The Journal of Nutrition Methodology and Mathematical Modeling Use of the Affinity/HPLC Method for Quantitative Estimation of Folic Acid in Enriched Cereal-Grain Products 1 Rosalia Po ´ o-Prieto, 2,4 David B. Haytowitz, 3 Joanne M. Holden, 3 Gail Rogers, 2 Silvina F. Choumenkovitch, 2 Paul F. Jacques, 2 and Jacob Selhub 2 * 2 Vitamin Metabolism Laboratory and Nutritional Epidemiology Program, Jean Mayer USDA Human Nutrition Research Center on Aging, Tufts University, Boston, MA and 3 Nutrient Data Laboratory, USDA-ARS, Beltsville Human Nutrition Research Center, Beltsville, MD Abstract 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 (a-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 mmol/L and 0.0125–0.25 mmol/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 ¼ 0.986). J. Nutr. 136: 3079–3083, 2006. Introduction In 1996, the United States Food and Drug Administration (FDA) issued a regulation, effective January 1998, to fortify all en- riched cereal-grain products (flour, rice, breads, rolls, buns, pasta, corn grits, corn meal, farina, macaroni, noodle products, etc.) with folic acid (1). The primary reason for this policy was the recognition that periconceptional folate supplementation significantly reduced the incidence of neural tube defects (2,3). A secondary benefit of fortifying foods with folic acid might be a reduction in the incidence of cardiovascular disease (4) and certain types of cancer (5). The amount of folic acid added to different products ranges from 95 to 309 mg/100 g of product. This range of fortification was selected on the basis of a target level of 140 mg of folic acid/ 100 g of the cereal-grain product. Initial estimates by the FDA anticipated an increase in folate intake of between 70 and 130 mg/d in adults depending on age and consumption patterns (6). This projection assumed that each enriched cereal-grain product con- tained the amount of folic acid required by the applicable regulation. However, actual measurements of total folate content in enriched cereal-grain products previously showed that a con- siderable proportion of these products contained total folate levels that were higher than the amounts required by regulations. These results could have resulted in part from the addition of excess folic acid to fortified foods to ensure that the product contains at least the minimum amount required by regulation throughout the shelf-life of the product. Another possible explanation for these higher values is the use of improved assay methods, which measure endogenous folate at levels consider- ably higher than those using older methods (7). With so much more folic acid in the American diet and the uncertainty regarding the amount of natural folate and added folic acid in enriched cereal-grain products, along with the need to explicitly establish the amount of folic acid in these foods to estimate folate intake as Dietary Folate Equivalents (8), it was deemed essential to develop a reliable analytical method to distinguish between folic acid and endogenous folates in enriched cereal-grain products. 1 Supported by the NIH through a grant from Office of Dietary Supplements and the National Institute of Diabetes and Digestive and Kidney Diseases (R01 DK058715) and through agreement Y1-HV-8116, and by the USDA, Agricultural Research Service (agreement 58-1950-4-401). Any opinions, findings, conclu- sion, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the USDA. 4 Present address: Dpto. de Nutricio ´ n, Bromatologı ´a y Tecnologı ´a de los Alimentos, Universidad San Pablo CEU, Madrid, Spain. * To whom correspondence should be addressed. E-mail: jacob.selhub@tufts. edu. 0022-3166/06 $8.00 ª 2006 American Society for Nutrition. 3079 Manuscript received 23 January 2006. Initial review completed 20 February 2006. Revision accepted 20 September 2006. by Pamela Pehrsson on December 18, 2006 jn.nutrition.org Downloaded from