Development and Validation of Liquid Chromatography Tandem Mass Spectrometry Method for Baloxavir Estimation in human plasma

A method for quantifying Baloxavir in human plasma using liquid chromatography-tandem mass spectrometry (LCMS/MS) with Baloxavir-d5 as an internal standard was detailed. Sample preparation involved liquid-liquid extraction, and chromatographic separation utilized an Acquity UPLC Peptide BEH C18 Column (300Å, 1.7µm, 2.1mm x 150mm). Positive mode monitoring of multiple reaction monitoring (MRM) transitions included m/z 484.1→247.0 for Baloxavir and m/z 489.1→252.1 for Baloxavir-d5. Sample concentrations were determined through linear regression analysis using Analyst software version 1.7.2, demonstrating an excellent linear response within the concentration range of 0.505 to 302.724 ng/ml for Baloxavir. Intra-day and inter-day precision were both within 3.95% for bulk spiked PA batches. The assay accuracy ranged from 97.08% to 105.51% for intra-batch and from 97.49% to 101.99% for inter-batch bulk spiked PA samples. Mean recoveries were 81.29% for Baloxavir and 92.76% for Baloxavir-d5. The limit of detection for Baloxavir was 0.127ng/ml. This methodology proved successful in a bioequivalence study.