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Analytical Profiles of Drug Substances and Excipients

Volume 21
Edited by

Harry G. Brittain
Bristol-Myers Squibb Pharmaceutical Research Institute New Brunswick, New Jersey

Founding Editor

Klaus Florey

ACADEMIC PRESS, INC.


Harcourt Brace Jovanovich, Publishers
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EDITORIAL BOARD

Abdullah A. Al-Badr Gerald S. Brenner Glenn A. Brewer Harry G. Brittain Klaus Florey

George A. Forcier Lee T. Grady David J. Mazzo Thomas W. Rosanske Timothy J. Wozniak

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AFFILIATIONS OF EDITORS AND CONTRIBUTORS

Mohummud A . Abounussf, Pharmaceutical Chemistry Department, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia Abdul Furruh A . A . Ajfy, Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia Iqbal Ahmad, Pharmaceutical Chemistry Department, Faculty of Pharmacy, University of Karachi, Karachi 75270, Pakistan Tuuqir Ahmud, Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Karachi, Karachi 75270, Pakistan Abdulluh A . Al-Budr, Pharmaceutical Chemistry Department, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia Fuhud J. Al-Shammury, Clinical Laboratory Sciences Department, College of Applied Medical Sciences, King Saud University, Riyadh 11433, Saudi Arabia Silvia Alessi-Severini, Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta T6G 2N8, Canada Syed Laik Ali, Zentrallaboratorium Deutscher Apotheker, 6236 Eschborn, Germany Adnun A . Budwun, The Jordanian Pharmaceutical Manufacturing Company, Naor, Jordan Gary Burberu, Bristol-Myers Squibb, Pharmaceutical Research Institute, New Brunswick, New Jersey 08903 Gerald S. Brenner, Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania 19486 Glenn A . Brewer, Bristol-Myers Squibb, Pharmaceutical Research Institute, New Brunswick, New Jersey 08903 Hurry G . Brirruin, Bristol-Myers Squibb, Pharmaceutical Research Institute, New Brunswick, New Jersey 08903 Marvin A . Brooks, Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania 19486 Robert A . Curr, Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta T6G 2N8, Canada Owen 1. Corrigan, School of Pharmacy, University of Dublin, Dublin 4, Ireland Ronald T. Coutts, Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta T6G 2N8, Canada

vii

Vlll

...

AFFILIATIONS OF EDITORS AND CONTRIBUTORS

Joseph D. DeMarco, Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania 19486 Joseph DeVincenfis, Bristol-Myers Squibb, Pharmaceutical Research Institute, New Brunswick, New Jersey 08903 Humeida A . El-Obeid, Pharmaceutical Chemistry Department, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia Dean K. Ellison, Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania 19486 Klaus Florey, Bristol-Myers Squibb Company, Lawrenceville, New Jersey 08543 George A . Forcier, Central Research Division, Pfizer, Inc., Groton, Connecticut 06340 Robert T. Foster, Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta T6G 2N8, Canada Lee T. Grady, The United States Pharmacopeia, Rockville, Maryland 20852 Dominic I? Zp,Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania 19486 Fakhreddin Jamafi, Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AlbertaT6G 2N8, Canada Eric C. Jensen, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285 Michael J. KauJinan, Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania 19486 David J. M a u o , Department of Analytical & Physical Chemistry, RhBnC-Poulenc Rorer Recherche-Development,92165 Antony Cedex, France Michael J. McLeish, School of Pharmaceutical Chemistry, Victorian College of Pharmacy, Monash University, Parkville, Victoria 3052, Australia Mohammad SafeemMian, Pharmaceutical Chemistry Department, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia Neelofur Abduf Aziz Mian, Clinical Laboratory Sciences Department, College of Applied Medical Sciences, and Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh 11433, Saudi Arabia Ann W. Newman, Bristol-Myers Squibb, Pharmaceutical Research Institute, New Brunswick, New Jersey 08903 CaifrionaM . ODriscoll, School of Pharmacy, University of Dublin, Dublin 4, Ireland Mahmoud A1 Omari, The Jordanian Pharmaceutical Manufacturing Company, Naor, Jordan Franco M . Pasutto, Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta T6G 2N8, Canada Thomas W Rosanske, Marion Merrell Dow, Inc., Kansas City, Kansas 64134 Charles M . Shearer, Wyeth-Ayerst Research, Rouses Point, New York 12979 Delores J. Sprankle, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285

AFFILIATIONS OF EDITORS AND CONTRIBUTORS

ix

K . Usmanghani. Department of Pharmacognosy, Faculty of Pharmacy, University of Karachi, Karachi 75270, Pakistan G.Michael Wall, Alcon Laboratories, Inc., Fort Worth, Texas 76134 Titnothy J. Wozniak, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285 Muhammad B . Zughul, Department of Chemistry, Faculty of Science, University of Jordan, Amman, Jordan

PREFACE

The profiling of drug compounds as to their physical and analytical characteristics has been the focus of the preceding twenty volumes in the Analytical Profiles series, and the need for this information is as important today as it was when the series was first initiated. The compilation of concise summaries of physical and chemical data, analytical methods, routes of compound preparation, degradation pathways, and the like, is a vital function to both academia and industry. Under the editorship of Klaus Florey, the Analytical Profiles has met this need over its twenty year history. With the publication of Volume 21, the editorship has been assumed by Harry Brittain. The focus of the chapters will remain unchanged, but the scope of the Analytical Projiles series has expanded to include profiles of excipient materials, and this has led to a modification of the series title. The series will henceforth be known as the Analytical Profiles o Drug Substances and Excipients. The first excipient f profile (anhydrous lactose) appeared in Volume 20, and a profile on titanium dioxide is included in the present volume. The success of the series will continue to be based on the contributions of the chapter authors, and on the quality of their work. We seek profiles of new drug compounds as they come to markets but we also wish to profile important older compounds that have escaped attention thus far. A complete list of available candidates can be obtained from the editor by any prospective author. We look forward to hearing from new and established authors and to working with the pharmaceutical community on the Analytical Profiles o Drug Substances and f Excipients.

Harry G . Brittain Editor

Klaus Florey Founding Editor

xi

ACETOHEXAMIDE

Abdullah A. Al-Badr and Humeida A. El-Obeid

Pharmaceutical Chemistry Department

College of Pharmacy
King Saud University

Riyadh, Saudi Arabia

ANALYTICAL PROFILES OF DRUG SUBSTANCES AND EXCIPIENTS VOLUME 21

Copyright 0 1992 by Academic Press, Inc. All rights o reproduction reserved in any form f

ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID

C O N T E N T S
1.

DESCRIPTION 1 . 1 Nomenclature 1.1.1 Chemical Names 1.1.2 Genermic Names 1.1.3 Trade Names 1 . 2 Formulae 1.2.1 Empirical 1.2.2 Structural 1.2.3 GAS No. 1.3 Molecular Weight 1 . 4 Elemental Composition 1 . 5 Appearance
PHYSICOCHEMICAL PROPERTIES 2 . 1 Melting Range 2.2 S o l u b i l i t y 2 . 3 Polymorphism 2 . 4 Thermal Analysis 2 . 5 X-ray Powder D i f f r a c t i o n 2.6 Spectral Properties 2.6.1 U l t r a v i o l e t Spectrum 2.6.2 I n f r a r e d Spectrum 2.6.3 Proton Nuclear Magnetic Resonance (PMR) Spectrum 2.6.4 lac-Nuclear Magnetic Resonance (SC-NMR) Spectrum 2.6.5 Mass Spectra

2.

3. 4.

SYNTHESIS
METHODS OF ANALYSIS 4 . 1 T i t r i m e t r i c Methods 4.1.1 Nonaqueous 4.1.2 Gravimetric 4.1.3 Campleximetric 4.2 Spectrometric Methods 4.2.1 Colorimetric 4.2.2 U1t r a v i o l e t 4.2.3 Infrared 4.2.4 Fluormetric 4.2.5 Proton Magnetic Resonance

ACETOHEXAMIDE

4.3

Chromatographic Methods 4.3.1 Thin-Layer Chromatography (TLC) 4.3.2 Gas-Liquid Chromatography (GLC) 4.3.3 High-Performance Liquid Chromatography (HPLC)

5.

PHARMACOKINETICS 5 . 1 Introduction 5.2 Mechanism o f Action 5.3 Onset and Duration o f Action 5.4 Absorption 5.5 Distribution 5.6 Metabol ism 5.7 Excretion 5.8 Half-Life

ACKNOWLEDGEMENT

REFERENCES

ABDULLAH A. AL-BADR AND HUMEIDA A . EL-OBEID

ACETOHEXAMIDE 1. DESCRIPTION 1 1 Nomenclature 1.1.1 fonamide Chemical Names

4-Acetyl-N-[(cyclohexylamino)carbonyl]benzenesull-[(pAcetylphenyl)sulfonyl]-3-cyclohexylurea. 3-Cyclohexyl-l-(pacetylphenylsulfonyl)urea.
1.1.2 Generic Names

N-(pAcetyl benzyl sul fonyl l-N -cyclohexyl urea.

Acetohexamide, Acetohexamidum 1 1.3

Trade Names

Cycl am1de , Dime 1in , Dime1o r , Dyme 1o r , Gamadiabet, Metaglucina, Ordimel, Tsiklamid. 1.2 Formulae 1.2.1. EmDlriCal

Ct sHzoNz04S

1.2.2

Structural

1.2.3

CAS No.

[968-81-01 1.3 Molecular Weight 324.42 (1) 1.4 Elemental ComDosltion


C 55.54%, H 6.21%, N 8.64%, 0 19.73%,

S 9.89% (1).

ACETOHEXAMIDE

1.5

Armearance

A w h i t e , c r y s t a l l i n e powder; o d o r l e s s o r almost odorless (2).

2.

PHYSICOCHEMICAL PROPERTIES
2.1

M e l t i n g Range

C r y s t a l s from 90% aqueous e t h a n o l m e l t between 188-190" ( 3 ) . Crystals from d i l u t e ethanol m e l t between 175-177 (4).
2.2

Solubility

Soluble i n p y r i d i n e , s l i g h t l y soluble i n alcohol and chloroform. I n s o l u b l e i n water and ether ( 1 ) .


2.3

P01YmOrDh'ism

The l i t e r a t u r e r e p o r t s i n d i c a t e t h a t acetohexamide e x i s t s as more t h a n one polymorphic forms ( 5 - 1 5 ) . G i rgis-Takla and Chroneos (5) prepared acetohexamide polymorphs A and B by h e a t i n g t h e drug ( 1 gm) w i t h g l a c i a l a c e t i c a c i d o r chloroform respectively, before c r y s t a l l i z a t i o n a t 1 0 5 ' and room t e m p e r a t u r e respectively. While acetohexamide polymorph A showed a m e l t i n g range o f 180"-183', t h e acetohexamide polymorph B melted a t 183'-185". D i f f e r e n t i a l scanning calorimetry and I R spectroscopy showed t h a t c r y s t a l s o f polymorph B were converted t o polymorph A by grinding. A c c o r d i n g l y , t h e s e r e s u l t s i n d i c a t e t h a t any i d e n t i f i c a t i o n t e s t u t i l i z i n g g r i n d i n g may f a i l to i d e n t i f y t h e two polymorphs. I n t h e i r phystco-chemical studies on t h e polymorphism o f acetohexamide, Kuroda e t a7 (6) obtained t h r e e polymorphs o f acetohexamide by r e c r y s t a l l i z a t i o n from d i f f e r e n t solvents. These are f o r m I,f o r m I 1 and CHC13-11. A l t h o u g h t h e X-ray d i f f r a c t i o n p a t t e r n s , I R s p e c t r a and d i f f e r e n t i a l scanning calorimeter curves o f t h e CHC13-I1 polymorph were i d e n t i c a l w i t h those o f polymorph 11, t h e CHC13-I1 t y p e c o n t a i n e d a C H C l j molecule which c o u l d n o t be removed by normal d r y i n g condition. Polymorph CHC13-I1 seemed t o be unsuitable f o r medicinal use. Form I 1 i s 1.2 times more soluble than form I .

ABDULLAH A. AL-BADR AND HUMElDA A. EL-OBEID

Burger ( 7 ) c h a r a c t e r i z e d t h e t h r e e p o l y m o r p h i c m o d i f i c a t i o n s o f acetohexamide by thermomicroscopy, d i f f e r e n t i a l scanning calorimetry and I R spectroscopy. The s o l u b i l i t y behavior o f the three modifications o f the drug i n butanol and buffer solutions i s described and d i s c u s s e d i n r e l a t i o n t o thermodynamics and pharmacological parameters such as b i o a v a i l a b i l i t y from t a b l e t s and USP X I X d i s s o c i a t i o n t e s t . M u e l l e r and L a g a s ( 8 ) h a v e c o n f i r m e d t h e e x i s t e n c e and characterized two polymorphic forms o f acetohexamide using d i f f e r e n t i a l scanning calorimetry, thermogravimetric analysis, scanning e l e c t r o n microscopy as we1 1 as I R , NMR and X-ray analysis. The study has pointed t o the u n s u i t a b i l i t y o f phosphate b u f f e r s o l u t i o n which i s sometimes prescribed f o r use i n the d i s s o l u t i o n t e s t s o f the drug since the s a l t o f the drug c r y s t a l l i z e s out during the t e s t . I n another study (9) the same authors reported t h a t form Idecomposed during melting and form I1 melted a t 180" and then r e c r y s t a l l i z e d t o form I A t . a heating r a t e o f lO'/minute melting points o f 193.6" and 180.5" were found f o r forms Iand 1 , respectively. 1 No morphological differences were observed between the two forms. I n s o l u b i l i t y and d i s s o l u t i o n r a t e studies i n sodium potassium b u f f e r , potassium acetohexamide c r y s t a l l i z e d e x h i b i t i n g a lower s o l u b i l i t y than acetohexamide. I n t h i s respect, form I 1 was transferred t o potassium acetohexamide more quickly than form I . Yokoyama e t a7 (10) calculated the thermodynamic values o f forms I and I 1 o f acetohexamide from s o l u b i l i t y measurements. The t r a n s i t l o n temperature and the heat o f t r a n s i t i o n were 154" and 230 cal/mole, respectively. I t i s found t h a t the polymorphic forms o f acetohexamide d i d n o t a f f e c t i t s b i o a v a i l a b i l i t y when i n v i v o absorption studies o f form I & I 1 were c a r r i e d out i n b e a g l e dogs. The p r e p a r a t i o n o f f o u r c r y s t a l l i n e modifications o f acetohexamide was reported (11). Their thermograms, I R s p e c t r a , X-ray d i f f r a c t i o n and s o l u b i l i t y are also reported. Two o f the forms reverted t o the most stable form on storage i n solution. S o l i d dispersion o f acetohexamide was studied by Graf e t a7 (12-14) u s i n g d i f f e r e n t polymers and v a r i o u s ratios. C o p r e c i p i t a t e s o f acetohexamide w i t h polyethylene g l y c o l (PEG 6000) were prepared by t h e s o l v e n t method w i t h ethanol ( c r y s t a l l i n e form I) r o with chloroform ( c r y s t a l 1ine form 111). Phase diagrams

ACETOHEXAMIDE

o f form I-PEG and form 111-PEG coprecipitates were o f the p e r i t e c t i c type and the molecular compounds were formed i n the r a t i o o f 1 mole o f acetohexamide t o 4 moles o f PEG. The e u t e c t l c t e m p e r a t u r e , e u t e c t i c composition and the end o f melting o f the two binary system were, however, d i f f e r e n t ( 1 2 ) . Both the s o l u b i l i t y and t h e s o l u t i o n r a t e were increased by PEG. S i m i l a r r e s u l t s were o b t a i n e d by s u b s t i t u t i n g p o l y ( v i n y l p y r r o 1 i d o n e ) (PVP) f o r PEG ( 1 3 ) . Also, c o p r e c i p i t a t e s o f acetohexamide-PVP ( i n e t h a n o l ) containing drug concentrations o f 60% o r more showed the same X-ray d i f f r a c t i o n pattern as t h a t o f form I . Increasing the PVP concentration ( > 55%) d i d n o t show any c r y s t a l behavior i n the X-ray analysis. I n another r e p o r t Graf e t a7 ( 1 4 ) d e s c r i b e d t h e methods o f p r e p a r a t i o n and t h e e f f e c t o f t h e s o l v e n t s on t h e acetohexamide-PVP coprecipi tates. They were obtained from ethanol o r chloroform by evaporating the solvent a t room temperature, under vacuum or by spray drying. Changing t h e s o l v e n t and/or i t s e v a p o r a t i o n r a t e affected the polymorphic form, the c r y s t a l l i n i t y and the s o l u t i o n r a t e o f acetohexamide i n coprecipitates containing less than 70% PVP. Kassem e t a7 (15) studied the enhancement o f the r a t e o f release o f acetohexamide from i t s t a b l e t s by t h e f o r m a t i o n o f s o l i d d i s p e r s i o n s w i t h each o f f o u r water-sol uble pol ymers prepared in d i f f e r e n t r a t i0s. The polymers were r a t e d i n t h e o r d e r o f decreasing r e l e a s e r a t e s as f o l l o w s : PEG 6 0 0 0 , PVP, hydroxypropylmethylcellulose, methylcellulose.
2.4

Thermal Analysis

The h e a t o f f u s i o n and m e l t i n g p o i n t o f acetohexamide were done u s i n g DuPont TA 9900 on t h e DSC- u n i t a t a temperature range i n d i c a t e d i n t h e thermogram (Figure 1). Sample i s done i n duplicate and the average o f t h e value i s reported as follows:

AHf
2.5

63.7 kJ/mOle

Purity

99.82%

Tm

187.45 C

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

The X-ray powder d i f f r a c t i o n p a t t e r n o f acetohexamide was determined using P h i l i p s f u l l automated X-ray d i f f r a c t i o n spectrogoniometer equipped w i t h PW1730/10

PURITY v l . l A
F i g u r e 1. Thermal cu rve o f acetohexamide.

ACETOHEXAMIDE

J
Figure 2 . X-Ray powder d i f f r a c t i o n p a t t e r n of acetohexamide.

10

ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID

generator. Radiation was provided by a copper t a r g e t (Cu anode 2000W, X = 1.5480 A), high I n t e n s i t y X-ray tube operated a t 40 kV and 35 mA. The monochromator was a curved s i n g l e c r y s t a l one (PW1752/00). Divergance s l i t and t h e r e c e i v i n g s l i t were 1 and 0.1 r e s p e c t i v e l y . The scanning speed o f t h e gonlometer (PW1050/81) used was 0.02 2 8 p e r second. The instrument i s combined w i t h P h i l i p s PM8210 p r i n t i n g r e c o r d e r w i t h b o t h analogue r e c o r d e r and d i g i t a l p r i n t e r . The goniometer was a l i g n e d using s i l i c o n sample before use. The X-ray pattern o f acetohexamlde I s presented i n Figure 2. The interplanar distance d(A) and r e l a t i v e i n t e n s i t i e s 1/10 are shown i n Table 1. 2.6 Spectral ProDerties 2.6.1 U l t r a v i o l e t Spectrum

The u l t r a v i o l e t a b s o r p t i o n s p e c t r u m o f acetohexamide i n methanol was obtained on a Cary 219 spectrophotometer. The spectrum, shown i n Figure 3, i s characterized by two maxima. The one w i t h a Xmax a t 247 n i s t y p i c a l o f s u b s t i t u t e d acetophenones. The m absorption a t X m a x 283 n represents a conjugated m aromatic r i n g system. 2.6.2 I n f r a r e d SDectrum

The i n f r a r e d absorption spectrum o f acetohexamide, obtained from a potassium bromide d i s p e r s i o n , was recorded on a Pye Unicam SP 1025 spectrometer and i s shown i n Figure 4. The assignment o f the c h a r a c t e r i s t l c bands are shown i n Table 2.

220

XO

nm

300

3 50

400

450

Figure 3 . U l t r a v i o l e t spectrum o f acetohexamide in methanol.

v,.
c
C. N

m.
-I

cn

I-

6. ,
m
a. l
Q.

z!
W

Y "

E N

c
t-.
V

Y W

0 c , W

tcl

'*0
L c ,
V W

f
0

*I

cn

v, )

fu

ACETOHEXAMIDE

13

Table 1: X-ray d i f f r a c t i o n p a t t e r n o f acetohexamfde d(A)


15.74 9.47 7.85 7.21 5.30 4.99 4.93 4.55 4.30 4.19 4.08 3.92 3.78 3.60 3.50 3.28 3.26 3.15 3.07 3.01 2.91 2.88 2.74 2.65 2.61 2.58

1/10
31.25 30.04 6.89 2.25 100.00 8.28 10.95 4.71 5.30 15.19 23.07 5.44 2.82 24.35 4.52 23.29 9.83 5.72 9.36 1.26 7.99 2.79 4.08 1.51 2.15 1.80

d(A)
2.55 2.49 2.40 2.36 2.31 2.29 2.27 2.24 2.18 2.15 2.13 2.09 2.04 1.99 1.95 1.94 1.89 1.81 1.77 1.72 1.66 1.64 1.61 1.57 1.47 1.35

11 /0 1.82 1.75 6.19 4.44 5.26 2.20 1.81 2.54 2.04 2.38 1.20 2.56 4.51 1.69 2.91 4.16 1.50 1.48 1.29 1.77 1 .oo 1.18 1.16 1.32 0.85 0.80

d 1/10

= Interplanner distance

r e l a t i v e i n t e n s i t y (based on highest i n t e n s i t y of
100).

14

ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID

Table 2:

I n f r a r e d c h a r a c t e r i s t i c bands and t h e i r assignments. Assignment Amide N-H s t r e t c h Aromatic C-H s t r e t c h


0

Frequency (cm3340, 3270 2980, 2940 1710, 1680 1602 , 1600 1455 1345 780, 760

Conjugated

E-

Aromatic C s t r e t c h
C

CH3 bending

Aromatic C-H out o f plane bending

2.6.3

Proton Nuclear Magnetic Resonance ( W R l Spectrum

Acetohexamide s o l u t i o n i n DMSO-de was used t o obtain the PMR spectrum on a Varian XL 200 MHr FT NWR spectrometer u s i n g TMS as i n t e r n a l reference. The spectrum i s shown i n Figure 5. The number o f protons i s established by both i n t e g r a t i o n o f the area under the curve and t h e m u l t i p l i c i t i e s o f t h e peaks. Table 3 a s s i g n s t h e chemical s h i f t s t o t h e i r r e s p e c t i v e protons. F u r t h e r evidence f o r p r o t o n assignment i s obtained fo the HETCOR pulse sequence (Figure 9). rm

Figure 5. PMR spectrum of acetohexamide i n DMSO-dG as internal reference.

using TMS

F i g u r e 6. 1 3 C NMR spectrum o f acetohexzmide i n DMSO-ds TMS as internal reference.

using

Figure 7.

1 3 C NMR spectrum o f acetohexarnide u s i n g DEPT ex P e r i ment

F i g u r e 8.

1 3 C I M R sDectrum of acetohexanide u s i n g APT expe r irnent .

Figure 9.

13C N M R spectrum of acetohexamide using HETCOR experiment.

20

ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID

Table 3:

Assignment o f the NMR chemical s h i f t s t o the d i f f e r e n t protons Multiplicity mu1ti p l e t Proton assignment Cyclohexyl ring3
0

Chemical s h i f t (6) 1.09 - 1.71

No. o f protons 11

2.66 6.45 8.06

singlet doublet mu1t ip l e t

-3-0 CH CH-NH
Aromat i d c

1 4

8.19

2.6.4

13C-Nuclear Magnetic Resonance SDect rum

(13C

NHR)

The 1 3 C NMR spectra o f acetohexamide i n DMSO-ds using TMS as i n t e r n a l reference are obtained using a V a r i a n XL 200 MHz p u l s e FT s p e c t r o m e t e r and a r e p r e s e n t e d i n F i g u r e s 6-9. The assignment o f t h e chemical s h i f t s and the degree o f carbon protonation, presented i n Table 4, are achieved u s i n g t h e DEPT (Figure 7) and APT (Figure 8) experiments as well as t h e HETCOR p u l s e sequence ( F i g u r e 9 ) and t h e approximate a d d i t i v e e f f e c t s o f substituents.
2.6.5

Mass SDectra

The 70 eV e l e c t r o n impact mass spectrum o f acetohexamide, presented i n Figure 10, was obtained on Varian MAT 311 mass spectrometer u s i n g i o n source pressure o f 10-0 Torr, i o n source temperature o f 180'C and an emission current o f 300 M. The molecular i o n i s detectable a t m/e 324 and the base peak a t m/e 56. A proposed fragmentation p a t t e r n and t h e mass/charge r a t i o s o f the major fragments are shown I n Scheme 1.

ACETOHEXAMIDE

21

Table 4:

Assignment o f the carbon chemical s h i f t s .

Chemical s h i f t (PHI
24.26 25.07 26.99 32.33 48.30 127.73 128.73 140.00 143.93 150.45 197.30

Carbon assignment

Number o f Protons attached


2 2 3 2 1 1 1

d
C

e b a

j
k

0
0

h
9 f

0 0

The chemical i o n i z a t i o n spectrum, shown i n Figure 11, was obtained on Finnigan 4000 mass spectrometer using methane gas as a reagent with ion electron energy o f 100 eV, ion source pressure o f 0 . 3 T o r r , i o n source temperature o f 150C and emission current o f 300 pA. The spectrum i s dominated by a quasimolecular i o n (M + 1 ) . Two peaks appearing a t m/e 353 and m/e 365 are a t t r i b u t a b l e t o the t r a n s f e r o f carbocations from the c a r r i e r gas. The mass s p e c t r a l assignment o f t h e

N N

F i g u r e 10. Electron impact mass spectrum o f

acetohexamide.

Figure 11. Chemlcal ionization mass spectrum o f acetohexarnide.

24

ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID

prominent ions under the chemical i o n i z a t i o n conditions are presented i n Table 5. Table 5: Mass spectral assignment o f acetohexamide using chemical ionization. Species
[M
[M [M
t t t

M/e
365 353 325 324

C3H5]+ CzHs]+ H (MH)1+

[MI+

3.

SYNTHESIS

Marshall e t a7 ( 4 ) reported a method o f synthesis o f acetohexamide which i n v o l v e s t h e r e a c t i o n o f t h e diazonium s a l t from paminoacetophenone w i t h s u l f u r dioxide t o a f f o r d the sulfonyl chloride which i s then converted t o the sulfonamide by reaction w i t h a m n i a . E l a b o r a t i o n v i a t h e carbamate w i t h cyclohexylamine a f f o r d s acetohexamide. Another r e p o r t e d method ( 1 6 ) uses p-chloroacetophenone as t h e s t a r t i n g m a t e r i a l . Both methods are o u t l i n e d i n Scheme 2.

ACETOHEXAMIDE

25

Scheme 1: Proposed mass fragmentation pattern o f acetohexamide

n
0
O
mle 3 2 4

W-QH

I
0-H
0
m/e 243

+
NH I1

26

ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID

Scheme 1 Continued ...

m l e 324

mle183

I -CH,-CO

m /el41

mle 324

m l e 200

0 C H3I;

- YN0,S

mle76

m l e 104

mle 119

ACETOHEXAMIDE

21

Scheme 1 Continued

...

1
O N H I +

m/e 324

II

2 68

28

ABDULLAH A . AL-BADR A N D HUMEIDA A. EL-OBEID

Scheme 3: Synthesis o f acetohexamide


Method 1 (4)

SO,-NH-C-NH

Method 2 (16)

0
C ,@ Ht

0
S03Na p0c13*

c H 3 - ! G so, CI

S , HON ,

ACETOHEXAMIDE

29

4 .

METHODS OF ANALYSIS

4.1

T i t r i m e t r i c Methods

4.1.1

Nonaaueous

A non-aqueous t i t r a t i o n method f o r t h e d r u g and other hypoglycemic and d i u r e t i c agents was reported by Agarwal and Walash (17). The drug i n t a b l e t o r pure form was d i s s o l v e d i n t e t r a m e t h y l urea and t i t r a t e d w i t h 0.1 N l i t h i u m m e t h o x i d e i n benzene-methanol medium. The end p o i n t was determined u s i n g 0.2% azo v i o l e t i n toluene as i n d i c a t o r . Recovery ranged from 98.8% t o 101.6%.

A n o t h e r non-aqueous t i t r a t i o n p r o c e d u r e , f o r t h e q u a n t i t a t i v e a n a l y s i s o f t h e d r u g and o t h e r h y p o g l y c e m i c s u l f o n y l u r e a s u s i n g HC104 t i t r a t i o n method, was a l s o reported (18).

4.1.2

Gravimetric

Amer and Walash (19, 20a) r e p o r t e d a method f o r t h e g r a v i m e t r i c d e t e r m i n a t i o n o f acetohexamide by treatment w i t h 2,4-dinitrophenylhydrazine t o p r e c i p i t a t e t h e h y d r a z o n e (19). A m i x t u r e o f acetohexamide, tolbutamide and chlorpropamide was a l s o determined g r a v i m e t r i c a l l y (20a).

4.1.3

Compleximetric

G u e r e l l o and Dobrecky (21) have d e s c r i b e d a procedure f o r t h e compleximetric e v a l u a t i o n o f medications w i t h hyoglycemic a c t i o n i n c l u d i n g acetohexamide. The procedure permits the determination o f t h e hypoglycemic sulphonylureas. A weighed amount o f drug was h y d r o l y s e d by h e a t i n g f o r 30 minutes w i t h d i l u t e aqueous sodium h y d r o x i d e and t h e s o l u t i o n n e u t r a l i z e d w i t h 0.1 N HC1, t r e a t e d w i t h 0.1 M CuSO4, then w i t h b u f f e r s o l u t i o n t o pH 6, and f i l t e r e d . The excess C U + ~ i n t h e f i l t r a t e was d e t e r m i n e d by complexometric t i t r a t i o n w i t h 0.02 M EDTA disodium s a l t u s i n g 1-(2-pyridylazo)-2-naphthol as i n d i c a t o r . The method i s applicable t o evaluate drugs i n t a b l e t .

30

ARDUI.1.AH A. AL-BADR AND HUMEIDA A. EL-OBEID

4.2

SDect romet r i c 4.2.1 Colorimetric

Reaction o f acetohexamide w i t h 2,4-dinitrophenylhydrazine t o produce t h e colored hydrazone was used by Amer and Walash (19) t o determine t h e drug c o l o r i m e t r i c a l l y . The c o l o r e d p r o d u c t was d i s s o l v e d i n KOH and determined a t 480 nm. The accuracy o f the method was claimed t o be 100%. A n i n h y d r i n c o l o r i m e t r i c method f o r some o r a l hypoglycemic agents was a l s o reported (20b). Meier e t a7 ( 2 2 ) analysed acetohexamide and o t h e r h y p o g l y c e m i c a g e n t s by d i s s o l v i n g t h e d r u g i n chloroform, adding calcium acetate (1% i n methanol), propylamine (5% i n methanol), d i l u t i n g w i t h chloroform and reading the absorbance a t 565 n a f t e r 15 minutes. m Pharmaceutical preparations may be estimated s i m i l a r l y . 4.2.2 U l t r a v i o l e t (UV)

Solomonova and D v o r n i t s k a y a ( 2 3 ) d e t e r m i n e d acetohexamide by measuring t h e absorbance a t 229 n i n m ethanol or 0.1 M sodium hydroxide. Other UV t e s t s f o r t h e drug are a l s o reported (24, 25). 4.2.3 Infrared (IR)

Acetohexamide and o t h e r s u l p h o n y l u r e a s were analysed by IR (22). A t e s t have a l s o been described (24). Lazaryan (26) determined t h e d r u g and o t h e r h y p o g l y c e m i c a g e n t s by i n f r a - r e d a b s o r p t i o m e t r i c determination. A sample i s t r e a t e d with chloroform and t h e s o l u t i o n from t h e t a b l e t sample i s f i l t e r e d . A p o r t i o n o f s o l u t i o n i s d i l u t e d w i t h chloroform and t h e absorbance i s measured a t 1722 t o 1715 cm-1 i n 0.25 m NaCl c e l l against chloroform. 4.2.4 F1uoromet r ic

G i r g i s - T a k l a and Chroneos ( 2 7 ) d e s c r i b e d a s e n s i t i v e method f o r t h e f l u o r o m e t r i c determination o f t h e d r u g i n plasma o r i n t a b l e t s by means of i t s r e a c t i o n w i t h 1 - m e t h y l n l c o t l n a m i d e . The l i m i t o f d e t e c t i o n was approximately 0.2 M o f t h e drug/mL and g t h e r e l a t i v e standard d e v i a t i o n was 31% f o r 2 Ng/ml i n

ACETOHEXAMIDE

31

plasma. The method i s s u i t a b l e f o r plasma samples containing 0.5-2.5 M o f the drug/ml. g 4.2.5 Proton Magnetic Resonance

Al-Badr and Ibrahim (28) described a simple, r a p i d and accurate method f o r t h e assay o f the drug and other hypoglycemic agents u s i n g p r o t o n magnetic resonance spectrometry. The pure drug o r i n t a b l e t form, can be determined using DMSO-ds as solvent and maleic a c i d as i n t e r n a l standard. The reported recovery i s 100 f 1.5% f o r pure drug and 98 t o 99.6 f 1.4% f o r t a b l e t s . 4.3 ChromatonraDhic Methods 4.3.1 Thin-Layer ChromatonraDhY (TLC]

Gergis-Takla and Josh1 (29) reported a TLC method f o r the i d e n t i f i c a t i o n , assay and p u r i t y determination o f t h e drug and other hypoglycemic agents i n powder and i n t a b l e t f o r m u l a t i o n . The d r u g was d e t e c t e d by d i s s o l v i n g powdered t a b l e t s o r powder i n dichloromethane-acetone m i x t u r e (2: 1) and chromatographing t h e s o l u t i o n on s i l i c a gel F 2 5 4 p l a t e s with cyclohexane-chloroform-acetic a c i d and e t h a n o l (10:7:2:1). For q u a n t i t a t i v e determination, t h e spots were s e p a r a t e d , e l u t e d w i t h m e t h a n o l i c sodium hydroxide, d i l u t e d w i t h m e t h a n o l i c HC1 and t h e absorbance was measured. Surborg and Roeder (30) have recommended c o n s t a n t b o i l i n g s o l v e n t m i x t u r e s f o r t h e development o f chromatograms on s i l i c a gel f o r acetohexamide and o t h e r a n t i d i a b e t i c drugs: propanol-cyclohexane (37:163), propanol-benzene-cyclohexene (9:14:27), and cyclohexane - isopropanol (177:23). The R f values o f t h e drugs a r e t a b u l a t e d , s p o t s were l o c a t e d by v i e w i n g i n 254 n m radiation. 4.3.2 Gas L i a u i d ChromatonraDhY (GLC)

Kleber e t a l . (31) determined acetohexamide and hydroxyhexamide i n b i o l o g i c a l f l u i d s u s i n g GLC. Tolbutamide was used as an i n t e r n a l standard and M-HC1 was added t o the sample o f plasma o r urine, the m i x t u r e

32

ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID

was shaken w i t h t o l u e n e and was c e n t r i f u g e d . The separated organic phase was shaken w i t h 7.5% KzC03 s o l u t i o n and centrifuged again. The aqueous phase was heated a t 6 0 " f o r 1 0 m i n u t e s w i t h methanol and dimethylsulphate, cooled and M-acetate b u f f e r s o l u t i o n was added t o a d j u s t t o pH 5.2. The m e t h y l a t e d sulphonylureas were e x t r a c t e d w i t h hexane and t h e e x t r a c t was evaporated t o dryness a t 50' i n a stream o f nitrogen. The residue was dissolved i n CS2-CHC13 (l:l), 25 u l and 2 p l was submitted t o GLC on a glass column (61 c X 3 mn) containing 0.5% o f PEG 20 M on Gas-Chrm m Q (80 t o 100 mesh) and the temperature was programed f o r 190 t o 240' a t 5 min-1, w i t h helium as c a r r i e r gas (90 m l min-1) and flame i o n i s a t i o n d e t e c t i o n . Peak heights were compared. A t concentrations o f 10 t o 40 ug m l - 1 i n plasma. The mean recoveries (8 determination) were : f o r acetohexamide 9.9 and 39.4 c(g m l - 1 ; f o r the metabolite hydroxyhexamide 14.1 and 40 c(g m l - 1 . Fricke (32) presented a G C method f o r t h e analysis o f L t h e drug and o t h e r drugs i n pharmaceuticals, u s i n g s i m p l e e x t r a c t i o n s and semiautomated g a s - l i q u i d chromatography, using Ddxil 300 as the l i q u i d phase and an automatic sample i n j e c t o r . Results by t h i s method and t h e o f f i c i a l and o t h e r a p p l i c a b l e methods a r e compared. Content uniformity analysis can be made by u s i n g t h i s procedure. The e x t r a c t i o n and chromatographic conditions were standardized t o make possible a successful interlaboratory study.
4.3.3

Hinh-Performance L i a u i d ChrmatoqraDhy ( HPLCl

A simple HPLC assay o f t h e drug i n plasma was developed by T a k a g i s h i e t a7 ( 3 3 ) . A sample was extracted w i t h a mixture o f benzene and e t h y l acetate a t pH 5 and t h e organic phase was evaporated. A 50% s o l u t i o n i n CH3CN o f the residue was chromatographed u s i n g a Lichrosorb RP-8 reversed-phase column and a mobile phase composed o f 0.2% a c e t i c a c i d - methyl c y a n i d e ( 1 : l ) . The m e t h o d c a n be u s e d f o r b i o a v a i l a b i l i t y and c l i n i c a l pharmacokinetic studies o f acetohexamide.

Beyer (34) used high speed l i q u i d chromatography f o r analysis o f the drug and other a n t i d i a b e t i c agents. The reocvery o f the drug from i n e r t t a b l e t ingredients by

ACETOHEX AM I DE

33

t h i s method was near 100%. A column (100 c X 2 . 1 mm) m packed w i t h 1% o f ethylene-propene copolymer on Zipax was used w i t h mobile phase o f 0.01 M disodium hydrogen c i t r a t e containing 15% o f methanol (pH 4 . 4 ) . Detection was c a r r i e d o u t a t 254 nm and pack a r e a s were integrated. Testosterone, chlorpropamide i n 95% ethanol were used i n t e r n a l s t a n d a r d s . The p r o c e d u r e was a p p l i e d t o compressed t a b l e t s , t h e powdered sample being e x t r a c t e d w i t h t h e i n t e r n a l standard s o l u t i o n . Recoveries o f added sulphonylurea were 98.9% t o 100.2%.
5. PHARMACOKINETICS
5.1

Introduction

Acetohexamide i s used as an o r a l a n t i d i a b e t i c agent f o r t h e t r e a t m e n t o f k e t o a c i d o s i s - r e s i s t a n t d i a b e t e s . I t i s an i n t e r m e d i a t e a c t i n g s u l f o n y l u r e a d e r i v a t i v e . The c l i n i c a l e f f e c t s o f lowering elevated blood glucose l e v e l s i s s i m i l a r f o r a l l o f t h e sulfonylurea d e r i v a t i v e s . Acetohexamide, however, i s t h e only one t o a l s o possess u r i c o s u r i c a c t i v i t y and t h e r e f o r e i s a p r e f e r a b l e agent t o t r e a t d i a b e t i c p a t i e n t s w i t h gout. The d u r a t i o n o f a c t i o n o f acetohexamide (12-24 hours) permits once o r t w i c e d a i l y dosage. The crossover study o f Fox e t a7. (35) conducted i n 36 p a t i e n t s w i t h m a t u r i t y onset diabetes m e l l i t u s i n d i c a t e d t h a t both chlorpropamide and acetohexamide gave s i m i l a r responses based on f a s t i n g blood sugar. Acetohexamide was used i n a dose range o f 500-3,000 mg/day and i t i s i n d i c a t e d t h a t primary f a i l u r e on acetohexamide i s more l i k e l y t o respond t o chlorpropamide and v i c e versa. Appropriate dosing r e q u i r e i n d i v i d u a l i z a t i o n o f therapy t i t r a t e d t o t h e d e s i r e d t h e r a p e u t i c e f f e c t . The usual PO dosage range i s 250-1500 mg/day i n s i n g l e o r d i v i d e d doses (36,37), w i t h a maximum recommended dose o f 1500 mg/day. The 250 mg dose o f acetohexamide i s equivalent t o 500 mg t o l b u t a m l d e , 100 mg tolazamide, o r 100 mg chlorpropamide (36). The o r a l a n t i d i a b e t i c agents prove more u s e f u l when d i e t a r y r e s t r i c t i o n and w e i g h t reduction accompany t h e i r use.

34

ABDULLAH A. AL-EADK AND HUMEIDA A. EL-OBEID

Acetohexamide i s l a r g e l y metabolized t o an a c t i v e metabolite which is excreted i n t h e u r i n e (see below). Therefore, dosage adjustment o r t o t a l avoidance i s necessary i n c e r t a i n cases. One such case i s t h e renal f a i l u r e . Azotenic p a t i e n t s may experience prolonged hypoglycemia. A t w i c e d a i l y dose i s recommended f o r p a t i e n t s w i t h m i l d r e n a l f a i l u r e and p a t i e n t s w i t h moderate t o severe renal f a i l u r e should not receive t h e drug (38,39). Dosage adjustment may a l s o be required i n p a t i e n t s with 1i v e r i n s u f f i c i e n c y since acetohexamide i s e x t e n s i v e l y metabolized i n t h e l i v e r . Prolonged hypoglycemia may r e s u l t i n p a t i e n t s w i t h severe l i v e r impairment (36). Dosage r e d u c t i o n may be r e q u i r e d i n e l d e r l y o r d e b i l i t a t e d p a t i e n t s , due t o renal o r l i v e r impairment o r hyperresponsiveness (36).
I t i s recommended by Bennett e t a7. (39) t h a t no dosage supplementatlon i s r e q u i r e d i n p a t i e n t s f o l l o w i n g p e r i t o n e a l d i a1y s i s

L i k e other o r a l a n t i d i a b e t i c agents, acetohexamide may be used i n combination w i t h i n s u l i n t o reduce i n s u l i n r e q u i r e m e n t s i n i n s u l i n dependent m a t u r i t y o n s e t d i a b e t i c s and t o r e d u c e t h e p o t e n t i a l f o r a hypoglycemic reaction.
5.2

Mechanism o f Action

Acetohexamide i s a sulfonylurea d e r i v a t i v e , t h a t produces i t s hypoglycemic e f f e c t by s t i m u l a t i n g t h e i s l e t t i s s u e t o s y n t h e s i z e and r e l e a s e endogenous i n s u l i n ( 4 0 ) . The h y p o g l y c e m i c e f f e c t s a r e a l s o a t t r i b u t e d t o an increased s e n s i t i v i t y o f i n s u l i n receptors as w e l l as improved peripheral u t i l i z a t i o n o f i n s u l i n (37).
A r e p o r t by Lebowitz and Feinglos (41) i n d i c a t e s t h a t , d u r i n g chronic administration, p a r t o f t h e hypoglycemic action o f the sulfonylureas i s e x t r a pancreatic. Peripheral t i s s u e s may become more s e n s i t i v e t o a f i x e d dose o f an a d m i n i s t e r e d hormone p o s s i b l y due t o an increase i n the number o f i n s u l i n receptors. A study on the mode o f a c t i o n o f t h e sulfonylureas (42)

has shown t h a t acetohexamide increased glucose uptake

ACETOHEXAMIDE

35

by r a t diaphragm, i n h i b i t e d the a c t i v i t y o f glucose-6phosphatase, triosephosphate isomerase and l i p o p r o t e i n 1ipase

5.3

Onset and Duration o f Action

B r e i d a h l e t a 7 . ( 4 3 , 4 4 1 r e p o r t e d a peak hypoglycemic e f f e c t t o occur between 8 t o 10 hour post ingestion o f acetohexamide.


A duration o f action o f 12 t o 24 hours i s reported by Breidahl et a7. (43,44) and Galloway et a7. (45) which i s s i m i l a r t o t h a t o f tolazamide (up t o 24 hours), less than t h a t o f chlorpropamide (60 hour) and greater than t h a t o f tolbutamide (6 t o 12 hours) (37).

The serum c o n c e n t r a t i o n s i n d i a b e t i c p a t i e n t s responding w e l l t o t h e drug had mean acetohexamide l e v e l s o f 3.7 mg/dL w i t h a ragne f o 2.5 t o 4 . 9 mg/dL f o l l o w i n g dosage regimens o f 0.5 t o 3 g/day (46). No good c o r r e l a t i o n between b l o o d c o n c e n t r a t i o n s o f acetohexamide and therapeutic e f f e c t i s established. However, f a s t i n g blood glucose concentrations a r e decreased i n a dose-dependent f a s h i o n i n t h e dosage range between 250 mg t o 1,000 m (47). g
5.4

AbsorDtion

O r a l l y a d m i n i s t e r e d acetohexamide i s almost completely absorbed (47). I t i s reported t o appear i n the blood w i t h i n 30 minutes a f t e r PO administration and peak l e v e l s occur a f t e r 3 t o 5 hours (43,44). Galloway et a7. (45) reported t h a t , f o l l o w i n g single PO doses o f 1 g o f acetohexamide, mean peak blood l e v e l s o f t h e drug t o be 47 mcg/ml and f o r hydroxyhexamide mean l e v e l s o f 60.3 mcg/ml were achieved. These peak l e v e l s occurred w i t h i n 1.5 t o 2 hours f o r the parent compound versus 2 t o 6 hours f o r th e a c t i v e metabolite, hydroxyhexamide.
5.5

Distribution

J u d i s ( 4 8 , 4 9 ) r e p o r t e d t h a t acetohexamide extensively binds t o plasma proteins t o the extent o f 65 t o 90%.

36

ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID

5.6

Hetabol ism

A c e t o h e x a m i d e is mainly metabolized b y hydroxylation reactions in the liver to inactive and active metabolites. The primary metabolite (47 to 60%) is hydroxyhexamide (47,501. It is an active metabolite and is reported (45,50) to be excreted unchanged in the urine, as well as metabolized to the inactive dihydroxyhexamide (38). Hydroxyhexamide, like acetohexamide, possesses both hypoglycemic and uricosuric properties (51,52), but it is 2.5 times as potent as its parent d r u g (36). Impairment of hydroxyhexamides el imination has been reported (51) to result in severe hypoglycemia. Kojima et a7. (53) investigated the effect of various drugs on the i n v i v o metabolic reduction of acetohexamide. Most of the nonsteroidal antiinflammatory drugs inhibited the acetohexamide reduction in liver, kidney and heart cytosol from rabbits. Ketone-containing drugs including warfarin also inhibited the reduction reaction in both the liver and the kidney; in the heart, acetohexamide reduction was inhibited only by warfarin. Species differences in the i n v i t r o metabolic reduction of acetohexamide were studied (54) in rabbit, guinea pig, hamster, rat and mouse. The rabbit exhibited the highest acetohexamide reductase activity in the cytosol of the liver and kidney among the species tested. The sensitivity to specific inhibitors of cytosolic acetohexamide reductase in the liver and kidney of the rabbit were different from those of the rat. Only rats and guinea pigs showed significant activity of acetohexamide reductase activity in the microsomes of the liver and kidney. Nagamine e t a7 (55) estimated the rates of available fraction for 4-acetamidoacetophenone, 4-acetylbenzenesulfonamide, and acetohexamide and their respective reduced compounds, 4-substituted a-hydroxyethylphenyl derivatives, in rats. The study indicated that the compounds are i n a reversible drug-metabolite relationship. The pharmacokinetic profiles o f the agents were studied after an intraportal administration

ACETOHEXAMIDE

37

i n comparison w i t h those a f t e r I . V . using an interconversion model.


5.7

administration

Excretion

Acetohexamide and i t s m e t a b o l i t e s a r e m a i n l y e x c r e t e d b y t h e k i d n e y s . The u r i n a r y r e c o v e r y o f radioactivity a f t e r the administration o f oral 14C-labeled acetohexamide averaged 71.6% i n 24 hours (45). Approximatley one-half t o two-third o f t h e drug was r e p o r t e d t o be excreted i n u r i n e as t h e a c t i v e metabolite, hydroxyhexamide (45,501. Fecal excretion o f r a d i o a c t i v i t y f o l l o w i n g o r a l administration o f t h e drug i n one p a t i e n t was 15%. Even a f t e r 1 g I . V . dose u r i n a r y recovery was o n l y 85% ( 4 5 1 , suggesting t h a t b i l i a r y e x c r e t i o n represents a secondary r o u t e o f e l i m i n a t i o n o f acetohexamide and/or i t s metabolites. However, more data are needed t o confirm the occurrence o f b l l i a r y excretion.
5.8

Half-Life

F o l l o w i n g o r a l a d m i n i s t r a t i o n o f 14C-labeled acetohexamide t o human subjects, a mean blood h a l f - l i f e o f t h e drug o f 1 . 6 hours was determined, using isotope d i l u t i o n a n a l y s i s , w i t h a range o f 0 . 8 - 2 . 4 hours
(45,56).

F i e l d e t a l . ( 5 1 ) , however, reported a range o f 21-70 minutes averaging t o a value o f 55.8 minutes. The combined h a l f - l l f e o f t h e parent compound and i t s a c t i v e metabolite, hydroxyhexamide, i s reported t o be 5.3 hours (43-45). The h a l f - l i f e o f acetohexamide i s reported be prolonged i n renal f a i l u r e ( 3 8 ) . The a c t i v e m e t a b o l i t e , hydroxyhexamide i s r e p o r t e d (45,561 t o have a mean h a l f - l i f e o f 5.3 hours with a range o f 3.7-6.4 hours. The average value o f 5.3 hours agrees w i t h t h e f i n d i n g o f F i e l d e t a l . ( 5 1 ) who reported a range o f 3.2-7.6 hours. The blood and u r i n e data reported by Galloway et a l . (45) agree w i t h those reported by Scheldon et a l . (46) and confirm the report by Smith e t a l . ( 5 6 ) , t h a t the combined half-1 i f e o f acetohexamide and hydroxyhexamide i s comparable w i t h t h a t o f tolbutamide.

38

ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID

AKO LGMN C N WE E E T The authors would l i k e t o thank M r . Tanvir A. B u t t f o r t y p i n g t h i s manuscript.


REFERENCES
1. 2. 3. 4.

The Merck Index, Tenth E d i t i o n , Rahaway, N w Jersey, 1983, page 9. e

Merck & Co.

Inc.,

The B r i t i s h PharmacoDoeia, HM S t a t i o n a r y O f f i c e , London, 1988, Vol. 1, page 18.


B r i t . Pat. 912, 789 (1962 t o L i l l y ) No. 1.

Through reference

F.J. M.A.

Marshall, M.V. Sigal, H.R. Sullivan, C. Cesnik and Root, J. Med. Chem., 5 , 60 (1963).

5.
6.

- 640 (1977). 29,

P. Girgis-Takla and

I. Chroneos, J. Pharm. Pharmacol.,


Takagishi,

K. Kuroda, T. Yokoyama, T. Umeda and V. Chem. Pharm. Bull., 26, 2565 (1978).
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7.
8.

Burger, Sci. Pharm.,

s, 207

(1978).

B.W. Mueller and M. 77 (1979). B.W.

Lagas, Pharm. Weekbl. Sci. Ed.,

1,

9.
10. 11. 12. 13. 14.

Mueller and M. Lagas,

u,1, 449

(1979).

T. Yokoyama, T . Umeda, K. Kuroda, K. S a t 0 and V. Takagishi, Chem. Pharm. Bull., 27, 1476 (1979).
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Abdallah,

A c t a Pharm.

E. Graf, C. Beyer and 0. Abdallah, @d 73 (1982). i 28, ,

E. Graf, C. (1982). E. G r a f , (1982). C.

Beyer and 0. A b d a l l a h , Beyer and 0. A b d a l l a h ,

ibid,

a,131
225

m, 28,

ACETOHEXAMIDE

39

15. 16. 17. 18. 19.

A.A.

Kassem, A.M. F o u l i , S. Said and E. Shehata, B u l l . Fac. Pharm. (Cairo Univ.), 19, 309 (1982). Chem.,

Mfg.

34, 454, 467 (1963).


I n d i a n J.

S u r a j P. Agarwal and Mohammed I W ash; . a Pharm., 3 4 ( 5 ) , 109-111 (1972).

Jose Dobrecky and Rogelio J . C a l l e j a ; Rev. Fac. Quim Farm., [Univ. Cent. Ecuador], El(16); 44 49 (1969).
M.M. Amer and M . I . Walash; B u l l . Fac. Pharm.. Univ., l 2 ( 2 ) , 199-209 (1973).

Cairo

20a. M.M.

Amer and M.I.

Walash: Walash;

W, l2(2),223-233 (1973).

20b. M.H. h e r and M.I. (Pub. 1975).

m, l 2 ( 2 ) ,

189-198 (1973)

21. 22.

L i l o 0. Guerello and Jose Dobrecky; Rev. Asoc. Bioauim. Argent. 33(178-1791, 185-8 (1968). Meier, S.O. Kohor, O.F. P i e r a r t , S . S . J . Cortes; Rev. Real. Acad. Cience. Exactas. Fis. Natur. Madrid, -( 3 ) , 653-674 (1971). 65

N.G.

23. 24. 25.

S.G.

Solomonova and L . Z . (Kiev), 2, 80-82 (1977).

Dvornitskaya;

Farm.

Zh.

Edward F. Salem and W.W. 385-386 (1967).

H i l t y ; J. Pharm. Sci.,

56(3),

M a r i a K u h n e r t - B r a n d s t a e t t e r , Adel h e i d K o f l e r , A. Vlachopoulas and A. Lobenwein; S c i . Pharm., 38(3) 154-163 (1970).

26. 27. 28. 29.

D.S.

Lazaryan, (1980).

Farmatsiya (Moscow),

29(2)

36-38,

Pamela Gergis-Takla and Ioannis Chroneos; Analyst, (1235) 117-123 (1979).


A.A.

14 0
378

Al-Badr (1982).

and S.E.

Ibrahim;

Pharmazie;

37(5),

Pamela Gergls-Takla and Shanta Raj Joshi; Anal. 1 ( 2 ) , 189-193 (1983).

J.

Biomed.

40

ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID

30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40.

K.H. Surborg and E. Roeder; Pharmazie, =(7), (1 97.3).

485-486,

u, 635-638 m(5),

J.W.

Kleber, J.A.

Galloway and B.E. (1977). Ass.


Off.

Rodda; J. Chem.,

Pharm.
55(6),

Fred L. F r i c k e , J. 1162-1 167 (1972).

Anal.

Yasushi Takagishi; K o j i Sato; Keizo Tomita and Teruo Sakamoto; Yakusaku Zasshi, B ( 9 ) , 961-963 (1979).

W i l l i a m F. Beyer; Anal. Chem., 44(7),


O.J. Fox e t a l , J. Med. Assoc. (1968). Product Information: Acetohexamide, Indianapolis, 1N: 1983.

1312-1314 (1972).

Alabama,

31,

1155

E l i L i l l y & Co.,

AMA Department o f Drugs: AMA Drug Evaluations, 4 t h ed. American Medical Association, Chicago, I L , 1980. B.D. Cohen, J.A. Galloway, R.E. Med. Sci., 254, 608 (1967).

McMahon et a l , Am. Morrison et a l , Am.

J.
J.

W.M. Bennett, G.R. Aronoff, G. Kidney Dis., 3, 155 (1983).


A.G.

Gillman, L.S. Goodman and A. Gillman; Goodman and Gillmans The Pharmacological Basis o f TheraDeutics, MacMillan & Co. N w York. 1980. e
H.E. Lebowitz and M.N. (1978).

41. 42. 43. 44. 45.

Feinglos, Diabetes Care, Kurup, Indian J. Biochem.,

1,

189

K.T. Augusti and P.A. 36 (1969).

6(1),

H.D. Breidahl, G.C. 79 (1972).


H.D. Breldahl, G.C. 204 (1972).

Ennis, F . I . Martin et a l , Drugs, Ennis, F.I. Martin et a l ,

3,

M, 3 ,
Marshall

J.A. Galloway, R.E. McMahon, H.W. Culp, F.J. and E.C. Young , Diabetes, 16, 118 (1967).

ACETOHEXAMIDE

41

46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56.

J. Scheldon, J. Anderson and L. Stoner,


(1965).

m, l4,

362

R.E.

J. Judis, J. Pharm. Sci., 6 l , 89 (1972).


J. Judis,

379 (1987).

Ferner and S.

Chaplin,

Clin.

Pharmacokinet.,

2,

w, 62,

232 (1973).

R . E . McMahon, F . J . Pharmacol. EXD. Ther.,

149, 272

M a r s h a l l and H.W.
(1965).

Culp,

J .

J.B.

Field, M. Ohta, C. 277, 889 (1967).

Boyle e t a l , N. Ensl. J. Med., Gutman, Metabolism,

T.F. Yu, L. Berger and A.B. (1968).

17, 309

Y. Kojima, Y. Imamura and M. Otagiri, Yakusaku Zasshi, 108(1), 66 (1988).

Y. Imamura, Y. Kojima and M. Bull., 36(1), 4199 (1988).


S.

Otagiri,

Chem.

Pharm.

36(11),

Nagamine, T . Otawa, H. Nakae and S. Asada,


4612 (1988).

M,

14,

D.L.

229 (1965).

Smith, T.J.

Vecchio and A.A.

Forist, Metabolism,

AMODIAQUINE HYDROCHLORIDE

Iqbal Ahmad and Tauqir Ahmad

Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Karachi, Karachi-75270, Pakistan.

K. Usmanghani
Department of Pharmacognosy, Faculty of Pharmacy, University of Karachi, Karachi-75270, Pakistan.

1. 2. 2.1 2.2 2.3 3. 4. 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 4.10 4.11 4.12 5. 5.1 5.2 5.3

INTRODUCTION DESCRIPTION Name, Formula, Molecular Weight Appearance, Color, Odor and Taste Proprietary Names SYNTHESIS PHYSICAL PROPERTIES Melting Point Solubility Completeness of Solution Acidity Water Content Residue on Ignition Chromatographic Purity Ultraviolet Spectrum Infrared Spectrum Nuclear Magnetic Resonance Spectrum Mass Spectrum Complex Formation QUALITATIVETESTS Identification Color Tests Field Test
43
Copyright G 1992 by Academic Press, Inc All rights of reproduction reserved in any form

ANALYTICAL PROFILES OF DRUG SUBSTANCES AND EXClPlENTS - VOLUME 21

44

IQBAL AHMAD, TAUQIR AHMAD. AND K . USMANGHANI

54 . 6 . 61 . 62 . 63 . 64 . 7 . 71 . 72 . 8 .

Impurity Test for 4-(7-Chloro-4-quinolylamino)phenol Hydrochloride METHODS OF ANALYSIS Titrimetric Analysis Spectrophotometric Analysis Fluorometric Analysis Chromatographic Analysis METABOLISM AND PHARMACOKINETICS Metabolism Pharmacokinetics

TOXICITY
ACKNOWLEDGEMENT REFERENCES

1.

INTRODUCTION

Amodiaquine is a congener of chloroquine and is employed for the treatment of overt malarial attacks and for suppression. Although it is n n more active than chloroquine both i vitro and i vivo against certain strains of Plasmodium fcipamm with decreased sensitivity to chloroquine, amodiaquine is not recommended for routine use in the treatment of such infections (1). It appears that phenolic hydroxyl is essential to the activity of amodiaquine since the removal of this group depresses, and its methylation completely destroys antibacterial activity (2). Amodiaquine has been synthesized and patented under the name of Camoquin by Parke, Davis and Company in 1949 (3). It is used medicinally in the form of its dihydrochloride.
2. DESCRIPTION

2.1 Name, Formula, Molecular Weight

Amodiaquine hydrochloride is 4-(7-chloro-4-quinolylamino)-2(diethylaminomethyl) phenol dihydrochloride dihydrate () 4.

AMODIAQUINE HYDROCHLORIDE

45

CaH2zCIN30,2HC1, 2H20

464.8

The CAS registry No. is 6398-98-7. Official monographs for amodiaquine hydrochloride are given in Argentinian (1966), British (1988), Brazilian (1977), Egyptian (1984), French (1982), Indian (1985), International (1981) and United States (1990) Pharmacopeias. 2.2 Appearance, Color, Odor and Taste
A yellow, odorless or almost odorless, crystalline powder with a bitter taste (5).

2.3 Proprietary Names


CAMAQI, Camoquin, Flavoquine, Miaquin (6,7).

3.

SYNTHESIS

Burckhaiter et al. (8) synthesized amodiaquine (111) in 1948 by condensing 4,7-dichloroquinoline (I) with 4-amino-2-diethylaminomethylphenol (11) in dilute hydrochloric acid (Figure 1). In a later method (9), the alkylamino group w s added as a last step. a

46

C1

c1
I

1dilute HCl 0 " 2 hours 0.

Figure 1. Synthesis of Amodiaquine

The free base was recrystallized from absolute ethanol and converted into the dihydrochloride by treating with hot concentrated hydrochloric acid.
4.

PHYSICAL PROPERTIES

4.1 Melting Point

It melts at about 158OC (7).


4.2 Solubility It is soluble in 22 parts of water and in 70 arts of ethanol (96%), practically insoluble in chloroform and ether &).

, q

IQBAL AHMAD. TAUQIR AHMAD, AND K. USMANGHANI

H2N a c H 2 N ( q H d 2

OH

II

,@2N(c2H5)2

11 1

OH

AMODIAQUINE HYDROCHLORIDE

47

4.3

Completeness of Solution A solution of 200 mg in 10 ml of water is clear (10).

4.4 Acidity The pH of a 2.0% w/v solution is 3.6 to 4.6 (4). 4.5 Water Content Not less than 7.0% and not more than 9.0% (10) 4.6 Residue on Ignition Not more than 0.2% (10) 4.7 Chromatographic Purity Chromatographic purity of amodiaquine hydrochloride can be examined on thin-layer plate coated with a 0.25 mm layer of silica gel G using solvent system chloroform (saturated with ammonium hydroxide): dehydrated alcohol (9: 1). Under short-wavelength ultraviolet light, the chromatogram shows principal spot at about the same Rf value, and no secondary spot, as obtained with the USP Amodiaquine Hydrochloride RS (10). 4.8 Ultraviolet (UV) Spectrum The ultraviolet spectra of amodiaquine and amodiaquine hydrochloride have been reported by Sunshine (12) and Clarke (7) respectively. The ultraviolet absorption characteristics are used for the identification of these drugs (4,10,13). The absorption spectrum of amodiaquine hydrochloride as a function of pH in the range 1-11.8 shows a hypsochromic effect at 343 n , a hyperchromic effect at 305 nm and the m i isosbestic point at 323 rm (14). The effect of solvents and substitution on the ultraviolet spectra of amodiaquine has been studied and the changes of absorption bands E,K, and B discussed in detail (15). The ultraviolet spectrum of amodiaquine hydrochloride in 0.1 M hydrochloric acid was recorded on a Shimadzu 240 UV-Visible spectrophotometer and is shown in Figure 2. The uv spectral data reported for amodiaquine and amodiaquine hydrochloride are listed in Table 1.

48

IQBAL AHMAD, TAUQIR AHMAD. AND K. USMANGHANI

1.50

!r
1

A
WAVELENGTH Cnml

3 .0

Figure 2.

Ultravlofet Spectrum of Amodluquinc Hydrochloride InO.l M HCI

AMODIAQUINE HYDKOCHLORIDE

49

Table 1

UV Spectral Data for Amodiaquine and Amodiaquine Hydrochloride


Compound Amodiaquine

Solvent
0.1 M HCl

Amax, nm
283 237 247 224 342 343 223 237 343 237 343 273 287 223 237 342.5

A (l%, Icm) 890 530 470 394410 366

Molar Absorptivity
41370 24630 21850 1831019060 17010

Ref. 12

Amodiaquine hydrochloride

Water

0.1 M HCI 0.1 M HCl

4 11

Aq. acid Aq. alkali 0.1 M HCl

366 600

17010 27890

7 7

836 489 369

38850 22710
17160

*Values determined by the authors.

4.9 Infrared (IR)Spectrum The infrared spectrum of amodiaquine has been determined in KBr disc ( ) The principal peaks in the infrared spectrum of amodiaquine 4. hydrochloride (KBr disc) are reported at 1565, 815, 1535, 1255, 869, 847 cm" ( ) Attenuated total reflectance infrared spectrum is used to detect 7. amodiaquine hydrochloride in the solid state as a layer of crystals on adhesive tape. The method has been applied to the identification of the drug in tablet formulations. Common excipients such as starch, and lactose (absorption in the 1000 to 1200 cm-' region) do not interfere with the method (16).
The infrared spectrum of amodiaquine hydrochloride as KBr disc was obtained with a Shimadzu IR 460 Infrared spectrophotometer and is shown in Figure 3. The assignments for characteristic bands are given in Table 11.

,
0 0
0 L

5.a ..
I

4.0
I

5
1

60
I

7.0
I

8.g
I

5.0
I

10 0
1

15.0

zoo
:oo.o

80.0
0

I
000

60-0
0

0 0

0 I D
0

40 0
0

*
0
0

I
I

20.0

3000

I 2000

Wave number (em-')

'1500

'I

00"

0.0
0

500

Figure 3. Infrared Spectrum of Amodiaquine Hydrochloride (KBr disc).

AMODIAQUINE HYDROCHLORIDE

51

Table I1 IR Spectral Assignments for Amodiaquine Hydrochloride


Frequency, cm1
Assignment

3420 3 170 1615 1585,1540,1505 1448 1265,1207 1095 852,840

- NH stretching - OH stretching
C = C stretching (aromatic) C =C, C = N stretching in disubstituted quinoline - CH2-N- deformation C-OH stretching (aromatic) C-Cl stretching (aromatic) isolated CH deformation in disubstituted quinoline

4.10 Nuclear Magnetic Resonance (NMR) Spectrum The 'H-NMR and 13C-NMR spectra of amodiaquine hydrochloride in DMSO-d6 were determined at 300 MHz and 75.4 MHz respectively on a Bruker AM-300 NMR spectrometer using tetramethylsilane as reference standard. The 'H-NMR determinations included spin decoupling experiments, 2D J-resolved and COSY-45 measurements (Figure 4-6). The 13C-NMR spectra comprised DEFT and hetero-nuclear (C-H correlation) measurements (Figure 7-9). The spectral assignments are listed in Table 111.

L
11

I0

..,. ,...9 0 ..

d,
.I' 11-1'
8 0

1 0

.. : .

P O

,.

.. ..
:

. . .. -*

. .

4
PTL

. ...

- rPPM ,

F l p r c I Homonuclear Chemlul Shin Cormlaled tCoS)-Ul ~ I I - N S I R . Spectrum or Amodisqulnc 1i)dmchloridc

e w

1
,

L
I

. . ,. . .. - , ._..
I!
1

Iv,

h ?,

, ,1, 1 , , , , , , ( ,
7.0

-r . 3

..

1 ,

, , , , , , , , r*~ , , , , , , , , ( , , , , , ,
5 0

, , , ,

a
2 0

BIO

PAI

6,O

4.0

I 0

1 0

LPr2

--

b u r r 6. Homonuclcar 211 J-Rcsolvcd N M R Spcclrum of Amodiaquinc It)drorhloridr..

CH: CHdCH2

l,,c.3tii
I ICI.
C.5

14
I I

1
C.ll..ll

cd

C.2

C.Y

I -

C4

11

A L

Figure R.

75 M l l r "c-NMR Off Resonance Decoupled Spectrum ofAmodiaquine Hydrofhloride

t'

-==i

7 '1
'4
I
i

t
t

L.

58

IQBAL AHMAD. TAUQIR AHMAD. AND K. USMANGHANI

Table I11

H- and 13C-NMR Chemical shifts and Coupling Constants for Amodiaquine Hydrochloride
H-NMR Chemical shift (PP4 Proton

coupling constant (J in Hz)

C-NMR Chemicalshift (PPd


156.04 138.87

Carbon

7.67 (1H,d) 7.35 (lH,dd) 4.22 (lH,d) 4.22 (lH,s) 3.09 (4H,q) 1.27 (6H,t) 8.44 (lH,d) 6.82 (1Hd) 8.% (lH,d) 7.78 (lH,dd) 8.17 (lH,d)

3
5 6 7 910 1112

25 8.62.5 8.6 6.7 1.2 7.0


7.0

l00 3.0 115.60 128.30 116.85 49.11 46.19 8.41


142.91 100.41 117.54 126.19 127.04

1 2 3 4 5 6 7 910 12 11 2 3 4 5
6 T 8

2 3 5
6

9.1 9.12.1 2.1

138.15
8
118.97

154.85
127.92 NH/OH

9
10

10.89

4.11 M s Spectrum as

The electron impact ionization spectrum of amodiaquine hydrochloride obtained at 70 eV using a solid probe insertion is shown in Figure 10. The spectrum was run on a Finnigan Mat 112s double focusing mass spectrometer connected to a PDP 11/34 (DEC) computer system. It shows a molecular ion peak M + at m/z 355. Since the molecule contains one chlorine atom, M+ 2 peak appears at m/z 357. The proposed fragmentation pattern and prominent ions are given in Table IV.

Figure 10. Electron Impact-Mass Spectrum of Amodiaquine Hydrochloride

60

IQBAL AHMAD. TAUQIR AHMAD. A N D K.USMANGHANI

Table 4 Proposed Fragmentation Pattern of Amodiaquine Hydrochloride


d z

Relative intensity %

Ion

355, 357

55.77, 16.83

283

43.05

NH

@CH2

OH

282

99.00

c'w
@H C OH
I-

253

43.56

179

8.18

177

5.81

AMODIAQUINE HYDROCHLORIDE

61

4.12 Complex Formation

Amodiaquine hydrochloride forms 1:l and 1:2 complexes with ferrous sulphate. The infrared spectra indicate that amodiaquine hydrochloride is bonded to iron via N and 0 and that water molecules are coordinated to iron (17). It forms a 1:2 complex with silver nitrate in alcoholic solutions. The average stability constant, log K, for the complex is 7.7 and A E is about 10.8 kcal/mol. (18). The formation of 1:l ion association complex between amodiaquine and Fast Green FCF or Orange I1 dye has been reported (19).
5.
QUALITATIVE TESTS

5.1

Identification (4)

5.1.1 Dissolve 0.1 g of amodiaquine hydrochloride in 10 ml of water and add 2 ml of 2 M sodium hydroxide. Extract with two 20 ml quantities of chloroform, wash the combined chloroform extracts with 5 ml of water, dry with anhydrous sodium sulphate and evaporate to dryness. Dissolve the residue in 2 ml of chloroform. The infrared absorption spectrum of the resulting solution is concordant with the reference spectrum of amodiaquine. 5.1.2 The light absorption in the range 240 to 360 nm of a 0.003% w/v solution of amodiaquine hydrochloride in 0.1 M hydrochloric acid exhibits a maximum only at 343 nm. The absorbance at 343 nm is about 1.1. 5.1.3 To 1 ml of a 2% w/v solution of amodiaquine hydrochloride add 0.5 ml of cobalt thiocyanate reagent. A green precipitate is produced. 5.1.4 Amodiaquine hydrochloride yields the reactions characteristic of chlorides. The identification tests of amodiaquine hydrochloride based on comparison of infrared and ultraviolet absorption spectra, and reactions of chloride are reported in USP (10). 5.2 Color Tests (7)

Amodiaquine hydrochloride gives a blue color with Folin-Ciocalteu reagent. The Liebermanns test yields a black color. An orange color is

62

IQBAL AHMAD. TAUQIR AHMAD, AND K. USMANGHANI

produced when amodiaquine hydrochloride is treated with Millon's reagent.

5 3 Field Test (20) .


Amodiaquine base is extracted from urine into amyl acetate immediately after alkalinization. The addition of bromophenol blue in 5% boric acid to the organic phase causes a green to blue coloring, depending on the concentration of the drug. The sensitivity of the test is 0.8 mg%. phenol Hydrochloride 5.4 Impurity Test for 4-(7-Chloro-4-quinolylamino) (4)Carry out thin-layer chromatography, using silica gel G as the coating substance, spread in a layer about 0.5 mm thick, and a solvent system chloroform: butan-2-one: diethylamine (50:40: 10). Apply separately to the chromatoplate 5 pl of each of two solutions in methanol containing (1) 10.0% w/v of the substance being examined and (2) 10.0% w/v of amodiaquine hydrochloride BPCRS and 0.020% w/v of 4-(7-chloro4-quinolylamino) phenol hydrochloride BPCRS. After development remove the plate, heat it at 105' for 10 minutes, spray with a freshly prepared mixture of equal volumes of a 10% w/v solution of iron (111) chloride and a 1% w/v solution of potassium hexacyanoferrate (111) and examine immediately. Any spot corresponding to 4-(7-chloro-4quinolylamino) phenol in the chromatogram obtained with solution (1) is not more intense than the spot with lower Rfvalue in the chromatogram as obtained with solution (2). 6.

METHODS OF ANALYSIS

6.1 Titrimetric Analysis 6.1.1 Nonaqueous titration The BP method (4) for the assay of amodiaquine hydrochloride as pure drug and in dosage forms is based on nonaqueous titration. A 0.2 g quantity of amodiaquine hydrochloride is dissolved in a suitable volume of anhydrous glacial acetic acid, 7 ml of mercury (11) acetate solution is added and the solution titrated with 0.1 M perchloric acid to a green end point using 1-naphtholbenzoin solution as indicator. In dosage forms, a

AMODIAQUINE HYDROCHLORIDE

63

quantity of the powdered material equivalent to about 0.2 g of amodiaquine hydrochloride is dissolved in 30 ml of water and 5 ml of 2 M sodium hydroxide is added. Amodiaquine base is extracted with three 30 ml quantities of chloroform, the combined chloroform extracts are washed with 10 ml of water and evaporated to a volume of about 10 ml. To the chloroform extracts, 40 ml of anhydrous glacial acetic acid is added and the solution titrated with 0.1 M perchloric acid using 1-naphtholbenzoin solution as indicator. Each ml of 0.1 M perchloric acid is equivalent to 0.02144 g of CaHzCIN30,2HCl. the nonaqueous titration of amodiaquine in dosage forms. A powdered sample of 5 milliequivalent weight is dissolved in 7 ml of N hydrochloric acid, made alkaline with 3 ml of 6 N sodium hydroxide, shaken with 30 ml of chloroform for 10 minutes and with 1 g of tragacanth for another 2 minutes, filtered through adsorbent cotton, and titrated (20 ml) with 0.1 N acetic perchloric acid to blue or green end point using crystal violet solution as indicator. For pure chemicals, the digestion with acid and alkali could be omitted. The results agree with those obtained by the official method.
6.1.2 Titration with brominating agents

Wu et d. (21) have described a simple, rapid and accurate method for

Amodiaquine can be determined in bulk and in dosage forms by a titrimetric method based on reaction with 1,3-dibromo-5,5dimethylhydantoin or N-bromosuccinimide as the titrant. The mixture is later treated with potassium iodide solution and the liberated iodine titrated with sodium thiosulphate solution. The recovery is about 100% (22).
A method for the determination of amodiaquine hydrochloride in tablets by titration with N-bromosuccinimide has been developed (23). The sample is dissolved in water, treated with an acetic acid solution of the reagent and mixed with potassium iodide. The iodine released is titrated with sodium thiosulphate solution. The relative standard deviation for the titration is 2.12% and the recovery is 99.4 - 101.0%.
6.1.3 Titration with vanadium (V)

The determination of amodiaquine hydrochloride by oxidation with ammonium metavanadate solution and back titration of the unconsumed reagent with acidic iron (11) ammonium sulphate solution, using

64

IQBAL AHMAD, TAUQlR AHMAD. A V D K. USMANGHANI

N-phenylanthranilic acid as indicator has been reported (24). The recovery of amodiaquine hydrochloride in the pure form and in pharmaceutical preparations is 99.83% (standard deviation 0.49%) and 99.69% (standard deviation 0.78%) respectively. The method is of general applicability and is quick and simple compared with the official methods. 6.2 Spectrophotometric Analysis 6.2.1 Ultraviolet spectrophotometry The USP assay (10) of amodiaquine hydrochloride in pure form and in tablets involves ultraviolet spectrophotometric determination. A quantity of the drug equivalent to about 300 mg is dissolved in dilute hydrochloric acid (1:lOO) to obtain a concentration of about 15 pg/ml. The absorbance of this solution, along with a solution of undried USP Amodiaquine Hydrochloride RS in the same medium having a known concentration of about 15 pg/ml, is determined at 342 nm using dilute hydrochloric acid (1:lOO) as the blank. The quantity, in mg, of CmH22CIN30, 2HC1 in the portion of amodiaquine hydrochloride taken is calculated by the formula 20C (Ad&), in which C is the concentration, in pg/ml, calculated on the anhydrous basis, of USP Amodiaquine Hydrochloride RS in the standard solution and AU and As are the absorbances of the solution of amodiaquine hydrochloride and the standard solution respectively. The same method is applied to the assay of amodiaquine hydrochloride in tablets after extraction of the base into chloroform and then re-extraction with dilute hydrochloric acid (1:lOO). Amodiaquine and primaquine can be quantitatively separated by selective precipitation with 4 N ammonium hydroxide, followed by determination of the two compounds at 342 and 282 nm respectively. The method is valid upto primaquine - amodiaquine ratio of 1:40. Recoveries of 98.30 - 100.11% have been reported (25). The presence of higher amounts of amodiaquine yields low results in respect of primaquine as on precipitation with ammonium hydroxide, the primaquine is trapped into the precipitate of amodiaquine (26). Hassan et a . (27) have developed a method for the simultaneous l determination of amodiaquine - primaquine mixtures in dosage forms. The drugs are extracted with 0.1 N hydrochloric acid and absorbance of the mixture is measured at 342 and 282 nm. The concentration of each compound is calculated by solving two simultaneous equations. Excellent

AMODlAQUlNE HYDROCHLORIDE

65

recoveries from authentic samples are obtained and the method is suitable for routine analysis. 6.2.2 Colorimetry Amodiaquine hydrochloride is determined colorimetricallyby complex formation, in aqueous solution, with bromophenol blue, bromocresol green, bromocresol purple, and methyl orange, respectively. The complex with bromophenol blue has the highest molar absorptivity. Recoveries are more than 98.6% for all complexes, and the absorbance is linear with concentration in the range 1-11 pglml. The absorption maxima for the complexes occur at 420 nm except for the bromocresol purple complex which exhibits maximum at 415 nm. The various complexes are extracted with chloroform and absorbance is measured at the respective maxima for quantitative determination (28).
A simple, sensitive, and selective method for the determination of amodiaquine hydrochloride in tablets has been developed. It is based on a color reaction with chloramine-T in the pH range 7.4- 8.0. The chromogen is extracted with chloroform and the absorbance is measured at 442 nm. Beers law is obeyed in the concentration range 1-200 p g / l . The coefficient of variation has been found to be 0.64% and the recovery ranges between 100.3 and 102.5%. Chloroquine phosphate or primaquine phosphate do not interfere with the method (29).

Amodiaquine reacts with cobalt and thiocyanate to yield stable ternary complexes. These complexes are readily extractable in nitrobenzene to give a greenish-blue color with maximum absorption at 625 nm that can be used for quantitative determination. The mean recoveries for authentic samples of amodiaquine hydrochloride are 100.81 & 1.77% (p = 0.05). Alternatively, determination of the cobalt content of nitrobenzene extract by atomic absorption spectroscopy provides an indirect method for the determination of the drug with a mean recovery of 99.99 2 2.16%. Both the methods have been successfully applied to the assay of the drug in pharmaceutical preparations (30).
A colorimetric method for the determination of amodiaquine in tablets or powders has been reported (31). The drug is dissolved in 0.1 N hydrochloric acid, treated with acidic ammonium reineckate, the precipitate dissolved in acetone, and the absorbance measured at 525 nm. The results compare favourably with those obtained by the official methods.

66

IQBAL AHMAD. TAUQlK AHMAD. AND K. USMANGHANI

Amodiaquine hydrochloride has been determined in tablets by dissolving it in water and treating with an acetic acid solution of N-bromosuccinimide. An orange-yellow color is produced, whose absorbance is measured at 450 nm. Beers law is obeyed in the concentration range 15-160 pglml. The relative standard deviation for the method is 1.44%, and the recovery is 99.7-100.9% (23). Amodiaquine hydrochloride tablets have been assayed by a method based on the reaction of the drug with 2,3-dichloro-S, 6-dicyanop-benzoquinone and measurement of the absorbance at 460 nm. The color attains its maximum intensity after five minutes and remains stable for at least one hour. Beers law is valid in the concentration range 1-4 mg/100 ml, and the recovery is 99.9-102.6% (32). Another colorimetric method for the determination of amodiaquine in tablets depends on its reaction with chloranilic acid in aqueous solution and measurement of the absorbance at 522 nm. The absorbance is linear over the concentration range 0.04 -0.20 mdml, and the recovery is 99.9-101.3% (33).
A simple, rapid and sensitive method for the colorimetric

determination of amodiaquine in bulk and in pharmaceutical preparations has been reported by Sastry et al. (34). It is based on the reaction of amodiaquine with potassium dichromate at pH 1.1 in the presence of sulphanilamide, and measurement of the absorbance of resulting solution at 510 nm. The color is stable for twenty-four hours. Beers law is obeyed in the concentration range 20-120pg/ml. The relative standard deviation of the method is 0.94%, and the recovery is 99.0-101.0%. Chloroquine present even in ten-fold excess does not interfere with the determination.
A highly sensitive method is based on the complexation of amodiaquine with ammonium molybdate. The bound molybdenum is converted into its thiocyanate, reduced, and the absorbance of the colored solution measured at 465 nm. The Beers law limits, molar absorptivity and Sandells sensitivity for the amodiaquine complex are 50-300pg/25 ml, 1.75 x lo4 M 1 cm-l and 0.026 &cm2 / 0.001 absorbance unit, respectively. Recovery ranges from 98-101%. The color obtained is stable for twenty-four hours and common excipients do not interfere with the method (35).

Amodiaquine forms a colored ion association complex with Fast Green FCF or Orange I1 dye. The stoichiometric ratio of the drug-dye complex has been shown to be 1:l. The method can be applied to the assay of amodiaquine in bulk and in pharmaceutical preparations. Sulphur

AMODIAQUINE HYDROCHLORIDE

67

containing drugs do not interfere with the determination (19).


6.3 Fluorometric Analysis
A fluorometric method for the determination of amodiaquine in serum, plasma, or red cells has been reported (36). Amodiaquine is extracted from alkalinized biological fluids, buffered, and heated to produce a species with marked increase in fluorescence, which could be measured. Standard curves prepared in serum and red cells are linear between 50 and 3000 pgll. Reproducibility of the assay and recovery of amodiaquine from serum and red cells is satisfactory. The specificity of the assay and the nature of the induced fluorophor are not known.
6.4

Chromatographic Analysis

6.4.1 Thin-layer chromatography (TLC)

Amodiaquine can be separated and identified on silica gel G plates using a number of solvent systems. The spots are visualized under short-wavelength ultraviolet light or by spraying with acidified iodoplatinate solution. The following R values (Table V) have been r reported (37). Table V Solvent Systems for TLC of Amodiaquine
Adsorbent Solvent system
Rr

Silica gel G F w dipped i 0.1 M KOH and dried n

Methanol: ammonia (1rn1.5) Cyclohexane :toluene : diethylamine (751510) Cbloroform:methanol ( 91) Acetone

0.62 0.08

0.40

0.37

The application of principal components analysis to the TLC behaviour of a large number of basic drugs including amodiaquine has been studied (38). A two-component model explains 77% of the total variance in four

68

IQBAL AHMAD, TAUQIR AHMAD. AND K. USMANCHANI

eluting mixtures. For the identification of unknowns, the method provides a drastic reduction of the range of possibilities to a few drugs. 6.4.2 High-performance liquid chromatography (HPLC)
A variety of HPLC packing materials have been prepared and their chromatographic properties evaluated for separating amodiaquine and other basic drugs using a single mobile phase. The three most promising packing materials are silica, a mercapto Pr modified silica and a Pr sulfonic acid modification (39). A simple and precise HPLC assay for quantitating amodiaquine in tablets and biological fluids involves acid extraction of the drug from tablets and chloroform extraction of its base from the biological fluids after treatment with ammonia. A p-Bondapak Ph column is employed for separation with a mobile phase comprising methanol : water: acetic acid (25:25:1) (pH 2.3), using quinidine as the internal standard. The mean recovery of the drug from tablets is 102.03%, while in the biological fluids, it ranges from 85.2 to 104.6%. Interference from tablet excipients or biological fluids is negligible (40). A column liquid chromatographic method for the simultaneous determination of chloroquine, amodiaquine and their monodesethyl metabolites in human plasma, red blood cells, whole blood and urine has been developed (41). The drugs and internal standards are extracted as bases with dichloromethane and then re-extracted into an acidic aqueous phase. Separation is achieved using a reversed-phase column and a mobile phase of phosphate buffer (pH 3.0) : methyl cyanide (88:12). The absorbance of the drugs is monitored at 340 nm with a sensitivity limit of 10 pmoVml. The mean overall recovery from each biological fluid is more than 75%. This method can be applied to therapeutic, pharmacokinetic, and epidemological studies.

7.

METABOLISM AND PHARMACOKINETICS

7.1 Metabolism Churchill et al. (42) have isolated four metabolites of amodiaquine in humans using a reversed-phase HPLC method. The two major metabolites have been identified as desethylamodiaquine and 2- hydroxy-

AMODIAQUINE HYDROCHLORIDE

69

desethylamodiaquine. The importance of these metabolites in the n antimalarial effect of amodiquine in humans and on the i vitro sensitivity of persons dosed with amodiaquine is discussed. 2-Hydroxydesethylamodiaquine has been isolated from urine and characterised by HPLC and NMR spectroscopy. The presence of three additional metabolites of this drug in humans has been suggested and chromatographic confirmation for one of these obtained. The i vitro n activity of 2-hydroxydesethylamodiaquine is shown to be 1% that of amodiaquine for two chloroquine sensitive Plasmodiumfdcipamm strains (43). The metabolism of 2-amino-4- quinoline derivatives of chloroquine and amodiaquine in humans has been compared by Pussard et al. (41). 7.2 Pharmacokinetics

Amodiaquine hydrochloride is readily absorbed from gastro-intestinal tract after oral administration, and higher concentrations occur in erythrocytes, kidney, liver, lungs and spleen than in the plasma. After absorption it is slowly released into the blood and excreted in the urine for at least seven days after a single dose. The rate of excretion is increased in n acid urine (5,7). Amodiaquine is altered rapidly i vivo to yield products which appear to be excreted slowly, and thus have a prolonged suppressive activity (44). Following a single oral dose of 10 mgkg of amodiaquine to five human subjects, serum concentrations of 0.30 to 0.68 pg/ml (mean 0.5) have been reported after four hours; the ratio of erythrocyte to serum concentration varies with time and between individuals, but erythrocyte concentrations are generally higher than the serum concentrations after forty-eight hours (36). The metabolic transformation of amodiaquine to monodesethylamodiaquine, and its pharmacokinetics in humans have been reported (41).
8. TOXICITY

Amodiaquine hydrochloride is an antimalarial of low toxicity and is three to four times as active as quinine as a suppressive drug against Plasmodium vivav and Plasmodium fakiparum infections (44,45,46). Jn therapeutic doses amodiaquine hydrochloride is generally well tolerated but may occasionally give rise to side-effects, including nausea, vomiting,

70

IQBAL AHMAD. TAUQIR AHMAD. AND K . USMANCHANI

diarrhoea, insomnia, vertigo, and lethargy (5). The prolonged use of amodiaquine hydrochloride in the dosages necessary to treat lupus erythematosus and rheumatoid arthritis is not recommended, for corneal opacities and retinopathy, peripheral neuropathy, fatal blood dyscrasias, and fatal hepatitis have been reported after these large dosages (47). Patients have experienced involuntary movements, usually with speech difficulty, after large but not excessive doses of amodiaquine (48). It may cause birth defects if taken during pregnancy (49). A method is described for evaluating the relative toxicity of amodiaquine in rats on the basis of effect on growth, lethal effects, production of pathological changes, and the concentration of drug in blood or plasma. The test can be completed in fourteen days (50).
ACKNOWLEDGEMENT

The authors wish to thank the United States Pharmacopeial Convention, Inc., for donating a sample of amodiaquine hydrochloride.
REFERENCES

1.

2. 3. 4. 5. 6.

7.
8.

Webster, L.T., Jr. (1985). In "Goodman and Gilman's The Pharmacological Basis of Therapeutics", 7th Edition (A.G. Gilman, L.S. Goodman, T.W. Rall and F. Murad, eds.), p. 1032, MacMillan Publishing Co., New York. Dyson, G.M. (1959). "May's Chemistry of Synthetic Drugs", 5th Edition, p. 538, Longmans, Green and Co., London. U.S. Patents (1949). 2,474,819; 2,474,821. "British Pharmacopoeia" (1988). pp. 37, 900, Her Majesty's Stationary Office, London. "Martindale, The Extra Pharmacopoeia" (1989). 29th Edition (J. E.F. Reynolds, ed.), p. 507, The Pharmaceutical Press, London. 'The Merck Index" (1983). 10th Edition (M. Windholz, ed.), p. 82, Merck and Co., Inc., Rahway, New Jersey. "Clarke's Isolation and Identification of Drugs" (1986). 2nd Edition (A.C. Moffat, ed.), p. 347, The Pharmaceutical Press, London. Burckhalter, J.H., Tendick, F.H., Jones, E.M., Jones, P.A., Holcomb, W.F. and Rawlins, A.L. (1948). J. Am. Chem. SOC. 70,1363.

AMODIAQUINE HYDROCHLORIDE

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9.
10.
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13.

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15. 16.
17.
18.

19. 20. 21. 22. 23. 24. 25. 26. 27. 28.

Burckhalter, J.H., DeWald, H.A., and Tendick, F.H. (1950). J. Am. Chem. SOC. 1024. 72, 'The United States Pharmacopeia" (1990). 22nd Revision 'The National Formulary" 17th Edition, p. 79, United States Pharmacopeial Convention, Inc., Rockville, Md. 'The Pharmaceutical Codex" (1979). 11th Edition, p. 36, The Pharmaceutical Press, London. Sunshine, I. (1981). "Handbook of Spectrophotometric Data of Drugs", p. 24, CRC Press, Inc., Boca Raton, Florida. Brandstaetter-Kuhnert, M., Kofler, A, Hoffmann, R. and Rhi, H.C. (1965). Sci. Pharm. 33,205. Cesaire, G., Fauran, F., Pellisier, C., Goudote, J. and Mondain, J. (1969). Bull. Mem. Fac. Mixte Med. Pharm. Dakar 17, 240; C.A. (1973) 79,97038k. Kracmar, J. and Kracmarova, J. (1974). Pharmazie 29,510. Kang, I.P.S., Kendall, C.E. and Lee, R.W. (1974). J. Pharm. Pharmacol. 26,201. Gupta, S.S., Siddique, S. and Kaushal, R. (1980). J. Indian Chem. SOC. 57,97. Gupta, S.S., Gupta, KK. and Kaushal, R. (1979). J.Sci. Res. (Bhopal, India) 1,125. Sastry, B.S., Rao, E.V., T u m u r u , M.K. and Sastry, C.S.P. (1986). Indian Drugs 24,105. Fuhrmann, G. and Werrbach, K. (1966). Tropenmed. Parasitol. 16, .. 269; C A (1966) 65,12725a. Wu, T.S., Sun, C.C. and Tang, T.H. (1958). Yao Hsueh Hsueh Pao 6, 253; C.A. (1959) 53,20691d. Walash, M.I., Rizk, M., Abou-Ouf, A.A. and Belal, F. (1983). Anal. Lett. 16, 129. Sastry, B.S., Rao, E.V. and Sastry, C.S.P. (1985). Indian Drugs 22, 550. Ahmad, S.J. and Shukla, LC. (1984). Analyst (London) 109,1103. Chatterjee, P.K., Jain, C.L. and Sethi, P.D. (1986). Indian Drugs 23, 563. Sethi, P.D. (1985). "Quantitative Analysis of Drugs in Pharmaceutical Formulations", p. 193, Unique Publishers, Delhi. Hassan, S.M., Metwally, M.E.S. and Abou-Ouf, A.A. (1983). J. Assoc. Off. Anal. Chem. 66,1433. Sane, R.T., Thombare, C.H., Anaokar, P.G.and Pandit, A.D. (1981). Indian J. Pharm. Sci. 43,22.

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29. Rao, G.R., Rao, Y.P. and Raju, I.R.K. (1982). Analyst (London) 107, 776. 30. Hassan, S.M., Metwally, M.E.S. and Abou-Ouf, A.A. (1982). Analyst (London) 107,1235. 3 1. Dalal, R.R., Bulbule, M.V., Wadodkar, S.G. and Kasture, A.V. (1982). Indian Drugs 19,361. 32. Issa, A.S., Mahrous, M.S., Salam, M.A.and Hamid, M.A. (1985). J. Pharm. Belg. 40,339. 33. Mahrous, M.S., Salam, M A , Issa, A.S. and Hamid, M.A. (1986). Talanta 33, 185. 34. Sastry, B.S., Rao, E.V. and Sastry, C.S.P. (1984). Indian J. Pharm. Sci. 46,186. 35. Sastry, B.S., Rao, E.V. and Sastry, C.S.P. (1986). Indian J. Pharm. Sci. 48,71. 36. Trenholme, G.M., Williams, R.L., Patterson, E.C., Frischer, H., Carson, P.E. and Rieckmann, K.H. (1974). Bull. Wld. Hlth. Org. 51, 431. 37. Stead, A.H., Gill, R., Wright, T., Gibbs, J.P. and Moffat, A.C. (1982). Analyst (London) 107,1106. 38. Musumarra, G., Scarlata, G., Romano, G., Clemente, S. and Wold, S. (1984). J. Chromatogr. Sci. 22,538. 39. Wheals, B.B. (1980). J. Chromatogr. 187,65. 40. Molokhia, A.M., El-Hoofy, S. and Dardiri, M. (1987). J. Liq. Chromatogr. 10,1203. 41. Pussard, E., Verdier, F. and Blayo, M.C. (1986). J. Chromatogr. 374, 111. 42. Churchill, F.C., Patchen, L.C., Campbell, C.C., Schwertz, I.K., Dinh, P.N. and Dickinson, C.M. (1985). Life Sci. 36,53. 43. Churchill, F.C., Mount, D.L., Patchen, L.C. and Bjoerkman, A. (1986). J. Chromatogr. 377,307. 44. White, A.I. (1977). In 'Textbook of Organic Medicinal and Pharmaceutical Chemistry", 7th Edition (C.O. Wilson, 0. Gisvold and R.F. Doerge, eds.), p. 258, J.B. Lippincott Co., Philadelphia. 45. Jenkins, G.L., Hartung, W.H., Hamlin, ICE., Jr. and Data, J.B. (1957). 'The Chemistry of Organic Medicinal Products", p. 361, John Wiley and Sons, Inc., New York. 46. Atherden, L.M. (1969). "Bentley and Driver's Textbook of Pharmaceutical Chemistry", 8th Edition, p. 638, Oxford University Press, London. 47. "New Drugs" (1966). p. 81, American Medical Association, Chicago.

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48. Akindele, M.O. and Odejide, A.O. (1976). Br. Med. J. 2,214. 49. 'The Physicians' and Pharmacists' Guide to Your Medicines",p. 18, United States Pharmacopeial Convention, Ballantine Books, New York. 50. Smith, C.C. (1950). J. Pharmacol. Exptl. Therap. 100,408.

CLOFAZIMINE

Caitriona M . ODriscoll and Owen I . Corrigan

University of Dublin Department of Pharmaceutics School of Pharmacy Trinity College, Dublin, Ireland

ANALYTICAL PROFILES OF DRUG SUBSTANCES AND EXClPlENTS - VOLUME 21

75

Copyright 0 1992 by Academic Press, Inc All rights of reproduction reserved in any form

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CAITRIONA M. O'DRISCOLL AND OWEN 1. CORRIGAN

CLOFAZIMINE Caitriona M. ODriscoll and Owen I. Corrigan University of Dublin, Department of Pharmaceutics, School of Pharmacy, Trinity College Dublin, Ireland.

2.

1.

3. 4,

5.

Introduction Description 2.1 Structural and Molecular Formulas and Molecular Weight 2.2 Nomenclature 2.3 Official Compendia 2.4 Other Compendia Synthesis Physical Properties Ultraiiolet Absorbance Spectrum 4.1 Infrared Absorbance Spectrum 4.2 Mass Spectrum 4.3 Proton Nuclear Magnetic Resonance Spectrum 4.4 Carbon-13 Nuclear Magnetic Resonance Spectrum 4.5 X-Ray Diffraction 4.6 Melting Point 4.7 Differential Scanning Calorimetry 4.8 Dissociation Constants 4.9 4.10 Solubilities 4.11 Par tition Coefficients Methods of Analysis 5.1 Elemental Analysis 5.2 Identification 5.3 Ultraviolet and Visible Spectrophotometry 5.4 Spectrofluorometric Analysis

CLOFAZIMINE

6. 7.

5.5 Thin Layer Chromatography 5.6 High Pressure Liquid Chromatography. Pharmacokinetics 6.1 Bioavailability Considerations 6.2 Distribution, Metabolism and Elimination Pharmacology 7.1 Mechanisms of Action 7.2 Structure - Activity Relationships 7.3 Toxicity 7.4 Dose Schedules Acknowledgements References

1. INTRODUCTION Clofazimine is active against Mycobacterium leprae and is used clinically to treat leprosy (Hansen's disease). It was synthesised in 1957 by Barry et al., Laboratories of the Medical Research Council of Ireland, Trinity College Dublin. The precise mechanism of the antileprotic action of clofazimine has not been established. The World Health Organisation classify clofazimine as an "essential drug" and recommend its use, in combination, with other agents to treat all cases of leprosy (WHO, 1982). Clofazimine is also used to treat Mycobacteriurn avium infections which frequently occur in patients with AIDS (acquired immunodeficiency syndrome), (Masur et al., 1987; Woods and Washington, 1987; Gangadharam et al., 1988; Lindholm - Levy and Heifets, 1988; Young, 1988). Clofazimine also displays anti inflammatory activity which is clinically useful in controlling erythema nodosum leprosum (ENL) reactions which occur in multibacillary forms of leprosy (Gidoh and Tsutsumi, 1979; Yawalkar and Vischer, 1979; Browne et al., 1981). A study using animal models of rheumatoid arthritis has indicated that clofazimine may be potentially useful to treat this disease (Currey and Fowler, 1972). Although the exact mechanism of clofazimine mediated anti-inflammatory activity is unknown, it may be related to the ability of the drug to increase

78

CAITRIONA M. ODRISCOU AND OWEN 1. CORRlGAN

the synthesis of anti-inflammatory immunosuppressive prostaglandin E2 (PGE2) by human polymorphonuclear leucocytes (Anderson, 1985;Zeis et al., 1987;Yawalkar, 1988).

2. DESCRIPTION

Clofazimine is a dark red or orange - red fine powder, odourless or almost odourless.
2.1 Structural and Molecular Formulas and Molecular Weight

Q
CI

Molecular Formula: Molecular Weight:

C27H22C12N4

473.4

CLOFAZIMINE

19

2.2 Nomenclature 2.21 Generic Name Clofazimine (BAN, USAN, rI") 2.22 Chemical Names

3-(4-chloroanilino)-10-(4-ehlorophenyl)-2,1 O-dihydrophenazin-2ylideneisopropylamine. N,5-Bis(4-chlorophenyl)-3,5-dihydro-3-[(l-methylethyl) iminol-2phenazinamine, or 3-(p-chloroanilino)-1O-(p-chlorophenyl)-2, 10-dihydro2(isopropylimino) phenazine, or 2-(4-chloroanilino)-3-isopropylimino-5-(4-chlorophenyl)-3,5dihydrophen azine, or 2-p-chloroanilino-5-p-chlorophenyl-3,5-dihydro-3isopropy liminophenazine.
2.23 Trade name Clofazimine is marketed by Ciba Geigy under the proprietary name "Lamprene". 2.24 Other Names, Abbreviations and Drug Codes Riminophenazine, 8663, G30320, NSC 141046, chemical abstracts service registry number (CAS no.) 2030 - 63-9. 2.3 Official Compendia A monograph on clofazimine is included in the British Pharmacoepia and the Indian Pharmacoepia. 2.4 Other Compendia Clofazimine is included in the Merck Index (19891, the Pharmaceutical Codex (1979), and in Martindale (1989). Clarke (1986) gives a useful summary of physical and chemical data.

80

CAITRIONA M. O'DRISCOU AND OWEN 1. CORRIGAN

+
NHz NHR

i, ii

NHR

iii
(3)
R = Ph, 4-CI-C6H4-

NHR

('1

R = aryl

iv

I V

Reagents: i, FeCl3, H+; ii, NH3; iii, R*NH2, alkyamines; iv, benzoquinone/carbonyl compound RkOR3; v, Pt @/H or Pt/C (lO%)/H2; vi, air; vii, Pd/C (lO%)/Hz.

Figure 1. Principal synthetic routes to riminophenazines (Hooper, 1987)


3. SYNTHESIS

The original synthetic routes to riminophenazines (Barry et al., 1956a; 1956b; 1957 and 1958 ) have been modified (O'Sullivan, 1984) to give reproducible high yields. The modifications have been summarised by Hooper (1987) (Figure I) as follows; N-aryl ortho -phenylenediamines (1) undergo regiospecific oxidative dimerization to yield the parent iminophenazines (2) which react further with alkylamines to give substituted iminophenazines (3). Alternatively, oxidation with benzoquinone in the presence of a

CLOFAZIMINE

81

carbonyl compound gives an imidazolophenazine (4) which may be reduced with cleavage of the imino substituent (5) followed by subsequent aerial oxidation to the parent iminophenazine (2). A more selective reduction results in an alternative cleavage of the imidazoline ring (6) which after oxidation gives a substituted iminophenazine (7). The type of catalyst used in the reduction of these compounds is crucial and allows full control of the reactions. 4. PHYSICAL PROPERTIES 4.1 Ultraviolet Absorbance Spectrum
The ultraviolet spectrum of clofazimine (0.001% w/v) is shown in Figure 2. The spectrum was obtained using a Hewlet Packard 845 2A diode array UV visible spectrophotometer and 1 em quartz cells. The spectrum, in the range 230 to 600nm, in 0.01m methanolic hydrochloric acid, exhibits two maxima, at 284nm and 486nm. The absorbance at 284nm is about 1.30 and at 486nm is about 0.64.

1
600

220

300

400

500

WAVELENGTH

Figure 2. Ultraviolet spectrum of clofazimine.

82

CAlTRlONA M. ODRISCOLL AND OWEN I. CORRIGAN

4.2 Infrared Absorbance Spectrum The infrared absorbance spectrum of clofazimine is shown in Figure 3. The spectrum was recorded with a Nicolet 5ZDX FT-IR spectrophotometer, from a compressed potassium bromide disc. Structural assignments for some of the characteristic absorption bands in the spectrum are listed in Table I. Table I. Infrared assignments for clofazimine

W avenumber (cm-l)
1587,1560,1510,1460,1300 1389,1360,1130

Assignment aromatic CH stretching CH(CH3)2 stretching

4.3 Mass Spectrum The mass spectrum of clofazimine, shown in Figure 4, was obtained using a Finnigan Quadrupole mass spectrometer, by electron - impact at 70 electron volts. The molecular ion (M-H) at m / z 473 was observed. Major peaks were detected at m / z (%) 474 (66.17),473 (36.22), 472 (1001,457 (93.04), 455 (70.87), 456 (24.13),431 (19.57), 414 (30.43), 380 (22.17), 345 (17.83), 331 (30.43). 4.4 Proton Nuclear Magnetic Resonance Spectrum (IH-NMR) The IH-NMR spectrum of clofazimine, shown in Figure 5, was obtained in deuterated chloroform containing tetramethylsilane (TMS) as internal standard, using a Joel GX- 270 MHz instrument. A 2D COSY spectrum was also obtained (Figure 6a and 6b). Figure 6b is an expansion showing coupling in the aromatic regions. 4.5 Carbon - 13 Nuclear Magnetic Resonance Spectrum The carbon-13 NMR spectrum of clofazimine was obtained in deuterated chloroform containing TMS as internal standard using a Joel GX-270 MHz instrument at a frequency of 67 MHz. The carbon-13 NMR spectrum, with DEPT, is shown in Figure 7.

CLOFAZIMINE

83

Figure 3.

Infrared spectrum of clofazimine.

Y
0
2000

. . .

I BOO

1so0

1400

1200 1000 W ~ v m u n b r r Cpm-1)

800

SO0

400

84

CAlTRlONA M . O'DRISCOLL AND OWEN 1. CORRIGAN

Figure 4.

Electron-impact mass spectrum of clofazimine.

CLOFAZIMlNE

85

Figure 5 .

Proton nuclear magnetic resonance spectrum of clofazimine.

86

CAITRIONA M. O'DRISCOLL AND OWEN I. CORRICAN

Figure 6a.

2-D proton nuclear magnetic resonance spectrum of clofazimine.

CLOFAZIMINE

87

Figure 6b.

2-D proton nuclear magnetic resonance spectrum of clofazimine.

88

CAITRIONA M. O'DRISCOLL AND OWEN I. CORRIGAN

Figure 7.

l3c1 nuclear magnetic resonance spectrum of clofazimine, with DEPT.

CLOFAZIMINE

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4.6 X-Ray Diffraction The powder X-ray diffraction pattern of clofazimine was obtained on a Siemens D-500 X-ray diffractometer, using a Cu X-ray tube, at 40 kV and 40 mA. The diffraction pattern is shown in Figure 8, indicating the crystalline nature of clofazimine. Rychlewska et al. (1985) reported two different crystalline forms of clofazimine, a monoclinic form with a density of 1.3 g cm-3, and a triclinic modification with a density of 1.29 g cm-3. The former was prepared by recrystallization from acetone, and the latter by recrystallization from 12 N-methylformamide/acetone. Cell constants were also calculated. The values obtained for the monoclinic form were a = 7.788 A, b = 22.960 A, c = 13.362 A, p = 98.580. The values for the triclinic form were a = 10.507 A, b = 12.852 A, c = 9.601 A, a = 95.960, p = 97.220, y = 69.730. 4.7 Melting Point Melting points reported in the literature are in the temperature range of 210 - 215OC, with degradation (Barry et al. 1956a; Clarke 1986; Merck Index 1989; Pharmaceutical Codex 1979). 4.8 Differential Scanning Calorimetry (DSC) The DSC thermogram of clofazimine obtained using a Mettler DSC 20, scan speed lOOC min-1, is shown in Figure 9. A single sharp melting endotherm was obtained with onset temperature at 214OC. This value is in good agreement with the melting points previously published (Section 4.7). The estimate of the heat of fusion (AH) was 740 joules/gram. However, with some samples there was evidence of degradation on melting. 4.9 Dissociation Constant The values for the dissociation constant reported for clofazimine are summarised in Table 1 . 1

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CAlTRlONA M . O'DRISCOLL AND OWEN 1. CORRIGAN

Figure 8.

X-ray powder diffraction pattern of clofazimine.

TWO

- THETA

IOEGREESI

CLOFAZIMINE

91

Figure 9.

Differential scanning calorimetry thermogram of clofazimine.

200.0-

2lO.O-

2 0 . 0 0 0 nU

220.0-

230.0-

240.0-

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CAITRIONA M. O'DRISCOLL AND OWEN 1. CORRIGAN

Table 11. Dissociation constant of clofazimine (pKa) PKa 8.35 8.37 8.51 Method of determination Not stated Potentiometric Spectropho tome tric Reference Morrison a n d Marley (1976a) Canavan et al. (1986) Fahelelbom et al. (1989)

4.10 Solubilities Clofazimine is practically insoluble in water, estimates in the range of 1.03 - 0.49 pg ml-1, at 37OC, have been reported (Fahelelbom, 1989; OReilly, 1991). It is soluble 1 in 700 of ethanol, 1 in 15 of chloroform, and 1 in 1000 of ether. It is also soluble in dilute acetic acid and dimethylformamide (Clarke, 1986).

PH
Figure 10. pH solubility profile of clofazimine.

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The effect of pH (range 5.15 - 7.8) on the solubility of clofazimine, shown in Figure 10 (OReilly, 19911, is consistent with the basic nature of the compound. The solubility of the drug is 5.68 and 0.278 mg ml-1 x 10 -3 at pH 5.15 and 7.8 respectively. Values for intrinsic solubility in the range of 2.0 - 2.3 x 10-5mg ml-1 (Fahelelbom, 1989; OReilly, 1991) have been reported. The solubility of clofazimine was enhanced in aqueous micellar systems, containing both naturally occuring surfactants e.g bile salts, and synthetic surfactants, e.g the non ionic Cremophor EL and Triton X100, and the anionic sodium dodecyl sulphate. The incorporation of fatty acids to form mixed micelles brought about a further enhancement in drug solubility in the case of naturally occuring surfactants (approximately 300 fold with sodium cholate: linoleic acid relative to buffer). In contrast, with synthetic surfactants this enhancement decreased (Fahelelbom et al., 1991; ODriscoll et al., 1991).
4.11 Partition Coefficients (Log P) Partition coefficients for clofazimine have been determined using different solvents and temperatures. The data is summarised in Table 111. Table 111. Partition coefficients of clofazimine Solvents Octanol: water Isooctane: buffer pH 5.15 N-octanol: buffer pH 5.15 N-octanol: buffer pH 5.15 N-octanol: buffer pH 5.15 N-octanol: buffer pH 5.15 Temp (OC) LogP Reference

20 20
37 45 55

+7.48* Morrison and Marley (1 976a,b) 5.01 Canavan et al. (1986) 4.30 Quigley et al. (1990) 4.40 Ibid 4.48 Ibid 4.54 Ibid

* Estimated

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CAITRIONA M.O'DRISCOLL AND OWEN 1. CORRIGAN

5. METHODS OF ANALYSIS 5.1 Elemental Analysis Carbon Hydrogen Nitrogen Chlorine 5.2 Identification The B.P. (1988) outlines three methods of identification: (A) By the infrared absorption spectrum, outlined in section 4.2. (B) The light absorption, the UV spectrophotometry is described in section 4.1. (C) A colour test, dissolve 2mg clofazimine in 3ml of acetone and add O.lml of hydrochloric acid, an intense violet colour is produced. Add 0.5ml of 5M sodium hydroxide, the colour changes to orange - red. 5.3 Ultraviolet and Visible Spectrophotometry Quantitative ultraviolet analysis of clofazimine has been performed, in a range of aqueous and nonaqueous media, at 280nm (Canavan et al., 1986; O'DriscolI et al., 1990a,b), and colorimetrically at 482nm (Quigley et al., 1990). A colormetric assay was developed by Barry et al. (1960) and modified by Mansfield (1974) to analyse plasma and tissue levels of clofazimine. The drug was extracted using benzene and concentrated hydrochloric acid, and the absorption read at 540nm. The limit of detection reported was 0.2pg/ml in plasma and O.lmg/gram in tissue. 5.4 Spectrofluorometric Analysis A fluorescent derivative of clofazimine was formed following reduction with titanous chloride (Dill et al., 1970). The fluorescence was measured at 366mp emission. The limits of
% Calculated

68.50 4.68 11.83 14.98

% Found

68.68 4.52 11.48 15.32

CLOFAZIMINE

95

detection reported for this method were in the range of 0.1 - 0.2 pg/ml in plasma (Banerjee et al., 1974; Levy, 1974). 5.5 Thin Layer Chromatography A thin layer chromatographic (TLC) system suitable for determination of clofazimine in plasma has been developed (Lanyi and Dubois, 1982). The plasma samples were acidified using acetate buffer pH 5 and extracted with toluene, evaporated to dryness under nitrogen, reconstituted in toluene and applied to the TLC plate. The adsorbent used was HPTLC silica gel 60. The plates were developed in toluene - acetic acid - water (50 : 50 : 4), allowed to stand for 30 min at room temperature, the Rf value of clofazimine was 0.36. Detection and quantitation is carried out using a densitometric method. The limit of detection reported for this method was 5ng/g. 5.6 High Performance Liquid Chromatography Gidoh et al. (1981) developed a high performance liquid chromatographic (HPLC) method with ultraviolet detection to separate and quantify clofazimine (287nm) from other antileprosy 1 drugs, dapsone and rifampicin, in serum on a pBondapak c 8 column. This method involved a complicated extraction procedure with the switching of 2 different mobile phase (i.e acetonitrile - water, 20 : 80; and tetrahydrofluran - water containing PIC B-5,50 : 50, the latter reagent contains 1 - pentanesulfonic acid and glacial acetic acid) in order to allow complete resolution of clofazimine from related components. The limit of detection for this method was long ml-1. Recently a modification of this technique was used to study clofazimine and its derivatives (O'Sullivan et al., 1990). Another HPLC method, was described by Peters et al. (1982), for measuring clofazimine in plasma, with a limit sensitivity of 10 ng ml-1. This method involved extraction of clofazimine into organic solvents and quantifation on a reversed-phase Ultrasphere - octyl column, using a mobile phase of 0.0425M phosphoric acid in 81% methanol and UV detection at 285nm. The gastrintestinal absorption of clofazimine, using a rat gut perfusion technique, was determined by HPLC (O'Driscoll et al., 1990a,b). The column used was Partisil lOPAC, the mobile phase

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CAITRIONA M. O'DRISCOLL AND OWEN 1. CORRIGAN

was ethanol : N-heptane (50 : 50) and detection was by UV at 283nm.The limit of sensitivity was 0.1pg ml-I.

6 . PHARMACOKINETICS
6.1 Bioavailability Considerations Clofazimine absorption following oral administration is incomplete and varies significantly from patient to patient. Following administration as coarse crystals only about 20% is absorbed, if however, the drug is given as a microcrystalline suspension in an oil wax base an absorption rate of 70% can be achieved (Yawalkar and Vischer, 1979). The gastrointestinal absorption of clofazimine in the anaestheised rat, using an in situ rat gut perfusion model (Komiya et al., 19801, was enhanced by co-administration of simple and mixed micellar systems (O'Reilly et al., 1988; O'Driscoll et al., 1990a,b). The simple micellar systems included various bile salts, and the synthetic emulgents, Cremophor EL (non ionic) and sodium dodecyl sulphate (anionic). The mixed micelles were formulated by the incorporation of various fatty acids. A mixed micellar system containing sodium cholate: linoeleic acid enhanced the rate of absorption of clofazimine by a factor of 840 compared to a buffered solution of the drug. The enhancements were due to a combination of increased solubility and increased membrane permeability. There is also evidence that clofazimine is transported in part via the lymphatic system (Barry et al., 1960; Atkinson et al., 1967). Clofazimine has a reported pKa of 8.35 and consequently it is highly ionised under physiological conditions. This high degree of ionization, together with its high molecular weight, may be significant factors in the poor oral bioavailability. Schaad - Lanyi et al. (1987) studied the pharmacokinetics of single oral doses of clofazimine over 11 days following administration. They examined the effect of food on the bioavailability. Following administration with food the area under the plasma concentration versus time curve (AUC) and the peak plasma concentration C , were 62 and 30% higher respectively compared to results obtained in the fasted state. The

CLOFAZIMINE

97

median time (tmx) to reach Cmaxwas 8 hours with food and 12 hours without food.
6.2 Distribution, Metabolism and Elimination

Plasma levels of the drug are approximately 0.5mg 1-1 but increase with the dose and at 300mg daily levels of 1.0 - 1.5 mg 1-1 have been achieved (Banerjee et al., 1974;Levy,1 7 ) 94. Administration of 50mg of clofazimine daily for 8 days did not achieve steady state (Schaad - Lanyi et al., 1 8 ) The time to reach 97. steady state has been theoretically estimated to be in the range of 30 - 70 days (Schaad - Lanyi et al., 1987; Holdiness, 1989). There is no data available on loading doses. Likewise, there is no information currently available on the pharmacokinetics of clofazimine following intravenous administration. The appearance of clofazimine in the plasma following absorption is short lived (Banerjee et al., 1 7 1 it rapidly passed 94, out of the circulation and is deposited in various tissues and organs, particularly the fatty tissue, the spleen, lymph nodes, and the cells of the reticulo - endothelial system. Concentrations of 2.15.3 mg g-1 have been reported in the subcutaneous fat (Mansfield, 1 7 ) and 0 6 1 0mg g in the spleen (Desikan and Balakrishnan, 94, .-. 1 1976;Mansfield, 1974). It is taken up by the macrophages throughout the body (Conalty et al., 1971;Yawalkar and Vischer, 1979). Electrophoretic studies of serum from orally treated mice have shown almost complete binding of clofazimine to the lipoproteins of the a and globulin fractions, these lipoprotein are 91. then phagocytosed by the macrophages (Conalty et al., 1 7 ) Clofazimine crystals have been found at autopsy in the small intestine and in the macrophages of mesenteric lymph nodes (Conalty et al., 1971;Aplin and McDougall, 1975;Desikan and Balakrishnan, 1976;Jopling, 1 7 ) Clofazimine does not appear to 96. cross the intact blood-brain barrier (Mansfield, 1974;Desikan and Balakrishnan, 1976). It does, however, appear to cross the placenta causing pigmentation of the foetus (Holdiness, 1989). There is no data available on the volume of distribution of clofazimine. Feng et al. (1981; 1982)have used mass, ultraviolet and visible spectrometry to identify three metabolites in the urine of leprosy patients (Figure 11). Metabolite I is the unconjugated compound 3 (p-hydroxyanilino)-lO-(p-chlorophenyl)-2, lO-dihydro-2-

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CAITRIONA M. ODRISCOLL AND OWEN 1. CORRIGAN

isopropyliminophenazine, the other two metabolites are


conjugated, metabolite 1 is 3-(P-D-glucopyransiduronic acid)-lO-(p1 chloropheny1)-2, 10-dihydro-2- isoproyliminophenazine), and metabolite 111 is 3 - (p-chlorani1ino)-10- (p-chlorophenyl) - 4, 10dihydro - 4 (PD-glucopyranosiduronic acid) -2isopropyliminophenazine. Metabolite I is reported to be formed 1 by a hydrolytic dehalogenation reaction, metabolite 1 by hydrolytic deamination followed by glucuronidation, and metabolite III by hydration followed by glucuronidation. Following administration of 300mg/day of clofazimine, 0.2% of metabolite I 0.25% of metabolite 1 , and 0.2% of metabolite I11 were , 1 recovered in the urine over 24 hours (Feng et al., 1981; 1982). No information is available on the pharmacological activity of these metabolites, or whether they are found in faeces or bile. The authors have shown that metabolite I11 may be produced in the laboratory through metabolism by liver enzymes. However, they were unable to demonstrate the same hepatic conversion of clofazamine to metabolites I and 11. In contrast, they suggest that these metabolites are produced by bacterial degradation in the intestine prior to absorption and urinary excretion. Clofazimine accumulates in certain tissues throughout the body (fatty tissue, skin, lymph nodes, macrophages etc.) and is eliminated very slowly. The kinetics of the drug has been described by both one and two compartment models. Data obtained with relatively low dose, short term administration indicated a one compartment model, with a a plasma tipof approximately 7 days (Levy, 1974; Hastings et al., 1976; Holdiness, 1989). A second compartment is evident with long term, high dose administration and appears to have a ttp of at least 70 days (Banerjee et al., 1974; Levy, 1974). Following oral administration of 50mg/day of clofazimine to health volunteers Schaad - Lanyi et al. (1987) predicted that steady state (SS) plasma concentrations would occur after approximately 30 days. They calculated an accumulation factor for the drug from the ratio of AUCss: AUC. A value of 4.85 was obtained suggesting a slow accumulation towards steady state. The authors suggest that this may be avoided by administering higher loading doses, followed by daily maintenance doses.

CLOFAZIMINE

99

CI

N.CH(CH,),

Metabolite I

N.CH(CH,),

deamination --- -- -- ---

1. Hydrolytic

2. Glucuronation

Clofazimine
Metabolite II

Metabolite 111

Figure 11. Metabolic pathways of clofazimine in humans (Feng et al., 1981; 19821)

Up to 50% of a dose of clofazimine is excreted unchanged in the faeces, indicative of poor oral absorption (Banerjee et al., 1974). However, high concentrations of the drug have been found in bile and in the gall bladder. This suggests that part of the ingested drug recovered from the faeces may represent excretion by means of the bile rather than simply the failure of absorption from the gastrointestinal tract (Mansfield, 1974). Urinary excretion in leprosy patients is negligable accounting for an average of 0.1%

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CAITRIONA M. O'DRISCOLL AND OWEN I. CORRIGAN

(range 0.01 - 0.43%)of the dose in 24 hours (Levy, 1974). A small amount of the drug is excreted in the sebum and sweat (Vischer, 1969).

7. PHARMACOLOGY 7.1 Mechanisms of Action

Although the precise mechanism of the antileprotic activity of clofazimine has yet to be determined several explanations have been proposed (Hooper 1987). (a) The drug has been shown to bind to cytosine - guanine DNA base pairs in vitro (Morrison and Marley, 1976a,b). The binding is specific for guanine residues only. The DNA of M. Zeprue has a high guanine - cytosine content, consequently this binding may disrupt the template function of the DNA, causing inhibition of protein synthesis. (b) The redox properties of clofazimine can divert up to 20% of cellular oxygen (Barry et al., 1957) and thus disrupt normal mitochondria1 oxidation processes (Rhodes and Wilkie, 1973). In addition, it has been suggested that cytotoxic oxygen species, hydrogen peroxide and superoxide, are generated as a result of the presence of the drug (Hooper and Purohit, 1983; Savage et al., 1989). If such a reaction occurred within the macrophages it will enhance the killing of the bacilli which are also found inside the macrophages. (c) In addition, it has been suggested that the antileprotic effect of clofazimine may be due to its action on the macrophage lysosomal apparatus (Sarracent and Finlay, 1984).
7.2 Structure Activity Relationships (SAR)

The earlier SAR studies, reviewed by Hooper and Purohit (1983), concentrated on three main areas of molecular modification (Figure 12). Firstly, the structure of clofazimine was varied by introducing additional chlorine atoms at positions 4, 7,8 and 9. This resulted in loss of activity, except for the 7- chloro derivative, with was equipotent with clofazimine. The second series was

CLOFAZIMINE

101

based on triaryl derivative. A variety of derivatives with a chloroor methoxy substituent in various positions showed only modest activity. The third series involved variations at R2 coupled with changes at R1 and R3, and the introduction of various substituents at positions 7 and 9. In general for optimum activity R2 had to be alkyl or cycloalkyl, and R1/ aryl or substituted aryl. When R3 hydrophilic salt forming groups were introduced at R2 activity was greatly reduced.

R
1

Figure 12. Basic structure of iminophenazines


An X-ray crystallographic study (Rychlewska et al., 1985) described the crystal and molecular structures of two crystal forms of clofazimine and of its inactive isomer, isoclofazimine (B3857). The geometric differences between clofazimine and isoclofazimine were probed by CND0/2 molecular orbital calculations. The geometry at the exocyclic amino nitrogen atom N(3) is f significantly different in isoclofazimine from that in both forms o clofazimine and in other active analogues (Figure 13). The authors suggest that the value for the intramolecular angle a at N(3) (defined by C(3) - N(3) - C(21)in clofazimine) may play a significant role in the activity. Molecules with values of a in the range 125.5 & 10 were inactive, while those with expanded a angles (i.e 131f 10) were active in vitro. The larger angle in the active compounds is thought to favour intramolecular hydrogen bonding between N(3)-H ... N(2). The capacity to form an intramolecular hydrogen bond was interpreted as evidence of a capacity for intermolecular hydrogen bonding in solution e.g between guanine in DNA and clofazimine.

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CAlTRlONA M. ODRISCOLL AND OWEN 1. CORRICAN

Figure 13. Crystal structure of clofazimine (Rychlewska et al., 1985)


A wide range of clofazimine analogues have been designed as follows; (a) to be active against resistant organisms, (b) not to accumulate in adipose or other tissues, (c) to be rapidly and adequately absorbed from the gastrointestinal tract, and (d) not to crystallize within cells (Barry et al., 1959; Franzblau and OSullivan, 1988; OSullivan et al., 1988; Byrne et al., 1989). These structural modifications generally involve substitution at the imino nitrogen atom by an unbranched alkyl or branched alkyl chain containing a primary, secondary, tertiary, or alicyclic amino group. Frequently the pKa values of these amine containing side chains are approximately 9.5 - 10.5 thus ensuring that these molecules will be substantially ionized under physiological conditions. To counter act this increased hydrophilicity the aliphatic part of the substituents usually contain 6 - 8 hydrophobic methylene groups (Hooper, 1987). A study, (Canavan et al., 19861, on the influence of lipophilic and stearic properties on the distribution of a range of clofazimine analogues to the spleen of mice following oral administration,

CLOFAZIMINE

I03

indicated that lipophilicity of the molecule is a significant factor whereas the stearic properties of the N2 - substituents are not. The structural features of phenazine derivatives which f contribute to stimulation o PGE2 production by polymorphonuclear leucocytes (Zeis et al., 1987) and pro-oxidative interactions with neutophils (Savage et al., 1989), have also been investigated. 7.3 Toxicity Clofazimine is a relatively non-toxic drug W.S. Leprosy panel, 1976). The acute LD50 was found to be >5 g/kg in mice rats and guinea pigs. It was 3.3 g/kg in the rabbit. Daily oral doses of 30 and 50 mg/kg given for six months were generally well tolerated by monkeys and rats. Reddish discolouration of the skin, faeces and urine was observed. Temporary diarrhoea was occassionally reported in rats (Stenger et al., 1970). Experimental studies in animals did not show any evidence that clofazimine possesses a primary embryotoxic or teratogenic action (Stenger et al., 1970). The drug does not exhibit mutagenic activity (Morrison and Marley, 1976a). A long term study on 51 patients receiving clofazimine for periods up to 8 years showed that, despite the deposition of the drug in various tissues, it appears to be remarkably free from serious side effects in clinical use (Hastings et al., 1976). Although clofazimine crosses the placenta, no evidence of teratogenicity has been found (Schulz, 1972). The most frequently reported side effects of clofazimine therapy are red-brown hyperpigmentation of the skin and conjunctiva, and abdominal pain (Hastings et al., 1976; Jopling, 1976; Yawalkar and Vischer, 1979; Granstein and Sober, 1981; Moore, 1983; Negrel et al., 1984; Venencie et al., 1986). Cutaneous pigmentation normally fades within 6 to 12 monthsGeneralised dryness of the skin (xeroderma) ichthyosis, puritis, phototoxicity, acneiform eruptions, exfoliative dermatitis and non specific skin rashes have been reported (Yalwalkar and Vischer, 1979; Pavithran, 1985). Discolouration of sweat, hair, sputum, urine and faeces have also been observed (Yalwakar and Vischer, 1979). Apart from subepithelial pigmentation in the cornea no other side effects on the eye were recorded. Clofazimine crystals were found in the tears of 82% of patients studied (Negrel et al., 1984).

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CAITRIONA M. O'DRISCOLL AND OWEN I. CORRIGAN

Gastrointestinal side effects, nausea, diarrhoea, anorexia, constipation and weight loss have also been reported (Hastings et al., 1976; Moore, 1983). These symptoms have been associated with the deposition of clofazimine crystals in the submucosa of the small intestine and in the mesenteric lymph nodes (Jopling, 1976; Harvey et al., 1977). The occurence of drug interactions involving clofazimine have also been investigated. Most of the studies show that clofazimine does not exert any effect on dapsone excretion in leprosy patients (Balakrishnan and Seshadri, 1981; Zuidema et al., 1986). Clofazimine has been shown to significantly reduce the absorption of simultaneously administered rifampicin, resulting in delayed time to reach peak serum concentration and increased t; . No significant changes were seen in C, or AUC (Mehta et al., 1986). , ,
7.4 Dose Schedules
A dose of 300mg once montly plus 50mg daily or lOOmg on alternative days has been recommended to treat multibacillary forms of leprosy (Martindale, 1989). The World Health Organisation (1982) has published guidelines for the treatment of leprosy. Dosage schedules are generally not based on serum/plasma concentrations, or pharmacokinetic data. Clofazimine is usually used in combination with other antileprotic agents e.g dapsone and rifampicin, to prevent the emergence of resistance. It is usually given with food in doses adjusted according to body weight and the activity of the disease. The therapeutic activity of clofazimine depends on the concentration of drug in the immediate environment of M.leprae in the tissues and not on the serum level. Since the drug in not evenly distributed through out the tissues it is impossible to calculate the minimal inhibitory concentration (MIC) in animals (Yawalkar and Vischer, 1979).

CLOFAZIMINE

10.5

ACKNOWLEDGEMENTS The authors wish to thank Dr. J. F. OSullivan, formerly of the Health Research Board, Trinity College, Dublin, Dr. Helen Sheridan, Department of Pharmacognosy and Dr. Mary Meegan, Department of Pharmaceutical Chemistry, Trinity College, Dublin for their advice and assistance, Ciba Geigy, England, for the supply of clofazimine, Ms. Mary Lally and Ms. Mary Reilly for technical assistance.

REFERENCES Alpin, R. T., and McDougall, A. C. (1975). Experientia, 31/4,468. Anderson, R. (1985). Lepr. Rev. 5 - 2 68 . Atkinson, A. J. Jr., Sheagren, J. N., Barba Rubio, J., and Knight, V. (1967). Int. J. Lepr.- 119. 5 3 Balakrishnan, S., and Seshadri, P. S. (1981). Lepr. India, 53, 17. Banerjee, D. K., Ellard, G. A., Gammon, P. T., and Waters M. F. R. (1974). Am. J. Trop. Med. Hyg. 23,1110. Barry, V. C., Belton, J. G., O'Sullivan, J. F., and Twomey, D. (1956a). J. Chem. Soc. 3347. Barry, V. C., Belton, J. G., OSullivan, J. F., and Twomey, D. (1956b). J. Chem Soc. 888. Barry, V. C., Belton, J. G. O'Sullivan, J. F., and Tomey, D. (1958). J. Chem. Soc. 859. Barry, V. C., Browne, J. G., Conalty, M. L., Denneny, J. M., Edward, D. W., O'Sullivan, J. F., Twomey, D., and Winder, F. (1957). Nature. 179,1013. Barry, V. C., Buggle, K., Byme, J., Conalty, M. L., and Winder, F. (1959). Bull. Internat. Union. Tuberc. 29,582. Barry, V. C., Buggle, K., Byrne, J., Conalty, M. L. and Winder, F. (1960). Irish J. Med. Sci. 416,345. British Pharmacopeia, (1988). British Pharmacopeial Commision London, HMSO, p. 145. Browne, S. G., Harman, D. J., Waudby, A., McDoughall, A. C. (1981). Int. J. Lepr. 49, 167. Byrne, J., Conalty, M.L., and OSullivan, J. F. (1989). Proc. R. Irish Acad. 89B, 115.

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Canavan, E. B., Esmonde, A. G., Feely, J. P., Quigley, J. M., and Timoney, R. F. (1986). Eur. J. Med. Chem. 21,199. Clarke, E. G. C. (1986). "Isolation and Identification of Drugs", 2nd Edn. The Pharmaceutical Press, London. p. 476. Conalty, M. L., Barry, V. C., and Jina, A. (1971). Int. J. Lepr. 39,479. Currey, H. L. F. and Fowler, P. D. (1972). Br. J. Clin. Pharmacol. 45. 676. Desikan, K. V., and Balakrishnan, S. (1976). Lepr. Rev. 47. 107. Dill, W. A., Chucot, L., and Glazko A. J. (1970). Int. J. Lepr. 38.355. Fahelelbom, K M. S. (1989). Ph.D. Thesis in Pharmaceutical Chemistry, School of Pharmacy, Trinity College Dublin, Ireland. Fahelelbom, K. M. S., Quigley, J. M., Timoney, R. F., and Corrigan, 0.I. (1989). Proc. R. Irish Acad. 89B, 287. Fahelelbom, K. M. S., Timoney, R. F., and Corrigan, 0. I. (1991). Pharm. Res. In press. Feng, D. C. C., Fenselau, C. C., and Jacobson, R. R. (1981). Drug Metab. Dispos. 521. Feng, P. C. C., Fenselau, C. C., and Jacobson, R. R. (1982). Drug. Metab. Dispos. 10.286. Franzblau, S. G., and OSullivan, J. F. (1988). Antimicrob. Agents Chemother. 32,1583. Gangadharam, P. R. J., Perumal, V. K., Podapati, N. R., Kesavalu, L. and Iseman, M. D. (1988). Antimicrob. Agents Chemother. 32, 1400. Gidoh, M., and Tsutsumi, S. (1979). Jap. J. Lepr. 48,7. Gidoh, M., Tsutsumi, S., and Takitani S. (1981). J. Chromatogr. 223,379. Granstein, R. D., and Sober, A. J. (1981). J. Amer. Acad. Derm. 5 1. Harvey, R. F., Harman, R. R. M., Read, A. E., et al. (1977). Brit. J. Derm. 96,19. Hastings, R. C., Jacobson, R. R., and Trautman, J. R. (1976). Int. J. Lepr. 44.287. Holdiness, M. R. (1989). Clin. Pharmacokin 16,74. Hooper, M. (1987). Chem. SOC. Rev. 16,437. Hooper, M., and Purohit, M. G. (1983). "The Chemotherapy of Leprosy" in Prog. Med. Chem., Ed. Ellis, G. P., and West, G. B. Vol 20, Elsevier, - North Holland. Indian Pharmacopeia, (1985). 3rd Edn. vol. 1. Government of India. Ministry of Health and Welfare, New Delhi. p. 127. Jopling, W. H. (1976). Lepr. Rev. 4-. 7 1

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Komiya, I., Park, J. Y., Kamani, A,, Ho, N. F. H., and Higuchi, W. I. (1980). Int. J. Pharm. 4 249. Lanyi, Z., and Dubois J. P. (1982). J. Chromatogr. 232.219. Levy,L. (1974). Am. J. Trop. Med. Hyg. 23,1097. Lindholm - Levy, P. J., and Heifets, L. B. (1988). Tubercle, 69.179. Mansfield, R. E. (1974). Am. J. Trop. Med. Hyg. 23. 1116. Martindale. The Extra Pharmacopoeia. (1989). 29th Edn. The Pharmaceutical Press, London. Masur, H., Tuazon, C., Gill, V., Grimes, G., Baird, B., Fauci, A. S., and Lane, H. C. (1987). J. Infect. Dis. (USA) 155.127. Mehta, J., Gandhi, I. S., and Sane, S. B. (1986). Lepr. Rev. 575.67. Merck Index. (1989). 11th Edn. Moore, V. J. (1983). Lepr. Rev. 54,327. Morrison, N. E., and Marley, G. M. (1976a). Int. J. Lepr. 44,475. Morrison, N. E., and Marley, G. M. (1976b).Int. J. Lepr. 44.133. Negrel, A. D., Chovet, M., Baquillan, G., Lagadec, R. (1984). Lepr. Rev. 55,349. ODriscoll, C. M., OReilly, J. R., and Corrigan, 0.1. (1991). Eur. J. Drug Metabolism and Pharmacokinetics. In press. ODriscoll, C. M., OReilly, J. R., and Corrigan, 0. I. (1990a). 17th Int. Symposium on Controlled Release of Bioactive Materials. Reno, Nevada, USA., Abstract S214. ODriscoll, C.M., O'Reilly, J. R., and Corrigan 0. I. (1990b). Fourth European Congress of Biopharmaceutics and Pharmacokinetics, Geneva, Abstract 125. f OReilly (1991). Ph.D. Thesis in Pharmaceutics School o Pharmacy, Trinity College Dublin, Ireland. In press. OReilly, J. R , ODriscoll, C. M., and Corrigan, 0.I. (1988). Third . Int. Conference on Drug Absorption, Edinburgh, Abstract 43. O'Sullivan, J. F. (1984). J. Chem. Res. (S), 52. OSullivan, J. F., Conalty, M. L., and Morrison, N. E. (1988). J. Med. Chem. 31,567. OSullivan, S., Corcoran, M., Byrne, M., McGrath, S., and OKennedy R. (1990). Biochem. Soc. Trans. 18.346. Pavithran, K. (1985). Int. J. Lepr. 53.645. Peters, J. H., Hamme, K. J., and Gordon, G. R. (1982). J. Chromatogr. 229,503. Quigley, J. M., Fahelelbom, K. M. S., Timoney, R. F., and Corrigan, 0. I. (1990). Int. J. Pharm. 58. 107. Rhodes, P. M., and Wilkie, D. (1973). Biochem. Pharmacol. 22, 1047.

I OX

CAITRIONA M. O'DRISCOLL AND OWEN 1. CORRIGAN

Rychlewska, U., Broom M. B. H., Eggleston, D. S., and Hodgson, D. J. (1985). J. Am. Chem. Soc. 107,4768. Sarracent, J., and Finlay, C. M. (1984). Int. J. Lepr. 52.154. Savage, J. E., OSullivan, J. F. Zeis, B. M., and Anderson, R. (1989). J. Antimic. Chemotherapy. 23,691. Schaad Lanyi, Z., Dieterle, W., Dubois, J. P., Vischer, T. W. (1987). Int. J. Lepr. - 9. 5 Schulz, E. J. (1972). Lepr. Rev. 42.178. Stenger, E. G., Aeppli, L., Peheim, E., and Thomann, P. E. (1970). Arzneim. Forsch. (Drug. Res). 20,794. The Pharmaceutical Codex (1979). 11th Edn. The Pharmaceutical Press, London U. S. Leprosy Panel (U. S. Japan Cooperative Medical Science Programme). (1976). Am. J. Trop. Med. Hyg. 25,437. Venencie, P. Y., Cortez, A., Orieux, G., Jost. J. L., Chomette, G. et al. (1986). J. Amer. Acad. Dermat. 15,290. Vischer, W. A. (1969). Lepr. Rev. 40, 107. 275. Woods, G. L., and Washington, J. A. (1987). Rev. Infect. Dis. World Health Organisation (1982). Chemotherapy of leprosy for control programs. Report of a WHO study group. World Health Organisation Technical Series number 675. Yawalkar, S J. (1988). "Lamprene in Leprosy", 3rd Edn. Ciba-Geigy . Ltd., Basle, Switzerland. Yawalkar, S. J., and Vischer, W. (1979). Lepr. Rev. 50. 135. Young, L. S.(1988). J, Infect. Dis. 157.863. 8 Zeis, B. M., Anderson, R., and OSullivan J. F, (1987). Lepr. Rev. 5 ,

Zuidema, J., Hilbers - Modderman, E. S. M., Merkus, F. W. H. M. (1986). Clin. Pharmacokin. I ,299. I

383.

CLONIDINE HYDROCHLORIDE

Mohamrnad A. Abounassif,' Mohammad Saleem Mian,' and Neelofur Abdul Aziz Mian'

(1)

Pharmaceutical Chemistry Department College of Pharmacy King Saud University Riyadh, Saudi Arabia

(2) Clinical Laboratory Sciences Department College of Applied Medical Sciences King Saud University Riyadh, Saudi Arabia

ANALYTICAL PROFILES OF DRUG SUBSTANCES AND EXCIPIENTS - VOLUME 21

109

Copyright 0 1992 by Academic Press, Inc. All rights of reproduction reserved in any form.

110

M.A. ABOUNASSIF, M.S. MIAN, AND N.A.A. MIAN

Contents
1, 2.

Introduction Description 2.1 Nomenclature 2 . 1 . 1 Chemical Names 2 . 1 . 2 Generic Names 2 . 1 . 3 Trade Names 2.2 Formulae 2 . 2 . 1 Empirical 2 . 2 . 2 Structural 2 . 2 . 3 CAS (Chemical Abstract Service Registry Number) 2.3 Molecular Weight 2.4 Elemental Composition 2.5 Appearance, Color, Odour and Taste Physical Properties 3.1 Melting Range Solubility 3.2 3.3 PH 3.4 Loss on drying 3.5 Sulphated Ash Clarity and Color of Solution 3.6 3.7 Stabi1 ity 3.8 PK 3.9 LD5 o 3 . 1 0 Action 3 . 1 1 Half Life Plasma 3 . 1 2 Volume of Distribution 3 . 1 3 Protein Binding 3 . 1 4 Storage 3 . 1 5 X-ray Powder Diffraction 3 . 1 6 Crystal Structure 3 . 1 7 Spectral Properties 3 . 1 7 . 1 Ultraviolet Spectrum 3 . 1 7 . 2 Infrared Spectrum 3 . 1 7 . 3 Nuclear Magnetic Resonance Spectra 3 . 1 7 . 4 Mass Spectrum Synthesis
5.1 5.2 5.3

3.

4.

5.

Phnrmacokirietics Absorption and Distribution Uses and Administration Adverse Effects

CLONIDINE HYDROCHLORIDE

5.4
6.

Precautions

Methods of Analysis 6.1 Identification 6.2 Colorimetric 6.3 Fluorimetric 6.4 Spectrophotometric Analysis 6.5 Radio-Immunoassay 6.6 Chromatographic Methods 6.6.1 Gas-Liquid Chromatography (GLC) 6.6.2 High-Performance Liquid Chromatography (HPLC)

7.
8.

Acknowledgements
References

112

M.A. ABOUNASSIF, M.S. MIAN, AND N.A.A. MIAN

Clonidine Hydrochloride
1.

Introduction Clonidine hydrochloride is an imidazoline derivative hypotensive agent ( 1 ) which is thought to act through the central nervous system to elicit a hypotensive response ( 2 ) . Although the locus of action in the central nervous system is unlcear, clonidine has been shown to be a potent a-adrenergic agonist in both central and peripheral systems ( 3 ) . The commercially available transdermal system of clonidine consists of an outer layer of pigmented polyester; a drug reservoir of clonidine, mineral oil, polyisobutylene, and colloidal silicon dioxide; a microporous polypropylene membrane that controls the rate of diffusion of the drug; and a final adhesive layer that provides an initial release of drug and contains those ingredients found in the reservoir. The adhesive layer is covered by a protective strip which is removed prior to application ( 1 ) .

2.

Description
2.1

Nomenclature
2.1.1

Chemical Names

hydrochloride (2,4); 2-(2,6-Dichloroanilino)-2-imidazoline hydrochloride ( 4 ) ; 2,6-Dichloro-N-(imidazolidine-2-ylidene)aniline hydrochloride (4); hydrochloride (5).


2.1.2

[2-(2,6-Dichlorophenylimino)imidazolidine

2-(2,6-Dichlorophenylamino)-2-imidazoline

Generic Names

Clonidine hydrochloride.

CLONIDINE HYDROCHLORIDE

I13

2.1.3 Trade Names Catapres, Catapresan, Clonistada; Dixarit, Drylon, Hyposyn, Ipotensium, Isoglaucon, Tenso-Timelets.

2.2

Formulae

2.2.1 Emoirical
CgHgClzN3

CsHsCIzN3 .HC1

(Clonidine). (Clonidine hydrochloride).

2.2.2 Structural

2.2.3 CAS (Chemical Abstract Service Registry Number 1


4205.90.7 (clonidine) (4). 4 2 0 5 . 9 1 . 8 (Clonidine hydrochloride) ( 4 ) .

2.3

Molecular Weipht

230.10 (Clonidine) ( 6 , 7 ) . 266.6 (Clonidine hydrochloride) ( 4 ) .


2.4

Elemental Composition

Clonidjne (7): C

46.98%; H 3.94%; 30.82%; N 18.26%. Clondine hydrochloride: C 40.51%; H 3.75% C1 3 9 . 9 8 % ; Nz 15.75%.

ci

I14

M.A. ABOUNASSIF, M.S. MIAN. AND N.A.A. MIAN

2.5

ADpearance, Color, Odour and Taste

A white o r almost white crystalline powder (8) which has a bitter taste ( 1 ) .
3.

Physical Properties
3.1.
Melting Range

Clonidine 130'C ( 7 ) . Clonidine hydrochloride 305'C ( 7 ) . Clonidine hydrochloride 300'C with decomposition (9).
3.2

Solubility

Soluble in 1 3 parts of water, soluble in absolute ethanol, slightly soluble in chloroform ( 8 ) . 1 g soluble in 6 m l H 2 O ( 6 0 ' C ) , about 13 m l H 2 0 ( 2 0 ' C ) , about 5 . 8 m l C:H3OH, about 2 5 ml C z H 5 0 H and about 5000 ml of CHCln (9). Practically insoluble in ether (6).

5% solution in
3.4

H2O

has a pH of 4 . 0 to 5 . 0 ( 8 ) .

Loss on Drying

When dried to constant weight at 1 O O ' C to 105-C, loses not more than 0 . 5 % of its weight use 1 g (8).
3.5

Sulphated Ash

Hot more than 1% (8).


3.6

Clarity and Color of Solution


A 5% w/v solution in carbon dioxide free water

is clear (8).

3.7

Stability Stable in light air and room temperature (9).

3.8

pk

The drug has a Pk of 8.2 ( 6 , 9 ) .

CLONIDLNE HYDROCHLORIDE

I I5

The acute toxicity for clonidine in related species is as follows: ( 7 ) Species


LD5o

(mg/kg)

Oral Mouse Rat Rabbit Dog Monkey


3.10

I.V.
18
29
45

328
270

80 30-100
150-267

Action

Clonidine hydrochloride is an antihypertensive agent, whose mechanism of action appears to be central a-adrenergic stimulation. This result in the inhibition of bulbar sympathetic cardioaccelarator and sympathetic vasoconstrictor centers, therapy causing a decrease in sympathetic outflow from the brain. Initially drug stimulates pheripheral a-adrenergic receptors producing transient vasoconstriction ( 5 ) .
3.11

Half-Life Plasma ( 6 ) Pl.asma half-life, 10 to 25 hours.

3.12

Volume of Distribution ( 6 )
2 to 4 litres/kg.

3.13

Protein Binding ( 6 ) About 20 to 40%.

3.14

Storage

The drug should be kept in a well-closed containers ( 8 ) and protect from sun light ( 4 ) .

116

M.A. ABOUNASSIF, M.S. MIAN, AND N.A.A. MIAN

3.15

X-ray Powder Diffraction

The X-ray diffraction pattern of clonidine hydrochloride was determined using philips full automated generator. Radiation was provided by a copper target (Cu annode 2000W, Y = 1.5480 A ) . High intensity x-ray tube operated at 40 kv and 35 Mv was used. The monochromator was a curved single crystal one (Pw 1752/00). Divergence slit and the receiving slit were 0 and 0 . 1 " , respectively. The scanning speed of the goniometer (Pw 1050/81) used was 0 . 0 2 - 20 per second. The instrument is combined with philips PM 8210 printing recorder with both analogue recorder and digital printer. The goniometer was aligned using silicon sample before use. The x-ray pattern of clonidine hydrochloride is presented in Fig. ( 1 ) . The values of scattering angle 2 0 interplanner distance dA and relative intensity 1/10 are shown in the table
(1). 3.16

Crystal Structure

Gudmund et a1 ( 1 0 ) have determined the crystal structure of clonidine hydrochloride by x-ray diffraction methods using 3209 observed reflections collected on a counter diffractometer. The crystals are monoclinic, space group ( 2 / c with unit cell dimensions a = 1 7 . 9 5 7 ( 2 ) A b = 1 1 . 9 5 0 ( 1 ) A , c = 13.664 ( 1 ) A and I3 = 128.64 ( 1 ) O ; (t = 18 k 1 . C ) ; V = 2290.2 A , M = 2 6 6 . 5 6 , Z 8 ; F(OOO) = 1088; Dcalc = 1 . 5 4 6 g cm-3; p = 7 . 6 7 cm-1, Selected interatomic distances and bond angles are listed in Table ( 2 ) . Perspective view of the molecule showing bond lengths is presented in Fig. ( 2 a ) . The stereoscopic view of the crystal structure of clonidine is shown in Fig, ( 2 b ) . Cody et al. (11) also determined the crystal structure of clonidine hydrochloride in order to determine the conformation of protonated clonidine and to el ucjdate the relationships between its structure and that required for binding to the a-adrenergic
re1,epl
UI",

CLONIDINE HYDROCHLORIDE

T a b l e ( 1 ) Characteristic lines of x-ray diffraction of

clonidine hydrochloride 29
9.281 9.779 12.463 13.032 14.625 16.801 17.676 19.723 22.232 22.684 23.093 24.634 25.308 25.872 26.334 27.032 28.182 29.088 29.828 30.660 30.889 33.515 33.813 34.788 35.335 36.073 36.754 37.214 38.105 38.104 39.260 39.861 40.252 40.851 41.889 42.175 42.471 43.419 43.747 44.235 44.586 45.623

d(A)
9.5283 9.0446 7.1020 6.7932 6.0569 5.2768 5.0176 4.5012 3.9986 3.9199 3.8514 3.6138 3.5191 3.4436 3.3842 3.2985 3.1664 3.0698 2.9953 2.9159 2.8948 2.6738 2,6509 2.5788 2.5401 2,4898 2.4452 2.4161 2.3616 2.3439 2.2947 2.2615 2.2394 2.2089 2.1566 2.1426 2.1284 2,0841 2.0692 2.0475 2,0322 1.9884

I/Io%

20.533 58,521 22.587 72.142 8.213 16,974 4.859 16.563 33,812 11.362 10.814 46.406 41.204 12.662 100 48.665 6.639 31.211 21.697 6.981 9.582 4.859 8.213 4.996 2.943 5,612 6.433 5,270 8.008 10.746 2.943 4.585 6.228 8.418 4.175 5.133 9.924 4.517 4.106 3.764 5.544 3.285

46.963 47.821 50.711 51.129 52.072 52.667 53.648 54.372 54.903 56.132 56.476 60.422 61.337 65.051 65.578 66.518 67.342 70.397 72.236 72.918

1.9347 1.9020 1.8002 1.7865 1.7563 1.7379 1.7084 1.6873 1.6722 1.6385 1.6293 1.5320 1.5114 1.4338 1.4235 1.4057 1.3905 1.3341 1.3078 1.2973

2.943 5.133 7.734 5.886 8,008 12.388 3.901 6.365 5.817 2.395 7.665 3.080 3.011 2.806 3.080 6,707 2.258 2.327 3.832 2.121

20 = scattering angle
dA = Interplanner distance.

I/Io

= relative intensity distance ( b a s e d on h i g h e s t i n t e n s i t y as 100.

(20 value)

Fig. (1)

The X-ray diffraction p a t t e r n o f Clonidine HC1.

CLONIDINE HYDROCHLORIDE

1 I9

Table (2). Distances ( A ) and angles ( " ) in the crystals of clonidine hydrochloride Bond Length Bond angles

Cl-C2 C2-C3 c3-c4 c4-c5 C5-C6 C6-C1 C1-N1 C2-Cl2 C6-Cl3 N1-C7 N2-C7 N3-C7 N2-C8 N3-C9 C8-C9

1.391 1.382 1.377 1.371 1.385 1.392 1,418 1.733 1.724 1 328 1.322 1.321 1.450 1.447 1.533

C6-Cl-C2 Cl-C2-C3 c2-c3-c4 C3-C4-C5 C4-C5-C6 C5-C6-C1 Cl-C2-C12 c3-c2-c12 Cl-CG-Cl3 C5-C6-C13 C2-C1-N1 C6-Cl-N 1 Cl-Nl-C'I Nl-C'?-NB Nl-C7-N2 C7-N2-C8 N2-C8-C9 C8-C9-N3 C9-N3-C7 N2-C7-N3

117.3 121.5 119.8 120.2 119.8 121.4 120.0 118.5 118.9 119.7 121.4 121.3 123.0 123.1 125.2 110.6 103.5 102.6 111.5 111.8

Torsional angles (positive for a clockwise rotation)

C2-Cl-Nl-C7 Cl-Nl-C7-N2 Cl-Nl-C7-N3 C6-Cl-bil-C7

76.5 0.0 178.1 105.2

Hydrogen bonds

Cll-Nl(i - X, - t t y, g C11-HN1 ( A 1 C11-HN1-N1 ( " ) Cll-NX(x,y,~)( A ) C11-HN2 ( A ) Cll-HN2-N2 ( " )

- Z) ( A )

3.094 2.25 161.2 3.193 2.38 163.4

I20

M.A. ABOUNASSIF. M.S. MIAN. AND N.A.A. MIAN

F i g (2b)

S t e r e o s c o p e v i e w o f t h e crystal s t r u c t u r e of C l o n i d i n e .

Fig (2a)

Perspective view o f Clonidine m o l e c u l e s h o w i n g b o n d lengths.

CLONIDINE HYDROCHLORIDE

121

grown by slow evaporation from aqueous solution. The crystals are of exceptional quality. A crystal of columnar shape, 0.2 x 0.2 x 0.6 mm, was screened o p t j cally and by X-ray Weissenberg photography for quality and assignment of space group. The refined cell constants, obtained by a least-squares fit of the I values of 73 high-angle reflections measured ( = 0.707 A ) automatically on a kappageometry diffractometer, are listed in table 3 along with other crystal data. Intensity data were measured in theQ-28 s c a n m o d e u s i n g Mo h'a r a d i a t i o n a n d a dispersion-corrected scan sidth of ( 0 . 8 t 0.2tane ) " to a. SinQ,/A maximum of 0.70 8 - l . Of the 3335 unique reflections measured, 2112 are greater than or equal to twice their estimated standard deviations. Table 3. Crystal data for clonidine HC1.

rophenylamino)-2-imidazoline HCl] , CgHioN3C13 , were

Crystals of clonidine hydrochloride [2,6-dichlo-

Molecular formula Molecular weight Crystal system Space group Cell dimensions
2

Cell volume Density (calc. ) (obs. ) Crystal size Final R index

CgHgN3Clz .CH1 266.56 Monoclinic C2/c 8 a = 17.962(3) A b = 11.976(2) W c = 13.672(2) A I3 = 128.62(1)' 2298.2 R 1.541 g ~ m - ~ 1.543 g cm-3 0.2 x 0.2 x 0.6 mm 0.05 (1223 data)

3.17

Spectral Properties
3.17.1 Ultraviolet SDectrun (UVY

The UV spectrum (12) of clonidine hydrochloride in H2O (8 mg%) was scanned from 200-600 nm (Fig. 3) using LKB 4054 UV/Vis spectrometer. Clonidine

0 0
( D

In

0 0

In

0
U

0 0
U

0 0

L n CJ

I 0 0

I 0
0

I 0
0
0

In

m
h l
(v
F

I 0 0 0
c

0 0

I n
0

I 0 0 0

0 0

cv
0

CLONIDINE HYDROCHLORIDE

123

hydrochloride exhibited the following U V (Table 4 ) . Table 4: UV data of clonidine hydrochloride \ax nm Absorbance Molar absorptivity (,E) cm-1 gm mol./L
8290,327

data

A(1%, 1 cm)

213 271 302

2.488 0.214 0.102

311 26.75 12.75

713.074
339.876

3.17.2

Infrared SDectrum

The I R spectrum ( 1 2 ) of clonidine hydrochloride as KBr disc was recorded on a Perkin Elmer 1310 Infrared spectrometer. Fig. (4) shows the infrared absorption spectrum of clonidine hydrochloride. The structural assignments of clonidine hydrochloride have been correlated with the following frequencies ) (Table 5 .
3.17.3

Nuclear Magnetic Resonance Spectra


4.17.3.1.

'H-NMR Spectrum

The 1 H - N M R spectrum ( 1 2 ) of clonidine hydrochloride in DMSO-ds (Fig. 5 - 6 ) was recorded on a Varian X L 200 M H z NMR spectrometer using TMS as an internal reference, The following structural assignments have been made (Table 6 ) .

0 0 to

0 .o
7

0
0

m
c

0 0 0
h l

8
m

1
3 3

JJ

aD

I 0
(0

A
U

I 0
h l

0 *

0 0

I24

Fig. (5)

PMR s p e c t r u m o f C l o n i d i n e HC1 i n DMS0.D6.

Fig. (6)

PMR spectrum o f Clonidine HCl in DMSO.D6

(DiO Each.)

CLONIDINE HYDROCHLORIDE

I21

Table 5:

IR characteristics of clonidine HC1.

Frequencies

Approximate description of vibrational modes

3320 3000-3080 1650, 1600, 1565 1440, 1400 1330, 1280 1190, 1100
790, 780

NH stretch
Chlorophenyl CH stretch Iaidazolidine ring stretch Phenyl ring stretch Chlorophenyl C-H planar bend Chlorophenyl C-C1 stretch bends.

Table 6: FMR characteristics of clonidine HC1 structure

Protons

6 (PPM)

Multiplicity singlet multiplet singlet

g, d (two protons)

8.618

a,b,c (three protons) 7.439-7.661

e,f

( four

protons)

3.672

I28

M.A. ABOUNASSIF. M.S. MIAN, ANDN.A.A. MIAN

3.17.3.2

13C-NMEI S p e c t m

13C-NMR spectrum (12) of clonidine hydrochloride Fig. (7-9) was recorded in DMSO-d6 by Varian XL-200 MHz NMR spectrometer. The multiplicity of the resonances was obtained from DEPT (Distortionless enhancement by polarization transfer) and APT (attached proton test), The carbon chemical shifts are presented in Table ( 7 ) .

Table 7: C-13 chemical shifts of clonidine HC1. Carbon assignment Chemical shift 6 (ppm) 42.647 129.121 133,987 130.772 130.262 157.919

cs,

c9

c1, c 3

CS, c 4

cz
c5

CI

3.17.4

Mass SDectrum

The mass spectrum ( 1 2 ) of clonidine hydrochloride obtained by electron impact ionization (Fig. 10) was recorded on a Finnigan MAT 90 mass spectrometer. The spectrum was scanned from 50 to 500 8.m.a. The electron energy W A S 70 ev. Emission current 1 mA and ion source pressure 10-6 t o r r . The spectrum shows a

Fig. (7)

13C.NMR spectrum of C l o n i d i n e HC1 in DMSO-D6.

Fig. (8)

13C,NMR spectrum of C l o n i d i n e H C 1 i n DMS0.D6

(APT)

Fig. (9)

13C.NMR

spectrum of Clonidine HC1 in DMS0.D6

(DEPT).

100.0

50.

100.01

50.

50

'

100

150

Fig.

(10)

M a s s s p e c t r u m o f C l o n i d i n e HCJ,

CLONIDINE HYDROCHLORIDE

I33

molecular ion M + at m/z 229 with a relative intensity 100%. The most prominent fragments and their relative intensities are listed in Table 8 .
4.

Synthesis Clonidine is synthesized (13) by the condensation of 2,6-dichloroaniline and imidazoline.

2,6-Dichloroani1ine
5.

Imidazoline

Clonidine

Pharmacokinetics 5.1 AbsorDtion and Dietribution Clonidine hydrochloride is readily absorbed by

to the eye, clonidine is well absorbed percutaneously following topical application of a transdermal system t o t h e arm or chest. Plasma clonidine concentrations of 2 ng/mL have been detected one hour after administration of a single 0.39 mg oral dose of radiolabeled drug. Peak plasma concentrations following oral administration occur in approximately 3-5 hours (1). Reduction in blood pressure is maximal at plasma clonidine concentrations less than 2 ng/mL. Blood pressure begins to decrease within 30-60 minutes after an oral dose of clonidine hydrochloride, the maximum decrease occurs in approximately 2-4 hours. The hypotensive effect lasts up t o 8 hours. Following administration of clonidine by slow intravenous injection in patients with acute hypertensive crises, a hypotensive effect occurred within minutes, peaked in 30-60 minutes and lasted more than 4 hours (1).

o r a l route with an absorption time of 2 to 4 hours (9). Drug is well absorbed from the gastro-intestinal tract. I t may also be absorbed when applied topically

I34

M.A. ABOUNASSIF, M.S. MIAN, AND N.A.A. MlAN

Table 8:

The mass fragments of clonidine HC1


Relative intensity % Fragment

m/z
230 229 221 207 200 196 194

65 100 10 12 20 22 52

193 186 174

17 11 45

172

54

165 147 124 109 73

20 18 18 20
17

CLONIDINE HYDROCHLORIDE

135

Animal studies indicate that clonidine is widely distributed into body tissues, tissue concentration of the drug are higher than the plasma concentration. After oral administration highest concentrations of the drug are found in the kidneys, liver, spleen, and GI tract. High concentrations of the drug also appear in the lacrimal and parotid glands. Clonidine is concentrated in the choroid of the eye and is also distributed into the heart, lungs, testes, adrenal glands, fat and muscle. The lowest conc. occurs in the brain. Clonidine is distributed in CSF. It is not known whether the drug crosses the placenta. Clonidine is distributed into milk (1). The plasma half life of clondine is 6-20 hours in patients with normal renal function. The half life in patients with impaired renal function has been reported t o range from 8-41 hours. The elimination half life of the drug may be dose dependent, increasing with increasing dose ( 1). The drug is metabolized in the liver. In humans, 4-metabolites have been detected but only one, the inactive p-hydroxylated derivative, has been identified ( 1 ) . In humans 65 % of administered dose of clonidine hydrochloride is excreted by the kidneys, 3 2 X as unchanged drug and the remainder as inactive metabolites. Approximatly 20 % of dose is excreted in feces, probably via entrohepatic circulation. Approximately 85 % of a single dose i excreted with s 72 hours and excretion is complete after 5 days (1).
5.2

Uses and Administration

Clonidine is an antihypertensive agent which appears to act centrally by stimulating az-adrenergic receptors and producing a reduction in sympathetic tone, resulting in a fall in diastolic and systolic blood pressure and a reduction in heart rate. It also acts peripherally, and this peripheral activity may be responsible for the transient increase in blood pressure seen during rapid intravenous administration as well as contributing to the hypotensive effect during chronic administration. Peripheral resistance i reduced during continuous treatment. Cardiovascular s

136

M.A. ABOUNASSIF, M.S. MIAN, AND N.A.A. MIAN

reflexes remain intact so postural hypotension occurs infrequently. When given by mouth its effects appear in about 30-60 minutes reaching a maximum after 2-4 hours as lasting up to 8 hours ( 4 ) . Clonidine hydrochloride is used in the treatment of grades of hypertension. The usual initial dose of clonidine hydrochloride is 50 to 100 pg orally thrice daily increased every second or third day according to the response of the patient. The usual maintenance dose is 0.3 to 1 . 2 mg daily but doses of up to 1.8 mg o r more daily may be required. To reduce side effects a similar dose of clonidine may be given in conjunction with a thiazide diuretic but combination w i t h a Ij-blocking agent should be avoided where possible clonidine may also be given in a sustainedrelease formulation which enables twice-daily dosage, or by a transdermal delivery system which is applied once a week and delivers 100-300 pg daily at a constant rate ( 4 ) . Drug may be given by slow intravenous injection in hypertensive crises usually in doses of 1 5 0 to 300 l.lg (419 It is also used in lower doses for the prophylaxis of migrane or recurrent vascular headaches and in the treatment of menopausal flushing ( 4 ) . Clonidine hydrochloride has been used topically to reduce intraocular pressure in the treatment of open angle (chronic simple) and secondry glaucoma and hemorrhagic glaucoma associated with hypertension (1). B e c a u s e of i t s GI e f f e c t s c l o n i d i n e hydrochloride has been used with some success in a limited number of patients for the management of diarrhea of various etiologies (e.g. narcotic bowel syndrom, idiopathic diarrhea associated with diabetes)
(1).

5.3

Adverse Effects

Serious toxic effects have been reported after ingestion of doses of 0.4 to 4 mg by children and 4 to 11 mg by adults. However, recovery is usually rapid
(6).

CLONIDLNE HYDROCHLORIDE

137

Drowsiness, dry mouth, dizziness and headache commonly occur during the initial stages of therapy with clonidine. Fluid retention is often transient but may be responsible for a reduction in the hypotensive effect during continued treatment. Constipation is also common and other adverse effects which have been reported include depression, anxiety, fatigue, nausea, anorexia, parotid pain, sleep disturbances, vivid dreams, impotence, urinary retention or incontinence, slight orthostatic hypotension, and dry itching or burning sensations in the eye. Rashes and pruritus may occur and are more common with the use of transdermal delibery systems. Less frequently, bradycardia, including sinus bradjcardia with atrioventricular block, hallucinations, and transient abnormalities in liver function tests have been reported large doses have been associat.ed with initial increases in blood pressure and persist during continued therapy ( 4 ) . Symptoms of overdosage include transient hypertension or profound hypotension, bradycardia, sedatjon, miosis, respiratory depression, and coma. Treatment consists of general supportive measures. An (1-adrenoceptor blocking agent may be given if necessary ( 4 ) , Clonidine withdrawal may result in an excess of circulating catecholamines. Therefore, caution should be exercised in concomitant use of drugs which effect the metabolism o r tissue uptake of these amines (monoamine oxidase inhibitors or tricyclic antidepressants, respectively) ( 1 ) .
5.4

Precautions

Clonidine should be used with caution in patients with cerebral, or coronary insufficiency, Raynauds disease or thromboangitis obliterans, or with a history of depression. The hypotensive effect may be antagonised by tricyclic antidepressants, and enhanced by thiazide diuretics. Clonidine cause drowsiness and patients should not drive or operate machinery where loss of attention could be dangerous. The effect of other cent.ra1 nervous system depressants mag be enhanced, withdrawal of clonidine therapy should be gradual as sudden discontinuation may cause rebound hypertension which may be severe. Agitation,

I38

M.A. ABOUNASSIF. M.S. MIAN, AND N.A.A. MIAN

sweating, tachycardia, headache, and nausea may also occur. 0-blockers can exacerbate the rebound hypertension and if clonidine is being given concurrently with a D-blocking agent, clonidine should not be discontinued until several days after the withdrawal of the B-blocker. It has been suggested that patients should be warned of the risk of missing a dose or stopping the drug without consulting their doctor and should carry a reverse supply of tablets (4). Although hypotension may o c c u r d u r i n g anaesthesia in clonidine-treated patients clonidine should not be given intravenously during the operation to avoid the risk of rebound hypertension. Intravenous injections o f clonidine should be given slowly to avoid a possible transient pressor effect especially in patients already receiving other antihypertensive agents such as guanethidine or reserpine ( 4 ) . Abrupt withdrawal of oral clonidine therapy may result in a rapid increase of systolic and diastolic blood pressure, with associated symptoms as nervousness, agitation, restlessness, anxiety, insomnia, headache, sweating, palpitation increased heart rate, tremor, hiccups, stomach pains, nausea, musc1.e pains, and increased salivation (1).
6.

Methods of Analysis
6.1

Identification

1) Dilute a volume containing 0.3 mg of clonidine hydrochloride to 5 m l with 0.01 M hydrochloric acid. The light absorption of the resulting solution in the range of 245 to 350 nm exhibits maxima at 272 nm and 279 nm and inflection at 265 nm ( 8 ) .
2) To a volume containing 0.15 mg of clonidine hydrochloride add 1 ml of a 10% w/v solution of ammonium reineckate and allow to stand for 5 minutes. A pink precipitate is produced (8).

3) The drug gives Libermanns color test yellow to orange at 100C ( 6 ) .


4)

Gives characteristics reaction of chlorides (8).

CLONlDlNE HYDROCHLORIDE

I39

5) The infrared absorption spectrum is concordant with the spectrum of clonidine hydrochloride (8).
6) Dissolve 0 . 2 g in 70 ml of ethanol ( 9 6 % ) and titrate with 0 . 1 M ethanolic sodium hydroxide vs determining the end point potentiometrically. Each ml of 0 . 1 M ethanolic sodium hydroxide vs is equivalent to 0.02666 g of C6HgClzN3.HCl ( 8 ) .
6.2

Colorimetric

Tawakkol et al, ( 1 4 ) developed a method for the colorimetric determination of colonidine in which clonidine reacts with sodium nitroprusside in presence of sodium hydroxide, and on treatment with saturated boric acid it gives a violet color which was measured a t 570 nm.
1 . 0 mg of powdered tablets were shaked with 10 ml of H20 and centrifuged, decant the clear solution into a 100 ml separating funnel. Repeat two times each Kith 1 5 m l of distilled H2O collecting in the same separating funnel, then add 1 ml of sodium carbonate and extract three times with chloroform, extract on a water bath and few drops of HC1 were added, evaporate and extract with few ml of distilled H2O. Then standard or test solution was treated with 0.8 ml of 1 N NaOH s o l u t i o n f o l l o w e d by 1 m l o f sod. nitroprusside, mixed and leave for 10 min, then 2 ml of 4% boric acid solution was added leave in an ice bath for 10-15 minutes. Complete up to the mark and measure the violet color of both the standard and the test at 570 nm.

Sane (15) developed a method for the estimation of clonidine hydrochloride in pharmaceutical preparations by ion pair extraction and colorimetric method. An acid-dye complexing method with bromophenol blue, broaocresol purple and methyl-orange was used for the ion-pa.ir extraction and colorimetric determination of clonidine hydrochloride in pharmaceuticals containing 100 mg of clonidine hydrochloride was 98.9% and relative standard deviation 0.89%.

I40

M.A. ABOUNASSIF, M.S. MIAN, AND N.A.A. MIAN

6.3

Pluorimetric

A very sensitive fluorimetric method ( 1 6 ) based on the reaction of clonidine hydrochloride with 1-dimethylaminonaphthalene-5-sulphonyl chloride (dansyl chloride) to give a highly fluorescent derivative.

Dissolve 50 mg of clonidine hydrochloride in a mixture of 10 ml of acetone and 40 ml of 0.5 M sod. carbonate solution. Transfer to a 50 ml flask make up to the mark with dansyl chloride and acetone was added and then 4-methyl pentan-2-one was added. Fluorescence intensity was measured after 10 minutes at 455 nm using an excitation wavelength of 345 nm.
6.4

Spectrophotometric Analysis
A

with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone to form a colored product with maximum absorption at 455 nm. Accurately weighed amount of clonidine hydrochloride equivalent to 50 mg of the base was dissolved in about 20 m l distilled HzO, made alkaline with few drops of 10% w/v NaOH solution and extract with five successive 10 ml portions of chloroform. Pass the chloroform extracts sequentially over anhydrous sod. sulphate and collect the combined chloroform extracts in 50 ml flask make up to volume with chloroform. Heat and dissolve in acetonitrile and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone and measured at 455 nm against. blank. An other simple and sensitive spectrophotometric method based 011 color reaction with bromocresol green WAS studied by Zivanov-Stakic et a l . ( 1 7 ) for the determination of colonidine hydrochloride in tablet form. Another method ( 1 8 ) reported the spectroscopic determination of clonidine hydrochloride.
6.5

( 1 6 ) based on the reaction of clonidine hydrochloride

simple and rapid spectrophotometric method

Radio-Immunoassag

Radio-immunoassay for clonidine in human plasma a.nd urine using a s o l i d phase second-antibody separation was studied by Farina et al. ( 1 9 ) . In which

CLONIDINE HYDROCHLORIDE

141

plasma was incubated for 1 8 hour at 4" with 0 . 1 M sodium phosphate buffer (pH 7.4). 1251-labeled 4-carboxyclonidine-tyrosine and antibodies raised in rabbit against 4-carboxyclonidine N-hydrosuccinimide ester conjugated with bovin serum albumin. After a second incubation with immuno beads of goat anti-rabbit immunoglobulin for 2 hours at room temperature, the mixture was centrifuged and radioactivity in the pallets was counted. The detection l i m i t was 10 pg per m l of clonidine, the within and between assay coefficient of variation were 2.8 to 9 and 10 to 13% respectively.

A newly developed and precise and sensitive radio-immunoassay f o r clonidine was done by Arndts et al. ( 2 0 ) . The antiserum is raised in rabbits injected with the pcarboxy derivative of clonidine and is used in the radioimmunoassay for clonidine in the residues of ethyl ether extracts (pH 9.5) of 0.2 ml of plasma [3H] clonidine being used as a tracer in a phosphate buffer medium (pH 7 . 4 ) . After incubation for 18 hours at 4'C unbound antigen is adsorbed on the dextran-coated charcoal, and the bound 3H is determined. A calibration graph is constructed for 0 . 1 to 10 ng per ml of clonidine in plasma.
Another method ( 2 1 ) of clonidine in rats as determined by radio-immunoassay. In which an antigen prepared by reacting 4-hydroxyclonidine with 4-carboxybenzene diazonium chloride and coupling the product t o bovin serum albumin was used to raise an antiserum in rabbits. [3Hl Clonidine or [ 1 4 C ! 1 clonidine was used as tracer and separation of the free and bound forms of the antigen was carried out. The sensitivity was 1 0 pg with f3H1 clonidine and 600 pg with [14C] clonidine. I t has been possible to measure plasma and tissue levels after the administration of rather high doses of radiolabelled clonidine to humans and animals
(22-25).
6.6

Chromatographic Methods
6.6.1 Gas-Liquid Chromatography (GLC)

1) Determination of submicrogram quantities of clonidine in biological fluids by Chu (26). Clonidine

I42

M.A. ABOUNASSIF, M.S. MIAN, AND N.A.A. MIAN

is extracted from plasma, extract is purified by solvent extraction and column chromatography and clonidine is converted to heptaf luorobutyryl derivative for g.1.c at 175' on a partially inactivated column cntaining 3% OV-17 on Chromosorb W AW DMCS with electron capture detection. The 4-methyl analogue of clonidine is used as internal standard. The limit of determination was 2 5 pg m l - 1 , and the coefficient of variation at the level of 60 pg ml-1 was approximately 8%.

2) The method (27) describes the measurement of clonidine in human plasma and urine by combined gas chromatography-mass spectrometry with ammonia chemical ionization. Addition of [2H4] clonidine to plasma or urine is followed by ethylacetate extraction of clonidine from alkaline medium, back extraction into acid extraction into ethyl ether from alkaline medium and evaporation of the extract to dryness. Trirnethylanilinium hydroxide is added to the residue, and dimethyl derivatives of clonidine are formed by on column methylation with an injection-port temperature of 250' for g.c. -70-eV m , s , , the glass column (1.8 m x 2 mm) packed with 3% of OV-17 on Gas-Chrom Q (100 to 120 mesh) is operated at 245". With He as carrier gas (15 m l min-l); NH3 is admitted to an ion-source pressure of 0.2 Torr, and ions are monitored at m/e 258 and 264. Graphs of peak height ratios (n/e 258 to 2 6 4 ) vs amounts of clonidine in urine (up to 4 0 ng ml-l) and in plasma (up to 5 ng ml-1) are rectilinear. The precision for assay of clonidine in plasma is 11% at 0.25 ng ml-1 and 5% at 0 . 5 ng ml-l and the lower limit of determination is 0.1 ng ml-1.
3) A simple and sensitive gas-liquid chromatographic method ( 2 8 ) has been developed for the quantitative determination of clonidine and some structurally related imidazolidines in rat brain tissues. The aqueous brain homogenates are first purified and t,hen extracted into benzene. Samples are injected directly to GLC column ( 2 m x 2 mm I.D.) pa.cked with 3% OV-17 on chromosorb 750 (80-100 mesh) was used at an oven temperature of 200-270' and an injector temperature of 280" the carrier gas was helium; flow rate 30 ml/min,

CLONIDINE HYDROCHLORIDE

143

A gas chromatography assay for clonidine in human plasma has been developed by per Olof Edlund (29). The buffered serum is extracted on silica columns, alkylated with pentrafluorobenzyl bromide, clertned up by extractions and analysed by glass-needle injection and electron-capture detection. The packed column (2 m x 3 m m I.D.) was silanized glass and was packed with 3% of OV-17 on 80-100 mesh. Gas-Chrom Q.

4)

Another method (30) for the determination of 2-(2,4-dichloroaniclonidine in plasma by G . L . C . line)-2-imidazoline is used as internal standard. The column (WCOT: 30 m x 0 . 3 5 mm) was operated at 250'C with H2 as carrier gas. Other methods used for the GLC determination of clonidine hydrochloride in biological materials (31,321. Recently GC was applied to the measurement of picogram levels of clonidine hydrochloride after derivatization ( 3 3 ) also by ( 3 4 - 3 6 ) . Advantages o f fused silica capillary gas chromatography (FSCC) for conventional GC method (37).
6.6.2.

5)

High-Performance Liauid ChmmatograDhp ( HPLC )

1) A rapid, reversed-phase high-performance liquid chromatography (HPLC) method (38) is described for the determination of clonidine in tablets. Individual tablets or composite samples were sonicated in water, diluted with methanol and filtered. Clonidine formulated at 0.1 or 0.2 mg/tablet was chromatographed on trimethylsilyl-bonded, 5 to 6-ym spherical silica with 65% methanol in pH 7.9 phosphate buffer as mobile phase detection at 254 nm. Mean recovery from 6 synthetic tablet samples was 99.7% (at 0 1 mg/tablet . level) with relative standard deviation of 1.55%.

2) A sensitive, selective and reproducible assay for clonidine hydrochloride in tablets and eye drops were described (39). A Nucleosil 5 Cis colum (125 mm x 4 . 6 mm - I.D.) with methanol-water 80:20 containing 0.005% of TEA as the mobile phase at a flow rate of 1 ml per minute at 240 nm U V detection and attenuation, 0.02 a . u . f . s . in tablets and 0.16 a.u.f.s. in eye drops and recorder chart speed, 0.5 cn min-l. r

144

M.A. ABOUNASSIF, M.S. MIAN. A N D N.A.A. MIAN

7.

Acknowledgements The authors are highly thankful to Mr. Babkir Awad Mustafa, College of Applied Medical Sciences f o r his efforts in drawing the spectras and figures, The authors also would like to thank Mr. Tanvir A. Butt, College of Pharmacy, King Saud University, for his valuable and professional help in typing the manuscript.

8.
1.

Beferences "Drug Information 90" American Society of Hospital Pharmacists Inc. 4630 Montgomery Avenue, Bethesda M.D. 20814, p. 910-915. Vivian Cody, presented in part at the American Crystallographic Association Meeting, Clemenson, South Carolina, January 1976, Goodman, L . S. and Gi lman, A . "The Pharmacological Basis of the Therapeutics", 4th ed., 1970, p. 7 3 5 . "Martindale", The Extra Pharmacopoeia, 29th Ed. Editor James E.F. Reynolds, The Pharmaceutical Press, London, p. 472 (1989). "Physician's Desk Reference", 42nd E d . , Economics Company, USA, p. 718 (1988). Medical

2.

3. 4.

5.

6.

"Clarke's Isolation and Identification of Drugs" 2nd ed., A.C. Moffat Edit., p. 481-482. 'The Pharmaceutical Press' London (1986). "The Merck Index", 11th Ed., Merck and C o . , Inc., Rahway, N.J., p 374 (1989). .
"The British Pharmacopoeia", Her Majestys Stationary Office, London, p. 147 (1988).

7.

a.
9.

"Remlngton's Pharmceutical Sciences", 16th ed., Mack Publishing Company Easton, Pennsylvania, 18042, p. 785 (1985). Gudmud Byre, Arvid Mostad and Christian Romming Acts Chemica Scandinavica, p. 843-846 (1976).

10

m,

CLONIDINE HYDROCHLORIDE

I45

11. 12.
13.

Vivan Cody and Corae J., Detitta, J. of Crystal and Molecular Structure, 9 ( 1 ) , p . 33-43 ( 1 9 7 9 ) . Mohammad Saleem Mian and Neelofur Abdul Aziz Mian, unpublished data ( 1 9 9 2 ) . "The Organic Chemistry of Drug Synthesis" Vol. 1, p. 2 4 1 , by Daniel Lednicer, Lester A. Mitscher, John Wiley dr Sons, New York. M.S. Tawakkol, A.I. Jado and H.Y. Arzniem.-Forsch 31, 1064-66 ( 1 9 8 1 ) .
(1981).

14.

Aboul-Enein,

15.
16. 17.

Sane, R.T. , Thombare, C.H. Indian D r u m , 1 8 ( 9 ) , 335-7 Fawzy, A,, El-Yazbi, Mona Badair, and Mohamed A. Analyst, Vol. III ( 1 9 8 6 ) . Zivanov-Stakic, D., Panic, L . J . and Agbaba, G . Farmaco
45, 381-383 ( 1 9 9 0 ) . 246 ( 1 9 8 4 ) .

18. 19.

Kovar, K.A. and Abdel-Hamed, M., Arch. Pharm., 317, Farina, P.R., Homon, C . A . , Chow, C.T., Keirns, J . J . , Zavorskas, P.A., Esber, H.J. Ther. Drug. Mointo.,
7 ( 3 ) , 344-350 ( 1 9 8 5 ) .

20.
21.

Arndts, D., Staehle, H. and Struck, Arznei,-Forsch., 2 9 ( 1 ) , 532-538 ( 1 9 7 9 ) .

C.J.,

Jarrot, Revyn, and Spectro, Sydney. J. Pharmacol. EXP. Ther. 2 0 7 ( 1 ) , 195-202 ( 1 9 7 9 ) . D. Rehbinder and W. Deckers. Arzneim.Forsch. (Drug Rea), l9, 169 ( 1 9 6 9 ) . D. Rehbinder, in A. Zahchett and M. Envico (Editors) Ipertension Arteriosa, Boehringer Ingelheim, p . 3
( 1973).

22.
23.

24.

J . P . Fillastre, D , Dubois and P. Brunelle, in A. Zanchett, and Enrico (Editors) IDertension Arteriosa Boehringer Ingelheim, p . 8 1 ( 1 9 7 3 ) .

I46

M.A. ABOUNASSIF, M.S. MIAN, AND N.A.A. MIAN

25.

S. Darda, in P. Millies and M. Safar (Editors). Recent Advances in Hypertension, Boehringer, Ingelheim, p .

375 ( 1 9 7 5 ) .

26. 27. 28. 29. 30. 31. 32,

Chu, L.C. , Bayne, W.F. , Tao, F.T., Schmitt, L.G. and Shaw, J.E. J. Pharm. Sci. 6 8 ( 1 ) , 72-74 ( 1 9 7 9 ) . Murray, S., Waddeil, K . A . and Davies, D.S. Biomed. Mass SDectrom, 8 ( 1 0 ) , 500-502 ( 1 9 8 1 ) . P.B.M.W.M. Timmermans, A. Brands and P.A. Van Zwieten. J. Chromatogr., 144, 215-222 ( 1 9 7 7 ) . Per Olof Edlund. J. Chromatogr., 187, 161-169 ( 1 9 8 0 ) . Hiltunen, R . , Maryola, M., Hirsjarvi, P. and Raisanen, S. Acta Pharm. Fenn., 8 8 ( 4 ) , 161-167 1 9 7 9 ) .
A.K.

(1969).

Cho and S.H. Curry, Biochem. Pharmacol., l8, 5 1 1

C.R.
S.

C.T. Dollery, D.S. Davies, G.H. Draffan, H.J. Dargie, Dean, J.L. Reid, R . A . Clare and '3. Murray, Clin. Pharmacol. Ther., l8, 11-17 ( 1 9 7 6 ) .
435 ( 1 9 8 4 ) .

33. 34. 35. 36.

Murray and D.S. Davies. Biomed. Mass Spectrom, 11,

P.O. Edlund and L . K . Paalzow, Acta Pharmacol. Toxicol., 40, 145-152 ( 1 9 7 7 ) . A . K . Cho and S.H. Curry. Biochem. Pharmacol. 18, 511 (1966).
p.

Hypertension, Boehringer, Ingelheim, p . 369 ( 1 9 7 5 ) .


37.
J . Settlage and 192-197 ( 1 9 8 4 ) .

A. Frydman, Y. Weiss, M. Safar and J.M. Alexandre, in P. Millies and M. Safar (Editors), Recent Advances in

H. Jaeger. J. Chromatogr. Sci. 22,

CLONlDINE HYDROCHLORIDE

I47

38.

&,

Walters, Stephen, M., Stonys, Dalia, B. J. Chromatom. 2 l ( l ) , 43-5 ( 1 9 8 3 ) .

39.

I. Wilezynska-Wojtulewicz and N. Sadlej-Sosnowska. Chromatogr., 367, 434-437 (1986).

JI

CYCLANDELATE

Charles M. Shearer

Wyeth-Ayerst Research Rouses Point, NY 12979

ANALYTICAL PROFILES OF DRUG SUBSTANCES AND EXCIPIENTS - VOLUME 21

149

Copyright Q 1992 by Academic Press, Inc. All rights of reproduction reserved in any form.

I50

CHARLES M.SHEARER

1. 2. 3.

4. 5. 6.

7. 8.

Description I . 1 Name Formul a Mol ecul ar Weight 1.2 Appearance, Color and Odor Synthesis Physical Properties 3.1 Nuclear Magnetic Resonance Spectra 3.2 Infrared Spectrum 3.3 Ultraviolet Spectrum 3.4 Mass Spectrum 3.5 Melting Point 3.6 Di fferential Scanning Calorimetry 3.7 Solubility 3.8 Crystal Properties Stability and Degradation Metabolism Analysis 6.1 Elemental Analysis 6.2 U1 traviolet Spectrophotometry 6.3 Titrimetry 6.4 Gas Chromatography 6.5 High-Performance Liquid Chromatography 6.6 Thin Layer Chromatography Identity References

CY CLAN DELATE

151

Description 1.1 Name. Formula. M o l e c u l a r Weiqht The name used by Chemical A b s t r a c t s f o r c y c l a n d e l a t e i s a-hydroxybenzeneacetic a c i d , 3,3,5trimethylcyclohexyl ester. It i s a l s o c a l l e d mandelic acid, 3 , 3 , 5 - t r i m e t h y l c y c l ohexyl e s t e r ; 3 , 3 , 5 - t r i m e t h y l c y c l ohexyl mandel ate; 3,3,5-trimethyl c y c l ohexyl amygdal a t e ; and 3,3,5t r i m e t h y l c y c l ohexanol a-phenyl -a-hydroxyacetate. Trade names in c l ude, Cycl ospasmol , Nat i1, Novodi 1 , P e r e b r a l , and Spasmocyclon (1). The Chemical A b s t r a c t s number i s 456-59-7.
1.

Appearance, C o l o r and Odor C y c l a n d e l a t e i s a w h i t e t o o f f - w h i t e amorphous powder w i t h a s l i g h t m e n t h o l - l i k e odor.

1.2

'17"24'3

M. W. 276.36

Synthesis T r i m e t h y l c y c l o h e x y l mandelate was f i r s t s y n t h e s i z e d by r e a c t i n g a - m a n d e l ic - a c i d w i t h 3 , 3 , 5 - t r i m e t h y l c y c l ohexanol ( c o n s i s t i n g o f c i s and t r a n s isomers) (2,3,4). C y c l a n d e l a t e i s now s y n t h e s i z e d u s i n g o n l y t h e l o w m e l t i n g ( c i s ) isomer o f 3,3,5-trimethylcyclohexanol (5,6). E s t e r s o f m a n d e l i c a c i d w i t h t h e h i g h e r m e l t i n g 3,3,5-trimethylcyclohexanol a r e t w i c e as t o x i c as t h o s e made w i t h t h e l o w m e l t i n g isomer ( 7 ) . The m a j o r s i d e r e a c t ion p r o d u c t , tri met h y l c y c l ohexyl phenyl g l y o x a l a t e , can be removed d u r i n g t h e s y n t h e s i s by t r e a t i n g t h e c r u d e c y c l a n d e l a t e w i t h aqueous sodium b o r o h y d r i d e (8) o r z i n c and h y d r o c h l o r i c a c i d ( 9 ) . T h i s s y n t h e s i s , u s i n g o n l y t h e c i s isomer, r e s u l t s i n f o u r isomers as d e s c r i b e d i n t h e n e x t s e c t i o n .

2.

152

CHARLES M.SHEARER

Figure 1 - Proton NMR Spectrum of Cyclandelate (Wyeth-Ayerst Reference Standard No. 1361) in deuterated chloroform

CYCLANDELATE

153

Figure 2 - Carbon -13 NMR Spectrum o f Cyclandelate (Wyeth-Ayerst Reference Standard No. 1361) in deuterated chloroform

I4 5

CHARLES M.SHEARER

Phvsical ProDerties 3.1 Nuclear Maanetic Resonance SDectra The f o u r isomers which make up c y c l a n d e l a t e a r i s e i n t h e synthesis from t h e r e a c t i o n o f a - m a n d e l i c a c i d w i t h cis-3,3,5-trimethylcyclohexanol and a r e described i n Table I (taken from Nakamichi (10)). Table 1 Isomers o f Cvcl andel a t e Isomer Absolute c o n f i g u r a t i o n a o f mandelic a c i d moiety Absol Ute c o n f i g u r a t i o n o f c y c l ohexanol moiety Position 1 Position 5
R

3.

A B

R R

S
R

a) The cyclohexanol m o i e t i e s o f A,C and B,D are l e v o r o t a t o r y and d e x t r o r o t a t o r y , r e s p e c t i v e l y (11). The absolute c o n f i g u r a t i o n o f ( - ) - c i s - 3 , 3 , 5 - t r i m e t h y l c y c l ohexanol i s assigned as R on t h e basis o f i t s chemical c o r r e l a t i o n w i t h p u l egone (12). The p r o t o n N R sample (Wyeth-Ayerst Reference Standard No. M 1361) was d i s s o l v e d i n deuterated c h l o r o f o r m c o n t a i n i n g tetramethyl s i l a n e as an i n t e r n a l standard. The spectrum was obtained (13) on a 400 MHz Bruker spectrometer and i s presented as Figure 1. The s p e c t r a l assignments are l i s t e d i n Table 11. The C-13 N R sample was a l s o prepared i n M deuterated chloroform and i t s spectrum obtained (13) on a 100 MHz Varian spectrometer. The spectrum i s presented as F i g u r e 2 and t h e s p e c t r a l assignments are l i s t e d i n Table 111. The spectra are i n agreement w i t h those o f Nakamachi (10). I n f r a r e d Soectrum The i n f r a r e d spectrum o f a K B r p e l l e t o f c y c l andel a t e (Wyeth-Ayerst Reference Standard No. 1361) was obtained (14) on a N i c o l e t 20 DX instrument and i s presented as Figure 3. The s p e c t r a l band assignments are g i v e n i n Table I V . 3.2

CYCLANDELATE

4000

3000

2000
Wavenumber

1500
(crn-1)

1000

500

Figure 3 - Infrared Spectrum o f Cyclandelate (Wyeth-Ayerst Reference Standard No. 1361) KBr pellet

156

CHARLES M. SHEARER

Table I 1 P r o t o n NMR S p e c t r a l Assisnments o f Cvcl a n d e l a t e Chemical S h i f t (ppm f r o m TMS) 5.10 d 4.95 m 3.47 exchangeabl e Number o f Protons 5 Assignment

7.4

2.1

0.6 0.94 s 0.88 s 0.84 d ( J 0.91 d (J

1 1 1 17

H-0

H-C-OH H-C-OC

Aromatic CH

= =

6)
6)

A l i p h a t i c CH, CH CH3 gem C I - J ~ (AB p a i g j gem CH3 (CD p a i r ) HC-CI-J3 (AB p a i r ) HC-Cti3 (CD p a i r )

Table I11 Carbon-13 NMR S p e c t r a l Assisnments f o r Cvcl andel a t e Carbon

PPm

1 2 3 4 6 7 8 9 1 2
5

2, 6 3, 5 4

73.3 43.7 (AB) 32.2 (AB) 47.3 27.0 (AB) 39.7 (AB) 32.9 (AB) 25.4 (AB) 22.0 (AB) 173.1 72.8 138.6 126.3 128.4 128.1

43.2 (CD) 32.1 (CD) 26.9 40.1 32.8 25.3 22.1 (CD) (CD)

(CD) (CD) (CD)

Table I V I n f r a r e d S p e c t r a l Assisnments f o r Cvcl andel a t e Wavenumber ( C m - l ) V i b r a t i o n Mode


OH s t r e t c h CH s t r e t c h CEO s t r e t c h C-0-C s t r e t c h o u t - o f - p l a n e bending of monosubstituted aromatic

3460 3100 - 2800 1730 1212, 1192 730, 695

CYCLANDELATE

157

U l t r a v i o l e t SDectrum The u l t r a v i o l e t spectrum o f c y c l a n d e l a t e (WyethA y e r s t Reference Standard No. 1361 r e c r y s t a l l i z e d t o remove 0.1% 3 , 3 , 5 - t r i m e t h y l c y c l ohexyl phenyl g l y o x a l a t e ) i n USP e t h a n o l i s presented as F i g u r e 4. The a b s o r p t i v i t i e s a r e as f o l l ows :

3.3

X max(nm) 269 258 251


3.4

a 0.57 0.73 0.59

1575 2020 1630

Mass SDectrum The mass spectrum o f c y c l a n d e l a t e was o b t a i n e d (15) by e l e c t r o n impact i o n i z a t i o n u s i n g a Finnegan MAT 8230 spectrometer and i s g i v e n as F i g u r e 5. I d e n t i f i c a t i o n o f t h e p e r t i n e n t masses i s presented i n Table V .

Table V Mass Spectrum Fraqmentation P a t t e r n o f Cvcl andel a t e m/e 276 125 107 83 Species
Mt

'9"17'

C6H5CHOHt
CH2CHCH2C ( CH3) *t '6"5' CH2CHCH2CHCH3t
( CH3) C C H 2 t

79 69
55

M e l t i n g Ranqe Observed (16) m e l t i n g range (USP I a ) f o r c y c l a n d e l a t e (Wyeth-Ayerst Reference Standard No. 1361) i s 55.0" - 56.5"C.

3.5

I58

CHARLES M. SHEARER

Figure 4 - Ultraviolet Spectrum o f Cyclandelate (Wyeth -Ayerst Reference Standard No. 1361) i n USP alcohol

CYCLANDELATE

20
111

50

100

150

200

250

mie

Figure 5

Mass Spectrum o f Cyclandelate (Wyeth-Ayerst Reference Standard No. 1361)

160

CHARLES M. SHEARER

The DSC thermogram (14) for cycl andel ate (WyethAyerst Reference Standard No. 1361) is presented as Figure 6. The thermogram was obtained at a heating rate of lO'C/minute in a nitrogen atmosphere utilizing a Perkin-Elmer DSC-2. The thermogram exhibits no endotherms or exotherms other than that associated with the melt.
3.7 Solubility The following s lubi ties at room temperature have been observed (16).

3.6

Di fferenti a1 Scanninq Calorimetry

USP Classificat ons : Sol vent Water Methanol Acetonitrile Ethyl acetate Di met hyl formami de To1 uene Chloroform

Solubil itv i nsoubl e very soluble freely soluble freely soluble freely soluble freely soluble very soluble

3.8 Crystal ProDerties The X-ray powder diffraction pattern of cycl andel ate (Wyeth-Ayerst Reference Standard No. 1361) obtained (14) with a Phillips diffractometer using copper Ka radiation is presented as Figure 7. The calculated "d" spacings are given in Table VI.

Table V I X-Ray Diffraction Pattern

m;

mo
100 4 5 40 34 15 21 13 9 21

d
4.72 4.56 4.42 3.99 3.90 3.85 3.77 3.71 3.57 2.65 69 11 14 32 15 13 17 15

19.04 11.72 9.55 7.80 7.34 6.77 6.11 5.59 5.27 4.97

8 8

CYCLANDELATE

161

20

40

60

80

100

120

Temperature
Figure 6

(C)

- Differential Scanning Calorimetric

Thermogram of Cycl andelate (Wyeth-Ayerst Reference Standard No. 1361)

I62

CHARLES M. SHEARER

13

DEGREES 2 THETA

22

31

40

Figure 7

X-Ray D i f f r a c t i o n Pattern o f Cyclandelate (Wyeth-Ayerst Reference Standard No. 1361)

CYCLANDELATE

163

4. Stability and Desradation Cycl andel ate can decompose by hydrolysis to mandel ic acid and 3,3,5-trimethylcyclohexanol (17). It is oxidized to 3,3,5-trimethyl cyclohexyl phenylglyoxal ate (18). A study of the formation of 3,3,5-trimethylcyclohexanol in cyclandelate capsules concluded that less than 5% of the cycl andel ate degraded in 66 months at ambient temperatures (17) *
5. Metabol i sm The metabolites of cyclandelate are mandelic acid, phenylglyoxyl ic acid and 3,3,5-trimethylcyclohexanol. These are detectable in the urine of rabbits and humans in less than two hours after oral administration (19,20). The ratio o f mandelic acid to phenylglyoxylic acid increases with increased dosage (21). Another metabolic study in humans showed that the maximum blood levels of mandelic acid were reached in 0.5 to 1.5 hours after oral dosing (22).
A pharmacokinetic study using tritiated cyclandelate shows that most organ specimens took up the radioactivity rapidly; usually reaching a maximum within one hour. The brain, diaphragm, stomach and vein specimem showed a maximum level at 24 hours. The levels gradually declined in a nonlinear manner over 28 days (23).

6. Analysis
6.1

Elemental Analysis Element Theory 73.88% 8.75% Found (24) 73.95% 8.55%

C
6.2

Ultraviolet SDeCtrODhOtOmetrY Di rect determination of cycl andel ate by UV spectrophotometry is not practical since the oxidative degradation product, 3,3,5-trimethyl cycl ohexyl phenylglyoxalate has about 55 times the absorptivity (25). Spectrophotometri c determinations of cycl andel ate after hydrolysis to mandel ic acid and oxidation to benzaldehyde have been reported (26,27).

164

CHARLES M.SHEARER

Titrimetrv Cycl andel ate can be determined by hydrolyzing the ester in 0.5 N NaOH under reflux for 0.5 hours, then backtitrating the excess base with 0.1 N HC1 (28,29).

6.3

6.4 Gas ChromatoqraDhv Gas chromatography has been cycl andel ate and to separate it from and impurities as well as from other VI gives column conditions and other various methods.

used to analyze its degradation products pharmaceuticals. Table necessary data for the

Table V I Gas ChromatoqraDhv of Cvcl andel ate Column 2 rn x 4 mm i.d.; 5% Q F - 1 on Chromosorb W(HP) 100/200 mesh
6 ft x 1/8 in; 3% QF 1 t 0.5% HiEFF 8BP on GasChrom Q

Temoera t ure
160"

Oven

Reference (30) (31)


(32)

200

25 m x 0.3 mm i.d.; deactivated, coated w/S E - 3 0


30 m x 0.28 mm i.d.; FFAP

125" for 13 min; 3'/min to 180", hold 1 min. 170"

(10)

6 f t x 1/4 in i.d.; 15% Dexsil 220' 300 on HP Chromosorb W 80/100 mesh

(33)

1 m x 3.2 mm; Tenax GC 60/80 mesh

140" for 5 min., 20'/min to 240", lO'/min to 280"


200
O

(34)

6 ft x 4 mm i.d.; 2.5% SE30 on 80/100 mesh Chromosorb G

(35)

CYCLANDELATE

I65

Hiqh-Performance L i a u i d ChromatoqraDhv An HPLC system c o n s i s t i n g o f a Microbondapak CN (30 x 0.39 cm.) column, 65% methanol, 35% sodium a c e t a t e b u f f e r , a d j u s t e d t o pH 3.7 as t h e e l u a n t : and 254 nm UV l i g h t f o r d e t e c t i o n has been used (36). 6.6 T h i n Laver ChromatosraDhy The f o l l owing TLC systems have been r e p o r t e d :
P1 a t e s

6.5

S o l v e n t Svstem Benzene

R f Value

Reference (37) (38) (39) (39)

S i l i c a Gel 254 S i l i c a Gel 254 Silica G Silica G


7.

Hexane 55 0.09 C h l o r o f o r m 45 Chloroform 4 Acetone 1 Ethyl Acetate 0.74

0.71

Identity C y c l a n d e l a t e can be i d e n t i f i e d amongst many o t h e r drugs, p o i s o n s and b i o g e n i c compounds by gas chromatography ( 3 3 ) . Several D e t a i l s f o r t h i s procedure a r e g i v e n i n S e c t i o n 6.4. odor and c o l o r i d e n t i f i c a t i o n t e s t s a r e g i v e n by Doorenboos and coworkers (28). 8. References

1.
2.
3.

The Merck Index, 1 1 t h ed., Merck and Co., Rahway NJ, (1989) page 421. N. V . K o n i n k l i j k e Pharmaceutische Fabrieken voor. Brocades-Stheeman & Pharmacia, Dutch P a t e n t 68,704.

K. J. H. van S l u i s , Chemical Products, 11, 374(1954).


Brocades-Stheeman & Pharmacia, B r i t i s h P a t e n t 707,227.

4. N. V . K o n i n k l i j k e Pharmaceutische F a b r i e k e n voorheen
A. B. H. Funcke, M. J . E. E r n s t i n g , R. F. Rekker, and W . Th. Natua, A r s n e i m i t t e l - F o r s c h . , 3, 503(1953).

5.

166

CHARLES M. SHEARER

6. N. V . K o n i n k l ij k e Pharmaceutische F a b r i e k e n voorheen Brocades-Stheeman & Pharmaci a, B r i t i s h P a t e n t 810,888.

7. N. V . K o n i n k l i j k e Pharmaceutische Fabrieken voorheen Brocades-Stheeman & Pharmacia, Dutch P a t e n t 88,249.


8. D. F l i t t e r , U n i t e d S t a t e s Patent 3,663,597. 9. H. Takahashi, U n i t e d S t a t e s P a t e n t 3,673,239. 10.

T. Amano, T. Kasahara and B u l l . , 3, 1106(1981),

H. Nakamachi, Chem. Pharm.

11. M. J . E. E r n e s t i n g and W. Th. Nauta, Rec. Trav. Chim. Pay-Bas., 8 l , 751 (1962). 12. N. L. A l l i n g e r and C . K. Riew, J . Org. Chern., - 1316 (1975). 40, 13. B. Hofmann, Wyeth-Ayerst L a b o r a t o r i e s , Personal Communication. 14. C. L o n g f e l l o w , Wyeth-Ayerst L a b o r a t o r i e s , Personal Communication, 15. J . Cantone, Wyeth-Ayerst L a b o r a t o r i e s , Personal Communication. 16.

D. Berg, Wyeth-Ayerst L a b o r a t o r i e s , Personal


Commun ic a t ion,

17. 3. R i c h a r d and G. Andermann, Pharm. A c t a Helv., - 116(1982). 57,


18.

M. J . E. E r n s t i n g , R. F . Rekker, J . H. Bos and


W . Th. Nauta, Pharm. Weekblad,

a,605(1953).

19. M. J . E. E r n s t i n g , R. F . Rekker, A. 6. H. Funcke, H. M. Tersteege, and W . Th. Nauta, A r s n e i m i t t e l Forsch, 6 , 245(1956).

20. M. J . E. E r n s t i n g , R. F. Rekker, H. M. Tersteege, and W. Th. Nauta, A r s n e i m i t t e l - F o r s c h , l2, 632(1962).

CYCLANDELATE

I67

21. M . J . E. E r n s t i n g , R. F . Rekker, H. M . Tersteege and W . Th. Nauta, A r s n e i m i t t e l - F o r s c h , l2, 853(1962). 22. K. Kojima, Y, Uezono, T. Takahashi and Y. Nakanishi, J . Chromatogr., 425, 203(1988). 23. A. O r r and J. R. W h i t t i e r , I n t . J . Nucl. Med. B i o l . , - 205( 1974). 4, 24. C. Kraml, Wyeth-Ayerst L a b o r a t o r i e s , Personal Communication. 25. R.F. Rekker, H. J . Doorenbos and W. Th. Nauta, Pharm. Weekbl ad, l 2 946( 1967). O, 26. L. Chafetz, J . Pharm. S c i . , 53, 1192(1964). 27. 6. Andermann, M. D i e t z , and D. Mergel, J. Pharm. B e l g . , 3 4 , 233( 1979). 28. H. J . Doorenbos, H. J . van d e r Pol, R. F . Rekker and W. Th. Nauta, Pharm. Weekblad, 100, 633(1965). 29. J . Zhou, and C . Zhou, Yiyao Gongye, l7, 369(1986) f r o m CA( 26) :232528t. 30. R.T. Sane, V.B. Malkar, and V . G . Nayak, I n d i a n Drugs, - 321(1985) f r o m CA103(16):129151z. 22, 31. D. Rodgers, Wyeth-Ayerst L a b o r a t o r i e s , Personal Communication. 32. G. Andermann and M. D i e t z , J 365 (1981) . 33. B. Kaempe, Arch. Pharm. Chem 145(1974). Chromatogr.,

223,

S c i . Ed.,

2,

34. M. D i e t z and G. Andermann, J . High. Resol. Chromatogr. & Chromatogr. Comm., 2, 635(1979). 35. B. S . F i n k l e , E. J . Cherry and D. M. T a y l o r , J . Chromatogr. S c i . , 9, 393(1971).

168

CHARLES M. SHEARER

36. R. T . Sane, S . V . Desai and R. S . Samant, Indian Drugs, 42(1986).

a,

37. M. He and X . L i , Yaowu Fenxi Zazhi, CAlOO(18): 1 4 5 0 7 5 ~ .

4, 40(1984) from

38. B. Kennedy, Wyeth-Ayerst L a b o r a t o r i e s , Personal Communication.


39. A. H. Stead, R. G i l l , T. Wright, J . P. Gibbs and A. C . Moffat, Analyst, 107, 1106(1982).

FLECAINIDE

Silvia Alessi-Severini, Ronald T.Coutts, Fakhreddin Jamali, and Franco M. Pasutto

Faculty of Pharmacy & Pharmaceutical Sciences University of Alberta Edmonton, Alberta, Canada, T6G 2N8

ANALYTICAL PROFILES OF DRUG SUBSTANCES AND EXCIPIENTS - VOLUME 21

169

Copyright D 1992 by Academic Press, Inc. All rights of reproductionresewed in any form.

SILVIA ALESSI-SEVERINI ET AL.

TABLE OF CONTENTS 1. Description 1.1 Nomenclature, Formula and Molecular Weight 1.2 Appearance, Color, and Odor 1.3 History

2. Synthesis 2.1 Synthesis of Flecainide Acetate 2.2 Preparative Separation of Flecainide Enantiomers 3. Physical Properties 3.1 Infrared Spectrum 3.2 NMR Spectra 3.3 Mass Spectrum 3.4 Ultraviolet Spectrum 3.5 Optical Rotation and Absolute Configuration 3.6 Melting Point 3.7 Ionization Constant 3.8 Distribution Coefficient 3.9 Solubility 3.10 Stability 4. Methods of Analysis 4.1 Elemental 4.2 Spectrophotofluorometry 4.3 Fluorescence Polarization lmmunoassay 4.4 Chromatographic Assays 4.4.1 Stereospecific 4.4.2 Non-stereospecific
5. Pharmacodynamics and Pharmacokinetics
6. References

FLECAlNlDE

171

1. DescriDtion

1.1 Nomenclature, Formula and Molecular Weiaht

(f)-Flecainide acetate, USAN, INN; described as R-818 in the early literature; ( f )-N-(2-piperidinylrnethyl)-2,5-bis(2,2,2trif1uoroethoxy)benzamide acetate. The terms flecainide and flecainide acetate refer to the respective racemates unless otherwise specified.
( Registry No.: ( f 1-flecainide acetate 99495-88-2;f 1flecainide free base 99495-87-1 ( )-flecainide acetate 99495; 93-9; + )-flecainide free base 99495-92-8; ( (-1-flecainide acetate 99495-94-0; (-)-flecainide free base 99495-90-6.

Free base Acetate

c17H20F6N203 c17H20F6N203 C2H402

M.W. 414.36 M.W. 474.40

1.2 Amearance. Color. and Odor


Free base: white granular solid from isopropanol/isopropyI ether; odorless. Acetate: white crystalline solid.

1.3 History
Flecainide acetate is a class Ic antiarrhythmic agent which was developed in the Riker Laboratories as part of a broad-based project investigating the effect of fluorine substitution on local anaesthetic or antiarrhythmic activity. The details concerning the development of this drug have been reported l .

I72

SILVIA ALESSI-SEVERINIET AL.

2. Svnthesig 2.1 Svnthesis of Flecainide Acetate (Fiaure 11 Trifluoroethylation of 2,5-dihydroxybenzoic acid affords 2,2,2-trifluoroethyl 2,5-bis(2,2,2-trifluoroethoxy)benzoate. This is slowly added to a solution of 2-aminomethylpyridine in glyme, under N2, at 25OC. After stirring for 20 hr the reaction mixture is refluxed (3 hr), cooled, and the solvent evaporated in vacuo. Recrystallization of the residue from benzene-hexane gave N42pyridylmethyl)-2,5-bis(2,2,2-trifluoroethoxy)benzamide as an offwhite solid (map103-105C) in 91 % yield. This was dissolved in acetic acid and reduced over Pt02 in a Parr hydrogenator. Filtration of the catalyst and evaporation of the filtrate gave a viscous syrup which solidified on trituration with isopropyl ether. Crystallization from isopropanol-isopropyl ether gave flecainide acetate as a white granular solid (m.p. 145-147OC) in 75% yield2.

FIGURE 1. Synthesis of Flecainide Acetate


2.2
PreDarative SeDaration of Flecainide Enantiomers

( )-Flecainide has been obtained as a salt of ammonium ( + )-ar-bromocamphor-7-sulfonate while (-1-flecainide was isolated

FLECAINIDE

I73

with ammonium (-)-a-bromocamphor-?r-sulfonate. In both cases salt formation was accomplished in methanol. The respective enantiopure flecainide free bases are readily recovered by treatment of the salts with dilute alkali. Subsequent reaction with acetic acid affords the enantiopure flecainide acetate$. Flecainide has also been similarly resolved by preparation of diastereomeric salts (in ethyl acetate) of enantiopure mandelic acids followed by fractional crystallization4,

3 Phvsical ProDertieg .
3.1
Infrared Soectrum

The infrared spectrum of flecainide free base (KBr disc) was recorded on a Nicolet 7199 Fourier Transform spectrometer and is presented in Figure 2. The principal absorption bands include (cm-l):

3427 3358 2927 1637 1606 1549 1500 1458

1291 1221 1169 1154 1083 978 863 657

Some suggested assignments include: broad peak centered at approximately 3400 cm-1 (N-H), 2927 (aliphatic CHI, 1637 (amide I band), 1606 (C=C), 1549 (amide II band), 1500 (C=C str). Aryl ether and CF3 absorptions appear in the same general region; CF3 groups typically show absorptions between 1120-1350 and 690-770. Ar-0-CH2 is usually evident as absorptions between 1200-1275 and 1020-1075 cm-1.

3.2 NMR SDectra


Proton, carbon-1 3, and fluorine-1 9 spectra of flecainide free base, in CDC13, were obtained on a Bruker AM-300 FT NMR spectrometer. The respective spectra are illustrated in Figures 3,

FIGURE 2. IR Spectrum of Flecainide Base. KBr Pellet.

FLECAINIDE

I75

4, and 5 and assignments presented in Tables 1, 2, and 3.


TABLE 1

300 MHz Proton NMR Spectrum of Flecainide Base in CDC13


Chemical Shift
(ppm from TMS)

Number of Protons
1 1 1 1 2

Assignment

7.76 broad 7.76 d (J= 3) 7.09 d,d (J= 9, 3) 6.9 d (J= 9) 4.44 q (JH,F = 8) 4.36 q ( ,, J,= 8) 3.48 m 3.30 m 3.06 m 2.75 m 2.62 m 2.1-1.08 m

2 1

1 1 1 7

CONH aromatic C6 H aromatic C4 j j aromatic C3 li c2 o c y 2 c5 ocy2 CONHCH2 CONHCH2 piperidine C H piperidine NCH2 piperidine NC& NH, piperidine (CH2)3

TABLE 2 75 MHz Carbon-13 NMR Spectrum of Flecainide Base in CDC13 a


14
16

OCH~CF~
16
17

Chemical Shift b

Assignment

24.34 26.49 30.57

2 or 4 2 or 4

FIGURE 3. Proton NMR Spectrum of Flecainide Base in CDCJ3.

PPM

FLECAINIDE

177

46.04 46.68 55.83

1 or6 1 or6

65.72 66.19 66.52 66.67 67.00 67.14 67.47 67.95

1 4 a n d 16 (C-C-F coupling)

77
114.88 1 1 7.22 120.49
1 17.47 1 1 7.67 121.15 121 -36 124.84 125.04 128.52 128.73

CDC13

12 9 11

(C-Fcoupling)

15 and 17

124.31 150.23 153.00 163.99


a

8 10 13 7

In Figure 4 the CH3 and CH groups are shown as signals possessing an anti-phase with respect to the CDC13 signal, while quaternary carbons, CH2 and carbonyls are in phase. ppmfromTMS carbon numbering as shown in structure

b
C

FIGURE 4. Carbon-13 NMR Spectrum of Flecainide Base in CDC13.

E9.S

89.0

88.5

8B.m

87.5

PPM

E7.m

86.5

86.8

85.5

FIGURE 5. Fluorine 19 NMR Spectrum of Flecainide Base in CDC13.

I80

SILVIA ALESSI-SEVERINI ET AL.

TABLE 3

282 MHz Fluorine-19 NMR Spectrum of Flecainide Base in CDC13


Chemical Shift a 87.1 1 87.14 87.17 87.44 87.46 87.49
a

Assignment

CF3

CF3

external standard CgFg

3.3 Mass SDectrum


The positive ion electron impact mass spectrum was recorded on a Kratos MS 50 double focusing magnetic sector mass spectrometer. Operating conditions: mass range 31.01 84415.1471, sampling rate 25, signal level threshold 1, minimum peak width 7, scan rate (sec/dec) 10.0, # of scans averaged 9. High resolution MS: M + calculated, 4 14.1378; found, 41 4.1351, Mass spectral data and suggested structures for fragment ions are shown in Figure 6. Significant Ions Measured Mass %Relative Abundance

c 1 7H19N203F6 c1 1H73F6 CgH1 1

413.1290 301.0295 97.0889

0.17
3.64

10.17

FLECAlNlDE

96.081 2 91.0546 84.0814 5 6.0520


CEO+

2.08

2.51

100.00
6.26

m h 301

mlz 97

0
N
H
m h 84

N
H mlz 5 6

FIGURE 6. Mass Spectral Data 3.4 Ultraviolet Saectrum


The ultraviolet spectrum of flecainide base in ethanol (0.0016 g m / l 0 0 ml) is shown in Figure 7. The absorptivities are: hmax
E (1%: 1 cml

205 230 (shoulder) 300


3.5
solvent)

521 219 59

Ootical Rotation and Absolute Confiauration Optical rotations (sodium D line, 1 dm cells, methanol as were obtained with a Perkin Elmer Model 241

182

SILVIA ALESSI-SEVERINI ET AL.

350.

4 68

FIGURE 7. Ultraviolet Spectrum of Flecainide Base.

FLECAINIDE

I83

polarimeter. Optical purity of flecainide free base enantiomers was >99% as determined by 100 MHz NMR using the chiral shift reagent tris[3-heptafluorobutyryl-d-camphoratoleuropium
11. 13

1-flecainide free base [a]26, (-1-flecainide free base


(

+3 . 4 O
+4.6O

-3.3O
-4.50

( +)-flecainide acetate [ I ' a2D

(-1-flecainide acetate [aI27,

The absolute configurations of flecainide enantiomers have been determined on the basis of CD spectra of the N-chloro derivatives. Thus, ( )-flecainide has the S-configuration while the antipode is R-(-)-flecainide, The optical rotations of hydrochloride salts were also reported4.

( 1-flecainide HCI [a12036s (- 1-f Ieca in ide HC I [a] 65 203

+ 20.0
-2O.OO

3.6 Meltina Points


base base (-1-flecainide free base
( f )-flecainide acetate ( + )-flecainide acetate
( f 1-flecainide free ( + 1-flecainide free

105-107C 104-105C 3 102-104OC 3 145-147OC 153-155OC 3 152.5-1 54OC 3 222-225OC 4 223-225OC

(-)-flecainide acetate
( + 1-flecainide HCI

(-1-flecainide HCI

3.7 Ionization Constanf


The pKa of flecainide acetate has been determined5 as

9.3.

184

SILVIA ALESSI-SEVERINI ET AL.

3.8 Distribution Coefficient


The octanoVwater partition coefficient was determined6 to be 11.4at pH 7.4and logP calculated7 as 4.50.

3 9 Solubility .
The solubility of flecainide acetate mg/ml in water and 300 mg/ml in alcohol5. 3.10 at 37C is 48.4

Stabilitv

A solution of flecainide acetate in water has been reported to be very stable at room temperatures. The stability in biological fluids seems t o be significantly decreased over a period of 3 months even under storage at -2O"Cg. The tablet formulation must be stored in light resistant containers at 15-30'
c5.

4.

f Anal
4.1

is

Elemental elemental analysis for flecainide

The calculated [Cl7H2oF$J2031: C H N

0
F

49.28% 4.87% 6.76% 11.58% 27.51%

The calculated elemental analysis for flecainide acetate [C17H20FgN203. C2H402I: C H N

0
F

48.10% 5.10% 5.91% 16.86% 24.03%

FLECAINIDE

185

A method for the determination of (*I-flecainide in plasma utilizes the natural fluorescence of the molecule. Flecainide is extracted from plasma with heptane after addition of 0.5 mol/L Na3P04 and triethylamine. The organic phase is reextracted with 0.25 mol/L NaH2P04 and the aqueous phase is read in the spectrophotofluorometer (300 nm excitation wavelength, 370 nm emission). The sensitivity is reported to be 25 ng/ml per 2 ml of plasmalo. 4.3 Fluorescence Polarization lmmunoassay

Direct determination of ( f )-flecainide in plasma is possible through the utilization of a commercially available fluorescence polarization immunoassay (Abbott). The reaction is based on the competitive binding of free and fluorescein-labeled flecainide to specific antibodies. Fluorescence polarization measurements are dependent upon the concentration of the free drug in the sample. The method can be performed on 5 0 pL of plasma, with a sensitivity of 0.1 pg/ml, and is very convenient for therapeutic drug monitoring l .
4.4

Chromatoarmhic Assavs
4.4.1 StereosDecific

Sample preparation conditions are summarized in Table 4. 4.4.2 Non-stereomecific Sample preparation conditions are summarized in Table 5.

and

chromatographic

and

chromatographic

5. Pharmacodvnamics and Pharmacokinetics The pharmacodynamics and pharmacokinetics of ( f )flecainide acetate have been studied extensively in animal models and in humans. This drug exhibits potent antiarrhythmic effects

TABLE 4: STEREOSPECIFIC ANALYTICAL METHODS


EXTRACTIONlDERlVATlZATION
( + 1-1-phenylethyl isocyanate

COLUMNlMOBlLE PHASE APPROXIMATE RETN TIME

DETECTION SENSITIVITY

a
1.19
1. 2. 3. 4.

R
2.37

REF

silica (250 x 4.6 mm) hexane:EtOAc (55:45) C18 MeOH:H20

4 12

1. tetra-O-acetyl-P-D-glucopyranosyl isothiocyanate 2. S-l-(l-naphthyl)ethyl isothiocyanate 3. R-l-(2-naphthyl)ethyl isothiocyanate 4. R-or-methylbenzylisothiocyanate

1.16 1.05 1.04

1.00

serum mixed with MeCN, supernatant C18 (100 x 3mm) evaporated; R( + 1-1 -phenylethyl isocyanate MeOH:H20:HOAc (60:40:1) 20 min plasma with butyl chloride:2-propanol silica (250 x 4.6 mm) (95:5); (-)-menthy1chloroformate hexane:EtOAc:Et3N(84:16:O.l) 22 min plasma and urine with diethyl ether; 1-1(4- C18 (300 x 3.9 mm) MeCN:H,O:Et,N (45:55:0.2) nitrophenyl)sulfonyll-L-prolyl chloride 3 0 min plasma with 1% 2-propanol in n-hexane; 1. RWphenylbutyric anhydride 2. R( + 1-1 -MeO-1(CF31phenylacetylchoride 3. N-trifluoroacetyl-L-prolyl chloride 4. f-butyloxycarbonyl-L-alanine SE 3 0 fused silica1 GC capillary column (25 m)

fluorescence (300ex. 370em); 0.05 mg/L fluorescence (305ex, 340em); 2.5 ng/ml UV (298); 40 ng/ml UV (280); 50 ng/ml

baseline resolved 1.08

13

14

1.07

1.25

15

negative ion chemical ionization mass spectrometer; 0.41 ng/ml

1. 1.39 2. 1.43 3.3.38 4. 1.14

16

plasma with 1% 2-propanol in n-hexane; pentafluoropropionic anhydride

Chirasil-L-Val fused silica GC capillary column (25 m); XE60-(R)-phenylethylamide glass capillary column (29 m) silica (250 x 4.6 mm); hexane: 2-butanol:MeCN(98.75:1:0.25) 20 min

negative ion chem- R = 1. l - 1 .6 ical ionization mass on either colspectrometer; < 0.4 umn ng/ml fluorescence (290ex, 340em); 25 ng/ml baseline resolved

16

urine with EtOAc; (-)-menthy1chloroformate

17

TABLE 5 : NON-STEREOSPECIFIC ANALYTICAL METHODS


EXTRACTlONlDERlVATlZATlON COLUMNlMOBlLE PHASE APPROXIMATE RETN TIME DETECTION SENSITIVITY REF

plasma deproteinized, pH adjusted, supernatant injected plasma or urine washed and extracted with hexane plasma or serum extracted with methyl rbutyl ether

C18 pBondapak (150 x 4 mm) ammonium phosphate buffer:MeOH (60:40); 6 rnin

fluorescence (300ex, 370em); 50 ng/ml UV (280)

18

UV (308); 22 ng/ml Zorbax TMS (150 x 4.6 mm) MeCN:l% HOAc in 0.01M pentanesulfonate (45:55); 5 min

19

fluorescence (200ex. 20 Spherisorb S5W silica (125 x 5 mm) MeOH:2,2,4-trimethylpentane (80:20) no emission filter); containing d-10-camphorsulfonic acid; 20 pg/L 4 rnin pBondapak phenyl (300 x 3.9 mm) MeCN:0.06% H,P04 (40:60); 5.5 rnin pBondapak phenyl (300 x 3.9 mm) MeCN:0.06% H,P04 (40:60); 5.5 rnin fluorescence (300ex. 370em); 3 ng/ml fluorescence (300ex. 370eml; 3 ng/ml UV (298); 50 ng/ml 21 22

plasma extracted with hexane solid phase extraction of plasma with C8 adsorbent

UV (214); <30 ng/ diethyl ether extraction of plasma then back pBondapak C18 (300 mm) MeCN:H20 (30:70) containing dibutyl- 0.5 ml extracted into dilute phosphoric acid amine phosphate; 7 min solid phase extraction of plasma with C8 adsorbent Radial-Pak C18 (100 x 8 mrn) MeOH:25% ammonia (99.9:O.l); min 7 fluorescence (293ex, 340em); 10 ng/ml

23

24

serum or plasma extracted with methyl butyl ether

r-

octyl microbore column (250 x 2 mm) UV (298); 80 pg/L 25 0.05% triethylamine in MeCN:O. 1M (250 pL plasma) sodium acetate (45:55); 11 min phenyl reverse phase (300 x 3.9 mm) UV (297); MeCN:20 mM sodium acetate mg/L (42:58); 10 min 0.033

plasma extracted with 1-chlorobutane then back extracted into dilute phosphoric acid

26

serum or plasma extracted with methyl t- octyl microbore column (250 x 2 mm) 0.05% triethylamine in MeCN:O.lM butyl ether sodium acetate (40:60); 8 rnin microscale protein precipitation of serum with Zn sulfate/MeCN, supernatant injected

fluorescence (300ex, 27 370em); 20 pg/L (100 pL plasma) 28

m
rD

fluorescence (285ex. Nucleosil 5 C18 (150 x 4.2 mm) 0.5 ml 370em); 30 ng/ml diethylamine in 1L H,O; 5 min

300 ml MeCN, 1 ml H,PO,,

plasma, urine or saliva extracted with diethyl 3% SP-2250 GC column (180 cm x 2 electron capture ether, back extracted into 0.5M HCI, mm); 16 min detection; 12.5 basified, derivatized with pentafluorobenzoyl ng/ml chloride, extracted into hexane

29

190

SILVIA ALESSI-SEVERINI ET AL.

by blocking the cardiac sodium channels and by stabilizing the myocyte membrane without affecting the repolarization30-34, It is used in the suppression and control of ventricular and supraventricwlar a r r h y t h m i a ~ 3 0 - ~ ~ it has shown some and The effectiveness in the treatment of atrial arrhythmia~3~-39. drug is not recommended in non-symptomatic post-infarction patients because of potentially life threatening t o ~ i c i t y . ~ 0 # ~ 1 Flecainide acetate is rapidly and almost completely absorbed from the GI tract following oral administration. Absolute bioavailability of the commercailly available tablets averages 8590%. First pass metabolism is negligible. Plasma concentrations must be kept within the 200-1000 ng/ml range in order t o maximize the therapeutic effect and minimize the risk of serious side e f f e c t ~ 3 0 - 3 ~Plasma levels and dose show a linear . correlation42, however considerable inter- and intra-individual variations have been observed and, most recently, evidence on non-linear kinetics has been reported43. After i.v. administration to humans, flecainide is rapidly and apparently widely distributed (Vd ranges from 5-13.4 L/kg; average 5.5-8.7 L/kg). After an oral dose the Vd has been determined t o be 10 L/kg44. After i.v. administration t o rats, flecainide is distributed extensively t o many tissues, including the heart, but only minimally into the CNS, and dose-dependent tissue uptake has been shown in rabbits after chronic a d m i n i ~ t r a t i o n ~ 5 , ~The in vitro protein 6. binding (mainly a1 -acid glycoprotein) is approximately 40-50% and is independent of the drug plasma concentration30-34, The pharmacokinetic profile after i.v. administration follows a twocompartment open model with tr/,ar = 3-6 min and a tr/,B = 1114 hr (range 7-19 hr)44. Elimination half-life is prolonged in arrhythmias ( 19 hr), in renal and hepatic impairment (26-49 hr), and in congestive heart failure (up to 50 hrl44f47-52. Urinary pH affects elimination half-life, prolonging it when alkaline (pH 7.28.3) and reducing it when acidic (4.4-5,8)53-55. Flecainide is extensively metabolized by the liver. The t w o major metabolites are m-0-dealkylated flecainide and the m-0dealkylated lactam derivative. These are formed by preferential 0-dealkylation at the meta-position of the benzamide ring and by subsequent oxidation of the piperidine ring of m-0-dealkylated flecainide, respectively. Both metabolites undergo extensive sulfate and glucuronide conjugation at the m-0-dealkylated

FLECAINIDE

191

position. About 80-90940 of a dose is recovered in the urine (30% as unchanged drug, the rest as metabolites and their conjugates). Only 5% is excreted in the faeces and 3% of the total excretion is attributed to at least three unidentified metabolites44. Hemodialysis removes about 1% of a dose as unchanged drug. Total apparent plasma clearance of flecainide, by healthy subjects, following oral administration, has been reported to be in the range of 4-20 ml/kg. Renal clearance of the drug is 2540% of total plasma clearance. Total apparent plasma clearance is decreased in arrhythmias, congestive heart failure and renal
impairment30-34,47-50,52,

There are comparatively few reports concerned with the stereoselective pharmacodynamics and pharmacokinetics of flecainide enantiomers. ln vivo studies have revealed equipotency and equiactivity for the two enantiomers in mouse and dog models of arrhythmia3. ln vitro tests on canine Purkinje fibres have also shown that the enantiorners have comparable electrophysiologic a~tivities5685~. The t w o enantiomers have also shown similar affinities to a receptor site associated with cardiac sodium channels in isolated rat cardiac m y o c y t e ~ ~ ~ . The pharmacokinetic patterns of the enantiomers following oral administration in humans appear to be essentially parallel. The plasma R/S ratio seems to range from 0.67-1.44 in different patient populationsl3#56,59, Stereoselective elimination has been suggested in healthy subjects, which have been classified as poor metabolizers of the sparteine debrisoquine type; R-flecainide is predominant in plasma after oral administration o f the racemate. The oral AUCs of the enantiomers as well as the elimination half-lives were slightly, but significantly, different. This has been interpreted as being the result of stereoselective hepatic metabolism1 7. 6. References

1. J.M. Hudak, E.H. Banitt, J.R. Schmid, Am. J Cardiol., 53, . 17B-20B (1984). 2. E.H. Banitt, W.R. Bronn, W.E. Coyne, J.R. Schmid, J Med. . Chern., 20, 821-826 (1977).

192

SlLVlA ALESSI-SEVERINI ET AL.

3. E.H. Banitt, J.R. Schmid, R.A. Newmark, J. Med. Chem., - 299-302 (1986). 29, 4. G. Blaschke, U. Scheidemantel, B. Walther, Chem Ber 1 8 1 461 6-4619 (1985). 5. American Formulary Service. Drug Information '88,
American Society of Hospital Pharmacists, Bethesda, MD,

832-840. 6. L. Lie-A-Huen, J.H. Kingma, Eur. J. Clin. Pharmacol., 35 8991 (1988). 7. Comprehensive Medicinal Chemistry, Vol. 6, pg. 526, C.

Hansch (Chairman, Editorial Board), Pergamon Press, Oxford, U.K., 1990. 8. Selvi-3M, Milan, Italy. Personal communication (1988). 9. Unpublished data from our laboratories. 10. S.F. Chang, A.M. Miller, J. Jernberg, R.E. Ober, G.J. Conard, Arzneim.-Forsch./Drug Res., 251-253 (1983). 11. R.E. Coxon, A.J. Hodgkinson, A.M. Sidki, J. Landon, G. Gallacher, Ther. Drug Monitor., 9 478-483 (1987). 12. D. Desai, S. Meyer-Lehnert, J. Gal, 193rd American Chemical Society National Meeting, Denver, CO, April 5-10, 1987, Abstract #I 99, Division of Analytical Chemistry. 13. L. Lie-A-Huen, R.M. Stuurman, F.N. Ijdenberg, J.H. Kingma, D.K.F. Meijer, Ther. Drug Monitor., 708-711 (1989). 14. J. Turgeon, H.K. Kroemer, C. Prakash, I.A. Blair, D.M. Roden, J.Pharm.Sci., 79 91-95 (1990). 15. S. Alessi-Severini, F. Jamali, F.M. Pasutto, R.T. Coutts, S. Gulamhusein, J. Pharm. Sci., 79 257-260 (1990). 16. C. Fischer, F. Schonberger, C.O. Meese, M. Eichelbaum, Biomed. Environ. Mass Spec., 1 .256-266 (1990). 9 17. A.S. Gross, G. Mikus, C. Fischer, R . Hertrampf, U. GundertRemy, M. Eichelbaum, Br. J. Clin. Pharmacol., 28 555-566

(1989). 18. J.W. DeJong, J.A.J. Hegge, E. Harmsen, P.Ph. DeTombe, J. Chromatogr., 229 498-502 (1982). 19. S.F. Chang, T.M. Welscher, A.M. Miller, R.E. Ober, J . Chromatogr., 272 341-350 (1983). 20. K.K. Bhamra, R.J. Flanagan, D.W. Holt, J Chromatogr., . 307 439-444 (1984). 21. S.F. Chang, A.M. Miller, J.M. Fox, T.M. Welscher, J. Liq. Chromatogr., 7 167-176 (1984).

FLECAINIDE

193

22. S.F. Chang, A.M. Miller, J.M. Fox, T.M. Welscher, Ther. Drug Monitor., 6 105-1 1 1 (1984). 23. J. Boutagy, F.M. Rumble, G.M. Shenfield,, J Li9. . Chromatogr., Z 2579-2590 (1984). 24. T.A. Plomp, H.T. Boom, R.A.A. Maes, J Anal. Toxicol., 10 . 102-106 (1986). 25. T. Annesley, K. Matz, J fig. Chromatogr., 11 891-899 . (1988). 26. N. Grgurinovich, J, Anal. Toxicol., 12 38-41 (1988). 27. T. Annesley, K. Matz, J. fig. Chromatogr., 11 1041-1049 (1988). 28. G. Malikin, M. Murphy, S. Lam, Ther. Drug Monitor., 11 210-213 (1989). 29. J.D. Johnson, G.L. Carlson, J.M. Fox, A.M. Miller, S.F. Chang, G.J. Conard, J. Pharm.Sci., Z3 1469-1471 (1984). 30. J.L. Anderson, J.R. Stewart, B.A. Perry, D.D. Van Hamersveld, T.A. Johnson, G.J. Conard, S.F. Chang, D.C. 473-477 (1981 1. Kvam, B. Pitt, New Engl. J. Med., 31. B. Holmes, R.C. Heel, Drugs, 29 1-33 (1 985). 32. S.L. Chase, G.E. Sloskey, Clim Pharm., 839-850 (1987). 33. R.W. Kreeger, S.C. Hammill, Mayo Clin. Proc., 62 10331050 (1987). 34. F. Furlanello, G. Vergara, R. Bettini, G. Mosna, L. Gramegna, M. Disertori, Eur. Heart J, 8 33-40 (1987). . 35. M. Epstein, R.M. Jardine, I.W.P. Obel, S. Afr. Med. J., 74 559-562 (1988). 36. V. Zeigler, P.C. Gillette, B.A. Ross, L. Ewing, Am. J. Cardiol., 818-820 (1988). 37. I.C. Van Gelder, H.J.G.M. Crijns, W.H. Van Gilst, C.D.J. De . Langen, L.M. Van Wijk, K.I. Lie, Am. J Cardiol., 63 112114 (1989). 38. M.J. Suttorp, J.H. Kingma, L. Lie-A-Huen, E.G. Mast, Am. J Cardiol., B 693-696 (1989). . 39. S.S. Wafa, D.E. Ward, D.J. Parker, A.J. Camm, Am. J. Cardiol., 1058-1064 (1989). 0 40. B.R. Winkelmann, H. Leinberger, Ann. Internal Med., 1 6 807-8 14 ( 1987). 41. D.S. Echt, P.R. Liebson, L.B. Mitchell, R.W. Peters, D. Obias-Manno, A.H. Barker, D. Arensberg, A. Baker, L. Fiedman, H.L. Greene, M.L. Huther, D.W. Richardson, and

I94

SILVIA ALESSI-SEVERINI ET AL.

the CAST Investigators, New Engl. J Med., 324 781-787 . (1991). 42. G.J. Conard, G.L. Carlson, J.W. Frost, R.E. Ober, A.S. Leon, D.B. Hunninghake, Clin. Therapeutics, 6 643-652 ( 1984). 43. G. Boriani, E. Strocchi, A. Capucci, R. Calliva, L. Frabetti, E. Ambrosioni, B. Magnani, Eur. J Clin. Pharmacol., 41 57-59 . (19911. 44. G.J. Conard, R.E. Ober, Am. J Cardiol., 53 41B-51B . (1984). 45. D. Piovan, R. Padrini, M. Furlanut, R. Moretto, M. Ferrari, Pharmacol. Res. Commun., 18 739-745 (1986). 46. R. Kannan, A. Matin-Asgari, Drug Metab. Disposit., l 228231 (1988). 47. J. Braun, J.R. Kollert, J.U. Becker, Eur. J. Clin. Pharmacol., - 71 1-714 (1987). 31 48. A.J. Williams, R.L. McQuinn, J. Walls, Clin. Pharmacol. Ther., 43 449-455 (1988). 49. S.C. Forland, R.E. Cutler, R.L. McQuinn, D.C. Kvam, A.M. Miller, G.J. Conard, S. Parish, J. Clin. Pharmacol., 28 727735 (1988). 50. S.C. Forland, E. Burgess, A.D. Blair, R.E. Cutler, D.C. Kvam, C.E. Weeks, J.M. Fox, G.J. Conard, J Clin. Pharmacol., 28 . 259-267 (1988). 51. R.L. McQuinn, P.J. Pentikainen, S.F. Chang, G.J. Conard, Clin. Pharmacol. Ther., 44 566-572 (1988). 52. A. Cavalli, A.P. Maggioni, S. Marchi, A. Volpi, R. Latini, Clin. Pharmacokinet., 14 187-188 (1984). 53. K.A. Muhiddin, A. Johnston, P. Turner, Br. J. Clin. Pharmacol., 1 447-45 1 (1984). 7 54. A. Johnston, S. Warrington, P. Turner, Br. J. Clin. Pharmacol., 20 333-338 (1985). 55. R. Hertrampf, U. Gundert-Remy, J. Beckmann, U. Hoppe, W. ElsaBer, H. Stein, Eur. J. Clin. Pharmacol., 41 61-63 (1991 1. 56. H.K. Kroemer, J. Turgeon, R.A. Parker, D.M, Roden, Clin. 584-590 (1989). Pharmacol. Ther., 57. J.K. Smallwood, D.W. Robertson, M.I. Steinberg, NaunynSchmiedeberg s Arch. Pharmacol. , 339 625-629 (1989).

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195

58. R.J. Hill, H.J. Duff, R.S. Sheldon, Molec. Pharmacol., 34 659-663 (1988). 59. S. Alessi-Severini, D.F. LeGatt, F.M. Pasutto, F. Jamali, R.T. Coutts, Clin. Chern., 1 11-1 12 ( 1991 ), Correction, C/in. Chern., aZ 886 ( 1991 1.

GLAFENINE

Adnan A . Badwan,' Muhammad B. Zughul,2 and Mahmoud A l Omari'

(I ) The Jordanian Pharmaceutical Manufacturing Co. Naor, Jordan

(2) Department of Chemistry Faculty of Science University of Jordan Amman, Jordan

ANALYTICAL PROFILES OF DRUG SUBSTANCES AND EXCIPIENTS - VOLUME 21

197

Copyright 0 1992 by Academic Press, Inc. All rights of reproducilon reserved In any form.

198

ADNAN A. BADWAN. MUHAMMAD B. ZUGHUL, AND MAHMOUD AL OMARI

- DESCRIPTION
1.1. 1.1.l. 1.1.2. 1.1.3. 1.1.4. 1.2. 1.2.1. 1.2.2. 1.2.3. 1.3. 1.4.

CONTENTS

Nomenclature. Chemical Names. Generic Name. Registry Number. Wiswesser Line Notation. Formulae. Emperical Formula. Molecular Weight. Structural Formula. Colour, Appearance and Odour. Therapeutic Use.

- SYNTHESIS
2.1. 2.2. 2.3. 2.4.

Synthetic Route (I). Synthetic Route (11). Synthetic Route (111). Synthetic Route (IV).

- PHYSIC0 - CHEMICAL PROPERTIES


3.1. 3.2. 3.3. 3.4. 3.5. 3.6. 3.6.1. 3.6.2. 3.6.3. 3.6.4. 3.6.5. 3.6.6. 3.6.7.

Melting Range. Differential Scanning Calorimetry. Solubility. DissociationConstant. Partition Coefficients. Spectral Properties. UltravioletSpectra. Fluorescence Spectrum. Single Photon Counting Spectrofluorometry. lnfra Red Spectrum. Nuclear Magnetic Resonance Spectrum. Mass Spectrum. X - Ray Powder Diffraction.

- METHODS OF ANALYSIS
4.1. 4.1.1. 4.1.2, 4.1 2 . 1 4.1.2.2 4.1.2.3.
I

GLAFENINE

199

Starting Material and PharmaceuticalDosage Forms. Elemental Composition. Related Materials. 4,7 - dichloroquinoline. Anthranilic acid esters. N - (7 - chloro - 4 - quinolyl) anthranilic acid (glafenic acid). Titrations. 4.1 -3. Non - Aqueous Titration. 4.1.3.1 4.1.3.2. Alkalimetric Titration. GravimetricAnalysis. 4.1.4. Spectrophotometric Methods. 4.1.5. 4.1 5 . 1 . Ultraviolet Absorption. 4.1.5.2. SpectrofluorometricAnalysis. Chromatographic Methods. 4.1.6. Thin Layer Chromatography. 4.1.6.1. 4.1-6.2. High Performance Liquid Chromatography. 4.2. Body Tissues and Fluids.

- STABILITY
5.1. 5.2.

Stability of The Solid. Stability in The Solution.

- PHARMACOKINETICS
6.1. 6.2. 6.3. 6.4. 6.5. 6.6.

Absorption. Bioavailability. Distribution. Metabolism. Excretion. Half - Life.

200

ADNAN A. BADWAN. MUHAMMAD B. ZUGHUL, AND MAHMOUD AL OMARl

1. DESCRIPTION
1.l. Nomenclature

1.1.1, Chemical names

1.1.1.1 a - glyceryl or 2', 3' dihydroxypropylN - (7 chloro 4 quinolyl) anthranilate. 1.1.1.2 2 , dihydroxypropyl2"-(7 - chloro - 4 aminoquinolyl) benzoate. ' 3' 1.1.1.3 4 - (2" (2', 3' - dihydroxypropyl carboxyphenyl) amine) 7 chloroquinoline.

- -

1.1.2. Generic name

Glafenine (Listed in The French Pharmacopoeia)


1.1.3. Registry number

CAS 3820 - 67 - 5
1.1.4. Wiswesser line notation

T66 BNJ EMR 8 V1 YQ1 Q and JG


1.2. Formulae 1.2.1. Emperical formula

1.2.2. Molecular weight

372.8

GLAFENINE

20 I

1.2.3. Structural Formula

1.3. Colour, Appearance and Odour.

Pale yellow, crystalline powder, odourless.

1.4. Therapeutic Use

Glafenine is an analgesic.

2. SYNTHESIS
Different routes of synthesis were proposed. A brief of these is described and a schematic presentation is illustrated.

2.1. Synthetic Route 1.

Reacting of isoatoic anhydride with glycerol to produce glyceryl anthranilate, followed by reacting with 4, 7 - dichloroquinoline to produce glafenine (1).

202

ADNAN A. BADWAN, MUHAMMAD B. ZUGHUL. AND MAHMOUD AL OMARl

Scheme (I)

GlafenineSynthesis, Route (I)

2.2. Synthetic Route II.

Condensation of 2 - chlorobenzoate, glyceryl ester with 7 - chloro - 4 aminoquinoline (1).

I--\

Scheme (/I)

Glafenine Synthesis, Route (11)

GLAFENINE

203

2 3 Synthetic Route 111. ..


Condensation of 4, 7 - dichloroquinoline with methylanthranilate. The resulting methyl ester is transesterified with glyceryl acetonide which is further hydrolysed to glafenine (1).

glvceryl acetonide

HN I

Scheme (111)

Glafenine Synthesis, Route (111).

204

ADNAN A. EADWAN. MUHAMMAD B. ZUGHUL, AND MAHMOUD AL OMARI

2.4. Synthetic Route IV

Esterification of 2 - nitrobenzoicchloride with glyceryl acetonide, followed by a reduction of the nitro group to the amino group. The resulted ester is condensed with 4, 7 - dichloroquinoline, followed by hydrolysis of acetonide (2).

HO OH

Scheme (IV)

Glafenine Synthesis, Route (IV).

GLAFENINE

205

3. PHYSIC0

- CHEMICAL PROPERTIES

3.1. Melting Range

The French Parmacopoeiaspecifies that the melting range of glafenine is between 170C - 174 "C, (3).
3.2. Differential Scanning Calorimetry

Glafenine was recrystallized from ethanol, butanol, hexanol and acetonitrile. Thermograms of these crystals were obtained using Mettler TA 3000 - DSC - 20 unit. The heating rate was 10"C. min-' and the sample size was ranging from 3 - 10mg. The recrystallized glafenine showed a single sharp peak without any decomposition at melting. Melting range of obtained crystals from different solvents was between 170 - 174C. Recrystallization from hexanol yielded sharper thermogram peak, figure (1). The heat of fusion of these crystals was in the vicinity of 43.8 KJ. mole-' (4).

Heat Flow Exothermal

l -

E
180

Figure ( 1 )

The DSC Profile of Glafenine Recrystallized from Hexanol

206

ADNAN A. BADWAN. MUHAMMAD B. ZUGHUL, AND MAHMOUD AL OMARI

3.3. Solubility

Equillibrium solubility of glafenine was determined by shaking an excess of glafeninewith the solvent requiredin a water bath at 30 C for 48 hours. Table ( I) presents glafenine solubility in commonly used solvents (4).

Table ( I )
Glafenine EquillibriumSolubility in Commonly Used Solvents
Solvent
Hexane Water Chloroform Acetone Ethanol 0.1N HCI

gm/lOOml at 30C

<0.001 001 .0 0.260 0.297 0.700 1.295

3.4. Dissociation Constant The pKa was determined spectrophotometricallyat 20 C in accordance with an earlier reported method (5). Stock solution of glafenine in 10 N HCI was prepared, and diluted with suitable buffer solutions rangingfrom pH 6 - 10 to obtain afinal glafenine concentrationof 1Oug. mL-. The absorbance of these solutions were measured at the maximumat 342.5nm. This method yielded 7.2 as pKa of glafenine at 20 C (4).

GLAFENINE

207

3.5. PartitionCoefficients

The partition coefficients of glafenine between n - decanol and aqueous buffers of different pH values were determined at room temperature. Different pH values were obtained by using 0.1 N HCI for pH 1.O, acetate buffers for pH 3.0,4.0,4.5 and 5.0 and phosphate buffers for pH 6.0,7.0 8.0. Figure (2) and shows the plot of pH against the partition coefficients (5).

20

15

10

PH

Figure (2)

The Plot of Partition Coefficient of Glafenine Against the pH

208

ADNAN A. BADWAN, MUHAMMAD B. ZUGHUL. AND MAHMOUD AL OMARI

3.6. Spectral Properties 3.6.1. Ultraviolet spectra


The absorption spectra of glafenine dissolved in methanol and 0.1 N HCI are shown in figure (3). It exhibits maximum at 356nm, 225nm and 255nm for methanol and maxima at 342nm, 223nm and a shoulder at 252 nm for 0.1N HCI. These were recorded using Beckman DU - 7 spectrophotometer.

Wavelength (nm)

Figure (3)

The UV absorption Spectra of Glafenine (70 ug.mL-) in Methanol (-) and 0.7N HCl (...).

GLAFENINE

209

Table (11) repesents the ultraviolet absorption spectral bands of glafenine in various commonly used solvents (4 , 7).

Table ( I ) 1
The UV Absorption Spectral Bands of Glafenine in Different Solvents
Solvent W8Velength (Extinction Coefficient) nm (M-' cm-')

Chloroform Benzene Methanol Ethanol Acetone


Ether

O.1NHCI

360 (22,900), 360 (14,900), 356 (21,OOO), 355 (19,600), 355 (18,700), 351 (19,300), 342 (18,300),

255 (18,100) 255 ( 4,100)

255 (1 8,800) 255 (1 7,000) 255( 5,600) 255 (1 9,000) 252 (Shoulder)

3.6.2. Fluorescencespectra The excitation and emission spectra of glafenine in ether is shown in figure (4). The excitation and emission maxima of glafenine in different solvents are presented in Table (Ill). Aqueous solutions having pH values lower while solutions having higher pH values exhibit than 4 do not fluoress fluorescence which intensifies with the pH increase. This intensity is maximized in solutions having pH 9 - 10. (7).

210

ADNAN A. BADWAN. MUHAMMAD 8 . ZUGHUL. AND MAHMOUD AL OMARI

.L.
u)

60

.9 c
Q 0

4-

C Q, C

40

20

Figure (4)

Wavelength (nrn) The Fluorescence Excitation and Emission Spectrum of Glafeninein Ether (10 ug. mL-'; Aexc= 250,327 and 7em = 400 nm).

0
Figure (5)

8 1 0 l 2 1 4 1 6

The Fluorescence Decay Curve of Glafeninein Ethanol at hexc=340nm and-hem=475nm.

GLAFENINE

21 I

Table ( 111 )
The Fluorescence Characteristics of Glafenine in Various Solvents (5 ug. mL-)
Solvent exc. (nm) em. (nm)
330 250b,327 250b,340 245b,336 340 245b,336 26!jb,355 330 273b,365 300 392 400 436 439 410 425 450 430 455 407

Intensitya
20 17 10 6

Benzene Ether Chloroform Ethanol 2 - Propanol - 10% (v/v) buffer pH9 (0.1N glycine-sodium hydroxide). Methanol 2 - Propanol - 10% (v/v) buffer, pH4 (0.1N citrate-hydrochloric acid). Acetone Methanol - 10% (v/v) ammonia (28% w/w). 2 - Propanol - 10% (v/v) buffer, pH1 (0.2N potassium chloride - hydrochloric acid). Sulfuric acid (10- 0.005N).

6
2 2 0.5 0.5 0.5

No signal

a: Referred to a solution of quinine sulfate in a concentration of 1ug.mL- in 0.5N sulfuric acid, of which the relative fluorescence intensity is 100, measured simultaneously.

b: Secondary excitation value with much less intensity.

3.6.3. Single photon counting spectrofluorometry. A single photon counting spectrofluorometry was used to measure the decay life time of glafenine in absolute ethanol (20ug. mL-). A hydrogen lamp was used as monochromatic radiation source while the excitation and emission wavelengths were 340nm and 475nm, respectively figure (5). The fluorscence decay curve was obtained using Edinibrugh Instruments single photon counting model 199 - spectrofluorometer. This figure shows that the decay life time of glafenine in ethanol is 0.88 n - sec.(8).

212

ADNAN A. BADWAN. MUHAMMAD 8. ZUGHUL. A N D MAHMOUD AL OMARI

3.6.4. Infrared spectrum

Glafenine infrared spectrum is shown in figure (6). This is obtained by screening glafenine - KBr dispersion disc using Perkin Elmer 598 spectrophotometer. Table (IV) shows the band assignments of infrared spectrum of glafenine (4). Table ( IV ) The I.R. Spectral Assignments of Glafenine
Wavenumber, cm-' Vibration Mode 0-H (stretching),N-H (stretching) C-H (stretching), aromatic C-H (stretching),aliphatic C =0 (stretching) C=C (stretching), aromatic C-0 (stretching), ester C - 0 (stretching), alcohol C-H (bending), aliphatic C-H (out of plane bending), aromatic

3500 - 3220 3 00 1 2900 1680 1620,1580,1530 1260,1050 1 100,980 1450 870,830,750,600

3.6.5. Nuclear magnetic resonance spectrum

NMR spectrum of glafenine is presented in figure (7). This spectrum was recorded on Varian T60 NMR spectrometer. Table (V) lists the spectral assignmentsof glafenine in DMSO (1,8). Table ( V ) The NMR Spectral Assignments of Glafenine
Chemical Shlft Number of Proton (Multiplicity) Assignment

1. 01 8.7- 7.0 4.4 3.8 3.5

Wavenumber (Cm-')

Figure (6)

The 1.R. Spectrum of Glafenine.

figure (7)

The N.M.R. Spectrum of Glafenine in DMSO.

GLAFENlNE

215

3.6.6. Mass spectrum.

The mass spectrum of glafenine was obtained using mass spectrometer MAT - 112. Some characteristic peaks are listed in Table (VI). Figure (8) representsthe mass spectrum of glafenine, (4).

Table ( VI ) The Mass Spectrum of Glafenine


m/z
Species
M+ I/ I,%

372 298 280 253 27 1 11 2 104 76 74

372 - (CHZ= CHOHCHZOH) 298 - (HZO) 280 - (CO) 253 - (CI) (C&COOH)' (C6H4CO)+ (c6H4)+ (CH2=CHOHCH20H)

40.0 27.1 62.9 71.4 34.3 40.0 100.0 70.0


15.7

I / lo = relative intensity (Eased on the highest intensityof 100.0).

The molecular ion peak appeared at m/z ratio of 372. Glafenine mass spectrum showed a distinct peak at m/z 298. This peak corresponds to Maclafferty rearrangement.The cleavage of oxygen - carbonyl bond is evident I at m/z 280. Further, the removal of C = 0 group and C atom is shown at m/z 253 and 217, respectively. The pattern of glafenine mass fragmentation is presented in scheme (V).

Figure (8)

The Mass Spectrum of Glafenine

GLAFENINE

217

-CHFCHOHC H, H O

mlz =74

C L U d

m/z = 372

QtJ
m/z=298

C L

cl$

m/z = 280

c,QI$

m/z = 253

QQ

m/z =29 8

m/z = 177

m/z =121

m/z =I21

m b =I 04

Scheme (V}

The Mass Fragmentation Pattern of Glafenine.

218

ADNAN A. BADWAN. MUHAMMAD B. ZUGHUL, A N D MAHMOUD AL OMARl

3.6.7. X ray powder diffraction

The x - ray powder diffraction of glafenine powder was determined using Phillips PW 1050 - 81 Goniometer with a PW 1729 Generator with Nickle filtered copper radiation ( = 1.5418nm) as a source of radiation. The scanning rate was 28. (2cm)-. min-. The interplanner distance and relative intensity of the major peaks are listed in Table (VII). Figure (9) illustrates the x ray powder diffraction pattern of glafenine recrystallizedfrom 1 - hexanol (4).

Table ( VII )

The X - Ray Powder Diffractionof Glafenine

5.6 7.6 9.9 11.4 14.9 15.5 16.6 17.6 19.2 20.4 21.2 22.2 22.8

15.781 11.632 8.9341 7.761 7 5.9454 5.7166 5.3402 5.0390 4.6226 4.3532 4.1907 4.0042 3.9001

2.8 17.3 5.6 70.9 16.8 30.7 51.4 4.5 5.6 79.9 14.0 50.3 22.3

23.4 24.1 25.3 26.0 27.6 29.8 30.9 34.1 35.8 37.9 40.6 41.7 44.1

3.8015 3.6926 3.5201 3.4269 3.2318 2.9980 2.8938 2.6292 2.5081 2.3738 2.2220 2.1659 2.0534

32.4 100.0 13.4 16.8 30.7 11.1 6.7 7.8 10.6 22.3 5.0 8.4 21.2

d = Interplanner Distance, l / l o = Relative Intensity (based on the highest intensity of 100).

44

38

32
Figure (9)

26 20 Diffraction Angle (28)

14

The Powder X - Ray Diffraction Pattern of Glafenine Recrystallized from Hexanol.

220

ADNAN A. BADWAN, MUHAMMAD B. ZUGHUL, AND MAHMOUD AL OMARI

4. METHODS OF ANALYSIS
4.1. Starting Material and PharmaceuticalDosage Forms 4.1 .l. Elemental Composition Calculated Percentage

C
H

61.21

4.60
9.51 7.51

C I
N

0
4.1.2. Related Materials

17.17

4.1.2.1. 4,7 - dichloro - quinoline : 10 mg glafenine are dissolved in 0.5rnLof hydrochloric acid solution (1 % v/v hydrochloric acid in ethanol). 0.5 rnLof paranitrophenyl hydrazine solution (0.1 % millimole in ethanol) is added. The mixture is cooled in air and then warmed in a water bath at 80Cfor one hour in darkness. The mixture is further cooled to 20 "Cf 1 "C. 0.25rnbftriethylamine is added, shaken and followed by the addition of 4 ml of dimethyformamide and shaken until a homogenous liquid is obtained. This solution is compared in colour intensity with a standard solution of 0.5rnLof 4,7 - dichlocoquinoline(10 ug permL)in 1 % v/v hydrochloric acid in ethanol. The percent concentrationof 4,7 - dichloroquinolinein glafenine should not exceed 0.05% (3). 4.1.2.2. Anthranilic acid esters: 50mg of glafenine are dissolved in 5mL of ethanol. 1mL of diluted sulphuric acid is added. The mixture is cooled for 2 minutes in an ice bath. 1mL of 1% millimole per volume of sodium nitrite solution is added and the mixture is left for 10 minutes in the ice bath. 1 mL of freshly prepared and filtered B - naphthol solution of 10% millimole per volume in concentrated ammonia, is added. The obtained solution is compared in colour intensity with a simultaneously prepared standard solution of a mixture of 4.8mLof ethanol and 0.2 mL of methyl anthranilate solution (0.05% millimole per volume in alcohol). The percent concentrationof anthranilic acid esters in glafenine should not exceed 0.2% (3).

GLAFENINE

22 I

4.1.2.3. N - (7 - chloro - 4 - quinolyl) anthranilic acid (glafenic acid): Thin layer chromatography can be used to detect glafenine and glafenic acid in the bulk material. The system consists of a plate of silica gel HF 254 impregnated with sodium acetate. The mobile phase is a mixture of chloroform - methanol glacial acetic acid and water mixed in volumetric proportions of 85:12:2:1, respectively. The developed spots are identified by exposure to U.V. lamp (254nm). Glafenine and glafenic acid solutions having a known concentration are prepared in a mixture of chloroform - methanol and water mixed in respectively. These solutions include volumetric proportions of 3.0:3.0:0.5, 0.5% w/v glafenine sample (solution a), 0.5%, 0.1 %, 0.0025% w/v glafenine standard solutions (b, c and d), 0.0025% w/v glafenic acid standard (solution e), a mixture of 0.5 w/v glafenine standard and 0.0025% w/v glafenic acid standard (solution f). If a secondary spot appears in the chromatogram of solution a with an Rf value slightly inferior to the principal spot of the same chromatogram, it should not be more intense than the principal spot in the chromatogram obtained from solution c. If a secondary spot appears corresponding to glafenic acid, it should not be more intense than the spot obtained from solution e. If secondary spots other than the previous two appear in the chromatogram of solution a, it should not be more intense than the principal spot of solution d. This test could be adopted only if glafenine and glafenic acid were separated in two spots in the developed chromatogram of solution f (3). 4.1.3. Titrations. 4.1.3.1. Non - aqueous titration: Glafenine (300 mg) is dissolved in 30 mL of acetic acid. The end point is detected potentiometrically. When glafenine hydrochloride is used, mercuric acetate is to be added to the titration medium. Each mL of 0.1 M perchloric acid volumetric solution is equivalent to 37.28mgof glafenine. This method is applied for the drugs determination in tablets and suppositories (9).

222

ADNAN A. BADWAN, MUHAMMAD B. ZUGHUL, AND MAHMOUD AL OMARI

4.1.3.2. Alkalimetric titration: Powdered tablets equivalent to 50 - 200 mg of glafenineare placed in 150 mL conical flask. 25 mL of 0.1N HCI are added and shaken for 2 - 3 minutes. The bromocresol green indicator is added to the mixture (2 - 3 drops) and titration is carried out with 0.1 N NaOH. The indicator colour changes from yellow to bluish green at the end point. This method could be adopted for different dosage forms determination. Each mL of 0.1 N HCI volumetric solution is equivalent to 37.28 mg of glafenine (10). 4.1.4. Gravimetricanalysis

Glafenine is determined in tablets and suppositories by adding 1 mL of 1N HCI, 10 mL of diluted acetic acid and 6 mL of 0.25M KBi14with stirring, to 10 mL portions of sample solution prepared from tablets or suppositories described, containing 50mg glafenine. After 30 minutes, the precipitate is filtered off on a sintered glass filter and washed with diluted acetic acid and water. The filter is then dried at 105C for 60 to 90 minutes, cooled and weighed. Each gram of the precipitate is equivalent to 0.3728 gm of glafenine(l1).
4.1.5. Spectrophotometricmethods 4.1 5 1 . Ultraviolet absorption: Glafenine (50 mg) is dissolved in 0.1 N HCI and the volume is made up to 250mL. 5 mL of this solution is further diluted to 100 mL with the same solvent. The solution exhibits a maximum absorption at about 343 nm. The specific absorbanceat this maximum is about 490 (4). 4.1 5.2. Spectrofluorometric analysis: Glafenine is determined in tablets by transfering a quantity of fine powder equivalent to 25 mg of glafenine into 500 mL conical flask. About 450 mL of ether are added and the mixture is stirred with a magnetic stirrer for 2 hours. After filtration on a paper filter, the filtrate is diluted to 500 mL with ether. Further 10 mL of this solution is dilutedto 100 mL with ether. An analogous standard solution having the same concentration of the sample solution is prepared. It is advisable to extract the tablets simultaneouslywith the preparationof the standard solution or during at least the same period to avoid incomplete extraction. Pure ether is used as the blank solution. Fluorometric measurement is performed at 327 nm excitation and 400 nm emission (7).

GLAFENINE

223

4.1.6. Chromatographic Methods 4.1.6.1. Thin - layer chromatography (TLC): Glafenine (I), glafenic acid (11) (major metabolite and major photoproduct in the solid state) and methyl N - (7 chloro - 4 - quinolyl) anthranilate (111) (minor photoproduct in the solid state) can be identifiedby TLC method using silica gel 60 F254 (2Ox20cm) with thickness of 0.2 mm as stationary phase. 1OuL of 0.20% and 0.01 % of glafenine and glafenic acid in chloroform are spotted. Methyl N - (7 - chloro - 4 - quinolyl) anthranilate is detected after storing a solution of 0.20% of glafenine in methanolfor 24 hours under ambient conditions. The system is equillibratedfor 15 minutes before the development. The development distance is 10 cm and the plate is air dried. The detection method is UV lamp (254 nm) or by naked eye (yellow colour spots). Table (VIII) lists the Rf values of glafenine and its photodegraded products in different mobile solvents (4).

Table ( Vlll ) The Thin layer Chromatography of Glafenine and its Photodegraded Products in the Solid State (4).
~ ~~~~

Mobile Phase

Rf Value of (1)

Rf Value of (11)
0.00 0.22 0.15

Rf Value of (111)
0.37 0.80

Ethylacetate - Chloroform (70/30by Volume) Chloroform- Methanol - Acetic Acid (85/12/3 Volume) by Ethylacetate- Methanol - 33% Ammonia (85/10/5 Volume) by Chloroform - Methanol (80/20 Volume) by

0.06 0.30
0.45

0.75

05 .7

0.24

0.77

224

ADNAN A. BADWAN. MUHAMMAD B. ZUGHUL, AND MAHMOUD AL OMARI

4.1.6.2. High performanceliquidchromatography:HPLC profile of glafenine is shown in figure (10). This profile was obtained using Beckman HPLC system. An R Sil C18column (150 mm x4.6 mm I.D.) with a particle size of 5 um was used. The mobile phase consisted of a mixture of methanol, water and acetic acid (64:27:9 by volume). The chromatographicsystem was operated at room temperature with an eluent flow rate of 1.OmL.min-. It has a sensitivity of 0.01 absorbance unit, attenuation of 64 and chart speed of 0.5 cm.min-. The wavelength of the detector was set at 344 nm. This method is stability indicating and may be used for tablets, capsules and suppositories (8).

r,

I
Figure (1 0)

Rt (min)
The HPLC Profiles of Glafenine Dissolved in Ethanol (10 ug. mL-).

GLAFENINE

225

4.2. Body Tissues and Fluids

Spectrophotometric methods for the determination of glafenine and its metabolitesin the body fluids were insensitive and unspecific. More recent and specific methods using HPLC were reported: Determinationof glafenineand its metabolitesinvolveda separation and extraction procedure in human plasma. Floctafenine was used as internal standard for both the drug and its metabolites. Chromatographic conditions were 5 um R - Sil Cle column, solvent mixture consisted of methanol - water acetic acid (67:23:10),respectively.pH was adjusted to 4.3 by the addition of ammonia. The flow rate was 0.5 mL.min- and the detector was set at 360 nm. For 1 mL plasma, the detection limit was 0.5mg. L- for glafenine and hydroxyglafenicacid, and 0.2mg. L for glafenic acid. This method allowedthe 1 deduction of some primary pharmacokineticparameters ( 2).

- For determination of glafenine (I): Plasma (1mL) containing 50uL of floctafenine (11) as internal standard solution, was made alkaline with glycine buffer of pH 11 (1 mL) and extracted with CHCl3 (2x5 mL).After centrifugation, the combined organic layer was evaporated to dryness at 37OC and the residue 1 was dissolved in the mobile phase ( 00 uL). For determinationof glafenic acid (111) plasma ( mL) containing (11) solution (50 uL) was acidified with 0.1 N HCI 1 (200uL) and extracted as above. Sample solutions (20uL) were analysed by HPLC on acolumn (1 5 cm x 4.6mm) of SpherisorbCe(5 um) with acetonitrile water - diethylamine (550:400:3) adjusted to pH 4 5with anhydrous acetic acid . and as mobile phase (1 mL.min-), detection was at 362,358 364 nm for I,II and 111, respectively. Calibration graphs were rectilinear from 0.05(detection limit) o to 2.5mg.L-, and 0.25(detection limit) t 2.0 mg. L for I and Ill, respectively. . The coefficients of variation (n = 10)were 8 1to 13.7and 7.7to 10.8% for Iand Ill, respectively (13).

226

ADNAN A. BADWAN, MUHAMMAD B. ZUGHUL, AND MAHMOUD AL OMARI

5. STABILITY
5.1. Stabillty of The Solid

Glafenine powder is stable against heat and moisture.The powdered drug was stable when stored at 40 "Cin the dark for 180 days (14). Glafenine in the solid form readily undergoes photodegradation when exposedto UV visible or solar radiation. The photodecomposed products are similar regardless of the method of radiation used. The two photodecomposition products were identified as N - (7 chloro 4 - quinolyl) anthranilic acid and methyl N - (7 chloro - 4 - quinolyl) anthranilate. The first was separatedand identifiedas solid while the second was identified in solution of isopropanol and was found to be present in trace amounts. These were cross checked with similar prepared photoproducts. It seems that intramolecular H - abstraction leads to the formation of these photoproducts. Glafenine formulated into solid dosage forms has to be guarded against sources initiating photochemical degradation(8).

5.2. Stability in The Solution

Glafenineis insoluble in water. Heating the drug's suspension at 50 "Cfor several hours produced no degradation. However, boiling the same solution yielded hydroysedforms of glafenine. In neutral alcoholic solution, galfenine is unstabletowards UV/visible radiationwhere it photodecomposes into two main products, namely glyceryl anthranilate and 7 - chloroquinoline. Glafenine photodegradation is suggested to occur via intermolecular H - abstraction in the presence of proton donor solvents. The rate of photodecomposition in neutralalcoholic solution was found to decrease with the polarity of the solvent, while the increase in viscosity of the solvent was found to be impededprobably due to the cage effect. In nonpolar solvents such as benzene, photodecomposition is very low. In acidic alcoholic and aqueous solutions, glafenine proved to be quite stable (8).

GLAFENINE

227

6. PHARMACOKINETICS
6.1. Absorption

Glafenine is well absorbed from the intestinalwall in gastrointestinaltract. Following oral administration of glafenine, peak concentration of the main metabolite, glafenic acid, is reached after about one hour, The decline in plasma concentration is multiphasic and incompatible with one compartment model. In view of the lack of free glafenine in the central compartment, a substantialfirst - phase elimination by liver or gut wall can be assumed. Rectal absorption of glafenine or glafenine hydrochloride is extremely slow and incomplete due to the slight water solubility of glafenine at the prevailing pH in the rectum lumen (15).
6.2. Bioavailability

Glafenine suspension was administered and compared with glafenine suppositories and enemas. It is clear from Table (IX) that rectal administration of micro - enemas or suppositories containing this drug is not bioequivalentwith oral dosage form (15).

6.3. Distribution

It seems that the glafenic acid is deposited in the kidney. Such deposition is manifested by yellow colouration which disappears with biochemical disturbances(16).

6.4. Metabolism

Comparative studies suggest that analgesic activity of glafenine is due to one of its metabolites. The glycerol liberated in vivo following administration of glafenine does not appear to be responsible for the effect of the drug since

228

ADNAN A. BADWAN, MUHAMMAD B. ZUGHUL, AND MAHMOUD AL OMARI

Table ( IX )
Absorption Characteristics, Relative Bioavailability and Urine Excretion Pattern of Glafenic Acid (Mean _+ S.D.), After Oral and Rectal Administrationof 400mg Glafenine (439mg Glafenine HCI).

Plasma concentation

(ug.mL-') at t: 30 (min) 60 120 1 80 240 300


Number Cll, ta mw AUCo.5
Frei

Urine concentation (mg) at t: 60 (min)

9.2f3.4 12.8k2.7 4.6k0.7 1.6k0.5 1.1k0.2 0.6k0.1 7 (ug.mL-') 12.6k3.1 (min) 50+27 (ug.rnin". mL-') 1308k68 1 .oo 31.126.7 44.2f8.2 41.0f6.7 1 . k3.1 21 9.20k2.7

0.20*0.02 0.36k0.05 0.40&0.07 0.37+0.05 0.34k0.06 0.30i~0.02 7 0.44k0.09 125&28 98+9 0.075

120 I80 240 300

3.8k0.4 5.1i-0.3 4.8k0.4 4.7f0.5 3.5f0.2

0.8k0.07 2 120.2 . 1.7k0.1 1 -5k0.2 0.2k0.04

GLAFENINE

229

equimolar doses of glycerol did not induce any of glafenine characteristic effects. It was reported (17) that glafenine occurs in human urine mainly as correspondingfree acid N - (7 - chloro - 4 quinolyl) anthranilic acid; the process o hydrotysis (enzymatic or not) being still unknown. Glafenine does not seem f to be metabolized into simpler molecules such as 4 - amino - chloroquinoline and anthranilic acid. The structure of glafenine and its metabolites are shown in metabolicpathway establishedin the rat scheme (VI). The excretion patterns in rat and human urine are very similar indicating that the metabolic pathway should be similar in the two species (17).

Scheme (vl)

The Metabolism Pathways of Glafeninein Rat.

230

ADNAN A. BADWAN, MUHAMMAD B. ZUGHUL, AND MAHMOUD AL OMARI

6.5. Excretion

In normal subjects 70% of the product is eliminated through the biliary tracts. The remaining 30% are eliminated in the urine. The urine elimination is early, the maximum is between 2 - 4 hours and it is also rapid as about 70% of the quantity eliminated in the urine is achieved in the first 6 hours. It is eliminated almost totally in the form of metabolites and the major metabolite is free glafenic acid. Glafenineis rapidly eliminated from the body even in cases of high doses of renal insufficiency,plasma half - life of glafenine though longer than in normal subjects stays sufficiently short. After the absorption of a 400 mg dose, the serum level after 24 hours is zero or extremely low (16).
6.6. Half Life

The distribution half - life was reported as 75 minutes (15) and the elimination half life was 3.03 hours (18).

GLAFENINE

23 1

REFERENCES
1 . Gilbert Mouzin, Henri Cousse and Jean Marie Autin; Synthesis, 1;54 55 (1980).

2 . Netherlands Appl. Patent 296,793 (CI. C07d), Roussel- Uclaf (1 965), C. A., 64,3504 (1966). e 3 . French Pharmacopeia. Glafenine Monograph. 4 . A.A. Badwan and M.M. AL - Omari; Unpublished Data. The Jordanian
PharmaceuticalManufacturingCompany, Jordan.
5

. Pamela Girgis Takla and Christos J. Dakas; Int. J. of Pharm., 43,225- 232
(1988).

6 . Nadia Ghazal; Unpublished Data, The Jordanian Pharmaceutical


ManufacturingCompany, Jordan.

7 . W. Baeyens and P. De Moerloose; J. of pharm. Sci., 66 ( 2),1771 - 1773 1 (1 977). 8 . M.M. Omari (1987); M.S. Thesis. Universityof Jordan, Jordan. 9 . Mostafa S.Tawakkol and Mohamed E. Mohamedi Analytical Letters14 (BlO), - 770 (1981). 763 10. Mostafa S.Tawakkol, Mohamed E. Mohamed and Mahmoud A. Ibrahim; Pharmazie, 36 (H.2), 163 (1981).
11. S. A. Ismaiel, Abdel - Moety, E.M.; Zentralbl. Pharm., Pharmakother 57 Laboratoriumsdiagn,127 (2), - 59 (1988).

12. Marie Christine Tournet, Catherine Girre and Pierre Etienne Fournier;J. of Chromatography, 224,348- 352 (1 981). 13. Ennachachibi, A,, Nicolas P., Fauvelle F., Perret G., Petitjean 0;J. 1 (J. 307 Chromatogr. Biomed. Appl., 3 June, 7 (2), Chromatog, (427), 314(1988). 14. A.A.Badwan; Stability Data on Glafenine, Unpublished Data, The
Jordanian PharmaceuticalManufacturing Company, Jordan.

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ADNAN A. BADWAN, MUHAMMAD B. ZUGHUL, AND MAHMOUD AL OMARI

15. F. Moolenaar, J. Visser and T. Huizinga; Int. J. Pharm. 4,195 - 203 (1980). 16. Pharmacology File on Glafenine - JPM. 17. J. Pottier, M. Busigny and J.P. Raynaudi Eur. J. Drug Metab. 4 (2) 109 115 (1979). 18. M. C. Tournet, S. Giudicelli, C. Girre, J. Crouzetle and P. E. Fournier; C. R. - Congr. Biopharm. Pharmaco Kinet. lst, 2,288 - 301, (1981). Editedby J. M. Aiache and J. M. Hirtz.

LISINOPRIL

Dominic P. Ip, Joseph D. DeMarco and Marvin A . Brooks

Merck Sharp & Dohme Research Laboratories West Point, PA 19486

ANALYTICAL PROFILES OF DRUG SUBSTANCES AND EXCIPIENTS- VOLUME 21

233

Copyright Q 1992 by Academic Press, Inc. All rights of reproduction W e N e d in any form.

234

DOMINIC P. IP, JOSEPH D. DEMARCO, AND MARVIN A. BROOKS

LlSlNOPRlL Dominic P. Ip, Joseph D. DeMarco and Marvin A. Brooks


1. History and Therapeutic Properties 2. Description 2.1

2.2 2.3

Nomenclature 2.1.1 Chemical Name 2.1.2 Generic Name 2.1.3 Laboratory Codes 2.1.4 Trade Names 2.1.5 CAS Registry Number Formula and Molecular Weight Appearance, Color, Odor

3. Synthesis

4. Physical Properties 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 4.10 4.1 1 4.12

Infrared Spectrum 'H - Nuclear Magnetic Resonance Spectrum I3C - Nuclear Magnetic Resonance Spectrum Ultraviolet Spectrum Mass Spectrum Specific Rotation Thermal Behavior Solubility Dissociation Constants Crystal Properties Hygroscopicity Partition Coefficient

LISINOPRIL

235

5 . Methods of Analysis
5.1 5.2 Elemental Analysis Chromatographic 5.2.1 Thin-Layer Chromatography 5.2.2 High Performance Liquid Chromatography 5.2.2.1 Bulk Drug Analysis 5.2.2.2 Lisinopril in Formulation Titration Other Methods Identification Tests

5.3 5.4 5.5

6. Stability 6.1 6.2 Solid State Stability Solution Stability

7. Determination in Body Fluids and Tissues 7.1 7.2 7.3 Radioimmunoassay Competitive Inhibitor Binding Assay Fluoroentymatic Assay

8. Drug Metabolic Products, Pharrnacokinetics and Bioavailability 9. References

236

DOMINIC P. IP, JOSEPH D. DEMARCO, AND MARVIN A. BROOKS

1. History and TheraDeutic ProDerties

Lisinopril, a lysine analogue of enalaprilat, is a long-acting angiotensin converting enzyme inhibitor which differs from captopril by lacking the sulfhydryl group. Lisinopril, discovered and developed by the Merck Sharp & Dohme Research Laboratories (l), is indicated for the treatment of hypertension and congestive heart failure. Several review articles give a detailed account of the history, design, chemistry and pharmacology of the drug (2-6).
2. Description

2.1

Nomenclature
2.1.1

Chemical Name (a) L-Proline, 1-[N2-(1-carboxy-3-phenylpropyl)-Llysyl -dihydrate, (S)(b) 1-[N -[(S)-1-carboxyl-3-phenylpropyl]-L-lysyl]-Lproline dihydrate

2.1.2

Generic Name Lisinopril

2.1.3

Laboratory Codes
L-l54,826-000T, MK-0521

2.1.4

Trade Names Prinivil, Zestril, Carace, Novatec

2.1.5

CAS Reqistry Number


93015-83-7

2.2

Formula and Molecular Weight


'21 H31 N35
' 2H20

Molecular Weight 441.52

LISINOPRIL

231

H CH2CH2

. - C- - N- - C.

T I T I I

O
2H20

-C-N H ?OOH

COOH (CH2)4 A NH2 AI

2.3

Appearance, Color, Odor Lisinopril is a white to off-white crystalline, odorless powder.

3. Synthesis

Lisinopril has been prepared by the scheme outlined in Figure 1 (7,8). The dipeptide, N,-trifluoroacetyl-L-lysyl-L-proline c1) is subjected to reductive alkylation with ethyl 2-oxo-4-phenylbutanoate 2 over Raney Nickel via a Schiff base 3 to yield a diastereomeric ( J (J mixture 4 (SSS and RSS). Hydrolysis of the N,-trifluoroacetyl moiety and saponification of the ethyl ester followed by crystallization in ethanol/water and final recrystallization in water yield lisinopril (SSS, 5 of greater than 98% purity in about 65% yield (based on 3. In J addition to this synthetic route, others have also been described in the literature (9-12). 4. Physical Properties 4.1 Infrared Spectrum (13) The infrared spectrum of lisinopril as shown in Figure 2 was obtained in a potassium bromide pellet using a Perkin-Elmer Model 281-B spectrophotometer. Assignments for the characteristic bands in the spectrum are listed in Table 1. 4.2

H-Nuclear Maanetic Resonance Spectrum (14)

The proton magnetic resonance spectrum of lisinopril is shown in Figure 3. The spectrum was obtained using a Bruker Instruments Model WM250 spectrometer and a 10% W N solution of lisinopril in , solution of deuterium chloride in l J deuterium oxide. The reference compound (internal) was p-

1
R=CF 3CONHCH 2(CH 2)3

N m W

Ph

*2H20

(1) OH-

(2) Crystallization

E~O,C

CO,H

- Lisinopril 5,
R'=H 2NCH ,(CH
2)3

Figure 1 Synthesis of Lisinopril

Wsvelenglh (rnlcrons)

239
N w 10

Frequency (CM.'I

Figure 2. Infrared Absorption Spectrum of Lisinopril

240

DOMINIC P. IP. JOSEPH D. DEMARCO, AND MARVIN A. BROOKS

dioxane. An expansion of the spectrum in the 0-6 ppm region is shown in Figure 4. Chemical shifts and assignments for the numbered structure shown below are tabulated in Table II.

COOH
1

Table I Lisinopril Infrared Band Assianmentsa Wavenumber (cm-')


3545

Assiqnment OH stretching vibrationb (dihydrate H 0) 06 stretching vibration C-H stretching region stretching region Asymmetric -C02: stretch Asymmetric -C02 stretch 'NH or NH, bending ' CH2%ending Symmetric -C02- stretch Not assigned Phenyl out-of-plane bending (2 bands plus -(CH2)4-r~~k)

Near 3370 813290 (broad) 3090 - 2860 -2800 - -2100 1655 1609 1570 1541 1450, -1 443 (strong) 1388 1340, 1299 741, 732,692
a

These bands are subject to a reading error range of 25 cm-' above 2000 cm-' and +3 cm-' below. These bands disappeared in a dehydration experiment monitored by infrared spectrometry.

Figure 3.

The Proton Magnetic Resonance Spectrum of Lisinopril

Figure 4.

The Proton Magnetic Resonance Expanded Spectrum of Lisinopril

LISINOPRIL

243

Table II Lisinopril, Proton Maqnetic Resonance Assiqnments Chemical Shift, SH (ppml 7.35 5.06 4.44 4.35 3.84 3.75 3.62 3.04 2.85 2.33 2.03 1.73 1.60 a b Assiqnmenta Phenyl ring protons HCI/HD0 C protonb , C protonb ,, C proton : , p-dioxane (reference) C protons , C6, protons C protons , C protons; C proton (one of , , the two)' C3, protons; C protons; C , proton (other of the twoyc C5, protons C protons ,,

Assignments refer to number structure above. As in the case of aqueous solutions of captopril (15) and enalapril (16), signals attributable to rotamers (rotation about the lysyl-proline tertiary amide bond) are observed in the spectrum of lisinopril. The triplets at 4.62 ppm and 4.13 ppm represent, respectively, the C and C2, protons in the minor , rotamer. Private communication with Dr. B. J. Woodhall, Pharmaceutical Division, Imperial Chemical Industry. I3C Nuclear Maqnetic Resonance Spectrum (12) The carbon-13 magnetic resonance spectrum of lisinopril shown in Figure 5 was obtained using a Varian Associates Model XL-1O A spectrometer and a 10% (WN) solution of O lisinopril in 1N deuterium chloride in deuterium oxide. The reference compound (internal) was p-dioxane. An expansion of the spectrum in the 12.5-75 ppm region is shown in Figure 6. Chemical shifts and assignments for the numbered structure shown below are tabulated in Table Ill.

4.3

244

Figure 5. The Carbon-13 Magnetic Resonance Spectrum of Lisinopril

3 3 N

rl)

N VI P

75

62.5

50

37.5
PPM

25

12.5

Figure 6.

The Carbon-13 Magnetic Resonance Expanded Spectrum of Lisinopril

246

DOMINIC P. IP, JOSEPH D. DEMARCO. AND MARVIN A. BROOKS

NH

COOH
1

Table Ill Lisinomil, Carbon-13 Maanetic Resonance Assianments Chemical Shift, 6 (ppmla , 21.57 (22.04) " 25.39 (22.51) 27.20 29.49 (31.38) 30.02 (30.58) 31.21 31.92 (32.24) 39.89 48.70 (48.1 6) 59.73 (60.29) 60.1 1 60.53 (59.64) 67.40 127.58 129.510~ 129.6Eid 140.53 (140.44) 167.32 (1 67.80) 171.44 (1715 5 ) 175.71 (175.14) Assianmentb-

:: ; :: ;
C , C .,
'6' c 5 c1 2

c3 4

C .,

p-dioxane (reference)

c2

crn
CIS

cP

: ;
c,
I.

Cl

Values in parentheses are due to a minor conformational isomer, and many of the assignments for this component are tentative. Assignments refer to numbered structure above. Private communication with Dr. 8 . J. Woodhall, Pharmaceutical Division, Imperial Chemical Industry. These assignments could be reversed.

LISINOPRIL

241

4.4

Ultraviolet Spectrum The ultraviolet absorbance spectra of lisinopril shown in Figure 7 (17) were obtained using a Perkin-Elmer Lambda 5 UV-VIS scanning spectrophotometer. The spectrum in 0.1N sodium hydroxide solution is characterized by low intensity maxima at -246 nm, -254 nm, -258 nm, -261 nm and -267 nm with respective A l % 1 cm values of -4.0, -4.5, -5.1, -5.1 and -3.7. The spectrum in 0.1N hydrochloric acid is characterized by maxima at -246 nm, -253 nm, -258 rim, -264 nm and -267 nm with respective A l % 1 cm values at -3.2, -3.9, -4.5, -3.0 and -2.8. The ultraviolet absorbance arises from the unconjugated phenyl ring in lisinopril molecule.

4.5

Mass Spectrum (18) The mass spectrum of lisinopril shown in Figure 8 was obtained by direct probe-electron impact (70 eV) method using an LKB Model 9000 mass spectrophometer. The spectrum shows no molecular ion peak. A pseudo-molecular ion peak at m/e 387.2160 is attributed to the diketopiperazine formed during vaporization. Mass fragment assignments are given in Table IV.

4.6

Specific Rotation (19) Lisinopril contains three chiral centers and is The specific rotation values [a]25 and [a] 405 nm 436 nrn 0.25M pH 6.4 zinc acetate) are respectively --120" and --96".

4.7

Thermal Behavior (20) Differential thermal analysis under vacuum (DTA heating rate = 2OoC/rnin.) as shown in Figure 9 indicated three endotherms with peak temperatures at -98"C, -1 22C and -1 82C. The first two endotherms correspond to loss of water of hydration and the one at 182C to melting.

0.80

0.64

e
2 !

0.48

0.32

0.16

220

240

260

280
Wavelength (nrn)

300

320

340

Figure 7.

The Ultraviolet Absorption Spectrum of Lisinopril in (a) 0.1NSodium Hydroxide; Concentration: 1.374 mg/ml and (b) 0.1NHydrochioric Acid; Concentration: 1.374 mg/ml

Figure 8. The Direct Probe-Electron Impact (70 eV) Mass Spectrum of Lisinopril

250

DOMINIC P. IP, JOSEPH D. DEMARCO, AND MARVIN A. BROOKS

Table IV Lisinopril, Mass Spectrum Assianments

M/e
387
C H2,N304 ,,

Assiqnment

(M' minus H ) , O

369 358 342/343 329 315/316 313 296 283 265 252 245

m/e 387 minus H O , m/e 387 minus CH3N m/e 387 minus CO,(H) m/e 387 minus C3H,N m/e 387 minus CH ,0 , 9N m/e 358 minus C0,H m/e 387 minus benzyl radical

1.
+

C,3H,1

N304 (m/e 387 minus styrene)

m/e 283 minus H20 m/e 296 minus CO,


CH&H?-N=CH(CH,),NH,

c=o
B

LISINOPRIL

25 I

Table IV (Continued) Lisinopril, Mass Spectrum Assiqnrnents Mle 224


1 1Hl ' a 2 N3 '
CH&HzCHzC&NH, CHCH$&CHzNH2

Assiqnment

d e 224

rnle 224

.,,dly"

CYCH,CH,CH,NH,

tj

we224

207

C, HO N, ,,

(rn/e 224 minus N , H )

179
H

Jaco-g
' e
H

84

QHH

252

DOMINIC P. IP, JOSEPH D. DEMARCO, AND MARVIN A. BROOKS

Table IV (Continued) Lisinopril, Mass SDectrum Assianments

M/e
70

Assianment

W"
n

4.7

Thermal Behavior (continued) The thermogravimetric analysis (TGA) curve of lisinopril shown in Figure 10 depicts three inflections corresponding to the loss of free water and the first and second moles of water of hydration. The excess unbound water was 0.6% over the theory of 8.2% for the dihydrate indicating lisinopril is somewhat hygroscopic.

4.8

Solubility (20) The following approximate solubility data were obtained at ambient temperature. Table V Solvent Water Methanol Ethanol Acetone Acetonitrile Chloroform N,N-Dimethylformamide Solubility (mq/ml)

97 14a <0.1 <o. 1 <0.1 <o. 1 <0.1

a Upon dissolution of lisinopril in methanol, changes in Xray diffraction patterns indicative of loss of water of hydration were observed. The solubility value obtained becomes dependent upon the water content of the solution.

~I

I I

I ~

04

00
c

-0.4 -0.8

g
c .

e 0

-1.2

0.

-16

20

-2 4
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 l 1 1 l l ~

Figure 9. DifferentialThermal Analysis (DTA) Curve of Usinopril

Temperelure ('C)

Figure 10. Thermogravimetric Analysis Curve of Llsinopril

LISINOPRIL

255

4.9

Dissociation Constants (20) Aqueous acidichasic potentiometric titration at 25C yielded four pKa values of 2.5, 4.0, 6.7 and 10.1 for lisinopril.

4.1 0 Crvstal ProDerties (20) Lisinopril is crystalline as determined by X-ray powder diffraction. A typical X-ray powder diffraction pattern obtained using a Philips APD 3720 X-ray powder diffractometer is shown in Figure 11. A monohydrate which is crystalline, also exists. The monohydrate is readily distinguishable from the dihydrate by its X-ray powder diffraction pattern (Figure 12). 4.1 1 Hvcrroscopicity (21) Lisinopril is slightly hygroscopic. A sample stored for three months at 98% relative humidity and room temperature showed an increase of 1.1Yo in the total volatiles content by TGA. 4.1 2 Partition Coefficient (17) The partition coefficient of lisinopril in the phosphate buffer (O.lM, pH 7)h-octanol system was determined to be 10.2 0.5 at room temperature.

5. Methods of Analvsis
5.1 Elemental Analysis (22) The elemental analysis found for a reference lot of lisinopril L154,826-00T031 was /Elemental) Analvsis
'21 H31 N35
' 2H20

% Theow*

% Found*

Carbon Hydrogen Nitrogen


*Anhydrous basis

57.13 7.99 9.52

56.89 7.69 9.49

256

Figure 11. Powder X-Ray Diffraction Pattern of Lisinopril

LU

Figure 12. Powder X-Ray Diffraction Pattern of Lisinopril Monohydrate

2%

DOMINIC P. IP, JOSEPH D. DEMARCO. AND MARVIN A. BROOKS

5.2

Chromatoqraphic Chromatographic procedures have been developed to separate lisinopril from its principal decomposition product, the diketopiperazine (DKP), a product of intramolecular dehydration. Since there are three optical centers in the molecule, isomers of lisinopril and its DKP degradate are possible. The sequence of thermal decomposition for lisinopril to form isomers of DKP is shown in Figure 13. The conversion of SSS DKP to SSR DKP has been demonstrated (23) by heating SSS DKP for 15 minutes at 160C. Chromatographic procedures also separate in some cases a process impurity, 4-phenyl-2-aminobutyric acid (APBA) and the RSS isomer of lisinopril (Figure 14).
5.2.1

Thin Laver Chromatoqraphy (TLCl(19) Four TLC systems have been developed and are listed below. Systems (I) and (11) separate lisinopril from its RSS isomer, and APBA. Systems (Ill) and (IV) are of somewhat limited value since they do not separate lisinopril from the RSS isomer. Systems (I) and (11) utilize E. Merck Silica Gel G-60 while systems (Ill) and (IV) use Analtech Silica Gel G-60. Visualization of spots is accomplished by reaction with ninhydrin.

Lisinopril (SSS)

Lisinopril SSS DKP

Lisinopril SSR DKP

Figure 13. Thermal Decomposition of Lisinopril to Its Diketopiperazine Isomers

H H O V H V I I HOOC- -C--N--C--C-N

CH2

(CH2)4 I

NH2

RSS Isomer

CHzCHpCHNH2

Ao2H

2-amino-4-phenyl butyric acid (AP BA)

Figure 14

LISlNOPRlL

26 I

Table VI TLC Systems Solvent Systems


(1)
Rf (Approximate)

Lisinopril

n- Butanol/tol uene/glacial acetic acid/water/acetone (1:l:l:l:l) n-Butanol/water/glacial acetic acid (3:l:l) n-Butanol/water/glacial acetic acid/ethyl acetate (1:l:l:l)

0.34

(11) (111)

0.22
0.43

(1V)

Chloroforrn/rnethanol/conc. ammonium hydroxide (4:4:1)

0.14 to 0.39

262

DOMINIC P. IP. JOSEPH D. DEMARCO. AND MARVIN A . BROOKS

5.2.2

Hiqh Performance Liquid Chromatoqraphv (HPLC) 5.2.2.1 Bulk Druq Analysis (24) Methods of analysis for lisinopril in bulk drug are summarized in Table VII. Method (I) which utilizes a linear gradient is capable of separating lisinopril from two potential process impurities, the RSS isomer of lisinopril and the 2-amino-4-phenylbutyric acid (APBA), and the diketopiperazine SSS and SSR isomer degradation products (see Figures 13 and 14). Under isocratic conditions, method (I) also separates lisinopril from its RSS isomer and APBA. Both gradient and isocratic procedures use a Zorbax (DuPont) RP-8 column at a pH of 5.0 and a column temperature of 50C. The isocratic procedure has been published in the USP (25). Lisinopril exhibits typical chromatographic behavior (peak broadening) attributed to rotational isomers (26) of proline-containing dipeptides. Higher column temperatures of 950C appears to be necessary to minimize this effect for acceptable chromatography. Method (11) which utilizes a PRP-1 column (Hamilton Co.) has also been used for the evaluation of lisinopril bulk drug. This method is, however, more cumbersome to use than method (I) which was found to offer better resolution for compounds of interest. Detection for all methods is by UV at 210 to 215 nm. 5.2.2.2 Lisinopril in Formulation (27) Methods for the analysis of lisinopril in dosage forms are summarized in Table VIII. Methods (I), (11) and (111) have been developed to separate lisinopril from its principle degradation product, the SSS diketopiperazine (DKP) and a process impurity APBA (see Section 5.2.1). Method

LlSlNOPRlL

263

(IV) was developed to include the separation of hydrochlorothiazide as well. These methods (I-IV) all utilize a Lichrosorb RP-8 column, 10 pm at 40C or 50C. Method (IV) uses a somewhat longer column (300 x 4.6 mm) and it was found that as a result of the increased length, column temperature could be reduced to 40C while maintaining good peak efficiency (see Section 5.2.2.1). This method is also capable of separating lisinopril diketopiperazine SSS from the SSR isomer as well.

The retention times of both lisinopril and SSS DKP have been found to be a function of the phosphate concentration in methanol (see Figures 15 and 16). Similar data have been obtained in acetonitrile. The retention of lisinopril and SSS DKP also decreased as the % organic modifier increased. Thus by varying both the molarity of phosphates and % organic modifier in the mobile phase, the resolution of these compounds can be optimized. Methods (V) and (Vl) were developed to quantitate lisinopril for content uniformity and dissolution. Detection in all of these methods as in the methods designed for the bulk drug is by UV at 210 nrn to 215 nm.
A stability-indicating HPLC method for lisinopril tablets has been published in the USP (28). This compendia1 method employs the same mobile phase composition as method (111) but contains an ion-pair reagent.

264

DOMINIC P.IP.JOSEPH D. DEMARCO. AND MARVIN A. BROOKS


Table VII

Merhcd
(1)

HPCC Systems for Lisinqxil Bulk DruQ Column


250 x 4.6 mm, 5 p n T = 50%

Chromatographic Conditions A:Acetonitrile B:0.02M NaH2P04 pH 5.0 Gradient: 0% A Isocratic: to 30% A linear over 35 minutes 96% Solvent B
4% Solvent A

Separation a.b,c,d,e

Zorbax (DuPont) RP-8

a,b,e

250 x 4.6 mm. 10 km, T = 50C

PRP-1 (Hamilton Co.)

A:O.O2M NaH2P04 at pH 6.8 B:O.O15M NaH2P04at pH 3.0 C:Acetonitrile

Isocratic: -

96% Solvent A 4% Solvent C

Gradient (11 Solvent NSolvent C ( 9 7 . 5 2 5 ) for 10 minutes, then linear gradient to Solvent NSolvent C (70:30) in 30 minutes Gradient [ZJ Solvent &Solvent C (95:s) for 10 minules then linear gradient to Solvent BlSolvent C (70:30) in 30 minutes.

Legend
a = Lisinopril b = RSS Isomer C = SSS DKP d = SSR DKP = APBA

LISINOPRIL
Table Vlll HPLC Systems for Lisinopril in Solid Dosage Formulations Purpose Stability Single entity Column Lichrosorb RP-8 (Hewlen Packard) 200 x 4.6 mm, 10 pm T = 50% Lichrosorb RP8 (Hewlen Packard) 200 x 4.6 mm. 10 pn T = 50C Lichrosorb RP-8 (Hewlea Packard) 200 x 4.6 mm. 10 pm T = 50C Lichrosorb RP-8 (E.S. Industries) 300 x 4.6 mm. 10pm T = 40% Hypersil ODs (Shandon) 50 x 4.6 mm, 5 pn T = 60C Lichrosorb RP-8 Hewlen Packard 200 x 4.6 mm, 10 pm T = 50C ChromatographicConditions Mobile Phase (Conditions) Acetonimle/0.004Mphosphate, pH 2.0 45:55

265

Separation a,c,e

Stability Single entity

MethanoVO.WMphosphate, pH 2.0 45:55

a.c.e

Stability Single entity

Acetonitrile/O.O3M phosphate, pH 2.0

a.c,e

2o:ao

Stability HCTZ Combination

AcetonitriIe/O.O05Mphosphate, pH 2.0 35:65

a.c,d,e.f

Content Uniformity Dissolution Single entity

MethanoVO.02M phosphate, pH 2.0 12:88

Content Uniformity Dissolution HCTZ Combination

Acetonitrile10.04M phosphate, pH 2.0 15:85

Legend
a = Lisinopril b = RSS Isomer C = SSS DKP d SSR DKP E = APf3A f = Hydrochlorothiazide(HCTZ)

Phosphate Molarity
Figure 15. Effect of Phosphate Concentration on Retention of Lisinopril

Figure 16. Effect of Phosphate Concentration on Retention of SSS DKP

268

DOMINIC P. IP, JOSEPH D. DEMARCO, AND MARVIN A. BROOKS

5.3

Titration (17) Lisinopril can be determined by potentiometric titration with aqueous sodium hydroxide and non-aqueous perchloric acid. The sodium hydroxide titration is carried out by titrating the lisinopril potentiometrically with carbonate free 0.1 N NaOH to one endpoint using a combination electrode. Lisinopril can also be determined by titration potentiometrically with 0.1 N perchloric acid in acetic acid to one endpoint. The electrode system consists of a glass electrode (such as a Metrohm Model EA 107 vs. a silver/silver chloride reference electrode . such as a Metrohm Model EA 432 filled with 0 1N lithium perchlorate in glacial acetic acid.

5.4

Other Methods Lisinopril has also been determined by radioimmunoassay (RIA), fluoroenzymatic assay (FEA) and competitive inhibitor binding assay (CIBA). These procedures are described in Section 7.

5.5

Identification Tests Identification of lisinopril can be carried out by infrared absorption (see Section 4.1), TLC (see Section 5.2.1) and by HPLC (see Section 5.2.2). Supportive evidence for identification can be obtained by differential thermal analysis (DTA) (see Section 4.7).

6. Stability
6.1

Solid State - Thermal (29) Lisinopril is stable as a solid at ambient temperatures (2025C). Degradation can be induced when the solid is stressed at the severe thermal stress conditions of 105C. HPLC studies have demonstrated that intramolecular dehydration to DKP is the primary degradate. The yield to the diketopiperazine as % of the total chemical loss of lisinopril is higher in nitrogen flushed closed container (80%) than in nonflushed closed container (50-60%) or in open container (6%). Lisinopril, when stressed under these severe conditions, yields

LISINOPRIL

269

additional degradates, the identity of which has not been determined. Studies have demonstrated that the lisinopril diketopiperazine isomer initially formed is the SSS isomer which can degrade further to the SSR isomer (see Section 5.2).

6.2 Solid State - Photochemical (30)


Very slight surface discolorations have been observed when lisinopril is exposed to intense UV radiation for 24 hours.
6.3

Solution Stability (29) The solution stability of lisinopril at a concentration of 0.2 mg/ml was studied at pHs ranging from 2.7 to 10.0 and at a constant ionic strength (p = 0.3). Figure 17 depicts a plot of the zero order rate constants from 60" and 80C data vs. pHs. Lisinopril decomposition proceeds rapidly in acidic media with the major decomposition product being the diketopiperazine. The rate of formation of the diketopiperazine was found to be linear with time. In neutral and basic >pH 7.0, the decomposition rate is minimal.

Figure 17. Plot of Rate Constants at 60" and 80C as a Function of pH

LISlNOPRlL

27 I

7. Identification and Determination in Body Fluids and Tissues The quantitation of lisinopril in biological fluids has been reported by standard radioimmunoassay (RIA), competitive inhibitor binding assay (CIBA) and fluoroenzymatic assay (FEA). 7.1 Radioimmunoassay Radioimmunoassay procedures for the drug in plasma and urine have been developed utilizing a polyclonal antiserum produced to a conjugate in which lisinopril is linked to albumin via a dinitrophenylene bridge (2,31) or succinoylated keyhole limpit haemocyanin (32). In the former procedure, the radiolabel was introduced via radio-iodination of a phydroxybenzamidine derived from lisinopril, whereas in the latter procedure, the radiotracer was prepared by acylation of the epsilon amino group of the lysyl side chain of lisinopril with N-~uccinimidyl-(2,3-~H)-proprionate. limit of sensitivity for The the RIA procedures is approximately 0.2-0.4 ng/ml (0.5-1nM). 7.2 Competitive Inhibitor Bindinq Assay Binding assays were first described as a technique for the measurement of angiotensin converting enzyme inhibitor (ACE inhibitor) activity (33). This procedure requires incubation (37C for 2 hours) of serum samples with 1251-labeledACE inhibitor (p-hydroxybenzamidinederivative of lisinopril) followed by a charcoal precipitation and gamma counting of the precipitate. The ACE value is then determined from a standard curve of inhibitor binding vs. ACE activity determined by an enzymatic kinetic assay (34). The principle of this assay was then extended to a competitive inhibitor binding assay in which the 1251-labeled inhibitor (see above) is displaced from isolated ACE by lisinopril in the biological sample (35-36). The free label is separated by adsorption onto charcoal and related to lisinopril drug concentration. The sensitivity of the assay is reported to be 2-4 ng/ml (35) and correlates well with specific radioimmuno-assays and ACE enzymatic activity (37). 7.3 Fluoroenzymatic Assay The determination of ACE enzymatic activity via the measurement of the rate of cleavage of the substrates hippuryl-L-histidyl-L-leucineor hippuryl-L-glycyl-L-glycine to

212

DOMINIC P. IP. JOSEPH D. DEMARCO. AND MARVIN A. BROOKS

hippuric acid with quantitation by fluorometric, radioassay, liquid chromatographic, radioimmunoassay and enzyme linked immunoassay methods has been reviewed (33). Recent assay procedures have incorporated ACE inhibitors into these enzymatic assays with radioassay (38) or fluorometric (39) measurement. The percent of ACE activity inhibited is correlated with drug (inhibitor) concentration. These procedures (38-39) require extraction of the drug from plasma or urine with methanol to separate the drug (inhibitor) from endogeneous ACE. The methanolic supernatant is evaporated, and then reconstituted in a solution of exogenous ACE containing the substrate. The fluorometric assay (39) utilizes the substrate N-benzoyloxycarbonyl-L-phenylalanyCLhistidyl-L-leucine which has been previously utilized to measure enalaprilat in biological fluids (40). The enzymatic reaction is quenched with ice, o-phthaldialdehyde added, and spectrofluorometric measurement of the derivative is performed at excitation/emission wavelengths of 365490 nm. A logit-log relationship between drug (inhibitor) concentration and percent enzyme inhibitor is used to calculate drug (inhibitor) concentration. The assay has demonstrated a sensitivity limit of 0.7 ng/ml with an RSD of 3-10% over the standard concentration range of 0.4-35 ng/ml for enalaprilat. The assay clearly has the advantage over RIA and ClBA as described of not requiring radiolabel technique to perform the assay. However, utilizing the lengthy sample preparation, assay capacity is limited.
8. Druq Metabolic Products, Pharmacokinetics and Bioavailability

Utilizing the RIA procedure for serum and urine and in vitro isotope dilution procedure for feces, the absorption and elimination profile of lisinopril was determined in 12 healthy male volunteers following oral administration of a 10 mg capsule (2). The observed peak serum concentration was 95 +. 55 nM with a time to peak of 7 +. 1 hours and an AUC (0-72 hours) of 1694 +. 808 nmol liter- hr. The serum concentration vs. time profile was polyphasic and the terminal half-life was approximately 30 hours. The renal clearance was 106 2 13 ml/min wit4 urinary and fecal recovery of 29% 2 15% and 69% 23%, respectively, indicating the drug was excreted unchanged. The half-life for the terminal phase (approximately 40 hours) was not predictive of steady state parameters when 10 daily doses of lisinopril were administered orally to healthy subjects. The mean effective

LISINOPRIL

213

half-life of accumulation was 12.6 hours. The mean accumulation ratio was 1.38 with steady state attained after the 2nd dose (41). The drug is not metabolized but is eliminated via the kidneys. Lisinopril probably undergoes glomerula filtration, tubular secretion and tubular reabsorption (42). The close correlation between serum concentration of drug and the degree of inhibition of ACE has been demonstrated. Furthermore, there was a close inverse relationship between plasma levels and the ratio of angiotensin II:i, the latter parameter being a measure of the conversion of angiotensin I to II (43). Age and cardiac failure are reported to be associated with reduced renal clearance of lisinoprii (44). The plasma concentration and drug half-life in patients with chronic renal failure (creatinine clearance 5 30 ml/min) are generally higher than those seen in patients with normally functioning kidneys (45). Food intake had no effect on the pharmacokinetics of lisinopril (42,46). Acknowledcrements The authors wish to thank Mrs. Laurie Rittle for typing the manuscript and Mrs. Florence Berg for conducting the literature search.

9. References
1. A.A. Patchett, E. Harris, E.W. Tristram, M.J. Wyvratt, M.T. Wu, D. Taub, E.R. Peterson, T.J. Ikeler, J. tenBroeke, L.G. Payne, D.L. Ondeyka, E.D. Thorsett, W.J. Greenlee, N.S. Lohr, R.D. Hoffsommer, H. Joshua, W.V. Ruyle, J.W. Rothrock, S.D. Aster, A.L. Maycock, F.M. Robinson, R. Hirschmann, C.S. Sweet, E.H. Ulm, D.M. Gross, T.C. Vassal and C.A. Stone, Nature 288, 280 (1980).

2. E.H. Ulm, M. Hichens, H.J. Gomez, A.E. Till, E. Hand, T.C. Vassil, J. Biollaz, H.R. Brunner and J.L. Schelling, Br. J. Clin. Pharmacol. 14, 357 (1982).
3. A.A. Patchett in "Hypertension and the Angiotensin System: Therapeutic Approaches", A.E. Doyle and A.G. Bearn, Editors, Raven Press, New York, NY 1984.

4. T.A. Noble and K.M. Murray, Clin. Pharm. 7, 659 (1988).

274

DOMINIC P. IP, JOSEPH D. DEMARCO. AND MARVIN A. BROOKS

5. C.S. Sweet and E.H. Ulm, Cardiovasc. Drua Rev. 6, 181 (1988).

6. H.J. Gomez, V.J. Cirillo and F. Moncloa, J. Cardiovas. Pharmacol. 9(SuDDI. 31, S27 (1987). 7. T.J. Blacklock, R.F. Shuman, J.W. Butcher, W.E. Shearin, Jr., J. Budavari and V.J. Grenda, J. Ora. Chem. 53, 836 (1988). 8. E.E. Harris, A.A. Patchett, E.W. Tristram and M.J. Wyvratt (Merck & Co., Inc.), U.S. Patent 4,374,829. 9. M.J. Wyvratt, E.W. Tristram, T.J. Ikeler, N. Lohr, H.Joshua, J.P. Springer, B. Arison and A.A. Patchett, 2816 (1984). 10. J.S. Kaltenbronn, D. DeJohn and U. Krolls, Ora. Prep. Proceed. Int. 15, 35 (1983). 11. H. Urback and R. Henning, Tetrahedron Lett. 25, 1143 (1984). 12. M.T. Wu, A.W. Douglas, D.L. Ondeyka, L.G. Payne, T.J. Ikeler, H. Joshua and A.A. Patchett, J. Pharm. Sci. 74, 352 (1985). 13. G. Bicker, Merck Sharp & Dohme Research Laboratories, Rahway, NJ. 14. A.W. Douglas, Merck Sharp & Dohrne Research Laboratories, Rahway, NJ. 15. D.L. Rabenstein and A.A. Isab, Anal. Chern. 54, 526 (1982). 16. D.P. Ip and G.S. Brenner, Analvtical Profiles of Druq Substance 1 ,207 (1987). 6

17. Merck Sharp & Dohme Research Laboratories, unpublished data.


18. G.A. Schonberg, Merck Sharp & Dohrne Research Laboratories, Rahway, NJ. 19. R.B. Waters, Merck Sharp & Dohrne Research Laboratories, Rahway, NJ. 20. J.A. McCauley, Merck Sharp & Dohme Research Laboratories, Rahway, NJ.

LISINOPRIL

215

21. R.J. Magliette, Merck Sharp & Dohme Research Laboratories, Rahway, NJ. 22. J. Perkins, Merck Sharp & Dohrne Research Laboratories, Rahway, NJ. 23. C. Bell, Merck Sharp & Dohme Research Laboratories, West Point, PA. 24. T. Novak, Merck Sharp & Dohme Research Laboratories, Rahway, NJ. 25. The United States Pharrnacopeia XXII, 2355 (1990). 26. W.R. Melander, J. Jacobsen and C. Horvath, J. Chrornatoqraphv - 269 (1982). 234, 27. J. DeMarco and P. Kusrna, Merck Sharp & Dohrne Research Laboratories, West Point, PA. 28. The United States Pharmacopeia XXII, 2475 (1991). 29. D.P. Ip, Merck Sharp & Dohrne Research Laboratories, West Point, PA. 30. J. DeMarco, Merck Sharp & Dohrne Research Laboratories, West Point, PA. 31. M. Hichens, E.L. Hand and W.S. Mulcahy, Liqand Quarterv 4, 43 (1981). 32. P.J. Worland and 6.Jarrott, J. Pharm. Sci 75, 512 (1986). 33. F. Fyhrquist, I. Tikkanen, C. Gronhagen-Riska, L. Hortling and M. Hichens, Clin. Chern. 30, 696 (1984). 34. J. Lieberrnan, Am. J. Med. 59, 365 (1975).
35. C. Gronhagen-Riska, I. Tikkanen and F. Fyhrquist, Clin. Chim. Acta 162, 53 (1987).

36. B. Jackson, R. Cubela, and C.I. Johnston, Biochem. Pharmacoloqy 36, 1357 (1987).

216

DOMINIC P. IP. JOSEPH D. DEMARCO, A N D MARVIN A . BROOKS

37. B. Jackson, R. Cubela and C.I. Johnston, J. Cardiovasc. Pharmacol. 9,699 (1987). 38. B.N. Swanson, K.L. Stauber, W.C. Alpaugh and S.H. Weinstein, Anal. Biochem. 148,401 (1985). 39. K. Sheplay, M.L. Rocci, H. Patrick and P. Mojaverian, J. Pharm. and Biomed. Awl. 6,241 (1988). 40. D.J. Tocco, F.A. deLuna, A.E.W. Duncan, T.C. Vassil and E.H. Ulm, Drua Met. Dispos. 10,15 (1982). 41. B. Beerman, A. Till, H.J. Gomez, M. Hichens, J.A. Bolognese, and I.L. Junggren, Biopharm. Druq Dispos. 10,397 (1989). 42. 8. Beerman, Am. J. Med. 85,25 (1988). 43.J. Biollaz, J.L. Schelling, J.L. descombes, D.B. Bruner, G.
Desponds, H.R. Brunner, E.H. Ulm and H.J. Gomez, Brit. J. Clin. Pharmacoloav 14,363 (1 982).

44.P.C. Gautam, E. Vargas and M. Lye, J. Pharm. Pharmacol. 39, 929 (1987).
45. J.G. Kelly, G.D. Doyle, M. Carmody, D.R. Glover and W.D. Cooper, Br. J. Pharm. Pharmacol. 25, 634p (1988). 46. P. Mojaverian, M.L. Rocci, P.H. Vlasses, C. Hoholick, R.A. Clementi and R.K. Ferguson, J. Pharm. Sci 75,395 (1986).

LOVASTATIN

Gerald S. Brenner, Dean K. Ellison, and Michael J . Kaufman

Merck Sharp & Dohme Research Laboratories West Point, PA 19486

ANALYTICAL PROFILES OF DRUG SUBSTANCES AND EXCIPIENTS - VOLUME 21

277

Copyright @ 1992 by Academic Press, Inc. All rights of reproduction reserved in any form.

278

GERALD S. BRENNER, DEAN K . ELLISON, AND MICHAEL J. KAUFMAN

LOVASTAT IN Gerald S. Brenner Dean K. Ellison Michael J. Kaufman

1. History and Therapeutic Properties


2. Description 2 1 Nomenclature . 2 1.l Chemical Name . 2.1.2 Generic Name (USAN) 2.1.3 Laboratory Codes 2.1.4 Trade Names 2.1.5 Trivial Names
2.1.6

2 2 Structure, Formula and Molecular Weight . 2.3 Appearance


3. Synthesis

Chemical Abstract Services (CAS)

4. Physical Properties 41 . 4.2 4.3 44 . 4.5 4.6 4.7 4.8


Infrared Spectrum Proton Nuclear Magnetic Resonance Spectrum Carbon-13 Nuclear Magnetic Resonance Spectrum Ultraviolet Spectrum Mass Spectrum Optical Rotation Thermal Behavior Solubility 4.9 Crystal Properties 410 Dissociation Constants . 4 11 Partition Behavior .

LOVASTATIN

219

5. Methods of Analysis

5.1 Elemental Analysis 5.2 Chromatography 5.2.1 Thin Layer Chromatography 5.2.2 High Performance Liquid Chromatography 5.3 Flow Injection Analysis 5.4 Identification Tests 6. Stability and Degradation 6.1 Solid State Stability 6.2 Solution Stability 7. Pharmacokinetics and Metabolism 7.1 Absorption and Distribution 7.2 Metabolism 7.3 Excretion
8. Determination in Biological Fluids

9. References

280

GERALD S . BRENNER, DEAN K. ELLISON, AND MICHAEL I. KAUFMAN

1. Historv and TheraDeutic ProDerties It was discovered by the Merck Sharp & Dohme Research Laboratories that a strain of Aspergillus terreus obtained from a soil sample produced the cholesterol lowering fungal metabolite lovastatin (initially named mevinolin). Details of the isolation, structural characterization and biochemical properties of lovastatin have been summarized by Alberts et al. (1). Lovastatin is identical to monacolin K isolated independently from Monascus ruber by Endo

(2).
Lovastatin is a prodrug. After oral administration, the inactive parent lactone is hydrolyzed to the corresponding hydroxyacid form. The hydroxyacid is the principle metabolite and a potent inhibitor of 3-

Lactone

Hydroxyacid

hydroxy-3-methylglutaryl-coenzyme A (HMG CoA) reductase. This enzyme catalyzes the conversion of hydroxymethylglutarate to mevalonate, which is an early and rate limiting step in the biosynthesis of cholesterol. The effectiveness of lovastatin in lowering cholesterol has been confirmed clinically and it is approved for the treatment of primary hypercholesterolemia. Several review articles give a detailed account of the discovery, preclinical evaluation, mechanism of action, biological profile, and clinical evaluation of the drug (3-7).

2. DescriDtion
2.1 Nomenclature 2.1.1 Chemical Name [l 1a R*),3a,7P,8P(2S*,4S*),8a~]]-2-Methylbutanoic S-[ ( acid 1,2,3,7,8,8a-hexahydro-3,7-dirnethyl-8-[2-

LOVASTATIN

28 I

(tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl)ethyl]-1naphthalenyl ester; (1S,3R,7S,8SI8aR)-1 ,2,3,7,8,8ahexahydro-3,7-dimethyl-8-[2-[(2R,4R)-tetrahydro-4hydroxy-6-0~0-2H-pyran-2-ylJethyl]1-naphthatenyl (S)2-methylbutyrate; 1,2,6,7,8,8a-hexahydro-P,6dihydroxy-2,6-dimethyl-8-(2-methyl-l -oxobutoxy)-1naphthaleneheptanoic acid Glactone; PP,Ga-dimethyl8a-(2-methyl-l -oxobutoxy)-mevinicacid lactone.

2.1.2

Generic Name (USAN1 Lovastatin

2.1.3

Laboratow Codes L-l54,803-000G MK-0803

2.1.4

Trade Names Mevacor; Mevinacor; Mevlor

2.1.5

Trivial Names Mevinolin Monacolin K 3-Methyl Compactin

2.1.6

Chemical Abstracts Services GAS1 Registry Number: 75330-75-5

2.2

Structure, Formula, and Molecular Weiaht Structure:

282

GERALD S . BREWER, DEAN K. ELLISON, AND MICHAEL I. KAUFMAN

Molecular Formula: C O H ,, Molecular Weiaht: 40&5

Lovastatin is a white, crystalline powder. 3. Synthesis There have been numerous approaches to the total synthesis of lovastatin (8-10); however, lovastatin is produced commercially via a multi-stage fermentation process which originates from cultures of a strain of Aspergilks ferreus. The complete details of the isolation and identification of lovastatin from the fermentation media have been described (1). Synthetic approaches have been reviewed (1 1).
4. Phvsical Properties 4.1

Infrared Spectrum The infrared spectrum of lovastatin is shown in Figure 1 (12). The spectrum was obtained as a potassium bromide pellet using a Nicolet Model 7199 FT-IR spectrophotometer. Assignments for the characteristic absorption bands are shown below.

Wavenumber (cm- 1 3542 3016 296 7 2929 2866 1725 1711 1700 1460 1384 1359 1260 1222 1072 1056 969 87 1

Assignment Alcohol 0-H stretch Olefinic C-H stretch Methyl C-H asymmetric stretch Methylene C-H asymmetric stretch Methyl and methylene C-H asymmetric stretch Lactone and ester carbonyl stretch (hydrogen bonded for 1711 and 1700 cm- ) Methyl asymmetric bend Methyl symmetric bend Methylene symmetric bend Lactone C-0-C asymmetric bend Ester C-0-C asymmetric bend Lactone C-0-C symmetric stretch Ester C-0-C symmetric stretch Alcohol C-OH stretch Trisubstituted olefinic C-H wag

LOVASTATIN

283

.49
a

37

'1 3
.25

.la
.12 .O6
0
1

4000 3600 3200 2800 2400 2000 1600 1200 800


Wavenumbers
Figure 1. Infrared Absorption Spectrum of Lovastatin

2R4

GERALD S. BRENNER. DEAN K. ELLISON. AND MICHAEL J. KAUFMAN

4.2

Proton Nuclear Mametic Resonance Spectrum The proton magnetic spectrum is shown in Figure 2 (13). This spectrum was obtained on a Bruker Instruments Model AM-300 NMR spectrometer using a 4% w h solution of lovastatin in deuterated chloroform. Chemical shifts (6) are expressed as ppm downfield from tetramethylsilane (internal standard). The tabulated signal assignments refer to the numbered structure of lovastatin shown below.

6 (mml 0.88 0.89 1.08 1.11 1.20-2.05


2.20-2.50 2.55-2.77 4.37 4.64 5.38 5.53 5.78
6.00 7.27

Multiplicity /J t/J = 7.6 HZ d/J = 7.3 HZ d/J = 7.4 HZ d/J = 7.0 HZ Overlapping Multiplets Overlapping Multiplets Overlapping Multiplets m m m Broad t d Of d/J = 6.1, 9.6 Hz dN = 9.6 HZ
S

'

Assianment

1 Multiplicity: s, singlet; d, doublet; t, triplet; m, multiplet

PPM
Figure 2. Proton Nuclear Magnetic Resonance Spectrum of Lovastatin

286

GERALD S. BRENNER, DEAN K . ELLISON, AND MICHAEL J. KAUFMAN

4.3

Carbon-13 Nuclear Maanetic Resonance Spectrum The carbon-13 nuclear magnetic resonance spectrum o f lovastatin shown in Figure 3 was obtained using a Bruker Instruments Model AM-300 NMR spectrometer and an approximately 4% w/v solution of the compound in deuterochloroform. Signal assignments are tabulated below and refer to the numbered structure shown in Section 4.2 Chemical Shift (61, Dpm
11.69 13.83 16.21 22.79 24.23 26.78 27.39 30.63 32.62 32.90 36.06 36.55 37.24 38.55 41.46 62.52 67.86 76.37 77.00 128.26 129.58 1315 3 133.03 170.50 176.88

Assianment

In recent publications, the 1H and 13C NMR spectra of lovastatin were fully assigned by the use o selective f homonuclear and heteronuclear decoupling and two dimensional techniques (14,15).

Figure 3. Carbon-13 Nuclear Magnetic Resonance Spectrum of Lovastatin

288

GERALD S. RRENNER. DEAN K . ELLISON. AND MICHAEL J . KAUFMAN

4.4

Ultraviolet Spectrum The ultraviolet (UV) absorption spectrum of lovastatin is characterized by absorption maxima at 231,238, and 247 nm with A l % values of 538, 629, and 424, respectively. The absorption maxima at 238 nm is typical for a trisubstituted heteroannular diene chromophore (16). A UV spectrum of lovastatin (c = 0.015 mg/mL in acetonitrile) is shown in Figure 4.

4.5

Mass Spectrum The mass spectrum of lovastatin is shown in Figure 5. This spectrum was obtained by the direct probe electron impact (90 eV) method using a Finnigan MAT 212 mass spectrometer (17). The spectrum exhibits a weak molecular ion signal at m/z = 404 (C H 0 , exact mass calculated = 404.2563; observed = 461.2%6lf. Other pertinent fragment ions are at m/z = 302, 284, and 159; these ions can be rationalized by the fragmentation pattern shown in Figure 6.

4.6

ODtical Rotation Lovastatin has eight chiral centers and is optically active. The specific rotation a5 [2 , ] is +330" for a 5.0 mg/mL solution in acetonitrile.

4.7

Thermal Behavior The differential scanning calorimetry (DSC) curve for lovastatin at a heating rate of 2"/min under a nitrogen atmosphere is shown in Figure 7. The thermogram is characterized by a single melting endotherm with an extrapolated onset temperature for melting of 175C which is independent of heating rate from 2-2O0C/min. In contrast, the DSC thermogram for lovastatin obtained at a heating rate of 2"/min in air (Figure 8) exhibits an exotherm at 154C which is attributed to oxidative reactions occurring in the non-inerted atmosphere. The thermal properties of lovastatin, in particular those derived from DSC experiments, have been used to assess the oxidative stability of the compound (18,19).

Wavelength (nm)
Figure 4. Ultraviolet Absorption Spectrum of Lovastatin

loo]

''{
20
0

I/.
60
105

159

15;

2 84

198
143

302

172

!85
200 224

hi
250

404

100

150

200

300

350

400

Figure 5 . Direct Probe Electron Impact Mass Spectrum of Lovastatin

LOVASTATIN

29 I

m/z 302

mlz 159

m/z 284

Figure 6. Proposed Fragmentation Pattern to Explain the Mass Spectrum of tovastastin

292

Figure 7.

DSC Therrnogram for Lovastatin under Nitrogen

a
L

C .t .c
c

3
>
J
L

E 2

m
0

r
l -

cd ?? 3

L L

0) .-

293

294

GERALD S . BRENNER. DEAN K. ELLISON, AND MICHAEL J. KAUFMAN

4.8

Solubility Lovastatin is insoluble in water, and is sparingly soluble in the lower alcohols (methanol, ethanol, and i-propanol). Solubility data obtained at room temperature are tabulated below (20). Solvent Acetone Acetonitrile n-Butanol i-Butanol Chloroform N,N-dimethylformamide Ethanol Methanol n-Octanol n-Propanol i-Propanol Water Solubility ImalmL)
47 28 7 14 350 90 16 28 2 11 20 0.4

4.9

Crvstal Properties Lovastatin is a white, crystalline, non-hygroscopic solid. Single crystal X-ray diffraction experiments on a sample crystallized from ethanol indicate that the space group is P2,2 2, with a = 5.974A, b = 17.337A, and c = 22.148A. The calculated density is 1.17 g/cm3. (1) The X-ray powder diffraction pattern for lovastatin is shown in Figure 9. This spectrum was obtained on a Phillips APD 3720 X-ray diffractometer using CuKa irradiation. No crystal forms (polymorphs) other than that represented by the X-ray pattern in Figure 9 have been observed.

4.1 0 Dissociation Constants

Consistent with the structure, lovastatin exhibits no acidhase dissociation constants. Potentiometric titration of a sample in 50% aqueous methanol revealed no observable buffering action in the pH range of 2-1 1.

d *
4
M
0
M
rc)

c!

9
m
N

I n
c

8
c

In
0

I
I
I

o m -

dc

o ~ o

o r

* r~ c o ? z ? r o

296

GERALD S. BRENNER, DEAN K. ELLISON, AND MICHAEL J. KAUFMAN

4.11 Partition Behavior

In the n-octanol/water test system, lovastatin partitions quantitatively into the organic phase. At room temperature, the = 1.2 x 104. The partition coefficient is approximately K partition coefficient for the hydroxyaci82erivative (opened lactone form of lovastatin) between n-octanol and a pH 7.4 phosphate buffer is K ,, = 14.1 (21). O
5. Methods of Analysis 5.1

Elemental Analysis Analysis of Merck Sharp & Dohme reference lot L-154,803000G102 for carbon and hydrogen gives values compared to calculated values as given below: Calculated Carbon Hydrogen
71.25 8.97

Found
71.26 9.18

5.2

Chromatoqraphy
5.2.1

Thin-Layer Chromatoaraphv Table I lists the thin layer chromatographic systems which have been used for the analysis of lovastatin.
Table 1

Thin-Layer ChromatographicSystems for Lovastatin (221 Solvent System Toluenehnethanol


70/30

Plate Type Analtech@ Silica Gel GF Analtech@ Silica Gel GF Analtech@ Silica Gel GF

Rf
0.48

System 1 2
3

0.77

Toluenelacetone 70BO

4:l:l

Cyclohexaneh-butanoI/ethyl acetate

0.43

Cyclohexane/chloroformlisopropanol 5:2:1

E. Merck Silica 0.60 Gel 60 F254 High Performance

LOVASTATIN

291

Visualization is either by viewing the developed plate under ultraviolet light or by spraying the developed plate with a dilute methanolic sulfuric acid solution and application of heat. System 4 with sulfuric acid spray detection is the most useful system because non-UV absorbing impurities are detectable.

5.3.2

Hiqh Performance Liquid Chromatoaraphv (HPLC)

A variety of gradient and isocratic reverse phase HPLC systems have been used to chromatograph lovastatin (see Table 2).
Table 2 Hiqh Performance Liquid Chromatoqraphic Systems Application Drug substance purity System No.
1

Column Whatrnan Partisil C-8

Mobile Phase A = Acetonitrile B = 0.1% (v/v%) H3P04 aqueous A:B 70:30

nm Detection

Ref
(23)

238

Measurement of low level impurities in drug substances

Whatman Partisil C-8

Gradient 238 and A = Acetonitrile 200 nrn B = 0.1% (vW/O) H3P04 aqueous

(24)

Measurement in plasma and bile

Sepralyte C-18 lsocratic and 238 nm Gradient A = 0.05 ( NH4)3P04 and 00 . 1 H3P04 Buffer B = acetonltrile A:B 5050 (isocratic)

(25)

An

Measurement of low levels in fermentation broth'

DuPont Zorbax C-8

A = acetonitrile B = methanol A:B:C

260 nrn
C = water

(26)

62229

Measurement in tablets

Waters

A = acetonitrile 238 nm B = water (0.04M KH2P04 pH s 4) 60:40 A:B

(27)

298

GERALD S. BRENNER. DEAN K. ELLISON, AND MICHAEL J . KAUFMAN

Table 2 (Cont'd) High Performance Liquid Chromatographic Systems Application Measurement in tablets System No.
6

Column Hypersil 5 micron ODS

Mobile Phase

nm Detection
230 nm

Ref
(28)

pH = 4 B = CH CN C = MebH 33:55:12 A:B:C Derivatization of lovastatin described.

A = 0.025M NaH2P04

5.3

Flow lniection Analysis

-- to simultaneously monitor lovastatin and antioxidants in et al.


tablets (29). 5.4 Identification Tests

A flow injection analysis system has been described by Mazzo

Three methods are routinely used to identify lovastatin: 1. the infrared spectrum; 2. the ultraviolet spectrum; and 3. the chromatographic retention time. 6. Stability and Degradation 6.1 Solid State Stability Crystalline lovastatin stored at room temperature yields with time trace amounts of oxidation products. The oxidative pathway for degradation has been supported with data generated by chromatography, degradate isolation, and identification, differential scanning calorimetry and heat conduction calorimetry. No products of nonoxidative degradation have been detected. HPLC and TLC studies have demonstrated that samples stored in air generate a complex mixture of largely unidentified trace polar products (30). These products are essentially absent and drug loss prevented in samples stored under nitrogen. For samples stored in air, all isolated and identified degradates result from oxidation and include the 4'-oxolactone which is the major

LOVASTATIN

299

degradate retaining the diene of the parent. The ultraviolet absorption spectra of air degradates indicate

Oxolactone that more than half of the mass has lost diene, suggesting that oxidation takes place primarily at this site (e.g., epoxidation and subsequent reactions of the resultant epoxides). Heat conduction calorimetry (19) and differential scanning calorimetry (18) also demonstrate the enhanced reactivity of the compound in an air vs. a nitrogen atmosphere.
6.2

Solution Stability The hydrolysis of the lactone ring of lovastatin occurs readily in aqueous solution especially under acidic or alkaline conditions (31). The acid catalyzed hydrolysis is reversible leading to a mixture of lactone and hydroxyacid, the equilibrium ratio of the two species being pH dependent. The rate to equilibrium is also pH dependent, being more rapid at acidic pH than near neutrality. In alkaline solution, the lactone ring is irreversibly converted to the hydroxyacid. Solutions of the hydroxyacid demonstrate good stability. Kaufman (32) has studied and determined the rate and equilibrium constants for the acid catalyzed hydrolysis of mevalonolactone, lovastatin and other structurally related HMG CoA reductase inhibitors in pH 2.0 buffer at 37C. Under these conditions lactone concentrations decrease with time but do not approach zero indicating that the hydrolysis is reversible. The equilibrium nature of the reaction was further confirmed by repeating the experiment with hydroxyacid as starting material in the same system and demonstrating that an equilibrium composition is achieved that is identical to that achieved starting with lactone. Kinetic points in all studies were carried out to 15 hours and data obtained indicate that

300

GERALD S. BRENNER, DEAN K . ELLISON. A N D MICHAEL J. KAUFMAN

there are no side reactions (e.g., oxidation) competing with hydrolysis/lactonization during this time frame. The solution phase oxidation of a number of HMG CoA reductase inhibitors, including lovastatin, was studied in aqueous surfactant solutions at 40C (33). Reaction rate constants were determined by monitoring oxygen consumption using an oxygen electrode. In the absence of a free radical initiator, there was no oxygen uptake indicating that the spontaneous rate of oxidation at 40C was too slow to be detected. With an initiator present, all analogs consumed oxygen with the exception of the one in which the diene is saturated, demonstrating the diene functionality to be most labile to oxidation. Oxidation of lovastatin in aerated ethylene dichloride solution at 35", containing a free radical initiator, has been monitored kinetically using HPLC (34). The degradates formed in this complex solution system, different from those in the solid state, are primarily oligomers, with peroxide groups within the backbone chain and hydroperoxide end groups. Also, some monomeric epoxides are formed. 7. Pharmacokinetics and Metabolism Lovastatin is an inactive prodrug which undergoes in vivo lactone hydrolysis to give the hydroxyacid derivative which is an inhibitor of HMG-CoA reductase. The pharmacokinetic and metabolic profile of lovastatin has been described in detail (33,3537). In the sections below, the absorption, distribution, metabolism, and excretion of lovastatin are briefly reviewed. For this discussion it is helpful to distinguish between active inhibitors (defined as the sum concentration of the hydroxyacid derivative of lovastatin plus other active hydroxyacid metabolites) and total inhibitors (the total concentration of active inhibitors plus lactones and conjugates). Active and total inhibitors can be separately quantitated by assaying samples before and after ex vivo hydrolysis of plasma samples. 7.1 Absorption and Distribution

In studies in laboratory animals, the absorption of lovastatin following oral administration is approximately 30% complete as estimated relative to an intravenous dose of the hydroxyacid. An intravenous formulation of lovastatin for human studies is

LOVASTATIN

30 I

not feasible due to its low aqueous solubility. In all species studied, lovastatin is converted to the hydroxyacid form in viva This conversion is apparently reversible since lovastatin is found in the biological fluids of rats and dogs following administration of the hydroxyacid. In animals, lovastatin is more efficiently extracted by the liver where it is converted to the active enzyme inhibitor. Accordingly, the systemic bioavailability of active inhibitors is less than 5% of an oral dose of lovastatin. The high hepatic extraction and low systemic availability are desirable features since the liver is the primary site of cholesterol biosynthesis. Peak plasma concentrations of both active and total inhibitors occur between 2-4 hours post dose, and the area under the curve (AUC) increases proportionally with dose. The hydroxyacid is rapidly cleared; plasma clearance and half-life range from 300-1248 mUmin and 1.1-1.7 hrs, respectively. When lovastatin is administered with food, a 50% increase in AUC for inhibitory activity is attained relative to administration in the fasted state. The plasma protein binding of lovastatin and the hydroxyacid form has been determined by equilibrium dialysis. Both forms are greater than 95% protein bound. 7.2 Metabolism Lovastatin is extensively metabolized to give both active and inactive compounds. The major active metabolites present in human plasma are the hydroxyacid of lovastatin and its 3hydroxy-, 3-hydroxymethyl, and 3-exomethylenederivatives. The 3-hydroxylated metabolite is approximately 70% as active as the non-hydroxylated metabolite. In human bile, the 3hydroxylated metabolite undergoes an allylic rearrangement to give the 6-hydroxy isomer which is inactive (38):

"3C

302

GERALD S. BRENNER. DEAN K . ELLISON. A N D MICHAEL J . KAUFMAN

All of the hydroxyacid metabolites also exist in their corresponding inactive lactone forms. After base hydrolysis to convert lactones to active inhibitors, about 80% of the total enzyme inhibitory activity in human plasma is accounted for by these four lactonelhydroxyacidpairs.

7.3

Excretion The excretion of lovastatin has been assessed following an oral dose of 14C-labeled compound in man. Total recovery of drug equivalents in urine and feces averaged 10% and 83%, respectively. A substantial amount of radioactivity is also recovered in the feces following intravenous dosing of 14Clabeled hydroxyacid, indicating that biliary excretion is an important elimination for orally administered lovastatin.

8. Determination in Bioloaical Fluids

An enzyme inhibition assay capable of measuring total HMG-CoA reductase inhibitors in biological fluids has been described in the literature (1). The basis of this assay is the in vitro inhibition of the HMG-CoA reductase catalyzed conversion of 14C-HMG-CoA to 14Cmevalonic acid. The concentration of inhibitors can be measured before and after base hydrolysis of plasma samples. The measurement before hydrolysis gives the concentration of inherently active species (active inhibitors). Base hydrolysis irreversibly converts inactive but potentially active species (lactones and conjugates) to their corresponding active forms; the inhibition assay of hydrolyzed samples thus provides the concentration of total inhibitors. The enzyme inhibition assay is sensitive (detection limit of ca. 5 ng/mL), but is not specific for lovastatin. The determination of lovastatin and its hydroxyacid metabolite in plasma and bile can be accomplished by high performance liquid chromatography (25). Plasma samples are prepared for analysis by solid phase extraction and are analyzed using isocratic elution on a C18 column. Bile samples do not require any sample clean-up prior to HPLC analysis, but do require the use of a gradient elution method to separate the compounds of interest. The HPLC assay has a limit of detection of 25 ng/mL. An analytical method for the determination of lovastatin in serum based on gas chromatography/massspectrometry has recently been reported (39).

LOVASTATIN

303

Acknowledclements The authors wish to thank Mrs. Laurie Rittle for typing the manuscript and Ms. Agnes Hendrick for performing the literature search.
9. References
1.

A.W. Alberts, J. Chen, G. Kuron, V. Hunt, J. Huff, C. Hoffman, J. Rothrock, M. Lopez, H. Joshua, E. Harris, A. Patchett, R. Monaghan, S. Currie, E. Stapley, G. Albers-Schonberg, 0. Hensens, J. Hirshfield, K. Hoogsteen, J. Liesch and J. 3957 (1980). Springer, Roc. Natl. Acad. Sci. USA 77, A. Endo, J Antibiot. 32, 852 (1979). . J.S. MacDonald, R.J. Gerson, D.J. Kornburst, M.W. Kloss, S. Prahalada, P.H. Berry, A.W. Alberts and D.L. Bokelman, Am. J Cardiol. 62, 16J (1988). .

2.

3.

4.
5.

E.E.Stater and J.S. MacDonald, Drugs 36 (Suppl. 3), 72 (1988).


J.M. McKenney, Clin. Pbarm. 7, 21 (1988).

6. 7. 8. 9. 10.

A.W. Alberts, Am. J. Cardiol. 62, 1OJ (1988). J.A. Tobert, Circulation 76, 534 (1987).

S.J. Hecker and C.H. Heathcock, J. Org. Chem. 50,5159


(1985).

M. Hirama and M. Iwashita, Tetrahedron Letters 24, 1811 (1983).


D.L.J. Clive, K.S.K. Murthy, A.G. Wee, J.S. Prasad, M. Majewski, P.C. Anderson, C.F. Evans, R.D. Hauger, L.D. Heerz and J.R. Barrie, J. Am. Cbern. SOC.112, 3018 (1990). T. Rosen and C.H. Heathcock, Tetrahedron 42, 4909 (1986).

11. 12.

R. Cervino, Merck Sharp & Dohme Research Laboratories, personal communication.

304

GERALD S.BRENNER, DEAN K. ELLISON, AND MICHAEL J. KAUFMAK

13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.

R. Reamer, Merck Sharp & Dohme Research Laboratories, personal communication.


J.K. Chan, R.N. Moore, T.T. Nakashima, J.C. Vederas, J Am. . Chem. SOC.105, 3334 (1 983). R.N. Moore, G. Bigam, J.K. Chan, A.M.Hogg, T.T. Nakashima, J.C. Vederas, J Am. Chem. SOC. . 107,3694 (1985). A.I. Scott, Interpretation of the Ultraviolet Spectra of Natural Products, Pergamon Press, Oxford, 1964. D. Zink, Merck Sharp & Dohme Research Laboratories, personal communication. J.P. Elder, Thermochim. Acta 134, 41 (1988). L.D. Hansen, E.A. Lewis, D.J. Eatough, R.G. Bergstrom, D. Degraft-Johnson, Pharm. Res. 6, 20 (1989). A.Y.S. Yang, Merck Sharp & Dohme Research Laboratories, personal communication. M.J. Kaufman, Merck Sharp & Dohme Research Laboratories, personal communication. A.Y.S. Yang, L. Pierson and J. Baiano, Merck Sharp & Dohme Research Laboratories, personal communication. A.H. Houck, Merck Sharp & Dohme Research Laboratories, personal communication. A.H. Houck, S. Thomas and D.K. Ellison, Pittsburgh Conference (1990),manuscript in preparation.

. R.J. Stubbs, M. Schwartz and W.F. Bayne, J Chromatog. 383, 438 (1986).
V.P. Gullo, R.T. Goegelrnan, I. Putter and Y. Lam, J. Chromatog. 212, 234 (1 98 1). L.L. Ng, Anal. Chem. 53, 1142 (1981).

LOVASTATIN

305

28. C.V. Bell and J.C. Wahlich, Merck Sharp & Dohme Research
Laboratories, personal communication.

29.
30.

D.I. Mazzo, S.E. Biffar, K.A. Forbes, C. Bell and M.A. Brooks, J Pharm. Biomed. Anal. 6, 271 (1 988). . M. Baum, G.Dezeny, L. DiMichele, R. Reamer and G.B. Smith, Merck Sharp & Dohme Research Laboratories, personal communication. A.Y.S. Yang, Merck Sharp & Dohme Research Laboratories, personal communication. M.J. Kaufman, Int. J. Pharm. 66, 97 (1990). M.J. Kaufman, Pharm. Res. 7, 289 (1990).

31.
32. 33. 34.

G. Dezeny and G.B. Smith, Merck Sharp & Dohme Research


Laboratories, personal communication.

35. J.J. Krukemeyer and R.L. Talbert, Pharmacotherapy 7, 198 (1 987). 36.
D.E. Duggan, I.W. Chen, W.F. Bayne, R.A. Halpin, C.A. Dunca, M.S. Schwartz, R.J. Stubbs and S. Vickers, Drug 1 Metab. Dispos. 17,166 ( 989). D.E. Duggan and S. Vickers, Drug Metab. Rev. 22, 333 (1990).
R.A. Halpin, K.P. Vyas, P. Kari, B.H. Arison, E.H. Ulrn and D.E. Duggan, Pharmacologist 29, 238 (1987).

37. 38.

39.

D. Wang-lverson, E. Ivashkiv, M. Jemal and A.I. Cohen, Rapid Comm. Mass. Spectrom. 3 , 132 (1989).

NAPHAZOLINE HYDROCHLORIDE

G . Michael Wall

Alcon Laboratories, Inc.

6201 South Freeway


Fort Worth, Texas 76134

ANALYTICAL PROFILES OF DRUG SUBSTANCES AND EXCIPIENTS - VOLUME 21

307

Copyright Q 1992 by Academic Press, Inc. All rights of reproduction reserved in any form.

308

G.MICHAEL WALL

NAPHAZOLINE HYDROCHLORIDE
1.

DESCRIPTION 1.1 Name, Formula and Molecular Weight 1.2 Appearance, Color, Odor 1.3 History 1.4 Pharmacology SYNTHESIS PHYSICAL PROPERTIES 3.1 Spectroscopy 3.1.1 Infrared Spectrum 3.1.2 Ultraviolet Spectra 3.1.3 Nuclear Magnetic Resonance Spectra 3.1.4 Mass Spectra 3.2 Thermal Properties 3.2.1 Melting Range 3.2.2 Differential Thermal Analysis 3.2.3 Thermogravimetric Analysis X-Ray Crystallography and Powder Diffractometry 3.3 3.4 Partition Coefficients 3.5 Ionization Constant, pKa 3.6 Solubility Solution Color, Clarity and pH 3.7 TYPICAL METHODS OF ANALYSIS 4.1 Identity 4.1.1 Infrared Spectrophotometry 4.1.2 Ultraviolet Spectrophotometry 4.1.3 Chloride Identity Test 4.1.4 Reaction with Bromine 4.2 Colorimetry 4.3 Elemental Analysis 4.4 Titrimetry 4.5 Chromatography 4.5.1 Thin-Layer Chromatography 4.5.2 High-pressure Liquid Chromatography 4.5.3 Gas Chromatography STABILITY-DEGRADATION 5.1 Potential Routes of Degradation 5.1.1 Characterization of 1-Naphthylacetylethylenediamine 5.1.1.1 Thin-Layer Chromatography 5.1.1.2 Liquid Chromatography 5.1.1.3 Synthesis of 1-Naphthylacetylethylenediamine

2.

3.

4.

5.

NAPHAZOLINE HYDROCHLORIDE

309

5.2 5.3
6.
7.

Physical/Chemical Properties of 1Naphthylacetylethylenediamine 5.1.2 Synthesis and Analysis of l-Naphthylacetic Acid Solid-state Stability Solution Stability 5.1.1.4

DISPOSITION AND TOXICITY ACKNOWLEDGEMENTS REFERENCES

8.

1. 1.1

DESCRIPTION
Name, Formula and Molecular Weight

Naphazoline hydrochloride is an u-adrenergic sympathomimeticagent used in topical nasal or ophthalmic pharmaceutical formulations. Naphazoline has been established as the International Nonproprietary Name (INN) by the World Health Organization for the chemical compound, (2-(l-naphthylmethyl)-2-imidazoline1$2, which is typically used as either the hydrochloride or nitrate salt. The hydrochloride salt has been given the USAN, naphazoline hydrochloride1. Other chemical names include: (a) 1H-imidazole, 4,sdihydro-2-(l-naphthalenylmethyl)-, monohydrochloridel, (b) 2-( l-naphthylmethyl)-2-imidazoline monohydrochloridel, and (c) 4,5-dihydro-2-(1naphthalenylmethy1)-1H-imidazole,monohydrochloride3. The CAS registry number for naphazoline hydrochloride is 550-99-21; the CAS number for the free base is 835-31-4l. Empirical Formulal: C14H14N2 * HC1 Molecular Weightl: Structure: 246.74

310

G . MICHAEL WALL

1.2

Appearance, Color and Odor

Naphazoline hydrochloride is a white to almost white, odorless, crystalline powdefl with a bitter taste5y6.
1.3

History

An investigation of the vasoconstrictor activity of substituted imidazolines by Fritz Uhlmann at Ciba in Basle, Switzerland during the early 1940s resulted in the introduction of the sympathomimetic drug, naphazoline; its analogs, xylometazoline and oxymetazoline, used as decongestants; and also the a-adrenoceptor antagonist, tolazoline798. Patents include: U.S Patent 2,161,938 (1939) and Danish Patent 62,889 (1944)6. Naphazoline has been marketed under a variety of trade names around the world29399, 1.4 Pharmacology

Naphazoline is a potent a-adrenergic sympathomimetic agent. It is a vasoconstrictor with a rapid and prolonged action in reducing swelling and congestion when applied to mucous membranes, hence, its use for the symptomatic relief of rhinitis and sinusitis. Rebound congestion and rhinorrhea are common after prolonged use. Nasal drops or spray are used as a 0.05% aqueous solution of the hydrochloride or nitrate, with a usual recommended dosage of 2 drops in each nostril every 3 hours. Aqueous solutions have also been used as ophthalmic conjunctival decongestants4. 2.

SYNTHESIS

Naphazoline hydrochloride has been prepared through a series of synthetic chemical steps beginning with (1-naphthy1)-acetonitrile,I (Figure l)3910. The starting material, I, is treated with ethanol and hydrochloric acid to obtain the naphthyl-(1)-acetiminoethylether hydrochloride, II1o. A solution is made of 2.7 parts II and 12 parts absolute alcohol3. One part of ethylenediamine is then added and the mixture is heated to gentle boiling with stirring under nitrogen until the evolution of ammonia ceases. The alcohol is then distilled and the residue is dissolved in 40 parts of benzene and 1.8 parts of caustic potash. The benzene is removed and the residue is recrystallized several times from toluene. Reaction with hydrochloric acid gives naphazoline hydrochloride, III3. The preparation of radiolabelled naphazoline with 14Cin the 2-position of the imidazoline ring has also been reported11 using a-chloromethylnaphazoline, potassium cyanide-14C, and ethylenediamine.

I11

Figure 1. Synthesis o naphzolinc hpirochbr&ie. f

312

G. MICHAEL WALL

3. 3.1

PHYSICAL PROPERTIES
Spectroscopy

3.1.1 Infrared Spectrum

The infrared spectrum of naphazoline hydrochloride was obtained. A mixture of the drug substance and potassium bromide was pressed into a pellet and analyzed using a Perkin-Elmer Model 1750 FTIR. The spectrum is shown in Figure 2. The major absorption bands for the infrared frequencies and the corresponding assignments are listed in Table I. 3.1.2 Ultraviolet Spectra The ultraviolet absorption spectra of naphazoline hydrochloride in absolute ethanol, pH 3 buffer (0.05M phosphate), pH 7 buffer (0.05M phosphate) and pH buffer (0.05M borate) were obtained using a Perkin-Elmer 559A W/VIS spectrophotometer and 1cm cells. A representative W spectrum in ethanol is shown in Figure 3. Samples of naphazoline hydrochloride in these solvents were scanned from 200 to 400 nm and the absorption coeffiI. cients at wavelengths of maximum absorption were calculated (Table I) 3.1.3 Nuclear Magnetic Resonance Spectra The lH-NMR spectrum (100 MHz) of naphazoline hydrochloride has been reported and the chemical shifts have been assigned for the methylene groups12. The IH-NMR spectrum (300 MHz) of naphazoline hydrochloride (143 mgmL DMSO-d6 at 1OOOC) was obained using a Vanan VXR 300 spectrometer (Figure 4). In order to assign all of the aromatic proton signals, a series of 2-D experiments were carried out: these spectra were not shown but the assignments are listed in Table III. The l3C-NMR spectrum (22.5 MHz) of naphazoline hydrochloride has been reported and the chemical shifts have been assigned for the methylene and imidazoline carbons12. The %NMR spectrum (75 MHz) of naphazoline hydrochloride (143 mg/mL DMSO-& at 1OOOC) was obtained using a Varian VXR 300 spectrometer (Figure 5 ) and the assignments are listed in Table III. The Attached Proton Test (APT)(Figure 5 ) and extensive 2-D studies were performed in order to assign aromatic carbons. The resonances for the methylene protons were shifted downfield for the HC1 salt compared to the base: h 6 (ppm) (+ indicates downfield shift compared to base); C&C&, +0.44 and aryl-C& +0.53)12. The reso-

Figure 2. In@zrd spctnm (KBr)O Mphotolinc hyhxhbnkk. f

3 14

G.MICHAEL WALL

I .E

0.c
200
I
I

250

300

350

600

Wavelength (nm)

ethanol).

Figure 3. UVspctrum o naphamline hydnxhloride (0.019mghL in f

NAPHAZOLINE HYDROCHLORIDE

315

Table I. Infrared spectral assignments for naphazoline hydrochloride. Wavelength (cm-I) 3150-2500 1618 1302, 1198 801, 765 602, 561, 525, 481 Assignment C-H and N-H stretch Amine salt N-H 20 N-H Imidazoline C-H Out of plane ring bend

Table I. Ultraviolet absorption of naphazoline hydrochloride. I Solvent Ethanol pH 3 Buffer pH 7 Buffer pH 9 Buffer 223nm 3622 3214 3246 3294 E (l%, cm) 1 270nm 280nm 239 246 238 236 286 287 279 274 287nm 196 198 193 191 291nm 198 193 188 187

I - - - . . - - -

- - - - 1 - - - .

. - - - I - . - .

..-.,.... ....,....

. . . . I . . . ,

...,,..., rrr ....,.,..

1)

F i R m 4. H-NMR Siuctn#n (300MHz) o Mphatolne h y h x h l o f i (143 m g h L in DMso-d6 a I W C ) . f t

Figun 5. JJC-NMRSpanun (75 MHz) o napAazoline hydrrnrhloride (143 m g h d inDMSOd6 a IcK)DC). f !

318

G . MICHAEL WALL

Table III. lH- (300 M H Z ) and 1% (75 MHz) N M R Data for Naphazoline HCl(l43 mg/mL in DMSO-& at 1OOOC).

1 2 3 4 5 6 7 8 9 10 11 12 14 & 15

7.64 (lH,d) 7.49 (lH,m) 7.91 (lH,d) 7.96 (lH,d) 7.54 (lH,m) 7.58 (lH,m) 8.15 (1H,d)

4.45 (2H,s) 3.82 (4H,s)

128.71 128.09 125.61 128.47' 128.66* 126.06 126.83 123.34 131.37 133.57 29.41 169.78 44.40

* Interchangeable assignments

NAPHAZOLINE HYDROCHLORIDE

319

nances for carbons attached to the imidazoline ring were shifted for the HC1 salt compared to the base: A 6 (ppm) (- and + indicate upfield and downfield shifts respectively compared to base); zIHgH2, -5.09; arylCH2, -3.63; and N-IZ-N, +3.9611. 3.1.4 Mass Spectra Mass spectra were obtained for naphazoline hydrochloride using a Finnegan MAT TSQ46 GC/MS/MS unit. A small amount of naphazoline hydrochloride was volatilized by heating at a linear rate of 5 mA/sec from 0 mA to about 500 mA and ionized by either chemical ionization (CI, 0.3 Torr pressure of isobutane) or by electron impact (EI) at 70 eV. The CI and EI mass spectra were presented in Figures 6 and 7 and the interpretation presented in Table IV.The fragmentation pattern was consistent with the chemical structure of naphazoline hydrochloride (Figure 8). 3.2 Thermal Properties

3.2.1 Melting Range The melting point of naphazoline hydrochloride has been reported as 257OC13 with a range of 255-60 (decomposition)5*6; melting point for the the base has been reported as 115-120OC13. 3.2.2 Differential Thermal Analysis A 2-mg sample of naphazoline hydrochloride drug substance was heated from 40OC to 3000C at a linear rate of 200C/min using a Perkin-Elmer DSC4. One single, sharp endotherm was observed with an onset of 259OC and a maximum of 261OC, corresponding to the melting range, after which decomposition occurred (Figure 9). 3.2.3 Thermogravimetric Analysis A 7-mg sample of naphazoline hydrochloride was heated using a PerkinElmer System 4 ThermogravimetricAnalyzer from 4OOC to 298OC at a linear rate of 200C/min. The drug substance exhibited a gradual weight loss near the melting range (Figure 10). 3.3 X-Ray Crystallography and Powder Diffractometry

Naphazoline hydrochloride exists as a crystalline powde8. Podder et a1.15 described the crystal structure: Mr = 246.73, monoclinic, P21/c, a = 11.895 (3), b = 9.228 (2), c = 12.820 (3) A, J3 = 117.18 (2)0, V = 1252 813, 2 = 4, D m 1.30, Dn= 1.29 Mg m-3, h (Cuka) = 1.5418 A,p = 2.48 mm-1, F(OO0) = 524, T = 277 (1) K. Final R = 0.040 for 1291 observed reflec-

P x

I
ii
d

E p

320

Figure Z E Mass spectrum o napfuwlituh@mhlon&. J f

322

C. MICHAEL WALL

Tablc I V . t I and CI Mass splrital Assignnrcnls for naphamline hydrochloridc


El

Rel.

~~~

Assignment

Rel.

CI

21 I 209

100
7 6 9 9 12

I95
181

I53
141 1 I5

--,-I

11s I

153 ----I

NAPHAZOLINE HCL
VT.

228 g

SCAN RATE.

20.00 w a i n

Figurc 9. DSC o naphazolinc hyhchloride. f

IT,

7.290. ag

RATE.

20.00 d.g/rln

m a
I#c .QJ T Q -84

P N

TEMPERATURE (C)

TC

Figwv 10. Z A o naphawline hydrochlode. f

. ( r

c
W Ln N

u)

U Y

10

15

28

2s
20

30

35

45

F i g u n 11. X-Ray pmvdPr difiaction pancrn o naphamline hyirochloride. f

326

G . MICHAEL WALL

Table V. X-Ray powder diffraction data for naphazoline hydrochloride obtained using CuKa radiation and indexed on the basis of a monoclinic cell: P2ljc, a = 11.895 (3), b 9.228 (2), c 12.820 (3) A, fl- 117.18
(2)O.

h k

Intensity

1 0 0
0 1 1 1 1 0 1 -1 1 1 )

10.7 7.21 6.97 6.38 5.71

47 29 13 41 3 1 2 1 3.760 18

- 2 1 3 1 1 2 2

-1 0 2
0 0 2 2 0 0 )
1 1 1

-3 1 2 3.598 -3 0 2 2 J

-2 0 -2 -2
1 1

I
I
.

5.28

8
0 1 3

'I

100

3.529 3.300 3.135 3.091 3.042 3.016

23 4 18 6

5.01 4.87 4.60

25 29 27

1 0

0 1 2 0 2 0 1 2 1 0 ) 0 2 1

-1 0 4 j

1 2 2 1 2 2

1 0 2 1

4.29

30

2 1 2 -2

8
14

1 4

- 3 0 2 i
1 1 2 1

3.881

NAPHAZOLINE HYDROCHLORIDE

327

tions. The bond lengths of the N-C-N group of the imidazoline ring were short and indicative of double bond character. One nitrogen atom was protonated and both nitrogen atoms participated in hydrogen bonding. Each chlorine atom was involved in two intermolecular hydrogen bonds of the form Nl-H-Cl-H-N2, that linked the molecules into continuous parallel chainsls. To obtain an x-ray powder diffraction pattern, a sample of the drug substance was irradiated using a Philips powder diffractometer equipped with a diffracted beam graphite monochronometer. CuKa (1 1.5405 A) radiation was used for obtaining the powder pattern (Figure 11).All of the diffraction lines could be assigned hkl indicies on the basis of the unit cell parameters proving that the material was single-phase (Table V).

3.4

Partition Coefficients

Partition coefficients were determined for naphazoline hydrochloride between pH 3 buffer (0.05 M phosphate), pH 7.0 buffer (0.05 M phosphate) and pH 9 buffer (0.05 M borate) versus l-octanol. All solutions were prepared using octanol-saturated buffers and buffer-saturated octanol. Tubes containing 100 mg of naphazoline hydrochloride, 10 ml of buffer and 10 ml of octanol were agitated for 2 hours at 23OC and allowed to partition overnight. Analysis (HPLC) of the aqueous phases of each mixture revealed the following partition coefficients: pH 3.0 = 0; pH 7.0 = 0; pH 9.0 = 7.4. 3.5 Ionization Constant, pKa

The pKa of naphazoline HC1 has been reported as 10.9 at 200C4, 10.35 k 0.02 at 25OC16, 10.13 ? 0.02 at 35*C16, and 9.92 f 0.03 at 450C16. 3.6 Solubility

The solubility of naphazoline hydrochloride in various solvents at room temperature is presented in Table VI. Table VI. Solubility of Naphazoline Hydrochloride Solvent Water Ethanol Chloroform Diethyl Ether Solubility
1i n 6 1 in 15 Very slightly soluble Practically insoluble

Reference 13 13
4 4

328

G . MICHAEL WALL

3.7

Solution Color, Clarity and pH

An aqueous solution (1 in 100) of naphazoline hydrochloride in carbon dioxide-free water is clear, colorless and exhibits a pH value between 5.0 and 6.617. 4.
4.1

TYPICAL METHODS OF ANALYSIS


Identity

4.1.1 Infrared Spectrophotometry The identity of naphazoline hydrochloride may be determined by comparison of its infrared spectrum (KBr) (see Figure 2) to an authentic reference standardl7.
4.1.2 Ultraviolet Spectrophotometry

The identity of naphazoline hydrochloride may be confirmed by comparison of its ultraviolet spectrum (1in 50,000) to that of an authentic standard and the observation of a maximum at 280 nm17.
4.1.3 Chloride Identity Test An aqueous solution of naphazoline hydrochloride (1 in 100) is treated with 6N ammonium hydroxide to precipitate naphazoline base. The filtrate then yields a white, curdy precipitate upon the addition of 0.1N silver nitrate. The precipitate is insoluble in nitric acid but is soluble in a slight excess of 6N ammonium hydroxide5~~~. 4.1.4 Reaction with Bromine
A 10-mL aliquot of an aqueous solution of naphazoline hydrochloride (1 in 100) when mixed with 5 mL of bromine-saturated water yields a yellow precipitate. Upon boiling, a deep purple color is produced5.

4.2

Colorimetry

Naphazoline has been analyzed by colorimetry using reagents such as sodium nitroprussidel8, ceric sulfatelg, chloranillg, bromocresol green20, bromophenol blue20, bromothymol blue20, methyl orange20, cobaltous acetate in chloroform-methanol21,iodine in chloroform22-25,and 2,6dichlorophenol-indophenolin CHC1326.

NAPHAZOLINE HYDROCHLORIDE

329

4.3

Elemental Analysis

Elemental analysis of a sample of naphazoline hydrochloride was performed: Anal. (C14H1&HCl) C (calcd 68.14; found, 68.52), H (calcd, 6.14; found, 6.28), N (calcd, 11.36; found, 11.48), C1 (calcd, 14.37; found, 14.54). 4.4 Titrimetry

Naphazoline hydrochloride drug substance may be titrimetrically assayed as described in the USP monographl7. The hydrochloride salt is dissolved in glacial acetic acid with mercuric acetate and titrated with dilute perchloric acid using crystal violet as the indicator. Also, nonaqueous titration of n a p hazoline hydrochloride in acetic anhydride/glacial acetic acid with dilute perchloric acid and potentiometric detection has been described as an official method in the Japanese Pharmacopoeias. In addition, nonaqueous titration with dioctylsulfosuccinate sodium salt using 3'3"5'5"-tetrabromophenolphthalein as the indicator has also been reported25.
4.5

Chromatography

4.5.1 Thin-Layer Chromatography The USP describes a TLC method for ordinary impurities in naphazoline hydrochloride drug substancel7. A sample of the drug substance dissolved in methanol (10 mg/mL) is spotted on a silica gel TLC plate, eluted with a mobile phase of methanol-glacial acetic acid-water (8:1:1, v/v/v), and visualized with iodoplatinate spray. 4.5.2 High-pressure Liquid Chromatography Naphazoline has been analyzed in ophthalmic preparations by HPLC using a 10 pm octadecylsilane column (3.9 X 300 mm), a mobile phase of 0.08 M HClO4 (PH 2.2)-methanol(7/3, v/v), a flow rate of 2 mL/min and UV detection at 265 nm28; in ear and eye drops using a 10 pm octadecylsilane column (4 X 250 mm), a mobile phase of methanol-water (40/60, vh), a flow rate of 2 mL/min and UV detection at 279 nm29; in ophthalmic formulations or raw material using a 5 pm cyano column (4.6 X 150 mm), a mobile phase of dilute phosphate solution (PH 3)/-acetonitrile (60:40, v/v), a flow rate of 2.0 mL/min and W detection at 225 nm30 or a 5pm octylsilane column (4.6 X 250 mm), a mobile phase of 0.05 M phosphate solution (pH 5,6)-acetonitrile (4:1, v/v) containing 0.07 M triethylamine, a flow rate of 1.5 mL,/min and W detection at 270 nm3O; in tablets and capsules using a 10 pm phenyl column (4 X 300 mm), a mobile phase of water-methanolglacial acetic acid (55:44:1, v/v/v) containing 0.005 M heptane sulfouic acid sodium salt, at a flow rate of 2.0 d / m h and W detection at 254 nrn3l.

330

G . MICHAEL WALL

4.5.3 Gas Chromatography Naphazoline has been analyzed by gas chromatography using various stationary phases including OV-l3%, OV-3 3%, OV-7 3%,OV-17 256, and QF-1 5%32*

5.
5.1

STABILITY-DEGRADATION
Potential Routes of Degradation

Naphazoline has been shown to be relatively stable in acidic or neutral solutions but readily prone to hydrolysis in basic solution. The first step in the hydrolytic reaction33 results in the formation of l-naphthylacetylethylenediamine which upon vigorous treatment34, undergoes further cleavage to form 1-naphthylacetic acid and ethylenediamine(Figure 12). The kinetics of this reaction have been describedl6. The major degradation products of naphaand zoline, 1-naphthylacetylethylenediamine 1-naphthylaceticacid, have been prepared33 and investigated30*33*34. Hydrochloride 5.1.1 Characterization of 1-Naphthylacetylethylenediamine
5.1.1.1 Thin-Layer Chromatographyof 1-Naphthylacetylethylenediamine
An adaptation of the USP TLC procedure17 for the determination of ordinary impurities in naphazoline hydrochloride allowed for the detection of 1naphthylacetylethylenediaminein the presence of naphazoline. Using silica gel 60 high-performance TLC plates (20 X 20 cm) and a mobile phase of methanol-glacial acetic acid-purified water (8:1:1, v/v/v), spots were visible after spraying with ninhydrin: naphazoline Rf= 0.54; l-naphthylacetylethylenediamine Rf 0,6330. A similar method has been described in the in European Pharmacopoeia for 1-naphthylacetylethylenediamine naphazoline nitrate35.

5.1.1.2 Liquid Chromatography of 1-Naphthylacetylethylenediamine

1-Naphthylacetylethylenediamine been quantitated in the presence of has naphazoline and 1-naphthylacetic acid using column chromatography followed by W assay33934. A modem HPLC procedure has been developed for the analysis of 1-naphthylacetylethylenediamine the presence of napin hazoline by HPLC using a 5 pm cyano column (4.6 X 150 mm), a mobile phase of 0.025 M Na2HPO4 buffer (pH 7,4)-acetonitrile (35:65, v/v), a flow rate of 2.0 mL,/min and W detection at 270 nm30. Retention times 3.1 were: naphazoline, 6.3 min; 1-naphthylacetylethylenediamine, min (Figure 13).

d ?
/ /

NH2

OHHC'

Naphazolinc HCI

1-Naphthylacetyluhylenadiamine

1-Naphthylacdc Acid

M y lmdiarnine

Figure 12. Lkgradaion producrs of nuphazolinc hydrochloridc undcr alkaline conditions.

332

G . MICHAEL WALL

Figure 13. HPLC of I-nriphrhylacerylerh~l~~diurnine (5.2 pg), L and HCI naphamtine HCl (1.2 ps), 2 I pm qano column, 4.6 X 150 nim, 5.025 M S phosphate bufler (pH 7.4)-acetonirrile ( 5 6 . vh). 2.0 mllinin, UV 2701. 3:S

NAPHAZOLINE HYDROCHLORIDE

333

5.1.1.3. Synthesis of 1-NaphthylacetylethylenediamineHydrochloride The synthesis of 1-naphthylacetylethylenediaminewas described by using a modification of previous work by Miescher et Schwartz et ~1.33 uP6. Five grams of naphazoline hydrochloridewere refluxed with 100 ml of 0.5N NaOH for 30 minutes. The mixture was then cooled, made alkaline, and extracted with CHC13. The CHCl3 extract was evaporated, leaving a yellowish oil which solidified upon chilling to give the base as an off-white solid recrystallized from CHC13-petroleum ether (1:l) mp 93-95OC. The base in chloroform was treated with HC1 gas to obtain the HCl salt as an off-white solid mp 142-8% 5.1.1.4. Physical/Chemical Properties of 1-Naphthylacetylethylenediamine Hydrochloride
A sample of 1-naphthylacetylethylenediamine hydrochloridewas prepared and evaluated30. The substance appeared as off-white powder with a melting point of 153.8-154.2OC. The material was not hygroscopic. The IR spectrum (Table W, Figure 14), U V spectrum (Figure 15), lH-NMR spectrum (Table VIII, Figure 16), 13C-NMR and APT spectra (Table WI, Figure 17), and mass spectrum (Table I , X Figure 18) were consistent with the proposed chemical structure. The DSC (Figure 19) of l-naphthylacetylethylenediaminehydrochloride was consistent with the metling range.

Table W. Infrared spectral assignments for l-naphthylacetylethylenediamine HC1. Wavelength (cm-1) 3392 3220-2400 1641 1599,1482,1438 1520 802,795,779 Assignment
10 amide N-H stretch C-H and N-H stretch amide C-0 aryl C-C stretch 20 amide N-H substituted aromatic

5.1.2. Synthesis and Analysis of 1-Naphthylacetic acid The synthesis of another naphazoline degradation product, 1-naphthylacetic acid, was described b Schwartz et aP3. Five grams of naphazoline HC1 were refluxed with 5(rml of 1N NaOH for 2 hours. The mixture was cooled

7 -

ma

1 2 1

CI.

Figun 14. Inrfrand spanun (KBr) of I-~phrhylaccrykthyktudicdiamirv HCI.

NAPHAZOLINE HYDROCHLORIDE

335

1.0

r
0

e
8 4

0.0

Figum 15. U V Spctrum o f l - M ~ h y l ~ ~ l ~ h HCI~(0.02 i ~ y M m m g M in ethanol).

01 W

Figure 16. IH-NMR (200 MHz) o l - ~ p h r h y l o c t r y l c t h y l e ~ i ~ i(20 in DMSOdd. f HCI ~ mg

4 W 1

338

G . MICHAEL WALL

Table WI. NMR assignments for 1-naphthylacetylethylenediamneHC1 (20 mg in DMSOQ6).

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

7.44-8.60 7.44-8.60 7.44-8.60 7.44-8.60 7.44-8.60

124.34J25.47, 125.59,125.96, 127.08,127.88, 128.31

NAPHAZOLINE HYDROCHLORIDE

339

and acidified with HCI, producing a flocculent white precipitate. The precipitate was filtered, washed with cold H20 and recrystallized from hot H20 mp 133-134OC; W spectrum, A max 283 pm, E (l%, 1 cm in CHC1-j) = 360. 1-Naphthylaceticacid has been quantitated in the presence of naphazoline and 1-naphthylacetylethylenediamineusing column chromatography followed by UV a~say3373~.

X EI Table I . Mass spectrum of 1-naphthylacetylethylenediamineHC1.


EI (We)
228 199 185 141 128 Relative Abundance (%) 4 14 73 100 14 Assignment

[M-NHCH$ [M-NHCH2CH2]+ [M-C3H7N20]+ [M-C4H8N2O]+

[MI+

5.2

Solid-state Stability

Naphazoline hydrochloride drug substance has been shown to be stable for at least 6 months under the conditions of room temperature, 4OC, 350C, 40OC at 75% relative humidity, 50OC and exposure to light (1000 foot-candles) at room temperature. The drug substance was found not to be hygroscopic. An investigation was performed to determine if naphazoline hydrochloride would exhibit polymorphism. Samples of the drug substance were treated with (a) heat at lOOOC for 4 hours or (b) vigorously ground with a mortar and pestle. Analytical data from IR, DSC, and x-ray powder diffractometry showed no changes compared to a control sample, suggesting no evidence of polymorphism for naphazoline hydrochloride30. 5.3 Solution Stability

The stability of naphazoline hydrochloride in aqueous buffers (PH 4.5,7.0, 9.0) and oxygen-saturated water was evaluated under the conditions room The temperature, 350C, 55OC, and light exp0sure3~. drug was relatively stable under all conditions at acidic and neutral pH and in oxygen-saturated water for at least 26 weeks. The alkaline solutions, however, turned a range of dark colors and showed severe degradation after only 4 weeks.

1m.a 7

n.00

1s

T
F i p n 18. El Mars s p c m m of 1-naphrhylacctyltthyknedim~ne Ha.

342

G . MICHAEL WALL

6.

DISPOSITION AND TOXICITY

Naphazoline hydrochloride is commercially available in concentrations of 0.01 to 0.1% as a nasal or ocular decongestant. Local effects are obtained after topical administration to the eye and nasal passages. Metabolic studies could not be found in the literature, despite the report of the synthesis of 14C-labelleddrug substance intended for that purpose1'. Dittgen et a1.37 studied the elimination of naphazoline from the isolated pig eye after topical application. Reports of systemic effects of poisoning with naphazoline have been scarce38. The LDSO in rats has been reported as 385 mgAcg6. S.C.

ACKNOWLEDGEMENTS
The author expresses his sincere thanks to the following persons who have provided data and/or information for this chapter: R.E. Hall for solid-state data; G. Havner for confirming the bromine ID test; D.D. Taylor for partition coefficients, UV, MS, and NMR data; R. Conroe and S. Spruill for the synthesis and B. Scott and P. Ritter for analytical data on 1naphthylacetylethylenediamine,all at Alcon Laboratories; Professor John Baker, Department of Medicinal Chemistry, University of Mississippi, Oxford, Mississippi for NMR data and interpretation; and Professor Hugo Steinfink, Department of Chemical Engineering, University of Texas, Austin, Texas for x-ray powder diffraction and crystallographic information.

REFERENCES
1.

Heller, W.M.; Fleeger, C.A. USAN and the USP Dictionary of Drug Names, United States Pharmacopeial Convention, Inc.: Rockville, Maryland; 1990, p. 405.

2.

Pharmacological and Chemical Synonyms, Ninth Edition, Marler, E.E.J., Ed., Elsevier: New York; 1990, p.380.
Sittig, M. Pharmaceutical Manufacturing Encyclopedia, Second Edition, Noyes Publications, Park Ridge, N.J., 1988, p. 1058.

3.
4.

Martindale: the Extra Pharmacopeia, Twenty-ninthEdition, Reynolds, J.E.F., Ed., The Pharmaceutical Press: London; 1989, 1470. The Pharmacopeia of Japan, Eleventh Edition (English Version), The Society of Japanese Pharmacopeia: Tokyo, Japan; 1986, p.761.

5.

NAPHAZOLINE HYDROCHLORIDE

343

6.

The Merck Index, Eleventh Edition, Budavari, S.; ONeil, M.J.; Smith, A.; Heckelman, P.E., Eds.; Merck & Co., Inc.: Rahway , N.J., 1989, p.1008.
Scholz, C.R. Ind. Eng. Chem., 1945,37, 120. Hartmann, M.; Isler, H. Arch. Exptl. Path. Pharmakol., 1939, 192, 141,

7.

8.
9.
10.

Japta List: Japanese Drug Directory, Third Edition, Japan Pharmacetucial Traders Association: Tokyo, Japan; 1987, p. 395.
Kleeman, V.A.; Engel, J. Pharmazeutische Wirkstoffe, Georg Thieme Verlag Stuttgart: New York; 1982, p. 620. Luu Duc, C.; Pera, M.H.; Fillion, H.; Delord, C.A. Bull. SOC. Chim. Fr., 1976, (3-4 Part 2), 555. Kountourellis, I.E. Pharmazie, 1988, 43, 26. Clarke, E.G.C. Isolation and Identification of Drugs, The Pharmaceutical Press: London; 1969, p. 435.

11. 12.

13.
14.

The United States Pharmacopeia, Twenty Second Revision, United States Pharmacopeial Convention: Rockville, Maryland; 1990, Naphazoline Ophthalmic Solution Monograph, p. 917.
Podder, A.; Mukhopadhyay, B.P.; Dattagupta, J.K.; Saha, N.N. Acta Cryst., 1983, C39, 495. Stern, M.J.; King, L.D.; Marcus, A.D. J . Am. Pharrn. Assoc., 1959, 48, 641. Reference 14, Naphazoline Hydrochloride Monograph, p. 916. Ismaiel, A.; Twakkol, M. Pharmazie, 1974, 29, 54. Belal, S.; Elsayed, A.H.; Abdel-Hamid, M.E.; Abdine, H. J . Pharm. Sci., 1981, 70, 127. Sane, R.T.; Sane, S. Indian Drugs, 1979,16, 239. Bult, A.; Klasen, H.B. Pharm. Weekbld., 1974, 109, 513. Kovar, V.K.A.; Abdel-Hamid, M. Arch. Pharm., 1984,317, 246. Lajosne, S. Acta Pharm. Hung., 1980,50, 130.

15. 16. 17. 18. 19. 20. 21. 22. 23.

344

G . MICHAEL WALL

24. 25. 26. 27. 28.

Lajosne, S . Acta Phurm. Hung., 1980,50, 270. Lajosne, S . Acta Pharm. Hung., 1982, 52, 61. Salam, M.A.; Issa, A.S.; Mahrous, M.S. Anal. Lett., 1986, 19, 2207.
11, 36.

Solimar, S.A.; Abdine, H.; Mocos, M. Can. J . Pharrn. Sci., 1976, Bauer, J.; Krogh, S . J . Pharm. Sci., 1983, 72, 1347.

29.
30. 31. 32. 33. 34. 35. 36. 37. 38.

Al-Kaysi, H.N.; Salem, M.S.; Al-Khalili, N . Dlrasat, 1985, 12, 101. Alcon Laboratories, Inc., Unpublished data on fire. Koziol, T.R.; Jacob, J.T.; Achari, N. 1. t'harm. Sci., 1979, 68, 1135. Quaglio, M.P.; Cavicchi, G.S.; Cavicchioni, G. Boll. Chim. Farm., 1973, 112, 760. Schwartz, M.; Kuramoto, R.; Malspeis, L. J . Am. ktiarm. Assoc., 1956,15, 814. Stern, M.J. Drug Standards, 1958,26, 158.

European Pharmacopoeia, Second Edition, Maisontleuve S .A.: Sainte-Ruffine, France; 1982, p. 147.
Miescher, K.; Marxer, A.; Urech, E. Helv. C h b . Acta, 1951,34, 1. Dittgen, M.; Oe.stereich, S.; Eckhardt, D. F'harmazie, 1991,46, 716. Montfrans, G.A.; van Steenwijk, R.P; Vyth, A.; Borst, C. Acta Med. Scand., 1981, 209, 429.

NAYKOXEN

Fahad I . Al-Shamnlary

.' Neelofur Abdul Aziz Mian.'

and Mcrhamrnad Saleem Mian'

( I ) Clinical Laboratory Sciences Department College of Applied Medical Sciences King Saud University Riyadh, Saudi Arabia

(2) Pharmaceutical Chemistry Department College of Pharmacy King Saud University Riyadh, Saudi Arabia

ANALYTICAL PROFILES OF DRUG SUBSTANCES AND EXCIPIENTS - VOLUME 21

345

Copyright c 1992 by Academic Press, Inc All rights of reproduction reserved in any form

346

F. I. AL-SHAMMAKY. N. A. A. MIAN. A N D M . S. MIAN

CONTENTS
1

Introduction Description

2.1 Nomenclature 2 1 .l Chemical Names . 2.1.2 Generic Names 2.1 .3 Trade Names 2.2 Formulae 2.2.1 Empirical 2.2.2 Structural 2.2.3 CAS (Chemical Abstract Service Registry Number) 2.2.4 Optical Rotation 2.3 Molecular Weight 2.4 Elemental Composition 2.5 Appearance, Colour, Odour and Taste
3
Physical Properties

3.1 3.2 3.3 3.4 3.5 3.6


3.7

3.8 3.9 3.10 3.11 312 .

Melting Range Solubility Dissociation Constant Loss on drying Half-life Volume of Distribution LD50 Action Sulphated Ash Stability X-Ray Powder Differaction Spectral Properties 3.12 1 . Ultraviolet Spectra (UV) 3.12.2 Infrared Spectrum 3 12.3 Nuclear Magnetic Resonance Spectra . 3.12.3.1 PMR Spectrum 3 12.3.2 13C-NMR Spectrum . 3 12.4 Mass Spectrum .

Synthesis

NAPROXEN

347

Pharmacokinetics

5.1 Absorption and Distribution 5.2 Adverse Effects and Precautions 5.3 uses
6 Methods of Analysis 6.1 6.2 6.3 6.4 Identification Methods Spectrophotometric Nuclear Magnetic Resonance Method Titrimetric 6.5 Polarometric 6.6 Fluorometric 6.7 Chromatographic Methods 6.7.1 Thin Layer Chromatography (TLC) 6.7.2 High Performance Liquid Chromatography(HPLC) Acknowledgements References

348

F. J. AL-SHAMMARY. N. A. A. MIAN. AND M. S. MIAN

NAPROXEN
1

INTRODUCTION

Naproxen, a propionic acid derivative, is a nonsteroidal antiinflammatory agent (NSAIA). The drug is structurally and pharmacologically related to fenoprofen and ibuprofen (1). Naproxen is a nonsteroidal compound that has anti-inflammatory, analgesic and antipyretic activities. The mode of action is unknown, except that inhibition of prostaglandian synthesis may have an action role. While various manifestations of anti-inflammatory and analgesic actions are evident in patients with rheumatoid arthritis under treatment with naproxen or its congeners, there is no evidence that the progressive source of the underlying disease is altered (2)
I

DESCRIPTION
2.1

1) (2) (3)
(4)

(S)-6-Methoxy- CY -methyl-2-naph?halene acetic d-2-(6-methoxy-2-napht hy1)propionic acid(3) 2-naphthalenacetic acid 6-methoxyCY

acid (3)

(2,4)

-methyl-,(+)(2,4) (5,6,7)

(5) (6)

(+)-2-(6-Methoxy-2-naphthyl)propionic acid

(+)-6-Methoxy- CY -methyl-2-naphthaleneacetic acid (2,8)


2.1.2

Generic Names Naproxen

NAPROXEN

349

2.1.3

Trade

Names

Axer; Bonyl; calasen; Diocadal, Dysrnenalgit N; Equiproxen; Floginex; Laraflex; Laser; Naixan, Napren, E; Naprux; Naxen; Prexan; Prirneral; Proxen; Reuxen; Veradol; Xenar.
2.2 2.2.1

I
Dirical

..

(3)

c14 H14 0 3
2.2.2

Structural

2.2.3

GAS (Chemical Abstracf Service Reaistrv Number)

(22204-53-11
2.2.4

Qptical

Rotat i o n

+63.0 - 6.' (6) 85


2.3

[ a ] +~65.5O (C = 1 in chloroforrn)(9) In 4% w/v solution in chloroform

Jvlo lecular Weiaht (3)


230.26

2.4

Elemental C o r n m s i t i o n (3)

73.03%

H = 6.13%

20.84%

350

F. J. AL-SHAMMARY. N. A . A. MIAN. AND M. S. MIAN

2 . 5 m e a r a n c e . Colour Odour and Taste

White to off white crystalline powder with bitter taste (2) odourless or almost odourless (6).

PHYSICAL PROPERTIES
3.1
152 - 1540 (3) about 156O (6,7) 155O (2) 155.30 (9)

3 . 2 Solubility
Practically insoluble in water. Soluble in 25 parts of ethanoI(96%), in 20 parts of methanol, in 15 parts of chloroform and in 40 parts of ether (6). Practically insoluble in water at pH 2; freely soluble in water at pH 8 or above, sparingly soluble in alcohol (2).

..

3 . 3 Dissociation ComtaqJ
Pka = 4.2 (25O) (7)

. .

3.4

Loss

0n

drvina (6.8)

Dry it at 105O for 3 hours; it loses not more than 0.5% of its weight.

3.5 Half-life
Plasma half fife, 10-20 hours (mean 14) (7)

3 . 6 Volume of Distribution (7)


About 0.1 litre/kg

3.7 LDgo(3)
In mice (mg/kg): In rats (mg/kg): 435 intraveinous; 1235 orally

575 ip; 534 orally

NAPROXEN

35 I

3.8

A C t i Q n (2,6)

Anti-inflammatory: analgesic and antipyretic activities.


3.9

Sulphated A s h (1 0)

Not more than 0.1%. Use 1.5 g and ignite at a temperature of about 6000. 3.1 0

Stabilitv

Commercially available preparations of naproxen should be stored at room temperature, exposure of suspension to temperature exceeding 4OoC should be avoided (1). It should be kept in well-closed containers, protected from light (10).
3.1 1

X-Rav

Powder Differaction

X-ray powder differaction data for Naproxen is determined by De Camp, W.H. (11). X-ray powder differaction pattern was obtained by the use of Cu K a radiation on a Philips goniometer. The X-ray tube was typically operated at 40 KV and 20 mA. Detection was effected with a Nal (TI) scintillation counter coupled to a pulse-height analyser.
3.1 2 3.12.1

Sr>ectral

PrsDertieS SDectra CUV)

Ultraviolet

UV spectra of Naproxen (12) in Ethanol (6 mg%) was scanned from 200-400 nm (Fig. 1) using LKB 4054 UV/vis spectrophotometer. Naproxen exhibited the following UV data (Table 1).
3.12.2

Infrared

SDec t r u m

The IR spectrum of Naproxen as KBr disc (12) was recorded on a Perkin Elmer 1210 infrared spectrometer and is presented in Fig. (2). The structural assignments of Naproxen have been correlated with the following frequencies (Table 2).
3.12.3

Nuclear Maanetic Resonance Spectra


3.12.3.1

PMR

Spectrum

The PMR spectra of Naproxen (12) in BMSO-d6 (Fig. 3, 4) was recorded on a varian XL 200 MHZ NMR spectrometer using TNS as an internal reference. The following structural assignments have been made (Table 3).

352

F.J. AL-SHAMMARY. N. A. A. MIAN. AND M. S. M I A N

Table 1:

UV Data of Naproxen in Ethanol


nrn
21 2 21 5 263 27 1 31 7 33 1

Absorbance

(E )ci'

Molar Absorptivity am moVL


10795.355 10791.51 7 51 61.66 5084.907 1792.189 1987.91

A :

2.81 3 2.812 1.345 1.325 0.467 0.518

468.833 468.666 224.1 66 220.833 77.833 86.333

Table 2:

1.R. Characteristics of Naproxen


Frequency cm-1
31 80 3000 2940, 2930

Assignment

(Carboxylic) -OH. Aromatic C=C stretch. Alipathic C-H stretch. 0

1730

(Carboxylic)

6-

II

1600

Aromatic stretch.

0 0 U

0 a 3

m
0

' W 0

a m
0
(v

m
0 ' 0

.%

. w

0
h l

- Y

0
h l

- 0
(v

-%_

-I
0 0

h l

-cz

.
m
0 0

o cv

0 0

Ln

0 0

0 Ln 0

0 0

TRANSMITTANCE
0 0 u3 0 0 03

0 0 0
0 0

2
0 0 4

0 0

l o

0 0

03
0 0 0

cy
0 0

.c.r
N

0
m

v ,
0 0
#
0

aJ Q

w
Y

0 0 L n

C I

cr,
0

0 0

LL
. t

Cj, .-

354

00
4 0
I

Fig. ( 3 ) PMR SPECTRUM OF NAPROXEN IN C D C l

. -

Fig.

( 4)

PMR SPECTRUM OF NAPROXEN ( D20 Exchange 1

NAPROXEN

3.51

h
9

C H,

CH-COOH b a

C H,O

Table 3:

'H-NMR Characteristics of Naproxen

Proton Assignment
6 H aromatics (at d, e f, g, h, i) 3 H Cj)
5 H (at a, b, c )

I Chemical Shift
I
7.087 - 7.136 (t) 7.656, 7699 (d)

6 (ppm)

3.869, 3.878 ( d )
1.552

1.590 (d)

douolet, t = triplet.
3.12.3.2 1 3C-NMR

Spectrum

n I3C-NMR spectrum of Naproxen (12) i DMSO-d6 (Fig. 5, 6) was recorded on varian XL-200 NMR-spectrometer. The multiplicity of the resonance was obtained from APT (Attached Proton Test) program. The BC-NMR spectrum displayed all the fourteen carbon resonances. The narrow resonance range of some of the carbons makes the spectrum rather complex. The carbon chemical shifts assignments are presented in (Table 4).
3.1 2.4

Mass SDectrurn

The mass spectrum of Naproxen (12) obtained by electron impact ionication (Fig. 7) was recorded on a Finnigen MAT 90 spectrometer. The spectrum wc?s scanned from 50 to 500 a m a . Electron energy was 70 ev. Emission current 1 mA and ion source pressure torr. The most prominant fragnents and their relative intensities are presented in (Table 5).

358

F. J. AL-SHAMMARY, N. A. A. MIAN,AND M.S.MIAN

3
7
5

14
10

12

Table 4:
~~~ ~~~

Carbon-13 Chemical Shifts of Naproxen


~~

Carbon Assianment

:hemica1 Shift 18.1 15 45.306 55.267 128.867 133.818 134.825 157.696 181.052 105.559 119.027 129.292

(wml

Linterchangable A
127.21 7

126.137, 126.174

180

160

140

1 3

100

80

60

40

2oPPM

Fig. ( 5 ) C -NMR

13

SPECTRUM OF NAPROXEN IN C D C l

-A- -

mlr
; I

I Ii I

I I

1-40

IIII I I 1 I I

I t ) ])IIIIIII I I I I I I l q I I I I l I I I I

Ill

1 1 )

I I I1

120

100

80

60

40

I II I I l i l l l 20 P P M

Fig. ( 6 1

13

C -NMR OF NAPROXEN I N C O C l

(APT)

100.0

23

Fig. ( 7 ) MASS SPECTRUM OF NAPROXEN

362

F. 1. AL-SUMMARY. N. A. A. MIAN. AND M.S.MIAN

Table 5:
rnlz

Mass Spectrum of Naproxen


Relative intensitv % ions

230.1

100

21 5

185

58

169.9

10.2

153.2

141.1

NAPROXEN

363

SYNTHESIS SCHEME
Naproxen is prepared (13) by the acylation of 6 substituted naphthalenes by AcCl forming the 2-acetyl derivative, which is further converted to 2-naphthyl acetic acid. Esterification and alkylation of 2-naphthyl acetic acid in the prescence of H2S04, MeOH, NaH, Me1 and with NaOH gave after hydrolysis the naphthyl propionic acid. Resolution of 2-(6-methoxy-2-naphthyl) propionic (Naproxen) was readily achieved by crystallization of the cinchonidine salt.

SCHEME

6-substituted Naphthalene

2-acetyl d e r i v a t i v e

(1) Morpholine, S

CH, &O bH C H30


Naproxene.

( i ) H2SO4, CH30H ( i i ) NaH, C H s I ( i i i ) NaOH


I

D CH,O

(6 s u b s t i t u t c d ) 2 - N a p h t h y l a c e t i c acid.

364

F. 1. AL-SHAMMARY. N. A. A. MIAN, AND M.S MIAN .

PHARMACOKINETICS

5 . 1 Absorption and Distribut i a

. .

When administered as the acid or the sodium salt, naproxen is completely absorbed from the gastrointestinal tract; the sodium salt is absorbed more rapidly than the acid (1). Peak plasma levels (about 55 pg/ml) are reached in 2 to 4 hours after a 500 mg dose, and steady state levels are attained after 4-5 doses at 12 hours intervals. More than 99% is bound to serum albumin. The mean plasma half life is about 13 hours (2). Approximately 95% of a dose is excreted in the urine, principally as conjugates of naproxen and it's inactive metabolite 6-desmethyl naproxen (2). The apparent volume of distribution of naproxen averaged abut 8.3 L in healthy adults and about 11.9 L in patients with severe renal failure (serum creatinine 5.4-1 2.5 mg/dl)(2). In healthy adults, plasma half-life of naproxen reportly ranges from 10-20 hours. After entering the stomach naproxen sodium readily dissolves in gastric juice and about 30% of dose of naproxen is metabolised in the liver to 6-desmethyl naproxen, which is inactive. Most of the drug is excreted in urine as unchanged naproxen (10%) and 6desmethyl-naproxen (5%) and their glucuronide or other conjugates (82%). Some data, however suggest that renal excretion of unchanged naproxen may be negligible or absent. In patients of with severe renal failure, total body clearance of naproxen may increase apparently because of decreased binding of the drug serum proteins. A small amount (less than 5%) of the drug excreted in feces (2), precipitates is out as fine particles of naproxen. These particles provide a greater surface area for dissolution than the larger particles that result from naproxen tablet disintegration (14). When using conventional tablet formulation of naproxen sodium and naproxen, the former consequently produces earlier and higher plasma conc. of naproxen (15). The same holds true during administration of suppositories of naproxen sodium and naproxen (16). Mean time to peak plasma conc. (tmax) is about 1 hour for naproxen sodium and 2 hours for naproxen when administered to fasting subjects (14). With respect to concomitant antacid administration past studies have shown that sodium bicarbonate enhances the rate of naproxen absorption, magnesium carbonate caused a slight reduction, and a mixture of magnesium oxide and aluminium hydroxide gave a clear reduction in the rate of naproxen absorption (17).

NAPROXEN

365

5 . 2 Adve rse Effects a nd Precautions

Adverse reactions to naproxen mainly involve the GI tract, constipation, heartburn, abdominal pain, and nausea occur in about 3-9% of patients recieving the drug, less frequently, dyspepsia, diarrhea, stomatitis, vomiting, anorexia, flatulence occur (1). Adverse effects of naproxen is similar to that of ibuprofen. The most frequent adverse effects occuring are gastrointestinal disturbances. Peptric ulceration and gastro-intestinal bleeding have been reported, other side effects include headache, dizziness, nervousness, skin rash, pruritus, tinnitus, oedema, depression, drowsiness, insomnia, and blurred vision and other occular reactions. Hypersensitivity reactions, abnormalities of liver function tests, impairment of renal function including interstitial nephritis or the nephrotic-syndrome, agranulocytosis, and thrombocytopenia have occasionally been observed (5). Naprosyn (Naproxen) should not be used conmitantly with the related rug anaprox (naproxen sodium) since they both circulate as the naproxen anion (4). All aspirin-sensitive asthmatic patients developed reactions such as rhinorrhoea, tightness of chest, wheezing, dyspnoea, after taking naproxen in doses of 40-80 mg (18). Gastrointestinal reactions tended to be more frequent and severe when naproxen dosage was increased from 750 mg/day to 1500 mglday in patients with rheumatoid arthritis. In studies involving almost 500 children with juvenile arthritis the incidence of rash and prolonged bleeding time was increased, gastrointestinal and CNS reactions were reported at approximately the same rate, and other adverse effects were observed less frequently in children than in adults. However naproxen was not associated with a higher frequency of adverse effects in elderly (> 65 years) patients with rhematoid arthritis or osteoarthritis compared with younger patients (19, 20). Naproxen should be given with care to patients with asthma or bronchospasm, bleeding disorders, cardiovascular disease, peptic ulceration or a history of such ulceration, renal failure, and in those who are recieving coumarin anticoagulants. Patients who are sensitive to aspirin should generally not be given naproxen (5). Naproxen may interfere with some tests for 17-ketogenic steroids

(5).

F. 1. AL-SHAMMARY, N. A. A. MIAN, AND M. S. MIAN

Naproxen has analgesic, anti-inflammatory, and antipyretic properties; it is an inhibition of prostaglandin synthetase. The drug is used in rheumatic disorders such as ankylosing spondylititis, osteo arthritis, and rhematoid arthritis, in mild to moderate pain such as dysmenorrhoea, migrane and some musclokeletal disorders, and in acute gout (43). Naproxen is used to relieve mild to moderately severe pain. The drugs are also used for anti-inflammatory and analgesic effects in the symptomatic treatment of mild to moderately severe, acute and chronic muscleskeletal and soft tissue inflammation (1). The usual dose of naproxen or naproxen sodium is the equivalent of 500 mg to 1 g of naproxen daily in 2 divided doses. A dose of 10 mg per kg body-weight daily of naproxens in 2 divided doses has been used in children over 5 years of age with juvenile rheumatoid arthritis ( 5 ) . In painful conditions such as dysmenorrhoea the usual initial dose is the equivalent of 500 mg of naproxen followed by 250 mg every 6 or 8 hours. In accute gout an initial dose equivalent to 750 mg of naproxen followed by 250 rng every 8 hours has been suggested (5). Rectal administration of naproxen is sometimes employed naproxen has also been used orally as the piperazine salt (5). Naproxen is comparable to aspirin in controlling disease symptoms, but with lesser frequency and severity of nervous system and milder gastrointestinal adverse effects (2). Naproxen has been used effectively to relieve pain, fever, redness, swelling and tenderness in patients with accute gouty arthritis (1). One study indicates that single oral dose of naproxen (2.5 or 7.5 mg/kg) was at least as effective as a single oral dose of aspirin (15 mg/kg) in the reduction of fever in children. The result of one study suggested that the combination of naproxen sodium and ampicillin was more effective than ampiciline alone in elleviating fever, dyspnea, and coughing associated with accute respiratory infections in children (1). A favourable antipyretic effect with naproxen 5 mg/kg twice daily in children with fever caused by infection (21). Naproxen 7.5 mg/kg twice daily in children (22) and 250-375 mg twice daily adults (23) has been shown to be extremely effective

NAPROXEN

367

for controlling fever associated with malagnancy. Naproxen has been proposed as a test to distinguish neoplastic fever from infectious fever in cancer patients with fever of unknown origin (23).

METHODS OF ANALYSIS
6 . 1 JDENTlFlCATION METHO DS

The infrared absorption spectrum of a potassium bromide dispersion of its exhibits maxima only at the same wavelengths as that of a similar preparation of USP Naproxen R.S. (8). It gives liebermann's test black-green; Marquistes-brown. Sulphuric acid-orange (7). The light absorption in the range of 230 to 350 nm of a 0.004% w/v solution in methanol, exhibits four maxima, at 262, 271, 316 and 331 nm. The absorbance at 262 nm is about 0.91, at 271 nm, about 0.92 at 316 nm, about 0.26 and at 331 nm, about 0.30 (6). It melts at 156OC (6). Dissolve about 500 mg of Naproxen, accurately weighed in a mixture of 75 ml of methanol and 25 ml of water that has been previously neutralized to the phenolphthalein end point with 0.1 N NaOH. Dissolve by gentle warming, add phenolphthalein and titrate with 0.1N NaOH VS. Each ml of 0.1N NaOH is equivalent to 23.03 mg of C14H1403 (8). 6 . 2 SPECTROPHOTOMETRIC Spectrophotometric determination of naproxen in tablets form was done by Tosunoglu, S. (24). A portion of the crushed tablets containing about 250 mgs of naproxen was shaken with 50 mi of 96% ethanol for 30 minutes and the mixture was diluted to 100 mi with 96% ethanol and filtered. A 1 ml portion of diluted solution was mixed with 4 mM-rosaniline in 20% ethanol (3 ml) and extracted with 5 mi of CHC13 and the absorbance was measured at 545 nm against blank. The calibration range was 1 to 15 p g per ml. Recovery was 99.9%. Quantitation of naproxen with other drugs in pharmaceutical dosage forms by first and second derivative uv spectrometry by

368

F. J. AL-SHAMMARY, N. A. A. MIAN. AND M. S. MIAN

Mahrous, M.S. et al (25). Derivative uv spectroscopy was used to determine the cited drugs in capsules and tablets. First derivative spectroscopy was used to determine indomethacin in 0.1 N-HzS04 solution, and second derivative spectroscopy was used to determine naproxen and iboprofen in O.1N-NaOH solution. The method is rapid and accurate. 3 Powdered tablets (26) equivalent to 50 mg of naproxen were dissolved in 10 ml of methanol and boiled under reflux with 20 ml of 5M-HCI for 45 mins. The solution was cooled and the excess of HCI was removed under vacuum. The residue was dissolved in 10 ml of methanol, adjusted to pH 7.0 with NaOH solution and diluted to 100 ml with H20. To a portion of this solution, were added 1 ml of 0.05% P-NN-dimethyl phenylenediammonium chloride and 1 ml of aq. 0.2% K2Cr207 and H 2 0 to 25 ml. The absorbance was measured after 10 minutes (but with in 1 h) at 600 nm vs, a reagent blank. Beer's law was obeyed from 5-40 pg ml-1 ( E = 2760).

6.3

Tosunoglu, S . ; et al (27) determined naproxen by NMR spectrometry. Powdered tablets equivalent to 85 mg of naproxen were mixed with acetanilide (40-45 mg; internal standard) and extracted with CHC13 (25 ml). After ultrasonic agitation for 30 minutes, the mixture was filtered and a 10 ml portion o the filtrate f was evaporated to dryness in vacuo. The residue was dissolved in 0.8 ml of CHC13 and the spectrum was recorded on a Bruker NMR spectrometer operated at 250 and 300 MHz with the probe of 370. Chemical shifts were measured relative to tetramethylsilane at 1.58 and 2.09 ppm for naproxen and the internal standard respectively.
6 . 4 TlTRlMETRlC DETERMINATION

The determination of naproxen involved oscillometric titration (28) of its solution in aq. 20% acetone (10 ml, 10 mM in naproxen and containing 2 ml of aq. 0.1 M-NH3) with O.1M-KOH, a Radelk is type OK-302 apparaturs being used for determining the end point. Results obtained for naproxen indicated the good precision of the oscillometric technique.
6.5

Polarometric determination (29) of naproxen by dissolving in various heterocyclic, aromatic and aliphatic basic solvents and the

NAPROXEN

369

optical rotation was determined at Na and Hg iines. The total specific rotation was calculated, the results obtained showed variations of up to 22O at the Na line and of 30 at the Hfg line with heterocyclic solvents with aromatic amines the values obtained were lower than those of published in U.S.P. and B.P; Negative values of optical rotation were determined when naproxen was dissolved in aliphatic amines.
6 . 6 FLUOAIMETR IC DETERMINAT l W

Naproxen contents in sugar coated tablets was determined by fluorescence (30) spectrophotometry. Naproxen tablets with their sugar-coating removed, were powdered and a sample equivalent to about 25 mg of naproxen was dissolved in 5 ml of 1% NaOH and diluted with Y20. A 0.5 ml portion of the supernatant solution was mixed with 1 ml of 1M-HCI and dilute to 100 ml with H20 and the fluorescence of the solution was measured at 356 nrn (excitation a t 274 nm) vs. 0.01 M-HCI. Recovery was 39.5% with a coeff. of variation of 0.8%
6 . 7 CHROMATOGRAPHIC METHODS

671 ..

Thin laver c h r o m c r t o w h v (TLQ

CHCl3:methanol (17:3) as mobile phase. Spots corresponding to naproxen and its 6-demethylmetabolite (Rf 0.54 and 0.41, respectively) were visualized under 254 nrn radiation, scraped off and extracted with aq. 95% ethanol (4 x 5 ml). The extracts were diluted to 25 ml, and the absorbance was measured at 232 nm. The calibration graph covered the range 0.2-0.3pg m1-l in the final solution. 6.7.2 Hiah Performa nce Liauid ChromatoaraDhy

Naproxen and its metabolites were measured (31)in urine. 12 ml ot sample was acidified with 0.1 rnl of O.1M-HCI and extracted with 5 rnl of CHCI3. The extract was evaporated, and the residue was dissolved in 100 jd of ethanol. 10 pI portions were spotted on to silica gel 60 F254 plates and TLC was carried out with

* A summary of the sum of the HPLC methods for the analysis of Naproxen are given in the Table (6).

ACKNOWLEDGEMENTS

The authors are highly thankful to Mr. Babkir Awad Mustafa, College of

TABLE ( 6 )

Summary of HPLC conditions of Naproxen.


Flow rate rnl/rnin. Ietectior
t t f

Sample 'lasma or blood

3ef. 32

240 nm

Dctadecy IsiIane
(10 cm x 4.6 mm) of

acetic acid (1 125:1375:8) 25 um.amm.phosphate buffer (pH 3.0) in 75% methanol acetonitrile-acetate buffc (pH 4.8 or 4.2) 5mM-aq. sod. phosphateH3P04 buffer of pH 2.6 (19:l) containing 0.9% acetonitrile
1
.**

254 nrn

'lasma

33

Brownlee RP 18 (5um)

(25 cm x 4 rnm) of Hypersil ODS (5 urn) (22 cm x 4.6 mm) of underivetized 5-urn Brownlee silica with 7-um silica pre-column (1.5 cm x 4.6)

240 nm

'lasrna or serur

34

ml/min.

uv

>apsules or ablets

35

Octadecylsilane column methanol.sod. acetate buffer (25 cm x 4.5 mm i.d)

1.5 ml/rnin.

235 nm

Plasma

36

NAPROXEN

37 1

Applied Medical Sciences for his efforts in drawing the spectrums and figures. The authors also would like to thank Liberty S . Matibag, College of Applied Medical Sciences for her valuable and professional help in typing the manuscript.

REFERENCES
1 2 3 4

"Drug Information 91" p. 1124. American Society of Hospital Pharmacists. "Pharmaceutical Sciences" p. 1059. Company Easton, Pennsylvania, U.S.A. (1980). "The Merck Index"
Mack publishing

11th ed. p. 1014 (1989).

Physician's Desk Reference 42nd ed. p. 2101 (988). "Martindale" "The extra pharmacopeia" 29th ed. p. 28. The Pharmaceutical Press, London (1 989). "British Pharmacopoeia" Office London p. 384 (1988).
Her Majesty's Stationary

5
6

7 8

"Clark's Isolation and Identification of Drugs" 2nd ed. The Pharmaceutical Press London (1986).

"T h e U n it ed States Ph a r ma cop o e ia" United States Pharmacopoeia1 Convention, Inc., 12601. Twinbrook Parkway, Rockville, M.D. 20882 p. 917 (1990).
"The Merck Index"
10th ed. p. 920 (1983). Her Majesty's Stationary

9 10 11 12 13

'* Br iti s h Pha rma co poe ia" Office London p. 300 (1980).

De Camp, W.H. 933 (1984).

J. Assoc. Off. Anal. Chem. 67(5) 9 2 7 unpublished

Mohammad Saleem Mian, Neelofur Abdul Aziz Mian data (1992).

Jan T. Harrison; Brian Lewis; Peter Nelson; Wendell Rooks; Adolph Rostkowski; Albert Tomolonis and John H. Fried. J. Med. Chem. 13 203-205 (1970). A, Moyer S.

14

Cephalgia 6 (Suppl. 4)

77-88 (1986).

372

F.J. AL-SHAMMARY. N. A. A. MIAN, AND M.S. MIAN

15 16

Sevelius H., Runkel R., Segre E., Bloomfield, S.S. British Journal of Clinical Pharmacology 10 259-263 (1980). Gamst, O.N., Vesje, A.K., Arabakke, J., International Journal of Clinical Pharmacology, Therepy and Toxicoloty 22 99-103 (1984). Weber, S.S., Bankhurst, A.D., Therapeutic Drug Monitoring A. Szczeklik, et. Mroszczak, E., Ding, T.L. 3 75-83 (1981).
231 (1977).

17 18 19 20 21 22

al.

Br. Med. J. 2

Geczy, M., Peltier, L., Wolbach, R. 348-354 (1987). Husby, G. (1986). American J. Medicine

J. Rheumatology
81(Suppl. S.B.)

14 6-10

Szmyd, L., Perry, H.D. 99 598 (1985).

American Journal of Bpthamology

Azeemuddin, S.K.; Vega, R.A.; Kim, T.H.; Ragab, A.H.; The Effect o f Naproxen on Fever in Children with Malignancies Cancers 59 1966-1968 (1987). Peter A. Todd and Stephen 137 (1990). Tosunoglu, S. (1 9 8 9 ) . Acta.

23 24 25 26 27 28 29

P. Clissold

Drugs
31(3)

40(1} p. 91-

Pharrn. Turc.

p. 119-122

Mahrous, M.S.; Abdel-Khalek, M.; Abdel-Hamid, M.E.; Assoc. Off. Anal. Chem. 68(3) 535-539 (1985).

J.

Sastry, C.S.P.; Prasad, Tipirneni, A.S.R.; Suryanavayana, M.V.; Aruna, M. India Drugs 26(11) 643-644 (1989). Tosunaglu, S.; Buyuktimkin, N. 149-152 (1989). Kolodziejska, T. (1 983)(Pol.). Acta. Acta. Pharm. Turc.
40(3) 31(4)

Pol. Pharm.

357-360

Ceccarin, G.; Maione, A.M. 1054 (1989).

J. Pharm. Sci.

78(12) 1053-

NAPROXEN

313

3 0 Tang, H.; Wang, K. (1990) (Chinese) 31 32 33

Yaown Fenxi Zazhi

lO(6) 358-359

Abdel.-Moety, E.M.; Al-Obaid, A.M.; Jado, A.I.; Lotfi, E.A. Eur. J. Drug Metab. Pharmokinet. 13(4) 267-271 (1988). Levine, B.; Caplan, Y.H. ( 1 985).
Clin. Chem.

31(2)

346-347
533

Kazernifard, A.G.; Moore, D.E.

132 (1990).

J. Chromatogr.

125-

3 4 Streete, P.J.
35

J. Chromatogr.
Stewart, J.T.;

495
J.

179-193 (1989).
Chromatogr.

381 -389

Larnpert, B.M.;

( 990). 1

504(2)

36

Shirnek, J.L.; Rao, N.G.S.; and Wahba Khalil, S.K. 71(4) 436-439 (1982).

J. Phar. Sci.

PERGOLIDE MESYLATE

Delores J . Sprankle and Eric C. Jensen

Lilly Research Laboratories Eli Lilly and Company Indianapolis, IN 46285

ANALYTICAL PROFILES OF DRUG SUBSTANCES AND EXClPlENTS -VOLUME 21

375

Copyright 0 1992 by Academic Press, Inc. All rights of reproduction reserved in any form,

316

DELORES J. SPRANKLE AND ERIC C. JENSEN

TABLE OF CONTENTS
1. Description Name, Formula, and Molecular Weight 1.1 1.2 Appekance and Color 1.3 History

2. Synthesis
3. Physical Properties 3.1 Confirmation of Structure 3.1 1 SpectroscopicData 3.12 Potential Isomerism 3.2 Infrared Spectrum 3.3 Nuclear Magnetic Resonance Spectrum 3.4 Mass Spectrum 3.5 Fluorescence Identification 3.6 Fluorescence Spectrum 3.7 Ultraviolet Spectrum 3.8 Melting Range 3.9 Differential Thermal Analysis 3.10 Thermogravimemc Analysis 3.11 Optical Rotation 3.12 Crystal Properties 3.13 Solubility 3.14 Partition Coefficient 3.15 Ionization Constant, pKa 3.16 Color Identification Test 4. Methods 4.1 Identity 4.2 Elemental Analysis 4.3 Ultraviolet Spectrum 4.4 Chromatography 4.41 Thin Layer 4.42 High Performance Liquid

5 . Stability - Degradation
5.2

5.1

Degradation Profile Stability in Dosage Form

6. Drug Metabolism and Pharmacokinetics

7. References
8 . Acknowledgments

PERGOLIDEMESYLATE

377

1. DESCRIPTION

1.1 Name, Formula, and Molecular Weight


Pergolide mesylate is marketed by Eli Lilly and Company under the trade name Permax@. It is also commonly referred to as LY 127809. Chemically, it is known as 8~-[(methylthio)methyl]-6-propylergoline, monomethane sulfonate. The CAS Registry Number is 66 104-23-2. Empirical Formula: Molecular Weight: Structure: C19H26N2SCQ03S

410.6

1 2 Appearance and Color .


Pergolide mesylate occurs as white to off-white crystals or a crystalline powder .

13 History .
Permax (pergolide mesylate) is a dopamine receptor agonist at both DI and D2 receptor sites.' Permax is indicated as adjunctive treatment to levodopalcarbidopa in the management of signs and symptoms of Parkinson's disease. Administration of Permax should be initiated with a 0.05 mg/day dosage for the first 2 days. The dosage should then be gradually increased by 0.1 or 0.15 muday every third day over the next 12 days of therapy. The dosage may then be increased by 0.25mg/day every third day until an optimum therapeutic dosage is achieved. Permax is usually administered in divided doses three times per day. During dosage titration, the dosage of concurrent I-dopalcarbidopa may be cautiously decreased. 2

DEWRES J. SPRANKLEAND ERIC C. JENSEN

2. SYNTHESIS

Pergolide mesylate can be synthesized from dihydroelymoclavine.3 In the first step of this method, dihydroelymoclavine is demethylated by von Braun cleavage. The resulting cyanamide intermediate I is then cleaved to intermediate I1 via sodium hydroxide. The Wallach reaction utilizing propionaldehyde and formic acid then propylates N-6 to give intermediate n1. A mesylate ester functional group is added to intermediate 1 1using 1 methanesulfonyl chloride in pyridine to give intermediate IV. Displacement of the mesylate ester with methanethiol and sodium methoxide results in sodium thiomethoxide to produce crude pergolide base, intermediate V, which is isolated, and using methanesulfonic acid, is converted into its final form as the crystalline mesylate salt from methyl alcohol. The isolated pergolide mesylate is finally recrystallized from methanol.

Dlhydrodymoclavlna

cn2cn2cn3
(IV)

CH,SH,

NaOCH.

DMF

Pergollde

Mesllste

Figure I . Chemical synthesis for pergolide mesylate

PERGOLIDE MESYLATE

379

3. PHYSICAL PROPERTIES 3.1 Confirmation of Structure 3.11 Spectroscopic Data The chemical structure of pergolide mesylate was determined from the data of synthetic method, elemental analysis, ultraviolet (UV) spectra, infrared (IR) absorption spectra, hydrogen (1H) and carbon (13C) nuclear magnetic resonance (NMR) spectra, and mass spectra. The following is a summary discussion of the spectroscopic data and potential isomerism of pergolide mesylate to support the confirmation of structure of this compound? Pergolide mesylate (I), is an ergoline alkaloid that is structurally related to lysergic acid and its derivatives.5-6 The structures of several lysergic acid derivatives have been determined by X-ray crystallography 6, and the proton NMR spectra of lysergic acid and iso-lysergic acid diakylamides have been reported.5 The proof of structure for pergolide mesylate is provided by spectroscopic data and supported by correlation with lysergic acid, its derivatives, and other ergoline alkaloids.
SCH 3

CH - SO 3 3

The proton-decoupled 13C NMR spectrum of pergolide mesylate indicates the presence of twenty carbons, which are further identified as four nonprotonated carbons, six methine carbons, and ten methyl and methylene carbons via a 1% Distortionless Enhancement by Polarization Transfer (DEFT) spectrum.7 The eight aromatic and olefinic carbon resonances in the 134 ppm to 106 ppm region have chemical shifts consistent with an indole structure (2).8 The 1H NMR spectrum contains four aromatic proton resonances, from 6.88 to 7.22 ppm, whose chemical shifts are also indicative of an indole-type structure. The assignment of these carbon and proton resonances were confirmed with IH correlation spectroscopy (COSY) and W-1H heteronuclear correlation spectroscopy (HETCORR). The UV spectrum also indicates the presence of an indole chromophore.

380

DELORES J. SPRANKLE AND ERIC C. JENSEN

The NMR data shows the molecule contains a methyl with carbon and proton shifts at 10.78 and 0.93 ppm, respectively. The COSY experiment identifies the coupling of these methyl protons to a methylene site, with 13C and 1H chemical shifts at 15.53 and 1.68 ppm, which in turn is coupled to another methylene site. The 13C and 1H shifts of this last site (53.83 and 3.36 ppm) indicate this methylene is attached to a heteroatom. Correlation experiments c o n f m the coupling to an NH+ site, and the IR absorption band at 2556 cm-1 is indicative of MI+,allowing us to propose substructure 3.

The COSY experiment shows the protonated amine of 3 residing in a ring system as shown in 4. The 13C, 'H, COSY, and HETCORR NMR data are consistent with the structure proposed in 3. The l3C data indicate a methylene carbon, located at 55.53 ppm, attached to a heteronuclear site. The corresponding protons, located at 2.95 and 3.59 ppm, were assigned from the HETCORR experiment. The COSY experiment shows that these two protons are coupled to a methine site, whose proton is coupled to protons located at 1.44 ppm, 1.55 ppm, and 2.8 ppm. These protons were assigned to the corresponding carbons using the HETCORR data, and these l3C resonances were identified as methylenes from the DEPT spectrum. The COSY and HETCORR experiments also show a methylene carbon whose protons are coupled to a methine carbon bonded to a heteroatom, based upon IH and 13C chemical shifts, 2.55 and 36.44 ppm, respectively.

PERGOLIDE MESYLATE

38 I

The MS spectrum of pergolide mesylate contains a fragment with m/z 267, which is consistent with the combination of substructures 2 and 4 to yield 5.
CH

MS spectrometry also indicates the presence of an -S-CH3 group from the m/z 285 fragment. The IH and l3C spectra also shows resonances, located at 2.08 and 15.19 ppm, which are consistent with the -S-CH3 group. f Attachment o the -S-CH3 group to a methylene follows from the 13C data of the methylene. Presence of the mesylate moiety was confirmed by the carbon resonance located at 39.64 ppm and its correlation to the protons at 2.36 ppm.8 This is supported by IR absorption bands for an S-0-C stretch at 1038 cm-1 and 0-S-0 stretches at 1157 cm -1 and 1331 cm-1. The structure for pergolide mesylate (1) is supported by other spectroscopic evidence. For example, aromaticity is observable in the IR spectrum and in fragments ( d z 144,267,285) of the mass spectrum. The mass spectrum fragmentation pattern also supports the connectivity of the ring systems of the molecule.

382

DELORES J. SPRANKLE AND ERIC C. JENSEN

3.12 Potential Isomerism Protonation of the tertiary nitrogen to produce the quaternary amine salt gives rise to the possibility of both an a and p isomer at the nitrogen position. The 1H NMR spectrum indicates the presence of two NH+ protons, confirmed by the results of a deuterium exchange experiment, which are isomers. These assignments are further consistent with 6-a (6) and -p (7) confirmed by the coupling of the 6-a-NH+ 6-p-NH+ protons to lH (the and protons attach to the carbon at the 1' position). In anhydrous solvents, such as dimethylsulfoxide-ds, it is possible to observe both isomers. In aqueous solvents, the rate of exchange is such that these isomers rapidly interconvert, resulting in only one NH+ resonance.
SCH 3

r2
N

SCH 3
-

3 3 CH-So

f"2

CH - SO 3 3

PERGOLIDE MESYLATE

383

3.2 Infrared Spectrum

The infrared spectrum for pergolide mesylate as a potassium bromide pellet is illustrated in Figure 2. The spectrum was recorded on a Nicolet Model 60SXB Fourier Transform infrared spectrophotometer. The infrared spectrum of pergolide mesylate is positively identified with maxima at the following approximate wave numbers: 3183 cm-l,2556 cm-1, 1456 cm-1, 1157 cm-l, 1038 cm-l, and 775 cm-l. The major absorption bands for the infrared frequencies and the corresponding assignments are listed in Table I.
Table I. Infrared Band Assignments for Pergolide Mesylate Wavenumber (cm-1)
3183 3040 2556
1619, 1606 1456, 1443
1331, 1157

Assignment
N-H pyrole with hydrogen bonding: N-Hstretch

Aromatic: C-Hstretch

NH+: N-H stretch


Aromatic: C-Cstretch Aliphatic: C-Hdeformation
RS03-: 03-0 stretch RS03-: S-0-C stretch

1038
794.775

N-Hpyrole with hydrogen bonding: N-H


deformation

00 1

08

33NWLlIWSNtJklL %

09

06

02

PERGOLIDE MESYLATE

385

3.3 Nuclear Magnetic Resonance Spectrum The 300 MHz 1H spectrum of pergolide mesylate (10 mglmL) in dimethylsulfoxide-dg (2.5ppm) is shown in Figure 3. The spectrum was obtained on a Varian Unity spectrometer using the following instrumental parameters: 5 mm 1H/13C dual probe; spectral width, 4416 Hz; 90" pulse; 64K time-domain data points; acquisition time, 7.421 seconds; 100 scans and . probe temperature, 35OC. The spectrum was provided with 0 1 Hz Lorentzian line broadening. The proton-decoupled 13C spectrum of pergolide mesylate (50mg/mL) in dimethylsulfoxide-& (39.5 ppm) is shown in Figure 4. These data were obtained using a 5 mm lH/13C dual probe; spectral width, 11 658.5Hz; 90" pulse width; 64K time-domain data points; acquisition time, 5.49 seconds; relaxation delay, 2.4 seconds; WALTZ- 1 proton decoupling; 4000 scans and 6 probe temperature, 35C. The spectrum was processed with 1.0 Hz Lorentzian line broadening followed by the addition of 64K zero-fill data points. Structural assignments for both proton and carbon NMR spectra are listed in Table 11. Assignments are based on 'H, l3C, lH-lH COSY, DEPT, and lH-13C HETCORR experiments.
8'

CHZ-S-CH,

13

1'

3'

N1

.Dm

Figure 3. IH NMR spectrum of pergolide msylate in dimethylsulfoxide-dg (35 "C)

m 4

i
IY

I U

I 1

" I

11,

,"

"

"

w(
1

e#

I.

F .

Figure 4 . l3C NMR spectrum of pergolide mesylate in dimethylsdfoxide-dg (35 "C)

388

DELORES 1. SPRANKLE AND ERIC C . JENSEN

Table 11. NMR Chemical Shift Assignments of Pergolide Mesylate

d: doublet s: singlet

m: multiplet q: quartet t: triplet bd: broad doublet bs: broad singlet

3.4 Mass Spectrum


The electron impact mass spectrum of pergolide mesylate is shown in Figure 5. The spectrum was obtained using a VG Model 7070E magnetic sector instrument operating at 70 eV ionizing potential. A molecular ion for the free base at m/z 314 was observed. Accurate mass measurements on these fragments support these assignments. Several of these fragment assignments are illustrated in Table 111.

Ik U
9s.

98.
65.

88. E.
78.

65.
68. 55.

5%.
15.
49.

35.

154

285

3%

4ie

Figure 5. Electron impact mass spectrum ofpergolide mesylate

390

DELORES J . SPRANKLE AND ERIC C. JENSEN

m/z

Table 111. Assignment of Ion Fragments of the EI-MS Spectrum


Elemental composition

- - Parent ion mass mmua D B E ~ relative - - - intensities


observec
314.181
1.1

Structure

314

8.0

100

$CHZ
I

SCH3
I

H N

-299 299.36C -2.1 8.5

&
S+
I

285 285.144

--1.7

N H
SCH3
I

8.5

34

Q $
H*

H C:

--

PERGOLIDE MESYLATE

39 I

Table 111. Assignment of Ion Fragments of the EI-MS Spectrum (continued)


Elemental composition

3bserved
mass

mmu

Parent ion relative intensities

267.183

3 .O

8.5

11

dNH*

Structure

25 1 C17H19N2

251.151

3.3

9.5

223

C15H15N2

223.123

0.7

9.5

@
N
H'

392

DELORES J. SPRANKLE AND ERIC C. JENSEN

Table 111. Assignment of Ion Fragments of the ELMS Spectrum (continued)


Elemental Zompositior

observed
mass

--

-0.4 9.5

mmua

Parent ion D B E ~ relative intensities

structure

c 14H13N2

209.107

-194.095
2.2

9.5

[@] [&]
H' CH2

c 12H 1oN
168.079
2.1

8.5

10

@
,

~~

-2.1

H'

167.071

9.0

12

PERGOLIDE MESYLATE

393

Table 111. Assignment of Ion Fragments of the EI-MS Spectrum (continued)


Elemental :ompositior

observec
mass

- - Parent ion --3.5


9 .O

miz -

mmua

D B E ~ rclative intensities

Structure

154

154.056

-33

H-

154

154.056

-0.1

4.0

144 144.069

-0.2 7.0 11

144 144.078

-2.8 6.5

iJH2

--

394

DELORES J. SPRANKLE AND ERIC C . JENSEN

Table 111. Assignment of Ion Fragments of the EI-MS Spectrum (continued)


structure

a: milli mass unit (difference between observed mass and elemental composition mass expressed in thousandths of mass units)

b double bond equivalents

3.5 Fluorescence Identification When pergolide mesylate is viewed under long wavelength UV light (366 nm), the product is observed to fluoresce. Pergolide mesylate has a characteristic, pale bluish-white fluorescence. 3.6 Fluorescence Spectrum The fluorescence spectrum of pergolide mesylate in methanol is shown in Figure 6. The fluorescence excitation and emission analyses of the compound were obtained using a Perkin-Elmer MPF-66 fluorometer.

Wavelength (nm)
Figure 6 . Fluorescence spectrum of pergolide mesylate

396

DELORES J. SPRANKLE AND ERIC C. JENSEN

3 7 Ultraviolet Spectrum .
The UV absorption spectrum of pergolide mesylate is consistent with the absorption pattern for compounds with an indole chromophore. The electronic transition of the indole chromophore of the pergolide mesylate molecule is observed as follows: the absorption bands at 280 and 274 nm are A to A* electronic transitions separated by l L b and 'La vibrational transitions. Additional vibrational transitions for the indole chromophore in pergolide mesylate are observed by the 0-0 'Lb transition at 291 nm and the 1B transition at 224 nm. The maximum absorptions (hm a ) and the molar absorption coefficients (E m a ) of pergolide mesylate agree with those observed in 3-methylindole? a model compound with an indole chromophore, and support the structure of pergolide mesylate. A maximum absorption at or about 280 nm, a shoulder at or about 290 nm, and a minimum at or about 244 nrn are useful for identification of an indole structure. The UV absorption data of pergolide mesylate and 3-methylindole are shown in Table IV. Table IV. Maximum Absorption and Molar Absorption Coefficients
Compounds Maximum absorption (nm) Molar absorption coefficient W-lcm-1)

29 1 280 274 224

474 1 5667 5290 26930

pergolide mesylate

290 282

4700

5640 35500

223 3-methylindole

PERGOLIDE MESYLATE

391

Spectral acquisition was performed on a Perkin Elmer Model Lambda 6 spectrophotometer using 1-cm quartz cells. The ultraviolet spectra of pergolide mesylate in water, methanol, and dehydrated ethanol are shown in Figure 7. UV absorption spectra of pergolide mesylate in water, methanol, and dehydrated ethanol exhibit different absorbances but similar spectral patterns. The ultraviolet spectra of pergolide mesylate in buffers of various pH values are shown in Figure 8. Nearly consistent spectra are observed from pH 2-6. No absorption pattern is observed for pH values above 6. This is due to the insolubility of pergolide mesylate in alkaline media.

Solvents
Water

1%

hmax (nm)
219 280
28 1

Elcm
155
170

E max
6385 6980 6993

Methanol
Dehydrated ethanol

170

3.8 Melting Range


Pergolide mesylate melts between 258-260C (decomposition).

3.9 Differential Thermal Analysis


The DTA thermogram for pergolide mesylate shows a large, sharp endotherm at 263OC, indicating a melt (decomposition).

3.10 Thermogravimetric Analysis


The TGA thermogram for pergolide mesylate shows a weight loss starting at 255C followed by a continuous weight loss, indicating decomposition.

1.500

ABS I .ooo

0.500

0.000

240

265

290
WAVELENGTH (nml

315

340

Figure 7. Ultraviolet absorption spectra of pergolide mesylate, in water, methanol, and ethanol

2.950

ABS
1.900

0.850

.200

240

265

290 WAVELENGTH Inn1

315

340

Figure 8. Ultraviolet absorption spectra of pergolide mesylate, obtained at pH 2-8

400

DELORES J. SPRANKLE AND ERIC C. JENSEN

3.1 1 Optical Rotation Pergolide mesylate contains three asymmetric carbon centers, all of which are chiral in the drug. The absolute configurations at the three asymmetric centers of the molecule, C5, C8, and C10, have been confirmed by anomalous x-ray dispersion techniques.3>10The centers at positions C5 and C10 are fixed in the starting material (dihydroelymoclavine)and are not changed during the course of the synthesis. The third chiral site occurs at position C8, where the methylthiomethyl side chain may occur in either an alpha or beta position. The stereochemistry at this position is known based on the known stereochemistry of dihydroelymoclavine, which contains a beta hydroxymethyl group in the C8 position. The synthetic chemistry involved in synthesis of the bulk drug is not expected to alter this configuration. The results of the structural determination by x-ray confirm these results.

CH3S03-

Because the stereochemistry is fixed at all three chiral centers of the molecule, one would expect to observe optical rotation by the molecule. The optical rotation of pergolide mesylate was measured with the sodium d line (589 nm) as the light source at a concentration of 10 mg/rnl in dimethylformamide @MF) in a 100 mm cell using a Perkin-Elmer Polarimeter Model 241MC. The specific rotation measured at 2OoC has been observed to be between - 18.0 and -23.0'.

3.12 Crystal Properties


The X-ray powder diffraction pattern of pergolide mesylate, crystal Form I, is shown in Figure 9. The pattern was obtained using a Nicolet powder diffractometer using copper Ka irradiation (1.5418 A) with a graphite monochrometer. Pergolide mesylate has been observed to demonstrate two different crystalline forms. Only one of these is observed routinely in the manufacture of the bulk drug substance and has been designated Form I. A mixture of

2
4

180160z
(0

CI

140-

-d

a , c,
H
Il l

120100BO60-

(0
(0

4J

0 U

C 3

201

. 1
15.. 0

5.0

10.0

20..0 Two-Theta

25.0

30.0

35.0

Figure 9. Powder X-ray diffraction pattern of pergolide mesylate (crystal form I )

402

DELORES J. SPRANKLE AND ERIC C. JENSEN

Form I and Form I1 has been observed experimentally in laboratory lots where the recrystallized solutions were cooled very rapidly. On larger scale equipment where the cooling takes place more slowly, only Form I is observed. Form I and a mixture of Form I and I1 have been studied by x-ray 0 diffraction and by IR spectroscopy. Figure 1 shows the IR spectrum for Form I. Figure 11 shows the IR spectrum for a mixture of Forms I and 11. An additional peak at angle 5.788 degrees is noted in a mixture of Forms I and I1 material, while this peak is absent in the pattern for the Form I material. Bands appear to be sharper in Form I material, and subtle differences are observed in the groups of peaks centered at about 775 cm-1,607 cm-l, and 544 cm-*. In the IR spectrum of Form I, these groups appear as a triplet, a doublet, and a triplet, respectively. In the IR spectrum from the mixture of Form I and 11, the groups of bands appear as a doublet, a singlet, and a doublet, respectively.

3.13 Solubility
The solubility properties of pergolide mesylate are listed in Table VI. The measurement was performed by adding an excess amount of sample to a solvent, shaking for thirty seconds at five-minute intervals for a total of thirty minutes, filtering the saturated solution, and determining the concentration of the drug in the filtered solution with a UVNIS spectrophotometer. (For dehydrated ethanol, ether, dimethylformamide, acetonitrile, dichloromethane, acetone, and chloroform, the filtered solutions were evaporated to dryness and reconstituted in methanol). Table VI. Solubility of Pergolide Mesylate

i
4000
3600

3iOO

2800

WAVENUMBER

2400

2bOO

1600

li00

800

4 00

Figure 10. Infrared spectrum of pergolide mesylate (crystalform I)

I0

3600

3200

2800

2400

2600

1600

1500

800

I 400

WAVENUMBER

Figure I I . Infrared spectrum o pergolide mesylute (crymlforms I & II) f

PERGOLIDE MESYLATE

405

3.14 Partition Coefficient

The partition coefficient was determined as follows. Saturated aqueous solutions of the product in various pH buffer solutions were prepared and an equal amount of chloroform was added and the solutions were shaken at 25C for thirty minutes. The aqueous and organic layers were separated and filtered. A portion of the organic layer was evaporated to dryness and reconstituted using a mixture of equal parts of methanol and a solution of methionine (0.01 mg/ml) in 0.01 N hydrochloric acid. A portion of the aqueous layer was prepared in a mixture of equal parts of methanol and a solution of methionine (0.01 m g / d ) in 0.01 N hydrochloric acid. The test solutions were assayed by subjecting them to the isocratic conditions described under high performance liquid chromatography (see Section 4.42). The chlorofodwater partition coefficients were calculated as the ratio of the concentration of pergoIide mesylate in the organic phase to the concentration of pergolide mesylate in the aqueous phase. The results are shown in Table VII. A partition coefficient was observed in the acidic buffers. No partition coefficient was observed in alkaline buffers due to the insolubility of the pergolide mesylate in alkaline media.

System pH 2.19 Buffer pH 4.02 Buffer pH 6.10 Buffer


pH 7.95 Buffer

Concentration in organic layer (rndml) 1.42 1.93 1.322

Concentration in aqueous layer (mdml) 0.23 0.02


0.00

Partition coefficient 6.14 119.6


-

0.02

0.00

3.15 Ionization Constant, pKa The pKa of pergolide mesylate in a 66% dimethylformamide solution was measured by potentiometric titration with 2 N potassium hydroxide. The pKa of the secondary amine is 7.8.

406

DELORES J. SPRANKLE AND ERIC C. JENSEN

3.16 Color Identification Test

Pergolide mesylate contains an indole ring system. Indole derivatives with free 2- or 3- positions in the pyrrole ring condense with p-dimethylaminobenzaldehyde a sulfuric acid solution to produce a deep in purplish blue color (Neubauer Rhode Reaction). Since the more reactive 3position in pergolide mesylate is blocked, the condensation reaction of the less reactive 2-position results in a slow color development. The addition of femc chloride catalyzes this color reaction. A 0.1 mg/ml solution of pergolide mesylate is prepared in 2.5 N sulfuric acid. The addition of 1 ml of a p-dimethylaminobenzaldehydeTS and 3 drops of ferric chloride TS to 2 ml of the pergolide mesylate solution results in the immediate formation of a dark purplish blue colored solution (the control solution remains a pale yellow solution).

4. METHODS 4.1 Identity

The identity of pergolide mesylate is determined using the specificity of infrared spectroscopy, which differentiates it from any synthetic intermediates, process related substances or degradation products. Pergolide mesylate is triturated with potassium bromide and pressed into a transparent pellet for spectroscopic analysis. The identity is confirmed by comparison to a reference standard spectrum obtained under similar conditions.
4.2 Elemental Analysis

The following elemental composition was obtained using a Controlled Equipment Corporation Elemental Analyzer Model 240XA. Table VIII. Elemental Analysis Element C H N S
% Calculated

9 Found 6
58.26 7.46 6.89 15.49

58.51 7.37 6.82 15.62

PERGOLIDE MESYLATE

407

4.3 Ultraviolet Spectrum The identity of pergolide mesylate can be determined by ultraviolet spectroscopic assay at 280 nm. Using a 1 cm cell, the procedure is done at a concentration of 0.026 mg/mL pergolide mesylate in a mixture of equal parts of methanol and a solution of methionine (0.01 mg/ml) in 0.01 N hydrochloric acid. 4.4 Chromatography 4.41 Thin Layer A TLC method can be used for the identity and purity of raw material for pergolide mesylate. Precoated silica gel 60 F254 TLC plates are used as the stationary phase and a tertiary solvent system consisting of chlorofomdrnethanolfethylacetate (90:10:10) is used as the developing solvent. Visualization is performed by viewing the plate under long wavelength UV light (366 nm) and also by exposing the plate to iodine vapors prior to viewing under short wavelength UV light (254 nm). This developing solvent system will resolve known process related substances and degradation products from pergolide mesylate. 4.42 High Performance Liquid Conditions for quantifying pergolide mesylate have been optimized using isocratic reversed-phase HPLC. The mobile phase consists of a 1/1 mixture of methanol and acetonimle added to an equal part of a 2 mg/ml octanesulfonic acid, sodium salt buffer (0.1% glacial acetic acid v/v). A DuPont Zorbax RX column (25 cm x 4.6 mm; 5 micron particle size) is used in conjunction with a flow rate of 1.5 mL/min. Detection is obtained with an UV detector set at 280 nm. The sample is analyzed at a concentration of approximately 0.065 mg/mL,. The method is stability-indicating as indicated by its ability to separate pergolide mesylate and known degradation products. Figure 12 shows the separation of pergolide mesylate and its primary known degradation product, the sulfoxide. Gradient reversed-phase HPLC methodology is used to quantify pergolide mesylate and its potential related substances (synthetic impurities and degradation products). A Supelco LC-18-DB column (25 cm x 4.6 mm; 5 micron particle size) is used in conjunction with a flow rate of 1 mL/min. Detection is obtained with a UV detector set at 280 nm. The mobile phase components are a 0.5% morpholine buffer (v/v) in water (pH 7.0 with phosphoric acid) (A) and HPLC-grade methanol/acetonitrile/tetrahydrofuran (1:1:1) (B). A linear gradient is initiated at 30% (B) and increased 2%/minute for 35 minutes to a final concentration of 100% (B); then returned to 30% (B) and re-equilibrated for 20 minutes. The sample is analyzed at a concentration of approximately 3 rngJmL.

Figure 12. HPLC chromatogramof stabiliry-indicating assayfor pergolide rnesylate and degradationproducts . Peak identification: ( 1 ) pergolide mesylate sulfoxide, (2)pergolide mesylate

PERGOLIDE MESYLATE

409

5. STABILITY - DEGRADATION

5.1 Degradation Profile


Pergolide mesylate is stable as the bulk drug substance stored at 25Cfor for up to 2 years and is also stable under stress conditions (100C 4 weeksklosed container, 3 x 106 lux-hr. light exposure, 6 ' for 3 months, 5C 40C/75% Wclosed container for 3 months, 25"C/75% Wclosed container for 3 months, and 4@C/75% RWopen container for 6 months).I4 A trace (the level of (8~)-8-[(methylsulfinyl)methyl]-6-propyl-D-ergoline sulfoxide, I) is observed to remain relatively constant under both long term and stress storage conditions. Pergolide mesylate was also subjected to degradation experiments involving water, acid, base, heat, and light to generate degradation profiles. In all of these studies, the compound was evaluated by specific HPLC or GC, and TLC procedures. The studies revealed the following resu1ts:ll
1. Pergolide mesylate is stable in base (a slurry in 0 1 N sodium . hydroxide at 4' for 7 days). 0C 2. Pergolide mesylate is stable in acid (a slurry in 0 1 N hydrochloric . acid at 4 ' for 7 days). 0C 3. Pergolide mesylate is considered unstable in water when exposed to severe light and heat. When exposed to 3 x 106 lux-hr., pergolide mesylate undergoes degradation to yield two degradation products, the sulfoxide (I) and (8~)-8-[(methylsulfonyl)methyl]6-propylergoline (the sulfone, 11). The sulfone probably results from further oxidation of the sulfoxide. When exposed to 40C for 7 days in a slurry of water, a slight increase in the sulfoxide is observed.

The following structures represent potential degradation products of pergolide mesylate:

Sulfoxide (I)

Sulfone (11)

410

DELORES J. SPRANKLE AND ERIC C. JENSEN

The conclusions of these studies demonstrate that, while some decomposition was observed under extreme conditions, pergolide mesylate bulk drug substance is very stable.

5.2 Stability in Dosage Form


Pergolide mesylate is marketed as 0.05,0.25, and 1 mg Permax tablets. The active ingredient in the formulated tablet for all three dosage strengths is stable for up to two years when stored in a cold-formed aluminum blister package. 12

6. DRUG METABOLISM AND PHARMACOKINETICS


Pergolide mesylate is a potent dopamine receptor agonist. Pergolide is 10 to 1,OOO times more potent than bromocriptine on a milligram per milligram basis in various in vitro and in vivo test systems. Pergolide mesylate inhibits the secretion of prolactin in humans; it causes a transient rise in serum concentrations of growth hormone and a decrease in serum concentrations of iuteinizing hormone. In Parkinsons disease, pergolide mesylate is believed to exert its therapeutic effect by directly stimulating postsynaptic dopamine receptors in the nigrostriatal system. Information on oral systemic bioavailability of pergolide mesylate is unavailable because of the lack of a sufficiently sensitive assay to detect the drug after the administration of single doses. However, following oral administration of 14Cradiolabeled pergolide mesylate, approximately 55% of the administered radioactivity can be recovered from the urine and 5 % from expired C&, suggesting that a significant fraction is absorbed. Nothing can be concluded about the extent of presystemic clearance, if any. Data on post absorption dismbution of pergolide are unavailable. At least 10 metabolites have been detected, including N-despropylpergolide, pergolide sulfoxide, and pergolide sulfone. Pergolide sulfoxide and pergolide sulfone are dopamine agonists in animals. The other detected metabolites have not been identified and it is not known whether any other metabolites are active pharmacologically. The major route of excretion is via the kidneys. Pergolide is approximately 90% bound to plasma proteins. This extent of protein binding may be important to consider when pergolide mesylate is coadministered with other drugs known to affect protein binding. Permax is indicated as adjunctive treatment to levodopa/carbidopa in the management of the signs and symptoms of Parkinsons disease.

PERGOLIDE MESYLATE

41 I

Evidence to support the efficacy of pergolide mesylate as an antiparkinsonian adjunct was obtained in a multicenter study enrolling 376 patients with mild to moderate Parkinsons disease who were intolerant to I-dopalcarbidopa as manifested by moderate to severe dyskinesia and/or onoff phenomena. On average, the sample of patients evaluated had been on I-dopdcarbidopa for 3.9 years (range, 2 days to 16.8 years). The administration of pergolide mesylate permitted a 5 to 30%reduction in daily dose of I-dopa. On average, these patients treated with pergolide mesylate maintained an equivalent or better clinical status than they exhibited at baseline.

412

DELORES 1. SPRANKLE AND ERIC C. JENSEN

7. REFERENCES

1.
2. 3. 4.

Package Insert, PermaxB, Eli Lilly and Company. Langtry, H.D. and Clissold, S.P. (1990). Drugs 39,493. Kornfeld, E.C., Bach, N.J. (1979). U.S. patent 4,166,182. McCune, K.A., Maple, S.R., Cooke, G.G., and Underbrink, C.D. (Eli Lilly and Company, Lilly Research Laboratories) Confirmation of Structure of Pergolide Mesylate internal report, 29 July 1991. Bailey, K., Grey, A.A. (1972). Can J. Chem. 2,3876. Baker, R.W., Chothia, C., Pauling, P., Weber, H.P. (1973). Mol. Pharm. 9, 23. Derome, A.D. (1987). in Modern Techniquesfor Chemistry Research; Chapter 6. Pergamaon Press, New York. Pretsch, E., Seibl, J., Simon, W., Clerc, T. (1989). in Tables of Spectral Data for Structure Determination o Organic Compounds; p. C160. f Springer-Verlag, New York.

5.

6.
7.
8.

9.

The Stadtler Handbook of Ultraviolet Spectra (1970). Stadtler Research Laboratories, Philadelphia.

10. Ma, L., Camerman, N., Swartzendruber, J. Jones, N., and Camerman, A. (1987). Can. J. Chem. 65, 256. 11. Jensen, E.C. et al. (Eli Lilly and Company, Lilly Research Laboratories) Physiochemistry of Pergolide Mesylate, internal report, 1990. 12. Jensen, E.C. er af. (Eli Lilly and Company, Lilly Research Laboratories) Permax Stability Reports, Japanese New Drug Application. 13. Jensen, E.C. et al. (Eli Lilly and Company, Lilly Research Laboratories) U.S. New Drug Application for Pergolide Mesylate, 1985. 14. Jensen, E.C. et al. (Eli Lilly and Company, Lilly Research Laboratories) Pergolide Mesylate Stability Reports, Japanese New Drug Application.

PERGOLIDE MESYLATE

413

8. ACKNOWLEDGMENTS
The authors wish to express their sincere thanks to the following individuals who have provided information for portions of this chapter: K.A. McCune, S.R. Maple, G.G. Cooke, C.D. Underbrink, and G. Stephenson for the spectroscopic data ; A.G. Wich and T. Wozniak for chapter review; and B.T. Farrell for method development.

PREDNISOLONE

Syed Laik Ali

Zentrallaboratorium Deutscher Apotheker

6236 Eschborn
Germany

ANALYTICAL PROFILES OF DRUG SUBSTANCES AND EXClPlENTS -VOLUME 21

415

Copyright D 1992 by Academic Press, Inc All rights of reproduction reserved In any form

416

SYED LAIK ALI

Prednisolone Syed Laik A l i


1.

H i story
Nomenc 1ature Description Name, Formula, Molecular weight Appearance I Colour Odour I Taste Svnthesis Phvsical properties Solubi 1 ity Loss on drying Melting point Specifical optical rotation Residue on ignition Selenium Light absorption Related impurities Colour reactions Ultraviolet spectrum Infrared spectrum Nuclear magnetic resonance spectrum Mass spectrum Crystal structure Po 1 ymorp h i sm Circu 1 ar dichroi sm Stability and deqradation

2. 3. 3.1 3.2

4.
5. 5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8 5.9 5.10 5.11 5.12 5.13 5.14 5.15 5.16 6.

PREDNISOLONE

417

7. 7.1
7.2 7.3 7.4

7.5 7.5.1
7.5.2 7.5.3 7.5.4

Methods of analysis Colorimetric and spectrophotometr ic determination Polarography Radiochemistry and radioimmunoassay NMR determination Chromatographic methods Thin layer chromatography High performance liquid chromatography Gas chromatography-mass spectrometry Supercritical fluid chromatography

a.
9.
10.

I n vitro dissolution
Pharmacokinetics and druq metabolism Acknowledqements References

11.

418

SYED LAlK ALI

Prednisolone

1.

History Hershberg and co-workers (1) observed at first that the dihydroderivative of hydrocortison, prednisolon, possesses a 4 to 5 times stronger antirheumatic and antia 1 1 erg i c activity , showing simu 1 taneous1 y 1esser undesired side effects. Nomenclature 1 ,2-Dehydrohydrocortisone; Pregna-1 ,4-diene-3,20-dione,11 , 17 21-tri- hydroxy-l1R-1
7a,21-Trihydro~y-l,4-pregnadien-3~2O-dion. formula is illustrated at the next page (Fig. 1).

2.

The

3.

Descriution Name, Formula, Molecular weiqht Prednisolone; C21H2805 360,45 (anhydrous) Appearance, Colour, Odour, Taste A white or almost white, crystalline hygroscopic, odourless powder with a bitter taste. Svnthesis Prednisolone can be obtained with a chemical dehydration of hydrocortisone with selendioxide in tertiary butanol (2) or microbiological ly through the dehydration action of corynebacterium simplex in Al,Z-position ( 3 , 4 . A methanolic solution of the ) substrate was mixed with a 24 hours old bacteria

3.1

3.2

4.

PREDMSOLONE

419

PREDNISOLON

ch20h i
C=O

&-QH

0Fig. 1
S t r u c t u r a l Formula

420

SYED LAIK ALI

culture (0.1 % yeast extract, buffer, pH 7.0) and was shaken for 3 to 34 hours at 28 "C. The contents were then extracted with chloroform and prednisolon could be crystalised from aceton in very good yield. Wettstein and Co-Workers have further found that an enzymatic introduction of a 1,Z-double bond in hydrocortisone could best be obtained with mushrooms of Genus Didy mella type ( 5 , 6). The 1,Z-double bond could also be introduced in the molecule of hydrocortisone chemically through 2,4-dibromination of 3-ketone and then the subsequent dehydrobromination ( 7 ) . The yield is only 10 - 15 %- The squibb company used this classical method for the production of 9 -Fluorprednisolon (8).
5. 5.1

Physical properties Solubility (9, 10) Prednisolone is very slightly soluble in water, soluble in 27 parts of absolute ethanol, in 30 parts of ethanol, in 50 parts o f acetone and in 1 0 parts 8 o f chloroform. It is soluble in dioxane and methanol.
Loss on dryinq (10. 111

5.2

Anhydrous prednisolone loses not more than 1.0 % of its weight when dried at 100 - 105 "C. Hydrous prednisolone loses not more than 7.0 % of its weight.
5.3

Meltina point (10) About 230 "C with decomposition.

PREDNISOLONE

42 1

5.4

Specific optical rotation (10) In a 1 % m/v Solution in 1,4-dioxanet +96 to +120 Residue on ignition (11) Negligible, from 100 mg. Selenium (11) 0.003 %, a 200 mg test specimen being used.

'.

5.5

5.6

5.7

Liqht Absorption (10) The A ( 1 %, 1 cm) in 96 % ethanol at the maximum of 240 nm i s between 400 to 430. Related impurities (10) Between 1 to 2 % using silica gel TLC plates containing a fluorescent indicator and a mixture of dichlormethane + ether + methanol + water, 77:15:8:1.2 as mobile phase and detection under UV 254 nm. Colour reactions The simplest reagent, used for more than 40 years in steroid analysis is concentrated sulfuric acid. They exhibit intense spectra in the range 220 - 600 nm. Prednisolone shows 2 hours after dissolution in concentrated sulphuric acid an absorption maximum at 470 nm with a specific extinction o f 89. Prednisolone gives after dissolving in conc. sulphuric acid ( 1 mg/ml) instantaneously a red colour which after dilution with water changes to violet-brown ( I ) . Prednisolone (1 mg in 5 ml nitromethane or nitrobenzene) reacts with aluminium

5.8

5.9

422

SYED LAIK 4 L I

chloride ( 4 g anhydrous aluminium chloride in 10 ml nitromethane or nitrobenzene, 2 ml reagent) to give a weak orange colouration (12).
5.10

Ultraviolet spectrum Prednisolone shows absorption maximum in methanol at 242 nm. The E 1 %, 1 cm in this solvent is reported to be 416 and the molecular extinction coefficient 15000 (13). The UV spectrum is shown in Fig. 2. Infrared spectrum The infrared spectrum i s given in Fig. 3. The spectrum was obtained with a Perkin-Elmer 1420 Ratio Recording Infrared Spectrophotometer from a KBr pel let. Nuclear maanetic resonance spectrum The nuclear magnetic resonance spectrum o f prednisolone was taken with a Varian 60 MHZ spectrometer i n deuterated dimethyl sulfoxide. The spectrum is reproduced in Fig. 4. Mass spectrum The mass spectrum was recorded with a Varian Mat 311 mass spectrometer using direct inlet in EI-mode at 80 ev and source temperature of 300C. The spectrum is illustrated in Fig. 5.

5.11

5.12

5.13

PREDNISOLONE

423

F i g . 2 (13)
UV S p e c t r u m o f P r e d n i s o l o n e i n Methanol

Fig. 3 IR Spectrum o f Prednisolone, KBr P e l l e t Perkin-Elmer 1420 Spectrophotometer

a, c, a, E c,
0

a,
v)

z
\o
C
*rl

w
W
C

4 0

-4

u)

+
0
E

k L

k c,

-tn
v)
-rl

mtx

LLZ

.z

a ,

425

426

SYED LAlK ALI

Fig. 5 Mass Spectrum o f P r e d n i s o l o n e

PREDNISOLONE

427

5.14 Crystal structure Inclusion complexation o f prednisolone with three Cyclodextrins, a p and -phomologues, in aqueous solution and in solid phase was examined using UV absorption, CD, C 13 NMR, C 13-CP/MAS-NMRI X-ray d iffractometry and thermal analysis. The spectroscopic data suggested the different inclusion mode of prednisolone within the three cyclodextrin cavities. X-ray diffraction patterns of the complexes differed significantly from those of the physical mixtures (14). 5.15

Polvmorphism Prednisolone shows the phenomenon of polymorphism (15, 16). The results o f investigation on po 1ymorph i sm and Pseudopo1 ymorph i sm (formation of solvates) of about 100 steroids including prednisolone is described. Prednisolone forms solvates with water and chloroform. These polymorphic forms have melting points between 218 234 "C and 210 - 225 "C. The hydrate always represents the most stable form. The analytical methods of thermomicroscopy, differential scanning calorimetry (DSC) and infrared spectrophotometry were applied for investigation (17, 18, 19, 20, 21). The therapeutic activity varies for the different polymorphs o f the same chemical substance. This behaviour is attributed to various factors but especially to the differences in the crystalline structure (22, 23, 24).

428

SYED LAIK ALI

Veiga and co-workers have isolated three forms I , 11, 111, of prednisolone which were identified by IR spectroscopy and characterized by scanning electron microscopy, X-ray powder diffraction and hot stage microscopy (25). The study by optical microscopy as well as scanning electron microscopy revealed that the crystals have a very different appearance. Form I has small, tridimensional crystals with irregular shape and granular surface. Crystals of form I 1 have a lamellar structure, well defined edges with smaller and very differently shaped crystals grown on their surfaces. Crystals of form I11 are reported to be similar in size to those of form 11; they have regular shape, but are formed by layers, so their edges are not well defined and are rather opaque (25). Diffractometer patterns are shown in fig. 6, interplanar spacings are presented in table 1 and unit cell parameters in table 2. The study was carried out by a Phillips PW 1010 powder diffactometer using the incident radiation Cu K (Y (X=1.5418A0) filterd with Nickel. The unit-cell parameters and the interplanar spacings were determined using a counter diffractometer and powdered samples containing si 1 icon powder as internal standard. Unit cell parameters have been assigned to forms I and 111, automatic indexation tests have failed to yield a solution for from I 1 (25). Table 3 shows the results of hot stage study. During the heating process forms I and I11 remained unchanged until the melting point was reached. Form I 1 showed in the

PREDNISOIBNE

429

FORM I
L)

c
Y

3
H

FORM I t

FORM 111

Fig. 6 (25) X-ray D i f f r a c t i o n P a t t e r n s o f P r e d n i s o l o n e

430

SYED LAIK ALI

10.9743 85840 6.3657 5.7120 5 m 3 5.4335 50924 5.0349 4.5484 4.1874 41)188 3.9139 35729 3.5173 5.4635 3.3985 52407
31)868

3M)55
2 m 5 2-7945 2.7526 2.7121

16.9SO3 10.9743 6.8044 6.1672 5.9806 56577 5.5546 50349 49239 4&670 4.5600 45709 4.3080 4m70 3.9224 38471 3.7124 36897 3.4635 3.4113 33483 331 I 6 3246s

10.9743 8.3S40 6:4116 5.7304


516043

5.4335 5.0349 4.5484 43392 4.1726 4.0188 3.9054 3.7825 3.7048 3.587 I 3.524 I 3.4615 3.3965 32465 3.1951
3.1079

258 .66
2.4693 23599 233018 22739 22176 2-1392 2.1 157 2M52 2fi5I 2.0128 I. % 90 I .979s I8827

37 m3 2 3 7 I2
2 m

2.8290
2.7363

2.6685 26766 261% 25129 2.4662 2.4275 2.-*2 22s22


2.~65 1

2.9956 2.9144 250m 2.5810 2.4959 25x29 23103 zm22 I S772

2.0107 I .%33 1.9240


1S226

1.7730

PREDNISOLONE

43 I

TABLE I1

(25)

Unit cell parameters.


Crystal
S>1ICm

Momclinic

Form I

&lonoclinic Form II

1 ID48 (A)

16.904 (A)
13.167

13.3%

6 3 I5 .
9(r

7.909

90s3m

9 1-76

90

90

TABLE 111

(25)
Form I
Form Ill

ram
Partial melting and recrystallization ienip. ("1
Melting point
(")

II

115-130

2 10224

205-218

2 10-220

432

SYED LAlK ALI

range of 115 - 130 "C a partial, intracrystalline melting with further recrystallization which then remains unchanged up to their melting point. Veiga and coworkers deduced from these facts that I and I 1 1 are anhydrous polymorphic forms of prednisolone while the form I 1 is a 1.5 hydrate and probably a twin (26). 5.16 Circular dichroism Circular dichroism (CD) is much more useful than any other spectroscopic technique in control 1 ing the sterospecificity of the reactions in the total synthesis of steroids. Prednisolone can be determined simultaneously along hydrocortisone. Hydrocortisone can be measured selectively at 326 nm where prednisolone does not show dichroism, while at 314 nm prednisolone can be measured on the basis of its selective dichroism (27, 2 ) 8.

6.

Stabilitv and deqradation One of the identified decomposition products formed during the anaerobic decomposition of prednisolone at pH 8 is 17-deoxyprednisolone. This decomposition product differs from prednisolone only in the side chain. The hydroxyl group at C 17 disappears thus giving 17-deoxyprednisolone (29). Gutman and Meister (30) found that with the dihydroxyacetone side chain of prednisolone two reactions predominate which yield the 17-ketosteroid and the hydroxy acid ( 3 0 ) . The isolation through TLC and HPLC methods and structural e 1 uci dat ion through po 1 arograph i c and

PREDNISOLONE

433

masspectrometric techniques is described. A decomposition mechanism of prednisolone leading to 17-deoxyprednisolone is postulated. Studies on the stability of corticosteroids and degradation patterns in aqueous solution are described ( 3 1 ) . Formation and degradation kinetics of 2 1 - d e h y d r o c o r t i c o s t e r o i d s , key intermediates in the oxidative decomposition of 21-hydroxycorticoids (32) , kinetics and mechanism of the acid-catalyzed degradation of corticosteroids are reported ( 3 3 ) . Another decomposition product formed during the anerobic decomposition of prednisolone is 17-deoxy-21-dehydroprednisolone. This can only be detected when the decomposition of prednisolone takes place at about pH 6 or lower pH-values. This product has been isotaled through chromatographic techniques and structure elucidated through mass spectrometry ( 3 4 ) . This product differs only from prednisolone in the side chain where the hydroxyl group at C 17 has disappeared and at C 21 the hydroxyl group has been changed to an aldehyde group (341. Another decomposition product, the 17-Ketosteroid where at C 17 the hydroxyl group has been converted to a keto group, is described. The isolation and the structural elucidation of 17-Ketosteroid is reported as well as decomposition mechanisms of prednisolone leading to the 17-ketosteroid are discussed ( 3 5 ) . The major decomposition product of prednisolone phosphate formed under anerobic decomposition conditions in

434

SYED LAIK ALI

aqueous solution at pH 8.3 i s identified as 17 a-h ydroxy- 1 7a- hydroxymethy 1 - 17- keto-Dhomosteroid phosphate. Isolation, structural elucidation and a mechanism leading to this compound are postulated (36). another publication the stability and interactions between prednisolone and urea in ointments and the influence of one component on the other are discussed (37). Factors influencing stabi 1 ity, the rate of disappearance of prednisolone from aqueous solution have been investigated (38). The rate exhibited a marked dependancy on buffer concentration. Prednisolone is susceptible to degradative reactions which involve the 17-dihydroxyacetone side-chain. Transformation and elimination of the side-chain have been shown to occur both in the presence and absence of oxygen. Autoxidation, however, appears to be the mode o f destruction which is most likely to be responsible for stability problems in drug products. The involvement of trace metals in catalyzing the autoxidation i s an obvious possiblity. Trace-metal impurities which were present in the buffer reagents were catalytically involved in the degradation. Rate constants were determined over a wide range o f pH in borate and phosphate buffers and in the presence and absence o f ethylenediamine tetraacetic acid. The rate of the apparent metal-catalysed reaction was pH dependant above pH 7 and below pH 5 and exhibited a first-order dependency on the hydroxide-ion in the

In

PREDNISOLONE

435

intermediate range. In the presence of EDTA, the rate of reaction was strongly dependant on OH-concentration above pH 8 but exhibited only slight dependency at lower pH values. Addition of EDTA provided a method to isolate and quantitate the rate o f the apparent metal-catalyzed reaction (38). The stability of prednisolone in an aqueous-organic solvent system (40 % lI2-propandiol + 38 % tetraglycol + 30 % water) under accelerated conditions has been studied. It was possible to demonstrate six different decomposition products. The four major products were identified by TLC. The accelerated stability tests were evaluated using a stability-indicating assay procedure. Although decomposition of prednisolone in solution is complex, stability prediction via Arrhenius plotting is possible. The degradation of prednisolone in this system is a first order kinetic reaction. The temperature dependence of degradation process in this organic-aqueous system is illustrated in fig. 7 and the Arrhenius plot of this diagramm is shown in fig. 8 (39).
7.
7.1

Methods of analvsis Colorimetric and spectrophotometric determination Blue tetrazolium (BT) and Phenylhydrazine H2SO4 (PH) reagents react with the intact side chain at C17 o f prednisolone while isonicotinic acid hydrazide (INH) and UV-absorption depend upon conjugation in Ring A at the other end of the prednisolone molecule. Since

I
100-

7.

90

w m

eo

f\sr
70.

-4

Fig. 7 (39) Temperature-Dependence of Degradation o f P r e d n i s o l o n e

0 4

a,

0 73
-4 5
v)

n
k
Lc

a, k

a,

E
k
0

2
+J 0

431

438

SYED LAIK ALI

the reactions in these four methods occur with different portions of the molecule, they can be used to detect and distinguish between decomposition products (40). The PH method is described by Silber and Porter (41) and the I N H method is given by Umberger (42). The Porter-Silber reaction for the colorimetric determination of corticosteroids is applicable only to steroids with a side chain at position 17 or their derivatives that are readily hydrolysed in the strongly acid medium (43). Prior to the determination by BT, PH, I N H and UV method a column chromatographic clean-up and removal of decomposition products and interfering substances has been performed (44). The method of tetrazolium assay of prednisolone consists of reaction of the substance (34 - 36 ug/ml) with a 0.5 % solution of triphenyl-tetrazolium chlorid (TTC) solution in aldehyde-free ethanol (96 %) and measuring the extinction at 485 nm (45). A colorimetric method of determination of prednisolone in powder and tablet forms using ammonium molybdate has been described. Prednisolone gives a blue colour with an absorption maximum at 655 nm. The range of sensitivity for prednisolone is given between 5 - 30 ug ( 4 6 ) . The prednisolone contents in low concentrated ointments and creams were measured with blue tetrazolium reaction after several steps of extraction and clean-up ( 4 4 ) . A large number of prednisolone dosage forms have been examined using tetrazolium blue method ( 4 8 ) . A review of methods of practical importance for the determination of steroids in

PREDNISOLONE

439

pharmaceutical formulations i s given (49). The most generally used method for the assay o f formulations containing unsaturated 3-Ketosteroids is their condensation with isonicotinoyl hydrazide ( I N H ) and measurement of the formed hydrazones in strongly acid medium at 410 nm (E 17000). For the determination of prednisolone in dosage forms on the basis of their side chain at C-17 tetrazolium methods based on the reducing properties of the side chain are stability indicating. Both the triphenyl tetrazol ium chloride and tetrazolium blue methods are fairly sensitive having molar absorptivities of 16200 and 24000 respectively (49, 50). Condensation of the glyoxal, obtained by cupric acetate oxidation of prednisolone, with aqueous phenylhydrazine reagent affords a near UV chromophore at 366 nm with a molar extinction coefficient of 17000 ( 5 1 ) . The TTC and BT methods have a relative standard deviation o f less than 1 % in the assay o f bulk corticosteroids and not more than 2 % for dosage forms ( 5 2 ) . The TTC method has been used for the determindtion of prednisolone in tablets (53, 54) , ointments (55, 56) and for the kinetic investigation of its decomposition in alkaline media (57). The sodium borohydride method has been appl ied by Gorog for the analysis of prednisolone in ointments (58). An alcoholic solution of ointment containing 10 - 15 mg prednisolone is treated with 1 N Sodium hydroxide followed by the addition of 100 mg sodium

440

SYED LAIK ALI

borohydride. The mixture is refluxed for 1 h, cooled and treated with 1 N HC1. The absorption of the resulting solution is measured at 243 nm against a corresponding blind solution. 7.2 Polaroaraphv The electoanalyt ical behaviour of predn i solone along with other corticosteroids has been studied in supporting electrolytes. Dependence of the peak potentials on the structure of the steroids at concentrations of 10-4 M in 0.03 M tetramethyl ammonium hydroxide (TMAH) in methanol, in Britton-Robinson buffer pH 10 (50 % V/V in methanol) and in 0.02 M TMAH in dimethyl formamide (87 % V/V) has been studied. In prednisolone the reduction of C-3 and C-20 keto groups takes place. Both reduction steps can be used for analytical purposes. The differential pulse peak height i s linear with the concentration down to 10-6 M. The wave pattern of the differential pulse polarography in methanol shows for prednisolon reduction at -1.60 V and an additional peak at -1.76 V versus SCE. The reduction in DMF is similar to that in methanol. The peak potential in a methanol-buffer mixture at pH 1 for 0 prednisolone is given as -1.50 V . Prednisolone has also been analysed by constant potential coulometry (59). The differential pulse polarographic determination of prednisolone in single component tablets is described. After extraction o f prednisolone with methanol from tablets it was

PREDNISOLONE

44 1

analysed in a supporting electrolyte of 0.03 M TMAH in methanol with a dropping mercury electrode, a Ag/AgCl reference electrode and a platinum wire auxiliary electrode. The concentration of prednisolone to be determined was in the range of 10-3 - 10-5 M (60). Prednisolone gives waves in d. C. and normal pulse polarography and peaks in differential pulse polarography which correspond to a one-electron uptake. Mechanism of polarographic electroreduction of prednisolone is described. The effect of pH using different buffers (acetate, phosphate, borate and ammonia buffers) on half-wave potential and limiting current for prednisolone is given. The half-wave potential of the prednisolone wave remains pH-independent up to pH 10.3 but is shifted towards more negative potentials at higher pH-values. Dependence of the peak-heights in pulse polarography on pH for the first wave for prednisolone closely resembles the pH dependence of these waves obtained by d-c-polarography. In linear sweep vol tammetric curves the dependence of the values of peak potentials of prednisolone on the logarithm of the scan rate was linear over a wide pH range (61).
A

differential pulse polarographic method for the determination of prednisolone in tablets is described. The method is more sensitive than dc polarography and the measurement of diffusion current is greatly simp1 if ied. Sorenson phosphate buffer pH 5.6 was used as the supporting

442

SYED LAlK ALI

electrolyte. The peak potential was found to be -1.19 V versus SCE for prednisolone. The position of the peak was independent of concentration and peak heights were linear over the 5 - 20 ug/ml range (62). Predni so 1 one and predn i sone can be determined in tablets using ethanol as an organic solvent, acetate buffer pH 5.6 as a supporting electrolyte and polarographing between -1 and -1.5 V ( 6 3 ) . 7.3 Radiochemistrv and radioimmunoassav Strict limits on allowable residual quantities of ethylene oxide and its major reaction products have been imposed due to their possible mutagenic and cancerogenic properties. Co-60 irradiation is a major goal o f a sterilization alternative programme. The cobalt 60 radiolytic degradation products have been identified for many corticoids including prednisolone. Two major types o f degradation processes have been identified: loss of the corticoid side chain on the D ring to produce the C-17 ketone and conversion of C 11 alcohol, if present, to C 11 ketone. Minor degradation products derived drom the other changes affecting the side chain are also identified. These compounds are frequently associated in corticoids as process impurities or degradation compounds. No new radiolytic compounds unique to Co-60 irradiation have been found. Through cobalt 60 radiolytic degradation pathway of prednisolone prednisone and llB-hydroxy-l,4-androstadiene 3,17-dione are formed.

PREDNISOLONE

443

Conditions for isolation of cobalt 60 radiolytic degradation products as well as paths and schemes of degradation are described. For prednisolone methylchloride was used as enrichment solvent, a HPLC Brownlee RP 18 column C 1 bonded phase and a 8 mobile phase o f methanol-water, 55:45 were used. The rate of radiolytic degradation in prednisolone is given as 0.7 %/Mrad (64). Radioimmunoassay has been used for the estimation of prednisolone after prednisone intake. Taking advantage of the similarity of structure of prednisone to cortisone and of prednisolone to cortisol , urinary prednisolone was estimated by radioimmunoassay for cortisol and found to be linearly correlated with the dose o f prednisone administered. Free prednisolone in urine was estimated by the "clinical Assay" R1A kit for cortisol, which gave a very high cross-reactivity with prednisolone. In estimating free prednisolone by RlA, cortisol cross reactivity was found to be 91,6 % in the range of 2-100 ug prednisolone. When RIA kits with differing specificity of the antibody to cortisol were used, the cross-reactivity also differed as expected. The less specific the antibody, the higher were the results obtained with the same blood sample (65). The predn i solone rad ioimmunoassay developed by Colburn and Buller (66) used an antiserum raised against prednisolone-21-hemisuccinate conjugated to

444

SYED LAIK ALI

bovine serum albumin (BSA) and showed roughly 10 % cross-reactivity with endogenous cortisol. The plasma samples were treated at 70 "C for 30 min prior to radioimmunoassay so as to eliminate interference by endogenous corticosteroid binding globulin with the binding of prednisolone to the antiserum ( 6 7 ) . The smallest amount of prednisolone which can be assayed by radioimmunoassay with confidence was 0.5 ng giving a usable range for the assay of 5 - 400 ng/ml in the plasma. The within-batch precision of the assay was between 3 - 5 %. The cross-reactivity of the antiserum with various metabolites of prednisolone and some endogenous steroids was between 6.4 and less than 1 %. The crossreaction with cortisol and corticosterone was 6.4 % and 3.8 % respectively (68). A method for measuring prednisolone using an antiserum raised against dexamethasone-21,-hemisuccinat-bovine serum albumin conjugate in sheep is reported, which reacts poorly with endogenous steroids. The results of radioimmunoassay were compared with those obtained by using competitive protein binding method (69). This method is based on the high affinity of predn i solone for p 1 asma corticosteroi d binding globulin (69, 70). The data show that binding of prednisolone to dexamethasone antiserum is sufficient for this to be used as the basis for a prednisolone radioimmunoassay as we1 1 as perfectly adequate for the measurement o f prednisolone plasma

PREDNISOLONE

445

levels in routine clinical investigations. Nevertheless, the technique is less sensitive than that which uses an antiserum raised against a predn i solone-21-hemi succinat-bov i ne serum a1 bumine conjugate. The dexamethasone antiserum used did not cross-react significantly with any of the several cortisol metabolites tested (68). Comparison of the results obtained by the competitive protein binding method and the radioimmunoassay method showed good agreement, although over the whole range of concentrations the latter technique always gave slightly lower values. This discrepancy was attributed either to the presence of cortisol in the pooled plasma used for preparing the standard curve or to some metabolite of prednisolone which cross-reacts in protein binding method. The method permits measurement o f prednisolone in the presence of prednisone, since the latter does not cross-react with the antiserum (68).

7.4

NMR determination A highly selective NMR method for the determ nation of prednisolone in tablets i s described. After extraction of prednisolone with 95 % ethano , the solvent is evaporated and the residue is dissolved in diemthyl sulphoxide containing fumaric acid as internal standard. The NMR spectrum of the resulting solution is recorded with a 60 MHZ instrument. The prednisolone content is calculated from the integral of the signal of the C 1 proton at 7.9 ppm w i t h the aid of the integral of the signal of internal standard at 6.9 ppm (71).

446

SYED LAIK ALI

7.5 7.5.1

Chromatographic methods Thin layer Chromatography (TLC) All important parameters of TLC separation are given in table 4. Prednisolone has been separated from its oxidation product through TLC on fluorescent silica gel plates. In some cases the quantity of the oxidation product could amount upto 2 %. By applying 100 ug of corticosteroid, the oxidation products can be detected at 0.4 % level and their presence is easily demonstrable at the 1 % level (72, 73). Knopp (74) applied three different mobile phases and HPTLC plates for the detection of decomposition products of prednisolone through UV 254 nm and INH reagent. 5 - 50 ul o f 0.1 % ethanolic solution of prednisolone were applied on TLC plates (74). Prednisolone could also be determined through densitometry at 250 nm ( 7 4 ) . A simple TLC screening procedure was developed for the detection of prednisolone as adulterant in Chinese herbal preparations. Depending on its complexity the sample may be directly extracted into aqueous ethanol, or stepwise fractionated into acidic, basic and neutral components. Extracts were analysed on silica gel TLC plates with fluorescent indicator with the aid of four solvent systems and detected under short and long wavelength UV light and iodine vapour (75). Prednisolone and chloramphenicol in oily 1 iquids could be separated through TLC (76). Quantitative HPTLC coupled with densitometry was developed for the determination of

PREDNISOLONE

441

prednisolone in human plasma, saliva and urine. For HPTLC analysis the residues after extraction were reconstituted in 10 ul acetone and 5 ul were applied on TLC plates and developed with two solvent systems. The plates were then sprayed with a mixture o f sulfuric acid-ethanol and heated at 60C for 45 min. The fluorescent intensity of the steroid bands were determined by densitometry at a wavelength of 598 nm, with excitation at 254 nm. The method allows simultaneous measurement of endogenous cort is01 in plasma following administration of prednisolone. The calibration curve was linear over a wide range of concentration in all biological fluids (0.025 - 4 ug/ml). The limit of detection was 10 ng/ml in plasma and saliva and 25 ng/ml in urine. The method was reproducible with an inter- and intra-assay coefficient of variation of < 10 %. No interference from endogenous steroids was found (77). TLC behaviour of prednisolone in creams is described. As the content of corticosteroids like prednisolone in creams is generally low, the detection of these active components can be a problem. Detection was not sufficiently sensitive for creams containing about 0.1 % or less of a particular corticosteroid (78). In B.P. 88 and european pharmacopeia a TLC method for identification of prednisolone and for the examination of related substances is given (79) K. Macek has tabulated the chromatographic data with number of mobile phases for the identification and separation of prednisolone from innumerable corticosteroids and related substances (80).

448

SYED LAIK ALI

Levarato has developed a series of quantitative methods for the determination of prednisolone along other corticoids after extraction from preparations and separation by TLC on silica gel plates and determination by spectrophotometric (absorption at 240 nm) or colorimetric methods (INH-hydrazone formation, tetrazol ium reaction) (81). A simple, fast and quantitative TLC-method for the determination of prednisolone in tablets is described. The method is stability-indicating with respect to accuracy, specificity, sensitivity and precision. The coefficient of variation was between 1.26 and 1.96 % and the sensitivity was about 25 ng. The chromatographic separation was performed on a silicagel plate using two step development of the plate ( 8 2 ) . A simple TLC method of separation of prednisolone from other corticosteroids is reported (83). Prednisolone and the dephosphorylated D-homosteroid can be separated on silanised silica gel TLC plates (36). Fluorimetry is a useful method for the direct determination of steroids on chromatographi c p 1 ate. The method is based on the measurement of the fluorescence produced on irradiation of the chromatogram with UV light. The quenching of the fluorescence of various activated layers by unsaturated ketosteroids 1 ike prednisolone can also be used for quantitative measurements. The basis of the quantitative evaluation is the difference between the fluorescence of the background and that

PREDNISOLONE

449

of the dark spots. A direct densitometric evaluation o f prednisolone has been performed (84) * The method for detecting prednisolone on TLC plates i s assigned according to the functional group involved in the reaction. The name and composition of the reagent, the colour o f fluorescence observable on spraying and the sensitivity is given. Detection of predn i solone with sulphur ic ac i d , orthophosphoric acid and antimony (111) chloride reagents is described ( 8 5 ) . The limit of detection of prednisolone on silica gel fluorescent TLC plates with a mobile phase acetone-cyclohexane-ethyl acetate, 1:l:l in UV light 254 nm is given as 0.3 ug (85). Compernolle and coworkers (86) reported on the purity checking of commercial prednisolone samples by TLC, where several impurities such as hydrocortisone, prednisone etc. could be detected. Excellent separations could be achieved by TLC with solvent mixtures such as dichlormethane-dioxanewater (2: 1 : l ) or dichlormethane-diethylethermethanol-water (77:15:8:1). The Rf-values for prednisolone 0.42 and for prednisone 0.62 were found with this system ( 8 7 ) .

Table 4 Stationary Phase Mobile Phase hRf -values Detect ion References

Silica gel 60 with fluorescence indicator

methylene chloridedioxane-water 100:50:50 prednisolone: 34 oxidation product: 77 same as above 120:30: 50 prednisolone: 13 oxidation product: 55 Methylene chloride-ethermethanol-water 77:15:8:1.2 prednisolone: 29 decomposition products: 0-71

UV 254 nm

VI P 3

same as above

UV 254 nm

(73)

HPTLC plates, Merck

UV 254 nm

(74)

pre washed with methanolchloroform (80+20); 15 cm

INH reagent

m
a , V

c
a ,

a, rc
p:

L
d
v
W

n
h

a ,

a ,
S

n
la
5

>

a ,

n
9 .
5

>
0
S

*r

0
wl

c, V
l0 -

m
a ,

>

c,

a ,

a ,

n
m m
*r

a ,

5 5
U 0
h Lo I

a ,
.r

N
h

d
7

a ,
7

m CI
c,

..
wl

..
Ln

n
5 +

c la r
h
N
L

a ,

c,

a ,

..
Q

U L

V S

U 0
D

c
Q
7

m
a ,

a
rc 0 L
0

E
2 .m ? o
m
*r
7

I
*r

a
c

a ,

la

.r

>

n rc
0 r
T

CL

, 2 ? ? G Ev a , N L a ,
e

o
m

2 o
- 0 0

..
Q

.e m
*r

h a ,

2s:
c , a ,

N e 0 m -

... .. ..
L

M N

.r

a , v

v m

n o

L a , Q U

a,u 5 L 3 Q

a ,
la T
Q

m
a ,

L
la
S
.r

0
la
wl

>

la

m
la

c,

c,

IU

a ,

5
wl

45 I

Table 4 Stationary Phase Mobile Phase hRf-values Detection References

same as above

ethyl acetate-toluene-formic acid-dimethyl formamide-water 75:75:2:4:4; prednisolone: 12 ethyl acetate- to1 uene-formi c acid-dimethyl formamide 75:75:2:4; prednisolone: 15 Ether; prednisolone: 1 1

same as above

(75)

same as above
P N VI

same as above

(75)

Silica gel UV 254

UW 254 nm

(76)

Table 4 Stationary Phase Mobile Phase hRf -values Detection References

HPTLC plates without fluorescent indicator BDH 10 x 20 cm

Chloroform-ethano 1 -water 90:10:2; prednisolone: 25

H2SOq-ethano1, 6.5:3.5 spray reagent; densitometric determination at 598 nm with excitation at 254 nm same as above (77)

e W ?n

same as above

Ch loroform-ethano 1 -water 45:5:7.5; prednisolone: 29 n-butan01-water-g1 ac i a 1 acetic acid, 20:5:2 prednisolone: 72

Silica gel 60 F HPTLC plates 10 x 1 cm Merck, 0 distance o f 7 cm under saturated conditions

UV 254

nm

(78)

Table 4 Stationary Phase Mobile Phase hRf-values Detect ion References

Silica gel 60 F 254 TLC plates

methyl ene ch 1 or i de-ethermethanol-water; 77:15:8:1.2

UV 254 nm, spraying

(79)

same as above
e VI P

ether-toluene-butano1 saturated with water, 80:15:5 a) to1uene-ether-acetone


18:19:3

with ethanolic sulfuric acid (20 % ) , heat at 120C for 10 min and UV 365 nm

(79)

Silica gel 60, 20 x 20 cm Merck without fluorescent indicator

250 nm, densitometry;

(82)

b) methylene chloride-ethyl acetate-ether-formic acid


10:10:10:0.3

prednisolone: 25

development first with mobile phase a, drying the plate with air and again development with mobile phase b

Table 4 Stationary Phase Mobile Phase hRf -values Detection References

Silica gel 60 F 254 plates Merck

methy 1 ene ch 1 or ide-acetone 75:25; prednisolone: 20

UV 254 nm, spraying

(83)

with 20 % H2S04 in ethanol and drying at 120C 10 min reddish brown spot
UV 254 nm; blue

% VI

Silanised silica plates 60 F 254 Merck, 0.25 nm

methanol-water - 0.4 M sodium phosphate solution 50:50:1 prednisolone: 54

colouration with tetrazolium blue

456

W E D LAlK ALI

7.5.2

Hiah performance liquid chromatoaraphy


A

sensitive, specific, HPLC procedure for the determination of prednisolone in plasma is described. The organic solvent extract from plasma is chromatographed on a silica gel column using a mobile phase of 0.2 % glacial acetic acid, 6 % ethanol and 30 % methylene chloride in n-Hexane at 254 nm. Quantitation of plasma samples containing 25 ng/ml prednisolone is reported. Metabolites and endogenous hydrocortisone do not interfere with prednisolone (88). For the simultaneous analysis of prednisone and prednisolone in plasma the internal standard was dexamethasone and the mobile phase consisted of glacial acetic acid-ethanolmethylene ch 1 or ide-n-hexane (0.2 :3.5 :30 :66.3) (89, 90). A brief discussion of the merits and limitations of HPLC relative to other chromatographic methods and special problems in the application to steroids are discussed (91). A sensitive, specific and reproducible HPLC assay for the simultaneous determination of prednisone, prednisolone and cortisol in biological fluids was developed with dexamethasone as the internal standard. Samples were extracted with methylene chloride, washed with sodium hydroxide and then water and chromatographed on a microparticulate silica gel column with a mobile phase methanol-methylene chloride, 3:97 at a flow rate of 2 ml/min and detected at 254 nm. Sensitivity was greater than 15 ng for all four steroids. A constant ratio of peak height of a steroid at the wavelengths

PREDNISOLONE

451

280 and 254 nm served as an added measure of specificity o f the assay. The 280:254 nm ratio for prednisolone was 0.09 ( 9 2 ) . While monitoring at dual wavelengths improves assay specificity, the 254 nm wavelength yields nearly optimum absorbance (92). Determination of prednisolone in plasma by HPLC using a water saturated mobile phase of equal volumes o f ethanol and methylene chloride and 1 % glacial acetic acid was used on a stainless steel 30 cm x 3.9 cm porasil column (10um porous silica) with a flow rate of 2 ml/min and detection at 254 nm. A 1inear relationship exists over the concentration range 25 - 150 ng/ml. The effects of sample storage on reproducibility of results were examined. The samples were stored at -20C for upto four weeks. The mean recovery was between 101.6 to 103.9 % for prednisolone concentrations between 20 and 100 ng/ml respectively (93). A normal phase micro-bore column packed with 1 um microsphere silica (50 cm x 1 mm 0 i .D.), mobile phase o f water-saturated butyl chloride-buty 1 ch lor ide-THF-met hano 1-g laci a1 acetic acid (450:450:105:53:44) , and the detection at 254 nm were used for the separation of prednisolone from other corticosteroids (94). HPLC retention values of prednisolone along various other corticoids on a Bondapak C18/corasil column using methanol or acetonitrile of different compositions in water at a flow-rate of 1.5 ml/min and detection at 254 nm were evaluated. The values for prednisolone lie between 1.06 t o 3.35 i n relation to acetone with a value 1.00 (95). An improved HPLC separation of

458

SYED LAIK ALI

decomposition products of prednisolone by adding sodium sulphite to the mobile phase is reported. To a methanol-water, 1:1, solution 1 % of 0.4 M sodium phosphate solution pH 7.0 was added. Sodium sulphite was optionally added t o give a concentration of 0.1 % w/w. The flow-rate was 1.0 ml/min and the detection was performed at 240 nm. A C18 M Bondapak co 1 umn was used. With this system 21-dehydro-prednisolone, a decomposition product of prednisolone is separated and detected (96). Prednisolone was separated along other corticosteroids in topical pharmaceuticals on a reversed phase microparticulate HPLC column Zorbax c8, Dupont (25 cm x 4.6 mm) with a mobile phase of THF-methanol-water, 25:12.5:62.5 with a flow-rate of 1 ml/min and at a detection wavelength of 254 nm. Prednisolone had a relative retention value of 0.94 with respect to hydrocortisone (value 1 .OO). Various commercial topical formulations of these corticosteroids were prepared by both simple dilution and by extraction for analysis by the proposed HPLC procedure, by the blue tetrazolium procedure and by the isoniazid procedure and/or by phenylhydrazine method (97). Prednisolone and chlorhexidine have been separated on a Nucelosil C18 1 um column, 30 cm x 4 mm along with a Vydac 201 RP 0 guard column 5 cm x 4 mm with a mobile phase methanol-water, 120:80 containing the PIC reagent 87, Waters with a flow-rate of about 1.35 ml/min at 50 "C and detected at 240 nm. Prednisolone and chlorhexidine dichloride were thus well separated in hydrophilic emulsions and lipophilic ointments (98).

PREDNISOLONE

459

normal phase HPLC method for the determination of prednisolone in tablets and bulk drugs was studied. The HPLC system consisted of the mobile phase methanol-water washed ethylene dichloride-acetic acid ( 6 + 94 + O . l ) , 25 cm x 4.6 mm column packed with 5-6 um porous spherical particles (Du Pont), flow-rate 1.5 ml/min, detection at 254 nm and injection volume 10 - 15 ul. The bulk drugs and tablets containing prednisolone were extracted with a mixture of methanol and methylene chlorid (4:96) and an internal standard of 1 mg/ml solution of f luoxymesteron was used. The coefficient of variation of the analysis results ranged from 1.34 % for bulk drugs to 2.14 % for tablets ( 9 9 ) . Extraction-monitoring and rapid flow fractionation for determination of serum corticosteroids is described. The HPLC system applied for the analysis of serum corticosteroids including prednisolone consisted of a mobile phase 0.1 % water, 4 % methanol, 30 % methylene chloride in n-hexane, Lichrosorb Si 60.5 um, 25 cm x 4 mm column pretreated with 5 % H2SO4, flow-rate 2 ml/min and 240 nm UV detection (100). A HPLC analysis of prednisolone and endogenous cortisol is described in plasma samples of kidney transplantation patients using dexamethasone as an internal standard. A glass column (15 cm x 3.1 r m filled with Separon Six, 5 n urn, Laboratorni Pristroe, Prag, CSSR), a mobile phase methylene chloride-methanol, 9 7 : 3 , flow-rate 1 ml/min and detection at 254 nm were the HPLC parameters. The calibration curve for prednisolone
A

460

SYED LAIK ALI

x) is linear up to 500 ng/ml and the detection limit is given between 2-5 ng/ml. The use of glass column permitted a higher sensitivity and less consumption of mobile phase (101).
(Y
=

0.0046

Application of HPLC procedure to the determination of binding of prednisolone to high-affinity binding sites in human serum is reported. Prednisolone binds to globulin and albumin in human serum. The binding affinity of the steroid for globulin is high whereas the capacity is low. In contrast, albumin has a low affinity for the drug but the binding capacity is high. The method describes a HPLC gel permeation procedure which a1 lows prednisolone bound to albumin to completly dissociate during chromatography while the binding of the drug to high affinity proteins is unaffected. The column (Bio-Sil TSK-250, 3 0 0 ~ 7 . 5mm, 10 um, Bio-rad Labs) with a molecular mass range of 1000-300,000 was preceded by a guard column. The mobile phase consisted of 0.1 M Sodium sulfate and 0.02 M Sodium phosphate monobasic adjusted to pH 6.8 with 0.1 M NaOH. The flow-rate was 5.4 ml/h and detection was performed at 280 nm. The data about the effect of prednisolone concentration on the binding in serum and comparison o f binding of prednisolone by HPLC and equilibrium dialysis is given (102). HPLC determination of prednisolone incorparated in gel ointment i s reported. The gel ointment is composed of carboxy vinyl polymer (1.3 % W/W) and a

PREDNISOLONE

46 1

large amount of an aqueous organic solvent. A methanol extraction system offered simultaneous advantages of the removal of the polymer and the recovery of active ingredients from the gel phase. The recovery of the drug was 100 %. The following chromatographic conditions were used: mobile phase methanol-water, 6:4, reversed phase, Bondapak C 18, 10 um column, 30 cm x 3.9 mm, flow-rate 1 ml/min, detection at 254 nm. The prednisolone content in gel ointment was well maintained for 3 months or longer when stored at 5 "C (103). Retention data of 12 corticosteroids including prednisolone is given on dynamically modified silica by cetyltrimethylammonium bromide added to the eluent with various organic modifiers. Separation factors between hydrocortisone and 11 other corticosteroids including prednisolone measured on 8 different silica columns and six different ODs-sil ica columns are presented. The variations in selectivity were found to be substantially smaller than those of chromatographic systems based on chemically bonded ODs-silicas from the same sources
(104).

method for the simultaneous determ nation of predn i so one a ong other corticosteroids in swine plasma i s described. Extraction of the steroid mixture from swine plasma with dexamethasone as internal standard was accompl ished by sol id-phase (SPE) extraction or by the more traditional
A HPLC

462

SYED LAIK ALI

liquid-liquid extraction (LLE) techniques. A Lichrosorb Si 60, 5 um silica column, 25 cm x 4.6 mm mobile phase methylene chloride-water chlor ide-tetrahydrofuransaturated methy 1ene (664.5 :300:10:25 :0.5) , met hano 1 -g 1 ac i a 1 acetic acid flow-rate 0.8 ml/min and detection at 254 nm were used. Calibration curves were found to be linear between 10 and 100 ng/ml by the LLE technique. Within-day and inter-day variability for the measurement of the plasma samples spiked with prednisolone is given (20 ng/ml, 10 % and 100 ng/ml, 8.4 % ) . The average recovery of prednisolone at 20 ng/ml is between 70 and 90 % (105). Cox et a1 (106) used a weak cation-exchange column and a mobile phase of 0.05 M ammonium formate in 2.5 % aqueous ethanol, flow rate 0.5 ml/min, detection 240 nm, to separate prednisolone from prednisone. The retention times were about 1 and 23 min for prednisone and 8 prednisolone respectively (106). Prednisolone and prednisone could not be resolved with a ODS 10 cm column, methanol-water (1:l) mobile phase, flow rate 1 ml/min, detection 240 nm (107). Retention values of prednisolone along with a number o f other steroids relative to acetone on a u Bondapak C 1 8 column using methanol-water and acetonitrile-water mobile phases o f different compositions at a flow rate of 1.5 ml/min are reported (108). The extraction efficiency of ethyl acetate, diethyl ether and dichlormethane in extracting prednisolone from pooled plasma is reported (109). Dichlormethane appears to be the best solvent for extraction of corticosteroids from plasma (109). Trefez et a1
I

PREDNISOLONE

463

showed that prednisolone could be separated from other corticosteroids in plasma samples on a 25 cm Zorbax Sil (Dupont) column. Two techniques, HPLC and HPTLC were developed for the determination o f prednisolone in human plasma, salvia and urine. Both methods shared a single and simple step o f an organic extraction procedure and separation using a normal phase column or HPTLC plates. A normal phase HPLC column (0.45 x 20 cm) packed with Zobrax SIL 5 um, mobile phase dichlormethane-methanol-acetic acid, 95:1:3:75, flow-rate 2.5 ml/min and detection at 254 nm were the chromatographic parameters. The method allows simultaneous measurement o f endogenous cortisol in plasma following administration of prednisolone and methyl prednisolone. The calibration curves of steroids in a1 1 biological fluids were linear over a range of concentration o f 0.025 - 4 ug/ml. The limit o f detection for prednisolone was 10 ng/ml in plasma and salvia and 25 ng/ml in urine. The method was reproducible with an inter- and intra-assay coefficient o f variation of < 10 % over a wide range of concentration in all biological fluids. No interference from endogenous steroids was found (111).
7.5.3 Gas chromatoaraphy

(110)

- mass spectrometrv

Pentafluorobenzyl hydroxylamine has been used as a derivatization reagent in the analysis of corti costeroids inc1 uding predn i solone by gas chromatography-negative ion chemical ionization mass spectrometry (NCI). The resulting pentaf luorobenzyloxime (PFBO) trimethylsi lyl (TMS)

464

SYED LAlK ALI

derivatives were generally formed in moderate yield but, despite this, the use of these derivatives resulted in a 10-fold improvement in the capability of identification of corticosteroids by GC/NCI mass spectrometry in comparison with the methoxime/TMS derivatives. The NCI mass spectra of PFBO/TMS dervatives were simple with most o f the ion current being carried by the (M-CfjF6CH2)- or (M-PFB)- - ion and by a reagent-specific peak at m/z 196. The PFBO/TMS derivatives are suitable for the analysis o f pi cogram quantities o f corti costeroi ds in biological media by GC/NCI mass spectrometry (112). The negative ion chemical ionization mass spectra of the methoxime-TMS dervatives of the corticosteroids including predni solone have been co 1 umn gas obtained using capi 1 lary chroamtography-mass spectrometry. Fig. 9 shows a NCI spectrum of the methoxim-TMS derivative of prednisolone. The spectra showed abundant diagnostic ions at m/Z greater than 300 allowing for clear discrimination between prednisolone and other steroid dervatives (113). A capi 1 lary GC-MS method using negative ion chemical ionization mass spectrometry has been developed to confirm the presence of the parent steroids in horse urine following the administration of proprietary preparations o f prednisolone and betamethasone. For this purpose standard steroids (40 ug) were treated with methoxylamine hydrochloride in dry pyridine (8 % w/v; 100 ul) and heated at 80C for 30 min. The solvent was removed under N2 and

Fig. 9 (113) N I Spectrum o f Methoxime C

TMS D e r i v a t i v e o f P r e d n i s o l o n e

441

512

300

400

466

SYED LAIK ALI

trimethylsilylimidazole (50 ul) was added and silylation was carried out at 80C for 2 hours. Excess dervatization reagents were removed by filtration through a 2 cm Sephadex LH-20 column using chloroform-n-hexane (1 :1) as eluent. The steroid MO-TMS derivatives were eluted in the first 2 ml of eluent. The solvent was removed under nitrogen and the residue was dissolved in n-hexane (100 ul) for analysis by GC and GC/MS. The base peak in the spectrum o f prednisolone-Mo-TMS occured at m/Z 457. The abundant diagnostic ions in higher mass regions of the spectra render these derivatives amenable to analysis by SIM. Capillary GC/MS-NCI analysis of a mixture of 1 ng derivatives of prednisolone Mo-TMS and dexamethasone Mo-TMS and monitoring ions 441, 457, 473 and 489 demonstrates the applicability of this technique. The sensitivity that can be achieved by this technique is 250 pg of each steroid derivative (113). The use of capillary GC-MS/NCI for the confirmatory analysis of corticosteroids in horse urine is more sensitive than the liquid chromatography-mass spectrometry method (114). The combination of HPLC and mass spectrometry for the analysis of prednisolone has been reported (115). Mass spectra for prednisolone has been obtained in the thermospray discharge mode. Thermospray is a reliable HPLC/MS interface. The spectra obtained are reproducible but fragmentation is not predictable. The sensitivity o f the technique i s compound-dependent and variable. Because of the dependence of ion production on solvent composition, it is not easy to use the interface with gradient elution (115).

PREDNISOLONE

461

7.5.4 Supercritical fluid chromatoqraphy (SFC) The coupling of supercritical fluid chromatography (SFC) with mass spectrometry (MS) seems to be easier than liquid chromatography (LC) and MS. This follows from consideration of the facility of the mass spectrometer vacuum system pumping excess carbon dioxide from SFC eluent rather than aqueous reversed-phase eluents from HPLC. The other important factor in SFC/MS is whether capillary or packed columns are used. A synthetic mixture of five corticosteroids including prednisolone was analysed by packed-column E 1 SFC/MS (Fig. 10). The separation was accomplished on a 2 mm x 250 mm, 3 um S3CN spherisorb column maintained at 70, a flow-rate of 0.8 ml/min 92:8 C02-methanol and an inlet head pressure of 3000 Psi. The corticosteroids are difficult to analyse even by GC/MS because they are relatively polar and thermally labile. Although they can be characterised by capillary GC/MS either as parent drugs or TMS derivatives, their long retention time and thermal instability suggest a need for alternative means of confirmation.
Prednisolone along with other corticosteroids could be separated within 6 min by EI SFC/MS. The packed column SFC/MS was applied for the analysis o f a TLC scrape of a urine sample collectd two hours after the intramuscular administration of 100 mg of prednisolone to a horse. The extracted ion current profile for the abundant fragment ion at m/z 122 and the molecular ion at m/z 360 easily identify the prednisolone in this sample. Packed-column SFC/MS

468

SYED LAIK ALI

PREONISONE H YDROCORT180N E 6 . PREOHl8OLONE 6- BETAUETHASONE


8.
4-

1- UELEWOESTROL 2. CORT180NE

ACEPITE

Fig. 1 (116) 0

1 2 3 4 5 6
MIN

Packed Column SFC/MS

Separation o f Prednisolone

100

80
60

80
100

Fig. 1 (116) 0
Mass Spectrum of Prednisolone

EI

40.

20.
0

720

140

160

180

200

220

80. 60

P R E O H t 8 0 LO H E

40.

20

0 ' 8

~.-

. .-

. . . .

PREDNISOLONE

469

with a two-stage momentum separator is feasible for obtaining E I mass spectra of compounds amenable to chromatographic separation by this route. The sensitivity afforded by this approach is nevertheless not suitable for trace analysis (116,
117).

Prednisolone has been analysed by capi 1 lary supercritical fluid chromatography in equine urine extract and was identified by matching retention time of pure standard. Supercritical fluid carbon dioxide was used as the mobile phase in conjunction with a methylpolysiloxane stationary phase capillary column and a flame ionization detector. SFC can thus be successfully applied for the estimation of prednisolone without derivatization (118).

8.

In vitro dissolution
X X I I requires that not less than 70 % of the labelled amount of prednisolone is dissolved in 30 minutes in dissolution medium water (900 ml) with paddle stirring element test apparatus (apparatus 2) at 50 rpm (119). I n vitro dissolution profiles of sustained release formulations of prednisolone are given. For each tablet formulation 20 tablets were placed in a 100 ml beaker of 5.5 cm diameter; 35 ml of distilled water were added and the contents were stirred for one hour, at 37C. Sustained-release formulation gives a more uniform blood level of prednisolone and avoids high peaks of plasma prednisolone (120).
USP

470

SYED LAIK ALI

Two nonporous and three porous-amorphous silicas were used as dispersion media to convert corticoid solutions into free flowing powders. Prednisolone was dissolved in N,N-dimethylacetamide- polyethylene glycol 400, 7:3, and their 10 % (w/v) solutions were mixed with silicas (1:3 V / W ) . Dissolution rate from such powdered solution was more rapid than those of their micronised powders in various aqueous media. Dissolution in simulated gastrointestinal media of solution of prednisolone dispersed on various silicas is reported (121). Prednisolone tablets, enteric coated with neutral ised hydroxypropyl methylcel luolsoe phthalate (HPMCP) were compared with Delta cortril tablets (Pfizer) by compendia1 in vitro testing. For this study tablets were tested using the disintegration and gastroresistance tests o f both the USP and of european pharmacopeia. The dissolution prednisolone from coated tablets followed the USP X X I procedure which involved monitoring drug release in a pH 6.8 phosphate buffer after two hours in 0.1 M HC1. Percent of drug (prednisolone) released in different pH media for neutral ised HPMCP coated tablets is illustrated in fig. 11. The dissolution performance closely reflected the disintegration characteristics and was independent o f coating weights between 5 and 25 mg for the neutralised HPCMP tab ets (122).
A

solid d spersion technique with PEG, PVP, urea, sorbitol , mannitol and cremophor has been used for

Percent of Drug Released i n Different pH Media for Neutralised HPMCP Coated Tablets

412

SYED LAIK ALI

improving prednisolone dissolution. The optimum di ssolution-rate composition was for dispersions containing 10 % w/w prednisolone (123). A marked increase in the dissolution rate of prednisolone in solid dispersion was observed compared with that of drug alone or with that of a physical mixture with a carrier (123). Prednisolone, which is poorly soluble in water, was chosen to prepare solid dispersion systems with water-soluble carriers. I t was further determined whether the quantities of these carriers and their chemical structure influenced the dissolution rate of prednisolone from such systems. The results of the studies showed that the dissolution rate of prednisolone from all solid dispersions increased markedly from those o f the physical mixture and the drug alone. Nevertheless, there was no observed relationship between the higher dissolution rate and chemical structure of the carriers, Nor was it possible to predict quantitatively to what extent any carrier would improve the dissolution rate o f the drug in solid dispersion. For example sorbitol and mannitol, which are chemically similar, produced different effects on dissolution rate. Results indicated that sorbitol was one of the better carriers and the maximum drug dissolved (100 %) was achieved after 3 h. Yet at the same time only 63.17 % prednisolone was dissolved from the mannitol solid dispersion. When PEG, PVP and urea were used as carriers, they gave similar results. The amount of prednisolone dissolved from the solid dispersions with above carriers was twice as great as that from the drug alone. Similar

PREDNISOLONE

413

investigations with physical mixtures showed that smaller amounts of drug were dissolved than solid dispersions. Comparison of these results with those of the dissolution rate of prednisolone in carrier solutions showed that a solubilizing effect had taken place and this had evoked a better dissolution rate. The mechanism of the enhanced dissolution properties of prednisolone in solid dispersion with different carriers could not be explained. The results showed that improved wettability of drug molecules and their solubilization by the carriers were not basic processes. Molecular dispersion of drug through the matrix o f the carriers was of greater importance. A1 though changes in crystalographical structure of the drug during preparation of solid dispersion were evident, x-ray diffraction studies nevertheless indicated an amorphous form of prednisolone in solid dispersion with PVP. In contrast the presence of identical prednisolone diffraction peaks in the spectrum of pure drug and solid dispersion systems with PEG, urea, mannitol, and sorbitol showed that these solid dispersions contained prednisolone in crystalline form (123). The dissolution behaviour of ground mixtures of prednisolone with chitin and chitosan were prepared by co-grinding in a ball mill. The x-ray diffraction patterns and results of differential scanning calorimetry suggested that the size of prednisolone crystals decreased in the ground mixtures. The dissolution rate o f prednisolone from the ground

414

SYED LAIK ALI

mixtures was significantly greater than that from the physical mixtures or from intact prednisolone powder. These results indicate that chitin and chitosan can improve the dissolution properties of prednisolone. The ground mixture with chitosan gave slightly greater dissolution than that with chitin and this difference reflected the reducing effect of chitosan on the relative enthalpy change o f prednisolone (124). The dissolution profiles of a model formulaion of prednisolone tablets containing different disintegrants have been investigated. Marked increase was observed in disintegration and dissolution rate with increased concentration of microcrystalline cellulose, methylcellulose, maize starch, whereby a decrease in dissolution rate was recorded with increasing concentration of sodium carboymethylcellulose and pregelatinized starch
(125).

9.

Pharmacokinetics and drua metabolism Prednisolone is efficiently absorbed through the gastrointestinal tract, with approximately 75 - 98 % o f the dose given being abosorbed. Inactivation of prednisolone is achieved mainly in the liver through reduction of the double bonds in ring A and the Keto groups to form tetrahydroprednisolone which conjugates with glucoronic acid and sulphate groups to form water-soluble compounds that are excreted in the urine (126, 127). Prednisolone plasma concentrations are commonly determined by either radioimmunoassay or competitive protein binding

PREDNISOLONE

415

techniques. Prednisolone is absorbed completely and rapidly after oral administration reaching peak plasma conentrations after 1 to 3 hours. The bioavailabiltiy of prednisolone afte oral prednisone administration is approximately 80 % of that after prednisolone. A wide intersubject variation in prednisolone concentration is evident, which may suggest impaired drug absorption in some individuals. Prednisolone shows dose-dependent pharmacokinetics, where an increase in dose leads to an increase in volume o f distribution and plasma clearance. This can be explained in terms of the non-linear binding of the drug to plasma proteins. The degree of binding will determine the distribution and clearance of free drug. Prednisolone pharmacokinetics is also dependent on age, the half-life being shorter in children. Liver disease prolongs the prednisolone half-life and also increases the percentage of unbound drug. In these cases prednisolone rather than prednisone is the drug of choice in active liver disease owing to the poor conversion of prednisone to prednisolone. However, the reduced plasma concentration of prednisolone in such patients is compensated for by delayed clearance. Thus, there is little advantage of one preparation over the other (126). Hepatic conversion o f prednisone to prednisolone i s extensive and the two compounds are generally considered to be therapeutically equivalent when used systemically. It is however suggested that orally dosed prednisone resulted in lower

416

SYED LAIK ALI

circulating prednisolone concentrations compared with equivalent oral doses of prednisolone. The results provided evidence that oral prednisone products may not be bioequivalent to oral prednisolone products and suggest that substitution of one drug from for another can result in marked changes in circulating concentrations o f active steroid (128). The half-life of prednisolone was found to be about 4.2 h , the apparent volume of distribution in the B-phase was about 0.6 l/kg and the systemic clearance about O.ll/l.h.kg (111). In Fig. 12 concentration-time profile of prednisolone in biological fluids is illustrated (111). Prednisolone is cleared from the body primarily by hepatic metabolism and greater than 90 % of radioacti vt i y admin i stered ora 1 1 y or intravenous1 y as 4-C14-prednisolone is recovered in urine (127, 128). Only approximately 7 - 15 % of an oral dose of prednisolone i s excreted as unchanged prednisolone in the urine, the rest being recovered as a variety of metabol ites (70). The plasma half-life of prednisolone following the oral administration of prednisone to normal subjects ranges form 2.5 to 3.5 h (126, 130, 131). Similar half-1 ife values for prednisolone were observed after oral prednisolone is administered (126, 132, 133, 134). Nugent et a1 (135) found after an intravenous dose of 1 mg/kg body weight of predn i so 1 one ( sodium succ i nate sa 1 t ) an average half-life o f 3.5 h. After an intravenous dose of 0.3 mg/kg prednisolone as phosphate the mean plasma

PREDNISOLONE

411

URINE

l o

12

TIME (hr) Fig. 12 ( 1 1 1 ) Concentration-Time Profile o f Prednisolone in Biological Fluids Following Intravenous Administration o f 64 mg o f Prednisolone

478

SYED LAIK ALI

half-life was 4.2 h, plasma clearance 97.3 ml/min/1.73 m2, volume of the central compartiment 28.2 1/1.73 m2 and that of peripheral compartiment 36.9 1/1.73 m2 (136). In another study eight subjects received an intravenous bolus dose of 12 mg prednisolone phosphate and four received 48 mg dose. Similar pharmacokinetic parameters were found. Neither half-life nor clearance was statistically different between the two dose levels (137). In a later study the mean metabolic clearance rate was measured as 1.16 ml/min/kg, mean half-life 3 . 2 h and the values for the apparent volume o f distribution were 0.14 l/kg for V and 0.15 l/kg for V2 (138). The plasma clearance, half-life and volume of distribution of prednisolone is reported to be independent in the range o f the doses 10, 20 and 30 mg prednisolone adminstered orally (139). Pickup et a1 (140) studied pharmacokinetics of prednisolone at different levels in ten subjects, four normal subjects and six patients with osteoarthritis after intravenous administration o f prednisolone. Average prednisolone half-lives were found to be between 2.6 to 3.8 h , mean volume distribution between 0.22 to 0.64 l/kg, and plasma clearance between 1.02 to 2.0 ml/min/kg following the tracer 0.15 mg/kg and 0.3 mg/kg doses. This data showed sginificant increases in volume of distribution and plasma clearance of prednisolone with inceasing dose. An increase in half-life was also observed. Pickup et al. (140) thus postulate that the observed dose-dependent kinetics is primarily due to the non-linearity in

PREDNISOLONE

479

plasma protein binding of prednisolone. The percentage of unbound prednisolone increases with increasing dose and results in a larger apparent volume of distribution and plasma clearance. The net effect of these changes causes the observed prolonged prednisolone half-life following larger doses (140). In another study the area under the plasma concentration-time curve for prednisolone for the 20 mg dose was 77.89 % of that calculated for the 10 mg dose. This change in area represented an increase in prednisolone clearance from 1,7 ml/min.kg to 2.2 ml/min.kg when the dose was increased (141). Rose et a1 . (142) found dose-dependent pharmacokinetics of prednisolone where the plasma half-life increased from 3 to 5 h as the oral dose of prednisone was increased from 5 t o 50 mg. T.anner et a1 (143) reported the pharmacokinetics of prednisolone at different dose levels in 43 subjects. Each subject received only a single dose, 5 - 200 rg of oral n prednisolone. Kinetic parameters of oral prednisolone are presented in table 5 and fig. 13 illustrates concentration-time profile of prednisolone. The mean half-life of prednisolone remained fairly constant between 3.4 to 3.8 h. Bioavailability of prednisolone was 98.5 2 4 %. Furthermore as the prednisolone dose increased, the area under the curve increased but not proportionally to the dose, such that a fivefold increase in dose from 20 to 100 mg resulted in only a two-to threefold increase in area under the curve.

Table 5

(143)

Kinetic parameters for oral prednisolone, 6 to 200 rng

a
0 W

I07 198 2 9 250 2 21 320 2 24 299 f 27 625,549 761 -c 177 676,661 I409

467 1,079 f 108 1,243 2 85 1,739 f 150 1,664 2 52 3 , ~ m s 4,361 f 1141 4,694,4186 8,692

2-9 3,7 2 0 , 2 3.8 t 0.2 3.5 2 0.2 3.7 2 3.3 3.4,3.4 3.7 2 0. I 3,6,3.6

99,109

66 f 6 58 2 5 I60 t I 1
1.72 5 40 134,146

45 49 ,c 5

10.7 9.3 f 0.8 12.1 2 0.9 11.5 t 0.9 30.0 2 2.6


22.9 t 5.2 26,28 23.0
19,23

3,8

I26

20 mo of thle preparation waa equlvalent to 18 mo prednleolone and 100 mg woe equlvslent to 90 mg prednleolone; numbere In Parentheeee lndlcate the number of eublects atudled a t that pertloular doaage and from whloh the data are derlved.

Fig. 13 (143) Concentration-Time Relationship o f Prednisolone and Prednisone Following the Oral Administration of 100 mg Prednisolone PREDN180LON 0- 0
PREDNISON

O--o

48 1

482

SYED LAIK ALI

The apparent volume of distribution divided by the extent of avai labi 1 ity (Vd/F) and clearance (CL/F) for prednisolone was found to increase with dose. For example Vd/F at 10 mg was 49 1 increasing to 132 1 if 100 mg dose was given and CL/F increased from 155 ml/rnin to 382 rnllrnin. The authors (143) postulated that since they found no change in the plasma protein binding of prednisolone over the dose range studied, the increase in volume may be due to increasing binding of prednisolone at extravascular sites. There was a constant amount of prednisolone bound to cortisol-binding-globulin (CBG; 145 2 16 ng/ml ) . It appears that prednisolone may exhibit dose-dependent pharmacokinetics, so that with increasing dose values volume of distribution, plasma clearance and half-life may increase. It is believed to be related to changes in the plasma protein binding of prednisolone. Prednisolone appears to bind to plasma proteins in a non linear manner over the range of doses used (131). It is believed that it is the free non-protein-bound fraction of the circulating steroid wich is biologically active and which is metabolised. Prednisolone has been shown to bind to albumin and to specific a- and P-globuline in plasma. Human corticosteroi d-bind ing g lobu1 in (CBG , transcortin) binds prednisolon with a high affinity but low capacity due to relatively low concentrations of

PREDNISOLONE

483

about 10-7 in plasma. Thus there is a saturation in protein binding as the steroid concentration increases above physiological levels. However, albumin, although it has a lower affinity for prednisolone, has a larger capacity for binding due to its higher plasma concentrations o f about 10-4 M. It is therefore suggested that changes in predn isolone plasma protein binding are responsible for the varience in volume of distribution, half-life and metabolic clearance (144). At low prednisolone concentrations binding to CBG is important, which becomes then saturated so that a higher concentration of albumin plays the major role (145). In another study it is suggested that variations in the level of circulating cortisol could cause variation in the protein binding o f prednisolone (146). Uribe et a1 (147) determined the effect of a liquid diet on the serum protein binding o f prednisolone in normal healthy subjects. It was observed that the percentage of prednisolone bound to plasma proteins measured at the time of peak levels was 80 %, whether administered with the meal or with water. Prednisone and prednisolone tablets are on the list o f drugs with high risk potential for therapeutic inequivalence because of differences in bioavailability. Levy et a1 reported a case of a patient with arthritis who was successfully treated with a proprietary brand o f prednisolone 5 mg tablets. Subsequently when prednisone 5 mg tablets

484

SYED LAIK ALI

as generic brand was given even at fourfold dose, but the response failed. It appears that prednisone tablets with low dissolution rates may be clinically ineffective (148). Gambertoglio et a1 (131) have given in a detailed review the pharmacokinetics of prednisolone in healthy volunteers, patients with different diseases as well as effect of other drugs such as barbiturates , phenytoi n , r ifampi n and oral contraceptives. Much attention has been focussed on comparisons between standard and susstained-release preparations (70, 149) and between standard and enteric-coated tablets (150). Enteric coated prednisolone has been introduced to reduce gastrointestinal distress (151). The ability of enteric coated and sustained-release preparations to produce prolonged plasma concentrations were considered to be of no greater value than conventional tablets (152). The distribution and elimination of prednisolone have been described in terms of a 2-compartiment open model, with rapid distribution within the first half-hour followed by a slower terminal elimination phase (126). The absolute bioavailability of prednisolone from a rectal capsule was tested in 12 healthy volunteers. the bioavailability from this dosage form was

PREDNlSOLONE

485

calculated with about 48 % compared to a short infusion. The maximum plasma levels occured between 1.2 to 3.4 h (153).

10.

Acknowledqement The author is indebted to Miss Michaela Schiavulli who has taken great pains in typing this manuscript. Mrs. Petra Grotsch kindly assisted in drawing the figures.

486

SYED LAIK ALI

11. (1)

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Sci. 2 333

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W. Avdovich and Coworkers, J. Pharm. Sci. 3 1164

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Lanouette and B. A. Lodge, J. Chromatogr. 129 475

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493

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(90) J .

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143 414

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J. Q.
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406 (1982)

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495

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H.

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Assoc.

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Anal. Chem. 47 674

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2614 (1984)

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632 (1987) (102) J.

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Ngoc,

J.

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(105) V . K. Prasad,

- 305 378

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78 97

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(108 (109

N.

W. Tymes, J. Chromatogr. Sci. 1 151 (1977) 5

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(110) F. K . (1975) (111) S .

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Biomedical

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Mass

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491

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Pharmacopeia XII, 2nd edition, Convention, Rockville, Md. 120852, P. 1130 (1990)
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L. S.

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J.

Morrison

and

Coworkers,

Br. J .

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(138) L. 6 . Petereit and A. Ther. 22 912 (1977)

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1 1131 9

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499

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(141) J .

C. K.

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736 (1978) (142) J . Q. Rose and Coworkers, In

Congress
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of

seventh International Pharmacology, Abstracts, Paris, P. 400

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R. Slaunwhite and A. A. Sandberg, J. 7 Endocrinol. Metab. 1 395 (1957)

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(146) A . Angeli 47 (1978) (147) M.

and Coworkers, Clin. Pharmacol. Ther. 23

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362

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Levy and Coworkers, Am. J. Hosp. Pharm. 21 402

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J.

Pharm. Sci 60 1028

500

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(150) C. G. Wilson and Coworkers, Bri. J. Clin. Pharmacol. - 703 (1977b) 4 (151) W.

Kammerer and Rheumatism L 122 (1958)


H.

Coworkers.

Arthritis

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Mehlhaus-Barlet, M. Therapiewoche 3 209 (1988) Kummer and K. Kunz,

(153) N.

SOTALOL

Robert T. Foster and Robert A. Carr

Faculty of Pharmacy & Pharmaceutical Sciences University of Alberta Edmonton, Alberta, Canada, T6G 2N8

ANALYTICAL PROFILES OF DRUG SUBSTANCES AND EXClPlENTS - VOLUME 21

501

Copyright 0 1992 by Academic Press, Inc. All rights of reproduction reserved in any form.

502

ROBERT T. FOSTER AND ROBERT A. CARR

1. Description 1.1 Nomenclature 1.1.1 Chemical Names 1.1.2 Nonproprietary Names 1.1.3 Proprietary Names 1.2 Formula 1.2.1 Empirical 1.2.2 structural 1.3 Molecular Weight 1.4 Appearance, Color and Odor 2. Synthesis

3. Physical Properties 3.1 Infrared Spectra 3.2 NMR Spectra 3.2.1 Proton NMR 3.2.2 13C N M R 3.3 Mass Spectra 3.4 Ultraviolet Spectra 3.5 Optical Rotation 3.6 Melting Point 3.7 Ionization Constants 3.8 Partition Coefficient 3.9 Solubility
4. Methods of Analysis 4.1 Elemental 4.2 Chromatographic 4.2.1 Thin-Layer 4.2.2 Gas 4.2.3 High-PerformanceLiquid

5. Pharmcokinetics 5.1 Absorption 5.2 Distribution 5.3 Metabolism 5.4 Excretion


6. References

SOTALOL

503

1. DESCRIPTION

1.1 Nomenclature 1.1.1 Chemical Names N-[4-[ 1-Hydroxy-2-[ (1-methylethyl)amino]ethyl]phenyl]methanesulfonamide; 4 ' -[1-hydroxy-2-(isopropylamino)ethyl]methanesulfonanilide (1,2); MJ-1999 (2). Chemical abstracts registry no. : 3930-20-9, sotalol; 959-24-0. sotalol hydrochloride (2). 1.1.2 Nonproprietary Name Sotalol (1) 1.1.3 Proprietary Names Beta-Cardone, Betacardone, Betades, Sotacor, Sotalex, Sotapor (192). 1.2 Formula 1.2.1 Empirical C ~ ~ H ~ O Nsotalol~base; C12H21ClN203S, sotalol ~O S, hydrochloride 1.2.2 structural Figure 1 depicts the structure of sotalol.

C Hg SO 2 N H

<ob*

! C HC HZN H C H ( C Hg ) 2 !

FIGURE 1.

Structure of sotalol, where the asterisk denotes the chiral center.

504

ROBERT T. FOSTER AND ROBERT A. CARR

1.3 Molecular Weight 272.36, sotalol base; 308.82, sotalol hydrochloride 1.4 Appearance, Color and Odor
An odorless, white, crystalline solid (1).
2. SyNntIESIs

The synthesis of several sulfonamidophenethanolamines, including sotalol, has been described previously (3). The synthesis of the sulfonamidophenethanolamine compounds relied upon firstly obtaining a 2-aminoacylsulfonanilideprecursor. The alcohol formed from the ketone precursor is via either palladium-catalyzed low-pressure hydrogenation or sodium borohydride chemical reduction (3). Two schemes outlining the synthesis of the 2aminoacylsulfonanilideprecursor have been reported (3). The first scheme (Figure 2) introduces the amino moiety last, whilst holding the suIfonamido constant. The second scheme (Figure 3) introduces the sulfonamido last, thus introducing the amine moiety first and holding it constant.

3. PEYSICAL PROPERTIES
3.1 Infrared Spectra The infrared spectrum of sotalol hydrochloride is depicted in Figure 4. The spectrum was obtained on a KBr disk using a Nicolet 20 SX Fourier Transform infrared spectrometer. Diagnostic peaks were observed at 3570 cm-l (secondary alcohol, free); 3410 cm-1 (secondary alcohol, H-bonded); 2700-2800 cm-l and 2950-3200 cm-1 (hydrochloride); 1325 cm-1 (S =O asymmetric stretch); 1154 cm-1 (S=O symmetric stretch). The peaks are presented in Table I.

SOTALOL

505

0 It

pczr2
NHS02R I
Br2

0
II

0 I1

R ,S02CI
NHS02R

0
II

NH2

FIGURE 2.

Synthetic Pathway for Ketone Precursor to Sotalol (from ref. 3). R1 =CH3; R2=H; R3Rq=CH(CH3)2

506

ROBERT T. FOSTER AND ROBERT A. CARR

0
H2N

HNR3R4

Fci:NR
3 4

0
II

NHSO R

2 1

FIGURE 3.

Synthetic Pathway for Ketone Precursor to Sotalol (from ref, 3). R1 =CH3; R2=H; R3Rq=CH(CH3)2

FIGURE 4.

Infrared Spectrum of Racemic Sotalol Hydrochloride. Instrument: Nicolet 20 SX Fourier Transform infrared spectrometer

508

ROBERT T. FOSTER AND ROBERT A. CARR

Table I. I.R. Spectrum of (+)-Sotalol HC1. KBr pellet. Instrument: Nicolet 20 SX Fourier Transform I.R.
Wavenumber cm2700-3 100
bl

Relative Intensit I
s broad s broad

1585 1508 1460 ,1393 1325 1225 1201 1071 1016 985 962 904

m
S

m m
S

m
W

m m m
W

m
W

864
837 ,793 773 689 655 1 635
8, s=strong; m=medium;

m
W

m
S
W

Iw

w=weak.

SOTALOL

509

3.2 NMR Spectra 3.2.1 Proton NMR


The 300 MHz proton NMR spectrum of (+)-sotalol in CD30D is described in Table 11. The spectrum was obtained on a Bruker AM-300 spectrometer. Instrumental settings were: time domain (data points), FT NMR 16K; aquisition time, 1.819 sec.; spectral width, 4504.51; receiver gain, 32; line broadening, 0.200. The spectrum is shown in Figure 5. The spectra for S( +)- and R(-)-sotalol are depicted in Figures 6 and 7, respectively. There is no difference between the spectra for either pure enantiomer of sotalol and that of the racemate (all run in CD30D). It is worth noting that the coupling of protons (e.g., -CHC&NH, -CH(C&)2, and -Cg(OH)CH2-) is altered probably as a function of the chiral center of sotalol. The D 2 0 exchange NMR spectrum of sotalol is shown in Figure 8. The exchangeable protons (OH and NH) are absent, and are replaced by a single HOD peak at 4.886.

3.2.2 13C NMR


The 300 MHz 13C NMR spectrum of sotalol in CD30D is described in Table 111. The spectrum was obtained on a Bruker AM-300 spectrometer. Instrumental settings were: time domain (data points), FT NMR 16K; aquisition time, 0.4424 sec.; spectral width, 18518.52; receiver gain, 400; line broadening, 2.00. The spectrum is shown in Figure 9.

3.3 Mass Spectra


Mass spectra were obtained on a AEI MS9 (Manchester, U.K.) instrument equipped with a fast atom bombardment source (Figures 10 and 11). The medium was either glycerol or Cleland and the sample was introduced by means of direct insertion. Instrument settings were: 92963; total scans in run, 4; sampling rate, 256; signal level threshold, 30; minimum peak width, 5; scan rate (sec/dec), 10.0.

510

ROBERT T. FOSTER AND ROBERT A. CARR

Table I . 300 MHz Proton NMR of (f)-Sotalol in CD30D. I

J-

i
r

1L ,
6
I

FIGURE 5 .

Proton NMR Spectrum of Racemic Sotalol. Instrument: Bruker AM-300 FT NMR spectrometer

Prn

JL 1
I
I

PPfl

FIGURE 6.

Proton N M R Spectrum of S +)-Sotalol. ( Instrument: Bruker AM-300 FT Nh4R spectrometer

-4
I

-I I
9

PPW

FIGURE 7.

Proton NMR Spectrum of R(-)-Sotalol. Instrument: Bruker AM-300 FT N M R spectrometer

!I

PPV

FIGURE 8.

Proton N M R Spectrum of Racemic Sotalol. D20 Exchange. Instrument: Bruker AM-300 FT N M R spectrometer

SOTALOL

515

Table III. 300 MHz l3C NMR of (+)-Sotalol in CD30D.

b Chemical Shift

139.22 140.81

1 1

c-4 c-1

wa

It

1mm

am PP 11

6m

rm

21

FIGURE 9.

l3C N M R Spectrum of Racemic Sotalol. Instrument: Bruker AM-300 FT NMR spectrometer

SOTALOL

517

Regardless of the medium, MH+ peaks were found at m/z 273. Both spectra also exhibited peaks at m/z of 545, corresponding to M2H+; at HC1; and at m/z of 255, m/z of 581, corresponding to M2H+ corresponding to MH+-H20. The spectrum with glycerol as the medium showed a base peak at m/z of 93, which suggests CH3NSOZ. Table IV summarizes the fast atom bombardment spectral data and suggests the structures for the fragments. Additionally, a positive ion electron impact mass spectrum was obtained on a Kratos MS 50 double focusing magnetic sector mass spectrometer. The sample was introduced by means of direct insertion. Instrument settings were: mass range, 5 1.0235-279.1606, total scans in run, 1; sampling rate, 25; signal level threshold, 1; minimum peak width, 7; scan rate (seddec), 10.0, number of scans averaged, 11. The calculated M+ is at m/z 272.1195; a M+ was found at m/z 272.1196. Furthermore, diagnostic peak (100% relative abundance) was found at m/z 72.0817 which suggests a C ~ H ~ O N fragment.

Table IV. FAB Mass Spectral Data of Sotalol HCl.

Ton
C12H21N203S C12H19N202S CH3NS02

Measured Mass

9% Relative Abundance
100.00 (Cleland) 97.05 (glycerol) 62.77 (Cleland) 56.46 (glycerol) 100.00 (glycerol)

272.93 272.93 255.01 255.02 93.04

Previously, spectral data for sotalol were reported using negative ion chemical ionization mass spectrometry (4). The base peak (M-79)corresponded to the loss of (-S02CH3). Other characteristic ions were found at m/z 163 (C2FsCOO)-; m/z 147 (C2F5CO)-; m/z 144 (C2F4COO)-; and at (M-147)- and (M-166)-. 3.4 Ultraviolet Spectrum Figure 12 depicts the ultraviolet spectrum of sotalol free base in chloroform. The spectrum was obtained using a Phillips PU8700 series UV/VIS scanning spectrophotometer (Cambridge, U.K.) Qualitative . results depict maximal wavelengths at 242.2 and 275.2 nm.

FIGURE 10. Positive Ion FAB M s Spectrum of Racemic Sotalol. as


Instrument: AEI, MS9. Medium: glycerol

80

60

255
119 33

20

I,,

.#.ul

155
./< I

177
l

195

213

238
1..

il.

I,

FIGURE 11. Positive Ion FAB Mass Spectrum of Racemic Sotalol.


Instrument: AEI, MS9. Medium: Cleland

520

ROBERT T. FOSTER AND ROBERT A. CARR

FIGURE 12. Ultraviolet Spectrum of Racemic Sotalol Base in Chloroform. Instrument: PU8700 series scanning UV/VIS spectrophotometer

SOTALOL

521

3.5 Optical Rotation Optical rotations of the two pure enantiomers of sotalol HC1 were obtained using a Perkin Elmer Model 241 polarimeter. The rotations were measured in a 10 cm cell (water as solvent) at the sodium D-line (589 nm). The optical rotations (specific rotations) of sotalol HCl were: (+)-Sotalol HC1 [ c Y ] ~ ~ D +35.80" (-)-SOtalol HC1 -34.75 " (5) as: The specific rotations of sotalol HC1 in methanol were reported (+)-sotal~i HCI (-)-SOtalol HC1 [ c Y ] ~ ~ D 3.6 Melting Points Utilizing a Uni-Melt capillary melting point apparatus (Arthur H. Thomas Company, Philadelphia, PA), the melting points of racemic sotalol HCl, S-and R-sotalol HCl were 218 to 219, 210 to 211 and 204 to 205 "C, respectively. The melting point of racemic sotalol HCl has previously been reported as being within the range of 206.5 to 207 (1). 3.7 Ionization Constants The pka values for sotalol are 9.8 and 8.3 for the amine and the sulfonamide, respectively (6). 3.8 Partition Coefficient The watedn-octanol partition coefficient (log P value) has been reported to be 0.24 (7). Using octan-1-ol/phosphate buffer @H 7.4) at 37" C, sotalol was reported to have a partition coefficient of 0.09 (8). 3.9 Solubility Sotalol HC1 is freely soluble in water and only slightly soluble in chloroform (1). +39.9" -36.3"

522

ROBERT T. FOSTER AND ROBERT A. CARR

4.1 Elemental
The elemental analysis of sotalol(1) is:

C H N

52.92% 7.40% 10.28% 17.62% 11.77%

4.2 Chromatographic Analysis 4.2.1 Thin-layer


A number of methods have been reported for the analysis of sotalol(9-1 ) These methods are summarized in Table V. 2.

Table V. Rf Values of Sotalol under Various Thin-Layer c Conditions Rf Value Solvent-System methano1:ammonium 01 962 1.5) where both hydroxide (100: constants cyclohexane: toluene:diethylamine represent principle (75: 1 :10) 5 chloroform:methanol component scores. (9: 1) acetone ethyl acetate:methanol: 30 % ammonia (85: 105)

Ref. 3,lO

continued.. .

SOTALOL

523

Table V continued.. .
Silica Gel 60 F254 ethyl acetate:methanol: ammonia (85: 105) methanol:ammonia (100:1.5) methanol:butanol (60:40 and 0.1 M NaBr) methanol:water:HCl (5050: 1) ethyl acetate:methanol: concentrated

22 56 75 (bad spot shape) 71 0.7

11

Polygram Sil N-HR UV 254

12

4.2.2 Gas
The use of gas chromatography has been reported by others (4, 13-15). Generally, these methods have only been utilized for the analysis of sotalol in urine. Table VI summarizes most gas chromatographic methods reported to date.

4.2.3 High-Performance Liquid


a. Nonstereospecific. Numerous HPLC methods have been reported for the analysis of sotalol (16-23). Generally, most nonstereospecific HPLC assays utilize reverse-phase chromatography with isocratic flow. Table VII summarizes the more recently reported methods.
b. Stereospecific. The enantiomers of sotalol have been reported utilizing HPLC methods (24-28). These methods employed either chiral stationary phases (24,25), or pre-column derivatization with a homochiral reagent and subsequent separation utilizing either reversephase (26) or normal-phase (27) chromatography. For the most part, however, chiral columns have primarily been used for preparative-scale

Table VI. Conditions of Reported Gas ChromatographicAnalyses. Extraction


diethyl ether; diethyl ether: dichloromethane (1:l)

Derivatization
pentafluoro-propionic anhydride:pyridine (2: 1) acetic anhydride:pyridine (3:2)

C o l d Retention Time capillary, fused silica (1=25 m, 0.32 mm i.d.) and SE-30 methylsilicone bonded phaseI8.64 min
capillary, cross-linked methyl-silicone (1 = 12 m, 0.2 mm i.d.), 0.33 pm film thickness/ ret. index, 2675 (see ref. 12)

electroncapture detector (ECD)

two extractions; dichloromethane:isopropanol :ethyl acetate (1:1:3)

flame ionization detector and a nitrogen-sensitive detector

13

continued.. .

Table VI continued.. . diethyl ether followed by tbutyl alcohol: diethyl ether (15 ) diethyl ether, followed by chloroformpentanol (3:l) Bond-Elut C2, C18 and CN solid-phase diethyl ether N-methyl-Ntrimethylsilyltrifluoracet-amide, followed by Nmethylbistrifluoracetamide trifluoroacetic anhydride:ethyl acetate (2: 1) dried 1-butaneboronic acid in ethyl acetate none capillary, J & W Durabond 1 (1=30 m, 0.25 mm i.d.), 25 pm film thickness/ 3.3 min.
~

Finnigan-MAT ion trap detector

14

Ln Ln N

capillary, fused silica, SE54 (1=25 m, 0.32 mm i.d.), 0.3 mm film thickness/ret. time not reported

Ion trap detector

15

gn>

Table VII. Conditions of Reported Non-Stereospecific High-Performance Liquid Chromatographic Analyses. Extraction Mobile Phase Composition; Flow Rate methano1:hexane (85:15) with 0.02 % perchloric acid (1.85 mM); 2mVmin Retention Time Detector; Ref. Sensitivity UV, 215 nm; 16 qualitative use
U V , 227nm;

no extraction, qualitative benzyl alcohol: chloroform (60:40)


1-butanol: chloroform (20: 60)

Baker- 10 SPE Octyl, elution with ethyl acetate: acetonitrile (1:2)

methanol:water: acetonitrile (55:45:20) with 1% acetic acid and 0.005 M dodecyl sodium sulphate; 1 mVmin 0.01 M phosphate buffer @H 10 min 3.2):acetonitrile (20:80) with 3 mM n-octylsodium sulphate; 1.5 ml/min water:methanol:acetonitrile: 0.1 8.3 min M dibasic ammonium phosphate (45:48:6: 1); 1.5 mVmin

rel. ret. time, 0.68 (rel. to prazepam, where rel. ret. time of 1.0=9.20 min) approx. 5 min.

10 ng/ml

17

W, 226 nm; 0.03 pmoV1

18

fluorescence, 19 240/310 nm (ex., em.); 10 ng/ml (plasma), 0.5 p g / d (urine) continued.. .

Table VII continued.. .


25 cm Altex, Baker-10 SPE Octyl, elution ODs-5-pm with acetonitrile: ethyl acetate (2: 1) 25 cm n-pentanol: Hypersil, chloroform (1:3) ODs-5-pm 0.01 M potassium phosphate dibasic buffer @H 2.4) containing 0.002 M nonylamine; 2 mYmin acetoniae: water:acetic acid (20:79: l), adjusted to pH 2.5 by NaOH; 0.005 mol/l heptanesulfonic acid and 0.0005 moYl sodium dodecylsulfate added; 1 mYmin 4.5 min diodearray, 235nm; 20 ng/ml 20

25 cm LiChrosorb, CN 10-pm 22 cm Brownlee Labs, ODS

1
ethyl acetate

approx. 5 min

5-pm

methanol:2-propanol: 1.16 M perchloric acid (75:25:0.5); 2.5 mYmin 1-pentanol: water:acetonitrile (60:40) with chloroform (1:3) 1% heptanesulfonic acid:glacial ! acetic acid (75); 1 ml/min

4.4 min 9.3 min

fluoreGnce, 21 235 nm/no emission filter; 50 ng/ml (plasma), 2 pg/ml (urine) fluorescence, 22 235/310 nm; 2 ng/ml fluorescence, 23 235 (excitation)/ no emission filter; 25 ng/ml

Table VIII. Conditions of Stereospecific High-Performance Liquid Chromatographic Analyses for Sotalol. Column

Extraction
No extraction; qualitative analysis

25 cm amylose tris (3,5-dimethylphenylcarbamate)


10 cm alphal-acid glycoprotein (Enantiopac, LKB)

Mobile Phase Composition; Flow Rate hexane:2propanol:diethylmine (80:20:0.1); 0.5 mYmin

Retention Time

(d);
sotalol at approx. 17 and 23 min, respectively (+)- and (-)sotalol at approx. 15 and 10 min, respectively. (-)- and (+)sotalol at approx. 28 and 30 min, respectively. sotalol at 7.5 and 8.7 min, respectively.
(+)- and (-)(+)- and (-)-

Detector; Sensitivity

Ref.
24

UV, h not

quantitation

acid in 0.02 M phosphate buffer @H


(60:40); 1 mYmin

specified; sensitivity not reported U V , 230 nm; sensitivity not reported

25

10 cm, Partisil ODS acetonitrile 5-pm

25 cm, Partisil5-pm

methanol (65:33: 2); 2 mYmin

fluorescence, 232 nm ex/no emission filter; sensitivity not reported fluorescence, 220 nm ex/no emission filter; I 20 ng/ml
~

26

27

SOTALOL

529

enantiomer separations of sotalol. Table VIII summarizes the stereospecific HPLC assays for sotalol.
5 . PaARMAcoKINEllcs

5.1 Absorption Although the lipid solubility of sotalol is relatively low compared with other P-blocking adrenoceptor drugs (28), oral bioavailability is deemed to be 100%. Sotalol is absorbed somewhat slower than most other @-blockers,with peak concentrations occuring within 2-3 hours (29). Although food may impair the absorption of sotalol (28), administration of either calcium carbonate or aluminum hydroxide antacids has little effect on absorption (30). After administration of a single 160 mg oral dose of sotalol, both enantiomers reached maximal plasma concentrations in approximately 3 hours (31) and, hence, did not exhibit stereoselective absorption. 5.2 Distribution Sotalol is only negligibly bound (28) to plasma proteins (albumin and alphal-acid glycoprotein). The volume of distribution of sotalol is 1.3 L/kg. As expected, the more lipophilic @-blocking drugs, including metoprolol and propranolol, have greater reported volumes of distribution of 5.5 and 2.8-5.5 Wkg, respectively (28). Interestingly, the volume of distribution appears to be somewhat reduced in elderly hypertensive subjects (32). For example, values of 3.55k0.51 and 2.22k0.28 Wkg were reported for healthy young and elderly hypertensive subjects, respectively. As sotalol has a very low lipid solubility compared with other P-blocking drugs, there is slow entry of drug into brain; the brain:plasma ratio was determined as 0.52 in anesthetized cats (29). At present, there is no evidence for stereoselective distribution of sotalol after administration of the racemate (31). 5.3 Metabolism Sotalol does not undergo first-pass metabolism after oral administration (29). Following intravenous administration of 3H-sotalol to dogs, over 90% of the drug was excreted renally; less than 1% of the drug was excreted in bile (33). In a stereospecific study of sotalol(31), nonrenal clearance constituted a mean of approximately 23% of the oral clearance. As sotalol does not a D m u to be metabolized in man. it was

530

ROBERT T. FOSTER AND ROBERT A. CARR

suggested that either biliary excretion and/or direct secretion of drug across gut wall may occur in humans (31). 5.4 Excretion Sotalol is excreted by glomerular filtration with approximately 75% of the drug being excreted within 72 hours (29). The reported elimination half-life ranges from 7-18 hours (29). As expected, reduced renal function (i.e., reduced creatinine clearance) results in reduced renal clearance values of sotalol. For example, renal clearance has been reported (34) to be reduced from a mean of 4.99 Wh (creatinine clearance > 80 ml/min) to a mean of 0.27 L/h (creatinine clearance C 10 ml/min). In fact, after chronic administration of sotalol, the serum half-life was reported to be 69 hours in an anuric patient (35). Although there is no difference in the enantiomeric clearance of sotalol (31), it has been suggested that the clearance of (+)-sotalol after administration of such may be reduced (36) as compared to its clearance when administered with an equal proportion of (-)-sotalol (i.e., when administered as racemate). The disposition of sotalol appear to be comparable between obese individuals and control subjects (37). In elderly hypertensive subjects, however, renal clearance was reduced from a value of 4.10f0.60 ml/min/kg which was observed in healthy young subjects, to 1.93f0.32 ml/min/kg (32). Presumably, the reduction in sotalol renal clearance in the elderly is a reflection of the changed physiology in the elderly (e.g., reduced glomeruIar filtration). Finally, sotalol is excreted in breast milk, whereby mi1k:serum concentration ratios ranged from 2.43-5.64 (38). Consequently, breastfed infants may be exposed to relatively large sotalol concentrations. 6. REFERENCES 1. Windholz M., editor. The Merck Index, 10th edition. Rahway, NJ, Merck & Co., Inc. 1983:1248. 2. Reynolds J.E.F., editor. Martindale: the Extra Pharmacopoeia, 29th edition. London, The Pharmaceutical Press. 1989:807-808. 3. Uloth RH, Kirk JR, Gould WA, Larsen AA: Sulfonanilides. I. Monoalkyl- and arylsulfonamidophenethanolamines.J. Med. Chem. 1966;9: 88-97. 4. Cartoni GP, Ciardi M, Giarrusso A, Rosati F: Detection of pblocking drugs in urine by capillary column gas chromatographynegative ion chemical ionization mass spectrometry. 1. High Resolution Chromatogr. Com. 1988;11528-532.

SOTALOL

531

5.

6. 7.

8.

9.

10.

11. 12.

13.

14.

15.

16.

and l-sotalol mandelate and hydrochloride salts by high performance liquic chromatography. J. Liquid Chromatogr. 1987;10:3015-3023. isn Doerge RF, Editor. W l o and Gisvold's Textbook of Organic Medicinal and Pharmaceutical Chemistry, 8th edition. Philadelphia, J.B. Lippincott Company. 1982:845. Burgot G, Serrand P, Burgot JL: Thermodynamics of partitioning in the n-octanol/water system of some &blockers. Internat. J . Pharmaceutics 1990;63:73-76. Jack DB, Hawker JL, Rooney L, Beerahee M, Lobo J, Pate1 P: Measurement of the distribution coefficients of several classes of drug using reversed-phase thin-layer chromatography. J. Chromatogr. 1988;452:257-264. Musumarra G , Scarlata G, Romano G, Clementi S, Wold S: Application of principal components analysis to TLC data for 596 basic and neutral drugs in four eluent systems. J. Chromatogr. Sci. 1984;221538-547. Musumarra G,Scarlata G,Cirma G,Romano G, Palazzo S, Clementi S, Giulietti G: Qualitative organic analysis: 1. identification of drugs by principal components analysis of standardized thin-layer chromatographic data in four eluent systems. J. Chromatogr. 1985;350:151-168. Ojanpera I, Vuori E: Thin layer chromatographic analysis of basic and quaternary drugs extracted as bis(2-ethylexy1)phosphate ionpairs. J. Liq. Chromatogr. 1987;10:3595-3604. Jack DB, Dean S, Kendall MJ, Laugher S: Detection of some antihypertensive drugs and their metabolites in urine by thin layer chromatography: 11. A further five beta blockers and dihydralazine. J. Chromatogr. 1980;196:189-192. Maurer H, Pfleger K: Identification and differentiation of betablockers and their metabolites in urine by computerized gas chromatography-massspectrometry. J. Chromatogr. 1986;382:147165. Leloux JS, De Jong EG, Maes M A : Improved screening method for beta-blockers in urine using solid-phase extraction and capillary gas chromatography-mass spectrometry. J. Chromatogr. 1989;488:357-367. Koppel C, Tenczer J, Peixoto-Menezes DM: Formation of formaldehyde adducts from various drugs by use of methanol in a toxicological screening procedure with gas chromatography-mass spectrometry. J. Chromatogr. 1991;563:73-81. Flanagan RJ, Storey GCA, Bhamra RK: High-performance liquid chromatographic analysis of basic drugs on silica columns using non-aaueous ionic eluents. J. Chromatogr. 1982:247:15-37.

Garrec LL, Delee E, Pascal JC, Jullien I: Direct separation of d-

532

ROBERT T. FOSTER AND ROBERT A. CARR

17. Lemmer B, Ohm T, Winkler H: Determination of the betaadrenoceptor blocking drug sotalol in plasma and tissues of the rat by high-performance liquid chromatography with ultraviolet detection. J. Chromatogr. 1984;309:187-192. 18. Karkkahen S: High-performance liquid chromatographic determination of sotalol in biological fluids. J. Chromatogr. 1984;336:3 13-319. 19. Bartek MJ, Vekshteyn M, Boarman MP, Gallo DG: Liquid chromatographic determination of sotalol in plasma and urine employing solid-phase extraction and fluorescence detection. J. Chromatogr. 1987;421:309-318. 20. Hoyer GL: Improved high-performance liquid chromatographic method for the analysis of serum sotalol. J. Chromutogr. 1988;427:181-187. 21. Gluth WP,Sorgel F, Gluth B, Braun J, Geldmacher-v. Mallinckrodt M: Determination of sotalol in human body fluids for pharmacokinetic and toxicokinetic studies using high-performance liquid chromatography. Anneim. Forsch. D u Res. 1988;38:1:408rg 411. 22. Poirier JM, Lebot M, Cheymol G: Rapid and sensitive column chromatographic determination of sotalol in plasma. J. Chromotagr. 1989;493:409-413. 23. Morris R: Improved liquid chromatographic fluorescence method rg for estimation of plasma sotalol concentrations. Ther. D u Mon. 1989;11 :63-66. 24. Okamoto Y, Aburatani R, Hatano K, Hatada K: Optical resolution of racemic drugs by chiral HPLC on cellulose and amylose tris(pheny1carbamate) derivatives. J. Liq. Chromatogr. 1988;11~2147-2163. 25. Le Garrec L, D e l e E, Pascal J-C: Direct separation of d- and 1sotalol mandelate and hydrochloride salts by high performance liquid chromatography. J. Liq. Chromutogr. 1987;10:3015-3023. 26. Mehvar R: Stereospecific liquid chromatographic analysis of racemic adrenergic drugs utilizing precolumn derivatization with (-)menthyl chloroformate. J. Chromatogr. 1989;492:402-408. 27. Carr RA, Foster RT, Bhanji NH: Stereospecific high-performance liquid chromatographic assay of sotalol in plasma. Phamz. Res. 1991;8: 1195-1198. 28. Riddell JG, Harron DWG, Shanks, RG: Clinical pharmacokinetics of 0-adrenoceptor antagonists: An update. Clin. Pharmacokinet. 1987;12:305-320. 29. Singh BN, Deedwania P, Nademanee K, Ward A, Sorkin EM: Sotalol: A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic use. Drugs 1987;34:311-349.

SOTALOL

533

Pharmacokinetics and pharmacodynamics in young normal and elderly hypertensive subjects: A study using sotalol as a model 173-181. drug. J. Pharmacol. &p. Ther. 1980;212: 33. Bourne GR: The metabolism of P-adrenoceptor blocking drugs. Progress in D u Metab. London, U . K . , John Wiley & Sons, rg 1981 ;6:77- 10. 1 34. Dumas M, D'Athis P, Besancenot JF, Chadoint-Noudeau V, Chalopin JM, Rifle G, Escousse A: Variations of sotalol kinetics in renal insufficiency. Znt. J. Clin. Pharmacol. Ther.Tox. 1989;27:486after chronic administration to patients with renal insufficiency. Eur. J. Clin. Pharmacol. 1980;18:321-326. 36. Carr RA, Foster RT: Enantiospecific study of sotalol in rats. Pharm. Res. 1991;8:S265. 37. Poirier JM, Le Jeunne C, Cheymol G, Cohen A, Barre J, Hugues FC: Comparison of propranolol and sotalol pharmacokinetics in obese subjects. J. Pharm. Pharmacol. 1990;42: 344-348. 38. Hackett LP, Wojnar-Horton RE, Dusci LJ,Ilett KF, Roberts MJ: Excretion of sotalol in breast milk. Br. J. Clin. Pharmacol. 1990; 277. 29:

30. Kahela P, Anttila M, Sundqvist H: Antacids and sotalol absorption. Acta Pharmacol. et Toxicol. 1981;49: 181-183. 31. Carr RA, Foster RT, Lewanczuk RZ, Hamilton PG: Pharmacokinetics of sotalol enantiomers in humans. J. Clin. Pharmacol. 1992; accepted. 32. Ishizaki T, Hirayama H, Tawara K, Nakaya H, Sat0 M, Sat0 K:

489. 35. Berglund G, Descamps R, Thomis JA: Pharmacokinetics of sotalol

THIOPENTAL SODIUM

Michael J . McLeish

School of Pharmaceutical Chemistry Victorian College of Pharmacy (Monash University) Parkville, Victoria, Australia

ANALYTICAL PROFILES OF DRUG SUBSTANCES AND EXCIPIENTS - VOLUME 21

535

Copyright 0 1992 by Academic Press, Inc. All rights of reproduction reserved in any form.

536

MICHAEL J. MCLEISH

1.

Description
1.1

1.2

1.3 1.4 1.5

Nomenclature 1.1.1 Chemical Names 1.1.2 Nonproprietary Names 1.1.3 Proprietary Names Formulae 1.2.1 Empirical 1.2.2 CAS Registry Numbers 1.2.3 Structural Molecular Weight Elemental Composition Appearance, Color and Odor

2.

Physical Properties
2.1 2.2 2.3 2.4 2.5 Melting Range Solubility Data Dissociation Constant pHRange Spectral Properties 2.51 Ultraviolet Spectrum 2.5.2 Infrared Spectrum 2.5.3 Nuclear Magnetic Resonance Spectrum M s spectrum as 2.5.4

3.
4.

Synthesis

Stability

THIOPENTAL SODIUM

531

5.

Methods of Analysis
5.1 5.2
Extraction Identification 5.2.1 USP Analysis BP Analysis 5.2.2 Colorimetric,Spectrophotometric and Fluorimetric Analysis Chromatography Paper, Thin-layer and Column Chromatography 5.4.1 5.4.2 G s Chromatography a 5.4.3 High Performance Liquid Chromatography Radioimmunoassay

5.3 5.4

5.5

6.

Metabolism
Uses, Administration and Contraindications

7.
8. 9.

Pharmacokinetics Acknowledgements References

10.

538

MICHAEL J. MCLEISH

1.
1.1

DESCRIPTION
Nomenclature
1.1.1 Chemical Names

(a) (rt)-5-Ethyldihydro-5-( 1-methylbutyl)-2-thioxo4,6(lH,5H)pyrimidinedione monosodium salt [1,2]


(b) (f)-S-Ethyl-5-(1-methylbutyI)-2-thiobarbituricacid sodium salt

[1,2,3,41 1.1.2 Nonproprietaw Names

Thiopental sodium, Thiopentone sodium, thionembutal, thiomembumal sodium, penthiobarbital sodium [lJ, soluble thiopentone [3J. 1.1.3 Proprietary Names

Pentothal Sodium, Nesdonal Sodium, Intraval Sodium, Trapanal, Thiothal Sodium, Farmotal, Hypnostan, Sandothal[ 1,3].

1.2

Formulae
1.2.1 Empirical
1lH17N2Na02S
cl 1H18N202S

(Thiopental sodium) (Thiopental)

1.2.2

CAS Registry Numbers 71-73-8 (Thiopental sodium) [1,2,3]

THIOPENTAL SODIUM

539

76-75-5 (Thiopental) [3] 1.2.3


H

structural
H

NaS N

y J
0

,CH, CHCH,CH,CH, CHCH,CH,CH,

CH,

CH,

Thiopental Sodium

Thiopental

1.3

Molecular Weight 264.31 242.33 (Thiopental Sodium) (Thiopental)

1.4

Elemental Composition

Thiopental Sodium:
C. 0.

49.98% 12.11%

H. Na.

6.48% 8.70%

N. S.

10.60% 12.13%

Thiopental:
C. 0.

54.52% 13.21%

H.

7.49%

N.

S.

11.56% 13.23%

S40

MICHAEL J. MCLEISH

1.5

Appearance, Color and Odor


Thiopental sodium is a yellowish-white, crystalline powder or pale greenish hygroscopic powder with an alliaceous, garlic-like odor [1,3,4]. Thiopental sodium for injection is a sterile mixture of thiopental sodium and anhydrous sodium carbonate as a buffer [1,2,4]

2.

PHYSICAL PROPERTLES Melting Range


The free acid (thiopental) melts at 158-160 O [5,6]. C

2.1

2.2

Solubility Data
At 20 "C, thiopental sodium i soluble in 1.5 parts water [3,4,5]. It is s partially soluble in alcohol [1,3,4,5] and is practically insoluble in ether [I ,3,4,5], benzene [l] and petroleum ether [ 1,3]. The partition coefficent of unionized thiopental between isoamyl alcohol and water at 37 "C, is 991 [7].

2.3

Dissociation Constant
pK, 7.6 at 20 O IS]. C

2.4

pH Range
An 8% solution for injection has a pH of 10.2 to 11.2 [2,5].

THIOPENTAL SODIUM

54 1

2.5

Spectral Properties
2.5.1 Ultraviolet Spectrum

The ultraviolet absorption spectra of thiopental sodium, in both 0.02M HCl and 0.02M NaOH, were obtained on a Shimadzu UV-160A recording UV-Vis spectrophotometer. The spectra (shown in Figure One) exhibited maxima at 295 and 305nm, respectively. The pH-dependence of the W absorption spectrum has also been determined 181. 2.5.2 Infrared Spectrum

The infrared spectrum of thiopental sodium and/or thiopental has undergone considerable investigation [S-1 11. The infrared spectrum of thiopental sodium and thiopental, both as KBr disks, were obtained on a Hitachi 270-30 infrared spectrophotometer. The spectra, presented as Figure Two and Three, respectively, display absorption characteristics in good agreement with those previously reported. Frequency assignments for some of the characteristic bands are given in Table 1.

Frequency cm-I
3270 2940 1720 1655 1525 1420 1350 1300 1220
T a b l e 1.

Infrared Assignment
N-H stretch C-H stretch C=O stretch C=O stretch N-C=S NCS stretch (asymm) C-N stretch C-N NCS stretch (symm)

IR characteristics of Thiopental

E
I . W

3
I

E z

m a
In

m
n, W
W

a m

6, 4

a
v)

a
m

..
m

542

0 0

0 0 W

0 0 m

0 0I . -

cy l r
h

:g
0

7 1

z f t) l
2 E

Q i
0 0

C 0,

0 N

0 0

N n

-\

0 0 F1 0

m ul

0 0
0

543

0 0 4

0 W 0

0 0 m

0
d 0

Y
h

8 'E 2s

04
d

::z

% E
C

2 3
0 0 0

m
(Y

0 0

0 0

* 0

THIOPENTAL SODIUM

545

2.5.3

Nuclear Magnetic Resonance Spectrum

The 'H nmr spectrum of thiopental in dimethyl SdphOXide-d6 has been recorded at 60 MHz [121. The spectrum showed considerable overlap which made assignment difficult. A later study, carried out in CDCL3, showed that lanthanide shift reagents could be used to simplify the spectrum [13]. The 'H nmr spectrum of thiopental sodium in DMSO-d6 has been recorded on a Bruker AMX300 nmr spectrometer and is presented in Figure Four. Spectral assignments for both thiopental sodium and thiopental are given in Table 2. Initial assignments were based on integrals and expected splitting patterns, and were later c o n f i i e d using two dimensional proton correlation spectroscopy (COSY). Figure Five shows the proton decoupled 13Cnmr spectrum of thiopental sodium in DMSO-d6. Spectral assignments for thiopental sodium and thiopental are provided in Table 3. The assignments are based on those of Fratiello et al. [14]. 2.5.4 Mass Spectrum

The fast atom bombardment (FAB) mass spectrum of thiopental sodium was recorded using a JEOL JMS-DX 300 mass spectrometer and is presented in Figure Six. The spectrum shows an (M+H)+peak a m/z 265 (relative intensity lo%), a peak at m/z 287 (45%) t corresponding to (M+Na)+,and a further peak at m/z 309 (28%) corresponding to (M+2Na-H)+. The base peak is at m/z 115. The electron impact (EI) mass spectrum of thiopental w s also a obtained, at an ionization voltage of 70eV, on the same instrument, and is presented in Figure Seven. The spectrum corresponds to that

Figure Four. '8 nmr spectiurn of thiopental sodium in D M S O - 4

ppm

12

10

'1

THIOPENTAL SODIUM

547

Table 2 .

' NMF4 Assignments H

CHCH,CH2CH,

I b

Thiopental Sodium 6 @pm)


0.583 0.785 0.825

Thiopental 6 (PPW 0.696


0.807

Multiplicity 'H (No. H) Assignment triplet (3H) triplet (3H) doublet (3H) multiplet (2H) muliplet (2H) H,
He
Hb

0.906 1.090 1.340 1.900 12.56

0.990
1.310

H, H,

1.730
10.42

approx quartet Ha, H, on multiplet (3H) broad singlet

Figure Five.

Proton &coupled

*C nmr spectrum of thiopental sodium

Y
I " ppm " I '
l ' ' ' ' l ~ ' i ' I

150

100

50

THIOPENTAL SODIUM

549

Table 3.

13C NMR Assignments


H

H
O
10

0 11

12

13

CHCH,CH,CH,

CH,

Carbon Assignment

Thiopental Sodium 6 (ppm)


185.6 177.0 176.5 58.3 41.3 33.6 27.4 20.5 14.3 14.1 9.8

Thiopental 6 @pm)
178.8 171.0 170.6 59.9 41.8 33.4 27.5 20.1 14.1 13.8 9.4

May be interchanged

Figure Six.

FAB mass spectrum of thiopental sodium

loo

115

(M-Na)
LA LA 0

287

(M+2Ya-H)+
309

100

150

200

250

300

350

M/Z

Figure Seven.

Electron impact (EI) mass spectrum of thiopental

100

50

172

157

50

100

150

200

250

300

M/Z

Figure Eight.

Fragmentation pathway for thiopental

H
I

CH3

m/z 242

% /
H
I

~-c5xlo
H

OH

+'

OH

OH

m/z 173

m/z 172

m/z 157

THIOPENTAL SODIUM

553

reported by Maurer [151. The most prominent ions were at m/z 242
(%), 173 (21%), 172 (42%), 157 (26%) and 29 (100%). A possible

fragmentation pathway is shown in Figure Eight.

3.

SYNTHESIS
Two methods of preparing thiopental are outlined in Figure Nine.

In method A, ethyl (1-methylbutyl) malonate (I) was condensed with thiourea (II) in the presence of sodium ethoxide in absolute alcohol. After heating at reflux for several hours the solvent was removed and the residue dissolved in cold water. Thiopental (III) w s precipitated a by the addition of dilute hydrochloric acid. Further purification could be accomplished by dissolving I I in dilute sodium hydroxide and I precipitating with carbon dioxide [6].

In method B, the appropriately substituted nitrile (IV) was condensed with thiourea (II), again in the presence of sodium ethoxide in absolute alcohol. Heating at reflux for several hours resulted in the 4-imino compound (V) which was hydrolysed to thiopental[16].
Thiopental sodium can be prepared by treating an alcoholic solution of thiopental with one equivalent of sodium hydroxide and removal of the solvent [6]. These two methods formed, respectively, the basis of the American and British patents for the production of thiopental[17]. Upon comparison of the two methods under laboratory conditions it was concluded that method A w s preferable [17]. a

Figure N i n e .

Synthesis of thiopental

Method A.

c H 3 c H 2 2 H 3 C,H HC , CHCH,CHzCH3 0C, H

- 'YJ
H
I

i) NaOCH2CH3

ii) H+

,H C, CHCH,CH,CH,

H '

11

III

C, H

Method B.

CH3CH20

CN CH,

IV

NaOCH,CH3
H '

H
I

CH,CH,

H20

'YJ
I

,H C,

N CHCH,CH,CH,
NH . .. .

H '

CHCH,CH,CH,

C, H

111

CH,

THIOPENTAL SODIUM

555

4.

STABLLITY
According to the Codex [5], aqueous solutions of thiopental sodium decompose upon standing and solutions should not be used if they have become cloudy or contain precipitates or crystals. A shelf life of 5 days has been reported for thiopental in glycine/NaOH buffer at pH 9.0 [18] while a thiopental sodium for injection solution (2.5%in water) is stable for at least 10 days at 25 O C [19].
In normal saline (0.9%), when stored in plastic infusion bags, thiopental sodium loses up to 23%of its activity in one day [20]. This

loss was attributed to sorption onto the polyvinyl chloride (PVC) of the infusion bags [20]. Furthermore, absorption onto the plastic matrix of an intravenous delivery system and concomitant loss of activity, has also been observed [21]. Conversely, a solution of thiopental sodium stored in disposable plastic syringes showed only negligible loss of potency after 5 days at 25 O and 45 days at 5 "C [22]. A recent study C has examined the interaction between thiopental sodium and infusion containers and found no decrease in potency when stored for 24 hours at 21 O in the dark [23]. The initial study [20] was carried out at pH C 6.0 while the more recent study was carried out at pH 9.1 [23]. The higher sorption rate at low pH was attributed to a greater fraction of thiopental being nonionized [23]. This is consistent with no loss of thiopental activity being seen in the study using plastic syringes 1221 which was also carried out at high (10.1) pH.

The effect of y-irradiation on thiopental has also been investigated 124). No evidence of decomposition was observed with a 2.5 Mrad radiation dose. It was concluded that thiopental, in powder form, may be sterilised by y-irradiation [24].

556

MICHAEL . . I MCLEISH

5. 5.1

METHODS OF ANALYSIS Extraction


Thiopentone sodium, although soluble in water, in acid solution is converted to the free acid (thiopental) which is water insoluble. The assay of thiopental sodium in both biological fluids and proprietary preparations takes advantage of these acid-base characteristics. Extraction is accomplished by acidification followed by shaking with organic solvents. Solvents commonly employed include chloroform [25-271, methylene chloride [28-311 and diethyl or petroleum ether [32-361. Less frequently used are benzene [37], toluene [38,39], ethyl acetate [40,41], n-hexane [15,28] and n-butyl chloride [42]. Additional selectivity and sensitivity was obtained by back-extraction into sodium hydroxide [25,28,35,36,42,43]. More recently an extraction procedure using a solid phase column (Bond-Hut C18)has been reported [MI.

5.2

Identification
5.2.1 USP Analysis 121

Dissolve about 500 mg thiopental sodium in 10 mL water in a separator, add 10 mL of 3 N hydrochloric acid, and extract the liberated thiopental with two 25 mL portions of chloroform. Evaporate the combined chloroform extracts to dryness. Add 10 mL of ether, evaporate again and dry at 105 O for 2 hours: the infrared absorption C spectrum of a potassium bromide dispersion of the residue so obtained exhibits maxima only at the same wavelengths as that of a similar preparation of USP Thiopental reference standard.

THIOPENTAL SODIUM

557

5.2.2

B.P. Analysis 141

A. Acidify 10 mL of a 10%w/v solution of thiopental sodium in carbon dioxide free water with 2 M hydrochloric acid. The solution effervesces. Shake the solution with 20 mL of ether, separate the ether layer, wash with 10 mL of water and dry over anhydrous sodium sulphate. Filter, evaporate the filtrate to dryness and dry the residue at 100 to 105 OC. The infrared absorption spectrum of the residue is concordant with the spectrum of thiopental EPCRS. B. Determine the melting point of the residue obtained in test A and of a mixture of equal parts of the residue and thiopental EPCRS. The difference between the melting points, which are about 160 OC, is not greater than 2 O C .

53 .

Colorimetric, Spectrophotometricand Fluorimetric Analysis


Early estimations of thiopental and other thiobarbiturates depended on color reactions with either cobalt [27] or copper [34]. These estimations were neither accurate nor particularly sensitive and were superseded by ultraviolet spectrophotometricmethods [33,35,45]. Thiopental has W absorption maxima at 290 nm and 305 nm in acidic and alkaline media, respectively. In contrast, the oxobarbiturates have absorption maxima at 220 nm and 255 nm, respectively 1461. These differences provided a means of distinguishing thiopental from its major metabolite, pentobarbital, and as a consequence most determinations were carried out at around 280 nm [33,35,45]. In one case greater selectivity w s provided by a change in extraction solvent, a which permitted the determination of the carboxylic acid metabolite of thiopental[35]. Additional sensitivity could be provided by the use of back-extraction methods (vide supra).

558

MICHAEL J. MCLEISH

The UV methods reported minimumdetectable limits of approximately 0.5 pg/mL. The development of a spectrofluorimetricmethod (hex= 305 nm,,A = 505 nm) lowered this limit to 0.1 pg/mL [36].
5.4

Chromatography
5.4.1 Paper, Column and Thin-Layer Chromatography

Raventh 1461 lists a number of methods using paper chromatography for the identification of thiopental and other barbiturates. These methods al show relatively poor sensitivity with a minimum of 50 pg l required for the positive identification of most barbiturates. Alumina column chromatography using 2% methanol in chloroform as eluant w s employed in the determination of thiopental in tissues, urine a and blood 1341. Thiopentone (>2 mg) showed as a dark band under ultraviolet light. Thin-layer chromatography using silica get has been used to isolate and identify thiopental[36,47]. Elution from silica gel was achieved with benzene-glacial acetic acid (1:9). The Rf under these conditions was 0.47, the recovery better than 95% and the sensitivity as low as 0.5 pg [36]. Other solvents have included chloroform-acetone(9:l) and dioxan, benzene and aqueous ammonia (20:75:5)[47]. Using a potassium permanganate spray, thiopental could be identified as a yellow spot on a purple background [47].
5.4.2

G s Chromatography a

Table 4, although by no means exhaustive, provides a summary of the numerous methods that have been developed for the gas

T a b l e 4.

GC Methods for the determination of thiopental


~~

COLUMN / SUPPORT
3% Neopental Adipate 3% Poly A-103 on G s Chrom Q a

DETECTOR
FID
Alkali - FID FID ECD

DEIUVAT'IZATION
none none

SENSITIVITY

REF.
25

37 38
40

3% SE-30 on HP Chromsorb WP
3% OV-17 on G s Chrom Q a 5% OV-1 on HP Chromosorb W 3% OV-17 on G s Chrom Q a 2% SP2110 - 1% 2510 DA on Supelcoport 5% OV-101 on HP Chromosorb G

TMPAH methylation
Th4AH methylation

m
NP-FID

none Iodomethane methylation none none

28 43
29

F!ID
FID

15

560

MICHAEL J. MCLEISH

chromatographicanalysis of thiopental [15,25,28,29,37,38,40,43]. Generally these methods have achieved much greater selectivity and sensitivity than colorimetric methods, with low nanogram levels being measured using alkali-flame or nitrogen-phosphorus detectors [37,43]. However, the lowest detection limit (100 pg) was obtained using an electron capture detector [40].

In most cases the extraction of thiopental was achieved using procedures described in section 5.1. When required, methylation was the favored method of derivatizationwith reagents including trimethylphenyl ammonium hydroxide F/IpAH, 381, trimethylaniliniumhydroxide [TMAH, 401 and iodomethane [43].

G s chromatography has also been combined with mass spectrometry a


to develop a computerised general screening procedure for barbiturates, including thiopental [151.
5.4.3

High Performance Liquid Chromatography

In recent times HPLC appears to have become the method of choice for the assay of thiopental. As detailed in Table 5 all the methods have employed reversed phase columns and ultraviolet detection. The variation in mobile phases and detector wavelength has been primarily to enable determination in different body matrices or to permit the simultaneous determination of thiopental and either its metabolites or another drug. For example, particular attention has been paid to the simultaneous measurement of thiopental and its active metabolite, pentobarbital [26,31,42,44,48 1.

When developing these assays much attention was focussed on sample preparation. For many of the methods sensitivity and selectivity were of prime importance and consequently extraction methods were

Table 5.

Conditions employed for the HPLC determination of thiopental

COLUMN
c18

MOBILE PHASE
MeCN / H,O (32 : 68)
( pH 7.7) / MeoH / THF (13 : 7 : 4)

DETECTOR

INTERNALSTANDARD Secobarbital

SENSITIVlTY REF.
1oong

-5p

w,254nm
W, 254nm

48

Nucleosil
c 8

-lop -lop

-04

none

125ng

44

Radial-Pak
cg

MeOH / H20 (60 : 40) NaP04 (0.05M, pH 4.6)MeCN (1 : 1) MeOH / KPO, (O.OlM, pH 4.4) (1 : 1) MeOH / H20 (60 :40)

W, 29Onm

Phenolphthalein

n.s.

30

c8 - 7 p 1 LiChroCart
c8 - 5 p 1 pBondapak
c18

W, 195nm

Hexobarbital

n.s.

39

W, 284nm

5-Ethyl-5-p-tolylbarbituric acid
Phenolphthalein

30Ong

31

5p

W, 280nm

30Ong

49
continued

Spheri-5

--------------

c -5 p 6 Spherisorb
c8 1

NaOAc (O.OlM,pH 3.6) / MeCN (70 :30)


NaP04 (0.16M, pH 6.6) /THF (86 : 14)

U V , 28Onm

Flunitrazepam Barbital

50Ong

50 42

-5

W, 24Onm

l0Ong

c8 1

@ondapak
ci8

- 1op-n - 5cUn

KP04(pH 7.8) / MeCN / THF (78 : 22 : 4)


Do4 (O.OMM, pH 6.5) / MeOH

W, 254nm

Pentobarbital

l0Ong

26

W, 28Onm

Phenolphthalein

n.s.

51

Spheri-5

(52 : 48) MeOH / H O , (1 : 1)


W, 2541x11

low Spherisorb
c18

Methohexitone

n.s.

32

Wondapak

KCL (0.2M, pH 2.0) / MeOH (1 : 1)

W, 254nm

none

90%

52

CIS- l o p NaCit (0.1%,pH 6.5) / MeOH Partisil10/25 (55 : 45) KPO4 (0.2M,pH 4.0) / MeCN (9: 1)

W, 254nm

Quinoline

50Ong

53

W, 205nm

Bupivicaine

5M
0

41

sil-x-1

THIOPENTAL SODIUM

563

favored. However, others have concentrated on the rapidity and ease

of sample preparation. In these cases the preparation was limited to the precipitation of plasma proteins with either acetonitrile [49-511 or ethanol [ 2 , 5 ] with the supernatant being injected directly on the column. In the most extreme example untreated plasma was also 5] hs injected directly onto the column [ 3 .Not unexpectedly, t i method suffered in that column efficiency was rapidly lost.
The sensitivity of most methods was 300 ng/mL, or better, which is ample for monitoring plasma levels during thiopental infusion. During continuous treatment plasma levels of even unbound thiopental are

51 generally greater than 500 n g h L [ 4 .


5.5

Radioirnmunoassay Flynn and Spector [55] developed a radioimmunoassay for a number of


barbiturates, including thiopental. The sensitivity for the latter was

100 ng, a figure tenfold higher than for its oxo-analogue, pentobarbital. This indicated that the urea portion of the ring w s critical in a %. determining antibody specificity [ I

6.

METABOLISM
The metabolism of thiopental and other thiobarbiturates has been

In reviewed extensively [46,56-581. mammals the biotransformation


of thiobarbiturates appears to take place by up to four different pathways [46,56-581: i) Side-chain oxidation Desulphuration Hydrolysis of the thiobarbiturate ring

ii) iii)

564

MICHAEL J . MCLEISH

iv)

N-dealkylation

In man, hydrolysis of the thiobarbiturate ring of thioperital does not occur [58], and biotransformation by the liver microsomal oxidase

system appears to be the main route of elimination from the body [59]. However, in spite of the many studies of thiobarbiturate metabolism, the fate of the majority of the administered dose is yet to be identified [58]. What is clear is that less than 0.5% of the dose is excreted as unchanged thiopental [35,60]. The major known pathways [58] of thiopental metabolism in humans are shown in Figure Ten. Of these, conversion to pentobarbital seems to be of minor significance, with only a small proportion of the administered dose being excreted as the desulphurated metabolite [25,60]. Oxidation of the side-chain appears to be the major pathway for metabolism in humans, 10-25% of the administered dose being excreted in urine as the carboxylic acid metabolite [35,60]. C r o l et arl al. 1601 also demonstrated the formation of the hydroxy metabolite, albeit in small amounts.

7 .

USES, ADMINISTRATION and CONTRAINDICATIONS


Thiopental sodium is a barbiturate which is administered intravenously for the induction of general anaesthesia or for the production of complete anaesthesia of short duration [3]. Other uses include the supplementationof regional anaesthesia or low potency agents such as nitrous oxide, the control of convulsive states and as a hypnotic [3,61]. In psychiatry it has found some use as an aid in diagnosis, and as a treatment of some disorders 1611. Thiopental sodium is administered intravenously as a 2.5% or 5%

Figure Ten.

Metabolism of thiopental in humans


H

ST-@---.
N
CH2CH3 H
0

CHCH2CH2CH3

oyJ
N H/
0

/ I \
CH3

H
I

H
I

H2CH3 CHCH2CH2CH3

syJ
N

H2CH3 CHCH2CH2COOH

sTJ
N

CH2CH3 CHCH2CH(OH)CH3

H/

H/

CH3

Pentobarbital

Thiopental Carboxylic Acid

Thiopental Alcohol

566

MICHAEL I. MCLEISH

solution, the dose for induction being 100-250mg administered over 10-20 seconds [3,61]. For longer operations it may be given as an intravenous drip, or additional injections of 50-100 mg may be given as required [3]. The physical incompatabilities of thiopental sodium which are sometimes observed [62] have been attributed to: a. acidic solutions that precipitate the free acid (thiopental) b. calcium or magnesium solutions that form insoluble carbonates c. amine salts that liberate the free base in alkaline solutions There are few absolute contraindicationsto the use of thiopental sodium, but porphyria is generally considered to be completely restrictive [61]. Extra care with both dosage and rate of administration is requited in cases of severe haemorrhage, burns dehydration, severe liver disease, status asthmaticus, severe anaemia, raised intracranial pressure, and some metabolic diseases such as thyrotoxicosis and diabetes 1611.

8.

PHARMACOKINETICS
Thiopental sodium is rapidly and efficiently absorbed following either oral or rectal administration [63,64]. However, clinically thiopental is administered as an intravenous injection whereupon it is extremely rapidly taken up by the brain. Equilibrium of brain and plasma thiopental is achieved within about one minute 165,661 and is followed by a speedy decrease in the brain concentration, approximately half the maximal concentration remaining after five minutes [65,66]. The rapid decline in brain and plasma concentration has been attributed to the redistribution of the drug to other body tissues [58, and references therein] and is responsible for its extremely short duration of action.

THIOPENTAL SODIUM

561

The plasma concentration curve for thiopental shows phases corresponding to its distribution to lean tissue, to adipose tissue and to its elimination from the body [67]. Although early studies showed the elimination half-life to be of the order of 2-5 hours [35,68,69], these studies were carried out using inadequate sampling times (less than three half-lives). Recent studies have determined the elimination half-life to be approximately 12 hours [67,70,71], although the half-life has been shown to be significantly longer in babies [72,73] and the elderly [74-761. Conversely, in patients aged between 5 months and 13 years the elimination half-life is considerably shorter than in adults [77], an observation attributed to children having a relatively higher hepatic mass [59].
To produce surgical anaesthesia, it has been seen that a plasma thiopental concentration of 39-42 pg/mL is necessary [78]. The average dose required for induction is essentially independent of age in patients between 20 and 60 years [59]. A reduction in dose may be required for patients over the age of 60; with severely deteriorated hepatic function, with moderately affected kidney function [59], or those heavily prernedicated with narcotics and other central depressants [61].

In some cases thiopental is used as a primary hypnotic and is administered as an infusion, over several days. If the plasma concentration does not exceed 15-20 p g / d the pharmacokinetics are essentially the same as those for bolus administration [54,79].

9.

ACKNOWLEDGEMENTS
The author would like to thank Abbott Australia Pty.Ltd. for providing

568

MICHAEL .. IMCLEISH

the sample of thiopental sodium (Lot No: 58355WB) used for spectral analyses. Thanks are also due to Denis Morgan and Malea Kneen for critical reading of the manuscript.

10.
1.

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THIOPENTAL SODIUM

569

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570

MICHAEL J. MCLEISH

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Raventtis J, Brit. J. Pharmacol. (1946) 1,210-214. Brodie BB, Mark LC, Papper EM, Lief PA, Bemstein E and Rovenstine EA, J. Pharmucol. Exp. Ther. (1950) 98,85-96. Dayton PG, Perel JM, Landrau MA, Brand L and Mark LC, Biochem. Pharmacol. (1967) 16,2321-2326. Sennello LT and Kohn FE, Anal. Chem. (1974) 46,752-755. Becker KE, Anesthesiology (1976) 45,656-660. Bjorkman S and Idvall J, J. Chromatogr. (1984) 307,481487.
Smith RH, MacDonald JA, Thompson DS and Flacke WE, Clin. Chem. (1977) 23,1306-1309.

Masoud AN, Scratchley GA, Stohs SJ and Wingard DW, J. Liq. Chrom. (1978) 1,607-616. Kelner M and Bailey DN, Clin. Chem. (1983) 29,1097-1100. Jung D, Mayersohn M and Perrier D, Clin. Chem. (1981) 27,113-1 15. Avram MJ and Krejcie TC, J. Chromatogr. (1986) 414,484-491. Goldstein A and Aronow L, J. Pharmacol. Exp. Ther. (1960) 128,
1-6.

45. 46. 47. 48. 49.

Raventh J, J. Pharm. Pharmacol. (1954) 6,217-235. Cochin J and Daly JW, Pharmacol. Exp. Ther. (1963) 139, J.
154-159.

Celardo A and Bonati M, J. Chromatogr. (1990) 527,220-225. Parviainen MT, Harmoinen A, Baer G and Jokela H, J. Liq. Chrom.
(1984) 7,2229-2235.

50.
51.

Premel-Cabic A, Turcant A, Cailleux A and M i P, J. Chromatogr. a n


(1983) 276,451-455.

Freeman DJ, Clin. Chem. (1981) 27,1942-1943.

THIOPENTAL SODILIM

57 1

52. 53. 54. 55. 56. 57.


58.

Christensen JH and Andreasen F, Acta Pharmucol. et Toxicol. (1979) 44,260-263. Blackman GL and Jordan GJ and P a d JD, J. Chromutogr. (1978) 145,492-495. Crankshaw DP, Edwards NE,Blackman GL, Boyd MD, Chan HNJ and Morgan DJ, Eur. J. Clin. Pharmucol. (1985) 28,543-552.

Flynn EJ and Spector S, J. Pharm. Exp. Ther. (1972) 181,547-554.


Mark LC, Clin. Pharmacol. Ther. (1963) 4,504-530. Dundee JW,International Anaesthetics Clinics (1974) 12, 121-133. Morgan DJ, In Sulphur Drugs and Related Organic Chemicals, Vol III (1989), LA Damani ed., Ellis Horwood Ltd. pp 89-120. Christensen JH, Danish Medical Bulletin, (1989) 36,2231-298. Carroll FI,Smith D, Mark LC, Brand L and Perel JM, Drug Metab. Dispos. (1977) 5,343-354. Prescription Products Guide Vol. II(1990), Australasian Pharmaceutical Publishing Co. Ltd. Melbourne, Australia. pp. 867-868,1245.

59. 60. 61.

62. 63.

In Handbook on Injectable Drugs 6th Edition, LA Trissel Ed. h e r .


SOC.Hosp. Pharm Inc., Bethesda, USA pp. 727-732.

Hogben CAM, Schanker LS, Tocco DJ and Brodie BB, J. Pharmucol. Exp. Ther. (1957) 120,450455.

64. Buchmann G, Acta Anaesthesiol. Scand. (Suppl.), (1966) 24,23-3 1.


65. 66. 67. 68. 69.
Price HL, Dundee JW and Comer EH, Anesthesiology (1957) 18, 171. Cassano GB, Ghetti B, Gliozzi E and Hansson E, Br. J. Anaesth. (1967) 39,ll-20. Morgan DJ, Blackman GL, Paull J and Wolf LI, Anesthesiology D (1981) 54,468-473. Plough IC, Waldstein SS, Bada TG and Goldbaum LR, J. Pharmacol. Exp. Ther. (1956) 116,486489.

Rahn E, Dayton PG and Frederickson EL, Br. J. Anaesth. (1969) 41,

512

MICHAEL J. MCLEISH

503-505. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79.

Burch PG and Stanski DR, Anesthesiology, (1983) 58, 146-152. Couderc E, Ferrier C, Haberer JP,Heme1 D and Duvaldestin P, Br. J. Anaesth. (1984) 56,1393-1397.
Scad. (1981) 25,174-179.

Christensen JH, Andreasen F and Jansen JA, Acta Anuesthesiol. Gaspari F, Marraro G, Penna GF, Valsecchi I and Bonati M, Eur. J. Clin. Pharmucol. (1985) 28,321-325.

Christensen JH, Andreasen F and Jansen JA, Br. J . Anaesth. (1981) 53,1189-1 195. Christensen JH, Andreasen F and Jansen JA, Anaesthesia (1982) 37,
398-404.

Jung D, Mayersohn M, Perrier D, Calkins J and Saunders R, Anesthesiology (1982) 56,263-268. Sorb0 S, Hudson RJ and Loomis JC, Anesthesiology (1984) 61, 666-670. Becker KE, Anesthesiology (1978) 49, 192-196.
67,32-41.

Crankshaw DP, Boyd MD and Bjorksten A, Anesthesiology (1987)

TICLOPIDINE HYDROCHLORIDE

Fahad J . Al-Shammary and Neelofur Abdul Aziz Mian

Clinical Laboratory Sciences Department College of Applied Medical Sciences King Saud University Riyadh, Saudi Arabia

ANALYTICAL PROFILES OF DRUG SUBSTANCES AND EXCIPIENTS - VOLUME 21

573

Copyright Q 1992 by Academic Press, Inc. All rights of reproduction reserved in any form.

514

F.J. AL-SHAMMARY AND N.A.A. MIAN

CONTENTS
1 2

Introduction Description 2.1 Nomenclature 2.1 .1 Chemical Names 2.1 .2 Generic Names 2.1.3 Properietary Names 2.2 Formulae 2.2.1 Empirical 2.2.2 Structural 2.2.3 CAS (Chemical Abstract Service Registry Nu mber) 2.3 Molecular Weight 2.4 Elemental Composition 2.5 Appearance, Colour and Odour 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 Melting Range Solubility Action Indications Partition Coefficient LD50 Compression Properties X-Ray Powder Differaction Spectral Properties 3.9.1 Ultraviolet Spectrum (UV) 3.9.2 Infrared Spectrum 3.9.3 Nuclear Magnetic Resonance Spectra 3.9.3.1 1H.NMR.Spectrum 3.9.3.2 13C .NMR .Spectrum 3.9.4 Mass Spectrum

3 Physical Properties

Synthesis

TICLOPIDINE HYDROCHLORIDE

575

Pharmacokinetics 5.1 Absorption and Distribution 5.2 Metabolism 5.3 Elimination and Excretion 5.4 Adverse Effects and Precautions uses 5.5 Methods and Analysis 6.1 Elemental Analysis 6.2 Spectrophotometric Determination 6.3 Chromatographic Methods 6.3.1 Gas Liquid Chromatography (GLC) 6.3.2 Thin Layer Chromatography (TLC) 6.3.3 High Performance Liquid Chromatography (HPLC) Acknowledgements References

576

F.J. AL-SHAMMARY AND N.A.A. MIAN

TICLOPIDINE HYDROCHLORIDE
1 INTRODUCTION

Ticlopidine ( l ) is an inhibitor of platelet action that has been used in the treatment of a variety of disease states in which platelet play a prominent role. Studies in animal and in man have demonstrated that ticlopidine is a potent inhibitor of platelet aggregation induced by adenosine diphosphate (ADP) and variably inhibits aggregation due to collagen, adrenaline (epinephrine), archidonic acid, thrombin and plate activating factor. Inhibition of platelet aggregation is both dose and time-related, with it's onsset of activity being 24 to 48 hours. It's maximal activity occuring after 3-5 days and its activity still being present 72 hours after a final dose. Ticlopidin (2) is potent and specific platelet aggregation inibitor and antithrombotic agent, exhibiting a sustained effect and wide spectrum of activity. Ticlopidine (3) is superior to aspirin and dipyridamole as anti-thrombotic agent towards different kinds of experimental thrombosis .
2

DESCRIPTION
2.1

m
2.1.1

e nclature Chemical Names

(a) (b)

(c)

5- [ (2-c h Ior op h e ny I)met hy I]-4 ,5,6,7tetra h y d ro ethieno [3,2-C] pyridine (2.4); 5- (0-chloro b e n t y I)- 4 ,5,6,7- r a h y dro t h ie no t et [3,2-C} pyridine (1, 2, 4) 5-(2-chlorobenzyl)-4,5,6,7-tetrahydrothieno [3,2-C] pyridinehydrochloride (5)
2.1.2

Generic Names

Ticlopidine hydrochloride, Ticlopidina.

TICLOPIDINE HYDROCHLORIDE

577

Anagregal, Aplaquette, Caudaline, Opteron, Panaldine, Ticlid, Ticlodix, Ticlodone, Tiklyd, Ticlosan.

2.2.1

E m D i r i c a L (4,5) (Ticlopidine) (Ticlopidine Hydrochloride)

C14H14CI N S C14HlqCI N S.HCI


2.2.2

Structural

Tic l o pidine Hydrochloride


2.2.3

(1)

CAS (Chemical AbsService Reaistrv Number1 (2, 5)

15 5 1 4 2 85 -3) [ 5 3 8 8 5 - 3 5 - 1]
2.3

(Ticlopidine) (Ticlopidine Hydrochloride)

Molecular Weiaht (Ticlopidine) (4) (Ticlopidine Hydrochloride) (5) Elemental Composition C

263.78 300.2 2.4

Ticlopidine:

CI S

63.75%

13.44% 12.1 5 %

5.35%

5.31 %

578

F.J.AL-SHAMMARY AND N.A.A. MIAN

Ticlopidine Hydrochloride: C 56.01 % CI 23.65% S 10.68%


2.5

H N

5.03% 14.006%

&lmneClrJ!3c.oo.~

Ticlopidine hydrochloride is a white, odorless crystalline powder.


3

PHYSICAL PROPERTIES
3.1

Meltina Range (4)


A c t i o n (2)

MP = 18900 for Ticlopidine Hydrochloride

3.2

Potent and specific platelet aggregation inhibitor and antithrombotic agent, exhibiting a sustained effect and wide spectrum of activity.

3.3

Jndicatlons (2)

Prevention and correction of platelet disorders in surgical patients undergoing extracorporal circulation and in long term hemodialysis patients.
3.4

p a r t i w n Coefficient The PKa of Ticlopidine is 7.64 (6)

..

..

3.5

LR50 ( 4 )
55 mg/kg/24 hrs (IV in mice). >300 mg/kg/24 hrs (orally in mice).

3.6

S o l u u

..

Almost soluble in water, soluble in 95% alcohol also soluble in methanol, chloroform and insoluble in ether. Comparative evaluations of aqueous film coated tablet formulations by high humidity aging was studied by Chowhan, Z. T; et a1.(7) Dissolution rate studies of 3 film coated formulations of ticlopidine. HCI compared by storage under

TICLOPIDINE HYDROCHLORIDE

579

95% relative humidity at 23 and 37% showed that tablets coated with a formulation containing Eudragit E 30D dissolved more slowly before storage and the dissolution became very slow after storage. Tablets coated with 10% hydroxypropyl Methyl cellulose or Ethyl. Cellulose colloidal dispersion also dissolved slower after storage except that the dissolution rate of tablets coaled with 10% hydroxy propyle-Me-Cellulose increased af'ter 12-15 week storage at 25%. In general the decrease in dissolution rate is related to the nature of the film coating temp. of storage, amount of moisture gain, and tablet core formulation. Thus to maintain good dissolution throughout the shelf life of film coated tablets they should be protected from high relative humidity.
3.7

Comp ression Proberties

Z.T. Chowhan and Y.P. Chow (8) studied the role of the granulation moisture content on compression properties of granules made with selected binders. The results suggested that at lower pressures, higher moisture containing granules were slightly more compressible than lower moisturecontaining granules. However at higher pressures, the reverse was true because of the water lubrication effect. At lower moisture levels, the crushing strength of the tablets was dependent on the binder, at higher moisture levels, binder differences became less significant.

3.8

X-rav

Powder Differaction

The X-ray differaction pattern of ticlopidine hydrochloride was determined using Philips full automated x-ray differPW 1730/10 action spectrogoniometer equipped with generator. Radiation was provided by a copper target (Cu annode 2000W, y = 1.5480 Ao). High intensity x-ray tube operated at 40 Kv and 35 Mv was used. The monochromator was a curved single crystal one (Pw 1752/00). Divergence slit and the receiving slit were 0 and 0.10 respectively. The scanning speed of the goniometer (Pw 1050/81) used was 0.02-2 0 per second. The instrument is combined with Philips PM 8210 printing recorder with both analogue recorder and digital printer. The goniorneter was aligned using silicon sample before use. The x-ray pattern of Ticlopidine hydrochloride is presented in Fig,(l). The values of scattering angle 2 8 interplanner distance dAO and relative intensity 1/10 are shown in the table
(1).

z e - VALUE)

Fig. ( 1 1 X-Ray powder Diffraction of Ticlopidine Hydrochloride

TICLOPIDINE HYDROCHLORIDE

58 I

Table (1):
20

Characteristic Lines of the X-ray Powder Diffraction of Ticlopidine Hydrochloride.


dA
I/lo%

20 36.859 37.725 38.192 39.461 40.261 40.710 42.240 42.960 43.496 44.423 44.824 46.72 48.418 48.719 50.099 50.288 52.095 52.61 53.453 53.773 55.35 56.273 56.99 58.879 61.613 64.749 70.693 77.876 78.818 83.013

dA

I/lo%

9.236 11.817 12.622 14.422 16.233 16.466 17.658 18.825 19.009 19.847 20.073 22.578 23.043 23.876 24.929 25.673 26.425 27.128 27.91 28.566 29.213 29.751 30.037 30.800 31.75 32.974 33.981 34.416 25.578 26.010 36.436

9.5746 7.4889 7.0127 6.1416 5.4602 5.3834 5.0227 4.7139 4.6686 4.4734 4.4234 3.938 3.8596 3.7268 3.5717 3.4698 3.3728 3.287 3.1966 3.1247 3.057 3.0028 2.975 2.9029 2.8183 2.7164 2.6381 2.6058 2.5233 2.494 2.4658

7.193 10.973 0.057 100 9.836 5.386 11.408 19.003 14.319 12.345 4.884 10.806 14.084 25.46 12.947 24.422 16.627 8.999 3.029 20.809 3.345 2.709 3.412 3.479 9.769 3.512 9.501 2.408 5.319 2.843 4.583

2.4385 2.3845 2.3564 2.2835 2.2400 2.2163 2.1395 2.1053 2.0805 2.0393 2.022 1.9442 1.8799 1.8690 1.8207 1.8143 1.7556 1.7396 1.7141 1.7047 1.6598 1.6347 1.6159 1.5685 1.5052 1.4397 1.3325 1.2266 1.2143 1.1 633

2.843 4.884 2.877 3.278 3.445 2.375 2.107 2.944 2.475 3.479 2.107 3.21 1 3.613 3.41 2 3.178 3.579 1.706 2.643 2.810 1.806 1.873 2.576 2.074 1.304 11.572 1.706 1.438 1.271 1.237 1.572

2 0 = scatterina anale. - -

I/lo%

dA = intemlanner distance. relative intensity based on highest as 100.

582

F.J.AL-SHAMMARY AND N.A.A. MIAN

3.9 3.9.1 Ultraviolet Spectrum

tuvl

The UV spectrum (9) of ticlopidine hydrochloride in H 2 0 (7 mg %) was scanned from 200 to 400 nm (Fig. 2) using LKB 4054 UV/Vis spectrophotometer. Ticlopidine hydrochloride exhibited the following UV data (Table 2).
Table
(2)
n.m max

UV Data of Ticlopidine

Absorbance

Molar Absorptivity cm-1 gm mol/L


9121.79 394.5 60.04

(1

A'

21 4
268

2.127
0.092
0.014

303.8 13.14 2

295

3.9.2

Infrared

Seect r u q

The 1R spectrum (9) of Ticlopidine hydrochloride as KBr disc was recorded on a Perkin Elmer 1210 Infrared Spectrometer. Fig. (3) shows the infrared spectrum of Ticlopidine hydrochloride. The structural assignments of Ticlopidine hydrochloride have been correlated with the following frequencies (Table 3).
3.9.3
Nuclear Maanetic Resonnance SDectra 3.9.3.1

PMR Spectrum

The PMR spectra (9) of Ticlopidine HCI in D M s 0 - d ~ (Fig. 4-6) was recorded on a varian XL 200 MHZ NMR spectrometer using TMS as an internal reference. The following structural assignments have been made (Table 4).

0
#

0 OD

E
-c
0

aJ

u 0 )

-s
m
0
(Y

r I a J
C .2 n
0 .A

0
0

I m
@
N
0

rc

E
0

lo
N
0 Y

a J c1 v,
7
N
c

l5

0
(Y (Y

c \

0 0 cy

.-

0 LL

jb
I

4 000

Fig. ( 3 1

2000 1500 1 boo WAVEN UM6 ER S Infra Red spectrum of Ticlopidine Hydrochloride
3000

6 0 r

V
I

LD

0
r/)

0
.C

U .L
d

aJ

U L 0

6.908
7

I -

.U .a .c
0
U

11 a J

%-

5
U

a ,

P
v)

a z z
7

I
CI

4
--c

m .u ,

Fig.

( 5

1 H-NMR Spectrum of Ticlopidine Hydrochloride

(Expansion of peak

m sw

4.J I

12
1

10

I I

0 PPM

Fig. ( 6 1 H-NMR spectrum of Ticlopidine Hydrochloride in DMSO- d 6 ( DzO Exchange )

588

F.J. AL-SHAMMARY AND N.A.A. MIAN

Table

(3)

IR Characteristics of Ticlopidine Hydrochloride


Assignment

Frequency cm-1
3400

NH stretch and plannar bend


Chlorophenyl CH Stretch C-S-C stretch Chlorophenyl ring stretch Pyridine methylene wag Methylene twist Chlorophenyl C-CI stretch and bends Pyridine ring stretch

3020, 3040 2260


1590, 1560
1430, 1425 1280

1220, 1200

1160

1080, 1060, 1020, 1000 Pyridine-methylene rock


750, 560, 720

Chlorophenyl spatial bend

3.9.3.2

l3C-NMR spectrum (9) of ticlopidine in DMSO-de (Fig. 7-9) was recorded on varian XL-200 NMR-spectrometer. The multiplicity of the resonances was obtained from APT (Attached Proton Test) and DEPT (Distortionless Enchancement by Polarization Transfer) programs. The 13CNMR spectrum displayed all the fourteen carbon resonances. The narrow resonance range of some of the carbons makes the spectrum rather complex. The carbon chemical shifts assignments are presented in table (5).

L ' J

I 180

160

140
(

120

100

80

60

40

20

P PM

Fig.

13

C- NMR spectrum of Ticlopidine Hydrochloride

065

Fig. ( 8 1

1 3

C - NMR Spectrum of Titlopidine Hydrochloride in D M SO -d 6 (APT)

CH 3

c MX

Fig, ( 9 ) C-NMR SPECTRUM OF TICLOPIOINE H d IN DMSO-d 6 (DEPTI

13

592

F.J. AL-SHAMMARY AND N.A.A. MIAN

TABLE (4 ): Structure

PMR Characteristics of Ticlopidine HCI

____------__________--__----__----____ Protons I (PPm) I Multiplicity


a,b g.h,i,j f d
C

8.086 - 8.131 7.457

m
m
d
S

7.577

6.908 - 6.934 4.609 4.277


3.424

S
S

The mass spectrum (9) of Ticlopidine HCI obtained by electron impact ionization (Fig. 10) was recorded on a Finnigen MAT 90 spectrometer. The spectrum was scanned from 50 to 500 a.rn.a. Electron energy was 70 ev. Emission current 1 mA and ion source pressure 10-6 torr. The most prominent fragments and their relative intensities are presented in Table (6 ).
4

SYNTHESIS
4.1

Scheme I

Ticlopidine Hydrochloride is prepared (10) by the treatment of 4, 5, 6,7-tetrahydrothieno [3,2-C] pyridine with

0 0 0
0 0

Fig. (10 1 Mass spectrum o f Ticlopidine Hydrochloride

594

F J AL-SHAMMARY AND N.A.A. MlAN ..

Table (5) Carbon-13 Chamical Shifts of TiclqpLQUle Chemical Shift

..

21.529 48.91 1 49.698 54.336 134.61 9 131.401 127.726 127.763 127.581 124.898 133.863 129.793 131.333 125.257

2-chlorobenzyl chloride in the presence of Pot. Flouride. The reaction mixture is stirred at 50% for three hours in THF. (Scheme I).
4.2

Scheme II

Yamanochi et al (11) developed a method for the preparation of 4, 5, 6, 74etrahydrothieno [3,2,-C] pyridine by treating 2-(24hienyl)ethylarnine and HCHO at 9OOC for three hours. The reaction mixture was extracted with CgHg which was recrystallised with CgHe/hexane mixture to give 1, 3, 5-

TICLOPIDINE HYDROCHLORIDE

595

tris(thieny1 ethyl) triazine. A solution of 1, 3, 5tris(thieny1 ethy1)triazine in (CH3)zCHOH was added dropwise to (CH3)zCHOH containing HCI at 50oC and reaction mixture was stirred at 50oC for 5 hours to give 81% of 1,3,5-tris(thienyI ethy1)triazine. (Scheme 11).
4.3

Scheme Ill

Ticlopidine HCI has also been synthesized (12) by the reaction of 4, 5, 6, 7-tetrahydrotheno[3,2-C] pyridine with O-CIC6H6COCI in CHCl3 - aq. NaOH at room temperature for overnight. Which was further treated with AIH(CH&HMe2)2 in toluene at 90-95OC for 2 hours. Ticlopidine has also been synthesised by other methods (1316). TABLE (6): The Mass fragments of Ticlopidine HCI
m/z
Relative Intensity Ions

110.4

100

125.2

25%

596

F.J. AL-SHAMMARY AND N.A.A. MIAN

Scheme I

OS) +

C I
&cH2c'

Scheme I1

Ticlopidine

r;Sf'ziW
for 90C for 3 h . , ~

4 , 5, 6, 7, tetrahydrothjeno [3,2-C] pyridine

TICLOPIDINE HYDROCHLORIDE

591

Scheme 111

0 5- (O-~hlorobenzoy1)-4,5,6,7, tetrahydrothieno [ 3,2-C] pyridine

in toluene

/ CH3

A1..H(CH2CH

'

CH3

12

Ticlopidine

598

F.J. AL-SHAMMARY A N D N.A.A. MIAN

5.1

Absorption and Distribution

About 80-90% of an oral dose of the drug absorbed after oral administration in rat or man (17, 18). After a single dose in rats or man, peak plasma concentrations occured at 1-3 hours (17, 18, 19, 20). In human volunteers and patients given single doses of 500 mg, peak plasma ticlopidine concentrations were 0.61 and 0.82 mg/L respectively. Accumulation was not noted in multiple-dose studies (18). In volunteers given a single oral dose of 1000mg, peak plasma concentrations were 2.13 mg/L those given repeated doses of 250 mg twice daily for 21 days. had peak concentrations of 0.90 mg/L (18). In rats given single or repeated doses, highest ticlopidine concentrations were measured in the liver, kidneys, duodenum and fat tissues. In pregnant rats, conc. in fetal blood were 4090% of those in maternal blood, and fetal, placental and amoniotic conc. were appreciable (20). Plasma protein binding has not been studied in vivo, but in rats 60% of circulating radiolabelled ticlopidine was distributed to plasma and 40% blood cells (1). Ticlopidine HCI (21) is readily absorbed from the gastrointestinal tract after oral dosing. The oral bioavailability of ticlopidine was increased by 20% when taken after a meal. In contrast, absorption of ticlopidine administered after antacid treatment was approximately 20% lower than under fasting conditons. Administration of drug with food is recommended to maximize gastrointestinal tolerance (22).
5.2

Metabolism

The metabolic disposition of ticlopidine is complex with at least four metabolites isolated in man and thirteen in rats (23). The rate of metabolism in man is rapid as even shortly after dosing, when ticlopidine concentrations are at their peak, only 22% of the total radioactivity in plasma represents unchanged ticlopidine (23), and by 15 hours past dose, unchanged ticlopidine represents 6% or less of the total dose in man (17).

TICLOPIDINE HYDROCHLORIDE

599

The main quantitative metabolic route in man is N.dealkylation, followed by oxidation with opening of the thiophene ring (17) but another metabolic pathway is responsible for the 2-keto derivative of ticlopidine called PCR-3787. This metabolite, which has been found in small concentrations in rat bile, has been found to be 5 to 10 times more potent than ticlopidine itself as an antiplatelet agent, although its potential contribution to ticlopidine's effect is as yet uncertain (24). Anne Tuang et al (21) has been studied the metabolism of Ticlopidine on rats, the compound was quickly absorbed as evaluated by the time of the peak plasma concentration. The metabolism of drug involved N-oxidation, cleavage of the N-C bond oxidation of aliphatic carbon followed by glycine conjugation. Urinary excretion took place essentially in the first 24 hours and biliary excretion was most pronounced in the hour following dosing. Small amounts of drug were excreted unchanged. The major urinary metabolites were 2chlorohippuric acid (16% of the dose) and tetrahydrothienopyridine (8%) while Tic1opidine.M predominated in the bile (2% in 0-5 h). The peak plasma concentration of Ticlopidine occured at 0-5 hour. The plasma concentrationAime curve displayed a biphasive profile and the terminal half life of Ticlopidine was tentatively estimated to be 6-10 h in the rat.
Metabolic Path of Ticlopidine

Unchanged Ticlopidine (25) and three metabolites Ticlopidine N-oxide, (T-NO), Tetrahydrothieno pyridine (THTP), and 2,chloro-hyppuric acid (CI-HPA) were isolated from rat urine by differencial solvent extraction and characterized by their behaviour on TLC and GLC. Their identities were confirmed by comparison with authentic standards. A fourth metabolite (T-M) gave rise upon acid hydrolysis to a compound, which co-chromatographed with authentic (T), both on TLC and GLC. The original structure of this metabolites is not yet elucidated. (T) and (T-M) were also found in bile wxtracts, whereas (T-NO), (THTP), and (CLHPA) were not detected in the bile, under the conditions used. Urine and bile samples were assayed for the supposed intermediates of (CI-HPA), i.e. 2-chlorobenzyl alcohol (CIBzOH), 2-chloro-benzaldehyde (CI-BzAld), and 2chlorobenzoic acid (CI-BzA), however, under the conditions used, only trace amounts of (CI-BzA) were detected by GLC.

F.J. AL-SHAMMARY AND N.A.A. MIAN

Glucuronides or sulphate conjugated metabolites may account for only insignificant amounts, as the TLC patterns of enzyme hydrolyzed and untreated samples were quite similar. Following acid hydrolysis, however, the TLC patterns showed the presence of several though minor compounds. Attempts to characterize these (except T-M) or to make derivatives suitable for GLC have so for failed. The possibility of hydroxylated metabolites was investigated using various TLC spray-techniques and spot-test reactions upon silicagel eluted materials, but no net reactions resulted. A scheme for the known metabolic pathway of Ticlopidine is shown in Fig. (11).
5.3

Elimination and Excretion

In man, approximately 60% a radiolabelled dose is recoverable in urine, and 25% infaeces following oral administration (17). Ticlopidine concentrations measured as detectable nitrogen by gas chromatography (thus, probably not specific for the parent compound) dropped rapidly from 0.70 mg/L at 2 hours post-dose to 0.15 mg/L at 6 hours post-dose following a single 500 mg oral dose (18). Plasma concentration of unchanged ticlopidine fell rapidly after oral administration of a single 750 mg dose in volunteers (18). After repeated doses of 250 mg twice daily for 21 days, peak concentrations of 0.90 L 0.18 mg/L fell to trough concentrations of 0.20 L 0.07 mg/L. Elimination half-lives of 24 2 7.5 hours and 33.2 f 3.8 hours have been reported (18). Ticlopidine plasma or blood concentrations do not correlate with ex vivo activity as an antiaggregant of platelets (17, 18). Anne Tuong et al (21) have studied that also high concentrations of unchanged ticlopidine were found in various organs (liver, kidney, and adipose tissues mainly) although only minute amounts of drug were excreted in urine (0.1% of the dose) and in bile (0.02% of the dose).
5.4

Adverse Effects and Precautions

Approximately 10-15% of patients receiving ticlopidine have experienced side effects, the most common of which have been

TICLOPIDINE HYDROCHLORIDE

60 I

Ticlopidine (T)

(T-NO)

-----, (THTP)

(C1 - B ZOH)

I
C1 -BzAld.

+ acid

Cl

C1-BzA

1
H O O C H2C H N O C
ti

+ Glycine

C1-HPA Fig. (11) Metabolic pathway of Ticlopidine.

602

F.J. AL-SHAMMARY AND N.A.A. MlAN

gastrointestinal complains and skin rash. About 10% experience gastrointestinal discomfort, dyspepsia, abdominal pain, nausea and diarrhoea (1). Gastro-intestinal disturbances and skin rashes are the most commonly reported side-effects associated with Ticlopidine therapy. Blood dsycrasias, particularly serious in elderly patients have been reports of vertigo and occasional reports of cholestatic jaundice (5)- Gastrointestinal distress may necessitate discontinuation of the medication but may be markedly reduced if the drug is given after meals (26). Bleeding during ticlopidine therapy is an unusual side effect, but is dangerous in patients who must undergo surgery or another invasive procedure (1). In patients undergoing AV access insertion, there has been no increase in bleeding (27). But in patients undergoing open heart surgery the risk of bleeding may be increased with ticlopidine (28). Agranulocytosis. neutropenia, thrombocytopenia, and erythroleu-kaemia have been reported during therapy with ticlopidine. Elevation of liver function tests are unusual with ticlopidine therapy, but occasionally cholestatic jaundice or hepatitis have been reported. Drug may increase total serum cholesterol, as well as LDL- and VLDL-cholesterol and other lipoproteins, without effecting HDL-cholesterol (1). Ticlopidine should not be administered to patients with haemorrhagic diathesis, gastrointestinal ulcers or severe liver dystfunction. It should not be given to patients receiving aspirin, anticoagulants or corticosteroids (5).
5.5

Uses

Ticlopidine is an inhibitor of platelet aggregation. It has been given in the treatment of atherosclerotic disease and intermittent claudication in doses of 250 mg once of twice daily by mouth, with meals. Regular haernotological monitoring has been recommended (5). Ticlopidine (29) is equally effective in both men and woman and also improves symptoms of claudication in patients with peripheral arterial disease and appears to reduce anginal pain. Patients with subarchnoid haemorrhage and sickle cell disease have shown some improvement with ticlopidine ad minist ration.

TICLOPIDINE HYDROCHLORIDE

603

Giuffetti, G; et al (30) studied the treatment with ticlopidine improved the neurologic outcome and the hemorheologic pattern in the postacute phase of ischemic stroke. The drug (31) is to be effective in influencing the rheological measures of red cell filteribility and membrane microviscosity filteribility was increased and microviscosity was decreased. Davi G., et al (32) concluded that in schemic hear disease patients the association of ticlopidine and low dose aspirin seems superior toe each drug alone in inhibiting platelet activity and according to Uchiyama S ; et al (33) combination of aspirin plus ticlopidine is a potent antiplatelet strategy, in ischemic attach or cerebral infarction. Balsano F; et al (34) concluded that long term treatment with ticlopidine improves walking ability and ankle systolic blood pressure in patients with claudication. 6 METHO DS OF ANA LYSlS
6.1

Elemental Analvsis

The elemental analysis of ticlopidine is as reported (4). Element C CI N S Composition 63.75% 5.35% 13.44% 5.31% 12.15%

For Ticlopidine Hydrochloride:


Element C H CI N Composition 56.01 % 5.03% 23.65% 4.01 y o 10.68%

6.2

SDectroDhotornetric

Determination

A spectrophotometric study of ticlopidine was carried out by Sanchez Perez (35). Ticlopidine reacts slowly with iodine in CHC13 forming a mol. complex with two change transfer bands

604

F.J. AL-SHAMMARY AND N.A.A. MIAN

(hmax = 295 nm and Amax = 360 nm) which was also observed after the extraction, into CHC13 of the ticlopidineiodine complex formed in aqueous solution. Two spectrophotometric methods for the determination of drug based on the formation of the ticlopidine-iodine complex were studied. The first involves the extraction of ticlopidine base from aqueous samples into CHC13 and addition of a solution of ticlopidine. The second involves the formation of the mol. complex in aqueous solution (pH = 7.0) over 90 minutes and its later extraction into CHC13. In both procedures Beer's Law was followed with the ticlopidine concentration rage of 1.6 x l o m 6 - 1.6 x 10-5 M for the two max.
6.3 6.3.1 Gas Liauid Chromatoaraphv (GLCL

1. Ticlopidine and its metabolites in biological fluids are being analysed by GLC. Ticlopidine (T), and Ticlopidine N-oxyde (T-NO), were simultaneously solvent extracted and separated by column chromatography (T-NO) was converted to (T) by reduction with SO2 (36) before analysis due to degradation of (T-NO) within the injection port of gaschromatograph. Ticlopidine-M (T-M) was processed as (T) after acid hydrolysis of the aqueous phase. GLC analysis was performed on Hewlett Packard model 5710 gaschromatograph equipped with a nitrogen-phosphorous detector and a HP 3352 data system using a 6 ft x 2 mm ID glass column, packed with 3% OV 17 on Chromosorb WHP 100/120. Injection port temp. 2500, detector temp. 3 0 0 0 , oven temp. 2000. He flow 25 ml/min. The retention times for Ticlopidine was 6.1 min and for internal standard was 3.2 min (25). 2. Another method used to analyse the drug and its metabolites ie. 2-chloro-hippuric acid (CI-HPA) after extraction and methylation of the dry residue with diazomethane, GLC analysis was performed on a Hewlett-Packard model 5830 gaschromatograph equipped with a flame-ionisation detector and an automatic sampler, using a 4 ft. x 2 mrn ID glass column, packed with 1% OV 25 on Chromosorb WHP 100/120, injection port temp. 2400, detector 2500, oven temp. 210,for 5 minutes then raised to 230 at 10/rnin.

TICLOPIDINE HYDROCHLORIDE

605

The methylated derivatives of CI-HPA and the internal standard had the reaction times of 4.0 min. and 8.4 min., respectively (25).
6.3.2 Jhin Laver Chromatoaraphv fTLC)

Folowing extraction and coupling in aqueous medium with sodium naphtoquinone-sulfonate (37) the tetrahydrothienopyridine (THTP) derivative extracted into methylene chloride. The residue after solvent evaporation was quantitatively applied on a silica gel plate (Merck 60F 254, 0.25). and the plate was developed in chloroformmethanol (9O:lO v/v). The orange coloured derivative migrated as a single spot (Rf 0.69) well resolved from coextracted, absorption measured at 480 nm (25). 2. Giuseppe Musumarra et al (38) analysed Ticlopidine HCI by T.L.C. The drug dissolved in methanol (5 ml) or extracted from an alkaline aqueous solution with ethyl acetate and prepared as a solution containing about 2 rng/ml of drug. The freshly made drug solution were applied approximately 1 cm apart to 20 x 10 cm silica gel 60 F254 HPTLC plates (Merck).
6.3.3 ah Performance L i a U ChrornatoaraDhvl [HPLC)

. .

An HPLC (39) method was developed for determination of the drug and its metabolites in human and rat bile. A stainlesssteel column (15 cm x 4.6 mrn I.D.) packed with LiChrosorb RP-8 (Pore size 5pm) or Nucleasil C i 8 (pore size 5pm) was used. The columns were packed by means of a balanced density slurry method specially developed for the ammonia elution system. Gradient elution was performed with water (0.005 M ammonia) to which methanol was added, according to the desired programme. The final elution was usually effected with 100% methanol. Flow rate was lml/min. A wavelength of 235 nm was found suitable for the detection of drug and its metabolites.
7

ACKNOWLEDGEMENTS

The authors are highly thankful to Liberty S. Matibag and Mr. Babikir Awad Mustafa, College of Applied Medical Sciences, King Saud University for their Secretarial and technical assistance respectively in preparing the manuscript.

606

F.J. AL-SHAMMARY AND N.A.A. MIAN

8
1

REFERENCES Emmanuel Saltiel and Alan Ward. Ticlopidine. A Review of it's Phamacodynamic and pharmacokinetic properties, and therapeutic efficacy platelet. Dependent disease states." Drugs 34 p. 222-262 (1987). "Annual Drug Data Report" J.R. Prous. Vol. Ill p. 249 (1981). Tomikawa M., Ashida S.1; Kakihata K., and Abiko Y; Ticlopidine. An Antiplatelet drug; Effects in human volunteers Thromb Res. 13 p. 245-254 (1978) "The Merck Index"
11th Ed. p. 1485. (1989).

2 3

5
6

"Marti ndale" The Extra Pharmacopeia" 29th Ed., p. 1623. The Pharmaceutical Press, London (1989).
338

Giovanni D.i. Minno et al. J. Clin. Invest. 75 p. 328(1981). Pharm. 8(5) p 713-37
1982.

7 Chowhan, Z.T.; Arnaro, A.A.; Chi, Li Hua. Drug Dev. Ind.


8 9 10

Z.T. Chowhan and Y.P. Chow. p. 1134-39 (1981).

J. Pharrna. Sci. 70(10)

Fahad J. Al-Shammary and Neelofur Abdul Aziz Mian Unpublished data (1992). hove, Kunimi; Yamad, Yoshiyuki; Tomioks, Shinji; Tarnsoki, Kentaro (Kyowa Hakko Kogyo Co., Ltd.) Jpn. Kokai Tokkyo JP 63,188,682[88,188,682]. 4 Aug. 1988 (CA 110: 173209t, (1989). Yarnanochi, Takenaga; Yarnane, Hiroyuki (Asahi Chemical Industry Co. Ltd.). Jpn. Kokai Kokkyo Koho JP 63,101,385 [88,101,385] 06 May 1988. (CA 109:92980 q) (1980). Yodhimots, Yoshifumi (Sanyo Kagaku Kenkyusho Co. Ltd.) Jpn, Kokai Tokkyo Koho JP 6388,186 [88 88, 1861 19 Apr. 1988 (CA 109: 149507~)(1988).

11

12

TICLOPIDINE HYDROCHLORIDE

607

13 14 15

Maffrand, J.P. and Eloy, F. Eur. J. Med. Chem. ChirnTher. 9( 5), 483-486 (1 974). Eloy, F.; Deryckere, A.; Maffrand, J.P. Eur. J. Med. Chern.-Chirn. Ther. 9(6), 602-6 (1974). Okada, Tsugio; Kawasaki, Hiroshi; Kikuchi, Toshio; Aoki Takao; Watanabe, Masahiro Jpn. Kokai Tokyyo Koho JP 62,164,683 [87,164,683] 21, Jul. 1987 (CA 110: 754771) (1 989). Yamanochi, Tekenaga Jpn. Kokai Tokyyo Koho JP 62,205,87 (7,205,0871Sep. 9, 1987 (CA 109:211033 Y) (1988). Bruno JJ, Molony BA. Ticlopidine, In Scriabine (Ed) New drugs anual, cardiovascular drugs p. 295-316. Raven Press New York (1983). Panak E. Maffrand JP, Picard-Fraire C, Vallee E, Blanchard J et. al Haernostasis 13 (Suppl. 1): 1-54, (1983). Knudsen JB, Gormsen J. The effect of ticlopidine on platelet function in normal volunteers and in patients with platelet hyperaggregability in virto. Thrombosis Research 16:

16 17

18 19

663-671, (1979). 20

349-361, (1980). 21

Takegoshi T, Ono K, Mutsubayashi K, Hasirnoto F, Sano M, Metabolic disposition of ticlopidine hydrochloride, a new anti-thrombotic agent, in rats, Pharrnacornetrics 19:

Anne Tuong; Anne Bouyssou; Josiane Paret; and Tuon Ghi Cuong. European Journal of drug metabolism and pharrnacokinetics 6(2) p. 91-98 (1981). Sha J; Fratis A; Ellis D; Murakami S; Teitelbaum P; J . Clin. Pharrnacol. 30(8), p 733-6 (1990). Picard-Fraire characteristics properties on supplements. C. Pharrnakokinetics and metabolic of ticlopidine in relation to its inhibitory platelet function. Agents and Actions Ticlopidine: Quo Vadis, 15(Suppl): 68-

22 23

75, (1984).

24

Aubert D, Bernat A, Ferrand JC, Maffrand JP, Szygenda E. et al. Pharmacological profile PCR 3787; a metabolite of

608

F.J. AL-SHAMMARY AND N.A.A. MIAN

ticlopidine. From the Seventh International Congress of Thrombosis, October, Valencia, Spain (1982).
I

25

Anne Tong, Anne Bouyssou, Josiane Paret and Tuaong Ghi Cuong, Eurp. J. of Drug Metab. and Pharmakokinetics, 6 (20) p 91-98 (1981). Goyan JE. Adverse reactions in man, Agents and Actions supplements, Ticlopidine; Quo vadis? 15. 116-1 25,
(1989).

26

27

Ell S, Mihindukulasuriya JCL, OBrien JR, Polak A, Vernham G, Ticlopidine in the prevention of blockage of fistuale and shunts. Abstract 332 from the 7th international congress on Thromasis, p. 180, Valencia, Spain, October 13-1 6,
(1982).

28

lnstalle E, Gonzalez M, Schoevaerdts JC, Tremouroux J. J. Cardiovascular Pharmacology 3: 1174-1 183


(1 9 8 1 ) .

29

40(2) p

McTavish D; Faulds D; Goa KL, "Drugs" United States


238-59 (1990).

30

Giuffetti, G; Aisa G; Meercuri M; Lombardini R; Paltriccia R; Neri C; Senin U; Angiology 41(7) p. 505-11 (1990). Caimi G; Lo Presti R; Serra A; Francavilla G; Catania A; Sarno A; J. Int. Med. Res. 18(2) p.161-3 (1990). Davi G; Catalano I; Spatola A; Alaimo P; Notarbartolo A; Cerbone AM; Strano A. Cardiologia. 344 p. 69-71
(1 9 8 9 ) .

31
32

33

(1989). 34

Uchiyama S; Sone R, Nagayama T; Shibagaki; Kobayashi I; Maruyama S; Kusakabe K: Stroke 20(12) p 1643-7

Balsano F; Coccheri S; Libretti A; Nanci GG; Catalano M, Fortunato G; Grasseli S; Violi F; Helemans H; Vanhove P. J. Lab. Clin. Med. 114(1), p. 84-91 (1989). Sanchez Perez A; Montero Garcia, J. Quim, Anal. (Barcelona) 6(2) 204-14 (1987). Breyer U. Urinary metabolites of 10-[3'-(4"-methylpiperaziny1)-propyll-pheno thiazine (perazine) in

35
36

TICLOPIDINE HYDROCHLORIDE

609

psychiatric patients. p. 777-788 (1969). 37

1.

Biochem.

Pharmacol., 18,

Pesez M and Bartos J Colorimetric and fluorimetric Analysis of Organic Compounds and Drugs. Chapter 4, Aliphatic Amines, p. 132, Marcel Dekker Inc. New York (1 974). Guiseppe Mausumarra; Giuseppe Scarlata and Gurseppe Cirma. J. of Chromtgr. 350 p. 151-168 (1985).

38 39

F. Overzet, A. Rurak, H. Vander Voet, B.F.H. Drenth, R.T. Ghijsen and R.A. De Zeeuw. J. of Chrom. 267 329-345 (1 9 8 3 ) .

VINBLASTINE SULFATE

(SUPPLEMENT)

Farid J . Muhtadi and Abdul Fattah A. A . Afify

Department of Pharmacognosy

College of Pharmacy

King Saud University


Riyadh, Saudi Arabia

ANALYTICAL PROFILES OF DRUG SUBSTANCES AND EXClPlENTS -VOLUME 21

611

Copyright 0 1992 by Academic Press. Inc. All rights of reproduction reserved in any form.

612

FARID J. MUHTADI AND ABDUL FATTAH A. A. AFIFY

V INB LASTINE SU LFAT E

Contents Foreword

1. Description
1.1 1.2 1.3 1.4 1.5 1.6 1.7
Nomenclature Empirical Formulae Molecular Weight Structure Elemental Composition CAS Registry Number Appearance, Color and Odor

2 Physical Properties
2.1 2.2 2.3 2.4 2.5 2.6 2.7
Melting Range Solubility Specific Optical Rotation pH Range Loss on Drying Dissociation Constant Spectral Properties

2.7.1 2.7.2 2.7.3 2.7.4 2.7.5

Ultraviolet Spectrum Infrared Spectrum 'H-NMR Spectrum Carbon-I3 Spectrum Mass Spectrum

3. Isolation of Vinblastine
4. Total Synthesis of Vinblastine 4.1 Total Synthesis of ( 2 ) - Vindoline 4.2 Total Synthesis of ( 2 ) - Catharanthine 4.3 Total Synthesis of Vinblastine 5. Biosynthesis of Vinblastine
6. Pharmacokinetics 6.1 Drug Absorption 6.2 Drug Distribution 6.3 Metabolism

VINBLASTINE SULFATE

613

6.4 Drug Excretion 6.5 Half-Life


7. Preparation and Preservation 8. Uses of Vinblastine Sulfate

8.1 Precautions 8.2 Contra-indications 9. Methods of Analysis 9.1 9.2 9.3 9.4 Identification Tests Titrimetric Determinations Voltametric Determination Spectrophotometric Determinations 9.4.1 UV Spectrophotometry 9.4.2 Colorimetric Determinations 9.5 Chromatographic Methods 9.5.1 9.5.2 9.5.3 9.5.4 Paper Chromatography Thin Layer Chromatography Gas Liquid Chromatography High Performance Liquid Chromatography

9.6 Radioimmunoassay Methods Acknowledgement References

614

FARID I. MUHTADI AND ABDUL FATTAH A. A. AFlFY

Foreword Vinblastine or vincaleukoblastine is an indole alkaloid obtained from Madagascan periwinkle, Catharanthus roseus G. Don., (FamiZy Apocynaceae) which has been formerly designated Vinca rosea L . Vinblastine is one of the antineoplastic agents and is mainly used for the treatment of Hodgkin's disease and other lymphomas as well as choriocarcinoma (1). It is used as vinblastine sulfate which is formulated as IV injections

1. Description
1.1 Nomenclature

Vinblastine; vincaleukoblastine; VBL; 29060 - LE. (The Base). Vinblastine sulfate; vincaleukoblastine sulfate; vincaleukoblastine sulfate ( 1 : 1) (salt) ; Exal; Velban; Velbe (The Salt).
1 . 2 Empirical Formulae

(Vinblastine) . C46H58N409 C46H58N409 .H2S04 (Vinblastine sulfate) .


1 . 3 Molecular Weight

810.98

909.06

(Vinblastine) (Vinhlastine sulfate).

1 . 4 Structure

The following is the absolute configuration of vinblastine ( 2 ) . The structure of vinblastine was deduced by a combination of chemical degradation and spectral data which indicated that the molecule is a dimeric indoleindoline (bisindole) and thus composed of two parts, vindoline which i connected through a carbon to ? carbon bond to 16 B-carbomethoxyvelbanamine.

VINBLASTINE SULFATE

615

The X-ray c r y s t a l - s t r u c t u r e d e t e r m i n a t i o n o f v i n c r i s t i n e methiodide d i h y d r a t e (3) defined t h e a b s o l u t e stereochemistry of v i n c r i s t i n e ; v i n b l a s t i n e should t h e r e f o r e h a s t h e above a b s o l u t e s t r u c t u r e i n view of t h e known r e l a t i o n s h i p between t h e s e two a l k a l o i d s . 1.5 Elemental Composition
C, 68.13%; H, 7.21%; N, 6.91%; 0, 17.75% ( V i n b l a s t i n e ) . C, 60.78%; H, 6.65%; N , 6 . 1 6 % ; S, 3.53%; 0, 22.88% (Vinblastine s u l f a t e ) ,

1.6

CAS R e g i s t r y Number

[ 865 -21 -4 ] [143-67-91


1.7

Vinb 1ast i n e Vinblastine sulfate.

Appearance, Color and Odor The base o c c u r s as s o l v a t e d n e e d l e s from methanol ( 4 ) . Small c o l o r l e s s n e e d l e s from e t h a n o l (5) o r a white c r y s t a l l i n e powder o r white t o s l i g h t l y yellow amorphous powder; o d o r l e s s ; very hygroscopic (1, 6) (The sulfate salt).

2.

Physical Properties

2.1

Melting Range V i n b l a s t i n e m e l t s a t 211-216' (4). V i n b l a s t i n e s u l f a t e m e l t s a t 284-285' (4,7).

616

FARID 1. MUHTADI AND ABDUL FATTAH A. A. A F I R

2.2

Solubility Vinblastine p r a c t i c a l l y insoluble i n water, s o l u b l e i n alcohols, acetone, e t h y l a c e t a t e and chloroform ( 4 ) . One p a r t of v i n b l a s t i n e s u l f a t e i s soluble i n 10 p a r t s of water; i n 50 p a r t s of chloroform; very s l i g h t l y soluble i n ethanol (96%); p r a c t i c a l l y insoluble i n e t h e r (6).

2.3

S p e c i f i c Optical Rotation [a]DZ6 + 42" ( i n CHC13) f o r v i n b l a s t i n e (4,7). The following data have been reported f o r v i n b l a s t i n e s u lf a t e : [a]D26

28'

( c = 1.01 i n methanol) (4,7).

[a]D - 28" t o (6)

35' i n a 2% w/v s o l u t i o n i n methanol

[u]D between 28' and 35', c a l c u l a t e d on t h e d r i e d b a s i s , determined i n a solution of methanol containing 200 m i n each 10 m l (8). g
2.4
PH Range (The s u l f a t e s a l t )

Between 3.5 and 5.0 i n a s o l u t i o n prepared by d i s s o l v ing 3 mg i n 2 m l of water (8). 3 . 5 t o 5 . 0 i n a s o l u t i o n of 0.15% w/v (6).
2.5

Loss on Drying
When v i n b l a s t i n e s u l f a t e i s d r i e d a t 6' a t a pressure 0 not exceeding 0.7 kPa f o r 16 hours, l o s e s not more than 17.0% of i t s weight (6). The USP (8) r e q u i r e s t h e determination t o be performed by thermogravimetric a n a l y s i s ,

2.6

Dissociation Constants pKa 5.4, 7.4 ( 1 , 7 ) .

VINBLASTINE SULFATE

617

2.7

Spectral Properties 2.7.1 Ultraviolet Spectrum (UV)

The UV absorbance spectrum of vinblastine sulfate in methanol was scanned from 200 to 400 nm using a PyeUnicum SP 8-100 Spectrophotometer. The spectrum is shown in Figure 1. Vinblastine sulfate exhibited the following absorptivity values (Table 1. ) Table 1 : UV Absorptivity Values
X max. nm

log
4.75

A (l%, lcm)

212 262 284 292

4.28 4.22 4.18

627.50 209.25 185.0 167.50

Other reported UV data for vinblastine Solvent Ethanol

X max. nm
214 259 288 296 (log E 4.74) (log E 4.22) (log 4*151 (log E 4.12)
= 176)

(Ref.) (7)

shoulder

Aqueous acid 2.7.2

268 (A

(9)

Infrared Spectrum (IR)

The IR absorption spectrum of vinblastine sulfate as a KBr-pellet (1%)was recorded on a Pye-Unicum SP 3300 Infrared Spectrophotometer. The spectrum is presented in Figure 2 . Assignment of the functional groups have been correlated with the following frequencies (Table 2 ) . Table 2 : IR Characteristics of Vinblastine Frequency cm

-1

Functional Group Free OH N-H stretch of indole ring C-H stretch Ester C=O (acetoxy)

3420 (very broad) 3035 2950 1725

618

FARID J. MUHTADI AND ABDUL FAITAH A. A. AFIFY

2
FIGURE 1 : UV SPECTRUM OF VINBLASTINE.

60 40.

20-

0*4000

3500

3000

2500

2000

1800

1600

ti00

1200

1000

800

600

400

20'0

FIGURE 2 : IR SPECTRUM O F VINBLASTINE.

620

FARID J. MUHTADI AND ABDUL FATTAH A. A. AFlFY

Frequency ern-' 1610 1580, 1500, 1455 1225

F u n c t i o n a l Group

Lactam C=O Aromatic C=C c-0-C

The f o l l o w i n g p r i n c i p a l peaks a t wave numbers 1227, 1136, 1111, 1724, 1176, 1613 cm-1 were reported f o r vinblastine sulfate as KBr d i s c (9). Other I . R . d a t a have been a l s o r e p o r t e d (10-12). 2.7.3
'H-NMR

Spectrum

The proton magnetic resonance spectrum of v i n b l a s t i n e s u l f a t e i s shown i n Figure 3. I t was o b t a i n e d on a Varian XL 200 NMR spectrophotometer f o r a s o l u t i o n i n D20. The proton chemical s h i f t s a r e p r e s e n t e d i n Table 3. Table 3 :
1

H-NMR Assignment t o V i n b l a s t i n e

Chemical S h i f t 6 (ppm) 7.18-7.48 (m)

Assignment

4 , H aromatic p r o t o n s o f c a t h a r a n t h i n e ( a t C9' ll', 1 2 3 .

6.68(s) 6.42 (s) 3.878(s) 3.867 ( s ) 3.692 (s) 2.755 (s) 2.123 (s)

H , aromatic proton of vindo l i n e ( a t Cg).

H, a r o m a t i c proton of v i n d o l i n e ( a t C12).
3H, e s t e r p r o t o n s of c a t h a r a n t h i n e ( a t Cl6/). 3H, methoxy p r o t o n s of vindoline ( a t C ) 11 3H, ester: p r o t o n s o f v i n d o l i n e ( a t C16).

3 H , N-methyl p r o t o n s of vindoline.

3H, ester p r o t o n s o f v i n d oline (at C ) . 17

s = s i n g l e t , m=multiplet 1 Other H-NMR s p e c t r a f o r v i n b l a s t i n e have been r e p o r t e d (13-16).

FIGURE 3 : 'H

- NMR SPECTRUM OF VINBLASTINE.

622

FARID J. MUHTADI AND ABDUL FA'ITAH A. A. AFlFY

2.7.4

13C-NMR

The carbon-13 NMR spectra of vinblastine and some derivatives have been exhaustively studied and complete assignments for all the 46 carbon atoms in the structure have been made (17-19). These are presented in Table 4. This table includes the reported 13C-chemical shifts of vinblastine, its sulfate salt and two of its derivatives i.e. the desacetylvinblastine and vinblastine N-oxide. Figure 4 represents the reported proton decoupled l3C-NMR spectrum of vinblastine which was measured on a Jeol PFT-100 Spectrometer (19).
a r - T r i t i a t e d v i n b l a s t i n e (C9,12,91,101,11 ',121-3H ) v i n b l a s 6 t i n e has been prepared and a n a l y z e d b y means of t r i t i u m NMR spectroscopy, this technique p r o v i d e s a r a p i d , nondestruct i v e and d i r e c t method for the a n a l y s i s of t r i t i u m on a v e r y small s c a l e and c a n be a p p l i c a b l e t o the a n a l y s i s of vinblast i n e r e c o v e r e d from animal t i s s u e s i n b i o l o g i c a l e x p e r i m e n t s
(20).

The NMR data f o r vinblastine are considered to be consistents with the conformation shown in structure below f o r the piperidine ring in the velbanamine residue (18).

'

HO

COOCH,

FIGURE 4 : 13C - NMR SPECTRUM OF VINBLASTINE.

624

FARID I. MUHTADI AND ABDUL FATTAH A. A. AFlFY

Table 4

: Carbon-13 Chemical Shifts o f Vinblastine and Derivatives 6 (ppm)

Carbon Vindoline Moiety c2 c3


c5

Vinblastine W B I 11) (8 83.1 50.0 50.0 44.3 52.8 122.6 123.1 120.4 157.8 93.8 152.5 124.3 129.7 79.3 76.1 8.1 30.5 42.3 65.2 170.6 51.8 55.3 38.0 171.4 20.7 2(19) 83.3a 50.2 50.2 44.6 53.2 122.6 123.5 121.1 158.0 94.2 152.5 124-4 129.9 79.7 76.4b 8.3 30.8 42.7 65.5 170.8 52.la 55.8a 38.3 171.6
21.1

VLB

Desacetyl VBL

H2S04
(19)
80.7 50.4 50.4 44.4 53.9 124.1 122.5 120.7 159.4 95.5 153.7 124,l 131.0 80.7 75.6 7.9 31.7 43.2 66.6 172.9 52.9 56.8 38.6 173.4 20.9

VLB

N-Oxide

82.8 50.4 49.8 44.7 53.2 122.8 123.9 120.9 158.0 93.9 152.5 124.2 130.0 80.7 74.1 8.6 32.9 42.4 66.4 173.1 52.8 55.8 38.6

83.0 50.4 50.4 44.5 53.2 123.6 123.1 120.5 157.7 93.8 153.0 124.6 130.0 79.7 76.4 8.1 30.7 42.7 65.5 170.9 52.2 55.8 38.0 171.6 21.1 contd

6 c7 c9

clo c1 1 2 3 1 4 1
15
6 1

7 1 c18 c19 c20

c21 COOCH3 COOCH3


ArOCH3 NCH3 OCOCH3 OCOCH3 -

... ,.

VINBLASTINE SULFATE

625

Table 4 contd... Carbon Velbanamine Moiety

.
VLB
H2S04

Vinblastine
WB)

Desacetyl
VLB

N-Oxide

VBL

ci
c ;

130.9 47.5 55.5 28.7 115.9 129.0 118.1 122.2 118.8 110.2 134.7 29.2

131.4 48.0 55.8 28.2 117.0 129.5 118.4 122.1 118.7 110.4 135.0 30.1 41.4 55.8 34.4 6.9 34.4 69.4 64.2 174.9 52.3

131.5 61.1 54.5 26.9 114.5 128.7

131.3 48.1 55.8 28.7 117.0 129.4 118.4 122.2 118.7 110.4 134.9 30.2 41.4 55.8 34.3 6.9 34.3 69.5 64.3 175.1 52.3

123.6 64.0 67.9 21.4 113.9 129.7 119.1 123.9 119.2 110.1 134.4 30.5 39.1 56.1 35.5 7.1 35.5 71.9 77.8 175.1 52.7

cs'
cs'
c;

cs'

121.1
121.1 119.0 112.3 135.9 35.8 45.8 56.1 34.8 6.9 36.1 68.9 61.1 175.2 52.8

clo' cli

cli
cl<
' 1 4 '

cis'
clB 1 ' ' 7

40.0
55.3

34.1
6.7 34.1 68.6 63.1 174.6 52.0

cli
c19' c2d

c21' COOCH3

Specific decoupling frequency (sdf) a = 3.76; h = 5.46 6;

c = 2.7 6.

626

FARlD J . MUHTADI AND ABDUL FATTAH A. A. AFIFY

2.7.5

Mass Spectroscopy

Conventional mass spectra (21) as well as high resolution mass spectra of vinblastine and vinblastine hydrazide have been reported (22). High resolution mass spectrometry has established the correct elemental composition of vinblastine, provided completely independent additional information regarding the point of attachment of the two parts (vindoline - velbanamine) and showed that this alkaloid is thermally labile. Some characteristic ion peaks, their corresponding element composition and element lost have been reported (22). 3. Isolation of Vinblastine Initial methods for the isolation of vinblastine from the periwinkle plants ( v i n c a r o s e a ) had been described (5,7,23-25) and well documented in several texts including the previous profile of vinblastine sulfate (12). Isolation of vinblastine and vincristine from C a t h a r a n t h u s r o s e u s continues to receive attention, and several procedures have been reported (mainly in the patent literature) for the isolation and separation of these alkaloids (24-29). Extracts of C a t h a r a n t h u s r o s e u s have been found to contain N-demethylvinblastine and this can be used to prepare vincristine by formylating the alkaloid mixture before separation and purification (30). In summary, vinblastine is extracted from C a t h a r a n t h u s r o s e u s plants with aqueous acid or with aqueous alcoholicacid, isolating the alkaloids from the extracts by the usual precipitation and solvent techniques, followed by purifying by chromatography (usually on alumina oxide columns), vinblastine is then obtained (31). Vinblastine sulfate Conversion to the (1:l) sulfate is effected by dissolving the alkaloid in an equimolar quantity of dilute sulfuric acid and either evaporating to dryness o r precipitating with a suitable organic solvent (31).

VINBLASTINE SULFATE

621

4.

T o t a l S y n t h e s i s of V i n b l a s t i n e S i n c e v i n b l a s t i n e i s a d i m e r i c a l k a l o i d , c o n s i s t s of v i n d o l i n e moiety and carbomethoxyvelbanamine p a r t , schemes f o r t h e t o t a l s y n t h e s i s of b o t h a r e r e q u i r e d followed by j o i n i n g t h e two monomeric u n i t s t o produce t h e d i m e r i c alkaloid. The t o t a l s y n t h e s e s o f v i n d o l i n e and d i h y d r o c a t h a r a n t h i n e ( a d e r i v a t i v e o f c a r b o m ethoxyvelbanamine) have been r e p o r t e d (32-34).
4.1

Total Synthesis of

(?)

-Vindoline (32)

6-Benzyloxyindole [ 11 underwent Mannich condensation with dimethylamine [ 2 ] and formaldehyde [3] i n aqueous a c e t i c a c i d t o g i v e t h e condensate [ 4 ] . T h i s a f t e r q u a t e r n i z a t i o n w i t h dimethyl s u l f a t e , was t r e a t e d with aqueous sodium c y a n i d e t o g i v e t h e n i t r i l e [ 5 ] . Methylation of [51 w i t h methyl iodide-sodium h y d r i d e i n dimethylformamide, followed by hydrogenation o v e r Pd/C i n methanolethyl a c e t a t e a t 50 p s i , gave t h e phenol [ 6 ] . T h i s was t r e a t e d with t o s y l c h l o r i d e sodium h y d r i d e i n t e t r a h y d r o f u r a n followed by hydrog e n a t i o n o v e r platinum i n aqueous e t h a n o l - e t h y l a c e t a t e c o n t a i n i n g h y d r o c h l o r i c a c i d t o produce t h e t r y p t a mine [ 7 ] . The h y d r o c h l o r i d e o f [7] was condensed with 1-chloro-3-ketobutene-1 i n e t h a n o l - t r i e t h y l a m i n e provided t h e l i q u i d Z-enamino ketone [ 8 ] ( i n 83% yield). [ 8 ] was converted t o i t s E-acetamide [ 9 ] by t r e a t m e n t with a c e t y l chloride-sodium h y d r i d e i n t e t r a h y d r o f u r a n ( i n 89% y i e l d ) . [9] was s u b j e c t e d t o c y c l i z a t i o n by h e a t i n g a t 90' i n boron t r i f l u o r i d e e t h e r a t e f o r 16 minutes t o a f f o r d t h e amine [ l o ] i n 89% y i e l d . The l a t t e r was t r e a t e d w i t h 20% potassium hydroxide i n methanol-water a t r e f l u x , t o g i v e t h e phenol which was h e a t e d with dimethyl s u l f a t e i n acet o n e o v e r suspended potassium c a r b o n a t e t o a f f o r d t h e methyl e t h e r [ l l ] i n q u a n t i t a t i v e y i e l d . Removal of t h e a c e t y l group i n [ l l ] was accomplished with t r i e t h y loxonium f l u o r o b o r a t e i n methylene c h l o r i d e a t room t e m p e r a t u r e o v e r suspended sodium b i c a r b o n a t e t o prov i d e t h e amine [ 1 2 ] i n 82% y i e l d . Condensation of [ 1 2 ] with a c r o l e i n i n methanol c o n t a i n i n g sodium metho x i d e followed by d e h y d r a t i o n with methanesulfonyl c h l o r i d e i n p y r i d i n e gave t h e u n s a t u r a t e d ketone [13] i n 60% y i e l d . E t h y l a t i o n of [13] w i t h e t h y l i o d i d e i n t e r t - b u t y l alcohol-dimethylformamide c o n t a i n i n g potassium t e r t - b u t o x i d e y i e l d e d t h e e t h y l u n s a t u r a t e d ketone [14] i n 53% y i e l d . Condensation o f t h e sodium

628

FARlD .. I MUHTADI AND ABDUL FATTAH A. A. AFIFY

Scheme I : Total Synthesis of (*)-Vindoline.

VINBLASTINE SULFATE

629

'15'

630

FARID J. MUHTADI AND ABDUL FAITAH A. A. AFlFY

h y d r i d e g e n e r a t e d e n o l a t e o f ketone [14] w i t h dimet h y l c a r b o n a t e gave t h e k e t o e s t e r [15]. Hydroxylation o f t h i s with 38% hydrogen p e r o x i d e i n t e r t - b u t y l alcohol-dimethoxyethane c o n t a i n i n g potassium t e r t butoxide a f f o r d e d t h e 8-hydroxy ketone [16] i n 76% y i e l d . [16] was t r e a t e d with aluminum c h l o r i d e (-2S0, t e t r a h y d r o f u r a n ) followed by r e d u c t i o n with sodium b i s (2-methoxyethoxy) aluminum h y d r i d e (-20) t o g i v e s i n g l e epimer a l c o h o l . A c e t y l a t i o n o f t h i s a l c o h o l w i t h a c e t i c anhydride-sodium acetate a f f o r d e d (?) v i n d o l i n e [ 171. T h i s s y n t h e s i s i s presented in scheme I .
4.2

T o t a l S y n t h e s i s of (+)-Dihydrocatharanthine (33,34) E t h y l 2-carbethoxy-4, 4-diethoxy b u t a n o a t e [ l ] (prepared from dimethylmalonate ( 35 ) underwent condensation with 0.5 molar excess of methyl-ae t h y l a c r y l a t e [ 21 (prepared from methyl -2-carboxybutan o a t e ( 36,37) i n t h e p r e s e n c e of f r e s h l y p r e p a r e d sodium e t h o x i d e as t h e c a t a l y s t t o g i v e t h e condensate, methyl-2-ethyl-4, 4-dicarbethoxy-6, 6-diethoxy hexano a t e [3] i n 86% y i e l d . T h i s was r e f l u x e d w i t h 1.5 e q u i v a l e n t s of d r y sodium cyanide i n dry dimethyl s u l f o x i d e t o a f f o r d [4] i n 70% y i e l d . Substance [ 4 ] was d i r e c t l y condensed with t r y p t a m i n e [S] by r e f l u x i n g i n aqueous acetic a c i d under Nz f o r 6 h o u r s t o produce t h e lactam e s t e r [63. Product [6] was reduced by r e f l u x i n g a s o l u t i o n o f i t i n t e t r a h y d r o f u r a n (THF) with LAH t o g i v e t h e amine a l c o h o l [ 7 ] . Mesylat i o n of [ 7 ] with anhydrous methane s u l f o n y l c h l o r i d e and trimethylamine in anhydrous e t h e r , followed by r e f l u x i n g t h e mesylate i n anhydrous a c e t o n i t r i l e f o r several hours t o y i e l d t h e q u a t e r n a r y s a l t [ 8 ] . T h i s s a l t was h e a t e d a t 200 KCN i n d i g o l , conversion t o 16-cyanodihydro cleavamine [9] was e f f e c t e d . Methano l y s i s of [ 9 ] under mild c o n d i t i o n s by u s i n g anhydr o u s methanol and bubbling d r y HC1 gas a t 25' a f f o r d e d (+)-16-methoxycarbonyldihydrocleavamine [ l o ] . S u b s t ance [ l o ] was s u b j e c t e d t o o x i d a t i o n with m e r c u r i c a c e t a t e t o g i v e (+)-dihydrocatharanthine [ l l ] . T h i s t o t a l s y n t h e s i s is presented i n scheme 1 . 1 Dihydrocatharanthine [ l l ] can be converted i n t o c a t h a r a n t h i n e [ 1 2 1 by an e s t a b l i s h e d method (38). Other s y n t h e s e s o f v i n d o l i n e (39,40) and o f c a t h a r a n t h i n e (41,42) have been r e p o r t e d .

VINBLASTINE SULFATE

63 I

( E t O ) 2CH

/\i

S c h e m e I1 T o t a l S y n t h e s i s of ( + ) - D i h y d r o c a t h a r a n t h i n e C02Et C02Et

+ H2C=C-C02CH3 I
Et

C02Et [I1

[21

(EtO) 2 C H q ^ ( c 0 2 c H 3 C02Et

NaCN DMSO

[31

PI

[41

LAH THF
1. C H 3 S 0 2 C 1
2 . CH3CN

q\T (-1
OS02CH3 H Dig KCN o i [81 MeOH/HCl
25'

CH2OH

[71

632

FARID I. MUHTADI AND ABDUL FATTAH A. A. AFIFY

4.3

T o t a l S y n t h e s i s of V i n b l a s t i n e (43-47) C a t h a r a n t h i n e [ l ] underwent o x i d a t i o n with m-chloroperbenzoic a c i d t o g i v e t h e N-oxide. C a t h a r a n t h i n e N-oxide [ 2 ] was t r e a t e d with v i n d o l i n e i n methylene chloride-trifluoroacetic anhydride a t 5 0 , c o u p l i n g o c c u r r e d , t o g i v e t h e immonium i o n [3] which w r,edu,ced with sodium borohydride t o p r o v i d e a t h e A15' q 2 0 1 2 0 - d e o x y v i n b l a s t i n e ( a n h y d r o v i n b l a s t i n e ) [ 4 ] . This upon t r e a t m e n t with t h a l l i u m t r i a c e t a t e followed by borohydride r e d u c t i o n a f f o r d e d v i n b l a s tine [ S ] . T h i s s y n t h e s i s i s p r e s e n t e d i n scheme III.
A h i g h l y e f f i c i e n t and commercially important s y n t h e s i s o f v i n b l a s t i n e from c a t h a r a n t h i n e and v i n d o l i n e h a s r e c e n t l y been d e s c r i b e d ( 4 8 ) . Other s y n t h e t i c methods have a l s o been r e p o r t e d ( 49,SO).

VINBLASTINE SULFATE

633

Scheme 111 : Total Synthesis of Vinblastine

-0

Vindol ine coup 1ing

/
Vindoline

[41

i) T l ( 0 A c ) ii) NaBH4

3.

H3C

H :
CH3

OCOCH3 ~ O ~ C H ~

H3COOC
/
/

/ Vindoline

[51

634

FARID I. MUHTADI AND ABDUL FATTAH A. A. AFIFY

5.

B i o s y n t h e s i s of V i n b l a s t i n e I t h a s long been proposed t h a t t h e i n d o l i c moiety of t h e i n d o l e a l k a l o i d s i s d e r i v e d from t h e aminoacid "tryptophan" ( 51-53). T h i s h a s been j u s t i f i e d when r a d i o a c t i v e t r y p t o phan o r t r y p t a m i n e (decarboxytryptophan) were i n c o r p o r a t e d i n t o s e v e r a l i n d o l e a l k a l o i d s ( 5 4 - 5 7 ) . I t h a s a l s o been p r e d i c t e d t h a t t h e non-tryptophan p o r t i o n s of t h e s e a l k a l o i d s a r e formed from two mevalonate u n i t s t o a f f o r d a c y c l o pentane monoterpenoid p r e c u r s o r (58,59). T h i s was proved upon f e e d i n g d l - [2-I4C] -mevalonic a c i d l a c t o n e , and sodium (?)-[2-14C] mevalonate i n t o V i n c a r o s e a p l a n t s and r e s u l t e d i n t h e i s o l a t i o n of r a d i o a c t i v e v i n d o l i n e , c a t h a r a n t h i n e and a j m a l i c i n e ( 60-64). I t was f u r t h e r p r e d i c t e d t h a t t h e monoterpenoid p r e c u r s o r c o u l d w e l l b e "the g l u c o s i d e loganin" ( 6 5 ) . I t i s now known t h a t l o g a n i n a r i s e s i n t h e p l a n t s from two mevalonate u n i t s . One of which i s transformed by a s e r i e s of s t e p s i n t o i s o p e n t e n y l d i p h o s p h a t e (66) and t h e o t h e r i n t o dimet h y a l l y l p y r o p h o s p h a t e ( 6 7 ) . Combination of t h e s e two u n i t s l e a d s t o g e r a n i o l (68-73), t h e n t o l o g a n i n (74-76) and f i n a l l y i n t o s e c o l o g a n i n (77, 7 8 ) . Evidence s u g g e s t s t h a t t r y p t a m i n e ( o r L-tryptophan) [ 11 r e a c t s w i t h s e c o l o g a n i n [2] t o form s t r i c t o s i d i n e ( i s o v i n c o s i d e ) [3] ( 66,79-84). I t h a s been observed t h a t l a b e l e d s t r i c t o s i d i n e [ 3 ] ; g e i s s o s c h i z i n e [4] ( 8 0 , 8 5 , 8 6 ) ; stemmadenine [7] ( 8 4 , 8 7 ) and t a b e r s o n i n e [7b,9] (86-88) were a l l i n c o r p o r a t e d i n t o b o t h c a t h a r a n t h i n e [8] and v i n d o l i n e [ 101 i n C a t h a r a n t h u s r o s e u s p l a n t s , i n d i c a t i n g t h a t t h e s e a r e t h e main p r e c u r s o r s i n t h e b i o s y n t h e t i c pathway t o t h e Aspidosperma-Iboga alkaloids. Other i n t e r m e d i a t e s such as g e i s s o s c h i z i n e o x i n d o l e [ 5 ] , preakuammicine [6] have been d e t e c t e d 28-40 h o u r s a f t e r germination of C. r o s e u s s e e d s (85,87,89) provided s t r o n g evidence f o r t h e f o r m a t i o n of c a t h a r a n t h i n e [8] and v i n d o l i n e [ l o ] as p r e s e n t e d i n schemes I and 11. Feeding r a d i o a c t i v e [8] as [3H-C02CH3] and [ l o ] as [14COCOCH31 i n t o a p i c a l c u t t i n g of 3-4 month-old C. r o s e u s p l a n t s a f f o r d e d low b u t d e f i n i t e i n c o r p o r a t i o n s of b o t h a l k a l o i d s i n t o v i n b l a s t i n e [12] d e m o n s t r a t i n g t h a t t h e s e monomeric a l k a l o i d s a r e t h e p r e c u r s o r s of [ 1 2 1 ( 9 0 ) . Feedi n g b o t h [acetyl-14C] v i n d o l i n e and [OC3H3] c a t h a r a n t h i n e t o 6 week-old d i f f e r e n t i a t e d c. r o s e u s p l a n t s f o r 6 d a y s , l a b e l l e d a n h y d r o v i n b l a s t i n e [ 111 was i s o l a t e d ( 91) . T h i s was i n c o r p o r a t e d i n t o v i n b l a s t i n e by c e l l - f r e e p r e p a r a t i o n s Of C a t h a r a n t h u s r o s e u s ( 9 2 , 9 3 J . Later it was found t h a t a n h y d r o v i n b l a s t i n e [ l l ] can b e conv e r t e d i n t o v i n b l a s t i n e [ 1 2 ] by c e l l - f r e e homogenates of

VINBLASTINE SULFATE

63.5

Scheme I :

B i o s y n t h e s i s of C a t h a r a n t h i n e

+-

[51

C02CH3
CH20H

636

FARID 1. MUHTADI AND ABDUL FATTAH A. A. AFIFY

Scheme 11:

B i o s y n t h e s i s of C a t h a r a n t h i n e and Vindoline

0-B
0N
(-)-form

COZCHS

[91

C02CH3

(+)-form
[7b1

VINBLASTINE SULFATE

Scheme 111:

Biosynthesis of Vinblastine

Catharanthine [S]

Vindoline [ l o ]

Cell-free extracts from Catharanthus roseus plants

Vindoline
Cell-free extracts leaves C. roseus
- Cell-free homoyenates or

of C. roseus cell suspension cultures

638

FARlD J. MUHTADI AND ABDUL FATTAH A. A. AFlFY

C . roseus

cell suspension cultures ( 3 4 ) , thus administration of [21a-3H] anhydro-VLB to a cell-free homogenate of suspension culture cells C. roseus afforded radioactive [3H]-vinblastine ( 94 ) .

The biosynthesis of vinblastine is presented i n scheme III.

6. Pharmacokinetics
6.1

Drug Absorption Vinblastine is poorly absorbed after oral administration (9,951. It is readily absorbed after intravenous administration (IV) or intraperitoneal injection (IP).

6.2

Drug Distribution Vinblastine is rapidly distributed with high tissue binding, readily binds to platelets, red blood cells and white blood cells; subject to enterohepatic circulation; volume of distribution, 86 to 111 liters ( 1,961. After IV radioactively labeled, vinblastine is detected mostly in the liver in less than an hour (97). Protein binding: It is highly protein bound ranging from 98 to 99.7% ( 9 8 ) . Binds in plasma to a- and 8 globulins (1,96). The drug does not penetrate the CNS o r other fatty tissues ( 99). Drug concentration levels: continuous infusions of vinblastine (2 mg/square meter) will produce cytotoxic concentrations of approximately 2 ng/mL ( 1 0 . 0) After an IV dose of 15 mg, a plasma concentration of about 16 ng/mL is obtained in 24 hours; an additional dose of 15 mg at this time produces a plasma concentration of about 55 ng/mL 4 hours later ( 1,96).

6.3 Metabolism

Vinblastine is metabolized in the liver. Metabolic reactions in rats is deacetylation to give desacetylvinblastine which is the major metabolite of vinblastine ( 1,9). A significant amount of vinblastine is metabolized in the liver to the active metabolite desacetylvinblastine ( 95).
6.4

Drug Excretion In 72 hours, 25 to 40% of an intravenous dose is excreted in the feces and 19 to 23% is excreted in the urine, most of the urinary excreted material is

VINBLASTINE SULFATE

639

unchanged, w h i l s t t h a t i n t h e f a e c e s i s i n t h e form of m e t a b o l i t e s (1,961. About 14% of a r a d i o a c t i v e l y l a b e l e d dose i s e x c r e t e d i n t h e u r i n e i n 72 hours and 10% i s e l i m i n a t e d i n t h e f a e c e s i n t h e same p e r i o d ( 9 ) . Following I V v i n b l a s t i n e d o s i n g depending upon t h e r a d i o a c t i v e l a b e l t e c h n i q u e used o n l y 13.6 t o 23% of t h e t o t a l dose was e x c r e t e d i n t h e u r i n e and t h a t e x c r e t e d i n t h e f e c e s ranged from 9.9 t o 41% w i t h 72 hours ( 101)

6.5

Half-Life Plasma h a l f - l i f e ( t o t a l a c t i v i t y ) , about 20 hours (9) * In whole blood, a - p h a s e , about 4 minutes and 8-phase, about 190 minutes, f o r drug p l u s m e t a b o l i t e s ( 1 ) . V i n b l a s t i n e f i t s a 3-compartment pharmacokinetic model w i t h a l p h a , b e t a and gamma ( t e r m i n a l p h a s e ) , h a l f - l i v e s of 0.062, 0.164 and 25 hours r e s p e c t i v e l y were o b t a i n e d ( 9 9 ) .

7.

P r e p a r a t i o n and P r e s e r v a t i o n V i n b l a s t i n e s u l f a t e should be s t o r e d i n a i r t i g h t c o n t a i n e r s , a t a t e m p e r a t u r e between 2" and 10" p r o t e c t e d from light. V i n b l a s t i n e s u l f a t e i s a d m i n i s t e r e d by t h e IV i n j e c t i o n o f a s o l u t i o n o f 1 mg p e r mL i n water f o r i n j e c t i o n o r i n sodium c h l o r i d e i n j e c t i o n ( p r e s e r v e d w i t h phenol o r benzyla l c o h o l ) . Usually t h e ampoule c o n t a i n s 10 m s t e r i l e v i n g blastine sulfate. The drug d i s s o l v e s i n s t a n t l y t o g i v e a c l e a r s o l u t i o n having a p i n t h e range of 3.5-5.0. H V i a l s of v i n b l a s t i n e s u l f a t e should be s t o r e d i n a r e f r i g e r a t o r between 2 O and 8C t o a s s u r e extended s t a b i l i t y . After r e c o n s t i t u t i o n w i t h 10 mL b a c t e r i o s t a t i c Sodium C h l o r i d e I n j e c t i o n USP ( p r e s e r v e d with b e n z y l a l c o h o l ) , s o l u t i o n may kept i n a r e f r i g e r a t o r a t 2" t o 8OC f o r 30 days without s i g n i f i c a n t l o s s of potency ( 1 0 2 ) . If t h e i n j e c t i o n c o n t a i n s no b a c t e r i o c i d e , i t should be used a s soon a s p o s s i b l e a f t e r p r e p a r a t i o n , and i n any c a s e w i t h i n 4 days. In t h e p r e s e n c e of a s u i t a b l e b a c t e r i o c i d e such a s 0.5% phenol, i t may be used f o r up t o a one month when s t o r e d a t 2 O t o 10C ( 1 ) .

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8.

Uses of Vinblastine Sulfate Vinblastine is an antineoplastic drug which apparentlyacts by binding to the microtubular proteins of the spindle and arresting mitosis at the metaphase. It also interferes with aminoacid metabolism and nucleic acid synthesis. It has some immunosuppressant activity, as it suppresses the immune response and in high doses it is neurotoxic. Like other cytotoxic drugs it is teratogenic (95). Vinblastine sulfate is mainly used in association with other antineoplastic agents, in the treatment of Hodgkin's disease and other lymphomas including mycosis fungoides. It is also of use in the treatment of some inoperable malignant neoplasms including those of the breast, female genital tract, testis, lung, gastrointestinal tract and in neuroblastoma, choriocarcinoma, Kaposi's sarcoma and histiocytosis X ( 9 5 ) . In the treatment of Hodgkin's disease, it is often given with cyclophosphamide or mustine, procarbazine and prednisone or with doxorubicin, bleomycin and dacarbazine. In carcinoma of testis, vinblastine is given with bleomycin and cisplatin (95). I n clinical dosage it depresses bone-marrow activity, affecting mainly the white cells, with relative sparing of the erythroid elements. The bone-marrow depression is reversible on stopping the drug (1).
8.1 Precautions

Vinblastine sulfate should be used with care in cachectic patients. Its use in pregnancy is not advised as it is teratogenic. Care should be applied when it is injected intravenously as perivenous infiltration may cause cellulitis, phlebitis and venous thrombosis ( 1).
8.2 Contra-indications

Vinblastine sulfate cell count is below bacterial infection is infiltrated with

should not be given if the white4000 per cubic millimeter, if is present, or if the bone-marrow neoplastic cells ( 1).

VlNBLASTlNE SULFATE

64 I

9.

Methods o f A n a l y s i s 9.1 I d e n t i f i c a t i o n Tests

The following identification tests are mentioned in the B P (6).

To lmg v i n b l a s t i n e s u l f a t e add 0.2 m l of a f r e s h l y p r e p a r e d 1%w/v s o l u t i o n of v a n i l l i n i n h y d r o c h l o r i c a c i d . A pink c o l o r i s produced i n about 1 minute ( d i s t i n c t i o n from v i n cristine sulfate). Mix 0.5 m v i n b l a s t i n e s u l f a t e w i t h 5 m of 4-dimethylaminog g benzaldehyde and 0.2 ml o f g l a c i a l a c e t i c a c i d and 0.2 ml of s u l f u r i c a c i d ; a reddish-brown c o l o r i s produced. Add 1 m l of g l a c i a l acetic a c i d ; t h e c o l o r changes t o p i n k .
The following identification tests are mentioned in the USP (8).

The i n f r a r e d a b s o r p t i o n spectrum o f a pot.assium d i s p e r s i o n of v i n b l a s t i n e s u l f a t e , p r e v i o u s l y d r i e d i n vacuum a t 60' f o r 16 h o u r s , e x h i b i t s maxima only a t t h e same wavelengths a s t h a t of a s i m i l a r p r e p a r a t i o n of USP v i n b l a s t i n e s u l f a t e RS Other i d e n t i f i c a t i o n t e s t : Marquis T e s t ( s u l f u r i c a c i d formaldehyde) g i v e s wine-red c o l o r w i t h v i n b l a s t i n e (103).

9.2

T i t r i m e t r i c Determinations Non-Aqueous T i t r a t i o n s

Q u a n t i t a t i v e d e t e r m i n a t i o n of t h e a l k a l o i d s i n c l u d i n g v i n b l a s t i n e i n d r u g s and e x t r a c t s a r e r e p o r t e d as f o l l o w s ( 104). A e r i a l p a r t s of Vinca r o s e a were ground, t r e a t e d w i t h 25% NH40H f o r 30 minutes, and e x t r a c t e d with MeOH. The e x t r a c t was evaporated and t h e r e s i d u e was d i s s o l v e d i n 2% H2SO4 on a w a t e r b a t h . The H2S04 e x t r a c t was a l k a l i n i z e d with NH40H, r e e x t r a c t e d w i t h CHC13, t h e e x t r a c t was d r i e d , and evaporat e d . The r e s i d u e was d i s s o l v e d i n HOAc and t i t r a t e d w i t h 0 . 1 ~ ~ 1 0 4 . A e r i a l p a r t s of v i n c a minor were t r e a t e d w i t h 25% NH40H f o r 30 minutes, e x t r a c t e d with CHC13, t h e c o n c e n t r a t e d e x t r a c t was r e e x t r a c t e d with 2% t a r t a r i c a c i d a d j u s t e d t o pH 9 . 0 with 25% NH40H, and a l k a l o i d s were e x t r a c t e d w i t h CHC13, d i s s o l v e d i n HOAc and t i t r a t e d w i t h H C l O 4 by u s i n g c r y s t a l violet indicator. - A mixture of v i n b l a s t i n e s u l f a t e and t e s t o l a c t o n e i s d e t e r m i ned by p r e c i p i t a t i o n a t pH 3.7 w i t h a measured volume o f Na t e t r a p h e n y l b o r a t e s o l u t i o n , and t i t r a t i o n of unconsumed r e a gent with hexadecylpyridinium c h l o r i d e u s i n g bromophenol b l u e a s an i n d i c a t o r ( 1 0 5 ) .

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9.3

Voltametric Determination

Ultrasensitive voltammetric measurements based on coupling hydrogen catalytic systems with controlled interfacial accumulation of the catalyst has been reported (106). By combining adsorptive stripping voltammetry with a catalytic hydrogen process, trace Pt can be measured with a detection limit of -1X10-12M. The supporting electrolyte (1.08 M H2SO4) contains 0.04% (wt./vol.) formaldehyde and 0.001% (wt./vol.) hydrazine. The adsorption of the Ptformazone complex (the catalyst) results in a well-defined catalytic H peak at - 0.92 V, with a peak half-width of 60 mV. The peak height increases rapidly with increasing preconcentration time, indicating a large enhancement of the complex on the surface of the hanging Hg drop electrode (SDS) and gelatin cause serious interference by competing with the catalyst on adsorption sites. By using 0.25 M ammoniacal buffer (pH 9 . 3 ) , the vinca alkaloids vinblastine and vincristine can be determined at subnanomol levels (106)

9.4 SDectroDhotometric Determinations


9.4.1 UV Spectrophotometry

The official methods for determination of vinblastine sulfate are UV spectrophotometric techniques(6,8). The BP ( 6 ) recommends the following procedure: Dissolve 10 mg of vinblastine sulfate in sufficient methanol to produce 500 ml and measure the absorbance of the resulting solution at the maximum at 267 nm. Calculate the content of C46H58N409, H2SO4 taking 185 as the value of A (1%, 1 cm) at the maximum at 267 nm. The USP (8) recommends the following procedure: Dissolve about 5 mg of vinblastine sulfate, accurately weighed in methanol and dilute quantitatively and stepwise with methanol to obtain a solution containing about 20 pg per ml on the dried basis. Dissolve an accurately weighed quantity of USP Vinblastine Sulfate RS in methanol and dilute quantitatively and stepwise with methanol to obtain a standard solution having a known concentration of about 20 pg per ml on the dried basis. Concomitantly determine the absorbances of both solutions in 1-cm cells at the wavelength of maximum absorbance at about 267 nrn, with a suitable spectrophotometer, using methanol as the blank. Calculate the quantity in mg of C46H58N40g. H2S04 in the portion of vinblastine sulfate taken by the formula

VINBLASTINE SULFATE

643

0.25 C (Au/As) , i n which C i s t h e c o n c e n t r a t i o n , i n pg p e r m l of USP V i n b l a s t i n e S u l f a t e RS i n t h e S t a n d a r d s o l u t i o n and Au and As a r e t h e absorbances of t h e s o l u t i o n o f v i n b l a s t i n e s u l f a t e and t h e Standard s o l u t i o n respectively. The USP r e q u i r e s t h a t t h e weighings t o be performed r a p i d l y and w i t h a minimum of exposure of t h e s u b s t a n c e t o a i r .
9.4.2

C o l o r i m e t r i c Determinations

A c o l o r i m e t r i c method was f i r s t deviced f o r t h e a s s a y of p u r e v i n b l a s t i n e s u l f a t e ( 107). The method depends on t h e f o r m a t i o n of a deep r o s e c o l o r upon h e a t i n g v i n b l a s t i n e s u l f a t e a t 80' w i t h a r e a g e n t c o n s i s t i n g of p y r i d i n e (35 ml) , c o n c e n t r a t e d s u l f u r i c a c i d (1 m l ) and a c e t i c anhydride ( 3 5 ml) c o n t a i n i n g 0.05% a c e t y l c h l o r i d e . The c o l o r so produced i s measured a t 574 nm. The s e n s i t i v i t y i s 5 t o 7 0 pg of v i n b l a s t i n e s u l f a t e p e r

To check p u r i t y o f t h e sample, t h e absorbances of t h e c o l o r produced a r e measured i n 1 c c e l l s a t 574 and m 538 n a g a i n s t w a t e r a s a r e f e r e n c e . The r a t i o of A574 m nm/A538 n should b e i n t h e range of 1.20 - 1.25 ( 1 0 7 ) . m Another c o l o r i m e t r i c method h a s been r e p o r t e d f o r t h e q u a n t i t a t i v e d e t e r m i n a t i o n of v i n b l a s t i n e and l e u r o s i n e i n Vinca r o s e a ( 1 0 8 ) . The s e q u e n t i a l s t a g e s used were - ( i ) s e l e c t i v e e x t r a c t i o n o f t h e a l k a l o i d s i n t o benzene o r t o l u e n e ( w e t t i n g t h e raw m a t e r i a l with aq. 5% Na a c e t a t e improved t h e e x t r a c t i o n ) , ( i i ) b a c k - e x t r a c t i o n of t h e a l k a l o i d s i n t o 2% c i t r i c o r t a r t a r i c a c i d , ( i i i ) adjustment of pH t o 6 . 0 w i t h aq. 5% NH3 and r e - e x t r a c t i o n of t h e a l k a l o i d s i n t o t o l u e n e , ( i v ) s e p a r a t i o n o f t h e a l k a l o i d s on LH-20 with methanol - CHC13 ( 7 : 3 ) , s e p a r a t i o n of t h e d i m e r i c a l k a l o i d f r a c t i o n on s i l i c a g e l , with CHC13 - benzene a c e t o n e - e t h y l a c e t a t e - methanol (20:20:15:5:3) ( v i ) e l u t i o n of t h e a l k a l o i d s w i t h 1%H C 1 , and ( v i i ) a d d i t i o n of 0.2 m l of 1%t r o p a e o l i n 000-1 (C.I. Acid Orange 20) and t h e n CHC13. The c o l o r so produced i s measured a t 490 nm.

ml .

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FARlD J . MUHTADl AND ABDUL FATTAH A. A. AFlFY

9.5 Chromatographic Methods 9.5.1 Paper Chromatography

Clarke ( 1 0 3 ) described the following system for the identification of vinblastine. System : Reversed phase, Whatman no. 1 or no. 3 paper chromatography, impregnated by dipping in 10% solution of tributyrin in acetone and drying in air. Sample : A 5 1-11 of 1 to 5% solution in ethanol o r chloroform. Solvent : Acetate buffer (pH 4.58). Location : Under UV light gives purple fluorescence. Location spray : Iodoplatinate. Rf value : 0.10. 9.5.2 Thin Layer Chromatography (TLC)

The following TLC systems were recommended for the identification and separation of vinblastine. Chromatograni

1. Silica gel G, 250 methanol-strong 1 1 thick, dipped -m ammonia (100:l.S) in or sprayed with Cyclohexane-toluene0.1 M KOH in diethylamine (75:15:10) 0.10 methanol and dried chloroform-methanol (90:10) 0.60 Oe60 2. Silica gel Gf 254 toluene-chloroformdiethylamine (80:40:6) 3 . Silica gel G ethylacetate-absolute alcohol (3 :1) 0.21 4. Silica gel G ethylacetate-absolute alcohol ( 1 :1) 0.33 5. Alumina ethylacetate-absolute 0.66 alcohol ( 3 : 1) 6. Silica Gel G n-butanol-acetic acidwater ( :1 :1) 4 0.19
7.

Solvent System

Rf value

Ref.
~~

(9 () 6

(1091

(110)
(110)

(109)
(109)

Silica Gel G

methano 1 chloroform-methanol (95 :5) chloroform chloroform-ethylacetate (1:l)

0.46

8. Silica gel G 9. Alumina

0.24 0.17 0.25

(111) (110) (110)

10. Alumina

VINBLASTINE SULFATE

645

Detection : The spots can be detected by: 1- Under short UV light ( 2 5 4 nm) 2- Spraying with: a) Dragendorff's reagent ( 103) b) Acidified iodoplatinate solution (103) s) 1% Ceric ammonium sulfate in 85% phosphoric acid (110) double development technique was recommended f o r the separation of vinblastine from the other dimeric catharanthus alkaloids on alumina layers(ll2). The loaded chromatoplates were first developed in the solvent ethylacetate, then after drying, a second development in ethylacetate-absolute alcohol (3: 1) was carried out. Vinblastine in this technique gave Rf value of 0.73 ( 112). - Two dimensional TLC technique was reported for the separation of more complex mixture of catharanthus alkaloids (115) . .- The BP (6) adopted a TLC technique to test for the presence of related alkaloids in vinblastine sulfate sample (checking the purity of the sample): TLC chromatoplates are coated with silica gel GF 254. 5 . l rt of the following three solutions in methanol are separately applied to one chromatoplate. 1- 1.0% w/v of the substance being examined. 2 - 0.02% w/v of standard vincristine sulfate BPCRS. 3- 1.0% w/v of standard vinblastine sulfate BPCRS. The chromatoplate is then developed in the solvent tolueneAfter development, the chloroform-diethylamine (80:40:6). plate is allowed to dry in air and examined under UV light (254 nm). Any secondary spot in the chromatogram obtained with solution (1) is not more intense than the spot obta2. ined with solution ( ) - A TLC - Densitometric determination of vinblastine was described. Two dimensional TLC is performed followed by densitometric scanning of the spots at 289 nm. The C O efficient variation of the method was 7.5% ( 114).
-A

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9.5.3

Gas Liquid Chromatography (GLC)

The following GLC system has been reported for the identification and separation of vinca alkaloids including vinblastine ( 115). Column condition: Glass column (lm x 3.2mm), precoated with hexamethyldisilazane and packed with 3% OV-101 on Gas chrom Q (80-100 mesh), with temperature programmed 0' from 2 0 to 300' at 5' min.-l Carrier gas: Nitrogen at a flow rate of 30 ml/min.-l Detection: F.1.D Condition: Vinca alkaloids including vinblastine were derivatized before application by heating for 5 minutes at room temperature with t r i f l u o r o b i s - ( T r i m e t h y l s i l y l ) acetamide-pyridine (1 : 1. )
9.5.4 High Performance Liquid Chromatography (HPLC)

Several HPLC methods have been employed to determine vinblastine and its metabolites in biological fluids and tissues. Some of these methods are as follows: System 1: The following system has been recommended for quantitative determination of vinblastine and other vinca alkaloids in plasma and urine Conditions:The drugs are extracted from biological materials by an ion pair extraction with sodium octylsulfate as counter ion at pH 3.0. The extracts are injected onto a reversed-phase system with a cyano column as stationary phase. Mobile MeCN -phosphate buffer pH 3.0 (65:35). phase : System 2: The following system is a reversed-phase with electrochemical detection. It is employed for quantitative determination of vinblastine and its metabolites in plasma and urine. Quantification of substances in human plasma and urine is possible down t o 1 ng/ml ( 117). Column : Hypersil ODs. Mobile Methanol - 10m M phosphate buffer pH 7.0. phase :
( 116).

VINBLASTINE SULFATE

641

System 3: This system is employed for the analysis of Catharanthus alkaloids including vinblastine (118). Column: A stainless steel (25cm x 4mm), packed with Li-Chrosorb RP-8; operated at ambient temperature. Mobile 0.01M ammonium carbonate-acetonitrile (53:47). phase : Flow rate: 1.5ml min.-l Retention 1 2 . 3 7 minutes forvinblastine. time : Detection: UV at 298 nm. This system has been employed f o r the separation, detection and correlation of plate height and molecular weight of vinblastine and other Vinca alkaloids (119). Column: 25cm x 4.6mm, packed with R SiL C1g HL-D octadecyl-silica gel. Mobile Gradient elution with aqueous 50 to 85% phase : methanol containing 0.1% ethanolamine. Flow rate: 2 ml min.-l System 4 : Detection: UV at 290 nrn. System 5: The following reversed-phase system has been used for the analysis of Catharanthus alkaloids including vinblastine by thermospray liquid chromatography-mass spectrometry (120). Column: p Bondapak C18 (30cm x 3.9mm), reversed-phase column. Mobile Isocratic solvent, 0.1M ammonium acetate (pH 7.2) - MeCN (51:49). phase : Flow rate: 1 ml min.-l Detection: Electrochemical and UV (The limit o f detection being 4 ng/injection or each alkaloid). System 6 : The following reversed-phase system has been reported f o r the determination of vinblastine and other alkaloids of Catharanthus roseus leaves ( 121). p Bondapak Cis. 0.1M diammonium hydrogen orthophosphate - MeCN (25:75), pH 7.0.

Column: Mobile phase :

Detection: UV at 254 and 280 nm.

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FARID J . MUHTADI AND ABDUL FATTAH A. A. AFIFY

System 7:

Column: Mobile phase : Internal standard : System 8 :

This reversed-phase system i s d e s c r i b e d f o r t h e d e t e r m i n a t i o n o f v i n b l a s t i n e and o t h e r Catharanthus a l k a l o i d 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 , t h e l i m i t of d e t e c t i o n and t h e r e c o v e r y were 1.63-3.52%, 1 0 ug/ml and 96.6-102% r e s p e c t i v e l y ( 1 2 2 ) . A c a r t r i d g e column packed w i t h Spheri-5RP.
2 g r a d i e n t systems c o n t a i n i n g MeOH, MeCN, 0.025M ammonium a c e t a t e and t r i e t h y l a m i n e i n different ratios. 5-Methoxytryptamine.

D e t e c t i o n : UV a t 280 and 254 nm. This i s a l s o a r e v e r s e d - p h a s e system which has been a p p l i e d f o r s e p a r a t i o n and q u a n t i t a t i o n o f a l k a l o i d s from c e l l suspension c u l t u res of Catharanthus r o s e u s i n c l u d i n g v i n b l a s t i n e (123). 1.1 Bondapak C18.
A mixture of methanol and (NH4)2HP04 i n w a t e r .

Column: Mobile phase :

D e t e c t i o n : UV a t 298 nm. Other HPLC systems f o r v i n b l a s t i n e have a l s o been r e p o r t e d (124-127). 9.6 Radioimmunoassav Methods

Q u a n t i t a t i v e d e t e r m i n a t i o n of v i n b l a s t i n e i n t i s s u e c u l t u r e s of Catharanthus r o s e u s by radioimmunoassay was r e p o r t e d ( 128). Antibody was o b t a i n e d by t h e immunization of r a b b i t s a g a i n s t a c o n j u g a t e of v i n b l a s t i n e w i t h bovine serum albumin. The a n t i b o d y had a h i g h a f f i n i t y (Ka = 1.2 x 109 L/mol) and s p e c i f i c i t y f o r v i n b l a s t i n e . The u s a b l e range o f s t a n d a r d curve f o r a s s a y was 0.5-10 ng/ml. Crude a l k a l o i d e x t r a c t s of t i s s u e c u l t u r e s c o u l d be assayed and many samp l e s could be p r o c e s s e d i n one time. The v i n b l a s t i n e cont e n t s o f m u l t i p l e shoot c u l t u r e s were lower t h a n t h a t of i n t a c t p l a n t s b u t much h i g h e r t h a n t h a t of c a l l u s c u l t u r e s . Another Radioimmunoassay method was r e p o r t e d f o r v i n b l a s t i n e and v i n c r i s t i n e a s f o l l o w s ( 1 2 9 ) : Radioimmunoassay developed f o r d e t e r m i n i n g t h e neoplasm i n h i b i t o r s v i n b l a s t i n e ( I ) and v i n c r i s t i n e (11) i n blood i n v o l v e s t h e u s e of antiserum r a i s e d i n a r a b b i t immunized with ( I ) bovine

VINBLASTINE SULFATE

649

serum albumin c o n j u g a t e . D e t e c t i o n l i m i t s (ng ml-1) o r 2 . 1 f o r ( I ) and 3.8 f o r (11) w i t h u s e o f t r i t i a t e d ( I ) under n o n - e q u i l i b r i u m a s s a y c o n d i t i o n s . The a n t i s e r u m showed no c r o s s - r e a c t i v i t y w i t h 25 o t h e r a l k a l o i d s and c y t o t o x i c drugs used t h e r a p e u t i c a l l y i n combination w i t h ( I ) and (11).
A t h i r d method o f Radioimmunoassay for Vinca a l k a l o i d s v i n b l a s t i n e and v i n c r i s t i n e was r e p o r t e d ( 130) a s f o l l o w s :

A n t i s e r a were r a i s e d i n r a b b i t s by immunisation a g a i n s t compounds p r e p a r e d by c o u p l i n g c a r b o x y l i c a c i d d e r i v a t i v e s of v i n b l a s t i n e and v i n c r i s t i n e t o human serum albumin. For a s s a y , a n t i s e r u m was i n c u b a t e d with t h e sample, e.g. p l a n t e x t r a c t and t h e a p p r o p r i a t e t r i t i a t e d a l k a l o i d f o r 1 h a t 37', and t h e m i x t u r e was allowed t o r e a c t w i t h a goat a n t i - r a b b i t serum o r w i t h polyoxyethylene g l y c o l o v e r n i g h t a t 4', and t h e n c e n t r i f u g e d ; t h e p r e c i p i t a t e was d i s s o l v e d i n NaOH s o l u t i o n f o r s c i n t i l l a t i o n c o u n t i n g . E i t h e r compound could be determined i n amounts down t o <1 pmol. The s p e c i f i c i t y o f t h e a n t i s e r a i s d i s c u s s e d ; t h a t r a i s e d a g a i n s t v i n c r i s t i n e bound t h i s a l k a l o i d 200 times more e f f e c t i v e l y t h a n it bound v i n b l a s t i n e . S e v e r a l o t h e r compounds, i n c l u d i n g a n t i n e o p l a s t i c d r u g s , showed no c r o s s r e a c t i v i t y . The a s s a y s were a p p l i e d t o t h e blood o f rabbi t s i n j e c t e d w i t h t h e d r u g s , and a l s o t o e x t r a c t s o f Vinca r o s e a a f t e r p r e l i m i n a r y f r a c t i o n a t i o n by HPLC.
A s e n s i t i v e radioimmunoassay method f o r v i n b l a s t i n e and v i n c r i s t i n e was a l s o r e p o r t e d a s f o l l o w s ( 131 ) : Antiserum which was used was r a i s e d i n r a b b i t s a g a i n s t a 4 - d e a c e t y l v i n b l a s t i n e carboxazide-bovine serum albumin c o n j u g a t e ; [3H] - v i n c r i s t i n e o r r3H] - v i n b l a s t i n e was used as r a d i o - l i g a n d . Rates of b i n d i n g of v i n c r i s t i n e and v i n b l a s t i n e t o t h e a n t i s e r u m were s i m i l a r . A f t e r incubat i o n f r e e and bound l i g a n d were s e p a r a t e d w i t h u s e o f a dextran - charcoal suspension; a f t e r c e n t r i f u g a t i o n t h e a c t i v i t y of t h e s u p e r n a t a n t s o l u t i o n was measured by l i q u i d s c i n t i l l a t i o n c o u n t i n g . S e n s i t i v i t y was improved by a s e q u e n t i a l s a t u r a t i o n procedure ( i n c u b a t i o n w i t h u n l a b e l l e d d r u g , followed by i n c u b a t i o n with r a d i o l i g a n d ) . O f t h e drugs t e s t e d , only bleomycin ( > 0 . 1 u n i t ) i n t e r f e r e d .

650

FARlD J. MUHTADI AND ABDUL FATTAH A. A. AFlFY

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ACKNOWLEDGEMENT The a u t h o r would l i k e t o thank Mr. Uday C . Sharma, Dept. o f Pharmacognosy, College of Pharmacy, Riyadh, Saudi Arabia f o r h i s v a l u a b l e and s i n c e r e e f f o r t s i n t y p i n g t h i s manuscript.

TITANIUM DIOXIDE

Harry C. Brittain, Gary Barbera, Joseph DeVincentis, and Ann W. Newman

Bristol-Myers Squibb Pharmaceutical Research Institute Bristol-Myers Squibb Company New Brunswick, NJ 08903

ANALYTICAL PROFILES OF DRUG SUBSTANCES AND EXClPlENTS - VOLUME 21

659

Copyright 0 1992 by Academic Press, Inc. All rights of reproduction reserved in any form.

660

HARRY G . BRlTTAlN ET AL.

CONTENTS
1.

Description 1.1 1.2 1.3 1.4

Name, Formula, and Molecular Weight Appearance General Chemical properties Uses and Applications

2.

Method of Preparation

3.

Physical Properties 3.1 Particle morphology 3.2 Crystallographic Properties 3.3 Thermal methods of analysis 3.4 Particle size distribution 3.5 Surface area 3.6 Density 3.7 Powder Flow characteristics 3.8 Hygroscopicity 3.9 Solubility 3.10 Spectroscopy

4. Methods of Analysis 4.1 Compendia1 Tests 4.2 Identification 4.3 Elemental Analysis 4.4 Spectrophotometric Methods of Analysis Thin Layer Chromatographic Methods of Analysis 4.5 Gas-Liquid Chromatographic Methods of Analysis 4.6 4.7 High Performance Liquid Chromatographic Methods of Analysis 5. Stability 5.1 5.2
Stability Incompatibilities with functional groups

6. References

TITANIUM DIOXIDE

66 1

1.

Description Name. Formula. and Molecular Weight

11 .

Titanium dioxide is the most stable oxide of titanium, and can be obtained from either natural or synthetic sources. The material exists naturally in three crystal modifications, known as rutile, anatase, and brookite [I]. The Chemical Abstracts identification number is CAS- 13463-67-7. In the United States, it is identified in the Color Index as 77891, and is denoted as C.I. Pigment White No, 6. It is also identified as EEC No. E 171 [2]. The chemical formula is TiO,, which corresponds to a formula weight of 79.90 Daltons. The elemental composition is Ti 59.95% and 0

40.05% .

1.2

Appearance

Naturally occurring titanium dioxide may appear red, or reddish brown to black. The color is normally due to the presence of iron, chromium, or vanadium contamination, which may amount to 10% of the total titanium content [3]. Synthetically purified titanium dioxide exists as a white, odorless, tasteless powder.

L3

General Chemical Properties

A very detailed description of the chemistry associated with titanium oxygen systems is available [4]. The material is very thermally stable, and extremely resistant toward chemical degradation. It can be partially reduced when heated in the presence of hydrogen or carbon monoxide, the products being either lower oxides or mixtures of titanium carbide and lower oxides. Reduction by active metals (Na, K, Ca, or Mg) can

662

HARRY G . BRIITAIN ET AL.

only be partially effected, and chlorination of the oxide phase is possible only in the presence of a reducing agent. The reactivity of titanium dioxide toward acids is very dependent on the temperature of the reaction mixture. It may be slowly dissolved by boiling concentrated sulfuric acid, with the dissolution rate being promoted by the addition of ammonium sulfate. Titanium dioxide may be readily dissolved by hydrofluoric acid. The material is completely insoluble in aqueous alkalies, but is readily dissolved in molten sodium (or potassium) hydroxide, carbonate, or borate. An equimolar molten mixture of sodium carbonate and sodium borate is partially effective as a dissolution medium.

The most important use of titanium dioxide is as a white pigment, owing to its very high reflectance at visible and ultraviolet wavelengths [ 5 ] . The refractive index of this material is so extremely high that fine particles scatter light with almost total efficiency. At the same time, the films are almost totally opaque. The ability of industry to produce titanium dioxide in appropriate particle size ranges has made it the most important white pigment in existence.
In ointments or lotions, titanium dioxide is a very efficient reflector of sunlight [6]. Its ability to act as a sunblock has led to its widespread use as a protective agent toward sunburn.

2.

Method of Preparation

Although titanium dioxide can be obtained naturally as one of three crystal polymorphs (anatase, rutile, or brookite), pharmaceutically acceptable material is produced synthetically. The bulk of pure titanium dioxide is obtained from purification of the abundant ore, illmenite (FeTiO,), using the su&m process [7]. In this

TITANIUM DIOXIDE

663

procedure, the raw ore is first digested with concentrated sulfuric acid. The product of this step is termed the "sulfate cake", which is then leached with water to produce a mixture of FeSO,, Fe;?(SOJ,, and TiOSO,. At this point, scrap iron is used to reduce all Fe(IT1) to Fe(II), whereupon the FeSO, is removed by filtration. The TiOSO, solution is boiled to hydrolyze the solute into a suspension of hydrated TiO,. This material is filtered, and finally calcined at 800-900C to produce the final product. When calcined at 8OO0C, hydrous oxide the normally converts to the anatase phase. The other important process for production of titanium dioxide is termed the chloride process [7]. The raw material used in this process is natural rutile, which is first heated at 950C in the presence of carbon (in the form of coke) and chlorine. This produces crude TiCI,, and this product is heated at 1 O " in the presence of oxygen to O OC produce the final titanium dioxide product. Under these conditions, the final product is the rutile phase. The chloride process accounts for over half of the TiOz production in the United States, and is the most economical when high-grade ores are available. The sulfate process cannot use rutile as its starting material, but is able to make use of low-quality ores (or slags remaining after iron processing is complete) as input materials. The physical properties of the titanium oxide pigments can be further improved by slurrying the products in water, and then selectively precipitating a surface coating of either S i 9 , A1203, or TiO, itself on the fine particles.

3.

Physical Properties

A concise summary of the physical properties of titanium dioxide is available in the Handbook of Excipients [S]. Also summarized in this publication are commercial availability, methods of manufacture, and various pharmacopeial specifications.

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HARRY G. B R I T A I N ET AL

For the present work, three representative samples of U.S.P. grade titanium dioxide were characterized. Two of these were obtained from Warner Jenkinson, and were described as being either oil ("Atlas White" material, lot 402239) or water ("Kowett" material, lot 401398). The third lot was obtained from Spectrum Chemical Co., and was also certified to be U.S.P. grade (lot HB098). In addition to these U.S.P. materials, a non-U.S.P. grade of titanium dioxide (obtained from Johnson Matthey, lot MTI50W) was also studied as part of the crystallographic characterization.

Photomicrographs of the titanium dioxide samples are shown in Figures 1-4. All materials were found to exist as aggregate species which were built up from the consolidation of exceedingly fine subparticles. When viewed at 2O,OOOx, the subparticles of the U.S.P. materials are uniformly round in nature, and appear to be approximately 100 nm in diameter. No difference in the subparticle size was noted for materials prepared for different applications, or obtained from alternate vendors. This would indicate that the subparticle size was determined by the method of manufacture. The aggregates formed from these subparticles were very compact in nature, indicating efficient close-packing of the subparticles. The particle size of the aggregate species did vary among vendors, and this difference will be discussed in a different section. The subparticles of the non-U.S.P. grade material were found to be much coarser. The size distribution of these was also quite variable, but averaged approximately 1 pm in diameter. These subparticles were also spherical in their appearance, and the aggregate species formed from these appeared to be looser than those of the U.S.P. materials.

Titanium dioxide is known to crystallize in three polymorphic forms, anatase, brookite, and rutile. While the rutile and anatase polymorphs are commonly encountered, the brookite phase is quite rare. The

TITANIUM DIOXIDE

665

Figure 1.

Scanning electron photomicrographs of U.S.P. grade titanium dioxide ("Atlas White" material, Warner Jenkinson), obtained at 500x (upper photo) and 3 0 0 0 ~ (lower photo).

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HARRY G . BRIlTAIN ET AL.

Figure 2.

Scanning electron photomicrographs of U.S.P. grade titanium dioxide ("Kowett" material, Warner Jenkinson), obtained at 500x (upper photo) and 3OOOx (lower photo).

TITANIUM DIOXIDE

661

Figure 3.

Scanning electron photomicrographs of U . S .P. grade titanium dioxide (Spectrum Chemical Company), obtained at 505x (upper photo) and 3OOOx (lower photo).

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HARRY G.BRI'ITAIN ET AL.

Figure 4.

Scanning electron photomicrographs of non-U .S.P. grade titanium dioxide (Johnson Matthey), obtained at SoOx (upper photo) and 3000~ (lower photo).

TITANIUM DIOXIDE

669

relative stabilities of rutile and anatase are almost equivalent, but it appears that rutile is the most stable polymorph of the two. The crystal structures of these three polymorphs are well known, and the important properties summarized in Table I 191. Although anatase and rutile are both tetragonal, they are not isomorphous. Anatase is usually obtained in near-regular octahedra, which has given it the alternate name of octahedrite. Rutile is found as slender prismatic crystals, which often grow as twins. The rutile structure has been discussed in great detail, since it is commonly taken as one of the prototype crystal structures [9]. The unit cell is depicted in Figure 5. The structure consists of chains of TiO, octahedra, in which each octahedron shares a pair of opposite edges and vertices with neighboring octahedra. Another way to envision the overall structure is to consider it as a slightly distorted hexagonally close packed array of oxygen atoms with half the octahedral interstices being occupied by titanium atoms. Powder x-ray diffraction can be used to easily differentiate between the polymorphs of titanium dioxide. For the anatase phase, the most intense diagnostic scattering peaks correspond to d-spacings of 3.52 and with relative intensities of 100:4. For rutile, the most intense 1.89 scattering peaks correspond to d-spacings of 1.69, 3.26, and 2.49 and exhibit relative intensities of 100:97:70. Although brookite is never encountered in synthetically produced samples of titanium dioxide, its three most intense scattering peaks would correspond to dspacings of 3.47, 2.90, and 1.88 A (relative intensities of 100:85:75).

A,

A,

The U.S.P. grade of titanium dioxide most commonly marketed is the anatase phase. The x-ray powder pattern for this material is shown in Figure 6, while the scattering angles, d-spacings, and relative intensities are found in Table 11. For comparison purposes, a powder pattern obtained for the rutile phase (the non-U.S.P. material described earlier) is shown in Figure 7, and its corresponding crystallographic information is presented in Table 111.

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HARRY C . B R I T A I N ET AL.

Table I Crystallographic Data for the Three Polymorphs of Titanium Dioxide Property Crystal system Space group Number Ti02 in unit cell Anatase orthorhombic Brookite tetragonal Pcab Rutile tetragonal P4/mnm
2

14, 4

8
0.5456 0.9182 0.5143 257.6 2.583 2.586 2.741 4.1 19

Cell dimensions (nm) a 0.3785 b C 0.9514 Cell volume (mL x i d 4 )

0.4594 0.2962 62.5 2.616 2.903 4.245

136.3

anisotropic refractive indices "1 2.554 n2 n3 2.493 density (glmL)

3.893

TITANIUM DIOXIDE

67 I

Figure 5 .

Structure of the unit cell of titanium dioxide, rutile phase.

- l

r\

@0
/

I I
I I

Oo

@ Ti

612

HARRY G.BRITTAIN ET AL.

Figure 6.

Powder x-ray diffraction pattern of titanium dioxide, anatase phase. The intensity scale is presented in arbitrary units.

0.0

10 .a

20.0

3 . 0 0

40.0

50.0

4
60.0

1
70.0

Degrees 2-8

TITANIUM DIOXIDE

673

Table II Crystallographic Data for the 10 Most Intense Scattering Peaks of Titanium Dioxide, Anatase Phase Angle (degrees 2-0) D-Spacing (Angstroms) Relative Intensity
wnax)

25.2625 36.9150 37.7600 38.5500 48.0100 53.8500 55.0200 62.6150 62.82OO 68.6800

3.5226 2.4330 2.3805 2.3335 1.8935 I .7011 I .6677 1.4824 1.4817 I .3655

100.00 7.12 27.59 8.41 42.40 28.61 28.32 23.58 11.98 11.14

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HARRY G . BRI'ITAIN ET AL.

Figure 7.

Powder x-ray diffraction pattern of titanium dioxide, rutile phase. The intensity scale is presented in arbitrary units.

0.0

10.0

20.0

3 .O 0

4 .O 0

50 .O

6 .O 0

70.0

Degrees 2-8

TITANIUM DIOXIDE

675

Table 111 Crystallographic Data for the 1 1 Most Intense Scattering Peaks of Titanium Dioxide, Rutile Phase Angle (degrees 2-8) 27.3025 36.0200 39.0275 41.1400 43.8625 54. I700 56.3075 62.7175 63.8100 68.7850 69.7850 D-Spacing (Angstroms) 3.2638 2.4914 2.3061 2.1924 2.0624 1.6918 1.6325 1.4802 1.4575 1.3637 1.3466 Relative Intensity (1 4naJ 97.07 69.69 4.64 37.65 10.34 100.00 23.69 23.69 13.60 37.29 30.43

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HARRY G . BRIITAIN ET AL

The rutile polymorph of titanium dioxide is refractory, and melts around 1850C. Melting points for the anatase and brookite polymorphs have not been established, since these materials convert to the rutile phase at elevated temperatures.

34 .

Particle size distribution

The particle size distributions of the three U.S.P. titanium dioxide samples were obtained using optical microscopy and image analysis. This method provides information on the size of the aggregate species only, and does not contain any data pertinent to the subparticles from which the aggregates are composed. Full particle size distributions for these three Ti@ lots are provided in Table IV. The particle diameters were obtained from analysis of the particle cross-sectional areas (provided by the image analysis system). All materials were found to be composed of very small particles, the basic units of which were smaller than 10 pm. The average particle size (shown in Table V) of the two Warner Jenkinson lots were equivalent, while the material obtained from Spectrum Chemical Co. was slightly coarser in nature.

3 2

s rface a r a u

The surface area of the three titanium dioxide lots was determined using a five-point B.E.T. analysis procedure, and the results of this study are found in Table V. The three materials exhibit fairly high surface areas (approximately 10 m2/g), and were found to be mutually equivalent.

The true density (measured by helium pycnometry) of titanium dioxide differs with the polymorphic state of the material. Rutile is the most dense (4.25 g/mL), followed by brookite (4.12 g/mL) and anatase (3.89 g/mL).

TITANIUM DIOXIDE

611

Table IV Particle Size Distributions for Various Titanium Dioxides, Obtained Using Optical Microscopy (aggregate species having diameters larger than 10 pm have been excluded) Band Size
(Pm)

401398

402239

HB098

0.0 - 0.5 . 0 5 - 10 . l . 0 - 1.5 1.5 - 2.0 . 2.0- 2 5 2 5 - 3.0 . 3.0- 3.5 3.5 - 4.0 4.0- 4.5 45- 50 . . 5.0 - 5.5 5.5 - 6.0 . 6.0 - 6 5 65- 7 0 . . 7.0 - 7.5 7.5 - 8 0 .

12.7 45.4 26.5 8.2 4.0 l .3 1.1


0.0 0.5
0.0 0.0 0.0 0.0 0.0 0.0

0.3

9.6 52.2 2. 67 5.4 36 . 1.1 0.0 0.5 0.0 0.4 0.2 0.0 00 . 0.2 0.0 0.0

8.0 1. 54 1. 41 14.6 1. 49 7.5 8.8 5.3 4.8 16 . 0.5 1.9 08 . 0.8 00 .


1.1

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HARRY 0. BRITTAIN ET AL.

Table V Micromeritic Properties Obtained for Various Titanium Dioxides

property

401398

402239

HB098

Average Particle Size (pm) Surface Area

1.05

1.02

2.19

(m2/s)

10.5

8.2

9.0

Bulk Density (g/mL) Tap Density

0.4

0.4

0.5

0.7

0.6

0.8

Compressibility 39 37

35

TITANIUM DIOXIDE

679

The bulk densities of the commercially supplied U.S.P. grade materials were found to average around 0.4 g/mL (Table V). The tap densities of these lots were found to increase to approximately 0.7 g/mL, corresponding to a compressibility factor of approximately 37%. No significant difference among the three lots studies was evident when comparing the micromeritic properties.

37 HVgroscoDicin! .
Titanium dioxide is not hygroscopic, and does not form true hydrate phases. It is possible to prepare hydrated titanium oxide materials through the addition of alkali-metal hydroxides to a solution of a Ti(I1) or Ti(II1) salt. The resulting titanium hydroxide precipitate is extremely unstable, is a powerful reducing agent, and rapidly converts to a hydrated oxide material [4].
If the precipitation is performed at room temperature, one obtains a compound known as orthotitanic acid, and which has the approximate formula of Ti0;2H20Ti(0H),. If the suspension is boiled, or if the precipitation is effected from a hot solution, a compound known as metatitanic acid is obtained. This less hydrated oxidic compound has the approximate formula of TiOz-H20TiO(OH)2,Metatitanic oxide is commonly obtained in the colloidal state, and is the preferred intermediate in the manufacture of titanium dioxide pigments.

Solubility

Titanium dioxide is completely insoluble in water, dilute acids, or common organic solvents. It can be dissolved in concentrated sulfuric or hydrofluoric acids at elevated temperatures, with the accompanying production of salt species.

322

Spectroscopy

Titanium dioxide transmits through the visible and near infrared regions

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HARRY G. BRlTTAlN ET AL.

of the spectrum, having no absorption bands in these regions. It becomes completely opa ue at wavelengths below 400 nm, and at energies below 2000 cm- . The reflectivity of TiO, is approximately 90% of that of a MgO standard [4].

4. Methods of Analysis

41 .

Compendia1 Tests

The U.S.P. compendia1 requirements [ 101 for titanium dioxide are that it cannot contain less than 99.0% and not more than 100.5% TiO,, when calculated on a dried basis. In addition, the material may not contain more than 0.001 % of lead, not more than 2 ppm of antimony, and not more than 1 ppm of mercury. The compound is tested as to its identification, loss on drying, loss on ignition, water-soluble substances, acid-soluble substances, and arsenic content. A full method is provided for the potency assay. The details of these tests are as follows: Identification: The compound is suspended in hot concentrated sulfuric acid, and diluted with water. Undissolved solid is filtered off, and a few drops of hydrogen peroxide test solution are added to the clear filtrate. The positive identification consists of an orange-red color which develops immediately.

Loss on drying: The general test method <731> is followed. After being dried at 105C for 3 hours, the material cannot lose more than 0.5% of its weight.

Loss on ignition: Following general test method <733 > , the material is ignited at 800 k 25C to constant weight. The material cannot lose more than 0.5% of its weight.
Water-soluble substances: The sample is suspended in water, mixed, and allowed to stand overnight. Ammonium chloride

TITANIUM DIOXIDE

68 1

test solution is used to clarify the suspension, which is then filtered. A 100 mL portion of the filtrate is collected, dried, and ignited to constant weight. The residue cannot amount to more than 0.25% of the original sample weight.

Acid-soluble substances: The solid is suspended in 0.5 N HCI, and heated on a steam bath. The suspension is filtered, with the filtered solids being washed with 0.5 N HCI. These washings are combined with the original filtrate, dried, and ignited to constant weight. The residue cannot weigh more than 0.5% of the original sample weight. Arsenic: The arsenic content is determined according to general test <211> . The solid is suspended in water, to which is added appropriate amounts of hydrazine sulfate, potassium bromide, sodium chloride, and sulfuric acid. Any evolved arsine is collected, and determined. The limit is 1 ppm. Assay: The initial sample is dissolved in a mixture of hot sulfuric acid and ammonium sulfate. After the dissolution is complete, the mixture is allowed to cool, and diluted with water. The suspension is then filtered, and neutralized with ammonium hydroxide. This filtrate is reduced in a Jones reductor (making use of a zinc amalgam), and then titrated with 0.1 N potassium permanganate volumetric reagent. IJnder these conditions, each mL of 0.1 N potassium permanganate reagent is equivalent to 7.988 mg of TiO,.

All identification tests for titanium dioxide first require solubilization of the oxide, typically by concentrated acid solutions. After production of aqueous solutions of titanium ions, a number of colorimetric reactions may be used for identification purposes.

Hydrogen peroxide causes a yellow color to develop in acidic solutions containing dissolved titanium [111. In solutions containing sulfuric

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HARRY G . BRITTAIN ET AL.

acid, the color is due to a complex peroxidic anion formed from free peroxodisulfatotitanic acid. This reaction is the one used for the cornpendial identification test since it is not interfered with by common metallic ions, Several other color reactions may be used for the identification of titanium dioxide, after solubilization of the material. Pyrocatechol yields a yellowish-red color with weakly acidified solutions of titanium salts [12]. When chromotropic acid is reacted with solubilized titanium between pH 2.5 and 5.0, wine red colored solution is obtained 1131. a An intense brown color is given by the addition of acidic solutions of 3,5,7,2,4-pentahydroxyflavone (morin) to titanium salts, although the composition of the product is unknown [ 141, Disodium- 1,2-dihydroxybenzene-3,5-disulfonate(tiron) yields a strong yellow color when reacted with titanium salts in the pH range of 4.39.6 [15]. Titanium salts also react with 5-sulfosalicylic acid at pH 3-5 to yield yellow solutions [16]. Other reagents which have found limited use in identification testing are thymol [17], gallic acid [18], and salicylic acid [ 191.

4J

Gravimetric Methods of Analysis

As one of the transition elements, titanium can be readily precipitated with ammonium, sodium, or potassium hydroxide. The hydrated oxide is ignited to constant weight at any temperature exceeding 350C. The method is not useful for complicated samples containing other cations which could be precipitated by hydroxide, but is very applicable to the analysis of U.S.P. grade titanium dioxide. Titanium salts can also be determined using cupferron (the ammonium salt of nitrosophenylhydroxylamine) [20] or tannin [21]. In addition, 2-hydroxy-4-methylpropiophenone oxime has been shown to form an insoluble 1:l complex with Ti salts [22].

TITANIUM DIOXIDE

683

4.4

Titrimetric Methods of Analysis

Most titrimetric methods for titanium depend on the reduction of Ti(1V) to Ti(III), followed by subsequent titration with a standard oxidizing solution [23]. The methods vary with the choice of reductant, the titrant, and with the method of detecting the endpoint. The standard dissolution procedure for titanium oxides is the ammonium sulfate-sulfuric acid mixture developed by Rahm [24]. The most commonly used method in industry is based on the use of metallic aluminum as the reductant, and ferric ammonium sulfate as the titrant. The use of ferric ion as the reagent is preferred, since relatively few species will interfere with its reaction with reduced titanium solutions. Other reductor systems can be used, which will yield equally satisfactory results [25]. These can be the Jones, lead, cadmium, iron, nickel, or bismuth reactors, with the Jones reactor being chosen for use in the compendia1 assay method. Liquid mercury amalgams can also be used as reductors, being prepared with zinc, cadmium, bismuth, lead, or tin. While the liquid amalgams are easier to handle, and are more rapid than are column reactors, none of these is as simple as the aluminum foil reductor. Besides ferric ammonium sulfate, permanganate solutions can be used to titrate the reduced titanium. Although many cations interfere with the permanganate titration, this reagent is still useful in the assay of highly purified titanium dioxide materials. Potassium dichromate can be used to titrate Ti(II1) solutions (with the aid of 0.2% indigo as the indicator), and Ce(1V) reagents can also be used as titrants.

Ethylenediaminetetraacetic acid can be used as a reagent for the titration of Ti(1V). However, the reaction proceeds slowly, and the Ti(1V) species tend to hydrolyze during the titration, so that a back-titration method is necessary to make the complexometric method work properly [261.

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HARRY G.BRI7TAIN ET AL.

Polarographic Methods of AM lysis

It has been demonstrated that the half-wave potential for the reduction of Ti(1V) to Ti(I1I) is -0.81 V (against the standard calomel electrode) in O.1M HCI [27]. The further reduction of Ti(II1) to Ti(l1) can be observed in alkaline media, but this reaction has no useful analytical significance. In these methods, oxalate, tartrate, or citrate buffer systems are used as supporting electrolytes to prevent the hydrolytic precipitation of hydrated titanium oxides. In the presence of tartrate buffer, well defined waves are obtained only at pH values less than 2, or between 6 and 7. The Ti(1V)-Ti(II1) couple is reversible only in tartrate buffer at pH values less than 1. When 0.4M citrate ion is used as the medium, the reduction is well defined at all pH values, but the reduction potential was found to vary with the solution pH [27]: PH
0.0 3.0

E,,,

V.S.

S.C.E.
-0.28 -0.80 -0.95 -1.49

11.5

7.0

Ethylenediaminetetraacetic acid has been found to be satisfactory as a supporting electrolyte [28]. It was demonstrated that the half-wave potential for a 0.4M solution of Ti(1V) in 0.25N EDTA varied from 0.22 V (vs. S.C.E.) at pH 3.0 to -0.82 V at pH 8.7. The half-wave potential was found to be independent of the solution acidity below pH 2.
Ti at a semistationary mercury drop electrode has been used for the direct determination of Ti [29]. This method has the distinct advantage of being able to tolerate large quantities of metal ion impurities. In another method, Ti salts were chelated with dihydroxyazo dyes, adsorbed onto a hanging mercury drop electrode, and then determined
AC voltammetry using the fundamental and second harmonic wave of

TITANIUM DIOXIDE

685

by cyclic voltammetry [30]

4.6

Atomic Spectroscopic Methods of Analysis

Historically, emission spectrography has been very important in the quantitative assay of titanium-containing materials. It should be pointed out that where Ti is a major constituent of the material (as would be the case for titanium dioxide), the spectrophotometric and titrimetric methods of analysis are more appropriate. Nevertheless, a variety of quantitative methods have been developed for the determination of Ti in oxidic phases. After spark-source excitation, the Ti lines at 3239.04 or 3349.41 A are determined against an internal standard. The 3239.04 Ti line is normally quantitated against the 3232.61 line of Li, while the 3349.41 Ti line is quantitated against the 3126.11 A line of Cu.

After dissolution of the oxide, Ti may be directly determined by atomic absorption using either the nitrous oxide-acetylene [3I] or oxygennitrogen-acetylene [32] flame systems. The method is straight-forward, and no interference has been noted from Cr, Co, Mn, Mo, Nb, W, Ta, or Cu [33]. An improvement in the determination of Ti using the nitrous oxide-acetylene flame system was noted when the analysis was performed in a buffered HF-boric acid mixture [34].

X-ray fluorescence has been found to be useful in the quantitation of titanium oxides, with internal standards also being used [35]. The and differ in methods all make use of the Ti K a emission at 2.750 their choice of internal standards.

A,

Atomic absorption spectroscopy has been used to determine the trace quantities of other metal contaminants in titanium dioxide pigments [36]. Auger electron spectroscopy has been used to directly determine the levels of Ti in oxide layers [37].

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HARRY G.BRITTAIN ET AL.

4.7

Spectrophotometric Methods of Analysis

Theoretically, any colorimetric method useful for identification purposes can be developed into a quantitative spectrophotometric assay. The number of reagents proposed for use in photometric assay methods is extensive, and interested readers should consult appropriate review articles. Although many of these reagents have been developed for use in areas relating to nonferrous metallurgy [38], they may be applied to the spectrophotometricassay of Ti in TiO,. Assays involving spectrophotometric reagents probably represent the most extensive range of methods developed for determination of Ti in any sample matrix. The peroxide method has proven to be the most useful for this purpose, owing to the high acidity of the medium in which the reaction is conducted. Interferences are observed only in the presence of V, Mo, or F, but these species are not normally present in U.S.P. grade titanium dioxide. In the spectrophotometric assay method, the absorption maximum at 410 nm is used to determine the titanium concentration after the oxide is dissolved [39]. The spectrophotometric endpoint of the peroxide method has been combined with flow injection analysis techniques to yield an automated procedure 1. Reagents containing oxygen-donor atoms (phenolic or alcoholic hydroxy groups) are most suitable as spectrophotometric reagents, but nitrogendonor functional groups can also be used. A detailed review of photometric reagents for Ti has been written by Sommer [41]. The yellow colors developed by titanium salts with tiron [15] and sulfosalicylic acid [16,42] have also been used to develop quantitative spectrophotometricassay methods. Other useful reagents include tichromin and dibromotichromin [43], chromotropic acid [MI, chlorophosphonazo I [45], and diantipyrylmethane [46]. The diantipyrylmethane reagent has also been used to measure Ti salts after these have been stripped off a silica gel column [47].

TITANIUM DIOXIDE

687

4J3

Chromatographic Methods of Analvsis

Chromatographic methods for titanium dioxide have not been investigated vigorously, owing to the wealth of alternate methods available. Spectrophotometric or atomic spectroscopic measurements can be conducted more rapidly, are more sensitive, and are not affected by the oxidation state of the metal. A chromatographic method would be appropriate only if the Ti species was to be separated from a complicated sample matrix, but this is not anticipated in the analysis of U.S.P. titanium dioxide.

In addition, Ti salts are prone to hydrolysis, and ultimately form insoluble hydrated oxides. For a chromatographic procedure to be developed, the eluent would have to contain strongly coordinating agents capable of preventing hydrolysis. Although such agents have been proposed for the ion chromatographic assay of other transition elements [48], they do not appear to have been applied to the analysis of titanium salts.
5. Stability Stability Titanium dioxide reacts only with hot, concentrated mineral acid solutions. It is completely stable with respect to light, oxidation, changes in pH of suspensions, and microbiological attack. It has been found to be stable at all temperature values up to its melting point (1850C). If heated strongly under vacuum, there is a slight loss of oxygen corresponding to a change in composition to TiO,.,. This product is dark blue, but reverts to the original white color when heated in air. Incompatibilities with functional srouDS Titanium dioxide is chemically unreactive, and its only chemistry takes place after the oxide has been dissolved in strong acids. It is a known

688

HARRY G . BRlTTAlN ET AL

catalyst, however, and is capable of inducing solid state chemical reactions under certain conditions. However, these reactions require the application of temperatures exceeding lOO"C, which should not be encountered in the normal range of pharmaceutical formulations.

6.

References
F.A. Cotton and G . Wilkinson, "Advanced Inorganic Chemistry", 4Ih ed., John Wiley & Sons, New York, 1980, pp.

I.

695-708. 2.

L.C. Pakenham and E.G. Murphy, "CTFA International Color Handbook, Cosmetic, Toiletry, and Fragrance Assoc., Washington, D.C., 1985, pp. 463-468.

3. 4.
5.

C.S. Hurlbut, " a a s Manual of Mineralogy", IS* ed., John Dn' Wiley & Sons, New York, 1949, pp. 200-201. "Kirk-Othmer Encyclopedia of Chemical Technology", John Wiley & Sons, New York, 1983, pp. 137-152.
A. Wade and J.E.F. Reynolds, "Martindale: The Extra Pharmacopoeia", 27"' ed., The Pharmaceutical Press, London, 1977, p. 458.
A.R. Gennaro, "Remington's Pharmaceutical Sciences", 17*

6.

ed., Mack Publishing Co., Easton, PA, 1985, p. 790.

7.
8.

N.N. Greenwood and A. Earnshaw, "Chemistry of the Elements", Pergamon Press, Oxford, 1984, pp. 11 17-1 126.

"Handbook of Pharmaceutical Excipients" , American Pharmaceutical Association, Washington, D.C., 1986, pp. 328330.

TITANIUM DIOXIDE

689

9.
10.

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CUMULATIVE INDEX

Bold numerals refer to volume numbers.

Acehutolol, 19. 1 Acetaminophen, 3, I; 14.551 Acetohexamide, 1.1; 2,573; I 21, Allopurinol, 7,1 Alpha-tocopheryl acetate, 3.111 Amantadine, 1 . 21 Amikacin sulfate, l2.37 Amiloride hydrochloride, IS, I Aminoglutethimide, 15,35 Aminophylline, 11, 1 Aminosalicylic acid, 10, 1 Amiodarone, 20.1 Amitriptyline hydrochloride, 3, 127 Amoharbital, 19,27 Amodiaquine hydrochloride, 21,43 Amoxicillin, 7,1 9 Amphotericin B,6,I; 7,502 Ampicillin, 2,I; 4.518 Apomorphine hydrochloride, 20,1 1 2 Ascorbic acid, ll,45 Aspirin, 8,1 Astemizole, 20,173 Atenolol, 13, 1 Atropine, 14,32 Azathioprine, 10,29 Azintamide, 18,1 Aztreonam, 17,l Bacitracin. 9,1 Baclofen, 14,527 Bendroflumethiazide, 5, I; 6,597 Benperidol, 14,245 Benzocaine, 12.73

Benzyl benzoate, 10.55 Betamethasone dipropionate, 6,43 Bretylium tosylate, 9.71 Bromazepam, 16.1 Bromocriptine methanesulfonate, 8,47 Bupivacaine, 19.59 Busulphan, 16.53 Caffeine, 15,71 Calcitriol, 8, 83 Camphor, 13,27 Captopril, ll, 79 Carbamazepine, 9,87 Cefaclor, 9,107 Cefamandole nafate, 9.125; 10,729 Cefazolin, 4,1 Cefotaxime, 11. 139 Cefoxitin, sodium, ll, 169 Ceftazidime, 19.95 Cefuroxime sodium, 20,209 Celiprolol hydrochloride, 20,237 Cephalexin, 4.21 Cephalothin sodium, 1,319 Cephradine, 5,21 Chloral hydrate, 2, 85 Chlorambucil, 16,85 Chloramphenicol, 4.47,518; 15,701 Chlordiazepoxide, 1, 15 Chlordiazepoxide hydrochloride, 1,39; 4,518 Chloroquine, 13,95 Chloroquine phosphate, 5, 6 1 Chlorothiazide, 18.33 Chloropheniramine maleate, 7.43

693

Chlorprothixene, 2, 63 Chlortetracycline hydrochloride, 8, 101 Chlorthalidone, 14.1 Chlorzoxazone, 16,119 Cholecalciferol, see Vitamin D, Cimetidine, U,127; 17, 797 Cisplatin. 14.77; L5,7% Clidinium bromide, 2,145 Clindamycin hydrochloride, 10.75 Clioquinol, 18.57 Clofazamine, 18,91 Clofazimine, 21,75 1 Clofibrate, 1 ,197 Clonazepam, 6,61 Clonidine hydrochloride. 21, 109 Clorazepate dipotassium, 4,91 Clotrirnazole, ll, 225 Cloxacillin sodium, 4, 113 Cocaine hydrochloride, 15,151 Codeine phosphate, 10,93 Colchicine, 10, 139 Cyanocobalamin, 10,183 Cyclandelate, 21,149 Cyclizine, 6, 83; 7, 502 Cyclobenzaprine hydrochloride, 17,41 Cycloserine, 1.53; 18,567 Cyclosporine, 16,145 Cyclothiazide, 1,66 Cypropheptadine, 9, 155 Dapsone, 5.87 Dexamethasone, 2,163; 4,519 Diatrizoic acid, 4, 137: 5,556 Diazepam, 1, 79: 4, 518 Dibenzepin hydrochloride, 9,181 Dibucaine and dibucaine hydrochloride, l2.105 Diclofenac sodium, 19, 123 Diethylstilbestrol, 19, 145 Diflunisal, 14,491 Digitoxin, 3.149 Digoxin, 9,207 Dihydroergotoxine methanesulfonate, 7 , 81 Diwtyl sodium sulfosuccinate, 2.199; 12,713 Diperodon, 6 , 9 9 Diphenhydramine hydrochloride, 3,173 Diphenoxylate hydrochloride, 7,149 Disopyramide phosphate, W , 183 Disulfiram, 4,168 Dobutamine hydrochloride, 8,139 Dopamine hydrochloride, 11,257 Doxorubicine, 9,245 Droperidol, 7, 171 Echothiophate iodide, 3, 233 Emetine hydrochloride, 10,289

Enalapril maleate, 16, 207 Ephedrine hydrochloride, 15, 233 Epinephrine, 7, 193 Ergonovine maleate, 11,273 Ergotamine tartrate, 6 , 113 Erythromycin, 8, 159 Erythromycin estolate, 1,101; 2,573 Estradiol, 15,283 Estradiol valerate, 4, 192 Estrone, 12, 135 Ethambutol hydrochloride, 7,231 Ethynodiol diacetate, 3,253 Etomidate, 12, 191 Etoposide, 18, 121 Fenoprofen calcium, 6, 161 Flecainide, 21, 169 Flucytosine, 5, 115 Fludrocortisone acetate, 3,281 Flufenamic acid, ll.313 Fluorouracil, 2,221: 18.599 Fluoxetine, 19, 193 Fluoxymesterone, 7,251 Fluphenazine decanote, 9,275; 10,730 Fluphenazine enanthate, 2,245; 4,524 Fluphenazine hydrochloride, 2,263; 4, 519 Flurazepam hydrochloride, 3,307 Folic acid, 19,221 Furosemide, 18, 153 Gentamicin sulfate, 9,295; 10,731 Glafenine, 21, 197 Glibenclamide, 10,337 Gluthethimide, 5, 139 Gramicidin, 8, 179 Griseofulvin, 8,219; 9,583 Guanabenz acetate, 15,319 Halcinonide, 8, 251 Haloperidol, 9 , 341 Halothane, 1,119; 2,573: 14,597 Heparin sodium, 12,215 Heroin, 10,357 Hexestrol, ll,347 Hexetidine, 7.277 Homatropine hydrobromide, 16,245 Hydralazine hydrochloride, 8,283 Hydrochlorothiazide, 10,405 Hydrocortisone, 12,277 Hydroflumethiazide. 7,297 Hydroxyprogesterone caproate, 4,209 Hydroxyzine dihydrochloride, 7,319 Impenem, 17.73 Imipramine hydrochloride, 14,37 Indomethacin, 13,211 Iodamide, 15,337 694

Iodipamide, 2,333 lodoxamic acid, 20,303 Ioparnidol. 17,115 Iopanoic acid, 14, 181 Iproniazid phosphate, 20, 337 Isocarboxazid, 2, 295 Isoniazide, 6,183 Isopropamide, 2,315; 12,721 Isoproterenol. 14,391 Isosorbide dinitrate, 4, 225; 5, 556 Ivermectin, 17,155 Kanamycin sulfate, 6, 259 Ketamine, 6,297 Ketoprofen, 10,443 Ketotifen, 13, 239 Khellin, 9, 371 Lactose, anhydrous, 20,369 Leucovorin calcium, 8,315 Levallorphan tartrate, 2,339 Levartereno1bitartrate, 1 ,4 9 ;2,573; 11,555 Levodopa, 5 , 189 kvothyroxine sodium, 5,225 Lidocaine base and hydrochloride, 14,207; 15, 76 I Lisinopril, 21, 233 Lithium carbonate, 15, 367 Lobeline hydrochloride, 19,261 Lomustine, 19.315 Loperamide hydrochloride, 19,341 Lorazepam, 9, 397 Lovastatin, 21, 277 Maprotiline hydrochloride, 15,393 Mebendazole, 16,291 Mefloquine hydrochloride, 14, 157 Melphalan, l3,265 Meperidine hydrochloride, 1, 175 Meprobamate, 1,209; 4,520; 11,587 6-Mercaptopurine, 7,343 Mestranol. ll, 375 Methadone hydrochloride. 3,365; 4,520; 9,601 Methaqualone, 4,245,520 Methimazole, 8,351 Methotrexate. 5, 283 Methoxamine hydrochloride, 20, 399 Methoxsalen, 9,427 Methyclothiazide, 5, 307 Methylphenidate hydrochloride, 10,473 Methyprylon, 2, 363 Metipranolol, 19, 367 Metoclopramide hydrochloride, 16.327 Metoprolol tartrate, 12,325 Metronidazole. 5.327 Mexiletine hydrochloride, 20,433 695

Minocycline, 6,323 Minoxidil, 17, 185 Mitomycin C, 16,361 Mitoxantrone hydrochloride, 17,221 Morphine, 17,259 Moxalactarn disodium, W , 305 Nabilone, 10,499 Nadolof, 9,455; 10,732 Nalidixic acid, 8,371 Naloxone hydrochloride, 14,453 Nalorphine hydrobromide, 18, 195 Naphazoline hydrochloride, 21,307 Naproxen. 21,345 Natamycin, 10,513 Neomycin, 8 ,3 9 9 Neostigmine, 16,403 Nicotinamide, 20,475 Nifedipine, 18,221 Nitrazepan], 9 ,4 8 7 Nitrofurantoin, 5, 345 Nitroglycerin, 9, 519 Nizatidine, 19,397 Norethindrone, 4,268 Norfloxacin, 20,557 Norgestrel, 4, 294 Nortriptyline hydrochloride, 1,233; 2,573 Noscapine, 11,407 Nystatin, 6,341 Oxamniquine, 20,601 Oxazepam, 3,441 Oxyphenbutazone, 13,333 Oxytocin, 10,563 Papaverine hydrochloride, 17,367 Penicillarnine, 10,601 Penicillin-G, benzothine, 11,463 Penicillin-G, potassium, 15,427 Penicillin-V, 1,249; 17,677 Pentazocine, W, 361 Pergolide mesylate, 21, 375 Phenazopyridine hydrochloride, 3 ,4 6 5 Phenelzine sulfate, 2,383 Phenformin hydrochloride, 4,319; 5,429 Phenobarbital, 7,359 Phenolphthalein, 20, 627 Phenoxymethyl penicillin potassium, 1,249 Phenylbutazone, l l .4 8 3 Phenylephrine hydrochloride, 3 ,4 8 3 Phenylpropanolarnine hydrochloride, 12,357; 13, 77 1 Phenytoin, 13,417 Physostigmine salicylate, 18,289 Phytonadione, 17,449 Pilocarpine, 12,385

Piperazine estrone sulfate, 5, 375 Pirenzepine dihydrochloride, 16,445 Piroxicam, 15,509 Polythiazide, 20,665 Pralidoxine chloride, 17,533 Prazosin hydrochloride, 18,361 Prednisolone, 21.415 Primidone, 2,409; 17,749 Probenecid, 10,639 h-ocainamide hydrochloride, 4,333 Procarbazine hydrochloride, 5,403 Promethazine hydrochloride, 5,429 Proparacaine hydrochloride, 6,423 Propiomazine hydrochloride, 2,439 Propoxyphene hydrochloride, 1,301; 4,520; 6, 598 Propylthiouracil, 6,457 Pseudoephedrine hydrochloride, 8,489 Pyrazinamide, 12,433 Pyridoxine hydrochloride, 13,447 Pyrimetharnine, 12,463 Quinidine sulfate, 12,483 Quinine hydrochloride, 12,547 Ranitidine, 15,533 Reserpine, 4,384;5,557; W,737 Riboflavin, 19,429 Rifampin, 5,467 Rutin, 12,623 Saccharin, 13,487 Salbutamol, 10,665 Salicylamide, 13,521 Scopolamine hydrobromide, 19,477 Secobarbital sodium, 1,343 Silver sulfadiazine, 13,553 Sodium nitroprusside, 6,487; 15,781 Sotalol, 21,501 Spironolactone. 4,431; 18,641 Streptomycin, 16,507 Strychnine, 15,563 Succinycholine chloride, 10,691 Sulfadiazine, ll.523 Sulfadoxine, 17,571 Sulfamethazine, 7,401 Sulfamethoxazole, 2,467; 4,521 Sulfasalazine, 5,515 Sulfisoxazole, 2,487 Sulfoxone sodium,19,553 Sulindac, 13,573 Sulphamerazine, 6,515 Sulpiride. 17,607 Teniposide, 19,575

Terazosin, 20.693 Terbutaline sulfate, 19,601 Terfenadine, 19,627 Terpin hydrate, 14,273 Testolactone. 5,533 Testosterone enanthate, 4,452 Tetracaine hydrochloride, 18,379 Tetracycline hydrochloride, W, 597 Theophylline, 4,466 Thiabendazole, 16,611 Thiamine hydrochloride. 18,413 Thiopental sodium, 21,535 Thioridazine and Thiondazine hydrochloride, 1 , 8 459 Thiostrepton, 7,423 Thiothixene, 18.527 Ticlopidine hydrochloride, 21,573 Timolol maleate, 16,641 Titanium dioxide, 21.659 Tolbutamide, 3,513;s.557;13,719 Trazodone hydrochloride, 16,693 Triamcinolone, 1,367; 2,571; 521,524; 4, ll,593 Triamcinolone acetonide, 1,397.416; 2,571; 4, 521; 7,501 ; U, 615 Triamcinolone diacetate, 1,423; 651 11, Triamcinolone hexacetonide, 6,579 Triclobisonium chloride, 2.507 Trifluoperazine hydrochloride, 9,543 Triflupromazine hydrochloride, 2,523; 4,521; 5, 557 Trimethaphan camsylate, 3,545 Trimethobenzamide hydrochloride, 2,551 Trimethoprim, 7,445 Trimipramine maleate. 12, 683 Trioxsalen, 10,705 Tripelennamine hydrochloride, 14,107 Triprolidine hydrochloride, 8,509 Tropicamide, 3, 565 Tubocurarine chloride, 7,477 nbamate, 4,494 Valproate sodium and valproic acid, 8,529 Verapamil, 17,643 Vidarabine, 15,647 Viblastine sulfate, 1,443; 21,611 Vincristine sulfate, 1,463 Vitamin D,, 13,655 Warfarin, 14,243 Xylometazoline hydrochloride, 14,135 Yohimbine, 16,731 Zidovudine, 24,729 Zomepirac sodium, 15.673

696