Combined affinity and ion pair column chromatographies for the analysis of food folate

Abstract

Folate in nature exists in a variety of forms that differ by the state of oxidation, one carbon substitution of the pteridine ring, and by the number of glutamate residues. A method was developed in our laboratory (Selhub, 1989) that uses affinity chromatography followed by ion-pair high performance liquid chromatography (HPLC) for the analysis of folate distribution in tissues with simultaneous information on the structural nature at both ends of the folate molecule. The purpose of this study was to determine if this (affinity/HPLC) method is also suitable for the analysis of food folates. A total of 10 food products were analyzed. The food items were suspended in 10 volumes of 2% ascorbate-10 mmol/L 2-mercaptoethanol at pH 7.8 and heat extracted in an autoclave for 30 min. After centrifugation, folate in the supernatant fraction was purified by affinity chromatography and analyzed by ion-pair reverse HPLC using a diode array UV detector. Results showed variability of folate distribution in the various products ranging from a single derivative of 5-methylH 4PteGlu found in egg yolk to more complex mixtures of pentaglutamyl folates in lima beans, a series of methylated tetrahydrofolates in banana, and a multiplicity of forms in yeast extracts. The method appears to be reliable, as the measured variability amounted to an average of 10%, while total folates obtained by integrating the concentration of individual folates were comparable to total folates estimated using the more traditional microbial assay method.