DIN EN 14164:2014-08

This European Standard specifies a method for the determination of vitamin B6 in foodstuffs by high performance liquid chromatography (HPLC). Vitamin B6 is the mass fraction of the sum of pyridoxine (PN), pyridoxal (PL) and pyridoxamine (PM) 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 EN 14663 by which the different vitamins of vitamin B6 (pyridoxal, pyridoxamine and pyridoxine) are separated and individually quantified. A third European Standard (EN 14166) determines the total vitamin B6 content by microbiological assay. In this standard, 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 fluorescence detection. This document has been prepared by Technical Committee CEN/TC 275 "Food analysis - Horizontal methods", the secretariat of which is held by DIN (Germany). The responsible German body is Working Committee NA 057-01-13 AA "Vitamine und Carotinoide" ("Vitamins and carotinoids") at the Food and Agricultural Products Standards Committee (NAL) at DIN.