Quantitative Determination of N7-Methyldeoxyguanosine and O6-Methyldeoxyguanosine in DNA by LC−UV−MS−MS

The N-7 and O-6 positions of 2'-deoxyguanosine are the predominant sites of methylation by N-methyl-N-nitrosourea (MNU), which is used to produce a variety of experimental cancers in animal models. Here we report the development of a highly sensitive quantitative assay based on high-performance liquid chromatography-UV-tandem mass spectrometry (LC-UV-MS-MS) to measure N7-methyl-2'-deoxyguanosine (N7-MedG) and O6-methyl-2'-deoxyguanosine (O6-MedG) in DNA hydrolysates. Since this assay was selective for deoxyribonucleosides, potential interference from methylated RNA was eliminated. Isotopically labeled analogues, [2H3]N7-MedG and [2H3]O6-MedG, were synthesized and added to the DNA hydrolysates as internal standards. In-line UV absorbance detection was used for the quantitative analysis of the native deoxyribonucleoside dG, and MS-MS was used for the determination of N7-MedG and O6-MedG. The limits of detection for N7-MedG and O6-MedG were determined to be 64 and 43 fmol, respectively. The limits of quantification were 0.13 pmol for N7-MedG and 0.085 pmol for O6-MedG. The stabilities of N7-MedG and O6-MedG were also investigated. Although O6-MedG was stable at room temperature for at least 11 days, the half-life of N7-MedG at room temperature was 2 days. Both adducts were stable at -20 degrees C. Calf thymus DNA and DNA from the livers of MNU-treated Sprague-Dawley rats were assayed using LC-UV-MS-MS, which was optimized for speed as well as for sensitivity. The levels of N7-MedG and 06-MedG in calf thymus DNA increased with MNU concentration and incubation time. The levels of N7-MedG and O6-MedG in the rat livers 2 h after treatment with a single dose of 50 mg/kg MNU were 95.2 N7-MedG/105 dG and 14.8 O6-MedG/105 dG. This LC-UV-MS-MS assay provides the sensitivity and speed required to evaluate the extent of methylated DNA lesions in animal models of cancer induced by the methylating agent MNU.