Determination of artemether and its metabolite, dihydroartemisinin, in plasma by high-performance liquid chromatography and electrochemical detection in the reductive mode

An analytical method for the determination of artemether (A) and its metabolite dihydroartemisinin (DHA) in human plasma has been developed and validated. The method is based on high-performance liquid chromatography (HPLC) and electrochemical detection in the reductive mode. A, DHA and artemisinin, the internal standard (I.S.), were extracted from plasma (1 ml) with 1-chlorobutane-isooctane (55:45, v/v). The solvent was transferred, evaporated to dryness under nitrogen and the residue dissolved in 600 mu l of water-ethyl alcohol (50:50, v/v). Chromatography was performed on a Nova-Pak CN, 4 mu m analytical column (150 mmx3.9 mm I.D.) at 35 degrees C. The mobile phase consisted of pH 5 acetate-acetonitrile (85:15, v/v) at a flow-rate of 1 ml/min. The analytes were detected by electrochemical detection in the reductive mode at a potential of -1.0 V. Intra-day accuracy and precision were assessed from the relative recoveries (found concentration in % of the nominal value) of spiked samples analysed on the same day (concentration range 10.9 to 202 ng/ml of A and 11.2 to 206 ng/ml of DHA in plasma). The mean recoveries over the entire concentration range were from 96 to 100% for A with CN, from 6 to 13%, from 92% to 100% for DHA (alpha-tautomer) with C.V. from 4 to 16%. For A, the mean recovery was 96% at the limit of quantitation (LOQ) of 10.9 ng/ml with a C.V. of 13%. For DHA, the mean recovery was 100% at the LOQ of 11.2 ng/ml with a C.V. of 16%.