LC-MS/MS-based metabolite quantitation of the antiviral prodrug baloxavir marboxil, a new therapy for acute uncomplicated influenza, in human plasma: Application to a human pharmacokinetic study.

Baloxavir marboxil (BXM) is a novel orally administrated prodrug for the treatment of acute uncomplicated influenza. In the present study, a bioanalytical LC-MS/MS method was developed and validated for the quantification of baloxavir acid (BXA), the active form of baloxavir marboxil in plasma of healthy volunteers using dolutegravir as an internal standard (IS) following plasma protein precipitation with acetonitrile. BXA and the internal standard were chromatographically separated using Waters Xterra® MS C column (5 µm, 4.6 × 50 mm) and a mobile phase comprised of 10.0 mM ammonium formate pH 3.5 and acetonitrile (80:20, v/v) delivered at a flow rate of 0.6 mL/min. The transitions of m/z 484.00 → 247.0 and 420.30 → 277.1 for BXA and IS, respectively in multiple reaction monitoring (MRM) mode in a positive ESI interface were used for quantitation through triple-quad mass spectrometry, API 4000. The method linearity was proven across the concentration range of 0.5-200.0 ng/mL, adjusted, and validated completely in accordance with the bioanalytical guidelines of the United States-FDA. Finally, the present method was effectively applied for the pharmacokinetic study of BXA in healthy human volunteers with accepted reproducibility and ruggedness.