Efficient procedure for isolating methylated catechins from green tea and effective simultaneous analysis of ten catechins, three purine alkaloids, and gallic acid in tea by high-performance liquid chromatography with diode array detection

Monomers of (−)-epigallocatechin (EGC), (−)-epigallocatechin gallate (EGCG), (−)-epicatechin (EC), (−)-epicatechin gallate (ECG), (−)-epigallocatechin 3-O-(3-O-methyl) gallate (EGCG3″Me) and (−)-3-O-methyl epicatechin gallate (ECG3′Me) (purity, >97%) were successfully prepared from extract of green tea by two-time separation with Toyopearl HW-40S column chromatography eluted by 80% ethanol. In addition, monomers of (−)-catechin (C), (−)-gallocatechin (GC), (−)-gallocatechin gallate (GCG), and (−)-catechin gallate (CG) (purity, >98%) were prepared from EC, EGC, EGCG, and ECG by heat-epimerization and semi-preparative HPLC chromatography. With the prepared catechin standards, an effective and simultaneous HPLC method for the analysis of gallic acid, tea catechins, and purine alkaloids in tea was developed in the present study. Using an ODS-100Z C18 reversed-phase column, fourteen compounds were rapidly separated within 15 min by a linear gradient elution of formic acid solution (pH 2.5) and methanol. A 2.5–7-fold reduction in HPLC analysis time was obtained from existing analytical methods (40–105 min) for gallic acid, tea catechins including O-methylated catechins and epimers of epicatechins, as well as purine alkaloids. Detection limits were generally on the order of 0.1–1.0 ng for most components at the applied wavelength of 280 nm. Method replication generally resulted in intraday and interday peak area variation of <6% for most tested components in green, Oolong, black, and pu-erh teas. Recovery rates were generally within the range of 92–106% with RSDs less than 4.39%. Therefore, advancement has been readily achievable with commonly used chromatography equipments in the present study, which will facilitate the analytical, clinical, and other studies of tea catechins.