Acidic Hydrolytic Stability of the Lifitegrast: Degradation, Method Development, Validation, Synthesis, LC–MS/NMR Characterization, In Silico and In Vitro Cytotoxicity

ABSTRACT Degradation products (DPs) in ophthalmic medications pose significant risks to ocular safety, necessitating comprehensive profiling and toxicological evaluation. This study presents a mass spectrometry‐compatible, stability‐indicating high performance liquid chromatography (HPLC) method for lifitegrast (LFT), utilizing 0.10% aqueous formic acid (A) and acetonitrile (B) with a gradient program: 0/30, 10/90, 15/90, 15.10/30, and 20/30 (time/%B). Acidic stress conditions yielded two major DPs: ( S )‐2‐(5,7‐dichloro‐1,2,3,4‐tetrahydroisoquinoline‐6‐carboxamido)‐3‐(3‐(methylsulfonyl)phenyl)propanoic acid (DP1) and benzofuran‐6‐carboxylic acid (DP2). The method was validated per International Conference on Harmonization (ICH) guidelines and is suitable for routine quality assessment of LFT. Quantum chemical calculations at the Becke, 3‐parameter, Lee–Yang–Parr (B3LYP)/6‐31+G(d) level evaluated the enthalpy of amide hydrolysis. DP1 was synthesized and confirmed by nuclear magnetic resonance spectrometry (NMR) (20 H, 20 C) and FT‐infrared (IR) spectroscopy (N─H: 3340.04 cm −1 , C─H: 3022.49 cm −1 , C═O: 1648.83 cm −1 ). DP2, procured and identified, showed six hydrogen and nine carbon atoms, with FT‐IR bands at 3435.45 cm −1 (O─H), 2925.75 cm −1 (C─H), and 1680.10 cm −1 (C═O). DEREK Nexus‐based toxicity predictions indicated structural alerts, and human corneal epithelial cell assays revealed cytotoxicity for DP2 at 8 µg/mL, whereas DP1 was toxic only at 1280 µg/mL. These findings highlight the importance of integrating degradation profiling with toxicological assessment to ensure ophthalmic drug safety.