Quantitative Analysis of Four Catechins from Green Tea Extract in Human Plasma Using Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry for Pharmacokinetic Studies

Abstract

Green tea is consumed as a beverage worldwide and has beneficial effects, such as a lower risk of cardiovascular disease and cancer. A quantitative analysis of the beneficial components in plasma is important for understanding the potential health benefits of green tea. Four catechins—epigallocatechin-3-gallate (EGCG), epicatechin-3-gallate (ECG), epigallocatechin (EGC), and epicatechin (EC)—which account for the majority of the components of green tea, were analyzed by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). In this study, a validated method was optimized to obtain the blood concentrations after the one-time ingestion of 630 mg green tea extract with digoxin and then after the ingestion of 630 mg green tea repeatedly for 15 days. The calibration curve, including the LLOQ, was constructed over 1⁻500 ng/mL for EGCG, ECG, and EGC and 0.1⁻50 ng/mL for EC. The method for inter- and intra-validation was applied, acceptable for both accuracy and precision. We successfully developed an appropriate UPLC-MS/MS method for human plasma with good reproducibility and sensitivity. Thus, this method could be applied for future preclinical and clinical studies on EGCG, ECG, EGC, and EC.

Keywords: epicatechin (EC); epicatechin-3-gallate (ECG); epigallocatechin (EGC); epigallocatechin-3-gallate (EGCG); green tea; ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS).

Figure 1

Chromatograms of epigallocatechin-3-gallate, epicatechin-3-gallate, epigallocatechin, epicatechin and ethyl gallate. EGCG, epigallocatechin-3-gallate; ECG, epicatechin-3-gallate; EGC, epigallocatechin; EC, epicatechin. The selected chromatographs were representative of the highest quantitation limit.

Figure 2

Representative chromatograms of (A) double blank human plasma and (B) spiked with EGCG, EGC, EGC, and EC at the LLOQ. EGCG, epigallocatechin-3-gallate; ECG, epicatechin-3-gallate; EGC, epigallocatechin; EC, epicatechin. Signal-to-noise ratios for EGCG, ECG, EGC, and EC were 76.93, 20.69, 14.16 and 54.70, respectively in Figure 2B.

Figure 3

Mean plasma concentration-time profiles of four catechins after oral administration of 630 mg of green tea extract to healthy volunteers given once and continuously administered for 15 days. EGCG, epigallocatechin-3-gallate; ECG, epicatechin-3-gallate; EGC, epigallocatechin; EC, epicatechin. Day 1: Orally administered 630 mg of green tea extract with 0.5 mg digoxin in sixteen volunteers; Day 15: Repeated daily administration for 15 days of 630 mg green tea extract with a single administration of digoxin in fifteen volunteers.