HPLC法同时测定大鼠血浆中茶多酚主要活性成分EGCG和ECG  

Simultaneous HPLC determination of the main active ingredients EGCG and ECG of TP in rat plasma

作　　者：梁骏[1] 付婷[1] 韩国柱[1] 吕莉[1] 李楠[2] 

机构地区：[1]大连医科大学临床药理教研室,大连116044 [2]大连理工大学分析化学教研室,大连116023

出　　处：《药物分析杂志》2009年第1期39-43,共5页Chinese Journal of Pharmaceutical Analysis

基　　金：大连市科技计划基金(2002B4NS044)

摘　　要：目的:建立HPLC法同时测定大鼠血浆中茶多酚(tea polyphenols,TP)主要活性成分表没食子儿茶素没食子酸酯(EGCG)和表儿茶素没食子酸酯(ECG)的浓度,为TP药代动力学研究提供测定方法学。方法:采用RP-HPLC法,使用Kro-masil-C18(4.6mm×250mm,5μm)色谱柱,预柱为Kromasil-C18保护柱(4.6mm×20mm,10μm),以乙腈-0.1%枸橼酸溶液(14:86)为流动相,流速1.5mL.min-1,检测波长280nm;血浆样品200μL加75μg.mL-1间苯二酚内标溶液10μL,乙酸乙酯400μL提取2次,N2流挥干后加10%乙腈水溶液100μL,漩混1min,4℃低温离心(7500r.min-1)10min后,取上清液60μL进样分析。结果:EGCG和ECG的保留时间分别为9.92min和29.51min,间苯二酚的保留时间为5.96min,三者之间达到基线分离,并不受空白血浆和代谢物以及茶多酚中其他成分的干扰。EGCG和ECG标准曲线的线性范围分别为0.5～300μg.mL-1(r=0.9999)和0.1～60μg.mL-1(r=0.9999);日内精密度RSD均小于5.7%,日间精密度RSD均小于11.2%;准确度为94.78%～110.6%;EGCG、ECG和内标的萃取回收率分别为85.73%～91.93%,79.08%～86.51%,91.88%～94.88%;血浆样品在-20℃保存下至少在21d内和室温保存下8h内稳定。大鼠ivTP100mg.kg-1后血浆样品应用上述方法测定血药浓度,表明TP药动学行为遵循二室模型一级动力学。结论:本文建立的大鼠血浆中TP主要活性成分EGCG和ECG的HPLC测定方法准确,专属性及重现性好,且快速简便,可用于TP注射液的非临床药代动力学研究。Objectlve：To develop an HPLC method for simultaneous determination of （ - ）epigallocatechin -3 - gallate （EGCG） and （ - ） epicatechin - 3 - gallate （ ECG）, the main active ingredients of tea polyphenols （ TP）, in rat plasma, in order to provide an analytical method to study pharmacokinetics of TP. Methods： EGCG and ECG in rat plasma were simultaneously determined by RP - HPLC with ultraviolet detection at 280 nm, using resorcinol as internal standard. Chromatographic separation was achived on a Kromasil C18 column （4. 6 mm × 250 mm, 5 μm） equipped with a Kromasil C18 pre -column（4. 6 mm ×20 mm,10 μm） ,using a mobile phase composed of acetonitrile - 0. 1% citric acid （ 14 ： 86 ） running at a flow rate of 1.5 mL · min^-1 The UV detector was set at 280 nm. 200 μL of rat plasma sample was transferred into an Eppendorff tube containing 10 μL each of resorcinol and 20% vitamin C solution. The resultant mixture was extracted twice with 400 μL each of ethyl acetate. The organic phase was evaporated to dryness under a gentle N2 stream at 50 ℃ in a water bath. The residue was reconstituted in 100 μL of a 10% acetonitrile aqueous solution ,60 μL of supernatant was injected onto chromatograph for analysis. Results： The HPLC resulted in base - line separation of resorcinol （ retention time 5.96 min）, EGCG（ retention time 9. 92 min）, ECG（ retention time 29.51 min）, and other components;There was no interference from blank plasma. The linear ranges were 0. 5 - 300 μg · mL^-1 for EGCG （ r = 0. 9999 ） and 0. 1 - 60 μg · mL^-1 for ECG （ r =0. 9999）. The intra - and inter - day precision（RSD） was better than 5.7% and 11.2% ,respectively,and the average accuracy was between 94.78% -110. 6%. The extraction recoveries of EGCG, ECG and internal standard were 85.73 % -91.93 % ,79.08% -86.51% and 91.88 % -94. 88%, respectively. The plasma samples were stable for at least 21 days at -20 ℃ and 8 h at room temperature. Application of the method to the determin

关 键 词：高效液相色谱 茶多酚(TP) 表没食子儿茶素没食子酸酯(EGCG) 表儿茶素没食子酸酯(ECG) 

分 类 号：R917[医药卫生—药物分析学]