High-Performance Liquid Chromatographic Analysis of 2′-Deoxynucleoside 5′-Monophosphate Using N-(Dansyl)Ethylenediamine as a Fluorescent Derivatizing Reagent

Abstract

A method for quantitative determination of 2′-deoxynucleoside 5′-mono-phosphate (dXmp) has been developed. This based on the preparation of fluorescent derivatives by the reaction of 5-dimethylaminonaphthalene-1-[N-(2-aminoethyl)] sulfonamide (dansyIEDA) with the phosphoric acid moiety of dXmp, and then the resolution of fluorescent derivatives by high-performance liquid chromatography with a spectrofluoro monitor. The fluorescent dansyIEDA derivatives of dXmp were separated more sharply and symmetrically from each other compared with the separation of non-derivatized dXmp. The detection limits for dansylEDA derivatives of dXmp were 6.4–6.7 pmol per 10-μI injection, and these values indicate that the fluorimetric analyses were approximately 10 to 20-times more sensitive than the UV-mouitoring method. The simplicity, sensitivity and selectivity make this method an attractive alternative to the established assay systems of dXmp.