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 (SIN=3) ranging from 1.28 x 10(-6) to 5.02 x 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.
Chen
Gang
G
Department of Chemistry, Fudan University, Shanghai, China. sgangchen@hotmail.com
Han
Xiaohui
X
Zhang
Luyan
L
Ye
Jiannong
J
D020374:Chromatography, Micellar Electrokinetic Capillary / Q000379:methods*; D004247:DNA / Q000737:chemistry*; D004563:Electrochemistry; D011687:Purines / Q000032:analysis*; D011743:Pyrimidines / Q000032:analysis*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D011687:Purines; D011743:Pyrimidines; D004247:DNA