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Development and Validation of a Quantification Method of Urinary Free Cortisol in 24 hours Urine by Mass Detection

Momal Alexe*, Dhesse Quentin, Goderniaux Nicolas, Colsoul Marie-Lise, Closset Mélanie

Introduction: Measurement of urinary excretion of corticosteroids and their metabolites is used to evaluate adrenal function in Cushing's syndrome or adrenal cancer. Quantification of Urinary Free Cortisol (UFC) is recommended for Cushing’s syndrome diagnosis.

Objective: To develop an easy, reproducible and specific assay technique to quantify UFC by High Performance Liquid Chromatography (UHPLC)

Methods: We developed a method based on an Ultra-High Performance Liquid Chromatography (UHPLC) assay coupled to a single Quadrupole mass (QDa) detector. Samples were extracted by Supported Liquid Extraction (SLE). Urines were pre-treated with 1% formic acid and the internal standard. Cortisol was eluted with dichloromethane. Solvent was evaporated and samples were reconstituted with water. The compounds were separated on a HSS T3 column with a gradient elution (water, 0.1% formic acid and acetonitrile). Injection volume was 1 µl and the flow rate 0.6 ml/min. We investigated the method regarding linearity, precision, recovery, accuracy and limits of detection and quantification.

Results: The linear range for this method was from 5 μµg/l to 1000 µg/l with a determination coefficient of 0.99. The precision was evaluated by the Relative Standard Deviation (RSD) for intra-assay and inter-assay and was below 9% for both of them. The method showed adequate cortisol recovery (>91%). The limit of detection was 0.28 µg/L and the limit of quantification was 0.75 µg/L.

Conclusion: The UFC UHPLC-QDa detection method with a SLE step shows adequate performances. It is easy, reproducible and suitable for routine laboratory use.