Sustainable Ultra‐High Performance Liquid Chromatographic Method Development for Mupirocin Loaded Nanosuspension by Analytical Quality by Design Approach

A reliable and eco‐friendly ultra‐high performance liquid chromatography method was developed for quantitative determination of mupirocin in bulk drug, nanosuspension formulations, and biological matrices using an Analytical Quality by Design approach. Risk assessment by an Ishikawa Fishbone diagram, followed by risk ranking and filtering approach and Taguchi orthogonal array design, identified critical risk parameters. Final optimization was achieved using a Box–Behnken design with three center points, evaluating organic phase composition, flow rate, and column temperature against responses including retention time, tailing factor, and theoretical plates. Optimized chromatographic conditions employed acetonitrile and 0.1% orthophosphoric acid (60:40 % v/v) as mobile phase, a flow rate of 1.0 mL/min, column temperature of 35°C, and detection at 222 nm using a C18 column (Phenomenex Luna, 150 mm × 4.6 mm, 5 µm). Efficient separation was achieved with retention time of 2.112 min. The method demonstrated linearity across 2–12 µg/mL, with validation confirming precision, accuracy, robustness, and compliance with ICH Q2 (R2) guidelines. The developed ultra‐high performance liquid chromatography method ensures stability, accuracy, sustainability, and bioanalytical applicability, with strong process capability, making it valuable for pharmaceutical research, quality assurance, and development.