High-performance liquid chromatographic determination of biotin in biological materials after crown ether-catalyzed fluorescence derivatization with panacyl bromide.
This paper reports a high-performance liquid chromatographic (HPLC) technique to determine biotin in biological samples. Biotin and the internal standard dethiobiotin are converted into fluorescent derivatives by using panacyl bromide [p-(9-anthroyloxy)phenacyl bromide] as a fluorescence label. Biotin is extracted from biological tissue with trichloroacetic acid and the extract is purified by a combination of solid-phase extraction on C18 cartridges, ion-exchange chromatography on DOWEX formate resin, and thin-layer chromatography. The purified sample extract is derivatized in the presence of a crown ether. The resulting panacyl esters can be separated on reversed-phase as well as on normal-phase HPLC. Normal phase HPLC is preferable because it provides higher sensitivity and demands less sample pretreatment. Analysis of rat intestinal tissue revealed that only about 13% of the biotin is present in free form whereas 87% is bound in proteins from which it can be released by hydrolysis. Biotin values determined by this method are comparable to those obtained by other techniques.
Stein
J
J
Institute of Nutrition, Justus-Liebig University, Giessen, Germany.
Hahn
A
A
Lembcke
B
B
Rehner
G
G
D000098:Acetophenones / Q000737:chemistry*; D000818:Animals; D001710:Biotin / Q000032:analysis*; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D004988:Ethers, Cyclic / Q000737:chemistry*; D005453:Fluorescence; D007421:Intestine, Small / Q000737:chemistry*; D008099:Liver / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D051381:Rats
D000098:Acetophenones; D004988:Ethers, Cyclic; D001710:Biotin; C051154:panacyl bromide