Identification of metabolites of adonifoline,a hepatotoxic pyrrolizidine alkaloid,by liquid chromatography/tandem and high‐resolution mass spectrometry

Hepatotoxic pyrrolizidine alkaloid (HPA)‐containing plants have always been a threat to human and livestock health worldwide. Adonifoline, a main HPA in Senecio scandens Buch.‐Ham. ex D. Don (Qianli guang), was used officially as an infusion in cases of oral and pharyngeal infections in China. In this study in vivo metabolism of adonifoline was studied for the first time by identifying the metabolites of adonifoline present in bile, urine and feces of rats using liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI‐MSⁿ) (ion trap) as well as liquid chromatography/electrospray ionization high‐resolution mass spectrometry (LC/ESI‐HRMS) (quadrupole‐time of flight). In total 19 metabolites were identified and, among them, retronecine‐N‐oxides were confirmed by matching their fragmentation patterns with their fully characterized synthetic compounds. These metabolites are all involved in both phase I and phase II metabolic processes and the principal in vivo metabolism pathways of adonifoline were proposed. Copyright © 2009 John Wiley & Sons, Ltd.     

Metabolism studies on prim‐O‐glucosylcimifugin and cimifugin in human liver microsomes by ultra‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometry

Prim‐O‐glucosylcimifugin (PGCN) and cimifugin (CN) are major constituents of Radix Saposhnikoviae that have antipyretic, analgesic and anti‐inflammatory pharmacological activities. However, there were few reports with respect to the metabolism of PGCN and CN in vitro. In this paper, we describe a strategy using ultra‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometry (UPLC‐Q‐TOF‐MS) for fast analysis of the metabolic profile of PGCN and CN in human liver microsomes. In total, five phase I metabolites of PGCN, seven phase I metabolites and two phase II metabolites of CN were identified in the incubation of human liver microsomes. The results revealed that the main phase I metabolic pathways of PGCN were hydroxylation and hydrolysis reactions. The phase I metabolic pathways of CN were found to be hydroxylation, demethylation and dehydrogenation. Meanwhile, the results indicated that O‐glucuronidation was the major metabolic pathway of CN in phase II metabolism. The specific UDP‐glucuronosyltransferase (UGT) enzymes responsible for CN glucuronidation metabolites were identified using recombinant UGT enzymes. The results indicated that UGT1A1, UGT1A9, UGT2B4 and UGT2B7 might play major roles in the glucuronidation of CN. Overall, this study may be useful for the investigation of metabolic mechanism of PGCN and CN, and it can provide reference and evidence for further pharmacodynamic experiments. Copyright © 2016 John Wiley & Sons, Ltd.     

Determination of triclosan metabolites by using in‐source fragmentation from high‐performance liquid chromatography/negative atmospheric pressure chemical ionization ion trap mass spectrometry

Triclosan is a widely used broad‐spectrum antibacterial agent that acts by specifically inhibiting enoyl–acyl carrier protein reductase. An in vitro metabolic study of triclosan was performed by using Sprague‐Dawley (SD) rat liver S9 and microsome, while the in vivo metabolism was investigated on SD rats. Twelve metabolites were identified by using in‐source fragmentation from high‐performance liquid chromatography/negative atmospheric pressure chemical ionization ion trap mass spectrometry (HPLC/APCI‐ITMS) analysis. Compared to electrospray ionization mass spectrometry (ESI‐MS) and tandem mass spectrometry (MS/MS) that gave little fragmentation for triclosan and its metabolites, the in‐source fragmentation under APCI provided intensive fragmentations for the structural identifications. The in vitro metabolic rate of triclosan was quantitatively determined by using HPLC/ESI‐ITMS with the monitoring of the selected triclosan molecular ion. The metabolism results indicated that glucuronidation and sulfonation were the major pathways of phase II metabolism and the hydroxylated products were the major phase I metabolites. Moreover, glucose, mercapturic acid and cysteine conjugates of triclosan were also observed in the urine samples of rats orally administrated with triclosan. Copyright © 2010 John Wiley & Sons, Ltd.     

Metabolite identification of tanshinol borneol ester in rats by high‐performance liquid chromatography combined with electrospray ionization quadrupole time‐of‐flight mass spectrometry

Tanshinol borneol ester (DBZ) is a potential drug candidate composed of danshensu and borneol. It shows anti‐ischemic and anti‐atherosclerosis activity. However, little is known about its metabolism in vivo. This research aimed to elucidate the metabolic profile of DBZ through analyzing its metabolites using high‐performance liquid chromatography combined with electrospray ionization quadrupole time‐of‐flight mass spectrometry. Chromatographic separation was performed on an Agilent TC‐C₁₈ column (150 × 4.6 mm, 5.0 μm) with gradient elution using methanol and water containing 0.2% (v/v) formic acid as the mobile phase. Metabolite identification involved analyzing the retention behaviors, changes in molecular weights and MS/MS fragment patterns of DBZ and its metabolites. As a result, 20 potential metabolites were detected and tentatively identified in rat plasma, urine and feces after administration of DBZ. DBZ could be metabolized to O‐methylated DBZ, DBZ‐O‐glucuronide, O‐methylated DBZ‐O‐glucuronide, hydroxylated DBZ and danshensu. Danshensu, a hydrolysis product of DBZ, could further be transformed into 12 metabolites. The proposed method was confirmed to be a reliable and sensitive alternative for characterizing metabolic pathways of DBZ and providing valuable information on its druggability.     

Metabolic profiling of quercetin in rats using ultra‐performance liquid chromatography/quadrupole‐time‐of‐flight mass spectrometry

Quercetin, a kind of major flavonoid found in many traditional chinese medicines, is an effective substance for treatments such as lowering blood lipids. However, the studies on quercetin have been mainly focused on its pharmacological effect; the treatment of diseases on a material basis, particularly the metabolites derived from quercetin in vivo , has not been evaluated. In this study, we determined the levels, distributions and types of quercetin's metabolites in plasma, urine, feces and bile of rats after a single oral administration of quercetin at a dose of 80 mg/kg, using ultra‐performance liquid chromatography/quadrupole‐time‐of‐flight mass spectrometry (UPLC‐Q‐TOF/MS). A total of 36 metabolites of quercetin were identified, including 11 metabolites in plasma, 34 metabolites in urine, 12 metabolites in feces and 21 metabolites in bile. The results showed that phase I metabolites were reduction metabolites and phase II metabolites mainly included glucuronidation, sulfation and methylation metabolites. These results provide important information on the metabolism of quercetin, which will be helpful for its further development and utilization.     

Mixed‐mode chromatography/isotope ratio mass spectrometry

Liquid chromatography coupled to molecular mass spectrometry (LC/MS) has been a standard technique since the early 1970s but liquid chromatography coupled to high‐precision isotope ratio mass spectrometry (LC/IRMS) has only been available commercially since 2004. This development has, for the first time, enabled natural abundance and low enrichment δ¹³C measurements to be applied to individual analytes in aqueous mixtures creating new opportunities for IRMS applications, particularly for the isotopic study of biological molecules. A growing number of applications have been published in a range of areas including amino acid metabolism, carbohydrates studies, quantification of cellular and plasma metabolites, dietary tracer and nucleic acid studies. There is strong potential to extend these to new compounds and complex matrices but several challenges face the development of LC/IRMS methods. To achieve accurate isotopic measurements, HPLC separations must provide baseline‐resolution between analyte peaks; however, the design of current liquid interfaces places severe restrictions on compatible flow rates and in particular mobile phase compositions. These create a significant challenge on which reports associated with LC/IRMS have not previously focused. Accordingly, this paper will address aspects of chromatography in the context of LC/IRMS, in particular focusing on mixed‐mode separations and their benefits in light of these restrictions. It aims to provide an overview of mixed‐mode stationary phases and of ways to improve high aqueous separations through manipulation of parameters such as column length, temperature and mobile phase pH. The results of several practical experiments are given using proteogenic amino acids and nucleosides both of which are of noted importance in the LC/IRMS literature. This communication aims to demonstrate that mixed‐mode stationary phases provide a flexible approach given the constraints of LC/IRMS interface design and acts as a practical guide for the development of new chromatographic methods compatible with LC/IRMS applications. Copyright © 2010 John Wiley & Sons, Ltd.     

In silico methods for predicting metabolism and mass fragmentation applied to quetiapine in liquid chromatography/time‐of‐flight mass spectrometry urine drug screening

Current in silico tools were evaluated for their ability to predict metabolism and mass spectral fragmentation in the context of analytical toxicology practice. A metabolite prediction program (Lhasa Meteor), a metabolite detection program (Bruker MetaboliteDetect), and a fragmentation prediction program (ACD/MS Fragmenter) were used to assign phase I metabolites of the antipsychotic drug quetiapine in the liquid chromatography/time‐of‐flight mass spectrometry (LC/TOFMS) accurate mass data from ten autopsy urine samples. In the literature, the main metabolic routes of quetiapine have been reported to be sulfoxidation, oxidation to the corresponding carboxylic acid, N‐ and O‐dealkylation and hydroxylation. Of the 14 metabolites predicted by Meteor, eight were detected by LC/TOFMS in the urine samples with use of MetaboliteDetect software and manual inspection. An additional five hydroxy derivatives were detected, but not predicted by Meteor. The fragment structures provided by ACD/MS Fragmenter software confirmed the identification of the metabolites. Mean mass accuracy and isotopic pattern match (SigmaFit) values for the fragments were 2.40 ppm (0.62 mDa) and 0.010, respectively. ACD/MS Fragmenter, in particular, allowed metabolites with identical molecular formulae to be differentiated without a need to access the respective reference standards or reference spectra. This was well exemplified with the hydroxy/sulfoxy metabolites of quetiapine and their N‐ and O‐dealkylated forms. The procedure resulted in assigning 13 quetiapine metabolites in urine. The present approach is instrumental in developing an extensive database containing exact monoisotopic masses and verified retention times of drugs and their urinary metabolites for LC/TOFMS drug screening. Copyright © 2009 John Wiley & Sons, Ltd.     

An online coupled peritoneal macrophage/cell membrane chromatography and high‐performance liquid chromatography/mass spectrometry method to screen for anti‐inflammatory components from the Chinese traditional medicine Chloranthus multistachys Pei

Cell membrane chromatography (CMC) is a chromatographic biological affinity method that uses specific cell membranes as the stationary phase. In this study, a novel peritoneal macrophage/cell membrane chromatography (PM/CMC)–online‐high performance liquid chromatography/mass spectrometry (HPLC/MS) method was established to screen for the anti‐inflammatory components from traditional Chinese medicines using hydrocortisone and dexamethasone as standards. The stationary phase of the CMC employed mouse peritoneal macrophage cell membranes. This method was applied to the purification and identification of components in extracts of Chloranthus multistachys Pei. The major component retained by CMC was identified as isofraxidin by HPLC/MS. In vitro experiments revealed that IF was able to inhibit the production of nitric oxide and tumor necrosis factor‐α in lipopolysaccharide‐stimulated mice and peritoneal macrophages in a dose‐dependent manner. The results demonstrated that the PM/CMC‐online‐HPLC/MS is an effective screening system for the rapid detection, enrichment, and identification of target components from complex samples. Copyright © 2013 John Wiley & Sons, Ltd.     

Characterization of metabolites in rat plasma after intravenous administration of salvianolic acid A by liquid chromatography/time‐of‐flight mass spectrometry and liquid chromatography/ion trap mass spectrometry

Salvianolic acid A (SalA) is one of the main active constituents in Salvia miltiorrhiza (Danshen). Although the pharmacokinetics of SalA in rats after intravenous (i.v.) administration of Danshen injection has been reported, the information relevant to the metabolites of SalA in vivo is absent so far. In this study, by means of liquid chromatography with time‐of‐flight mass spectrometry (LC/TOFMS) and liquid chromatography with ion trap mass spectrometry (LC/MSⁿ) techniques, the unknown metabolites of SalA in rat plasma after i.v. administration of the purified SalA at the dose of 20 mg/kg body weight were identified. A liquid‐liquid extraction method was established to separate the metabolites from the plasma and the chromatographic separations were performed on a Xterra MS C₁₈ column (100 mm × 4.6 mm i.d., 3.5 µm) with acetonitrile/methanol/water/formic acid (20.5:19.5:64: 0.05, v/v/v/v) as the mobile phase at a constant flow rate of 0.2 mL/min. Based on the data obtained from the LC/TOFMS determination (the total ion chromatograms, MS spectra and extracted ion chromatograms), in combination with the characteristic fragment ions acquired from the LC/MSⁿ determination, five metabolites were identified as SalA‐monoglucuronide, monomethyl‐SalA‐monoglucuronide, mono‐methyl‐SalA, dimethyl‐SalA and dimethyl‐SalA‐monoglucuronide, and the possible chemical structures were deduced. The results indicated that SalA might mainly undergo two metabolic pathways in vivo in rats, which were methylation and glucuronidation. The present studies have laid a solid foundation for the metabolic mechanism of SalA in vivo. Copyright © 2009 John Wiley & Sons, Ltd.     

Chemometrics comparison of gas chromatography with mass spectrometry and comprehensive two‐dimensional gas chromatography with time‐of‐flight mass spectrometry Daphnia magna metabolic profiles exposed to salinity

The performances of gas chromatography with mass spectrometry and of comprehensive two‐dimensional gas chromatography with time‐of‐flight mass spectrometry are examined through the comparison of Daphnia magna metabolic profiles. Gas chromatography with mass spectrometry and comprehensive two‐dimensional gas chromatography with mass spectrometry were used to compare the concentration changes of metabolites under saline conditions. In this regard, a chemometric strategy based on wavelet compression and multivariate curve resolution–alternating least squares is used to compare the performances of gas chromatography with mass spectrometry and comprehensive two‐dimensional gas chromatography with time‐of‐flight mass spectrometry for the untargeted metabolic profiling of Daphnia magna in control and salinity‐exposed samples. Examination of the results confirmed the outperformance of comprehensive two‐dimensional gas chromatography with time‐of‐flight mass spectrometry over gas chromatography with mass spectrometry for the detection of metabolites in D. magna samples. The peak areas of multivariate curve resolution–alternating least squares resolved elution profiles in every sample analyzed by comprehensive two‐dimensional gas chromatography with time‐of‐flight mass spectrometry were arranged in a new data matrix that was then modeled by partial least squares discriminant analysis. The control and salt‐exposed daphnids samples were discriminated and the most relevant metabolites were estimated using variable importance in projection and selectivity ratio values. Salinity de‐regulated 18 metabolites from metabolic pathways involved in protein translation, transmembrane cell transport, carbon metabolism, secondary metabolism, glycolysis, and osmoregulation.     

Screening anti‐tumor compounds from Ligusticum wallichii using cell membrane chromatography combined with high‐performance liquid chromatography and mass spectrometry

Tyrosine 367 Cysteine‐fibroblast growth factor receptor 4 cell membrane chromatography combined with high‐performance liquid chromatography and mass spectrometry was developed. Tyrosine 367 Cysteine‐HEK293 cells were used as the cell membrane stationary phase. The specificity and reproducibility of the cell membrane chromatography was evaluated using 1‐tert‐butyl‐3‐{2‐[4‐(diethylamino)butylamino]‐6‐(3,5‐dimethoxyphenyl)pyrido[2,3‐d]pyrimidin‐7‐yl}urea, nimodipine and dexamethasone acetate. Then, anti‐tumor components acting on Tyrosine 367 Cysteine‐fibroblast growth factor receptor 4 were screened and identified from extracts of Ligusticum wallichii. Components from the extract were retained on the cell membrane chromatographic column. The retained fraction was directly eluted into high‐performance liquid chromatography with mass spectrometry system for separation and identification. Finally, Levistolide A was identified as an active component from Ligusticum wallichii extracts. The 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐tetrazolium bromide‐formazan colorimetric assay revealed that Levistolide A inhibits proliferation of overexpressing the mutated receptor cells with dose‐dependent manner. Phosphorylation of fibroblast growth factor receptor 4 was also decrease under Levistolide A treatment. Flex dock simulation verified that Levistolide A could bind with the tyrosine kinase domain of fibroblast growth factor receptor 4. Therefore, Levistolide A screened by the cell membrane chromatography combined with high‐performance liquid chromatography and mass spectrometry can arrest cell growth. In conclusion, the two‐dimensional high‐performance liquid chromatography method can screen and identify potential anti‐tumor ingredients that specifically act on the tyrosine kinase domain of the mutated fibroblast growth factor receptor 4.     

A gas chromatography/mass spectrometry method for the determination of sildenafil,vardenafil and tadalafil and their metabolites in human urine

Sildenafil (SDF), vardenafil (VDF) and tadalafil (TDF) are phosphodiesterase type 5 enzyme inhibitors (PDE5Is), used in the treatment of erectile disorders and to improve breathing efficiency in pulmonary hypertension. The increasing incidence of their use among young athletes has drawn the attention of the anti‐doping authorities to the possible abuse of PDE5Is by athletes due to their pharmacological activities. This paper describes a method for the determination in urine of PDE5Is and their metabolites by gas chromatography/mass spectrometry (GC/MS) after liquid/liquid extraction of the analytes from urine and derivatisation to obtain trimethylsilyl derivatives. The metabolic profile was studied on real samples collected from subjects taking PDE5Is (Viagra®, Levitra® or Cialis®); the main urinary metabolites were identified and their MS fragmentation characterized. The sample pre‐treatment and GC/MS conditions for the detection of the metabolites have been optimised. A method for their preliminary screening and subsequent confirmation is described that takes into account the general requirements of a routine doping analysis to be used for the screening of large numbers of samples. The main metabolites identified can be included in a general purpose screening method and all the metabolites in a more specific confirmation method. The method developed has been applied for the screening of PDE5Is in 5000 urine samples. Based on the obtained results, the proposed method appears to be of practical use in analytical and forensic toxicology, including doping analysis. Copyright © 2010 John Wiley & Sons, Ltd.     

Identification and structural characterization of in vivo metabolites of ketorolac using liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS)

In vivo metabolites of ketorolac (KTC) have been identified and characterized by using liquid chromatography positive ion electrospray ionization high resolution tandem mass spectrometry (LC/ESI‐HR‐MS/MS) in combination with online hydrogen/deuterium exchange (HDX) experiments. To identify in vivo metabolites, blood urine and feces samples were collected after oral administration of KTC to Sprague–Dawley rats. The samples were prepared using an optimized sample preparation approach involving protein precipitation and freeze liquid separation followed by solid‐phase extraction and then subjected to LC/HR‐MS/MS analysis. A total of 12 metabolites have been identified in urine samples including hydroxy and glucuronide metabolites, which are also observed in plasma samples. In feces, only O‐sulfate metabolite and unchanged KTC are observed. The structures of metabolites were elucidated using LC‐MS/MS and MSⁿ experiments combined with accurate mass measurements. Online HDX experiments have been used to support the structural characterization of drug metabolites. The main phase I metabolites of KTC are hydroxylated and decarbonylated metabolites, which undergo subsequent phase II glucuronidation pathways. Copyright © 2012 John Wiley & Sons, Ltd.     

Identification of bupropion urinary metabolites by liquid chromatography/mass spectrometry

Human urinary metabolism of the antidepressant bupropion was studied using liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS). A total of 20 metabolites were detected and identified. The phase I metabolism included formation of morpholinohydroxybupropion, threo- and erythrohydrobupropion, aromatic hydroxylation, butyl group hydroxylation with ketone hydrogenation and dihydroxylation. These metabolites were detected either as the free form or as glucuronide and/or sulphate conjugates. In addition also m-chlorohippuric acid was detected. Of the phase I metabolites, a dihydroxylation to the aromatic ring and to the methyl group in the middle of the substrate molecule was reported here for the first time, as well as eight of the glucuronide conjugates (to hydroxy, dihydroxy, hydroxy and hydrogenation metabolites) and three of the sulphate conjugates (to aromatic hydroxy and hydroxy and hydrogenation metabolites).     

Preventive doping control screening analysis of prohibited substances in human urine using rapid‐resolution liquid chromatography/high‐resolution time‐of‐flight mass spectrometry

Unification of the screening protocols for a wide range of doping agents has become an important issue for doping control laboratories. This study presents the development and validation of a generic liquid chromatography/time‐of‐flight mass spectrometry (LC/TOFMS) screening method of 241 small molecule analytes from various categories of prohibited substances (stimulants, narcotics, diuretics, β₂‐agonists, β‐blockers, hormone antagonists and modulators, glucocorticosteroids and anabolic agents). It is based on a single‐step liquid‐liquid extraction of hydrolyzed urine and the use of a rapid‐resolution liquid chromatography/high‐resolution time‐of‐flight mass spectrometric system acquiring continuous full scan data. Electrospray ionization in the positive mode was used. Validation parameters consisted of identification capability, limit of detection, specificity, ion suppression, extraction recovery, repeatability and mass accuracy. Detection criteria were established on the basis of retention time reproducibility and mass accuracy. The suitability of the methodology for doping control was demonstrated with positive urine samples. The preventive role of the method was proved by the case where full scan acquisition with accurate mass measurement allowed the retrospective reprocessing of acquired data from past doping control samples for the detection of a designer drug, the stimulant 4‐methyl‐2‐hexanamine, which resulted in re‐reporting a number of stored samples as positives for this particular substance, when, initially, they had been reported as negatives. Copyright © 2010 John Wiley & Sons, Ltd.     

Identification of metabolites of Helicid in vivo using ultra‐high performance liquid chromatography‐quadrupole time‐of‐flight mass spectrometry

Helicid is an active natural aromatic phenolic glycoside ingredient originating from a well‐known traditional Chinese herbal medicine and has the significant effects of sedative hypnosis, anti‐inflammatory analgesia and antidepressant. In this study, we analyzed the potential metabolites of Helicid in rats by multiple mass defect filter and dynamic background subtraction in ultra‐high‐performance liquid chromatography–quadrupole time‐of‐flight mass spectrometry (UHPLC‐Q‐TOF‐MS). Moreover, we used a novel data processing method, ‘key product ions’, to rapidly detect and identify metabolites as an assistant tool. MetabolitePilot™ 2.0 software and PeakView™ 2.2 software were used for analyzing metabolites. Twenty metabolites of Helicid (including 15 phase I metabolites and five phase II metabolites) were detected by comparison with the blank samples. The biotransformation route of Helicid was identified as demethylation, oxidation, dehydroxylation, hydrogenation, decarbonylation, glucuronide conjugation and methylation. This is the first study simultaneously detecting and identifying Helicid metabolism in rats employing UHPLC‐Q‐TOF‐MS technology. This experiment not only proposed a method for rapidly detecting and identifying metabolites, but also provided useful information for further study of the pharmacology and mechanism of Helicid in vivo. Furthermore, it provided an effective method for the analysis of other aromatic phenolic glycosides metabolic components in vivo.     

In vitro metabolism study of Strychnos alkaloids using high‐performance liquid chromatography combined with hybrid ion trap/time‐of‐flight mass spectrometry

In this report, the in vitro metabolism of Strychnos alkaloids was investigated using liquid chromatography/high‐resolution mass spectrometry for the first time. Strychnine and brucine were selected as model compounds to determine the universal biotransformations of the Strychnos alkaloids in rat liver microsomes. The incubation mixtures were separated by a bidentate‐C₁₈ column, and then analyzed by on‐line ion trap/time‐of‐flight mass spectrometry. With the assistance of mass defect filtering technique, full‐scan accurate mass datasets were processed for the discovery of the related metabolites. The structural elucidations of these metabolites were achieved by comparing the changes in accurate molecular masses, calculating chemical component using Formula Predictor software and defining sites of biotransformation based upon accurate MSⁿ spectral information. As a result, 31 metabolites were identified, of which 26 metabolites were reported for the first time. These biotransformations included hydroxylation, N‐oxidation, epoxidation, methylation, dehydrogenation, de‐methoxylation, O‐demethylation, as well as hydrolysis reactions. Copyright © 2013 John Wiley & Sons, Ltd.     

Structural elucidation of metabolites of ginkgolic acid in rat liver microsomes by ultra‐performance liquid chromatography/electrospray ionization tandem mass spectrometry and hydrogen/deuterium exchange

Ginkgolic acids have been shown to possess allergenic as well as genotoxic and cytotoxic properties. The question arises whether the metabolism of ginkgolic acids in the liver could decrease or increase their toxicity. In this study, the in vitro metabolism of ginkgolic acid (15:1, GA), one component of ginkgo acids, was investigated as a model compound in Sprague‐Dawley rat liver microsomes. The metabolites were analyzed by ultra‐performance liquid chromatography coupled with photodiode array detector/negative‐ion electrospray ionization tandem mass spectrometry (UPLC‐PDA/ESI‐MS/MS) and hydrogen/deuterium (H/D) exchange. The result showed that the benzene ring remained unchanged and the oxidations occurred at the side alkyl chain in rat liver microsomes. At least eight metabolites were found. Among them, six phase I metabolites were tentatively identified. This study might be useful for the investigation of toxicological mechanism of ginkgolic acids. Copyright © 2009 John Wiley & Sons, Ltd.     

New clostebol metabolites in human urine by liquid chromatography time‐of‐flight tandem mass spectrometry and their application for doping control

In this study, clostebol metabolic profiles were investigated carefully. Clostebol was administered to one healthy male volunteer. Urinary extracts were analyzed by liquid chromatography quadrupole time‐of‐flight mass spectrometry (MS) using full scan and targeted MS/MS techniques with accurate mass measurement for the first time. Liquid–liquid extraction and direct injection were applied to processing urine samples. Chromatographic peaks for potential metabolites were found by using the theoretical [M–H]^? as target ion in full scan experiment, and their actual deprotonated ions were analyzed in targeted MS/MS mode. Fourteen metabolites were found for clostebol, and nine unreported metabolites (two free ones and seven sulfate conjugates) were identified by MS, and their potential structures were proposed based on fragmentation and metabolism pathways. Four glucuronide conjugates were also first reported. All the metabolites were evaluated in terms of how long they could be detected and S1 (4ξ‐chloro‐5ξ‐androst‐3ξ‐ol‐17‐one‐3ξ‐sulfate) was considered to be the long‐term metabolite for clostebol misuse detected up to 25 days by liquid–liquid extraction and 14 days by direct injection analysis after oral administration. Five conjugated metabolites (M2, M5, S2, S6 and S7) could also be the alternative biomarkers for clostebol misuse. Copyright © 2015 John Wiley & Sons, Ltd.     

A liquid chromatography/tandem mass spectrometry method for detecting UGT‐mediated bioactivation of drugs as their N‐acetylcysteine adducts in human liver microsomes

The detection of the reactive metabolites of drugs has recently been gaining increasing importance. In vitro trapping studies using trapping agents such as glutathione are usually conducted for the detection of reactive metabolites, especially those of cytochrome P450‐mediated metabolism. In order to detect the UDP‐glucuronosyltransferase (UGT)‐mediated bioactivation of drugs, an in vitro trapping method using N‐acetylcysteine (NAC) as a trapping agent followed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) was developed in this study. After the test compounds (diclofenac and ketoprofen) had been incubated in human liver microsomes with uridine diphosphoglucuronic acid (UDPGA) and NAC, the NAC adducts formed through their acyl glucuronides were analyzed using LC/MS/MS with electrospray ionization (ESI). The NAC adduct showed a mass shift of 145 units as compared to its parent, and the characteristic ion fragmentations reflected the parent. This is a concise and high‐throughput method for evaluating reactive metabolites by UGT‐mediated bioactivation. Copyright © 2009 John Wiley & Sons, Ltd.