2009 Annual Report 1a.Objectives (from AD-416) To develop new analytical methods for water-soluble vitamins and vitamin D to replace existing, outdated methods. 1b.Approach (from AD-416) For the water-soluble vitamins, use liquid chromatography-mass spectrometry for optimized individual methods, multiple vitamin methods, and stable isotope dilution methods. For vitamin D, use liquid chromatography-mass spectrometry for an optimized individual method and a stable isotope dilution method. 3.Progress Report During the final 5 months of the project, a series of samples collected for the USDA NFNAP program consisting of four regional composites of each of 12 separate cereal brand-name products was analyzed for water soluble vitamin content. Samples of NIST Standard Reference Material #1849 (infant/adult supplement formula) were analyzed to contribute to setting the certified value for vitamin D for this reference material published in July 2009. In addition, vitamin D analyses were conducted or initiated on whole egg, milk, and seafood samples from the National Food and Nutrient Program (NFNAP) for incorporation into the National Nutrient Database for Standard Reference by the Nutrient Data Laboratory. Over the five years of the project, methods were developed to simultaneously determine water soluble vitamins in dietary supplements and fortified foods using HPLC with multiple diode array ultraviolet and fluorescence detection. A series of samples collected for the USDA NFNAP program consisting of four regional composites of each of 12 separate cereal brand-name products was analyzed for water soluble vitamin content. The new multivitamin method was used to determine vitamins for assigning nutrient values for the new Standard Reference Material 1849 Adult/Infant Formula released in 2009 by the National Institute of Standards and Technology. A definitive analytical method developed using isotope dilution-liquid chromatography/mass spectrometric (ID-LC/MS) for the analysis of individual water soluble vitamins (thiamin, niacin, pyridoxine, folic acid, biotin, pantothenic acid, and ascorbic acid) in dietary supplements was extended to fortified foods. This required custom synthesis of previously unavailable multiple isotope-labeled vitamins for use as internal standards for these methods. This methodology was used to obtain an alternate confirming set of data used in assigning final values to the new SRM 1849 Adult/Infant Formula released by the NIST in 2009. In preparation for the project to develop analytical values for vitamin D, an improved method of vitamin D analysis was developed. The best aspects of two methods to analyze vitamin D from the Association of Official Analytical Chemists (AOAC) (Method 2002.05 and Method 992.26) were incorporated into a single method that has been found to be effective for a wide range of food sample matrices. Conditions for the two chromatographic separations in the method and for atmospheric pressure chemical ionization mass spectrometry (APCI-MS) of vitamin D have been optimized. FCMDL then participated in a round robin collaborative study for the analysis of five representative foods for vitamin D: skim milk, multi-grain cereal, orange juice, Alaskan red sockeye salmon, processed cheese food, and a diluted vitamin D standard from the U.S. Pharmacopoeia. The analytical results from the FCMDL demonstrated the lab’s ability to analyze samples for the National Nutrient Databank; FCMDL has undertaken analysis of additional samples as identified by the NDL and the Food Surveys Research Group. 4.Accomplishments 1. The Food Composition and Methods Development Laboratory (FCMDL) method for simultaneous determination of water soluble vitamins in multivitamin dietary tablets was chosen by an AOAC Expert Review Panel for inclusion in “World Class” methods being developed by AOAC under an NIH/FDA/AOAC contract for methods for vitamin analysis in dietary supplements. Of over 50 reviewed methods, 12 were chosen for final evaluation and the best aspects of different methods chosen for further development, including the chromatography/detection aspects of the FCMDL method. FCMDL was subsequently chosen to carry out optimization and a formal Single Laboratory Validation (SLV) of this combined method, with financial support from the NIH. A successful SLV would subsequently lead to a full collaborative study and acceptance of the FCMDL method as an AOAC Official Method of Analysis. 2. Food Composition and Methods Development Laboratory (FCMDL) results for water soluble vitamins and vitamin D were used to assign values for the Certificate of Analysis for the SRM® 1849 Infant/Adult Nutritional Formula, issued by the National Institute of Standards and Technology (NIST) in July 2009. Providing high quality measurements that are accepted to assign certified values to SRMs is a direct transfer of the technology developed in FCMDL to make these measurements. This SRM will be available internationally to be used by laboratories to validate their analytical measurements in food samples. Review Publications Byrdwell, W.C., Devries, J., Exler, J., Harnly, J.M., Holden, J.M., Holick, M., Hollis, B., Horst, R., Lada, M., Lemar, L.E., Patterson, K.K., Phillips, K., Tarrago-Trani, M., Wolf, W.R. 2008. Analyzing vitamin D in foods and supplements: Methodological challenges. American Journal of Clinical Nutrition. 88:554S-557S. Byrdwell, W.C. 2009. Comparison of analysis of vitamin D3 in foods using ultraviolet and mass spectrometric detection. Journal of Agricultural and Food Chemistry. 57(6):2135-2146.