Objectives: Twenty-four-hour urinary free cortisol (UFC) measurement is the initial diagnostic test for Cushing's syndrome (CS). We compared UFC determination by four new immunoassays using Autobio A6200, Mindray CL-1200i, Snibe MAGLUMI X8 and Roche 8000 e801 with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Additionally, we evaluated the value of 24-h UFC measured by four direct immunoassays for diagnosing CS.
Methods: Residual 24-hr urine samples of 94 CS and 243 non-CS patients collected from previous cohort were used. A laboratory-developed LC-MS/MS method was used as reference. UFC was measured by immunoassays using Autobio, Mindray, Snibe and Roche platforms. Method was compared using Passing-Bablok regression and Bland-Altman plot analyses. Cut-off values for each assay and corresponding sensitivities and specificities were calculated by ROC analysis.
Results: All four immunoassays showed strong correlations with LC-MS/MS (Spearman coefficient r = 0.950, 0.998, 0.967, and 0.951, respectively). All immunoassays showed proportionally positive bias. The areas under the curve were 0.953 for Autobio, 0.969 for Mindray, 0.963 for Snibe, and 0.958 for Roche. The cut-off values varied from 178.5 to 272.0 nmol/24 h). Assay sensitivity and specificity ranged from 89.66 % to 93.10 % and from 93.33 % to 96.67 %, respectively.
Conclusions: Four newly available direct immunoassays for measuring UFC show good analytical consistency compared to LC-MS/MS. The elimination of organic solvent extraction simplifies workflows while maintaining high diagnostic accuracy. Additionally, they exhibited similarly high diagnostic accuracy for CS identification. Future multi-center studies are needed to validate our findings and establish method-specific UFC cut-off values to enhance clinical utility.
Keywords: Cushing's syndrome; Immunoassay; Liquid chromatography-tandem mass spectrometry; Method comparison; Urinary free cortisol.
© 2025 The Authors.