Simultaneous Determination of Cortisol, Cortisone, and 6β-Hydroxycortisol in Human Urine by UPLC with UV Detector and Application to Determine Diurnal Variations

In the present studies, a simple rapid ultra performance liquid chromatography (UPLC) method with UV detection for the simultaneous determination of cortisol, cortisone and 6 beta-hydroxycortisol in human urine was developed. The three analytes and the internal standard dexamethasone were separated on a Waters Acquity UPLC-Tunable UV system with an Acquity UPLC BEH C18 column (50 x 2.1 mm ID, 1.7 mu m) using a gradient elution of methanol and water (containing 0.01% formic acid) with a run time of 7 min. The method was accurate and precise, over the ranges of 5-200 ng mL(-1) for cortisol, and 10-1,000 ng mL(-1) for both cortisone and 6 beta-hydroxycortisol, and showed good linearity (r (2) > 0.999). This method was applied for the measurement of cortisol, cortisone and 6 beta-hydroxycortisol in samples collected over different periods as a tool to assess the activity of CYP3A and 11 beta-hydroxysteroid dehydrogenase type 2 enzymes.