液相色谱检测乌龙茶水浸提物及其单宁酶水解产物中的儿茶素

HPLC Analysis of Catechins from Oolong Tea Infusion Treated by Tannase

目的:建立测定乌龙茶水浸提物中儿茶素的高效液相色谱(HPLC)方法,研究单宁酶对儿茶素成分的影响。方法:通过优化液相色谱的流动相组成,改善儿茶素的分离效果,缩短分析时间。利用液-质联用及全波长扫描鉴定乌龙茶水浸提物中的儿茶素,用回收率及相对标准偏差评价方法的适用性。以儿茶素成分的变化为指标,分析单宁酶对乌龙茶水浸提物的影响。结果:选用symmetry C18(3.0mm×250mm,5μm)色谱柱,在流速0.5mL/min,柱温30℃,检测波长278nm,进样体积20μL的条件下,优化得到的流动相为0.5%乙酸(A)和甲醇(B),梯度洗脱条件为0min:88%A;5min:88%A;16min:81%A;28min:76%A;32min:70%A;40min:88%A;45min:88%A;样品分析时间为45min。乌龙茶水浸提物中含有表没食子儿茶素没食子酸酯(EGCG)、表没食子儿茶素(EGC)、没食子酰儿茶素(EC)、没食子儿茶素没食子酸酯(GCG)、没食子儿茶素(GC)、表儿茶素没食子酸酯(ECG)等儿茶素以及没食子酸(GA)和咖啡因。用该方法检测乌龙茶中儿茶素组分,以上各种儿茶素的最低检出限在0.006～0.197μg/mL之间,定量检出限在0.019～0.656μg/mL之间,RSD在0.17%～1.57%之间,回收率96%～103%。经单宁酶作用后,乌龙茶水浸提物中酯型儿茶素(EGCG、GCG、ECG)被完全水解,同时GA和非酯型儿茶素(EGC、GC、EC)含量分别增加了2059.7%、66.4%、39.1%、54.3%。结论:本试验所建立的HPLC方法能高效分离乌龙茶水浸提物中的儿茶素,具有灵敏度高,精密度和准确性好的优点。单宁酶可高效水解乌龙茶水浸提物中酯型儿茶素。

The aim of this study was to develop a simple high performance liquid chromatographic analysis of tea eatechins for Oolong tea infusion which was treated by tannase. Liquid chromatograph was used to separate the components of Oo]ong tea infusion. Chromatographic conditions of the mobile phase composition, proportion and analysis time were optimized. We also identified the the catechins in Oolong tea infusion using LC-MS analysis system and a Agilentl200 liquid chromatography to evaluated the kinds of catechins and its spectral features through comparing with sev- eral kinds of catechin standards. At the same time, the applicability of this method was evaluated by its recovery and relative standard deviation（RSD） and the effect of tannase addition in Oolong tea infusion was also investigated. The op- timum separation system consisted of a symmetry Cis reversed-phase column（3.0x250 mm, 5 μm）, a gradient elution system of water （0 min, 88% A-5 min, 88% A-16 min, 81% A-28 rain, 76% A-32 min, 70% A-40 min, 88% A-45 min, 88% A） with（0.5%） acetic acid（A） and methanol（B） at 0.5 ml/min and 30℃ for 45 min and detective wavelength was 278 nm. EGCG, EGC, EC, GCG, GC, ECG, GA and caffeine were found in Oolong tea infusion. We using this method to indentify eatechins in Oolong tea infusion, and its minimum detection limits were from 0.006 μg/mL to 0.197 μg/mL for the components of tea infusion. Meanwhile, its quantitative detection limits were from 0.019 μg/mL to 0.656 μg/mL, RSDs were 0.17%-1.57% and the recoveries were 96%-103%. The gallated catechin （EGCG, GCG, ECG） were totally hydrolyzed while adding tannase in tea infusion accompanying with the generation of ungallted catechin（EGC, GC, EC） and GA. Tile results indicated we had established an effective HPLC method which had the advantages of high sensitivity and accuracy to separate the catechins in Oolong tea infusion. Tannase could hydrolyze the gallate catechins in tea infusion, effectively.