Chemiluminescence–High-Performance Liquid Chromatographic Determination of Tea Catechin, (−)-Epigallocatechin 3-Gallate, at Picomole Levels in Rat and Human Plasma

Abstract

A method of chemiluminescence detection–high-performance liquid chromatography (CL–HPLC) for the highly specific determination of tea catechin, (−)-epigallocatechin 3-gallate (EGCg), present in rat and human plasma has been newly developed. The CL–HPLC system consists of reversed-phase HPLC and chemiluminescence detector, in which separated EGCg generates chemiluminescence at post column successively reacting with the following two chemiluminescence cocktails; 8.2macetaldehyde in 50 mmphosphate buffer (pH 7.4, contained 108 mg horseradish peroxidase/L) and 8.8mhydrogen peroxide aqueous solution. The plasma EGCg was extracted by methanol. This method enables the detection of EGCg in the free form selectively at as low as 2 pmol with recovery of 84%. The EGCg concentration in fasted rat plasma was initially below the detection limit (<2 pmol/ml), but increased to maximum level (2284 pmol/ml plasma, 1047 ng/ml; calculated 0.012% of ingested EGCg) 30 min after a single oral supplementation of 56 mg EGCg per rat. The EGCg concentration in fasted human plasma was also initially below the detection limit and increased to 341 pmol/ml (156 ng/ml; calculated 0.32% of ingested EGCg) at 60 min after a single oral intake of 97 mg EGCg per subject. The results indicated that tea catechin, EGCg, is absorbed from the digestive tract into the rat and human body and that the CL–HPLC method reported here should be a powerful tool for studying the metabolic fate and bioavailability of EGCg.