Analytical Quality by Design‐Guided Optimisation and Validation of a Short‐Run Stability‐Indicating Reverse‐Phase Ultra‐Fast Liquid Chromatography (RP‐UFLC) Method for the Quantitation of Nonivamide

Abstract

Nonivamide (NON), a capsaicin analogue with similar pharmacological effects, is used in pain management, yet limited information exists on its accurate quantification by reverse-phase liquid chromatography. A cost-effective, efficient and precise RP-HPLC method was developed, optimised using response surface methodology (central composite design), validated and assessed for greenness. Naproxen served as the internal standard. Acetonitrile (ACN) with a potassium phosphate buffer achieved separation, with detection at 285 nm. Critical analytical attributes included retention times, resolution and solvent composition. Optimised conditions were 41.0% v/v of ACN, a flow rate of 1.5 mL/min and a buffer of pH 4. Under these conditions, NON eluted at 6.8 min with a resolution of 13 and %RSD < 5%, aligning with model predictions, confirming robustness and system suitability. Greenness evaluation (AGREE) yielded a score of 0.72 (good-very good). Method validation followed the ICH guidelines: linearity of 2-200 μg/mL, LOD of 0.67 μg/mL and LOQ of 2 μg/mL. Precision and accuracy were within acceptable limits (< 5%). Stress studies revealed NON stability under most conditions, except alkaline and oxidative stress, whereas specificity was confirmed by detection in the presence of degradants. Overall, the developed method is robust, reproducible, reliable and environmentally considerate, enabling accurate NON quantification from pharmaceutical bulk.