Determination of 17-oxosteroid glucuronides and sulphates in urine by liquid chromatography using 2,4-dinitrophenylhydrazine as a prelabelling reagent for spectrophotometric detection.

A direct, convenient method using high-performance liquid chromatography with spectrophotometric detection has been developed for the determination of conjugated 17-oxosteroids in urine without hydrolysis. Conjugated 17-oxosteroids are extracted with a Sep-Pak C18 cartridge, prelabelled with 2,4-dinitrophenylhydrazine in trichloroacetic acid-benzene solution and then separated by high-performance liquid chromatography on a reversed-phase C18 column using a mobile phase of 70% methanol in a buffer consisting of 50 mM sodium acetate in 2% (v/v) acetic acid. The eluate is monitored by a spectrophotometer at 380 nm and a linear response was found for absorbance readings (peak heights) from amounts of various conjugated oxosteroids between 25 and 250 ng. The method provides a sensitive, reliable technique for the analysis of urinary 17-oxosteroid conjugates.