Determination of 5-methyldeoxycytosine and oxidized derivatives by nano-liquid chromatography with zwitterionic...

DNA methylation is one of the epigenetic modifications at the 5-carbon of cytosine to form 5-methyl-2'-deoxycytidine (5mdC). In mammalian cells, 5mdC can be transferred to 5-hydroxymethyl-2'-deoxycytidine (5hmdC) by ten-eleven translocation (TET), and 5hmdC is further oxidized to 5-formyl-2'-deoxycytidine (5fodC) and 5-carboxyl-2'-deoxycytidine (5cadC). In the present work, we developed a highly sensitive nano liquid chromatographic method for the determination of 5mC and 5hmC with zwitterionic monolithic capillary column. The conditions for the preparation of zwitterionic monolithic capillary column were systematically optimized. The monolithic capillary column displayed high column efficiency for nucleoside dA (190,000 plates/m) and allowed the baseline separation of 10 standard nucleosides in HILIC mode. The detection sensitivity was improved significantly by using the large volume injection combined with sample stacking onto the head of the column when sample was dissolved in high content organic solvent (ACN: H₂O = 97:3). The limit of detection for 5mdC and 5hmdC were determined as 4 nM and 3 nM, respectively, and the corresponding limit of quantification were determined as 12 nM and 10 nM, respectively. Further, the zwitterionic monolithic capillary column can be easily utilized in nano-LC and mass spectrometry coupling for qualitative analysis of 5mdC, 5hmdC, 5fodC and 5cadC in real mouse brain sample. The whole genomic DNA methylation levels in mouse brain sample can be easily determined with UV detection.