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United States Department of Agriculture

Agricultural Research Service

Research Project: Fingerprinting and Profiling Methods for Characterization of Foods and Dietary Supplements

Location: Food Composition and Methods Development Lab

2012 Annual Report

1a.Objectives (from AD-416):
Objective 1: Develop and evaluate procedures for quantitative extraction and/or fractionation of food materials by polarity. Sub-objective 1.A.: Develop an extraction procedure for sequential fractionation of the major groups of components from plant materials. Sub-objective 1.B.: Develop optimized extraction procedures for accurate quantification of individual phytochemicals in plant materials.

Objective 2: Develop and evaluate spectral fingerprinting and chromatographic profiling methods to characterize components in lipid soluble, water soluble, and intermediate fractions of food materials. Sub-objective 2.A.: Develop spectral fingerprinting methods for identification of plant materials and individual components using direct analysis (no prior chromatographic separation) and pattern recognition algorithms. Sub-objective 2.B.: Develop chromatographic profiling methods for identification and quantification of individual components in plant materials.

Objective 3: Develop methods to determine variability of biologically active components in food materials through profiles and/or fingerprints.

Temporary Objectives:

Objective 1: Conduct an independent and outside evaluation of the IT capabilities of the Food Composition and Methods Development Laboratory coordinated with the Nutrient Data Laboratory and the Food Surveys Research Group; include an assessment of whether cloud computing or local data storage is the optimal approach for the next 5-10 years of anticipated data collection, storage, and dissemination.

Objective 2: Implement changes to update and modernize the IT infrastructure of the Food Composition and Methods Development Laboratory that links to the Nutrient Data Laboratory and Food Surveys Research Group.

1b.Approach (from AD-416):
Objective 1: Methods will be developed for the quantitative extraction of macro and micro components from plant materials using commercial, high pressure/temperature extraction instrumentation. Soybeans will be tested initially as they contain both lipid soluble and highly polar molecules of health interest. Sequential and parallel extraction will be investigated. Extracted materials will be characterized using liquid chromatography with diode array and electrospray ionization/mass spectrometric detection (LC-DAD-ESI/MS). The new method will be applied to foods and botanical materials. In addition, optimized methods will be developed for specific families of compounds such as water-soluble vitamins, lipid-soluble vitamins, phenolic acids, and flavonoids.

Objective 2: Spectral fingerprinting methods will be developed based on ultraviolet and visible molecular absorption (UV/Vis), infrared (IR), near-infrared (NIR), and mass spectrometric (MS) detection. The overlapping complex spectra will be interpreted using pattern recognition programs. The patterns will be used to determine the sensitivity of the different detection systems for discriminating between plant materials based on genera, species, variety, growing year, growing site, and processing conditions. These methods will be developed using 3 food materials and 3 botanical supplement materials. Repeat samples will be examined over a period of years to determine the stability of the spectra and the ability to compare spectra of new materials to archived spectra. The phenolic and vitamin content of the plant materials will also be determined using chromatographic profiling using LC-DAD-ESI/MS. This will make it possible to determine which compounds are contributing most to differences arising from the various growing factors.

Objective 3: The spectral fingerprints can be used with nested analysis of variance to determine the relative variance contributed by each growing factor: species, variety, site, year, plant-to-plant variation, and analytical uncertainty. Samples will be obtained from collaborators across the country and representing a variety of foods and botanical supplements. UV/Vis, IR, and NIR spectra will provide variance data for the integrated chemical composition of the plant materials and MS will provide variance data for specific masses and, with the assistance of chromatographic profiling, specific compounds of health interest.

Temporary Approaches:

Approach 1: Submit names of individuals to the BHNRC with expertise to evaluate existing and future needs for evaluating food composition data arising from analysis in the laboratory. Assessment will include how to work more effectively in sharing compositional information across the various laboraties within the BHNRC.

Approach 2: Replace as needed hardware and software needed to evaluate food composition information generated by the FCMDL and for sharing quality compositional information with the NDL and the FSRG.

3.Progress Report:
In collaboration with Thermo Scientific and support from the Office of Dietary Supplements (NIH), FCMDL is establishing an in-house database for phytochemical compounds, such as anthocyanins and other flavonoids, using high resolution mass spectrometry. To date, more than 300 compounds have been positively identified using the scientific literature and authentic reference materials. The new database contains their exact mass, chemical formula, structure, full mass spectra, and molecular absorption spectra. The database will be used to facilitate identification of chemical components in future analysis of food materials and will save significant time and resources.

Proanthocyanins are found most notably in chocolate, cranberries, and grape seed and have purported health benefits. Data suggests that the proanthocyanin metabolites are absorbed in the large intestine following digestion by gut bacteria. In this study, pigs were fed diets supplemented by cranberry or grape seed extracts. The extracts have been carefully analyzed and metabolomics analysis of the urine and blood samples is proceeding.

Whole wheat flour from five wheat cultivars was evaluated for phenolic, carotenoid, and tocopherol compositions as well as anti-inflammatory and antiproliferative activities against HT-29 cells. The ferulic acid content ranged from 452 to 731 µg/g among the five cultivars and was primarily present in a bound insoluble form. The results showed that whole wheat flours of these five cultivars varied significantly with respect to their content of these bioactive compounds.

1. Detection of Germander in Skullcap. Skullcap (Scutellaria lateriflora)is used as a dietary supplement and is an ingredient in numerous herbal products. It is routinely contaminated or adulterated with Germander (Teucrium canadense or Teucrium chamaedrys) due to the morphological similarities between the two genera. Unfortunately, Germander contains a compound that is hepatotoxic. A fast and reliable analytical method was developed using flow-injection mass spectrometry that can authenticate the purity of Skullcap in 1 minute. A brief survey of skullcap based dietary supplements revealed that 4 out of 13 were contaminated with germander. The project revealed that the commercially available skullcap based dietary supplements sold in the U.S. market are not safe and offer an easy and reliable method to identify unsafe products.

2. Identification of Wisconsin grown ginseng. American ginseng (Ginseng quinquefolius) is known worldwide as the purest, finest ginseng and is very important to the economy of Wisconsin. Unfortunately, American ginseng grown in China is being used to counterfeit Wisconsin ginseng. FCMDL has developed the first analytical method in the world, based on liquid chromatography, mass spectrometry, and pattern recognition mathematics that can differentiate between American ginseng grown in Wisconsin and China. This method can identify counterfeit American ginseng and can provide significant economical benefits to Wisconsin ginseng farmers.

3. Quantification of flavonoids. There are more than 8,000 flavonoid compounds in the plant kingdom. While current technology can identify these compounds it is impossible to obtain standards or maintain a standard inventory for quantifying all these compounds. FCMDL has developed an analytical scheme based on the common molecular absorption patterns of these compounds that allows quantification using a few inexpensive, commercially standards and easily determined molar relative response factors. The relative accuracy of the method is plus/minus 15%. High resolution mass spectrometry can readily identify all the flavonoids and this quantification scheme can determine their concentration.

4. Phenolic content of eggplant. Phenolic acids are ubiquitous in the plant world and contribute positively to human health and negatively to the palatability of foods. A rapid screening method for phenolic acid concentration in eggplant pulp was developed based on a simple water/methanol extraction and UV spectrophotometric analysis. The total phenolic acid concentration, as determined by the sum of the individual phenolic acids as analyzed by high performance liquid chromatography, correlated well with the UV analyses. These results demonstrated that a single UV absorption reading can be used to estimate the concentration of phenolic acids in eggplant pulp extracts and can be used to identify cultivars and growing conditions that influence phenolic acid content in eggplant samples.

5. Phenolic content of corn by-products for cattle. Distillers dried grains with solubles (DDGS), a by-product of breweries, and now ethanol production plants, is commonly used as fodder for livestock. The phenolic acids in corn are relatively unaffected by the distillation process and become concentrated in the DDGS fraction. The phenolic acids increase the nutrient content of the feed but reduce its palatability. In this study, corn samples were collected from three commercial plants and analyzed for individual phenolic acids by high performance liquid chromatography coupled with diode array and/or mass spectrometry. Five common phenolic acids composed approximately 80% of the total phenolic acid content. The relative phenolic acid concentration of DDGS was 3 times higher than that of the raw corn. Good agreement was found for the phenolic content of the 3 plants. Results from this study will be valuable to bioethanol manufacturers and feed industry.

Review Publications
Chen, P., Luthria, D.L., Harrington, P.B., Harnly, J.M. 2011. Discrimination among Panax species using spectral fingerprinting. Journal of the Association of Official Analytical Chemists. 94:1411-1421.

Sun, J., Chen, P. 2011. Profiling the indole alkaloids in yohimbe bark with ultra-performance liquid chromatography coupled with ion mobility quadrupole time-of-flight mass spectrometry. Journal of Rapid Communications in Mass Spectroscopy. 25:2591-2602.

Lin, L., Harnly, J.M., Zhang, R., Chen, H., Fain, Z. 2011. A general approach to quantification of hydroxycinnamic acid derivatives and flavones, flavonols, and their glycosides by UV spectrophotometry. Journal of Agricultural and Food Chemistry. 60:544-553.

Harnly, J.M., Applequist, W., Caspar, S., Harrington, P., Hill, N., Labudde, R., Neal-Kababick, J., Harbaugh-Reynaud, D., Roman, M., Roman, S., Sullivan, D., Titlow, B., Wehling, P. 2012. Guidelines for validation of botanical identification methods. Official Methods of Analysis of AOAC International. 95:268-272.

Labudde, R., Harnly, J.M. 2012. Probability of identification (POI): a statistical model for the validation of qualitative botanical identification methods. Official Methods of Analysis of AOAC International. 95:273-285.

Lin, L., Sun, J., Chen, P., Harnly, J.M. 2011. UHPLC-PDA-ESI/HRMS/MSn analysis of anthocyanins, flavonol glycosides, and hydroxycinnamic acid derivatives in red mustard green (Brassica juncea (L) Coss variety). Journal of Agricultural and Food Chemistry. 60:544-553.

Whent, M., Huang, H., Lutterodt, H., Zhouhong, X., Lu, Y., Fuerst, E.P., Morris, C.F., Yu, L., Luthria, D.L. 2012. Phytochemical Composition, Anti-inflammatory, and Antiproliferative Activity of Whole Wheat Flour. Journal of Agricultural and Food Chemistry. 60:2129-2135.

Lv, J., Whent, M., Huang, H., Lu, Y., Charles, D., Liu, L., Yu, L., Luthria, D.L. 2012. Phenolic composition, anitproliferative and anti-inflammatory properties of conventional and organic cinnamon and peppermint. Food Chemistry. 132:1442-1450.

Sun, X., Chen, P., Cook, S., Jackson, G., Harnly, J.M., Harrington, P. 2012. Classification of cultivation locations of panax quinquefolius L samples using high performance liquid chromatography-electrospray ionization mass spectrometry and chemometric analysis. Analytical Chemist. 84:3628-3634.

Lin, L., Harnly, J.M. 2011. Quantitation of flavanols, proanthocyanidins, isoflavones, flavanones, dihydrochalcones, stilbenes, and benzoic Acid derivatives after identification by LC-MS. Journal of Agricultural and Food Chemistry. 60:5832-5840.

Lin, L., Harnly, J.M. 2012. LC-PDA-ESI/MS Identification of the Phenolic Components of Three Compositae Spices: Chamomile, Tarragon, and Mexican Arnica. Natural Product Communications. 7:749-753.

Memon, A.A., Menon, N., Luthria, D.L., Pitafi, A.A., Bhanger, M.I. 2012. Phenolic compounds and seed oil characterization of Ziziphus Mauritiana L. fruit grown in Pakistan. Polish Journal of Food and Nutrition Sciences. 62:15-21.

Luthria, D.L. 2012. A simplified 96-well method for the estimation of phenolic acids and antioxidant activity from eggplant pulp extracts using UV spectral scan data. Phytochemical Analysis. 4:238-242.

Chanthai, S., Prachakool, S., Ruangviriyachai, C., Luthria, D.L. 2012. Influence of extraction methodologies on the analysis of five major volatile aromatic compounds of citronella grass (Cymbopogon nardus) and lemongrass (Cymbopogon citratus) grown in Thailand. Journal of the Association of Official Analytical Chemists. 95:763-772.

Last Modified: 4/18/2014
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