Development and Validation of a Novel Bioanalytical Method for Estimating Epigallocatechin 3 Gallate in Wistar Rat Plasma by RP-HPLC Employing Gradient Elution Techniques

Year 2023, Volume: 27 Issue: 3, 1039 - 1055, 28.06.2025

Varsha Mane , Suresh Killedar Harinath More Sachin Salunkhe Harshal Tare

Abstract

The goal of this study was to provide a new, easy, accurate, cost-effective, exact, sensitive, specific, robust, and rugged method for quantifying Epigallocatechin 3 gallate in wistar rat plasma using reverse phase high-performance chromatography (RP-HPLC). The stationary phase was a Zorbax SB C18 5μ (4.6*150) mm column, while the mobile phase was water with 0.1 percent formic acid (A) and acetonitrile (ACN) with 0.08 percent formic acid (B). The experiment was conducted at 30°C with a flow rate of 1.0 ml/min with PDA detectors at 274 nm. With an r2 of 0.9999, the method was shown to be linear in the concentration range of 0.2–25 μg/ml. At the same retention time (Rt) of epigallocatechin 3 gallate, no interference of co-eluting peaks of endogenous chemicals from the biological matrix was found. The intraday and interday precision RSD (%) was found to be within acceptable limits (less than 2%). The overall mean recovery percentage was determined to be 96.92 %. The LOD and LOQ were determined to be 0.0682 ± 0.0011 μg/ml and 0.205 ± 0.004 μg/ml, respectively. In short-term and long-term stability tests, auto sampler, bench-top, and freeze-thaw stability tests were found to be stable. The developed approach reported was determined to be well within acceptable limits. As a result, in the future, this method can be successfully employed in clinical laboratories to estimate epigallocatechin 3 gallate alone or in conjunction with other analytes or markers in pharmacokinetic, bioequivalence, and therapeutic drug monitoring.

Keywords

Epigallocatechin 3 gallate , Wistar rat plasma , RP-HPLC , gradient elution , bioanalytical method , validation

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