Simple high-performance liquid chromatographic method with electrochemical detection for the simultaneous determination of artesunate and dihydroartemisinin in biological fluids.

A simple, rapid, sensitive, selective and reproducible high-performance liquid chromatographic method with reductive electrochemical detection is described for the simultaneous quantification of artesunate (ARS) and dihydroartemisinin (DHA) in plasma. The procedure involved the extraction of ARS, DHA and the internal standard (artemisinin, ARN) with a mixture of dichloromethane and tert.-methyl butyl ether (8:2, v/v). Chromatographic separation consisted of the mobile phase (acetonitrile-water containing 0.1 M acetic acid, pH 4.8; 45:55, v/v) running through the column (Nova-Pak C18, 150 cm x 3.9 mm I.D., 5 microm particle size). The retention times of alpha-DHA, beta-DHA, ARS and ARN were 2.9, 4.2, 4.5 and 6.0 min, respectively. The average recoveries of ARS, alpha-DHA and ARN in the concentration range of 10-800 ng/ml were 81.9, 88.2, 101.1 and 84.3%, respectively. The coefficients of variation (precision and repeatability) were below 10% for all three compounds at concentrations of 50, 200, 400 and 800 ng/ml, and below 20% at a concentration of 10 ng/ml. The limits of quantification for both ARS and alpha-DHA in spiked plasma samples were 5 and 3 ng/ml, respectively. The method was found to be suitable for application to pharmacokinetic studies of both ARS and DHA.