An Ion-Pair HPLC Method for Simultaneous Determination of Exogenous Phosphocreatine and Its Metabolite Creatine and Related ATP in Rabbit Plasma and RBC: Application to a Pharmacokinetic Study

Abstract

A specific, precise and accurate ion-pair HPLC-UV method has been developed and validated for simultaneous determination of phosphocreatine (PCr), and its metabolite creatine (Cr) as well as related ATP in plasma and red blood cell (RBC) of rabbits. After addition of TMP as IS, the samples were deproteinized with 6% PCA. The analytes were separated on a Kromasil C18 column using a tertiary gradient mobile phase composed of buffer A (0.2% KH 2 PO 4 + 0.08% tetrabutyl ammonium hydrogen sulphate, pH 3.0), buffer B (buffer A adjusted to pH 7.5 with 1 mol/L NaOH) and MeOH. Detection wavelengths were set at 210 nm for PCr and Cr and 260 nm for ATP and TMP. Some blank samples were initially run for baseline subtraction. The linear detection responses were obtained for PCr concentration over a range of 10 - 7500 m g/ml (plasma) and 5 - 2500 m g/ ml (RBC) and for both Cr and ATP concentrations of 10 - 1500 m g/ml (plasma) and 5 - 750 m g/ml (RBC) (r > 0.99). The QC samples of 3 analytes showed intra-day and inter-day precisions (RSD) of <10%, accuracy of 97% - 107%. The method was successfully used to simultaneously determine plasma and RBC concentrations of the 3 analytes and to study pharmacokinetics after iv administration of PCr to rabbits.