Rapid Detection and Characterization of in Vitro and Urinary N

The polarity switching function of the hybrid Q-Trap instrument enables the two data acquisition processes to be achieved in a single run. ...... Shoc...
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Rapid Detection and Characterization of in Vitro and Urinary N-Acetyl-L-cysteine Conjugates Using Quadrupole-Linear Ion Trap Mass Spectrometry and Polarity Switching Wenying Jian,†,‡ Ming Yao,§ Duxi Zhang,† and Mingshe Zhu*,§ Bioanalysis and DiscoVery Analytical Research and Biotransforamtion, Pharmaceutical Research and DeVelopment, Bristol-Myers Squibb, Princeton, New Jersey 08543 ReceiVed January 29, 2009

The present study describes a novel methodology for the rapid detection and structural characterization of unknown N-acetyl-L-cysteine (NAC) conjugates using polarity switching of triple quadrupole mass spectrometry. This method utilizes a negative neutral loss (NL) scan of 129 Da or multiple reaction monitoring (MRM) from predicted m/z values to product ions derived from the NL of 129 Da as a survey scan to trigger the acquisition of enhanced product ion (EPI) spectra in the positive ion mode. Thus, selective detection of NAC conjugates and acquisition of fragment-rich MS/MS spectra were accomplished in a single LC/MS run. The utility of this methodology was evaluated through analysis of NAC conjugates of acetaminophen in human urine after an oral dose. The MRM-EPI approach, which showed better sensitivity than the NL-EPI approach in analyzing urine samples, revealed three NAC-acetaminophen conjugates in the human urine, including two minor NAC conjugates that were derived from hydroxyl acetaminophen and methoxy acetaminophen. In addition, the methodology was applied to screening for reactive metabolites of clozapine and diclofenac using NAC as a trapping agent. Results showed reactive metabolite profiles comparable to those obtained from glutathione (GSH) trapping experiments, while MS/MS spectra of NAC conjugates provided more valuable structural information than those of GSH adducts. The study demonstrates that NAC trapping followed by NL-EPI analysis is a useful approach for high-throughput screening of reactive metabolites and that the MRM-EPI method is well-suited for analysis of low levels of NAC conjugates in urine. Introduction Xenobiotics, including drugs and environmental contaminates, are metabolized by P450 and other enzymes to more hydrophilic metabolites that are rapidly eliminated from the body in urine or bile. However, the metabolism of some xenobiotics can lead to chemically reactive metabolites or intermediates such as quinoneimines, nitrenium ions, imine methides, and arene oxides (1-3). The majority of reactive metabolites are electrophilic species that can covalently bind to macromolecules to form protein and DNA adducts. GSH, an endogenous cysteinecontaining tripeptide, exits at high levels in hepatocytes and other cells (up to 10 mM) (4). As a major scavenger of electrophiles, GSH reacts with a variety of reactive metabolites to form drug-GSH adducts that are much more hydrophilic than their parent drugs and the oxidative metabolites (5-7). GSH adducts are either directly excreted into bile or undergo a series of biotransformation steps mediated consecutively by γ-glutamylcysteine transpeptidase, dipeptidase, and N-acetyl * To whom correspondence should be addressed. Tel: 609-252-3324. E-mail: [email protected]. † Bioanalysis and Discovery Analytical Research. ‡ Current address: BA/DMPK, Pharmaceutical Research and Development, Johnson & Johnson, Raritan, NJ 08869. § Biotransforamtion, Pharmaceutical Research and Development. 1 Abbreviations: CID, collision-induced dissociation; EPI, enhanced product ion scan; ER, enhanced resolution; FDA, Food and Drug Administration; IDA, information-dependent acquisition; LC/MS, liquid chromatography/mass spectrometry; NAC, N-acetyl-L-cysteine; NL, neutral loss; MRM, multiple reaction monitoring; PI, precursor ion; TIC, total ion current.

transferase to give rise to N-acetyl-L-cysteine (NAC)1 conjugates (mercapturic acid conjugates) that are commonly excreted into urine. Reactive metabolites are considered mediators of druginduced toxicity (8). To minimize the potential for reactive metabolite formation in humans, detection, structural characterization, and quantitative estimation of reactive metabolites of lead compounds or new chemotypes followed by structural modification have become an integral part of the drug discovery process (9). In vitro GSH trapping experiments combined with liquid chromatography/mass spectrometry (LC/MS) analysis are often employed to examine the bioactivation potential of drug candidates and structures of reactive metabolites in early drug discovery (5). Many in vivo biotransformation reactions catalyzed by P450 enzymes are often predictable based on observations with liver microsome incubations. However, it is difficult to predict in vivo bioactivation mediated by non-P450 enzymes or that associated with multiple biotransformation reactions based on data from in vitro experiments. For example, the presence of large amounts of urinary NAC conjugates of valproic acid (10) and felbamate (11) in humans demonstrated that these drugs underwent extensive bioactivation in humans, but in vitro reactive metabolite screening in liver microsomes did not find any reactive metabolites of them. In these cases, the analysis of NAC conjugates in urine and/or GSH adducts in bile can provide valuable information on the extent and chemical nature of metabolic activation in vivo in humans and animals. Recently, the Food and Drug Administration (FDA) published a guidance document regarding safety testing of drug metabolites

10.1021/tx900035j CCC: $40.75  2009 American Chemical Society Published on Web 06/15/2009

Detection and Characterization of NAC Conjugates Scheme 1

that highlighted the need for quantitative analysis of plasma metabolites in humans and toxicological species, especially those with pharmacological activity and structural alerts (12, 13). Most

Figure 1. LC/MS/MS analysis of clozapine conjugates in rat liver microsome incubation. (A) TIC of negative NL scan of m/z 129 for analysis of clozapine NAC conjugate, (B) TIC of positive EPI triggered by negative NL scan of m/z 129 for analysis of clozapine NAC conjugate, (C) TIC of negative MRM scan with 40 transitions for analysis of clozapine NAC conjugate, (D) TIC of positive EPI triggered by negative MRM scan with 40 transitions for analysis of clozapine NAC conjugate, and (E) TIC of negative PI scan of m/z 272 for analysis of clozapine GSH conjugate.

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reactive metabolites and their GSH or NAC conjugates are not present in the circulation, and human bile is very difficult to collect. Therefore, the assessment of the exposure of reactive metabolites in humans relies on the identification and quantitative measurement of stable products from further metabolism of reactive metabolites or GSH-trapped reactive metabolites in human urine, such as drug-NAC conjugates (5, 14-16). Furthermore, urinary NAC conjugates of xenobiotics are important markers that reflect the electrophilic burden of environmental chemicals on humans (17). For example, urinary NAC conjugates of acrylamide and glycidamide are considered as biomarkers for acrylamide exposure (18). Although a variety of LC/MS methodologies (5, 19), including neutral loss (NL) scan (20), precursor ion (PI) scan (21-23), multiple reaction monitoring (MRM) (24, 25), mass defect filtering (26), background subtraction analysis (27, 28), isotope pattern filtering (29), and NL/product ion filtering (30, 31), have been developed for screening and structural characterization of GSH adducts formed in either GSH trapping experiments or animal bile samples, there are limited mass spectrometric methods available for screening for NAC conjugates (5, 32). The majority of LC/MS methods employed in the analysis of urinary NAC conjugates are associated with target analysis and quantification of specific NAC conjugate(s) using MRM methods on triple quadrupole instruments. Scholz et al. developed a new LC/MS methodology for detecting synthetic NAC conjugates in vitro and those spiked in rat urine with a hybrid triple quadrupole instrument (Q-trap 2000) (33). In the approach, the negative NL scan of 129 Da or MRM from expected molecular masses to product ions raised from a NL of 129 Da was employed as the survey scan that triggers acquisition of negative enhanced product ion spectra in the trap (EPI scan). The methodology was also applied to metabonomics and biomarker discovery studies via analyzing patterns of endogenous NAC conjugates in urinary samples from human subjects (17, 34). Results from these studies proved that the NL or MRM analysis is particularly useful in confirming the presence of targeted NAC conjugates. However, MS/MS spectra of NAC conjugates acquired in the negative ion mode displayed the same NL fragmentation with little other fragment information, making structural elucidation of NAC conjugates very difficult. Recently, updated triple quadrupole-linear ion trap instruments (Q-trap 4000 and Q-trap 5500) (35-37) have been introduced, which not only increase analytical sensitivity but also enable polarity switching between the survey scan and the EPI (38). The information-dependent polarity switching technology has been successfully employed for screening for oxidative metabolites (39) and reactive metabolites trapped by GSH (22) and GSH derivatives (23). The major goal of the current study was to develop new methods capable of selectively detecting unknown NAC conjugates and acquiring fragment-rich MS/MS spectra for structural elucidation by using the polarity switching capabilities of the Q-trap instruments. The effectiveness of these methods was evaluated by analyzing NAC conjugates of acetaminophen in human urine after a single oral dose. Additionally, the use of NAC as a trapping agent with adduct detection carried out by these methods was explored for reactive metabolite screening.

Experimental Procedures Materials. The following chemicals were purchased from Sigma-Adrich (St. Louis, MO): clozapine, diclofenac, NAC,

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Figure 2. Tandem MS spectra and proposed structures of clozapine NAC conjugates acquired by NL-directed EPI scan. (A) Negative tandem MS spectra for CNM2 acquired by negative NL-directed EPI without polarity switch, (B) positive tandem MS spectra for CNM2 and CNM4 acquired by negative NL-directed EPI with polarity switch, (C) negative tandem MS spectra for CNM3 acquired by negative NL-directed EPI without polarity switch, and (D) positive tandem MS spectra for CNM3 and CNM6 acquired by negative NL-directed EPI with polarity switch.

Table 1. Summary of NAC Conjugates Identified by the Negative NL Scan/Positive MS/MS and GSH Conjugate Identified by Negative Precursor Scan/Positive MS/MS compound (MH+)

NAC conjugate

MH+ and major fragments of NAC conjugate

clozapine (327)

CNM1 CNM2 CNM3 CNM4 CNM5 CNM6 CNM7 DNM1 DNM2

506 (488, 406, 377, 364, 301, 277, 245) 474 (432, 345, 328, 302, 285, 276) 488 (446, 431, 359, 328, 302, 285, 276) 474 (432, 345, 328, 302, 285, 276) 504 (486, 460, 404, 375, 357, 331, 328, 302, 288, 285, 275, 247) 488 (446, 431, 359, 328, 302, 285, 276) 504 (486, 460, 404, 375, 357, 331, 328, 302, 288, 285, 275, 247) 439 (421, 403, 379, 375, 333, 315, 292, 288, 262, 230) 473 (455, 437, 413, 367, 350, 326, 324, 294)

diclofenac (296)

compound (MH+)

GSH conjugate

MH+ and major fragments of GSH conjugate

clozapine (327)

CGM1 CGM2 CGM3 CGM4 CGM5 CGM6 CGM7 CGM8 DGM1 DGM2 DGM3

646 (628, 571, 517, 499, 414, 373) 650 (632, 575, 521, 503, 421, 377, 301, 275) 618 (600, 489, 432, 345, 328, 302, 276) 632 (503, 446, 359, 328, 302) 648 (630, 573, 559, 519, 501, 419, 375, 357, 302, 288) 618 (600, 489, 432, 345, 328, 302, 276) 632 (503, 446, 359, 328, 302) 648 (630, 573, 559, 519, 501, 419, 375, 357, 302, 288) 581 (563, 506, 434, 416, 331, 296) 583 (565, 508, 490, 454, 436, 419, 333, 315, 308, 292, 262) 617 (599, 542, 488, 470, 452, 350, 367, 342, 331, 296)

diclofenac (296)

reduced glutathione, and β-nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt (NADPH). Oasis solidphase extraction (SPE) cartridges were purchased from Waters (Milford, MA). Rat liver microsomes were obtained from BD Biosciences (Woburn, MA). Acetaminophen was purchased as Tylenol Extra Strength (500 mg of acetominophen, McNeil Consumer Products). All other chemicals and solvents were of reagent grade or better. Microsomal Incubations. Test compounds (30 µM) were incubated with rat liver microsome (1.0 mg/mL), NADPH (1.0 mM), and NAC (1.0 mM) or GSH (1.0 mM) in potassium phosphate buffer (100 mM, pH 7.4) for 30 min. The total incubation volume

postulated conjugate composition P+NAC+ O P+NAC-2H-CH2 P+NAC-2H P+NAC-2H-CH2 P+NAC+ O-2H P+NAC-2H P+NAC+ O-2H P+NAC+ O-HCl P+NAC+ O-2H

postulated conjugate composition P+GSH+ O-4H P+GSH+ O P+GSH-2H-CH2 P+GSH-2H P+GSH+ O-2H P+GSH-2H-CH2 P+GSH-2H P+GSH+ O-2H P+GSH+ O-HCl-2H P+GSH+ O-HCl P+GSH+ O-2H

was 2.0 mL. The incubation reaction was initiated by addition of NADPH solution after 5 min of preincubation at 37 °C. The control samples contained no test compounds. The incubation was conducted in a 37 °C water bath with shaking. After incubation, the samples were centrifuged at 10000g for 10 min at 4 °C, and the supernatants were subjected to SPE. Urine Sample Collection. Urine samples were collected from a healthy volunteer after a single oral administration of acetaminophen (500 mg) at predose, 0-2, 2-4, 4-6, 6-10, and 24 h. The samples were immediately frozen at -20 °C and thawed before extraction. A 2 mL portion of each sample was subjected to SPE.

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Figure 3. Product ion spectra and proposed structures of clozapine conjugates. (A) Positive product ion spectrum of CNM7 acquired by negative NL-directed EPI with polarity switch and (B) positive product ion spectrum of CGM8 acquired by negative PI-directed EPI with polarity switch.

SPE. SPE was performed using Oasis SPE cartridges packed with 60 mg of C18 sorbent. The cartridges were conditioned with 2 mL of methanol and 2 mL of water. After the samples were loaded, the cartridges were washed with 2 mL of water, followed by elution with 2 mL of methanol. The eluent was dried under nitrogen gas and reconstituted with 300 µL of 30:70 (v:v) methanol and water. Aliquots (20 µL) of the reconstituted solutions were injected into the LC/MS system. LC/MS. The HPLC system consisted of Shimadzu LC10ADvp pumps (Columbia, MD) and an HTC PAL autosampler (LEAP, technologies, Carrboro, NC). An Atlantic dC18 column (2.1 mm × 150 mm, 5 µm, Waters) was employed. The HPLC mobile phase A was 10 mM ammonium acetate with 0.1% (v:v) formic acid in water, and the mobile phase B was 0.1% (v:v) formic acid in acetonitrile. The gradient started at 5% B for 2 min, ramped to 50% B over 20 min, increased to 90% B in 1 min, held at 90% B for 1 min, and then returned to 5% B in 1 min. The HPLC flow rate was 0.3 mL/min. The HPLC system was interfaced with an API4000 Q-Trap hybrid triple quadrupole-linear ion trap mass spectrometer (Applied Biosystems, Foster City, CA). For NL-EPI analysis, the NL loss scan of 129 Da was run in the negative mode at a scan range from 300 to 700 with 1.0 Da step size with a 5 ms pause between mass ranges and a 2 s scan rate. The operating parameters were optimized and set at the following values: curtain gas, 25; collision gas medium; ion spray, 4500 V; temperature, 300 °C; ion source gas, 1:50; ion gas source, 2:50; declustering potential, -80 V; entrance potential, -10 V; collision energy, -30 eV; and collision cell exit potential, -12 V. Nitrogen was used as the nebulizer gas and auxiliary gas. Enhanced resolution (ER) scan in negative mode with a scan time of 0.008 s was utilized before the information-dependent acquisition (IDA). IDA

was used to trigger acquisition of EPI spectra for ions exceeding 3000 cps with exclusion of former target ions after three occurrences for 10 s. The EPI scan was run under positive mode using a scan range from m/z 100 to m/z 800. The EPI scan was operated in profile mode with step size of 0.08 Da. The scan rate was 1000 Da/s with a 5 ms pause between mass ranges. The other operating parameters were the same as those in NL scan except for the switch in polarity. A collision energy spread of (15 eV was applied. For PI-EPI analysis, the precursor scan of m/z 272 was run in the negative mode at a scan range from 400 to 700 with 1.0 Da step size with a 5 ms pause between mass ranges and a 2 s scan rate. For MRM-EPI analysis, the MRM protocol contained parent ions from -40 to +40 Da around the m/z value of the drug-NAC conjugate in steps of 2 mass units (totally 40 channels). The product ions corresponded to the loss of 129 from deprotonated molecules of NAC conjugates. The dwell time was 20 ms, and the interscan pause time was 5 ms. For PI-EPI and MRMEPI, the enhanced resolution, IDA and EPI experiments were operated under the same condition as that of NL-EPI. Determination of Matrix Effects of Human Urine. Reconstituted clozapine rat liver microsome incubation samples from SPE were diluted 10 times with 30:70 (v:v) methanol and water. Various amounts of human urine (0, 10, and 30 µL) were spiked into the diluted samples (5 µL). NAC adducts in these samples were analyzed by NL-EPI and MRM-EPI methods.

Results Analytical Strategy. The analytical strategy employed for the detection and characterization of NAC conjugates of drugs using the hybrid triple quadrupole-linear ion trap LC/MS (Q-

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Figure 4. LC/MS/MS analysis of diclofenac conjugates in rat liver microsome incubation. (A) TIC of negative NL scan of m/z 129 for analysis of diclofenac NAC conjugate, (B) TIC of negative MRM scan with 40 transitions for analysis of diclofenac NAC conjugate, and (C) TIC of negative precursor scan of m/z 272 for analysis of diclofenac GSH conjugate.

Trap) is illustrated in Scheme 1. The NAC conjugates include those formed in vivo and present in urine and NAC-trapped reactive metabolites formed in live microsome incubations. Polarity switching is applied between the detection of NAC conjugates using NL scan or MRM in the negative ion mode and the information-dependent EPI acquisition of the detected NAC conjugates in the positive ion mode. In the NL-EPI experiment, the negative NL of 129 Da, corresponding to a single, dominant fragmentation pathway of NAC conjugates, served as the survey scan. Once a NAC conjugate is detected by the NL scan, an ER scan is acquired, which further triggers the acquisition of EPI in the positive ion mode via polarity switching. In the MRM-EPI experiment, MRM with multiple transitions, each of which corresponds to the loss of 129 Da from a parent ion to a daughter ion, is used as a survey scan that triggers EPI acquisition in the positive mode. Two sets of LC/MS data are generated from either the NL-EPI or the MRMEPI experiment: (1) a total ion chromatogram (TIC) of the NL scan or MRM, from which PI spectra that display deprotonated molecules of detected NAC conjugates can be retrieved, and (2) a TIC from the dependent EPI acquisition, from which positive MS/MS spectra of the conjugates can be recovered. The current analytical methodology takes advantage of selectivity and sensitivity of negative NL scan or MRM in detecting NAC conjugates and usefulness of positive ion MS/MS spectra in structural characterization (33). The polarity switching function of the hybrid Q-Trap instrument enables the two data acquisition processes to be achieved in a single run. Detection of NAC Conjugates Formed in Rat Liver Microsome Incubations. An incubation sample of clozapine with rat liver microsomes and excessive amounts of NAC was analyzed using the NL-EPI scan (Scheme 1). As a result, a total

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of seven NAC conjugates were revealed in the TIC of the NL scan of 129 Da (Figure 1A) and the TIC of the NL-directed EPI scan (Figure 1B), in which there were no or a few minor false positive peaks. These conjugates were arbitrarily designated as CNM1 (13.4 min), CNM2 (13.5 min), CNM3 (13.9 min), CNM4 (14.1 min), CNM5 (14.3 min), CNM6 (14.5 min), and CNM7 (15.1 min). Positive MS/MS spectra of these conjugates were acquired by EPI scan in the same LC/MS injection (Figure 2B,D and Table 1). This clozapine sample was subjected to MRM-EPI analysis, and resultant MRM (Figure 1C) and EPI (Figure 1D) chromatograms displayed seven NAC conjugates identical to those characterized by the NL-EPI scan (Figure 1A,B and Table 1). There was no significant difference between NLEPI and MRM-EPI analyses of the in vitro incubation sample in terms of signal intensity and detection selectivity (Figure 1). The NL-directed positive MS/MS spectrum of the most abundant conjugate CNM6 showed an [M + H]+ ion at m/z 488, corresponding to the direct addition of NAC to clozapine (P + NAC - 2H), and multiple informative fragmentation ions (Figure 2D and Table 1). CNM3 had an identical molecular ion and MS/MS spectrum (Table 1) as those of CNM6. On the basis of the MS/MS spectral data, CNM3 and CNM6 were tentatively assigned as a pair of stereoisomers derived from the addition of NAC to a clozapine nitrenium ion (26) (Figure 2D). The same GSH adduct analogues were previously observed in liver microsome incubations of clozapine supplemented with GSH (22, 26). CNM2 displayed an [M + H]+ ion at m/z 474 corresponding to the addition of NAC to N-demethylclozapine that was a major metabolite of clozapine identified in human liver microsomal incubation (40). The NL-directed MS/MS spectrum of CNM2 was consistent with the proposed structure of this conjugate (Figure 2B). CNM4 showed an essentially identical spectrum as that of CNM2. Thus, they were designated as a pair of isomers resulting from direct addition of NAC to the different positions on the chlorophenyl ring of a Ndemethylclozapine nitrenuim ion structure (26). CNM5 and CNM7 represented a third pair of NAC conjugate isomers observed in the incubation sample. Their protonated molecular ion at m/z 504 (P + NAC + O - 2H) and product ions at m/z 288, 302, 331, and 404 suggested that CNM5 and CNM7 were NAC conjugates of clozapine N-oxide (Figure 3A and Table 1), which could be formed via the nitrenuim intermediate of clozapine N-oxide. The characteristic product ion at m/z 285 confirmed that the NAC moiety was attached to the chlorophenyl group of CNM5 and CNM7 (Figure 3A). The formation pathways and structures of multiple nitrenuim reactive metabolites from clozapine were discussed in detail in the literature (22, 26). For the purpose of comparison, negative MS/MS spectra of CNM2 and CNM3 were acquired by Q-trap. As shown in Figure 2A,C, CNM2 and CNM3 underwent only one significant fragmentation pathway in the negative ion mode that led to an ion at m/z 343 and 357, respectively. The NL-EPI analysis of a rat liver microsome incubation of diclofenac with NAC revealed two major NAC conjugates, DNM1 (16.9 min) and DNM2 (17.9 min) (Figure 4A). The positive MS/MS spectrum of DNM1 exhibited an [M + H]+ ion at m/z 439, corresponding to the addition of NAC to a diclofenac metabolite derived from a combination of dechlorination and mono-oxidation (P + NAC + O - HCl) (Figure 4A and Table 1). The product ions at m/z 288 and 230 (Figure 5A) confirmed that the sites of the metabolic modifications occurred on the dichlorophenyl ring. Most likely, DNM1 was formed via the initial formation of 1′-4′ quinone imine intermediate from 4′-hydroxydiclofenac, followed by the re-

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Figure 5. Tandem MS spectra and proposed structures of diclofenac conjugates. (A) Positive tandem MS spectra for DNM1 acquired by negative NL-directed EPI with polarity switch and (B) positive tandem MS spectra for DGM2 acquired by negative PI-directed EPI with polarity switch.

placement of 2′-chlorine via the attack of NAC on the quinone imine intermediate. A GSH adduct analog (P + GSH + O HCl) possibly derived from the same reactive intermediate was previously characterized in human liver microsome incubations by LC/MS and/or NMR (22, 26, 41). DNM2 had an [M + H]+ ion at m/z 473 (P + NAC + O - 2H) and was designated as a NAC conjugate of a mono-oxidation product of diclofenac (Table 1). DNM2 could be formed via quinone imine intermediates of diclofenac, which led to the formation of similar GSH adducts (P + GSH + O - 2H) in human liver microsome incubations fortified with GSH (22). The TIC of MRM-EPI for the same incubation sample showed DNM1 and DNM2 as the major metabolites (Figure 4B) similar to that of the NL-EPI analysis (Figure 4A). Detection of GSH Conjugates Formed in Rat Liver Microsome Incubation. Negative precursor scan at m/z 272 coupled with positive EPI was employed to detect and characterize GSH-trapped reactive metabolites of clozapine and diclofenac in rat liver microsome incubations. Figure 1E shows the total ion chromatogram of the negative PI scan of m/z 272 obtained from a clozapine incubation sample. A total of eight conjugates were detected and designated arbitrarily as CGM1 (10.5 min), CGM2 (11.4 min), CGM3 (11.5 min), CGM4 (11.6 min), CGM5 (11.8 min), CGM6 (12.4 min), CGM7 (12.6 min), and CGM8 (13.0 min). Their MS/MS spectral data acquired by the dependent EPI scan in the positive ion mode are listed in Table 1. The most abundant conjugate CGM7 showed an [M + H]+ of m/z of 632 corresponding to (P + GSH - 2H) (Table 1). CGM4 had an identical MS/MS spectrum as that of CNM7 and was assigned as a positional isomer of CGM7. CGM3 and CGM6 showed same protonated molecule at m/z 618 (P + GSH

- 2H - CH2) and MS/MS spectrum and were assigned as a pair of positional isomers of GSH conjugates of N-demethylclozapine (Table 1). CGM5 and CGM8, a third pair of isomers, gave rise to a protonated molecule at m/z 648, which corresponded to the addition of GSH and an oxygen atom to clozapine (P + GSH + O - 2H). The product ions at m/z 288 and 302 confirmed that they were products of GSH trapping of a N-oxide clozapine nitrenium intermediate (Figure 3B). GSH adducts of clozapine, which had the same molecular masses and MS/MS spectra with those of CGM1, CGM2, and CGM4 were previously detected in human liver microsome incubations (22). Three GSH adducts of diclofenac, DGM1 (13.5 min), DGM2 (14.5 min), and DGM3 (15.0 min), were identified in a rat liver microsome incubation of diclofenac with GSH (Figure 4C and Table 1). DGM2 had an [M + H]+ at m/z 583 (P + GSH + O - HCl) (Figure 5B) and DGM3 showed an [M + H]+ ion at m/z 617 (P + GSH + O - 2H) (Table 1). These GSH adducts were previously observed as major GSH adducts in human liver microsome incubations (22, 26). DGM1 had an [M + H]+ at m/z 581 (P + GSH + O - HCl - 2H). This GSH adduct has not been reported previously, and its structure remains to be determined. Detection of NAC Conjugates of Acetaminophen in Human Urine. The NL-EPI analysis of the urine samples collected from a healthy volunteer after an oral administration of 500 mg of acetaminophen revealed a single predominant component, AM2, in all urine samples, including the sample collected at 2-4 h (Figure 6A). In addition to AM2, two minor NAC conjugates of acetaminophen, AM1 and AM3, were found by the MRM-EPI analysis (Figure 6B-E). On the basis of

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Discussion

Figure 6. LC/MS/MS analysis of acetaminophen NAC conjugates in a human urine sample collected at 2-4 h. (A) TIC of negative NL scan of m/z 129, (B) TIC of negative MRM scan with 40 transitions, (C) extracted MRM transition for AM1 (m/z 327 > 198), (D) extracted MRM transition for AM2 (m/z 311 > 182), and (E) extracted MRM transition for AM3 (m/z 341 > 212).

positive MS/MS spectra (Figure 7), AM2 (MH+ at m/z 313) was tentatively assigned as the NAC adduct of acetaminophen (P + NAC - 2H) (Figure 7B). AM1 (MH+ at m/z 329) and AM2 (MH+ at m/z 343) were tentatively identified as NAC adducts of 3-hydroxyl acetaminophen (P + NAC + O - 2H) (Figure 7A) and 3-methoxyl acetaminophen (P + NAC + OCH2 - 2H) (Figure 7C), respectively. Matrix Effect of Human Urine. To evaluate the matrix effect of urine on the performance of NL-EPI and MRM-EPI in the detection of NAC conjugates, NAC conjugates of clozapine (5 µL of rat liver microsome incubation solution) were mixed with various volumes of human urine (0-30 µL) and analyzed. In the absence of urine, the total ion chromatogram of both NL-EPI and MRM-EPI showed similar profiles, in which CNM1-CNM7 were clearly displayed without significant false positives (Figure 8A,D). With increasing amounts of urine injected, the background noise levels and intensities of the interference peaks were elevated in the TIC of NL-EPI. As a result, only the most predominant peak CNM6 was detected when 15 µL of urine was added (Figure 8B), and none of the conjugates were detected when 30 µL of urine was added (Figure 8C). In contrast, the TIC of MRM demonstrated almost identical profiles of NAC conjugates in the absence or presence of urine (Figure 8D-F). In addition, no significant decrease in signal intensity was observed when increasing amounts of urine were added.

As illustrated in the product ion spectra of NAC conjugates of clozapine and N-demethylclozapine (Figure 2A,C), the NL of 129 Da resulting from a cleavage of the thioether bond was the single major fragmentation pathway of NAC conjugates in the negative ion mode. This unique fragmentation pattern has been observed for all types of NAC conjugates regardless of their structures and molecular masses (17, 33, 34). In contrast, positive product ion spectra of the same conjugates displayed several smaller fragments associated with the drug moieties (Figure 2B,D), which provided much more structural information for NAC conjugates than negative MS/MS spectra (Figure 2A,C). By taking advantage of the distinct fragmentation behaviors of NAC conjugates in both the negative and the positive ion modes, an improved analytical methodology using the polarity switching function of new hybrid triple quadrupolelinear ion trap mass spectrometers has been developed for detection and structural characterization of unknown NAC conjugates (Scheme 1). Results from the analysis of in vitro incubations (Figures 1 and 4) and human urine (Figure 6) demonstrate that it enables the detection of NAC conjugates and acquisition of MS/MS spectra in a single LC/MS run. Additionally, the NL or MRM-dependent acquisition methodology provides a quick and sensitive means for recording of MS/ MS spectra with rich fragment ions and no mass cutoff (Figures 2, 3, 5, and 7). The NL-EPI approach is well-suited for rapid screening and structural characterization of NAC conjugates formed in NAC trapping experiments (Scheme 1). In the NL-EPI analysis, a simple, generic acquisition method is employed so that the set up of an acquisition protocol for sample analysis takes minimal time and mass spectral data acquisition can be performed continuously. Additionally, data processing in the NL-EPI experiment is straightforward and can be carried out in parallel with data acquisition. As shown in the analyses of NAC-trapped reactive metabolites of clozapine and diclofenac, NAC conjugates were clearly detected as major components in the NL chromatograms with few false positives (Figures 1A and 4A). On the other hand, the positive MS/MS spectra acquired with polarity switching provided rich information for the structural identification of the conjugates. The results also indicate that trapping efficiency of NAC and GSH toward reactive metabolites is comparable. Among the eight GSH-clozapine conjugates identified in the GSH incubation, seven of the major ones showed a corresponding NAC conjugate in the NAC incubation (Figure 1 and Table 1). The only one that showed no corresponding NAC conjugate peak was CGM1 (P + GSH + O 4H), a minor metabolite that has not been fully characterized. In addition to screening for reactive metabolites formed in liver microsomes supplemented with NAC, the NL-EPI approach was proved to be effective in the fast analysis of urinary NAC conjugates, such as the detection of the major NAC-acetaminophen adduct, AM2, in human urine samples (Figure 6A). One of the applications of this LC/MS method would be rapid screening of major reactive metabolites of lead compounds or clinical candidates in urine samples collected in pharmacokinetic or toxicity studies in animals. The major advantage of the NAC-trapping experiment over the GSH-trapping experiment is that NAC conjugates may have the potential to generate more fragments from cleavages of the drug moiety than those from GSH conjugates, providing critical information on structures of the reactive metabolites. This is due to the fact that NAC is a smaller molecule with fewer bonds susceptible to collision-induced dissociation (CID) fragmentation

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Figure 7. Positive tandem MS spectra and proposed structures of acetaminophen NAC conjugates acquired by negative MRM-directed EPI scan with polarity switch. (A) AM1, (B) AM2, and (C) AM3.

Figure 8. Analysis of clozapine NAC conjugates with or without addition of human urine by NL or MRM scan. (A) TIC of NL scan of a 5 µL sample with no urine, (B) TIC of NL scan of a 5 µL sample with 15 µL of urine, (C) TIC of NL scan of a 5 µL sample with 30 µL of urine, (D) TIC of MRM scan of a 5 µL sample with no urine, (E) TIC of MRM scan of a 5 µL sample with 15 µL of urine, and (F) TIC of MRM scan of a 5 µL sample with 30 µL of urine.

than GSH and the collision energy applied has more chance to distribute to the drug moiety itself instead of the trapping groups as shown in the current study. For example, CNM7, an NAC conjugate of N-oxide clozapine, generated more informative fragments from the EPI scan (Figure 3A) than those from GSH adduct of N-oxide clozapine (Figure 3B). The product ion at

m/z 331 derived from CNM7 confirmed unambiguously that the mono-oxidation occurred on the nitrogen of the methylpiperazine ring, and the product ion at m/z 285 suggested that NAC was attached on the chlorine-substituted phenyl ring (Figure 3A). However, these key fragmentation pathways were not observed in the MS/MS spectrum of the GSH adduct analogue (Figure

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3B). In a similar fashion, the CID of DNM1, an NAC conjugate of dechlorinated/mono-oxidized diclofenacclozapine, produced two informative fragments at m/z 288 and 230, the latter of which corresponded to the chlorophenyl ring containing the monohydroxyl group and a part of NAC moiety derived from a cleavage of the carbon-nitrogen bond (Figure 5A). Consequently, the bioactivation pathway leading to the DNM1, proposed to involve an epoxidation on the diclorophenyl ring followed by NAC attack and a sequential loss of HCl, was confirmed. In contrast, the MS/MS spectra of DGM2, a GSH adduct of dechlorinated/mono-oxidized clozapine, did not show any fragmentations associated with the cleavage of the carbon-nitrogen bond (Figure 5B). Therefore, the exact locations of the hydroxyl group and GSH moiety cannot been determined on the basis of the mass spectral data. As compared to the NL-EPI approach, the MRM-EPI method provided better sensitivity and selectivity for analyzing NAC conjugates in urine. As illustrated in Figure 6B-E, the MRMEPI analysis clearly determined the identities of AM1 and AM3, two minor NAC conjugates of acetaminophen. The two acetaminophen metabolites were not detected by the NL-EPI method in the urine samples (Figure 6A). Acetaminophen is mainly catalyzed by human CYP2E1 to N-acetyl-p-benzoquinone imine (NAPQI) (42), a reactive intermediate, resulting in protein adducts and GSH adducts. This drug is also converted by P450 enzymes to 3-hydroxyl acetaminophen that was observed as a major oxidative metabolite in human liver microsomes (43) and human urine (44). 3-Hydroxyl acetaminophen is further converted to 3-methoxyl acetaminophen and the sulfate conjugate of 3-hydroxyl acetaminophen. These metabolites and the glucuronide (44, 45) as well as a sulfate conjugate of 3-methoxyl acetaminophen (44, 46) were observed in human urine. The results from the analysis of NAC conjugates in urine demonstrate that like acetaminophen, 3-hydroxyl acetaminophen and 3-methoxy acetaminophen undergo bioactivation, presumably mediated by P450 enzymes, to corresponding quinone and quinone imine intermediates, respectively, leading to GSH adducts (47) in humans. The major limitation of the MRM-EPI approach is the lack of the fast analysis capability. In the MRM-EPI experiment, data acquisition protocols are compound-dependent, in which MRM transitions are calculated based on the molecular mass of each test compound. Therefore, it is a time-consuming process to set up acquisition protocols for a large of number of samples. Additionally, in the current study, approximately 40 MRM transitions were set to cover a (40 Da range around the m/z value of the drug-NAC conjugate. The clozapine and acetaminophen examples demonstrated that the MRM scan is capable of finding all NAC conjugates that have similar molecular masses as the total masses of drug + NAC. However, some NAC conjugates, such as a NAC conjugate of a dealkylation product, could be significantly smaller than the corresponding NAC conjugate of the parent drug. To detect these NAC conjugates, additional MRM-EPI analyses, each of which performs 40 MRM transitions and covers a different m/z range, would be required. Alternatively, MRM-EPI analysis with a more advanced Q-trap instrument (Q-trap 5500) would enable one to perform a much larger number of MRM transitions to cover wider m/z ranges with comparable sensitivity. Interestingly, the NL-EPI and MRM-EPI approaches were shown to have comparable sensitivity for detecting in vitro NAC conjugates (Figures 1 and 4), while the MRM-EPI approach was much more sensitive than the PI-EPI method in analyzing urinary NAC conjugates (Figure 6). This

Jian et al.

observation suggests that endogenous components and other matrixes present in urine significantly affected the performance of the NL-EPI analysis. To further investigate the nature of the matrix effect, NAC conjugates of clozapine from rat liver microsome incubation were mixed with different volumes of human urine (0-30 µL) and analyzed using these two LC/MS methods. Results from the experiment showed that the signal intensities of NAC conjugates in the TIC of NL scan remained unchanged and the background noise levels significantly increased with the increase of urine injected (Figure 8), indicating the elevation of the noise levels rather than ion suppression by matrixes caused the sensitivity reduction of the NL analysis. The background noise could come from endogenous NAC conjugates and other components that were picked up by the negative NL scan analysis (34). In contrast, because the MRM analysis was much more selective, the noise levels in the MRM chromatograms did not change with the increase of urine injected. In summary, a novel LC/MS methodology using NL-EPI and MRM-EPI combined with polarity switch has been developed for the analysis of NAC conjugates. By taking advantage of the unique fragmentation patterns of NAC conjugates in the negative and positive ion modes and information-dependent polarity switching of the hybrid Q-trap instrument, the methodology enables the selective detection of NAC conjugates and sensitive acquisition of informative MS/MS in a single LC/MS run. The NL-EPI method uses a generic data acquisition protocol and enables the detection of various types of NAC conjugates without multiple injections. Therefore, it is well-suited for higher throughput analysis of NAC conjugates formed in vitro and present in urine. The MRM-EPI approach provides superior sensitivity in analysis of NAC conjugates in urine samples but requires a compound-dependent acquisition protocol. Therefore, this method is especially useful in the target analysis or confirmation of low levels of urinary NAC conjugates of drugs in the first in human studies when high analytical sensitivity and selectivity are required. Acknowledgment. We greatly appreciate Dr. W. Griffith Humphreys for a critical reading of the manuscript and Dr. Qian Ruan for helpful discussions during the course of this study.

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