Determination of fortified and endogenous folates in food-based Standard Reference Materials by liquid chromatography-tandem mass spectrometry

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The National Institute of Standards and Technology (NIST) is developing a wide variety of Standard Reference Materials (SRMs) to support measurements of vitamins and other nutrients in foods. Previously, NIST has provided SRMs with values assigned for the folate vitamer, folic acid (pteroylglutamic acid), which is fortified in several foods due to its role in prevention of neural tube defects. In order to expand the number of food-based SRMs with values assigned for folic acid, as well as additional endogenous folates, NIST has developed methods that include trienzyme digestion and isotope-dilution liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis. Sample preparation was optimized for each individual food type, but all samples were analyzed under the same LC-MS/MS conditions. The application of these methods resulted in folic acid values for SRM 1849a Infant/Adult Nutritional Formula and SRM 3233 Fortified Breakfast Cereal of (2.33 ± 0.06) μg/g and (16.0 ± 0.7) μg/g, respectively. In addition, the endogenous folate vitamer 5-methlytetrahydrofolate (5-MTHF) was detected and quantified in SRM 1849a Infant/Adult Nutritional Formula, candidate SRM 1549a Whole Milk Powder, and candidate SRM 1845a Whole Egg Powder, resulting in values of (0.0839 ± 0.0071) μg/g, (0.211 ± 0.014) μg/g, and (0.838 ± 0.044) μg/g, respectively. SRM 1849a Infant/Adult Nutritional Formula is the first food-based NIST SRM to possess a reference value for 5-MTHF and the first certified reference material to have an assigned 5-MTHF value based on LC-MS/MS. The values obtained for folic acid and 5-MTHF by LC-MS/MS will be incorporated into the final value assignments for all these food-based SRMs.