High-Performance Liquid Chromatography−Tandem Mass Spectrometry Measurement of Radiation-Induced Base Damage to Isolated and Cellular DNA

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Abstract
A method involving high-performance liquid chromatography (HPLC) separation associated with tandem mass spectrometry (MS/MS) detection in the multiple-reaction monitoring mode was set up for the assessment of radiation-induced degradation products of DNA bases. This sensitive and specific assay is aimed at assessing six oxidized 2‘-deoxyribonucleosides and two modified purine bases within both isolated and cellular DNA. For this purpose, stable isotopically labeled internal standards were prepared and used for isotope dilution mass spectrometry measurements. The latter method was validated through a comparison with two other assays, including HPLC associated with electrochemical detection and gas chromatography coupled to mass spectrometry. Using the specific and sensitive HPLC−MS/MS approach, 5,6-dihydroxy-5,6-dihydrothymidine, 5-hydroxy-2‘-deoxyuridine, 5-(hydroxymethyl)-2‘-deoxyuridine, 5-formyl-2‘-deoxyuridine, 8-oxo-7,8-dihydro-2‘-deoxyadenosine, 8-oxo-7,8-dihydro-2‘-deoxyguanosine, 4,6-diamino-5-formamidopyrimidine, and 2,6-diamino-4-hydroxy-5-formamidopyrimidine were quantified within both isolated and cellular DNA upon exposure to γ-radiation.

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