Validated green ultra-fast UPLC-MS/MS method for the quantification of fedratinib in an HLM matrix: application to in vitro and in silico 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 in vitro 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 in silico 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 in vitro half-life (t_1/2) was determined to be 23.26 min. In silico 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 in silico FDB ADME properties and metabolic stability is important for the progression of novel drug discovery aimed at improving metabolic stability.