Measurement of urinary free cortisol by LC–MS–MS: adoption of a literature reference range and comparison with our current immunometric method. (Saba A., Agretti P., co-last authors)

Purpose: One of the best indicators of adrenal gland dysfunction is the level of free cortisol measured in the 24-h urine (UFC) which faithfully reflects the level of biologically active serum cortisol not subjected to circadian variations. Liquid chromatography coupled with tandem mass spectrometry (LC–MS–MS) is a sensitive, accurate and precise method recently available in routine laboratories that could remedy interference problems of immunoassays. Methods: In this study, a literature reference range for UFC measured by LC–MS–MS was verified, and UFC values measured by LC–MS–MS and immunoassay were compared. Immunometric UFC measurement was performed by ACCESS CORTISOL assay without preliminary extraction, using Beckman Coulter UniCel DxI 600 highly automated platform. Liquid chromatography–tandem mass spectrometry UFC measurement was performed by a home-made validated method using cortisol-D 4 as internal standard with preliminary deproteinization of urinary samples by centrifugal filter and injection on reverse-phase column. Cortisol was analyzed in positive ion mode with an ESI interface. Results: The reference interval from literature (11–70 μg/day) was confirmed by results obtained for healthy study group. Comparison study of the two methods highlighted a constant and proportional systematic error with a general tendency to overestimate results for the in-use method. Conclusions: In conclusion, the direct immunometric method overestimates UFC results with respect to liquid chromatography–tandem mass spectrometry which represents the reference method. The literature reference range 11–70 μg/day was confirmed and can be adopted by our lab that will shift all UFC tests performed in routine to the mass spectrometry-based method, satisfying clinicians’ request.