2008 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(from AD-416) To develop new analytical methods for water-soluble vitamins and vitamin D to replace existing, outdated methods. 1b.Approach (from AD-416) For the water-soluble vitamins, use liquid chromatography-mass spectrometry for optimized individual methods, multiple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 A previously reported method for the simultaneous determination of water soluble vitamins in dietary supplements using HPLC with multiple diode array UV and fluorescence detection was extended to fortified foods. A series of samples collected for the USDA NFNAP program consisting of four regional composites of each of 12 separate cereal brand products has been analyzed for water soluble vitamin content and a publication is being prepared. The new multivitamin method was used to determine vitamins for a new standard reference material (SRM 1849 Adult/Infant Formula) being developed by the National Institute of Standards and Technology (NIST). A previously reported isotope dilution-liquid chromatography/mass spectrometric (ID-LC/MS) method for the analysis of individual water soluble vitamins (thiamine, niacin, pyridoxine, folic acid, biotin, pantothenic acid, and ascorbic acid) in dietary supplements has been extended to fortified foods. Previously obtained stable isotope labeled-vitamins were used as internal standards for these methods. This methodology was used to obtain an alternate confirming set of data to help assign final values to the new SRM 1849, Adult/Infant Formula, being developed by the NIST. We developed a modified method for vitamin D based on the combination of AOAC 2002.05 and AOAC 992.26. Initial work with the Vitamin D working group, made up of members from NDL, FCMDL, the Beverage Institute for Health & Wellness (an affiliate of Coca-Cola company), the Food Analysis Laboratory Control Center (FALCC) at Virginia Polytechnic Institute and State University, Boston University, Medical University of South Carolina, Heartland Assays, established values for 5 control materials (dry cereal, orange juice, processed cheese, salmon, and skimmed milk) (see second Accomplishment). This method has been used to quantify vitamin D in 37 orange juice samples, 25 salmon samples, and 5 shrimp samples submitted by NDL and FALCC. Analysis of the salmon samples required modification of the method to include internal standards at 3 levels of concentration to account for the greater variability of the vitamin D concentrations. Samples of NIST SRM 1849, Adult/Infant Formula, were analyzed and will be reported shortly. A further modification of the method was developed that used 2 chromatographic columns in series to provide improved resolution between vitamin D2 and D3. This eliminated an interfering species that precluded accurate quantification by UV detection. With the new chromatographic method, the interferent is now resolved. Our excellent results on control materials and low standard deviations have made us the de facto reference laboratory. NDL has requested the analysis of a small numbers of additional samples, including processed cheese, milk, and yogurt, as a cross-check of the analyses performed by other members of the collaborative working group. This research falls under National Program 107 - Human Nutrition, Component 1: Composition of Foods, and Component 3: Nutrition Monitoring. 4.Accomplishments 1. A method for the simultaneous determination of multiple water-soluble vitamins. Expediting the analysis of vitamins in foods is necessary to allow the USDA nutrient database to keep up with the changing food supply and to allow the FCMDL to support the development of vitamin reference materials. A method has been developed for the simultaneous determination of niacin, thiamine, pyridoxine, riboflavin, folic acid, biotin, pantothenic acid, and ascorbic acid in dietary supplements and fortified foods. This method uses reversed phase liquid chromatography (LC) and detection with a combination of photodiode array/fluorescence in tandem with mass spectrometry. Single laboratory method validation of this method for dietary supplements has been conducted, and validation for fortified foods is in progress. Having a simple, single-run LC method for determining multiple vitamins in dietary supplements and foods will have a significant impact on the capability to generate valid data to populate the USDA Databases, i.e., the Dietary Supplement Ingredient Database currently being assembled in collaboration with the Office of Dietary Supplements (National Institutes of Health) and NDL (ARS). This research falls under National Program 107 - Human Nutrition 2004-2008, Component 1: Composition of Foods, and Component 3: Nutrition Monitoring. 2. Method and control materials for vitamin D. The analytical methodology for vitamin D was highly suspect and reference materials were not available. Based on supplemental support from the Beverage Institute for Health & Wellness (a division of the Coca-Cola Company), NDL and FCMDL established a vitamin D analytical working group, made up of members from NDL, FCMDL, Coca-Cola, Virginia Tech, Boston University, Medical University of South Carolina, and Heartland Assays. This group enlisted the Food Analysis Laboratory Control Center (FALCC) at Virginia Tech University to develop 5 control materials (dry cereal, orange juice, processed cheese, salmon, and skimmed milk). These control materials were analyzed by FCMDL, Boston University, Medical University of South Carolina, Heartland Assays, and 2 commercial labs using 6 different analytical methods. Initial results were discouraging. However, a careful examination of the data and exchange of primary standards between labs eliminated major discrepancies and established acceptable values for all samples except the salmon (which is still being analyzed). Availability of these control materials will have a major impact on the analysis of vitamin D. The project results have also established FCMDL as the de facto reference laboratory for vitamin D analysis. The results of the project have been published. This research falls under National Program 107 - Human Nutrition 2004-2008, Component 1: Composition of Foods, and Component 3: Nutrition Monitoring. 5.Significant Activities that Support Special Target Populations None. 6.Technology Transfer Review Publications Dwyer, J., Holden, J.M., Andrews, K., Roseland, J.M., Zhao, C., Schweitzer, A., Perry, C., Harnly, J.M., Wolf, W.R., Picciano, M., Fisher, K., Saldanha, L., Yetley, E., Betz, J., Coates, P., Milner, J., Whitted, J., Burt, V., Radimer, K., Wilger, J., Sharpless, K., Hardy, C. 2007. Measuring vitamins and minerals in dietary supplements for nutrition studies in the USA. Analytical and Bioanalytical Chemistry. 389(1):37-46. Phillips, K., Byrdwell, W.C., Exler, J., Harnly, J.M., Holden, J.M., Holick, M., Hollis, B., Horst, R., Lemar, L.E., Patterson, K.K., Tarrango-Trani, M., Wolf, W.R. 2008. Evaluation and harmonization of measurements of vitamin D in selected food materials. Journal of Food Composition and Analysis. 21:527-534.