Simultaneous determination of the major active components of tea polyphenols in rat plasma by a simple and specific HPLC assay

A simple and specific HPLC assay for simultaneous determination of two major active components (−) epigallocatechin-3-gallate (EGCG), and (−) epicatechin-3-gallate (ECG) of tea polyphenols (TP) in rat plasma was developed and validated. Following addition of resorcinol as internal standard (IS) the analytes were isolated from rat plasma by liquid–liquid extraction with ethyl acetate. The chromatographic separation was achieved on a reversed-phase C18 column using an isocratic mobile phase consisting of 0.1% citric acid + CH3CN (86:14, v/v) running at flow rate of 1.5 mL/min. The effluent was monitored at a wavelength of 280 nm. EGCG, ECG and IS were well separated from each other and free from interference from blank plasma and other components in TP as well as metabolites post-dosing. The calibration curve was constructed by plotting peak area ratio of analytes to IS vs. concentration. The method showed good linearity over range of 0.5–300 μg/mL for EGCG and 0.1–60 μg/mL for ECG (r > 0.999). The intra- and inter-day precision (R.S.D.) was better than 6 and 12%, respectively. Assay accuracy was better than 94.78% for both compounds. Extraction recovery at QC samples was between 85.73 and 91.93% for EGCG and 79.08 and 86.51% for ECG. The developed method was successfully used to simultaneously measure plasma concentrations of EGCG and ECG after intravenous administration of TP to rats and yielded two typical biexponential decay concentration–time curves.