Development and Evaluation of an Electrochemical Method for

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Chem. Res. Toxicol. 2007, 20, 821-831

821

Development and Evaluation of an Electrochemical Method for Studying Reactive Phase-I Metabolites: Correlation to in Vitro Drug Metabolism Kim G. Madsen,*,† Jørgen Olsen,‡ Christian Skonberg,† Steen H. Hansen,† and Ulrik Jurva§ Department of Pharmaceutics and Analytical Chemistry, Faculty of Pharmaceutical Sciences, UniVersity of Copenhagen, Denmark, Exploratory ADME Diabetes, NoVo Nordisk A/S, DK-2760 MåløV, Denmark, and DMPK & Physical Chemistry, Lead Generation, AstraZeneca R&D Mo¨lndal, Mo¨lndal, Sweden ReceiVed January 23, 2007

A reactive metabolite may react covalently with proteins or DNA to form adducts that ultimately may lead to a toxic response. Reactive metabolites can be formed via, for example, cytochrome P450-mediated phase 1 reactions, and in this study, we report the development and evaluation of an electrochemical method for generating reactive metabolites. Paracetamol was used as a test compound to develop the method. The stability of the electrochemically generated N-acetyl-p-benzoquinoneimine (NAPQI) from paracetamol was investigated at 37 °C at pH 5.0, 7.4, and 9.0. The highest stability of NAPQI was observed at pH 7.4. The reaction rate between NAPQI and glutathione (GSH) was studied with cyclic voltammetry. NAPQI reacted quantitatively with GSH within 130 ms. The reactivity of NAPQI toward other nucleophiles was investigated, and for the reaction with N-acetyltyrosine, a time-dependent formation of a conjugate with N-acetyltyrosine was observed from 0 to 4 min. The applicability of the method was evaluated with compounds that were able to form quinone imines (amodiaquine), quinones (3-tert-butyl4-hydroxyanisole and p-cresol), imine methides (3-methylindole; trimethoprim), quinone methides (3,5di-tert-butyl-4-hydroxytoluene), and nitrenium ions (clozapine). The compounds were oxidized in an analytical electrochemical cell, and the formed reactive metabolites were trapped with GSH. The samples were then analyzed by LC-MS and LC-MS/MS. For comparison, all compounds were incubated with GSH in rat and human liver microsomes, and the formation of GSH conjugates was compared with that observed by electrochemical oxidation. Furthermore, the electrochemical method was used to synthesize a GSH conjugate of clozapine, which made it possible to obtain structural information by NMR. In summary, a high degree of similarity was observed between the conjugates identified from electrochemical oxidation and GSH conjugates identified from incubation with liver microsomes. In conclusion, we have developed a method that is useful for studies on reactive metabolites and furthermore can be scaled up for the synthesis of GSH conjugates for NMR. Introduction The toxicities of a number of drugs, for example, paracetamol and diclofenac, have been associated with their ability to form reactive metabolic intermediates (1). The majority of these reactive metabolic intermediates are formed by oxidation of the parent compound to an electrophilic intermediate, which subsequently can react with nucleophilic functional groups in cellular biomacromolecules such as proteins and DNA. Some reactive metabolic intermediates, for example, some epoxides and acyl glucuronides, have a relatively long half-life and can be detected in biological samples. Other reactive metabolites, for example, many quinones and quinone imines, have a short half-life in biological samples and can often only be identified indirectly as reaction products formed with endogenous molecules such as glutathione conjugates (2, 3). The utility of electrochemistry for mimicking biologic reactions such as oxidative drug metabolism is well documented (4-9). Electrochemistry has been used to mimic different phase-I reactions such as aromatic hydroxylation, dehydrogenation, O-, and N-dealkylation, by introducing the compound

into an electrochemical cell and applying a potential to the solution. Another electrochemical method used for studying fast reactions, which can be related to metabolic reactions, is cyclic voltammetry. Cyclic voltammetry has been used to obtain information about reaction rates between an electrochemically generated electrophile and a nucleophile (10-12). Such applications indicate that electrochemistry has a potential in the studies of the formation of reactive metabolic intermediates. In this study, we report the development of a method for generating and studying reactive oxidative phase I metabolites using electrochemical methods. The methods were developed using paracetamol as the test substrate, but the utility of the methods were further evaluated with a range of compounds (Scheme 1), reported to form quinone imines, imine methides, quinones, quinone methides, and nitrenium ions (13-19). Finally, the results from the electrochemical studies were compared to what was observed for the metabolism of the compounds in human and rat liver microsomes.

Experimental Procedures * Corresponding author. Tel: [email protected]. † University of Copenhagen. ‡ Novo Nordisk A/S. § AstraZeneca R&D Mo ¨ lndal.

+45-35-30-64-63.

E-mail:

Chemicals and Reagents. Paracetamol (APAP1), amodiaquine (AQ), cresol, clozapine (CLZ), 3,5-di-tert-butyl-4-hydroxytoluene (BHT), 3-tert-butyl-4-hydroxyanisole (BHA), 4-methylcatechol,

10.1021/tx700029u CCC: $37.00 © 2007 American Chemical Society Published on Web 04/21/2007

822 Chem. Res. Toxicol., Vol. 20, No. 5, 2007 Scheme 1. Compounds Used to Develop and Evaluate the Method for the Electrochemical Generation of Reactive Metabolic Intermediates

glutathione (GSH), nicotinamide adenine dinucleotide phosphate (NADPH), phosphoric acid, N-acetyllysine, N-acetylserine, Nacetylcysteine, and N-acetyltyrosine were obtained from SigmaAldrich (St. Louis, MO). p-Acetamidobenzoic acid was obtained from Acros Organics (New Jersey). 3-Methylindole (3-MI) was obtained from Lancaster Synthesis (Alfa Aesar, Lancashire, England). Ascorbic acid, sodium dihydrogenphosphate, and disodium hydrogenphosphate were obtained from Merck (Darmstadt, Germany). Trimethoprim (TMP) and tert-butylhydroquinone (TBHQ) were obtained from Fluka (Sigma-Aldrich (St. Louis, MO)). Water used for mobile phases and solutions for the electrochemical oxidations was purified with a Milli-Q de-ionization unit (Millipore, USA). All solvents used for mobile phases and electrochemical oxidations were of HPLC-grade. Instrumentation. LC-MS and LC-MS/MS analysis was performed using either an Agilent 1100 Series LC/MSD Trap (Agilent, Germany) or a Thermo Finnigan Surveyor LC system coupled to a Thermo Finnigan TSQ Quantum Ultra AM triple quadrupole mass 1 Abbreviations: APAP, paracetamol; AQ, amodiaquine; BHA, 3-tertbutyl-4-hydroxyanisole; BHT, 3,5-di-tert-butyl-4-hydroxytoluene; CLZ, clozapine; HLM, human liver microsomes; 3-MI, 3-methylindole; MeCN, acetonitrile; NAC, N-acetylcysteine; NAPQI, N-acetyl-p-benzoquinoneimine; NAT, N-acetyltyrosine; RLM, rat liver microsomes; TBHQ, tert-butylhydroquinone; TMP, trimethoprim.

Madsen et al. spectrometer with an ESI interface (Thermo Finnigan). LC-MS/ MS analysis for comparison of the electrochemical and microsomal samples was performed on a Shimadzu LC system coupled to a Sciex API 4000 Q-Trap mass spectrometer. NMR-analysis was performed on a Bruker 400 MHz instrument. Preparative HPLC was performed on a Shimadzu system equipped with an LC-10AD pump and an SPD-10A UV/vis detector (Shimadzu, Japan) or on an Agilent 1100 Series LC system with a fraction collector. Electrochemical oxidations were performed in an ESA 5011 analytical electrochemical cell equipped with a porous graphite working electrode, a Pd counter electrode, and a Pd/H2 reference electrode. The cell was controlled by an ESA Coulochem potentiostat (Model 5100A, ESA Bioscience, Inc.). The electrochemical reactions were performed at 37 °C. Cyclic voltammograms were recorded by the use of a threeelectrode system controlled by a computer-controlled potentiostat (Autolab, Netherlands). A carbon electrode (Radiometer, Denmark) was used as the working electrode, a platinum wire as the counter electrode and a homemade silver/silver chloride electrode as the reference electrode. Chromatography. The incubation samples from studies of the stability of N-acetyl-p-benzoquinoneimine (NAPQI) and the formation of the paracetamol-N-acetyltyrosine conjugate were all analyzed by LC-MS using a Luna C18(2), 3 µm, 100 × 2 mm column (Phenomenex) and gradient elution using mobile phase A consisting of 5% MeOH and 0.02% TFA in water and mobile phase B consisting of MeOH with 0.02% TFA added. All other LC analyses were performed with a Luna C18(2), 3 µm, 100 × 2 mm column (Phenomenex) using a gradient elution with mobile phase A consisting of 5% MeCN and 0.2% HCOOH in water and mobile phase B consisting of MeCN and 0.2% HCOOH. To study the oxidation of reduced glutathione in the electrochemical cell, an Aqua C18, 3 µm, 150 × 2 mm column (Phenomenex) was used. The mobile phase consisted of 20 mM potassium phosphate buffer (pH 2.7) and MeCN (99:1). Glutathione (oxidized and reduced) was detected at 210 nm. Synthesis of 5-(3-(5-Acetamido-2-hydroxyphenylthio)-1-(carboxymethylamino)-1-oxopropan-2-ylamino)-2-amino-5-oxopentanoic acid (APAP-SG). The APAP-SG conjugate was synthesized according to the method by Thatcher and Murray (20). APAP (425 mg; 2.8 mmol) was suspended in chloroform (50 mL), and freshly prepared silver oxide was added (2.2 g; 9.5 mmol). The reaction mixture was stirred for 2 h at room temperature. The suspension was then filtered through a paper filter into a solution containing GSH (857 mg; 2.8 mmol) in a 0.1 potassium phosphate buffer (pH 7.4). The solution was stirred for an additional 1 h at room temperature. The reaction mixture was then transferred to a separating funnel, and the organic layer was discarded. The aqueous phase was washed twice with chloroform (10 mL). The aqueous phase was then lyophilized, and the APAP-SG conjugate was purified by preparative chromatography using an XTerra Prep MS C18 column, 10 µm (100 × 19 mm). 4% MeCN in water containing 0.2% formic acid was used as the mobile phase. MS: m/z: 457.2 ([APAP-SG + H]+). MSMS: m/z 382.1 ([APAP-SG - glycine + H]+); m/z 208.0 ([APAP-SG - glutamic acid - glycine - CO - 2H + H]+); m/z 182.0 ([APAP-SG glutamic acid - glycine - C3H7NO - 2H + H]+); m/z 166.0 ([APAP-SG - glutamic acid - glycine - C3H7NO - H2O + H]+); m/z 140.0 ([APAP-SG - glutamic acid - glycine - C3H7NO CH3CO + H]+) and m/z 129.1 (Glutamine - H2O]+) (Figure 1A). 1H NMR (D O): δ 7.46 (d; J ) 2.5 Hz; Ar-H); δ 7.22 (dd; J ) 2 8.7 Hz & 2.5 Hz; Ar-H); δ 6.94 (d; J ) 8.7 Hz; Ar-H); δ 4.47 (dd; J ) 4.8 Hz and 3.5 Hz; CH (cysteine)); δ 3.80 (s; CH2 (glycine)); δ 3.76 (t; J ) 6.4 Hz; CH (glutamic acid)); δ 3.37 & 3.23 (2 × dd; J ) 14.5 Hz and 4.8 Hz; CH2(cysteine)); δ 2.44 (t, J ) 7.2 Hz, CH2(glutamic acid)); δ 2.14 (s, CH3); δ 2.07 (q; J ) 7.8 Hz; CH2 (glutamic acid)). Synthesis of 2-Acetamido-3-(4-(5-acetamido-2-hydroxyphenoxy)phenyl)propanoic Acid (APAP-NAT). APAP (0.70 g; 4.6 mmol) was suspended in chloroform (25 mL). Freshly prepared silver oxide (1.4 g; 6.0 mmol) was then added, and the solution

ReactiVe Phase-I Metabolites

Figure 1. MS/MS spectra of paracetamol-SG at m/z 457.2 ([APAPSG + H]+). (A) Synthetic standard. (B) APAP-SG formed from electrochemical oxidation of 10 µM paracetamol at +600 mV and collection of the sample in an aqueous solution of GSH. (C) Incubation of 10 µM paracetamol with human liver microsomes (HLM), GSH, and NADPH.

was stirred in the dark for 1 h. The suspension was filtered through a paper filter, and the yellow filtrate was added to N-acetyltyrosine (NAT) (0.2 g; 1.0 mmol) in 0.1 M phosphate buffer (pH 7.4) (25 mL) and stirred for 2 h. The organic phase was discarded, and the aqueous phase was washed with chloroform (2 × 10 mL). The aqueous phase was lyophilized, and APAP-NAT was purified by preparative chromatography using a Luna C18(2) column, 3 µm (150 × 10 mm). 10% MeCN in water containing 0.2% formic acid was used as the mobile phase. MS: m/z: 373.0 ([APAP-NAT + H]+). MSMS: m/z 355.0 ([APAP-NAT - H2O + H]+); m/z 337.0 ([APAP-NAT - 2 × H2O + H]+); m/z 331.0 ([APAP-NAT - CH3CO + 2H]+); m/z 327.0 ([APAP-NAT - COOH + H]+); m/z 313.0 ([APAP-NAT - CH3-

Chem. Res. Toxicol., Vol. 20, No. 5, 2007 823 CO - H2O + H]+); m/z 285.0 ([APAP-NAT - CH3CO - COOH + H]+) and m/z 243.0 ([APAP-NAT - 2 × CH3CO - COOH + H]+). (For the MS/MS-spectrum, see Supporting Information 1.) Internal Standard. Several compounds were investigated for their potential use as an internal standard (IS) that would be stable under the conditions used for the electrochemical studies of paracetamol. APAP (25 µM) and the IS (25 µM) candidate in 0.1 M phosphate buffer were oxidized at +600 mV. The sample was analyzed by LC-MS before and after oxidation to examine the stability of the IS candidate toward electrochemical oxidation at the applied potential. Investigation of the Stability of Electrochemically Generated NAPQI. Twenty-five micromolar APAP was oxidized at +600 mV in a 0.1 M phosphate buffer with 25 µM p-acetamidobenzoic acid added as the internal standard. The experiments were performed at pH 3.0, 5.0, 7.4, and 9.0. The flow through the electrochemical cell was 50 µL/min. The oxidized samples were collected from the outlet of the electrochemical cell for 30 s (∼25 µL), and 90 µL of 0.1 M phosphate buffer was added. The samples were then incubated at 37 °C. At selected time points (pH 5: 0, 1, 2.5, 5, 10, 15, 20, 30, and 45 min; pH 7.4: 0, 5, 10, 20, 30, 45, 60, and 120 min; pH 9: 0, 1, 2, 3, 5, 7.5, 10, 15, and 20 min), 10 µL of 62.5 mM glutathione in 0.1 M phosphate buffer was added to an incubation sample to react with the remaining NAPQI. The final reaction mixtures were analyzed by LC-MS, and the levels of APAP, APAP-SG, and p-acetamidobenzoic acid were determined. Determination of the Reactivity of NAPQI toward Other Nucleophiles. Twenty-five micromolar APAP was oxidized at +600 mV in a 0.1 M phosphate buffer with 25 µM p-acetamidobenzoic acid added as the internal standard. The experiments were performed at pH 5.0, 7.4, and 9.0. The flow through the electrochemical cell was 50 µL/min. The oxidized sample was collected from the outlet of the electrochemical cell for 30 s (∼25 µL), and 90 µL of 0.1 M phosphate buffer and 10 µL of 62.5 mM N-acetylcysteine, Nacetyltyrosine, N-acetylserine, or N-acetyllysine in 0.1 M phosphate buffer were added. The sample was then incubated at 37 °C. At selected time points (N-acetyltyrosine: 0, 0.17, 0.5, 1, 2, 3, 5, 10, 15, and 20 min; N-acetyllysine and N-acetylserine: 0, 10, 20, 30, 45, and 60 min), the reaction was stopped by the addition of 10 µL of 500 µM ascorbic acid to the reaction mixture. The samples were analyzed by LC-MS, and the levels of APAP, APAP conjugate, and p-acetamidobenzoic acid were determined. Determination of the Reactivity of NAPQI with Glutathione. The reactivity of NAPQI with glutathione was investigated at pH 5.0, 7.4, and 9.0 using cyclic voltammetry. Cyclic voltammograms were recorded for solutions containing 0.5 mM paracetamol and 5 mM glutathione in 0.1 M phosphate buffer. The scanning speed was varied between 20 and 15000 mV/s. Evaluation of the Electrochemical Method with Other Compounds. Several compounds for which metabolic oxidation has been reported to be responsible for the formation of reactive metabolic intermediates in liver microsomes were used to evaluate the method. A 10 µM solution of the test compound in a 0.1 M phosphate buffer (pH 7.4) and 25% MeCN was oxidized in intervals of 100 mV from 0 to +1500 mV versus the Pd/H2 reference electrode. Acetonitrile was added to enhance the solubility of the test compounds. From the outlet of the electrochemical cell, the sample was collected in a vial containing glutathione (final concentration was 10 mM), and the samples were analyzed by LC-MS and LC-MS/ MS for identification of glutathione conjugates. If no glutathione conjugates could be detected, oxidation was performed again, but this time glutathione (10 mM) was added to the sample before it was oxidized by passing through the electrochemical cell. Incubations in Human and Rat Liver Microsomes. All compounds tested in the electrochemical system, including APAP, were incubated with rat and human liver microsomes (0.5 mg protein/mL). The final concentration of the tested compound was

824 Chem. Res. Toxicol., Vol. 20, No. 5, 2007 10 µM, the GSH concentration was 3.3 mM, and the NADPH concentration was 0.66 mM. All incubations were performed in 0.1 M phosphate buffer at pH 7.4 at 37 °C for 30 min. The incubations were terminated by adding one volume of cold acetonitrile (containing 0.2% formic acid) to the incubation mixture. The samples were centrifuged (Eppendorf Centrifuge 5810R) for 10 min at 4 °C at 2900g. The supernatant was diluted 1:1 with water and was analyzed by LC-MS.

Results Internal Standard. To test the stability of NAPQI and the time-dependent formation of the APAP-NAT conjugate, an internal standard was needed to account for differences in volumes collected from the electrochemical cell. For this purpose, a wide range of compounds were tested for their potential use as an internal standard. Besides being electrochemically stable, the internal standard should also have a suitable retention in the chromatographic systems, that is, have a polarity similar to that of APAP-NAT. The MS response of p-acetamidobenzoic acid was constant before and after cooxidation with APAP at +600 mV, showing that p-acetamidobenzoic acid was not oxidized at any significant extent at +600 mV and, thus, was a suitable compound to use as an internal standard. Stability of Electrochemically Generated NAPQI. NAPQI was not analyzed directly by LC-MS/MS because the quinone imine was expected to be relatively unstable in aqueous solution. Because GSH reacts quantitatively and rapidly with NAPQI, as described in the Cyclic Voltammetry section, the presence of NAPQI was indirectly determined by adding GSH to the oxidized solution, and the resulting amount of GSH conjugate was used as a measure of NAPQI. The conjugate formed from the reaction of NAPQI with GSH eluted at 2.9 min and was detected at m/z 457.2 ([APAP-SG + H]+). The MS/MS spectrum contained the following fragment ions: m/z 382.1 ([APAP-SG - glycine + H]+); m/z 208.0 ([APAP-SG - glutamic acid - glycine - CO - 2H + H]+); m/z 182.0 ([APAP-SG - glutamic acid - glycine - C3H7NO - 2H + H]+); m/z 166.0 ([APAP-SG - glutamic acid - glycine - C3H7NO - H2O + H]+); m/z 140.0 ([APAP-SG - glutamic acid - glycine - C3H7NO - CH3CO + H]+); and m/z 129.1 ([glutamine - H2O]+) (Figure 1B). The retention time and fragmentation pattern of the conjugate formed from the reaction of electrochemically generated NAPQI with GSH was identical to those of a synthesized standard of APAP-SG. The stability of NAPQI was measured at selected time points at pH 3.0, 5.0, 7.4, and 9.0. At pH 3.0, it was not possible to detect any formation of APAP-SG, indicating that NAPQI was highly unstable. Preliminary experiments showed that NAPQI was hydrolyzed to the quinone in the electrochemical cell at pH 3.0 and that the quinone reacted with GSH (data not shown). At pH 5.0, 7.4, and 9.0, NAPQI was degraded by apparent firstorder kinetics (r2 > 0.988 at all pH values, data not shown). The stability of NAPQI was highest at pH 7.4, where the halflife was 47 min. At pH 5.0 and 9.0, the half-lives were 8 and 5 min, respectively. Oxidation of GSH. An alternative approach to trap electrochemically generated reactive intermediates involves the addition of GSH to the solution of the compound prior to electrochemical oxidation. In such an experiment, GSH is present inside the electrochemical cell during oxidation. Therefore, the oxidation of GSH to GSSG was investigated at the maximal oxidation potential used in the experiments. Five millimolar GSH was subjected to +1500 mV in 0.1 M phosphate buffer at pH 5.0,

Madsen et al.

7.4, and 9.0 and analyzed by HPLC using UV detection by which GSH and GSSG could be separated and detected. At pH 5.0, approximately 65% of GSH remained in the reduced form, whereas only approximately 30% GSH remained in the reduced form at pH 7.4 and 9.0. Oxidation of APAP in the Presence of GSH. APAP was oxidized at +600 mV with GSH present inside the electrochemical cell during oxidation. Three different products were detected at m/z 381.5 ([APAP-(SG)2 + 2H]2+), corresponding to the m/z value of the double charged diglutathione conjugate of paracetamol. In MS/MS experiments, the following fragment ions were detected: m/z 373.0 ([APAP-(SG)2 - NH3 + 2H]2+); m/z 344.0 ([APAP-(SG)2 - glycine + 2H]2+); m/z 317.0 ([APAP-(SG)2 - glutamic acid + 2H]2+); m/z 308.5 ([APAP(SG)2 - glutamic acid - NH3 + 2H]2+); and m/z 130.0 ([glutamic acid - H2O + H]+). Another GSH conjugate was identified at m/z 534.1 ([APAP-(SG)3 + 2H]2+) corresponding to the double charged triglutathione conjugate of paracetamol. By MS/MS fragmentation, the following fragment ions were detected: m/z 525.0 ([APAP-(SG)3 - H2O + 2H]2+); m/z 496.5 ([APAP-(SG)3 - glycine + 2H]2+); m/z 469.5 ([APAP-(SG)3 - glutamic acid + 2H]2+); m/z 460.5 ([APAP-(SG)3 - glutamic acid - H2O + 2H]2+); m/z 432.0 ([APAP-(SG)3 - glycine glutamic acid + 2H]2+); and m/z 405.0 ([APAP-(SG)3 - 2 glutamic acid + 2H]2+). The monoglutathione conjugate of paracetamol at m/z 457.2 could not be detected in this experiment. Reactivity of NAPQI toward N-Acetylcysteine, N-Acetyltyrosine, N-Acetyllysine, and N-Acetylserine. To investigate the reactivity of NAPQI toward different nucleophilic functional groups in proteins, the electrochemically generated NAPQI was incubated with N-acetylcysteine, N-acetyltyrosine, N-acetyllysine, and N-acetylserine at pH 5.0, 7.4, and 9.0 at 37 °C for different time intervals (see Experimental Procedures). To investigate whether there was a time-dependent formation of conjugates, it was important to be able to stop the reaction between NAPQI and the nucleophile. Ascorbic acid at a final concentration of 37 µM proved to be efficient at quenching the reaction, whereas higher concentrations of ascorbic acid affected the retention times of paracetamol, paracetamol-SG conjugate, and p-acetamidobenzoic acid in the LC-system. NAPQI reacted rapidly with N-acetylcysteine (NAC) to form the corresponding NAC-S-thioether conjugate, whereas it was not possible to detect N-acetylated amino acid conjugates following the incubation of NAPQI with N-acetyllysine and N-acetylserine. When NAPQI was incubated with N-acetyltyrosine, a time-dependent formation of a paracetamol-N-acetyltyrosine conjugate was observed (Figure 2), which reached a constant level after 4 min. Estimation of the Reactivity of NAPQI toward Thiols. Because of the very fast reaction between NAPQI and GSH, it was not possible to directly study the time-dependent formation of the APAP-SG adducts from the reaction of electrochemically generated NAPQI and GSH. Instead, another electrochemical method, cyclic voltammetry, was used for these studies, as this technique provides a rapid scanning method, which is more suitable for studying fast reactions (10-12). A cyclic voltammogram at a scan speed of 15 V/s at pH 7.4 is shown in Figure 3. An oxidation peak at +400 mV (oxidation to NAPQI) and a reduction peak at -370 mV (reduction of NAPQI to APAP) were observed for APAP. When GSH was present in the solution, it was not possible to detect a reduction peak, indicating that NAPQI was not reduced back to APAP. The returning potential in this experiment was +1000 mV,

ReactiVe Phase-I Metabolites

Figure 2. Time-dependent formation of the paracetamol-N-acetyltyrosine conjugate (APAP-NAT) at 37 °C in a 0.1 M phosphate buffer at pH 5.0 ((), 7.4 (9), and 9.0 (2) (n ) 4) following the incubation of electrochemically generated N-acetyl-p-benzoquinone imine with Nacetyltyrosine. p-Acetamidobenzoic acid was used as the internal standard (IS).

Figure 3. Cyclic voltammograms of 0.5 mM paracetamol (black) and 0.5 mM paracetamol in the presence of GSH (gray). Both voltammograms were recorded with a scan speed of 15 V/s in a 0.1 M phosphate buffer (pH 7.4).

meaning that the 600 mV from the oxidation peak of APAP to the scan direction was reversed. From reversing the potential to the reduction peak, there was 1370 mV, giving a scanned potential distance between the oxidation peak and reduction peak of 1970 mV. At a scanning speed of 15000 mV/s, this distance corresponds to approximately 130 ms, which indicates that NAPQI reacts quantitatively with GSH within 130 ms. It was not possible to identify any difference in reactivity among the experiments performed at pH 5.0, 7.4, and 9.0. Evaluation of the Method with Other Classes of Compounds. The formation of reactive metabolites was studied electrochemically for representative compounds (Scheme 1) reported to form quinone imines, imine methides, quinones, quinone methides, and nitrenium ions (Table 1) (13, 15, 1719, 21, 22). Furthermore, the same compounds were incubated with human and rat liver microsomes in order to compare electrochemical oxidation with the oxidative metabolism observed in a biological system. Samples obtained from electrochemical oxidation and from microsomal incubations were analyzed by LC-MS and LC-MS/ MS, and retention times and fragmentation patterns of the

Chem. Res. Toxicol., Vol. 20, No. 5, 2007 825

identified GSH conjugates were compared. For identification of conjugates formed at a low concentration in the microsomal incubations, a selective SRM transition for the GSH conjugate was used. Quinone Imines. As shown above, paracetamol was oxidized electrochemically to the corresponding quinone imine, which was efficiently trapped with GSH when the oxidized sample was collected at the outlet of the electrochemical cell. When paracetamol was incubated with HLM and GSH, it was also possible to detect a GSH conjugate at the same retention time (2.9 min) as the conjugate formed from electrochemical oxidation, indicating that the same product was formed in the electrochemical system as well as in human liver microsomes. As shown in Figure 1, similar MS/MS spectra were obtained for a synthetic standard of APAP-SG and APAP-SG formed electrochemically and in microsomes. Amodiaquine (AQ) is another compound for which metabolic oxidation to a quinone imine metabolite has been reported (13). By electrochemical oxidation of AQ at +600 mV, followed by collection of the oxidized sample in an aqueous solution of GSH, three different conjugates with GSH were detected at m/z 661.3 ([AQ-SG + H]+) with retention times of 3.9, 4.1, and 4.2 min. Fragmentation of m/z 661.3 gave the following fragment ions: m/z 588.1 ([AQ-SG - N(CH2CH3)2 + H]+) and m/z 315.0 ([AQ-SG - glutamic acid - glycine - C3H7NO - N(CH2CH3)2 + H]+). From incubation of AQ with HLM and RLM, only one conjugate at m/z of 661.3 was detected. The retention time was 4.3 min, and the fragmentation pattern was identical to that observed for the coeluting conjugate from the electrochemical experiment. (For chromatograms and MS/MS data, see Supporting Information 2 and 3.) Quinone Methides. Electrochemical oxidation of 3,5-di-tertbutyl-4-hydroxytoluene (BHT) at +400 mV, followed by collection of the oxidized sample in an aqueous solution of GSH, gave a product at m/z 526.5 ([BHT-SG + H]+) with a retention time of 5.9 min. (For the chromatogram, see Supporting Information 4.) Fragmentation of m/z 526.5 by MS/MS gave the following fragment ions (Figure 4): m/z 308.0 ([BHT-SG - BHT + H]+); m/z 233.0 ([BHT-SG - glycine - glutamic acid - NH3- H2O - tert-butyl + 3H]+); m/z 218.9 ([BHT-SG - GSH - 2H + H]+); m/z 215.2 ([BHT-SG - BHT - glycine - H2O + H]+); m/z 203.2 ([BHT-SG - GSH - H2O]+); m/z 179.2 ([BHT-SG - BHT - glutamic acid]+); m/z 162.0 ([BHTSG - BHT - glutamic acid - NH3 + H]+); m/z 143.9 ([BHTSG - BHT - glutamic acid - NH3 - H2O + H]+); and m/z 115.9 ([BHT-SG - BHT - glutamic acid - NH3 - CH3S]+). A product with the same m/z, retention time and fragmentation pattern was identified from the incubation of BHT with HLM and RLM (Figure 4 and Supporting Information 4 and 5) indicating that the same conjugate was formed in microsomes and in the electrochemical experiment. In HLM and RLM, a metabolite was observed at m/z 542.4, but this metabolite could not be identified in the electrochemical oxidation of BHT. The MS/MS spectrum of m/z 542.2 contained the following fragment ions: m/z 308.0 ([OH-BHT-SG - OHBHT + H]+); m/z 235.1 ([OH-BHT-SG - GSH - 2H + H]+); m/z 217.2 ([OH-BHT-SG - GSH - H2O - 2H + H]+); m/z 179.0 ([OH-BHT-SG - OH-BHT - glutamic acid + H]+); m/z 162.0 ([OH-BHT-SG - OH-BHT - glutamic acid - NH3 + H]+); and m/z 143.8 ([OH-BHT-SG - OH-BHT - glutamic acid - NH3 - H2O + H]+). (For the spectrum see Supporting Information 6.) As indicated by the assignment of the fragment ions, this spectrum was consistent with a GSH conjugate of hydroxy-BHT.

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Table 1. Comparison of the GSH Conjugates Formed from the Reaction of GSH with Reactive Intermediates from Electrochemical Oxidation (EC) and with Those Formed in Liver Microsomes (LM)

ReactiVe Phase-I Metabolites

Figure 4. MS/MS spectra of the BHT-SG conjugate at m/z 526.3 ([BHT-SG + H]+) from (A) electrochemical oxidation of 10 µM BHT at +400 mV and collection of the sample in an aqueous solution of GSH. (B) Incubation of 10 µM BHT with HLM, GSH, and NADPH.

Quinones. When BHA was oxidized in the electrochemical cell at +900 mV and the oxidized sample was collected in an aqueous solution of GSH, a product was detected at m/z 472.3 ([TBHQ-SG + H]+) at a retention time of 5.2 min. This m/z value corresponded to the glutathione conjugate of demethylated BHA. By MS/MS fragmentation of m/z 472.3, the following fragment ions were observed: m/z 397.2 ([TBHQ-SG - glycine + H]+); m/z 343.1 ([TBHQ-SG - glutamic acid + H]+); m/z 306.2 ([TBHQ-SG - TBHQ + H]+), m/z 251.0 ([TBHQ-SG - glycine - glutamic acid - NH3 + H]+); m/z 240.2 ([TBHQ-

Chem. Res. Toxicol., Vol. 20, No. 5, 2007 827

SG - glycine - glutamic acid - CO + H]+); m/z 223.1 ([TBHQ-SG - glycine - glutamic acid - CO - NH3 + H]+); m/z 197.0 ([TBHQ-SG - glutamic acid - glycine - C3H7NO + H]+); m/z 166.9 ([TBHQ-SG - GSH + H]+); m/z 163.1 ([TBHQ-SG - TBHQ - glutamic acid - H2O + 2H + H]+); m/z 130.0 ([glutamic acid - H2O + H]+); and m/z 112.0 ([glutamic acid - 2 H2O + H]+). (For chromatograms and MS/ MS data, see Supporting Information 7 and 8.) All fragment ions were in accordance with the product proposed in Scheme 2. The proposed mechanism for the electrochemical formation of the conjugate is shown in Scheme 2. The first two steps involve abstraction of an electron followed by deprotonation. The resulting radical then undergoes another one-electron abstraction. The formed carbocation rearranges, and the positively charged carboxy-carbon undergoes an ipso attack by water. Elimination of methanol gives a quinone that immediately reacts with GSH, resulting in the observed conjugate (23). To confirm the structure of this conjugate, tert-butylhydroquinone (TBHQ) was also employed in the studies. When a solution of TBHQ was oxidized in the electrochemical cell at +600 mV and collected in an aqueous solution of GSH, a product with the same retention time and MS/MS fragmentation pattern, as shown from the electrochemical oxidation of BHA, was detected. In human and rat liver microsomes, BHA and TBHQ both formed a GSH conjugate, which had the same retention time and MS/MS fragments as those observed for the product formed by electrochemical oxidation. (For chromatograms and MS/MS data, see Supporting Information 7 and 8.) When p-cresol was oxidized in the electrochemical cell in the presence of GSH at +1000 mV, a product eluting at 4.5 min was detected at m/z 430.3, corresponding to the addition of 321 Da to p-cresol. It is reasonable to assume that this conjugate originated from the hydroxylation of p-cresol resulting in the formation of 4-methylcatechol. The catechol was then oxidized to the o-quinone, which was trapped with GSH. Fragmentation of m/z 430.3 by MS/MS gave the following fragment ions: m/z 355.1 ([4-methylcatechol-SG - glycine + H]+); m/z 256.9 ([4-methylcatechol-SG - 4-methylcatechol SH - NH3 + H]+); m/z 209.2 ([4-methylcatechol-SG - glycine - glutamic acid - NH3 + H]+); m/z 198.2 ([4-methylcatecholSG - glycine - glutamic acid - CO + H]+); m/z 181.2 ([4methylcatechol-SG - glycine - glutamic acid - CO - NH3]+); m/z 163.1 ([4-methylcatechol-SG - 4-methylcatechol - glutamic acid - H2O + 2H]+); m/z 154.9 ([4-methylcatechol-SG glycine - glutamic acid - C3H7NO - 2H + H]+); m/z 130.2 ([glutamic acid - H2O + H]+); and m/z 111.8 ([glutamic acid

Scheme 2. Proposed Mechanism for the Formation of tert-Butylbenzoquinone from the Electrochemical Oxidation of BHA

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Madsen et al.

Figure 5. Extracted ion chromatograms of the 3-methylindole-SG conjugate at m/z 437.3 ([3-MI-SG + H]+) from (A) electrochemical oxidation of 10 µM of 3-methylindole at +600 mV and collection of the sample in an aqueous solution of GSH and (B) incubation of 10 µM 3-methylindole with HLM, GSH, and NADPH.

Figure 6. MS/MS spectra of the 3-methylindole-SG conjugate at m/z 437.3 ([3-MI-SG + H]+). (A) Electrochemical oxidation of 3-methylindole at +600 mV and collection of the sample in an aqueous solution of GSH. (B) Incubation of 3-methylindole with HLM, GSH, and NADPH.

- 2 H2O + H]+). (For the spectrum, see Supporting Information 9A.) All fragment ions were in accordance with the proposed structure of the 4-methylcatechol-SG conjugate. To further study the oxidation of p-cresol, a standard of 4-methylcatechol was oxidized at +600 mV. This resulted in the formation of a GSHconjugate with the same retention time and the same MS/MS characteristics as those described above, which was consistent with the proposed electrochemical oxidation of p-cresol. (For the spectrum, see Supporting Information 9.) Following microsomal incubations of p-cresol and 4-methylcatechol in HLM and RLM in the presence of GSH, the same conjugate at m/z 430.3 was detected. On the basis of the MS response, the amount of GSH conjugate formed from the incubation of p-cresol was relatively low, compared to the amount of GSH conjugate formed from the incubation of 4-methylcatechol, and could only be detected by SRM (430.3 f 163.1). (For chromatograms, see Supporting Information 10.) This showed that an identical reactive metabolite was formed in the electrochemical cell and in the microsomes. Imine Methides. When 3-methylindole (3-MI) was oxidized at +600 mV and the oxidized sample from the electrochemical cell was collected in an aqueous solution of GSH, a conjugate was detected at m/z 437.1 ([3-MI-SG + H]+), consistent with an addition of GSH (305 Da) (Figure 5). The MS/MS spectrum of m/z 437.1 contained the following fragment ions: m/z 308.2 ([3-MI-SG - 3-MI + H]+); m/z 233.2 ([3-MI-SG - glycine glutamic acid + H]+), m/z 231.1 ([3-MI-SG - 3-MI - glycine

- 2H + H]+); m/z 216.0 ([3-MI-SG - glycine - glutamic acid - NH3 + H]+); m/z 205.2 ([3-MI-SG - glutamic acid - glycine - CO + H]+); m/z 199.1 ([3-MI-SG - 3-MI - SH glycine]+); m/z 188.0 ([3-MI-SG - glutamic acid - glycine CO - NH3 + H]+); m/z 177.1 ([3-MI-SG - 3-MI - glutamic acid - 2H + H]+); m/z 164.0 ([3-MI-SG - glycine - glutamic acid - C3H7NO + H]+); m/z 162.0 ([3-MI-SG - 3-MI glutamic acid - NH3 + H]+); m/z 153.0 ([3-MI-SG - 3-MI glycine - CO - H2S - H2O + H]+); and m/z 145.0 ([3-MISG - 3-MI - glutamic acid - 2 H2O+ H]+) (Figure 6A). From incubations of 3-MI in HLM and RLM, two GSH conjugates were detected at m/z 437.1 at a retention time of 5.0 and 5.2 min. Both conjugates had almost identical MS/MS spectra. One of these conjugates had the same retention time (5.2 min) and MS/MS spectrum as that observed by electrochemical oxidation (Figure 6). (MS/MS data of the other conjugate is shown in Supporting Information 11.) Following electrochemical oxidation of trimethoprim (TMP) at +1000 mV in the presence of GSH, a GSH conjugate was observed at m/z 596.2 ([TMP-SG + H]+) corresponding to the addition of GSH (305 Da). This conjugate was not detected when the outlet from the electrochemical cell was collected in an aqueous solution of GSH, indicating high reactivity of the intermediate. The MS/MS spectrum of m/z 596.2 gave the following fragment ions: m/z 467.1 ([TMP-SG - glutamic acid + H]+); m/z 323.0 ([TMP-SG - glutamic acid - glycine -

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Figure 8. Aromatic region of the NMR spectrum of the clozapineSG conjugate. The conjugate was obtained from the purification of the sample from the electrochemical oxidation of clozapine in the presence of GSH at +600 mV.

is shown in Figure 8. The aliphatic region is not shown because of the complexity of signals from the glutathione moiety. When clozapine was oxidized in the electrochemical cell and the oxidized sample was collected in an aqueous solution of GSH, no GSH conjugate could be detected, indicating that the nitrenium ion is highly unstable.

Discussion

Figure 7. MS/MS spectra of the clozapine-SG conjugate at m/z 632.3 ([CLZ-SG + H]+). (A) Electrochemical oxidation of 10 µM clozapine in the presence of GSH at +600 mV. (B) Incubation of 10 µM clozapine with HLM, GSH, and NADPH.

C3H7NO + H]+), m/z 321.0 ([TMP-SG - glutamic acid glycine - C3H7NO - 2H + H]+); and m/z 254.1 ([TMP-SG TMP - 3 H2O + 3H]+). (For the spectrum, see Supporting Information 12.) TMP-SG could not be detected following incubations in microsomes. (For chromatograms, see Supporting Information 13.) Nitrenium Ion. When clozapine (CLZ) was oxidized at +600 mV in the presence of GSH, a product was detected at m/z 632.3 ([CLZ-SG + H]+), consistent with an addition of GSH (305 Da). Fragmentation of m/z 632.3 by MS/MS gave the following fragment ions: m/z 614.2 ([CLZ-SG - H2O + H]+); m/z 575.1 ([CLZ-SG - C3H7N(piperazine ring) + H]+); m/z 503.1 ([CLZSG - glutamic acid + H]+); m/z 446.1 ([CLZ-SG - glutamic acid - C3H7N(piperazine ring) + H]+); m/z 359.0 ([CLZ-SG - glutamic acid - glycine - C3H7NO(GSH-moiety) + H]+); and m/z 302.0 ([CLZ-SG - glutamic acid - glycine - C3H7NO(GSH-moiety) - C3H7N(piperazine ring) + H]+). All fragment ions were consistent with the CLZ-SG conjugate shown in Figure 7. A product with the same m/z, retention time, and fragmentation pattern was identified from incubations of clozapine in both HLM and RLM, indicating that a reactive intermediate identical to that in microsomes was electrochemically formed. A 1H NMR spectrum was also obtained for the electrochemically generated clozapine-SG conjugate. The aromatic region is shown in Figure 8. The chemical shifts of the observed signals were δ 6.87 ppm (1 H, d, J ) 2 Hz); δ 7.05 ppm (2 H, m); δ 7.13 (1 H, d, J ) 2.4 Hz); δ 7.22 (1 H, d, J ) 7.2 Hz); and δ 7.39 (1 H, t, J ) 7.2 Hz). This coupling pattern correlates with the C6-glutathionyl-clozapine conjugate, which

It has previously been reported that electrochemical methods can be used to mimic simple metabolic reactions such as N-, O-, and S-dealkylations and aromatic hydroxylations (Jurva et al.) (4, 5). Furthermore, Getek et al. and van Leeuwen et al. have shown that electrochemical methods can be used to generate reactive metabolites, such as the quinone imine of paracetamol (6) and the nitrenium ion of clozapine (7). In this study, a method, which enabled electrochemical formation of reactive metabolites, is presented. The method is an off-line approach, which utilizes experimental conditions that are similar to the physiological conditions used in in Vitro metabolism studies. The reactive metabolic intermediates that were formed using the electrochemical method reacted with GSH, and the resultant GSH conjugates were in accordance with those observed in in Vitro metabolism studies with rat and human liver microsomes (Table 1). Two different electrochemical approaches for studying reactive metabolic intermediate formation were investigated. The first approach involved oxidation of the analyte in the electrochemical cell followed by addition of glutathione after the cell to trap the reactive metabolic intermediate. The second approach was to trap the reactive metabolic intermediate inside the electrochemical cell by including GSH in the analyte solution, which proved to be a useful approach for unstable or very reactive intermediates, such as those of clozapine and trimethoprim. GSH was also shown to be oxidized, but at least 30% remained of the reduced form when the maximal potential (+1500 mV) was applied. Because GSH was added in great excess, the remaining part (>3 mM) was still sufficient to trap reactive intermediates. Electrochemical oxidation of APAP in different buffers showed that pH was an important factor for the stability of the quinoneimine (NAPQI). At pH 3.0, APAP-SG formed from the reaction of NAPQI with GSH was not detected, which may be due to a rapid hydrolysis of NAPQI under acidic conditions as

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reported by Novak et al. (24). At pH 5.0 and 9.0, the stability of NAPQI increased relative to pH 3.0, and at pH 7.4, NAPQI showed the highest stability. For the other test compounds, the stability of the reactive intermediate at different pH values was not investigated, but pH 7.4 was chosen because of the physiological relevance. Cyclic voltammetry was used to study the rate of the reaction between NAPQI and GSH at pH 5.0, 7.4, and 9.0. In these studies, it was shown that NAPQI and GSH quantitatively reacted within 130 ms. The reaction time was in the same order of magnitude as that previously proposed by Brookes et al. (12). NAPQI also formed conjugates with N-acetyltyrosine and N-acetylcysteine, whereas N-acetyllysine and N-acetylserine conjugates were not detected. It took about 4 min before the reaction between NAPQI and N-acetyltyrosine was complete, indicating that this reaction was more than 2000-fold slower than the reaction between NAPQI and GSH. The initial rate constants were almost the same at the three different pH values for the formation of APAP-NAT. However, the amount of APAP-NAT conjugate differed, which may be due to the relatively rapid degradation of NAPQI at pH 5.0 and 9.0 compared to that at pH 7.4. These results showed that NAPQI reacts very rapidly with thiols but that NAPQI may also react with tyrosine groups in proteins. In general, the conjugates formed after electrochemical oxidation of the compounds were also identified in microsomal incubations. This method is therefore well suited to study reactive metabolic intermediates and their reaction with any chosen nucleophile. In the case with clozapine, the method also proved to be useful for the synthesis of GSH conjugates, which otherwise can be quite time consuming if they have to be synthesized in a traditional manner. The nitrenium ion of clozapine reacted with GSH inside the electrochemical cell and was formed in amounts that made it possible to obtain NMR data of the conjugate. The NMR data suggested that the most reactive position of the clozapine intermediate is the C-6 atom. The electrochemical method showed some limitations because there were some CYP-mediated reactions, which could not be mimicked electrochemically. OH-BHT-SG was detected in the microsomal incubations, whereas this conjugate could not be detected by electrochemical oxidation of BHT. BHT is most likely hydroxylated on an aliphatic carbon (17), a reaction that was not mimicked electrochemically, probably because of the high energy required for aliphatic hydroxylation and thus a very high potential, which could not be obtained in the electrochemical cell used in these experiments. p-Cresol has been reported to form a quinone methide in microsomes (18), but the corresponding conjugate was detected neither in the samples from electrochemical oxidation nor in microsomes. Instead, p-cresol was found to be hydroxylated, resulting in the formation of 4-methylcatechol, which was immediately further oxidized to the o-quinone and trapped with GSH. BHT, which forms a quinone methide electrochemically as well as in microsomes, has two tert-butyl groups in orthopositions to the phenol, which block the hydroxylation in this position. 3-Methylindole formed only one GSH conjugate after electrochemical oxidation, whereas two GSH conjugates were identified in the microsomal incubations. One of the conjugates that was formed in microsomal incubation coeluted with the conjugate from electrochemical oxidation, which may have originated from the reaction of GSH with the imine methide metabolite. The other conjugate, which appeared in the microsomal samples, may originate from the reaction of GSH with

Madsen et al.

an epoxide or hydroxyindolenine followed by the loss of water, as proposed by Skordos et al. (25). A trimethoprim-SG conjugate was readily identified from the electrochemical oxidation of trimethoprim in the presence of GSH. This conjugate may have resulted from the reaction of an electrochemically generated imine methide with GSH. In this study, it was not possible to identify the conjugate in microsomal incubations, although this conjugate has previously been reported by Lai et al. (15). This may be due to the difference in substrate concentration in microsomal incubations, as 10 µM was used in the present study in contrast to 400 µM in the study by Lai et al. In conclusion, a method for generating reactive intermediates by electrochemical oxidation has been developed. A wide range of reactive metabolites can be formed with this method, including quinone imines, quinones, imine methides, quinone methides, and nitrenium ions. The reactive metabolites formed by electrochemical oxidation were trapped with GSH, and the resultant GSH conjugates were for many of the test compounds similar to what was seen in microsomal incubations in the presence of GSH. This may be a useful tool for studies on reactive metabolic intermediates such as reactivity measurements, studies with different nucleophiles, or large-scale synthesis of GSH conjugates for NMR analysis. Acknowledgment. We thank Henrik Jensen (Department of Pharmaceutics and Analytical Chemistry at The Faculty of Pharmaceutical Sciences, University of Copenhagen) for help with the cyclic voltammetry experiments and Claus Cornett (Department of Pharmaceutics and Analytical Chemistry at The Faculty of Pharmaceutical Sciences, University of Copenhagen) for recording the obtained NMR-spectra. Furthermore, we thank Tove Johansson at Discovery DMPK & Analytical Biochemistry, AstraZeneca R&D Mo¨lndal, Sweden for help with the microsomal incubations. The Danish Medical Research Council Grant No. 22-02-0340, The Danish Technical Research Council Grant No. 56-01-0014, and Lundbeckfonden Grant No. 142/02 are acknowledged for financial support. Supporting Information Available: Supporting information 1 to 13 contains selected chromatograms and MS/MS-spectra. This information is available free of charge via the Internet at http:// pubs.acs.org.

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