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Thevis M.,German Sport University Cologne | Thomas A.,German Sport University Cologne | Kohler M.,German Sport University Cologne | Beuck S.,German Sport University Cologne | And 7 more authors.
European Journal of Mass Spectrometry | Year: 2010

Efficient and comprehensive sports drug testing necessitates frequent updating and proactive, preventive anti-doping research and the early implementation of new, emerging drugs into routine doping controls is an essential aspect. Several new drugs and drug candidates with potential for abuse, including so-called Rycals (ryanodine receptor calstabin complex stabilizers, for example, S-107), hypoxia-inducible factor (HIF) stabilizers and peroxisome-proliferator-activated receptor (PPAR) δ agonists (for example, GW1516), were studied using different mass spectrometry- and ion mobility-based approaches and their gas-phase dissociation behaviors were elucidated. The detailed knowledge of fragmentation routes allows a more rapid identification of metabolites and structurally related, presumably "tailor-made", analogs potentially designed for doping purposes. The utility of product ion characterization is demonstrated, in particular, with GW1516, for which oxidation products were readily identified in urine samples by means of diagnostic fragment ions, as measured using high-resolution/high- accuracy mass spectrometry and higher energy collision-induced dissociation (HCD). © IM Publications LLP 2010.

Thevis M.,German Sport University Cologne | Moller I.,German Sport University Cologne | Thomas A.,German Sport University Cologne | Beuck S.,German Sport University Cologne | And 4 more authors.
Analytical and Bioanalytical Chemistry | Year: 2010

Since January 2009, the list of prohibited substances and methods of doping as established by the World Anti-Doping Agency includes new therapeutics such as the peroxisome-proliferator-activated receptor (PPAR)-delta agonist GW1516, which is categorized as a gene doping substance. GW1516 has completed phase II and IV clinical trials regarding dyslipidemia and the regulation of the lipoprotein transport in metabolic syndrome conditions; however, its potential to also improve athletic performance due to the upregulation of genes associated with oxidative metabolism and a modified substrate preference that shifted from carbohydrate to lipid consumption has led to a ban of this compound in elite sport. In a recent report, two presumably mono-oxygenated and bisoxygenated urinary metabolites of GW1516 were presented, which could serve as target analytes for doping control purposes after full characterization. Hence, in the present study, phase I metabolism was simulated by in vitro assays employing human liver microsomal fractions yielding the same oxygenation products, followed by chemical synthesis of the assumed structures of the two abundant metabolic reaction products. These allowed the identification and characterization of mono-oxygenated and bisoxygenated metabolites (sulfoxide and sulfone, respectively) as supported by high-resolution/high-accuracy mass spectrometry with higher-energy collision-induced dissociation, tandem mass spectrometry, and nuclear magnetic resonance spectroscopy. Since urine samples have been the preferred matrix for doping control purposes, a method to detect the new target GW1516 in sports drug testing samples was developed in accordance to conventional screening procedures based on enzymatic hydrolysis and liquid-liquid extraction followed by liquid chromatography, electrospray ionization, and tandem mass spectrometry. Validation was performed for specificity, limit of detection (0.1 ng/ml), recovery (72%), intraday and interday precisions (7.7-15.1%), and ion suppression/enhancement effects (<10%). © 2009 Springer-Verlag.

Krotov G.,Moscow Anti Doping Center | Nikitina M.,Moscow Anti Doping Center | Rodchenkov G.,Moscow Anti Doping Center
Drug Testing and Analysis | Year: 2014

Homologous blood transfusion is a prohibited method of blood manipulation that can be used to increase the number of erythrocytes circulating in the blood stream resulting in an increased oxygen transport capacity. In doping controls, homologous blood transfusions are determined by means of a procedure based on the detection of red blood cell phenotypes by flow cytometry. In the past six years, no adverse analytical findings concerning homologous blood transfusions were reported. One explanation for that phenomenon, assuming that athletes have not completely given up this kind of manipulation, would be a more careful selection of potential donors. If such a donor has the same set of minor erythrocyte antigens as the recipient, the established methodology to detect homologous transfusion would fail. We have hypothesized that any athlete can be a potential donor for teammates with the same RhD factor and AB0 blood group. Having analyzed the phenotype of erythrocytes of 535 Russian athletes in various endurance sports, several pairs of athletes with the same phenotype were observed. Based on the frequency of occurrence of red blood cell antigens, the theoretical probability of finding a donor within a team with exactly the same phenotype was calculated, and the existing number of occurrences where two individuals share the same phenotype in the same sport was in fact five times higher than the theoretical probability. © 2014 John Wiley & Sons, Ltd.

Revelsky A.I.,Moscow State University | Samokhin A.S.,Moscow State University | Virus E.D.,Moscow Anti Doping Center | Rodchenkov G.M.,Moscow Anti Doping Center | Revelsky I.A.,Moscow State University
Drug Testing and Analysis | Year: 2011

The method of high sensitive gas chromatographic/time-of-flight mass-spectrometric (GC/TOF-MS) analysis of steroids was developed. Low-resolution TOF-MS instrument (with fast spectral acquisition rate) was used. This method is based on the formation of the silyl derivatives of steroids; exchange of the reagent mixture (pyridine and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA)) for tert-butylmethylether; offline large sample volume injection of this solution based on sorption concentration of the respective derivatives from the vapour-gas mixture flow formed from the solution and inert gas flows; and entire analytes solvent-free concentrate transfer into the injector of the gas chromatograph. Detection limits for 100 μl sample solution volume were 0.5-2 pg/μl (depending on the component). Application of TOF-MS model 'TruTOF' (Leco, St Joseph, MO, USA) coupled with gas chromatograph and ChromaTOF software (Leco, St Joseph, MO, USA) allowed extraction of the full mass spectra and resolving coeluted peaks. Due to use of the proposed method (10 μl sample aliquot) and GC/TOF-MS, two times more steroid-like compounds were registered in the urine extract in comparison with the injection of 1 μl of the same sample solution. © 2010 John Wiley & Sons, Ltd.

Samokhin A.S.,Moscow State University | Perevozchikova D.V.,Moscow State University | Revelsky A.I.,Moscow State University | Virus E.D.,Moscow Anti Doping Center | And 3 more authors.
Journal of Analytical Chemistry | Year: 2013

An approach to determining the yield of derivatization reaction is based on a comparison of chromatographic peak areas of the derivative and native (underivatizied) compound. In contrast to the previous publication [J. Anal. Chem., 2011, vol. 66, no. 12, pp. 1186-1189], ratio of the sensitivity coefficients of the derivative and native forms of the analyte was calculated using only experimental data obtained upon varying the derivatization conditions (solution containing equal amounts of underivatized compound and a respective derivative was analyzed previously). The approach was used to investigate the influence of the reaction time and the type of an external action on the yield of the derivatization (silylation) reaction for some anabolic steroids (methyltestosterone, methandienone, oxandrolone and oral-turinabol) containing a hindered tertiary hydroxyl group at C17. The amounts of the derivatized steroids were equal to about 20-60 ng (depending on the component). Steroids were derivatized with a mixture of pyridine and N,O-bis(trimethylsilyl) trifluoroacetamide (BSTFA) containing 1% trimethylchlorosilane. The derivatization reaction was carried out for 15, 30 or 45 min under conventional heating, sonication at room temperature, and sonication at elevated temperature. © 2013 Pleiades Publishing, Ltd.

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