Highly sensitive liquid chromatographic analysis of artemisinin and its derivatives after pre-column derivatization with 4-carboxyl-2,6-dinitrobenzene diazonium ion

A highly sensitive and reproducible isocratic liquid chromatographic method has been developed for the analysis of artemisinin and its three commonly used derivatives (artesunate, dihydroartemisinin, and artemether). The method involves a precolumn derivatization reaction with 4-carboxyl-2,6-dinitrobenzene diazonium ion to produce azo adducts that are UV-active. The critical parameters for the derivatization such as temperature, reaction time, and reagent concentrations were studied and optimized. The chromatographic separations were carried out on a C-18 column with mobile phase consisting of acetonitrile-0.1% acetic acid (60:40) at a flow rate of 1 mL min(-1). UV detection was set at 254 nm. Dynamic linear calibration range was obtained at concentrations of artemisinins ranging from 0.26 to 1.44 mu g mL(-1). The low limits of detections of artemisinin, artesunate, dihydroartemisinin, and artemether were found to be 0.091, 0.0125, 0.0489, and 0.0128 ng mu L-1, respectively. The developed methods were precise (RSD <3%) and accurate (% error <5%). The developed methods may find application in dosage form analysis and pharmacokinetic studies.