Journal of Endocrinological Investigation

, Volume 42, Issue 11, pp 1299–1305 | Cite as

Measurement of urinary free cortisol by LC–MS–MS: adoption of a literature reference range and comparison with our current immunometric method

  • L. Bianchi
  • B. Campi
  • M. R. Sessa
  • G. De Marco
  • E. Ferrarini
  • R. Zucchi
  • C. Marcocci
  • P. Vitti
  • L. Manetti
  • A. Saba
  • P. AgrettiEmail author
Original Article



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.


Urinary 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

Informed consent was obtained from all individual participants included in the study.


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Copyright information

© Italian Society of Endocrinology (SIE) 2019

Authors and Affiliations

  • L. Bianchi
    • 1
  • B. Campi
    • 2
  • M. R. Sessa
    • 1
  • G. De Marco
    • 3
  • E. Ferrarini
    • 3
  • R. Zucchi
    • 4
  • C. Marcocci
    • 3
    • 5
  • P. Vitti
    • 3
    • 5
  • L. Manetti
    • 5
  • A. Saba
    • 4
    • 6
  • P. Agretti
    • 1
    Email author
  1. 1.Laboratory of Chemistry and EndocrinologyUniversity Hospital of PisaPisaItaly
  2. 2.Institute of Clinical PhysiologyNational Research CouncilPisaItaly
  3. 3.Section of Endocrinology, Department of Clinical and Experimental MedicineUniversity of PisaPisaItaly
  4. 4.Department of Surgical, Medical and Molecular Pathology and Critical Care MedicineUniversity of PisaPisaItaly
  5. 5.Endocrinology UnitsUniversity Hospital of PisaPisaItaly
  6. 6.Laboratory of Clinical PathologyUniversity Hospital of PisaPisaItaly

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