Chemical modification of deoxyribonucleic acids: Quantitation of 3-methylthymidine and O4-methylthymidine by tandem mass spectrometry

Abstract Quantitation of 3-methylthymidine and O 4 -methylthymidine generated in the reaction of calf thymus DNA with methyl methanesulfonate (MeMS) and 1-methyl-1nitrosourea (MeNU) by mass spectrometry is reported. Quantitative precision of 7% or better is achieved on samples of 10 −12 −10 −13 mole in the HPLC and a final stage of separation before quantification by tandem mass spectrometry using desorption chemical ionization. Synthetic CD 3 -labeled nucleosides were used as internal standards for mass spectral quantification. A unique mass spectrometric scanning procedure, which allowed simultaneous MS—MS product ion analysis of both the analyte and the internal standard, was utilized to enchance precision and accuracy in these low level determinations. MeNU (a potent carcinogen) resulted in 18&%; 3-methylation and 0.17% O 4 -methylation of deoxythymidine whereas MeMS (a weak carcinogen) produced only 6.8% 3-methylation and 0.005% of deoxythymidine. These results demonstrate that the sensitivity and accuracy of this method should be adequate for the detection and quantification of methyl-nucleosides at the sub-picomole level at which mutation is induced in cell cultures.