B-366 Simultaneous LC–MS/MS Measurement of a Urinary Panel of Oxidative Stress Biomarkers

Abstract

Oxidative stress results from an imbalance between reactive oxygen and nitrogen species and antioxidant defenses, leading to cumulative molecular damage involving nucleic acids, lipids, and proteins. This process plays a central role in aging, inflammation, chronic diseases, and physiological responses to environmental and lifestyle factors. Urinary biomarkers reflecting oxidative damage pathways—such as oxidized nucleosides, isoprostanes, and modified amino acids—are widely used in clinical and translational research due to their non-invasive sampling and ability to capture systemic oxidative processes. These biomarkers provide complementary information on oxidative damage affecting DNA/RNA, lipid peroxidation, and protein oxidation/nitration mechanisms. However, current analytical approaches are typically limited to single-analyte measurements performed using heterogeneous methodologies, including immunoassays and isolated chromatographic methods. This fragmentation reduces analytical specificity, limits inter-study comparability, and prevents comprehensive evaluation of oxidative damage across multiple molecular pathways. A panel-based analytical strategy enabling simultaneous quantification of complementary oxidative stress biomarkers within a single workflow may support a more robust and comprehensive assessment of systemic oxidative status in laboratory research settings. A quantitative LC–MS/MS panel was developed for the simultaneous determination of six urinary oxidative stress biomarkers: 2-deoxyguanosine (2-dG), 8-hydroxy-2'-deoxyguanosine (8-OHdG), 8-hydroxyguanosine (8-OHGuo), 8-iso-prostaglandin F2a, 2,2'-dityrosine, and 3-nitrotyrosine. Urine samples were prepared using a dilution-based protocol with early addition of stable isotope-labelled internal standards. Chromatographic separation and detection were performed using LC–MS/MS in multiple reaction monitoring mode. Quantification relied on matrix-matched calibration curves prepared in lyophilized human urine, with independent quality control materials used to assess analytical performance. All biomarkers were successfully separated and quantified within a single analytical run, showing stable chromatographic performance across batches. Calibration curves demonstrated excellent linearity for all analytes, with correlation coefficients =0.995 across validated concentration ranges. Precision studies at multiple concentration levels showed within-run and between-run coefficients of variation below 17%. Limits of quantification ranged from approximately 1.5–2.5 ng/mL for nucleic acid oxidation markers, and nitrotyrosine, while dityrosine exhibited a limit of quantification of 11.1 ng/mL. Matrix-matched calibrators and controls demonstrated recoveries close to expected values, supporting method robustness and reproducibility. This LC–MS/MS panel enables simultaneous and reliable quantification of complementary urinary biomarkers reflecting nucleic acid oxidation, and protein oxidation/nitration within a single analytical workflow. The approach may facilitate comprehensive multi-marker assessment of oxidative stress in clinical and translational research applications.