Simultaneous Determination of Mono-, Di-, and Trinucleotides by High-Performance Liquid Chromatography Using N-(Dansyl)Ethylenediamine as a Fluorescent Derivatizing Reagent

Abstract

A method for simultaneous determination of mono-, di- and trinucleotides with high sensitivity has been developed. This based on the fluorescent derivatization of nucleotides by the reaction of 5-dimethylaminonaphthaIene-l-[N-(2-aminoethyl)]sulfonamide (dansylEDA) with the phosphoric acid moiety of nucleotide, and then the resolution of fluorescent derivatives by high-performance liquid chromatography with a spectrofluoro monitor. The fluorescent dansylEDA derivatives of nucleotides were separated sharply each other. The detection limits for dansylEDA derivatives of nucleotides were between 4.7 and 20.3 pmol per 10-μ1 injection. This method was applied to analyze the yeast RNA after the generation of ribonucleoside 5′-monophosphate by nuclease P1 digestion. As a result, no more than 40 ng of RNA was needed for one analysis, and so such sensitivity of this method gives an advantage to analyze the RNA in a small scale.