title: Simultaneous quantification of ethylpurine adducts in human urine by stable isotope dilution nanoflow liquid chromatography nanospray ionization tandem mass spectrometry.
abstract: Ethylating agents contained in cigarette smoke can damage DNA producing ethylated DNA adducts, including N(3)-ethyladenine (3-EtAde) and N(7)-ethylguanine (7-EtGua). In this study, a highly specific and sensitive assay based on stable isotope dilution nanoflow liquid chromatography nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS) was used to measure 3-EtAde and 7-EtGua in human urine. These urinary adducts were enriched by a polymeric reversed phase solid-phase extraction column before the nanoLC-NSI/MS/MS analysis. The on-column detection limits (S/N≥3) of 3-EtAde and 7-EtGua were 15fg (92amol) and 10fg (56amol), respectively, while the lower quantification limits of 3-EtAde and 7-EtGua were 930 and 840 amol, respectively. Urinary concentrations of 3-EtAde and 7-EtGua in 21 smokers were 68.6±29.4 and 18.7±13.8pg/mL, respectively. In 20 nonsmokers, concentrations of 3-EtAde and 7-EtGua were 3.5±3.8 and 2.4±3.0pg/mL, respectively. The urinary concentrations of 3-EtAde and 7-EtGua were statistically significantly higher in smokers than in nonsmokers (p<0.0001). Moreover, 3-EtAde and 7-EtGua concentrations are significantly correlated with the number of cigarettes smoked per day and with the smoking index. This highly specific and sensitive assay based on stable isotope dilution nanoLC-NSI/MS/MS assay should be clinically valuable in assessing the possibility of measuring urinary ethylpurines as noninvasive biomarkers for smoking-related cancers in humans.
authors.affiliation: Department of Chemistry and Biochemistry, National Chung Cheng University, 168 University Road, Ming-Hsiung, Chia-Yi 62142, Taiwan. Electronic address: chehjc@ccu.edu.tw.
authors.forename: Hauh-Jyun Candy
authors.initials: HJ
authors.lastname: Chen
authors.forename: Chao-Ray
authors.initials: CR
authors.lastname: Lin
mesh_terms: D002247:Carbon Isotopes; D002853:Chromatography, Liquid / Q000379:methods*; D018736:DNA Adducts; D006801:Humans; D007201:Indicator Dilution Techniques; D057230:Limit of Detection; D009587:Nitrogen Isotopes; D011687:Purines / Q000652:urine*; D052616:Solid Phase Extraction; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D002247:Carbon Isotopes; D018736:DNA Adducts; D009587:Nitrogen Isotopes; D011687:Purines; C030985:purine
keywords: 3-Ethyladenine; 7-Ethylguanine; DNA adduct; Mass spectrometry; Nanoflow LC; Urine