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J. L. Ward and P. r. SheWryCentre for Crop Genetic Research,

Rothamsted ResearchHertfordshire, United Kingdom

The HEALTHGRAIN program is one of several initiatives to increase the

consumption and benefits of wholegrain cereals at the international, national, and local levels. Perhaps the most important of these is the HarvestPlus program of the Consultative Group on International Agri-cultural Research (CGIAR), which in-cludes increasing iron, zinc, and provitamin A in wheat among a range of targets to improve the nutrition of the poor, particu-larly those in developing countries (www.harvestplus.org) (11). In contrast, the EU FP6 HEALTHGRAIN project (2005–2010) (www.healthgrain.org) focuses on health benefits for consumers in developed coun-tries, including reduced incidences of type 2 diabetes, cardiovascular disease, and cer-tain types of cancer. Similarly, the Austra-lian Food Futures Flagship project of CSIRO (www.csiro.au/org/FoodFutures-Flagship.html) includes grain composition and human health as one of a number of targets in food quality.

All of these programs require the analy-sis of multiple samples in order to identify variation in composition in germplasm col-lections, lines from breeding programs, grain fractions, and food products. To fa-cilitate this, the methods used to determine bioactive components in the HEALTH-GRAIN program and in other leading labo-ratories worldwide have been brought together in a new analytical handbook pub-lished by AACC International, HEALTH-

GRAIN Methods: Analysis of Bioactive Components in Small Grain Cereals. All of the methods described are “tried and test-ed,” but many of them are time consuming and require specialized equipment. Fur-thermore, separate analyses are required for most groups of compounds, which may each require different skills and equipment. It is therefore necessary to simplify and combine analyses for bioactive compo-nents to facilitate the development of cereal cultivars and food products with enhanced nutritional benefits.

Simplification of AnalysesNear infrared (NIR) spectroscopy is

widely used in the plant breeding and food industries, pro viding robust high through-put analyses for grain components, such as starch, pro tein, and water. NIR calibrations can be developed for arabinoxylans, which are present at lower levels in the grain. How ever, these components account for up to 3% of white flour and more than 20% of bran and we do not yet know whether ro-bust calibrations can be developed for mi-nor components such as sterols and tocols (less than 100 and 1,000 µg/g wholemeal, respectively) (16).

Other options are to develop specific antibody- or enzyme-based kits. Con-siderable success has been achieved in de-veloping and marketing enzyme-based kits for major grain components such as β-glucans, total starch, and the proportion

of amylose (see, for example, kits marketed by Megazyme, Bray, Ireland), and there is no doubt that this range could be extended if sufficient demand was demon strated.

Combining Analyses: MetabolomicsMetabolomics can be defined as a com-

prehensive analysis where all of the small metabolites present in a tissue are analyzed in an unbiased manner. The ultimate aim is to determine the whole complement of metabolites in a single analysis al though this is not currently feasible due to differ-ences in the concentrations and chemical properties of components. In practice, two systems are widely used, based on nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) with the sample being introduced directly into the mass spectrometer or after sepa ration by gas or liquid chromatography. These two approaches are to some extent complemen-tary and their principles, advantages, and drawbacks are discussed by Hall (9) and Ward et al. (15).

Baker et al. (2) described the application of NMR fingerprinting to deter mine the substantial equivalence of field-grown, ge-netically modified, and parental wheat lines but also used GC-MS for additional quantitative amino acid analyses. This study illustrated the current status in the application of metabolomics technoloogies to wheat, in that the spectra of fractions extracted with deuterated methanol/water

Future Prospects for the Analysis of Bioactive Components in Cereal Grain1

➤ Whole grain cereal products have established health benefits in reducing the incidence of

chronic diseases in developed countries.

➤ The EU FP6 HEALTHGRAIN program aims to understand and maximize these benefits by developing improved cereal varieties and products.

➤ The development of improved varieties and products requires rapid, high-throughput analytical systems, combining analyses for multiple groups of bioactive compounds.

➤ FEATURE

1 This article is modified from Chapter 20 of the new AACC International PRESS title, HEALTHGRAIN Methods: Analysis of Bioactive Components in Small Grain Cereals.

Photograph of grain section courtesy of VTT Techni-cal Research Centre of Finland.

doi:10.1094 / CFW-55-2-0071

© 2010 AACC International, Inc.

72 / MARCH-APRIL 2010, VOL. 55, NO. 2

were dominated by the major soluble com-ponents (mainly carbohydrates) with minor peaks corresponding to the most abundant free amino acids (Fig. 1). The only “bioac-tive” components which are extracted un-der these conditions and are present in sufficient amounts to be detected are the methyl donors, choline, and betaine, and NMR, therefore, provides an excellent sys-tem for these to be determined in high throughput analyses (Fig. 2). In our own laboratory, these analyses can be carried out at a rate of six samples/hour using ro-botics for weighing and extraction and au-tomated injection into a 600 MHz NMR instrument. The data files from the analysis can also be used for multivariate analysis

Fig. 1. Typical 1H nuclear magnetic resonance (NMR) spectrum (400 MHz) of wheat flour fol-lowing extraction by 80:20 D2O:CD3OD. (a) full spectrum; (b) central region of the spectrum, dominated by overlapping carbohy drate signals; (c) aliphatic region of the spectrum dominated by aliphatic amino acids. Reprinted from Baker et al. (2) with permission.

to compare the fingerprints of different lines, treatments, or fractions (Fig. 3).

However, the real challenge for the application of metabolomics technol-ogy, whether MS- or NMR-based, to biologically active components in grain is to develop systems for the simultaneous extraction of multiple groups of compo-nents and to remove (either chemically or by data processing) the signals originating from the major components to allow trace amounts of bioactive components to be identified and quantified. Although a full metabolomic analysis of bioactive compo-nents may ultimately not be achievable, we are confident this approach will at least be applicable to groups of components with

similar chemical properties, for example, terpenoids or phenolic components.

Extraction Solvents for Metabolomics

The majority of techniques utilized in the handbook still rely on an initial sol-vent extraction being made to release the metabolites from the biological tissue and the careful selection of this initial extrac-tion solvent is paramount in dictating the final complement of metabolites present in the final sample. Polar metabolites can be released using isopropanol, ethanol, methanol, acidic methanol, acetonitrile (1), water, methanol/water (12), or other mix tures of these solvents, while more lipophilic metabolites can be extracted by chloroform or ethyl acetate. The advan-tage of extracting samples with mixtures of water/methanol/chloroform or ethyl ac-etate is the generation of a biphasic sample and the fractionation of the metabolites into polar aqueous and lipophilic organic fractions, which can then be analyzed separately (6).

While a large number of studies employ extraction with a single solvent, as out lined above, the key to developing a method for the simultaneous detection of a range of phytochemicals may be to employ a tiered or multilevel extraction strategy and to “stitch” the data together to form a “vir-tual metabolomic signature.” Although this approach is more complex by design and yields multiple analytical samples per biological sample, it may be the only way to obtain data on a range of diverse me-tabolites, which previously have required either nonpolar extractions, polar extrac-tions, or saponification methodologies to release them from the raw material. This approach has recently been applied to the analysis of rice grain with the analysis of four different solvent fractions providing information on a range of metabolites from nonpolar lipids to common carbohydrates, amino acids and or ganic acids through to tocols, phenolics, and sterols (13).

InstrumentationThe most successful metabolomics

studies employ a broad range of analytical techniques and integrate the data to give the broadest coverage of the metabolome. For quantitation of metabolites with a wide range of polarities, LC-MS may offer the best way forward. The use of modern multi-sector instruments, set up to moni tor multiple target compounds by using the technique of multiple reaction moni toring (MRM), provides significantly increased sensitivity and selectivity by effec tively screening out all of the “unwanted” ions and concentrating on only the metabolite

CEREAL FOODS WORLD / 73

signals of interest (8). Coupling this sys-tem with ultra performance liquid chro-matography (UPLC) technologies would speed up sample throughput by shortening chromatography run times and thus make the technology amenable to high through-put screens of large sample numbers such as quality trait analysis or complex geno-type × environment (G × E) experiments. While many studies use combinations of gas chromatography (GC)-MS or liquid chromatography (LC)-MS, the use of mass spec trometry without prior chromatog-raphy is becoming increasingly popular and pro vides a fast screening method for all of the ionizable metabolites present in the sample. This technique has been suc-cessfully used to profile a number of dif-ferent biological systems including, more recently, polyphenolics in fruit (14). The use of an initial polar solvent extraction makes it possible not only to capture in-formation on primary metabolites, such as carbohydrates and amino acids, but also to determine changes in secondary metabo-lites such as phenylpropanoids, anthocya-nins, and flavonoids, which are antioxidant molecules present in plant tis sues includ-ing cereals. Fourier transformation ion cyclotron resonance mass spec trometry (FT-ICR-MS) provides an extension to the “nominal mass” direct infusion technique and is capable of producing a fingerprint of unparalleled resolution and accurate mass data which is sufficient to confidently sug-gest chemical formulae and thus help to identify the large number of “unknowns” present in any typical metabolomics sam-ple (3). The restriction, however, is that unlike other techniques, FT-MS cannot distinguish between isomeric compounds and may need to be coupled to liquid chro-matography to achieve this.

Laser desorption/ionization (LDI) MS and matrix-assisted laser ionization/de-sorption (MALDI) MS have not been widely used in plant metabolomics to date generally because of difficulties in gener-ating ions from the relatively hydrophobic plant tissue and the problems of utilizing a chemical matrix which itself gives rise to many low molecular weight ions. That said, a number of successful studies using these techniques have been reported. In cereals, MALDI-MS imaging has recently been used to profile a variety of metabo-lites in wheat seeds using a conventional matrix (4) whilst LDI MS imaging ap-proaches have been used for the direct profiling of plant carotenoids (7), bark tannins (10), phosphatidylcholine (18) and, via the use of graph ite as an assist-ing material (GALDI MS), lipids, organic acids, and flavonoids from plant tissues (5,19). The obvious advantage of these

strated by the utilization of 1H NMR analysis of the intact cell wall, via solubiliza tion, to yield important informa-tion on the difficult to analyze cell wall bound phe nolics, lignin, and fiber compo-nents (17). Although in its infancy, this technique obviously offers the potential to

Fig. 3. Principal component analysis (PCA) of 1H nuclear magnetic resonance (NMR) data from field grown wheat flour demon strating ability of the technique to cluster samples from different environments and harvest times. Reprinted from Baker et al. (2) with permission.

Fig. 2. Analysis of triplicate samples of 31 cereal bran lines by 1H nuclear magnetic resonance (NMR), demonstrating ability to analyze methyl donors, such as betaine and choline, within a typical NMR fingerprinting screen.

techniques is their ability to spatially pro-file metabolites in intact tissues without the need for an extraction protocol which will inevitably discriminate against cer tain classes of chemical compound.

An extension of the philosophy of analy-sis without extraction is further demon-

74 / MARCH-APRIL 2010, VOL. 55, NO. 2

analyze a large number of biologi cally im-portant metabolites, using one technique, which have previously relied upon a vari-ety of methods involving, in some cases, a lengthy and complex sample preparation protocol.

ConclusionsIn recent years, there has been much

development of analytical instrumentation with new equipment offering increased sensitivity, dynamic range, and detection limits. While this no doubt will assist in the

analysis of complex mixtures of bioac tive compounds, there remains the problem of trying to attain the holy grail of en-capsulating all of the desired information on a range of complex phytochemicals in a single analytical run. It is not anticipated that this goal will be reached in the very near future, but developments in sample preparation strategies, the use of multiple extraction procedures, or the utilization of techniques which bypass solvent extrac-tion completely, may increase number and diversity of the metabolites under study in

any given sample and this, coupled with the ever evolving and advancing tech-nology, whether mass spectrometry or NMR, may allow researchers, particularly those involved in the analysis of bioactive components in cereals, to combine and simplify their methods, whilst increasing the range of components analyzed.

AcknowledgmentsThis publication is financially supported by the

European Commission in the Communities 6th Framework Program, Project HEALTHGRAIN (FOOD-CT-2005-514008). It reflects the authors’ views and the community is not liable for any use that may be made of the information contained in this publication. Rothamsted Research receives grant-aided support from the Biotechnology and Bio logical Sciences Research Council of the United Kingdom.

References 1. Aharoni, A., Ric de Vos, C. H., Verhoeven, H.

A., Maliepaard, C. A., Kruppa, G., Bino, R., and Goodenowe, D. B. (2002). Nontargeted metabolome analysis by use of Fourier trans-form ion cyclo tron mass spectrometry. Omics 6:217-234.

2. Baker, J. M., Hawkins, N. D., Ward, J. L., Lovegrove, A., Napier, J. A., Shewry, P. R., and Beale, M. H. (2006). A metabolomic study of substantial equivalence of field-grown ge-netically modified wheat. Pl. Biotech. J. 4:381-392.

3. Brown, S. C., Kruppa, G., and Dasseux, J. L. (2005). Metabolomics applications of FT-ICR mass spectrometry. Mass Spectrom. Rev. 24:223-31.

4. Burrell, M. M., Earnshaw, C. J., and Clench, M. R. (2007). Imaging matrix assisted laser desorption ionization mass spectrometry: a technique to map plant metabolites within tis-sues at high spatial resolution. J. Exp. Bot. 58:757-763.

5. Cha, S., Zhang, H., Ilarslan, H., Wurtele, E. S., Brachova, L., Nikolau, B. J., and Yeung, E. S. (2008). Direct profiling and imaging of plant metabolites in intact tissues by using colloidal graphite-assisted laser desorption iionization mass spectrometry. Plant J. 55:348-360.

6. Fiehn, O., Kopka, J., Trethewey, R. N., and Willmitzer, L. (2000). Identification of uncom-mon plant metabolites based on calculation of elemental compositions using gas chromatog-raphy and quadru pole mass spectrometry. Anal. Chem. 72:3573-3580.

7. Fraser, P. D., Enfissi, E. M. A., Goodfellow, M., Eguchi, T., and Bramley, P. M. (2007). Metabolite profiling of plant carotenoids using the matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Plant J. 49:552-564.

8. Kitteringham, N. R., Jenkins, R. E., Lane, C. S., Elliott, V. L., and Park, B. K. (2008). Mul-tiple reaction monitoring for quantitative bio-marker analysis in proteomics and metabolomics. J. Chromatogr. B. 877:1229-1239.

9, Hall, R. D. (2006). Plant metabolomics: from holistic hope, to hype, to hot topic. New Phy-tol. 169:453-468.

10. Ishida, Y., Kitagawa, K., Goto, K., and Ohtani, H. (2005). Solid sampling technique for direct detection of condensed tannins in bark by ma-

A Perten ad appeared here in the printed version of the journal.

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Jane L. Ward is the manager of the National Centre for Plant and Microbial Metabolomics, located at Rothamsted Re-search. She has 19 years of experience in synthetic and ana-lytical chemistry techniques and has helped to develop the institute’s metabolomics program. Ward also manages the MeT-RO metabolomics service which provides a comprehen-sive metabolite profiling service to U.K. researchers on a fee-for-service basis, and as such, has participated in many large-scale metabolomics projects addressing key questions in plant science. She can be reached at jane.ward@bbsrc.ac.uk.

Peter R. Shewry has more than 30 years of experience in research on wheat and other cereal grains, ranging from grain structure and functionality to molecular genetics. He is the author of more than 350 papers in international journals and editor or coeditor of 18 books. He has been awarded the Os-borne Medal by AACC Intl. (2000), the Rank Prize for Nutrition (jointly with Don Kasarda) (2002), and a Fellowship of the ICC Academy (2009). He and Khalil Khan are coeditors of the re-cently published fourth edition of Wheat: Chemistry and Tech-nology. His current research focuses on the biosynthesis and deposition of protein and cell wall components that determine wheat grain quality. Shewry can be reached at peter.shewry@bbsrc.ac.uk.

trix-assisted laser desorption/ionization mass spectrometry. Rapid Comm. Mass Spectrom. 19:706-710.

11. Ortiz-Monasterio, J. I., Palacios-Rojas, N., Meng, E., Pixley, K., Trethowan, R., and Peña, R. J. (2007). Enhancing the mineral and vita-min content of wheat and maize through plant breeding. J. Cereal Sci. 46:293-307.

12. Roessner, U., Wagner, C., Kopka, J., Trethewey, R. N., and Willmitzer, L. (2000). Technical advance: Simultaneous analysis of metabolites in potato tuber by gas chromatography-mass spectrometry. Plant J. 23:131-142.

13. Shu, X-L., Frank, T., Shu, Q-Y., and Engel, K-H. (2008). Metabolite profiling of germinating rice seeds. J. Agric. Food Chem. 56:11612-11620.

14. Stewart, D., McDougall, G. J., Sungurtas, J., Verrall, S., Graham, J., and Martinussen, I. (2007). Me tabolomic approach to identifying bioactive compounds in berries: advances toward fruit nutritional enhancement. Mol. Nutr. Food Res. 51:645-651.

15. Ward, J. L., Baker, J. M., and Beale, M. H. (2007). Recent applications of NMR spectroscopy in plant metabolomics. FEBS J. 274:1126-1131.

16. Ward, J. L., Poutanen, K., Gebruers, K., Piironen, V., Lampi, A.-M., Nyström, L., Anderson, A. A. M., Åman, P., Boros, D., Rakszegi, M., Bedo, Z., and Shewry, P. R. (2008). The HEALTHGRAIN cereal diversity screen: concept, results and prospects. J. Agr. Food Chem. 56:9699-9709.

17. Yelle, D. J., Ralph, J., and Frihart, C. R. (2008). Characterization of nonderivatized plant cell walls using high-resolution solution-state

NMR spectroscopy. Magn. Reson. Chem. 46:508-517.

18. Zabrouskov, V., Al-Saad, K. A., Siems, W. F., Hill, H. H., and Knowles, N. R. (2001). Analysis of plant phosphatidylcholines by matrix assisted laser desorption/ionization time-of-flight mass spectrometry. Rapid

Commun. Mass Spectrom. 15:935-940. 19. Zhang, H., Cha, S. W., and Yeung, E. S. (2007).

Colloidal graphite assisted laser desorption/ionization MS and MSn of small molecules. 2. Direct profiling and MS imaging of small metabolites from fruits. Anal. Chem. 79:6575-6584.

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