Markers of mesterolone abuse in sulfate fraction for doping control in human urine.

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-3ß-ol-17 one, M2 is 1α-methyl-5α-androstan-3α-ol-17 one, M4 is 1α-methyl-5a-androstan-3ß, 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.