Determination of chloroquine and desethylchloroquine in plasma and blood cells of Plasmodium vivax malaria cases using liquid chromatography

Abstract

Chloroquine and de-ethylchloroquine were extracted from plasma and blood cells by addition of 80 µl internal standard [2.5 µg/ml of 4-(4-dimethylamino-1-methylbutylamino)-7-chloroquinoline], 1 ml 1M-NaOH and 6 ml diethyl ether with shaking for 15 min. After centrifugation at 1000 g for 10 min, the ether layer was evaporated under N2 and the residue was dissolved in mobile phase. HPLC was performed on a 5 µm µPorasil column ( 30 cm × 3.9 mm i.d.) with dichloromethane/methanol/1M-perchloric acid (100:9:1.2) as mobile phase (1 ml/min) and UV detection at 343 nm or fluorimetric detection at 380 nm (excitation at 340 nm). Calibration graphs were linear from 0.025-1 µg/ml and the detection limit was 5-10 ng. Within-day and day-to-day RSD (n=5) in plasma for chloroquine were 2.3-2.76% and 2.83-3.36% for de-ethylchloroquine. Recoveries were 89.7-91.05%.