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Objective To develop a simple and specific reversed-phase HPLC-UV method for simultaneous determination of(-)Epigallocatechin-3-gallate(EGCG)and(-)Epicatechin-3-gallate(ECG),the main active ingredients of tea polyphenols(TP),in rat plasma.Methods EGCG and ECG were eluted on a Kromasil C18 analytical column(150 mm×4.6 mm,5 μm)protected by a C18 pre-column(4.6 mm×20 mm,10 μm)with a linear gradient mobile phase composed of CH3CN(A)-0.1% citric acid(B),which was run from initial 14% A and 86% B to 20% A and 80% B at a flow rate of 1.0 mL·min-1 in 14 min,then changed to 14% A and 86% B at a gradient flow rate of 1.0-1.5 mL·min-1 during 14-18 min,and then maintained until 22 min at a gradient flow rate of 1.5-1.0 mL·min-1.The UV detector was set at 280 nm.Plasma samples(200 μL each)were prepared by liquid-liquid extraction procedure with double volumes of EtoAc and then evaporation of organic phase under N2 stream to dry,followed by reconstitution with 100 μL of 20% CH3CN aqueous solution.The peak area ratios of analytes to vanillin as internal standard vs concentration of analytes to construct calibration curves.Results The HPLC resulted in base-line separation of vanillin,EGCG,ECG and other components;there was no interference from blank plasma.The linear range was 0.5-300 μg·mL-1 for EGCG(r=0.9999)and 0.1-60 μg·mL-1 for ECG(r=0.9999).The intra-and inter-day precision(RSD)was better than 6.1% and 12.6%,respectively,and the average accuracy was between 86.25%-103.14%.The extraction recovery of EGCG and ECG was 79.80%-84.64% and 75.22%-91.39%,respectively.The plasma samples were stable for at least 30 days at-20 ℃ and 8 h at room temperature;EGCG,ECG and IS stock solutions 2 months at-20 ℃,and the EtoAc-extracted plasma samples 24 h at 4 ℃.Application of the method to the determination of EGCG and ECG in plasma of rats receiving iv 100 mg·kg-1 of TP showed that these 2 compounds pharmacokinetically behaved as the two-compartment model and first-order kinetics,with t1/2β 122.9 min and 59.2 min,Vd 7.96 L·kg-1 and 1.22 L·kg-1,CL 0.044 L·kg-1·min-1 and 0.015 L·kg-1·min-1 for EGCG and ECG,respectively.Conclusions The method developed in the present study is highly specific,precise,accurate,and suitable for the non-clinical pharmacokinetic study of the TP in rats.
Objective To develop a simple and specific reversed-phase HPLC-UV method for simultaneous determination of (-)Epigallocatechin-3-gallate (EGCG) and (-)Epicatechin-3-gallate (ECG), the main active ingredients of tea polyphenols ( TP), in rat plasma.Methods EGCG and ECG were eluted on a Kromasil C18 analytical column (150 mm×4.6 mm, 5 μm)protected by a C18 pre-column (4.6 mm×20 mm, 10 μm) with a linear gradient Mobile phase composed of CH3CN(A)-0.1% citric acid(B), which was run from initial 14% A and 86% B to 20% A and 80% B at a flow rate of 1.0 mL·min-1 in 14 Min,then changed to 14% A and 86% B at a gradient flow rate of 1.0-1.5 mL·min-1 during 14-18 min, and then maintained until 22 min at a gradient flow rate of 1.5-1.0 mL·min -1.The UV detector was set at 280 nm.Plasma samples(200 μL each)we prepared prepared by liquid-liquid extraction procedure with double volumes of EtoAc and then evaporation of organic phase under N2 stream to dry,followed by reconstitution with 100 μL Of 20% CH3CN aqueous so The lution. The peak area ratios of analytes to vanillin as internal standard vs concentration of analytes to construct calibration curves.Results The HPLC resulted in base-line separation of vanillin,EGCG,ECG and other components;there was no interference from blank plasma.The The linear range was 0.5-300 μg·mL-1 for EGCG (r=0.9999) and 0.1-60 μg·mL-1 for ECG (r=0.9999). The intra-and inter-day precision (RSD) was better than 6.1 % and 12.6%,respectively,and the average accuracy was between 86.25%-103.14%.The extraction recovery of EGCG and ECG was 79.80%-84.64% and 75.22%-91.39%,respectively.The plasma samples were stable for at least 30. Days at-20 °C and 8 h at room temperature;EGCG,ECG and IS stock solutions 2 months at-20 °C,and the EtoAc-extracted plasma samples 24 h at 4 °C.Application of the method to the determination of EGCG and ECG In plasma of rats receiving iv 100 mg·kg-1 of TP test that these 2 compounds pharmacokinetically behaved as the two-compartment model and first-order k Inetics,with t1/2β 122.9 minutes and 59.2 minutes, Vd 7.96 L·kg-1 and 1.22 L·kg-1, CL 0.044 L·kg-1·min-1 and 0.015 L·kg-1·min-1 for EGCG and ECG, respectively. Conclusions The method developed in the present study is highly specific, precise, accurate, and suitable for the non-clinical pharmacokinetic study of the TP in rats.