Simultaneous Determination of Black Tea-Derived Catechins and Theaflavins in Tissues of Tea Consuming Animals Using Ultra-Performance Liquid-Chromatography Tandem Mass Spectrometry

Abstract

The bioavailability, tissue distribution and metabolic fate of the major tea polyphenols, catechins and theaflavins as well as their gallated derivatives are yet to be precisely elucidated on a single identification platform for assessment of their relative bioefficacy in vivo. This is primarily due to the lack of suitable analytical tools for their simultaneous determination especially in an in vivo setting, which continues to constrain the evaluation of their relative health beneficiary potential and therefore prospective therapeutic application. Herein, we report a rapid and sensitive Ultra-Performance Liquid Chromatography Tandem Mass Spectrometry (UPLC-MS/MS) based method for the simultaneous determination of the major catechins and theaflavins in black tea infusions as well as in different vital tissues and body fluids of tea-consuming guinea pigs. This method allowed efficient separation of all polyphenols within seven minutes of chromatographic run and had a lower limit of quantification (LLOQ) of ~5 ng/ml. Using this method, almost all bioactive catechins and theaflavins could be simultaneously detected in the plasma of guinea pigs orally administered 5% black tea for 14 days. Our method could further detect the majority of these polyphenols in the lung and kidney as well as identify the major catechin metabolites in the urine of the tea-consuming animals. Overall, our study presents a novel tool for simultaneous detection and quantitation of both catechins and theaflavins in a single detection platform that could potentially enable precise elucidation of their relative bioavailability and bioefficacy as well as true health beneficiary potential in vivo. Such information would ultimately facilitate the accurate designing of therapeutic strategies utilizing high efficacy formulations of tea polyphenols for effective mitigation of oxidative damage and inflammation in humans as well as prevention of associated diseases.

Fig 1. Detection of Catechins and Theaflavins in Plasma Spiked with Individual Polyphenols.

Representative MRM chromatograms of epigallocatechin (EGC), epicatechin (EC), epigallocatechin-3-gallate (EGCG), epicatechin-3-gallate (ECG), theaflavin (TF), theaflavin-3-monogallate (TF3G), theaflavin-3,3'-digallate (TF33'diG), theaflavin-3'-monogallate (TF3'G), in blank plasma (control) spiked with the polyphenol standards to the final concentration of their LLOQ, showing the retention time (labelled below the analyte name) and response (labelled below the retention time) of the analytes (analyte names highlighted against their corresponding chromatograms). The right-hand top labels on the chromatograms indicate their respective MRM transitions and peak heights. Data are representative of three independent experiments done under similar conditions.

Fig 2. Detection and Identifiction of Catechin Metabolites in the Urine of Tea-Consuming Guinea Pigs.

Representative chromatograms for the identification of catechin metabolites in the urine of tea consuming guinea pigs analysed by precursor ion scan showing epigallocatechin glucuronide (EGC-glucuronide), epicatechin glucuronide (EC-glucuronide), o-methyl epigallocatechin glucuronide (o-methyl EGC glucuronide), epigallocatechin sulphate (EGC-sulphate), epicatechin sulphate (EC-sulphate), o-methyl epigallocatechin sulphate (o-methyl EGC-sulphate), o-methyl epicatechin sulphate (o-methyl EC-sulphate) as the primarily identifiable metabolites. The chromatograms also depict the retention times (RT) (highlighted corresponding to the monitoring ions) and the intensity count (highlighted at the right hand side of the chromatograms) of the identified metabolites. Data are representative of three independent experiments done under similar conditions.