SIMULTANEOUS HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC DETERMINATION OF 6-BETA-HYDROXYCORTISOL AND CORTISOL IN URINE WITH FLUORESCENCE DETECTION AND ITS APPLICATION FOR ESTIMATING HEPATIC DRUG-METABOLIZING ENZYME-INDUCTION

A simple and sensitive high-performance liquid chromatographic method is described for the simultaneous determination of 6 beta-hydroxycortisol (6 beta-OHF) and cortisol (F) in urine. Urine (1 ml) containing fludrocortisone as the internal standard is extracted with ethyl acetate. The extract is washed successively with sodium hydroxide solution and water, and subsequently dried under a stream of nitrogen. The residue is redissolved in methanol. The 6 beta-OHF, F and fludrocortisone in the methanol solution are oxidized by cupric acetate and the resulting glyoxal compounds are converted into fluorescent derivatives with 1,2-diamino-4,5-methylenedioxybenzene (DMB). The DMB derivatives of the corticosteroids are separated within 70 min on a reversed-phase column, L-Column ODS, using stepwise elution with methanol-acetonitrile-0.5 M ammonium acetate and detected fluorimetrically at 350 nm (excitation) and 390 nm (emission). The lower limits of detection for 6 beta-OHF and F are 1.8 pmol (680 pg) and 2.4 pmol (950 pg)/ml urine (0.6 pmol and 0.8 pmol/100 mu l injection volume), respectively, at a signal-to-noise ratio of 3. This method can be applied to the determination of urinary 6 beta-OHF, and the ratio of 6 beta-OHF to F in humans and in rhesus monkeys treated orally with phenobarbital as a hepatic drug-metabolizing enzyme inducer.