Quantitative Analysis of Multiple Exocyclic DNA Adducts in Human Salivary DNA by Stable Isotope Dilution Nanoflow Liquid Chromatography–Nanospray Ionization Tandem Mass Spectrometry

Exocyclic DNA adducts, including 1,N 2-propano-2′-deoxyguanosine derived from acrolein (AdG) and crotonaldehyde (CdG) and the three lipid peroxidation-related etheno adducts 1,N 6-etheno-2′-deoxyadenosine (εdAdo), 3,N 4-etheno-2′-deoxycytidine (εdCyt), and 1,N 2-etheno-2′-deoxyguanosine (1,N 2-εdGuo), play an important role in cancer formation and they are associated with oxidative-stress-induced DNA damage. Saliva is an easily accessible and available biological fluid and a potential target of noninvasive biomarkers. In this study, a highly sensitive and specific assay based on isotope dilution nanoflow LC–nanospray ionization tandem mass spectrometry (nanoLC–NSI/MS/MS) is developed for simultaneous detection and quantification of these five adducts in human salivary DNA. The levels of AdG, CdG, εdAdo, εdCyd, and 1,N 2-εdGuo, measured in 27 human salivary DNA samples from healthy volunteers, were determined as 104 ± 50, 7.6 ± 12, 99 ± 50, 72 ± 49, 391 ± 198 (mean ± SD) in 108 normal nucleotides, respectively, starting with 25 μg of DNA isolated from an average of 3 mL of saliva. Statistically significant correlations were found between levels of εdAdo and εdCyd (γ = 0.8007, p < 0.0001), between levels of εdAdo and 1,N 2-εdGuo (γ = 0.6778, p = 0.0001), between levels of εdCyd and 1,N 2-εdGuo (γ = 0.5643, p = 0.0022), between levels of AdG and 1,N 2-εdGuo (γ = 0.5756, p = 0.0017), and between levels of AdG and εdAdo (γ = 0.3969, p = 0.0404). Only 5 μg of DNA sample was analyzed for simultaneous quantification of these adducts. The easy accessibility and availability of saliva and the requirement for the small amount of DNA samples make this nanoLC–NSI/MS/MS assay clinically feasible in assessing the possibility of measuring 1,N 2-propano-2′-deoxyguanosine and etheno adducts levels in human salivary DNA as noninvasive biomarkers for DNA damage resulting from oxidative stress and for evaluating their roles in cancer formation and prevention.