A hydrophilic interaction ultraperformance liquid chromatography (HILIC-UPLC) method for genomic DNA methylation assessment by UV detection

摘要

A hydrophilic interaction chromatography-based method, in combination with 1.7 μm ethylene bridged hybrid particle packed column (100 mm × 2.1 mm I.D.) and ultraperformance liquid chromatography, has been developed to measure cytosine (C) and methylcytosine (mC) in order to evaluate the extent of DNA methylation. Separation of cytosine and methylcytosine was achieved with good resolution and in fairly short times (5.5 min) by using isocratic elution with a mixture of 97:3 (v/v) acetonitrile/10 mM ammonium acetate as a mobile phase. The determination coefficients of C and mC were high (R ~2 > 0.999) within the range tested. The %RSD for intraday and interday were respectively 2.2% and 2.5% for C and 3.5% and 3.8% for mC. The limit of detection was 0.52 μM (0.52 fmol on-column) both for C and mC while the limit of quantification was 1.72 μM (1.72 fmol on-column) both for C and mC. The smallest amount of purified DNA that yielded a measurable level of C and mC was 10 μg. On the whole, this method is simple, rapid, sensitive, and precise.