30] High-performance liquid chromatographic determination of biotin in biological materials after crown ether-catalyzed fluorescence derivatization with panacyl bromide

Abstract

This chapter aims to quantify biotin by high-performance liquid chromatography (HPLC) after precolumn derivatization of the carboxylic group with fluorescent panacyl bromide [ p -(9-anthroyloxy)phenacyl bromide] in the presence of crown ether. The analytical procedure should be applicable to biological samples containing biotin in picomolar amounts and in free as well as protein-bound form. To characterize the panacyl esters of biotin and dethiobiotin, 1 H nuclear magnetic resonance (NMR) spectra of the derivatization product is recorded. The substances are dried under a nitrogen stream and subsequently dissolved in [ 2 H]chloroform. From the spectra of the panacyl esters of biotin and dethiobiotin, it could be concluded that only the carboxylic function of biotin participates in the formation of the panacyl ester whereas the amide functions of the molecule could still be detected at 5.8 and 6.3 ppm. Both reversed-phase and normal-phase HPLC are well suited to achieve complete separation of the panacyl esters of biotin and dethiobiotin within 7 min. The fluorescence maxima of both derivatives are found to be 380 and 470 nm for excitation and emission wavelengths, respectively, in both mobile phases.