高效液相色谱法同时测定茶叶中表儿茶素及其氧化二聚体、没食子酸和咖啡碱的含量

张凯新; 孔祥慧; 徐卫奇; 陈贵杰; 曾晓雄

doi:10.13386/j.issn1002-0306.2022120093

高效液相色谱法同时测定茶叶中表儿茶素及其氧化二聚体、没食子酸和咖啡碱的含量

Simultaneous Determination of Epi-Catechins and Their Oxidized Dimers, Gallic Acid and Caffeine in Tea by HPLC

摘要

摘要: 目的：本研究旨在建立同时分析茶叶中4种表儿茶素（表儿茶素、表儿茶素没食子酸酯、表没食子儿茶素和表没食子儿茶素没食子酸酯）及其氧化二聚体（4种茶黄素（Theaflavins，TFs）：茶黄素、茶黄素双没食子酸酯、茶黄素-3-没食子酸酯和茶黄素-3’-没食子酸酯）、3种聚酯型儿茶素（Theasinensins，TSs）（TSA、TSB和TSC）、没食子酸和咖啡碱的高效液相色谱（High performance liquid chromatography，HPLC）方法。方法：首先利用化学合成的方法制备TSA、TSB和TSC；然后参照文献对HPLC流动相做适当调整和对梯度洗脱条件进行优化，色谱柱为Cosmosil 5C₁₈-AR-Ⅱ色谱柱（250 mm×4.6 mm，5 m），流动相为纯水（A）、50 mmol/L磷酸（B）和乙腈（C），检测波长为280 nm，流速为0.8 mL/min，柱温为30 ℃，进样量为20 L；最后根据建立的HPLC方法对绿茶、乌龙茶、红茶和黑茶中的上述13种物质含量进行测定。结果：在最优梯度洗脱条件下4种表儿茶素、4种TFs、3种TSs、没食子酸及咖啡碱可在66 min内得到较好地分离，同时各物质在0.01~1.00 mg/mL的质量浓度范围内与其峰面积的线性关系良好（R²均在0.9904~0.9966范围内），重复性相对标准偏差（The relative standard of deviation，RSD）为1.034%~4.008%，精密度RSD为1.882%~4.835%，稳定性RSD为0.349%~3.994%，三种浓度的加标回收率在90.106%~107.331%之间且RSD<3.268%。此外，上述13种物质含量在不同茶叶样本间存在显著差异，且所有检测值均在标准曲线有效测定范围内。结论：本方法操作简单高效、结果准确可靠，可用于茶叶中表儿茶素、茶黄素、聚酯型儿茶素、没食子酸和咖啡碱的同时分析。

Abstract: Objective: The present study was aimed to develop the simultaneous determination of four kinds of epi-catechins ((-)-epicatechin, (-)-epicatechin gallate, (-)-epigallocatechin and (-)-epigallocatechin gallate) and their oxidized dimers (four kinds of theaflavin (TF): theaflavin, theaflavin-3-gallate, theaflavin-3'-gallate and theaflavin-3,3'-digallate, three kinds of theasinensin (TS): TSA, TSB and TSC), gallic acid and caffeine by high performance liquid chromatography (HPLC). Methods: Firstly, TSA, TSB and TSC were chemically synthesized. Then, the gradient elution conditions for HPLC analysis were obtained according to the reported methods with some modifications. Chromatographic separation was achieved on a Cosmosil 5C₁₈-AR-Ⅱ column (250 mm×4.6 mm, 5 μm). Ultrapure water (eluent A), 50 mmol/L H₃PO₄ (eluent B) and acetonitrile (eluent C) were used as the mobile phases. The DAD acquisition wavelength was set at 280 nm. The flow rate was 0.8 mL/min and oven temperature was set at 30 ℃. The volume of injection was 20 μL. Finally, the conents of these componds mentioned above in green, oolong, black and dark teas were determined by the developed HPLC method. Results: Under the optimized conditions, 4 kinds of epicatechins, 4 kinds of theaflavins, 3 kinds of theasinensins, gallic acid and caffeine could be separated within 66 min. At the same time, in the range of 0.01~1.00 mg/mL, the substances and their peak areas showed a good linear relationship (R², 0.9904~0.9966). The relative standard of deviation (RSD) for repeatability was 1.034%~4.008%. The RSD of the precision and the stability were 1.882%~4.835% and 0.349%~3.994%, respectively. The recovery test of three concentrations was in the range of 90.106%~107.331% and the RSD was less than 3.268%. In adition, it was found that the contents of these 13 compounds were significantly different among different tea samples, and all the detected values were within the effective determination range of the standard curve. Conclusion: The developend method is simple, accurate, and reproducible, it can be used for the simultaneous determination of epi-catechins, theaflavins, theasinensins, gallic acid, and caffeine in tea samples.