HPLC for Separation and Quantification of Deoxyribonucleic Acid Fragments and Measurement of Deoxyribonucleic Acid Degradation

Abstract

DNA quantification has made its mark in pharmaceutical analysis and the life sciences. In particular, in the quality control of nucleic acid drugs and the detection and quantification of genetically modified organisms, evaluation of the DNA degradation rate has become imperative. In this study, by using high-performance liquid chromatography with an anion-exchange column, we established a method for the separation and quantification of DNA fragments in mixed DNA samples. By using a NaCl concentration gradient, DNA fragments in mixed DNA sample were separated well. A calibration curve from 0.05 to 12.4 ng μL⁻¹ was obtained with high linearity and the correlation coefficient was 0.9999. The limit of detection was 0.02 ng μL⁻¹ and the limit of quantification was 0.06 ng μL⁻¹ for S/N = 3 or S/N = 10, respectively. The relative standard deviation was less than 2 % in the measurement of peak area repeatability. The recovery of approximately 1 ng μL⁻¹ of a specific DNA spiked in a mixed DNA sample was 99.9 ± 3.6 %. The method was able to measure the degradation rate of 600 bp DNA with a variation of approximately 1 %.