Journal of Chromatography & Separation Techniques
Open Access

Abstract

Development and validation of a quantification method of urinary free cortisol in 24 hours urine by mass detection.

Momal Alexe, Dhesse Quentin, Goderniaux Nicolas,Closset Mélanie, Zihao Wang2, Colsoul Marie-Lise*

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, reproductible and specific assay technique to quantify UFC by High
Performance Liquid Chromatography (UHPLC) and mass detection.
Methods: We developed a method based on an UHPLC assay coupled to a single quadrupole mass (QDa)
detector. Urines were pre-treated with 1% formic acid and the internal standard before the Supported Liquid
Extraction (SLE). Elution was performed with dichloromethane that was then evaporated. Samples were
reconstituted with water. The compounds were separated on a HSS T3 column with a gradient elution (0.1% formic
acid in water and acetonitrile). Injection volume was 1 μl and the flow rate 0.6 ml/min. We investigated the
method regarding linearity, precision, recovery, accuracy, limits of detection and quantification, robustness, and
carry-over.
Results: The linear range of the method was from 5 μg/l to 1000 μg/l with a determination coefficient of 0.999. The
precision was evaluated by the Relative Standard Deviation (RSD) for intra- and inter- assay and was below 9% for
both of them. The method showed adequate cortisol recovery (> 90%). The bias between the mean of between-day
results and the target value was < 4.7% while the biases between measured and expected values from an external
quality control program were <6%. The limit of detection was 0.284 μg/L and the limit of quantification was 0.755
μg/L. The variation representing the method robustness was 4.1% that is lower than the between-day variation. The
carry-over was <0.02%.
Conclusion: The UHPLC-QDa method with a SLE step for UFC quantification shows adequate performances. It is
easy, reproductible and suitable for routine laboratory use.

Published Date: 2025-07-07; Received Date: 2025-06-05