2008 Annual Report 1a.Objectives (from AD-416) To develop analytical methods for the determination of phenolic acids and glycosylated flavonoids and to develop fingerprinting methods, based on phenolic compounds, for the identification of plant and botanical cultivars, growing sites, and growing conditions. 1b.Approach (from AD-416) Due to the existence of thousands of phenolic compounds in plant and botanical materials, sophisticated extraction, separation, detection, and data processing methods will be used for this project. Reversed phase-high performance liquid chromatography (RP-HPLC), with acid and base hydrolysis pre-treatment, will be used to analyze phenolic acids. A screening method based on a standardized extraction and RP-HPLC separation method with tandem ultraviolet (UV) and mass spectrometry (MS) detection will be developed for the identification and quantification of glycosylated flavonoids and phenolic compounds in plant and botanical materials. Fingerprinting methods will be developed based on infrared, near infrared, UV, and MS detection of un-separated extracts (no chromatography) of the materials. Information will be elucidated from the highly overlapping spectra using pattern recognition software. These methods will be supported by the development of sample preparation methods for quantitative removal of all the phenolic compounds from the plant matrices. 3.Progress Report Mass spectral (MS) fingerprints of broccoli extracts (with no separation)which were used for UV analyses (see first Accomplishment) were obtained. With analysis of variance-principal component analysis, it was possible to discriminate between both cultivars and all seven treatments. The relative variance due to cultivar and treatment matched values obtained by UV analysis although the analytical variance with MS was greater. Compounds present that enabled discrimination between cultivar and treatment were organic acids, amino acids, and carbohydrates. A paper is currently in press. Using the standardized method for the determination of phenolic compounds developed at FCMDL, phenolic profiles were obtained and permitted identification of more than 80 components in 66 tea varieties and samples obtained from around the world. Based on the profiles, it was possible to divide the teas into 6 major groups. More than 43 phenolic compounds were identified in 16 pear skins and 4 categories were established that followed taxonomic lines. In cowpeas and blackeye peas, 28 phenolic components were identified, and 22 phenolic components identified in long beans established their phenolic component profile. This was the first report of hydrolysable hydroxycinnamates in these peas and beans. Papers are in press or have been submitted for publication for all 3 projects. A chromatographic method, based on the use of 2 columns and FCMDL's standardized method for phenolic compounds, was developed for the identification of hydroxylcinnamoylquinic isomers in plant materials. Arnica and burdock root were used as model systems. A paper has been submitted for publication. In collaboration with the American Herbal Pharmacopoiea, a chromatographic method for the detection of adulteration of skullcap (Scutellaria lateriflora) by germander (Teucrium canadense and T. chamaedrys), which contains hepatotoxins, was developed. This method was based on FCMDL's standardized method for phenolic compounds and has been submitted for publication. Collaborations have been established with ARS Scientiests in the Food Safety Lab, Beltsville; the Sustainable Agricultural Systems Lab, Beltsville; The Sustainable Perennial Crops Lab, Beltsville; and the Water Management Research Unit, Fresno, CA). FCMDL is developing methods for efficient extraction and identification of phytochemical compounds in spinach, lettuce, eggplant, tomatoes, broccoli, and prickly pears, examining pressurized liquid extraction with acid and base hydrolysis and microwave assisted digestion. This research falls under National Program 107 - Human Nutrition, Component 1: Composition of Foods, and Component 3: Nutrition Monitoring. 4.Accomplishments 1. UV spectral fingerprints of broccoli. Methods are needed for the rapid identification and categorization of foods and dietary supplements. Two cultivars of broccoli treated 7 different ways (organic and conventional farming and different levels of selenium (Se) fertilization) were analyzed by direct extraction, ultra-violet light (UV) analysis, and data processing with analysis of variance-principal component analysis (ANOVA-PCA). Cultivars and treatments could be distinguished by their chemical patterns. it was possible to determine the relative variance associated with each variable (cultivar, treatment, and analytical repeatability). This methodology will allow foods to be discriminated based on species, variety, growing conditions, and processing conditions. This research falls under National Program 107 - Human Nutrition 2004-2008, Component 1: Composition of Foods, and Component 2: Nutrition Monitoring. 2. Identification of phenolic compounds in Ginkgo biloba. Ginkgo biloba is one of the most widely used herbal products in the world and little is known about its chemical composition. A standardized profiling method (developed at FCMDL) based on aqueous methanol extraction, liquid chromatography, and diode array and mass spectrometric was used to identify 62 phenolic compounds. Of these, more than 20 were reported for the first time. Ten biflavones which might have health benefits are present in the raw leaves but not in commercial products prepared by alcoholic extraction. This composition data will allow researchers to better evaluate the health promoting benefits of Ginkgo biloba. This research falls under National Program 107 - Human Nutrition 2004-2008, Component 1: Composition of Foods, and Component 3, Nutrition Monitoring. 3. Phenolic compounds in dry beans. Common beans are one of the most important pulse crops in the world. They contain significant amounts of phenolic compounds that are potentially health promoting. A standardized profiling method (developed at FCMDL) based on aqueous methanol extraction, liquid chromatography, and diode array and mass spectrometric was used to identify more than 37 phenolic compounds in 17 varieties of beans. Several of the phenolic components were reported for the first time. Based on this data, dry beans can be organized into six groups. The value of the standardized method (with data archival) was proven by the identification of many of the phenolic acids in beans by data for naval orange peels. This composition data provides specific data on the phenolic composition of bean varieties and can be used to establish potential health benefits. This research falls under National Program 107 - Human Nutrition 2004-2008, Component 1: Composition of Foods, and Component 3, Nutrition Monitoring. 6.Technology Transfer Number of Non-Peer Reviewed Presentations and Proceedings 3 Review Publications Lin, L., Chen, P., Ozcan, M., Harnly, J.M. 2008. Chromatographic profiles of Ginkgo biloba leaves and selected products. Journal of Agricultural and Food Chemistry. http://dx.doi.org/10.1021/jf800488x. He, Q., Luo, Y., Chen, P. 2008. Elucidation of the mechanism of enzymatic browning inhibition by sodium chlorite. Journal of Food Chemistry. 110(4):847-851. Lin, L., Harnly, J.M., Pastor Corrales, M.A., Luthria, D.L. 2008. The polyphenolic profiles of common bean (Phaseolus vulgaris L.). Journal of Food Chemistry. 107:399-410. Luthria, D.L., Mukhopadhyay, M., Finley, J., Banuelos, G.S., Harnly, J.M. 2008. UV spectral fringerprinting and analysis of variance-principal component analysis: a tool for characterizing sources of variance in plant materials. Journal of Agricultural and Food Chemistry. http://dx.doi.org/10.10.21/jf0734572.