| Objective:(1) To develop an ion-par HPLC-UV method for simultaneous determination of exogenous phosphocreatine (PCr) and its metabolite creatine (Cr) and Pcr-related ATP in plasma and RBC of rats. In order to provide a bio-analytical methodology for study of pharma-cokinetics and metabolic disposition of exogenous PCr in rats. (2) To research the characteristic of pharmacokinetics in plasma samples and bioavailability after rats iv and po PCr. (3) To research metabolic disposition and the characteristic of pharmacokinetics after rats iv PCr. (4) To research the characteristic of pharmacokinetics in RBC samples after rats iv PCr.Methods:The PCr, Cr and ATP was chromatographically separated on a Kromasil C18 column using a tertiary gradient mobile phase composed of 0.2%KH2PO4+0.08%tetrabutylammonium hydrogen sulphate (pH3.0) (A), A adjusted to pH 7.5 with 1N NaOH (B) and MeOH (C) coupled with a detection wavelength of 210 nm for PCr and Cr and 260nm for ATP. A blank plasma and RBC was initially run for baseline subtraction. For quantification TMP was used as internal standard. Plasma and RBC were deproteinized with 6%PCA, followed by neutralization to pH7.0 with K2CO3 prior to HPLC. The peak area rations of analytes to TMP as internal standard vs concentration of analytes to construct calibration curves. Use the method of baseline subtraction to calculate the exogenous PC,Cr,and ATP. Rats were randomly divided into many groups receiveing iv administration via caudal vein and po of PCr and Cr respectively. Plasma and RBC were deproteinized with 6%PCA followed by neutrallyzation for IP-HPLC to determinate the concentration of PCr,Cr,and ATP. Calculate the parameter of pharmacokinetics and bioavailability.Results:The method validation including specificity, linearity, precision, accuracy, recovery and stability was conducted by routine procedures as descryibed in guiding principles of PK study issued by SFDA. It was shown to be specific without interference from matrix, a good linearity was obtained over a wide range of 10-7500μg/ml (plasma) and 5-2500 ug/ml (RBC) for PCr, and 10-1500 ug/ml (plasma) and 5-750 ug/ml (RBC) for both Cr and ATP (r＞0.99). The QC samples of 3 analytes showed intra-day and inter-day precisions (RSD) of≤8.98%and≤8.42%(plasma) and≤8.02%and≤6.41%(RBC), accuracy of 97.86～104.68%(plasma) and 97.73～105.04%(RBC), and in the plasma and RBC the extraction recovery of> 90%. The PK behavior of PCr in the plasma after iv PCr could be described by two-compartment model and first-order kinetics, with t1/2β22.5～23.3 min, Vd 0.9564～0.9786 L/kg and CL 0.029 L/(kg·min). After iv PCr, in the plasma it immediately apper Cr, it could be described by one-compartment model and first-order kinetics, with tmax 20 min, t1/2k(m) 40.6-42.7 min, it is longer than PCr. Through iv PCr and iv Cr, With the AUC of Cr could calculate f(m) 60～76%. After po PCr in the plasma, it not apper PCr, but could find Cr, t1/2k(m) 56.0-57.7min, tmax 90-95min. Through iv PCr and iv Cr, With the AUC of Cr could calculate bioavailability Fm 55.02～62.31%. After iv and po PCr in the plasma, it not apper ATP, but it could survy ATP in the RBC, after iv PCr. In the RBC the ATP tmax 68～83min, t1/2p 49～52min. In the plasma, when the concentration of PCr reduces obviously the concentration of ATP in the RBC could still maintain a high level, and the high level could maintain more than 300 min. After iv PCr it not apper PCr in the RBC, but could survy Cr, tmax 120min, ti/2k(m) 70min.Conclusion:The method was successfully used to simultaneously determine plasma and RBC concentrations of the 3 analytes after iv administration of PCr to rats and yielded a typical biexponential decay C-T curve for PCr and an extra-vascular absorption-like curve for both Cr and ATP. The PK behavior of PCr in the plasma after iv PCr could be described by two-compartment model and first-order kinetics, t1/2β＜0.5h, after iv administration of PCr, the rapid appearance of the hydrolytic metabolite Cr was evident, the percent conversion is 50%-75%. The elimination rate of hydrolytic metabolite Cr is slower than PCr, it belong to elimination rate limitation (ERL), iv administration of PCr, the level of ATP is raise evident in RBC, and in the plasma, when the concentration of PCr reduce obviously the concentration of ATP and Cr in the RBC could still maintain a high level, and the high level could maintain more than 300 min, it provide suspensory proof for hemorheology. After po administration PCr, it could not survy PCr, but could survy Cr, calculate bioavailability Fm from Cr 55-62%.
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