High performance liquid chromatography: a rapid isocratic method for determination of creatine compounds and adenine...

Previous methods for high performance liquid chromatography (HPLC) measurements of myocardial high energy phosphate (HEP) compounds have used either two different assays or a gradient technique in order to determine both creatine compounds (creatine (Cr), creatine phosphate (CrP)), and adenine nucleotides (ATP, ADP, AMP). An isocratic ion-pair reversed-phase assay has been developed which allows for the rapid (single run within 10 min) measurement of these compounds in extracts from freeze-clamped and freeze-dried myocardial tissue. The HPLC system made use of a RP18 column and a spectrophotometer, set at 206 nm. The mobile phase consisted of KH2PO4 (215 mM), tetrabutylammonium hydrogen sulphate (2.3 mM) and acetonitrile (3.5%) at pH 6.25. A distinct separation (confirmed by enzymatic and other methods) of HEP compounds was achieved. Standard curves were linear and passed through the origo for all examined concentrations. In isolated rat hearts subjected to control aerobic perfusion the following values were obtained (mumol/g dry wt, mean +/- S.E.M.): ATP 26.6 +/- 0.4, ADP 4.6 +/- 0.1, AMP 1.4 +/- 0.1, CrP 35.5 +/- 1.0, Cr 46.2 +/- 1.1. Values for HEP compounds of hearts undergoing anoxia +/- reoxygenation, and ischemia +/- reperfusion are presented. The importance of the procedure for extraction of HEP from freeze-dried cardiac tissue is highlighted.