A-Ring reduced 17β-hydroxymethyl-13-ene metabolites for further extension of long-term detectability of 17-methyl steroids

Code: 20A06MP 

Since the detection of the latest long-term metabolite of metandienone a lot of effort is put in the uncoverage of similar metabolites with 17β-hydroxymethyl-17α-methyl-13-ene structure for long-term detection of other 17-methyl steroids. The closely related 4-chlorometandienone (Oral Turinabol, DHCMT) was found to be excreted as analogous metabolites, with A-ring reduced metabolites being even longer detectable.

Thus, in the current project we aim to investigate the excretion of metandienone with special respect to A-ring reduced metabolites as well. Their integration in doping control analysis may further extend the detection window of a misuse of metandienone and open new possibilities to catch cheaters that adjusted their habits to experiences after the integration of 17β-hydroxymethyl-17α-methylandrosta-1,4,13-triene-3-one into routine screening. Deduced from DHCMT metabolism, it is expected that also after metandienone administration a potentially further increased detection window may be achieved from screening for the analogue metabolite 17β-hydroxymethyl-17α-methyl-18-norandrost-13-en-3-ol.

Main findings

Since the detection of the latest long-term metabolite of metandienone a lot of effort is put in the discovery of similar metabolites with 17β-hydroxymethyl-17α-methyl-13-ene structure for long-term detection of other 17-methyl steroids. Thereby the closely related 4-chlorometandienone (Oral Turinabol, DHCMT) was found to be excreted as analogous metabolites, with A-ring reduced metabolites being even longer detectable. Thus, in the current project, the elimination of metandienone with special respect to A-ring reduced metabolites was investigated as well. Various diastereomers of 17-hydroxymethyl-17-methyl-18-norandrost-13-en-3-ol (20OHNorTHMT) were chemically synthesized as well as diastereomers of 17-methylandrostanediol (THMT) for analytical method optimization, and characterized by high resolution mass spectrometry (HRMS) and nuclear magnetic resonance (NMR) spectroscopy. Their excretion in human urine after a single dose administration of metandienone was monitored using GC-MS. Both THMT and 20OHNorTHMT were detected in the post administration urines. Final excretion data and excretion kinetics was evaluated for the various metabolites in six volunteers. As diastereomers 3α,5β-20OHNorTHMT, 3α,5β-THMT and 3α,5β-epiTHMT as well as 3α,5β-NorTHMT were unambiguously confirmed in the post-administration urines, besides the classical metandienone metabolites. Stereochemical assignments were achieved by comparison of retention times and mass spectral data with authentic reference material. While the commonly considered metandienone long-term metabolite, 20OHNorMD was detectable for up to 34 days in the GC-QQQ-MS analysis, the above-mentioned metabolites with fully reduced A-ring were detectable for much shorter post-administration windows (approximately 5-9 days post-administration). Thus, the reduced metabolites are obviously not amenable for long-term detection of a metandienone administration, which is in contrast to its closely related chlorinated analog DHCMT. However, a promising new candidate long-term metabolite was assigned to the 1-ene analogue of 3α,5β-20OHNorTHMT (17-hydroxymethyl-17-methyl-18-nor-5β-androst-1,13-dien-3α-ol). While its quantifier transition allowed for detection for up to 38 days after administration using GC-QQQ-MS in one volunteer its confirmation will most likely require improved clean-up procedures or optimized chromatographic separation to eliminate interferences from the urinary matrix.

In addition to further assembling the picture of human metabolism of AAS with newly discovered metabolites, NorTHMT turned out to be a promising candidate for a new long term marker in anti doping analyses. However, the current method did not allow for proper confirmation in the late excretion urines due to matrix interferences. Thus, method adaptations have to be evaluated in the future to provide a reasonable method for later routine use as confirmatory procedure.