High-performance liquid chromatographic determination of 6.BETA.-hydroxycortisol in urine.

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Abstract
A simple, rapid, sensitive and reproducible reversed-phase high-performance liquid chromatographic (HPLC) method was developed for the quantitative determination of urinary 6.beta.-hydroxycortisol (6.beta.-OHF) as a non-invasive indicator of drug-metabolizing enzyme activity in man. The urine (10 ml) containing 6.beta.-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 243.5 nm. The analytical recovery of added 6.beta.-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 6.beta.-OHF was 214 .mu.g/d (S.D. = 41.4; n = 10) and the mean ratio of 6.beta.-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 6.beta.-OHF in clinical studies.

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