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

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Abstract

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-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.

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.

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Author information

Author notes

  1. L. Bianchi and B. Campi contributed equally to this work (co-first authors).

  2. A. Saba and P. Agretti designed and supervised equally the experimental work (co-last authors).

Authors and Affiliations

  1. Laboratory of Chemistry and Endocrinology, University Hospital of Pisa, Via Paradisa 2, 56124, Pisa, Italy

    L. Bianchi, M. R. Sessa & P. Agretti

  2. Institute of Clinical Physiology, National Research Council, Via Moruzzi 1, 56124, Pisa, Italy

    B. Campi

  3. Section of Endocrinology, Department of Clinical and Experimental Medicine, University of Pisa, Via Paradisa 2, 56124, Pisa, Italy

    G. De Marco, E. Ferrarini, C. Marcocci & P. Vitti

  4. Department of Surgical, Medical and Molecular Pathology and Critical Care Medicine, University of Pisa, Via Savi 10, 56126, Pisa, Italy

    R. Zucchi & A. Saba

  5. Endocrinology Units, University Hospital of Pisa, Via Paradisa 2, 56124, Pisa, Italy

    C. Marcocci, P. Vitti & L. Manetti

  6. Laboratory of Clinical Pathology, University Hospital of Pisa, Via Roma 67, 56126, Pisa, Italy

    A. Saba

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  1. L. Bianchi
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  2. B. Campi
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  10. A. Saba
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Corresponding author

Correspondence to P. Agretti.

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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.

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Bianchi, L., Campi, B., Sessa, M.R. et al. Measurement of urinary free cortisol by LC–MS–MS: adoption of a literature reference range and comparison with our current immunometric method. J Endocrinol Invest 42, 1299–1305 (2019). https://doi.org/10.1007/s40618-019-01050-5

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  • DOI: https://doi.org/10.1007/s40618-019-01050-5

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