Fluorescence HPLC Analysis of Teriflunomide in Human Plasma Following Derivatization with 4-Chloro-7-Nitrobenzofurazan: Method Development and Application to a Prototype Pharmacokinetic Evaluation

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–liquid 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 µm, 4.6 × 150 mm) at 30 °C 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 λex = 465 nm and λem = 535 nm. The method meets European Medicines Agency (EMA) guidelines for bioanalytical validation and was successfully applied to pharmacokinetic studies, including AUC0–t, AUC0–∞, Cmax, Tmax, and t½. Results: Teriflunomide showed a retention time of 2.55 ± 0.01 min. The method exhibited linearity in the range of 0.01–30 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.