Purity determination of yunaconitine reference standard using HPLC with experimental design and response surface optimization

Experimental design and response surface methodology have been used for the development of the stability‐indicating HPLC method for the purity determination of yunaconitine reference standard. Significant factors including the contents of ACN, perchloric acid, triethylamine (TEA), and column temperature were optimized using a Box–Behnken design. A mixture of crude yunaconitine extract and degradation solutions of yunaconitine under stress conditions was chromatogramed. The normalized peak area of total impurities, the retention time of yunaconitine, and the resolutions between yunaconitine and its adjacent peaks were selected as optimization criteria. Derringer desirability function of the multicriteria and the tested factors were used to establish 3‐D response surfaces. The optimal condition was achieved with a mobile phase of ACN/water (30:70, containing 0.125% perchloric acid and 1.0% TEA) at a column temperature of 37.5°C. The method was validated and shown comparable to that of phase solubility analysis. As a result, the newly developed method can be used to determine the chromatographic purity and stability of the yunaconitine reference standard.