Determination of chloroquine and desethylchloroquine in biological samples using perfusion chromatography and fluorescence detection

A high-speed reversed phase HPLC method for the determination of chloroquine and desethylchloroquine in biological samples is presented. perfusion chromatography, based on the use of now-through particles made of cross-linked polystyrene/divinylbenzene, allowed to increase the flow-rate of the mobile phase to 4 mL/min, resulting in base-line separation of bis-desethylchloroquine, desethylchloroquine, chloroquine, and the internal standard in less than 3 minutes. As the column material withstands prolonged exposure to high pH conditions, the pH of the mobile phase could be adjusted to 9.5, enabling direct determination of chloroquine and desethylchloroquine by fluorescence detection after elution; therefore, no post-column alkalinization to obtain the pH of optimal fluorescence was required. Interday as well as intraday reproducibility for chloroquine concentrations ranging from 31.3 to 500 ng/mL in plasma was lower than 7%. The applicability of the method is illustrated by measuring blood concentrations of chloroquine and desethylchloroquine in blood during 1 week after oral intake of 500 mg chloroquine.