Sustainable RP‐HPLC Method Development for Simultaneous Estimation of Fampridine and Monomethyl Fumarate in Lipid Nanocarriers and Biological Fluids: Application of AQbd Principles

ABSTRACT Analytical quality by design (AQbD) provides a systematic framework for developing reliable pharmaceutical analytical methods. In this study, environmentally conscious RP‐HPLC method was developed for fampridine and monomethyl fumarate, two disease‐modifying agents used in multiple sclerosis therapy. Critical risks were mapped by Fishbone diagram, prioritized by ranking and filtering, screened via Taguchi design, and optimized using Box–Behnken design to evaluate the influence of organic phase concentration, flow rate, and column temperature on critical chromatographic responses, namely retention time, tailing factor, and theoretical plates for both fampridine and monomethyl fumarate. Seventeen experimental trials identified optimal conditions consisting of a mobile phase of water with 0.1% ortho phosphoric acid and methanol (85:15% v/v), a flow rate of 1.0 mL/min, column temperature of 35°C, and a Phenomenex Luna C18 column (5 µm, 150 × 4.6 mm). Under these settings, retention times were 3.492 min for fampridine and 9.482 min for monomethyl fumarate. The method demonstrated excellent linearity across 2–12 µg/mL ( R 2 = 0.9992 for fampridine; R 2 = 0.9989 for monomethyl fumarate), with low detection and quantification limits. Recovery studies from lipid nanocarriers yielded values between 96.13% and 98.75%. Forced degradation experiments confirmed sensitivity to acidic, oxidative, and thermal stress, while application to biological matrices produced recoveries above 95%, underscoring bioanalytical applicability.