LC–MS/MS simultaneous quantitation of 2-hydroxyethylated, oxidative, and unmodified DNA nucleosides in DNA isolated from tissues of mice after exposure to ethylene oxide

• We developed a novel LC/MS–MS method for quantitative analysis of various DNA adduct biomarkers from EO exposure.
• 2-Hydroxyethylated and oxidized DNA nucleoside adducts and DNA nucleosides (dA and dG) were simultaneously quantified.
• This method will provide insight into the mode of action of genotoxicity caused by EO exposure.

2-Hydroxyethylated and oxidative DNA nucleosides (DNA adduct biomarkers), such as O6-(2-hydroxyethyl)-2′-deoxyguanosine (O6HEdG), N6-(2-hydroxyethyl)-2′-deoxyadenosine (N6HEdA), 1-(2-hydroxyethyl)-2′-deoxyadenosine (N1HEdA), and 8-hydroxy-2′-deoxyguanosine (8-OHdG), N2,3-etheno-2′-deoxyguanosine (N2,3-ethenodG), α-methyl-γ-hydroxy-1,N2-propano-2′-deoxyguanosine (CrotondG), are important proposed biomarkers for exploring the genotoxicity mechanism of ethylene oxide (EO) in vivo. A liquid chromatography–tandem mass spectrometric method was developed for the simultaneous determination of O6HEdG, N6HEdA, N1HEdA, 8-OHdG, CrotondG, and N2,3-ethenodG together with regular 2′-deoxyguanosine (dG), and 2′-deoxyadenosine (dA) nucleosides in the DNA extracted from mouse lung tissues for the assessment of exposure to EO after inhalation. The lower limits of quantitation for 8-OHdG, CrotondG, N2,3-EthenodG, O6HEdG, N1HEdA, N6HEdA, dG, and dA were 0.025, 0.00125, 0.025, 0.00125, 0.025, 0.01, 2342, and 2500 ng/mL, respectively. The linearity of the calibration curves for all analytes were >0.989. The intra-day assay precision relative standard deviation (RSD) values for quality control (QC) samples for all analytes were ≤13.5% with accuracy values ranging from 86.5% to 111%. The inter-day assay precision (RSD) values for all analytes were ≤18.8% with accuracy values ranging from 87.9% to 119%. This method was used for simultaneous determination of the levels of 8-OHdG, CrotondG, N2,3-EthenodG, O6HEdG, dG, N1HEdA, N6HEdA, and dA in DNA enzymatic hydrolysates from DNA extracted from mouse lung after 12 weeks’ inhalation exposure to EO at atmospheric concentrations of 0, 100, and 200 ppm. Overall, N2,3-ethenodG was not detected in any samples. 8-OHdG, CrotondG, dG, and dA were all quantifiable in all samples. O6HEdG, N1HEdA, and N6HEdA were quantifiable in most samples and the ratio of the corresponding adduct versus their corresponding DNA base (dG or dA) [×10 (e6)] was increased as the EO exposure concentration increased.