{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,1]],"date-time":"2026-07-01T17:48:55Z","timestamp":1782928135742,"version":"3.54.5"},"reference-count":31,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2026,6,26]],"date-time":"2026-06-26T00:00:00Z","timestamp":1782432000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pharmaceuticals"],"abstract":"<jats:p>Background\/Objectives: Teriflunomide is an active metabolite of leflunomide and acts as a selective and reversible inhibitor of dihydroorotate dehydrogenase, a key enzyme in de novo pyrimidine biosynthesis. It exhibits immunomodulatory activity by reducing the proliferation of activated T and B lymphocytes and is widely used in the treatment of rheumatoid arthritis and relapsing multiple sclerosis. This study aimed to develop a rapid, accurate, and simple high-performance liquid chromatography (HPLC) method with fluorometric detection for quantifying teriflunomide in human plasma. Methods: Plasma samples were prepared by liquid\u2013liquid extraction followed by pre-column derivatization with NBD-Cl. Teriflunomide was derivatized with 4-chloro-7-nitrobenzofurazan (NBD-Cl) and separated using a reversed-phase C18 column (5 \u00b5m, 4.6 \u00d7 150 mm) at 30 \u00b0C with isocratic elution. The mobile phase consisted of acetonitrile and 0.1% orthophosphoric acid (80:20, v\/v) at a flow rate of 1.1 mL\/min. Fluorescence detection was performed at \u03bbex = 465 nm and \u03bbem = 535 nm. The method meets European Medicines Agency (EMA) guidelines for bioanalytical validation and was successfully applied to pharmacokinetic studies, including AUC0\u2013t, AUC0\u2013\u221e, Cmax, Tmax, and t\u00bd. Results: Teriflunomide showed a retention time of 2.55 \u00b1 0.01 min. The method exhibited linearity in the range of 0.01\u201330 ng\/mL (r2 = 0.9998), with a limit of detection and quantification of 0.003 and 0.01 ng\/mL, respectively. The relative standard deviation was 3.27%. Conclusions: This work introduces a novel, cost-effective, and highly sensitive HPLC with fluorescence detection (HPLC-FL) method for the determination of teriflunomide in human plasma, providing an efficient alternative to LC-MS\/MS for routine pharmacokinetic and bioequivalence studies.<\/jats:p>","DOI":"10.3390\/ph19070987","type":"journal-article","created":{"date-parts":[[2026,7,1]],"date-time":"2026-07-01T16:50:33Z","timestamp":1782924633000},"page":"987","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Fluorescence HPLC Analysis of Teriflunomide in Human Plasma Following Derivatization with 4-Chloro-7-Nitrobenzofurazan: Method Development and Application to a Prototype Pharmacokinetic Evaluation"],"prefix":"10.3390","volume":"19","author":[{"given":"Meltem","family":"Cayci","sequence":"first","affiliation":[{"name":"Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Harran University, Sanl\u0131urfa 63050, Turkey"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Burhan","family":"Ceylan","sequence":"additional","affiliation":[{"name":"Department of Pharmacognosy, Faculty of Pharmacy, Harran University, Sanl\u0131urfa 63050, Turkey"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5840-7386","authenticated-orcid":false,"given":"Cem","family":"Onal","sequence":"additional","affiliation":[{"name":"Department of Analytical Chemistry, Faculty of Pharmacy, Istanbul Health and Technology University, Istanbul 34445, Turkey"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,6,26]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Yang, B., Ma, D., Zhao, X., Zhu, X., Wang, Y., Xu, K., and Zhang, L. 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