Ultra performance liquid chromatography analysis of adenine nucleotides and creatine derivatives for kinetic studies

Abstract

A rapid method for simultaneous quantification of compounds participating in energy metabolism of cardiac muscle cells (creatine (Cr), phosphocreatine (PCr), ADP, and ATP) is described where a conventional ion-pair reversed phase HPLC separation has been improved by introducing the method based on the recently developed ultra performance liquid chromatography (UPLC) technique. In the 0.005-1 mM concentration range, the calibration curves for Cr, ADP, and ATP as pure standard compounds were fitted by the polynomic relationship y = y 0 + ax - bx2 at a high confidence level (R2 0.999 in all cases) and that for PCr by a simple linear relationship with R 2 7> 0.998. The method was applied for the study of the kinetics of PCr production by permeabilized cardimyocytes due to the cellular oxydative phosphorylation reactions. The determined steady-state levels of ADP and ATP as well as the rate of PCr production in different conditions can be used for the verification of the results of mathematical modelling of cardiomyocyte functioning.