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 N-7-methyl-2'-deoxyguanosine (N-7-MedG) and O-6-methyl-2'-deoxyguanosine (O-6-MedG) in DNA hydrolysates. Since this assay was selective for deoxyribonucleosides, potential interference from methylated RNA was eliminated. Isotopically labeled analogues, [H-2(3)]N-7-MedG and [H-2(3)]O-6-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 N-7-MedG and O-6-MedG. The limits of detection for N-7-MedG and O-6-MedG were determined to be 64 and 43 fmol, respectively. The limits of quantification were 0.13 pmol for N-7-MedG and 0.085 pmol for O-6-MedG. The stabilities of N-7-MedG and O-6-MedG were also investigated. Although O-6-MedG was stable at room temperature for at least 11 days, the half-life of N-7-MedG at room temperature was 2 days. Both adducts were stable at -20 degreesC. 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 N-7-MedG and O-6-MedG in calf thymus DNA increased with MNU concentration and incubation time. The levels of N-7-MedG and O-6-MedG in the rat livers 2 h after treatment with a single dose of 50 mg/kg MNU were 95.2 N-7-MedG/10(5) dG and 14.8 O-6-MedG/10(5) 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.