Development and Validation of a Robust LC-MS/MS Method for Quantitation of a Novel Kinase Inhibitor, Ritlecitinib, in Rat Plasma: Application in Pharmacokinetic Study

Background Ritlecitinib (RIT) is a novel kinase inhibitor with promising therapeutic applications in the treatment of autoimmune disease alopecia areata. Accurate quantification of RIT in biological matrices is essential for pharmacokinetic studies and drug development.Objectives This study aims to develop and validate a sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantitation of RIT in rat plasma and to apply this method in a pharmacokinetic study.Materials and Methods A sensitive and selective LC-MS/MS method was developed in a AB Sciex QTRAP 5500 coupled with Shimadzu Prominence LC-20AD UFLC (ultra-fast liquid chromatography) System using an C18 column (Xselect HSS T3 2.5 mu m, 2.1x150 mm) with a mobile phase consisting of 0.1% formic acid in high-performance liquid chromatography (HPLC) grade water and 0.1% formic acid in acetonitrile (70:30, v:v). RIT (m/z 286.1 down arrow 105.7) and [ 13 C4]-ritlecitinib (internal standard, IS) (m/z 290.1 down arrow 109.7) were detected in a positive mode by electrospray ionization (ESI) using multiple reaction monitoring (MRM) and extracted from rat plasma samples through protein precipitation We validated the method according to United States Food and Drug Administration (U.S. FDA) guidelines, assessing parameters such as linearity, accuracy, precision, selectivity, sensitivity, recovery and stability. The validated method was then applied to a pharmacokinetic study in rats following the administration of RIT.Results On rat plasma, the LC-MS/MS method has shown exceptional linearity across the concentration range of 5-100 ng/mL. The FDA's approved standards of +/- 15% matched the accuracy and precision measurements. The matrix effect was negligible, and the mean recovery was about 93%, indicating that RIT was fully extracted from plasma. Different experimental conditions found RIT to be stable. RIT is quickly absorbed, with a peak plasma concentration (Cmax) of 43.82 ng/mL occurring 0.5-0.75 hours after the dose. It then drops in concentration in a way that is biexponential, with a half-life at the end of 1.5 hours.Conclusion A highly sensitive and specific LC-MS/MS method for quantitation of RIT in rat plasma was successfully developed and validated. The method was effectively applied to a pharmacokinetic study, providing critical data on the absorption and elimination characteristics of RIT in rats. This established approach may support additional preclinical pharmacokinetic investigations of RIT.