2007 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 variety of phenolic compounds that exist in plant based foods (nearly 8000), our method development investigations are structured into three major objectives: determination of glycosylated (conjugated) flavonoids, determination of phenolic acids, and development of a fingerprinting method for total phenolics content. Our approach, with respect to the glycosylated flavonoids, will be to develop a high performance liquid chromatography (HPLC) method with both UV-Vis and mass spectrometry (MS) detection for a series of groups of flavonoids based on polarity spectrum of the molecules. For the phenolic acids, our approach is to develop a single reversed-phase HPLC/UV system for the separation and quantification of prominent food phenolic acids. For the total phenolics fingerprint, our approach is to use, IR, Near IR and MS data collected on unseparated fractions and use chemometric analysis to establish a classification process for various cultivars of foods high in phenolic compounds. Developing analytical methods for these measurements includes not only choice of analytical instrumentation for separation, detection and quantitation but also factors that stabilize, liberate, extract and identify phenolic acids and flavonoids from the complex food matrix. Hence, part of our approach is to develop, concurrent with method development, quantitative sample preparation conditions (e.g. extraction parameters and hydrolysis conditions). 3.Progress Report Research continued on the development of analytical methods for the determination of phenolic phytonutrients in foods. The significance of sample preparation in developing analytical methodologies for accurate determination of phenolic phytochemicals in foods was evaluated. Analytical procedures for extraction of isoflavones from soybean samples were compared and optimized. The evaluation of pressurized liquid extractor parameters for the extraction of phenolic phytochemicals from parsley was completed. The project identifying phenolic compounds present in 17 common bean samples was completed. It was demonstrated that simple spectral fingerprinting approaches (mass, molecular absorption, infrared, and near infrared spectrometry) could be used to classify two varieties of broccoli (Majestic and Legacy) grown under seven experimental conditions. The spectral fingerprints of the broccoli were analyzed using analysis of variance-principal components analysis (ANOVA-PCA). It was demonstrated that ANOVA-PCA provides a simple, visual method for determining chemical differences between cultivars and treatments. It was demonstrated that the sources of variance (cultivar and treatment) can be quantified using the same data matrices constructed for ANOVA-PCA. Work was completed on the development of a chromatographic profiling method for phenolic compounds in plant materials (see Accomplishments). The method was used to profile the phenolic compounds in a wide variety of plant materials and botanicals: beans, celery, Mexican oregano, different varieties of peas, cashew apples, chrysanthemum flowers, Ginkgo biloba, Scutellaria, and Teucrium. Research was started on the development of a general quantitative method for glycosylated flavonoids and an in-depth determination of the phenolic compounds in tea. 4.Accomplishments A Profiling Method for Phenolic Compounds: Phenolic compounds are purported to be beneficial to human health. Accurate assessment of their health value requires analysis of the more than 6,000 phenolic compounds found in foods. A profiling method for most phenolic compounds was developed that uses a standardized approach based on liquid chromatography with diode array and mass spectrometric detection. This method allows rapid determination of the most common phenolics and archiving of data for comparison to future results. This method will allow the beginning of rapid and systematic identification of thousands of phenolic compounds in plant food materials. This accomplishment addresses National Program 107 Component 1: Composition of Foods. Sample Preparation for Phenolic Compounds: A systematic evaluation of different sample preparation parameters and their influence on extraction of different phenolic phytochemicals for different food matrices is necessary to insure accurate analyses. An approach for optimization of extraction of phenolic phytochemicals was developed and reported in 2006. A manuscript entitled “Influence of Sample Preparation on the Assay of Phenolic Acids from Eggplant” published in the Journal of Agricultural and Food Chemistry was featured on the American Chemical Society (ACS) Publications website in 2007 as a most-cited article in 2006. Dionex Inc. has published an application note based on the research reported in this paper. This work will lead to more accurate determination of phenolic compounds in foods and will allow more accurate assessment of the health impact of these compounds. This accomplishment addresses National Program 107 Component 1: Composition of Foods. 5.Significant Activities that Support Special Target Populations None 6.Technology Transfer Number of active CRADAs and MTAs 3 Number of non-peer reviewed presentations and proceedings 10 Number of newspaper articles and other presentations for non-science audiences 1 Review Publications Luthria, D.L., Biswas, R., Natarajan, S.S. 2007. Comparison of extraction solvents and techniques used for the assay of isoflavones from soybean. Food Chemistry. 105:325-333. Luthria, D.L. 2006. Significance of sample preparation in developing analytical methodologies for accurate estimation of bioactive compounds in functional foods. Journal of the Science of Food and Agriculture. 86:266-2272. Harnly, J.M., Doherty, R.F., Beecher, G.R., Holden, J.M., Haytowitz, D.B., Bhagwat, S.A., Gebhardt, S.E. 2006. Flavonoid content of U.S. fruits, vegetables, and nuts. Journal of Agricultural and Food Chemistry. 54:966-9977. Lin, L., Lu, S., Harnly, J.M. 2007. Detection and quantification of glycosylated flavonoid malonates in celery seed,(Apium graveolens L.) Chinese celery and celery by LC-DAD-ESI/MS. Journal of Agricultural and Food Chemistry. 55:1321-1326. Erdman, J., Balentine, D., Arab, L., Beecher, G., Dwyer, J., Folts, J., Harnly, J.M., Hollman, P., Keen, G.J., Messina, M., Messina, M., Scalbert, A., Vita, J., Williamson, G., Burrowes, J. 2007. Flavonoids and Heart Health. Journal of Nutrition. 137:718S-737S. Lin, L., Mukhopadhyay, S., Robbins, R.J., Harnly, J.M. 2007. Indentification and quantification of flavnoids of Mexican oregano (Lippia graveolents) by LD-DAD-ESI/MS analysis. Journal of Food Composition and Analysis, 20:361-369. Chen, P., Ozcan, M., Harnly, J.M. 2007. Chromatographic Fingerprints Analysis for Evaluation of Ginkgo Biloba Products. Analytical and Bioanalytical Chemistry. Available on-line doi: 10.1007/s00216-007-1386-9. Harnly, J.M., Bhagwat, S.A., Lin, L. 2007. Profiling methods for the determination of phenolic compounds in foods and dietary supplements. Analytical and Bioanalytical Chemistry. Available: doi:10.1007/s00216-007-1424-7. Holden, J.M., Harnly, J.M., Beecher, G. 2006. Food Composition. In: Bowman, B.A., Russell, R.M., editors. Present Knowledge in Nutrition, Ninth Edition, Volume 1. Washington, D.C.:International Life Sciences Institute. p. 781-794.