Quantifying Plasma Androgens by LC-MS/MS

Plasma androgens (testosterone and androstenedione) were analyzed by adapting a previously described method (17 (link)). Briefly, steroids were extracted from plasma (500 μL) by solid-phase extraction on an HLB Oasis (60-mg, 3-cc columns; Waters UK, Elstree, UK) with 10 ng ¹³C₃-testosterone and ¹³C₃-androstenedione (Sigma Aldrich, Dorset, UK) as internal standards. Extracted steroids were separated using liquid chromatography on a UPLC column (2.1 mm × 50 mm, 1.7 μm; Acquity UPLC BEH C18; Waters, MA). All steroids were analyzed in positive ion mode using electrospray ionization on an AB Sciex QTrap 5500 operating in triple quadrupole mode for testosterone (m/z 289 → 97, 25 V) and androstenedione (m/z 287 → 97, 25 V). Linear regression analysis of calibration standards, calculated using peak area ratios of analytes to internal standard, was used to determine the concentration of the analytes in the samples.