The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent systemic lung carcinogen. Upon metabolic activation, NNK forms reactive metabolites that  react with DNA bases to form pyridyloxobutyl (POB)-DNA adducts. Upon acid hydrolysis, the majority of POB-DNA adducts decompose to release 4-hydroxy-1-(3-pyridyl)-1-butanone(HPB).  Studies in cellular and rodent systems demonstrate the importance of HPB-releasing adducts in carcinogenesis by NNK.  Therefore, HPB-releasing DNA adducts represent an important biomarker to study NNK-induced DNA damage in smokers, and its relationship to cancer risk. We previously developed a sensitive and selective method for the analysis of HPB-releasing DNA adducts in human oral cells; however, the original analytical procedure was not optimized for the robust application in large clinical trials.  Therefore, our purpose in this study was to modify oral cell DNA extraction, hydrolysis, and purification procedures with further analysis of the prepared samples by the highly sensitive and selective liquid chromatography (LC)-nanoelectrospray ionization (NSI)-high resolution tandem mass spectrometry (HRMS/MS). The developed protocol employs  solid-phase purification using Hypercarb cartridges and analysis by  LC-NSI-HRMS/MS using higher energy collision dissociation cell at isolation width of 1 amu, mass resolution of 30,000, and an extracted ion mass tolerance of = 5 ppm. The method is characterized by high accuracy and precision and an on-column limit of detection of 70 amol.  Furthermore, the optimized method incorporates a novel procedure for the purification and analysis of guanine by LC-electrospray ionization-MS/MS for DNA quantitation in the acid hydrolysates.  This unique and robust method is being applied in an ongoing clinical trial aimed at characterizing inter-individual differences in NNK metabolic activation in smokers and understanding the role of these differences in cancer risk

Novel high resolution tandem mass spectrometry method for the analysis of 4-hydroxy-1-(3-pyridyl)-1-butanone-releasing DNA adducts in human oral cells

248th National Meeting (2014)

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