Development Of A Quantitative Liquid Chromatography/Electrospray Mass Spectrometric Assay For A Mutagenic Tobacco Specific Nitrosamine-Derived Dna Adduct, O-6-[4-Oxo-4-(3-Pyridyl)Butyl]-2 '-Deoxyguanosine

Liquid chromatography with electrospray tandem mass spectrometry (LC/ESI-MS/MS) was employed to quantify O-6-[4-oxo-4-(3-pyridyl)butyl]-2'-deoxyguanosine (O-6-pobdG), a mutagenic adduct formed by pyridyloxobutylating nitrosamines. Selected reaction monitoring (SRM) of the neutral loss of the sugar from protonated molecules of the adduct, [M + H - 116](+), was utilized for detection of O-6-pobdG in pyridyloxobutylated DNA from both in vitro and in vivo sources. Quantitation was based on isotope dilution with synthetic O-6-[1,2,2-H-2(3)-4-oxo-4-(3-pyridyl)butyl]-2'-deoxyguanosine. The detection limits in this study were less than 5 fmol of pure standard and 50 fmol in 1.5 mg of DNA. This method was validated by comparing adduct levels measured with the LC/ESI-MS/MS method to those obtained with radiochemical methods in DNA alkylated with the model pyridyloxobutylating agent, [5-H-3]4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone ([5-3H]NNKOAc). The pyridyloxobutyl 2'-deoxyguanosine adduct coeluting with the deuterated standard disappeared when NNKOAc-treated DNA had been reacted with the repair protein, O-6-alkylguanine-DNA alkyltransferase. This result confirms that the coeluting peak is solely O-6-pobdG. Preliminary studies with liver DNA isolated from NNKOAc-treated mice demonstrated that this method can be used to quantify O-6-pobG in DNA from in vivo sources. The improved sensitivity and specificity of adduct detection afforded by this LC/ESI-MS/MS method will allow us to explore the role of O-6-pobdG in the toxicological properties of pyridyloxobutylating nitrosamines.