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Food Nutr Res. 2012;56. doi: 10.3402/fnr.v56i0.5409. Epub 2012 Apr 2.

All-enzymatic HPLC method for determination of individual and total contents of vitamin B(6) in foods.

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Faculty of Agriculture and Agricultural Science Program, Graduate School of Integrated Arts and Sciences, Kochi University, Nankoku, Kochi, Japan.



There is a need for a reliable and accurate method for quantification of each of the seven individual vitamin B(6) compounds including pyridoxine-β-glucoside in foods.


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.


By specific enzymatic treatment, each of the seven vitamin B(6) 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.


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 B(6) compounds. The PNG was only found in low amounts; that is, 17-29nmol vitamin B(6)/g in the plant-derived foods, carrot and garlic. Only egg white showed a lower content, 3nmol/g. Overall the content in chicken liver, chicken white meat, and egg yolk had a total content of vitamin B(6) between 42 and 51nmol/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 B(6) in the egg yolk was PLP. The dried anchovy contained high amounts of PLP and PMP and a total content of 144nmol/g.


The enzymatic-based HPLC method was applied for the determination of seven vitamin B6 compounds in foods. Their distribution in the foods varied significantly.


phosphates; pyridoxal; pyridoxamine; pyridoxine; pyridoxine-β-glucoside

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