Comparison of a two-dimensional liquid chromatography/mass spectrometry approach with a chip-based nanoelectrospray device for structural elucidation of metabolites in a human ADME study using a quadrupole time-of-flight mass spectrometer

The study of the metabolic fate of drugs is essential for the safety assessment of new compounds in the drug development process. However, the characterization and structural elucidation of metabolites from in vivo experiments is still a very challenging task. In this paper, we compare a two-dimensional liquid chromatography/mass spectrometry (LC/MS) approach using either a capillary LC/MS system or the recently introduced chip-based nanoelectrospray/MS system (Nanomate) as the second dimension for structural elucidation of metabolites by MS. More than 30 radioactive fractions of a chromatographic separation from a human urine sample were analyzed and 54 metabolites could be identified. The long persisting and stable nanoelectrospray enabled the search for unknown metabolites by precursor-ion scanning experiments followed by product-ion scanning experiments of potential metabolites using a quadrupole time-of-flight (qTOF) mass spectrometer. The number of fragments produced by nanoelectrospray with product-ion scanning was significantly higher compared to LC/MS experiments with in-source fragmentation. Therefore, the assignment of possible modifications in metabolites to certain moieties of the drug could be investigated with higher accuracy. The capillary LC/MS system for the second dimension was more sensitive in the case of low abundant metabolites. These metabolites could not be detected by direct nanoelectrospray infusion, which limits the application of the Nanomate for trace metabolites.     

Metabolite identification in rat brain microdialysates by direct infusion nanoelectrospray ionization after desalting on a ZipTip and LTQ/Orbitrap mass spectrometry

Analyzing brain microdialysate samples by mass spectrometry is challenging due to the high salt content of the artificial cerebral spinal fluid (aCSF), low analyte concentrations and small sample volumes collected. A drug and its major metabolites can be examined in brain microdialysates by targeted approaches such as selected reaction monitoring (SRM) which provides selectivity and high sensitivity. However, this approach is not well suited for metabolite profiling in the brain which aims to determine biotransformation pathways. Identifying minor metabolites, or metabolites that arise from brain metabolism, remains a challenge and, for a drug in early discovery, identification of metabolites present in the brain can provide useful information for understanding the pharmacological activity and potential toxicological liabilities of the drug. A method is described here for rapid metabolite profiling in brain microdialysates that involves sample clean‐up using C18 ZipTips to remove salts followed by direct infusion nanoelectrospray with an LTQ/Orbitrap mass spectrometer using real‐time internal recalibration. Full scan mass spectra acquired at high resolving power (100 K at m/z 400) were examined manually and with mass defect filtering. Metabolite identification was aided by sub‐parts‐per‐million mass accuracy and structural characterization was accomplished by tandem mass spectrometry (MS/MS) experiments in the Orbitrap or LTQ depending on the abundance of the metabolite. Using this approach, brain microdialysate samples from rats dosed with one of four CNS drugs (imipramine, reboxetine, citalopram or trazodone) were examined for metabolites. For each drug investigated, metabolites, some of which not previously reported in rat brain, were identified and characterized. Copyright © 2009 John Wiley & Sons, Ltd.     

Evaluation of relative LC/MS response of metabolites to parent drug in LC/nanospray ionization mass spectrometry: potential implications in MIST assessment

There is an increasing demand for quantitative data on metabolite exposure triggered by regulatory guidances. This contribution describes the accuracy of nanoelectrospray ionization mass spectrometry response of drug compounds and their metabolites from biological matrices compared with radiometric quantification. This is a comprehensive investigation of a set of real-life pharmaceutical compounds in relevant matrices such as urine, bile, feces and plasma. The data suggest that nanoelectrospray mass spectrometry can be used for semi-quantitation of metabolites in the absence of reference standards. Therefore, this approach is suitable to screen out non-relevant metabolites early in development as long as an adequate analytical error margin is applied thus balancing risks and resources.     

Use of liquid chromatography/time-of-flight mass spectrometry and multivariate statistical analysis shows promise for the detection of drug metabolites in biological fluids

The process of metabolite identification is essential to the drug discovery and development process; this is usually achieved by liquid chromatography/tandem mass spectrometry (LC/MS/MS) or a combination of liquid chromatography/mass spectrometry (LC/MS) and nuclear magnetic resonance (NMR) spectroscopy. Metabolite identification is, however, a time-consuming process requiring an experienced skilled scientist. Multivariate statistical analysis has been used in the field of metabonomics to elucidate differences in endogenous biological profiling due to a toxic effect or a disease state. In this paper we show how a combination of liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS) and multivariate statistical analysis can be used to detect drug metabolites in a biological fluid with no prior knowledge of the compound administered.     

MSE with mass defect filtering for in vitro and in vivo metabolite identification

Metabolite identification studies involve the detection and structural characterization of the biotransformation products of drug candidates. These experiments are necessary throughout the drug discovery and development process. The use of high-resolution chromatography and high-resolution mass spectrometry together with data processing using mass defect filtering is described for in vitro and in vivo metabolite identification studies. Data collection was done using UPLC coupled with an orthogonal hybrid quadrupole time-of-flight mass spectrometer. This experimental approach enabled the use of MS(E) data collection (where E represents collision energy) which has previously been shown to be a powerful approach for metabolite identification studies. Post-acquisition processing with a prototype mass defect filtering program was used to eliminate endogenous interferences in the study samples, greatly enhancing the discovery of metabolites. The ease of this approach is illustrated by results showing the detection and structural characterization of metabolites in plasma from a preclinical rat pharmacokinetic study.     

Liquid chromatography/mass spectrometry‐based plasma metabolic profiling study of escitalopram in subjects with major depressive disorder

Liquid chromatography‐mass spectrometry (LC‐MS) method revealed the plasma metabolite profiles in major depressive disorder patients treated with escitalopram (ECTP) (n = 7). Depression severity was assessed according to the 17‐item Hamilton Depression Rating Scale. Metabolic profiles were derived from major depressive disorder subject blood samples collected after ECTP treatment. Blood plasma was separated and processed in order to effectively extract metabolites, which were then analyzed using LC‐MS. We identified 19 metabolites and elucidated their structures using LC‐tandem MS (LC‐MS/MS) combined with elemental compositions derived from accurate mass measurements. We further used online H/D exchange experiments to verify the structural elucidations of each metabolite. Identifying molecular metabolites may provide critical insights into the pharmacological and clinical effects of ECTP treatment and may also provide useful information informing the development of new antidepressant treatments. These detailed plasma metabolite analyses may also be used to identify optimal dose concentrations in psychopharmacotherapeutic treatment through drug monitoring, as well as forming the basis for response predictions in depressed subjects.     

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.     

Characterization of metabolites of Celecoxib in rabbits by liquid chromatography/tandem mass spectrometry

The metabolism of the anti-inflammatory drug Celecoxib in rabbits was characterized using liquid chromatography (LC)/tandem mass spectrometry (MS/MS) with precursor ion and constant neutral loss scans followed by product ion scans. After separation by on-line liquid chromatography, the crude urine samples and plasma and fecal extracts were analyzed with turbo-ionspray ionization in negative ion mode using a precursor ion scan of m/z 69 (CF(3)) and a neutral loss scan of 176 (dehydroglucuronic acid). The subsequent product ion scans of the [M - H] ions of these metabolites yielded the identification of three phase I and four phase II metabolites. The phase I metabolites had hydroxylations at the methyl group or on the phenyl ring of Celecoxib, and the subsequent oxidation product of the hydroxymethyl metabolite formed the carboxylic acid metabolite. The phase II metabolites included four positional isomers of acyl glucuronide conjugates of the carboxylic acid metabolite. These positional isomers were caused by the alkaline pH of the rabbit urine and were not found in rabbit plasma. The chemical structures of the metabolites were characterized by interpretation of their product ion spectra and comparison of their LC retention times and the product ion spectra with those of the authentic synthesized standards.     

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.     

An algorithm for thorough background subtraction from high-resolution LC/MS data: application to the detection of troglitazone metabolites in rat plasma, bile, and urine

Interferences from biological matrices remain a major challenge to the in vivo detection of drug metabolites. For the last few decades, predicted metabolite masses and fragmentation patterns have been employed to aid in the detection of drug metabolites in liquid chromatography/mass spectrometry (LC/MS) data. Here we report the application of an accurate mass-based background-subtraction approach for comprehensive detection of metabolites formed in vivo using troglitazone as an example. A novel algorithm was applied to check all ions in the spectra of control scans within a specified time window around an analyte scan for potential background subtraction from that analyte spectrum. In this way, chromatographic fluctuations between control and analyte samples were dealt with, and background and matrix-related signals could be effectively subtracted from the data of the analyte sample. Using this algorithm with a +/- 1.0 min control scan time window, a +/- 10 ppm mass error tolerance, and respective predose samples as controls, troglitazone metabolites were reliably identified in rat plasma and bile samples. Identified metabolites included those reported in the literature as well as some that had not previously been reported, including a novel sulfate conjugate in bile. In combination with mass defect filtering, this algorithm also allowed for identification of troglitazone metabolites in rat urine samples. With a generic data acquisition method and a simple algorithm that requires no presumptions of metabolite masses or fragmentation patterns, this high-resolution LC/MS-based background-subtraction approach provides an efficient alternative for comprehensive metabolite identification in complex biological matrices. Copyright (c) 2008 John Wiley & Sons, Ltd.     

Isobaric metabolite interferences and the requirement for close examination of raw data in addition to stringent chromatographic separations in liquid chromatography/tandem mass spectrometric analysis of drugs in biological matrix

In addition to matrix effects, common interferences observed in liquid chromatography/tandem mass spectrometry (LC/MS/MS) analyses can be caused by the response of drug-related metabolites to the multiple reaction monitoring (MRM) channel of a given drug, as a result of in-source reactions or decomposition of either phase I or II metabolites. However, it has been largely ignored that, for some drugs, metabolism can lead to the formation of isobaric or isomeric metabolites that exhibit the same MRM transitions as parent drugs. The present study describes two examples demonstrating that interference caused by isobaric or isomeric metabolites is a practical issue in analyzing biological samples by LC/MS/MS. In the first case, two sequential metabolic reactions, demethylation followed by oxidation of a primary alcohol moiety to a carboxylic acid, produced an isobaric metabolite that exhibits a MRM transition identical to the parent drug. Because the drug compound was rapidly metabolized in rats and completely disappeared in plasma samples, the isobaric metabolite appeared as a single peak in the total ion current (TIC) trace and could easily be quantified as the drug since it was eluted at a retention time very close to that of the drug in a 12-min LC run. In the second example, metabolism via the ring-opening of a substituted isoxazole moiety led to the formation of an isomeric product that showed an almost identical collision-induced dissociation (CID) MS spectrum as the original drug. Because two components were co-eluted, the isomeric product could be mistakenly quantified and reported by data processing software as the parent drug if the TIC trace was not carefully inspected. Nowadays, all LC/MS data are processed by computer software in a highly automated fashion, and some analysts may spend much less time to visually examine raw TIC traces than they used to do. Two examples described in this article remind us that quality data require both adequate chromatographic separations and close examination of raw data in LC/MS/MS analyses of drugs in biological matrix.     

Metabolite identification of artemether by data-dependent accurate mass spectrometric analysis using an LTQ-Orbitrap hybrid mass spectrometer in combination with the online hydrogen/deuterium exchange technique

Artemether (ARM), the O-methyl ether prodrug of dihydroartemisinin (DHA), is a first-line antimalarial drug used in areas of multi-drug resistance. Artemisinin drugs can be metabolized extensively in vivo and this seems related to their autoinduction pharmacokinetics. In the present study, the metabolite identification of ARM was performed by the generic data-dependent accurate mass spectrometric analysis, using high-resolution (HR) liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) and tandem mass spectrometry (MS/MS) LTQ-Orbitrap hybrid mass spectrometer in conjunction with online hydrogen (H)/deuterium (D) exchange for rapid structural characterization. The LC separation was improved allowing the separation of ARM parent drugs and their metabolites from their diastereomers. A total of 77 phase I metabolites of ARM were identified in rat liver microsomal incubates and rat urine, including dihydroartemisinin and artemisinin. In rat bile, 12 phase II metabolites were found. Accurate mass data were obtained in both full scan and HR-MS/MS mode to support assignments of metabolite structures. Online H/D exchange LC/HR-ESI-MS experiments provided additional evidence in differentiating dihydroxylated deoxy-ARM from mono-hydroxylated ARM. The results showed the main phase I metabolites of artemether are hydroxylated, dehydro, demethylated and deoxy products, and they will undergo subsequent phase II glucuronidation processes. Most metabolites were reported for the first time. This study also demonstrated the effectiveness of high-resolution mass spectrometry in combination with an online H/D exchange LC/HR-MS(n) technique in rapid identification of drug metabolites.     

Identification of metabolites of Danggui Buxue Tang in rat urine by liquid chromatography coupled with electrospray ionization time‐of‐flight mass spectrometry

A method coupling liquid chromatography with electrospray ionization time‐of‐flight mass spectrometry (LC/ESI‐TOF/MS) has been developed for rapid and sensitive analysis of rat urinary metabolite profile of Danggui Buxue Tang (DBT), a well‐known Chinese herbal formula. After oral administration of DBT, urine samples were collected during 0–24 h, and then pretreated by solid‐phase extraction. A total of 68 compounds including 13 parent compounds and 55 metabolites were detected in the drug‐containing urines compared with blank urines. The total analytical time was less than 20 min. Metabolites of DBT were identified using dynamic adjustment of the fragmentor voltage to produce structure‐relevant fragment ions. By using this approach, the mass accuracy of precursor and fragment ions was typically within ±5 ppm of the theoretical values, and enabled the identification of 43 metabolites including 27 isoflavanoid and 16 phthalide metabolites. Our results indicated that glucuronidation and sulfation were the major metabolic pathways of isoflavonoids, while glutathione conjugation, glucuronidation and sulfation were the main metabolic pathways of phthalides. No saponin‐related metabolites were detected. The results of the present study provided important structural information relating to the metabolism of DBT. Furthermore, this work demonstrated the potential of the LC/ESI‐TOF/MS approach for identification of metabolites from Chinese herbal medicines in urine. 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.     

Strategy of using microsome-based metabolite production to facilitate the identification of endogenous metabolites by liquid chromatography mass spectrometry

One of the challenges in metabolomic profiling of complex biological samples is to identify new and unknown compounds. Typically, standards are used to help identify metabolites, yet standards cannot be purchased or readily synthesized for many unknowns. In this work we present a strategy of using human liver microsomes (HLM) to metabolize known endogenous human metabolites (substrates), producing potentially new metabolites that have yet to be documented. The metabolites produced by HLM can be tentatively identified based on the associated substrate structure, known metabolic processes, tandem mass spectrometry (MS/MS) fragmentation patterns and, if necessary, accurate mass measurements. Once identified, these metabolites can be used as references for identification of the same compounds in complex biological samples. As a proof of principle, a total of 9 metabolites have been identified from individual HLM incubations using 5 different substrates. Each metabolite was used as a standard. In the analysis of human urine sample by liquid chromatography MS/MS, four spectral matches were found from the 9 microsome-produced metabolite standards. Two of them have previously been documented as endogenous human metabolites, the third is an isomer of a microsome-metabolite and the fourth compound has not been previously reported and is also an isomer of a microsome-metabolite. This work illustrates the feasibility of using microsome-based metabolism to produce metabolites of endogenous human metabolites that can be used to facilitate the identification of unknowns in biological samples. Future work on improving the performance of this strategy is also discussed.     

Identification of new minor metabolites of penicillin G in human serum by multiple-stage tandem mass spectrometry

Liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) were applied to characterize drug metabolites. Although these two methods have overcome the identification and structural characterization of metabolites analysis, they remain time‐consuming processes. In this study, a novel multiple‐stage tandem mass spectrometric method (MSⁿ) was evaluated for identification and characterization of new minor metabolism profiling of penicillin G, one of the β‐lactam antibiotics, in human serum. Seven minor metabolites including five phase I metabolites and two phase II metabolites of penicillin G were identified by using data‐dependent LC/MSⁿ screening in one chromatographic run. The accuracy masses of seven identified metabolites of penicillin G were also confirmed by mass spectral calibration software (MassWorks?). The proposed data‐dependent LC/MSⁿ method is a powerful tool to provide large amounts of the necessary structural information to characterize minor metabolite in metabolism profiling. Copyright © 2010 John Wiley & Sons, Ltd.     

Mass defect profiles of biological matrices and the general applicability of mass defect filtering for metabolite detection

Recent examples have demonstrated that the high-resolution liquid chromatography/mass spectrometry (LC/MS)-based mass defect filtering (MDF) technique was effective in selectively detecting drug metabolites regardless of their molecular weights or fragmentation patterns. The main objective of the current study was to evaluate the general applicability of MDF for drug metabolite detection in typical biological matrices. Mass defect profiles of commonly used biological matrices including plasma, urine, bile, and feces were obtained using an LTQ FT mass spectrometer and were compared with those of 115 commonly prescribed drugs. The mass defect profiles were presented as two-dimensional Y-X plots with the determined mass defects of components on the y-axis versus the corresponding m/z values on the x-axis. The mass defect profiles of the matrices appeared to be similar for each type of matrix across species, yet marked differences were apparent between matrices of a given species. The mass defect profiles of components in plasma, bile, and feces showed significant separation from most of the 115 drugs. The mass defect profiles of urine did not show such clean separation from that of the 115 drugs. The results suggest that MDF has a broad applicability for selective detection of drug metabolites in plasma, bile and feces although the selectivity for detecting urinary drug metabolites is not as good as in the other matrices. In addition, the mass defect profiles of the biological matrices allow for prediction of the effectiveness of MDF for certain applications, and for designing specific MDF windows for selective detection of drug metabolites.     

The combination of liquid chromatography/tandem mass spectrometry and chip-based infusion for improved screening and characterization of drug metabolites

An approach has been developed for drug metabolism studies of non-radiolabeled compounds using on-line liquid chromatography/tandem mass spectrometry (LC/MS/MS) combined with chip-based infusion following fraction collection. The potential of this approach, which improves the data quality compared with only LC/MS analysis, has been investigated for the analysis of in vitro metabolites of tolcapone and talinolol, two compounds with well-characterized metabolism. The information-dependent LC/MS/MS analysis enables the characterization of the major metabolites while the chip-based infusion is used to obtain good product ion spectra for lower level metabolites, to generate complementary MS information on potential metabolites detected in the LC/MS trace, or to screen for unexpected metabolites. Fractions from the chromatographic analysis are collected in 20 second steps, into a 96-well plate. The fractions of interest can be re-analyzed with chip-based infusion on a variety of mass spectrometers including triple quadrupole linear ion trap (QqLIT or Q TRAP) and QqTOF systems. Acquiring data for several minutes using multi-channel acquisition (MCA), or signal averaging while infusing the fractions at approximately 200 nL/min, permits about a 50 times gain in sensitivity (signal-to-noise) in MS/MS mode. A 5-10 microL sample fraction can be infused for more than 30 min allowing the time to perform various MS experiments such as MS(n), precursor ion or neutral loss scans and accurate mass measurement, all in either positive or negative mode. Through fraction collection and infusion, a significant gain in data quality is obtained along with a time-saving benefit, because the original sample needs neither to be re-analyzed by re-injection nor to be pre-concentrated. Therefore, a novel hydroxylated talinolol metabolite could be characterized with only one injection.     

High-throughput, accurate mass liquid chromatography/tandem mass spectrometry on a quadrupole time-of-flight system as a 'first-line' approach for metabolite identification studies

Throughput for drug metabolite identification studies has been increased significantly by the combined use of accurate mass liquid chromatography/tandem mass spectrometry (LC/MS/MS) data on a quadrupole time-of-flight (QTOF) system and targeted data analysis procedures. Employed in concert, these tools have led to the implementation of a semi-automated high-throughput metabolite identification strategy that has been incorporated successfully into lead optimization efforts in drug discovery. The availability of elemental composition data on precursor and all fragment ions in each spectrum has greatly enhanced confidence in ion structure assignments, while computer-based algorithms for defining sites of biotransformation based upon mass shifts of diagnostic fragment ions have facilitated identification of positions of metabolic transformation in drug candidates. Adoption of this technology as the 'first-line' approach for in vitro metabolite profiling has resulted in the analysis of as many as 21 new chemical entities on one day from diverse structural classes and therapeutic programs.     

A strategy for metabolite identification using triple-quadrupole mass spectrometry with enhanced resolution and accurate mass capability

Using a single platform of a triple-quadrupole mass spectrometer equipped with enhanced resolution and accurate mass capabilities, a strategy for metabolite identification of a drug in a biological matrix has been demonstrated. The strategy is based on first screening for metabolites via neutral loss and precursor ion scan schemes, devised as the result of the product ion spectrum of a matrix-free standard of the drug. The accurate masses of the precursor ions identified via the two scan schemes plus the precursor ions of structurally likely metabolites are then determined by enhanced resolution, accurate mass (AM) selected ion monitoring (SIM). The identities of the metabolites are further established by determining the accurate masses of the product ions via enhanced resolution AM selected reaction monitoring (SRM). The feasibility of the strategy was demonstrated using a liver microsome incubation sample of nefazodone, an antidepressant drug. The neutral loss and precursor ion screening runs were able to identify most of the metabolites of nefazodone. The subsequent SIM and SRM experiments gave mass accuracy of better than +/-0.003 u for the masses of the precursor and product ions of nefazodone and all the metabolites. The ability to perform metabolite screening by using the scan features followed by accurate mass determinations on the same instrument is an attractive feature of using a triple-quadrupole mass spectrometer with enhanced resolution and accurate mass capability.