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.