Capillary electrophoretic separation, immunochemical recognition and analysis of the diastereomers quinine and quinidine and two quinidine metabolites in body fluids.

Abstract

The capillary electrophoretic separation and immunochemical recognition of the two naturally fluorescing, cationic diastereomers quinine (QN) and quinidine (QD), their hydroderivatives and two major QD metabolites (3-hydroxyquinidine and quinidine-N-oxide) was investigated. Plain aqueous phosphate buffers and an alkaline buffer containing dodecyl sulfate micelles are shown to be incapable of resolving the two diastereomers. However, incorporation of an additional chemical equilibrium (with beta-cyclodextrin) in the case of capillary zone electrophoresis (CZE) and the presence of a small amount of an organic solvent as buffer modifier (2-propanol) in dodecyl sulfate based micellar electrokinetic capillary chromatography (MECC), were found to provide separation media which lead to complete resolution of QN, QD and the other compounds of interest. Furthermore, for MECC- and CZE-based immunoassay formats, a commercially available antibody against QD was found to be a perfect discriminator between QD and QN. It was determined to recognize QD and the two QD metabolites (cross reactivity of 20--30%) but not QN. MECC and CZE with laser induced fluorescence (LIF) detection are shown to be suitable to determine QD and metabolites in urine and plasma (quinidine-N-oxide only) collected after single dose intake of 50 mg QD sulfate and of QN in urine, saliva and serum samples that were collected after self-administration of 0.5 l of quinine water (25 mg of QN). With direct injection of a body fluid, MECC with LIF was found to provide 10 ng/ml detection limits for QD and QN. This ppb sensitivity is comparable to that obtained in HPLC assays that are based upon drug extraction. Furthermore, MECC and CZE assays with UV detection are shown to provide the ppm sensitivity required for therapeutic drug monitoring and clinical toxicology of QD and QN.