Unique Quality by Design Approach for Developing High‐Performance Liquid Chromatography and Estimation of Rifaximin and Its Degradants by Mass Spectrometry

ABSTRACT A rapid and simple stability‐indicating assay method for High‐performance liquid chromatographic methods has been developed by studying the quality by design (QbD) principles and coupled with mass spectrometry for degradant product characterization. The method was systematically designed using the QbD approach and optimized via Box–Behenken design. Rifaximin is a nonabsorbable antibiotic belonging to the rifamycin class and is structurally similar to Rifampin. It inhibits Ribonucleic Acid (RNA) synthesis by interacting with the β‐subunit of bacterial deoxyribose nucleic acid‐dependent ribonucleic acid polymerase. A stability‐indicating assay method for rifaximin was developed and validated per the International Council for Harmonization (ICH) guidelines. Mass spectrometry was used for further characterization of formed degradants. Rifaximin and its degradation products were separated in isocratic elution mode. The mobile phase was methanol and acetonitrile in a (60:40 v/v ratio), 0.001% Triethyl amine was used to adjust pH 6, 1.0 mL/min was the flow rate, and all separations were detected at 293 nm. The Box–Behnken design identified and optimized critical method parameters (CMP), including the methanol percentage and pH. All the method validation parameters are within the acceptable range. The drug tR was found at 2.6 min. The statistical analysis shows a p value of 0.05, which means the model is significant, and the optimized model demonstrated a desirability of 1. The present study proved a specific, linear, precise, and accurate method in determining rifaximin and characterization of its degradation products.