HPLC with ultraviolet detection for the determination of chloroquine and desethylchloroquine in whole blood and finger-prick capillary blood dried on filter paper.

A simple, sensitive, selective and reproducible method based on liquid chromatography was developed for the determination of chloroquine (CQ) and its active plasma metabolite desethylchloroquine (DECQ) in finger-pricked capillary blood spot onto filter paper (DBS) and whole blood samples. Both were separated from the internal standard quinine on a reversed phase C18 column, with the mobile phase consisting of a mixture of 1% diethylamine, acetonitrile and methanol (20:55:25, v:v:v) running at a flow rate of 1.0ml/min. Retention times of QN, DECQ and CQ were 4.5, 5.7 and 6.4min, respectively. Ultraviolet detection was at the wavelength 256nm. Sample preparation was done by extraction with hexane and tert-butyl methyl ether (1:1, v:v). Good precision and accuracy were obtained for both within-day repeatability and day-to-day reproducibility. Limit of quantification (LOQ) for both CQ and DECQ was accepted as 50ng/ml using 80μl DBS sample and 25ng/ml using 150μl whole blood sample. The mean recoveries for CQ, DECQ and internal standard for both whole blood and DBS were between 74 and 87%. The method was successfully applied for a pharmacokinetic study of CQ and DECQ in patients with Plasmodium vivax. Excellence correlation (r=0.997) was observed between the analysis of both CQ and DECQ in paired whole blood and DBS samples.