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This manuscript describes the direct detection of mesteroloe sulfo-conjugated metabolites by liquid chromatography/quadrupole/time of flight mass spectrometry (LC/Q/TOFMS) with special focus on evaluation of their retrospective detectability and their structure elucidation. A comparison of their long-term detectability, with the mesterolone main metabolite (1α-methyl-5α-androstan-3α-ol-17-one) excreted in glucuronide fraction and detected by gas chromatography/high resolution mass spectrometry (GC/HRMS), is also presented. Studies on mesterolone were performed with samples obtained from two excretion studies after single oral administration of Proviron© by healthy volunteers. Potential sulfate metabolites were detected in post administration samples after liquid-liquid extraction (LLE) with ethyl acetate and LC/TOFMS analysis, in negative mode. Twelve mesterolone sulfate metabolites from the first excretion study and nine from the second were subsequently confirmed by LC/Q/TOFMS. Finally, six mesterolone sulfate metabolites were considered important taking into account their abundance and long-term detectability, encoded as M1, M2, M4, M5, M6 and M7. The proposed mesterolone sulfate metabolites M1, M2, M4 and M5 (excreted as sulfates) have the same retrospectivity with the main mesterolone metabolite, excreted in glucuronide fraction. For metabolite characterization, LC fractionation was performed. The metabolites were identified and characterized by GC/MS, after solvolysis and derivatization. Combined mass spectra data from trimethyl-silyl (TMS), TMS-enolTMS and methoxime-TMS derivatives were taken into account for the characterization of these metabolites. It was concluded that M1 is 1α-methyl-5α-androstan-3β-ol-17 one, M2 is 1α-methyl-5α-androstan-3α-ol-17 one, M4 is 1α-methyl-5a-androstan-3β, 16z-diol-17-one, M5 is 1α-methyl-5α-androstan-17z,4ξ-diol-3one, M6 is 1α-methyl-5α-androstan-3z,6z-diol-17-one and M7 is 4z-hydroxy-1α-methyl-5α-androstan-3,17-dione.
The abuse of unknown designer androgenic anabolic steroids (AAS) is considered to be an issue of significant importance, as AAS are the choice of doping preference according to World Anti-doping Agency statistics. In addition, unknown designer AAS are preferred since the World Anti-doping Agency mass spectrometric identification criteria cannot be applied to unknown molecules. Consequently, cheating athletes have a strong motive to use designer AAS in order to both achieve performance enhancement and to escape from testing positive in anti-doping tests. To face the problem, a synergy is required between the anti-doping analytical science and sports anti-doping regulations. This Review examines various aspects of the designer AAS. First, the structural modifications of the already known AAS to create new designer molecules are explained. A list of the designer synthetic and endogenous AAS is then presented. Second, we discuss progress in the detection of designer AAS using: mass spectrometry and bioassays; analytical data processing of the unknown designer AAS; metabolite synthesis; and, long-term storage of urine and blood samples. Finally, the introduction of regulations from sports authorities as preventive measures for long-term storage and reprocessing of samples, initially reported as negatives, is discussed.
This paper presents the development and validation of a high-resolution full scan (FS) electron impact ionization (EI) gas chromatography coupled to quadrupole Time-of-Flight mass spectrometry (GC/QTOF) platform for screening anabolic androgenic steroids (AAS) in human urine samples. The World Antidoping Agency (WADA) enlists AAS as prohibited doping agents in sports, and our method has been developed to comply with the qualitative specifications of WADA to be applied for the detection of sports antidoping prohibited substances, mainly for AAS. The method also comprises of the quantitative analysis of the WADA's Athlete Biological Passport (ABP) endogenous steroidal parameters. The applied preparation of urine samples includes enzymatic hydrolysis for the cleavage of the Phase II glucuronide conjugates, generic liquid-liquid extraction and trimethylsilyl (TMS) derivatization steps. Tandem mass spectrometry (MS/MS) acquisition was applied on few selected ions to enhance the specificity and sensitivity of GC/TOF signal of few compounds. The full scan high resolution acquisition of analytical signal, for known and unknown TMS derivatives of AAS provides the antidoping system with a new analytical tool for the detection designer drugs and novel metabolites, which prolongs the AAS detection, after electronic data files' reprocessing. The current method is complementary to the respective liquid chromatography coupled to mass spectrometry (LC/MS) methodology widely used to detect prohibited molecules in sport, which cannot be efficiently ionized with atmospheric pressure ionization interface.
The population based Steroid Profile (SP) ratio of testosterone (T) and epitestosterone (E) has been considered as a biomarker approach to detect testosterone abuse in '80s. The contemporary Antidoping Laboratories apply the World Antidoping Agency (WADA) Technical Document (TD) for Endogenous Androgenic Anabolic Steroids (EAAS) in the analysis of SP during their screening. The SP Athlete Biological Passport (ABP) adaptive model uses the concentrations of the total of free and glucuronide conjugated forms of six EAASs concentrations and ratios measured by GC/MS. In the Antidoping Lab Qatar (ADLQ), the routine LC/MS screening method was used to quantitatively estimate the sulfate conjugated EAAS in the same analytical run as for the rest qualitative analytes. Seven sulfate EAAS were quantified for a number of routine antidoping male and female urine samples during screening. Concentrations, statistical parameters and selected ratios for the 6 EAAS, the 6 sulfate EAAS and 29 proposed ratios of concentrations from both EAAS and sulfate EAAS, which potentially used as SP ABP biomarkers, population reference limits and distributions have been estimated after the GC/MSMS analysis for EAAS and LC/Orbitrap/MS analysis for sulfate EAAS.
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