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Carcinogenesis Advance Access originally published online on July 6, 2005
Carcinogenesis 2006 27(1):146-151; doi:10.1093/carcin/bgi177
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Carcinogenesis vol.27 no.1 © Oxford University Press 2005; all rights reserved.

Quantitative determination of urinary N7-ethylguanine in smokers and non-smokers using an isotope dilution liquid chromatography/tandem mass spectrometry with on-line analyte enrichment

Mu-Rong Chao, Chien-Jen Wang 1, Louis W. Chang 1 and Chiung-Wen Hu 2, *

Department of Occupational Safety and Health, Chung Shan Medical University, No. 110, Sec.1, Chien-Kuo N Road, Taichung 402, Taiwan and 1 Division of Environmental Health and Occupational Medicine, National Health Research Institutes, 100 Shih-Chuan 1st Road, Kaohsiung, Taiwan and 2 Department of Public Health, Chung Shan Medical University, no. 110, Sec. 1, Chien-Kuo N Road, Taichung 402, Taiwan

* To whom correspondence should be addressed. Tel: +886 4 24730022, ext. 11835; Fax: +886 4 23248179; Email: windyhu{at}csmu.edu.tw

Previous studies demonstrated the presence of unknown direct-acting ethylating agents arising from cigarette smoke. We hypothesized that such agents would also lead to ethylation of guanine in DNA followed by depurination/repair and excretion of N7-ethylguanine (N7-EtG) in urine. In this study, a highly specific and sensitive liquid chromatographic/tandem mass spectrometric (LC/MS/MS) method was firstly developed for measuring urinary N7-EtG. With the use of an isotope internal standard (15N5-N7-EtG) and on-line enrichment techniques, the detection limit of this method was estimated as 0.59 pg/ml (0.33 pmol) on-column. This method was then applied to measure urinary samples obtained from 35 non-smokers and 32 smokers with dietary control. The results showed that the mean urinary levels of N7-EtG were 85.5 ± 105 and 28.1 ± 19.4 pg/mg creatinine for smokers and non-smokers, respectively. Smokers had about three times higher level of N7-EtG than non-smokers (P < 0.005). It was further noted that the urinary level of N7-EtG was significantly associated with cotinine for smokers (r = 0.49, P < 0.005). Taken together, this is the first study that demonstrated the presence of N7-EtG in urine, and that cigarette smoke was highly responsible for the increased urinary excretion of N7-EtG. This non-invasive measurement of urinary N7-EtG would be useful for the surveillance of ethylating agent exposure and its associated cancer risk in the future.


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C.-W. Hu, C.-J. Wang, L. W. Chang, and M.-R. Chao
Clinical-Scale High-Throughput Analysis of Urinary 8-Oxo-7,8-Dihydro-2'-Deoxyguanosine by Isotope-Dilution Liquid Chromatography-Tandem Mass Spectrometry with On-Line Solid-Phase Extraction
Clin. Chem., July 1, 2006; 52(7): 1381 - 1388.
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