SIMULTANEOUS DETECTION OF NUCLEOTIDES, NUCLEOSIDES AND OXIDATIVE METABOLITES IN MYOCARDIAL BIOPSIES

A new simple, simultaneous matrix HPLC methodology was developed to fa cilitate 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, hyp oxanthine, xanthine, adenosine, inosine, NAD, and NADH, which are used to establish myocardial cellular energy status and effectiveness of c ardioprotection. Their detection was achieved using a 4-mu m spherical bead, 300 x 3.9 mm I.D. Nova-Pak C-18 column in a 12% methanol mobile phase solvent selection, ion-pairing reagents 1.47 mM TBAP (tetrabuty lammonium phosphate) and 73.5 mM KH2PO4, at a pH of 4.0. The extractio n method was modified for rapid determination to ensure diminished aci d labile NADH effects Comparisons of peak retention (k), resolution (R (s)) of solvents of varying concentrations and pH adjustment facilitat ed this method. This isocratic single run determination allows for sim ple, simultaneous rapid quantification and identification of alteratio ns in high-energy phosphates, nucleoside degradation products and NAD/ NADH levels associated with myocardial ischemia, with excellent reliab ility.