Development, validation, and mass characterization of enalapril and its impurities with integrated in silico safety assessment

Enalapril is one of the most commonly used angiotensin converting enzyme inhibitors. However, there is little information available on impurities of enalapril in terms of their chromatographic properties, stability behavior, safety prediction, and greenness of the analytical methods used for such characterization. Hence, the current work was intended to develop an analytical approach for the identification and characterization of impurities of enalapril. The AQbD-based stability-indicating RP-HPLC assay was developed for the simultaneous determination of enalapril, enalapril impurity B and enalapril Impurity F in tablets. The method employed a mobile phase consisting of 10 mM phosphate buffer (pH 4.5) and acetonitrile in a 75:25 (v/v) ratio. Forced degradation studies were conducted under various stress conditions, and the resulting degradation products were characterized by mass spectrometry using positive electrospray ionization (ESI) mode to elucidate their structures. Moreover, in silico ADME and toxicity studies were carried out through SwissADME and ProTox-II respectively. The RP-HPLC method formulated was able to provide good resolution of enalapril and the impurities with reasonable validation parameters. The forced degradation study helped determine the selectivity and stability-indicating nature of the method, whereas mass spectrometric analysis gave insight into the structure of impurity B and impurity F. In silico analysis helped predict the physicochemical properties and pharmacokinetic properties of the identified impurities, where the ProTox-II analysis gave warnings about potential toxicity for nephrotoxicity and respiratory toxicity pathways for some impurities. The suggested analytical workflow combines chromatographic optimization, impurity identification, computational toxicity prediction, and sustainability analysis into one workflow. This method allows obtaining a holistic approach to the identification of enalapril impurities and can be used for the analysis of quality attributes of drugs and their associated risks.