Imatinib metabolite profiling in parallel to imatinib quantification in plasma of treated patients using liquid chromatography-mass spectrometry

Besides affecting the systemic bioavailability of the parent drug, drug metabolizing enzymes (DMEs) may produce bioactive and/or toxic metabolites of clinical interest. We have investigated the capability to analyze simultaneously the parent drug and newly identified metabolites in patients' plasma by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The anticancer drug, imatinib, was chosen as a model drug because it has opened a new area in cancer therapy and is given orally and chronically. In addition, resistance and rare but sometimes severe side effects have been reported with this therapy. The quantification of imatinib and the profiling of its metabolites in plasma were established following three steps: (1) set-up of a generic sample extraction and LC-MS/MS conditions, (2) metabolite identification by LC-MS/MS using either in vitro incubations performed with human liver microsomes (HLMs) or patient plasma samples, (3) the simultaneous determination of plasma levels of imatinib and 14 metabolites in the plasma samples of 38 patients. Partial or cross method validation has been done and revealed that precise determinations of metabolite levels can be performed whereas pure standards are not available. Preliminary results indicate that the disposition of imatinib and its metabolites is related to interindividual variables and that outlier metabolite profiles can be revealed. This article underscores that, in addition to usual therapeutic drug monitoring (TDM), LC-MS/MS methods can simultaneously record a complete drug metabolic profile enabling various correlation studies of clinical interest.     

Rapid screening and characterization of drug metabolites using multiple ion monitoring dependent product ion scan and postacquisition data mining on a hybrid triple quadrupole‐linear ion trap mass spectrometer

Multiple ion monitoring (MIM)‐dependent acquisition with a triple quadrupole‐linear ion trap mass spectrometer (Q‐trap) was previously developed for drug metabolite profiling. In the analysis, multiple predicted metabolite ions are monitored in both Q1 and Q3 regardless of their fragmentations. The collision energy in Q2 is set to a low value to minimize fragmentation. Once an expected metabolite is detected by MIM, enhanced product ion (EPI) spectral acquisition of the metabolite is triggered. To analyze in vitro metabolites, MIM‐EPI retains the sensitivity and selectivity similar to that of multiple reaction monitoring (MRM)‐EPI in the analysis of in vitro metabolites. Here we present an improved approach utilizing MIM‐EPI for data acquisition and multiple data mining techniques for detection of metabolite ions and recovery of their MS/MS spectra. The postacquisition data processing tools included extracted ion chromatographic analysis, product ion filtering and neutral loss filtering. The effectiveness of this approach was evaluated by analyzing oxidative metabolites of indinavir and glutathione (GSH) conjugates of clozapine and 4‐ethylphenol in liver microsome incubations. Results showed that the MIM‐EPI‐based data mining approach allowed for comprehensive detection of metabolites based on predicted protonated molecules, product ions or neutral losses without predetermination of the parent drug MS/MS spectra. Additionally, it enabled metabolite detection and MS/MS acquisition in a single injection. This approach is potentially useful in high‐throughout screening of metabolic soft spots and reactive metabolites at the drug discovery stage. Copyright © 2009 John Wiley & Sons, Ltd.     

Mass defect filter technique and its applications to drug metabolite identification by high‐resolution mass spectrometry

Identification of drug metabolites by liquid chromatography/mass spectrometry (LC/MS) involves metabolite detection in biological matrixes and structural characterization based on product ion spectra. Traditionally, metabolite detection is accomplished primarily on the basis of predicted molecular masses or fragmentation patterns of metabolites using triple‐quadrupole and ion trap mass spectrometers. Recently, a novel mass defect filter (MDF) technique has been developed, which enables high‐resolution mass spectrometers to be utilized for detecting both predicted and unexpected drug metabolites based on narrow, well‐defined mass defect ranges for these metabolites. This is a new approach that is completely different from, but complementary to, traditional molecular mass‐ or MS/MS fragmentation‐based LC/MS approaches. This article reviews the mass defect patterns of various classes of drug metabolites and the basic principles of the MDF approach. Examples are given on the applications of the MDF technique to the detection of stable and chemically reactive metabolites in vitro and in vivo. Advantages, limitations, and future applications are also discussed on MDF and its combinations with other data mining techniques for the detection and identification of drug metabolites. Copyright © 2009 John Wiley & Sons, Ltd.     

Rapid screening and characterization of drug metabolites using a multiple ion monitoring-dependent MS/MS acquisition method on a hybrid triple quadrupole-linear ion trap mass spectrometer

A novel LC/MS/MS method that uses multiple ion monitoring (MIM) as a survey scan to trigger the acquisition of enhanced product ions (EPI) on a hybrid quadrupole-linear ion trap mass spectrometer (Q TRAP) was developed for drug metabolite identification. In the MIM experiment, multiple predicted metabolite ions were monitored in both Q1 and Q3. The collision energy in Q2 was set to a low value to minimize fragmentation. Results from analyzing ritonavir metabolites in rat hepatocytes demonstrate that MIM-EPI was capable of targeting a larger number of metabolites regardless of their fragmentation and retained sensitivity and duty cycle similar to multiple reaction monitoring (MRM)-EPI. MIM-based scanning methods were shown to be particularly useful in several applications. First, MIM-EPI enabled the sensitive detection and MS/MS acquisition of up to 100 predicted metabolites. Second, MIM-MRM-EPI was better than MRM-EPI in the analysis of metabolites that undergo either predictable or unpredictable fragmentation pathways. Finally, a combination of MIM-EPI and full-scan MS (EMS), as an alternative to EMS-EPI, was well suited for routine in vitro metabolite profiling. Overall, MIM-EPI significantly enhanced the metabolite identification capability of the hybrid triple quadrupole-linear ion trap LC/MS.     

High-throughput cytochrome P450 (CYP) inhibition screening via a cassette probe-dosing strategy. VI. Simultaneous evaluation of inhibition potential of drugs on human hepatic isozymes CYP2A6, 3A4, 2C9, 2D6 and 2E1

The inhibition potential of drugs towards five major human hepatic cytochrome P450 (CYP) isozymes (CYP2A6, 3A4, 2C9, 2D6, and 2E1) was investigated via cassette dosing of the five probe substrates (coumarin, midazolam, tolbutamide, dextromethorphan, and chlorzoxazone) in human liver microsomes using a 96-well plate format. After microsomal incubations had been terminated with formic acid, the five marker metabolites (7-hydroxycoumarin, 1'-hydroxymidazolam, 4-hydroxytolbutamide, dextrorphan, and 6-hydroxychlorzoxazone) were simultaneously quantified using direct injection/online guard cartridge extraction/tandem mass spectrometry (DI-GCE/MS/MS). Several advantages resulted from the use of a short C(18) guard cartridge (4 mm in length) for DI-GCE/MS/MS, including minimal sample preparation, fast online extraction, short analysis time (2.5 min), and minimal source contamination. In addition, this method demonstrated an inter-day accuracy range from -8.7 - 7.4% with a precision less than 8.3% for the quantification of all the marker metabolites. The inhibition assay for the five CYP isozymes was evaluated using their known selective inhibitors via individual and cassette dosing of the probe substrates. The IC(50) values measured via cassette dosing were consistent with those observed via individual dosing, which were all in agreement with the reported values. In addition, the validated assay was used to evaluate the inhibitory potential of 23 generic drugs (randomly selected) towards the five CYP isozymes. The results suggest the integration of the cassette dosing strategy and the DI-GCE/MS/MS method can provide a reliable in vitro approach to screening the inhibitory potential of new chemical entities, with maximal throughput and cost-effectiveness, in support of drug discovery and development.     

Determination of imatinib and its active metabolite N-desmethyl imatinib in human plasma by liquid chromatography/tandem mass spectrometry

Imatinib is a first-line treatment for chronic myelogenous leukaemia (CML). The pharmacokinetics of imatinib in patients with CML are characterised by large interpatient variability. Concentration monitoring of imatinib and its active metabolite N-desmethyl imatinib (DMI) is considered necessary to enhance the safe and effective use of imatinib. A rapid, simple and sensitive liquid chromatography/tandem mass spectrometry assay was developed for the simultaneous determination of imatinib and its metabolite DMI in human plasma. After proteins were precipitated with acetonitrile, imatinib, DMI and the internal standard D8-imatinib were resolved on a Gemini-NX 3?μm C18 column using gradient elution of 0.05?% formic acid and methanol. The three compounds were detected using electrospray ionisation in the positive mode. Standard curves of imatinib and DMI were adequately fitted by quadratic equations (r?>?0.999) over the concentration range of 10 to 2,000?ng/mL which encompasses clinical concentrations. Bias was ≤±8.3?%, intra- and inter-day coefficients of variation (imprecision) were ≤8.0?% and the limit of quantification was 10?ng/mL for both imatinib and DMI. The assay is being used successfully in clinical practice to enhance the safe and effective use of imatinib.     

Fragmentation study on the phenolic alkaloid neferine and its analogues with anti-HIV activities by electrospray ionization tandem mass spectrometry with hydrogen/deuterium exchange and its application for rapid identification of in vitro microsomal metabolites of neferine

The application of mass spectrometry in drug discovery, especially in drug metabolites, is very important. This present paper is at first focused on the elucidation of fragmentation patterns of the phenolic bisbenzyltetrahydroisoquinoline alkaloid, neferine, together with its analogues isoliensinine and liensinine with anti-HIV activities using electrospray ionization tandem mass spectrometry (ESI-MS/MS) and hydrogen/deuterium (H/D) exchange. All title compounds displayed major diagnostic fragments that formed by the cleavage of the C1'--C9' bond resulting in positive group CD, and the loss of 4-ethyl-1-phenol or 4-ethyl-1-methoxybenzene following rearrangements. Their ESI-MS/MS spectra also showed the relatively stable fragment ions formed by the elimination of H2O, CH3NH2, CH3OH, and CH3-N==CH2. Secondly, the metabolites of neferine from dog hepatic microsomal incubations were analyzed and characterized by high-performance liquid chromatography (HPLC) and data-dependent ESI-MS/MS. Based on fragmentation patterns and compared with their retention times in LC, molecular weights and ultraviolet (UV) absorbances with standard compounds, six metabolites were identified as isoliensinine, liensinine and four novel bisbenzyltetrahydroisoquinoline alkaloids named as 6-O-desmethylneferine, 2'-N-desmethylneferine, 2'-N-6-O-didesmethylneferine, and 6,13-O-didesmethylneferine. All metabolites were desmethyl or didesmethyl products of neferine. The possible metabolic pathways for neferine have been proposed. The results suggest that N-demethylation and O-demethylation are two important metabolic pathways of neferine in dog hepatic microsomal incubations. This is critical for screening and development of phenolic bisbenzyltetrahydroisoquinoline alkaloids with anti-HIV activities such as neferine and its analogues isoliensinine and liensinine.     

High-throughput cytochrome p450 inhibition assays by ultrafast gradient liquid chromatography with tandem mass spectrometry using monolithic columns

A generic method employing ultrafast liquid chromatography with tandem mass spectrometry (LC/MS/MS) was developed and employed for routine screening of drug candidates for inhibition of five major human cytochrome p450 (CYP) isozymes, CYP3A4, CYP2D6, CYP2C9, CYP2C19, and CYP1A2. The method utilized a monolithic silica rod column to allow fast flow rates to significantly reduce chromatographic run time. The major metabolites of six CYP-specific probe substrates for the five p450 isoforms were monitored and quantified to determine IC(50) values of five drug compounds against each p450 isozyme. Human liver microsomal incubation samples at each test compound concentration were combined and analyzed simultaneously by the LC/MS/MS method. Each pooled sample containing six substrates and an internal standard was separated and detected in only 24 seconds. The combination of ultrafast chromatography and sample pooling techniques has significantly increased sample throughput and shortened assay turnaround time, allowing a large number of compounds to be screened rapidly for potential p450 inhibitory activity, to aid in compound selection and optimization in drug discovery.     

An experimental approach to enhance precursor ion fragmentation for metabolite identification studies: application of dual collision cells in an orbital trap

Recent advancements in mass spectrometry including data-dependent scanning and high-resolution mass spectrometry have aided metabolite profiling for non-radiolabeled xenobiotics. However, narrowing down a site of metabolism is often limited by the quality of the collision-induced dissociation (CID)-based precursor ion fragmentation. An alternative dissociation technique, higher energy collisional dissociation (HCD), enriches compound fragmentation and yields 'triple-quadrupole-like fragmentation'. Applying HCD along with CID and data-dependent scanning could enhance structural elucidation for small molecules. Liquid chromatography/multi-stage mass spectrometry (LC/MS(n) ) experiments with CID and HCD fragmentation were carried out for commercially available compounds on a hybrid linear ion trap orbital trap mass spectrometer equipped with accurate mass measurement capability. The developed method included stepped normalized collision energy (SNCE) parameters to enhance MS fragmentation without tuning for individual compounds. All the evaluated compounds demonstrated improved fragmentation under HCD as compared with CID. The results suggest that an LC/MS(n) method that incorporated both SNCE HCD- and CID-enabled precursor ion fragmentation afforded comprehensive structural information for the compounds under investigation. A dual collision cell approach was remarkably better than one with only CID MS(n) in an orbital trap. It is evident that such an acquisition method can augment the identification of unknown metabolites in drug discovery by improving fragmentation efficiency of both the parent compound and its putative metabolite(s).     

Assessment and validation of the MS/MS fragmentation patterns of the macrolide immunosuppressant everolimus

Everolimus (40-O-(2-hydroxyethyl)rapamycin, Certican) is a 31-membered macrolide lactone. In lymphocytes, it inhibits the mammalian target of rapamycin (mTOR) and is used as an immunosuppressant after organ transplantation. Due to its instability in pure organic solvents and insufficient HPLC separation, NMR spectroscopy analysis of its metabolite structures is nearly impossible. Therefore, structural identification based on tandem mass spectrometry (MS/MS) and MS(n) fragmentation patterns is critical. Here, we have systematically assessed the fragmentation pattern of everolimus during liquid chromatography (LC)-electrospray ionization (ESI)-MS/MS and validated the fragment structures by (1) comparison with structurally identified derivatives (sirolimus), (2) high-resolution mass spectrometry, (3) elucidation of fragmentation pathways using ion trap mass spectrometry (up to MS(5)) and (4) H/D exchange. In comparison with the structurally related immunosuppressants tacrolimus and sirolimus, our study was complicated by the low ionization efficiency of everolimus. Detection of positive ions gave the best sensitivity, and everolimus and its fragments were mainly detected as sodium adducts. LC-ESI-MS/MS of everolimus in combination with collision-induced dissociation (CID) resulted in a complex fragmentation pattern and the structures of 53 fragments were identified. These detailed fragmentation pathways of everolimus provided the basis for structural elucidation of all everolimus metabolites generated in vivo und in vitro.     

Metabolic profile of glyburide in human liver microsomes using LC‐DAD‐Q‐TRAP‐MS/MS

The sulfonylurea urea drug glyburide (glibenclamide) is widely used for the treatment of diabetes milletus and gestational diabetes. In previous studies monohydroxylated metabolites were identified and characterized for glyburide in different species, but the metabolite owing to the loss of cyclohexyl ring was identified only in mouse. Glyburide upon incubation with hepatic microsomes resulted in 10 metabolites for human. The current study identifies new metabolites of glyburide along with the hydroxylated metabolites that were reported earlier. The newly identified drug metabolites are dihydroxylated metabolites, a metabolite owing to the loss of cyclohexyl ring and one owing to hydroxylation with dehydrogenation. Among the 10 identified metabolites, there were six monohydroxylated metabolites, one dihydroxylated metabolite, two metabolites owing to hydroxylation and dehydrogenation, and one metabolite owing to the loss of cyclohexyl ring. New metabolites of glyburide were identified and characterized using liquid chromatography–diode array detector–quadruple‐ion trap–mass spectrometry/mass spectrometry (LC‐DAD‐Q‐TRAP‐MS/MS). An enhanced mass scan–enhanced product ion scan with information‐dependent acquisition mode in a Q‐TRAP‐MS/MS system was used to characterize the metabolites. Liquid chromatography with diode array detection was used as a complimentary technique to confirm and identify the metabolites. Metabolites formed in higher amounts were detected in both diode array detection and mass spectrometry detection. Copyright © 2012 John Wiley & Sons, Ltd.     

Liquid chromatography/tandem mass spectrometric determination of inhibition of human cytochrome P450 isozymes by resveratrol and resveratrol-3-sulfate

trans-Resveratrol, a phenolic phytoalexin occurring in grapes, wine, peanuts, and cranberries, has been reported to both have anticarcinogenic, antioxidative, phytoestrogenic, and cardioprotective activities, and to be a weak inhibitor of cytochrome P450 (CYP)3A4, which might have significance for drug-drug interactions. Since trans-resveratrol is rapidly converted in vivo to primarily trans-resveratrol-3-sulfate, a rapid, selective, and sensitive method using liquid chromatography/tandem mass spectrometry (LC/MS/MS) was developed to investigate human cytochrome P450 inhibition by trans-resveratrol-3-sulfate. Effects of trans-resveratrol and trans-resveratrol-3-sulfate on the metabolism of selective cytochrome P450 substrates (CYP1A2/ethoxyresorufin, CYP2C9/diclofenac, CYP2C19/(S)-mephenytoin, CYP2D6/bufuralol, CYP3A4/testosterone) were monitored using cDNA-expressed human recombinant isozymes. For method validation, LC/MS/MS was used to measure the inhibition of various cytochrome P450 isozymes by different concentrations (0-50 microM) of known selective inhibitors. IC(50) values of 3.2, 1.4, 8.9, 0.2, and 0.3 microM were obtained for the standard isozyme inhibitors CYP1A2/furafylline, CYP2C9/sulfaphenazole, CYP2C19/tranylcypromine, CYP2D6/quinidine, and CYP3A4/ketoconazole, respectively, which were in good agreement with literature values. trans-Resveratrol showed IC(50) values of 11.6 microM for CYP2C19 and 1.1 microM for CYP3A4, but the IC(50) values exceeded 50 microM for all the other CYP isozymes, which indicated no inhibition. No enzyme inhibition was observed for trans-resveratrol-3-sulfate. Our results indicate that trans-resveratrol is a marginal inhibitor of CYP3A4 and a weak inhibitor of CYP2C19, but its major metabolite trans-resveratrol-3-sulfate is not an inhibitor of any of the cytochrome P450 isozymes investigated.     

A UPLC-MS/MS assay of the "Pittsburgh cocktail": six CYP probe-drug/metabolites from human plasma and urine using stable isotope dilution

The efficiency of drug metabolism by a single enzyme can be measured as the fractional metabolic clearance which can be used as a measure of whole body activity for that enzyme. Measurement of activity of multiple enzymes simultaneously is feasible using a cocktail approach, however, analytical approach using different assays for drug probes can be cumbersome. A quantitative ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) based method for the rapid measurement of six cytochrome P450 (CYP) probe drugs and their relevant metabolites is described. The six specific probe substrates/metabolites are caffeine/paraxanthine (CYP1A2), flurbiprofen/4'-hydroxyflurbiprofen (CYP2C9), mephenytoin/4'-hydroxymephenytoin (CYP2C19), debrisoquine/4-hydroxydebrisoquine (CYP2D6), chlorzoxazone/6'-hydroxychlorzoxazone (CYP2E1) and dapsone/N-monoacetyldapsone (NAT2). These probes were quantified by stable isotope dilution from plasma and urine. The present workflow provides a robust, fast and sensitive assay for the "Pittsburgh cocktail", and has been successfully applied to a clinical phenotyping study of liver disease. A representative group of 17 controls and patients with chronic liver disease were administered orally caffeine (100 mg), chlorzoxazone (250 mg), debrisoquine (10 mg), mephenytoin (100 mg), flurbiprofen (50 mg) and dapsone (100 mg). Urine (0 through 8 h) and plasma (4 and 8 h) samples were analyzed for drug/metabolite amounts by stable isotope dilution UPLC-MS/MS. The phenotypic activity of drug metabolizing enzymes was investigated with 17 patient samples. Selected reaction monitoring (SRM) was optimized for each drug and metabolite. In the method developed, analytes were resolved by reversed-phase by development of a gradient using a water/methanol solvent system. SRM of each analyte was performed in duplicate on a triple quadrupole mass spectrometer utilizing an 8 min analytical method each, one with the source operating in the positive mode and one in the negative mode, using the same solvent system. This method enabled quantification of each drug (caffeine, chlorzoxazone, debrisoquine, mephenytoin, flurbiprofen, and dapsone) and its resulting primary metabolite in urine or plasma in patient samples. The method developed and the data herein demonstrate a robust quantitative assay to examine changes in CYP enzymes both independently or as part of a cocktail. The clinical use of a combination of probe drugs with UPLC-MS/MS is a highly efficient tool for the assessment of CYP enzyme activity in liver disease.     

High-throughput cytochrome P450 (CYP) inhibition screening via cassette probe-dosing strategy. I. Development of direct injection/on-line guard cartridge extraction/tandem mass spectrometry for the simultaneous detection of CYP probe substrates and their metabolites

A highly efficient direct injection/on-line guard cartridge extraction/tandem mass spectrometry (DI-GCE/MS/MS) method utilizing electrospray polarity switching was developed for the simultaneous detection of probe substrates and marker metabolites of seven human hepatic cytochrome P450 (CYP) isozymes: CYP1A2, 2A6, 3A4, 2C9, 2C19, 2D6 and 2E1. Microsomal incubations were terminated with formic acid, centrifuged, and the resulting supernatants were injected for analysis by DI-GCE/MS/MS. This method employed an extremely short C(18) cartridge (4 mm in length) which allowed rapid cleanup of sample matrices while retaining the analytes an appropriate time (2. 0-2.2 min). From 1.5 to 2.7 min the effluent was directed to the mass spectrometer for detection otherwise diverted to waste. As a result of the efficient on-line extraction, matrix (e.g., salts and proteins) suppression was minimized. In addition, no visible source contamination was observed and system performance (chromatographic and mass spectrometric) did not significantly deteriorate after 500 consecutive injections. Electrospray polarity switching was strategically executed on a Micromass Quattro II mass spectrometer by establishing dummy ion transitions to protect the analytes from the interference of the overwhelming noise which was unavoidable for the first transition scanned following each polarity switch. This unique strategy led to the simultaneous detection of seven CYP probe substrates and seven corresponding marker metabolites (12 by positive mode and 2 by negative mode).     

Characterization of metabolites of a NK1 receptor antagonist, CJ-11,972, in human liver microsomes and recombinant human CYP isoforms by liquid chromatography/tandem mass spectrometry

The in vitro metabolism of CJ-11,972, (2-benzhydryl-1-aza-bicyclo[2.2.2]oct-3-yl)-(5-tert-butyl-2-methoxybenzyl)amine, an NK1 receptor antagonist, was studied in human liver microsomes and recombinant human CYP isoforms. Liquid chromatography/mass spectrometry (LC/MS) and tandem mass spectrometry (LC/MS/MS) coupled to radioactive detection were used to detect and identify the metabolites. CJ-11,972 was extensively metabolized in human liver microsomes and recombinant human CYP 3A4/3A5 isoforms. A total of fourteen metabolites were identified by a combination of various MS techniques. The major metabolic pathways were due to oxidation of the tert-butyl moiety to form an alcohol (M6) and/or O-demethylation of the anisole moiety. The alcohol metabolite M6 was further oxidized to the corresponding aldehyde (M7) and carboxylic acid (M4). Two unusual metabolites (M13, M17), formed by C-demethylation of the tert-butyl group, were identified as 2-{3-[(2-benzhydryl-1-aza-bicyclo[2.2.2]oct-3-ylamino)methyl]-4-methoxyphenyl}propan-2-ol and (2-benzhydryl-1-aza-bicyclo[2.2.2]oct-3-yl)-(5-isopropenyl-2-methoxybenzyl)amine. A plausible mechanism for C-demethylation may involve oxidation of M6 to form an aldehyde metabolite (M7), followed by cytochrome P450-mediated deformylation leaving an unstable carbon-centered radical, which would quickly form either the alcohol metabolite M13 and the olefin metabolite M17.     

Accurate mass filtering of ion chromatograms for metabolite identification using a unit mass resolution liquid chromatography/mass spectrometry system

Acceleration of liquid chromatography/mass spectrometric (LC/MS) analysis for metabolite identification critically relies on effective data processing since the rate of data acquisition is much faster than the rate of data mining. The rapid and accurate identification of metabolite peaks from complex LC/MS data is a key component to speeding up the process. Current approaches routinely use selected ion chromatograms that can suffer severely from matrix effects. This paper describes a new method to automatically extract and filter metabolite-related information from LC/MS data obtained at unit mass resolution in the presence of complex biological matrices. This approach is illustrated by LC/MS analysis of the metabolites of verapamil from a rat microsome incubation spiked with biological matrix (bile). MS data were acquired in profile mode on a unit mass resolution triple-quadrupole instrument, externally calibrated using a unique procedure that corrects for both mass axis and mass spectral peak shape to facilitate metabolite identification with high mass accuracy. Through the double-filtering effects of accurate mass and isotope profile, conventional extracted ion chromatograms corresponding to the parent drug (verapamil at m/z 455), demethylated verapamil (m/z 441), and dealkylated verapamil (m/z 291), that contained substantial false-positive peaks, were simplified into chromatograms that are substantially free from matrix interferences. These filtered chromatograms approach what would have been obtained by using a radioactivity detector to detect radio-labeled metabolites of interest.     

Identification and fragmentation pathways of caffeine metabolites in urine samples via liquid chromatography with positive electrospray ionization coupled to a hybrid quadrupole linear ion trap (LTQ) and Fourier transform ion cyclotron resonance mass spectrometry and tandem mass spectrometry

Liquid chromatography (LC) with positive ion electrospray ionization (ESI+) coupled to a hybrid quadrupole linear ion trap (LTQ) and Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS) was employed for the simultaneous determination of caffeine and its metabolites in human urine within a single chromatographic run. LC/ESI‐FTICRMS led to the unambiguous determination of the molecular masses of the studied compounds without interference from other biomolecules. A systematic and comprehensive study of the mass spectral behaviour of caffeine and its fourteen metabolites by tandem mass spectrometry (MS/MS) was performed, through in‐source ion trap collision‐induced dissociation (CID) of the protonated molecules, [M+H]⁺. A retro‐Diels‐Alder (RDA) process along with ring‐contraction reactions were the major fragmentation pathways observed during CID. The base peak of xanthine precursors originates from the loss of methyl isocyanate (CH₃NCO, 57 Da) or isocyanic acid (HNCO, 43 Da), which in turn lose a CO unit. Also uric acid derivatives shared a RDA rearrangement as a common fragmentation process and a successive loss of CO₂ or CO. The uracil derivatives showed a loss of a ketene unit (CH₂CO, 42 Da) from the protonated molecule along with the loss of H₂O or CO. To assess the potential of the present method three established metabolite ratios to measure P450 CYP1A2, N‐acetyltransferase and xanthine oxidase activities were evaluated by a number of identified metabolites from healthy human urine samples after caffeine intake. Copyright © 2009 John Wiley & Sons, Ltd.     

Improved detection of reactive metabolites with a bromine‐containing glutathione analog using mass defect and isotope pattern matching

Drug bioactivation leading to the formation of reactive species capable of covalent binding to proteins represents an important cause of drug‐induced toxicity. Reactive metabolite detection using in vitro microsomal incubations is a crucial step in assessing potential toxicity of pharmaceutical compounds. The most common method for screening the formation of these unstable, electrophilic species is by trapping them with glutathione (GSH) followed by liquid chromatography/mass spectrometry (LC/MS) analysis. The present work describes the use of a brominated analog of glutathione, N‐(2‐bromocarbobenzyloxy)‐GSH (GSH‐Br), for the in vitro screening of reactive metabolites by LC/MS. This novel trapping agent was tested with four drug compounds known to form reactive metabolites, acetaminophen, fipexide, trimethoprim and clozapine. In vitro rat microsomal incubations were performed with GSH and GSH‐Br for each drug with subsequent analysis by liquid chromatography/high‐resolution mass spectrometry on an electrospray time‐of‐flight (ESI‐TOF) instrument. A generic LC/MS method was used for data acquisition, followed by drug‐specific processing of accurate mass data based on mass defect filtering and isotope pattern matching. GSH and GSH‐Br incubations were compared to control samples using differential analysis (Mass Profiler) software to identify adducts formed via the formation of reactive metabolites. In all four cases, GSH‐Br yielded improved results, with a decreased false positive rate, increased sensitivity and new adducts being identified in contrast to GSH alone. The combination of using this novel trapping agent with powerful processing routines for filtering accurate mass data and differential analysis represents a very reliable method for the identification of reactive metabolites formed in microsomal incubations. Copyright © 2010 John Wiley & Sons, Ltd.     

Use of stable isotope labeled probes to facilitate liquid chromatography/mass spectrometry based high‐throughput screening of time‐dependent CYP inhibitors

Inhibition curve shift is a commonly used approach for screening of time‐dependent CYP inhibitors which requires parallel paired incubations to obtain two inhibition curves for comparison. For the control incubation, a test compound is co‐incubated with a probe substrate in human liver microsomes (HLM) fortified with NADPH; for the time‐dependent incubation (TDI), the test compound is pre‐incubated with NADPH‐fortified HLM followed by a secondary incubation with a probe substrate. For both incubations, enzyme activity is measured respectively by liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis of the CYP‐specific metabolite, and a TDI inhibitor can be readily identified by inhibition curve shifting as a result of CYP inactivation by the test compound during the pre‐incubation. In the present study, we describe an alternative approach to facilitate TDI screening in which stable isotope labeled CYP‐specific probes are used for the TDI, and non‐labeled substrates are included in the control incubation. Because CYP‐specific metabolites produced in the TDI are stable isotope labeled, two sets of incubation samples can be combined and then simultaneously analyzed by LC/MS/MS in the same batch run to reduce the run time. This new method has been extensively validated using both a number of known competitive and TDI inhibitors specific to five most common CYPs such as 1A2, 2C9, 2C19, 2D6, and 3A4. The assay is performed in a 96‐well format and can be fully automated. Compared to the traditional method, this approach in combination with sample pooling and a short LC/MS/MS gradient significantly enhances the throughput of TDI screening and thus can be easily implemented in drug discovery to evaluate a large number of compounds without adding additional resource. Copyright © 2010 John Wiley & Sons, Ltd.     

Rapid determination of six metabolites from multiple cytochrome P450 probe substrates in human liver microsome by liquid chromatography/mass spectrometry: application to high-throughput inhibition screening of terpenoids

A rapid liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for the determination of six cytochrome P450 (CYP) probe substrate metabolites including paracetamol (PAR) for CYP1A2, 4-hydroxytolbutamide (OHTOL) for CYP2C9, 5-hydroxyomeprazole (OHOMe) for CYP2C19, dextrorphan (DEXM) for CYP2D6, 6-hydroxychlorzoxazone (OHCHL) for CYP2E1 and dehydronifedipine (DNIF) for CYP3A4. The triple-quadrupole mass spectrometer was operated in both positive and negative modes, and selective reaction monitoring was used for quantification. The method was validated over the concentration ranges (0.075/0.04/0.05/0.02/0.1/0.0625 microM to 4.8/2.56/3.2/1.28/6.4/4.0 microM) for PAR/OHTOL/OHOME/DEXP/OHCHL/DNIF analytes with acceptable accuracy and precision. The inhibitory effect on the six CYP enzymes has been verified with their known specific inhibitors. This high-throughput inhibition screening approach has been successfully applied to study the inhibitory effects of 18 terpenoids on CYP enzymes. Among them, tanshinone IIA and cryptotanshinone are found to be potent inhibitors to CYP1A2, while artemisinin is a marginal inhibitor to CYP1A2 and glycyrrhetic acid is a weak inhibitor to CYP2C9.