Simultaneous determination of dehydroepiandrosterone and its 7-oxygenated metabolites in human serum by high-resolution gas chromatography--mass spectrometry.
A highly sensitive and specific method has been developed for the simultaneous measurement of free (unconjugated) or sulfate-conjugated forms of dehydroepiandrosterone (DHEA), 7alpha-hydroxy-DHEA (7alpha-OH-DHEA), 7beta-hydroxy-DHEA (7beta-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, 7alpha-OH-DHEA, 7beta-OH-DHEA and 7-oxo-DHEA were 1.0, 0.5, 0.5 and 2.0pg, 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, 7alpha-OH-DHEA, 7beta-OH-DHEA and 7-oxo-DHEA levels ranged between 0.21-3.55, 0.001-0.194, 0.003-0.481, and 0.000-0.077ng/ml, respectively, and the sulfate-conjugated steroid levels of these metabolites ranged between 253-4681, 0.082-3.001, 0.008-0.903, and 0.107-0.803ng/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.
Matsuzaki
Yasushi
Y
Department of Gastroenterology and Hepatology, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan. ymatsuzaki-gi@umin.ac.jp
Yoshida
Shigemasa
S
Honda
Akira
A
Miyazaki
Teruo
T
Tanaka
Naomi
N
Takagiwa
Aya
A
Fujimoto
Yoshinori
Y
Miyazaki
Hiroshi
H
D000328:Adult; D000368:Aged; D002138:Calibration; D005591:Chemical Fractionation; D003687:Dehydroepiandrosterone / Q000031:analogs & derivatives / Q000097:blood* / Q000378:metabolism; D003903:Deuterium; D005260:Female; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods* / Q000592:standards; D006801:Humans; D008297:Male; D008875:Middle Aged; D015203:Reproducibility of Results
D003687:Dehydroepiandrosterone; D003903:Deuterium