SIMULTANEOUS DETERMINATION OF CHLOROQUINE, PROGUANIL AND THEIR METABOLITES IN HUMAN BIOLOGICAL-FLUIDS BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

A reversed-phase ion-pair high-performance liquid chromatographic method with ultraviolet detection is described for the simultaneous measurement of chloroquine, proguanil and their major metabolites in human plasma, erythrocytes and urine. After a liquid-solid extraction on a Bond Elut(R) C8 cartridge, the compounds are separated on a C8 Lichrospher 60 RP select B column by isocratic elution; the mobile phase is water-acetonitrile-methanol (78:28:4, v/v/v) with 0.5 M ammonium formate and 0.075 M perchloric acid. The eluent is monitored with an ultraviolet detector at 254 nm. The lower limits of quantification in plasma are near 6.0 ng ml-1 for chloroquine and near 9.0 ng ml-1 for proguanil. No chromatographic interference can be detected from endogenous compounds or from other antimalarial drugs. The method is accurate and precision is good with inter- and intra-assay relative standard deviations lower than 6.8% for plasma samples. N-(2-6 dichlorobenzylidene amino)guanidine is used as an internal standard. The chromatographic procedure takes 35 min and can be used for therapeutic drug monitoring and clinical studies.