Novel Dual-wavelength Monitoring Approach for the Improved Rapid Separation and Estimation of Adenine Nucleotides and Creatine Phosphate by High-performance Liquid Chromatography

A rapid and improved method for the simultaneous quantification of adenine nucleotides, including adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, as well as creatine phosphate by high-performance liquid chromatography is described. A programmable multi-wavelength UV detector was used to monitor the effluent initially at 210 nm for 4 min to measure creative phosphate, and then at 259 nm for 6 min to quantitate adenosine mono-, di- and triphosphate. The method is, thus, not only rapid, but also extremely sensitive because of the utilization of corresponding absorption maxima for creatine phosphate and adenine nucleotides, rather than measuring all of them at a single wavelength. In addition, computer-generated standard curves were used to estimate these compounds, thereby improving the accuracy of the measurements. Since myocardial high-energy phosphate levels are widely measured because they are reliable parameters for myocardial preservation during ischemia, these compounds were assayed in pig heart prior to and following ischemia. The results were compared with those obtained by conventional high-performance liquid chromatographic assay methods in order to examine the validity of this method. Our results indicate that this is indeed a novel method, which is more rapid as well as sensitive and accurate compared with other methods.