Abstract
A fluorescence high-performance liquid chromatographic method is described for the determination of 17-oxosteroids in biological fluids. 17-Oxosteroids in urine samples are extracted with dichloromethane after enzymatic hydrolysis (β-glucuronidase—sulfatase), and dehydroepiandrosterone sulfate in serum samples is solvolysed with sulfuric acid in ethyl acetate. 17-Oxosteroids are labeled with dansyl hydrazine in trichloroacetic acid—benzene solution, and then chromatographed on the microparticulate silica gel column using dichloromethane—ethanol—water (400:1:2) as the mobile phase. The eluate is monitored by a fluorophotometer at 365 nm (excitation) and 505 nm (emission). Linearities of the fluorescence intensities (peak heights) with the amounts of various 17-oxosteroids were obtained between 60 and 1000 pg. The assay proved satisfactory with respect to sensitivity, precision and accuracy. The results obtained by a radioimmunoassay and this method were in good agreement ( r = 0.964, n = 81) for serum dehydroepiandrosterone sulfate. This method is also useful for the simultaneous determination of individual 17-oxosteroids in serum and urine.
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