Simultaneous quantification of methylated purines in DNA by isotope dilution LC-MS/MS coupled with automated solid-phase extraction

Since methylation at the N-7 and O⁶ positions of guanine and the N-3 position of adenine in DNA are the predominant reaction sites, N ⁷-methylguanine (N ⁷-MeG), O ⁶-methylguanine (O ⁶-MeG), and N ³-methyladenine (N ³-MeA) have been suggested as good biomarkers for assessing exposure to methylating agents. Here, we report the development of a sensitive and selective assay based on liquid chromatography–tandem mass spectrometry (LC-MS/MS) to simultaneously measure N ⁷-MeG, O ⁶-MeG, and N ³-MeA in DNA hydrolysates. With the use of isotope internal standards (¹⁵N₅-N ⁷-MeG, d ₃-O ⁶-MeG, and d ₃-N ³-MeA) and online solid-phase extraction, DNA hydrolysates can be directly analyzed within 12 min without prior sample purification. The limits of detection were 0.02, 0.002, and 0.01 ng/mL on-column (6.1, 0.6, and 3.4 fmol) for N ⁷-MeG, O ⁶-MeG, and N ³-MeA, respectively. Inter- and intraday imprecision (CV) were 3.6–9.6% and 2.7–13.6%, respectively. Mean recoveries were 96–109%. This method was then applied to quantitate the amounts of methylated purines in calf thymus DNA treated with methyl methanesulfonate (MMS). The levels of N ⁷-MeG, O ⁶-MeG, and N ³-MeA in calf thymus DNA increase with MMS concentration and incubation time. The ratio of relative yields of N ⁷-MeG, O ⁶-MeG, and N ³-MeA in MMS-treated DNA was found to be 1.00:0.0032:0.119, respectively. This LC-MS/MS assay provides the sensitivity and high throughput required to evaluate the extent of methylated lesions in DNA induced by methylating agents.