Liquid chromatography clean-up method to improve identification of anabolic agents in human urine by gas chromatography–mass spectrometry Original Research Article

A confirmatory procedure to allow the unequivocal identification of the presence of urinary anabolic steroids and metabolites banned for use in sport by athletes is presented. A single liquid chromatography (LC) procedure was developed to selectively isolate the metabolites of the most abused anabolic agents. Five millilitre urine samples, with trenbolone added as a retention time marker, were concentrated by solid-phase extraction, enzymatically hydrolysed and directly injected onto the LC system. Chromatographic separation was carried out on a 150 mm×4.6 mm I.D. column, packed with 3 μm Hypersil BDS-C18 with gradient elution and UV absorbance detection (340 nm). Fractions containing compounds of interest were collected and evaporated. After solvent evaporation and derivatisation to form trimethylsilyl (TMS) derivatives, fractions were analysed by GC/MS in full scan mode with an external ionisation ion trap mass spectrometer. A validation procedure for qualitative analysis was performed for the anabolic agents with special sensitivity requirements according to the International Olympic Committee (IOC) and the World Anti-Doping Agency (WADA). Appropriate precision, accuracy and recoveries were found. The selectivity of the method showed that no interfering peaks were observed at the retention time of the analytes, allowing obtaining full scan spectra even at the low concentrations required (2 ng/ml).