Reverse Phase-High-Performance Liquid Chromatography (RP-HPLC) Method Development and Validation Using Analytical...

The primary objective of the present investigation is to develop and validate a simple, robust, and cost-effective isocratic reverse phase-high-performance liquid chromatography (RP-HPLC) method for determining isoliquiritigenin (ISL) in both bulk and biological samples using an analytical quality-by-design (AQbD) approach. The central composite design was employed for method optimization using Design Expert® software, by taking mobile phase ratio and flow rate as independent variables and peak area, retention time, tailing factor, and theoretical plates as dependent variables. The design suggested the use of a mobile phase consisting of acetonitrile:0.2% ortho-phosphoric acid (75:25, v/v) and a flow rate of 0.9 mL/min as optimal chromatographic conditions. The detection of ISL was performed at 364 nm. The optimized method was validated in accordance with International Conference on Harmonization (ICH) Q2(R1) guidelines. The method showed excellent linearity, limit of detection, limit of quantification, accuracy, precision, robustness, and system suitability. All validation parameters fell within the acceptable limits set by ICH. Additionally, the applicability of the method in biological samples were analyzed. In conclusion, the results suggest that the developed and validated AQbD-based RP-HPLC method was well-suited for the estimation of ISL in bulk and biological sample.