Simultaneous Determination of Free Cortisol, Cortisone and their Tetrahydrometabolites in Urine by Single Solvent Extraction and Liquid Chromatography–Tandem Mass Spectrometry

Abstract

A fast, efficient and low-cost high performance liquid chromatography–tandem mass spectrometry methodology was developed and validated for the simultaneous determination of free urinary cortisone, cortisol and their tetrahydro-metabolites. The developed method comprises a simple liquid-liquid extraction with CH2Cl2, followed by reversed-phase liquid chromatography–tandem mass spectrometry (LC–MS/MS) with electrospray ionization (ESI) in positive mode. The baseline chromatographic separation of the analytes, including the stereoisomers tetrahydrocortisol (THF) and allo-THF, was achieved on a Hypersil Gold C18 column with a mobile phase consisting of 0.05%v/v formic acid in water—acetonitrile, using a gradient elution program. The influence of the mobile phase composition and the ESI parameters on the sensitivity of the method was extensively studied. Sample preparation was also optimized, testing two techniques: solid phase extraction (SPE) and liquid-liquid extraction (LLE). Recoveries ranged from 74.7% (a-THF) to 93.5% (cortisol) and the method limits of detection (MLD) ranged from 0.34 ng mL−1 (cortisol) to 1.37 ng mL−1 (THF). Intra- and inter-day coefficient of variation of the assay varied from1.5% (allo-THF) to 13% (tetrahydrocortisone) and from 3.6% (allo-THF) to 14.9% (tetrahydrocortisone), respectively. The method was applied for the analysis of urine samples from 53 healthy individuals with a mean age of 13.96 years in order to estimate the concentration of the five corticosteroids and the ratio of the metabolites. Associations between urinary cortisol/cortisone and serum cortisol/cortisone values were also characterized.