Fluorescence detection of biotin using post-column derivatization with OPA in high performance liquid chromatography

A sensitive and selective high-performance liquid chromatographic method with post-column derivatization, using o-phthalaldehyde (OPA) and 3-mercaptopropionic acid (3-MPA), is described for the determination of biotin in pharmaceutical preparations. The use of 3-MPA gives intense fluorescent derivative and improves the stability of biotin fluorophore towards oxidation to the picomole level. The fluorophore was detected at 453 nm (excitation at 342 nm). The calibration graph was linear for 20-200 ng per injection. The detection limit of biotin under these conditions was about 10 ng per injection. The RSDs were 1.9-3.4%. This method could be applied to pharmaceutical preparations without interference of other compounds.