To provide a bio-analytical methodology for study of pharmacokinetics and metabolic disposition of exogenous PCr in rats, ion-pair HPLC-UV method was developed for the simultaneous determination of exogenous phosphocreatine(PCr) and its metabolite creatine(Cr) and related ATP in plasma and RBC of rats. By changing flow rate and detection wavelength, the analytes were separated and detected on a Kromasil C18 column with a tertiary gradient mobile phase composed of(A) 0. 2％KH2PO4 +0. 08％ tetrabutylammonium hydrogen sulphate(pH 3.0), (B) the abovementioned solution whose pH value was adjusted to 7.5 with 1 mol/L NaOH, and(C) MeOH. The blank plasma and RBC sample was initially run for baseline subtraction. For quantification, TMP was used as internal standard. Plasma and RBC were deproteinized with 6％ PCA prior to HPLC. The method was shown to be specific without interference from matrix. A good linearity was obtained over a wide range of 10-7500 mg/L(plasma) and 10-2500 mg/L(RBC) for PCr, and 10-1500 mg/L(plasma)and 10-750 mg/L(RBC) for both Cr and ATP(R2＞0.99). The samples of 3 analytes showed that intra-day and inter-day precisions(RSD) were ＜10 ％, and the extraction recovery was ＞92％. The method was successfully used to determine plasma and RBC of the 3 analytes after intravenous administration of PCr to rats simultaneously. The results indicated this method meet the requirement of PCr pharmacokinetics study and metabolic disposition of rats.%建立了离子对反相高效液相色谱(IP-RPHPLC)同时测定大鼠血浆和红细胞(RBC)中磷酸肌酸(PCr)及其代谢产物肌酸(Cr)以及相关三磷酸腺苷(ATP)浓度的方法.采用Kromasil-C18色谱柱(250 mm×4.6 mm,5 μm),流动相A:0.2%KH2PO4+0.08%四丁基硫酸氢铵混合溶液(pH 3.0)；流动相B:0.2%KH2PO4+0.08%四丁基硫酸氧铵混合溶液(以1 mol/NaOH调至pH 7.5)；流动相C:甲醇.梯度洗脱,同时改变流速和检测波长.血浆及RBC样品以HClO4沉淀蛋白予以净化.选用甲氧苄啶(TMP)为内标,以峰面积比内标法进行定量分析,以基线扣除法计算外源性PCr、Cr和相关ATP浓度.在上述色谱条件下,血浆及RBC中PCr、Cr、ATPrn及TMP达基线分离,不受基质干扰,PCr在10～7500 g/L(血浆)、10～2500 mg/L(RBC),Cr和ATP在10～1500 mg/L(血浆)、10～750 mg/L(RBC)范围内线性关系良好(r2>0.99)；3种分析物QC样品的日内和日间精密度(RSD)均小于10%；回收率均大于92%.采用本方法测定了静脉和口服给药PCr后大鼠血浆和RBC中3种分析物浓度,结果满意.
展开▼
