Liquid Chromatography–Electrospray Ionization Tandem Mass Spectrometry Analysis of 7-Ethylguanine in Human Liver DNA

Abstract

Considerable evidence in the literature demonstrates the exposure of humans to an unknown ethylating agent. Previous studies have demonstrated the presence of 7-ethyl-Gua and 3-ethyl-Ade in urine, 7-ethyl-dGuo and O4-ethyl-dThd in human lung, and ethylvaline in hemoglobin. Some studies also report higher levels of ethyl adducts in smokers than in nonsmokers, and there is convincing evidence for an uncharacterized ethylating agent in cigarette smoke. To further investigate this question, we have developed a liquid chromatography-electrospray ionization tandem mass spectrometry-selected reaction monitoring method for analysis of 7-ethyl-Gua in human liver DNA. To our knowledge, there are no previous reports of MS analyses of 7-ethyl-Gua in human tissues. [15N 5]7-Ethyl-Gua was synthesized and used as the internal standard. Human liver DNA was heated to release 7-ethyl-Gua. After partial purification by solid-phase extraction, analysis was carried out using the transition m/z 180 [M+H]+-->m/z 152 [Gua+H]+ for 7-ethyl-Gua and m/z 185-->m/z 157 for the internal standard. The method was accurate and precise. The detection limit was approximately 8-9 fmol/micromol Gua, starting with 1-2 mg of DNA. Clear coeluting peaks for 7-ethyl-Gua and the internal standard were observed in the human liver DNA samples. Twenty-six human liver DNA samples (0.77+/-0.40 mg) were analyzed, and 25 were positive for 7-ethyl-Gua. The mean level of 7-ethyl-Gua was 42.2+/-43.0 fmol/micromol Gua (8.4+/-8.6 adducts per 10(9) nucleotides). These results demonstrate that 7-ethyl-Gua is a common DNA adduct in human liver with likely endogenous sources that require further investigation.