Quantification of dehydroepiandrosterone, 17β-estradiol, testosterone and their sulfates in mouse tissues by LC/MS/MS

Abstract: We report a high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) assay to quantify without derivatizaton dehyroepiandrosterone (DHEA), 17β-estradiol (E2), testosterone (T), and their sulfates in serum and tissues. This assay functions well with multiple adipose depots, a previously unattained analysis. To delipidate and facilitate recovery, tissues were homogenized in acetonitrile and the homogenate was frozen. The supernatant was evaporated, resuspended in an aqueous acetate buffer, and extracted with hexane to separate free (unconjugated) from sulfated steroids. Sulfated steroids in the aqueous medium were then hydrolyzed with sulfatase and extracted with hexane. Each extract was analyzed separately. HPLC resolution combined with the sensitivity and specificity of MS/MS allowed quantification of DHEA, E2, and T with 10, 10 and 5 fmol lower limits of quantification and linear ranges to 1 pmol. Application of the method to mouse serum and tissues reveals ranges of DHEA, E...