Competitive-binding approach to liquid chromatographic postcolumn reactions with fluorimetric detection

A model mixture consisting of biotin and biocytin was used to test the feasibility of applying a competitive-binding approach in the development of liquid chromatographic (LC) postcolumn reactions with fluorimetric detection. Specifically, the effluent from the LC column was merged with a reagent stream containing the biological binder avidin, binding sites of which were occupied by the fluorescent probe 2-anilinonaphthalene-6-sulfonic acid (2,6-ANS). The probe was displaced by the analytes from the avidin-2,6-ANS complex with a concomitant decrease in the fluorescence intensity at 438 nm. This decrease was proportional to the concentration of biotin and biocytin and constituted the analytical signal. The procedure was optimized with respect to LC separation conditions, reagent concentrations and flow-rate of the reagent solution. Analytical characteristics of the method were determined and compared with those for LC determinations of biotin and biocytin with absorptiometric detection. Finally, the proposed reaction detection system was validated by using it for the determination of biotin in vitamin tablets and a horse-feed supplement.