High Performance Liquid Chromatographic Determination of Ribonucleotides by Postcolumn Derivatization Involving Oxidation Followed by Fluorescence Reaction, and Its Application to Human Erythrocyte Sample

Abstract

A selective method for the determination of ribonucleotides is described which employs high performance liquid chromatography with on-line postcolumn fluorescence derivatization. Ribonucleotides are separated on a reversed-phased column (TSK gel ODS-80_TM) and automatically converted into fluorescent derivatives by reaction with 1, 2-bis(4-methoxyphenyl)ethylenediamine in an acidic medium after periodate oxidation. The detection limits (S/N=3) for ribonucleotides are 14-67pmol in a 100μl injection volume. The quantification of ribonucleotides in human erythrocytes is also described.