Isocratic separation of ATP and its degradation products from biological fluids by automated liquid chromatography

Two groups of metabolites (a) IMP, AMP, ADP, ATP, and cAMP in extracts of fibroblasts and erythrocytes and (b) hypoxanthine, xanthine, adenosine, and inosine in plasma and urine have been separated by ion-pairing reversed-phase chromatography on a microBondapak C18 column, with use of the following reagents: 60 mmol/L KH2PO4, 0.45 mmol/L tetrabutylammonium phosphate, and 1.26 mol/L acetonitrile, pH 3.2 (at 23 degrees C) (group a) and 20 mmol/L KH2PO4, 0.45 mmol/L tetrabutylammonium phosphate, and 0.35 mol/L acetonitrile, pH 2.70 (at 24 degrees C) (group b). Under both sets of conditions, the compounds are completely separated in less than 15 min. The separation is isocratic, so the method is easily adaptable to automation.