ARTICLE pubs.acs.org/crt
Evaluation of Deuterium Labeled and Unlabeled Bis-methyl Glutathione Combined with Nanoliquid Chromatography-Mass Spectrometry to Screen and Characterize Reactive Drug Metabolites Daniel Defoy,† Patrick M. Dansette,§ Witold Neugebauer,† J. Richard Wagner,‡ and Klaus Klarskov*,† †
Department of Pharmacology, and ‡Department of Nuclear Medicine and Radiobiology, Universite de Sherbrooke, 3001, 12e Avenue Nord, Sherbrooke, QC, Canada § Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, UMR 8601 CNRS, Universite Paris Descartes, 45 rue des Saints-Peres, 75270 Paris Cedex 06, France
bS Supporting Information ABSTRACT: We present a reactive metabolite detection assay based on the use of deuterium labeled/unlabeled bis-methyl glutathione (GSH) esters (GSH(CH3/CD3)2) and nanoliquid chromatography coupled online with electrospray ionization tandem mass spectrometry (nLC-ESI-MS/MS). Compared with glutathione, neutralization of the carboxylic acid groups by esterification introduced a mass difference of 6, which facilitated the identification of trapped metabolites and improved the intensity of the mass spectrometry signal in positive ionization mode. The peptides allowed for the trapping of soft electrophilic reactive metabolites generated in vitro by incubation with acetaminophen, carbamazepine (CBZ), NADPH, and microsomes.
’ INTRODUCTION Metabolic transformation of drugs to compounds that are more hydrophilic than the parent drug is the organism’s way of eliminating both foreign and intrinsic molecules. As a consequence of biotransformation, many drugs are converted into reactive metabolites that can react with scavenger molecules, such as gluthathione (GSH), proteins, and DNA.1,2 A relationship between the covalent modification of macromolecules and the induction of adverse drug reactions (ADR) is strongly suspected. Therefore, much effort is invested in the continuous development of improved rapid approaches to screen for reactive metabolites, before drugs are evaluated in clinical trials and eventually commercialized. ADR limits the therapeutical use and can cause the potential withdrawal of important drugs from the health care industry. To reduce the risk of ADR, drug biotransformation pathways are extensively scrutinized, and new approaches facilitating their studies, in particularly those that are focused on the detection of reactive metabolites, are under current development. In this context, mass spectrometry is suitable and well-adapted for the detection of stabilized (trapped) reactive drug metabolites.3-6 Although GSH is the preferred compound for trapping reactive metabolites, the presence of two carboxylic acid groups and only one amino group in the peptide backbone provides a modest ionization response in positive ion mode. To overcome this problem, we prepared two low cost methyl esters of GSH. The two carboxylic acid groups of GSH were methylated using an adapted, simple, and well-known reaction, involving the treatment r 2011 American Chemical Society
of samples with anhydrous deuterated or nondeuterated methanolic hydrochloride as previously described in the derivatization of fatty acids for gas chromatography.7 The deuterium label introduces an easily detectable mass difference of m/z 6 between labeled and unlabeled bis-methyl GSH esters, which facilitate the recognition of trapped metabolites in the LC-ESI-MS analysis. The capacity of bis-methyl GSH esters to trap reactive drug metabolites in microsome incubations were evaluated using two well-known drugs (acetaminophen and carbamazepine).
’ MATERIALS AND METHODS All reagents and solvents used in this study were of HPLC grade or of the highest available purity. Glutathione, trichloroacetic acid (TCA), iodoacetamide, silver(I) oxide, acetylchloride, carbamazepine, and acetaminophen were purchased from Sigma-Aldrich (St. Louis, MO). Phenobarbital induced rat microsomes were obtained from Celsis International (Chicago, US). Deuterated methanol was obtained from CDN Isotopes (Point-Claire, Canada). Derivatization of GSH. Methyl esterification was carried out by dissolving 20 mg of GSH in 800 μL of anhydrous methanol or deuterated methanol, cooled on ice followed by the dropwise addition of 160 μL of acetyl chloride. The samples were incubated for 2 h, lyophilized, and dissolved in 100 μL of methanol as described above. After the addition of 20 μL of acetyl chloride, the samples were further incubated for 30 min. The extent of derivatization was verified by Received: October 29, 2010 Published: March 04, 2011 412
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Chemical Research in Toxicology ESI-MS. Both samples were pooled and purified by preparative HPLC to remove excess of reagents and incompletely derivatized GSH. The GSH methyl ester (20 mg) was purified on an Agilent HPLC with an XDBC18 column using the following gradient (% solvent B/min): 0/0 70/25 100/30 35/0 and a flow of 1.5 mL/min. Solvent A was 0.1% aqueous TFA, and solvent B was acetonitrile with 0.1% TFA. The final product identity was confirmed by MS/MS using a Q-TOF-2 (Waters Corp., ON, Canada). Quantification of Peptides. Quantification of GSH(CH3/CD3)2 was performed with few modifications using 4,40 -dithiodipyridine (4DPS) following a procedure described elsewhere.8 A linear calibration curve in the range of 0 to 300 μM was established using GSH. The peptides were dissolved in 50 mM ammonium acetate buffer at pH 4.5 containing 0.36 mM 4-DPS and incubated for 30 min. The 4-TP compound was detected at 324 nm using a 96 well plate reader. Stability of Bis-methyl Glutathione Esters. The stability of the bis-methyl GSH esters during microsomal incubation was verified using GSH(CH3/CD3)2 modified with iodoacetamide (CAM) or N-ethylmaleimide (NEM). GS(CH3/CD3)2-CAM was prepared by incubating 10 mg of GSH with 100 mg of iodoacetamide in 1 mL of 200 mM ammonium bicarbonate buffer at pH 8 for 1 h in the dark. After dividing the sample into two equal aliquots and lyophilizing, carbamidomethylated GSH was then methylated and purified by HPLC as described above. Derivatization of GSH(CH3/CD3)2 (185 μg) with NEM was performed in 100 mM potassium phosphate buffer containing 40% (v/v) MeOH (pH 6.0) and initiated by the addition of a 50-fold molar excess of NEM. After 60 min, the MeOH concentration was reduced by drying under vacuum, and GS(CH3/CD3)2-NEM was subsequently purified on a SepPak C18 cartridge using the conditions described below. The derivatized bis-methyl GSH esters were incubated with microsomes following the protocol described below.
Preparation in Situ of N-Acetyl-p-benzo-quinone Imine (NAPQI). Acetaminophen (1 mg) was incubated with silver(I) oxide
(10 mg) in 1 mL of acetonitrile. Following sonication in a water bath for 10 min, 50 μL was diluted in duplicate with 100 μL of buffer containing 100 mM potassium phosphate and 50 mM EDTA, pH 7.4, to which 10 nmol (10 μL) of GSH or GSH(CH3)2 were added, as determined by the 4-DPS assay. The mixtures were incubated for 1 h at 37 °C, and the reaction was then stopped by the addition of 200 μL of 0.2% aqueous formic acid. An equal volume of the two samples was mixed and diluted in water containing 0.2% formic acid prior to the analysis by nLC-ESI-MS. Microsome Incubations. Microsomal incubations were performed in a total volume of 500 μL. The buffer was 100 mM potassium phosphate, with pH adjusted to 7.4. The concentration of microsomes was 1 mg/mL. Following 2 min of preincubation in the presence of 40 μM drug and 1 mM trapping peptide mixed at an approximate ratio of 1:1 GSH(CH3/CD3)2, NADPH was added (1 mM final concentration) to start the reaction. Drug stock solutions (10 mM) were prepared in methanol. The final methanol concentration was below 0.8%. After incubation at 37 °C for 1 h, proteins were precipitated by adding 250 μL of 10% (W/V) TCA and centrifuging for 10 min in a Heraeus biofuge (Kendro laboratory products). Microsome incubations of carbamidomethylated and NEM derivatized bis-methyl GSH esters were performed for 2 h as described above except that a 2 μg/μL protein concentration was used instead. An aliquot (75 μL) was withdrawn at time 0, 60, and 120 min and purified by SepPak (see below) prior to analysis by nLC-ESI-MS. SepPak and ZipTip Extractions. Immediately after protein precipitation with 10% TCA, the sample was loaded onto a methanol prewetted and water equilibrated SepPak C18 cartridge (Waters Corporation, MA, USA). After washing the SepPak cartridge with 2 mL of water, the sample was eluted with 1050 μL of methanol that was then separated into five 200 μL aliquots and lyophilized. Immediately before
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nLC-ESI-MS analysis, the samples were dissolved in 45 μL of water containing 20% MeOH.
Online Nanoliquid Chromatography-Electrospray Ionization Mass Spectrometry. nLC-ESI-MS/MS analyses were performed on a Q-TOF-2 coupled online to a CapLC pump (Waters Corp., ON, Canada) equipped with three separate syringe pump modules, an autoinjector, a 10 port valve, and a 0.1 25 mm precolumn. Separations were performed on a 0.1 100 mm capillary column. Both columns were packed with Aqua C18 (5 μm, 125A) resin (Phenomenex, Montreal, Canada). Analytes were eluted at a flow rate of approximately 0.25 μL/min and a linear gradient of solvent B in solvent A: (% B/min) 20/0.1 20/18 60/48 80/50 20/55 20/63. Solvent A and solvent C (used to load the sample onto the precolumn) consisted of 1% acetonitrile, 1% isopropanol, and 0.2% aqueous formic acid, and solvent B was 80% acetonitrile containing 10% isopropanol, 9.8% water, and 0.2% formic acid. The Q-TOF mass spectrometer was calibrated in MS/MS mode by infusing a solution of 10 picomol/μL of Sar1,Leu8 angiotensin II dissolved in 35% acetonitrile containing 0.2% formic acid. The cone voltage was set to 30 V if not otherwise stated, and the capillary voltage was 2000 V. Survey mass and MS/MS spectra were acquired over the m/z ranges 200-900 and 50-900 in 2 s, respectively. After the analysis, appropriate segments of spectra were combined and visually inspected for m/z with a delta mass of 6 a.m.u, before creating the corresponding selected ion chromatograms shown in the figures. The collision energy (CE) used for MS/MS was determined by the m/z of the parent ion using the following linear m/z/CE gradient: 200/14 V 700/28 V.
’ RESULTS Comparison of the mass spectrometric response of equal amounts of GSH(CH3/CD3)2 and GSH demonstrated approximately 26-fold gain using the bis-methyl GSH ester compared to GSH (Figure S1, Supporting Information). A similar gain in MS response was also obtained after adduct formation in situ using the reactive metabolite of acetaminophen, N-acetyl-p-benzoquinone imine (NAPQI) (Figure S2, Supporting Information). To determine whether bis-methyl GSH esters were prone to esterase activity during microsome incubation, we derivatized GSH with iodoacetamide (GS(CH3/CD3)2-CAM) and N-ethylmaleimide (GS(CH3/CD3)2-NEM). Time-course analysis by nLC-ESI-MS of microsomal incubations with GS(CH3/CD3)2-NEM and GS(CH3/CD3)2-CAM indicated that although a loss in the ion intensity of CAM-GS(CH3/CD3)2 was observed after 1 and 2 h of incubation, the bis-methylated GSH esters remained stable over an incubation period of 2 h with a 2-fold higher concentration of microsomal proteins (2 μg/μL) than that commonly used (Figure S3, Supporting Information). The analgesic antipyretic drug acetaminophen (Tylenol) is metabolized mainly by cytochrome P450 3A4 to NAPQI, which is commonly used to evaluate mass spectrometry approaches to capture reactive metabolites.4,9-21 To determine if reactive metabolites of acetaminophen may be trapped in microsome incubations, parallel incubations were carried out in the presence of either GSH or GSH(CH3/CD3)2. Analysis by nLC-ESI-MS revealed the m/z values corresponding to the nucleophilic conjugation of glutathione with acetaminophen of mass 457 and 485/491 (GSH(CH3/ CD3)2), respectively. The selective ion chromatograms of the corresponding m/z values are shown in Figure 1. In addition, with GSH(CH3/CD3)2 as the nucleophile, one additional pair of m/z 517/523 was observed, corresponding to a tentatively identified adduct of dihydroxyacetaminophen (DþNþ2O-2H), as suggested by tandem mass spectrometry (Figure 1). Interestingly, this adduct was not detected with GSH, although it was previously observed 413
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Figure 1. Analyses by nLC-ESI-MS of Sprague-Dawley rat male microsome incubations with acetaminophen in the presence of GSH or GSH(CH3/ CD3)2 as trapping agents. Selected ion chromatograms (SIC) of (A) m/z 457 (GSHþD-2H), (B) 517 (GSH(CH3)2þDþ2O-2H), and (C) 485 (GSH(CH3)2þD-2H) are shown. The corresponding MS/MS and MS survey spectra are shown in the inserts. The MS/MS spectra were obtained from separate analyses.
with human microsomes using a method with polarity switching and neutral loss scanning.22 In the latter work, an additional GSH adduct corresponding to (DþNþO-2H) was also observed that was not detected with either GSH or GSH(CH3/CD3)2. The discrepancy between these and our data is likely due to the use of dissimilar microsomes (phenobarbital-induced rat versus human
microsomes), mass spectrometry instrumentation (polarity switching on a triple quadrupole linear iontrap versus positive acquisition mode with quadrupole-TOFMS), or different conditions for incubation, i.e., drug concentration. We did not observe a significant improvement in the ion intensity as observed with in situ conjugation. The sum of intensities for the two GSH(CH3/CD3)2 414
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Table 1. Summary of Trapped Reactive Metabolites of Carbamazepine Formed in Phenobarbital-Induced Male Rat Microsome Incubations in the Presence of GSH or GSH(CH3/CD3)2 and NADPH label
GSH; GSH(CH3/CD3)2 observed [MþH]þ
ion count ratioc
calculated [MþH]þa
composition
CBZ1
556.1
584.2/590.2
2.5x
556.2/584.2
DþNþO-4H
CBZ2 CBZ3
558.1 558.1
586.2/592.2 586.2/592.2
13.4x 11.6x
558.2/586.2 558.2/586.2
DþNþO-2H DþNþO-2H
CBZ4
558.1
586.2/592.2
4.8x
558.2/586.2
DþNþO-2H
CBZ5
560.1
588.2/594.2
7.1x
560.2/588.2
DþNþO
CBZ6
560.1
588.2/594.2
6.9x
560.2/588.2
DþNþO
CBZ7
NDb
588.2/594.2
ND
560.2/588.2
DþNþO
CBZ8
572.1
600.2/606.2
4.8x
572.1/600.2
DþNþ2O-4H
CBZ9
572.1
600.2/606.2
2.3x
572.1/600.2
DþNþ2O-4H
CBZ10 CBZ11
574.1 574.1
602.2/608.2 NDb
1.5x ND
574.2/602.2 574.2/602.2
DþNþ2O-2H DþNþ2O-2H
CBZ12
576.1
604.2/610.2
0.23x
576.2/604.2
DþNþ2O
CBZ13
576.1
604.2/610.2
0.25x
576.2/604.2
DþNþ2O
Calculated [MþH]þ for GSH and GSH(CH3)2 conjugates. b Not detected. c The ion count ratio was calculated by dividing the counts for GSH(CH3)2 conjugates with the corresponding ones for GSH. The ion counts were obtained from Figure S4, Supporting Information. a
conjugates m/z 485 and 517 corresponded to that detected for the GSH conjugate m/z 457, albeit they were each present at half of the concentration used in incubations with GSH (Figure 1). Tandem mass spectrometry of GSH(CH3)2-NAPQI m/z 485 resulted in fragments of m/z 396, 225, 208, 182, 166, 144, and 140 that could all be readily assigned to the molecular structure (Figure 1). These fragments are in excellent agreement with recently obtained data using ion mobility TOF-MS and GSH as the trapping nucleophile.10 Carbamazepine (CBZ) is widely used in the treatment of convulsive disorders in spite of a report that as many as 50-70% of treated patients may develop adverse drug reactions.23 Whereas most of these reactions are transient and relatively mild, about 5% may be classified as idiosyncratic reactions involving complications, such as hepatitis, skin rash, and blood disorders.24 It has been postulated that the formation of reactive carbamazepine metabolites may be involved in the adverse reactions.25 NanoLC-ESI-MS analysis of reactive carbamazepine metabolites generated with microsomes isolated from phenobarbital-induced rats in the presence of NADPH, GSH, or GSH(CH3/CD3)2 showed a total of 13 captured species (Table 1). Representative nLC-ESI-MS chromatograms are depicted in Figure S4 (Supporting Information). Although the ion intensity in most cases was very low and, in the case of CBZ9, near the limit of detection, a total of 12 potential GSH conjugation products was observed: (DþNþO-4H), (DþNþO-2H), (DþNþO), (DþNþ2O-4H), (DþNþ2O2H), and (DþNþ2O). When using the corresponding bis-methyl esters of GSH, the same number of reactive metabolites were trapped with an approximate gain in ion intensity for those observed by both nucleophiles, i.e., CBZ1-10 (except CBZ7 and 9), ranging from 1.5- to 13.3-fold (Table 1). Two conjugates (CBZ12 and 13) exhibited slightly lower ion intensities with GDH(CH3/CD3)2. The tandem mass spectrometry of CBZ5 (Table 1) mass 560 (DþGSHþO) and 588 (DþGSH(CH3)2þ O) are shown in Figure 2. The tandem mass spectra showed a few but confirmative fragment ions. Compared to the GSH conjugate, remarkably improved tandem mass spectra were obtained for the bis-methyl GSH conjugate, likely due to the higher intensity of parent ions. Moreover, except for the GSH conjugate CBZ1 and CBZ12 (m/z 576), ion abundances of other GSH conjugates were
Figure 2. nLC-ESI-MS/MS of Sprague-Dawley rat microsome incubations with carbamazepine in the presence of (A) GSH (parent m/z 560) and (B) GSH(CH3/CD3)2 (parent m/z 588). The tandem mass spectra were obtained from separate nLC-ESI-MS/MS analyses of individual incubations with Sprague-Dawley rat male microsomes. The structures are tentative, while the m/z values of undetected fragment ions are indicated by asterisks. 415
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too low to obtain adequate MS/MS data, allowing structural analysis. The ion intensity of the conjugates with GSH(CH3/ CD3)2 CBZ1, 2, 3, 5, and 6 was appropriate for their analysis by tandem mass spectrometry (data not shown).
intermediates using isotopic pattern triggered data-dependent highresolution accurate mass spectrometry. Rapid Commun. Mass Spectrom. 22, 1295–1311. (5) Ma, L., Wen, B., Ruan, Q., and Zhu, M. (2008) Rapid screening of glutathione-trapped reactive metabolites by linear ion trap mass spectrometry with isotope pattern-dependent scanning and postacquisition data mining. Chem. Res. Toxicol. 21, 1477–1483. (6) Leblanc, A., Shiao, T. C., Roy, R., and Sleno, L. (2010) Improved detection of reactive metabolites with a bromine-containing glutathione analog using mass defect and isotope pattern matching. Rapid Commun. Mass Spectrom. 24, 1241–1250. (7) Hornstein, I., Elliott, L. E., and Crowe, P. F. (1959) Gas chromatographic separation of lingchain fatty acid methyl esters on polyvinyl acetate. Nature 184 (Suppl. 22), 1710–1711. (8) Hansen, R. E., Ostergaard, H., Norgaard, P., and Winther, J. R. (2007) Quantification of protein thiols and dithiols in the picomolar range using sodium borohydride and 4,40 -dithiodipyridine. Anal. Biochem. 363, 77–82. (9) Potter, D. W., and Hinson, J. A. (1987) Mechanisms of acetaminophen oxidation to N-acetyl-P-benzoquinone imine by horseradish peroxidase and cytochrome P-450. J. Biol. Chem. 262, 966–973. (10) Chan, E. C., New, L. S., Yap, C. W., and Goh, L. T. (2009) Pharmaceutical metabolite profiling using quadrupole/ion mobility spectrometry/time-of-flight mass spectrometry. Rapid Commun. Mass Spectrom. 23, 384–394. (11) Jian, W., Yao, M., Zhang, D., and Zhu, M. (2009) Rapid detection and characterization of in vitro and urinary N-acetyl-Lcysteine conjugates using quadrupole-linear ion trap mass spectrometry and polarity switching. Chem. Res. Toxicol. 22, 1246–1255. (12) Laine, J. E., Auriola, S., Pasanen, M., and Juvonen, R. O. (2009) Acetaminophen bioactivation by human cytochrome P450 enzymes and animal microsomes. Xenobiotica 39, 11–21. (13) Wen, B., Chen, Y., and Fitch, W. L. (2009) Metabolic activation of nevirapine in human liver microsomes: dehydrogenation and inactivation of cytochrome P450 3A4. Drug Metab. Dispos. 37, 1557–1562. (14) Zhu, M., Ma, L., Zhang, H., and Humphreys, W. G. (2007) Detection and structural characterization of glutathione-trapped reactive metabolites using liquid chromatography-high-resolution mass spectrometry and mass defect filtering. Anal. Chem. 79, 8333–8341. (15) Zheng, J., Ma, L., Xin, B., Olah, T., Humphreys, W. G., and Zhu, M. (2007) Screening and identification of GSH-trapped reactive metabolites using hybrid triple quadruple linear ion trap mass spectrometry. Chem. Res. Toxicol. 20, 757–766. (16) Soglia, J. R., Contillo, L. G., Kalgutkar, A. S., Zhao, S., Hop, C. E., Boyd, J. G., and Cole, M. J. A. (2006) Semiquantitative method for the determination of reactive metabolite conjugate levels in vitro utilizing liquid chromatography-tandem mass spectrometry and novel quaternary ammonium glutathione analogues. Chem. Res. Toxicol. 19, 480–490. (17) Mutlib, A., Lam, W., Atherton, J., Chen, H., Galatsis, P., and Stolle, W. (2005) Application of stable isotope labeled glutathione and rapid scanning mass spectrometers in detecting and characterizing reactive metabolites. Rapid Commun. Mass Spectrom. 19, 3482–3492. (18) Soglia, J. R., Harriman, S. P., Zhao, S., Barberia, J., Cole, M. J., Boyd, J. G., and Contillo, L. G. (2004) The development of a higher throughput reactive intermediate screening assay incorporating microbore liquid chromatography-micro-electrospray ionization-tandem mass spectrometry and glutathione ethyl ester as an in vitro conjugating agent. J. Pharm. Biomed. Anal. 36, 105–116. (19) Zhang, T., Zhu, Y., and Gunaratna, C. (2002) Rapid and quantitative determination of metabolites from multiple cytochrome P450 probe substrates by gradient liquid chromatography-electrospray ionization-ion trap mass spectrometry. J. Chromatogr., B 780, 371–379. (20) Thatcher, N. J., and Murray, S. (2001) Analysis of the glutathione conjugate of paracetamol in human liver microsomal fraction by liquid chromatography mass spectrometry. Biomed. Chromatogr. 15, 374–378.
’ DISCUSSION In summary, our results demonstrate that bis-methyl GSH esters in combination with nLC-ESI-MS are suitable for trapping and identifying reactive drug metabolites with, in most cases, a significant gain in detection. However, not all trapped metabolites are detected with the same increase in ion counts. This discrepancy may be explained by glutathione-S-transferase (GST) activity in microsome incubations, which contribute to the formation of GSH conjugates. Bis-methyl GSH esters likely remain transparent to GST. It was earlier mentioned by Soglia et al. that GSH containing a modified γ-glutamyl residue with a permanently positive charged quaternary ammonium group affects GSH binding to GST.16 Thus, the uncatalyzed compared to the catalyzed reaction may give lower amounts of adduct formation of reactive intermediates arising from certain drugs. Nevertheless, from a toxicological point of view, reactive metabolites that do not require GST activity could be the more intriguing ones because these are likely to spontaneously form conjugates with proteins and other macromolecules. However, experimental evidence on which reactive metabolites are the most toxicologically important, if any, and the biological impact of the modifications remain to be investigated. ’ ASSOCIATED CONTENT
bS
Supporting Information. Spectra showing the gain in sensitivity and stability of GSH(CH3/CD3)2 during microsome incubations including representative nLC-MS chromatograms of trapped reactive metabolites of carbamazepine. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected]. Funding Sources
The Natural Sciences and Engineering Research Council of Canada and PROTEO are greatly acknowledged for their financial support.
’ ACKNOWLEDGMENT Ibrahim Hasibu and Tiphany Grisin are acknowledged for help with the preparation of glutathione methyl esters and microsome incubations. ’ REFERENCES (1) Miller, J. A. (1970) Carcinogenesis by chemicals: An Overview. G. H. A. Clowes memorial lecture. Cancer Res. 30, 559–576. (2) Hanzlik, R. P., Fang, J., and Koen, Y. M. (2009) Filling and mining the reactive metabolite target protein database. Chem.-Biol. Interact 179, 38–44. (3) Yan, Z., and Caldwell, G. W. (2004) Stable-isotope trapping and high-throughput screenings of reactive metabolites using the isotope MS signature. Anal. Chem. 76, 6835–6847. (4) Lim, H. K., Chen, J., Cook, K., Sensenhauser, C., Silva, J., and Evans, D. C. A. (2008) Generic method to detect electrophilic 416
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(21) Dahlin, D. C., and Nelson, S. D. (1982) Synthesis, decomposition kinetics, and preliminary toxicological studies of pure N-acetyl-pbenzoquinone imine, a proposed toxic metabolite of acetaminophen. J. Med. Chem. 25, 885–886. (22) Wen, B., Ma, L., Nelson, S. D., and Zhu, M. (2008) Highthroughput screening and characterization of reactive metabolites using polarity switching of hybrid triple quadrupole linear ion trap mass spectrometry. Anal. Chem. 80, 1788–1799. (23) Pellock, J. M. (1987) Carbamazepine side effects in children and adults. Epilepsia 28 (Suppl. 3), S64–S70. (24) Askmark, H., and Wiholm, B. E. (1990) Epidemiology of adverse reactions to carbamazepine as seen in a spontaneous reporting system. Acta Neurol. Scand. 81, 131–140. (25) Shear, N. H., and Spielberg, S. P. (1988) Anticonvulsant hypersensitivity syndrome. In vitro assessment of risk. J. Clin. Invest. 82, 1826–1832.
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