High-Performance Liquid Chromatographic Determination of 6β-Hydroxycortisol in Urine

Abstract

A simple, rapid, sensitive and reproducible reversed-phase high-performance liquid chromatographic (HPLC) method was developed for the quantitative determination of urinary 6β-hydroxycortisol (6β-OHF) as a non-invasive indicator of drug-metabolizing enzyme activity in man. The urine (10ml) containing 6β-hydroxycortisone as an internal standard was loaded onto a Sep-Pak C₁₈ column and eluted with ethyl acetate. The samples were analyzed on a Radial-Pak Nova-Pak C₁₈ column, and the compounds were eluted with a mixture of 0.01 M potassium dihydrogen phosphate-acetonitrile-methanol (935 : 55 : 10) containing 0.005% trichloroacetic acid as a mobile phase and quantified by ultraviolet absorbance measurement at 243.5nm. The analytical recovery of added 6β-OHF was almost complete. The reproducibility assessed by repeated analysis was satisfactory, that is to say, the coefficient of variation ranged from 2.06 to 2.25% (within-run) and from 2.82 to 3.11% (between-run). In healthy adult subjects (23-41 years), the mean excretion rate of 6β-OHF was 214 μg/d (S.D.=41.4; n=10) and the mean ratio of 6β-OHF to 17-hydroxycorticosteroids was 0.0310 (S.D.=0.00662; n=10).The present HPLC method was found to be applicable to the routine assay of urinary 6β-OHF in clinical studies.