Class-Selective Detection System for Liquid Chromatography Based on the Streptavidin-Biotin Interaction

Abstract

A protein binding assay (BA) was coupled with high-performance liquid chromagraphy (HPLC) to provide a highly sensitive postcolumn reaction detection system for the biologically important molecule biotin and its derivatives biocyhn, biotin ethylenediamine, 6-(biotinoylamino)caproic acid, and 6-(biotinoylamino)caproic acid hydrazide. This detection system is selective for the biotin moiety and responds only to the class of compounds that contain biotin in their molecules. In this assay, a conjugate of streptavidin with fluorescein isothiocyanate (streptavidin-FITC) was employed. Upon binding of the analyte (biotin or biotin derivative) to streptavidin-FITC, an enhancement in fluorescence intensity results. This enhancement in fluorescence intensity can be directly related to the concentration of the analyte and thus serves as the analitycal signal. The HPLC-BA system is more sensitive and selective than either the BA or HPLC alone. With the described system, the detection limits for biotin and biocytin were found to be 97 and 149 pg, respectively. Finally, the analysis of an infant formula and several cell culture media, with little or no sample preparation, demonstrated the analytical utility of this system