An Improved approach for Creating and Validating a Bioanalytical Method to Quantify Baloxavir Marboxil in Human Plasma using LC-API-MS/MS

This study describes a new method for accurately measuring baloxavir marboxil (BXM) in human plasma using LC-API-MS/MS. We employed baloxavir marboxil d4 as an internal standard for precise quantification. BXM and the internal standard were isolated from plasma using tert-butyl methyl ether (TBME) extraction. The LC system separated BXM and the internal standard on a Zorbax SB C18column, and tandem mass spectrometry (MS/MS) detected them with minimal interference from human plasma components. The method showed excellent linearity (R²≥0.9956) across a wide concentration range (10.610–1229.080ng/mL). BXM stability was evaluated under various conditions, including room temperature, injector storage, freeze-thaw cycles, and long-term storage at -70°C. The method was validated according to USFDA guidelines, demonstrating its effectiveness for quantifying BXM in human plasma for pharmacokinetic studies.