Detection and Quantification of 1,N6-Ethenoadenine in Human Urine by Stable Isotope Dilution Capillary Gas Chromatography/Negative Ion Chemical Ionization/Mass Spectrometry

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Abstract
1,N6-Ethenoadenine (εAde) is a promutagenic lesion detected in tissue DNA; it has been shown that εAde can be repaired by human DNA glycosylases, and it is expected to be excreted in urine. In this paper, we present for the first time detection and accurate quantification of εAde in human urine samples by a highly sensitive and specific stable isotope dilution gas chromatography/negative ion chemical ionization/mass spectrometric assay (GC/NICI/MS). Analysis by GC/NICI/MS includes adduct enrichment by a solid phase extraction column, followed by electrophore labeling and postderivatization cleanup. Using selective ion monitoring mode, the assay allows quantification of 0.5 pg of εAde in as little as 0.1 mL of the urine sample, which is equivalent to corresponding concentration quantification limit of 31 pM. Using this assay, concentrations of εAde in the 24 h urine samples of 23 healthy individuals were determined, which ranged from 0 to 124 pg/mL. After we adjusted for creatinine, a statistically significant correlation was found between εAde excretion and cigarette smoking in males (p = 0.03). Thus, this stable isotope dilution GC/NICI/MS assay offers a sensitive and accurate quantification of urinary εAde as a potential biomarker for oxidative damage of DNA and repair.

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