Validated HPLC Method for the Simultaneous Quantification of Hydrochlorothiazide and Metoprolol in Pharmaceutical Tablets

Abstract The development and validation of a robust, accurate and precise Reverse Phase High Performance Liquid Chromatography (RP-HPLC) method for Simultaneous Estimation of Hydrochlorothiazide and Metoprolol in tablet formulations has been made. Hypersil ODS C18 column (250 mm × 4.6 mm, 2.5 µm particle size) and mobile phase of acetonitrile and 20 mM ammonium acetate buffer (pH 3.5, 45 and 55 parts of volume) at isocratic condition was used for chromatographic separation. Finally, the hydrochlorothiazide and metoprolol retention times were found to be 3.12 min and 4.93 min, respectively. The method was shown to have excellent linearity (R² > 0.999), precision (<1.18% RSD), accuracy (99.65–101.72%) and robustness under minor chromatographic parameters variation as per ICH guidelines. A statistical analysis using design of experiments (DoE) was performed to systematically evaluate the effect of critical chromatographic factors on system suitability parameters. The reliability of the method was supported by the values of % RSD, tailing factor (<1.22), and theoretical plates (>5600). At 80%, 100% and 120% spiked levels recovery studies yielded values within acceptable limits, and the quantification was found to be accurate. The method is proven to be a good routine quality control, stability analysis, and formulation development method for pharmaceutical products consisting of Hydrochlorothiazide and Metoprolol. Keywords: RP-HPLC, Hydrochlorothiazide, Metoprolol, Method Validation, Pharmaceutical Analysis, Stability Study.