Chiral Separations by Liquid Chromatography - American Chemical

Chapter 12

Direct Stereochemical High-Pressure Liquid Chromatographic Separation of Aminoglutethimide and Its Major Metabolite

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Its Applications to Biological Fluids Hassan Y. Aboul-Enein and M. Rafiqul Islam Radionuclide and Cyclotron Operations, Drug Development Laboratory, King Faisal Specialist Hospital and Research Centre, P.O. Box 3354, Riyadh 11211, Kingdom of Saudi Arabia A direct, isocratic, sensitive and precise liquid chromatographic method is developed for the enantiomeric separation of aminoglutethimide (AG) and its acetylated metabolite (AcAG) using cellulose tris-3,5-dimethylphenyl carbamate (Chiralcel OD) column and cellulose tris-(4-methylphenyl beonzoate) ester (Chiralcel OJ) column in series. The enantiomeric elution order is determined by chromatographing the racemate aminoglutethimide and racenate acetylated aminoglutethimide separately and their enantiomers under the similar conditions. This method has been applied to determine and identify the enantiomers of AG and AcAG in the urine sample collected from a metastatic breast cancer patient after 24 hours administration of (±)AG. Large amounts of (+)-R-aminoglutethimide are excreted unchanged in the urine together with a smaller quant i t i e s of its (+)-R-acetylated metabolites, while most of the (-)-S-aminoglutethimide is metabolically converted into (-)-S-acetylated aminoglutethimide. In most cases chiral drug enantiomers have different biological and pharmacological activities due to different interactions on the receptors (which also have chiral centers), posses different pharmacokinetics and metabolic pathways. Drug chirality is becoming an important issue facing the pharmaceutical industry and pharmaceutical research in developing of new drugs (1,2). A rapid survey of the 700 most frequently prescribed drugs shows that 75% of them are marketed and administered as racemic mixture. It is therefore important during the development of drugs to be able to isolate the enanticmers (if chiral centers exist) to assess which is responsible for the potency, the toxicity and for the side effects. For these reasons, there is a great demand in the pharmaceutical industry for the developments of effective analytical techniques and preparative separation of a variety of enantiomeric compounds that are known to have different physiological activities. 0097-6156/91/0471-0203$06.00/0 © 1991 American Chemical Society Ahuja; Chiral Separations by Liquid Chromatography ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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D i f f e r e n t chromatographic techniques have been developed over the past f i f t e e n t o twenty years t o separate stereoisomeric compounds. But the e f f i c i e n c y , speed, wide a p p l i c a b i l i t y , and r e p r o d u c i b i l i t y of high performance l i q u i d chromatography (HPLC) have made i t the method of choice. There are now more than 40 c h i r a l stationary phases (CSP's) a v a i l a b l e commercially f o r d i r e c t separation and resolution of wide range of drug enantiomers by HPLC(3-6). One highly promising i s the cellulose-based CSPs (O-Type) described by Ichida, e t . a l . , ( 7 ) . The primary mechanism of these phases are the formation of the solute-CSP complex through a t t r a c t i v e interactions but where i n c l u s i o n complexes a l s o play an important r o l e . C h i r a l c e l OD (Cellulose tris-3,5-dimethylphenyl carbamate) (Scheme 1) and C h i r a l c e l OJ [Cellulose tris-(4-methylphenyl benzoate) ester] (Scheme 2), are two of these ptoses which were used i n s e r i e s i n t h i s study f o r enantiomeric separation of aminoglutethimide (AG) and i t s major metabolite acetylated aminoglutethimide (AcAG) using a s i n g l e chromatographic conditions. Aminoglutethimide, chemically known as (±)-3-(aminophenyl)-3ethyl-2,6-piperidinedione (AG) (Scheme 3) i s currently c l i n i c a l l y used f o r the treatment of metastatic breast cancer and adrenocort i c a l tumors and Gushing's syndrcme (8,9). Metabolic studies i n man have shown that, a f t e r s i n g l e doses of AG, large amounts are excreted unchanged i n the urine together with smaller quantities of i t s acetylated metabolite, N-acetyl aminoglutethimide (AcAG), accounting f o r 4-25% o f an o r a l dose of AG(10,11). Kamblawi e t . al.(12) developed a simple HPLC method f o r the quant i t a t i v e determination of AG and AcAG i n urine i n order t o study the i n t e r - i n d i v i d u a l v a r i a t i o n i n the excretion of AG and i t s acet y l a t i o n p r o f i l e i n man and other species. Several methods have been developed f o r the assay of AG i n b i o l o g i c a l f l u i d s and has been reviewed by Aboul-Enein(13). I t was reported that the (+)-R-isomer had the most stereodogenesis i n h i b i t o r y a c t i v i t y (two to three times more potent than the racemate), while the (-)-S-isomer had very l i t t l e a c t i v i t y a t dose l e v e l s 10-folds higher(14). Resolution o f aminoglutethimide, therefore, provided the compound (+) -R-aminoglutethimide with e s s e n t i a l l y a l l of the s t e r o i d synthesis i n h i b i t i n g a c t i v i t y of the racemate. Resolution of racemic aminoglutethimide adopting chemical methods was reported i n e a r l i e r works (14). A d i r e c t resolution of racemic aminoglutethimide (AG) and i t s acetylated metabolite (AcAG) was obtained using a 100-mm long ax-acid glycoprotein column(15) Racemic aminoglutethimide and i t s acetylated metabolite were a l s o separated using c h i r a l c e l OD and c h i r a l c e l OJ column (16). Maximum and symmetrical stereochemical resolution (R) obtained f o r AG was on C h i r a l c e l OD column. Aminoglutethimide was a l s o resolved on C h i r a l c e l OJ column, but the peak obtained was not symmetrical. #

Ahuja; Chiral Separations by Liquid Chromatography ACS Symposium Series; American Chemical Society: Washington, DC, 1991.


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ABOUL-ENEIN & ISLAM

Scheme study.

Scheme study.

HPLC Separation of Aminoglutethimide205

1. T h e structure of the chiral stationary p h a s e ( O D - C S P ) u s e d in this

2. T h e structure

of the chiral stationary phase ( O J - C S P ) used in this

NH

(-)-S- aminoglutethimide

2

(+)-R- aminoglutethimide

Scheme 3. The absolute configuration of (-)-S and (+)-R aminoglutethimide.


206



Maximum and symmetrical stereochemical resolution (R) with basel i n e separation was obtained f o r acetylated aminoglutethimide on C h i r a l c e l OJ column using hexane and 2-propanol (50:50). Acetyl a t e d aminoglutethimide was a l s o resolved on C h i r a l c e l OD column, but the e l u t i o n order was reversed. Taking i n consideration of a l l the above-cited observations i t was decided t o develop a method where aminoglutethimide (AG) and i t s major metabolite (AcAG) could be simultaneously separated and analysed i n b i o l o g i c a l f l u i d s e.g. urine, using one s i n g l e i s o c r a t i c solvent system, thus making i t more r a p i d and convenient i s described i n t h i s paper.

EXPERIMENTAL Apparatus The Waters (Waters Associates, M i l f o r d , Mass., 01757, U.S.A.) LC system consisted of a Model M-45 pump, a U6K i n j e c t o r , and a Lamda-Max Model 481 LC spectrophotometer UV detector operated at 257nm. The stationary phase of C h i r a l c e l CD a n a l y t i c a l column of c e l l u l o s e tris-3,5-dimethylphenyl carbamate and C h i r a l c e l OJ a n a l y t i c a l column of c e l l u l o s e tris-(4-methylphenyl benzoate) ester (25cm x 0.46cm, I.D., D a i c e l Chemical Industries, Tokyo, Japan) coated on s i l i c a g e l with p a r t i c l e s i z e 10 urn were used. Chemicals Racemic aminoglutethimide (±AG) (Lot No. 800383), (+)-R-aminoglutethimide (+AG) (CGS-2396), and a racemic acetylated aminoglutethimide Gb AcAG) (Ba 17873) were supplied by Ciba Geigy, Basle, Switzerland. HPLC grade hexane was obained from Fisher S c i e n t i f i c , New Jersey, U.S.A. HPLC grade 2-propanol was obtained from Romil Chemicals, Ltd., England. (+)-R-AAG was prepared as previously described method by Aboul-Enein and Islam (15). Sample Pretreatment: Urine F i f t y ml of urine sample was c o l l e c t e d from a female patient with metastatic breast cancer, receiving AG f o r treatment. I t was d i l u t e d t o 100ml with water, and then extracted with methylene d i c h l o r i d e (25ml x 3). The volume was reduced under reduced pressure and d r i e d with nitrogen flow. The residue was redissolved i n an appropriate volume of methanol and injected t o the column. Chromatographic Conditions The maximum and symmetrical stereochemical resolution of AG and AcAG enantiomers i n urine were obtained using hexane and 2-propanol (50:50) on C h i r a l c e l CD followed by C h i r a l c e l OJ


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columns i n s e r i e s . The flow rate was 0.7ml/min and chart speed was 0.25cnVmin. Temperature was maintained a t 23°C. Detection was obtained a t UV 257nm with s e n s i t i v i t y range 0.01 AUFS. The sample amount i n j e c t e d was 5 nmole f o r racemate AG, 2.5 nmole f o r (+)-R-AG enantiomer, 1 nmole f o r racemate AcAG, 0.5 nmole f o r (+)-R-AcAG enantiomer.


Determination of Enantiomeric E l u t i o n Order The enantiomeric e l u t i o n order was determined by chromatographing the racemate AG and racemate AcAG separately and t h e i r respective enantiomers under the s i m i l a r conditions. Thus racemate AcAG was found to be eluted e a r l i e r than racemate AG. In case of racemate AG, the peak that eluted e a r l i e r was i d e n t i f i e d as (-)-S-AG and i n case of racemate AcAG, the peak that eluted e a r l i e r was found t o be (+)-R-AcAG.

RESULTS & DISCUSSION Aboul-Enein and Islam(15) recently described a d i r e c t stereochemic a l r e s o l u t i o n of racemic aminoglutethimide and i t s acetylated metabolite using a 100-irm long a i - a c i d glycoprotein column. However, the r e s o l u t i o n obtained with t h i s column was not symmetrical and without base l i n e separation f o r AcAG. The method was a l s o found to be lengthy, and required a c a r e f u l consideration of a number of parameters such as preparation of phosphate buffer and pH adjustment, s a l t e f f e c t , concentration of organic modifier and temperature c o n t r o l . Two new methods f o r stereochemical resol u t i o n of AG and AcAG using C h i r a l c e l OD and C h i r a l c e l OJ column are described by Aboul-Enein and Islam(16). I t was found that the maximum stereochemical r e s o l u t i o n obtained f o r AG was on C h i r a l c e l OD column while f o r AcAG was on C h i r a l c e l OJ column using d i f f e r e n t solvent systems. This paper describes another developed method where AG and AcAG could be resolved and ident i f i e d simultaneously i n b i o l o g i c a l f l u i d s using C h i r a l c e l OD and C h i r a l c e l OJ columns i n s e r i e s using one i s o c r a t i c solvent system, thus making i t more r a p i d and convenient. Using t h i s method, enantiomers of AG and AcAG were a l s o i d e n t i f i e d i n the urine sample c o l l e c t e d from a female metastatic breast cancer p a t i e n t . The optimization of separation were achieved using d i f f e r e n t concentration of 2-prcpanol as a mobile phase i n room temperature. The chromatogram of enantiomer separation of aminoglutethimide (AG) on C h i r a l c e l OD and C h i r a l c e l OJ i n s e r i e s i s shown i n F i g . 1. Compared with the chromatogram of (+) -R-aminoglutethimide ( F i g . 2) the peak that eluted with a lower capacity factor was i d e n t i f i e d as (-)-S-AG and the peak that eluted with a higher capacity f a c t o r was i d e n t i f i e d as (+)-R-AG. Maximum stereochemic a l r e s o l u t i o n (R) obtained was 13.68. The chromatogram of enantiomer separation of AcAG on C h i r a l c e l OD and C h i r a l c e l OJ i n s e r i e s i s shown i n Figure 3. Compared with the chromatogram of (+)-R-AcAG ( F i g . 4 ) , the peak that eluted with a higher capacity factor was i d e n t i f i e d as (-)-S-AcAG. Maximum stereochemical resol u t i o n obtained was 4.39.


208


S


3 (-)-S-AG

F i g . 1. Enantiomeric separation o f racemic AS. Columns: C h i r a l c e l OD and C h i r a l c e l OJ (250 x 4.6 mm, I.D.) i n s e r i e s ; mobile phase: hexane and 2-propanol (50:50); flow rate: 0.7 ml/min.; chart speed: 0.25 cm/min.; temperature: 23°C; detector: UV 257 nm; s e n s i t i v i t y : 0.01 AUFS; sample amount: 5 nmole.



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F i g , 2. Chromatogram of (+)-R-AG. Conditions were same as i n Figure 1, except the sample amount was 2.5 nmole.


CHIRAL SEPARATIONS BY LIQUID C H R O M A T O G R A P H Y


210

P i g , 3. E n a n t i o m e r i c s e p a r a t i o n o f r a c e m i c AcAG. C o n d i t i o n s were same a s i n F i g u r e 1, e x c e p t sample amount i n j e c t e d was 1 nmole.



ABOUL-ENEIN & ISLAM

HPLC Separation of Aminoglutethimide

(+)-R-AcAG

F i g . 4. Chromatogram of (+)-R-AcAG. Conditions were same as i n Figure 1, except sample amount injected was 0.5 nmole.


211

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CHIRAL SEPARATIONS BY LIQUID C H R O M A T O G R A P H Y

(-)-S-AG


(+)-R-AcAG

(+)-R-A3

F i g . 5. Enantiomeric separation of mixture of racemic AG & AcAG. Conditions were same as i n Figure 1, except sample amount injected was 5 nmole f o r AG and 1 nmole f o r AcAG.

Enantiomeric separation of mixture of racemic AG and AcAG i s shown i n F i g . 5. A l l the four peaks could be i d e n t i f i e d separately by comparing i t with the chromatograms of F i g s . 1-4. The enantiomeric separation of a methylene chloride extract of racemic AG and AcAG i n a urine sample c o l l e c t e d from a metastatic breast cancer patient a f t e r 24 hours administration of AG i s shown i n Figure 6. A l l the peaks were e a s i l y i d e n t i f i e d from retention times comparing them with F i g . 5.

CONCLUSION D i r e c t stereochemical separation of aminoglutethimide (AG) and i t s acetylated metabolite (AcAg) was achieved on commercially a v a i l a b l e c e l l u l o s e tris-3,5-dimethylphenyl carbamate ( C h i r a l c e l OD) and c e l l u l o s e t r is-(methylphenyl benzoate) ester ( C h i r a l c e l Ahuja; Chiral Separations by Liquid Chromatography ACS Symposium Series; American Chemical Society: Washington, DC, 1991.


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ABOUL-ENEIN & ISLAM

HPLC Separation of Aminoglutethimide 213

F i g . 6. Chromatogram obtained from the methylene chloride extract of 50 ml of urine sample c o l l e c t e d from a metastatic breast cancer patient a f t e r 24 hours administration of racemic AG. Chromatographic conditions were same as i n F i g . 1.

OJ) columns i n s e r i e s using one s i n g l e i s o c r a t i c solvent system, hexane and 2-propanol (50:50) a t 23^C with flow rate 0.7ml/min. The method was applied to determine and i d e n t i f y the enantiomers of AG and AcAG i n the urine sample c o l l e c t e d from a metastatic breast cancer patient a f t e r 24 hours administration of AG. The e f f e c t s of s t e r e o s e l e c t i v i t y on drug action and metabolic d i s p o s i t i o n has been recently reviewed(17). I t was observed from t h i s determination that the metabolism of AG racemate i n human i s s t e r e o s p e c i f i c . A large amount of (+)-R-AG i s excreted unchanged i n the urine together with a smaller q u a n t i t i e s of (+)-R-AcAG, while most of the (-)-S-AG i s metabolically converted i n t o Ahuja; Chiral Separations by Liquid Chromatography ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

214


(-)-S-AcAG. This simple method could also be applied to determine and quantitate the AG enanticmers and its acetylated metabolite AcAG in biological fluids. Furthermore, this method could be used for determination of the acetylation phenotype of patients receiving AG.


ACKNOWLEDGMENT

The authors thank the administration of King Faisal Specialist Hospital and Research Centre for their continuous support to the Drug Development Research Program. This investigation was supported financially, under Project No. 88-0015 by the King Faisal Specialist Hospital and Research Centre. LITERATURE CITED 1.

Holmstedt, B . ; Frank, H. and Testa B. Eds., in Chirality and Biological Activity: Alan R. Liss, Inc., NY, NY, 1990.

2. Wainer, I.W.; Drayer, P . E . , Eds., in Drug Stereochemistry: Analytical Methods and Pharmacology; Marcel Dekker, NY, NY, 1988. 3.

Stevenson, D. and Williams, G . , in Chiral Separations; edited by Stevenson, D. and Wilson, I.D.; Plenum Press, NY, NY, 1988, pp. 1-9.

4.

Wainer, I.W.; Rose, M.S. and Chu, Y . Q . , in Chiral Separations; edited by Stevenson, D. and Wilson, I.D.; Plenum Press, NY, NY., 1988, pp. 11-21.

5.

Krstulovic, A.M. (1988) J. Pharm. Biomed. Anal. 6, 641-656.

6.

Aboul-Enein, H.Y., and Islam, M.R. (1988) Labmedica, 5, 27-36.

7.

Ichida, A . ; Shibata, T . ; Okamoto, I . ; Yuki, Y . ; and Namikashi, H . ; Toga, Y. (1984) Chromatographia 19, 280-284.

8.

Nagel, G.A. and Santen, R . J . eds.; in Aminoglutethimide as an Aromatase Inhibitor in the Treatment of Cancer; Hans Huber Publishers, Berne, Switzerland, 1984.

9.

Lonning, P.E. and Kvinnsland. (1988).

Drugs, 35, 685-710.

10. Douglas, J . S . , and Nicholls P . J . (1972) J. Pharm. Pharmacol. 24 Suppl., 150 P. 11. Pourgholami, M.H.; Brain, K.R. and Nicholls, P . J . (1988). J . Chromatogr. 424, 163-169, and references were cited therein. 12. Kamblawi, M.O.; Stevens, R.G.; and Nicholls, P . J . (1984). Chromatogr. 309 431-435.


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HPLC Separation of Aminoglutethimide

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13. Aboul-Enein, H.Y. "Amnoglutethimide" a chapter in "Analytical Profile of Drug Substances, K. Florey, Editor, Academic Press Inc., Orlando, F l a . , 1986, V o l . 15, pp. 35-69 and the references cited therein. 14. Finch, N . ; Dziemian, R.; Cohen, J.; and Steinetz, B.G. (1975) Experientia (CH) 31(9), 1002-1003.


15. Aboul-Enein, H.Y.; and Islam, M.R. (1988). J. Chromatogr. S c i . 26, 616-619. 16. Aboul-Enein, H.Y.; and Islam, M.R. (1990). 30, 223-227.

Chromatographia

17. Jamali, F . ; Mehvar, R.; and Pasutto, F.M. (1989) J. Pharm. S c i . 78 695-715. RECEIVED January 30, 1991


Chiral Separations by Liquid Chromatography - American Chemical

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