Detection time comparison of non‐hydrolysed sulphated metabolites of metenolone, mesterolone and 17α‐methyltestosterone analysed by four different mass spectrometric techniques

Abstract

The frequent detection of anabolic androgenic steroids (AAS) indicates their popularity among rule-breaking athletes. The so called long-term metabolites play a crucial role in their detection, and non-hydrolysed sulphated metabolites have gained renewed interest, as research has demonstrated their extended detection time compared to the more conventional markers (e.g., for metenolone and mesterolone).

New long‐standing metabolites of 17α‐methyltestosterone are detected in HepG2 cell in vitro metabolic model and in human urine

“…Nevertheless, other minor metabolites such as 17α-hydroxy-17β-methylandrost-4-ene-3-one (epimethyltestosterone, Μ2) and 17β-hydroxy-17α-methylandrost-4,6-dien-3-one (6-enemethyltestosterone, Μ3) are detected by LC-MS/MS, 30 while the 3α-sulfate derivative of 17β-methyl-5α-androstane-3α,17α-diol was proven to have important potential for the long-term detection of methyltestosterone misuse by LC-MS/MS. 11,31 Hydroxylated metabolites of methyltestosterone in positions C-2, C-4, and C-6 were also detected, but proven to be minor ones, according to the results of both in vitro and in vivo experiments. 32 Recently, a metabolite featuring a fully reduced A-ring with a rearranged D-ring, that is, the 17α-hydroxymethyl-17β-methyl-18-nor-5β-androst-13-en-3α-ol, was detected in post-administration samples of methyltestosterone along with the 17βmethyl-5β-androstane-3α,17α-diol (M7), which was also detected.…”

“…In comparison, specific examples of partially reduced A-ring steroids with 17,17-dimethyl-18-nor-Δ13 structure are known for other steroids like the methandienone metabolite, 17,17-dimethyl-18-nor-5β-androsta-1,13-dien-3α-ol, known as well as normetenol. 37 These metabolites, however, might be the missing links of the metabolic chain between the 17β-methyl-5β-androstane-3α,17α-diol (M6), 17β-methyl-5αandrostane-3α,17α-diol (Μ7), 11,[30][31][32][33][34] and the recently reported 17αhydroxymethyl-17β-methyl-18-nor-5β-androst-13-en-3α-ol and its 5α analog. 33 Synthesis of the speculated metabolites with reduced A-ring and 17,17-dimethyl-18-nor-Δ13 structure for methyltestosterone was conducted on a microscale, and the synthesized standards were characterized by GC-MS/(MS).…”

“…[3][4][5][6][7] AASs are metabolized extensively to both phases I and II metabolites in humans, and despite the in-depth studies on their biotransformation routes performed in previous decades, the need for more efficient markers of abuse justifies ongoing studies using state-of-the-art instrumentation. [8][9][10][11] It is important to note that for the efficient monitoring of AASs misuse in the frame of sports drug testing, the most abundant metabolites are not necessarily the best markers of AASs administration, but instead, the best markers are those which provide the longest period of detectability (long-term metabolites).…”

“…32 Recently, a metabolite featuring a fully reduced A-ring with a rearranged D-ring, that is, the 17α-hydroxymethyl-17β-methyl-18-nor-5β-androst-13-en-3α-ol, was detected in post-administration samples of methyltestosterone along with the 17βmethyl-5β-androstane-3α,17α-diol (M7), which was also detected. 33 An overview on the metabolism of methyltestosterone metabolism in humans as described in the literature 11,[30][31][32][33][34] is presented in Figure 1.…”

“…The above metabolites 35 have been confirmed by another study in equine urine, which proposed similar metabolic routes for methyltestosterone and mestanolone. 36 A review of the human metabolism of 17α-methyltestosterone 11,[30][31][32][33][34] reveals that the epimerization of the 17α-methyl substituent results in further A-ring reduced metabolites with long-term detection potential. These metabolites are, most probably, produced 34 by spontaneous hydrolysis of the 17β-sulfate group, formed during the phase II metabolism.…”