2009 Annual Report 1a.Objectives (from AD-416) To develop comprehensive methods for the determination of S and Se in proteins and free amino acids in foods. Of particular interest are the secondary metabolite (non-protein) amino acids that have been implicated in the prevention of chronic disease. 1b.Approach (from AD-416) Proteins and free amino acids will be separated from other food components. Proteins will be hydrolyzed chemically and/or enzymatically and methionine, cysteine, selenomethionine, and selenocysteine will be determined using high performance liquid chromatography (HPLC) or reaction with cyanogen bromide. Free amino acids will be separated and derivatized for separation by HPLC or gas chromatography (GC). S and Se-containing compounds will be identified using inductively coupled plasma-mass spectrometry (ICP-MS) or pulsed flame photometric detection (PFPD). The organic component of the non-protein amino acids will be characterized using HPLC-MS with electrospray ionization. 3.Progress Report Methods for total selenium (Se) and selenomethionine (SeMet) were developed by the Food Composition and Methods Development Laboratory (FCMDL) that were continuously refined over the course of this project and were used to characterize a variety of samples and certified reference standards. The Se content of foods is of critical interest since diets high in Se appear to reduce the risk of prostate cancer. In 2004, Se and SeMet were characterized for wheat gluten, Durum wheat, hard red spring wheat, and soft winter wheat. Further analysis showed that, despite large differences in the total Se concentration, SeMet was a constant fraction of the total Se. This led to collaboration with the National Research Council of Canada (Ottawa, Canada) to assign values for SeMet in a Certified Reference Material (SELM-1). FCMDL also contributed values for SeMet to the U.S. National Institute of Standards and Technology to support development of a wheat Standard Reference Material. In 2007 and 2008, FCMDL participated in two international studies:. 1)the international pilot study CCQM-P86 “Analysis of total Se and SeMet in pharmaceutical supplements” organized by the Laboratory of the Government Chemist (London, UK), and. 2)the international pilot study CCQM-P86.1 “Total Se and Se speciation analysis of Se-rich wheat flour” organized by the International Committee of Weights and Measures (Paris, France). FCMDL developed a method for sulfur (S)- and Se-containing secondary amino acids using gas chromatography with flame ionization detection based on a commercial kit using ethylchloroformate derivatization for determining primary amino acids, identified conditions for detecting methionine, SeMet, cysteine, selenocysteine, methylselenocysteine, and alliin (the active sulfur compound in garlic). This method was used to characterize the free amino acid and alliin content of 11 sub-species of garlic and a broccoli cultivar grown with 4 levels of Se fertilization (0, 5, 100, and 1000 mg/liter). This research project demonstrated that Se enrichment not only affected the Se-containing amino acids but also disrupted the total relative and total concentration of all the primary amino acids.