CHIUNG-WEN HUMU-JUNG CHAO2026-10-162026-10-162012-01-01Pure ID: 14976705Pure UUID: fc7c36e3-154f-4057-90fb-7a703e4874eb10.1007/s00216-011-5559-1https://www.scopus.com/pages/publications/84856218049https://scholars.csmu.edu.tw/handle/123456789/28637Since methylation at the N-7 and O 6 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 ( 15N 5-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.10enjournal article