HPLC analysis of vitamin B-6 in foods

The objective of this work was to evaluate the methods for determination of vitamin B-6 in foods. To achieve this, the literature review focused on sa mple treatment and liquid chromatographic analysis of vitamin B-6 related c ompounds. In the experimental part, the chosen sample pretreatment and the high-performance liquid chromatographic (HPLC) method were validated, and u sed to produce vitamin B-6 data on various food items commonly consumed in Finland. The main emphasis of the sample treatment was on the extraction ef ficiency and the maintenance of the original concentration profile of the v itamers. Several acid extraction procedures were tested for this purpose. P erchloric acid was chosen as the extraction agent. Routine food analysis wa s then performed using dilute ice-cold perchloric acid extraction followed by an internally standardized ion-paired reversed-phase liquid chromatograp hy. Food samples were hydrolyzed with beta-glucosidase and alkaline phospha tase enzymes, phosphorylated and glycosylated vitamers were quantitated bef ore and after the enzymatic digestion. This procedure enabled the extractio n of vitamin B-6 compounds in their intact forms, and the measurement of fr ee, phosphorylated and glycosylated forms. The maintenance of the concentra tion profile of the vitamers was verified by using C-14 -labeled pyridoxal- 5'-phosphate in the examination of the extraction procedure. The extraction efficiency and laboratory performance were confirmed by interlaboratory st udies. Up-to-date data on vitamin B-6 content of about fifty common food it ems was produced. The data includes the results from meat and poultry, fish and fish product, dairy product, cereal and vegetable, and ready-to-eat fo od samples. Free and phosphorylated vitamin B-6 compounds were measured in all food groups, and the glycosylated vitamer fraction was analyzed in all plant-derived foods. The results obtained in this work showed that vitamin B-6 content of nearly all foods of plant origin was mainly comprised of gly cosidically bound pyridoxine derivatives. These bound analytes are normally not taken into account in traditional analytical methods, and food composi tion tables lack the data of glycosylated pyridoxine. The role of the glyco sylated pyridoxines need to be clarified in terms of their analytical and p hysiological nature, if, as it is currently assumed, the availability of th e bound forms is limited for humans, the role of vegetables, cereals and ot her foods of plant-origin as a source of vitamin B-6 as well as the analyti cal methods should be reassessed.