Concurrent quantification of multiple biomarkers indicative of oxidative stress status using liquid chromatography-tandem mass spectrometry

• LC-MS/MS method developed to simultaneously measure 8-OHdG, 8-NO2Gua, HNE-MA, and 8-isoPGF2α.
• Biomarker levels almost constant in urine samples collected from control subjects.
• Results reveal that these biomarkers could be constantly generated in vivo.
• Applications in epidemiological studies to examine oxidative and nitrative stress.

8-Hydroxy-2-deoxyguanosine (8-OHdG), 8-nitroguanine (8-NO2Gua), 8-iso-prostaglandin F2α (8-IsoPGF2α), and N-acetyl-S-(tetrahydro-5-hydroxy-2-pentyl-3-furanyl)-L-cysteine (HNE-MA) are well-studied and representative biomarkers for oxidative DNA damage, inflammation, and lipid peroxidation; all of which have been associated with increases in risks of various diseases and cancers. A rapid and highly sensitive isotope-dilution liquid-chromatography tandem mass spectrometry (LC-MS/MS) method was developed to simultaneously quantify the aforementioned biomarkers in urine. Upon validation, this method shows excellent feasibility, sensitivity (0.008–0.03 ng/mL) and satisfactory recoveries (88.7–95.4%); the calibration curves displayed excellent linearity with coefficients of determination (R2) greater than 0.998. Additionally, low variations were observed in the relative standard deviation for intra- and inter-day measurements for the four analytes. The relative matrix effects for all four analytes ranged from 2.04 to 3.27%, which signaled that interferences from endogenous levels of the analytes were deemed statistically insignificant. This study successfully developed an analytical method capable to simultaneously quantify urinary 8-OHdG, 8-NO2Gua, 8-IsoPGF2α, and HNE-MA. This analytical protocol can be applied towards conducting epidemiological studies to reveal the mechanisms related to disease development, and thus evaluate the associated risks of diseases.