LC-MS/MS Identification of Ozanimod Degradation Products and Development of a Green HPLC Method for Impurity Quantification

A green high-performance liquid chromatography (HPLC) method was developed and validated for the quantification of Ozanimod and its impurities, addressing the lack of environmentally friendly approaches in the literature. The method utilizes ethanol and aqueous trifluoroacetic acid (TFA) (70:30 v/v) as the mobile phase, with a Waters XBridge C18 column (150 × 4.6 mm, 5 μm), a 0.7 mL/min flow rate, and 270 nm detection. Method validation demonstrated acceptable linearity, accuracy, precision, and sensitivity for Ozanimod and its impurities. A forced degradation study identified three degradation products (DPs) under acidic and basic stress conditions, designated as DP 1, DP 2, and DP 3. Structural elucidation using LC-MS/MS and MSⁿ fragmentation analysis confirmed their identities. The green assessment of the method was conducted using GAPI (Green Analytical Procedure Index) and AGREE (Analytical GREEnness) tools, confirming a significant reduction in hazardous solvent usage while maintaining excellent chromatographic performance. This study introduces a validated, eco-friendly HPLC method for the quantification of Ozanimod and its impurities, along with structural characterization of its DPs, making it a sustainable and reliable approach for routine pharmaceutical analysis and stability studies.