The analytical quality by design (AQbD) approach is utilized for developing and validating the simple, sensitive, cost-effective reverse-phase high performance liquid chromatographic method for the estimation of xanthohumol (XH) in bulk and nanoformulations. The Box-Behnken design (BBD) is applied for method optimization. The mobile phase ratio, pH and flow rate were selected as independent variables, whereas retention time, peak area, peak height, tailing factor, and theoretical plates were selected as dependent variables. The chromatogram of XH obtained under optimized conditions has given optimum conditions such as retention time (5.392 min), peak area (1,226,737 mAU), peak height (90,121 AU), tailing factor (0.991) and theoretical plates (4446.667), which are contoured in the predicted values shown by BBD. Validation of the method has been performed according to ICH Q2(R1) recommendations, using optimized conditions for linearity, limit of detection (LOD) and limit of quantification (LOQ), accuracy, precision, robustness and system suitability. All the values of validation parameters lie within the acceptable limits prescribed by ICH. Therefore, the developed and validated method of XH by the AQbD approach can be applied for the estimation of XH in bulk and various nanoformulations.
Keywords: AQbD; BBD; RP-HPLC; anticancer; beer; humulus; validation.