A Validated Stability-Indicating RP-HPLC Assay Method for Amsacrine and its Related Substances

Abstract

A validated specific stability indicating reversed-phase high-performance liquid chromatography method was developed for the quantitative determination of Amsacrine as well as its related substances determination in bulk samples, in presence of degradation products, and its process related impurities. Forced degradation studies were performed on bulk samples of Amsacrine as per International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human use (ICH) prescribed stress conditions using acid, base, oxidative, thermal stress, and photolytic degradation to show the stability indicating power of the method. Significant degradation was observed during basic hydrolysis, slight degradation was observed in oxidative and thermal stress, and no degradation was observed in other stress conditions. The chromatographic method was optimized using the samples generated from forced degradation studies and the impurity spiked solution. Good resolution between the peaks corresponds to process-related impurities and degradation products from the analyte were achieved on Inertsil ODS column using the mobile phase consists a mixture of 1.0% triethyl amine in 20 mM potassium dihydrogen orthophosphate, with pH adjusted to 6.5, with ortho phosphoric acid in water and acetonitrile using a simple linear gradient. The detection was carried out at wavelength 248 nm. The mass balance in each case was in between 99.4% to 99.9%, indicating that the developed method was stability-indicating. Validation of the developed method was carried out as per ICH requirements. The developed method was found to be suitable to check the quality of bulk samples of Amsacrine at the time of batch release and also during its stability studies.