Validated green ultra-fast UPLC-MS/MS method for the quantification of fedratinib in an HLM matrix: application to <i>in vitro</i> and <i>in silico</i> metabolic stability studies

Fedratinib (INREBIC®; FDB), an orally administered selective Janus kinase 2 (JAK-2) inhibitor, has been approved by the FDA for the treatment of intermediate-2 or high-risk primary or secondary myelofibrosis in adult patients. This study established a sensitive, fast, green, and dependable UPLC-MS/MS approach for quantifying FDB in human liver microsomes (HLMs); moreover, this approach was employed to assess the <i>in vitro</i> metabolic stability of FDB in HLMs. The validation steps of the UPLC-MS/MS approach adhered to the US-FDA principles for bioanalytical method validation. The StarDrop program, incorporating the DEREK and P450 modules, was employed to monitor the FDB chemical structure alerts and <i>in silico</i> metabolic lability, respectively. FDB and encorafenib (ENB as the IS) were analyzed using the isocratic mobile phase method on an Eclipse Plus C18 column. The developed UPLC-MS/MS method exhibited an ultra-fast (1 min), wide linearity range (1.0-3000 ng mL^-1) with good separation of the target analytes and was accurate and reproducible in the absence of HLM matrix effects. The current study evaluated the precision and accuracy of the UPLC-MS/MS method for intra-day and inter-day assessments, varying from -5.33% to 5.56% and -9.00% to 6.67%, respectively. The intrinsic clearance (Cl_int) of FDB was measured at 34.86 mL min^-1 kg^-1, whereas the <i>in vitro</i> half-life (<i>t</i>_1/2) was determined to be 23.26 min. <i>In silico</i> screening indicated that minor structural modifications to the pyrrolidine moiety in the process of drug design could increase metabolic stability and enhance safety relative to FDB. The assessment of <i>in silico</i> FDB ADME properties and metabolic stability is important for the progression of novel drug discovery aimed at improving metabolic stability.