Determination of theaflavins including methylated theaflavins in black tea leaves by solid-phase extraction and HPLC analysis.
A quantitative method for four theaflavins and two methylated theaflavin derivatives in black tea leaves was developed by solid-phase extraction and a high-performance liquid chromatographic method with photodiode array detection. The theaflavins in black tea leaves were extracted three times with 40 vol 50% aqueous ethanol (mg dry tea powder/mL) containing 2% ascorbic acid. The ethanol extracts were diluted 4-fold with distilled water. All diluted extracts were directly applied to the solid-phase C18 cartridge column without concentration. The fraction of theaflavins was obtained by 40% ethanol extraction after rinsing with water followed with 15% ethanol extraction. An aliquot of theaflavins after concentration was injected onto an ODS C18 reversed-phase column, and four theaflavins and two methylated theaflavins were sufficiently separated by a linear gradient system using distilled water and acetonitrile with 0.5% acetic acid. This analytical method is sensitive for the determination of a small amount of methylated theaflavins, since various interfering substances produced during the fermentation process were eliminated in advance by solid-phase extraction. Using this analytical method, we also demonstrated that methylated theaflavins were easily produced during the manufacture of black tea.
Nishimura
Masami
M
Division of Food Science and Nutrition, Nagoya Women's University, 3-40 Shioji-Cho Mizuho-ku, Nagoya 467-8610, Japan.
Ishiyama
Kinuko
K
Watanabe
Akiko
A
Kawano
Setsuko
S
Miyase
Toshio
T
Sano
Mitsuaki
M
D044946:Biflavonoids / Q000032:analysis* / Q000737:chemistry; D028241:Camellia sinensis / Q000737:chemistry*; D002392:Catechin / Q000032:analysis* / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D000431:Ethanol; D005285:Fermentation; D008745:Methylation; D018515:Plant Leaves / Q000737:chemistry*
D044946:Biflavonoids; C056068:theaflavin; D000431:Ethanol; D002392:Catechin