A new potential biomarker for 1-testosterone misuse in human urine by liquid chromatography quadruple time-of-flight mass spectrometry

Abstract

In this study, 1-testosterone urinary metabolic profiles were investigated by liquid chromatography quadruple time-of-flight mass spectrometry (LC-QTOF-MS) using full scan and targeted MS/MS techniques with accurate mass measurement. 1-Testosterone was administered to two healthy male volunteers. Liquid–liquid extraction (LLE) and direct-injection were applied for processing urine samples. Chromatographic peaks for potential metabolites were found by using the theoretical [M − H]⁻ as the target ion in the full scan experiment and their actual deprotonated ions were analyzed in targeted MS/MS mode. Four free reported metabolites (M1, M2, M3 and M4) were found for 1-testosterone together with an unreported sulfate-conjugated metabolite (S1). Due to the absence of useful fragment ions to illustrate the sulfate structure, a gas chromatography mass spectrometry (GC-MS) instrument was applied to obtain structural details of the trimethylsilylated phase I metabolite released after solvolysis. Thus its potential structure was proposed particularly by a combined MS approach. All the metabolites were evaluated in terms of how long they could be detected and S1 was considered to be the long term metabolite for 1-testosterone misuse detected up to 15 days by LLE and 10 days by direct-injection analysis after oral administration.