DNA methylation plays an important role in both normal physiological and disease processes. In this study, a sensitive and precise liquid chromatography-electrospray ionization tandem mass spectrometry method has been optimized for quantification of global DNA methylation levels in human peripheral blood by using multiple reaction monitoring (MRM) mode. This method was then used to analyze age-dependent association of DNA methylation. The results showed that the limit of detection was 1 pg for 5-methyl-2′-dexycytidine (5mdC) (S/N = 5.6). The linear calibration curves with correlation coefficients higher than 0.9990 were ranged 0.75–10 ppm and 0.0375–0.5 ppm for 2′-dexycytidine (dC) and 5mdC, respectively. The relative standard derivatives (RSD) of inter-day, intra-day and parallel samples were less than 5%. The analysis results of the blood samples from 60 healthy individuals showed that the child group had the highest mean 5mdC content while the adult group had the lowest values.
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