Analytical Evaluation of High Energy Phosphate Determination by High Performance Liquid Chromatography in Myocardial Tissue

High performance liquid chromatography (HPLC) is an established method for the determination of myocardial high energy phosphates (HEP). Quantification of HEP compounds in small tissue specimens obtained by endomyocardial biopsy technique requires maximal sensitivity without impairment of precision. Employing isocratic ion-pair reversed-phase HPLC, high sensitivity and precision were obtained by running analyses for adenonucleotides and creatine compounds separately at detection wavelengths of 254 and 210 nm, respectively. Further reasons for separate runs were given by the necessity for different sample preparation as remaining perchloric ion after deproteinizing and pH in the samples had various effects on adenonucleotides and creatine compounds. Mechanical homogenization for 20s in 0.42 mol/l perchloric acid ensured a consistent myocardial HEP extraction. Sample preparation directly following biopsy sampling is preferable since HEP compounds were labile in tissue within days at -80°C even though an initial metabolic inhibition in liquid nitrogen had been induce. Following extraction and neutralization. HEP compounds were stable for up to 3 months at -20°C.