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DNA N6-methyladenine(6mA)modification is commonly found in microbial genomes,and plays important functions in regulating numerous biological processes in bacteria.Here we show that 6mA is present in Drosophila genome.A stability method for identification and quantification of 6mA by means of high-performance liquid chromatography–tandem quadrupole mass spectrometry(HPLC–MS/MS)was developed.In this method,extremely low concentration formic acid was employed in the mobile phase to maintain the high ionization efficiency.This assay use C18 reverse-phase column followed by electrospray ionization and the resulting ions were detected using triple-quadrupole mass spectrometry in multiple reaction monitoring mode.The result display that the detection of 6mA with a high sensitivity.This HPLC–MS/MS method was successfully applied to determine these modified nucleotides and the relative standard deviations of the measurements were able to meet our need.