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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 establish a reverse phase HPLC method for the simultaneous determination of four catechins, epigallocatechin gallate (EGCG), epicatechin gallate (ECG), epigallocatechin (EGC) and epicatechin (EC) provide an assay methodology for the TP study of excretion of urine and bile from rats. METHODS: The mobile phase consisted of acetonitrile-0.1% citric acid solution with Kromasil-C18 column (4.6 mm × 150 mm, 5 μm) using RP-HPLC-UV method with compositional gradient and flow rate gradient. The detection wavelength 20 μL of vitamin C20 μL and 10 μg.mL-1 vanillin internal standard solution (IS) were added to 200 μL of urine or bile samples, followed by liquid-liquid extraction of 500 μL of ethyl acetate twice. After the N2 stream was evaporated to dryness, 20% of acetonitrile 100μL aqueous solution was dissolved, the supernatant was taken 60μL injection analysis. Results: The retention times of EGCG, ECG, EGC, EC and IS were 11.81, 18.02, 6.21, 10.42 and 16.36 min, respectively. Baselines were separated from each other and were not disturbed by blank matrix, metabolites and other components in TP . The linear range of urine / bile standard curves of the four catechins were 0.5 ~ 10μg · mL -1 / 0.2 ~ 5μg · mL -1 (EGCG), 0.125 ~ 5μg · mL -1 / 0.2 ~ 5μg · (ECG), 2.5 ~ 50μg · mL -1 / 0.8 ~ 20μg · mL -1 (EGC) and 1 ~ 20μg · mL -1 / 0.4 ~ 10μg · mL -1 (r> 0.9977). The intra-day and inter-day RSD of QC samples were all less than 10% and the accuracy was between 90% and 110%. The recovery rates of EGCG, ECG, EGC, EC and IS in urine and bile were more than 60% and 80% . The cumulative excretion fraction of urine and bile 8h after intravenous administration of TP100mg · kg-1 was 1.57% (EGCG), 2.00% (ECG), 8.33% (EGC) and 10.67% (EC), respectively. Conclusion: RP-HPLC-UV method established in this paper is specific and reproducible, accurate and reliable, and can be used for the simultaneous determination of 4 catechins in rat urine and juvenile gall bladder and their in vivo excretion.