Optimization and Validation of Reversed Phase High Performance Chromatography Method for Rapid Estimation of Theaflavin

Abstract

The manuscript deals with the optimization of simple, rapid, sensitive and economic HPLC method for estimation of Theaflavin in Camellia sinensis extract and Theaflavin loaded nanoformulation. The preliminary screening was performed employing fractional factorial (2^5-1) design with mobile phase pH, flow rate, column temperature, injection volume, and acetonitrile concentration as independent variables while tailing factor as the dependent variable. The separation was achieved using the isocratic system as mobile phase, flow rate of 1 ml/min, and ultraviolet detection at 275 nm. Upon further evaluation, the method revealed a high degree of linearity, accuracy, precision, sensitivity and robustness, while, forced degradation studies indicated stability of Theaflavin at pH 3-6 in an air tight, light-protected container. The method when applied towards estimation of Theaflavin in C. sinensis extract and Theaflavin loaded nanoformulation, indicated no significant change in retention time. Thus, a HPLC method for rapid estimation of Theaflavin with an understanding of influencing variables for enhancing the method performance was developed.

Keywords:

• Theaflavin
• HPLC
• fractional factorial design
• isocratic method
• nanoformulation