Simultaneous determination of O6-methyl-2′-deoxyguanosine, 8-oxo-7,8-dihydro-2′-deoxyguanosine, and 1,N6-etheno-2′-deoxyadenosine in DNA using on-line sample preparation by HPLC column switching coupled to ESI-MS/MS

O6-Methyl-2′-deoxyguanosine (O6-mdGuo), 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo), and 1,N6-etheno-2′-deoxyadenosine (ɛdAdo) are promutagenic DNA lesions originating from both endogenous and exogenous agents and actions (methylation, hydroxylation, lipid peroxidation products). A highly sensitive quantitative method was developed to measure these DNA adducts simultaneously, using liquid chromatography tandem mass spectrometry with column switching. Deuterated O6-[2H3]mdGuo was synthesized and used as internal standard. The limits of quantification for O6-mdGuo, 8-oxodGuo, and ɛdAdo were 24, 98, and 48 fmol on column, respectively. The method showed linearity in the range 0.24–125 pmol/ml, 0.98–125 pmol/ml, and 0.49–62.5 pmol/ml for the three adducts, respectively. The inter-day precision in the linear concentration range was between 1.7 and 9.3% for O6-mdGuo, 10.6 and 28.7% for 8-oxodGuo, and 6.2 and 10.4%, for ɛdAdo. In DNA isolated from liver of untreated 12-week-old female F344 rats, O6-mdGuo was above the limit of detection (37 adducts per 109 normal nucleosides) but could not be quantified. 8-oxodGuo and ɛdAdo showed background levels of 500 and 130 adducts per 109 normal nucleosides, respectively. DNA analyzed 1 h after treatment of rats with dimethylnitrosamine by oral gavage of 50 μg/kg b.wt. did not affect the levels of 8-oxodGuo and ɛdAdo but resulted in 200 O6-mdGuo adducts per 109 normal nucleosides. The method developed will be of use to study the biological significance of exogenous DNA adducts as an increment to background DNA damage and the role of modulating factors, such as DNA repair.