Development and validation of a UHPLC-MS/MS method for the quantification of total conjugated 3-bromotyrosine and investigation of its association with asthma severity.

A robust and sensitive UHPLC-MS/MS method was developed and validated for quantifying urinary total conjugated bromotyrosine (TCB). The method showed excellent linearity (R2 = 0.9995) across the validated concentration range, with a limit of detection (LOD) of 13.6 pM and a lower limit of quantification (LLOQ) of 45.2 pM. Results were systematically normalized to urinary creatinine to correct the dilution effects. Method validation according to regulatory guidelines showed good selectivity, specificity, and robustness. The rapid method with a run time of 6.51 min enabled efficient analysis, and application to healthy control samples established an upper reference limit of 2.84 pmol/mmol creatinine. This method was applied to assess the clinical relevance of urinary TCB in asthma. In the DIVE cohort (n = 25; NCT05992519), urinary TCB showed limited ability to discriminate airway hyperresponsiveness (AUC = 0.65; 95% CI: 0.43-0.88). However, in the PRISMA cohort (n = 26; NCT05870215), higher baseline TCB levels were significantly associated with greater improvement in lung function following oral corticosteroid therapy at day 7. Multivariable modeling adjusting for blood eosinophils and fractional exhaled nitric oxide showed that urinary TCB remained independently associated with functional improvement, while associations for conventional inflammatory biomarkers (blood eosinophils and exhaled nitric oxide) were no longer significant. Although exploratory and based on secondary analyses, TCB may represent a non-invasive predictive biomarker of oxidative stress-related tissue injury and therapeutic response in asthma.