DETERMINATION OF ARTEMETHER AND ITS METABOLITE, DIHYDROARTEMISININ, IN PLASMA BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY AND ELECTROCHEMICALDETECTION IN THE REDUCTIVE MODE

An analytical method for the determination of artemether (A) and its m etabolite dihydroartemisinin (DHA) in human plasma has been developed and validated. The method is based on high-performance liquid chromato graphy (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). Chromatogr aphy was performed on a Nova-Pak CN, 4 mu m analytical column (150 mmx 3.9 mm I.D.) at 35 degrees C. The mobile phase consisted of pH 5 aceta te-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 fr om the relative recoveries (found concentration in % of the nominal va lue) of spiked samples analysed on the same day (concentration range 1 0.9 to 202 ng/ml of A and 11.2 to 206 ng/ml of DHA in plasma). The mea n 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 lim it of quantitation (LOQ) of 10.9 ng/ml with a C.V. of 13%. For DHA, th e mean recovery was 100% at the LOQ of 11.2 ng/ml with a C.V. of 16%.