Rapid and accurate determination of deoxyribonucleoside monophosphates from DNA using micellar electrokinetic chromatography with a cationic surfactant additive |
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Authors: | Nan-Sook Hong Lian Hua Shi Ji Seon Jeong Inchul Yang Sook-Kyung Kim Sang-Ryoul Park |
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Institution: | (1) Center for Bioanalysis, Korea Research Institute of Standards and Science, Daejeon, 1 Doryong-dong, Yusung-gu, Daejeon, 305-340, South Korea;(2) University of Science and Technology, Daejeon, 305-701, South Korea; |
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Abstract: | A micellar electrokinetic chromatography (MEKC) method for rapid and accurate determination of 2′-deoxyribonucleoside 5′-monophosphates
(dNMPs), four structural elements of DNA, is described. MEKC separation at an optimized pH enabled complete separation of
four dNMPs. The use of a cationic surfactant additive for MEKC led to the reversal of EOF, which enhanced the migration velocities
of the negatively charged dNMPs. Under the optimized condition, full-baseline separation of the four dNMPs assuring accurate
peak integration was obtained within 5 min. For the given separation condition, pH-mediated on-column sample stacking was
optimized and applied to enhance sensitivity up to 6-fold. Analytical precision was improved by spiking iothalamate as an
internal standard. The accuracy of dNMP quantitation was ensured with dNMP standard solutions determined by inductively coupled
plasma-optical emission spectroscopy that measured phosphorous quantity. Performance of the proposed method was ultimately
proven by accurate quantitation of a DNA oligonucleotide that was enzymatically hydrolyzed prior to dNMP analysis. The proposed
MEKC method turned out to be a reliable analytical method for dNMPs that features high speed, high sensitivity, and high precision,
and could be utilized for high-accuracy determination of the amount of DNA as well as the base composition of DNA. |
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