Isolation of Xenobiotic Conjugates - ACS Publications - American

5 Isolation of Xenobiotic Conjugates W. Muecke

Downloaded by EAST CAROLINA UNIV on September 22, 2015 | http://pubs.acs.org Publication Date: January 24, 1986 | doi: 10.1021/bk-1986-0299.ch005

Ciba-Geigy Ltd., Basel, Switzerland Procedures and instruments are available for the isolation of virtually all types of low-molecular, polar xenobiotic conjugates. Most isolation procedures involve the use of liquid chromatography (anion exchange and partition mode), high performance liquid chromatography (partition mode), and thin layer chromatography (adsorption mode). A dedicated isolation strategy must be designed, after taking into account the properties of the conjugates under study, the nature of the matrix and the features of available methods with regard to capacity, selectivity, and resolution. The Vail symposium in 1975 on bound and conjugated pesticide residues (1) summarized the state of the art on methods and instru ments that were available for the isolation of conjugates at that time. Although dedicated to pesticides, the presentations reviewed the frontiers of methodology available for the isolation of all types of xenobiotics conjugates (2,3,4,5,6,7)* When summarizing the symposium, Geissbuehler (8) stated, "HPLC has been mentioned as a possibility for speeding up and improving separations ... and ought to be utilized without delay". In the last decade HPLC has become the most powerful tool the metabolism chemist has for isolating xenobiotic conjugates. During this period , many improvements have also been made in older traditional techniques. Noteworthy here is the positive impact HPLC had on conventional liquid chromatography regarding equipment and stationary phases and the significantly enhanced sensitivity of the radioactivity detectors for monitoring column effluents. Dramatic improvements in instrumentation and methods used for the identification of xenobiotic conjugates (and other metabolites), notably proton nuclear magnetic resonance and mass spectroscopy, have significantly influenced the strategies for the isolation of conjugates. The recently developed soft ionization techniques make it possible to obtain useful mass spectral information for a wide variety of intact underivatized xenobiotic conjugates. The state of 0097-6156/86/0299-0108S06.00/0 © 1986 American Chemical Society

In Xenobiotic Conjugation Chemistry; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

Isolation of Xenobiotic Conjugates


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the a r t NMR s p e c t r o m e t e r s g i v e i n t e r p r e t a b l e s p e c t r a from sample amounts as low as about 10 yg. C o n s e q u e n t l y methods w i t h l i m i t e d c a p a c i t y , but h i g h r e s o l u t i o n can be u t i l i z e d a t an e a r l i e r s t a g e o f the i s o l a t i o n p r o c e d u r e . Thus a w i d e r v a r i e t y o f p u r i f i c a t i o n systems can now be s e l e c t e d from. These developments i n s p e c t r o s copy a r e thus c h a n g i n g the i s o l a t i o n s t r a t e g y and t h e r e b y s i g n i f i c a n t l y i m p r o v i n g the economy o f the whole p r o c e s s . D u r i n g the r e c e n t y e a r s s e v e r a l r e v i e w s on the a n a l y t i c a l methodology and s t r a t e g y i n v o l v e d i n i s o l a t i n g x e n o b i o t i c m e t a b o l i t e s , i n c l u d i n g c o n j u g a t e s have been p u b l i s h e d , (9,10,11,12,13)* T h i s p r e s e n t a t i o n w i l l not be a review, but w i l l attempt to p l a c e w i t h i n a frame of the p r e s e n t l y used t e c h n i q u e s and methods my v e r y p e r s o n a l e x p e r i e n c e s and views. T h i s d i s c u s s i o n w i l l be l i m i t e d to the " c l a s s i c a l " p q l a r c o n j u g a t e s . Very d i f f e r e n t methodology i s r e q u i r e d to p u r i f y the non p o l a r " l i p o p h i l i c " conjugates. F u r t h e r m o r e , o n l y methods t h a t r e s u l t i n the i s o l a t i o n o f i n t a c t c o n j u g a t e s i n amounts adequate f o r m u l t i p l e - m e t h o d s t r u c t u r a l i d e n t i f i c a t i o n s t u d i e s w i l l be d i s c u s s e d ( i . e . combined methods such as GC-MS and HPLC-MS and d e r i v a t i z a t i o n t e c h n i q u e s w i l l not be c o v e r e d ) . Prerequisites F o r the sake o f c o n v e n i e n c e the i s o l a t i o n p r o c e s s may be a r b i t r a r i l y subdivided i n t o s e v e r a l interdependent steps. The x e n o b i o t i c c o n j u g a t e s p r e s e n t i n s o l i d m a t r i c e s must be s o l u b i l i z e d and s e p a r a t e d from the b u l k o f o t h e r m a t e r i a l s c o n c o m i t t a n t l y e x t r a c t e d o r p r e s e n t i n the o r i g i n a l l i q u i d sample (enrichment). T h e r e a f t e r the m i x t u r e s o f x e n o b i o t i c c o n j u g a t e s must be s e p a r a t e d i n t o i n d i v i d u a l f r a c t i o n s ( s e p a r a t i o n ) which i n t u r n must be ultimately purified ( p u r i f i c a t i o n ) for spectroscopic i d e n t i f i c a t i o n . As the i n v e s t i g a t o r moves from the enrichment v i a s e p a r a t i o n t o p u r i f i c a t i o n s t e p s the methods r e q u i r e l e s s c a p a c i t y and s e l e c t i v i t y , but g r e a t e r r e s o l u t i o n . S u i t a b l e methods have to be s e l e c t e d and a r r a n g e d i n a m e a n i n g f u l sequence i n o r d e r to o b t a i n an adeq u a t e , ( i . e . a s u c c e s s f u l and e c o n o m i c a l ) i s o l a t i o n s t r a t e g y . The o v e r a l l s t r a t e g y s h o u l d take i n t o c o n s i d e r a t i o n the n a t u r e o f the m a t r i x and the p r o p e r t i e s o f the m e t a b o l i t e s p r e s e n t . Relevant p r o p e r t i e s , to be i n v e s t i g a t e d by a p p r o p r i a t e a n a l y t i c a l p r o c e dures i n c l u d e h y d r o l y t i c a l s t a b i l i t y , v o l a t i l i t y , l i p o p h i l i c i t y , s o l v e n t p a r t i t i o n p r o p e r t i e s , e l e c t r i c a l charge and s u s c e p t i b i l i t y towards d e f i n e d enzymes. A r e l i a b l e a n a l y t i c a l system p r o v i d i n g e f f i c i e n t s e p a r a t i o n o f i n d i v i d u a l m e t a b o l i t e s p r e s e n t , needs to be e s t a b l i s h e d as e a r l y i n the i s o l a t i o n p r o c e d u r e as p o s s i b l e . This a n a l y t i c a l system i s used to m o n i t o r the r e s u l t o f each s i n g l e i s o l a t i o n s t e p , and - o f e q u a l importance - t o r e c o g n i z e any a r t i f a c t f o r m a t i o n which may o c c u r d u r i n g the e x e c u t i o n o f t h a t s t e p . Twod i r e c t i o n a l TLC and r e v e r s e d phase HPLC a r e f a s t and, t h e r e f o r e , commonly used methods f o r t h i s purpose. E n r i c h m e n t Methods The e x t r a c t i o n o f p o l a r c o n j u g a t e s from s o l i d samples ( e . g . p l a n t m a t e r i a l , a n i m a l t i s s u e , and f e c e s ) i s most commonly c a r r i e d out



110

XENOBIOTIC CONJUGATION CHEMISTRY

with polar solvents, such as methanol, acetone and a c e t o n i t r i l e and mixtures thereof with water. These solvents w i l l , when adequately applied, s o l u b i l i z e a l l low molecular conjugates, including glutathionates. After reconstituting these extracts i n water, the s o l u b i l i z e d metabolites may be processed as aqueous samples. P a r t i t i o n i n g of the aqueous solution with water non-miscible organic solvent i s frequently used to remove l i p o p h i l i c metabolites and contaminants as well. The extraction of polar conjugates from aqueous solution with organic solvents at or below t h e i r pk-values or at t h e i r i s o e l e c t r i c points has been used, occasionally, but usually does not result i n a complete p a r t i t i o n i n g into the organic solvent. Ion pair extraction of ionizable conjugates has never aquired a widespread use f o r preparative i s o l a t i o n s , because optimation of the extraction conditions with regard to the selection and concentration of the counter ions, the pH-value, etc. i s tedious. Therefore, this method of enriching a complex mixture of metabolites i s competitive i n only special cases (14). Currently the most powerful and commonly used enrichment technique i s s o l i d extraction applying non-ionic hydrophobic resins, such as Amberlite XAD-2, Amberlite XAD-4, Porapak Q and t h e i r analogs. These resins extract a wide variety of conjugates from aqueous phases providing a hydrophobic moiety i s present i n the molecule. Extractions of v i r t u a l l y a l l types of conjugates from various matrices have been reported [e.g. 0-, S- and N-glucuronides (15-24), 0- and N-glucosides (25-27), sulfates (17, 28), amino acid conjugates including glutathionates, homoglutathionates and taurinates (17,21,24, 29-32)]. Controlling the pH value of the aqueous solution, (to maintain the conjugates i n t h e i r non-ionized forms) and/or increasing the ionic strength by adding s a l t s (preferably buffer) are very useful tools to enhance adsorption of polar xenobiotic conjugates to hydrophobic resin columns. Usually the adsorbed metabolites are quantitatively desorbed from the column by elution with watermiscible organic solvents, such as methanol, acetone or acetonit r i l e . However, unreasonably low recoveries are sometimes obtained, even when columns such as those prepared from Amerlite XAD are sequentially eluted with polar, non-polar, a c i d i f i e d and a l k a l i n i z e d solvents. We have found that this type of adsorption i s not a problem i f s i l i c a bound reversed phase columns (e.g. RP 8 or RP 18) are used instead of XAD-2 and XAD-4 r e s i n columns. As these materials are now available as bulk material at reasonable prices and p a r t i c l e sizes allowing low pressure chromatography, they represent an a t t r a c t i v e alternative to the forementioned copolymers Amberlite XAD-2, Amberlite XAD-4 and Porapak Q (33). Under favourable circumstances these reversed phase enrichment methods can be - i n a preparative scale - extended to the separation of i n d i v i d u a l metabolites. Other s o l i d extractants, e.g. ion exchange resins (34,35), charcoal (36-38), Sephadex LH 20 (39,40) were used f o r the recovery of conjugates from crude aqueous extracts of b i o l o g i c a l samples. Even though these s o l i d extractants are not recommended f o r general use, however, an excellent separation of conjugates i n the urine of rats dosed with of o-toluidine on Sephadex LH-20 was published by Son et a l . (40) and i s demonstrated i n Figure 1.


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L i q u i d Column Chromatography C o n v e n t i o n a l l i q u i d chromatography, i n i t s d i f f e r e n t o p e r a t i o n modes, s t i l l r e p r e s e n t s a v e r s a t i l e and p o w e r f u l t e c h n i q u e . The development o f HPLC d u r i n g the l a s t decade has a l s o made a s i g n i f i cant impact on the "hardware" used w i t h t r a d i t i o n a l column chromatography ( i . e . s o l v e n t d e l i v e r y pumps, g r a d i e n t programmers, sample a p p l i c a t i o n systems, c o n t i n u o u s f l o w UV and r a d i o a c t i v i t y d e t e c t o r s , have been g r e a t l y improved). These advances make i t p o s s i b l e t o c a r e f u l l y c o n t r o l the c o n v e n t i o n a l l i q u i d chromatographic c o n d i tions. Furthermore, the i n c r e a s i n g commercial a v a i l a b i l i t y o f s y n t h e t i c s t a t i o n a r y phases, o r i g i n a l l y developed f o r HPLC, but adapted f o r low p r e s s u r e chromatography ( p a r t i c l e - f o r m , - s i z e and - d i s t r i b u t i o n ) has widened the a p p l i c a b i l i t y o f t h i s t r a d i t i o n a l t e c h n i q u e and the t e c h n i c a l d i f f e r e n c e s between t h e s e methods a r e d i m i n i s h i n g

(41,42). A l t h o u g h a l l modes o f column chromatography have been used i n the i s o l a t i o n o f u n d e r i v a t i z e d p o l a r c o n j u g a t e s , i o n exchange and p a r t i t i o n chromatography can be c o n s i d e r e d as modes o f f i r s t c h o i c e . C e l l u l o s e and d e x t r a n based weakly b a s i c a n i o n exchangers, such as the DEAE-type, a r e the most v e r s a t i l e and most commonly used i o n exchange columns. Two decades ago Knaak e t a l . (43) demonstrated e x c e l l e n t s e p a r a t i o n o f v a r i o u s g l u c u r o n i c a c i d and s u l f a t e e s t e r c o n j u g a t e s i n the u r i n e o f c a r b a r y l t r e a t e d r o d e n t s ( F i g u r e 2 ) . G r a d i e n t s formed by i n c r e a s i n g i o n i c s t r e n g t h ( c o n s t a n t pH) a r e p r e f e r a b l e to g r a d i e n t s formed by changing the pH. V o l a t i l e buff e r s , such as formates and a c e t a t e s o f p y r i d i n e and ammonium a r e u s u a l l y used because they can be e a s i l y removed from the e l u t e d fractions. However, the use o f n o n - i o n i c h y d r o p h o b i c r e s i n s and the s i l i c a bonded a l k y l phases o f f e r a c o n v e n i e n t a l t e r n a t i v e t o remove a l s o n o n - v o l a t i l e b u f f e r s . The s e p a r a t i o n o f c o n j u g a t e s from the u r i n e o f r a t s dosed w i t h the f u n g i c i d e p y r o q u i l o n i s p r e s e n t e d i n F i g u r e 3 (44). Chromatography on p o l y g l y c o s i d e based DEAE and QAE a n i o n exchangers has a l s o been used s u c c e s s f u l l y f o r the i s o l a t i o n o f v a r i o u s g l u t a t h i o n a t e pathway m e t a b o l i t e s (45-48). F i g u r e 4 demons t r a t e s the s e p a r a t i o n o f t h i s type o f m e t a b o l i t e s o f m e t h i d a t h i o n produced by r a t l i v e r i n - v i t r o experiments (47). C a t i o n exchange chromatography has been used o c c a s i o n a l l y , m a i n l y f o r amino a c i d c o n j u g a t e s , such as g l u t a t h i o n a t e s , g l u t a m y l c y s t e i n a t e s , m a l o n y l c y s t e i n a t e s , N - a c e t y l c y s t e i n a t e s and c y s t e i n a t e s (49-51). However, L a r s e n e t a l . (52) a l s o used i t f o r i s o l a t i n g S- and 0 - g l u c u r o n i d e s by e x p l o i t i n g the p r o p e r t i e s o f the c o r r e s p o n d i n g exocons. I n c o n t r a s t to a n i o n exchange chromatography changing the pH i s the most common way o f f o r m i n g a g r a d i e n t f o r the e l u t i o n o f c a t i o n exchange columns. I t s h o u l d be noted t h a t s t r o n g l y a c i d i c d i v i n y l b e n z e n e c r o s s - l i n k e d p o l y s t y r e n e based r e s i n s , such as Dowex 50, tend t o g e n e r a t e a r t i f a c t s when used to i s o l a t e a c i d l a b i l e conjugates. R e c e n t l y S j S v a l l e t a l . (53) proposed the use o f l i p o p h i l i c i o n exchangers f o r group s e p a r a t i o n o f c o n j u g a t e s . These exchangers can be e l u t e d w i t h o r g a n i c s o l v e n t s and s h o u l d be e s s e n t i a l l y d e v o i d


112


SAMPLE:

URINE (METABOLITES ENRICHED BY PRECIPITATION OF NATURAL INGREDIENTS)

COLUMN:

SEPHADEX LH-20 (2 SERIELLY CONNECTED COLUMNS: 2.6 x 57 AND 2.6 x 130 CM )

ELUENT:

CH OH / H 0 / 0.5 M TRIETHYLAMINE 3

C0

2

2

BUFFER/ PH 9.2


(100 / 100 / 2

l

230

v/v)

r — r — r

250

F i g u r e 1• LC o f m e t a b o l i t e s i n the u r i n e o f r a t s dosed w i t h l C] toluidine. (Reproduced w i t h p e r m i s s i o n from R e f . 40. C o p y r i g h t 1980, T a y l o r & F r a n c i s L t d . ) lh

F i g u r e 2. LC o f m e t a b o l i t e s i n t h e u r i n e o f g u i n e a - p i g s and r a t s dosed w i t h [ ^ C ] c a r b a r y l . (Reproduced w i t h p e r m i s s i o n from R e f . 43* C o p y r i g h t 1965, American C h e m i c a l S o c i e t y )


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SAMPLE;

SUBCELLULAR FRACTIONS OF RAT LIVER INCUBATED WITH METHIDATHION (ENRICHED BY PARTITION WITH C H C I 3 / FREEZE DRYING OF THE H 0 PHASE/ R E C O N S T I T U T E 2

IN H 0 )


2

COLUMN:

QAE-SEPHADEX A-25 (1.7 x 25 CM)

ELUENT:

LINEAR GRADIENT, KBr (0

+ 1 .0 M)

7600r

7200 -

DESMETHYL METHIOATHION

lk

Figure 4. LC of metabolites of l C] methidathion produced by rat l i v e r i n - v i t r o . (Reproduced with permission from Ref. 47. Copyright 1981, Academic Press Inc.)



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of non-ionic interactions. This very i n t e r e s t i n g approach has s t i l l to be validated under p r a c t i c a l conditions. Besides ion exchange, p a r t i t i o n chromatography i s the most powerful technique for i s o l a t i n g polar conjugates. The older technique f o r preparing a p a r t i t i o n column involved coating a deactivated c a r r i e r with the stationary phase. This usually resulted i n a rather l a b i l e system that was tedious to operate and i s now considered to be obsolete. The formation of a reversed phase p a r t i t i o n system i n - s i t u involves washing an appropriate c a r r i e r such as Amb e r l i t e XAD-2 with a polar solvent containing a small amount of a l i p o p h i l i c solvents. The l i p o p h i l i c solvent i s p r e f e r e n t i a l l y taken up by the resin, thus forming a stationary phase, that i s s t a b i l e , and gives high resolution reversed phase chromatographic separations (54,55)* Figure 5 i l l u s t r a t e s the use of this type of stationary phase for the separation of several glucuronides including two enantiomers i n the urine of man (55)* Correspondingly, using a polar c a r r i e r such as s i l i c a and washing with an unpolar solvent containing small amounts of a hydrophilic solvent leads to a "normal" phase p a r t i t i o n system. To date the l i t e r a t u r e contains only a few examples of the use of the non-ionic resins (e.g. Amberlite XAD-2, Amberlite XAD-4) and especially the organic phases covalently bound to porous s i l i c a f o r the preparative separation of conjugates by conventional l i q u i d column chromatography. A l k y l , amino, d i o l , n i t r i l , phenyl moieties have an inherently high potential f o r the preparative separation of polar conjugates. The use of corresponding HPLC systems to aid i n the optimation of the separation conditions for the low pressure chromatography i s an additional and very advantageous feature of these systems. Figure 6 i l l u s t r a t e s the use of RP 18 on a preparative scale to separate conjugates i n the urine of rats o r a l l y dosed with the safening agent 4,6-dichloro-2-phenyl-pyrimidine (44)* In the past size exclusion chromatography, u t i l i z i n g hydrophil i c as well as l i p o p h i l i c gels, such as Sephadex 6-10 and Sephadex LH-20 were used quite extensively for the penultimate and ultimate p u r i f i c a t i o n of i n d i v i d u a l conjugates. Some excellent separations of complex mixtures have been accomplished using these types of columns and reported i n the l i t e r a t u r e (39,40,56). However, the separation mode of these systems i s probably best described as part i t i o n chromatography. Thus these techniques have been e f f e c t i v e l y replaced by reversed phase chromatography on l i p o p h i l i c resins or s i l i c a bound phases. High Performance Liquid Chromatography Although HPLC was developed as an a n a l y t i c a l t o o l i t has proven to be the most powerful and v e r s a t i l e method invented during the l a s t decade f o r the i s o l a t i o n of metabolites, including polar conjugates, i n preparative amounts. The commercial a v a i l a b i l i t y of a large variety of stationary phases allows the application of a l l modes of l i q u i d chromatography. When used i n combination with adequate e l u ting solvents chromatographic systems f o r v i r t u a l l y a l l types of conjugates are provided. Increasing the size of the chromatography column and/or r e p e t i t i v e sample injections are used to overcome the



URINE

SAMPLE:

(METABOLITES ENRICHED BY AMBERLITE XAD-2/


COARSE GRADE) COLUMN:

AMBERLITE XAD-2/ MICRONIZED ( 1 2 pn) (1.3 x 33 CM)

ELUENT:

CH OH / H 0 / C H C I C H C I / CH3COOH 2

3

2

( 6 0 / 3 8 / 1 / 1

2

v/v)

n

m ^

NH-CH

3

Y ^ O - G L U C . AC.

II

- s (+)

I I I = R (-)

I

'

1

0

20

'

1

40

'

1

1

1

60 80 Retention volume (ml)

Figure 5. LC of metabolites i n the urine of man closed with [ 0] oxaprotiline. (Reproduced with permission from Ref. 55. Copyright 1984, Taylor & Francis Ltd.) lk



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5.

: . 1 : ...__._.].....

1 i.

1

!

i

i

1

!

:

I

:

'



118


rather low capacity of the method. High s e l e c t i v i t y can be achieved by the proper selection of the HPLC column and high resolution i s an inherent feature of this technique. Commercially available equipment i s designed to allow convenient and precise control of the chromatographic parameters. Therefore, the chromatographic conditions can be quickly and e f f i c i e n t l y optimized. Because the HPLC system i s a closed system, usually operated under mild conditions at ambient temperature with exclusion of l i g h t and a i r , a r t i f a c t format i o n i s usually not a serious problem. Therefore, despite the high price of the equipment, the technique i s highly economical. In order to maximize the u t i l i z a t i o n of these advantages, HPLC should be u t i l i z e d as early as possible i n the i s o l a t i o n process* Occas i o n a l l y , l i p o p h i l i c contaminants can be removed from the sample by solvent p a r t i t i o n , and the polar metabolites (enriched by non-ionic r e s i n extraction) can be d i r e c t l y applied to a HPLC column. However, i n complex mixtures or i n very crude samples, separation of i n d i v i d u a l metabolites or groups of metabolites u t i l i z i n g high capac i t y methods p r i o r to HPLC i s usually advisable f o r the sake of economy. The high resolution afforded tjy HPLC usually make i t the method of choice f o r the ultimate p u r i f i c a t i o n step. Reversed phase HPLC i s the most popular chromatographic method f o r p u r i f i c a t i o n of polar conjugates. The reversed phase technique i s commonly performed on s i l i c a bonded phases, such as RP 8 and RP 18. When these systems are used control of a variety of parameters i s of paramount importance. The s p e c i f i c properties of conjugates can be i n d i v i d u a l l y addressed i n the design of the chromatographic procedure. The control of the pH of the eluent i s of importance i n maintaining ionizable conjugates (and/or the other ionizable contaminants present i n the sample) i n the desired status to optimize separation. Precisely c o n t r o l l i n g the ionic strength of the eluent i s also important i n maintaining the metabolites i n the appropriate form to maximize resolution. By applying these p r i n c i p l e s i n a dedicated manner, v i r t u a l l y a l l low-molecular, polar conjugates can be separated and/or p u r i f i e d using these reversed phase systems. This also applies to the separation of isomers, including enantiomers. Reviewing the l i t e r a t u r e on the i s o l a t i o n of conjugates, reveals that the advantages of reverse phase HPLC has been exploited i n nearly a l l studies reported i n recent publications. S i l i c a bonded phases with functional groups (e.g. CN, NH , Diol) which can be operated as "normal" and "reversed" phase systems have not been extensively used f o r preparative purposes. Some preliminary experiments i n our laboratory indicated that complex conjugate mixtures can be separated using these types of columns but that the capacity and the resolution i s lower compared to RP 18 columns. When operating i n the "normal" phase mode, polar conjugates are eluted with e s s e n t i a l l y non-aqueous solvents. Unfortunately, polar compounds i n non-aqueous solvents have a strong tendency to s t i c k to the glass beads s c i n t i l l a t o r of the r a d i o a c t i v i t y detector causing unpleasant memory e f f e c t s . On the basis of the limited information availabe no s i g n i f i c a n t advantage of these "normal" phase systems i s obvious, however, more experience i s necessary to make a sound judgement on the usefulness of these systems for polar conjugates. 2


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Adsorption HPLC on s i l i c a phases has been occasionally used to i s o l a t e polar conjugates (57), but i t i s not generally recommended, as the desorption of polar compounds requires highly polar (mostly aqueous) solvents, which tend to deteriorate the stationary phases.


Other Methods The thin layer chromatography technique, now more than 30 years old, continues to deserve i t s reputation as a preparative t o o l f o r the i s o l a t i o n of polar conjugates. Reasons f o r i t s continuous popularity include that i t i s easy to use, the technique i s f a s t , the excellent p r e d i c t i b i l i t y of the results from a n a l y t i c a l runs, the low price of the equipment, and the high resolution of the method. Preparative TLC i s commonly performed using s i l i c a as the support, but other supports have been occasionally used as well (58). The method i s generally used for p r e p u r i f i c a t i o n rather than f o r separation of complex mixtures; generally an additional ultimate p u r i f i c a t i o n step i s required to remove the contaminants coextracted from the support. Preparative TLC has been used for a l l types of polar compounds, but precautions must be taken with l a b i l e conjugates, as the method i s prone to a r t i f a c t formation. Paper chromatography was occasionally used to i s o l a t e polar conjugates u n t i l recently, but the usage of this technique i s now diminishing. Gas chromatography has never gained recognition as a common tool for the i s o l a t i o n of polar conjugates. However, one laboratory ( i . e . the Metabolism and Radiation Research Laboratory, Fargo, N.D., USA) has had the patience and the eagerness to acquire the experience to successfully apply this technique to glucuronic acid, amino acid and glucose conjugates a f t e r appropriate d e r i v a t i zation (21,24,27,52,59). Nevertheless, looking through t h e i r recent publications, i t appears that these colleagues are now tending to replace gas chromatography by more common methods. The recently developed counter current chromatography, o r i g i n a t i n g from the counter current d i s t r i b u t i o n techique, i s i n i t s various technical modifications, p o t e n t i a l l y suitable for the i s o l a t i o n of polar xenobiotic conjugates. The f r a c t i o n a t i o n of polar plant constituents, including glycosides has been demonstrated (60-62). Also, separation of a mixture of the glucuronic acid, s u l f u r i c acid and glucose conjugate of p-nitrophenol using this approach has been reported (63). Although the high capacity of the method i s a very a t t r a c t i v e feature of counter current chromatography, e s p e c i a l l y for crude plant extracts, the comparatively low resolution of this method w i l l probably not allow i t to successfully compete against other established methods. The Ultimate P u r i f i c a t i o n After most of the contaminating materials have been removed from a compound an ultimate p u r i f i c a t i o n step i s required p r i o r to spectroscopic analysis. For polar conjugates t h i s most commonly implies HPLC i n the reversed phase mode. However, even when the f i n a l chromatographic procedure y i e l d s the metabolite i n highly p u r i f i e d form s p e c i a l considerations must be taken i n this f i n a l step including the succeeding transfer procedure.



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In addition to mass spectral analysis, proton NMR i s usually required f o r the unequivocal structure elucidation of most xenob i o t i c conjugates. Modern NMR instruments w i l l produce interpretable spectra from samples of about 10 yg. In contrast to MS, which may act as a separation technique tjy sequential v o l a t i l i z a t i o n of components i n a mixture, the NMR spectrum w i l l d i r e c t l y r e f l e c t the purity of the sample i n the NMR tube. This means, that impurities i n the sample w i l l jeopardize the assignment of a structure to the xenobiotic conjugate. Based on my experience, there are often more impurities brought into the sample during the ultimate p u r i f i c a t i o n and transfer procedure, than concomittantly isolated from the matrix. Having this i n mind, I w i l l devote the remainder of t h i s presentation to a discussion of procedures used i n our laboratory to minimize contamination of the sample during these f i n a l procedures. The manufacturers of the high quality solvents commonly used i n HPLC guarantee

Isolation of Xenobiotic Conjugates - ACS Publications - American

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