Cortisol and cortisone ratio in urine: LC-MS/MS method validation and preliminary clinical application

Background: The determination of urinary cortisol/cortisone ratio is of clinical utility in cases of Cushing's syndrome, apparent mineralocorticoid excess, and also provides information on 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) type 2 activity. It is therefore of utmost importance to ensure accurate cortisol and cortisone measurement and establish appropriate reference ranges. Methods: After the isotopic dilution of urine, sample cleanups were obtained with on-line solid-phase extraction and cortisol and cortisone, separated using a Zorbax Eclipse XDB-C18 HPLC analytical column, were analyzed by tandem mass spectrometry with an electrospray ionization source in positive ion mode operation. Results: The method was linear, with concentrations of up to 625 and 1125 nmol/L and lower limit of quantitation (LLOQ) of 5 and 6 nmol/L, for cortisol and cortisone, respectively. Within-run and between-run coefficients of variation were <5% and 6% for cortisol and 6% and 8% for cortisone, respectively. No ion suppression was observed. The non-parametric reference range for the cortisol/cortisone ratio was 0.14-1.09. Conclusions: A simple and sensitive liquid chromatography tandem mass spectrometry method was developed and validated for the measurement of cortisol and cortisone in urine. Our findings indicate that the proposed analytical method is suitable for routine purposes and useful in many pathological conditions.