Quantification of testosterone and metabolites released after alkaline treatment in human urine

A rapid, accurate, and sensitive method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the quantification of the testosterone (T) fraction released under basic conditions and its metabolites 1,4-androstadien-3,17-dione (1,4-AD), 4,6-androstadien-3,17-dione (4,6-AD) and 4,6-androstadien-17β-ol-3-one (6-T) obtained in the same way has been developed and validated. Urine samples (5 ml) were alkalinized and the released analytes were extracted by liquid-liquid extraction. The chromatographic separation was performed in 8 min. MS/MS determination was performed under selected reaction monitoring mode using electrospray ionization in positive mode. The method was shown to be linear from 0.05 to 50 ng/ml for 1,4-AD, 6-T and T while for 4,6-AD the linearity from 0.5 to 500 ng/ml was demonstrated (r > 0.99). Limits of detection below 0.05 ng/ml were obtained for all analytes. Intra-assay precision and accuracies, evaluated at three concentrations levels (0.1, 1 and 20 ng/ml) were below 20%. The applicability of the analytical method was confirmed by analysis of several samples collected from healthy volunteers and samples collected after oral testosterone undecanoate administration. The analytical method was found to be able to quantify the metabolites either at the low concentrations levels present in the urine from untreated volunteers or at the high concentrations found after T administration. Short analysis time, simple sample preparation, and satisfactory quantitative parameters make this method potentially useful for anti-doping control purposes.