Stereoselective determination of hydroxychloro‐quine and its major metabolites in human urine by solid‐phase microextraction and HPLC

The enantioselective analysis of hydroxychloroquine (HCQ) and its major metabolites was achieved by HPLC and solid‐phase microextraction. The chromatographic separation was performed on a Chiralcel OD‐H column using hexane/methanol/ethanol (96:2:2, v/v/v) plus 0.2% diethylamine as the mobile phase, at the flow rate of 1.3 mL/min. The main extraction parameters were optimized. The best condition was achieved by the addition of 10% NaCl and 1 mL phosphate buffer 1 mol/L pH 11 to 3 mL human urine. The extraction was conducted for 40 min at 25°C and the desorption time was 3 min using methanol (100%). PDMS‐DVB 60 μm fiber was used in this study. The mean recoveries were 9.3, 9.2, and 14.4% for HCQ, desethylhydroxychloroquine (DHCQ), and desethylchloroquine (DCQ), respectively. The method was linear over the range of 50–1000 ng/mL for HCQ enantiomers and over the range of 42–416 ng/mL for DCQ and DHCQ enantiomers. Within‐day and between‐day precision and accuracy assays for HCQ and its metabolites were lower than 15%. The preliminary 48 h urinary excretion study performed in human urine showed to be stereoselective. The amount of (+)‐( S )‐enantiomer excreted was higher than its antipode.