Simultaneous determination of global DNA methylation and hydroxymethylation levels by hydrophilic interaction liquid chromatography-tandem mass spectrometry.

Methylation of DNA at the 5-position of cytosine (Cyt) is a well-studied epigenetic pathway implicated in gene silencing and embryogenesis. Recently, in addition to 5-methylcytosine (5mC), substantial amounts of 5-hydroxymethylcytosine (5hmC) have been detected in certain mammalian tissues. Here, we developed and validated a hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method for the simultaneous determination of Cyt, 5mC, and 5hmC levels in biological samples. DNA was extracted with phenol-chloroform, hydrolyzed using 88% formic acid at 140 °C, separated using a bridged ethylene hybrid HILIC column, and analyzed by tandem MS. The linearity was established over the concentration range of 1 to 500 ng/mL for Cyt, 0.2 to 100 ng/mL for 5mC, and 0.1 to 50 ng/mL for 5hmC, and the correlation coefficients were all >0.99. Limits of detection were 1 pg/mL for Cyt, 45 pg/mL for 5mC, and 57 pg/mL for 5hmC, and the limit of quantification values for Cyt, 5mC, and 5hmC were 2 pg/mL, 90 pg/mL, and 100 pg/mL, respectively. The relative standard deviation (RSD) of the intraday precision ranged from 1.87% to 4.84% and the interday precision from 2.69% to 4.98%. The recovery of the method varied from 88.25% to 104.39%. The method was then applied to the analysis of DNA from biological samples, establishing its potential for helping researchers understand the roles of modified nucleobases in DNA.