Cyanide Trapping of Iminium Ion Reactive Intermediates Followed by

Sep 22, 2005 - Secondary and tertiary alicyclic amines are widely found in pharmaceuticals and environmental compounds. The formation of iminium ions ...
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Chem. Res. Toxicol. 2005, 18, 1537-1544

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Cyanide Trapping of Iminium Ion Reactive Intermediates Followed by Detection and Structure Identification Using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Dayana Argoti,† Li Liang,‡ Abdul Conteh,‡ Liangfu Chen,‡ Dave Bershas,‡ Chung-Ping Yu,‡ Paul Vouros,† and Eric Yang‡ The Barnett Institute and Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts 02115, and Drug Metabolism and Pharmacokinetics, GlaxoSmithKline Pharmaceuticals, 709 Swedeland Road, King of Prussia, Pennsylvania 19406 Received June 16, 2005

Secondary and tertiary alicyclic amines are widely found in pharmaceuticals and environmental compounds. The formation of iminium ions as reactive intermediates in the metabolic activation of alicyclic amines has previously been investigated in radiometric assays where radiolabeled cyanide is typically employed. In this paper, we report a relatively high throughput LC-MS/MS method for the detection of the nonradiolabeled cyanide adduct formed in rat or human liver microsomal incubations via constant neutral loss scan followed by structural characterization using product ion scan on a triple quadrupole mass spectrometer. A total of 14 alicyclic amine compounds were investigated with the cyanide trapping LC-MS/MS screen and also with the glutathione (GSH) trapping screen, a well-established and commonly employed technique for reactive metabolite screening. Our results are found to be in general agreement with the previous metabolism reports for these compounds, demonstrating the effectiveness, speed, and simplicity of the cyanide trapping LC-MS/MS method to study the iminium ion intermediates from alicyclic amines and its complementarities to GSH trapping method for reactive metabolite screenings.

Introduction Over the years, reactive metabolites are believed to have played an important role in the safety profile of pharmaceuticals. Cytochrome P450-mediated bioactivation of the drug to reactive metabolites has been reported to be the first step in many adverse drug reactions (1). The covalent binding theory has linked toxicity to the formation of reactive metabolites, where bioactivation is a critical step in the process leading to an electrophilic metabolite that can bind to cellular macromolecules, for example, proteins and DNA (2). The resulting drugprotein adduct may cause partial or complete loss of the biochemical function of the protein or act as a hapten that elicits an immune response and results in cellular and organ damage. The drug-DNA conjugates are potentially carcinogenic and may lead to undesired effects on gene expression and gene mutation. Currently, no animal model can accurately predict adverse drug reactions in humans. Adverse drug reactions usually have a low occurrence rate and typically do not surface until a large population has been studied, most likely during or after phase III clinical trials (3). Therefore, reactive metabolite detection has become a vital topic of interest for early screening to help avoid safety issues leading to compound failure at a later stage. A few methods have been introduced in the preclinical phase of drug development to help identify compounds from a large library that carry such potential risk. One of the most commonly used techniques is gluta* Corresponding author. Tel, 610-270-6291; fax, 610-270-4971; e-mail, [email protected]. † Northeastern University. ‡ GlaxoSmithKline Pharmaceuticals.

thione (GSH) trapping screen with hepatic subcellular fractions in the presence of cytochrome P450 cofactor NADPH (4-6). In this method, GSH is used to trap electrophilic metabolites via the cysteine sulfhydryl group to form a conjugate. The resulting GSH conjugate, when fragmented by collision-induced dissociation (CID), gives a common loss of the pyroglutamic acid moiety from the structure, which can be easily detected and characterized by a neutral loss scan of 129 Da using liquid chromatography coupled with tandem mass spectrometry (LC-MS/ MS) (7-8). Reported reactions of GSH with electrophilies include the nucleophilic substitution at saturated and aromatic carbons as well as oxirane ring, addition to a carbonium and nitroso ions, and Michael addition (9). However, there are a few drawbacks associated with the GSH trapping screen. Not all GSH adducts afford the neutral loss of 129 Da upon CID. Aliphatic and benzylic thioether conjugates may eliminate the GSH (307 Da) as a neutral, while thioester conjugates typically lose glutamic acid (147 Da) from the protonated GSH adducts (10-11). It was recently reported that scanning in the negative ion mode for the precursors of the ion at m/z 272, corresponding to deprotonated glutamyl-dehydroalanyl-glycine, may provide a more generally applicable technique for the detection of GSH adducts (12). Moreover, the GSH trapping screen cannot detect some types of reactive intermediates, for example, iminium ions, acyl glucuronides, and aldehydes which generally do not form stable adducts with GSH, perhaps due to the reversibility of the reaction and also exchange reactions with other nucleophiles. Metabolic activation of alicyclic amines often generates a number of oxidative products including N-dealkylation,

10.1021/tx0501637 CCC: $30.25 © 2005 American Chemical Society Published on Web 09/22/2005

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ring hydroxylation, R-carbonyl formation, N-oxygenation, and ring opening metabolites that can be formed through an iminium ion intermediate (13). Therapeutic pharmaceuticals and their metabolites containing an alicyclic amine structure have the potential to form iminium ions that are reactive toward nucleophilic macromolecules. An extensively studied example is nicotine, which has been shown to form an iminium ion that can mediate covalent binding of nicotine to cellular macromolecules (13). The iminium ion of mianserin has also been reported as the reactive intermediate that causes agranulocytosis (1). The metabolism of compounds containing an alicyclic amine structure has been studied with radiolabeled cyanide in radiometry assays, where radiolabeled cyanide was used to react with the iminium ion intermediates (14). The method was relatively time-consuming, and no structural information was directly available. Recently, it was reported by Evans et al. that the cyanide conjugates could be detected using the neutral loss scan of 27 and 29 Da by LC-MS/MS, using a mixture containing equal amounts of unlabeled and isotopically labeled cyanide (3, 15). We present in this paper a relatively high-throughput LC-MS/MS method for screening iminium ion formation in rat and human liver microsomes using potassium cyanide as the trapping reagent. The cyanide trapping experiments, along with GSH trapping, were performed on 14 compounds containing an alicyclic amine structure. Furthermore, the MS/MS fragmentation pathways of cyanide adducts of nefazodone and prochlorperazine detected in the microsome incubations were investigated, and the structures of the trapped iminium ion intermediates were proposed.

Experimental Procedures Chemicals and Reagents. All test compounds, potassium cyanide (KCN, K13C15N) and glutathione (GSH) were obtained from Sigma-Aldrich Corporation (MO). Pooled mixed-gender human and rat liver microsome were purchased from XenoTech, LLC (KS). Methanol (HPLC grade) was purchased from JT Baker (NJ), and ammonium acetate was purchased from Sigma/ Aldrich (MO). Distilled water was purified “in-house” using a MilliQ system Millipore (MA). Other chemicals and reagents were of the highest quality available. Microsomal Incubations. For the cyanide trapping experiments each test compound was incubated with rat or human liver microsomes in the presence of an NADPH regenerating system and cyanide in 50 mM potassium phosphate buffer (pH 7.4). Stock solutions of test compounds were prepared in DMSO and added to the microsomal incubation with a final DMSO concentration of 0.5% (v/v). Two separate incubation sets were performed, one with KCN and the other with K13C15N. Each incubation set included three controls, the first without test compound, the second without NADPH, and the third without KCN. The incubations were conducted in 96-deepwell refill tubes (Micronic North America, PA) at 37 °C in a water bath while shaking at 45 rpm. The initial incubation mixture consisting of test compound, microsomes, and KCN/K13C15N was preincubated for 3 min in a total volume of 400 µL. The reaction was initiated by the addition of 100 µL of NADPH-generating system consisting of 2.22 mM NADP+, 27.65 mM glucose-6-phosphate, and 6.0 units/mL glucose-6-phosphate dehydrogenase together with 15 mM MgCl2 in phosphate buffer. The final volume in incubation was 500 µL. The final concentrations of microsome, test compound, and KCN/K13C15N in the incubation mixture were 1 mg/mL, 100 µM, and 1 mM, respectively. The incubation was terminated after 90 min by the addition of 100 µL of acetonitrile containing 5% ammonium hydroxide. The samples were placed on ice for approximately 10 min, followed by centrifugation at 2250g at 4 °C for 20 min. The supernatant

Argoti et al. was directly injected onto LC-MS/MS. The same experiments described above for cyanide incubations were also performed for GSH trapping, except that 10 mM GSH (final concentration) was used to replace KCN as the trapping agent, and the quenching solution consisted of 100 µL of acetonitrile containing 6% of acetic acid. Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS). LC was performed on a Flux Instruments Rheos 2000 (Leap Technology, NC) and CTC HTS PAL autosampler (Leap Technology). The separations were performed on a 2.1 mm × 50 mm Waters YMC ODS-AQ C18 5 µm column. A gradient elution was employed on the column at 300 µL/min with mobile phase A (10 mM ammonium acetate, pH adjusted to 4 with acetic acid) and mobile phase B (methanol) running a linear gradient in which the percent of mobile phase B was held at 4% for 0.5 min, then was linearly increased to 90% during the next 2.5 min. The mobile phase B was held at 90% for 2 min and then immediately returned to 4%. The analysis time was 8 min per sample. The typical injection volume was 5 µL. All the mass spectra were collected on an Applied Biosystems/ MDS Sciex API-4000 triple quadrupole mass spectrometer (ON, Canada). The TurboIonSpray (TIS) interface was operated in positive ion mode. The mass spectrometric conditions for cyanide trapping samples were optimized using the nicotine cyanide conjugate. The MS/MS experiments performed included the neutral loss scan (NL scan) of 27 (or 29) and the product ion scan of the cyano adducts detected. The mass spectrometric conditions for GSH conjugates detection using the NL scan of 129 were optimized using the benzoquinone-GSH adduct, formed by mixing benzoquinone and GSH solutions directly. The TurboIonSpray source temperature was maintained at 600 °C. The ionspray voltage was set at 4000 V. The curtain gas was set at 20 and the declustering potential (DP) at 56 V, and the nebulizer (GS1) and TIS (GS2) gases were set at 50 and 70 psi, respectively. The CID gas was set at 4, and the collision energy was set at 29 eV for the NL scan experiments. Unit mass resolution was used in all the experiments. LC-MS/MS data were acquired using Analyst Software Version 1.1 (MDS Sciex, Canada).

Results and Discussion The experimental conditions for cyanide incubation were modified from the established GSH trapping method (7, 14). Using nicotine as the test compound, we optimized the final KCN/K13C15N concentration in the incubation mixture at 1 mM, which yielded the highest cyano adduct amount. Acetonitrile containing 5% ammonium hydroxide was chosen as the quenching solution to prevent the potential formation of toxic HCN gas. Two separate cyanide incubations were performed for each study, one with KCN and the other with K13C15N, to firmly establish the formation of cyano adducts detected in the incubations. The cyanide conjugates were detected by LC-MS/MS analysis via a constant neutral loss scan (NL) of 27 and 29 Da on a triple quadrupole mass spectrometer for incubations with KCN and K13C15N, respectively. A second injection was then made to acquire MS/MS product ion spectral of the detected cyano adducts to confirm the proposed structures for the trapped iminium ion intermediates. Each incubation set entailed three controls, one without test compound to rule out any potential interference/ contamination from endogenous compounds, the second without NADPH, a cofactor for cytochrome P450 activities, to assess the metabolic dependence of cyano adduct formation, and the third without KCN/K13C15N. No cyano

Cyanide Trapping of Iminium Ion Reactive Intermediates

Chem. Res. Toxicol., Vol. 18, No. 10, 2005 1539 Table 1. Summary of Cyanide and GSH Trapping Results for 14 Compounds Containing Alicyclic Amine Structurea

drug nicotine nefazodone

Figure 1. Iminium ion intermediate formation through hydrogen or electron abstraction from an alicyclic amine by cytochrome P450 isozyme in liver microsome and subsequent trapping via cyanide adduction of the carbonium ion.

prochlorperazine

CN adduct detected (proposed metabolic reaction)

Pb NDc Pb P + 16 (monooxygenation)b P + 32 (dioxygenation)b NDc P - 112 + 14 (dealkylation + unknown)b P - 112 + 30 (dealkylation + mono-oxygenation + unknown)b b P

P + 16 (monooxygenation)b P + 2 (demethylation + mono-oxygenation)b P + 30 (monooxygenation + unknown)b b triprolidine P P + 14 (R-carbonyl formation)b mianserin Pb P + 16 (monooxygenation)b indinavir Pb P - 77 (ring opening)b P - 77 + 16 (ring opening + mono-oxygenation)b ketoconazole Pb P + 16 (monooxygenation)b ciprofloxacin P + 16 (monooxygenation)b thioridazine P + 2 (demethylation + mono-oxygenation)b P + 16 (monooxygenation)b P + 32 (dioxygenation)b P + 48 (trioxygenation)b clozapine P - 14 (demethylation)b Pb P + 16 (monooxygenation)b phencyclidine NDc clemastine NDc rifampin

Figure 2. Total ion chromatogram (TIC) of neutral loss scan (NL) of 27 (panel A) and 29 Da (panel B) LC-MS/MS analysis for cyano adducts of nicotine formed in human liver microsomes with KCN and K13C15N, respectively. The two peaks on the chromatogram reflect the formation of two iminiun ion intermediates and are subsequently correlated by the formation of two isomeric cyano adducts.

adducts were detected from the “no compound” and “no NADPH” controls. However, cyano adducts at lower intensity were found in some of the “no cyanide” control incubation samples. Cyanide in these samples was suspected to be generated by the metabolically active microsomes and derived from the cytochrome P450dependent oxidative metabolism of acetonitrile which was added as the quenching solution. Acetonitrile is substrate for this reaction and was known to quickly metabolize into cyanide in microsomes (16). Nicotine. A scheme for the formation of a iminium ion, in equilibrium with the carbonium ion, through hydrogen or electron abstraction by cytochrome P450 isozymes and the subsequent trapping with cyanide is shown in Figure 1 (7). Nicotine has often been used as the standard in these investigations due to its thoroughly studied cyano adduct formation using radiolabeled cyanide (14, 17). The LC-MS/MS chromatograms from the NL scan of 27 and 29 Da for incubations of nicotine in

NDc ticlopidine

GSH adduct detected (proposed metabolic reaction)

NDc

NDc

Pb NDc

NDc

P - 2 (unknown)b NDc

NDc

P NDc NDc m/z 971 (unknown) m/z 953 (unknown) P - 16 (unknown)b P - 14 (demethylation)b P + 16 (mono-oxygenation)b P + 16 (mono-oxygenation)b

a

The results from human and rat microsomes are found to be similar. b P: parent drug. c ND: not detected.

human liver microsome with KCN and K13C15N are shown in Figure 2, panels A and B, respectively. The detection of the two isomeric cyano adducts of nicotine reflects the likely formation of two iminium ion intermediates resulting in direct cyanide addition to the carbons alpha to the endocyclic nitrogen. The mass-tocharge (m/z) ratio for the protonated cyanide adduct was found to be 188 for incubation with KCN and 190 for incubation with K13C15N. The difference in m/z ratio corresponded to the mass difference between KCN and K13C15N. This observation is in agreement with the previous knowledge on the metabolic activation of the compound (14, 17). Nicotine was therefore chosen as a positive control compound included in each set of cyanide trapping studies described in this paper. On the other hand, in a parallel incubation of nicotine with GSH followed by an NL scan of 129 Da LC-MS/MS experiment, few GSH adducts were detected, thus, confirming that iminium ion intermediates were in fact trapped much more efficiently by cyanide (Table 1).

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Figure 3. TIC of NL 27 LC-MS/MS analysis of nefazodone KCN trapping incubation in rat liver microsome. The cyano adducts of parent (NM6) and several metabolites (NM1-NM5) are detected in this sample.

Nefazodone. Nefazodone, an antidepressant drug with known hepatotoxicity (18) and has been reported to be extensively metabolized via N-dealkylation and hydroxylation, typical of alicyclic amines (19). Although negative in GSH screen performed in this study, a GSH adduct of this drug was detected and characterized by Kalgutkar et al. (20). As shown in Figure 3, LC-MS/MS analysis of the rat liver microsome incubation sample using an NL scan of 27 Da has detected the cyano adducts of parent nefazodone (NM6) and several metabolites (NM1-5). On the basis of the mass of protonated cyano adducts detected in the NL scan and the subsequent product ion scan experiments described below, structures for the alicyclic amine metabolites are proposed (Figure 4). The product ion mass spectrum of the parent drug nefazodone (Figure 5A) was used as a template to characterize the structures of the various nefazodone metabolites. Comparison of MS/MS spectra of the parent compound with those of the mono-oxygenated cyano ad-

Argoti et al.

ducts with [M + H]+ values of m/z 511 revealed two mono-oxygenated isomers, NM5 and NM4. The prominent fragment at m/z 290 in the MS/MS spectrum of mono-oxygenated metabolite, NM5 (Figure 5B), reflects a 16 Da shift from the m/z 274 ion in the MS/MS spectrum of parent drug, which is consistent with mono-oxygenation on the portion of the molecule containing the phenoxyethyl triazolone (Figure 4, NM5). As a result of the neutral loss of HCN (27 Da) from the cyano adduct of the mono-oxygenated metabolite under collision-induced dissociation (CID) condition, the fragment ion at m/z 484 was observed. Subsequent further fragmentation of the ion at m/z 484 gave rise to the fragment at m/z 194 in the MS/MS spectrum of NM5. Additionally, MS/ MS spectra of the cyano adduct of NM5 formed in the presence of K13C15N did not show any 2 Da shifts in the fragmentation pattern, indicating the immediate neutral loss of H13C15N (29 Da) from the cyano adducts under CID conditions (Figure 5C). Further investigation of the MS/MS spectrum of NM5 using m/z 513 (the 37Cl isotope of the protonated molecule) as a precursor ion (Figure 5D) confirmed the presence of a chlorine atom in the fragment ion at m/z 194 in NM5 from the respective shift to m/z 196. The structure of the second mono-oxygenated metabolite NM4 can be inferred by comparison of its MS/MS spectrum with that of NM5. The fragment of NM5 at m/z 194 shifted by 16 Da, to m/z 210, in the MS/MS spectrum of NM4 (Figure 6A), suggesting likely mono-oxygenation of the chlorophenyl piperazine in NM4. As indicated by ions at m/z 210 and 290 shown in Figure 6B, NM3 (m/z 527) was found to be the dioxygenated metabolite with oxygenation sites on the phenoxyethyl triazolone and chlorophenyl piperazine (Figure 4). The NM2 metabolite (m/z 399) corresponded to N-dealkylation plus a 14 Da mass addition to the piperazine ring, possibly due to a R-carbonyl formation, a methylation, or a double bond

Figure 4. Proposed structures of the cyano adducts of nefazodone and metabolites with bioactivation sites indicated in blocks. Key fragment ions detected in MS/MS experiments are shown in Figures 5 and 6. NM1, cyano adduct of N-dealkylated/mono-oxygenated metabolite with an addition of 14 Da to the piperazine ring (structure unknown); NM2, cyano adduct of N-dealkylated metabolite with an addition of 14 Da to the piperazine ring (structure unknown); NM3, cyano adduct of dioxygenated metabolite; NM4 and NM5, cyano adduct of mono-oxygenated metabolite; NM6, cyano adduct of the parent compound.

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Figure 5. (A) MS/MS spectrum of nefazodone (m/z 470); (B) MS/MS spectrum of NM5 (m/z 511) formed in KCN incubation; (C) MS/MS spectrum of NM5 (m/z 513) formed in K13C15N incubation; and (D) MS/MS spectrum of 37Cl isotope of NM5 (m/z 513) formed in KCN incubation.

Figure 6. (A) MS/MS spectrum of mono-oxygenated cyano adduct NM4 (m/z 511); (B) MS/MS spectrum of dioxygenated cyano adduct NM3 (m/z 527); (C) MS/MS spectrum of N-dealkylated plus 14 Da cyano adduct NM2 (m/z 399); and (D) MS/MS spectrum of N-dealkylated/mono-oxygenated metabolite plus 14 Da cyano adduct NM1 (m/z 415).

formation in conjunction with a hydroxylation. Comparison of panels C and D of Figure 6 shows the shift of the product ion at m/z 274 ion for NM2 to m/z 290 for NM1, indicating mono-oxygenation on the phenoxyethyl triazolone portion of the molecule in NM1 (m/z 415). These results are in agreement with the previously published data on the metabolites of nefazodone (19). Prochlorperazine. Prochlorperazine is a currently marketed drug used to treat dizziness due to labyrinthine disorder. It contains the characteristic alicyclic amine moiety and has been reported to form a number of metabolites via N-oxidation, N-alkylation, sulfoxidation, and

aromatic ring hydroxylation (21). The neutral loss scan (NL) of 27 Da LC-MS/MS analysis of prochlorperazine in microsome incubations revealed a number of metabolites, including two mono-oxygenated metabolites with [M + H]+ value of m/z 415 (PM1 and PM4), one monooxygenated plus N-demethylated metabolite at m/z 401 (PM2), one mono-oxygenated plus 14 Da metabolite at m/z 429 (PM3), and a cyano adduct of prochlorperazine parent at m/z 399 (PM5) (Figure 7). The proposed structures and key MS/MS fragments of the cyano adducts detected are shown in Figure 8.

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Figure 7. TIC for NL scan of prochlorperazine KCN trapping incubation in human liver microsome. The cyano adducts of parent (PM5) and several metabolites are detected in this sample (PM1-PM4).

Figure 8. Proposed structures of the cyano adducts of prochlorperazine and metabolites with key fragment ions detected in MS/MS experiments shown in Figures 9 and 10. PM1 and PM4, cyano adduct of mono-oxygenated metabolite; PM2, cyano adduct of N-demethylation/mono-oxygenated metabolite; PM3, cyano adduct of a mono-oxygenated metabolite plus 14 Da (structure unknown); and PM5, cyano adduct of parent compound.

A strategy similar to that for nefazodone was used to determine the structures of the cyano adducts of metabolites detected in NL scans. The CID mass spectrum of the parent drug, as shown in Figure 9A, was used as a template to determine sites of biotransformation of prochlorperazine. The structure of the cyano adduct of the parent drug at m/z 399, PM5, was characterized as follows. A comparison of the MS/MS spectrum for the parent compound and its cyano adduct, PM5 (Figure 9, panels A and B), reveals a 25 Da shift of the fragment ions at m/z 113 and 141 to m/z 138 and 166, respectively, indicative of cyanide addition on the piperazine ring (Figure 8, PM5). This assignment was further confirmed by examination of the MS/MS spectra of PM5 formed by incubation in the presence of K13C15N and of the 37Cl isotope of PM5 (Figure 9, panels C and D). Specifically, the MS/MS data for the cyano adduct formed in the presence of K13C15N showed a 27 Da mass shift for the fragments at m/z 113 and 141 to m/z 140 and 168,

Argoti et al.

respectively (Figure 9, panels A and D). This shift confirmed the presence of isotopically labeled cyanide on the piperazine ring. The MS/MS spectrum of the 37Cl isotope of the protonated parent ion at m/z 401, shown in Figure 9C, subsequently correlated the presence of Cl on parent fragment m/z 246 by exhibiting a 2 Da shift to m/z 248. This information in turn further confirmed the presence of a cyano adduct on the piperazine-ring portion of the parent structure. For structural characterization of the mono-oxygenated metabolite PM1 (m/z 415) at retention time of 3.94 min, a comparison of the MS/MS spectrum of the parent drug with that of PM1 showed that a major parent fragment ion at m/z 141 had shifted by 25 Da to a minor, yet significant, fragment ion at m/z 166 indicative of cyanide presence on the piperazine ring (Figure 10A). The prominent fragment ion at m/z 139 observed in Figure 10A was formed by a loss of HCN from the fragment ion at m/z 166. This information also suggested that the mono-oxygenation occurred on the phenothiazine. For metabolite PM4 at retention time of 4.49 min, the fragment ion at m/z 388 was formed by loss of HCN (27 Da) from the cyano adduct of the mono-oxygenated metabolite m/z 415 (Figure 10B). The major fragment ion at m/z 155 was generated by the loss of the phenothiazine from m/z 388. The subsequent loss of 17 Da from m/z 388 yielded a fragment ion at m/z 371, suggesting the formation of N-oxide on the piperazine ring. The remaining cyano adducts of prochlorperazine, the mono-oxygenated plus 14 Da, and the demethyl plus mono-oxygenated metabolites, were characterized in a similar manner, and proposed modification sites are shown in Figure 8 for PM3 and PM2, respectively. However, the biotransformation pathways corresponding to the 14 Da increase in PM3 cannot be accurately ascertained using the methodology described in the paper. Summary of Results from Cyanide and GSH Trapping Experiments. Cyanide and GSH trapping experiments have also been performed on 11 additional compounds with the characteristic alicyclic amine core. The neutral loss scans (NL) of 27 Da and of 129 Da were performed to detect the formation of cyanide adducts and GSH conjugates, respectively. In general, cyanide and GSH trapping results were found to be similar between rat and human microsome incubation samples for all 14 compounds tested. Results from both experiments and the proposed structures for the detected adducts are summarized in Table 1 (see Supporting Information for compound structures). In addition to nicotine, nefazodone, and prochlorperazine, cyano adducts have also been detected in seven other compounds listed in Table 1. The cyano adducts of the iminium ion intermediate formed directly from parent drug were observed from triprolidine, mianserin, indinavir, ketoconazole, and clozapine. A few metabolitebased cyano adducts were also detected with structures in agreement with those metabolism reports for these compounds (22-30). This corresponded to the metabolic activations of N-dealkylation (thioridazine and clozapine), possible R-carbonyl formation (triprolidine), oxygenation (mianserin, indinavir, ketoconazole, ciprofloxacin, thioridazine, and clozapine), and ring opening (indinavir). Cyanide trapping LC-MS/MS screen has thus proven to be an important method for the detection and identification of iminium ion intermediates from the alicyclic amine compounds.

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Figure 9. (A) MS/MS spectrum of prochlorperazine (m/z 374); (B) MS/MS spectrum of cyano adduct, PM5 (m/z 399); (C) MS/MS spectrum of 37Cl isotope ion of cyano adduct, PM5 (m/z 401); and (D) MS/MS spectrum of cyano adduct, PM5, in K13C15N incubation (m/z 401).

cyanide (cyanide was reported to inhibit the metabolism of phencyclidine in a microsomal system (31)), or that the amount of cyano adducts formed is below the detection limit of the LC-MS/MS instrument employed. For a majority of the compounds presented in Table 1, no or few GSH adducts were detected. Triprolidine and clozapine were found to conjugate with GSH directly. While no cyano adducts were detected from rifampin and ticlopidine incubations, several electrophilic metabolites were trapped with the GSH screen. On the other hand, cyanide has the capability to trap reactive iminium ion intermediates previously not detected with GSH. For example, in clozapine, reactive metabolites resulting from the formation of radical intermediates of oxidative processes were detected with GSH (28, 29). Other metabolites reported for the metabolism of clozapine include a demethylation, an N-oxidation, and hydroxylation. These metabolites were detected in the cyanide trapping screen and thus are presumed to form iminium ion intermediates. In this case, all metabolites related to the metabolism of clozapine were detected with the combination of both GSH and cyanide trapping screen, demonstrating the complementarities of the GSH and cyanide trapping screen for reactive metabolite screenings in alicyclic amines. Figure 10. MS/MS spectra for cyano adducts of prochlorperazine mono-oxygenated metabolite isomers (m/z 415), PM1 and PM4. (A) PM1 in KCN incubation and (B) PM4 in KCN incubation.

No cyano adducts were detected in 4 of the 11 compounds listed in Table 1 using the trapping method reported in this paper. Negative results for these compounds may be due to the fact that not every alicyclic amine drug/metabolite forms an iminium ion intermediate, that our incubation conditions are not optimal for the reaction of these iminium ion intermediates with

Conclusions The utility of the cyanide trapping LC-MS/MS screen for iminium ion intermediate screenings and its complementarities to GSH trapping screen has been demonstrated above. Although the GSH trapping screen can detect a large number of electrophilic phase I metabolites, the cyanide trapping screen has proven to be very effective in detecting those metabolites that have the potential to form the reactive iminium ion intermediates. The complementarities of both trapping screens are demonstrated in clozapine, triprolidine, and ketoconazole,

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where oxidative reactive metabolites were detected with GSH and those metabolites forming iminium ion intermediates were trapped with cyanide. Finally, in addition to the fact that the method does not require the use of radioactive material, there are several major advantages to the cyanide trapping technique in comparison to radiometry assays. First, the throughput of the screen is improved with LC-MS/MS analysis at 8 min per sample. In comparison, longer separations, >20 min per sample, are required to separate the excess cyanide from the cyano adducts and also to profile the cyanide adducts for the detection of iminium ion intermediates in radiometry assays. A second considerable advantage over radiometric techniques is the ability to characterize the structures of the cyano adducts with MS/MS experiments and also to determine the sites of bioactivation where reactive iminium ion intermediates are formed. However, unlike the radiometric cyanide trapping assay, the LC/MS/MS screen does not provide the quantitative information on the amount of cyanide adducts formed.

Acknowledgment. The authors thank Matthew Cyronak, Jacob Dunbar, Harma Ellens, Steve Clarke, Frank Hollis, Stephanie North, and Roberto Tolando at Drug Metabolism and Pharmacokinetics, GlaxoSmithKline Pharmaceuticals, for their support and help with this project. Partial support (P.V.) from the National Cancer Institute is also acknowledged (1R01CA69390). Supporting Information Available: Structures for the compounds investigated.

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