2009 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 During the final five months of this project, the standardized screening method for phenolic compounds (developed at FCMDL) was used to identify flavonoids and phenolic acids in green and fermented teas, pear skins, arnica flowers, burdock roots, Scutellaria lateriflora, and 2 species of Teucrium. In another project, spectral fingerprints were obtained using direct injection (no chromatographic separation) mass spectrometry permitting discrimination between the two species of broccoli. Additional mass spectral fingerprints permitted discrimination between genera (American ginseng, bitter orange, black cohosh, Chinese ginseng, cranberries, Echinacea, germander, golden seal, kava kava, licorice, pygeum, red clover, skullcap, St. Johns Wort, and milk thistle) and species (5 species of black cohosh, 3 species of skullcap, and 3 species of teucrium) of selected botanical materials. Over the 5-year period for this project, the standardized screening method was developed to determine glycosylated flavonoids and phenolic acids based on cross-comparison of compounds from samples, standards, and samples reported in the literature and resulted in the positive identification of most common phenolics and tentatively identified the remainder of the phenolics. This method was used to analyze more than 350 plant materials and 200 standards. In addition, a method was developed to determine 16 phenolic acids (PAs) in food materials; its use in collaboration with other USDA scientists in Kansas, Washington, North Dakota, and here at Beltsville led to the determination of PAs in wheat, selenium-enriched broccoli, dry beans, and tomatoes. Fingerprinting methods based on either infrared or near-infrared analysis of dried, finely powdered samples or direct ultra-violet and mass spectrometric analysis of sample extracts were developed that, when combined with analysis of variance-principal component analysis (ANOVA-PCA), differentiated between samples based on genus, species, sub-species, and growing year, location, and conditions. This work was supported in part by an interagency agreement with the Office of Dietary Supplements at the National Institutes of Health. In addition, methods were developed for the extraction of phenolics (flavonoids and PA) from foods and botanical materials using accelerated solvent extraction (ASE), and verified in broccoli, black cohosh, parsley, soybeans, and eggplants. This work was supported in part by a CRADA which provided the lab with expendable supplies over a 4-year period, along with testing a new ASE system manufactured by Dionex in 2007. 4.Accomplishments 1. A reference material for evaluating anti-oxidant activity. A mixture containing multiple phenolic phytochemical standards was developed for use as a reference material to permit the accurate quantification of antioxidant activity in various matrices. This reference material is currently being used in single-laboratory validation studies by the Association of Official Analytical Chemists (AOAC International) for total phenolics using the Folin-Ciocalteu assay and for total antioxidant capacity using the oxygen radical absorbing capacity (ORAC) assay. 2. Identification of hydroxycinnamoylquinic acids of arnica flowers and burdock roots using a standardized liquid chromatorgraphy-diode array detector, electrospray ionization/mass spectrometric (LC-DAD-ESI/MS) profiling method. These phenolic acid derivatives are ubiquitous, complex, and difficult to separate (many overlapping chromatographic peaks. A method was developed that permits identification of all of the isomers using two separate chromatographic columns and solvent programs. The key to method development was the acquisition of a full set of the hydroxycinnamoylquinic acid isomers. This method will permit identification of these compounds using LC with diode array detection and eliminates the need for expensive MS/MS (two mass spectrometers in tandem) equipment. Review Publications Lin, L., Harnly, J.M., Upton, R. 2009. Comparison of the phenolic component profiles of skullcap (Scutellaria lateriflora) and germander (Teucrium canadense and T. chamaedrys), a potentially hepatotoxic adulterant. Phytochemical Analysis. 20(4):298-306. Luthria, D.L., Lin, L., Robbins, R.J., Finley, J.W., Banuelos, G.S., Harnly, J.M. 2008. Discriminating between cultivars and treatments of broccoli using mass spectral fingerprinting and analysis of variance-principal component analysis. Journal of Agricultural and Food Chemistry. 56(21):8130-8140. Lin, L., Harnly, J.M. 2008. Phenolic compounds and chromatographic profiles of pear skins (Pyrus spp.). Journal of Agricultural and Food Chemistry. 56(19):9094-9101. Lin, L., Harnly, J.M. 2008. New phenolic components and chromatographic profiles of green and fermented teas. Journal of Agricultural and Food Chemistry. 56(17):8130-8140. Lin, L., Harnly, J.M. 2008. A systematic approach to the identification of common hydroxycinnamoylquinic acids in plant materials. Journal of Agricultural and Food Chemistry. 56:10105-10114. Pal, M., Talawar, S., Deshmukh, P., Vishwanathan, C., Khetarpal, S., Kumar, P., Luthria, D.L. 2008. Effect of elevated CO2 on growth and photosynthetic characteristics of chickpea (Cicer arietinum L.). Indian Journal of Plant Physiology. 13:4. Harnly, J.M., Pastor Corrales, M.A., Luthria, D.L. 2009. Variance in the chemical composition of dry beans determined from UV spectral fingerprints. Journal of Agricultural and Food Chemistry. 57(19):8705-8710.