Summary - B6 by HPLC
EN 14164:2008 Foodstuffs - Determination of vitamin B6 by HPLC:
This European Standard specifies a method for the determination of vitamin B6 in foodstuffs by high performance liquid chromatography (HPLC). Vitamin B6 is a mass fraction of the sum of pyridoxine, pyridoxal, pyridoxamine including their phosphorylated derivatives determined as pyridoxine. The β-glycosylated forms are not taken into account. These can be determined with the method given in EN14164 by which the different vitamer of vitamin B6 (pyridoxal, pyridoxamine and pyridoxin) are separated and individually quantified. A third European Standard (EN 14166) determines the total vitamin B6 by microbiological assay.
Pyridoxal, pyridoxamine and pyridoxine are extracted from food by acid hydrolysis and dephosphorylated enzymatically using acid phosphatase.
By reaction with glyoxylic acid in the presence of Fe2+ as a catalyst, pyridoxamine is transformed into pyridoxal, which is then reduced to pyridoxine by the action of sodium borohydride in alkaline medium. Pyridoxine is then quantified in the sample solution by HPLC with a fluorometric detection.