Stability-Indicating Method Development and Validation for Quantitative Estimation of Assay and Organic Impurities of anti-viral drug Baloxavir-Marboxil in Drug Substance and Pharmaceutical Dosage Form using HPLC and LC-MS Methods.

Baloxavir-Marboxil (BXM) is a polymerase-acidic-endonuclease inhibitor used as an anti-viral drug. A simple, reliable, and robust liquid chromatographic method was developed and validated per ICH Q2(R1) for estimating the assay and impurities of BXM in drug substance and pharmaceutical formulations. The chromatographic separation was carried-out on C 18 (100 x 4.6 mm, 5-μm) with binary solvent delivery system (A:0.1% trifluoroacetic-acid in water; B:0.1% trifluoroacetic-acid in acetonitrile) along with detection wavelength of 260 nm, column temperature of 57°C, flow of 1.2-mL/min and injection volume of 10-μL. All five known impurities and unknown impurities were separated well with resolution >1.7, and were estimated accurately without any interference. Recovered values and regression value were 99.5-101.2% and R 2 >0.999, respectively. The recovery and linearity studies covered from 50-150% for assay, and quantitation limit (QL) - 120% for five BXM impurities. Stability-indicating property of the HPLC developed method was assessed from the forced degradation studies. The mass spectral data of unknown impurity formed under oxidation stress condition was discussed. The developed method was also successfully utilized for stability samples analysis of drug substance and tablets dosage form.