Validated LC–MS/MS Method for the Quantitative Determination and Pharmacokinetic Profiling of Fedratinib (TG101348), an Oral JAK2 Inhibitor, in CD1 Mice Plasma

Fedratinib, a selective Janus kinase 2 (JAK2) inhibitor approved by the FDA for the treatment of myeloproliferative neoplasms, particularly myelofibrosis (MF), has clinical relevance. Despite this, no liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been reported for its quantification in biological matrices. This study aimed to develop and validate an LC-MS/MS method for quantifying fedratinib in mice plasma and assessing its pharmacokinetics. The method employed a Sciex 4500 triple quadrupole mass spectrometer with a Shimadzu LC system in positive ionization mode. Chromatographic separation was achieved using a Phenomenex Kinetex C18 column (2.1 × 50 mm, 5.0 μm) with a gradient mobile phase of 5 mM ammonium formate in 0.1% formic acid and acetonitrile at a flow rate of 0.8 mL/min. Telmisartan was used as the internal standard (IS). The method was validated in accordance with USFDA M10 bioanalytical guidelines. Fedratinib and IS were detected at m/z 525.5 → 468.9 and m/z 515.2 → 276.0, respectively. The assay demonstrated excellent sensitivity (LLOQ: 0.5 ng/mL), linearity (0.5 to 1000.0 ng/mL, r² = 0.99), and accuracy (95.80-104.52%). Stability studies confirmed the analyte's integrity across multiple freeze-thaw cycles. The developed LC-MS/MS method is selective, sensitive, fully validated, and was successfully applied to pharmacokinetic studies.