High-Performance Liquid Chromatographic Assay for Chlorquine and its Two Major Metabolites, Desethylchloroquine and Bidesethylchloroquine in Biological Fluids

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Abstract
A high-performance liquid chromatography (HPLC) method for the determination of chloroquine and its two major metabolites in biological fluids is described. Hydroxychloroquine is used as an internal standard (I.S.). Drug, metabolites and I.S. were extracted as bases with diethyl ether by a single step procedure. After drying and evaporation of the organic phase, the residue was dissolved into the mobile phase and injected into the chromatographic system. Separation was performed using a normal phase column (Inertsil sill with mixture of acetonitrile, methanol and ammonia as mobile phase. The detection was carried out by fluorescence measurement : excitation wavelength was set at 325 nm and emission at 380 nm. The limit of detection was near 3.7 ng ml−1 for chloroquine and metabolites. No chromatographic interference could be detected by endogenous compounds or other antimalarial drugs. Because of the good accuracy of the method, concentrations were determinated with a relative standard deviation lower than 7% at the 25 ng ml−l level for all substances. An excellent precision was obtained over the range of concentrations tested, 25–1000ng ml−l. This method can be applied to therapeutic, pharmacokinetic and epidemial studies.

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