Optimized and Validated Box–Behnken Design Combined Response Surface Methodology to Quantify Acetic Acid Content in Potassium Clavulanate Using High–Performance Liquid Chromatography

Abstract

A simple and robust high-performance liquid chromatography method with UV detection was developed to quantify acetic acid in potassium clavulanate. The chromatographic separation was performed on the Hypersil BDS C18 column (250×4.6 mm, 5 μm) with optimized response surface methodology combined Box–Behnken experimental design. The mobile phase comprises of a buffer prepared by ammonium dihydrogen orthophosphate (20 mM) containing 0.01% triethylamine, maintained pH between 2.8–3.2 using phosphoric acid and % acetonitrile as per Box–Behnken factorial design. The detection was performed at 235 nm with a mobile phase flow rate of 1 ml/min. The method was validated as per the ICH guidelines. The linearity of the proposed method was rectilinear within the range of 2.5–24.8 μg/ml. The value of the limit of detection (LOD) & limit of quantitation (LOQ) by the validated method was found to be 1.9 and 5.8 μg/ml, respectively. The robustness and ruggedness studies were conducted by small changes with parameters in the proposed method. The % RSD values were within the accepted limit of criteria. This developed method can quantify routine acetic acid content analysis in potassium clavulanate pharmaceutical formulations.