Determination of ATP and its metabolites released from rat caudal artery by isocratic ion-pair reversed-phase high-performance liquid chromatography.

A sensitive and selective assay method for adenine compounds (purines) using high-performance liquid chromatography with fluorescent detection was developed. The 1,N6-ethenoderivatives of adenine, adenosine, AMP, ADP, and ATP formed by reaction with chloroacetaldehyde at 80 degreesC were separated by ion-pair reversed-phase chromatography within 15 min under isocratic conditions. alpha,beta-Methylene adenosine 5'-diphosphate could be used as an internal standard for the determination of purines. The calibration graphs constructed with peak area ratios against amounts were linear between 0.1 and 10.0 pmol of each purine. The repeatability and intermediate precision were less than 6% (RSD, n = 5) and 8% (RSD, n = 3), respectively, at 0.5 pmol of each purine. A method for calculation of each purine amount which considers hydrolysis by derivatization is described. The optimized method was applied to determine the purines released from the rat caudal artery stimulated by noradrenaline.