Optimization of the separation and determination of nitidine and chelerythrine in Zanthoxylum nitidum by high-performance liquid chromatography with fluorescence detection.

A new ion-pair high-performance liquid chromatography method with fluorescence detection was developed for the determination of nitidine and chelerythrine in Zanthoxylum nitidum. To optimize the separation of the two compounds in reversed-phase liquid chromatography, a response surface method (Box-Behnken designs) was used. Three important factors: concentration of ion pair agent, mobile phase composition and buffer pH, were studied for their contribution to the analytes' response, leading to a total of 17 experiments performed on a Kromasil C18 column. The experimental responses were fitted into a second-order polynomial to predict the best conditions. The optimal mobile phase conditions were predicted to be acetonitrile-sodium dodecyl sulphate (17.8 mM, 20 mM citric acid, pH 2.98, 57:43, v/v). The proposed method was validated according to International Conference on Harmonization guidelines and it is suggested to be appropriate for the routine quality control analysis of nitidine and chelerythrine in Zanthoxylum nitidum.