Challenges in the diagnosis and follow-up of Cushing's syndrome: Evaluating LC-MS/MS as a confirmation method for urinary free cortisol analyses.

24-h urinary free cortisol (UFC) measurements are fundamental in the diagnosis and follow-up of Cushinǵs syndrome (CS) and immunoassays (IA) are the most widely used tests for its quantification in clinical laboratory practice. However, their suitability has been questioned mainly due to their limitations concerning analytical specificity. The aim of this research project was to evaluate a novel algorithm for CS diagnosis and follow-up in the clinical laboratory, based on the combination of IA tests with liquid chromatography-tandem mass spectrometry (LC-MS/MS) for UFC quantification. A quantitative LC-MS/MS approach based on liquid-liquid extraction for sample preparation was developed and fully characterized. A population-based reference range was established and the level of agreement for UFC values when compared to IA approach was assessed in patients under CS follow-up or clinical suspicion for hypercortisolism. Significantly higher UFC values were observed for IA when compared to LC-MS/MS approach, therefore population-based reference range was established for the latter (i.e. 4 - 41 µg/day). The clinical application of the herein presented LC-MS/MS approach to be used as a confirmation procedure for CS management was assessed and a high level of agreement with IA UFC values, except in the case of IA undetectable results, was observed. However, IA potential false negative (FN) and false positive (FP) findings were also noted. Higher clinical sensitivity for CS diagnosis / follow-up was observed for LC-MS/MS when compared to IA, supporting the advantage and necessity of implementing LC-MS/MS as a confirmation procedure in the clinical laboratory.