Abstract: A hydrophilic interaction liquid chromatographic (HILIC) method has been developed for the determination of global DNA methylation in tissues. The DNA was extracted by phenol-chloroform, hydrolyzed with 88% formic acid at 140 ℃, evaporated under nitrogen at 60 ℃, and reconstituted in a mixture of acetonitrile/water (90:10, v/v); the separation was achieved on a Waters bridged ethylene hybrid (BEH) HILIC column (100 mm×2.1 mm, 1.7 μm). The cytosine (Cyt) and 5-methylcytosine (5-mCyt) were separated in a fairly short time (3.5 min) by isocratic elution with a mixture of acetonitrile/10 mmol/L ammonium formate (94:6, v/v) as the mobile phase. Under the optimized conditions, calibration standard curve showed a good linearity in the range 1~900 μmol/L for Cyt and in the range 1~64 μmol/L for 5-mCyt with correlation coefficients of 0.9999 and 0.9998, respectively. The limit of detection (LOD) was 54 nmol/L (0.54 pmol on-column) both for Cyt and 5-mCyt, and the limit of quantification was 250 nmol/L (2.5 pmol on-column) both for Cyt and 5-mCyt. The recoveries of Cyt at the spiked levels of 90, 450, 900 μmol/L and 5-mCyt at the spiked levels of 5, 16, 64 μmol/L all ranged from 94.7% to 100.5% with a relative standard deviations less than 1.48%. The method was applied to the analysis of DNA from colon cancer tissue, and the average degree of methylation was 4.0%. The method is rapid, simple, sensitive, reliable, and suitable for the determination of global DNA methylation.

Key words: colon cancer, global DNA methylation, tissues, hydrophilic interaction liquid chromatography (HILIC)