Oxidative stress biomarkers have been associated with both acute and chronic health outcomes. However, traditional methods analyze different biomarkers separately, resulting in complex sample preparation, high sample consumption, lengthy processing time, and limited comparability. In this study, we presented a newly developed and validated method for the simultaneous determination of oxidative stress from multiple perspectives (DNA, lipids, and antioxidants). Using a one-step solid-phase extraction and ultra-high-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (UHPLC-QqQ-MS/MS), we measured four oxidative stress related biomarkers in urine simultaneously, within a run time of only 12 min. These biomarkers included 8-hydroxy-2'-deoxyguanosine (8-OHdG), 6-sulfatoxymelatonin (aMT6s), 8-isoprostaglandin-F (8-isoPGF), and 11-dehydro thromboxane B (11-DH-TXB). The calibration curves showed wide linear ranges (0.4-800 ng/mL for 8-OHdG, 0.2-600 ng/mL for aMT6s, 0.4-600 ng/mL for 8-isoPGF, and 0.4-600 ng/mL for 11-DH-TXB), with r values above 0.9932 for all analytes. The method demonstrated excellent sensitivity, with detection limits below 0.12 ng/mL, and good precision, with intra- and inter-day coefficients of variation ranging from 1.2 % to 14.4 %. We applied this method to urine samples from two populations living at different altitudes and found significantly higher levels of both 8-OHdG and 11-DH-TXB in the high-altitude group, likely due to hypobaric hypoxia. In the future, this new method could be applied in large-scale epidemiological studies to investigate biological mechanisms of oxidative stress in health risks or for clinical diagnosis.
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