Analytical and semi-preparative high-performance liquid chromatographic separation and assay of hydroxychloroquine enantiomers

Abstract

(±)-Hydroxychloroquine (HCQ) is an antimalarial and anti-arthritic drug which is administered as the racemate. An accurate, precise and sensitive high-performance liquid chromatographic assay was developed for the determination of HCQ enantiomers in samples from human plasma, serum, whole blood, and urine. After addition of (±)-chloroquine (internal standard), samples of blood component (0.5 ml) or urine (0.1 ml) were alkalinized and extracted with 5 ml of diethyl ether. After solvent evaporation the residues were derivatized with (+)-di-O-acetyl- l-tartaric anhydride at 45°C for 30 min. The resulting diastereomers were then resolved using a C 8 analytical column with a mobile phase consisting of 0.05 M KH 2PO 4 (pH 3)—methanol—ethanol—triethylamine (78:22:1:0.08). The ultraviolet detection wavelength was set at 343 nm. The derivatized HCQ enantiomers eluted in less than 40 min, free of interfering peaks. Excellent linear relationships ( r 2 > 0.997) were obtained between the area ratios and the corresponding plasma concentrations over a range of 12.5–500 ng/ml. The diastereomers could be hydrolysed using microwave energy and neutral pH, which enabled us to resolve the enantiomers on a semi-preparative (C 18 column) scale. The method was suitable for the analysis and semi-preparative separation of HCQ enantiomers.