AQbD Enabled Stability Indicating Simultaneous Quantification of Berberine, Rutin and Eugenol in Herbal Formulation by RP-HPLC

The present research describes a stability indicating RP-HPLC technique for simultaneous quantification of Berberine, Rutin and Eugenol in herbal formulation by using Analytical Quality by Design (AQbD) approach. AQbD based HPLC method is crucial for quality evaluation of herbal formulation over traditional method of HPLC, to develop more robust method by considering simultaneous influence of multiple factors on the developed method. The Analytical target profile (ATP) and Critical Method Parameters (CMPs) were determined. Risk assessment and factor screening studies enabled to detect Critical Method Responses (CMRs). Three factors at three levels were considered such as % Organic phase (CMP1), Flow rate (CMP2) and pH (CMP3) were assessed by Central Composite Design (CCD) for method optimization. Statistical data (ANOVA, Regression coefficient) and graphical analysis of data such as normality plots, contour plots and 3D response surface plots to assess the individual and combined interaction effects of CMPs on CMRs. The optimized mobile phase was Methanol: 0.1% Orthophosphoric acid pH 2.5 (50:50% v/v) with 0.9ml/min rate of flow, at 217nm. Berberine was eluted at 2.958+0.1 min, Rutin and Eugenol were 3.845+0.2 min and 4.587+0.1min respectively with 8 min run time. The developed method was validated in compliance with ICH Q2 R1 guidelines and performed stress degradation studies, all the parameters were within the limits. Moreover, the established HPLC technique was effectively utilized to quantify the Berberine, Rutin and Eugenol in an herbal formulation (Clear stone drops).