Development and Validation of a Gradient RP-HPLC Method for Quantitative Determination of Potential Manufacturing Impurities in New Drug Apronalide and Pharmaceutical Formulations

Abstract

Three impurities in apronalide drug substance were detected and quantified using a simple isocratic reverse phase HPLC method. The chromatographic separation was achieved on a Thermo Hypersil gold-CN, 100 A⁰ 5 µm, and 250X4.6 mm using a mobile phase consisting of phosphate buffer (pH-4.6) - Methanol with UV detection at 210 nm and flow rate of 0.8 ml/min. The column temperature was maintained at 25⁰C throughout the analysis. This method was rapid, simple for separation of apronalide and potential manufacturing impurities from each other in a 30 min run time.  To the best of our knowledge, none of the method has been reported in the any pharmacopeia and literature for separation of apronalide from the given set of related substances. The proposed method has been validated as per international guidelines on method validation and can be used for the routine quality control analysis of apronalide as active pharmaceutical ingredient (API) and pharmaceutical formulations. Also the method was determined to be robust with regards to the following parameters: mobile phase apparent pH; mobile phase organic content; detection wavelength and time dependence of sample and standard stability. 

Keywords: Apronalide, related Substances, HPLC, pharmaceutical formulation, drug