Xenobiotic Conjugation Chemistry - American Chemical Society

7 Glucuronic Acid, Sulfate Ester, and Glutathione Xenobiotic Conjugates Analysis by Mass Spectrometry 1

Catherine Fenselau and Lauren Yellet

Downloaded by UNIV LAVAL on September 23, 2015 | http://pubs.acs.org Publication Date: January 24, 1986 | doi: 10.1021/bk-1986-0299.ch007

Department of Pharmacology, Johns Hopkins University School of Medicine, Baltimore, MD 21205

The potential of the newer desorption ionization methods f o r analysis of glucuronic acid, sulfate ester, and glutathione xenobiotic conjugates by mass spectrometry i s discussed. Synthesis and analyses of six new glucuronides conjugated through oxime or carbinolamine functional groups are presented by way of demonstrating the strengths and limitations of fast atom bombardment mass spectrometry.

Historically mass spectrometric analysis has required that samples be v o l a t i l e . This has limited the application of this important analytical technique in structure studies of conjugated metabolites to v o l a t i l e derivatives. The widely realized potential of electron impact ionization, chemical ionization, and gas chromatography mass spectrometry for the analysis of glucuronides derivatized as acetates, methyl ethers and t r i m e t h y l s i l y l ethers has been reviewed (1). Derivatized sulfate esters (2,3) and glutathione conjugates (T,5) have been analyzed by these techniques only rarely. Attempts to analyse underivatized sulfate and glutathione conjugates by electron impact or chemical ionization, (including so-called direct chemical ionization) have resulted in analysis of pyrolysis products. 'Current address: A. D. Little, 15 Acorn Park, Cambridge, MA 02138

0097-6156/ 86/ 0299-0159506.00/ 0 © 1986 American Chemical Society

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

160

XENOBIOTIC CONJUGATION CHEMISTRY


In r e c e n t y e a r s , s e v e r a l t e c h n i q u e s have been developed f o r mass s p e c t r o m e t r y , whereby samples are i o n i z e d and a n a l y s e d from a condensed phase, w i t h o u t p r i o r v o l a t i l i z a t i o n . These d e s o r p t i o n t e c h n i q u e s have p e r m i t t e d t h e e x t e n s i o n o f mass s p e c t r o m e t r i c a n a l y s e s t o s u l f a t e and g l u t a t h i o n e c o n j u g a t e s , as w e l l as t o u n d e r i v a t i z e d and l a b i l e g l u c u r o n i c a c i d c o n j u g a t e s . Primary among t h e s e t e c h n i q u e s a r e f i e l d d e s o r p t i o n ( 6 ) , plasma d e s o r p t i o n (_7), l a s e r d e s o r p t i o n ( 8 ) , f a s t atom bombardment ( o r secondary i o n mass s p e c t r o m e t r y w i t h a l i q u i d sample m a t r i x ) (9) and thermospray i o n i z a t i o n ( 1 0 ) . The l a t t e r can a l s o s e r v e t o c o u p l e high p r e s s u r e l i q u i d chromatography and mass s p e c t r o m e t r y f o r a n a l y s i s o f i n v o l a t i l e and t h e r m a l l y l a b i l e samples. A number o f a u t h o r s have p o i n t e d out t h a t s p e c t r a a c q u i r e d u s i n g v a r i o u s d e s o r p t i o n t e c h n i q u e s have many f e a t u r e s i n common (11-14). G e n e r a l l y t h e i o n s d e t e c t e d are even e l e c t r o ^ i o n | : m o l e c u l a r i o n s formed by p r o t o n a t i o n o r a d d i t i o n o f NH- , Na , e t c . , and fragment i o n s formed by e l i m i n a t i o n o f n e u t r a l m o l e c u l e s . I t i s not c l e a r t o what e x t e n t p y r o l y s i s and s o l v o l y s i s r e a c t i o n s augment t h e c o n t r i b u t i o n s o f u n i m o l e c u l a r gas phase d e c o m p o s i t i o n s to spectra obtained using the various desorption techniques. G l u c u r o n i c A c i d Conjugate D e s o r p t i o n F e a t u r e s common t o t h e s p e c t r a o f g l u c u r o n i c a c i d c o n j u g a t e s a n a l y s e d by FAB, l a s e r and f i e l d d e s o r p t i o n were summarized s e v e r a l y e a r s ago ( 1 5 ) . These appear t o hold as w e l l as f o r plasma d e s o r p t i o n and thermospray s p e c t r a more r e c e n t l y examined. The s i t u a t i o n w i t h thermospray i s somewhat more c o m p l i c a t e d as w i l l be discussed l a t e r . Generally speaking, p o s i t i v e ion spectra contain p r o t o n a t e d , n a t M a t e d or analogous m o l e c u l a r i o n s s p e c i e s , and u s u a l l y (M+H-176) i o n s formed by t h e e l i m i n a t i o n o f n e u t r a l dehydroglucuronic a c i d . The (M-H)~ a n i o n s d e t e c t e d i n n e g a t i v e i o n d e s o r p t i o n s p e c t r a a l s o e l i m i n a t e d e h y d r o g l u c u r o n i c a c i d t o from (M-H-176)" o r (M-177)" i o n s . Anions c o m p r i s i n g t h e g l u c u r o n i c a c i d moiety (mass 193) a r e a l s o commonly o b s e r v e d . Examples o f l a s e r d e s o r p t i o n (16) and plasma d e s o r p t i o n s p e c t r a o f u n d e r v a t i z e d g l u c u r o n i c a c i d c o n j u g a t e s are shown i n F i g u r e s 1 and 2. The n a t u r e o f t h e anions and c a t i o n s observed i n FAB s p e c t r a formed by c l e a v a g e s i n the a c e t a l o r g l y c o s i d i c f u n c t i o n a l group have been found t o c o r r e l a t e w i t h the n a t u r e o f t h e a g l y c o n bond c o n j u g a t e d ( 1 5 ) . These c o r r e l a t i o n s a r e proposed i n scheme 1. D e s p i t e o u t s t a n d i n g FD measurements i n l a b o r a t o r i e s at Mainz and a few o t h e r p l a c e s , a n a l y s i s o f g l u c u r o n i c a c i d c o n j u g a t e s by FAB i s g e n e r a l l y more r e l i a b l e than FD a n a l y s e s . However, FAB i s a l s o not c o m p l e t e l y r e l i a b l e . We have worked w i t h some g l u c u r o n i c a c i d c o n j u g a t e s which p r o v i d e d n e i t h e r an anion o r c a t i o n spectrum from a v a r i e t y o f FAB m a t r i c e s . In one o f t h e s e cases t h e a n a l y s i s has been o b t a i n e d s u c c e s s f u l l y by thermospray. In s e v e r a l o t h e r i n s t a n c e s l a s e r d e s o r p t i o n has been s t r a i g h t f o r w a r d . Plasma d e s o r p t i o n u s i n g f i s s i o n fragments from Cf-252 has a l s o proven s u c c e s s f u l i n some i n s t a n c e s where FAB has failed. In t h e i n s t a n c e s where l a s e r and plasma d e s o r p t i o n were more s u c c e s s f u l , i t i s p o s s i b l e t h a t t h e FAB a n a l y s i s was



FENSELAU AND YELLET

Analysis of Glucuronic Acid by MS

M/Z F i g u r e 1. L a s e r d e s o r p t i o n mass spectrum o f t h e g l u c u r o n i c a c i d c o n j u g a t e o f 1 - n a p h t h y l a c e t i c a c i d ( 6 6 ) . The spectrum p r o v i d e d by Dr. Robert C o t t e r .



V O H

?

HO-^^T^Ô—CH-CH*0

\ H ,

1001 Downloaded by UNIV LAVAL on September 23, 2015 | http://pubs.acs.org Publication Date: January 24, 1986 | doi: 10.1021/bk-1986-0299.ch007

NH-CH

H

260 (MH-176)

«0 e

>

50 436 (fHO*

. 1

LA

,

m/z F i g u r e 2. Plasma d e s o r p t i o n mass spectrum o f g l u c u r o n i c a c i d conjugates of propranolol glucuronides. The spectrum i s p r o v i d e d by Dr. Robert C o t t e r .

Fast atom bombardment COOH

0 OH

O

Alcohols Phenols Carboxy Acids Quaternary Amines

MH-176

M-177

MH-192

m/z 193

Carbinolamlnes

MH-176 MH-192

M-177 M-193 m/z 193

Oxlmes N-hydroxy Amides

OH

COOH -o

^-OH OH

OH

BOTH LOSSES

t

Scheme 1


7. FENSELAU AND YELLET


163

confounded by s o l u t i o n phenomena, p r o b a b l y t h e absence ot s u f f i c i e n t sample m o l e c u l e s on t h e s u r f a c e o f t h e l i q u i d m a t r i x . In the second h a l f o f t h i s c h a p t e r we d i s c u s s i n more d e t a i l t h e high p o t e n t i a l o f f a s t atom bombardment, d e m o n s t r a t i n g a p p l i c a t i o n s t o a n a l y s i s o f some new g l u c u r o n i c a c i d c o n j u g a t e s r e c e n t l y s y n t h e s i z e d i n our l a b o r a t o r y .


Sulfate

Desorption

E a r l y d e s o r p t i o n s t u d i e s o f s u l f a t e e s t e r s (17) and t h e s e v e r a l f i e l d d e s o r p t i o n papers (18-22) c o n f i r m t h a t c a t i o n i z e d m o l e c u l a r i o n s can p r o v i d e m o l e c u l a r weight i n f o r m a t i o n , but a l s o suggest t h a t m u l t i p l y charged i o n s , s a l t c l u s t e r s , p y r o l y s i s and i r r e p r o d u c e a b i l i t y confound a n a l y s i s o f desorbed c a t i o n s . Fast atom bombardment has been r e p o r t e d t o be more e f f e c t i v e (23-26) a l t h o u g h c a t i o n i z a t i o n by a l k a l i e a r t h m e t a l s as w e l l as p r o t o n a t i o n produces redundant m o l e c u l a r i o n s p e c i e s i n p o s i t i v e i o n s p e c t r a . One group o f a u t h o r s (23) d e s c r i b e p o s i t i v e i o n s p e c t r a as p o o r l y r e p r o d u c e a b l e and "of low i n t e n s i t y , and agreement seems t o e x i s t t h a t n e g a t i v e i o n s p e c t r a are s i m p l e r , independent o f t h e presence o f m i x t u r e s o f a l k a l i e a r t h c o u n t e r i o n s and more r e p r o d u c e a b l e . Background s u b t r a c t i o n t e c h n i q u e s have been a p p l i e d t o i n c r e a s e s e n s i t i v i t y ( 2 3 , 2 4 ) . D e r i v a t i z a t i o n has been suggested as a way t o i n c r e a s e t h e m o l e c u l a r weight o f t h e s u l f a t e and move i t away from g l y c e r o l m a t r i x i o n s ( 2 3 ) . Sample p r e p a r a t i o n has been found t o be i m p o r t a n t t o c o n t r b T s i g n a l s u p p r e s s i o n by i m p u r i t i e s i n a c h i e v i n g f u l l scans on 15 ng samples and q u a n t i t a t i o n w i t h s t a b l e i s o t o p e l a b e l l e d i n t e r n a l standards ( 2 3 ) . The FAB spectrum o f t h e mixed a n h y d r i d e s u l f a t e e s t e r c o n j u g a t e o f indomethacin i s shown i n F i g u r e 3. Glutathione

Desorption

F i e l d d e s o r p t i o n has p r o v i d e d s a t i s f a c t o r y a n a l y s e s o f a number o f g l u t a t h i o n e c o n j u g a t e s ( 2 2 , 27-34) i n c l u d i n g a d i g l u t a t h i o n e formed i n the metabolism o f c h o l o r f o r m , ( 2 8 ) ^ Abundant molecular^ i o n s p e c i e s are o b s e r v e d , v a r y i n g among M , (M+H) and (M+Na) . Class c h a r a c t e r i s t i c f r a g m e n t a t i o n o c c u r s on one s i d e o r t h e o t h e r o f t h e t h i o e t h e r l i n k a g e , o r b o t h , g e n e r a t i n g some or a l l o f t h e c a t i o n s shown i n scheme 2. Onkenhout and c o l l e a g u e s have p o i n t e d out t h a t t h e s e same decomposition r e a c t i o n s a l s o occur during p y r o l y s i s (33). C e r t a i n l y t h e p o s s i b i l i t y o f p y r o l y t i c c o n t r i b u t i o n s can not be excluded i n most c a s e s . However t h e o b s e r v a t i o n o f a t l e a s t one o f t h e s e r e t r o - M i c h a e l f r a g m e n t a t i o n s induced by c o l l i s i o n a l a c t i v a t i o n s (22) a l s o argues f o r t h e i r occurence independent o f pyrolysis. F r e q u e n t l y cleavage^ i n t h e C y s - G l u bond produces c a t i o n s o f mass 130 and/or (MH-130) , which can be viewed as c o n f i r m i n g t h e presence of a g l u t a t h i o n e m o i e t y . One r e p o r t suggests 200 ng as a r e a s o n a b l e sample s i z e ( 3 0 ) . G l u t a t h i o n e c o n j u g a t e s are a l s o analysed r e a d i l y by f a s t atom bombardment and by plasma d e s o r p t i o n , as e i t h e r anions o r c a t i o n s ( 3 5 - 3 9 ) . In a d d i t i o n t o abundant m o l e c u l a r i o n s ( F i g . 4) f a s t atom



164


F i g u r e 3. F a s t atom bombardment mass spectrum o f indomethacin a c y l hydrogen s u l f a t e ( 6 7 ) .

R-S-G

R-s-H

^

^H-S-C (m/z 307)?

(M-273)?

RH (MH-306)* Scheme 2

6SCH2CH2 ^

847 (M + H)

____

r-X2.5

N-^V-CHgCHCOON GSCH CHo 9

^

NH

2

I

883

I 869 I 500 M/Z

550

600 800

850

F i g u r e 4 . Fast atom bombardment mass spectrum o f t h e d i g l u t a t h i o n e c o n j u g a t e o f p h e n y l a l a n i n e mustard ( 6 8 ) .




165


bombardment promotes some f r a g m e n t a t i o n analogous t o f i e l d d e s o r p t i o n , n o t a b l y f o r m a t i o n o f c l a s s c h a r a c t e r i s t i c (MH-306) cations (38). The complementary c a t i o n s o f mass 308 are sometimes formed as w e l l , and i n n e g a t i v e i o n s p e c t r a anions o f mass 306 are o b s e r v e d , r e f l e c t i n g t h e presence o f t h e g l u t a t h i o n e m o i e t y . A comparison o f the e f f i c a c y o f s e v e r a l l i q u i d m a t r i c e s has l e d t h e a u t h o r s t o recommend t h e a n a l y s i s o f p o s i t i v e i o n s from t h i o g l y c e r o l as a r e l i a b l e general method. Fast atom bombardment has a l s o been used s u c c e s s f u l l y w i t h m e t a b o l i t e s r e l a t e d t o o r d e r i v e d from g l u t a t h i o n e c o n j u g a t e s , e . g . a homoglutathione soybean m e t a b o l i t e (40) and mammalian mercapturates and c y s t e i n y l c o n j u g a t e s (24, 4 1 - 4 4 ) . Ion E v a p o r a t i o n and LCMS Among t h e s e v e r a l types o f i n t e r f a c e s c o m m e r c i a l l y a v a i l a b l e f o r c o u p l i n g high p r e s s u r e l i q u i d chromatography and mass s p e c t r o m e t r y , t h e moving b e l t s (45) d i r e c t l i q u i d i n j e c t i o n (46) and thermospray ( i o n e v a p o r a t i o n ) ~(T4,47) systems have been u s e S T s u c c e s s f u l l y t o analyze underivatized glucuronic acid conjugates. S i n c e moving b e l t and d i r e c t i n j e c t i o n i n t e r f a c e s r e q u i r e at l e a s t m a r g i n a l l y v o l a t i l e samples, t h e y have been l e s s r e a d i l y a p p l i c a b l e t o s u l f a t e and g l u t a t h i o n e c o n j u g a t e s . G l u t a t h i o n e and i t s c o n j u g a t e s have been i o n i z e d by thermospray (38,48) and f i e l d induced i o n evaporation (49). Thermospray ( 1 0 ) , e l e c t r o s p r a y (50) and f i e l d induced i o n e v a p o r a t i o n ( 4 9 ) , varTants on t h e same mechanism ( 5 1 , 5 2 ) , appear t o o f f e r a t r u e d e s o r p t i o n t e c h n i q u e , where i o n s are formed i n the condensed phase and s u b s e q u e n t l y evaporated i n t o t h e gas phase. A s e p a r a t i o n o f d i a s t e r e o m e r i c p r o p r a n o l o l g l u c u r o n i d e s by r e v e r s e d phase HPLC i s shown i n F i g u r e 5. The chromatogram recorded by UV d e t e c t i o n can be compared w i t h chromatograms recorded by a thermospray i n t e r f a c e d mass s p e c t r o m e t e r . The p o t e n t i a l uses o f t o t a l i o n c u r r e n t chromatograms, mass chromatograms and s e l e c t e d i o n m o n i t o r i n g p a r a l l e l s t h e i r e s t a b l i s h e d u t i l i t y i n GCMS ( 5 3 ) . Both p o s i t i v e and n e g a t i v e i o n s are produced. G l u c u r o n i d e s were used t o i l l u s t r a t e t h e p o i n t t h a t the s e n s i t i v i t y o f thermospray (and l i k e l y , e l e c t r o s p r a y and f i e l d induced i o n e v a p o r a t i o n as w e l l ) v a r i e s from compound t o compound (47). Subsequently s e n s i t i v i t y f o r p o s i t i v e i o n s has been c o r r e l a t e d w i t h proton a f f i n i t y i n t h e gas phase ( 1 4 ) . Some o f t h e fragment ions may be formed by h i g h t e m p e r a t u r e r e a c t i o n s w i t h t h e v o l a t i l e b u f f e r ( u s u a l l y ammonium a c e t a t e ) r e q u i r e d f o r thermospray i o n i z a t i o n (10). C o n s i s t e n t w i t h t h e f r a g m e n t a t i o n scheme general t o d e s o r p t i o n t e c h n i q u e s , (M+NH.-176) i o n s are i m p o r t a n t i n t h e c a t i o n s p e c t r a , as w e l l as t h e c l a s s c h a r a c t e r i s t i c i o n o f mass 194, ammoniated g l u c u r o n i c a c i d . Less f r a g m e n t a t i o n i s observed i n anion s p e c t r a . The e x t e n t o f f r a g m e n t a t i o n o f g l u c u r o n i d e s i n thermospray i s dependent i n p a r t on t h e b u f f e r c o n c e n t r a t i o n and on the temperature. In a comparison o f thermospray w i t h FAB ( 1 4 ) , ammonium a c e t a t e was used i n t h e FAB m a t r i x as w e l l as tHe thermospray buffer. I t s presence s i g n i f i c a n t l y a l t e r s the c a t i o n FAB s p e c t r a +


166


P R O P R A N O L O L

-• jd —

D - G L U C U R O N I D t

( 2 Dl ASTEREOMERS)

ULTR ASPHERE OOS 15 cm 4 5 ° / M E 0 H : 5 5 % AMMONIUM ACETATE o

PROTONATED


M/Z 260

.05M

AGLYCON

*C-OH H O - ^ ^ V ^ ^ O — CH-CHÔH 16

.20

4 7 % M E 0 H : 53/o AMMONIUM ACETATE

0.1 M

I ML /MINUTE

OMIN.

20

F i g u r e 5. High p r e s s u r e l i q u i d chromatograms o f p r o p r a n o l o l g l u c u r o n i d e s r e c o r d e d by thermospray mass s p e c t r o m e t r y and u l t r a v i o l e t spectroscopy (47). Upper p a n e l : s e l e c t e d i o n p r o f i l e o f (MH-176) . MidHTe p a n e l : s e l e c t e d i o n p r o f i l e o f (MH) . Lower p a n e l : p r o f i l e o f a b s o r p t i o n at 280 nm.


7.

FENSELAU AND YELLET


167

of glucuronic a c i d conjugates of non-basic aglycons, providing (M+NH4) i o n s w i t h improved s i g n a l t o n o i s e r a t i o s and s e n s i t i v i t i e s compared t o (M+H) i o n s generated i n g l y c e r o l o r t h i o g l y c e r o l w i t h o u t ammonium a c e t a t e ( 5 4 ) .


FAB A n a l y s i s o f G l u c u r o n i d e s o f C a r b i n o l a m i n e s and Oximes. Numerous s t u d i e s have been conducted t o i n v e s t i g a t e t h e m e t a b o l i c o x y g e n a t i o n o f C-N s y s t e m s , due t o t h e widespread o c c u r r e n c e o f t h i s type o f s t u c t u r e i n p h a r m a c e u t i c a l agents and p e s t i c i d e s , and a l s o because o f t h e p o t e n t i a l t o x i c o l o g i c a l and p h a r m a c o l o g i c a l p r o p e r t i e s which N-oxygenated compounds p o s s e s . O x i d i z e d m e t a b o l i t e s i n c l u d e h y d r o x y l a m i n e s , oximes and c a r b i n o l a m i n e s . A l l o f t h e s e can be c o n j u g a t e d w i t h g l u c u r o n i c a c i d , which may r e s u l t i n d e t o x i f i c a t i o n . However, some o f t h e s e N-O-glucuronides are v e r y r e a c t i v e , w i t h the g l u c u r o n i c a c i d moiety a c t i n g as a good l e a v i n g group. In o r d e r t o examine t h e pharmacology o f t h e s e g l u c u r o n i d e s , r e p r e s e n t a t i v e C-N c o n t a i n i n g m e t a b o l i t e s have been o b t a i n e d and conjugated w i t h g l u c u r o n i c a c i d ; t h e s t a b i l i t y , chromatographic and mass s p e c t r a l c h a c t e r i s t i e s o f t h e s e compounds have been s t u d i e d . A s t u d y o f the O - g l u c u r o n i d e s o f a s e t o f hydroxy a c e t y l a r y l a m i n e s has been p u b l i s h e d ( 5 5 ) . The c h a r a c t e r i z a t i o n by f a s t atom bombardment o f c a r b i n o l a m i n e g l u c u r o n i d e s d e r i v e d from t h e h e r b i c i d e diphenamid (56) t h e a n t i t u m o r agent hexamethylmelamine (57) and t h e Q h o l T n e s t e r a s e r e a c t i v a t o r 2 - p r a l i d o x i m e (58) i s d i s c u s s e d h e r e , along w i t h t h e c h a r a c t e r i z a t i o n o f g l u c u r o n i d e s o f phenyl acetone oxime (a m e t a b o l i t e o f amphetamine) (59,60) acetophenone oxime (61,62) and t h e a n t i v i r a l agent e n v i r o x i m e ( 6 3 ) . The f i r s t two c a r b i n o l a m i n e s named were s y n t h e s i z e d u s i n g s e q u e n t i a l c a t a l y s i s by cytochrome P-450 oxygenase and g l u c u r o n y l t r a n s f e r a s e i m m o b i l i z e d from r a b b i t l i v e r microsomes onto sepharose beads (64,65) as shown i n scheme 3. 2 - P r a l i d o x i m e c h l o r i d e was a l l o w e d t o decompose at pH 7.4 i n t h e presence o f i m m o b i l i z e d UDP-glucuronyl t r a n s f e r a s e and the c o f a c t o r u r i d i n e diphosphoglucuronic a c i d to obtain the conjugated c a r b i n o l i m i n e shown i n the scheme. C o n j u g a t i o n o f t h e t h r e e oximes shown i n scheme 4 was c a t a l y s e d by i m m o b i l i z e d t r a n s f e r a s e enzymes ( 6 5 ) . Conjugates were p u r i f i e d by e x t r a c t i o n and chromatography. The e l e c t r o n impact spectrum o f t h e v o l a t i l e p e r ( t r i m e t h y l ) s i l y l a t e d d e r i v a t i v e o f e n v i r o x i m e g l u c u r o n i d e i s shown i n F i g u r e 6. The m o l e c u l a r i o n i s v i s i b l e , p r o v i d i n g a m o l e c u l a r weight which can be c o r r e c t e d f o r t r i m e t h y l s i y l groups i n t h e case o f an unknown sample by a n a l y z i n g a second p o r t i o n o f t h e sample d e r i v a t i z e d w i t h d g - t r i m e t h y l s i l y l groups { ! ) . The i n t e n s e peak at m/z 375 i s c h a r a c t e r i s t i c o f t r i m e t h y l s i l y l a t e d g l u c u r o n i d e s as a c l a s s ( 1 ) . The l o s s o f 481 mass u n i t s i s c h a r a c t e r i s t i c o f t r i m e t h y l s i l y a t e d g l u c u r o n i c a c i d l i n k e d to a hydroxyl or other o x y g e n - c o n t a i n i n g f u n c t i o n a l group. The analogous fragment l o s t from an amine l i n k e d g l u c u r o n i d e would have a d i f f e r e n t mass. This argues f o r c o n j u g a t i o n o f t h e hydroxyl groups o f e n v i r o x i m e and not t h e amiô group. The presence o f (M-481) i o n s and t h e absence o f (M-392) i o n s i s s u s t a i n e d i n s p e c t r a o f t h e o t h e r two oxime g l u c u r o n i d e s as w e l l , and may t h e r e f o r e be i n d i c a t i v e o f oxime c o n j u g a t i o n . T h i s e l e c t r o n impact a n a l y s i s does r e q u i r e


168


HOOC .CH, P-ASO OXYGENASE

CHj

N


hexamethylmelamine

dyphenamid

DEGRADATION

HON-C-

P

H

7

'

HOOC GLUCURONYL TRANSFERASE

t

4

CH cr 3

HO^N

H O ^ - O ^ g

CH CI

S

3

OH

2-pralidoxime chloride Scheme 3

acetophenone oxime glucuronide

amphetamine oxime glucuronide Scheme 4


HjCl




169

d e r i v a t i z a t i o n o f the g l u c u r o n i d e . However, t h i s approach i s a l s o c o m p a t i b l e w i t h gas chromatography and combined gas chromatography mass s p e c t r o m e t r y . P o s i t i v e and n e g a t i v e i o n s p e c t r a o f u n d e r i v a t i z e d e n v i r o x i m e g l u c u r o n i d e o b t a i n e d by f a s t atom bombardment mass s p e c t r o m e t r y are shown i n F i g u r e s 7 and 8. These were run u s i n g a g l y c e r o l m a t r i x . T y p i c a ^ o f many FAB s p e c t r a , t h e c a t i o n spectrum r e v e a l s (M+H) (M+Na) at m/z 557 and (M+K) a t m/z 573, a l l even e l e c t r o n molecular ion species. Even e l e c t r o n fragment i o n s a r e observed n o t a b l y (M+H-176) a t m/z 359 and (M+H-192) a t m/z 343. The anion spectrum c o n t a i n s an (M-H) peak at m/z 533 unaccompanied by a n a t r i a t e d s a t a l l i t e . T h i s and o t h e r peaks i n t h e anion spectrum appear t o correspond t o t h e same i o n s seen i n t h e c a t i o n s p e c t r u m , however two mass u n i t s l i g h t e r . The (M-H-176)" o r (m-177) ion r e f l e c t s a c l a s s c h a r a c t e r i s t i c , f r a g m e n t a t i o n . Ions o f mass 193 presumeably comprise anions o f t h e g l u c u r o n i c a c i d m o i e t y . Anions were recorded w i t h g r e a t e r s e n s i t i v i t y than c a t i o n s i n t h i s c a s e . F i g u r e 9 c o n t a i n s t h e FAB c a t i o n spectrum ( g l y c e r o l ) o f t h e g l u c u r o n i c a c i d c o n j u g a t e o f t h e h y d r o l y s i s product o f 2 - p r a l i d o x i m e . In a d d i t i o n t o being a c a r b i n o l i m i n e , t h i s m e t a b o l i t e a l s o has q u a t e r n a r y ammonium c e n t e r . T h i s permanent charge r e s u l t s i n f a c i l e d e t e c t i o n o f m o l e c u l a r c a t i o n s . Anions a r e much h a r d e r t o r e c o r d . I n t e r e s t i n g l y no fragment i o n s are formed under the c o n d i t i o n s used t o r e c o r d F i g u r e 9. S p e c t r a o f p o s i t i v e and n e g a t i v e i o n s formed from t h e g l u c u r o n i d e o f hydroxydiphenamid (a c a r b i n o l a m i d e ) , by f a s t atom bombardment w i t h g l y c e r o l m a t r i c e s , are shown i n F i g u r e s 10 and 1 1 . Again the anion spectrum can be recorded w i t h l e s s sample. As i n t h e case o f e n v i r o x i m e g l u c u r o n i d e , a n i o n s c o m p r i s i n g t h e g l u c u r o n i c a c i d moiety a r e d e t e c t e d (mass 1 9 3 ) , and t h e l o s s o f the g l u c u r o n i c a c i d , (M+H-192) i s found i n t h e c a t i o n spectrum. Both o f t h e s e are c l a s s c h a r a c t e r i s t i c i o n s . Cleavage r e s u l t i n g i n r e t e n t i o n o f both oxygen atoms by t h e g l u c u r o n i c a c i d m o i e t y (mass 192,193 or 194) combined w i t h t h e absence o f mass 177 or (M+H-176) i o n s may be c h a r a c t e r i s t i c o f c a r b i n o l a m i n e c o n j u g a t i o n . Enzymatic oxygenation i n t h e d i p h e n y l m e t h i n e m o i e t y can be r u l e d out by o b s e r v a t i o n o f t h e peak at m/z 167 i n t h e anion specrum. The l a s t FAB s p e c t r u m , F i g u r e 12, i s t h a t o f hydroxyhexamethylmelamine g l u c u r o n i d e measured w i t h a t h i o g l y c e r o l m a t r i x . The p r o t o n a t e d m o l e c u l a r i o n i s o b s e r v e d , as w e l l as t h e c l a s s c h a r a c t e r i s t i c i o n (M+H-194) o f mass 20£. When t h i s a n a l y s i s was run from a g l y c e r o ^ m a t r i x , (M-H) i o n s were desorbed i n g r e a t e r abundance than (M+H) ions. In a d d i t i o n t o p r e s e n t i n g new o b s e r v a t i o n s on t h e a n a l y s i s o f some novel g l u c u r o n i d e s c o n j u g a t e d t o oxime and c a r b i n o l a m i n e f u n c t i o n a l groups, t h i s d i s c u s s i o n i s intended to i l l u s t r a t e the p o t e n t i a l and l i m i t a t i o n s o f ^ f a s t atom bombardment, t h e most w i d e l y used of the new d e s o r p t i o n mass s p e c t r o m e t r y t e c h n i q u e s . Molecular weights may be o b t a i n e d and c o n f i r m e d by redundant s p e c i e s . Class c h a r a c t e r i s t i c f r a g m e n t a t i o n may support i d e n t i f i c a t i o n as a glucuronide. In some cases f r a g m e n t a t i o n w i t h i n t h e aglycon may p r o v i d e an i n d i c a t i o n o f t h e s i t e o f m e t a b o l i c o x y g e n a t i o n and conjugation. I t appears t h a t c o n j u g a t i o n w i t h d i f f e r e n t f u n c t i o n a l groups l e a d s t o d i f f e r e n t p a t t e r n s o f c l e a v a g e w i t h i n the


170


SMT-OOC SMTO

N

100 -1

O-TMS

375

217

-X

10


1 50

*

-1

-XÂ / A ^ / r l 220

240

270

290

370

//Xr-J//A 380

410

430

500 510

trX780

790

820

830

870

M/Z

F i g u r e 6. E l e c t r o n impact mass spectrum o f t r i m e t h y l s i l y l a t e d enviroxime glucuronide. >NH

359 100'

(M^-175) 359 235

x2

GH+ -106-194)

535

50-J

253

0^-106-176)

2

(ttf-192) 343 (!U*-106)

J

-

• iJllllllll^JjlJl, 250

T// 290

557 573

429

i — r * r 340

380

420

440

530

55G

570

F i g u r e 7. P o s i t i v e i o n f a s t atom bombardment mass spectrum o f enviroxime glucuronide.


2


7.


FENSELAU AND YELLET

160

200

230

260

350

370

171

390

430

530

F i g u r e 8. Negative i o n f a s t atom bombardment mass spectrum of enviroxime glucuronide.

100

100

150

ZC0

250

F i g u r e 9. Fast atom bombardment mass spectrum o f t h e g l u c u r o n i d e o f t h e c a r b i n o l a m i n e d e r i v e d from p r a l i d o x i m e chloride.


540

172


inn

240

xlO

H


50

3 eg

452

i 210

I

I

T

I

i

i

i

i

f~~~i 400

i

i

i 450

500

M/Z

F i g u r e 10. P o s i t i v e i o n f a s t atom bombardment mass spectrum o f hydroxydiphenamide g l u c u r o n i d e .

167

100

OH

430

^

193

to

I

50 _

i

H

1^

T

1

T—I

250

200

M/Z

F i g u r e 11. Negative i o n f a s t atom bombardment mass spectrum o f hydroxydiphenamide g l u c u r o n i d e .


1 450

FENSELAU AND YELLET


100-1


-X20

H00C

403

50(MH -194) 209

—I—i—i—i—nfn—r —i—i 250 340 M/Z 1

200

1—i—i—r»—i—r 400

F i g u r e 12. Fast atom bombardment mass spectrum o f hydroxyhexamethylmelamine g l u c u r o n i d e .


174


glycosidic acetal group. When both can be obtained, anion and cation spectra provide complimentary and reinforcing information. Summary


All of the more common conjugated xenobiotic metabolites are now known to be susceptible to mass spectral analysis using one or another of the desorption techniques. An investigator can expect to obtain molecular weight information and, in most cases, some structural information from fragmentation. Sensitivities currently range between 1 ng and 1 ug. Liquid chromatography mass spectrometry can be utilized as well as gas chromatography mass spectrometry. Acknowledgments We thank Donald Delong, L i l l y Research Laboratories, for a sample of enviroxime, Patrick Callery, University of Maryland, School of Pharmacy, for a sample of the oxime of phenyl acetone; Robert J . Cotter, Dan Liberato, Gordon Hansen, Jeff Honovich, Ron Robbins, Carol Lisek, Mehrshid Alai and Deanne Dulik for assistance with mass spectra, especially those in Figures 1-5. This research was supported by USPHS grants NIH GM-21248 and GM07626. Literature Cited 1. Fenselau, C.; Johnson, L.P. Drug Metab. Disp., 1980, 8, 274-283. 2. Paulson, G.; Bakke, J.; Giddings, J.; Simpson, M. Biomed. Mass Spectrom. 1978, 5, 128-132. 3. Paulson, G.; Simpson, M.; Giddings, J.; Bakke, J.; Stolzenberg, G.; Biomed. Mass Spectrom. 1978, 5, 413-417. 4. Damon, M.; Chavis, C.; Godard, P.; Michel, F.B.; Crastes de Paulet, A. Biochem. Biophys. Research Commun. 1983, 111, 518-524. 5. Maas, R.L.; Lawson, J.A.; Brash, A.R.; Oates, J.A. Adv. Prostag. Thrombox. and Leukotr. Res. 1983, 11, 229-234. 6. Giessman, U.; Rollgen, F.W. Int. J . Mass Spectrom. Ion Phys. 1981, 38, 267-279. 7. MacFarlane, R.D. Acc. Chem. Res. 1982, 15, 268-275. 8. Cotter, R.J. Anal. Chem. 1984, 56, 485A-504A. 9. Barber, M.; Bordoli, R.S.; E l l i o t , G.J.; Sedgwick, R.D.; Tyler, A.N. Anal. Chem. 1982, 54, 645A-657A. 10. Blakely, C.R.; Vestal, M.L. Anal. Chem. 1983, 55, 750-754. 11. Ens, W.; Standing, K.; Chait, B.T.; Field, F.H. Anal. Chem. 1981, 53, 1241-1244. 12. Wunsch, L.; Benninghoven, A.; Eicke, A.; Heinen, H.J.; Ritter, H.P.; Taylor, L.C.E.; Veith, J. Org. Mass Spectrom. 1984, 19, 176-182. 13. Balasunmugan, K.; Hercules, D.M.; Cotter, R.J.; Heller, D.; Benninghoven, A.; Sichterman, W.; Anders, V.; Keough, T.; MacFarlane, R.D.; McNeal, C.J. Anal. Chem. 1984, 56, 5759-5762.


7.

FENSELAU AND YELLET

14. 15. 16. 17. 18.


19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40.


175

Fenselau, C.; Liberato, D.J.; Yergey, J.A.; Cotter, R.J.; Yergey, A.L. Anal. Chem. 1984, 56, 5759-5762. Fenselau, C., Yelle, L.; Stogniew, M.; Liberato, D.; Lehman, J.; Feng, P.; Colvin, M. J r . Intern. J . Mass Spectrom. Ion Phys. 1983, 46, 411-414. van Breemen, R.B.; Tabet, J.C.; Cotter, R.J. Biomed. Mass Spectrom. 1984, 11, 278-283. Mumma, R.O.; Vastola, F.J. Org. Mass Spectrom. 1972, 6, 1373-1376. Games, D.E.; Games, M.P.; Jackson, A.H.; Olavesen, A.H.; Rossiter, M.; Winterburn, P.J. Tetrahed Let. 1974, 2377-2380. Schulten. H.R.; Lehmann, W.D. Anal. Chim. Acta. 1976, 87, 103-112. Matcham, G.W.J.; Dodgson, K.S. Biochem. J. 1977, 167, 717-722. Deutsch, J.; Gelboin, H.V. Biomed. Mass Spectrom. 1982, 9, 99-102. Nelson, S.D.; Vaishnav, Y.; Kambara, H.; Baillie, T.A. Biomed. Mass Spectrom. 1981, 8, 244-251. Gaskell, S.J.; Brownsey, B.G.; Brooks, P.W.; Green, B.N. Biomed. Mass Spectrom. 1983, 10, 215-219. Ackermann, B.L.; Watson, J.T.; Newton, J . F . ; Hook, J.B.; Braselton, W.E. J r . Biomed. Mass Spectrom. 1984, 11, 502-511. Jardine, I.; Scanlan, G.F.; Mattox, V.R.; Kumar, R. Biomed. Mass Spectrom. 1984, 11, 4-9. Fenselau, C.; Cotter, R.J.; In "IUPAC Frontiers of Chemistry"; Laidler, K.J., Ed.; Pergammon: Oxford, 1982, p. 207-216. Tunek, A.; P l a t t , K.L.; Przybylski, M.; Oesch, F. Chem. - B i o l . Interactions 1980, 33, 1-17. Pohl, L.R.; Branchflower, R.V.; Highet, R.J.; Martin, J . L . ; Nunn, D.D.; Monks, T . J . ; George, J.W.; Hinson, J.A. Drug Metab. Disp. 1981, 9, 334-339. Przybylski, M.; Cysyk, R.L.; Shoemaker, D.; and Adamson, R.H. Biomed. Mass Spectrom. 1981, 8, 485-491. Meerman, J.H.N.; Beland, F.A.; Ketterer, B.; Srai, S.K.S.; Bruins, A.P.; Mulder, G.J. Chem.-Biol. Interactions 1982, 39, 149-168. Moss, E.J.; Judah, D.J.; Przybylski, M.; Neal , G.E. Biochem. J. 1983, 210, 227-233. Gandich, K.; Przybylski, M. Biomed. Mass Spectrom. 1983, 10, 292-299. Onkenhout, W.; Vermeulen, N.P.E.; Luijten, W.C.; deJong, H.J. Biomed. Mass Spectrom. 1983, 10, 614-619. Przybylski, M.; Luderwald, I.; Kraas, E.; Voelter, W.; Nelson, S.D. Z. Naturforsch. Teil 1979, 34, 736-743. Murphy, R.C.; Mathews, W.R.; Rokach, J.; Fenselau, C. Prostaglandins, 1982. 23, 201-206. Larsen, G.L.; Ryhage, R. Xenobiotica 1982, 12, 855-860. Ross, D.; Larsson, R.; Norbeck, K.; Ryhage, R.; Moldeus, P. Molecular Pharmacology 1985, 27, 277-286. Pallante, S.L.; Lisek, C.A.; Dulik, D.M.; Fenselau, C. Drug Metab. Disp., 1986, in press. Fairlamb, A.H.; Blackburn, P.; Ulrich, P.; Chait, B.T.; Cerami, A. Science, 1985, 227, 1485-1487. Frear, D.S.; Swanson, H.R.; Mansager, E.R. Pest. Biochem. Physiol. 1985, 23, 56-65. In Xenobiotic Conjugation Chemistry; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

176



41.

Bakke, J.E.; Bergman, A.L.; Larsen, G.L. Science 1982, 217, 645-647. 42. Stogniew, M.; Fenselau, C. Drug Metab. Disp. 1982, 10, 609-613. 43. Logan, C.J.; Cottee, F.H.; and Page, J.A. Biochem. Pharmacol. 1984, 33, 2345-2346. 44. Hutson, D.H.; Logan, C.J.; Regan, P.D. Drug Metab. Disp. 1984, 12, 523-524. 45. Games, D.E.; Lewis, E. Biomed. Mass Spectrom. 1980, 7, 433-436. 46. Kenyon, C.N.; Goodley, P.C.; Dixon, D.J.; Whitney, J.O.; F a u l l , K.F.; Barchas, J.D. American Laboratory, 1983, January, 38-49. 47. Liberato, D.J.; Fenselau, C.C.; Vestal, M.L.; Yergey, A.L. Anal. Chem. 1983, 55, 1741-1744. 48. Fenselau, C.; Larsen, B.S. In "Drug Metabolism"; Siest, G., Ed.; Pergamon Press: Oxford, 1985. 49. Thomson, B.A.; Iribarne, J.V.; Dziedzic, P.J. Anal. Chem. 1982, 54, 2219-2224. 50. Whitehouse, C.M.; Dreyer, R.N.; Yamashita, M.; Fenn, J.B. Anal. Chem. 1985, 57, 675-679. 51. Iribarne, J.V.; Thomson, B.A. J . Chem. Phys. 1976, 64, 2287-2294. 52. Thomson, B.A.; Iribarne, J.V. J . Chem. Phys. 1979, 71, 4451-4463. 53. Fenselau, C. Anal. Chem. 1977, 49, 563A - 570A. 54. Fenselau, C. In "Mass Spectrometry in Health and Life Sciences"; Burlingame, A.L. and Castagnoli, N.J., Ed.; Elsevier: Amsterdam, 1985. 55. Feng, P.C.; Fenselau, C.; Colvin, M.; Hinson, J . Drug Metab. Disp. 1981, 9, 521-524. 56. McMahon, R.E.; Sullivan, H.R. Biochem. Pharm. 1965, 14, 1085-1092. 57. Gescher, A,; Hickman, J.A.; Stevens, F.G. Biochem. Pharmacol. 1979, 28, 3235-3238. 58. Brown, N.D.; S t r i c k l e r , M.P.; Sleeman, H.; Doctor, B.P. J . Chromatog. 1981, 212, 361-365. 59. Cho, A.K.; Wright, J . Life Science, 1978, 22, 363-371. 60. Hucker, H.B.; Michniewicz, B.M.; Rhodes, R.E. Biochem. Pharmacol. 1971, 20, 2123-2127. 61. Sternson, L.A.; Hes, J . Pharmacology 1975, 13, 234-240. 62. Sternson, L.A.; Hincal, F.; Bannister, S.J. J . Chromatog. 1979, 144, 191-200. 63. Wikel, J.H.; Paget, C.J.; Delong, D.C. J . Med. Chem. 1980, 23, 368-372. 64. Lehman, J.P.; Ferrin, L.; Fenselau, C.; Yost, G.S. Drug Metab. Disp. 1981, 9, 15-18. 65. Yelle, L., M.A. Thesis, Johns Hopkins University, Baltimore, MD 1982. 66. Hamar-Hansen, C.; Fournel, S.; Magdalou, J.; Boutin, J.A.; Siest, G. submitted. 67. van Breemen, R.B.; Fenselau, C.; Dulik, D.M. In "Biological Reactive Intermediates"; Snyder, R.; Witmer, C.M.; Jollow, D.J. and Kocsis, J.J. Eds.; Plenum Press: New York, 1986. 68. Dulik, D.M.; Hilton, J.; Fenselau, C.; Colvin, M.; Biochem. Pharm. i n press. RECEIVED October 30, 1985 In Xenobiotic Conjugation Chemistry; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

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