New drostanolone metabolites in human urine by liquid chromatography time-of-flight tandem mass spectrometry and their application for doping control

• Drostanolone metabolic profile was first investigated by LC–QTOFMS.
• Sulfate-conjugated metabolites were first reported for drostanolone.
• New potential biomarkers S4 and G1 were confirmed for drostanolone.
• Direct analysis of intact metabolites was first reported.
• Eleven urinary metabolites were characterized and identified.

Drostanolone is one of the most frequently detected anabolic androgenic steroids in doping control analysis. Here, we studied drostanolone urinary metabolic profiles using liquid chromatography quadruple time of flight mass spectrometry (LC–QTOF-MS) in full scan and targeted MS/MS modes with accurate mass measurement. The drug was administered to one healthy male volunteer and liquid–liquid extraction along with direct-injection were used to analyze urine samples. Chromatographic peaks for potential metabolites were identified with the theoretical [M−H]− as a target ion in a full scan experiment and actual deprotonated ions were analyzed in targeted MS/MS mode. Eleven metabolites including five new sulfates, five glucuronide conjugates, and one free metabolite were confirmed for drostanolone. Due to the absence of useful fragment ions to illustrate the steroid ring structure of drostanolone phase II metabolites, gas chromatography mass spectrometry (GC–MS) was used to obtain structural details of the trimethylsilylated phase I metabolite released after enzymatic hydrolysis and a potential structure was proposed using a combined MS approach. Metabolite detection times were recorded and S4 (2α-methyl-5α-androstan-17-one-6β-ol-3α-sulfate) and G1 (2α-methyl-5α-androstan-17-one-3α-glucuronide) were thought to be new potential biomarkers for drostanolone misuse which can be detected up to 24 days by liquid–liquid extraction and 7 days by direct-injection analysis after intramuscular injection. S4 and G1 were also detected in two drostanolone-positive routine urine samples.

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