Objective: Ion-exchange high-performance liquid chromatography (HPLC) was investigated for the analysis of cAMP hydrolysis catalyzed by rabbit liver cyclic nucleotide phosphodiesterase (PDE). Methods: The HPLC column was Shim-Pak WAX-1

mobile phase was 30 mmol/L potassium phosphate at pH 4.0 eluted at 0.5 ml/min

detection was absorbance at 254 nm

and quantification was performed with peak area. Results: cAMP and AMP were well-resolved and quantified by peak area under this HPLC condition. Rabbit liver PDE was partially purified by ammonium sulfate fractionation. In this PDE reaction mixture

residual contaminants after extraction were all baseline-resolved from both cAMP and AMP. The time course of rabbit PDE reaction showed that cAMP continuously dropped but AMP increased to a plateau before dropping. Analysis of cAMP dropping by the integrated Michaels-Menten rate equation of mono-substrate irreversible reaction lead to negative Michaelis constant. Conclusion: This ion-exchange HPLC method was desirable for the analysis of cAMP hydrolysis by PDE without phosphatase interference.