Analysis of blood and urine samples for hydroxychloroquine and three major metabolites by high-performance liquid chromatography with fluorescence detection.

A high-performance liquid chromatographic (HPLC) method using fluorescence detection is described for the quantification of hydroxychloroquine (HCQ) and three of its metabolites in blood and urine samples. The method is selective, permitting quantification of analytes without interferences from chloroquine or quinine in the sample. Detection limits for HCQ, desethylhydroxychloroquine, desethylchloroquine, and bisdesethylchloroquine are 10, 30, 5, and 5 ppb, respectively, for a 100-microliters blood or urine sample. The internally standardized method requires only one extraction step and utilizes normal-phase HPLC conditions including an amine modifier in the mobile phase. These conditions facilitate fluorescence detection, selective separation, and acceptable peak shapes. A mobile phase of 0.5% n-butylamine in methanol-hexane-methyl tert. butyl ether (1:1:1) is used in the analysis. Analysis of blood and urine samples from two healthy volunteers given 400 mg of Plaquenil (310 mg of HCQ base) weekly for four weeks provided data on HCQ metabolism for the two persons during the recommended chemoprophylactic regimen for malaria.