IMID‐2, a newly identified piperazine‐based anticancer molecule, has been shown to be cytotoxic against various cancer cell lines. The primary aim of this research was to identify and characterize possible metabolites of the molecule formed during biotransformation. A metabolite identification study was first executed using an in silico tool to predict the possible metabolism sites of IMID‐2. Thereafter, metabolites generated in vitro (rat liver microsomes, rat S9 fractions and human liver microsomes) and in vivo (rat plasma, urine and feces) were identified and characterized employing UPLC–QTOF–MS/MS. A total of eight metabolites, among which were six in phase I and two in phase II reactions, were recognized. The plausible structure of the metabolites and probable metabolic pathway have been established based on the mass fragmentation pattern, mass ppm error, ring double bond calculation and nitrogen rule. The majority of phase I metabolites were generated by N‐oxidation, hydroxylation, oxidative deamination followed by reduction, oxidative dechlorination, N‐dearylation, and N‐dealkylation. Glucuronidation played a significant role in the formation of phase II metabolites of the molecule.

Aim: The current research aims to establish a stability-indicating analytical method (SIAM) for the quantification of evodiamine (EVO), characterization of its degradation impurity, and establishment of possible degradation pathways. Background: None of the degradation impurities of EVO is known and the mechanism of their formation has not been reported in any literature to date. Moreover, a SIAM for EVO is not available in any public domain. Objective: The objective of this study is to characterize the degradation impurity of EVO by LC-MS/MS, proposing its molecular structure, identifying possible degradation pathways of generation of its impurity, and establishing a SIAM. Method: To assist future product development, a degradation study of EVO was performed and an RP-HPLC-based SIAM was developed. The major degradation product was characterized by LC-Q-TOF-MS/MS. In addition, in silico toxicity prediction was performed using the ProTox-ІI toxicity predictor. Result: The method was found to be linear, accurate, precise, and robust over the range of 12.5 to 100 µg /mL of EVO. The method met all the acceptance criteria as specified in the ICH guideline. Only one degradation product (9% of the drug area) of EVO was generated in acidic hydrolytic conditions. The degradation product was found to be potentially inactive for hepatotoxicity and immunotoxicity, with a confidence score of more than 0.7 (70%). Moreover, the confidence score for carcinogenicity, mutagenicity, and cytotoxicity was less than 0.7, indicating it was moderately inactive for these toxicities. Conclusion: The molecule was found to be stable in the majority of the tested stress conditions. However, the degradation product generated in acidic hydrolytic stress was characterized using LC-Q-TOF-MS/MS, which was unknown to date. The novelty of this research can be justified by the unavailability of any SIAM of EVO and the absence of any report on its susceptibility to degradation in the presence of different potential stressors. Moreover, the potential toxicity of the molecule and its impurity was not known previously. The reported degradation impurity may be useful to set the quality control acceptance criteria for EVO. Additionally, pharmaceutical industries and research laboratories may use the developed method for the analysis of quality control and stability samples of EVO.