Direct quantification of steroid glucuronides by liquid chromatography tandem mass spectrometry

A method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the direct quantification of glucuronides of testosterone (TG), epitestosterone (EPG), androsterone (AG) and etiocholanolone (ETG) has been developed. The method allowed for the direct determination of these analytes avoiding hydrolysis and derivatization, which are usual steps in commonly used methods based on gas chromatography-mass spectrometry (GC-MS). Two different approaches have been tested for sample preparation: direct injection after filtration and acidic liquid-liquid extraction (LLE) with ethyl acetate. The values obtained by both approaches were satisfactory for accuracy and precision with limits of detection lower than 1ng/ml for TG and EPG. The applicability of the method has been checked by the analysis of 100 urine samples. The results were compared with those obtained with the common GC-MS method. Results showed a good correlation between both methods. Introduction The abuse of T is normally controlled by the ratio between urinary concentrations of T and epitestosterone (E) (T/E ratio). Other ratios including concentrations of androsterone and etiocholanolone can also be useful to provide evidence of T, dihydrotestosterne (DHT), dehydroepiandrosterone (DHEA) and alfa reductase inhibitor abuse [1]. In order to establish these ratios accurately, quantitative methods for these analytes are needed. Endogenous steroids are normally excreted in urine after conjugation in phase II metabolism reactions, of which the most common one is glucuronidation. The determination of these endogenous steroids is normally performed by GC-MS after enzymatic hydrolysis, liquidIn: W Schänzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent Advances In Doping Analysis (16). Sport und Buch Strauß Köln 2008