A.

Acknowledgement The author wishes t o thank Mrs. M. Watson f o r her invaluable secretarial help.

FLUPHENAZINE DECANOATE

293

8.

References Squibb Institute, p r i v a t e commun-

M.S.Puar and P.T.Funke; ication. P.R.Wood; P.Dondzila; H.L.Yale, (1963).

Squibb Institute, p r i v a t e communicaton. Squibb Institute,private communication. A.1.Cohen and F.Sowinski; J.Med.Chem.6, - 347,

H.K ad in; Squ ibb Inst it u t e ,private communication.

R.D.G.Woolfenden;Squibb B.J.Millard;School cation. Institute,private communication.
of Pharmacy, London, p r i v a t e communi-

J.Dreyfuss, J.J.Ross and E.C.Schreiber; 829,( 1971). H.L.Ya1e and R.C.Merril1; J.A.Hill;Squibb U.S.Patent

J.Pharm.Sci.,

60,

U.Timm and S.Pfeifer;Pharmazie a,11,(1973).

1 194,733(1965).

Institute,private communication.

H.Kadin; Squibb Institute,private communication.

L.Cavatorta;J.Pharm.Pharmacol,c 49(1959).
G.A.Brewer jnr;Squibb Institute,private communication.

M.Parr y; Squibb Inst i t u t e ,pr ivat e com municat ion.

H.R.Roberts;Squibb Inst itute,private communication. Inst itute,private communiS. Shand;Squi bb Inst itute,pri vat e communication. M.Parry and 1.M.Jackson;Squibb cation. C.L.Kroll;Squibb W.F.Heyes;Squibb T.Cowen;Squibb

C . .Hug hes ;Squ ibb Inst itut e ,pr iv a t e communication. G Inst itute,private communication. Institute,private communication. Inst itute,private communication. W .F. H eyes;Squi bb Inst itut e,private communication.

M. Parr y; Squ i bb Inst itute,pr iv a t e com municat ion. W.F.Heyes and J.R.Sa1mon;J.Chromatog.E W.F.Heyes;Squibb

309(1978)

Institute,private communication.

P.Y eh ;Squ ibb Inst itute,pr iv a t e communication.

E.C. Schre ibe r and M.L.Gro z ier;T hera p i e

3441( 1973).

294

GEOFFREY CLARKE

- 294(1973). 75

M.I.Kelsey,A.Keskiner

and E.A.Moscatelli; J.Chromatog. and P.G.Geddes;

E.C.Dinovo,L.A.Gotlschalk,B.Naudi J. Phar m. Sci .65 66 7(197 6). -

A.B.Smulevitch,E.l.Minsker, N.A.Mazayyena, R.P. Volkora and S.K.Lukanina; Comprehensive Psych. 227(1973).

14

C.P.Chien, T.L.Chan, D.Daniano and K.Chung; Abstracts, 10th congress C.I.N.P., Quebec(1976). (33)

32(10)1276(1973).

F.Qui t ken, A. R it k i n and D.F.Klei n., A r c h .Gen .Psychat.

- 869(1976). 28

(34)
(35) (36) (37)

R.Whelpton and S.H.Curry;J.Pharm.Pharmacol.

U.R.Tjaden, J.Laukelma, H.Poppe and R.G. Muusze; J.Chromatog.125 275(1976). N.J.Gaertner, U.Breyer and G.Liornin; Biochem.Pharm.23 303.
L

J. Phar m Sci - 2) 144( 1966). 55(

R. J W arren, I. B. Eisdorf er, W .E. Thompson and .

. .

J. E. Zarern bo;

GENTAMICIN SULFATE Bernard E. Rosenkruntz, Joseph R. Greco, John G. Hoogerheide, and Edwin M . Oden
1.

2.

3. 4. 5. 6. 7.

8.

9. 10.

11.

12.

Description 1 . 1 Drug Properties 1.2 Chemical Properties and Structure 1.3 Appearance, Color, Odor 1.4 The USP Standard Physical Properties 2.1 Infrared Spectrum 2.2 Ultraviolet Spectrum 2.3 NMR Spectrum 2.4 Mass Spectrum 2.5 Thermal Properties (DSC, TGA) 2.6 Electrometric Titration-pK Value 2.7 Optical Rotation 2.8 X-Ray Diffraction 2.9 Solubility 2.10 Countercurrent Distribution Biosynthesis Isolation and Purification Processes Drug Metabolism and Pharmacokinetics Stability Methods of Analysis 7.1 Identification 7.2 Determination of Sulfate 7.3 Loss on Drying and Moisture Content 7.4 Determination of Component Ratios 7.5 Microbiological Assay Chromatographic Analysis 8.1 Paper Chromatography 8.2 Thin Layer Chromatography 8.3 Ion Exchange Chromatography 8.4 Gas- Liquid Chromatography 8.5 High Pressure Liquid Chromatography Electrophoresis Determination in Body Fluids 10.1 Microbiological Assay 10.2 Fluoroimmunoassay 10.3 Radioimmunoassay 10.4 Radioenzyme Assay 10.5 High Pressure Liquid Chromatography Acknowledgments References
295

296 296 296 298 298 298 298 300 300 302 302 310 310 310 313 314 314 314 315 315 316 316 316 317 317 320 320 320 32 1 326 326 326 327 330 330 330 330 331 33 1 333 334
Copynght @ 1980 by Academic Ress. Inc. AU rights of reproduction i any form IXSSNC~. n ISBN: 0-12-260809-7

Analytical Rofiles of Drug Substances, 9

296

BERNARD E. ROSENKRANTZ et al.

Gentamicin S u l f a t e

1.

Description

1.1

Drug P r o p e r t i e s

Gentamicin i s an important member of t h e aminoglyc o s i d e class of a n t i b i o t i c s u b s t a n c e s t h a t w a s f i r s t i s o l a t e d i n 1963 by W e i n s t e i n e t a l . 1 from two p r e v i o u s l y undescribed s p e c i e s of Micromonospora. I s o l a t i o n and prel i m i n a r y chemical s t u d i e s 2 demonstrated t h a t i t is a m i x t u r e of b a s i c , water s o l u b l e a n t i b i o t i c s c o n t a i n i n g t h e a m i n o c y c l i t o l 2-deoxystreptamine and 2 a d d i t i o n a l amino sugars. Chromatographic s e p a r a t i o n of t h e g e n t a m i c i n complex showed i t t o c o n s i s t of 3 major components d e s i g n a t e d The g e n t a m i c i n complex is used a s as C1, C2 and C l a . 3 9 4 t h e s u l f a t e s a l t i n v a r i o u s dosage forms i n c l u d i n g i n j e c t a b l e and t o p i c a l p r e p a r a t i o n s , and is e f f e c t i v e a g a i n s t a wide v a r i e t y of gram-negative and gram-positive organisms.

1.2

Chemical P r o p e r t i e s and S t r u c t u r e

Each of t h e t h r e e major components of t h e gentamic i n complex c o n t a i n s f i v e b a s i c amino f u n c t i o n s . A s i s t y p i c a l of t h i s c l a s s of a n t i b i o t i c s , 6 g e n t a m i c i n s u l f a t e i s o b t a i n e d a s a h y d r a t e d amorphous s o l i d w i t h o u t c h a r a c t e r i s t i c m e l t i n g p o i n t , o r UV a b s o r p t i o n . The e l u c i d a t i o n of t h e s t r u c t u r e and s t e r e o c h e m i s t r y of t h e components of t h e g e n t a m i c i n complex a r e d e s c r i b e d i n p u b l i c a t i o n s by Cooper e t al.7-11 and Daniels.12 The s t r u c t u r a l formulae, m o l e c u l a r w e i g h t s and t h e nomenclature of t h e amino s u g a r u n i t s comprising t h e g e n t a m i c i n complex are g i v e n i n F i g u r e 1; t h e common s u g a r u n i t has been named garosamine and t h e d i s s i m i l a r 2,6-diamino s u g a r s have been named purpurosamine A , B and C , corresponding t o g e n t a m i c i n s C1, C 2 , and Cia, r e s p e c t i v e l y .
A number of i n v e s t i g a t o r s have r e p o r t e d on minor components t h a t are coproduced w i t h gentamicin. In addition t o g e n t a m i c i n A and g e n t a m i c i n B which were noted i n t h e o r i g i n a l p a p e r by W e i n s t e i n e t al.1, t h e s e minor components i n c l u d e gentamicins B1, X, C2a, A summary of t h e methods used t o i s o l a t e and s e p aand e t ese is g i v e n i n a rat r e c e n t review.13

c2k

PURPUROSAMINE
/ 0 /

2-DEOXYSTREPTAMINE

GENTAMICIN C1 GENTAMICIN C2 GENTAMICIN Cia

R = R1 = CH3 R = CH3; R, = H R = R) = H

C21 H 4 3 N 5 0 7 (M.W. 477.6) C20H41 N507 (M.W. 4 6 3 . 6 ) C19 H 3 9 N 5 0 7 (M.W. 4 4 9 . 5 )

Figure 1:

Structural Formula of Gentamicin Complex.

298

BERNARD E. ROSENKRANTZ

el

al.

1.3

Appearance, C o l o r , Odor

Gentamicin s u l f a t e is a w h i t e t o b u f f c o l o r e d , o d o r l e s s , h y g r o s c o p i c powder. 1.4 The USP S t a n d a r d

The b i o l o g i c a l a c t i v i t y of b u l k g e n t a m i c i n s u l f a t e i s e x p r e s s e d i n mcg g e n t a m i c i n p e r mg g e n t a m i c i n s u l f a t e based on a p o t e n c y of 1000 mcg p e r mg ( d r i e d b a s i s ) o r i g i n a l l y a s s i g n e d t o t h e master s t a n d a r d b a s e . The c u r r e n t USP S t a n d a r d of g e n t a m i c i n s u l f a t e h a s a p o t e n c y of 650 mcg/mg on t h e d r i e d b a s i s and t h e minimum a c c e p t a n c e l i m i t on potency f o r g e n t a m i c i n s u l f a t e b u l k s u b s t a n c e i s 590 mcg/mg ( d r i e d b a s i s ) . FDA c e r t i f i c a t i o n a l s o r e q u i r e s compliance w i t h s p e c i f i c a t i o n s f o r i d e n t i t y , pH, l o s s on d r y i n g , o p t i c a l r o t a t i o n and g e n t a m i c i n C component r a t i o s . 1 4

2.

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

The i n f r a r e d s p e c t r u m of a p o t a s s i u m bromide (KBr) p e l l e t of Gentamicin S u l f a t e USP R e f e r e n c e S t a n d a r d is g i v e n i n F i g u r e 2. I t was o b t a i n e d u s i n g a P e r k i n E l m e r 180 g r a t i n g s p e c t r o p h o t o m e t e r . The i n f r a r e d band a s s i g n ments a r e g i v e n below.l5 I t s h o u l d b e n o t e d t h a t bands are n o t p r e s e n t which would p e r m i t d i f f e r e n t i a t i o n from s i m i l a r aminoglycoside a n t i b i o t i c s .

-1 Wavenumber (cm )
3500-2500 ( s , v b r )

Assignment
OH, NH3

+, NH2 +

stretch

1620 (m)
1525 (m) 1150-1000 ( v s , b r ) 610 ( s ) Notation:

NH3
NH3

+, NH2 +

symmetric bend bend

+, NH2 + symmetric
HS04

C-0,

- stretch

SO2 bend

w = weak, m = medium, s = s t r o n g , vs = v e r y s t r o n g , b r = b r o a d , v b r = v e r y broad.

m
U

id

oa,
C

n -la,

ua, ar-r

-lw ala,

a&

cv

..

300

BERNARD E. ROSENKRANTZ ef (11.

2.2

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

The g e n t a m i c i n complex does n o t p o s s e s s u l t r a v i o l e t l i g h t a b s o r b i n g p r o p e r t i e s ; b o t h t h e f r e e b a s e and s u l f a t e show end a b s o r p t i o n only. 2.3 Nuclear Magnetic Resonance S p e c t r a 2.3.1 Proton Magnetic Resonance Spectrum

An 80 M z p r o t o n NMR spectrum of a s o l u t i o n H of Gentamicin S u l f a t e USP Reference S t a n d a r d 15% w/v i n D20 i s g i v e n i n F i g u r e 3. It was o b t a i n e d u s i n g a Varian CFT-20 s p e c t r o m e t e r a t ambient t e m p e r a t u r e and sodium 2,2-dimethylY 2-silapentane-5-sulfonate (DSS) as t h e i n t e r n a l r e f e r e n c e . The s p e c t r a l assignments g i v e n below a r e i n ppm ( 6 ) downfield from DSS.I5

Protons 5-CH

Chemical S h i f t s ( 6) 1.30 1.35 4CH2 1.75-2.5

Multiplicity doublets (J=7.5 Hz) singlet broad singlet singlet doublet (J=11.0 H z ) mu1t i p l e t d o u b l e t of d o u b l e t (J=11.0, 4.0 Hz) doublet (5-4.0 Hz) ove r l a p p i n g doublets
C1,

Oripin components of C1 and C 2

(a3)

4-CH3
2, 3,

5-CH ( CH3)NHa3 3-NHCH3 3-H 5-CH20 eq

2.7 5
2.95 3.48 4.0 4.25
5.16

1 2 la 1 2
la

2-H 1-H
1-H

C 2 , Cla

5c2
1 2
la

5.88

A d d i t i o n a l d i s c u s s i o n of NMR s p e c t r a l a s s i g n ments f o r g e n t a m i c i n i s g i v e n by Cooper e t a1.8,10,11

GENTAMICIN SULFATE

/i
I

"

'

"

'

'

"

.I

I " '

I "

" ' I

F i g u r e 3:

PMR Spectrum of G e n t a m i c i n S u l f a t e USP R e f e r e n c e S t a n d a r d .

30 1

302

BERNARD E. ROSENKRANTZ

el

al.

2.3.2

Carbon-13 Magnetic Resonance Spectrum

A carbon-13 NMR s p e c t r u m of a s o l u t i o n of Gentamicin S u l f a t e USP R e f e r e n c e S t a n d a r d (80 mg/0.50 m l i n D20) i s g i v e n i n F i g u r e 4. It was o b t a i n e d u s i n g a V a r i a n XL-100 s p e c t r o m e t e r a t ambient t e m p e r a t u r e and d i o x a n e as t h e i n t e r n a l r e f e r e n c e . The chemical s h i f t a s s i g n m e n t s g i v e n i n T a b l e 1 a r e i n ppm ( 6 ) w i t h r e f e r e n c e t o i n t e r n a l d i o x a n e t a k e n as 67.40 ppm down from e x t e r n a l t e t r a m e t h y l ~ i l a n e . ' ~
A d i s c u s s i o n of C-13 NMR s p e c t r a l d a t a of t h e g e n t a m i c i n C components C C 2 , and C1 i s g i v e n by la' Morton e t a1.I6

2.4

Mass Spectrum

The mass spectrum of g e n t a m i c i n f r e e b a s e , p r e p a r e d by n e u t r a l i z a t i o n of Gentamicin S u l f a t e USP R e f e r e n c e It w a s o b t a i n e d u s i n g S t a n d a r d i s g i v e n i n F i g u r e s 5 and 5a. a V a r i a n MAT CH-5 medium r e s o l u t i o n s i n g l e f o c u s i n g s p e c t r o 0 meter a t a probe t e m p e r a t u r e of 170 C and a s o u r c e t e m e r a t u r e of 250C. The m R s s a s s i g n m e n t s are g i v e n i n T a b l e 2. 11;

metry -et al.

~5,fegntamicinr fiist t gei vt e anl .by9 Cooper and P a 1

2.5.1

A d d i t i o n a l d i s c u s s i o n r e l a t i n g t o t h e mass s p e c t r o e t a1.8,11, D a n i e l s Thermal P r o p e r t i e s (TGA, DSC) Thermogravimetric A n a l y s i s (TGA)

2.5

A t h e r m o g r a v i m e t r i c a n a l y s i s c u r v e w a s obt a i n e d f o r Gentamicin S u l f a t e USP R e f e r e n c e S t a n d a r d ( s e e F i g u r e 6 ) u s i n g a DuPont Nodel 950 Thermogravimetric A n a l y z e r equipped w i t h a Model 900 Programmer-Recorder. The a n a l y s i s w a s performed a t a h e a t i n g rate of 10C/minute, u n d e r a n i t r o g e n atmosphere.

The t h e r m o g r a v i m e t r i c a n a l y s i s of t h e USP R e f e r e n c e S t a n d a r d i n d i c a t e s loss of a p p r o x i m a t e l y 12% water from ambient t o 125OC. Decomposition s t a r t s a t 22OoC and p r o c e e d s s t e p w i s e u n t i l 33OoC; above 330 a d d i t i o n a l dec o m p o s i t i o n o c c u r s , y i e l d i n g a f i n a l r e s i d u e of a b o u t 30% which is a t t r i b u t a b l e t o t h e s u l f a t e s a l t . 1 5

'80 30

3500

3000

2500

2000

CJ

1800

1600

1400

1200

lo00

800

!-

.-2

:0 625

Wavelength urn

Figure 4:

Carbon-13 NMR Spectrum of Gentamicin Sulfate USP Reference Standard.

304

BERNARD E. ROSENKRANTZ et al.

Carbon-13 chemical s h i f t assignments of Gentamicin S u l f a t e USP Reference Standard i n ppm (6) w i t h r e f e r e n c e t o i n t e r n a l dioxane t a k e n as 67.40 ppm down from e x t e r n a l t e t r a m e t h y l s i l a n e (see Figure 4). 1 Chemical S h i f t s Carbon 1
2

50.6 28.5 49.5 76.7 75.3

4
5

6 1'
2'

84.4
95.4, 49.5 21.4 24*0(C2,C1) , 26*3(CIa) 70.0, 43.5 50.4 (13.1) 58.3 (10.1) (32.0) c2
C1

95.3,

95.0*

3'

4'
5' 5'-CH2NH2 5'-CH ( CH3) NH2 5'-CH(CH3)NH(CH3) 1" 2"

69.6*

102.0
67.1 64.3 70.8 68.7 35.4 21.8

3"

4"
5"

3"-NHCH3 4"-CH3

'The o p e r a t i n g frequency of t h e s p e c t r o m e t e r was 25.2 MHz ( 1 3 C ) ; 8 K d a t a p o i n t s were a c q u i r e d w i t h a s p e c t r a l w i d t h of 5500 Hz a p u l s e w i d t h of 15.0 p s e c , y i e l d i n g a f l i p a n g l e b of 60 and a r e p e t i t i o n r a t e of 0.8 s e c . * M u l t i p l i c i t y i s due t o t h e m i x t u r e of C components.

100
90

> 80
W

5 60
2
40
J W

5 70 z

J 50
E

30

50

100
MASSICHARGE

Figure 5:

Mass Spectrum of Gentamicin Base (mass range 0 to 600).

300

350

400

450

500

550

MASS/CHARGE

F i g u r e 5a:

Mass Spectrum of Gentamicin Base (mass r a n g e 300 t o 5 5 0 ) -

CENTAMICIN SULFATE

307

TABLE 2

Mass Spectral Assignments of Gentamicin Base Masses (amu) Ions (M+l)+

(M-l7)+

' l a 450 (NH3)

0

c2 464 446 35 0 333 322 304 19 1

160

c1

47 8 46 0 35 0 347 322 30 4 191

160

432
0 '
= CHOH

@-

-@ -

350 319 322 30 4 191

160

@@-

0 0

- @ - 0+ = CHOH
-@- 0+H2
B]+

[@- 0 HO

@-

O+ = CHOH

@ OH]+

See the following page for the definition of A

+@

15 7 145

143
145

129 145

,B

and

C .

308

BERNARD E. ROSENKRANTZ ei al.

T a b l e 2 (Continued)

-51

1 I

I I

".
I

1 .

. . I , , . . I

I , . . , I .
I " "

I , . .
"

I , , , , 1 .
I "
" ' ' 7

'

'

' I , ,

+,, I

" I

'

h i

I '

'

" I

'

"

'

Figure 6:

Thermogravimetric Analysis (TGA) Curve of Gentamicin Sulfate USP Reference Standard.

310

BERNARD E. ROSENKRANTZ et al.

2.5.2

D i f f e r e n t i a l Scanning C o l o r i m e t r y (DSC)

A d i f f e r e n t i a l scanning calorimetry curve ( s e e F i g u r e 7) w a s o b t a i n e d f o r Gentamicin S u l f a t e USP R e f e r ence S t a n d a r d u s i n g a DuPont Model 990 Thermal A n a l y z e r equipped w i t h a Model 910 C e l l Base. The s c a n was performed 0 a t a t e m p e r a t u r e program r a t e of 10 C h i n u t e , u n d e r a n i t r o g e n atmosphere a g a i n s t an empty aluminum sample pan.

The d i f f e r e n t i a l s c a n n i n g c a l o r i m e t r y c u r v e of t h e USP R e f e r e n c e S t a n d a r d h a s a b r o a d e n d o t h e r m i c peak around 75OC due t o l o s s of water and a l a r g e endotherm a t 25OoC c o r r e s p o n d i n g t o m e l t i n g decomposition. ' 5 2.6 E l e c t r o m e t r i c T i t r a t i o n CurveIApparent pKa Value

Each of t h e t h r e e m a j o r g e n t a m i c i n C components cont a i n s 5 b a s i c amino groups. Because of t h e i r s i m i l a r b a s i c s t r e n g t h , t h e e l e c t r o m e t r i c t i t r a t i o n c u r v e P o ( F i g u r e 8) g i v e s one t i t r a t i o n "break" correspondilng t o f i v e e q u i v a l e n t s of n a c i d consumed. A a p p a r e n t pKa v a l u e ( h a l f n e u t r a l i z a t i o n ) of 7.9 i s d e r i v e d from F i g u r e 8. T h i s c u r v e w a s o b t a i n e d w i t h a Mettler a u t o m a t i c t i t r a t i o n s y s t e m ( c o n s i s t i n g of modules D V 1 1 , D K l O and DV103) and a Corning semimicro combination pH e l e c t r o d e . About 180 mg g e n t a m i c i n b a s e w a s d i s s o l v e d i n water and t i t r a t e d w i t h 0.5N h y d r o c h l o r i c a c i d . A pKa v a l u e of 8.2 f o r g e n t a m i c i n w a s r e p o r t e d by Done2' and a l s o by Newton and K l u z a . 2 2

2.7

Optical Rotation

Allowable l i m i t s f o r t h e s p e c i f i c r o t a t i o n of g e n t a m i c i n s u l f a t e are +107O t o +121 as g i v e n i n t h e Code of F e d e r a l R g u l a t i o n s (CFR)23 as w e l l as i n t h e B r i t i s h Pharmacopoeia.2' The CFR s t a t e s t h a t t h e measurement s h o u l d be performed on a 1%aqueous s o l u t i o n a t 25OC, w h i l e t h e B r i t i s h Pharmacopoeia s t a t e s t h a t a 10% aqueous s o l u t i o n s h o u l d b e measured a t 2OoC. The s p e c i f i c r o t a t i o n of t h e USP Gentamicin S u l f a t e R e f e r e n c e S t a n d a r d w a s found t o b e +115.9' when measured as a 0.3% aqueous s o l u t i o n i n a Bendix Series 1100 P o l a r i m e t e r a t 26OC. 25 2.8
X-Ray

Diffraction

X-ray powder d i f f r a c t i o n s t u d i e s 2 6 show t h a t g e n t a m i c i n s u l f a t e i s e s s e n t i a l l y a n amorphous s u b s t a n c e ; no s p e c t r a l bands were observed when t h e USP R e f e r e n c e S t a n d a r d w a s r u n on a P h i l i p s APD-3500 u t i l i z i n g Cu Ka r a d i a t i o n (1.54182).

Figure 7:

Differential Scanning Calorimetry (DSC) Curve of Gentamicin Sulfate USP Reference Standard.

312

BERNARD E. ROSENKRANTZ er (11.

9-

07PH 6-

54-

32-

F i g u r e 8:

E l e c t r o m e t r i c T i t r a t i o n Curve of Gentarnicin Base.

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2.9

Solubility Gentamicin s u l f a t e i s f r e e l y s o l u b l e i n w a t e r ,

0.1N h y d r o c h l o r i c a c i d , 0.1N sodium h y d r o x i d e (>1 g/ml i n

e a c h of t h e s e aqueous media). It is i n s o l u b l e i n a l c o h o l and most o t h e r o r g a n i c s o l v e n t s . A s p a r t of a comprehensive s t u d y of 5 1 a n t i b i o t i c compounds Marsh e t a1.W r e p o r t e d t h e s o l u b i l i t y of g e n t a m i c i n s u l f a t e i n 26 s o l v e n t s a t room temperature. Some of t h e s e d a t a are p r e s e n t e d below: S o l u b i l i t y a t 28+4'C Gentamicin S u l f a t e (mdml) >20 >20 6.332 0.678 0.200 0.072 0.045 0.042 0.028 0.028 0.025 0.0 0.0 0.0 0.0

Solvent Ethylene Glycol Formamide Propylene Glycol Chloroform Methanol Dimethyl S u l f o x i d e Is0p r opano 1 Acetone Carbon D i s u l f i d e Pyr i d i n e Ethyl Acetate Benzene Carbon T e t r a c h l o r i d e Is0 oct a n e Diethyl Ether

Gentamicin b a s e complex was found v e r y s o l u b l e i n water (>1 /ml) and i s more s o l u b l e t h a n t h e s u l f a t e s a l t i n a number 2 8 of o r g a n i c s o l v e n t s . Some of t h e s e d a t a are t a b u l a t e d below: S o l u b i l i t y a t 25+loC Gentamicin Base (mnlml) >25 >25 >25 >25 >25 2.6 2.4 2.1 0.2

Solvent Methanol n-But a n o l Ethanol Chloroform Acetone 2-Butanone Toluene Ethyl Acetate Cyc l o h exane

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2-10

Countercurrent Distribution

I n 1977, Byrne e t a1.28 r e p o r t e d on t h e s e p a r a t i o n of t h e g e n t a m i c i n C complex i n t o f i v e components by C r a i g d i s t r i b u t i o n . I n a d d i t i o n t o t h e t h r e e m a j o r components C1, C 2 , and C l a y t h e s e w o r k e r s s e p a r a t e d two a d d i t i o n a l comand CZb. Gentamicin CZa w a s i d e n t i f i e d as t h e ponents, C 2a 6'-C-epimer of g e n t a m i c i n C , w h i l e g e n t a m i c i n C2b w a s ident i f i e d a s 6'-N-methylgentam$cin C1 The s e p a r a t i o n s were c a r r i e d o u t i n a 1 0 2 0 - c e l l automat% C r a i g d i s t r i b u t i o n a p p a r a t u s of 10 m l f i x e d lower p h a s e volume, u s i n g a c h l o r o f o r m : methanol:17% ammonia ( 2 : l : l ) s o l v e n t system.

3.

Biosynthesis

The b i o s y n t h e s i s of a m i n o c y c l i t o l a n t i b i o t i c s , i n c l u d i n g g e n t a m i c i n , i s d i s c u s s e d i n a r e c e n t comprehensive review.29 Glucose h a s been shown t o p r o v i d e t h e s k e l e t o n s of a l l subu n i t s of t h e a n t i b i o t i c s s o f a r s t u d i e d ; however, d e t a i l s o f t h e s t e p s i n v o l v e d a r e s t i l l unknown i n a l m o s t a l l cases. Of t h e deoxystreptamine-containing a n t i b i o t i c s , t h e b u l k of t h e e f f o r t h a s b e e n d i r e c t e d toward t h e b i o s y n t h e s i s of neomycins. Gentamicins d i f f e r from t h e neomycins, kanamycins, and paromomycins i n t h a t t h e y c o n t a i n b o t h C-methyl and N-methyl s u b s t i t u e n t s ; most s t u d i e s on g e n t a m i c i n s have been aimed a t d e t e r m i n i n g t h e s o u r c e of t h e methyl g r o u p s . Studies carried o u t by Lee e t a1.30 i n d i c a t e a h i g h e f f i c i e n c y of L-methionine i n c o r p o r a t i o n i n t o gentami i n s . L a b e l l i n g experiments u s i n g 5 13C-methyl m e t h i o n i n e and H-methyl m e t h i o n i n e have shown t h a t a l l of t h e m e t h y l g r o u p s i n g e n t a m i c i n are d e r i v e d from m e t h i 0 n i n e . 3 ~ A d d i t i o n a l work by L e e e t a1.32 shows t h a t when 13 C-methyl-methionine w a s added a t t h e o n s e t of b i o s y n t h e s i s of t h e g e n t a m i c i n components, i n c o r p o r a t i o n of l a b e l i n t o t h e minor components preceded i n c o r p o r f k i o n i n t o t h e m a j o r compon e n t s . D e g r a d a t i o n o c c u r r e d when C-methyl g e n t a m i c i n major components w e r e added t o t h e g e n t a m i c i n - p r o d u c i n g c u l t u r e medium and shaken.

4.

I s o l a t i o n and P u r i f i c a t i o n P r o c e s s e s

I n 1963 R o s s e l e t 3 3 and co-workers f i r s t r e p o r t e d o n t h e i s o l a t i o n of t h e g e n t a m i c i n complex u s i n g ion-exchange chromat o g r a p h y . V a r i o u s ion-exchange p r o c e d u r e s c o n t i n u e t o be u s e d e x t e n s i v e l y f o r t h e s e p a r a t i o n and p u r i f i c a t i o n of g e n t a m i c i n on a p r e p a r a t i v e s c a l e . A commonly used p r o c e d u r e is t o adj u s t t h e whole b r o t h t o pH 2 w i t h s u l f u r i c a c i d , f o l l o w e d by

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filtration. After a d j u s t m e n t t o pH 7 , t h e n e u t r a l i z e d f i l t r a t e i s p a s s e d t h r o u g h a n IRC-50 r e s i n column i n t h e ammonium c y c l e , and t h e a n t i b i o t i c i s t h e n e l u t e d w i t h 2 N aqueous ammonia. The g e n t a m i c i n C complex may be i s o l a t e d from co-produced minor components u s i n g a Dowex 1x2 column (OH-f o rm) - 5

5.

Drug Metabolism and P h a r m a c o k i n e t i c s

Gentamicin s h a r e s w i t h many o t h e r a m i n o g l y c o s i d e a n t i b i o t i c s t h e i m p o r t a n t p r o p e r t y of b e i n g s t a b l e i n b i o l o g i c a l s y s t e m s . When a d m i n i s t e r e d t o man o r a n i m a l s , t h e m a j o r p o r t i o n i s e x c r e t e d i n t h e u r i n e by g l o m e r u l a r f i l t r a t i o n . 34. Gentamicin i s n o t absorbed i n a p p r e c i a b l e amounts from t h e i n t a c t g a s t r o i n t e s t i n a l t r a c t . A f t e r i n t r a m u s c u l a r a d m i n i s t r a t i o n , p e a k serum concent r a t i o n s u s u a l l y o c c u r between 30 and 60 m i n u t e s and serum levels a r e m e a s u r a b l e f o r s i x t o e i g h t hours. When g e n t a m i c i n i s a d m i n i s t e r e d by i n t r a v e n o u s i n f u s i o n o v e r a two-hour p e r i o d , t h e serum c o n c e n t r a t i o n s are s i m i l a r t o t h o s e o b t a i n e d by i n t r a m u s c u l a r a d m i n i s t r a t i o n . P r o t e i n b i n d i n g s t u d i e s have i n d i c a t e d t h a t t h e d e g r e e of g e n t a m i c i n b i n d i n g i s low, between 0 and 3O%.35

6.

Stability

Gentamicin s u l f a t e powder is v e r y s t a b l e when s t o r e d i n t i g h t l y c l o s e d c o n t a i n e r s a t room t e m p e r a t u r e . Gentamicin s u l f a t e is s t a b l e f o r a t least f i v e years with r e s p e c t t o p o t e n c y , s p e c i f i c r o t a t i o n and pH. Gentamicin w a s a l s o s t a b l e i n b o i l i n g aqueous b u f f e r s of p 2 t o 14.36 It is p a r t i c u l a r l y r e s i s t a n t t o a t t a c k by H a l k a l i , and h a s b e e n r e f l u x e d i n 2 N sodium h y d r o x i d e f o r 2 h o u r s w i t h no a p p a r e n t l o s s i n a c t i v i t y . 3 7 More r e c e n t s t u d i e s on g e n t a m i c i n c o n f i r m i t s e x c e l l e n t s t a b i l i t y i n Under h ghm o d e r a t e l y a c i d t o s t r o n g l y b a s i c aqueous media. l y s t r e s s e d c o n d i t i o n s ( h e a t i n g i n 1N s u l f u r i c a c i d f o r 5 0 d a y s a t 60 C ) , a p p r o x i m a t e l y a 30% l o s s i n p o t e n c y w a s f 0 u n d . 3 ~ Gentamicin s u l f a t e w a s a l s o shown t o be s t a b l e n i n f u s i o n s o l u t i o n s 3 9 and i n a r t i f i c i a l t e a r s o l u t i o n s . 4 0 Gentamicin s u l f a t e e x h i b i t s e x c e l l e n t s t a b i l i t y i n v a r i ous p h a r m a c e u t i c a l dosage forms. I n p a r e n t e r a l s o l u t i o n s and t o p i c a l o i n t m e n t s i t h a s b e e n shown t o be s t a b l e f o r a t l e a s t 0 f i v e y e a r s u n d e r normal s t o r a g e c o n d i t i o n s ( 2 t o 30C).

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7.

Methods of A n a l y s i s

7.1

Identification

Gentamicin is c o n v e n i e n t l y i d e n t i f i e d by t h i n - l a y e r chromatography (TLC). Gentamicin is r e s o l v e d i n t o i t s 3 components and a l s o can b e s e p a r a t e d from most o t h e r r e l a t e d a n t i b i o t i c s u s i n g TLC. R e f e r t o t h e d i s c u s s i o n i n s e c t i o n 8.2 and e s p e c i a l l y Wilson e t a l . 4 l a n d Pauncz42 f o r r e l a t e d discussion. I n a t y p i c a l TLC method about 50 t o 100 ug of gentamicin s u l f a t e is a p p l i e d t o a s i l i c a g e l TLC p l a t e and developed u s i n g t h e lower phase of a m i x t u r e of e q u a l volumes of chloroform, methanol and c o n c e n t r a t e d aqueous ammonia. 41 The s p o t s a r e t y p i c a l l y v i s u a l i z e d w i t h n i n h y d r i n r e a g e n t o r w i t h i o d i n e vapors. R e s u l t s a r e compared w i t h t h o s e o b t a i n e d from a s i m i l a r l y chromatographed r e f e r e n c e s o l u t i o n . Paper chromatography i s a l s o u s e f u l f o r i d e n t i f i c a t i o n (see s e c t i o n 8.1). The B r i t i s h Pharmacopoeia 1973, p. 216 d e s c r i b e s a method where t h e s o l v e n t system chloroform: methano1:concentrated aqueous ammonia:water (10:5:3:2) i s used a l o n g w i t h n i n h y d r i n s p r a y d e t e c t i o n . The BP a l s o d e s c r i b e s a method where a UV s p e c t r u m is o b t a i n e d a f t e r t r e a t m e n t w i t h s u l f u r i c a c i d . No maximum is o b t a i n e d f o r g e n t a m i c i n , which d i s t i n g u i s h e s i t from kanamycin, neomycin and paromomycin. The Code of F e d e r a l R e g u l a t i o n s (444.20) d e s c r i b e s an i n f r a r e d spectrophotometric technique using a K r disc. B The i n f r a r e d spectrum of g e n t a m i c i n s u l f a t e , however, is v e r y similar t o t h a t of o t h e r aminoglycoside a n t i b i o t i c s and i s t h e r e f o r e of l i m i t e d v a l u e as a n i d e n t i f i c a t i o n test.

7.2

Determination of S u l f a t e Content

A s d e s c r i b e d i n s e c t i o n 1 . 2 , g e n t a m i c i n s u l f a t e is composed of t h r e e major components- S i n c e each component h a s 5 b a s i c n i t r o g e n s , 5 e q u i v a l e n t s of s u l f u r i c a c i d are req u i r e d p e r mole of g e n t a m i c i n base. The l i m i t s f o r s u l f a t e c o n t e n t g i v e n i n t h e B r i t i s h Pharmacopoeia 1973 are 31.0 t o 34.0% (anhydrous b a s i s ) .

The g r a v i m e t r i c p r o c e d u r e d e s c r i b e d i n t h e B&3 i n v o l v e s p r e c i p i t a t i o n of barium s u l f a t e by t h e 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 and barium c h l o r i d e t o a n aqueous s o l u t i o n

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of t h e a n t i b i o t i c , f o l l o w e d by washing, i g n i t i n g and weighing the residue. Each gram of r e s i d u e is e q u i v a l e n t t o 0.4116 gram of s u l f a t e . 7.3 Loss on Drying and M o i s t u r e Content

Gentamicin s u l f a t e is an amorphous, h y g r o s c o p i c powder which t y p i c a l l y c o n t a i n s 10 t o 15% water. The U . S . Government Code of F e d e r a l R e g u l a t i o n s (CFR) a l l o w s a maximum of 18% l o s s on drying.44 I n t h e CFR method45 t h e g e n t a m i c i n s u l f a t e sample i s h e a t e d a t a t e m p e r a t u r e of 110 C f o r 3 h r i n a vacuum (5 5mm mercury). The B r i t i s h Pharmacopoeia s p e c i f i c a t i o n f o r water c o n t e n t is 15%*4 F i s c h e r t i t r a t i o n and e l e c t r o n i c e n d p o i n t d e t e c t i o n .
It has b e e n shown t h a t l o s s on d r y i n g r e s u l t s and Karl F i s c h e r t i t r a t i o n r e s u l t s are i n good agreement.47

7.4

D e t e r m i n a t i o n of Component R a t i o s

A number of methods have been used f o r t h e determiBrief d e s c r i p t i o n s n a t i o n of g e n t a m i c i n C component r a t i o s . of n i n e of t h e s e methods are p r e s e n t e d i n t h i s s e c t i o n .

7.4.1 For U.S. c e r t i f i c a t i o n , a l l b a t c h e s of gentam i c i n s u l f a t e must conform t o t h e f o l l o w i n g r e q u i r e m e n t s f o r component r a t i o s d e s c r i b e d i n t h e Code of F e d e r a l Regulat ions :
C1: C : C ? :

Not l e s s t h a n 25% n o r more t h a n 50% Not l e s s t h a n 15% n o r more t h a n 40% Not l e s s t h a n 20% n o r more t h a n 50%

The o f f i c i a l method g i v e n i n 2 1 CFR 444.20 a ( b ) ( 8 ) i s based on a p a p e r chromatographic p r o c e d u r e r e p o r t e d by Kantor and S e l z e r . 4 8 I n t h i s method, two i d e n t i c a l sample chromatograms are developed i n t h e lower p h a s e of chloroformmethanol-17% ammonium hydroxide ( 2 : l : l ) . One chromatogram i s sprayed w i t h n i n h y d r i n r e a g e n t t o l o c a t e t h e p o s i t i o n s of t h e C components Cl , C 2 and C , which have approximate The s p o t s i n R v a l u e s of 0.35, 8.50 and 0.$5 r e s p e c t i v e l y . t h s s t r i p are used t o l o c a t e t h e c o r r e s p o n d i n g zones i n t h e second s t r i p . The zones are c u t o u t , e l u t e d w i t h pH 8.0 0.1M phosphate b u f f e r , and a s s a y e d u s i n g t h e CFR microbiologi c a l a g a r d i f f u s i o n assay. Wagman e t a1.49 r e p o r t e d a d i f f e r e n t i a l chro7.4.2 matographic b i o a s s a y f o r t h e g e n t a m i c i n complex. The t h r e e gentamicins are s e p a r a t e d u s i n g t h e same p a p e r chromatography

318

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system d e s c r i b e d i n s e c t i o n 7.4.1. A f t e r chromatographic development, t h e p a p e r s t r i p s are d r i e d and p l a t e d a g a i n s t Staphylococcus a u r e u s ATCC 6538P and t h e zones of i n h i b i t i o n are q u a n t i t a t e d u s i n g s t a n d a r d zone r e s p o n s e c u r v e s .
I n a n o t h e r m o d i f i c a t i o n u s i n g t h e same p a p e r 7.4.3 chromatography s y s t e m d e s c r i b e d i n S e c t i o n 7 . 4 . 1 , t h e d e v e l oped p a p e r s are s p r a y e d l i g h t l y w i t h d i l u t e 2 , 4 , 6 - t r i n i t r o b e n z e n e s u l f o n i c a c i d (TNBSA) t o d e t e c t t h e g e n t a m i c i n C components, which a p p e a r as y e l l o w zones. The zones a r e c u t o u t , a d d i t i o n a l TNBSA i n a pH 9.4 b u f f e g is added and t h e chromophore i s allowed t o develop a t 30 C f o r one h o u r . The amount of each g e n t a m i c i n C component is q u a n t i t a t e d by m comparison of t h e a b s o r b a n c e s o b t a i n e d a t 420 n w i t h t h o s e o b t a i n e d from a s i m i l a r l y t r e a t e d chromatogram of t h e r e f e r e n c e s t a n d a r d . 25

Wagman e t a1.50 r e p o r t e d a d i f f e r e n t i a l 7.4.4 n i n h y d r i n p a p e r chromatographic a s s a y f o r t h e g e n t a m i c i n complex. After development, t h e p a p e r s t r i p s a r e d r i e d and sprayed w i t h ninhydrin reagent. The c o l o r i s developed w i t h h e a t and t h e c o l o r i n t e n s i t i e s are r e a d on a n i n t e g r a t i n g s c a n n e r . The p r o p o r t i o n s of t h e t h r e e g e n t a m i c i n C compon e n t s i n t h e s a m p l e are c a l c u l a t e d by comparison t o s t a n d a r d s of t h e t h r e e i n d i v i d u a l C components s i m i l a r l y t r e a t e d . Anhalt e t a1.51 r e p o r t e d a h i g h p r e s s u r e 7.4.5 l i q u i d chromatographic method f o r g e n t a m i c i n C component determination. I n t h i s method a r e v e r s e d p h a s e LC column s e p a r a t e s t h e t h r e e g e n t a m i c i n C components by p a i r e d - i o n chromatography. The s e p a r a t e d components are d e r i v a t i z e d w i t h o-phthalaldehyde t o g i v e f l u o r e s c e n t p r o d u c t s . Results from t h i s p r o c e d u r e compare f a v o r a b l y w i t h a s s a y r e s u l t s by t h e CFR m i c r o b i o l o g i c a l a g a r d i f f u s i o n method. S e e S e c t i o n 8.5 f o r a n expanded d i s c u s s i o n of t h i s HPLC t e c h n i q u e . 7.4.6 K a b a s a k a l i a n e t a1.52 r e p o r t e d a method f o r t h e d e t e r m i n a t i o n of t h e g e n t a m i c i n components i n f ermentat i o n b r o t h by i n - s i t u f l u o r i m e t r i c measurements of 4-chloro7-nitrobenzo-2-oxa-l,3-diazole (NBD) d e r i v a t i v e s . I n t h i s method, f e r m e n t a t i o n b r o t h samples are a c i d i f i e d , c e n t r i f u g e d , a d j u s t e d t o pH 12, s p o t t e d on TLC p l a t e s and developed. The d r i e d p l a t e s are dipped i n m e t h a n o l i c NBD c h l o r i d e , h e a t e d , c o o l e d and rechromatographed i n methanol. The f l u o r e s c e n t s p o t s are scanned and i n t e g r a t e d u s i n g a d e n s i t o m e t e r . This method p r o v i d e s a r a p i d means of f o l l o w i n g changes i n component r a t i o s d u r i n g t h e c o u r s e of t h e f e r m e n t a t i o n .

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7 . 4 . 7 Calam e t a1.53 r e p o r t e d a method f o r c o n t r o l and m o n i t o r i n g t h e p r o p e r t i e s of t h e t h r e e g e n t a m i c i n C components by lH n u c l e a r m a g n e t i c r e s o n a n c e s p e c t r o s c o p y . T h i s method i n v o l v e s measurement of t h e peak h e i g h t s of s i g n a l s f o r N-methyl and C-methyl g r o u p s p r e s e n t i n a l l t h r e e components and of t h o s e p r e s e n t i n C1 and C2 o n l y . The peak h e i g h t r a t i o s a r e c a l c u l a t e d . The r e s u l t s a r e used t o c o n t r o l and m o n i t o r c o m p o s i t i o n w i t h i n c e r t a i n l i m i t s and n o t t o d e t e r m i n e t h e a c t u a l % c o m p o s i t i o n of each component. The l i m i t s and t h e method a p p e a r i n t h e g e n t a m i c i n s u l p h a t e monograph of t h e B r i t i s h Pharmacopoeia 1973, Addendum 1975.
using

. The l 3 C NMR

g e n t a m i c i n C components have been m i t o r e d o by 2 4 hour a c c u m u l a t i o n of ~ p e c t r a . 5 ~

7 . 4 . 8 Thomas and Tappin55 r e p o r t e d a n ion-exchange method w i t h d i r e c t o p t i c a l r o t a t i o n measurement t h a t i s u s e f u l f o r examining C component d i s t r i b u t i o n i n g e n t a m i c i n sulfate. I n t h i s method, 80 m s a m p l e s of g e n t a m i c i n s u l f a t e g are d i s s o l v e d i n 0 . 5 m l 2 M sodium c h l o r i d e and added t o t h e t o p of a column (0.9 x 15 cm) f i l l e d w i t h c e l l u l o s e p h o s p h a t e P-11 ion-exchange material. A g r a d i e n t mixer d e l i v e r s sodium c h l o r i d e s o l u t i o n i n i n c r e a s i n g m o l a r i t y t o t h e column. The column e l u a t e i s monitored by a p o l a r i m e t e r w i t h a flowthrough microc e l l . The o u t p u t i s p l o t t e d on a p o t e n t i o m e t e r r e c o r d e r . The peak areas are d e t e r m i n e d w i t h a p l a n i m e t e r and e x p r e s s e d as p e r c e n t of t h e t o t a l area.
See Thomas56 f o r a comparison of r e s u l t s obt a i n e d by t h e CFR m i c r o b i o l o g i c a l method, t h e lH NMR method, and t h e above ion-exchange method.

7 . 4 . 9 Wilson e t a1.57 r e p o r t e d a g e n t a m i c i n C component a s s a y method u s i n g t h i n - l a y e r chromatography f o l l o w e d by d i r e c t d e n s i t o m e t r y . Gentamicin s u l f a t e is s p o t t e d on s i l i c a g e l TLC p l a t e s f o l l o w e d by development i n t h e lower p h a s e of chloroform-methanol-concentrated ammonium h y d r o x i d e (1:l:l). A f t e r d r y i n g , t h e p l a t e s are s p r a y e d w i t h n i n h y d r i n r e a g e n t y i e l d i n g magenta s p o t s o n a w h i t e background. The s p o t s a r e examined by d i r e c t d e n s i t o metry and q u a n t i t a t e d w i t h a d i g i t a l i n t e g r a t o r . The a u t h o r s c l a i m t h a t t h i s method i s f a s t e r and o f f e r s t h e s a m e p r e c i s i o n as m i c r o b i o l o g i c a l methods.

320

BERNARD E. ROSENKRANTZ er al.

7.5

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

d i s c - p l a t e a g a r d i f f u s i o n assay using Staphylococcus aureus ATCC 6538P as t h e t e s t o r g a n i s m f o r t h e a n a l y s i s of gentamic i n r a w materials. In t h i s p a p e r a s t a n d a r d c u r v e c y l i n d e r p l a t e a s s a y u t i l i z i n g B a c i l l u s s u b t i l i s ATCC 6633 w a s a l s o r e p o r t e d f o r t h e d e t e r m i n a t i o n of g e n t a m i c i n i n s e r u m samples. ( r e f e r t o s e c t i o n 10.1) F a c t o r s a f f e c t i n g t h e a s s a y r e s u l t s u s i n g t h e s e a s s a y p r o c e d u r e s , s u c h as t h e e f f e c t of s a l t s in t h e a g a r media, a r e d e s c r i b e d . The c u r r e n t o f f i c i a l m i c r o b i o l o g i c a l a s s a y proced u r e d e s c r i b e d i n t h e U.S. Code of F e d e r a l R e g u l a t i o n s (CFR)59 f o r t h e s u b s t a n c e and dosage f o r m s i s a c y l i n d e r p l a t e a s s a y u s i n g S t a p h y l o c o c c u s e p i d e r m i d i s ATCC 2228 as t h e t e s t organism. The B r i t i s h Pharmacopoeia u t i l i z e s 6 0 a c y l i n d e r p l a t e a s s a y and B a c i l l u s p u m i l u s NCTC 8241 as t h e t e s t organism. D e t a i l e d p r o c e d u r e s f o r c a r r y i n g o u t t h e a s s a y s are g i v e n i n t h e compendia. The minimum p o t e n c y r e q u i r e d by b o t h t h e CFR and BP f o r a c c e p t a n c e of b u l k commercial g e n t a m i c i n s u l f a t e i s 590 mcg p e r mg o n t h e d r i e d (anhydrous) b a s i s .

In 1963 Oden e t al.5 8 d e s c r i b e d a s t a n d a r d c u r v e

8.

Chromatographic Analysis 8.1

P a p e r Chromatography

Gentamicin c a n be s e p a r a t e d i n t o i t s t h r e e components (Cla,C2,C1) by d e s c e n d i n g p a p e r chromatography u s i n g t h e s o l v e n t s y s t e m c h l o oform:methanol:17% aqueous ammonia (2: 1: 1, lower p h a s e ) T h i s is a m o d i f i c a t i o n of t h e s y s t e m g i v e n by Ikekawa e t a1.61 The a p p r o x i m a t e R v a l u e s r e p o r t e d f o r t h e t h r e e g e n t a m i c i n C components62fare : Component

.'

Rf
0.21
0.40

c2

0.67

GENTAMICIN SULFATE

321

Other paper chromatography systems have been r e p o r t e d b u t p r o v i d e l i t t l e o r no s e p a r a t i o n of t h e t h r e e gentamicin c components.62-65 Gentami i n can be d e t e c t e d by s p r a y i n g w i t h n i n h y d r i n reagent' (0.25% n i n h y d r i n i n p y r i d i n e - a c e t o n e 1 : l ) followed by h e a t i n g a t 105O f o r s e v e r a l minutes. The s p o t s produced a r e purple-blue i n c o l o r a g a i n s t a w h i t e background. Ninhydrin r e a g e n t p r e p a r e d by d i s s o l v i n g 1 gram of n i n h y d r i n and 0.1 gram of cadmium a c e t a t e i n a s o l u t i o n of 3 m l water, 1.5 m l g l a c i a l a c e t i c a c i d and 100 m l of g-propanol has a l s o been used.48,66 A bioautography method may a l s o be used where t h e paper s t r i p is p l a c e d on a g a r seeded w i t h Staphylococcus a u r e u s ATCC 6538P. The Rf v a l u e s of t h e r e s u l t i n g zones of i n h i b i t i o n a r e t h e same a s t h e s p o t s produced with n i n h y d r i n d e t e c t i o n . Table 3 i s a summary of paper chromatography systems f o r gentamicin and c o n t a i n s r e f e r e n c e s t o q u a l i t a t i v e and q u a n t i t a t i v e methods. A expanded d i s c u s s i o n of q u a n t i n t a t i v e methods I s g i v e n i n S e c t i o n 7.4.

8.2

Thin-Layer Chromatography

Thin-layer chromatography i s an e f f e c t i v e means of i d e n t i f y i n g and s e p a r a t i n g t h e components of t h e g e n t a m i c i n complex. Table 5 g i v e s a l i s t of TLC systems t h a t have been used f o r gentamicin. R e v i e w s concerning TLC of g e n t a m i c i n and r e l a t e d a n t i b i o t i c s are availabIe.62,65,67,70
TLC i s v e r y u s e f u l f o r s e p a r a t i n g gentamicin from o t h e r r e l a t e d aminoglycoside a n t i b i o t i c s . I t o e t a l e 6 8 l i s t s Rf v a l u e s f o r gentamicin and 14 o t h e r b a s i c water s o l u b l e a n t i b i o t i c s u s i n g s o l v e n t system C i n Table 5. Pauncz42 s e p a r a t e d gentamicin from s e v e r a l o t h e r deoxys t r e p t a m i n e c o n t a i n i n g a n t i b i o t i c s and t h e i r decomposition p r o d u c t s u s i n g s o l v e n t system E. This system d i d n o t , howe v e r , s e p a r a t e g e n t a m i c i n i n t o i t s t h r e e components.

Kabasakalian e t al.52 r e p o r t e d a q u a n t i t a t i v e TLC method f o r t h e g e n t a m i c i n complex u s i n g f l u o r i m e t r i c d e t e c tion. See S e c t i o n 7.4 f o r an expanded d i s c u s s i o n of t h i s method

Table 3 Paper Chromatography Systems or Gentamicin

Method Type Qualitative Qualitative Qualitative

N W N

Paper Whatman No. 1 Whatman No. 1 Whatman No. 1 Whatman No. 1 Whatman No. 1 Whatman No. 1 Whatman No. 4 S & S NO. 5 8 9

Solvent See Table 4

A

Detection See Table 4

1
0.59*

Reference
1

B
C
D
E

1 1 1 12

0.26*
0.10*

1
1

Qualitative Qualitative Quantitative Quantitative Quantitative

0.30*

1
4 49 48 50

E E E

3
2

*Gentamicins C

la

C2 and C 1 are not separated.

Table 4 Paper Chromatography Solvent Systems for Gentamicin A . B. C.

D.
W W w

Methano1:water (4:l) + 3% sodium chloride vs. paper buffered with 0.95 sodium sulfate + 0.05 M sodium bisulfate.
Propano1:pyridine:acetic

Iy

acid:water (15:10:3:12).

Propano1:water:acetic acid (50:40:5). Aqueous phenol, 80%. Lower phase of chloroform:methanol:l7% ammonium hydroxide ( 2 : l : l ) Paper Chromatography Detection Methods for Gentamicin

E.

1.
2 .
3.

Bioautography vs. Staphylococcus aureus ATCC 6538P. Spray with 0.25% ninhydrin in pyridine:acetone (1:l). several minutes giving purple to blue spots. Heat at 105OC for

Spray with Modified Barrollier reagent. Add 3 ml water and 1 5 ml glacial . acetic acid to 1 g ninhydrin and 0 1 g cadmium acetate and shake. Add to . 100 ml n-propanol and shake until solution is complete.

Table 5 Thin-Layer Chromatography Systems for Gentamicin Plate Medium (see below) Solvent (see below)
A

Detection (see below) 1 1,2,3 1 132

1

Bf Values

Cla, C2, C1

Reference 41
4

C1a<C2<C 1 1 a' 2 1 ' < " 0.20, 0.28, 0.35

B
C
W
h) P

68 69
42

b
d

D E

0.69, 0.76, 0.71

0.05*

*Gentamicins Cla, C2, and C1 are not separated. Plate Medium

a . b.
c.

d .

Silica Gel 60, 0.25 mm thickness, E M Laboratories .. Silica Gel G MN cellulose powder 300, 250 micron thickness Dowex 50 x 8 ion-exchange resin-coated plate

4 4

4
$4 (d

.. ..
d

E!
0
$4

u a

a
*rl 0

a,

t?

h c
(d

a
a, M

$4

u e
L l
a,

$
w
0

0
d
.rl
\D

P
m
(d

a
u
(d $4

a,

(d

a
V
V
W

a,

co m m

PI

u
0
0
03

&

a, U
0

U I

5
$4

z
w a,
$4 P) (d

m aJ U G

2
aJ
$4

1
h
(d

a,

1
&

(d

m
L) L)

b
v

$4
rc(

a, a,

0 d

L)

a, M
(d $4

a,

&
4

2 a d $4 (d u a cn a 0
m
U
P)

a,

5
w
0

a,

c a
$4

$4

G 5 d m Y ch 3 a u
a,
*i t

(d $4

$4

00

a,

8
.rl

a
1

a
0

d F4

% &
m

(d

325

326

BERNARD E. ROSENKRANTZ et al.

8.3

Ion-Exchange Chromatography

Ion-exchange chromatography h a s b e e n u s e d e x t e n s i v e l y f o r t h e p r e p a r a t i v e s e p a r a t i o n of g e n t a m i c i n components. Gentamicins may be i s o l a t e d from f e r m e n t a t i o n media by p a s s a g e o v e r ion-exchange r e s i n s ; 20-50 mesh A m b e r l i t e IRC-50, sodium form33, A m b e r l i t e IRC-50, ammonium form69, and m - 2 - 7 ~ ~ ~ r e s i n s have been used. The g e n t a m i c i n C components may be s e p a r a t e d from t h e minor A, B y and X components on a s t r o n g l y b a s i c r e s i n , Dowex 1x2 w i t h d e i o n i z e d water as e l ~ e n t ~ 9 , 7 ~ . B a s i c i o n exchange r e s i n s such as A m b e r l i t e IRA-400 (OH-) a r e used t o c o n v e r t g e n t a m i c i n s u l f a t e s t o t h e c o r r e s p o n d i n g f r e e b a s e s .33 An ion-exchange s e p a r a t i o n h a s b e e n s u g g e s t e d as a q u a n t i t a t i v e measure of C component r a t i o s and c o n t e n t i n g e n t a m i c i n samples.56 See S e c t i o n 7.4 f o r a d d i t i o n a l d i s c u s s i o n of t h i s paper. 8.4 Gas Chromatography

S i n c e g e n t a m i c i n h a s r e l a t i v e l y low v o l a t i l i t y , a l l g a s chromatographic a n a l y s e s i n v o l v e d e r i v a t i z a t i o n . Cunningham and Matsen73 h y d r o l y z e d serum g e n t a m i c i n w i t h 6 HCL and a n a l y z e d t h e r e s u l t i n g 2-deoxystreptamine as i t s t r i f l u o r o a c e t y l d e r i v a t i v e on a 3% OV-101 column a t 150C w i t h f l a m e i o n i z a t i o n d e t e c t i o n . Mayhew and G ~ r b a c h75 ~ , ~ determined serum g e n t a m i c i n by a two s t e p d e r i v a t i z a t i o n w i t h N-trimethylsilylimidazole and N-heptafluorobutyrylimidazole; q u a n t i t a t i v e measurements w e r e made by means of a n e l e c t r o n c a p t u r e d e t e c t o r f o l l o w i n g chromatography on 3% OV-101 supp o r t e d on Chromosorb W AW DMCS. 8.5 High P r e s s u r e L i q u i d Chromatography

High p r e s s u r e l i q u i d chromatography (HPLC) h a s been used a s a n a s s a y method b o t h f o r t o t a l g e n t a m i c i n s and f o r i n d i v i d u a l components. Samples which have been a n a l y z e d inc l u d e serum and u r i n e specimens a s w e l l as t h e b u l k d r u g material.
A major o b s t a c l e t o g e n t a m i c i n HPLC a s s a y s h a s b e e n t h e problem of d e t e c t i o n . Gentarnicin e x h i b i t s no s i g n i f i c a n t UV bands above 190 nm and h a s no n a t i v e f l u o r e s c e n c e . In add i t i o n , a s s a y l e v e l s a r e g e n e r a l l y t o o low f o r t h e u s e of t h e r e f r a c t i v e i n d e x d e t e c t o r . Hence, t h e several methods which have been developed i n v o l v e d e r i v a t i z a t i o n of t h e drug.

GENTAMICIN SULFATE

321

Peng, e t a d 6 d e t e c t e d g e n t a m i c i n as i t s d a n s y l d e r i v a t i v e . Following d e p r o t e i n i z a t i o n of serum, g e n t a m i c i n w a s d e r i v a t i z e d w i t h d a n s y l c h l o r i d e and e x t r a c t e d i n t o e t h y l a c e t a t e . The sample w a s t h e n chromatographed on a m i c r o p a r t i c u l a t e r e v e r s e d phase column by u s i n g a n aqueous a c e t o n i t r i l e e l u e n t and f l u o r e s c e n c e d e t e c t i o n . Maitra e t a1.77,78 s e p a r a t e d g e n t a m i c i n from serum by means of a s i l i c i c a c i d column. The d r u g w a s t h e n r e a c t e d w i t h o - p h t h a l a l d e h y d e , chromatographed on a WBondapak C18 column w i t h a methano1:water (79:21) m o b i l e p h a s e cont a i n i n g t r i p o t a s s i u m EDTA and d e t e c t e d by f l u o r e s c e n c e r e a d o u t . Continuous-flow post-column d e r i v a t i z a t i o n of e n t a m i c i n w i t h o - p h t h a l a l d e h y d e w a s performed by Anhalt. 79,88 The d r u g w a s d e t e r m i n e d by f l u o r e s c e n c e r e a d o u t a f t e r s e p a r a t i o n column w i t h a m o b i l e p h a s e of methanol: on a pBondapak C water (3:97) ~ i t k ~ 0 g2 N a SO . 0.02 g sodium p e n t a n e s u l f o 2 4 n a t e and 0.1% ( v / v ) a c e t i c a c i d . A n h a l t , e t al.51 compared t h i s method t o a normal p h a s e s e p a r a t i o n on a P a r t i s i l (30 cm x 3.9 mm i . d . ) column w i t h a diethy1amine:methanol:water ( 0 . 5 : 4 0 : 6 0 ) m o b i l e p h a s e and r e f r a c t i v e i n d e x d e t e c t i o n . An e l a b o r a t i o n on t h e method of Anhalt i n c l u d e s t h e u s e of n e t i l m i c i n as i n t e r n a l s t a n d a r d and r e s o l v e s minor S e p a r a t i o n s are obi m p u r i t i e s i n t h e bulk drug substance.81 t a i n e d on a n E.M. Merck RP-8 column, mobile p h a s e of 0.2 g sodium s u l f a t e , 0.02 sodium p e n t a n e s u l f o n a t e , 0.1% ( v / v ) a c e t i c a c i d , and p o s t column d e r i v a t i z a t i o n w i t h o - p h t h a l a l d e hyde. T h i s method g i v e s a l i n e a r r e s p o n s e t o g e n t a m i c i n o v e r a wide c o n c e n t r a t i o n r a n g e , up t o 0.15 mg/ml f o r e a c h component. I t is s p e c i f i c , a c c u r a t e and p r e c i s e , and h a s b e e n u s e d e f f e c t i v e l y t o d e t e r m i n e t h e g e n t a m i c i n c o n t e n t of b u l k s a m p l e s as w e l l as f i n i s h e d p r o d u c t s . 9. Electrophoresis

E l e c t r o p h o r e s i s h a s been u s e d t o s e p a r a t e g e n t a m i c i n from o t h e r a n t i b i o t i c d r u g s i n serum and i n u r i n e . V a r i o u s s y s t e m s which have b e e n u s e d f o r t h e s e s e p a r a t i o n s are g i v e n i n W h i t e l e y and co-workersg2 have u s e d a n electroT a b l e 6. p h o r e t i c s e p a r a t i o n t o demonstrate an in-vitro i n t e r a c t i o n between g e n t a m i c i n and c e p h a l e x i n .

Table 6 Conditions for Electrophoresis of Gentamicin Detection Method Gentamicin Mobility Relative to Neomycin Reference

Medium Filter Paper (Whatman No. 4 ) Filter Paper (Whatman No. 4 )

W W N

Buffer
1
0.005

in n-PrOH:H20 (5:95) pH 1.8

g formic acid g p-toluenesulfonic acid

Microbiological

0.95

83

0.8 g formic acid 0.005 g p-toluenesulfonic acid in n-PrOH:H20 (5:95) pH 1.9 0.8 0.005

Microbiologica1

83

Filter Paper (Whatman No. 4 ) Filter Paper (Whatman No. 4 )

in H20 pH 1.9 0.2 NH OH

g p-toluenesulfonic acid

formic acid

Microbiological

83

0.005 3 AaOH 0.01 g p-toluenesulfonic acid in n-PrOH:H20 (1:9) pH 10.8

Microbiological Microbiological Microbiological

1.74 1.43

83 83 83

Filter Paper (Whatman No. 4 ) Filter Paper (Whatman No. 4 )

0.2 NH OH, 0.0025 in H20 pA 11.5

0.05

g NaOH,

pH 12.2

g NaOH in H20

1.02

Table 6

(Continued)

C o n d i t i o n s f o r E l e c t r o p h o r e s i s of Gentamicin Detection Method Gentamicin Mobility Relative t o Neomycin

Medium F i l t e r Paper (Whatman 3 b@f)

Buffer formic a c i d : a c e t i c a c i d :

Reference

water ( 6 : 2 4 : 170)

0.25% n i n h y d r i n , 0.01% i s a t i n , 1%l u t i d i n e i n

acetone

1
0.85

84 85

0.9% a g a r o s e i n pH 5.6 buffer F i l t e r Paper (Whatman No. 1)

T r i s - (hydr oxyme t h y 1) aminomethane, m a l e i c a c i d i n H20 pH 5.6

Bio-autographical

0.05 NH40Ac, 0.05 NH40H i n H20 p 9.4 H

Ninhy d r in

n.a.

82

330

BERNARD E. ROSENKRANTZ et al.

10.

D e t e r m i n a t i o n i n Body F l u i d

Assays f o r g e n t a m i c i n i n body f l u i d s have been performed u s i n g a number of methods; t h e s e i n c l u d e m i c r o b i o l o g i c a l a s s a y s , immunoassays, radioenzyme a s s a y s and h i g h p r e s s u r e l i q u i d chromatographic a s s a y s .

10.1

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

The m i c r o b i o l o g i c a l a s s a y of g e n t a m i c i n is an a g a r d i f f u s i o n a s s a y b a s e d upon a comparison between t h e growth i n h i b i t i o n zones produced by t h e t e s t sample and t h o s e produced by g e n t a m i c i n s t a n d a r d of known p o t e n c i e s d i l u t e d i n a p p r o p r i a t e body f l u i d . Oden e t al.58 d e s c r i b e d a s t a n d a r d c u r v e c y l i n d e r - p l a t e a s s a y u t i l i z i n g B a c i l l u s s u b t i l i s ATCC 6633 as t h e t e s t o r anism w i t h a s e n s i t i v i t y of 0.05 mcg/ml. A l c i d and Seligmangg r e p o r t e d t h e u s e of a m u l t i p l e a n t i b i o t i c r e s i s t a n t s t r a i n of S t a p h y l o c o c c u s e p i d e r m i d i s ATCC 27626 as t h e t e s t organism. Gentamicin c o n c e n t r a t i o n s are e s t i m a t e d u s i n g t h i s t e s t organism i n t h e p r e s e n c e of o t h e r a n t i b i o t i c s w i t h o u t t h e u s e of enzymes, r a d i o a c t i v e material, o r e l a b o r a t e equipment and t e c h n i q u e s . 10.2 Fluoroimmunoassay

A f l u o r o m e t r i c irnmunoassay f o r g e n t a m i c i n i n serum w a s developed by Watson and co-workers87, who h a v e p r e p a r e d a f l u o r e s c e i n i s o t h i o c y a n a t e d e r i v a t i v e of t h e d r u g . The p r i n c i p l e upon which t h e a s s a y i s b a s e d i s t h a t a complex of t h e d e r i v a t i z e d g e n t a m i c i n w i t h a n t i b o d y would s c a t t e r i n c i d e n t p o l a r i z e d l i g h t more t h a n t h e smaller uncomplexed d e r i v a t i v e molecule. The d e g r e e of a n t i b o d y b i n d i n g t h u s c a n b e d e t e r m i n e d from changes i n t h e f l u o r e s c e n c e i n t e n s i t y .

10.3

Radioimmunoassay

Radioimmunoassay f o r g e n t a m i c i n i s b a s e d on t h e b i n d i n g of t h e a n t i b i o t i c by a n a p p r o p r i a t e a n t i b o d y . By adding b o t h a n t i b o d y and r a d i o - l a b e l l e d d r u g t o a sample cont a i n i n g g e n t a m i c i n , one e s t a b l i s h e s a n e q u i l i b r i u m between t h e bound and f r e e l a b e l l e d and u n l a b e l l e d g e n t a m i c i n molec u l e s . Following s e p a r a t i o n of t h e bound and f r e e f r a c t i o n s , c o u n t i n g of t h e bound g e n t a m i c i n p r o v i d e s a measure of t h e p r o p o r t i o n of bound d r u g which i s r a d i o a c t i v e l y l a b e l l e d . T h i s measurement l e a d s t o t h e c a l c u l a t i o n of t h e amount of g e n t a m i c i n i n t h e o r i g i n a l sample.

GENTAMICIN SULFATE

331

S e v e r a l of t h e methods which have been developed f o r g e n t a m i c i n radioimmunoassay a r e o u t l i n e d i n T a b l e 7 . G r i f f i t h s and co-workers88 have compared t h e 3Hg e n t a m i c i n , 1251-gentamiciny radioenzyme, and m i c r o b i o l o g i c a l a s s a y methods. Jonsson89 s t u d i e d t h e s p e c i f i c i t y of t h e 1251-gentamicin radioimmunoassay and compared t h e r e l a t i v e r e s p o n s e s of t h e i n d i v i d u a l g e n t a m i c i n components t o t h e Similar r e s p o n s e s of several a m i n o g l y c o s i d e a n t i b i o t i c s . comparisons have b e e n made f o r t h e 3H-gentamicin r a d i o i m u n o a s s a y .go, 91992

10.4

Radioenzyme Assay

D e t e r m i n a t i o n of g e n t a m i c i n i n serum by r a d i o e n zyme a s s a y i s b a s e d on t h e enzyme-catalyzed d e r i v a t i z a t i o n of t h e d r u g w i t h a l a b e l l e d s u b s t i t u e n t group. D e r i v a t i z e d g e n t a m i c i n is a d s o r b e d o n t o some s t a t i o n a r y medium t o s e p a r a t e i t from u n r e a c t e d components. The r a d i o a c t i v i t y of t h e a d s o r b e n t p l u s g e n t a m i c i n t h u s p r o v i d e s a measure of t h e amount of d r u g p r e s e n t i n t h e sample. Smith e t a1.97yg8 employed a n R-factor-mediated enzyme t o a d e n y l y l a t e g e n t a m i c i n w i t h I 4 C - l a b e l l e d ATP s e r v i n g as t h e s o u r c e of t h e a d e n y l group. A d e n y l y l a t e d d r u g , b u t n o t ATP, w a s adsorbed o n t o Whatman P-81 phosphoc e l l u l o s e paper. F u r g e r e t a1.99 used a s i m i l a r t e c h n i q u e where t h e l a b e l l e d material w a s 3H-ATP. Smith and coworkers100 a l s o r e p o r t e d t h e i s o l a t i o n , p a r t i a l p u r i f icat i o n , and c h a r a c t e r i z a t i o n of t h e g e n t a m i c i n a d e n i n e monon u c l e o t i d e t r a n s f e r a s e , a s w e l l as com a r i n g methods i n which t h e s o u r c e s of t h e a d e n y l group were IEC-ATP and c~J~P-ATP. The 1 4 C - l a b e l l e d enzyme a s s a y h a s a l s o been compared t o t h e m i c r o b i o l o g i c a l assay.100,101,102
O ' N e i l l e t a l e 1 0 3 as w e l l a s B r o u g h a l l and Reeves104 have compared enzyme a s s a y s i n v o l v i n g a d e n y l y l a t i o n t o t h o s e employing a c e t y l a t i o n . Two a d d i t i o n a l p a p e r s l o 5 , l o 6 have d e s c r i b e d radioenzyme a s s a y s i n which t h e s u b s t i t u e n t g r o u p was a c e t y l d e r i v e d from 1 4 C - a c e t y l coenzyme A.

10.5

High P r e s s u r e L i q u i d Chromatography.

Anhalt e t a 1 . 7 9 ~ 8 0 d e s c r i b e d a h i g h p r e s s u r e l i q u i d chromatography (HPLC) p r o c e d u r e f o r t h e a s s a y of g e n t a m i c i n i n serum. The t e c h n i q u e i n v o l v e s t h e s e p a r a t i o n of g e n t a m i c i n from i n t e r f e r i n g compounds i n serum o n a CM-

Table 7 Radioimmunoassay Methods f o r Gentamicin Source of Antibody Rabbit Rabbit Separation Method dextran:charcoal adsorption second antibody precipitation

Carrier Protein Human serum albumin Human serum albumin bovine serum albumin porcine thyroglobulin keyhole limpet hemocyanin Human serum albumin

Isotope 3H, 1251 3H

Sensitivity

Reference
88

--2 ng

93,94,95

Rabbit

killed protein-A containing Staphylococcus aureus dextran:charcoal dextran:charcoal adsorption dextran:charcoal adsorption

1251

89

Bovine serum albumin Bovine thyroglobulin Human serum albumin

Rabbit Rabbit Rabbit

3H 3H 3H

80 Pg

90,96 91 92

10 ng 0 0 ug/ml .1

GENTAMICIN SULFATE

333

Sephadex column followed by a n a l y s i s u s i n g reverse p h a s e i o n - p a i r chromatography. 1-N-acetylgentamicin C may be used a s i n t e r n a l s t a n d a r d f o r t h e serum a s s a y . bontinuousflow, post-column d e r i v a t i z a t i o n w i t h 2-phthalaldehyde i s used t o form f l u o r e s c e n t p r o d u c t s f o r d e t e c t i o n . The HPLC t e c h n i q u e , when compared t o m i c r o b i o l o g i c a l a s s a y s , o f f e r s advantages w i t h r e g a r d t o r a p i d i t y , s p e c i f i c i t y and p r e c i s i o n .
11.

Acknowledgements

The a u t h o r s wish t o thank t h e Schering C o r p o r a t i o n Research L i b r a r y , Microbiology and P h y s i c a l and A n a l y t i c a l Chemistry s t a f f s , i n p a r t i c u l a r M r . Stephen Gruber, D r . Mohindar S. Puar, M r s . J e a n e t t e S a b a t i n o , and D r . M.D. Yudis f o r t h e i r a s s i s t a n c e i n t h e p r e p a r a t i o n of this analytical profile.

334

BERNARD E. ROSENKRANTZ e af. f

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Smith, J. I n f e c t . D i s . ,

3 391 ,

A.L. Smith, J.A. Waitz, D.H. Smith, E.M. Oden and B.B. Emerson, Antimicrob. Agents Chemother. , 5, 316 (1974).
E. Ten Krooden and J.H. 452 (1974).

102. 103. 104. 105.

Darrell, J. C l i n . Path.,

27,

M.A. O ' N e i l l , B.M. Maxwell, A.D. B l a i r , A.W. F o r r e y , and R.E. C u t l e r , C l i n . Chem., 20, 897 (1974).
J.M. B r o u g h a l l and D.S. 140 (1975).

Reeves, J.Clin.

P a t h . , 28,

J . M . B r o u g h a l l and D.S. Reeves, Proc. 9 t h I n t e r n a t . Cong. Chemother., London, J u l y 1975, i n Chemotherapy 2, 159 (1975).

106.

P. Berman and P. Botha, S . Afr. Med. J., (1977) , A b s t r a c t .

51,

930

HALOPERIDOL
Casirnir A . Janicki and Chan Yan KO
1.

2.

3.
4.

5. 6.

7. 8.

Description 1 . 1 Name, Formula, Molecular Weight I .2 Appearance, Color, Odor Physical Properties 2.1 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 Solubility 2.8 pKa 2.9 X-ray Diffraction Synthesis Stability-Degradation Drug Metabolic Products Methods of Analysis 6.1 Elemental Analysis 6.2 Non-aqueous Titrimetric Analysis 6.3 Colorimetric Analysis 6.4 Spectrophotometric Analysis 6.5 Thin- Layer Chromatographic Analysis 6.6 Gas Chromatographic Analysis 6.7 High- Performance Liquid Chromatographic Analysis 6.8 Differential Scanning Calorimetric Analysis 6.9 Polarographic Analysis 6.10 Fluorescence Analysis Determination in Biological Fluids Determination in Pharmaceuticals

342 342 342 342 342 344 346 346 35 1 35 1 35 1 353 353 353 355 356 357 357 357 359 359 359 36 1 361 363 363 364 364 365

Analytical F'rofiles of Drug Substances, 9

34 1

Copyright 1980 by Academic Ress. Inc. All rights of reproduclionin any form reserved.
ISBN: 012-260809-7

342

CASlMlR A . JANICKI AND CHAN YAN KO

1.

Description
1.1

Name, Formula, Molecular Weight

Haloperidol is 4-[4-(4-chlorophenyl)-4hydroxy-l-piperidinyl]-l'-(4-fluorophenyl)1-butanone. The trademark of the manufactured dosage form is HALDOLa.

OH

The molecular weight is 3 7 5 . 8 7 C21H23C1FN02

1.2

Appearance, Color, Odor White to faintly yellowish amorphous or microcrystalline odorless powder.

2.

Physical Properties 2.1 Infrared Spectrum

The infrared spectrum is presented in Figure 1. The spectrum was obtained from a potassium bromide dispersion using a PerkinElmer Model 283 grating infrared spectrophotometer. A list of the assignments made for some of the characteristic bands is given in Table I (1,2). Table I IR Spectral Assignment for Haloperidol 3125 2953 2918
2839

-OH -CH2

2822

0 - 0

0 - 0

- 0
(Y

-I

-8 ;
K

$ I

> a

0 - 0
N

0 3

0 0 -0
P )

..
E

[%I

33NVlllWSNVHl

344

CASIMIR A. JANICKI A N D CHAN YAN KO

Table I (cont'd)
1681 1597 1500 1454 1482 1410
1221 1156

-c=o
Substituted Aromatic Ring

-CH2 -OH -CH deformation of

F substituted aromatic ring

995 827 541 2.2

-C1 substltuted
-CH deformation of

aromatic ,ring p-substituted aromatic ring

-c=o

Nuclear -____

Magnetic Resonance Spectrum ___

The 9 0 MHz spectrum of haloperidol presented in Figure 2 was obtained in deuterated dimethylsulfoxide (d6) at a concentration of 8 2 mg/ml with tetranethylsilane as the internal standard. Spectral assignments are listed in Table I1 ( 2 ) .

c
1 0
9
8

5
PPM 161

F I G U R E 2: The NMR of Haloperidol in Deutcrated

Dimethylsulfoxide (d ) with TMS as the Internal Standard. Pnstrument : PerkinElmer R-32.

346

CASIMIR A. JANICKI A N D CHAN Y A N KO

Table I1 NMR Spectral Assignments for Haloperidol Chemical Shift ( ppm 1

8.05 7.32 7.31 4.77 2.98 2.20-2.70 1.38-1.97
2.3

Multiplicity mu 1tip1et singlet mu 1tip1et singlet triplet mu 1 tip1et mu 1tip 1et

Characteristic of proton a,b 1 ,m,n,p c,d

e , h,i

f,j,k

Ultraviolet Spectrum

The ultraviolet absorption spectrum of haloperidol obtained from a 9:l 0.1 M hydrochloric acid: methanol solution is &own in Figure 3. Two absorption maxima, were observed at 245 nm and 221 nm, with molar absorptivities of about 13,300 and 15,000 respectively.
2.4

Mass Spectrum

The EI mass spectrum, is given in Figure 4, and the fragmentation pattern is presented in Table 111. The fragmentation patterns were discussed in further detail in papers by Blessington ( 3 ) , Leferink and Moes (4), and Diding and Co-workers ( 5 ) . The chemical ionization mass spectrum obtained on a Finnigan model 3300 CI mass spectrometer with a model 6100 data system using methane as reagent gas is presented in Figure 5 ( 6 ) . A strong hydrogenated molecular ion was observed. The fragmentation pattern is presented in Table IV.

H A LOPE R1 DO L

347

OX

u z
a
a
m
0 v) m

0.6

0.4

0.2

0 210

260

310

360

WAVELENGTH I n m I

FIGURE 3: The U V Absorption Spectrum of Halop e r i d o l in 3:l 0 . 1 M Hydrochloric a c i d : Methanol.

..

j4
rLJ

= 3

..

3
Erc

fx

348

HALOPERIDOL

Direct I n l e t

1 CI

methane 1

60

100

150

200

250

300

350

400

450

500

ml e

F I G U R E 5: The Mass Spectrum of Haloperidol,

Chemical Ionization with Methane Instrument: Finnigan Model 3300 D.

350

CASIMIR A. JANICKI A N D CHAN YAN KO

Mass F r _ a q m e n t a t i o n P a t t e r n of H a l o p e r i d o l o n EI-MS

T a b l e I11

b
I
I I 4

4
a

I 1

375 237 224 206 165 123 95

Table I V

M+ C-H e

d d-H2 0 b a

Major I o n s and F r a g m e n t a t i o n of t h e CI-MS of H a l o p e r i d o l Methane as t h e R e a c t a n t G a s .

F ~ ~ - C H ~ T C H ~ T C H ~ - N I I I I

lo

AC
/ /

HALOPERIDOL

35 1

m/e
418 404 376 358 280 264 237 224

Ion
M*C H 7+ M*C H M a H M. H + - Ho ~ f

z5

C - H d

was : 1 , The isotopic ratio of ~ 1 ~ ~ : ~ 1 ~ ~ , 3observed. 2.5 Melting Range

The melting range of a haloperidol sample, determined after drying in vacuum at 6' 0 C for 3 hours, is between 147'-152OC according to the USP X I X class I procedure. No polymorphs of haloperidol have been reported to date. 2.6 Differential Scanning Calorimetry (DSC)

The DSC of haloperidol is shown in Figure 6. A melting endotherm is observed around 423OK using a temperature program of 5 /minute ( 7 1 . ' 2.7 Solubility

The approximate solubilities obtained at room temperature are listed in Table V (2, 7, 11).

426'

423"

420'

417'

414"

411"

QUALITATIVE DSC THERMOGRAMS OF MIXTURES OF HALOPERIDOL WITH AN IMPURITY, 5"PER MINUTE

0

0
\

v) v)

2
w K

2 A

s a
rl
f

a Reference standard b Mixture, purity = 98.75 c I# ,, = 97.33 d II = 94.84 e ,, = 92.73 f I, ,, = 89.93

Mole % Mole % Mole % Mole % Mole %

TEMPERATURE,

O K

FIGURE 6: DSC Thermogram of Haloperidol. Instrument: Perkin-Elmer DSC-2.

HALOPERIDOL

353

Table V Solubility Data of Haloperidol at Room Temperature Approximate Solubility (g/lOO ml)
1.1 6.6 1.3 1.7 0.5 1.8 0.5 100 1.8 <0.1
<0.01

S o Ivent

Acetone Benzene Chloroform Citric Acid (0.1 M ) Ethanol Ether Ethyl Acetate n-Hexane Lactic Acid Methanol n-Octano 1 2-Propanol Tartaric Acid (0.1 M ) Water (pH 5.9)
2.8

2.0

1.2

0.5

pKa

The pKa of haloperidol is 8.3 calculated by linear extrapolation using potentiometric titration in 158, 258, 358, 45% methanolwater (v/v) with 0.005 NaOH as titrant (1). 2.9 X-ray Diffraction

Reed and Schaefer (8) determined the crystal and molecular structure of haloperidol by single crystal X-ray diffraction techniques.
3.

Synthesis

Haloperidol is synthesized by heating a mixture of 4-chloro-l-(4-fluorophenyl)-l-butanone, potassium iodide, and 4-(4-chlorophenyl)-4piperidinol in a toluene solvent to about 1 0 0 C , in a closed vessel, (9,lO).

354

CASIMIR A. JANICKI A N D CHAN Y A N KO

OH Foi-CH2CH2CH2-CI

The 4 - c h l o r o - l - ( 4 - f l u o r o p h c n y l ) - l - b u t a n o n e was obtained by a Friedel Crafts reaction using fluorobenzene whereas the 4-(4-chlorophenyl)-4piperidinol was obtained in three steps from ctmethylstyrene.
A

major impurity that has been isolated and identified is 4-(4-(4-chlorophenyl)-4-hydroxy-lpiperidinyl)-l-(4-(4-(4-chlorophenyl)-4-hydroxy4-piperidinyl)phenyl)-l-butanone The structure is given as:
OH

OH

HALO PERIDO L

355

Diding and co-workers identified deschlorohaloperidol as an impurity in a sample of haloperidol hydrochloride by GC-MS ( 5 ) . The structure is given as:

OH

4.

Stability Degradation

Haloperidol is a relatively stable compound. Samples have been found to be stable for up to five years stored at room temperature in amber glass containers. Storage for up to one year at 4 5 C in amber glass containers did not adversely affect the drug substance. Haloperidol discolors and de-grades when exposed to natural sunlight for long periods. However, no degradation was noted when the drug substance was exposed to 2 0 0 0 foot candles for two weeks. The drug is only very slightly hygroscopic ( 2 , 7 ) . Haloperidol suspensions (1 mg/ml) were refluxed for 2 4 hours in water, 0.1 N sodium hydroxide, and 1 sodium hydroxide. No degradation was observed. Under the same reflux condition but in 0.1 N hydrochloric acid solution and 1 N hydrochloric acid solution approximately 108 and 5 0 % degradation was ob-served respectively (7). The hydrolysis products have not been identified.
A pH stability profile has been obtained from pH 2 to pH 8 using citrate-phosphate buffers. Good stability was observed at room temperature, 4 0 C and 6OoC for up to 2 weeks (11).

Pharmaceutical solutions in lactic acid with a pH of about 3 have been found to be stable for up to 5 years at room temperature, 2 years at 4 0 C

356

CASIMIR A . JANICKI A N D CHAN YAN KO

and 6 months at 6OOC. However, when the solutions were exposed to natural sunlight they became cloudy and discolored. A drop in haloperidol content was observed by assay and TLC. Pharmaceutical tablets have been found to be stable for up to 5 years at room temperature, 2 years at 4OoC and even 6 months at 6 O o C (7). The stability depends on the particular tablet formulation. An example was reported where haloperidol was found to be incompatible with 5(hydroxynethyl)-2-furfuraldehyde, an impurity in anhydrous lactose (12). The adduct of haloperidol with the furfural is shown below:

OH

5.

Drug Metabolic Products

The excretion and metabolism of haloperidol have been studied in rats after the administration of tritium labeled haloperidol (13,14). Oxidative N-dealkylation represents the major metabolic pathway (14). The major urinary metabolite of haloperidol is N-(2-(4-fluorophenyl)acetyl)glycine (d) (13,141. Other metabolites found were 4fluoro-v-oxobenzenebutanoic acid (a) (13,14) and 4-fluorobenzeneacetic acid ( c ) (14). Only traces of unmetabolized haloperidol were found in the urine. Since the tritium label was located on the fluorophenyl ring, the metabolism of the piperidine part of the molecule was not followed.

HALOPERIDOL

357

In man, haloperidol is metabolized to 4fluoro-y-oxobenzenebutanoic acid and N-(2-(4fluoropheny1)acetyl)glycine ( 1 5 ) . This demonstrates that the metabolism in man follows the same general pattern as that in the rat. No measurable amounts of any glutamine or glucuronic acid derivatives could be demonstrated. A trace of unmetabolized haloperidol was found in the urine. Recently a "reduced" form of haloperidol ( e ) has been detected and identified in serum and urine of patients on high doses of haloperidol (16). Hydroxylation was also proposed as another The proposed metabolic pathway in pathway ( 1 7 ) . man for haloperidol is summarized in Figure 7, with oxidative N-dealkylation representing the major metabolic path.
6.

Methods of Analysis
6.1

Elemental Analysis Element Carbon Hydrogen Chlorine Fluorine Nitrogen Oxygen

8 Theory

67.10 6.17 9.43 5.05 3.73 8.51

6.2

Non-aqueous Titrimetric Analysis

The non-aqueous titration procedure is the official method listed in the United States Pharmacopeia XIX ( 1 8 ) for the drug substance. An accurately weighed sample of haloperidol is dissolved in glacial acetic acid. After the addition of 3 drops of p-naphtholhenzcin TS, the solution is perchloric titrated with standardized 0.05 acid to the end point.

I 0

n I

0,

0=0

0
O+ f

"\
0
E
U
H

I 0=0

0
! i

i .

..

HA LOPERl DOL

359

6.3

Colorimetric Analysis

A general procedure for the quantitative determination of butyrophenones was described by Haemers and Van Den Bossche (19). Their procedure involves the reaction of butyrophenones with 3,5-dinitrobenzoic acid in an alkaline medium resulting in the formation of a red colored complex. Using this procedure, haloperidol was determined in pharmaceutical solutions and tablets. Haloperidol also forms a chloroform soluble methyl orange complex at a pH of 5, which is suitable for quantitative work (1,12). Another colorimetric procedure involves the use of potassium iodoplatinate (20). None of the colorimetric procedures have been found to be suitable for stability work since they lack the necessary specificity.

6.4

Spectrophotometric Analysis __-

The official USP XIX analysis of haloperidol tablets is a spectrophotometric analysis. A portion of powdered tablets is weighed and extracted with chloroform and 0.1 - sodium hydroxide. A portion of the N chloroform layer is extracted with 0.1 N sulfuric acid and the U V spectrum recorzed. The sample is compared against a reference standard diluted to the same final concentration in 0.1 N sulfuric acid, at the maximum about 2 7 5 nm. For solutions containing parabens, the same assay described above can be used, except that the final acid layer is extracted twice with diethyl ether. This removes any UV interference in the sulfuric a c i d solution due to the parabens ( 7 ) .
6.5

Thin-Layer Chromatoqraphic Analysis

Some TLC methods for haloperidol are given in Table VI, along with the various detection methods. Braun and co-workers published a method to follow the metabolites of haloperidol in urine (13).

360

CASIMIR A. JANICKI A N D CHAN Y A N KO

TLC Methods ___-____

Ad sor ben t
1; Silica GF

Table VI for Haloperidol Ref. (2)

Solvent System Ethyl Acetate: Ch1oroform:Methanol: Sodium Acetate Buffer ( p H 4.7) (54:23:18:5) Ch1oroform:Methanol (92:8)

Rf 0.64

2. Silica GF

0.40 0.50 0.61

(2) (2) (7)

3. Alumina GF Ch1oroform:Ethanol (99:l) 4. Silica GF Ch1oroform:Methanol Ammonium Hydroxide (91:8:l) Ethano1:lN hydrochloric acid (95:5) Acetone Benzene:Acetone: Petroleum ether: Ammonium hydroxide (10:10:10:2) Acet0ne:Petroleum ether (7:3) n-butano1:isopropanol: Acetic Acid:Water (3:3:2:4) Methano1:Acetone (12:88)

5. Silica G 6. Silica G 7. Silica G

0.57 0.60 0.90

(7)
(21) (21)

8. Silica G 9. Silica G

0.32 0.86

(21)
(21)

10. Silica GF with Na2C03

0.50

(22)

HALOPERIDOL

11. Silica GF with Na2C03 12. Silica GF 13. Silica GF

Ethano1:Chloroform (16:84)

0.74

Ch1oroform:Methanol: Formic Acid (85:10:5) 0.51 Ch1oroform:Methanol: 25% ammonium hydroxide (85:14:1) Ethy1acetate:isopropano1:Ammonium hydroxide (70:25:4)

0.87

14. Silica GF

0.81

15. Silica GF Cyc1ohexane:diethylamine:benzene (80:15:5) 0.18 6.6 Gas Chromatographic Analysis

Haloperidol has been assayed using a glass column packed with the following: 2% OV-1 on Chromosorb W HP with a column temperature of Z l O O C (25); a mixture of 0.3% Versamid and 0.6% OV-17 on Gas Chrom Q with a column temperature of 23OOC (26); 3% OV-17 on Gas Chrom Q with a column temperature of 28OOC (27); and 3% OV-1 on Gas Chrom Q with a column temperature of 24OOC (28). All of these reported methods u s e an electron capture detector to have the necessary sensitivity for determining haloperidol in body fluids. Bianchetti and Morselli used a 3 % OV-17 on Chromosorb W at a column temperature of 285OC along with a Nitrogen-Phosphorous detector (29). 6.7 High-Performance Liquid Chromatographic Analysis

A HPLC method was described in the literature (30). A reverse phase column was used with a solvent mixture of 44% tetrahydrofuran and 0.75% phosphoric acid in

362

CASIMIR A. JANICKI AND CHAN YAN KO

water. An UV detector at 254 nm was used. A retention time of 5.5 minutes was reported, but no specificity data were given.
A HPLC method has been developed, which gives the necessary specificity, that can be used to follow the stability of haloperidol in tablets and oral and injectable solutions (31). A 10 mg equivalent of haloperidol is transferred to a 120 ml bottle. A 50 m l aliquot of chloroform and 25 m l of 0.1 N sodium hydroxide are added. The bottle-is shaken for 30 minutes, centrifuged, and the aqueous layer discarded. A 15 ml aliquot of the chloroform layer is evaporated to dryness in a stream of nitrogen. The residue is dissolved in 4 ml of the intefnal standard solution (0.6 mg/ml oxatomide in methanol). A 5 1 1 sample is injected into the coluru?. .1

The chromatographic conditions a c e as follows: Column: LiChrosorb RP-18; 25 cm x 2.1 mm id stainless steel column M Mobile Phase: 30% 0.1 - ammonium carbonate: 70% methanol Flow Rate: 1 ml per min Wavelength: 270 nm Retention times: Haloperidol - 3 . 3 min Oxatomide - 10.0 min The specificity of the method is demonstrated by the data given in Table V I I .
~ ~~

1- 3- [ 4 - (diphenylmethyll-l-piperazinyllpropyl 1,3-dihydro-2H-bcnzinidazol-2-one.

HALOPERIDOL

363

Table VII Separation of impurities, degradation products and preservatives from haloperidol by HPLC. Compound Retention Time (minutes)

1.0 4-Fluorobenzoic acid 4-fluoro-y-oxobenzenebutanoic acid 1.1 Methylparaben 1.3 Propylparaben 1.9 4-(4-Chlorophenyl)-4-piperidinol 2.0 1-(4-Fluorophenyl)-4-(4-hydroxy4-phenyl-l-piperidinyl)-l-butanone 2.5 4-chloro-l-(4-fluorophenyl)-lbutanone 2.9 3.9 Haloperidol Oxatomide 10.0 4-(4-(4-chlorophenyl)-4-hydroxy1-piperidiny1)-l-(4(4-chlorophenyl)-4hydroxy-4-piperidinyl). pheny1)-1-butanone 11.3

4-[4-(4-Chlorophenyl)-3,6-dihydro-1(2H)-pyridyl]-4'-fluorobutyrophenone

22.0

6.8

Differential Scanning Calorimetric Analysis

A quantitative analysis of the purity of haloperidol can be obtained by DSC ( 7 ) , using the method by Plato and Glasgow (32). Qualitative DSC thermograms of mixtures of haloperidol with an impurity are given in Figure 6 to show the specificity of the technique.
6.3

Polarographic Analysis

A polarographic analysis of haloperidol was described by Volke et a1 using a dropping mercury electrode (33). A half-wave potential of -1.28 volts was obtained in a

364

CASIMIR A. JANICKI AND CHAN YAN KO

solvent of 1:l v/v acetate buffer, pH 5.3.; DMF, and -1.64 volts using 1:l v/v 0 . 2 5 potassium hydroxide: ethanol as the solvent.
6.10 Fluorescence Analysis

Haloperidol exhibits weak fluorescence in methanol, ethanol, and 2-propanol with an excitation wavelength at 310 nm and emission wavelengths at 410 nm, 3 7 5 nm and 390 nm respectively ( 3 4 ) . Haloperidol is converted into a strongly fluorescent derivative by the action of potassium permanganate on its alcoholic solution in an acid medium. An excitation wavelength of 3 0 5 nm produces emission at 3 8 3 nm ( 3 5 ) .
7.

Determination in Biological Fluids

Several gas chromatographic assay methods have been published for the determination of haloperidol in biological fluids ( 2 5 , 2 6 , 2 7 , 2 8 , 2 9 ) and they were given in Section 6 . 6 . Problems have been reported with the various gas chromatographic assays, for example, as the column temperature increased, the haloperidol dehydrated. As the temperature reached about 285OC, the haloperidol peak decreased in size while the dehydrated product peak greatly increased. At 2 8 5 O , the large dehydrated-haloperidol peak was lost in the solvent peak ( 3 6 ) . It is important that the temperature of the column stay about 240OC. The GC assay methods often lack the required sensitivity or specificity to obtain reproducible plasma data from clinical studies. So a sensitive and specific GC/CI-MS method was developed for the determination of haloperidol in plasma ( 3 7 ) . The procedure involves the addition of the internal standard (the chloro substituted analog of haloperidol), alkalinization of the sample with sodium hydroxide and extraction with heptane containing 1 . 5 % isoamyl alcohol. Prior to injection, the

HALOPERlDOL

365

organic layer is evaporated to dryness and reconstituted in methanol containing 5 % triethylamine. The GC conditions are as follows: Instrument: A gas chromatograph equipped with all glass-lined transfer lines Column: 10% OV-1 on Chromosorb WHP-0.3 m x 2 mm id Column Temperature: 260 Carrier: Methane at 25 ml/min Detector: Quadrupole mass spectroveter set to monitor ion m/$ 376 (MH of haloperidol and MH -H20 of the internal standard ) Haloperidol has a retention time of 0 . 7 minutes and the internal standard of 1.1 minutes. The response curve was linear between 0.2 ng and 6.0 ng Radioimmunoassays have been published (17, 38,391. A radioimmunoassay kit is also available (17) which is specific for haloperidol in the presence of the metabolites a,b,c,d, and e given in Figure 7 . The limit of detection of the assay is reported to be 0.02 ng contained in 0.5 ml of plasma. 8. Determination in Pharmaceuticals

Demoen (1) proposed a spectrophotometric assay. However, no specificity data were given. A method that has been found to be suitable for stability work is that given in the U.S.P. XIX for tablets (18). The method has been described in Section 6.4. Janicki and Almond (12) used that method to determine the haloperidol content in direct compression tablets that contained an unexpected degradation product of haloperidol. The method can also separate and quantitate haloperidol in the presence of the following compounds considered model degradation products: Institut National Des Radioelements, 6220 Fleurus, Belgium

366

CASIMIR A . JANICKI A N D CHAN YAN KO

4-chloro-l-(4-fluorophenyl)-l-butanone; 4-fluoro-0xobenzene butanoic acid; 4-fluorobenzoic acid; and 4-(4-chlorphenyl)-4-piperidinol.

The method cannot separate the dehydrated product of haloperidol from haloperidol. The structure of the dehydrated product is given as:

The dehydrated product of haloperidol is a theoretical degradation product and has not been found to be an actual degradation product in dosage forms to date (7). However, TLC solvent systems 2 and 4 in Section 6.5 can separate the dehydrated product from haloperidol, which runs ahead of haloperidol in both systems. The HPLC assay may be used for stability analysis. The method and specificity were described in Section 6.7. The methyl orange procedure described by Janicki and Almond (12), the colorimetric procedure using 3,5-dinitrobenzoic acid described by Haemers and Van Den Bossche (191, and the colorimetric determination using potassium iodoplatinate described by Pawelezyk and Plostkowiak (20) cannot be used for stability work since they all lack the necessary documentation of specificity. The fluorescence method of Baeyens and De Moerloose (35) has been applied to dosage forms but no specificity data were presented.

HALOPERIDOL

361

References

1.
2.

P. J . A. W . (1961).

Demoen, J . Pharm. S c i . ,

50,

350

P. J . A. W . Demoen, J a n s s e n P h a r m a c e u t i c a , Beerse, B e l g i u m , u n p u b l i s h e d d a t a .
B.

3.

B l e s s i n g t o n , Org. Mass S p e c t r o m . , (1971).

2,

1113

4.

J. G . L e f e r i n k , a n d R . A . A . Moes, J . ASSOC. O f f . Anal. Chem., 60, 2 1 ( 1 9 7 7 ) .

E.

5.
6. 7.

D i d i n g , H. S a n d s t r o m , J . O s t e l i u s , a n d B . K a r l e n , A c t a Pharm. S u e c . , 13, 55 ( 1 9 7 6 ) .

C . Y. KO, M c N e i l L a b o r a t o r i e s , F o r t W a s h i n g t o n , PA, u n p u b l i s h e d d a t a . C. A. J a n i c k i , M c N e i l L a b o r a t o r i e s , F o r t W a s h i n g t o n , PA, u n p u b l i s h e d d a t a .

L . L. Reed, a n d J . P. S c h a e f e r , A c t a C r y s t a l l o g r . , S e c t B . , 2, 1 8 8 6 ( 1 9 7 3 ) .

a.
9.

P. A. J . J a n s s e n , U . (1969).
M.

S. P a t e n t 3 , 4 3 8 , 9 9 1

10.

P. A . J. J a n s s e n , C . Van D e W e s t e r i n g h , A. H. J a g e n e a u , P. J . A. Demoen, B. K . E. Hermans, G . H. P. Van Daele, K . H. L. S c h e l l e k e n s , C. A . M. Van D e r E y c k e n , a n d C . J. E. Niemegeers, J. Med. Pharm. C h e m . , 281 (1959).

A,

11.
12.

W.

D . W a l k l i n g , M c N e i l Laboratories, F o r t W a s h i n g t o n , PA, u n p u b l i s h e d d a t a . C . A. Sci.,

2, 1 4

J a n i c k i a n d H. R. (1974).

Almond J r . , J . Pharm. S o u d i j n , Eur.

13.

G. A . B r a u n , G.
J. P h a r m a c o l . ,

L,

I. P o o s ,

a n d W. 58 ( 1 9 6 7 ) .

368

CASIMIR A . JANICKI AND CHAN YAN KO

14. 15.

W. Soudijn, I. Van Wijngaarden, and F. Allcwijn, Eur. J. Pharmacol., I,4 7 ( 1 3 6 7 ) . A. Forsman, G. Folsch, M. Larsson, and R. Ohman, Curr. Ther. Res., Clin. Exp., 21, 6 0 6
(1977).

16. 17.

A. Forsman and M. Larsson, Curr. Ther. Res., Clin. Exp., 24, 5 6 7 ( 1 9 7 8 ) .

J. Heykants, Symposium on Modern Trends in Psychopharmacology and Psychiatry, Kollekolle, Denmark, Sept. 1 9 7 8 .

18.
19. 20. 21. 22. 23. 24. 25. 26.

United States Pharmacopeia, XIX Revision, Mack Publishing Co., Easton, PA, 2 2 7 ( 1 3 7 5 ) .
A. Hacmers, and W. Van Den Bossche., J. Pharm. Pharmacol., 2 , 5 3 1 ( 1 9 6 9 ) .

E. Pawelczyk, and Z. Plotkowiak, Chem. Anal. (Warsaw), 17, 1 3 3 3 ( 1 9 7 2 ) .

M. P. Quaglio, G. S. Cavicchi, and M. Muraglia, Boll. Chim. Farm., 110, 3 8 5 ( 1 9 7 1 ) . E. Roder, E. Mutschler, and H. Rochelmeyer, J. Chromatogr., 42, 1 3 1 ( 1 9 6 9 ) .

R. A. Egli,

2.

Anal. Chem., - 2 7 7 ( 1 9 7 2 ) . 259,

Von S. Lauffer, E. Schmid, and F. Weist, 1 Arzncim. - Forsch., 3, 3 6 5 ( 1 9 6 9 ) .

I. A . Zingales, J. Chromatogr.,
(1971).

54,

15

A. Forsman, E. Martensson, G. Nyberg, and R. Ohman, Naunyn - Schmiedeberg's Arch. Pharmacol., 386, 1 1 3 ( 1 9 7 4 ) .
F. Marcucci, L. Airoldi, E. Mussini, and S. Garattini, J. Chromatogr., 53, 1 7 4 ( 1 9 7 1 ) .

27.

HALOPERIDOL

369

28.

J. N. S. Tam, and W. A. Cressman, McNeil Laboratories, Fort Washington, PA, unpublished data.

29. 30. 31.

G. Bianchetti, and P. L. Morselli, J. Chromatogr., 153, 2 0 3 ( 1 9 7 8 ) . D. S. Greene, Drug Dev. and Ind. Pharm.,
127 (1979).

L. T. Olszewski, C. M. Moral, and J. Ranweiler, McNeil Laboratories, Fort Washington, PA, unpublished data.
330 ( 1 9 6 9 ) .

S.

32. 33. 34. 35. 36.

C. Plato, and A. P. Glasgow, Anal. Chem., 41,

J. Volke, L. Wasilewska, and A. Ryvolova Kejharova, Pharmazie, 26, 3 9 9 ( 1 9 7 1 ) .

W. Raeyens, Analyst, 1 0 2 , 5 2 5 ( 1 9 7 7 ) .

-, 32

W. Baeyens, and P. DeMoerloose, Pharmazie,

764 ( 1 9 7 7 ) .

H. R. Almond Jr., and J. A. Meschino, McNeil Laboratories, Fort Washington, PA, unpublished data.

37.

K. T. Ng, and J. J. Kalbron, 25th National Meeting of the Academy of Pharmaceutical Sciences, Hollywood, FL, Nov. 1 9 7 8 .
B. R. Clark, B. B. Tower, and R. T. Rubin, Life Sci., 20, 3 1 9 ( 1 9 7 7 ) .
M. Shostak and J. M. Perel, Fed. Proc.,
531 (1976).

38. 39.

35

KHELLIN
Muhmoud A . Hassan and Muhammad Uppal Zubair
1.

2.

3. 4. 5. 6.

Description 1.1 Nomenclature 1.2 Formulae I .3 Molecular Weight 1.4 Elemental Composition I .5 Appearance, Color, Taste, Odour Physical Properties 2.1 Crystal Properties 2.2 Solubility 2.3 Identification 2.4 Spot Tests 2.5 Microcrystal Tests 2.6 Spectral Properties Isolation Biosynthesis Synthesis Methods of Analysis 6.1 Modified Zeisel-Viebock Method 6.2 Colorimetry 6.3 UV Spectrophotometry 6.4 Thin Layer Chromatography 6.5 Two Dimensional Thin Layer Chromatography 6.6 PMR Spectrometry References

372 372 372 373 373 373 373 373 374 374 374 375 375 380 382 382 386 386 386 387 387 388 388 393

Analytical Rofiles of D u Subslancss, 9 rg

37 1

Copyright @ 1980 by Academic Press, Inc. All rights of reproduction in any form E S ~ N ISBN 0-12-260809-7

~ .

372

MAHMOUD A. HASSAN A N D MUHAMMAD UPPAL ZUBAIR

1.

Description 1.1 Nomenclature

1.1 1 Chemical Names

a.
h.

4,9-Dimethoxy-7-methyl-5 H-furo[3,2-g] - 1 benzopyran-5-one. [] 5,8-Dimethoxy-2-methyl-4,5-furo-6,7chromone.

5,8-Dimethoxy-2-methyl-6,7-furano-

c.

chromone.

d.
e.

4,9-Dimethoxy-7-methyl-5-oxofuro [3,2-g] 1,2-chremone 4,9-Dirnethoxy-7-methyl-5-ox0furo [3,2-g] [l] benzopyran.

4,9-Dimethoxy-7-methyl-5-0~0-1,8dioxabenz [ f] indene.

f.

The CAS Registry No. is [82-02-01.

1..1 2 Generic Name

Khellin

1.1 3 Trade Names

Ammincardine, Amicardine, Ammipuran, Ammivin Ammivisnagin, Benecardin, Corafurone, Cardiokhellin, Ceronin, Eskel, Kelamine, Kelicorin, Kelicor, Keloid, Khellin, Gynokhellin, Khelfren, Lynamine, Methafrone, Norkel, Simes Kellina, Visaminin, Visnagin, Visnagalin, Vasokellina, Viscardin.
1.2

Formulae 1.2 1 Empirical 14

H

12 5

KHELLIN

373

1.2 2 Structural

OCH3 The structure of Khellin has been elucidated by degradative methods as well as by its partial synthesis by reconstruction of the chromone ring starting from Khellinone (1) and also by its total synthesis. 1.2 3
*

Wiswesser Cine Notation

T C 566 DO JV MOJ B01 HO 1L

1.3 Molecular Weight

260.24 1.4 Elemental ComDosition C,64.61%; H, 4.65%; 0, 30.74%. 1.5 Appearance, Color, Taste, Odour White odourless crystals sometimes with slight yellowish tinge, and with a bitter taste.
2.

Physical Properties 2.1 Crystal Properties: 2.1 1 X-Ray Diffraction: Khellin forms monoclinic-Prismatic crystals with forms C 0 0 1 ~ { 1 0 0 ~ { 2 0 1 ) ( 0 1 0 ) { 1 0 1 ~Fro? . measurements a:b:c = 2.462:1:2.681; B=102 53-. Cleavage parallel (100) good, (010) less perfect. Optical constants: a= 1.478; B=1.741; y =1.785. Orientation a=b; Optical axes observed on (OlO), dispersion 6)8.25 ( f o r

3 14

MAHMOUD A. HASSAN A N D MUHAMMAD UPPAL ZUBAIR

yellow light2 48' 52 , angle c: y=6O(red),7' (yellow) , 84 (green) ,lO$O(blue); 1 ' v o e ) 2(ilt. Unit, cell for orientation a :bo:Co=2,007:1: 1.608; B=93' 39:a 14.49, b 7f22, C 11.61 A; Z=4, space group C 5h - PZl/n (derived from Polany rotation an$ Weissenberg diagram) ( 2 ) . 2.1 2 Melting Range Khellin melts at 1 4 1 5 . 5-5' at 0.05 mm Hg. 2.2 Solubility 25 mg/100 ml of water, 2.6 g/lOO ml of methanol, 1.25 g/100 ml of isopropanol, 0.5 g/100 ml of ether, 0.15 g/100 ml of skellysolve B, and much more soluble in hot water and hot ethanol ( 3 ) . 2.3 Identification 1. Khellin gives a red-violet color with NaOH o r KOH or m-dinitrobenzene in alkaline solution(4-6). 2. A solution containing khellin 0.5-1 ml is heated to boiling with 0.3 ml of 2,4-dinitrophenylhydrazine (0.5% solution in 1.5 N hydrochloric acid) f o r 30 minutes, after cooling, 0.5 ml 30% potassium hydroxide and 1-2 ml o f ethanol are added. The resulting red-violet stable color is proportional to the amount of khellin present. The 2,4-dinitrophenyl hydrazone of khellin was prepared and it5 m.p. is, 284-28S0(7).
3 . Add 2 ml o f phosphoric acid to 0.1 g of khellin,

Boils at 180-200'

orange-red crystals are formed, which dissolve on heating. To the viscous orange solution of 0.5 g of khellin in 1 ml phosphoric acid, gradually add dry ethylacetate with trituration to obtain yellow crystalline precipitate of the oxonium phosphate; m.p. 1 6 2' with decomposition ( ) 8.

2.4

Spot Tests 1. To a few crystals of khellin on a white porcelain . plate add 2 drops of phosphoric acid.(sp.gr. 8. 1.75), an orange-colored crystals are formed ( )

KHELLIN

375

2. To a few crystals of khellin add few crystals

of alloxan, 5 drops of sulphuric acid and triturate with a thin glass rod till the solids dissolve, an initial orange color is formed which changes to violet color with an orange 8. edge after 20 minutes ( )

3 . This is

a more specific test for khellin and is not given by compounds devoid of 5-OH and 8-OCH3 substituents. The reaction is carried out by dissolving a few crystals of khellin in HN03, and then destroying the oxonium salt by diluting with alkali to yield a color, which i s due to the quinone formed by oxidation with HNC3 (9,lO).

25 .

Microcrystal Tests Khellin gives oxonium salt compounds when the following reagents are added to the dry substance (11) :
1. I -KI reagent, gives trimorphic crystallisation. 2 In the outer zone of the precipitation there are rather coarse needles of good bire fringence, those broad enough showing a yellow to dark red dichroisy; then a zone of small grains, some dichroic, yellow to dark, mostly nondichroic, red after the test has stood a little while, at the central concentrations fine needles in purple masses, some of these needles in outer tufts showing dark blue to light brown dichroism. This is a sensitive test and probably specific.
2. HC104, gives yellow rods and needles.
3 . HAuBr4 in dil HC104-H9AC gives fine needles,

and at central concentration large tablets, chips and bars and fans of fairly large needles, with yellow to orange dichroism.

2.6

Spectral Properties
2 . 6 1 Infrared Spectrum

The infrared spectrum of khellin is recorded as a nujol mull on a Unicam SP 1025 spectrophotometer and is shown in Fig. 1 . The assignments

c
n

c :

a l

Lk

P
E:
3
h

r'

.. M
.A

LL

KHELLIN

311

for the characteristic bands in the infrared spectrum are listed in table 1. Table 1 Frequency cm-1 1690 1650,1640 1600 1580 1250,1230 1190,1160 1090,1070 870,820 790,740 720. Assignment

C = C (aromatic)

c = o c = o

ethylenic linkage

c-0-c
-CH out of plane

deformation.

Other fingre print bands characteristic of khellin are 13913,1370, 1050, 1040, 1000, 960, 920 and 910. 2.6 2 Ultraviolet Spectrum (UV) The UV spectrum of khellin in ethanol was scanned using Pye Unicam SP 800; from 400200 my, three maxima and three minima were observed. The maxima are located at 220, 244 and 328 nm. The minima occur at 232, 272 and 300 nm. The spectrum is shown in Fig.2. The UV spectral data of khellin and analogues have also been reported (12). 2.6 3 Nuclear Magnetic Resonance Spectrum (NMR) Proton Spectrum The proton NMR spectrum of khellin in deuterated chloroform i s shown in Fig.3. It was recorded on a Varian T-60AY60MHz N M R Spectrometer, using tetramethylsilane as an internal reference. It is to be noted that both natural and synthetic khellin are currently used in pharmaceutical formulations. Natural khellin might contain variable amounts of visnagin due to incomplete purification.

378

a 3UEqxo s qv

ra,

Id4

Fig. 2: Ultraviolet spectrum of khellin in ethanol.

350

300

250

200

I 4.0

1'

PARTS PLH ?lILLIOE;.

rig.

3:

YFIK

spectrum of khellin and TMS in CDCl

3'

380

MAHMOUD A. HASSAN A N D MUHAMMAD UPPAL ZUBAIR

Therefore, the PMR spectrum of visnagin in deuterated chloroform using tetramethylsilane is shown in Fig. 4. The PMR spectral assignments of khellin and visnagin are given in Table 2 (13). Table 2: PMR Comparison of Khellin and Visnagin Chemical shifts(6) 2-CH (s) Khe11in Vi snagin 2.40 2.33 3-H (s) 6.05 6.03 5-OCHs
(5)

8-OCH3 8-H (SJ (s) 4.05

2-H (d)

- 3-H
(d)
7.00 7.00

4.20

7.18

7.63 7.63

4.20

( s ) = singlet,

(d) = doublet.

2.6 4 Mass Spectrum The mass spectrum of khellin obtained by conventional elect-fonimpact ionization shows+a molecular ion M at m/e 260.07 ( 4 . The M ion 1) peak is the base peak and is shown in Fig. 5. The fragmentation of khellin and other furanochromones have been reported (15).

Fig. 5:

Mass Spectrum of Khellin

3. Isolation Khellin [5,8-Dimethoxy-2-methyl-4,5-furo-6,7-chromone] is obtained as the main chromone constituent from the fruits of Ammi visnaga (Fam. Umbelliferae) (16-19).

~ ~ . . . . ~ . . . . I . . . . I I . . . . I . . . 1 . f . . . 1. . .
I

I.

I
* .
1 1

a .

14

'43

L D

1.0

t.b

Fig. 4: PMR Spectrum o f visnagin and TYS i n C D C l

3 82

MAHMOUD A. HASSAN A N D MUHAMMAD UPPAL ZUBAIR

4.

Biosynthesis Geissman (20) on the basis of the striking similarity between the furocoumarin isopimpinellin and the furanochromone khellin, suggests that the two heterocyclic ring systems have a common origin, namely cinnamic acid and they are formed by the shikimic acid - phenylalanine pathway. Extension of the-cinnamic acid side-chain, possibly at the orthohydroxylated intermediate as glucoside, by the addition of two carbon fragment, as shown+ However, Chen et al(21) has reported that blpnthesis of radio-active khellin and visnagin from C -acetate by Ammi visnaga plants. Their results support the hypothesis that furanochromones are biosynthesised via an acetate condensation pathway rather than by the phenylalanine-shikimic acid route as is the case for the very closely related furocoumarins.

5.

Synthesis Several synthetic routes to khellin and derivatives have been reported (22-35). Two of them are illustrated. Route - I : Involves the condensation of 5,7-Dihydroxy-2 methylchromone ( I ) with bromoethyl acetate, followed by nuclear oxidation of the product with alkaline persulfate to give quinol (111) (36). The partially methylated quinol ( I V ) is condensed with hexamine to give the aldehyde (V) which on complete methylation and hydrolysis with dilute alkali gives aldehydic acid ( V I ) , which on boiling with sod. acetate and acetic anhydride afforded khellin ( V I I I ) . Route - 11: Describes total synthesis of khellin starting with 2,5-dimethoxyresorcinol ( I ) , (37,38). ( I ) is converted into the coumaranone (11) by means of Hoesch Reaction, using chloroacetanilide. Acetylation of (11) and reduction yielded ( I V ) . After removal of the acetyl group of ( I V ) it was subjected to a Hoesch Raction with acetonitrite to yield dihydrokhellinone ( V I ) . Acetylation of V I and reaction with N-Bromosuccinimide in carbon tetrachloride and purification yielded pure khellinone ( V I I I ) . Khellinone was condensed with ethyl-acetate in presence of sodium hydride to give a diketone ( I X ) which is then cyclised to give k I l e l l i n

KHELLIN

383

2-methyleehromone Biosynthesis of a 2-methylchromone.

384

MAHMOUD A . HASSAN A N D MUHAMMAD UPPAL ZUBAIR

II

Ill

IV

VI

KHELLIN

385

ROUTE I 1

3 86

MAHMOUD A. HASSAN A N D MUHAMMAD UPPAL ZUBAIR

6.

Methods of Analysis 6.1 Modified Zeisel-Viebock Method (39) Malysz et a1 (40) have applied this method for determining methoxy groups by using a special apparatus. The sample containing equivalent of 2-5 mg of methoxy group was mixed with 0.5 g of phenol, 2.4 g of potassium iodide and 4 ml of phosphoricoacid , then heated in an atmosphere of C02 at 150 for 1.5 hours. The iodomethane produced was distilled off in a stream of CO and absorbed in 10 ml of bromine solution (dissolve 10 g of potassium acetate in 100 ml of anhydrous acetic acid and add 4 ml of Br2). The absorbent solution was then mixed with 10 rnl of 2.5% aqueous sodium acetate and diluted with 100 ml of water and the excess of bromine was destroyed with 3 drops of formic acid. The colorless solution was acidified with 10 ml of 2 N sulfuric acid, 1 g of potassium iodide was added and after 5 minutes the liberated iodine was titrated with 0.05N sodium thiosulfate solution and starch as indicator. This method has been used to determine khellin in the pure form and in pharmaceutical preparations. The results were within 20.1+1% of those obtained by various pharmacopoeia1 methods.
6.2

Colorirnetry Different colorimetric methods have been used for the determination of khellin, based on color reaction with sulphuric acid (41,42), phosphoric acid (43,45) and m-dinitrobenzene and potassium hydroxide (45,47). A colorimetric method based on the color developed by treating khellin with nitric acid followed by sodium hydroxide is officially adopted by E.P. 1972 ( 8 . The method 4) is based on the oxidation of khellin with nitric acid to produce quinone derivative which gives violet color with sodium hydroxide solution. The reaction was favourably carried out at room 0 temperature (20-30 ) , lower or higher temperature, either slow the reaction or enhance it, respectively. The absorbance of the resulting violet color is measured at Xmax 540 nm within 15 minutes

KHELLIN

387

CH3

Khellin

OCH3.

Khellin-Quinone

after the addition of Na0I-I solution. The formed color obeys Beer's Law in a concentration range of 200-1200 11 of khellin. This method has been used .8 to determine khellin in fruits and extracts of Ammi visnaga and in pharmaceutical formulations (49)

6.3

U\I

Spectrophotometric Method

Khellin can be analyzed spectrophotometrically. The sample is dissolved in ethanol (95%) to give a concentration of about 12 Ug/ml, and the absorbance of the solution so produced is measured at 244 nm. The l o g E values are given in table 3 (SO). Table 3 Xmax (nm)
220 244 328

Log

&

Ash (nm)
228 280

Log

4.453 4.513 3.614

4.400 3.678

6.4

Two Dimensional Thin Layer Chromatography Different solvent systems have been used for the separation of khellin from other natural chromones.

388

MAHMOUD A. HASSAN AND MUHAMMAD UPPAL ZUBAIR

Table 4 Solvent System Xylene-Acetone(4:1) Xylene-Ethyl AcetateAcetic Acid (15:s: 1 ) Xylene-Ethyl AcetatePyridine (6:18:1) Xylene-Pyridine-Formic Acid (23:5:2). Ether-Formamideethanol (93: 2: 5 . ) Chloroform-Tetrahydrofuran-formamide (50: 50: 6.5). Ethylacetate-BenzeneWater (50:50:50). Ethylacetate-Benzenewater (50:75:50) (Fig.6). Rf Value
0.25

Reference 51
51

0.31

0.62 0.63
0.86

51 51
52

0.67

52

0.50

52

0.31

52

6.5

Two Dimensional Thin Layer Chromatography Karawya et a1 (53), reported two dimensional TLC technique on silica gel G plates using ethylacetate as developing solvent (Fig.7). This technique has offered a better separation of khellin from visnagin than the unidimensional multiple-run technique and was used in the quantitative recovery of the two constituents and their subsequent colorimetric estimation. It is also used for the determination of khellin in pharmaceutical formulations.

6.6

PMR Spectrometry

Hassan and Aboutabl (13) has published a rapid, accurate and specific PMR method for the determination of khellin in bulk drug and pharmaceutical

KHELLIN

389

Ethyl acebfe Benzene Water 50/75/50

.-, .__.

gr b r g <.-..*
,% .

;;::
..:
A

,,-1

\.

____._--

Fig. 6: A:0.1 cc alcoholic extract of Ammi visnaga f r u i t s .

IROV l U 1 U

EXttUCT

6.conA

- + run

Fig.

7 :

Two dimensional T I L u s i n g e t h y l acetate as the developing system.

390

MAHMOUD A . HASSAN A N D MUHAMMAD UPPAL ZUBAIR

formulations. It also furnishes a specific means of identification of khellin as well as simultaneous detection and determination of the less potent 8demethoxy analogue, visnagin. Acetanilide exhibiting three proton singlet at 2.30 ppm in CDC13 assigned to its methyl group, is employed as an internal standard. The two singlets at 4.2 and 4.05 ppm (in CDCl ) assigned to the 5-and 8-methoxy protons of khelqin respectively, were chosen for its quantitative analysis (Fig.8). However, the presence of other ingredients in injectables interfere with the precise integration of the 5- and 8-methoxy signals. For this reason the 2-methylprotons singlet appearing at 2.4 ppm (in CDCl ) was used for assay of khellin in injectables. zthanol-free chloroform was used as a solvent, as its proton singlet of 7.25 ppm does not interfere with upfield protons of both compounds. The method is rapid, accurate and precise, with standard deviations of 2 0.76% in synthetic mixtures and 0.94% in tahlets and injectables respectively. No interference from tablet excipients could be observed. Visnagin shows in deuterated chloroform a very similar PMR spectrum to that of khellin, except f o r the presence of an aromatic proton singlet at 7.18 ppm and three proton singlet at 2.33 ppm assigned to its 8-H and 2-CH 3 group. This has allowed facile detection of visnagin in khellin in bulk drug and formulations. Moreover, the ratio determination of visnagin to khellin is achieved by integration measurements of the 2-methyl protons singlets at 2.33 and 2.40 pprn respectively (i.). Fg:)

*r >

Ii

.0.

rlu

**

@-coNHI

T S M

-a

l
I

..
.

.,

..
.

so

**a.

<.I

Fig. 8:

PMR spectrum of khellin, acetanilide M and T S in ethanol-free chloroform.

, i

.W

"I

wrt

T S M

I . . . . I . . . . I . . . .
I 0

I*

,4

Fig. 9:

PMR spectrum of khellin, v i s n a g i n and T S in CDC13. M

KHELLIN

393

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1.
2.

E.Spath and W.Gruber, Chem. Ber.

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KHELLIN

395

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C l a r k e and A . Robertson, J. Chem. SOC., 302 (1949). Klimova and K.S. ( l ) , 109 (1963). Zabrodina, Zhur, Anal. Khim,

18,

A.V.

D. Malysz, E . Perkowski, W. Turska and T. Walicka, Acta P o l . Pharm. , 24(3) 307 (1967). I . R . Fahmy, N. Badran and F. Messeid, J. Pharm. Pharmac o l . , 1_, 529 (1949). 16, - 329 (1968). H. Supneiwska, E. Zarpo, rest), S.I. Balba, A . Y. Zaki and S.M. A.bdelwahab, P l a n t a Med;

42. 43. 44. 45. 46. 47. 48. 49.

SO.

Diss. Pharm.,

S,

63 (1956).

10,737

0. Contz and M. (1962).

Gheorghini, Pharmacia (Bucha19 - (41,

M.S. Karawya, 368 (1971).

G . Nour and A. S i n a , P l a n t a Med.,

G . Honstmann, K u l t u r p f l a n z e ,

10, 132

(1962).

W. Messere,

A r z e n e i For. - 1359 (1965). 15, The Amiriah Press, C a i r o , 1972,

J. Pharm.

Egyptian Pharmacopoeia, p.547.

M.S. Karawya, M.A. E l Kiei and G . Now, U.A.R. Sci. , No.2, 273 (1970).

11,

M. Uppal Zubair and M.M.A.

Hassan, Unpublished R e s u l t s .

51.

M.M. Badawi and M.B.E. (1967).

Fayez, P l a n t a Mod., 15, 1.40 -

396

MAHMOUD A . HASSAN A N D MUHAMMAD UPPAL ZUBAIR

52. 53.

S . I . Balba, A . Y . Zaki and S.M. Abdel Wahab, P l a n t a Med. - 329 (1968). 16,

M.S. Karawya, M.A. El-Kiei, A . S i n a and G . Nour, J. Pharm. S c i , 59, 1025 (1970).

LORAZEPAM
Jay G. Rutgers and Charles M . Shearer
1. Description 1 . 1 Name, Formula, Molecular Weight 1.2 Appearance, Color, Odor 2. Physical Properties 2.1 Infrared Spectrum 2.2 Nuclear Magnetic Resonance Spectrum 2.3 Ultraviolet Spectra 2.4 Mass Spectra 2.5 Melting Range 2.6 Differential Scanning Calorimetry 2.7 Solubility 2.8 Crystal Properties 2.9 Dissociation Constants 2.10 Protein Binding 3. Synthesis 4. Stability and Degradation 5 . Metabolism and Pharmacokinetics 5.1 Metabolism 5.2 Pharmacokinetics 6. Identity 7. Methods of Analysis 7. I Elemental Analysis 7.2 Phase Solubility Analysis 7.3 Direct Spectrophotometric Analysis 7.4 Colorimetric Analysis 7.5 Electrochemical Analysis 7.6 Titrimetric Analysis 7.7 Chromatographic Analysis 8. References
398 398 398 398 398 400 400 404 404 407 407 407 407 409 409 411 412 412 414 414 415 415 415 415 416 416 417 417 424

Analytical Rofiks of Drug Substances, 9

397

Copyright 0 1980 by Academic Ress, Inc. All rights of nproduction in any form ~ ~ e ~ e d . ISBN: 0-12-260809-7

398

JAY G . RUTGERS A N D CHARLES M. SHEARER

1. Description
Name, Formula, Molecular Weight The name used by Chemical Abstracts for lorazepam is 7-chloro-5-(2-chlorophenyl)-1,3-d~hydro-3-hydroxy-2H-l, 4-benzodiazepin-2-one. The Chemical Abstracts Registry Number is 846-49-1. 1.1

15H10C12N202

Mol. Wt. = 321.2

1.2 Appearance, Color, Odor Lorazepam is a white or nearly white, practically odorless, crystalline powder.
2.

Physical Properties

Infrared Spectrum An infrared absorption spectrum of a potassium bromide dispersion of lorazepam (Wyeth Reference Standard Lot C-10684) is presented in Figure 1. The spectral band assignments (1) are listed in Table I. Table I Infrared Spectral Assignments of Lorazepam Wave number (cm-l)
3500 to 2700

2.1

Vibration Mode OH,NH stretch C=O stretch C=N stretch Aromatic C=C stretch Out of plane CH deformation of 1,2,4 substituted aromatic Out of plane CH deformation of ortho disubstituted aromatic 1,2,4 substituted aromatic and ortho disubstituted aromatic. Unequivocal assignment cannot be made.

1690 1610 1565 and 1475 825 730

750 and 695

2.5

WAVELENGTH (MICRONS) 6 7

10

12

14

16 18 20

25 30 40 50

01 4ooo

3500

Moo

2500

1800

1 m

1400

1200

loo0

800

600

400

2GfJ

FREQUENCY (CM-ll

Figure 1

I n f r a r e d Spectrum of Lorazepam (Wyeth Reference Standard, L o t C-10684) KBr p e l l e t

JAY G. RUTGERS AND CHARLES M. SHEARER

2.2 Nuclear Magnetic Resonance Spectrum The nuclear magnetic resonance spectrum sample (Wyeth Reference Standard, Lot C-10684) was prepared by dissolving 75 mg of it in 0.5 ml of deutero-dimethylsulfoxide containing tetramethylsilane as internal reference. The spectrum was obtained on a 100 MHz Varian XL-100 spectrometer and is presented as Figure 2. Deuteration or irradiation of the OH reduces the methine doublet to a sharp singlet. The spectral assignments ( 2 ) are listed in Table
11.

Table I1 NMR Spectral Assignments of Lorazepam Proton (No. A1 iphatic C-H(1) 0-H( 1 ) Aromatic C-H(4) Aromat ic C-H( 1 ) Aromatic C-H( 1) Aromatic C-H( 1 Chemica1 Shift (ppm) 4.88 6.38 7.4 to 7.7 6.97 7.6 7.30 10.96

ZYE
Doublet Doublet Mu1t ip let Doublet

(in Hz) 7.5

Doublet of Doublet Doublet Broad singlet

N-H(~)

Ultraviolet Spectra The ultraviolet spectrum of lorazepam in methanol is presented in Figure 3. The spectra of lorazepam in 1N NaOH and in 1N HC1 are presented in Figure 4. The absorptivities and maximum wavelengths are given in Table 111. These values agree with published data (3,4,5). Levillain (6), has studied the relationship of structure and the UV absorption characteristics of a series of 1,4-benzodiazepines, including lorazepam, considering the electronic distribution of the various substitutes and the stereochemistry. The spectrum of lorazepam is consistent with that of other benzodiazepines with similar structure.

2.3

I
I

-I 1
I,
II

ppm

Figure 2

NMR Spectrum of Lorazepam (Wyeth Reference Standard,

Lot C-10684) i n deutero dimethylsulfoxide

402

JAY G. RUTGERS AND CHARLES M. SHEARER

0. t

0.5

c r

U m

0.4

0 m

2 0.3
0.2

0.1

0.0
WAVELENGTH (nm)

Figure 3

U l t r a v i o l e t Spectrum of Lorazepam (Wyeth Reference Standard Lot C-10684) S o l v e n t - methanol

LORAZEPAM

403

0.1

220

0.0

'

240

260

280

300 320 340 WAVELENGTH (nm)

360

380

400

Figure

U l t r a v i o l e t S p e c t r a of Lorazepam (Wyeth Reference S t a n d a r d , L o t C-10684) S o l v e n t A , 1N N d H ; Solvent B , 1 N HC1

404

JAY G. RUTGERS A N D CHARLES M . SHEARER

Table 111 Ultraviolet Spectral Characteristics Solvent methanol 1N NaOH 1N HCI A M a x (nm) 320 229 347 233 368 287 237 Absorptivity 6.1 116

8.4

92 8.0 25 94

2.4 Mass Spectra The mass spectrum o f lorazepam (Wyeth Reference Standard, Lot C-10684) was obtained with Kratos DS-50-S Data System coupled with a MS-902 double focusing, high resolution mass spectrometer (7). The ionizing electron beam energy was at 70 eV. Figure 5 is a bar graph of the mass spectrum with the molecular ion at m/e 320. Identification of the pertinent masses is presented in Table IV. A chemical ionization spectrum showed the parent peak at M+1, 321. Table IV Mass Spectrum Fragmentation Pattern of Lorazepam
__ mle

Species M+
M+

320 302 291 274 263 239

2.5

M+ M+ M+ M+

H20 CHO H20 - CO HCO - CO H20 - CO - C1

Melting Range The following melting range temperatures have been reported. OC Reference 167 170 (d) 4 166 - 168 (d) 8 Trace amounts of certain acidic impurities,including 6-chloro-4-(~-chlorophenyl)-2-qu~nazoline~rboxyl~c acid (a possible degradation product), benzoic acid and salicylic acid, will markedly depress the decomposition temperature of lorazepam ( 9 ) .

3
a

a
(A

$4 (d

cl

4J

U QJ

(A

n
W

0 X

50 Figure 6

100
O C

150

200

Differential Thermal Analysis Spectrum of Lorazepam (Wyeth Reference Standard, Lot C-10684)

LOR A ZE PA M

407

2.6 Differential Scanning Calorimetry The DSC thermogram (10) of lorazepam (Wyeth Reference Standard, Lot C-10684) is shown in Figure 6 . The thermogram was obtained at a heating rate of 10C/min in a nitrogen atmosphere using a Perkin-Elmer DSC-2. The thermogram exhibits no endotherms or exotherms other than that associated with the decomposition melt. Solubility The following solubilities at room temperature have been reported. 2.7

Solvent Alcohol Water Propylene glycol Chloroform Ethyl Acetate

Solubility (mg/ml) 14 0.08

Reference 11 11 12 13 13

16 3 30

2.8 Crystal Properties The X-ray powder diffraction pattern of lorazepam (Wyeth Reference Standard, Lot C-10684) ,obtained (10) with a Phillips diffractometer using CuKd radiation is presented in Figure 7. The calculated "d" spacings are presented in Table V. It is possible for lorazepam to form solvates and other crystal forms (9). The crystal and molecular structure of the ethanol adduct of lorazepam have been characterized by X-ray analysis (14). The asymmetric unit consists of one ethanol and two lorazepam molecules linked together by hydrogen bonds. The crystalsoare monoclini; with cell dimensions of a=13.446A, b=19.259A and c=13.789A. The 0 angle i s 116.80O. The heterocyclic seven-membered ring adopts a boat configuration. The two phenyl rings are planar and the obtuse angles between them are 106.6O and 99.1. 2.9 Dissociation Constants Two pKa's are observed for lorazepam (15,5). The pKa values, determined spectrophotometrically in aqueous buffers, are 1.3 and 11.5. Polarographically, the first pKa was found ( 1 6 ) to be 1.8. Barret et a1 (5) have proposed that three species, a protonated, a neutral, and a deprotonated form, are involved i n the equilibria. Protonation at low pH occurs at the nitrogen in the 4 position. Deprotonation occurs at high pH with the loss of the hydrogen atom from the 3-hydroxyl group.

5 a
al N

3
li-4 0

td

LORAZEPAM

49 0

Hagel et a1 (17) have studied the nonaqueous titration of lorazepam with tetrabutylammonium hydroxide and perchloric acid. Their findings indicate that protonation does occur at the N-4 position. However, they propose that the deprotonation occurs at the N-1 position rather than at the 3-substituent. Table V X-Ray Powder Diffraction Pattern d 13.5 7.31 6.71 5.68 5.47 4.96 4.86 4.56 4.46 4.40 4.10 3.99 3.87 3.83 3.72
__

I/I ,

d 3.65 3.54 3.44 3.38 3.23 3.12 3.02 2.97 2.88 2.82 2.77 2.66 2.36 2.33 .31 1 .oo .18 .13 .40 .20 .23 .29 .07 .15 .17 .10 .10 .ll

.46 .26 .20

.10

.29 .79 .60 .53 .36 .45 .19 .33 .42 .12 .07

2.10 Protein Binding The protein binding of lorazepam and other benzodiazepines have been studied extensively by Mueller and Wollert (18-24)using circular dichroism and gel filtration techniques. The binding to albumin is decreased by the addition o f chlorine in the ring 2' position as evidenced by the fact that oxazepam binding is greater than that o f lorazepam. The binding is relatively independent of pH (pH 6.60 to 8.20).

Synthesis One synthetic route for lorazepam is shown in Figure 8 beginning with 2-amino-2',5-dichlorobenzophenone (I). The benzophenone is first converted to its oxime (11) with

3.

410

JAY G. RUTGERS AND CHARLES M. SHEARER

a CI

'c=q
H R

2 NHZOH HCI . -.

CI

'
N

Z C=NOH \R

R'

2-AMINO-Z', 5-DICHLOROBENZOPHENONE

2-AMINO-Z', 5-D ICHLOROBENZOPHENONE OX IME

6-CHCORO-Z-CHLOROMETHYL-4(Z-CHLOROPHENYL)-QUINAZOLlNE 3-OX IDE

3-ACETOXY -7-CHLORO-S-(Z-CHLOROPHENYL) 1, 3-DIHYDRO-2H-1, 4-BENZODIAZEPIN-PONE

7-CHLORO-5-(2-CHLOROPHENYL) 1,3-DlHYDRO-ZH-l, 4-BENZOD IAZEPIN-2ONE 4-OXIDE

I! s

R= R LORAZEPAM

bC1

!l l

Figure 8

Synthesis of Lorazepam

LORAZEPAM

41 1

hydroxylamine. Reaction of the oxime with chloroacetyl chloride produces (111) the quinazoline 3-oxide ( 2 5 ) . Ring enlargement to the benzodiazepin-2-one &-oxide (IV) is accomplished by treatment with sodium hydroxide ( 6 . 2) Reaction with acetic anhydride and subsequent hydrolysis of the ester (V) with base produces (VI) lorazepam (8,27). A variation of this procedure is to react (IV) with isopropenyl acetate to form (V) which is hydrolyzed with base to produce lorazepam ( 8 . 2) In another synthetic procedure the benzophenone antioxime is reacted with 2,2-diacetoxyacetyl chloride to produce a dihydroxyacetanilide derivative. This intermediate is cyclized with base and then hydrogenated to yield lorazepam
(29).

Stability and Degradation Lorazepam can lose a molecule of water and rearrange to form 6-chloro-4-(~-chlorophenyl)-2-quinazolinecarboxaldehyde (0. 3)

4 .

This quinazolinecarboxaldehyde can disproportionate and be oxidized orreducedto form the corresponding quinazolinecarboxylic acid or quinazoline alcohol respectively.

412

JAY G . RUTGERS A N D CHARLES M. SHEARER

cl%cl

*YCozH
\

", '
\

Acid hydrolysis of lorazepam ultimately produces 2-amino-2',5-dichlorobenzophenone which is the basis for numerous GLC, TLC and colorimetric analyses of lorazepam. In base lorazepam rearranges ( 3 1 ) to 7-chloro-5-(2-chlorophenyl~-4,5-d~hydro-2H-l,4-benzod~azepin-Z,3~lH)-dione. -

5. Metabolism and Pharmacokinetics

5.1 Metabolism The metabolites of lorazepam which have been characterized in human and animal studies are shown in Figure 9. In man the major metabolite is the glucuronide ( 3 2 , 3 3 , 3 4 ) . I4C labeled studies have shown that 88% of the administered radioactivity was recovered in the urine and 7% in the stool. The glucuronide comprised 86% of the urinary activity. Minor metabolites are II,V,VI and VII. Characterization of the metabolites was made by mass spectrometry and by thin-

CI @co / JoH f

CI @yCOOH

LORAZEPAM GLUCURONIDE Ill

HYDROXYLORAZEPAM Ill1

~-CHLORO-~-(O-CHLOROPHENY Ll2-QUINAZOLINECARBOXYLIC ACID IV)

(VII

CI &O HYDROXYMETHOXYLORAZEPAM I 1 I 11 LORAZEPAM DIHYDRODIOL f I V I

CI

mNH2
c=o

,HCOH CI OR

6-CHLORO-4-lo-CHLOROPHENYLl-2~lHlQU INAZOLINE IVII)

2-AMINO-2'. 5 - 0 ICHLOROBENZOPHENONE IVIIII

LORAZEPAM D IHY DROD IOL [ IVI

LORAZEPAM DIHYDRODIOL I I V I

Figure 9-

M t a b o l i t e s of Lorazepam

414

JAY G. RUTGERS AND CHARLES M. SHEARER

layer chromatographic comparison to authentic samples. The glucuronide of lorazepam, in particular, has been characterized extensively by chemical analysis and infrared and mass spectroscopy of the trimethylsilyl derivative (35). The glucuronide of lorazepam is also the major metabolite in miniature swine, dogs and cats (32,33,36,37). Minor metabolites in these species are II,III,V,VII and VIII. The metabolic transformation in rats is quite different from that in other animals investigated. Significant amounts of metabolite are found in plasma, bile and tissue. Compounds II,III,IV,V and VII have been identified as metabolic products (37).
A review of the metabolism of lorazepam has been written by Elliot (38).

5.2 Pharmacokinetics The pharmacokinetics of lorazepam has been studied by Greenblatt et a l . (33). A 2 mg oral dose of I 4 C lorazepam was administered to eight male adults. Blood samples were collected for a period of 96 hours and urine and feces samples 120 hours after administration. The various fractions were examined by means of a liquid scintillation spectrometer and gas chromatography. Data obtained on pooled plasma samples indicate that there is a lag time of about 35 minutes before the beginning of absorption. The apparent half-life of the absorption process is about 15 minutes for free lorazepam and 39 minutes for the glucuronide conjugate. Maximum plasma levels observed were 16.9 mg/ml for free lorazepam at 2 hours and 29.9 mg/ml for the conjugate at 4 hours. The apparent elimination half-lives are approximately 12 and 16 hours respectively. 88% of the total radioactivity administered was eventually recovered in the urine predominantly in the form of the conjugate. AR additional 7% was recovered in the stool. Identity Kuhrent-Brandstaetter (4 ) has described several qualitative tests based on melting point or formation of color which can be used to identify lorazepam. A sample warmed in a phenylhydrazine solution forms crystals slowly when cooled. The crystals melt at 88-92OC. Upon continued heating the melt recrystallizes to orange-yellow crystals which remelt at 6.

LOR A ZE PA M

415

205-207OC. Heating a mixture of lorazepam and benzidine to the melting point produces an orange-brown melt. A method has been developed for the detection of lorazepam in urine (39). A urine sample is extracted with ether and the ether extract examined under longwave UV light. A blue fluorescence due to the quinazolinone metabolite is indicative of lorazepam. The residue from the ether extract is then heated in 6N hydrochloric acid to produce the benzephenone derivative. A blue color developed with BrattonMarshall reagent is a l s o indicative of lorazepam. However, the test is not specific for lorazepam. Tetrazepam is reported to give the same positive tests. Infrared spectroscopy can be used directly on the drug substance for its identification.
7.

Methods of Analysis 7.1

Elemental Analysis The elemental analysis of lorazepam (Wyeth Reference Standard, Lot C-10684) is presented below.

Elemen t
C H N c1

% Calculated

% Reported (7)

56.10 3.14 8.72 22.08

56.05 2.99 8.65 21.77

7.2 Phase Solubility Analysis Phase solubility analysis (9 ) on lorazepam (Wyeth Reference Standard, Lot C-10684) using isopropanol as the solvent gave a purity of 99.8 f 0.2%. Direct Spectrophotometric Analysis Seitzinger ( 3 ) has described an ultraviolet spectrophotometric method for the analysis of lorazepam in tablets. A sample equivalent to 5 mg of lorazepam is weighed into a 100-ml volumetric flask, 50 ml of alcohol is added and warmed in a steam bath. After cooling, the sample is diluted to volume with alcohol. The sample is filtered and a 10.0 ml aliquot of the filtrate diluted to 100 ml with alcohol. The absorbance is determined at 228 nm using alcohol as a blank and compared with the absorbance of a standard solution of lorazepam. 7.3

416

JAY G. RUTGERS AND CHARLES M. SHEARER

The adaption of the spectrophotometric method to automated analysis has been reported (40). 7.4 Colorimetric Analysis Lorazepam can be hydrolyzed with hydrochloric acid to form 2-amino-2',5-dichlorobenzophenone. The aromatic amine is diazotized with nitrous acid and the diazonium salt coupled to N-(1-naphthy1)ethylenediamine. The absorbance of the resulting colored solution i s determined at 5 5 5 nm. A standard lorazepam solution is subjected to the same reactions for comparison. The procedure was applied to several tablet dosage forms of lorazepam. The tablets were extracted initially with chloroform and a portion of the chloroform extract evaporated for color development. Results obtained by the colorimetric procedure were in good agreement with those obtained by the spectrophotometric methods (3). Polarographic Analysis Lorazepam is reducible at the dropping mercury elec trode over a wide pH range. In the pH range of 0 to 6 a well defined wave is obtained. Above pH 6 the wave becomes strongly affected by absorption of the reducible species on the mercury electrode resulting in a distorted wave (41). The optimum pH range for analytical applications is considered to be 3 to 6 . The diffusion current is linear with concentration in the range of to 10-4M (16). Several analytical procedures for lorazepam tablet dosage forms employing methanolic acetate buffer (pH5) have been reported (16,421. The procedure can be adapted to differential pulse polarography(43,44,45). The polarographic technique has also been adapted to automated analysis by interfacing a polarographic analyzer with a continuous flow system (46,471. Polarographic analysis is stability indicating for the major route of degradation ( 3 0 ) . Oelschlager (48) has investigated the reduction o f lorazepam and found that it consumes four electrons in three steps to form 7-chloro-5-(q-chlorophenyl)-l,3,4,5-tetrahydro-2H-1,4-benzodiazepin-2-one. The first step in the postulated mechanism is the reduction of the 4,f-N=C double bond with the consumption of two electrons. Water is eliminated with the formation of the aldimine. The aldimine is subsequently reduced with the consumption of two additional electrons.
7.5

LORAZEPAM

417

7.6 .-Titrimetric Analysis The tetrabutylammonium hydroxide titration procedure for oxazepam (NF-XIV, 1975) was shown to be applicable to the titration of lorazepam. The titration is considered to proceed through the deprotonation at the N-1 position. Titration of lorazepam with perchloric acid in glacial acetic acid resulted in poorly defined potential breaks (17). 7.7 Chromatographic Analysis 7.71 Thin-Layer Chromatography Lorazepam may be evaluated on a thin-layer plate as the intact drug or, frequently, as the acid hydrolysis product, 2-chloro-2',5-dichlorobenzophenone. There are certain cases where it is advantageous to develop lorazepam as its hydrolysis product. Conversion to the benzophenone may be achieved by hydrolyzing in solution before spotting (49) or hydrolyzing directly on the plate after spotting (50). One method of detection is also based on conversion of lorazepam to the benzophenone after development ( 3 2 ) . The various solvent systems used for thinlayer chromatography of lorazepam on silica gel plates are given in Table VI. The table indicates those cases where the material was developed as the benzophenone. Table VII lists the methods of detection used for lorazepam on thinlayer chromatograms. 7.72 Gas Gas metabolic and technique can the low doses Chromatography chromatography has been used extensively in pharmacological studies of lorazepam. This provide the sensitivity which is required for usually administered.

Lorazepam is not thermally stable. A number of investigations (55-58) have shown that under gas chromatographic conditions lorazepam can lose a water molecule and rearrange to form 6-chloro-4-( 2'-chlorophenylquinazoline) -2-carboxaldehyde. Consequently, in any gas chromatographic procedure where lorazepam is injected directly this rearrangement must be considered. Another consideration is that in metabolic studies the major metabolite is excreted as the glucuronide and a preliminary enzymatic incubation i s usually employed. However, Marucci (59) was able to chromatograph the glucuronide directly by preparation of a derivative. The conjugate was first reacted with diazomethane to methylate the uronic acid carboxyl group and also the N position. The methyl derivative was then silylated with hexamethyldisilazane. The mass spectrum was consistent with a dimethyltrisilyl derivative. The procedure was

Table VI Thin Layer Chromatopraphy for Lorazepam Solvent Benzene Chloroform-AcetoneDiethylamine ( 5 0 : 50: 10) Hexane - 25% diethylamine in ethanol (75: 2 5 )
e

Rf x 100 46(as benzophenone) Rm=80

(vs. meprobamate)

Application Separation of oxazepam and lorazepam Identification of drugs in biological media

11

Reference
49
52

Rm=5 9 (vs. nitrazepam) Rm=5 (vs. thioridazine)

33

52

To1uene-acetone
(80:20)(Tank contains ammonia vapor) Chloroform-ethanolacetone ( :1: 1 8 )

II

52

Netabolic studies
I1
11

32

E thy1 acetate-ethano1conc. ammonia ( 5 : 5: 1 )

63

32

Chloroform-methanol (lo: 1) Benzene Heptane-chloroformethanol ( 5 0 : 5 0 : 5 )

36
41 11

Identity in tablets Separation of 1,4-benzodiazepines Separation of 1-4-benzodiazepines

53
51

Table VI (continued) Solvent 1,2Ethyl acetate dichloroethane - 25% amnonium hydroxide

Rf x 100 25

Application Separation of 1-4-benzodiazepines

Reference

51

(80:20:5)

Ethyl acetate-ethanol25% ammonium hydroxide (50: 50: 10) Heptane-chloroform-ethanol (50:50: 10)
,P a

61

11

51

24

II

51

1,2-dichloro- 20 Ethyl acetate ethane 25% ammonium hydroxide ( 0 20:10) 8:

II

51

Cyclohexane-ethyl acetateethanol - 25% ammonia (20:20: 7:O.l)

42 (as benzophenone)

Identification of 1,4-benzodiazepines in urine

54

420

J A Y G. RUTGERS A N D CHARLES M . SHEARER

Table V I I

TLC D e t e c t i o n Methods f o r Lorazepam

Method Quenching of Phosphorescence on a phosphorescent p l a t e under shortwave W light Conc. HCl s p r a y , h e a t , followed by Bratton-Marshall spray Conc. I.r, S , O, s p r a y , observe under longwave W light Mercuric c h l o r i d e diphenylamine spray

Color
Dark s p o t a g a i n s t a green background

Detect ion L i m i t (pg)

Reference

0.1

B 1ue - v i o le t

0.02

Green

0.01

51

Blue

52

Data n o t a v a i l a b l e

LORAZEPAM

42 1

applied to glucuronide levels in urine from humans and animals. An alternate technique is conversion of lorazepam to its benzophenone derivative prior to injection. An example of this is the procedure developed by Knowles et al. ( 6 0 ) for determination of free and conjugated lorazepam in serum,urine and feces. The biological sample is adjusted to pH 7 and extracted with ether to remove free lorazepam. The aqueous phase is adjusted to pH 4 . 5 and incubated overnight with$ -glucuronidase to cleave the conjugate. The aqueous phase is again adjusted to pH 7 and extracted with ether. Lorazepam is re-extracted into 12N sulfuric acid and then heated at looo to form the benzophenone. The samples are dissolved in toluene prior to analysis. The limit of detection was 0.01 ug/ml. The conditions for various methods are given in Table V I I I .
7.73 High Performance Liquid Chromatography

Gonnet ( 6 4 ) has developed an isocratic elution technique for separating lorazepam from a series of other benzodiazepines (medazepam, diazepam, nitrazapam, chloazepate, oxazepam and chlordiazepoxide). Separations were achieved on a 20 cm x 4 . 6 mm column packed with Lichrosorb SI 6 0 , 5 micron, at a pressure of 1090 psi and a flow rate of 2 . 6 ml/min. The mobile phase was dichloromethane-isopropanol ( 9 6 : 4 ) saturated with water. de Silva (65) discusses the high performance liquid chromatography of lorazepam and other benzodiazepines. A reverse phase column (a Bondapak C - 1 8 ) was used with an eluant consisting of methanol (500 ml); isopropanol (50 ml); pH 3.25 1 M potassium phosphate buffer (1 ml): diluted to 1000 ml with distilled, deionized water. A normal phase system ( 1 0 Partisil silica gel) had an eluant of methylene ~ chloride (95):methanol (5). The reverse phase column was the better. Lorazepam can be separated (12) from its degradation products (see Section 4 ) by an eluant consisting of acidic aqueous acetonitrile on a reverse phase column. High performance liquid chromatography has been used to analyze for benzodiazepines including lorazepam in tissue ( 6 6 ) . An eluant of 60% methanol (v/v) in phosphate buffer (pH 7.8) eluted lorazepam in 4 . 2 ml from a column of Spherosorb-5-ODS (150 mm x 4 . 6 mm id).

Table V I I I Gas Chromatographic Systems for Lorazepam Injected As Lor azepam Lor a zepam Column Packing umn l m x 2mm glass 4 ft x 4mm boros i licate glass ColCarrier Gas Nitrogen a t 42 ml/min Argon-methane (90: 10) 120 ml/min Helium 30 ml/min Argon-me t hane (90: 10) 75 ml/min
A r gon-me thane (90: 10) 75 ml/min

Column Temp. 255C 240C

or -

Detect

Ref. -

3% O V 1 7 on Gas Chrom Q 3% O V 1 7 on Gas Chrom Q ( 60/80)

Electron capture Electron capture Mass Spectrometer Electron capture Electron capture

55
61

e
N

Lor a z e pam

3% O V 1 7 on Gas Chrom Q (100-120)

3% O V 1 7 on Gas Chrom Q ( 60/80) 3% OV17 on Gas Chrom Q (100-120)

2m x Pmm

280C

56

Lor a ze Pam

4 f t x 4mm borosilicate glass

230,
210%

62

Trimethyls i l y l deriv a t i v e of Lor a zepam

4 f t x 4rnm boros i licate glass

2 10"C

62

Table V I I L ( c o n t i n u e d ) Injected As
M e t h y 1t r i methyls i l y l deriv a t i v e of Lorazepam glucuronide

Column Packinq

l m glass

Column

Carrier Gas Nitrogen 30 ml/min

Column Temp. 320C

Detector Flame ionization

Ref. -

3% OV17 on G a s Chrom Q (100-120)

59

Benzophenone
w N

3% O V 1 7 on Chromosorb W (80/100)
SE 30

10 f t x 2im s t a i n less s t e e l
2 glass m

Helium 20 ml/min Nitrogen 40 ml/min

2sooc
240" C
24OOC

E l e c t r on capture
F1 m e a ionization Electron capture

60

Benzophenone Benxophenone

53

6% QF-1 on Anakrom ABS (80/90)

6 ft x 1/8"
stainless steel 2 x 4mm m glass

H e 1ium 40 ml/min

34

Lorazepam

3% OV-7 on Varaport 30 80/100

Argon 80 ml/min

25OOC

Electron capture

63

424

JAY G. RUTGERS A N D CHARLES M. SHEARER

8 References .
1.

2 .

3.
5.

4 .

6 .
7 .

8 .
9 .

1. 0
11.

12.

13.
14. 15. 16. 17. 18.
1. 9 20. 21. 22. 23.

24.

Bellmy, L. J., "The Infra-Red Spectra of Complex Molecules'1,2nd Ed., John Wiley & Sons, Inc., New York 1964. Hoffman, B., Wyeth Laboratories, Inc., personal communication. Seitzinger, R.W.T., Pharm. Weekbl., 110,1109 (1975). Kuhnert-Brandstaetter, M., Kofler, A., and Friednich-Sander, G., Sci. Pharm., 42, 234 (1974). Barrett, J., Smyth, W.F., and Davidzn, I.E., J. Pharm. Pharmac., 2, 387 (1973). Levillain, P., Bertucat, M . , and Perrot, B., Eur. J. Med. Chem.-Chem. Therapeutica, 10,433 (1975). Sellstedt, J., Wyeth Laboratories, Inc., personal communic ation. Bell, S.C., McCaully, R.J., Gochman, C., Childress, S.J., and Gluckman, M.I., J. Med. Chem., 2, 457 (1.968). DeAngelis, N.J., Wyeth Laboratories, Inc., personal communic ation. Sivieri, L., Wyeth Laboratories, Inc., personal communication. Kyriakopoulos, A.A., in "Pharmacokinetics of Psychoactive Drugs", Edited by Gottschalk, L A .. and Merlis, S., John Wiley and Sons, New York 1976. Snodgrass-Pilla, C., Wyeth Laboratories, Inc., personal communication. Bissinger, J.M., Wyeth Laboratories, Inc., personal communication. Bandoli, G . and Clemente, D.A., J. Chem. SOC., Perkin Trans., 11,413 (1976). Davidson, I E . . and Smyth, W.F., Proc. SOC. Anal. Chem., 9, 209 (1972). Goldsmith, J.A., Jenkins, H.A., Grant, J., and Smyth, W.F., Anal. Chem. Acta, 66, 427 (1973). Hagel, R B .. and DebesisTE.M., ibid., 78,439 (1975). Mueller, W and Wollert, U , Arch. Pharmacol., 278, . . 301 (1973). . 229 (1973). Mueller, W and Wollert, U., ibid.,=, Mueller, W and Wollert, U., ibid.,=, . R68 (1974). . Mueller, W and Wollert, U., ibid.,283, 6 7 (1974). 17 (1975). . Mueller, W and Wollert, U., ibid.,=, Mueller, W and Wollert, U., Biochem. Pharmacol., 25, . 141 (1976). Mueller, W. and Wollert, U., ibid.,%, 147 (1976).

LORAZEPAM

425

25.
26. 27. 28. 29. 30.

31.
32.

33.

34.
35. 36.

37.
38 39.

40.

41.
42.

43. 44.
45. 46.

47.

S t e r n b a c h , L.H., K a i s e r S., and Reeder, E., J. Am. Chem. SOC . , 82, 475 (1960). B e l l , S.C., Sulkowski, T.S., Gochman, C., and C h i l d r e s s , S.J., J. Org. Chem., 27, 562 (1962). B e l l , S.C. and C h i l d r e s s , S.J., i b i d . , - 1691 27, ( 1962). B e l l , S.C., U.S. P a t e n t 3,296,249, Jan. 3, 1967. McCaully, R.J., U.S. P a t e n t 3,926,952, Dec. 16, 1975. Wyeth L a b o r a t o r i e s , I n c . , unpublished R u t g e r s , J.G., results. Nudelman, A . , McCaully, R.J., and B e l l , S.C., J. Pharm. S c i . , 2, 1880 (1974). S c h i l l i n g s , R.T., S h r a d e r , S.R., and R u e l i u s , H.M., Arzneim.-Forsch., 21, 1059 (1971). Greenblatt, D.J., S c h i l l i n g s , R.T., Kyriakopoulos, A.A., Shader, R . I . , Sisenwine, S.F., Knowles, J.A., and R u e l i u s , H.W., Clin. Pharmacol. Ther., 329 (1976). E l l i o t , H.W., Nomof, N., Navarro, G., R u e l i u s , H.W., i b i d . , - 468 12, Knowles, J.A., and Comer, H.W., (1971). Chang, T.T.L., Kuhlman, C.F., S c h i l l i n g , R.T., Sisenwine, S.F., T i o , C.P., and R u e l i u s , H.W., E x p e r i e n t i a , 2, 653 (1973). Sisenwine, S.F., Schwartz, M.H., S c h i l l i n g s , R.T., and R u e l i u s , H.W., Drug Metabolism and D i s p o s i t i o n , -, 85 (1975). 3 S c h i l l i n g s , R.T., Sisenwine, S.F., and R u e l i u s , H.W., ibid., 5, 425 (1977). E l l i o t , H.W., B r . J. Anesth., - 1017 (1976). 48, Lafarque, P., J. Eur. Toxicol., 6, 136 (1973). C u l l e n , L.F., S c h l e i f e r , A . , B r i n d l e , M.P., and Anal. Chem., - 1936 (1975). 47, P a p a r i e l l o , G.J., C l i f f o r d , J.M. and Smyth, W.F., Z. Anal. Chem., &, 149 ( 1 9 7 3 ) . O e l s c h l a g e r , H. and Sengun, F.I., Pharmazie, 770 (1974). d e S i l v a , J.A.F., Bekersky, I., and Brooks, M.A., J. Pharm. S c i . , 1943 (1974). Van Doorne, P . , Pharm. Weekbl., 149 (1975). and Tan, S.B., Smyth, W.F., Smyth, M.R., Groves, J.A., A n a l y s t , log, 4 9 7 ( 1 9 7 8 ) . C u l l e n , L.F., B r i n d l e , M.P., and P a p a r i e l l o , G.J., J. Pharm. S c i . , 62, 1708 (1973). C u l l e n , L.F., B r i n d l e , M.P., and P a p a r i e l l o , G . J . , Adv. Autom. Anal., Technicon I n t . Congress, 2, 9 (1972).

20,

2,

2,

110,

426

J A Y G. RUTGERS A N D CHARLES M. SHEARER

48.

O e l s c h l a g e r , H. and Sengun, F.I., Chem. Ber., 3303 (1975). 183 S c h u l t z , C. and S c h u l t z , H., Arch. Tox., 49. (1973). 50. S c h u l t z , C. and S c h u l t z , H., Z. K l i n . Chem. Klin. Biochem. , l0, 528 (1972). Dreijer-Van Der Glas, S.M., and 51. Meeles, M.T.H.A., Kho, G.L., Pharm. Weekbl., 492 (1975). 52. F l o u v a t , B., Roux, A., I r o n d e l l e , B., and Sanchez A . , Eur. J. T o x i c o l . , 8, 305 (1975). Arch. T o x i c o l . , 32, 341 53. Maier, R.D. and Wehr, K.H., (1974). , Pharm. Weekbl. , 54. Krugers Dagneaux, P.G.L.C. 1025 (1973). 55. Marucci, F., M u s s i n i , E . , A i r o l d i , L., G u a t a n i , A., G a r a t t i n i , S., J. Pharm. Pharmacol., &, 6 3 (1972). and B e l v e d e r e , G., Anal. 56. F r i g e r i o , A . , Baker, K.M., Chem. , 45, 1846 (1973). 57. Forgione, A., F r i g e r i o , A., M a r t e l l i , P., Proc. I n t . Symp. Gas Chromatogr. Mass Spectrom., 213 (1972). 709 (1972). 58. F r i g e r i o , A., Boll. Chim. Farm., 59. Marucci, F. , B i a n c h i , R. , A i r o l d i , L. , Salmona, M., F a n e l l i , R., Chiabrando, C., F r i g e r i o , A . , M u s s i n i , E. , and G a r a t t i n i , S. , J. Chromatogr. , 1 0 7 , 2 8 5 (1975). Comer, W.H., and R u e l i u s , H.W., 60. Knowles, J.A., Arzneim.-Forsch. , 2 l , 1055 (1971). Bekersky, I . , and P u g l i s i , C.V., 61. d e S i l v a , J.A.F., J. Chromatogr. S c i . , 547 (1973). Bekersky, I . , P u g l i s i , C.V., Brooks, 62. d e S i l v a , J.A.F., M.A., and Weinfeld, R.E., Anal. Chem., 10 (1976). 439 ( 1 9 7 7 ) 63. R u t h e r f o r d , D.M. , J. Chromatogr., ibid., 419 (1976). 64. Gonnet, C., and Rocca, J.L., S t r o j n g , N . , and P u g l i s i , C.V., 65. d e S i l v a , J.A.F., Anal. L e t t e r s , 135 (1978). Hammond, M.D., and T w i t c h e t t , P.J., 66. O s s e l t e n , M.D., J. Pharm. Pharmacol., 29, 460 (1977).

z,

108,

110,

108,

111,

11,

48, 137, 120,

g,

METHOXSALEN
Mohammed A . Lou& and Mahmoud A . Hassan
1.

2.

3.

4.
5. 6. 7.

8.

Description 1.1. Nomenclature 1.2 Formulae 1.3 Molecular Weight I . 4 Elemental Composition 1.5 Appearance, Color, Taste, Odor Physical Properties 2.1 Crystal Properties 2.2 Solubility 2.3 Identification 2.4 Spectral Properties Isolation Biosynthesis Synthesis Metabolism Methods of Analysis 7.1 Colorimetry 7.2 Spectrophotometry 7.3 Fluorimetry 7.4 TLC-Fluonmetry 7.5 Time- resolved Phosphorimetry 7.6 Electrophoresis 7.7 Chromatography 7.8 PMR Spectrometry References

428 428 428 429 429 429 429 429 429 430 430 435 437 437
440 440 440

441 441 441 442 442 443 447 450

AnaIyTicfd Rofiles of Drug Substances, 9

427

Copyright @ 1980 by Academic Rcss, Inc. AU rights of reproduction in any form rexrved. ISBN: 0-12-260809-7

428

MOHAMMED A . LOUTFY A N D MAHMOUD A . HASSAN

1.

Description 1.1 Nomenclature

1.11

Chemical Names 9-Methoxy-7H-furo 13 ,3-gl 111 benzopyran-?-one; 6 -Lactone of 6-hydroxy-7-methoxy-5-benzofuranacryJi: acid; 8-Methoxy [ furano-3,2, : 6,7 - coumarin]; 8-Methoxy-4, 5 : ' 6,7-furanocoumar in.

1.12

Generic Names Ammoidin,Xanthotoxin, 8-Methoxypsoralen, Methoxsalen.

1.13

Trade Names Meladinin, Meloxine, 8-MOPr 8-MP, Methoxa-Dome, New-Meladinin, Oxsoralen.

1.2

Formulae 1.21 Empirical C12H804 1.22 Structural

METHOXSALEN

429

1.23

Wiswesser Line Notation T C566 DO LV OJ BOI

1.3

Molecular Weight 216.18

1.4

Elemental Composition
C,

66.67%; H, 3.73%; 0, 29.60%.

1.5

Appearance, Color, Taste, odor Silky fluffy needles or long rhombic prisms, white to cream-colored, bitter taste followed by tingling sensation, odorless.

2.

Physical Properties
2.1

Crystal Properties 2.11 X-Ray Diffraction The crystallographic properties of methoxsalen has been reported(1). 2.12 Melting Point Methoxsalen melts between 143 and 148O(2).

2.2

Solubilitv Methoxsalen is practically insoluble in cold water, sparingly soluble in boiling water and freely soluble in chloroform. It is soluble in boiling alcohol, acetone, and acetic acid. It is also soluble in aqueous alkalis with ring cleavage, reconstitution occurs upon neutralisation.

430

MOHAMMED A. LOUTFY AND MAHMOUD A . HASSAN

OCH3

OCH3

2.3

Identification a- An alcoholic solution of methoxsalen gives a deep-violet color with 8amino-5-hydroxy-2-methyl-furo-4; 5 6, 7 - chromone, in the presence of alkali (3). The test is sensitive to 0.02 mg of the drug. b- The UV absorption spectrum of a 1 in 125,000 solution in alcohol exhibits maxima and minima at the same wavelengths as that of a similar solution of USP Methoxsalen Reference Standard, concomitantly measured (4)

c- Dissolve, by heating, about 10 mg in 5 ml of diluted nitric acid, the solution turns yellow. Render the solution alkaline with sodium hydroxide, the solution turns brown (4). 2.4 SDectral ProDerties
2.41

Ultraviolet Spectrum Methoxsalen exhibits a characteristic UV Spectrum (Fig.l), in 95% ethanol, with the following electronic absorption bands ( 5 ) :

1Max

219 245 249 262 301

Log E 4.48 4.44s. 4.46 4 -23s. 4.16

A min
232 262 276 s.= Shoulder

Log E 4.23 4.23s. 3.90

METHOXSALEN

43 1

432

MOHAMMED A . LOUTFY A N D MAHMOUD A . HASSAN

Other UV spectral data for methoxsalen and other psoralens have been reported (6-9). 2.42 Infrared Spectrum The IR spectrum of methoxsalen has been determined in nujol on a Unicam SP - 200 (Fig.2). The structural assignments have been correlated with the following band frequencies: Frequency Cm-1 Assignment 3110, 3080, 3040 1705 1620 1580 , 1540 1150 875 800 CH C=O ( a-pyrone) C=C (aromatic and a-pyrone) C=C (aromatic) Furan ring Isolated H (penta substituted aromatic)

c-0-c

These findings are in agreement with reported data (5,9). Other finger print bands characteristic of methoxsalen are: 1400, 1380, 1340, 1300, 1220, 1180, 1120, 1100, 1020, 1000,820 and 760 cm-l 2.43 Nuclear Elagnetic Resonance Spectrum The proton magnetic resonance spectra of methoxsalen and other furocoumarins have been investigated (5,9,10,11). A typical PMR spectrum of methoxsalen is shown in Fig.3. The sample was dissolved in CDC13 and the spectrum was recorded on a T60A NMR spectrometer, using tetramethylsilane as a reference standard.

.
0 0 0 , r l

8.0

. .

. . I .
7.0

6.0

. ( . _ , I , I ,
5.0
4.0

I
L .

30 .

. . I .
2.0

I
,
,
,

PARTS PEIt M I L L I O N ,

6
TMS, tetrartgthylsilane

Fig. 3

- PMR swtm of Methoxsalen and

i deutrated chlorofom. n

METHOXSALEN

435

The PMR d a t a of m e t h o x s a l e n a n d i t s b i o l o g i c a l l y i n a c t i v e isomer, b e r g a p t e n ( 5 - m e t h o x y p s o r a l e n ) are i l l u s t r a t e d i n t a b l e I.

T a b l e I: PMR c h a r a c t e r i s t i c o f M e t h o x s a l e n and Bergapten.

Chemical s h i f t s ( 6 )
5-ocH 8 a 3 3-H 4-H sa3 s dk d

5-H s

8-H d

4'-H d

5'-H d

Methoxsalen

4.25

4.35 6.38 7.75 7.33

7.25

6.83

7.66

Bergap

ten

6.23 8.10

7.03

7.53

A l s o o t h e r PMR s t u d i e s o n metho-

x s a l e n and a n a l o g u e s have been published (5,9,10).

2.44

Mass S p e c t r u m
The mass s p e c t r u m of m e t h o x s a l e n , o b t a i n e d by c o n v e n t i o n a l e l e c t r o n i m p a c t i o n i z a t i o n , shows a molec u l a r i o n M+ a t m / e 216.04 (12,131. The M+ i o n p e a k i s t h e b a s e p e a k (Fig.4). The f r a g m e n t a t i o n p a t t e r n s of m e t h o x s a l e n a n d o t h e r p s o r a l e n d e r i v a t i v e s have been reported (5,13,14 1

3.

Isolation Fahmy and Abu-Shady ( 1 5 - 1 7 ) h a v e r e p o r t e d t h e i s o l a t i o n of m e t h o x s a l e n f r o m t h e f r u i t s o f E g y p t i a n , u m b e l l i f e r o u s Ammi r n a j u s ( L ) plant. The d r i e d powdered Ammi m a j u s ( L ) (Fam.

..
'0

-W
.N
-r+

~0

.N

--!
N 0

0 03
c

W
4

d
d

I
W

m I

2 0
L

i; i"

u )

N N
0
u )
4

'in

0 0 0 0 0 ' 5 -

o r n w w ~

METHOXSALEN

431

umbelliferae) fruits are exhaustively extracbd with petroleum ether (60-80). The deep green extract is concentrated and kept for overnight and the greenish resinous crystalline deposit is filtered and crystallised from ethanol. The yield is 0.25%. The isolation of methoxsalen and other furocoumarins have been also described by other co-workers (18-26). 4. Biosynthesis The exact biogenetic pathway leading to the formation of methoxsalen is uncertain. One of the problems still to be resolved concerns the mechanism by which ?near furocoumarins are isopropyl formed from their 2 , 3-dihydro-2counterparts (27). It seems certain that marmesin is directly involved and tracer experiments indicate that (+) - ( S ) - form, rather than the ( - 1 - ( R ) - (nodakenetin), is preferentially incorporated (28,29). The sequence of further substitution of furanocoumarins has - a1 been studied by Caporale, et - (30, 31). The most recent suggestion is that further substitution,hydroxylation followed by methylation, occurs after furan ring formation. Scheme I illustrates the formation of methoxsalen starting from trans-cinnamic acid.
5.

Synthesis The synthesis of methoxsalen has been achieved by two main routes. Route I: Benzofuraneroute (32-34), which involves the hydrogenation of 6,7-dihydroxybenzofuran (I) or 6,7-dihydroxycoumaran-3-one (11) to afford 6,7-dihydroxycoumaran (111) I11 was then converted, by Pechman? reaction with malic acid, to 8-hydroxy-4,5 -dihydro6,7,3,2-f~ran0~0~rnarin (IV). Methylation of IV followed by dehydrogenation gives methoxsalen ( V ) .

Route 11: Umbelliferore route (35), involves the use of a coumarin ring instead of a benzofuran derivative. Although it is an improvement on the previous route, it gives a low

438

MOHAMMED A. LOUTFY A N D MAHMOUD A. HASSAN

Scheme I.

Biosynthesis of methoxsalen from trans-cinnamic

acid.

HO

glucose

METHOXSALEN

439

Route I (Benzofuran route):

I1

OH

I11

OH

R o u t e I1 ( U m b e l l i f e r o n e r o u t e ) :

I1

BrCH2COOEt

HO
OCH3 OH

QCH3

RH2C nCH

v
R = COOEt

VI

440

MOHAMMED A. LOUTFY A N D MAHMOUD A . HASSAN

yield (33%).In this route, 7-hydroxy-8-methoxycoumarin (111) is the key intermediate for the synthesis of methoxsalen. 111 is converted into the 6-formyl derivative which in turn is cyclised, using ethyl bromoacetate to effect ring closure, to VII. This approach is attractive in that an umbelliferone is an intermediate rather than 6-hydroxycoumaran. Umbelliferones can be more easily synthesised and are more readily available from natural sources. Other methods for the synthesis of methoxsalen and analogues are also reported (19,36-39). 6. Metabolism Very little is known about the metabolism of photosensitising furocoumarins (40). By contrast no data are so far available about the metabolismof methoxsalen. However studies have been performed in mice and human volunteers on the absorption, metabolism and excretion of psoralen and trimethylpsoralen (41,42). 7. Methods of Analysis

7.1.

Colorimetry Colorimetric determination of methoxsalen in the Ammi majus fruits and tablet formulation has been described (43,44). The method involves the addition of 0.5M potassiumhydroxide solution to the drug, after 30 minutes, diazotized sulfanilic acid solution (a 1:l - mixture of 0.64% sulfanilic acid solution in dilute hydrochloric acid and 0.4% aqueous sodium nitrite) is added. The extinction of the solution is then measured and referred to acalbbration curve prepared by a standard methoxsalen solution. et a1 Schonberg, - - (3) have described another colorimetric procedure for the determination of methoxsalen and other psoralens. The method involves the addition of 1 ml of a 0.1% solution of 8-amino-5-hydroxy-2-methylfuro (5, 4, 6,7) chromone to 2 ml of an alcoholic solution of the psoralen. The reaction

METHOXSALEN

441

mixture is made alkaline with 10 ml of a buffer solution (pH 11.61, a violet color is gradually developed and can be measured. 7.2 Spectrophotometry Methoxsalen and bergapten have been determined in the plant material, by measuring the absorbance of the chloroform extract at 300 and 311 nm, respectively (45). Fedorin and Georgrievskii (46) have described a spectrophotometric procedure for the estimation of methoxsalen and bergapten in Beroxan preparations. Yeargers and Auqenstein (47) have described the absorption and emission spectra of methoxsalen and psoralen in powders and in solutions.

_ a1 Chakrabarti , et - (48 ) have developed a spectrophotometric method for the estimation of methoxsalen in the plasma. The method is based on extraction of the plasma, acidified with hydrochloric acid, with benzene-ethyl acetate mixture (9:l) and the solvent is evaporated, to dryness. The residue is dissolved in xylene and the absorbance is measured at 300 nm. The drug extracted from plasma is characterised by TLC, UV absorption spectrum and GC/MS fragmentation pattern. The U.S.P. method for the assay of methoxsalen is spectrophotometric (4) one.
7.3 Fluorimetry The assay of methoxsalen and bergapten in Beroxan preparations has been achieved fluorimetrically (46).

7.4

TLC

Fluorimetry

Chakrabarti, _ - (49) have described et a1 a rapid and sensitive method for the determination of methoxsalen in the plasma. The plasma samples are acidified with hydrochloric acid and heated in a boiling water-bath to release the plasma-

442

MOHAMMED A. LOUTFY AND MAHMOUD A. HASSAN

bound drug. The drug is extracted by a solvent system (benzene-ethylacetate, 9:l). The extract is evaporated to dryness and the residue is dissolved in methylene chloride and is spotted on thin layer plates and developed with the same solvent system. The plates are visualized under UV light (320-400 nm) and scanned. The method is sensitive up to 2 0 ng of methoxsalen. The overall recovery of the drug from the plasma is 84%. The identity of the recovered drug was confirmed by GC/MS (Fig.5). 7.5 Time-resolved phosphorimetry Phosphorescence spectra for methoxsalen and psgralen have been recorded for 300K and 77 K. The peaks for 300 fluoresceme excitation spectra obtained from a "front face" cell agreed with peaks in the absorption spectra, when correlations have been made for the output of the exciting lamp. At 770K the phosphorescence lifetimes vary from 0.4 to 1.1 seconds (47). 7.6 ElectroDhoresis Berlingozzi and Parrini ( 5 0 ) have described a method for separation of methoxsalen from other coumarin derivatives by circular paper electrophoresis.The compounds are first subjected to circular paper chromatography in water-acetic acidbutylene glycol (86:10:6). The Rf values found in strips, complete circle, and a 9 0 0 sector of circle, and the color of fluorescence in Wood's hight are, respectively 0.602, 0.779 and 0.786, light green colour for methoxsalen. The electrophoretic experiments are conducted in a buffer (pH9) of sodium barbital, sodium acetate, potassium oxalate,O.l N hydrochloric acid, and water. The travelling distances for methoxsalen and other coumarins are reported. A better separation has been obtained by means of electrophoresis than chromagography.

METHOXSALEN

443

7.7.

Chromatography
7.71

Paper chromatography Methoxsalen and other psoralens have been separated by paper chromatography. The following solvent systems have been used, in a unidimensional ascendinq method (51): water; water-methyethylketone (17:3 ) ; water-ethanolmethylethylketone ( 1 5 : 3 : 2 ) i waterformamide-methylethyl ketone water ( 8 : 5 : 3 ) and butanol-aceticacid-water ( 4 :1 :1)
(9:3:2));

formarnide-ethylacetate-

Grujie-Vasie- ( 5 2 ) has described a paper chromatographic separation of methoxsalen and some psoralens, using the following solvent systems: water-saturated ammonium hydroxide; butanol-ethanolconcentrated ammonium hydroxidewater ( 4 : 4 : 1 : 1 ) : and propanolwater ( 9 0 : 1 0 , 8 0 : 2 0 , 7 0 : 3 0 , and
20:80).

Beyrich ( 4 5 ) has reported a method for the quantitative determination of methoxsalen, bergapten, and imperatorin in the dried plant material. The powdered plant is extracted with chloroform in a Soxhlet, the extract is evaporated to dryness and the residue is dissolved in toluene. An aliquot (10-100 1 1 )is applied to a paper -1 imprignated with .dimethylformamide and developed with heptanebenzene mixture ( 4 : 2 ) . The separated methoxsalen is eluted with chloroform and determined spectrophotometrically. Heptane-benzene mixture ( 4 : l and 7 : 3 ) has been also used ( 5 3 , 5 4 ) on paper impriqnated with formamide, for the detection of methoxsalen and other

444

MOHAMMED A. LOUTFY A N D MAHMOUD A . HASSAN

psoralens. The detection has been achieved by their fluorescence alone, and after treatment with 0.5N ethanolic potassium hydroxide, by the diazoreaction, and by the use of Emerson phenazone-potassium ferricyanide reagent.

- - ( 4 3 ) have deset a1 Lutomski, cribed a paper chromatographic method for quantitation of methoxsalen in Ammi majus fruits and their preparations. The powdered sample is extracted with methanol in a Soxhlet and the extract is applied to Whatman No.1 paper, previously imprignated with dimethylformamide-ethanol mixture (2:3 ) . The chromatogram is then developed with heptane-benzene (7:3 ) by the descending-solvent technique. The spot of methoxsalen is eluted with ethanol and then determined colorimetrically.
__.____

7.72

Thin Layer Chromatography The isolation and detection of methoxsalen from other coumarins, by TLC methods, have been reported (55,56). The chromatoplates are prepared in the usual manner ( 5 7 ) using silica gel G as the adsorbant. Development has been carried out in different solvent systems (Table 11). The developed plates are dried and then observed under UV light (yellowish green). The plates are finally sprayed with a 0.5% Iodine-potassium iodide solution, the colors are observed in daylight (reddish brick-red) and under UV light, and the Rf values are determined (Table 11).

METHOXSALEN

445

Table I1 Solvent system a- Toluene-ethylformateformic acid ( 5 : 4 : 1) b- Benzene-ethylacetate ( 9 : 1 ) c- Benzene-acetone ( 9 : 1 )

Rf 0.65

0.39

0.71

A better resolution has been effected by two-dimensional chromatography on silica gel thin layers (Fig.61, and a l s o by the use of wedge-shaped ( 5 6 ) chromatogram (Fig.7).

7.73

Gas Liquid Chromatography Stewart, and Shyluck ( 5 8 ) have developed a GLC method for the separation of methoxsalen and certain coumarins. The relative retention time of methoxsalen, relative to herniarin, is 3 . 6 under the following conditions: SEG column of copper tubing ( 0 . 6 1 m X 5mm 0.d.) packed with succinate-ethyleneglycol polyester on a support of 6 0 - 8 0 mesh chromosorb W; column temperature, 208O; helium flow rate, 100 ml/min; injector temperature, 2 4 5 0 ; and recorder sensitivity, 1 mv.

7.74

High Performance Liquid Chromatoqraphy

A HPLC method has been reported ( 5 9 ) for the estimation of methoxsalen in the blood.
Another method for its determination in tablet dosage form has been also described ( 6 0 ) . It

446

MOHAMMED A. LOUTFY AND MAHMOUD A. HASSAN

Fig. 5
No. 114

Cursory Nos

m/e
4 Ib)

4 (a1

I ! m
c

n " '
A -

Methoxsalen (a and b ) .
Fig. 6

Fig. 7

M*sional TLC shminq a-Methoxsalen and b-&rgapten.

Wedge-shaped TLC showing, a-*thoxsalen and b-Bergapten.

METHOXSALEN

447

i n v o l v e s t h e u s e of a l o w volume p o s i t i v e d i s p l a c e m e n t pump, u n i v e r s a l i n j e c t o r , and a s i n g l e w a v e l e n g t h d e t e c t o r (254 nm) A uBondapak C18 s t a i n l e s s s t e e l column ( 3 . 8 mm x 3 0 c m ) i s u s e d . The column t e m p e r a t u r e i s a m b i e n t , t h e o p t i c a l d e n s i t y i s set a t 0.05 a . u . f . s . , t h e recorderis set a t 1 0 mv f u l l s c a l e , and t h e c h a r t s p e e d i s 0.25 c m p e r m i n u t e . The s o l v e n t ( m o b i l e p h a s e ) i s methanol-water m i x t u r e ( 6 0 : 4 0 ) , and t h e f l o w r a t e i s c o n t r o l l e d a t 2 m l p e r minute. A t y p i c a l chromatogram o f m e t h o x s a l e n , u s i n g Khellin a s an i n t e r n a l s t a n d a r d , i s shown i n F i g . 8 . The r e c o v e r y o f m e t h o x s a l e n h a s been f o u n d t o b e 86-95% of t h e s t a t e d amount.

7.8

PMR S D e c t r o m e t r v

L o u t f y and Hassan (11) h a v e rep o r t e d a r a p i d and s i m p l e PMR p r o c e d u r e f o r t h e e s t i m a t i o n of m e t h o x s a l e n i n b u l k d r u g and i n p h a r m a c e u t i c a l f o r m u l a t i o n s . The method i s b a s e d on t h e integration of t h e t h r e e methyl p r o t o n s s i n g l e t of t h e 8-methoxy g r o u p o f m e t h o x s a l e n a p p e a r i n g a t 4.35 ppn. Acetanilide, exihibiting three methyl p r o t o n s s i n g l e t a t 2.16 ppm, h a s been employed as a n i n t e r n a l s t a n d a r d ( F i g . 9 ) Ethan o l - f r e e c h l o r o f o r m h a s been u s e d a s a s o l v e n t i n t h e a s s a y . The method i s a c c u r a t e , w i t h a s t a n d a r d d e v i a t i o n of 2 2 . 4 , and a n a v e r a g e r e c o v e r y of 9 8 . 3 % , i n t h e t a b l e t d o s a g e form. Themethod proved t o be r e l i a b l e f o r t h e d e t e c t i o n of t h e b i o l o g i c a l l y i n a c t i v e isomer, b e r g a p t e n . T h i s i s a t t r i b u t e d t o t h e presence of 4 - H p r o t o n d o u b l e t of b e r g a p t e n a p p e a r i n g a t 8 . 1 ppm. ( F i g . 1 0 )

TMS

L
8C .

7.0

l . . I . . I . . . . I . . . . I . . . . 1 . . . . 1 . . . . 1 , . . . 6.0 5 0 PPM . 1 4.0 3.0 I2.0 $ 0

PARTS PER MILLION,

Fig. 9

- PMR Spectrum of Wthmsalen,

Acetanilide and TMS i CL?C13. n

449

450

MOHAMMED A . LOUTFY A N D MAHMOUD A . HASSAN

This finding has contributed greatly to the specificity of the met hod. 8. References

1.
2.

K. John, Anal. - - 389 (1951). - Chem., 23,

"Remington's Pharmaceutical Sciences" 15th ed., Mack Publishing Co., Easton, Pennsylvania, p. 726 (1975). J. - Am. A. Schonberg, N. Badran, and N.A. Starkowsky, Chem. SOC., 77,5438 (1955). -The United States Pharmacopeia, 19th Ed., Mack Publishing Co., Easton, Pa., p.317 (1975).
K. Lee & T.O. Soine, - - J. Pharm. Sci., 681 (1969).

3.

4.

5. 6. 7. 8. 9.

58,

G. Rodighiero and C. Antonello, Ann. - Chim. - 960 (1956). 46,

Chem., - -23,

M.E. Brokke and B.E. Christensen, - J. Org. 589 (1958 D.K. Chatterjee, R.M Chatterjee, and K. Sen, - - - Chem., - 2467 (1964). J. Org. 29,

A. Mustafa,"Furopyrans and Furopyrones", Interscience Publishers,John Wiley and Sons, (London), p.14 (1967). E.A. Abu-Mustafa and M.B.E. Fayez, Canad. 45, J. Chem. , - 325 (1967). - M.A. Loutfy and M.M.A. - 55 (1979). 12, Hassan, Spect. Lett.,

10.

11.
12.

E. Stenhagen, S. Abrahamsson, and F.W. McLafferty "Registry of Mass Spectral Data", Vol. 2, John Wiley and Sons Inc. , (N.Y. ) , p. 1172 (1974). C.S. Barnes and J.L. Occolowitz, Austral. 17, J. Chem. , - 975 (1964). - -

13.

METHOXSALEN

45 1

14.

E.A. Abu-Mustafa, F.K.A. El-Bay, and M.B.E. Fayez, - _ _ Trav. Chim. Pays-Bas, 87, 925 Rec. _ _ _ _ _ _ _ - (1968). I.R. Fahmy and H. Abu-Shady, Quart. J. Pharm. Pharmacol., - 281 (1947). 20, 21, Ibid, - 499 (1948). I.R. Fahmy, H. Abu-Shady, A. Schonberg, and A. Sina, Nature, 160, 468 (1947).

15. 16. 17. 18. 19. 20. 21.

A. Schonberg and A. Sina, J. Am. Chem. SOC.,. - 4826 (1950). 72,

M.A. Loutfy, M.M.A. Hassan, and H. Abu30 Shady,Pharmazie, -, 748 (1975)

E.Spath, - Ber., - 83 (1937). Chem. - 70A, F.M. Dean, "Fortschritte der Chemie Organischer Naturstoffe", Vol 9, Ed. L. Zechmeister, Wien Springer , Verlag , Austria, p.225 (1952). F.M. Dean, "Naturally occurring Oxygen Ring Compounds", Butterworth, London, p. 176 (1963).
L. Reppel, Pharmazie, - 278 (1954). 9, W. Karrer, "Konstitution und Vorkommen der Organischen Pflanzenstoffe", Birkenhauser, Verlag, Base1 und Stuttgart, p.531 (1958).
T.O. Soine, - Pharm. ~ , - 231 (1964). J. Sci. 53, -

22.

23. 24.

25. 26.
27.

S.M. Sethna and N.M. 1 (1945).

Shah, ~Chem. Rev.,

36,

A.I. Gray and F.G. Waterman, Phytochemistry, - 845 (1978). 17,

~Canad.

28.

S.A. Brown, M.El-Dakhakhny, and W. Steck, J. Biochem., - 863 (1970). 48,

452

MOHAMMED A. LOUTFY A N D MAHMOUD A . HASSAN

29. W. Steck and S.A. Brown, ~Canad. J. Biochem., 49., 1213 (1971).
c

30. G. Caporale, F. Dall' Acqua, A. Capozzi, S. Marciani, and R. Crocco, 2. Naturforsch. , 26B, 1256 (1971). 31. F. Dall' Acqua, G. Innocenti, and G. Caporale, Planta Medica, 27, 343 (1975). 32. E. Spath and M. Pailer, Chem. Ber., (1936).

--69,

767

33. E. Spath and T Vierhapper, Chem. Ber., . - 248 (1937). 70, 34. R.C. Esse and B.E. Christensen, J. Org. Chem., 25, 1565 (1960). - 35. G. Rodighiero and C. Antonello, - Ann. Chim. (Rome), 46, 960 (1956); Chem. Abstr., 51, 6616 (199). 36. C. Antonello, - Chim. - 88, 415 Gazz. - Ital., (1958); -- -, 20046 (L959). Chem. Abstr., 5 3 37. M.A. Loutfy, M.M.A. Hassan, A.I. Jado, and H. Abu-Shady, Pharmazie, - 819 (1976). 31, 38. T.R. Seshadri and M.S. Sood, Indian - 291 (1963). 1,

2,

Chem.

39. M.A. Loutfy and H. Abu-Shady, Pharmazie, 573 (1976). 40.

G . Rodighiero and F. Dall'Acqua, Phytochemistry and Photobiology, - (1976). 23

31,

41. M.A. Pathak, F. Dall'Acqua, G. Rodighiero, and J.A. Parrish, 3. Investig. Dermatol., - 347 (1974). 62, 42. M.A. Pathak, B.B. Mandula, J. A. Parrish, and T.B. Fitzpatrick, Commun. Presented at VII Inter. Congress on Photobiology, Rome, 1976.

METHOXSALEN

453

43. 44. 45. 46.

12, Herba Pol., -A.P.

J. Lutomski, H. Szostak, and H. Speichert; 200 (1966). Prokopenko and C.O. Torasenko, Farmatsevt. g.(Kiev), . , 18 (1962). 7 l

T. Beyrich, Pharmazie, - 700 (1964). 19, G.F. Fedorin and V.P. Georgievskii, Zh. Prikl. Spektrosk., 22, 1120 (1975) Anal. Abstr. 31, 4E57 (1976). E . Yeargers and L. Auqenstein, - Investiq. J. 44, Derxnatol, - 181 (1965).
S . G . Chakrabarti, D.A. Gooray, W.L. Ruff, and J.A. Kenney, - - - 1170 Clin. Chem., 23, (1977).

47. 48.

49. 50. 51. 52. 53. 54.

55.

S.G. Chakrabarti, D.A. Gooray, and J.A. Kenney, Clin. - - 1155 (1978). - Chem., 24,
S. Berlingozzi and V. Parrini, Sperimentale, 6 Sez. Chim. biol., -, 59 (1956). - - -

D.P. Chakrabarti, B.K. Barman, and K.R. Guha; Trans. Bose Res. Inst., 24, 211(1961).
J. Grujie
/

Vasie, Monatsh., - 236 (1961). 92,

T. Beryrich, - Chromat., J.

13, 181

(1964).

J. T. Beyrich and H. Poser, - Chromat., 87 (1968).

32,

E.A. Abu-Mustafa, B.A.H. El-Tawil, and M.B. E. Fayez, Phytochemistry, 3, 701 (1964).
F.M. Abdel-Hav, E.A. Abu-Mustafa, B.A.H. El-Tawil , and-M. B. E. Fayez , Planta Medica , - 91 (1965). 13,
G.

56.

57.
58.

E. Stahl , "Thin Layer Chromatography", Allen and Unwin Ltd., (London), 1969.

Anal. Chem., A.B. Stewart and J.P. Shyluck, - -, 1058 (1962). 34

454

MOHAMMED A. LOUTFY AND MAHMOUD A. HASSAN

59.

C.V. Puglisi, A.F. de Silva, and J.C. Yeyer; Anal. - - 39 (1977). - Lett., 10,

60.

A.H. Hikal, A.R.M. Morad, and S. P1-Houfy, Chromatoqraphia (in press).

NADOLOL
Lidia Slusarek and Klaus Florey
1.

2. 3.

4.

5. 6. 7. 8.

Introduction 1 . 1 History 1.2 Name, Formula, Molecular Weight 1.3 Appearance, Color, Odor Synthesis Physical Properties 3.1 Spectral Properties 3.2 Solid State Properties 3.3 Racemate Composition 3.4 Solution Data Analytical Tests and Methods 4.1 Elemental Analysis 4.2 Identification Tests 4.3 Spectrophotometric Analysis 4.4 Titrimetric Methods 4.5 Gas Chromatography/Mass Spectrometry 4.6 Chromatographic Methods Stability-Degradation 5.1 Solid State Stability 5.2 Solution Stability Analysis of Body Fluids Drug Metabolism References

456 456 456 456 456 459 459 470 47 5 416 477 477 477 477 478 479 479 480 480 48 1 48 1 482 483

Analytical Profiles of D u Substances, 9 rg

455

Copynght Q 1980 by Acackmic Fress. Inc. All nghts of reproduction m any form reserved. ISBN: 0-12-260809-7

456

LIDIA SLUSAREK AND KLAUS FLOREY

Introduction 1.1 History Nadolol, a "B-blocking" antiarrhythmic agent, was synthesized,' tested2 and developed in the laboratories of the Squibb Institute for Medical Research. Name, Formula, Molecular Weight Nadolol (SQ 11,725) is 2,3-cis-1,2,3,4tetrahydro-5-(2-hydroxy-3-(tert-butylam~no~propoxy)2,3-naphthalenediol; CAS:42200-33-9. 1.2

1.

CH3 I OCH -CH-CH2-NH-C-CH3

I

bH

CH 3

17H27N04 1.3

Molecular Weight 309.41

Appearance, Color, Odor Nadolol is a white to off-white crystalline, odorless powder. Synthesis The multistep synthesis of nadolol is presented in Figure 1: 5,8-Dihydronaphthol (1) via its acetyl derivative (2) is converted to cis-5,6,7,8tetrahydro-l16,7-naphthalene trio1 (3). This in turn is converted with or without intermediate formation of the acetonide (4) to 2,3-cisI 1,2,3,4tetrahydro-5-(2,3-epoxypropoxy)-2,3-naphthalene diol ( 5 ) which forms nadolol ( 6 ) on the addition of tertiary butylamine. For details, see reference 1. It can also be prepared by attaching t-butylamine to 5,8 dihydro-l-(2,3-epoxypropoxy) naphthalene (7) to form (8) and subsequent oxidation of the double bond and resolution to obtain nadolol ( 6 v f 4 2.

NADOLOL

451

Figure 1

Synthetic Pathways to Nadolol

OH

[wj
OCOCH3

(1)

r
L
(4)

OH
(3)

OH

HO
I

(5)

CH HO
I

OCH -CH-CH2-NH-C-CH3 2 1 I OH CH3

(6)

l 3

OCH2 -CH-CH2

\/

7 6 9 WAVELENGTH IMICRONSI

II

12

1 3

I5

Sq 11,725 Curve 70207

Mineral Oil Mu13

Figure 2.

IR Spectrum of Nadolol, Mineral Oil Mull. Instrument: Perkin Elmer Model 21

NADOLOL

459

Physical Properties 3.1 Spectral Properties 3.11 Infrared Spectrum The infrared spectrum of nadolol taken as a mineral oil mull is shown on Figure 2. The following assignments have been made for structurally significant bands:'
3.

Frequency (cm-l)
3280 1570 1260 1240) 1090) 1060

Assignment sharp 0 - H stretch broad OH stretch aromatic ring C = C stretch =c-0 stretch of aromatic ether C - 0 stretch of hyd roxy1s

3510

An infrared assay has been developed5 for the determination of the racemates of nadolol (Section 3.3). Nuclear Magnetic Resonance Spectra Figures 3 and 4 show the 100 MHz nuclear magnetic resonance spectra of nadolol and its corresponding D20 exchange in dimethyl sulfoxide - d ~ .The proton assignments are listed ~ in Table I.
3.12

Table I

Figure 3.

NMR Spectrum of Nadolol in DMSO-d6. Instrument: Internal Standard: Tetramethylsilane

Varian XL-100-15

NADOLOL

46 1

Table I (continued) Proton P o s i t i o n

T Value* ( C o u p l i n g C o n s t a n t , J , i n Hz)

6'; 7 7' 8

2 3 4 (3 protons) 5 6

6' '

3.3211 ( 8 . 0 Hz) 3 . 0 0 t (8.0 Hz) 3.24d ( 8 . 0 Hz) 5.53 6.75 7.28m 7.28m 6.15b 6.15b 8.97
b-broad

*d-doublet;

t-triplet; m-multiplet,

An i n s p e c t i o n o f t h e D20 e x c h a n g e s p e c t r u m ( F i g u r e 4 ) shows d i s a p p e a r a n c e of t w o r e s o n a n c e s a t ~ 5 . 5 3a n d ~ 6 . 7 5 . They c o r r e s p o n d t o t h e four interchangeable protons: t h r e e hydroxyls a n d o n e NH p r o t o n . The C-13 NMR d a t a of n a d o l o l i n d i m e t h y l s u l f o x i d e -dg ( F i g u r e 5 ) and i t s t e t r a b e n z o a t e d e r i v a t i v e i n CDC13 ( F i g u r e 6 ) a r e compared i n Table II.6 T a b l e I1 C-13 NMR P a t t e r n of Nadolol a n d i t s Tetrabenzoate Derivative

RO
OCH2-CH-CH2-N-C I I OR R
( CH3)

t, "

Figure 4.

NMR Spectrum of Nadolol in DMSO-d6, D20 exchange. Instrument: Varian XL-100-15 Internal Stardard: Tetramethylsilane

I/

L
40

Figure 5.

C-13 NMR Spectrum of Nadolol in DMSO-d6. Instrument: Varian XL-100-15, operated at 25.2 MHz

1""""'d"'

Figure 6.

C-13 NMR Spectrum of Tetrabenzoate Derivative of Nadolol in CDC13. Instrument: Varian XL-100-15, operated at 25.2 MHz.

NADOLOL

465

Table I1 (continued) Carbon Nadolol R=H Tetrabenzoate R=COC 6H5

Chemical Shifts, ppm.

c-1 c-2 c-3 c-4 C-4a c-5 C- 6 c-7 C- 8 C- 8a OCH2 CHO CH2N *C (CH3) C (*CH333
* n.a.

34.38 67.82 67.82 28.81 122.94 156.13 107.91 125.92** 120.71"" 135.49 70.33 68.98 45.25 49.39 28.81

31.04, 31.27 69.86** 71.19** 26.60, 26.77 121.12 155.37 107.91 n.a. 121.46 138.70 65.82, 66.23 69.10, 69.21 47.47, 47.76 56.82 28.76

** -

indicates to which carbon the shift is ascribed. these carbons may be interchanged. not assigned.

The chemical shift assignments are made on the basis of non-decoupled spectra and known substituent effects. The data of the tetrabenzoate derivative show double resonances for side chain carbons (OCH2CH(OR)CH2N) as well as the tetrahydro carbons (>CH2). This can be ascribed to the presence of two racemates A and B in nadolol (see Sec. 3.3). Mass Spectra The low resolution mass spectrum of nadolol is shown on Figure 7. The high resolution mass spectrum yields a molecular ion at m/e 309.1956 with the formula C1 H27NO4 consistent with the assigned structure.7 3.13

t-butyl

Typical of compounds containing groups is the loss of 15 a.m.u. from the

1.5 92 11s 99
18 92 19 92
1h 92

12 92
d
d

I12 39

5g I
1 1, 9 9
1h I 9

a l a z
u-4
0 k
4J

52 1

a9 1 1
1 9 8
19 9
5h

52

m m
3

m r

m n

m r

m n

m ~

m m

m ~

m m

hlISN31NI 3 A I l V 1 3 t l

NADOLOL

467

molecular ion to yield a peak at m/e 294. The loss of 44 a.m.u. to give the peak at m/e 265 (formula C15H23N03) is interpreted as the elimination of CH2 = C H - OH from the tetrahydronaphthalenediol portion of the molecule (see schematic below).

m/e 309

m/e 265+CH2=CH-OH

Fragmentation of the aliphatic side chain yields ions at m/e 252, 222 and 180, the latter two accompanied by the proton transfer shown below:

OCH -CH-CH2-NH-C (CH3) 2 1 OH

/
HO
HO

Nadolol

\
+

\
OH

I'Ofq HO

OCH CHCH2NH 2 1 OH m/e 252

m/e 180

OCH CH
21
0

m/e 222

468

LIDlA SLUSAREK AND KLAUS FLOREY

A fragment ion at m/e 116, corresponding to C6H14N0, can arise from the side chain cleavage depicted below:

HO
I

m/e 116
OH

OCH2-\- CHCH2NHC ( CH3)

Ultraviolet Spectrum The ultraviolet spectrum of nadolol in methanol' is shown on Figure '8. It depicts a shoulder at 218 nm (concentration 0.017 mg/ml) and two well-defined peaks at 270 and 278 nm (concentration 0.171 mg/ml). The table below lists the absorbances in methanol: 1% X nm Elcm 219 (shoulder) %275
3.14 270 278 37.5 39.1

Inspection of this table reveals that the UV absorbance of nadolol is very low. It should also be noted that an appropriate blank correction would be necessary for the true value at 219 nm (see also 4.31). lcm Fluorescence Spectroscopx Nadolol has no native fluorescence in 95% ethanol, aqueous 0.1N sodium hydroxide or 0.1N hydrochloric acid. It can, however, be induced by heating samples at 100C. in concentrated sulfuric acid (excitation maximum at 260 nm; Nevertheless, this emission maximum at 400 nm).' approach has not been utilized for analytical purposes because of interferences and variations in fluorescence.9
3.15

In
rl

470

LIDIA SLUSAREK AND KLAUS FLOREY

3.2

Solid State Properties 3.21 Melting Range Nadolol : Racemate A: Racemate B:

3.22 124-136; C. 134-1360 C. 1 0 148-157 C.

Differential Thermal Analysis (DTA) Nadolol shows a single endotherm at about 130 C. The twooracemic com ounds A and B showed endotherms at 140 C. and 1468 C., respectively, which correlate with their melting ranges (see Section 3.21). The differential thermal analysis curves were recorded on a DuPont 900 Thermoanalyzer with a temperature rise of 15O C. per minute.

Differential Scanning Calorimetry (DSC) Attempts were made to determine the purity of nadolol by differential scanning calorimetry. However, the results obtained were difficult to interpret due to the complex melting behavior exhibited by racemic mixtures.
3.23

Polymorphism No polymorphism has been reported for nadolol. However, it was observed that an amorphous form of nadolol can be obtained by lyophilizing an aqueous solution of the compound. The amorphous nature of the lyophilate was ascertained through x-ray and thermal analysis. The amorphous form exhibited diffused melting behavior at 50 C. and was at least ten times as soluble in water at room temperature as the crystalline form.
3.24

X-Ray Powder Diffraction The x-ray powder diffraction patterns of nadolol (Table 111, Figure 9) and racemiccompounds A (Figure 10) and B (Figure 11) are presented.
3.25

The diffraction patterns of the A and B racemates are quite different as can be seen from Figures 10 and 11. Consequently an X-ray powder diffraction method was developed to measure the percentages of racemate A and racemate B in samples of nadolol. The range of concentrations measurable

N A DO LO L

47 1

by this technique is 30% to 70% with an accuracy of - 5% (see Section 3.3). + Table I11 X-Ray Powder Diffraction Pattern of Nadolol* 28 (Deg.) 6.32 7.34 10.57 13.12 15.07 15.33 15.67 17.03 18.64 19.41 19.83 21.36 21.79 22.38 22.72 23.32 24.08 24.93 26.12 27.06 28.76 30.29 30.80 37.85 "28 d (Ao) 13.98 12.04 8.37 6.75 5.88 5.78 5.66 5.21 4.76 4.57 4.48 4.16 4.08 3.97 3.91 3.81 3.70 3.57 3-41 3.30 3.10 2.95 2.90 2.38
1/10

1.000 0.433 0.123 0.303 0.293 0.316 0.346 0.349 0.437 0.170 0.140 0.358 0.584 0.434 0.508 0.436 0.222 0.153 0.125 0.117 0.200 0.133 0.107 0.094

Twice the angle of incidence or reflection. d - Interplanar distance. 1/10 - Relative peak intensity based on highest intensity as 1.000 Single Crystal X-Ray Diffraction Single crystal x-ray diffraction data of the hydrobromide-salt of racemate A have been collected.'4 Crystals of the hydrobromide salt were large, well formed rods of the triclini system. The unit cell dimensions were a = 7.822 b = 12.535 8, c = 9.712 8, a = 101.80,B = 93.10, 6 = 89.2O. There are two molecules in the unit cell, centrosymmetrically related. 3.26

1,

5 N

I0

Figure 9.

X-Ray Powder Diffraction Pattern of Nadolol. Instrument: Phillips 120-101-11

5 W

Figure 10.

X-Ray Powder Diffraction Pattern of Racemate A of Nadolol. Instrument: Phillips 120-101-11

Figure 11.

X-Ray Powder Diffraction Pattern of Racemate B of Nadolol. Instrument: Phillips 120-101-11

NADOLOL

475

Racemate Composition Nadolol consists of two sets of enantiomers. They are present as two racemic compounds: racemate A and racemate B.I4 The following table illustrates the relative optical activity: Table IV Enantiomers of Nadolol

3.3

Optical Rotation

Racemate -

The composition of the racemates can be determined by infrared spectroscopy of mineral oil mulls, powder x-ray diffraction (Section 3 . 2 5 ) or by NMR techniques. The infrared spectroscopic method5 is based on the presence of specific absorption bands for racemate A and for racemate B: A: 1260 cm-l (7.9 p ) B: 1240 cm-l ( 8 . 0 5 p ) and 3 5 8 0 cm- ( 2 . 8 p ) These bands are recognizable in mixtures of A and B in the range from 30%A - 70%B to 70%A 308B. Either one or both racemates may be measured + independently with an accuracy of about - 5 % . The NMR method of analysis is based upon the chemical shift difference of t-butyl groups of the tetrabenzoates of racemate A 7 6 1.60) and racemate B (6 1.57).6 Quantitation of each racemate was obtained with - 2 % accuracy for samples contain+

476

LIDIA SLUSAREK AND KLAUS FLOREY

ing 20 to 7 0 % of racemate B. Structural assignments were made on the basis of europium shift reagent studies. Kiralshift reagent allowed for the separation of t-butyl group resonances of the d,l isomers of theside chains. Use of this reagent permits the determination of the optical purity of the side chain of racemate A and B or of the mixture of racemates. An x-ray powder diffraction method for the quantitation of racemates was also developed' (see Section 3.25 and Figures 10 and 11). An attempt was also made to separate racemates A and B by thin-layer chromatographic procedures. I 5 Sufficient separation was not achieved, however, even after 202 solvent systems and 14 chromatographic adsorbents were examined. 3.4 Solution Data 3.41 Solubility Solubility data are summarized in Table V Solubility of Nadolol Solvent 0.1N HC1 pH 5.0, 0.2M citrate p 5.0 , 0.2M phosphate H pH 7.0, 0.2M phosphate Propylene glycol 50% Aq. PEG 400 Methylene Chloride Methanol Isopropanol l,l,l-Trichloroethane 95% Ethanol Chloroform Acetone Benzene Ethyl Ether Hexane Temp. CO 37 R.T.
II

Table V . 1 2 ~ 1 6 t 1 7

Solubility mg/ml 42.5 40.1 40.2 30.4 97.5 46.0 2.0 >200.0 Inso lub1e Freely soluble Slightly soluble Insoluble Inso 1uble Insolub1e Insoluble
5.0

37
II

R.T.
fl

N ADOLOL

477

A pKa value of 9.67 was determined potentiometricaIly.16 Partition Coefficient The partition coefficient of nadolol was determined in the octanol/Krebs buffer system at room temperature. The composition of Krebs buffer is the following: KC1-5mM; KH2P04-lmM; NaHC03 - 26mM and NaC1-122mM. The table below shows the results obtained: Partition Coefficient 0.25 1.3
4.

3.42

pKa

3.43

PH 8.1 8.7

Analytical Tests and Methods 4.1 Elemental Analysis The followincr results were obtained on a Squibb Research Standird: Element C H N 4.2
%

Theory 66.99 8.80 4.53

8 Found

65.92 8.76 4.38

Identification Tests Identification of nadolol in tablet formulations is based on a color reaction of the oxidized The drug with phenylhydrazine and ferricyanide. cis-hydroxy groups are first oxidized to aldehydes with periodate and then reacted with phenylhydrazine to form a hydrazone. In acid solution, the hydrazone gives a red color with potassium ferricyanide. Thin-layer chr~rnatography~ (Section 4.61) and infrared spectroscopy (Section 3.11) have also been used to identify the drug. Spectrophotometric Analysis 4.31 Ultraviolet Analysis Nadolol displays three absorption peaks in the ultraviolet region at about 218, 270 and 278 nm (Section 3.14). Although the molar absorptivity of nadolol is quite low, it is adequate 4.3

478

LIDIA SLUSAREK AND KLAUS FLOREY

for the study of dissolution rates of nadolol tablets.20 Beers law is obeyed up to at least 4 mg of nadolo1/100 ml, as measured in pH 1.2 hydrochloric acid at 277 Colorimetric Methods Complexation of the amino group of nadolol with bromophenol blue in chloroform yields a yellow color with an absorption maximum at 414 nm. This is of potential usefulness for a quantitative assay of nadolol in formulation.
4.32

A colorimetric assay for the determination of nadolol in tablet formulation is based on a hydrazone absorption at 352 nm in chloroform?2 The two vicinal hydroxyl groups are oxidized to the corresponding dialdehyde, which is condensed with 2,4-dinitrophenylhydrazine yielding the hydrazone. Fluorescence Spectrophotometric Analysis Although nadolol does not exhibit native fluorescence, it can be modified to yield a strongly fluorescent derivative. A fluorometric assay for the quantitation of nadolol in serum and urine at nanogram and microgram levels has been described.23
4.33

The drug is oxidized with periodic acid to the corresponding dialdehyde and coupled with o-phenylenediamine to produce a fluorescent compound. Using a suitable filter, the emission peaks of the reagents and nadolol derivative are well separated ( A excitation = 305 nm and X emission = 445 nm). Titrimetric Methods 4.41 Reaction with Chloramine-T* Nadolol is oxidized with ChloramineT and the excess reagent is reacted with potassium iodide. The liberated iodine is titrated with sodium thiosulfate. The mechanism of the reaction of the drug with Chloramine-T is not known. This reaction can be used for the determination of nadolol in tablet formulations. However, the more readily controlled colorimetric method is preferable (Section 4.32).
4.4

N ADO LO L

419

Nonaqueous Titrations By virtue of the presence of an amino group, titration with acetous perchloric acid can serve to quantitate nadolol. 2 4 Quinaldine red or crystal violet indicators are used to determine the end-point. The amino group is titrated indirectly?' First, an ammonium salt of nadolol is formed with glacial acetic acid. Then, the released acetate ion is titrated with perchloric acid to the endpoint monitored potentiometrically or with an internal indicator. The method has good precision and the results obtained using both indicators were comparable. It was used to develop bulk, batching and formulation assays.
4.42

4.5

tion of nadolol by selected ion monitoring (SIM) and gas chromatography/mass spectrometry (GC/MS) of the tri(trimethylsily1) ether derivative has been described.26 The drug is extracted from serum and a known amount of internal reference, N-methylnadolol, is added. After lyophilization of the acidic extract, the resulting solid is reacted with N-trimethylsilylimidazole. The m/e 8 6 fragment ion of nadolol and the m/e 100 ion of the internal reference N-methyl-nadolol are monitored to establish the relative concentration ratio.

A method to determine the serum concentra-

Gas Chromatography/Mass Spectrometry

The detection level of this method is 2 . 6 ng/ml. No interferences are detected from extracts of fresh human serum at the relatively low mass ions of m/e 8 6 and 100. However, significant interferences were observed with several commercial serum samples at these masses. They probably result from contamination by plastic or rubber components used during the serum processing. Parallel measurements by spectr~fluorometry~~ (Section 4 . 3 3 ) on duplicate samples, demonstrate a correlation coefficient of
0 . 9.

Chromatographic Methods 4.61 Thin-Layer Chromatography A-thin-layer- chromatographic method has been developed15 to measure quantitatively the purity of nadolol samples. The TLC separation is achieved on silica gel GF plates using the solvent system acetone-chloroform-2N ammonium hydroxide

4.6

480

LIDIA SLUSAREK A N D KLAUS FLOREY

(80:lO:lO). The position of the nadolol zone is located under short-wave ultraviolet light (maximum at %254 nm). The isolated zone is eluted with 95% ethanol and the absorbance of the eluate is measured at 278 nm. This procedure provides an excellent separation of ultraviolet absorbing impurities and allows for the quantitative measurement of the drug. This assay has been adapted for measuring the stability of nadolol in tablet formulations.

As mentioned in Section 3.3, attempts to separate the two racemates of nadolol by TLC were unsuccessfu1.l5
Gas Chromatography A gas chromatographic method has been developed2 for the quantitative measurement of nadolol in solutions. The drug is extracted with dichloromethane, filtered and evaporated together with added brompheniramine maleate as an internal standard. After evaporation to dryness, the trimethylsilyl derivative is formed. The GC parameters are as follows: oven temperature is 210 C. and the circular glass column is 1.7 m with 3 mm i.d., packed with 3% (w/w) OV-17 on 60-80 mesh Gas Chrom Q (silanized). Retention times of a typical run are: nadolol-8.5 min and brompheniramine standard 4.5 min. High Pressure Liquid Chromatography An HPLC method for the quantitative determination of nadolol has been developed. * a A reverse phase ethylsilane column was used, operated at pressures of 200 to 2,000 psi and equipped with a precision loop injector and a fixed wavelength (254 nm) or variable wavelength (220 nm) detector. As mobile phase, a 35% methanol-65% aqueous 0.0005M hydrochloric acid-0.05M sodium chloride solution was used. Stability - Degradation 5.1 Solid State. - _... _ ~ _ Stabilitv _ ~ _ _ ~ Nadolol exhibits excillent stabilitv as a There was no apparent degradation of the solid. bulk samples which were held at high temperatures for prolonged periods. The same TLC patternowas obtained for samples held at 5O C. and at 50 C. for 5. 4.63 4.62

NADOLOL

481

over two years." Results of a light stability study" shows that nadolol and its racemic composition are stable under 9 0 0 foot candle light. Visual examination of a sample exposed to liqht for 6 months showed slight discbloration. Solution Stability Lyophilized sterile solutions of nadolol in 0.1M, pH 7.4 sodium phosphate buffer, showed no evidence of decomposition when held at room temper-' ature for 51 days. ' In unbuffered solutions, samples prepared at various H's were stable after 3 months' storage at 50 c. 38 A very slight discoloration was noted in some samples after 3 months at 50 C. Storage of nadolol solutions at 80 C. for 2 months produces degradation and discoloration at most pH's. Exposure to intense light results ir, discoloration of solutions at pH 2, 2 . 9 2 and 9 . 8 , after 2 weeks'storage. Variation in the pH values with temperature and time are below 1 pH unit for most solutions with the exception of those stored at 80' C.
5.2

Analysis of Body Fluids A sensitive fluorometric method, capable of measuring microgram or nanogram levels bf nadolol in human urine and serum has been developedz3 (Section 4.33). There is no interference in this assay from: dialyzing medium used during the clinical study, the diuretics hydrochlorothiazide and furosemide, and epinephrine and norepinephrine.3 This fluorometric method has been adapted for nadolol determinations in human bile at levels from 0.005 to 5 ug/ml.'
6.

Another technique, Selected Ion Monitoring Gas Chromatography/Mass Spectrometry, is described in Section 4.5, for application to nadolol quantitation in serum.'6 Suitable detection levels are obtained and no interferences from blood components or other administered drugs are observed. The SIM-GC/MS method shows lower detection limit and better sensitivity than the spectrofluorometric assay. Both SIM-GC/MS and fluorometric methods, in the absence of fluorescing metabolites, yield equivalent results. The fluorometric method is more adaptable to processing a large number of samples while the SIM-GC/MS method should be selected where specifi-

482

LIDIA SLUSAREK AND KLAUS FLOREY

city is required or where the serum levels are extremely low. Drug Metabolism Metabolic studies with n a d ~ l o l - ~ ~ C were carried out in patients at a dose that could safely be given both orally and intravenously. Maximum concentrations of radioactivity were attained in plasma 2 to 4 hours after drug administration. When given intravenously, concentrations of radioactivity decreased rapidly during the first hour after drug administration, reflecting distribution of radioactivity into tissues. Terminal plasma half-times are an average 12.2 hours after oral and 9.8 hours after intravenous administration. After oral doses, an average of 24.6% and 76.9% of the dose is excreted in urine and feces, respectively, whereas, after intravenous doses, an average of 72.9% and 23.3% of the dose was excreted by the same route. The radiolabeled drug is excreted unchanged in the urine and feces after either oral or intravenous administration indicating no biotransformation of the drug. The metabolism of nadolol has also been studied in rats, dogs and monkeys.32,33 7.

N ADO LO L

483

8. 1.

References M.E. Condon, C.M. Cimarusti, R. Fox, V.L. Narayanan, J. Reid, J.E. Sundeen and F.P. Hauck, J. Med. Chem., 21, 913 (1978).
R.J.

2.
3.

D.B. Evans, M.T. Peschka, R.J. Lee and Laffan, Eur. J. Pharmacol., 35, 17 (1976).

F.P. Hauck and C.M. Cimarusti, Gen. Pat. 2,421,549 (see also Drugs of the Future, Vol.1, No. 9, 434 (1976)). F.P. Hauck, C.M. Cimarusti and V.L. U.S. Patent 3,935,267 (1976). Narayanan,

4. 5.
6.

B. Toeplitz, Squibb Institute, personal communication. M.S. Puar, Squibb Institute, personal communication. P.T. Funke, Squibb Institute, personal communication.
E. Ivashkiv, Squibb Institute, personal communication. K. Bush, Squibb Institute, personal communication.

7.
8.

9. 10. 11. 12. 13. 14. 15.

G. Brewer, Squibb Institute, personal communication. H. Jacobson, Squibb Institute, personal communication.
D. Wadke, Squibb Institute, personal communication.

Q. Ochs, Squibb Institute, personal communication. J.Z. Gougoutas, B. Toeplitz, Squibb Institute, personal communication.

F.P. Targos, Squibb Institute, personal communication.

484

LIDIA SLUSAREK A N D KLAUS FLOREY

16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.

V. Valenti, Squibb Institute, personal communication.

A. Weiss, Squibb Institute, personal communication.

P. Valatin, Squibb Institute, personal communication. H.R. Roberts, Squibb Institute, personal communication.
M.D.

Ward, Squibb Institute, personal communication.

C. Papastephanou, Squibb Institute, personal communication.

E. Ivashkiv, J. Pharm. Sci., - 1 0 2 4 ( 1 9 7 8 ) . 67, E. Ivashkiv, J. Pharm. Sci.,

66,

1168 (1977).

J. Alicino, Squibb Institute, personal communication.

D.B.

Whigan, Squibb Institute, personal communication.

P.T. Funke, M.F. Malley, E. Ivashkiv, A. Cohen, J. Pharm. Sci., 67, 6 5 3 ( 1 9 7 8 ) . J.R. Salmon, Squibb Institute, personal communication.
B. Pate1 and J. Kirschbaum, Squibb Institute, personal communication.

27.
28. 29. 30. 31.

C.R. Bennett, Squibb Institute, personal communication. I.S. Gibbs, Squibb Institute, personal communication.

-, 17

J. Dreyfuss, L.J. Brannick, R.A. Vukovich, J.M. Shaw, D.A. Willard, J. Clin. Pharmacol.,
300 (1977).

N ADO LO L

485

32.

K . K . Wong, J. Dreyfuss, J.M. Shaw, J.J. Ross and E.C. Schreiber, Pharmacologist, - 245 15,

(1973).

33.

J.M.

Shaw and J. Dreyfuss, Fed. Proc., 35, 365 (1976).

NITRAZEPAM
Hussun Y. AbouE-Enein, Ahmud I . Judo, and Mohummed A . L o u ~
I, Description 1 . 1 Nomenclature 1.2 Formulae I .3 Molecular Weight 1.4 Elemental Composition 1.5 Appearance, colour, odour Physical Properties 2.1 Crystal Properties 2.2 Solubility 2.3 Identification 2.4 Spectral Properties Synthesis Stability and Decomposition Products Metabolism Methods of Analysis 6.1 Titrimetry 6.2 Spectrophotometry 6.3 Chromatography 6.4 Polarography Acknowledgement References
488 488 488 488 488 489 489 489 489 489 490 496 497 498 500 500 500 504 51 1 513 514

2.

3.
4.

5. 6.

7. 8.

Analytical Profiles of Drug Substances, 9

487

Copyright Q 1980 by Academic Press, Inc. All rights of nproduction i any fonn ~ e ~ e ~ e d . n ISBN: 0-12-260809-7

1. D e s c r i p t i o n 1.1. Nomenclature 1.11

Chemical Names
1,2-Dihydro-7-nitro-~-oxobenzodiazepine.

1,3-Dihydro-7-nitro-5-phenyl-2H-l, diazepin-2-one.

4-benzo-

1.12

Generic N a m e Nitrazepam

1.13

Trade N a m e s B e n z a l i n , Calsmin, E u n o c t i n , Megadon, Mogadon, Mogadan, Nelbon, N i t r e n p a x , Paxisyn, P e l s o n , Radedorm, R e l a c t , Sonebon, Sonnolin.

1.2

Formu l a e

1.2 1

Empirical

1.22

Structural

1.3

M 1 u 1a r weight o ec

281.26 1.4
Elemental Composition C,64.05%; H , 3.94%; N , 14.94%; 0, 17.07%

488

NITRAZEPAM

489

1.5

Appearance, c o l o r , odor
A y e l l o w , c r y s t a l l i n e powder, o d o r l e s s .

2.

Physical properties

2.1

Crystal properties 2.11 Crystallinity Parch and Lapysh (1) had d e s c r i b e d microcrystallographic reaction, for the detection of n i t r a z e p a m ( d e t e c t i o n l i m i t 0.1 ug) and o t h e r benzodiazepine d e r i v a t i v e s . T h i s i s based on e v a p o r a t i n g a s o l u t i o n of t h e sample, on a w a t c h - g l a s s , t h e r e s i d u e i s k e p t f o r 5 t o 1 0 m i n u t e s a f t e r adding one d r o p of 0 . 1 N-HC1, t h e n one drop of a s u i t a b l e r e a g e n t s o l u t i o n i s added and t h e m i x t u r e i s s e t a s i d e i n a m o i s t atmosphere. The v a r i o u s t y p e s of c r y s t a l s formed have been d e s c r i b e d . 2.12 Melting P o i n t 224-226'C (2) ; 226-229'C (3)

2.2

Solubility Nitrazepam is s o l u b l e i n a l c o h o l , a c e t o n e , c h l o r o form, and e t h y l a c e t a t e ; i n s o l u b l e i n water, e t h e r , benzene, and hexane ( 3 , 4 ) .

2.3

Identification
B.P. 1973 (3) s p e c i f i e s t h e f o l l o w i n g i d e n t i f i c a t i o n tests f o r nitrazepam:

a ) The i n f r a r e d a b s o r p t i o n spectrum e x h i b i t s m a x i m a which are o n l y a t t h e same wavelengths a s , and have s i m i l a r r e l a t i v e i n t e n s i t i e s t o , t h o s e i n t h e spectrum of n i t r a z e p a m a u t h e n t i c specimen. b) The l i g h t a b s o r p t i o n , i n t h e r a n g e 230 t o 250 nm, o f a 2-cm l a y e r of a 0.0005% w/v s o l u t i o n , i n a m i x t u r e of 1 volume of N h y d r o c h l o r i c a c i d and 9 volumes of methyl a l c o h o l , e x h i b i t s a maximum o n l y a t 280 nm; e x t i n c t i o n a t 280 nm, about 0.91.

490

HASSAN Y. ABOUL-ENEIN er al.

c ) To 1 0 mg add 5 m l of h y d r o c h l o r i c a c i d and 1 0 m l of water, h e a t on a w a t e r - b a t h f o r 1 5 m i n u t e s , and f i l t e r . To t h e clear f i l t r a t e add 1 m l of a 0.1% w / v s o l u t i o n of sodium n i t r i t e , a l l o w t o s t a n d f o r 3 m i n u t e s and add 1 m l of a 0.5% w / v s o l u t i o n of s u l f a m i c a c i d . Allow t o c o o l f o r 3 m i n u t e s and add 0.1% w/v s o l u t i o n of N-(1-naphthy l ) ethylenediamine hydrochloride, a red colour is produced. 2.4 Spectral properties 2.41 U l t r a v i o l e t Spectrum: Nitrazepam, i n n e u t r a l methanol s o l u t i o n , shows maxima a t 218, 258 nm, and a n i n f l e c t i o n a t a b o u t 308 nm ( F i g . 1). Nitrazepam, i n e t h a n o l , e x h i b i t s ( 4 ) maxima a t 218, 260 nm; minimum a t a b o u t 242 nm. I n 0.1N s u l p h u r i c a c i d , t h e d r u g shows a maximum a t 2 7 7 . 5 n E l % lcm = 1500 and a n i n m f e c t i o n a t a b o u t 340 nm. The UV a b s o r p t i o n s p e c t r u m of n i t r a z e p a m i s used a s a mean of i d e n t i f i c a t i o n and a s s a y of t h e d r u g i n t a b l e t f o r m u l a t i o n i n B.P. 1973 ( 3 ) . 2.42 I n f r a r e d spectrum The I R spectrum of n i t r a z e p a m i s shown i n F i g . 2 . The spectrum w a s o b t a i n e d from N u j o l m u l l . The s t r u c t u r a l a s s i g n m e n t s have been c o r r e l a t e d w i t h t h e f o l l o w i n g band frequencies:

-1 Frequency (cm )

Assignment

1.680 c=o 1600 C=C a r o m a t i c 1370 NO2 Clarke (4) has c i t e d t h e following characteri s t i c f i n g e r - p r i n t bands f o r n i t r a z e p a m when d e t e r m i n e d i n p o t a s s i u m bromide d i s c : 1352, 1692, 702, and 1615 cm-

NITRAZEPAM

49 1

Fig. 1 - Ultraviolet spectrum of Nitrazepam i n methanol

lOOL

. !loo . 80 . 60
* 40

5 LLI

80-

2
+
IT

2 5

k -/
40-

60-

20
0

WAVENUMBER (CM-)

- 20

Fig. 2 - Infrared spectrum of Nitrazepam in nujol mull.

NITRAZEPAM

493

2.43

N u c l e a r Magnetic Resonance Spectrum

A t y p i c a l NMR spectrum o f n i t r a z e p a m i s shown i n F i g . 3 . The sample w a s d i s s o l v e d i n d e u t e r a t e d c h l o r o f o r m (CDC1 The spectrum w a s d e t e r m i n e d on a 3Varian T-60A N M R s p e c t r o m e t e r w i t h TMS as t h e r e f e r e n c e standard.

>.

The f o l l o w i n g s t r u c t u r a l a s s i g n m e n t s have been made f o r F i g . 3 : Chemical S h i f t (6) Assignment

4.4 ( s i n g l e t ) 7.2 ( s i n g l e t ) 7.4 ( d o u b l e t )

CH2 a t C3
9 Five aromatic protons of t h e phenyl group a t
C-H aromatic at C

c5. 8.2 ( s i n g l e t )
1 0 . 1 (broad singlet)
Two a r o m a t i c p r o t o n s a t C6 and C

N - H

2.44

Mass s p e c t r u m and fragmentometry

The l o w r e s o l u t i o n m a s spectrum of n i t r a zepam i s shown i n F i g . 4. I t was o b t a i n e d on a F i n n i g a n 1015 L q u a d r u p o l e m a s s s p e c t r o m e t e r of a n i o n i s a t i o n p o t e n t i a l of 7 0 e V . The s p e c t r u m shown w a s o b t a i n e d by d i r e c t i n s e r t i o n of n i t r a z e p a m . I t shows a m o l e c u l a r i o n M+ a t m / e 281 ( r e l a t i v e i n t e n s i t y 42.8%) and M+ 1 a t m / e 282 ( r e l a t i v e i n t e n s i t y 8.1%). Some of t h e most prominent i o n s are g i v e n i n T a b l e I .

Table I

& m

Fragment M-H

280 264 254 253 252 235

M-OH M-(H,CO) M-N02

M-HCN M- (H,HCN)

. . .
a
1 '

. . . .
8

. . . .
1

. . . .
I

, . . .
I '

. . . .

, . . .
I t 00

. .

300

200

i *I
. U

P W P

Fig. 3

NMR spectrum of Nitrazepam in CDCl

as an internal standard.

containing TMS

1' :?A

*
495

496

HASSAN Y. ABOUL-ENEIN el al.

m/e
234 207 206

Fragment M- (H ,NO2 M-(N02-CO) M-(H-NO~-CO)

3.

Synthesis The two most f r e q u e n t l y used methods, w i t h good y i e l d s ( 5 , 6 ) , f o r t h e s y n t h e s i s of s i m p l e b e n z o d i a z e p i n o n e s a r e shown i n Scheme 1. Scheme 1
R I X-CO-CH-X (X=halogen) 't

x@NHco:"'

p=0

Pyr i d i n e , heat

ROCOCHR.HC1 I

NH2
H

x?i?-oR
heat

- a:_.

NH3

c =O j H - R
NH2

y@

A s c a n b e s e e n , i n b o t h c a s e s , 2-aminobenzophenones a r e used as s t a r t i n g m a t e r i a l s . Treatment of t h e a p p r o p r i a t e l y s u b s t i t u t e d aminobenzophenone w i t h a h a l o a c e t y l h a l i d e y i e l d s a compound I1 which, on t r e a t m e n t w i t h ammonia, g i v e s t h e b e n z o d i a z e p i n o n e I V v i a am amino d e r i v a t i v e 111. T h i s method g e n e r a l l y g i v e s b e t t e r o v e r a l l y i e l d s of up t o 70-80%, a l t h o u g h i t i n v o l v e s more s t e p s . Another e x t e n s i v e l y used method i s t h e t r e a t m e n t of 2-aminobenzophenone w i t h a n amino a c i d e s t e r h y d r o c h l o r i d e i n p y r i d i n e , l e a d -

NITRAZEPAM

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i n g d i r e c t l y from 1 t o IV. O t h e r r o u t e s f o r t h e c o n s t r u c t i o n of t h e 7-membered r i n g , which have been developed s u b s e q u e n t l y , i n v o l v e t h e u s e of intermediates possessing a protected or potential glycine moiety ( 7 , 8 ) . Nitrazepam h a s been p r e p a r e d by t h e f o l l o w i n g method ( 9 ) : Anhydrous h y d r o c h l o r i c a c i d i s p u t i n t o a s t i r r e d m i x t u r e c o n t a i n i n g 2-amino-5-nitrobenzophenone, g l y c i n e , and p y r i d i n e . The r e a c t i o n m i x t u r e i s r e f l u x e d f o r more t h a n 48 h o u r s , a t i n t e r v a l s , and t h e n c o n c e n t r a t e d undervacuum. The r e s i d u e i s p a r t i t i o n e d between benzene and water. The benzene l a y e r i s washed w i t h water and d r i e d o v e r anhydrousmagnesium s u l p h a t e and t h e n c o n c e n t r a t e d u n d e r vacuum t o g i v e t h e d r i e d p r o d u c t . Nitrazepam i s a l s o p r e p a r e d by t h e t r e a t m e n t of 2-amino5-nitrobenzophenone w i t h a f+acylaminoethyl h a l i d e ( 1 0 ) .

02N C6H5 C6H5

4.

S t a b i l i t y and Decomposition P r o d u c t s : Beyer and Sadee (11) have p u b l i s h e d a monograph g i v i n g t h e a n a l y t i c a l d a t a on l Y 4 - b e n z o d i a z e p i n e d e r i v a t i v e s , i n c l u d i n g n i t r a z e p a m , c o n c e r n i n g t h e s t a b i l i t y of t h e d r u g i n s o l u t i o n . Nitrazepam i s a r e l a t i v e l y s t a b l e d r u g a t room t e m p e r a t u r e . However, 2-amino-5-nitrobenzophenone i s cons i d e r e d a s a d e c o m p o s i t i o n p r o d u c t . The B.P. 1973 ( 3 ) d e s c r i b e s a method f o r t h e d e t e c t i o n of t h i s d e c o m p o s i t i o n p r o d u c t , u s i n g TLC. Genton and K e s s e l r i n g ( 1 2 ) have s t u d i e d t h e e f f e c t of t e m p e r a t u r e and r e l a t i v e h u m i d i t y on t h e s t a b i l i t y of n i t r a z e p a m i n t h e s o l i d s t a t e . The d r u g and i t s d e c o m p o s i t i o n p r o d u c t s have been d e t e r m i n e d i n a 1% i l u t i o n i n m i c r o c r y s t a l l i n e c e l l u l o s e . The d The extract sample i s e x t r a c t e d by s h a k i n g w i t h methanol. i s chromatographed by TLC on K i e s e l g e l GF 254 u s i n g benz e n e - e t h y l a c e t a t e - a c e t i c a c i d (15:9:1) a s a d e v e l o p i n g

498

HASSAN Y. ABOUL-ENEIN et al.

s o l v e n t . The d i f f u s e r e f l e c t a n c e of t h e s p o t s are measured a t 265, 365 and 295 n f o r n i t r a z e p a m , 2-amino-5-nitrom benzophenone and 3-amino-6-nitro-4-phenyl-4H-quinoline-2one, r e s p e c t i v e l y . Meyer, e t a 1 (13) have p u b l i s h e d a r e p o r t on t h e s t a b i l i t y and a n a l y s i s of t h e h y d r o l y t i c p r o d u c t s of n i t r a z e p a m . T h e drug i s hydrolysed i n t o 5-nitro-2-aminobenzophenone and 3-amino-6-nitro-4-phenyl-4H-quinoline-2-one ( r i n g c o n t r a c t i o n ) . These h y d r o l y t i c p r o d u c t s can b e determined s e p a r a t e l y by UV a b s o r p t i o n a f t e r f r a c t i o n a t i o n by TLC on a K i e s e l g e l PF 254 u s i n g benzene-isopropanol (9:l) as a s o l v e n t ; o r by means of t h e a b s o r p t i o n of t h e i r diazonium salts. A l t e r n a t i v e l y , t h e hydrolytic products can a l s o be determined t o g e t h e r by means of a (dead-stop) t i t r a t i o n w i t h 0 . 0 1 N sodium n i t r i t e s o l u t i o n . Meyer e t a1 (14) H have a l s o s t u d i e d t h e e f f e c t of p on t h e s t a b i l i t y of 1,4-benzodiazepine d e r i v a t i v e s i n i n j e c t i o n f o r m u l a t i o n s . 5. Metabolism The m e t a b o l i t e s of n i t r a z e p a m i n man and rat i s shown i n F i g . 5 . With t h e e x c e p t i o n of s u b s t a n c e IV, which w a s d e s c r i b e d by Beyer and Sadee ( 1 5 ) , and s u b s t a n c e X , which i s s t i l l h y p o t h e t i c a l , t h e o t h e r compounds l i s t e d have been proved by Rieder and Wendt (16) t o b e b i o t r a n s f o r mation p r o d u c t s of t h e d r u g a p p e a r i n g i n t h e u r i n e . They have been i s o l a t e d by v a r i o u s p r o c e d u r e s of e x t r a c t i o n , column chromatography, and t h i n - l a y e r chromatography, and t h e i r chemical s t r u c t u r e s have been e l u c i d a t e d by chemical r e a c t i o n s , comparison w i t h a u t h e n t i c samples, m a s s spect r o m e t r y , n u c l e a r magnetic r e s o n a n c e s p e c t r o m e t r y , and, i n t h e c a s e s of I1 and 111, also by u l t r a v i o l e t and i n f r a r e d s p e c t r o m e t r y . The main m e t a b o l i c pathway i n man and rat i n d i c a t e s ( F i g . 5 ) t h e r e d u c t i o n of t h e n i t r o group t o t h e c o r r e s p o n d i n g amine I1 and - by a c e t y l a t i o n of I1 - t o t h e 7-acetamido d e r i v a t i v e 111, which i s t h e major m e t a b o l i t e . A s m a l l p r o p o r t i o n of I1 and 111 i s hydroxylated i n p o s i t i o n 3 , y i e l d i n g compounds IV and V. Another m e t a b o l i c pathway c o n s i s t s of t h e c l e a v a g e of t h e benzodiazepine r i n g , w i t h t h e f o r m a t i o n of t h e benzophenone d e r i v a t i v e s VI, VII, and VIII. There i s someevidence t h a t t h e a c i d X is formed d u r i n g t h e g e n e r a t i o n of t h e benzophenones, which can b e c a l l e d an opened lactam. The end p r o d u c t of t h i s l i n e , i n man, t h e 2-amino-3-hydroxy5-nitro-benzophenone VII and, i n r a t , t h e 2-amino-5notro-4-hydroxy-benzophenone VIII. P a r t of compound VIII

w
0
ffl

I
O "

h rd

5
(d

-d 4

e
u
rd

a,

a
a l

500

HASSAN Y. ABOUL-ENEIN et al.

may b e p o s s i b l y d e r i v e d from t h e 4-hydroxylated n i t r a z e Pam-IX, which h a s been found i n t h e u r i n e of r a t , b u t n o t i n man ( 1 6 ) . The p h e n o l i c s u b s t a n c e s VII, V I I I , and I X are e x c r e t e d a l m o s t e x c l u s i v e l y ; I1 and V I o n l y t o a minor p a r t i n c o n j u g a t e d form. The d i s t r i b u t i o n , e x c r e t i o n and p h a r m a c o k i n e t i c s of n i t r a zepam have been d i s c u s s e d by R i e d e r and Wendt ( 1 6 ) . 6. Methods of A n a l y s i s 6.1 Titrimetry 6.11 Aqueous Blaszek-Bodo, e t a 1 ( 1 7 ) have d e s c r i b e d a d i a z o m e t r i c method f o r t h e d e t e r m i n a t i o n of n i t r a z e p a m i n p u r e form and p h a r m a c e u t i c a l f o r m u l a t i o n s . The method i s based on d i a z o t i s a t i o n r e a c t i o n i n which t h e d r u g i s f i r s t hydrolysed with h y d r o ch lo r ic a c i d i n t h e p r e s e n c e of z i n c t o a f f o r d 2,5-diaminobenzophenone. T h i s p r o d u c t i s t i t r a t e d a g a i n s t s t a n d a r d sodium n i t r i t e s o l u t i o n . The method proved t o b e a c c u r a t e and t h e r e i s no i n t e r f e r e n c e from t h e d r u g e x c e p i e n t s . 6.12 Non-aqueous
A non-aqueous t i t r a t i o n method h a s been described ( 3 ) f o r the quantitative analysis of n i t r a z e p a m a s t h e p u r e d r u g . The d r u g i s t i t r a t e d by p e r c h l o r i c a c i d i n a c e t i c a c i d and t h e e n d p o i n t i s d e t e r m i n e d p o t e n t i ometrically

6.2

Spectrophotometry 6.21 Colorimetry C o l o r i m e t r i c methods have been used f o r t h e d e t e r m i n a t i o n of n i t r a z e p a m i n v a r i o u s p r e p a r a t i o n s . Wassel and Diab (18) have developed t h e f o l l o w i n g p r o c e d u r e s f o r t h e d e t e r m i n a t i o n of n i t r a z e p a m i n pharmaceutic a l f o r m u l a t i o n s and u r i n e samples:

NITRAZEPAM

501

a ) F e r r o u s hydroxamate p r o c e d u r e :
To a n e t h a n o l i c s o l u t i o n (1 m l 0.2 t o 5 mg of n i t r a z e p a m ) , add f i l t e r e d Goddu r e a g e n t 112.5% m e t h a n o l i c hydroxylammoniurn c h l o r i d e - 12.5% m e t h a n l o i c sodium h y d r o x i d e (:) 11] ( 3 m l ) . The s o l u t i o n i s h e a t e d a t 45OC f o r 50 m i n u t e s t h e n c o o l e d and t h e f e r r o u s r e a g e n t [ (NH4) 2 S04Fe2 (S04) 3 . 24H20) ( 2 0 gm) d i s s o l v e d i n 70% aqueous p e r c h l o r i c a c i d (10 ml) i s added. Shake t h e s o l u t i o n and d i l u t e t o 25 m l w i t h a c e t a t e b u f f e r s o l u t i o n ( 0 . 1 M sodium a c e t a t e a d j u s t e d t o pH 1 . 5 w i t h 70% aqueous p e r c h l o r i c a c i d ) . Measure t h e e x t i n c t i o n a t 550 n m and o b t a i n t h e amount of n i t r a z e p a m by r e f e r e n c e t o a c a l i b r a t i o n g r a p h , which i s r e c t i l i n e a r from 0 . 1 t o 5 mg of n i t r a zepam.

Treat u r i n e samples (200 t o 400 ml) w i t h ammonia ( t o pH 10) and e x t r a c t n i t r a z e p a m and i t s m e t a b o l i t e s w i t h c h l o r o f o r m (4x100 m l ) . Wash t h e combined e x t r a c t s w i t h water, add anhydrous e t h a n o l (5 ml) and e v a p o r a t e t o d r y n e s s i n vacuo a t 500. D i s s o l v e t h e r e s i d u e i n anhydrous e t h a n o l ( 5 ml) , add t h e Goddu r e a g e n t ( 3 ml) and c o n t i n u e a s above. b) C i t r i c a c i d method
To t h e e t h a n o l i c s o l u t i o n ( l m l up t o 50 ug of n i t r a z e p a m ) , add 5 m l of c i t r i c a c i d r e a g e n t [ c i t r i c a c i d ( 2 gm) d i s s o l ved i n e t h a n o l (10 ml) and anhydrous a c e t i c a c i d (90 m l ) ] and h e a t t h e s o l u t i o n a t 7O-8O0C f o r 20 m i n u t e s . D i l u t e t h e s o l u t i o n t o 25 m l w i t h e t h a n o l and measure t h e e x t i n c t i o n a t 510 nm. T h i s procedure is s u i t a b l e f o r r o u t i n e analyses.

Diab (19) h a s developed a c o l o r i m e t r i c method f o r t h e a n a l y s i s of t h e d r u g i n f o r m u l a t i o n s and i t s m e t a b o l i t e s i n blood and u r i n e . The method d e p e n d s on t h e c o l o r r e a c t i o n of P o r t e r ( 2 0 ) f o r t h e

502

HASSAN Y . ABOUL-ENEIN er al.

a r o m a t i c n i t r o compounds. The method i s is e s s e n t i a l l y as follows: Transfer 5 m l of s t a n d a r d s o l u t i o n ( 2 mg of n i t r a z e p a m i n 100 m l e i t h e r dimethylformamide o r a c e t o n e ) i n t o s e p a r a t e t e s t - t u b e s . Add 0 . 1 m l of 10% tetraethylammonium hydrox i d e s o l u t i o n t o t h e d i m e t h y l formamide s o l u t i o n o r 0 . 1 m l of 10% sodium hydrox i d e s o l u t i o n t o t h e acetone s o l u t i o n , s h a k e t h e m i x t u r e s and measure t h e e x t i n c t i o n s a t 410 nm. The c o l o r formed by e i t h e r r e a c t i o n is s t a b l e f o r more t h a n two h o u r s . For t h e a s s a y o f t a b l e t s , a q u a n t i t y of powdered sample i s e x t r a c t e d w i t h e t h a n o l . The combined f i l t e r e d e t h a n o l extracts are d i l u t e d t o a c e r t a i n volume and 1 m l of t h e s o l u t i o n i s evapor a t e d t o d r y n e s s on a steam b a t h . The r e s i d u e is d i s s o l v e d i n e i t h e r dimethylformamide o r a c e t o n e and proceed a s f o r t h e s t a n d a r d s o l u t i o n . For samples of blood and u r i n e , n i t r a z e p a m i s e x t r a c t e d w i t h benzene and d e t e r m i n e d a s above. M e t a b o l i t e s a r e d e t e r m i n e d w i t h 4dimethylaminobenzaldehyde r e a g e n t (0.125 g i n 100 m l of 65% s u l p h u r i c a c i d p l u s m 0 . 1 m l of 5% aqueous f e r r i c c h l o r i d e s o l u t i o n ) and measurement of t h e e x t i n c t i o n a t 420 nm. Raber and Gruber ( 2 1 ) have d e s c r i b e d a p h o t o m e t r i c method f o r e s t i m a t i o n of n i t r a z e p a m and o t h e r 1 , 4 - b e n z o d i a z e p i n e d e r i v a t i v e s . Nitrazepam i s h y d r o l y s e d w i t h h y d r o c h l o r i c a c i d and t h e r e s u l t i n g 2-aminobenzophenone d e r i v a t i v e i s d i a z o t i s e d and c o u p l e d w i t h 1 - n a p h t h o l . t h e e x t i n c t i o n of t h e s o l u t i o n i s t h e n measured a t 607 nm. The method i s a p p l i c a b l e f o r e s t i m a t i o n of a m i x t u r e of n i t r a z e p a m and o t h e r d e r i v a t i v e s . Also, t h e p r o c e d u r e i s a p p l i c a b l e f o r t h e d e t e r m i n a t i o n of n i t r a z e p a m i n t a b l e t o r capsule formulations

NITRAZEPAM

503

Beyer and Sadee (15) have e s s e n t i a l l y a p p l i e d t h e same p r i n c i p l e used b e f o r e for t h e d e t e r m i n a t i o n of n i t r a z e p a m (and o t h e r 5-phenyl-1,4-benzodiazepines) and f o r i n v e s t i g a t i o n s on t h e metabolism of n i t r a z e p a m . The diazonium s a l t b e i n g t r e a t e d w i t h 2% sulphamic a c i d s o l u t i o n and t h e d i a z o compound is t h e n c o u p l e d w i t h N-1-naphthylethylenediamine d i h y d r o c h l o r i d e . The e x t i n c t i o n of t h e r e s u l t i n g a z o d y e i s measured a t 533 t o 535 n m and r e f e r r e d t o c a l i b r a t i o n g r a p h s . R e c e n t l y , Blaszek-Bodo, e t a 1 (22) have r e p o r t e d a method f o r e s t i m a t i o n of n i t r a z e p a m . The drug i s h y d r o l y s e d and s i m u l t a n e o u s l y reduced and t h e r e s u l t i n g 2,5-diaminobenzophenone i s d i a z o t i s e d and coupled w i t h N-l-naphthylethylened i a m i n e

Egg (23) h a s d e v i s e d a q u a l i t a t i v e method f o r t h e d e t e c t i o n of n i t r a z e p a m and o t h e r d e r i v a t i v e s by t h e c o l o u r r e a c t i o n of S a w i c k i and Johnson. B e n z o d i a z e p i n e s c a n b e d e t e c t e d a f t e r TLC s e p a r a t i o n by s p r a y i n g t h e chromatogram w i t h 1% , 5 2 dimethoxytetrahydrofuran s o l u t i o n i n a c e t i c a c i d and d r y i n g f o r 5 t o 1 0 m i n u t e s a t 1OOOC; a r e d d i s h - v i o l e t s p o t i s produced on r e - s p r a y i n g w i t h 2% 4dimethylaminobenzaldehyde s o l u t i o n i n a c e t i c a c i d - conc. h y d r o c h l o r i c a c i d (17:3). The s e n s i t i v i t y of t h e c o l o u r r e a c t i o n i s dependent on t h e s u b s t i t u e n t s i n t h e benzodiazepine molecule. 6.22 Spectrofluorimetry T h i s method h a s been used f o r t h e i d e n t i f i c a t i o n i n urgent t o x i c o l o g i c a l a n a l y s i s of 1 , 4 - b e n z o d i a z e p i n e s used i n t h e r a p e u t i c t r e a t m e n t ( 2 4 ) . The g a s t r i c f l u i d i s made n e u t r a l o r weakly a c i d and t h e d r u g i s e x t r a c t e d by e t h e r . The e x t r a c t i s e v a p o r a t e d t o d r y n e s s and t h e r e s i d u e i s t h e n d i s s o l v e d i n HCl04, H3PO4 o r H2SO4.

504

HASSAN Y . ABOUL-ENEIN et al.

6.23

Ultraviolet The determination of nitrazepam and other benzodiazepines in solutions, injections, tablets and syrups pharmaceutical formulations by UV spectrophotometry has been described (25). Nitrazepam is determined at 259 nm in neutral 96% ethanol. Another report (26) has been published for the spectrophotometric determination of nitrazepam in methanolic solution at 259 and 309 nm in concentration ranges 0.2 to 2 and 0.4 to 3 mg dl-, respectively. The B.P. 1973 ( 3 ) describes a method for the assay of nitrazepam tablets depending on acid hydrolysis and the resulting 2-amino5-nitrobenzophenone is measured spectrophotometrically at a maximum of about 280nm (Elcm 1% = 910).

6.3

Chromatography 6.31 Thin Layer Chromatography A compilation of qualitative colour and precipitation reactions, spectrophotometric and TLC data (useful for identification purposes and for quantitative assay methods) related to nitrazepam has been reviewed by Dobrecky, et a1 (27). Several Reports had been published concerning the chromatographic identification and separation of nitrazepam and its metabolites as shown in Table 2. Table 2

Solvent System

Absorbent

Detection

Reference 28

Toluene-acetone-conc. Kieselgel aq. ammonia GF254 (50 : 50 : 1)

-UV at 254 or356 nm -d iazotisation and coupling

NITRAZEPAM

505

Solvent System Ethyl acetate-propanol-diethyl-amine.

Absorbent

Detection -reduction by Na2S204 to give coloured d er ivat ive

: ierence e

(70 : 30 :

1)

Methanol-1,2-dichloroethane-conc. aqu. ammonia

(10 :

90: 1) Lieselgel
:F

Toluene-diethylamine

-UV at 254 nm

29 30

(4

1)

254

Ethyl acetate-methanol-acetic acid

(9 0 : 10 : 1)

jilica gel -UV at 254 nm

Heptane-chloroformethano1

jilica gel
:F

31

254

(5 : 5 : 1)
Ethyl acetate-1,2dichloroethane-25% aq. ammonia

(8 : 2 : 1 )

-UV at 254 nm -Spraying with conc. H SO4,HC1 H3P04, Hc104 t i the color of the fluorescene in radiation at 254 and 366 nm is noted.
32

Ciesel gel -fluorescence Dioxane-benzenehexane-onc. aq.ammonii ;F 254 -spray with Dragendorff reagent ( 9 : 10 : 14 : 1) -1% 2-furaldehyde solution i acetone n chloroform-acetonesolution of 10 gm tetrahydrofuran H2SO4 in 90 ml ( 9 : 1 : 1) acetone

506

HASSAN Y . ABOUL-ENEIN ei al.

Solvent system

AbsorEent

Detection -Dragendorff reagent diluted 1:lO with 10% HC1

.ef erence 33

Shellsol A -methanol- Silica gel 25% aq. ammonia G (85 : 15 : 1 ) Chloroform-benzeneether-tetrahydrofuran-acetone-acetic acid (35: 15: 16: 10: 5:3) Chloroform-ether (3:2)

34

(UV at 230-300
I

Silica gel -UV at 254 nm and G sprayed with ethanolic 0.01% N-l-naphthylethylenediamine Merck Aluminium
Oxide (type

35

Chloroform-tolueneethano1 (20 : 30: 1 )

F354

-UV at 254 nm -Spraying with K2PtI6

IChloroform-ethanol

Whatman SG 8 1 Aluminium oxide F254

Benzene-chloroform (3:l) Chloroform-ethanol f29:l)

-UV at 254 nm
-Fluorescent spot at 366 nm -diazot isation followed by spray ing with 0.1% aq N-1-naphthyl-NNd iethylpropane-1 2-d iamine hydrochloride and hea at 50OC.

24

Negritescu et a1 (37) have described a TLC separation method of the reaction products formed during synthesis of nitrazepam by nitration of 2,3-dihydro-5-phenyl-lH-l,4-

NITRAZEPAM

507

benzodiazepine-2-one. Nitrazepam i s formed a l o n g w i t h a d i n i t r o d e r i v a t i v e . The l a t t e r

can be s e p a r a t e d from t h e reac%ion m i x t u r e by TLC on K i e s e l g e l H , u s i n g 6 s o l v e n t s y s t e m s , namely:

1) benzene-n-butanol-formic a c i d (50: 28:8) 2) d i b u t y l e t h e r - e t h y l a c e t a t e - f o r m i c a c i d (25 :75 :8 ) 3) benzene-ethyl a c e t a t e - formic a c i d (25:75:5 ; 25:75:10; 25:75:15; 25:75:20)
A f t e r d r y i n g t h e chromatograms a r e sprayed w i t h HN03 (0.15 m l of conc. HNO3 i n 1 0 m l of e t h a n o l ) and t h e p l a t e s a r e t h e n examined under UV r a d i a t i o n . Schuetz (38) h a s developed a chromatographic method f o r t h e d e t e c t i o n of n i t r a z e p a m and i t s major m e t a b o l i t e s . The sample ( e . g . u r i n e e x t r a c t ) i s s u b j e c t e d t o TLC, withbenzene-isopropyl alcohol-25% aq. ammonia (80:20:1) a s t h e s o l v e n t . The d r u g and i t s m e t a b o l i t e s , are t h e n hydrolysed and reduced by s p r a y i n g w i t h a c i d i c T i C 1 3 s o l u t i o n . The p l a t e i s t r e a t e d w i t h gaseous ammonia t o c o n v e r t any amine s a l t s i n t o t h e f r e e b a s e s . A second development a t r i g h t a n g l e s w i t h t h e same m o b i l e phase is t h e n c a r r i e d o u t . D i a z o t i s a t i o n followed by c o u p l i n g w i t h N-ln a p h t h y l e t h y l e n e d i a m i n e makes i t p o s s i b l e t o d e t e c t amounts a s low a s 0.02 ug p e r s p o t .

508

HASSAN Y. ABOUL-ENEIN er al.

6.32

Column Chromarography Golovenko, e t a l . (39) have r e p o r t e d a method f o r t h e s e p a r a t i o n of n i t r a z e p a m and i t s m e t a b o l i t e s from r a t u r i n e . P o r t i o n s (10 t o 100 ug each) of n i t r a z e p a m and i t s p o s s i b l e m e t a b o l i t e s , d i s s o l v e d i n chloroform-hexane (l:l), a r e a p p l i e d t o a column (10 cm x 0 . 5 cm)of KSK-1 S i l i c a g e l (76 mesh) and t h e column i s washed w i t h 1 0 m l of hexane; c l e a n s e p a r a t i o n a r e o b t a i n e d by s t e p w i s e change of e l u e n t . The c o l l e c t e d 1 - m l f r a c t i o n s a r e e v a p o r a t e d i n vacuum, e a c h r e s i d u e i s d i s s o l v e d i n 4 m l of e t h a n o l , and t h e absorba n c e s of t h e r e s u l t i n g s o l u t i o n s a r e measured a t t h e a p p r o p r i a t e w a v e l e n g t h s . The o r d e r of e l u t i o n of compounds i n v e s t i g a t e d i n model m i x t u r e s and e l u e n t s used ( a s 10-ml p o r t i o n s ) are a s f o l l o w s : Nothing e l u t e d i n C C 1 4 ; n i t r a z e p a m , hexanea c e t o n e (4:1); 7-amino-l,2-dihydro-5-phenyl3H-1,4-benzodiazepin-2-one, chloroforma c e t o n e ( 4 : l ) ; and 7-acetamido-1,2-dihydro5-phervl-3H-1, 4-benzodiazepin-2-one, c h l o r o form-acetone (4:l). Sawada, e t a 1 (30) have d e s c r i b e d a method f o r t h e i s o l a t i o n and i d e n t i f i c a t i o n of n i t r a z e p a m and i t s m e t a b o l i t e s i n r a b b i t u r i n e . The method i n v o l v e s t h e s o r p t i o n on a column of XAD-2 r e s i n , which i s e l u t e d by methanol and e t h y l a c e t a t e - m e t h a n o l - a c e t i c a c i d (9O:lO:l). Conjugated m e t a b o l i t e s i n t h e e l u a t e from t h e column a r e h y d r o l y s e d e n z y m i c a l l y , and t h e l i b e r a t e d compounds a r e extracted into ethyl acetate. Missen (40) h a s r e p o r t e d a p r o c e d u r e f o r e x t r a c t i n g n i t r a z e p a m from t h e b l o o d , by column chromatography. The p r o c e d u r e i n v o l v e s t h e a b s o r p t i o n of t h e d r u g on a c t i v a t e d c h a r c o a l , A m b e r l i t e XAD-2 r e s i n and C e l i t e e l u t i n g with chloroform.

6.33

High Performance L i q u i d Chromatography Moore, e t a1 ( 4 1 ) have r e p o r t e d a HPLC method f o r t h e a n a l y s i s of n i t r a z e p a m and i t s m e t a -

NITRAZEPAM

509

b o l i t e s , i n u r i n e . The u r i n e sample i s a d j u s t e d t o pH7 w i t h a c e t a t e b u f f e r s o l u t i o n and e x t r a c t e d w i t h e t h y l acetate. The comb i n e d e x t r a c t s are e v a p o r a t e d t o d r y n e s s and t h e r e s i d u e i s d i s s o l v e d i n e t h y l acetate. P o r t i o n s of t h i s s o l u t i o n a r e s u b j e c t e d t o HPLC on a s t a i n l e s s s t e e l column (50 cm x 2mm) packed w i t h Zipax SAX ( 3 0 um) and o p e r a t e d w i t h hexane-ethyl acetate (7:3) as m o b i l e p h a s e , a t a r a t e of 1 m l p e r m i n u t e , and d e t e c t i o n a t 260 nm. T h i s method i s s u i t a b l e f o r t h e d e t e r m i n a t i o n of t h e d r u g and of i t s 7-amino-and 7-acetamido-metabolites up t o 700 ng of e a c h i n j e c t e d . D e t e c t i o n l i m i t s r a n g e from 20-100 ng and t h e r e c o v e r y of t h e added compounds i s 80%. H a r z e r and B a r c h e t ( 4 2 ) have d e s c r i b e d a method f o r t h e a n a l y s i s of n i t r a z e p a m and o t h e r b e n z o d i a z e p i n e s and t h e i r h y d r o l y s i s p r o d u c t s , namely; benzophenones, by r e v e r s e d p h a s e HPLC. The method i s a p p l i e d t o t h e a n a l y s i s of e x t r a c t s from blood and u r i n e . The method i s based on t h e s e p a r a t i o n , by HPLC on a column (25 cm x 4 mm) of LiChrosorb SI-100 ( g r a i n s i z e 10 um), o p e r a t e d a t room t e m p e r a t u r e and 750 p . s . i . w i t h aqueous methanol ( 6 0 t o 100% of methanol) a s t h e m o b i l e p h a s e a t t h e r a t e of 0.75 m l p e r m i n u t e . Another p r o c e d u r e h a s been r e p o r t e d ( 4 3 ) f o r t h e a n a l y s i s of b e n z o d i a z e p i n e s , i n c l u d i n g n i t r a z e p a m , and t h e i r m e t a b o l i t e s , by enzymic d i g e s t i o n and high-performance l i q u i d chromatography. The p r o c e d u r e i n v o l v e s t h e l i b e r a t i o n and e x t r a c t i o n of t h e d r u g s a n d / o r m e t a b o l i t e s , w i t h e t h e r . The e t h e r e x t r a c t i s d r i e d and e v a p o r a t e d and t h e r e s i d u e i s d i s s o l v e d i n anhydrous e t h a n o l . Few m i c r o l i t r e s of t h e e t h a n o l i c s o l u t i o n a r e s u b m i t t e d t o HPLC on a a column (150 mm x 4.6 mm) packed w i t h Spherisorb-5-ODS and o p e r a t e d w i t h 0.025 M Na2HP0 -methanol ( 2 : 3 ) , 4 a d j u s t e d t o pH 7 . 8 , as m o b i l e p h a s e , a t a r a t e of 1 m l p e r m i n u t e . The d e t e c t i o n i s done by UV s p e c t r o s c o p y a t 254 nm.

510

HASSAN Y. ABOUL-ENEIN et al.

6.34

Gas L i q u i d Chromatography GLC h a s been e x t e n s i v e l y used a s a method f o r t h e d e t e r m i n a t i o n of n i t r a z e p a m i n pharmaceutical preparation; a l s o f o r t h e determ i n a t i o n of t h e d r u g and i t s m e t a b o l i t e s i n b i o l o g i c a l f l u i d s and t i s s u e s . F u r t h e r m o r e , GLC i s one of t h e most c o n v e n i e n t methods f o r t h e d e t e c t i o n and d e t e r m i n a t i o n of n i t r a zepam i n t o x i c o l o g i c a l s c r e e n i n g . The d r u g is chromatographed w i t h o u t d e r i v a t i s a t i o n o r a f t e r a c i d h y d r o l y s i s i n t o 2-amino-5-nitrobenzophenone. The g a s l i q u i d chromatographic c o n d i t i o n s are given i n Table 3 .

Table 3 Stationary phase Detect o r : a r r i e r Zolumn temper:as 0 i t u r e ,C

--Jr
Remarks* Refer-

2% of OV-17 on Flame Chromosorb G-Hr i o n i s a t i o n 3% of OV-1 on Flame ionisat ion Chromosorb Q (60 t o 8 0 mesh) 3% of OV-1 on Chrom Q ( l O 0 t o 120 mesh) Flame ionisat ion

N2
N2

260 245

45

N2

250

For t h e d r u g & i t s metabolites For t h e hydrolysis products After acid hydrolysis

46

210

~~~

3% of OV-17 on 6 3 ~elec- ,r-CH 4 Diatomate CQ(8( t r o n c a p t u r e 9:l) t o 100 mesh)

245

47

3% of OV-17 o r Sp-2250 on shromosorb W o r Supelcoport (100 t o 120 mesh)

63Ni elect r o n captur e

245 275

a s hydrolyt i c product

48

- - - - - L

NlTRAZEPAM

511

'
Stationary phase Refer5nce 49 3% of OV-17 on 6 3 N i e l e c t - A r Gas-Chrom Q ( 6 0 r o n capture t o 80 mesh) 235 a f t e r acid h y d r o 1y s i s

3.8% o f SE-30

Flame ionisat ion

He

240

a f t e r acid hydrolysis

50

c hromosorb

2% of OV-17

on

W.H.P. (80 t o 1 0 0 mesh)

6 3 N i e l e c t - He ron capture

27 5

51

U n l e s s o t h e r w i s e s t a t e d i n t h e r e m a r k s , t h e d r u g h a s been determined underivatised. Lafargue,eta1(24)have r e p o r t e d a gas c h r o m a t e g r a p h i c metFod f o r t h e i d e n t i f i c a t i o n of n i t r a z e p a m i n t h e g a s t r i c f l u i d . The l a t t e r i s e x t r a c t e d a f t e r b e i n g made n e u t r a l o r weakly a c i d i c , w i t h e t h e r . The extract i s t h e n examined by G C i n a 2-metre column L packed w i t h 3% of OV-17 on G a s Cbrom Q ( 1 g O t o 120 mesh) and o p e r a t e d a t 250 ( o r 210 f o r the hydrolysis products). 6.4 PolarographyS e v e r a l methods have been p u b l i s h e d f o r t h e d e t e r m i n a t i o n of n i t r a z e p a m and r e l a t e d d e r i v a t i v e s i n pharmaceutical formulations as w e l l as i n biologic a l f l u i d s , ( b l o o d , u r i n e , and serum). O e l s c h l a e g e r , e t a 1 (52) had r e p o r t e d a p r o c e d u r e f o r t h e d e t e r m i n a t i o n of n i t r a z e p a m a f t e r t h e d r u g i s s e p a r a t e d by a TLC on s h e e t s h a v i n g s i l i c a g e l o r a l u m i n a a s a d s o r b e n t on a polyethylenetetrap h t h a l a t e b a c k i n g w i t h o u t removal of t h e p l a s t i c f o i l

512

HASSAN Y . ABOUL-ENEIN

el

al.

'Ihe p l a s t i c f o i l is s t a b l e t o all s o l v e n t s used and tk binder and t h e s o r b e n t d o n o t i n t e r e f e r e w i t h t h e development of t h e c u r r e n t v e r s u s p o t e n t i a l c u r v e . Any z i n c - c o n t a i n i n g i m p u r i t i e s d o , however, i n t e r f e r e and must b e marked w i t h EDTA b e f o r e measurement. A dropping-mercury e l e c t r o d e i s used i n t h e determina t i o n i n which n i t r a z e p a m and i t s 7-amino r e d u c t i o n product are determined. Dimethyl s u l f o x i d e and dimethylformamide a r e used as s o l v e n t s . The r e c o v e r y is more t h a n 95%. For t h e amino compound, good r e c o v e r y i s achieved o n l y i f t h e s o r b e n t i s removed; t h i s i s not necessary f o r nitrazepam.

E l l a i t h y , e t a 1 ( 5 3 ) , had r e p o r t e d t h e d e t e r m i n a t i o n of some b e n z o d i a z e p i n e s , among which n i t r a z e p a m i s i n c l u d e d , by d i f f e r e n t i a l p u l s e polarography w i t h a dropping-mercury i n d i c a t o r e l e c t r o d e and a s a t u r a t e d mercuric sulphate r e f e r e n c e electrode. The c a l i b r a t i o n g r a p h of peak c u r r e n t v e r s u s drug conc e n t r a t i o n i s r e c t i l i n e a r f o r c o n c e n t r a t i o n down t o 0.14 ug p e r m l . Nitrazepam i s d i s s o l v e d i n acet o n i t r i l e and t h e s o l u t i o n is b u f f e r e d a t p 4 . 8 . H The method i s a p p l i c a b l e f o r t h e d e t e r m i n a t i o n of n i t r a z e p a m and some o t h e r b e n z o d i a z e p i n e s i n u r i n e ( 2 ml) w i t h o u t p r i o r e x t r a c t i o n . Halvorsen, e t a1 (54) have r e p o r t e d t h e e l e c t r o r e d u c t i o n and p o l a r o g r a p h i c d e t e r m i n a t i o n of n i t r a zepam i n serum. The e l e c t r o - r e d u c t i o n of nitrazepam h a s been s t u d i e d by p o l a r o g r a p h y , c y c l i c v o l t a mmetry, chromopotentiometry and c o n t r o l l e d - p o t e n t i a l coulometry. I n a phosphate b u f f e r s o l u t i o n of p H 6.9 t h e r e a r e two r e d u c t i o n s t e p s ; t h e f i r s t g i v i n g a well-defined p o l a r o g r a p h i c wave b e i n g a f o u r e l e c t r o n r e d u c t i o n of t h e n i t r o - g r o u p and t h e second b e i n g a two-electron r e d u c t i o n . The o x i d i s e d form of n i t r a z e p a m i s s t r o n g l y adsorbed on t h e e l e c t r o d e s u r f a c e and t h u s i t i s p o s s i b l e t o d e t e r mine n i t r a z e p a m i n t h e p r e s e n c e of p r o t e i n s . The p o l a r o g r a p h i c d e t e r m i n a t i o n of n i t r a z e p a m i n whole blood, i n a c u t e p o i s o n i n g , h a s been r e p o r t e d (55). The procedure i s based on a d m i n i s t r a t i o n of n i t r a z e p a m t o r a t s and t h e homogenised blood samples a r e d i l u t e d w i t h an e l e c t r o l y t e c o n s i s t i n g of 1:l m i x t u r e of methanol w i t h Britton-Robinson b u f f e r s o l u t i o n of p 2 . 2 t o 3.3. H The s o l u t i o n s a r e examined p o l a r o g r a p h i c a l l y i n t h e r a n g e 0.0 to0.6V.

NITRAZEPAM

513

The p o l a r o g r a p h i c and s p e c t r a l b e h a v i o u r of 7-amino and 7-acetamido n i t r a z e p a m m e t a b o l i t e s have been u t i l i s e d t o e f f e c t s e p a r a t i o n s of m i x t u r e s ( 5 6 ) . Changes of UV a b s o r p t i o n s p e c t r a w i t h pH in s o l u t i o n a r e used t o d e t e r m i n e pKa values f o r n i t r a z e p a m m e t a b o l i tes 7 -Ace t a m i d o -n it r a z epam gives t w o pKa v a l u e s , c o r r e s p o n d i n g t o p r o t o n a t i o n i n a c i d and d e p r o t o n a t i o n of t h e n e u t r a l m o l e c u l e i n a l k a l i n e media. 7 - h i n o n i t r a z e p a m g i v e s t h r e e pKa v a l u e s , t h e t h i r d one being due t o a d d i t i o n a l p r o t o n a t i o n i n a c i d media. The s p e c t r a a r e e x p l a i n ed by c o n s i d e r i n g them t o b e superimposed s p e c t r a of t h e two benzene r i n g s , one m o n o s u b s t i t u t e d , and o n e t r i s u b s t i t u t e d w i t h i n t h e molecule. D i f f e r e n c e s i n t h e pK v a l u e s o r t h e p o l a r o g r a p h i c b e h a v i o u r b e t w e e n n i t r a z g p a m and i t s m e t a b o l i t e s a r e used t o e f f e c t n o v e l s e p a r a t i o n a f t e r s o l v e n t e x t r a c t i o n s from aqueous b u f f e r e d s o l u t i o n s .

ACKNOWLEDGEMENT

The a u t h o r s w i s h t o t h a n k M r . Dennis Charkowski, o f t h e Toxicology C e n t e r , The U n i v e r s i t y of Iowa, Iowa C i t y , Iowa 52242, U.S.A., f o r d e t e r m i n i n g t h e mass spectrum of n i t r a zepam, and M r . E s s a m A. L o t f i f o r h i s h e l p i n t h e l i b r a r y research.
A sample of nitrazepam-R04-5360/000, w a s k i n d l y d o n a t e d by D r . R. Amrein and D r . S. Kessler of F. Hoffmann - L a Roche & Co. L i m i t e d , B a d e , S w i t z e r l a n d .

514

HASSAN Y. ABOUL-ENEIN er ai.

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27. 3. Dobrecky, B. Gonzalez, and E. M o l i n a r i , Revta Asoc. bioquim. a r g e n t . , 36, 214 (1971) - t h r o u g h Anal. A b s t r . , -, 3062 ( 1 9 7 3 ) . 24 28. K.H. Beyer and W. Sadee, Arch. Pharm. Berl., 3 0 2 , 1 5 2 18, (1969) - t h r o u g h Anal. A b s t r . - 4315 (1970). 29. D. K r u g e r s , Pharm. Weekbl. Ned., 1 8 1 0 2 5 (1973) 0, t h r o u g h Anal. A b s t r . 27, 247 ( 1 9 7 4 ) . 30. H. Sawada, A. Hara, S.Asano, and Matsumoto, C l i n . Chem., - 1 5 9 6 ( 1 9 7 6 ) - t h r o u g h Anal. A b s t r . -, 3 D 7 1 ( 1 9 7 8 ) . 22, 34 31. M.T.H.A.
Meeles, S.M. Dreijer-Van Glas, and G.L. Kho, Pharm. Weekbl., 110, 492 (1975) - t h r o u g h Anal. A b s t r .

30,

1E34 (1976).

516

HASSAN Y. ABOUL-ENEIN er al.

3 2 . B. U n t e r h a l t , B. K e i l , and K. Reinhold, D t . Apothztg.,&l, 1 7 7 5 ( 1 9 7 1 ) - through Anal. A b s t r . , 789 (1972). 3 3 . J . Dobrecky, Revta Farm., B . A i r e s , through Anal. A b s t r . , 2, 1453 ( 1 9 7 1 ) .

23, 112,1 1 5

(1970)

3 4 . S . Ebel and H. Kussmaul, Arch. Pharm., Berl., 3 0 7 , 2 3 0 ( 1 9 7 4 ) - through Anal. A b s t r . , 2196 (1974).

27,

3 5 . S.N.

T e w a r i and Shukla; Pharmazie,

31,

893 (1976).

3 6 . H.M. S t e v e n s and R.W. J e n k i n s , J. Forens. S c i . Soc., 1 8 3 ( 1 9 7 1 ) - through Anal. A b s t r . , 2645 ( 1 9 7 2 ) .

22,

11,

3 7 . S. N e g r i t e s c u , E . Mihalas, M. S t e r e s c u , and C . Pharmazie, 31, 8 2 3 ( 1 9 7 6 ) . 3 8 . H. Schuetz, J . Chromat.,

Ioan;

94,1 5 9

(1974).

3 9 . N.Y. Golovenko, V.G. ZinKovskii, T.L. Karaseva, and A.V. B o g a t s k i i , F a r m a t s i y a , 2, 3 9 ( 1 9 7 7 ) - through Anal. A b s t r . , 34, 2D 58 ( 1 9 7 8 ) . 4 0 . A.W.

Missen, C l i n . Chem.,

22, 927

(1976).

4 1 . B. Moore, G . N i c k l e s s , C . H a l l e t t , and A.G. Chromat., 215 ( 1 9 7 7 ) .

137,

Howard, J .
(1977).

4 2 . K. Harzer and R. B a r c h e t , J. Chromat., 4 3 . M.D. O s s e l t o n , M.D. Pharm. Pharmacol.,

132, 83

2,6 0 4

Hammond, and P. J . T w i t c h e t t , J . (1977). Franch; J . Anal.

4 4 . R.C. Basatt, C.B. S t e w a r t , and S . J . Toxicol., 10 (1977).

1,

4 5 . Marcucci, R. F a n e l l i , and E . M u s s i n i , J. Chromat., 318 ( 1 9 6 8 ) . 4 6 . P. Lafargue, P . P o n t , and J. Meunier; Annls. Pharm. -, 477 ( 1 9 7 0 ) . 28 4 7 . K.M.

37,
fr.,

J e n s e n , J. Chromat.,

4 8 . L. Kangas, J . Chromat., 4 9 . G.P. B e h a r r e l l , D.M. -, 45 ( 1 9 7 2 ) . 70

111,389 ( 1 9 7 5 ) . 136,259 ( 1 9 7 7 ) .
McLaurin, J.Chromat.,

H a i l e y , and M.K.

NITRAZEPAM

517

50. T.A. Rejent and K . C . Wahl, Clin. Chem. (Winston - Salem, N.C.), 22, 8 8 9 ( 1 9 7 6 ) .

51. I.A. Zingales, J. Chromat.,

61,

237 ( 1 9 7 1 ) .

52. H. Oelschlaeger, S. Lumbantoruan, J . Volke, and G. Kraft, Z. Analyst., Chem., 279, 257 ( 1 9 7 6 ) .

53. M.M. Ellaithy, J. Volke, and 0. Manousek, Talanta, 137 (1977).

5 4 . S. Halvorsen and E. Jacobsen, Analytica -, 127 ( 1 9 7 2 ) . 59

24,

Chim. Acta.,

55. A. Kobilela-Krzyanowska, Pharmazie,

31,

649 ( 1 9 7 6 ) .

56. J. Barret, W.F. Smyth, and J.P. Hart, col., 26, 9 ( 1 9 7 4 ) .

J. Pharm. Pharma-

NITROGLYCERIN
Edward F. McNiff, Peter S. K. Yap, and Ho- Leung Fung
1.

2.

3. 4. 5.

6.

7.

8.

Description 1.1 Name, Formula, Molecular Weight 1.2 Appearance, Color, Odor Physical Properties 2.1 Nuclear Magnetic Resonance Spectrum 2.2 Infrared Spectrum 2.3 Ultraviolet Spectrum 2.4 Mass Spectrum 2.5 Vapor Pressure and Boiling Point 2.6 Melting and Crystal Properties 2.7 Density 2.8 Viscosity 2.9 Solubility Synthesis Stability 4.1 Chemical Stability 4.2 Physical Stability Metabolism 5.1 Biochemistry 5.2 Site of Metabolism 5.3 Metabolic Fate Pharmacokinetics 6.1 Tissue Distribution 6.2 Intravenous Administration 6.3 Oral and Topical Administration Methods of Analysis 7.1 Official Methods 7.2 Spectrophotometnc 7.3 Thin Layer chromatography 7 . 4 Polarography 7.5 Gas Chromatography 7.6 High Performance Liquid Chromatography References

520 520 520 520 520 520 523 523 523 523 523 523 524 524 525 525 527 527 527 528 529 53 1 53 1 532 532 533 533 533 534 534 535 535 531

Analytical Profiles of Drug Substances, 9

519

AU

Copyrighi1 0 1960 by Academic Ress, Inc. rights of reproduction i any form reserved. n ISBN: &12-260809-7

5 20

EDWARD F. McNIFF, et u1.

1 . Description
1 . 1 Name, Formula, Molecular Weight Nitroglycerin (glyceryl trini t r a t e , t r i n i t r o g l y c e r o l ) i s 1,2,3-propanetriol t r i n i t r a t e .

C3H 5N309 M.W. 227.09

CH20N02 ~HONO~ CH20N02

1.2 Appearance, Color, Odor Pale yellow, odorless, o i l y liquid w i t h a sweet, burning t a s t e .
2.

Physical Properties

2.1 Nuclear Magnetic Resonance Spectrum An NMR sDectrum of nitroalvcerin i s shown i n Fia. 1 . The sample was isolated from a l a h o s e adsorbate by eth& extraction. After solvent evaporation, the sample was p u r i fied by hexane elution from a neutral s i l i c a column, followed by gentle hexane evaporation under a stream of nitrogen. A CDC13 solution spectrum was r u n on a Varian T-60A spectrometer using trimethylsilane as the internal reference. The multiplet a t - 4.8 6 i s assigned t o the four protons a t the 1 and 3 carbon atoms, and t h a t a t - 5.5 6 i s assigned t o the proton a t the C - 2 position. Small s i n g l e t s appearing a t - 1 . 5 6 and - 7 . 2 6 a r e apparently due t o t r a c e impurities of hexane residue and non-deuterated chloroform, respectively. The integrated areas c o r r e l a t e well w i t h the structural assignments. No attempts were made t o i n t e r p r e t the s p l i t t i n g patterns.
2 . 2 Infrared Spectrum The IR spectrum, F i g . 1 , was obtained on a PerkinElmer model 272B infrared spectrophotometer. Nitroglycerin was isolated and purified as described i n 2.1. Sample m o u n t i n g was by formation of a c a p i l l a r y film of neat n i t r o glycerin between NaCl plates. The band a t 850 cm-l i s found i n a l l organic and inorganic n i t r a t e s . Bands a t 1650 and 1280 cm- a r e attributed t o symmetrical stretching and deformation vibrations of the NO2 group, respectively. T h i s spectrum compares well w i t h one t h a t was reported along w i t h a large number of other n i t r a t e s by Pristera e t a l l .

80

1 . . . . 1 . . . . 1 . . . . 1 . . . . 1 . , . . 1
70

I . .

. . .

.
. O

LO

50

ppu

10

. . . .. . . l
1

. . . . I . . . . I ,
I 0

'

20

F i g . 1: NMR Spectrum of n i t r o g l y c e r i n

ii

h l

..

NITROGLYCERIN

523

2.3 U 1 t r a v i o l e t Spectrum Nitroglycerin, i n a solution of neutral pH, has no appreciable absorbance i n the near u l t r a v i o l e t and v i s i b l e region2.

2.4 Mass Spectrum The mass spectra of 21 nitrate e s t e r s , including nitroglycerin, were run on an A.E.I. MS2H single focusing mass spectrometer, operating a t 70 ev3. The base peak o f M/e 46 (NO$) i s c h a r a c t e r i s t i c o f the lower n i t r a t e s . Other major peaks, w i t h t h e i r corresponding structural assignments, a r e shown i n Table I :
TABLE
13

Mass Spectral Characteristics o f Nitroglycerin

M/ e
28 29 30 43 46 76

Relative Intensity
6 15 24 5 100 9

S tructura 1 Assi gnment

CHO' NO'

cot

2.5 Vapor Pressure and Bioling P o i n t The vapor pressure of nitrogl cerin a t Z O O , 25O and 37O has been reported4y5 as 2.6 x lo-', 5.5 x and 2 . 2 x Torr, respectively. The gravimetric Knudson effusion technique has been used t o study the vapor pressure of nitroglycerin i n molded t a b l e t s 6 . Pure nitroglycerin has an a parent boiling p o i n t of 1450 C ( w i t h violent decomposition) .

1:

2.6 Melting and Crystal Properties A t low temperatures, nitroglycerin e x i s t s i n two crystal forms. I t freezes t o form a s t a b l e dipyramidal polymorph which melts a t 13.20 C . Under some conditions, an unstable t r i c l i n i c crystal (m.p. 2 . 2 0 C ) may form. This l a b i l e polymorph will convert into the more s t a b l e form upon standi n g l . 2.7 Density The density of nitroglycerin i s 1.601 a t 15' C4.

2.8 Viscositv7

524
-

EDWARD F. McNIFF, et

N/

Viscosity ( c P ) 35.5 21 .o 9.4

6.8

Temperature
20 30

(OC)

50 60

2.9 Solubility The following information i s available from r e f e r ence 7: nitroglycerin has an aqueous s o l u b i l i t y o f 1.73 and 2.46 mg/ml a t 200 and 600 C respecti'vely; ethanol dissolves nitroglycerin t o the extent of 375 mg/gm a t Oo and 540 mg/gm a t 200; h o t ethanol i s miscible w i t h nitroglycerin i n a l l proportions; other solvents completely miscible w i t h n i t r o glycerin are: acetone, ether, glacial a c e t i c acid, ethylacetate, benzene, toluene, phenol, ni trobenzene, chloroform, ethylene chloride and n i t r i c e s t e r s . Additionally, i t has been reported4 t h a t nitroglycerin i s miscible with pyridine and ethylene bromide, b u t i s only sparingly soluble in petroleum ether, liquid petrolatum and glycerol. The solub i l i t y in methanol and carbon d i s u l f i d e i s 56 mg/gm and 8.3 mg/gm respectively - Using the- aqueous sol u b i 1 i ty o f nitroglycerin and partitioning data, Horhota and Fung8 calculated n i t r o glycerin s o l u b i l i t y i n d i f f e r e n t water-polyethylene g ycol 400 co-solvent systems. For instance, the calculated sol ub i l i t y of nitroglycerin i n a 90% (w/v) polyethylene g ycol 400-water mixture was estimated a t 135 mg/ml.

Synthesis Organic n i t r a t e synthesis i s commonly accomplished by e s t e r i f i c a t i o n of the corresponding a l c ~ h o l , ~ o, .~ In the l case of nitroglycerin, the n i t r a t i n g mixture consists of equal volumes of n i t r i c and s u l f u r i c acids. A small amount o f urea o r urea n i t r a t e i s added as a scavenger f o r any excess nitrous acid present. Esterification i s carried out by slow addition of glycerol t o the mixed acids.

3.

NO;

+ R-OH

R-O-N02 + H

Careful control o f temperature and r a t e of addition reduces or eliminates the side reaction of alcohol oxidation. The e s t e r can be separated by p o u r i n g the reaction mixture i n t o cold water o r by careful d i s t i l l a t i o n .

NITROGLYCERIN

525

4.

Stability 4.1 Chemical S t a b i l i t y

4.11 Hydrolysis The s tabi 1 i ty of nitroglycerin i n a1 coho1 i c solutions as a function of pH has been studied by Arnshlerll. The compound i s r e l a t i v e l y s t a b l e i n neutral and weakly acidic solutions b u t degrades very rapidly i n the presence o f a1 kal i* 3 3 . Alkaline hydrolysis o f n i t r a t e e s t e r s can proceed v i a three possible mechanisrnsl4: ( a ) Nucleophilic substitution ( S N ~ )
CH2R

0HniH2-

0.NO2-+R.CH2-CH2-OH + NO;

(alcohol t nitrate)

( b ) 6-hydrogen elimination ( E 2 )

RCH-CH2--U.N02+H20

+ R C H : C H ~ + NO; ( o l e f i n +
nitrate)

OH

nH

( c ) a-hydrogen elimination (ECo2)

1 3

RCH2*CH-0-NO2+

H20

RCH2 * C H O + NO;

(carbonyl t n i t r i t e )

The i n i t i a l step of alkaline hydrolysis of nitroglycerin involves a-elimination a t the secondary n i t r a t e g r o u p resulting in the formation o f n i t r i t e ion and a carbonyl . T h i s electronegative carbonyl g r o u p causes e i t h e r of the remaining primary n i t r a t e s t o be more suscept i b l e t o nucleophilic attack. A slower reaction on the primary n i t r a t e , producing the alcohol and n i t r a t e ion, becomes more important w i t h increasing r a t i o s o f hydroxide ion t o nitroglycerin2. Alkaline degradation of n i t r o glycerin i s accompanied by the appearance and subsequent disappearance of an u l t r a v i o l e t absorption peak near 335 nm due, presumably, t o the monocarbonyl intermediate. The maximum absorbance and the peaking time of this chromophore are dependent upon i n i t i a l concentrations o f nitroglycerin and hydroxide ion. T h i s reaction i s the basis f o r a kinetic

526

EDWARD F. McNIFF, e r a / .

assay procedure f o r n i t r o g l y c e r i n l 5 - 1 7 which i s discussed 1a t e r Acid c a t a l y z e d h y d r o l y s i s o f n i t r o g l y c e r i n was found t o occur a t a much slower r a t e than t h a t o f a l k a l i n e h y d r o l y s i s l ' + , 1 * . I n c u b a t i o n o f n i t r o g l y c e r i n a t 370 f o r 15 minutes i n 4 N NaOH r e s u l t e d i n e s s e n t i a l l y complete d e n i t r a t i o n , w h i l e i n 4 N HC1, n i t r o g l y c e r i n was degraded o n l y 28% a f t e r 6 h o u r s l s . Under a c i d c o n d i t i o n s , t w i c e as much g l y c e r y l - 1 , 2 - d i n i t r a t e i s formed compared t o g l y c e r y l - l , 3 d i n i t r a t e l g , suggesting t h a t t h e i n i t i a l r e a c t i o n s i t e i s on t h e p r i m a r y n i t r a t e . The k i n e t i c s o f n i t r o g l y c e r i n h y d r o l y s i s i n n i t r i c a c i d a t 200 t o 800 C has a l s o been studied20. Klason and Carlson21 observed t h a t a1 k a l i n e degradation o f n i t r o g l y c e r i n i n t h e presence o f phenylmercaptan r e s u l t e d i n t h e f o r m a t i o n o f d i p h e n y l d i s u l f i d e and g l y c e r o l . I t was l a t e r shown t h a t reduced g l u t h a t h i o n e (GSH) r e a c t s w i t h n i t r o g l y c e r i n t o produce i n o r g a n i c n i t r i t e ions22. Subsequent s t ~ d i e s c h a r a c~ e~i z e d t h e r e a c t i o n as: ~ ~ , t r

C3H5(0N02)3 f 2GSH-+C3H5(0N02)2

OH + GSSG

HN02

T h i s r e d u c t i o n process was found t o be r e l a t i v e l y slow. With equal and 10 molar e q u i v a l e n t s o f GSH, 0 and 22% o f n i t r o g l y c e r i n were degraded w i t h i n 1 hour a t 37O C, r e s p e c t i v e l y . B i o t r a n s f o r m a t i o n o f n i t r o g l y c e r i n i n t h e body i s a p p a r e n t l y i _ iv c l o s e l y r e l a t e d t o t h e above r e a c t i o n . The -n v_o process, however, i s a much f a s t e r r e a c t i o n because i t i s enzymatica l l y c a t a ysed. 4 12 P h o t o l y t i c and Thermal S t a b i l i t y Although i t was suggested t h a t n i t r o g l y c e r i n i s t~ suscepti b e t o p h o t ~ l y s i s ~ h e,r e i s no s u p p o r t i n g evidence i n t h e li e r a t u r e . I n aqueous s o l u t i o n , exposure t o l i g h t does n o t ead t o a c c e l e r a t e d disappearance o f n i t r o g l y c e r i$ 6 . The thermal decomposition o f n i t r o g l y c e r i n i s h i g h l y dependent on t h e r a t i o o f n i t r o g l y c e r i n mass t o t h e volume o f t h e r e a c t i o n presumably due t o p r o d u c t i n h i b i t i o n by NO2. W i t h i n t h e temperature range o f 140 t o 160' and a mass t o volume r a t i o o f 3.5 x vapor g m phase degradation f o l l o w s f i r s t o r d e r k i n e t i c s and obeys t h e Arrhenius r e l a t i o n s h i p w i t h an energy o f a c t i v a t i o n (Ea) o f approximately 36 kcal/mole. D e v i a t i o n from f i r s t o r d e r k i n e t i c s i s observed i n t h e l i q u i d phase, and i s p r o b a b l y due t o a u t o c a t a l y t i c e f f e c t s 2 7 . Below 1400, t h e decomposit i o n r e a c t i o n s a r e a l s o a f f e c t e d by a u t o c a t a l y s i s 2 * .

NITROGLYCERIN

527

4.2 P h y s i c a l S t a b i l i t y I n s t a b i l i t y o f n i t r o g l y c e r i n i n pharmaceutical dosage forms can g e n e r a l l y be a t t r i b u t e d t o two processes, v i z : ( a ) v o l a t i z a t i o n l e a d i n g t o l o s s o f d r u g t o t h e atmosphere,and ( b ) s o r p t i o n o f d r u g t o p l a s t i c s . The a p p r e c i a b l e v o l a t i l i t y o f n i t r o g l y c e r i n a t room temperatures has been shown t o be a m a j o r cause o f l o s s o f potency and i n t e r t a b l e t m i g r a t i o n o f drug d u r i n g storage o f u n s t a b i l i z e d sub1 i n g u a l tablet^^,^^. T h i s problem has been somewhat a l l e v i a t e d by t h e a d d i t i o n o f p o l y e t h y l e n e g l y c o l 400 and povidone as s t a b i 1 izers 3 0 - 3 4 . Drug 1oss due t o s o r p t i v e phenomena has been imp1 i c a t e d when n i t r o g l y c e r i n t a b l e t s a r e s t o r e d i n p l a s t i c c o n t a i n e r s and u n i t dose s t r i p packa g e ~ ~ ~ ' ~ e ~ u .l a t i o n s (promulgated i n 1972)36 r e q u i r e FDA r g t h a t n i t r o g l y c e r i n t a b l e t s be packaged i n t i g h t c o n t a i n e r s , p r e f e r a b l y o f g l a s s w i t h metal screw caps, and dispensed i n t h e o r i g i n a l , unopened c o n t a i n e r w i t h a s p e c i a l warning l a b e l . No more t h a n 100 t a b l e t s s h o u l d be dispensed i n each container. Problems o f s t a b i l i t y and potency r e l a t i n g t o extemporaneously prepared n i t r o g l y c e r i n i n f u s i o n s have r e c e n t l y been p o i n t e d Extensive loss o f n i t r o g l y c e r i n from i n t r a v e n o u s s o l u t i o n s s t o r e d i n p l a s t i c i . v . bags can be a t t r i b u t e d t o s o r p t i ~ n ~ ~ , i~ c~ -i n t a c,t d r u g can be s n e ~ ~ recovered from t h e c o n t a i n e r 4 0 . P l a s t i c t u b i n g used f o r t h e a d m i n i s t r a t i o n o f intravenous n i t r o l y c e r i n s o l u t i o n s a l s o causes d r u g l o s s due t o s ~ r p t i o n ~ ~ High d e n s i t y p o l y e t h y , ~ ~ . l e n e t u b i n g , however, i s n o n - a d s o r p t i ~ e ~ ~ . Metabolism The metabol ism o f n i t r o g l y c e r i n and o t h e r o r g a n i c n i t r a t e s has been e x t e n s i v e l y r e ~ i e w e d ~ ~ Only~ a summary o f t h e - ~ . m a j o r f i n d i n g s r e g a r d i n g t h e metabolism o f n i t r o g l y c e r i n i s presented here. 5.1 B i o c h e m i s t r Heppel and i i l m o e 2 2 showed t h a t t h e spontaneous r e a c t i o n between n i t r o g l y c e r i n and GSH t o be c a t a l y z e d by a hog 1 i v e r microsomal enzyme. I n i t i a l c h a r a c t e r i z a t i o n o f t h e enzymatic process u s i n g p a r t i a l l y p u r i f i e d hog l i v e r acetone powder demonstrated t h a t t h e system i s anaerobic, has an o p t i m a l pH o f 7-8, i s i n h i b i t e d by c u p r i c s u l f a t e and s t i m u l a t e d by cyanide. Subsequent i n v e s t i g a t i o n s showed t h a t t h e l i v e r enzymes, p u r i f i e d f r o m r a t and guinea p i g l i v e r and named o r g a n i c n i t r a t e reductases (ONR), c o n s i s t e d o f 2 d i s t i n c t fragments w i t h d i f f e r e n t a c t i v i t y f o r n i t r o g l y c e r i n and o t h e r o r g a n i c n i t r a t e s 4 7 ,4*. The two d i f f e r e n t enzymes were e s t i m a t e d t o have a m o l e c u l a r w e i g h t o f 14,000 and

5.

528

EDWARD F. McNIFF, er al.

43,700 r e s p e c t i v e l y . Needleman and Hunter23 developed a r a p i d and s e n s i t i v e enzymatic assay t o q u a n t i f y t h e r e l a t i v e a c t i v i t i e s o f r a t l i v e r ONR toward d i f f e r e n t o r g a n i c n i t r a t e s . T h i s assay measures t h e disappearance o f reduced tri phosphopyridi ne n u c l e o t i d e (TPNH), which i s consumed f o r t h e p r o d u c t i o n o f GSH, which i n t u r n i s r e q u i r e d f o r t h e d e n i t r a t i o n o f t h e organic n i t r a t e ( F i g . 3 ) . ORGANIC

ORGANIC NITRATE REDUCTASE

GLUTATH IONE REDUCTME

F i g u r e 3. Biochemical r e a c t i o n s i n v o l v e d i n t h e d i n i t r a t i o n o f organic n i t r a t e s . The maximum v e l o c i t i e s o f t h e enzymatic r e a c t i o n o f d i f f e r e n t organic n i t r a t e has been r e p o r t e d 2 3 . P o l y n i t r i c e s t e r s a r e r a p i d l y metabolized by l i v e r ONR, i n t h e o r d e r o f manni t o 1 hexani t r a t e >> e r y t h r i t o l t e t r a n i t r a t e >> n i t r o g l y c e r i n . Replacement o f a n i t r a t e group w i t h a hydrogen atom o r a hydroxy group, o r i n t r o d u c t i o n o f an e t h e r l i n k a g e i n t o a l i n e a r c h a i n n i t r a t e compound, decreases t h e r a t e o f enzymatic t r a n s f o r m a t i o n . Branched c h a i n a l c o h o l n i t r a t e s a r e also s i g n i f i c a n t l y l e s s s u s c e p t i b l e t o organic n i t r a t e reductase degradation. I n v i t r o s t u d i e s have a1 so demons t r a t e d t h a t t h e metabolism o f n i t r o g l y c e r i n i n l i v e r homogenates can be enhanced o r depressed upon p r e t r e a t m e n t o f t h e experimental animals w i t h b a r b i t u r a t e s and bromobenzene r e s p e c t i ~ e l y ~ ~ I t was suggested51 t h a t pheno,~~. b a r b i t a l pretreatment caused increase i n t h e amount o f reduc tase enzyme as w e l l as t h e a c t i v i t y o f GSH g e n e r a t i n g capacity. 5.2 S i t e o f Metabolism Needleman and H a r k e ~ ~ ~ compared t h e r a t e o f degradat i o n o f n i t r o g l y c e r i n i n i s o l a t e d perfused r a t l i v e r t o t h e -n v_ o b i o t r a n s f o r m a t i o n r a t e s . The i n v i t r o h a l f t i m e o f i _ iv 2 minutes was comparable t o t h a t observed i n i n t a c t e x p e r i mental animals. I n e v i s c e r a t e d r a t s , t h e b i o l o g i c a l h a l f -

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l i f e o f n i t r o g l y c e r i n was 7 t o 8 minutes as compared t o l e s s than 2 minutes i n c o n t r o l s 5 2 . These experiments c l e a r l y e s t a b l i s h t h e importance o f h e p a t i c metabolism o f n i t r o g l y c e r i n i n experimental animals. Recently, Maier e t a l S 3 showed t h a t a r e l a t i o n s h i p e x i s t s between i n v i v o n i t r o g l y c e r i n b i o a v a i l a b i l i t y (100 mg/kg o r a l l y 7 n x s ) and t h e --t r o l i v e r ONR a c t i v i t y i n i n d i v i d u a l animals. i n vi Glutathione-dependent ONR a c t i v i t y was found o n l y i n t r a c e q u a n t i t i e s i n t h e kidney and was absent i n t h e lung, small i n t e s t i n e , h e a r t and s k i n . These d a t a suggested t h a t f i r s t pass metabolism of o r a l l y administered n i t r o g l y c e r i n occurred primarily i n the l i v e r . Under p h y s i o l o g i c a l c o n d i t i o n s , r a t serum a l s o hydrolyzed n i t r o g l y c e r i n t o d i n i t r a t e s and mononitrates, b u t a t a much slower r a t e . The h a l f - l i f e o f serum degradation The e f f e c t s was found t o be 15 t o 20 minutes a t 37O C 2 4 9 5 4 . of c o n c e n t r a t i o n , temperature, r e d blood c e l l hemolysi s and s i l v e r n i t r a t e a d d i t i o n on n i t r o g l y c e r i n s t a b i l i t y i n human and r a t plasma have a l s o been examineds5. W i t h i n t h e temperat u r e range o f -200 t o 370 C, degradation was shown t o obey t h e Arrhenius r e l a t i o n s h i p w i t h an apparent energy o f a c t i v a t i o n (Ea) o f 24.1 and 19.0 kcal/mole f o r human and r a t plasma, r e s p e c t i v e l y . Depending w o n t h e temperature, n i t r o g l y c e r i n i s degraded 10-50 times f a s t e r i n r a t plasma compared t o human plasma. Hepatic and blood metabol ism o f n i t r o g l y c e r i n has been demonstrated i n o t h e r animal speciess6. Human 1 i v e r biopsy samples were shown t o c o n t a i n a g l u t a t h i o n e dependent ONR capable o f r a p i d b i o t r a n s f o r m a t i o n o f n i t r o g l y c e r i n t o i t s lower n i t r a t e s 5 7 . The s i t e and mechanism o f o x i d a t i o n o f n i t r o g l y c e r i n t o carbon d i o x i d e has a l s o been i n v e s t i gated58. I n v i t r o experiments demonstrated t h a t homogenates o f the liv%,kidney, b r a i n and s k e l e t a l muscle o x i d i z e d g l y c e r o l , a m e t a b o l i t e o f n i t r o g l y c e r i n , t o C02 b u t c o u l d n o t o x i d i z e n i t r o g l y c e r i n t o C02. E v i s c e r a t i o n o f r a t s i n h i b i t e d C02 p r o d u c t i o n a f t e r t h e a d m i n i s t r a t i o n o f n i t r o g l y c e r i n b u t n o t g l y c e r o l . Pretreatment o f t h e rodents w i t h p h e n o b a r b i t a l o r SKF 525A had no e f f e c t on n i t r o g l y c e r i n o x i d a t i o n , nor was t h e r e an enhancement o f C02 r e l e a s e i n n i t r o g l y c e r i n t o l e r a n t animals. Thus, C02 p r o d u c t i o n may have r e s u l t e d from b i o t r a n s f o r m a t i o n a t e x t r a h e p a t i c s i t e s subsequent t o hepatic d e n i t r a t i o n o f n i t r o g l y c e r i n . 5.3 Metabolic Fate Upon o r a l a d m i n i s t r a t i o n o f 10 mg/kg o f 1,3-C14 n i t r o g l y c e r i n t o r a t s s 9 20% o f t h e l a b e l e d dose was e x p i r e d as carbon d i o x i d e w i t h an equal amount o f t h e r a d i o a c t i v i t y e x c r e t e d i n t h e u r i n e a t t h e end o f 4 hours. TLC-radio-

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chromatographic analysis revealed t h a t the cumulative urinary excretion consisted of 7% glycerol , 1 % glyceryl-1 , Z - d i n i t r a t e , 0.5% glyceryl-1 , 3 - d i n i t r a t e Y 4% glyceryl mononitrates and 8% of unidentified water soluble metabolites. Needleman e t a1 5 * administered a smaller dose ( 5 mg/kg) of radioactive nitroglycerin subcutaneously t o rats. They observed t h a t 17% of the dose was eliminated as expired C O Z Y w i t h urinary excretion accounting f o r another 50% of the r a d i o a c t i v i t y in 0-24 hours. The major urinary metabolites were the glyceryl mononitrates (32% of dose). The sum of the mononitrates and water soluble metabol i t e s (unidentified) accounted f o r 80% of the excreted l a b e l . A small f r a c t i o n of the labelled dose ( 1 .3%) was excreted as unchanged nitroglycerin. I n a more recent study, Hodgson and Lee60 administered a very h i g h oral dose, 180 mg/kg ( L D lo%), of n i t r o glycerin t o r a t s . Radioactive C02 accounted f o r 26% of the dose and 40% of the label was eliminated i n the urine w i t h i n 24 hours. These authors showed (Table 11) t h a t the major urinary metabolites a r e glyceryl d i n i t r a t e gl ucuronide (14% of dose) glyceryl mononi t r a t e (1 1 % ) and glycerol ( 7 % ) . T h i s study was the f i r s t w h i c h showed t h a t conjugation plays a major r o l e in the metabolism of nitroglycerin. TABLE 1160 Metabol i t e s of Nitroglycerin i n Rat Urine 24 Hours After Oral Administration of IL4C1 Nitroglycerin (180 mg/kg) Metabolite Nitroglycerin Glyceryl-l,3-dini t r a t e Glyceryl-1 , Z - d i n i t r a t e Glyceryl mononi t r a t e Glyceryl-l,3-dinitrate glucuronide Glyceryl-1 ,2-di n i t r a t e gl ucuronide Glyceryl mononi t r a t e glucuronide G ycerol 1 Unidenti f ied a Mean - SE of three r a t s +
% of Administered Dose

0.1 0.4 + O.Za 0.7 0.4 10.6 1.3 3.5 0.4 10.0 0.7 1 . 5 T 0.2 6.9 - 0.8 =-6

The metabolic f a t e of nitroglycerin i n the r a t can, therefore, be schematically summarized a s follows ( F i g . 4):

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Nitroglycerin
I

G l y c e r y l - l , 3 - D i n i t r a t e Glucuronide

G1 ycogen Proteins Lipids RNA & DNA Fig. 4

co2 (Expired Air)

Polar Components

Bile

M e t a b o l i c Fate o f N i t r o g l y c e r i n

The m e t a b o l i c f a t e o f n i t r o g l y c e r i n i n man has n o t been s t u d i e d i n g r e a t d e t a i l . So f a r , o n l y g l y c e r y l monon i t r a t e s have been i d e n t i f i e d a s t h e major u r i n a r y m e t a b o l i t e o f n i t r o g l y c e r i n i n man61. 6. Pharmacokinet i c s

6.1 Tissue D i s t r i b u t i o n N i t r o g j y c e r i n i s r a p i d l y and e x t e n s i v e l y d i s t r i b u t e d i n t h e body. F o l l o w i n g intravenous a d m i n i s t r a t i o n o f r a d i o l a b e l e d n i t r o g l y c e r i n i n t h e r a t , Needleman and c o - ~ o r k e r s ~ ~ found t h a t t h e apparent d i s t r i b u t i o n phase o f unchanged n i t r o g l y c e r i n from blood has a h a l f - l i f e o f l e s s than 20 seconds. T i ssue r a d i o a c t i v i t y was not, however, measured i n t h e study. D i C a r l o e t a1.59, s t u d i e d t h e d i s t r i b u t i o n o f CC141 a f t e r o r a l a d m i n i s t r a t i o n (10 mg/kg) o f I C 1 4 ) n i t r o g l y c e r i n i n t h e same species. They measured t h e dioxane e x t r a c t a b l e and n o n - e x t r a c t a b l e r a d i o a c t i v i t y i n t h e t i s s u e s as a f u n c t i o n o f time. The l i v e r and carcass appeared t o be t h e major s i t e s o f d i s t r i b u t i o n o f absorbed r a d i o a c t i v i t y . The h e a r t , lung, kidney and spleen took up o n l y small q u a n t i t i e s o f t h e r a d i o - l a b e l . S i g n i f i c a n t accumulation o f none x t r a c t a b l e r a d i o a c t i v i t y was shown i n t h e carcass, l i v e r and G I t r a c t , suggesting t h a t n i t r o g l y c e r i n and/or i t s b i o t r a n s f o r m a t i o n products m i g h t be i n c o r p o r a t e d i n t o t h e t i s s u e s .

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This o b s e r v a t i o n agrees w i t h t h a t o f a l a t e r study62 which showed t h a t t h e r a d i o a c t i v i t y from { 1 4 C ) n i t r o g l y c e r i n c o u l d be i n c o r p o r a t e d i n t o r a t l i v e r glycogen, l i p i d , p r o t e i n , RNA and DNA. Hodgson and Lee60 a l s o s t u d i e d t h e d i s t r i b u t i o n o f { 1 4 C ) i n t h e r a t a f t e r o r a l a d m i n i s t r a t i o n o f 180 mg/kg o f r a d i o a c t i v e n i t r o g l y c e r i n i n peanut o i l . They found however, no accumulation o f absorbed r a d i o a c t i v i t y i n t h e carcass a f t e r 4 hours p o s t dosing. A t t h e same time, about 60% o f t h e r a d i o a c t i v i t y was detected i n t h e GI t r a c t . 6.2 Intravenous A d m i n i s t r a t i o n The pharmacokinetics o f n i t r o g l y c e r i n a r e c h a r a c t e r i z e d by an extremely r a p i d plasma c l e a r a n c e o f drug. Followi n g intravenous a d m i n i s t r a t i o n i n r a t s (0.35-2.5 mg/k ) , In plasma n i t r o g l y c e r i n clearance i s about 0.6 L/min/kg6 man, plasma n i t r o g l y c e r i n c l earance has been r e p o r t e d as 23.6 and 28 L/min f o l l o w i n g intravenous i n f u s i o n 6 4 and subl i n g u a l a d m i n i s t r a t i o n , r e ~ p e c t i v e l y ~ Since these values ~. a r e i n excess o f l i v e r blood flow, i t has been suggested65 t h a t t h e r e must be s u b s t a n t i a l e x t r a - h e p a t i c e l i m i n a t i o n . Plasma drug clearance i s a f u n c t i o n o f t h e apparent volume o f d i s t r i b u t i o n and t h e e l i m i n a t i o n r a t e constant, both o f which a r e q u i t e h i g h f o r n i t r o g l y c e r i n . An apparent volume o f d i s t r i b u t i o n o f about 3 L/kg has been c a l c u l a t e d f o r n i t r o g l y c e r i n i n r a t s 6 6 , which i s c o n s i s t e n t w i t h t h e e x t e n s i v e t i s s u e d i s t r i b u t i o n discussed e a r l i e r . F o l l o w i n g doses o f 0.7 mg/kg i n r a t s 6 6 and t h e r a p e u t i c doses i n man64, 6 5 , plasma e l i m i n a t i o n appears monoexponenti a1 w i t h an e l i m i n a t i o n h a l f - 1 i f e o f approximately 3-4 minutes. Admini s t r a t i o n o f h i g h e r doses (2.5 and 3.5 mg/kg) i n r a t s r e 15 s u l t e d i n an apparent b i e x p o n e n t i a l decay w i t h a t + B I t i s p o s s i b l e t h a t a t t h e r a p e u t i c doses, m u l t i min.63. exponential d i s p o s i t i o n cannot be c h a r a c t e r i z e d because o f a n a l y t i c a l u n c e r t a i n t i e s encountered w i t h t h e extremely low plasma c o n c e n t r a t i o n s ( < 1 ng/ml) found.

9.

6.3 Oral and Topical A d m i n i s t r a t i o n The r a t i o n a l e f o r o r a l use o f o r g a n i c n i t r a t e s has been a c o n t r o v e r s i a l s u b j e c t . F o l l o w i n g o r a l a d m i n i s t r a t i o n and p o r t a l v e i n i n f u s i o n o f n i t r o g l c e r i n t o r a t s , i n doses up t o 0.5 mg/kg, Needleman e t a1 5 7 observed no blood pressure response and n e g l i g i b l e blood c o n c e n t r a t i o n s o f i n t a c t drug. These authors a l s o observed human l i v e r b i o p s y samples t o have m e t a b o l i c c a p a c i t y f o r o r g a n i c n i t r a t e s s i m i l a r t o t h a t found i n r a t s , and t h e y concluded t h a t t h e systemic a v a i l a b i l i t y o f n i t r o g l y c e r i n f o l l o w i n g o r a l admini s t r a t i o n i s n e g l i g i b l e . C l i n i c a l s t u d i e s demonstrating e f f i c a c y o f o r a l l y administered n i t r o g l y c e r i n 6 7 imply,

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however, t h a t systemic a v a i l a b i l i t y o f n i t r o g l y c e r i n may be s i g n i f i c a n t i n man, s i n c e t h e m e t a b o l i t e s o f n i t r o g l y c e r i n are considerably less active. The use o f t o p i c a l n i t r o g l y c e r i n has been shown t o g i v e s u s t a i n e d hemodynamic e f f e c t s i n man68. F o l l o w i n g t o p i c a l a d m i n i s t r a t i o n o f a 2% n i t r o g l y c e r i n ointment, e q u i v a l e n t t o 16 mg o f n i t r o g l y c e r i n , plasma c o n c e n t r a t i o n s were s i m i l a r t o t h o s e seen a f t e r o r a l a d m i n i s t r a t i o n o f a 6.5 mg s u s t a i n e d r e l e a s e capsule69. S i t e dependency i n t h e p e r cutaneous a b s o r p t i o n o f n i t r o g l y c e r i n has been observed i n man70 and i n r a t s 8 b u t n o t i n t h e rhesus monkey71. The s u r f a c e area o f a p p l i c a t i o n has a l s o been shown t o be an import a n t f a c t o r i n t o p i c a l a b s o r p t i o n when assessed by hemodynamic e f f e c t s 7 2 . 7. Methods o f A n a l v s i s

7.1 O f f i c i a l Methods The " O f f i c i a l Methods o f A n a l y s i s " p u b l i s h e d by t h e A s s o c i a t i o n o f O f f i c i a l A n a l y t i c a l Chemists73, d e s c r i b e s two methods f o r t h e d e t e r m i n a t i o n o f n i t r o g l y c e r i n . The f i r s t i n v o l v e s e t h e r e x t r a c t i o n f o l l o w e d by t h e r e d u c t i o n o f n i t r o gen t o ammonia and subsequent d e t e r m i n a t i o n by t i t r a t i o n w i t h a c i d . A second method u t i l i z e s t h e i n f r a r e d a b s o r p t i o n peak near 7.89 pm and r e q u i r e s a n i t r o g l y c e r i n r e f e r e n c e standard f o r q u a n t i t a t i o n . The assay f o r n i t r o g l y c e r i n developed by Hohman and Levine7!+ i s t h e b a s i s f o r t h e o f f i c i a l USP75 procedure. T h i s technique uses column chromatography t o separate n i t r o g l y c e r i n from i t s d e g r a d a t i o n p r o d u c t s f o l l o w e d by a c i d h y d r o l y s i s t o n i t r a t e i o n and subsequent 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 n i t r a t e d p h e n o l d i s u l f o n i c a c i d . Potassium n i t r a t e i s used as a r e f e r e n c e standard. Both t h e AOAC reduct i o n method and t h e USP procedure a r e u s e f u l as p r i m a r y s t a n d a r d i z i n g procedures f o r up t o m i l l i g r a m q u a n t i t i e s o f nitroglycerin.

7.2 Spectrophotometric Q u a n t it a t i o n o f n i t r a t e and n i t r i t e i o n f o l l o w i n g hydrolysis o f organic n i t r a t e s i s possible using colorimetric methods. Spectrophotometric measurement o f n i t r o x y l e n o l formed from t h e r e a c t i o n o f h y d r o l y z e d o r g a n i c n i t r a t e w i t h e i t h e r 2,4-xylenol o r 2,6-xylenol i s t h e b a s i s of t h e x y l e n o l procedure76y77. A p p l i c a t i o n o f t h e G r i e s s r e a c t i o n and v a r i ous m o d i f i c a t i o n s have been used i n b i o l o g i c a l work f o r n i t r a t e rr~easurement~~-~O. However, t h e s e methods do n o t possess t h e r e q u i s i t e s e n s i t i v i t y f o r t h e a n a l y s i s o f n i t r o g l y c e r i n i n b i o l o g i c a l f l u i d s d u r i n g d r u g therapy.

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Several spectrophotometric methods a r e a v a i l a b l e f o r qua1 i t y c o n t r o l d e t e r m i n a t i o n s o f n i t r o l y c e r i n i n dosage forms. I n t h e assay described by Be1 1 89 3 8 2 , n i t r o g l y c e r i n i s hydrolyzed w i t h s t r o n t i u m hydroxide t o form n i t r i t e i o n . F o l l o w i n g d i a z o t i z a t i o n w i t h N-(1-naphthyl) e t h y l e n e diamine d i hydrochloride, q u a n t i t a t i o n i s achieved b y c o l o r i m e t r i c d e t e r m i n a t i o n o f t h e azo dye. The use o f s t r o n t i u m h y d r o x i d e i s s a i d t o reduce t h e i n t e r f e r e n c e due t o l a c t o s e . Since t h e conversion o f n i t r o g l y c e r i n t o n i t r i t e i s n o t s t o i c h i o m e t r i c , absolute quanti t a t i o n requires a n i t r o g l y c e r i n reference standard. T h i s method has been a ~ t o m a t e d ~ ~ , @ ~ .o f t e t r a Use methyl ammonium hydroxide t o hydrolyse n i t r o g l y c e r i n has been r e p o r t e d 8 4 t o produce s t o i c h i o m e t r i c conversion o f 2 moles o f n i t r i t e per mole o f n i t r o g l y c e r i n as p r e d i c t e d by Hay85. I t should t h e r e f o r e be p o s s i b l e t o use potassium n i t r i t e as a r e f e r e n c e standard f o r t h e B e l l assay w i t h t h i s m o d i f i c a t i o n . A k i n e t i c method has been developed which i s s u i t a b l e f o r t h e a n a l y s i s o f s i n g l e dosage u n i t s l 5 - I 7 . T h i s assay i s based upon t h e stepwise degradation o f n i t r o g l y c e r i n i n a l k a l i n e a l c o h o l i c s o l u t i o n s , w i t h t h e f o r m a t i o n o f a chromop h o r i c i n t e r m e d i a t e . The absorbance maximum a t 328 nm was shown t o be p r o p o r t i o n a l t o t h e i n i t i a l n i t r o g l y c e r i n concent r a t i o n present i n t h e r e a c t i o n . The s p e c i f i c i t y o f t h e USP, B e l l and k i n e t i c assays was examined by Morrison and FungE6. They found t h e USP and k i n e t i c assay procedures t o be s t a b i l i t y - i n d i c a t i n g whereas t h e B e l l assay i s p r e d i c t a b l y i n t e r f e r e d w i t h by i n o r g a n i c n i t r i t e . However, under t h e r e a c t i o n c o n d i t i o n s o f t h e B e l l method, t h e d i n i t r a t e s , mononitrates and i n o r g a n i c n i t r a t e d i d not i n t e r f e r e .
7.3 T h i n Layer Chromatography T h i n l a y e r chromatoqraphy has been used t o separate - * " t h e 14C-glyceryi n i t r a t e s ( n i t r o g l y c e r i n and i t s m e t a b o l i t e s ) p r i o r t o q u a n t i t a t i o n o f t h e r a d i o a c t i v i t y . The system r e p o r t e d by Crew and DiCarlo18 (Table 111) i s r e p r e s e n t a t i v e o f o t h e r ~ t~ a t ,have been r e p o r t e d . h~ ~ ~

7.4 Polargraphy The p o l a r g r a p h i c behavior o f n i t r o g l y c e r i n , pentae r y t h r i t o 1 t e t r a n i t r a t e and e t h y l e n e g l y c o l d i n i t r a t e has been s t u d i e d i n an ethanol-water system based on t h e r e d u c t i o n o f n i t r a t e a t t h e dropping mercury e l e c t r o d e . Tetramethyl ammonium c h l o r i d e was used as t h e s u p p o r t i n g e l e c t r o l y t e . The e f f e c t s of pH, number o f n i t r a t e groups, mercury column h e i g h t , b u f f e r s and s o l v e n t on t h e half-wave p o t e n t i a l (measured a g a i n s t t h e s a t u r a t e d calomel e l e c t r o d e (El vs. S.C.E.) and t h e d i f f u s i o n c u r r e n t ( i . d . ) was examinedE9?

NITROGLYCERIN

535

Using t h i s technique f o r the assay of s i n g l e sublingual t a b l e t s , Flann88 reported a E% of -0.91 v o l t s (vs. S.C.E.) and the i . d . t o be dependent on nitroglycerin conc e n t r a t i o n . A non-aqueous polargraphic method has a l s o been described by Woodson and A1 berE9.

TABLE 11118
T h i n-Layer Chromatography of Nitroglycerin
TLC p l a t e s : Sol vent: Rf values:
250 1-1 s i l i c a gel G bound w i t h calcium s u l f a t e benzene:ethylacetate:acetic acid (16:4:1) nitroglycerin 0.60 glyceryl-1 , 3 - d i n i t r a t e 0.45 glyceryl-l,2-dinitrate 0.30 glyceryl-l-mononitrate 0.10 glyceryl-2-mononitrate 0.10 g 1ycerol 0.00

7.5 Gas Chromatography Several GC procedures have been described f o r the analysis of organic n i t r a t e s . This technique i s e s p e c i a l l y s u i t a b l e f o r determination of nitroglycerin i n biological f l u i d s a f t e r d r u g administration. T h e use of the e l e c t r o n capture d e t e c t o r gives the necessary s e n s i t i v i t y . Table IV gives chromatographic conditions t h a t have been u t i l i z e d f o r nitroglycerin determination. 7.6 High Performance L i q u i d Chromatography Several HPLC methods have been reported f o r the assay of nitroglycerin i n dosage forms. Two normal phase methods a r e a ~ a i l a b l e b u ~ data~ i s lacking on t h e i r s p e c i f i c i t y . ~ t , ~ Table V l i s t s the chromatographic conditions of two procedures shown t o be s p e c i f i c f o r nitroglycerin i n the presence o f degradation products.

TABLE I V
GC C o n d i t i o n s f o r N i t r o g l y c e r i n

Reference 90 91 92 93
94

Column 3.5% QF - 1 on 60-80 Gas Chrom Q . 3% SP-2401 on 100-120 Supel c o p o r t . 3% SE-30 on 50-60 Anakrom AB15. 0.4% OV-17 on 60-80 g l a s s beads 3% SE-30 on 100-120 Gas Chrom Q. 10% OV-101 on 100-120 Chromosorb W-HP 30% SE-30 on 80-100 Chromosorb W-HP 3.8% OV-101 on 80-100 Gas Chrom Q; 2.5% OV-210 on Chromosorb W-HP; 1.1% OV-225 on Gas Chrom Q. 3% XE-60, 3.5% QF-1 on 60-80 Gas Chrom Q.

Detector ECD ECD TCD EC D

ECD

Temperature (OC) ( I = I n j e c t i o n port, C = Column, D = D e t e c t o r )

I= D I= D C =
= 180

Sample Ana 1yzed 5 m l human plasma 0.2 m l r a t / human plasma Tab1 e t extract 2 m l human blood or urine 3 m l human blood 4 m l human p l asma 5 m l human plasma \li t r o c e l l u l o s e propellants

Sensi t i v ity (ng/ml) 0.5

0.1

160, C = 120,

160, C = 140, = 180 130, D = 192

-0.1-2
?

65 95 96

ECD ECD

FID

= 120, D = 150 I = 200, C = 150, D = 175 I = 150, C = 130, D = 200 I = 150, C = 130, D = 210 I = 70, C = 70-220 @ 60/min, D = 225

I = 150, C

0.5

--

97

FID ECD

I = 160, C

D = 200
=

= 150,

D = 260

160 C = 120,

solvent mixtures

NITROGLYCERIN

531

TABLE V HPLC C o n d i t i o n s f o r Assay o f N i t r o g l y c e r i n

_ _f_o o Re l
Column M o b i l e Phase Flow r a t e (ml / m i n) Detection Detection 1i m i t R e t e n t i on time

Reflol A1 k y l phenyl bonded t o s i l i c a gel A c e t o n i t r i l e-Tetrahydrofuran-Water (26:64:10) 2.0 u . v 218 nm 50 ng on column 10 m i n

C18 m i c r o p a r t i c u l a t e
60% MeOH 2.0 u.v 200 nrn 30 ng on column 4 rnin

Acknowledgement Supported i n p a r t by N I H g r a n t 22273. W thank D r . e Dinesh Gala f o r r u n n i n g t h e i n f r a r e d and nmr s p e c t r a . 8. References 1. 2. F. P r i s t e r a , M. H a l i k , A. C a s t e l l i and W. F r e d e r i c k s , Anal.Chem. 32, 495 (1960). W.M. Ayres, G.C. Whitnack and R.T. Merrow, NAVWEPS r e p o r t 7608, NOTS T e c h n i c a l P u b l i c a t i o n 2604 (1961) U.S.Nava1 Ordinance T e s t S t a t i o n , China Lake, C a l i f . ; Chem. Abs t. 60, 5266f ( 1964) R.T.M. F r a s e r a n d N.C. Paul, J . Chem. SOC. (B), 659 (1968). M. Windholz (ed.), The Merck Index, 9 t h ed., Merck & Co., Rahway, N. J. , 1976. M.J. P i k a l , A.L. Lukes and L.F. E l l i s , J. Pharm. S c i . - 1278 (1976). 65, M.J. P i k a l and A.L. Lukes, J. Pharm. S c i . - 1269 65, ( 1 976). H. F. Mark (ed. ) , Kirk-Othmer E n c r c l o p e d i a o f Chemical Technology, 2nd ed., v o l . 8, I n t e r s c i e n c e P u b l i s h e r s (1965). S.T. Horhota and H.-L. Fung, J . Pharm. S c i . - 608 68, (1979). E.M. Johnson J r . i n "Organic N i t r a t e s " , P. Needleman ( e d . ) , S p r i n g e r - V e r l a g , New York (1975) p. 17. R. Boschan, R.T. Merrow and R.W. Van Dolah, Chem. Rev. - 485 (1955). 55,

3. 4. 5. 6. 7.
8

9. 10.

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11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38.

U V. Amshler, Pharmazeutische Z e i t i n g , - 1077 119, ( 974); Chem. A b s t r . 81, 1267Op (1974). R T.M. Fraser, U.S. GGt. Res. Developm. Rep. ( 968); Chem. A b s t r . 70, 86755e (1969). R.T.M. Fraser, Chem. X d . 1117 (1968). J.W. Baker and D.M. Easty, J. Chem. SOC. 1193 (1952). H.-L. Fung, P. D a l e c k i , E. Tse and C.T. Rhodes, J. Pharm. S c i . 62, 696 (1973). S.K. Yap, C . T . Rhodes and H.-L. Fung, Anal. Chem. 47, 1183 (1975). S.K. Yap, C.T. Rhodes and H.-L. Fung, Am. J. Hosp. Pharm. 32, 1039 (1975). M.C. C r e w and F.J. D i C a r l o , J. Chromatogr. 35, 506 ( 1968) . M.T. Rosseel, M.G. Bogaert and D. DeKeukeleire, B u l l . SOC. Chim. Belg. 83, 211 (1974). B.S. S v e t l o v and K P . Shelaputina, Zh. F i z . Khim. - 2889 (1966). 40, P. Klason and T. Carlson, Chem. Ber. 39, 2725 (1906). L.A. Heppel and R.J. Hilmoe, J. B i o l . C h e m . 183, 129 ( 1950). P. Needleman and F.E. Hunter J r . , Molec. Pharmacol. 1, 77 (1965). F.J. D i C a r l o and M.D. Melgar, Biochem. Pharmacol. 19, 1371 (1970). G.F. Burchand N.P. DePasquale, Am. H e a r t J. 72,842 (1972). J.K. Sturek, T.D. Sokoloski, W.T. Winsley and P.E. Smith, Am. J. Hosp. Pharm. 35, 537 (1978). C.E. Waring and G. Krastins,J.Phys. Chem. 74, 999 ( 1970) . S.Z. R o g i n s k i i , Phys. Z. Sowjetunion, 1, 640 (1932). S. F u s a r i , J . Pharm. S c i . 62, 122 (1977). H.-L. Fun S.K. Yap and C T . Rhodes, J. Pharm. S c i . - 1810 919974). 63, H.-L. Fung, S.K. Yap and C.T. Rhodes, J. Pharm. S c i . - 558 (1976). 65, S.A. F u s a r i , J. Pharm. S c i . 62, 2012 (1973). F.W. Goodhart, H. G u c l u y i l d i z R . E . Daly, L. 65, C h a f t e z and F.C. Ninger, J. Pharm. S c i . - 1466 (1976). D. Banes, J . Pharm. Sci. 57, 893 (1968). B. Dorsch and R. ShangrawrAm. J. Hosp. Pharm. 32, 795 (1975). Federal R e g i s t e r 37, 15858 (Aug. 5, 1972). H.-L. Fung, Am. J r H o s p . Pharm. 35, 528 (1978). W.G. Crouthamel, B. Dorsch and R. Shangraw, N. Engl. J. Med. - 262 (1978). 296,

NITROGLYCERIN

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D. J . Ludwig and C.T. Ueda, Am. J . Hosp. Pharm. 35, 541 ( 1 9 7 8 ) . 40. B . L . McNiff, E.F. McNiff and H.-L. Fung, Am. J . Hosp. Pharm. 36, 173 (1979). 41 . P.-H. YuTn, S.L. Denman, T.D. Sokoloski, A.M. Burkman, J . Pharm. S c i . 68, 1163 (1979). 42. D.M. Baaske, A.H. Amann,D.M. Wagenknecht, M. Mooers, J.E. C a r t e r , H.J. Hoyt and R . G . S t o l l , Am. J . Hosp. Pharm. 37, 201 (1980). 43. P.A. CoEum, M.S. Roberts, A.J. G a l b r a i t h and G.W. Boyd, Lancet 2, 349 ( 1 9 7 8 ) . 44. M . H . L i t c h f i e f d , J . Pharm. S c i . 1599 ( 1 9 7 1 ) . 45. F.J. DiCarlo, Drug Met. Rev. 4 , 1 (1975). 46. P. Needleman ( e d . ) , Organic N T t r a t e s , S p r i n g e r e Verlag, Nw York (1975). 47. F. Posades del Rio, Fed. Proc. 29, 412 ( 1 9 7 0 ) . 48. F. Posades del Rio and F . F . Hunter, Fed. Proc. 32, 733 (1973). 49. P . Needleman and A . B . Harkey, Biochem. Pharmacol. 20, 1867 (1971). 50. P. Needleman and J.C. Krantz J r . , Biochem. Pharmacol. - 1225 (1965). 14, 51. N . H . Lee and F.M. B e l p a i r e , Biochem. Pharmacol. 21, 3171 (1972). 52. S. Lang, E.M. Johnson J r . and P. Needleman, Biochem. Pharmacol . 21, 422 (1972). 53. G . A . M a i e r , C . Arena and H.-L. F u n g , Biochem. Pharmacol . 29, 646 (1980). 54. F.J. D i C a r l o a n d M.D. Melgar, Proc. SOC. B i o l . Med. 131, 406 (1969). 55. G.A. Maier, A. P o l i s z c z u k and H.-L. Fung, I n t . J . Pharmaceut. 4 , 75 ( 1 9 7 9 ) . 56. N . H . Lee, Biochem. Pharmacol. 22, 3122 (1973). 57. P. Needleman, S. Lang and E.M.Johnson J r . , J . Pharma c o l . Exp. Ther. 181, 489 (1972). 58. P. Needleman, DJlh , ..e m A.B. Harkey, E.M. Johnson J r . and S . Lang, J . Pharmacol. Exp. Ther. 179,347 (1971 ) . 59. F. J . DiCarlo, M.C. Crew, L . J . Haynes, M. D . Flelgar and R . L . Gala, Biochem. Pharmacol. 17, 2179 (1968). 60. J.R. Hodgson and C.-C. Lee, Tox. A p p . Pharmacol. 34, 449 (1975). 61. M.G. Bogaert, M.T. Rosseel and F.M. B e l p a i r e , Arch. I n t . Pharmacodyn. Ther. 192, 198 (1971). 62. F.J. DiCarlo, J . Viau a n d . D . Melgar, Biochem. Pharmacol. 1 8 , 965 ( 1 9 6 9 ) . 63. G. Maier, HTOgata and H.-L. Fung, unpublished r e s u l t s 64. V.M.S. Oh and P . R . Reid, Pharmacologist - 202 (1979) 21,
39.

a,

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65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 73. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90.

P.W. Armstrong, J.A. Armstrong and G.S. Marks, C i r c u l a t i o n 3, 585 (1979). P.S.K. Yap and H.-L. Fung, J. Pharm. S c i . - 584 67, (1979). T. Winsor and H.J. Berger, Am. H e a r t J. 90, 661 (19751. J . O . Parker, R. J . Augustine, J . R. Burton, R.O. West and P.W. Armstrong, Am. J . C a r d i o l . 38, 162 (1976). H.P. Blumenthal, H.-L. Fung, E.F. M c K f f and P.S.K. Yap, Br. J. C l i n . Pharmacol. 4, 241 (1977). M.S. Hanson, Am. J. C a r d i o l . T2, 1061 (1978). P.K. Noonan and R.C. Wester, Pharm. S c i . 69, 365 ( 1988) S.G. M e i s t e r , T.R. Engel, N. Guiha, C.M. F u r r , G.S. F e i s t o s a , K. H a r t and W.S. F r a n k l , D r . H e a r t J . - 1031 (1976). 38, W. H o r w i t z (ed. ) " O f f i c i a l Methods o f A n a l y s i s " , v01.12, Assoc. o f O f f i c i a l A n a l y t i c a l Chemists, Washington, D.C. (1975). J.R. Hohman and J. Levine, J. A. 0. A. C. - 471 47, (1964). "The U n i t e d S t a t e s Pharmacopeia" , 2 0 t h r e v . , U n i t e d S t a t e s Pharmacopeial Convention, Inc., R o c k v i l l e , Md. (1979). H. Yagada, I n d . Eng. Chem. 15, 27 (1943). D.W.W. Andrews, A n a l y s t 8 9 , 7 3 0 (1964). J.L. Lambert and F.Zitomer, Anal. Chem. - 1684 32, ( 1960) L.J. Cass, W.S. F r e d e r i k and H. Delucia, A n g i o l o g y - 469 (1962). 13, O.J. L o r e n z e t t i , A. Tye and J.W. Nelson, J. Pharm. S c i . 55, 105 (1966). F.K. Ell, J.J. O ' N e i l l and R.M. Burgison, J. Pharm. S c i . 52, 637 (1963). F.K. Ell, J . Pharm. S c i . 53, 752 (1964). M.H. L i t c h f i e l d , A n a l y s t 9 2 , 132 (1967). C.E. Wells, H.M. M u l l e r a n d Y.H. Pfabe, J . A. 0. A. C. - 579 (1970). 53, M. Hay, M o n i t . S c i . P a r i s 15, 424 (1885). R.A. M o r r i s o n and H.-L. F u K , J . Pharm. S c i . - 1197 68, (1979). G.C. Whitnak, J.M. N i e l s o n and E.S. Gantz, J. Am. Chem. SOC. 76, 4711 (1954). B.C. F l a n n , J . Pharm. S c i . 122 (1969). A.L. Woodson and L . L . A l l e r , J . A. 0. A. C. - 847 52, ( 1 969). M.T. Rosseel and M.G. Bogaert, J. Pharm. S c i . 62, 754 (1973).

z,

NITROGLYCERIN

541

91. 92. 93. 94. 95. 96. 97. 98. 99.

100.
101.

S.K. Yap, E.F. M c N i f f and H.-L. Fung, J. Pharm. S c i . 67, 682 (1978). E.T. E s s e l , J . Gas Chromatog. 179 (1965). G.B. Neurath and M. Dunger, Drug Res. 27, 416 (1977). Y. G i v a n t and F.G. Sulman, E x p e r i e n t i a 3 4 , 643 (1978). J.Y. Wei and P.R. Reid, C i r c u l a t i o n 59, 588 (1979). B.J. A l l e y and H.W.H. Dykes, J . C h r o K t o g r . - 23 71, (1972). M.-T. 'Rosseel and M.G. Bogaert, J. Chromatogr. 64, 364 (1972). C.D. Chandler, G.R. Gibson and W.T. B o l l e t a r , J. Chromatogr. 100, 185 (1974). J.O. D o a l i andA.A. Juhasz, J. Chromatogr. S c i . 12, 51 (1974). W.G. Crouthamel and B. Dorsch, J. Pharm. Sci. 68, 237 (1979). D.M. Baaske, J.E. C a r t e r and A.H. Amann, J . Pharm. S c i . - 481 (1979). 68,

L i t e r a t u r e reviewed up t o 4/1/80.

TRIFLUOPERAZINE HYDROCHLORIDE
Alex Post, Richurd J . Wurren, und John E . Zarembo
Description I . 1 Nomenclature I .2 Formula, Molecular Weight, Structure 1.3 Appearance, Color, Odor Physical Properties 2. I Spectral Properties 2.2 X-Ray Diffraction Pattern 2.3 Solubility 2.4 Apparent Partition Coefficients 2.5 pKa 2.6 Thermal Properties Synthesis Identification 4.1 Derivatives 4.2 Color Reactions 4.3 Microscopy 4.4 Miscellaneous Identification Tests Stability and Degradation Metabolism 6.1 Metabolic Products 6.2 Biological Half-Life 6.3 Protein Binding Methods of Analysis 7.1 Elemental Analysis 7.2 Titrimetric Analysis 7.3 Complexometric Analysis 7.4 Spectrophotometnc Analysis 7.5 Spectrofluorometric Analysis 7.6 Chromatographic Methods of Separation Miscellaneous 8. I Adsorption Phenomena 8.2 Surface Activity References
544

544

544
544

545 545
55 1

3. 4.

5. 6.

7.

8.
9.

555 556 556 556 558 559 559 559 560 56 1 561 562 562 565 565 565 565 566 567 567 568 568 578 578 578 579

Analytical RofiLs of Drug Subsfnnces. 9

543

Copyri%I 0 1980 by Acadcrnic R s s . Inc. All rights of reproduction i any form reserved n ISBN: 0-12-260809-7

544

ALEX POST et ul.

I.

Description
I. I

Nomenclature 1.11 Chemical Names

Several chemlcal names have been used t o denote t r i f I uoperazi ne hydroch l o r i de: (a) IOH-Phenothl azlne. IO-[3-(4-methyI-l-p i p e r a z i n y 1 )propy I]-

2- ( t rif I uoromethy I )-d ihydroch Io r id e l

( b ) lO-f3-(4-methyl-l phenothiazine d i h y d r o c h l o r i d e l -p p e r a z i n y l Ipropy 1 l - Z - ( t r I f Iuoromethy I ) 0-~3-(4-methyIplperazine-I-yI

(c1 D l h y d r o c h i o r i d e o f 2-trl f Iuoromethylphenothia~ine~ (d) phenothiazinej I. 12 Terfluzine 1.2 Trade Names

)propyl]-

2-TrlfIuor~lethyI-I0-~3(I-methyI-4-piperazinyI)propyI]

I a t r o n e u r a l , J a t r o n e u r a l , Eskazinyl, Eskazine, S t e l a z i n e 4 @

Formula, Molecular Weight, S t r u c t u r e

1.21

E m p i r i c a l Formula, Molecular Weiqht C~~H~L,FSJN~S-~HCI 400.420

1-22 S t r u c t u r e

n CH2CHzCH2-N
I

.2HCI

wN-CH3

1.3 b p e a r a n c e . Color, Odor

Both t h e National Formulary1 and t h e B r i t i s h Pharmacopoeia2 d e s c r i b e T r i f l u a p e r a z i n e Hydrochloride.as f o l l o w s :
A w h l t e t o o f f - w h l t e (cream colored) c r y s t a l l i n e powder w i t h l i t t l e o r no odor.

TRI FLUOPERAZINE HYDROCHLORIDE

545

2.

Phys i ca I P r o p e r t i e s

2. I

Spectra I P r o p e r t i e s

2. I I

I n f r a r e d Spectra

F i g u r e 1 i s t h e i n f r a r e d spectrum o f t r i f l u o p e r a z i n e f r e e base and F i g u r e 2 i s t h e i n f r a r e d spectrum o f t h e h y d r o c h l o r i d e s a l t o f t r i f l u o perazine taken i n mineral 011 d i s p e r s i o n from 4000-625 cm-l on a Perkin-Elmer Model 457A. The s i g n i f i c a n t bands I n t h e spectra are assigned as f o l l o w s : Free Base Wave I ength cm-l Assipnment

HCI S a l t
Wavelength cm-l 2700-2 I00 1600, 1570, 1470 1115 Assionmnt

1600, 1575,
1330, 830 750

1500

c=c, aromatic
CF3 I ,2,4-trl subs t i t u t e d aromatic
1,2-substltuted aromat ic

NH+
C=C, a r o m a t i c

1250, 1130

1320, 1340,

CF,

a29
760

I ,2,4-trisubs t l t u t e d aromatic
1,2-substituted aromat i c

The i d e n t i f i c a t i o n and d i f f e r e n t i a t i o n o f phenothiazine t y p e t r a n q u i l i z e r s by t h e I R spectra o f s a l t s as d e r i v a t i v e s has been r e p ~ r t e d . ~ ~ ~ 2.12 U l t r a v i o l e t Spectrum

The u l t r a v i o l e t a b s o r p t i o n spectrum o f t r l f l u o p e r a z i n e i n 95% ethanol 1s shown I n F i g u r e 3. Maxima a t 258 n ( l o g E 4.50) anj3307.5 n ( l o g m m 3.50) a r e bands c h a r a c t e r i s t i c of a 2 - s u b s t i t u t e d phenothiazine

The u l t r a v i o l e t spectrum o f t r i f l u o p e r a z i n e has been used i n t h e a n a l y s i s o f b i o l o g i c a l s p e ~ i m e n s ~ * ~ ~ w~ l~ as i n t h e a n a l y s i s Of as e l t h e drug i t s e l f and i t s d e r i v a t i v e s . 8 The importance of c a r e f u l c o n t r o l of instrumental Parameters such as s l i t wtdth and t h e absorption o f UV and v i s i b l e I i g h t by phenothiazines has a t s o been r e p o r t e d . l l

2.13

Nuclear Magnetic Resonance Srjectra

2.131

Proton Spectrum

The p r o t o n NMR spectrum (Figure 4 ) was obtalned on a deuterochlorofonn s o l u t l o n c o n t a i n i n g approximately 100 mg/ml o f t r l f l u o p e r a z l n e and t e t r a m e t h y l s l l a n e as i n t e r n a l reference standard. The spectrum was obtained on a Perkin-Elmer R32 NMR. The NMR s i g n a l s a r e assigned as follows:

546

ALEX POST c ' f

ti/.

Protons

Chemical S h i f t , ppm

M I tI c i p Ib u mu I t 1 p l e t sing l e t

:3

I .94 2.25
2.3

d, d'
e

c, c '

- 2.6 -

broad m u l t l p l e t , s i g n a l s over I app i ng t r i plet broad m u l t i p l e t , s i g n a l s overlapping

g ( a l i aromatics)
2.132

3.96 6.75 7.30

13C Spectrum

The NMR Spectrum ( F i g u r e 5 ) was obtalned on a deuterochloroform s o l u t i o n o f t r i f l u o p e r a z i n e w i t h t e t r a m e t h y l s i l a n e as a reference. The spectrum was obtained on a Varian Associates Model FT-80 spectrometer. The NMR s i g n a l s a r e assigned as follows:

8.8'

d'

eJe

e,e' d

Figure 1 : I n f r a r e d Spectrum of T r i f l u o p e r a z i n e Free Base

or

548

- - - - - - ---

Trifluoperazine Sulfoxide Sulfone

vt

W P

220

240

260

280

300

320

_''.... , ......
I

--__ ...: .
..

340

360

380

400

F i g u r e 3-

U l t r a v i o l e t A b s o r p t i o n S p e c t r u m of T r i f l u o p e r a z i n e i n 95% E t h a n o l

TRI FLUOPERAZINE HYDROCHLORIDE

55 i

Table I ( c o n t ' d ) m/e -

266

248

,141

127

I13

99

70

An a lys i s and i d e n t i f i c a t i o n o f drugs by mass spectroscopy has been reported. l4

2.2

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

The X-Ray d i f f r a c t i o n p a t t e r n of t r l f luoperazine dihydr cchlor ide i s presented I n T ab l e 2 .

TIU FLUOPERAZINE HYDROCHLORIDE

553

Carbon

Chemical S h i f t , ppm

a
C

f
i
k
9 h i

d, d' e, e '

118.83 122.96 123.84 127.54 127.39 127.30 129.56 ( d o u b l e t c e n t e r ) 129.79 144.23 145.68 124.29 ( q u a r t e t c e n t e r )
Mass Spectrum

115.88

24.15 45.23 45.95 53.26 55.16 111.80

2.14

The mass spectrum o f t r l f l u o p e r a r i n e wss obtained by d i r e c t i n s e r t l o n i n t o an H f t a c h i Perkin-Eimer R4U-6E low r e s o l u t i o n mass spectrometer. The r e s u l t s a r e presented i n t a b u l a r form i n Table I and as a b a r graph I n Figure 6 Table I Ion m/e -

M +

407 392

(M

CH3)+

307
294 280

TRIFLUOPERAZINE HYDROCHLORIDE

555

Table 2

X-Rav D i f f r a c t i o n P a t t e r n o f T r i f I uoperaz i ne D I hydroch t o r i de

2 0 -

1/10 -

d(Ao 1

17-30

10.10 13.80 15.30 15.60

20.50 21 -90 23.20 23.50 23.80 27.60 27.80 30.30 32 -50

I00

33 1 2 98 58 8

8 24 24 8
12

1 6 1 6
12

8.75 6.44 5.79 5.68 5.12 4.33 4.06 3.03 3.78 3.74 3.23 3.21 2.95 2.75

d = I n t e r p l a n a r spacing ( d i s t a n c e ) .

1/10 = R e l a t i v e i n t e n s i t y based on h i g h e s t i n t e n s i t y o f 103.

23 .

Solubility
So I vent

Grams/100 ml

Reference
15
2 15 15

water water

66 59 >66
i nsol ub l e

02 HCi .E

0.2g NaOH
pH 7.4 b u f f e r
ethanol (95%) ethyl ether chloroform benzene water
( a ) f r e e base

0.0014 14.5
insoluble

16 1s

1
15 1

I .9 i nsol ub I e

0.0013

17

ALEX POST ('t

trl

2.4

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

(K)

I n an attempt t o c o r r e l a t e s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s , several i n v e s t i g a t o r s determined t h e apparent p a r t i t i o n c o e f f i c i e n t s i n a v a r i e t y o f hydrophobic-hydrophilic systems. R e s u l t s o f these s t u d i e s a r e l i s t e d i n the following table. OrganidAqueous Phase dodecane/pH 7.0 b u f f e r ( 0 C 3') chloroform/water (pH I .O) chIoroform/pH 7.6 b u f f e r chIoroform/O.l! HCI n-octanoI/pH 7 b u f f e r n - o c t a n o l / O . l 2 5 ~ KCI 2.5 Apparent pKa K 97 0.37 Reference
18 19 19

>5000
0.7
>to5

20
21 18

49.3

The a a r e n t pKal and pKa2 have been determined using t i t r i m e t r i c and solubi I i t y " measurements. As reported by many o f these i n v e s t i g a t o r s , t h e d e t e n i n a t i o n o f t h e pKa o f phenothiazines, i n general, a r e d i f f i c u l t t o o b t a i n because of t h e i r poor water s o l u b i l i t y . Thus t h e use o f t h e term apparent pKa. However, Green16, u s i n g s o l u b i I ity measurements, d i d indeed c o n f i r m t h a t apparent pKa2 f o r t r i f l u o p e r a z i n e i s approximately 8.1 confirming t h e r e s u l t s obtained by t i t r i m e t r i c measurements.

Apparent pKa

pKal
3.9 3.9 4.10

PKal
8.4 8. I 8.36 8. I 8.3

* Procedure
tl t r i m e t r l c ti trimtri c ti trl mtri c s o l u b l I ity TLC ( a )

Reference 22 ia 23
16
24

( a ) Thin l a y e r chromatography 2.6 Thermal P r o p e r t i e s 2.61 M e l t i n g Range

T r i f l u o p e r a z i n e d i h y d r o c h l o r i d e melts a t about 242OC w i t h decomposition' using t h e c a p i l l a r y m e l t i n g tube method. A m e l t i n g range o f 243244' was r e p o r t e d by Anderson, e t a1 .25 2.62 D l f f e r e n t l a l Scannlno Calorlmetrv

The DSC thermgram f o r t r l f l u o p e r a z i n e d l h y d r o c h l o r l d e 15 shown i n Fiqure 7 . I t i s e v l d e n t t h a t m e l t i n g does appear t o s t a r t a t approximately I 80C w i t h subsequent r a p i d deconposi t ion a t about 250C.26 The thermogram was obtained a t a h e a t i n g r a t e o f 20'C per minute.

TRIFLUOPERAZINE HYDROCHLORIDE

557

OC

Figure 7

- Differential

Scanning Calorimetry Curve of Trifluoperazine

Hydrochloride (USPI

558

A L E X POST e t a l .

3. Synthesis
The detailed synthesis of trifluo erazine dihydrochloride i s described by Craig, e t a I 2 a n d Anoerson, et a l g 5 . The schematic i s illustrated below.

TRI FLUOPERAZINE HYDROCHLORIDE

559

4.

Identification
4.1

Derivatives

Several salts have been prepared thbt can be used for identification purposes (Table 3). As the preparation of the sulfoxide of trifluoperazine can be easily orepared, it has also been used for the identification of the parent compound. (refer to Figure 3) Table 3 Salts of Trifluoperazine Salt Dihydrochioride Pi crate Re i neckate D i ma I eate Dimethylsulfonate Difumarate Disuccinate Pmoate
tienzcphenonedicarboxylate

Melting Range
c *42'(0)

Reference
1

242.0 (decomp) 183.5 193

196

160 8.'

(decomp)

257

14 9' 17 9'
258"

29
38

38 38

215'
130

152 158 -

12 3'
17 6'
:61'

38 38
38

Dipyromellitate Dimethiodide Salt Trif luoperazine (free base)

T r i fI

>240'

162
'L

38

163"
Ibi 175'

40

80"

38
27

uoperaz i ne Su I foxide Di h y a r o c h lor i de

173

(a) Refer to Section 2 6 .

( 6 ) With 3 M I S H20

4.2

Color Tests

Reactions with color reagents has been the method of choice for differentiating trifluoperazine, its degradation products, and metabolites efter a prel imirary saparation bv thin layer and paper c h r o m a t ~ g r a p h y . ~ ~ * ~ ~ - ~ A listing of several of these color reagents are given in Table 4 .
3 6 j 3 7

5 60

A L E X POST ei a / .

Table 4 I d e n t i f i c a t i o n o f T r i f l u o p e r a z i n e w i t h Color Reagents Reagent Bromine water + H2S04 Selenious Acid Concentrated HNO3 68% HNO3 1 % Cobalt a c e t a t e + isopropylamine 10% aq. Chloramine-7 Pal ladium c h l o r i d e Uran i urn n i t r a t e A m n i u vanadate m S i I ver n i t r a t e K e l l e r T e s t (Fee13 2% aq. F e C l j Ceric s u l f a t e 40-50% H2SO4 Folin-Ciocalteau FF" Reagent kbnde I i n Cinnamylaldehyde Furfura I FeCl3 Res Donse cher r y - r e d amber brown-qreen p i n k y e 1 low p u r p l e y e 1 low I i g h t b I ue creamy b I ue orange brown orange ye1 low brown creamy w h i t e pink-mrange red-wiolet red+coiorless orange cameo pink flesh flesh cameo amber Reference
28 28

6.28 29
6

6 6 6 6 6
29 31 30

31,33,34
32 32 32 32 32

4.3

Microscopy

was successful i n u s i n s m i c r o c r v s t a l I ine r e a c t i o n s t o d i f f e r e n t i a t e between phenothiazine t y p e t r a n q u i I i z e t s . Table 5 c o n t a i n s the r e s u l t s f o r t r i f l u o p e r a z i n e o n l y . Table 5 Reaaent Ammonium reineckate Picrlc acid Stannous C h l o r i d e Description of C v s t a I s amorphous yellow, b i r e f r i n g e n t rosettes c o l o r I ess. weak I v b l refringen? irregular r o s e t t e s and long needles amorphous reddish-brown. weakly b i r e f r i n g e n t needles, dense r o s e t t e s Sensitivily o f Detection ( u g h I )

--

0 I . 0 I .

P I a t i num b rom ide

Gold c h l o r l d e

-0 I .

TRI FLCIOPE R A Z l NE HYDROCHLORIDE

S6 I

In a subsequent col laborative. study6, Andres recommended that stannous chloride was the reagent of choice to identify trif luoperazlne. F ~ l t o n ~ ~ , extensive study o f phenothiazines, was able to characterize in an and distinguish trifluoperazine from other phenothiazines via the color of crystals formed with gold, platinum, and palladium reaaents (Tabte 6).
Table 6

Reaaent

Color of Trifluoperazine in Microcrystal Tests

Color Obtained

HAuC14 in (I+I)H2S0, H2PtCI6 aq(l0). to (2+1) acetic acid solution, to dryness H2PdCI4 in (I+35)H2SO4, to (2+1) acetic acid solution; then evaporated H2PtC16 in diluted HClO~+-acetic acid H2PdCI4 In diluted HCIOt,-acetic acid

orange ourple and violet light salmon deep purple brigh sky blue red (purple to pale orange)

4.4

Miscellaneous Identification Tests

Ultraviolet and infrared absorption have been used as identity the Rf and Rt of thin layer chromatography and gas liquid and high performance liquid chromatography, respectively. have also been used. These will be cited in subsequent sections. Proton and C-13 nuclear magnetic resonance spectra .. and mass spectrometry are currently in use. Coupled techniques. i e GLC/mass spec, GLC/IR and HPLC/mass spec38, are now more comnplace in identifying components from biological tissues.
5.

Stabi I ity and Deqradation

Trifluoperazine I s subject to air and light induced oxldative degradation. The r n e ~ h a n i s m ~ ~ > be considered a two-step reaction involvlng the intercan ~ ~ mediate formation of a semiquinone free radical which I then oxidized to the s s u 1 fox Ide.

ALEX POST et al.

The formation o f t h e s u l f o x i d e can be r e a d i l y followed spectrophotoAs t h e o x i d a t i o n proceeds,the wavelength maximum, 255 nm, f o r n-&ricaIIy. t r i f l u o p e r a z i n e f a l l s w i t h t h e concomitant increase i n t h e wavelength maximum f o r t h e s u l f o x i d e a t 278 nm. S i m i l a r l y , degradation can be r e a d i l y followed by many o f t h e methods c i t e d i n Section 7.6 and/or q u a n t i f i e d by methods l i s t e d i n Sectlon 7.63-7.65.

Aqueous a c i d s o l u t i o n s o f t r i f l u o p e r a z i n e , f l u s h e d w i t h n i t r o g e n , and However, i n t h e l i g h t and k e p t i n t h e dark, a r e s t a b l e f o r several days. e s p e c i a l l y UV l i g h t , degradation occurs r a p i d l y . W i t h i n 15 minutes and under I n ethanol o r a c i d i f i e d UV l i g h t , d i s c o l o r a t i o n o f t h e s o l u t i o n i s evldent. ethanol, no such degradation i s observed w i t h i n 48 hours3*.
T r i f luoroperazine d i h y d r o c h l o r i d e s t o r e d a t r w m temperature f o r up t o

two years d i d n o t show degradation3.

Lever and Hague4 observed t h a t on d i l u t i n g concentrated s o l u t i o n s o f t r i fluoperazine w i t h c o m n d i l u e n t s used under c l i n i c a l s i t u a t i o n s l i . e . c o l a , coffee, tea, grape and apple j u i c e ) t h e r e was a c o l o r change and t u r b i d i t y and They recommended d i l o r p r e c i p i t a t i o n w i t h i n two hours a t room temperature. u t i o n s be made f r e s h l y and w i t h d i s t i l l e d water only.

6.

Metabolism

6. I

Metabolic Products

The i n v i v o and i n v i t r o metabolism of t r i f l u o p e r a z i n e have been e x t e n s i v e l y s t u d i e d by man i n v e s t i g a t o r s . .The f o l l o w i n g schematic a b s t r a c t e d from several pub1 i n d i c a t e s several pathways t o t h e m e t a b o l i c A summary of t h e f i n d i n g s along w i t h t h e l n d e n t i f l c a t i o n products i d e n t i f i e d . and/or q u a n t i t a t i v e techniques used t o e s t a b l i s h t h e amounts p r e s e n t a r e l i s t e d i n Table 7 . The c l t e d references c o n t a i n Information r e g a r d i n g t h e pharmacokinetics o f t h i s compound as it r e l a t e s t o t h e mode of a d m i n i s t r a t i o n , and the amounts of t h e m e t a b o l i t e s present i n t h e various t i s s u e s .

TRI FLUOPE RAZ IN E HYDROCHLORIDE

563

Table 7 Metabo I i t e l d e n t i f i e d and/ o r Quantlf ied Ana I y t i ca I Technlques Used TLC',

M S ~

Animal Rat

T Issue Liver K I dney Braln U r Ine Urine Bra I n P I asma L l v e r Mlcrosomes B r a In Llver Lungs K i dney L i v e r Mlcrosornes

Re f e r en ce
40.49

I, IV, VII, VIII I, IV, VII, VIT I, VII X

I, VII, I, IV I, IV

Man

TLC, MS

40.49

SFe
S Fe

Rat Rat

I , 111, v, VI, VII,

G L C ~ , MS

50
51

VI I I, 11, IV, VII, VIII, XI1 I. 111, VII I, 111, VII

I One d e - a i k y l a t e d analogue Two h y d r o x y l a t e d analogues

TLC. UVc

Rat

TLC, UV

52

Rat Rat Rat

Ur I ne
L I v e r M i crosomes

I . I1

TLC, UV T LC TLC, UV

53
54 12

VT I
111, XI

Ur I ne

( a ) T h i n l a y e r chromatography ( b ) Mass s p e c t r o m e t r y
(el
(c) U l t r a v i o l e t a b s o r p t i o n ( d ) Gas l i q u i d chromatography
Spectrofiuorometry

TRIFLUOPE RAZl N E HYDROCHLORIDE

565

Using 3 5 S tagged trlfluoperazlne, Flanagan, et a155 showed that only about 11-136 of the o r a l l y admlnlstered dose was detected In the urine of nonfasted rats after 96 hours. Of this amunt, 80-855 was found in the 24 hour urine collectlon. Using fasted rats, only about 96 of the total dose was detected with about 90% found In the 24 hour collectlon. Using 24 hour urine collections, West, et aIs6 found that trlfluoperazine was extensively metabolized by man with unchanged trifluoperazine accounting for tess then I6 of the dose; the sulfoxide was 1-65 of the dose; and that the excretion of the trlfluoperazine and Its metabolite was dose dependent.

62 .

Biological Hal f-Life

Schrnalzing and &eyer5' showed that when trlf luoperazine is administered intravenously tomale rats, the biological half-life for the trifluoperazine in brain, lung, kidney, and plasma was approximately 2.5 hours, and much longer In the liver. After o r a l administration, the concentration in the liver was the same as after the intravenous dosage. 63 . Protein Eindlnq

Nambu and Nagar59~tudied the binding of trif luoperazine t o bovine serum albumin using an equillbrium dialysis method and a gel flltration procedure. They showed that binding increased with pH, the order of increase was dependent on the ion species with citrate,succinatsphosphate>acetate, was correlated with surface activity, and increased with the partition coefficient in dadecane/water system. Binding to B.9 in 1/30 g phosphate at (The results suggested that a hydrophobic pH 7.00 at 10C was 82-87s. Zla and Price60apparently reached inferaction takes part I n the binding.) the same conclusion when they used 2-(4'-hydroxybenzeneazo)benzoic acid as a spectrophotometric probe and measured the dlfference absorption spectra with binding of trifluoperazine to bovine serum albumin. Gabay61and Huanglostudied the binding of trif luoperazine t o human serum albumln, as well as that from the dog, rat, rabblt, pig. horse, sheep, goat, and chlcken. They also used a UV difference spectrophotometric method and an intrinsic protein fluorescence quenching method. From the shapes of the W difference spectra, whlch were essentially ldeptical, indicated that the overall binding s i t e environment (hydropholicl of the ten species were s i m l lar.

7 .

Methods of Analysis

71 .

Elemental Analysls

Conventional procedures for the determination of C, H. N, S, CI, and F yielded the following results on a sample which passed NF X I V spec I f i cat i ons. 62

566

A L E X POST e t a /

E I ement
C H

Found 52.26 5.31 8.72 6.12 14.87 I I .87

Theory 52.50 5.46 8.75 6.67 14.76 I I .86

N S

CI F
7.2 T i t r i m e t r i c Analysis

Several t i t r i m e t r i c procedures have been r e p o r t e d f o r t h e assay o f t r i f I uoperazine d i h y d r o c h l o r i de: 7.21 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 i n g l a c i a l a c e t i c a c i d i s apparently t h e most f r e q u e n t l y used.63 The sample i s d i s s o l v e d i n g l a c i a l a c e t i c acid, mercuric .: acetate T.S. i s added and the t i t r a t i o n e f f e c t e d w i t h standardized 01 p e r c h l o r i c a c i d i n g l a c i a l a c e t i c a c i d t o t h e blue-green end-point o f c r y s t a l v i o l e t . Each ml of O.IN p e r c h l o r i c a c i d i s e q u i v a l e n t t o 24.02 mo o f t r i fluoperazine dihydrochloride. The end-point can a l s o be determined potent i o m e t r i ca I I y us i ng g I ass-ca lome I e I ectrodes. 64 7.22 T i t r a n t : Ceric Sulfate Detection: Photometric Endpoint

Agarwal and Blake30 employed a photometric t i t r a t i o n procedure. They t i t r a t e d an a c i d s o l u t i o n of t h e sample w i t h 0.02N c e r i c s u l f a t e , d e t e c t i n g t h e endpoint p h o t o m e t r i c a l l y a t t h e wavelength o f maximum absorbance of c e r i c s u l f a t e , 420 nm. An e x c e l l e n t c o r r e l q t i o n w i t h t h e method noted i n Section 7.21 was obtained. 7.23 Titrant: Ceric Sulfate Detection: C o l o r l e s s Endpoint

The sample, dissolved i n d i l u t e s u l f u r i c a c i d i s t i t r a t e d w i t h c e r i c s u l f a t e t o a c o l o r l e s s endpoint. The equivalence p o i n t corresponds t o t h e a d d i t i o n o f two moles o f c e r i c i o n per mole o f t r i f l u o p e r a z i n e dihydrochloride. The a c i d - s t a b i l l z e d c o l o r e d f r e e r a d i c a l i s discharged when t h e o x i d a t i o n t o t h e ' s u l f o x i d e ' i s completed.65

TRIFLCIOPERAZINE tlYDROCHLORIDE

7.3

Comp I exometrtc Analysis

Preclpltation of the trifluoperazine as its mono-Reineckate salt with an excess of the precipitating reagent and titratin the excess bromometrically is the basis of the method proposed by Olech.;10 The method is rapid, requiring only mi I iaram amounts o f samole and with s n error o f ~~ f1.0 1-59. G a j e ~ s k aused an excess of lead, cadmium, copper, or zinc picrate to precipitate trifluoperazine. The lead picrate forms an insoluble complex, while the others form 5:3 complexes. Titration of the excess cation is made with standardized EDTA.

7.4

SDectrophotometric Analysis

Trifluoperazine dihydrochloride can be assayed by ultraviolet spectrophotometry in dilute hydrochloric acid at its maximum wavelength ( Q 5 5 nm) or As, via a two point analysis ( b , , nm- Abs278 nm). 38 Several approaches to the assaying of trifluoperazine in Various CM)mercial preDarations have been reported. The British Pharma~opoeia3~ and the National methods involve an extraction followed by the UV readout o f suitably diluted solutions; the former report a method for tablets, the latter for tablets, injection, and syrup. Alternate procedures were Droposed by Watson, et a l : 8 for the analysis of trifluoperazine in fablets. In their first procedure, they partitioned a t r i f l u o p e r a z i n e - b r o m o c r e s o l purple complex between an aqueous pH 6 buffer and benzene-isoamyl alcohol and measured the absorbance of the yellow colored In the second method, a I %hydrochloric acid extract organic phase at 410 nm. through an a I ka I i ne di atomaceous earth col umn, and the tri f I bocleri azine eluted with chloroform. The chlorofom extract is nixed with methanolhydrochloric acid and the solution measured at 259 nm. These procedures eiim~na+edpotential interferences not accomodated by the British Pharmacopoeia procedure.

A di fferentiaI spectrophotometric method was developed by Davi dson6' which precluded interferences from the photochemical decomposition product (sulfoxide) and excipients including the conventional coloring and flavoring agents. The sample i treated with peroxyacetic acid to rapidly and quants itatively convert the trifluoperazine to its sulfoxide. The difference absorption maximum at 353 nm is a measure of the trifluoperazine. This procedure has been used with sustained-release capsules, as well as other conventional dosage f orms.
A highly specific procedure for the shenothiazine nucleus in biological tissues was reported by Wal lach and Biggs7. A characteristic oxidation product is obtained when the alkaline-extracted phenothiazine i treated with cobalt s ( I l l ) ion and is stable in the hexane-tertiary butyl alcohol used. The wavelength maximum occurs at 272 nm and the assay is linear over a range of 0 . 5 50.0 mcg/ml.

Huang and Bhansal i 5 3 separated the trifluoperazine and its sulfoxide in urine using thin layer chromatography and after a quantitative elution from the plate determined the amount of each present spectrophotometricall y . Using Oeproteinated human blood and liver (with 5tj HCI), followed by extraction of alkal inzed solution, Stevens. et a 7 quantified the amunt of trifluoperazine I' spectrophotonetrical l y . Recoveries of 60-76% were obtained.

5 68

A L E X POST

cf trl

U s i n g Sephadex LH-20, Malcolm71 separated t h e t r i f l u o p e r a z i n e and then determined t h e amount p r e s e n t i n t h e v a r i o u s f r a c t i o n s s p e c t r o p h o t o Reference samples were s i m i l a r l y m e t r i c a l l y t o determine t h e c o n c e n t r a t i o n . treated. 7.5 Spectrof lucrometric Analysis

The f l u o r e s c e n c e spectrum of a p h e n o t h i a z i n e i s unique and t h u s can be used t o q u a n t i f y t h i s s p e c i f i c compound i n b i o l o g i c a l t i s s u e s , s o l u t i o n s and t a b l e t s .

Me1 I i n g e r and Keeler72 showed t h a t when t r i f l u o p e r a z i n e i s t r e a t e d w i t h KMNO,, i s a c i d , t h e f l u o r e s c e n c e s h i f t s t o s h o r t e r wavelengths w i t h an increase i n i n t e n s i t y and c o n c o m i t a n t l y , t h e e x c i t a t i o n spectrum changes t o form a c h a r a c t e r i s t i c wavelength p a t t e r n o f f o u r d i s t i n c t peaks. These a u t h o r s used t h i s procedure f o r t h e q u a l i t a t i v e i d e n t i f i c a t i o n o f pheno0.8 pg/ml of body f l u i d c o u l d be d e t e c t e d . T h i s t h i a z i n e s showing t h a t 0.6 was about a f i v e - f o l d i n c r e a s e o v e r u l t r a v i o l e t a b s o r p t i o n procedures. A subsequent r e p o r t by t h e s e same a u t h o r s 7 3 showed t h a t s p e c t r o f I u o r o m e t r i c a n a l y s i s c o u l d be used t o q u a n t i t a t e t r i f l u o p e r a z i n e i n b i o l o g i c a l t i s s u e s , ampuls, and t a b l e t s a t t h e f i n a l c o n c e n t r a t i o n o f 2 t o 20 ng/ml.

Ragland and K e n r ~ s s - W r i g h t found t h a t i f t h e o x i d a t i o n was e f f e c t e d ~~ w i t b hydrogen p e r o x i d e i n 50% a c e t i c a c i d , t h e f l u o r e s c e n c e spectrum was more s t a b l e and more i n t e n s e . They subsequently used t h i s procedure t o q u a n t i f y nanogram q u a n t i t i e s of t h e p h e n o t h i a z i n e i n b l o o d serum. b r a i n t i s s u e , and ~~ l i v e r . 7 5 T ~ m p s e t tconfirmed t h e a o p i i c a b j l i t y and r e l i a b i l i t y of t h i s method f o r t h e q u a n t i t a t i o n o f t r i f l u o p e r a z i n e i n b l o o d serum. West, e t a156 used s p e c t r o f Iuorometry t o determine b o t h t r i f l u o p e r a z i n e and i t s s u l f o x l d e i n u r i n e . plasma, and b r a i n . Recoveries o f 68-80% T h e i r r e s u l t s were i n e x c e l l e n t were o b t a i n e d on 10 pg/ml o f u r i n e s o l u t i o n s . agreement of t h o s e o b t a i n e d by Spano, e t at7 who used a s p e c i f i c r a d i o i s o t o p e procedure. 7.6 Chromatographic Methods o f Seoaration 7.61 Paper Chromatography

Chromatography on paper and m o d i f i e d papers u s i n g an a s s o r t ment o f m b i l e phases has been used t o separate t r i f l u o p e r a z i n e and i t s metabolites. Several o f t h e m o b i l e phases and s t a t i o n a r y phases a r e l i s t e d i n Table 8 and d e t e c t i o n methods i n Table 9. Paper chromatography has been o used i n ana I y z i ng b i l o g i c a i t i s s u e s . 3 3 s 81 p a 2

TRI FL UOPE RAZ I N E HY D ROCH LO I DE U

569

Table p Paper Chromatoaraphv o f T r i f i u o p e r a z i n e M o b i l e Phase !I Sodi um Formate S t a t i o n a r y Phase Whatman 3MM Whatman 3 M M Whatman 3 M M Whatman 3 1 M Whatman 3MM Whatman 3 I M Whatman 3M 4 Whatman # I impregnated w i t h 5% sodium d i hydrogen c i i r a t e Whatman # I impregnated w i t h 10% t r i b u t y r i n Whatman # I impregnated w i t h 105 t r i b u t y r i n Whatnlan # I impregnated w i t h c i t r i c acid-phosphate b u f f e r , pH 4.0 Whatman # I impregnated w i t h c i t r i c acid-phosphate b u f f e r , pH 4.0 Whatman # I impregnated w i t h c i t r i c acid-phos. p h a t e b u f f e r , pH 4 0 or 4

Rf -

Reference
33

0.27
0.25 0.25
0.60

IN Sodium Forrnateo-propanol (90:lO) IN Sodium F o n a t e I N ammonia (9O:lO)

33

33

95%Formic

I N Sodium Formatea c i d (97:3)

33 33

IN Sodium A c e t a t e
I N Sodium A c e t a t e n-propanol ( 9 0 : 1 0 )

0.17

0.35 0.55
0.34

33

Sodium C h l o r i d e n-proDaqoI ( 9 2 : 8 ) autanoi-hater-Ci t r i c Acid (870:130:4.8 g ) pH 4.58 A c e t a t e B u f f e r , r u n a t 95'~ pH 7.4 Phosphate B u f f e r , r u n a t 86OC n-Butan c I -HC I -Water (6:I :7.5) n - B u t a n o l - A c e t i c AcidWater (6:I :7.5) lsobutyl alcohol-2ropionic a c i d - w a t e r (lO:1:4.5)

33 36

0.06,0.09 0.03

36981

36

0.91

78

0.88

78

0.91

70

55 Amronium S u l f a t e s a t urated w i t h Isobutanol Cyclohexane-Benzene (9:l)

S A S #57, Whatman

#I

0.23

79

Several papers imDregnated w i t h formamide5% a m n i u m f o n a t e

0.78

80

570

A L E X POST era1

Table 9 Spray Reagents f o r D e t e c t i o n o f T r i f I u o p e r a z i n e (Paper Chromatography 1

Reaae nt

Co Io r orange purple red red orangered red red+ row n red-orange d a r k green bluish yellow p u r p l e yellow

Reference
33

40% H2SO4
D r a g e n d o r f f ' s potassium l o d o p l a t i n a t e Mod i f i ed' Modified' Modified' Mod i f i ed' conc. H2S04 Marquis Mandelin Mec ke

genera I I y used

81
81 81
81

Modi f ieda Frohde Palladium Chloride ( 1 % ) Bromine w a t e r UV, 263 nm UV, 254 nm

81
81

81
33
81

fa) T r e a t e d w i t h sodium s u l f a t e t o reduce r a t e o f r e a c t i o n .

7.62
T h i n Layer Chromatography

A s i g n i f l c a n t number o f m o b i l e phases have been used t o chromatograph t r i f l u o p e r a z i n e on s i l i c a g e l . m d i f i e d s i l i c a g e l , and aiumina, and a r e l i s t e d i n T a b l e 10 a l o n g w i t h t h e r e s p e c t i v e R f s o b t a i n e d . S i m i l a r l y , a l a r g e number o f d e t e c t i o n reagents, i n c l u d i n g spray reagents, have been used t o d e t e c t t r i f luoperazine.
T h i n l a y e r chromatographic s e p a r a t i o n s have been used t o separate t r i f l u o p e r a z i n e from i t s m e t a b o l i t e s and a l l subsequently i d e n t i f i e d u s i n g d i f f e r e n t i a l spray r e a g e n t s ( T a b l e 10). s p e c t r o p h o t o m e t r i c p r o cedures, and mass spectrometry. F u r t h e r , on i s o l a t i o n o f t h e s e m a t e r i a l s , t h e y were q u a n t i f i e d u s i n g s p e c t r o p h o t o m t r i c procedures. T a b l e I I l i s t s t h e t i s s u e s s t u d i e d and t h e ' r e a d - o u t ' used for t h e q u a l i t a t i v e and/or q u a n t i t a t i v e analysis.

TRIFLUOPERAZINE HYDROCHLORIDE

57 I

T a b l e 10 TLC Systems M o b i l e Phase Cyc I ohexane-BenzeneD i e t h y l a m i n e (75:15:10) Methano I Aceione Methanol Ethanol ( 9 5 % ) Ethylacetate-MeThanoIAmmnia (85:10:5) Ammn i urn Acetate-Methanol (10 ml 15%:40) Methanol-121 Ammonium Hydroxide (100: I .5) Adsorbent Rf 0.45 0.49 0.19 Reference
83

0.13 KOH

S i l i c a Gel-

S i l i c a Gel0. ltj KOH S i l i c a GelO.IM KOH

83

83

S i I i c a Gel0: KHsot, S i I i c a Gel0.13 KHSO,

S i l i c a Gel S i I i c a Gel S i I i c a Gel S i l i c a Gel

0.10
0.02 0.12

83

83

84

0.63
0.57 0.54 0.12

85

32

Cyclohexane-DiethylamineBenzene (75:20:15) Ace tone

32

S l I i c a Gel

32 32 32

Ch lorofonn-Methanol

(90:10)

S i l i c a Gel S i I i c a Gel

0.52
0.56
0.44

Benzene-Ethanol-I2N Arnmnium Hvdroxide (95:15:5) Arnmon i urn Hyd r o x i de i n Ethanol (90:45:4) Ethanol-Water-Acetic (20:20: I ) Chloroform-Methanol

Ethylacetate-Acetone-1:l

S i l i c a Gel

86

Acid

S i I i c a Gel
S i l i c a Gel S i l i c a Gel

0.28 0.45

07

(100: 10)

88 89

ethyl acetate-Ch1oroformMethanol-O.IbJ Sodium Acetate pH 4.7 b u f f e r (54:23:18:5)

0.33

A L E X POST

('I

crl.

Table 10 (continued) Mobi l e Phase Benzene-Dioxane-Ammonia ( 60: 35 :5 ) Ethanol-Acetic acid-Water ( 50: 30 :20 ) Methanol-Butanol (60:40)
Ad so rbent Rf -

Reference 31

S i l i c a Gel S i l i c a Gel

0.69

0.33
0.40 0.18 0.33

31

Si I i c a Gel
S i l i c a Gel

31 33

t-Buty i a I coho I -IF Ammon i a

(90: 10)

n-Propanol-IN Ammonia (88:12) Ether, saturated w i t h water

S i I ica Gel

33
33 33 33

Si I i c a Gel
S i I i c a Gel

0.09
0.34

70% Methanol 85% n-propanol

n-Butanol saturated w i t h IN Ammonia

S i I i c a Gel
S i l i c a Gel S i l i c a Gel

0.12

0.5 I
0.61

33
51

I sopropano I -Ch I o r o f o r m I .3N Ammonia water (l6:E:I:l)

Acetone-lsopropanol-I! Ammonia (27:21:12) 1.2-DicholoroethaneEthylacetate-Ethanol-Acetic

S i l i c a Gel S i I i c a Gel

0.77/0.8 I 0.5 I /O .32

12/51

12/51

acid-Water(l5:26:12:8:7.5)

I sopropanol-Ch loroform-25% A m n i a - W a t e r (32: l6:25: I )

Ch I o r o f o rm- Ethano I-Arnmon i a (80:20: 1 )

S i l i c a Gel

0.83
0.80

12

S i I i c a Gel
S i l i c a Gel

52

Benzene-Dioxane-DiethylamlneEthanol (50 :40: 5: 5 1 Acetone-Ethyl acetate-Ethanol (5:4:l) s a t u r a t e d w i t h Amnonium l a c t a t e pH 3 Acetone-Ethyl acetate-Ethanol (5:4: I ) saturated w i t h Amnonlum l a c t a t e pH 7

0.85
0.10

37 90

S i I i c a Gel

S i I i c a Gel

0.30

90

TRl FLVOPE RAZINE HYDROCHLORIDE

513

Table 10 (continued) Mcbi le Phase Acetone-Ethyl acetate-Ethanol ( 5 : 4 : I ) saturated with A m m n i u m lactate pH 9 Benzene-Acetone 95:5) Benzene-Acetone 95:5) Benzene-Acetone 90: 10) Water-Acetone (70:30) Toluene-Chloroform-MethanolAmmonium Hydroxide
(60:40: 1O:O.Z)

Adsorbent Silica Gel

Rf
0.17

Reference
90

Alumina
A 1 u m i na

neutral 1 basic) basic)

0.20
0.40

91 91 91
91

A I umi na
Cel lulose
S i I ica Ge

0.53 c.35
0.44

92

Ethyl acetate-n-Propan6iAmmnia (70:25:4) Ethyl acetate-DichloroethanePmmon i a ( 8 0 : 20: 5)

Cyclohexane-Diethylamine-

S i I ica Ge

0.53
0.47

43
93

Si I ica Gel
S i I ica Gel

Benzene (80:15:5)

0.43

93

m
N

x
X

x x x x x x x x

a
x

0
I a l

> m -c

TRI FLUOPERAZINE HYDROCHLORIDE

515

7.621

D e t e c t i o n Methods

Numerous d e t e c t i o n methods, i n c l u d i n g s p r a y r e a g e n t s , have been used t o v i s u a l i z e t r l f l u o p e r a z i n e on t h i n l a y e r p l a t e s . A listing o f them can be found i n T a b l e 12. T a bl e 12 D e t e c t i o n Methods Used i n TLC o f T r i f l u o p e r a z i n e Reagent Response ( c o l o r ) yellow-brown cameo (tan) R eference
86

+ Ferric chloride
Folin-Ciocalteau

Phosphomolybdic a c i d

F e r r i c c h l o r i d e i n HCIOb t HN03 A m n i u vanadate m Cinnamylaldehyde

p in k-.o range
flesh cameo pink cameo violet orange-p i n k mauve-purp le brown+yellow

32 32.97 32 32 32 32
85
94
94

p-Dimethylaminobenzaldehyde
Fu r f u r a I l o d o p t a t i n a t e s p ray Bromine A n i l i n e vapor f o l l o w e d by bromine Te t r a c y a n o e t h y le ne i n a c e t o n i t r i le

95

2 , 4 , 7 - T ri n i t r0 - 9 -f l u o r e n o n e aceton i tri l e
1,3,5-Trinitrobenzene

in

grey brown orange orange orange blue orange brown brown-yel low orange

95 95
96 97

i n toluene

Vanadi um pentoxide-HzSOb

p-Dimethylaminobenzaldehyde
40% HzSO,, + h e a t 0. I%Bromocresol p u r p l e 5% HgSO,+-ethanol
HC104 N i t r i c acid

92
98
31

99
99

5% A m n i u m p e r s u l f a t e
7.63

52

H igh Performance L i q u i d Chromatography (HPLC)

A d s o r p t i o n . l o 0 i o n - e x ~ h a n g e , r~ v e r s e ~ ~ ~ l C 1 and i o n e ~ ~ ~ phase, p a i r i n g r e v e r s e phase102 systems have been used t o e v a l u a t e t r i f l u o p e r a z i n e . The p r o c e d u r e i s r a p i d , y i e l d i n g good r e s o l u t i o n of s e v e r a l p h e n o t h i e z i n e s .

Table 13

HPLC Parameters f o r T r i f I u o p e r a z l z
Flow Rate (ml / m l n)

Co Iumn
SII-x-I (Perk In-E I mer 1
ION-X-SC

t b b l l e Phase

Detector

R t (mln) (approx)

Re fererice

Ch1orobutane:Iso-octane conta I n I n g I % d i e t h y lami no

0.01M ( H )2ttPO4 I n methanol : NI , H20 (2:3), adJusted t o pH 9 . 0

I I

UV (254 nm)

16

LOO

UV (254 nm)

e
5

100

U I trasphere-IP (Altex) Bondapak C-18 (Waters)

0.01M %POI, + 0.01M Nonylamlne, adJuTted t o pH 3 . 0 , + 35% acetonltrlle

2.0

uv

10 I

)1

10%0.25M Camphorsulfonlc acid, 60% methanol, 30% water, a d j u s t e d t o pH 3.0

Methanol-O.5M Ammonlum N i t r a t e (pH 6.0) (4:T)

UV (262 nm) UV (254 nm)

30
10

I02

Alkylsulfonic Acid s t r o n g cation exchanger

103

TRiFLUOPERAZlNE HYDROCHLORIDE

577

7.64

Gas L i q u i d Chromatography

Gas l i q u i d chromatography continues t o p l a y an i m p o r t a n t r o l e i n t h e d e t e c t i o n and d e t e r m i n a t i o n of t r i f l u o p e r a z i n e and i t s d e t e c t i o n and d e t e r m i n a t i o n o f t r i f l u o p e r a z i n e and i t s m e t a b o l i t e s i n b i o l o g i c a l m a t e r i a l s . I n Table 14 a r e l i s t e d a number o f s u b s t r a t e s and o t h e r parameters used t o chromatograph t r i f I uoperaz i ne. Using p y r o l y s i s techniques, Fontan, e t a t T t 1 c o u l d r e a d i l y i d e n t i f y t r i f l u o p e r a z i n e and d i f f e r e n t i a t e i t from its m e t a b o l i t e s and o t h e r De LeenheerllZ coupled p r e p a r a t i v e gas I i q u i d chromatography phenothiazines. w i t h m i c r o - i n f r a r e d spectroscopy f o r t h e i d e n t l f i c a t i o n of t r i f l u o p e r a z i n e and o t h e r phenothiazines. T r i f l u o p e r a z i n e was separated on a 1% FFAP column a t 23OoC. Table 14 Gas L i q u i d Chromatography Parameters f o r t h e GLC of T r i f I uoperazi ne Column ABS, Co I umn Temperature Carrier Gas Detector FID Ref. (min) - -

Rt

5% QF-l on Anakrom 100/110

210' f o r 18 min. program4' med t o 2 0

N2
He

22

104

Chrom Q,

3.5% XE-60 on Gas

100/120

25 3' 235O 20 3' 20 3' 210

2200
205'
245'

FID FID FID FID FID FID

%

9.0

104 I04
9
9
85

3% OV-17 on Gas Chrom Q

He
N2 N2
He

22.5
6.9 13.6

5% Ov-1 on Diatop o r t S,

80-100 mesh

2% FFAP on Diatoport S , 80-100 mesh

Q,

3% SE-30 on Gas Chrom

80-100 mesh 1% HI-EFF-8BP + 10% SE-52 on Gas Chrom Q, 80-100 mesh 2% SE-30 on Gas Chrom Q, 80-100 mesh

8.6

N2

120

105

Argon
N2

90SR

42

106

3%OV-l on Gas Chrom Q,

80-100 mesh
5% SE-30 on D i a t o p o r t 60-80 mesh

FID .FID FID FID FID

3. I

107
108

S,

270
245O

5.5
6.9

10% SE-30 and I % t r l s t e a r i n on Gas C h r m W 1 % HI-EFF-86 on S l l a n i z e d Gas Chrom P

1% HI-EFF-8B o n S i l a n i z e d Gas C h r m P

N2
N2

109

2208
250

10.9
I. 6

110 I10

N2

ALEX POST ei ul.

7.65

E I e c t rophores i s

Paper e l e c t r o p h o r e s i s on b u f f e r e d Whatman 3 M paper (pH 3.3 M t o 9.3) s e p a r a te d t r i f l u o p e r a z i n e and i t s s u l f ~ x i d e . ~ d e n t i f i c a t i o n was l ~ made from t h e r e s p e c t i v e m i g r a t i o n d i s t a n c e and by t h e response t o a s u l f u r i c a c i d spray r e a g e n t and by i t s f l uo resce nce . Migration ( i n cml

f!E
4.7 6.0 1.2 8.0 9.3

T r i f I uoperaz i ne

Su I f o x i de 7.2 6 -2
5.8 5.4 5.4 5.9

3.3

7.0 4.4 4.7 4.7 3.2 1.7

8.

M is c e lla n e o u s

8. I

A d s o r p t i o n I sot he rm

A d s o r p t i o n isotherms o f t r i f l u o p e r a z i n e by carbon b l a c k , g r a p h i t e , s i I i c a g e l , and p o l y e t h y l e n e determined by Nogami, e t a1 'I4 showed a r e l a t i o n The amount absorbed was s h i p t o i t s n e u r o l e p t i c and h a e m o l y t i c a c t i v i t y . r e l a t e d t o t h e m l e c u l a r volume o f R a t t h e 1 0 - p o s i t i o n , t h e b u l k i n e s s of t h e s u b s t i t u e n t a t t h e 2 - p o s i t i o n , t h e pH o f t h e b u f f e r s o l u t i o n , and t h e determined t h e p a r t i t i o n c o e f f i c i e n t i n CHCt3/0. I& HCI. Sorby, e t They showed a d s o r p t i o n is o th e r m s by k a o l i n , t a l c , and a c t i v a t e d carbon. t h a t t h e a d s o r p t i o n by k a o l i n and t a l c was aependent upon t h e pH o f t h e medium whereas t h i s WBS n o t t h e case w i t h a c t i v a t e d carbon.

8.2

S u r fa c e A c t i v i t y

S e v e r a l p h eno t h i azi n es have been e v a l u a t e d f o r t h e i r e f f e c t on s u r f a c e a c t i v i t y as an e x p l a n a t i o n for t h e i r p h y s i o l o g i c a l a c t i v i t y . Zografi and Munski116 showed t h a t t r i f l u o p e r a z i n e was many t i m e s more e f f e c t i v e than c hl orpro m a z ln e i n l o w e r i n g t h e s u r f a c e t e n s i o n of a pH 5.0, i o n i c s t r e n g t h 0.1, s o l u t i o n a t 25OC. Seernan and B i a l y l l ' a t t r i b u t e d t h e a c t i v i t y o f t r a n q u l I l z e r s t o t h e lowering of t h e surface tension a t the e r y t h r o c y t e surface v i a t h e a d s o r p t i o n o f a m n o m l e c u l a r l a y e r o f t h e p h e n o t h i a z i n e analog. Trifluopera z i n e was shown t o be s i g n i f i c a n t l y m r e e f f e c t i v e i n l o w e r i n g t h e s u r f a c e t e n s i o n t h a n c h lo r p r oma zi ne , and, t h e r e f o r e , a more p o t e n t t r a n q u i I i z e r .

TRI F L U OPE R A Z I N E HY D R O C H LORI DE

579

9.

References N a t i o n a l Formulary X I V , p . 736, S r i t i s h Pharmacoooeia, p . 483 (1973). For t h e f r e e base: Cha?ten, L.G. and H a r r i s , L.E., Anal. Chem., 1495 (19621, Brand o f T r i f l u o p e r a z i n e H y d r o c h l o r i d e : Smith K I i n e & French Labs, P h i l a d e l p h i a , PA. Thompson, W.E. e t al., J . Pharm. S c i . , 54, 1819 (1965) Yung, D.K. and Pernarowski, M., J . P h a r K 5 c i . , 52, 365 (1963). J. Pharm. Sci., 6 r 1346 ( 1 9 7 1 ) . Wallace, J.E. and Biggs, J.D., De Leenheer, A., J. Assoc. O f f . Anal. Chem., 55 1 6 0 , ( 1973). De Leenheer, A . , J . Phann. Sci., 63, 389 ( 1 9 7 f i . Huang, P.C. and Gabay, s., Biochem. Pharmacol.. - 957 (1974). 23, J. Pharmacol 433 ( 1 9 6 4 ) . Rogers, A.R., Gaertner, H.J. e t a l Biochem. Pharmacol 23, 303 ( 1 9 7 4 ) . Warren, R.J. et a l J. Phann. Sci., 14471966). S a f e r s t e i n , R. e t a l . , J. F o r e n s i c Sci., 463 (1974). A t 24OC. Smith K I i n e 8 French Labs, P h i l a d e l p h i a , PA. Green, A.L., J . Pharm. Pharmacol 2 . 1 0 (1967). Green, A.L., Smith KI i n e 8 F:ench Research I n s t i t u t e . Murthy, K.S. and Z o g r a f i , G., J. Pharm. sci.. 59, 1281 (1970). Baur, E.Pd., J Pharm. Exper. Therap., 2 1 9 7 1 9 7 1 ). Mao, T.S.S. and hoval, J.J., Biochem. Pharmacol., 501 (1966). Frisk-Holmours, e t al., Eur. J . Pharmacol., Is, 139 (19711. Chatten, L.G. and H a r r i s , L.E., Anal. Chem., 34, 1495 (1962). Scrby, 2.L. e t al., J. Pharm. Sci., 785 (1966!. Kraus, L. and Dumont, E., J. Chromatog., 56, I 5 9 ( I971 ) Anderson, E.L. e t al., Arzneim-Forsch., 12, 937 (1962). J . W a l t e r h a m i l l , personal comrnunicationFSmith K l i n e & French Labs. C r a i g , P.N. e t a l . , J. Org. Chem., 709 ( 1 9 5 7 ) . Lucas, G.H.W. and F a b i e r k i e w i c z , D., J. F o r e n s i c Sci., 8, 462 ( 1 9 6 3 ) . F i t z g e r a l d , T.J. and Walaszek, E.J., C1 in. T o x i c o l a , 5 9 9 (1973). 1011 ( 1 9 6 9 ) . A g a m a l , S.P. and Blake, M.I., J . Pharm. Sci., Cochin, J . and Daly, J.W., J. Pharmacol. Exp. Therap., 160 ( 1 9 6 3 ) . Zingales, I., J. Chromatog., 405 ( 1 9 6 7 ) . M e l l i n g e r , T.J. and Keeler, C.E., J. Pharm. Sci., 1169 (1962). B r i t i s h Pharmecopoeia, p. 484 (1973). F a b i e r k i e w i c z , C.K. e t al., J. F o r e n s i c Sci., lo, 308 ( 1 9 6 5 ) . M o f f a t , A.C. and Smalldon, K.W., J. Chromatog., 90, 9 (1974). G a r r i o t t , J.C. and Stolman, A., C I i n . Toxicol., 225 ( 1 9 7 1 ) . David S t a i g e r , personal comnunicatiop. Smith K I i n e & French Labs. Merck Index, 9 t h Ed. No. 9353. Huang, C.L. and Chang. C.T., J. Pharm. Sci., 1895 ( 1 9 7 1 ) . Andres, C.N., J. Assoc. O f f . Anal. Chem., 1020 ( 1 9 6 8 ) . i b i d . , 2, 824 ( 1 9 7 0 ) . Andres, C.N., Modern M i c r o c r y s t a l T e s t s f o r Drugs, Chap. XIX, WileyF u l t o n , C.C., I n t e r s c i e n c e , N.Y. (19691, Borg, D.C. and C o t z i a s , G.C., Proc. Nat. Acad. Sci., 617 (1962). Borg, D.C. and C o t z i a s , G.C., ibid.. 623 (1962). Borg, D.C. and C o t z i a s , G.C., ibid., 643 (1962). Lever, G.P. and Hague, J.R., Am. J. P s y c h i a t r y , I000 (1964).

2,

a
la

l2 l3

l4 l5
l6 17
18

.,

.,

., 16,

., 2, 2,

.,

19
20

17 7.

2,

21
22

23
24

55,

25 26

27
28 29

22,

30
31
32

E,

.,

33 34

2,

a,

139,

35 36
37 38 39
40 41

4,

2,

60,

42

43
44 45
46

48,

2,

2,

47

g,

580

A L E X POST

ct

(I/.

48 Breyer, U. and Gaertner. H.J., The P h e n o t h i a z i n e s ana S t r u c t u r a l l y R e l a t e d Drugs, e d i t e d by 1,s. Forres;, e t a l . , Raver. Press, N.Y., p. 167 (1974). 49 Breyer, U. e t al., Biochem. Pharmacol., 313 (19741. 50 S p i r t e s , M.A., The P h e n o t h i a z i n e s and S T r u c r u r a l l y R e l a t e d Drugs, F o r r e s t , e t al., Raven Press, N.Y., p. 399 (1974). e d i t e d by I.S. 51 Breyer, U. and Schmalzing, G., Drug Metab. Disoos., 2, 97 (1977). 52 Robinson, A.E., J, Pharm. Pharmacol., Is, 1 (1966). 9 5 3 Huang, C.L. and B h a n s a l i , K.G., J. Pharm. Sci., 57, 1511 (1968). 54 B l c k e l , M.H. e t a l Nauyn-Schmiedebergs Arch. Pharmacol. U. Exp. Path.,

23,

55 5 6 Schmalzing,

256, - 360 (1967).t a l . , Flanagan, T.L. e 293 u p p 1. - ( s N.R. i . e t

.,

G.

J. Pharm. Sci., 996 (1962). and Breyer, U., Nauyn-Schmjedebergs Arch. Pharmacol.,

51.

a l . , J. Phann. Sci., 417 (1974). 58 2eferences l i s t e d i n S e c t i o n 7.5 c o n f i r m t h i s i a e n t i f i c a t i o n . 59 Nambu, N. and Nagai, T., Chem. Phann. Bull., 2463 (1972). 6 0 Zia, H. and P r i c e , J.C., Anal. Chem., 1177 (1975). 6 1 Gabay, S. and Huang, P.C., The P h e n o t h i a z i n e s and S t r u c t u r a l l y R e l a t e d Drugs, a d l t e d by 1.5. F o r r e s t , e t a l . , Raven Press, N.Y. (1974). 6 2 E.R. Reich, personal communication, Smith K I i n e & French Labs. 6 3 N a t i o n a l Formulary XIV, p. 737. 6 L Eno p o i n t d e t e c t i o n used a t t h e Smith K I i n e 8 French L a b o r a t o r i e s . 6 5 Chatten, L.G. e t al., J. Pharm. Sci., 588 (1971). 66 Olech, A., Acta. Polon. Pharm., 64 (1972). 67 Chemia .Analit., Is, 651 (1973). Ana!. Abct. #465 (January 19741. 68 Watson, J.R. e t a l . , J. Pharm. Sci., 391 (1970). 69 Davidson, A.G., J. Pharm. Pharmacol., 795 (1976). 70 Stevens, H.M. e t a l . , J. F o r e n s i c Sci., 169 (1977). ?I Palcolm, H.M., ibid., 1 , 57 (19771, 1 72 M e l l i n g e r , T.J. and Keeler, C.E.. Anal. Chem., 554 (1963). 73 M e l l i n g e r , T.J. and Keeler, C.E., ibid., 1840 (19641. 7 4 Ragland, J.B. and Kenross-Wright, V.J., i b i d . , 2, 1356 (1964). 75 Ragland, J.B. e t al., Anal. Biochem., 60 (1965). 76 Tompsett, S.L., Acta. Pharmacol. e t . Toxicol., 298 (1968). 77 Spano, P.F. e t al.. J. Pharmacol. Exp. Therap., 174, 20 (1970). 78 Eagelson, D.A., J. C I l n . Pathol., 39, 648 (3 1) 9. 6 79 Nadeau, G. and Sobollewski, E., J.Thromatog., 544 (1959). 80 Macek, K. e t al., Pharmazie, 605 (1965). 8 1 S t r e e t , H.V., Acta. Pharmacol. e t . Toxicol., 3, 312 (19621. 82 Heyman. J.J. e t al., Am. J. Psychiat., 117, 1108 (19601. 83 F i ke, W.W., Anal. Chem., 2, 1697 ( 1 9 6 6 r 84 Neesby, T., C l i n . Chem., 19, 356 (1973). 85 Kofoed, J. e t al., J. C h r z a t o g . , 23, 410 (1966). 86 Korczak-Fabierkiewicz, C. and Cimbpa, G., ibid., 53 413 (1970). 8 7 Clarke, V. and Cole, E.R., ibid., 24, 259 (19661..-' 8 8 Kiger, J.L. and Kiger, J.G., A n n a l z Pharmaceutiques Francaises,
5 7 West,

63,

64,

2,

2,

60,

2, 2,

17,

a, 35, u, 2.
2,

20,

89 Pluym, A., J. P h a n . Sci., 1050 (1979). 90 Margasinski, 2 . e t al., Acta. Polon. Pharm., 5 (1964). 9 1 Eiden, F. and Stachel, H.D., Deut. Apoth. Ztg.. 121 (1963). 92 P e t e r Begosh, personal connnunication, Smith K I i n e b French Labs.

489 (19651,

2 .

68,

2,

103,

TRI FLUOPE RAZ I N E HYDROCHLORIDE

58 I

9 3 L a u f e r , V.S. e t al., Arzneim-Forsch., 19, 1965 (1969). 9 4 C l a r k e , V. and Cole, E.R., 3. Chromatog,, 413 ( 1 9 7 0 ) 95 F o r r e s t , J.E. and Heacock. R.A., ibid., 156 (1973). 96 N o i r f a l i s e , h . , i b i d . , Z, & 61 (1965). 9 7 De Leenheer, A., ibid., 156 (1979). q 8 Dernoen, P.J., J. Pharm. S c i , 50, 350 ( I961 ) . 99 Eberhardt, H. e t a l . , Arzneim-Ersch.. 804 ( 1 9 6 3 ) . 100 Rogers, D.H., J . Chrom. Sci., 742 (1974). I o 1 Cooke, N.H.C. and O l s e n , J., American Lab., Aug. 1979, p . 45. 10: E.R. White, p e r s o n a l communication, Smith K I i n e & French Labs. l o 3 Wht:aIs, B.B., J, Chromatog., 263 (1979). lo4 Procisc, H.F. and Lohmann, H.J., C I in. Chem., 222 (1971 ). 15 Rader, 6.R. and Aranda, E.S., J. Phann. Sci., 847 (1968). l o 6 Anoers, M.W. and Mannering, G.J., J. Chromatog., 7, 258 ( 1962). Io7 Wells, J. e t al., J. F o r e n s i c Sci., 382 (1975r, M a r t i n , H.F. e t al., Anal. Chem.. 35,. 1901 (1963). l o g McMartin, C. and S t r e e t , H.V., J . Chromatog., 22, 274 (1966). Microchern. J., 12, z 9 ( 1 9 6 7 ) . 110 Jain, N.C. and K i r k , P.L., Fontan, C.R. e t a l . , Mikrochim. Acta, 1 9 6 $ . 3 6 4 . 11* De Leenheer, h . , J. Chromatog., 35 (1972). 1 1 3 Gudzinow;cz, B.J., Gas Chromatographic A n a l y s i s o f Drugs and P e s t i c i d e s , (1967). Marcel Dekker, Inc., N.Y. 1643 ( 1 9 7 0 ) . 114 Nogami, H. e t a l . , Chem. Pharm. B u l l . . 785 ( 1 9 6 6 ) . 1 1 5 Sorby, D.L. e t a l . , J. Pharm. Sci., 116 Z o g r a f i , G. and Munski. M.V., ibid., 59, 819 (1970:. 1181 (15633. 117 Seeman, P.M. and B i a l y , H.S., BiochemFPharm?coI.,

53, 75,

75,

12,

z .

177,

2.
57,

0,

74,

2,

,s,

2,

GRISEOFULVIN
Mahrnoud A . Hassan and Elsayed A . Aboutabl

1.

2.

3. 4.

Description I . 1 Nomenclature 1.2 Formulae Physical Properties 2.1 Crystal Properties 2.2 Dissolution 2.3 Spectral Properties Synthesis Methods of Analysis 4.1 Time- Resolved Phosphorimetry 4.2 Liquid Chromatography 4.3 Isotope Dilution 4.4 PMR Spectrometry 4.5 Microbiological References

584 584
584 585 585 587 587 592 594 594 594 594 595 597 599

Analytlcal Rofiles of D u Substances, 9 rg

583

Copyright @ 1980 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN:0-12-260809-7

584

MAHMOUD A. HASSAN A N D E. A. ABOUTABL

GRISEOFULVIN 1. Description 1.1 Nomenclature 1.1.1 1.2 Other names: Curling Factor.

Formulae 1.2.1 Structural

s
Various structural formulae have been proposed or griseofulvin, but the currently accepted is that suggested by Grove -- ( ) According to this formuet a1 1 . lae the molecule of the antibiotic contains three rings: the aromatic benzene ring ( ) a 5-membered heterocyclic ring A, with an atom of oxygen (B) and a hydroaromatic 6-membered ring (C). The A 6 B

rings are condensed forming a coumaronone system. Carbon atom 2, which is common rings B & C is an assymetrical carbon atom giving griseofulvin its spiran structure and causing its optical activity, to

GRISEOFULVIN

5 85

which the assymetrical 6 carbon also contri -butes. The C ring may be regarded as the methyl ether of the enol form of 2,4 diketone o r as the methyl ether of 6-methyl dihydroresorcinol. 1.2.2 Wiswesser Line Notation
C - & D L 6 V DX BUTJ CO1 E l

T56 BOXVJ F01 H01 1G

1.2.3

Conformation The preferred conformation of griseofulvin in solution is that shown in the stereostructure given before ( 2 ) . This is based on the finding o f relatively strong coupling ( = 13.5 H z ) between the 6ca and the J

/ 5-a-protons. This relies on the application

o f an NMR shift reagent [Tris

(Dipevalo-

methanato) Europium], to the spectrum o f a partially deuterated sample of griseofulvin. 2. Physical Properties:
2.1

Crystal Properties: 2.1.1 Crystallinity Griseofulvin crystallizes from benzene as stout octahedra o r rhombs. The crystals

5 86

MAHMOUD A . HASSAN A N D E. A. ABOUTABL

are generally up to 5 nm in maximum dimension, although larger particles which may occasionally exceed 30nm may be present. Crystal size affects the absorption of griseofulvin when administered orally. Microsize griseofulvin may be administered in significantly smaller doses than the conventional size powder to obtain the same effect. The U.S.P. specifies that the offi-

cial product is the "Microsize" powder ( 3 ) . Brown and Sim (4) carried out a quantitative X-ray study of 5-bromogriseofulvin in order to define unambiguously the stereochemical relationship of the 2- and the 6 /centre. Crystals of 5-bromogriseofulvin belong to the monoclinic system, space 2 group P2 (C ), with two molecules of C17 1 2
H

BrClO in a unit cell of dimensions a = 6 16

= 108'30.
/

10.96, b = 8.61, c = 10.27 A',

Initial phase determination was based on the bromine and the chlorine atom and several three dimensional Fourier syntheses were evaluated, followed by least squares refinement of the atomic parameters. The

GRISEOFULVIN

587

final discrepancy R over the 1 1 2 9 observed reflexions is 14%.

2.2

Dissolution The dissolution rate of griseofulvin had been significantly enhanced by solid dispersion in succinic acid. This had been initially attributed to the extensive formation of a solid solution of griseofulvin in succinic acid (5). Later, it was shown by X-ray diffraction and differential thermal analysis methods that solid solubility was negligible and such a binary system could be classified more adequately as a simple eutectic mixture ( 6 ) . The dissolution profile of the griseofulvin-succinic acid eutectic mixture system was evaluated using the powder and constant surface area tablet methods ( 7 ) . Contrary to the original proposal of Sekigushi -- (8), dissoet a1 lution rates of griseofulvin from solid dispersions were found to be markedly affected by their particle size.

2.3

Spectral Properties
2.3.1

Ultraviolet Spectrum
In ethanolic solution, griseofulvin exhi-

bits a characteristic UV spectrum (Fig.1)

588

MAHMOUD A. HASSAN A N D E. A. ABOUTABL

1 I

---

CH; 0

CH; 0
C L

50,000
10,
1 1 I
1 1 1

45,000
,
1 I 1

40,000
I ,

I 1

cm-

35,000
1

3(
I I I

Fig. 1

UV Spectrum of Griseofulvin in Ethanol.

CRISEOFULVIN

589

with maxima at 325, 292 and 236 nm. The spectrum of isogriseofulvin is similar to that of griseofulvin differing only by the presence of a fourth maximum at 263 nm.

En ;, !

at 292 nm = 686. The W spectral data

of griseofulvin analoges have also been reported (9-11).

2.3.2

Nuclear MagnetiL Resonance Spectra 2.3.2.1

PMR The proton magnetic resonance spectra of' griseofulvin and its derivatives have been investigated(l2, 1) 4.
A typical PMR spectrum of griseo-

fulvin is shown in Fig.2. The sample was dissolved in deuterated chloroform (CDC1 ) . The spectrum
3

was recorded on a Varian T-60A NMR spectrometer with TMS as the reference standard. The following structural assignments have been made (15)

Fig. 2

PMR Spectrum of Griseofulvin in CDC13 and TMS.

GRISEOFULVIN

591

Chemical Shift ( 6 )
0.97 (doublet)

Assignment
6/ - CH3 5/-,6/- H
2 / - OCH3

2.70 (multiplet)
3.60 (singlet) 3.97 (singlet)

4 6

OCH3

4.00 (singlet) 5.50 (singlet)

6.13 (singlet)

OCH3

3/ - H 5 - H

The PMR spectrum o f griseofulvin

- 5,

5/-d exhibits only one ali2

phztic proton appearing as a quartet at 2.75 6 ascribable to the

6/ -a-proton ( 2 ) . On stirring with

neutral alumina in chloroform this compound undergoes stereoselective partial replacement of the 5/ -6deuterium substituent with hydrogen to give griseofulvin 5-a-d. The
PMR spectrum of a mixture of the

2 compounds (Fig. 3-A) shows no peaks

in the region of 2.3

(501-H) but

exhibits a complex band at 2.7

2.96 (1,4H) representing the coup-

led and closely spaced 5/ -B- and

592

MAHMOUD A. HASSAN AND E . A. ABOUTABL

6 -a proton signals.

A
spectrum

strikingly altered PMR

(Fig. 3) was obtained on application of Eu (DPM)3. Proton

signals are shifted downfield in general proportion to their closeness to the C-4 carbonyl oxygen. The signals due to
6/ -CH3 (1.48), 6/ C-H ( - 3 . 9 8 ) ,

and 5 / B-H (5.36) constitute a first order (A3MX) system in which the doublet at 5.36 gives J 5 / B-6 / a-13.5 Hz. A vicinal coupling of this magnitude must be due to trans diaxial hydrogen substituent. The PMR spectrum of griseofulvin is DMSO D6 has been reported (16). 3 Synthesis . Several synthetic routes to griseofulvin have been reported
(17-20).

GRISEOFULVIN

593

I
Fig. 3 - B

' 1111

.I11
I

. I

Fig. 3-A : NMR (100) Spectrum of Griseofulvin -5 / , / d2 and its stereoselective Hydrogen Exchange Product in CDC13. Fig. 3 - B : The same with 0.4 molar equivalent of Eu (DPM)3 in CDC13.

594

MAHMOUD A. HASSAN AND E. A. ABOUTABL

4.

Methods of 4nalysis 4.1 Time-resolved Phosphorimetry Phosphorescene life times of griseofulvin and dechlorogriseofulvin are shown to be 0,11 sec, and 1.16 sec. respectively ( 2 2 ) . This 10-fold difference was shown t o enable the use of timeresolved phosphorimetry for the determination of griseofulvin in mixtures with dechlorogriseofulvin
4.2

Liquid Chromatography
4.2.1

Column Chromatography A liquid solid chromatographic method was reported (23) or the direct analysis of griseofulvin is complex fermenter brothes. The method is tedious and time consuming.

4.3

Isotope Dilution Ashton (24) described an isotope dilution method or the assay of griseofulvin based on the estimation of the radioactivity employing griseofulvin labelled with radioactive 36Cl. McNall (25,26) reported, another method using tritium-labelled griseofulvin

GRISEOFULVIN

595

5.4

PMR Spectrometry

A rapid, accurate and specific PMR method for the determination of griseofulvin in bulk drug and pharmaceutical formulations has been developed in our laboratory (15). From Fig.2, it is evident that griseofulvin exhibits, among other peaks, two singlets at 3.97 and 4.00 ppm (in CDCl,) assigned to the 4/ - and 6/ - methoxy protons respectively, Since the integration of these two peaks gives the largest region f o r measurement, they are chosen for the quantitative analysis of griseofulvin
LI

Acetanilide, exhibiting a three protons signlet at

2.30 ppm (in CDC13), assigned to its methyl groups

is employed as internal standard. The determination is based on the integration of the 4and

6 / -methoxyprotons of griseofulvin relative to

that of the methylprotons of acetanilide, Accurate determination is achieved, since the signals chosen for griseofulvin are widely separated from that of acetanilide. Ethanol-free chloroform is used as the solvent, its proton siTglet at
7.25 ppm does not interfere with the upfield

protons of both compounds. Fig. 4,

500

400

300

200

100

0;-

Fig. 4

PMR spectrum of Griseofulvin, acetanilide and TMS in ethanol-Eree chloroform.

GRISEOFULVIN

597

Assay of a series of known standard mixtures of griseofulvin and acetanilide by this PMR technique established the accuracy and precision of the method with an average recovery of 99.55%. The results of estimation of griseofulvin in tablets and drysuspension powders are in agreement with pharmacoepial requirements, No interference from excipients could be observed.
4,s

Microbiological Dittmer ( 2 7 ) reported on the determination o f microbiological activity of griseofulvin in body fluids by dilution methods in liquid or solid media using Tricophyton mentagrophyte as the test organism. Mrtek et a1 (28) developed the microculture slide technique of Elliott -- (29). The assay et a1 system consists of a suspension of Microsporum gypseum macroconidia in Sabouraud liquid medium containing nanogram quantities of griseofulvin. Antifungal activity is determinzd on specially prepared microculture slides by measuring changes
in the rate of hyphal elongation. A liner rela-

--

tionship of log dose to hyphal growth rate is

598

MAHMOUD A. HASSAN AND E. A. ABOUTABL

observed in the range of 0.001

0.01 mcg/ml gri-

seofulvin. This technique exhibited precision at least equivalent to that of the agar cup procedure

GRlSEOFULVlN

599

REFERENCES 1. 2. 3. J.F. Grove, J. Macmillan, T.P.C. Mulholland and M.A. Thorold Rogers, J. Chem. SOC., 3977 (1952). S.G. Levine and R.E. Hicks, Tetrahedron, Lett., (4),311, (1971). Wilson, 0 . Gisvold, R.F. Doerge, "Textbook of Organic Medicinal and Pharmaceutical Chemistry", 7th Ed., J.B. Lippincott Co., Philadelphia, p.343 (1977).
C.O.

4.

WA ..

Brown, G.A. Sim, J. Chem. SOC., 1050 (1963). Kanig, J. Pharm., Sci.

5.
6.

A.H. Goldberg, M. Gibaldi and J . L . 55, - 487 (1966).

W.L. W.L.

Chiou and S Niazi, J. Pharm. Sci., . Chiou and S. Niazi, J. Pharm. Sci.,

7.
8 . 9.

62, 65,

498 (19731.

1212 (1976).

N. Obi., Sekiguchi and Y. Ueda, Chem. Pharm. Bull., 866 (1961).

J. Chem. SOC., 1260, (1962).

9,

V. Arkley, J. Attenburrow, G.I. Gregory and T. Walker,

G.I. Gregory, P.J. Holton, H. Robinson and T. Walker, J. Chem. SOC., 1269 (1962).

10.

. 11. V. Arkley, G.I. Gregory and T Walker, J. Chem. SOC., 1603 (1963).
12. G.F.H. Green, J.E. Page and S.E. Staniforth, J. Chem. SOC., 144 (1964).

13. S.G. Levine E R.E. Hicks, Tetrahedron Lett., 5409 (1968).

14. S.G. Levine 6 R.E. Hicks, Tetrahedron Lett., 311 (1971).

15. 16.

E. Aboutabl and M.M.A.

Hassan, Talanta, (in press).

Edward R. Townley, ftAnalytical Profile of Griseofulvin" a chapter in Analytical Profile of Drug Substances, Vol. 8, Edited by K . Florey, Academic Press, Newyork, Newvork. 1979 n.224.

600

MAHMOUD A. HASSAN AND E. A. ABOUTABL

17. C.H. Kuo, R.D- Hoffsommer, H.L. Slates, D. Taub and N.L. Wendler, Chem. Ind., 1627 (1960). 18. G. Stork, M. Tomasz, J. Am. Chem. SOC., ibid., 86, 471 (1964).

84,

310 (19621,

. . 13, 19. T Fields, H Newman and R.B. Angier, J.Med. Chem., 1242 (1970).
20. T. Fields, H. Newman and R.B. Angier, J. Med. Chem., 767 (1971).

14,

. 21. Y. Sato, T. Oda and S Urano, Tetrahedron Lett., (31), 2695 (1976).
54, .. 22. J.R. Meduffie and W C Neely, Anal. Biochem. - 507, (1973).

23. A. Holbrook, F. Bailey and G.M. Bailey, J. Pharm. Pharma -co~., 2, 274 T. (1963). 24.
G.C. Ashton, Analyst, - 288 (1956). 81,

60, 25. E.G.McNal1, Antibiotics Annal., - 674 (1959).

26. 27.

E.G.McNaI1,

Arch. D e n . Chicago, - 657 (1960). 81,

W. Dittmer, Intern. Congr. Chemotherapy, Proc., Stutgart 1963 (1) , 728-32 (1964).

.. 28. M.B. Mrtek, L J Lebeau, F.P. Siege1 and R.G. Mrtek, J. Pharm. Sci., - 1363 (1969). 58, 29. H.E. Elliott, L.J. LeBeau and M. Novak, Bacteriol. Proc., 56, 55 (1956).

METHADONE HYDROCHLORIDE
Muhmoud A . Hassun and Abdulluh A . Al-Budr

1.

2.

3.

Description 1 . 1 Nomenclature 1.2 Conformation Physical Properties 2.1 Optical Rotatory Dispersion Spectrum 2.2 Circular Dichroism Spectrum 2 . 3 Crystallographic Properties Methods of Analysis 3.1 Gravimetric Analysis 3.2 Ultraviolet Analysis 3.3 Ion-Exchange Chromatography 3.4 Radio-Immunoassay 3.5 Thin Layer Chromatography 3.6 Gas Chromatography 3.7 High Pressure Liquid Chromatography References

602 602 602 602 602 605 606 607 607 607 607 609 609 610 61 I 614

Analytical Rofiks of D u Substances. 9 rg

60 1

Copyright @) 1980 by Academic Press. h c . n All rights of reproduction i any form reSerVed ISBN: 012-260809-7

602

MAHMOUD A. HASSAN A N D ABDULLAH A . AL-BADR

METHADONE HYDROCHLORIDE
1. Description 1.1 Nomenclature

1.11 Chemical Name N,N-Dimethyl-1, 1-diphenyl-1-propan-1-one-methyl propylamine hydrochloride ( ) 1. 1.12 Generic Name Methadone hydrochloride; Metadone hydrochloride. 1.13 Trade Name Tussal.

1.14 Wiswesser Line Notation

2VXR&RhlY&N1&1 &GH DL 1.2 Conformation based upon crystallographic (2) and spectroscopic evidence ( 3 ) , is shown in Fig.1. This conformation is stabilised by a hydrogen-bonding interaction as has 4 . Further evibeen suggested by Beckett and Casy ( ) dence of such conformation was also obtained by the et a1 5 . work of Henkel - - ( )
2. Physical Properties
A probable conformation of methadone hydrochloride,

2.1 Optical Rotatory Despersion Spectrum The ORD characteristics of (+) and (-)-methadone have been reported (1) and given below. The ORD curves are shown in Fig. 2.
(+) Methadone :

METHADONE HYDROCHLORIDE

603

Q
H

Me
\

PROBABLE CONFORMATION OF METHADONE

FIG, 1

604

MAHMOUD A. HASSAN AND ABDULLAH A. AL-BADR

16

12

4
I I
I
I I I

$+

a
m

e 4

,'7

!
I

200

300

400

500

600

S(mp)

Fig, 2: ORD Curves of (+ 1-methadone (a) and (-)-methadone(b)

(-> Methadone :

[a]*36" (Cyclohexane). RD (C,O,ll;Dioxanne) : [@]600-185'; [ @ 1 5 0 0 - 1 8 5 ~ [ @ ] 4 0 0 - 9 3 " ; [@]37s+O0; [ @ ] 3 1 6 + 4 066O; ; [@]29e+0; [@]274- 1 4 974"; [@]270-12 796'; [@]26813611'; [@]251+-11433";[@]228- 1 1 9 8 0 ' .

UV : X -

250 nm (log
(log
E =

=3,01) (inflexion), (log

E =

286 nm

2 , 6 2 ) , 296 nm

2,62).

METHADONE HYDROCHLORIDE

605

'1

:I -.Ll
'"1
F i g . 3 : CD spectra of (+) - (6s) -methadone : 0.1% solutions in CH30H ( - ) ,
(---)y

hexane(*-.), and CH3CN(-*-.

3'"

2.2 Circular Dichroism Spectrum

The CD spectral characteristics f o r (+)-methadone and (+)-methadone hydrochloride have been reported (5) and are given below. The CD spectra are shown in Fig. 3, and Fig. 4.

606

MAHMOUD A. HASSAN A N D ABDULLAH A . AL-BADR

hydroFig. 4 : CD spectra of (+)-(6s)-methadone chloride, 0.025% solutions in CH30H (-) and CHCl ( - - - ) . 3

2.3 Crystallographic Properties Hanson and Ahmed (2) have reported the crystal structure and absolute configuration of monoclinic form of d-methadone hydrobromide. The crystal is monoclinic, probably P2 , a = 10.69, b = 8.74, c = 10.74 1

METHADONE HYDROCHLORIDE

607

A " , B = 9 4 . 6 " , 2 = 2. The structure determination, which was essentially three-dimensional, was begun by the heavy atom method, and completed by means of differential syntheses. The absolute configuration of the molecule was determined by measuring the effect on two selected sets of reflexions of the imaginary part of the dispersion of copper radiation by the bromine atom. A projection of a single molecule along a convenient direction is shown in Fig.5. The absolute configuration is that of the (+)-isomer. The bromine atom, which is not shown, lies near the apex of the pyramid formed by the nitrogen atom and its neighbours.

3. Methods of Analysis: 3.1 Gravimetric Analysis:

Loucas et a1 (6) have published a gravimetric method for the determination of methadone hydrochloride in flavoured syrup formulation, by mixing the sample (equivalent to 10-20 mg of the drug) with lOml of 1% Molypdophosphoric acid solution, collecting the precipitate on a Millipore membrance-filter and drying it at 60"; lmg of the precipitate = 0.4mg of the drug. Nitrogenous bases, particularly alkaloids interfered, being co-precipitated with the drug.
3.2 Ultraviolet Analysis:

Caddy et a1 (7) described an oxidative analytical procedure for the determination of certain drugs contairdng the diphenylmethylene group in blood and urine. The method is based on the oxidation of the drug with alkaline potassium permanganate to form benzophenone. For calibration, a standard solution of the drug salt is heated with alkaline potassium permanganate solution and heptane, and the extinction of the organic layer is measured vs heptane at (247 nm). Beers Law is obeyed for up to 20 u g of benzophenone per ml of heptane solution. Urine or blood samples (adjusted to pH 10.5) are extracted with ethyl ether the extract is washed with N H C 1 , and the concentrated acid solution is treated as for standard solution.
3.3 =Exchange

Chromatography:

Knox et a1 ( 8 ) have described a chromatographic method for the separation of methadone from mixtures of other

608

MAHMOUD A . HASSAN A N D ABDULLAH A . AL-BADR

Carbon

Fig. 5 : The d-methadone molecule, a s i t occurs i n the monoclinic form of the bromine derivative. The orientation t r i p l e t i s composed of 1 A.'vectors, i n the directions of the principal axes.

METHADONE HYDROCHLORIDE

609

drugs. The method was carried out on a stainless steel column (lm x 2.lmm) which was packed with Zipax SCX (37-44 um), and sample was injected through a septum and was eluted with aqueous borate buffer under pressures of 500-1500lb per square inch; the elute was passed through an 8 p.1 flow-cell and its extinction was measured. Methadone was separated with buffer solution of pH 9.8. 3.4 Radio-Immunoassay: Cleeland et a1 (9) published a review dealing with the analysis of urine, blood, saliva and tissues for methadone with other drugs of abuse using radio-immunoassay. 3.5 Thin Layer Chromatography (TLC): Gupta et a1 ( 0 have described a TLC method for scre1) ening of the major methadone metabolites and methyl amphetamine in urine. Urine lml is placed in a screwcapped PTFE-lined culture tube and 0.25 M CuSO4 (lml), saturated aqueous sodium bicarbonate (lml) and chloroform (5ml) and added. The aqueous layer after centrifugation is aspirated off and the organic layer is decanted into a test-tube to which is added 4-chloro7-nitrobenzofurazan chloride in chloroform. The solution is evaporated to dryness and the residue is dissolved in chloroform. The solution is subjected to TLC on silica gel (0.25 mm thick) by development The methadone metabowith ethylether-benzene(1:l). lite 2-ethylidene-1,5-dimethyl-3,3-diphenyl pyrrolidine produces a blue-green and purple spots (% 0.94 and 0.84, respectively) with the above reagent. Jain -- (11) have reported another TLC method et a1 far the separation of methadone and its primary metabolite in the presence of other drugs in urine specimens. The sample was treated with conc. aqueous ammonia and extracted with chloroform-ethyl acetate-methanol (3:l:l). The organic layer was filtered through phase-separating paper and evaporated at 70" under N The residue was dissolved in methanol and applie8 to a silica gel E or FG precoated plates. The best solvent systems were ethyl acetatedichloromethane-conc. aqueous ammonia (90:10:0.9), ethyl acetate-octanol-conc. aqueous ammonia (93:7:1) and ethylacetate-isopropylether-water-conc. aqueous ammonia (90:lO:l.l). Spots were detected with iodoplatinate spray reagent. Methadone and its primary metabolite 2-ethylidene-1, 5-dimethyl-3,

610

MAHMOUD A. HASSAN A N D ABDULLAH A . AL-BADR

3-diphenyl pyrrolidine were well separated from each other. The limit of detection was 0.25 pg/ml both for methadone and for its metabolite. Davis et a1 (12) have reported an improved thin layer chromatographic system for methadone and its metabolites in biological samples using the Gelman instant thin layer chromatography (ITLC) system. The ITLC was modified by applying a thicker layer of silica gel to the base of the imprignated fiber-glass strip, so as to reduce the tendency to over load when working with biological extracts. The technique described is illustrated by the application to the separation of labelled methadone and metabolites (pyrrolidine and the N-oxide) in a kidney extract by the following solvent systems: a) ethylacetate-methanol-aqueous ammonia

17
b)

benzene-ethylacetate

19 :
c)

benzene-ethylacetate-methanol-aqueous ammonia
800
:

2000

12

followed by radiometric coating 3.6 Gas Chromatography: Gas liquid chromatography systems for determination of methadone in sustained-release tablets (13). The method involves the extraction of a tablet at 37" with successive portions of dissolution medium (mixtures of gastric fluids and intestinal fluids of pH increasing from 1.2-7.5). Each extract is made alkaline to phenolphthalein and 10 ml portions were extracted with chloroform (50 m ) l. Each chloroform extract was dried over sodium sulfate and a 10 .nl portion was evaporated with a chloroform solution of atropine (internal standard). The residue was dissolved in chloroform (2 ml) and a 1-2 1 1 portion was subjected .1 to GLC on a spiral siliconized glass column (3 ft. long x 2 mm packed 3% of SP 2250-DP on Supelcophrt (100-120 mfsh) and operated at 235" with a Helium 35 ml min- as a carrier gas and flame ionization detection. The amount of methadone was calculated

METHADONE HYDROCHLORIDE

61 1

from the peak height and molar response ratios relative to atropine. Lynn et a1 ( 1 4 ) has reported a new gas-chromatographic assay for determination of methadone in man and animals ( ) The internal standard.,2-dimethyldno-4 6. 4-diphenylnonane-5-one is added to the specimen containing the drug and then extracted with chlorobutane at pH 9.8. Then it is extracted into 0.5M H SO4 and and after alkalinization is extracted into choroform. The extract was analysed on a column (6ft x 2mm) of 1.5% OV-101 on Gas-Chrom Q (100-120 mesh). Thf temperature is programmed from 170-250' at 1 min , with N2 as carrier gas (30ml min-l) and a H-flame ionization detector. The peak area ratio of the standard and the drug was obtained by electronic integration. Tracer studies with (+)-14C-methadone showed that the recovery was 9 3 + 2 % for the extraction and > 99% in subsequent stages. 3.7 High Pressure Liquid Chromatography (HPLC): Knox and Jurand (15) have applied a high-speed liquid chromatography or the determination of methadone and other narcotics. The chromatographic behaviour of the narcotics studied has been investigated on a glass or stainless steel column (80-100 cm x 2mm) packed with Zipax Pellicular resins (37-44 m) and operated at room temperature, with UV detection. Conditions are outlined for rapid determination of methadone on a column of strong anion exchange resin. The eluted compound was identified by its W absorption and mass spectrum. Trinler and Renland (16) have reported a rapid screening of methadone and other narcotics by reverse phase HPLC. The column (2ft x 0.125 inch, 0.d.) packed with Bondapak c18 - Corasil; detection is by W spectrometry (254 nm). The eleuent is acetonitrile-water ( 9 : l ) and the fractions are collected for analysis by W or IR spectrometry. Goodman et a1 (17) have tried a combination of HPLC and tritium exchange for the determination of common drugs of abuse and their metabolites including methadone. The HPLC effluent is passed through the tritium exchange system, which consisted of a PTFE-lined stainless-steel column packed with a trituim exchange

612

MAHMOUD A. HASSAN A N D ABDULLAH A. AL-BADR

polymer followed by an ionization chamber detector. The method was partially successful. Hsieh et a1 (18) have recently reported a high-performance liquid chromatographic analysis of methadone in sustained release formulations. HPLC separation of methadone was carried out using a reversed-phase 1-1 Bondapak c18 column. The column temperature was ambient. The electrometer was set at 0.01 a.u.f.s. with a recorded chart speed of 2 in. per 10 min. The volume of the samples introduced into the column was 10 1-11. The solvent (mobile phase) flow rate was controlled at l.Om/min. A stock solution of 0.1 mg/ml anthracene in methanol was used as an internal standard. The sodium salt of 1-pentanesulfonic acid was used as an ion-pair agent. Fig. 6a represents a typical chromatogram of methadone hydrochloride using a mobile phase of methanol-water (75:25), while Fig. 6b illustrates the response of the same solution when the ion-pair agent is present in the mobile phase. It is seen that the ion-pair agent increases the absorption and the resolution of the methadone peak. The high sensitivity and the low quantities (us) of drug detected by this method indicates that this method may be successfully used for the in vivo determination of methadone (Table 1. ) Recovery data of methadone from sustained release tablets. Weight of sample (mg>
10
20
I

Methadone in sample (mg)

1.1 2.2 3.3 4.4
I

Methadone recovered (mg)

1.020

Recovery f 5%
93 93 94 93
I

15

2.050
4.090
I

3.100

METHADONE HYDROCHLORIDE

613

Fig. 6: (a) Typical chromatogram of methadone hydrochloride in a methanol-water (75 :25) solution. (b) Chromatogram of methadone hydrochloride in the presence of an ion-pair agent (sodium salt of 1-pentanesulfonic acid). M = Methadone; S = internal standard.

614

MAHMOUD A . HASSAN A N D ABDULLAH A . AL-BADR

REFERENCES

1. P. Crabbe, P. Demoen and P. J a n s s e n , B u l l . SOC. Chim. (France) 10,2855 (Fr) 1 9 6 5 .

2. 3. 4. 5. 6. A.W. Hanson and F.R. Ahmed, Acta C r y s t . , 724 (1958). A.F. Casy, J. Chem. SOC. B , 1158 (1966). B e c k e t t and A.F. Casy, J. Pharm. Pharmacol.,

11,

- 986 (1954). 6,
J.G.

A.H.

Henkel, K.H.

J. Wd. Chem.

1 7

(l), 1 2 4 (1974).

B e l l and P.S. P o r t o g h e s e ,

S.P. Loucas, R.L. F e i n b e r g , P.A. Gunning, F.F. Hartmann and B. Mehl, Am. J . Hosp. Pharm., 2, (12) , 1193-1197 (1974).
B. Caddy, F. F i s h , P.W. Mullen and J. T r a n t e r , J . Forens. S c i . SOC., (2), 127-135 (1973).

7.

1 3

8.
9.

- (2) , 398-401 82

John H. Knox and Jadtriga J u r a n d ; J . Chromat., (1973).

R. C l e e l a n d , J . C h r i s t e n s o n , M. Usategni-Gomez, J . Heveran; R. Davis and E. Grunberg, C l i n . Chem, - (6), 712-725 (1976). 22

10. R.N. Sci

12 (2),

Gupta, B.G. C h i t t i m and P.M. 67 (1974).

Keane, J. Chromat.

11. Naresh C. J a i n , Wai J. Lenng, Robert D. Budd and Thomas C. S n e a t h , J. Chromat., 1 3 (l), 85 (1975). 0
1 2 . C.M. David and D.C. 193 (1975).
Ferimore, J. Chromat.,

1 4 (I), 0

13. N i c o l a s , H. C h o u l i s and Harry Papadopoulas, J. Chromat. , 1 6 (1), 180-183) (1975). 0

1 4 . R.K.

Lynn; R.M. Leger, W.P. and N. Gerber, J. Chromat.

131,329

Gordon, G.D. Olsen (1977).

15. John H. Knox and Jadwiga J u r a n d , J. Chromat., - 95 (1973). 87,

METHADONE HYDROCHLORIDE

615

16. W.A. T r i n l e r , D . J . Sci. Soc., 1 (2), 5

Reuland, J. Forens. 153 (1975).

1 7 . P . Goodman, A . R e n n e r t and J. Downs, Rep. Atom. Energy Commn. U . S . , 100-2292-1, (1974).

18. J. H s i e h , J.K.H. Ma, J.P.O. Donne11 and N.H. C h o u l i s , J. Chromat. 1 6 1 (ll), 366 (1978).

CUMULATIVE INDEX
Italic numerals refer to volume numbers Acetaminophen, 3, I Acetohexamide, 1. 1; 2. 573 Allopurinal, 7, 1 Alpha-tocopheryl acetate, 3, 11 1 Amitriptyline hydrochloride, 3, 127 Amoxicillin, 7, 19 Amphotericin B, 6, 1; 7, 502 Ampicillin, 2, 1; 4. 517 Aspirin, 8, 1 Bacitracin, 9, 1 Bendroflumethiazide, 5 , 1; 6 . 597 Betamethasone dipropionate, 6, 43 Bretylium Tosylate, 9, 71 Bromocriptine methanesulfonate, 8, 47 Clacitriol, 8, 83 Carbamazepine, 9. 87 Cefaclor, 9, 107 Cefamandole Nafate, 9, 125 Cefazolin*; 4, 1 Cephalexin. 4. 21 Cephalothin sodium, I. 319 Cephradine*, 5 , 21 Chloral hydrate, 2, 85 Chloramphenicol, 4 , 47, 517 Chlordiazepoxide, I, 15 Chlordiazepoxide hydrochloride, I, 39; 4, 517 Chloroquine phosphate, 5, 61 Chlorpheniramine maleate, 7. 43 Chloroprothixene, 2. 63 Chlortetracycline hydrochloride, 8, 101 Clidinium bromide, 2, 145 Clonazepam, 6, 61 Clorazepate dipotassium, 4 , 91 Cloxacillin sodium, 4 , 113 Cyclizine, 6, 83; 7, 502 Cycloserine, I, 53 Cyclothiazide, I, 66 Cyproheptadine, 9, 155 Dapsone, 5 , 87 Dexamethasone, 2, 163; 4, 518 Diatrizoic acid, 4, 137; 5 , 556 Diazepam, I, 79;4, 517 Dibenzepin hydrochloride, 9, 181 Digitoxin, 3, 149 Digoxin, 9, 207 Dihydroergotoxine methane sulfonate, 7, 8 1 Dioctyl sodium sulfosuccinate, 2, 199 Diperodon, 6, 99 Diphenhydramine hydrochloride, 3, 173 Diphenoxylate hydrochloride, 7, 149 Disulfiram, 4, 168 Dobutamine hydrochloride, 8, 139 Doxorubicin, 9, 245 Dmperidol, 7, 171 Echothiophate iodide, 3, 233 Epinephrine, 7, 193 Ergotamine tartrate, 6, I 13 Erythromycin, 8, 139 Erythromycin estolate, I, 101; 2, 573 Estradiol valerate, 4, 192 Ethambutol hydrochloride, 7, 231 Ethynodiol diacetate, 3, 253 Fenoprofen calcium*, 6, 161 Flucytosine, 5, 115 Fludrocortisone acetate, 3, 281 Fluorouracil, 2, 221 Fluoxymesterone, 7, 251 Fluphenazine decanoate, 9, 275 Fluphenazine enanthate, 2, 245; 4, 523 Fluphenazine hydrochloride, 2, 263; 4. 518 Gentamicin Sulfate, 9, 295 Gluthethimide, 5, 139

*Monographs in Pharmacological and Biochemical Properties of Drug Substances M. E. Goldberg, D. Sc., Editor American Pharmaceutical Association.

617

618
Gramicidin, 8. 179 Griseofulvin, 8, 219, 9, 583 Halcinonide, 8, 251 Haloperidol, 9, 341 Halothane, I , 119; 2, 573 Hexetidine, 7, 277 Hydralazine hydrochloride, 8, 283 Hydroflumethiazide, 7, 297 Hydroxyprogesterone caproate, 4, 209 Hydroxyzine dihydrochloride, 7, 3 19 Iodipamide, 3, 333 Isocarboxazid, 2, 295 Isoniazide, 6, 183 Isopropamide, 2, 315 Isosorbide dinitrate, 4, 225; 5 , 556 Kanamycin sulfate, 6, 259 Ketamine, 6, 297 Khellin, 9, 371 Leucovorin Calcium, 8, 315 Levarterenol bitartrate, I, 49; 2, 573 Levallorphan tartrate, 2, 339 Levodopa, 5, 189 Levothyroxine sodium, 5 , 225 Lorazepam, 9, 397 Meperidine hydrochloride, I, 175 Meprobamate, I, 209; 4 , 519 6-Mercaptopurine, 7, 343 Methadone hydrochloride, 3, 365;4, 519.9, 60 1 Methaqualone, 4, 245, 519 Methimazole, 8, 351 Methotrexate, 5 , 283 Methoxsalen, 9, 427 Methyclothiazide, 5 , 307 Methyprylon, 2, 363 Metronidazole, 5 , 327 Minocycline, 6, 323 Nadolol, 9, 455 Nalidixic Acid, 8, 371 Neomycin, 8. 399 Nitrazepam, 9, 487 Nitrofuraptoin, 5 , 345 Nitroglycerin, 9, 519 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

CUMULATIVE INDEX 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 Rocarbazine hydrochloride, 5. 403 Promethazine hydrochloride, 5. 429 Pmparacaine hydrochloride, 6, 423 Ropiomazine hydrochloride, 2, 439 Propoxyphene hydrochloride, I , 301 ; 4, 5 19; 6, 598 hpylthiouracil, 6, 457 Pseudoephedrine hydrochloride, 8, 489 Reserpine, 4 , 384; 5 , 557 Rifampin, 5 , 467 Secobarbital sodium, I, 343 Spironolactone, 4, 431 Sodium nitroprusside, 6, 487 Sulphamerazine, 6, 515 Sulfamethazine, 7, 401 Sulfamethoxazole, 2, 467; 4, 520 Sulfasalazine, 5 , 515 Sulfisoxazole, 2, 487 Testolactone, 5 , 533 Testosterone enanthate, 4 , 452 Theophylline, 4 , 466 Thiostrepton, 7, 423 Tolbutamide, 3, 513; 5 , 557 Triamcinolone, I, 367; 2, 571; 4. 520, 523 Triamcinolone acetonide, I, 397, 416; 2, 571; 4 , 520; 7 Triamcinolone diacetate, I. 423 Triamcinolone hexacetonide, 6, 579 Triclobisonium chloride, 2, 507 Trifluoperazine hydrochloride, 9, 543 Triflupromazine hydrochloride, 2, 523; 4, 520; 5 , 557 Trimethaphan camsylate, 3, 545 Trimethobenzamide hydrochloride, 2, 551 Trimethoprim. 7, 445 Triprolidine hydrochloride, 8. 509 Tropicamide, 3, 565 Tubocurarine chloride, 7, 477 Tybarnate, 4, 494 Valproate Sodium and valproic acid*, 8. 529 Vinblastine sulfate, 1. 443 Vincristine sulfate, 1. 463