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. 2019 Nov;42(11):1299-1305. doi: 10.1007/s40618-019-01050-5. Epub 2019 Apr 22.

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.

Keywords: Antibody cross-reactivity; Immunometric assay; Interference from steroid similar molecules; LC–MS–MS; Reference range; Urinary free cortisol.

Publication types

  • Comparative Study

MeSH terms

  • Adolescent
  • Adult
  • Aged
  • Chromatography, Liquid / methods*
  • Female
  • Healthy Volunteers
  • Humans
  • Hydrocortisone / urine*
  • Immunoassay / methods*
  • Male
  • Middle Aged
  • Reference Values
  • Tandem Mass Spectrometry / methods*
  • Young Adult

Substances

  • Hydrocortisone