A novel, simple, specific, sensitive and reproducible high-performance liquid chromatography (HPLC) assay method has been developed and validated for the estimation of tofacitinib in rat plasma. The bioanalytical procedure involves extraction of tofacitinib and itraconazole (internal standard, IS) from rat plasma with a simple liquid-liquid extraction process. The chromatographic analysis was performed on a Waters Alliance system using a gradient mobile phase conditions at a flow rate of 1.0mL/min and C18 column maintained at 40±1°C. The eluate was monitored using an UV detector set at 287nm. Tofacitinib and IS eluted at 6.5 and 8.3min, respectively and the total run time was 10min. Method validation was performed as per US Food and Drug Administration guidelines and the results met the acceptance criteria. The calibration curve was linear over a concentration range of 182-5035ng/mL (r2=0.995). The intra- and inter-day precisions were in the range of 1.41-11.2 and 3.66-8.81%, respectively, in rat plasma. The validated HPLC method was successfully applied to a pharmacokinetic study in rats.
- method validation
- rat plasma