Metabolite profile of sibutramine in human urine: a liquid chromatography-electrospray ionization mass spectrometric study

We present a detailed experimental approach to detection and subsequent structural characterization of unknown metabolites of sibutramine, using liquid chromatography-mass spectrometric techniques. The full-, precursor ion, and constant neutral loss scan modes of a triple quadrupole mass spectrometer were used for screening sibutramine metabolites in human urine. The structural assessment of unknown metabolites was based on MSn ion trap mass spectrometric analysis and comparison of MSn spectra between the standards and compounds detected. Two phase-I (M1 and M2) and eight phase-II (M3-M6) metabolites of sibutramine were found in human urine. Metabolites M1 and M2, which were found as minor metabolites, originated from N-demethylation of sibutramine. Carbamoyl glucuronides formed from metabolites M1, M2, and their hydroxylated analogs were the main metabolites of sibutramine and were characterized by tandem mass spectrometric analysis and by the chemical modification of their structure. We demonstrate the usefulness of the chemical derivatization approach for estimation of the site of glucuronidation and propose the formation of hydroxylated regioisomers of metabolites M4 and M6.     

Metabolism of olaquindox in rat and identification of metabolites in urine and feces using ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry

Olaquindox (OLA), N-(2-hydroxyethyl)-3-methyl-2-quinoxalincarboxamide-1,4-dioxide, is an antimicrobial and growth-promoting agent for animals, which has been banned or allowed only limited use for its potential toxicity. To thoroughly understand the metabolic pathways, metabolism of OLA in rat was studied using ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry with MS(E) and mass defect filtering techniques. Twenty metabolites (M1-M20) were detected in rat feces and urine, of which nine phase I metabolites (M6, M7, M11-M16) and four phase II metabolites (M17-M20) were found in vivo for the first time. The structures of metabolites were reliably characterized on the basis of accurate mass and fragment ions in MS(E) spectra. The major metabolic pathways reported previously in pigs, including reduction of N→O groups, oxidation of the alcohol and hydrolysis, were also confirmed in this study. In addition, hydroxylation of the methyl group, N-dehydroxyethylation and glucuronidation were also proved to be the important metabolic pathways, which contribute to improving our knowledge about in vivo metabolism of OLA.     

Application of a linear ion trap/orbitrap mass spectrometer in metabolite characterization studies: Examination of the human liver microsomal metabolism of the non-tricyclic anti-depressant nefazodone using data-dependent accurate mass measurements

We report herein, facile metabolite identification workflow on the anti-depressant nefazodone, which is derived from accurate mass measurements based on a single run/experimental analysis. A hybrid LTQ/orbitrap mass spectrometer was used to obtain accurate mass full scan MS and MS/MS in a data-dependent fashion to eliminate the reliance on a parent mass list. Initial screening utilized a high mass tolerance ( approximately 10 ppm) to filter the full scan MS data for previously reported nefazodone metabolites. The tight mass tolerance reduces or eliminates background chemical noise, dramatically increasing sensitivity for confirming or eliminating the presence of metabolites as well as isobaric forms. The full scan accurate mass analysis of suspected metabolites can be confirmed or refuted using three primary tools: (1) predictive chemical formula and corresponding mass error analysis, (2) rings-plus-double bonds, and (3) accurate mass product ion spectra of parent and suspected metabolites. Accurate mass characterization of the parent ion structure provided the basis for assessing structural assignment for metabolites. Metabolites were also characterized using parent product ion m/z values to filter all tandem mass spectra for identification of precursor ions yielding similar product ions. Identified metabolite parent masses were subjected to chemical formula calculator based on accurate mass as well as bond saturation. Further analysis of potential nefazodone metabolites was executed using accurate mass product ion spectra. Reported mass measurement errors for all full scan MS and MS/MS spectra was <3 ppm, regardless of relative ion abundance, which enabled the use of predictive software in determining product ion structure. The ability to conduct biotransformation profiling via tandem mass spectrometry coupled with accurate mass measurements, all in a single experimental run, is clearly one of the most attractive features of this methodology.     

大鼠血液中溴莫普林的代谢产物核磁共振氢谱和质谱研究

用核磁共振氢谱和质谱法研究大鼠血浆中溴莫普林（BDP)及其代谢物。首先用固相萃取法将血液中内源性物质除去,然后经核磁共振氢谱和质谱检测大鼠血液中BDP及其代谢物。大鼠服用溴莫普林后20h时血浆中检测到两个代谢产物：脱甲基－溴莫普林葡萄糖醛酸和溴莫普林硫酸。结果证明核磁共振氢谱和质谱法可以快速、方便地用于血液中药及其代谢产物的含量与结构检测的研究。     

Metabolism of olaquindox in rat liver microsomes: structural elucidation of metabolites by high-performance liquid chromatography combined with ion trap/time-of-flight mass spectrometry

Olaquindox (N-(2-hydroxyethyl)-3-methyl-2-quinoxalincarboxamide-1,4-dioxide) is a growth-promoting feed additive for food-producing animals. Its toxicity is closely related to the metabolism. The complete metabolic pathways of olaquindox are not revealed. To improve studies of the metabolism and toxicity of olaquindox, its biotransformation in rat liver microsomes and the structure of its metabolites using high-performance liquid chromatography combined with ion trap/time-of-flight mass spectrometry (LC/MS-ITTOF) were investigated. When olaquindox was incubated with an NADPH-generating system and rat liver microsomes, ten metabolites (M1-M10) were detected. The structures of these metabolites were identified from mass spectra and comparison of their changes in their accurate molecular masses and fragment ions with those of the parent drug. With the high resolution and good mass accuracy achieved by this technique, the elemental compositions of the metabolites and their fragment ions were exactly determined. The results indicate that the N --> O group reduction is the main metabolic pathway of olaquindox metabolism in rat liver microsomes, because abundant 1-desolaquindox (M2), 4-desolaquindox (M1) and bisdesoxyolaquindox (M9) were produced during the incubation step. Seven other minor metabolites were revealed which were considered to be hydroxylation metabolites, based on the position of the quinoxaline ring or 3-methyl group and a carboxylic acid derivative on the side chain at position 2 of the quinoxaline ring. Among the identified metabolites, five new hydroxylated metabolites (M3-M7) were found for the first time in rat liver microsomes. This work will conduce to complete clarification of olaquindox metabolism, and improve the in vivo metabolism of olaquindox in food animals.     

Metabolite identification of a new antitumor agent icotinib in rats using liquid chromatography/tandem mass spectrometry

Icotinib, 4-[(3-ethynylphenyl)amino]-6,7-benzo-12-crown-4-quinazoline, is a new antitumor agent. The metabolic pathway of icotinib in rats was studied using liquid chromatography/tandem mass spectrometry (LC/MS(n)) analysis. Full scan and selected ion monitoring modes were used to profile the possible metabolites of icotinib in rat urine, feces and bile samples. Four phase I metabolites (M1-M4) and two phase II metabolites (M5, M6) were detected and characterized. Multiple-stage mass spectrometry and nuclear magnetic resonance (NMR) spectrometry were employed to elucidate structures of metabolites. Icotinib was metabolized to open the crown ether ring to form the main phase I metabolites. During metabolism, a reactive metabolite was formed. Using semicarbazide as a trapping agent, an intermediate arising from opening of the crown ether ring was detected as an aldehyde product by LC/MS/MS. These data indicated that ring opening of the crown ether was triggered by hydroxylation at the 8'-position of the ring to form a hemiacetal intermediate, which was further oxidized or reduced. Finally, the metabolic pathway of icotinib in rats was proposed.     

Complete profiling and characterization of in vitro nefazodone metabolites using two different tandem mass spectrometric platforms

This paper describes the complete profiling and characterization of in vitro metabolites of the antidepressant agent nefazodone (NEF) generated by human liver microsome (HLM). Two new metabolic pathways (biotransformation) for NEF have been discovered by the characterization of three new metabolites, including two new metabolites (M24, M25) formed due to the N-dealkylation reaction that occurred between the triazolone and propyl units, and one new metabolite (M26) formed due to the O-dearylation reaction that occurred on the phenoxyethyl unit. These metabolites were initially detected by a 4000 Q-Trap instrument and then confirmed by exact mass measurement using an LTQ-Orbitrap. Both instruments proved to be capable of providing complete in vitro metabolite information in a single liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis, although each had its advantages and disadvantages. One noticeable disadvantage of the 4000 Q-Trap was the reduced quality of isotopic pattern in the enhanced mass scan (EMS) spectrum when it was used as survey scan to trigger multiple dependent product ion scans. The problem was especially exacerbated for minor metabolites with low signal intensity. On the other hand, the LTQ-Orbitrap maintained excellent isotopic pattern when used as a full scan survey scan. Twenty-six metabolites were detected and identified. The formation of these new metabolites was also confirmed by analyzing duplicate incubations at different time points.     

Metabolism of eupatilin in rats using liquid chromatography/electrospray mass spectrometry

Eupatilin (5,7-dihydroxy-3',4',6-trimethoxy flavone) is an active ingredient of an ethanol extract of Artemisia asiatica (DA-9601) that is used in the treatment of gastritis. In vitro and in vivo metabolism of eupatilin in the rats has been studied by LC-electrospray mass spectrometry. Rat liver microsomal incubation of eupatilin in the presence of NADPH and UDPGA resulted in the formation of four metabolites (M1-M4). M1, M2, M3 and M4 were tentatively identified as 3'- or 4'-O-demethyl-eupatilin glucuronide, eupatilin glucuronide, 6-O-demethyleupatilin and 3'- or 4'-O-demethyl-eupatilin, respectively. Those metabolites from in vitro study were also characterized in bile, plasma or urine samples after an intravenous administration of eupatilin to rats. In rat bile, plasma and urine samples, eupatilin glucuronide (M2) was a major metabolite, whereas M3, M4 and M4 glucuronide (M1) were the minor metabolites.     

Comparative analysis on microbial and rat metabolism of ginsenoside Rb1 by high-performance liquid chromatography coupled with tandem mass spectrometry

Ginsenoside Rb1 is an active protopanaxadiol saponin from Panax species. In order to compare the similarities and differences of microbial and mammalian metabolisms of ginsenoside Rb1, the microbial transformation by Acremonium strictum and metabolism in rats were comparatively studied. Microbial transformation of ginsenoside Rb1 by Acremonium strictum AS 3.2058 resulted in the formation of eight metabolites. Ten metabolites (M1-M10) were detected from the in vivo study in rats and eight of them were identified as the same compounds as those obtained from microbial metabolism by liquid chromatography-tandem mass spectrometry analysis and comparison with reference standards obtained from microbial metabolism. Their structures were identified as ginsenoside Rd, gypenoside XVII, 20(S)-ginsenoside Rg3, 20(R)-ginsenoside Rg3, ginsenoside F2, compound K, 12beta-hydroxydammar-3-one-20(S)-O-beta-d-glucopyranoside, and 25-hydroxyl-(E)-20(22)-ene-ginsenoside Rg3, respectively. The structures of the additional two metabolites were tentatively characterized as 20(22),24-diene-ginsenoside Rg3 and 25-hydroxyginsenoside Rd by HPLC-MS/MS analysis. M7-M10 are the first four reported metabolites in vivo. The time course of rat metabolism of ginsenoside Rb1 was also investigated.     

A new strategy for the discovery of epimedium metabolites using high-performance liquid chromatography with high resolution mass spectrometry

In this paper, a new strategy of drug metabolite discovery and identification was established using high-performance liquid chromatography with high resolution mass spectrometry (HPLC–HRMS) and a mass spectral trees similarity filter (MTSF) technique. The MTSF technique was developed as a means to rapidly discover comprehensive metabolites from multiple active components in a complicated biological matrix. Using full-scan mass spectra as the stem and data-dependent subsequent stage mass spectra to form branches, the HRMS and multiple-stage mass spectrometric data from detected compounds were converted to mass spectral trees data. Potential metabolites were discovered based on the similarity between their mass spectral trees and that known compounds or metabolites in a mass spectra trees library. The threshold value for match similarity scores was set at above 200, allowing approximately 80% of interference to be filtered out. A total of 115 metabolites of five flavonoid monomers (epimedin A, epimedin B, epimedin C, icariin, and baohuoside I) and herbal extract of epimedium were discovered and identified in rats via this new strategy. As a result, a metabolic profile for epimedium was obtained and a metabolic pathway was proposed. In addition, comparing to the widely used neutral loss filter (NLF), product ion filter (PIF), and mass defect filter (MDF) techniques, the MTSF technique was shown superior efficiency and selectivity for discovering and identifying metabolites in traditional Chinese medicine (TCM).     

Human urinary metabolite profile of tea polyphenols analyzed by liquid chromatography/electrospray ionization tandem mass spectrometry with data-dependent acquisition

Tea is rich in polyphenols and has a variety of biological activities. In order to better understand the biological effects of tea constituents on human health, markers for their exposure and their metabolic fates are needed. Previously, we have characterized several catechin metabolites in the blood and urine, but more information on the metabolite profile of tea polyphenols is needed. In the present study, the human urinary metabolite profile of tea polyphenols was investigated using liquid chromatography/electrospray ionization tandem mass spectrometry with data-dependent acquisition. With data-dependent MS/MS analysis by collecting the MS2 and MS3 spectra of the most intense ions in the sample, we identified more than twenty metabolites of tea polyphenols from human urine samples. (-)-Epigallocatechin (EGC) glucuronide, methylated EGC glucuronide, methylated EGC sulfate, (-)-epicatechin (EC) glucruronide, EC sulfate, methylated EC sulfate, as well as the glucuronide and sulfate metabolites of the ring-fission metabolites of tea catechins, 5-(3',4',5'-trihydroxyphenyl)-gamma-valerolactone (M4), 5-(3',4'-dihydroxyphenyl)-gamma-valerolactone (M6) and 5-(3',5'-dihydroxyphenyl)-gamma-valerolactone (M6'), were the major human urinary metabolites of tea polyphenols. To our knowledge, this is the first report of the direct simultaneous analysis of the human urinary metabolite profile of tea polyphenols using single sample analysis. This method can also be used for thorough investigations of the metabolite profiles of many other dietary constituents.     

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.     

In vivo metabolite detection and identification in drug discovery via LC-MS/MS with data-dependent scanning and postacquisition data mining

An important aspect in drug discovery is the early structural identification of the metabolites of potential new drugs. This gives information on the metabolically labile points in the molecules under investigation, suggesting structural modifications to improve their metabolic stability, and allowing an early safety assessment via the identification of metabolic activation products. From an analytical point of view, metabolite identification still remains a challenging task, especially for in vivo samples, in which they occur at trace levels together with high amounts of endogenous compounds. Here we describe a method, based on LC-ion trap tandem MS, for the rapid in vivo metabolite identification. It is based on the automatic, data-dependent acquisition of multiple product ion MS/MS scans, followed by a postacquisition search, within the entire MS/MS data set obtained, for specific neutral losses or marker ions in the tandem mass spectra of parent molecule and putative metabolites. One advantage of the method is speed, since it requires minimum sample preparation and all the necessary data can be obtained in one chromatographic run. In addition, it is highly sensitive and selective, allowing detection of trace metabolites even in the presence of a complex matrix. As an example of application, we present the studies of the in vivo metabolism of the compound MEN 15916 (1). The method allowed identification of monohydroxy ([M + H](+) = m/z 655), dihydroxy ([M + H](+) = m/z 671), and trihydroxy ([M + H](+) = m/z 687) metabolites, as well as some unexpected biotransformation products such as a carboxylic acid ([M + H](+) = m/z 669), a N-dealkylated metabolite ([M + H](+) = m/z 541), and its hydroxy-analog ([M + H](+) = m/z 557).     

Characterization of rat liver microsomal metabolites of AM-630, a potent cannabinoid receptor antagonist, by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry

The in vitro metabolism of AM-630 was studied by high-performance liquid chromatography coupled with tandem mass spectrometry. AM-630 is an aminoalkylindole analogue that behaves primarily as a potent CB2-selective antagonist. In this study, 17 metabolic products were identified that resulted from the incubation of AM-630 in rat liver microsome preparations. Six metabolic pathways were proposed to account for all detected metabolites: (1) o-demethylation of the methoxyphenyl group, (2) morpholinyl ring opening, (3) hydroxylation on the methoxy/hydroxyl phenyl ring, (4) hydroxylation on the indole ring, (5) hydroxylation on the morpholine ring and (6) loss of the morpholine ring leading to metabolites containing either a hydroxylated or a carboxylated alkyl terminal. Three metabolites were identified as morpholinyl ring-opening products: M1, M6 and M13. Six metabolites (M2-M5, M7 and M8) were proposed to be the products of o-demethylation, hydroxylation on the methoxyphenyl group or the morpholinyl ring, dehydration following morpholinyl ring monohydroxylation, or a combination of the above metabolic pathways. The remaining eight metabolites were attributed to a pathway involving the loss of the morpholine ring at various points during the metabolic processes.     

Screening of pesticides in blood with liquid chromatography–linear ion trap mass spectrometry

In clinical or forensic toxicology, general unknown screening procedures are used to identify as many xenobiotics as possible, belonging to numerous chemical classes. We present here a general unknown screening procedure based on liquid chromatography coupled with use of a single linear ion trap mass spectrometer, and focus on the identification of pesticides and/or metabolites in whole blood. After solid-phase extraction (SPE), the compounds of interest were separated using a reversed-phase column and identified by the mass spectrometer operated first in the full-scan mass spectrometry (MS) mode, in the positive and negative polarities, followed by MS² and MS³ scanning of ions selected in data-dependent acquisition. The total scan time was 2.45 s. Two mass spectral libraries (MS² and MS³), each of 450 spectra, were created for the 320 pesticides and metabolites detected after injection of pure solutions. Robustness of the spectra and matrix effects were studied and were satisfactory for the present application. Detection limits for the 320 compounds were studied by extracting 1 mL spiked blood at concentrations between 10 μg/L and 10 mg/L. If necessary, it was possible to decrease the detection limits of some compounds by 10–100-fold by scanning MS² in only one polarity, owing to a shorter total scan time. However, at the same time, the detection specificity decreased as no confirmation could be recorded in the following MS³ scan and no information could be registered in the other polarity. So, in these rare cases, confirmation by another method was required.     

High-Throughput Metabolic Soft-Spot Identification in Liver Microsomes by LC/UV/MS: Application of a Single Variable Incubation Time Approach

CYP-mediated fast metabolism may lead to poor bioavailability, fast drug clearance and significant drug interaction. Thus, metabolic stability screening in human liver microsomes (HLM) followed by metabolic soft-spot identification (MSSID) is routinely conducted in drug discovery. Liver microsomal incubations of testing compounds with fixed single or multiple incubation time(s) and quantitative and qualitative analysis of metabolites using high-resolution mass spectrometry are routinely employed in MSSID assays. The major objective of this study was to develop and validate a simple, effective, and high-throughput assay for determining metabolic soft-spots of testing compounds in liver microsomes using a single variable incubation time and LC/UV/MS. Model compounds (verapamil, dextromethorphan, buspirone, mirtazapine, saquinavir, midazolam, amodiaquine) were incubated at 3 or 5 µM with HLM for a single variable incubation time between 1 and 60 min based on predetermined metabolic stability data. As a result, disappearances of the parents were around 20–40%, and only one or a few primary metabolites were generated as major metabolite(s) without notable formation of secondary metabolites. The unique metabolite profiles generated from the optimal incubation conditions enabled LC/UV to perform direct quantitative estimation for identifying major metabolites. Consequently, structural characterization by LC/MS focused on one or a few major primary metabolite(s) rather than many metabolites including secondary metabolites. Furthermore, generic data-dependent acquisition methods were utilized to enable Q-TOF and Qtrap to continuously record full MS and MS/MS spectral data of major metabolites for post-acquisition data-mining and interpretation. Results from analyzing metabolic soft-spots of the seven model compounds demonstrated that the novel MSSID assay can substantially simplify metabolic soft-spot identification and is well suited for high-throughput analysis in lead optimization.     

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.     

Use of on-line hydrogen/deuterium exchange to facilitate metabolite identification

Biotransformation studies performed on an investigational compound (I, represented by R1-CH(NH(2))-CO-N(R2)-CH(2)-S-R3) led to the identification of five metabolites (M1-M5). Based on LC/MS (liquid chromatography/mass spectrometry) analysis which included the use of H(2)O and D(2)O in the mobile phases, they were identified as the sulfoxide (M1), sulfone (M2), carbamoyl glucuronide (M3), N-glucuronide (M4), and N-glucoside (M5) metabolites, respectively. The structure of M3, a less commonly seen carbamoyl glucuronide metabolite, was established using on-line H/D (hydrogen/deuterium) exchange experiments conducted by LC/MS. H/D exchange experiments were also used to distinguish the S-oxidation structures of M1 and M2 from hydroxylation. Herein, the application of deuterium oxide as the LC/MS mobile phase for structural elucidation of drug metabolites in biological matrices is demonstrated.     

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.     

Qualitative–(semi)quantitative data acquisition of artemisinin and its metabolites in rat plasma using an LTQ/Orbitrap mass spectrometer

Artemisinin (QHS) is one of the first‐line antimalarials, and autoinduction of CYP‐mediated metabolism can result in its reduced exposure. To better understand the autoinduction of QHS, we evaluated the pharmacokinetics of QHS and its phase I metabolites in rats using an liquid chromatography‐high resolution mass spectrometry (LC‐HRMS) method. The LC separation was improved, allowing the separation of QHS and its metabolites from their diastereomers, and seven metabolites of QHS with relatively high exposure were identified in rat plasma, including deoxyartemisinin (DQHS), three monoyhydroxylated plus deoxyl metabolites (M1–M3) and three monohydroxylated metabolites (M4–M6). For detection, a high‐resolution LTQ/Orbitrap mass spectrometer with an electrospray ionization (ESI) inlet in the positive ion mode was used. High‐resolution extracted ion chromatograms for each analyte were obtained by processing the full‐scan MS dataset with 10 ppm mass tolerance. The plasma samples were pretreated by protein precipitation with acetonitrile. The standard curve was linear (r² > 0.99) over the QHS and DQHS concentration range of 5.0–200.0 ng/ml in 50 µl of plasma, which offered sufficient sensitivity and accuracy for the determination of QHS and its metabolites. A 3‐day validation approach was used for absolute quantitation of QHS and DQHS. The other six metabolites of QHS were semiquantified based on the calibration curve of QHS. The present method was applied to the pharmacokinetic study of QHS in rats after a single oral administration. The data shown here also suggest that this type of mass analyzer will be capable of a quantitative–qualitative workflow. Copyright © 2012 John Wiley & Sons, Ltd.