Box Behnken Design Assisted RP-HPLC Method Development and Validation of Phytomarker Zingerone in Polyherbal Formulation

Abstract

A robust, precise, and accurate reverse-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the quantification of zingerone using an Analytical Quality by Design (AQbD) approach. The Box-Behnken Design (BBD) was employed to optimize three critical method parameters: mobile phase composition, flow rate, and injection volume. The dependent variables Retention time, Theoretical plate and tailing factor were considered as critical method response. The chromatographic separation was achieved using a C18 column (150mm x 4.5mm x 5µm) with a mobile phase consisting of Acetonitrile: Ortho phosphoric acid 0.1% pH 3 (28:72), UV detection wavelength optimized at 282 nm. The developed method was validated according to ICH Q2 (R2) guidelines and demonstrated excellent analytical performance. The method showed good linearity over the concentration range of 50–150 µg/mL with a correlation coefficient (R²) of 0.9968. Precision studies showed % RSD less than 2%, indicating good repeatability and reproducibility. Accuracy was confirmed through recovery studies, with mean recovery of 100.83%. The method also showed good robustness under small deliberate variations in chromatographic conditions. The limits of detection and quantification were found to be 0.532 µg/mL and 1.613 µg/mL respectively, indicating high sensitivity. This study confirms that the developed method is suitable for routine analysis of zingerone in herbal formulations.