Rapid, isocratic separation of purine nucleotides using strong anion-exchange high-performance liquid chromatography

A method is presented for the rapid, isocratic separation of purine nucleotides using strong anion-exchange high-performance liquid chromatography at ambient temperature. The last peak of interest guanosine 5'-triphosphate (GTP) is eluted within 30 min and immediate reinjection is possible. All adenine and guanine nucleotides can be assayed with a single injection without the use of a gradient for elution. The procedure is particularly useful for the assay of NTP:AMP phosphotransferase reactions and/or the determination of changes in size of cellular purine nucleotide pools and computation of energy charges. An Altex Ultrasil AX prepacked column was used, and virtually identical results were obtained under similar conditions with the Whatman Partisil-10 SAX column. The eluting solution was 200 mM potassium phosphate, pH 6.85.