Development and Validation of a Stability-Indicating RP-HPLC Method for Simultaneous Determination of Metronidazole Benzoate, Diloxanide Furoate, Methylparaben and Propylparaben in Pharmaceutical Suspensions

Abstract

A validated reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed for the simultaneous identification and quantification of metronidazole benzoate, diloxanide furoate, methylparaben, and propylparaben in combined pharmaceutical suspensions. Chromatographic separation was achieved using a C18 column with an isocratic mobile phase consisting of ammonium acetate buffer (pH 7.0), methanol, and acetonitrile in an optimised ratio, with photodiode array detection at 254 nm. All analytes were efficiently separated within 12 min, with satisfactory peak symmetry and resolution. The method was validated according to the ICH Q2 (R1) guidelines. Excellent linearity was obtained over the investigated concentration ranges, with correlation coefficients (R²) greater than 0.999. The method demonstrated high accuracy, with recovery values ranging from 98% to 102%, and good precision, with percentage relative standard deviation (%RSD) values of ≤1.1%. The limits of detection (LOD) and quantification (LOQ) ranged from 0.000075 to 0.009530 mg/mL and from 0.000226 to 0.028576 mg/mL, respectively, confirming the adequate sensitivity of the method. Specificity was demonstrated by the absence of interference from formulation excipients and the successful separation of analyte peaks from degradation products during forced degradation studies. The developed method demonstrated reliable analytical performance in terms of accuracy, precision, robustness, and stability-indicating capability. Therefore, the method provides a simple, rapid, and practical approach for the routine quality control and stability assessment of pharmaceutical suspensions containing metronidazole benzoate, diloxanide furoate, methylparaben, and propylparaben.