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Wilhelm Schanzer - One of the best experts on this subject based on the ideXlab platform.
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unexpected contribution of cytochrome p450 enzymes cyp11b2 and cyp21 as well as cyp3a4 in xenobiotic androgen elimination insights from Metandienone metabolism
Toxicology Letters, 2012Co-Authors: Gregor Fusholler, Mariakristina Parr, Nils Schlörer, Matthias Bureik, Andy Zollner, Georg Opfermann, Mirela Zorio, Wilhelm SchanzerAbstract:Abstract The metabolism of a variety of anabolic steroids frequently misused for doping purposes has been investigated in the last years. This research mainly focused on main and long-term metabolites suitable for detection, but detailed clearance mechanisms have rarely been elucidated. Recent studies on Metandienone focused on the identification of 17β-hydroxymethyl-17α-methyl-18-norandrosta-1,4,13-trien-3-one (20βOH-NorMD) as long-term metabolite, however, the metabolic pathway of its generation remained unclear. Metandienone and its Wagner–Meerwein rearrangement product 17,17-dimethyl-18-norandrosta-1,4,13-trien-3-one (NorMD) were hydroxylated by different human cytochrome P450 enzymes (CYPs). Some of their hydroxylation products were chemically synthesized and characterized by mass spectrometry to allow for their trace detection in urine samples. Following oral administration of Metandienone or NorMD in one human volunteer each the post administration urines were checked for the presence of those hydroxylated metabolites using GC–MS/MS analysis. The human mitochondrial steroid hydroxylating enzymes CYP11B1 and CYP11B2 were capable to metabolize Metandienone leading to the formation of 11β-hydroxyMetandienone and 18-hydroxyMetandienone. Following Wagner–Meerwein rearrangement, the resulting products could be assigned to 20βOH-NorMD and 11βOH-NorMD. The contribution of CYP11B1 and CYP11B2 in human metabolism of Metandienone was confirmed by analysis of post-administration samples of Metandienone and NorMD. Combined with the results from a previous study, enzymatic pathways were identified that involve CYP21 and CYP3A4 in the hydroxylation of NorMD, while CYP21, CYP3A4 and CYP11B2 take part in 20βOH-NorMD generation from MD. The current study represents a valuable contribution to the elucidation of clearance mechanisms of anabolic steroids and also indicates that mainly non-liver CYPs seem to be involved in these processes
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sensitive determination of prohibited drugs in dried blood spots dbs for doping controls by means of a benchtop quadrupole orbitrap mass spectrometer
Analytical and Bioanalytical Chemistry, 2012Co-Authors: Andreas Thomas, Wilhelm Schanzer, Hans Geyer, Catharina Crone, Markus Kellmann, Thomas Moehring, Mario ThevisAbstract:In the present study, a new type of mass spectrometer combining a quadrupole mass filter, a higher collision dissociation (HCD) cell and an Orbitrap detector, was evaluated for the analysis of dried blood spots (DBS) in doping controls. DBS analysis is characterized by the necessity to detect prohibited compounds in sub-nanogram-per-milliliter levels with high identification capacity. After extraction of DBS with an organic solvent and liquid chromatographic separation (using a regular C18-RP-analytical UHPLC-column) of target analytes, mass spectrometry is performed with a high-resolution full scan in positive and negative mode by means of electrospray ionisation. Single-product ion mass spectra are acquired using the data-dependent analysis mode (employing an inclusion list) for previously selected precursors of known prohibited compounds with fixed retention time ranges. Besides, a sensitive screening in a targeted approach, non-targeted analysis for retrospective data evaluation is thus possible. The chosen experimental design enables the determination of various drugs from different classes with one generic sample preparation which is shown for 26 selected model compounds (Δ9-tetrahydrocannabinol (THC), tetrahydrocannabinol-9-carboxylic acid (THC-COOH), methylhexaneamine, methylphenidate, cocaine, nikethamide, 3,4-methylenedioxyamphetamine, N-methyl-3,4-methylenedioxyamphetamine, strychnine, mesocarb, salbutamol, formoterol, clenbuterol, Metandienone, stanozolol, bisoprolol, propranolol, metoprolol, anastrazole, clomiphene, exemestane, dexamethasone, budesonide, selective androgen receptor modulator (SARM) S4 (andarine), SARM S1, hydrochlorothiazide). Generally, only qualitative result interpretation was focussed upon, but for target analytes with deuterium-labelled internal standards (salbutamol, clenbuterol, cocaine, dexamethasone, THC-COOH and THC) quantitative analysis was also possible. Especially the most challenging analytes, THC and its carboxy metabolite, were detected in DBS at relevant concentrations (<0.5 ng/mL) using targeted HCD experiments. The method was validated for the parameters: specificity, linearity (0–20 ng/mL), precision (<25%), recovery (mean 60%), limit of detection/quantification, ion suppression, stability and accuracy (80–120%). Six isotope-labelled analogues used as internal standards facilitate a quantitative result interpretation which is of utmost importance especially for in-competition drug sports testing.
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cyp21 catalyzed production of the long term urinary Metandienone metabolite 17beta hydroxymethyl 17 alpha methyl 18 norandrosta 1 4 13 trien 3 one a contribution to the fight against doping
Biological Chemistry, 2010Co-Authors: Andy Zollner, Wilhelm Schanzer, Mariakristina Parr, Nils Schlörer, Călinaurel Drăgan, Stefan Dras, Frank T Peters, Hans H Maurer, Matthias BureikAbstract:Anabolic-androgenic steroids are some of the most frequently misused drugs in human sports. Recently, a previously unknown urinary metabolite of Metandienone, 17beta-hydroxymethyl-17 alpha-methyl-18-norandrosta-1,4,13-trien-3-one (20OH-NorMD), was discovered via LC-MS/MS and GC-MS. This metabolite was reported to be detected in urine samples up to 19 days after administration of Metandienone. However, so far it was not possible to obtain purified reference material of this metabolite and to confirm its structure via NMR. Eleven recombinant strains of the fission yeast Schizosaccharomyces pombe that express different human hepatic or steroidogenic cytochrome P450 enzymes were screened for production of this metabolite in a whole-cell biotransformation reaction. 17,17-Dimethyl-18-norandrosta-1,4,13-trien-3-one, chemically derived from Metandienone, was used as substrate for the bioconversion, because it could be converted to the final product in a single hydroxylation step. The obtained results demonstrate that CYP21 and to a lesser extent also CYP3A4 expressing strains can catalyze this steroid hydroxylation. Subsequent 5 l-scale fermentation resulted in the production and purification of 10 mg of metabolite and its unequivocal structure determination via NMR. The synthesis of this urinary Metandienone metabolite via S. pombe-based whole-cell biotransformation now allows its use as a reference substance in doping control assays.
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combination of liquid chromatography tandem mass spectrometry in different scan modes with human and chimeric mouse urine for the study of steroid metabolism
Drug Testing and Analysis, 2009Co-Authors: Oscar J Pozo, Wilhelm Schanzer, Mariakristina Parr, Leen Lootens, Philip Meuleman, Peter Van Eenoo, Geert Lerouxroels, Koen Deventer, F T DelbekeAbstract:Anabolic steroids are among the most frequently detected compounds in doping analysis. They are extensively metabolized and therefore an in-depth knowledge about steroid metabolism is needed. In this study, a liquid chromatography tandem mass spectometry (LC-MS/MS) method based on a precursor ion scan with a uPA-SCID mouse with humanized liver (a chimeric mouse) was explored for the detection of steroid metabolism. Methandienone was used as a model compound. The application of the precursor ion scan method in positive human samples and chimeric mice samples after methandienone administration allowed the detection of most steroid metabolites without any structural restriction. Three hitherto unreported metabolites were found using this approach. These metabolites were characterized using LC-MS/MS and feasible structures were proposed. The structure of one of them, 6-ene-epimethandienone, was confirmed by the synthesis of the reference compound. A selected reaction monitoring (SRM) method for the specific detection of all these metabolites has been developed. The application of this method to several human and chimeric mouse samples confirmed that more than 80% of the steroid metabolites were found in both samples. Only metabolites that are poorly detectable by LC-MS/MS were not detected in some urine samples. The metabolic nature of the unreported metabolites was also confirmed. A global strategy for the detection of steroid metabolites combining both human and chimeric mouse urine is proposed. Copyright (C) 2009 John Wiley & Sons, Ltd.
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liquid chromatographic mass spectrometric analysis of glucuronide conjugated anabolic steroid metabolites method validation and interlaboratory comparison
Journal of Mass Spectrometry, 2008Co-Authors: Laura Hintikka, Wilhelm Schanzer, Mario Thevis, Antti Leinonen, Tiia Kuuranne, John M Halket, David A Cowan, Joachim Grosse, Peter Hemmersbach, Michel W F NielenAbstract:Liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method for simultaneous and direct detection of 12 glucuronide-conjugated anabolic androgenic steroid (AAS) metabolites in human urine is described. The compounds selected were the main metabolites detected in human urine after dosing of the most widely abused AAS in sports, e.g. methandienone, methenolone, methyltestosterone, nandrolone and testosterone, and certain deuterium-labeled analogs of these metabolites. Sample preparation and the LC-ESI-MS/MS method were optimized, validated, and the overall process was implemented and the results between seven laboratories were compared. All the metabolites were extracted simultaneously by solid-phase extraction (SPE) and analyzed by LC-ESI-MS/MS with positive ionization mode and multiple reaction monitoring (MRM). Recovery of the SPE for the AAS glucuronides was 89-100% and ten out of twelve compounds had detection limits in the range of 1-10 ng/ml in urine. The results for inter/intraday repeatability were satisfactory and the interlaboratory comparison with authentic urine samples demonstrated the ease of method transfer from one instrument setup to another. When equivalent triple quadrupole analyzers were employed the overall performance was independent from instrument manufacturer, electrospray ionisation (ESI) or atmospheric pressure chemical ionization (APCI) and liquid chromatohraphic (LC) column, whereas major differences were encountered when changing from one analyzer type to another, especially in the analysis of those AAS glucuronides ionized mainly as adducts.
Mariakristina Parr - One of the best experts on this subject based on the ideXlab platform.
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drug drug interaction and doping effect of non prohibited drugs on the urinary excretion profile of methandienone
Drug Testing and Analysis, 2018Co-Authors: Monica Mazzarino, Xavier De La Torre, I. Fiacco, Mariakristina Parr, Franziska L Khevenhullermetsch, Francesco BotrèAbstract:The potential consequences of drug-drug interactions on the excretion profile of the anabolic androgenic steroid methandienone (17β-hydroxy-17α-methylandrosta-1,4-dien-3-one) are discussed. More specifically, we have evaluated by in vitro and in vivo experiments the effects of 7 non-prohibited drugs (fluconazole, ketoconazole, itraconazole, miconazole, fluoxetine, paroxetine, and nefazodone) on the main metabolic pathways of methandienone. These are selected among those most commonly used by the athletes. The in vitro assays were based on the use of human liver microsomes, specific recombinant enzyme isoforms of cytochrome P450 and uridine 5'-diphospho-glucuronosyl-transferase. The in vivo study was performed by analyzing urines collected after the oral administration of methandienone with and without the co-administration of ketoconazole. Methandienone and its metabolites were determined by liquid chromatography-mass spectrometry-based techniques after sample pretreatment including an enzymatic hydrolysis step (performed only for the investigation on phase II metabolism) and liquid/liquid extraction with t-butyl methyl-ether. The results from the in vitro experiments showed that the formation of the hydroxylated and dehydrogenated metabolites was significantly reduced in the presence of itraconazole, ketoconazole, miconazole and nefazodone, whereas the production of the 18-nor-hydroxylated metabolites and glucuronidation reactions was reduced significantly only in the presence of ketoconazole and miconazole. The analysis of the post-administration samples confirmed the in vitro observations, validating the hypothesis that drug-drug interaction may cause significant alterations in the metabolic profile of banned drugs, making their detection during doping control tests more challenging.
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unexpected contribution of cytochrome p450 enzymes cyp11b2 and cyp21 as well as cyp3a4 in xenobiotic androgen elimination insights from Metandienone metabolism
Toxicology Letters, 2012Co-Authors: Gregor Fusholler, Mariakristina Parr, Nils Schlörer, Matthias Bureik, Andy Zollner, Georg Opfermann, Mirela Zorio, Wilhelm SchanzerAbstract:Abstract The metabolism of a variety of anabolic steroids frequently misused for doping purposes has been investigated in the last years. This research mainly focused on main and long-term metabolites suitable for detection, but detailed clearance mechanisms have rarely been elucidated. Recent studies on Metandienone focused on the identification of 17β-hydroxymethyl-17α-methyl-18-norandrosta-1,4,13-trien-3-one (20βOH-NorMD) as long-term metabolite, however, the metabolic pathway of its generation remained unclear. Metandienone and its Wagner–Meerwein rearrangement product 17,17-dimethyl-18-norandrosta-1,4,13-trien-3-one (NorMD) were hydroxylated by different human cytochrome P450 enzymes (CYPs). Some of their hydroxylation products were chemically synthesized and characterized by mass spectrometry to allow for their trace detection in urine samples. Following oral administration of Metandienone or NorMD in one human volunteer each the post administration urines were checked for the presence of those hydroxylated metabolites using GC–MS/MS analysis. The human mitochondrial steroid hydroxylating enzymes CYP11B1 and CYP11B2 were capable to metabolize Metandienone leading to the formation of 11β-hydroxyMetandienone and 18-hydroxyMetandienone. Following Wagner–Meerwein rearrangement, the resulting products could be assigned to 20βOH-NorMD and 11βOH-NorMD. The contribution of CYP11B1 and CYP11B2 in human metabolism of Metandienone was confirmed by analysis of post-administration samples of Metandienone and NorMD. Combined with the results from a previous study, enzymatic pathways were identified that involve CYP21 and CYP3A4 in the hydroxylation of NorMD, while CYP21, CYP3A4 and CYP11B2 take part in 20βOH-NorMD generation from MD. The current study represents a valuable contribution to the elucidation of clearance mechanisms of anabolic steroids and also indicates that mainly non-liver CYPs seem to be involved in these processes
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cyp21 catalyzed production of the long term urinary Metandienone metabolite 17beta hydroxymethyl 17 alpha methyl 18 norandrosta 1 4 13 trien 3 one a contribution to the fight against doping
Biological Chemistry, 2010Co-Authors: Andy Zollner, Wilhelm Schanzer, Mariakristina Parr, Nils Schlörer, Călinaurel Drăgan, Stefan Dras, Frank T Peters, Hans H Maurer, Matthias BureikAbstract:Anabolic-androgenic steroids are some of the most frequently misused drugs in human sports. Recently, a previously unknown urinary metabolite of Metandienone, 17beta-hydroxymethyl-17 alpha-methyl-18-norandrosta-1,4,13-trien-3-one (20OH-NorMD), was discovered via LC-MS/MS and GC-MS. This metabolite was reported to be detected in urine samples up to 19 days after administration of Metandienone. However, so far it was not possible to obtain purified reference material of this metabolite and to confirm its structure via NMR. Eleven recombinant strains of the fission yeast Schizosaccharomyces pombe that express different human hepatic or steroidogenic cytochrome P450 enzymes were screened for production of this metabolite in a whole-cell biotransformation reaction. 17,17-Dimethyl-18-norandrosta-1,4,13-trien-3-one, chemically derived from Metandienone, was used as substrate for the bioconversion, because it could be converted to the final product in a single hydroxylation step. The obtained results demonstrate that CYP21 and to a lesser extent also CYP3A4 expressing strains can catalyze this steroid hydroxylation. Subsequent 5 l-scale fermentation resulted in the production and purification of 10 mg of metabolite and its unequivocal structure determination via NMR. The synthesis of this urinary Metandienone metabolite via S. pombe-based whole-cell biotransformation now allows its use as a reference substance in doping control assays.
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combination of liquid chromatography tandem mass spectrometry in different scan modes with human and chimeric mouse urine for the study of steroid metabolism
Drug Testing and Analysis, 2009Co-Authors: Oscar J Pozo, Wilhelm Schanzer, Mariakristina Parr, Leen Lootens, Philip Meuleman, Peter Van Eenoo, Geert Lerouxroels, Koen Deventer, F T DelbekeAbstract:Anabolic steroids are among the most frequently detected compounds in doping analysis. They are extensively metabolized and therefore an in-depth knowledge about steroid metabolism is needed. In this study, a liquid chromatography tandem mass spectometry (LC-MS/MS) method based on a precursor ion scan with a uPA-SCID mouse with humanized liver (a chimeric mouse) was explored for the detection of steroid metabolism. Methandienone was used as a model compound. The application of the precursor ion scan method in positive human samples and chimeric mice samples after methandienone administration allowed the detection of most steroid metabolites without any structural restriction. Three hitherto unreported metabolites were found using this approach. These metabolites were characterized using LC-MS/MS and feasible structures were proposed. The structure of one of them, 6-ene-epimethandienone, was confirmed by the synthesis of the reference compound. A selected reaction monitoring (SRM) method for the specific detection of all these metabolites has been developed. The application of this method to several human and chimeric mouse samples confirmed that more than 80% of the steroid metabolites were found in both samples. Only metabolites that are poorly detectable by LC-MS/MS were not detected in some urine samples. The metabolic nature of the unreported metabolites was also confirmed. A global strategy for the detection of steroid metabolites combining both human and chimeric mouse urine is proposed. Copyright (C) 2009 John Wiley & Sons, Ltd.
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mass spectrometric identification and characterization of a new long term metabolite of Metandienone in human urine
Rapid Communications in Mass Spectrometry, 2006Co-Authors: Wilhelm Schanzer, Hans Geyer, Gregor Fusholler, Natalia Halatcheva, Maxie Kohler, Mariakristina Parr, Sven Guddat, Andreas Thomas, Mario ThevisAbstract:Anabolic-androgenic steroids are some of the most frequently detected drugs in amateur and professional sports. Doping control laboratories have developed numerous assays enabling the determination of administered drugs and/or their metabolic products that allow retrospectives with respect to pharmacokinetics and excretion profiles of steroids and their metabolites. A new metabolite generated from Metandienone has been identified as 18-nor-17beta-hydroxymethyl,17alpha-methyl-androst-1,4,13-trien-3-one in excretion study urine samples providing a valuable tool for the long-term detection of Metandienone abuse by athletes in sports drug testing. The metabolite was characterized using gas chromatography/(tandem) mass spectrometry, liquid chromatography/tandem mass spectrometry and liquid chromatography/high-resolution/high-accuracy (tandem) mass spectrometry by characteristic fragmentation patterns representing the intact 3-keto-1,4-diene structure in combination with typical product ions substantiating the proposed C/D-ring structure of the steroid metabolite. In addition, structure confirmation was obtained by the analysis of excretion study urine specimens obtained after administration of 17-CD(3)-labeled Metandienone providing the deuterated analogue to the newly identified metabolite. 18-Nor-17beta-hydroxymethyl,17alpha-methyl-androst-1,4,13-trien-3-one was determined in Metandienone administration study urine specimens up to 19 days after application of a single dose of 5 mg, hence providing an extended detection period compared with commonly employed strategies.
Oscar J Pozo - One of the best experts on this subject based on the ideXlab platform.
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lc ms ms detection of unaltered glucuronoconjugated metabolites of Metandienone
Drug Testing and Analysis, 2017Co-Authors: Argitxu Esquivel, Cristina Gomez, Oscar J Pozo, Lorena Garrostas, Georgina Balcells, Aristotelis Kotronoulas, Jesus Joglar, Rosa VenturaAbstract:The aim of this study was to evaluate the direct detection of glucuronoconjugated metabolites of Metandienone (MTD) and their detection times. Metabolites resistant to enzymatic hydrolysis were also evaluated. Based on the common mass spectrometric behaviour of steroid glucuronides, three liquid chromatography-tandem mass spectrometry (LC-MS/MS) strategies were applied for the detection of unpredicted and predicted metabolites: precursor ion scan (PI), neutral loss scan (NL), and theoretical selected reaction monitoring (SRM) methods. Samples from four excretion studies of MTD were analyzed for both the detection of metabolites and the establishment of their detection times. Using PI and NL methods, seven metabolites were observed in post-administration samples. SRM methods allowed for the detection of 13 glucuronide metabolites. The detection times, measured by analysis with an SRM method, were between 1 and 22 days. The metabolite detected for the longest time was 18-nor-17β-hydroxymethyl-17α-methyl-5β-androsta-1,4,13-triene-3-one-17-glucuronide. One metabolite was resistant to hydrolysis with β-glucuronidase; however it was only detected in urine up to four days after administration. The three glucuronide metabolites with the highest retrospectivity were identified by chemical synthesis or mass spectrometric data, and although they were previously reported, this is the first time that analytical data of the intact phase II metabolites are presented for some of them. The LC-MS/MS strategies applied have demonstrated to be useful for detecting glucuronoconjugated metabolites of MTD, including glucuronides resistant to enzymatic hydrolysis which cannot be detected by conventional approaches. Copyright © 2016 John Wiley & Sons, Ltd.
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a new sulphate metabolite as a long term marker of Metandienone misuse
Steroids, 2013Co-Authors: Cristina Gomez, Oscar J Pozo, Lorena Garrostas, Jordi Segura, Rosa VenturaAbstract:Abstract Metandienone is one of the most frequently detected anabolic androgenic steroids in sports drug testing. Metandienone misuse is commonly detected by monitoring different metabolites excreted free or conjugated with glucuronic acid using gas chromatography mass spectrometry (GC–MS) and liquid chromatography tandem mass spectrometry (LC–MS/MS) after hydrolysis with β-glucuronidase and liquid–liquid extraction. It is known that several metabolites are the result of the formation of sulphate conjugates in C17, which are converted to their 17-epimers in urine. Therefore, sulphation is an important phase II metabolic pathway of Metandienone that has not been comprehensively studied. The aim of this work was to evaluate the sulphate fraction of Metandienone metabolism by LC–MS/MS. Seven sulphate metabolites were detected after the analysis of excretion study samples by applying different neutral loss scan, precursor ion scan and SRM methods. One of the metabolites (M1) was identified and characterised by GC–MS/MS and LC–MS/MS as 18-nor-17β-hydroxymethyl-17α-methylandrost-1,4,13-triene-3-one sulphate. M1 could be detected up to 26 days after the administration of a single dose of Metandienone (5 mg), thus improving the period in which the misuse can be reported with respect to the last long-term Metandienone metabolite described (18-nor-17β-hydroxymethyl-17α-methylandrost-1,4,13-triene-3-one excreted in the glucuronide fraction).
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comparison between triple quadrupole time of flight and hybrid quadrupole time of flight analysers coupled to liquid chromatography for the detection of anabolic steroids in doping control analysis
Analytica Chimica Acta, 2011Co-Authors: Oscar J Pozo, Rosa Ventura, Peter Van Eenoo, Koen Deventer, Hisham Elbardissy, Susana Grimalt, Juan V Sancho, Felix Hernandez, Frans DelbekeAbstract:Triple quadrupole (QqQ), time of flight (TOF) and quadrupole-time of flight (QTOF) analysers have been compared for the detection of anabolic steroids in human urine. Ten anabolic steroids were selected as model compounds based on their ionization and the presence of endogenous interferences. Both qualitative and quantitative analyses were evaluated. QqQ allowed for the detection of all analytes at the minimum required performance limit (MRPL) established by the World Anti-Doping Agency (between 2 and 10 ng mL(-1) in urine). TOF and QTOF approaches were not sensitive enough to detect some of the analytes (3'-hydroxy-stanozolol or the metabolites of boldenone and formebolone) at the established MRPL. Although a suitable accuracy was obtained, the precision was unsatisfactory (RSD typically higher than 20%) for quantitative purposes irrespective of the analyser used. The methods were applied to 30 real samples declared positives either for the misuse of boldenone, stanozolol and/or methandienone. Most of the compounds were detected by every technique, however QqQ was necessary for the detection of some metabolites in a few samples. Finally, the possibility to detect non-target steroids has been explored by the use of TOF and QTOF. The use of this approach revealed that the presence of boldenone and its metabolite in one sample was due to the intake of androsta-1,4,6-triene-3,17-dione. Additionally, the intake of methandienone was confirmed by the post-target detection of a long-term metabolite.
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combination of liquid chromatography tandem mass spectrometry in different scan modes with human and chimeric mouse urine for the study of steroid metabolism
Drug Testing and Analysis, 2009Co-Authors: Oscar J Pozo, Wilhelm Schanzer, Mariakristina Parr, Leen Lootens, Philip Meuleman, Peter Van Eenoo, Geert Lerouxroels, Koen Deventer, F T DelbekeAbstract:Anabolic steroids are among the most frequently detected compounds in doping analysis. They are extensively metabolized and therefore an in-depth knowledge about steroid metabolism is needed. In this study, a liquid chromatography tandem mass spectometry (LC-MS/MS) method based on a precursor ion scan with a uPA-SCID mouse with humanized liver (a chimeric mouse) was explored for the detection of steroid metabolism. Methandienone was used as a model compound. The application of the precursor ion scan method in positive human samples and chimeric mice samples after methandienone administration allowed the detection of most steroid metabolites without any structural restriction. Three hitherto unreported metabolites were found using this approach. These metabolites were characterized using LC-MS/MS and feasible structures were proposed. The structure of one of them, 6-ene-epimethandienone, was confirmed by the synthesis of the reference compound. A selected reaction monitoring (SRM) method for the specific detection of all these metabolites has been developed. The application of this method to several human and chimeric mouse samples confirmed that more than 80% of the steroid metabolites were found in both samples. Only metabolites that are poorly detectable by LC-MS/MS were not detected in some urine samples. The metabolic nature of the unreported metabolites was also confirmed. A global strategy for the detection of steroid metabolites combining both human and chimeric mouse urine is proposed. Copyright (C) 2009 John Wiley & Sons, Ltd.
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upa scid mouse with humanized liver as a model for in vivo metabolism of exogenous steroids methandienone as a case study
Clinical Chemistry, 2009Co-Authors: Leen Lootens, Oscar J Pozo, Philip Meuleman, Peter Van Eenoo, Geert Lerouxroels, F T DelbekeAbstract:Background: Adequate detection of designer steroids in the urine of athletes is still a challenge in doping control analysis and requires knowledge of steroid metabolism. In this study we investigated whether uPA+/+-SCID mice carrying functional primary human hepatocytes in their liver would provide a suitable alternative small animal model for the investigation of human steroid metabolism in vivo. Methods: A quantitative method based on liquid chromatography–tandem mass spectrometry (LC-MS/MS) was developed and validated for the urinary detection of 7 known methandienone metabolites. Application of this method to urine samples from humanized mice after methandienone administration allowed for comparison with data from in vivo human samples and with reported methandienone data from in vitro hepatocyte cultures. Results: The LC-MS/MS method validation in mouse and human urine indicated good linearity, precision, and recovery. Using this method we quantified 6 of 7 known human methandienone metabolites in the urine of chimeric mice, whereas in control nonchimeric mice we detected only 2 metabolites. These results correlated very well with methandienone metabolism in humans. In addition, we detected 4 isomers of methandienone metabolites in both human and chimeric mouse urine. One of these isomers has never been reported before. Conclusions: The results of this proof-of-concept study indicate that the human liver–uPA+/+-SCID mouse appears to be a suitable small animal model for the investigation of human-type metabolism of anabolic steroids and possibly also for other types of drugs and medications. .
Mario Thevis - One of the best experts on this subject based on the ideXlab platform.
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(www.interscience.wiley.com) DOI: 10.1002/jms.1452 Review
2014Co-Authors: Mario ThevisAbstract:Since 1999 several groups have analyzed nutritional supplements with mass spectrometric methods (GC/MS, LC/MS/MS) for contaminations and adulterations with doping substances. These investigations showed that nutritional supplements contained prohibited stimulants as ephedrines, caffeine, methylenedioxymetamphetamie and sibutramine, which were not declared on the labels. An international study performed in 2001 and 2002 on 634 nutritional supplements that were purchased in 13 different countries showed that about 15 % of the nonhormonal nutritional supplements were contaminated with anabolic-androgenic steroids (mainly prohormones). Since 2002, also products intentionally faked with high amounts of ‘classic ’ anabolic steroids such as Metandienone, stanozolol, boldenone, dehydrochloromethyl-testosterone, oxandrolone etc. have been detected on the nutritional supplement market. These anabolic steroids were not declared on the labels either. The sources of these anabolic steroids are probably Chinese pharmaceutical companies, which sell bulk material of anabolic steroids. In 2005 vitamin C, multivitamin and magnesium tablets were confiscated, which contained cross-contaminations of stanozolol and Metandienone. Since 2002 new ‘designer ’ steroids such as prostanozol, methasterone, androstatrienedione etc. have been offered on the nutritional supplement market. In the near future also cross-contaminations with these steroids are expected. Recently a nutritional supplemen
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sensitive determination of prohibited drugs in dried blood spots dbs for doping controls by means of a benchtop quadrupole orbitrap mass spectrometer
Analytical and Bioanalytical Chemistry, 2012Co-Authors: Andreas Thomas, Wilhelm Schanzer, Hans Geyer, Catharina Crone, Markus Kellmann, Thomas Moehring, Mario ThevisAbstract:In the present study, a new type of mass spectrometer combining a quadrupole mass filter, a higher collision dissociation (HCD) cell and an Orbitrap detector, was evaluated for the analysis of dried blood spots (DBS) in doping controls. DBS analysis is characterized by the necessity to detect prohibited compounds in sub-nanogram-per-milliliter levels with high identification capacity. After extraction of DBS with an organic solvent and liquid chromatographic separation (using a regular C18-RP-analytical UHPLC-column) of target analytes, mass spectrometry is performed with a high-resolution full scan in positive and negative mode by means of electrospray ionisation. Single-product ion mass spectra are acquired using the data-dependent analysis mode (employing an inclusion list) for previously selected precursors of known prohibited compounds with fixed retention time ranges. Besides, a sensitive screening in a targeted approach, non-targeted analysis for retrospective data evaluation is thus possible. The chosen experimental design enables the determination of various drugs from different classes with one generic sample preparation which is shown for 26 selected model compounds (Δ9-tetrahydrocannabinol (THC), tetrahydrocannabinol-9-carboxylic acid (THC-COOH), methylhexaneamine, methylphenidate, cocaine, nikethamide, 3,4-methylenedioxyamphetamine, N-methyl-3,4-methylenedioxyamphetamine, strychnine, mesocarb, salbutamol, formoterol, clenbuterol, Metandienone, stanozolol, bisoprolol, propranolol, metoprolol, anastrazole, clomiphene, exemestane, dexamethasone, budesonide, selective androgen receptor modulator (SARM) S4 (andarine), SARM S1, hydrochlorothiazide). Generally, only qualitative result interpretation was focussed upon, but for target analytes with deuterium-labelled internal standards (salbutamol, clenbuterol, cocaine, dexamethasone, THC-COOH and THC) quantitative analysis was also possible. Especially the most challenging analytes, THC and its carboxy metabolite, were detected in DBS at relevant concentrations (<0.5 ng/mL) using targeted HCD experiments. The method was validated for the parameters: specificity, linearity (0–20 ng/mL), precision (<25%), recovery (mean 60%), limit of detection/quantification, ion suppression, stability and accuracy (80–120%). Six isotope-labelled analogues used as internal standards facilitate a quantitative result interpretation which is of utmost importance especially for in-competition drug sports testing.
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liquid chromatographic mass spectrometric analysis of glucuronide conjugated anabolic steroid metabolites method validation and interlaboratory comparison
Journal of Mass Spectrometry, 2008Co-Authors: Laura Hintikka, Wilhelm Schanzer, Mario Thevis, Antti Leinonen, Tiia Kuuranne, John M Halket, David A Cowan, Joachim Grosse, Peter Hemmersbach, Michel W F NielenAbstract:Liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method for simultaneous and direct detection of 12 glucuronide-conjugated anabolic androgenic steroid (AAS) metabolites in human urine is described. The compounds selected were the main metabolites detected in human urine after dosing of the most widely abused AAS in sports, e.g. methandienone, methenolone, methyltestosterone, nandrolone and testosterone, and certain deuterium-labeled analogs of these metabolites. Sample preparation and the LC-ESI-MS/MS method were optimized, validated, and the overall process was implemented and the results between seven laboratories were compared. All the metabolites were extracted simultaneously by solid-phase extraction (SPE) and analyzed by LC-ESI-MS/MS with positive ionization mode and multiple reaction monitoring (MRM). Recovery of the SPE for the AAS glucuronides was 89-100% and ten out of twelve compounds had detection limits in the range of 1-10 ng/ml in urine. The results for inter/intraday repeatability were satisfactory and the interlaboratory comparison with authentic urine samples demonstrated the ease of method transfer from one instrument setup to another. When equivalent triple quadrupole analyzers were employed the overall performance was independent from instrument manufacturer, electrospray ionisation (ESI) or atmospheric pressure chemical ionization (APCI) and liquid chromatohraphic (LC) column, whereas major differences were encountered when changing from one analyzer type to another, especially in the analysis of those AAS glucuronides ionized mainly as adducts.
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mass spectrometric identification and characterization of a new long term metabolite of Metandienone in human urine
Rapid Communications in Mass Spectrometry, 2006Co-Authors: Wilhelm Schanzer, Hans Geyer, Gregor Fusholler, Natalia Halatcheva, Maxie Kohler, Mariakristina Parr, Sven Guddat, Andreas Thomas, Mario ThevisAbstract:Anabolic-androgenic steroids are some of the most frequently detected drugs in amateur and professional sports. Doping control laboratories have developed numerous assays enabling the determination of administered drugs and/or their metabolic products that allow retrospectives with respect to pharmacokinetics and excretion profiles of steroids and their metabolites. A new metabolite generated from Metandienone has been identified as 18-nor-17beta-hydroxymethyl,17alpha-methyl-androst-1,4,13-trien-3-one in excretion study urine samples providing a valuable tool for the long-term detection of Metandienone abuse by athletes in sports drug testing. The metabolite was characterized using gas chromatography/(tandem) mass spectrometry, liquid chromatography/tandem mass spectrometry and liquid chromatography/high-resolution/high-accuracy (tandem) mass spectrometry by characteristic fragmentation patterns representing the intact 3-keto-1,4-diene structure in combination with typical product ions substantiating the proposed C/D-ring structure of the steroid metabolite. In addition, structure confirmation was obtained by the analysis of excretion study urine specimens obtained after administration of 17-CD(3)-labeled Metandienone providing the deuterated analogue to the newly identified metabolite. 18-Nor-17beta-hydroxymethyl,17alpha-methyl-androst-1,4,13-trien-3-one was determined in Metandienone administration study urine specimens up to 19 days after application of a single dose of 5 mg, hence providing an extended detection period compared with commonly employed strategies.
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lc ms ms detection of unaltered glucuronoconjugated metabolites of Metandienone
Drug Testing and Analysis, 2017Co-Authors: Argitxu Esquivel, Cristina Gomez, Oscar J Pozo, Lorena Garrostas, Georgina Balcells, Aristotelis Kotronoulas, Jesus Joglar, Rosa VenturaAbstract:The aim of this study was to evaluate the direct detection of glucuronoconjugated metabolites of Metandienone (MTD) and their detection times. Metabolites resistant to enzymatic hydrolysis were also evaluated. Based on the common mass spectrometric behaviour of steroid glucuronides, three liquid chromatography-tandem mass spectrometry (LC-MS/MS) strategies were applied for the detection of unpredicted and predicted metabolites: precursor ion scan (PI), neutral loss scan (NL), and theoretical selected reaction monitoring (SRM) methods. Samples from four excretion studies of MTD were analyzed for both the detection of metabolites and the establishment of their detection times. Using PI and NL methods, seven metabolites were observed in post-administration samples. SRM methods allowed for the detection of 13 glucuronide metabolites. The detection times, measured by analysis with an SRM method, were between 1 and 22 days. The metabolite detected for the longest time was 18-nor-17β-hydroxymethyl-17α-methyl-5β-androsta-1,4,13-triene-3-one-17-glucuronide. One metabolite was resistant to hydrolysis with β-glucuronidase; however it was only detected in urine up to four days after administration. The three glucuronide metabolites with the highest retrospectivity were identified by chemical synthesis or mass spectrometric data, and although they were previously reported, this is the first time that analytical data of the intact phase II metabolites are presented for some of them. The LC-MS/MS strategies applied have demonstrated to be useful for detecting glucuronoconjugated metabolites of MTD, including glucuronides resistant to enzymatic hydrolysis which cannot be detected by conventional approaches. Copyright © 2016 John Wiley & Sons, Ltd.
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a new sulphate metabolite as a long term marker of Metandienone misuse
Steroids, 2013Co-Authors: Cristina Gomez, Oscar J Pozo, Lorena Garrostas, Jordi Segura, Rosa VenturaAbstract:Abstract Metandienone is one of the most frequently detected anabolic androgenic steroids in sports drug testing. Metandienone misuse is commonly detected by monitoring different metabolites excreted free or conjugated with glucuronic acid using gas chromatography mass spectrometry (GC–MS) and liquid chromatography tandem mass spectrometry (LC–MS/MS) after hydrolysis with β-glucuronidase and liquid–liquid extraction. It is known that several metabolites are the result of the formation of sulphate conjugates in C17, which are converted to their 17-epimers in urine. Therefore, sulphation is an important phase II metabolic pathway of Metandienone that has not been comprehensively studied. The aim of this work was to evaluate the sulphate fraction of Metandienone metabolism by LC–MS/MS. Seven sulphate metabolites were detected after the analysis of excretion study samples by applying different neutral loss scan, precursor ion scan and SRM methods. One of the metabolites (M1) was identified and characterised by GC–MS/MS and LC–MS/MS as 18-nor-17β-hydroxymethyl-17α-methylandrost-1,4,13-triene-3-one sulphate. M1 could be detected up to 26 days after the administration of a single dose of Metandienone (5 mg), thus improving the period in which the misuse can be reported with respect to the last long-term Metandienone metabolite described (18-nor-17β-hydroxymethyl-17α-methylandrost-1,4,13-triene-3-one excreted in the glucuronide fraction).
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comparison between triple quadrupole time of flight and hybrid quadrupole time of flight analysers coupled to liquid chromatography for the detection of anabolic steroids in doping control analysis
Analytica Chimica Acta, 2011Co-Authors: Oscar J Pozo, Rosa Ventura, Peter Van Eenoo, Koen Deventer, Hisham Elbardissy, Susana Grimalt, Juan V Sancho, Felix Hernandez, Frans DelbekeAbstract:Triple quadrupole (QqQ), time of flight (TOF) and quadrupole-time of flight (QTOF) analysers have been compared for the detection of anabolic steroids in human urine. Ten anabolic steroids were selected as model compounds based on their ionization and the presence of endogenous interferences. Both qualitative and quantitative analyses were evaluated. QqQ allowed for the detection of all analytes at the minimum required performance limit (MRPL) established by the World Anti-Doping Agency (between 2 and 10 ng mL(-1) in urine). TOF and QTOF approaches were not sensitive enough to detect some of the analytes (3'-hydroxy-stanozolol or the metabolites of boldenone and formebolone) at the established MRPL. Although a suitable accuracy was obtained, the precision was unsatisfactory (RSD typically higher than 20%) for quantitative purposes irrespective of the analyser used. The methods were applied to 30 real samples declared positives either for the misuse of boldenone, stanozolol and/or methandienone. Most of the compounds were detected by every technique, however QqQ was necessary for the detection of some metabolites in a few samples. Finally, the possibility to detect non-target steroids has been explored by the use of TOF and QTOF. The use of this approach revealed that the presence of boldenone and its metabolite in one sample was due to the intake of androsta-1,4,6-triene-3,17-dione. Additionally, the intake of methandienone was confirmed by the post-target detection of a long-term metabolite.