SIMULTANEOUS HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC DETERMINATION OF6-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-hydroxycorti sol (6 beta-OHF) and cortisol (F) in urine. Urine (1 ml) containing fl udrocortisone as the internal standard is extracted with ethyl acetate . The extract is washed successively with sodium hydroxide solution an d water, and subsequently dried under a stream of nitrogen. The residu e is redissolved in methanol. The 6 beta-OHF, F and fludrocortisone in the methanol solution are oxidized by cupric acetate and the resultin g 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 a nd 0.8 pmol/100 mu l injection volume), respectively, at a signal-to-n oise ratio of 3. This method can be applied to the determination of ur inary 6 beta-OHF, and the ratio of 6 beta-OHF to F in humans and in rh esus monkeys treated orally with phenobarbital as a hepatic drug-metab olizing enzyme inducer.