VALIDATION OF AN IMPROVED REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY ASSAY WITH REDUCTIVE ELECTROCHEMICAL DETECTION FOR THE DETERMINATION OF ARTEMISININ DERIVATIVES IN MAN

For the determination of artemisinin (ART) and analogs, a reversed-pha se high-performance liquid chromatography method using reductive elect rochemical detection (ED) was set up with some important modifications as compared to previously published assays. A different technique of deoxygenating resulted in a factor 2-3 lower background current. A Spe ctroflow 400 liquid chromatograph in combination with a Triathlon auto injector coupled to a Decade electrochemical detector was used. The de tector was operated in the reductive mode as a closed system under chr omatography grade helium to exclude any access of oxygen. The Decade h as a glassy carbon electrode and a reference Ag/AgCl electrode. Infreq uent electropolishing was required implicating a very stable system. B y increasing acetonitril or lowering the pH of the mobile phase, the v arious derivatives could be determined in the same chromatogram. The a ssay was validated using artemether (ATM) and dihydroartemisinin (DHA) as test substances. In the concentration range seen in people after u sual doses (5 to 220 ng/ml), the assay performs with adequate accuracy and precision. The interassay and intraassay precision are <6% for AT M. For DHA, the interassay and intraassay precision are <9%. The accur acy expressed as the deviation from the expected concentration varies from -1% to +4.5% for the intraassay ATM-determinations and from +1% t o +6.3% for the interassay measurements. For DHA, the accuracy is some what less, varying from -0.3% to -9.5% for the intraassay measurements and -0.6% to +2.6% for the interassay measurements. The reproducibili ty of the assay, measured over a time period of 3 months, is good for ATM and DHA with an interassay precision of <18% in 70 repetitive samp les and an accuracy varying from -0.6% to +7.6%. In a cross-check with two other reference laboratories who used comparable methods of deter mination, a strong correlation (correlation coefficient >0.98) was ach ieved. The method was applied in a study in which artemether was admin istered orally to healthy white subjects. We consider high-performance liquid chromatography with electrochemical detection an accurate and precise method for quantitative determination of artemisinin derivativ es in pharmacokinetic studies.