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First bioanalytical method being reported for quantification of defactinib in mice plasma on LC–MS/MS.Method was validated as per regulatory guidelines.The method is specific, precise, accurate and no matrix effect was observed and linear from 0.13 to 106 ng/mL.The method was successfully used in a mice pharmacokinetic study and for the first time pharmacokinetic parameters for defactinib in mice.A sensitive, specific, selective and rapid LC-ESI–MS/MS method has been developed and validated for the quantification of defactinib in mice plasma using 13C3,15N-tofacitinib as an internal standard (I.S.). Sample preparation was accomplished through a liquid–liquid extraction process. Baseline chromatographic resolution of defactinib and the I.S. was achieved on an Atlantis dC18 column using an isocratic mobile phase comprising 0.2% formic acid in water and acetonitrile (25:75, v/v) delivered at a flow rate of 0.5 mL/min. Defactinib and the I.S. eluted at ˜1.59 and 0.99 min, respectively. The total chromatographic run time was 2.50 min. A linear response function was established in the concentration range of 0.13–106 ng/mL. Method validation was performed as per regulatory guidelines and the results met the acceptance criteria. The intra- and inter-day accuracy and precision were in the range of 5.57–13.3 and 8.63–12.1%, respectively. Defactinib was found to be stable under various stability conditions. This novel method has been applied to a pharmacokinetic study in mice.