This study aimed to evaluate whether it is analytically possible to distinguish the transdermal absorption of methyltestosterone from oral administration. The urinary 17α-methyl-5α-androstane-3α,17β-diol to 17α-methyl-5β-androstane-3α,17β-diol ratio (5α-THMT/5β-THMT) may serve as a valuable indicator for the administration route. For dried blood spot analysis, the detection window was shorter than that of tetrahydro-metabolites in urine.

ABSTRACT

Unintentional doping violations have been reported after secondary skin exposure, including partner contact or contact sports. Methyltestosterone (MT), an anabolic androgenic steroid on the World Anti-Doping Agency Prohibited List, is readily available in Japan as an over-the-counter topical hair-growth preparation and as oral tablets. This study evaluated whether transdermal absorption of MT can be distinguished from oral administration using human urine and dried blood spot (DBS) analyses. Ten men received MT once daily for five consecutive days (five oral, five transdermal). The urinary tetrahydro-metabolites 17α-methyl-5α-androstane-3α,17β-diol (5α-THMT) and 17α-methyl-5β-androstane-3α,17β-diol (5β-THMT) were detectable within hours after administration for both routes. Following transdermal administration, 5α-THMT remained detectable up to 97 h after the final administration, whereas, after oral administration, 5β-THMT persisted longer, up to 265 h. Transdermal application produced a marked increase in 5α-THMT, consistent with high 5α-reductase activity in skin, yielding a significantly higher 5α-THMT/5β-THMT ratio than after oral administration. Nonetheless, individuals with inherently high 5α-reductase activity may exhibit elevated ratios even after oral dosing, warranting careful interpretation. In DBS after oral dosing, parent MT was generally detectable up to 24 h, providing a shorter detection window than urinary tetrahydro-metabolites. Overall, combining urine (tetrahydro-metabolites) and DBS (parent MT) analyses enables effective detection of MT use and supports differentiation of administration routes.