Asian Journal of Pharmaceutical Analysis. 14(4): October - December, 2024 241 ISSN 2231–5667 (Print) 2231–5675 (Online) DOI: 10.52711/2231-5675.2024.00043 Vol. 14 | Issue-04| October - December | 2024 Available online at www.anvpublication.org www.asianpharmaonline.org Asian Journal of Pharmaceutical Analysis Home page www. ajpaonline.com RESEARCH ARTICLE A High-Performance Liquid Chromatography Method Development for Leflunomide Analysis and Validation Siddhant M. Sawant, Prashant J. Burange*, Maithili P. Amale, Pankaj H. Chaudhary, Adnya D. Bahurupi Department of Pharmaceutical Quality Assurance, P. R. Pote Patil College of Pharmacy, Sant Gadge Baba Amravati University, Amravati 444602, Maharashtra, India. *Corresponding Author E-mail: prashantburange@gmail.com ABSTRACT: In this study, we developed and validated a rapid and precise RP-HPLC method to quantify leflunomide in bulk and tablet dosage forms. The analysis was performed using an Inertsil-ODS C18 column (250x4.6mm, 5μm) with a mobile phase composed of 95:5 methanol/water at a flow rate of 1mL/min. Leflunomide demonstrated a linear range between 20-70μg/ml with a high correlation coefficient (R² = 0.995). Adhering to ICH guidelines, the method was designed to be accurate, precise, sensitive, selective, repeatable, and robust. The validation parameters included system suitability, specificity, linearity, accuracy, precision, detection limit, quantitation limit, robustness, and stability. The results indicated excellent accuracy and precision, with recoveries close to 100% and relative standard deviations well within acceptable limits. The method's sensitivity allowed for reliable detection and quantification at low concentrations, making it ideal for routine quality control. Additionally, robustness studies confirmed the method's reliability despite minor changes in experimental conditions. This HPLC method is both efficient and cost-effective, enabling faster analysis while maintaining high quality. The validated method was successfully applied to the analysis of leflunomide in pharmaceutical formulations, proving its suitability for routine use in quality control laboratories. Overall, this method offers a comprehensive solution for accurately estimating leflunomide in various dosage forms. KEYWORDS: RP-HPLC, Leflunomide, Validation, Accurate, Robust, Precise. Received on 08.06.2024 Revised on 11.09.2024 Accepted on 19.10.2024 Published on 10.12.2024 Available online on December 30, 2024 Asian Journal of Pharmaceutical Analysis. 2024; 14(4):241-246. DOI: 10.52711/2231-5675.2024.00043 ©Asian Pharma Press All Right Reserved This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License. INTRODUCTION: Leflunomide [N-[4',5'-trifluoromethylphenyl]-5- methylisoxazole-4-carboxamide] is an isoxazole derivative used as an anti-rheumatic medication, with a molecular weight of 270.2 (fig. 1) 1 . Its mechanism of action involves the selective inhibition of dihydro-orotate dehydrogenase 2,3 , a crucial enzyme in the de novo synthesis of pyrimidines, which subsequently suppresses the production of ribonucleic acid and deoxyribonucleic acid 4 . Leflunomide is particularly effective against activated T cells, which primarily produce pyrimidines through the de novo