Simultaneous determination of androstenedione, 11β-hydroxyandrostenedione, and testosterone in human plasma by stable isotope dilution mass spectrometry

This study describes a GC–MS method for the simultaneous determination of androstenedione (AD), 11β-hydroxyandrostenedione (11β-OHAD), and testosterone (TS) in human plasma. [19,19,19-2H3]Androstenedione (AD-2H3), 11β-hydroxy-[1,2,4,19-13C4]androstenedione (11β-OHAD-13C4), and [1,16,16,17-2H4]testosterone (TS-2H4) were used as internal standards. Pentafluoropropionic (PFP) derivatization with good GC behavior was employed for the GC–MS analysis of the three steroids. The detection limit of the present GC–MS–SIM method was found to be 1 pg per injection for AD (S/N ratio = 4.5), 5 pg for 11β-OHAD (S/N ratio = 5.0), and 1 pg for TS (S/N ratio = 4.4), respectively. Calibration curves were linear from 0.22 to 2.80 ng/mL (r = 0.9998) for AD, from 0.56 to 3.19 ng/mL (r = 0.9996) for 11β-OHAD, and from 2.05 to 10.3 ng/mL (r = 0.9996) for TS. The intra- and inter-day assay reproducibilities in the amounts of the three androgens determined were in good agreement with the actual amounts added, the relative errors (R.E.) were −3.1 to 2.4%. The inter-assay relative standard deviation (R.S.D.) was less than 5.3%. The present method provides a sensitive and reliable technique for the simultaneous determination of AD, 11β-OHAD, and TS in plasma. The method can be applied to pharmacokinetic and metabolic studies of androgens with a particular interest in evaluating the conversion of AD to 11β-OHAD and the interconversion of AD and TS in humans.