Use of the affinity/HPLC method for quantitative estimation of folic acid in enriched cereal-grain products

In 1998, the United States introduced mandatory fortification of enriched cereal-grain products with folic acid to reduce the incidence of neural tube defects. As a consequence, substantial amounts of folic acid, the synthetic form of folate, were added to the American diet, and the ability to assess folic acid intake took on greater importance. The purpose of the current study was to separate and quantify folic acid and 5-methyltetrahydrofolate, the most prominent naturally occurring folate in fortified foods, with a reliable and robust method. Folates were heat-extracted from food samples. A trienzyme treatment (alpha-amylase, rat plasma conjugase, and protease) was applied to the extracts followed by purification by affinity chromatography. Folic acid and 5-methyltetrahydrofolate were separated and quantified by reversed-phase HPLC with fluorescence and UV detection. A gradient elution with phosphate buffer and acetonitrile was used to separate the different forms of folates. The method gave a linear response in a range of 0.1-3 mumol/L and 0.0125-0.25 mumol/L for folic acid and 5-methyltetrahydrofolate, respectively. These ranges were similar to the expected levels in the samples. The CV of the peak areas of folic acid and 5-methyltetrahydrofolate for 5 commercial wheat flour samples extracted and run separately on the same day was 2.0 and 5.7% and, run over 5 consecutive days, was 7.2 and 7.3%, respectively. Total folate values in 45 samples of fortified food measured by HPLC and by the traditional microbiological assay demonstrated a high correlation (r(2) = 0.986).