Journal of Pharmacology and Pharmacotherapeutics, Ahead of Print.
BackgroundRitlecitinib (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.ObjectivesThis 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 MethodsA 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 µm, 2.1×150 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 ↓ 105.7) and [C4]-ritlecitinib (internal standard, IS) (m/z 290.1 ↓ 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.ResultsOn 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.ConclusionA 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.