Capillary electrophoretic chiral separation of hydroxychloroquine and its metabolites in the microsomal fraction of liver homogenates

A rapid, selective, and low‐cost chiral capillary electrophoretic method was developed for the simultaneous analysis of hydroxychloroquine (HCQ) and its three chiral metabolites: desethylchloroquine (DCQ), desethylhydroxychloroquine (DHCQ), and bisdesethylchloroquine (BDCQ) in the microsomal fraction of liver homogenates. After liquid–liquid extraction using toluene as extracting solvent, the drug and metabolites were resolved on a fused‐silica capillary (50 μm ID, 50 cm total length, and 42 cm effective length), using 100 mmol/L of Tris/phosphate buffer, pH 9.0 containing 1% w/v sulfated‐β‐CD and 30 mg/mL hydroxypropyl‐β‐CD. Detection was carried out at 220 nm. The extraction procedure was efficient in removing endogenous interferents, and low values (≤15%) for CVs and deviation from theoretical values were demonstrated for both within‐day and between‐day assays. The quantitation limit was 125 ng/mL with linear response over the 125–2000 ng/mL of concentration range for all metabolites. After validation, the method was used for an in vitro metabolism study of HCQ. The major HCQ metabolite formed by microsomal enzymes was (−)‐(R)‐DHCQ.