Association between Cigarette Smoking and Urinary Excretion of 1,N2-Ethenoguanine Measured by Isotope Dilution Liquid Chromatography-Electrospray Ionization/Tandem Mass Spectrometry

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Abstract
Levels of the promutagenic 1,N2-ethenoguanine (1,N2-εGua), an etheno DNA adduct derived mainly from lipid peroxidation, in experimental animals are associated with risk of cancer formation. Since 1,N2-εGua can be repaired by human glycosylases, it is possible to use it as a biomarker for cancer risk assessment in humans. In the present study, a highly sensitive and specific stable isotope dilution liquid chromatography-electrospray ionization/tandem mass spectrometry (LC-ESI/MS/MS) was developed for accurate quantification of 1,N2-εGua in human urine. The sample pretreatment involved a consecutive strong cation exchange solid-phase extraction (SPE) and reversed phase SPE chromatography. The pretreated sample was analyzed by LC-ESI/MS/MS under multiple reaction monitoring mode (MRM) using a triple quadrupole mass spectrometer. The detection limit of 1,N2-εGua using this LC-ESI/MS/MS assay was 1.0 pg (5.8 fmol) injected on-column. Levels of urine samples collected from healthy volunteers were found to range from 0 to 199 pg/mL, and levels as low as 5.0 pg/mL (29 pM) could be accurately quantified. After adjusting for creatinine levels and body weight, an statistically significant association was observed between urinary levels of 1,N2-εGua and cigarette smoking (p = 0.0006). This highly specific and sensitive assay should be valuable in measuring urinary 1,N2-εGua as a potential noninvasive biomarker for oxidative DNA damage.

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