Sensitive and selective liquid chromatographic postcolumn reaction detection system for biotin and biocytin using a homogeneous fluorophore-linked assay

A homogeneous fluorophore-linked assay was used to develop a posteolumn reaction detection system for high-performance liquid chromatography (HPLC). Biotin and biocytin were chosen as the model analytes. The effluent from the HPLC column was merged with a reagent stream containing avidin that was labeled with fluorescein isothiocyanate (avidin-FITC). The binding of the separated analytes by the labeled avidin was accompanied by an enhancement of the fluorescence intensity at 520 nm. This increase in fluorescence was proportional to the concentration of the analytes and constituted the analytical signal. The procedure was optimized with respect to the reagent concentration and the flow-rate of the reagent solution. Analytical characteristics of the method were determined. The procedure was highly selective for biotin and its derivatives. The detection limits for biotin and biocytin were 89 and 94 pg, respectively, for 20-μl injections. The developed postcolumn reaction detection system was validated by determining biotin in a liquid vitamin preparation and a horse-feed supplement.