Determination of 17-oxosteroids in serum and urine by fluorescence high-performance liquid chromatography using dansyl hydrazine as a pre-labeling reagent.
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 (beta-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 use for the simultaneous determination of individual 17-oxosteroids in serum and urine.
D015068:17-Ketosteroids / Q000097:blood* / Q000652:urine; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003619:Dansyl Compounds; D005260:Female; D005453:Fluorescence; D006801:Humans; D006834:Hydrazines; D008297:Male; D012016:Reference Values
D015068:17-Ketosteroids; D003619:Dansyl Compounds; D006834:Hydrazines; C027913:dansyl hydrazine