Determination of 17-oxosteroids in serum and urine by fluorescence high-performance liquid chromatography using dansyl hydrazine as a pre-labeling reagent

J Chromatogr. 1981 Nov 13;226(1):1-12.

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 (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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • 17-Ketosteroids / blood*
  • 17-Ketosteroids / urine
  • Chromatography, High Pressure Liquid / methods
  • Dansyl Compounds*
  • Female
  • Fluorescence
  • Humans
  • Hydrazines
  • Male
  • Reference Values

Substances

  • 17-Ketosteroids
  • Dansyl Compounds
  • Hydrazines
  • dansyl hydrazine