A comparison and column selection of Hydrophilic Interaction Liquid Chromatography and Reversed-Phase...

5-methylcytosine (5mC) is an epigenetic mark which has a profound effect on various fundamental processes in cells. In present study, at first Hydrophilic Interaction Liquid Chromatography (HILIC) was compared with Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) based on their selectivity (α), retention factor (k), and resolution (R) for cytosine (C) and 5mC nucleobases. We tried to justify the separation mechanism on the basis of mobile phase and solute polarity, structural characterization of solute and stationary phases, log Do/w, and pk_a under both modes. Then, these two modes were compared in order to select the best column for measurement of methylation level in two real samples with less analytical complexity (i.e. animal and bacteria) and a highly complex sample (i.e. plant), after chemical hydrolysis of DNA. In this favor, diol and cyano (CN) columns in HILIC mode as well as C₈ and C₁₈ in RP-HPLC were investigated. Optimum separation and the best validation parameters were obtained for CN column with Limit of Detection (LOD) of 1.4 pmol and Limit of Quantification (LOQ) of 4.8 pmol for 5mC. When the CN column was used in HILIC-UV procedure, separation of 5mC and C bases was achieved in all types of hydrolyzed DNA solutions.