The Experts below are selected from a list of 303 Experts worldwide ranked by ideXlab platform
Mario Thevis - One of the best experts on this subject based on the ideXlab platform.
-
complementing the characterization of in vivo generated n glucuronic acid conjugates of Stanozolol by collision cross section computation and analysis
Drug Testing and Analysis, 2015Co-Authors: Mario Thevis, Andreas Thomas, Georg Opfermann, Josef Dib, Sebastian Hoppner, Andreas Lagojda, Dirk Kuehne, Mark Sander, Wilhelm SchanzerAbstract:Detailed structural information on metabolites serving as target analytes in clinical, forensic, and sports drug testing programmes is of paramount importance to ensure unequivocal test results. In the present study, the utility of collision cross section (CCS) analysis by travelling wave ion mobility measurements to support drug metabolite characterization efforts was tested concerning recently identified glucuronic acid conjugates of the anabolic-androgenic steroid Stanozolol. Employing travelling-wave ion mobility spectrometry/quadrupole-time-of-flight mass spectrometry, drift times of five synthetically derived and fully characterized steroid glucuronides were measured and subsequently correlated to respective CCSs as obtained in silico to form an analyte-tailored calibration curve. The CCSs were calculated by equilibrium structure minimization (density functional theory) using the programmes ORCA with the data set B3LYP/6-31G and MOBCAL utilizing the trajectory method (TM) with nitrogen as drift gas. Under identical experimental conditions, synthesized and/or urinary Stanozolol-N and O-glucuronides were analyzed to provide complementary information on the location of glucuronidation. Finally, the obtained data were compared to CCS results generated by the system's internal algorithm based on a calibration employing a polyalanine analyte mixture. The CCSs ΩN2 calculated for the five steroid glucuronide calibrants were found between 180 and 208 A(2) , thus largely covering the observed and computed CCSs for Stanozolol-N1'-, Stanozolol-N2'-, and Stanozolol-O-glucuronide found at values between 195.1 and 212.4 A(2) . The obtained data corroborated the earlier suggested N- and O-glucuronidation of Stanozolol, and demonstrate the exploit of ion mobility and CCS computation in structure characterization of phase-II metabolic products; however, despite reproducibly measurable differences in ion mobility of Stanozolol-N1'-, N2'-, and O-glucuronides, the discriminatory power of the chosen CCS computation algorithm was found to be not appropriate to allow for accurate assignments of the two N-conjugated structures. Using polyalanine-based calibrations, significantly different absolute values were obtained for all CCSs, but due to a constant offset of approximately 45 A(2) an excellent correlation (R(2) = 0.9997) between both approaches was observed. This suggests a substantially accelerated protocol when patterns of computed and polyalanine-based experimental data can be used for structure elucidations instead of creating individual analyte-specific calibration curves.
-
expanding analytical possibilities concerning the detection of Stanozolol misuse by means of high resolution high accuracy mass spectrometric detection of Stanozolol glucuronides in human sports drug testing
Drug Testing and Analysis, 2013Co-Authors: Wilhelm Schanzer, Hans Geyer, Andreas Thomas, Sven Guddat, Georg Opfermann, Mario ThevisAbstract:Anabolic-androgenic steroids (AAS) represent one of the most frequently detected classes of prohibited substances in doping controls. Due to their long-lasting beneficial effects on athletic performance, utmost retrospectivity via urine analysis is desirable and accomplished by targeting long-term metabolites of the respective drugs. In case of Stanozolol, a substantial variety of metabolites has enabled the identification of numerous adverse analytical findings in the past, and recent studies concerning complementary phase-I and phase-II metabolites has further expanded the windows of opportunity for detecting the abuse of Stanozolol. In this study, the utility of liquid chromatography-high resolution/high accuracy (tandem) mass spectrometry (LC-MS/MS) for the detection of 3’-OH-Stanozolol glucuronide in sports drug testing is presented and the identification of two additional and so far unreported metabolites is shown. The structures of the complementary glucuronic acid conjugates were attributed to Stanozolol-N-glucuronide and 17-epiStanozolol-N-glucuronide. By means of chemical synthesis, Stanozolol-N-glucuronide was prepared and used to corroborate the suggested structures. The 3’-OH-Stanozolol glucuronide and the newly identified target compounds were implemented into routine sports drug test assays consisting of direct injection LC-MS/MS or solid-phase extraction (SPE) followed by LC-MS/MS. A considerably expanded detection window for Stanozolol abuse was demonstrated compared to the use of conventional phase-I metabolites and methodologies based on, for example, low resolution LC-MS/MS or gas chromatography-tandem mass spectrometry (GC-MS/MS). The commercial availability of 3’-OH-Stanozolol glucuronide has been of great value for confirmatory purposes, and 17-epiStanozolol-N-glucuronide was found to be a favourable long-term metabolite for doping controls as it was observed up to 28 days post-administration of the drug. Applying the established methodology over a period of six months to 659 routine sports drug testing samples, a total of 85 adverse analytical findings was uncovered, 72 of which would have remained undetected using earlier employed GC-MS/MS approaches. Copyright © 2013 John Wiley & Sons, Ltd.
-
Expanding analytical possibilities concerning the detection of Stanozolol misuse by means of high resolution/high accuracy mass spectrometric detection of Stanozolol glucuronides in human sports drug testing
Drug Testing and Analysis, 2013Co-Authors: Wilhelm Schanzer, Hans Geyer, Andreas Thomas, Sven Guddat, Georg Opfermann, Mario ThevisAbstract:Anabolic-androgenic steroids (AAS) represent one of the most frequently detected classes of prohibited substances in doping controls. Due to their long-lasting beneficial effects on athletic performance, utmost retrospectivity via urine analysis is desirable and accomplished by targeting long-term metabolites of the respective drugs. In case of Stanozolol, a substantial variety of metabolites has enabled the identification of numerous adverse analytical findings in the past, and recent studies concerning complementary phase-I and phase-II metabolites has further expanded the windows of opportunity for detecting the abuse of Stanozolol. In this study, the utility of liquid chromatography-high resolution/high accuracy (tandem) mass spectrometry (LC-MS/MS) for the detection of 3’-OH-Stanozolol glucuronide in sports drug testing is presented and the identification of two additional and so far unreported metabolites is shown. The structures of the complementary glucuronic acid conjugates were attributed to Stanozolol-N-glucuronide and 17-epiStanozolol-N-glucuronide. By means of chemical synthesis, Stanozolol-N-glucuronide was prepared and used to corroborate the suggested structures. The 3’-OH-Stanozolol glucuronide and the newly identified target compounds were implemented into routine sports drug test assays consisting of direct injection LC-MS/MS or solid-phase extraction (SPE) followed by LC-MS/MS. A considerably expanded detection window for Stanozolol abuse was demonstrated compared to the use of conventional phase-I metabolites and methodologies based on, for example, low resolution LC-MS/MS or gas chromatography-tandem mass spectrometry (GC-MS/MS). The commercial availability of 3’-OH-Stanozolol glucuronide has been of great value for confirmatory purposes, and 17-epiStanozolol-N-glucuronide was found to be a favourable long-term metabolite for doping controls as it was observed up to 28 days post-administration of the drug. Applying the established methodology over a period of six months to 659 routine sports drug testing samples, a total of 85 adverse analytical findings was uncovered, 72 of which would have remained undetected using earlier employed GC-MS/MS approaches. Copyright © 2013 John Wiley & Sons, Ltd.
-
Detection of Stanozolol and its major metabolites in human urine by liquid chromatography- tandem mass spectrometry
Chromatographia, 2006Co-Authors: Mario Thevis, Gregor Fußhöller, Hans Geyer, G. Rodchenkov, Ute Mareck, Gerd Sigmund, Anja Koch, Andreas Thomas, W. SchänzerAbstract:The determination of Stanozolol and its metabolites in human urine has been of particular interest in sports drug testing due to its frequently revealed misuse. A simple and rapid sample preparation procedure based on consecutive solid-phase and liquid–liquid extraction with subsequent re-extraction followed by liquid chromatography and electrospray ionization tandem mass spectrometry was established. It allowed the determination of Stanozolol and its metabolic products 16β-OH-Stanozolol and 4β-OH-Stanozolol in human urine at detection limits of 0.1, 0.2 and 0.2 ng mL−1, respectively, with recoveries ranging from 5 to 38%. The robust nature of the assay and the efficient removal of interfering biological matrix provides excellent signal-to-noise ratios, and, thus, a rapid alternative to established procedures utilizing multiple solid-phase extraction or immunoaffinity chromatography strategies. More than 15 doping control urine specimens tested positive for Stanozolol during the last 12 months have been confirmed using the described approach.
-
mass spectrometry of Stanozolol and its analogues using electrospray ionization and collision induced dissociation with quadrupole linear ion trap and linear ion trap orbitrap hybrid mass analyzers
Rapid Communications in Mass Spectrometry, 2005Co-Authors: Mario Thevis, Alexander Makarov, Stevan Horning, Wilhelm SchanzerAbstract:Mass spectrometric identification and characterization of growth-promoting anabolic-androgenic steroids in biological matrices has been a major task for doping control as well as food safety laboratories. The fragmentation behavior of Stanozolol, its metabolites 17-epiStanozolol, 3'-OH-Stanozolol, 4alpha-OH-Stanozolol, 4beta-OH-Stanozolol, 17-epi-16alpha-OH-Stanozolol, 16alpha-OH-Stanozolol, 16beta-OH-Stanozolol, as well as the synthetic analogues 4-dehydroStanozolol, 17-ketoStanozolol, and N-methyl-3'-OH-Stanozolol, was investigated after positive electrospray ionization and subsequent collision-induced dissociation utilizing a quadrupole-linear ion trap and a novel linear ion trap-orbitrap hybrid mass spectrometer. Stable isotope labeling, H/D-exchange experiments, MS3 analyses and high-resolution/high mass accuracy measurements of fragment ions were employed to allow proposals for charge-driven as well as charge-remote fragmentation pathways generating characteristic product ions of Stanozolol at m/z 81, 91, 95, 105, 119, 135 and 297 and 4-hydroxylated Stanozolol at m/z 145. Fragment ions were generated by dissociation of the steroidal A- and B-ring retaining the introduced charge within the pyrazole function of Stanozolol and by elimination of A- and B-ring fractions including the pyrazole residue. In addition, a charge-remote fragmentation causing the neutral loss of methanol was observed, which was suggested to be composed by the methyl residue at C-18 and the hydroxyl function located at C-17.
Wilhelm Schanzer - One of the best experts on this subject based on the ideXlab platform.
-
complementing the characterization of in vivo generated n glucuronic acid conjugates of Stanozolol by collision cross section computation and analysis
Drug Testing and Analysis, 2015Co-Authors: Mario Thevis, Andreas Thomas, Georg Opfermann, Josef Dib, Sebastian Hoppner, Andreas Lagojda, Dirk Kuehne, Mark Sander, Wilhelm SchanzerAbstract:Detailed structural information on metabolites serving as target analytes in clinical, forensic, and sports drug testing programmes is of paramount importance to ensure unequivocal test results. In the present study, the utility of collision cross section (CCS) analysis by travelling wave ion mobility measurements to support drug metabolite characterization efforts was tested concerning recently identified glucuronic acid conjugates of the anabolic-androgenic steroid Stanozolol. Employing travelling-wave ion mobility spectrometry/quadrupole-time-of-flight mass spectrometry, drift times of five synthetically derived and fully characterized steroid glucuronides were measured and subsequently correlated to respective CCSs as obtained in silico to form an analyte-tailored calibration curve. The CCSs were calculated by equilibrium structure minimization (density functional theory) using the programmes ORCA with the data set B3LYP/6-31G and MOBCAL utilizing the trajectory method (TM) with nitrogen as drift gas. Under identical experimental conditions, synthesized and/or urinary Stanozolol-N and O-glucuronides were analyzed to provide complementary information on the location of glucuronidation. Finally, the obtained data were compared to CCS results generated by the system's internal algorithm based on a calibration employing a polyalanine analyte mixture. The CCSs ΩN2 calculated for the five steroid glucuronide calibrants were found between 180 and 208 A(2) , thus largely covering the observed and computed CCSs for Stanozolol-N1'-, Stanozolol-N2'-, and Stanozolol-O-glucuronide found at values between 195.1 and 212.4 A(2) . The obtained data corroborated the earlier suggested N- and O-glucuronidation of Stanozolol, and demonstrate the exploit of ion mobility and CCS computation in structure characterization of phase-II metabolic products; however, despite reproducibly measurable differences in ion mobility of Stanozolol-N1'-, N2'-, and O-glucuronides, the discriminatory power of the chosen CCS computation algorithm was found to be not appropriate to allow for accurate assignments of the two N-conjugated structures. Using polyalanine-based calibrations, significantly different absolute values were obtained for all CCSs, but due to a constant offset of approximately 45 A(2) an excellent correlation (R(2) = 0.9997) between both approaches was observed. This suggests a substantially accelerated protocol when patterns of computed and polyalanine-based experimental data can be used for structure elucidations instead of creating individual analyte-specific calibration curves.
-
expanding analytical possibilities concerning the detection of Stanozolol misuse by means of high resolution high accuracy mass spectrometric detection of Stanozolol glucuronides in human sports drug testing
Drug Testing and Analysis, 2013Co-Authors: Wilhelm Schanzer, Hans Geyer, Andreas Thomas, Sven Guddat, Georg Opfermann, Mario ThevisAbstract:Anabolic-androgenic steroids (AAS) represent one of the most frequently detected classes of prohibited substances in doping controls. Due to their long-lasting beneficial effects on athletic performance, utmost retrospectivity via urine analysis is desirable and accomplished by targeting long-term metabolites of the respective drugs. In case of Stanozolol, a substantial variety of metabolites has enabled the identification of numerous adverse analytical findings in the past, and recent studies concerning complementary phase-I and phase-II metabolites has further expanded the windows of opportunity for detecting the abuse of Stanozolol. In this study, the utility of liquid chromatography-high resolution/high accuracy (tandem) mass spectrometry (LC-MS/MS) for the detection of 3’-OH-Stanozolol glucuronide in sports drug testing is presented and the identification of two additional and so far unreported metabolites is shown. The structures of the complementary glucuronic acid conjugates were attributed to Stanozolol-N-glucuronide and 17-epiStanozolol-N-glucuronide. By means of chemical synthesis, Stanozolol-N-glucuronide was prepared and used to corroborate the suggested structures. The 3’-OH-Stanozolol glucuronide and the newly identified target compounds were implemented into routine sports drug test assays consisting of direct injection LC-MS/MS or solid-phase extraction (SPE) followed by LC-MS/MS. A considerably expanded detection window for Stanozolol abuse was demonstrated compared to the use of conventional phase-I metabolites and methodologies based on, for example, low resolution LC-MS/MS or gas chromatography-tandem mass spectrometry (GC-MS/MS). The commercial availability of 3’-OH-Stanozolol glucuronide has been of great value for confirmatory purposes, and 17-epiStanozolol-N-glucuronide was found to be a favourable long-term metabolite for doping controls as it was observed up to 28 days post-administration of the drug. Applying the established methodology over a period of six months to 659 routine sports drug testing samples, a total of 85 adverse analytical findings was uncovered, 72 of which would have remained undetected using earlier employed GC-MS/MS approaches. Copyright © 2013 John Wiley & Sons, Ltd.
-
Expanding analytical possibilities concerning the detection of Stanozolol misuse by means of high resolution/high accuracy mass spectrometric detection of Stanozolol glucuronides in human sports drug testing
Drug Testing and Analysis, 2013Co-Authors: Wilhelm Schanzer, Hans Geyer, Andreas Thomas, Sven Guddat, Georg Opfermann, Mario ThevisAbstract:Anabolic-androgenic steroids (AAS) represent one of the most frequently detected classes of prohibited substances in doping controls. Due to their long-lasting beneficial effects on athletic performance, utmost retrospectivity via urine analysis is desirable and accomplished by targeting long-term metabolites of the respective drugs. In case of Stanozolol, a substantial variety of metabolites has enabled the identification of numerous adverse analytical findings in the past, and recent studies concerning complementary phase-I and phase-II metabolites has further expanded the windows of opportunity for detecting the abuse of Stanozolol. In this study, the utility of liquid chromatography-high resolution/high accuracy (tandem) mass spectrometry (LC-MS/MS) for the detection of 3’-OH-Stanozolol glucuronide in sports drug testing is presented and the identification of two additional and so far unreported metabolites is shown. The structures of the complementary glucuronic acid conjugates were attributed to Stanozolol-N-glucuronide and 17-epiStanozolol-N-glucuronide. By means of chemical synthesis, Stanozolol-N-glucuronide was prepared and used to corroborate the suggested structures. The 3’-OH-Stanozolol glucuronide and the newly identified target compounds were implemented into routine sports drug test assays consisting of direct injection LC-MS/MS or solid-phase extraction (SPE) followed by LC-MS/MS. A considerably expanded detection window for Stanozolol abuse was demonstrated compared to the use of conventional phase-I metabolites and methodologies based on, for example, low resolution LC-MS/MS or gas chromatography-tandem mass spectrometry (GC-MS/MS). The commercial availability of 3’-OH-Stanozolol glucuronide has been of great value for confirmatory purposes, and 17-epiStanozolol-N-glucuronide was found to be a favourable long-term metabolite for doping controls as it was observed up to 28 days post-administration of the drug. Applying the established methodology over a period of six months to 659 routine sports drug testing samples, a total of 85 adverse analytical findings was uncovered, 72 of which would have remained undetected using earlier employed GC-MS/MS approaches. Copyright © 2013 John Wiley & Sons, Ltd.
-
mass spectrometry of Stanozolol and its analogues using electrospray ionization and collision induced dissociation with quadrupole linear ion trap and linear ion trap orbitrap hybrid mass analyzers
Rapid Communications in Mass Spectrometry, 2005Co-Authors: Mario Thevis, Alexander Makarov, Stevan Horning, Wilhelm SchanzerAbstract:Mass spectrometric identification and characterization of growth-promoting anabolic-androgenic steroids in biological matrices has been a major task for doping control as well as food safety laboratories. The fragmentation behavior of Stanozolol, its metabolites 17-epiStanozolol, 3'-OH-Stanozolol, 4alpha-OH-Stanozolol, 4beta-OH-Stanozolol, 17-epi-16alpha-OH-Stanozolol, 16alpha-OH-Stanozolol, 16beta-OH-Stanozolol, as well as the synthetic analogues 4-dehydroStanozolol, 17-ketoStanozolol, and N-methyl-3'-OH-Stanozolol, was investigated after positive electrospray ionization and subsequent collision-induced dissociation utilizing a quadrupole-linear ion trap and a novel linear ion trap-orbitrap hybrid mass spectrometer. Stable isotope labeling, H/D-exchange experiments, MS3 analyses and high-resolution/high mass accuracy measurements of fragment ions were employed to allow proposals for charge-driven as well as charge-remote fragmentation pathways generating characteristic product ions of Stanozolol at m/z 81, 91, 95, 105, 119, 135 and 297 and 4-hydroxylated Stanozolol at m/z 145. Fragment ions were generated by dissociation of the steroidal A- and B-ring retaining the introduced charge within the pyrazole function of Stanozolol and by elimination of A- and B-ring fractions including the pyrazole residue. In addition, a charge-remote fragmentation causing the neutral loss of methanol was observed, which was suggested to be composed by the methyl residue at C-18 and the hydroxyl function located at C-17.
-
Metabolism of Stanozolol : identification and synthesis of urinary metabolites
Journal of Steroid Biochemistry, 1990Co-Authors: Wilhelm Schanzer, Georg Opfermann, Manfred DonikeAbstract:Urinary metabolites of Stanozolol (17 alpha-methyl-17 beta-hydroxy-5 alpha-androst-2-eno(3,2-c)-pyrazole) following oral administration were isolated by chromatography on XAD-2 and by preparative high-performance liquid chromatography (HPLC) and identified by gas chromatography-mass spectrometry (GC/MS) with electron impact (EI)-ionisation. Stanozolol is excreted as a conjugate but is metabolized to a large extent. All identified metabolites are hydroxylated, namely at C-3' of the pyrazole ring and at C-4 beta, C-16 alpha and C-16 beta of the steroid. Less than 5% of the metabolites are found in the unconjugated urine fraction: 3'-hydroxy-Stanozolol (II) and 3'-hydroxy-17-epiStanozolol (III). Conjugated excreted metabolites are 3'-hydroxyStanozolol (II), Stanozolol (I), 4 beta-hydroxy-Stanozolol (IV), 16 beta-hydroxyStanozolol (V), 16 alpha-hydroxyStanozolol (VI), two isomers of 3',16-dihydroxyStanozolol (VII, VIII), two isomers of 4 beta, 16-dihydroxyStanozolol (IX, X) and a 3',?-dihydroxyStanozolol (XI). 3'-HydroxyStanozolol, 4 alpha-hydroxyStanozolol, 4 beta-hydroxyStanozolol, 16 alpha-hydroxy-, 16 alpha-hydroxy-17-epi- and 16 beta-hydroxyStanozolol were synthesised to confirm the structural assignment of the main metabolites.
Declan P. Naughton - One of the best experts on this subject based on the ideXlab platform.
-
Inhibitory Effects of Diclofenac on Steroid Glucuronidation in Vivo Do not Affect Hair-Based Doping Tests for Stanozolol
2017Co-Authors: Gergely Zachar, James Barker, Andrea Petróczi, Nawed I. K. Deshmukh, Andrea D. Székely, Iltaf Shah, Declan P. NaughtonAbstract:In vitro studies show that diclofenac inhibits enzymatic steroid glucuronidation. This study was designed to investigate the influence of diclofenac on the excretion of Stanozolol and 3'-hydroxyStanozolol via analyses in hair, blood and urine in vivo in a rat study. Brown Norway rats were administered with Stanozolol (weeks 1-3) and diclofenac (weeks 1-6). Weekly assessment of steroid levels in hair was complemented with spot urine and serum tests. Levels of both Stanozolol and 3'-hydroxyStanozolol steadily increased in hair during Stanozolol treatment and decreased post-treatment, but remained readily detectable for 6 weeks. In contrast, compared to control rats, diclofenac significantly reduced urinary excretion of 3’-hydroxyStanozolol which was undetectable in most samples. This is the first report of diclofenac altering steroid metabolism in vivo, detrimentally affecting detection in urine, but not in hair which holds considerable advantages over urinalysis for anti-doping tests.
-
determination of Stanozolol and 3 hydroxyStanozolol in rat hair urine and serum using liquid chromatography tandem mass spectrometry
Chemistry Central Journal, 2012Co-Authors: Nawed Deshmukh, James Barker, Andrea Petróczi, Gergely Zachar, Andrea Szekely, Declan P. NaughtonAbstract:Anabolic androgenic steroids, such as Stanozolol, are typically misused by athletes during preparation for competition. Out-of-competition testing presents a unique challenge in the current anti-doping detection system owing to logistic reasons. Analysing hair for the presence of a prohibited drug offers a feasible solution for covering the wider window in out-of-competition testing. To assist in vivo studies aiming to establish a relationship between drug levels detected in hair, urine and blood, sensitive methods for the determination of Stanozolol and its major metabolite 3′-hydroxyStanozolol were developed in pigmented hair, urine and serum, using brown Norway rats as a model system and liquid chromatography tandem mass spectrometry (LC-MS/MS). For method development, spiked drug free rat hair, blood and urine samples were used. The newly developed method was then applied to hair, urine and serum samples from five brown Norway rats after treatment (intraperitoneal) with Stanozolol for six consecutive days at 5.0 mg/kg/day. The assay for each matrix was linear within the quantification range with determination coefficient (r2) values above 0.995. The respective assay was capable of detecting 0.125 pg/mg Stanozolol and 0.25 pg/mg 3′-hydroxyStanozolol with 50 mg hair; 0.063 ng/mL Stanozolol and 0.125 ng/mL 3′-hydroxyStanozolol with 100 μL of urine or serum. The accuracy, precision and extraction recoveries of the assays were satisfactory for the detection of both compounds in all three matrices. The average concentrations of Stanozolol and 3′-hydroxyStanozolol, were as follows: hair = 70.18 ± 22.32 pg/mg and 13.01 ± 3.43 pg/mg; urine = 4.34 ± 6.54 ng/mL and 9.39 ± 7.42 ng/mL; serum = 7.75 ± 3.58 ng/mL and 7.16 ± 1.97 ng/mL, respectively. The developed methods are sensitive, specific and reproducible for the determination of Stanozolol and 3′-hydroxyStanozolol in rat hair, urine and serum. These methods can be used for in vivo studies further investigating Stanozolol metabolism, but also could be extended for doping testing. Owing to the complementary nature of these tests, with urine and serum giving information on recent drug use and hair providing retrospective information on habitual use, it is suggested that blood or urine tests could accompany hair analysis and thus avoid false doping results.
-
Detection of Stanozolol in environmental waters using liquid chromatography tandem mass spectrometry
Chemistry Central Journal, 2011Co-Authors: Nawed Deshmukh, James Barker, Andrea Petróczi, Declan P. NaughtonAbstract:Owing to frequent administration of a wide range of pharmaceutical products, various environmental waters have been found to be contaminated with pharmacologically active substances. For example, Stanozolol, a synthetic anabolic steroid, is frequently misused for performance enhancement as well as for illegal growth promoting purposes in veterinary practice. Previously we reported Stanozolol in hair samples collected from subjects living in Budapest. For this reason we initiated this study to explore possible environmental sources of steroid contamination. The aim of this study was to develop a method to monitor Stanozolol in aqueous matrices using liquid chromatography tandem mass spectrometry (LC-MS/MS). Liquid-liquid extraction using pentane was found to be an efficient method for the extraction of Stanozolol from water samples. This was followed by direct detection using LC-MS/MS. The method was capable of detecting 0.25 pg/mL Stanozolol when only 5 mL water was processed in the presence of Stanozolol D3 as internal standard. Fifteen bottled waters analysed were found to be negative for Stanozolol. However, three out of six samples from the Danube river, collected from December '09 to November '10, were found to contain Stanozolol at concentrations up to 1.82 pg/mL. In contrast, only one sample (out of six) of urban tap water from Budapest city was found to contain Stanozolol, at a concentration of 1.19 pg/mL. The method developed is efficient, rapid, reproducible, sensitive and robust for the detection of Stanozolol in aqueous matrices.
-
analysis of anabolic steroids in human hair using lc ms ms
Steroids, 2010Co-Authors: Nawed I. K. Deshmukh, James Barker, Andrea Petróczi, Iltaf Hussain, Declan P. NaughtonAbstract:New highly sensitive, specific, reliable, reproducible and robust LC-MS/MS methods were developed to detect the anabolic steroids, nandrolone and Stanozolol, in human hair for the first time. Hair samples from 180 participants (108 males, 72 females, 62% athletes) were screened using ELISA which revealed 16 athletes as positive for Stanozolol and 3 for nandrolone. Positive samples were confirmed on LC-MS/MS in selective reaction monitoring (SRM) mode. The assays for Stanozolol and nandrolone showed good linearity in the range 1 to 400 pg/mg and 5 to 400 pg/mg respectively. The methods were validated for LLOD, interday precision, intraday precision, specificity, extraction recovery and accuracy. The assays were capable of detecting 0.5 pg Stanozolol and 3.0 pg nandrolone per mg of hair, when approximately 20mg of hair were processed. Analysis using LC-MS/MS confirmed eleven athletes' positive for Stanozolol (5.0 pg/mg to 86.3 pg/mg) and 1 for nandrolone (14.0 pg/mg) thus avoiding false results from ELISA screening. The results obtained demonstrate the application of these hair analysis methods to detect both steroids at low concentrations, hence reducing the amount of hair required significantly. The new methods complement urinalysis or blood testing and facilitate improved doping testing regimes. Hair analysis benefits from non invasiveness, negligible risk of infection and facile sample storage and collection, whilst reducing risks of tampering and cross contamination. Owing to the wide detection window, this approach may also offer an alternative approach for out-of-competition testing.
Georg Opfermann - One of the best experts on this subject based on the ideXlab platform.
-
complementing the characterization of in vivo generated n glucuronic acid conjugates of Stanozolol by collision cross section computation and analysis
Drug Testing and Analysis, 2015Co-Authors: Mario Thevis, Andreas Thomas, Georg Opfermann, Josef Dib, Sebastian Hoppner, Andreas Lagojda, Dirk Kuehne, Mark Sander, Wilhelm SchanzerAbstract:Detailed structural information on metabolites serving as target analytes in clinical, forensic, and sports drug testing programmes is of paramount importance to ensure unequivocal test results. In the present study, the utility of collision cross section (CCS) analysis by travelling wave ion mobility measurements to support drug metabolite characterization efforts was tested concerning recently identified glucuronic acid conjugates of the anabolic-androgenic steroid Stanozolol. Employing travelling-wave ion mobility spectrometry/quadrupole-time-of-flight mass spectrometry, drift times of five synthetically derived and fully characterized steroid glucuronides were measured and subsequently correlated to respective CCSs as obtained in silico to form an analyte-tailored calibration curve. The CCSs were calculated by equilibrium structure minimization (density functional theory) using the programmes ORCA with the data set B3LYP/6-31G and MOBCAL utilizing the trajectory method (TM) with nitrogen as drift gas. Under identical experimental conditions, synthesized and/or urinary Stanozolol-N and O-glucuronides were analyzed to provide complementary information on the location of glucuronidation. Finally, the obtained data were compared to CCS results generated by the system's internal algorithm based on a calibration employing a polyalanine analyte mixture. The CCSs ΩN2 calculated for the five steroid glucuronide calibrants were found between 180 and 208 A(2) , thus largely covering the observed and computed CCSs for Stanozolol-N1'-, Stanozolol-N2'-, and Stanozolol-O-glucuronide found at values between 195.1 and 212.4 A(2) . The obtained data corroborated the earlier suggested N- and O-glucuronidation of Stanozolol, and demonstrate the exploit of ion mobility and CCS computation in structure characterization of phase-II metabolic products; however, despite reproducibly measurable differences in ion mobility of Stanozolol-N1'-, N2'-, and O-glucuronides, the discriminatory power of the chosen CCS computation algorithm was found to be not appropriate to allow for accurate assignments of the two N-conjugated structures. Using polyalanine-based calibrations, significantly different absolute values were obtained for all CCSs, but due to a constant offset of approximately 45 A(2) an excellent correlation (R(2) = 0.9997) between both approaches was observed. This suggests a substantially accelerated protocol when patterns of computed and polyalanine-based experimental data can be used for structure elucidations instead of creating individual analyte-specific calibration curves.
-
expanding analytical possibilities concerning the detection of Stanozolol misuse by means of high resolution high accuracy mass spectrometric detection of Stanozolol glucuronides in human sports drug testing
Drug Testing and Analysis, 2013Co-Authors: Wilhelm Schanzer, Hans Geyer, Andreas Thomas, Sven Guddat, Georg Opfermann, Mario ThevisAbstract:Anabolic-androgenic steroids (AAS) represent one of the most frequently detected classes of prohibited substances in doping controls. Due to their long-lasting beneficial effects on athletic performance, utmost retrospectivity via urine analysis is desirable and accomplished by targeting long-term metabolites of the respective drugs. In case of Stanozolol, a substantial variety of metabolites has enabled the identification of numerous adverse analytical findings in the past, and recent studies concerning complementary phase-I and phase-II metabolites has further expanded the windows of opportunity for detecting the abuse of Stanozolol. In this study, the utility of liquid chromatography-high resolution/high accuracy (tandem) mass spectrometry (LC-MS/MS) for the detection of 3’-OH-Stanozolol glucuronide in sports drug testing is presented and the identification of two additional and so far unreported metabolites is shown. The structures of the complementary glucuronic acid conjugates were attributed to Stanozolol-N-glucuronide and 17-epiStanozolol-N-glucuronide. By means of chemical synthesis, Stanozolol-N-glucuronide was prepared and used to corroborate the suggested structures. The 3’-OH-Stanozolol glucuronide and the newly identified target compounds were implemented into routine sports drug test assays consisting of direct injection LC-MS/MS or solid-phase extraction (SPE) followed by LC-MS/MS. A considerably expanded detection window for Stanozolol abuse was demonstrated compared to the use of conventional phase-I metabolites and methodologies based on, for example, low resolution LC-MS/MS or gas chromatography-tandem mass spectrometry (GC-MS/MS). The commercial availability of 3’-OH-Stanozolol glucuronide has been of great value for confirmatory purposes, and 17-epiStanozolol-N-glucuronide was found to be a favourable long-term metabolite for doping controls as it was observed up to 28 days post-administration of the drug. Applying the established methodology over a period of six months to 659 routine sports drug testing samples, a total of 85 adverse analytical findings was uncovered, 72 of which would have remained undetected using earlier employed GC-MS/MS approaches. Copyright © 2013 John Wiley & Sons, Ltd.
-
Expanding analytical possibilities concerning the detection of Stanozolol misuse by means of high resolution/high accuracy mass spectrometric detection of Stanozolol glucuronides in human sports drug testing
Drug Testing and Analysis, 2013Co-Authors: Wilhelm Schanzer, Hans Geyer, Andreas Thomas, Sven Guddat, Georg Opfermann, Mario ThevisAbstract:Anabolic-androgenic steroids (AAS) represent one of the most frequently detected classes of prohibited substances in doping controls. Due to their long-lasting beneficial effects on athletic performance, utmost retrospectivity via urine analysis is desirable and accomplished by targeting long-term metabolites of the respective drugs. In case of Stanozolol, a substantial variety of metabolites has enabled the identification of numerous adverse analytical findings in the past, and recent studies concerning complementary phase-I and phase-II metabolites has further expanded the windows of opportunity for detecting the abuse of Stanozolol. In this study, the utility of liquid chromatography-high resolution/high accuracy (tandem) mass spectrometry (LC-MS/MS) for the detection of 3’-OH-Stanozolol glucuronide in sports drug testing is presented and the identification of two additional and so far unreported metabolites is shown. The structures of the complementary glucuronic acid conjugates were attributed to Stanozolol-N-glucuronide and 17-epiStanozolol-N-glucuronide. By means of chemical synthesis, Stanozolol-N-glucuronide was prepared and used to corroborate the suggested structures. The 3’-OH-Stanozolol glucuronide and the newly identified target compounds were implemented into routine sports drug test assays consisting of direct injection LC-MS/MS or solid-phase extraction (SPE) followed by LC-MS/MS. A considerably expanded detection window for Stanozolol abuse was demonstrated compared to the use of conventional phase-I metabolites and methodologies based on, for example, low resolution LC-MS/MS or gas chromatography-tandem mass spectrometry (GC-MS/MS). The commercial availability of 3’-OH-Stanozolol glucuronide has been of great value for confirmatory purposes, and 17-epiStanozolol-N-glucuronide was found to be a favourable long-term metabolite for doping controls as it was observed up to 28 days post-administration of the drug. Applying the established methodology over a period of six months to 659 routine sports drug testing samples, a total of 85 adverse analytical findings was uncovered, 72 of which would have remained undetected using earlier employed GC-MS/MS approaches. Copyright © 2013 John Wiley & Sons, Ltd.
-
Metabolism of Stanozolol : identification and synthesis of urinary metabolites
Journal of Steroid Biochemistry, 1990Co-Authors: Wilhelm Schanzer, Georg Opfermann, Manfred DonikeAbstract:Urinary metabolites of Stanozolol (17 alpha-methyl-17 beta-hydroxy-5 alpha-androst-2-eno(3,2-c)-pyrazole) following oral administration were isolated by chromatography on XAD-2 and by preparative high-performance liquid chromatography (HPLC) and identified by gas chromatography-mass spectrometry (GC/MS) with electron impact (EI)-ionisation. Stanozolol is excreted as a conjugate but is metabolized to a large extent. All identified metabolites are hydroxylated, namely at C-3' of the pyrazole ring and at C-4 beta, C-16 alpha and C-16 beta of the steroid. Less than 5% of the metabolites are found in the unconjugated urine fraction: 3'-hydroxy-Stanozolol (II) and 3'-hydroxy-17-epiStanozolol (III). Conjugated excreted metabolites are 3'-hydroxyStanozolol (II), Stanozolol (I), 4 beta-hydroxy-Stanozolol (IV), 16 beta-hydroxyStanozolol (V), 16 alpha-hydroxyStanozolol (VI), two isomers of 3',16-dihydroxyStanozolol (VII, VIII), two isomers of 4 beta, 16-dihydroxyStanozolol (IX, X) and a 3',?-dihydroxyStanozolol (XI). 3'-HydroxyStanozolol, 4 alpha-hydroxyStanozolol, 4 beta-hydroxyStanozolol, 16 alpha-hydroxy-, 16 alpha-hydroxy-17-epi- and 16 beta-hydroxyStanozolol were synthesised to confirm the structural assignment of the main metabolites.
Andreas Thomas - One of the best experts on this subject based on the ideXlab platform.
-
complementing the characterization of in vivo generated n glucuronic acid conjugates of Stanozolol by collision cross section computation and analysis
Drug Testing and Analysis, 2015Co-Authors: Mario Thevis, Andreas Thomas, Georg Opfermann, Josef Dib, Sebastian Hoppner, Andreas Lagojda, Dirk Kuehne, Mark Sander, Wilhelm SchanzerAbstract:Detailed structural information on metabolites serving as target analytes in clinical, forensic, and sports drug testing programmes is of paramount importance to ensure unequivocal test results. In the present study, the utility of collision cross section (CCS) analysis by travelling wave ion mobility measurements to support drug metabolite characterization efforts was tested concerning recently identified glucuronic acid conjugates of the anabolic-androgenic steroid Stanozolol. Employing travelling-wave ion mobility spectrometry/quadrupole-time-of-flight mass spectrometry, drift times of five synthetically derived and fully characterized steroid glucuronides were measured and subsequently correlated to respective CCSs as obtained in silico to form an analyte-tailored calibration curve. The CCSs were calculated by equilibrium structure minimization (density functional theory) using the programmes ORCA with the data set B3LYP/6-31G and MOBCAL utilizing the trajectory method (TM) with nitrogen as drift gas. Under identical experimental conditions, synthesized and/or urinary Stanozolol-N and O-glucuronides were analyzed to provide complementary information on the location of glucuronidation. Finally, the obtained data were compared to CCS results generated by the system's internal algorithm based on a calibration employing a polyalanine analyte mixture. The CCSs ΩN2 calculated for the five steroid glucuronide calibrants were found between 180 and 208 A(2) , thus largely covering the observed and computed CCSs for Stanozolol-N1'-, Stanozolol-N2'-, and Stanozolol-O-glucuronide found at values between 195.1 and 212.4 A(2) . The obtained data corroborated the earlier suggested N- and O-glucuronidation of Stanozolol, and demonstrate the exploit of ion mobility and CCS computation in structure characterization of phase-II metabolic products; however, despite reproducibly measurable differences in ion mobility of Stanozolol-N1'-, N2'-, and O-glucuronides, the discriminatory power of the chosen CCS computation algorithm was found to be not appropriate to allow for accurate assignments of the two N-conjugated structures. Using polyalanine-based calibrations, significantly different absolute values were obtained for all CCSs, but due to a constant offset of approximately 45 A(2) an excellent correlation (R(2) = 0.9997) between both approaches was observed. This suggests a substantially accelerated protocol when patterns of computed and polyalanine-based experimental data can be used for structure elucidations instead of creating individual analyte-specific calibration curves.
-
expanding analytical possibilities concerning the detection of Stanozolol misuse by means of high resolution high accuracy mass spectrometric detection of Stanozolol glucuronides in human sports drug testing
Drug Testing and Analysis, 2013Co-Authors: Wilhelm Schanzer, Hans Geyer, Andreas Thomas, Sven Guddat, Georg Opfermann, Mario ThevisAbstract:Anabolic-androgenic steroids (AAS) represent one of the most frequently detected classes of prohibited substances in doping controls. Due to their long-lasting beneficial effects on athletic performance, utmost retrospectivity via urine analysis is desirable and accomplished by targeting long-term metabolites of the respective drugs. In case of Stanozolol, a substantial variety of metabolites has enabled the identification of numerous adverse analytical findings in the past, and recent studies concerning complementary phase-I and phase-II metabolites has further expanded the windows of opportunity for detecting the abuse of Stanozolol. In this study, the utility of liquid chromatography-high resolution/high accuracy (tandem) mass spectrometry (LC-MS/MS) for the detection of 3’-OH-Stanozolol glucuronide in sports drug testing is presented and the identification of two additional and so far unreported metabolites is shown. The structures of the complementary glucuronic acid conjugates were attributed to Stanozolol-N-glucuronide and 17-epiStanozolol-N-glucuronide. By means of chemical synthesis, Stanozolol-N-glucuronide was prepared and used to corroborate the suggested structures. The 3’-OH-Stanozolol glucuronide and the newly identified target compounds were implemented into routine sports drug test assays consisting of direct injection LC-MS/MS or solid-phase extraction (SPE) followed by LC-MS/MS. A considerably expanded detection window for Stanozolol abuse was demonstrated compared to the use of conventional phase-I metabolites and methodologies based on, for example, low resolution LC-MS/MS or gas chromatography-tandem mass spectrometry (GC-MS/MS). The commercial availability of 3’-OH-Stanozolol glucuronide has been of great value for confirmatory purposes, and 17-epiStanozolol-N-glucuronide was found to be a favourable long-term metabolite for doping controls as it was observed up to 28 days post-administration of the drug. Applying the established methodology over a period of six months to 659 routine sports drug testing samples, a total of 85 adverse analytical findings was uncovered, 72 of which would have remained undetected using earlier employed GC-MS/MS approaches. Copyright © 2013 John Wiley & Sons, Ltd.
-
Expanding analytical possibilities concerning the detection of Stanozolol misuse by means of high resolution/high accuracy mass spectrometric detection of Stanozolol glucuronides in human sports drug testing
Drug Testing and Analysis, 2013Co-Authors: Wilhelm Schanzer, Hans Geyer, Andreas Thomas, Sven Guddat, Georg Opfermann, Mario ThevisAbstract:Anabolic-androgenic steroids (AAS) represent one of the most frequently detected classes of prohibited substances in doping controls. Due to their long-lasting beneficial effects on athletic performance, utmost retrospectivity via urine analysis is desirable and accomplished by targeting long-term metabolites of the respective drugs. In case of Stanozolol, a substantial variety of metabolites has enabled the identification of numerous adverse analytical findings in the past, and recent studies concerning complementary phase-I and phase-II metabolites has further expanded the windows of opportunity for detecting the abuse of Stanozolol. In this study, the utility of liquid chromatography-high resolution/high accuracy (tandem) mass spectrometry (LC-MS/MS) for the detection of 3’-OH-Stanozolol glucuronide in sports drug testing is presented and the identification of two additional and so far unreported metabolites is shown. The structures of the complementary glucuronic acid conjugates were attributed to Stanozolol-N-glucuronide and 17-epiStanozolol-N-glucuronide. By means of chemical synthesis, Stanozolol-N-glucuronide was prepared and used to corroborate the suggested structures. The 3’-OH-Stanozolol glucuronide and the newly identified target compounds were implemented into routine sports drug test assays consisting of direct injection LC-MS/MS or solid-phase extraction (SPE) followed by LC-MS/MS. A considerably expanded detection window for Stanozolol abuse was demonstrated compared to the use of conventional phase-I metabolites and methodologies based on, for example, low resolution LC-MS/MS or gas chromatography-tandem mass spectrometry (GC-MS/MS). The commercial availability of 3’-OH-Stanozolol glucuronide has been of great value for confirmatory purposes, and 17-epiStanozolol-N-glucuronide was found to be a favourable long-term metabolite for doping controls as it was observed up to 28 days post-administration of the drug. Applying the established methodology over a period of six months to 659 routine sports drug testing samples, a total of 85 adverse analytical findings was uncovered, 72 of which would have remained undetected using earlier employed GC-MS/MS approaches. Copyright © 2013 John Wiley & Sons, Ltd.
-
Detection of Stanozolol and its major metabolites in human urine by liquid chromatography- tandem mass spectrometry
Chromatographia, 2006Co-Authors: Mario Thevis, Gregor Fußhöller, Hans Geyer, G. Rodchenkov, Ute Mareck, Gerd Sigmund, Anja Koch, Andreas Thomas, W. SchänzerAbstract:The determination of Stanozolol and its metabolites in human urine has been of particular interest in sports drug testing due to its frequently revealed misuse. A simple and rapid sample preparation procedure based on consecutive solid-phase and liquid–liquid extraction with subsequent re-extraction followed by liquid chromatography and electrospray ionization tandem mass spectrometry was established. It allowed the determination of Stanozolol and its metabolic products 16β-OH-Stanozolol and 4β-OH-Stanozolol in human urine at detection limits of 0.1, 0.2 and 0.2 ng mL−1, respectively, with recoveries ranging from 5 to 38%. The robust nature of the assay and the efficient removal of interfering biological matrix provides excellent signal-to-noise ratios, and, thus, a rapid alternative to established procedures utilizing multiple solid-phase extraction or immunoaffinity chromatography strategies. More than 15 doping control urine specimens tested positive for Stanozolol during the last 12 months have been confirmed using the described approach.