Separation of purine and pyrimidine bases by ion chromatography with direct conductivity detection

A simple, rapid and accurate method for the simultaneous determination of four purine and pyrimidine bases (cytosine, 5-methylcytosine, adenine and N6-methyladenine) has been developed. The quantitative determination of these bases was accomplished by ion chromatography (IC) with direct conductivity detection (CD) based on their ionization in acidic medium without chemical suppression. The recovery of cytosine, 5-methylcytosine, and adenine in calf thymus DNA was more than 98% (n=3) and the relative standard deviation (RSD, n=5) less than 2.4%. In a single chromatographic run, the four bases could be separated and determined in less than 10 min. The detection limits were found to be 0.05 microg/mL for cytosine, 0.08 microg/mL for 5-methylcytosine, 0.07 microg/mL for adenine, and 0.07 microg/mL for N6-methyladenine. Linear ranges were 0.2-95.1 microg/mL for cytosine (r2=0.9996), 0.3-196.6 microg/mL for 5-methylcytosine (r2=0.9994), 0.3-105.5 microg/mL for adenine (r2=0.9998), and 0.3-159.1 microg/mL for N6-methyladenine (r2=0.9999). With the proposed method, purine and pyrimidine bases could be successfully detected in calf thymus DNA. We also determined these bases in calf thymus DNA using RP-HPLC. Compared to RP-HPLC, the IC method offers advantages such as high selectivity and simple mobile phase.