Simultaneous determination of cortisol, cortisone, and 6 β-hydroxycortisol in human urine by UPLC with UV detector and application to determine diurnal variations

Abstract

In the present studies, a simple rapid ultra performance liquid chromatography (UPLC) method with UV detection for the simultaneous determination of cortisol, cortisone and 6β-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 × 2.1 mm ID, 1.7 μm) 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 6β-hydroxycortisol, and showed good linearity (r ² > 0.999). This method was applied for the measurement of cortisol, cortisone and 6β-hydroxycortisol in samples collected over different periods as a tool to assess the activity of CYP3A and 11β-hydroxysteroid dehydrogenase type 2 enzymes.