Comprehensive Forced Degradation Study of Pemigatinib and Structural Elucidation of Its Degradation Products Using Liquid Chromatography–High‐Resolution Mass Spectrometry and Nuclear Magnetic Resonance

ABSTRACT Pemigatinib (PEMAZYRE) is a selective fibroblast growth factor receptor 2 inhibitor approved for the treatment of cholangiocarcinoma. Understanding its degradation behavior is essential to ensure product quality, safety, and regulatory compliance. In the present study, the forced degradation behavior of pemigatinib (PMT) was systematically investigated under hydrolytic (acidic, basic, and neutral), oxidative, thermal, and photolytic stress conditions in accordance with International Council for Harmonization Q1A and Q1B guidelines. Before experimental evaluation, an in‐silico degradation profile of PMT was predicted using Zeneth software to support degradation pathway assessment. Chromatographic separation of PMT and its degradation products was achieved using a reversed‐phase high‐performance liquid chromatography (HPLC) method on a Phenomenex Gemini C18 column (250 × 4.6 mm, 5 µm) with a gradient elution employing methanol and 10 mM ammonium formate buffer (pH 6.5) as the mobile phase. Structural characterization of the degradation products was carried out using LC coupled with high‐resolution mass spectrometry (LC–HRMS). Pemigatinib was found to be stable under hydrolytic and thermal stress conditions, whereas significant degradation was observed under oxidative (hydrogen peroxide) and photolytic conditions, leading to the formation of five novel degradation products. Accurate mass measurements and tandem MS fragmentation data were used to propose the chemical structures and plausible degradation pathways of these degradation products. One major oxidative degradation product (DP‐3) was isolated and fully characterized using nuclear magnetic resonance spectroscopy. In addition, the oxidative degradation kinetics of PMT were evaluated and were found to follow second‐order reaction kinetics. Furthermore, in silico toxicity and mutagenicity assessments of the identified degradation products were performed using PROTOX‐3.0, SARAH Nexus, and DEREK Nexus to evaluate their potential safety risks. Overall, this study provides a comprehensive degradation profile of PMT and contributes valuable information for its stability assessment and quality control.