Urinary detection of conjugated and unconjugated anabolic steroids by dilute-and-shoot liquid chromatography-high resolution mass spectrometry

Anabolic androgenic steroids (AAS) are an important class of doping agents. The metabolism of these substances is generally very extensive and includes phase‐I and phase‐II pathways. In this work, a comprehensive detection of these metabolites is described using a 2‐fold dilution of urine and subsequent analysis by liquid chromatography‐high resolution mass spectrometry (LC‐HRMS). The method was applied to study 32 different metabolites, excreted free or conjugated (glucuronide or sulfate), which permit the detection of misuse of at least 21 anabolic steroids. The method has been fully validated for 21 target compounds (8 glucuronide, 1 sulfate and 12 free steroids) and 18 out of 21 compounds had detection limits in the range of 1–10 ng mL−1 in urine. For the conjugated compounds, for which no reference standards are available, metabolites were synthesized in vitro or excretion studies were investigated. The detection limits for these compounds ranged between 0.5 and 18 ng mL−1 in urine. The simple and straightforward methodology complements the traditional methods based on hydrolysis, liquid‐liquid extraction, derivatization and analysis by gas chromatography–mass spectrometry (GC‐MS) and liquid chromatography‐mass spectrometry (LC‐MS). Copyright © 2014 John Wiley & Sons, Ltd. A validated DS‐LC‐HRMS method allows to detect 32 conjugated and unconjugated anabolic steroids in urine. The detection limits of 30 of them were in the range 0.5‐20 ng mL‐1. The application to in vitro experiments and excretion urine permitted the extension of the scope to compounds not commercially available as well as improving knowledge of the metabolism finding better targets compounds. This approach complements more traditional methodologies and sets the stage for future screening methods in doping control.