Simultaneous high-performance liquid chromatographic determination of 6β-hydroxycortisol and cortisol in urine with fluorescence detection and its application for estimating hepatic drug-metabolizing enzyme induction

Abstract

A simple and sensitive high-performance liquid chromatographic method is described for the simultaneous determination of 6β-hydroxycortisol (6β-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 6β-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 6β-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 μl injection volume), respectively, at a signal-to-noise ratio of 3. This method can be applied to the determination of urinary 6β-OHF, and the ratio of 6β-OHF to F in humans and in rhesus monkeys treated orally with phenobarbital as a hepatic drug-metabolizing enzyme inducer.