2012 Annual Report 1a.Objectives (from AD-416): 1. Measure in vivo pharmacokinetics and biological actions of citrus phytonutrients in human and small-animal trials. 2. Study factors that influence the in vivo pharmacokinetics of citrus phytonutrients. 3. Characterize the beneficial actions of citrus phytonutrients in in vivo systems. 1b.Approach (from AD-416): 1. Administer citrus juices to test subjects and to monitor targeted biological actions and the presence of these compounds or their metabolites in biological sample extracts, primarily of blood and urine, over set periods of time. 2. Extract phytonutrients and main metabolites from biological tissue extracts, and analyze by high performance liquid chromatography-mass spectrometry (HPLC-MS). 3.Progress Report: This project is related to Objective 1 of this in-house project: Identify and characterize biologically active compounds in citrus peel and associated processing byproducts for potential as value-added products to promote human health. Future investigations into the influences of different methods of juice production on orange juice phytonutrient bioavailabilities were planned. Cooperator visited ARS location for these planning purposes on three occasions. Preliminary experiments on the production of different styles of orange juice were performed, and initial chromatographic isolations of potential citrus flavonoid metabolites were achieved. Extensive analyses of metabolites of the bioactive citrus polymethoxylated flavones by high performance liquid chromatography-mass spectrometry (HPLC-MS) were continued. The structural diversity of these metabolites were evaluated by mass spectroscopy, and quantitations of these compounds in rat blood serum have been partially completed. Additional methods to detect these compounds by fluorescence spectroscopy linked by reversed phase HPLC were evaluated and optimized. Preparations and plans were made for future polymethoxylated flavone feeding trials and bioavailability studies.