DEVELOPMENT AND VALIDATION OF AN LC-MS/MS ASSAY FOR THE QUANTIFICATION OF FEDRATINIB IN HUMAN PLASMA AND DRIED BLOOD SPOT MATRICES
DOI:
https://doi.org/10.22159/ajpcr.2026v19i1.57007Keywords:
Fedratinib, Dried blood spot, Bioanalytical method validation, Bioanalysis, Clinical study, Liquid chromatography-mass spectrometry/ mass spectrometry, MyelofibrosisAbstract
Objectives: 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 Janus kinase 2 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 36–3,600 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, auto sampler, 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 lower-quality control (QC), middle-QC, and high-QC 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
Downloads
References
1. Pardanani M. Fedratinib, a selective JAK2 inhibitor: An emerging therapeutic option for myelofibrosis. Leuk Res. 2016;46:15-8.
2. Harrison J, Schaap N, Vannucchi AM, Kiladjian JJ, Verstovsek S. Risk mitigation in fedratinib therapy: Revisiting Wernicke’s encephalopathy concerns. J Hematol Oncol. 2020;13(1):1-8.
3. Wang J, Aubry D. Bioanalytical LC-MS/MS method validation and its application to pharmacokinetic studies. Bioanalysis. 2011;3(16):1819-32.
4. Spooner A, Lad J, Barfield M. Dried blood spots as a sample collection technique for the determination of pharmacokinetics in clinical studies. Bioanalysis. 2009;1(1):81-90.
5. Li R, Brown M, Smith K. Advantages and limitations of dried blood spot analysis: The past, present, and future. TrAC Trends Anal Chem. 2018;102:248-56. 6. Deshmukh P, AQ4 et al. Evaluation of DBS sampling in pharmacokinetic studies. Int J Appl Pharm (IJAP). 2022;14(5):45-53.
7. De Kesel PM, Capiau S, Lambert WE, Stove CP. Current strategies for coping with the hematocrit problem in dried blood spot analysis. Bioanalysis. 2014;6(14):1871-4. doi: 10.4155/bio.14.151, PMID 25158957
8. Li W, Tse FL. Dried blood spot sampling in combination with LC-MS/ MS for quantitative analysis of small molecules. Biomed Chromatogr. 2010;24(1):49-65. doi: 10.1002/bmc.1367, PMID 20017122
9. Rowland M, Emmons GT. Use of dried blood spots in drug development: Pharmacokinetic considerations. AAPS J. 2010;12(3):290-3. doi: 10.1208/s12248-010-9188-y, PMID 20383669
10. Abu-Rabie P, Denniff P, Spooner N, Chowdhry BZ, Pullen FS. Investigation of different approaches to incorporating internal standard in DBS quantitative bioanalytical workflows and their effect on nullifying hematocrit-based assay bias. Anal Chem. 2015;87(9):4996-5003. doi: 10.1021/acs. analchem.5b00908, PMID 25874899
11. Koster RA, Botma R, Greijdanus B, Uges DR, Kosterink JG, Touw DJ, et al. The performance of five different dried blood spot cards for the analysis of six immunosuppressants. Bioanalysis. 2015;7(10):1225-35. doi: 10.4155/bio.15.63, PMID 26045003
12. International Council for Harmonisation (ICH). M10: Bioanalytical Method Validation Guideline. Geneva: ICH; 2022. Available from: https://www.ich.org
13. Bland JM, Altman DG. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet. 1986;1(8476):307-10. PMID 2868172
14. Passing H, Bablok W. Comparison of several regression procedures for method comparison studies and determination of sample sizes. Application of linear regression procedures for method comparison studies in clinical chemistry, Part II. J Clin Chem Clin Biochem. 1984;22(6):431-45. doi: 10.1515/cclm.1984.22.6.431, PMID 6481307
15. Bablok W, Passing H. Application of statistical procedures in analytical instrument testing. J Automat Chem. 1985;7(2):74-9. doi: 10.1155/ S1463924685000177, PMID 18925074
16. Ramesh D, Habibuddin M. Application of validated RP-HPLC method for simultaneous determination of metaxalone and diclofenac potassium in plasma. Int J Curr Pharm Res. 2024;16(4):89-94. doi: 10.22159/ ijcpr.2024v16i4.5039
17. Neelima CH, Hemasri M, Susmitha P, Srilakshmi S. A novel method development and validation for the quantification of nefopam hydrochloride in parenteral dosage form by RP-HPLC method. Int J Curr Pharm Res. 2022;14(4):42-50. doi: 10.22159/ijcpr.2022v14i4.1982
18. Jemal M, Xia YQ. Bioanalytical method validation with structurally different internal standards. Rapid Commun Mass Spectrom. 2000;14(23):2219-26. doi: 10.1002/1097-0231(20001215)14:23<2219: AID-RCM155>3.0.CO;2-Y
19. Balakrishnan P, Vaidyanathan S, Natarajan J, Nageswari A. LC-MS/MS quantification of small molecules using surrogate internal standards. J Chromatogr B. 2014;961:45-52. doi: 10.1016/j.jchromb.2014.05.019
20. Linnet K. Comparison of quantitative analytical methods: Regression techniques for bias estimation. Clin Chem. 1993;39(3):424-32. doi: 10.1093/clinchem/39.3.424, PMID 8448852
21. Pawar A, Jadhav V, Deshmukh S, et al. Development and validation of LC-MS/MS method for quantification of drug in dried blood spots. Int J Appl Pharm. 2023;15(2):77-85. doi: 10.22159/ijap.2023v15i2.48010
22. Pucci V, Di Palma S, Alfieri A, Bonelli F, Monteagudo E, Rossi CE. LC=MS/MS analysis of kinase inhibitors in dried blood spots. J Chromatogr B. 2018;1083:82-90. doi: 10.1016/j.jchromb.2018.02.010
23. Jadhav S, Patil P, Desai M, et al. Application of microsampling in drug analysis: A review. Int J Pharm Pharm Sci. 2022;14(3):12-20. doi: 10.22159/ijpps.2022v14i3.46888
Published
How to Cite
Issue
Section
Copyright (c) 2025 Atharva Bhalerao
This work is licensed under a Creative Commons Attribution 4.0 International License.
The publication is licensed under CC By and is open access. Copyright is with author and allowed to retain publishing rights without restrictions.