Simple high-performance liquid chromatographic method for the determination of all seven vitamin B6-related compounds

A simple, sensitive, isocratic high-performance liquid chromatographic method has been developed for the separation of all seven vitamin B6-related compounds. The separation is accomplished using an ODS column and a mobile phase of 0.15 M sodium dihydrogenphosphate, adjusted to pH 2.5 with perchloric acid. The concentration of the compounds is determined with a fluorescence detector (excitation, 290 nm; emission, 389 nm). Isopyridoxal is used as an internal standard. The fluorescence intensity of pyridoxal-5'-phosphate is enhanced by post-column derivatization with sodium bisulfite. All seven compounds are separated in less than 20 min at a flow-rate of 1 ml/min. Applications of this method to yeast cell-free culture media, baker's yeast extract, egg and milk are presented.