A new simple, simultaneous matrix HPLC methodology was developed to facilitate better peak separability and resolution for the determination of levels of myocardial tissue nucleotides, nucleosides and oxidative metabolites. The components of interests were ATP, AMP, ADP, IMP, hypoxanthine, xanthine, adenosine, inosine, NAD, and NADH, which are used to establish myocardial cellular energy status and effectiveness of cardioprotection. Their detection was achieved using a 4-microns spherical bead, 300 x 3.9 mm I.D. Nova-Pak C18 column in a 12% methanol mobile phase solvent selection, ion-pairing reagents 1.47 mM TBAP (tetrabutylammonium phosphate) and 73.5 mM KH2PO4, at a pH of 4.0. The extraction method was modified for rapid determination to ensure diminished acid labile NADH effects. Comparisons of peak retention (k), resolution (Rs) of solvents of varying concentrations and pH adjustment facilitated this method. This isocratic single run determination allows for simple, simultaneous rapid quantification and identification of alterations in high-energy phosphates, nucleoside degradation products and NAD/NADH levels associated with myocardial ischemia, with excellent reliability.