Chromatography of Pharmaceuticals - American Chemical Society

Department, MBC-03, King Faisal Specialist Hospital and Research. Centre, P.O. ... effects were reported during AG administration e.g., sedations, CNS...
0 downloads 0 Views 681KB Size
Chapter 9

Direct Enantiomeric Separation and Analysis of Some Aromatase Inhibitors on Cellulose-Based Chiral Stationary Phases Downloaded by PENNSYLVANIA STATE UNIV on August 2, 2012 | http://pubs.acs.org Publication Date: October 29, 1992 | doi: 10.1021/bk-1992-0512.ch009

Hassan Y. Aboul-Enein Drug Development Laboratory, Radionuclide and Cyclotron Operations Department, MBC-03, King Faisal Specialist Hospital and Research Centre, P.O. Box 3354, Riyadh 11211, Kingdom of Saudi Arabia

The stereoisomeric composition of pharmaceuticals is rapidly becoming one of the key issues in the development of new drugs. Several chiral stationary phases (CSP's) are now available to effect direct separation and analyses of drug enenatiomers and racemates. Cellulose CSP's are one of these commonly employed phases which have been successfully used in separation, enantiomeric purity determination and analysis of several aromatase inhibitors belonging to the glutarimide group namely aminogluthethimide (1), pyridogluthethimide (2), cyclohexylaminogluthethimide(3). A comprehensive presentation of baseline resolution of these drugs is presented.

Aminogluthethimide (AG, 1) was first used as anticonvulsant, but was observed later to cause adrenal insufficiency which led to its withdrawal from the market. The drug inhibits several enzymes in the pathway of steroidogenesis, mainly the desmolase enzyme system which is responsible for the cholesterol side chain cleavage, i.e., conversion of cholesterol to pregnenolone (1), and the aromatase enzyme system which is responsible for the aromatization of androstene-3,17-dione and testosterone to esterone and estradiol, respectively (2). Thus, AG can interfere with the biosynthesis of mineralocorticoids, glucocorticoids and sex hormones and effectively perform a physiological adrenalectomy. Aminogluthethimide has been used as a racemic mixture in the treatment of metastatic estrogen-dependent breast cancer as a reversible nonsteroidal aromatase inhibitor (3,4). However, due to its inhibitory effect on desmolase, corticosteroid production is depleted, and consequently patients receiving AG 0097-6156/92/0512-0111$06.00/0 © 1992 American Chemical Society In Chromatography of Pharmaceuticals; Ahuja, S.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

112

CHROMATOGRAPHY OF PHARMACEUTICALS

require hydrocortisone replacement therapy to prevent the reflex rise in adrenocorticotropic hormone (ACTH) which can counteract the initial blockade of desmolase. Furthermore, several side effects were reported during AG administration e.g., sedations, CNS depression, ataxia and neurotoxic effects (5).

Downloaded by PENNSYLVANIA STATE UNIV on August 2, 2012 | http://pubs.acs.org Publication Date: October 29, 1992 | doi: 10.1021/bk-1992-0512.ch009

It is of interest to mention that the (+)-R enantiomer of AG is 30 times more potent than the (-)-S enantiomer in aromatase inhibition, whereas the (+)-R enantiomer is 15 times less potent than the (-)-S isomer in inhibiting desmolase (6,7). Several analogs of AG were developed through substitution of the ethyl group by other bulkier groups or by replacing the p-aminophenyl group by other basic functionalities in order to design specific and selective aromatase inhibitors with l i t t l e or no desmolase inhibitory activity. Subsequently, the need for hydrocortisone supplement administration will not be required. Foster et al.(8) reported the synthesis of pyridogluthethimide (PG, 2) which is considered a bioisoster of AG. Pyridogluthethimide exhibited a strong, selective and competitive inhibitory activity against aromatase while i t did not inhibit the desmolase enzyme system. Pyridogluthethimide is administered as a racemic mixture, although i t is reported that the (+)-R-enantiomer is more active than the (-)-S-enantiomer (9). Cyclohexylaminogluthethimide (ChAG, 3 J , another active analog of AG, first synthesized by Hartmann et a l . (10) showed a strong and selective inhibitory activity (123-fold in vitro and 10-fold in vivo and compared to AG) against aromatase, while the inhibition against the desmolase enzyme system was greatly reduced. Hartmann et a l . (11) also reported that the (+)-S-enantiomer proved to be responsible for the aromatase inhibition and is 30 times more active than the (-)-R-enantiomer. Furthermore, (+)-S-ChAG showed no CNS depressive activity and possessed a weaker effect regarding the inhibition of the desmolase compared to its (-)-R-enantiomer. The chemical structures of these drugs are shown in Figure 1. Compound

Name

R,

R

1

Aminoglutethimide (AG)

C H

2

Pyridoglutethimide (PG)

C H

3

Cyclohexylaminoglutethimide (ChAG) C H

2

2

6

5

2

~^J^-NH

2

~^^N

5

n

-^^-NH

2

* Asterisk denotes chiral carbon.

Figure 1.

The chemical structures of various glutarimide aromatase inhibitors.

Thus, from the previous introduction, i t would be clinically more useful to administer these aromatase inhibitors as pure enantiomers namely: (+)-R-aminogluthethimide (+)-R-pyridogluthethimide and (+)-S-cyclohexylaminogluthethimide rather than other racemate mixtures. This will reduce the side effects encountered by the

In Chromatography of Pharmaceuticals; Ahuja, S.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

9. ABOUL-ENEIN

Enantiomeric Separation of Aromatase Inhibitors 113

o t h e r e n a n t i o m e r s and a l s o a l l o w t h e use o f a p p r o p r i a t e d o s e s , t h u s a c h i e v i n g more e f f e c t i v e t h e r a p y .

smaller

Downloaded by PENNSYLVANIA STATE UNIV on August 2, 2012 | http://pubs.acs.org Publication Date: October 29, 1992 | doi: 10.1021/bk-1992-0512.ch009

T h i s paper d e s c r i b e s a method f o r the d i r e c t s e p a r a t i o n o f the racemates o f t h e s e aromatase g l u t a r i m i d e i n h i b i t o r s t o t h e i r corresponding enantiomers using commercially a v a i l a b l e c e l l u l o s e based c h i r a l s t a t i o n a r y phases ( C S P ' s ) namely C h i r a l c e l OD ( c e l l u l o s e t r i s - 3 , 5 - d i m e t h y l p h e n y l carbamate) and/or C h i r a l c e l OJ ( c e l l u l o s e t r i s - 4 - m e t h y l b e n z o a t e ) . The optimum chromatog­ r a p h i c c o n d i t i o n s f o r t h e s e p a r a t i o n o f t h e s e drugs on t h e s e C S P ' s are a l s o s t u d i e d .

EXPERIMENTAL Apparatus The Waters L i q u i d Chromatography System used (Waters A s s o c i a t i o n , M i l f o r d , MA) c o n s i s t e d o f a Model M-45 pump, a U6K i n j e c t o r , and a Lambda-Max Model 481 LC s p e c t r o p h o t o m e t e r d e t e c t o r o p e r a t e d at 257 nm. C h i r a l c e l OD and C h i r a l c e l OJ c o a t e d on s i l i c a g e l w i t h p a r t i c l e s i z e o f lOum were used as s t a t i o n a r y phases (25cm χ 0.46cm i . d . , Dai e e l Chemical I n d u s t r i e s , T o k y o , J a p a n ) . Chemicals Racemic a m i n o g l u t e t h i m i d e (±AG) ( L o t No. 8 0 0 3 8 3 ) , ( + ) - R - a m i n o g l u t e t h i m i d e (+AG) (CGS-2396) were s u p p l i e d by C i b a - G e i g y ( B a s l e , Switzerland). Racemic p y r i d o g l u t e t h i m i d e (±PG), and i t s c o r r e s ­ ponding (+)-R and ( - ) - S - e n a n t i o m e r s were k i n g l y s u p p l i e d by D r . M. Jarman o f the I n s t i t u t e o f C a n c e r R e s e a r c h , S u t t o n , S u r r e y , U.K. Racemic c y c l o h e x y l a m i n o g l u t h e t h i m i d e (±ChAG) and i t s c o r r e s p o n d i n g ( - ) - R - e n a n t i o m e r were k i n d l y s u p p l i e d by P r o f e s s o r R.W. Hartmann, U n i v e r s i t y o f S a a r l a n d , S a a r b r u c k e n , Germany. HPLC grade hexane and HPLC grade 2-propanol were p u r c h a s e d from Fisher S c i e n t i f i c , Fairlawn, NJ. Chromatographic

Conditions

The r e s o l u t i o n o f AG, PG and ChAG were o b t a i n e d on t h e C h i r a l c e l OD column u s i n g hexane: 2-propanol as a m o b i l e phase i n p r o p o r t i o n s shown i n F i g u r e 2, 3 and 4 , r e s p e c t i v e l y . Racemic AG and PG c o u l d a l s o be r e s o l v e d s u c c e s s f u l l y on an OJ column u s i n g hexane: 2-propanol (50:50) as a m o b i l e phase as shown i n F i g u r e s 5 and 6, r e s p e c t i v e l y . Temperature was m a i n t a i n e d a t 23°C t h r o u g h o u t the e x p e r i m e n t s . Determination of Enantiomeric

Elution

Order

The e n a n t i o m e r i c e l u t i o n o r d e r was d e t e r m i n e d by chromatographing the s e p a r a t e i n d i v i d u a l e n a n t i o m e r s under s i m i l a r conditions. T h u s , i n t h e c a s e o f AG on both t h e C h i r a l c e l 0D and 0J columns the peak t h a t e l u t e d f i r s t was i d e n t i f i e d as ( - ) - S - A G w h i l e the second peak was i d e n t i f i e d as (+)-R-AG

In Chromatography of Pharmaceuticals; Ahuja, S.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

Downloaded by PENNSYLVANIA STATE UNIV on August 2, 2012 | http://pubs.acs.org Publication Date: October 29, 1992 | doi: 10.1021/bk-1992-0512.ch009

114

CHROMATOGRAPHY OF PHARMACEUTICALS

Ε (-hS-AG

F i g u r e 2.

LC s e p a r a t i o n o f r a c e m i c AG (I). Column: C h i r a l c e l OD (250 X 4.6mm, I . D . ) ; m o b i l e p h a s e , hexane: 2-propanol ( 6 0 : 4 0 ) ; flow r a t e : 0.7 ml/min; c h a r t s p e e d : 0 . 2 5 cm/mi η; t e m p e r a t u r e : 2 3 ° C ; d e t e c t o r : UV 257nm; s e n s i t i v i t y 0 . 0 1 AUFS; sample amount: 2 . 5 nmol.

In Chromatography of Pharmaceuticals; Ahuja, S.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

Downloaded by PENNSYLVANIA STATE UNIV on August 2, 2012 | http://pubs.acs.org Publication Date: October 29, 1992 | doi: 10.1021/bk-1992-0512.ch009

ABOUL-ENEIN

Figure 3.

Enantiomeric Separation of Aromatase Inhibitors

LC s e p a r a t i o n o f r a c e m i c PG (2). Chromatographic c o n d i t i o n s were the same as i n F i g u r e 2, e x c e p t m o b i l e p h a s e , hexane: 2 - p r o p a n o l ( 6 5 : 3 5 ) ; sample amount 2.8 nmol.

In Chromatography of Pharmaceuticals; Ahuja, S.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

116

CHROMATOGRAPHY OF PHARMACEUTICALS

(-)-R-ChAG

Downloaded by PENNSYLVANIA STATE UNIV on August 2, 2012 | http://pubs.acs.org Publication Date: October 29, 1992 | doi: 10.1021/bk-1992-0512.ch009

(+)-S-ChAG

1 Figure 4.

LC s e p a r a t i o n o f r a c e m i c ChAG {3). Chromato­ g r a p h i c c o n d i t i o n s were t h e same as i n F i g u r e 2, e x c e p t m o b i l e p h a s e , hexane: 2-propanol ( 5 0 : 5 0 ) ; f l o w r a t e , 1.0 m l / m i n ; c h a r t s p e e d , 0 . 2 cm/min; sample amount, 2 . 5 nmol.

Ε

S 7 (+)-R-AG

Figure

5.

LC s e p a r a t i o n o f r a c e m i c AG (_1). Column: C h i r a l c e l OJ (250 X 4.6mm, I . D . ) ; m o b i l e p h a s e , hexane: 2-propanol ( 5 0 : 5 0 ) . Other c h r o m a t o g r a p h i c c o n d i t i o n s were t h e same as i n F i g u r e 2, e x c e p t sample amount: 3 nmol.

In Chromatography of Pharmaceuticals; Ahuja, S.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

Downloaded by PENNSYLVANIA STATE UNIV on August 2, 2012 | http://pubs.acs.org Publication Date: October 29, 1992 | doi: 10.1021/bk-1992-0512.ch009

9. ABOl^ENEIN

Enantiomeric Separation of Aromatase Inhibitors 117

(•)-R-PG CM ν·

8 (-)-S-PG

F i g u r e 6.

LC s e p a r a t i o n o f r a c e m i c PG {2). Column: C h i r a l c e l OJ (250 X 4 . 6 mm, I . D . ) ; o t h e r c h r o m a t o g r a p h i c c o n d i t i o n s were t h e same as F i g u r e 5, e x c e p t c h a r t speed: 0.2 cm/min; sample amount: 2.8 nmol.

In Chromatography of Pharmaceuticals; Ahuja, S.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

in

CHROMATOGRAPHY OF PHARMACEUTICALS

118

In t h e c a s e o f t h e r e s o l u t i o n o f t h e PG e n a n t i o m e r s on t h e C h i r a l c e l OD c o l u m n , t h e peak t h a t e l u t e d w i t h a lower c a p a c i t y f a c t o r was the ( - ) - S - P G and t h e peak t h a t e l u t e d w i t h a h i g h e r c a p a c i t y f a c t o r was i d e n t i f i e d as ( + ) - R - P G . However, t h e e l u t i o n o r d e r o b t a i n e d f o r t h e r e s o l u t i o n o f (±) PG on OJ column was r e v e r s e d .

Downloaded by PENNSYLVANIA STATE UNIV on August 2, 2012 | http://pubs.acs.org Publication Date: October 29, 1992 | doi: 10.1021/bk-1992-0512.ch009

In t h e c a s e o f ChAG, i t was found t h a t t h e peak e l u t e d w i t h a lower c a p a c i t y f a c t o r was i d e n t i f i e d as (-)-R-ChAG and t h e peak t h a t e l u t e d w i t h a h i g h e r c a p a c i t y f a c t o r was i d e n t i f i e d as (+)-S-ChAG.

RESULTS AND DISCUSSION The s t e r e o c h e m i c a l c o m p o s i t i o n o f p h a r m a c e u t i c a l s i s becoming an i m p o r t a n t i s s u e n o t o n l y i n t h e c o u r s e o f drug d e v e l o p m e n t , b u t a l s o drug a p p r o v a l and c l i n i c a l u s e . A t p r e s e n t , t h e p h a r m a c e u t i c a l i n d u s t r y i s r a p i d l y moving towards p r e c i s e l y t a r g e t e d , z e r o - r i s k drug t h e r a p i e s . The f a s t development o f c h i r a l s t a t i o n a r y phases i n r e c e n t y e a r s had demonstrated t h e v e r s a t i l i t y and c o n v e n i e n c e o f t h e t e c h n i q u e i n c h i r a l s e p a r a t i o n o f drug racemates t o t h e i r corresponding enantiomers. C e l l u l o s e d e r i v a t i v e s f u n c t i o n as c h i r a l a b s o r b e n t s and e x h i b i t good performance comparable t o o t h e r p h a s e s . C e l l u l o s e CSP's b e l o n g t o t h e h e l i c a l polymer p h a s e s , namely t h e c e l l u l o s e e s t e r s and carbamates whose c h i r a l i t y a r i s e s from h e l i c i t y . V a r i o u s t y p e s o f c e l l u l o s e and d e r i v a t i v e s c a r r y i n g v a r i o u s s u b s t i t u e n t s have been d e v e l o p e d ( 1 2 ) . The c e l l u l o s e d e r i v e d C h i r a l c e l OD column has been s u c c e s s f u l l y used t o d i r e c t l y s e p a r a t e several S-adrenergic blockers (13-18). C h i r a l c e l OJ has a l s o been used t o s e p a r a t e drugs c h e m i c a l l y r e l a t e d t o t h e g l u t a r i m i d e r i n g s y s t e m , e . g . , t h a l i d o m i d e (19) and m e p h o b a r b i t a l ( 2 0 ) . In t h i s s t u d y C h i r a l c e l OD and C h i r a l c e l OJ were s u c c e s s f u l l y used i n t h e r e s o l u t i o n o f t h e r a c e m i c m i x t u r e o f A G , PG, and ChAG. O p t i m i z a t i o n o f t h e s e p a r a t i o n o f t h e s e drugs was a c h i e v e d u s i n g d i f f e r e n t c o n c e n t r a t i o n s o f 2-propanol i n hexane as a m o b i l e phase ( s e e T a b l e I). S i n c e good s t e r e o c h e m i c a l r e s o l u t i o n was o b t a i n e d f o r t h e s e d r u g s , t h i s method i s c u r r e n t l y a p p l i e d f o r p r e p a r a t i v e s e p a r a t i o n o f the p h a r m a c o l o g i c a l l y a c t i v e e n a n t i o m e r s (eutomers) i n l a r g e quantities. F u r t h e r m o r e , t h e method c o u l d be used f o r e n a n t i o m e r i c and o p t i c a l p u r i t y d e t e r m i n a t i o n o f t h e s e drugs i n b u l k fonn and pharmaceutical f o r m u l a t i o n s . The d e s c r i b e d method has an advantage o f being f a s t and r e q u i r e s no d e r i v a t i z a t i o n . Work i s i n p r o g r e s s i n t h i s l a b o r a t o r y t o v a l i d a t e t h e method f o r a n a l y s i s o f t h e s e drugs i n b i o l o g i c a l f l u i d s . In c o n c l u s i o n , methods f o r t h e d i r e c t various

non-steroidal

glutarimide

enantiomeric

separation o f

aromatase i n h i b i t o r s

developed using c e l l u l o s e - b a s e d CSP's

(Chiralcel

have been

OD and

OJ c o l u m n s ) .

In Chromatography of Pharmaceuticals; Ahuja, S.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

9. ABOUL-ENEIN

Enantiomeric Separation of Aromatase Inhibitors 119

Downloaded by PENNSYLVANIA STATE UNIV on August 2, 2012 | http://pubs.acs.org Publication Date: October 29, 1992 | doi: 10.1021/bk-1992-0512.ch009

T a b l e I . O p t i m i z e d parameters o f c a p a c i t y f a c t o r ( k ' ) , s t e r e o c h e m i c a l s e p a r a t i o n f a c t o r ( a ) and s t e r e o c h e m i c a l r e s o l u ­ t i o n (R) o f AG, PG and ChAG on C h i r a l c e l OD and C h i r a l c e l OJ columns

Compound

Solvent

Column

AG AG PG PG ChAG

A Β C B Β

OD OJ OD OJ OD

Chromatographic

K

1

6.61 2.35 2.76 3.15 2.51

2

α R

9.96 8.78 3.69 4.74 8.18

α 1.51 3.74 1.34 1.50 3.26

R 8.87 10.34 0.96 1.56 4.89

Conditions

S o l v e n t system A = hexane: 2 - p r o p a n o l S o l v e n t system Β = hexane: 2-propanol S o l v e n t system C = hexane: 2 - p r o p a n o l D e t e c t o r : UV257nm; t e m p e r a t u r e 23°C. k^ = 1 s t e l u t e d peak k'

k'

=

2nd e l u t e d

= =

stereochemical stereochemical

(60:40) (50:50) (65:35)

peak factor resolution

ACKNOWLEDGEMENTS The a u t h o r would l i k e t o thank the a d m i n i s t r a t i o n o f K i n g F a i s a l S p e c i a l i s t H o s p i t a l and R e s e a r c h C e n t r e f o r t h e i r c o n t i n u o u s s u p p o r t t o t h e Drug Development Research Program. This i n v e s t i ­ g a t i o n was s u p p o r t e d f i n a n c i a l l y under P r o j e c t No. 88-0015 by King F a i s a l S p e c i a l i s t H o s p i t a l & Research C e n t r e . The a u t h o r wishes t o thank D r . K. S c h e i b l i , C i b a - G e i g y , B a s l e , S w i t z e r l a n d , D r . M. Jarman, I n s t i t u t e o f C a n c e r R e s e a r c h , S u t t o n , S u r r e y , U . K . and D r . R.W. Hartmann, I n s t i t u t e o f P h a r m a c e u t i c a l C h e m i s t r y , U n i v e r s i t y o f S a a r l a n d , S a a r b r u c k e n , Germany f o r p r o v i d i n g t h e samples o f drugs used i n t h i s s t u d y .

LITERATURE CITED: 1.

Lonning, P. E. and Kvinnsland, S. (1988). Drugs 35, 685-710.

2.

Dexter, R. N., Fishman, L. M., Ney, R. L. and Liddle, J. (1967), J. Clin. Endocrinol 27, 374-480.

3.

Aboul-Enein, H. Y., (1988). Drug Design & Delivery, 2, 221-226, and references cited therein.

4.

Katzung, B. G., (1989). Basic & Clinical Pharmacology 4th Ed. Appleton & Lange, East Norfolk, CT (USA), p. 480.

5.

Salhanick, Η. Α., (1982). Cancer Res. (Suppl.), 42, 3315S-3321S. In Chromatography of Pharmaceuticals; Ahuja, S.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

Downloaded by PENNSYLVANIA STATE UNIV on August 2, 2012 | http://pubs.acs.org Publication Date: October 29, 1992 | doi: 10.1021/bk-1992-0512.ch009

120

CHROMATOGRAPHY OF PHARMACEUTICALS

6.

Uzgiris, V. I., Whipple, C. A. and Salhanick, Η. Α., Salhanick, (1977). Endocrinology, 101, 89-92.

7.

Finch, N., Dziemian, R., Cohen, J. and Steinetz, B. G., (1975). Experientia, 31, 1002-1003.

8.

Foster, A. B. Jarman, M., Leung, C. S., Rowlands, M. G., Taylor, G. N., Plerey, P. G. and Sampson, P., (1985). J. Med. Chem., 28, 200-204.

9.

Clissold, D. W., Jarman, M., Mann, J., McCague, R., Neidle, S., Rowlands, M. G., Thickitt, C. P. and Webester, G., (1989). J. C. S. Perkin Trans. I. 196-198.

10.

Hartmann, R. W., Batzl, C., Mannschreck, A. and Seydel J. K. (1989),. Trends in Medicinal Chemistry '88; van der Goot, H., Domany, G., Pallos, L., Timmermann, T. (Eds.), Elsevier Science Publishers, Amsterdam, The Netherlands, pp 821-838.

11.

Hartmann, R. W., Batzl, C., Mannschreck, Α., Pongratsz, T., (1990). Chirality and Biological Activity; Holmstedt, B., Frand, H., Testa, B. (Eds).; Alan B. Liss: NY, USA, pp 185-190.

12.

Aboul-Enein, H. Y., Islam, M. R., (1990). J. Liq. Chromatogr., 13, 485-492., and references cited therein.

13.

Okamoto, Y., Kawashima, M., Aburatani, R., Hatada, K., Nishiyama, T., Matsuda, M., (1986), Chem. Lett., 1237-1240.

14. Aboul-Enein, H. Y. and Islam, M. R., (1990). J. Chromatogr., 511, 109-114. 15.

Aboul-Enein, H. Y. and Islam, M. R., (1989). Chirality, 1, 301-304.

16.

Aboul-Enein, H. Y. and Islam, M. R., (1990), Anal. Lett. 23, 83-91.

17.

Aboul-Enein, H. Y. and Islam, M. R., (1990), Anal. Lett. 23, 973-980.

18.

Krstulovic, A.M., Fouchet, M. H., Burke, J. T., Gillet, G. Durand, Α., (1988). J. Chromatogr., 452, 477-483.

19.

Aboul-Enein, Η. Y. and Islam, M. R., (1991). J. Liq. Chromatogr., 14, 667-673.

20.

Okamoto, Y., Aburatani, R., Hotada, K., (1987). J. Chromatogr., 389, 95-102. RECEIVED February 24, 1992

In Chromatography of Pharmaceuticals; Ahuja, S.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.