Box-Behnken Design-Assisted Development and Validation with Stability Study of RP-HPLC Method for Analyzing ۱-(۴-Chlorophenyl) pyrrolidine-۲,۵-dione Using Analytical Quality-by-Design Approach

ABSTRACT-Herein, we present the development and validation of a Reverse Phase High-Performance Liquid Chromatography (RP-HPLC) method for the analysis of ۱-(۴-chlorophenyl) pyrrolidine-۲,۵-dione (CPS), an important intermediate and active pharmaceutical ingredient. CPS, also known as chlorophenyl pyrrolidine succinimide, consists of a chlorophenyl group, which enhances lipophilicity and receptor binding; a pyrrolidine ring, contributing to structural stability; and a succinimide moiety, which facilitates biological activity. The Box–Behnken Design (BBD), a type of response surface methodology, was employed to optimize chromatographic conditions, including the mobile phase composition, flow rate, detection wavelength, and other parameters, with a strong focus on sensitivity, clarity, accuracy, and robustness. The optimized RP-HPLC method achieved a favorable retention time of ۴.۵۶۷ minutes for CPS at a detection wavelength of ۲۲۲ nm. The method demonstrated excellent linearity (r² = ۰.۹۹۸۸), good accuracy (recovery range: ۹۸–۱۰۲%), and precision (%RSD < ۲%). The specificity of the method was confirmed by the absence of interference from excipients and degradation products. The limits of detection (LOD) and quantification (LOQ) were calculated to be ۰.۱۸۳۷ ppm and ۰.۵۵۶۹ ppm, respectively. Stability studies under various stress conditions demonstrated that the method is stability-indicating and capable of separating CPS from its degradation products. This study highlights the effectiveness of the Box–Behnken Design in the systematic development of an RP-HPLC method and underscores the importance of the Quality by Design (QbD) approach in ensuring method reliability and regulatory compliance. Accurate, precise, and reproducible results were obtained by strictly adhering to ICH guidelines.

کلیدواژه ها:

RP-HPLC method ، Box-Behnken design ، Stability-Study ، Method Development ، Validation ، Succinimide