Quantitative profiling of cortisol metabolites in human urine by high-resolution accurate-mass MS

Abstract

Aim, materials & methods: Urinary cortisol profile has the potential as a diagnostic biomarker. We therefore developed a stable-isotope dilution ultraperformance chromatography multistage MS-based method to quantify cortisol and 16 metabolites in human urines. Results & conclusion: The LOD for cortisol and its metabolites ranges from 0.02 to 5.81 pg/μl urine. The inter- and intraday variations were 3.7-12.9% and 3.5-15.6%, respectively. Among the metabolites analyzed, significant person-to-person heterogeneity was observed, demonstrating the need for comprehensive metabolite profiling in diagnosis. Nevertheless, the glucuronides of dihydrocortisol, dihydrocortisone, tetrahydrocortisol, allo-tetrahydrocortisol and tetrahydrocortisone are the major ones. The sum of the glucuronidated and free forms constitute >93% of the metabolites analyzed, which is termed as total cortisol equivalent. Total cortisol equivalent may serve as a surrogate of cortisol secretion. Clinical trial registration number: NCT02500472.

Keywords: cortisol metabolism; high-resolution accurate-mass MS; stress; tobacco; urine biomarker.

Conflict of interest statement

Financial & competing interests disclosure

The work was funded by the University of Minnesota Masonic Cancer Center pilot grant, University of Florida Medicinal Chemistry Department, College of Pharmacy Frank Duckworth Endowment, University of Florida Health Cancer Center Startup fund and NIH R01CA193286. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Figures

Figure 1.. Major metabolic pathways of cortisol in human.

Figure 2.. Product ion mass spectra of (A) cortisol (m/z 363.2 >) and (B) [2H4]-cortisol (m/z 367.2 >) at MS2 scan stage.

Figure 3.. Reconstructed ion chromatograms of (A) cortisol and (B) cortisone metabolites in the urine samples of human nonsmokers and smokers.

The mass extraction window was ±5 p.p.m.

Figure 3.. Reconstructed ion chromatograms of (A) cortisol and (B) cortisone metabolites in the urine samples of human nonsmokers and smokers.

The mass extraction window was ±5 p.p.m.

Figure 4.. Estimates of total cortisol equivalent in the urines of nonsmokers and smokers.

Unpaired two-tailed t-test. TCE: Total cortisol equivalent.