Simultaneous determination of 6β-hydroxycortisol and cortisol in human urine by liquid chromatography with ultraviolet absorbance detection for phenotyping the CYP3A activity determined by the cortisol 6β-hydroxylation clearance

Abstract This study describes a high-performance liquid chromatographic (HPLC) method for the simultaneous determination of 6β-hydroxycortisol (6β-OHF) and cortisol in human urine using either methylprednisolone or beclomethasone as internal standard. Separation was achieved on a reversed-phase phenyl column by a gradient elution of 0.05 M KH 2 PO 4 –0.01 M CH 3 COOH (pH 3.77) and 0.05 M KH 2 PO 4 –0.01 M CH 3 COOH with acetonitrile (4:6, v/v). 6β-Hydroxycortisol and cortisol were monitored by UV absorption at 239 nm. The lower quantitation limits of the present HPLC method were 21.5 ng/ml for 6β-OHF and 5.0 ng/ml for cortisol in urine. The within-day reproducibilities in the amounts of 6β-OHF and cortisol determined were in good agreement with the actual amounts added, the relative error being less than 1.59%. The inter-assay precisions (R.S.D. values) were less than 7.91% for 6β-OHF and cortisol. The method was compared with the GC/MS method by measuring 6β-OHF in the same urine samples. A good correlation was found between the amounts determined by the two methods. The regression equations for the HPLC ( y ) and GC/MS ( x ) methods were: y =1.0701 x +17.389 ( r =0.9772) for methylprednisolone as internal standard and y =1.0827 x +6.1364 ( r =0.9794) for beclomethasone as internal standard.