Simultaneous determination of dehydroepiandrosterone and its 7-oxygenated metabolites in human serum by high-resolution gas chromatography–mass spectrometry

Abstract

A highly sensitive and specific method has been developed for the simultaneous measurement of free (unconjugated) or sulfate-conjugated forms of dehydroepiandrosterone (DHEA), 7α-hydroxy-DHEA (7α-OH-DHEA), 7β-hydroxy-DHEA (7β-OH-DHEA), and 7-oxo-DHEA (7-oxo-DHEA) in human serum. This method is based upon a stable isotope-dilution technique by gas chromatography–selected-ion monitoring mass spectrometry. Free steroids were extracted from serum with an organic solvent and the sulfate-conjugated steroids remained in aqueous phase. Free steroids were purified by solid-phase extraction, while sulfate-conjugated steroids were hydrolyzed by sulfatase and deconjugated steroids were purified by solid-phase extractions. The extracts were treated with O-methylhydroxylamine hydrochloride and were subsequently dimethylisopropylsilylated. The resulting methyloxime-dimethylisopropylsilyl (MO-DMIPS) ether derivatives were quantified by gas chromatography–selected-ion monitoring mass spectrometry in a high-resolution mode. The detection limits of MO-DMIPS ether derivatives of DHEA, 7α-OH-DHEA, 7β-OH-DHEA and 7-oxo-DHEA were 1.0, 0.5, 0.5 and 2.0 pg, respectively. Coefficients of variation between samples ranged from 10.6 to 22.9% for free 7-oxygenated DHEA to less than 10% for DHEA and sulfate-conjugated 7-oxygenated DHEA. The concentrations of these steroids were measured in 18 sera samples from healthy volunteers (9 males and 9 females; aged 23–78 years). Free DHEA, 7α-OH-DHEA, 7β-OH-DHEA and 7-oxo-DHEA levels ranged between 0.21–3.55, 0.001–0.194, 0.003–0.481, and 0.000–0.077 ng/ml, respectively, and the sulfate-conjugated steroid levels of these metabolites ranged between 253–4681, 0.082–3.001, 0.008–0.903, and 0.107–0.803 ng/ml, respectively. The free DHEA-related steroid concentrations were much lower than those previously measured by RIA and low-resolution GC–MS. The present method made it possible to determine simultaneously serum DHEA-related steroid levels with sufficient sensitivity and accuracy.