"Reverse-Phase HPLC Method Development And Validation For The Analysis Of Teriflunomide In Oral Dosage Forms"
Abstract
Objective: This study aimed to develop and validate a simple, precise, accurate, and robust reverse-phase high-performance liquid chromatography (RP-HPLC) method for the quantification of Teriflunomide, an immunomodulatory drug primarily used in the treatment of relapsing forms of multiple sclerosis (MS).
Methods: Chromatographic separation was optimized using a Shim-pack XR-ODS C18 column (150 × 4.6 mm, 5 µm) with a mobile phase composed of acetonitrile and phosphate buffer (75:25 v/v), adjusted to pH 3.6 with orthophosphoric acid. The flow rate was maintained at 1.2 mL/min, the injection volume was 10 µL, and detection was performed at 289 nm. The method was validated following ICH guidelines, assessing parameters such as accuracy, precision, linearity, limit of detection (LOD), limit of quantification (LOQ), robustness, specificity, and system suitability.
Results: Teriflunomide eluted with a retention time of 3.5 minutes, showing symmetrical peaks and acceptable system suitability metrics. The method exhibited excellent linearity over the range of 20–100 µg/mL (R² = 0.998). Accuracy was confirmed with recovery rates between 100.08% and 100.76% across three concentration levels. Intraday and Interday precision yielded %RSD values of 1.13% and 1.27%, respectively. LOD and LOQ were determined to be 9.58 µg/mL and 29.05 µg/mL. The method remained robust under small, deliberate changes in chromatographic conditions.
Conclusion: The developed RP-HPLC method is reliable, sensitive, and reproducible, making it suitable for routine quality control analysis of Teriflunomide in pharmaceutical dosage forms.
References
2. Göttle, P., Manousi, A., Kremer, D., Reiche, L., Hartung, H., & Küry, P. (2018). Teriflunomide promotes oligodendroglial differentiation and myelination. Journal of Neuroinflammation, 15(1). https://doi.org/10.1186/s12974-018-1110-z
3. Khan, O., Rieckmann, P., Boyko, A. N., Selmaj, K., & Zivadinov, R. (2013). Three times weekly glatiramer acetate in relapsing–remitting multiple sclerosis. Annals of Neurology, 73(6), 705-713. https://doi.org/10.1002/ana.23938
4. Nazareth, T., Rava, A. R., Polyakov, J. L., Banfe, E. N., Waltrip, R. W., Zerkowski, K., … & Herbert, L. B. (2018). Relapse prevalence, symptoms, and health care engagement: patient insights from the multiple sclerosis in america 2017 survey. Multiple Sclerosis and Related Disorders, 26, 219-234. https://doi.org/10.1016/j.msard.2018.09.002
5. Scalfari, A., Neuhaus, A., Degenhardt, A., Rice, G. P., Muraro, P. A., Däumer, M., … & Ebers, G. C. (2010). The natural history of multiple sclerosis, a geographically based study 10: relapses and long-term disability. Brain, 133(7), 1914-1929. https://doi.org/10.1093/brain/awq118
6. Scalfari, A., Neuhaus, A., Däumer, M., DeLuca, G. C., Muraro, P. A., & Ebers, G. C. (2013). Early relapses, onset of progression, and late outcome in multiple sclerosis. JAMA Neurology, 70(2), 214. https://doi.org/10.1001/jamaneurol.2013.599
7. Chitnis, T., Tardieu, M., Amato, M. P., Banwell, B., Bar‐Or, A., Ghezzi, A., … & Wassmer, E. (2013). International pediatric ms study group clinical trials summit. Neurology, 80(12), 1161-1168. https://doi.org/10.1212/wnl.0b013e318288694e
8. Bar‐Or, A., Pachner, A. R., Menguy-Vacheron, F., Kaplan, J., & Wiendl, H. (2014). Teriflunomide and its mechanism of action in multiple sclerosis. Drugs, 74(6), 659-674. https://doi.org/10.1007/s40265-014-0212-x
9. Wostradowski, T., Prajeeth, C. K., Gudi, V., Kronenberg, J., Witte, S., Brieskorn, M., … & Stangel, M. (2016). In vitro evaluation of physiologically relevant concentrations of teriflunomide on activation and proliferation of primary rodent microglia. Journal of Neuroinflammation, 13(1). https://doi.org/10.1186/s12974-016-0715-3
10. Ambrosius, B., Faissner, S., Guse, K., Lehe, M. v., Grünwald, T., Gold, R., … & Chan, A. T. (2017). Teriflunomide and monomethylfumarate target hiv-induced neuroinflammation and neurotoxicity. Journal of Neuroinflammation, 14(1). https://doi.org/10.1186/s12974-017-0829-2
11. Ciardi, M. R., Zingaropoli, M. A., Pasculli, P., Perri, V., Tartaglia, M., Valeri, S., … & Mastroianni, C. M. (2020). The peripheral blood immune cell profile in a teriflunomide-treated multiple sclerosis patient with covid-19 pneumonia. Journal of Neuroimmunology, 346, 577323. https://doi.org/10.1016/j.jneuroim.2020.577323
12. Ochoa‐Repáraz, J., Colpitts, S. L., Kircher, C., Kasper, E., Telesford, K., Begum-Haque, S., … & Kasper, L. H. (2016). Induction of gut regulatory cd39 + t cells by teriflunomide protects against eae. Neurology Neuroimmunology &Amp; Neuroinflammation, 3(6). https://doi.org/10.1212/nxi.0000000000000291
13. Tilly, G., Cadoux, M., García, A., Morille, J., Wiertlewski, S., Pecqueur, C., … & Degauque, N. (2021). Teriflunomide treatment of multiple sclerosis selectively modulates cd8 memory t cells. Frontiers in Immunology, 12. https://doi.org/10.3389/fimmu.2021.730342
14. Zmira, O., Gofrit, S. G., Aharoni, S. A., Weiss, R., Shavit‐Stein, E., & Chapman, J. (2022). Teriflunomide normalizes anti-anxiety effect in anti-anxa2 aps mice model teriflunomide in anti-anxa2 mice model. Lupus, 31(7), 855-863. https://doi.org/10.1177/09612033221095150
15. Prabhakara, K. S., Kota, D. J., Jones, G., Srivastava, A. K., Cox, C. S., & Olson, S. D. (2018). Teriflunomide modulates vascular permeability and microglial activation after experimental traumatic brain injury. Molecular Therapy, 26(9), 2152-2162. https://doi.org/10.1016/j.ymthe.2018.06.022
16. Wolinsky, J. S., Narayana, P. A., Nelson, F., Datta, S., O’Connor, P., Confavreux, C., … & Freedman, M. S. (2013). Magnetic resonance imaging outcomes from a phase iii trial of teriflunomide. Multiple Sclerosis Journal, 19(10), 1310-1319. https://doi.org/10.1177/1352458513475723
17. Papp, V., Buron, M. D., Siersma, V., Rasmussen, P. V., Illés, Z., Kant, M., … & Magyari, M. (2021). Real-world outcomes for a complete nationwide cohort of more than 3200 teriflunomide-treated multiple sclerosis patients in the danish multiple sclerosis registry. Plos One, 16(5), e0250820. https://doi.org/10.1371/journal.pone.0250820
18. Nwankwo, E., Allington, D., & Rivey, M. P. (2012). Emerging oral immunomodulating agents – focus on teriflunomide for the treatment of multiple sclerosis. Degenerative Neurological and Neuromuscular Disease, 15. https://doi.org/10.2147/dnnd.s29022
