Abstract

Cross reacting antibodies can cause an overestimation of the results of immunoassays. Therefore, alternative methodsare needed for the accurate quantification of steroids. Gas chromatography combined with isotope-dilution mass spectrometry(GC-IDMS) is developed to quantify urinary active androgens, testosterone, epitestosterone and dihydrotestosterone, which areclinically relevant androgens to both hair-loss and prostate diseases. The method devised involves enzymatic hydrolysis with β-glucuronidase, solid-phase extraction, liquid-liquid extraction using methyl tert-butyl ether and subsequent conversion to pentafluorophenyldimethylsilyl-trimethylsilyl (flophemesyl-TMS) derivatives for sensitive and selective analysis in selected-ionmonitoring mode. Flophemesyl-TMS derivatization not only eliminates matrix interference but also has a good peak resolutionwithin a 6 min-run. A selective and sensitive GC technique with flophemesyl-TMS derivatives also allows accurate quantitativeanalysis of three active androgens when combined with IDMS. The limit of quantification of the three analytes was <50 pg/mL,and extraction recoveries ranged from 91.9 to 102.1%. The precision and accuracy were 1.2~6.5% and 89.0~106.7%, respectively. This GC-IDMS method can be useful for evaluating the drug efficacy and monitoring the biological processes responsiblefor male-pattern baldness and prostate diseases.