High-throughput quantitation method for amodiaquine and desethylamodiaquine in plasma using supported liquid extraction technology.

Amodiaquine is a drug used for treatment of malaria and is often used in combination with artesunate in areas where malaria parasites are still susceptible to amodiaquine. Liquid chromatography tandem-mass spectrometry was used to quantify amodiaquine and its active metabolite, desethylamodiaquine, in plasma samples. A low sample volume of 100 µl, and high-throughput extraction technique using a supported liquid extraction (SLE+) technique on an automated liquid handler platform for faster sample processing are some of the advantages of this method. Separation of amodiaquine from desethylamodiaquine was achieved using a reversed phase Zorbax SB-CN 50 mm × 4.6 mm, I.D. 3.5 µm column with acetonitrile and 20 mM ammonium formate with 1% formic acid pH ~ 2.6 (15-85, v/v) as mobile phase. The absolute recoveries of amodiaquine and desethylamodiaquine were 66% to 76%, and their isotope label internal standard were in the range of 73% to 85%. Validation results of the developed method demonstrated intra-batch and inter-batch precisions within the acceptance criteria range of ± 15.0%. There were no matrix or carry-over effects observed. The lower limit of quantification was 1.08 ng/ml for amodiaquine and 1.41 ng/ml for desethylamodiaquine. The method showed robust and accurate performance with high sensitivity. Thus, the validated method was successfully implemented and applied in the evaluation of a clinical trial where participants received artemether-lumefantrine plus amodiaquine twice daily for three days (amodiaquine dose of 10 mg base/kg/day).

[Preparation of monodesethylamodiaquine from human urine]. / Préparation de monodéséthylamodiaquine à partir d'urines humaines.

60 mg of monodesethylamodiaquine, the active metabolite of amodiaquine were prepared by extraction from human urine, at pH 11-12, using ethyl acetate. The purification was carried out by medium pressure liquid chromatography. The identity and the purity of the compound were checked by thin layer chromatography, 1H nuclear magnetic resonance and mass spectrometry.

Isolation, characterization and standardization of a major metabolite of amodiaquine by chromatographic and spectroscopic methods.

The amodiaquine metabolite 2-hydroxydesethylamodiaquine (designated metabolite II), one of the two major human metabolites of this antimalarial prodrug, is characterized by chromatographic and spectroscopic methods. This metabolite has been isolated in milligram quantities from the urine of an amodiaquine-dosed individual by extraction and preparative high-performance liquid chromatography (HPLC) and standardized using nuclear magnetic resonance spectroscopy with internal standardization. Aliquots of this standard provided accurately known amounts of the compound for spectroscopic characterization, for use as an HPLC standard and for assessment of in vitro activity against malaria parasites. Knowledge of the structure of the two major metabolites of amodiaquine (the other is desethylamodiaquine) permits speculation as to the presence of three additional human metabolites, chromatographic confirmation for one of which is demonstrated. The in vitro activity of metabolite II is shown to be 1% that of amodiaquine for two chloroquine-sensitive Plasmodium falciparum strains. Should this relationship hold generally, desethylamodiaquine is the only chemical species resulting from oral dosing with amodiaquine which contributes significantly to antimalarial activity in the blood.