TITLE:
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
AUTHORS:
Li Lv, Heng Xi, Guozhu Han
KEYWORDS:
Ion-Pair HPLC; Phosphocreatine; Creatine; ATP; Plasma; RBC; Pharmacokinetics; Rabbit
JOURNAL NAME:
Journal of Analytical Sciences, Methods and Instrumentation,
Vol.3 No.3A,
September
26,
2013
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% KH2PO4 + 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 mg/ml (plasma) and 5 - 2500 mg/ml (RBC) and for both Cr and ATP
concentrations of 10 - 1500 mg/ml (plasma) and 5 - 750 mg/ml (RBC) (r > 0.99). The QC samples of 3 analytes
showed intra-day and inter-day precisions (RSD) of - 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.