离子对反相液相色谱同时测定家兔血浆中的外源性磷酸肌酸及其代谢产物肌酸

摘要

建立了离子对反相高效液相色谱法( IP-RP-HPLC)同时测定家兔血浆中外源性磷酸肌酸(PCr)及其代谢产物肌酸(Cr)的方法,用于研究外源性PCr在家兔体内的药代动力学.以含离子对试剂四丁基硫酸氢铵(TBA)的磷酸盐缓冲液-甲醇为流动相,在Kromasil-C18色谱柱上进行梯度洗脱.采用内标法定量、以基线扣除法计算外源性PCr和Cr的浓度.PCr和Cr的线性范围分别为10 ～7 500 mg/L和10～1 500 mg/L;日内和日间精密度均≤6.2％,准确度分别为99.7％ ～ 102.2％和96.5％～102.4％；萃取回收率均大于92％.静脉注射PCr后,血浆中PCr的消除为二室模型,消除半衰期为(20.4±2.7) min;表观分布容积为(0.179±0.037)L/kg；清除率为(0.019±0.002) L/(kg·min);静脉注射PCr后血浆中迅即出现降解产物Cr,其达峰时间为30 min;消除半衰期为(43.7±4.5) min.本方法的专属性强,准确度和精密度高,能特异性地测定家兔血浆中的PCr和Cr.实际应用结果表明,该方法完全符合PCr药代动力学生物分析方法学的要求.%A method for simultaneous determination of exogenous phosphocreatine (PCr) and its metabolite creatine (Cr) in rabbit plasma was developed by using an ion-pair reversed-phase high performance liquid chromatography (IP-RP-HPLC). The pharmacokinetics (PK) of PCr was also investigated. In the IP-RP-HPLC method, a Kromasil C,, column was used with meth-anol and phosphate buffer containing tetrabutylammonium hydrogen sulfate (TBA, ion-pair reagent) as the mobile phases in a gradient elution mode, while changing detection wavelength and flow rate. The internal standard method was used to quantify PCr and Cr, and the baseline subtraction method was applied. The calibration curves showed good linearity ranged from 10 to 7 500 mg/L for PCr and from 10 to 1 500 mg/L for Cr, and the correlation coefficients (r) were greater than 0.999. The methodology validation showed high specificity, precision and recovery with the intra-day and inter-day relative standard deviations (RSDs) of not more than 6. 2%, accuracies of 96. 5% - 102.4%, and extraction recoveries of more than 92%. After intravenous injection of PCr, the concentration-time profile can be best described by two-compartment model with elimination half time of (20. 4 ±2. 7) min, apparent volume of distribution of (0. 179 ±0.037) L/kg and clearance rate of (0.019 ±0.002) L/(kg ■ min). The Cr appeared rapidly with time to maximal concentration of 30 min, elimination half time of (43. 7 ± 4. 5 )min. The results of practical application showed that this bio-analytical method can completely meet the requirements for PK study of PCr in rabbit plasma.