eng Oriental Scientific Publishing Company Biosciences Biotechnology Research Asia 0973-1245 2026-06-24 23 2 59267 article Mayur Bhosale 1 Rahul Khaire 2 Mohit Wagh 1 Dheeraj Chechare 3 Yogesh Thombare 3 Department of Pharmaceutical Chemistry, Pravara Rural College of Pharmacy, Loni, India Department of Pharmaceutical Chemistry, PRES’s College of Pharmacy (For Women), Nashik, India Department of Pharmaceutical Chemistry, PRES’s College of Pharmacy (D.Pharm), Nashik, India To develop and validate a robust, stability-indicating RP-HPLC method for safinamide mesylate quantification employing systematic Quality by Design approach for pharmaceutical quality control and regulatory applications in Parkinson's disease therapy.Box-Behnken design with 17 experimental runs optimized three critical parameters: mobile phase composition (45-55% methanol), flow rate (0.8-1.2 mL/min), and column temperature (33-37°C). Chromatographic separation was achieved on Inertsil ODS-3V C18 column with UV detection at 226 nm. Response surface methodology established design space boundaries, followed by comprehensive validation per ICH Q2(R1) guidelines including specificity, linearity, accuracy, precision, robustness, LOD/LOQ, solution stability, and filter compatibility.Optimal conditions (47.5% methanol, 0.8 mL/min, 35°C) yielded retention time 3.56 minutes with excellent system suitability (% RSD 0.04%, theoretical plates 4366, asymmetry 1.11). Quadratic models demonstrated strong predictive capability (R² >0.88, relative errors <2%). The method exhibited exceptional linearity (r² = 0.9999, 2-30 μg/mL), high sensitivity (LOD 0.079 μg/mL, LOQ 0.240 μg/mL), excellent accuracy (99.81% recovery), superior precision (repeatability 0.86% RSD, intermediate 1.05% RSD), and robust performance under deliberate parameter variations. Solutions remained stable for 24 hours with both PVDF and Nylon filters compatible. https://www.biotech-asia.org/vol23no2/analytical-method-development-and-validation-for-safinamide-mesylate-using-rp-hplc-with-quality-by-design-approach/ Box-Behnken design; ICH Q2(R1); Method validation; Parkinson's disease; Quality by Design; RP-HPLC; Safinamide mesylate