Use of LC-MS/MS for the Open Detection of Steroid Metabolites Conjugated with Glucuronic Acid

Fabregat, Andreu; Pozo, Óscar J.; Marcos, Josep; Segura, Jordi; Ventura, Rosa

doi:10.1021/ac4001749

Cited by 96 publications

(115 citation statements)

References 39 publications

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“…The product ion spectrum confirmed the presence of the glucuronide moiety by the typical loss of 176 Da in positive ionization mode and the occurrence of the ion at m/z 113 in negative ionization mode associated with the glucuronide functionality (Fabregat et al, 2013). In addition, the product ion spectrum helped in the assignation of the hydroxylation site ( Supplemental Fig.…”

Section: Mass Spectrometric Results For Mephedrone Metabolitesmentioning

confidence: 66%

Mass Spectrometric Evaluation of Mephedrone In Vivo Human Metabolism: Identification of Phase I and Phase II Metabolites, Including a Novel Succinyl Conjugate

et al. 2014

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In recent years, many new designer drugs have emerged, including the group of cathinone derivatives. One frequently occurring drug is mephedrone; although mephedrone was originally considered as a "legal high" product, it is currently banned in most Western countries. Despite the banning, abuse of the drug and seizures are continuously reported. Although the metabolism of mephedrone has been studied in rats or in vitro using human liver microsomes, to the best of our knowledge, no dedicated study with human volunteers has been performed for studying the in vivo metabolism of mephedrone in humans. Therefore, the aim of this study was to establish the actual human metabolism of mephedrone and to compare it with other models. For this purpose, urine samples of two healthy volunteers, who ingested 200 mg mephedrone orally, were taken before administration and 4 hours after substance intake. The discovery and identification of the phase I and phase II metabolites of mephedrone were based on ultra-high-performance liquid chromatography coupled to hybrid quadrupole time-of-flight mass spectrometry, operating in the so-called MS E mode. Six phase I metabolites and four phase II metabolites were identified, four of them not previously reported in the literature. The structure of four of the detected metabolites was confirmed by synthesis of the suggested compounds. Remarkably, a mephedrone metabolite conjugated with succinic acid has been identified and confirmed by synthesis. According to the reviewed literature, this is the first time that this type of conjugate is reported for human metabolism.

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Section: Mass Spectrometric Results For Mephedrone Metabolitesmentioning

confidence: 66%

Mass Spectrometric Evaluation of Mephedrone In Vivo Human Metabolism: Identification of Phase I and Phase II Metabolites, Including a Novel Succinyl Conjugate

et al. 2014

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“…The internal standards were b-boldenone sulphate-d3 for the sulphate forms, b-boldenoned3 for the free forms (LGC Standards, Teddington, UK) and epitestosterone (EpiT) glucuronide-d3 for the glucuronate forms (National Measurement Institute, Pymble, NSW, Australia). The sulphate and glucuronate forms of a-boldenone, provided by research partners, were prepared with a two-step synthesis procedure, in which b-boldenone (Steroid SpA, Cologno Monzese, Milan, Italy) was epimerized using a modified Mitsunobu protocol, according to Dodge and Lugar [23,24], which was followed by sulphation, according to Sanaullah and Bowers [25], or glucuronation, according to Casati et al [26]. Cortisone, cortisol, prednisone, prednisolone and dexamethasone were purchased …”

Section: Chemicals and Reagentsmentioning

confidence: 99%

Detection of boldenone, its conjugates and androstadienedione, as well as five corticosteroids in bovine bile through a unique immunoaffinity column clean-up and two validated liquid chromatography–tandem mass spectrometry analyses

Chiesa

Nobile

Panseri

et al. 2014

Analytica Chimica Acta

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The presence of β-boldenone II phase metabolites and prednisolone in urine samples, owing to endogenous or natural origin or illicit treatment, is under debate within the European Union. The detection of β-boldenone conjugates, α-boldenone conjugates at concentrations higher than 2 ng mL(-1) and prednisolone above the cut-off level of 5 ng mL(-1) in urine have been, until now, critical in deciding if illegal drug use has occurred. The use of urine sometimes is not entirely satisfactory, especially when the drug is administrated at low doses or when its metabolic conversion is very fast. This subsequently would hamper its detection in urine. The introduction of a new, advantageous matrix where the illicit treatment can be investigated would be highly appreciated. In this study, we have developed and validated a simple and unique immunoaffinity clean-up procedure, which was applied to bovine bile samples, followed by two different analytical liquid chromatography-electrospray-tandem mass spectrometry methods. The first method tests androstadienedione, α- and β-boldenone sulphate, glucuronate and related free forms, while the other method assays prednisolone, prednisone, dexamethasone, cortisone, and cortisol. The methods were validated according to European Commission Decision 2002/657/EC. The evaluated parameters were linearity, specificity, precision (repeatability and intra-laboratory reproducibility), recovery, decision limit and detection capability. The decision limits (CCα) were between 0.38 and 0.45 ng mL(-1) for anabolic steroids, and 0.13 and 0.15 ng mL(-1) as far as corticosteroids were concerned. Intra- and inter-day repeatability was below 15.8 and 19.9% for all analytes, respectively. The methods were applied to the analysis of some bile samples collected from untreated young bulls in order to investigate the presence of the studied steroids in this matrix.

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“…The discovery of alternative markers which might complement the steroid profile is currently an active research topic in the doping control field. Several alternative T metabolites have been proposed for this purpose 18,19 . The application of target metabolomic approaches has been described as a useful tool for the selection of new markers 20,21 .…”

Section: Introductionmentioning

confidence: 99%

Untargeted Metabolomics in Doping Control: Detection of New Markers of Testosterone Misuse by Ultrahigh Performance Liquid Chromatography Coupled to High-Resolution Mass Spectrometry

et al. 2015

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ABSTRACT:The use of untargeted metabolomics for the discovery of markers is a promising and virtually unexplored tool in the doping control field. Hybrid quadrupole time-of-flight (QTOF) and hybrid quadrupole Orbitrap (Q Exactive) mass spectrometers, coupled to ultra-high pressure liquid chromatography are excellent tools for this purpose. In the present work, QTOF and Q Exactive have been used to look for markers for testosterone cypionate misuse by means of untargeted metabolomics. Two different groups of urine samples were analyzed; collected before and after the intramuscular administration of testosterone cypionate. In order to avoid analyte losses in the sample treatment, samples were just two-fold diluted with water, and directly injected into the chromatographic system. Samples were analyzed in both positive and negative ionization modes. Data from both systems were treated under untargeted metabolomic strategies using XCMS application and multivariate analysis. Results from the two mass spectrometers differed in the number of detected features, but both led to the same potential marker for the particular testosterone ester misuse. The in-depth study of the MS and MS/MS behavior of this marker allowed for the establishment of 1-cyclopentenoylglycine as feasible structure. The putative structure was confirmed by comparison with synthesized material. This potential marker seems to come from the metabolism of the cypionic acid release after hydrolysis of the administered ester. Its suitability for doping control has been evaluated.

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Use of LC-MS/MS for the Open Detection of Steroid Metabolites Conjugated with Glucuronic Acid

Cited by 96 publications

References 39 publications

Mass Spectrometric Evaluation of Mephedrone In Vivo Human Metabolism: Identification of Phase I and Phase II Metabolites, Including a Novel Succinyl Conjugate

Mass Spectrometric Evaluation of Mephedrone In Vivo Human Metabolism: Identification of Phase I and Phase II Metabolites, Including a Novel Succinyl Conjugate

Detection of boldenone, its conjugates and androstadienedione, as well as five corticosteroids in bovine bile through a unique immunoaffinity column clean-up and two validated liquid chromatography–tandem mass spectrometry analyses

Untargeted Metabolomics in Doping Control: Detection of New Markers of Testosterone Misuse by Ultrahigh Performance Liquid Chromatography Coupled to High-Resolution Mass Spectrometry

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