Control of anabolic steroids misuse in sport : potential of direct detection of phase II metabolites

Anabolic androgenic steroids (AAS) are widely used by athletes to enhance sports performance. In this thesis, different studies were performed to evaluate the potential of the direct detection of phase II metabolites by LC-MS/MS to improve the detection of AAS misuse in sport. First, the direct detection of glucuronide metabolites was investigated using metandienone, allowing the detection of thirteen glucuronides, one of them resistant to enzymatic hydrolysis. The proposed analytical strategy provides a comprehensive approach and it is recommended to be used for metabolic studies of other AAS. Second, the detection of bisglucuronide conjugates of AAS was investigated. The mass spectrometric behavior was studied using synthesized AAS bisglucuronides to develop strategies for their detection. The formation of bisglucuronides was demonstrated in samples collected after norandrostenediol administration. Finally, the role of sulfate metabolites to detect endogenous AAS administration was evaluated. A LC-MS/MS method was developed for the direct quantitation of fourteen endogenous steroid sulfates. The use of some ratios between steroid sulfates improved the retrospectivity after oral and intramuscular testosterone administration. Steroid sulfates should be included in the steroid profile to complement the current markers. The results of the thesis demonstrate the increased role of the direct analysis of phase II metabolites of AAS in doping control.Los esteroides anabolizantes androgénicos (EAA) son ampliamente usados por los atletas para mejorar el rendimiento deportivo. En esta tesis se han desarrollado diferentes estudios en los que se evalúa el potencial de la detección directa de metabolitos de fase II por CL-EM/EM para mejorar la detección del abuso de los EAA en el deporte. Primeramente, se ha evaluado la detección directa de metabolitos glucurónido para la metandienone, permitiendo la detección de trece glucurónidos, uno de ellos resistente a la hidrolisis enzimática. La estrategia analítica propuesta ofrece un estudio exhaustivo y se recomienda su uso para estudios metabólicos de otros EAA. En segundo lugar, se ha evaluado la detección de conjugados bisglucurónidos de EAA. Se ha estudiado la ionización y fragmentación usando bisglucurónidos de EAA sintetizados con el fin de desarrollar estrategias para detectarlos. Estos conjugados se han detectado en muestras de orina recogidas tras la administración de norandrostenediol. Finalmente, se ha evaluado la utilidad de los metabolitos conjugados con sulfato para detectar la administración de EAA endógenos. Se ha desarrollado un método CL-EM/EM para la cuantificación directa de catorce esteroides endógenos sulfato. El uso de algunos ratios entre sulfatos mejora la capacidad de detección tras la administración oral e intramuscular de testosterona. Los esteroides sulfato deberían incluirse en el perfil esteroidal como complemento a los marcadores convencionales. Los resultados de esta tesis demuestran el aumento del protagonismo del análisis directo de metabolitos de fase II de los EAA en el control de dopaje

Screening for anabolic steroids in sports: analytical strategy based on the detection of phase I and phase II intact urinary metabolites by liquid chromatography tandem mass spectrometry

In order to improve the detection capabilities of anabolic androgenic steroids (AAS) in sports, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) screening method for the simultaneous detection of AAS phase I and phase II intact urinary metabolites (glucuronides and sulfates) was developed. A total of 36 metabolites (7 unconjugated; 19 glucuronides and 10 sulfates) corresponding to 15 of the most reported AAS were included. Analytes were extracted from urine using C18 cartridges. LC and MS conditions were studied in-depth to determine the most sensitive and selective conditions for each analyte. A selected reaction monitoring method was set up. The optimization of the experimental parameters for 13 metabolites not available as standards was performed using excretion study urines. Extraction recoveries were above 77% for all 23 validated analytes. Intra-day precision was lower than 21%, and LODs were in the range 0.25-4ng/mL for 18 of the 23 analytes. Matrix effect was evaluated using post column infusion and ranged from 92 to 147%. The method was successfully applied to excretion study urines of different exogenous AAS. The suitability of the strategy was demonstrated with methyltestosterone and stanozolol excretion study urines by achieving detection times of 22 and 21 days, respectively. The method is compliant with the World Antidoping Agency requirements for most of the studied compounds. It represents a cost-effective approach that improves the detection capabilities of AAS by increasing the sensitivity for some metabolites and by including recently described phase II long-term metabolites not detectable using the current screening strategy.Grants by Ministerio de Economía y Competividad (Gobierno de Espana) ˜ (Project number DEP2012-35612) and Generalitat de Catalunya (Consell Català de l’Esport and DIUE 2014 SGR 692) are gratefully acknowledge

Screening for anabolic steroids in sports: analytical strategy based on the detection of phase I and phase II intact urinary metabolites by liquid chromatography tandem mass spectrometry

In order to improve the detection capabilities of anabolic androgenic steroids (AAS) in sports, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) screening method for the simultaneous detection of AAS phase I and phase II intact urinary metabolites (glucuronides and sulfates) was developed. A total of 36 metabolites (7 unconjugated; 19 glucuronides and 10 sulfates) corresponding to 15 of the most reported AAS were included. Analytes were extracted from urine using C18 cartridges. LC and MS conditions were studied in-depth to determine the most sensitive and selective conditions for each analyte. A selected reaction monitoring method was set up. The optimization of the experimental parameters for 13 metabolites not available as standards was performed using excretion study urines. Extraction recoveries were above 77% for all 23 validated analytes. Intra-day precision was lower than 21%, and LODs were in the range 0.25-4ng/mL for 18 of the 23 analytes. Matrix effect was evaluated using post column infusion and ranged from 92 to 147%. The method was successfully applied to excretion study urines of different exogenous AAS. The suitability of the strategy was demonstrated with methyltestosterone and stanozolol excretion study urines by achieving detection times of 22 and 21 days, respectively. The method is compliant with the World Antidoping Agency requirements for most of the studied compounds. It represents a cost-effective approach that improves the detection capabilities of AAS by increasing the sensitivity for some metabolites and by including recently described phase II long-term metabolites not detectable using the current screening strategy.Grants by Ministerio de Economía y Competividad (Gobierno de Espana) ˜ (Project number DEP2012-35612) and Generalitat de Catalunya (Consell Català de l’Esport and DIUE 2014 SGR 692) are gratefully acknowledge

Detection and characterization of clostebol sulfate metabolites in Caucasian population.

Anabolic androgenic steroids (AAS) are synthetic testosterone derivatives which undergo extensive metabolism in man. Differences in the excretion of phase II metabolites are strongly associated with inter-individual and inter-ethnic variations. Sulfate metabolites have been described as long-term metabolites for some AAS. Clostebol is the 4-chloro derivative of testosterone and the aim of the present study was the evaluation of clostebol sulfate metabolites in Caucasian population by LC-MS/MS technology. Clostebol was orally administered to four healthy Caucasian male volunteers, and excretion study urines were collected up to 31 days. Several analytical strategies (neutral loss scan, precursor ion scan and selected reaction monitoring acquisitions modes) were applied to detect sulfate metabolites in post-administration samples. Sixteen sulfate metabolites were detected, five of them having detectability times above 10 days (S1a, S2a, S3b, S3g and S4b). Interestingly, metabolite S1a could be detected up to the last collected sample of all excretion studies and it was characterized by LC-MS/MS and GC-MS as 4ξ-chloro-5α-androst-3β-ol-17-one 3β-sulfate. Thus, monitoring of S1a improves the detection time of clostebol misuse with respect to the commonly monitored metabolites, excreted in the glucuronide fraction. Importantly, this new metabolite can be incorporated into recently developed LC-MS/MS screening methods base on the direct detection of phase II metabolites.The financial support received from the World Anti-Doping Agency WADA (12A21RV), Ministerio de Economía y Competividad (Gobierno de Espana)(Project number DEP2012-35612) and Generalitat de Catalunya (Consell Català de l’Esport, DIUE 2014 SGR 692) is gratefully acknowledged. Spanish Health National System is also acknowledged for O. J. Pozo contract (MS10/00576)

LC-MS/MS detection of unaltered glucuronoconjugated metabolites of metandienone

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.Grants by Ministerio de Economía y Competitividad (Gobierno de España) (Project number DEP2012-35612) and Generalitat deCatalunya (Consell Català de l’Esport and DIUE 2014 SGR 692) are gratefully acknowledge