Analysis of 8-oxo-78-dihydro-2′-deoxyguanosine by ultra high pressure liquid chromatography–heat assisted electrospray ionization–tandem mass spectrometry

摘要

Increased amounts of reactive oxygen species (ROS), generally termed oxidative stress, are frequently hypothesized to be causally associated with many diseases. Analyses of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dG) in DNA and urine are widely used biomarkers for oxidative stress. Over the years it became clear that analysis of 8-oxo-dG in DNA is challenging due to artifactual formation during sample work up. The present study demonstrates that 8-oxo-dG can be measured reliably and accurately when appropriate precautions are taken. First, the presence of an antioxidant, metal chelator, or free radical trapping agent during sample preparation improves reproducibility. Second, sample enrichment by HPLC fraction collection was used to optimize sensitivity. Third, heat assisted electrospray ionization (HESI) eliminated potential interferences and improved assay performance and sensitivity. Subsequently, the UPLC–HESI–MS/MS method was applied to show the biphasic dose response of 8-oxo-dG in H2O2-treated HeLa cells. Application of this method to human lymphocyte DNA (n = 156) gave a mean±SD endogenous amount of 1.57±0.88 adducts per 10⁶ dG, a value that is in agreement with the suggested amount previously estimated by European Standard Committee on Oxidative DNA Damage (ESCODD) and others. These results suggest that the present method is well suited for application to molecular toxicology and epidemiology studies investigating the role of oxidative stress.