Bioanalysis of Stress Biomarkers through Sensitive HILIC-MS/MS Method: A Stride toward Accurate Quantification of MDA, ACR, and CTA.

Quantifying reactive aldehyde biomarkers, such as malondialdehyde, acrolein, and crotonaldehyde, is the most preferred approach to determine oxidative stress. However, reported analytical methods lack specificity for accurately quantifying these aldehydes as certain methodologies may produce false positive results due to harsh experimental conditions. Thus, in this research work, a novel HILIC-MS/MS method with endogenous histidine derivatization is developed, which proves to have higher specificity and reproducibility in quantifying these aldehydes from the biological matrix. To overcome the reactivity of aldehyde, endogenous histidine is used for its derivatization. The generated adduct is orthogonally characterized by NMR and LC-HRMS. The method employed a hydrophilic HILIC column and multiple reaction monitoring (MRM) to accurately quantify these reactive aldehydes. The developed method is an unequivocal solution for quantifying stress in in vivo and in vitro studies.