DEVELOPMENT AND VALIDATION OF AN LC-MS/MS ASSAY FOR THE QUANTIFICATION OF FEDRATINIB IN HUMAN PLASMA AND DRIED BLOOD SPOT MATRICES

Abstract

Objective: The objective of the study was to develop and validate a highly sensitive and robust LC–MS/MS method for the quantification of Fedratinib (FRB), a selective JAK2 inhibitor used in the treatment of myelofibrosis, in both human plasma and dried blood spot (DBS) samples as per ICH M10 guidelines.

Methods: Quantification was performed using a liquid chromatography–tandem mass spectrometry system equipped with electrospray ionization and multiple reaction monitoring. DBS samples were prepared using Whatman 903 cards, with FRB extracted efficiently to enable low-volume, stable, and decentralized sampling. Calibration was established over the range of 36–3600 ng/mL.

Results: The method demonstrated excellent selectivity, precision, and accuracy, with recoveries of 63.1% (plasma) and 77.1% (DBS) for FRB, and 60.9% (plasma) and 87.4% (DBS) for the internal standard. Stability assessments confirmed robustness under bench-top, autosampler, and long-term conditions. Comparative analysis of plasma and DBS matrices showed strong agreement: Passing–Bablok regression indicated a slope close to 1 with negligible intercept, and Bland–Altman analysis revealed a mean bias of approximately –2%, well within the bioanalytical acceptance limits (<15%).

Conclusion: DBS demonstrated equivalence with plasma for FRB quantification across LQC, MQC, and HQC levels, confirming its suitability as a reliable alternative to plasma for pharmacokinetic and bioequivalence studies. This validated method provides a practical and sensitive tool for FRB determination, particularly advantageous in decentralized and resource-limited settings.