Characterization of competitive binding, fluorescent drug immunoassays based on micellar electrokinetic capillary chromatography

Abstract

This paper characterizes competitive binding, electrokinetic capillary-based immunoassays for various drugs in human serum using reagents which were commercialized for fluorescence polarization immunoassays. After incubation of serum with the reactants, a small aliquot of the mixture is applied onto a fused-silica capillary and tracers (fluorescein-labeled drugs) and the antibodytracer complexes are separated and analyzed by micellar electrokinetic capillary chromatography with on-column laser-induced fluorescence detection. Examples studied include serum assays for theophylline, ethosuximide, paracetamol, salicylate and quinidine. With these assays, concentration-dependent peaks produced by the free tracers or the antibody-tracer complexes serve as the basis for quantitation. The sizes of the peaks produced are shown to be dependent on the applied power and the proportions of the reactants and serum employed. The separation medium permits effective characterization of tracers and antibody selectivities. Based on the high selectivity of the antibodies employed, the feasibility of the simultaneous performance of different immunoassays is demonstrated. For capillaries of 50 μm internal diameter (ID), separations are best performed at electric fields < 500 V/cm, this resulting in electrokinetic analyses within 4 to 10 min (capillaries of 20 to 50 cm effective length).