Validation of high performance liquid chromatography-electrochemical detection methods with simultaneous extraction procedure for the determination of artesunate, dihydroartemisinin, amodiaquine and desethylamodiaquine in human plasma for application in clinical pharmacological studies of artesunate-amodiaquine drug combination.

With the expanded use of the combination of artesunate (AS) and amodiaquine (AQ) for the treatment of falciparum malaria and the abundance of products on the market, comes the need for rapid and reliable bioanalytical methods for the determination of the parent compounds and their metabolites. While the existing methods were developed for the determination of either AS or AQ in biological fluids, the current validated method allows simultaneous extraction and determination of AS and AQ in human plasma. Extraction is carried out on Supelclean LC-18 extraction cartridges where AS, its metabolite dihydroartemisinin (DHA) and the internal standard artemisinin (QHS) are separated from AQ, its metabolite desethylamodiaquine (DeAQ) and the internal standard, an isobutyl analogue of desethylamodiaquine (IB-DeAQ). AS, DHA and QHS are then analysed using Hypersil C4 column with acetonitrile-acetic acid (0.05M adjusted to pH 5.2 with 1.00M NaOH) (42:58, v/v) as mobile phase at flow rate 1.50ml/min. The analytes are detected with an electrochemical detector operating in the reductive mode. Chromatography of AQ, DeAQ and IB-DeAQ is carried out on an Inertsil C4 column with acetonitrile-KH(2)PO(4) (pH 4.0, 0.05M) (11:89, v/v) as mobile phase at flow rate 1.00ml/min. The analytes are detected by an electrochemical detector operating in the oxidative mode. The recoveries of AS, DHA, AQ and DeAQ vary between 79.1% and 104.0% over the concentration range of 50-1400ng/ml plasma. The accuracies of the determination of all the analytes are 96.8-103.9%, while the variation for within-day and day-to-day analysis are <15%. The lower limit of quantification for all the analytes is 20ng/ml and limit of detection is 8ng/ml. The method is sensitive, selective, accurate, reproducible and suited particularly for pharmacokinetic study of AS-AQ drug combination and can also be used to compare the bioavailability of different formulations, including a fixed-dose AS-AQ co-formulation.