Sulfate metabolites improve retrospectivity after oral testosterone administration.

The detection of testosterone (T) misuse is performed using the steroid profile that includes concentrations of T and related metabolites excreted free and glucuronoconjugated, and ratios between them. In this work, the usefulness of fourteen endogenous steroid sulfates to improve the detection capabilities of oral T administration has been evaluated. Quantitation of the sulfate metabolites was performed using solid-phase extraction and analysis by liquid chromatography-tandem mass spectrometry. Urine samples were collected up to 144 h after a single oral dose of T undecanoate (120 mg) to five Caucasian male volunteers. Detection times (DTs) of each marker were estimated using reference limits based on a population study and also monitoring the individual threshold for each volunteer. High inter-individual variability was observed for sulfate metabolites and, therefore, better DTs were obtained using individual threshold. Using individual threshold limits, epiandrosterone sulfate (epiA-S) improved the DT with respect to testosterone/epitestosterone (T/E) ratio in all volunteers. Androsterone, etiocholanolone and two androstanediol sulfates also improved DTs for some volunteers. Principal component analysis was used to characterize the sample cohort, obtaining thirteen ratios useful for discrimination. These ratios as well as the ratio epiA-S/dehydroepiandrosterone sulfate were further examined. The most promising results were obtained using ratios between sulfates of epiA, androsterone or androstanediol 1 and E, and also sulfates of epiA or androstanediol 1 and dehydroandrosterone. These selected ratios prolonged the DT of oral T administration up to 144h, which corresponded to a significantly higher retrospectivity compared to those obtained using concentrations or the conventional T/E ratio.