Simultaneous quantitative analysis of N3-ethyladenine and N7-ethylguanine in human leukocyte deoxyribonucleic acid by stable isotope dilution capillary liquid chromatography–nanospray ionization tandem mass spectrometry

Abstract

Cigarette smoke contains ethylating agents which damage DNA producing ethylated DNA adducts, such as N3-ethyladenine (3-EtAde), N7-ethylguanine (7-EtGua), and regioisomers of ethylthymine. Among them, 3-EtAde and 7-EtGua are present in human urine and their levels are higher in smokers than in nonsmokers. The amount of ethylated DNA adducts in tissue DNA represents the steady-state levels of DNA adducts resulting from the ethylating agent after repair in vivo. In this study, we have developed a highly sensitive, accurate, and quantitative assay for simultaneous detection and quantification of 3-EtAde and 7-EtGua by stable isotope dilution capillary liquid chromatography–nanospray ionization tandem mass spectrometry (capLC–NSI/MS/MS). Under the highly selective reaction monitoring (H-SRM) mode, the detection limit of 3-EtAde and 7-EtGua injected on-column was 5.0fg (31amol) and 10fg (56amol), respectively. The quantification limit for the entire assay was 50 and 100fg of 3-EtAde and 7-EtGua, corresponding to 4.7 and 8.6 adducts in 109 normal nucleotides, respectively, starting with 20μg of DNA isolated from <1mL of blood and injecting an equivalent of 4μg of DNA on-column. The mean (±SD) levels of 3-EtAde and 7-EtGua in leukocyte DNA from 20 smokers were 16.0±7.8 and 9.7±8.3 in 108 normal nucleotides, respectively, which were statistically significantly higher than those of 5.4±2.6 3-EtAde and 0.3±0.8 7-EtGua in 108 normal nucleotides from 20 nonsmokers (p<0.0001). The levels of 3-EtAde and 7-EtGua in these 40 leukocyte DNA samples are positively correlated (γ=0.6970, p<0.0001). Furthermore, there are statistically significant associations between the number of cigarettes smoked per day, as well as the smoking index, and the levels of 3-EtAde and 7-EtGua. Levels of 3-EtAde and 7-EtGua are compared to those of ethylthymidine adducts. To our knowledge, this is the first assay for simultaneous quantification of 3-EtAde and 7-EtGua in the same DNA sample and is the first report of 3-EtAde in human DNA. This highly sensitive and specific stable isotope dilution capLC–NSI/MS/MS assay should be useful in measuring 3-EtAde and 7-EtGua in human leukocyte DNA as potential biomarkers for smoking-related cancers.