Selective and sensitive liquid chromatographic assay of amodiaquine and desethylamodiaquine in whole blood spotted on filter paper

Abstract

We have developed a sensitive, selective and reproducible reversed-phase HPLC method with ultraviolet detection (340 nm) for the simultaneous quantification of amodiaquine (AQ) and its major metabolite, desethylamodiaquine (AQm) in a small volume (200 microl) of whole blood spotted on filter paper. The method involves liquid-liquid extraction with diethyl ether followed by elution from a reversed-phase phenyl column with an acidic (pH 2.8) mobile phase (25 mM KH2PO4-methanol; 80:20% (v/v) +1% (v/v) triethylamine). Calibration curves in spiked whole blood were linear from 100-2500 ng/ml (r2 > or = 0.99) for AQ and 200-2500 ng/ml (r2 > or = 0.99) for AQm. The limit of detection was 5 ng for AQ and 10 ng for AQm. The relative recovery at 150 ng/ml of AQ (n = 6) was 84.0% and at 300 ng/ml of AQm the relative recovery was 74.3%. The intra-assay coefficients of variation at 150, 600 and 2250 ng/ml of AQ and 300, 600 and 2250 ng/ml of AQm were 7.7, 8.9 and 6.2% (AQ) and 10.1, 5.4 and 3.9% (AQm), respectively. The inter-assay coefficient of variation at 150, 600 and 2250 ng/ml of AQ and 300, 600 and 2250 ng/ml of AQm were 5.2, 8.1 and 6.9% (AQ) and 3.3, 2.3 and 4.6% (AQm). There was no interference from other commonly used antimalarial and antipyretic drugs (chloroquine, quinine, sulfadoxine, pyrimethamine, artesunate, acetaminophen and salicylate). The method is particularly suitable for pharmacokinetic studies in settings where facilities for storing blood/plasma samples are not available.