AN LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY BIOANALYTICAL METHOD DEVELOPMENT AND VALIDATION FOR SIMULTANEOUS QUANTIFICATION OF GLECAPREVIR AND PIBRENTASVIR IN HUMAN PLASMA
TL;DR
The validated LC–MS/MS method was selective, sensitive, accurate, precise, and robust for the simultaneous quantification of GPV and PBV in human plasma, making it suitable for pharmacokinetic, bioavailability, bioequivalence, and therapeutic drug monitoring studies.
Abstract
Objective: This study aimed to develop and validate a rapid, sensitive, and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) bioanalytical method for the simultaneous quantification of glecaprevir (GPV) and pibrentasvir (PBV) in human plasma. Methods: Chromatographic separation was achieved on a Phenomenex C18 column (50×4.6 mm, 5 μm) maintained at 35°C using an isocratic mobile phase consisting of 0.1% formic acid and acetonitrile (ACN) (15:85, v/v). Quantification was performed using positive electrospray ionization in multiple reaction monitoring (ESI–MRM) mode with daclatasvir (DCV) as the internal standard. Plasma samples were processed by protein precipitation with ACN, evaporated under nitrogen, and reconstituted with 200 μL of mobile phase before LC-MS/MS analysis. The method was validated according to the US Food and Drug Administration (US-FDA) bioanalytical method validation guideline for selectivity, carry-over, linearity, accuracy, precision, matrix effect, extraction recovery, dilution integrity, and stability. Results: The method demonstrated excellent linearity over concentration ranges of 29.85–1194 ng/mL for GPV and 5.5–220 ng/mL for PBV using weighted (1/x²) linear regression, with regression equations of y=0.002073x+0.001400 (r²=0.99984) for GPV and y=0.016048x+0.002243 (r²=0.99969) for PBV. No endogenous interference or significant carry-over was observed. Extraction recovery ranged from 95.14% to 102.52% for GPV and 96.97% to 98.03% for PBV, with acceptable precision. Matrix effect was negligible, dilution integrity satisfied the acceptance criteria, and intra- and inter-day accuracy and precision met US-FDA requirements. Stability studies confirmed that both analytes remained stable under short-term, long-term, freeze–thaw, bench-top, autosampler, wet extract, and dry extract storage conditions. Conclusion: The validated LC–MS/MS method was selective, sensitive, accurate, precise, and robust for the simultaneous quantification of GPV and PBV in human plasma. The method exhibited satisfactory extraction recovery, negligible matrix effect and carry-over, acceptable dilution integrity, and excellent stability, making it suitable for pharmacokinetic, bioavailability, bioequivalence, and therapeutic drug monitoring studies.