Dosage du cortisol libre urinaire par chromatographie liquide

In our laboratory, a specific assay technique of urinary free cortisol was developed in liquid chromatography using methylprednisolone as the internal standard. Before the chromatography, sample were extracted with double extraction liquid-liquid followed by C18 solid-phase extraction. The compounds were separated on a Zorbax Eclipse® XDB-C18 column with a mobile phase of water-acetonitrile. Linearity, precision, recovery and accuracy of the method were established. The limit of quantification was 5 ng/mL of cortisol, and the total coefficients of variation were <8%. The chromatography method was compared first with an automated immunoassay using chemiluminescent label (Advia-Centaur®) then with a reference laboratory. The results obtained in immunology showed an over-estimate of the excretion of free cortisol compared to the chromatographic method. The evaluation compared to the reference was satisfactory. At the end of study, the chromatographic technique allowed us to reconsider the reference range of normal cortisol excretion from 3.9 to 58.5 μg/24 hours.