CHAPTER 22: Enzymatic HPLC Assay for all Six Vitamin B₆ Forms

A method for quantitating all six (three free forms and three phosphoester forms) vitamin B₆ forms and pyridoxine-β-glucoside (PNG) in foods and human-derived samples has been developed. These compounds are specifically converted into the highly fluorescent 4-pyridoxolactone (4-PLA), which is then measured by high-performance liquid chromatography (HPLC). Pyridoxal 4-dehydrogenase (PLDH) converts pyridoxal into 4-PLA. Pyridoxine 4-oxidase and PLDH converts pyridoxine (PN) into 4-PLA. Pyridoxamine-pyruvate aminotransferase (PPAT) and PLDH converts pyridoxamine (PM) into 4-PLA. The three phosphoester forms (PLP, PMP and PNP) are first converted into their free forms by alkaline phosphatase and then converted into 4-PLA; PNG is first converted into PN by β-glucosidase. Thus, seven sets of reaction conditions are used to quantitate the seven compounds. A sample preparation method for this method is also described. The results of food analysis showed that the PN content was lower than those of the other vitamin B₆ forms. Thus, this method makes it possible to examine the mechanisms underlying the absorption, circulation, metabolism and excretion of vitamin B₆ when animals and human consume usual foods.