Ultrasensitive UPLC–MS/MS method for analysis of etheno-DNA adducts in human white blood cells

Abstract

Etheno-DNA adducts are generated by interaction of cellular DNA with exogenous environmental carcinogens and end products of lipid peroxidation. It has been determined that 1,N6-etheno-2’-deoxyadenosine (εdA) and 3,N4-etheno-2’-deoxycytidine (εdC) adducts formed in human white blood cells can be used to serve as biomarkers of genetic damage mediated by oxidative stress. In this study, we developed an ultrasensitive ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method used to detect and quantify εdA and εdC adducts in human white blood cells. The percent recoveries of εdA and εdC adducts were found to be 88.9% ± 2.8 and 95.7% ± 3.7, respectively. The detection limits were ∼1.45 fmol for εdA and ∼1.27 fmol for εdC in 20 μg of human white blood cell DNA samples, both εdA and εdC adducts could be detected using only ∼ 5 μg of DNA per sample. For validation of the method, 34 human blood cell DNA samples were assayed and the results revealed a significant difference (P < 0.01) between levels (fmol/μg DNA) of 0.82 ± 0.83 (standard deviation [SD]) (range: 0.15–3.11) for εdA, 3.28 ± 3.15 (SD) (range: 0.05–9.6) for εdC in benzene-exposed workers; and 0.04 ± 0.08 (SD) (range: 0.0–0.27) for εdA and 0.77 ± 1.02 (SD) (range: 0.10–4.11) for εdC in non-benzene-exposed workers. Our method shows a high sensitivity and specificity when applied to small amounts of human white blood cell DNA samples; background levels of εdA and εdC could be reproducibly detected. The ultrasensitive and simple detection method is thus suitable for applications in human biomonitoring and molecular epidemiology studies.