Pharmacokinetics and tissue distribution study of PA-824 in rats by LC-MS/MS

A simple, sensitive and rapid LC-MS/MS method has been developed and validated for determination of PA-824 in rat biological samples using darunavir as internal standard. Chromatographic separation was achieved on an Inertsil®ODS3 C18 column (150 mm × 4.6 mm, 5 μm) using gradient elution of methanol-0.1% ammonia in water (90:10, v/v) with fast gradient elution at a flow rate of 0.6 mL/min and run time of 5 min. The mass spectrometer was run in positive electrospray ionization (ESI) mode using multiple reaction monitoring (MRM) to monitor the mass transitions. The optimized ion transition pairs for quantitation were m/z 360.1 → m/z 175.0 for PA-824, m/z 548.5 → m/z 504.2 for IS. The method was validated for accuracy, precision, linearity, range, selectivity, lower limit of quantification (LLOQ), recovery, matrix effect and robustness. All validation parameters met the acceptance criteria according to regulatory guidelines. The LLOQ was 0.05 μg/mL. The calibration curves showed a good linearity over the concentration range of 0.05-50 μg/mL. The calibration curves for all biological samples showed good linearity (r2 > 0.9978) over the concentration ranges tested. The recoveries obtained for PA-824 were ≥88.8%. The developed method was successfully applied to investigate the pharmacokinetics and tissue distribution of PA-824 in rats following oral administration. It was also the first study to investigate the tissue distribution of PA-824 in rats following oral administration.