A HPLC Method for the Separation and Quantification of the Enantiomers of Hydroxychloroquine and Its Three Major Metabolites

Abstract

Abstract A two step HPLC method for analysis of the enantiomers of Hydroxychloroquine (HCQ) and its three major metabolites, Desethylhydroxychloroquine (DHCQ), Desethylchloroquine (DCQ) and Bisdesethylchloroquine (BDCQ), was developed. Fluorescence detection was used at λex=230nm and λem=385nm with a 370nm cut-off filter. This method has higher sensitivity and better resolution of the parent drug and its three metabolites when compared to published methods. In the first step, a cyano column was used to separate and collect fractions containing HCQ and its three metabolites. The mobile phase was 20% pH 6.0 (0.015M K2HPO4) buffer, 30% methanol and 50% acetonitrile at a flow rate of 2 ml/min and 50°C. This method was linear over the concentration ranges of 2–20, 20–300 and 200–2000 ng/ml of blood (r>0.99) and was used for quantitation. In a second step, chiral separation was performed on a chiral-AGP column using a mobile phase of 94% pH 7.0 (0.05M NH4H2PO4, 0.005M dihexylamine) buffer, 5% isopropanol and 1% acetonitrile at a flow rate of 1 ml/min and 35°C. Baseline separation was obtained for the enantiomers of the parent drug and its metabolites. Resolution of the enantiomers on the chiral column for HCQ, DHCQ, DCQ and BDCQ was 3.2, 3.2, 2.9 and 2.5, respectively. The limit of detection for HCQ and its metabolites was less than 1 ng for each enantiomer.