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 O positions of guanine and the N-3 position of adenine in DNA are the predominant reaction sites, N -methylguanine ( N -MeG), O -methylguanine ( O -MeG), and N -methyladenine ( N -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 -MeG, O -MeG, and N -MeA in DNA hydrolysates. With the use of isotope internal standards (N- N -MeG, d - O -MeG, and d - N -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 -MeG, O -MeG, and N -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 -MeG, O -MeG, and N -MeA in calf thymus DNA increase with MMS concentration and incubation time. The ratio of relative yields of N -MeG, O -MeG, and N -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. [ABSTRACT FROM AUTHOR]