Florey Vol 3

Analytical Profiles of

Drug Substances Volume 3

Edited by

Klaus Florey The Squibb Institute for Medical Research

New Brunswick, New Jersey

Contributing Editors

Norman W. Atwater Olenn A. Brewer, Jr. Lester Chafetz Boen T. Kho Jack P. Comer

Salvatore A. Fusari Erik H. Jensen

Gerald J. Papariello Bernard 2. Senkowski

Compded under the auspices of the Pharmaceutical Analysis and Control Section

Academy of Pharmaceutical Sciences

Academic Press New York and London 1974 A Subsidiary of Harcourt Brace Jovanovich, Publishers

EDITORIAL BOARD

Norman W. Atwater Glenn A. Brewer, Jr. Lester Chafetz Boen T. Xho Edward M. Cohen Jack P. Comer Klaus Florey Bernard Z. Senkowski Salvatore A. Fusari Frederick Tishler

David E. Guttmaa Erik H. Jensen

Arthur F. Michaelis Oerald J. Papariello

COPYRIGHT 1974, BY THE AMERICAN PHARMACEUTlCAL ASSOCIATION ALL RIGHTS RESERVED NO PART OF THIS BOOK MAY BE REPRODUCED IN ANY FORM, BY PHOTOSTAT, MICROFILM, RETRIEVAL SYSTEM, OR ANY OTHER MEANS, WITHOUT WRITTEN PERMISSION FROM THE PUBLISHERS.

ACADEMIC PRESS, INC. 111 Fifth Avenue, New York, New York 10003

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

Library of Congress Cataloging in Publication Data Main entry under title:

Analytical profiles of drug substances.

Compiled under the auspices of the Pharmaceutical Analysis and Control Section, Academy of Pharmaceutical Sciences.

Includes bibliographical references. 1. Drug-Collected works. 2. Chemistry, Medical

and pharmaceutical-Collected works, I, Florey, Klaus, ed. 11. Brewer, Glenn A. 111. Academy of Pharma- ceutical Sciences. Pharmaceutical Analysis and Control Section. [DNLM: 1. Drugs-Analysis-Yearbooks. QV740 AA1 ASS] RM300 .AS 6 615'.1 7 0-187 259 ISBN 0-12-260803-5 (v. 3)

PRINTED IN THE UNITED STATES OF AMERICA

AFFILIATIONS OF EDITORS, CONTRIBUTORS, AND REVIEWERS

N. W. Atwater, Searle and Company, Chicago, Illinois

W. F. Beyer, The Upjohn Company, Kalamazoo, Michigan

K. W. Blessel, Hoffmann-LaRoche, Inc., Nutley, New Jersey

R. H. Bishara, Eli Lilly and Company, Indianapolis, Indiana

C. A . Brewer Jr., The Squibb Institute for Medical Research, New Brunswick, New Jersy

L . Chafetz, Warner-Lambert Research Institute, Morris Plains, New Jersey

E. M . Cohen, Merck, Sharp and Dohme, West Point, Pennsylvania

J. P. Comer, Eli Lilly and Company, Indianapolis, Indiana

R. D. Daley, Ayerst Laboratories, Rouses Point, New York

J. E. Fairbrother, The Squibb Institute for Medical Research, Moreton, Wirral, England

K. Florey, The Squibb Institute for Medical Research, New Brunswick, New Jersey

R. I. Fryer, Hoffmann-LaRoche, Inc., Nutley, New Jersey

S. A . Fusari, Parke, Davis and Company, Detroit, Michigan

C. A . Caglia, Jr., Warner-Lambert Research Institute, Morris Plains, New Jersey

vii

AFFILIATIONS OF EDITORS, CONTRIBUTORS, AND REVIEWERS

D. E. Guttman, School of Pharmacy, University of Kentucky, Lexington, Kentucky

I. J . Holcombe, Parke, Davis and Company, Detroit, Michigan

I. M. Jakovljeric, Eli Lilly and Company, Indianapolis, Indiana

E. H. Jensen, The Upjohn Company, Kalamazoo, Michigan

B. T. Kho, Ayerst Laboratories, Rouses Point, New York

E. P. K. Lau, Searle and Company,Chicago, Illinois

H. H. Lerner, The Squibb Institute for Medical Research, New Brunswick, New Jersey

A . F. Michaelis, Sandoz Pharmaceuticals, East Hanover, New Jersey

G. J. Papariello, Wyeth Laboratories, Philadelphia, Pennsylvania

C. R. Pilla, Wyeth Laboratories, Philadelphia, Pennsylvania

E. L. Pratt, The Sterling-Winthrop Research Institute, Rensselaer, New York

B. C. Rudy, Hoffman-LaRoche, Inc., Nutley, New Jersey

B. 2. Senkowski, Hoffmann-LaRoche, Inc., Nutley, New Jersey

C. M . Shearer, Wyeth Laboratories, Philadelphia, Pennsylvania

viii

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 o r 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 Pharmaceuti- cal Sciences, has undertaken a cooperative venture t o compile and publish Analytical Profiles of Drug Substances in a series of volumes of which this is the third.

Reviews and comments received so far have reinforced our belief that the series fills a need and they have strengthened our determination to con- tinue. The enthusiasm and cooperative spirit of our contributors have made these profiles possible. All those who have found the profiles useful are earnestly requested t o contribute a monograph of their own. The editors stand ready to receive such contributions.

Beginning with Volume 2 a cumulative index has been added, t o facili- tate the correction of errors and to encourage the addition or relevant new information.

The concept of analytical profiles is taking hold not only for com- pendial drugs but, increasingly, in the industrial research laboratories. Ana- lytical profiles are being prepared and periodically updated t o provide physico-chemical and analytical information on 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 com- pendial status.

Klaus Florey

ix

ACETAMINOPHEN

John E. Fairbrother

JOHN E. FAIRBROTHER

CONTENTS

1. Description 1.1 Name, Formula, Molecular Weight 1 . 2 Appearance, Color

2 . 1 Spectra 2. Physical Properties

2 . 1 1 Infra-red Spectrum 2 . 1 2 Ultra-violet Spectrum 2 . 1 3 Fluorescence Spectrum 2 . 1 4 N.M.R. Spectrum 2 . 1 5 Mass Spectrum

2.2 Physical Properties of the Solid 2 . 2 1 Melting Characteristics 2.22 Density 2 . 2 3 Vapor Pressure and T.G.A. 2 . 2 4 D.T.A. and D.S.C. 2 . 2 5 Crystal Characteristics 2.26 X-ray Diffraction

2 . 3 1 Static Charge 2 . 3 2 Flow Properties 2 . 3 3 Compression Characteristics 2.34 Surface Area and Porosity

2 . 4 1 Solubility in Aqueous Solvents 2 . 4 2 Solubility in Water Miscible

2 . 4 3 Solubility in Solvents Immisc-

2.44 Rate of Dissolution

2 . 5 1 Cryoscopy 2 . 5 2 Ionisation and pH 2.53 Dipole Moment 2 . 5 4 Refractive Index 2 . 5 5 Adsorption from Solution 2 . 5 6 Partition Coefficients

2.3 Powder Characteristics

2 . 4 Solubility

Solvents

ible with Water

2 . 5 Physical Properties of Solutions

3 . Molecular Complexes

2

ACETAMINOPHEN

4.

5.

6.

Synthesis and Purification 4 . 1 Chemical Synthesis

4.11 Synthetic Routes 4 . 1 2 Purification 4.13 Impurity Profile 4.14 Reference Standards

4 . 2 1 Metabolism of Phenacetin and

4 . 2 2 Prodrugs 4.23 Microbial Biosynthesis

4 . 2 Biosynthesis

Ace tan i 1 ide

S tabi 1 ity 5.1 Stability to Light 5.2 Stability of Solid Acetaminophen to

5.3 Heat Stability of Solutions of Acetamino- - phen

Materials

5 . 4 Stability to Oxidation 5 .5 Compatibility with Excipient

5.6 Compatibility with Aspirin 5.7 Physical Incompatibilities

6 . 1 Identity Tests 6.2 Methods of Analysis

Analytical Chemistry

6 . 2 0 Gravimetric Procedures 6 . 2 1 6.22 6.23

6.24 6 . 2 5

6 . 2 6

6 . 2 7

6.28

6 .29

T itr ime t r ic Procedures Polarographic Procedures U.V. Spectrophotometric Pro- cedures Photocolorimetric Procedures Ion-Exchange Chromatrographic Procedures Partition Chromatographic Pro- cedur es Paper and Thin-Layer Chromato- graphic Procedures Vapor' Phase Chromatographic Procedures High - Pressure Liquid Chrom- atographic and Gel Filtration Procedures

6.3 Automated Procedures 6.4 Radiochemical Procedures

3

JOHN E. FAIRBROTHER

6.5

6.6

Determination Degradation Products

7 of Acetam

of Trace Impurities and

i nophen and Luids and

Tissues 6.61 Determination in Urine 6.62 Determination in Serum, Plasma

and Whole Blood 6.63 Determination in Tissues and

Orsans 7. Metabolic Transzormations

7.1 Metabolism in Man 7.11 Adults 7.12 Newborn Infants

7.2 Metabolism in Animals

8.1 Pharmacokinetics 8.2 Protein Binding 8.3 Interactions with Other D,rug Sub-

8 . 4 Biopharmaceutics

8. Drug Availability

stances

9 . Toxicity References

4

ACETAMINOPHEN

1. Description

1.1 Name, Formula, Molecular Weight

1 Generic names - Acetaminophen , Paracetamol and Acetophenum2.

Chemical names - 4 - Hydroxyacetan- ilide; p-hydroxyacetanilide; p- acetamidophenol; p-acetaminophenol; p-acetylaminophenol; N-acetyl-p- aminophenol.

1

Mol. wt. 151.16 ‘gHgN02

1.2 Appearance, Color, Odor, Taste

White, odorless, crystalline powder, possessing a bitter taste.

2. Physical Properties

2.1 Spectra

2.11 Infrared Spectrum

Infrared spectra of solid dis ersions of acetaminophen in potassium bromide3 t 7 and in Nujo16, have been recorded. In the solid state the carbonyl stretching band appears at 1659 c m - I (1650 cm-1; ref. 3 ) , the N-H stretching band at 3326 cm-1 and a broad 0-H stretching band at 3162 cm-1. In solution the C=O,N-H and 0-H stretching bands occur at higher fre- quencies.

6

5

JOHN E. FAIRBROTHER

c =o N-H 0 -H

Band Band Band Solvent Stretching Stretching Stretching

-1 ( 9 ) Chloroform 1686cm

Dichloro- 1690cm 3435cm (6) 3588cm methane

-1 (8)

-1 (8)

-1(8

1700cm

1,4-Dioxan 1692cm

10,11,12,15,16 Several other authors report infrared spectra of acetaminophen. The infrared spectra of acetaminophenl4 in KBr and in a mineral oil mull are presented in figures 1 and 213.

2.12 Ultraviolet Spectrum

The U.V. spectrum of acetaminophen has been recorded in a number of solvents, showing two bands in each. The long wavelength band corresponds to the A l g + B2u transition while the short wavelength band corresponds to the IT^ + T * transition1’. co

6

Fig . 1. Inf rared spectrum of acetaminophen (KBr p e l l e t )

Fig. 2. Infrared spectrum of acetaminophem (Mineral Oil Mull)

ACETAMINOPHEN

TABLE 1 Absorption maxima of

acetaminophen in neutral solvents

Solvent K band B band Referenca

Me thano 1 Ethanol (abs. )

n-Butanol iso-Propanol Cyclo-

Cyclo- hexane

hexane Ether Ether (dry) Water

248-249mp. 249-250mu.

250mv. 2 50mp. 244-245mi.l.

278mp.

264mi.l. 247mp.

243.5mp. 242.5-

3.18 about 290mi.l. 4, 8,19

20 19 19

8

19 about 283mi.l. 8 about 283mp. 8,19,23

The addition of acid to aqueous and alcoholic solutions does not give any observable change in the sition of the maximum of the

alkali acetaminophen ionises to give the p- acetamidophenolate ion and the maximum of the main band is shifted bathochromically, in aqueous solution from 243 mu. to about 258 mu. 19,20,22,23 anpSin methanolic solution from 248 mp. to 262 mp. .

main band 7 I 16r~~,19r21,22* In 10-1 M caustic

9

JOHN E. FAIRBROTHER

TABLE 2

acetaminophen in different solvents Molar absorptivities ( c ) of

E . References Solvent Wavelength - Ethanol

Me thano 1 Ethanol/O.lN Hydrochloric Acid Water (pH 2 to 3) Water ( p H 7.2) (Clark and Lubs Buffer) 0.1N Sodium Hydroxide 0.01N Sodium Hydroxide Water (pH 10 to 11 1

249 mu.

288 mu.

249 mu. 249 mu.

242 mp.

242.5mp.

257 mp.

258 mu.

258 mu.

13,090 to

2,000 to 2,120 13 I 600 13 , 750

14,000

ca .11 , 000 10,037

10 , 820

10 , 830

ca. 10 , 500

4,8,19,24

19,24

3 26

25

8

21

24

25

The ultraviolet spectra of acetaminophen in ethanol (95%) and in 0.01N Sodium hydroxide (aqueous) are presented24 in figures 3a and 3b.

10

FIGURE 3a. Ultraviolet Spectrum of Acetaminophen (e thanol 95%)

11

FIGURE 3b. Ultraviolet Spectrum of Acetamino- phen (0.01N sodium hydroxide)

12

ACETAMINOPHEN

2.13 Fluorescence Spectrum

Acetaminophen has been reported 25 , 27 to exhibit fluorescence in neutral and acidic solution (excitation at 330 mp. and emission peak at 400 mp.) . fluorescence in aqueous alkaline conditions (excitation at 315 mp. and emission peak at 430 mu.). However, recent attempts 24,28 to confirm these findings have been unsuccessful and it has been suggested28 that the earlier observation~~~ ' 27 could have resulted from the Raman emission of water in conjunction with poorly aligned monochromator systems. Acet- anilide is not fluorescent436 and it is unlikely that the introduction of a para-hydroxylgroup into the molecule would change this characteristic.

Nang et a1.25 also observed

2 . 1 4 N . M . R . Spectrum

Puar and Funke 201 recorded the N . M. R. spectrum of acetaminophen in dimethylsulphoxide - d6 (see figure 4) and assigned the observed chemical shifts in the following manner.

69.07 69.58 62.00

0

\& NH - C-CH HO 5 -

6 6.70d ( 9.0)

II

67.35d(9.0)

Theriault and Longfield181 used the N . M . R . spectrum as determined in deuterated acetone to identify acetaminophen, formed by Amanita muscaria as a conversion product of acetanilide.

13

ACETAMINOPHEN

2.15 Mass Spectrum

The effects of substituents on the mass spectral fragmentation of para-substituted acetanilides has been studied in detail29 ,30,446 but unfortunately examination of the p-hydroxy com ound was omitted in each case. Burtis et al.y1 give the main peaks of the mass spectrum of acetaminophen as m/e 151, 135, 121, 109, 95, 81 and 55. The molecular ion undergoes a mass loss of 42 to give the base peak of m/e 109. This results from the re-arrangement of a proton of the acetyl group to the phenyl ring, followed by cleavage of the amide bond with the loss of CH CO (m/e 42). This metastable transition gi6es a strong diffuse peak.

Fales, Milne and Law 444 recorded the mass spectrum of acetaminophen, reporting the most abundant peaks as m/e 109, 151, 43, 80 and 81. The relative abundancies for m/e 40 to 152 are tabulated444.

Puar and Funke 201 have also recorded the high-resolution mass spectrum of acetaminophen (see figure 5 ) and suggest the following scheme of fragmentation:-

15

JOHN E. FAIRBROTHER

The occurrence of all three alternative modes of fragmentation of m/e 109 is supported by observation of corresponding metastable ions and high resolution data.

Milne, Fales and Axenrod 445 have recorded the isobutane chemical ionisation mass spectrum of acetaminophen indicating the peaks found to be m/e 152, 153 and 151.

16

ACETAMINOPHEN

1595 ACETAMINOPHEN

1 d

' I h )

L-J: 35 5 I

38 2 I

Z 25 0 -

J

0 t

t Z

28 2

8

3

MASS/CHARCE TOTAL UNSEALED I N T E N S I T Y . - 692QN B A S E PEAK IS '13.93 PERCENT OF TOTAL

F i g . 5 . Mass spectrum of acetaminophen

17

JOHN E . FAIRBROTHER

2 . 2 Physical P rope r t i e s of t h e So l id

2 . 2 1 Melting C h a r a c t e r i s t i c s

31 The melt ing po in t f i r s t quoted f o r acetaminophen (179OC) appears t o be erron- eous. Subsequent determinat ions gave melt ing po in t s of 165 t o 168OC f o r r e l a t i v e l y unpur i f ied ma te r i a l 32 t o 39 and a mel t ing r an e of 1 6 8 t o 1690C f o r p u r i f i e d m a t e r i a l 40 to 49. r ecen t ly improved p u r i f i c a t i o n procedures have been developed g iv ing m a e r i a which m e l t s i n t h e range 1 6 9 t o 1 7 1 0 C . 4i to i8. I t is t h i s mel t ing range t h e r e f o r e which is quoted i n t h e cu r ren t re ference books3 t 4 and o f f i c i a l compendia 1 4 , 2 1 .

More

49 ,50 has Kuhnert-Brandsttitter recorded t h e melt ing p o i n t using a Kofler h o t s t age a s 1 6 7 t o 169OC and c a r e f u l l y desc r ibes the melt ing process . From 14OoC t o t h e mel t ing poin t g r a i n s , hexagonal prisms and rhomboids s u b l i m e . Residual c r y s t a l s i n t h i s temp- e r a t u r e range grow i n t o hexagonal t o polyhedral g ra ins and prisms. The m e l t s o l i d i f i e s t o a g l a s s and g ives uns tab le columnar aggregates a t l l O ° C on which r ec t angu la r prisms of t h e s t a b l e modif icat ion a r e induced from about 1 4 0 O C . This uns tab le modif icat ion (11) melts i n t h e range 154 t o 156OC.

2 . 2 2 Density

Fels4' repor ted t h e s p e c i f i c g rav i ty of acetaminophen a t 2 1 O C a s 1.293.

2.23 Vapor Pressure and T.G.A.

Thermogravirnetric Analysis (T.G.A.) f a i l e d t o d e t e c t any loss of v o l a t i l e s from a sample of acetaminophen N . F . 30.

Jaecke l and P e ~ e r l e ~ ~ ~ measured t h e dependence of t h e condensation c o e f f i c i e n t

18

ACETAMINOPHEN

on the partial pressure over an evaporating crystal face of acetaminophen. Measurements of the vapor pressures on single crystal faces as functions of the partial pressure were made using a torsion balance.

2.24 D.T.A. and D.S.C.

Differential Thermal Analysis D.T.A.) of a sample of acetaminophen N.F. gave436 a sharp melting endotherm at 171OC. Examinati0x-1~~~ of a sample of B.P. grade material by Differential Scanning Calorimetry (D.S.C.) similarly gave an endotherm at 171OC. On cooling the sample and rescanning a different pattern was obtained showing the sample melting at 157OC and also an exotherm occurred at 67OC. data a value of 6.8 K cals./mol. was obtained for the Latent Heat of Fusion.

From the D.S.C. 431

2.25 Crystal Characteristics

Morse31 in a paper describing the first reported synthesis of acetaminophen, recorded that it crystallised in the form of white monoclinic prisms.

Kuhnert-Brandstatter 49r50 has described visual changes which take place in crystalline acetaminophen durin the melting process (see Section 2.21). arently isomorphous crystalline forms of acetaminophen on recrystallisation from ethanol. From his optical examination of these crystals, Fels4O assigns them to the monoclinic system with symmetry 2/m; C2h

a: b: c = 1.3688 : 1 : 1.5103

F e l ~ ~ ~ obtained two app-

6 = 115O 49.5'

40 For the two isomorphous forms he makes the following face assignments:-

19

JOHN E. FAIRBROTHER

Form 1

and Form 2 m =

c = f = p3

The observed angles between these f aces a r e given and i n some cases compared with cal- cu la ted va lues . Form l i s r epor t ed t o be gap- a b l e of undergoing t ransformat ion ( t o 001/010/ 100) bu t Form 2 does not .

This da t a has been systematised i n t h e

Dispersion of t h e o p t i c a l axes i s very

Barker Index of Crys ta l s434 .

s t rong i n acetaminophen, r < v. A very s t rong negat ive b i r e f r ingence i s exhib i ted .

t i o n of acetaminophen from a wide range of so l - ven t s gave e s s e n t i a l l y two types of c r y s t a l h a b i t . Hexagonal prisms (by c r y s t a l l i s a t i o n from a lcoho l s , esters, ketones, water, dioxan and a c e t o n i t r i l e ) and s lender rhombohedra1 needles (by c r y s t a l l i s a t i o n from benzene, t o lu - ene, d ich loroe thane and s e v e r a l o t h e r ch lor in- a t ed s o l v e n t s ) . Examination of t h e s e two c r y s t a l types by D . S . C . , i . r . and x-ray d i f f r a c - t i o n (powder) f a i l e d t o show any evidence of polymorphism.

2 . 2 6 x-ray D i f f r a c t i o n

Coy and Ochs433 have recorded t h e x-ray

F a i r b r ~ t h e r ~ ~ ’ found t h a t c r y s t a l l i s a -

powder d i f f r a c t i o n p a t t e r n f o r a sample of acetaminophen N.F. (see Fig. 6 and Table 3 ) .

20

ACETAMINOPHEN

TABLE 3 X-ray Powder Diffraction Pattern of Acetaminophen (7032-LKR-242)

Interplanar Distances

- d (8) 7.36 6.42 5.78 5.30 4.90 4.70 4.38 3.81 3.68 3.37 3.29 3.21 3.08 2.75 2.48 2.44 2.34

Relative Intensities

i/i1 0.26 0.20 1.00 0.13 0.66 0.19 0.34 0.65 0.90 0.74 0.11 0.06 0 .09 0.20 0.07 0.11 0.07

2.3 Powder Characteristics

2.31 Static Charge

Acetaminophen particles flowing through a hopper acquire a negative static charge51. This charge is reduced by the addition of tablet lubricants and by small quantities (0.5%) of water.

2.32 Flow Properties

The bulk density of acetaminophen granules falls with increasing water content. This represents a rise in internal cohesion and causes a deterioration in flow properties52r 53. 0nization~~9 of granules for tablet compression

Spher-

21

Fig. 6. X-ray powder-diffraction pattern of acetaminophen

ACETAM I NOPH EN

improves granulation flow rate. The presence of water in acetaminophen granules increases the angle of repose52.

2.33 Compression Characteristics

Uniaxial compression of crystalline acetaminophen ives a pressure cycle typical of a Mohr's body5zr59 producing capping and lamin- ating compacts. The effects moisture content and granulation have on the com ression characteristics have been studied 541 E 5,58,59,60,456.

2.34 Surface Area and Porosity

The surface area of acetaminophen powder compacts has been studied by the Brunauer- Emmett-Teller 1B.E.T.) low-temperature nitrogen adsorption pr~cedure~~. surface area with changes in moisture content and/or compression pressure of the compacts has been studied57158.

The change of this

2.4 Solubility

2.41 Solubility in Aqueous Solvents

The solubility of acetaminophen in distilled water has been described by several authors.

Temperature Solubility (mg./ml.)

2O0C about 11.3 about 14.5

2 5OC 11.66 13.85

37OC about 19 about 20

l0O0C about 5 2

References

61 3,21 I 63 8,64 65 66 67 3,61,62,63

In pH 6.0 buffer solution at 37OC its solubility has been recorded68 as 23.8 mg./ml.

23

JOHN E. FAIRBROTHER

Paruta and showed the solub- i l i t y p r o f i l e of acetaminophen i n dioxan-water mixtures to correlate inve r se ly w i t h t he polar- i t y (dielectr ic cons tan t ) p r o f i l e of t h e s o l v e n t f o r mixtures conta in ing more than 30% water .

ro se s o l u t i o n s ( a s so lven t s ) gave t h e oppos i te e f f e c t , t h e s o l u b i l i t y of acetaminophen dec- r eas ing w i t h decreasing d i e l e c t r i c cons t an t of t h e so lvent ( i .e. w i t h i nc reas ing sucrose concen- t r a t i o n ) .

A s i m i l a r study65 conducted w i t h suc-

Goldberg e t a1.66 examined t h e solub- i l i t y of acetaminophen i n aqueous urea s o l u t i o n s and found a l i n e a r i nc rease i n acetaminophen sol- u b i l i t y with increas ing urea concent ra t ion . T h i s increased t h e s o l u b i l i t y a t 37OC from about 1 9 mg./ml. ( i n water) t o about 31 m g . / m l . ( i n 3.0 Molar urea s o l u t i o n ) . The authors66 a t t r i b u t e t h e s o l u b i l i z i n g effect t o an i n t e r a c t i o n occurring i n s o l u t i o n between urea and t h e acetaminophen. The s o l u b i l i t y of acetaminophen i n water is r e a t l y increased i n the presence of phenazone 7 O f 7 1 by a process thought t o involve hydro en

t h e presence of c a f f e i n e but theophyl l ine has been shown435 t o reduce t h e s o l u b i l i t y of ace ta -

bonding. A s i m i l a r s i t u a t i o n is observed 435 i n

minophen.

2 . 4 2

Solvent

Ethanol Ethanol ( 9 5 % ) Ethanol Methanol Acetone A c e tone

S o l u b i l i t y i n Water Miscible Solvents

1 i n 10 6 3 1 i n 7 2 1 1 i n 8 6 1 1 i n 10 61,63 1 i n 13 21,63 1 i n 20 6 1

Propylene Glycol 1 i n 9 2 1 (cont ' d)

24

ACETAMINOPHEN

Solvent Reference

Propylene Glycol 1 i n 10 63 Propylene Glycol 1 i n 50 61 Glycerol 1 i n 40 21,63 Glycerol 1 i n 50 61

The fol lowing s o l u b i l i t i e s have been determined under c o n t r o l l e d condi t ions .

Solvent Temperature S o l u b i l i t y Ref. (OC) (mg . / m l . )

Water conta in ing 2% e thanol 26.5 23.9 72 Propylene Glycol 37 156 68 Dioxan 25 90 69

2.43 S o l u b i l i t y i n Solvents Immiscible with Water

Solvent

Chloroform 1 i n 50 Benzene Inso l . E t h e r In so l . Petroleum Ether Inso l . Pentane I n s o l .

Reference

3,61,63 4,61,63 3,61,63 4 4

The following s o l u b i l i t i e s have been determined under c o n t r o l l e d condi t ions:-

Solvent S o l u b i l i t y a t 37OC Reference (mg . /ml 1

Cyclohexane 0.0015 Theobroma O i l 2.16

67 68

2.44 Rate of Dissolu t ion

Disso lu t ion ra te s t u d i e s conducted by Goldberg e t a 1 . 6 6 examined t h e d i s s o l u t i o n of

25

JOHN E. FAIRBROTHER

monoparticulate layers73 of acetaminophen, alone, and in fused and physical mixtures with urea. The dissolution of samples of pure acetaminophen followed pseudo-zero order kinetics over a 5 min. period. Coarse particles (50-60 mesh) gave data in reasonable agreement with the "cube root law" 7 4 thus representing a system requiring correction for the decrease in surface area during dissolution. Finer material (100-120 mesh) gave data more in agreement with a planar surface dissolution model. The eutectic and physical mixtures with urea gave biphasic dissolution curves, the rate constant of the first part being approximately twice that for pure acetaminophen of similar particle size, while the second rate constant closely resembled that for pure acetaminophen. This suggests the urea is leached out leaving a matrix of effective surface area comparable with that of the pure acetaminophen.

75 Mattok, McGilveray and Mainville studied the dissolution of eight different lots of formulated acetaminophen tablets using the USP XVIII - NF XI;i,jrotating basket) method and two other methods . None of the methods gave complete correlation with the blood and urine profiles obtained with the same samples. All of the recently manufactured samples required less than 15 mins. for 50% dissolution but stored samples showed greatly diminished dissolution rates in some cases.

Chow and studied the dissolution rate of acetaminophen and its physical mixtures and complexes with caffeine and theophylline. The anhydrous form and the monohydrate of the 1:l acetaminophen-caffeine complex showed more than two and a half times the dissolution rate of acetaminophen. However, the hexahydrate of the acetaminophen-caffeine complex and the 1:l acetaminophen-theophylline complex both showed a reduction in acetaminophen dissolution rate relative to pure acetaminophen.

26

ACETAMINOPHEN

2.5 Physical Properties of Solutions

2.51 Cryoscopy

Several eutectics of acetaminophen have been described in the literature:-

Eutectic Eutectic Eutec tic Reference with Temperature Composition

(OC) ( % Acetaminophen)

Phenacetin 115 Benzanilide 136 Urea 115 Acetylsalic- ylic Acid 118.2 Phenazone 83

10 4

37 85 28.5 6 59.5 6

The cryoscopic properties of acetaminophen in naphthalene have been reported by Auwers 86.

2.52 Ionisation and DH

Acetaminophen is a weak acid its saturated aqueous solution having a pHi4 of 5.3 to 6.5 at 25OC. pKa values for acetaminophen have been quoted betwe also recently as 10.15995. Two papers describe the determination of the pKa value of acetaminophen b spectrophotometric procedures. Talukdar et al.15 obtained a value of 9.35 + 0.05 (uncor- rected for ionic strength) at 25%: procedure described by Roth and Bunnett Dob68 et al.79 a value of 9.55 + 0.03 (at 25OC) using the procedure described by Albert and Ser jeant82.

9.078 and 9.580 and

2.53 Dipole Moment

The dipole moment of acetaminophen has been determined8 in 1,4-dioxan solution using a Dipolmeter DMOl (heterodyne beat apparatus) and

21

JOHN E. FAIRBROTHER

the molecular dipole moment ca ulated using the method described by Hedestrand . k5

This result is in good agreement with the value of 3.96D reported by Lutskii et al.84.

entation Polarisation (P2-) of 325.4658 cm3.

(i.e. 1.1 obs. - LI calc.) of - 0.53 D .

Tomlinson's8 value yields a Molar Ori-

Lutskii et a1.84 quote a value for 1 . 1 ~ ~

2.54 Refractive Index

Microscopic studies with the Kofler hot stage49tSO showed melts of acetaminophen to have a refractive index of 1.5403 at 174oC (for red light) and at 181 - 182OC (for sodium light). Using an Abbe refractometer solutions of acetaminophen in 1,4-dioxan and in methyl alcohol show linear increase of refractive index with concentration up to 3.6% w/w and 10.8% w/w res- pectively94.

From the equation:

n (observed) =n (acetaminophen) + "(methanol) ( 1-4

(x = weight fraction of acetaminophen in solutim)

a refractive index for acetaminophen of 1.608 (21°C, white light) was calculated94 (methyl alcohol solution).

Measurement of the nD in ethanol has been used87 to quantitatively determine the concentration of acetaminophen in two component mixtures.

28

ACETAMINOPHEN

2.55 Adsorption from Solution

The quantitative adsorption of acetaminophen from 2% ethanol solution was investigated for the solid adsorbents, nylon, cellulose triacetate and cellulose by Ward and Upchurch72. The influence of temperature, time, solubility and solvent were examined.

Cellulose did not adsorb acetaminophen while nylon adsorbed almost twice as much as cellulose triacetate. Desorption studies indicated that adsorption occurred through hydrogen bond formation, the preferred mechanism being through the amido hydrogen of the acetaminophen and the carbonyl oxygen of the adsorbent. Brook and Munday89 have examined the adsorption of acetaminophen on a dextran gel (methylated Sephadex (LH-20)) and suggest a similar mechanism of hydrogen bond formation.

2.56 Partition Coefficients

Acetaminophen is preferentially extrac ted into ether from acid and weakly alkaline a ueous solutionsg0 g1 I 92. Brodie and Axelrod 9Yi92 examined the effect of pH on the partition of acetaminophen between ether and aqueous solution saturated with NaC1.

PH Volume Fraction Extracted

- (ether/water) (ref. 9 2 ) (ref. 91) ratio in ether phase

4.0 5 7.0 5 9.0 S 10.0 5 11.0 5 13.0 5

- 0.88 0.91 0.88 0.85 0.89 0.61 0.79 0.57 0.62 0.0 0.0

Partition coefficients for acetaminophen between other organic phases and water have been described.

29

JOHN E. FAIRBROTHER

Organic Partition Co- Phase efficient

(PI

Cyclo- hexane 0.000075 Chloroform/ Ethanol about 0.44 l-Octanol - l-Octanol (part it ion with pH 7.2 buffer) 6.237 +

2 .o%-

Log P Hansch Hy- Ref. drophobic Substituent Constant

- (TI)

n.a. n. a. 67

n.a. n.a. 93 0.55 -0.61 88

0.795 -0.36 8

Similar information may also be derived from the R values obtained in specially designed reversed phase silica gel thin layer chromatographic systems. Tomlinson8 employed two systems of this kind. (see Section 6.27)

3. Molecular Complexes

Acetaminophen has been reported to interact with chloral95 and with sorbitolg6tg7r 98. Possibly these interactions may result in molecular complexes but insufficient data is available for any interpretation of this kind. A molecular complex of acetaminophen with pyramidon (1:l) has been made99 and acetaminophen is known to hydrogen bond onto the surfaces of nylon72 and rayon72.

30

ACETAMINOPHEN

Lach and CohenlOO demonstrated the solubilis- ation of acetaminophen with alpha - and beta - cyclodextrins (Schardinger dextrins) . The cyclodextrins exist in the form of cyclic chains having a relatively large open space within each molecule ( 6 a for alpha - and 82 for beta - cyclodextrin) . The interaction of acetaminophen with the cyclodextrins produces non stoichiometric inclusion complexes of the clathrate typelol. Beta - cyclodextrin solubilises acetaminophen to a greater extent than alpha-cyclodextrin, the respective slopes of the interaction isotherms being 1.100 and 0.395.

acetaminophen was reported yo2 to give a syrupy mass. Ridgway and Johnson70 independently found that phenazone solubilised acetaminophen in water and that an equimolar molecular complex crystallised from solution. The complex was also obtained from alcoholic or acetone solution and from melts7l. melting point (109.5 to 110.50C)71 and a hydrggen bonded structure has been proposed by Dearden for the complex.

plexes of acetaminopheq with caffeine and with theophylline by a process of crystallisation from aqueous solution. These complexes were shown to exist in several hydrated forms:-

When mixed with henazone (antipyrine) ,

It showed a congruent

Chow and prepared (1:l) com-

Acetaminophen (1 : 1) Complex

with

Caffeine Caffeine Caffeine Theophylline

Degree of M.pt. K1:l Heat of Hydration (OC) (l.mole) Solutim

at 25OC (K cal./

mole) anhydrous ca.145 59.4 6.5

hexahydrate 42-50 anhydrous 192-195 16.1

monohydrate 75-80 - 10.1 - - -

Theobromine does not form a complex with acetaminophen in aqueous solution435.

31

JOHN E. FAIRBROTHER

4. Synthesis and Purification

4.1 Chemical Synthesis

4.11 Synthetic Routes

Acetaminophen was first synthesised by Morse31 in 1878 by reduction of p-nitrophenol with tin in glacial acetic acid. The p-aminophenol produced by the reducing action of the tin was not isolated, being acetylated in situ by the acetic acid. Tingle and W i l l i a m s 3 3 w e d the Morse synthesis but found it necessary to increase the acetic acid concentration to 100% by the addition of acetic anhydride.

Vignololo3 simplified the synthesis by employing p-aminophenol a6 his starting material which he acetylated with acetic acid. Friedlander32 modified this process slightly by acetylating the p-aminophenol (from p-nitrophenol) with acetic anhydride in place of acetic acid. Many preparative methods have since been described employing the acetylation of p-amino henol with acetic acid and/or acetic anh dride33 I s 4 I 36 I

some cases anh drous sodium acetate also has been added34 I 3% I 37 I 41 I 42. has been produced in numerous ways includin the electrolytic reduction of nitrobenzene4% I the

37 I 39 I 41 I 42 I 4 3 I 44 I 48 I104 I 105 I106 I187 I108 ; in

The p-aminophenol

direct o alytic hydrogenation of p-nitrophe- no 1 4 3 I 10s I gG , the sulphide reduction of p-nitrosophenolLu'.

A typical43 reaction sequence is : -

32

ACETAMINOPHEN

NH,HCI N02 0 zr1{5"to p.s.i.g.) 3ob 0 OH /40H Neutralise to

Pd/C

OH

NHCOCH, NH2 0 . Acetic 0 OH OH

Anhydride

In some processes the p-aminophenol is not isolated but is acetylated in situ as it is formed45,47,109,110. An alternative to the acetylation procedure uses the action of ketene 111 in p-aminophenol.

Other synthetic pathwa s involve the saponification of esters36 I 112 I lY3 such as 4- acetamidophenylacetate,the hydroxylation of anilides b chemica1114 I electrolytic115 or enzymat ic116 processes, and the decomposition of diazo compounds such as p-AcNHC H N BFq117. Acetaminophen has also been synt gide8 from p-hydroxyacetophenone hydrazone 2% .

4.12 Purification

Crude acetaminophen is in most cases

Coloured impurities are removed furified by recrystallisation from hot water39 I

during the recrystallisation by treatment with 5 t 48t110.

33

JOHN E. FAIRBROTHER

activated charcoal 39'48 and oxidation is supp- ressed the addi on of small quantities of NaHS031f8, Na2S204" or hydro~ulphite~~. One process48 controls the pH to 6.5 during the recrystallisation by the addition of ammonia.

A number of processes seek to purify the p-aminophenol intermediate in a similar manner with activated carbon and Na2S204 43,44, 437,438 and in one case44 also by extraction of the aqueous p-aminophenol with an organic solvent such as benzene, toluene, hexane etc., to remove impurities such as azoxybenzene and azobenzene.

A patent by Hahn and Q ~ i n n ~ ~ deals specifically with the purification of acetaminophen and related compounds made from crude discoloured intermediates (p - aminophenol). The discoloured acetaminophen is dissolved in hot water, acidified (pH 1 to 5) with a non-oxidising mineral acid and kept in a non-oxidising atmosphere (H2,C020r S02). The solution is agitated with activated charcoal, filtered and allowed to crystallise in the presence of an alkaline reducing sulphite, bisulphite, or hydrosulphite.

4.13 Impurity Profile

The following impurities have been detected in acetaminophen:-

Substance Origin Amount Reference in commercial

(pharmaceutical) grade material

p-Nitrophenol Synthetic - 119 precursor

in termed ia te p-Aminophenol Synthetic t 0.025% 7,14(first

p-Ch loro Impurity +lo p.p.m. 7,14 acetanilide cont ' d

suppl) ,21

34

ACETAMINOPHEN

Substance Origin Amount Reference in commercial

(pharmaceutical) grade material

0-Acetyl para- Impurity - 119 cetamol ( DAPAP )

Azobenzene Azoxybenzene

Guinone Quinonimine meri-Quinon- imine

Inorganic Chloride

I nor gall i c Su 1 $ate

Inorganic Sulphide

Water

from over- none detected 120 12 1

of para- ce t amo 1

acetylationl- 1 to 1.3%

By-products - of reduction - of nitrobenzene (precursor)

44 44

Oxiuation Give a bluish 46 of p-amin- or greyish color 46 ophenol to acetaminophen 46

intermediate) (synthetic

- 4 0.02% 7,14

- Not detected 7,14

- 4 0.5% 7 I 14,21

4.14 Reference Standards

A National Formular Reference Stan- dard exists for acetaminophen Y4 .

4.2 Biosynthesis

4.21 Metabolism of Phenacetin and Acetanilide

35

JOHN E. FAIRBROTHER

Acetaminophen is the main metabolite of both acetanilide and phenacetin (acetophenetidin) in man and in animals.

Acetanilide was introduced by Cahn and Hepp12’ as an analgesic and antipyretic in 1887. Investigating the metabolism of acetanilide, Mdrner (1889) 122 isolated potassium p-acetamido- phenyl sulphate as a double salt with potassium ethyl oxalate from human urine. He also isolated a glucuronide tentatively identified as a conjugated p-acetamidophenol. Confirmation of this work was provided by Greenberg and Lester 123,124 and shortly after by Smith and Williams 12’ Who demonstrated that in the rabbit 70% of the administered dose was excreted in the urine as the glucuronide conjugate of acetaminophen and 12% as acetaminophen sulphate. The metabolic fate o aypanilide has since been studied in detail12 r 1 .

128

similarly isolated acetaminophen sulphate and the conjugated glucuronide from human urine. Smith and Williams130 showed that 54% of the dose administered to rabbits was recovered in the urine as conjugated acetaminophen, (47% glucuronide and 7% sulphate). In man, Brodie and Axelrodgl found up to 82% of the administered dose in the urine as conjugated acetaminophen and about 3 % as free acetaminophen. More recent papers231, 132 give an essentially similar picture. A comparative study133 of the availability of acetaminophen administered orally as such and as pnenacetin gave availability ratios (acetarninophen/phenacetin) in two studies as 1.04 and 1.06.

In the case of phenacetin MBrner

4.22 Prodrugs

Prodrugs are defined134 as having physico-chemical properties different from the parent drag but retaining qualitatively identi- cal pharmacologic effects and reverting to the parent drug in the body.

36

ACETAMINOPHEN

c t ophen forms numerous ester prodrugs 67 , 13' Kotenko and MokhortlEj5 describe an ethoxyphenylmethylacrylamide homopolymer and its copolymer with o-carboxyphenylmethacryl- amide which as analogs of phenacetin may be con- sidered as prodrugs of acetaminophen. Extensive studv has been made of the release of acetamino-

4.23 Microbial Biosynthesis

Theriault and Longfield 18' studied the microbial conversion of acetanilide to acetaminophen. An unidentified Streptomyces species RJTS- 539 gave a peak yield of 405mg./litre of acetamirr ophen from 1000mg./litre of acetanilide.

Amanita muscoria F-6 gave a mixed yield of acetaminophen and 2'-hydroxyacetanilide.

5. Stability

5.1 Stability to Light

Acetaminophen is slightly light sensi- tive in solution63 and may degrade by a mechanism involving pre-dissociation of the N-C bond as in the case of acetanilide1711172.

5.2 Stability of Solid Acetaminophen toHeat

Dry, pure, acetaminophen is very stable at temperatures up to at least 45OC. Should it however, be contaminated with traces of p-aminophenol or be exposed to humid conditions such that hydrolysis to p-aminophenol takes place, then further oxidative degradation of the p- aminophenol occurs121 characterised by a gradual color change through pink to brown and eventually to black. This involves the breakdown of the p- aminophenol to quinonimine and related compounds 46.

37

JOHN E. FAIRBROTHER

5.3 Stability of Solutions of Acetaminophen

The degradation of acetaminophen in aqueous solution appears to be both an acid catalysed and a base catalysed reaction173 I 174. It is first order with respect to the concentration of acetaminophen and first order with respect to the hydrogen and hydroxyl ion con- centrationl73.

Koshy and proposed reaction mechanisms for the acid and base catalysed hydrolysis of acetaminophen and determined the specific reaction constants (k') over the pH range 2 to 9.

pH k' (hours-' x - Ea t\ at 25OC

35OC 7OoC 9ooc (K (years) - - 2 2.52 29.13 3 - 7.40 4 5 6 - 2.56 7 8 - 6.58 9 - 19.02

- - - -

- -

168.3 31.03 10.76 8.37 6.98 13.16 25.37 66.62

16.69 0.78 17.99 5.83

- 15.39 - 19.78

17.42 21.80 - 12.59 17.99 7.13 17.42 2.28

The above studies17 were carried out isother- mally. Zoglio et a1.175 repeated part of the study (pH 2 buffer) but used a nonisothermal linear temperature programmed technique. The results obtained were in good agreement with those of Koshy and Lach yielding a value for Ea of 17.0 K cal./mole and a value for k' (at 35OC) of 1.95 x 10-4 hr.-1. Zoglio et al. calculated the activation energy by comparing analytical data with Arrhenius model degradation curves using a digital computer. This approach was further improved by Kay and Simon176 who re- calculated the data of Zoglio et al. using an analog computer system.

38

ACETAMINOPHEN

5 . 4 Stability to Oxidation

Acetaminophen is relatively stable to aerial oxidation unlike its hydrolysis product p-aminophenol. Acetaminophen has been used as an antioxidant for carotene in mineral o i l solution177, a heat stabilizer for p~lyamidesl~~ and as an antioxidant, stabilizer and short-stop- ping agent for synthetic rubber latexesl79.

5.5 Compatibility with Excipient Materials

The compatibility of acetaminophen with a wide ran e of excipient materials has been re- ported156 20 170.

5.6 Compatibility with Aspirin

Acetaminophen has been formulated in numerous commercial tablet preparations with aspirin. In some cases a third active drug substance such as caffeine, codeine phosphate or salicylamide is also present.

Acetaminophen is known85 to form a eutectic product with aspirin (m.p. 118.2OC) and there is also some evidence to suggest that the two substances interact chemically to produce salicylic acid and diacetyl-p-aminophenol (p- ace t oxyace t an i 1 ide ) .

Koshy et al. 180 found up to 4mg./tablet of diacetyl-p-aminophenol (DAPAP) in commercial products and studied the formation of DAPAP in laboratory prepared mixtures of acetaminophen and aspirin after storage for up to 1 month at 5OoC. They also noted that magnesium stearate appeared to accelerate the formation of DAPAP.

Boggiano, Drew and Hancock12'in a later study confirmed the formation of DAPAP in formulations containing acetaminophen and aspirin (compressed tablets and uncompressed mixtures) on storage at elevated temperatures ( 6 0 O C ) . They

39

JOHN E. FAIRBROTHER

also suggested that codeine phosphate and magnesium stearate both accelerate the formation of DAPAP .

Kalatzis 12' refutes the findings of both the previous authors180,120, and shows DAPAP to be present as a synthetic impurity in commercial grades of acetaminophen and conse- quently is present in conunercial products containing acetaminophen. In stability evaluation experiments with mixtures of acetaminophen and aspirin stored at 45OC for up to 2 months no DAPAP was formed. Samples of the acetaminophen/ aspirin mixtures spiked with DAPAP in fact showed a gradual decline in DAPAP content if stored under humid conditions at elevated temperature.

5.7 Physical Incompatibilities

Acetaminophen shows physical incompat- ibility with antipyrine, Irgapyrin, Irgaphen, 2- phenylquinoline-4-carboxylic acid and diphenhydramine hydrochloridelo2 , mixtures with these substances becoming sticky on mixing.

Rheological examination68 of acetaminophen in microcrystalline cellulose-carboxymeth- ylcellulose gels shows some evidence of an interaction between the acetaminophen and MCC - CMC.

Under humid conditions and at elevated temperatures acetaminophen discolours in the presence of codeine phosphate or caf f einelzl.

6. Analytical Chemistry

6.1 Identity Tests

Acetaminophen may be identified by its melting point50 (see Section 2.21) and its eutectic temperatures with phenacetin50, benzan- ilide50 or urea66. measurement of hysical parameters such as infrared spectrum14rs1 or G.L.C. retention time182.

It may be identified by

40

ACETAMINOPHEN

Acetaminophen yields numerous derivatives many of which have clearly defined melting points:-

Reagent Derivative M.p. Ref. (OC 1

Benzoyl chloride - KOH 4- Ni trobenzoy 1 chloride-pyridine

Succinyl chloride -pyr idine

Phthaloyl chloride -pyridine Et2S04- alkali

0- Benz oy 1 -acetaminophen 0- (4-Nitroben- zoyl) -acetamin- 'ophen bis (p-acetam- inopheny 1) succinate 0-Phthaloyl -acetaminophen Phenacet in

Ally1 bromide 0-Allyl- acetaminophen

1-Fluoro-2,4- p- (2,4-dinitro dinitrobenzene phenoxy ) -ace tan-

ilide) Br /C HC 1 2 , 6-dibromo-

acetaminophen conc.HN03/conc. 2-nitro- H2S04 (-5oC) acetaminophen

171 62,183

210 21

225- 135 227

235- 137 237 134- 106 136 93 42

197- 184 .198

17 4 41

158 185,186

Diazotised aniline m-acetamino- 226 34,36 - HC1 o-hydroxyazo- benzene

l-Nitroso-2- 10-acetamido- 337.5 190 naphthol-HN03 SH-benzo- [a]

phenoxa- zonium nitrate

Acetaminophen gives a characteristic violet-blue color reaction with a ferric chloride test solution3 ,14' 21 and may be distinguished from phenacetin by the color formed with Liebermann's reagent3,Zl. T h i s involves oxidation of the acetaminophen with acid dichromate to slowly give a violet coloration in contrast to phenacetin which gives a red coloration.

41

JOHN E. FAIRBROTHER

Feig1187 describes a spot test for acetaminophen claimed to have a sensitivity of lpg. The test uses a procedure involving the nitrosylation of the amine group followed by its hydrolysis to a diazonium group which is subsequently coupled with 1-naphthol to give a red precipitate.

Le Perdriel et al. 186 found that in the case of acetaminophen the initial nitrosylation reaction proposed by Feigl did not occur but 2-nitro-4-acetaminophen01 is being formed instead.

Acidification of the final Feigl test solution (containing 1-naphthol) (as applied to acetaminophen) produces a yellow-orange coloured solution whereas in the contrasting cases of acetanilide, phenacetin and p-aminophenol; red, violet and black precipitates are formed.

Paper and thin-layer chromatography have been used extensively to separate acetaminophen from other substances and the combination of Rf value and chromogenic response to spray reagents may be used as an identity test. Of particular note are the papers by Gumprecht and Schwartzenburg1B8 (paper chromatography of isomeric monosubstituted phenols) and by Goenechea 189 (thin-layer chromatography of analgesics related to acetanilide).

6.2 Methods of Analysis

6.20 Gravimetric Procedures

Poethke and Kdhne 184 describe the quantitative precipitation of acetaminophen with l-fluoro-2,4-dinitrobenzene in a sodium bicarbonate-dimethylformamide medium to give p-(2,4- dinitrophenoxy) acetanilide. The precipitation is carried out over a 4 hour period and is claimed to give a precision of + 0 . 3 % . Caffe- ine, phenazone, 4-aminophenazone, phenacetin and codeine phosphate do not interfere.

42

ACETAMINOPHEN

6.21 Titrimetric Procedures

Acetaminophen may be determined by titration with sodium nitrite after prior acid hydrolysis of the acetaminophen to p-amin p

end-points have been used. oxidises acetaminophen thus rendering it possible to titrate acetaminophen with 0.1N Ce(S04)~ in an ethanolic HC1 medium1g3 I lg4.

Both visual511911440 and potentiometric 1 9 s , P J S O l

Ce4+ quantitatively

Chatten and Orbecklg5 attempted to titrate acetaminophen with perchloric acid in various acetic anhydride based solvents but were unable to obtain an end-point.

Acetaminophen may be successfully titrated in a dimethylformamide medium with 0.1N sodium methoxide (in benzene-methanol) . The end-point may be determined visually using anlg6 azo violet indicator1g2 or potentiometrically .

Laurentlg7 also using dimethylformamide solution tit-rated acetaminophen visually to a thymol blue end-point employing 0.1N Me4NOH (in benzene-methanol) as titrant. Fogg et al. 2o employed a similar system with 0.1M Bu4NOH as titrant, a N2 atmosphere and potentiometric end- point detection using a calomel reference electrode filled with EtqNBr saturated dimethylformamide.

6.22 Polarographic Procedures

The anode polarographic behaviour of acetaminophen has been studiedl98 at the wax- impregnated graphite electrodelgg. This system employs a solution of acetaminophen in aqueous ethanol/phosphate buffer (l:l), pH 7.1 and gives a value for ES vs. S.C.E. of 333mV. Brockelt 2oo describes a cathode polarographic procedure for the determination of acetaminophen after nitration with 5N HNO3. The solution containing nitrated acetaminophen is treated with potassium hydroxide and phosphoric acid to give a

43

JOHN E. FAIRBROTHER

solution pH of 5.8 and examined polarographically (E4 versus S.C .E . - 0.38V).

Shearer et al. 441 found that with the use of a glassy carbon electrode, acetaminophen could be determined polarographically with a peak potential of about + 0.5V versus S.C .E . This procedure is capable of selectively determining acetaminophen in the presence of p- aminophenol ( E # versus S.C.E. + 0.2V) and thus may be used as a stability-indicating assay. The water content of the acetate-acetic acid- methanol supporting electrolyte significantly alters the measured peak current for a given concentration of acetaminophen and thus has to be limited.

6.23 U.V. Spectrophotometric Proc-

The British Pharmacopoeia 1963223 and U . S . National Formulary XI62 both adopted U.V. spectrophotometric procedures for the determination of acetaminophen in acetaminophen tablets. In both procedures the tablets are extracted with an anhydrous alcoholic solvent (B.P.- ethanol and N.F.-methanol), the extract acidified with a small amount of dilute hydrochloric acid and then further diluted with the alcohol. The acetaminophen concentration is determined by spectrophotometric measurement of the extinction of the solutions(249 mu.) and its content calculated against, a standard E (1 percent 1 cm.) ( B . P . method) or, the extinction given by a sample of the N.F. Reference Standard.

official B.P.223 method on the grounds of cost of the solvent and the use of a standard extinction coefficient. They proposed the adoption of an alternative procedure employing 0.01N NaOH as both extractant and spectrophotometric solvent (extinction measured at 257 mu). This procedure was subsequently adopted for use in

edures

Brown and Gwilt26 challenged the

44

ACETAMINOPHEN

the Addendum 1964224 to the B.P. 1963 and has been continued in the B.P. 196821.

width on the precision of the B.P.224t21 assay and calculated that for an extinction error of 0 . 2 % a maximum half-intensity slit width of 1.7mp. may be used (calculated for Hilger and Watts, Uvispek H.700 and Unicam SP. 700 spectro- photometers) .

and XIII14 editions) retains the use of the acidified methanol solvent but employs a revised extraction procedure in which the acetaminophen is extracted from an aqueous suspension of the ground tablet with a mixture of chloroform and ethanol (3:l). Ivashkiv93 has studied the parameters of this14 procedure as applied to the assay of Squibb Acetaminophen Tablets and reports the (75:25) ratio of chloroform to ethanol to be critical. Also examined were the partition coefficient of acetaminophen between the solvent phases (see Section 2.56) and the effect of the grinding procedure used, on the extraction of the acetaminophen. The results obtained indicate micro-milling should not be employed in the sample preparation.

Rogers 202 examined the effects of slit-

203 The U . S . National Formulary (XI1

Acetaminophen has been determined spectrophotometrically (250 mp.) after partition into n-butan0120 from sodium bicarbonate solution. This facilitates the determination of acetaminophen in the presence of aspirin20. manner acetaminophen may be determined in formulated tablets of the acetaminophen-phenazone complex204 (see Section 3) by selective retention of the acetaminophen in 0.1N sodium hydroxide solution after partitioning with chloroform. The alkaline solution containing the acetaminophen is acidified with hydrochloric acid and the acetaminophen determined spectrophotometrically at 245 mp.

In a similar

45

JOHN E. FAIRBROTHER

Differential spectrophotometry has been used to determine acetaminophen in mixtures with other drug substances. determined acetaminophen in mixtures with salicylamide by differential spectrophotometric measurement at two wavelengths (255 and 301 mu).

Hdberli and Bgguin205

Shane and X~wblansky’~ used differential spectrophotometry to determine acetaminophen in the presence of aspirin, salicylamide and caffeine in analgesic tablets. Their procedure is based on the observation that the subtraction spectrum obtained by measuring the U.V. absorption of the p-acetamidophenolate ion (pH 10) against p-acetamidophenol (pH6) yields an absorption maximum near to the isobestic point of zero absorbance for salicylamide (263.5 mu.). Under the same subtraction conditions caffeine and aspirin (which is converted to sodium salicylate) do not exhibit any absorbance from 255 to 340 mu.

Routh et al. 206 employ a similar procedure for the determination of acetaminophen in the presence of aspirin and salicylic acid.

Acetaminophen has been determined spectrophotometrically in mixtures with other drug substances by several procedures involving preliminary ion-exchange (see Section 6.25) or partition chromatographic (see Section 6.26) separation of the acetaminophen.

6.24 Photocolorimetric Procedures

The majority of the published colorimetric methods for the determination of acetaminophen are based on one of three systems. These are nitration, oxidation or hydrolysis to p- aminophenol followed by diazotisation and phenolic coupling.

Gi~zardl~~ nitrated acetaminophen with a mixture of nitric and sulphuric acids at -5OC

46

ACETAMINOPHEN

to yield 2-nitro-4-acetamidophenol. Horn225 described the nitration of phenacetin with nitric acid and Brockelt20° applied this procedure to the nitration of acetaminophen. This involved nitration of an aqueous solution of acetaminophen with 5N nitric acid at room temperature for 20 minutes. Brockelt found no absorption maximum for the nitrated acetaminophen in the range 380 to 750 my. and decided to use the color formed in an assay procedure measuring the light absorption at 428 mp. (mid-point of the straight part of the light absorption curve).

re- Fe ig 1 and also Rosenthaler ported that amides such as acetaminophen undergo nitrosylation with nitrous acid to yield an N- nitroso compound which may be saponified to give a diazonium compound suitable for phenolic coupling. with sodium nitrite, acetaminophen gives a yellow color which changes to an orange color on rendering alkaline and uses this color to quantitatively determine acetaminophen. Le Perdriel et a1.186 found that acetaminophen did not form a nitroso compound on reaction with sodium nitrite and dilute hydrochloric acid but the 2-nitro-4-acetamidophenol described by Girard. They found that this reaction could be used as the basis of a colorimetric assay procedure. The solution containing nitrated acetaminophen is made alkaline with sodium carbonate solution and the light absorption measured at the absorption maximum occurring between 440 and 445 mp.

Inamdar and Kaji227 employ a similar procedure but do not make the final solution alkaline. This yields a solution of nitrated acetaminophen giving absorption maxima at 375 and 395 mp., the latter wavelength being used for quantitative measurement.

the work of Le Perdriell86 studying the mechanism of the nitration of acetaminophen and modified the spectrophotometric assay procedure.

226

Koen5 noted that in acidic medium

Hanegraaff and Chastagner continued

47

JOHN E. FAIRBROTHER

They greatly increased the concentration of acid employing a very strong mixture of nitric and sulphuric acids in addition to the use of sodium nitrite and measured the extinction at 375 mu.

The method of Le Perdriel ha thoroughly evaluated by Chafetz et al. have attempted to optimize the reaction parameters and claim the procedure has an excellent precision and is well suited to automated tech- niques442. This procedure229 is claimed to be specific for the determination of acetaminophen in the presence of a range of excipient materials and drug substances commonly found in formulations containing acetaminophen.

phen were prepared by Tin le and Williams35 and

cently the absorption spectra of the 4-nitro- benzoic acid230 and the 2,4- and 3,5-dinitro- benzoic231 acid esters of acetaminophen (as well as the trans-4-nitrocinnamic acid ester232) have been thoroughly studied and may represent useful colorimetric reagents for the determination of acetaminophen.

The p-nitrobenzoyl esters of acetamino-

by Reverdin and Cuisinier 7 83 in 1906. More re-

Oxidative reactions have been used in the determination of acetaminophen. Basu250 hydrolysed acetaminophen with hydrochloric acid to give p-aminophenol which was then oxidised with 0.1N potassium dichromate to give a violet coloured oxidation product. This dye was quantitatively extracted into isobutyl alcohol and its absorbance measured spectrophotometrically at 550 mu.

Brodie and Axelrodg2 found that p- aminophenol from the acid hydrolysis of acetaminophen could be oxidised with sodium hypobromite and the oxidation product coupled with phenol to form an indophenol dye (absorption maximum 620 to 630 mp.) This procedure has been used to determine acetaminophen in biological material

48

ACETAMINOPHEN

92r251 and has also been used to determine the level of free p-aminophenol in acetaminophen621 203 , 252.

The Brodie-Axelrod procedureg2 requires the neutralisation of an acid solution of p-aminophenol with alkali and should the neutralisation point be passed to give an excess of alkali, degradation of the p-aminophenol results. This roblem has been overcome by Murfin and Wragg253, 554 who have been able to remove the need for preliminary hydrolysis of the acetaminophen to p-aminophenol. In their manual procedure254 the acetaminophen solution is added to a hydrochloric acid-sodium hypochlorite mixture (pH 3.4; ca. 0.25% available chlorine) and the excess hypochlorite is removed with sodium arsenite. The quinone chlorimide thus formed is then coupled with phenol in the presence of a borate buffer (pH 9.9) to give a stable blue indophenol dye (A max. 625 mp.). The procedure yields results with good precision, the mean relative standard deviation obtained by the authors being 0.36%.

Nin~miya~’~ oxidised acetaminophen directly with potassium ferricyanide in sodium hydroxide solution at OOC and then formed an indophenol dye ( A max. 635 mu.) by coupling with phenol. The coloured product was tentatively identified by Ninomiya as N-(p-hydroxypheny1)- p-benzoquinone imine.

Sakurai and Umeda2560xidised acetaminophen with chloramine T in the presence of 2,4- dinitrophenyl hydrazine to give a coloured p- benzoquinone imine 2,4-dinitrophenylhydrazone. The color produced can probably be used in a similar manner to that described in a further paper by Umeda257 which describes the oxidation of acetaminophen with ceric ammonium sulphate in acidified ethanolic solution and is subsequently reacted with 3-methyl-2-benzothiazoline hydrazone. This reaction mixture on neutralisation with tri-

49

J O H N E . F A I R B R O T H E R

ethanolamine yields a blue violet color (A max. 580 mp.) which facilitates quantitative spectrophotometric measurement.

Routh et al. 206 employed a stable free radical, diphenylpicrylhydrazyl, to abstract a hydrogen atom from acetaminophen (in ethylene dichloride solution) thereby promoting a process of radical coupling. This results in a reduction of the violet color of the diphenylpicrylhydrazyl ( A max. 527 mp. ) with the formation of yellow diphenylpicrylhydrazine. The decrease in the intensity of the violet color is used to measure the concentration of acetaminophen.

Dedicoat and Symonds 443 found that in a pH 8.0 borate buffer acetaminophen reduces Folin and Ciocalteau's reagent to give a stable blue color (A max. 700 mp.) . This was best produced by heating the reaction mixture at 100°C for 10 minutes and could be used to determine acetaminophen in the presence of several other analgesic drugs.

A number of procedures are based on the acid hydrolysis of acetaminophen to p- aminophenol which is then coupled with a suitable agent. A much used procedure employs the reaction of the p-aminophenol with - naphthol in alkaline solution, extraction of the coloured reaction product into n-butanol followed by measurement of the extinction at 635 mp. This procedy55,i54based on the work of Greenberg and

and the later papers of Kosh and Lester LachZ09 and Gwilt, Robertson and McChesney 3 3 , the latter authors employing an 0: - naphthol reagent containing potassium dichromate.

Y

Brodie and Axelrod 92 modified the procedure and diazotised the p-aminophenol before reacting it with alkaline - naphthol to give a dye exhibiting an h max. at 510 mp. This procedure was also employed by Carlo et a1.234 after slight modification.

50

ACETAMINOPHEN

Kos 235 reacted the p-aminophenol (obtained by acid hydrolysis of acetaminophen) with alkaline B - naphthol (without diazotisation) to give a green color having an absorption maximum at 420 mu.

Mouton and Mason 236 used trichloro- acetic acid to hydrolyse acetaminophen to p- aminophenol which they then diazotised and coupled with N1-diethyl-N-1-naphthyl-propylene- diamine. The dye formed was extracted into amyl alcohol and the extinction determined at 590 mp. This procedure was modified slightly by Heirwegh and Fevery237 who substituted N- (1-naphthyl) ethylenediamine as coupling agent ( A max. 596mp.). I v a ~ h k i v ~ ~ ~ has also examined this procedure critically, finding at least 4 hours incubation at room temperature is required for complete color development.

Other procedures have been described where the p-aminophenol obtained has been cou led with alkaline anisaldehyde239, acid vanillin2go and other reagents241,247.

It is possible to couple diazotised reagents directly with acetaminophen but they react on1 s l 0 w l y 7 9 ~ ~ ~ ~ . Dobsg, Stgrba and

reactions and propose a reaction mechanism. VebeFLa79, s 42 have studied the kinetics of these

Acetaminophen has also been shown to couple with the diazotised reagents shown in Table 4 thus presenting the opportunity for possible spectrophotometric measurement.

Kondo et al. 246 have examined the absorption spectra (and bathochromic shift) produced by the reaction of acetaminophen in alkaline methanolic solution with an p-nitrobenzene diazonium fluoroborate reagent. Acetaminophen has been shown to produce colors of potential spectrophotometric use by interaction with 1- nitroso-2-naphthol, 2-nitroso-1-naphthol and

5 1

JOHN E. FAIRBROTHER

disodium-3-h dr -4-nitros0-2~7-naphthalenedi- sulphonate 2x8 5%.

TABLE 4 Diazotised Reasents CaDable of Coupling wit; Acetaminophen

Reagent Color of Ref,

Diazotised aniline (acid) Product Yellow 7%

Diazotised 1,4-Napthyl- amino-sulphonic acid (alkali) Red 243

Diazotised 2-Naphthyl- Yellow- 243 amino-8-sulphonic acid(alka1i) brown

Diazotised lf4-Amido-aceto- Red 243 naphthalide-6-sulphonic acid (alkali)

Diazotised 4-Nitro-6-chloro- Olive 244 2-aminophenol brown

Diazotised 4,6-Dinitro- 2-aminophenol

- 245

6.25 Ion-Exchange Chromatographic

Ion-exchange column chromatography has

Procedures

been used to separate acetaminophen from its decomposition product, p-aminophenol, from mixtures containing other medicinal agents and from dosage forms (see Table 5) .

Street and Niyogi 211f 213 separated acetaminophen from phenacetin (acetophenetidin) phenobarbitone and salicylic acid by two dimensional chromatographic development on diethyl- aminoethylcellulose ion-exchange paper (Whatman DE 20). Initial separation was by simple development in a 0.2N ammonium hydroxide solvent. This was followed by ionophoretic development at right angles in the same solvent (5mA; 250V.).

52

TABLE 5 Ion-Exchange Chromatographic Separation of Acetaminophen

Separation Ion-Exchange Elutrient Quant itation Reference from medium

Elixir formulation Dowex 1 - X8, 20% glacial Titrimetry in DMF 200-400 mesh acetic acid solution with sodium 196 (hydroxide form) in ethanol methoxide

Phenobarbitone Dowex 1 - X1 70% methanolU .V. Spectro- photometry (249mu. ) 207

0.1N HC1 in Differential U.V. 70% ethanol Spectrophotometry 205

metry (244 mv.) 208 [ Chlorobenzoxazolin f:fyite IR-120 Water metry (244 mu.) 209

VI p-Aminophenol Amber 1 ite Water U.V. Spectrophoto-

p-Aminopheno 1 U.V. Spectrophoto-

Sulphadimethoxine Caffeine Theophorin Tartrat

Phosphate Cation Exchange Water U.V. Spectrophoto- 210 Phenylephrine HC1 Resin, Alginic rnetry (249 mu.) Pyrilamine Maleate acid, Mesh 40-100 1 (B.D.H.)

JOHN E. FAIRBROTHER

6.26 Partition Chromatographic

Koshy213 separated acetaminophen from

Procedures

admixture with caffeine and aspirin by partition chromatography employing consecutive sulphuric acid and sodium bicarbonate impregnated Celite columns. Caffeine was retained on the acid column and aspirin on the alkaline column. Acetaminophen which is not ionised under either set of conditions passed through the columns in the diethyl ether solvent.

Levine and Hohmann 214 noted that the above system was unable to separate acetaminophen from neutral or weakly acidic compounds. To overcome this weakness they replaced the sulphuric acid by hydrochloric acid and the sodium bicarbonate by a sodium carbonate-sodium bicarbonate buffer (pH 10.1). The sample containing acetaminophen is incorporated into the acid impregnated Celite before it is packed into the first column which is placed directly above the second alkaline column.

The two columns are washed with chloroform and then the acetaminophen is eluted with ether. The acetaminophen is determined spectrophotometrically in acid methanol solution (249mp.) following evaporation of the ether. This procedure will successfully allow the determination of acetaminophen in the presence of many co- administered drug substances.

This rocedure214 was slightly modified by HohmannSlS who contained the two packing materials in a single column as separated segments. The modified procedure shown to be satis- factory for the determination of acetaminophen in liquid preparations was adopted by the NFXIII for the determination of acetaminophen in Aceta- minophen Elixir.

Both procedures ’ 215 have been

54

ACETAMINOPHEN

s u c c e s s f u l l y a p p l i e d 2 1 6 to 219 t o t h e de te rmin- a t i o n of acetaminophen i n o t h e r formula ted pro- d u c t s . column p rocedure i n d i c a t e d t h a t optimum r e s u l t s were o b t a i n e d if 1N h y d r o c h l o r i c a c i d and 0 . 5 % e t h a n o l i n ch loroform a r e used i n p l a c e o f t h e c o n c e n t r a t e d h y d r o c h l o r i c a c i d and ch loroform.

F u r t h e r e v a l u a t i o n 2 2 0 of t h e s i n g l e

H a m i 1 t o n 221 h a s d e s c r i b e d a f u r t h e r m o d i f i c a t i o n t o t h e two-column procedure i n which t h e columns are s e p a r a t e d a f t e r t h e ch lo ro - form wash and acetaminophen is e l u t e d from t h e a c i d column w i t h water-washed e t h y l acetate.

column sys tem (based on t h e Levine222 system) t o s e p a r a t e diacetyl-p-aminophenol from t a b l e t f o r m u l a t i o n s c o n t a i n i n g acetaminophen, a s p i r i n and c a f f e i n e .

Koshy e t a l . have used a t h r e e

6.27 Paper and Thin Layer Chromato- g r a p h i c Procedures

A number of t h i n l a y e r and paper chrom- a t o g r a p h i c methods have been found s u i t a b l e 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 acetaminophen. The q u a l i t a t i v e a s p e c t s of t h e s e methods a r e summarised i n Tab les 6 t o 11.

Reversed phase chromatography w a s used by Tomlinson8 i n a s t u d y des igned t o c o r r e l a t e Rf c h a r a c t e r i s t i c s w i t h chemica l s t r u c t u r e f o r a series o f s u b s t i t u t e d a c e t a n i l i d e s i n c l u d i n g acetaminophen. I n t h i s s tudy8 t w o s e p a r a t e s t a t i o n a r y phases were used on s i l i c a g e l G p l a t e s .

55

JOHN E. FAIRBROTHER

Stationary Mob i 1 e Rf at 20 + 0.5OC Phase Phase (av. of lo-results)

l-octanol acetone/water 0.758

liquid acetone/water 0.713

(B.P.)

(1:9) (0.740 to 0.779)

paraffin (2 : 8) (0.710 to 0.716)

Semi-quantitative procedures relying on the visual comparison of sample spot size and intensity with standards have been described by Klutch and Bordunl31 and by Shand267. B k h et a1.270 described a quantitative procedure in which the acetaminophen is acid hydrolysed to p-aminophenol which is then separated (lO-lOOug./ spot) by thin layer chromatography on a Silica Gel G layer. The p-aminophenol is eluted with 0.5N sodium hydroxide solution and determined spectrophotometrically at2zj0 mu. A further paper by the same authors employs chromatographic separation of the acetaminophen (without prior hydrolysis) on a layer of Silica Gel GF followed by elution with methanol and spectrophotometric determination at 245 mu. The procedure may be used to determine acetaminophen (60-3OOpg. /spot) in serum with a precision of + 5%. Cummings, King and Martin265 describe a similar quantitative procedure that employs elution of the acetaminophen from the silica gel with water rather than with methanol.

6.28 Vapor Phase Chromatographic Procedures (G.L.C., V.P .C. )

Acetaminophen presents difficulties for quantitative G.L.C. determination as a result of the pronounced elution peak tailing caused by its polar hydroxyl group.

Koibuchi et al. 278 overcame this prob- lem by acetylating the hydroxyl group to give N,O-diacetyl-p-aminophenol (DAPAP) which gave a good symmetrical peak after elution from a 1%

56

TABLF: 6 Paper Chrmtographic Systems for Acetaminophen

paper Direction

W h a m 3MM Two dimensional development, ascending

Whatman No. 1 ascending whatmatman No.1

Matman No. 1

whatmatman No. 1 ascending impregnated with t r b u t y r i n

II

ln 4 11

Solvent System F+ Value

a) Isopropanol/aq. 0; 83 mnia/water (8:l:l)

acid/water (loo0 : 700:41) Water 0.83 Mineral spir i ts saturated with water 0.00 Toluene saturated 0.03 w i t h water Phosphate buffer 0.80 (pH 7.4)

b) Benzene/propionic 0.24

Wha- No. 1 ascending Benzene/gl . acetic n. a. acid/water/n-butanol (38 : 38: 17 : 7)

Grade FN1 ascending n-Butanol/lO% aq. 0.61 (VEB Spezial- m n i a (2:l) papierf abrik) Grade FN1 ascending Benzene/acet i c 0.04 impregnated w i t l i acid/water (4:4:2) 4% sodium bicarbonate (pH9)

- U s e Reference

Characteris- ation in human urine

Chromatographic study of isomeric phenols

I t II

Identity test

Isolation from microbial cul- cultures Separation from other analgesics

Separation from other analgesics

481

188

II

I I

3

181

2

2

TABLE 7 Thin Layer Chromatographic Systems for Acetaminophen (Neutral Systems)

Absorbent

Silica Gel GF

Silica Gel G

Silica Gel GF

Silica Gel HF Silica Gel GE'

Silica Gel G

Silica G e l G Silica G e l G

Silica Gel G

Silica G e l GE'

Silica Gel G

solvent system Rf Value

&k?thanol 0.50

Methyl ethyl ketone 0.50

&k?thyl ethyl ketone 0.70

Butanone 0.44 Chlorofom/diethyl ether 0.00 (85:15) Chlorof orm/acetone ( 90 : 10) 0.09

Chlorofom/methanol (80: 20) 0.75 BenZene/acetone (20 : 10) 0.33

Chlorofobzene/ 0.33 acetone ( 65 : 10 : 25) Acetone/n-butanol/ 0.92 water (50:40:10)

Two dimnsional develop-ent a) Chloroform/acetone ( 90 : n . a.

10) b) Chlorof orm/benzene/ n.a.

acetone ( 65 : 5 : 30)

Use - Separation from phenazone Separation from chlorpromzine Separation frcan phena-

Identity test Separation from phenazone Separation from butc- barbitone Identity test Separation from other analgesic metabolites as abwe

zone

Determination of acetaminophen and mtabolites in serum

Reference

Determination of acetaminophen and metabolites in urine

259

260

259

2 68 259

191

261 262

189

2 63

264

TABLE 8

Thin Layer Chromtographic Systems for Acetaminophen (Alkaline Systems)

Adsorbent Solvent system Rf Value U s e Reference - Sil ica Gel GF Chlorofom/95% rraethanol/ammnia 0.47

(85: 15: 1)

35% aq. m n i a (45:45:10) Si l ica Gel GF chlorofom/iso-propanol/ 0.80

Si l ica Gel GF Chloroform/iso-propanol/ - 33% aq. ammnia (80:15:5) lower phase/methanol ( 90 : 5 ) 0.79

2 Sil ica G e l G Butylacetate/acetone/n- butanol/lO% aq. m n i a ( 5 0 : 40: 30: 10: )

pyridine (20:60:5)

0.67 Si l ica Gel c;F Cyclohexane/chloroform/ 0.05

0.06

Isolation from micro- b i a l cultures Determination of acetaminophen and metabolites i n serum

as above

Identity test Separation from phenazone Identity test

181

263

263

269 259

268

m 0

mLlE 9 Thin Layer Chromtographic Systems for Acetaminophen (Acidic Systems)

Adsorbent Silica Gel G

Silica Gel G

S i l ica G e l GF

S i l i c a Gel GF

Si l ica G e l (F

Br inlaMn Al oxide GF

Brinlrman Al oxide GF

B r inlaMn A1 oxide GI?

solvent system Rf Value Chloroform/ethanol/acetic Chrmtogratn acid (88:10:2) diagram Chloroform/acetone/acetic as above acid (80:18:2) &nzene/nethanol/acetic 0.58 acid (45:8:4)

Mhy1 acetate/nethanol/ 0.82 water/acetic acid ( 60 : 30: 9 : 1) Double developxent a) Benzene/diethyl ether/ Chrmtogratn

acetic acid/rraethanol illustrated (120: m: 18 : 1)

ether (80:20) b) E t h y l acetate/diethyl

Toluene/benzene/water/ 0.10 acetic acid (2:2:1:2) (upper phase) Chloroform/mthanol/water/ 0.35 acetic acid (20:10:20:1) (lmer phase) Cyclohexane/n-propano 1/ 0.84 water/acetic acid (20: 20:20:1) (upper P h - 4

U s e Reference SeparationTFom WAP 121 and other analgesics as above 121,180

Determination of 2 63 acetaminophen and metablites in serum Determination of 265,266 acetaminophen and metabclit=s in urine

Separation from W A P 120 and other analgesics

Separation from other 154 roetabolites

as above 154

Separation of 131 phenacetin metabolites

cont'd ......

?IABLE 9 (cont'd) Thin Layer Chramatographic Systems for Acetaminophen (Acidic Systems)

Adsorbent Solvent system Rf Value - Use Reference

Brinlcman Ethylene dichloride/methanol/ 0.40 Separation of 131 A1 oxide GF water/acetic acid (20:10:20:1) phenacetin metabolites

minlanan Ethylene dichloride/nrethanol/ 0.16 as above 131 Al oxide GF water/acetic acid (30:5:10:3) Silica Gel G Butyl acetate/chloroform/ 0.46 Identity test 2 69

S i l i c a Gel W Dichloroethane/ethyl acetate/ 0.65 as above 263

(1- phase)

85% formic acid (60:40:20)

98% formic acid (60:20:20)

TAEu;E 10 Reagents for Papa Chrm-aphic Visualisation of Acetaminophen

Reagent Color

1. U.V. - Fluorescence Blu-rey 2. U.V. - Fluorescence after Y e l l o w

spraying w i t h 0.5% ethanolic oxine

3. Diazotised p-nitroaniline Violet 4. 5% ethanolic ferric chloride Violet 5. 15% aq. ferric chloride/

1% aq. potassium ferricyanide (1:l)Deep blue

ferricyanide/Millon's reagent (2: 2: 2)

O, 6. Ferric chloride/potassium V i o l e t h,

7. Amnoniacal silver nitrate (0. W) Black 8. Rromine/starch/potassium iodide Blue 9. C e r i c m n i u m nitrate purple

Sensitivity

20 1

(pg . acetaminophen)

< 1 20

Reference

2 2

2,258 2,181

2 2

2 3

188

TABLE 11 Reagents for Thin Layer Chranatographic Visualisation of Acetaminophen

Reagent

1. U.V. (254mp.) - fluorescence

2. 5% aq. si lver n i t ra te 3. 10% aq. si lver n i t ra te 4. 10% fer r ic chloride and 0.5%

5. 5% aq. fe r r ic chloride 6. Folin and Ciocalteu reagent 7. pDimethylaminabenzaldehyde/

w hydrochloric acid 8. Iodine vapor 9. Pauly reagent

quencl ling

potassium ferricyanide in w a t e r

o\

10. Diazotised o-dianisidine 11. Diazotised sulphanilic acid

Color

Dark spot

Black Black Dark blue

Grey blue Blue Yellcw

n.a. n.a. n.a. n.a.

Sensitivity

0.5 ( ug . acetaminophen)

0.25-0.5 0.2 0.1

< 5 0.5 1

n.a. n. a. n. a. n.a.

Reference

267

259, 262,267 189, 264 181, 267

189 263 189

180 131 131 120

JOHN E. FAIRBROTHER

DEGS column. The acetaminophen was acetylated with a pyridine-acetic anhydride reagent employing strictly controlled reaction conditions to suppress the formation of N,N,O-triacetyl-p- aminophenol (TAPAP), a secondary product of the reaction. Quantitation was effected by peak height ratio measurement using an internal standard and almost theoretical recoveries were obtained from laboratory prepared mixtures (standard deviation 0.4 to 0 . 5 % ) .

Pr escott has more recently employed a similar procedure for the determination of acetaminophen in plasma (standard deviation about 3 . 5 % ) .

cetaminophen may be readily silylated 286 to 29Q to form derivatives suitable for quantitative G.L.C. determination2791280,2811292. Pr e Scott2 used a N- tr ime thy 1 s i ly 1 imida zo 1 e (TMSI) reagent to selectively silylate the hydroxyl group. In a separate procedure he280 used a N,O-bis (trimethylsilyl) acetamide (BSA) reagent which produces a di-TMS derivative by silylating both the hydroxyl group and the amide nitrogen. He reports280 near theoretical recoveries (TMSI procedure) for acetaminophen from plasma and urine with standard deviations of 1.8 and 2.8% respectively (calculation from peak height ratio with an internal standard).

The direct G.L.C. determination of acetaminophen has been described27212741275 but the accuracy seems generally to be inferior to that of the indirect procedures and the working range for sample size is narrower as a result of poor peak symmetry.

Table 12 gives details of the various G.L.C. systems described for the separation, identification and quantitative determination of acetaminophen.

64

TAJ3LE 12 G.L.C. (V.P.C.) Determination of Acetaminophen

Colunm Column Column Support Stationary Temp.

P h a s e

C h r m s o r b W 10% E-W-982 195OC (AWDE.r=S) 80/100 (mesh) Anakr0111 AS 1% SE-30 plus 20O0C

20M 80/90 1% carbowax

6% QF-1-0065 16OoC 60/70

o\ VI

C h r o m s o r b W 2% SE-30 plus 180°C (Awl 0.1% Triste-

Gas Chror~i 0 3% OV-17 165OC 100/120 Aeropak 30 2% FFAP 2 4OoC 70/80 (in silanised Column) Ana?-xm AS 0.5% SE-30 190°C m / 9 0 plus 0.5%

arin

Carbcwax 2 0 M

R e t e n t i o n Detector Internal T y p of Reference Time S y s t e m Standard Deter-

mination

ca.1 min. F.I.D.

3 min. E l e c t r o n C a p t u r e (Sr-90)

C a p t u r e (Tritium Foil)

ca.3 min. E l e c t r o n

3.5&. F.I.D.

A m i t r i p t y - Analgesic 272 line hydro-Preparations chloride External Metabolic 1 5 4 Standard

- Qualitative- 2 5 8 C l i n i c a l

- Toxicological 273

ca.5 min. KCl-T.I.D.AmbarbitalPhannaceutica1 274

6.5 min. F.I.D. D i p h e n y l bktabolic 275 phthalate

Preparations

10 mins. E l e c t r o n External Metabolic 131 C a p t u r e Standard (Sr-90)

TABLE: 12 (cont'd) G.L.C. (V.P.C.) Determination of Acetamhaphen

Column Column Column Retention Detector SUPpo* Stationary Temp. Time system

Phase

Gas Chrom Q 1% HI-EFF-8BP 22OoC ~a.17 min. F.I.D. 8O/l00 plus 10% SE-52 Chrcdrosorb W 10% Apiezon L 2lOoC 2.4 rel- F.I.D. (AWHQS) ative t o

Cklrmsorb W 5% SE-30 or 190°C n.a. n.a. 6o/m 3% Neopentyl or (silanised) glycol succhate

Chmrmsorb G 5% Carhowax 225 C n.a. F.I .D.

Gas C h r o m Q 3% HI-EFF-8BP 22OoC 3 . m . F.I .D.

barbitme

poljjester 2003

70/8O 2oM

100/120 (as 0-acetyl

Chrmsorb W 1% Diethylene- 180°C ca. 8 m i n . 60/80 glycol succin- (as 0-acetyl (AW-silanised) ate polyester acetamino-

as cilrom Q 5% Apiezon L MOOC 9 min. (as F.I.D. 100/120 T.M.S.

acetaminophen)

F. I .D.

phen)

ether of

Internal Type of Reference standard D e t e r -

mination

External Pharmaceutical 182 Standard Preparations

- Qualitative 3

n.a. Pharmaceutical 2 7 6 Preparations

External Analgesic 180 Standard mixtures N-butyryl- Metabolic 277 p-aminoph- en01 (as 0- acetyl deriv.) N,@diiso- Antipyretic 278 butyryl-p- Preparations aminupherd

n-Hexade- Pharmaceutical 279 cane Preparations

Column support

Gas Chrom Q 80/100

Gas Worn Q 80/100

TAE3LE 12 (cont’d) G.L.C. (V.P.C.) Determination of Acetaminophen

Column Column Retention Detector Internal Type of Ref.

Phase mination -

5% ov-1 l55OC ca.15.6 m i n . (as F.I.D. p-Bram- Metabolic 280

Stationary T ~ I T ~ . Time System Standard Deter-

B.S.A.* deriv.) acetanilide (as J3.S.A.” deriv . )

10% OV-17 200OC ca.17.6 min. (as F.I.D. p-chloro- Metabolic 280 T.M.S. I. **) acetanilide

(as T.M.S.I. ** deriv.)

Chrmsorb W 5% OV-101 Program- 10.9 m i n . (as (W 80/1m med from B.S.T.F.A.***

100 to deriv.) 3oooc a t 10 deg./ min.

Gas Ckrom Q 3% OV-1 160’~ n.a.(as B.S.T. F.A. *** deriv. )

Gas Chrom Q OV-17 1woc 3 . 3 min. (as H .M. D . s . +

deriv. )

F.I.D. - Qualitative 11 Pathological

F . I .D . p-Bram- Metabolic 281 acetanil ide (as E.S.T.F. A.*** deriv.)

F.1.D- ~ooosane (as bktabolic 447 H.M.D.S. -k

deriv. )

JOHN E. FAIRBROTHER

* B.S.A. - N,O-bis (trimethylsilyl) acetamide

* * T.M.S.I. - N-trimethylsilylimidazole *** B.S.T.F.A. - bis (trimethylsilyl) trifluo-

roacetamide (Regisil)

methyl silane -t L.M.D.S. - hexamethyl disilazane/trichloro

6.29 High-pressure Liquid Chrom- atographic and Gel Filtration Procedures

.,- Burtis, Butts and Rainey'l first des-

cribed the use of high-pressure liquid chromatography in the determination of acetaminophen and its glucuronide metabolite in urine. Their procedure employed a high-pressure anion exchange chromatographic system293 with a U.V. detector and gave very long retention times in excess of 16 hours.

Anders and Latorre 295 have more recently developed an improved procedure capable of resolving acetaminophen and its glucuronide and sulphate conjugates present in urine within a total elution time of 40 min.

Henry and S ~ h m i t ~ ' ~ described a rapid high-pressure anion exchange chromatographic procedure for the determination of acetaminophen in analgesic tablets using a peak area ratio measurement with an internal standard. A plot of peak area versus concentration was linear for both acetaminophen and the salicylamide3interna1 standard over a dynamic range of 5 x 10 . This gave a possible range for acetaminophen determination of 3 mg. to about 50 pg. per sample inject ion.

Stevenson and bur ti^^'^ have improved on this procedure and describe a rapid high-pressure liquid chromatographic assay for acetaminophen in a wide range of analgesic tablets claim- ing a precision giving a relative % standard

68

ACETAMINOPHEN

deviation of 0.79 (using an external standard). Details of the procedures are given in Table 13.

Jagenburg, Nagy and Re)djer297 separated the conjugated metabolites of acetaminophen by a gel filtration technique using Sephadex G-10 (Pharmacia) but no mention is made of the elution of acetaminophen.

Brook and Munday8’ studied the interaction of a series of compounds including acetaminophen with Sephadex G-10 and Sephadex LH-20 eluting with 0 . 1 N sodium hydroxide solution.

6.3 Automated Procedures

Ederma et a1.282 automated the Brodie and Axelrodg2 procedure for the determination of acetaminophen in serum.

The ether extraction of acetaminophen from the serum and its backwashing into dilute caustic soda remained as manual procedures but the conversion of the acetaminophen to p-aminophenol and the colorimetric determination were automated using an Auto Analyser system, A later paper283 describes the application of basically the same system to the determination of acetaminophen in whole blood.

Shane and X~wblansky~~ automated their differential U.V. spectrophotometric procedure (see Section 6.23) for the determination of acetaminophen in the presence of aspirin, salicylamide and caffeine. Alber and O ~ e r t o n ~ ’ ~ also determining acetaminophen in the presence of salicylamide and caffeine used a G.L.C. system with automated peak height measurement and calculation. This system employed an amplified KC1 thermionic detector system with a direct feed into the analog-to-digital converter of a PDP 1 2 A LINC System computer. It is suggested that the sample preparation could also be automated.

Daley, Moran and Chafetz 442 automated

69

TAFLE 13 High-Pressure L i q u i d Chrmtographic Determination of Acetaminophen

Insizxumnt Column Size C o l m Temp. F l w Elution R e t e n t i o n Ref. - and Packinq and Pressure - Rate T k 2 - (ml . /ii. 1

"W-ANALYSER" 0.45x2OQ~1. 25uC inmwbg (Oak Ridge t o 6WC after National Lab- Dowex 1-X8 16 hr. oratory) w i t h (5 t o 10~) 1-2000 p.s.i.g. P n o t m t e r Detector (260 and 29Qnl.l.)

Du Pant We1 0.2UooOCm. T e n m a t u r e 820 L i q u i d n. a. Chrmatograph Zipax coated 1200 p.s.i.g. w i t h Model 410 w i t h strong Photare!ter anion ex- Detector change resin ( 2 5 4 ~ . 1

-.I C

30 ~Amnxlium Chlo- Acetaminophen 11, ride-Acet ic 16.5 hr. 293 Acid Buffer pH 4.4 Acetaminophen 0 . 0 1 5 M (38Qnl. ) glucwonide 1.OM (37cknl.) 22.4 hr. 4.0M (36cknl.) 6.0M (525m1.)

Gram-Pac) pH 9.2 containing 0 . 0 0 5 M m n i u m nitrate

90 Buffer (Fisher ca. 2 mins. 294

(cont'd.. . . .)

TABLE 13 (cont'd) High-pressure Liquid ChrOIMtographic Determination of Acetaminophen

Instrurrent Column Size Column Temp. and Packing and Pressure

V a r i a n WS-1030 O.lOx25Ocm. 80°C with Photometer Detector LSF pellicular -loo0 (254 nu.) anion ex- p.s. i.g.

change resin

4 r

varian ~ ~ - 1 0 0 0 0.10xmcm. 60Oc with Photorreter LSF pellicular 925-1030 Detector anion exchange p.s.i.9. (254 nip.) resin

Flow Elution Retention Ref.

X, 1 O . W formic Acetaminophen 295 acid (pH3) con- 3.6 min . taining 1.OM Acetaminophen potassium c h l o r - l g l m m i d e ide (gradient 2.7 min. system also Acetaminophen given) sulphat e

9.5 min.

649 secs. (+ 1.08%)

8.6 1.OM Tris Acetaminophen 296 Buffer (pH 9.0) -


the colorimetric procedure of Chafetz et al. 229 (see Section 6.24).

Murf in253 has automated a colorimetric procedure based on the chromogenic reaction of acetaminophen with an acid hypochlorite - alkaline phenol reagent system.

single tablet assay of acetaminophen, alone or in combination with aspirin and codeine phosphate is based on a Technicon 25-channel system preceded by a sampling unit and a Technicon continuous filter. The complete procedure from commencement of sampling to the recording of the maximum color takes only 11 mins. and yields results with a coefficient of variation of about 0.4%. The sampling time takes 2 min. 15 secs. followed by a wash time of 45 secs. thus permitting the examination of up to 20 samples per hour on a continuous basis.

The procedure which may be used for

6.4 Radiochemical Procedures

Davison et al. 284 described the preparation of N-(114C-Acetyl)-p-aminophenol from sodium hydrosulphite washed, p-aminophenol and (acetyl 14C) acetic anhydride giving a product with an activity of 0.88pC/mg. prepared quantities of acetaminophen labelled on the nucleus or acetyl side chain.

N-Acetyl-2, 6-14C-p-aminophenol was prepared by the reaction of sodium nitromalondialde- h de monohydrate with 1,3-14C-acetone to give 2,6- 1XC-p-nitrophenol. concurrently acetylated to give the required product.

N- ( l-14C-Acetyl) -p-aminophenol was produced by reacting p-aminophenol in peroxide free tetrahydrofuran with l-14C sodium acetate.

the or ans of Wistar-Rats dosed with either of the lagelled compounds was carried out employing

Koss et a1.285

This was then reduced and

The determination of radioactivity in

72

ACETAMINOPHEN

a Packard Scintillation Counter. Samples were dissolved in a benzalkonium chloride solution, decolorised with hydrogen peroxide and a scin- tillator solution added which contained naphthalene, PPO and POPOP.

Radiolabelled metabolites were estimated after thin layer chromatographic separation using a Berthold T.L.C. Radioactivity Scanner.

6.5 Determination of Trace Impurities and Deqradation Products

The impurity profile of acetaminophen has already been discussed (see Section 4.13).

The early compendia1 procedures for the determination of p-arninophenol in acetaminophen relied on colorimetric limit tests employing either a sodium nitro-prusside reagent21 or the phenol-hypobromite reaction62. The latter procedure was shown252 to be capable of quantitative use having a precision of about + 5% for a p-arninophenol level in acetaminophen-of 0.012%.

More recently the NF XI11 l 4 I 7 has adopted a thin layer ch;omatographic procedure for the determination of traces ( 4 0.025%) of p- aminophenol in acetaminophen. The procedure uses silica gel (HR grade) plates and a methyl ethyl ketone/acetic acid (9:l) solvent system. Visualisation is achieved with an acid p-dimeth- ylaminocinnamaldehyde spray reagent and the size and intensity of the sample spot is compared with a standard spot.

p-Chloroacetanilide levels in acetaminophen were determined by Savidge and Wraggllg using a thin layer chromatographic separation which employed a solvent mixture of cyclohexane/ acetone/diisobutylketone/methanol/water (100:80: 30:S:l). This procedure was designed for a p- chloroac tanilide limit test of 0.03%. The 1JF XIIIlg limits the level of p-chloroacetan-

73

JOHN E. FAIRBROTHER

ilide to 10 p.p.m. and describes a thin layer chromatographic procedure (solvent-chloroform/ benzene/acetone (65:10:25)) capable of this sen- sit ivity .

Savidge and Wragg showed their T.L.C. procedure to be capable of separating O-acetyl- acetaminophen (DAPAP) from acetaminophen and using this procedure found levels of up to 0.09% DAPAP in commercial samples of acetaminophen.

Several other T.L.C. procedures have been described12° ,121,180 for the determination of DAPAP in acetaminophen and quantitative determinations have also been made using a G.L.C. systemlb0. (see Section 5.6).

The limitation of the content of quin- oniraine type oxidation products has been achieved mainly by close control of the white color of acetaminophen.

6.6 Determination of Acetaminophen and its Metabolites in Body Fluids and Tissues

6.61 Determination in Urine

Tne majority of the published work centres on the determination of free and conjugated acetaminophen in human and animal urine.

Lester and G~eenberg’~~ determined the metabolites of acetanilide by colorimetric reaction with a - naphthol of the p - aminophenol produced after acid hydrolysis. Acetaminophen was selectively determined by the same colorimetric procedure after extraction into ethylene dichloriae from urine, salted out with dibasic potassium phosphate.

Smith and Williams125 examining rabbit urine containing acetanilide metabolites, hydrolysed etrier extracted urine by heating with acid thus lherating p-aminophenol from the acetam-

14

ACETAMINOPHEN

inophen conjugates. The p-aminophenol was determined gravimetrically after isolation.

Brodie and Axelrodg2 determined free acetaminophen in urine by a procedure involving the extraction of the acetaminophen from sodium chloride saturated urine into a solvent mixture of isoamyl alcohol and diethyl-ether. The extracted acetaminophen was then acid hydrolysed to p-aminophenol and determined colorimetrically after diazo coupling with a - naphthol. Con- jugated acetaminophen was calculated by differ- ence from total acetaminophen determined as total p-aminophenol obtained by direct acid hydrolysis of the urine. In this case the p-aminophenol was determined by the phenol/hypobromite colorimetric procedure.

and GreenEz$y24 procedure separating the p- aminophenol from acid hydrolysed urine on an ion- exchange column prior to colorimetric determin-

have used slight modifications of the two procedures described by Brodie and Axelrodg2 for the determination of free and conjugated acetaminophen in both human and rabbit urine. Levy and Yamada3O0 used the Brodie and Axelrod92 procedure but deconjugated the metabolites by incubation with an enzyme mixture rather than re- 1 ing on acid hydrolysis. Heirwegh and Fevery 2y7 retained the acid hydrolysis procedure for deconjugation of the tabolites but substituted the Bratt~n-Marshall~~' diazotisation procedure for the diazo coupling with = - naphthol.

and Lach 208 modified the Lester

ation. Several authors91,126,251,264,298,299

Lower, Murphy and Bryan302 employed both enzyniic hydrolysis and the Bratton-Marshall colorimetric procedure for the assay of acetaminophen glucuronide in urine. In this case the sample preparation involved a preliminary fract- ionation step on a cation-exchange resin.

A third colorimetric procedure303 has also been described involving diazo coupling of

75

JOHN E. FAIRBROTHER

p-aminophenol (from acid hydrolysis of acetaminophen and metabolites) with o-cresol.

Welch et al. 304 examining the metabolism of acetaminophen in animals determined the conjugated metabolites in urine (after B - glucuronidase and sulphatase hydrolysis) by a colorimetric procedure involving the formation of ion- pairs of acetaminophen with methyl orange.

Acetaminophen and its conjugated metabolites have been determined in urine after thin- layer chromato raphic se aration by U.V. spectro- photometryl31 I 363 I 265 I 267 I 270 and by measurement of radioa~tivity~~~.

Vapor phase chromatography has been used extensively to measure acetaminophen and its metabolites in urine. Grove275 determined free acetaminophen in urine by a direct G.L.C. procedure employing an ether extract of treated urine. Klutch and B0rdun1~~r154 also determined conjugated acetaminophen using a preliminary enzymic (6-qlucuronidase) hydrolysis step.

Prescott, Steel and Ferrier 292 describes a procedure for the determination of both free and conjugated acetaminophen in urine. Their procedure requires the formation of the trimethylsilyl derivative of acetaminophen which is then chromatographed. Conjugated metabolites are enzymically hydrolysed to give free acetaminophen suitable for trimethylsilylation. Improve me t pproach have also been described 277 , 38$f2k!??f4$.

High pressure liquid chromatography has been used to determine acetaminophen, acetaminophen glucuronide and acetaminophen sulphate direct without hydrolysis or derivative formation l1thq5. Similarly gel filtration procedures297 may be used but the chromatographic separation is tedious.

76

ACETAMINOPHEN

S- (1-acetamido-4-hydroxyphenyl) cysteine and 1-acetamido-4-hydroxyphenylmercapturic acid, minor metabolites of acetaminophen have been determined in urine by a gel filtration procedure 297. mined305 in urine following ion-exchange chromatographic separation by a ninhydrin colorimetric procedure.

The cysteine compound has also been deter-

6.62. Determination in Serum, Plasma and Whole Blood

The procedures for the determination of acetaminophen and its metabolites in blood are essentially similar to those described above for its determination in urine.

Lester and Greenberg124 treated both blood and plasma samples with tungstic acid to precipitate proteins and determined the acetaminophen derivatives using the same procedure as described for urine. Gwilt, Robertson and Mc Chesney306 described a procedure for the determination of free and total acetaminophen in plasma and in whole blood which is essential1 a modification of the Lester and Greenberg134 procedure. In this procedure whole blood is tri- turated with anhydrous sodium sulphate to give a dry friable mass from which free acetaminophen is extracted with 1.5% isopentanol in diethyl- ether. Acetaminophen is back washed with alkali, hydrolysed with acid to give p-aminophenol which is coupled with alkaline a-naphthol as in the Lester and Greenberg procedure. However, the green solution so produced is then saturated with potassium chloride and the chromophore extracted into butanol for spectrophotometric measurement. This is claimed to increase the sensitivity of the procedure 2% times.

126'307 for plasma and serum are essentially as described for urine after suitable sample preparation. These procedures have also been auto- mated282 I 283 for the determination of acetamin-

91,92, The Brodie and Axelrod procedures

77

JOHN E. FAIRBROTHER

ophen in blood.

The Heirwegh and F e ~ e r y ~ ~ ~ procedure which employs the Bratton-Marshall colorimetric system has been used for determinations in serum as described for determinations in urine. This procedure has also been used by Ivashkiv308 who critically evaluated the reaction parameters.

Routh et al. 206 employed two procedures for the determination of acetaminophen in serum or plasma, one employing differential U.V. absorption spectrophotometry and the other the decolorisation of diphenylpicrylhydrazyl dye.

Bdch, Pf leger and R ~ d i g e r ~ ~ ~ determined acetaminophen in serum by a quantitative thin- layer chromatographic procedure, the acetaminopkn eluted from the sample spot being quantified by a U.V. spectrophotometric procedure. Koss et al. 2a5 used quantitative thin layer chromatography to determine radiolabelled acetaminophen and its metabolites in human serum, measurement being made with a radio-autography scanner.

Free acetaminophen has been determined in plasma by vapor phase chromatography by several authors275~277,280,2a1~2g2. The chromatographic procedures in each case are those described for the determination in urine. The samplz7Yreparation however, differs slightly. Grove extracts the acetaminophen into ether from plasma saturated with solid ammonium sulphate. acetaminophen into ether but buffer the plasma to pH 7.4 with phosphate buffer and then satur- ate the solution with sodium chloride.

Thomas and Coldwell281 also extract the

In all the papers by Prescott and co- w0rkers27~ I 280 1 292 the plasma is buffered to pH 7.4 with phosphate buffer and the acetaminophen extracted into ethyl acetate. Amsel and Davison 447 also use extraction into ethyl acetate.

78

ACETAMINOPHEN

6.63 Determination in Tissue and Organs

Brodie and Axelrodg2 determined acetaminophen and total conjugated p-aminophenol in homogenised tissue (emulsified in acid1309 essentially using the same procedures they described for similar determinations in urine. Gwilt, Robertson and McChesney306 used a very similar procedure to Brodie and Axelrod homogenising the tissue in 0 . 1 N hydrochloric acid, neutralising and buffering to pH 6.6 before extracting the free acetaminophen.

285 Davison et a1 284 and Koss et al. describe the radioassay of total acetaminophen in tissue and organ homogenates using radiolabelled acetaminophen and also describe the separation of free acetaminophen and the individual conjugates in bile by a radioautographic procedure.

7. Metabolic Transformations

7.1 Metabolism in Man

7.11 Adults

Lester and Greenberg124 and Brodie and Axelrod911921126 established that acetaminophen is the main metabolite of acetanilide and acetophenetidin (phenacetin). Thus the main metabolites excreted in the urine after administration of acetanilide, acetophenetidin, bucetin310 or acetaminophen are the glucuronide and ether sulphate conjugates of acetaminophen124 I 125 I 1 2 6 .

Acetaminophen sul hate had already been isolated in 1889 by MBrnerls2 from the urine of patients who had received acetanilide. Smith and Williams1251130 isolated crude acetaminophen lucuronide from rabbit urine and Shibaski et al.

3 6 6 purified this isolated material and also produced it synthetically.

Minor metabolites have been identified

79

JOHN E. FAIRBROTHER

by Jagenburg and Toczko305 and by Jagenburg, Nagy and Rddjer297. These are the cysteine and mercapturic acid conjugates of acetaminophen. The recent findings of Nery311 of four new metabolites of acetophenetidin suggest that the list of acetaminophen metabolites may not et be complete

a metabolite of acetophenetidin isolated from dog urine as 4-hydroxy-3-methylthioacetanilide. This substance may also be a metabolite of acetaminophen. In the same ~ t u d y ~ 4 ~ the S - ( 1 - acetamido-4-hydroxyphenyl) cysteine found by Jagenburg and Toczko305 was tentatively identified more correctly as 3-[(5-acetamido-2-hydroxy- phepyl) thio] alanine.

Burtis et al.ll described the formation

329. Focella, Heslin and Teitel4 5; 8 identified

of 3-methoxy-acetaminophen (by a girl with a neuroblastoma) after treatment with acetophenetidin. They11 ascribe the formation of this metabolite and its excretion as the glucuronide to an induced activity of the hydroxylase and catechol 0-methyl transferase enzyme systems caused by the high level of acetaminophen (see also refs. 331 and 332).

The metabolic routes are summarised in Fig. 7.

The relative amounts of free acetaminophen and its sulphate and glucuronide conjugates excreted in the urine var with the individual. Typical results265,29Y,300,312 for a dose of 1 to 2 gm. acetaminophen show 75 to 90% of the dose is excreted in the urine with the acetaminophen and its metabolites distributed (approximately) in the following manner:-

Free acetaminophen 2 to 5% (of total excreted) Acetaminophen glucuronide 55 to 75% (but some

Acetaminophen sulphate 20 to 40% Acetaminophen 3-cysteine 0.5 to 7% (only 3 resula Acetaminophen 3-mercapturic acid 5 to 7% (only 3

results are much lower)

results).

80

ACETAMINOPHEN

HO acetaminophen glucuronide

OH a c y i n o p h e n

NHCOCH NHCOCH3

$SC12y HC O2 H @scH2c I HC02H

xyphenyl) thiolalanine

OH H2 HNCOCH

3 OH 3-[(5-acetamido-2-hydro-

l-acetamido-4- hydroxyphenyl-mercapturic acid

FIGURE 7 - Metabolic Pathways of Acetaminophen

81

JOHN E. FAIRBROTHER

Patients suffering with chronic hepa- titis and with liver cirrhosis show a decrease in the blood serum and urine levels of acetaminophen glucuronide and increased levels of free acetaminophen. This results from a corresponding decrease in the activity of lucuron ltransferase in the pathologic l i v e r s 2 3 q , 3 1 3 1 3 1 ~ , 3 1 5 . 3 1 6 , 3 1 7 . Renal insufficiency does not effect the ratio of free to conjugated acetaminophen in the plasma but through a decrease in glomerular filtration it may increase the plasma level of total acetaminophen by as much as 4-fold318. ism of acetaminophen to its sulphate can be blocked by salicylamide which competes with the acetaminophen for sulphate3O0 I 319. can be counteracted by L-c steine, a well absorbed source of sulphate350. This may be due to sulphate availability being the capacity- limiting factor3001320. Salicylamide also decreases the excretion of acetaminophen glucuronide possibly by a similar mechanism. Salicylic acid has no significant effect on the formation of acetaminophen sulphate and g l u ~ u r o n i d e ~ ~ ~ .

The metabol-

This effect

7.12 Newborn Infants

Vest and co-workers 307r323 found that in newborn infants acetaminophen (produced by the administration of acetanilide) is much more slowly conjugated to the glucuronide than in older children and adults. Similar results have been obtained after the administration of acetamino hen324,325,3261327 and it has been sugges- ted32g1327 that the urinary excretion and blood levels of acetaminophen conjugates depend on the maturity of the glucuronide-forming enzyme system (glucuronic acid transferase and uridine dipho- sphate glucuronic acid) and the development of renal tubular function.

7.2 Metabolism in Animals

Clark 328 demonstrated that the metabolic pathways of acetaminophen in man and dog were

82

ACETAMINOPHEN

similar. It has been shown in rats285 that about30% of the dose was secreted with the bile in 4 hr. The acetaminophen in the rat bile was shown to be almost completely conjugated to the glucuronide (83%) and the sulphate (14%) and only about 2.5% free acetaminophen was still available.

Cats dosed with acetaminophen metabolise the drug in a different manner from man, dog, rabbit and rat, in that less than 6% of the dose was excggied in the urine as acetaminophen glucuronide and less than 2% as acetaminophen sul- phate304. It has been reported3331334 that the cat has an impaired ability to form glucuronides, and this defect has been attributed to the lack Q35the glucuronyl transferase enzyme in the liver. . The cat, however, does excrete acetaminophen in the urine, in a conjugated form (capable of enzymic hydrolysis with 6-glucuronidase) and it has been suggested by Welch et a1.304 that it may be conjugated with cysteine.

These same authors 304 found that 10 to 13% of the dose administered to cats appears in the urine as an aromatic primary amine but this does not appear to be p-aminophenol.

Acetaminophen is metabolised in the rat and rabbit in a similar manner to that in ma with different ratios of the metabolites 263,330,

12 5 , Y29Yt

8. Drug Availability

8.1 Pharmacokinetics

Many authors have described various aspects of the harmacokinetics of acetaminophen 68 80 91 124 127,148 , 234,285,292,298,299 , 300,306 , 325,336 t o 347

83

JOHN E. FAIRBROTHER

G w i l t e t a l . 336 examined t h e a b s o r p t i o n o f a c e t - amino hen i n man fo l lowing o r a l a d m i n i s t r a t i o n . They336 found t h e h i g h e s t ave rage blood l e v e l o f t o t a l acetaminophen was reached a f t e r between 30 and 9 0 min. depending on t h e i n d i v i d u a l . The e f f e c t s of food and s l e e p on t h e a b s o r p t i o n and e x c r e t i o n of acetamino en have been examined by

lowed t h e a d m i n i s t r a t i o n (100 mg./kg.) of l ab - e l l e d acetaminophen i n r a t s , showing t h a t r a p i d a b s o r p t i o n occurs i n t h e f i r s t h a l f hour , o n l y about 4 0 % of t h e dose r each ing t h e s m a l l i n t e s t - i n e . T h i s g r a d u a l l y r eaches t h e l a r g e i n t e s t - i n e where t h e con t inued a b s o r p t i o n appea r s t o be compensated f o r by b i l i a r y s e c r e t i o n of acetaminophen (about 30% of t h e d o s e ) .

McGilveray and Mattok 4 s 2 . Koss e t a l . 2 8 5 fol-

The plasma h a l f l i v e s r e p o r t e d v a r y as shown i n Table 15 .

TABLE 1 5 Acetaminophen Plasma Half - L i f e i n Man

Author Ref. P l a s m a h a l f - l i f e ( t f ) - (hour s 1

Brodie and Axelrod 1 . 5 1 2 6

Carlo e t a l . 2.3 234 G w i l t e t a l . 2 . 7 336 Prescot t e t a l . 2.03 34 7

Heald and Evans 2 . 9 4 322

P r e s c o t t e t a l . 2 . 0 + 0.1 317

2 . 4

(mean of 8 s u b j e c t s )

(mean of 10 s u b j e c t s )

( 1 7 s c b j e c t s ) McGilveray e t al. 3.02 + 0 . 3 339 Careddu e t a l . 2.23 - 0 . 5 315

(unchanged) acetaminophen and i t s con juga ted m e t a b o l i t e s f o r man have been de termined by sev- e ra l au thors265,312,319,339 ,348 ,454.

The e l i m i n a t i o n ra te c o n s t a n t s f o r f r e e

84

ACETAMl NOPHEN

8.2

cellulose

Protein Binding

The bindin of acetaminophen to n lon triacetateq2 and to dextran gels84; has

72

been described in Section 2.56. Hansch and Helmer88 related this work to the octanol-water partition coefficient and ultimately to the binding of acetaminophen to natural polymers such as proteins.

Dearden and T o m l i n ~ o n ~ ~ have examined the binding of acetaminophen to bovine serum albumin (BSA) using a dynamic dialysis method finding the association constant to be sufficiently low to give a fairly high free drug concentration in the bloodstream over a relatively long time period.

Koss et al. 285 measured the binding of radiolabelled acetaminophen onto serum protein using a Sephadex filtration technique and found that about 18% of the acetaminophen is bound to the serum albumin over a wide acetaminophen concentration range. amount of acetaminophen bound to the plasma proteins as about 25%.

Hartshorn 349 quotes the

8.3 Interactions with Other Drug Substances

The analgesic activity of acetaminophen has been claimed to be enhanced by the co-administration of a number of other analgesics and

521,322,350-356, harmacologically active drug substances2t285 I 300,

Levy and Yamada300 examined the effects of salicylamide on the pharmacokinetics of acetaminophen, showing that salicylamide retards the excretion rate of acetaminophen conjugates. This was shown to be accompanied by a competitive in- hibition of the formation of acetaminophen and salicylamide conjugates in the blood implying increased therapeutic availability of free acetaminophen.

85

JOHN E. FAIRBROTHER

Niwa and N a k a ~ a m a ~ ~ l found that acetaminophen and ant ipyrine (phenazone) mutually in- hibit the metabolism of each other in the rat and rabbit and showed that penetration of acetaminophen and antipyrine through excised intestine is mutually inhibted by the other drug substance.

Heald and Evans 322 determined the effect of antipyrine (as acetaminophen-antipyrine complex) on the plasma level of free acetaminophen in man (10 subjects). Their results suggest that antipyrine prolongs the peak plasma level of free acetaminophen.

Acetaminophen has been re orted to be anta onistic to a number of drugs3251 349 350,353 3551361. It has also been reported to show synergism of anti-inflammator c iv wi h other anti-inflammatory drugs to 367.

3 5 4 t o st8 an$ 362

8.4 Biopharmaceutics

Assessment of the bioavailability of acetaminophen has been made using both in vitro measurement of dissolution rate and in vivo pharmacokinetic methods.

Goldberg, Gibaldi and Kanig66 used dissolution rate measurement to evaluate the potential increase in the bioavailability of acetaminophen after fusing it with urea to form eutectic mixtures. Lach and CohenlOO carried out similar studies employing alpha and beta cyclodextrins to increase the dissolution rate of acetaminophen (see also Section 3).

Many authors have used the measurement of acetaminophen plasma levels and/or urinary levels to demonstrate ‘ts bioavailability. Mattok and co-~orkers~~r 339 I 345 have used both in vivo and in vitro procedures and attempted to correlate the results. under the acetaminophen plasma concentration vs. time curvesI to estimate the comparative systemic

Levy133 used the areas

86

ACETAMINOPHEN

availabilities of acetaminophen when administered orally as such and when administered as acetophenetidin (phenacetin) .

75,339,345 used Mattok and co-workers these techniques to compare the bioavailability of acetaminophen in eight commercial tablet formulations, a formulated elixir and a simple laboratory prepared solution and showed no significant differences between them. Gwilt et a1.336 examined the plasma levels given by seven acetaminophen formulations and later also examined an eighth formulation containing acetaminophen and sorbitol. The plasma levels of free acetaminophen.given by this acetaminophen-sorbitol formulation were significantly higher than those given by the seven other formulations.

Sorbitol was claimed336 to potentiate the absorption of acetaminophen and thus is claimed to enhance the antipyretic and analgesic effects of the drug96~97198.

Walter s 385 has critically examined these claims using in vitro methods and concludes that sorbitol does not form an absorbable complex with acetaminophen and that the enhanced activity of acetaminophen in tablets containing sorbitol may result solely from the improved dissolution rate.

Bloor and Morrison455 examined the effects of solubilization of acetaminophen by Tween4' (a polyoxyethylene sorbitan monopalm- itate) on its rate of diffusion.

Carlo et al. 234 examined the effect on bioavailability obtained by formulating acetam-. inophen in an effervescent tablet form. The effervescent formulation gave higher and more rapidly attained plasma concentrations of acetaminophen than an ordinary non-effervescent formulation but did not maintain plasma levels as efficiently as the latter. Bru452 makes similar

87

JOHN E. FAIRBROTHER

claims.

The effect of vehicle composition on the rectal absorption of acetaminophen from su pository formulations has been examined6812981 455.

Incorporation of enzymes having hyalur- onidase and chrondrosulphatase activity into acetaminophen formulations has been claimed to enhance acetaminophen absorption from and rectally administered dosage forms 371~372~3731374. These claims were later refu- ted in a study by Brustier et a1.375.

3sb!?3?6;1 ly

The administration of acetaminophen by percutaneous absorption from s lution in dialkyl sulphoxides has been reported 376 . Modification of drug availability can be effected by formulation as a sustained release (timed-release) dosage form. Several such formulations have been described for acetaminophen377 I 3781 379 1380 381,382. formulation of the drug in a microencapsulated form. This method of presentation has also been used to mask the taste of acetaminophen383138~.

Timed release may also be effected by

9. Toxicity

The acute and chronic oral toxicity of acetaminophen in man and animals has been well reported3~8r67~386 to 398. Overdosin a e - amino hen can cause hepatic necrosis 317,996,s92 I 3961 go 405 and also in a few cases of heav overdosin 414 421 91 I 124,304 ,%b:s9k:?f!$? fff5e%s4%ve been discussed

renal insufficiency3181347 I 386,396 1x06 to

88

ACETAMINOPHEN

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Aftalion H., K e i m N. and Sterescu M., Rev. Chim. (Bucha res t ) 11, 49 (1960) Kalinmska Z x . and Hasztar H., Farm. Pol. 23 (5-6), 447-450 (1967)

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196.

197. 198.

199.

200. 201.

202. 203.

204. 205.

206.

207.

208.

209.

210.

211.

212.

213.

214.

215.

216. 217. 218.

219. 220.

H u n t J. , Fthcdes H.J. and B l a k e M.I . , Can. J. Phann. Sci. - 6 (11, 20-21 (1971) Lament 0. , J. P h m . Belg. 25, 157-159 (1970) N a s h R.A., Skauen D.M. and G d y W.C., J. Am. Pharm. Assoc. 47, 433-435 (1958) G a y l o r V.F. , C o n r a d A.L. and Lander1 J.H., Anal. Chem. 29, 224 (1957) EirockeE G. , Pharmazie 20 (3), 136-140 (1965) puar M.S. and m e P.T= sq-uibb private C c m m i c a t ion Wers A.R., J. Pharm. Phannacol. 17, 325 (1965) N a t i o n a l Fomlary, 12th Ed.. , AmerEan Pharma- ceutical A s s o c i a t i o n , Washhgton, D.C., 1965,

Hoyland D. , Squibb Private C a m n u n i c a t i o n

65-78 (1959) Fauth J.I., Shane N.A., Arrendondo E.G. and Paul W.D., C l h . Chm. 14 ( 9 ) , 882-889 (1968) DFbbem H.W. and S z o l z G., Arch. Pharm. -1 298 175-184 (1965) Koshy K.T. and Lach J.L., Drug Standards, 28, 53-56 (1960) K o s h y K.T. and Lach J.L., Drug Standards, 2, 85-87 (1960) De Fabrizio F., J. Phann. Sci. - 57 (4) , 644-645 (1968) Stree t H.V. and N i y o g i S.K., N a t u r e , - 190, 537, 718 and 1199 (1961) Street H.V. and N i y c g i S.K. , Analyst, 86, 671-673 (1961) Koshy K.T. , J. Phann. Sci. 53 (10) , 1280-1282 (1964) Levhe J. and Hohruann J.R. , J. Assoc. O f f i c . Anal. C h e m i s t s 49 (3), 533-536 (1966) Hohmnn JX., J. A s s o c . O f f i c . Anal. C h e m i s t s - 53 (3), 591-594 (1970)

V a l a t i n P., Squibb Private Comunication V a l a t i n P., Squibb Private C a m n u n i c a t i o n Ivashkiv E., and V a l a t i n P., Squibb Private Comroylicatian Ivashkiv E. , Squibb Private C o m n u n i c a t i o n Ivashkiv E., Squibb Private C o m n u n i c a t i o n

pp. 1e12.

mli E. and win E.1 Ph-. ACta HelV. - 34.

-

98

ACETAMINOPHEN

221.

222.

223.

224.

225. 226.

227.

228.

229.

230.

231.

232.

233.

234.

235. 236.

237.

238. 239.

240. 241. 242.

243.

244. 245.

Hamiltm J.L., J. A s s o c . Offic. Anal. Chemists 54 ( 4 ) , 895-899 (1971) Levine J., J. Am. Pharm. ASSOC., Sci. a. - 46, 687 (1957) Brit ish Pharmacopoeia, 1963, Pharrn. Press, London, p. 558. Bri t i sh Pharmacopoeia, 1963, Addendum 1964, Pharm. Press, London, p. 55-56. Horn D. , Pharm. Zentralhalle, 90, 296 (1951) Rosenthaler L., Pharm. A c t a Helv. 25, 365-367 (1950) I n a d a r M.C. and K a j i N . N . , Indian J. Pharm. - 3 1 (3) I 79-81 (1969) Haneg-raaff C. and Chastagner N. , Ann. P h m . Fr. - 27 (U) , 663-672 (1969) Chafetz L., Daly R.E., Schriftman H. and Lcanner J.J., J. Pharm. Sci. 60 (3) , 463-466 (1971) Belotsvetov A.V. , Gluzdcov V.A. and Larina M.K., Zh. Obshch. Khh. 36 (7) , 1198-1201 (1966) L a r i n a M.K., BelotGetov A.V. , Zh. Obshch. K h h . - 37 (12), 2664-2699 (1967) Belotsvetov A.V., Ehina T.F. and Larina M.K., Zh. Obshch. K h h . 36 (7) , 1202-1204 (1966) G w i l t J .R. , Robertson A. and PkChesney E.W., J. Pharm. Pharmacol. 15, 440-444 (1963) Car10 P.E., CambosG N.M., Feeney G.C. and Smith P.K., J. Am. P h m . AsSOC., Sci. Ed., - 44 (7) 396-399 (1955) Kos J. , Farm. Glasnik 22 (2) , 51-53 (1966) Mouton M.M. and Mason K, Ann Pharm. Fr. 18 (10) I

759-762 (1960) Heirwegh K.P.M. and Fevery J., C l i n . Chem. 2 (31, 215-219 (1967) Ivashkiv E. , Squibb Private Cormmication D'Sowa A.A. and Shenoy K.G., Can. J. Pharm. Sci. 3 (4), 90-92 (1968) vaughan J .B. , J. Pharm. Sci. 58 (41, 469-470 (1969) Mhllin H. , Pharmazie 22 (1) ,27-29 (1967) Dobas I., &&ba V. m-VerEefa M. , Chem. Ind. (London) 1968 (51), 1814 Deut. Reich. Patent 146,265 (Nov. 9, 1903) t o Dahl and Co. in Barmen Pa ten t , Swiss 286,500 (Feb. 16, 1953) t o C i b a Ltd. Patent, G e r . (East) 12,865 (Mar. 5, 1957) to VEB Fabenfabrik Wolfen

-

-

99

JOHN E. FAIRBROTHER

246.

247.

248. 249. 250. 251.

252. 253. 254.

255.

256.

257. 258.

259.

260.

261. 262.

263.

264. 265.

266.

267. 268.

269. 270.

271.

Tatsuo Kondo and Iwao Kawashiro,. Shokuhin Eiseigaku Zasshi, 6 (51, 433-436 (1965) Inoue Tekio, Tatsuzawa Masayoshi, Hojo Mash and Okawara Akira, E i s e i Kagaku, 16 (11, 24-27 (1970) Masuo umda, Yakugaku Zasshi, 84 (9), 836-838 (1964) Masuo Umda, Yakugaku Zasshi, 84 (9) , 839-845 (1964) Easu A.P., Indian J. Pharm., g ( 1 2 ) , 280 (1968) Welch R.M. and C m e y A.H. , C l i n . Chm. - 11 (12) , 1064-1067 (1965) Hoyland D., Squibb Private Ccarmunication Murfin J .W. , Analyst 2, 663-669 (1972) Mwfh J .W. and Wragg J.S., Analyst 97, 670-675 (1972) ~eruo Ninaniya, yakugaku Zasshi - 85 (51, 394-399 (1965) Hiroshi Sakurai and Masuo m a , Yakugaku Zasshi 82, 1282-1286 (1962) Gsuo Umeda, Yakugaku Zasshi 83, 951-956 (1963) Shinsaku Imashuku and La Brosz E.H., C l i n . Chem. - 17 (2), 122-124 (1971) Fairbrother J.E. and Johnson B.A., Squibb Private Ccmrmnication Datta D.D. and Ghosh D. , Indian J. Phm. - 28 (51, 133-134 (1966) C i e r i U.R., J. Phm. Sci. 58 (12), 1532-1535 (1969) Shun-Ichi Naito and Kazuo F&i, J. Phm. Sci. - 58 (lo), 1217-1220 (1969) Blich H., Pfleger K. and Rildiger W. , 2. Klin. Chm. - 5 (3) , 110-114 (1967) Goenechea S., 2. Klin. Chm. 7 (4), 346-349 (1969) Cumnings A. J. , King M.L. , and-hartin B.K. , Brit. J. Pharmacol. Chemther. 29 (21, 150-157 (1967) Juichim Shibasaki, Etsuko Sadakane, Ryoji Konishi and Tarmtsu Koizuni, Chm. Pharm. Bull. 2 (U), 2340-2343 (1970) Shand S., Squibb Private Camnulication Stahl E., "Thin Layer Chranatography" Springer, Berlin-Gdttingm-Heidelberg, 1965. Zarnack J. and Pfeifer S., Pharmazie 19, 216 (1964) glich H., Hauser H., Pfleger K. and Rmger W., 2. Klin. Chgn. 4 (6), 288-290 (1966) Lindberg N.OT, Aktiebolaget Draco, Lund, Sweden, Private Comnmication

100

ACETAMINOPHEN

272. 273.

274.

275. 276.

277.

278.

279.

280.

281.

282.

283.

284.

285.

286.

287.

288.

289.

290.

291.

Nieminen E. , Bull. Narcot. 23 (l), 23-28 (1971) McMartin C. and street H.V.,J. Chromatog. - 22 (21, 274-285 (1966) Alber L.L. and Overton M.W., J. Ass . Offic. Anal. Chm. 54 (3) , 620-624 (1971) Grove TI J. Chranatog. 59 (2), 289-295 (1971) Ryabtseva I .M., Kuleshom-M.I., Rudenko B.A. and Kucherov V.F., Izv. Akad. Nauk SSSR, S e r . Khim. (12) I 2676-2680 (1970) Prescott L.F., J. Pharm. Pharmacol - 23, 807-808 (1971) Masanobu Koibuchi , Toshio Shibazaki, Tsutanori Minamikawa and Yukio Nishimra, Yakugaku Zasshi - 88 (7), 877-881 (1968) Kipping S.A.B., The Boots C m y Ltd. , Nottingham, Private Cammication Presoott L.F., J. Pharm. Pharmacol. 23 (21, 111-115 (1971)

- Thanas B.H. and Coldwell B.B., J. Pharm. Pharmacol. - 24, 243 (1972) Ederma H.M., Skerpac J., Cotty V.F. and Sterbenz F., Automation in Analytical Chemistry, Technicon Symposium, 1966, p. 288-231. Ederrna H.M. , Cotty V.F. and M e m K.J., Advan. AutcaMt. Anal. , Technicon Int. Congr. (Chicago) 1969 (pub. 1970), 2, 179-181 Davison C. , Guy J.E., Levitt M. and Smith P.K., J. Phamacol. Eqtl. Therap. 134, 176-183 (1961) Koss F.W., Mayer D. , Haindn. and Kabbara T. , Arzneh.-Forsch. - 20 (9) , 1218-1222 (1970) Reikhsfel'd V.O., Tr. Konf. po Probl. Primeneniya Korrelyatsion. Uravnenii v Organ. Khim., Tartusk. Gos. Univ., Tartu (1962) (11, 214-249 Reikhsfel'd V.O. , Prokhorova V.A. and Pmia V.A., Kinetika i Kataliz 6 (11, 171-176 (1965) Reikhsfel'd V.O. a d Korol'ko V.V. , Reaksionnaya Sposobnost. Organ. Scedin., Tartusk. Gos. Univ. - 2, (2) , 77-87 (1965) Reikhsfel'd V.O. and Prokhorova V.A., Zh. Obshch. K h h . 35 (10) I 1826-1829 (1965) ReikhsGl'd V.O. , Intern. Symp. Organosilicon Chen. Sci. Comnun., Suppl., Prague (1965) 34-40 Reikhsfel'd V.O., Khim. Prakt. Primen. Krerrmiiorg. Win., Tr. Sovesch. (19661, 7-23

101

JOHN E. FAIRBROTHER

292.

293.

294.

295.

296.

297.

298.

299.

300.

301.

302.

303.

304.

305.

306.

307.

308. 309.

310.

311. 312.

313.

Prescott L.F., S teel R.F. and Ferr ie r W.R., C l i n . Pharmac01. Ther. 11 (4) I 496-504 (1970) Burtis C.A. and Wzren K.S., C l i n . Chem. 14 (4) , 2 9 e 3 0 1 (1968) H e n r y R.A. and Schnit J .A. , C h r c m a t c g r a p h i a - 3, 116- 120 (1970) Anders M.W. and Latorre J.P. , J. C h r o m t o g . - 55, 409-413 (1971) Stevenson R.L. and Burtis C.A., J. ChrcaMtog. - 61, 253-261 (1971) Jagenburg R., Nagy A. and RcMjer S., Scand. J. C l i n . Lab. Invest. 22 (11, 11-16 (1968) Hobel M. and TalebiK M. , Arzneimittel - Forsch. - 10, 653-656 (1960) Juichiro Shibasaki, R y o j i Konishi, Yoshinori T a k e d a and T m t s u Koizumi, Chem. P h m . Bul l . (Tokyo) - 1 9 (9), 1800-1808 (1971) Levy G. and Y m d a H. , J. Pharm. Sci. 60 (2) , 215-221 (1971) B r a t t o n A.C., Marshall E.K., Babbitt D. and Hendrickson A.R., J. B i o l . Chem. 128, 537 (1939) Lower G.M. , Murphy S.B. and B r y a n T T . , C l i n . Chim. A c t a . 29 (31, 421-427 (1970) Tonpsez S.L. , Ann. C l i n . Biochem. - 6, (41, 81-82 (1969) Welch R.M., Conney A.H. and Bums J.J., Biochem. Pharrnacol. 15 (5), 521-531 (1966) Jagenbmg O x . and Toczko K. , Biochem. J., 92, 639- 643 (1964) W i l t J .R. , R o b e r t s o n A. and McChesney E.W., J. Pharm. Pharmacol. 15, 440-444 (1963) V e s t M.F. , S t r e i f f y . R . , A.M.A. J. D i s e a s e s of C h i l d r e n , 98, 688-693 (1959) Ivashkiv E T Squibb Private C c m m n i c a t i o n Brodie B.B., Udenfriend S. and Baer J .C. , J. B i o l . Chem. 168, 299 (1947) Shibasaki J., K o i z u n i T. , Tanaka T. and N a k a t a n i M., Chem. B u l l (Tokyo) 16, 1726 (1968) N e r y R., Biochem. JY - 122 (3) , 317-326 (1971) Levy G. and -&dh C.- G., J. P h m . Sci. 9, (4) 608-611 (1971) Hart'umr C.H., and P r e l l w i t z W., K l i n . Wochenschr . - 44 (17) I 1010-1014 (1966)

-

102

ACETAMINOPHEN

314.

315.

316.

317.

318.

319.

320.

321.

322.

323.

324.

325.

326.

327.

328.

329.

330.

331.

332. 333.

334. 335.

V e s t M.F. and Fr i tz E., J. C l i n . Pathol. 14, 482 (1961)

Careddu P. , Mereu T. and Apolloni T. , Minerva Pedi& . 13, 1619-1622 (1961) Every J. and D e G r o o t e J. , Acta Hepato-Splenol. - 16 (1) , 11-18 (1969) F’rescott L.F., Roscoe P., Wright N. and Brown S.S., Lancet (1971) , 519-522 Dubach U.C. I K l h . Wochenschr. 46 (5) I 261-264 (1968) Levy G. and Yamada H., J. Pharm. Pharmacol. 22, 964-965 (1970) Buech H., R m l W., Pfleger K., Eschrich C. and T e x t e r N. , Naunyn-Schmiedebergs Arch. Pharmakol. Exp. Pathol. 259 (31, 276-289 (1968) Niwa H. and N Z y m T. , Yakugaku Zasshi, - 88 (5) , 542-548 (1968) Heald A.F. and Evans R. , Squibb Private Ccarmunic- at ion V e s t M.F., Schweiz.med. hbchenschr. 89, 102-105 (1959)

Careddu P., Mereu T. and Apollonio T., Boll. SOC.

i t a l . biol. sper. 37, 359-363 (1961) Careddu P. , Pacenizereni L. , Apollonio T. and Mereu T. , Minerva Pediat. 14 (40), 1047-1049 (1962) Mereu T. , Apollonio T. , PaGi-Sereni L. and Careddu P., Lancet (1962 - I) 1300 V e s t M.F. and Rossier R. , Ann. N.Y. Acad. Sci. - 111 (1) , 183-197 (1963) Clark B.B., Symposium on N-acetyl-p-minophenol. p. 23-34, Ins t i tu te for the Study of Analgesic and Sedative D r u g s , Elkhart, 1951 Klutch A., Harfenist M. and Conney A.H., J. Med. Chem. 9 , 63 (1966) Bray HTG. , Thorpe W.V. and White K. , Biochem. J. , - 52, 423-430 (1952) Inscoe J.K., Daly J. and Axelrod J., Biochem. Ph-col. - 14 (8) I 1257-1263 (1965) Axelrod J., Science 140 (3566) , 499-500 (1963) Wbinson D. and W i l l 5 R.T. , Biochem. J., 68, 23P (1958) Borrell S., Biochem. J., 70, 727 (1958) Dutton G. J. and Greig C . G Y Biochem. J. , 66, 52P (1957)

-

-

-

-

103

JOHN E. FAIREROTHER

336.

337.

338.

339.

340. 341.

342.

343. 344.

345.

346.

347.

348.

349.

350.

351.

352. 353.

354.

355.

356.

357.

G w i l t J .R. , Robertson A., Go- L. and Blanchard A.W., J. Phann. Pharmacol. 15, 445-53 (1963) Ja f f e J .M. , Colaizzi J.L. a Barry H. , J. Pharm. Sci. 60, (ll), 1646-1650 (1971) WeikerJ.H. and Lish P.M., Arch. In t . Phanradyn. "her. 119, 398-408 (1959) McGilv=y I.M. , Mattok G.L., Fooks J .R. , Jordan N. and Cook D., Can. J. P h m . Sci. 5 (2), 38-42 (1971) Stricker H., Pharm. Ind. 31 (111, 794-799 (1969) Dearden J.C. and Tcsnlinsm-E., J. Pharm. Pharmacol. - 23, Suppl., 68s-72s (1971) Dearden J.C. and Tcanlinson E., J. Phann. Pharmacol. - 23, Suppl., 73s-76s (1971) Salzn?ann K., Therapiemche - 12, 1034 (1962) D i t t e r t L.W. and Adams H.J . , J. Phann. Sci. 2, 1269 (1968) Mattok G.L., W i l v e r a y I .M. and Cook D., Can. J. Pharm. Sci. - 6 (21, 35-38 (1971) Wikel J .H . , J. Am. Pharm. Assoc., Sci. Ed. 2, 477-479 (1958)

Prescott L.F., Sansur M., Levin W., Conney A.H., C l i n . Pharmacol. Therap. 9, 605-614 (1968) N e l s o n E. and Morioka T., J. Pharm. Sci. 52. (91, 864-868 (1963) H a r t s h o r n E.A., Drug Intell igence and C l i n i c a l Pharmacy 6, 50-54 (1972) Grotto M., D f i s t e h S. and Sulman F.G., Arch. Int. Pharmacody~. 155 (2), 365-372 (1965) Takagi K., Tajiii-pagi I., Kayaoka S., Okabe S. , Shigenobu K. , Fukao T., Kawashh K. and Taga F., Yakugaku Zasshi 88 (61, 779-783 (1968) Grosto M., Harefa 73 (31, 90-93 (1967) Boreus L.O. and S&g F., A c t a Physiol. Scand. - 28, 266-271 (1953) Botha D., Mueller F.O., Krueger F.G.M., Melnitzky H., Vemak L. and buw L., Em. J. Pharmacol. 6 (3), 312-321 (1969) Grotto M., Harokeach H a i v r i 11, 152/172-157/167 (1965)

- Patent, U.S., 3,439,094 (15 Apr. 1969) to Wle J . F . (Warner-Lambert Pharmaceutical Co . ) Patent, U.S. 3,482,021 (2 D e c . 1969) t o Gosling R.H. (Geigy Chemical Corp.)

104

ACETAMINOPHEN

358.

359.

360.

361.

362.

363.

364.

365.

366.

367.

368.

369.

370.

371. 372. 373.

374.

375.

376.

377.

378.

Patent, Ger. Offen. 2,058,893 (9 Jun. 1971) to Wilhelmi G. (CIBA-Geigy A<) Lechat.P., Delaeu D. and Bunot O., Therapie - 20 (41, 867-877 (1965) Lechat P., Delaeu D., Fontagne J. and m o t O., Therapie, - 21 (31, 565-570 (1966) Bush H., Gerhards W., Pfleger K., Rueaiger W. and Ihnmrel W., Biochan. Pharmacol. - 16 (81, 1585-1599 (1967) Patent, B r i t . 901,674 (25 July, 1962) t o Johnson W.J . (F.W. Homer Ltd.) Patent, U.S. 3,053,737 (Sept. 11, 1962) to Johnson W. J. Corte G. and Johnson W . J . , Proc. Soc. Exptl. B i o l . W. 97 751-755 (1958) MacK&ie D.H.H. and smythe H.A., Can. Conf. Res. Rhemat. Diseases, 2nd., Toronto - 1960, 148-149 (Pub. 1961). Levillain R., Cluzan R., David G. and Cayeux Ph., Therapie 2 (21, 539-548 (1965) Weichselbaum T.E. and Margraf H.W., Proc. Soc. Exptl. B i o l . Med. 107, 128-131 (1961) Niwa H. and Nakaym-T., Yakugaku Zasshi 88 (71, 843-846 (1968) Patent, Belg. 668,418 (Dec. 16, 1965) t o Riviere J. (Laboratoire Solac) Patent. Fr . M 4,196 (July 4, 1966) t o Riviere J. (Laboratoire Solac) Patent, Fr. M 4,825 (Mar. 20,1967) to Riviere J. Patent, Fr. M 4,519 (Nov. 21,1966) to Riviere J. Patent, F'r. M 5,575 (Jan. 2,1968) to C e n t r e d'Etudes pour 1 ' Industr ie Pharmaceut ique Patent, Fr. M 5,022 (May 29, 1967) t o C e n t r e d ' Etudes pur 1'Industrie Pharmaceutique Brustier V., Aubert D., Amselem A., Gazz. Int . Med. Chir. 74 (41, 357-367 (1969) Patent, Bric 1,043,104 (Sept. 21, 1966) t o Senior N. and Madinaveitia J.L. (Imperial Chemical Industries Ltd. ) Patent, U.S. 3,133,863 (May 19, 1964) t o Tansey R.P. (Strong Cobb A m e r Inc. ) Patent, Brit . 1,019,146 (Feb. 2, 1966) t o Hermelin V.M.

105

JOHN E. FAIRBROTHER

379. Patent, B r i t . 1,021,924 (Mar. 6, 1966) to W t h ,

380. Patent, N e t h . Appl. 6,512,130 (Mar. 21, 1966) t o

381. Patent, U.S. 3,279,998 (Oct . 18, 1966) to Raff A.M.

K l i n e and French Laboratories

Gaunt W.E.

and Svedres E.V. (Smith, Klhe and French Laboratories)

M. (Key Pharmaceuticals Inc.)

P-11 T.C. and Anderson J.L. (National Cash Register Co.)

384. Bakan J.A. and Sloan F.D., D r u g and Cosmetic Industry, March 1972, 34-38, 902, 9OD, 117-121

385. Walters V., J. Pharm. Pharmacol. - 20, Suppl. 228s- 231s (1968)

386. Ebyer T. and muff S., J. Am. Med. A s s o c . - 218, 440-441 (1971)

387. Boxill G.C., Nash C.B. and Wheeler A.G., J. Am. Pharm. ASSOC., Sci. Ed. 47, 479-487 (1958)

388. Renault H., Rohrbach P. and D u g n i o l l e J., Therapie 11, 3e306 (1956)

389. z t c h f i e l d J.T. and Wilcoxon F., J. Phannacol. Exptl. Therap. 96, 99 (1949)

390. Burn J.H. I Finn= D.J . and Goodwin L.G., "Biological Standardisation", 1950, Oxford Press, London, p.114.

391. S t m r G.A., McLean S. and Thorns J., 'Ibxicol. Awl. P h m c o l . 19 (l), 20-28 (1971)

392. Manolov P., Suwemenna Med. 14 (101, 33-37 (1963) 393. Miller M.M., Squibb Private C&munication 394. Boyd E.M. and Bereczky G.M., Brit. J. Pharmacol.

Chamtherapy - 26 (31, 606-614 (1966) 395. Swinyard E.A., Brown W.C. and Goo&nan L.S., J.

Pharmacol. Exptl. Therap. 106,319 (1952) 396. Boyd E.M. and H o g a n S.E., can. J. Physiol. Pharmacol.

397. &d E.M., J. C l i n . P h m c o l . 10 (4), 222-227 (1970) 398. Boys E.M. I C l h . P-ml. 4 (2), 205-213 (1971) 399. Dimn M.F., N i ~ m J. and Prezcott L.F., J. Pathol.

400. E i d s m D.G.D. and Eastham W.N., B r i t . M e d . J.

401. Thomson J.S. and Prescott L.F., B r i t . Med. J.

382. Patent, Brit. 1,125,882 (Sept. 5, 1968) t o Shepard

383. Patent, Ger. Offen. 1,917,930 (Nov. 6, 1969) to

46 (2), 239-245 (1968) ,

103, 225-229 (1971)

(19661, g, 497

(1966),g, 506

106

ACETAMINOPHEN

402.

403.

404. 405.

406.

407. 408.

409.

410.

411.

412.

413.

414.

415.

416. 417.

418.

419.

420.

421.

422.

423.

424.

Pimstone B.L. and Uys C.J., S. Afr. M e d . J., 42, 259 (1968) Maclean D. , P e t e r s T.J . , Brown R.A.G. , mathie M., mines G.F. , Robertson P.G.C., Lancet (19681, 849 Rose P.G. , Brit. Med. J. , (19691, i,381 Toghill P.J., W i l l i a m s R. , Stepheng J.D. and C a r r o l l J . D . , Gastroenterology 56, 773 (1969) P r e s c o t t L.F., J. P h a n n ~ P h a r m c o l . 18, 331-353 (1966) Prescott L.F., Lancet (19651, ii, 91-95 C a l d e r I.C. , Funder C.C. , G r e e n C.R., Ham K.N. and Tange J .C. , Brit. Med. J. (1971) 4 , 518-521 Smith P.K., Davison C. and Scdd M.A. , (1956) , Proceedings of the International P h y s i o l o g i c a l Congress, B r u s s e l s , p. 836. E i s a l o A. and T a l a n t i S., A c t a Med. Scand. 169, 655-660 (1961) Angervall L. , Lehmnn L. and L i n c o l n K. , A c t a P a t h o l , Microbiol. Scand. 154, 274-282 (1962) Angervall L. , LehsMnn L. a n T L i n c o l n K., A c t a P a t h o l . Microbiol. Scand. 154, 283-286 (1962) Angervall L., LehTMnn L. a n ? L i n c o l n K. , A c t a P a t h o l . Microbiol. Scand. 154, Suppl. 61-63 (1962) Angervall L. , Lem- L. and Bengtsson U. , A c t a Med. S c a d . 175 ( 2 ) , 155-160 (1964) K i e s e M. and-zel H. , Arch. Exptl. P a t h o l . Pharmakol. - 242, 551-554 (1962) Schwerd W., Med. W l t (1962) 2348-2349 Heubne.r W., Arch. Ekptl. P a t h o l . Pharmakol. 72 239-247 (1913) S c h n i t z e r B. and Smith E.B., Arch. Pa thol . 81 ( 3 ) , 264-267 (1966) J3aader H., G i r g i s S., Kiese M., Menzel H. and S k r o h t L., Arch. Expt l . P a t h o l . Pharmakol. 214, 317-334 (1961) L e s t e r D., J. P h a m c o l . Expt l . Therap. - 77, 154-159 (1943) D o l l G. and Hackenthal E., Arzneimittel-Forsch. 13, 68-71 (1963) Bums J.J. and Conney A.H., Proc. Eur. Soc. Study D r u g T o x i c i t y , 6, 76-82 (1965) Dern R.J . , B e u t i e r E. and A l v i n g A.S. , J. Lab. C l i n . Med. 45, 30-39 (1955) C l a u s e n E. and Larsen O.A., A c t a Pharmacol. Toxicol . - 22 ( 2 ) , 135-140 (1965)

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-

-

-

-

107

JOHN E. FAIRBROTHER

425.

426.

427.

428.

429. 430. 431. 432.

433. 434.

435.

436.

437.

438.

439.

440.

441.

442.

443.

444.

445.

446.

b y d M. and Sheppard E.P., B r i t . J. Pharmacol. C h e m t h e r a p y - 27 (21, 497-505 (1966) D i k s t e i n S., G r o t t o M., Zor U., T m r i M. and S u h F.G., J. Endccrinol. 36 (3), 257-262 (1966) Thanas J .M. , Nakaue H.S. and Reid B.L., Poultry Sci. - 46 ( S ) , 1216-1219 (1967) D i k s t e i n S., Zor U., Ruah D. and S u b a n F.G., poultry Sci. 45 ( 4 ) , 744-746 (1966) Lloyd T.W., G c e t (1961), i, 114 Jacobson H., Squibb Private Comnunication C l a r k e G., Squibb Private C m i c a t i o n Jaeckel R. and Peperle W., 2. N a t u r f o r s c h . s, 171-172 (1960) Coy N.H. and Ochs Q., Squibb Private C c g n n u n i c a t i o n Porter M.W. and Spiller R.C., The Barker Index of C r y s t a l s , Vo l . 2, P a r t 2 , 1956, H e f f e r and Sons Ltd. , C a m b r i d g e (M. 1769A and M. 1769B). Chaw Y.P. and Repta A.J . , J. Pharm. Sci. 61 ( 9 ) , 1454-1458 (1972) W i l l i a m s R.T. and Bridges J . W . , J. C l i n . Pathol. - 17, 371-394 (1964) Patent, G e r . ( E a s t ) 81,119 (12 Apr. 1971) to H i n s d o r f G.K. Patent, Ger. O f f e n . 2,121,164 (11 Nov. 1971) to Schulman H.L. , Baron F.A. and Minberg A.E. ( H a l l , Howard and Co.) Jalal I.M., M a l i n w s k i H.J. and Smith W.E., J. P h m . Sci. 61 ( 9 ) , 1466-1468 (1972) Elste U. and Duda H., B u t . Apth.-Ztg. 112 (19) , 711-715 (1972) Shearer C.M., C h r i s t e n s o n K. , Mukherii A. and Paperiello G i J . , J. P h m . Sc i . - 6 1 (iO), 1627-1630 (1972)

D a l y R.E., Mran C. and C h a f e t z L., J. Pharm. Sci. - 61 ( 6 ) , 927-929 (1972) Dedicoat H. and Symnds D.C., J. Assoc. Publ. Anal. - 10, 14-17 (1972) Fales H.M., Milne G.W.A. and Law N.C., Arch. Mass Spectral Data 2 ( 4 ) , 6-1 (1971) Mihe G.w.A., Fales H.M. and menrod T., -1. C h m . 43 (13), 1815-1820 (1971) Lageoty., L i m u z i n Y. and Maire J.C. , J. Organmtal. Chem. 38, 23-27 (1972)

108

ACETAMINOPHEN

447.

448.

449. 450.

451.

452.

453. 454.

455.

456.

Amsel L.P. and Davison C., J. Pharm. Sci. 61 ( 9 ) , 1474-1475 (1972) Fccella A . , Heslin P. and Teitel S. , Can. J. Chem. c 50, 2025-2030 (1972) Fairbrother J .E. , Squibb Private Comica t ion Patent, G e r . 1,493,727 (31Aug. 1972) t o Koenig C., Schmitz P. and P e l s t e r H. (Bayer A.-G.) Peters G., Baechtold-Fowler N. , Bonjour J., et al., Arch. ToxFkol. 28 ( 4 ) , 225-269 (1972) Patent, Fr. m-de 2,092, 893 (3 Mar. 1972) t o Bru J. R.P. Scherer Ltd. , Capsule News lo ( 2 ) , 3 (1972) McGilveray I.J. and Mattok G.L., J. Pharm. Pharmacol - 24, 615-619 (1972) Blmr J.R. and Mrrison J.C., J. Phann. Pharmacol. - 24, 927-933 (1972) Patent, B r i t . 1,287,431 (31 Aug. 1972) t o Lakenorris W. and Slater J.E. (Aspro-Nicholas Ltd. )

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This profile a t t q t s t o cover the published l i terature on acetaminophen up to Chemical Abstracts, V o l m 77, Issue 21

109

dl-ALPHA-TOCOPHERYL ACETATE

Bruce C. Rudy and Bernard 2. Senkowski

BRUCE C. RUDY AND BERNARD 2. SENKOWSKI

1.

2.

3.

4 .

5 .

6.

7 .

8.

INDEX

A n a l y t i c a l P r o f i l e - dl-Alpha-Tocopheryl Acetate

D e s c r i p t i o n 1.1 Name, Formula, Molecular Weight 1 . 2 Appearance, Color , Odor 1 . 3 I somer ic Forms

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 2 . 2 Nuclear Magnetic Resonance Spectrum 2.3 U l t r a v i o l e t Spectrum 2.4 Mass Spectrum 2.5 O p t i c a l R o t a t i o n 2.6 Mel t ing Range 2.7 D i f f e r e n t i a l Scanning Calor imet ry 2.8 Thermal Gravimetr ic Analys is

S y n t h e s i s

S t a b i l i t y Degradat ion

Drug Metabol ic Products

Methods of Analys is 6 .1 Elemental Analys is 6.2 Thin Layer Chromatographic Analys is 6.3 Gas-Liquid Chromatographic Analys is 6.4 Direct Spectrophotometr ic Analys is 6.5 C o l o r i m e t r i c Analys is 6 .6 S p e c t r o f l u o r o m e t r i c Analys is 6.7 T i t r i m e t r i c Analys is

Acknowledgements

References

112


1. Description

1.1 Name, Formula, Molecular Weight dl-Alpha-tocopheryl acetate is a racemic mixture

of 2,5,7,8-tetramethy1-2-(4',8',12'-trimethy1tridecy1)-6- chromarol acetate.

Molecular Weight: 472.76 '31'52'3

1.2 Appearance, Color, Odor

nearly odorless, clear, viscous oil. dl-Alpha-tocopheryl acetate occurs as a yellow,

1.3 Isomeric Forms There are four possible enantiomeric pairs of

diastereoisomers which result from the three asymmetric centers present in the alpha-tocopheryl acetate molecule (the asymmetric centers are marked with a small circle in the above structural formula). dl-Alpha-tocopheryl acetate contains an equimolar mixture of the eight isomers.


2 - 1 Infrared Spectrum The infrared spectrum of dl-alpha-tocopheryl

acetate is presented in Figure 1 (1). The spectrum was measured with a Perkin-Elmer 621 Spectrophotometer on a capillary layer of the liquid between KBr discs. The assignments for the characteristic bands in the infrared spectrum are listed in Table I (1).

Table I

Infrared Assignments for dl-Alpha-Tocopheryl Acetate

Frequency (cm-1) Characteristic of

2943 and 2861 CH3 stretching vibrations

113

PI U

rd L)

Ll L)

M a CJY

a

al h

rd Ll w

C

H

33

NV

lllWS

NW

l K

8 OD

0

0-

-

-

114

dl-ALPHA-TOCOPHERY L ACETATE

2920 and 2850 CH2 s t r e t c h i n g v i b r a t i o n s 1752 C=O s t r e t c h i n g v i b r a t i o n s 1456 CH2 and CH3 de fo rma t ions 1363 CH3 symmetric de fama t ions 1206 C-0-C s t r e t c h i n g v i b r a t i o n s

2.2 Nuclear Magnetic Resonance Spectrum (NMR) The spectrum shown i n F i g u r e 2 w a s o b t a i n e d on a

J e o l c o 60 MHz NMR by d i s s o l v i n g 92.4 mg of dl-alpha- t ocophery l acetate i n 0 . 5 m l of C D C 1 3 c o n t a i n i n g tetra- m e t h y l s i l a n e as a n i n t e r n a l r e f e r e n c e ( 2 ) . The s p e c t r a l a s s ignmen t s are g i v e n i n Tab le I1 ( 2 ) .

Tab le I1

NMR Assignments € o r dl-Alpha-Tocopheryl Acetate '

Number of Chemical S h i f t P ro ton P ro tons (ppm) Mu1 t i p l i c i t y

a 12 0.85 Doublet (Ja-d = 5 HZ) b 3 1 . 2 1 S i n g l e t

e 2 1 . 7 5 T r i p l e t (Je-h = 6.8 Hz) f 9 1 .96,2.00,2.07 Three Overlapping singlets g 3 2.29 S i n g l e t h 2 2.57 T r i p l e t (Jh-e = 6 .8 Hz)

c&d 21 1.0-1.6 Complex M u l t i p l e t

2 .3 U l t r a v i o l e t Spectrum (UV) When t h e UV spectrum of d l - a lpha - tocophery l

a c e t a t e w a s scanned from 350 t o 220 n m , two maxima and two minima were observed. One maximum is l o c a t e d a t 285 n m (E = 2.24 x l o 3 ) w i t h a s h o u l d e r a t 287 nm ( E = 2 .21 x l o 3 ) and t h e o t h e r maximum o c c u r s a t 279 n m (E = 1 .98 x l o 3 ) . The minima are l o c a t e d a t 281 nm and 253 nm. The spectrum shown i n F i g u r e 3 w a s o b t a i n e d from a s o l u t i o n of 12.411 mg of d l - a lpha - tocophery l acetate p e r 100 m l of cyclohexane(3).

115


F i g u r e 2

NMR Spectrum of dl-Alpha-Tocopheryl Acetate

l ~ ~ [ ' ~ ~ ~ l ~ ~ ~ ~ l l ~ l l ~ l l ~ l l , ~

240 180 I20

t

116


.8

.7

.6

; .5- a m a

m a % 4-

.3

.2

.I

Figure 3

Ultraviolet Spectrum of dl-Alpha-Tocopheryl Acetate

-

-

-

-

-

-

0-

1 117

BRUCE C. R U D Y A N D BERNARD 2. SENKOWSKI

2.4 Mass Spectrum The mass spectrum of dl-alpha-tocopheryl a c e t a t e

was obta ined u s i n g a CEC 21-110 mass spec t rometer w i t h a n i o n i z i n g energy of 70 e V . The o u t p u t from t h e mass spec t rometer w a s analyzed and presented i n t h e form of a b a r graph, shown i n F i g u r e 4, by a Var ian 100 MS d e d i c a t e d computer system ( 4 ) . The mass spectrum of dl-alpha- tocophery l acetate i s c h a r a c t e r i z e d by t h e absence of numerous f ragmenta t ion p r o c e s s e s . The molecular i o n peak occurs a t m / e 472. t h e l o s s of ke tene from t h e molecular i o n . Fragmentat ion of t h e non-aromatic r i n g occurs , y i e l d i n g a peak a t m / e 207 (with t h e a c e t a t e group p r e s e n t ) and a peak a t m / e 165 ( a f t e r l o s s of t h e k e t e n e group) . An in-depth a n a l y s i s of t h e mass s p e c t r a of tocophero ls has r e c e n t l y been publ i shed by Scheppele e t a l . (5).

The b a s e peak a t m / e 430 occurs from

2.5 O p t i c a l R o t a t i o n dl-Alpha-tocopheryl acetate i s an equimolar

mixture of t h e o p t i c a l isomers of a lpha-tocopheryl a c e t a t e and t h e r e f o r e e x h i b i t s no o p t i c a l r o t a t i o n .

2.6 Mel t ing Range dl-Alpha-tocopheryl a c e t a t e is an o i l a t room

temperature . It s o l i d i f i e s a t a tempera ture of -27.5OC (6).

2.7 D i f f e r e n t i a l Scanning Calor imetry (DSC) A DSC s c a n w a s run by c o o l i n g t h e head of t h e DSC

which h e l d t h e sample pan c o n t a i n i n g ;he dl-alpha-tocopheryl a c e t a t e t o -9OOC. f o r 30 minutes , t h e tempera ture w a s i n c r e a s e d a t a r a t e of 10°/minute. Only a n extremely weak endothermic t r a n s i t i o n w a s observed s t a r t i n g a t about -44OC. It appears t h a t when dl-alpha-tocopheryl a c e t a t e s o l i d i f i e s i t forms a g l a s s i n s t e a d of a c r y s t a l l i n e m a t e r i a l ( 7 ) .

A f t e r h o l d i n g t h e tempera ture a t -9OOC

2 . 8 Thermogravimetric Analysis (TGA) The TGA of dl-alpha-tocopheryl a c e t a t e i n a

n i t r o g e n atmosphere showed no weight l o s s from ambient t o 210OC. A s i n g l e weight l o s s cor responding t o 100% of t h e sample occurred between 210' and 37OoC ( 7 ) .

118

a, u

(d u

a, U

4

rl h

k

a, F: a

0

U

I .

3R

a

m

hF

:

1a

M

rl

*rl

4

kl

I

rl a

w 0

BRUCE C. RUDY A N D BERNARD Z . SENKOWSKI

3 . S y n t h e s i s

scheme shown i n F igure 5. densed w i t h racemic i s o p h y t o l t o y i e l d dl-alpha-tocopherol which is then a c e t y l a t e d ( 8 ) .

dl-Alpha-tocopheryl a c e t a t e i s prepared by t h e r e a c t i o n Trimethylhydroquinone is con-

4. S t a b i l i t y Degradat ion dl-Alpha-tocopheryl a c e t a t e i s prac t ica l ly u n a f f e c t e d

by t h e o x i d i z i n g i n f l u e n c e of a i r and u l t r a v i o l e t l i g h t ( 6 ) . When i t is r e f l u x e d i n a c i d i c and and b a s i c s o l u t i o n s i n t h e absence of oxygen, t h e molecule is hydrolyzed t o t h e f r e e dl-alpha-tocopherol . I f oxygen is p r e s e n t , t h e d l - a lpha-tocopherol , once formed, w i l l o x i d i z e r a p i d l y t o t h e quinone. The ra te of o x i d a t i o n i s much f a s t e r i n t h e b a s i c s o l u t i o n .

5 . Drug Metabol ic Products dl-Alpha-tocopheryl a c e t a t e i s metabol ized as o u t l i n e d

i n F i g u r e 6 (9, lO). The ester i s r e a d i l y conver ted i n t h e animal t o f r e e a lpha-tocopherol which is then f u r t h e r metabol ized to a lpha-tocopherol quinone and an a lpha- tocopherol dimer. The alpha-tocopherol quinone may b e reduced t o t h e corresponding hydroquinone o r f u r t h e r ox id ized t o a l p h a tocopheronic a c i d (9,10,11).

6. Methods of Analys is

6 .1 Elemental Analys is The resu l t s from t h e e lementa l a n a l y s i s of d l -

a lpha-tocopheryl a c e t a t e a r e l i s t e d below (12).

E l emen t % Theory % Found

C 78.76 78.55 H 11.09 11.05

6.2 Thin Layer Chromatographic Analys is (TLC) A TLC system which can be used t o s e p a r a t e d l -

a lpha-tocopheryl a c e t a t e from i t s major m e t a b o l i t e s i s as fo l lows . The sample is a p p l i e d t o a S i l i c a G e l G p l a t e and s u b j e c t e d t o ascending chromatography u s i n g cyc1ohexane:di- e t h y l e t h e r ( 4 : l ) as t h e developing s o l v e n t . A f t e r t h e s o l v e n t has ascended 10 t o 1 5 c m , t h e p l a t e is a i r d r i e d , sprayed w i t h c o n c e n t r a t e d s u l f u r i c a c i d , and warmed i n an oven a t 105OC f o r 5 minutes . The approximate Rf v a l u e s

120

Figure 5

Synthesis of dl-Alpha-Tocopheryl Acetate

a 3

TRIMETHYLHYDROQUINONE RACEMIC ISOPHYTOL

Acetic Anhydride

C"3

dl - ALPHA-TOCOPHEROL

dl- ALPHA-TOCOPHERYL ACETATE

Figure 6

Metabolic Products of dl-Alpha-Tocopheryl Acetate

c w w

ALPHA-TOCOPHERYL ACETATE

deestwificatian I ALPHA- TOCOPHEROL / (major)

O+

% \


are l i s t e d below (13).

a lpha tocopherol 0.5 alpha tocopheryl acetate 0.7 a lpha tocopherol quinone 0.9

6 . 3 Gas-Liquid Chromatographic Analysis (GLC) A r ecen t c o l l a b o r a t i v e s tudy has shown dl-alpha-

tocopheryl a c e t a t e may r e a d i l y be separa ted and assayed by GLC ( 1 4 ) . The p e r t i n e n t experimental condi t ions as w e l l a s t h e r e t e n t i o n t i m e s a r e given i n Table 111.

Table I11

GLC Method f o r dl-Alpha-Tocopheryl Acetate

Column: 4-8 f e e t , 2-3 mm i . d . , Pyrex

Support: S i l a n i z e d Diatomaceous Ea r th S ta t iona ry Phase: 5-10% SE-30 Detector : Hydrogen flame i o n i z a t i o n Temperature (OC)

o r s t a i n l e s s steel

I n j e c t i o n Por t : 300 Column : 280 Detector : 300

Car r i e r Gas Flow Rate: $40 ml/min. of n i t rogen Quan t i t i e s In jec ted : c10 mcg i n n-hexane Retent ion Time (minutes)

a lpha-tocopheryl ac id

alpha-tocopheryl

do t r iacontane

alpha-tocopherol 2 2

suc c ina t e 23

a c e t a t e 26

( i n t e r n a l s tandard) 30

6 . 4 Direct Spectrophotometr ic Analysis Direct spectrophotometry may be c a r r i e d ou t on

dl-alpha-tocopheryl a c e t a t e provided t h e r e a r e no i n t e r - fe rences present . The maxima f o r dl-alpha-tocopheryl a c e t a t e a r e dependent on the choice of so lven t used. I f cyclohexane is used as the so lven t , t h e maximum a t 285 nm may be used f o r q u a n t i t a t i o n .

I23

BRUCE C. RUDY A N D BERNARD 2 . SENKOWSKI

6.5 Color imet r ic Analysis An i n d i r e c t method f o r t h e a n a l y s i s of dl-alpha-

tocophery l a c e t a t e u t i l i z e s t h e Emmerie-Engle c o l o r i m e t r i c procedure (15) . The dl-alpha-tocopheryl a c e t a t e is b a s e hydrolyzed i n anhydrous e t h a n o l t o t h e f r e e tocophero l . The s o l u t i o n i s a c i d i f i e d t o prevent a i r o x i d a t i o n , water i s added, and t h e dl-alpha-tocopherol i s e x t r a c t e d i n t o di- e t h y l e t h e r . t h e r e s i d u e is immediately d i s s o l v e d i n anhydrous e t h a n o l . F e r r i c c h l o r i d e i s added along wi th 2 , 2 ' - b i p y r i d i n e , b o t h i n anhydrous e t h a n o l . The mixture i s shaken v i g o r o u s l y and timed f o r 10 minutes . The absorbance of t h e r e d s o l u - t i o n i s measured a t 520 nm (16 ,17) . A review of t h e Emmerie-Engle and c e r r i c s u l f a t e t i t r i m e t r i c methods f o r Vitamin E was publ i shed by Lehman (18).

The e t h e r is evapora ted under n i t r o g e n and

6.6 S p e c t r o f l u o r o m e t r i c Analysis The i n t e n s e u l t r a v i o l e t f l u o r e s c e n c e e x h i b i t e d by

alpha-tocopherol has provided t h e b a s i s f o r a s imple and extremely s e n s i t i v e method f o r t h e d e t e r m i n a t i o n of f r e e a lpha-tocopherol and alpha-tocopheryl acetate i n plasma (19 ,20) . and 4 ml of e t h a n o l and then e x t r a c t e d w i t h 8 ml of hexane. The f r e e alpha-tocopherol i s determined by d i l u t i n g a 1 ml a l i q u o t of t h e hexane phase w i t h 3 ml of e t h a n o l and mea- s u r i n g t h e i n t e n s i t y of t h e f l u o r e s c e n c e produced a t 340 nm by e x c i t i n g t h e sample a t 295 nm. The a l p h a tocophery l a c e t a t e i s determined by d i f f e r e n c e a f t e r hydro lyz ing a 4 ml a l i q u o t of t h e hexane phase w i t h LiAlH4 t o c o n v e r t any acetate t o a lpha-tocopherol and measuring t h e i n t e n s i t y of t h e f l u o r e s c e n c e as above. A p l o t of f l u o r e s c e n c e v e r s u s c o n c e n t r a t i o n of a lpha tocophero l was l i n e a r over t h e range of 0 .6 ug/ml through 40 pg/ml (20) . The l i m i t of d e t e c t i o n i s about 0.01 pg/ml ( 1 9 ) .

Two m l of plasma are d i l u t e d w i t h 2 ml of w a t e r

6.7 T i t r i m e t r i c Analysis dl-Alpha-tocopheryl a c e t a t e (about 250 mg) i s d i s -

so lved i n 25 m l of anhydrous e thanol , 20 m l of 5 N e t h a n o l i c s u l f u r i c a c i d is added and t h e s o l u t i o n r e f l u x e d f o r 3 hours t o e f f e c t complete h y d r o l y s i s t o t h e f r e e dl-alpha- tocopherol . After t h e s o l u t i o n is cooled , i t is t r a n s - f e r r e d t o a 200-ml v o l u m e t r i c f l a s k and d i l u t e d t o volume w i t h 50 m l of 0.5N e t h a n o l i c s u l f u r i c a c i d and 20 m l of water. Two drops of diphenylamine T .S . are added and t h e

124


s o l u t i o n is t i t r a t e d wi th 0.01N ceric s u l f a t e u n t i l a b l u e end po in t i s reached which p e r s i s t s f o r 10 seconds. A blank is run and any necessary volume c o r r e c t i o n made. Each ml of 0.01N c e r i c s u l f a t e i s equiva len t t o 2 .3638 mg of dl-alpha-tocopheryl acetate ( 1 7 ) .

7 . Acknowledgments

typ ing many of t h e Ana ly t i ca l P r o f i l e s and M r s . L. B. Rubia f o r drawing and l e t t e r i n g many of t h e f i g u r e s . The h e l p a f forded by t h e S c i e n t i f i c L i t e r a t u r e Department and t h e Research Records Of f i ce of Hoffmann-La Roche Inc . i n t h e l i t e r a t u r e sea rches also is g r a t e f u l l y acknowledged.

The au thors wish t o acknowledge Mrs. A. M. Ormsby f o r

125


8. References

1.

2.

3,

4.

5.

6. 7.

8.

9.

10.

11.

12.

13.

14.

15. 16. 17. 18. 19.

20.

Hawrylyshyn, M., Hoffmann-La Roche Inc., Personal Communication. Johnson, J. , Hoffmann-La Roche Inc., Personal Communication. Rubia, L. B., Hoffmann-La Roche Inc., Personal Communication. Benz, W., Hoffmann-La Roche Inc. , Personal Communication. Scheppele, S . E., Mitchum, R. K., Rudolph, C. J., Kinneberg, K. F. , and Odell, G. V. , Lip ids , 1, 297 (1972). Merck Index, Eight Edition, p. 1115, 1968. Moros, S., Hoffmann-La Roche Inc., Personal Communication. Surmatis, J., and Weber, J., U . S . Patent 2,728,278 (1955). Gallo-Torres, H. E., Miller, 0. N., Hamilton, J. G., and Tratnyek, C., L i p i d s , 5, 318 (1971). Draper, H. H., and Csallany, A . S . , "Metabolism of Vitamin E", DeLuca, H. F., and Suttie, J. W. (editors) , flae Fat-SoZubZe Vitamins, The ZTniversity of Wisconsin Press, pp. 347-353, 1970. Simon, E. J., Eisengart, A . , Sundheim, L., and Milhorat, A . T., J . B i o l . &em.. 221, 807 (1956). Scheidl, F., Hoffmann-La Roche Inc., Personal Communication. Bolliger, H., and KGnig, A . , Hoffmann-La Roche Inc., Unpublished Data. Rudy, B. C., Mahn, F. P., Senkowski, B. Z., Sheppard, A. J. , and Hubbard, W. D. , J . A . 0. A. C., November, 1972 (In press). Emmerie, A., and Engel, C., Nature, 142, 873 (1938). United S ta t e s Pharmacopeia XVIII, p. 914, 1970. National Formulary X I I , p. 760, 1970. Lehman, R. W., J . Pham. S c i . , 53, 201 (1964). Duggan, D. E., Arch. Biochem. Biophys., 9, 116 (1959). Hansen, L. G., and Warwick, W. J., Am. J . Clin. Path., 46, 133 (1966).

126

AMITRIPTYLINE HYDROCHLORIDE

Kenneth W. Blessel, Bruce C. Rudy, and Bernard Z. Senkowski

KENNETH W. BLESSEL, BRUCE C. RUDY, AND BERNARD 2. SENKOWSKI

1.

2 .

3 .

4 .

5 .

6 .

7 .

8 .

INDEX

Analytical Profile - Amitriptyline Hydrochloride

Description 1.1 Name, Formula, Molecular Weight 1.2 Appearance, Color, Odor

Physical Properties 2 . 1 Infrared Spectrum 2 . 2 Nuclear Magnetic Resonance Spectrum 2 . 3 Ultraviolet Spectrum 2 . 4 Fluorescence Spectrum 2.5 Mass Spectrum 2.6 Optical Rotation 2.7 Melting Range 2.8 Differential Scanning Calorimetry 2.9 Thermogravimetric Analysis 2.10 Solubility 2 . 1 1 X-ray Crystal Properties 2.12 Dissociation Constant

Synthesis

Stability Degradation

Drug Metabolic Products

Methods o f Analysis 6 . 1 Elemental Analysis 6 . 2 Phase Solubility Analysis 6 . 3 Thin Layer Chromatographic Analysis 6 . 4 Gas-Liquid Chromatographic Analysis 6 . 5 Colorimetric Analysis 6 . 6 Fluorescence Analysis 6.7 Non-Aqueous Titration

Acknowledgements

References

128

AMlTRlPTYLlNE HYDROCHLORIDE

1. Descr ip t ion

1.1 Name, Formula, Molecular Weight Amit r ip ty l ine hydrochlor ide i s lO,ll-dihydro-N,N-

dimethyl-5H-dibenzo [ a ,d ] cycloheptene-A5, '-propylamine hydrochlor ide .

C20H23N*HC1 Molecular Weight: 313.87

1 .2 Appearance, Color, Odor Arni t r iptyl ine hydrochlor ide i s an odor l e s s , o f f -

white c r y s t a l l i n e powder.

2 . Phys ica 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 a m i t r i p t y l i n e hydro-

ch lo r ide is shown i n F igure 1 (1). The sample w a s d i spersed i n f luo ro lube f o r t h e reg ion of 4000-1350 cm-I and i n mineral o i l t o record t h e spectrum i n the reg ion of 1350-600 cm-1. The fo l lowing assignments of bands i n t h e spectrum have been made (1).

- Band Assignment

3057 cm-l Aromatic CH s t r e t c h 2949 and 2825 c m - l

2921 and 2852 cm-l

2545-2428 c m - l

767 and 757 cm-l

Asymmetric and symmetric CH3

Asymmetric and symmetric CH2

C h a r a c t e r i s t i c of H C 1 s a l t of

Four ad jacen t hydrogens on

s t r e t c h

s t r e t c h

t e r t i a r y amine

benzene r i n g

129

Figure 1

c w 0

Infrared Spectrum of Amitr ip ty l ine Hydrochloride

WAVELENGTH (MICRONS) 7 8 9 10 12 15 20 25 3 4 5 6

I I I I I I I I I I I I 100 100 I I I I I I I I

80 80 - -

-60

- 40 a a

$ 2 0 - - 20

I I I I I I I OL I 0 4OOO 3500 3OOO 2500 ZOO0 1700 1400 1 1 0 0 800 500

k

FREQUENCY (CM-')


2.2 Nuclear Magnetic Resonance Spectrum (NMR) The NMR spectrum of amitriptyline hydrochloride

is shown in Figure 2 ( 2 ) . a solution of 54.7 mg/0.5 ml in CDC13. signments are shown in Table I ( 2 ) .

The spectrum was recorded using The spectral as-

Table I

NMR Spectral Assignments for Amitriptyline Hydrochloride

Proton Total No. Chemical Shift Identification of Each (ppm) Multiplicity

N,N-dimethyl 6 2.65 Singlet

Protons on 7 - membered ring and Multiplet methylene protons 8 2.75-3.34 (unresolved)

Methine pro tons 1 5.8 Triplet

Aromatic protons 8 7.2 Mu1 t iple t

Proton of Hydro- Singlet chloride 1 12.5 (broad)

2 . 3 Ultraviolet Spectrum The ultraviolet spectrum of amitriptyline hydro-

chloride is shown in Figure 3 ( 3 ) . corded on a solution which contained 1.00 mg in 100 m l of 0.1N HC1. A maximum was observed at about 239 nm (E = 1.4 x 104) and a minimum at 228-231 nm.

The spectrum was re-

2 . 4 Fluorescence Spectrum The excitation and emission spectra of amitrip-

tyline hydrochloride are shown in Figure 4 (4). The sample was dissolved in methanol at a concentration of 1 mg/ml and the spectra were recorded using a Farrand MK-1 recording spectrofluorometer. emission with a maximum at 357 nm.

Excitation at 302 nm produced

2.5 Mass Spectrum The low resolution mass spectrum of amitriptyline

is shown in Figure 5 (5 ) . The spectrum was recorded on a CEC 21-110 mass spectrometer using an ionizing energy of 70 eV, which was interfaced with a Varian data system

131

L

132

Figure 3

Ultraviolet Spectrum

of Amitriptyline Hydrochloride

I .(

O.!

0. f

0. ;

0.c

0

z

a

m

Iz: 0

.:

m 0

in

U 0.4

0.3

0.2

0. I

0.0 210

2 5

0

300

3 50

NA

NO

ME

TE

RS

133

AlIS

N3

1N

I

0 z

U z

134

.7

I I

I 1

I I

I I

I I

88

88

8 0

AlIS

N31N

I 3A

11Vl3Y

l

as

e

135

KENNETH W. BLESSEL, BRUCE C. RUDY, AND BERNARD Z . SENKOWSKI

100 MS. The d a t a system accepted t h e output of the spectrometer , ca l cu la t ed t h e masses, compared t h e i r in ten- s i t i es t o t h e base peak and p l o t t e d t h i s d a t a as a series of l i n e s whose he igh t s were propor t iona l t o t h e in tens i t ies .

The molecular i on w a s measured a t m / e 277 ( f r e e base) . The c h a r a c t e r i s t i c f e a t u r e of t h i s compound is i t s s t rong tendency t o l o s e N i n order t o a t t a i n a romat ic i ty and/or r i n g c losure . The main fragments are t h e loss of N(CH3)2 and CH2N(CH3)2 from t h e s i d e cha in t o form m / e 233 and 219 r e spec t ive ly . Each of these spec ie s then l o s e s hydrogens, g iv ing rise t o m / e 231, 217, and 215. Complete l o s s of t h e s i d e chain g ives rise t o m / e 192 and m / e 85. The base peak occurs a t m / e 58 and is due t o CH2N(CH3)2 (5) . p a t i b l e wi th t h e low r e s o l u t i o n scan.

A high r e so lu t ion spectrum w a s found t o b e f u l l y com-

2.6 Op t i ca l Rota t ion Amit r ip ty l ine hydrochlor ide does no t e x h i b i t

o p t i c a l a c t i v i t y .

2 . 7 Me1 t i n g Range The melt ing range repor ted i n USP X V I I I i s

195-198OC when a Class I procedure i s used (6) .

2 . 8 D i f f e r e n t i a l Scanning Calorimetry (DSC) The DSC curve f o r a m i t r i p t y l i n e hydrochlor ide a t

a scan r a t e of 10°C/min. i s shown i n F igure 6 ( 7 ) . The curve was recorded with a Perkin E l m e r DSC-1B under an atmosphere of n i t rogen . A s i n g l e endotherm was observed, t h e ex t rapola ted onse t of mel t ing occurr ing a t 193.0 5 0.2OC and the peak a t 197.5 5 0.2OC. cor rec ted . f o r t he m e 1 t i n g endo therm.

A l l temperatures a r e The va lue ca l cu la t ed f o r AHf was 6.7 kcal/mole

2.9 Thermogravimetric Analysis (TGA) The TGA curve f o r r e fe rence s tandard ami t r ip-

t y l i n e hydrochlor ide exhib i ted no l o s s of weight from 30-195OC. range of 195-317OC which accounted f o r 100% of t h e sample weight (7) .

A s i n g l e weight l o s s occurred i n t h e temperature

2.10 S o l u b i l i t y The s o l u b i l i t y d a t a obtained f o r re ference s t an -

dard a m i t r i p t y l i n e hydrochlor ide is l i s t e d i n Table I1 ( 8 ) .

136

AM1 TR IPTY LINE H Y DROCH LOR I DE

F i g u r e 6

DSC Curve of A m i t r i p t y l i n e Hydroch lo r ide

I I I I i

A Hf =6.7 kcal / m o l e

Endothermic

t I

4

Exothermic

I I I I I

OC

I70 I80 190 200 210

137

KENNETH w. BLESSEL, BRUCE c. RUDY, AND BERNARD z . SENKOWSKI

Table I1

S o l u b i l i t y of A m i t r i p t y l i n e Hydrochloride

Solvent S o l u b i l i t y (mg/ml)

petroleum e t h e r d i e t h y l e t h e r water 2 -propano1 3A a lcohol chloroform 95% ethanol benzene methanol

(30-60°C) 0.30 0.50 >SO0 -

53 *

313 - >500. >SO0 - 5.0

>500

2 . 1 1 Crys t a l P rope r t i e s The x-ray powder d i f f r a c t i o n d a t a f o r a sample

of re ference s tandard a m i t r i p t y l i n e hydrochlor ide is given i n Table I11 (9). The opera t ing parameters of the hstru- ment are given below.

Instrumental Conditions

General E l e c t r i c Model XRD-6 Spectrogoniometer

Generator: 50 KV, 12-112 MA Tube t a r g e t : Copper Radiat ion : o p t i c s : 0.1' Detector s l i t

CU Ka = 1.542 8

M.R. S o l l e r s l i t 3' Beam s l i t 0.0007" N i f i l t e r 4O t ake of foangle

Goniometer: Scan a t 0.2 20 per minute Detector : Amplifier ga in - 1 6 course,

8.7 f i n e Sealed propor t iona l counter

tube and DC vo l t age a t p l a t eau

Pulse he igh t s e l e c t i o n EL - 5 v o l t s

Eu - o u t Rate meter T.C. 4 2000 CIS f u l l s c a l e

138

AMlTRl PTY LINE HY DROCH LORlDE

Recorder: Chart speed 1 inch per 5

Samples : minutes

temperature Prepared by grinding at room

Table I11

Interplanar Spacings in Amitriptyline Hydrochloride from Powder Diffraction Data

28 - 11-50 12.78 13.46 14.86 15.66 16.06 16.34 16.72 17.72 18.58 18.90 19.26 20.24 20.94 21.34 21.87 22.94 23.42 24.32 24.88 25.78 26.62 27.30 27.58 28.16 29.00

d&)*

7.69 6.93 6.58 5.96 5.66 5.52 5.42 5.30 5.01 4.78 4.70 4.61 4.39 4.24 4.16 4.06 3.88 3.80 3.66 3.58 3.46 3.35 3.27 3.23 3.17 3.08

IITo% 21 10 4 16 9 4 15 18 1 74 13 36 9

100 10 4 60 7 9 3 19 16 9 3 9 4

28

29.36 29.52 30.10 30.96 31.28 31.74 31.98 32.46 32.76 33.80 34.12 34.98 35.30 36.00 38.26 39.02 39.66 40.04 40.54 41.22 41.88 42.06 43.24 43.88 44.22 44.70 45.30

- 3.04 3.03 2.97 2.89 2.86 2.82 2.80 2.76 2.73 2.65 2.63 2.57 2.54 2.49 2.35 2.31 2.27 2.25 2.23 2.19 2.16 2.15 2.09 2.06 2.05 2.03 2.00

*d = (interplanar distance) - nh 2 Sin e

3 2 4 4 7 6 3 5 7 6 3 2 3 3 3 6 2 1 1 2 1 2 3 2 2 2 2

**I/ = relative intensity (based on highest I0 intensity of 100)

139


2.12 Dissociat ion Constant The d i s soc ia t ion constant f o r a m i t r i p t y l i n e

hydrochloride was determined using a graphica l meihod involving the pH dependence of t he water s o l u b i l i t y . The value f o r t he pKa determined by t h i s method was 9.4 (10).

3. Synthesis

Figure 7. The f i r s t , r eac t ion sequence I (11,12), involves t h e addi t ion of a Grignard reagent followed by dehydrat ion wi th HC1 o r a c e t y l ch lo r ide , while I1 (13) uses a cyclo- propyl Grignard reagent followed by r ing opening wi th dimethylamine t o form t h e des i r ed compound. A number of a l t e r n a t i v e syntheses have been descr ibed i n t h e l i t e r a t u r e

Two s y n t h e t i c routes t o a m i t r i p t y l i n e are shown i n

(14-17).

4 . S t a b i l i t y Degradation The s t a b i l i t y of ami t r ip ty l ine hydrochlor ide, i n t he

bulk form, was s tudied under condi t ions of e levated temper- a t u r e o r exposure t o l i g h t (18). It was found t o be s t a b l e a t room temperature and a t 45OC f o r a per iod i n excess of two months. It showed some decomposition a f t e r two months a t 100°C, and when exposed t o l i g h t , a s evidenced by the formation of a brownish d i sco lo ra t ion of t he powder. A 1% aqueous s o l u t i o n was found t o be s table a t O°C, room temperature, and 45OC, a s w e l l as f o r a per iod of 60 hours a t I O O O C .

5. Drug Metabolic Products Hucker and Por t e r (19) demonstrated t h a t very l i t t l e of

a dose of ami t r ip ty l ine is excreted unchanged i n humans. Other i n v e s t i g a t o r s (20-23) have demonstrated t h e presence of t he major metabol ic products shown i n Figure 8. Facino and Corona (20) have demonstrated metabol i tes I, 111, IV, t h e two isomers of V, and VI i n t he organs of r a b b i t s . Eschenoff and Rieder (21) demonstrated metabol i tes I, 11, and V and i n addi t ion repor ted the ex i s t ence of t he N-oxide metabol i te ( V I I ) i n s t u d i e s on rats and humans. They reported t h a t t h e metabolism of a m i t r i p t y l i n e i n these two species i s near ly i d e n t i c a l ( 2 4 ) . I n add i t ion , Facino and Corona (25) l a t e r reported the ex is tence of an a c i d i c metabol i te t o which they ascr ibed t h e s t r u c t u r e of t h e carboxyl ic ac id which would be formed by oxida t ion deamina- t i o n of t he drug.

140


Figure 8

Metabolic Products of Amitriptyline Hydrochloride

CH(CH1)zN(CHJ2


f

CI

0 - t

I V

142

AMlTRl PTY LINE HYDROCHLORIDE

6. Methods of Analys is

6 .1 Elemental Analysis The r e s u l t s of an e l e m e n t a l a n a l y s i s of a sample

of r e f e r e n c e s t a n d a r d a m i t r i p t y l i n e h y d r o c h l o r i d e i s pre- s e n t e d i n Table I V below (32).

Element Theory % Found %

C 76.53 76.44 H 7.70 7.79 N 4.47 4.50 c 1 11.30 11 .26

6.2 Phase S o l u b f l i t y Analys is

h y d r o c h l o r i d e is shown i n F i g u r e 9. a c e t o n e and t h e e x t r a p o l a t e d s o l u b i l i t y w a s 17.81 mg/g (8).

A phase s o l u b i l i t y a n a l y s i s f o r a m i t r i p t y l i n e The s o l v e n t used w a s

6.3 Thin Layer Chromatographic Analys is A number of t h i n l a y e r chromatographic sys tems

f o r a m i t r i p t y l i n e h y d r o c h l o r i d e have been d e s c r i b e d i n t h e l i t e r a t u r e . A r e p r e s e n t a t i v e number o f t h e s e systems are shown i n Table V. Methods of d e t e c t i o n were n o t inc luded s i n c e t h e s e are many times determined by t h e i n d i v i d u a l p r e f e r e n c e s of t h e a n a l y s t o r t h e p a r t i c u l a r s e p a r a t i o n d e s i r e d (26).

Table V

TLC Systems f o r A m i t r i p t y l i n e Hydrochlor ide

Adsorbent Solvent System & Reference

S i l ica G e l

S i l i c a G e l

S i l i c a Gel

benzene:dioxane:NH3 0.74 27 (60 : 35 : 5)

e thano1:ace t ic a c i d : 0.65 27 H20 (50 : 30 : 20)

methanol : b u t a n o l 0.37 27 (60 :40)

S i l i c a G e l + 0.1M NaOH cyc1ohexane:benzene: 0.72 28 d i e thylamine (75 : 1 5 : 10)

143

Figure 9

c

P P

I- z W > J 0 v)

0 \

W I- 3 -I 0 v)

LL 0 w E

z - 2 t v) 0 0. I 0 V

z

I- 3 J 0 v)

0

2 5

2 0

15

10

5

0

PHASE SOLU B I LI TY AN ALY S I S

Sample: Amitriptyline Solvent = Acetone Slope : 0.04 o/o Equilibration : 20 hrs at 25OC Extrapolated Solubility * 17.81 mg/g of Acetone

l l l r l l ~ ~ ~ ~ ~ ~ ~ ~ ~ r , r l

0 25 50 75 100

SYSTEM COMPOSITION : mq OF SAMPLE PER g SOLVENT

AMITR IPTY LINE HYDROCHLORIDE

S i l i c a G e l + 0.1M NaOH methanol 0.50 28

S i l i c a Gel + 0.1M NaOH acetone 0.34 28

S i l i c a G e l + 0.1M KHSO4 methanol 0 .41 28

S i l i c a Gel + 0.1M KHS04 95% e thanol 0 . 2 8 28

6 . 4 Gas-Liquid Chromatographic Analysis (GLC) A GLC method f o r t he q u a n t i t a t i v e de te rmina t ion

of a m i t r i p t y l i n e has been descr ibed (29). The method w a s developed f o r measuring t h e a m i t r i p t y l i n e conten t of plasma. The lower l i m i t of d e t e c t i o n is 20 ng/ml. The chromatographic condi t ions a r e given below.

Column - Support - Liquid Phase -

Detect ion - Oven Temperature - Detector

Temperature - I n j e c t i o n Por t

Temperature - C a r r i e r Gas -

5 f t . x 114 i n . s i l a n i z e d

Chromosorb W (80-100 mesh) 1% polyvinyl pyr ro l id inone

Flame i o n i z a t i o n 205OC

g l a s s

and 3% Versamid 900

240°C

Maximum Nitrogen, 50 ml/min.

6 . 5 Color imet r ic Analysis Amit r ip ty l ine hydrochlor ide can be determined

co lo r ime t r i ca l ly using the methyl orange r eac t ion . The method involves bu f fe r ing t h e s o l u t i o n conta in ing t h e compound a t a pH va lue of 4 .3 , adding the methyl orange and e x t r a c t i n g the r e s u l t i n g complex i n t o e thylene d i c h l o r i d e . The absorbance of t h e e thylene d i c h l o r i d e e x t r a c t i s measured a t about 430 nm and t h e amount of a m i t r i p t y l i n e cal- cu la ted by comparison wi th a c a l i b r a t i o n curve prepared from pure a m i t r i p t y l i n e . This method can be used t o determine a m i t r i p t y l i n e i n t h e presence of i t s N-demethylated metabol i tes by t h e a d d i t i o n of acetic anhydride be fo re ex- t r a c t i o n s i n c e primary and secondary amines do no t r e a c t with methyl orange i n t h e presence of a c e t i c anhydride (30).

6 . 6 Fluorescence Analysis A s e n s i t i v e f l u o r i m e t r i c assay has been developed

f o r a m i t r i p t y l i n e hydrochlor ide i n b i o l o g i c a l samples (31).

KENNETH W. BLESSEL, BRUCE C. RUDY, AND BERNARD 2 . SENKOWSKI

The b io log ica l material t o be analyzed is homogenized and an equal volume of methanol added. 15 m l of heptane and 1 m l of d i s t i l l e d water i s added t o a 1 m l a l i q u o t of the methanolic sample. The heptane l aye r a f t e r s epa ra t ion , is then ex t rac ted wi th pe rch lo r i c ac id . The ac id e x t r a c t is then heated i n a b o i l i n g water bath f o r 10 min. and cooled. The f luorescence of t h e carbonium ion generated by t h e hea t ing process is then measured a t 555 nm using an a c t i v a t i o n wavelength of 305 nm. The f luorescence i n t e n s i t y was found to be l i n e a r with a m i t r i p t y l i n e concen t r a t ion i n the range of 0.05-5.0 mcg/ml.

Then 0.2 g of borax,

6.7 Titrimetric Analysis A non-aqueous t i t r a t i o n with Derchlor ic acid i n

acetic ac id is the p re fe r r ed method f o r ' t h e ana lys i s of bulk a m i t r i p t y l i n e hydrochloride. i n g l a c i a l a c e t i c ac id . Then mercuric a c e t a t e T.S. and crystal v i o l e t T.S. are added. The s o l u t i o n is t i t r a t e d with 0.1N pe rch lo r i c ac id to a green end-point. 0.1N perch lo r i c acid i s equivalent t o 31.39 mg of ami t r ip- t y l i n e hydrochloride ( 6 ) .

The sample is d isso lved

Each m l of

7. Acknowledgments The authors wish t o acknowledge t h e a s s i s t ance of t h e

Research Records Off ice and the S c i e n t i f i c L i t e r a t u r e Department of Hof fmann-La Roche Inc.

146


8. References

1.

2 .

3.

4.

5.

6.

7.

8.

9.

10. 11.

12. 13.

14.

15.

16. 17.

18.

19.

20.

21.

22. 23.

Hawrylyshyn, M,, Hoffmann-La Roche Inc., Personal Communication. Johnson, J. H., Hoffmann-La Roche Inc., Personal Communication. Rubia, L. B., Hoffmann-La Roche Inc., Personal Communication. Boatman, J., Hoffmann-La Roche Inc., Personal Communication. Benz, W., Hoffmann-La Roche Inc., Personal Communication. The United S ta tes Pharmacopeia XVIII, pp. 38-40 (1970). Moros, S., Hoffmann-La Roche Inc., Personal Communication. MacMullan, E., Hoffmann-La Roche Inc., Personal Communication. Hagel, R., Hoffmann-La Roche Inc., Personal Communication. Green, A. L., J . Pharm. Pharmac.,E, 10 (1967). Hoffmann-La Roche, F. and Co., A . G., Belgian Patent 577,057 (1959). Merck and Co. Inc., Belgian Patent 584,061 (1960). Hoffsommer, R. D., Taub, D., and Wendler, N. L., J. Org. Chem., 2, 1829 (1962). Protiva, M., et al., J . Med. Pharm. Chern.,&, 411 (1961). Villani, F. J., Ellis, C. A . , Teichman, C., and Bigos, C . , J . Med. Pharm. Chem.,?, 373 (1961). Winthrop, S . , et al., J . Org. Chern.,Z, 230 (1962). Merck and Co, Inc., United States Patent 3,205,264 (1965) . Schmidli, B., Hoffmann-La Roche Inc., Unpublished Data. Hucker, H. B. and Porter, C. C., Federation Proc., - 20, 172 (1961). Facino, R. M. and Corona, G. L., J . Pharm. Sci., 58, 764 (1969). Eschenhof, E. and Rieder, J., ArzneimitteZ-Forsch , - 19, 957 (1969). Hucker, H. B., PharmaeoZogis$ k, 171 (1962). Diamond, S., Current Therap. Res., - 7, 170 (1962).

147

KENNETH W. BLESSEL. BRUCE C. RUDY, AND BERNARD 2. SENKOWSKI

24.

25.

26.

27.

28. 29.

30. 31.

32.

Eschenhof , E. and Rieder, J., Deut. Apotheker-Ztg., - 108, 1202 (1968). Facino, R., Santagostino, G. and Corona, G., Biochem. PharmacoZ., 2 , 1503 (1970). Comer, J. P. and Comer, I., J . Pham. Sci . , 56, 413 (1967) . Cochin, J. and Daly, J. W., J. PhamacoZ. ExptZ. Therap., 139, 160 (1963). Fike, W. W., AnaZ. Chem.,E, 1697 (1966). Braithwaite, R. A . and Widdop, B., CZin. Chim. Acta , - 35, 461 (1971). Silverstein, R. M., AnaZ. Chem.,g, 154 (1963). Eschenhof, E. and Rieder, J. , Hoffmann-La Roche Inc. , Unpublished Data. Scheidl, F., Hoffmann-La Roche Inc., Personal Communication.

148

DIGITOXIN

Ivan M . Jakovljevic

IVAN M. JAKOVLJEVIC

CONTENTS

1. DESCRIPTION 1.1 R e g i s t e r e d Names 1 . 2 Chemical Name 1.3 Formula, S t r u c t u r e , Molecu la r Weight 1 . 4 Appearance

2 . PHYSICAL PROPERTIES 2.1 I n f r a r e d Spec t rum 2 . 2 Nuc lea r Magnet ic Resonance Spectrum 2.3 U l t r a v i o l e t Spec t rum 2 . 4 Mass Spectrum 2.5 O p t i c a l R o t a t i o n 2.6 O p t i c a l R o t a t o r y D i s p e r s i o n (ORD) and

2.7 Mel t ing Range 2 . 8 X-Ray D i f f r a c t i o n P a t t e r n 2.9 Po la rography 2 . 1 0 S o l u b i l i t y

C i r c u l a r Dichroism (CD)

3. SYNTHESIS 4. STABILITY 5. METABOLISM, P R O T E I N BINDING A N D

C L I N I C A L ASSAYS 5 .1 Metabol ism 5.2 P r o t e i n Binding 5 .3 De te rmina t ion i n Blood

5.3.1 Chemical Methods 5.3.2 P h y s i c a l Methods 5.3.3 Radioimmunoassay (Deuter ium and

T r i t i u m Labeled D i g i t o x i n )

6. METHODS O F ANALYSIS 6 .1 I d e n t i f i c a t i o n Tests 6.2 E lemen ta l A n a l y s i s 6 . 3 Chromatography

6 .3 .1 Column Chromatography 6.3.2 Thin Layer Chromatography 6 .3 .3 Pape r Chromatography 6.3.4 Gas Chromatography 6 . 3 . 5 High Speed Liquid Chromatography

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DIG I TOX I N

6.4 Colorimetric Analysis 6.5 Fluorometric Analysis 6.6 Electrophoresis 6.7 Automated Assay

7. CLEAVAGE OF CARDIAC GLYCOSIDES 8. BIOLOGICAL ACTIVITY

8.1 Characteristic Structural Features 8.2 Bioassay

9 , ACKNOWLEDGMENT

10. REFERENCES

15 1

IVAN M. JAKOVLJEVIC

1. DESCRIPTION

from D i g i t a l i s p u r p u r e a LinnQ, D i g i t a l i s l a n a t a Digitoxin is a cardiotonic glycoside obtained

Ehrhart , leaves,

1.1

names:

and other suitable species of D i g i t a l i s

Registered Names

Digitoxin is designated by the following -

C A R D I G I N (Nat , Drugs) , C R Y S T O D I G I N (Li 1 ly) , D I G I C O R Y L (Roussel) , D I G I L O N G (Roehringer) , D I G I - JYERCK (Merck) , D I G I P A N , D I G I S I D I N (Winthrop) , D I - G I T A L I N E N A T I V E L L E (Varick), D I G I T O R A (Upjohn), D I G I T O X I N (Sandoz) , D I G I T O X O S I D E (W.H.0,) , D I G I - T R I N (Astra) , L A N A T O X I N (Beiersdorf) , P U R O D I G I N (Wyeth) , P U R P U R E N , P U R P U R I D (Promonta),

1 . 2 Chemical Name - 3B-(D-Digitoxosyl-D-digitoxosyl-D-digito-

xosyl-oxy)-14~-hydroxy-5~-card-2O(2Z)-enolide,

1.3 Formula, Structure, Molecular Weight

c 4 1H6 4 0 1 3 Mol.Wt. 764.94

C18H3109 (tridigitoxose)

C23H3404 digitoxigenin

THE CONFORMATIONAL ARRANGEMENT

Digitoxin belongs to the cardenolide series, which consists of a steroid nucleus with a 5-membered unsaturated lactone ring at C-17.

present in the six-membered ring form (pyranoid As in most other glycosides, the sugar is

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DIGITOXIN

form) w i t h t h e c h a i r c o n f o r m a t i o n .

a t e i t h e r e n d o f t h e m o l e c u l e , which i s a n impor- t a n t s t e r i c r e q u i r e m e n t f o r c a r d i o t o n i c a c t i v i t y . S a t u r a t i o n of t h e l a c t o n e r i n g g r e a t l y r e d u c e s t h e c a r d i o t o n i c a c t i v i t y . The u n s a t u r a t e d l a c t o n e r i n g must b e a t t a c h e d i n t h e B - c o n f i g u r a t i o n . E p i m e r i z a - t i o n r e d u c e s p h a r m a c o l o g i c a l a c t i v i t y by a t l e a s t 400 times. Opening t h e l a c t o n e r i n g by a l k a l i n e hy- d r o l y s i s a l s o r e s u l t s i n l o s s o f a c t i v i t y ' ,

The s t e r o i d framework i s c o n s i d e r a b l y b e n t

1 . 4 Appearance

Very s m a l l e l o n g a t e d , r e c t a n g u l a r p l a t e s f rom d i l u t e d e t h a n o l , o r m i c r o c r y s t a l l i n e powder , w h i t e o r p a l e b u f f , o d o r l e s s , v e r y b i t t e r t a s t e .

2. PHYSICAL PROPERTIES

2.1 I n f r a r e d S p e c t r u m

The i n f r a r e d ( I R ) s p e c t r u m o f d i g i t o x i n , USP r e f e r e n c e s t a n d a r d , i s g i v e n i n F ig .1 . The I R s p e c t r u m was t a k e n i n a K B r p e l l e t on a Beckman IR-12 s p e c t r o m e t e r .

a s f o l l o w s 2 :

W a v e l e n g t h of V i bra ti on Mode 8

A b s o r p t i o n (CM-') t

IR s p e c t r a l a s s i g n m e n t s o f d i g i t o x i n a r e

3575 -OH s t r e t c h Inon-hydrogen bonded)

3440 - O H s t r e t c h ( h y d r o g e n bonded)

2960, 2935 C H s t r e t c h , C H 3 , C H 2

1740 CEO s t r e t c h (a,B u n s a t u -

1630 CH s t r e t c h [ c o n j u g a t e d

1448, 1403, 1 3 7 9 , 1367, 1348 C H d e f o r m a t i o n (CH3, C H 2 ,

s t r e t c h

r a t e d l a c t o n e ]

d o u b l e bond)

C - C H J , C H )

153

IVAN M. JAKOVLJEVIC

1162 1125 1075

1060

1040

3 O - O H d e f o r m a t i o n Z O - O H d e f o r m a t i o n C - 0 - s t r e t c h ( g l y c o s i d i c

e t h e r ) C - 0 - s t r e t c h ( c y c l i c e t h e r

o x y g e n s ) 1°-OH d e f o r m a t i o n

The IR s p e c t r a o f 36 g l y c o s i d e s a n d t h e i r a g l y c o n e s w e r e s t u d i e d . G l y c o s i d e s were c h a r a c t e r - i z e d b y a d o u b l e t i n t h e r e g i o n 1099-1031 a n d 1066-1013 cm-'.'

x i n r e v e i l e d t h e p r e s e n c e o f two p o l y m o r p h s . One o f t h e p o l y m o r p h i c c r y s t a l s was o b t a i n e d by r e c r y s - t a l l i z a t i o n f rom 85% e t h a n o l a n d t h e o t h e r f r o m t h e c o l d e t h a n o l e v a p o r a t i o n ,

2 . 2 N u c l e a r M a g n e t i c Resonance S p e c t r u m

The N M R s p e c t r u m o f d i g i t o x i n i s complex e v e n a t 220 MHz, however 1 7 p r o t o n s i g n a l s may b e s e e n t o low f i e l d o f 2.5 ppm b o t h i n C D C 1 3 nnd CD3SOCD3 a f t e r e x c h a n g e w i t h D,O t o remove t h e s i g n a l s f o r O H , They may b e a s s i g n e d w i t h r e a s o n - a b l e c e r t a i n t y f r o m c h e m i c a l s h i f t a n d c o u p l i n g c o n s t a n t s , The 6 v a l u e s a r e l i s t e d a s f o l l o w s :

p r o t o n a t 1 7 ; 3 .20 , 3.23, 3 .27 ( o y e r l a p p i n g ) , Jaa= 10 .0 Hz,

( n u m b e r i n g f rom t h e a n o m e r i c p r o t o n away f rom t h e oxygen i n e a c h s u g a r r i n g s e q u e n t i a l l y s t a r t i n g w i t h p o s i t i o n 3 on t h e s t e r o i d ) ; 3 .77 u n r e s o l v e d p r o t o n s 5 ' , l l ' , 1 7 ' ; 4 . 0 1 , p r o t o n 3 ; 4.10, p r o t o n 1 5 ' ; 4 . 2 2 , u n r e s o l v e d p r o t o n s 3 ' a n d 9 ' ; 4 .78 , 4 .97 , Jgem- -18 .0 Hz, J z 1 - 2 2 = 1 .8 , C H 2 a t 2 1 ;

4 .84 , 4 .88 , 4 .91 , ( o v e r l a p p i n g ) Jaa- 1 0 , p r o t o n s

l ' , 7 ' , 1 3 ' ; 5 .88 , p r o t o n a t 2 2 . 5 S e e F i g . 2 , P o s i t i o n s o f t h e a c e t y l g r o u p s i n p a r t i a l l y

a c e t y l a t e d c a r d e n o l i d e s were e s t a b l i s h e d by t h e a n a l y s i s o f N M R s p e c t r a . Chemica l s h i f t s o f p r o t o n s b e l o n g i n g t o s p e c i f i c a c e t y l g r o u p s d i f f e r s u f f i - c i e n t l y t o serve as d i a g n o s t i c e h a r a c t e r i s t i c s .

E x a m i n a t i o n o f t h e I R s p e c t r a o f d i g i t o -

1

I n C D C 1 , : 2 .79 ppm ( b r o a d e n e d t r i p l e t ) ,

= 2.8 , p r o t o n s 4 , l o ' , 1 6 ' Jae

154

DIGITOXIN

FREOUENCY 3000 2500 2000 1600 1400 1200 1000 900 850 800 750 700 65

I I 1 0 1 I I

I I I I I I I I I I I

3 4 5 6 7 8 9 10 11 12 13 14 15 WAVELENGTH

I I

F i g . 1 I R S p e c t r u m of D i g i t o x i n , U S P R e f e r e n c e S t a n d a r d . K B r P e l l e t . I n s t r u m e n t : Beckman I R - 1 2 S p e c t r o p h o t o m e t e r .

500 400 300 200 100 I I I 1 I

8 0 7 0 6 0 5 0 4 0 3 0 2 0 1 0 P P M ( 6 )

L

Fig. 2 NHR S p e c t r u m of D i g i t o x i n , U S P R e f e r e n c e S t a n d a r d . I n s t r u m e n t : V a r i a n T-60A.

155

IVAN M. JAKOVLJEVIC

The s i g n a l s o f t h e e q u a t o r i a l p r o t o n s o f d i g i t o x o - se m o l e c u l e s a r e a l s o o f d i a g n o s t i c v a l u e a s t h e i r p o s i t i o n c h a n g e s when t h e a d j a c e n t a x i a l g r o u p i s a c e t y l a t e d 6 .

2 . 3 U l t r a v i o l e t S p e c t r u m

The u l t r a v i o l e t c u r v e o f a s o l u t i o n i n methanol shows a peak a t 218 nm, E 17.4 x

2.4 Mass S p e c t r u m - Mass s p e c t r o m e t r i c d a t a were o b t a i n e d

u s i n g e l e c t r o n i m p a c t and low r e s o l u t i o n on a C . E . C . 110 mass s p e c t r o m e t e r . The f r a g m e n t a t i o n p a t t e r n i s t y p i c a l o f t h e s t e r o i d p o r t i o n o n l y . The h i g h e s t s i g n i f i c a n t mass i s a t m/e 357 which r e p r e s e n t s t h e d i g i t o x i g e n i n f r a g m e n t .

2.5 O p t i c a l R o t a t i o n

I n v e s t i g a t o r s h a v e d e t e r m i n e d t h e o p t i c a l r o t a t i o n u n d e r d i f f e r e n t c o n d i t i o n s :

[a];'= +4.8' (c31 .2 i n d i o x a n e ) '

[a];'= a b o u t +18 ' (cp2 .5 i n c h l o r ~ f o r m ) ~

2 0 = a b o u t +21' ( c x l . 0 i n c h l o r ~ f o r m ) ~

[ a ] ; ' = a b o u t +13' (cl.1.0 i n m e t h a n o l ) '

2 .6 O p t i c a l R o t a t o r y D i s p e r s i o n ( O R D ) and

C i r c u l a r D i c h r o i s m ( C D )

The c i r c u l a r d i c h r o i c (CD) s p e c t r a were r e c o r d e d on a Cary 60 s p e c t r o p o l a r i m e t e r , e q u i p p e d w i t h Model 6002 C D u n i t . The O R D c u r v e shows a p o s i t i v e [a] of 1 3 . 1 and a p o s i t i v e C o t t o n e f f e c t of [ a ] 12.3 x l o - ' a t 254 nm a s do many s t e r o i d s . See F i g . 3. The C D c u r v e shows a p o s i t i v e e f f e c t o f [ a ] 11.1 x 1 O - j a t 238 nm. These s tudies were done i n m e t h a n o l . 7

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DIGITOXIN

S t e r e o c h e m i c a l e f f e c t s a r e f o u n d i n car- d e n o l i d e s w i t h a - k e t o l g r o u p s i n t h e 11 ,12 p o s i - t i o n ” .

Fig. 3 O R D and CD S p e c t r a of D i g i t o x i n , USP R e f e r e n c e S t a n d a r d . I n s t r u m e n t : C a r y 6 0 S p e c t r o - p o l a r i m e t e r , e q u i p p e d w i t h Model 6 0 0 2 C D U n i t .

.?.

300 350 250 nm

200

2.7 M e l t i n g Range

The m e l t i n g p o i n t o f d i g i t o x i n is 256 - 2 5 7 ° C . ( a n h y d r o u s ) . e D i g i t o x i g e n i n h a s a m e l t i n g p o i n t 25OoC.”

a s a) immers ing t h e s u b s t a n c e u n d e r s i l i c o n e o i l , b ) u n d e r an a t m o s p h e r e o f n i t r o g e n and c ) i n a n e v a c u a t e d , s e a l e d c a p i l l a r y t u b e were s t u d i e d on a m i c r o s c o p e h o t s t a g e , The m e l t i n g p o i n t s w h i c h a r e o b t a i n e d u n d e r s u c h c o n d i t i o n s a r e o f t e n h i g h e r 1 2 .

The e f f e c t o f p r o t e c t i v e e n v i r o m e n t s s u c h

2.8 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 r a c t i o n p a t t e r n o f d i g i t o x i n

conforms t o t h e f o l l o w i n g p a t t e r n ” .

dA 1/11 dA I / I ’ dA I / I ’

15 7 6 . 2 6 40 4 . bO 4 0 9.07 2 0 5.95 I00 4 . 6 2 40 8 . 0 1 7 5 . 6 3 50 4 . 3 2 70 7 . 2 b 3 0 5 . 3 2 1 3 4 . 1 0 3 0 7.00 3 0 5.40 40 3 . 9 3 3 0

157

I V A N M. JAKOVLJEVIC

dA

3 . 7 5 3 . 6 2 3 . 5 2 3 . 3 9 3 . 3 0 3 . 2 3

2 . 9

1/1 ' dA I/I' dA 1/11

3 0 3 . 0 6 7 2 . 3 7 3 7 2 . 8 9 7 2 . 2 0 3 7 2 . 7 5 7 2 . 1 0 3 7 2 . 6 0 3 2 . 0 5 3

2 0 2 . 5 2 3 2 . 0 1 3 7 2 . 4 3 3 1 . 9 5 3

Polarography

A study of the polarographic characteristics of digitoxin in 50% ethanolic solution containing tetraethylammonium hydroxide as electrolyte, showed an average half-wave potential of -1,965 volts. The diffusion current wave height versus concentration graph indicated that quantities as low as 2 mcg could be determined. The method has been successfully applied to the tinc- ture of digitali~'~) 1 5 .

2.10 Solubility

Digitoxin is practically insoluble in water (1 g dissolves in about 100 liters at 20'C). One gram dissolves in about 40 m l chloroform, 60 m l ethanol, and 400 m l ethyl acetate. It is also soluble in ether, petroleum ether, benzene and vegetable oils.

3. SYNTHESIS

cardenolide family has been synthesized using as the starting material methy l -3B-ace toxy-14B-hydroxy-Sf3-etinate by a seven-step sequence16. In the last step a solution ofa,B-unsaturated ester was treated with SeO2 by boiling under reflux for 10 hours. The filtrate was poured into water and the product isolated with ether. Acid hydrolysis of digitoxigenin acetate yielded digitoxigenin (M.p. 246-249'C and [ a ] +19' in ethanol).

Digitoxigenin, a typical member of t h e

158

DIGITOX IN

4 . STABILITY

The s t a b i l i t y o f two l i q u i d e x t r a c t s f rom t h e l e a v e s o f D i g i t a l i s p u r p u r e a was examined . Both p r o d u c t s c o n t a i n e d d i g i t o x i n and g i t o x i n . The a c - t i v i t y o f e a c h d r u g was d e c r e a s e d by more t h a n 1 0 % o f t h e i n i t i a l v a l u e i n less t h a n t h r e e months a t 20". The r a t e of d e c o m p o s i t i o n was g i t o x i n > d i - g i t o x i n " .

S t o r a g e of d i g i t o x i n p r e p a r a t i o n s for o n e y e a r d i d n o t s i g n i f i c a n t l y d e c r e a s e t h e i r p o t e n c y s t o r - ed a t t e m p e r a t u r e s up t o 3OoC. The p o t e n c y was c h e c k e d by B a l j e t c o l o r i m e t r i c a s s a y and by t h e b i o l o g i c a l method a c c o r d i n g t o t h e S w e d i s h P h a r - macope ia XI

No breakdown o f d i g i t o x i n i n t a b l e t s , i n j e c - t i o n s o r s o l u t i o n s was f o u n d when s t o r e d f o r 5 y e a r s i n t h e d a r k up t o 3OoC. l 9

5. METABOLISM, P R O T E I N B I N D I N G A N D

CLINICAL ASSAY

5 . 1 M e t a b o l i s m

D i g i t o x i n i s c o m p l e t e l y a b s o r b e d f o l l o w - i n g o r a l i n g e s t i o n a n d i t s f u l l e f f e c t a p p e a r s a s r a p i d l y a s by i n t r a v e n o u s i n j e c t i o n * ' . The l i v e r i s t h e main s i t e o f d e t o x i f i c a t i o n o f d i g i t o x i n , I t m e t a b o l i z e s v e r y r a p i d l y . One m e t a b o l i t e h a s been i d e n t i f i e d : digoxigenin-di-digitoxoside, p r o - duced by h y d r o x y l a t i o n a n d t h e l o s s o f o n e mole- c u l e o f s u g a r .

Ten d a y s a f t e r an i n j e c t i o n , h a l f o f t h e d o s e i s s t i l l p r e s e n t i n t h e body , and some re- mains a f t e r 2 0 d a y s . F o l l o w i n g t h e a d m i n i s t r a t i o n o f m a i n t e n a n c e d o s e s o f 0 .1 t o 0 . 3 mg d a i l y , 1 0 % o f t h e d o s e i s e x c r e t e d u n c h a n g e d .

An e x p e r i m e n t a l method h a s b e e n d e v e l o p e d i n o r d e r t o s t u d y t h e m e t a b o l i c d e g r a d a t i o n o f d i - g i t o x i n and d i g o x i n and c h a n g e s i n l i p i d s o l u b i l - i t y o f t h e r a d i o a c t i v e m a t e r i a l i n p l a s m a 2 ' , The c h a n g e s o c c u r a f t e r t h e a d m i n i s t r a t i o n o f r a d i o - a c t i v e l y l a b e l l e d g l y c o s i d e s i n t o an i s o l a t e d p e r - f u s e d l i v e r s y s t e m ( g u i n e a p i g ) o r t o i n t a c t r a b - b i t s , The m e t a b o l i c d e g r a d a t i o n of d i g i t o x i n a n d d i g o x i n i n t h e l i v e r r e s u l t s i n t h e f o r m a t i o n o f

159

I V A N M. JAKOVLJEVIC

t h e mono- a n d b i s - d i g i t o x o s i d e s o f d i g o x i g e n i n a n d o f c o n j u g a t e d p r o d u c t s , T h e s e m e t a b o l i t e s a r e more p o l a r t h a n o r i g i n a l g l y c o s i d e s . An i n c r e a s e d p l a s - m a - c h l o r o f o r m c o e f f i c j e n t i n d i c a t e s a c h a n g e i n t h e r a t i o o f p o l a r / n o n p o l a r s u b s t a n c e s i n t h e e x - t r a c t e d medium.

5 .2 P r o t e i n B i n d i n g

The p r o t e i n b i n d i n g o f d i g i t o x i n i s t h o u g h t t o a c c o u n t f o r t h e h i g h e r p l a s m a l e v e l s . The r e s u l t s were o b t a i n e d by t h e R b E 6 u p t a k e i n - h i b i t i o n t e c h n i q u e , s u g g e s t i n g i t s p r o b a b l e v a l u e as a c l i n i c a l l y a p p l i c a b l e q u a n t i t a t i v e me thod f o r t h e d e t e c t i o n o f commonly u s e d d i g i t a l i s g l y c o s i d e s i n p l a s m a . Com a r i s o n r e s u l t s b y two l a b o r a t o r i e s were p r e s e n t e d 2 ! .

The p r o t e i n b i n d i n g c a p a c i t y o f poor ly s o l u b l e c a r d e n o l i d e s i n wa te r i s d e t e r m i n e d f r o m t h e s a t u r a t i o n c o n c e n t r a t i o n o f t h e s e s u b s t a n c e s b o t h i n p r o t e i n s o l u t i o n and i n t h e i r u l t r a f i l - t r a t e i n m i c r o s c a l e . F i g u r e s a b o u t t h e b i n d i n g o f d i g i t o x i n , d i g o x i n e t c . t o human s e r u m p r o t e i n , and t h e b i n d i n g o f d i g i t o x i n t o s e r u m p r o t e i n s o f d i f f e r e n t s p e c i e s , a r e p r e s e n t e d . The c a r d e n o l i d e - s e r u m p r o t e i n b i n d i n g i s a f f e c t e d b y c a l c i u m i o n s 2 3 .

5 . 3 D e t e r m i n a t i o n i n B lood

5 . 3 . 1 C h e m i c a l Methods

D i g i t o x i n c o n c e n t r a t i o n i n t h e b l o o d o f o r a l l y d i g i t a l i z e d p a t i e n t s was q u a n t i - t a t i v e l y d e t e r m i n e d e m p l o y i n g a c o m b i n a t i o n o f T L C a n d a f l u o r o m e t r i c me thod :

30 m l v e i n - b l l d was d i l u t e d t o 300 m l w i t h w a t e r , a n d t h e h a e m o l y s a t was ex t r ac t ed w i t h c h l o r o f o r m , The c h l o r o f o r m e x t r a c t was e v a p - o r a t e d u n d e r m i l d c o n d i t i o n ( t e m p . n o t e x c e e d i n g 40OC.). The r e s i d u e was d i s s o l v e d i n 5 0 % a q u e o u s m e t h a n o l a n d t h e n e x t r a c t e d w i t h p e t r o l e u m e t h e r . R e m a i n i n g m e t h a n o l was e x t r a c t e d w i t h c h l o r o f o r m , The r e s i d u e a f t e r c h l o r o f o r m e v a p o r a t i o n ( r e d i s - s o l v e d i n a n e x a c t amoun t o f c h l o r o f o r m ) was a p p l i e d t o K i e s e l g e l G p l a t e s . Mob i l p h a s e : m e t h y l e n c h l o - ride/isopropanol/formamide, 8 0 : 1 9 : 1 . S p r a y r e a g e n t :

160

DIG ITOX IN

a m i x t u r e o f c h l o r a m i n e and t r i c h l o r o a c e t i c a c i d , After s p r a y i n g t h e p l a t e s were h e a t e d a t 115OC. f o r 10 min. O p t i m a l f l u o r e s c e n c e i n UV l i g h t was a t 365 nm24 .

t e r m i n e d by enzyme p - e s t e r h y d r o l a s e and ATP-ase i n h i b i t i o n t e c h n i q u e 2 ' .

D i g i t o x i n i n b l o o d p l a s m a was d e -

5.3.2 P h y s i c a l Methods C a r d i a c g l y c o s i d e s c a n b e d e t e r -

mined i n b i o l o g i c a l f l u i d s f rom c o n c e n t r a t i o n s o f 1 ng/ml by t h e i r i n h i b i t i o n o f t h e u p t a k e o f Rb by r e d b l o o d c e l l s . The g l y c o s i d e e x t r a c t was i n - c u b a t e d a t 3 7 ' C . f o r 2 h o u r s w i t h d i m e t h y l s u l f o - x i d e , r e d b l o o d c e l l s , and a s o l u t i o n o f RbC1. The Rb r e m a i n i n g i n t h e s u p e r n a t a n t i s m e a s u r e d by a t o m i c a b s o r p t i o n s p e c t r o m e t r y 2 6 ,

5.3.3 Radioimmunoassay ( D e u t e r i u m and T r i t i u m L a b e l e d D i g i t o x i n )

A l l t h r e e p r o - t o n s i n t h e u n s a t u r a t e d b u t e n o l i d e r i n g c a n b e e x c h a n g e d i n a b a s e - c a t a l y z e d p r o c e s s . The e x - c h a n g e t a k e s p l a c e e v e n u n d e r v e r y m i l d c o n d i t i o n s and t h e r i n g d o e s n o t open . D i g i t o x i n was t r e a t e d w i t h t r i e t h y l a m i n e and d e u t e r i u m o x i d e , and U V , IR and N N R d a t a showed t h a t t h e compound formed c o r r e s p o n d s t o t h e 21,21,22-trideuterodigi- t o x i n . Similar r e a c t i o n t a k e s p l a c e w i t h t r i t i u m o x i d e . The e x c h a n g e i s l i m i t e d t o t h e t h r e e p r o - t o n s i n t h e u n s a t u r a t e d b u t e n o l i d e r i n g 2 ' .

C l i n i c a l l y a p p l i c a b l e r ad io immuno- a s s a y t e c h n i q u e s f o r measu remen t o f s e rum d i g i t o x - i n h a v e b e e n u s e d t o d e t e r m i n e l e v e l s o f t h i s d r u g

se rum d i s p l a c e s t r i t i a t e d d i g i t o x i n ( a d d e d i n v i t - r o ) f rom s p e c i f i c a n t i b o d y b i n d i n g s i t e s . The p r o - c e d u r e r e q u i r e s o n e h o u r 2 ' .

i n man h a v e b e e n s t u d i e d u t i l i z i n g a s e n s i t i v e (0.2 n g / m l ) s p e c i f i c r a d i o i m m u n o a s s a y . P a t i e n t s r e c e i v i n g 0 . 1 mg o f d i g i t o x i n d a i l y had a mean se rum d i g i t o x i n l e v e l o f 25 n g j m l , and 4 4 ng /ml w a s d e t e c t e d i n p a t i e n t s r e c e i v i n g 0.2 mg d a i l y 2 ' .

f rom 2 5 0 p a t i e n t s . U n l a b e l e d d r u g i n t h e p a t i e n t s 8

The pha rmacodynamics o f d i g i t o x i n

161

IVAN M. JAKOVLJEVIC

I n a n o t h e r s t u d y u n l a b e l e d d i g i - t o x i n i n t h e unknown s a m p l e c o m p e t e s w i t h a t r i - t i a t e d d i g i t o x i n t r a c e r f o r b i n d i n g s i t e s o f h i g h a f f i n i t y r a b b i t a n t i b o d i e s t o a human s e r u m a l b u - men-d igox in c o n j u g a t e . Free l a b e l e d d i g i t o x i n was s e p a r a t e d f rom t h e a n t i b o d y - b o u n d f r a c t i o n b y a d - s o r p t i o n t o d e x t r a n - c o a t e d c h a r c o a l . The method i s s e n s i t i v e t o 2 ng/ml o r l e s s ” .

6 . METHODS O F ANALYSIS 6 . 1 I d e n t i f i c a t i o n T e s t s

D i s s o l v e a b o u t 1 m g of d i g i t o x i n i n 2 m l o f a s o l u t i o n p r e p a r e d b y m i x i n g 0 . 3 m l o f a 9 % a q u e o u s f e r r i c c h l o r i d e s o l u t i o n a n d 5 0 m l o f g l a c i a l a c e t i c a c i d , and u n d e r l a y w i t h 2 m l o f s u l f u r i c a c i d : a t t h e zone o f c o n t a c t o f t h e two l i q u i d s a brown c o l o r i s p r o d u c e d , and i t g r a d - u a l l y c h a n g e s t o l i g h t g r e e n , t h e n t o b l u e , and f i n a l l y t h e e n t i r e a c e t i c l a y e r a c q u i r e s a b l u e c o l o r 3 l ,

D i s s o l v e a b o u t 0 .2 mg o f d i g i t o x i n i n 2 m l o f a f r e s h l y p r e p a r e d 1 i n 100 s o l u t i o n o f m - d i n i t r o b e n z e n e i n e t h a n o l , and a l l o w t o s t a n d f o r 1 0 min, w i t h f r e q u e n t s h a k i n g . Add 2 m l o f a m i x t u r e o f 1 volume o f a 1 0 % t e t r a m e t h y l a m m o n i u m h y d r o x i d e and 200 volumes of e t h a n o l , and m i x : a r e d - v i o l e t c o l o r d e v e l o p s s l o w l y a n d t h e n f a d e s ” .

6 . 2 E l e m e n t a l A n a l y s i s

E l e m e n t a l a n a l y s i s o f d i g i t o x i n a s ‘4 l H 6 4’1 3 :

C - 6 4 . 4 %

H - 8 . 4 %

0 - 27.2%

6 . 3 Chromatography

6 . 3 . 1 Column C h r o m a t o g r a p h y Aluminum o x i d e , s i l i c e o u s e a r t h ,

and S e p h a d e x a r e a d s o r b e n t s commonly u s e d f o r t h e s e p a r a t i o n o f c a r d e n o l i d e g l y c o s i d e s a n d t h e i r m e t a b o l i t e s ,

162

DIGITOX IN

The USP X V I I I employs a column o f s i l i c e o u s e a r t h p r e v i o u s l y c l e a n 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 n a c t i v a t e d a t 500°Cc. Formamide i s added a s t h e s t a t i o n a r y p h a s e . D i g i t o x i n i s e l u t e d w i t h a m i x t u r e o f b e n z e n e / c h l o r o f o r m , 3 : l .

Aluminum o x i d e d e a c t i v a t e d w i t h 3% wa te r and p a c k e d i n a column o f 1.5 cm d i a m e t e r t o a height o f 10 cm h a s b e e n u s e d s u c c e s s f u l l y f o r t h e s e p a r a t i o n o f d i g i t o x i n f rom i t s m e t a b o l i t e s . E l u t i o n i s a c h i e v e d w i t h 100 m l o f c h l o r o f o r m f o l - lowed by 35 m l o f 2 % e t h a n o l i n c h l o r o f o r m , a n d f i n a l l y w i t h 2 5 0 m l o f 10% e t h a n o l i n c h l o r o f o r m . D i g i t o x i n and i t s m e t a b o l i t e s a r e f o u n d i n t h a t p o r t i o n o f e l u a t e b e t w e e n 275 and 425 m l ( t h i s i n c l u d e s t h e c h l o r o f o r m p r e w a s h ) 1 2 .

S e p h a d e x G-200, s w e l l e d w i t h a m i x t u r e o f w a t e r / m e t h a n o l , 7 : 3 , was employed f o r t h e s e p a r a t i o n o f d i g i t o x i n and i t s m e t a b o l i t e s . Sephadex was p a c k e d i n a column o f 1 cm d i a m e t e r t o a h e i g h t o f 15 cm. The s a m p l e was e l u t e d u s i n g t h e above m i x t u r e . C a r d e n o l i d e s were i n t h e f r a c - t i o n be tween 1 0 and 2 5 m 1 3 ’ .

6 . 3 .2 T h i n L a y e r Chromatography A r a p i d s e p a r a t i o n o f d i g i t o x i n

from d i g o x i n a n d a c e t y l d i g i t o x i n c a n b e a c h i e v e d a p p l y i n g 2p1 o f a 0 .01% s a m p l e s o l u t i o n i n c h l o - r o f o r m / m e t h a n o l , 1 : l t o K i e s e l g e l G p l a t e s . A s t h e e l u e n t a c h l o r o f o r m / m e t h a n o l , 9 : l m i x t u r e was u s e d , D e t e c t i n g a g e n t : h y d r o c h l o r i c a c i d . The s p o t s o f d i g i t o x i n (Rf 0 . 3 3 ) , d i g o x i n (Rf 0.24) and a c e t y l d i g i t o x i n (Rf 0.48) were d a r k brown a f t e r 5 min. Dry ing a t l l O ° C . f o r 5 min and exam- i n a t i o n u n d e r u l t r a v i o l e t l i g h t (365 nm) showed brown s p o t s f o r d i g i t o x i n and a c e t y l d i g i t o x i n , w h i l e d i g o x i n s p o t was b l u e . The l i m i t o f d e t e c - t i o n i s 0 . 0 5 mcg3’ .

D i g i t o x i n was s e p a r a t e d f rom d i - g o x i n on S i l i c a Gel G p l a t e s w i t h c h l o r o f o r m / m e t h - a n o l , 88:12 d e v e l o p i n g s o l v e n t . The z o n e s were l o c a t e d by s p r a y i n g w i t h 1% i o d i n e i n c h l o r o f o r m , t h e n removed from t h e p l a t e and e x t r a c t e d w i t h c h l o r o f o r m / m e t h a n o l , 1 : l m i x t u r e . A f t e r c e n t r i - f u g a t i o n , a 7 m l a l i q u o t of s u p e r n a t a n t was e v a p -

163

I V A N M. J A K O V L J t V l C

o r a t e d t o d r y n e s s . The r e s i d u e was d r i e d and t h e n t r e a t e d w i t h d i x a n t h y l u r e a r e a g e n t and t h e c h r o - mophor r e a d a t 535 nm34) 35.

A method f o r t h e d i r e c t q u a n t i t a - t i v e e v a l u a t i o n o f d i g i t o x i n , d i g o x i n and a c e t y l - d i g i t o x i n on T L C u s i n g s p e c t r o f l u o r o m e t r y was i n - v e s t i g a t e d . The o n l y r e a g e n t u s e d was h y d r o c h l o - r i c a c i d , L i n e a r s t a n d a r d c u r v e s were o b t a i n e d when t h e a r e a u n d e r t h e f l u o r o m e t r i c c u r v e was c o r r e l a t e d w i t h t h e amount o f g l y c o s i d e s a p p l i e d . The o p t i m a l r a n g e f o r t h e s p e c t r o f l u o r o m e t r i c d e - t e r m i n a t i o n o f t h e s e t h r e e g l y c o s i d e s was a b o u t 0 .25 m c g j 6 .

S e p a r a t i o n o f t h e c a r d i a c g l y c o - s i d e s d i g i t o x i n and d i g o x i n from t h e i r 2 0 , 2 2 - d i - h y d r o d e r i v a t i v e s c a n b e a c h i e v e d b y m u l t i p l e TLC on c e l l u l o s e f i lms ’.

An u l t r a m i c r o f l u o r e s c e n t s p r a y r e a g e n t f o r d e t e c t i o n and q u a n t i t a t i o n o f d i g i - t o x i n and o t h e r c a r d i o t o n i c g l y c o s i d e s on T L C was d e s c r i b e d . The s p r a y r e a g e n t c o n s i s t s o f a s c o r b i c a c i d , m e t h a n o l , h y d r o c h l o r i c a c i d and h y d r o g e n p e r o x i d e . The l imi t s o f d e t e c t i o n were 0.01 mcg3’ ,

A p p l i c a t i o n of d i f f u s i o n and f l u - o r e s c e n c e t o t h e d i r e c t d e t e r m i n a t i o n of d i g i t o x - i n was a c h i e v e d by c o n v e r t i n g d i g i t o x i n i n t o a f l u o r e s c e n t d e r i v a t i v e by means o f a r e a g e n t con- t a i n i n g p - t o l u e n e s u l f o n i c a c i d , h y d r o c h l o r i c a c i d , a s c o r b i c a c i d and h y d r o g e n p e r o x i d e . S e n s i t i v i t y : 0 .3-1 rncg3’.

p l a t e s h a s b e e n d e v e l o p e d u s i n g as t h e m o b i l e s o l v e n t a m i x t u r e o f m e t h y l e n e c h l o r i d e / m e t h a n o l / formamide, 80 :19 :1 , and s p r a y i n g t h e p l a t e s w i t h a c i d - f e r r i c c h l o r i d e j l . The same t e c h n i q u e can b e u s e d f o r t h e i d e n t i f i c a t i o n o f d i g i t o x i n , d i g o x i n and a c e t y l d i g i t o x i n , and f o r t h e d e t e r m i n a t i o n o f a n y g i t o x i n p r e s e n t i n t h e i r d r u g f o r m u l a t i o n s , D igox in i s n o t a c t i v a t e d t o v i s i b l e f l u o r e s c e n c e a t room t e m p e r a t u r e by a c i d - f e r r i c c h l o r i d e re- a g e n t . T h e r e f o r e any f l u o r e s c e n c e p r e s e n t immedi- a t e l y a f t e r s p r a y i n g i s d u e t o g i t o x i n a l o n e , H e a t i n g t h e p l a t e a t 100°C. d e s t r o y s t h e g i t o x i n f l u o r e s c e n c e and c o n v e r t s t h e d i g o x i n t o a f l u o -

A T L C s y s t e m on s i l i c a g e l G

164

DIGITOXIN

r e s c e n t a n h y d r o d e r i v a t i v e w h i c h may b e seen u n d e r b o t h U V a n d v i s i b l e l i g h t " ' .

6 . 3 . 3 P a p e r C h r o m a t o g r a p h y

Af te r s e p a r a t i o n by p a p e r c h r o m a - t o g r a p h y i n f o r m a m i d e s a t u r a t e d m e t h y l e t h y l k e t o n e / x y l e n e , 1: l m i x t u r e , d i g i t o x i n and d i g o x i n were d e t e r m i n e d w i t h x a n t h y d r o l ( 1 0 - f o l d excess o f r e - a g e n t i n a c e t i c a c i d / h y d r o c h l o r i c a c i d , 9 9 : l m i x - t u r e ) . I t was n e c e s s a r y t o h e a t t h e r e a c t i o n m i x - t u r e f o r 2 0 min . a t 60'C. A 1 : l c o m p l e x ( A m a x 535 nm) , s t a b l e f o r 3 h o u r s was f o r m e d . Beer's law was o b e y e d o v e r t h e r a n g e 1 -20 m ~ g / m l " ~ * " * .

A b u t e n o l i d e r i n g s p e c i f i c me thod o f q u a n t i t a t i v e p a p e r c h r o m a t o g r a p h i c a n a l y s i s o f d i g i t o x i n u s i n g 2,4,2',4'-tetranitrodiphenyl i s d e s c r i b e d . P a p e r : S c h l e i c h e r a n d S c h U l l 2043b i m - p r e g n a t e d w i t h f o r m a m i d e . D e v e l o p i n g s o l v e n t : methylethylketone/xylene, 1:l s a t u r a t e d w i t h f o r - mamide. The c h r o m a t o g r a m was d r i e d f o r 1 5 min. a t 60'C. "'

6.3 .4 Gas C h r o m a t o g r a p h y

T r i m e t h y l s i l y l e t h e r d e r i v a t i v e s o f d i g i t o x i n , d i g o x i n a n d g i t o x i n h a v e b e e n shown t o b e r e s o l v a b l e on a g a s C h r o m a t o g r a p h i c co lumn p a c k i n g c o n t a i n i n g a s a l i q u i d p h a s e 2 .5% O V - 1 o r OV-17 on Chromosorb W. Gas c h r o m a t o g r a p h y was p e r - fo rmed on a Barbe r -Co lman 5000 s e r i e s i n s t r u m e n t e q u i p p e d w i t h h y d r o g e n f l a m e d e t e c t o r . D u r i n g i s o - t h e r m a l o p e r a t i o n i n j e c t i o n p o r t a n d co lumn b a t h t e m p e r a t u r e s were i d e n t i c a l , D e t e c t o r t e m p e r a t u r e was m a i n t a i n e d a t 34OoC. I n t h e c a s e o f t e m p e r a - t u r e p r o g r a m m i n g , i n j e c t i o n p o r t t e m p e r a t u r e was i d e n t i c a l t o t h e s t a r t i n g t e m p e r a t u r e : 240 'C.""

c h r o m a t o g r a p h i c i d e n t i f i c a t i o n o f d i g i t a l i s c a r - d e n o l i d e s as t h e i r a n h y d r o d e r i v a t i v e s h a s b e e n d e v e l o p e d , r e s u l t i n g i n g r e a t l y r e d u c e d r e t e n t i o n t imes and e n h a n c e d r e s o l u t i o n . R e t e n t i o n d a t a o f 18 c a r d e n o l i d e s on t h r e e l i q u i d p h a s e s a r e r e p o r t - e d . S p e c t r a l e v i d e n c e i s p r e s e n t e d s h o w i n g t h a t t h e t e r t i a r y 14B-OH g r o u p i s n e i t h e r a f f e c t e d b y e s t e r i f i c a t i o n n o r e t h e r i f i ~ a t i o n ~ ~ .

An i m p r o v e d me thod f o r t h e g a s

165

IVAN M. JAKOVLJEVIC

6.3.5 High Speed Liquid Chromatography High speed liquid chromatography

has been used to examine steroids and steroid conjugates such as digitoxin or digoxin. In these studies reverse phase liquid partition chromatography was employed. The columns consisted of a cyanoethylsilicone polymer (Dupont ZipaxR ANH) and the mobile phase was a mixture containing 2.5% methanol and 97.5% water, The compounds were detected by means of a 254 nm photometerk6.

6.4 Colorimetric Analysis

The methods for the determination of cardiac glycosides can be divided into three general groups based on: 1- the sugar moiety, 2 - the butenolide moiety, and 3 - the steroid part of the molecule.

methodk7 was published using equal amounts of sulfuric acid and ethanol with the addition of ferric chloride. Others4’ used a solution of ferric sulfate in concentrated sulfuric acid, or added ferric chloride to a solution of glycoside in acetic acid and then underlaid the Kiliani reagent“’. Many methods employ xanthydrol as the reagent for digitoxoseSo.

2- The reagent employing picric acid in alkaline ethanol is the most frequently used5’. There are many modifications and applications of

1- As far back as 1885, a colorimetric

this reactions2# 5 3 ) 54? 5 5 ? The official U S P XVIII method depends on

a chromatographic separation on siliceous earth in the presence of formamide, and a reaction in the butenolide side chain by picric acid in alkaline solution.

The application of m-dinitrobenzene for the quantitative determination of cardiac glycosides is very successfuls6, as well as 1,3,5-tri- nitrobenzene in alkaline medium5’. Some authors use 2,4-dinitrodiphenylsulfone in alkaline ethanol”. The other rea ents used are 2-naphtho- quinone -4 -sul f onat e ’ ‘ , and 2 , 2 , 4.4 - t e t ran i t ro- diphenyl ’.

166

DIG ITOX IN

The r e a g e n t s b a s e d upon t h e r e a c t i o n i n b u t e n o l i d e r i n g h a v e a w i d e a p p l i c a t i o n i n c a r d i - a c g l y c o s i d e s m e t a b o l i s m s t u d i e s , T h e s e r e a g e n t s r e a c t w i t h a n y g l y c o s i d e o r i t s m e t a b o l i t e t h a t s t i l l c o n t a i n s t h e i n t a c t b u t e n o l i d e r i n g .

t h e s t e r o i d m o i e t y a r e f o r t h e most p a r t f l u o r o - m e t r i c ,

3- Methods b a s e d upon t h e r e a c t i o n i n

6 . 5 F l u o r o m e t r i c A n a l y s i s

Methods b a s e d upon t h e r e a c t i o n i n t h e s t e r o i d m o i e t y . ' a r e m a i n l y d e h y d r a t i o n t y p e of re - a c t i o n s s u c h as t h a t u s i n g s y r u p y p h o s p h o r i c a c i d 6 2 , o r e q u a l amoun t s o f h y d r o c h l o r i c a c i d and g l y c e r o l as t h e d e h y d r a t i n g a g e n t s 6 3 . Hydro e n

a m i x t u r e o f s u l f u r i c a n d p h o s p h o r i c a c i d s w i t h t h e a d d i t i o n o f f e r r i c c h l o r i d e 6 6 a r e a l s o u s e d .

a c e t i c a n h y d r i d e , a c e t y l c h l o r i d e a n d t r i f l u o r o - a c e t i c a c i d , s u p p o r t s t h e t h e o r y , b a s e d on N M R , I R and f l u o r e s c e n c e a c t i v a t i o n s p e c t r a l d a t a , t h a t a low y i e l d o f a h i g h l y c o n j u g a t e d f l u o r o p h o r O f s u b s t i t u t e d 3 .4 -benzpyrene i s o b t a i n e d 6 7 .

p e r o x i d e , h y d r o c h l o r i c a c i d and m e t h a n o l 6 4 ' 8 , , o r

The f l u o r o p h o r obtained with a mixture of

6 . 6 E l e c t r o D h o r e s i s ~ ~~ ~~

D i g i t o x i n may be d e t e c t e d and e s t i m a t e d i n human a u t o p s y t i s s u e s by p a p e r e l e c t r o p h o r e s i s a c c o r d i n g t o a n a u t h o r 6 * who u s e d a m i x t u r e o f o x a l i c a c i d , b o r i c a c i d and e t h a n o l t o d e v e l o p d i f f e r e n t c o l o r s d e p e n d i n g upon t h e compound. The l i m i t f o r i d e n t i f i c a t i o n was a b o u t 15 mcg f o r d i - g i t o x i n , and 1 0 mcg f o r d i g i t o x i g e n i n . The d i g i - t o x i n was t o t a l l y d e g r a d e d i n t h e t i s s u e s f o l l o w - i n g p u t r e f a c t i o n f o r t h r e e m o n t h s 6 ' ,

6 . 7 Automated Assay

An a u t o m a t e d p r o c e d u r e u s i n g a s t a n d a r d T e c h n i c o n a u t o m a t i c a n a l y z e r s y s t e m is d e s c r i b e d f o r t h e u n i t dose a n a l y s i s o f d i g i t o x i n and d i - g o x i n i n t a b l e t s 7 ' . The t e c h n i q u e i s b a s e d on t h e f l u o r o m e t r i c measu remen t o f t h e d e h y d r a t i o n p r o d - u c t s of t h e c a r d i o t o n i c s t e r o i d s r e s u l t i n g f rom t h e i r r e a c t i o n w i t h h y d r o g e n p e r o x i d e and h y d r o -

167

IVAN M. JAKOVLJEVIC

c h l o r i c a c i d , T h e a u t o m a t e d s y s t e m a s d e s c r i b e d i s c a p a b l e o f a n a l y z i n g 1 2 t a b l e t s p e r h o u r ,

7 . C L E A V A G E O F C A R D I A C GLYCOSIDES

m a t e r i a l i s made d i f f i c u l t by t h e l a c k o f r e l i a b l e methods t h a t w i l l m a i n t a i n t h e g e n i n s i n t a c t a f t e r h y d r o l y s i s o f t h e i r g l y c o s i d e s .

A p r o c e d u r e i s d e s c r i b e d f o r c l e a v i n g t h e s u g - a r bond o f d i g i t o x i n i n o r g a n i c media and u n d e r m i l d a c i d c o n d i t i o n s . Maximum y i e l d s were o b t a i n e d i n 30 min. o f r e a c t i o n t ime a t 5OoC: i n a medium c o n s i s t i n g o f a n h y d r o u s t e t r a h y d r o f u r a n made 0 .002 N w i t h r e s p e c t t o p e r c h l o r i c a c i d . D i g i t o x i g e n i n i s s t a b l e u n d e r t h e s e condition^^^.

The i s o l a t i o n o f c a r d i a c a g l y c o n e s from p l a n t

8 . B I O L O G I C A L A C T I V I T Y 8 . 1 C h a r a c t e r i s t i c S t r u c t u r a l F e a t u r e s

From t h e s t u d i e s o f b i o l o g i c a l a c t i v i t y 7 2 f o u r c h a r a c t e r i s t i c s t r u c t u r a l f e a t u r e s o f t h e g e n i n s c a n b e e a s i l y i d e n t i f i e d as e s s e n t i a l f o r c a r d i a c a c t i v i t y :

1- The L a c t o n e Ring: The d o u b l e bond o f t h e l a c t o n e r i n g i s a p p a r e n t l y n e c e s s a r y f o r c a r - d i a c a c t i o n . The r u p t u r e o f t h e l a c t o n e r i n g r e - s u l t s i n a loss o f c a r d i a c a c t i o n .

2 - The Hydroxy Group on C - 1 4 Atom: T h i s hydroxy g r o u p i s i m p o r t a n t and i t s m o d i f i c a t i o n r e s u l t s i n a s i g n i f i c a n t l o s s o f a c t i v i t y . I f t h i s g r o u p i s removed, t h e i m p o r t a n t s t e r e o c h e m i c a l re- l a t i o n s h i p i s d e s t r o y e d s o t h a t l o s s o f a c t i v i t y c o u l d b e d u e e i t h e r t o t h e loss o f t h e 1 4 - h y d r o x y l g r o u p , o r t o t h e a l t e r a t i o n o f t h e c i s C / D r i n g a r r a n g e m e n t 3 ,

a g l y c o n e , t h e a t t a c h m e n t o f o n e o r more s u g a r s a t C-3 u s u a l l y r e s u l t s i n i n c r e a s e d a c t i v i t y .

4 - S t e r e o c h e m i c a l A r r a n g e m e n t s : A l l c i s l t f u s i o n o f t h e C and D r i n g s i s n e c e s s a r y f o r a c - t i v i t y . O t h e r t y p e s o f n a t u r a l s t e r o i d s h a v e t h e " t r a n s " c o n f i g u r a t i o n . The p r e s e n c e o f two h y d r o x - y l g r o u p s a t C - 1 2 and C-16 i n t h e m o l e c u l e o f d i -

3- S u g a r s a t C-3 Atom: I n t h e c a s e o f t h e

DIGITOXIN

gitoxigenin diminishes the activity hy two-thirds. 14~~,15u-epoxy-14-anhydrodigitoxigenin is practically inactive.

8.2 Bioassay

Experiments were made t o examine the suktability of young chicks for the assay of digitalis glycosides, The jugular vein was cannu- lated and a volume of the test solution was in- fused. The dose was repeated at 5 min. intervals until cardiac arrest was noticed. The order of susceptibility of tested animals was as follows: pigeon > chick> rat74.

9. ACKNOWLEDGMENT The author acknowledges the invaluable help

of Miss Adele Hoskin for her assistance in t h e 1 it e rat ure search.

l o . REFERENCES

1. Chen,K.K.,Ann.Rev.Physiol. 7,677 (1945). 2. Kossoy,A.D. and Underbrink,F.D.,Eli Lilly and

Company, personal communication, 3 . Khalique,A. et al., Sci.Res.Pakistan 3,177

(1966). 4. Takuma,O. et al., Bunseki Kagaku, 17(1),53

5. Boaz,H.E.,Eli Lilly and Company, personal

6. Voigtlaender,H.W. et al., Arch.Pharm. 301,208

7. Beasly,F.W., Eli Lilly and Company, personal

8 . The Merck Index, VIII Edition, 1968. 9. British Pharmacopoeia, 1968.

(1968).

communi cat i on.

(1968).

communication.

10, Djerassi,C. et al., Helv.Chim.Acta 41,250 (1958).

11, Fieser and Fieser, Steroids, Reinhold Publish.

12. Mueller,L. , Microchem.J., z(1) ,20 (1968).

-

Corp., 1959.

IVAN M. JAKOVLJEVIC

13.

14. 15.

16. 17. 18. 19.

20.

21. 22. 23.

24.

25. 26.

27.

28.

29. 30.

31. 32.

Parson et al., Henry Ford Hosp.Med.Bull.,

Hilton,J.G,, Science, 110,526 (1949). Hilton,J.G., J.Pharmacol,Exptl.Therap.

100,258(1950). Danieli,N. et al., Tetrahedron 22,3189(1966). Lutomski, J. , Herba Pol. 12,243 (1966). Samuelsson,G., J.Mond,Pharm. 4,112 (1961). Samuelsson,G., Acta Pharm.Suecica - 1(6),227 Clarke,E.G.C., Isolation and Identification

Glover,A., Klin.Wochenschr. 49,1062 (1971). Shapiro,W., Circulation 4O0supp1.3,l84(1969). Pfordte,K. et al., Acta Biol.Med.Germ. 25,

19 (1970). Seipe1,H. et al., Klin.Wochenschr. - 46,1257

Bentley,J.D., Circulation 41(1),67 (1970). Bourdon,R. et al., Ann.BiorClin.(Paris) 27

6,365 (1958).

(1964).

of Drugs, London, 1969.

(1968).

(10-12) ,651 (1969). Haberland,G. et al. , Naturwissensch, 56(10) ,

516 1196n. Smith,T.W. et al., Circulation 4O(Suppl.3),

Morrison,J. et al., Clin.Res. 18(4),668(1970). Smith,T.W., J.Pharrnacol.Exptl.merap.l75(2),

1 8 8 n969).

352 (1970). The United States Pharmacopeia, XVIII. Pfordte,K. et al., Z.Med.Labortechn. - 11,272

(1970). 33. Hoeke,M. et al., Pharm.Weekhlad 104,877

34. Rican-Fister et al., J-Chromatogr. 41(1),91

35. Johnston,E. J. et a1 . , J. Pharm.Sci. 55 ( 5 ) ,531

36. Frijns,J.M.G,J., Pharm.Weekblad 105,209

37. Rabitzsch,G., J.Chromatogr, 35,122 (1968) * 38. Jelliffe,R.W. et a l . , J.Chromatagr. - 27,172

39. Eide-Juergensen et al., Plants Med.98

- (1969).

(1969).

(1966) . (1970).

- -

(1967).

(Suppl.5) ,112 (1971).

DIG ITOX IN

40. Dow,M.L. et al., J.Pharm.Sci. %(2),298 (1971).

41. Dzyuba,N.P. et al., Pharm.Zh. (Kiev) 26(3) ,42 - f 1971).

42. Dzyuba,N.P. et al,, Khim.Farm.Zh. S(llj,Sl - (1971).

43. Rabitzsch,G. et al., J.Chromatogr. 4l,96 (1969).

44.

45. 46.

47. 48. 49. 50. 51. 52.

53.

54. 5 5 .

56. 5 7 . 58. 59.

60.

61.

Wilson,W.E. et al., Analyt.Chem. 41(6),810

Tan,L., J.Chromatogr. 45,68 (1968). Henry,R.A. et al., J.Chromatogr.Sci. 9,513

Lafon,P., Compt.Rend. 100,1463 (1885). Kiliani,H., Arch.Pharm. 234,273 (1896). Keller,C., Ber.Pharm.Ges. 5,275(1895). Pesez,M., Ann.Pharm.Franc.-ED104 (1952). Baljet,H., Schweiz.Apoth.Ztg. s,71,84 (1918). Bel1,F.K. et al., J.Am.Pharm.Assoc.,Sci.Ed.

37,297 (1948). Bel1,F.K. et al., J.PharmaZl.Exptl.Therap.

88,14 (1946). Dyer,F.J., Quart.J.Pharm. 5,172 (1932). Kennedy,E.E., J.Am,Pharm.Assoc.,Sci.Ed.

39,25 (1950). Morel,A., Bull.Soc.Chim.FrGce 5 ( 2 ) ,949(1935). Kimura,M., J. Pharm.Soc. Japan 27991 (1951). Tattje,D.H.E., Pharm,Weekblad 93,245 (1958). Warren,A. T. et al. , J.Am. Pharmxssoc. ,Sci .Ed. Doelker E. et al., Pharm.ActaHelv. 44,647

Rabitzsch,G. et al. , Pharmatie a ( 5 ) ,262

- (1969).

(1971).

-

37,186 (1948).

(1969).

(1969). 62. Petit,A. et al., Bull.Soc.Chim.France-~,288

(1950). 63. Jensen,K.B., Acta Pharmacol.Toxico1. 8,101

T1952).

64. Jensen,K.B., Ibid. 9,66 (1953). 65. Wells,D. et al., J.Fharm.Pharmaco1. 13,389

66. Tattje,D.H.E., J.Pharm.Pharmaco1, 6,476(1954). 67. Jakovljevic,I.M., Anal.Chem. 35(10),1513

(1961).

(1963).

171

IVAN M. JAKOVLJEVIC

68. Dyes,H.P., Pharm.Weekblad 95,682 (1960). 69, Bors,G. et al., Farmacia (Bucharest) 15(5),

70. Cullin, L.F. et al. , J.Pharm.Sci. 59(5),697 (1970).

71. Frey,M.J. et al., Anal.Biochem. 36,78 (1970). 72. Henderson,F.G., Digitalis, G r u n e E Stratton

Publ.,New York-London 1969. 73. Wright,S.E., The Metabolism of Cardiac Glyco-

sides, Charles C. Thomas Publ. 1960. 74. Fukuda,H. et al,, J.Pharm.Soc.Japan 89(6),

269 (1967).

-

866 (1969).

***********

During the preparation of these analytical profiles, the following, most recent, papers on different topics of digitoxin have been found:

1- Gisvold,O. Acetyldigitoxin and acetyldigoxin from Digitalis lana- ta. J.Pnarm.Sci. 61, 1320 (1972).

Extraction of digitalis leaves with the aid of ultra- sonics. Pharmazie 27, 615 (1972).

3- Watson, E. et al. Identification of submicrogram amounts of digoxin, digitoxin ..... Isolation by chromatography., .. J. Chromatogr. - 69, 157 (1972).

4- Potter, H. et al. TLC analysis of digitalis glycosides. Pharmazie 27, 315 (1972).

Assay of digitalis in the blood. Prog.Cardiovasc.Dis. - 14, 571 (1972).

Determination of digoxin and digitoxin in the blood.. . Klin.Wochenschr. - 51, 57 (1973).

- 2- suss, w.

-

- 5- Butler, V.P.,Jr.

6- Bodem, G. et al.

THE L l T E R A T U R E SEARCff WAS CONDUCTEV UP TO MAY 1 9 7 3 .

172

DIPHENHYDRAMINE HYDROCHLORIDE

Ira J. Holcomb and Salvatore A. Fusari

IRA J. HOLCOMB AND SALVATORE A. FUSARI

1. Description

1.1 Name, Formula, Molecular Weight 1 . 2 Appearance, color, Odor

2 . physical properties

2 . 1 2.2 2 - 3 2 -4 2 -5 2 .6 2 - 7 2 .8 2.9

2.10 2 . 1 1 2.12 2.13

Infrared Spectrum Nuclear Magnetic Resonance ultraviolet Spectrum Mass Spectrum optical Rotation Me It ing Range Differential Thermal Analysis Solubility crystal Properties 2 . 9 1 optical crystal Properties 2.92 X-Ray Diffraction

Distribution Coefficients Aggregation: Micelle Formation PK; values Metal Complex Formation and Binding

3 . Synthesis

4. Stability - Degradation 5 . Drug Metabolic Products - Pharmacokinetics

6. Identification: Microchemical Tests

7 . Methods of Analysis

7.1 Elemental Analysis 7.2 Spectrophotometric Analysis

174


7.21 Elemental Analysis 7.22 Separation Methods Prior to

7.23 Methods Based on Conversion to Spectrophotometric Assay

Benzophenone Prior to Spectro- photometric Assay

Chloranilic Acid Prior to Spectrophotometric Assay

7.24 Method Based on Conversion to

7.3 Colorimetric Analysis 7.31 Ion-Pair Extraction Methods 7.32 Ammonium Reineckate Methods 7.33 Picric Acid Method 7.34 Method Based on Molle Reaction 7.35 Miscellaneous Colorimetric Methods

7.4 Titrimetric Analysis 7.41 Direct Methods of Titration 7.42 Separation Prior to Titration

7.421 Reineckate salt Formation 7.422 Complexometric Method 7.423 Slurry Method 7.424 Ion Exchange Method 7.425 Extraction Method

7.43 Miscellaneous Titrimetric Methods

7.5 Fluorometric Analysis 7.6 Automated Analysis 7.7 Biological Assay 7.8 Gravimetric Analysis 7.9 Chromatography

7.91 Paper Chromatography 7.92 Thin Layer chromatography 7.93 Gas Chromatography

175

IRA J. HOLCOMB A N D SALVATORE A. FUSARI

7.931 Direc t Methods on N e u t r a l columns

7.932 Di rec t Methods on B a s i c columns

7.933 o x i d a t i o n to Benzophenone P r i o r t o G a s Chromato- graphy

7.94 Column chromatography 7.95 Electrophoresis

8.0 References

176


1. Descr ip t ion 1.1 N a m e , Formula, Molecular Weiqht

Diphenhydramine hydrochlor ide i s 2- (dipheny1methoxy)-N,N-dimethylethylamine hydroch lor ide -1 Six a d d i t i o n a l chemical names are l i s t e d i n The Merck Index2, along wi th twelve t r a d e names. One a d d i t i o n a l name i s Dimedrol.

The empir ica l formula i s C17H21NO'HCl with a molecular weight of 291.82.

. HC1

The CAS Regis t ry Number is 58-73-1 for 2- (diphenylmethoxy) -N, N-dimethylethylamine and f o r the hydrochlor ide , 147-24-0.

1.2 Appearance , c o l o r , odor 1 White, odor l e s s , c r y s t a l l i n e powder.

2. Physical P rope r t i e s 2 . 1 I n f r a red Spectrum

The i n f r a r e d spectrum of diphenhydramine hydrochlor ide i s presented i n Figure 1. The S a d t l e r Reference Number i s 9382. The spectrum is used f o r c o n t r o l pur oses .1 i3 Spectra a r e presented by de ~ o o s 'I4 and Wallace. 5

The i n f r a r e d band assignments a r e given i n Table I .

177

Y C 0

E c - .-

Frequency (cm-1)

Fig. 1. In f rared Spectrum of Diphenhydramine Hydrochlor ide, U.S.P. Parke-Davis 8 Co.

Lot No. 563463. Instrument: Perkin-Elmer 621, Phase: KBr, 1:300.

c 0 -I4 4J a

h

&

Q

u-4 0

c 0 ul m

0

m

E c, aJ &

U

in id k

m

rl

V

3: N

h

Z 1

-rl 3

rl

a 4J aJ rl

aJ

% tn c -4

&

id E 0

k

m

F

c .I4 a E: Q

) n

F

c a E: al

n

i?

u

tn c .d

4J a, k

4J m

% U

I 0 I

u

aJa G

@

mu

.d

179


2.2 Nuclear Maqnetic Resonance In Figure 2 the nuclear magnetic

resonance spectrum of diphenhydramine hydrochloride is presented. The spectral peak assignments7 are presented in Table 11. The Sadtler NMR Reference Number is 14360.

2.3 ultraviolet Spectrum The ultraviolet spectrum of diphenhy-

dramine hydrochloride is presented in Figure 3. The absorptivities at 258 nm. listed in Table 111 gompare well with the literature values of 15.4 in methanol and 16.59 in an aqueous system.

11 2.4 Mass Spectrum, LOW Resolution A plot of the relative intensities vs.

mass/charge ratio is presented in Figure 4 and summarized in Table IV. The ionization potential is 70 electron volts.

2.5 Optical Rotation Diphenhydramine hydrochloride is not

optically active.

2.6 Melting Range

the range 167O to 172°C.’ which the compound melts is usually less than 2OC. The melting point is affected by the rate of heating1* as shown in Table v.

Diphenhydramine hydrochloride melts in The actual range in

Data was obtained from melting point scans using the Mettler FP-1.

180

c W c

Fig. 2. Nuclear Magnetic Resonance Spectrum of Diphenhydramine Hydrochloride, U.S.P.,

Parka-Davis & Co. l o t No. 5 6 3 4 6 3 .

Instrument: Varian A-60. Solvent. D20. Sweep Offset: 0 cps.

x7 167 Y Y 0 0

d

m .

&i

u I 0

In

?

m

I m

?

‘c!

182

Fig. 3. Ultraviolet Spectrum of Diphenhydramine Hydrochlor ide, U.S.P., Parke-Davis & Co.

Lot NO. 563463. Instrument: Cory 14.

IRA J. HOLCOMB A N D SALVATORE A. FUSARI

Table 111. Absorptivities'O of D iphenhydr amine Hydrochloride , Parke, Davis & Co., Lot N o . 563463

Aqueous Medium (pH 3):

wavelength a (l%, 1 cm.)

267 nm(s) 9.3

263 nm(s) 12.7

257.5 nm(s) 16.3

252 nm 13.95

Methanol, absolute:

268 nm 7.95

264 nm 12.1

258 nm 15.5

252 nm 12.8

e

270

370

476

4 06

-

232

353

452

374

184

W

BC

- 70 K - 0

C

-u C

Y

8 60

a n * 5 0

: 4c

> 0 .- + -

30

m

lc

I X I 0

L 1 X l o o

r/l 200 A 250

Fig. 4. Mass Spoctrum of Diphenhydramine Hydrochloride, U.S.P., Parke-Davis & CO. Lot NO. 563463.

lnstru men t: F i n n i g a n Qua dr u pol e Mass Spectr o mete r, M ode I 101 5.


Table Iv Low Resolution Mass Spectrum Assignments

f o r Diphenhydramine Hydrochloride

Measured Re la t ive Mass I n t e n s i ty S t r u c t u r a l Assignments

183

16 7

256 10.67

10 -0

30 .O

16 5

15 2

52.67

( O \ H

(oyc -

a

H -CH2 -N

4-

,CH3 CH3

4-

0 C13H110

+

C13Hll

+

22.00pJ-$J1+

C13H9

C12H8

186


Table Iv( cont .)

Measured Relative Mass Intensity Structura l Assiqnments

58

45

100

12

C3H8N

2H7

187

Table V. Melting Poin t and R a n g e 1 2 of D i p h e n h y d r a m i n e

H y d r o c h l o r i d e , Parke, D a v i s & co . , L o t N o . 563463

Star t Temp. ( O c .) H e a t i n g R a t e R a n q e Mid-Point

163

158 O

1 OC/min. 167 -7-168 -6 (0 -9) 168.1 168.2 167 -7-168 -7 (1 - 0 )

3 OC/min . 168 -7-169.3 (0 -6) 169 -0 169 -4 168 -7-170 -0 (1 - 3 )


2 . 7 D i f f e r e n t i a l Thermal Analysis The DTA curve obtained using a Met t le r

TA 2000 is shown i n Figure 5 . The percent p u r i t y found f o r t h e sample, Diphenhydramine Hydro- ch lo r ide , USP, Parke, Davis & co., Lot N o . 593125, i s 99 .62%.13

2.8 S o l u b i l i t y The s o l u b i l i t y of diphenhydramine i n

water is 0.7 rng./m1.lo hydramine hydrochlor ide have been determined14 and a r e presented i n Table VI.

S o l u b i l i t i e s of diphen-

2 .9 c r y s t a l P rope r t i e s The o p t i c a l c r y s t a l l o raphic cons t an t s

have been repor ted by Keenan .'? Diphenhydramine hydrochloride i s descr ibed as c o l o r l e s s , mostly s ix-s ided p l a t e s wi th lengthwise cleavage. The n20 va lues are: a , 1.602; p , 1.625: and y , 1.630; a f l 5 0.002. t i n c t i o n is p a r a l l e l and t h e s i g n of e longat ion i s negat ive . c rys t a l log raph ic p r o p e r t i e s and gave t h e d e n s i t y a s 1.189.

I n p a r a l l e l po lar ized l i g h t , ex-

Shel l16 a l s o repor ted on o p t i c a l

2.92 x-Ray D i f f r a c t i o n The X-Ray d i f f r a c t i o n da ta f o r

The compound has been run by Krcl* diphenh dramine hydrochlor ide were reported by Gadret''. and t h e d i f f r a c t i o n p a t t e r n i s presented i n Figure 6 .

The ca l cu la t ed I'd I' spacings18 f o r t h e d i f f r a c t i o n p a t t e r n a r e given i n Table VII. The 28 angles were co r rec t ed on the d i f f r a c t i o n p a t t e r n using known values f o r c a l c i t e added t o a sample.

Fig. 5 Diphenhydramine Hydrochloride, D.T.A Curve, Parke-Davis & CO. Lot NO. 593125

Instrument: Met t le r T A 2000 AHz7.495 kcal. Mel t ing point: 1 6 8 . 3 O C .


Table V I . S o l u b i l i t y 1 4 of Diphenhydramine

Hydrochloride i n Various Solvents

Solvent S o l u b i l i t y , mq ./ml.

Water 85 8

Methanol 599

Alcohol, 95% 408

Chloroform 3 94

Isopropyl Alcohol 35

Acetone 16

191

z 2 - 0

z c

C

m E

2 c ua C

DlPH EN HY DRAM IN E HYDROCHLORIDE

Table VII . Diphenhydramine Hydrochloride :

calculated 'Id" Spacings and I/I, Values

Radiation: C u G , h 1.5418

Filter: N i

d (Ao)

8.56 7.97 7.19 6.74 5.70 5.42 5.30 4.78 4.59 4.37 4.05 3.85 3.59 3.40 3.35 3 -33 2.98 2 .91

7 2.86 4 (1 2.78 1 10 0 2 -75 (1

2 2.70 1 (1 2.60 2

5 2.58 (1 (1 2 -53 (1 1 3 2.46 (1

5 2.42 (1 20 2.29 1 24 2.19 1

2 1.98 1 12

7 d 7.19 4.05 4.37 8.56

6 4 3

l1 1/11 100 24 20 7

193

IRA J. HOLCOME AND SALVATORE A. FUSARI

2.10

behavior

D i s t r i b u t i o n Coef f i c i en t s Doyle” has determined t h e d i s t r i b u t i o n of- diphenhydramine hydrochlor ide i n

ch1oroform:water and e t h e r :water. A logar i thmic d i s t r i b u t i o n diagram i s presented i n the s tudy f o r s e l e c t i o n of p a r t i t i o n chromatographic systems . I9 hydrochloride between ch1oroform:water as a funct ion of aqueous p- toluenesulfonic ac id w a s a l s o s tud ied by Doyle .20 e f f e c t on the p a r t i t i o n of amines, i n gene ra l , has been studied.21

Konyushko22 examined t h e e f f e c t of pH on the d i s t r i b u t i o n of diphenhydramine between water and chloroform. The e x t r a c t i o n of diphenhydramine with chloroform i n presence of W F , KF, K C 1 , KBr, K I and KSCN a s s a l t i n out agents

The d i s t r i b u t i o n of diphenhydramine

The so lvent composition

a t 20° and pH 3 has been repor ted . 29

2 . 1 1 Aggregation - Micelle Formation Diphenhydramin hydrochloride forms

aggregates i n s o l u t i o n . ’‘ Attwood2’ has determined the c r i t i c a l mice l le concent ra t ion using s c a t t e r i n g a t an angle of 90° t o t h e inc iden t beam and the p l o t

2 . 1 2

constant

determining t h e i n f l e c t i o n po in t s i n versus the molal concent ra t ion .

PKA values AndrewsLo determined t h e i o n i z a t i o n of diphenhydramine hydrochloride a t 0 O ,

p d = 9.67, and 25 O , pKA = 9.12 i n water . values compare w e l l with those obtained by L ~ r d i ~ ~ of pKA = 9.00 i n water. determined t h e p& a t 2 0 ° t o be 9.06 i n water .

These

deRoos28 has

194


The pK4 of Diphenhydramine H drochlor ide , USP, Lot 563463, has been determined2S; i n a water:methanol (1: 1) system t o be 8.4. Since the p& v a r i e s s l i g h t l y wi th t h e alcohol con ten t , the value obtained i s acceptab le .

2.13 Metal complex Formation and Binding Diphenhydramine hydrochlor ide forms

complexes26 wi th var ious m e t a l ions such as Cu++, co++, and N i + + .

Evidence f o r i n t e r a c t i o n of diphenhydramine hydrochlor ide wi th s t rene-maleic30 and sodium carboxymethylcelluloseYl has been r epor t ed .

3 . Synthesis

syn thes i s of diphenhydramine w a s by Rieveschl i n 1947, ass igned t o Parke, Davis & C o . The genera l method involves t h e r e a c t i o n of bromo- diphenylmethane with the appropr ia te dialkylamino a lcohol i n the presence of anhydrous sodium carbonate . The dialkylamino a lcohol used i s dimethylamino e thano l (see Figure 7 ) . The diphenhydramine base t h a t i s formed i s then converted t o the H C 1 s a l t .

32 The f i r s t method patented f o r t h e

A v a r i e t y of s y n t h e t i c methods have appeared i n t he l i t e r a t u r e . 3 3 , 34, 35 In the major i ty of methods, t he base , diphenhydramine i s formed f i r s t and then converted t o the hydroch lo r ide . In some ins t ances , diphenhydramine hydrochlor ide may be formed d i r e c t l y by rearrangement of a quaternary ammonium s a l t 3 6 , 37 (see Figure 8 ) . The f r e e base can a l s o be formed as the r e s u l t of a decarboxylat ion react ion38 (Figure 9 ) .

195

Q CHZ + Brz I

Q H C - O C H ~ C H ~ -

I


hv -

HCI 7 HC-0 -cH~-cH~-N',

CH3

(b2 150-165')

Fig. 7. Synthesis of Diphenhydramine Hydrochloride.

196

m

2 I

uu

\

/

z uN I

U

- U

?

I97

Fig. 8. Synthesis of Diphenhydramine Hydrochloride: Rearrangement Reaction

\ C H z C ~ - O - C - C O z N a

/

230° -300° c

A

t

A H 3

/ ‘ C Y HC-0-CHZCHZ-N HCI

Fig. 9. Synthesis of Diphenhydramine Hydrochloride : Decarboxylation Reaction

DIPHENHY DRAMINE HYDROCHLORIDE

4. stability - Deqradation The earliest published detailed work on

the stability - decomposition o diphenhydramine hydrochloride is that of Nogami in 1961. The kinetics of the decomposition was examined in an acidic and alkaline medium. In an acidic medium, diphenhydramine undergoes fairly rapid decomposition, whereas the compound is fairly stable in an alkaline solution. The decomposition in an acid medium is due to hydrolysis of the ether linkage. The rate determining step is first order and catalyzed by hydrogen ion. The principle degradation products are benzhydrol and 2- (dimethylamino) ethanol.

59

Earlier observations on the decomposition of diphenhydramine hydrochloride were in relation

ultraviolet light on the compound. The decomposition products with hydrogen peroxide are toluene, benzophenone, benzyl alcohol, benzoic acid and phenolic substances in addition to dimethylaminoethanol. The benzhydrol under the conditions used undergoes further reactions. Under ultraviolet irradiation, the principle decomposition products are benzhydrol and dimethylaminoethanol .

to the effect of h drogen peroxide 40t 41 and 43

The work by Nogami3’ on the stability of diphenhydramine hydrochloride was confirmed in part by d e R ~ o s ~ ~ in 1963 in a study on the stability of the ether bond in a series of benzhydryl ethers.

199

I R A J. HOLCOME A N D SALVATORE A. FUSARI

5 . Drug Metabolic Products - Pharmacokinetics I n t h e i n i t i a l work by Glazko and co-

workers 44, 45 on t h e metabolic f a t e o f diphenhydramine hydrochlor ide, ra t s and guinea p i g s w e r e examined a t d e f i n i t e t i m e s a f t e r sub- cutaneous i n j e c t i o n s . The h ighes t concent ra t ions of diphenhydramine w e r e found i n t h e lungs, wi th lower concent ra t ions i n t h e sp leen , l i v e r and muscles. Peak concent ra t ions occurred i n about one hour wi th a f a i r l y r a p i d drop over a s i x hour per iod . Diphenhydramine w a s demonstrated i n human u r i n e i n s m a l l amounts by e x t r a c t i o n and u l t r a v i o l e t absorp t ion .

The r e s u l t s obtained using r a d i o a c t i v e carbon incorporated i n t o t h e ci p o s i t i o n of t h e benzhydryl group of diphenhydramine agree wi th t h e chemical ana lys i s .45 I n rats t h e maximum rate of exc re t ion occurred i n t h e f i r s t seven hours . Radioautographs prepared from u r ine samples showed a t least s i x d i f f e r e n t radio- a c t i v e compounds p resen t , one of which was d i phe nh yd r a m i ne .

K i k k a ~ a ~ ~ i d e n t i f i e d benzhydrol and dimethylaminoethanol a s metabolic products i n v i t r o and i n v ivo . An a c i d i c compound w a s a l s o detected, bu t not i d e n t i f i e d .

D r a ~ h ~ ~ , 48 using t r i t i u m labe led diphenhydramine i n rhesus monkey plasma found t h e major metabol i te t o be a deaminated carboxyl ic acid d e r i v a t i v e of diphenhydramine, (diphenylmethoxy) ace t ic acid. The a c i d , t h e

mono- and d i - dealykylated d e r i v a t i v e s of diphenhydramine and the N-oxide d e r i v a t i v e w e r e i d e n t i f i e d chromatographically. The diphenyl-

200

DIPHENHY DRAMINE HY DROCHLORI DE

methoxyacetic ac id i s excre ted a s t he glutamine conjugate .

Kinke14’ examined plasma l e v e l s of diphenhydramine a f t e r s i n g l e dose o r a l adminis- t r a t i o n of diphenhydramine hydrochlor ide capsules a t d i f f e r e n t l e v e l s i n human vo lun tee r s . Peak plasma l e v e l s were obtained 2 t o 3 hours following t h e dose. I n a mul t ip l e dose s tudy, a r e l a t i v e l y cons tan t plasma l e v e l i s obtained a f t e r t h r e e days.

50 Addi t iona l work has been done on t h e

metabol i tes i n r e l a t i o n t o d i f f e r e n c e s observed between s p e c i e s . The major metabol i te i n a l l spec ie s s tud ied , except t h e r a t , w a s (diphenylmethoxy) a c e t i c a c i d . This metabol i te i s conjugated with glutamine i n t h e monkey and g lyc ine i n the dog.

6 . I d e n t i f i c a t i o n : Microchemical T e s t s

t es t s f o r d e t e c t i o n and i d e n t i f i c a t i o n of diphenhydramine w a s descr ibed by ~ a l e y ’ l i n 1948. The r e a c t i o n s and r e s u l t s are summarized i n Table V I I I .

The f i r s t c o l l e c t i o n of microchemical

~ o l l e ~ ~ descr ibed an a d d i t i o n a l co lo r r e a c t i o n i n 1950 i n which the reagent , H2SO4, 90%, and HNO3, lo%, reacts t o g ive a red-v io le t co lo r changing slowly t o yellow. The r e s u l t i n g mixture i s d i l u t e d with water t o g ive an orange- yellow color and then t h e t u r b i d mixture becomes a v io l e t - rose . chloroform is added and mixed w e l l : the separa ted chloroform layer is v i o l e t and t h e aqueous l aye r becomes c o l o r l e s s . The co lor r e a c t i o n i s due t o benzhydrol.

201

Table V I I I . Identification of Diphenhydramine

51 Hydrochloride: Microchemical Tests

Observation

Definite crystalline form

Mandelin reagent Red oily globules

Marquis reagent h) 0 h)

Mecke reagent

Canary yellow, reddish-orange then chocolate brown

canary yellow then reddish-orange

Frohde reagent Canary yellow to orange-red

H2SO4, conc. orange

K2Cr207- H2SO4 Yellow

Resorcinal - H2SO4 Orange then reddish-orange and wine color on dilution

Furfural, 1%, over H2S04 Orange brown and yellow-green on shaking

Table VIII (cont - )

Reagent observat ion

chromic ac id , 5% orange-red p r e c i p i t a t e

Foucery reagent Cherr y-r ed

Name Reagent Compositions:

Mandelin reagent N 0 W

Marquis reagent

Mecke reagent

Frohde reagent

Foucery reagent

Ammonium vanadate, 1 g . i n LOO m l . concentrated s u l f u r i c a c i d

s u l f u r i c acid-formaldehyde. Two m l . of a 40% s o l u t i o n of formaldehyde mixed wi th 45 m l . of w a t e r and 55 ml. of concentrated s u l f u r i c a c i d

Selenous a c i d , 0 .5 g . , i n s u l f u r i c a c i d , 100 a

Ammonium molybdate, 0 .l% i n concentrated s u l f u r i c acid

m l

Quinone, 1 g . i n acetic acid:alcohol(S : 100)


Addi t iona l microchemical tests have been descr ibed by A ~ t e r h o f f ~ ~ , 0 ~ 0 1 ~ ~ and N e ~ h o f f ~ ~ *

Clarke56 i n 1957 gave, i n add i t ion t o observa- t i o n s , t h e s e n s i t i v i t y of t he t e s t s used (Table 1x1.

Clarke57 h a s l d e s c r i b e d a method f o r t h e rap id d e t e c t i o n of b a s i c drugs i n u r i n e t h a t u t i l i z e s many of t h e react ions descr ibed i n procedures t h a t require a m i n i m u m of equipment.

Addi t iona l reagents which form c r y s t a l - l i n e p r e c i p i t a t e s with diphenhydramine are f l a v i a n i c acid58, 4,4 I -dibromodibenzenesulf on- amide59, and 8 h droxy-7-iodoquinoline-5-

-68 sulphonic a c i d .


7.1 Elemental Analysis The e l emen ta l a n a l y s i s of Diphenhydramine

Hydrochloride, USP, Lot 563463, i s presented below :

Element % c a l c u l a t e d ReportedG1 C 69.97 69.99 H 7.60 7.56 N 4.80 4.84 c1 1 2 . 1 5 12.09

7.2 Spectrophotometric Assay

7 . 2 1 D i rec t Methods Methods i n which t h e sample i s

~

d i l u t e d t o the proper concentration for absorbance reading i n t h e u l t r a v i o l e t region have been repor ted by Setniker62 and D e m i r 6 3 . using or thogonal func t ions f o r i r r e v e l a n t

Correc t ions

204

T a b l e IX. Microchemical Identification Tests

56 and Sensitivity

Reagent

Gold bromide/HCl

Potassium triiodide

Formaldehyde-sulfuric acid (Marquis)

Ammonium vanadate

Ammonium molybdate

Selenium dioxide

Observations

Needles, some curved

plates

yellow color

ye 1 l o w

ye 1 low

yellow

Sensitivity

0.1 pg

0.1 pg

0.1 pLJ

0.1 w

0.1 pg

0.1 pg


absorption in tablets64 has been applied with a recovery of 99.2 2 1.8%.

7.22 Separation Methods Prior to Spectrophotometric Assa column chromatography 0: alumina65

thin layer chromatography with an alumina layer66, direct extraction from a basic solution67, ion exchange chromatography68, and extraction from a 5% hydrochloric acid ~olution~~r 7o have been used prior to the spectrophotometric assay.

7.23 Methods Based on Conversion to ~enzophenone Prior to Spectro- photometric As say Diphenhydramine hydrochloride is

oxidized to benzophenone b either dichromate in a sulfuric acid medium’’, y2 or permanganate in a basic medium73. steam distilled or separated by extraction into hexane or heptane and determined spectrophotometrically.

The benzophenone is either

7.24 Conversion to Chloranilic Acid Prior to Spectrophotometric Assay Diphenhydramine has been deter-

mined in drugs by conversion to chloranil with FeC13 in hydrochloric acid and hydrogen peroxide . The chloranil formed is extracted and hydrolyzed to chloranilic acid (2,5-dichloro-3,6-dihydroxy- p-benzoquinone) with potassium hydroxide. The absorbance measured at 331 nm. The conversion to chloranilic acid is constant, but not 100%.

74

206

DIPHENHY DRAMlNE HYDROCHLORIDE

7.3 Colorimetric Assay 7.31 lon-Pair Extraction Methods

routine control procedures is based on the extraction of diphenhydramine with methyl orange into chloroform. The procedure was initially studied by Dill and G l a z k ~ ~ ~ for use in the determination of diphenhydramine in body tissues. A recent modification involves the addition of methanol after the complex is completely extracted into chloroform to prevent adsorption of the methyl orange-diphenhydramine ion-pair onto the walls of the fla~k7~.

The method commonly used for

Other dyes that have been used for the colorimetric assay are bromocresol green77 I bromocresol p~rple7~, bromothymol blue78, er iochrome blue SE79 I tetrabromophenolphthale in ethyl ester*O and eosin811 8 2 .

7.32 Ammonium Reineckate Methods

dramine as the reineckate salt followed by solution of the salt in acetone and colorimetric estimation at 525 nm. A very comprehensive paper on the identification and determination of nitrogenous bases with ammonium reineckate was presented by KU~n-Tatt~~ in which the mole composition of diphenhydramine reineckate is given as ~21~28cr~7OSq. The salt decomposes at 178 - 180 O .

Bandelin83 separated diphenhy-

7.33 Picric Acid Method Picric acid has been used for the

colorimetric determination of diphenhydramine in the urine of rabbits and man85.

207


7.34 Method based on Molle Reaction Horn86 examined s e v e r a l d i f f e r e n t

procedures f o r t he determinat ion of diphenhydramine hydrochlor ide, one of which i s based on the M O l h r eac t ion descr ibed e a r l i e r . The compound i s reac ted wi th a mixture of s u l f u r i c ac id and n i t r i c ac id (9 :1) , d i l u t e d with water and t h e colored compound formed e x t r a c t e d wi th chloroform. The absorbance of t h e chloroform l aye r w a s then determined wi th a f i l t e r type instrument .

7.35 Miscellaneous Color imet r ic Methods D iphe nh ydr a m ine has been d e t e r -

mined by e x t r a c t i o n of a chloroform so luble complex wi th coba l t thiocyanate87, 88, *9. Diphenhydramine reacts i n a 2 : l mole r a t i o and i n chloroform i s measured i n t h e reg ion 590 t o 625 nm.

Diphenhydramine can be ex t r ac t ed from an acetate b u f f e r , p H 5 , conta in ing iod ide wi th a 0.5% I 2 so lu t ion i n e thylene d ich lor idegO.

condi t ions f o r t h e complex forma- t i o n of diphenhydramine with H ( T 1 Br4) have been examined wi th subsequent displacement by b r i l l i a n t green”.

The c o l o r r eac t ion wi th d i e t h y l oxalateg2 and th ioba rb i tu r i cg2 acid w a s used t o determine diphenhydramine i n p i l l s g 3 .

7.4 T i t r i m e t r i c Analysis 7.41 Direc t Methods of T i t r a t i o n

The o f f i c i a l methodl f o r t h e assay of diphenhydramine hydrochlor ide i s by

208

DIPHENHY DRAM INE HYDROCHLORIDE

nonaqueous t i t r a t i o n wi th 0.1N perch lo r i c a c i d i n t he presence of mercury (11) a c e t a t e using c r y s t a l v i o l e t a s i n d i c a t o r .

Work on t h e nona ueous t i t r a t i o n method w a s repor ted by Ekablad" and a c e t o n i t r i l e w a s examined as a so lven t by Mainvi1leg5. Severa l a c i d s a l t s of diphenhydramine were t i t r a t ed with pe rch lo r i c acid i n g l a c i a l acetic a c i d using t h e g lass -g lass re ta rded poten t iomet r ic method f o r d e t e c t i o n of t he endpointg6. The endpoint has been de tec t ed conductimet- r i c a l l y g 7 . Diphenhydramine and ac id sa l t s can be t i t r a t e d d i r e c t l y i n anhydrous propionic ac id with p e r c h l o r i c a c i d using glass-calomel e lec t rodes98 .

Diphenhydramine can a l s o be de te r - mined using a 0.004M s o l u t i o n of sodium l a u r y l s u l f a t e o r sodium d i o c t y l su l fosucc ina te a s t h e t i t r a n t g g , 100. The r a t i o of t h e an ion ic surface- a c t i v e agent t o t h e base i s not i n t e g r a l , bu t approximate and cons t an t .

7.42 sepa ra t ions p r i o r t o T i t r a t i o n 7.421 Reineckate S a l t Formation The re ineckate of diphenhydramine

may be decomposed by hea t ing i n an a l k a l i n e medium and a Volhard t i t r a t i o n performed t o determine t h e th iocyanate content '''# lo2. formed may a l s o be determined b r o m a t ~ m e t r i c a l l y ~ ~ ~ , r e s u l t s are about 5% low. The chromium (111) content can be determined wi th very ood accuracy f 0.5% using a chela tomet r ic method 184 .

The s a l t

209


7.422 Complexometric Method

inso lub le s a l t wi th bismuth which, from t h e reagent repared, releases an equiva len t amount

0.lM ZnSO4 a t pH 9.1 using eriochrome b lack T as i n d i c a t o r .

Diphenhydramine forms an

of EDTA 1g5 . The l i b e r a t e d EDTA is t i t r a t e d w i t h

7.423 S l u r r y Method c 1 a i r l u b and chattenlO7

presented s l u r r y methods f o r t h e sepa ra t ion of ant ihis tamines f r o m tablet or capsule material. The sample i s simply s l u r r i e d wi th chloroform and f i l t e r e d . The f i l t r a t e i s t i t r a t e d wi th acetous pe rch lo r i c ac id a f t e r g l a c i a l a c e t i c ac id i s added. magnesium oxide and s i l i c e o u s e a r t h for pre- t reatment of an aqueous i n j e c t i o n followed by washing wi th warm chloroform i n t o g l a c i a l a c e t i c a c i d . The base is then t i t r a t e d wi th 0.1N per- c h l o r i c a c i d using p-naphtholbenzein as i n d i c a t o r .

Tuckermanlo8 used a mixture of

7.424 Ion Exchanqe Method Ion exchange columnsl09, 110

have been used i n t h e determinat ion of a n t i - his tamines with subsequent t i t r a t i o n of t h e e f f l u e n t .

7.425 Ex t rac t ion Method A c o l l a b o r a t i v e s tudy on

t h e e x t r a c t i o n method was repor ted by Heiml l l . The f r e e base is e x t r a c t e d wi th e t h e r and de te r - mined by t i t r a t i o n . Recoveries w e r e 99-101%.

7.43 Miscellaneous T i t r i m e t i c Methods p-Toluenesulfonic ac id i n chloro-

form112 and methanesulfonic ac id i n g l a c i a l

210


ace t ic a c i d l l 3 have been used as t i t r a n t s f o r diphenhydramine w i t h v i s u a l i n d i c a t o r s I n aqueous s o l u t i o n , s i l i c o t u n g s t i c acidl i4 wi th me tan i l yellow or con o r ed as i n d i c a t o r w a s recommended by ~ramml” and t h e compound has a l s o been t i t r a t e d wi th 0 .W n i t r a n i l i c acid using a po la ro raph f o r d e t e c t i o n of t h e endpoint i n 0.01~ K C 1 11% .

7.5 Fluorometr ic Analysis Weak f luo rescen t i n t e n s i t y w a s observed

f o r diphenhydramine when t r e a t e d wi th 3% H202 1 1 7 , b u t no a n a l y t i c a l use w a s made of t h i s observa- t i o n . Mar t in l l8 t r e a t e d a r e s idue conta in ing diphenhydramine w i t h concent ra ted s u l f u r i c a c i d and perchloric acid t o ob ta in f luorescence a t 525 nm. w i t h e x c i t a t i o n a t 375 nm. L i m i t of d e t e c t i o n observed w a s 0.02 w./ml.

Glazko119 used f luo rescen t dyes t o extract diphenhydramine as an ion-pair and increased the s e n s i t i v i t y of d i r e c t e x t r a c t i o n methods s e v e r a l hundred fo ld over t h e use of methyl orange and co lor imet ry .

7.6 Automated Analysis Robertson120 has presented an automated

method of a n a l y s i s f o r amine drugs based on acid-dye methods. B r O m O C r e S O l purp le i s used f o r diphenhydramine. The automated and manual method agree q u i t e w e l l wi th a 0.4% l a b e l c la im d i f f e r e n c e . Fusa r i has presented an u l t r a v i o l e t method for conten t un i formi ty of diphenhydramine samplesl21.

21 1

IRA J. HOLCOMB A N D SALVATORE A . FUSARI

7.7 Biological Assay Chen122 used isolated guinea pig ileum

for the assay of histamine and diphenhydramine in vitro. A linear relationship is observed between dose and effect.

7.8 Gravimetric Analysis U ~ e n o ~ ~ ~ used the picrate method to

determine diphenhydramine gravimetrically. The picrate is filtered, washed with water and ether, dried and weighed.

7.9 Chromatography 7.91 paper chromatography

paper are summarized in Table X for diphenhydramine hydrochloride.

The results of chromatography on

7.92 Thin Layer Chromatography An excellent review of the thin

layer chromatography methods for diphenhydramine was presented by Comer130 in 1967. Additional mobile phases used on silica gel prior to 1967 are presented by Kampl31, (1) CC14:BUOH:MeOH:25% W O H (40:30:30:1), and (2) Petroleum ether:ether:EtzNH (20 :80:1) and by F ~ w a l ~ ~ , CHC13 :MeOH:NqOH (98 : 1 : 1) . Diphen- hydramine has also been chromatographed on thin layers of alumina using @H:EtOH (9:1) or (9:1.5)l33; 6H:EtOH :HOAc ( 3 : 1.2 : 0 - 5 ) 133; CHC13:BuOH (98:2)134; CHC13:Me2CO and 6H:EtOH (9:1)134.

212

Table x. Paper Chromatoqraphy of

Mobile Phase

Diphenhydramine Hydrochloride

Rf L.

Pretreatment Ref crence

n-BuOH:HOAc:H20 (40 :10 : 5 0 ) 0.85 none 124

n-BUOH:HCl, 0.5N (90:30) 0 -68 none 124

I sopropyl alc. :HCl, 0.5N h, (90 :30) 1.00 none 124

EtOH:H2O:QOH (55 :43 :2) 0 -31 Impregnated with soh. of 125

+ w

petroleum (180-215 O C ) and petroleum ether

EtOH:H20:NH40H (95:3:2) 0 -76 Impregnated with soh. of 125 petroleum (180-215 Oc) and petroleum ether

n-BUOH sat.. with 1N HC1 0 -60 C6H6:HOAC:H20 (4:4:1) 0 -70

none none

126 126

127 n-BuOH sat. with pH 3 buffer 0.63 Treated with pH 3 buffer

Table x( cont .)

Mobile Phase - Rf Pretreatment Reference

n-BUOH s a t . with pH 5 buffer 0.63 Treated with pH 5 buffer 127

n-BuOH s a t . with pH 6 . 5 buffer 0.67 Treated with pH 6.5 buffer 127

n-BUOH sat. with pH 7 - 5 buffer 0.91 Treated w i t h pH 7.5 buffer 127

n-BUOH:H20 (50:50) with 1 g . c i tr ic acid (use Dipped i n 5% sodium 128 upper layer) - dihydrogen citrate

n-BUOH s a t . with 1 N HC1 0 -96 Whatman No. 4 129


Additonal systems for the thin layer chromatographic examination and detection of diphenhydramine are presented in Table XI.

The reverse phase paper chromatographic s stem developed by Vecerkova has been modifiedlT2 to run on thin layers of silica gel in about 3 hours and will separate diphenhydramine from bromodiphenhydramine.

Thin layer chromatography has been used prior to assays by the spot-area method143 and by the acid-dye reaction after elution from the plate.144

7.93 Gas chromatography 7.931 Direct Methods on Neutral

columns The majority of published

gas chromatographic systems for diphenhydramine hydrochloride involve injection of the free base on columns differing in polarity.

MacDonald 14’ used a 6 ft . column of 1% SE-30 on 100-120 mesh Gas Chrom P. Retention time for diphenhydramine was 6.3 min. : column temperature 173 Oc . ; injection port, 256 Oc; argon flow rate 60 ml./min. using an argon @-ray ionization detector.

146 Kazpk presented

additional data on 1% SE-30 columns at different temperatures.

14 7 MacDonald compared four

columns in 1964 and found 0.08% PDEAS on a 120/ 170 glass bead column to be most successful for

215

N

m c

T a b l e XI. Thin Layer chromatography of Diphenhydramine Hydrochloride

Mobile Phase Sorbent

CHC13 :Me2CO (9 : 1) Kieselgel G w/ fluorescent indicator

MeOH I1

CHC13:EtOH (9: 1) I 1

CHC13:EtOH (8:2) I 1

EtOAc:MeOH:QOH(85:10:5) Silica Gel G

CHC13 :MeOH (9 : 1) Silica Gel G

1 sopropyl ether :EtOH (8 : 2)

MeOH : NH40H ( 100 : 1 -5 )

ISOprOpyl ether:Me2CO(l:l) Kieselgel G or GF

Rf

0.08

-

0.22

0.31

0.33

0.90

0.76

0 -11

0.77

0.55 s 0.48 f

Reference

El Gendi135

II

I 1

Davidow 136

Bastos 137

I 1

II

138 Eiden

Table XI. (cont .)

Mobile Phase

Isopropanol

Benzene :dioxane :HOAc (50:40 : 10)

Cyc10hexane:EtOAc:Et~NH 4 z (65 :30 :5)

Sorbent

Kieselgel c7 or GF

Rf Reference - 0.50 s 0.50 f Eiden

0.07 s II

I 1

0.51

13 9 Et0Ac:cyclohexane:dioxane: Gelman silica MeOH : H2 0: OH (50:50:10 :lo: 1.5 :O .5) fiber sheets

g e l glass micro- 0.71 Kaistha

Et0Ac:cyclohexane: Gelman silica ge l NH40H :MeOH : H20 glass microfiber 13 9 (70:15:2:8:0.5) sheets 0 -86 Kaistha

EtOAc : cyclohexane :MeOH: NH4OH (70 :15: 10: 5) 0.91

I 1

0

i?

3.r k

0

c, m al

9

5: x

9'

00

4Jm

w-

Y

X 0

dn **

N

x **

003

om

c

sE

218


the antihistamines. Diphenhydramine has a retention time of 2.5 min. on a 6' column at 175OC. with an argon flow rate of 60 ml./min.

Jain148 used 1% Hi-Eff-8B on 100/120 mesh silanized Gas Chrom P. Retention times of diphenhydramine given are relative to methapyrilene; 0.43 at 160Oc. and 0.46 at 190". Gas chromatography was used for the determination of diphenhydramine in blood after an extraction with acetone-ether .

A mixture of Hi-Eff-8BP, 1%, and 10% SE-52 on Gas Chrom Q was used by ~ a d e r l ~ ~ in application to single and multiple component drugs. The column temperature was 22OOc. Relative retention time was 0.83 to pentobarbital.

presented a general article on gas chromatography in which diphenhydramine was chromatographed on 3% Phenyl Methyl Silicone (OV 17) on Gas Chrom Q at 175O (6 ft., 4 mm. I.D.).

A rapid, direct analysis of antihistamines was reported by Reiss151 in which the sample is dispersed in water, diluted to volume, filtered and injected. Relative retention times to chlorpheniramine maleate are reported on two columns both 4 ft . x 0.25 in. 0.d. glass: 2% SE-30 and 2% Carbowax 20M on 80/100 mesh DlatOpOrt S, 0.52; and 10% silicone oil DC-200 on 60/80 mesh Diatoport S, 0.63. column temperature was 210 OC.

219


7.932 D i r e c t Methods on B a s i c Columns S t e e l e reported152 on t h e

use of a column wi th 5% Apiezon L and 4.5% potassium hydroxide. column temperature w a s 138O f o r t he f i r s t 6 min. a f t e r i n j e c t i o n and then r a i s e d a t 6Oc./min. t o 275°C. Retent ion t i m e f o r diphenhydramine w a s 3.64 min.

7.933 Oxidation t o Benzophenone P r io r t o G a s Chromatoqraphy Oxidation wi th 0.033M

Cr2O3 f o r 60 min. conver t s diphenhydramine t o benzophenone which can be determined i n nanogram amounts wi th a p rec i s ion of 1.5% using e l e c t r o n capture gas c h r ~ m a t o g r a p h y l ~ ~ .

7.94 Column chromatography ~ e v i n e l b 4 repor ted on the par-

t i t i o n i n g of diphenhydramine between 2 N HC1 and chloroform on a C e l i t e column. The diphenhydramine i s e lu t ed wi th a mixture of 90 m l . chloroform containing 1 m l . of a c e t i c ac id a f t e r a prewash of t h e column wi th d i e t h y l e t h e r .

Doyle’’ has examined d i s t r i b u t i o n diagrams and se l ec t ed C e l i t e p a r t i t i o n chromatographic systems f o r var ious sepa ra t ions on t h e b a s i s of t h e diagrams. The e f f e c t s of so lven t composition on t h e column p a r t i t i o n chromato- graphr5gf amines has a l s o been examined by Doyle and some information on diphenhydramine was presented.

7.95 Elec t rophores i s The e l ec t rophores i s of diphen-

124 hydramine has been carried out by B a r u f f i n i

220


i n d i f f e r e n t pH b u f f e r s a t 7 volts/cm. f o r 3 hours . Migration i s opt imal a t low pH wi th 8 2 mm. displacement toward t h e cathode a t pH 2 .l.

Der li kowski 156 examined the e l ec t rophores i s of diphenhydramine i n 1965. zones w e r e detected wi th a Dragendorff reagent .

The l i t e r a t u r e has been reviewed through 1972.

Acknowledgment

The most capable a s s i s t a n c e of M r s . L u c i l l e Kelly, Information S p e c i a l i s t , parke, Davis & C o . , is g r a t e f u l l y acknowledged. The au thors a l s o wish t o thank M i s s Beverly Jozwiak f o r h e r pa t ience i n t he prepara t ion and co r rec t ion of t h i s manuscript .

22 1


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Pharm. S c i . , 53, 154-7 (1964). c . A . - 60, 10476d (lZ4) Kashima, T. , C h e m . Pharm. B u l l . , - 6 , 229-34 (1958) E l l a r t , H., S e l l , E . , Farmacja pol . 21, 167-70 (1965) Anal . Abstr., 2, 5804 (1966) Kashima, T., Kano, K., J. Pharm. SOC.

Japan, 7 2 , 931-5 (1956). c . A . - 51, 1541g (1957) R O j a S , L. A . Araya, Anales Fac. Quim. Farm., Univ. chile, 15, 101-6 (1963) . C . A . 62, 12976c ( 1 9 E ) Roushdi, I . M . , Shafik, R. M., J . Pharm.

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Mont iqui , R., Fernandez de Valderrama, E . , Farm. Nueva, 3, 221-6 (1961) . C . A . - 55, 27770h (1961) I r i t a n i , N . , Miyahara, T . , Takahashi, I . , J. Pharm. SOC. Jap. 89, 432-35 (1969) Roushdi, I . M . , Shaf ik, R . M. , J . Pharm.

1 1 3 0 3 3 ~ (1970) c l a i r , E . G . , Chat ten, L. G . , J. Pharm. Sc i . , 50, 848-53 (1961) c h a t t e n , L . G . , i n " T i t r i m e t r i c Methods", fed . D. S . Jackson) , Plenum Press, N.Y.,

S c i . U.A.R. 9, 65-71 (1968) . C. A.

SCi U.A.R., 9, 57-63 (1968). C. A . 73,

1961, p . 129-70

228


108.

109.

110.

111.

1 1 2 .

113.

114.

115.

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1 1 7 .

118.

119.

1 2 0 .

1 2 1 .

T u c k e r m a n , M . M . , A c e l . , K., B i c a n - F i s t e r , T . , B i l l o w , J . , G i b b s , I . S . , J. Pharm. S c i . , 61, 448-9 ( 1 9 7 2 ) K u d a k o v a , N. A., A p t e c h n o e D e b , 4, 34-7 ( 1 9 5 5 ) . C . A . 50, 535131 ( 1 9 5 6 )

B l a u g , S. M., Z o p f , L . C . , J . Am. Pharm.

4460d ( 1 9 5 6 ) H e i m , H. C. , J. A s s o c . O f f i c . A g r , Chem. ,

ASSOC., 45, 9-12 ( 1 9 5 6 ) . C . A. 50,

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BOUW, P . S . , Suara P h a r m . ~ a d j a l a h , 4 , 41-4 ( 1 9 5 9 ) . C . A. 58, 1 2 3 6 9 d ( 1 9 6 3 )

E l l e r t , H. , se l l , E . , Farm. pol . , 2 1 , 739-42 ( 1 9 6 5 ) . C . A. - 65, 2 0 6 8 d ( 1 E 6 ) G r a f , E. , F fed ler , E., A r c h . Pharm., - 2 8 6 , 401-16 ( 1 9 5 3 ) . C . A . 49, 4 2 3 9 h ( 1 9 5 5 ) Gramm, H . W . , Pharmazie, 145-310-13 ( 1 9 5 9 ) . C . A . 54, 5 0 1 2 b ( 1 9 6 0 ) K r a c m a r , J., zyka, J. , C e s k o s l a v . farm.,

Jensen, R . E., Pflaum, R. T. , J. pharm.

9359d (1964) Mart in , E . A. , C a n . J . Pharm. Sc i . , 2 , 95-96 ( 1 9 6 7 ) . A n a l . Abstr ., 16, 2 6 9 8 ( 196 9 )

G l a z k o , A . J., D i l l , W . A. , Fransway, R . L. , Fed. Proc., 3, 195 ( 1 9 6 2 ) R o b e r t s o n , D . L . , Matsui, F . , French, W . N . , C a n . J . Pharm. S c i . , 1, 47-50 ( 1 9 7 2 ) Fusa r i , S. A., D i t t m a r , D., perr izo, C . H . i n " A d v a n c e s i n A u t o m a t e d A n a l y s i s 'I, Vol . 11, T h u r m a n A s s o c i a t e s , M i a m i , F l a . ,

- 7 , 246-9 ( 1 9 5 8 ) . C. A., 55, 2 7 7 8 2 h ( 1 9 6 1 )

S c i . , 53, 835-7 ( 1 9 6 4 ) . C . A . 61,

1971, p . 2 4 1 - 2 4 5

229


1 2 2 .

123.

124.

125.

126.

1 2 7 .

128.

129.

130.

131.

132.

133.

134.

135.

136 .

Chen, G ., Ensor , c . R . , C l a r k e , I . G . , J. Pharmacol. E x p t l . Therap., 92, 90-7 (1948) . Uyeno, S. , O i s h i , H . , J . Pharm. SOC., Japan , 72, 443-6 (1952). c . A. - 46, 7708g (1952) B a r u f f i n i , A. , Farmaco ( P r a c t . E d . ) , 1 3 , 459-65 (1958) . C . A . 53, 16473b ( 1 9 5 9 r Vecerkova, J., Sulcova, M. , K a c l , K. , J . Chromatog., 7 , 527-34 (1962) . C. A . - 58, 232633 (1963y I t a i , T., Kamiya, S., Yakuzaigaku, 17 , 249-51 (1957). C . A . 52, 11356b (1z8) Goldbaum, L. R . , K a z y a c L . , Anal. Chem., - 2 8 , 1289-90 (1956) . C . A . , 51, 673g (1957) Curry , A . S . , Powell , H., Nature , - 173,

B e l l e s , G . C . , S i e v e r t , H. W . , J. Lab. C l i n . Med ., 46, 628-40 (1955) Comer, J. P . , Comer, I . J., pharm. S c i . ,

Kamp, W., Onderberg, W . J . M . , v a n s e t e r s , W . A . , pharm. weekblad. 98, 993-1007 (1963) .

Fuwa, T., Kido, T. , Tanaka, H. , Yakuzaigaku, 25, 138-42 (1965) . c . A . - 64, 6405h ( 1 9 G ) Moldaver, B . L . , Sakovan, T . M . , Farm. Zh. ( K i e v ) , 23, 28-33 (1968) . c . A . 69, 109839f (1968) Shcherbakova, M. N., Aptechn. Delo, 13, 41-4 (1964). C. A . 2, 1518b (1965) E l Gendi, S . , K i s s e r , W . , Machata, G . , Microchim. A c t a , 1965 , 125 Davidow, B . , L i P e t r i , N . , Quame, B . , h e r . J. C l i n . pa th . , 50, 714-19 (1968)

C . A . - 42, 2635f (19485-

1143-4 (1954) . C . A., 48, 1 1 7 3 1 ~ ( 1 9 5 4 )

- 56, 413-36, (1967)

C . A. - 61, 1 7 0 7 f 7 1 9 6 4 )

230

DIPHENHY DRAMlNE HYDROCHLORIDE

137.

138.

139.

140.

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143.

144.

145.

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147.

148.

149.

150.

151.

152.

Bastos , M . L . , Kananen, G . E . , Young, R. M., M O n f O r t e , J. R., Sunshine, I. , C l i n . Chem., 16, 931-40 (1970) Eiden, F . , Kammash, G . , pharm. Z e i t . ,

Ka is tha , K. K. , J a f f e , J. H., J . Pharm. Sc i . , 61, 679-689 (1972) Boonen, F . , J . pharm. Belg. , 27,233-40 (1972) . C . A . 7 7 , 2 4 8 6 0 ~ (19z)

Quan, J. H. , parke, Davis & co., Personal Communication T e r h a l l e , M . L . , Parke, Davis & co . , Personal Communication Morrison, J . c . , Chat ten, L . G . , J . pharm. sci . , - 53, 1205-1208 (1964) Matsu i , F., Watson, J. R. , French, W . N . , J . Chromatog., - 44, 109-115 (1969) MacDonald, Jr ., A . , Pflaum, R . T ., J . pharm. Sc i . , 52, 816 (1963). c . A . - 59, 97388 (1963) Kazyak, L . , Knoblock, E . c . , Anal. Chem., - 35, 1448-52 (1963) . c . A . - 59, 9737a (1963)

MacDonald, Jr . , A. , Pflaum, R . T. , J. Pharm. Sci., 53, 887-91 (1964) . c . A . - 60, 11852h (1964) J a b , N. C., Kirk, P. L., Microchem.

Rader, B . R . , Aranda, E . S., J. Pharm. S c i . , 57, 847-51 (1968) Patel, J. A., Am. J. H o s p . Pharm., 27,

R e i s s , T . J. , J. Assoc. Off. Anal. Chem. , - 53, 609-11 (1970) . Anal. Abstr., 20, 3345 (1371) S t e e l e , J. W., B O l a n , M., Eyolfson, J. K. , Can. J . Pharm. S c i . , 2, 107-111 (1970)

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J., 2, 242-48 (1967)

411-14 (1970)

23 1


153. vessman, J., H a r t v i g , P . , Stromberg, S . , A c t a P h a r m . Suecica, 7, 373-88 (1970). C. A . , 2, 12355733 (1970)

(1965) 154. L e v i n e , J. , J. Pharm. Sci ., 54, 485-488

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156. D e r l i k o w s k i , J. , N a r b u t t - M e r i n g , A . B . , P e r k o w s k i , E . , W e g l o w s k a , w . , Pota j alo- G u l i n s k a , J a r A c t a Polon. P h a r m . , 21,

- 54, 364-372 (1971)

9-18 (1964) . C. A . 62, 8935~ (1965)

232

ECHOTHIOPHATE IODIDE

Raymond D. Daley

RAYMOND D. DALEY

1. Description 1.1 Name, Formula, Molecular Weight 1.2 Appearance, Color, Odor

2. Physical Properties 2.1 Infrared Spectra 2.2 Nuclear Magnetic Resonance Spectra 2.3 Ultraviolet Spectra 2.4 Mass Spectra 2.5 Dif fe ren t i a l Thermal Analysis 2.6 Solub i l i t y 2.7 Crystal Properties 2.8 Melting Point

3. Synthesis

4 . S t a b i l i t y -- Degradation

5. Drug Metabolic Products

6 . Methods of Analysis 6 . 1 Elemental Analysis 6.2 Ultraviolet Spectrophotome t r ic Analysis

6.21 Direct Ultraviolet Absorption Measurement 6.22 Ind i r ec t Ultraviolet Absorption Method

6.3 Titrimetric Assay Method 6 . 4 Thin Layer Chromatography 6 . 5 Other Tests

234

ECH OTH I OPHATE I OD I DE

1. Description


The Chemical Abstracts name for echothiophate iodide is 2-[ (diethoxyphosphiny1)thiol -N,N,N-trimethyl ethanaminium iodide, starting with Volume 76 (1). Previously the Chemical Abstracts name was (2-mercapto- ethy1)trimethylauunonium iodide S-ester with 0,O-diethyl phosphorothioate. The CAS Registry No. is [ S U - l O - O ] . The British Pharmaceutical Codex lists the compound as ecothiophate iodide (2), and The Merck Index lists several other names (3).

C 9H23 IN03PS Mol. Wt.: 383.23


White crystalline pwder, with a slight mercaptan-like odor.


2.1 Infrared Spectra

Figure 1 is an infrared spectrum of one of the crystalline forms of echothiophate iodide. This form will be designated as Form I in Section 2.7, Crystal Properties. The spectrum was run in several sections: to 540 cm” as a mineral oil mull on polyethylene; (b) from 470 to 1360 cm” as a mineral oil mull between potassium bromide plates, with the 900 to 1055 cm’ and 1220 to 1275 cml regions run in two thicknesses; (c) from 1360 to 4000 cm” as a perfluorinated oil mull between potassium bromide plates. The spectrum was obtained with a Beckman IR-12 spectrophotometer.

(a) from 200

Some of the absorption bands can be assigned as follows ( 4 ) :

235

Figure 1. Infrared spectrum of echothiophate iodide, perfluorinated and mineral o i l mull.

ECH OTH I OPH ATE I 0 0 I DE

3000 cm' C-H Stretching 1240 Cm" P=O Stretching 1157 cm' P-0-Ethyl Vibration

975 cm' P-0-C (Alkyl) Vibration

2.2 Nuclear Magnetic Resonance Spectra

' b o proton magnetic resonance spectra of echothiophate iodide are shown in Figures 2 and 3. These were run in D 0 and in CDCl , on a Varian A-60A 60 MHz NMR spectraneger, with a te2ramethylsilane reference ( 5 ) .

The NMR spectra of organic phosphorus compounds are complicated by coupling of the proton signals with that of phosphorus. This coupling causes readily observable splitting of the lines from methylene protons in the groups P-0-CH constants of 9 Hz (6 f .

and P-S-CH2, with JpH coupling

The proton NMR spectral assignments are given in Table I (5).

A phosphorus NMR scan indicates a chemical shift of about -28 ppm for the phosphorus in echothiophate iodide in aqueous solution, relative to a phosphoric acid reference ( 7 ) . This is consistent with literature values for this structure (8 ) .

2.3 Ultraviolet Spectra

Figure 4 shows the ultraviolet absorption spectrum of echothiophate iodide, run on a Cary Model 14 spectrophotometer. The sample was dissolved in water. The maximum at 226 nm has an absorptivity of 1.34 x 10 liters per mole cm. This absorption is essentially that of the iodide ion (the ultraviolet spectrum of a potassium iodide solution exhibits a imum at 226 nm with an absorptivity of about 1.35 x 3 liters per mole cm) .

4

2.4 Mass Spectra

Attempts to obtain the mass spectrum of echothiophate iodide were unsuccessful; the compound apparently

237

N w 00

Figure 2. Proton NMR spectrum of echothiophate iodide, D20 solution.

bJ w W

Figure 3. Proton NMR spectrum of echothiophate iodide, CDC13 solution.

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Figure 4 . Ultraviolet spectrum of echothiophate iodide in aqueous solution vs water, 1 an ce l l s ; 25.0 mcg/ml, 250 mcg/ml, 2.50 mg/ml.

RAYMOND D. DALEY

decomposed i n the heated i n l e t of the mass spectrometer (5).

2.5 Dif fe ren t ia l Thermal Analysis

Figure 5 shows the d i f f e r e n t i a l thermal analysis curve of echothiophate iodide, run a t 10°C per minute on a Dupont Model 900 instrument. The only thermal event below 2OO0C is the melting point, which occurs a t 122OC on t h i s scan. melting endotherm was observed a t 125.5OC ( 9 ) .

When the mater ia l was run a t 2OoC pe r minute, the

2.6 Solubi l i ty

The s o l u b i l i t y of echothiophate iodide a t room temperature is as follows:

Solvent

Water Methanol Ethanol (952) 2-Propanol Ace t oni t r i l e Chlorof orm Acetone Die thy1 Ether Petroleum Ether Benzene Ethyl Acetate

Approximate Solubi l i ty , w I m l

> 500 > 250 > 120

4 25

250 8

< 1 < 1 < 1 < 1

2.7 Crystal Properties

Two c r y s t a l forms of echothiophate iodide have been observed. given i n Table 11. diffractometer, using nickel-f i l tered copper IUr radiation.

The x-ray powder d i f f r a c t i o n patterns a r e These were obtained with a Norelco

2.8 Melting Point

The following melting points have been reported:

124-4.5OC (10) 138 (11)

242

N P w

Figure 5. Differential thermal analysis scan of echothiophate iodide.

RAYMOND D. DALEY

TABLE I1

X-Ray Puwder Diffraction Data for Echothiophate Iodide

Form I 1/11 - d , A' -

10.54 8.00 6.14 5.90 5.49 5.30 5.01 4.86 4.75 4.49 4.33 4.21 4.16 4.10 4.01 3.94 3.82 3.69 3.61 3.54 3.49 3.45 3.40 3.28 3.24 3.20 3.16 3.12 3.06 3.01 2.97 2.95 2.92 2.89 2.82

23 9

99 66 22 41 10

100 52 10 25 56 66 8

46 13 60 21

2 70 92 22 33 11 6

22 4

15 9 3 8

13 15 12 31

Form I1

1/11 - d, A' - 10.00 6.79 6.68 5.40 4.99 4.64 4.45 4.37 4.21 4.06 4.00 3.95 3.81 3.73 3.60 3.41 3.34 3.28 3.22 3.14 3.07 2.96 2.92 2.86 2.81 2.77 2.71 2.67 2.43 2.34

64 24 15

100 40 47 56 56 22 28 17 92 24 20 30 35 30

4 24 13 12 24 13

7 24 6

2 1 7

13 11

244


TABLE I1 (Cont'd.)

d , A' - 2.74 2.70 2.67 2.58 2.53 2.50 2.48 2.44 2.40 2.37 2.31

d , A' - 1/11 - 10 16 25 13 4 6 4 14 3 4 4

1/11 -

2.27 7

3. Synthesis

Two methods f o r preparing echothiophate iodide have been published. The react ions a r e shown i n Figure 6.

In the f i r s t method ( l o ) , the sodium sal t of dimethylaminoethyl mercaptan is prepared by t rea t ing dimethylaminoethyl mercaptan hydrochloride with sodium. The product is treated with diethylchlorophosphate t o yield 0,O-diethyl-S-$ -dimethylaminoethyl thiophosphate. This mater ia l is t reated with methyl iodide t o make echothiophate iodide.

I n the second method ( l l ) , a mixture of diethylchlorophosphate, dimethylaminoethyl mercaptan, and triethylamine i n e ther is refluxed. The mixture is f i l t e r e d t o remove the insoluble triethylammonium chloride and d i s t i l l e d t o obtain the 0,O-diethyl-S-6 -dime thylaminoe thy1 thiophosphate. This material is t reated with methyl iodide t o make echothiophate iodide.

4. S t a b i l i t y -- Degradation

Hussain e t a 1 (12) have shown t h a t echothiophate iodide decomposes by a t l e a s t two mechanisms. I n the pH range 2.4 t o 5, the major react ion is hydrolysis of one of the C-0 bonds t o form ethanol and the monoethyl e s t e r .

245

+ + 1. (a) (CH3l2NH -CH2CH2-SH + 2Na * (CH3)2N-CH2CH2S- + 2Na + H2

0 0 + 9 (b) (CH3)2N-CH2CH2S- + Cl-P(OC2H5)2- (CH3)2N-CH2CH2-S-P(OC2H5)2 + C1-

0 0 f +

(c) ( c H ~ > ~ N - c H ~cH~-s-P(oc~H~)~ + C H ~ I I: (cH~)~N+cH~cH~-s-P(oc~H~)~-J I-

0 t

2. (a) (CH3)2N-CH2CH2-SH + C1-P(OC2H5)2 + (C2H5)3N - 0 9

(cH~)~N-cH~cH~-s-P(oc~H~)~ + [ ( c ~ H ~ ) ~ N + ~ c ~ -

0 + t C H ~ ) ~ N - C H ~ C H ~ - S - + ( - C ~ H ~ ) ~ + C H ~ I - I: (cH~)~N C H ~ C H ~ - S - P C O C ~ H ~ ) ~ J I-

N P rn

(b

Figure 6. Synthetic Methods for Echothiophate Iodide.

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241

RAYMOND D. DALEY

In the pH range 9.5 to 12, the major reaction is hydrolysis of the S-P bond to yield (2-mercaptoethyl) trimethylammonium iodide and diethylphosphoric acid. These reactions are shown in Figure 7. At intermediate pH, both react ions occur.


No metabolic products have been reported.

6. Methods of Analysis

6.1 Elemental Analysis

The elemental composition of echothiophate iodide is as followst

- Element - 'K Theory

Carbon 28.21 Hydrogen 6.05 Iodine 33.11 Nitrogen 3.65 Oxygen 12.52 Phosphorus 8.08 Sulfur 8.37

6.2 Ultraviolet Spectrophotometric Analysis

6.21 Direct Ultraviolet Absorption Measurement

Although the ultraviolet absorption at 226 nm has been used in hydrolysis studies (12), it was useful only because it increased as the echothiophate iodide hydrolyzed. The iodide ion is the principal absorbing species at this wavelength in echothiophate iodide solutions (see Section 2.3), so that this maximum can be used only indirectly to measure the echothiophate cation concentration.

6.22 Indirect Ultraviolet Absorption Method

An ultraviolet assay for echothiophate cation is possible, using hydrolysis to thiocholine, followed by reaction with 4,4'-dithiopyridine to form an

248

ECHOTH IOPHATE lODl DE

ultraviolet absorbing material (13). The method is based on that of Grassetti and Murray (14). The ultraviolet quantitation is essentially an alternative to the titration described in Section 6.3, but requires less sample.

The echothiophate iodide is hydrolyzed quantitatively to thiocholine in 20 minutes in pH 12.0 phosphate buffer. The hydrolyzed sample is then treated with a solution of 4,4'-dithiopyridine in pH 2.3 phosphate buffer. The final solution has a pH of 6.2, and the 4.4I-dithiopyridine reacts with thiocholine to form 4-thiopyridone. 4-Thiopyridone has an absorption maximum at 323 nm. portion of the original echothiophate iodide solution, before hydrolysis, with a solution of 4,4'-dithiopyridine. Echothiophate iodide assayed by the titration procedure is used as a standard (13).

A blank is prepared by mixing a

6.3 Titrimetric Assay Method

The USP method for assay of raw material and

In dosage forms is iodimetric titration of the thiocholine formed by hydrolysis of the echothiophate iodide. the USP XVIII procedure, the sample is hydrolyzed with sodium hydroxide (15). It has been shown recently that greater specificity is obtained when hydrolysis is conducted with a pH 12 buffer (16). the pH to be as high as 12 in order that the hydrolysis be completed in 20 minutes. a pH increases interference from possible impurities (16).

It is necessary for

On the other hand, too high

6.4 Thin Layer Chromatography

The following systems have been found useful for separating echothiophate iodide from possible degradation products: (a) Silica Gel G (E. Merck) with methanol- water-concentrated ammonium hydroxide (2: 2: 1) developing solvent and iodine vapor detection (17); (b) Cellulose F (E. Merck) with butanol-acetic acid-water (4:1:5) developing solvent and iodine vapor detection (18).

249

RAYMOND D. DALEY

6.5 Other Tests

A microscopic identity test for echothiophate iodide has been reported. Echothiophate iodide in aqueous solution forms a crystalline precipitate with amnonium reineckate (19).

7. Acknowledgments

The writer wishes to thank Dr. B. T. Kho for his review of the manuscript, Dr. G . Schilling of Ayerst Research Laboratories and Dr. W. E. Krueger of the State University of New York at Plattsburgh for their NMR data and interpretation, the library staff for their literature search, the numerous other contributors who provided information for this profile, and Mrs. Kay Mannan for typing the profile.

250


REFERENCES

1. 2.

3.

4.

5.

6.

7.

80

9.

10 . 11. 12

13.

14.

15.

16.

17.

18.

19.

C. A. 76, 2946 (1972), Chemical Abstracts Index Guide. British Pharmaceutical Codex 1968, Supplement 1971, The Pharmaceutical Press, London, 1971. The Merck Index, 8th Ed., Merck and Co., Inc., Rahway, N. J., 1968. L. J. Bellamy, The Infra-red Spectra of Complex Molecules, 2nd Ed., John Wiley and Sons, Inc., New York, 1958. G. Schilling, Ayerst Research Laboratories, personal communi ca ti on. H. Babad, W. Herbert, and M. C. Goldberg, Anal. Chim. Acta 41, 259-68 (1968). W. E.Krueger, State University of New York at Plattsburgh, personal communication. V. Mark, C. H. Dungan, M. M. Crutchfield, and J. R. Van Wazer, Top. Phosphorus Chem. 2, 227-457 (1967). F. Q. Gemmill, Ayerst Laboratories, Inc., personal communica ti on. H. M. Fitch, U. S. Patent 2,911,430; C. A. 54, 4386h. L-E. Tammelin, Acta Chem. Scand. ll, 1340-9 (1957). A. Hussain, P. Schuman, V. Peter, and G. Milosovich, J. Pharm. Sci. 57, 411-8 (1968). N. G. Nash and F. DiBernardo, Ayerst Laboratories, Inc., personal communication. D. R. Grassetti and J. F. Murray, Jr., Arch. Biochem. Biophys. 119, 41-9 (1967). Echothiophate iodide monographs, Pharmacopeia of the United States of America, 18th Revision, Mack Printing Co., Easton, Pa., 1970, pp. 220-1. C. Warner, F. DiBernardo, A. Bylw, A. Hussain, and B. T. Kho, J. Pham. Sci. 60, 1548-9 (1971). A. Bylow, Ayerst Laboratories, Inc., personal communication. G. R. Boyden, Ayerst Laboratories, Inc., personal communication. L. G. Chatten, A. C. Napper, and P. J. Barry, J. Pharm. Sci. 56, 834-8 (1967).

The above references cover the literature through 1972.

25 1

ETHYNODIOL DIACETATE

Edward P. K. Lau and John L . Sutter

EDWARD P. K. LAU AND JOHN L. S U T E R

Contents

1. Description

1.1 Name, Formula, Molecular Weight. 1.2 Appearance, Color, Odor.


2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9

Infrared Spectrum Nuclear Magnetic Resonance Spectrum Ultraviolet Spectrum Mass Spectrum Optical Rotation Melting Range Differential Scanning Calorimetry Thermogravimetric Analysis Solubility

3. Synthesis

4. Stability and Degradation

5.


Drug Metabolic Products and Phannacokinetics

6.1 Phase Solubility 6.2 Spectrophotometric Analysis 6.3 Colorimetric Analysis 6.4 Fluorometric Analysis 6.5 Titrimetric Analysis 6.6 Chromatographic Analysis

6.61 Column Chromatography 6.62 High Pressure Liquid Chromatography 6.63 Thin Layer Chromatography

7. Acknowledgments

8. References

254


1. Description


Ethynodiol Diacetate is 19-Nor-17a-pregn-4-en- 20- yne-3B, 17-diol Diacetate.

Weight: 348.52

1.2 App earance, Color, Odor

Ethynodiol diacetate is a white to off-white, essentially odorless powder.


2.1 Infrared Spectrum

The infrared absorption spectrum of an ethynodiol diacetate reference standard compressed in a KBr pellet is shown in Figure 1. the same infrared spectrum in chloroform solution. Tne following assignments have been made for absorption bands in Figure 1 ,'

The compound exhibits essentially

Qn. -1 Assignment

3315

1740

C r . ( 3 I : Acetylenic C-H stretching

C=O : Acetate Carbonyl stretching

1670 C=C : Ethylenic stretching

255


n I1

CH -C -0 - : Acetate C-0 3 stretching

1275, 1028

2.2 Nuclear Magnetic Resonance Spectrum

The NMR spectrum of ethynodiol diacetate in deuterated chloroform is shown in Figure 2. Spectral assignments are as follows:2

Chemical Shift (PPm) Type

5.00- 5.41 Broad Singlet

2.58 Singlet

2.03

0.90

Sing 1 e t

Sing 1 e t

Ass ignment

Protons at C-3 and c- 4

C G C H : Ethynyl pro ton

0 -0-C-CH : Acetyl

methgl protons

Aethyl protons -a : C-18

2.3 Ultraviolet Spectrum

Ethynodiol diacetate does not absorb between 420 nm and 210 nm. A peak is observed at 204 nm which is not con- venient for quantitative determination.

The USP XVIII assay procedure involves acid hydrolysis of the compound in methanolic 0.7 N HC1, for 10 minutes on a steam bath. The resulting soTution of diene exhibits the absorption spectrum sham in Figure 3, with maxima at about 229 nm, 236 nm and 244 nm. The peak at 236 nm is used for quantitative determination.3

2.4 Mass Spectrum

The low resolution mass spectrum of ethynodiol diacetate shown in Figure 4 was obtained with an AEI Ihdel M5-30

251

0 0

CI)

0

m

N

0

co N

0

b

N

0

10 N

0

In

N

0

4

PJ

0

m

N

0

N

N

259

FIG. 3: ULTRAVIOLET SPECXRLIM OF ETHYNODIOL DIACETATE

h) w UY

Wavelength (mp)

H n

A

'LNI

'13

M I


mass spectrometer. 384. The base peak in the spectrum was at m/e 43, COT- responding to (31 a+. Structure assignments are summarized below: '

A molecular ion was observed at m/e

m/e

384

Assignment % Relative Intensity

M'f 1 . 0

369 M- CH3' 1 .4

342 M- m2C0 0.2

324 M- C H 3 0 € I 8.5

1 .6 30 9 M- (QI + M3COOH) 3

282 M- (CHzCO + M3COOH) 2.2

2 64 M- (2 CH3cooH) 4.0

249 M- (2 M3COCH + M,') 1.4

43 c)13co+ 100.0

2.5 Optical Rotation

The following specipc rotation values in chloroform have been reported.

261

EDWARD P. K. LAU AND JOHN L. SU7TER

2.6 Melting Range

"lie melting range given in the USP XVIII is 126'to 132OC.

2 . 7 Differential Scanning Calorimetry

The DSC tliermogram of ethynodiol diacetate obtained a t a heating rate of S°C/minute is shown in Figure 5 . Tne endothermic change observed a t about 126OC corresponds to the melting of the drug. temperature is 228OC. ' The decomposition

2.8 Thennogravimetric Analysis

"lie TGA spectrum of ethynodiol diacetate i n Figure 6 was produced under a nitrogen sweep a t a heating rate of 10°C/minute. A rapid weight loss was observed from about 21OoC t o 26OOC. Another Fapicl weight loss was seen s ta r t ing a t about 400 C.

2.9 Solubili ty

Solubi l i t ies i n various solvents a t 25OC are given in the following table:

Solvent Solubili ty, mg ./d.

Water 0.0014

Methanol >so

Ethanol >50

Chloroform )SO

Heptane 18

262


80 100 120 140 160

TEMPERATURE oc

263

EDWARD P. K. LAU AND JOHN L. SUTTER

FIG, 6: TGA SPECIRJM OF FI1NNODlC)L DIAEI'XTE

100

80

60 s .-. 9 0"

40

20

100 200 300 400 500

TEGW?ATJRE OC

264


3. Synthesis

Ethynodiol diacetate has been synthesized by routes utilizing both estradiol 3-methyl ether (I) and 38- hydroxyandrost-5-en-17-one (11) as starting materials.

In the former method, 9 9 l o , l'outlined in Figure 7 , estradiol 3-methyl ether (I) is reduced by the Wills- Nelson modification of the Birch procedure12,to give the 1,4-dihydro derivative (111). Oppenauer oxidation of (111)13 yields the 17-ketone (IV), which is then ethynylated,14 giving the enol ether intermediate, (V). Reaction of (V) with dilute acetic acid produces norethynodrell with aqueous mineral acid gives norethindrone (VII) , which is then converted to ethynodiol (VIII) by reduction with sodium borohydridelO, 16. The diol is then diacetylated with acetic anhydride and pyridine, yielding ethynodiol diacetate (IX) .

(VI) . Treatment of either (V) or (VI)

Alternatively, as shown in Figure 8, peracid treatment of 3fi-hydroxyandrost-5-en-17-one (11) yields the 5,6 a-epoxide (X) . Perchloric acid cleavage of (X) results in the 5,6-diol (XI); acetylation then gives the 3, 5,6-triacetate (XII) , which reacts selectively with bicarbonate to give the 3fi,68-diol-5a-acetate (XIII) , Selective acetylation at C-3 followed by lead tetraacetate and iodine functionalization of C-19 then yields the 68, 19-oxide (XIV). Bicarbonate hydrolysis of (XIV) followed by chromic acid oxidation of the resulting alcohol affords the key intermediate (XV), which, when treated with zinc and zinc chloride in methanol gives 19-hydroxyandrostene- dione (XVI). Treatment of (XVI) with chromic acid affords the acid (XVII), which on heating in pyridine is decarboxy- lated to give the 5 (10)-dehydro derivative (XVIII). Selective ketalization of (XVIII) at C-3 is accomplished by treatment with weak acid in methanol, yielding (XIX). Ethynylation at C-17 then gives the 3-dimethyl ketal of norethynodrel (XX), Weak acid cleavage of (XX) gives norethynodrel (VI) , while more vigorous acid treatment gives norethindrone (VII) , Conversion of (VII) to ethynodiol diacetate (IX) is accomplished as previously described,

265


FIG. 7: SYNTHESIS OF E"ODI0L DIACETAE

a3 && I 013 I11

Q13 &;3&-c=m V

&:;&-a @' d

d

I/ IV

&;&--ca

VI VI I

HO VIII

266


FIG. 8: SyN?HESIS OF ETHYNODIOL DIACETATE

X 0 I1

XI I

I( XI

H Ac6

Aco OH XIV XI11

267


FIG. 8: (CONT.)

XVI I W I I I

268


4. Stabi l i tv and Demadation

Ethynodiol diacetate appears to be very s table as a solid. The degradation of ethynodiol diacetate in both acidic and basic alcoholic solutions is s h m i n Figure 9. acidic alcohol solution, the primary degradation product was found to be the diene (I). In basic alcohol solution, the primary degradation product was found to be the diol

In the

(11). l 9

5. Drug hletabolic Products and Pharmacokinetics

The major metabolites of ethynodiol diacetate in urine a re shown i n Figure 10. These metabolites were ;$en- t i f i e d by Kishimoto, Kraychy, Ranney and G a n t t , metabolism of ethynodiol diacetate by rat and human l i ve r was reported by Freudenthal, Cook, Forth, Rosen- fe ld and Wall, *' They found tha t the biotransformation reactions involved i n the in v i t ro metabolism include deacetylation, saturation 3 r ing A, aromatization of ring A, formation of 3-ketone and an6-bond formation. A method of analysis of very low levels of the metabolite norethindrone has been developed by Freudenthal , Cook and Wall. 2 2 The principle of t h i s method is t o convert the cold noreth'

3,17- B-diol .

The

-

rone by enzyme reduction i n the presence of NADF'H-4 e t o t r i t i a t e d 17-a-ethynylestrane-

The pharmacokinetic prof i le of the t o t a l tritium label and metabolic composition in the plasma a t e r an oral ad-

subject was studied by Karim, Ranney, Cook and Bres~ le r .~3 The ab orption rate constant (k) of the t o t a l label was

a f t e r 3 hours. The elimi at ion r a t e constant (K) of the

The volume o f dis t r ibut ion (V) was found to be 33L and the metabolic clearance rate (MCR) 21.9L per day. On chloroform extraction of the pooled plasma, 20% of the radioactivity was obtained as a f ree f ract ion which on TLC analysis gave two major spots tentatively identified as saturated dihydroxy metabolites and norethindrone. Eighty percent of the pooled plasma radioactivity was pres

ministration of ethynodiol diacetate-6,7- \ I to a human

0.79% % per hour, the peak plasma level being attained

to t a l label was 0.0276% % per hour (ha l f - l i f e 25 hours).

269

FIG. 9: CEGRADATION OF ETHYNODIOL DIACETATE IN ACIDIC F7 BASIC SOLUTION


FIG. 10: MAJOR METABOLITES OF ETHYNODIOL DIACETATE IN URINE

H

27 1


ent as water-soluble conjugates which on acidic hydrolysis furnished two major aglycones having chromatographic mo- b i l i t i e s similar t o the two major spots. In a plasma sample taken one hour after administration of the labeled drug, 58% of the radioactivity was associated with the conjugated metabolites, 12.5% with the spot identified as saturated dihydroxy metabolites and 19.7% with the spot identified as norethindrone.


6.1

6.2

6.3

Phase Solubili ty

Phase so lubi l i ty analysis can be carried out by equilibrating the drug substance i n hexane a t 25%. Figure 11 shows the phase so lubi l i ty diagram of a reference standard run.

Spectrophotometric Analysis

Ethynodiol diacetate does not have a useful spectrum for direct U.V. analysis. The solution of diene re- sul t ing from acid treatment has an absorbance maximum a t about 236 nm. The USP XVIII assay is based on t h i s reaction.

Colorimetric Analysis

A variety of colorimetric methods have been developed t o detect and t o determine ethynodiol diacetate.

6.31 Reaction of ethynodiol diacetate with antimony t r ichlor ide in dry chloroform containing 1% acetic anhydride produces a v io le t color. The absorbance of the solution a t 565 nm. is l inear w i t h ethynodiol concen- t ra t ion over a range of 5-60 mcg./5 ml. Tne method has been adapted for the analysis of ethynodiol dosage forms.25 None of the other steroids comonly found in oral estro- gen-progestin combination dosage forms interfere . A chloroform solution of a n t h n y

212


WIPE: ETHYNODIOL DIACETAE S O L W : H E M SLOPE: 0.0%

EQUILIBRATICN: 24 hrs. at 2S°C EXTRAPOLATED S O ~ I L I T Y : 27.9 mg./g. solvent

FIG. 11:

PHASE SOLUBILITY

- & = Q s - n

V W

40 00 llu 100 120 u 20

27 3

EDWARD P. K. LAU AND JOHN L. S U l T E R

6.32

6.33

trichloride has also been proposed as a spray reagent for ethynodiol diacetate in quantitative thin layer chromatography.

Reaction of ethynodiol diacetate with 52% sulfuric acid for 5 minutes at room temperature yields a solution having an absorbance maximwn at 484 nm. This method may be used for quantitative determination of ethynodiol diacetate, provided that a preliminary separation from other steroids is made,

Reaction of ethynodiol diacetate with hy- droxylamine hydrochloride and ferric chloride produces a deep red color which serves to distinguish the compound from steroids having no ester group, The color is not sufficiently stable for use in a quantitative determination. *

6 . 4 Flwrometric Analysis

Ethynodiol diacetate can be quantitatively determined by flwrometry in 65% sulfuric acid solution, with an activation wavelength of about 458 nm. and measuring fluorescence at about 520 nm. sary due to their interference. sitivity of the method is 4 mcg./lOO d.”

Separation from other steroids is neces- The limit of sen-

6 . 5 Titrimetric Analysis

6.51 Ethynyl Titration - ethynodiol diacetate reacts stoichiometrically with silver nitrate in tetrahydrofuran. produced can be titrated with sodium hydroxide, either potentiometrically or using phenolphthalein as indicator. One equivalent of the compound is titrated.

6 . 5 2 Ester Saponification - ethynodiol diacetate may be saponified with a h o r n amount of standardized alcoholic potassium hydroxide.

The nitric acid

274


6.6

The excess base is then titrated with hydrochloric acid, either potentiometrically or using phenolphthalein as indicator. 2 9 One equivalent of the compound is saponified.

Chromatographic Analysis

6.61 Column Chromatography - the quantitative separation of ethynodiol diacetate and mes- tranol in dosage f o m on Sephadex LH-20 has been reported.

6.62 H i g h Pressure Liquid Chromatography - ethynodiol diacetate can be separated from its possible degradation products and quanti tat ivel y determined by reverse- phas e high pressure liquid chromatography, using a W o n t ODs column and methanol-water eluants. 3 1

6.63 Thin Layer firomatography - TLC systems and corresponding Rf values of ethynodiol diacetate are summarized in the following table:

Thin Layer Chromatography of Ethynodiol Diacetate

Solvent System Adsorbent Detection 3 Reference

cyclohexane : SG 1, 2 0.40 32 isopropanol

(97 : 3)

benzene :methanol SG (95:s)

benzene:acetone SG (80:20)

chloroform : SG methanol

(90: 10)

1, 2 0.77 33

3, 4 0.68 33

3, 4 0.76 33

27 5

EDWARD P. K. LAU AND JOHN L. SUlTER

methylene chlor- SG 3, 4 0 .84 33 ide :methanol :water

(150 : 9 : 0.5)

SG = Silica gel.

Detection: 1. Spray with 50% H2S04, heat at 8OoC for 10 minutes.

2 . Spray with phosphomolybdic acid.

3. Spray with concentrated H SO4; heat at 100°C for 38 mmutes.

4. Observe under short wave U.V.

7 . Acknowledgments

The authors wish t o express their appreciation to Dr. N. W. Atwater, Dr. R. Bible, Dr. F. Colton, Mr. A. J. Damascus and Dr. J. Hribar for their help in preparing sections of the manuscript. tarial assistance of Miss Mia Mulder is also grate- fully acknowledged, as is Mrs. Lorraine Wearley's aid in preparing the figures,

The expert secre-

276

ETH Y N OD1 OL D I ACETATE

8. References

1. Damascus, A. J., Searle Laboratories, personal communication.

2 . Bible, R., Searle Laboratories, personal cmuni- cation.

3. Searle Laboratories Method of Analysis No, RS- 24-817.

4. Hribar, J., Searle Laboratories, personal communication.

5. Damascus, A. J., Searle Laboratories, personal communication.

6. "The United States Phannacopeia" XVIII, p. 259 (1970).

7. Carey, S . and Anthony, G . , Searle Laboratories,

8. Chow, A. and Marshall, S., Searle Laboratories,

9. Colton, F. B., U.S. Pats, 2,691,028 (1954);

personal c o m i c a t i o n .

personal c o m i c a t i o n .

2,725,389 (1955).

10. Colton, F. B., U.S. P a t , 2,843,609 (1958).

11. Klimstra, P. D. and Colton, F. B., Steroids - 10 411 (1967).

12 . Birch, A. J. and Smith, H., J. Chem. SOC. -' 1951 1882.

13.

14.

15. Colton, F . B., U.S. Pats, 2,655,518 (1952);

Oppenauer, R. V., Org. Syn. - 21, 18 (1941),

Stavely, H. E., J. Am. Chem. SOC. - 61, 79 (1939).

2,691,028 (1954); 2,725,378 (1955).

277

EDWARD P. K. LAU AND JOHN L. SUlTER

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27-

28.

29.

Sondheimer, F, and Klibansky, Y., Tetrahedron 5, 1 5 (1959)

Pappo, R. and Nysted, L . , U.S. Pat. 3,176,014 (1965).

Hagiwara, H., Noguchi, S., and Nishikawa, M., &em. Pharm. Bull. (Tokyo), - 8, 84 (1960).

Bollweg, M. and Baier, M., Searle Laboratories, personal comunication.

Kishimoto, Y . , Kraychy, S., Ranney, R. E. and Gantt, C. L . , Xenobiotica - 2, 237-52 (1972)

Freudenthal, R. I., Cook, C. E., Forth, J., Rosenfeld, R. and Wall, M. E , , J. Phannacol. Exp. Ther. 177, 468-73 (1971).

Freudenthal, R. I . , Cook, C. E. and Wall, M, E., "Progress Report No. 2", RTI Project No. CN-385, Research Triangle Ins t i tu te , Research Triangle Park, N. C., 1971.

Karin, A,, Ranney, R. E. , Cook, C. E. and Bres- sler, R., "Pharmacokinetics and Plasma Metabol- i t e s of SC-11800 (Ethynodiol Diacetate) in a Human Subject", Searle Laboratories Progress Re- port.

Root, A. and Chow, R., Searle Laboratories, personal c o m i c a t i o n .

-

Pasini, R. and Gavazzi, G . , J. Pharm. Sci. 58 -' 872-4 (1969).

Keay, G. R., Analyst - 93, 28 (1968).

Seul, C. J., Searle Laboratories, personal corn- munication.

Jack, M., Searle Laboratories, personal comunicat ion.

Brown, V., Searle Laboratories, personal communication.

278

ETH YNODl OL DI ACETATE

30. Fernandez, A. L . , and Noceda, V. T., J. Pharm. Sci. 58 740 (1969).

Wood, N . , Searle Laboratories, personal comunication.

-9

31.

32. Smith, B., Searle Laboratories, personal comunicat ion.

33. Simard, M. B. and Lodge, B. A., J. Chromatog. - 51, 517 (1970).

279

FLUDROCORTISONE ACETATE

Klaus Florey

KLAUSFLOREY

CONTENTS

1.

2.

3. 4. 5.

6.

7.

9. a.

Description 1.1 Name, Formula, Molecular Weight 1.2 Appearance, color, Odor Physical Properties 2.1 Infrared Spectra 2.2 Nuclear Magnetic Resonance Spectrum 2.3 Ultraviolet Spectrum 2.4 Mass Spectrum 2.5 Optical Rotation 2.6 Melting Range 2.7 Differential Thermal Analysis 2.8 Solubility, Dissolution, Partition

2.9 Crystal Properties Synthesis Stability, Degradation Drug Metabolism 5.1 Pharmacokinetic 5.2 Metabolic Products 5.3 Microbiological Transformations Methods of Analysis 6.1 Elemental Analysis 6.2 Direct Spectrophotometric Analysis 6.3 Colorimetric Analysis 6.4 Polarographic Analysis 6.5 Chromatographic Analysis

Coefficient

6.51 Paper 6.52 Thin Layer

6.6 Bioassay 6.7 Other Determination in Body Fluids and Tissues. Determination in Pharmaceutical Preparations References

282

F LUDROCORTISON E ACETATE

1. Description 1.1 Name, Formula, Molecular Weight

Fludrocortisone Acetate is 9a-fluoro- llf3, 17a,21-trihydroxy-4-pregnane-3,2O-dione, 21- acetate: also 9a-fluorohydrocortisone acetate: 9a-fluoro-17-hydroxycortisone 21-acetate;ga- fluorocortisol 21-acetate; fluodrocortisone 21-acetate; fluohydrisone, 21-acetate, fluohydro- cortisone 21-acetate, SQ 9321.

21 HzOCOCH3

L O

'2 3H3 lF06 M.W. 422.48


crystalline, odorless substance. Fludrocortisone Acetate is a white

2. Physical Properties 2.1 Infrared Spectra

Mesley' has reported four polymorphic forms and their infrared spectra. ~

Pharmacopoeia.

solution at room temperature followed by heating at l o o o for 15 minutes.

Form C - (amorphous) - usually obtained by evaporation of chloroform or acetone solution

Form A - as received from the British

Form B - evaporation of chloroform

KLAUS FLOREY

a t room tempera ture .

c r y s t a l l i z a t i o n from f o r m C.

c h a r a c t e r i s t i c a b s o r p t i o n peaks ( c m - l ) Form A: 1412 1339 1274 1239 ( s h ) 1022 958 940 819 777 680 Form B: 1418 1339 1272 1226 1195 1020 958 945 868 782 678 Form C: 1418 (sh) 1267 1236 1198 1023 959 936 870 782 680 Form D:

Form D - may be o b t a i n e d by spontaneous

These forms g i v e the fo l lowing

1406 1344 1267 1232 1198 1020 955 942 869 - 678

The i n f r a r e d f r e q u e n c i e s of m o d i f i c a t i o n s and s o l v a t e d e s c r i b e d by Kuhner t -Brands tae t t e r and Gasser2 (see a l so S e c t i o n 2.9) are p r e s e n t e d i n Tab le 1.

Tab le 1 9a-Fluorohydrocor t i sone A c e t a t e

F requenc ie s ( c m - l ) Form OH CIO and C l C M o d i f i c a t i o n I 3440 1738,1716,1651,1629 (w)

3350

3460

3370 1650,1611 ( w ) 3300 ( s h )

3500 (w) 3350

I1 3500 (sh) 1756,1720,1650,1617

V 3510 1760,1745,1730,1721

VI 3525 1761,1750,1730,1647

Methyl a c e t a t e s o l v a t e 3510 1761,1748,1738,1722

3320 1643 3230 ( s h )

E thy l a c e t a t e 3515 1759,1738,1725,1652 s o l v a t e 3360

284


Benzene s o l v a t e 3500 1761,1749,1736,1721, 1644

3320 D i m e thylforma-

mide s o l v a t e 3360 1740,1721,1660,1624 3320 ( s h )

The i n f r a r e d spectrum of Squibb House S tanda rd ( b a t c h #48004-001) i n f i g u r e 1 r e p r e s e n t s m o d i f i c a t i o n I. (Form A ) 3

2 . 2 Nuclear Magnetic Resonance Spectrum The NMR spectrum of f l u o d r o c o r t i s o n e

a c e t a t e is p resen ted i n f i g u r e 2 . The s t r u c t u r a l d a t a p r e s e n t e d in Tab le 2 a g r e e w i t h t h e a s s igned s t r u c t u r e 4 . p ro tons a t C 1 1 and C 1 7 exchange wi th deuterium.

The two hydroxyl

Table 2 NMR S p e c t r a l Assignments of SQ 9321a

Proton a t

c4 c-11 C- 18 c-19 c-21 c-21

Ch e m i c a 1 S h i f t , 6 (ppm)

5.64 s 4.10 b 0.77 s 1 .49 s

4 * 7 8 ABq;J=17.0 Hz 5.06

C - 2 1 A c e t a t e 2.10 Ox(Exchangeab1e) 5.00 b, 5.43 s

a = DMs0-d~ s= s i n g l e t b= broad ABq = AB q u a r t e t

285

Figure 1. Infra Red Spectrum of Fludrocortisone Acetate (Squibb House Standard batch 48004-001) from KBr/Chloro€orm. 1nstrument:Perkin Elmer 21.

Figure 2. NMR Spectrum of Fludrocortisone acetate (batch 76682) in deuterated DMSO (1nstrument:Varian XL-100).

KLAUS FLOREY

2.3 U l t r a v i o l e t Spectrum F r i e d and Sabo5 r e p o r t e d max 238 nm;

c C = 16,800 i n e t h a n o l . 2.4 Mass Spectrum

The low-reso lu t ion mass spectrum o f SQ 9,321 (see f i g u r e 3 ) shows the expec ted M+ a t m/e 422. C o r t i c o s t e r o i d s g e n e r a l l y show fragmen- t a t i o n p a t t e r n s r e s u l t i n g from the loss of D-ring s u b s t i t u e n t s (cf A n a l y t i c a l P r o f i l e s , Triamcino- lone , Triamcinolone Acetonide, Triamcinolone 16 , 17-diacetate) . I n a d d i t i o n , f l u o r i n a t e d steroids a l s o have f r agmen ta t ion pathways i n v o l v i n g the loss of HF. Thus, t h e f r agmen ta t ion pathways shown below d e p i c t the losses o f t h e s e groups.

m / e 422 M+

m / e 363 -C0CH20Ac m / e 362

I H2 34 2

m / e 292 m/e 303

m/e 283 m / e 301 1 -HF m / e 344

The base peak of m/e 42 i s from the a c e t y l p o r t i o n of t h e 21 -ace ta t e . Although n o t a s i n t e n s e a s t h o s e from A-ring d ienones , t h e m / e 121-123 (CgHg-110)and the m / e 135-137 (C Hll-130) i o n s s u p p o r t t h e p re sence of t h e A-ring enone group. The mass spectrum i s c o n s i s t e n t w i t h t h e proposed s t r u c t u r e 6 .

288

F i g u r e 3 . Low r e s o l u t i o n mass spectrum of F l u d o r c o r t i s o n e Acetate. (Squibb s t a n d a r d batch 48004-001) Ins t rument : AEI-MS-902.

KLAUS FLOREY

2.5 O p t i c a l R o t a t i o n

L d i ~ Solven t +143O Chloroform +127O Acetone +149O Dioxane +145- 15O0Dioxane

2.6 Meltinq Range

233-234' 230° ( decomp. ) 220-233O ( decomp. )

Ref 5 5 7 8

-

Ref 5 7 8

-

I t was n o t e d 5 t h a t o c c a s i o n a l samples s t a r t e d t o m e l t a t 205-208° , r e so l id i f i ed and e v e n t u a l l y melted a t 226-228 (See s e c t i o n 2 . 1 0 ) . The me l t ing behav io r by h e Kof l e r method has been d e s c r i b e d a s fo l lows : 5 A t 210° d r o p l e t s s t a r t t o form. The r e s i d u a l c r y s t a l s grow t o g r a i n s , squa res , and hexagons t h a t f i n a l l y aggrega te t o a mosaic. Three o r f o u r d i f f e r e n t forms a r e produced a t 1600 i n t h e g l a s s y s o l i d i f i e d m e l t . The bu lk cons is t s of long s t a l k e d s p h e r u l i t e s of Form I1 t h a t m e l t a t 208-212O and l e a f y , p a r t i a l l y f a n l i k e r a d i a t e s p h e r u l i t e s of Form I11 t h a t m e l t a t 205-208O and e x h i b i t low-order i n t e r f e r e n c e c o l o r s . Form I V appears o n l y r a r e l y a s f i b r o u s - t w i s t e d s p h e r u l i t e s . The m e l t becomes brown i n c o l o r . The e u t e c t i c tempera ture w i t h pheno lph tha le in i s 202O. (For t h e me l t ing behavior of polymorphic forms see a l s o s e c t i o n 2.9. )

2.7 D i f f e r e n t i a l Thermal Ana lys i s Squibb S tanda rd (ba t ch 48004-001)

e x h i b i t s a s h a r p endotherm a t 23OoC1O.

290


2 . 8 Solubili ty ' '

I n water: 0 .04 mg/ml; i n acetone 56 mg/ml;

The d i s s o l u t i o n behavior of c r y s t a l l i n e i n chloroform 20 mg/ml; i n ether 4 mg/ml.

f ludrocor t insone a c e t a t e and i t s pentanol and e t h y l ace ta te lgolva tes were s t u d i e d by Shef t e r and Higuchi . The i n i t i a l d i s s o l u t i o n rates of t h e s o l v a t e w e r e s i g n i f i c a n t l y h ighe r than t h e non- so lva ted form. Flynn determined t h e p a r t i t i o n c o e f f i c i e n t between ether and water a s 45.7.i3

2.9 C r y s t a l Properties The opt ical c r y s t a l l o g r a p h i c p r o p e r t i e s

of f ludrocor t i sone a c e t a t e (probably modi f ica t ion A) and f l u d r o c o r t i n one i t s e l f have been presented as fol lows by B i l e s T4 .

System Crystal Optic Axial

+ Habit S iqn

Fludrocortisone Orthorhombic Columnar Fludrocortisone acetate Tetraqonal Columnar - 00

Refractive Indexes optic Orientation a ( w ) B ( 5 ) Y

Fludrocortisone xxll c 1.575 1.588 1.646

~ l l a 2 2 I1 b

acetate w II a t IIC

Fludrocortisone 1.604 1.538 --

Photomicrographs of the two crystals also were presentedl4.

29 1

FLAUS FLOREY

The existance of several polymorphs has already been reported in section 2.1. As many as six may exist, according to Kuhnert-Brandstaetter and Gasser15 but classification of the polymorphic conditions proved very difficult since except for modification I (m. p. 225-233OC), all other modifications show only very slight differences in their melting temperatures which are in the 205-215O range. Modification I11 and IV were not obtained in pure form. A further complica- tion is the formation of solvates from a variety of solvents (see section 2.8). The powder X-ray diffraction pattern of fludrocortisone acetate (Form A ) is presented in table 3l6:

.d-

12.40 9.10 8.70 7.40 6.80 6.50 6.30 6.20 5.78 5.62 5.49 5.15 4.80 4.62 4.50 4.33 4.20 4.12

Table 3 Relative Re la tive Intensity* * d Intensity

0.07 3.77 0.23 0.16 3.70 0.16 0.10 3.56 0.16 0.15 3.53 0.13 0.28 3.44 0.12 0.59 3.29 0.32 0.65 3.20 0.13 0.98 3.10 0.18 0.40 3.03 0.18 1.00 2.89 0.13 0.17 2.81 0.18 0.35 2.69 0.13 0.35 2.55 0.15 0.35 2.45 0.12 0.63 2.39 0.09 0.15 2.32 0.20 0.18 *d= (interplanar distance)ni'L 0.29 Asin 0 -

3 4.02 **based on highest intensit 3.94 0.12 of 1.00 Radiation: k a l an 3.83 0.17 Ka, Copper

1nstrument:Phillips

292

F LUDROCORTISON E ACETATE

3 . Synthes is

synthesized by Fr ied and Sabo’ by t rea tment of t h e epoxide 111 with hydrogen f luo r ide . Compound V I I hydrocort isone a c e t a t e ) was found a s a byproduct of t h e r eac t ion5 , 7 9 4 2 . Other approaches repor ted a r e in t roduc t ion of t h e 4-double bond v ia bromination ( I V and V ) , a l b e i t i n low yield17, and osmium t e t r o x i d e oxida t ion of t h e A17(20) precursor ( V I ) 1 8 . I t can be p u r i f i e d from V I I v i a the benzene adduct19. A method f o r t h e production of dense c r y s t a l has been patentedz0. I t can be deace ty la ted t o f luorohydrocort isone (11) 5. I t can se rve a s s t a r t i n g m a t e r i a l f o r 9a-fluoro-prednisolone (cf. r e f . 2 1 ) . For microbio logica l conversion t o t r iamcinolone see sec t ion 5 .2 .

F ludrocor t i sone Aceta te (Fig. 4) was f i r s t

4. S tab i l i ty -Degrada t ion

s o i i d . I n aqueous and a l c o h o l i c s o l u t i o n s the a -ke to l s idechain , a s i n a l l such c o r t i c o s t e r o i d q i s prone t o ox ida t ive rearrangement and degrada- t i o n a t a l k a l i n e pH* s.

F ludrocor t i sone a c e t a t e i s very s t a b l e a s a

I t has been repor ted22 t h a t hydrocort isone and prednisolone, when exposed t o u l t r a v i o l e t l i g h t o r o rd ina ry f l u o r e s c e n t l abora to ry l i g h t i n g i n a l c o h o l i c s o l u t i o n s , undergo p h o t o l y t i c degradat ion of t he A-ring. S ince f lud rocor t i sone a c e t a t e has t h e same A-ring a s hydrocort isone it probably a l s o i s l a b i l e under these condi t ions .

5. Drug Metabolism 5 . 1 Pharmocokinetics

The d i s t r i b u t i o n i n r a t t i s s u e s and organs was s t u d i e d with t r i t i u m l abe led f ludrocor t isone23. The k i n e t i c s of metabolism were determined i n man, dog, r a t , monkey, and guinea

293

CH2OCOCH3

c=o CH20COCH3 I

H20COCH3

I11

VI

CH OCOCH3

L O

w \D P

Br 0

I R=COCH3 Br

VI I V I1 R=H

Figure 4

CH2OCOCH3

c=o CH20COCH3 I

H20COCH3

I11

VI

CH OCOCH3

L O

w \D P

Br 0

I R=COCH3 Br

VI I V I1 R=H

Figure 4


pig after I . V . and intraduodenal administration. Depending on species,50% or more of the steroid remained unchanged 30 minutes after adminis tration24. Fludrocortisone and it' s acetate had the same pharmocokinetic profile in dogs. The blood level reached a peak between 4 and 8 hours25. of fluorine at position 9 prolonged the plasma half-life and depressed urinary excretion after oral and I . V . administration to dogs as compared to hydrocortisone. Disappearance of fludrocortisone acetate after incubation with rat liver slices27-31 or perfusion of rat liver32 was a l s o studied.

Silber26 found that introduction

W

5.2 Metabolic Products After incubation of fludrocortisone

ith rat liver slices S ~ h r i e f e r s ~ ~ identified 9a-f luoro-5@-pregnan-l1@, 17a, 21-trihydroxy- 3,20-dione and 9a-fluoro-5@-pregnan-3@, ll@, 17a, 21-tetrahydroxy-20-one. There was no evidence for 5a-or 20-hydroxy metabolites. Bush and Mahesh3' identified the following metabolites in human urine: 9a-Fluoro-3a, lip, 17a, 20,21 pentahydroxy-5@- pregnane 9a-Fluoro-3a,ll~,17a,20,21 pentahydroxy-58- pregnane 9a-Fluorote trahydrocortisol 9a-Fluoroallotetrahydrocortisol 9a-Fluoro-20,20-dihydrocortisol 9a-F 1 uoroco r t i so 1 9a-Fluoro-ll@-hydroxyetiocholanolone 9a-Fluoro-11B-hydroxyandrostanone

Bush and M a h e ~ h ~ ~ noted the far greater proportion of 5a-(H) steroids than found with the halogen-free parent steroid. The expected 11-ketone steroids were completely absent.

295

KLAUS FLOREY

5.3 Mic rob io log ica l Transformat ion The fo l lowing m i c r o b i o l o g i c a l t r ans -

formations of f l u d r o c o r t i s o n e and i t ' s a c e t a t e have been re o r t e d : 1 -hydroxy la t i on35, l-dehydro- gena t ion 9 ', 6 -hydroxy 1 a t i on 38, 16 -hydroxy- l a t i ~ n ~ ~ (see a l s o under Triamcinolone, A n a l y t i c a l P r o f i l e s of Drug Subs tances , V o l . l ) , and 20-carbonyl r e d u c t i o n t o 20-hydroxy140. For t r ans fo rma t ion by mixed c u l t u r e s see r e f . 41.

6. Methods of Ana lys i s 6 . 1 Elemental Ana lys i s

r

Element % Theory Repor t ed3 C 65.39 65.32 H F

7.39 7.26 4.52 4.50

6 .2 Di rec t Spec t ropho tomet r i c Assay

238 nm ( s e e 2 . 3 ) i s due t o t h e a,@ u n s a t u r a t e d ketone of t h e A-ring. The absorbance i s u s e f u l a s a measure of p u r i t y from ex t r aneous m a t e r i a l s and h a s been so used8, a l b e i t a t 242 nm.

The u l t r a v i o l e t a b s o r p t i o n band a t

6 . 3 C o l o r i m e t r i c Methods A number of c o l o r i m e t r i c methods f o r

i d e n t i f i c a t i o n , d i f f e r e n t a t i o n from o t h e r s t e r o i d s and q u a n t i t a t i o n have been a p p l i e d t o f l u d r o c o r t i s o n e a c e t a t e . Based on react ion w i t h t h e A-ring a r e t h e i ~ o n i a z i d ~ ~ ( k max 382 nm i n e t h a n o l ) and 2,4-dinitrophenylhydra~ine~~methods. Based on r e d u c t i o n of t h e dihydrox a c e t o n e s idecha in a r e t h e b l u e t e t r azo l iumYO, P o r t e r - S i lber3O, and Nessler' s reagent44 methods. A b l u e chromogen ( k m a x 625 nm) i s produced by r e a c t i n g f l u d r o c o r t i s o n e a c e t a t e w i t h

Reac t ions wi th a phenol, hydroquinone,phosphoric- s u l f u r i c a c i d mixture (amber c o l o r ) 4 6 , p - n i t r o so-

2,6-di-tert-butyl-p-cresol i n a l k a l i n e s o l u t i o n 45 .

296


d i m e t h y l a n i l i n e ( 2 max 650 nm) *’ and a s u l f u r i c acid, f r u c t o s e , c y s t e i n e mixture ( 2 max 548 nm) have been desc r ibed . The l a s t r e a c t i o n h a s a l s o been used t o determine sgs idua l f l u d r o c o r t i n s o n e i n f e r m e n t a t i o n b r o t h s . Chromo ens are a l so formed i n concen t r a t ed s u l f u r i c 53 and phosphor ic acids. 5 1

6 . 4 P o l a r p g r a p h i c Ana lys i s CohenJL s u b j e c t e d f l u d r o c o r t i s o n e t o

p o l a r a g r a p h i c r e d u c t i o n i n dime t h y 1 f ormamide and found two reduc ing waves:

Wave 1 Wave 2 E 1/2 (Volts vs.

I d ( D i f f u s i o n

n (Apparant number

mercury poo l anode) 1.66 2 .10

c u r r e n t c o n s t a n t ) 1 . 4 1 .8

of e l e c t r o n s t r a n s f e r r e d 0 .0 3 0 . 6 9

6.5 Chromatographic Ana lys i s

6.51 Paper Chromatographic Ana lys i s For pape r chromatographic

sys tems, see Table 4.

297

Table 4

System #

1 2 3

4 5 6

7 8

9 10

11

12

Solvent System Developing Time Rf Values (hrs)

Formamid/Chloroform 18 - Methanol/Water/Benzene 1:1:2 Methanol/Water/Ethylacetate/ Benzene 25:25:2.5:47.5 Propylene glycol/Toluene Benzene/Formamide Toluene/Heptane, Methanol/Water 5:5:7:3 Benzene/Methanol/Water 2:l:l Petroleum ether (b. p. 100-120°) Toluene/Methanol/Water 67:33 :85: 15 Benzene/Ethanol/Water 2:1:2 Toluene/Petr. ether (b. p. 30-60°), Methanol/Water 12:8:13:7 Benzene/Petr. ether (b. p. 90-looo), Methanol/Water 5:5:7:3 Methyl isobutyl ketone/Formamide 20:l

4

4 96 --

-- --

-- 5

2 - 1/2

2-1/2

2-1/2

-

- - -

0.27 0.9

- 0.9

0.35

0.18

0.87

Ref.

33 33

33 29 53

53 53

53 54

54

54

55


The following detection systems have been reported: Detection Systems Ref. u. v. 33 Tetrazolium 3 3 , 3 4 Phosphoric acid fluorescence 3 3 2,4 Dinitrophenylhydrazine 5 3 Tollen's Reagent 5 3 Isonicotinic acid hydrazide 55

System #12 can be used to separate fludrocortisone acetate (Rf 0.87) from fludrocortisone (Rf 0.68) and 16a-hydroxyf ludrocortisone (Rf 0 . 3 0 ) . It can be used for the quantitative determination of f ludrocortisone acetates5 by dissolving the ground tablets in dimethylformamide, spotting approx 100 mcg. on filter paper impregnated with formamide-methanol 20 :80, developing with methyl isobutylketone-formamide 20:1, elution, reaction with isonicotinic acid hydrazide and determination of the absorbance at 4 1 5 nm against a standard, us inz6 the genera 1 procedure of Roberts and Florey .

6.52 Thin Layer Chromatographic Analysis Experience with the thin layer

chromatography of fludrocortisone acetate is summarized in Table 5.

299

Table 5 Rf or "running distance" values

(for explanation of individual values, see below)

Sys tem 1 A B C D E a b C

Fludrocortisone Acetate 0.87 1.031.14 1.16 2.6 0.62 0.48 0.42 0.31

-- -- Fludrocortisone - 0.24 0.33 0.63 0.70 0.01 -- S y s tem I I1 I11 IV V VI Fludrocortisone

Fludrocortisone 0.49 0.46 0.29 0.71 -- 0.76

System 157: Kieselguhr G plate; Dichloroethane/methylacetate/water 2: 1: 1: Spray reagent:Alkaline 2,5-diphenyl-3(4-styrylphenyl)tetrazolium solution; ''Running distances" values related to cortisone acetate = 1.00; Systems A-E58:Kieselguhr GF 254 plates; Spray reagent:Tetrazolium blue: "Running distance" values: A,B,C,E related to hydrocortisone acetate=1.00 D related to hydrocortisone = 1.00 Solvent systems: A- 1,2-Dichloroethane:methanol:water 95:5:0.2

Acetate -- -- -- -- 0.55 0.90

w 0

B- 1,2-Dichloroethane:2-methoxyethyl acetate:water 80:20:1 C- Cyc1ohexane:ethylacetate:water 25:75:1 D- Stationary phase: 20% v/v formamide in acetone

E- Stationary phase: 25% v/v formamide in acetone Mobile phase:Chloroform:ether:water 80:20:0.5

Mobile phase:Cyclohexane:tetrachloroethane:water 50:50:0.1


Systems a-c59: K iese lguhr G p l a t e s : Spray r e a g e n t : Te t r azo l ium b l u e . Values g iven a r e Rf va lues .

S o l v e n t systems: a - methylene ch1or ide : to luene 60:40

b - methylene c h l o r i d e : t o l u e n e 50:50

c - chloroform: t o l u e n e 25: 75

Systems I - 1760: S i l i c a g e l p l a t e s , Spray r e a g e n t : V a n i l l i n - p e r c h l o r i c a c i d sprayed o v e r t e t r a z o l i u m r e a g e n t . Values g iven a r e Rf va lues . S o l v e n t system I - E t h y l a c e t a t e

I1 - Methylene ch1oride:dioxan: wa te r

(80:25:0.5) on formamide p l a t e

V - E t h e r

I11 - Chloroform-ether-water

I V - Amylacetate-acetone 1:1

System ~ 1 ~ 5 , S i l i c a g e l G F P l a t e , U .V. d e t e c t i o n o r e l u t i o n and r e a c t i o n wi thNydraz id . So lven t : Ether-dimethylformamide, ace tone , methanol 88:8:2:2. c o r t i s o n e a c e t a t e h a s an R va lue of 0.83 i n r e l a t i o n t o f l u d r o c o r t i s o n e a c e t a t e .

I n t h i s system A8~14-hydro-

6 .6 Bioassay A s e n s i t i v e b i o a s s a y i s based on t h e

u r i n a r y Na+/K+ r a t i o , exp res sed a s p e r c e n t of t h e c o n t r o l v a l u e a f t e r i n j e c t i o n of f l u d r o c o r t i s o n e a c e t a t e i n t o ad rena lec tomized r a t s 6 1 .

30

FLAUS FLOREY

6.7 Other Bismuth oxidation to the corresponding

17-ketosteroid has a l s o been used as the basis for an analytical method62.

7. Determination in Body Fluids and Tissues References mentioned earlier, can be

summarized as follows:

References : Thin Layer Chromatography 2 Paper Chromatography 29,33,34 Colorimetric 25,30,31,49 Bioassay 61

8 . Determination in Pharmaceutical Preparations

mention analysis in pharmaceuticals. The following references specifically

Paper Chromatography Colorimetric

References : 55 8,45,47

302


R e f e r e n c e s 9.

1.

2.

3.

4.

5.

6 .

7.

8.

9.

10.

11. 12.

13. 14. 15.

16.

17.

18.

19.

20.

R. J. Mesley, S p e c t r o c h i m i c a Acta 22 ,889 ( 1 9 6 6 ) .

M. K u h n e r t - B r a n d s t a e t t e r a n d P. Gasser , Microchem. J. 16, 577 (1971) . B. Toepl i tz , T G Squ ibb I n s t i t u t e f o r Medica l Resea rch , P e r s o n a l Communication. M. S . Puar , The Squ ibb I n s t i t u t e f o r Medical Research, P e r s o n a l Communication. J. F r i e d and E . F. Sabo, J. Am. Chem. SOC. - 76, 1455 (1954) and i b i d . 79, 1130 (1957) . A. I . Cohen, The Squibb I n s t i t u t e f o r Medica l Resea rch , P e r s o n a l Communication. R. F. Hirschmann,R. M i l l e r , J. Wood and R. E . J o n e s , J. Am. Chem. SOC. 78 ,4956 (1956) . James B. Kottemann, Drug Stand’ards 26, 38 (1958). M. K u h n e r t - B r a n d s t a e t t e r , E. J u n g e r and A . K o f l e r , Microchem. J. 53, 105(1965) . H. J acobson , The Squ ibb I n s t i t u t e f o r Medica l Research , P e r s o n a l Communication. The Merck Index , 8 t h E d i t i o n 1968. E . S h e f t e r and T. H iguch i , J. Pharm. S c i . - 52, 781 (1963) . G. L . F lynn , J. Pharm. Sci. 60, 345(1971) . J. A . B i l e s , J. Pharm. S c i . 50, 4 6 4 ( 1 9 6 1 ) . M. K u h n e r t - B r a n d s t a e t t e r and P. Gasser, Microchem J. l.6, 577 (1971) . Q. Ochs, The Squ ibb I n s t i t u t e f o r Medica l Resea rch , P e r s o n a l Communication. J. E l k s , G. H. P h i l l i p s and W. F. Wal l , J. Chem. SOC. 1958, 4001. J. A . Hogg and F. H. L i n c o l n Jr . , U.S. P a t e n t 2 ,875,200 (1959) K.G. F l o r e y and J. F r i e d , U . S . P a t e n t 2 ,809 ,977 ( 1 9 5 7 ) . R. P. Graber and C. S. Snoddy,u. S. P a t e n t 2 ,957 ,013 (1960) .

303

KLAUS FLOREY

21.

22 .

23.

24. 25.

26.

27. 28.

29.

30.

31 .

32,

33,

34.

35.

36.

J. F r i e d , K. F lorey , E . F. Sabo, J. H. Herz, A . R. Restivo,A. Borman and F. M. S i n g e r , J. Am. Chem. SOC. 77, 4181 (1955). W. E . Hamlin, T. Chulsk i , R. H. Johnson and J. G. Wagner, J .Arn. Pharm.Assoc., SCi. Ed, - 49 253(1963) and D. R. Bar ton and W. C. Taylor , J . A m . Chem.Soc. 80, 244(1958) , J. Chem. SOC. 1958 2500. H. Wenzl, A . Garbe, H, Nowak, Arzneim.- Forschung 1971, 1123. H. Wenzl, Arzneim. -Forsch. 1971,1127. H. Wenzl, A . Garbe, H. Nowak, Arzneim.- Forsch. 1971,1115. R. H. S i l b e r and E. R. Morgan, C l i n . Chem. -3 2 170(1956) . G. M. Reaven, Endocrinology 57, 580(1955). E. M. Glenn, R. 0. S t a f f o r d , S. C. L y s t e r and B. J. Bowman, Endocrinology 61,128 (1957). H. S c h r i e f e r s , W. Korus, and W. D i r s c h e r l , A c t a Endocrinol. 26, 331 (1957). J.H.U.Brown and A . Anason, Endocrinology - 62, 103 (1958). W. Korus and H. L. Krdskemper, K l i n . Wochschr. 38, 938 (1960). H. S c h r i e f e r s and W. Korus, Z . Phys io l . Chem. 318, 239(1960). H. S c h r i e f e r s , J. Phys io l . Chem. 324, 188 (1961) . I. E. Bush and V. B. Mahesh, Biochem. J. 93,236 (1964). W. J. McAleer, M. A . Kozlowski, T. H. S t o u d t and J. M. Chemerda, J. Org. Chem. 23, 508 (1958).

-’

C. J. S i h , Biochim. Biophys. Acta 62,541 (1962) .

37. G. M. S h u l l , U. S. P a t e n t 2,776,927(1957). 38. L. L. Smith, J. J. Goodman, H, Mendelsohn,

J. P. Dusza and S. B e r n s t e i n , J. Org. Chem. 26,974 - (1961).

304

F LUDROCORTISONE ACETATE

39.

40.

41.

42.

43.

44.

45.

46. 47.

48.

49. 50.

51.

52. 53. 54.

55.

56.

57.

58.

R. W. Thoma, J. F r i e d , S. Bonanno, and P. Grabowich, J. Am. Chem. SOC. 79, 4818 (1957). L. L. Smith, T. F o e l l and T. J. Goodman, Biochemis t ry L, 353 (1962). D. Y. Ryu, B. K. Lee, R. W.Thoma and W. E . Brown, B io techno l , Bioeng. 1969,1255. N. L. Wendler, R. P . Graber , C. S . Snoddy Jr and F. W. B o l l i n g e r , J. Am. Chem. SOC. - 79,4476 (1957) . L. L. Smith and Th. F o e l l , Anal. Chem. 31. 102 (1959) . C. Monder and A . White, Endocrinology E, 159 (1961). E. P. Schulz and J. D. N e u s s , Anal. Chem. 29,1662 (1957) . E. Ivashk iv , J. Pharm. S c i . 51,698( 1962) . J. Verd ie r , Ann. Pharm. Franc.18,795 - (1960; C . A . 55, 14826g (1961). C. J. S i h , S. C. Pan and R. E. Bennet, Anal. Chem. 32, 669(1960) . E. I vashk iv , Anal. Chem. 33,1051 (1961) . L. L. Smith and W. H. M u l l e r , J.Org.Chem. -’ 2 3 960 (1958) . W. J .Nowaczinski and P. R. Steyermark, Can. J. Biochem. and Phys io l . 34,592 (1956) . A . I. Cohen, Anal. Chem. 3 5 , 1 2 8 (1963) . L. M. Reineke, Anal. Chem. 28 ,1853(1956) . L. L. Smith, Th. F o e l l , R. deMaio and M. Halwer, J. Am. Pharm. Assoc. S c i . Ed. - 48, 528 (1959) . H. R. Rober t s , The Squibb I n s t i t u t e f o r Medical Research, Pe r sona l Communication (1967) .

H. R. Roberts and K. F lo rey , J. Pharm. S c i . , 51 ,794(1962) . X H a l l , J. Pharm. Pharmacol. S u p p l . 16, 9T (1964) . C. J. C l i f f o r d , J. V. Wilkinson and

305

KLAUS FLOREY

59.

60.

61.

62,

J. S. Wragg, J. Pharm. Pharmacol. Suppl. - 16, 11T (1964). D. Sonanin i , R. H o f s t e t t e r , L. Anker and H. Mdhlemann, Pharm. Acta. - 40, 302 (1965). M. S. Moss and H. J. Rylance, J. Pharm. Pharmacol. l8, 13 (1965). J. G. Llaurado, K l i n . Wochschr. 34 669 (1956). H. S c h r i e f e r s and W. Korus, J. Phys io l . Chem. 313,66 (1958).

L i t e r a t u r e surveyed through July 1972.

306

FLURAZEPAM HYDROCHLORIDE

Bruce C. Rudy and Bernard Z. Senkowski

Chemistry reviewed by R. I. Fryer.

BRUCE C. RUDY A N D BERNARD Z . SENKOWSKI

1.

2 .

3 .

4 .

5 .

6 .

7 .

8.

INDEX

Analytical Profile - Flurazepam Hydrochloride

Description 1.1 Name, Formula, Molecular Weight 1.2 Appearance, Color, Odor

Physical Properties 2.1 Infrared Spectrum 2.2 Nuclear Magnetic Resonance Spectrum

2.21 Proton Spectrum 2.22 19F Spectrum

2.3 U1 traviolet Spectrum 2.4 Fluorescence Spectrum 2.5 Mass Spectrum 2.6 Optical Rotation 2.7 Melting Range 2.8 Differential Scanning Calorimetry 2.9 Thermal Gravimetric Analysis 2.10 Solubility 2.11 X-ray Crystal Properties 2.12 Dissociation Constant

Synthesis

Stability Degradation


Methods of Analysis 6 . 1 Elemental Analysis 6.2 Fluorine Analysis

6.21 Organically Bound Fluorine Analysis 6.22 Free Fluoride Analysis

6.3 Thin Layer Chromatographic Analysis 6.4 Gas-Liquid Chromatographic Analysis 6.5 Polarographic Analysis 6.6 Direct Spectrophotometric Analysis 6.7 Colorimetric Analysis 6.8 Fluorimetric Analysis 6.9 Titrimetric Analysis

Acknowledgement

References

308

F LURAZEPAM HYDROCHLORIDE

1. Descr ip t ion

1.1 Name, Formula, Molecular Weight Flurazepam hydrochlor ide is 7-chloro-1-(2-

[diethylamino] e t h i l ) -5~(o-fluorophenyl)-1,3-dihydro-2_TI-l,4- benzodiazepin-2-one dihydrochlor ide.

C21H23C1FN30.2HC1 Molecular Weight: 460.83

1 . 2 Appearance, Color, Odor Flurazepam hydrochlor ide occurs a s an off-white

t o yellow, nea r ly odor less , c r y s t a l l i n e powder.

2 . Phys ica l P rope 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 flurazepam hydrochlor ide

is presented i n Figure 1 (1). The spectrum was measured with a Perkin-Elmer 621 Spectrophotometer i n a KBr p e l l e t containing 1.0 mg of flurazepam hydrochloride/300 mg of KBr. The assignments f o r t h e c h a r a c t e r i s t i c bands i n t h e i n f r a r e d spectrum a r e l i s t e d i n Table I (1) .

Table I

In f ra red Assignments f o r Flurazepam Hydrochloride

Frequency (cm-l) C h a r a c t e r i s t i c of

3066

2500

1683 1560 and 1483 748

aromatic CH s t r e t c h i n g

hydrochlor ide of t e r t i a r y

C-0 s t r e t c h i n g v i b r a t i o n s aromatic r i n g 4 adjacent H ' s on phenyl r i n g

v i b r a t i o n s

amine

3 09

&I

&I-

s- m- cu-- 0

- a--

a-

Ic--

a--

m--

t--

- m

- - ?:

310


2.2 Nuclear Magnetic Resonance Spectrum (NMR)

2.21 Proton Spectrum The pro ton NMR s p e c t r a shown i n F igure 2

were run on a Jeo lco 60 MHz NMR us ing t e t r ame thy l s i l ane as an i n t e r n a l r e fe rence (2 ) . The flurazepam hydrochlor ide spectrum, F igure 2A, was obta ined by d i s so lv ing 59.0 mg of sample i n 0.5 m l of methanol-d4. The s p e c t r a l ass ignments are l i s t e d i n Table I1 (2) . d4 occurs about 3.27 ppm and i n t e r f e r e s wi th t h e ass ignments i n t h a t reg ion . Therefore , t h e spectrum of f lu raze - Pam base (54.2 mg/0.5 m l CDC13), shown i n F igure 2B, w a s determined and t h e s p e c t r a l assignments presented i n Table

The s o l v e n t peak f o r methanol-

I1 (2 ) .

2 . 2 2 19F Spectrum The 1yF spectrum shown i n F igure 2C w a s

ob ta ined wi th a Jeo lco C-60 HL ins t rument wi th a 19F module c r y s t a l modified t o a f requency of 56.446 MIz. Two hundred mg of flurazepam hydrochlor ide were d i s so lved i n 0 .5 m l of methanol con ta in ing CC13F as t h e i n t e r n a l r e f e r e n c e (2 ) . The spectrum c o n s i s t s of a q u i n t e t a t -108 ppm. of CC13F as t h e i n t e r n a l r e fe rence along wi th the ass ignment of -108 ppm is i n accordance wi th Bovey (3 ) .

The choice

Table I1

NMR Assignments f o r Flurazepam and Flurazepam Hydrochloride

No. of Chemical S h i f t Proton Protons (ppm) M u l t i p l i c i t y

Flurazepam Hydrochloride a 6 1.40 T r i p l e t

b 6 ~ 3 . 4 2 Mu 1 t i p 1 e t C 2 ' ~ 4 . 5 0 T r i p l e t

(JH,-H~ = 7Hz)

311


Figure 2

A . B. C.

NMR Spectrum of Flurazepam Hydrochloride NMR Spectrum of Flurazepam Base 19F NMR Spectrum of Flurazepam Hydrochloride

I

C

3 12


No. of Chemical S h i f t Pro ton Pro tons (pprn) Mu1 t i p 1 i c i t y

d 2 ~ 4 . 5 0 e 2* 4.92 Sing 1 e t f 7 7.30-8.15 Mu1 t i p l e t

Flurazepam Base a 6 0.95 T r i p l e t

b 6 2.50 Quartet

C 2 3.47-4.55 Mu 1 t i p l e t d 2 3.76-4.85 Two sets of

double ts (J = 11 Hz)

( J H ~ - H ~ = 7.5Hz)

( J H ~ - H ~ = 7 . 5 ~ 2 )

f 6.95-7.80 Mu1 t i p l e t

I9F Spectrum of Flurazepam Hydrochloride -108 Quinte t * Also any H20 presen t i n methanol-d4

When t h e W spectrum of flurazepam hydrochlor ide 2.3 U l t r a v i o l e t Spectrum (UV)

w a s scanned from 450 t o 210 nm, t h r e e maxima and t h r e e minima were observed. The maxima a r e loca t ed a t 362 nm (E = 3.7 x lO3), 284 nm (E = 1.2 x 1041, and 239 nm (E = 2.8 x lo4) . The m i n i m a occur at 333 nm, 263 nm, and 219 nm. The spectrum shown i n Figure 3 was obtained from a s o l u t i o n of 1.006 mg of flurazepam hydrochlor idef100 m l of a c i d i f i e d methanol (2.8 m l of concentrated H2SO4 d i l u t e d t o 100 m l wi th anhydrous methanol) (4).

2.4 F1uorescenc.e Spectrum The e x c i t a t i o n and emission s p e c t r a f o r f lu raze -

Pam hydrochlor ide (1 mg/ml of methanol) a r e shown in Figure 4 (5). One maximum appears i n t h e e x c i t a t i o n spectrum a t 378 nm and one maximum i n t h e emission spectrum a t 492 nm.

2.5 Mass Spectrum The mass spectrum of flurazepam hydrochlor ide w a s

obtained us ing a CEC 21-110 mass spectrometer wi th an i o n i z i n g energy of 70 ev. The output from t h e mass spec tm- meter was analyzed and presented i n t h e form of a b a r

313

A 11 S N3lN I

tn a

w

I- w I

0

z

4 z

315

ar a

.?I

k 0

rw 0

I

t si R P 7 fl A H E w 8 0

316


graph, shown i n F i g u r e 5 , by a Varian 100 MS d e d i c a t e d com- p u t e r system. Due t o t h e extreme i n t e n s i t y of t h e base peak a t m / e 86, t h e r e l a t i v e i n t e n s i t y of t h e peaks a t t h e h i g h e r mass u n i t s are v e r y weak. Therefore , t h e peaks from m / e 150 up were s u b j e c t e d t o a t e n f o l d s c a l e expansion ( F i g u r e 5 - i n s e r t ) ( 6 ) . The p a r e n t peak (+) a t m / e 387 is due t o t h e free f lurazepam b a s e . The base peak a t m / e 86 i s due t o t h e (C2H5)2NCH2 fragment . can b e a t t r i b u t e d t o s t e p w i s e f ragmenta t ion of t h e p a r e n t i o n ; i . e . , M+ - (C2H5)zN = 315, 315 - CH2N = 287 ( 6 ) .

The o t h e r major peaks

2.6 O p t i c a l R o t a t i o n Flurazepam h y d r o c h l o r i d e e x h i b i t s no o p t i c a l

a c t i v i t y .

2.7 Mel t ing Range Flurazepam h y d r o c h l o r i d e m e l t s w i t h decomposi t ion

w i t h i n a 5 O range between 208' and 218OC when t h e USP X V I I I Class Ia procedure is used ( 7 ) .

2.8 D i f f e r e n t i a l Scanning Calor imet ry (DSC)

i n t h e m e l t i n g r e g i o n are v e r y dependent on previous thermal h i s t o r y . t h e e x t r a p o l a t e d o n s e t of a n endothermic t r a n s i t i o n occurred a t 215 . a 0 C fol lowed immediately by t h e exothermic t r a n s i t i o n due t o decomposi t ion a t 229.5OC (Figure 6 ) . A t a s c a n ra te of 10°C/min., a small endothermic t r a n s i t i o n o c c u r s a t 203.3OC fol lowed by sample decomposi t ion a t 2 1 7 . 5 O C . Due t o t h e sample i n s t a b i l i t y i n t h e r e g i o n of t h e m e l t , t h e AHf was n o t determined ( 8 ) .

The thermal p r o p e r t i e s of f lurazepam hydrochloride

Using a tempera ture program of 20°C/min.,

2.9 Thermal Gravimet r ic Analys is (TGA) A TGA performed a t a s c a n rate of 10°C/minute

showed l i t t l e weight loss f o r f lurazepam h y d r o c h l o r i d e from ambient t o 190°C. t h e sample o c c u r r e d between 190 and 345OC (8).

A weight l o s s amounting t o about 70% of

2 . l o S o l u b i l i t y The s o l u b i l i t y d a t a f o r f lurazepam h y d r o c h l o r i d e

obta ined by e q u i l i b r a t i o n f o r 20 hours a t 25OC are g iven i n Table 111 ( 9 ) .

3 17

2

Figure 6

DSC Scan for Flurazepam Hydrochloride

d 3 240 230 220 210 200 190 I80 170 I

TEMPERATURE 'C

318


Table I11

S o l u b i l i t y P r o f i l e f o r Flurazepam Hydrochlor ide

Solvent

3A a l c o h o l benzene chloroform 95% e t h a n o l d i e t h y l e t h e r m e thano 1 petroleum e t h e r 2-propanol water

S o l u b i l i t y (mg/ml)

28.3 0.4

11.1 2 60 0.2 340

14.6 >500

(3Oo-6O0) 0 . 2

2 . 1 1 X-ray C r y s t a l P r o p e r t i e s The x-ray powder d i f f r a c t i o n p a t t e r n of f l u r a z e -

Pam h y d r o c h l o r i d e i s presented i n Table I V (10). The i n s t r u m e n t a l c o n d i t i o n s are g iven below.

I n s t r u m e n t a l Condi t ions


Generator : Tube t a r g e t : Radia t ion : o p t i c s :

Goniometer : D e t e c t o r :

Recorder:

50KV-12-1/2 MA Copper Cu K, = 1.542 1 0.1' D e t e c t o r s l i t M.R. S o l l e r s l i t 3' B e a m s l i t 0.0007 inch N i f i l t e r 4' t a k e o f f a n g l e Scan a t O.Zo 28 p e r minute Amplif ier g a i n - 1 6 c o u r s e ,

8 . 7 f i n e Sea led p r o p o r t i o n a l c o u n t e r

t u b e and DC v o l t a g e a t p l a t e a u

P u l s e h e i g h t s e l e c t i o n EL -

Rate meter T .C . 4 2000 CIS f u l l s c a l e Chart Speed - 1 inch p e r 5

5 v o l t s ; EU - o u t

minutes

Samples prepared by g r i n d i n g a t room tempera ture .

319


20

8.32 9.04

11.62 12.49 12.86 15.50 16.40 16.78 17 38 17.94 18 .65 19.16 20.05 20.38 20.75 21.66 23.14 23.61 24.32 24.50 25.18 25.96 26.18 26.40 26.96 27.44 27.94 28.46 28.78 29.96 30.54 31.00

-

Tab le I V

X-ray Powder D i f f r a c t i o n P a t t e r n of Flurazepam Hydroch lo r ide

d (8) * 10.6

9.78 7.62 7.09 6.88 5.72 5.40 5.28 2.10 4.94 4.76 4.63 4.43 4.36 4.28 4.10 3.84 3.77 3.66 3.63 3.54 3.43 3.40 3.38 3.31 3.25 3.19 3.14 3.10 2.98 2.93 2.88

I / T E 3

20 37 1 5 11 62 11

9 7 5

50 100

26 20 19 1 2 82 39 40 36 98 1 2 33 24 1 2 1 5 18

5 5

1 9 4

1 5

28 - 31.40 32.36 32.92 33 -30 33.88 34.24 34.87 35.20 35.77 36.18 37.28 37.72 38.17 38.78 39.47 40.18 40.86 41.28 41.86 42.20 42.94 43.58 44.24 44.70 46.06 47.00 47.52 48.32 49.08 50.00 50.26 50.84

*d - ( i n t e r p l a n a r d i s t a n c e ) nX 2 S i n 0

d (8) * 2.85 2.77 2.72 2.69 2.65 2.62 2.57 2.55 2.51 2.48 2 .41 2.38 2.36 2.32 2.28 2.24 2.21 2.19 2.16 2.14 2.11 2.08 2.05 2.03 1.97 1 .93 1 . 9 1 1.88 1 . 8 6 1 . 8 2 1 .82 1 .80

I/TE

1 38

7 8 6 7

11 1 4

5 10

5 11 11 1 9

3 3 5 2 5 8 9 9 4 5 7 4 2 5 3 3 3 4

**I/Io = 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 of 1.00)

320


2.12 Dissociation Constant The pKa's for flurazepam were determined spectro-

photometrically and found to be 1.90 5 0.05 and 8.16 5 0.05 (11). titration curve in a 2-propano1:water (1:l) mixture and found to be 7.0 5 0.1 (11). In water, the trialkylamino type compounds are stronger bases, on the average, by 0.9 pK units (11,12). Therefore, the estimated pKa2 in water would be 7.9 which is in good agreement with that found spectrophotometrically.

The apparent pKa2 has also been determined from the

3. Synthesis Flurazepam hydrochloride may be prepared by the re-

action scheme shown in Figure 7. phenyl)-1,3-dihydro-2H-l,4-benzodiazepin-2-one is reacted with diethylaminoethyl chloride in the presence of sodium methoxide to yield flurazepam which is then converted to flurazepam hydrochloride by the addition of hydrochloric acid (13). A complete review of the chemistry of benzodiazepines by Archer and Sternbach presents several pathways to arrive at the basic benzodiazepine ( 1 4 ) .

7-Chloro-5-(o-fluoro-

4 . Stability Degradation When sealed amber ampuls with dilute solutions of

flurazepam hydrochloride in 0.1N HC1, water, and 0.1N NaOH: 3A alcohol (1:l) were heated in a boiling water bath for one hour, the degradation products shown in Figure 8 were observed by thin layer chromatography (15). In 0.1N HC1 solution the main hydrolysis product was 5-chloro-2-(2- diethylaminoethylamino)-2'-fluorobenzophenone hydrochloride. In aqueous solution the main degradation product was 7- chloro-5-(o-fluorophenyl)-ly3-dihydro-2H-l,4-benzodiazepin- 2-one. Finally, in the 0.1N NaOH:3A alcohol (1:l) solution, the main degradation products were 5-chloro-2-(2- diethylaminoethylamino)-2'-fluorobenzophenone ,and 2-chloro- l0-(2-diethylaminoethyl)-9-acridone. When a solution of flurazepam hydrochloride in water in irradiated with light from a high pressure U.V. lamp for 3 hours, some hydrolysis to 5-chloro-2-(2-diethylaminoethylamino)-2'-fluorobenzophenone occurs (16). Flurazepam hydrochloride, when stored in well closed, light resistant containers, is quite stable.

321

Figure 7

Synthes is f o r Flurazepam Hydrochloride

H

Naoc% + ClCH CH N( _____)

CH2CH3

CI * CH2CH3

7-CHLORO-5-(&FLUORO- PHENY L )-I ,3-DI HYDRO- 2 H -1.4- BENZODIAZEPINE- 2-ONE

CI K T C H ="

FLURAZEPAM

DIE THYLAMINO- ETHYLCHLORIDE


F i g u r e 8

Major Degradat ion Products f o r Flurazepam Hydrochlor ide i n Acid, Basic, and Aqueous S o l u t i o n s

FLURAZEPAM HYDROCHLORIDE &\ CH CH N(C2H5)2 1 2 2

CH2CH2NlC2H&, I

$H2CHZ"C2H32

&?.ti2 CI a- + CI fJ?n 6 C bf 6 8

nw ACRIDONE BENZOPHENONE 7-CHLORO-54 O-FLUOR& BENZOPMENONE

PHENYLkt3-DIHYDRO -2 H- I.4-BENZWUZEPIN-2-ONE


5. Drug Metabolic Products The metabolic pathways of flurazepam in dog and man are

shown in Figure 9 (17-22). Initially, five metabolites plus the intact drug were found in the urine of dogs. Four of these metabolites were identified as monodesethyl- flurazepam, didesethylflurazepam, flurazepam-N1-ethanol, and N1-desalkyl-3-hydroxyflurazepam (17,ZO). The fifth metabolite was tentatively identified as a phenolic derivative of N1-desalkyl-flurazepam by mass spectrometry (17). The major metabolite found in the dog urine is flurazepam- N1-acetic acid (17,19). N1-ethanol was the major metabolite along with smaller quantities of the mono- and didesethyl-flurazepam and N1- desalkyl-3-hydroxyflurazepam (17-22).

In human urine, the flurazepam-


6.1 Elemental Analysis The results from the elemental analysis are

listed in Table V (23).

Table V

Elemental Analysis of Flurazepam Hydrochloride

Element X Theory % Found

C 54.74 54.73 H 5.47 5.46 N 9.12 9.11 F 4.12 4.22 c1 7.69 7.86 Cl-(ionic) 15.38 15.42

6.2 Fluorine Analysis

6.21 Organically Bound Fluorine Analysis There are several methods available to

determine the amount of carbon-bonded fluorine. One of the earlier methods employed the Schoniger Combus tion technique followed by thorium nitrate or cerous chloride titration using sodium alizarin sulfonate or murexide as the indicator (24).

With the advent of good specific ion

324

Figure 9

Metabolic Products of Flurazepam Hydrochloride

H

NI-DESALKYL- FLURAZEPAM FLURUEPAY Nl-ETHAWL

CHpCOOH I N-C-0

Cl &:o" cgfa a ac=;.I. OH 8"

- \

N I -DESALKYL FLURAZEPAY

&F

N,-DESALKYL-3- HYDROXYFLURAZEPAM FLURAZEPAM PHENOL NI-ACETIC ACD


e lec t rodes , methods were developed t o l i b e r a t e t h e bound f l u o r i n e and d i r e c t l y measure the f l u o r i d e concent ra t ion . The r eagen t , sodiumbiphenyl , followed by oxida t ion wi th hydrogen peroxide, is used t o l i b e r a t e t h e o rgan ica l ly bound f l u o r i n e i n flurazepam hydrochlor ide. s p e c i f i c e l ec t rode i s used, i n the presence of a high- ion ic-s t rength b u f f e r s o l u t i o n , f o r d i r e c t measurement of t he l i b e r a t e d f l u o r i d e (25).

ana lys i s of t he carbon-bonded f l u o r i n e i n flurazepam hydroch lo r ide is 19F Nuclear Magnetic Resonance spectrometry (2). A flurazepam hydrochlor ide r e fe rence s t anda rd and an i n t e r - n a l s tandard , reagent grade o-fluorobenzoic a c i d , a r e d i s - solved i n methanol and t h e I9F spectrum obtained and in- tegra ted . conversion f a c t o r can b e ca l cu la t ed and used t o determine the amount of f l u o r i n e present i n a sample of flurazepam hydrochlor ide t h a t i s run i n a similar manner (26) .

A f l u o r i d e ion

The las t method t o be presented f o r t h e

From t h i s d a t a an i n t e r n a l s t anda rd f l u o r i n e

6.22 Free F luo r ide Analysis The de termina t ion of any f r e e f l u o r i d e

present i n flurazepam hydrochlor ide bulk samples i s car - r i e d o u t by d i r e c t measurement us ing a f l u o r i d e s p e c i f i c ion e l ec t rode . The measurements are made i n an a c e t a t e bu f fe r s o l u t i o n (pH 5 . 3 ) . The e l e c t r o d e response w a s found t o be l i n e a r throughout t h e working range of 0.08 t o 0.20 mg of F-/100 m l of s o l u t i o n (27 ) .

6 . 3 Thin Layer Chromatographic Analysis (TLC) Several TLC systems f o r t h e sepa ra t ion of

flurazepam hydrochlor ide from its me tabo l i t e s and similar s t r u c t u r e d compounds are given i n Table V I . I n each case the sample i s spo t t ed on a s i l i c a g e l GF p l a t e * which is allowed t o develop i n a s a t u r a t e d tank u n t i l t he s o l v e n t f r o n t has ascended about 15 cm. The p l a t e is then removed, a i r d r i e d , and viewed under shortwave and longwave U.V. r a d i a t i o n .

* If t h e sample s o l u t i o n is too a c i d i c , an a r t i f a c t appears a t t he po in t of a p p l i c a t i o n due t o t h e quenching of t h e phosphor i n the s i l i c a g e l GF p l a t e by the ac id .

326


Table VI

Rf Values for Flurazepam in Various Developing Solvents

Solvent System - R Value Reference

diethyl ether:diethylamine (75:2) 0.6 28

methylene ch1oride:ethyl ether: methano1:conc. ammonium hydroxide (240:360:8:3) 0.2 28

ethyl acetate:conc. ammonium hydroxide (200: 1) 0.14 20

ethyl acetate: ethanol: conc. ammonium hydroxide (100 : 10: 0.3) 0.38 20

benzene:methanol:glacial acetic acid (9: 1: 1) 0.26 20

chloroform: acetone (17 : 3) 0.00, 0.05* 20

* When the plate is developed two times in the same system

6 . 4 Gas-Liquid Chromatographic Analysis (GLC)

flurazepam and its metabolites has been used by deSilva et al. (29) to prepare the respective benzophenones as volatile derivatives for gas chromatography. This method is an adaptation of the method developed for GLC of diazepam and its metabolites (30). When the benzophenones were chromatographed at 21OoC on a 2 feet x 114 inch column containing 2% Carbowax 20M-TPA, they showed an excellent response to detection by electron capture which was linear between 10 and 40 ng. The main disadvantage of hydrolysis to the benzophenone is the lack of specificity for a given benzodiazepine.

A method recently published by Sine et al. (31) for chromatographing flurazepam directly, utilizes a 3 feet x 2 mm glass column packed with 3.8% SE-30 on Chromosorb W (AW-DMCS, 80-100 mesh). The GLC is equipped with a hydrogen flame ionization detector and the column temperature is about 23OoC. The patient's serum is adjusted to pH 7.4 and extracted with chloroform. The chloroform is evaporated, the residue is dissolved in acidic methanol (1 ml HCl/liter methanol) and chromatographed.

The acid hydrolysis of blood extracts containing

327


This method w i l l s epa ra t e flurazepam from diazepam and chlordiazepoxide.

6.5 Polarographic Analysis Polarographic a n a l y s i s of f lurazepam hydro-

ch lo r ide has been c a r r i e d o u t i n Britton-Robinson Buffer a t pH 4.4. a s i l v e r / s i l v e r ch lo r ide r e fe rence e l e c t r o d e and is pro- po r t iona l to concent ra t ion . reduct ion of t h e azomethine (,C=N) func t iona l group and v a r i e s wi th pH ( 3 2 ) .

The halfwave p o t e n t i a l occurs a t -0.78 V.versus

This wave is a t t r i b u t e d t o the

6.6 Direct Spectrophotometric Analysis Direct spectrophotometr ic a n a l y s i s is used t o

determine t h e quan t i ty of flurazepam hydrochlor ide p re sen t i n capsules. A quan t i ty of t h e capsule conten ts is at- cu ra t e ly weighed and t h e flurazepam is ex t r ac t ed i n t o a c i d i f i e d methanol ( see s e c t i o n 2 . 3 ) . The methanol solu- t i o n is f i l t e r e d and appropr i a t e subd i lu t ions made t o y i e l d a f i n a l s o l u t i o n conta in ing 1 .0 mg of flurazepam hydroch lo r ide pe r 100 m l of a c i d i f i e d methanol. The absorbance of t h i s s o l u t i o n along wi th a s o l u t i o n of flurazepam hydroch lo r ide r e fe rence s tandard s i m i l a r l y prepared is measured versus a c i d i f i e d methanol a t t h e 239 NO maximum. From t h i s da t a t h e concent ra t ion of flurazepam hydrochlor ide i n t h e capsules is ca lcu la t ed ( 3 3 ) .

6.7 Colorimetr ic Analysis Flurazepam hydrochlor ide forms a ion-pair complex

wi th bromocresol green i n a pH 5.3 b u f f e r . complex is ext rac ted i n t o chloroform and i ts absorbance measured a t t h e 415 nm maximum. versus absorbance is l i n e a r from 0 t o 2.5 mg of flurazepam hydrochlor ic per 100 m l of chloroform (34) .

This colored

A p l o t of concent ra t ion

6.8 F luor imet r ic Analysis A f l u o r i m e t r i c a n a l y s i s f o r t h e de te rmina t ion of

flurazepam hydrochlor ide and i t s metabol i tes i n blood and u r i n e has been descr ibed by de S i lva and St ro jny ( 2 0 ) . This a s say involves s e l e c t i v e e x t r a c t i o n i n t o d i e t h y l e t h e r from blood buffered t o pH 9 o r u r ine made b a s i c with NaOH, then back-extracted i n t o 4N H C 1 , and hydrolyzed t o t h e r e spec t ive benzophenones. cyc l ized t o t h e 9-acridone d e r i v a t i v e s i n dimethylformamide

The benzophenones are then

328


i n t h e presence of K2CO3. These d e r i v a t i v e s a re sepa ra t ed by TLC, e l u t e d from t h e s i l i c a g e l , and t h e i r f l uo rescence determined i n methanol:O.lN HC1 ( 4 : l ) . This method a l lows q u a n t i t a t i o n i n t h e range of 0.003 t o 10.0 mcg of compound/ ml of blood o r u r i n e (20) .

6.9 T i t r i m e t r i c Analysis Flurazepam hydrochlor ide is assayed by d i s s o l v i n g

about 0 .6 gm of sample i n g l a c i a l acet ic ac id , adding ex- c e s s mecuric acetate, and t i t r a t i n g with 0.1N p e r c h l o r i c ac id i n g l a c i a l a c e t i c ac id . The end-point is determined po ten t iome t r i ca l ly us ing a glass-calomel e l e c t r o d e system. Each m l of 0.1N p e r c h l o r i c a c i d is equ iva len t t o 23.04 mg of C21H23C1FN30*2HC1.

7 . Acknowledgment The au thors would l i k e t o thank D r . P. S o r t e r and t h e

S c i e n t i f i c L i t e r a t u r e Department as w e l l as t h e Research Records Department of Hoffmann-La Roche Inc . f o r t h e i r h e l p i n t h e l i t e r a t u r e sea rch f o r t h i s Ana ly t i ca l P r o f i l e .

329

BRUCE C. R U D Y A N D BERNARD Z . SENKOWSKI

8 . References

1.

2 .

3.

4 .

5.

6.

7. 8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18. 19.

20.

21.

Hawrylyshyn, M., Hoffmann-La Roche Inc., Personal Communication. Johnson, J. H., Hoffmann-La Roche Inc., Personal Communication. Bovey, F. A . , NucZear Magnetic Resonance Spectroscopy, Academic Press, New York City, pp.

Rubia, L. B., Hoffmann-La Roche Inc., Personal Communication. Boatman, J . , Hoffmann-La Roche Inc., Personal Communication. Benz, W., Hoffmann-La Roche Inc., Personal Communication. United S t a t e s Pharmacopeia XVIII, 935 (1970). Moros, S . , Hoffmann-La Roche Inc., Personal Communication. MacMullan, E., Hoffmann-La Roche Inc., Personal Communication. Hagel, R. B., Hoffmann-La Roche Inc., Personal Communication. Toome, V. and Raymond, G., Hoffmann-La Roche Inc., Unpublished Data. Gutbezahl, B. and Grunwald, E., J . Amer. Chem. Soc., - 75, 559 (1953). Sternbach, L. H., Archer, G. A., Earley, J. V., Fryer, R. I., Reeder, E., Wasyliw, N., Randall, L., and Banziger, R., J. Med. Chem., 8_, 815 (1965). Archer, G. A. and Sternbach, L. H., Frem. Review., 68, 747 (1969). Senkowski, B. Z., Hoffmann-La Roche Inc., Unpublished Data. Fryer, R. I., Hoffniann-La Roche Inc., Unpublished Data. Schwartz, M. A . , Vane, F. M., and Postma, E., J . Med. Chem., 11, 770 (1968). Usdin, E., PsychopharmaeoZ. BUZZ., 5, 4 (1970). Schwartz, M. A. and Postma, E., J . .?harm. S c i . , 59, 1800 (1970). deSilva, J. A . F. and Strojny, N., J . Pharm. Sei . , - 60, 1303 (1971). I

Randall, L. O., In t . Symp. Benzodiazepines, Sum., Milan, Italy, 1971:Z.

211-214.

330


22.

23.

24.

25.

26.

27 .

28.

29.

30.

31.

32.

33.

34.

Schwartz , M. A . , I n t . Symp. Benzodiazepines, Sum., Milan, I t a l y , 1971:3. S c h e i d l , F., Hoffmann-La Roche I n c . , P e r s o n a l Communication. Steyermark, A , , Quantitative Organic MieroanaZysis, 2nd Ed., Academic Press, New York, N. Y., 1961,

J o n e s , B. C . , Heveran, J. E . , and Senkowski, B . Z., J . Pharm. Sc i . , 60, 1036 (1971). Rudy, B. C. and Senkowski, B. 2. "Analy t ica l P r o f i l e of F l u o r o u r a c i l " , accepted f o r p u b l i c a t i o n i n AnaZyticaZ ProfiZes of Drug Substances, V o l . 2, 1972. Jones , B. C . and Heveran, J . E . , Hoffmann-La Roche I n c . , Unpublished Data. Hochhauser, L . , Hoffmann-La Roche I n c . , Unpublished Data. d e S i l v a , J. A . F. , Bader, G . , and Kaplan, J . , Hoffmann-La Roche Inc . , Unpublished Data. d e S i l v a , J. 4. F . , Schwartz , M. A . , S t e f a n o v i c , V., Kaplan, J . , and D'Arconte, L . , AnaZ. Chem., 36, 2099 (1964). S i n e , H. E . , McKenna, M. J . , Law, M. R. , and Murray, M. H . , J . Chromatog. S c i . , 10 297 (1972) . Levin, M. , Hof fmann-La Roche I n c . , Unpublished Data. G u a s t e l l a , J . , Hoffmann-La Roche I n c . , Unpublished Data. Houghton, R. E . , Hoffmann-La Roche I n c . , Unpublished Data.

pp. 326-332.

331

IODIPAMIDE

Hyam Henry Lerner

HYAM HENRY LERNER

Table of Contents

1. Description

1.1 Name, Formula, Molecular Weight 1.2 Appearance, Color, Odor


2.1 Spectra

2.11 Infrared Spectra 2.12 Nuclear (Proton) Magnetic Resonance 2.13 Ultraviolet Spectra 2.14 Mass Spectrometry


2.21 Differential Thermal Analysis 2.22 Thermal Gravimetric Analysis 2.23 Melting Range 2.24 X-Ray Powder Diffraction

2.3 Solution Data

2.31 So lub i l i t y 2.32 2.33 I so ton ic i ty 2.34 pKa 2.35 pH 2.36 Index of Refraction 2.37 Phys icochemical Data

Apparent Molecular Weight i n Solution

3. Synthesis

4. S t a b i l i t y

5. Purif icat ion and Analysis f o r Impurities

5.1 Gel F i l t r a t i o n 5.2 Complexometric Methods of Separation 5.3 Countercurrent Distribution 5.4 Free Iodine and Free Halide 5.5 Free Amino Compounds

334

IODIPAMIDE

5. (Cont'd.)

5.6 Free Adipic Acid 5.7 Determination of Water and Conditions f o r

Drying


6.1 Elemental Analysis 6.2 I d e n t i f i c a t i o n Tests 6.3 Direct Spectrophotometric Analysis 6.4 Organically Bound Iodine 6.5 Polarograpliy 6.6 Chromatographic Analysis

6.61 Paper Chromatography 6.62 Thin-Layer Chromatography 6.63 Elec t rophore t ic Analysis

X-Ray and B-Particle Dispersion Methods 6.7 6.8 Flame Photometry

7. Drug Metabolism

8. References

335

HYAM HENRY LERNER

1. Description


Iodipamide is N,N'-adipyl bis (3-amin0-2~4~6 triiodobenzoic acid) . the heading benzoic acid, 3,3' (adipyldiimino) bis [2,4,6 triiodol. (3-carboxy-2,4,6 triiodoanilide; N,N'-di- (3-carboxy-2,4,6- triiodopheny1)-adipamide and 3,3'- (adipoy1diimino)-bis t2, 4,6-triiodobenzoic acid].

Chemical Abstract listings are under

Other derived chemical names are adipic acid di-

Among the generic and trivial names for this com- Common trade names pound are iodipamic acid and adipiodon.

are Biligrafin and Cholografin.

Iodipamide was officially recognized in "National Formulary XI." United States Pharmacopoaeia XVIII continues this name in a monograph for Meglumine Iodipamide Injection.

COOH YOOH

C20H1416N206 Mol. Wt. 1,139.7


Iodipamide is a white, odorless and tasteless crystalline powder1*11,12. The disodium salt has a sweet, metallic taste followed by a bitter aftertastd2.

336

I OD I PAM I DE


2.1 Spectra

2.11 Infrared Spectra

The spectra of iodipamide i n Figures la and l b were determined on a Perkin-Elmer Model 621 grat ing in- f rared spectrophotometer. Samples of iodipamide were dispersed i n a potassium bromide p e l l e t or i n mineral

by ToeplitzS0 on t h e spectrum obtained from the sample dispersed i n mineral o i l (Figure lb) :

The following spec t ra l assignments were made

- cm-1 Assignment

3200 N-H 2500, 1900 N-H and OH of amido acid

1690 C=O of carboxyl group 1610 C=O of amide 1530 secondary amide 1280 C-OH of carboxyl group

The spectra shown i n Figures l a and are dissimilar. An explanation was advanced by Toeplitzi’, who suggested t h a t iodipamide might be react ing with potassium bromide.

Herrmannl published an infrared spectrum ob-

Neudert and RUpke3 ta ined on a potassium bromide dispersion t h a t agrees quali- t a t i v e l y with t h e spectrum i n Figure la. published an infrared spectrum t h a t does not agree with the spectrum i n e i t h e r Figure la or Figure lb.

2.12 Nuclear (Proton) Magnetic Resonance Spectrum

w a s determined on a Varian XL-100 NMR spectrometer2 at ambient probe temperature (z. 31O). i n deuterated dimethylsulfoxide containing tetramethylsilane as an in te rna l reference (Me4Si = 0 ppm). ments of the peaks are recorded in Table I.

The MlR spectrum of iodipamide i n Figure 2

The sample was dissolved

Spectral assign-

337

w w m

Figwe la. Infrared Spectnxn of Iodipamide, Squibb Iot 03122, from KBr pellet. Instrun-ent: PE -1 621 Infrared spectm&oixm=@r

WAVELENGTH m)

Figure lb, Infrared Spectrum of Iodipamide, Sguibb Lot 03122, from minerdl o i l mull. Instnarent: PE -1 621 Infrared Sp-olxmeter

zdw I

1000

f 4

Figure 2. NMR Spectrum of Iodipamide, Squibb Lot 03122 in lXSO-%. Instrumnt: V a r i a n - m 0 0 NMR Speetrmeter

IODIPAMIDE

In deuterated water and deuterated sodium hydroxide, the peak at 69.86 w a s absent, indicating exchange of the amine proton. were not located, probably because of hydrogen bonding. High-field methylene resonance indicated the absence of other groups attached t o the methylene groups.

The carboxylic acid protons

Table I

NMR Spectral Assignments

Chemical Shif t Assignment (Ppm , 6) No. of Protons

-cI12-(312- 1.78 (s)

2.36 (m) aromatic 8.33 (3) -NH 9.86 (s) -COW not located

f i -C-CHz-

4

s = singlet ; m = nult iplet

2.13 Ultraviolet Spectra

The following ul t raviolet spectral data have been reported for iodipamide :

E Reference Solvent Amax, nm - 0.01N NaOH 238 0.1N-NaOH 236 0.lT NaOH 236 0,lR KOH 237 M e t b o l 238 Methanol 239 0.15M N a C l 238 0.lSR Phosphate 238 Bufrer (pH 5.8)

70,700 72,000 71,800 72,400 68,000 71,800 73,200 73,200

48 48 1 4 8 4 5 5

Neudert and RtJpke3 reported the E value of the disodium sal t i n methanol, at the 239 nm maximum, t o be 74,700. Ostrow and LevyS reported t h e i r data in terms of absorbance per micromole of iodine. Sodium iodide, which peaks a t 226 NB, has the same absorbance pe r rnicromole of

341

HYAM HENRY LERNER

iodine as does iodipamide, which suggests t h a t the u l t r a - v i o l e t absorption of iodipanide r e s u l t s from the presence of iodine chromophores .

2.14 Mass Spectrometry

No w l e c u l a r ion is observed for iodipamide because of its low v o l a t i l i t y and because of the thermal degragation of the compound. Funke consistent with the replacement of four protons by four trimethyl s i l y l groups. t a t ion pa t te rn are depicted below:

Per-trimethyl s i l y l a t i o n by yielded a compound with a molecular ion of m/e 1428,

The s t ruc ture and major fragmen-

COOH YOOH

NH-c -(C%),- C -HN

I 0 0 I

Other fragments tha t have been found are due t o loss of I o r ti1 and include:

m/e 1428+m/e 1301 + I m/e 7 4 2 4 m/e 614 + H I m/e 728 3 m/e 601 + I m/e 728+m/e 600 + H I


2.21 Different ia l Thermal Analysis

on a Du Pont 900 Themnoanalyzer at a temperature rise of 15O per min.

Valenti7 determined t h e DTA of iodipamide

A single ondothem at 308O and a s ingle

342

1001 PAM I DE

exotherm a t 314" were detected. duced in Figure 3.

The thermogram is repro-

2.22 Thermal Gravimetric Analysis

Valenti' determined the TGA of iodipamide on a Du Pont Thennogravimetric Analyzer. was heated a t a rate of 15" per minute under nitrogen sweep, no weight loss w a s observed below 250°,

When the compound

2.23 Melting Range

W i l l i ~ ~ ~ reported a melting range f o r iodipamide of 306.5 - 308", with decomposition, as determined on a Thomas-Hoover Ca i l l a r y Melting Point apparatus. Priewe and Rutkowskilj reported the meltin! range t o be 306O - 308*, with decomposition. Herrmann reported t h a t the compound decomposes above 280O.

Hoevel-Kestermann and Muhlemng determined the melting range on a Kofler Microblock (Reichert) and reported the melting range t o be 289 - 290°, with decomposition. This l a t t e r value appears t o be i n e r ror , when compared with t h e previously c i t e d DTA da ta (Section 2.21) and measurements made with the capi l la ry melting point apparatus.

2.24 X-Ray Powder Diffraction

OchslO obtained the X-ray powder d i f f rac- t ion spectrum of iodipamide on a Phi l l ips X-Ray Powder D i f - fractometer, a t a voltage of 45 kv and a current of 15 ma. The sample w a s i r rad ia ted by a copper source a t 1.54A. Diffraction da ta f o r Squibb Lot 03122 are recorded i n Table 11.

343

P

T. OC (CHROMEL: ALUMEL)* WE IlS,""CIIO* I.*"./ roll I C l l l COIIIIILIId.

figure 3. IICA Themxqram of IOaipami.de, Squibb Iat 03122. Instrumnt: Du Pant 900 ThermDanalyzer

IODIPAMIDE

Table I1 ~

X-Ray Powder Diffraction Pattern o f Iodipamide, Squibb Lo t 03122

d (Ao)*

8.80 7.40 5.70 5.5 5.10 4.46 4.40 4.29 4.23 4.16 4.09 3.93 3.80 3.67 3.64 3.47 3.44 3.37 3.30 3.25 3.17 3.14 3.10 2.99 2.96 2.92 2.90 2.84 2.66 2.61 2.56 2.51 2.43 2.32

Relative In tens i ty**

0.25 0.18 0.13 0.19 0.19 0.34 0.60 0.51 0.39 0.34 1.00 0.69 0.15 0.25 0.18 0.16 0.16 0.10 0.15 0.56 0.28 0.39 0.40 0.29 0.43 0.20 0.20 0.14 0.22 0.17 0.25 0.17 0.13 0.13

*d = ( in t e rp l ana r d i s tance) nX 2 s i n 0

where X = 1.539A ** Based on h ighes t i n t e n s i t y of 1.00

345

HYAM HENRY LERNER

2.3 Solution Data

2.31 Solubi l i ty

The following data were reported for the solubi l i ty of f ree acid of iodipamide a t room temperature:

Solvent

Acetone Ethanol, 95% Ether Chloroform Methanol Water 0.1N sodium hyxroxi de

n-hexane Benzene Propylene glycol 0.1N hydrochloric acrd

Ethylene glycol Tet rahydro f uran Tet rahydrofurfury 1

a1 coho 1

Solubili ty (mrg /lo0 m l )

Ref.12 Ref.8 a t 20° Refa7

200 - < 20 - - <20 100 - < 20 - - < 20 800 440 -

ins o lub 1 e 46 <20

- -

- - 5,240 - - < 20 insoluble - <20 - - <20

- 8,200 - Neudert and R8pke8 also reported the solu-

b i l i t y of iodipamide i n acetamide, urethan and phenol, a t the melting point of the solvents, t o be 3 g, 0.5 g, and 0.3 g per 100 g of solvent, respectively.

The so lubi l i ty at 20° of th disodium and dilithium salts of iodipamide were reported Q :

346

IODIPAMIDE

Solubi l i ty (g/100 ml)

Disodium Dilithium Solvent S a l t S a l t

Water 350 450 Methanol 14 65 Tetrahydro furan 4 3 Tetrahydro furfuryl alcohol 4 -

2.32 Apparent Molecular Weight i n Solution

According t o Neudert and R(lpke8, iodipamide forms micelles and has soap-like properties. Colloidal solut ions contain molecular aggregates with an apparent molecular weight 16 times that of the empirical formula of iodipamide.

2.33 I so tonic i ty

A 14.6% (w/v) solut ion of t h e disodium sal t of iodipamide (0.1233M) is isotonic with physiological salt solutionl2*24.

2.34 pKa

The pKa of the f ree acid of iodipamide w a s r e p ~ r t e d ~ r ~ ~ t o be 3.5. pKa1 and pKa2, s ince both dissociat ion constants can be expected t o be similar i n value.

This value may be a composite of

2.35 pH

The pH of a 1% suspension of iodipamide w a s Herrmannl proposed limits f o r a sat- reportedg t o be 3.95.

urated solut ion of 3.5 - 3.9.

Iodipamide neutral ized with sodium hydroxide w a s reported8 t o have a pH of 7.4.

2.36 Index of Refraction

The refractive index of iodipad.de at 21,5O, i n methanol12, is given i n Table 111. reported t h e refractive index of the disodium sal t , a t a

Neudert and R1Spke8

341

HYAM HENRY LERNER

concentration of 35 el l00 i n water, and measured with the D-line of sodium, t o be 1.4016.

Table I11

Refractive Index of Iodipamide at 21.5OC i n Methanol12

"D $/lo0 m l - 0.0 0.116 0.263 0.348 0.445

1.3278 1.3283 1.3288 1.3292 1.3294

2.37 Physicochemical Data

The freezing-point depression (-AT) , degree of dissociat ion (a), and osmotic pressure (Po) of t h e disodium salt of iodipamide i n aqueous solut ion were re- ported8*12, and are recorded i n Table IV.

Table IV

Physicochemical Data8,12 for the Disodium S a l t of Iodipamide :

Freezing-Point Depression (-AT), Degree of Dissociation (a), and Osmotic Pressure (Po)

Po - a - -AT - &/lo0 m l Molarity

2.0 0.0170 0.095 1.00 1.13 5.0 0.0422 0.226 0.94 2.69

10.0 0.0844 0.425 0.85 5.04 14.6 0.1233 0.557 0.72 6.64 21.6 0,1825 0.720 0.56 8.56

3. Synthesis

Iodipamide is prepared by the react ion of 2,4,6- tr i iodo-3 aminobenzoic acid with adipic acid dichloride13. The former is dissolved i n chlorobenzene and heated t o 110 - 130%. The adipic acid dicliloride is added dropwise,

348

IODIPAMIDE

resu l t ing i n the evolution of hydrochloric acid. evolution of HC1 has ceased, the precipi ta ted crude product is f i l t e r e d , with suction, while still hot. The crude pre- c i p i t a t e is washed with chlorobenzene, and t h e residual chlorobenzene is extracted by boi l ing with methanol. p rec ip i ta te is dissolved i n caus t ic methanol, f i l t e r e d through charcoal, and precipi ta ted with d i l u t e hydrochloric acid.

When the

The

An a l t e r n a t e solvent f o r the reaction of 2,4,6-tri- iodo-3 aminobenzoic acid with adipic acid dichlor ide is toluenel4.

COOH

* 110"-130° 0

2 'elz + II C1-C-(CH,),-C-Cl II - 2 HCl + C,H,Cl

i COOH COOH

NH - C - (CH2),- C -HN

I I


Iodipamide is chemically s t a b l e at room temperature. By extrapolation t o room temperature of the data f o r a c t i - vation energy and frequency constant of the reaction between 70 and l l O o , the decomposition at the N-acyl bond w a s calculated t o be 0.1% i n 50 yr.33 a t 90° from very pure iodiparnide i n solut ion at pH 9 is less than 3% i n 75 hr. under-iodinated compounds , enhances d e c o m p ~ s i t i o n ~ ~ .

Evolution of iodine

The presence of impurit ies such as

Formulated neutralized (pli 7.2) solutions containing iodipamide (192 mg/ml) sodium citrate (3.2 mg/ml) a and sodium edetate (0.28 mg/ml), show l i t t l e o r no loss of po- tency a f t e r storage for 5 yr. at room temperaturel5.

5. Purif icat ion and Analysis for Impurities

Purif icat ion procedures used f o r analysis a re described under Cliromatographic Analysis (Section 6.6).

349

HYAM HENRY LERNER

5.1 Gel F i l t r a t i o n

Ostrw and Levys attempted t o i s o l a t e and purify iodipamide on 1.0- x 12.5-cm columns prepared from dextran gel (Sephadex G-10 and G-25, Pharmacia) and polyacrylamide gel (Bio-Gel P-2, Bio-Rad). Samples o f 0.2 m l , containing 0.3 t o 8.6 pmoles of iodipamide, were eluted with 0.15M sodium phosphate buffer (pi4 5.8). The eluted fract ions rere moasured spectrophotometrically at 226, 238, and 255 nm. Results are shown i n Table V. Due t o adsorption of iodipamide on the gel, recoveries were not quant i ta t ive. Con- taminants c losely r e l a t ed t o iodipamide were shown, by paper chromatography of t h e iodipamide f r ac t ions , t o be present even after chromatography on Sephadex Gel G-10.

Table V

Gel F i l t r a t i o n of Iodipamide

P ro f i l e on Three Different Gels, with pH 5.8 Phosphate Buffer as Elution Solvent3

Sephadex Gel Sephadex Gel Bio Gel Component G-25 G- 10 P-2

Iodipamide 8-32 m l e luted a t void 8-50 m l (peak a t 20 ml) volume - ta i l - (peak a t

ing till 50 m l 16 ml)

Sodium Iodide 10-24 m l 28-50 m l 11-25 m l (peak a t 15 ml) (peak at 37 ml) (peak at

18 ml)

5.2 Complexometric Methods of Separation

Hentrich and Pfeiferd4 described methods f o r the precipi ta t ion of ten contrast agents as the metallic salts o r metallic complex salts. Iodipamic acid can be precipi- t a t e d quant i ta t ively by s i l v e r n i t r a t e , cadmium s u l f a t e with thiourea, cadmium s u l f a t e with pyridine, copper sulfate with thiourea, and copper s u l f a t e with pyridine. Chelatometric methods are a l s o described f o r the t i t r a t i o n of excess precipi tant , after separation of t h e p rec ip i t a t ed salt by f i l t r a t i o n . The formulas of t he p rec ip i t a t ed salts and complexes, t h e i r molecular weights, melting ranges, and

350

IODIPAMIDE

t he equivalent weight o f t h e iodipamide p rec ip i t a t ed by 1 m l of 0.1M so lu t ion of t he inorganic p rec ip i t an t are given i n Table m.

Table VI

S a l t Complexes of Iodipamic Acid34

Equ iv. Weight t o 1 m l of

Precip- Mol . Formula M . W . of Melting 0.1M i t a n t of Salt S a l t Range Prec ipr tan t

CdS04- Cd[(NH2)2CSl4*- 1,554.7 237O-238O 0.1140 g

Cd[(CsH N)4]*- 1,586.6 290 O 0.1140 g

CsSO4- <Cu[(NH2)2C- 1,569.4 167O 0.0570 g Thiourea S l 2 ) 'C20H12-

CuSO - [CU(C~H N)2]'- 1,359.5 202O-203O 0.1140 g Pyr i j i ne ~ 2 0 ~ 1 2 5 6 ~ 2 0 6

Thiourea C20H1216N206

i g % n e C2oH12f6N206

IgN2'6

5.3 Countercurrent Dis t r ibu t ion

S t r i c k l e r -- et a120 separated iodipamide and o t h e r con t r a s t agents from sera by countercurrent d i s t r ibu t ion . They used a solvent system composed o f - sec-butanol: d i l u t e aqueous ammonia (1:l). Both a 30-tube manual procedure and a 200-tube automatic procedure are described.

5.4 Free Iodine and Free Halide

Free iodine can be de tec ted by bo i l ing iodipamide with water f o r 2 min, f i l t e r i n g , and observing a blue co lor a f t e r treatment with s ta rch . another por t ion of f i l t r a t e with d i l u t e n i t r i c a c i d and treatment of it with s i l ve r n i t r a t e test so lu t ion , t h e presence o f free ha l ide ions can be detected by t h e

After a c i d i f i c a t i o n o f

35 1

HYAM HENRY LERNER

occurrence of opalescence o r turbidity1 a2 '. Hartmann and RUpke33 quantitated free iodide by

t i t r a t i n g potentiometrically with 0.001 - N s i l v e r nitrate, under a protective cover of nitrogen.

5.5 Free Amino Compounds

Hartmann and RUpke33 determined iodinated impuri- t i e s having a free amino group, by a k i n e t i c method based on t h e more rapid r e a c t i v i t y of these impurit ies with elemental bromine i n acetic acid solution than is shown by iodipamide. pounds quant i ta t ively s p l i t off iodine, which is then oxidized t o iodate by the bromine. excess bromine, the iodate is reduced t o iodine and ti- t ra ted with sodium th iosu l fa te , t o a s ta rch endpoint. Free-iodide compounds a l so react with bromine and would give posi t ive r e s u l t s by t h i s method.

Bratton-Marshall colorimetric reaction f o r the detection of f ree amino groups. Marshall reaction t o quant i ta te f ree amino compound impuri- t i e s .

Under these conditions, t h e free amino com-

After destruction of the

Hoevel-Kestermann and hluhlemann9 described a

Hartmann and R8pkd3 used the Eratton-

5.6 Free Adipic Acid

Hoevel-Kestermann and Wlemanng described a thin-layer chromatographic procedure f o r adipic acid, after its cleavage from iodipmic acid. scribed i n Section 6.2 and can be adapted t o determine free adipic acid.

This procedure is de-

5.7 Determination of Water and Conditions f o r h y i n g

Herrmannl dried iodipamide at 1 0 5 O f o r 4 hr. Hoevel-Kestermann and Muhlemanng dr ied iodipamide over phosphorus pentoxide f o r 24 hr. In the "National Formulary X I , ' @ water is determined by t h e Karl Fischer titrimetric method.

352

lODlPAMlDE



Element % Theory % Reported25

C 21.075 21-16 n 1.238 1.35 I 66.806 67.28 N 2.458 2.30 0 8.422 -

6.2 Ident i f ica t ion Tests

Infrared (Section 2.11) , paper chromatography (Section 6-61), and thin-layer chromatography (Section 6.62) have been used t o ident i fy iodipamide.

S h a m o t i e n k ~ ~ ~ ident i f ied iodipamide by boi l ing it

Iodipamide gives a cloudy yellow solut ion with with t r ich loroace t ic acid and a 5% aqueous solut ion of chloramine. a yellow prec ip i ta te .

The evolution of intense v i o l e t fumes of l iber - ated iodine can be observed by heating a sample of iodipamide over an open flame1*9a11.

Ident i f ica t ion of t h e bound m i n e group can be made by f i r s t ascer ta ining t h e absence of free amino groups (Section 5.6) and then cleaving the molecule by refluxing i n base and repeating the Bratton-Marshall reactiong.

Iodipamide may be hydrolyzed by refluxing with hydroiodic acid t o l i b e r a t e adipic acid. The adipic acid can be extracted with e ther and chromatographed on s i l ica gel G plates. acid (65:25:29) w a s used. Adipic acid (Rf 0.64) can be detected visual ly by spraying the developed p l a t e with bromo- c w s o l greeng.

The solvent system benzene:dioxane:acetic

6.3 Direct Spectrophotometric Analysis

The strong u l t r a v i o l e t band of iodipamide a t 238 2 2 nm i n bas ic solut ion and methanol (Section 2.13) can be used f o r d i r e c t spectrophotometric analyses4. The

353

HYAM HENRY LERNER

absorption band is s a i d t o r e s u l t from the iod ine chromo- #ores5. chromatographic separa t ions are e a s i l y accomplished by using this band f o r analyses.

Detection and quan t i t a t ion of e lua t e s from

6.4 Organically Bound Iodine

Under r e f l w conditions, t he iod ine i n iodipamide can be reduced and replaced by hydrogen, generated by t h e reac t ion of powdered z inc and sodium hydroxide. The iodide is determined by t i t r a t i o n with standardized s i l v e r n i t r a t e i n ac id so lu t ion i n t h e presence of tetrabromophenol- phthalein e thyl ester ind ica to r solution1

l i n e p e n ~ a n g a n a t e ~ ~ ,28. ganate with sodium n i t r i t e and ac id , they t i t r a t e d the l ibora ted iod ine with 0.1N - sodium th iosu l f a t e .

Yakatan and TuckenaanZg reviewed fou r methods f o r decomposing con t r a s t agents t o l i b e r a t e organica l ly bound iodine: A. Parr bomb (fusion with sodium peroxide); B. alka l ine permanganate reduction; C. zinc-sodium hydroxide reduction; and D. oxygen f l a sk (Schbniger) combustion. Meth- od D is recommended as a general technique f o r a l l iod i - nated organic compounds because of i t s r ep roduc ib i l i t y , s impl ic i ty , and rap id i ty . For compounds t h a t have a l l the iodine atoms ortho o r para t o t h e e lec t ronegat ive carbox- y l i c acid on t h e aromatic ring, e g iodipamide, Method C (zinc-sodium hydroxide reduction$ is recommended.

Krasnova’O recornended a modification of th

Hoevel-Kestermann and hhlemann9 reviewed t h r e e methods f o r l i b e r a t i n g organica l ly bound iodine i n con t r a s t agents: A. Parr banb (fusion with sodium peroxide); B. c a t a l y t i c reduction with sodium borohydride; and C. zinc- sodium hydroxide reduct ion. They recommend t h e sodium boro- iiydride reduction because of i t s s impl i c i ty and s h o r t assay time. The sodium borohydride reduction w a s o r i g i n a l l y proposed by EgliS1.

Ates and Ama126 decomposed iodipamide with alka- After decoloration of the pennan-

oxygen-flask combustion method of Yakatan and hickermann 39 .

354

I OD1 PAM I DE

6.5 Polarography

Vaskel i s et al.32 determined contrast agents

Iodipamide yieids a s ingle waveD with a half-wave polarographically irOm potassium chloride containing gelatin. potent ia l of - 1 . 2 ~ vs SCE. prepared c a l i b r a t i o r c u r v e s .

Results were quant i ta ted from

6.6 Chromatographic Analysis

6.61 Paper Chromatography

Many descending paper chromatographic methods have been found su i tab le f o r the i s o l a t i o n and detec t ion of iodipamide. These are summarized i n Table VII. Some of t h e references c i t e d give sample preparation techniques and methods of e lu t ing the drug from the developed chromatogram t o permit quant i ta t ion by u l t r a v i o l e t spectrophotometry (Section 2.13) o r o ther means. Pileggi -- e t a1.22 described a method f o r separating 19 organic iodide compounds from blood serum.

Table VII

Paper Chromatographic Systems f o r Iodipamide

Solvent S ys t em Paper Detection Reference Method of

I Whatman No. 1 A 35 I1 Whatmen No. 4 A 36 I11 Whatman No. 1 B 5 I V Whatman No. 3 B # C 26 39 V Whatman No. 3 B #C 26 39 V I Whatman No. 3 D 22

Solvent Systems

I - n-butanol:lc ammonium hydroxide:ethanol (5:2:1)

I1 - H20:n-butano1:ethanol (5:4:1) ;upper phase used for development lower phase t o equi l ibra te chamber.

355

HYAM HENRY LERNER

Table VII (Cont’d)

I11 - n-butanol:O.SN- ammonium hydroxide:ethanol:H20 (20:20:2:1); upper phase used f o r development, lower phase + 20 m l o f upper phase used t o equ i l ib ra t e chamber.

I V - Ethanol:25% ammonia ( r a t i o o f solvents not given)

V - Methanol:2N - acetic acid ( r a t i o of solvents not given)

V I - sec-butano1:enmronia 4% (3:l) - Methods of Detection

A. Long-wave u l t r a v i o l e t l i gh t .

9. Spray with 10% ceric s u l f a t e and 5% sodium arse- n i t e , both prepared i n 1 N - s u l f u r i c acid37.

C. Short-wave u l t r a v i o l e t l i gh t .

D. Spray with mixture of c e r i c ammonium s u l f a t e and arsenious acid, followed b spraying with 0.5% solut ion of methylene blue i 2 . 6.62 Thin-Layer Chromatography

Thin-layer chromatographic methods found su i t ab le f o r t he separation and detection of iodipamide are summarized i n Table VIII. Some of t h e references c i t e d present sample preparation techniques and methods for e lu t - ing the drug from the developed p l a t e t o p e r m i t q u a n t i t a - t i on by u l t r a v i o l e t spectrophotometry (Section 2.13) o r other means.

Hol lingsworth e t a l .41 separated iodoamino acids and r e l a t ed compounds onTemulose p l a t e s , with a solvent system composed of tert.-butanol:2N ammnia:chloroform (376:70:60). They d id not report usiiig t h i s technique on iodipamide. However, it seems reasonable t o expect t h a t t h i s system w i l l separate iodinated con t r a s t agents. S t ah l and Pfeifle38 reported on s i x systems used t o separate 17 iodinated compounds and Hocvel-Kestermann and Muhlemann’ separated 8 contrast agents with one system.

-

356

IODIPAMIDE

Table VIII

Thin-Layer Systems for Separation of Iodipamide

Solvent Detect ion System Plate Sys t em Rf Reference

I1 BDC a, C not given 26,39 I11 BBC a,c not given 26 , 39 IV A a,b 0.27 38 V A a Bb 0.33 38 VI A a D b 0.33 38 VI I D a 0.20 40

- I A a,b 0.09 9

Solvent Systems

I - Ethyl acet8te:isopropyl alcoho1:ammonia 25% (11:7:4)

I1 - Methanol:amrPonia, 25% (2:l)

I11 - Methanol:2N - acetic acid (1O:l)

IV - Acetone:isopropyl alcohol:ammonia, 25% (2:2:1)

V - Isopropyl alcohol:ammonia, 25% (4:l)

VI - Ethy1acetate:methanol :diethylamine (5 :4: 2) VII - n-butano1:ethanol:lg ammonia (5:1:2)

Plate - A. 30 g of silica gel HF254, 70 ml of H20 and 0.5 g

of starch.

B. Silica Gel G (Merck).

C. Silica Gel H254-366 (blerck).

D. Eastman tThromogramtg #6061 silica gel, plastic plates.

3 5 1

HYAM HENRY LERNER

Table VIII (Cont'd)

Detection System

A.

B.

C.

Short-wave u l t r a v i o l e t l i g h t (254 m).

Spray with 50% solution of acetic acid, followed by i r rad ia t ion a t 254 MI f o r 10 lain t o give blue- v io le t spots. peated t o increase i n t e n s i t y of the spots.

Spraying and i r r a d i a t i o n may be re-

Spray with 1:l solution of 10% ceric s u l f a t e and 5% sodium arsenite, both i n 1 N - s u l f u r i c acid3'.

6.63 Electrophoretic Analysis

Ardoino and P a ~ o n e ~ ~ reported on t h e elec- t rophoret ic analysis of contrast agents- i n biological secre- t ions from the l iver and kidney. Iodipamic acid migrates at the speed of albumin and with albumin.

6.7 X-Ray and B-Particle Dispersion Methods

Holynska and J a n k i e w i c ~ ~ ~ used X-ray fluorescence and absorption techniques t o determine iodine i n various contrast agents. was obtained by i r rad ia t ion of the sample, from a 2 4 1 h source of 5 mCi act ivi ty . The fluorescence of the K series of iodine (28.5 keV) was measured with a s c i n t i l l a t i o n counter having a 6-mm thick NaI/T1 crystal . separated by means of a single-channel, pulse-amplitude analyzer covering the t o t a l width of the K-peak iodine. t4ea- surement time was 1 min. A cal ibrat ion curve of the whole range of iodine contents investigated w a s made from standard samples of iod ic acid (HI03).

241Am and the energy w a s 60 keV. t i l l a t i o n counter with a 6-mm thick NaI/T1 crystal . t ion measurements were made by means of a single-channel, pulse-amplitude analyzer i n the energy channel of 5 keV covering 60 keV. Calibration curves were again prepared from su i tab le concentrations of iod ic acid. The authors claim the fluorescence technique is the method of choice.

I n t h e X-ray fluorescence work, exci ta t ion

Energy of exci ta t ion w a s 60 keV,

The c h a r a c t e r i s t i c radiat ion of iodine was

In the absorption method the X-ray source w a s The detector w a s a scin-

Absorp-

358

l OD1 PAMI DE

Analyses take less than 5 min, and the r e l a t i v e e r r o r is claimed t o be 1-3%, depending on iodine content.

persion of be ta par t ic les t o assay contrast media. %l, with about 3 V C i a c t i v i t y deposited on a r ing was used as the source of radiation. A ca l ibra t ion curve showing t h e number of sca t te red electrons VS. concentration w a s established. with determinations made by conventional metliods.

Mikolajek e t a1,44 used t h e method of retrograde -T

Results by t h i s method are i n good agreement

6.8 Flame Photometry

S h m ~ o t i e n k o ~ ~ determined sodium iodipamide i n pharmaceutical preparations by measuring t h e sodium content by flame photometry. Pr ior t o analysis, t h e iodipamic acid w a s precipi ta ted by acidifying the solut ion with 2N hydrochlor ic acid, and w a s then separated by fi l tration: De- terminations f o r the f i l t ra te were evaluated from a c a l i - bration curve of sodium chloride i n the range of 4.5 - 8.5 mg 0 .

This procedure lacks spec i f ic i ty , s ince it is dependent on t h e content of an atom not associated with t h e a c t i v i t y of iodipamide. Many formulations are a l s o pH adjusted with sodium hydroxide and contain o ther sodium com-

sodium c i t r a t e , sodium edetate , as excipi- ents. In "%' t ose cases, t h i s method would give erroneously high values,

7. Drug Metabolism

Iodipamide has been demonstrated t o be excreted large- ly i n the unchanged form12r16,17. Langecker e t a1.12 recovered 70% of t h e unchanged compound in the K l r o f rab- b i t s within 6 h r after dosing. (3.5)24 and high molecular weight, iod i amide is not reab- sorbed after its excretion i n the b i l e J*19. S t r i c k l e r et al. 2o reported evidence f o r the metabolic conversion of iodipamide t o an unident i f ied product.

Because of its low pKa

-- Hydro1 sis of t h e

amide linkago was postulated as a p o s s i b i l i t y 2 1 . Deiodination of iodipamide w a s s tudied i n man21 and

Pileggi was found t o be less than 1% of the given dose.

359

HYAM HENRY LERNER

et a1 .22 described a papor chromatographic screening test F r - & . e m i n a t i o n of iodipamide and iod ide i n sera (Section 6.61). A method fo r t h e removal o f iodipamide from blood sera by Craig countercurrent d i s t r i b u t i o n w a s described by S t r i c k l e r e t a1.20 (Section 5.3). --

M ~ C h e s n o y ~ ~ reviewed t h e literature through February 1968 i n a chapter e n t i t l e d , l"llie Biotransforrnation of Iodi- nated Itadiocontrast Agents.

(1) C. tlermann, Drug Stand., 2, 169 (1957).

(2) M. Puar, Squibb I n s t i t u t e , personal communica- t ion.

(3) W. Neudert and H. Rbpke , s. E., 89, 845 (19%).

(4) T. Pomazonska and W. Zyzynski, Diss. Pharm. , 17, 319 (1965).

(5) J. D. Ostrow and R. P. Levy, Gastroenterology, 54, 1085 (1968). -

( G ) P. Funke, Squibb I n s t i t u t e , personal comunica- t ion .

(7) V. Valenti, Squibh I n s t i t u t e , personal comunication.

(8) W. Neudert and H. Hbpke, Chem. Ber 87 659 - -*I -, (1954).

(9) kl. tloevel-Kestermann and H. Muhlemann, Pharm.

(10) Q. Ochs, Squibb I n s t i t u t e , personal communica-

Acta Helv 47 394 (1972). --*' -*

tion.

(11) National Formulary X I , p. 176 (1960).

360

IOOIPAMIDE

(12) H. Langecker, A. Harwart and K, JUnkmann, Arch. Pathol. Pharmakol., 220, 195 (1953). - E x p o -

(13) H. Prime and R. Rutkowski, U. S. Patent 2,776,241 (1957).

(14) H. Casselbaum and R. Drux, German (East) Patent 33738 (1964); Chem. Abstr 63 11441 b (1965).

(15) P. Kallos , Squibb Corp. , personal communication.

(16) E. Clerc, Aerztl. Wochenschr., 10, 1156 (1955).

(17) B. H. Bi l l ing, Q. Maggione and M. A. Carter, &. N. Y. Acad, =., E, 319 (1963).

- -.’-’ -

- - - (18) H, W. Fischer, Radiology, 84, 483 (1965).

(19) K. H. Kimbel, W. Bbrner and E. Heise, Fortschr. Roentgenprax.,

(20) H. S t r i c k l e r , E. Saier , E. Kelvington, J. Kempic, E, Campbell and R. Graven, 2. Clin. ocrinol. Metab 24 15 (1964). -*’ -’

(21) H. Langecker, A. Hartwart, K. H. Kolb and M. Kramer, Arch. 9, Pathol. Pharmakol., 247, 493 (1964).

(22) V. J. Pil leggi , K. J. Henry, M. Segalove and G. C. k lami11 , C l i n . Chem., 2, 647 (1962).

E. W. McChesney i n t g I n t e n a t i o n a l Encylopedia of Pharmacology and- Therapeutics P. K. Knoefel, Ed., Pergamon Press, New York, 1971, Vol. I ,

(23)

Sect, 76, pp. 147-163.

(24) P. K. Knoefel, Wadiopaque Diagnostic Agents , I ’

Charles C. Thomas, Springfield, Ill., 1961, pp. 41-44.

(25) J, Hydro, Squibb I n s t i t u t e , personal communication.

361

HYAM HENRY LERNER

0. Ates and H. Amal, Istanbul Univ. Eczac i l ik . Fak. Mecm., 2, 82 (1966) ; Chem. Abstr., 66, 11889-1965i). - National Formulary X I , p. 173 (1960).

- - --

United S ta t e s Pharmacopoaeia XV, p. 356 (1955).

G. J. Yakatan and M. M. Tuckerman, J. Pham. Sci., 55, 532 (1966).

- - - - bi. A. Krasnova, Farmatsi a (Moscow), 18, 57 (1969); - Chem. - * ’ - Abstr *24-(196v. - R. E g l i , Z. Anal. Chem., 3, 39 (1969).

A. Vaskelis, Y. Gaule and S. Chausovskii, Farmatsiya (Moscow 17 54 (1968); Chem. Abstr., - 70, 60890~ - (m’ -’ E. Hartmann and H. Rbpke, 2. Anal. Chem., 232, 268 (1967).

- - -

--

- - -

K. Hentrich and S. P fe i f e r , Pharmazie, 21 , 296 (1966); Chem. Abstr., 65, 8672f (1966).

T. Soh, Squibb Corp., personal communication.

- - --

L. Chow, Squibb Corp., personal communication.

S. L i s s i t z k e y , Bull. SOC. Chim. Biol., 37, 89 (1955).

---- E. Stahl and J. P fe i f l e , Z. Anal. Chem., 200, 377 (1964).

- - - 0. Ates and H. Amal, Istanbul Univ. Eczacilik. Fak. Mecm., 4 , 36 (1968);m=str. , 70, co886-9 - 697.

T. Soh, Squibb Corp. , personal communication.

D. R. Hollingsworth, M. Di l lard and P. K. Bondy, J. Lab. Clin. Med 62 346 (1963). - - - -*’-’

-- -

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IODlPAMlDE

(42) R. H. Mandl and R. J. Block, Arch. Biochem. - Biophys, 1 81, 25 (1959).

(43) B. Holynska and J. Jankiewicz, Chem. Anal. (Warsaw), 14, 219 (1969); Chem. Abstr., 2, 2=(1969). - --

(44) E. Mikolajek, J. Kormicki and 2. Kawalczyk, e. Pharm. Pharmecol., 2, 523 (1966); Chem. Abstr., 6449b-m. -2 -

(45) G. D. Chem. -

(46) L. A. Biol. m 9

Shamotienko, Khim. Farm. Zh., L, 32 (1968); -0, Abstr -’ 69 S m y W8)T

Ardoino and M. Pavone, Boll. Soc. Ital.

( I Y O U J

(47) G. D. Shamotienko, (1970); Chem.

(48) S, Willis, Squibb I n s t i t u t e , personal communica- t ion.

(49) A. Hoffmann, Squibb I n s t i t u t e , personal communication.

(50) B. Toeplitz, Squibb I n s t i t u t e , personal c o m n i - cat ion.

3 63

METHADONE HYDROCHLORIDE

Rafik H. Bishara

RAFlK H. BISHARA

CONTENTS

1. D e s c r i p t i o n 1.1 Nomenclature 1.2 Formula 1.3 Molecular Weight 1 .4 S t r u c t u r e 1.5 Appearance, Color, Odor, and Taste 1 .6 P r o p r i e t a r y N a m e s

2 . P h y s i c a l P r o p e r t i e s 2 . 1 Melt ing Range 2 .2 S o l u b i l i t y 2 .3 O p t i c a l R o t a t i o n 2.4 pH Range 2 .5 D i s s o c i a t i o n Cons tan t (pKa) 2 .6 P a r t i t i o n C o e f f i c i e n t 2 .7 D i f f e r e n t i a l Thermal A n a l y s i s 2 . 8 Thermogravimetr ic A n a l y s i s 2 .9 O p t i c a l and C r y s t a l l o g r a p h i c P r o p e r t i e s 2 .10 X-Ray Powder D i f f r a c t i o n 2 . 1 1 U l t r a v i o l e t Spectrum 2.12 I n f r a r e d Spectrum 2.13 Nuclear Magnetic Resonance Spectrum 2.14 Mass Spectrum

3. S y n t h e s i s , S t r u c t u r e and R e s o l u t i o n 3.1 S y n t h e s i s and Conf i rmat ion of S t r u c t u r e 3 .2 R e s o l u t i o n

4 . R e a c t i v i t y and S t a b i l i t y

5 . Drug Metabol ic P roduc t s and Pharmacokine t ics 5.1 Absorp t ion 5.2 D i s t r i b u t i o n 5.3 Metabolism 5.4 E x c r e t i o n

6 . I d e n t i f i c a t i o n

7. Microchemical Reac t ions

366


8 . Methods of A n a l y s i s 8.1 Elemental A n a l y s i s 8 . 2 T i t r a t i o n

8 .2 .1 Non-Aqueous T i t r a t i o n 8 .2 .2 Direct T i t r a t i o n

8.3 C h l o r i d e De te rmina t ion 8.4 U l t r a v i o l e t A n a l y s i s 8 . 5 Fluoromet r i c A n a l y s i s 8 . 6 I n f r a r e d A n a l y s i s 8 . 7 Colorimetric A n a l y s i s 8 . 8 Polarography 8.9 Bioassay 8.10 S p i n Immunoassay 8.11 R a d i o t r a c e r Techniques 8.12 Column Chromatography 8.13 Paper Chromatography 8.14 Thin Layer Chromatography 8.15 Gas Chromatography 8.16 Combined G a s Chromatography-Mass

Spec t romet ry 8.17 High P r e s s u r e L iqu id Chromatography

9. Ext rac t ion from B i o l o g i c a l F l u i d s

10. De te rmina t ion i n T i s s u e s

11. Bib l iog raphy

12 . Acknowledgements

13. Refe rences

367

RAFlK H. EISHARA

1. D e s c r i p t i o n

1.1 Nomenclature

heptanone hydroch lo r ide .

2- bu t anone hydroch lo r ide . heptanone hydroch lo r ide .

one hydroch lo r ide .

6-Dimethylamino-4,4-diphenyl-3-

1, l -Diphenyl - l - (2-dimethylaminopropy1)-

4,4-Diphenyl-6-dimethylamino-3-

6-Dimet hylamino-4,4-diphenylheptan-3-

1.2 Formula -- CBlH2,NO*HCl

1.3 Molecular Weight 43.92

1.4 S t r u c t u r e

0

1.5 ApEearance, Color, Odor, and T a s t e -- --- WhiFe, e s s e n t i a l l y o d o r l e s s powder.

B i t t e r t a s t e fo l lowed by s t i n g i n g s e n s a t i o n .

1 . 6 P r o p r i e t a r y N a m e s - - dl-Form: Adanon hydroch lo r ide ; Algidon;

Algolys in ; Amidon hydroch lo r ide ; AN-148; Bu ta lg in ; Depr idol ; Diadone; Diaminon hydrochlor ide ; Dolophine hydroch lo r ide ; Eptadone; Fenadone; Heptadon hydrochlor ide ; Hoechst 10,820; Ke ta lg in hydrochlor ide ; Mecodin; Mephenon;

368


Miadone; Moheptan; Phenadone hydroch lo r ide ; Physeptone hydroch lo r ide ; Polamidon hydrochlor ide ; Symoron; Z e f a l g i n .

1-Form: Levadone; Levothyl .

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

2 . 1 Mel t in Range 8Idk--e- 235.0'C1 mp 232.5 - 233.0°C2 mp 233.0 - 236.0°C3 mp 236.0 - 236.5"C4

mp 245.0 - 246.OoC4 1-Form: mp 241.O'Cl

2 .2 S o l u b i l i t ~ ~ ~ ~ ~ ~ ~ ~ =-metha= hvdrochlor i d e is verv

soluble i n water (12 g/100 mi), s o l u b l e i n a l c o h o l ( 8 g/100 ml), i n i sop ropano l (2 .4 g/ 100 m l ) , and i n ch loroform; p r a c t i c a l l y i n s o l u b l e i n e t h e r and i n g l y c e r i n e .

has s imi la r s o l u b i l i t y t o t h e racemic form i n a l c o h o l , i n ch lo ro fo rm and i n e t h e r .

The 1-form o f methadone hydroch lo r ide

2.3 O p t i c a l R o t a t i o n ----- No o p t i c a l r o t a t i o n is observed w i t h

t h e racemic methadone hydroch lo r ide . For t h e 1-form t h e fo l lowing o p t i c a l rotat ions are r e p o r t e d :

-145' (C = 2.5) 1

and

[a], 2 0 -169" (C = 2 . 1 i n a l c o h o l ) 1 9 4 .

2.4 pH Range T h e m o f a 1% s o l u t i o n is between 4 . 5

and 6.5.1,3,5

2.5 D i s s o c i a t i o n Cons tan t (pKa) L e v i e t al.8 r e p o r t e d t h e pKa of -- -- ----

methadone h y d r G h E r i d e , i n water a t 2OoC., t o

369

RAFlK H. BISHARA

be 8.25. Other data on t h e d i s s o c i a t i o n c o n s t a n t of methadone are r e p o r t e d by Marshall' and Beckett. *

2.6 P a r t i t i o n C o e f f i c i e n t F m o n coef f icGZs of d l -met hadone

i n heptane/p]C! 7.4 b u f f e r and chloroform/pH 7.4 b u f f e r a t 25 C . a r e 0.84 and 1fb56 r e s p e c t i v e l y . 9 Misra and Mule r e p o r t e d 57.3 and 28.3 t o be t h e p a r t i t i o n c o e f f i c i e n t s of t h e 1- and d-isomers, r e s p e c t i v e l y , i n o c t a n o l / pH 7.4 b u f f e r . No e x p e r i m e n t a l d e t a i l s are g i v e n t o e x p l a i n t h i s d i f f e r e n c e between t h e two o p t i c a l isomers.

2.7 D i f f e r e n t i a l Thermal T Z T f f e r e n t i a l h e r m a l

of methadone h y d r o c h l o r i d e was performed- u s i n g a DuPont 900 D i f f e r e t t i a l Thermal Ana lyze r a t a h e a t i n g ra te of 20 C. p e r min. and a n i t r o g e n a tmosphere . endotherm a t 235 C. , w h i c h appears t o be a m e l t , f o l lowed immediately by decompos i t ion .

Theothermogram ( F i g u r e 1) shows a n

2.8 Thermogravimet r ic Ana lys i s " A-GagravTmetric-anaysis, TGA , of

methadone h y d r o c h l o r i d e w a s performed u s i n g a DuPont 950 Thermogravimet r ic Ana lyze r a t a h e a t i n g rate of 5 C. per min. and a n i t r o g e n atmosphere. The thermogram ( F i g u r e 2) shows a weight loss beg inn ing a t 156.C. and c o n t i n u i n g th rough decompos i t ion .

2 . 9 0 t i c a l and C r y s t a l l o g r a p h i c P r o p e r t i e s T h E E of methadone hydrocklEFTaE

p repa red by Huback and 3ones12 f o r o p t i c a l examinat i o n and i d e n t i f i c a t i o n were o b t a i n e d by c o o l i n g a w a r m s a t u r a t e d aqueous s o l u t i o n of t he compound. The s i n g l e c rys ta l s were mounted onl a s t a g e goniometer t o measure t h e p r i n c i p a l r e f r a c t i v e i n d i c e s . A ro ta tory s t a g e w a s used t o measure a l l a n g l e s . Diamond-shaped c r y s t a l s r e s t i n g on a n end face are r e p o r t e d . These c r y s t a l s show symmet r i ca l e x t i n c t i o n , g i v e a n

370

I I I I I I I 20 40 5 9 79 98 117 137 157 177 197 217

T, "C (CHROMEL: ALUMEL) 217 236 256 276

Figure 1. lYl?A-thenrogram of methadone hydro&loride taken on a W o n t 900 Different ia l Thermal Analyzer

100 4 90

* 80- I--

!2 5 70-

I

60

50

-

-

- I 1 I I I I I I I I

Figure 2. TGA-themogram of mthadone hydrochloride taken on a DuPont 950 Thenrcgravk&xic Analyzer


i n t e r f e r e n c e f i g u r e t h a t shows t h e o b t u s e b i s e c t r i x a t one Zdge of t h e f i e l d , and t h e i r a c u t e a n g l e of 62 is r e l i a b l e f o r c h a r a c t e r i s - t i c d i a g n o s i s . Table 1 summarizes t h e o p t i c a l and c r y s t a l l o g r a p h i c d a t a o f racemic methadone hydroch lo r ide . methadone hydroch lo r ide , t a b u l a t e d by Barnes and Forsy th ,13 i n d i c a t e t h a t t h e s p a c e group o f t h i s compound is Cc or C2/c. a = 16.26, b = 9.76, and c = 25.74 9. The monocl in ic a n g l e was measured as 74 ( i .e. , I3 = 106'). The s p a c e group e x t i n c t i o n o f C2/c is f avored by t h e p resence of e i g h t molecules p e r c e l l (z = 8 m o l e c u l e s / c e l l ) . A d e n s i t y o f 1.178 g./ml. (average o f 8 measurements) w a s observed for c r y s t a l s from d i f f e r e n t p repa ra - t i o n s (Pca lcd . = 1.171 g./ml.).

The s i n g l e c r y s t a l d a t a f o r d l -

The a u t h o r 9 l i s t e d

2.10 X-Ray Powder D i f f r a c t i o n The x- ray d i f? rx t Ion-powder d a t a of

dl-methadone hydroch lo r ide o b t a i n e d by Barnes and Sheppard14 u s i n g f i l t e r e d CoKa (A = 1.790 A ) r a d i a t i o n are i n very good agreement w i t h t h o s e o b t a i n e d by Hubach and Jones ,12 fo r a sample from a d i f f e r e n t commericgl sou rce , w i t h f i l t e r e d CuKa ( A = 1.542 A ) r a d i a t i o n . With copper r a d i a t i o n , t h e s p a c i n g s of t h e 3 s t r o n g e s t l i nes ,o f t h e p a t t e r n are 7 46 A (v .v .s . ) , 4 .55 A (v .v . s . ) , and 6.45 A (v .s . ) w i t h cobal t r a d i a t i o n t h g 3 s t r o n g e s t pac ings o f t h e p a t t e r n are 4 .57 A (1001, 7.50 1 (901, and 6.48 A (701, t h u s merely i n t e r c h a n g i n g t h e " f i r s t " and "second" l i n e s .

Barnes and Sheppard14 drew t h e a t t e n - t i o n t o t h e f a c t t h a t w h i l e t h e p a t t e r n o f t h e dl-methadone h y d r o c h l o r i d e is no t t h e same a s t h a t of t h e d- and t h e 1- isomers, t h e p a t t e r n s of t h e free base i n d-, 1-, and d l - forms are i d e n t i c a l . The x-ray powder d i f f r a c t i o n d a t a of dl-methadone hydroch lo r ide r e p o r t e d by these a u t h o r s 1 4 are shown below:

373

TABLE 1

OPTICAL AND CRYSTALLOGRAPHIC PROPERTIES OF RACEMIC METHADONE HYDROCHLORIDE*

C r y s t a l sys tem

O p t i c o r i e n t a t i o n

5 R e f r a c t i v e i n d i c e s , P 5893A. ; 25.C.

Op t i c ax ia l a n g l e

Observed

Calcd. from s i n V = s i n E 1.623

Observed

Monocl inic , C l a s s ?, on ly a p l a n e of symmetry; a c u t e angle B = 74

t3 v i b r a t i o n d i r e c t i o n is p a r a l l e l t o c r y s t a l l o g r a p h i c axis b . P l a n e of symmetry c o n t a i n s ax i a l p l a n e . a d i r e c t i o n is a c u t e b i s e c t r i x which is n e a r l y p e r p e n d i c u l a r t o c r y s t a l l o g r a p h i c a x i s c .

a = 1.5713 0.0005, B = 1.6232 f 0.0005, y = 1.6360 * 0.0005, a' = 1.5760 f 0.0005 from c r y s t a l s r e s t i n g on an end f a c e

2E = 90° f 1. by c a l i b r a t e d micrometer e y e p i e c e

2 V ' = 52.

2V = 52. by r o t a t i n g from one o p t i c a x i s t o t h e o t h e r on goniometer

( con t inued . . . )

n

a

'C

0

d

v)

v)

*rl

a, a

h

P

E

2 0 0 W

d

0

fi c, 0

P

E 0

cu Lo

II

* cu

a, 3

.d

c, cd M

tr a, L)

0

cd tr cd * 0 r(

cd 0

d

* a 0

a

a, 0

5 a 0

fi a

a,

I*

IE

r: 375

RAFlK H. BISHARA

d (1) 12.4

- 8.25 7.87 7.50 6.48 6.20 5.92 5.70 4.72 4.57 4.34 4.20 4.14 4; 00 3.87 3.71 3.49 3.33 3.20 3.14

1/11 - 25 10

5 00 70 1

20 5

30 100

40 3

20 20 20

5 BB 20 B

5 25

1 B

d (A) - 3.10 3.03 2.97 2.92 2.84 2.75 2.68 2.60 2.53 2.48 2.30 2.23 2.16 2.11 2.08 2.04 1.99 1.92 1.67

I/I 1 - 30 3

10 3 5

20 15 3 B

20 2 5 1 2 8 3

15 2 3 2

2.11 Ultraviolet Spectrum A scan of methadone-hydrochloride

i n e thano l a t a concen t r a t ion of 0.27 mg./ml., (7.8 x 10'' M) on a Cary 15 spectrophotometer, from 400 t o 210 nm. (Figure 3) shows maxima a t 254, 250, 265, and 203 nm. The corresponding molar a b s o r b t i v i t i e s ( e of t h e s e maxima are 410, 485, 460, and 470 r e s p e c t i v e l y .

Hubach and Jones12 r epor t ed that an a l c o h o l i c s o l u t i o n of methadone hydrochlor ide exh ib i t ed t w o c h a r a c t e r i s t i c e l e c t r o n i c abso rp t ion bands a t hmax = 294 nm. ( E = 460) and t h e aromatic band a t Amax = 259 nm. (6 = 480). In water s o l u t i o n , t h e long wave- l eng th maximum is s h i f t e d t o 292 nm. and t h e molar a b s o r p t i v i t y is increased t o E. = 520. The spectrum of t h e f r e e base, methadone, us ing e thano l or hexane as a s o l v e n t , is e s s e n t i a l l y t h e same i n t h e r eg ion of t h e 294 nm. band,

376


20

18

16

14 N

52 ; 12-

t g 10-

5

k 5

v)

4 m

a

I 0

-

-

-

-

8-

6-

A max 254 ( t = 410)

Amax 2 5 9 ( c = 485)

A max 265 ( e = 460)

X m a x 293 ( e = 470)

0 200 250 300 350 400

WAVELENGTH. nm

F i g u e 3. Ultraviolet spectrum of mthadone hydrochloride i n ethanol taken on a Cam 15 Spectroeotcmter

377

RAFlK H. BISHARA

However, t h e molar a b s o r p t i v i t i e s are E = 760 i n a l c o h o l and E = 830 i n hexane.

data o f Mule15 on methadone i n 0.1 N h y d r o c h l o r i c acid showed Xmax a t 292 nm, ( E = 554) , Xmin a t 275 nm. ( E = 3 7 2 ) . Data o b t a i n e d i n e t h y l e n e d i c h l o r i d e c o n t a i n i n g 25% i s o b u t a n o l (v/v) are Xmax a t 295 nm. ( 6 = 433) and Xmin a t 280 nm. ( E = 3 9 0 ) .

The 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 a

Ul t rav io le t a b s o r p t i o n s p e c t r a a t reduced t e m p e r a t u r e s of methadone n i t r i l e and isomethadone n i t r i l e are p r e s e n t e d by S inshe imer e t a l . l 6 - --

2.12 I n f r a r e d Spectrum Ascanof-methaa&e hvdroc h l o r i d e i n a

po ta s s ium bromide p e l l e t on a Beckman IR-12 spec t ropho tomete r is shown i n F i g u r e 4 . Underbrink" a s s i g n e d t h e f o l l o w i n g bands t o t h e methadone h y d r o c h l o r i d e I R s p e c t r u m :

a .

b.

C .

d .

e.

710-770 c m . ' l character is t i c f o r aromatic carbon- hydrogen o u t of p l a n e bending .

due t o s k e l e t a l f r e q u e n c i e s and aromatic carbon- hydrogen i n p l a n e bending .

methylene and me thy l bending .

900-1200 cm." f i n g e r p r i n t r e g i o n ;

13 00- 1500 c m . - c h a r a c t e r i s t i c f o r

1450, 1490, c h a r a c t e r i s t i c f o r 1580, and aromatic r i n g 1600 c m . - 1 f r e q u e n c i e s . 1708 cm." c h a r a c t e r is t i c f o r

carbony 1 s t r e t c h i n g .

378

100 n

o l , , l ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ I I I I I , , ~ 4000 3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1900 1800 1700 1600 1500 1400 1300 1200 1100 10.00 900 800 700 600 500 4nl:

WAVENUMBEA CM-’

Figure 4. Infrared spectrum of methadone hydrochloride taken in a KE3r pellet on a BeduMn IR-12 Spectrqhotorneter

RAFIK H. BISHARA

c h a r a c t e r i s t i c f o r t e r t i a r y amine h y d r o c h l o r i d e

a l i p h a t i c carbon- hydrogen s t r e t c h i n g .

aromatic carbon- hydrogen s t r e t c h i n g .

-1 f . 2400 c m .

g . 2810-3000 cm." c h a r a c t e r i s t i c f o r

h . 3000-3080 c m . -' c h a r a c t e r i s t i c f o r

2 .13 Nuc lea r Magnet ic Resonance Spec t rum

The NMR s p e c t r u m o f methadone

---

h y d r o c h l o r i d e i n CDC1, c o n t a i n i n g t e t r a m e t h y l s i l a n e as i n t e r n a l s t a n d a r d on a Varian Associates HA-100 is shown i n F i g u r e 5 . The s p e c t r a l ass ignments1* are summarized i n T a b l e 2 . The chemica l s h i f t s are measured i n p.p.m. downf ie ld from t e t r a m e t h y l s i l a n e . The m u l t i p l i c i t y of t h e peaks , and t h e a p p r o x i m a t e c o u p l i n g c o n s t a n t s (J) are g i v e n i n Hz where a p p r o p r i a t e .

Group 3 and Group 4, r e s p e c t i v e l y , were i d e n t i f i e d by d e c o u p l i n g . The t w o methylene p r o t o n s of Group 6 are n o n e q u i v a l e n t g r o b a b l y because o f c o n f o r m a t i o n a l e f f e c t s . "9 c h e m i c a l s h i f t s are a s s i g n e d a t a p p r o x i m a t e l y 2 .30 and 3.15 p.p.m. by t h e p r o c e s s o f e l i m i n a - t i o n and i n t e g r a t i o n . The p r o t o n s o f t h e N-methyl g r o u p s are n o n e q u i v a l e n t due t o t h e r e l a t i v e p r o x i m i t y of e a c h me thy l g r o u p t o t h e d e s h i e l d i n g cone o f t h e pheny l g r o u p and a p p e a r as a p a i r of d o u b l e t s (J" 4 Hz) c e n t e r e d a t 2 . 7 5 p.p.m.

methadone and some r e l a t e d s u b s t a n c e s , and t h e a p p l i c a t i o n of t h i s t e c h n i q u e f o r s t e r i o c h e m i c a l and o p t i c a l i somer ism problems are found i n t h e l i t e r a t u r e . 1 9 - 2 3

The me thy lene and me th ine p r o t o n s o f

T h e i r

D i s c u s s i o n o f t h e NMR and PMR o f

3 80

381

figure 5. Nuclear magnetic resomce spectnm of mthadone hydrochlori& in CDCl3 taken on a varian Associates HA-100 Spectmmter

TABLE 2

NMR SPECTRAL ASSIGNMENTS OF METHADONE HYDROCHLORIDE

Group Multiplicity Chemical Shift (p.p.m.1 J(H=)

1. %-CH- Doublet I

N (CH3 1 2

0

Triplet II

2. CH3-CH2-C-

N (CH~ 1 I

Mult iplet

5. CH3-CH- Mu 1 t i p let

4. CHs-CJ-CH2-

1 N (CH3 2

0.70 6.5

0.83

Approx. 2.30

Approx. 3.04

2.75

7

(continued . . .

nl

n

P

d

0

C 0

0

W

4 E

a,

a,

C

E

00

c, a,

.I4 u1

P

(d 0

h

m

383

RAFlK H. BISHARA

2.14 Mass S ec t rum There -f-- a t i v e mass f r a g m e n t a t i o n p a t t e r n

of methadone was ob ta ined24 u s i n g a n LKB-9000 combined gas chromatograph-mass s p e c t r o m e t e r (GCMS). A f o u r - f o o t s i l i c o n i z e d glass column (2.5 mm. I . D . ) packed w i t h 1% W-98 m e t h y l v i n y l s i l i c o n gum r u b b e r on 80-100 mesh G a s Chrom Q w a s employed as t h e O K column. The column t e m p e r a t u r e was 1 7 0 C . and t h e carr ier gas (he l ium) flow w a s 40 ml./min. An e l e c t r o n ene rgy of 70 e V w a s used f o r i o n i z a t i o n . The computer ized mass f r a g m e n t a t i o n p a t t e r n shown i n F i g u r e 6 was o b t a i n e d by u s i n g a H e w l e t t Packard 2100 computer i n t e r f a c e d d i r e c t l y t o t h e EMS. The a s s ignmen t s and c o m p o s i t i o n s are as follow:

c- m/e

3 09

2 94

26 5

223

165

72

57

44

29

Assignment -

M+

(M-CH3 > + (M-N ( C H ~ ) ) +

+ (M-CHz-CH-CH,

I N ( C H 3 ) 2

C o m D o s it i o n

c2 l H 2 7NO

c2 O H 2 4NO

c1 gH2 lo

C l g H 1 5 0

C 1 3 H 9

C4H1 ON

c3 H5O

C 2 H 6 N

C2H5

384

r 12

I

I I I

294 I E

- 0 z

10 120 110 220 210 m l e

Figure 6. Relative mass fragmntation pattern of mthadone abtained by using an LKB-9000 ccxbined gas chrangogra&-mass spectrcmter ((336) . courtesy of Sullivan, H. R.

mta

RAFlK H. BISHARA

The above data are i n good agreement w i t h t h e h igh r e s o l u t i o n mass spec t rum25 of methadone hydroch lo r ide o b t a i n e d by u s i n g a CEC21-11OA mass spectrometer w i t h p h o t o p l a t e r e c o r d i n g . The ass ignments of t h e prominent i ons are g i v e n i n Table 3.

The mass spec t rum of methadone, t h e most abundant peaks and t h e metastable i o n s are r e p o r t e d by Fales e t a1.26 of methadone by i s s u s n e chemical i o n i z a t i o n mass spec t romet ry is p resen ted by Milne - e t -- a l . 2 7

3. S y n t h e s i s , S t r u c t u r e and R e s o l u t i o n -

The i d e n t i f i c a t i o n

3.1 S n t h e s i s and Conf i rmat ion of S t r u c t u r e b t of t h e United States Depart-

ment of Commerce28 about Amidone (methadone), t h e new German a n a l g e s i c drug no. 10820, i n c l u d e s t h e method g i v e n by t h e German chemists f o r its s y n t h e s i s . I n t h i s method, 1-chloro-2- propanol is added t o a n aqueous s o l u t i o n of d i e thy lamine and sodium hydroxide t o p r e p a r e 1-dimethylamino-2-propanol [l]. To [ 1 3 a s o l u - t i o n of t h i o n y l chlor ide i n benzene is added t o form 1-dimethylamino-2-chloropropane [2 1. Compound [21 is t h e n dropped on a cool m i x t u r e of sodamide and d i p h e n y l a c e t o n i t r i l e and t h e t empera tu re is allowed t o rise. The s o l u t i o n is r e f l u x e d for 15 minutes , cooled, poured on water, and t h e water is removed. The benzene s o l u t i o n is a c i d i f i e d w i t h hydroch lo r i c acid, t h e aqueous acidic l a y e r is made a l k a l i n e w i t h sodium hydroxide, and t h e product , 1-d imet hy laminopropy 1-2-d ipheny lacet o n i t r i l e [3 1, is d i s s o l v e d i n x y l e n e and added t o a s o l u t i o n of ethylmagnesium bromide, heated, and poured over a c i d i f i e d water t o s e p a r a t e t h e hydrobromide of t h e ke tone . The l a t t e r compound is d i s s o l v e d i n w a r m water and made a l k a l i n e t o y i e l d a n o i l y base, methadone, which is c r y s t a l l i z e d from methanol. The hydroch lo r ide of methadone is prepared by d i s s o l v i n g t h e base i n alcohol, alcoholic hydrogen ch lo r ide is added, and upon coo l ing , t h e material c r y s t a l l i z e s .

--

386

TABLE 3

w W 4

HIGH RESOLUTION MASS SPECTRUM ASSIGNMENTS

Calculated Mass

3 09.2061 294.1854 265.1586 236.1433 223.1121 179.0846 178.0779 165.0699 117.0711 115.0543 91.0552 85.0896 72.0814 71.0745 70.0653

Theore t ica l Mass

309.2093 294.1858 265.1592 236.1439 223.1123 179.0861 178.0782 16 5.0704 117.0704 115.0548 91.0548 85.0892 72.0813 71.0735 70.0657

OF METHADONE HYDROCHLORIDE

Emperical Formula

C

21 20 19 17 16 14 14 13

9 9 7 5 4 4 4

I

H

27 24 21 18 15 11 10 9 9 7 7

11 10 9 8

- 0 N

1 1 1 1 0 1 1 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 1 0 1 0

- -

RAFlK H. BISHARA

I t was no ted28 ,29 t h a t t h e above- ment ioned s y n t h e s i s would n o t be e x p e c t e d t o l e a d t o methadone [41, b u t t o t h e f o r m a t i o n of t h e isomeric s t r u c t u r e of i somethadone [5]:

S c h u l t z e t a l . 2 9 es tab l i shed and proved s t r u c t u r e [41 f o r T e n a d o n e . I n t h e i r s y n t h e s i s , when d i p h e n y l a c e t o n i t r i l e is r e a c t e d w i t h 1-dimethylamino-2-chloropropane [2 1 e i ther i n t h e p r e s e n c e of sodamide28 or p o t a s s i u m t - b u t o x i d e , t h e p r o d u c t is a m i x t u r e of a p p r o x i m a t e l y e u a l amounts of t h e isomeric n i t r i l e s [3] and [ 6 7 . The 2,2-d ipheny l-4-d i m e t hy laminopen tanen i t r i l e [6] r eac t s smooth ly w i t h e thylmagnesium bromide t o g i v e methadone [41. Trea tment o f 2,2- diphenyl-3-methyl-4-dimethylaminobutanenitrile [ 3 ] w i t h t h e G r i g n a r d ' s r e a g e n t does n o t g i v e t h e methadone isomer C5l b u t a d i b a s i c p r o d u c t C71 w h i c h a p p e a r s t o be t h e c o r r e s p o n d i n g k e t i m i n e . Al though t h e k e t i m i n e s t r u c t u r e is s u p p o r t e d by a n a l y t i c a l d a t a , t h e o r d i n a r y c o n d i t i o n of h y d r o l y s i s f a i l s t o g i v e t h e k e t o n e . 3 0 The s t r u c t u r e of t h e isomeric n i t r i l e s , and hence t h e s t r u c t u r e o f methadone, were e s t a b l i s h e d by decompos i t ion of t h e q u a t e r n a r y b a s e s d e r i v e d from t h e m e t h i o d i d e s of t h e n i t r i l e s by t r e a t m e n t w i t h s i l v e r oxide. A summary of t h i s s y n t h e s i s is i l l u s t r a t e d i n F i g u r e 7.

Brode and H i l l 3 1 r e p o r t e d t h e r ea r r angemen t of t h e isomeric 1 ,2- dimethylaminochloropropanes, d e r i v e d from t h e ch lor i n a t i o n of 1-dimethy lamino-2-propanol [ 8 ) and 2-dimethylamino-1-propanol [91, t h r o u g h t h e

388


C H /CH3

C6H5 / I 5‘CH-CN + CI-CH-CH2-N

KOCaHS-t

C H C H

C&’l 5‘C-CN V C - C N

+ C6H5’1 /CH3 CH-CH2-N CH2-CH-N

I ‘CH3 1 ‘CH3 CH3 0

C2HglYlgBr

@

Figure 7 . Synthesis of adone according to Scf iu l tz et aL TF --

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RAFlK H. BISHARA

e t h y l e n e immonium i o n . 3 2 t h e c o n v e r s i o n of [81 t o C91 i n l o w y i e l d s .

To a v o i d t h e t e c h n i c a l d i f f i c u l t i e s due t o t h e f o r m a t i o n o f t h e i s o m e r i c a m i n o n i t r i l e s , a new s y n t h e s i s w a s deve loped by E a s t o n e t a l . 33 i n which d i p h e n y l a c e t o n i t r i l e is condensed a t h p r o p y l e n e o x i d e i n t h e p r e s e n c e o f sodium amide t o y i e l d 3,3-diphenyl-5-methyltetrahydro-2- fu ranone imine [ l o ] . When [ l o ] is treated w i t h phosphorus t r i b r o m i d e , t h e p r o d u c t is 4-bromo- 2,2-diphenylpentanenitrile [111* On condens ing [111 w i t h d imethylamine , Compound [6 1 is formed. Methadone [ 4 1 is p r e p a r e d from [ 6 ] by t h e a c t i o n of ethylmagnesium bromide a s d i s c u s s e d p r e v i o u s l y . 28 The y i e l d s of t h e a m i n o n i t r i l e from t h e h a l o n i t r i l e is below 107;. The major p r o d u c t a lways formed is a n u n s a t u r a t e d n i t r i l e , presumably 2 ,2 -d ipheny l -3 -pen tene - n i t r i l e [ 1 2 ] or a m i x t u r e of [12 ] and 2,2- diphenyl-4-pentenenitrile 113 1.

The s t r u c t u r e o f methadone e s t a b l i s h e d by S c h u l t z e t a1.29 w a s con f i rmed by t h e f o l l o w i n g series of r e a c t i o n s . Compound [ 6 3 is degraded by e x h a u s t i v e m e t h y l a t i o n (methyl i o d i d e , s i l v e r oxide, and h e a t i n g ) t o an u n s a t u r a t e d n i t r i l e [121, [131 or a m i x t u r e which is hydrolyzed w i t h o u t p u r i f i c a t i o n t o y i e l d t h e l a c t o n e of 2 ,2-d iphenyl -4-hydroxy- p e n t a n o i c a c i d [14]. The h y d r o l y s i s o f [13] forms t h e same l a c t o n e [141. Long s t a n d i n g o f t h e h y d r o c h l o r i d e of Compound [ l o ] i n aqueous s o l u t i o n g i v e s t h e l a c t o n e [141. These facts are accoun ted f o r by t h e s t r a i g h t s t r u c t u r e of t h e a m i n o n i t r i l e [6]. F i g u r e 8 shows t h e s y n t h e s i s and c o n f i r m a t i o n o f s t r u c t u r e a c c o r d i n g t o E a s t o n e t a l . 3 3

The p r e p a r a t i o n of some isomers, ana logs ,35 -39 and re la ted subs t ances40-44 t o methadone is r e p o r t e d i n t h e l i t e r a t u r e . T o l b e r t e t a l . 4 5 s y n t h e s i z e d dl-methadone labe led w i t h 1 4 C - i n e i ther t h e 1 or 2 p o s i t i o n .

T h e i r da ta a l so show

--

--

I-


C6H5 /O\ ‘CH-CN + CH2-CH-CH3 /

C6H5

Figure 8. Synthesis and confirmation of mthadone s t ructure according t o Easton e t al, 33 --

39 1

RAFlK H. BISHARA

3.2 R e s o l u t i o n -_-- Methadone h a s one a s s y m e t r i c c a r b o n atom

and t h e r e f o r e c a n e x i s t a s dext ro or l e v 0 forms or as racemic m i x t u r e . The o p t i c a l r e s o l u t i o n of methadone is r e p o r t e d by Brode and H i l l , 4 e L a r s e n et e.,47 and Thorp e t -- a l . 4 8 t h r o u g h t h e u s e o f d - ta r ta r ic ac id . Howe and S l e t ~ i n g e r , ~ ~ and Howe and T i s h l e r 5 ' r e s o l v e d dl-methadone, or its h y d r o c h l o r i d e , by fo rming t h e e a s i l y p u r i f i e d , w a t e r - i n s o l u b l e d-a-bromocamphor-n-sulfonate of t h e d- isomer. P u r e d-met hadone is p r e c i p i t a t e d by slow a d d i t i o n o f water. The 1-form is o b t a i n e d , f rom t h e mother l i q u o r , by forming t h e d - t a r t r a t e s a l t . When t h e 1-methadone is d e s i r e d , t h e d-isomer is removed from a s o l u t i o n i n b u t y l a l c o h o l as t h e p - n i t r o b e n z o y l - l - glutamate. The u s e o f a-bromocamphor-n-sulfonic ac id and p-nitrobenzoyl-L-glutamic ac id a s t h e r e s o l v i n g a g e n t r e d u c e s t h e e x c e s s i v e c r y s t a l l i z a t i o n time and s u b s t a n t i a l l y i n c r e a s e s t h e y i e l d s . ZauggS1 p a t e n t e d a s p e c i a l a p p a r a t u s t o p r o v i d e a new p h y s i c a l method f o r s i m u l t a n e o u s r e s o l u t i o n of b o t h o p t i c a l isomers o f d l - methadone. Zaugg e x p l a i n s t h a t " t h i s i n v e n t i o n is based on t h e knowledge t h a t a seed c r y s t a l of t h e d e x t r o - r o t a t o r y isomer w i l l a t t r a c t t h e d- i somer i n s a t u r a t e d s o l u t i o n , and when t h e d e g r e e of s a t u r a t i o n of t h e s o l u t e i n t h e s o l u t i o n is i n c r e a s e d , t h e d - i s o m e r w i l l t e n d t o c r y s t a l l i z e o u t on t h e d- i somer seed c r y s t a l . A t t h e same t i m e , a p o r t i o n o f t h e 1-isomer w i l l t e n d t o c r y s t a l l i z e o u t on t h e 1- i somer seed c r y s t a l . T h i s p r o c e s s w i l l c o n t i n u e s o l o n g as t h e s o l u t i o n is s u p e r s a t u r a t e d w i t h t h e composi- t i o n or s o l u t e and s e e d e d c r y s t a l s w i l l grow t o s u b s t a n t i a l s ize . A t t h e c o n c l u s i o n o f t h e o p e r a t i o n , i t w i l l be found t h a t r e l a t i v e l y p u r e c r y s t a l s o f t h e d-isomer and 1-isomer w i l l have been grown on t h e seed c r y s t a l s . "

4 . R e a c t i v i t y and S t a b i l i t y me r e l a t i v e l y l o w r e a c t i v i t y of t h e c a r b o n y l g roup of methadone [11 is i n d i c a t e d by n o t g i v i n g

------

t h e semica rbazone unde r t h e u s u a l c o n d i t i o n s and-

392


r e s i s t i n g r e d u c t i o n w i t h aluminum i s o p r o p o x i d e or sodium amalgam.36 [2] is formed w i t h p l a t i n u m o x i d e . A c e t y l a t i o n o f [ 2 ] y i e l d s t h e 0 - a c e t y l d e r i v a t i v e [3-a]. R e a c t i o n of [ 2 ] w i t h c h l o r i n a t i n g a g e n t s ( t h i o n y l c h l o r i d e o r phosphorus p e n t a c h l o r i d e ) leads t o t h e f o r m a t i o n of a m i x t u r e o f 6-d i m e t hy l a m ino- 4,4- d ipheny l-2- h e p t e n e [4 1 and 3-chloro-6-dimethylamino-4,4-diphenylheptane [51. A l k a l i n e c l e a v a g e o f t h e e t h y l k e t o g r o u p r e s u l t s i n t h e f o r m a t i o n o f 3-d imethylamino-1 , l - d i p h e n y l b u t a n e [6 1. Compound [6] is a l so formed by r e f l u x i n g 4-dimethylamino-2,2- d i p h e n y l p e n t a n e n i t r i l e [ 7 ] w i t h po ta s s ium hydrox ide and t r i e t h y l e n e g l y c o l . Hydrogenat ion of t h e r e s u l t i n g o l e f i n C91 from t h e Hofmann d e g r a d a t i o n of [8], t h e me th iod ide of [6l (which is a l s o formed by a l k a l i t r e a t m e n t of t h e me th iod ide of [ 111, g i v e s 1 , l - d i p h e n y l b u t a n e [ lo ] . The l a t t e r compound is a l s o p r e p a r e d f rom e t h y l b u t y r a t e [ll] v i a 1 , l - d i p h e n y l - 1 - b u t a n o l [12] which is hydrogenated t o [ l o ] w i t h pa l l ad ium-c h a r c o a l o r pa l l ad ium-ba r ium s u l f a t e c a t a l y s t i n t h e p r e s e n c e of acetic a c i d c o n t a i n i n g traces o f p e r c h l o r i c ac id . A l k a l i t r e a t m e n t of a,a-diphenylvaleronitrile [131 g i v e s a l o w y i e l d o f [ l o ] a l o n g w i t h a,a- d i p h e n y l v a l e r i c a c i d [14] . F i g u r e 9 shows t h e s e r e a c t i o n s . I n a later r e p o r t by May and P e r ~ - i n e ~ ~ t h e s t r u c t u r e s of [41 and [53 were proven t o be 6-d i m e t hylamino-3,4-d iphenyl -3- hep tene [15 I, and 4-chloro-6-dimethylamino-3,4- d i p h e n y l h e p t a n e [16], r e s p e c t i v e l y . T h i s is due t o t h e Wagner's r ea r r angemen t of [21 t o g i v e 6-dimethylamino-3,4-diphenyl-4-heptanol [ l7] , [151 and [161 depending upon t h e reaction c o n d i t i o n s .

I r r a d i a t i o n w i t h gamma r a y s (cobal t -601 , u l t r a v i o l e t , or t h e r m a l n e u t r o n s h a r d l y a f f e c t s t h e m e l t i n g p o i n t of m e t hadone h y d r o c h l o r i d e . 5 3 , 5 4 However, t h e i r r a d i a t i o n produces a brown color, c h a n g e s of pH i n s o l u - t i o n , decreases s p e c i f i c r o t a t i o n , and m o d i f i e s t h e i n f r a r e d spec t rum. A d d i t i o n a l s p o t s a p p e a r

The c o r r e s p o n d i n g carbinol

393

394

w W P

I I I C3H7C0ZCZH5 t PhzC(OH)CH2CHzCH3 PhzCC3H7 -Ph2CC3H7

0 0 63 0

Figure 9. S o m reactions of nethadone. 36 Reproduced by permission.


on t h e t h i n l a y e r chromatogram of t h e i r radiated sample. I r r a d i a t i o n is less d e s t r u c - t i v e t o t h e s o l i d material t h a n t o t h e aqueous s o l u t i o n .

P h o t o l y s i s and r a d i o l y s i s of methadone h y d r o c h l o r i d e s o l u t i o n r e s u l t in t h e formation of 3 ,3 -d ipheny l -2 -e t hy l idene-5- m e t hy 1 t e t r a hyd ro f u r a n and 3 - d i m e t hy l a m i no - 1,l- d i p h e n y l b u t e n e , r e s p e c t i v e l y . " 9 56

free base a t 30 C . shows t h e f o r m a t i o n of methadone-N-oxide by t h i n l a y e r chromatography and combined gas chromatography-mass s p e c t r o m e t r y a n a l y s e s . 5 7 The re la t ive concen- t r a t i o n of t h e chemical o x i d a t i o n p roduc t , methadone-N-oxide, i n c r e a s e s w i t h t i m e of s t o r a g e .

S t o r a g e of $n o r g a n i c s o l u t i o n of methadone

5. Drug Metabo l i c P r o d u c t s and P h a r m a c o k i n e t i c s ----- -------.---

5.1 A b s o r p t i o n Absorp t ion of methadone is r e l a t i v e l y

prompt . Expe r imen t s w i t h "C-methadone show appreciable c o n c e n t r a t i o n s of 14C i n plasma58 and b i l e 5 9 w i t h i n 10 m i n u t e s after s u b c u t a n e o u s i n j e c t i o n of t h e labeled d r u g . Fo l lowing subcu taneous i n j e c t i o n of methadone i n rats, 47% of the dose rema ins a t t he i n j e c t i o n s i te a f te r 1 h o u r , 6 o 10-15$ a f t e r 2-3 hours , 3% a f t e r 5 hours , and v i r t u a l l y none is p r e s e n t a f t e r 24 hours .61 a d m i n i s t e r e d by stomach t u b e t o fasted rats disappears w i t h i n 2 h o u r s from t h e gastro- i n t e s t i n a l t rac t .'j2

Seven ty p e r c e n t of a methadone dose

5.2 D i s t r i b u t i o n Methadone ma in ly localizes i n t h e l i v e r ,

k idneys , l ungs , and s p l e e n . Blood, heart, b r a i n , and musc le show o n l y l o w level^.^'-^^ Methadone is a l so c o n c e n t r a t e d i n t h e a d r e n a l s and t h y r o i d . 5 8 , 6 1 9 6 5 Using s e n s i t i v e tracer t e c h n i q u e s , 6 5 methadone l e v e l s of 0.6 t o 0.9 p g . / g . of v a r i o u s segments of t h e c e n t r a l ne rvous s y s t e m are found 30 minu tes a f t e r

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subcu taneous a d m i n i s t r a t i o n of 3 mg./kg. T h i s correlates w e l l w i t h t h e i n t e n s i t y and d u r a t i o n of t h e a n a l g e s i c effect a s demons t r a t ed by t h e r e s c t i o n t i m e of t h e r a t t a i l t o the rma l s t i m u l u s . E l l i o t t et -- a l .61 reported h i g h concen- t r a t i o n of methadone i n t h e g a s t r o i n t e s t i n a l t r ac t a f t e r subcu taneous a d m i n i s t r a t i o n of t h e d rug . C o n s i d e r a b l e amounts of r a d i o a c t i v i t y are found i n t h e p l a c e n t a e and f e t u s e s of t h e p regnan t r a t af ter t h e a d m i n i s t r a t i o n of 1 4 C labeled methadone.61 The methadone c o n c e n t r a - t i o n i n t h e b r a i n of t h e f e t u s is 2-3 times t h e c o n c e n t r a t i o n found i n t h e m a t e r n a l b r a i n . "

t o t i s s u e p r o t e i n . 6 7 However, accumula t ion of t h e d r u g does n o t o c c u r t o any great e x t e n t . A large p a r t of t h e methadone p r e s e n t i n t h e whole a n i m a l is found i n t h e carcass ma in ly t h e s k e l e t o n , muscle , and bone. '9 61'

man6' shows t h a t t h e blood c o n c e n t r a t i o n of t h e d r u g is less t h a n b i l e and u r i n e c o n c e n t r a t i o n s . The k idney and l i v e r c o n c e n t r a t i o n s are approx ima te ly e q u i v a l e n t . B r a i n t i s s u e is t h e p o o r e s t s o u r c e of methadone and lung t h e r i c h e s t . Binding of methadong9to human plasma a lbumin is reported by Ol sen .

Methadone appears t o be f i r m l y bound

A d i s t r i b u t i o n s t u d y of methadone i n

5.3 Metabolism !i%6 i n d i c a t i o n t h a t t h e first two

ca rbon atoms of methadone are n o t removed by o x i d a t i o n w a s demons t r a t ed by E l l i o t t e t a l . ' l No 1 4 C 0 2 is e l i m i n a t e d by r a t s g i v e n mZhadone labeled w i t h l 4 C i n p o s i t i o n 2 . C o n t r a r y t o these e a r l y data, t h e r e c e n t work by S u l l i v a n 2 4 shows t h a t abou t 1s of t h e dose of 14C labeled methadone i n p o s i t i o n 2 is e l i m i n a t e d as 1 4 C 0 2 . The p r e s e n c e of 4-dimethylamino-2,2- d i p h e n y l v a l e r i c acid i n u r i n e of humans is a l s o a n i n d i c a t i o n of t h e s i d e c h a i n o x i d a t i o n , 5 7 The l i v e r a p p e a r s t o be t h e o r g a n c h i e f l y r e s p o n s i b l e for t h e metabolism of methadone. 9 O- 73

396


The major metabolites of methadone i n humans are shown i n F i g u r e 10.57,74 metabolite of methadone [11 is formed by N-demethylation t o y i e l d t h e u n s t a b l e N-desmethylmethadone [2 3 which is ~ y c l i z e d ~ ~ t o 1,5-dimethyl-3 3-d iphenyl -2-e thyl idene p y r r o l i d i n e [3>. forms 2 - e t hy 1- 5-met hy l-3,3-d ipheny 1- 1- p y r r o l i n e [el. Both [3] and [4 ] and t h e i r co r re spond ing r i n g hydroxyla ted ana logs , 2 -e thyl idene- l ,5 - d i m e t hyl-3- (p-hydroxyphenyl -3-phenylpyrro l i d i n e [: 5 3 and 2- e t hy 1- 5-me t hy 1- 3 - ( p- hy d r oxy p heny 1 - 3 - phenyl -1-vgrro l ine [S 1, are detected i n human u r i n e .76-

I n a minor pathway, t h e keto group of methadone is enzymat i ca l ly reducedSB t o form methadol [ 7 ] which is N-demethylated t o y i e l d normethadol [81 which is excreted i n t h e u r i n e . 8 0 I n a r e l a t i v e l y minor pathway t h e s i d e c h a i n of methadone is o x i d i z e d t o form 4-dimethylamino- 2 ,2 -d ipheny lva le r i c acid 191 which subsequen t ly N-demethylates, i n p a r t , t o a non- i so l a t ed in t e rmed ia t e , 4-met hy lamino-2,2- d ipheny l v a l e r i c acid [lo]. Ring c l o s u r e ( c y c l i z a t i o n ) of t h e i n t e r m e d i a t e [ lo ] y i e l d s 1,5-dimethyl-3,3- d ipheny l-2- p y r r o l idone [ 11 1.

In a d d i t i o n t o t h e p r e v i o u s l y mentioned pheno l i c metabolites [53 and c61, r i n g hydroxyla ted methadone [12] is also found i n t h e u r i n e of s u b j e c t s main ta ined o n methadone.57 Methadone N-oxide [13] is found i n u r i n e from s u b j e c t s r e c e i v i n g a s i n g l e dose of methadone, i n u r i n e from a d d i c t s be ing treated w i t h t he drug, 74 and in u r i n e of rats. 1 0 However, t he work of S u l l i v a n and Due57 i n humans i m p l i e s t h a t t h e r e is some q u e s t i o n as t o whether t h e methadone N-oxide is a t r u e m e t a b o l i t e o r a n a r t i f ac t caused by o x i d a t i o n ,

The primary

F u r t h e r N-demethylation of [3 1

5.4 E x c r e t i o n --I--

Way and Adler" i n d i c a t e d t h a t less t h a n 10% of methadone is e x c r e t e d unchanged i n t h e u r i n e and i n t h e feces. Ur inary e x c r e t i o n s tud ies show v a r i o u s c o n c e n t r a t i o n s of methadone

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RAFlK H. BISHARA

57,74 Figure 10. Major mtabolites of mthadone in h-.

398


i n u r i n e . Recovery of 4 s t o 35% of t he admin i s t e red dose is repor ted .82-85 Twenty-four hours af ter a d m i n i s t r a t i o n of methadone to rats, t h e unchanged drug found i n u r i n e and feces is 4-11s and 19-24s r e s p e c t i v e l y from t h e admin i s t e red dose.6a u t i l i z i n g coun te r - cu r ren t t echn iques , showed t h a t t h e p rev ious v a l u e s are h igh and recommended a f a c t o r of 0.8 and of 0.25 for t h e c o r r e c t i o n of t h e u r i n a r y and fecal e x c r e t i o n r e s p e c t i v e l y .

impor tan t avenue for t h e e l i m i n a t i o n of methadone and its b i o t r a n s f o r m a t i o n

However, Way e t g. ,82

B i l a r y e x c r e t i o n is r e p o r t e d t o be a n

p r o d u c t s , 5 9 ~ 6 3 - 6 5 ~ 8 2

The r e s u l t s of Baselt and Casarett' demonstrated that , i n man, r e n a l e l i m i n a t i o n may become t h e major e x c r e t o r y pathway after d a i l y doses g r e a t e r t h a n 55 mg. S i x t y pe rcen t of a 160 m g . dose of methadone p e r day is e x c r e t e d as unchanged drug i n u r i n e . These r e s u l t s are i n c o n f l i c t w i t h t h o s e of S u l l i v a n and Due5' who r e p o r t e d t h a t a r e l a t i v e l y small p o r t i o n of a n 80 mg. dose of methadone w a s found unchanged i n t h e u r i n e of h e r o i n maintenance s u b j e c t s . Ur inary methadone e x c r e t i o n is markedly enhanced by a c i d i f i c a t i o n of the u r ine . ' A sex d i f f e r e n c e i n t h e p a t t e r n of e x c r e t i o n of methadone and its metabolites is found and is related t o t h e rate of b i o t r a n s f o r m a t i o n of t h e drug.

F i g u r e 101, are p r e s e n t i n s u f f i c i e n t l y h igh c o n c e n t r a t i o n i n human sweat t o s u g g e s t t h a t sweat may be a s i g n i f i c a n t r o u t e of e l i m i n a t i o n of t h i s d rug .86

The mean a p p a r e n t ha l f - l i f e of orally- admin i s t e red methadone is 15 hours." Fol lowing in t r amuscu la r a d m i n i s t r a t i o n , t h e h a l f - l i f e is 7.3 hours.88 S u b j e c t s of a methadone maintenance program who r e c e i v e a large o ra l dose of 100 or 120 m g . show a mean appa ren t h a l f - l i f e of methadone t o be 2 5 hours.''

Methadone, metabolites C33 and L43 (See

399

RAFlK H. EISHARA

A n a l y t i c a l p r o c e d u r e s for i s o l a t i o n , d e t e r m i n a t i o n , and i d e n t i f i c a t i o n of methadone and its metabolites from b i o l o g i c a l f l u i d s and t i s s u e s i n c l u d e colorimetric and p h o t o m e t r i c t e c h n i u e s f o l l o w i n g i n t e r a c t i o n w i t h i n d i c a t o r

chromatography, 9, 6 8 , 7 4 9 76-78, chromatography, c o u n t e r - c u r r e n t t r a n s f e r , 8 1 radiotracer methods, 4 5 9 5 8 - 6 1 , 659 72, 8 0 i n rare 76 81 ,92

combined gas chromatography-mass s p e c t r o m e t r y . 579 749 77-80

6’3 e2 , 6 4 , 6 7 9 70, 82-84 pape r chromato column chromato r a hy, 79,8i g a s 7 2 , 9 0 , 9 1

raphy, 9i2 t h i n l a y e r

9, “9 54, “ 9 “9 70 repeated

n u c l e a r magnet ic r e sonance , 75, 76, g h , 95 and

6 . I d e n t i f i c a t i o n

v i r t u e of its characterist ic x - ray powder d i f f r a c t i o n p a t t e r n , W, I R , and NMR spectra (See 2.10, 2.11, 2.12, and 2 . 1 3 ) . ter5st ic m e l t i n g p o i n t of a b o u t 160 C . , or a b o u t 180 C . of t h e c r y s t a l s formed by p i c r o l o n i c acid and methadone is a l s o u s e f u l as a n i d e n t i t y t e ~ t . 3 , ~ A d d i t i o n of 2 m l . of me thy l o range test s o l u t i o n 5 t o a 0.5% methadone h y d r o c h l o r i d e s o l u t i o n forms a y e l l o w p r e c i p i t a t e . The m e l t i n g p o i n t of t h e water-washed r e s i d u e , formed by add ing excess s o d i u m hydrox ide s o l u - t i o n 3 t o a 5% s o l u t i o n of methadone, is a b o u t 76’C. Methadone h y d r o c h l o r i d e reacts p o s i t i v e l y t o t h e ch lo r ide characterist ic t es t w i t h s i l v e r n i t r a t e . 3 ~ 5

E m a d o n e hydFoch lo r ide c a n be i d e n t i f i e d by

The charac-

7. Microchemical React i o n s

th rough t h e f o r m a t i o n of cr s t a l s w i t h c e r t a i n r e a g e n t s has been reported. ?12,93-97 Hubach and Jones12 l i s t e d s e v e n r e a g e n t s which produced c h a r a c t e r i s t i c m i c r o s c o p i c c r y s t a l s from d i l u t e aqueous m e t hadone h y d r o c h l o r i d e s o l u t i o n s . Equa l d r o p s of t h e r e a g e n t and t h e methadone s o l u t i o n are mixed on a microscopic s l i d e and a l lowed t o s t a n d u n t i l c r y s t a l s d e v e l o p or a l l l i q u i d e v a p o r a t e s . The c r y s t a l s are t h e n

--- The microchemical i d e n t i f i c a t i o n of methadone

400

METHADONE HY DROCH LORlDE

observed and examined m i c r o s c o p i c a l l y . The r e a g e n t s used and t h e lowest c o n c e n t r a t i o n o f methadone hydroch lo r ide from which c r y s t a l s are o b t a i n e d are p r e s e n t e d i n Table 4 a l o n g w i t h t h e d e s c r i p t i o n of t h e formed c r y s t a l s .

t i o n s g i v e t u r q u o i s e color w i t h methadone.@8 A s o l u t i o n cons i s t ing of 0.8% w/v c o b a l t (11) t h i o c y a n a t e i n a (2 :3) v/v mix tu re of methanol and 1% or thophosphor i c a c i d ( sp . g r . 1.75) g i v e s a c o l o r r e sponse w i t h i n 5 seconds from t h e a d d i t i o n of t h e r e a g e n t .

8. Methods of Ana lys i s

F ive modi f ied c o b a l t (11) t h i o c y a n a t e s o l u -

8.1 Elementa l A n a l y s i s (As C21H2,NO*HC1) -

47,459 '$ Determined z

- ---------- E l e m e n t Theory 1 ----- ---

C 72.91 73.14 72.77, 73.06, 72.90 72.95

H 8 .16 8 .03 7.98, 8.23, 8.36 7.99

N 4 .05 3 .96 3.88 4 .07

0 4.63

c1 10.25

8.2 T i t r a t i on ------ 8 . 2 . 1 Non-Aqueous T i t r a t i o n

methadone hydroch lo r ide is d i r e c t l y t i t r a t e d i n non-aqueous medium, g l a c i a l acet ic a c i d , i n t h e p re sence of mercu r i c acetate t o t i e up t h e c h l o r i d e ion . The t e c h n i q u e of t h e non-aqueous t i t r a t i o n of t h e drug u s i n g v i s u a l i n d i c a t o r s ,

_I--------

The t e r t i a r y amine group of

40 1

RAFlK H. BISHARA

TABLE 4

Reagent

MICROCHEMICAL CRYSTALLIZATION OF METHADONE HYDROCHLORIDE

Concent ra - t i o n o f

Methadone HC1 - R e a c t i o n

Po ta s s ium i o d i d e , 5% 1 : 1,000 Colorless c r y s t a 1s

Po tas s ium f e r r o c y a n i d e , 1 : 500 C o l o r l e s s 5% ( f r e s h s o l u t i o n ) c r y s t a l s

Po ta s s ium f e r r i c y a n i d e , 1 : 500 Y e l l o w 5% ( f r e s h s o l u t i o n ) c r y s t a l s

Marme' s r e a g e n t 1 : 10,000 Colorless ( f r e s h s o l u t i o n ) c r y s t a 1s

Mayer's r e a g e n t

Wagner's r e a g e n t

1 :20,000 C o l o r l e s s

1 : 1,000 P a l e

c r y s t a l s

brown c r y s t a l s

Lanthanum n i t r a t e , 20% 1 :20 Color less c r y s t a l s

402


c r y s t a l v i o l e t 3 j 5 or me thy l v i o l e t j 6 has been d e s c r i b e d i n de t a i l . Each 1 m l . of 0.1 N 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 34.59 m g . of C21H2,NO*HC1. P o t e n t i o m e t r i c end p o i n t detec- t i o n has a lso been used . 9 9

8 . 2 . 2 Direct T i t r a t i o n

r a p i d d i r e c t t i t r ime t r i c method, u s i n g a n e x t r a c t i v e end p o i n t , for d e t e r m i n a t i o n of methadone i n p h a r m a c e u t i c a l p r e p a r a t i o n s . Chloroform is added t o t h e o r g a n i c base d i s s o l v e d i n pH 2 . 8 acetate b u f f e r s o l u t i o n s o t h a t t h e r a t i o of chloroform to t h e aqueous phase is a b o u t 3 t o 1. T i t r a t i o n is per formed u s i n g sodium d i c o t y l s u l f o s u c c i n a t e and Dimethyl Y e l l o w s c r e e n e d w i t h Oracet B lue as i n d i c a t o r . The change of c o l o r of t h e c h l o r o f o r m phase from g r e e n t o p i n k i n d i c a t e s t h e end p o i n t . The a c c u r a c y of t h e method is f 1%.

J o h n s o n a n d K i n g l o o deve loped a

8.3 C h l o r i d e D e t e r m i n a t i o n (Mercur ic _-----___ ________-_ Nitrate T i t r a t i o - A s E r n x e o P m e Z k H a o n e h y d r o c h l o r i d e

c o n t a i n i n g a t l eas t 2 mg. of c h l o r i n e is d i s s o l v e d i n 80 m l . of water -methanol ( 2 0 : 6 0 ) m i x t u r e and three d r o p s of d i p h e n y l c a r b a z o n e s o l u t i o n ( 5 mg./ml. methanol ) are added . The sample s o l u t i o n is t h e n t i t r a t e d w i t h s t a n d a r d 0.5 N m e r c u r i c n i t r a t e s o l u t i o n t o t h e first s i g n of rose color, u s i n g a one-ml. m i c r o b u r e t t e .

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

mL m e r c u r i c n i t r a t e x n o r m a l i t y X 35.5 X 100 mg. sample

____--- _--------

p e r c e n t p u r i t y of m e t hadone h y d r o c h l o r i d e =

p e r c e n t c h l o r i n e - f o u n d X 100 __-___ _--- - -I-..._- p e r c e n t c h l o r i n e - t h e o r y

403

RAFlK H. BISHARA

8 .4 U l t r av io l e t A n a l y s i s Wallace -- et al.101 oxidized methadone

w i t h bar ium p e r o x i d e t o benzophenone which was e x t r a c t e d i n hep tane and measured spectro- p h o t o m e t r i c a l l y a t 247 nm., E = 18,713. T h i s r e p r e s e n t s a n i n c r e a s e i n molar a b s o r b a n c e of approx ima te ly 34 times o v e r t h a t of methadone i n 0.1 N h y d r o c h l o r i c acid, g = 554, a t 292 nm. The method is s u c c e s s f u l l y used f o r d e t e r m i n i n g methadone i n b i o l o g i c a l spec imens , namely u r i n e , l i v e r , l ungs , k idneys , stomach, and i n t e s t i n e s .

8.5 F l u o r o m e t r i c A n a l y s i s ------- The f o r m a t i o n of a f l u o r o p h o r e when

methadone is hea ted i n a s o l u t i o n of formaldehyde and c o n c e n t r a t e d s u l f u r i c ac id followed by a d d i t i o n of water is reported by McGonigle. The f l u o r e s c e n c e is recorded a t a n e x c i t a t i o n wavelength of 270 nm. and a n e m i s s i o n wave leng th of 450 nm. The aqueous f l u o r o p h o r e is s t ab le f o r a t l e a s t 1.5 h o u r s a t room t e m p e r a t u r e . T h i s p r o c e d u r e is s u i t a b l e fo r measur ing microgram q u a n t i t i e s of methadone. Morphine, he ro in , code ine , and c o c a i n e d o n o t i n t e r f e r e . However, p r i o r s e p a r a t i o n of amphetamine, meper id ine , and q u i n i n e is r e q u i r e d s i n c e t h e y f l u o r e s c e under t h e c o n d i t i o n s of t h e a s s a y and i n t e r f e r e w i t h t h e d e t e r m i n a t i o n of methadone. The r e l a t i v e s t a n d a r d d e v i a t i o n of t h e methadone a s s a y is 2.2%.

chromatography t o mass d r u g s c r e e n i n g is p r e s e n t e d by S a n t i n g a . l o 3 The smallest detectable qufB4i ty of methadone is 1 p g . / m l . L a w l e r e t a l . used s p e c t r o p h o t o f l u o r o m e t r y for t h e d e t e r m i n a t i o n of methadone i n t i s s u e s .

1 0 2

A p p l i c a t i o n of f l u o r e s c e n c e and gas

8.6 I n f r a r e d A n a l y s i s !3 - The sample of methadone h y d r o c h l o r i d e

s o l u t i o n is made a l k a l i n e w i t h (1:l) sodium hydroxide i n water. The p r e c i p i t a t e d base is e x t r a c t e d w i t h chloroform. The extracts are d r i e d o v e r anhydrous sodium s u l f a t e a n d t h e chloroform is e v a p o r a t e d u s i n g steam heat and

404


a c u r r e n t of a i r . The r e s i d u e is d i s s o l v e d i n chloroform and t h e abso rbance , v e r s u s a chloroform b lank , is de te rmined a t 5.87 u s i n g 0.1 mm. ce l l s and a Beckman IR spectrophotometer. The abso rbance of t he s t a n d a r d is de te rmined i n t h e same manner and t h e m g . methadone h y d r o c h l o r i d e is c a l c u l a t e d from t h e r e l a t i v e r a t io . U l t r a v i o l e t i r r a d i a t i o n of a m e t h a n o l i c s o l u t i o n of methadone h y d r o c h l o r i d e , for 1 .5 hours , c a u s e s 74% d e t e r i o r a t i o n as shown by t h i s method.

8.7 Colorimetric A n a l y s i s Drugs c o n t a i n i n g b a s i c g roups form a

colored complex w i t h s u l f o n i c acid i n d i c a t o r d y e s s u c h as Methyl Orange and Bromcresol Green. lo' from t h e e x c e s s dye by e x t r a c t i o n i n t o chloroform or a s u i t a b l e o r g a n i c s o l v e n t and t h e q u a n t i t y of t h e complexed d r u g is estimated s p e c t r o p h o t o m e t r i c a l l y .

The methadone-dye complex is formed u s i n g bromthymol b l u e , 83 bromcresol g reen , 8 4

bromphenol b l u e , 8 4 bromchlorophenol b l u e , 84 c h l o r o p h e n o l r e d , 8 4 bromcresol p u r p l e , 8 4 , 1 0 7 and methyl orange.62982 With these dyes, any basic amine which c a n form a n o r g a n i c s o l v e n t - s o l u b l e dye complex would react as methadone, hence p r e c a u t i o n s need t o be t a k e n t o e l i m i n a t e i n t e r f e r i n g s u b s t a n c e s . Fo r t h e d e t e r m i n a t i o n of methadone i n t i s s u e s , R icka rds e t al.GO liberated t h e compound by d i s i n t e g r a t i o n of t h e t i s s u e w i t h s t r o n g a lka l i , ether e x t r a c t i o n , n i t r a t i o n of t he pheny l radicals i n methadone, c o l o r development w i t h e t h y l me thy l k e t o n e and measur ing t h e color a t 565 nm. The r e p r o d u c i b i l i t y and s e n s i t i v i t y are f 5% and 1 p g . of methadone. However, i t is t o be n o t i c e d tha t a n y methadone metabolic f ragment r e t a i n i n g t h e pheny l and amine g roups would react as t h e p a r e n t s u b s t a n c e . 8 i N i t r a t i o n of methadone w i t h a m i x t u r e of HN03/H2S04 (1:l) and t h e s u b s e q u e n t photometric d e t e r m i n a t i o n of t h e

The colored complex is t h e n s e p a r a t e d

--

405

RAFlK H . BISHARA

colored d e r i v a t i v e u s i n g f i l t e r S42 (428 nm.) is p r e s e n t e d by S k o r a . l 0 * The method is used f o r d e t e r m i n a t i o n of methadone i n ampoules , tablets , and b io logica l media (blood, l i v e r and u r i n e ) .

t i o n of methadone h y d r o c h l o r i d e i n d i s s o l u t i o n samples w a s deve loped by Bechtel and B r i c k l e y . l o 9 The d i s s o l u t i o n samples a r e s e q u e n t i a l l y sampled by an AutoAnalyzer and i n j e c t e d i n t o a n a i r - segmen ted stream of pH 1 .2 b u f f e r ( U . S . P . ) which is pumped t h r o u g h a glass mixing c o i l , Bromcresol p u r p l e (BCP) dye s o l u t i o n is t h e n added t o t h e stream of t h e methadone sample and t h e s o l u t i o n s are mixed i n a T e f l o n c o i l f o r t h e f o r m a t i o n of t h e dye- complex. The methadone-dye complex is extracted i n t o e t h y l e n e d i c h l o r i d e and t h e color i n t e n s i t y is measured a t 420 nm. The r e l a t i v e s t a n d a r d d e v i a t i o n (R.S.D.) and t h e r e l a t i v e error (R.E.) are f 1.48% and + 0.05% r e s p e c t i v e l y .

u r i n e by CM-cel lu lose column chromatography u s i n g c o n t i n u o u s drug-dye complex e x t r a c t i o n as a d e t e c t i o n sys t em is d e s c r i b e d by McMartin e t a l , lo6

An automated method f o r t h e de t e rmina -

The specif ic a s s a y of basic d r u g s i n

-- 8.8 Po la rography ---

The keto g r o u p of methadone is n o t p o l a r o g r a p h i c a l l y r e d u c i b l e because t h e d o u b l e bonds are n o t c o n j u g a t e d . T h e r e f o r e , a n e l e c t r o a c t i v e n i t r o d e r i v a t i v e is prepared. S k o r a l o 8 n i t r a t e d methadone u s i n g a m i x t u r e of n i t r i c a c i d / s u l f u r i c acid (1:l). N i t r a t i o n is completed i n 30 minu tes a f t e r h e a t i n g on a b o i l i n g water bath. The m i x t u r e is t h e n cooled, d i l u t e d w i t h 5 m l . d i s t i l l e d water and made a l k a l i n e , pH 10, w i t h 5 N NaOH. Three drops of 0.5% g e l a t i n s o l u t i o n are added and t h e s o l u t i o n is po la rographed , af ter deoxygena t ion , i n a p o t e n t i a l r a n g e of -0.4 t o -1.2 V. The h a l f - wave p o t e n t i a l of t h e n i t r a t e d methadone is -0 .64 V r e l a t i v e t o a s a t u r a t e d calomel electrode. A s t r a i g h t l i n e is o b t a i n e d when t h e c o n c e n t r a t i o n of t h e n i t r a t e d methadone

406


d e r i v a t i v e is p lo t t ed ve r sus t h e d i f f u s i o n cu r - r e n t ( h e i g h t of t h e r e d u c t i o n wave) fo r concen- t r a t i o n s of 2 0 , 30, 40, 50, and 60 p g . / m l . C o n c e n t r a t i o n s lower t h a n 5 Ccg./ml. can be de termined . The r e d u c t i o n wave of methadone n i t r a t e is d i s t i n c t and t h i s method is used for a s s a y i n g methadone i n ampoules and t a b l e t s . A p p l i c a t i o n of t h e method t o a s s a y f o r t h e drug i n blood, l i v e r and u r i n e is d i s c u s s e d .

Cathode r a y polarography of methadone N-oxide i n Walpole 's a c e t a t e b u f f e r pH 5 g i v e s a r e d u c t i o n wave which has a peak p o t e n t i a l of -1 .21 V . Reduct ion of t h e same s o l u t i o n w i t h T i C l , / H C l g i v e s methadone. 74

8 .9 Bioassay -- S ~ h a u m a n n ~ ~ r e D o r t e d a bioassay

procedure fo r methadone ;sing i so l a t ed guinea- p ig g u t . The method is s e n s i t i v e t o methadone a t a c o n c e n t r a t i o n as low as lo-* M.

8.10 Spin Immunoassay F r e e - r a d i c a l t echnology is combined

w i t h immunoassay t o y i e l d t h e new method of " s p i n immunoassay" used f o r d e t e c t i o n and a s s a y of small molecules i n b io logica l f l u i d s . l l 0 - 1 1 2 An a n t i b o d y is made a g a i n s t t h e haptene t o be a s s a y e d . The a n t i g e n is first prepared by c o u p l i n g t h e hap tene t o bovine serum albumin and immunizing rabbits or goats. Ammonium s u l f a t e is used t o p r e c i p i t a t e t h e y -g lobu l in f r a c t i o n of t h e serum, The haptene is s p i n labeled u s i n g a s table n i t r o x i d e rad ica l . The s p i n of t he unpai red e l e c t r o n of t h e labeled compound produces a magnet ic moment which is detected and measured i n an e l e c t r o n s p i n r e sonance (ESR) spectrometer. Very broad s p e c t r a l peaks a r e observed when t h e complex of s p i n - l a b e l e d haptene w i t h a n t i b o d y i n a c a p i l l a r y t u b e is placed i n t h e c a v i t y of t h e ESR spectrometer. T h i s re f lec ts t h e immobi l i za t ion of t h e free r a d i c a l a t or nea r t h e a n t i b o d y s i te . Sharp peaks r e s u l t from t h e d isp lacement of t h e s p i n - labeled hap tene by free hap tene (as by methadone

407

RAFlK H. BISHARA

i n u r i n e or s a l i v a ) . The a m p l i t u d e s of t h e s h a r p peaks measure q u a n t i t a t i v e l y t h e number of t h e f r e e r a d i c a l mo lecu le s tumbl ing f r e e l y i n s o l u t i o n and is a d i r e c t measure of t h e h a p t e n e c o n c e n t r a t i o n , A c o n c e n t r a t i o n greater t h a n 5 X M of methadone h y d r o c h l o r i d e or methadone c y c l i c metabolites is r e q u i r e d t o produce s p i n immunoassay r e s p o n s e s e q u i v a l e n t t o 0.5 p g . / m l . (1.8 X M ) o f morphine. The advan tages , d i s a d v a n t a g e s and compar ison o f t h i s t e c h n i q u e t o t h i n l a y e r chromatography are d i s c u s s e d by Leu te -- e t al.''O

8.11 R a d i o t r a c e r Techniques l 4 C and 3 H a r e a - = label methadone.

The 1 4 C a c t i v i t y is a s s a y e d as a t h i n l a y e r of barium c a r b o n a t e mounted on aluminum d i s c s and coun ted under a b e l l c o u n t e r u s i n g a t h i n mica window5' and a Geiger -Mul le r c o u n t e r . r e c e n t y e a r s , l i q u i d s c i n t i l l a t i o n c o u n t e r s have been used f o r d e t e r m i n i n g t h e r a d i o a c t i v i t y of l 4 C and 3 H l a b e l e d samples.10,66,114-116

I n

8 .12 Column Chromatography ----------_- A p p l i c a t i o n of CM-cel lu lose column

chromatography fo l lowed by c o n t i n u o u s dye complex e x t r a c t i o n is used f o r t h e a s s a y of methadone i n human u r i n e . '06

i n t r o d u c e d on Amberlite XAD-2 r e s i n column and e l u t e d w i t h methanol , 7 9 9 '17, '" methanol- ammonia (300: 51, '' chloroform- i s o p r o p a n o l (3:1), ' '9 and waterso p r i o r t o f u r t h e r a n a l y s i s . The XAD-2 r e s i n is a s t y r e n e - d i v i n y l b e n z e n e copolymer and has t h e c a p a b i l i t y of a d s o r b i n g many w a t e r - s o l u b l e o r g a y & compounds p r i n c i p a l l y by Van d e r Waal forces.

The e f f e c t i v e n e s s of r e c e n t l y deve loped r e s i n s BRX-SM-1, -2, -4, and Porapak t y p e Q are compared w i t h XAD-2 r e s i n by Bastos -- e t a1.12* - The r e c o v e r y of 3H-methadone is somewhat similar f o r e a c h r e s i n and r a n g e s from 45.4 t o 56.0%.

Samples c o n t a i n i n g methadone are

408


Methadone is r e s o l v e d from o p i a t e s and d i l u e n t s i n i l l i c i t n a r c o t i c m i x t u r e s by u s i n g a column of SE-Sephadex C-25 ion-exchanger . 12'

D i s p o s a b l e ch romatograph ic columns are used t o ex t r ac t methadone from ur ine .122

8.13 P a p e r Chromatograph Paperchromatograp 8 y o n b u f f e r e d

Whatman N o . 1 is used by A x e l r o d g o t o p rove t h e N-demethylat i o n of methadone.

t og raphy s y s t e m s for methadone. T a b l e 5 summarizes t h e pape r chroma-

8 .14 Thin Layer Chromatography (TLC) A compar ison of t h e t h i n laser chroma-

tog raphy of methadone on s e v e n commerc ia l ly a v a i l a b l e s i l i c a g e l c o a t e d f i l m s and sheets w i t h s i l i ca gel coated glass p l a t e s u s i n g chloroform/n-butanol/ammonium hydrox ide (70:40:5) , and benzene/dioxane/ethanol/ammonium hydrox ide ( 5 0 : 5 0 : 5 : 5 ) is p r e s e n t e d by Schweda. lZ9 The hand-coa ted s i l i ca ge l l a y e r on t h e g l a s s p l a t e s is t h e most v u l n e r a b l e l a y e r . The f i l m s are s u p e r i o r t o i t . The s i l i c a g e l s h e e t s r e q u i r e c a r e f u l h a n d l i n g . t h i n l a y e r p l a t e s (3 X 3 c m . ) to detect t h e p r e s e n c e of methadone, 100 p g . / m l . , i n a n unhydrolyzed u r i n e sample . The d e v e l o p i n g t i m e is u s u a l l y 1.5 min. Copenhaver and B l o s e l 3 l u s e s l i d e s t o p r e p a r e t h i n l a y e r m i c r o p l a t e s f o r fas t d e t e c t i o n of methadone and o the r d r u g s of a b u s e i n u r i n e . Gupta l32 u s e s d i s p o s a b l e p l a s t i c bags t o r u n t h e t h i n l a y e r chromatography o f methadone, methadol , normethadol , and a c e t y l m e t h a d o l . I d e n t i c a l r e s u l t s are o b t a i n e d from p l a t e s deve loped i n t h e p l a s t i c bag or i n a g l a s s t a n k . Dole -- et a1.133-135 u s e ion-exchange pape r e x t r a c t i o n p r i o r t o TLC. Two-dimensional TLC is used fo r i d e n t i f i c a t i o n of t h e metabolites of methadone.80 F i s h e r - et al.136 chromatograph methadone on p r e c o a t e d , f l e a b l e t h i n l a y e r sheets. The s e n s i t i v i t y of TLC for d e t e c t i o n o f methadone is d i s c u s s e d by Gorodetzky . l37

Ho e t al .130 u s e m i n i

409

T A B U 5

PAPER CHROMATOGRAPHY SYSTEMS FOR METHADONE

- Solven t System

te r t . - A m y 1 a l c o h o l / n -buty l e the r /wa te r (80: 7: 13 1

f Butanol/formic ac id / 0 water (12 : 1 : 7)

D i c h l o r o e t hane/ g l a c i a l acetic a c i d / water (20:8:2)

n-Butanol/c i t r i c a c i d / water (50: 1 : 50)'

Paper

S c h l e i c h e r & S c h u l l , #591-C,

pH 3.0 pH 4.0 pH 5.0 pH 6.0

S c h l e i c h e r & S c h u l l , #2045b

Whatman No. 1 dipped i n 5% s od i urn hy d r og e n c i t ra t e

R € D e t e c t i o n * ( x 100)

IP

35 49 22 56

BCG 7 8

D

75

74

Reference

123

124

91, 124

12 5

(cont h u e d . . . )

TABLE 5 (concluded)

So lven t System

Acetate b u f f e r ,

---- 2

pH 1.00

pH 3.30 pH 4.58

pH 7.40

M/15 Phosphate b u f f e r , pH 7.40

'Upper layer was used

2 127 According t o Vogel

Rf Paper D e t e c t i o n * - ( x 100)

Whatman No. 3 IP impregnated 86' 93 w i t h t r i b u t y r i n 95" 88 (1% v/v i n 86" 76 a c e t o n e ) 86" 75

95" 6 7 95" 59 86O 1 95" 2

86" 0

Reference -

126 126 126 126 126 128 126 126

12 8

*Key - BCG : bromcresol green , D : Dragendorf f , IP : i o d o p l a t i n a t e

TABLE 6

THIN LAYER CHROMATOGRAPHY SYSTEMS FOR METHADONE

Solvent System --- Rf

Adsorbent* Detection* ( X 100) Reference -- - -- Benzene/ethyl acetate/ SG RS 58 10 methanol/ammonium hydroxide SG IP 68 123 (80 :20 : 1.2 : 0.1)

Et hy 1 ace t a t e/me t hano 1/ SG ammonium hydroxide (85:10:5) SG

SG SG SG SG SG SG SG SG

Dioxane/ethanol/pyridine/ water (25 : 50 : 20 : 5)

Ethanol/glacial acetic acid/ water (60:30:10)

DPNA IP IP IP IP IP IP IP IP BC G

95 13 8 96 117 65 118 91 13 0 96 133 77 134,135 82 13 6 96.9 i37 99 13 9 84 140

C IP 34 15

C IP 59 15 SG D 46 76

(continued . . .)

TABLE 6 (Continued)

Solvent System

Benzene/dioxane/ethanol/ ammon ium hydroxide ( 5 0 : 4 0 : 5 : 5

Benzene/n-butanol/methanol/ water (10 : 15 :60 : 15)

-

tert .-Amy1 alcohol/n-butyl ether/water (80 : 7 : 13

water (4:1:2) $ n-Butanol/glacial acetic acid/

n-Butanol/conc . hydrochloric acid, saturated with water

Ethyl acetate/methanol/ ammonium hydroxide (85 : 10:3

Ethanol/hexane (8:92)

(90 : 10)

Ethyl acetate/methanol/ ammonium hydroxide (85:lO:l)

R f Adsorbent * Detect ion* ( X 100) Reference --

C SG

C SG

SG

SG SG

C SG

SG

SG

SG

IP D

IP D

IP

IP IP

IP IP

IP

IP

IP + D

99 15 75 76

17 15 15 76

17 76

55 76 64 14 1

62 15 57 14 1

96 142

31 142

94 143

(continued . . . )

TABLE 6 (Continued)

So lven t System

Carbon t e t r a c h l o r i d e

---

I s o p r o p y l e t h e r

Benzene

E thy lene d i c h l o r i d e

Met hy lene c h l o r i d e

Chloroform 2 P

E t h y l e t h e r

E t h y l acetate

n-Butyl a l c o h o l

Isopropy 1 a l c o h o l

Acetone

M e t hano 1

Rf Adsorbent * Detect i o n * ( X 100) Reference --

S G I P 11 144'

S G IP 27 144'

SG IP 42 144'

S G IP 3 7 144 ' SG IP 58 144'

S G IP 55 144 ' S G I P 6 7 144'

S G I P 73 144 ' S G I P 6 1 144 ' SG IP 63 144'

SG IP 79 144'

SG IP 77 144 ' ( con t inued . . .

c, C

a, P

Ll

0

om

n

4.

0

.

w-

rl

4

Lo

co

Q

, c

aw

w

r(

rl

Lo L

oc

o

w

wv

r(

lid

m

w

r(

P

a, 7

c *A

Q

,c

,

co

c

0

0

v

0

ma

Q,

cow

0

co 0

Lo a

m

Q,

wt

-

co

* ct 01

B 2

2B

2 2

n

P

Q, 7

c 0

00

m

mm 0

m

.r(

L1

h 0 u

n

0

rl

Lo 03

rl 0

c cd

.. v

5

n

0

rl

0

r(

003

..

‘2;;;

E

5

n

0

cv 0

Q,

.. v

4

k

Q,

c,

c 0

E

E

n

rl

v ..

v

cd 3

0

C

‘2; .. 0

c .. 0

C

b

5 U cd 5

5 k 0

w

id 9

a, 0

cd

rl h

s

c, w

a, o

E

.. 0

w 0

k 0

4

n

om

.r( ..

c,W

a,

**

o

rl

4-

0

k 0

rl

ox

k

0

ii u J

i

u

415

Sol vent System

Ethyl acetate/cyclohexane/ p-dioxane/methanol/water/ ammonium hydroxide (50: 50: 10: 10: 1.5 : 0.5)

TABLE 6 (Continued) Rf

Adsorbent* - Detection* ( X 100) Reference

SG SA 83 150

Ethyl acetate/cyclohexane/ p-dioxane/methanol/water/ ammonium hydroxide (50:50:10:10:0.5:1.5)

5 Ethyl acetate/cyclohexane/ methanol/water/ammonium hydroxide (70:15:8:0.5:2)

Ethyl acetate/cyclohexane/ ammonium hydroxide (50:40:0.1)

Benzene/cyclohexane/ diethylamine (15:75:10)

Ethyl acetate/dimethylformamide (3:l)

m

tert.-Amy1 alcohol/n-butyl ether/water (14: 7: 1)

SG

SG

SG

SG

SG

SG

IP 91 150

ASA 94 150

BCG 98 150

IP 99 123

IP 99 123

IP 55 123

(continued . . .


Solvent Sys tem - Adsorbent * Detection* ( x 100) Reference

tert.-Amy1 alcohol/n-butyl ether/wa ter (80 : 7 : 13

Chloroform/methanol/ammonium hydroxide (85 : 10 : 1)

Ethyl acetate/methanol/ ammonium hydroxide (85: 10: 1.5)

Chloroform/methanol (9:l) z 4

Acetone

Acetone/ammonium hydroxide (99:l)

Methanol

Chloroform/methanol (50:50)

Chloroform/methanol/ammonium hydroxide (47.5 :47.5 : 5)

Chloroform/glacial acetic acid/ methanol (47.5:5:47.5)

SG

SG3

SG3

SG SG

SG

SG

SG

SG

SG

SG

IP

IP

IP

IP IP

IP

IP

IP

IP

IP

IP

86 123

80 151

67 151

17 146 32 152

20 146

59 146

16 146

20 146

80 146

54 146

(continued . . .


Solvent System Adsorbent* - Detection* - ( x 100) Reference

Benzene/dioxane/ethanol/ ammonium hydroxide (50:40:5 :5)

Methanol/l2 N ammonium hydroxide (100:1.5)

n-Butano l/e t hy 1 acetate/ E ethanol/ammonium hydroxide O0 (2:28:14:0.4)

Benzene/ether (10:l)

D ic h lor ome t ha ne/e t her ( 10 : 2 1

Benzene/diethylamine/dioxane/ ethanol (50 : 5 : 40 : 5 1

Dimethylformamide/ethyl acetate (1:3)

Ethanol/isopropyl ether (20:80)

SG

SG SG SG SG

SG

A

A

SG

SG

SG

IP

IP IP IP

D + IP IP

IP

IP

IP

IP

IP

98 153

42 153 53 152 37 12 8 97 154

71 133

20 155

35 155

91 156

86 157

11 152

(cont hued . . .

TABLE 6

Solvent System

Acetoacetic ester/chloroform (1: 1)

-

Acetone/chloroform/formic acid (4:16:1)

Cyclohexane/diethylamine (9: 1)

Benzene/chloroform/ diethylamine (6 :3 : 1)

Benzene/l,4-dioxane/ethanol/ ammonium hydroxide (100:80:10:11)

f \b

Acetic acid/chloroform/ met hano 1 (10 : 3 5 : 65 1

Benzene/ethyl acetate/ ammonium hydroxide (35:60:5)

Acetone/chloroform/ diethylamine (2:88:10)

(Continued )

Adsorbent * Detection* ( X 100) Reference Rf

--- cc

A

SG

SG

SG

SG

SG

SG

S W + D

D

D

D

D + IP

S W

S W

D

88 158

59 159

71 159

88 159

97 154

53 16 0

75 16 0

72 92

(continued . . .


Adsorbent* Detection* ( X 100) Reference -- Solvent System -- Benzene/diethylamine/methanol SG (75 : 10 : 15)

D 74 92

Benzene/n-butanol/methanol/ water/ammonium hydroxide (10:15:60:10:5)

SG D 79 92

Ethanol/ethyl acetate/ SG ammonium hydroxide (50:45:5)

D 73 76

$ Chloroform/dioxane/ethyl 0 acetate/ammonium hydroxide

(25 :60 : 10: 5 )

SG I, D, PP 73 16 1

lIn an ammonium hydroxide atmosphere

2Top layer was used

Silica gel GF with 1% CaSO4.&H2O 3

*Key - A: alumina, ASA: ammonical silver nitrate and heat, BCG: bromcresol green, C : cellulose, D: Dragendorff, DPNA : diazotized

(continued . . .

42 1

TABLE 6 (Concluded)

p-nitroaniline, I: 1% iodine, IP: iodoplatinate, PP: 0.1% potassium permenganate, RS: radioscanning, SA: 0.5% sulfuric acid, SG: silica gel, SUV: short ultraviolet light

RAFlK H. BISHARA

Table 6 c o n t a i n s t h e most commonly used t h i n l a y e r chromatography sys tems f o r methadone.

8.15 G a s Chromatography --- (GC) ----_- G a s chromatography s y s t e m s for

m e t hadone are r e p o r t e d - i n - Table 7. i o n i z a t i o n detector is used i n a l l r e f e r e n c e s u n l e s s otherwise i n d i c a t e d .

A f lame

8.16 Combined --- G a s Chromatography-Mass Spec t roscopy ~ G C I ~ E J - - - - ~ The GCMS c o n d i t i o n s and t h e mass

f r agmen ta t ion p a t t e r n have been p r e v i o u s l y d i s c u s s e d (See 2 .14) . T h i s t e c h n i q u e is used f o r t he i d e n t i f i c a t i o n of methadone m e t a b o l i t e ~ ~ 9 , 7 ~ , 7 7 - ~ ~ and f o r t h e s c r e e n i n g and i d e n t i f i c a t i o n of t h e dangerous d r u g s of abuse. 171, 1 7 2

8 .17 High P r e s s u r e Liquid Chromatography ---- (HPU: 1 -- High p r e s s u r e l i q u i d chromatography is

used by Lorenz1T3 f o r t h e d e t e r m i n a t i o n of isomethadone i n methadone. A r e v e r s e phase system is used , The column, 1 meter long x 2 mm. i . d . is packed w i t h DuPont Permaphase ETH packing material. The mobile l i q u i d c o n s i s t s of 1% r e a g e n t ammonium hydroxide, 15% methanol, and 84% water. The column is o p e r a t e d a t a f low ra te of 50 m l . / h r . A 254 nm. d e t e c t o r is used for moni tor ing t h e column e l u e n t . The r e t e n t i o n volumes t o e l u t e isomethadone and methadone are 6 m l . and 17 m l . r e s p e c t i v e l y .

9. E x t r a c t i o n from B i o l o g i c a l F l u i d s

drugs i n b i o l o g i c a l f l u i d s i n v o l v e three s t e p s , namely s o l v e n t e x t r a c t i o n , c o n c e n t r a t i o n , and d e t e c t i o n o r a s s a y i n g . Each of these s t e p s is time-consuming and d rug losses due t o a d s o r p t i o n on t o g lassware , incomple te t r a n s f e r of s o l v e n t s and e v a p o r a t i o n of v o l a t i l e compounds may lower t h e r ecove ry and hence t h e s e n s i t i v i t y . Ramsey and Campbell163 d e s c r i b e d

Most methods u s e d f o r t h e d e t e r m i n a t i o n of

422

Column --

TABLE 7

GAS CHROMATOGRAPHY SYSTEMS FOR METHADONE

C a r r i e r Flow Rate Colum: R e t e n t i o n Refer- ml./min. Temp. C . Time, min. ence -- - G a s --

3 f t . long x 0.125 i n . N2 3 0 190 7.40 68 O.D. , 3% OV-17 on 100/ 120 mesh G a s Chrom Q

6 f t . long x 3 mm. I . D . , A 47 215 3.32 15 2% SE-30 on 80/100 mesh G a s Chrom S

P W h, 6 f t . long X 2 mm. I .D . , He

3% SE-30 on 8 0 / l O O mesh G a s Chrom Q

2 M long X 0.125 i n . O.D., N2 2.5% E - 3 0 1 on 80/100 mesh Chromosorb G

4.75 f t . long x 0.128 i n . - diameter, SE-30 on 70/80 mesh DMCS

1.22 M long X 5.5 mm. I .D . , 3% OV-17 on 100/ 200 mesh G a s Chrom Q

N2

32

3 0

-

50

2 00 2 .80 162

2 00 5.20 163

- 14 .8 149

240 7.7 164

(cont inued . . . I

TABLE 7 (Continued)

Column C a r r i e r Flow R a t e Column R e t e n t i o n Refer- Gas m l . / m i n . Temp. OC. Time, m i n . e n c e -- --

3% ov-1 - - 220 2 .6 165

3% OV-17 - - 22 0 1 . 7 165

- 220 1.8 165

5 f t . long X 0.125 O.D., N2 3 0 . 7 23 0 4 . 7 166 5% SE-30 on 60/80 mesh C h r omosorb W

3% ov-210 -

P w + 6 f t . long X 4 mm. I . D . , A 65 180 12 .1 167l

1% SE-30 on 100/200 m e s h A 56 2 00 4 .9 167 Anakrom ABS A 70 210 3.3 16 7

2% OV-225 on G a s Chrom Q N2 35 190 2 153

1.2 M long X 3 mm. I . D . , N2 35 23 5 1 .2 168 3 . 5 % UCW98 on 80/lOO m e s h C hr omos or b W -A W- DMCS

1.2 M long X 4 mm. I . D . , H e 75 2 10 4 160 3% SE-30 on 8 0 / l O O mesh G a s Chrom Q

(cont inued . . .

TABLE 7 (Continued)

Column C a r r i e r Flow Rate Colum$ R e t e n t i o n Refer -

Gas -- ml./min. Temp. C. Time, min. ence

4 f t . long X 2 . 5 mm. I.D., H e 6 0 16 5 4 57,78 1% W-98 on 8 0 / l O O mesh G a s Chrom Q

2 M long X 0.25 i n . O.D., N2 65 195 12 92 3% OV-17 on 60/80 mesh a c i d washed, DMCS t r e a t e d G a s Chrom Q

1 M long X 0.125 i n . O.D., N2 36 180 12 92

mesh a c i d washed, DMCS t r e a t e d Chromosorb G

’ 2% Carbowax on 80/100

6 f t . long X 0.25 i n . , 5% Nz OV-1 on 100/120 mesh Chromosorb W

1.3 M long X 6.25 mm. O.D., - 3.8% UC-W98 on D i a t o p o r t S

2 M long x 0.25 i n . O.D., N2 2% SE-30 on SO/lOO mesh Chromosorb G

50

-

16

23 5 3.3 9

190 6 . 4 77

180 14.2 76

(cont inued . . .

TABLE 7 (Continued)

Column C a r r i e r Flow R a t e C o l u m t R e t e n t i o n Refer -

ml./min. Temp. C. Time, min. - -- ence -- -- G a s

1 M long x 0.125 i n . O.D. , Nz 14 185 7.9 76 5% KOH and 2% Carbowax 20 M

4 f t . long X 3 mm. I . D . , N2 3.8% W98 on 80/100 mesh Dia topor t S

6 f t . long x 3 mm. I . D . , NZ fi 1% cyclohexane dimethanol

s u c c i n a t e on 100/120 mesh Dia tomi te CQ

2 f t . long x 4 mm. I . D . , N2 2.5% SE-30 on S O / l O O mesh Chromosorb G

6 f t . long X 0.25 i n . NZ I . D . , 3% OV-1 on S O / l O O mesh Chromosorb WHP

80

80

40

6 0

175

185

2 00

255

5.4 68

4 .6 6 8

1 . 7 169

1.3 170

(cont inued . . .

Column

TABLE 7 (Concluded)

Carr ier Flow Rate C o l u m t R e t e n t i o n Refer- G a s ml./min. - Temp. C . Time, - min. e n c e --

6 f t . l o n g X 0.25 i n . N2 4 0 2 05 5.25 145 O.D. , 3% SE-30 on loo/ 200 mesh G a s C h r o m Q

' S t r o n t i u m 90 a r g o n i o n i z a t i o n d e t e c t o r

RAFlK H. BISHARA

a r a p i d method fo r e x t r a c t i o n of methadone i n which t h e complete a n a l y s i s is carried ou t i n one v e s s e l and o m i t t i n g t h e e v a p o r a t i o n s t e p , t o overcome t h e v o l a t i l i t y problems. L y o p h i l l i z a - t i o n and l i q u i d - s o l i d e x t r a c t i o n are used t o detect d rugs of abuse i n u r i n e . l r 2

f o r drugs of abuse i n a u r i n e s c r e e n i n g program and d rug e x c r e t i o n data is p resen ted by Ka i s tha and Jaffe.174 exchange r e s i n loaded paper and t h e i o n paper is e x t r a c t e d a t p H 1 w i t h CHC13 and t h e n chromatographed on t h i n l a y e r p l a t e s . The c o n c e n t r a t i o n of methadone i n t h e u r i n e by i o n exchange paper p r o v i d e s a c l e a n e r e x t r a c t t h a n by d i r ec t e x t r a c t i o n of t h e ur ine .147 Dole e t -- a l . - describe b u f f e r e l u t i o n of t h e ion exchange paper135 and t h e a p p l i c a t i o n of Amber l i t e IR-120 c a t i o n exchange paper pr ior t o t h i n l a y e r chromatography.l34 Comparison of three u r i n e e x t r a c t i o n t e c h n i q u e s is r e p o r t e d by K a i s t h a and Jaf fe. 175

for t h e d e t e r m i n a t i o n of t h e d rugs of abuse are r e v i e w e d by Sohn e t a l . l76

drug abuse s c r e e n i n g programs, d e t e c t i o n procedures , development costs, s t r e e t - s a m p l e a n a l y s e s and f i e l d tests.

R e l i a b i l i t y of i d e n t i f i c a t i o n t e c h n i q u e s

The drug is absorbed on c a t i o n -

"Clean-up" p rocedures fo r u r i n e and blood

Kaistha -- et a1.177 r e c e n t l y e v a l u a t e d t h e

10. Determina t ion i n T i s s u e s

mina t ion of methadone i n blood samples , u r i n e samples and u r i n e s c r e e n s . However , L a w l e r e t a l . 1 0 4 su rvey t h e q u a n t i t a t i v e a s s a y s of methadone i n t i s s u e s . The drug i n t h e t i s s u e is examined by spec t ropho to f luo romet ry fo l lowing formaldehyde t r e a t m e n t and u s i n g e x c i t a t i o n a t 270 nm. and emiss ion a t 450 nm. G a s l i q u i d chromatography is a lso used, after s i l y l a t i o n , on a 3% OV-17 column packed on 100/120 mesh G a s Chrom Q and hea ted a t 22OoC.

-_-_-----_---_----__- The p rev ious methods emphasize t h e deter-

-- -

428


11. Bib l iog raphy A comprehensive b i b l i o g r a p h y o f "Methadone,

1929-1971" has been pub l i shed i n t w o p a r t s , l 7 8 , l 7 9 Langrodlso compiled a b i b l i o g - raphy of methadone main tenance t r e a t m e n t of h e r o i n addic t ion . The reader is r e f e r r e d t o t h e s e t h r e e lists of r e f e r e n c e s f o r complete l i t e r a t u r e abou t methadone.

p r o f i l e w a s conducted th rough A p r i l of 1973. The l i t e r a tu re s e a r c h f o r p r e p a r i n g t h i s

12. Acknowledgments Theauthorwishes t o thank t h e Merck and

Co., Inc., Rahway, N, J., f o r g r a n t i n g t h e pe rmis s ion t o u s e some of t h e i n f o r m a t i o n about methadone hydroch lo r ide from t h e Merck Index, 8 t h e d i t i o n . Thanks are a l so due t o Mr. C . E. Hubach and A n a l y t i c a l Chemistry, and t o D r . E . L. May and T h e m 5 6 T Organic - Chemistry f o r t h e i r pe rmis s ion t o r ep roduce Table 1 and F i g u r e 9 r e s p e c t i v e l y .

The a u t h o r e x p r e s s e s h i s a p p r e c i a t i o n t o D r . A . D. Kossoy, M r . C . D. Underbrink, D r . D. E. Dorman, Mr. H. R . S u l l i v a n , and M r . J. L. Occolowitz of E l i L i l l y and Company f o r a s s i s t a n c e i n p r e p a r a t i o n and d i s c u s s i o n of t h e i n t e r p r e t a t i o n of v a r i o u s s p e c t r a l d a t a i n t h i s monograph.

Mrs. N e l l V. Ward, and Mrs. JoAnn Spencer f o r t h e i r h e l p i n t h e s e a r c h of t h e voluminous l i t e ra ture .

The a u t h o r is v e r y g r a t e f u l t o Mrs. J a n i s A . Brown f o r he r i n v a l u a b l e secretar ia l h e l p i n deve lop ing t h e format of t h i s p r o f i l e and t y p i n g t h e manuscr ip t .

t i o n t o h i s c o l l e a g u e s a t E l i L i l l y and Company, w i t h a s p e c i a l r e f e r e n c e t o Mr. C . D. Wentling, f o r t h e i r he lp , c r i t i c i sm, and s u g g e s t i o n s t o improve t h i s a n a l y t i c a l p r o f i l e .

S p e c i a l t h a n k s go t o Miss Adele Hoskin,

The a u t h o r e x t e n d s h i s s i n c e r e a p p r e c i a -

429

RAFlK H. BISHARA

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Dole, V. P., Crowther, A . , Johnson, J . , Monsalvatge, M. , B i l l e r , B. , and Nelson, S. S. , N . Y . S t a t e J . Med., 72, 471 (1972). Dole, V . P., K i m , W . K . , a n d E g l i t i s , I . , Psychopharmacol. B u l l . , 3, 45 (1966 1. Dole, V . P. , K i m , W . K . , and E g l i t i s , I . , J . A m e r . Med. A s s . , - 198, 115 (1966) . F i s h e r , W . T. , B a i t s h o l t s , A . D . , and Grau, G. S., J . Chromatogr. S c i . , - 10, 303 (1972 1. Gorodetzky, C . W . , Tox ico l . Appl. Pharmacol. , 23, 511 (1972). Neesby, T., m i n . Chem., 19, 356 (1973). Davidow, B. , P e t r i , N . L., and Quame, B . , A m e r . J. C l i n . P a t h o l . , 50, 714 (1968) . Heaton, A . M. , and Blumberg, A . G. , J. Chromatogr., 41, 367 (1969) . Wallace, J . ET, Biggs, J. D. , Merritt, J . H., Hamilton, H. E . , and Blum, K . , J. Chromatogr., 71, 135 (1972). Broich, J . R. , Hs fman , D. B., Goldner, S. J., Andryauskas, S. , and Umberger, C . J., J. Chromatogr. , 63, 309 (1971). Broich, J . R. , Hoffman, D. B., Andryauskas, S., Galante , L., and Umberger, C . J., J. Chromatogr. , - 60, 95 (1971) . Emmerson, J. L., and Anderson, R . C . , J. Chromatogr. , 17, 495 (1965) . Mule, S. J. , J . Ckromatogr., 55, 255 (1971) . N o i r f a l i s e , A . , and Mees, G. , J . Chromatogr. , 31, -- 594 (1967). B a s e l t , R . C . , and C a s a r e t t , L. J. , J . Chromatogr., -- 57, 139 (1971) .

-

-

431

RAFlK H. BISHARA

148.

149.

150.

151.

152.

153.

154.

155.

156.

157.

158.

159. 160.

161.

162.

163.

164.

165.

166.

Jansen, G. , and Bickers , I . , Southern Med. J. , - 64, 1072 (1971). Baden, M. M . , Valanju, N . N . , V e r m a , S. K., and Nalanju, S. N. , A m e r . J. C l i n . Pa tho l . , - 57, 43 (1972). Kais tha , K. K., and J a f f e , J . H., J. Pharm. S c i . , 61, 679 (1972). Kenison, L. TY Loveridge, E . L., Gronlund, J . A . , and Elmowafi, A . A . , J . Chromatogr., 71, 165 (1972). Bastos, M . L , Kananen, G . E . , Young, R . M . , Monforte, J . R . , and Sunshine, I . , C l i n . Chem., -- 16, 931 (1970). B e r r y , D. J . , and Grove, Y . , Y . Chromatogr., - 61, 111 (1971). Pa rke r , K . D . , and Hine, C . H . , Psychopharmacol. Bu l l . , 3, - 18 (1966). Rummler, R. , and Haller, R . , Arzneim. Forsch . , 20, - 281 (1970). G a r r i o t t , J. C., and Stolman, A , , C l i n . Toxicol . , 4, - 225 (1971). Eberhard t , H., and Norden, O. , Arzneim. Forsch . , -- 14, 1354 (1964). Ebel, S., Bahr, E . , and P l a t e , E . , J . Chromatogr., 59, 212 (1971). Vidic, E . , A r c h . T o x i k o l . , 27, 19 (1970). Chou, K. M . , p e r sona l commuxcation, E l i L i l l y and Company, I n d i a n a p o l i s , Ind. 462 06. Choul i s , N . H., J . Pharm. Sc i . , -- 62, 113 (1973 I . I n t u r r i s i , C . E . , and Verebely, K. , J. Chromatogr., 65, 361 (1972). Ramsey, J. , and Campbell, D. B., 3 . Chromatogr., 63, 303 (1971). V a l e n t i n e , J .L . , Wiegert, P. E . , and Char l e s , R. L., J . Pharm. S c i . , 61, 796 (1972). Goldbaum, L. R. , Sant inga , P., and Dominguez, A . M., C l i n . Toxicol . , 5, 369 (1972). Pa rke r , K. D., Fontan, C . R . , and Kirk, P. L., Anal. Chem., - 35, 356 (1963).

-

3

438


167.

168.

169.

170.

171.

172.

173.

174.

175.

176.

177.

178.

179.

180,

Kazyak, L., and Knoblock, E . C., Anal. Chem., 35, 1448 (1963). Van der-looten, E. P. J., Van d e r H e l m , H. J., and Geer l ings , P. J., J. Chromatogr., - 60, 131 (1971). F ink le , B. S., Cherry, E. J., and T a y l o r , D. M . , J. Chromatogr. S c i . , 9, 393 (1971). Moore, J. M . , and Bena, F. ET, Anal . Chem., - 44, 385 (1972). Law, N. C. , Aandahl, V . , Fa l e s , H. M., and Milne, G. W . A., C l i n . Chim. Acta, 32; 221 (1971). F ink le , B. S., Taylor , D. M . , and B o n e l l i , E . J;, J. Chromatogr. S c i . , - 10, 312 (1972). Lorenz, L. J. , p e r s o n a l communication, E l i L i l l y and Company, I n d i a n a p o l i s , Ind. 46206. Kais tha , K. K., and J a f f e , J. H., J . Pharm. S c i . , - 61, 305 (1972). Kais tha , K. K., and J a f f e , J. H., J. Chromatogr., - 60, 83 (1971). Sohn, D., Simon, J., Hanna, M. A , , and Gha l i G . , J. Chromatogr. S c i . , - 10, 294 (19723. Kais tha , K. K. , J. Pharm. S c i . , 61, 655 (1972). Methadone : A Bib l iography, 1929-1971, P a r t I, I n t . J . Addic t . , 6, 329 (1971). Methadone : A Bib l iography, 1929-1971, P a r t 11, I n t . J. Addict . , 6, 677 (1971). Langrod, J., I n t . J. AddicF., - 5, 581 (1970).

439

OXAZEPAM

Charles M . Shearer and Caesar R . Pilla

CHARLES M. SHEARER AND CAESAR R . PILLA

CONTENTS 1. Descr ip t ion

1.1 Name, Formula, Molecular Weight 1.2 Appearance, Color , Odor

2.1 In f r a red Spec t ra 2.2 Nuclear Magnetic Resonance Spec t ra 2.3 U l t r a v i o l e t Spec t ra 2.4 Mass Spec t ra 2.5 Melting Range 2.6 D i f f e r e n t i a l Thermal Analysis 2.7 S o l u b i l i t y 2.8 Crys t a l P r o p e r t i e s 2.9 Di s soc ia t ion Constant

2. Phys ica l P r o p e r t i e s

3. Synthes is 4. S t a b i l i t y 5. Metabolism 6. Methods of Analysis

6.1 Elemental Analysis 6.2 Gravimetr ic Analysis 6.3 D i r e c t Spectrophotometr ic Analysis 6.4 Color imet r ic Analysis 6.5 Fluorometr ic Analysis 6.6 T i t r i m e t r i c Analysis 6.7 Polarographic Analysis 6.8 Chromatographic Analysis

6.81 Paper Chromatography 6.82 Thin Layer Chromatography 6.83 Gas Chromatography 6.84 Column Chromatography

7. References

442

OXAZEPAM

1. Desc r ip t ion


The name used by Chemical Abs t r ac t s and the Nat ional Formulary XI11 f o r oxazepam i s 7-chloro-1,3- dihydro-3-hydroxy-5-phenyl-2&1,4 benzodiazepin-2-one.

C H C 1 N 2 0 2 Mol. W t . : 286.72

1 . 2 Appearance, Color , Odor 15 11

Oxazepam is a creamy whi te t o p a l e yellow powder having p r a c t i c a l l y no odor.

2. Phys ica l P r o p e r t i e s

2.1 In f r a red Spec t ra

An i n f r a r e d absorp t ion spectrum of a potassium bromide d i s p e r s i o n of oxazepam (NF Reference Standard m a t e r i a l ) i s presented i n F igure 1. with publ ished s p e c t r a l , 2 * 3 b ments a r e l i s t e d i n Table I.

This spectrum agrees The s p e c t r a l band ass ign-

Table I In f r a red Spec t r a l Assignments of Oxazepam

Wavelength, u Vibra t ion Mode Reference 3.05 t o 3.20 OH, NH s t r e t c h 4 5.79 and 5.86 C=O s t r e t c h 5

6.19 C=N-s t r e t ch 5 6.35 and 6.72 Aromatic C=C deformations 6

12.07 Out of p lane CH 6 deformation of 1 , 2 , 4 s u b s t i t u t e d aromatic

deformation of mono s u b s t i t u t e d aromatic

13.39 and 14.35 Out of p lane CH 6

443

&

M m

w 0

OXAZEPAM

2.2 Nuclear Magnetic Resonance

The 60 MHz NMR spectrum, shown i n F igure 2 , was obtained by d i s so lv ing oxazepam (NF Reference Standard ma te r i a l ) i n deutero dimethylsulfoxide containing t e t r a - methyls i lane a s i n t e r n a l re ference . The s p e c t r a l ass ignments a r e l i s t e d i n Table 11.

Table 11 NMR Spec t r a l Assignments of Oxazepam

Chemical S h i f t ( & I Protons S p l i t t i n g 4.82 a l iDha t i c C-H Doublet 6.35 7.52

10.78

0-H' Doublet aromatic CH N-H S i n g l e t

Mu1 t i p 1 e t

- Both the hydroxyl and amino protons exchanged wi th D 0. Sadee8 repor ted a s i n g l e t f o r t h e a l i p h a t i c C-H proton, however, a s i n g l e t was observed i n these l a b o r a t o r i e s only a f t e r D 0 exchange.

2

2 2.3 U l t r a v i o l e t Spec t ra

The u l t r a v i o l e t s p e c t r a of oxazepam i n 0.1Nhydro- c h l o r i c ac id , 0.1N sodium hydroxide, and i n pH 7 bu f fe r a r e presented i n Figure 3. The spectrum of oxazepam i n alcohol i s presented i n F igure 4. The spectrum and absorp- t i v i t i e s of oxazepam i n a lcohol agree wi th publ ished v a l u e ~ l , ~ , ~ - a r e presented i n Table 111.

The a b s o r p t i v i t i e s and maximum wavelengths

Table I11 U l t r a v i o l e t Spec t r a l C h a r a c t e r i s t i c s

Absorp t iv i ty Solvent max (nm) 0.1N HC1 236 111

PH 7

284 362

230 315

236 342

230 318

41 13

131 9

111 11

126 9

0.1N NaOH

alcohol

445

w 0

E 2 I N

446

OXAZEPAM

0.7

0.6

0.5

w 0.4

3 0.3

m Q 0.2

u z

a

0.1

0 .o

1

WAVE LENGTH (nrn)

Fig . 3 - U l t r a v i o l e t S p e c t r a of Oxazepam (NF Refe rence S tanda rd ) Solvent-0.1N H C l - - - pH 7 b u f f e r , 8 I t 0,lNNaOfi.

WAVE LENGTH (nrn)

Fig. 4 - U l t r a v i o l e t Spectrum of Oxazepam (NF Refe rence S tanda rd ) So lven t - a l coho l .

447

CHARLES M. SHEARER AND CAESAR R. PlLLA

2.4 Mass Spectra

The mass spectrum of oxazepam (NF Reference Stan- dard ma te r i a l ) w a s obtained by d i r e c t i n s e r t i o n of the sam- p l e i n t o the MS-902 double focusing, high r e so lu t ion mass spectrometer. The sample w a s run a t 200°C. and 1.0 x 10- t o r r wi th the ion iza t ion e l ec t ron beam energy a t 70 e.v. The high r e so lu t ion d a t a was compiled and tabula ted wi th the a i d of the PDP-8 D i g i t a l computer. A l i n e graph of the mass spectrum i s shown as Figure 5 and the major high

10 r e so lu t ion da ta i n Table IV

Table IV Mass Spectrum of Oxazepam

6

Measured Mass Calculated Mass Formula C H O N C 1

1 5 11 2 2 286.0484 286.0509

270.0372 270.0321 C15H902NC1

269.0474 269.0481

268.0436 268.0402

267.0319 267.0325

259.0427 259.0399

257.0450 257.0481

241.0461

239.0379 239.0376

233.0715 233.0714

229.0526 229.0532

205.0738 205.0765

194.0849 194.0844

C H ON C 1 15 10 2

C H ON C 1 15 9 2

C H ON C 1 15 8 2

C H 0 N C 1 14 10 2

C H ON C 1 14 10 2

241.0420 1 5H1 0'''

14H8N

'1 gH90N2

C13H10N2C1

C14H9N2

C13H10N2

11 This spectrum i s i n agreement with t h a t presented by Sadee.

The molecular ion was a t rn /= 286. 257, i s generated by loss of a formyl r a d i c a l , which may be preceeded by a hydride migrat ion from C-3 to C-5. t i o n a l loss of CO generated an indazole a tm/e 229, which then el iminated C 1 50 give m/e 194. peaks a r e m/g 269(M - OH) and m/e 259(M -CHN).

The base peak, m/g

Addi-

OthFr predominant

448

n

a

9

E

W 0

(I)

2 I

ln

00 -r

l FLI .

CHARLES M. SHEARER AND CAESAR R. PILLA

2.5 Melting Range

The following melt ing po in t temperatures have been reported:

O C . Reference 203 - 204 5 205 - 206 1 2

20 6 13,14

2.6 D i f f e r e n t i a l Thermal Analysis

A s tudy by d i f f e r e n t i a l thermal ana lys i s (DTA) revea ls t h a t be melt ing endotherm i s a func t ion of t he hea t ing r a t e . This i s i l l u s t r a t e d i n Table V. A DTA curve of oxazepam (NF Reference Standard ma te r i a l ) ob ta ined a t a hea t ing r a t e of 10°/min. i s included a s Figure 6.

15

Table V D i f f e r e n t i a l Thermal Analysis of Oxazepam

melt ing endotherm OC. OC.

heat ing r a t e - min. 5

10 20

188 193 201

2.7 S o l u b i l i t y

The following s o l u b i l i t y d a t a were obtained a t uncontrol led room temperature:

4.5 mg./ml. i n 95% ethanol 0.03 mg./ml. i n water 4 mg./ml. i n chloroform

These values agree wi th t h e s o l u b i l i t i e s given i n the National Formulary XIII.

2.8 Crys ta l P rope r t i e s

The X-ray d i f f r a c t i o n p a t t e r n of oxazepam (NF Reference Standard ma te r i a l ) was obta ined with a P h i l i p s d i f f rac tometer using Cu KG r a d i a t i o n 15. spacings of t h e d i f f r a c t i o n p a t t e r n a r e presented i n Table VI.

The ca l cu la t ed d

450

0 50 100 150 201) 250 300 350 400 450 T. OC (CORRECTED FOR CHROMEL ALUMEL THERMOCOUPLES)

Fig. 6 - DTA Spectrum of Oxazepam (NF Reference Standard)

1


28 * 6.6 12.4

14.8 16.6 17.4 17.7 18.5 19.4

39.8 20.0

13.2

Table V I X-Ray-Powder, D i f f r a c t i o n P a t t e r n

Sample: Oxazepam: NF Reference Standard Source: C u K S - d 2& d 2 0 d 13.4 >G5.3 4T3 74 31.0 2T885

- 7.13 6.70 5.98 5.34 5.096 5.010 4.796 4.576 4.482 4.440

21.1 21.5 22.6 23.3 24.3 25.4 26.5 27.2 29.8 30.4

4.210 31.4 2.849 4.132 32.2 2.780 3.934 33.3 2.690 3.817 34.0 2.636 3.662 34.4 2.607 3.506 36.8 2.442 3.363 38.7 2.326 3.279 39.0 2.310 2.998 *Most i n t e n s e peaks 2.940

~ ~~ ~~ ~

2.9 Dissoc ia t ion Constant

The pKa's o f oxazepam i n aqueous so lu t ions were determined spectrophotometr ical ly t o be 1.8, a t which a proton i s gained; and 11.1, a t which a proton is l o s t .

3. Synthesis

One s y n t h e t i c rou te f o r the product ion of oxazepam i s given i n F igure 7. The quinazol ine 3-oxide can be prepared by r eac t ing ch lo race ty l ch lo r ide wi th 2-amino-5-chlorobenzophenone oximel6. The r ing expansion s t e wi th sodium hydroxide was descr ibed by Bell and coworkersq7. Treat- ment of t h e r e s u l t i n g benzodiazepin -2-one 4-oxide wi th a c e t i c anhydride and hydro lys is of t h e ester wi th base y i e l d s oxazepam5. of benzodiazepines i n genera l a r e d iscussed i n review ar t ic les18 ,19 ,20 ,21 .

Other s y n t h e t i c rou te s and the chemistry


19 Oxazepam i s s t a b l e a s a s o l i d o r i n a n e u t r a l so lu t ion . Acid hydro lys is produces 2-amino-5-chlorobenzophenone 22. The itnino-carbinol s t r u c t u r e l eads t o a number of r ea r r an e- ments (F igure 8) upon t reatment wi th a c e t i c acid5 o r bassg.

452

OXAZEPAM

'gH!5

2-amino- 5- chlorobenzophenone

H

c1 a$ c6H5

2-amino- 5-chloro- 6- chloro- 2- benzophenone oxime chloromethyl-

4-pheny l- quin- a z o l i n e 3 oxlde \1 NaOH

0 H II

c6H5 3-acetoxy-7-chloro-5-phenyl-l,3- 7- chloro- 5-phenyl- dihydro- 2E- 1,4-benzodiazepine-3 one 1,3-dihydro-2&-1,

4-benzodiazepine- 2 one 4 oxide

NaOH

6H5 oxa zep am

Figure 7 - Synthe t ic Route €or Oxazepam

453

H

H

H

7l

u

m

%

z 1 0

8

'd

V

454

OXAZEPAM

5. Metabolism

The major metabolite (greater than 95%) of oxazepam in man has been determined to be oxazepam glucuronide22. Minor metabolites are as follows: quinazol inone; 2- amino- 5- chlorobenzophenone ; 2 '-ben~oyl-4~- chloro- 2,2-dihydroxyacetanil ide; 2' -benzoyl-4khloro- 2- hydroxy- 2-ureidoacetanil ide ; 7-chloro- I, 3-dlhydro-Shydr~xy- 5-(phydroxyphenyl)-2&1,4-benzodiazepin-2-one; and 7- chloro-l,3-dihydro-3-hydroxy-5-[ 3 (or 4)-hydroxy-4-(or 3) methoxyphenyll-2~-1,4-benzodiazepin-2-one. The first two minor metabolites exist only in the unconjugated state in urine while the othe 8 are present as conjugates as well as in the free form

6-chloro-4-phenyl-24 1H)-

25



The following data were obtained on NF Reference Standard material7: Element % Theorv % Found

C 62.94 62.71 H N c1

3.85 9.79 12.41

3.99 9.62 12.47

6.2 Gravimetric Analysis

Oxazepm, can be precipitated out of a very dilute The precipitate acidic solution with silicotungstic acid.

is collected, washed with water, dried at 7OOC. and weighed This method has been applied to dosage forms2.

When a solution of Reinecke's salt is added to a dilute solution of oxazepam, a bright rose-violet colored precipitate is formed. The precipitate is then washed, dried, and weighed 2;Z4.

6.3 Direct Spectrophotometric Analysis

Salim and coworkers' described an ultraviolet spectrophotometric method of analysis which was applicable to capsules of oxazepam. In this method a sample equiva-

45 5

CHARLES M. SHEARER AND CAESAR R. PlLLA

l e n t t o about 50 mg. oxazepam is ex t r ac t ed wi th alcohol through a s i n t e r e d g l a s s funnel. t ed t o ob ta in a f i n a l concent ra t ion of about 4 mcg./ml. i n alcohol. The absorbance i s read on a spectrophotometer a t 229 nm. using alcohol a s a blank and compared wi th t h e absorbance of a s tandard s o l u t i o n of oxazepam. This i s a l s o the method spec i f i ed i n the Nat ional Formulary XIII.

A completely automated system capable of d i s i n t e - g ra t ing a whole t a b l e t or capsule , d i s so lv ing the a c t i v e cons t i t uen t , f i l t e r i n g i t , d i l u t i n g a po r t ion of t h e clear f i l t r a t e t o a des i r ed volume an obta in ing a complete UV- v i s i b l e scan, has been reportedg5. The accuracy of t he automated method i s comparable t o t h a t of t h e manual spec- t rophotometr ic method f o r oxazepam.

This s o l u t i o n i s d i lu -

6.4 Colorimetr ic Analysis

When a so lu t ion of Reinecke 's sa l t i s added t o a d i l u t e so lu t ion of oxazepam a b r i g h t ro se -v io l e t colored p r e c i p i t a t e i s formed. solved i n acetone and i t s cen t r a t ion determined spectro-

This can be i s o l a t e d and then d i s -

photometr ical ly a t 525 nm 2yt . Oxazepam can be hydrolyzed wi th hydrochlor ic ac id

t o form g lyc ine and 2-amino-5- chlorobenzophenone. The aromatic amine can be d i azo t i zed wi th n i t r o u s ac id and coupled t o naphthylene diamine26, N-alpha naphthy1-N- d ie thy p r ~ p y l e n e d i a m i n e ~ ~ , N-(l-naphthyl)ethylenediamine.2 HC122138, o r a lpha naphthol29. The concent ra t ion of t h e oxazepam i n the r e s u l t i n g colored s o l u t i o n can be de te r - mined spectrophotometr ical ly i n the v i s i b l e region.

6.5 Fluorometr ic Analysis

Walkens t e i n and coworkers22 appl ied f luorometry t o the determinat ion of oxazepam i n b io log ica l f l u ids . Oxazepam w a s ex t r ac t ed from the body f l u i d s by e thylene d i ch lo r ide and the f luorescence measured on a f luorometer equipped with a high pressure mercury lamp, a 200-400 m p broad band primary f i l t e r and a B540 (520-660 m p ) broad band secondary f i l t e r .

Braun and coworkers30 developed a f luorometr ic method f o r determining oxazepam which had a s e n s i t i v i t y of

456

OXAZEPAM

0.005 pg./ml. I n t h i s method an e t h a n o l i c s o l u t i o n of oxazepam i s heated i n phosphoric ac id , producing a very i n t e n s e f luorescence. The f luorescence is measured a t an e x c i t a t i o n wavelength of 360 nm. and an emission wavelength of 475 nm. dosage form ana lys is31 .

This method has a l s o been used f o r

S t e id inge r and S ~ h m i d ~ ~ developed two methods f o r determining oxazepam, combining t h i n l a y e r chromatography and fluorometry. In one method, t h e oxazepam is scraped o f f t he developed TLC p l a t e and ex t r ac t ed wi th methanol. Pe rch lo r i c ac id i s added t o t h i s s o l u t i o n and i t is heated. The r e s u l t i n g f luorescence can then be determined wi th a f luorometer . i s t o treat the i n t a c t p l a t e wi th t r i c h l o r o a c e t i c a c i d , h e a t the p l a t e , and determine the f luorescence d i r e c t l y by use of a t h i n l a y e r chromatographic p l a t e scanner.

The o t h e r method, a l s o repor ted by L a u f f l e l , 3

6.6 T i t r i m e t r i c Analysis

Oxazepam can be d isso lved i n dimethylformamide,

t o a po ten t iomet r ic and t i t r a t e d wi th 0.1N tetrabutylammonium hydroxide [ prepared i n benzene:methanol (9: 1 ) 1 endpoint using glass vs. calomel electrodes1.

Beyer and Sadee34 determined oxazepam by d i s s o l - ving it i n g l a c i a l a c e t i c ac id and t i t r a t i n g wi th 0.05N p e r c h l o r i c acid. The endpoint w a s determined e i t h e r poten- t i o m e t r i c a l l y o r v i s u a l l y , using c r y s t a l v i o l e t a s an ind i - ca tor . Acet ic anhydride w a s a l s o used as a so lven t f o r oxazepam, g iv ing comparable r e s u l t s .

6.7 Polarographic Analysis

The polarographic behavior of oxazepam has been discussed i n many papers. Fazzar i and Riggleman350btained wel l -def ined ca thodic waves a t t h e dropping-mercury elec- t rode i n a mixture of methylene chloride-methyl a lcohol wi th a support ing e l e c t r o l y t e of 0.1M tetraethylammonium bromide. The halfwave p o t e n t i a l of oxazepam i n t h i s system i s about -1.02V and t h e d i f f u s i o n cu r ren t i s l i n e a r wi th concentrat ion. This method w a s appl ied t o capsules of oxazepam.

Oelschlager and coworkers36 a l s o found a l inear

451


r e l a t i o n between concent ra t ion and d i f f u s i o n cu r ren t i n an a c e t a t e bu f fe r system conta in ing 20% dimethylformamide. This procedure w a s used t o assay t a b l e t s containing oxazepam. They a l s o co r re l a t ed half-wave p o t e n t i a l versus a sa tu ra t ed calomel e l ec t rode , wi th pH as shown below. The pH w a s determined i n a Britton-Robinson bu f fe r conta in ing 20% dimethylformamide.

PH 2.3 3.4 5.1 6.0 7.2 8.2 9.2 E % ( V ) -,695 -.825 -.955 -1.005 -1.08 -1.125 -1.185

Oeschlager and coworkers37 f u r t h e r i nves t iga t ed the e lec t rochemis t ry of oxazepam and found t h a t i n ac id b u f f e r s it i s reduced wi th t h e uptake of 4 e l e c t r o n s t o form 7- chloro-5-phenyl-1,3,4,5-tetrahydro-2~-1,4-benzodiazepin- 2-one a t the dropping mercury e lec t rode . However, i n a l k a l i n e b u f f e r s t he uns t ab le 4 ,5 dihydro d e r i v a t i v e which i s formed f i r s t by consuming 2 e l e c t r o n , reacts f u r t h e r forming a c y c l i c aldehyde-ammonia adduct which undergoes reduct ion t o form the same product as is formed i n a c i d i c so lu t ion .

6.8 Chromatographic Analysis

Many of the more common chromatographic technique6 have been appl ied t o oxazepam.

6.81 Paper Chromatography

Oxazepam has been chromatographed on Whatman %1 paper wi th e i t h e r butanol , e thanol , water (17:3:20) upper shase o r butanol , pyr id ine , water (6:4:3) as the e luan t 2.

6.82 Thin Layer Chromatography

The var ious e l u a n t systems used f o r t h i n l a y e r chromatography on s i l i c a g e l p l a t e s f o r oxazepam are given i n Table V I I . Table V I I I g ives spray r eagen t s used f o r t he de t ec t ion of oxazepam on t h i n l a y e r chromatographs.

6.83 Gas Chromatography

Gas chromatography has been used t o analyze oxazepam. The necessary d a t a of the var ious methods are given as Table IX.

458

OXAZEPAM

!k 0.00 0.03

0.04 0.08

0.08 0.13 0.13

0.14 0.16 0-21

0.23 0.29 0.29 0-35 0.37 0.46 0.48 0.50 0.50 0.51 0.52 0.55 0.57 0.58 0.59 0.60 0.60 0.66 0.68 0.68 0.73

0.82 0.84

0.88

Table V I I

Eluant Reference Thin Layer Chromatography System for Oxazepam

chloroform: e t h a n o m l ) 27 cyc1ohexane:diethylamine:benzene (75:20:15) benzene:ethyl acetate (5:l) benzene: ethanol: ammonium hydroxide (95: 15: 5) chloroform: to1uene:methanol (10: 9: 1) heptane: chloroform: ethanol (10: 10: 1) ch1oroform:cyclohexane:diethylamine (40: 50: 10) to1uene:nitromethane:methanol (11: 8: 1) carbon tetrach1oride:methanol (90: 10) benzene:methanol:ammonium hydroxide

to1uene:diethylamine (80: 20) benzene: acet0ne:diethylamine (70: 20: 10) isopropanol: isopropyl ether (16: 84) ch1oroform:methanol (10: 1) ethanol :water ( 96: 4) methanol: acetone (12: 88) methanol :methyl acetate (18: 82) benzene: ethano1:diethylamine (5: 1:0.5) ch1oroform:ethanol: acetone (8:l: 1) cyclohexane: diethylamine (85: 17) carbon tetrach1oride:methanol (75: 25) acetone methanol : ammonium hydroxide (100: 1,5) chloroform: acetone:methanol (70: 20: 10) heptane: ch1oroform:ethanol (5:5:2) ethyl acetate: 1,2 dichloroethane (80: 20) isopropanol: ammonium hydroxide (20: 1) benzene: acetone:methanol (55: 35: 10) isopropano1:methanol (30: 70) ch1oroform:methanol: acetic acid (88: 10: 2) isopropano1:ammonium hydroxide:water (75:17:18) chloroform: acet0ne:diethylamine (50:40: 10) ethyl acetate:methanol:acetic acid (80: 20: 10) chloroform: acetic acid:methanol (15:114)

(9O:lO: 1)

40

41 40

42 43 44

42 44 13

44 43 45 42 45 45 45 46 41 44 44 40 40 44 47 44 43 44 44 44 44

44 44

41

459

CHARLES M. SHEARER AND CAESAR R . PlLLA

Sadee and Van d e r Kleijn38 found t h a t oxazepam rearranged t o the quinazol ine carboxaldehyde (F igure 8, St ruc tu re IV) during gas chromatography.

6.84 Column Chromatography

Sco t t and B ~ m m e r ~ ~ desc r ibe a h igh pressure l i q u i d chromatography system which w a s used f o r s eve ra l benzodiazepines, including oxazepam. The column was 100 cm. x 1 mm. i.d. s t a i n l e s s s teel packed wi th Waters Asso- c i a t e s Durapak rrOPN", 36-75 p m. p a r t i c l e diameter. The e luan t w a s a mixture of hexane and isopropanol. An u l t r a - v i o l e t de t ec to r w a s used.

Table V I I I TLC Spray Reagents f o r t h e Detect ion of Oxazepam

Color Light* Reference - Reagent

Bromine water Reinecke so lu t ion Iodine so lu t ion S u l f u r i c ac id Dragendorf f Chlor ine-o- toluidine Cerric sulfate-Dragendorff Diphenylcarbazone S i lve r a c e t a t e Mercuric s u l f a t e Cinnamaldehyde Furfura l reagent Zinc (11) chlor ide-

hydrochlor ic ac id 70% perch lo r i c ac id Cerium (IV) s u l f a t e Formaldehyde-hydrochloric

40% o-phosphoric ac id Antimony (111) chlor ide-

Van i l l i n - su l fu r i c ac id

ac id

a c e t i c ac id

Source orange v i s . 13 - r o s e brown yellow yellow v i o l e t red-or ang e l i g h t purp le blue-purple lavender b lue green

be ige yellow-orange yellow

l i g h t b lue 1 i g h t ye1 low l i g h t b lue yellow b lue yellow

I 1

11

II

I 1

I t

II

II

II

I 1

uv I t

II

v i s . uv 1 1

v i s . uv

v i a uv

v i s .

13 13 13 46 42 42 48 48 48 40 40

44 44 44

44 44 44 44 44 44

*vis. = v i s i b l e UV = u l t r a v i o l e t

460

Table I X

Column Packing

3% OV 1 on G a s Chrom Q (60180)

3% OV 1 7 on Gas Chrom Q (60180)

1.5% OV 1 on fi HP Chrom G z

3% OV 1 7 on Gas Chrom W

3% OV 1 7 on G a s Chrom W

G a s Chromatographic System f o r Oxazepam

Column

2 m, x 2 mm. g l a s s

4 f t . x 4 mm. g l a s s

3 f t . x 118 in . g l a s s

6 f t . x 118 in . s t a i n l e s s s tee l

6 it. x 118 in. s t a i n l e s s s teel

Carrier G a s Column Temperature

Nitrogen @ 22 ml./min.

245QC.

Argon-me thane ( 90 : 10) @ 100 mlJmin,

230OC.

H e l i u m @ 20 200 - 280°C. pro- m l . Imin. grammed

Nitrogen @ 70 ml./min.

245°C.

Argon-me thane ( 20 : 1 ) @ 70 ml./min.

245OC.

Detector Ref*

flame ioni - 49 za t ion

e l e c t r o n 50 capture

t o t a l ion 38 cu r ren t

flame ioni - 38 za t ion

e l ec t ron 38 capture


7. References 1. E. F. Salim, J. L. Deuble and G. Papa r i e l lo ,

J. Pharm. Sci. 2, 311 (1968). 2. J. Bal tazar and M. M. Ferreira Brager, Rev. Portug.

Farm. 1 7 , 109 (1967)) C - A = 67, 102845e. 3. F. R Fazzar i , M. F. Sharkey, C. A. Y a c i w and W. 1.

Brannon, J. A s s . Off ic . Anal. Chem., 5l,1154(1968). 4. N. B. Calthup, L. H. Daly and S. E. Wiberly, "In-

--

t roduct ion to In f r a red and Raman Spectroscopy," Academic Press, New York, 1964, Chapter 13. S. C. Bell and S. J. Chi ldress , J. Org. Chem., 27, 1691 (1962).

MOlecules," John Wiley 6 Sons, New York, 1954, Chapter 5.

7. B. Hofmann, Wyeth Laborator ies Inc., personal communication.

8. W. Sadee, Arch. Phann., 302, 769 (1969), C.A. 72, 95079f.

9. M. F. Sharkey, C. N. Andres, S. W. Snow, A. Major, T. Krm, V. Warner, T. G. Alexander, J. A s s . Of f iS Anal. Chem., 2, 1124 (1968).

personal commun i c a t ion.

5.

6. L. J. Be l lmy , "The Inf ra red Spectra of Complex

10. T. Chang and C. Kuhlman, Wyeth Laborator ies Inc.,

11. 12.

13.

14.

15.

16.

1 7 .

18.

19.

20 .

W. Sadee, J. Med. Chem., 13, 475 (1970). Po G. Stecher , Ed., "The Merck Index,'' Eighth Edi t ion , Merck & Co., Inc., Rahway, New Jersey, (19681, p. 772. L. D. Rodrigues and M. A. P. Alvee, Rev. Port . Farm 15, 309 (19651, C.A. 64L 11030f. F. Berte, L. Manzo, M. DeBernardi and G. Benzi, Farmaco (Pavia) Ed. P r a t . , 25, 1 7 7 (1970). N. DeAngelis, Wyeth Laborator ies Inc. , personal communication. L. H. Sternbach, S. Kaiser and E. Reeder, J. Am. Chem. SOC. 82, 475 (1960). S. C. B e l l , T. S. Sulkowski, C. Gochman and s. J.

Chi ldress , J. Org. Chem., 2, 562 (1962). F. D. Popp and A. C. Noble, Advan. Hererocycl ic m, 8, 21 (1967). S. J. Childress and M. I. Gluckman, J. Pharm. Sci . , 53, 577 (1964). L. H. Sternbach, Angew Chem. Intern. Ed., 2, 34 (1971).

.) -

-

462

OXAZEPAM

21. G, A. Archer and L. H. Sternbach, Chem. Rev., 68, 747 (1968).

22. S. S. Walkenstein, R. Wiser, C. H. Gudmundsen, H. B. K i m m e l and R. A. Corradino, J. Pharm Sci . , 53, 1181 (1964).

23. S. F. Sisenwine, C. 0. Tio, S. R. Shrader and H. W- Ruel ius , Arzneim. Forsch., 22, 682 (1972).

24. B. Grecu and S. Barbu, Farmacie (Bucharest) , l6 , 199 (1968) C. A. 69, 12,950x.

25. A. J. Khoury and L. J. C a l i , Ann. N. Y- Acad. Sci., 153, 456 (1968).

26. D. Halo t , Prod. Probl. Pharm., 25, 175 (19701, && - 73, 485672.

27. P. Lafargue, P, Pont and J. Meunier, Ann. Pharm. Franc., 28, 343 (19701, C. A. 73, 133950~.

28. G. Kamm and R. K e l m , Arzneim. Forsch., 2, 1659

29. H. Pe l ze r and D. Maass, w., 19, 1652 (19691, C. A. , 7 2 , 2082P.

30. J. Braun, G. Cai l le and E. A. Martin, Can. J. Pharm. Sc i . , 3, 65 (19681, Q., 3, 317184.

31. G, C a i l l e , J. Braun and J. A. Mockle, Can. J. Pharm. Sc i . , 5, 78 (19701, C. A., 74, 25020~.

32. J. Ste id inger and E. Schmid, Arzneim. Forsch., 2, 1232 (1970), C. A., 73, 129198g.

33. S. Lauffer , E. Schmid and F. Weist, i b id . , 2,1965 (19691, C. A = , 72, 47413~ .

34. K. H. Beyer and W. Sadee, Arch. Pharm., 2, 667 (19671, C. A., 68, 72284~.

35. F. R. Fazzar i and 0. H.Riggleman, J. Pharm. Sci . , 58, 1530 (1969).

36. H. Oelschlager , J. Volke, G. T. Lim and R. Spang, Arch. Pharm., 302, 946 (19691, C. A., 2, 93359 y.

37. H. Oelschlager, J. Volke, G. T. Lim and U. Bremer, i b i d 303, 364 (19701, c. A = , 2, 20844~. .* -

38. W. Sadee, E. Van der K le i jn , J. Pharm. sci., 60, 135 (1971).

39. C. G. Sco t t and P. Bommer, J. Chromatogr. Sci., 8, 446, (1970).

40. I. Zingales , J. ChromatoR., 31, 405 (1967). 41. F. J. DiCarlo and J. P. Viau, J. Pharm. Sci., 2,

322 (1970).

-

(19691, C - A * , 72, 20201f.

-

463


42.

43.

44.

45.

46.

47.

48.

49.

50.

H. D. Beckstead and S. J. Smith, Arzneim. Forsch., 18, 529 (1968)) C. A * , 69, 54319d. M. A. Schwartq B. A. Koechlin, E. Postma, S-Palmer and G. Krol , J. Pharmacol. E x p t l . Therap., 149, 423 (1965). F. R. Weist, Arzneim. Forsch., 2, 87 (19681, C . 0

- 68, 85880~ . E. Roder, E. Mutschler and H. Rochelmeyer, z. Anal. .) Chem - 244, 45 (1969). T. S. Glor ia , Rev. Fac. Farm. Bioquim. Univ. Sao Paulo, 2, 391 (1966), C. A., 67, 8 4 9 1 7 ~ . M. A. Schwartz, P. Bommer, and F. M. Vane, Arch. Biochem. Biophysics, 121, 508 (1967). K. K. K a i s t h a a n d J . H. J a f f e , J. Pharm. s c i . , 3, 679 (1972).

-

F. Marcucci, R. F a n e l l i and E. Mussini, J. Chroma- tog., 37, 318 (1968). J. A. F. deSi lva and C. V. P u g l i s i , Anal. Chem., 42, 1725 (1970). -

464

PHENAZOPYRIDINE HYDROCHLORIDE



INDEX

Analytical Profile - Phenazopyridine Hydrochloride 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 U1 traviolet Spectrum 2.4 Fluorescence Spectrum 2.5 Mass Spectrum 2.6 Optical Rotation 2.7 Melting Range 2.8 Differential Scanning Calorimetry 2.9 Thermogravimetric Analysis 2.10 Solubilities 2 , I1 X-ray Crystal Properties

3. Synthesis

4 . Stability Degradation


6. Methods of Analysis 6.1 Elemental Analysis 6.2 Phase Solubility Analysis 6.3 Thin Layer Chromatographic Analysis 6.4 Direct Spectrophotometric Analysis 6.5 Coulometric Analysis 6.6 Titrimetric Analysis

7. Acknowledgments

8 . References

466

PHENAZOPYRIDINE HYDROCHLORIDE

1. D e s c r i p t i o n

1.1 N a m e , Formula, Molecular Weight Phenazopyridine h y d r o c h l o r i d e is 2,6-diamino-3-

(pheny1azo)pyridine monohydrochloride.

Cl1HI1N5 * H C 1 Molecular Weight: 249.70

1 . 2 Appearance, Color , Odor

r e d , o d o r l e s s c r y s t a l l i n e powder.


Phenazopyridine h y d r o c h l o r i d e i s a l i g h t t o d a r k

2.1 I n f r a r e d Spectrum The i n f r a r e d spectrum of a sample of r e f e r e n c e

s t a n d a r d phenazopyridine h y d r o c h l o r i d e is shown i n F i g u r e 1 (1). The ins t rument used w a s a P e r k i n E l m e r Model 621 record ing spec t rophotometer . The sample was d i s p e r s e d i n minera l o i l i n o r d e r t o record t h e spectrum. The f o l l o w i n g assignments have been made f o r t h e bands i n F i g u r e 1 (1).

Band

3329 and 3275 2 5 c m - 1 3065 2 5 cm-1 1601 and 1500 5 5 c m - l 1638 If: 5 cm-1 815 2 5 cm-1

713 2 5 cm'l 682 5 c m - l

Assignment

N-H s t r e t c h a romat ic C-H s t r e t c h a romat ic r i n g v i b r a t i o n s NH2 deformat ions o u t of p l a n e deformat ion of

o u t of p l a n e bending of NH2 o u t of p l a n e deformat ions of

H o n p y r i d i n e r i n g

H on benzene r i n g

2.2 Nulear Magnetic Resonance Spectrum (NMR) The NMR spectrum of phenazopyridine h y d r o c h l o r i d e

is shown i n F i g u r e 2 ( 2 ) . t h e i n t e r n a l r e f e r e n c e w a s t e t r a m e t h y l s i l a n e and t h e

The s o l v e n t used was DMSO-d6,

467

a, 7)

.d k 0

4 0 k

TI & aJ E

'd

7

) 'rl U

6 8 8 E B (D

W 0

k

u

V

a, a

v)

a

a, k

(D k

W C

I4

f

33NV

lllWS

NV

Ul '10

468

I

469


solution concentration was 30.9 mg in 0.5 ml of solvent. The spectral assignments are given in Table I (2).

Table I

NMR Spectral Data for Phenazopyridine Hydrochloride

No. of Chemical Coup1 ing Pro ton Each Shift (ppm) Multiplicity Constant

Proton on C5 1 6.36

Protons meta and para to phenylazo

Protons ortho to phenylazo nitrogen 2 7.85-8.02

Proton on C4 1 8.29

nitrogen 3 7.25-7.65

Amino protons 4 -8.7

doublet J H ~ - H ~ = ~ O Hz

mu1 tip 1 e t

mu1 t iplet

doublet

singlet (broad)

2.3 Ultraviolet Spectrum The ultraviolet spectrum of phenazopyridine

hydrochloride (0.5 mg/100 ml of acidified 3A alcohol) in the region of 210-450 nm exhibits two maxima and three mini- m. The maxima occur at 238-240 nm (E = 2.2 x lo4) and 390-392 nm (E = 2.4 x lo4), while the minima are at 220 nm, 272 nm and 296 nm respectively (3). in Figure 3.

The spectrum is shown

2.4 Fluorescence Spectrum The excitation and emission spectra o f phena-

zopyridine hydrochloride (10 pg/ml in methanol) are shown in Figure 4 (4). recording spectrofluorometer. 341 nm produced an emission spectrum with a maximum at 380 nm.

The instrument used was a Farrand MK-1 An excitation wavelength of

2.5 Mass Spectrum The low resolution mass spectrum of phenazo-

pyridine is shown in Figure 5 (5). tained using a CEC 21-110 spectrometer w i t h an ionizing voltage of 70 eV, which was interfaced with a Varian data

The spectrum was ob-

470

47 1

F i g u r e 3

U l t r a v i o l e t Spectrum of Phenazopyridine Hydrochlor ide

W V z 4

(r

0 m

4

m

m

NANOMETERS

412

Figure 4

Fluorescence Spectra of Phenazopyridine Hydrochloride

t

u) z W t

t

z

NANOMETERS

L1

I

I I

I I

I I

I 3

0

0

0

0

1 (D

0

*

el

AllS

N3

1N

I 3

AllV

l3L

I

47 3

KENNETH W. BLESSEL, BRUCE C. RUDY, AND BERNARD 2.SENKOWSKI

system 100 MS. The d a t a system accepted t h e o u t p u t of t h e spec t rometer , c a l c u l a t e d t h e masses, compared t h e i n t e n s i - ties t o t h e b a s e peak and p l o t t e d t h i s in format ion as a series of l i n e s whose h e i g h t s were p r o p o r t i o n a l t o t h e i n - t e n s i t i e s .

The molecular i o n of t h e f r e e b a s e was measured a t m / e 213. m / e 214, corresponding t o (MSH), m / e 184, which cor responds t o t h e l o s s of HN2 from t h e molecular i o n , m / e 136, t h e l o s s of a phenyl r i n g from t h e p a r e n t mass, and m / e 108 which is t h e 2,6-diamino-pyridinium moiety ( 5 ) . A h i g h r e s o l u t i o n s c a n confirmed t h e r e s u l t s of t h e low r e s o l u t i o n

Other c h a r a c t e r i s t i c masses were observed a t

spectrum.

2.6

a c t i v i t y . 2.7

Opt ica l R o t a t i o n Phenazopyridine hydrochlor ide e x h i b i t s no o p t i c a l

Melt ing Range Phenazopyridine hydrochlor ide m e l t s wi th decom-

p o s i t i o n a t approximately 235OC when a class Ia procedure is used ( 6 ) .

2 .8 D i f f e r e n t i a l Scanning Calor imetry (DSC) The DSC s c a n of phenazopyridine h y d r o c h l o r i d e is

shown i n F i g u r e 6 ( 7 ) . Perk in E l m e r DSC-1B Calor imeter . The tempera ture program used w a s 10°C/min. i n an atmosphere of n i t r o g e n . t h e s e c o n d i t i o n s , t h e exothermic decomposi t ion o f phenazo- p y r i d i n e hydrochlor ide occurs a t approximately 239OC.

The curve 'kas o b t a i n e d w i t h a

Under

2.9 Thermogravimetric Analysis (TGA) The TGA s c a n showed no loss of weight as t h e

temperature was r a i s e d from ambient t o 115OC a t a rate of 10°C/min. ( 7 ) .

2.10 S o l u b i l i t y The s o l u b i l i t y d a t a obta ined f o r a sample of

r e f e r e n c e s t a n d a r d phenazopyridine h y d r o c h l o r i d e a t 25OC is shown in Table I1 (8). The e q u i l i b r a t i o n t i m e w a s 20 hours a t 25OC.

474

a, 'd

*rl k

0

rl c V

0

h a

3 a,

a, 3

k

1

u

475

Figure 6

DSC Curve €or Phenazopyridine Hydrochloride

TEMPERATURE "C


Table I1

Phenazopyridine Hydrochloride - S o l u b i l i t i e s

Solvent

3 A a lcohol benzene chloroform 95% ethanol d i e t h y l e the r 2 - propanol m e t hano 1 petroleum e t h e r (30°-600) water 1N H C 1 a c i d i f i e d 3 A a lcohol

S o l u b i l i t y (rnglml)

2.7 0.3 0.4 3.5 0.2 2.1 2.7 0.1 3.2 0.3 3.2

2.11 Crys ta l P rope r t i e s The x-ray powder d i f f r a c t i o n d a t a from phenazo-

pyr id ine hydrochlor ide is presented i n Table I11 ( 9 ) . The opera t ing condi t ions of t h e instrument are given below.

Instrument Conditions

General Electric Model XRD-6 Spectrogoniometer

Generator: Tube t a r g e t : Radiat ion: o p t i c s :

Goniometer: Detector:

50 KV, 12-112 MA Copper Cu Ka = 1.542 0.10 Detector s l i t M.R. S o l l e r s l i t 3' Beam s l i t 0.0007" Ni f i l t e r 4' t ake o f f angle Scan a t O.2O 28 per minute Amplifier g a i n - 16 coa r se ,

8.7 f i n e Sealed propor t iona l counter

tube and DC vo l t age a t p l a t eau

Pulse he igh t s e l e c t i o n EL - 5 v o l t s ;

Eu - Out

476

PHENAZOPYR I DINE HYDROCHLORIDE

Rate meter T.C. 4 2000 C/S f u l l scale

Recorder: Char t Speed 1 inch p e r 5

Samples: minutes

tempera ture Prepared by g r i n d i n g a t room

Table I11

I n t e r p l a n a r Spacings i n Phenazopyridine Hydrochlor ide from X-ray Powder D i f f r a c t i o n Data

d* - ** 28 - 28 - d* uIo- - 8.68 9.12 10.22 12.88 13.68 14.46 18.10 19.44 20.40 21. 82 22.10 23.06 24.06 25.74 26.38 27.10

10.20 6 27.88 3.20 9.70 30 29.28 3.05 8.66 84 30.64 2.92 6.87 10 31.32 2.86 6.47 8 32.84 2.73 6.13 100 34.38 2.61 4.90 20 36.06 2.49 4.57 32 36.58 2.46

4.07 25 40.02 2.25 4.02 21 41.14 2.19 3.86 10 42.32 2.14 3.70 10 43.78 2.07 3.46 32 47.00 1.93 3.38 85 48.75 1.87 3.29 46

4.35 19 37.58 2.39

UIo3 85 18 23 18 10 6 9 5 6 4 3 8 2 4 3

nA 2 Sin 8

*d = ( i n t e r p l a n a r spac ing)

**I/I,= re la t ive 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 of 100)

3. S y n t h e s i s Phenazopyridine may b e prepared by coupl ing benzene

diazonium c h l o r i d e w i t h a,a-diamino p y r i d i n e (10) .

4 . S t a b i l i t y Degradat ion Phenazopyridine has been found t o be s t a b l e i n d i s -

t i l l e d water and 0.1N sodium hydroxide when r e f l u x e d f o r one hour on a steam bath . Some d e g r a d a t i o n o c c u r s i n 0.1N

417

KENNETH W. BLESSEL, BRUCE C. RUDY, AND BERNARD A. SENKOWSKI

h y d r o c h l o r i c a c i d s o l u t i o n a t r e f l u x temperature . A f t e r one hour a t r e f l u x i t w a s found, by q u a n t i t a t i v e d e n s i t o - metry on a t h i n - l a y e r chromatographic p l a t e , t h a t 20-25% of t h e i n i t i a l amount of m a t e r i a l had degraded. The de- g r a d a t i o n products were ye l low b u t no i d e n t i f i c a t i o n w a s a t tempted.

5. Drug Metabol ic Products

has been s t u d i e d i n t h e r a b b i t and i n man (11). When a n o r a l dose of 600 mg w a s g iven t o humans, about 80% w a s e l i m i n a t e d i n t h e u r i n e w i t h i n 24 hours . Of t h i s amount, 7 .6% appeared as a n i l i n e , 19.9% as N-acetyl-p-aminophenol, 27.1% as p-aminophenol, 45.4% as t h e unchanged drug as w e l l as a t r a c e amount of o-aminophenol. Tr iaminopyr id ine a l s o was d e t e c t e d b u t n o t measured.

The metabol ic f a t e of phenazopyridine h y d r o c h l o r i d e


6 . 1 Elemental Analysis The r e s u l t s of an e lementa l a n a l y s i s of a sample

of r e f e r e n c e s t a n d a r d phenazopyridine hydrochlor ide are presented i n Table I V (12).

Table I V

Elemental Analys is of Phenazopyridine Hydrochlor ide

Element T h e o r e t i c a l % Found %

C 52.91 52.89 H 4.84 4.76 N 28.05 28.00 c1 14.20 14.23

6.2 Phase S o l u b i l i t y Analys is Phase s o l u b i l i t y a n a l y s e s have been c a r r i e d ou t

f o r phenazopyridine hydrochlor ide . An example i s shown i n Figure 7 (8) . The s o l v e n t used w a s methanol w i t h an e q u i l i b r a t i o n t i m e of 20 hours a t 25OC.

6 . 3 Thin Layer Chromatographic Analys is Phenazopyridine can b e d e t e c t e d i n AZO GANTRISIN

t a b l e t s u s i n g t h e fo l lowing TLC procedure. The t a b l e t s

478

Figure 7

5

I- 2

2 w 4 -

t o m m

I'

z g 2 - o m k l L

0 0 l - ' " E

2 3

g 0 3 '

s!

3 0

0

1 1 l ] l l l l ~ l l l l ~ l ~ l ~

-

- " .. " n A - 0 PHASE SOLUBILITY ANALYSIS

Sample : Phenazopyridine Hydrochloride - Solvent : Methanol

Slope: 0.0% Equilibration: 20 hrs at 25OC Extrapolated Solubility : 2.99 mg/g

-

I l r l l l r l l l r l l l l l l l l

KENNETH W. BLESSEL, BRUCE C. RUDY, A N D BERNARD 2. SENKOWSKI

are ground t o a powder and a p o r t i o n d i s s o l v e d i n ace tone . This i s a p p l i e d t o t h e p l a t e ( s i l i c a g e l GF) and developed f o r a t least 10 cm w i t h t h e s o l v e n t mixture chloroform: heptane:3A a l c o h o l (45:45:10) i n a paper - l ined , pre- s a t u r a t e d tank . as a yel low s p o t a t a n Rf of 0.4 (13).

Phenazopyridine can be d e t e c t e d v i s u a l l y

6.4 Direct Spectrophotometr ic Analys is The spec t rophotometr ic d e t e r m i n a t i o n o f phena-

zopyr id ine hydrochlor ide i n 1.ON H C l is, accord ing to t h e Nat iona l Formulary, t h e method of c h o i c e f o r t h e a s s a y of t h e b u l k drug (14). and t h e absorbance determined a t t h e maximum a t about 480 nm. The q u a n t i t y of phenazopyridine hydrochlor ide is c a l c u l a t e d by comparison wi th a sample of r e f e r e n c e s t a n - dard m a t e r i a l prepared and measured i n a s i m i l a r way.

The above method is s u b j e c t t o t h e d isadvantage of t h e low s o l u b i l i t y of phenazopyridine hydrochlor ide i n 1.ON HC1. The s u b s t i t u t i o n of a c i d i f i e d 3A a l c o h o l as t h e s o l v e n t would g i v e approximately a ten- fo ld i n c r e a s e i n t h e s o l u b i l i t y and eliminate t h e n e c e s s i t y of h e a t i n g t h e s o l u t i o n i n o r d e r t o d i s s o l v e t h e r e q u i r e d amount of phenazopyr id ine hydrochlor ide . The maximum i n t h i s s o l v e n t occurs about 390 nm.

The sample i s d i s s o l v e d i n 1 . O N H C 1

6.5 Coulometric Analys is Phenazopyridine hydrochlor ide may b e determined

c o u l o m e t r i c a l l y i n a n a c i d i f i e d water-acetone s o l u t i o n , u t i l i z i n g a mercury pool e l e c t r o d e . The sample is reduced a t a p o t e n t i a l of -0.40 v o l t s u n t i l t h e observed c e l l c u r r e n t drops t o 1/1000, of i t s i n i t i a l va lue . A b lank de- t e r m i n a t i o n is c a r r i e d o u t and any c o r r e c t i o n s made. Each coulomb of e l e c t r i c i t y is e q u i v a l e n t t o 646.9 mcg of phenazopyr id ine hydrochlor ide (6) .

6.6 Titr imetr ic Analys is Phenazopyridine hydrochlor ide may b e assayed by

a p o t e n t i o m e t r i c t i t r a t i o n w i t h HClO4. solved i n water which i s made b a s i c w i t h 10% NaOH and t h e l i b e r a t e d base i s e x t r a c t e d i n t o c h l o r o f o r m . I t i s then t i t r a t e d p o t e n t i o m e t r i c a l l y w i t h 0.01N HClO4, i n dioxane, u s i n g a glass-calomel ( s l e e v e type) e l e c t r o d e combination. Each ml of 0.01N HClO4 i s e q u i v a l e n t t o 2.497 mg of phenazopyridine hydrochlor ide (15).

The sample i s d i s -

480

PHENAZOPYR I DI NE HYDROCHLORIDE

7. Acknowledgments

L i t e r a t u r e Department and t h e Research Records Off i ce of Hoffmann-La Roche Inc. f o r a s s i s t a n c e i n t h e l i t e r a t u r e search f o r t h i s Analy t ica l P r o f i l e .

The au thors wish t o acknowledge the S c i e n t i f i c


8 . References

1.

2.

3 .

4 .

5 .

6 . 7.

8.

9.

10.

11.

12.

13.

1 4 .

1 5 .

Hawrylyshyn, M., Hoffmann-La Roche Inc., Personal Communication. Johnson, J. H., Hoffmann-La Roche Inc., Personal Communication. Rubia, L. B., Hoffmann-La Roche Inc., Personal Communication. Boatman, J. , Hoffmann-La Roche Inc., Personal Communication. Benz, W., Hoffmann-La Roche Inc., Personal Communication. The National Formulary XIII, pp. 538-540 (1970). Moros, S. , Hoffmann-La Roche Inc., Personal Communication. MacMullan, E. , Hoffmann-La Roche Inc., Personal Communication. Hagel, R. , Hoffmann-La Roche Inc. , Personal Communication. Chichibabin, Zeide, J. Russ. Phys. Chern. SOC., 46, 1216 ( 1 9 1 4 ) . Johnson, J. W. and Burba, J., Federation Proc.. g, 734 ( 1 9 6 6 ) . Scheidl, F. , Hoffmann-La Roche Inc. , Personal Communication. Sokoloff, H., Hoffmann-La Roche Inc., Unpublished Results . The National Formulary XIII, First Supplement, p. 24 (1970). Senkowski, B., Hoffmann-La Roche Inc., Unpublished Results.

482

PHENY LEPHRINE HYDROCHLORIDE

Charles A . Gaglia, Jr.

Reviewed by E. L. Pratt and L. Chafetz

CHARLES A. GAGLIA. JR.

CONTENTS

A n a l y t i c a l P r o f i l e - P h e n y l e p h r i n e H y d r o c h l o r i d e

1 . Desc r ip t ion 1 . 1 Name, F o r m u l a , Molecular Weight 1 . 2 Appearance , C o l o r , O d o r , T a s t e

2 . P h y s i c a l P r o p e r t i e s 2 . 0 1 M e l t i n g Range 2 . 0 2 S o l u b i l i t y 2 . 0 3 pK 2 . 0 4 O p t i c a l R o t a t i o n 2 .05 U l t r a v i o l e t S p e c t r u m 2 . 0 6 I n f r a r e d S p e c t r u m 2 . 0 7 N u c l e a r M a g n e t i c Resonance S p e c t r u m 2 . 0 8 Mass S p e c t r u m 2 . 0 9 D i f f e r e n t i a 1 The rma l A n a l y s i s 2 . 1 0 Thermal G r a v i m e t r i c A n a l y s i s

3 . S y n t h e s i s 4 . S t a b i l i t y - D e g r a d a t i o n 5. D r u g M e t a b o l i c P r o d u c t s 6 . Methods of A n a l y s i s

6 . 1 Di rec t S p e c t r o p h o t o m e t r i c A n a l y s i s 6 . 2 Co 1 o r i me t ri c Ana l y s i s

6 . 2 1 I n d o p h e n o l Dye 6 . 2 2 C o u p l i n g w i t h p - N i t r o a n i l i n e 6 . 2 3 C o u p l i n g w i t h 4 - A m i n o a n t i p y r i n e 6 . 2 4 C o m p l e x a t i o n 6 . 2 5 C o u p l i n g w i t h Nitrous Acid 6 . 2 6 I d e n t i f i c a t i o n 6 . 2 7 Other Methods

6 . 3 C h r o m a t o g r a p h i c Methods o f A n a l y s i s 6 . 3 1 P a p e r C h r o m a t o g r a p h y 6 . 3 2 T h i n L a y e r C h r o m a t o g r a p h y 6 . 3 3 L i q u i d - L i q u i d C h r o m a t o g r a p h y 6 . 3 4 Gas C h r o m a t o g r a p h y 6 . 3 5 Ion Exchange C h r o m a t o g r a p h y

6 . 4 Spe c t ro f 1 u o r ome t r i c a n d P hosp h or i met r i c A n a l y s i s

6 . 5 Other Methods o f A n a l y s i s 7 . R e f e r e n c e s

484


1 . D e s c r i p t i o n

1 . 1 Name, Formula , M o l e c u l a r Weight P h e n y l e p h r i n e H y d r o c h l o r i d e i s l-rn-Hy-

d r o xy -a- [ ( me t h y 1 am i n o ) me t h y 1 ] be n zy 1 a 1 co h o 1 h y - d r o c h l o r i d e ( 1 ) . I t i s a l s o known a s l - a - h y d r o x y - B -me t hyl a m i no- 3 -hyd roxy -1 -e t h y 1 benzene hydro - c h l o r i d e ; m-methylaminoethanolphenol h y d r o c h l o - r i d e ; Neo S y n e p h r i n e h y d r o c h l o r i d e ; Meta-Syn- e p h r i n e h y d r o c h l o r i d e ; A d r i a n o l ; rn-Sympatol ; Meta-Sympatol ; Neophryn; I s o p h r i n H y d r o c h l o r i d e ; O f t a l f r i n e ; L e x a t o l ( 2 ) .

O H CsHl4ClN02 Mol. W t . 203 .67

1 . 2 A p p e a r a n c e , C o l o r , Odor , T a s t e White o r n e a r l y w h i t e , o d o r l e s s c r y s t a l s

h a v i n g a b i t t e r t a s t e .


2.01 M e l t i n g Range P he n y 1 e p h r i n e H C1 140 - 145°C ( 1 ) * - .

139 - 143°C ( 3 ) P heny 1 eph r i n e b a s e 170 - 177°C ( 1 ) "

170 - 171OC ( 3 ) * U S P S p e c i f i c a t i ons

2 .02 S o l u b i l i t y F r e e l y s o l u b l e i n water and i n a l c o h o l

2 . 0 3 pK pK, = 8 . 7 7 ( 4 ) p K 2 = 9 . 8 4

2 . 0 4 O p t i c a l R o t a t i o n [a];' = -46 .2 t o - 4 7 . 2 " ( c = l ) ( 2 )

485

Fig. 1 Phenylephrine hydrochloride - IR spectrum o f 1 3 mm. KBr p e l l e t from 1 mg. drug dispersed in 200 mg. KBr - Instrument: Perkin-Elmer 621

PHENYLEPHRINE HYDROCHLORIDE

2 . 0 5 U l t r a v i o l e t S p e c t r u m S o l u t i o n X max. n m .

0 . 0 5 N HC1 216 2 7 4 2 7 9 s

0 . 0 5 N N a O H 2 39 2 9 2 . 5

I s o s b e s t i c 2 2 2 . 5 P o i n t s 2 6 0

2 7 8 . 5

x 1 0 - 3

5 . 9 1 1 . 8 1 1 . 6 5 8 . 9 5 3.04 4 . 2 8 6 . 6 6 1 . 6 7

2 . 0 6 I n f r a r e d S p e c t r u m ( F i g u r e 1 ) The s p e c t r u m i n F i g u r e 1 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 6 2 1 , I n f r a r e d S p e c t r o p h o t o - meter, A 1 3 m m . KBr p e l l e t c o n t a i n i n g 1 mg. p h e n y l e p h r i n e HC1 a n d 2 0 0 mg. KBr was u s e d . Char- a c t e r i s t i c b a n d a s s i g n m e n t s a r e l i s t e d b e l o w .

B,and cm” A s s i gnmen t

3 4 2 0 , 3 4 5 0 2 4 0 0 - 2 8 0 0 1 5 9 0 1 2 7 0 1 0 8 0

9 0 0

780

6 9 0

- O H N H* a r o m a t i c C-0 s t r e t c h a r o m a t i c C-0 s t r e t c h s e c o n d a r y a l c o h o l a r o m a t i c o u t o f p l a n e b e n d s i n g l e

a r o m a t i c o u t o f p l a n e b e n d meta

a r o m a t i c o u t o f p l a n e b e n d m e t a

CH

d i s u b s t i t u t e d

d i s u b s t i t u t e d

2 . 0 7 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 u m S o l v e n t : DMSO I n s t r u m e n t : V a r i an A60 C o n c e n t r a t i o n : A p p r o x i m a t e l y 8%

48 I


P h e n y l e p h r i n e H y d r o c h l o r i d e ( F i g u r e 2 ) ppm. ( f r o m TMS) I n t e g r a l I d e n t i f i c a t i on

2 . 6 ( S ) 3 N - C H 3

2 . 8 - 3 . 2 ( M ) 2 C H 2 - N 1 CH 1 CHOH 4 a r o m a t i c

27 [!I* 6 . 7 - 7 . 5 ( M ) 9 - 9 . 8 ( B ) * 3 p h e n o l i c O H ,

NH':

P h e n y l e p h r i n e Base ( F i g u r e 3 )

ppm. ( f r o m TMS) I n t e g r a l I d e n t i f i c a t i o n 2 . 4 ( S ) 3 N - C H 3

2 . 5 5 ( D ) 2 CH 2 - N

4 . 5 ( T ) 1 C H 4 . 8 - 5 . 8 (B)* 3 OH ( 2 ) , NH 6 . 5 - 7 . 5 ( M ) 4 a r o m a t i c * D i s a p p e a r on D20 e x c h a n g e

2 . 0 8 Mass S p e c t r u m The low r e s o l u t i o n mass spectrum was

d e t e r m i n e d on an MS 902 mass s p e c t r o m e t e r . The s a m p l e was i n t r o d u c e d by a h e a t e d d i r e c t i n s e r - t i o n p r o b e a t a t e m p e r a t u r e o f 150°C f o r t h e h y - d r o c h l o r i d e a n d o f 1 0 0 ° C f o r t he b a s e .

Mass No.

167 1 4 8 1 3 3 1 2 1 1 0 7

9 5 77 65 44 3 8 36

I n t e n s i t y HC1

1 0 2 1 5 2 4 8 4

1 0 0 8

26

I n t e n s i t y Base 38

6 4

l o 1 0 20 1 5 3 0

100 ---

488

Fig. 2 Time averaged NMR spectrum o f phenylephrine hydrochloride, 8% i n DMSO - Instrument: Varian A-60

n

W

v)

l-0 o F i g . 3 Time averaged N M R spectrum o f pheny lephr ine base , a b o u t 8% i n

DMSO - Ins t rument : Varian A - 6 0


2 .09 D i f f e r e n t i a1 T h e r m a l A n a l y s i s

Onse t o f Me1 t i n g Peak E n d o t h e r m

P h e n y l e p h r i ne HC1 142°C 144°C P h e n y l e p h r i ne Base 172°C 174°C

2 .10 Therma l G r a v i m e t r i c A n a l y s i s No s i g n i f i c a n t w e i g h t l o s s u n t i l decom-

p o s i t i o n a t 230°C f o r p h e n y l e p h r i n e HC1.

3. S y n t h e s i s

L e g e r l o t z was t h e f i r s t t o p r e p a r e p h e n y l - e p h r i n e h y d r o c h l o r i d e b y t h e h y d r o g e n a t i o n o f m- hydroxy-w-methylamino-acetophenone i n t h e p r e s - ence o f c o l l o i d a l p a l l a d i u m ( 5 ) . Bergmann and S u l z b a c h e r ( 6 ) s y n t h e s i z e d p h e n y l e p h r i n e by t r e a t i n g 5-(3'-benzyloxyphenyl)-3-methyl-2-oxa- z o l i d o n e w i t h 40% h y d r o c h l o r i c a c i d s o l u t i o n . R u s s e l l and C h i l d r e s s ( 7 ) a c h i e v e d t h e same end by r e f 1 u x i n g 3 - b e n z y l o x y - w - m e t h y l mandel ami de w i t h L i A1H4 i n t e t r a h y d r o f u r a n (THF) t o p r o d u c e 3 - b e n - z y 1 o x y - a-me t h y 1 ami n o-me t h y 1 b e n z y 1 a1 coh o l h y d r o - c h l o r i d e . T h i s compound was t h e n h y d r o g e n a t e d i n t h e p r e s e n c e o f 5% p a l l a d i u m - C c a t a l y s t u n t i l one e q u i v a l e n t o f H i s consumed. The h y d r o g e n a t i o n o f 3-benzyloxy-a-methylamino-methyl b e n z y l a l c o h o l was a l s o u s e d b y B r i t t e n ( 8 ) and, mos t r e c e n t l y , R i z z i ( 9 ) as t h e l a s t s t e p s o f t h e i r s y n t h e s e s o f p h e n y l e p h r i n e .

4 . S t a b i l i t y - D e g r a d a t i o n

P h e n y l e p h r i n e h y d r o c h l o r i de i s s t a b l e as a s o l i d . The d e g r a d a t i o n o f aqueous s o l u t i o n s has been s t u d i e d b y E l - S h i b i n i e t aZ. ( 1 0 , l l ) . The compound i s s t a b l e b e l o w pH 7 . Above pH 7 , d e g - r a d a t i o n o c c u r s and a p p a r e n t l y i n v o l v e s t h e s i d e c h a i n w i t h l o s s o f t h e s e c o n d a r y amine f u n c t i o n . The p h e n o l i c g r o u p r e m a i n s i n t a c t . The decompo- s i t i o n p r o d u c t s have n o t been i d e n t i f i e d b u t 5 - h y d r o x y - N - m e t h y l i n d o x y l has been p r o p o s e d . The p r e s e n c e o f h e a v y m e t a l s , p a r t i c u l a r l y c o p p e r was f o u n d t o c a t a l y z e t h e d e c o m p o s i t i o n .

49 1

CHARLES A. GAGLIA, JR

T r o u p and Mitchner (12 ) c h a r a c t e r i z e d t h e a c e t y l a t i o n of phenylephrine i n t h e presence o f a s p i r i n . The r e a c t i o n i s appa ren t ly a c c e l e r a t e d by t he presence of phenylephrine base a n d t h e a v a i l a b i l i t y of a c e t a t e . The amine g r o u p a c e t y - l a t e s p r e f e r e n t i a l l y . The hydroxyl g r o u p s become a c e t y l a t e d a f t e r prolonged exposure t o a s p i r i n .

Luduena e t aZ. ( 1 3 ) s t u d i e d t h e e f f e c t o f u l t r a v i o l e t i r r a d i a t i o n on phenylephr ine s o l u - t i o n s . Epinephrine was i d e n t i f i e d a s t h e product. Luduena p o s t u l a t e d t h e ep inephr ine could f u r t h e r r e a c t t o produce o t h e r compounds. The f i n d i n g s of West a n d Whi t te t ( 1 4 ) suppor t t h i s p o s t u l a t e .

Schrif tman ( 1 5 ) found from 1 2 t o 28% decompo- s i t i o n of unbuffered phenylephr ine s o l u t i o n s i n one week a t var ious t empera tu res . He a l s o found u p t o f i v e decomposi t ion products . The secondary amine f u n c t i o n was absent in a t l e a s t one of t he products .

Broadly a n d Roberts ( 1 6 ) found a second com- p o u n d p r e s e n t i n s t r o n g ac id (10 N hydroch lo r i c a c i d ) s o l u t i o n s of phenylephr ine . The compound was n o t i d e n t i f i e d .

Misgen ( 1 7 ) determined t h e phys ica l compati- b i l i t y of phenylephrine with twenty-seven common in t ravenous admixtures . He found on adding a s o l u t i o n o f phenylephrine t o a s o l u t i o n of d i l a n - t i n sodium a p r e c i p i t a t e formed w i t h i n two h o u r s .

be s t a b l e i n b rown g l a s s b o t t l e s , g ive 1 % decom- p o s i t i o n a f t e r 11 days in low d e n s i t y polye thylene b o t t l e s a n d decompose t o 81% o f i n i t i a l when s t o r e d f o r 130 days i n nylon b o t t l e s .

P e t r a g l i a a n d Dick ( 1 9 ) r epor t ed t h e s t a b i l i - z a t i o n of phenylephrine s o l u t i o n s t o s u n l i g h t by a d d ’ i n g 0 . 2 % sodium m e t a b i s u l f i t e a n d 0 .1% t a r t r a - z i n e .

F a g a r d ( 1 8 ) found phenylephr ine s o l u t i o n s t o

E l -Shib in i e t aZ. ( 1 1 ) i n d i c a t e d t h e s t a b i -

492


l i z i n g e f f e c t o f E D T A on b a s i c s o l u t i o n s o f p h e n y l e p h r i ne.

Kisbye and Bols ( 2 0 ) found t h a t n o r a c e m i z a - t i o n o f p h e n y l e p h r i n e o c c u r s a s a f u n c t i o n o f p H . P r a t t ( 2 1 ) found p h e n y l e p h r i n e o p t i c a l l y s t a b l e i n s o l u t i o n s a t pH 3 . 0 and 6 .0 when r e f l u x e d f o r 3 h o u r s .

Fourneau et aZ. ( 2 2 ) r e p o r t e d t h e r e a c t i o n o f p h e n y l e p h r i n e w i t h a l d e h y d e s u n d e r " p h y s i o l o g i c a l c o n d i t i o n s " t o produce a m i x t u r e o f 4 , 6 - and 4 , 8 - d i hydroxy-2-methyl -1 y 2 , 3 , 4 - t e t r a h y d r o i s o q u i n o - 1 i n e s .

5. D r u g M e t a b o l i c P r o d u c t s

t h e m e t a b o l i s m o f p h e n y l e p h r i n e . Bruce ( 2 3 ) r e - p o r t e d p h e n y l e p h r i n e i s e x c r e t e d i n u r ine a l m o s t e n t i r e l y a s t h e s u l f a t e c o n j u g a t e . Bruce r e - p o r t e d 82% a v e r a g e t o t a l r e c o v e r y o f p h e n y l - e p h r i n e from a t a b l e t f o r m u l a t i o n i n 24 h o u r s . T y p i c a l u r ine sample c o n t a i n e d from 86 t o 98% o f t h e r e c o v e r e d p h e n y l e p h r i n e a s t h e s u l f a t e con- j u g a t e .

Bogner and Walsh ( 2 4 ) , and C a v a l l i t o e t aZ. ( 2 5 ) showed b l o o d l e v e l and u r i n e e x c r e t i o n p a t - t e r n s f o r p h e n y l e p h r i n e h y d r o c h l o r i d e and p h e n y l - e p h r i n e t a n n a t e . Thei r work i n v o l v e d tr i t ium l a b e l e d d r u g . M e t a b o l i c p r o d u c t s were n o t i d e n - t i f i e d .

R e l a t i v e l y l i t t l e work has been r e p o r t e d on

R u b i n and Knot t ( 2 6 ) r e p o r t e d a f l u o r o m e t r i c

Dombrowski and P r a t t ( 2 7 ) r e p o r t a p r o c e d u r e

p r o c e d u r e f o r d e t e r m i n i n g p h e n y l e p h r i n e i n s e r u m .

f o r d e t e r m i n i n g u n m e t a b o l i z e d p h e n y l e p h r l n e i n p lasma.

6 . Methods o f A n a l y s i s 6 . 1 D i r ec t S p e c t r o p h o t o m e t r i c A n a l y s i s

The u l t r a v i o l e t a b s o r p t i o n band a t 2 7 2

493

CHARLES A. GAGLIA, JR.

nm. i s due t o t h e p h e n o l i c s t r u c t u r e . The a b - s o r b a n c e caL be u s e d t o q u a n t i t a t e p h e n y l e p h r i n e d i r e c t l y (28 ,29 ,30 ) o r a f t e r e x t r a c t i o n ( 3 1 ) . S h i f t i n g t h e maxima t o 290 nm. i n b a s i c s o l u t i o n has a l s o been u s e d as a d i r e c t a s s a y as w e l l as a d i f f e r e n t i a l t e c h n i q u e t o q u a n t i t a t e p h e n y l - e p h r i n e ( 3 2 , 3 3 ) . O x i d a t i on o f p h e n y l e p h r i ne t o m - h y d r o x y b e n z a l d e h y d e a n d m e a s u r i n g a b s o r b a n c e a t 257 nm. a n d / g r 315 nm. i n a c i d i c o r n e u t r a l s o l u t i o n s o r a t 237 , 267 and 357 nm. i n b a s i c s o l u t i o n h a s a l s o been c a r r i e d o u t ( 3 4 ) . The o x i d a t i o n a l s o o f f e r s i n c r e a s e d s e n s i t i v i t y o v e r d i r e c t U . V . U . V . i s a common d e t e c t i o n t e c h n i q u e f o r t h i n l a y e r , p a p e r a n d c o l u m n c h r o m a t o g r a p h i c t e c h n i q u e s .

6 .2 Col o r i r n e t r i c A n a l y s i s P h e n v l e D h r i ne has been i d e n t i f i e d a n d

q u a n t i t a t e d b y a’ v a r i e t y o f c o l o r i m e t r i c t e c h - n i q u e s .

6 .21 I n d o p h e n o l Dye I n d o p h e n o l dye i s f o r m e d b y t h e

r e a c t i o n o f p-Me2NC6H4NH2C1 a n d K 3 F e ( C N ) 6 w i t h p a r a unsubs t i t u t e d p h e n o l s i n a1 k a l i n e medi a ( 3 5 36 ,37) . The dye r e s u l t i n g f r o m t h e r e a c t i o n w i t h p h e n y l e p h r i n e has an a b s o r b t i on maximum a b o u t 620 nm.

6.22 C o u p l i n g w i t h p - N i t r o a n i l i n e P h e n y l e p h r i n e may be c o u p l e d w i t h

d i a z o t i z e d p - n i t r o a n i l i n e i n a c i d s o l u t i o n ( 3 8 , 3 9 ) . The r e s u l t i n g compound i s made b a s i c and d e t e r m i n e d a t 495 nm.

6 .23 Coup1 i n g w i t h 4-Ami n o a n t i p y r i ne P - a m i n o a n t i p y r i n e i s a s e l e c t i v e

c o u p l i n g a g e n t f o r p h e n o l s w i t h t h e p a r a p o s i t i o n f r e e . The r e a c t i o n i s c a r r i e d o u t i n a l k a l i n e b u f f e r s o l u t i o n pH = 9 i n t h e p r e s e n c e o f K3Fe- (CN),. The r e s u l t i n g a b s o r b t i o n maximum a t 460 nm, i s q u a n t i t a t i v e f o r p h e n y l e p h r i n e (40,41 , 4 2 ) .

6 .24 C o m p l e x a t i o n P h e n y l e p h r i n e f o r m s c o m p l e x e s w i t h

494

PHENY LEPHR IN E HYDROCHLORIDE

v a r i o u s s u l f o p h t h a l e i n dyes i n n e u t r a l t o s l i g h t - l y b a s i c s o l u t i o n . The r e s u l t i n g complexes a r e t h e n e x t r a c t e d i n t o a p o l a r o r g a n i c s o l v e n t a n d t h e co l o r d e t e r m i n e d s p e c t r o p h o t omet ri c a l l y ( 4 3 , 4 4 )

6 . 2 5 C o u p l i n g w i t h N i t r o u s Acid When s o l u t i o n s o f P h e n v l e P h r i n e " .

a r e h e a t e d w i t h mercury s a l t s then c o u p l e d w i t h n i t r o u s a c i d , a red c o l o r d e v e l o p s . The peak a t 495 n m . has been used t o q u a n t i t a t e p h e n y l e p h r i n e ( 4 5 , 4 6 ) . D e t a i l e d s t u d i e s o f t h e r e a c t i o n c o n d i - t i ons have been c o n d u c t e d ( 4 7 , 4 8 ) .

6 . 2 6 I d e n t i f i c a t i o n P h e n y l e p h r i n e undergoes many c o l or

r e a c t i o n s . S e v e r a l schemes f o r i d e n t i f y i n g p h e n y l e p h r i n e a l o n e and i n t h e p r e s e n c e o f o t h e r d r u g s have been d e v e l o p e d ( 4 9 , 5 0 , 5 1 , 3 ) .

6.27 Other Methods Many o t h e r q u a n t i t a t i v e c o l o r re-

a c t i o n s have been k e p o r t e d ' i n t h e l i t e r a t u r e . The r e a c t i o n p r o d u c t w i t h i o d i c a c i d i s d e t e r - m i n e d a t 420 n m . ( 5 0 ) . P h e n y l e p h r i n e r e a c t s w i t h 2 , 6 - d i c h l o r o q u i n o n e i n n e u t r a l s o l u t i o n and i s d e t e r m i n e d a t 625 n m . ( 5 2 ) . The o x i d a t i o n o f p h e n y l e p h r i n e t o an a l d e h y d e f o l l o w e d by r e a c t i o n w i t h t h i o b a r b i t u r i c a c i d ( 5 3 ) o r 3 - m e t h y l b e n t o - t h i a z o l i n - 2 - o n e ( 5 4 ) i s a l s o q u a n t i t a t i v e . N i n - h y d r i n r e a c t s w i t h p h e n y l e p h r i n e t o p r o d u c e a p i n k c o l o r w i t h a m a x i m u m a b s o r b a n c e a t 440 n m .

6 .3 Chromatographic Methods o f A n a l y s i s

( 5 5 )

6.31 Paper Chromatography P a p e r chromatoqraDh.y has been used

t o i s o l a t e p h e n y l e p h r i n e f r o m - i t s d e c o m p o s i t i o n p r o d u c t s ( 1 5 , 5 6 ) and from o t h e r sympathicomi - m e t i c s ( 5 7 , 5 8 , 5 9 , 6 0 , 3 7 ) . T a b l e 6 .31 -1 summarizes t h e 1 i t e r a t u r e f o r p a p e r c h r o m a t o g r a p h i c s e p a r a - t i o n o f p h e n y l e p h r i n e .

495


TABLE 6.31 -1

Sol vent Method o f Sys tern V i s u a l i z a t i on R f x 100 Reference

A n i n h y d r i n 57 57

B d i a z o t i z e d p - 63 1 5 sul f a n i l i c d r a g e n d o r f f U . V . d e n i t o m e t r y

C n i n h y d r i n 67 56

D n o t a v a i 1 a b l e - - 60

E indophenol 1 0 37

A = n - b u t a n o l / a c e t i c a c i d / w a t e r 4:5:1

B = n - b u t a n o l / a c e t i c a c i d / w a t e r 5:l :3

C = p h e n o l c o n t a i n i n g 15% v / v 0 , I N H C l

D = b u t a n o l / t o 1 u e n e / a c e t i c a c i d / w a t e r 100 : 100: 50:50

E = benzyl a l c o h o l / a c e t i c a c i d / w a t e r 5:2:5

496


TABLE 6 . 3 2 - 1

S y s t e m

I

I 1

I 1 1

I V

V

V I

V I I

I

I 1

1 1 1

I V

V

M e t h o d o f R f x 100 D e t e c t i on R e f e r e n c e

5 s p r a y 1% I 2 i n 61 m e t h a n o l a n d / o r d r a g e n d o r f f ' s r e a g e n t

21 I1 61

I I 3 3 61

II 60 61

I1 45 61

- - p o t a s s i um 62 f e r r i c y a n i d e 0 .6% w/v i n 0 . 5 % w/v NaOH quan . U V . den- si tome t r y

50 n i n h y d r i n 63

S i l i c a Ge l P l a t e s C o a t e d w i t h D e v e l o p i n g S o l v e n t

0 . 1 NaOH c y c l ohexane /benzene / d i e t h y l a m i n e 75 :15 :10 ( v / v )

0.1 M NaOH

0 . 1 M NaOH

0 . 1 M KHSOI,

0 .1 M KHSOI,

m e t h a n o l

a c e t o n e

m e t h a n o l

9 5 % e t h a n o l

497


TABLE 6.32-1 (continued)

S i l i c a Gel Plates Coated with Developing Sol vent

VI c e l l u l o s e 2 5 0 1-1 n-butanol /acet i c acid/water 4 : l : 5 v / v organic phase as the developing s o l - vent

VII s i l i c a ge l G n-butanol l a c e t i c aci d/water 1 2 :1 :7 v / v organic phase as the developing solvent

498


6.32 T h i n L a y e r Chromatog raphy The t h i n l a y e r c h r o m a t o g r a p h i c R f

v a l u e s f o r p h e n y l e p h r i n e i n a number o f s o l v e n t sys tems a r e g i v e n i n T a b l e 6.32-1

6 .33 L i q u i d - L i q u i d Chromatog raphy P h e n y l e p h r i ne l e n d s i t s e l f r e a d i l y

t o l i q u i d - l i q u i d c h r o m a t o g r a p h y , The d i f f i c u l t y i n e x t r a c t i n g p h e n y l e p h r i n e f r o m aqueous s o l u - t i o n s has been u s e d t o a d v a n t a g e t o remove o t h e r compounds f r o m phen l e p h r i n e . L e v i n e and D o y l e (64 ,65) and Cox ( 6 6 7 p r e s e n t e d t h e t h e o r e t i c a l a s p e c t s o f l i q u i d - l i q u i d p a r t i t i o n s y s t e m s . T h e i r work i n c l u d e s t h e p a r t i t i on c o e f f i c i e n t s o f many d r u g s and i s p r e s e n t e d s o t h a t op t imum c o n - d i t i o n s f o r p a r t i c u l a r s e p a r a t i o n s may be s e l e c t - ed . T a b l e 6 .33 -1 summar izes t h e p r a c t i c a l a p p l i- c a t i ons o f 1 i qu i d - 1 i q u i d c h r o m a t o g r a p h i c s e p a r a - t i o n s o f p h e n y l e p h r i n e .

6 .34 Gas Chromatog raphy Gas c h r o m a t o g r a p h y has been u s e d

t o s e p a r a t e , i d e n t i f y and q u a n t i t a t e p h e n y l - e p h r i n e . A summary o f t h e gas c h r o m a t o g r a p h i c d a t a i s p r e s e n t e d i n T a b l e 6 .34-1 .

6 .35 I o n Exchange Chromatog raphy T a b l e 6 .35-1 summar izes t h e l i t e r -

a t u r e on i o n exchange s e p a r a t i o n o f p h e n y l e p h r i n e .

6 .4 S p e c t r o f l u o r o m e t r i c and P h o s p h o r i m e t r i c

p r o p e r t i e s . U d e n f r i e n d et Q Z . (80) r e p o r t e d 270 nm. as t h e w a v e l e n g t h o f e x c i t a t i o n w i t h 305 nm. b e i n g t h e w a v e l e n g t h o f e m i s s i o n . The f l u o r e s - cence o c c u r s i n aqueous a c i d s o l u t i o n w i t h a r e - p o r t e d s e n s i t i v i t y o f 0.04 pg . /m l . R u b i n and K n o t t ( 2 6 ) used a f l u o r o m e t r i c p r o c e d u r e t o d e t e r - mine p h e n y l e p h r i n e i n serum. W i n e f o r d n e r (81 ) d e t e r m i n e d p h e n y l e p h r i ne b y i t s p h o s p h o r e s c e n t p r o p e r t i e s a t l i q u i d n i t r o g e n t e m p e r a t u r e s i n e t h a n o l i c s o l u t i o n s . The w a v e l e n g t h s o f e x c i t a - t i o n a r e 290 and 2 4 0 nm. w i t h p h o s p h o r e s c e n c e

A n a l y s i s P h e n y l e p h r i n e has n a t i v e f l u o r e s c e n t

499


T A B L E 6 . 3 2 - 1

Sys tem

I

I 1

I 1 1

I V

V

V I

V I I

I

I 1

I 1 1

I V

V

Method o f R f x 1 0 0 D e t e c t i o n R e f e r e n c e

5 s p r a y 1 % I 2 i n 5 8 m e t h a n o l a n d / o r d r a g e n d o r f f I s

r e a g e n t

I I 21 5 8

I1 33 5 8

II 60 5 8

II 45 5 8

- - p o t a s s i um 59 f e r r i c y a n i d e 0 . 6 % w/v i n 0 . 5 % w/v NaOH quan. U V . d e n - s i t o m e t r y

50 n i n h y d r i n 60

S i l i c a Ge l P l a t e s C o a t e d w i t h D e v e l o p i n g S o l v e n t

0 . 1 M NaOH Cyc 1 o hex a n e / b e n z e n e / d i e t h y l a m i n e 7 5 : 1 5 : 1 0 (v /v )

0 . 1 M NaOH m e t h a n o l

0 . 1 M NaOH a c e t o n e

0 . 1 M K H S O k me t han o l

0 . 1 M K H S O k 95% e t h a n o l

500


T A B L E 6.32-1 (continued)

Silica Gel Plates Coated with Developing Solvent

V I c e l l u l o s e 250 1.1 n-butanol lacetic acidlwater 4 :1 :5 v/v organic phase as the developing solvent

V I I silica gel G n-butanollacetic acid/water 1 2 : 1 : 7 v / v organic phase as the developing s o l - vent

501

TABLE 6 . 3 3 - 1

Col umn S t a t i o n a r y S u p p o r t P h a s e Mobi l e P h a s e

c e l i t e 5 4 5 a c e t i c a c i d c h l o r o f o r m NaCL ( s a t ' d ) wash then

e t h e r e l u t i o n

c e l i t e 5 4 5 pH 5 . 8 b u f f e r c h l o r o f o r m pH 5.1 b u f f e r wash then e l u t e

w i t h 2 . 4 % v /v D E H P i n c h l o r o - form

Other Compounds Present Not I n t e r f e r i n g R e f e r e n c e

c o d e i n e 67 d e x t rometh o r p h a n phenyl p r o p a n o l amine

c h l o r p h e n i r a m i n e p h e n i r a m i n e p y r i l a m i n e doxy1 amine s u c c i n a t e t r i p e 1 ennamine ci t r a t e p h e n y l t o 1 oxamine

d i hydrogen c i t r a t e a s p i r i n

pheny l p r o p a n o l ami ne 6 8

d e x t rome t h o r p han H B r g l y c e r y l g u a i a c o l a t e a c e t a m i n o p h e n c h l o r p h e n i r a m i n e pheny l t o l o x a m i ne c i t r a t e a s p i r i n p h e n o l p h t h a l e i n p y r i 1 amine ma1 e a t e g u l f i i ;oxgzol e

HC1

HC1

romp en1 r a m i n e ma1 e a t e

Column Support

c e l i t e 545 acid washed

ul c e l i t e 545 aci d washed 0

w

c e l i t e 545 aci d washed

S t a t i o n a r y Phase

sodi u m bora te

T A B L E 6.33-1 (cont inued) Other ComDounds

s o d i u m bora te

var ious ac ids a n d bases

Mobile Phase

chloroform wash then ace ty l a t e and e l u t e ace ty- 1 a ted phenyl- ephri ne w i t h chloroform saponi fy

same a s above

c h 7 o rof orm wash e l u t e w i t h e thanol

Present Not I n t e r f e r i n g Ref e rence

codeine s u l f a t e 69 me t h a p y r i 1 en e H C1 pyr i lamine maleate d-amphetamine s u l f a t e

codeine phosphate 70 chl orpheni ramine

ma1 e a t e

chlorpheniramine 71

pyri lamine maleate codeine phosphate phenyl p r o panol ami ne

ma1 e a t e

H C 1

Method of Q u a n t i t a t i o n - U . V .

TABLE 6.34.1

I ns trumen t a1 Cond i t ions D e r i v a t i v e Reference

c o l . temp. 135"C, f low base 7 2 r a t e 30 m l . / m i n . i n l e t a c e t o n e p r e s . 31 p s i

c o l . temp. 135"C, i n l e t a c e t o n e 72 pres. 30 psi B- ioni - z a t i on d e t e c t o r

butanone

Column Condi t i ons

8 f t . , 3 m m . I .D. , SE- 30 1 .15% on gas chrom- P 100-140 mesh

6 f t . , 3 m m . I .D., QFI -0065 (Dow Corni n g ) 2.8% on chromsorb 60- 80 mesh

2 M g l a s s , 4 m m . I . D . , 0.1% s i l i c o n e o i l ( D C - 710) on 60-80 mesh d i m e t h y l - d i c h l o r o s i l a n e t r e a t e d g l a s s beads

VI 0 P

6 f t . , 4 mm. I.D., 10% F-60 (methyl p o l y s i l o - xane) on gas chrom-P 80-100 mesh

i n j . 300"C, d e t c . 260°C t r i f l u o r o - 73 a f lame i o n i z a t i o n d e t c . a c e t i c a c i d he1 i urn/hydrogen/ai r f low r a t e 80/80/450 m l J m i n . , resp. program col. 100°C t o 200°C a t l O " C / m i n .

d e t c . 300°C f lame i o n i - HMDS (hexa - z a t i on t e m p . program methyld i s i 1 - 100"C-2OO0C 8 1.5"C/ azane ) m i n . n i t r o g e n 12 psi a c e t o n e a i r 40 p s i , hydrogen cycl obutanone 20 psi

74

a = assayed t a b l e t s and s y r u p s

Res i n

Amberl i t e IR -45

Dowex

Dowex

Dowex

Dowex

Dowex

Dowex

Amberl i t e

50-X-1

50-X-2

50-X-8

50-X-12

50-X-16

50-W-X-1

I R 120

Type

w e a k l y b a s i c

s u l f o n i c a c i d

H+ f o r m

TABLE 6 .35-1

Method o f M o b i l e Phase Quan.

75% e t h a n o l t i t r a t i o n

w a t e r ash azo t h e n e u t e coup1 i ng w i t h 0.5 N M i l l o n ' s HCI r e a g e n t

Compounds P r e s e n t N o t I n t e r f e r i n g R e f e r e n c e

c o d e i n e phos - 75 p h a t e

g u a i a c o l - s u l f o n a t e

p o t a s s i um

camphor m e n t h o l

A P A P t heny 1 d i ami ne

c a f f e i n

39 H C 1

T A B L E 6.35-1

Resin Type Mobile Phase

Type A P01Y - g r a d i e n t pH

s u l f o n i c M t o 0 .37 M acid N H t N H b O H form

s t y r e n e 10-12 c r 0 . 1 5

(cont inued) Compounds

Method of Present Not Q u a n . I n t e r f e r i n g Re f e ren ce

U.Y. met anep h ri ne 76 p -synephri ne + 13 o t h e r com-

pounds

A G 5OW-X-4 s t r o n g 1 N HC1 i n 50% U . Y . codeine phosphate 77 ca t ion met ha no1 chl orpheni rami ne 78

maleate prome t h a z i ne HC1 methapyrilene HC1 dext romet h o r p h a n H B r

A1 g i n i c c a t i o n 0 . 0 1 N HC1 Acid

U . Y . p y r i l ami ne ma1 e a t e 79 codeine phosphate acetaminophen


o c c u r r i n g a t 390 n m .

6 .5 Other Methods o f A n a l y s i s A non-aqueous t i t r a t i o n o f p h e n . y l e p h r i n e

t o a c r y s t a l v i o l e t e n d p o i n t u s i n g p e r c h l o r i c a c i d i n d i o x a n e - a c e t i c a c i d medium h a s been r e - po r t ed ( 8 2 ) .

e p h r i ne u s i n g bromine w a t e r ( 8 3 ) and coul ometri - c a l l y g e n e r a t e d bromine ( 8 4 ) .

Bromi n a t i on has been used t o d e t e r m i n e phenyl-

T h e i n c r e a s e i n b lood pressure o f bo th r a t s and g u i n e a p i g s i s t h e b a s i s o f a b i o l o g i c a l a s s a y o f p h e n y l e p h r i n e ( 8 5 ) . S a l i c y l a m i d e , N - a c e t y l -p-aminophenol and c h l o r p h e n i ramine m a l e a t e d o n o t i n t e r f e r e . Sample s i z e s o f 1 . 5 t o 80 u g . have been d e t e r m i n e d .

I n t e r f e r e n c e r e f r a c t o m e t r y ( 8 6 ) has been u s e d a s a q u a n t i t a t i v e m i c r o m e t h o d o f p h e n y l e p h r i n e a n a l y s i s .

7 . R e f e r e n c e s

1 .

2 .

3 .

4 .

5 .

6 .

7 .

8.

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CHARLES A. GAGLIA, J R .

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3 -46

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R. C . D'Alessio d e C a r n e v a l e B o n i n o , Reu. A S S O C . Bioquirn. A r g . , 3 0 ( 1 5 7 ) , 115-20 ( 1 9 6 5 ) . H . Wachsmuth a n d L . van Koeckhoven , J . Pharm. BeZg., 1 7 ( 7 - 8 ) , 2 2 7 - 2 3 0 ( 1 9 6 2 ) , CA 61 : 5 4 6 0 d . P . M e s n a r d , G . Devaux a n d J . F a c q u e t , J . B U Z Z . SOC. Pharm. Bordeaux, 1 0 S ( l ) , 1 3 - 20 ( 1 9 6 5 ) , C A 6 3 : 1 2 9 7 5 e . M . P a y s and 0 . D a n l o s , Ann. Pharm. F r . , 2 5 ( 7 - 8 ) , 533-36 ( 1 9 6 7 ) . H . Wachsmuth and L . van Koeckhoven , J . Pharm. BeZg., 1 8 ( 5 - 6 ) , 2 8 0 - 8 3 ( 1 9 6 3 ) . K . J . B r o a d l e y and D . J . R o b e r t s , J . Pharm. PharmacoZ., 1 8 ( 3 ) , 1 8 2 - 8 7 ( 1 9 6 6 ) . R . P o h l o u d e k - F a b i n i and K . KSn ig , Pharmazie, 13, 1 3 1 - 3 5 ( 1 9 5 8 ) . H . C h . S k a l i ks, Arzne imi t t eZ -Forsch . , 7, 386-7 ( 1 9 5 7 ) . R . P o h l o u d e k - F a b i n i and K . Ki inig, Pharmazie, 1 5 , 61 -70 ( 1 9 6 0 ) . J . v a n Espen, MededeZ VZaam. Chem. V e r . , 15, 66-71 ( 1 9 5 3 ) , CA 4 7 : 1 1 6 6 1 ~ . W . W . F i k e , AnaZ. Chem., 3 8 ( 1 2 ) , 1 6 9 7 - 1 7 0 2 , ( 1 9 6 6 ) . N . H . C h o u l i s and C . E . C a r e y , J . Pharm.

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510


69 .

70 .

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72 .

73.

74.

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86.

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b e r 1 9 7 1 ) .

6 4 4 - 4 5 ( 1 9 6 8 ) .

511


Acknowledgment

The a u t h o r w i s h e s t o a c k n o w l e d g e t h e a s s i s t a n c e o f Dr. R. C . Greenough i n p r e p a r i n g and i n t e r p r e t i n g s p e c t r a l d a t a i n t h i s p r o - f i l e .

512

TOLBUTAMIDE

William F. Beyer and Erik H. Jensen

WILLIAM F. BEYER AND ERIK H. JENSEN

CONTENTS

1. Description 1.1. Name, Formula, Molecular Weight 1.2. Appearance, Color, Taste, Odor

2. Physical Properties 2.1. Solubility 2.2. Melting Range 2.3. Crystal Properties

2.31. Crystal Morphology 2.311. System and Class 2.312. Axial Ratio 2.313. Interfacial Angles 2.314. Habit

2.321. Refractive Indicies 2.322. Molecular Refraction 2.323. Optic Axial Angle 2.324. Dispersion 2.325. Optic Orientation 2.326. Common Crystal Orientation 2.327. Optic Sign

2.32. Optical Properties

2.33. Fusion Properties 2.34. X-Ray Diffraction

2.4. Infrared Spectrum 2.5. Nuclear Magnetic Resonance Spectrum 2.6. Mass Spectrum 2.7. Ultraviolet Spectrum 2.8. pKa 2.9. Differential Scanning Calorimetry

3. Synthesis 4 . Stability 5. Drug Metabolites 6 . Methods of Analysis

6.1. Elemental Analysis 6.2. Phase Solubility 6.3. Titr ime tric 6.4. Ultraviolet Spectrophotometric 6.5. Colorimetric

5 14

TOLBUTAMIOE

6.6. Gas Chromatographic 6.7. Liquid Chromatographic 6.8. Paper Chromatographic 6.9, Thin Layer Chromatographic 6.10. Coulometric

7. Pharmacokinetics and Toxicity 8. Identification 9. References Cited

515


1. Description

1.1. Name, Formula, Molecular Weight To 1 but amid e is 1 -But y 1 - 3- (p - to 1 y 1s ul f ony 1 ) urea '.

N-(4-methyl-benzenesulphonyl)- It is also referred to as: N'-n-butyl-urea2, to1 lsulfonylbutylurea~, 3- (p-tolyl-4-

butylurea3. and Rastinon; 14 additional are listed in the Merck Index 8th Edition3. Tolbutamide is a sulfonamide but it is not a sulfanilamide derivative,

sulfony1)-1-butylurea 3 , N-(sulfonyl-p-methylbenzene)-N'-n- The most commonly used trade marks are Orinase

0 - - S O 2 - $" - 4 C - N - CH3 I c4H9

1 2H 1 BN 2' 3' Mol. Wt. 270.35

1.2. Appearance, Color, Taste, and Odor

It has a slightly bitter taste and is Tolbutamide is a white, or practically white,

crystalline powder, practically odorless 1 . 2. Physical Properties

2.1. Solubility Practically insoluble in water but forms water-

soluble salts with alkalies. It is soluble in alcohol and in chloroform1. It is soluble to the extent of 7.8 mg/ml in toluene and 4.4 mg/ml in ethyl acetate:heptane (1:3)4.

516

TOLBUTAMIDE

2.2. Melt ing Range The melt ing range of tolbutamide has been r e -

ported a s 126-132'l and 128.5-129.5'3.

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

2.31. Crys t a l Morphology5

2.311. System and Class Orthorhombic, rhombic pyramidal,

MM2

2.312. Axial Rat io a:b:c = 0.4504: 1:0.3864

2,313. I n t e r f a c i a l Angles

(101) A ( io i ) = 810; (011) A ( o i l ) = 1380; ( i20)A ( i i o ) = 960

2,314. Habi t Tabular {OlO) wi th {lOl) , { O l l ) ,

[i20) . with the composition plane and r e e n t r a n t angles v i s i b l e .

Supplementary twinning is common, but u sua l ly

2.32. Opt ica l Proper t ies5

2,321. Ref rac t ive I n d i c i e s (5893A) Nx = 1.544; Ny = 1.550; Nz =

1.604; geometric mean = 1.562

2,322. Molecular Ref rac t ion Observed = 69.4; ca l cu la t ed = 70.4

2,323. Optic Axial Angle (58938) 2V = 38' ca l cu la t ed from

r e f r a c t i v e i n d i c i e s ; 42' us ing Mal la rd ' s cons tan t .

2.324. Dispersion 1: > v, s t rong .

517


2.325. O p t i c O r i e n t a t i o n a = Y ; b = X

2.326. Common Crys t a l Or i en ta t ion (010) showing centered obtuse

b i s e c t r i x i n t e r f e r e n c e f igu re .

2.327. Optic Sign Pos i t i ve

2.33. Fusion P rope r t i e s When tolbutamide i s melted and slowly

cooled, an uns tab le form c r y s t a l l i z e s which, upon r ehea t ing , slowly undergoes a s o l i d - s o l i d phase t ransformat ion t o the s t a b l e form5.

2.34. X-ray D i f f r a c t i o n Precession and Weissenberg photographs of

the X-ray d i f f r a c t i o n p a t t e r n of s i n g l e c r y s t a l s of USP Tolbutamide were made in a study t o update the powder d i f - f r a c t i o n f i l e 6 . The c r y s t a l s were found t o be orthorhombic, and systematic absences uniquely determined the space group a s P nma. The un i t c e l l parameters measured were i n good agreement with the o r i g i n a l determinat ion of Qhel l5 . The l abe l ing of the u n i t c e l l axes given by S h e l l has, however , been permuted t o agree w i t h t h e I n t e r n a t i o n a l Tables - fo r X-ray Crystal lography convention f g r space grgup P nma. The new c e l l dimensions a re : a = 20.14A; b = 9 . 0 7 ~ ; and c = 7.781.

The DIFMAX computer program was used t o a r r i v e a t the indexed r e f l e c t i o n s , 2 8 values , and d spacings of Table I. I n space group P nma, the f o l l m i n g condi t ions l i m i t poss ib le r e f l e c t i o n s :

0, k, l : k + 1 m u s t be even

h , k, o:h m u s t be even

Accordingly, d e l e t i o n s of s s t ema t i ca l ly absent r e f l e c - t i o n s were made i n Table I. g

518

TOLBUTAMIDE

TABLE I

Possible X-ray Diffraction Maxima for Tolbutamide

H

2 1 2 2 0 1 2 3 4 0 3 4 4 2 0 1 4 1 2 2 5 1 3 4 2 3 6 5 3 6 4 6 4 0 1 6

K

0 0 1 0 1 1 1 0 0 2 1 1 0 2 0 2 1 0 2 0 0 1 2 2 1 0 0 1 1 1 2 0 0 2 2 1

L

0 1 0 1 1 1 1 1 0 0 1 0 1 0 2 1 1 2 1 2 1 2 1 0 2 2 0 1 2 0 1 1 2 2 2 1

519

2 Theta

8.78 12.19 13.13 14.38 15.00 15.63 17.40 17.44 17.61 19.57 20.02 20.17 21.01 21.48 22.86 23.12 23.21 23.28 24.37 24.53 24.89 25.30 26.33 26.45 26.45 26.48 26.55 26.79 28.28 28.35 28.87 28.96 29.00 30.26 30.60 30.63

D

10.0608 7.2507 6.7332 6.1510 5.8998 5.6614 5.0893 5.0780 5.0304 4.5308 4.4299 4.3981 4.2231 4.1312 3.8864 3.8423 3.8278 3.8159 3.6480 3.6253 3.5737 3.5168 3.3807 3.3666 3.3659 3.3627 3.3536 3.3245 3.1526 3.1451 3.0893 3.0792 3.0755 2.9499 2.9187 2.9155

WILLIAM F. BEYER AND ERIK H . JENSEN

H K

4 1 2 3 2 2 0 3 5 2 5 0 1 3 2 3 3 2 7 0 6 2 5 1 3 3 4 3 7 1 I 0 6 2 4 2 6 0 8 0 2 0 0 1 1 1 4 3 6 1 8 1 2 1 3 0 8 0 5 2 1 3 3 1 2 3 7 2 8 1 7 0 5 3 4 0 0 4

TABLE I (Cont inued)

L

2 0 2 1 1 2 1 1 2 1 0 2 1 0 1 3 1 2 2 0 3 3 3 1 2 0 3 3 1 2 2 3 2 1 1 2 1 3 0

520

2 T h e t a

30.66 30.87 31.57 31.75 31.86 31.98 32.06 33.00 33.14 33.19 33.20 33.51 34.51 34.60 34.68 34.88 35.20 35.23 35.31 35.66 35.75 36.01 36.30 36.53 36.72 37.05 37.14 37.16 37.54 37.77 37.95 38.51 38.76 38.83 38.88 38.93 39.00 39.06 39.75

D

2.9123 2.8930 2.8307 2.8154 2.8059 2.7956 2.7883 2.7113 2,7002 2.6960 2.6955 2.6713 2.5960 2.5896 2.5841 2.5697 2.5467 2.5446 2.5390 2.5152 2.5091 2.4911 2.4722 2.4568 2.4448 2.4235 2.4181 2.4169 2.3930 2.3791 2.3683 2.3352 2.3206 2.3169 2.3137 2.3110 2.3069 2.3034 2.2654

TOLBUTAMIDE

Figure 1 gives the X-ray powder diffraction pattern for USP Tolbutamide obtained with a General Electric XRD-5 Diffractometer using C u K d 1, 50 KVP, 16 MA, and tray mount6,

2.4. Infrared Spectrum Tolbutamide can be identified bv means of its

infrared spectrum (Figure 2). The spectrLm of USP Tolbut- amide7 was obtained from a Nujol mull using a Perkin-Elmer Model 421 spectrophotometer. The principal peaks are assigned as follows: 3320, 3190 (urea, NH stretch), 2920, 2850 (alkane, CW stretch including Nujol), 1700, 1660 (urea, C=O stretch), 1600, 1500 (aromatic, C=C stretch), 1555 (urea, amide 11), 1460, 1375 (alkane, CH deformation including Nujol), 1335, 1160 (sulfonamide, S=O stretch), and 815 cm-1 (aromatic, CH deformation for para substi- tution). Other peaks occur at 1320, 1310, 1250, 1220, 1190, 1120, 1095, 900, 740, 725, and 660 cm-l.

2.5. Nuclear Magnetic Resonance Spectrum The nuclear magnetic resonance (NMR) spectrum of

tolbutamide was obtained using a Varian instrument Model A-60 D. Figure 3 gives the spectrum8. The tolbutamide was dissolved in deutero chloroform with tetramethylsilam as the internal reference. The NMR proton spectral assignments are given in Table I18.

2.6. Mass Spectrum Tolbutamide mass-spectral data are given in

Table III9. The data were obtained using an Atlas CH4 instrument. The l o s s of 64 mass units from the molecular ion is attributed to the loss of S02, a unique loss of these elements from the middle of the tolbutamide molecule. The molecular ion was observed at mfe 270. At 70 ev the most intense peak was observed at m/e 91 (15.4% of total ionization). This peak can be represented as a C7H7+ion which yields CgHg+(m/e 65; metastable at 46.4) upon expulsion of an acetylene molecule after decomposition.

521

i

0

0

m

0

0

b

00

00

00

00

06

o

ma

bu

,u

,a

n~

-

33NW

lllHISN

WM

l %

a, a

W

0

523

a \I

\-

Figure 3. Nuclear magnetic resonance spectrum of tolbutamide

TOLBUTAMIDE

TABLE I1

NMR Spectral Assignments for Tolbutamide

CH3-CgHq- - CH3-CH2-CH2-CH2-

-N_H-CH2-

-

H

CH3 QS

H

CH3 9 S

H

-02S-NK-CO-

Shape

Distorted Triplet

Broad Multiplet

Singlet

Quartet

Triplet

Apparent Doublet

Apparent Doublet

Broad Singlet

Chemical Shift J (Hz)

0.88 6.5

2.43

3.23

6.57

7.33

7.83

9.7

6.0 (av.)

6.0 (av.)

8.0 (av.)

8.0 (av.)

2.7. Ultraviolet Spectrum The UV spectrum of USP Tolbutamide is shown in

Figure 4 . The spectrum was obtained with a Cary 15 spectrophotometer using a 15 mcg/ml solution of tolbutamide in anhydrous ethanol. The spectrum, obtained with a 1-cm cell, shows A max at 228 nm.

525


0.3

WAVELENGTH (nm)

Figure 4 . Ultraviolet spectrum of tolbutamide

526

TOLBUTAMIDE

TABLE 111

Mass Spectral Data for Tolbutamide

% total ionization 19 ev ml e 70 ev

2 70 2.2 7.5 255 0.2 241 0.3 227 1.1 215 0.4 206 6.5 35.7 184 0.2 171 0.6 0.5 163 0.3 155 7.1 0.4 139 0.6 115 2.0 1.5 i o a 14.0 19.4 107 5.3 11.5 99 3.2 3.3 91 15.4 0.1 73 3.2 3.1 72 2.3 0.5 65 4.6 30 12.6 5.7

- -

2.8. p ~ a The pKa' of tolbutamide by two separate pro-

cedures was 5.43 at 25OC and 5.32 at 37.5OC10.

2.9. Differential Scanning Calorimetry The absolute purity of tolbutamide can be deter-

mined using differential scanning calorimetry. The purity of Tolbutamide USP Reference Standard with this technique was 99 12. A Perkin-Elmer Differential Scanning Calorimeter Model 1 - B was used at a scan speed of 1.25'1 min. at a sensitivity of 2 m callsec f u l l scale.

521


W. F. Beyer and E. H. Jensen

3. Synthesis The patent procedure for tolbutamide2 gives the

following example for its preparation: 50 gms of n-butyl isocyanate are stirred at RT into a suspension of 96 gms of sodium 4-methyl-benzenesulfamide in 120 ml of dry nitrobenzene, and the mixture is then heated for 7 hours at 100°C. After being cooled, the reaction mixture, which is a thick magma, is diluted with methylene chloride or ethyl acetate, and the sodium salt of the sulfonylurea formed is separated by centrifuging. The centrifuged crystalline residue freed from organic solvents is dissolved in 500-600 ml of water heated at 5OoC and decolor- ized with charcoal. The precipitate obtained by acidification with dilute hydrochloric acid is dissolved in an equivalent quantity of dilute ammonia solution (about 1:20), again treated with charcoal and reprecipitated with dilute hydrochloric acid. In this manner tolbutamide is obtained in analytically pure form in a yield of 70-80 percent of theory. Menzer et all3 list p-tolylsulfonamide and p-tolylsulfonylurea as the primary impurities to be expected in the synthesis of tolbutamide.

4 . Stability

common to antibacterial sulfonamides, tolbutamide cannot be acetylated. Its p-methyl group, however, renders tolbutamide susceptible to oxidation, occurring chiefly in biological systems.

Because of the absence of p-amino groups, which are

Thermal decomposition of tolbutamide has been reported by Menzer et al13, with reformation of p-tolylsulfonamide and synthesis of butylisocyanate. The latter then reacts with unconverted butylamine and ammonia to form N,N-dibutylurea and N-butylurea. The authors also isolated four additional by-products of tolbutamide, one identified as p-tolylsulfonylbiuret.

The hydrolysis of tolbutamide in an acid environment was investigated by Vogtl4 and in alkaline solution by Haussler and Hajduls. butamide to p-toluenesulfonamide and n-butyl isocyanate was reported by Ulrich and Sayighl6 to take place in inert solvent at 160 to 180°C. A significant degradation

A quantitative dissociation of tol-

528

TOLBUTAMIDE

occurred in some o/w creams when the drug wa dissolved in the oil phase of the emulsion at 70 to 80 C 17. No such loss occurred when the sulfonylurea was incorporated in the base at room temperature. The authors concluded that the instability of tolbutamide in the oil phase of the emulsion was due to components containing hydroxyl groups. The investigations were expanded to study the dissociation of tolbutamide at 80OC in twelve primary alcohols and in polyethylene glycol 40018. dissociate to give butylamine and p-toluenesulfonyl isocyanate. N-(p-toluenesulfonyl) carbamate, formed by the reaction of the sulfonylisocyanate with the alcohols, was present in the equilibrium mixture. Bottari et all9, in studies investigating the reaction products of tolbutamide and other N-substituted sulfonylureas in alcohols, water, and amines, concluded that dissociation, rather than solvolysis, was the most likely mechanism by which sulfonylureas undergo breakdown at rela ively low temperatures. A report by Chubb and SimmonsZS indicates that tolbutamide reacts with refluxing methanol to form the butylamine salt of methyl p-tolylsulfonylcarbamate. They subscribe to a mechanism of methanolysis for the reaction rather than one of pyrolysis.

Tolbutamide was shown to

5. Drug Metabolites

appears to be the principal manner of degradation of tolbutamide in man. The p-methyl group is oxidized to form a carboxyl group, converting tolbutamide into its principal metabolite, 1-butyl-3-p-carboxyphensulfony- lurea (carboxytolbutamide)21s22.

Oxidation of tolbutamide through its p-methyl group

The tolbutamide metabolite is highly soluble over the critical acid range of urinary pH values, and its solubility increases with an increase in pH. The measured solubility at pH 5 is 2.8 mg/ml, increasing to 20 mg/ml at pH 5.5; atpH6.0 by extrapolation, the solubility becomes 300 mg/m123. the following carboxyto1butamide:tolbutamide solubility ratios were determined: pH 5.0, 13:l; pH 5.5, 50:l; pH 6.0, 350:11°. A pKa1 of 3.54 at 37.5OC has been determined for the metabolitelo.

At 37.5OC and at various pH ranges

529

WILLIAM F. BEYER A N D E R I K H. JENSEN

The amount of m e t a b o l i t e i n u r i n e can be determined by measuring t h e c o l o r found when amyl a c e t a t e - e x t r a c t e d u r i n e i s added t o 0.1% d i n i t r o f l u o r o b e n z e n e and hea ted a t 150'24.

6 . Methods of Ana lys i s

6.1. Elemental A n a l y s i s of t o lbu tamide4 ,

E 1 e men t % Theory % Reported

Carbon 53.31 53.43 Hydrogen 6 .71 6.99 Ni t rogen 10.36 10.29 S u l f u r 11.86 11.88

6.2. Phase S o l u b i l i t y Ana lys i s Phase s o l u b i l i t y p r o f i l e s o f t o lbu tamide were

ob ta ined w i t h s o l v e n t systems of t o l u e n e and e t h y l a c e t a t e : heptane (1 :3 ) , g i v i n g s o l u b i l i t i e s of 7.79 k0.15 mg/gm and 4 .41 k0.08 mg/gm r e s p e c t i v e l y 4 . of t h e to lbu tamide sample based on i t s s o l u b i l i t y p r o f i l e i n t o l u e n e was 100.1 k0.70% and i n e t h y l a c e t a t e : h e p t a n e (1:3) i t was 99.6 k0.41%.

The c a l c u l a t e d p u r i t y

6.3. Ti t r imetr ic The procedure of Franchi25 depends on t i t r a t i o n

o f t o lbu tamide w i t h sodium methoxide i n anhydrous p y r i - dine-chloroform-methanol.

I n a r e p o r t e d t i t r i m e t r i c method of a s say f o r t o l - butamide i n non-aqueous mediaz6, 50 t o 150 mg i s d i s s o l v e d i n 10 m l of anhydrous ace tone o r p y r i d i n e and t i t r a t e d w i t h 0 . 1 1 sodium methoxide t o a p h e n o l p h t h a l e i n e n d p o i n t , I n a mix tu re of benzene and methanol (2:1) , thymol b l u e can be used a s i n d i c a t o r . A s i m i l a r procedure was r e p o r t e d by Dave and Pa te127 .

Simionovici and ConuZ8 developed a d i r e c t t i t r a t i o n method whereby approximately 300 mg of

530

TOLBUTAMIDE

t o lbu tamide i s d i s s o l v e d i n 25 m l of ace tone p r e v i o u s l y n e u t r a l i z e d t o c r e s o l r e d and t i t r a t e d w i t h 0.1; sodium hydroxide t o a r o s e - v i o l e t c o l o r .

I n a procedure depending on h y d r o l y s i s , t h e previous a u t h o r s 2 8 t r e a t e d approximately 300 mg of t o lbu tamide w i t h 10 m l o f e t h a n e d i o l and 2 m l of conc. h y d r o c h l o r i c a c i d , and a p p l i e d 120-122'C h e a t f o r 30 min. The mixture was then d i l u t e d w i t h w a t e r , t r e a t e d i n a K j e l d a h l f l a s k w i t h 15 g sodium hydroxide and t h e amine was d i s t i l l e d o f f i n t o 25 m l o f 0.1N h y d r o c h l o r i c a c i d , t h e excess determined by t i t r a t i o n . Assay v a r i a t i o n of +1.0% was r e p o r t e d .

P a r i k h and Mukherji2' developed a t i t r a t i o n procedure f o r tolbutarnide i n which to lbu tamide was converted i n t o i t s sodium s a l t , combining i t q u a n t i t a t i v e l y w i t h s i l v e r n i t r a t e t o form an i n s o l u b l e s i l v e r s a l t . F e r r i c ammonium s u l p h a t e was used a s t h e i n d i c a t o r and 0.05; ammonium t h i o c y a n a t e a s t h e t i t r a n t .

Beyer and Houtman3' d e s c r i b e d automated t i t r i - m e t r i c p rocedures f o r t h e a n a l y s i s o f t o lbu tamide t a b l e t s u s ing a modified F i s c h e r T i t r a l y z e r . The r e l a t i v e s t a n d a r d d e v i a t i o n f o r t h e procedure was approx ima te ly 1%.

The USP a s s a y p rocedure1 f o r t o lbu tamide depends upon t h e t i t r a t i o n of t h e d rug i n n e u t r a l i z e d aqueous a l c o h o l w i t h sodium hydroxide a s t h e t i t r a n t , and phenol- p h t h a l e i n as i n d i c a t o r .

6.4. U l t r a v i o l e t Spec t ropho tomet r i c S p i n g l e r and Ka i se r JL determined to lbu tamide i n

serum a f t e r l y o p h i l i z a t i o n , e x t r a c t i o n w i t h a c i d i f i e d e t h y l a c e t a t e , r e d u c t i o n t o d r y n e s s , and f i n a l l y d i s s o l - u t i o n i n methanol. Absorbances a t 228 nm and 280 nm were u s e d i n q u a n t i t a t i n g to lbu tamide .

F o r i s t e t a132 developed an a n a l y t i c a l procedure f o r t h e d e t e r m i n a t i o n of t o lbu tamide i n plasma. The

531

WILLIAM f. BEYER A N D ERIK H . JENSEN

procedure depends on ch lo ro fo rm e x t r a c t i o n of weakly a c i d i f i e d plasma, c o n c e n t r a t i o n o f t h e e x t r a c t i o n t o d ry - n e s s , d i s s o l u t i o n of t h e d r y r e s i d u e i n e t h a n o l , t r e a t - ment of t h e s o l u t i o n w i t h c h a r c o a l , and measurement of t h e absorbance of an a l c o h o l i c s o l u t i o n a t 228 nm. Experiments w i t h human and dog plasma gave r e c o v e r i e s of added to lbu tamide of about 98-99%. R e f i n ments i n t h e method were re o r t e d by Bladh and Norden3', and D e l a v i l l e and P a l a z z o l i 34 .

A procedure f o r t h e automated a n a l y s i s of t o lbu tamide t a b l e t s h a s been r e p o r t e d u s i n g Technicon Corpora t ion ' s AutoAnalyzer equipment35. The a n a l y s i s was c a r r i e d o u t a t a wavelength of 263 nm a t a sampling r a t e of 201hour. A c o e f f i c i e n t o f v a r i a t i o n of approximately 1% was o b t a i n e d .

The USP X V I I I l procedure f o r t o lbu tamide t a b l e t s depends upon t h e UV absorbance of e x t r a c t e d t a b l e t s i n chloroform a t a wavelength of 263 nm. A UV d i s s o l u t i o n r a t e t e s t f o r t o lbu tamide t a b l e t s i s d e s c r i b e d i n t h e 1st supplement t o t h e USP X V I I I u s ing t r is(hydroxymethy1) aminomethane b u f f e r a t pH 7.6 and a s t i r r i n g r a t e of 150 rpm. F i l t e r e d samples a r e read a t a wavelength of 226 nm. The t e s t s p e c i f i e s t h a t t h e t ime r e q u i r e d f o r 50% of t h e l a b e l e d amount of t o lbu tamide i n t a b l e t s t o d i s s o l v e i s n o t more than 45 minutes ,

6.5. C o l o r i m e t r i c McDonald and S a ~ i n s k i ~ ~ developed a c o l o r i -

me t r i c method i n v o l v i n g t h e r e a c t i o n between to lbu tamide , 2-naphthol , sodium n i t r i t e , and c o n c e n t r a t e d s u l f u r i c a c i d forming a red c o l o r . The method i s r e p o r t e d t o be a p p l i c a b l e over t h e c o n c e n t r a t i o n r ange of approximately 50 mcg t o 10 mg of t o lbu tamide p e r m l of s o l u t i o n .

C h ~ l s k i ~ ~ d e s c r i b e d a method f o r t h e d e t e r - minat ion of t o lbu tamide i n serum by e x t r a c t i n g a c i d i f i e d serum w i t h chloroform. A f t e r r educ ing t h e ch lo ro fo rm e x t r a c t t o d ryness , an a l c h o l i c s o l u t i o n o f p-N-dimethyl- aminobenzaldehyde i s added and t h e s o l u t i o n reduced t o d ryness . The d ry r e s i d u e i s heated €or 2-112 hour s a t

532

TOLBUTAMIDE

7 O o C and a l c o h o l i s added. Absorbance o f t h e s o l u t i o n s i s measured a t 395 nm. The ave rage r e c o v e r y o f t o lbu tamide from serum was r e p o r t e d t o be n e a r 100% w i t h a s t a n d a r d d e v i a t i o n o f approximately 5%.

The c o l o r i m e t r i c t e r m i n a t i o n o f serum t o l b u t - amide developed by Spingler" depends on t h e r e a c t i o n of t o lbu tamide and d i n i t r o f l u o r o b e n z e n e a t 15OoC f o l l o w i n g e x t r a c t i o n o f a c i d i f i e d serum w i t h amyl a c e t a t e . The absorbance i s determined a t abou t 380 nm. A f t e r a s t u d y of t h e method of S ~ i n g l e r 3 ~ , Pignard3g suggested improve- ments t h a t were r e p o r t e d t o i n c r e a s e s p e c i f i c i t y and range of u s e f u l n e s s . Among t h o s e suggested were p u r i f i - c a t i o n of r e a g e n t s and l e n g t h e n i n g t h e h e a t i n g t i m e from 5 t o 30 minutes a t a t empera tu re of 100°C i n s t e a d o f 15OoC.

D ~ r f m ~ l l e r ~ ~ r e p o r t e d t h e r e a c t i o n of t o l b u t - amide i n a l k a l i n e media w i t h diacetylmonoxime and N-phenyl- a n t h r a n i l i c a c i d , fol lowed by a c i d i f i c a t i o n and h e a t and t h e a d d i t i o n of sodium p e r s u l f a t e and sodium a c e t a t e t o form a b l u e c o l o r .

Mesnard and Crocke t t41 extended t h e work o f R ich te r4* , which involved t h e d e t e r m i n a t i o n of a l i p h a t i c amino a c i d s , t o t h e a n a l y s i s of t o lbu tamide i n b i o l o g i c a l f l u i d s . The method i s based on t h e s t r o n g ye l low c o l o r a - t i o n of s u b s t i t u t e d ammonium p i c r a t e s i n s e l e c t e d anhy- d rous s o l v e n t s , i n which p i c r i c a c i d i s e s s e n t i a l l y c o l o r - less. Of t h e two absorbance maxima observed (355 nm and 412 nm) t h e maximum a t 412 nm w a s used t o avo id a b s o r p t i v e m a t e r i a l s i n blood and u r i n e t h a t cou ld i n t e r f e r e a t t h e 355 nm wavelength. The a u t h o r s a l s o reviewed o t h e r methods f o r t h e d e t e r m i n a t i o n of t o lbu tamide and o t h e r non-amino hypoglycemic su I f ~ n a m i d e s ~ ~ 9 4 4 .

Kern45 r e p o r t e d a procedure f o r t h e d e t e r - mina t ion of t o lbu tamide i n blood fo l lowing e x t r a c t i o n w i t h e t h y l e n e chloride a t pH 5. A f t e r n i t r a t i o n , d i a z o - t i z a t i o n and c o u p l i n g w i t h N(1-naphtyl) e t h y l e n e diamine, an azo dye i s produced t h a t i s measured a t 547 nm.

533


A number o f o t h e r i n v e s t i g a t o r s have d e s c r i b e d co l o r ime t r i c procedure s f o r t o 1 but amid e46-49 .

6.6. Gas Chromatographic A g a s - l i q u i d chromatographic method f o r t h e

d e t e r m i n a t i o n of to lbu tamide i n b lood , u r i n e , and t a b l e t s was r e p o r t e d by Sabih and SabihSO. u r i n e invo lved e x t r a c t i o n o f t o lbu tamide from a c i d i f i e d plasma o r u r i n e and c o n v e r s i o n t o t h e methyl d e r i v a t i v e w i t h d i m e t h y l s u l f a t e i n t h e p re sence o f base . A g a s chromatograph (F & M 5755B) w i t h a f lame i o n i z a t i o n d e t e c t o r and f i t t e d w i t h a s t a i n l e s s s t e e l column was used. The column was packed w i t h diatomaceous e a r t h (Gas chrom Q) and c o a t e d w i t h 5% DC-200. Temperatures o f 205-210' f o r t h e column, 320' f o r t h e d e t e c t o r , and 330' f o r t h e i n j e c t i o n p o r t were used. A d d i t i o n a l GLC methods f o r t o l - butamide i n blood have been r e p o r t e d by P r e s c o t t and Red- man51 and Simmons e t a152 a l s o i n v o l v i n g m e t h y l a t i o n w i t h d ime thy l s u l f a t e .

A method f o r blood and

6.7. L iqu id Chromatographic A l i q u i d chromatographic a s s a y p rocedure f o r

t o lbu tamide i n t a b l e t s ( a l s o a p p l i c a b l e t o bu lk d rug) was d e s c r i b e d by B e ~ e r ~ ~ . graph w i t h a n HCP column ( s t a i n l e s s s teel) 1 M long x 2.1 mm I D and a mobile phase of pH 4.4 monobasic sodium c i t r a t e b u f f e r i n 15% methanol a t a f low r a t e of 0 .36 m l / min were used f o r t h e a n a l y s i s . The r e l a t i v e s t a n d a r d d e v i a t i o n w a s less t h a n 2% and r e c o v e r y was q u a n t i t a t i v e ,

A duPont Model 820 Liquid Chromato-

6.8. Paper Chromatographic Chakraba r t i54 r e p o r t e d a paper chromatographic

a n a l y s i s o f t o lbu tamide i n v a r i o u s s o l v e n t systems. The developed pape r i s r e a c t e d w i t h phenyl hydraz ine and sprayed w i t h a s o l u t i o n of ammoniacal Ni2+ g i v i n g p i n k t o v i o l e t s p o t s , depending on t h e c o n c e n t r a t i o n of t o l b u t - amide s o l u t i o n s .

Hentr ichf j5 d e s c r i b e d a paper chromatographic s e p a r a t i o n o f t o lbu tamide by chromatographing t h e b u t y l amine produced when to lbu tamide i s r e a c t e d w i t h a modif ied F o l i n r e a g e n t . A f t e r h e a t i n g t h e paper a t 180-200', a brown s p o t i s produced.

534

TOLBUTAMIDE

Abdel-Wahab and E l - A l l a ~ y ~ ~ repor ted a procedure u t i l i z i n g paper chromatography f o r r a d i o i so topes and t h e de te rmina t ion of tolbutamide. The au tho r s employed var ious deve lopers and co lo r ing reagents such a s ninhydrin. Radio-ac t iva t ion of d r i e d , developed chromatograms, us ing 1131was appl ied t o determine Rf va lues . S35 l abe led tolbutamide showed t h a t paper chromatography, accompanied by radioscanning o r autoradiography could be used f o r t he sepa ra t ion , d e t e c t i o n , and de termina t ion of tolbutamide.

I n v e s t i g a t i o n s of

An i n f r a r e d i d e n t i f i c a t i o n o f tolbutamide i n human serum employing paper chromatography was r epor t ed by K r i v i s and For i s t57 . The procedure depends upon ex- t r a c t i o n of serum wi th chloroform, eventua l r educ t ion t o dryness , d i s s o l u t i o n i n methylene c h l o r i d e , and a p p l i c a t i o n t o prewashed Whatman No. 1 paper. Af t e r development i n a butanol-water-piperidine (81: 17: 2) system by descending chromatography, t he tolbutamide zone was e lu t ed w i t h water . A potassium bromide micropel le t prepared from a methylene ch lo r ide e x t r a c t i o n of t he aqueous e l u a t e was then examined by i n f r a r e d spectrophotometry.

6.9, Thin Layer Chromatographic S t r ick landb8 descr ibed TLC procedures f o r t h e

sepa ra t ion and d e t e c t i o n of microgram amounts of t o l b u t - amide i n the presence of acetohexamide, chlorpropamide, and phenformin HC1. Various so lvent systems and spray r e - agents were used t o determine r e l a t i v e Rf va lues . A so lvent system c o n s i s t i n g of acetone-benzene-water (65:30:5) separa ted tolbutamide and the o the r t h r e e a n t i - d i a b e t i c agents . The l i m i t of d e t e c t i o n f o r tolbutamide using UV was about 1 mcg.

A TLC procedure f o r t he d e t e c t i o n of tolbutamide i n blood and u r ine was repor ted by Baumler and Rippstein59. The d r u g i s ex t r ac t ed w i t h e t h e r and chromatographed us ing Kiese lge l - ce l lu lose ( 1 : l ) a s t he TLC support and developed wi th benzene-methanol ( 4 : 1). Tolbutamide appears a s a v i o l e t spot when the developed p l a t e i s sprayed wi th ninhydrin-s tannous ch lo r ide and heated, then sprayed wi th a c i d i f i e d n inhydr in and heated again.

535


Guven e t a160 i d e n t i f i e d tolbutamide amongst o the r a n t i - d i a b e t i c drugs by TLC on s i l i c a g e l us ing a so lvent system composed of bu tano l - ace t i c acid-water (10:2:1). A s o l u t i o n of copper s u l f a t e (10%) ammonia (2%) (5 : l ) was used t o d e t e c t t he spo t s , wi th tolbutamide e x h i b i t i n g a green co lo r .

A r e p o r t by Hutzul and Wright61 f o r t he d e t e c t i o n of small amounts of impur i t i e s ( f o r example, 0.05% p- to luenesul fonylurea) i n tolbutamide, makes use of a "Moscow" method of TLC developed by Mistryukov62. The p l a t e , 5x8x1/8" f ros t ed window pane, i s covered wi th adsorbent and maintained h o r i z o n t a l l y whi le t h e developing s o l u t i o n i s fed t o one edge by c a p i l l a r y a c t i o n . E-Toluenesulfonylurea is separated from tolbutamide us ing Davison s i l i c a g e l a s adsorbent , benzene-acetone- methanol-acet ic acid (70: 20: 9: 1) a s developing so lven t , and v i sua l i zed using mists of hypochlorous a c i d , e thano l , and an aqueous s o l u t i o n of a c e t i c ac id wi th iod ide and sa tu ra t ed wi th benzidine. toluenesulfonamide, an adsorbent of aluminum oxide and a developing so lvent of benzene-acetone-methanol (70:20:10) was used. Agents f o r v i s u a l i z a t i o n were the same a s those f o r p t o l u e n e s u l f o n y l u r e a .

For the d e t e c t i o n of 1-

Menzer e t a l l 3 u s e d a developing system com- p r i sed of ch1oroform:glacial acetic ac id (99:l) and s i l i c a g e l H p l a t e s , 0.25 mm t h i c k t o sepa ra t e t o l b u t - amide from i t s by-products. Evaluat ions were made under UV l i g h t a f t e r spraying wi th xanthydrol s o l u t i o n . Their work a l s o d isc losed a new by-product i n the syn thes i s of tolbutamide: p t o l y l s u l f o n y l b i u r e t . Table I V summarizes t h e i r r e s u l t s .

536

TOLBUTAMIDE

TABLE IV

Relative TLC Rf Values for Tolbutamide and By-products Using a Ch1oroform:Glacial Acetic Acid (99:l)

Developing Solution

R f - Compound

Tolbutamide 0.85 N,N'-Dibutylurea 0.56 P- Toly lsu 1 f onamide 0.47 P- Toly lsu 1 f ony lurea 0.26 P-Tolylsulfonylbiuret 0.13 N-Butylurea 0.12

Reisch et a163 reported five TLC developing systems using n-butanol in combination with two to three other organic solvents. Spots were detected on silica gel G plates when sprayed with visualizing agents. TLC was applied by Neidlein et a164 and Glogner et d5 in separating tolbutamide from other sulfonamides.

6.10. Coulometric A Mercurocoulometric method for the determin-

ation of tolbutamide has been reported by Kalinowski and Korzybski66 and V O ~ C U ~ ~ .

7. Pharmacokinetics and Toxicity

amide given as a single dose following an overnight fast is reported by McMahon et a168 to be 5.7 hours. The LD50 f o r tolbutamide administered orally to rats is 2,344 mg/ kgm and in mice injected IP is 1,232 mg/kgm68.

Inactivation of tolbutamide to carboxytolbutamide occurs in man and is rapidly excreted in urine as the principal metabolite.

The half-life in human subjects for 1 gm of tolbut-

537


8. I d e n t i f i c a t i o n

U.S.P. X V I I I 2 . The t e s t s depend upon: a ) t h e in f r a red adsorp t ion spectrum of a mineral o i l d i s p e r s i o n of t he drug i n the range o f 2 t o 1 2 p; b) formation of an orange-red co lo r a f t e r t he drug i s re f luxed wi th d i l u t e s u l f u r i c ac id , steam d i s t i l l e d i n d i l u t e hydrochlor ic acid a f t e r being made s t rong ly a l k a l i n e , made a l k a l i n e w i t h a c e t a t e and bora te b u f f e r , and r eac t ed i n an i c e ba th w i t h p - n i t r o a n i l i n e and sodium hydroxide; and c ) pro- duc t ion of p-toluenesulfonamide (melt ing between 136' and 141') by r e f lux ing i n d i l u t e s u l f u r i c a c i d , cool ing t h e so lu t ion , c o l l e c t i n g and pu r i fy ing the c r y s t a l s .

I d e n t i f i c a t i o n t e s t s f o r tolbutamide a r e given i n

538

TOLBUTAMI DE

9. References

1. "United States Pharmacopeia," 18th Ed., Mack Printing Co., Easton, Pa.

2. Ruschig, H., W. Aumiiller, G. Korger, H. Wagner, J. Scholz, and A. BPnder, U.S. Patent 2,968,158, January 17, 1961 (assigned to The Upjohn Co.).

3 . The Merck Index, 8th Edition, Merck and Co., Inc. Rahway, N.J. (1968).

4. Humphrey, L. M., The Upjohn Co., unpublished data.

5. Shell, J. W., Anal. Chem., 30, 1577 (1958).

6. Zipple, K. G., C. G. Chidester, C. G. Waber, and D. J. Duchamp, The Upjohn Co., unpublished data.

7. Muelman, P. A. and M. L. Knuth, The Upjohn Co., unpublished data.

8. Slomp, G., The Upjohn Co., unpublished data.

9. Grostic, M. F., R. J. Wnuk, and F. A. MacKellar, J. Am. Chem. SOC., g,(1966).

10. Forist, A. A , , T. Chulski, Metabolism, 3, 807 (1956).

11. Bowman, P. B., The Upjohn Co., unpublished data.

12. Perkin-Elmer Trade Publication, Thermal Analysis News Letter No. 5.

13. Menzer, M., J. Presewowski, and U. Haug, Pharmazie - 26, 738 (.i97i).

14. Vogt, H., Pharm. Zentralh, 98, 651 (1959).

15. Haussler, A . and P. Hajdu, Arch, Pharm., 295, 471 (1962).

539

WILLIAM F. BEYER A N D ERIK H. JENSEN

16.

17 .

18.

19.

20.

21.

22.

23 .

24.

25.

2 6 .

27.

28.

U l r i c h , H. and A.A.R. Sayigh, Angew. Chem. I n t . Ed . Engl. 2, 724 (1966).

B o t t a r i , F., M. F. S a e t t o n e , B. G i a n n a c c i n i , and M. E. LoBrut to , Rass. Dermatol, S i f i l o g r . , 20, 235 (1967).

B o t t a r i , F., M. Manne l l i , and M. F. S a e t t o n e , J. Pharm. S c i . , 2, 1663 (1970).

B o t t a r i , F., B. G i a n n a c c i n i , E. N a n n i p i e r i , and M. F. S a e t t o n e , J. Pharm. S c i . , 61, 602 (1972).

Chubb, F. L. and D. L. Simmons, Can. J. Pharm. S c i . , 1, 28 (1972).

L o u i s , L. H . , S. S , F a j a n s , J . W . Conn, W , A . S t r u c k , J. B. Wright , and J. L. Johnson, J. Am. Chem. SOC. 78, 5701 (1956).

P h y s i c i a n s Desk Refe rence , 26 E d . , Medical Economics, I n c . , 1972.

Tucker, H. A., "Oral A n t i d i a b e t i c Therapy 1956 1965: w i t h P a r t i c u l a r Re fe rence t o Tolbutamide (Or inase ) , " C. C. Thomas, P u b l i s h e r , 1965.

Nelson, E . , I. O ' R e i l l y , and T. Chu l sk i , C l i n . Chim. Acta, 3 , 774 (1960).

F r a n c h i , G . , A t t i . Accad. F i s i o c r i t . S i e n a 6, 61 (1956-57).

Kracmarova, J . , J. Kracmar, Ceskos l . Farm., 1, 566 (1958).

Dave, J. B. , and J. L. Pa t e l , I n d i a n . J. o f Pharm. Z l , 226 (1959).

S imionov ic i , R., and I. Conu, Rev. Chim. Bucha- r e s t , Lo, 107 (1959).

540

TOLBUTAM I DE

29.

30.

31.

32.

33.

34.

35.

36.

37.

P a r i k h , P. M. , S. P. Mukherj i , I n d i a n J. of Pharrn. , 2l, 110 (1959).

Beyer, U. F. and R. L. Houtman, Ann. N.Y:Acad. S c i . , 130, 539 (1965).

S p i n g l e r , H. and F. K a i s e r , Arzne i rn i t t e l -For sch , I 6 , 760 (1956).

F o r i s t , A.A., W. L. Mil ler ,Jr . , J. Krake, W. A . S t r u c k , Proc. SOC. E x p t l . B i o l . Med., 96, 180 (1957).

Bladh, E . and A. Norden, Acta . Pharrnacol. Tox ico l . - 14, 188 (1958).

D e l a v i l l e , G . , and M. P a l a z z o l i , Ann. B i o l . C l i n . ( P a r i s ) , l6, 481 (1958) .

Beyer, W. F. and R. L. Houtman, Ann. N.Y. Acad. S c i . , 130, 535 (1965).

McDonald, H. and V. Sawinski , Texas Rep. B i o l . Med., l6, 479 (1958).

Chu l sk i , T., J. Lab. and C l i n . Med. 53, 490 (1959) .

38. S p i n g l e r , H . , K l i n . Wochschr,

39. P igna rd , P., Ann. B i o l . C l i n . (1958) .

- 35, 533 (1957).

( P a r i s ) , l6, 471

40. Dorfrnul ler , T . , "Das A r t z l i c h e Laboratorium" (1957).

41. Mesnard, P. and R. C r o c k e t t , Rev. Esp. F i s i o l , - 16 , 163 (1960).

42. R i c h t e r , D. , Biochem. J., 32, 2 (1938).

WILLIAM F. BEYER A N D ERIK H . JENSEN

43.

44

45.

46.

47.

48.

49.

50.

51.

52

53.

54.

55.

56.

57.

Mesnard, P. and R , Crockett., Chim. Anal., 9, 346 (1960).

Mesnard, P. and R. Crockett, m, 42, 381 (1960).

Kern, W., Anal. Chem. 35, 50 (1963).

Wermuth, C. G., and P. Morand, Trav. SOC. Pharm. Montpellier, 20, 234 (1960).

Jung, L. M., C. G. Wermuth, and P. Morand, x, - 21, 170 (1961).

Alessandro, A., R. Emer, and G. Abbondanza, G, Med. Mil., 116, 827 (1966).

Kaistha, K. K. and W. N. French, J. Pharm. Sci., - 57, 459 (1968).

Sabih, K. and K. Sabih, J. Pharm. Sci., 59, 782 (1970) . Simmons, D. L., R. J. Ranz, and P. Picotte, J. Chromatog.71, 421 (1972).

Prescott, L. F. and D. R. Redman, J. Pharm. Pharmac., 24, 713 (1972).

Beyer, W. F., Anal. Chem., 44, 1312 (1972).

Chakrabarti, J. K., J. Chromatog., 8, 414 (1962).

Hentrich, K., Pharmazie, l8, 405 (1963).

Abdel-Wahab, M. F. and R. M. El-Allawy, Isoto- penpraxis, 4, 371 (1968).

Krivis, A. F. and A. A. Forist, Microchem, J. - 5, 553 (1961).

542

TOLBUTAMIDE

58. Strickland, R. D., J. Chromatog., 24, 455 (1966).

59. Baumler, J. and S . Rippstein, Dt. ApothZtg. 107, 1647 (1967).

60. Gcven, K. C., S. Gecgil, and 0. Pekin, Eczacilik BElteni, 2, 158 (1966).

61. Hutzul, M. and G. Wright, Canadian J. Pharm. S t i . , - 3, 4 (1968).

62. Mistryukov, E. A,, Collection Czech. Chem. Commun. 26, 2072 (1961).

63. Reisch, J., H. Bornfleth, and G. L. Tittel, Pharm. Ztz Apotheker Ztg, 109, 74 (1964).

64. Neidlein, R., G. KlGgel, and U. Lebert, Pharm. Ztg. Ver. Apotheker Ztg, 110, 651 (1965).

65. Glogner, P., H. Lange, and R, Pfab, Med. Welt, 52, 2876 (1968).

66. Kalinowski, K. and R. Korzybski, Acta Polon. Pharm. 2, 221 (1963).

67. Voicu, A., Farrnacia l0, 399 (1962).

68. McMahon, F. G., H. L. Upjohn, 0. S. Carpenter, J. B. Wright, H. Oster, and W. E. Dulin, Current Therapeutic Research, 4, 330 (1962).

This monograph is based to a great extent on a preliminary compilation of analytical information on tolbutamide by Dr. Arlington A. Forist. His background data facilitated the preparation of the monograph. Mrs. Betty Breseman deserves special recognition for preparation of the manuscript in its final form. The authors also wish t o acknowledge the valuable secretarial support of Mrs. Mari- lynn K. Nelson.

543

TRIMETHAPHAN CAMSYLATE


KENNETH W. BLESSEL, BRUCE C. RUDY, A N D BERNARD 2 . SENKOWSKI

INDEX

1. Description

1.1 Name, Formula, Molecular Weight 1.2 Appearance, Color, Odor 1.3 Isomeric Forms

2 . Physical Properties

2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 2.10 2.11

Infrared Spectrum Nuclear Magnetic Resonance Spectrum Ultraviolet Spectrum Fluorescence Spectrum Mass Spectrum Optical Rotation Melting Range Differential Scanning Calorimetry Thermogravimetric Analysis Solubility X-ray Crystal Properties

3. Synthesis

4. Stability Degradation

5 . Drug Metabolic Products


6 . 1 Elemental Analysis 6.2 Phase Solubility Analysis 6.3 Thin Layer Chromatographic Analysis 6.4 Direct Spectrophotometric Analysis 6.5 Colorimetric Analysis 6.6 Non-Aqueous Titration

7. Acknowledgment

8. References

546

TRIMETHAPHAN CAMSY LATE

1. Description

1.1 Name, Formula, Molecular Weight Trimethaphan camsylate is (+)-1.3-dibenzyldeca-

hydro-2-oxoimidazo~4,5-c] thieno [ 1,2-a] -thiolium 2-bxo-10- bornane sulfonate.

Molecular Weight: 596.81. C32H40N205S 2

1.2 Appearance, Color, Odor Trimethaphan camsylate is a white crystalline

powder which is odorless or has a slight odor.

1.3 Isomeric Forms Trimethaphan camsylate has four possible isomers,

grouped in two pairs of enantiomers.


2.1 Infrared Spectrum The infrared spectrum of a sample of reference

standard trimethaphan camsylate is shown in Figure 1 (1). The spectrum was recorded using a 5% w/v solution in chloroform, utilizing a Perkin Elmer 621 Spectrophotometer equipped with 0.1 mm NaCl liquid cells. The following assignments of some of the bands in the spectrum have been made which are shown in Table I below (1).

Table I

Band Assignment - 1735 cm'l

1700 c m - l

C=O stretch in the camphor- sulfonic acid moiety C=O stretch of the trimethaphan moiety

547

8 In

8 8 - 8 3-

I f

00

e;: 0

8: R

W

8'

v)

8 8 (u

0

M

In

M

8 U 33N

WlllW

SN

WU

l Yo

548


1447 cm-l CH2 and CH3 deformations

t h e monosubs t i t u t e d benzene r ings .

701 cm-1 out-of-plane deformations of

2.2 Nuclear Magnetic Resonance Spectrum (NMR) The NMR spectrum of a sample of re ference stan-

dard trimethaphan camsylate i s shown i n Figure 2 ( 2 ) . The so lvent used was DM!SO-d6 and the concent ra t ion of the trimethaphan camsylate was 54.8 mg/0.5 m l . complex na tu re of the spectrum, a cons iderable amount of spin-spin decoupling w a s necessary i n order t o ob ta in t h e assignments shown i n Table I1 ( 2 ) . An a r b i t r a r y set of numbers was used on the s t r u c t u r a l formula, shown below, f o r ease i n present ing t h e assignments.

Due t o the

Table I1

NMR S p e c t r a l Assignments f o r Trimethaphan Camsylate

Total No. of Chemical Coup1 ing

Proton Protons S h i f t (ppm) M u l t i p l i c i t y Constant

c18 protons 3 0.73 Sing le t

C19 protons 3 1.03 Sing le t

C14 and C15 4 1.10-1.60 Mul t ip l e t pro tons

c16 and C20 3 -2.3 Mu1 t i p l e t protons

C12 protons 2 -3.0 T r i p l e t

Cg and Cll 4 4.0-4.3 2 p a i r s of J(C.:i)-16 Hz protons 4.6-5.0 Doublets

549


c1,c2 , c3 ,C4’ 11 2.0-3.8 Two sets of

C6,C7 and C8 4.5-5.9 Mul t ip l e t s

pro tons

aromatic 10 7.34 Sing le t protons

2.3 U l t r a v i o l e t Spectrum The u l t r a v i o l e t spectrum of r e fe rence s tandard

trimethaphan camsylate (a) is shown i n Figure 3 (3). The concent ra t ion w a s 1.00 mg/ml i n chloroform. shows a maximum a t 258 nm (E = 3.9 x lo2) having two shoulders on t h e r i s i n g po r t ion of t h e curve. Also shown on t h e same f i g u r e is t h e b a s e l i n e scan (b) and t h e u l t r a - v i o l e t spectrum of t h e bromocresol green complex ( c ) wi th tr imethaphan camsylate which is used f o r co lo r ime t r i c determinat ion.

The curve

2.4 Fluorescence Spectrum TrimethaPhan camsylate (1 mg/ml i n methanol) has

extremely weak e x c i t a t i o n and emission s p e c t r a which are shown i n Figure 4 (4). The instrument used w a s a Farrand MK-1 record ing spec t rof luorometer . Exc i t a t ion a t 290 nm produced an emission spectrum having a maximum a t 416 nm.

2.5 Mass Spectrum The low r e s o l u t i o n mass spectrum of a sample of

re ference s tandard trimethaphan camsylate i s shown i n Figure 5 ( 5 ) . The spectrum was obtained wi th the a i d of a CEC 21-110 mass spectrometer a t an ion iz ing energy of 70 eV, i n t e r f a c e d with a Varian d a t a system 100 MS. The da ta system accepted t h e output of t h e spectrometer , cal- cu la ted t h e masses, compared t h e i n t e n s i t i e s t o t h a t o f t he base peak and p l o t t e d t h e i n t e n s i t i e s as a series of l i n e s whose h e i g h t s were p ropor t iona l t o t h e i n t e n s i t i e s .

as such has very low v o l a t i l i t y , t he re fo re , a h ighly c h a r a c t e r i s t i c mass spectrum cannot b e expected. The h ighes t mass observed by low r e s o l u t i o n was m / e 488. base peak was observed a t m / e 91, corresponding t o C H5-CH20. m f e 578 which probably arises from the l o s s of H20 from the molecular ion . probably due t o t h e l o s s of SO from t h e parent mass and

Trimethaphan camsylate is a thiophanium sa l t and

The

A high r e s o l u t i o n scan showed masses up t o

Other masses were observed a t m / e 548,

551

KENNETH W. BLESSEL, BRUCE C. RUDY, AND BERNARD Z. SENKOWSKI

Figure 3

Ultraviolet Spectra

(a) Trimethaphan Camsylate (b) Solvent ( c ) Trimethaphan Camsylate - Bromocresol Green Complex

I .c

r

8: .i

C 7 I I I I I !!m x)o 350 400 450 500 5

WAVELENGTH, nm.

552

0

0

rp

8 In

v)

W

+ a

ow

4 a

0

0

rn

0

0

N

553

ts

--I:

554


m / e 545, due t o t h e l o s s of SH and H20 from the molecular ion. These peaks are r a t h e r p e c u l i a r i n t h a t they appear t o a r i s e from fragments conta in ing both the a c i d and base po r t ions of t he molecule ( 5 ) .

2.6 Op t i ca l Rota t ion The va lue repor ted f o r t h e s p e c i f i c r o t a t i o n of

trimethaphan camsylate i n t h e United S t a t e s Pharmacopeia XVIII i s "not less than +20° and not more than +23' d e t e r - mined i n a s o l u t i o n conta in ing 400 mg i n each 10 m l " ( 6 ) . A graph of s p e c i f i c r o t a t i o n as a func t ion of wavelength i s a l s o presented i n F igure 6 (7 ) . The d a t a were obta ined by conver t ing r o t a t i o n va lues from a Jasco ORD-UV 5 i n s t r u - ment t o s p e c i f i c 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 w a s zero a t 294 nm and 225 nm. It can be seen t h a t t h e s p e c i f i c r o t a t i o n changes r a p i d l y a t wavelengths below 300 nm.

2 . 7 Melt ing Range Trimethaphan camsylate melts wi th decomposition

over a range of 230-235'C when a Class I a procedure is used (6 ) .

2.8 D i f f e r e n t i a l Scanning Calorimetry (DSCl The DSC curve f o r a sample of r e fe rence s t anda rd

trimethaphan camsylate is shown i n F igure 7 (8 ) . This curve was obta ined a t a scan r a t e of 10°C/min. i n a n i t r o - gen atmosphere u t i l i z i n g a Perk in E l m e r DSC-1B. The ex- t r apo la t ed o n s e t of t h e mel t ing endotherm, accompanied by decomposition, is 234.9 f. 0.loC and t h e peak is a t 238.4 2 0 . 4 O C . A l l temperatures have been co r rec t ed . Because of t he decomposition during the m e l t , t h e AHf cannot be cal- cu la t ed wi th much r e l i a b i l i t y , however, i t s va lue is approximately 1 2 kcal /mole (8) .

2.9 Thermogravimetric Analysis (TGA) The TGA curve f o r r e fe rence s tandard t r imethaphan

camsylate showed no weight l o s s from ambient temperature t o 265OC a t a hea t ing ra te of 10°C/min. A t about 265OC, weight l o s s began which amounted t o approximately 70% of t h e weight a t 3 4 5 O C (8) .

555

KENNETH W. BLESSEL, BRUCE C. RUDY, A N D BERNARD 2.SENKOWSKI

Figure 6

Specific Rotation vs Wavelength f o r Trlmethaphan Camsylate

+ 400 -

I - 7 1200- -

1600 - 2000 - 2400 - 2800 - 3200 -

- - - - -

3600 - - 4000 - - 4400 - - 4800 - -

556


Figure 7

DSC Curve of Trimethaphan Camsylate

I I I I I

Endothermic

1 Exothermic

\ 234.9'C

I I I I I 2 5 0 2 4 0 2 3 0 2 2 0 210

"C

557

KENNETH W. BLESSEL. BRUCE C. RUDY, AND BERNARD 2 . SENKOWSKI

2.10 S o l u b i l i t y The s o l u b i l i t y d a t a shown i n Table I11 was ob-

t a i n e d a t 25OC f o r r e f e r e n c e s t a n d a r d t r imethaphan cam- s y l a t e ( 9 ) .

Table I11

S o l u b i l i t y Data f o r Trimethaphan Camsylate

Solvent S o l u b i l i t y (rng/ml)

d i e t h y l e t h e r 0.01 petroleum e t h e r (30-60') 0.05 2 - propanol 20.15 3A a l c o h o l 89.06 chloroform >500. 95% e t h a n o l 175.80 benzene 2.59 methano 1 >500. water >500.

2 . 1 1 C r y s t a l P r o p e r t i e s The x-ray powder d i f f r a c t i o n d a t a € o r a sample

of r e f e r e n c e s t a n d a r d t r imethaphan camsylate is g iven i n Table I V (10) . The o p e r a t i n g parameters of t h e i n s t r u m e n t are g iven below.

Ins t rumenta l Condi t ions


Generator : 50 KV, 12-112 MA Tube t a r g e t : o p t i c s : 0.1' D e t e c t o r s l i t

Cu K, = 1 .542 8

M.R. S o l l e r slit 3' Beam s l i t 0.0007" N i f i l t e r 4' t a k e o f f a n g l e Scan a t 0.2' 28 p e r minute Goniometer :

Detect o r : Amplif ier g a i n - 1 6 c o u r s e , 8 .7 f i n e

Sea led p r o p o r t i o n a l c o u n t e r t u b e and DC v o l t a g e a t p 1 a t eau

558


P u l s e h e i g h t s e l e c t i o n EL - Eu - o u t Rate meter T.C. 4 2000 CIS f u l l scale

5 v o l t s

Recorder: Chart Speed 1 i n c h p e r 5

Samples : minutes

temperature . Prepared by g r i n d i n g a t room

Table I V

I n t e r p l a n a r Spacings i n Trimethaphan Camsylate from Powder

28

7.04 10.94 12.00 12.26 12.52 12.76 14.14 14.84 16.22 17.54 18.68 19.24 20.78 21.28 21.94 22.34 22.82 23.28 24.50 24.86 25.20 25.58 25.94

- d (1) * 1 2 . 5

8.09 7.38 7.22 7.07 6.94 6.26 5.97 5.46 5.06 4.75 4.61 4.27 4.18 4.05 3.98 3.90 3.82 3.63 3.58 3.53 3.48 3.43

D i f f r a c t i o n Data

28 - I/1***

100 26.94 14 28.06 13 28.86 1 9 29.92 53 30.42 23 31.12 52 31.66

7 32.54 96 34.54 81 35.10 44 35.86 98 36.96 57 37.44 48 37.84 20 38.96 2 1 39.56 11 40.40 10 41.00 25 41.76 11 43.00 9

1 3 4

nX 2 S i n 8

*d = ( i n t e r p l a n a r d i s t a n c e )

d (1) * 3.31 3.18 3.09 2.99 2.94 2.87 2.83 2.75 2.60 2.56 2.50 2.43 2.40 2.38 2 .31 2.28 2.23 2.20 2.16 2.10

ZL1,- ** 16 18 14 25

2 4 4 8

1 0 6 3 3 6 4 4 3 4 1 3 4

**I/ = re la t ive 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 I0 of 100)

559

KENNETH W. BLESSEL, BRUCE C. RUDY, A N D B E R N A R D Z . SENKOWSKI

3 . S y n t h e s i s

i n t h e s y n t h e s i s of b i o t i n . The s y n t h e t i c r o u t e s t o b i o t i n have been r e p o r t e d i n s e v e r a l p a t e n t s (11-13).

Trimethaphan camsy la t e can b e o b t a i n e d as a by-product

4 . S t a b i l i t y Degrada t ion

c a r r i e d o u t by h e a t i n g t h e material i n t h e d r y form f o r d i f f e r e n t p e r i o d s of t i m e . The s o l i d w a s d i s s o l v e d i n double d i s t i l l e d water a t a c o n c e n t r a t i o n of 5% and a n estimate of s t a b i l i t y was made by n o t i n g t h e d e g r e e o f t u r b i d i t y caused by decomposi t ion p r o d u c t s . It w a s ob- s e rved t h a t on h e a t i n g a t 100°C f o r p e r i o d s up t o 2 h o u r s , only a s l i g h t t u r b i d i t y was noted i n t h e s o l u t i o n ( 1 4 ) . The decomposi t ion of t r ime thaphan camsy la t e under extreme c o n d i t i o n s w a s a l s o s t u d i e d . It was found t h a t r e f l u x i n g a 5% aqueous s o l u t i o n f o r 90 h o u r s caused approx ima te ly 60% decompos i t ion , c a l c u l a t e d from t h e amount of l i b e r a t e d f r e e a c i d ( 1 5 ) .

A s t u d y o f t h e s t a b i l i t y of t r ime thaphan camsy la t e w a s

5 . Drug Metabo l i c P roduc t s

b o l i c p r o d u c t s o f t r ime thaphan camsy la t e were unknown. A s e a r c h of t h e l i t e r a t u r e from t h a t p o i n t up t o t h e p r e s e n t d i d n o t add t o t h e c u r r e n t knowledge p e r t a i n i n g t o metabo l i sm of t h e drug. S i n c e t h e p h y s i o l o g i c a l a c t i o n of t h e d rug commences and c e a s e s v e r y r a p i d l y , t h e e f f e c t o r n a t u r e of t h e m e t a b o l i t e s i s u n c e r t a i n .

S c u r r and Wyman ( 1 9 ) r e p o r t e d i n 1954 t h a t t h e meta-


6 . 1 Elemental Ana lys i s The r e s u l t s of a n e l e m e n t a l a n a l y s i s of a sample

o f r e f e r e n c e s t a n d a r d t r ime thaphan camsy la t e are p r e s e n t e d i n Tab le V ( 1 6 ) .

Tab le V

Elemental Ana lys i s of Trimethaphan Camsylate

E l emen t % Theory % Found C 64.40 6 4 . 3 9 H 6 . 7 6 6 . 8 1 N 4 . 6 9 4 . 7 6 S 1 0 . 7 4 10 .78

560


6.2 Phase S o l u b i l i t y Analysis The r e s u l t s of a phase s o l u b i l i t y a n a l y s i s a s an

ind ica t ion of p u r i t y i s shown i n Figure 8 f o r a r e fe rence s tandard sample of tr imethaphan camsylate (9 ) . The so lven t used was acetone wi th an e q u i l i b r a t i o n t i m e of 20 hours a t 25OC. r e s u l t s a r e shown i n F igure 8.

The remainder of t he experimental condi t ions and

6.3 Thin Layer Chromatography A t h i n l a y e r chromatographic system has been de-

veloped f o r t h e sepa ra t ion of hydro lys i s products of trimethaphan camsylate from t h e pa ren t subs tance (17). The type of p l a t e used w a s s i l i c a g e l G, wh i l e t h e developing solvent was methanol: 10% aq. H2SO4 (90 : 10) . After t h e sol- t kont h a ascended f o r 15 cm the p l a t e is a i r d r i e d and sprayed wi th modified Dragendorff reagent . The Rf value of tr imethaphan camsylate was 0.5 while t h a t of t he major hydro lys i s product was 0 .7 .

6.4 Direct Spectrophotometric Analysis Trimethaphan camsylate , In i n j e c t a b l e s o l u t i o n ,

can be assayed d i r e c t l y by a UV absorp t ion procedure. This procedure involves the d i l u t i o n of 5 m l of ampul s o l u t i o n (50 mg/cc) t o one l i t e r . The absorbance of t h i s s o l u t i o n i s measured a t wavelengths from 254-259 nm. The maximum absorpt ion i n t h i s range i s used t o c a l c u l a t e t h e amount of tr imethaphan camsylate present i n t he ampul by comparison with a sample of r e fe rence s tandard ma te r i a l prepared and measured i n a similar way (18).

6 .5 Color imet r ic Analysis Trimethaphan camsylate can be determined c o l o r i -

m e t r i c a l l y by formation of t h e bromocresol green ion p a i r followed by e x t r a c t i o n as descr ibed i n t h e fol lowing procedure. A volume of s o l u t i o n equiva len t t o a b o u t 100 m g of tr imethaphan camsylate is d i l u t e d t o o n e l i t e r . pH of 5 .3 by the a d d i t i o n of a phosphate bu f fe r . Then 5-ml of a bromocresol green s o l u t i o n i n t h e phosphate buf- f e r i s added, a f t e r which the aqueous s o l u t i o n is ex t r ac t ed wi th two 25-ml po r t ions of chloroform. The combined chloroform e x t r a c t s a r e d i l u t e d t o 100 m l and the absorbance determined a t about 420 nm. The quan t i ty of t r imethaphan camsylate present i s ca l cu la t ed by comparison with a

A 10-ml a l i q u o t of t h i s s o l u t i o n is buffered a t a

561

Figure 8

5

e z

z?!lLI 4 - 2 3 k $

z : 3 -

O E : -

Q I

v)

= u z ; z' - I 2 -

3 L -lo +

I l l 1 ~ l l l l ~ l l l l ~ l l l l

- n n A n - " 0 - s

PHASE SOLUBILITY ANALYSIS - Sampte : Trimethophan Camsylate Solvent : Acetone Slope : 0.19 Ole - Equilibration : 20 hrs at 25'C Extrapolated Solubility : 3.72 mg/q

-: -


known concent ra t ion of re ference s tandard material similar- l y prepared and measured (6).

6.6 Non-Aqueous T i t r a t i o n The non-aqueous t i t r a t i o n descr ibed i n the USP

XVIII is t h e accepted method f o r t he ana lys i s of t r imethaphan camsylate i n the bulk form (6) . The sample i s d is - solved i n a c e t i c anhydride and t i t r a t e d wi th 0.1N H C l O 4 u t i l i z i n g a potent iomet r ic end-point. HC104 is equivalent t o 59.68 mg of trimethaphan camsylate.

7 . Acknowledgment

Research Records Of f i ce of Hoffmann-La Roche Inc. f o r t h e i r l i t e r a t u r e search.

Each m l of 0.1N

The au thors wish t o acknowledge t h e a s s i s t a n c e of t h e

563


8. References

1.

2 .

3 .

4 .

5.

6 .

7 .

8.

9 .

10 *

11. 1 2 . 13. 1 4 . 15 .

16.

1 7 .

1 8 .

1 9 .

Hawrylyshyn, M., Hoffmann-La Roche Inc., Personal Communication. Johnson, J. H., Hoffmann-La Roche Inc., Personal Communication. Rubia, L. B., Hoffmann-La Roche Inc., Personal Communication. Boatman, J., Hoffmann-La Roche Inc., Personal Communication. Benz, W., Hoffmann-La Roche Inc., Personal Communication. The United States Phamacopeia XVIII, p p . 755-757 ( 1 9 7 0 ) . Toome, V., Hoffmann-La Roche Inc., Personal Communication. Moros, S., Hoffmann-La Roche Inc., Personal Communication. MacMullan, E., Hoffmann-La Roche Inc., Personal Communication. Hagel, R., Hoffmann-La Roche Inc., Personal Communication. United States Patent 2,489,232-2 , November, 1 9 4 9 . United States Patent 2,489,238, November, 1 9 4 9 . United States Patent 2 , 5 1 9 , 7 2 0 , August, 1 9 7 0 . Rubin, S., Hoffmann-La Roche Inc., Unpublished Data. Sternbach, L., Hoffmann-La Roche Inc., Unpublished Data. Scheidl, F., Hoffmann-La Roche Inc., Personal Communication. Sokoloff, H., Hoffmann-La Roche Inc., Unpublished Results . Keller, C., Hoffmann-La Roche Inc., Personal Communication. Scurr, C. F. and Wyman, J. P., Lancet, 266, 338 ( 1 9 5 4 ) .

5 64

TROPICAMIDE



1.

2.

3 .

4 .

5.

6 .

7.

8 .

INDEX

Analy t ica l P r o f i l e - Tropicamide

Descr ip t ion 1.1 N a m e , Formula, Molecular Weight 1 . 2 Appearance, Color, Odor

Phys ica l P r o p e r t i e s 2 . 1 In f r a red Spectrum 2.2 Nuclear Magnetic Resonance Spectrum 2.3 U l t r a v i o l e t Spectrum 2.4 Fluorescence Spectrum 2.5 Mass Spectrum 2.6 Op t i ca l Rota t ion 2 . 7 Melting Range 2.8 D i f f e r e n t i a l Scanning Calorimetry 2.9 Thermogravimetric Analysis 2.10 S o l u b i l i t i e s 2 . 1 1 X-ray Crys t a l P r o p e r t i e s 2 . 1 2 D i s soc ia t ion Constant

Synthes is

S t a b i l i t y Degradation


Methods of Analysis 6.1 Elemental Analysis 6.2 Phase S o l u b i l i t y Analysis 6 .3 Thin Layer Chromatographic Analysis 6.4 Direct Spectrophotometric Analysis 6.5 Non-Aqueous T i t r a t i o n

Acknowledgments

References

5 66

TROPICAM I DE

1. Description

1.1 Name, Formula, Molecular Weight Tropicamide is N-ethyl-2-phenyl-N-(4-pyridyl-

methyl)-hydracrylamide. OH

C17H20N2N2 Molecular Weight: 284.36

1 .2 Appearance, Color, Odor Tropicamide is a white crystalline odorless

powder.

2 . Physical Properties

2.1 Infrared Spectrum The infrared spectrum of a sample of reference

standard tropicamide is shown in Figure 1 (1). The spectrum was recorded on a KBr pellet containing 0.5 mg of tropicamide and 300 mg of KBr, using a Perkin Elmer 621 Spectrophotometer. The following assignments have been made of the bands in Figure 1 (I).

- Band Assignment

3396 cm-’ OH stretch 1620 cm-1 CEO stretch 1595 and 1493 cm-l Aromatic Ring Vibrations 810 cm-1 Monosubs tituted Pyridine

760 and 707 cm-l Ring

Monosubstituted Benzene Ring

2.2 Nuclear Magnetic Resonance Spectrum (NMR) The NMR spectrum of a sample of reference stan-

dard tropicamide is shown in Figure 2 (2). The sample solution contained 62.5 mg of tropicamide per 0.5 ml o f C D C l 3 . Due to the complex spectrum observed, extensive spin decoupling experiments were carried out in order to evaluate the coupling constants and chemical shifts. Con- secutive irradiations were performed at 58.8 Hz, 65.4 Hz, 196 Hz, and 200 Hz which simplified the spectrum to the

561

KENNETH W. BLESSEL, BRUCE C. RUDY, AND BERNARD Z. SENKOWSKI

ex ten t t h a t t h e fol lowing assignments could b e made (2 ) .

Table I

NMR Spec t r a l Data f o r Tropicamide*

No. of Chemical Each S h i f t M u l t i p l i c i t y

C5 protons 3 0.98,1.09 (2) J [ CH3-CH2-N] = t r i p 1 e ts 7Hz

7Hz C protons 2 3.27 o c t e t J [ N - C H Z - C H ~ ] =

OH-pro ton 1 3.60 s i n g l e t (broad)

c(5 protons 2 3.75 m u l t i p l e t

C 1 and C2 protons 3 Q3.8-4.9 m u l t i p l e t

cg and Cl1 protons 2 7.05 t r i p l e t JQ-H~ = 5 ~ z

ar0rnati.c protons on t r o p i c 5 7.35

acid moiety

Cg and Cl0 protons 2 8.50 doublet J H ~ - H ~ = 5Hz

4

* Arbi t ra ry numbers were assigned t o the atoms i n t h e s t r u c t u r e f o r ease i n p re sen ta t ion of da t a .

2.3 U l t r a v i o l e t Spectrum The u l t r a v i o l e t spegtrum of tropicamide i n the

region of 200-400 nm is shown i n Figure 3- ( 3 ) . spectrum shows a maximum a t 254 nm (E = 5.1 x 103) and a minimum a t 235-237 nm. The s Q l u t i o n concent ra t ion was 0.025 mg/ml i n 0.1N H C 1 .

The

TROPICAM I DE

Figure 3

Ultraviolet Spectrum of Tropicamide

210 250 300 350

NANOMETERS

571


2 . 4 Fluorescence Spectrum An e x c i t a t i o n axid emission scan were c a r r i e d o u t

with a methanol s o l u t i o n of r e fe rence s tandard t ropicamide. There w a s , however, no f luorescence observed (4).

2.5 Mass Spectrum The low-resolut ion mass spectrum of t ropicamide

is shown i n F igure 4 (5 ) . a CEC 21-110 spectrometer w i th an i o n i z i n g v o l t a g e of 70 eV, which was i n t e r f a c e d wi th a Varian d a t a system 100 MS. The d a t a system accepted t h e ou tpu t of t h e spec t rometer, ca l cu la t ed t h e masses, compared t h e i r i n t e n s i t i e s t o t h e base peak and p l o t t e d t h i s in format ion as a series of l i n e s whose he igh t s were p ropor t iona l t o t h e i n t e n s i t i e s .

The molecular i on was measured a t m / e 284. The base peak a t m / e 254 arises due to t h e l o s s of CH2 = 0 from t h e molecular i on by a McLafferty rear range- ment. rearrangement of m / e 254 leading t o t h e loss of HCO. Cleavage between t h e benzyl carbon and t h e carbonyl group g ives r ise t o t h e i o n a t m / e 163. t he ni t rogen-containing tropylium ca t ion , C6H614+ (5).

The spectrum w a s ob ta ined us ing

The ion a t m / e 225 probably arises via a s k e l e t a l

The ion a t m / e 92 is

2 .6 Op t i ca l Rota t ion Tropicamide does not e x h i b i t o p t i c a l a c t i v i t y .

2.7 Melting Range The mel t ing range repor ted i n t h e United S t a t e s

Pharmacopeia X V I I I f o r t ropicamide is 96-1OO0C when a Class I procedure is used ( 6 ) .

2 .8 D i f f e r e n t i a l Scanning Calor imetry (DSC) The DSC scan of a sample of r e f e r e n c e s tandard

t ropicamide is shown i n Figure 5 ( 7 ) . The temperature w a s r a i sed a t a ra te of 10°C/min. i n an atomosphere of f lowing n i t rogen . A s i n g l e endotherm w a s observed, t h e extrap- o l a t ed onse t of which w a s 95.5 & 0.2OC. melt ing endotherm w a s observed a t 98.6 5 0.2OC. a t u r e s are cor rec ted . endotherm was ca l cu la t ed t o be 8.8 kcal/mole.

The peak of t h e All t e m p e r

The va lue of AHf f o r t h e mel t ing

2.9 Thermogravimetric Analysis (TGA) The r e s u l t s of a TGA scan of t ropicamide ind i -

cated t h a t no weight loss occurred from ambient temperature

572

Figure 4

Mass Spectrum of Tropicamide


F i g u r e 5

DSC Curve f o r Tropicamide

1 I I I AHf = 8.8 kcal / mole

I I I I

OC

80 90 too 110 I :0

574

TROPICAM I DE

t o 15OoC. 15OoC and cont inuing t o 334OC a t which p o i n t 100% of t h e sample weight had been l o s t ( 7 ) .

A s i n g l e weight l o s s w a s observed beginning a t

2.10 S o l u b i l i t y The s o l u b i l i t y d a t a shown i n Table IIwas obta ined

f o r a sample of r e f e r e n c e s t a n d a r d t ropicamide a t a temper- a t u r e of 25OC (8).

Table I1

S o l u b i l i t i e s of Tropicamide

Solvent S o l u b i l i t y (mg/ml)

3A a l c o h o l benzene chloroform 95% e t h a n o l d i e t h y l e t h e r 2 - propanol methanol petroleum e t h e r (30-60') water

235.0 25.9

>500. 321.5

3.9 112.4 >500.

0 .2 5.7

2 . 1 1 C r y s t a l P r o p e r t i e s Table I11 g i v e s i n t e r p l a n a r spac ings from x-ray

powder d i f f r a c t i o n d a t a f o r t rop icamide ( 9 ) . The o p e r a t i n g parameters of t h e ins t rument are g iven below.

Ins t rumenta l Condi t ions :


Genera tor : 50 KV, 12-112 MA Tube t a r g e t : Copper Radia t ion : o p t i c s : 0.1' Detec tor s l i t

Cu Ka = 1.542 8

3' B e a m s l i t 0.0007" N i f i l t e r 4O t a k e o f f a n g l e Scan a t 0.2' 28 p e r minute Goniometer:

575

KENNETH W. BLESSEL. BRUCE C. RUDY, AND BERNARD 2. SENKOWSKI

D e t e c t o r : Ampl i f i e r g a i n - 1 6 c o u r s e , 8 .7 f i n e

Sea led p r o p o r t i o n a l c o u n t e r t u b e and DC v o l t a g e a t p l a t e a u

P u l s e h e i g h t s e l e c t i o n EL - 5 volts

Eu - o u t Rate meter T.C. 4 2000 c/s f u l l scale

Recorder: Cha r t speed 1 i n c h p e r 5

Samples : minu tes

temper a t u r e P repa red by g r i n d i n g a t room

Tab le 111

I n t e r p l a n a r Spacings from Powder D i f f r a c t i o n Data

28 - 10.54 13 .76 14.60 18.14 18.94 19.84 20.40 20.88 21.88 22.60 23.22 24.30 25.38 26.10 26.46 27.54 28.00 28.94

d*

8.39 6.44 6.07 4.89 4.69 4.47 4.35 4.25 4.06 3 .93 3.83 3.66 3.51 3.41 3.37 3.24 3.19 3.09

- I/Io- ** - 9

75 63 27 47 1 2 67 72

100 28 1 3 1 9 1 2

4 4

30 5

4 1

28 - 29.74 30.06 31.42 32.00 33.06 34.46 35.18 35.52 37.04 37.54 39.00 39.82 40.22 40.62 41.66 42.24 42.70 45 * 54

- d* I / T V * *

3 .OO 11 2.97 1 2 2 .85 4 2.80 8 2.71 5 2.60 9 2.55 8 2.53 1 5 2.43 4 2.40 4 2 .31 7 2.26 4 2.24 8 2.22 4 2.17 2 2.14 4 2.12 3 1 . 9 9 6

* d = ( i n t e r p l a n a r s p a c i n g ) nX 2 S i n 8

** I/Io = r e l a t i v e i n t e n s i t y (based o n h i g h e s t i n t e n s i t y of 100)

576

TROPICAMIDE

2.12 D i s s o c i a t i o n Cons tan t The pKa of t rop icamide was determined by s p e c t r o -

pho tomet r i c a n a l y s i s and by a p o t e n t i o m e t r i c t i t r a t i o n . The v a l u e observed w a s 5 .2 by spec t ropho tomet ry and 5 . 3 by po ten t iome t ry (10 ) .

3. S y n t h e s i s

e t h y l - ( y - p i c o l y l ) -amine w i t h t r o p i c a c i d c h l o r i d e , i n the p resence o f b a s e , c a r r i e d o u t i n anhydrous ch lo ro fo rm (11).

4 . S t a b i l i t y Degradat ion A s t u d y h a s been c a r r i e d o u t i n which t h e s t a b i l i t y o f

t rop icamide i n op tha lmic s o l u t i o n was determined (12 ) . A 3% s o l u t i o n of t rop icamide i n op tha lmic s o l u t i o n w a s main- t a i n e d a t t empera tu res r a n g i n g from O°C t o 45OC f o r p e r i o d s of t i m e up t o 1 2 weeks. I n o r d e r t o g a i n i n f o r m a t i o n abou t p o s s i b l e breakdown p r o d u c t s of t rop icamide , pH measurements, t u r b i d i t y d a t a and a d i r e c t s p e c t r o p h o t o m e t r i c a s s a y was performed a t t h e s t a r t and a f t e r 3 , 6 and 1 2 weeks. No ev idence of decomposi t ion w a s found a € t e r p e r i o d s of up t o 12 weeks a t each of t h e above t empera tu res (12 ) .

Tropicamide may b e p repa red by t h e condensa t ion o f

5. Drug Metabo l i c P roduc t s Tropicamide i s used e x c l u s i v e l y € o r op tha lmic s o l u t i o n s

i n t h i s c o u n t r y , and is a p p l i e d t o p i c a l l y . F o r t h i s r e a s o n no m e t a b o l i c s t u d i e s have been pursued.

6. Methods of Ana lys i s

6.1 Elemental Ana lys i s The r e s u l t s of a n elemental a n a l y s i s of a sample

of r e f e r e n c e s t a n d a r d t rop icamide are p r e s e n t e d i n Tab le I V (13 ) .

E l emen t % Theory % Found

C 71.81 71.87 H 7.09 7 . 1 3 N 9.85 9.93

6.2 Phase S o l u b i l i t y Ana lys i s Phase s o l u b i l i t y a n a l y s e s have been c a r r i e d o u t

f o r t rop icamide t o e s t i m a t e t h e - p u r i t y of t h e sample. An


example is shown i n F i g u r e 6 ( 8 ) where t h e s o l v e n t used w a s t o l u e n e and t h e e q u i l i b r a t i o n t i m e was 20 hours a t 25%.

6 . 3 Thin Layer Chromatographic Analys is A TLC system h a s been developed which has proved

t o b e u s e f u l f o r a n a l y s i s of t ropicamide. The adsorbant f o r t h e system is s i l i c a g e l and t h e deve loping s o l v e n t is ch1oroform:methanol:concentrated ammonium hydroxide (90:10:2). The s o l v e n t f r o n t is allowed t o t r a v e l f o r about 15 cm i n a p r e - s a t u r a t e d tank. The p l a t e is a i r dried and then sprayed w i t h iodine-modified Dragendorff r e a g e n t . The approximate Rf of t rop icamide i n t h i s system is 0 . 6 5 (14) .

6.4 Direct Spectrophotometr ic Analys is Tropicamide may b e assayed s p e c t r o p h o t o m e t r i c a l l y

i n opthalmic s o l u t i o n a f t e r an e x t r a c t i o n i n t o chloroform and a back e x t r a c t i o n i n t o d i l u t e s u l f u r i c a c i d . The absorbance of t h i s s o l u t i o n is measured a t t h e wavelength of maximum absorbance a t about 253 nm. The amount of t r o p i c - amide i n t h e opthalmic s o l u t i o n i s c a l c u l a t e d by comparison w i t h a r e f e r e n c e s t a n d a r d sample of t rop icamide measured i n a similar way ( 6 ) .

6.5 Non-Aqueous T i t r a t i o n The non-aqueous t i t r a t i o n d e s c r i b e d i n t h e USP

X V I Z I i s t h e p r e f e r r e d method f o r t h e a n a l y s i s of t r o p i c a - mide i n t h e b u l k form ( 6 ) . The sample is t i t r a t e d i n g l a c i a l a c e t i c a c i d w i t h O . 1 N H C l O 4 i n acet ic a c i d , u s i n g c r y s t a l v i o l e t as t h e i n d i c a t o r . One m l of 0.1N HClO4 is e q u i v a l e n t t o 28.44 mg of t ropicamide.

7 . Acknowledgments The a u t h o r s wish t o acknowledge t h e S c i e n t i f i c Litera-

t u r e Department and t h e Research Records O f f i c e of Hoffmann-La Roche I n c . f o r t h e i r a s s i s t a n c e i n t h e l i t e r a - t u r e s e a r c h f o r t h i s a n a l y t i c a l p r o f i l e ,

578

1 I I I I 1 1 1 1 1 I 1 I '

- - - n n n - n

.# - -

PHASE SOLUBILITY ANALYSIS - Sample : Tropicomide Solvent : Toluene Slope : .150/0 - Equilibrotian : 20 hrs at 25OC - Extrapolated Solubility : 12.35 mq/g

-

- - - - - -

1 1 1 1 1 1 1 1 , ) 1 1 1 1 l l l l d

0 25


8. References

1.

2.

3.

4.

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Hawrylyshyn, M., Hoffmann-La Roche Inc., Personal Communication. Johnson, J. H., Hoffmann-La Roche Inc., Personal Communication. Rubia, L. B., Hoffmann-La Roche Inc., Personal Communication. Boatman, J., Hoffmann-La Roche Inc. , Personal Communication. Benz, W., Hoffmann-La Roche Inc., Personal Communication. The United States Pharmacopeia XVIII, pp. 762-763 (1970) . Moros, S., Hoffmann-La Roche Inc., Personal Communication. MacMullan, E., Hoffmann-La Roche Inc., Personal Communication. Hagel, R., Hoffmann-La Roche Inc., Personal Communication. Heveran, J., Hoffmann-La Roche Inc., Unpublished Results. Hoffmann-La Roche Inc. , United States Patent 2,726,245 (1955) . Bollinger, A., Hoffmann-La Roche Inc., Unpublished Data. Scheidl, F., Hoffmann-La Roche Inc., Personal Communication. Sokoloff , H. , Hoffmann-La Roche Inc. , Unpublished Results.

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CUMULATIVE INDEX Italic numerals refer to Volume numbers.

Acetaminophen, 3, 1 Acetohexamide, I, 1; 2,573 Alpha-Tocopheryl Acetate, 3, 11 1 Amitriptyline Hydrochloride, 3, 127 Ampicillin, 2. 1 Chlorprothixene, 2, 63 Chloral Hydrate, 2, 85 Chlordiazepoxide, 1, 15 Chlordiazepoxide Hydrochloride, 1, 39 Clidinium Bromide, 2, 145 Cycloserine, I, 5 3 Cyclothiazide, 1, 66 Dexamethasone, 2, 163 Diazepam, 1. 79 Digitoxin, 3, 149 Dioctyl Sodium Sulfosuccinate, 2, 199 Diphenhydramine Hydrochloride, 3, 17 3 Echothiophate Iodide, 3, 233 Erythromycin Estolate, I, 101; 2, 573 Ethynodiol Diacetate, 3, 253 Fludrocortisone Acetate, 3. 281 Fluorouracil, 2, 221 Fluphenazine Enanthate, 2, 245 Fluphenazine Hydrochloride, 2, 263 Nurazepam Hydrochloride, 3, 307 Halothane, I, 119; 2, 573 Iodipnmide, 3, 333 Isocarboxazid, 2, 295 Isopropamide, 2, 3 15 Levallorphan Tartrate, 2, 339

Lavatesenol Bitartrate, 1, 149; 2, 573 Meperidine Hydrochloride, 1. 175 Meprobamate, I, 209 Methadone Hydrochloride, 3, 365 Methyprylon, 2, 363 Nortriptyline Hydrochloride, 1, 233; 2, 573 Oxazepam, 3, 441 Phenazopyridine Hydrochloride, 3 ,465 Phenelzine Sulfate, 2, 383 Phenylephrine Hydrochloride, 3, 483 Potassium Phenoxymethyl Penicillin, 1, 249 Primidone, 2, 409 Propiomazine Hydrochloride, 2, 439 Propoxyphene Hydrochloride, I , 301 Sodium Cephalothin, 1, 319 Sodium Secobarbital, I, 343 Sulfamethoxazole, 2, 467 Sulfisoxazole, 2, 487 Tolbutamide, 3, 513 Triamcinolone, 1, 367; 2, 571 Triamcinolone Acetonide, I, 397; 2, 571 Triamcinolone Diacetate, I, 423 Triclobisonium Chloride, 2, 507 Triflupromazine Hydrochloride, 2, 523 Trimethaphan Camsylate, 3, 545 Trimethobenzamide Hydrochloride, 2, 55 1 Tropicamide, 3, 565 Vinblastine Sulfate, I, 443 Vincristine Sulfate, 1, 463

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