All-enzymatic HPLC method for determination of individual and total contents of vitamin B6 in foods

Background : There is a need for a reliable and accurate method for quantification of each of the seven individual vitamin B6 compounds including pyridoxine-β-glucoside in foods. Objective : To determine pyridoxal (PL), pyridoxamine (PM), pyridoxine (PN), pyridoxal 5′-phosphate (PLP), pyridoxamine 5′-phosphate (PMP), pyridoxine 5′-phosphate (PNP), and pyridoxine-β-glucoside (PNG) in foods. Design : By specific enzymatic treatment, each of the seven vitamin B6 compounds was all converted into 4-pyridoxolactone, which is a highly fluorescent compound. In total, seven separate, enzymatic steps were performed for each sample. Separation and quantification were performed with reversed-phase high performance liquid chromatography (HPLC) coupled with fluorescence detection. For each sample type the result was corrected for the recovery based on spiked samples. The method was applied for analyses of chicken liver, chicken white meat, egg yolk, egg white, dried anchovy, carrots, and garlic. Results : The recovery varied from 14 to 114% in chicken liver, chicken white meat, egg yolk, egg white, dried anchovy, carrot, and garlic. Each food showed a characteristic distribution of the seven vitamin B6 compounds. The PNG was only found in low amounts; that is, 17–29 nmol vitamin B6/g in the plant-derived foods, carrot and garlic. Only egg white showed a lower content, 3 nmol/g. Overall the content in chicken liver, chicken white meat, and egg yolk had a total content of vitamin B6 between 42 and 51 nmol/g. Both PM and PMP were high in the chicken liver. In contrast, PL and PLP were high in the chicken white meat. The main vitamin B6 in the egg yolk was PLP. The dried anchovy contained high amounts of PLP and PMP and a total content of 144 nmol/g. Conclusions : The enzymatic-based HPLC method was applied for the determination of seven vitamin B6 compounds in foods. Their distribution in the foods varied significantly.