Development of a quantitative liquid chromatography/electrospray mass spectrometric assay for a mutagenic tobacco specific nitrosamine-derived DNA adduct, O⁶-[4-oxo-4-(3-pyridyl)butyl]-2′-deoxyguanosine

Liquid chromatography with electrospray tandem mass spectrometry (LC/ESI-MS/MS) was employed to quantify O⁶-[4-oxo-4-(3-pyridyl)butyl] -2′-deoxyguanosine (O⁶-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 O⁶-pobdG in pyridyloxobutylated DNA from both in vitro and in vivo sources. Quantitation was based on isotope dilution with synthetic O⁶-[1,2,2- ²H₃-4-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- ³H]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, O⁶-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 O⁶-pobdG in the toxicological properties of pyridyloxobutylating nitrosamines.