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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 r ecently 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 = y0 + ax - bx 2 at a high confidence level ( R2 > 0.999 in all cases) and that for PCr by a simple linear relationship with R2 > 0.998. The method was applied for the study of the kinetics of PCr production by permeabilized cardimyocytes due to the cellular oxydative phosphorylation r eactions. 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 res ults of mathematical modelling of cardiomyocyte functioning.

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