VALIDATED HPLC METHOD FOR SIMULTANEOUSQUANTIFICATION OF APREPITANT ANDITSEPIMPURITIES WITH LC-MS/MS ELUCIDATIONANDTOXICITY PREDICTION OF ITS FORCEDDEGRADATIONPRODUCTS

This present study is a validated, sensitive, and robust RP-HPLC method for real-time quantification of Aprepitant as well as its European Pharmacopoeia impurities A and B, and the proposed method was applied for acomprehensive forced degradation study through Liquid Chromatography Mass Spectrometry. Chromatographicseparation was optimized utilizing a Hypersil ODS C18 (250 × 4.6 mm, 5 µm) column with equal volume isocraticelution of solvent A (45 % acetonitrile, water and solvent B (70:30 v/v composition of 0.1% formic acid (aqueous) and methanol). This method achieves effective separation of aprepitant (Rt = 6.02 min), impurity A (Rt = 3.95 min), and impurity B (Rt = 8.02 min). The method exhibits excellent sensitivity and can detect impurities up to 0.045µg/mL. Forced degradation studies reveal that aprepitant was stable in most conditions but underwent significant degradation under alkaline stress, with the formation of five degradation products. The method resolves these newlyformed DPs at retention times of 1.907, 3.140, 4.740, 5.365, and 6.923 minutes, and these DPs were designatedasDP 1 to 5, respectively. This proposed method was applied to LC-MS/MS for structural characterization of DP1(487.45 m/z), DP 2 (509.39 m/z), DP 3 (412.31 m/z), DP 4 (396.31 m/z), and DP 5 (438.35 m/z) basedonfragmentation patterns. The in-silico toxicity assessment of characterized DPs indicates that the DPs possess varyingdegrees of organ-specific toxicities, such as neurotoxicity and respiratory toxicity. The study result highlights theneed for cautious monitoring, structural characterization, and control of these impurities during formulationdevelopment and stability studies to ensure patient and environmental safety.