Determination of 17-hydroxycorticosteroids in urine by fluorescence high-performance liquid chromatography using Dns-hydrazine as a pre-column labeling reagent.

A method is described for the determination of urinary 17-hydroxycorticosteroids using fluorescence high-performance liquid chromatography. After enzymatic hydrolysis, 17-hydroxycorticosteroids were extracted using an Extrelut column and then labeled with Dns-hydrazine in hydrochloric acid-ethanol solution. The labeled steroids were chromatographed on a microparticulate silica gel column, the mobile phase was dichloromethane-ethanol-water (900:60:40). The eluate was monitored on a fluorophotometer at 365 nm (excitation) and 505 nm (emission). Linearity of the fluorescence intensities (peak heights) of various 17-hydroxycorticosteroids were obtained between 60 pg and 20 ng. The assay was sensitive, precise and accurate. Comparison with the results obtained by radioimmunoassay gave correlation coefficients of 0.932 for tetrahydrocortisol and 0.930 for tetrahydrocortisone. The proposed method is clinically useful for the routine analysis of urinary 17-hydroxycorticosteroids.