Studies on steroids. CC. Determination of 17-ketosteroid sulphates in serum by high-performance liquid chromatography...

A new sensitive method is described for the determination of 17-ketosteroid sulphates, particularly dehydroepiandrosterone sulphate, in human serum by high-performance liquid chromatography with electrochemical detection. The 17-ketosteroid sulphates in serum were extracted with acetonitrile and derivatized with p- nitrophenylhydrazine in trichloroacetic acid--benzene solution. The p- nitrophenylhydrazones were separated by high-performance liquid chromatography on a mu Bondapak C18 column using methanol--0.5% ammonium dihydrogen phosphate (8:3) as a mobile phase. The proposed method proved to be applicable to the quantitation of 17-ketosteroid sulphates with satisfactory sensitivity and reliability, providing a quantitation limit of 80 ng/ml and coefficient of variation of 4%. A good correlation was observed between the values obtained by the present method and radioimmunoassay for dehydroepiandrosterone sulphate in serum.