Chemiluminescence-high-performance liquid chromatographic determination of tea catechin, (-)-epigallocatechin 3-gallate, at picomole levels in rat and human plasma

Analytical Biochemistry
K Nakagawa, T Miyazawa

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; 8.2 M acetaldehyde in 50 mM phosphate buffer (pH 7.4, contained 108 mg horseradish peroxidase/L) and 8.8 M hydrogen 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...Continue Reading

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