Study of the Fragmentation Mechanism of Protonated 6

ions indicated possible aromatic ring opening for the first two intense product ... Giovanna Nardo , Andrea Fantuzzi , Anastasia Sideri , Paola Pa...
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Anal. Chem. 2003, 75, 469-478

Study of the Fragmentation Mechanism of Protonated 6-Hydroxychlorzoxazone: Application in Simultaneous Analysis of CYP2E1 Activity with Major Human Cytochrome P450s M. Reza Anari,* Ray Bakhtiar, Ronald B. Franklin, Paul G. Pearson, and Thomas A. Baillie

Department of Drug Metabolism, Merck Research Laboratories, RY80L-109, Rahway, New Jersey 07065

The application of liquid chromatography tandem mass spectrometry for simultaneous analysis of major human cytochrome P450 activities via a single atmospheric pressure ionization (API) LC/MS/MS method has been hampered by the preferred detection of 6-hydroxychlorzoxazone (HCZ), the metabolite of the CYP2E1 probe, chlorzoxazone, under negative API. An initial simulation of the dissociation constants suggested the potential ionization of the enol form of HCZ at low pH, and the accurate mass measurements confirmed the presence of the protonated HCZ signal under (+) ESI at pH 3. However, the CID spectrum of the protonated HCZ resulted in a few intense, but uncommon, fragment ions that could be utilized for specific selected reaction monitoring (SRM) transitions. The deduced elemental compositions of these fragment ions indicated possible aromatic ring opening for the first two intense product ions at m/z 130 and 115, as well as chlorine radical loss for the third ion at m/z 151. Further precursor and product ion scan studies, along with the deuterium ion exchange in solution, revealed the involvement of three distinct pathways of fragmentation. The m/z 186 f 130 transition, which was shown to be specific in human plasma and rat hepatic microsomes, was further combined with the SRM transition of reserpine (internal standard) and eight probe substrates for human cytochrome P450 isoforms. This led to the development of a full LC/MS/MS method capable of analyzing a total of nine human P450 activities within 3 min, including CYP2E1, using a single assay in the (+) ESI mode. The HCZ assay showed excellent linearity with a coefficient of determination (R2) greater than 0.98 at dynamic range of 0.05 (LOQ) to 40 µM. Preliminary data from the three-day validation of the HCZ assay indicated that the accuracy and precision for quality control samples was within (15% of the spiked concentration at all levels. Cytochrome P450 2E1 (CYP2E1) is a major constitutive mammalian liver enzyme that is also expressed at significant levels in extrahepatic tissues such as brain, lung, kidney, and intestine.1,2 * Corresponding author. Telephone: E-mail: [email protected]. 10.1021/ac026157m CCC: $25.00 Published on Web 01/03/2003

732-594-6184. Fax:

732-594-4390.

© 2003 American Chemical Society

Levels of human hepatic CYP2E1 vary by 1 order of magnitude with reported intraindividual variations as high as 20-fold.3-6 Chronic exposure to ethanol, acetone, and other low molecular weight solvents3,7 as well starvation, obesity, and diabetes can contribute to this variation by induction of CYP2E1.8-12 The level of CYP2E1 can affect the metabolism and toxicity profile of drugs such as acetaminophen, isoniazid, methoxyflurane, isoflurane, enflurane, sevoflurane, and chlorzoxazone that are oxidized predominantly by CYP2E1.13-16 The 6-hydroxylation of chlorzoxazone, a centrally acting muscle relaxant drug, has been used widely as a probe to determine the activity of human CYP2E1 both in vitro and in vivo.16-20 The study of the potential induction or inhibition of cytochrome P450 enzymes, such as CYP2E1, by new chemical entities is a key aspect of preclinical drug development.21,22 Assays based on the analysis of 6-hydroxychlorzoxazone (HCZ) by HPLC or (1) Ding, X. X.; Koop, D. R.; Crump, B. L.; Coon, M. J. Mol. Pharmacol. 1986, 30, 370-378. (2) de Waziers, I.; Cugnenc, P. H.; Yang, C. S.; Leroux, J. P.; Beaune, P. H. J. Pharmacol. Exp. Ther. 1990, 253, 387-394. (3) Guengerich, F. P.; Kim, D. H.; Iwasaki, M. Chem. Res. Toxicol. 1991, 4, 168-179. (4) Guengerich, F. P.; Turvy, C. G. J. Pharmacol. Exp. Ther. 1991, 256, 11891194. (5) Porter, T. D.; Khani, S. C.; Coon, M. J. Mol. Pharmacol. 1989, 36, 61-65. (6) Kim, R. B.; O’Shea, D. Clin. Pharmacol. Ther. 1995, 57, 645-655. (7) Coon, M. J.; Koop, D. R. Arch. Toxicol. 1987, 60, 16-21. (8) Funae, Y.; Imaoka, S.; Shimojo, N. Biochem. Int. 1988, 16, 503-509. (9) Salazar, D. E.; Sorge, C. L.; Corcoran, G. B. Biochem. Biophys. Res. Commun. 1988, 157, 315-320. (10) Tindberg, N.; Ingelman-Sundberg, M. Biochemistry 1989, 28, 4499-4504. (11) Morimoto, M.; Hagbjork, A. L.; Nanji, A. A.; Ingelman-Sundberg, M.; Lindros, K. O.; Fu, P. C.; Albano, E.; French, S. W. Alcohol 1993, 10, 459-464. (12) O’Shea, D.; Davis, S. N.; Kim, R. B.; Wilkinson, G. R. Clin. Pharmacol. Ther. 1994, 56, 359-367. (13) Zand, R.; Nelson, S. D.; Slattery, J. T.; Thummel, K. E.; Kalhorn, T. F.; Adams, S. P.; Wright, J. M. Clin. Pharmacol. Ther. 1993, 54, 142-149. (14) Kharasch, E. D.; Thummel, K. E. Anesthesiology 1993, 79, 795-807. (15) Garton, K. J.; Yuen, P.; Meinwald, J.; Thummel, K. E.; Kharasch, E. D. Drug Metab. Dispos. 1995, 23, 1426-1430. (16) Peter, R.; Bocker, R.; Beaune, P. H.; Iwasaki, M.; Guengerich, F. P.; Yang, C. S. Chem. Res. Toxicol. 1990, 3, 566-573. (17) Mishin, V. M.; Rosman, A. S.; Basu, P.; Kessova, I.; Oneta, C. M.; Lieber, C. S. Am. J. Gastroenterol. 1998, 93, 2154-2161. (18) Lucas, D.; Menez, J. F.; Berthou, F. Methods Enzymol. 1996, 272, 115123. (19) Dreisbach, A. W.; Ferencz, N.; Hopkins, N. E.; Fuentes, M. G.; Rege, A. B.; George, W. J.; Lertora, J. J. Clin. Pharmacol. Ther. 1995, 58, 498-505. (20) Bachmann, K.; Sarver, J. G. Pharmacology 1996, 52, 169-177. (21) Rodrigues, A. D.; Lin, J. H. Curr. Opin. Chem. Biol. 2001, 5, 396-401.

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radiochemical means have been validated and utilized widely to probe the CYP2E1 activity.23-26 Assays that determine individual cytochrome P450 activities facilitate selection of lead structures that exert low inhibition or induction of CYP2E1 or other human P450 enzymes, thereby minimizing the chance of developing drugs with potential to cause clinical drug-drug interaction.27 The classical cytochrome P450 (P450) probe assays, based on HPLC/ UV or radiodetection, suffer from a lack of specificity as well as low throughput.28,29 This lack of analytical specificity necessitates the individual analysis of each probe for accurate estimation of the drug-drug interaction potentials.21,28 Thus, a variety of individual assays are required, e.g., nine specific assays for human drug-metabolizing cytochrome P450 isozymes 1A2, 2A6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4, and 4A11, for sufficient assessment of a single test compound with respect to its drug interaction liability. Cytochrome P450-mediated drug interaction investigations are therefore an integral part of drug discovery and development, though such studies using conventional analytical approach can be tedious and time-consuming. Ayrton and colleagues reported the first application of tandem mass spectrometry, as a highly specific and universal detector, to determine the activity of major human cytochrome P450 activities.30 Using fast gradient chromatography, the activity of six human P450 enzymes was determined simultaneously within 10 min under positive atmospheric pressure chemical ionization (APCI) mass spectrometry. However, the CYP2E1-mediated 6-hydroxylation of chlorzoxazone activity has historically been quantified using a separate method under negative mode of ionization.30,31 To address this analytical inconvenience, the strategy of polarity switching has been implemented,32 along with the use of a dummy transition to protect the analyte ion signal from the overwhelming switching noise; this resulted in a method for analysis of seven human P450 activities in a single run.32 However, this strategy cannot be applied to many commercially available triple quadruple mass spectrometers due to hardware electronic incompatibility or the slow switching of the polarity. Earlier studies on the possible ionization and detection of HCZ under positive API indicated the formation of protonated HCZ ion at m/z 186 under acidic mobile phases, although the responses were lower than those observed under the (-) ESI or (-) APCI modes.30,32 Our preliminary simulation of the apparent dissociation constants for various HCZ functional groups indicated that the nitrogen heteroatom in the benzoxazolone ring could be protonated and thereby detected under positive electrospray ioniza(22) Davit, B.; Reynolds, K.; Yuan, R.; Ajayi, F.; Conner, D.; Fadiran, E.; Gillespie, B.; Sahajwalla, C.; Huang, S. M.; Lesko, L. J. J. Clin. Pharmacol. 1999, 39, 899-910. (23) Lucas, D.; Berthou, F.; Girre, C.; Poitrenaud, F.; Menez, J. F. J. Chromatogr. 1993, 622, 79-86. (24) Frye, R. F.; Stiff, D. D. J. Chromatogr., B 1996, 686, 291-296. (25) Draper, A. J.; Madan, A.; Latham, J.; Parkinson, A. Drug Metab. Dispos. 1998, 26, 305-312. (26) Tanaka, E. J. Pharm. Biomed. Anal. 1998, 16, 899-904. (27) Weaver, R. J. Xenobiotica 2001, 31, 499-538. (28) Miller, V. P.; Stresser, D. M.; Blanchard, A. P.; Turner, S.; Crespi, C. L. Ann. N. Y. Acad. Sci. 2000, 919, 26-32. (29) Crespi, C. L. Pharm. Sci. Technol. Today 1999, 2, 119-120. (30) Ayrton, J.; Plumb, R.; Leavens, W. J.; Mallett, D.; Dickins, M.; Dear, G. J. Rapid Commun. Mass Spectrom. 1998, 12, 217-224. (31) Scott, R. J.; Palmer, J.; Lewis, I. A.; Pleasance, S. Rapid Commun. Mass Spectrom. 1999, 13, 2305-2319. (32) Bu, H. Z.; Magis, L.; Knuth, K.; Teitelbaum, P. Rapid Commun. Mass Spectrom. 2000, 14, 1619-1624.

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tion (see Supporting Information). The collision-induced dissociation (CID) spectra of the protonated HCZ showed a number of intense, but unexpected, fragment ions that could be used for developing a specific LC/MS/MS assay for HCZ in positive mode of ionization. Although detailed understanding of the fragmentation route of analytes is not necessary for quantitative mass spectrometry purposes, the observation of three major unusual fragments for HCZ was indeed intriguing to further study the fragmentation pathways of this small molecule. The present study describes a detailed investigation of the fragmentation pathways of the protonated HCZ using a combination of complementary techniques such as accurate mass measurements, precursor ion scans, and deuterium ion exchanges, in addition to the product ion scan tandem mass spectrometry. The major product ion of the protonated HCZ has been utilized further to develop an LC/ MS/MS assay for simultaneous analysis of nine human cytochrome P450 activities. EXPERIMENTAL SECTION Chemicals. 1-Hydroxymidazolam, (()-1-hydroxybufuralol, (()4′-hydroxymephenytoin, 4′-hydroxydiclofenac, 6R-hydroxypaclitaxel, and 12-hydroxylauric acid were obtained from Ultrafine Chemicals (Manchester, U.K.). HCZ, 7-hydroxycoumarin, acetaminophen, reserpine, NADPH, dibasic sodium phosphate dihydrate, dibasic potassium phosphate, and magnesium chloride hexahydrate were purchased from Sigma Aldrich Inc. (St. Louis, MO). HPLC-grade acetonitrile was obtained from J. T. Baker (Phillipsburg, PA). Formic acid was purchased from GFS Chemicals (Columbus, OH), and HPLC-grade water was obtained from Mallinckrodt Baker, Inc. (Paris, KY). D2O, methanol-d4, and DCl were purchased from Cambridge Isotope Laboratories. Drug-free human plasma (containing EDTA as the anticoagulant) was obtained from Bioreclamation Inc. (Hicksville, NY). Individual human liver microsomes were purchased from Gentest (Woburn, MA). Safety Considerations. Human plasma and liver microsomes pose a safety risk from potential pathogenic contamination. Every batch of samples was handled by wearing appropriate protective clothing, restriction of work area access, and disinfection of exposed surfaces. Orthogonal Quadrupole Time-of-Flight Accurate Mass Spectrometry. Accurate mass measurements were carried out using a quadrupole orthogonal acceleration time-of-flight mass spectrometer (Q-ToF II, Micromass UK Ltd.), which was equipped with the LockSpray dual-electrospray ion source. The instrument was operated in the positive ion electrospray mode with both the reference and the sample sprays set at 3 kV. The source and desolvation temperatures were set to 80 and 200 °C, respectively. The nitrogen desolvation and nebulizer gas flow rates were set to 400 and 90 L/h, respectively. For the MS or MS/MS studies, the cone voltage was set to 40 V, while for the in-source CID experiments, the cone voltage was set to a higher value of 65 V. The collision energy ranged from 20 to 35 eV, depending on the analyte. Prior to performing all experiments, the instrument was calibrated over a 50-300-Da mass range using a 0.1 ng/L solution of PEG 200 dissolved in acetonitrile/10 mM ammonium acetate (50:50 v/v). The ion m/z 195.123 25 from the PEG 200 calibration solution was used as the reference peak to provide exact mass measurement in the positive ESI mode. This solution was infused

at ∼5 µL/min through the reference spray via a Harvard 22 syringe pump (Harvard Apparatus Inc., South Natick, MA). The flow rate was adjusted to give a reference spectrum of ∼100 counts/s. The reference was sampled every 5 s with the data acquisition rate set to 1 spectrum/s for all analytes. For accurate mass studies of the protonated HCZ, a solution of HCZ (10 nM) in acidic mobile phase (aqueous 0.1% formic acid/methanol, 50: 50 v/v) was infused at a flow rate adjusted to give an analyte spectrum of ∼100 counts/s. For deuterium exchange studies, HCZ was dissolved in a solution of D2O/methanol-d4 (50:50 v/v) containing 0.1% DCl. Data were acquired in a continuum mode and accurate mass measurements and elemental compositions were carried out using the MassLynx software (v3.4). The absolute mass measurement deviations, in parts per million (ppm), were calculated by combining several spectra of the analyte data and applying the correction factor obtained from the reference data. To calculate the correction factor from the reference stream, five spectra were combined to produce an averaged spectrum at the same time point as the analyte data to be measured. For the insource CID studies that utilized Q-TOF to obtain information on the secondary products of each in-source fragment ion, no daily calibration and accurate mass measurement procedure were carried on. High-Performance Liquid Chromatography (HPLC). The HPLC system consisted of a Leap Technologies HTS CTC-PAL autosampler (Carrboro, NC) and two Perkin-Elmer series 200 micropumps (Norwalk, CT). The fast gradient chromatographic separation was carried out using a Phenomenex Synergi 4-µm Max-RP column (2 × 50 mm, 4 µm, 80 Å; Torrance, CA). The mobile phase flowing through the column consisted of two eluants, solvent A (95% H2O/5% acetonitrile, containing 0.1% formic acid, v/v) and solvent B (95% acetonitrile/5% H2O, containing 0.1% formic acid, v/v) with a flow rate of 400 µL/min. The column was maintained at initial conditions of 85% B for 6 s, followed by a linear fast gradient to 90% B over 84 s. This condition was maintained for 30 s, then returned to the initial conditions over 30 s, and maintained until the end of a 3-min run. The CTC-PAL Leap cooling unit was set at 10 °C. The volume of injection onto the column was 10 µL, and the injector syringe was prewashed (100 µL) twice with water prior to each injection. The syringe, injection loop, and switching valve were postwashed (100 µL) five times by two wash solutions, sequentially. The first wash solution was water, and the second wash solution was acetonitrile containing formic acid (0.1%, v/v). During the LC/MS/MS analysis, the first 40 s of the flow was diverted to waste using a switching valve while the rest of eluant (400 µL/min) was directly forwarded to the mass spectrometer without splitting. Quadrupole Tandem Mass Spectrometry. The analytes were detected using a PE Sciex API 4000 (Toronto, Canada) triple quadrupole mass spectrometer equipped with a turbo ion spray source operated in the positive selected reaction monitoring (SRM) mode. All the source and instrument parameters were optimized by infusing a solution of individual metabolites of CYP P-450 probe substrates (10 µM in 50% aqueous methanol) and reserpine into the mass spectrometer. The solutions were introduced at 10 µL/min by an infusion pump (Harvard Apparatus) teed into the mobile-phase mixture (50% B) at flow rate of 400 µL/min. Both Q1 and Q3 were operated under unit mass

resolution (0.7 Da at full width half-maximum). The specific SRM transitions for 6-hydroxychlorzoxazone (CYP2E1),16-20 1′-hydroxymidazolam (CYP3A4),33 (()-1-hydroxybufuralol (CYP2D6),34 (()-4′-hydroxymephenytoin (CYP2C19),35 4′-hydroxydiclofenac (CYP2C9),36 6R-hydroxypaclitaxel (CYP2C8),37 12-hydroxylauric acid (CYP4A11),38 7-hydroxycoumarin (CYP2A6),30 acetaminophen (CYP1A2),39 and reserpine (internal standard) were monitored at m/z 186 f 130, 342 f 203, 278 f 186, 235 f 150, 312 f 231, 871 f 105, 217 f 69, 163 f 107, 152 f 110, and 609 f 195, respectively. The dwell time for each transition was set to 50 ms. Nitrogen was used as the curtain (setting 10), drying gas 1, (setting 50), drying gas 2 (setting 50), and collision gas (setting 6), which was delivered from a nitrogen generator with the back pressure maintained at 80 psi. The electrospray voltage was set at 4.5 kV, and the turbo ion spray interface was maintained at 500 °C. The data were collected and quantified using PE Sciex Analyst software (v 1.2). Microsomal Chlorzoxazone 6-Hydroxylase Assay. Microsomal incubations were performed using a Boekel Industries Jitterbug microplate incubator/shaker (Philadelphia, PA). All incubations were done under conditions shown to be linear with respect to time and protein and substrate concentrations. Triplicate reactions were performed in 96-well plates in a total volume of 0.7 mL. Each incubation contained 1 mg/mL human hepatic microsomal protein, 10 mM MgCl2, 100 mM potassium phosphate buffer (pH 7.4), and 40 µM chlorzoxazone (approximate Km). Samples were preincubated for 5 min at 37 °C in a shaking water bath. The reactions were initiated by addition of NADPH to a final concentration of 2 mM. The reaction was sampled at 0.5, 5, 10, 20, 30, and 40 min, by removing 100 µL of the incubation mixture and mixing it with 0.2 mL of iced-cooled acetonitrile containing 0.1% formic acid and 10 nM reserpine (internal standard). Samples were centrifuged at 3000 rpm for 10 min at 10 °C to pellet the denatured protein. The aqueous acetonitrile supernatants were transferred to the 96-well conical plates, and 10 µL was injected for the LC/MS/MS analysis. Method Validation. Three-day validations were performed to validate the chlorzoxazone 6-hydroxylase LC/MS/MS assay. Ten calibration standard concentrations at 0, 0.1, 0.2, 0.5, 1, 2, 5, 10, and 20 µM were used for the CYP2E1 assay. The quality control samples (QCs) were prepared at three concentrations of 0.2, 1, and 5 µM (low, medium, and high; n ) 3) for each assay. The acceptance criterion for specificity was defined as the absence of any detectable SRM LC/MS/MS ion currents at the retention time regions of 6-hydroxychlorzoxazone in blank plasma or microsomes (double blanks). The accepted criterion for linearity was a coefficient of determination (R2) of greater than 0.98, using at least (33) Kronbach, T.; Mathys, D.; Umeno, M.; Gonzalez, F. J.; Meyer, U. A. Mol. Pharmacol. 1989, 36, 89-96. (34) Gut, J.; Gasser, R.; Dayer, P.; Kronbach, T.; Catin, T.; Meyer, U. A. FEBS Lett. 1984, 173, 287-290. (35) Goldstein, J. A.; Faletto, M. B.; Romkes-Sparks, M.; Sullivan, T.; Kitareewan, S.; Raucy, J. L.; Lasker, J. M.; Ghanayem, B. I. Biochemistry 1994, 33, 17431752. (36) Leemann, T.; Transon, C.; Dayer, P. Life Sci. 1993, 52, 29-34. (37) Rahman, A.; Korzekwa, K. R.; Grogan, J.; Gonzalez, F. J.; Harris, J. W. Cancer Res. 1994, 54, 5543-5546. (38) Kawashima, H.; Kusunose, E.; Kikuta, Y.; Kinoshita, H.; Tanaka, S.; Yamamoto, S.; Kishimoto, T.; Kusunose, M. J. Biochem. (Tokyo) 1994, 116, 74-80. (39) Distlerath, L. M.; Reilly, P. E.; Martin, M. V.; Davis, G. G.; Wilkinson, G. R.; Guengerich, F. P. J. Biol. Chem. 1985, 260, 9057-9067.

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Table 1. Accurate Mass Measurements of the Major Ions Observed in the Electrospray CID Mass Spectrum of Protonated 6-Hydroxychlorzoxazone Figure 1. Keto-enol tautomers of 6-hydroxychlorzoxazone. obsd m/z

calcd m/z

error (ppm)

suggested elemental composition

assignment

185.9961 158.0006 151.0267 142.9900 142.0059 130.0059 114.9952 106.0288 102.0104

185.9958 158.0009 151.0269 142.9900 142.0060 130.0060 114.9951 106.0293 102.0111

1.4 -1.9 -1.8 0.3 -0.6 -0.8 1.0 -4.5 -6.8

C7H5NO3 C6H5NO235Cl C7H5NO3 C6H4O235Cl C6H5NO35Cl C5H5NO35Cl C5H4O35Cl C6H4NO C4H5N35Cl

+ H]+ - CO - 35Cl - CONH - CO2 - (CO + CO) - (CO + CONH) - (CO2 + H35Cl) - (CO+CO+CO)

187.9933 159.9976 151.0261 144.9866 144.0021 132.0025 116.9914 106.0281 104.0071

187.9928 159.9979 151.0269 144.9870 144.0030 132.0030 116.9921 106.0293 104.0081

1.5 -1.8 -5.7 -3.1 -6.2 -3.8 -5.9 -11.6 -10.0

C7H5NO337Cl C6H5NO237Cl C7H5NO3 C6H4O237Cl C6H5NO37Cl C5H5NO37Cl C5H4O37Cl C6H4NO C4H5N37Cl

[37ClM + H]+ - CO - 37Cl - CONH - CO2 - (CO + CO) - (CO + CONH) - (CO2 + H37Cl) - (CO + CO + CO)

35Cl

[35ClM

Figure 2. Product ion spectra of the 6-hydroxychlorzoxazone following protonation of 35Cl m/z 185.99 (A) and 37Cl m/z 187.99 (B) isotopic variants.

six sets of standard concentrations. The combined triplicate set of standards, based on a linear regression and 1/X2 weighting, was used in all experiments for the quantification of samples and QCs. The target values for intra- and interassay mean accuracy of each QC sample were within (15% of the known spiked concentration. RESULTS AND DISCUSSION Electrospray Ionization of Protonated 6-Hydroxychlorzoxazone. 6-Hydroxychlorzoxazone has a 5-chloro-1,3-benzoxazole core structure with two-hydroxyl substituents at carbons six and two that result in two tautomeric structures (Figure 1). The positive electrospray mass spectrum of HCZ showed two major molecular ions at m/z 185.9961 and 187.9931 with abundance ratio of 3:1, respectively (Table 1). Accurate mass measurements using the PEG 200 ion peak, m/z 195.123 25, as the reference mass revealed the deduced elemental compositions (mass errors 50 ppm. The formation of these major product ions and the relationship between their intensity and the laboratory collision energy (ELab) were verified using a Sciex API 4000 triple quadrupole mass spectrometer (Figure 3). The most abundant ions in the CID spectrum of the 35Cl showed the m/z 130 and 115 at an optimum E Lab value of

Figure 4. Precursor ion spectra for the product ions m/z (A) 158, (B) 151, (C) 143, (D) 142, (E) 130, (F) 115, (G) 106, and (H) 102 of protonated [35Cl]6-hydroxychlorzoxazone.

∼35 eV, followed by the two fragments m/z 158 and 151 with ELab values of 27-29 eV (Figure 3), consistent with the Q-ToF CID spectrum. Based on the results of accurate mass measurements of the protonated HCZ, the most abundant ion m/z 130 seemed likely to be the product of two consecutive losses of CO from the protonated HCZ (Table 1). The ion at m/z 158 was likely to be the product of the first loss of CO from m/z 186 at low ELab (27 eV) (Figure 3). The second loss of CO would require the opening of an aromatic ring, a process that is not thermodynamically

favored and not common in CID mass spectrometry.40 The second intense fragment ion at m/z 115 was consistent with consecutive losses of CONH and CO (Table 1). The neutral loss of CONH, which is a common pathway of collision-induced dissociation,41 resulted in the formation of the intermediate fragment ion at m/z 143 seen at the lower ELab (27 eV) (Figure 3). However, the (40) McLafferty, F. W., Turecek, F., Eds. Interpretation of mass spectra, 4th ed.; University Science Books: Sausalito, CA, 1993. (41) Cerny, R. L.; Tomer, K. B.; Gross, M., L,; Grotjahn, L. Anal. Biochem. 1987, 15, 175-182.

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473

Table 2. Accurate Mass Measurements of the Major Ions Observed in the Electrospray CID Mass Spectrum of Deuterated 6-Hydroxychlorzoxazone obsd m/z

Figure 5. Fragmentation pathways of protonated 6-hydroxychlorzoxazone 35Cl and 37Cl isotopic variants.

subsequent loss of CO observed at the higher ELab (35 eV) again required an unfavored aromatic ring opening.40 The third intense fragment ion m/z 151 was likely to be the product of chlorine 35 loss from the protonated 35Cl-HCZ isotopic variant (Table 1). The formation of this ion from the protonated 37Cl-HCZ confirmed the direct loss of chlorine without any hydrogen rearrangement. This is also not a common pathway of collision-induced dissociation, since it requires the loss of chlorine radical instead of the favored neutral loss of HCl,42 leaving a radical cation m/z 151 as a major fragment ion (Table 1). A series of precursor ion scan studies were conducted (Figure 4) to gain insight into the fragmentation pathways of these major, yet uncommon, CID fragments from the protonated HCZ. Precursor ion scans of m/z 130 and 158 confirmed the direct elimination of CO from ions at m/z 158 or 186, respectively. In addition, the ion m/z 102, which was found to be the product of CO loss from m/z 130 (Table 1) at the higher ELab (Figure 3), revealed common precursors at m/z 158 and 186 in addition to m/z 130 (Figure 4). This further confirmed that the major fragmentation pathway of protonated HCZ involved two consecutive losses of CO to give rise to the product ion m/z 130 (Figure 5), which was consistent with the elemental composition results (Table 1). The second intense fragment ion at m/z 115 showed precursor ions at m/z 143 and 186 (Figure 4), confirming the neutral losses of CO and CONH observed by the accurate mass measurements (Table 1). The study of the precursor ion at m/z 143 revealed that this ion might be generated also by the direct loss of NH3 and CO from m/z 160 and 171, respectively. However, this pathway seemed to be a minor route of decomposition compared to the direct neutral loss of CONH, as judged by the relative intensity of the precursor ions (Figure 4). Thus, the results of these precursor ion studies were consistent with the accurate mass measurements (Table 1) and identified a pathway of fragmentation that involved consecutive losses of CONH and CO from protonated HCZ to produce m/z 115 (Figure 5). The precursors of the third intense ion at m/z 151 showed parent ions at m/z 186 and 188, corresponding to the direct loss (42) McLafferty, F. W., Turecek, F., Eds. Interpretation of mass spectra, 4th ed.; University Science Books: Sausalito, CA, 1993; pp 348-350.

474 Analytical Chemistry, Vol. 75, No. 3, February 1, 2003

calcd m/z

error (ppm)

189.0146 161.0191 154.0452 145.0245 145.0028 133.0245 117.0075 108.0416 105.0299

189.0146 161.0197 154.0458 145.0248 145.0025 133.0248 117.0076 108.0418 105.0299

191.0120 163.0163 154.0449 147.0215 146.9993 135.0216 119.0045 108.0415 107.0274

191.0100 163.0168 154.0458 147.0218 146.9996 135.0218 119.0047 108.0418 107.0269

suggested elemental composition

assignment

-0.1 -3.9 -3.4 -2.0 1.9 -2.0 -0.8 -1.9 0.6

35Cl

C7H2D3NO3 C6H2D3NO235Cl C7H2D3NO3 C6H2D3NO35Cl C6H2D2O235Cl C5H2D3NO35Cl C5H2D2O35Cl C6H2D2NO C4H2D3N35Cl

+ D]+ - CO - 35Cl - CO2 - COND - (CO + CO) - (CO + COND) - (CO2 + D35Cl) - (CO + CO + CO)

1.7 -2.6 -5.4 -2.4 -2 -1.9 -1.4 -3.2 4.3

C7H2D3NO337Cl C6H2D3NO237Cl C7H2D3NO3 C6H2D3NO37Cl C6H2D2O237Cl C5H2D3NO37Cl C5H2D2O37Cl C6H2D2NO C4H2D3N37Cl

[37ClM + D]+ - CO - 37Cl - CO2 - COND - (CO + CO) - (CO + COND) - (CO2 + D37Cl) - (CO + CO + CO)

[35ClM

Figure 6. Product ion spectra of the in-source fragment ions m/z (A) 158, (B) 151, (C) 142, (D) 130, and (E) 115 of the protonated 35Cl-6-hydroxychlorzoxazone.

of 35Cl and 37Cl from protonated HCZ (Figure 4). The m/z 106, which was likely to be the product of a minor route of protonated HCZ decomposition (Figure 3), also showed the loss of chlorine, based on accurate mass measurements (Table 1). However, this ion had precursors at m/z 142, 150, 186, and 188, corresponding to the neutral losses of HCl and CO2 (Figure 4), which are common pathways of collision-induced dissociation.42 The intensity of the product ions at m/z 106 and 142 under optimum ELab was much less than the m/z 151 ion (Figure 2), which was formed via the loss of a chlorine radical. Therefore, based on the relative intensity of the intermediate precursor and product ions involved in m/z 151 generation, the fragmentation path by way of the direct loss of chlorine without hydrogen rearrangement was favored (Figure 5). Tandem Mass Spectrometry of the Deuterated 6-Hydroxychlorzoxazone. The deuterium isotope exchange studies were

Figure 7. Product ion spectra of the in-source fragment ions m/z (A) 161, (B) 154, (C) 145 (D) 133, and (E) 117 of the deuterated [35Cl]6hydroxychlorzoxazone.

conducted in conjunction with accurate mass measurement tandem mass spectrometry to gain insights into the mechanism of formation and chemical nature of uncommon HCZ product ions. The positive electrospray accurate mass studies of HCZ in deuterated methanol/D2O showed two major molecular ions at m/z 189.0146 and 191.0120 with an abundance ratio of 3:1, corresponding to the chlorine 35 and 37 isotopes, respectively (Table 2). The accurate mass studies indicated the presence of three exchangeable hydrogens for both chlorine isotopic variants (Table 2). The m/z 133, 117, and 154 were the most abundant ions seen in the product ion spectrum of the 35Cl following deuterium exchange, consistent with the fragmentation pattern of 37Cl isotopic variant. Further evidence for the chemical nature of each ion was obtained by the CID product ion scan studies on the major ions, generated by the in-source fragmentation of the protonated and deuterated HCZ at high cone voltage (65 V) using a Q-ToF mass spectrometer (Figures 6 and 7). The ion at m/z 133, the most intense fragment with three exchangeable hydrogens (Table 2), was confirmed to be the product of the second loss of CO from m/z 161 (Figure 7). The product of m/z 133 (MD+-d3 - C2O2) showed additional losses of CO and ND3, indicating the presence of deuterated nitrogen and a carbonyl moiety (Figure 7), consistent with the pattern of m/z 130 product ion spectra (Figure 6). The formation of the major product ion of the protonated (m/z 130) or deuterated (m/z 133) HCZ might be explained by an earlier report of two consecutive losses of CO from the electron ionization (EI) mass spectra of the anthraquinones.43 Such eliminations are relatively common under EI conditions for compounds with two aromatic rings.44 A proposed mechanism for

Figure 8. Proposed mechanism for fragmentation of deuterated 35Cl-HCZ to the ion [MD+ - C O ] m/z 133. rH, charge site hydrogen 2 2 rearrangment; i; inductive cleavage.

the formation of ion m/z 133 from the deuterated HCZ is shown in Figure 8. A charge site rearrangement to the ionized keto Analytical Chemistry, Vol. 75, No. 3, February 1, 2003

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Figure 9. Proposed mechanism for fragmentation of deuterated 35Cl-HCZ to the ions [MD+ - COND] m/z 145 and [MD+ - CO COND] m/z 117. rH, charge site hydrogen rearrangment; i; inductive cleavage.

tautomer followed by the inductive cleavage of the N-C bond was proposed for the formation of the first intermediate ion m/z 161, seen at the lower ELab (27 eV) (Figure 3). A prime driving force for such elimination reactions is known to be the stability of the product ions, although the loss of small stable molecules such as CO is also thermodynamically favorable.40 The tentative structure shown for the ion m/z 161 is not aromatic; however, this appears to be the most stable resonance form (Hf ∼ 5.35 eV) that could be favored on an energetic basis (see Supporting Information). This may further explain the second loss of CO by the inductive cleavage of the nonaromatic C-C bond at higher ELab (35 eV for m/z 130, Figure 3). The concomitant loss of CO and cyclization could generate a stable aromatized product ion at m/z 133 (Figure 8). In the case of anthraquinone fragmentation, the second CO loss was also shown to be associated with the formation of a new bond between two rings, which leads to a stable product ion.43 The fragmentation of the in-source ion m/z 145 (MD+-d2 COND) confirmed the loss of CO to the ion m/z 117, the second most intense product ion of HCZ with two exchangeable hydrogens (Table 3). Other small peaks at m/z 108 and 116 found in the product mass spectrum of m/z 145 (145.0248, C6H2D3NOCl) could be the fragment ions of m/z 145.0025 (C6H2D2O2Cl), detected under nominal in-source CID mass spectrometry, which was shown to be a minor pathway of fragmentation (Figure 3). (43) Beynon, J. H. Mass Spectrometry and Its Applications to Organic Chemistry; Elsevier Science: Amsterdam, 1960. (44) Wszolek, P.; McLafferty, F. W.; Brewster, J. H. Org. Mass Spectrom. 1968, 1, 127.

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Figure 10. Proposed mechanism for fragmentation of deuterated 35Cl-HCZ to the ions [MD+ - Cl] m/z 154 and [MD+ - DClCO ] m/z 2 108. rH, charge site hydrogen rearrangment; rr, radical resonance; i; inductive cleavage.

The product of ion m/z 117 showed an additional loss of CO to m/z 89, which suggested the presence of one carbonyl group in the molecule, as proposed in Figure 9. The neutral loss of CONH is a common pathway of collision-induced dissociation.41 The charge site rearrangements followed by an inductive cleavage of the C-N bond could form the tentative structure m/z 145 at the lower ELab (28 eV for m/z 143, Figure 3). The subsequent loss of CO at higher collision energy (ELab 35 eV for m/z 115) by the inductive cleavage of C-C bond and ring condensation could form the tentative ion m/z 117 (Figure 9). The product of the in-source ion m/z 154 (MD+-d2 - 35Cl.) showed fragment ions m/z 126, 108, and 98, indicating the presence of two carbonyl groups and one carboxyl moiety in the molecule (Figure 7), similar to the pattern of HCZ fragmentation. However, the product of the in-source ion m/z 151 (MH+ - 35Cl.) showed fragment ions m/z 124, 108, and 96 (Figure 6), which may indicate a similar fragmentation pattern if one accounts for one hydrogen atom rearrangement. The product ion spectrum of neither m/z 151 nor 154 showed the ion m/z 106 or 108, observed

Figure 11. Reconstructed ion chromatograms for the specific SRM transitions of probes used to monitor CYP2E1 (A), CYP2C19 (B), CYP2C8 (C), CYP2A6 (D), CYP4A11 (E), CYP1A2 (F), CYP3A4 (G) CYP2C9 (H), CYP2D6 (I), and reserpine (J) obtained from the analysis of a test mixture under (+) ESI-LC/MS/MS.

in the CID product ion spectrum of protonated or deuterated HCZ, respectively (Tables 1 and 2). The two mass unit higher values for the ions m/z 108 and 110 suggest further hydrogen rearrangement and transfer must have occurred during the process of CO2 elimination in CID tandem mass spectrometry of intermediate insource ions m/z 151 and 154 (Figures 6 and 7). Based on these results, a tentative mechanism for the formation of ions m/z 154, 145, and 108 from the deuterated HCZ is proposed in Figure 10. A charge site rearrangement to the keto tautomer followed by the inductive cleavage of the N-C bond was anticipated to initiate this fragmentation route. The major path to the product ion m/z 154 is proposed to occur via charge site rearrangement and hemolytic cleavage of the C-Cl bond, which has been reported under EI conditions.40 The tentative structure shown for the ion m/z 154 is not aromatic; however, this ion again appears to be the most stable resonance structure (Hf ∼ 6.09 eV) that could be favorable on an energetic basis (see Supporting Information). The inductive cleavage of the C-O bond of radical cation m/z 154 at higher collision energy (ELab 34 eV for the m/z 106, Figure 3) is

proposed to result in loss of CDO2 radical to the product ion m/z 108 (Figure 10). On the other hand, the minor fragmentation path to the product ion m/z 145, formed by neutral loss of CO2, is proposed to occur via charge site rearrangement and inductive cleavage of the C-O bond (Figure 10). Although CO2 elimination is common under CID mass spectrometry,40 the intermediate ion m/z 145 formed through this pathway appears to be less stable (Hf ∼ 8.05 eV) than that of the ion m/z 154 (Hf ∼ 6.09 eV) formed via chlorine radical loss (see Supporting Information), consistent with the fact that the stability of the product ion is a prime driving force in each elimination pathway.40 Simultaneous Analysis of CYP2E1 with Major Human Cytochrome P450s. The SRM transition from the protonated 35Cl-HCZ at m/z 186 to its most intense product ion m/z 130 was selected for the specific detection of 6-hydroxychlorzoxazone. The turbo ion spray, collision cell, and gas parameters were optimized using the m/z 186 f 130 transition on the Sciex API 4000 triple quadrupole instrument. A complete LC/MS/MS method was developed using a fast gradient reversed-phase chromatographic Analytical Chemistry, Vol. 75, No. 3, February 1, 2003

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separation, which was optimized for the m/z 186 f 130 transition for HCZ as described in the Experimental Section. This transition was further combined with eight other transitions known to be specific for metabolites of human cytochrome P450 probe substrates,45 in addition to reserpine that was added as an internal standard. A representative reconstructed ion chromatogram for each transition following addition of 0.1 µM concentrations of each metabolite to human hepatic microsomes is shown in Figure 11. The full validation of LC/MS/MS methods based on the specific transition of the metabolites of other human P450 probes has already been reported.31,32 The preliminary results from the validation of HCZ transition indicated that the m/z 186 f 130 SRM is specific as no interfering signal was observed at the retention time of the HCZ (1.59 min) from the control human liver microsomes or human plasma extracts (data not shown). The HCZ assay showed excellent linearity with a coefficient of determination (R2) greater than 0.98 at dynamic range of 0.05 (LOQ) to 40 µM. Preliminary data from the three-day validation of HCZ assay indicated that the accuracy and precision for quality control samples was within (15% of the spiked concentration at all levels. The estimated CYP2E1 activities for the five human hepatic microsomal preparations showed that the rate of chlorzoxazone 6-hydroxylation was within (15% of the characterized rates reported using classical HPLC assays, i.e., average rate of 1500 pmol. mg-1 min-1.46 CONCLUSIONS A selective and sensitive quantitative assay, capable of analyzing major human P450 enzyme activities, has significant applications in drug discovery and development to evaluate the potential (45) Wilkinson, G. R. In Drugs and Pharmaceutical Sciences: Drug-Drug Interactions; Rodrigues, A. D., Ed.; Marcel Dekker: New York, 2002; Vol. 116, pp 439-504. (46) Fairbrother, K. S.; Grove, J.; de Waziers, I.; Steimel, D. T.; Day, C. P.; Crespi, C. L.; Daly, A. K. Pharmacogenetics 1998, 8, 543-552.

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drug-drug interaction of new chemical entities. Earlier studies on the possible ionization of HCZ under acidic conditions were encouraging for study of the ionization and detection of HCZ under positive API mass spectrometry. However, the preliminary CID studies of the protonated HCZ showed a number of intense but unexpected fragment ions that could be used for developing a specific LC/MS/MS assay for HCZ in positive mode of ionization. The accurate mass spectrometry and precursor ion scan studies revealed the precursor-product ion relationship for the major routes of HCZ fragmentation. Collectively, these data along with the results of deuterium ion exchange and in-source CID tandem mass spectrometry proposed the presence of three distinct pathways of fragmentation for protonated HCZ. The m/z 186 f 130 SRM combined with transitions of major metabolites of eight cytochrome P450 probe substrates led to the development of an LC/MS/MS method capable of analyzing nine human P450 activities in a single 3-min run. ACKNOWLEDGMENT We are grateful to Professor Simon Gaskell, Institute of Technology, University of Manchester, and Dr. Xue-Zhi Qin, Department of Pharmaceutical Research, Merck Research Laboratories, for their helpful discussions with respect to the proposed structures of the fragment ions. We are grateful to Mrs. Honeah Sohail for assistance with the validation of HCZ assay. We thank Mr. Koppara Samuel for his expertise in conducting the microsomal 6-hydroxychlorzoxazone assays. SUPPORTING INFORMATION AVAILABLE Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review September 20, 2002. Accepted December 5, 2002. AC026157M