Sensitive high-performance liquid chromatographic analysis for chloroquine in body fluids. Application to studies of drug resistance in Plasmodium falciparum.

A high-performance liquid chromatographic method has been developed for the sensitive determination of chloroquine in body fluids. THe method has been applied to quality-control assay of World Health Organization (WHO) In-Vitro, Macro-Test Kits for the assessment of susceptibility of Plasmodium falciparum to chloroquine. Experiments utilizing [14C] chloroquine demonstrated that water was not capable of efficiently desorbing chloroquine from the inside surfaces of kit vials. The addition of blood to the vials effectively desorbs chloroquine. Subsequent addition of the blood to aqueous base followed by hexane extraction permits quantitation by reversed-phase, ion-pair high-performance liquid chromatography utilizing ultraviolet detection at 344 nm. The method is capable of determining as little as 20 ng of chloroquine per vial. This method, utilizing the methyl ether of 9-anthra cenemethanol as internal standard, can quantify chloroquine in 1 ml of blood or urine with a minimum detection limit of 20 ppb (ng/ml). Measurement of blood levels of chloroquine in persons contracting falciparum malaria while following a prophylactic regimen complements in-vitro drug susceptibility measurements in characterizing resistant strains of the parasite.