High-performance liquid chromatography determination of 5-methyl-2'-deoxycytidine, 2'-deoxycytidine, and other deoxynucleosides and nucleosides in DNA digests.

This work has undertaken liquid chromatographic separation of nucleosides and deoxynucleosides. Two different columns with three mobile phases (A, deionized water; B, 50 mM phosphate buffer (pH 4.0); C, methanol) and slightly different gradient programs were used. The elution order was as follows: cytidine (C), 2'-deoxycytidine (dC), uridine (U), 5-methyl-2'-cytidine (5mC), 5-methyl-2'-deoxycytidine (5mdC), guanosine (G), deoxyguanosine (dG), 2'-deoxythymidine (dT), adenosine (A), and 2'-deoxyadenine (dA). Using a Luna C18 Phenomenex column (150 x 4.6 mm, 5 microm), the separation was performed at 40 degrees C with a total flow rate of 1 ml/min and a run time of 10 min. The second column was an Agilent C18 (50 x 3 mm, 1.8 microm), for which the run time was 4.5 min with a flow rate of 0.6 ml/min (25 degrees C). In application to the DNA digests from human THP-1 cells, the quantification of C, dC, U, 5mC, 5mdC, G, dG, and A was performed. The percentages of global methylation were evaluated based on the 5mdC and dC concentrations (c(5mdC) / [c(5mdC)+c(dC)], where c is concentration in microg/ml) and compared with those calculated from the respective peak areas (A(5mdC) / [A(5mdC)+A(dC)], where A is peak area at 254 nm). For peak area measurements, excellent agreement was obtained with the results reported previously in the same cell line. In the quantitative approach, the results of DNA methylation were higher but consistent with the previous data obtained using mass spectrometric detection. Comparing the analytical features of the two procedures, the use of a smaller column could be recommended because it provides efficient separation (capacity factors in the range of 1.29-10.66), a short run time, and feasibility of nucleoside and deoxynucleoside quantification in real-world samples and because it also minimizes the use of reagents.