HPLC法同时测定大鼠尿和胆汁中4种儿茶素类活性成分  

HPLC method for simultaneous determination of 4 eatechins of tea polyphenols in rat urine and bile

作　　者：李秋莎[1] 邹玲莉[1] 付婷[1] 吕莉[1] 林娅芳[1] 韩国柱[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》2010年第3期372-378,共7页Chinese Journal of Pharmaceutical Analysis

基　　金：大连市科技计划基金(2002B4NS044)

摘　　要：目的:建立一种反相HPLC法同时测定大鼠尿和胆汁中茶多酚(TP)的4种儿茶素类活性成分表没食子儿茶素没食子酸酯(EGCG)、表儿茶素没食子酸酯(ECG)、表没食子儿茶素(EGC)和表儿茶素(EC),为TP自大鼠尿和胆汁的排泄研究提供测定方法学。方法:采用RP-HPLC-UV法,使用Kromasil-C18(4.6mm×150mm,5μm)色谱柱,以乙腈-0.1%枸橼酸溶液为流动相,洗脱方式采用组成梯度和流速梯度,检测波长为280nm;尿或胆汁样品200μL中加入20%维生素C20μL以及10μg.mL-1香草醛内标溶液(IS)20μL,乙酸乙酯500μL液-液萃取2次,N2流挥干后加20%乙腈水溶液100μL溶解,取上清液60μL进样分析。结果:EGCG、ECG、EGC、EC和IS的保留时间分别为11.81,18.02,6.21,10.42,16.36min,各组分之间达基线分离且不受空白基质、代谢物和TP中其他成分的干扰。4种儿茶素类活性成分的尿/胆汁标准曲线的线性范围分别为0.5～10μg·mL-1/0.2～5μg·mL-1(EGCG)、0.125～5μg·mL-1/0.2～5μg·mL-1(ECG)、2.5～50μg·mL-1/0.8～20μg·mL-1(EGC)和1～20μg·mL-1/0.4～10μg·mL-1(EC)(r>0.9977)。QC样品的日内和日间精密度RSD均小于10%;准确度均在90%～110%;EGCG、ECG、EGC、EC及IS在尿和胆汁中的萃取回收率均大于60%和80%。应用上述方法测定大鼠静脉注射TP100mg·kg-1后尿和胆汁8h总累积排泄分数为1.57%(EGCG)、2.00%(ECG)、8.33%(EGC)和10.67%(EC)。结论:本文建立的RP-HPLC-UV法专属性及重现性好,准确可靠,可用于大鼠尿和汁胆中4种儿茶素类活性成分的同时测定及其体内排泄研究。Objective：To develop an RP-HPLC-UV method for simultaneous determination of （-）epigallocatechin-3-gallate（EGCG）,（-）epicatechin-3-gallate（ECG）,（-）epigallocatechin（EGC）,and（-）epicatechin（EC）as main active ingredients of tea polyphenols（TP）,in rat urine and bile in order to provide an analytical method for the study of excretion from urine and bile of TP.Methods： EGCG,ECG,EGC and EC in urine and bile samples were separated on a Kromasil-C18 column（4.6 mm×150 mm,5 μm） protected by a Kromasil-C18 pre-column（4.6 mm×20 mm,10 μm）,using a gradient mobile phase composed of acetonitrile-0.1% citric acid and a gradient flow rate（1.0~1.5 mL·min-1）.The effluent was detected at 280 nm.An aliquot of 200 μL of rat urine or bile sample spiked with 10 μg·mL-1,vanillin（van）as IS in presence of 20 μL of 20% vitamin C solution was extracted twice with 500 μL of ethyl acetate（EtoAc）each.The collected EtoAc fraction was evaporated to dryness under N2 stream,followed by reconstitution in 100 μL of a 20% acetonitrile aqueous solution,50 μL of supernatant was injected onto chromatograph for analysis. Results： The HPLC resulted in base-line separation of 4 eatechins and IS.There was no interference from blank urine and bile.The linear range of urine / bile samples was 0.5-10 μg·mL-1 / 0.2-5 μg·mL-1 for EGCG,0.125-5 μg·mL-1 / 0.2-5 μg·mL-1 for ECG,2.5-50 μg·mL-1 / 0.8-20 μg·mL-1 for EGC,and 1-20 μg·mL-1/0.4-10 μg·mL-1 for EC.The intra-day and inter-day precision（RSD）of QC samples was better than 10%,and the average accuracy was between 90%-110%.The extraction recovery of EGCG,ECG,EGC,EC and IS in urine and bile was better than 60% and 80%,respectively.The above-stated method was used to determine the concentration of the 4 eatechins in urine and bile of rats receiving iv 100 mg·kg-1 TP and showed the 8 h total cumulative excretion of 163.44 μg（EGCG）,73.39 μg（ECG）,123.30 μg（EGC）and 253.21 μg（EC）in urine and bile.Conclusion：The

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

分 类 号：R917[医药卫生—药物分析学]