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.     

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.     

A generic method to detect electrophilic intermediates using isotopic pattern triggered data-dependent high-resolution accurate mass spectrometry

A need still exists for a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method that can detect broad classes of glutathione (GSH) conjugates and provide characterization of their structures. We now describe the development of a method that multiplexes high-resolution accurate mass analysis with isotope pattern triggered data-dependent product ion scans, for simultaneous detection and structural elucidation of GSH conjugates within a single analysis using a LTQ/Orbitrap. This method was initially developed to detect GSH conjugates generated from incubating 10 microM test compound with pooled human liver microsomes fortified with NADPH-regenerating system and a 2:1 ratio of 5 mM glutathione and [(13)C(2) (15)N-Gly]glutathione. The GSH conjugates were detected by isotope search of mass defect filtered and control subtracted full scan accurate MS data using MetWorks software. This was followed by elucidation of reactive intermediate structures using chemical formulae for both protonated molecules and their product ions from accurate masses in a single analysis. The mass accuracies measured for the precursor and product ions by the Orbitrap were <2 ppm in external mass calibration mode. Successful detection and characterization of GSH conjugates of acetaminophen, tienilic acid, clozapine, ticlopidine and mifepristone validated this method. In each case, the detected GSH conjugates were within the top five hits by isotope search. This method also has a broader detection capability since it is independent of the collision-induced dissociation behavior of the GSH conjugates. Furthermore, this method is amenable to a broad class of reactive intermediate trapping agents as exemplified by the simultaneous detection and structural elucidation of the cyano-N-methylene iminium ion conjugates of verapamil and its O-desmethyl metabolites, which we report for the first time. In addition to the chemically tagged reactive intermediates, this method also provides information on stable metabolites from the full scan accurate MS data.     

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.     

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.     

An integrated method for metabolite detection and identification using a linear ion trap/Orbitrap mass spectrometer and multiple data processing techniques: application to indinavir metabolite detection

A new strategy using a hybrid linear ion trap/Orbitrap mass spectrometer and multiple post-acquisition data mining techniques was evaluated and applied to the detection and characterization of in vitro metabolites of indinavir. Accurate-mass, full-scan MS and MS/MS data sets were acquired with a generic data-dependent method and processed with extracted-ion chromatography (EIC), mass-defect filter (MDF), product-ion filter (PIF), and neutral-loss filter (NLF) techniques. The high-resolution EIC process was shown to be highly effective in the detection of common metabolites with predicted molecular weights. The MDF process, which searched for metabolites based on the similarity of mass defects of metabolites to those of indinavir and its core substructures, was able to find uncommon metabolites not detected by the EIC processing. The high-resolution PIF and NLF processes selectively detected metabolites that underwent fragmentation pathways similar to those of indinavir or its known metabolites. As a result, a total of 15 metabolites including two new indinavir metabolites were detected and characterized in a rat liver S9 incubation sample. Overall, these data mining techniques, which employed distinct metabolite search mechanisms, were complementary and effective in detecting both common and uncommon metabolites. In summary, the results demonstrated that this analytical strategy enables the high-throughput acquisition of accurate-mass LC/MS data sets, comprehensive search of a variety of metabolites through the post-acquisition processes, and effective structural characterization based on elemental compositions of metabolite molecules and their product ions.     

Detection and characterization of ticlopidine conjugates in rat bile using high‐resolution mass spectrometry: applications of various data acquisition and processing tools

Ticlopidine, an antiplatelet drug, undergoes extensive oxidative metabolism to form S‐oxide, N‐oxide, hydroxylated and dealkylated metabolites. However, metabolism of ticlopidine via conjugation has not been thoroughly investigated. In this study, multiple data acquisition and processing tools were applied to the detection and characterization of ticlopidine conjugates in rat bile. Accurate full‐scan mass spectrometry (MS) and collision‐induced dissociation (CID) MS/MS data sets were recorded using isotope pattern‐dependent acquisition on an LTQ/Orbitrap system. In addition, mass spectral data from online H/D exchanging and high collision energy dissociation (HCD) were recorded. Data processes were carried out using extracted ion chromatography (EIC), mass defect filter (MDF) and isotope pattern filter (IPF). The total ion chromatogram displayed a few major conjugated metabolites and many endogenous components. Profiles from EIC and IPF processes exhibited multiple conjugates with no or minimal false positives. However, ticlopidine conjugates that were not predictable or lost a chorine atom were not found by EIC or IPF, respectively. MDF was able to detect almost all of ticlopidine conjugates although it led to a few more false positives. In addition to CID spectra, data from HCD, H/D exchanging experiments and isotope pattern simulation facilitated structural characterization of unknown conjugates. Consequently, 20 significant ticlopidine conjugates, including glucuronide, glutathione, cysteinylglycine, cysteine and N‐acetylcysteine conjugates, were identified in rat bile, a majority of which are associated with bioactivation and not previously reported. This study demonstrates the utility and limitation of various high‐resolution MS‐based data acquisition and processing techniques in detection and characterization of conjugated metabolites. Copyright © 2013 John Wiley & Sons, Ltd.     

Characterization of glutathione conjugates of chloroacetanilide pesticides using ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry and liquid chromatography/ion trap mass spectrometry

Glutathione S-transferases (GSTs) isolated from maize were used to catalyze the conjugation of glutathione (GSH) with chloroacetanilide herbicides, producing stable conjugates that were structurally characterized using ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC/QqToF-MS) and liquid chromatography/ion trap mass spectrometry (LC/IT-MS). Enzyme-mediated dechlorination of alachlor, metolachlor, and propachlor resulted during GSH conjugation as revealed by the mass spectra of the conjugates, which was confirmed by the loss of the chlorine isotopic signature and from high accurate mass measurements. Several fragmentation patterns in the mass spectra of the chloroacetanilide-GSH conjugates can be used to verify the identities of the enzyme reaction products, such as characteristic ions corresponding to the neutral loss of glutamic acid residue (129 Da) and water (18 Da) observed in the product ion spectrum. For the first time, data are presented showing detection of chloroacetanilides that are conjugated with two GSH molecules, in addition to the known single GSH conjugates.     

Feasibility of different mass spectrometric techniques and programs for automated metabolite profiling of tramadol in human urine

The purpose of the study was to determine the advantages of different mass spectrometric instruments and commercially available metabolite identification programs for metabolite profiling. Metabolism of tramadol hydrochloride and the excretion of it and its metabolites into human urine were used as a test case because the metabolism of tramadol is extensive and well known. Accurate mass measurements were carried out with a quadrupole time-of-flight mass spectrometer (Q-TOF) equipped with a LockSpray dual-electrospray ionization source. A triple quadrupole mass spectrometer (QqQ) was applied for full scan, product ion scan, precursor ion scan and neutral loss scan measurements and an ion trap instrument for full scan and product ion measurements. The performance of two metabolite identification programs was tested. The results showed that metabolite programs are time-saving tools but not yet capable of fully automated metabolite profiling. Detection of non-expected metabolites, especially at low concentrations in a complex matrix, is still almost impossible. With low-resolution instruments urine samples proved to be challenging even in a search for expected metabolites. Many false-positive hits were obtained with the automated searching and manual evaluation of the resulting data was required. False positives were avoided by using the higher mass accuracy Q-TOF. Automated programs were useful for constructing product ion methods, but the time-consuming interpretation of mass spectra was done manually. High-quality MS/MS spectra acquired on the QqQ instrument were used for confirmation of the tramadol metabolites. Although the ion trap instrument is of undisputable benefit in MS(n), the low mass cutoff of the ion trap made the identification of tramadol metabolites difficult. Some previously unreported metabolites of tramadol were found in the tramadol urine sample, and their identification was based solely on LC/MS and LC/MS/MS measurements.     

Rapid detection and characterization of reactive drug metabolites in vitro using several isotope‐labeled trapping agents and ultra‐performance liquid chromatography/time‐of‐flight mass spectrometry

Reactive metabolites are believed to be one of the main reasons for unexpected drug‐induced toxicity issues, by forming covalent adducts with cell proteins or DNA. Due to their high reactivity and short lifespan they are not directly detected by traditional analytical methods, but are most traditionally analyzed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) after chemical trapping with nucleophilic agents such as glutathione. Here, a simple but very efficient assay was built up for screening reactive drug metabolites, utilizing stable isotope labeled glutathione, potassium cyanide and semicarbazide as trapping agents and highly sensitive ultra‐performance liquid chromatography/time‐of‐flight mass spectrometry (UPLC/TOFMS) as an analytical tool. A group of twelve structurally different compounds was used as a test set, and a large number of trapped metabolites were detected for most of them, including many conjugates not reported previously. Glutathione‐trapped metabolites were detected for nine of the twelve test compounds, whereas cyanide‐trapped metabolites were found for eight and semicarbazide‐trapped for three test compounds. The high mass accuracy of TOFMS provided unambiguous identification of change in molecular formula by formation of a reactive metabolite. In addition, use of a mass defect filter was found to be a usable tool when mining the trapped conjugates from the acquired data. The approach was shown to provide superior detection sensitivity in comparison to traditional methods based on neutral loss or precursor ion scanning with a triple quadrupole mass spectrometer, and clearly more efficient detection and characterization of reactive drug metabolites with a simpler test setup. Copyright © 2009 John Wiley & Sons, Ltd.     

A ferrocene-based reagent for the conjugation and quantification of reactive metabolites

In the present study, a method for the analysis of reactive metabolites via liquid chromatography (LC) with inductively coupled plasma–mass spectrometry (MS) was developed. A ferrocenyl-modified glutathione (GSH) reagent, consisting of GSH and succinimidyl-3-ferrocenylpropionate, was synthesized. Derivatization of the tripeptide was performed at the N-terminus, leaving the nucleophilic thiol group vacant for the attack of electrophilic compounds. The potential of ferrocenylpropionate (FP)-GSH as a trapping agent for reactive metabolites was investigated using an electrochemical flow-through cell for metabolism simulation coupled online to a LC system with electrospray ionization mass spectrometric detection. The pharmaceuticals amodiaquine, an antimalarial agent, and clozapine, an antipsychotic compound, served as model substances. By proving the successful adduct formation between the reactive metabolite and ferrocene-labeled GSH, it could be shown that FP-GSH is an effective trapping agent which eases routine reversed-phase LC analyses. In contrast to GSH, which is usually used for the conjugation of reactive metabolites and where the resulting adducts often show no or only very little retention, FP-GSH facilitates the detection of the corresponding metabolite adducts due to higher retention times.     

An integrated method for degradation products detection and characterization using hybrid ion trap/time-of-flight mass spectrometry and data processing techniques: Application to study of the degradation products of danofloxacin under stressed conditions

A new strategy using hybrid ion trap/time-of-flight mass spectrometry coupled with high-performance liquid chromatography and post-acquisition data mining techniques was developed and applied to the detection and characterization of degradation products of danofloxacin. The degradation products formed under different forced conditions were separated using an ODS-C18 column with gradient elution. Accurate full-scan MS data were acquired in the first run and processed with the combination of extracted ion chromatograms and LC-UV chromatograms. These processes were able to find accurate molecular masses of possible degradation products. Then, the accurate MS/MS data acquired through data-dependent analysis mode in another run facilitated the structural elucidations of degradation products. As a result, a total of 11 degradation products of danofloxacin were detected and characterized using the developed method. Overall, this analytical strategy enables the acquisition of accurate-mass LC/MS data, search of a variety of degradation products through the post-acquisition processes, and effective structural characterization based on elemental compositions of degradation product molecules and their product ions. The ability to measure degradation products via tandem mass spectrometry coupled with accurate mass measurement, all in only two experimental runs, is one of the most attractive features of this methodology. The results demonstrate that use of the LC/MS-IT-TOF approach appears to be rapid, efficient and reliable in structural characterization of drug degradation products.     

Direct Flavonoid-Focused Chemical Comparison among Three Epimedium Plants by Online Liquid Extraction–High Performance Liquid Chromatography–Tandem Mass Spectrometry

It is usually a tedious task to profile the chemical composition of a given herbal medicine (HM) using high performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) due to the time-consuming sample preparation and laborious post-acquisition data processing procedures. Even worse, some labile compounds may face degradation risks when exposed to organic solvents for a relatively long period. As one of the most popular HMs, the promising therapeutic benefits of Epimedii Herba (Chinese name: Yinyanghuo) are well defined; however, the chemical profile, and in particular those flavonoids that have been claimed to be responsible for the efficacy, remains largely unknown. Attempts are devoted here to achieve direct LC–MS measurement and efficient post-acquisition data processing, and chemome comparison among three original sources of Epimedii Herba, such as Epimedium sagittatum (Esa), E. pubescens (Epu), and E. koreanum (Eko) was employed to illustrate the strategy utility. A home-made online liquid extraction (OLE) module was introduced at the front of the analytical column to comprehensively transfer the compounds from raw materials onto the LC–MS instrument. A mass defect filtering approach was programmed to efficiently mine the massive LC–MS dataset after which a miniature database was built involving all chemical information of flavonoids from the genus Epimedium to draw a pentagonal frame to rapidly capture potential quasi-molecular ions (mainly [M–H]⁻). A total of 99 flavonoids (66 in Esa, 84 in Eko, and 66 in Epu) were captured, and structurally annotated by summarizing the mass fragmentation pathways from the mass spectrometric data of authentic compounds and an in-house data library as well. Noteworthily, neutral loss of 144 Da was firstly assigned to the neutral cleavage of rhamnosyl residues. Significant species-differences didn’t occur among their chemical patterns. The current study proposed a robust strategy enabling rapid chemical profiling of, but not limited to, HMs.     

The application of high-resolution mass spectrometry-based data-mining tools in tandem to metabolite profiling of a triple drug combination in humans

Patients are usually exposed to multiple drugs, and metabolite profiling of each drug in complex biological matrices is a big challenge. This study presented a new application of an improved high resolution mass spectrometry (HRMS)-based data-mining tools in tandem to fast and comprehensive metabolite identification of combination drugs in human. The model drug combination was metronidazole-pantoprazole-clarithromycin (MET-PAN-CLAR), which is widely used in clinic to treat ulcers caused by Helicobacter pylori. First, mass defect filter (MDF), as a targeted data processing tool, was able to recover all relevant metabolites of MET-PAN-CLAR in human plasma and urine from the full-scan MS dataset when appropriate MDF templates for each drug were defined. Second, the accurate mass-based background subtraction (BS), as an untargeted data-mining tool, worked effectively except for several trace metabolites, which were buried in the remaining background signals. Third, an integrated strategy, i.e., untargeted BS followed by improved MDF, was effective for metabolite identification of MET-PAN-CLAR. Most metabolites except for trace ones were found in the first step of BS-processed datasets, and the results led to the setup of appropriate metabolite MDF template for the subsequent MDF data processing. Trace metabolites were further recovered by MDF, which used both common MDF templates and the novel metabolite-based MDF templates. As a result, a total of 44 metabolites or related components were found for MET-PAN-CLAR in human plasma and urine using the integrated strategy. New metabolic pathways such as N-glucuronidation of PAN and dehydrogenation of CLAR were found. This study demonstrated that the combination of accurate mass-based multiple data-mining techniques in tandem, i.e., untargeted background subtraction followed by targeted mass defect filtering, can be a valuable tool for rapid metabolite profiling of combination drugs in vivo.     

'All-in-one' analysis for metabolite identification using liquid chromatography/hybrid quadrupole time-of-flight mass spectrometry with collision energy switching

The removal of bottlenecks in discovery stage metabolite identification studies is an ongoing challenge for the pharmaceutical industry. We describe the use of an 'All-in-One' approach to metabolite characterization that leverages the fast scanning and high mass accuracy of hybrid quadrupole time-of-flight mass spectrometry (QqToFMS) instruments. Full-scan MS and MS/MS data is acquired using collision energy switching without the preselection, either manually or in a data-dependent manner, of precursor ions. The acquisition of 'clean' MS/MS data is assisted by the use of ultrahigh-performance chromatography. Data acquired using this method can then be mined post-acquisition in a number of ways. These include using narrow window extracted ion chromatograms (nwXICs) for expected biotransformations, XICs for the product ions of the parent compound and/or expected modification of these product ions, and neutral loss chromatograms. This approach has the potential to be truly comprehensive for the determination of in vitro biotransformations in a drug discovery environment.     

A high-throughput liquid chromatography/tandem mass spectrometry method for screening glutathione conjugates using exact mass neutral loss acquisition

Chemically reactive metabolites may cause hepatotoxicity and as a result liver failure or other adverse side reactions. Therefore, this is a vital topic of interest because early reactive metabolite screening may prevent compound failure at a later stage. In order to address this issue, a screening assay has been developed to detect the formation of reactive metabolites by using glutathione as a trapping reagent, which will allow us to search for phase I metabolites and also glutathiones during in vitro metabolite screening using liquid chromatography/tandem mass spectrometry (LC/MS/MS) with exact mass. Glutathione conjugations when fragmented by the mass spectrometer give a common loss corresponding to the pyroglutamic acid moiety, which can be monitored. Until recently, this work has been carried out with triple quadrupole technology using nominal mass. The advantage of the hybrid quadrupole time-of-flight mass spectrometer is the selectivity and sensitivity that can be achieved. Exact neutral loss detection is achieved via sequential low- and high-energy MS acquisitions. After detection of the loss of the pyroglutamic acid moiety, using a window of +/-20 mDa on the high-energy scan, MS/MS is carried out on the parent mass of interest to confirm the common neutral loss.     

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.     

Urinary exposure marker discovery for toxicants using ultra-high pressure liquid chromatography coupled with Orbitrap high resolution mass spectrometry and three untargeted metabolomics approaches

Human biomonitoring is the assessment of actual internal contamination of chemicals by measuring exposure markers, chemicals or their metabolites, in human urine, blood, serum, and other body fluids. However, the metabolism of chemicals within an organism is extremely complex. Therefore, the identification of metabolites is often difficult and laborious. Several untargeted metabolomics methods have been developed to perform objective searching/filtering of accurate-mass-based LC-MS data to facilitate metabolite identification. In this study, three metabolomics data processing approaches were used for chemical exposure marker discovery in urine with an LTQ-Orbitrap high-resolution mass spectrometry (HRMS) dataset; di-isononyl phthalate (DINP) was used as an example. The data processing techniques included the SMAIT, mass defect filtering (MDF), and XCMS Online. Sixteen, 83, and 139 probable DINP metabolite signals were obtained using the SMAIT, MDF, and XCMS procedures, respectively. Fourteen probable metabolite signals mined simultaneously by the three metabolomics approaches were confirmed as DINP metabolites by structural information provided by LC-MS/MS. Among them, 13 probable metabolite signals were validated as exposure-related markers in a rat model. Six (m/z 319.155, 361.127, 373.126, 389.157, 437.112 and 443.130) of the 13 exposure-related DINP metabolite signals have not previously been reported in the literature. Our data indicate that SMAIT provided an efficient method to discover effectively and systematically urinary exposure markers of toxicant. The DINP metabolism information can provide valuable information for further investigations of DINP toxicity, toxicokinetics, exposure assessment, and human health effects.     

Identification of glucuronide conjugates of ketobemidone and its phase I metabolites in human urine utilizing accurate mass and tandem time-of-flight mass spectrometry

The glucuronide conjugates of ketobemidone, norketobemidone and hydroxymethoxyketobemidone were identified in human urine post-intravenous administration of Ketogan Novum. The human urine was extracted on a mixed-mode solid-phase micro-column before analysis with liquid chromatography/electrospray ionization time-of-flight mass spectrometry (LC/ESI-TOF-MS) and tandem MS (MS/MS). Accurate mass and collision-induced dissociation product ion spectra were used for identification of the glucuronide conjugates. Two different TOF mass spectrometers were used and the accurate mass measurements were performed on three separate days with each instrument. The accuracy of the mass measurements was better than 2.1 ppm for two out of three conjugates and the inter-day relative standard deviation was within +/-0.00049%. The MS/MS fragmentation patterns of the conjugates were in accordance with those of the synthetic aglycones and included peaks originating from the [M + H](+) ion of the respective aglycone.