Determination of human urinary cortisol, cortisone and relative phase II metabolites by dilute-and-shoot LC-MS/MS analysis: an application to adrenal disease.

BACKGROUND

Cortisol and its primary metabolite, cortisone, play key roles in managing stress responses and maintaining balance via the hypothalamic-pituitary-adrenal axis. Traditional immunoassays, often hindered by cross-reactivity, can yield inaccurate results. This study introduces a novel dilute-and-shoot using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the rapid and accurate analysis of urinary free cortisol, cortisone, and their intact phase II metabolites.

METHODS

A new LC-MS/MS method was developed to quantify five analytes, including intact glucuro- and sulfo-conjugated cortisol and cortisone metabolites. The method underwent validation assessments in accordance with ISO 17025 standards for quantitative methods. Its performance was compared with a commercial LC-MS/MS assay used in clinical laboratories, analyzing free cortisol and cortisone in 79 24-hour urine samples. Additionally, the novel method was applied to authentic urine samples from 168 adrenal disease patients.

RESULTS

The validation protocol assessed method's selectivity, matrix effects, quantitative performance and carry-over. The comparison showed excellent correlation with the commercial LC-MS/MS method for urinary free cortisol and cortisone measurement. Pathological sample analysis indicated that monitoring intact phase II metabolites, particularly sulfoconjugates, could aid in differentiating adrenal diseases.

CONCLUSION

The developed and validated dilute-and-shoot LC-MS/MS method demonstrated satisfactory performance, offering a faster, cost-effective solution for adrenal hormone analysis in clinical settings. This research also provided insights into adrenal function by examining the urinary excretion of target analytes in a pathological cohort, enhancing diagnostic precision for related disorders.