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

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。

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-Ⅱcolumn(250 mm×4.6 mm,5μm).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 20μL.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.