Analysis of 8-oxo-7,8-dihydro-2'-deoxyguanosine by ultra high pressure liquid chromatography-heat assisted electrospray ionization-tandem mass spectrometry

Abstract

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 H(2)O(2)-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 10(6) 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.

Fig. 1

Schematic outline of analysis protocol.

Fig. 2

Control rat liver DNA spiked with authentic 8-oxo-dG (negative control, n = 9; positive control, n = 3) was analyzed for 8-oxo-dG by UPLC–HESI–MS/MS as described in materials and methods.

Fig. 3

Biphasic dose response in H₂O₂-treated HeLa cells (n = 6). HeLa cells were treated with different concentrations of H₂O₂ for 15 min and amounts of 8-oxo-dG in DNA were measured by UPLC–HESI–MS/MS as described in materials and method.

Fig. 4

Extracted ion chromatogram of 8-oxo-dG and [¹⁵N₅]8-oxo-dG in rat liver DNA analyzed by (A) capillary LC–MS/MS or (B) UPLC–HESI–MS/MS. Shown are the transitions of m/z 284.1 → 168.0 and m/z 289.1 → 173.0 for 8-oxo-dG (upper traces) and [¹⁵N₅]8-oxo-dG (lower traces), respectively.

Fig. 5

Endogenous 8-oxo-dG in human peripheral blood lymphocytes and snap frozen rat liver DNA measured by UPLC–HESI–MS/MS. The mean±SD amount of 8-oxo-dG in human lymphocytes was 1.57±0.88 8-oxo-dG/10⁶ dG (n = 156).