DEVELOPMENT AND VALIDATION OF A SENSITIVE LC-MS/MS ANALYTICAL METHOD FOR QUANTIFYING AXITINIB WITH AXITINIB D3 AS AN INTERNAL STANDARD

Objective: An efficient, highly sensitive, and robust LC-MS/MS method has been optimized, validated for accurately measuring Axitinib concentrations in human plasma, utilizing Axitinib D3 as an internal standard. Methods: A liquid–liquid extraction approach was utilized to isolate both the analyte and the internal standard from the plasma matrix. An Eclipse Phenyl column of 100 × 3.0 mm size with 5 µm particles operated using a mobile phase containing methanol mixed with 0.1% formic acid in 10 mM ammonium formate at a ratio of 60:40 (v/v) following the advanced bioanalytical framework for method establishment and validation. Results: The complete chromatographic run was accomplished in 4.0 minutes, with the analyte and internal standard eluting at approximately 1.89 minutes. The assay demonstrated a validated linear response across the concentration range of ~ 0.2 to 125 ng/mL. The developed method was thoroughly validated for key parameters including precision, accuracy, selectivity, matrix effects, and stability. All validation results met the predefined specifications, confirming method's reliability. Conclusion: The validated method for quantifying Axitinib in human plasma complies with regulatory guidelines set by the FDA and EMA, making it appropriate for pharmacokinetic studies in human subjects.