Identification and Structural Characterization of Degradation Products of Tivozanib Using LC‐Q‐TOF‐MS/MS and NMR: An In Silico Approach for Degradation and Toxicity Prediction

Rationale The present study investigated the degradation behaviour of tivozanib (TVZ), a potent tyrosine kinase inhibitor, under various stress conditions, including hydrolytic (acid, base, and neutral), oxidative, thermal, and photolytic (UV and visible light) conditions. The resulting degradation products (DPs) were characterized by LC‐Q‐TOF‐MS/MS and NMR. Additionally, in silico toxicity assessment and mutagenicity studies were accomplished by using DEREK Nexus and SARAH Nexus for TVZ and its DPs. Methods The in silico degradation profile of TVZ was predicted using Zeneth software. Separation of the DPs was executed by RP‐HPLC with gradient elution mode using Shim‐pack Solar C18 column (250 × 4.6 mm, 5 μm). The mobile phase consists of solvent A as 10 mM ammonium acetate buffer (pH 3.8) and solvent B as a mixture of acetonitrile:methanol (70%:30% v/v). Structural characterization was performed for TVZ and its DPs using LC‐Q‐TOF‐MS/MS and NMR spectroscopy. Results TVZ was sensitive to hydrolytic (acid, base, and neutral), oxidative, and photolytic (UV light) degradation conditions, wherein seven DPs were observed and separated by RP‐HPLC. A plausible degradation pathway of TVZ was proposed based on mass spectrometry data. The preparative HPLC was used to isolate DP‐III, DP‐IV, and DP‐VI, and the structures were confirmed by NMR spectroscopy. Furthermore, the 2D NMR data helped to differentiate the chemical structure of DP‐IV, which had the same molecular mass as TVZ. Conclusion Forced degradation studies were conducted in accordance with ICH Q1A (R2) and Q1B guidelines. Totally, seven DPs were observed in various stress conditions. DP‐IV, a structural isomer of TVZ, was detected in basic hydrolytic conditions. The developed RP‐HPLC method was validated following the ICH Q2(R1). This study helps in the stability testing and routine quality control analysis during the formulation development of TVZ.