Separation of twenty isomers of ribonucleotides and deoxyribonucleotides by reversed-phase ion-pairing highperformance liquid chromatography

A procedure is described for the separation of 20 isomers of ribonucleotides and deoxyribonucleotides by reversed-phase ion-pairing high-performance liquid chromatography using a Waters mu Bondapak C-18 10 mu m column and a gradient elution system consisting of phosphate buffer (0.05 M, pH 5.45) and methanol. The method is relatively fast, taking 40 min for each run, and quantitative. Nucleosides and nucleobases were eluted earlier in the system and did not interfere with the separation of nucleotides. Sample preparation is simple and prior group separation is not necessary. The method has been used successfully for the identification and quantification of nucleotides produced from nucleic acid degradation during yeast autolysis.