Simultaneous determination of 7-methylguanine and O6-methylguanine in DNA by liquid chromatography with postcolumn fluorescence derivatization using 3,4-dimethoxyphenylglyoxal

3,4-Dimethoxyphenylglyoxal was found to react sensitively and selectively with guanine-containing compounds to yield their fluorescent derivatives. This reaction and postcolumn fluorescence derivatization liquid chromatography were used to determine the production of 7-methylguanine and O6-methylguanine in DNA after alkylation with N-methyl-N-nitrosourea. In this method, nucleic acid bases produced by acid hydrolysis were separated on a reversed phase column (TSK-gel ODS-120T) and subjected to postcolumn derivatization involving demethylation followed by fluorescence reaction with 3,4-dimethoxyphenylglyoxal. The detection limits (S/N =3) for 7-methylguanine and O6-methylguanine were 6.7 and 0.7 pmol respectively in a 20 μl injection volume.