Chapter 8
Liquid Chromatography/Mass Spectrometry Downloaded from pubs.acs.org by YORK UNIV on 12/17/18. For personal use only.
Analysis of Xenobiotic Conjugates by Thermospray Liquid Chromatography/Mass Spectrometry Deanne M. Dulik, George Y. Kuo, Margaret R. Davis, and Gerald R. Rhodes Department of Drug Metabolism, Smith, Kline and French Research Laboratories,Swedeland,PA19479
Positive ion thermospray LC/MS and LC/MS/MS analysis of xenobiotic conjugates obtained either from biological fluids or from enzymatic/chemical synthesis provides important information for the structure elucidation of this class of polar compounds. Conjugate metabolites amenable to thermospray LC/MS analysis include sulfate esters, glucuronides, taurine and carnitine conjugates, and mercapturic acid pathway conjugates. Thermospray ionization of these metabolites is best achieved in acidic buffers such as ammonium acetate; molecular ions are observed as [M+H] or [M+NH ] . Fragment ions are typically formed by loss of the conjugate moiety. Further structural information may be obtained through fragmentation of sample ions by collision-activated dissociation. +
+
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H e p a t i c m e t a b o l i z i n g enzymes p l a y a k e y r o l e i n t h e biotransformation of xenobiotics t o polar, ionized species w h i c h a r e more r e a d i l y e x c r e t e d b y v a r i o u s routes o f elimination. The r o u t e s o f h e p a t i c b i o t r a n s f o r m a t i o n a r e designated generally as t h e Phase I reactions of f u n c t i o n a l i z a t i o n (oxidation, reduction, and h y d r o l y s i s ) and P h a s e I I r e a c t i o n s o f c o n j u g a t i o n . The P h a s e I I metabolites arise from conjugation with several substrates, including sulfonic acid, glucuronic acid, g l u t a t h i o n e , amino a c i d s , t a u r i n e , a n d c a r n i t i n e . F o r
0097-6156/90/0420-0124$06.00/0 © 1990 American Chemical Society
8. DULIK ET AL.
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many years, the mass spectrometric a n a l y s i s o f t h i s c l a s s of conjugates was hindered due to both t h e i r high p o l a r i t y and thermal lability ( 1 ) . More r e c e n t l y , LC/MS techniques, p a r t i c u l a r l y by thermospray i o n i z a t i o n mode, have proven t o be s u c c e s s f u l f o r t h e s t r u c t u r a l c h a r a c t e r i z a t i o n o f conjugate metabolites from both in vitro and in vivo sources (2-8). P o s i t i v e ion thermospray LC/MS and LC/MS/MS methods c a r r i e d out on a t r i p l e quadrupole mass spectrometer have been s u c c e s s f u l l y u t i l i z e d i n our l a b o r a t o r y t o e l u c i d a t e the s t r u c t u r e s o f m e t a b o l i t e s o f s e v e r a l compounds c u r r e n t l y undergoing development as drug candidates. Samples were obtained from both in vivo sources (urine, b i l e , or plasma) and analyzed d i r e c t l y from the b i o l o g i c a l f l u i d or from in vitro enzymatic/chemical methods. In a l l cases, b u f f e r i o n i z a t i o n mode u s i n g ammonium acetate i n the HPLC mobile phase was employed f o r i o n i z a t i o n o f the metabolites o f i n t e r e s t . MATERIALS AND METHODS Chemicals SK&F 96148, SK&F 86466, and SK&F 82526 were obtained from Drug Substances and Products, Smith K l i n e and French Laboratories, Swedeland, PA. C-SK&F 96148 and C-SK&F 864 66 used f o r in vivo metabolism studies were provided by the Department o f Radiochemistry, Smith K l i n e and French L a b o r a t o r i e s , Swedeland, PA. Menadione (2-methyl-l,4naphthoquinone) and i t s glutathione and N-acetyl c y s t e i n e conjugate were prepared by chemical synthesis under b a s i c c o n d i t i o n s and were k i n d l y p r o v i d e d by Thomas Jones, Ph.D., Department o f Pathology, U n i v e r s i t y o f Maryland School of Medicine. The r e g i o i s o m e r i c p h e n o l i c glucuronides o f SK&F 8252 6 were synthesized by r e a c t i o n with rabbit liver microsomal uridine 5'diphosphoglucuronyltransferases immobilized onto cyanogen bromide a c t i v a t e d Sepharose 4B according t o a p u b l i s h e d method (9). The products of the enzymatic incubation were p u r i f i e d u s i n g s o l i d phase e x t r a c t i o n b e f o r e LC/MS analysis. 14
14
Animal Studies B i l e duct cannulated Sprague-Dawley r a t s (male and female) were administered C-SK&F 96148 d i l u t e d with nonlabeled compound at a t o t a l dose of 100 mg/kg o r a l l y . B i l e was c o l l e c t e d at s e v e r a l time i n t e r v a l s up t o 24 hrs post dose, a c i d s t a b i l i z e d with 10% g l a c i a l a c e t i c a c i d and f r o z e n immediately a f t e r c o l l e c t i o n . Female beagle 14
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SPECTROMETRY
14
dogs were a d m i n i s t e r e d C - S K & F 86466 d i l u t e d with nonlabeled compound at a t o t a l dose of 10 mg/kg o r a l l y . U r i n e samples were c o l l e c t e d f o r 24 h r s and frozen immediately. LC/MS and LC/MS/MS A n a l y s i s LC/MS and LC/MS/MS spectra were acquired on a t r i p l e quadrupole mass spectrometer (TSQ-45, Finnigan MAT) using a c o n v e n t i o n a l F i n n i g a n thermospray i o n source. No e l e c t r o n filament or discharge electrode was employed. In g e n e r a l , i o n source c o n d i t i o n s f o r the p o l a r conjugate metabolites were: block temperature 200°-250°C; vaporizer temperature 120°-130°C; r e p e l l e r voltage 40-50 v. Spectra were recorded i n 1.95 second scans over the mass range 120-650 amu. LC/MS/MS analyses were performed with an e l e c t r o n m u l t i p l i e r voltage of 1700, c o l l i s i o n energy -25 t o -30 eV, and argon c o l l i s i o n gas pressure of 1.0 mtorr. Reversed phase HPLC c o n d i t i o n s were optimized f o r each compound of i n t e r e s t . In a l l cases, the flow r a t e was 1.1 ml/min, o p t i m i z e d for ionization in the thermospray i n t e r f a c e . A Kratos Spectroflow Model 783 UV d e t e c t o r was p l a c e d i n l i n e before the mass spectrometer and the e l u e n t monitored at 237 or 254 nm f o r a l l compounds. For SK&F 96148 b i l i a r y m e t a b o l i t e s , the f o l l o w i n g c o n d i t i o n s were employed: Brownlee RP-300 (100 χ 4.6 mm, 7 μπι) ; s o l v e n t A, ammonium a c e t a t e (0.1 M) adjusted to pH 5.3 with g l a c i a l a c e t i c a c i d , solvent B, a c e t o n i t r i l e ; l i n e a r gradient c o n d i t i o n s , time 0 min 25% solvent B, time 25, 70% solvent B. This HPLC method was a l s o employed f o r c h a r a c t e r i z a t i o n o f the s u l f a t e conjugate of SK&F 86466 i n dog u r i n e and the Na c e t y l c y s t e i n e conjugate of menadione, except t h a t the l i n e a r gradient conditions were 0-50% solvent Β i n 20 min. A loop i n j e c t i o n technique was employed f o r a n a l y s i s of the g l u t a t h i o n e conjugate of menadione, with an RP-300 guard column (30 χ 4.6 mm, 7 μπι) and e i t h e r 25% or 75% i s o c r a t i c s o l v e n t B. In the case of the g l u c u r o n i d e conjugates of SK&F 8252 6, solvent Β was methanol under 20% i s o c r a t i c c o n d i t i o n s ; the HPLC column was an A l t e x Ci8 (150 χ 4 .6 mm,
RESULTS AND
5 μπι) .
DISCUSSION
B i l i a r y Metabolites of SK&F 96148 The t o t a l ion chromatogram f o r male r a t b i l e (50 μΐ d i r e c t i n j e c t i o n ) a f t e r o r a l dosing with C-SK&F 96148 i s shown i n F i g u r e 1. F i v e d r u g - r e l a t e d components (as 14
8. D U L I K E T A K
Analysis ofXenobiotic Conjugates
10:00 13:20 time (min)
16:40
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F i g u r e 1. Thermospray LC/MS t o t a l i o n chromatogram f o r male r a t b i l e a f t e r o r a l dosing with SK&F 96148 (100 mg/kg). Major drug r e l a t e d components are designated M1-M5. L i n e a r LC g r a d i e n t i n d i c a t e d by dashed l i n e (%B i s percent a c e t o n i t r i l e ) .
127
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SPECTROMETRY
c o n f i r m e d by m o n i t o r i n g o f C by f r a c t i o n c o l l e c t i o n and l i q u i d s c i n t i l l a t i o n c o u n t i n g ) were o b s e r v e d , designated as M1-M5. The t h e r m o s p r a y LC/MS and LC/MS/MS s p e c t r a f o r compounds M1-M5 a r e shown i n F i g u r e s 2-6. M e t a b o l i t e Ml gave a p r o t o n a t e d m o l e c u l a r i o n a t m/z 477, 479 and an ammonium a d d u c t a t m/z 494, 496 ( F i g . 2a) . The CAD d a u g h t e r i o n s p e c t r u m o f m/z 4 94 showed d i a g n o s t i c f r a g m e n t i o n s f o r l o s s o f t h e t a u r i n e m o i e t y a t m/z 126 and l o s s o f t h e t e m i n a l c a r b o x y l g r o u p from t h e d e h y d r a t e d p a r e n t s t r u c t u r e a t m/z 306 ( F i g . 2 b ) . These o b s e r v a t i o n s ( a l o n g w i t h p r o t o n NMR d e c o u p l i n g d a t a , n o t shown) were c o n s i s t e n t with the proposed s t r u c t u r e o f Ml as the t a u r i n e conjugate of a hydroxylated metabolite of the parent (M5) . In a d d i t i o n , the a s s i g n e d p o s i t i o n of h y d r o x y l a t i o n was s u p p o r t e d by d e h y d r a t i o n i n t h e MS/MS mode t o g i v e a s t r o n g f r a g m e n t i o n a t m/z 459 and f u r t h e r d e c a r b o x y l a t i o n o f t h e p a r e n t s t r u c t u r e t o g i v e a fragment a t m/z 334. M e t a b o l i t e M2 gave a p r o t o n a t e d m o l e c u l a r i o n a t m/z 4 61, 4 63 and a b a s e peak ammonium a d d u c t a t m/z 478, 480 ( F i g . 3a) . The m i n o r f r a g m e n t i o n a t m/z 387 may arise from cleavage o f the e t h a n e s u l f o n i c a c i d moiety; the i o n o b s e r v e d a t m/z 533 i s most l i k e l y t h e ammonium a d d u c t o f t h e endogenous b i l e a c i d , t a u r o c h o l i c a c i d . The CAD d a u g h t e r i o n s p e c t r u m o f m/z 478 p r o d u c e d f r a g m e n t s a t m/z 12 6 c o r r e s p o n d i n g t o t h e l o s s o f t a u r i n e and a t m/z 308 due t o s u b s e q u e n t d e c a r b o x y l a t i o n o f t h e p a r e n t ( F i g . 3 b ) . T h i s i n f o r m a t i o n i s c o n s i s t e n t w i t h t h e a s s i g n m e n t o f M2 as t h e t a u r i n e c o n j u g a t e o f t h e p a r e n t . M e t a b o l i t e M3 was t h e m a j o r b i l i a r y m e t a b o l i t e o f SK&F 96148. T h e r m o s p r a y LC/MS a n a l y s i s o f M3 showed an ammonium a d d u c t a t m/z 547, 549 and a p r o m i n e n t f r a g m e n t i o n f o r t h e a g l y c o n e p a r e n t a t m/z 371, 373 ( F i g . 4a) . The i o n a t m/z 413 c o r r e s p o n d s to thermal a c e t y l a t i o n of the parent a f t e r glucuronide h y d r o l y s i s i n the ion source and i s o b s e r v e d f o r o t h e r g l u c u r o n i d e s . The CAD daughter ion spectrum of m/z 547 produced fragment ions c o r r e s p o n d i n g t o a l o s s o f g l u c u r o n i c a c i d (m/z 354, 356, and 371, 373), a base peak fragment due t o d e c a r b o x y l a t i o n of the parent s t r u c t u r e a t m/z 308, and o t h e r minor fragments of the parent molecule ( F i g . 4b) . These d a t a s u p p o r t e d t h e s t r u c t u r e a s s i g n m e n t o f M3 as t h e acyl glucuronide conjugate of the parent. Subsequent p r o t o n NMR a n a l y s i s (not shown) c o n f i r m e d t h e s t r u c t u r e as t h e 10-acyl glucuronide. M e t a b o l i t e M4 was a n a l y z e d b y t h e r m o s p r a y LC/MS ( F i g . 5a) and p r o d u c e d a p r o t o n a t e d m o l e c u l a r i o n a t m/z 497, 4 99 ( c o n f i r m e d by f a s t atom bombardment mass s p e c t r a l analysis) . T h i s mass s p e c t r u m , a s w e l l a s p r o t o n NMR analysis (not shown) was consistent with the acyl c a r n i t i n e conjugate of the parent. Fragmentation occurred v i a l o s s o f t h e c a r n i t i n e m o i e t y t o g i v e m/z 371, 373; t h e f r a g m e n t i o n a t m/z 455, 457 could correspond to an
129
Analysis of Xenobiotic Conjugates
8. DULIKETAL.
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