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A simple method for simultaneous determination of androstenediol 3-sulfate (Adiol-3S) and dehydroepiandrosterone sulfate (DHEA-S) in human serum using isotope diluted liquid chromatography-electrospray ionization-ion trap-mass spectrometry (LC-ESI-ion trap-MS) was developed. After addition of deuterated internal standards ([H-2(5)]Adiol-3S and [H-2(4)]DHEA-S), human serum (100 mul) was deproteinized with acetonitrile and then applied to a solid-phase extraction cartridge, Oasis HLB. The obtained steroid sulfates fraction was washed with hexane and then analyzed by LC-ESI-MS operated in the negative ion mode. The quantification ranges of Adiol-3S and DHEA-S were 10-400 ng/ml and 0.05-8 mug/ml, respectively. The method does not require the chemical or enzymatic hydrolysis of the conjugates and purification with high-performance liquid chromatography, and shows satisfactory reproducibility and accuracy. The concentrations of these sulfates in the sera of healthy male volunteers (n = 14) were 19.2-245.3 mg/ml (Adiol-3S) and 0.175-5.16 mug/ml (DHEA-S), and those of patients with prostate cancer (n = 19) were 15.3-182.7 ng/ml (Adiol-3S; four samples, not detectable) and 0.110-2.421 mug/ml (DHEA-S). (C) 2003 Elsevier B.V. All rights reserved.