ONLINE IMMUNOCHEMICAL DETECTION IN LIQUID-CHROMATOGRAPHY USING FLUORESCEIN-LABELED ANTIBODIES

A postcolumn immunochemical detection system for on-line coupling to HPLC is described. The effluent from a reversed-phase LC column is mixed with fluorescein-labelled antibodies that are added via a mixing union. Antigenic analytes react with the antibodies to form strongly fluorescent immunocomplexes. In a second step, free antibodies are removed prior to fluorescence detection via passage through a small column packed with an antigen-bound support. The performance of the immunochemical reaction system was investigated using digoxin and its metabolites as analytes and fluorescein-labelled Fab fragments of polyclonal anti-digoxigenin as immunoreagent. This system tolerates up to 95% methanol or 45% acetonitrile in the LC eluent, allowing the separation of digoxin and its metabolites. The immunoreaction sequence is in equilibrium after ca. 1 min resulting in peak broadening comparable to that in standard postcolumn derivatization systems. The detection limits obtained for digoxin and digoxigenin after separation on a C18 column are 200 and 50 fmol, respectively. The applicability of the method is demonstrated for the bioanalysis of digoxin and digoxigenin. Owing to the high selectivity of the immunodetection system, sample pretreatment can be reduced to deproteination and dilution of plasma and urine samples. Detection limits in both matrices (100-mul injections) are 1 . 10(-9) M for digoxigenin and 4 . 10(-9) M for digoxin.