Alternative markers for Methylnortestosterone misuse in human urine

Methylnortestosterone is a progestin and synthetic androgenic anabolic steroid, prohibited by WADA. Methylnortestosterone misuse is commonly detected by monitoring the parent compound and its main metabolites, 17 alpha-methyl-5 alpha-estrane-3 alpha, 17 beta-diol (M1) and 17 alpha-methyl-5 beta-estrane-3 alpha, 17 beta-diol (M2), in the glucuronide fraction. In the current study, a direct detection of methylnortestosterone sulfo-conjugated metabolites after ethyl acetate extraction and analysis by LC/Q/TOF-MS in negative ionization mode was performed, detecting two main sulfate metabolites (S1, S2). For the characterization of metabolites, samples from the excretion study, were additionally analyzed by GC-MS, after solvolysis and per TMS derivatization. RT and MS data collected, were compared with RT and MS data from metabolites of 17z-methyl-5 alpha/beta-estrane-3 alpha/beta, 17z-diols structures with prefixed stereochemistry at 3 and 5 positions, synthesized through Grignard reaction from 19-noretiocholanolone, 19-norandrosterone and 19-norepiandrosterone. Confirmed sulfate metabolites were S1, 17 alpha-methyl-5 alpha-estrane-3 alpha, 17 beta-diol 3 alpha sulfate (detected up to 72 h) and S2, 17 alpha-methyl-5 beta-estrane-3 alpha, 17 beta-diol 3 alpha sulfate (detected up to 192 h). Furthermore, applying targeted analysis based on RT and MS data of the synthesized metabolites two additional metabolites M3, 17 beta-methyl-5 beta-estrane-3 alpha, 17 alpha-diol and M4, 17 beta-methyl-5 alpha-estrane-3 alpha, 17 alpha-diol were detected in the glucuronide fraction and one more metabolite (S3) 17 beta-methyl-5 beta-estrane-3 alpha, 17 alpha-diol was detected in the sulfate fraction in lower abundance until the end of the excretion study (192 h). Interestingly, S2 could also be detected after the direct analysis of non-hydrolyzed steroid by GC-MS/MS as artifact, following normal ProcIV anabolic steroid procedure and using diethylether as extraction solvent.