Development and validation of a liquid chromatographic method for the analysis of squaric acid dibutyl ester and its...

A simple, fast and selective stability indicating liquid chromatographic method has been described for the simultaneous determination of squaric acid dibutyl ester and its impurities. The chromatographic separation was achieved on a C₂ column (250mm×4.6mm i.d., 5μm) using a mobile phase consisting of 0.15% phosphoric acid - acetonitrile - methanol (30:60:10, v/v/v). Isocratic elution was performed at a flow rate of 1.0mLmin^-1. The analytes were detected by UV at 252nm. The method was validated according to the ICH guidelines and satisfactory results were obtained. The specificity of the developed method was tested using forced degradation solutions of the drug substance. Characterization of squaric acid dibutyl ester and its forced degradation products was achieved by coupling mass spectrometry (MS) to the liquid chromatographic (LC) system. The method was successfully applied for quality control purposes including assay and determination of related compounds as required by regulatory guidelines to ensure its safety and efficacy since no monograph is available in official compendia.