New Insights into the Metabolism of Methyltestosterone and Metandienone: Detection of Novel A-Ring Reduced Metabolites

Abstract

Metandienone and methyltestosterone are orally active anabolic-androgenic steroids with a 17α-methyl structure that are prohibited in sports but are frequently detected in anti-doping analysis. Following the previously reported detection of long-term metabolites with a 17ξ-hydroxymethyl-17ξ-methyl-18-nor-5ξ-androst-13-en-3ξ-ol structure in the chlorinated metandienone analog dehydrochloromethyltestosterone ("oral turinabol"), in this study we investigated the formation of similar metabolites of metandienone and 17α-methyltestosterone with a rearranged D-ring and a fully reduced A-ring. Using a semi-targeted approach including the synthesis of reference compounds, two diastereomeric substances, viz. 17α-hydroxymethyl-17β-methyl-18-nor-5β-androst-13-en-3α-ol and its 5α-analog, were identified following an administration of methyltestosterone. In post-administration urines of metandienone, only the 5β-metabolite was detected. Additionally, 3α,5β-tetrahydro-epi-methyltestosterone was identified in the urines of both administrations besides the classical metabolites included in the screening procedures. Besides their applicability for anti-doping analysis, the results provide new insights into the metabolism of 17α-methyl steroids with respect to the order of reductions in the A-ring, the participation of different enzymes, and alterations to the D-ring.

Keywords: 17-hydroxymethyl-17-methyl-18-nor; 17α-methyl steroids; D-ring alteration; doping control; gas chromatography-mass spectrometry; long-term metabolites; metabolism.

Figure 1

Adverse analytical findings of methyltestosterone and metandienone between 2003 and 2018, according to [4].

Figure 2

Chemical structures of phase I metabolites of metandienone reported in the literature.

Figure 3

Chemical structures of phase I metabolites of methyltestosterone reported in the literature.

Figure 4

Reaction scheme for 17α-hydroxymethyl-17β-methyl-18-nor-5ξ-androst-13-en-3ξ-ol steroids.

Figure 5

Mass spectrum (GC-EI-QTOF-MS, 70 eV) of 17α-hydroxymethyl-17β-methyl-18-nor-5β-androst-13-en-3α-ol (8), bis- trimethylsilyl) (TMS (x-axis: m/z; y-axis: relative abundance).

Figure 6

Synthesis route for 17β-methyl-5β-androstane-3α,17α-diol (11).

Figure 7

Mass spectrum (GC-EI-QTOF-MS, 70 eV) of 17β-methyl-5β-androstane-3α,17α-diol (11), bis TMS (x-axis: m/z; y-axis: relative abundance).

Figure 8

GC-QQQ-MS chromatograms (MRM, m/z 345 → 173).

Figure 9

Chromatograms (MRM, m/z 450 → 345); black: positive urine sample after intake of methyltestosterone; red: synthesized substances.

Figure 10

Potential ways of A-ring reduction.

Figure 11

Proposed metabolism of methyltestosterone (black, 18) and metandienone (red, 12) to the found metabolites 17α-hydroxymethyl-17β-methyl-18-nor-5β-androst-13-en-3α-ol (8), 17α-hydroxymethyl-17β-methyl-18-nor-5α-androst-13-en-3α-ol (8a), and 17β-methyl-5β-androstane-3α,17α-diol (11) except last step of metandienone. The question marks represent reactions whose enzymes have not been elucidated yet.