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

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.