Androstanediol and 5-androstenediol profiling for detecting exogenously administered dihydrotestosterone, epitestosterone, and dehydroepiandrosterone: Potential use in gas chromatography isotope ratio mass spectrometry

Abstract

The basis of a potential method for confirming intake of four natural androgens (testosterone, epitestosterone, dihydrotestosterone, and dehydroepiandrosterone is presented. The method relies on isolating from urine a steroid fraction containing androstenediol and androstanediol metabolites of these natural steroids and analyzing their 13C content by gas chromatography, combustion, isotope ratio mass spectrometry. The steroids were recovered from urine by conjugate hydrolysis with a Helix pomatia preparation (sulfatase and β-glucuronidase), Girard T reagent separation to obtain a nonketonic fraction, and Sephadex LH-20 chromatography for purification. Metabolites appropriate for all of the natural steroids could be separated (as diacetates) by gas chromatography on a DB-17 capillary column viz.: 5α (and β)-androstane-3α,17α-diol (epitestosterone as precursor); 5α (and β)-androstane-3α,17β-diol (testosterone as precursor); 5-androstene-3β,17β-diol (dehydroepiandrosterone precursor); and 5α-androstane-3α,17β- (and 17α-) diol (dihydrotestosterone precursor). Measurement of the 13C content of the specific analytes after ingestion of the androgen precursors demonstrated a lowering of δ 13 C‰ value compared to normal values. Typically, in the male individual studied, δ 13 C‰ values for all components were −26 to −27 before drug administration and −29 to −30 at 6 h after, the latter values reflecting those obtaining for commercial synthetic steroid compared to in vivo synthesized steroid. While generally the metabolism of the steroids was as expected, this was not the case for 5α-dihydrotestosterone. A major metabolite was 5α-androstane-3α,17α-diol, which had presumably been formed by 17β 17α isomerization, a process previously known for unnatural anabolics but not for natural hormones. The isolation, purification, and isotope ratio mass spectrometry techniques described may form the basis of a general method for confirming natural steroid misuse by sports participants.