Stress Degradation Behavior of Entacapone and Development of LC Stability-Indicating Related Substances and Assay Method

A new, sensitive, stability indicating gradient RP-LC related substances and assay method has been developed for the quantitative determination of entacapone in bulk drugs. Efficient chromatographic separation was achieved on a Cl 8 stationary phase with simple mobile phase combination of buffer and acetonitrile. Buffer consisted of 0.1% orthophosphoric acid, delivered in a gradient mode and quantitation was carried out using ultraviolet detection at 220 nm with a flow rate of 1.5 mL min(-1). In the developed LC method the resolution (R-s) between entacapone and its three potential process impurities were found to be > 2.0. Regression analysis showed an r(2) value (correlation coefficient) >0.99 for entacapone and its three potential impurities. This method was capable to detect all three process impurities of entocapone at a level of 0.003% with respect to test concentration of 0.5 mg mL(-1) for a 20 mu L injection volume. The inter- and intra-day precision valves for all three impurities and for entacapone was found to be within 2.0% RSD. The method has shown good and consistent recoveries for entacapone in bulk drugs (99.2-101.5%) and its three impurities (99.5-102.2%). The test solution was found to be stable in diluent for 48 h. The drug substances were subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. Considerable degradation was found to occur in acid stress, base stress and oxidative conditions. The stressed test solutions were assayed against the qualified working standard of entacapone and the mass balance in each case was close to 99.7% indicating that the developed method was stability-indicating. The developed RP-LC method was validated with respect to linearity, accuracy, precision and robustness.