Ultra high-Performance Liquid Chromatography (UHPLC) method development and validation for the identification of oxidized product of Epigallocatechin-3-Gallate (EGCG).

Abstract

In the present study we reported oxidation of epigallocatechin-3-gallate (EGCG) and validation of oxidized product by a validated ultra high-performance liquid chromatography (UHPLC) method. Two hundred milligrams of EGCG was oxidized in 5mL of hydrogen peroxide (H2O2) and was identified by a validated UHPLC method with precision and robustness. Confirmation of parameters like C-H stretching and mass was carried out using infrared spectroscopy and mass spectroscopy, respectively. Identification of oxidized EGCG (O-EGCG) was done by UHPLC. The infrared spectroscopy chromatograms observed less intensity C-H stretching as compared to O-EGCG. The mass of EGCG and O-EGCG were 459.09 and 915.16, respectively. Structure elucidation revealed a loss of one proton in O-EGCG as compared to EGCG. Validation of the developed method was specific, with linear correlation coefficient 0.9981 and 0.9917, respectively for EGCG and O-EGCG, the accuracy rate of 95.2%-99.6% for EGCG, and 99.18%-101.5% for O-EGCG. Together, the results of this study demonstrate the formation of a dimer also the UHPLC method developed for identification of both EGCG and O-EGCG is validated as per the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines.