Determination of artemether and its major metabolite, dihydroartemisinin, in plasma using high-performance liquid chromatography with electrochemical detection

A rapid, selective, sensitive and reproducible HPLC with reductive electrochemical detection for quantitative determination of artemether (ART) and its plasma metabolite, dihydroartemisinin (DHA: alpha and beta isomers) in plasma is described. The procedure involved the extraction of ART, DHA and the internal standard, artemisinin (ARN) with dichloromethane-tert.-methylbutyl ether (1:1, v/v) or n-butyl chloride-ethyl acetate (9:1, v/v). Chromatographic separation was performed with a mobile phase of acetonitrile-water (20:80, v/v) containing 0.1 M acetic acid pH 5.0, running through a mu Bondapak CN column. The method was capable of separating the two isomeric forms of DHA (alpha, beta). The retention times of alpha-DHA, beta-DHA, ARN and ART were 4.6, 5.9, 7.9 and 9.6 min, respectively. Validation of the assay method was performed using both extraction systems. The two extraction systems produced comparable recoveries of the various analytes. The average recoveries of ART, DHA and ARN over the concentration range 80-640 ng/ml were 86-93%. The coefficients of variation were below 10% for all three drugs (ART, alpha-DHA, ARN). The minimum detectable concentrations for ART and alpha-DHA in spiked plasma samples were 5 and 3 ng/ml, respectively. The method was found to be suitable for use in clinical pharmacokinetic study.