Determination of purine and pyrimidine bases in DNA by micellar electrokinetic capillary chromatography with electrochemical detection

A method based on micellar electrokinetic capillary chromatography with electrochemical detection was developed for the determination of cytosine, 5-methylcytosine (5-MC), thymine, adenine, and guanine in the hydrolysates of DNA. The working electrode was fabricated in a novel self-positioning carbon disc electrode system that can align the capillary outlet with the working electrode without a three-dimensional micromanipulator. The five analytes could be well separated within 10 min in a 40 cm length capillary at a separation voltage of 9 kV in a 40 mmol/l borate buffer (pH 10.0) containing 100 mmol/l sodium dodecyl sulfate. Good linearity was observed between peak current and concentration of bases over three orders of magnitude with the detection limits (S/N=3) ranging from 1.28·10−6 to 5.02·10−6 mol/l. This proposed method demonstrated long-term stability and reproducibility with relative standard deviations of less than 5% for both migration time and peak current (n=7). It has been successfully applied to determine bases including 5-MC in the hydrolysates of fish sperm DNA, calf thymus DNA, and DNA isolated from spleen cells of female mice.