Measurement of urinary free cortisol by LC–MS–MS: adoption of a literature reference range and comparison with our current immunometric method
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.
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-D4 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.
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.
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.
KeywordsUrinary free cortisol LC–MS–MS Immunometric assay Interference from steroid similar molecules Antibody cross-reactivity Reference range
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
Statement of human rights
The study has been performed in accordance with the ethical standards as laid down in the 1964 Declaration of Helsinki, and its later amendments or comparable ethical standards.
Informed consent was obtained from all individual participants included in the study.
- 4.Wood L, Ducroq DH, Fraser HL, Gillingwater S, Evans C, Pickett AJ, Rees DW, John R, Turkes A (2008) Measurement of urinary free cortisol by tandem mass spectrometry and comparison with results obtained by gas chromatography–mass spectrometry and two commercial immunoassays. Ann Clin Biochem 45:380–388CrossRefGoogle Scholar
- 11.European Medicines Agency (2015) Guideline on bioanalytical method validation. http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2011/08/WC500109686.pdf. Accessed 21 July 2011
- 15.Henny J, Vassault A, Boursier G, Vukasovic I, Mesko Brguljan P, Lohmander M, Ghita I, Andreu FA, Kroupis C, Sprongl L, Thelen MH, Vanstapel FJ, Vodnik T, Huisman W, Vaubourdolle M, Working Group Accreditation and ISO/CEN standards (WG-A/ISO) of the EFLM (2016) Recommendation for the review of biological reference intervals in medical laboratories. Clin Chem Lab Med 54:1893–1900CrossRefGoogle Scholar
- 23.McCann SJ, Gillingwater S, Keevil BG (2005) Measurement of urinary free cortisol using liquid chromatography–tandem mass spectrometry: comparison with the urine adapted ACS:180 serum cortisol chemiluminescent immunoassay and development of a new reference range. Ann Clin Biochem 42:112–118CrossRefGoogle Scholar