Quantifying endogenous androgens, estrogens, pregnenolone and progesterone metabolites in human urine by gas chromatography tandem mass spectrometry

A method for the quantitation of 22 urinary steroids (androgens, estrogens and the main pregnenolone and progesterone metabolites) by means of gas chromatography tandem mass spectrometry wing a triple quadrupole analyzer has been developed. Two different enzymatic hydrolysis protocols were investigated; one capable of releasing steroids present as both sulfates and glucuronides (total fraction), and another with beta-glucuronidase activity only. After selecting adequate internal standards and choosing the optimal instrumental parameters, i.e. chromatographic separation and ion transition conditions, the method was fully validated using both hydrolysis protocols. The method was shown to be linear (r > 0.99) in the range of endogenous concentrations for all studied steroids with extraction recoveries higher than 80%. The use of labeled internal standards allowed for both a correct quantification and the evaluation of the rate of deconjugation for sulfates and glucuronides in every sample. In general, the sensitivity of the method was suitable for the detection of the endogenous levels, with limits of quantification ranging from 0.1 to 20 ng/mL. Accuracies ranging from 80% to 120%, and relative standard deviations below 25% in intra- and inter- assay experiments were found for most of the analytes. The applicability of the validated method was tested by quantifying twenty-two metabolites in 24-h urine samples collected from healthy individuals. The ranges for the excretion of steroids in the total and glucuronide fractions obtained with the new method were compared with those available in the literature. By comparing the figures in both fractions, an estimation of the percentage that the sulfation represents for each steroid was also calculated. The presence of side enzymatic activities and the utility of the method for clinical studies as well as for doping control analysis is discussed.