Application of high-performance liquid chromatography to the measurement of cortisol secretion rate

After administration of [3H] cortisol, the specific radioactivity of the urinary cortisol metabolites is determined by chemical conversion of the 17-hydroxycorticosteroid conjugates to 17-ketosteroids, extraction with Sep-Pak C18 cartridges, derivatisation with 2,4-dinitrophenylhydrazine and isocratic reversed-phase high-performance liquid chromatography. This allows on-line measurement of ultra-violet absorbance; fractions are collected and 3H is measured by scintillation counting. Alternatively extraction and chromatography are done after enzymic hydrolysis of the steroid conjugates, and the major metabolites are determined colorimetrically in the fractions. The results from the two methods were consistent with each other and with published data.