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Journal of Chromatography A, 1000 (2003) 657–691

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Review

Strategies for the determination of bioactive phenols in plants, fruit
and vegetables
Kevin Robards*
School of Science and Technology, Charles Sturt University, Locked Bag 588, Wagga Wagga, NSW 2678, Australia

Abstract
Analytical strategies dealing with bioactive phenols in plants and foods are reviewed. These depend on the purpose of the
analysis which may be classified as studies where the principal purpose is biological screening, phytochemical and / or
chemical screening. Nevertheless, extraction of the phenol from the sample matrix is common and methods of achieving a
suitable extract are assessed. Advances in the separation sciences and spectrometry are exploited for identification and
quantification of isolated phenols. The various procedures are summarized and some typical ‘‘case studies’’ are presented.
Two important areas are introduced briefly. Thus, plant phenols are reactive species and their ultimate fate has been
relatively neglected. Studies of bioactive compounds generate a considerable volume of data making data handling and
informatics important topics that warrant a separate review.
 2003 Elsevier Science B.V. All rights reserved.
Keywords: Reviews; Plants; Fruit; Bioactivity; Phenols

Contents
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3.
4.

Introduction ............................................................................................................................................................................
Sample collection and storage ..................................................................................................................................................
Screening of bioextracts...........................................................................................................................................................
Phytochemical studies .............................................................................................................................................................
4.1. Sample preparation .........................................................................................................................................................
4.2. Analyte recovery.............................................................................................................................................................
4.2.1. Recovery of flavan-3-ols and anthocyanins............................................................................................................
4.2.2. Fresh versus processed samples ............................................................................................................................
4.3. Clean-up of extracts ........................................................................................................................................................
4.4. Quantification .................................................................................................................................................................
4.4.1. Chromatographic methods ...................................................................................................................................
4.4.1.1. Detection.............................................................................................................................................
5. Chemical screening .................................................................................................................................................................
5.1. Structural characterization ...............................................................................................................................................
5.1.1. Mass spectrometry ..............................................................................................................................................
5.1.1.1. Applications of mass spectrometry ........................................................................................................
5.1.2. Nuclear magnetic resonance spectrometry .............................................................................................................
*Fax: 161-2-6933-2737.
E-mail address: krobards@csu.edu.au (K. Robards).
0021-9673 / 03 / $ – see front matter  2003 Elsevier Science B.V. All rights reserved.
doi:10.1016 / S0021-9673(03)00058-X

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K. Robards / J. Chromatogr. A 1000 (2003) 657–691

6. Degradation products of antioxidants ........................................................................................................................................
7. Informatics and data handling ..................................................................................................................................................
8. Conclusions ............................................................................................................................................................................
9. Nomenclature..........................................................................................................................................................................
References ..................................................................................................................................................................................

1. Introduction
Epidemiological studies over the last three decades
have consistently correlated certain diets, specific
foods and disease expression. At the same time, the
number of bioactive compounds has increased
dramatically and a new diet-health paradigm has
evolved that emphasizes the positive aspects of diet.
The terms ‘‘phytochemical’’, ‘‘nutraceutical’’ and
‘‘functional food’’ have been introduced [1] to
describe various aspects of this development. Databases detailing the presence and amount of bioactive
compounds in foods have recently been reviewed
[2]. Bioactive compounds included a range of compounds with diverse chemical structures such as
plant sterols, carotenoids, v-3-fatty acids, indoles
(benzopyrroles) and phenols. The number and diversity of these compounds preclude an exhaustive
coverage of their determination and analytical strategies are illustrated in this review with specific
reference to plant phenols.
Plant phenols (Fig. 1) embrace a wide range of
secondary metabolites that are synthesized from
carbohydrates via the shikimate pathway. This is the
biosynthetic route to the aromatic amino acids and is
restricted to microorganisms and plants. Thus, phenolic compounds are ubiquitous in the plant kingdom
being found in all fruits and vegetables in virtually
all parts of the plant but with quantitative distributions that vary between different tissues of the plant
and within different populations of the same plant
species [3]. The phenolic component of plants constitutes a complex mixture, and only a small number
of plants have been examined systematically for their
phenolic content. Thus, the data on phenolic content
of plants, fruits and vegetables are incomplete.
Nevertheless, both qualitative and quantitative data
have been summarized in various reviews [4–11]
although quantitative data are not reliable [8] due to
the wide diversity of extraction and quantification
procedures.

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The determination of phenols encompasses a
number of distinct aspects and the analytical strategy
will depend on the sample, analyte and nature of the
problem. Given the diversity of analytes and sample
types and the number of permutations of the three,
there is no global strategy that will suffice for a
given phenol in all situations although a number of
generalisations can be made. Thus, the general
analytical strategy involves recovery of the phenol
from the sample matrix followed by separation,
identification and measurement. For most phenols,
the recovery step typically involves solvent extraction using a range of solvents. Special considerations
apply to some phenols such as the anthocyanins and
oligomeric species. Polymeric phenols introduce a
new range of considerations and are not considered
in this review. Separation is commonly achieved by
HPLC although GC is used in some instances. The
most common mode of separation exploits reversedphase systems typically with a C 18 column and
various mobile phases. Detection is routinely
achieved by ultraviolet absorption often involving a
photodiode array detector although the versatility of
the latter often appears to have been neglected.
Coupled techniques particularly various mass spectral methods are being used increasingly for routine
work although analyte collection using preparativescale HPLC and off-line identification are often still
needed for non-routine samples.
Considering the nature of the analytical problem,
several roles can be identified although there is no
rigid distinction and an investigation may encompass
aspects of each one. In the first role, screening of
bioextracts for biologically active natural products
played a strategic role in the phytochemical investigation of crude plant extracts [12,13]. The
primary strategy for the discovery of bioactive
natural products through the 19th century and into
the 20th century was the structure elucidation of
active ingredients of plants with reported biological
properties. Methods of characterization and identifi-

K. Robards / J. Chromatogr. A 1000 (2003) 657–691

Fig. 1. Structure of typical phenols.

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Robards / J. A 1000 (2003) 657–691 Fig. 1. (continued) .660 K. Chromatogr.

For instance. there is a clearly defined desired outcome. Robards / J. quercetin. Such studies demonstrate the importance of isomerism as a feature of bioactivity. A separate clean-up step may be mandatory in some cases. This role is being reversed and chemotaxonomy is generating bioactive compounds of the same or related molecular structures. This is clearly an area which requires closer examination to establish guidelines and definitive procedures. The isolation of new bioactive compounds from plants can be directed by bioassays [31].30]. In this role. bioguided fractionation. chemical ecology and plant anatomy [28]. in some instances. The availability of specific in vitro bioassays has facilitated the screening of numerous bioactivities of natural products. Alternatively. the second role employs ‘‘chemical screening’’ using coupled techniques at the earliest stage of separation on crude extracts. . 2. However. Microwave thawing produced the most reliable results and was also the most practical approach for routine analyses. Screening has uncovered new pharmaceuticals and structure–activity relationships which has provided leads for design of new drugs. Screening of bioextracts Current strategies for choosing candidate plant species or tissues for isolation of bioactive components are based on ethnobotany. both phenylpropanoid glycosides. Such investigations are conveniently termed phytochemical studies and involve diverse approaches. The ultimate goal of such studies is the identification of a compound with a high level of bioactivity and a low degree of toxicity plus optimal pharmacokinetic properties. extract preparation is followed by isolation of the phenol(s) and structure elucidation. cinnamic acid derivatives generally occur as the trans 661 isomers but verbascoside and lantanaside. isolation and structure elucidation was the usual approach where complete characterization was required. A third application area involves the determination of previously identified substances as in the quality control of the active principles in herbal and other products [15– 18]. Hence. definitive procedures for collection and storage have not been established although the limited data clearly indicate the importance of this step.K. However. caffeic. With this approach. analysis is carried out simply to profile the phenolic content of the plant or food. The thawing method (refrigerator. p-hydroxybenzoic. new uses of compounds can be identified when known compounds are tested in new bioassays. the antioxidant activity of flavonoids is closely related to the position and degree of hydroxylation of the molecule [11. the number of biological assays that are available in a given laboratory is often limited and the range of activities screened is thus restricted. Structure–antiviral activity effects [22] have been assessed using three series of 3-methoxyflavones. biological screening. room temperature or microwave) showed differential effects on the level of various phenols. gallic and ellagic acids) from frozen non-grape berries [24–27]. Chromatogr. In the first three application areas. This efficient and targetted isolation of compounds permits an optimization of an investigation and avoids the time-consuming and costly isolation of ‘‘trivial’’ natural products.21]. the analysis is simplified to the preparation of an extract and quantification of the analyte. Sample collection and storage is relevant to all application areas and is treated briefly. myricetin) and phenolic acids ( p-coumaric. Phytochemicals such as flavonoids have been used historically to identify plants in chemotaxonomy [29. A further application area that is not treated in this review involves use of quantitative structure–activity relationships (QSAR) [19] and molecular modeling of bioactive species [20] to assess structure–activity relationships. For instance. 3. Lantanaside contains a cis-caffeoyl moiety instead of trans-caffeoyl as in verbascoside. This review examines each of these application areas and covers the period from approximately 1990. As a complementary approach. A 1000 (2003) 657–691 cation of plant phenols followed those in general use for natural products [14]. Three extraction and hydrolysis procedures were examined for the recovery of flavonols (kaempferol. ferulic. preparation of an extract. Sample collection and storage In many instances. were isolated [23] from the polar fraction of a methanolic extract of the leaves of Lantana camara.

catechins) also function as good browning substrates in fruit and vegetables [11]. the crude alkaloid fraction exhibited no in vitro activity whilst the pure alkaloid showed pronounced cytotoxicity in the same test [33]. Each has its advantages depending on the objectives and these have been discussed by Duke et al. Their antioxidant activity means that phenolic compounds (e. bound vs. The action of phenols as antioxidants is viewed as beneficial in both foods and the body where phenols are oxidized in preference to other food constituents or cellular components and tissues. Other factors include synergistic effects. For example. However. oligomeric or polymeric species) impact on the choice of a method. [28]. solvents often appear as the culprits.29diphenyl-1-picrylhydrazyl radical scavenging method. other phenols. Moreover. total phenols. they continue in use because of their simplicity. They are functional as antioxidants at relatively low concentrations while at higher concentrations.40]. in the isolation of leurosine. The nature of both the sample and analyte (e. Caution is necessary to avoid artefacts ranging from the obvious such as antioxidants added to certain solvents used in measurement of antioxidant activity to the less obvious. since they themselves are susceptible to oxidation. the trends in antioxidant activity of phenols differed [46] according to whether hydroperoxide formation (peroxide value) or decomposition (hexanal and volatiles) was measured in accelerated stability tests on olive oil. low cost and the potential to generate activity data of direct relevance. they can behave as prooxidants due to their involvement in initiation reactions. Middleditch [37] has compiled a list of commonly encountered artefacts. Chromatogr.g. Thus. For example. chemical changes during extraction and cancellation of activity by certain concentrations of substances. free phenols. The following presents an overview of this area but with an emphasis on the sample type. Nevertheless. It is sufficient to state that methods show extreme diversity [42. Three methods widely employed in the evaluation of antioxidant activity. 4. other factors must also be considered in designing an optimal sample handling strategy that will ensure a sample extract uniformly enriched in all components of interest and free from interfering matrix components. static headspace gas chromatography and bcarotene bleaching test. However. have been compared with regard to their application in the screening of plant extracts of differing polarity [47]. Activity in each assay was affected by the complex composition of the extracts and partition phenomena. caffeic esters. These results emphasize the need to measure at least two oxidation parameters to better evaluate antioxidant activity. Triacontanol is bioactive and is a known contaminant of some filter papers. Phytochemical studies 4. Robards / J. the determination of antioxidant potential (to determine level of food protection) or physiological activity using in vitro [38] or in vivo tests [39. Plasticizers are ubiquitous and their exclusion from extracts is a challenging problem [35.36]. specific phenolic classes such as flavonone glycosides or individual compounds.1. Many factors can complicate results when using bioassays or bioassayguided fractionation. . The selection of solvent must be considered carefully in relation to the nature of the bioassay in order to avoid false results. The most studied bioactivity of the phenols is their antioxidant status. o-diphenols vs. the active ingredient was isolated as a hemiacetal from alcoholic solvents [34] rather than the naturally occurring dialdehydic species. Methods of measuring antioxidant activity have been reviewed elsewhere [41] and are not re-examined here. Sample preparation Sample handling strategies have been reviewed recently [48].662 K. Solvent is important as many bioextracts have limited solubility [32]. namely 2. A 1000 (2003) 657–691 Bioassays can range from molecular assays to whole-organism assays. monomeric. measurement of antioxidant activity of a phenol or mixture of phenols (as in a juice extract) has been applied in two situations.g. they do not distinguish between members of a class of bioactives and provide only semi-quantitative measurements of substances detected. In the isolation of an oleoside derivative from olive. The problem with bioassays is that they provide no data on individual compounds.43] and that activity depends on the analytical technique [44] and substrate [45].

2). solvent and Soxhlet extraction. commercial processing of many plant-derived foods now involves alkali-treatment and the stability of plant phenols under these conditions becomes of interest [75]. Two forces have driven the use of alkaline hydrolysis. SPE and headspace analysis. Clear fruit juices [89] and wines [86] often fall into . solvent extraction). There is considerable variation in the lability of the glycosidic bond under hydrolytic conditions and this was exploited in the HPLC analysis of a flower extract (Fig. In a number of instances. Traditional procedures include homogenization. It appears that acid hydrolysis is seen to more closely reflect dietary intakes although it is evident that absorption. 4. Analyte recovery Some liquid samples are amenable to direct analysis requiring no treatment other than centrifugation. Derivatization of the analyte may also be incorporated in the recovery step. solid-phase microextraction. which will invariably result in artefactual changes involving hydrolysis. filtration and / or dilution as summarized in Table 2. an hydrolysis step has been included to minimize interferences in subsequent chromatography [55] and to simplify chromatographic data [56–59] particularly in instances where appropriate standards are commercially unavailable [60]. Isolation of the phenolic compound(s) from the sample matrix is generally a pre-requisite to any comprehensive analysis scheme although enhanced selectivity in the subsequent quantification step may reduce the need for sample manipulation. The introduction of SFE. 3) were used to identify the 663 components in the crude extract as shown in Fig. Chemical treatment dominates applications (Table 1) because it is more exhaustive and less selective. On-line spectra (Fig. oxidation [49. Care is necessary as structural rearrangements can occur as seen in the case of flavanones with appropriate hydroxyl-substitution that can be easily converted to isomeric chalcones in alkaline media (or vice versa in acidic media) [63]. Hydrolysis has also been used as an aid to structural elucidation and characterization of phenolic glycosides [61] and phenolic choline esters [62]. Thus. filtration / centrifugation. Robards / J.g. 2a. Firstly. A 1000 (2003) 657–691 Thus. indicating that these were C-glycosides. and hence extreme care must be taken to ensure correct extraction. For instance. Acyl groups were removed by alkaline hydrolysis and confirmed the identity of late eluting peaks as acyl derivatives of compound 3. conventional techniques continue to dominate this application area. devoid of chemical modification. distillation. Chromatogr. Luteolin and kaempferol were obtained by acid hydrolysis (Fig. an inert atmosphere and addition of an antioxidant stabilizer was used as a routine precaution (Table 1) whereas the use of an inert atmosphere was an essential precaution in other cases [80] due to poor stability of some phenolic acids in alkaline ambient conditions. 2c) which cleaved O-glycosides but peaks assigned to apigenin glycosides remained. many phenols and particularly the phenolic acids exist in a wide range of conjugated forms and the free phenols are liberated following alkaline hydrolysis. coffee [81]. metabolism and bioavailability of plant phenols are complex and that knowledge of these is still very limited. Some procedures have limited application such as headspace analysis whereas others are more broadly applicable (e. cereals [82]. The task of recovery is further complicated as many foods and plants have a high enzyme activity. oilseeds [83] and medicinal plants [85] in order to determine ‘‘bound’’ phenols. grape and cherry juices [80]. Nevertheless. In many instances. Secondly. The loss of o-diphenols by oxidation via the corresponding quinones [84] is a concern under alkaline conditions. pressurized liquid or fluid extraction [52].2.K. the major characteristic phenols of olive are secoiridoids and their reactivity in alkali has been examined [77].50] and / or isomerization [51]. structural diversity of the phenols affects physicochemical behaviour such as solubility and partitioning behaviour and makes optimisation of the recovery system difficult in all but the simplest cases. membrane extraction and surfactant cloud point extraction [54] have met the increasing demand for new extraction techniques. microwave-assisted extraction [53]. amenable to automation with reduced solvent consumption. Acid hydrolysis has been the traditional approach to measurement of aglycones and phenolic acids from flavonoid glycosides and phenolic acid esters.67–74]. alkaline conditions have been employed in the isolation of phenolic acids from samples such as citrus (juices) [79]. respectively [59.

In: L.). Bloor. Methods in Enzymology.J. Packer (Ed. a luteolin glycoside (4). Academic Press. showing these are apigenin C-glycosides. p. a set of apigenin glycosides (2). Overview of methods for analysis and identification of flavonoids.664 K. a chalcone (6). Vol. 2000. (b) the alkaline hydrolysis product of the same mixture shows a large relative increase in peak 3 and loss of the acylated kaempferol glycoside peaks. 2. Robards / J. Source: S. Peaks due to apigenin glycosides are still present. London. . and (c) the acid-hydrolyzed mixture showing luteolin and kaempferol [8]. HPLC chromatograms [absorbance at 352 nm vs. A 1000 (2003) 657–691 Fig. a set of acylated kaempferol glycosides (5). Flavonoids and Other Polyphenols. and luteolin (7). Chromatogr. 10 [64]. a kaempferol diglycoside (3). time (min)] of (a) a flavonoid mixture showing seven different groups of compounds: a kaempferol triglycoside (1). 335.

Chromatogr. Aqueous percolation or infusion is useful for samples such as coffee and tea. Flavonoids and Other Polyphenols. Academic Press. 2A. caution is necessary as some flavan-3-ols (catechins) are unstable [98] in neutral or alkaline solutions but were precipitated with aluminium chloride [99] which reduced pH and stabilized the extract. London.K. Vol. Freezing of the sample with liquid nitrogen is often a convenient first step in sample preparation [143. p. Cloudy juices such as citrus juices are also amenable to direct analysis following filtration and centrifugation [35. Source: S. In: L. No single solvent will provide optimum recovery of all phenols or even a limited range of phenols. 3. endocellular material is also extracted.97].135] samples are typically extracted with a variety of solvents. However. Methods in Enzymology. Overview of methods for analysis and identification of flavonoids. Packer (Ed. The frozen sample can be ground and then freeze-dried or extracted directly. In many instances. respectively where the extracts provide data on probable dietary intakes [96. For example. and sometimes sequentially with solvents of increasing polarity. 2000.90–92] although poor recoveries have been attributed to low solubility of certain phenolics and / or to sorptive losses on the filtration medium [48]. A 1000 (2003) 657–691 665 Fig. Tunichromes are air. Further treatment of the extracts was carried out as shown in Fig. Bloor. Plant phenols are ionizable with typical pKa . Liquid extraction represents a simple and convenient alternative that has been widely used (Table 3).). oxygen-free argon in the presence of an antioxidant stabilizer. Fresh. 335. freeze-dried [130.J. In either case.and water-sensitive and thus sampling was carried out under a current of dry. this category.132] or airdried [133. Peak numbers as in Fig. The advantages of liquid extraction versus direct injection have been demonstrated [100] for HPLC analysis of a wine sample. 11 [64]. vanadocytes were recovered [141] from the tunicate Ascidia nigra after treatment with liquid nitrogen and freeze drying. On-line UV spectra of selected peaks from Fig. simple filtration is ineffective in recovering a broad range of phenols and alternative strategies are necessary (Table 3). 4 to recover tunichrome B. the reducing blood pigment. Robards / J.144]. Extraction method and solvent choice are generally critical [107] as are extraction time and temperature [120] reflecting the conflicting actions of solubilization and analyte degradation by oxidation for example. 2A.

329 and 370 nm (flavonols) and electrochemical array detector (catechins) [70] UV at 365 and 280 nm [71] UV using PDA [72] Acid hydrolysis in sodium diethyldithiocarbamate stabilized aqueous methanol Acid hydrolysis in ascorbic acid stabilized aqueous methanol under nitrogen and reflux [67] K. tea. acidic hydrolysis in TBHQ stabilized solution and liquid extraction Acidic hydrolysis under nitrogen of ascorbic acid stabilized methanolic extract. 280 nm. naringenin and hydroxycinnamic acids Blueberries and blackberries Phenolic acids. 316 nm. 280. hydroxycinnamates. catechins.g. wine. acidic hydrolysis in ascorbic acid stabilized aqueous methanol Hydrolysis in acidified aqueous methanol containing TBHQ Acidic hydrolysis of flavonoid glycosides [62] Persimmon fruit Phenolic acids GC–MS of oxime TMS derivatives FID and EI-MS [65] Berries Flavonoids Colorimetry. kaempferol. Robards / J. Free flavonoids and phenolic acids fractionated by SPE into neutral and acidic groups Acid hydrolysis in BHA stabilized aqueous methanol. 360 nm (flavonols) [24. A 1000 (2003) 657–691 Sample . 320 nm (hydroxycinnamic acids). flavones (luteolin. fruits (9) Three extraction and hydrolysis procedures using freeze dried berries (e. 365 nm. kaempferol. flavones Rapeseed Phenolic choline ester fragments RPLC using formic acid/acetonitrile (gradient) RPLC using C 18 and acetonitrile/phosphate buffer (isocratic) RPLC on C 18 using aqueous methanol/ phosphate buffer (gradient) UV at 360 nm.26] Berries Flavonols Flavonols. Chromatogr. myricetin). fruits Catechins. followed by aqueous methanol or aqueous acetone extraction [58] Acid hydrolysis RPLC Freeze-dried. PDA and LC–MS (ion trap) UV at 370 nm (PDA used to confirm peak identity) UV at 210 nm [27] Vegetables (28). quercetin SPE of methanolic extract of defatted rapeseed followed by alkaline hydrolysis Petroleum ether extraction of powdered mesocarp. 280 nm (catechins).666 Table 1 Hydrolysis conditions used in preparation of various samples for analysis of phenols Analyte Hydrolysis conditions Quantification Detection Ref Berries Flavonols. anthocyanins. flavonols. 520 nm [66] Various foods Flavonoid aglycones representing five major sub-classes Flavonols (quercetin. HPLC using ternary mobile phase Flavan-3-ols and benzoic acid derivatives. myricetin. hydroxybenzoic and hydroxycinnamic acids RPLC using C 18 and acetonitrile/phosphate buffer (gradient) 260 nm (ellagic and p-hydroxybenzoic acids). ethyl acetate extraction of catechins RPLC UV detection [68] RPLC on C 18 using water/methanol (containing acetic acid) (gradient) UV at 280 and 360 nm [69] RPLC using ODS3 and acetonitrile/ phosphate buffer (gradient: flavonols or isocratic: catechins) RPLC on C 18 using water/methanol (containing trifluoroacetic acid) (gradient) RPLC on C 18 using water/methanol (containing formic acid) (gradient) UV at 270. flavonols Tomato Quercetin. kaempferol. apigenin) Flavonoids and phenolic acids Fruits Cranberry juice Berries. alkaline hydrolysis and ethyl acetate extraction (Enzymatic pectinase extraction). vegetables.

ethyl acetate extraction. flavonoids Effect of sample treatment including alkaline hydrolysis Juices Green coffee Phenolic acids Phenolic acids Alkaline hydrolysis and solvent extraction Rye Phenolic acids plus ferulic acid dehydrodimers Rapeseed and canola meals Total phenolic acids Cherry laurel fruits Phenolic acids Enzymatic hydrolysis of starch followed by alkaline hydrolysis under nitrogen and ethyl acetate extraction Refluxing with acidic acetone. followed by alkaline hydrolysis of residue under nitrogen and ethyl acetate recovery RPLC using C 18 column (gradient) UV at 280 and 340 nm [73] RPLC using a phenyl column with acetonitrile/water (isocratic) UV spectrophotometry UV at 249. Total acids: alkaline hydrolysis in dark and ethyl acetate extraction Ethanol and sodium metabisulfite added to sample. filtered. spinach 667 . 259 and 343 nm. Robards / J. Chromatogr. washed with hexane and extracted with ethyl acetate Acid hydrolysis in aqueous methanol Orange Phenolic and hydroxycinnamic acids. rutin Blood orange juice Hydroxycinnamic acids Olive fruit and brines Oleuropein and derivatives Fruits. LC–MS with APCI and ESI in positive and negative ion modes UV absorption UV at 320 nm using PDA [79] [80] [81] UV at 280 nm [82] Colorimetry Folin-Denis reagent [83] GC of TMS derivatives FID and GC–MS [84] RPLC using isocratic elution RPLC on C 18 using water/methanol (containing formic acid) (gradient) RPLC on C 18 using methanol/phosphate buffer (gradient) [75] K. A 1000 (2003) 657–691 Onion. Spectra recorded from 200 to 380 nm [77] UV at 370 nm [78] UV at 252 and 284 nm. LC–MS–MS with heated nebulizer interface in positive mode UV spectra from 190–690 nm [74] RPLC on C 18 using tetrahydrofuran/water/ acetic acid (gradient) UV at 280 nm [76] RPLC on C 18 using water/acetonitrile/phosphoric acid (gradient) RPLC on C 18 and acetonitrile/phosphate buffer plus methanol/phosphate buffer (isocratic) RPLC on C 18 using methanol/phosphate buffer (gradient) UV at 280 nm and 330 nm. extraction with ethyl acetate/ethyl ether Petroleum ether extraction of powdered mesocarp. Acid hydrolysis under nitrogen and reflux Acidic hydrolysis using HCl at reflux versus phosphoric acid at lower temperatures Stability of various phenols in base and when added to apple juices Free acids: acid hydrolysis and. vegetables. flavones (glycosides) Plant material freeze-dried and mixed with ascorbic acid stabilized aqueous methanol.Flavonoid (glycosides) Soybean and soybean products Isoflavones (Apple juice and cider) Phenolic acids. beverages Flavonols. catechins. alkaline hydrolysis of esterified phenolic acids followed by acidification.

quercetin. beer. Chromatogr. tea Catechins.668 Sample Analyte Sample preparation Quantification Ref Wines Red wines Fortified wines Cis.and trans-resveratrol Rutin. theaflavins Flavanols. flavonol aglycones Phloretin glucosides Flavan-3-ols Nil Nil Nil RPLC. HPLC-PDA. 280 nm and post-column reactor with absorption at 640 nm [89] [90] Flavan-3-ols Flavanone glycosides Flavanones. 340 nm (flavones and hydroxycinnamic acids) RPLC. apple cider. 280 nm RPLC. 290 nm (flavanones). 280 nm RPLC. flavones and hydroxycinnamic acids Filtration Heat and centrifuge Soluble fraction centrifuged. gallic acid. Robards / J. 520 nm Colorimetry. 308 nm (trans-isomer) RPLC-PDA RPLC-PDA [86] [87] [88] Centrifuge and adjust sugar content Filtration RPLC. 280 and 320 nm [91] [92] [93] RPLC and capillary electrophoresis RPLC [96] [97] Apple juice Red wine. and sour cherry and blackthorn fruit liqueurs Wine Orange juice Orange juice Pomegranate juice Grape juice Anthocyanins Black and green teas Coffee. 285 nm RPLC. flavonol glycosides Percolation or infusion [94] [95] K. A 1000 (2003) 657–691 Table 2 Representative examples of the use of simple dilution / filtration for the recovery of phenols from plants and foods . insoluble fraction extracted with dimethyl sulfoxide Filtration Filtration except for procyanidins (isolation on Sephadex LH-20 column) HPLC-PDA. 288 nm (cis-isomer). t-resveratrol Phenolic acids. flavan-3-ols. coumarins.

Thus. which are widely distributed in plants. methanol. However. in the case of extractants using aqueous mixtures. Extraction and collection variables were optimized and revealed that the use of methanol as modifier was mandatory. various extractants have been used [146–148]. Their levels in three model foods: apples. The choice of stabilizer can be influenced by the subsequent procedure as co-elution with plant phenols can occur [26]. black grapes. with dried materials. fruits and vegetables [126. 4. the extraction yield obtained was only 45% of that obtained with liquid methanol. However. Many extraction procedures incorporate the use of an antioxidant as a stabilizer and compounds that have been used for this purpose include BHA [70].2. Some practical illustrations of this diversity are seen in the need to optimize the alcohol content of aqueous alcoholic extractants for phenols of diverse structures [24–27]. Thus. oil [104. Maximum recovery required a minimum of 70% methanol in the extractant and this was attributed to the need to inactivate polyphenol oxidases. and canned kidney beans were not affected [145] by sample drying processes but recoveries were dependent on the type (ethanol. solvent strength can be controlled by varying the pressure thus facilitating sequential extraction of phenols of increasing polarity. the required proportion of water in the extractant is lower with fresh than with dried samples.1.K. Thus. soy [117] and spices [123]. Supercritical fluid extraction offers some advan- 669 tages relative to conventional extraction particularly for chemically or thermally labile compounds. different recovery procedures may be required for the range of phenols encountered in a single sample [124]. they exhibit considerable diversity in terms of acidity as well as polarity ranging from hydrophobic to hydrophilic in character. trans-resveratrol and salicylic acid while mean recoveries of more polar phenolic acids and flavonoids were between 30 and 70%. TBHQ [142] and ascorbic acid [24. ethanol [136] or acetone [144] are often the solvent(s) of choice for recovery of a wide range of phenols from diverse sample types including oats [111]. ethoxyethane and ethyl acetate.26].5 [121]. Chromatogr. Furthermore. Recovery of flavan-3 -ols and anthocyanins Extraction of some phenolic groups warrants special consideration. the relative proportion of endocellular and exocellular components may be determined by solvent choice. Robards / J. A 1000 (2003) 657–691 values ranging from 8 to 12 and oil / water partition coefficients ranging from 6310 24 to 1. in pH control to ensure favourable partitioning behaviour during extraction [121]. low polarity solvents and ethyl acetate will simply leach the sample whereas alcoholic solvents presumably rupture cell membranes and enhance the extraction of endocellular materials. Aqueous methanol was chosen for extraction from diverse food samples [146] because . In many instances. Moreover. Thus. Moreover. There are some important distinctions between fresh and dried samples. The extraction behaviour of phenolic compounds covering a range of polarities has been modelled using supercritical carbon dioxide and an inert support as a sample matrix [128]. The monomeric flavan-3-ols (or catechins) predominantly (1)-catechin and (2)epicatechin are found mainly in brewed tea and red wine. The range of physicochemical behaviours must be considered when determining sample handling strategies as. The effectiveness of added stabilizer will inevitably depend on its concentration and activity relative to indigenous phenols.134].119]. The situation with respect to pH-dependent equilibria is especially complex in the case of anthocyanin extraction [122]. analyte recovery can be reduced at higher concentrations of stabilizer due to the pro-oxidant action of the latter [73]. In the case of the oligomeric and polymeric proanthocyanidins and procyanidins which are based on the flavan-3-ols. or acetone) and concentration (40–100% in water) of extraction solvent. quantitative recovery was limited to the less hydroxylated compounds such as p-coumaric acid. Ethyl acetate [110] and dimethyl sulfoxide [133] have been used as extractants but aqueous mixtures of methanol [135]. extraction yield varies with the solvent system used and also the polymerization degree of the analyte. Dynamic SFE produced clean extracts with higher recoveries of total phenols from dried olive leaf [129] than sonication in liquid solvents such as n-hexane. for example. Aqueous acetone [144] generally gives the best yields although a variable proportion of proanthocyanidins resist extraction particularly in aged or oxidized tissues [149].

gallic acid. flavonols. luteolin-7-O-glucoside. LC–MS–MS ion trap in positive and negative ion modes RPLC. sonicated at low temperature. A 1000 (2003) 657–691 Orange Olive oil Pear Extraction of powdered samples with 80% aqueous ethanol at low temperature followed by SPE Hydroxybenzoic and hydroxycinnamic acids. thiolysis. apricot) Olive oil Red wine Sage Olive fruit RPLC. 325 nm (hydroxycinnamic acids). evaporation and dissolution in methanol Phenols Extraction with methanol and isopropanol/methanol Chlorogenic acid. 540 nm (procyanidins). 280 nm RPLC-PDA. 280 and 340 nm RPLC. followed by SPE Polymethoxylated flavones Extraction with benzene. rosmarinic acid. 280. dissolution in acetonitrile and hexane washing Flavanols. 340 nm [114] [115] FT-ion cyclotron resonance-ESI-MS RPLC-PDA. flavonols and glycosides Homogenized with methanol (containing formic acid). 360 nm (flavonols) Colorimetry. rutin. clean-up by liquid liquid extraction Procyanidins Freeze dried. dihydrochalcones. Chromatogr. 374 nm RPLC. ferulic acid. 280 and 316 nm. evaporation to derivatives. 340 and 365 nm [120] Not applicable [121] Not applicable RPLC. peach. 360 nm [100] and exc. HPLC. LC–ESI-MS negative ion mode RPLC-PDA. flavonols. flavan-3-ols. Juice concentrated at controlled temperature in under 40 min. oleuropein and derivatives Examined partitioning behaviour after removal of phenolics with aqueous methanol Anthocyanins Examined pH-dependent equilibria Caffeic acid. 330 nm. 280 nm (flavanols). HPLC-PDA. flavonoids Petroleum ether wash followed by aqueous methanol extraction and fractionation by column chromatography Anthocyanins. Extraction of ground samples with ethanol apigenin. UV at 280 nm. chalcones. 280 nm [113] RPLC-PDA full scan mode. 320 and 350 nm [122] [123] RPLC-PDA [124] K. 240 nm RPLC. 278 nm. flavones Extraction in an ultrasonic bath in the dark with acidified aqueous methanol containing BHT Phenolic acids. LC–ESI-MS RPLC. epicatechin Aqueous ethanol extraction of powdered fruit. tyrosol. 280 nm. hydroxycinnamic acids. grape. 280 nm (other phenols). em. oleuropein and Extraction of oil with aqueous methanol. 280 nm [102] [103] [104] [105] [106] [107] [108] RPLC. 280. successive extractions with methanol and aqueous acetone. 210–360 nm [116] [117] [118] GC and GC–MS of TMS derivatives.670 Table 3 Examples of procedures used for the recovery of phenols from plants and foods Sample Analyte Recovery Quantification Wines Phenolic acids and aldehydes. flavonols esculin Dilution with aqueous methanol Isoflavone Extraction with 80% ethanol Benzoic and cinnamic acids. PDA [101] Dried plums Cider apple tissues Red beetroot peel Chinese medicine Oat groats Pollen Oats Lentils and beans Olive oil Rose hip Grapevine leaf Wine Soy Peach and apple purees and concentrates Olive oil Fruit juices (apple. flavonols. em. flavonoids Addition of acid to lower pH and extraction with diethyl ether Soy-based foods Isoflavones RPLC. flavanones. 254. flavonol glycosides Extraction with diethyl ether and then ethyl acetate. dried and extracted with flavan-3-ols ethyl acetate Derivatives of tyrosol and oleuropein Methanol extraction Benzoic and hydroxycinnamic acids. LC–ESI-MS–MS negative (and positive) ion modes HPLC. hydroxytyrosol. residue dissolved in aqueous methanol Tyrosol. Spectra recorded 210–350 nm [109] [110] [111] [112] RPLC. Robards / J. Homogenized in aqueous methanol. 280 and 350 nm RPLC RPLC. LC–ESI-MS negative (and positive) ion modes HPLC–DAD. evaporation and ethyl acetate extraction Hydroxycinnamic acids Extraction with methanol Flavonoid aglycones Extraction with ethyl acetate Phenolic acids Extraction with aqueous ethanol Benzoic and cinnamic acids. Butanol/hydrochloric acid hydrolysis for procyanidins p-Coumaric acid. fluorescence at exc. pineapple. Dried plums homogenized in 80% aqueous methanol at low chlorogenic acids. 232 and 278 nm [119] RPLC. cirsimaritin Nine phenols Two extraction methods used Ref . dried and redissolved Caffeic acid. hispidulin. orange. anthocyanins temperature. ligstroside dryness. flavones. flavonoids Extraction with aqueous methanol Isoflavones Soxhlet extraction of ground powder with aqueous methanol. pear.

catechin.Table olives. flavonoids Olive leaves Phenols Flavonols. 280. e. proanthocyanidins. frozen berries extracted with acetone/methanol/water/formic acid followed by SPE Freeze-dried and sequentially extracted with hexane. flavonol glycosides. spectrophotometry. 4 Freeze-dried samples refluxed under nitrogen in acidified aqueous methanol containing TBHQ Sample freeze-dried. flavones and naringenin Flavonoids and glycosides Flavanones and flavanone glycosides Lemon verbena leaves Flavonoids Olive pulp Oleuropein (derivatives). 280. catechins Spiked diatomaceous earth Phenolic acids. 280 nm. 313 nm (phenolic acids) RPLC. 280 nm (proanthocyanidins). tyrosol. dihydrochalcones Apple and grape Flavan-3-ols Eucalyptus Procyanidins Extraction of dried. anthocyanins. flavonols. 525 nm [128] [129] [130] RPLC. green tea Flavonol glycosides White grape seeds Gallic acid. Robards / J. 280 nm. freeze-dried and extracted with methanol Ground. flavonols Tunichrome B Anthocyanidins. ground fruit with dimethyl sulfoxide Extraction of ground apple peel with acidified methanol 671 . 280. ground leaves with aqueous ethanol Pulp frozen in liquid nitrogen. parsley. methanol evaporated and partitioning into diethyl ether Extraction with aqueous ethanol of dried. 350 nm [136] [137] RPLC. catechins. extraction of oil with aqueous hydrogen carbonate and SPE for (hydroxy)tyrosol Infusion (tea) or aqueous methanol extraction (onion) of freeze-dried sample followed by SPE Sequential SFE using carbon dioxide and adding methanol as a polar modifier SFE using carbon dioxide with methanol modifier SFE using carbon dioxide with methanol modifier of dried. blackberries Chocolate Anthocyanins. 280 nm [127] RPLC Colorimetry using Folin Ciocalteu reagent and ESI-MS negative ion mode screening RPLC. ground leaves with aqueous methanol.g. 370 nm RPLC. flavan-3-ols. 355 nm. 530 nm (anthocyanins). UV [131] RPLC-PDA HPLC. ethyl acetate. 520 nm. ion trap negative ion mode [143] RPLC. 350. LC–ESI-MS positive ion mode LC–MS. A 1000 (2003) 657–691 Apple Different procedures depending on analyte. ground leaves Frozen in liquid nitrogen. phenolic acids Phenolic acids. methanol and acetone Extraction with aqueous methanol LC–APCI-MS–MS [125] MALDI–TOF-MS positive (and negative) ion modes [126] RPLC. frozen. methanol Extraction of dried. flavonoids Grape skin Ascidia nigra Onions. ground fruit Extraction with methanol (glycosides) or acid hydrolysis (aglycones) Room temperature extraction of dried. 535 nm [140] [141] Kinetics method (RPLC. ionspray with heated turboprobe (negative ion mode). 340 nm [139] [138] HPLC. chlorogenic acid Blueberries Anthocyanins Tart cherries Flavonoids Grapefruit and pummelo Apple skin Sour orange Orange juice Flavanones Flavonols. 520 nm versus MALDI–TOF-MS (complementary role) RPLC. 320 nm [144] RPLC. phloridzin. and extracted sequentially with hexane then acetone/water acetic acid Tissues isolated while spraying with aqueous formic acid. UV (270 nm) or fluorescence (280/310 nm excitation/emission) [145] K. 520 nm) [142] LC–ESI-MS(-MS). 325 nm [133] [134] [132] [135] RPLC. freeze-dried and extracted sequentially with hexane. 350 nm (flavonols). flavonols. RPLC. Chromatogr. ground and aqueous ethanol extraction followed by liquid–liquid partitioning Aqueous methanol extraction Refer to Fig. olive oil Phenolic acids. 290 nm. oleuropein derivatives Yellow onion. flavones Cider apple tissues Hydroxycinnamic acid derivatives.

their extraction procedure involved acid hydrolysis (at reflux) in TBHQstabilized aqueous methanol under which conditions anthocyanidins and catechins are unstable thus limiting the applicability of the method. Their chemistry is complicated by various pH-dependent equilibria and this was exploited in traditional strategies for their recovery as the flavylium cation form by extraction with cold methanol containing hydrochloric acid [154.2. The replacement of hydrochloric acid with weaker acids. With the most labile anthocyanins. 4. Robards / J. the use of nonacidified solvents is probably a sensible precaution. catechins and anthocyanidins). Fresh versus processed samples Recovery methods for use with processed products generally exploit the same principles as for fresh and / or dried materials but allowing for any differ- . Isolation of tunichrome B complex from Ascidia nigra showing the structure of one of the components. 4. either formic or acetic acid [156] will overcome this problem and both grape and cherry anthocyanins were extracted [51. [141].150–153].2.672 K. Chromatogr. Anthocyanins are widely distributed and comprise a significant portion of the phenolic content of many fruits (e. For instance.155]. the acylated anthocyanins are frequently labile under such conditions [67]. Source: Adapted from Ref. of its efficiency in recovering flavanols of low polymerization degree.157] at room temperature using a mixture of formic acid in aqueous methanol. A 1000 (2003) 657–691 Fig.g. Merken and Beecher [67] developed an HPLC system that separated major phenolics from each of the five sub-classes of flavonoids (flavones. They are acylglycosides and glycosides of the anthocyanidins of which six are commonly encountered. flavanones. However. However. flavonols. dark-coloured berries) [51.

Phenolic acids were determined [186] in four fruit juices after pre-concentration by SPE using a combination of reversed-phase and ionexchange cartridges in series.194] exhibit extreme diversity. catechin and procyanidins plus flavonols present as the glycosides of quercetin and kaempferol) by processing on Sep-Pak C 18 cartridges. carotenoids and chlorophyll). sequential extraction or liquid liquid partitioning and / or SPE. Quantification The number. enzymatic activity in green olive drupes [158] was inhibited by refluxing in boiling methanol for 30 min. A 1000 (2003) 657–691 ences in moisture content and / or enzyme activity. Robards / J. In orange . Enzymatic activity can produce qualitative and quantitative changes in the phenolic content of fresh samples [12. flavonoids were not detected in cultivated mushrooms [195] whilst they were present in orange juice at levels up to 500 mg l 21 [196]. Gallic acid was concentrated on the latter but was not retained on the reversed-phase. Various strategies have been designed to cope with this situation. Chromatogr. interfering sugars can be eluted with aqueous methanol prior to elution of phenols with methanol [167]. The aqueous extract following removal of methanol was exhaustively extracted with ethyl acetate and purified using reversed-phase TLC. C 8 ) [185] remain the most popular. The acids were eluted with 0. The sample was filtered and the organic solvent removed under vacuum. Hydroxycinnamic acid derivatives.3. frozen. Thus. the alcohol content can be reduced and the phenols 673 partitioned into a non-polar solvent [135]. 4. Phenols in the filtered ethanolic extract were quantified by ultraviolet derivative spectrometry. oils [172–178] and extracts [179–183] can be achieved by SPE or traditional polyamide columns [184]. Residual oil is removed by overnight storage at subambient temperature [119]. With reversed-phase cartridges.130]. A similar approach was used for tissues of mature cider apples [144]. namely. flavonols and dihydrochalcones were identified in the extracts. type and concentrations of phenols in plants [24. Clean-up of extracts Many extracts contain significant quantities of carbohydrates and / or lipoidal material that potentially interfere with subsequent quantification. The aqueous residue was analysed by HPLC without further clean-up.1 M HCl and methanol after washing of the cartridges with water. ground and defatted with hexane in an ultrasonic bath [143] before extraction of catechins and procyanidins with an acetone / water acetic acid extractant. freezedried and extracted sequentially with hexane (to remove lipids. Procyanidins were the predominant phenolic constituents in the fruits. Several SPE formats are commercially available ranging from the original cartridges to disks in a range of sorbents although C 18 and other reversed-phase materials (e. An example of sequential extraction (Table 3) is provided by the analysis of chocolate which was freeze dried.50] to effect further clean-up. flavan-3-ols. interfering species. In some cases. Representative examples of the approaches used for clean-up are summarized in Table 4.4. It follows that the phenolic profile of processed products typically differs from that of the fresh material [160] arising from the effects of enzymatic activity during processing.4-dihydroxybenzoic acids) and weakly acidic materials (epicatechin. Extraction with boiling ethanol (5 min) followed by aqueous ethanol (1 h) has also been applied [159] and the authors noted that boiling inactivated enzymes and aided in phenolic recovery. caffeic and 3. by centrifugation [115] or by further extraction with hexane although Sephadex column chromatography has also been used [49. The tissues were isolated while spraying with aqueous formic acid to avoid oxidation. methanol (sugars. In the case of aqueous alcoholic extracts. organic acids and low molecular mass phenols) and acetone (polymeric phenols).187–191] as illustrated by the analysis of kiwifruit juice [192] which was fractionated into strongly acidic (derivatives of coumaric. Simultaneous sample clean-up and pre-concentration of juices and wines [162–171]. Samples such as olive oil are typically dissolved in hexane [161] or ethoxyethane [119] followed by liquid– liquid extraction using various mixtures of water and alcohol in order to isolate the desired analytes from unsaturated.g. much of them corresponding to highly polymerized structures. Fractionation of the phenolic components is readily achieved by SPE [69. 4.K. preparativescale HPLC has been used [193] in sample preparation. For example.

o-diphenols Raspberry juice Hydroxybenzoic and hydroxycinnamic acids. 285 nm [168] RPLC RPLC. 15) Total phenols. flavonol glycosides. 254 nm [169] [170] RPLC. overnight refrigeration Oil dissolved in hexane and phenols partitioned into aqueous methanol Acid and base hydrolysis followed by SPE on C 18 cartridge eluting with acidified methanol SPE Solvent extraction and two SPEs SPE Bond Elute SPE eluting with acidified methanol SPE using phenyl boric cartridges Dealcoholized (wines) and SPE to remove sugars [60] RPLC RPLC RPLC FT-IR (mid-infrared) and UV–Vis spectra RPLC Colorimetry [162] [163] [164] [165] [166] [167] HPLC-PDA. 239 nm. 332 nm. Robards / J. methanol evaporated and phenols partitioned into diethyl ether Oil dissolved in diethyl ether and extracted with methanol or aqueous methanol. 336 nm GC [174] [178] FT-IR. catechins. flavones and naringenin Phenols (ca. nm. Chromatogr. ligstroside Spinach Flavonoid aglycones and glycosides Blood orange Flavanone glycosides and t-cinnamic acid Extraction with methanol/dimethyl sulfoxide.674 Table 4 Techniques used for extraction of phenols and clean-up of extracts Analyte Sample extraction/clean-up Quantification Ref. 280 nm.50] Eucalyptus RPLC. 280 nm. SPE concentration for tert. vegetables Berry and fruit wines and liquors Citrus tissues Flavonoids (25) Citrus juices Fruit of Libanotis Flavanones.-cinnamic acid [161] K. ‘‘SPE’’ on C 18 Dilution in dimethylformamide/ammonium oxalate solution and centrifugation. Olive oil Hydroxytyrosol derivatives RPLC. EI-MS. flavones. 278 nm Colorimetry. SPE on C 18 cartridge SPE Refluxed with methanol followed by SPE on C 18 microcolumn and then SPE on quaternary amine microcolumn SPE using amino phase and diol phase Comparison of SPE on C 18 cartridge and liquid liquid extraction Various extractants and methods (including Soxhlet) compared. flavonols Anthocyanins Anthocyanins Proanthocyanidins Phenols Flavones. RPLC. 370 nm (o-diphenols) RPLC. ground leaves with aqueous methanol. 325 nm [135] Olive oil Phenolic acids. 24. flavonols Phenolic acids Olive oil Olive oil Phenols (ca. RPLC at 260 and 315 nm [180] HPLC. 280 nm [182] Fruit juices and syrups Purple passion fruit Grapes and wine Red wine Red wine. 278 nm [49. 320 nm. 260 nm. 15) Oleuropein. 360 nm [119] Olive oil Various procedures compared. 725 nm (total phenols). flavonols Phenols GC–MS after derivatization. A 1000 (2003) 657–691 Sample . Extraction with various solvents direct from oil or from oil dissolved in various solvents Extraction of dried.

100–500 mg l 21 ) were present in much higher concentrations than polymethoxylated flavones with typical values of 1 mg l 21 [196. total flavanol contents varied from non-detectable in most vegetables to 1840 mg kg 21 in a broad bean sample [146]. The concentration of 1.114.82.K. an unidentified substance in pineapple juice was quantified [200] as p-coumaric acid whilst kaempferol and quercetin glycosides were calculated as the corresponding aglycones [135]. gallic acid [205] or quercetin [135]. This variability in the quantitative distribution of phenols coupled with the wide variation in relative sensitivity of detectors often limits the ability to measure more than a restricted range of phenols in a single analysis.50] for the preliminary separation / clean-up of sample extracts. 148. A disadvantage is the interference of reducing substances such as ascorbic acid. For instance. in part. The blue colour formed after 15–60 min is measured at 725–735 nm [206] and results are expressed in terms of molar equivalents of a commonly occurring phenol.199] of the relevant compounds.193. However. for example. For instance. In many instances. direct method for determination of total phenols using catechol as the reference standard.209] and exploited [49. isocratic elution [193] has provided adequate resolution due to selec- . Robards / J. somewhat surprisingly.1.205]. Post-harvest and processing-induced changes further modify the phenol contents. all spectrophotometric measurements lack specificity and give an over-estimation of ‘‘phenolic’’ content.135. flavanone glycosides (e.g. The typical system involves RPLC comprising a C 18 stationary phase [71. The limited volatility of many phenols has restricted the application of GC to their separation. The contents of phenolic acids and flavonoids in flowerheads were quantified by HPLC using quercetin and cynarin as internal standards [201]. HPLC currently represents the most popular and reliable technique for analysis of phenols [71. Traditional methods for the determination of the phenolic component relied on colorimetric measurement of total phenols using one of a number of reagents of varying selectivity.177.199. by synthesis [119.211–216]. Chromatogr. Nevertheless. 4. Chromatographic methods The need for profiling and identifying individual phenolic compounds has seen traditional methods replaced by high-performance chromatographic analyses. with suitable derivatization (e.2-diphenols is determined [204] with molybdate by measurement at 350 675 nm whilst Folin-Ciocalteu reagent is the classic reagent recommended for total phenols [135. it requires that the sample details are fully specified and characterized [198].193.203] is based on the reaction of dilute alkali with flavanones to form the corresponding yellow chalcones that are measured at 470 nm. However. quantification is carried out by reference to one or more appropriately selected reference compounds. The availability and use of standard reference materials and certified methods of analysis will greatly enhance the confidence in analytical data. the Davis Test [202. Specificity can be enhanced by derivative spectrometry or by preliminary separation.197].185. Columns are commonly 100 to 300 mm in length with 10 mm or increasingly 5 mm packings thus favouring the shorter columns.g.4. trimethylsilylation) they are amenable to GC and GC–MS [119.232] chemistry. They are generally semi-quantitative at best and.231] or other alkyl [205.114. All phenols absorb radiation in the ultraviolet and this provides the basis for an alternative measurement of total phenols [207]. For instance. The diversity of phenolic compounds means that selection of a reagent and / or absorbing wavelength will be a compromise although this is less of a problem where a single class of phenols predominates. A 1000 (2003) 657–691 juice. On a practical level. In some instances. TLC methods have been devised [135. high-performance TLC [210] has not been widely used for phenols. Merken and Beecher [234] have presented a comprehensive review on the analytical chemistry of food flavonoids in which they present detailed tabulations of columns and mobile phases used in HPLC. hesperidin. the relevant compounds isolated by preparative scale chromatography can serve as reference standards. measurement based on the second derivative of the absorbance at 278 nm [208] provided a rapid. there are significant quantitative differences between cultivars of a single species and between tissues of a single plant. In another study.217–233]. Moreover. Alternatively. The limited availability of suitable reference standards for quantification is a problem that has been overcome.

The complementary nature of fluorescence detection [35. Alternatively. Robards / J. acetonitrile) of the mobile phase [118] although gradient elution has usually been mandatory [71. followed by those of decreasing polarity.77. electrochemical array detection [59. A 1000 (2003) 657–691 tivity effects of one or more components (e. This creates problems in quantification as discussed by Tsimidou et al. . The linearity.135. for example.235] in recognition of the complexity of the phenolic profile of most samples.cinnamic acids. simple phenol. Chromatogr. The popularity of PDA in HPLC attests to the value of UV spectra and can be combined with the use of post-column shift reagents [250]. polarity is increased most by hydroxy groups at the 4-position. Furthermore. fraction collection and characterization offline have been used particularly in preparative scale separations. In cinnamic and phenolic acids.118. Their UV spectra are particularly informative providing considerable structural information that can distinguish the type of phenol (e.246–248] is a powerful tool for the selective as well as sensitive detection of phenolic compounds. Numerous mobile phases have been employed but binary systems comprising an aqueous component and a less polar organic solvent such as acetonitrile or methanol remain common. [242] who classified the various phenols into four groups and used a single calibration standard for the members of each group. xanthone. Ternary phases offer greater flexibility and will likely increase in popularity [236].245] was based on reaction with p-dimethylaminocinnamaldehyde to form coloured derivatives absorbing at 640 nm. Chemical reaction detection of catechins [90.237] although overlap of the individual members of different classes is inevitable because of the diversity of compounds.240] in the case of anthocyanins.172.1.114.g.g. formic or phosphoric acid) is usually added to both components to maintain constant acid concentration during gradient runs.205. visible radiation [239.243] and dual wavelength detection [49] has been applied.205] or.168. followed by those at the 3. an elution order can be developed as phenolic acids. phenolic acids) elute first. An optimization and validation strategy has been developed [112. All phenols possess a strong chromophore system. spectra of eluting peaks obtained at.205. polar compounds (e.1. Identification of the eluted phenols in GC and HPLC is usually based on correspondence of retention data with an appropriate standard. there are significant differences in absorption maxima and molar absorptivities [242] of even the major phenols identified in a single fruit. In contrast.231] although detection at other wavelengths [77.676 K.193] at 225 nm have been demonstrated but problems associated with high background absorption of typical mobile phases in RPLC have limited its use. that is. precision and limits of detection have been compared for HPLC of benzoic and cinnamic acids using UV. Acid (acetic. Detection Routine detection in HPLC is typically based on measurement of UV absorption [193. The method provided an alternative provisional identification of unknown phenols prior to their full characterization.and 2-positions.238. flavone) and the oxidation pattern [249].g.4.244] has been demonstrated and used in series with a UV detector for catechin detection [145]. Compound elution is typical of reversed-phase LC. No single wavelength is ideal for all classes of phenols since they display absorbance maxima at distinctly different wavelengths [241]. Hence. 176. The elution pattern for flavonoids containing equivalent substitution patterns [238] is flavanone glycoside followed by flavonol and flavone glycosides and then the free aglycones in the same order. Indeed.flavonoids [80. the apex and both inflexion points of the peak can be compared and used as an indicator of purity. 4. Methoxy and acrylic groups reduce polarity and hence increase retention times. The most commonly used wavelength for routine detection has been 280 nm which represents a suitable compromise [71.251] for the HPLC analysis of representative phenols from different food sources. The important feature of this strategy was the selection of the representative phenols and food extracts and the rapid analysis time. The advantages of low wavelength detection [177. Evaporative light scattering detection [15] offers the opportunity for non-selective ‘‘universal’’ detection. less commonly. electrochemical and mass spectrometric detection [247].

253] by the efficient and targetted isolation of bioactive compounds. thermolabile and high molecular mass molecules such as plant phenols . The moving belt method [278] represented an early attempt to provide data under both EI and CI conditions but the first real success was achieved with the thermospray interface that dominated applications in the 1980s and was still in use through part of the 1990s (Table 7). non-volatile and thermolabile phenols such as glycosides). elemental formula and substitution patterns.1. non-volatile compounds. API is a soft ionization source suitable for the analysis of polar. The latter has been used successfully to analyse a number of phenolic classes in various foods (Table 5). Chemical screening The on-line coupling of chromatography with spectrometry has been the single-most important advance in analysis of bioactive compounds facilitating optimization of an investigation [252. In coupled mode. the GC–MS data exhibited the same typical fragmentation patterns but with slight differ- 677 ences in intensities [254. tandem MS has enjoyed limited popularity in off-line mode. For instance. However. A 1000 (2003) 657–691 5.g. Despite the obvious successes of GC– MS. the mass spectrum will assist in structural elucidation although other techniques such as NMR are required for a definitive structural assignment. chemical derivatization usually involving silylation may overcome these limitations (Table 6) but can introduce further difficulties by increasing the molecular mass of the analyte possibly beyond the range of the mass analyzer. HPLC distinguished anthocyanin isomers whereas MALDI–MS was more rapid in the identification and quantification of anthocyanins with different masses.263. Mass spectrometry Mass spectrometry can be carried out either online in combination with chromatographic or electrophoretic techniques or off-line. Games and Martinez [278] predicted that LC-FAB-MS using the moving belt interface might provide improved detection performance for more polar. LC–MS interfacing has been achieved in a number of ways. In less favourable situations. the preparation of a proper matrix and sample preparation procedure are very important [261].1. [271–277]). flavanones and chalcones without derivatization by GC–MS in EI mode. Robards / J. In instances where the classical mass spectrometric gas phase ionization techniques such as EI and CI are unsuitable (e.212] in chamomile flowers [211]. Somewhat surprisingly. The analyte fragmentations in EI mass spectra may provide sufficient information to determine molecular mass. This is a major consideration for glycosidic species with numerous hydroxyl groups although permethylation or perdeuteromethylation offer suitable alternatives. Structural characterization 5. Compared with direct inlet mass spectra. It is interesting to note that in 1989. Derivatization also often produces mixtures of partially derivatized compounds [199] from a single analyte.K. MS is potentially a powerful tool for elucidating phenolic structures but the number of applications involving direct inlet introduction of analytes to the mass spectrometer was limited (Table 5) until the advent of MALDI– MS. non-volatile. it is the hyphenation of liquid chromatography with MS that has revolutionized the analysis of non-volatile species as evidenced by the number of reviews (e. the analysis and identification of the phenolic metabolites of the ascomycete Eutypa lata was performed by GC–MS of their trimethylsilyl ether derivatives [193] whilst individual compounds were quantified by analytical HPLC and separated by preparative HPLC. Both techniques generally provided comparable quantitative profiles for anthocyanin contents. the mass spectrometer may function simply as a highly selective detector but it is in qualitative analysis that it excels providing unsurpassed opportunities for compound ‘‘identification’’. Chromatogr. 5. [262] analysed 49 flavones. flavonols. it was with the advent of API techniques that LC–MS came of age.1. with polar.g. Refs.270]. propolis [212] and rapeseed leaves [213]. However. MALDI–MS and HPLC have been compared [131] for the analysis of blueberries for anthocyanins. Schmidt et al. On-line applications of MS are extensive and growing at an increasing rate with GC–MS now well established (Table 6) as a routine technique carried out with either EI or CI sources for the introduction of volatile compounds such as phenolic acids [213] and flavonoids [211.

MS MS MS MS MS MS. hesperitin (and glycosides)—no derivatization Caffeoyl esters Anthocyanins Anthocyanins. fruit juice Soy products Naringenin. flavonols esculin Flavonol glycosides Anthocyanins Anthocyanins Isoflavones [258] MS–MS FT-ion cyclotron resonance-MS FT-ion cyclotron resonance-MS Blood/urine following ingestion of citrus products Echinacea roots Wines Wine (and negative) ion ion ion ion [259] [111] [116] [126] [131] [260] [261] K. Chromatogr. A 1000 (2003) 657–691 Table 5 Conditions used in the mass spectrometric analysis of plant phenols .678 System Ionization Sample Analyte Ref. catechins—no derivatization Polymethoxylated flavones—no derivatization Phenolic acids and glucosides—isolation by HPLC [129] [191] [254] [255] [256] [257] MS–MS ESI (negative ion) EI EI. Robards / J. 15)—no derivatization ‘‘Tyrosol ester’’—no derivatization Flavonoids—no derivatization Gallic acid. CI (positive ion) EI EI ESI and FAB (positive and negative ion) EI. CI (positive and negative ion) Fast atom bombardment (negative ion) ESI (positive and negative ion) ESI (positive and negative ion) MALDI–TOF-MS MALDI–TOF-MS MALDI–TOF-MS MALDI–TOF-MS Positive Positive Positive Positive Yellow onion. green tea Blueberries Red wine. MS–MS Olive leaf Olive oil Not applied Evening primrose seeds Tangerine oils Soybean root nodules Phenols (ca. hydroxycinnamic acids.

1. EI EI EI EI EI EI. the fragmentation behaviour varied between the two papers illustrating the need for additional fundamental studies on fragmentation mechanisms.137].36. CI (positive ion) EI EI EI EI EI EI.1. Moreover.293. HPLC with tandem mass spectrometry (LC–MS–MS) and negative and positive ESI was used [114] for analysis of phenols in rose hip extract. LC–MS–MS [114] and LC–NMR to rapid detection of biologically active natural products has been reviewed [300]. Although API has revolutionized the application of LC–MS. appropriate choice of interface parameters distinguished structural isomers of flavonoid glycosides differing in the nature of the disaccharide linkage [35. A related gas phase system is the heated nebulizer APCI interface and each of these techniques has been applied to a range of phenols and sample types (Table 7).1. or deprotonated molecular ions. LC–MS can provide data sufficient for full structure analysis [201] but more generally it is used to determine molecular mass and to establish the distribution of substituents on the phenolic ring(s).287. 15) Acetylenic phenols Phenolic acids Phenolic and secoiridoid aglycones Gallic acid. [M1H] 1 or sodium adduct ions in positive ion mode. CI (negative ion) EI EI Persimmon fruit Cherry laurel fruits Olive oil Eutypa lata Mushrooms Olive oil Soybean root nodules Flowers Not applied Citrus and grape juices Wines Passionfruit juice or peel Olives Corn. For instance. 5. [M-H] 2 in negative ion mode with few fragment ions and thus have a low structure information content. some problems remain. However. flavonoids Glycosides of methyl salicylate and eugenol Phenolic acids. wheat. Thus.297–299]. API-based interfacing systems which are liquid-based are ESI and ISI. The use of multiple approaches in phytochemical research is illustrated by the identification of an unknown peak in the chromatogram of apple fruit extracts [294].K.136.235. flavonoids Dehydrodimers of ferulic acid Phenolic acids [65] [84] [119] [193] [195] [199] [257] [262] [263] [264] [265] [266] [267] [268] [269] [35. resveratrol. it is difficult to optimize conditions for a typical extract containing a broad range of analytes although many instruments now have provision for programmed operation of spectral conditions. generation of mass spectral libraries is difficult. Similarly. On rare occasions. Robards / J. Applications of mass spectrometry API mass spectra typically comprise protonated molecular ions. molecular masses of the separated phenols were obtained through prominent [M-H] 2 ions for most of the compounds and M 1 ions for the anthocyanins whilst CID of the [M-H] 2 (or M 1 ) precursor ions yielded product ions that determined the molecular mass of the aglycones.226. HPLC with PDA indicated that the unknown peak was an isorhamnetin glycoside having the same retention time as isorhamnetin 3-O-gluco- . The application of LC–MS. Negative ESI was more sensitive for the majority of phenols with the exception of anthocyanins that were more sensitive in positive ion mode. structural information about the molecules can be obtained from CID processes [293]. gallic acid methyl ester Flavonoid aglycones Polymethoxylated flavones Flavanones following hydrolysis of glycosides Phenolic acids. the major limitation being the strong dependency of the response on the nature of the analyte plus the mobile phase. Thus. A 1000 (2003) 657–691 679 Table 6 Conditions used in the GC–MS analysis of plant phenols Ionization Sample Derivatization Analyte Ref. the unconjugated phenols were identified by in-source fragmentation followed by CID of the resulting deprotonated aglycone [A-H]. However. rice Distilled alcoholic beverages Oxime / trimethylsilylation Trimethylsilylation Trimethylsilylation Trimethylsilylation ? Trimethylsilylation Trimethylsilylation Nil Nil Trimethylsilylation Trimethylsilylation Trifluoroacetylation Trimethylsilylation Trimethylsilylation Silylation Phenolic acids Phenolic acids Phenols (ca. Chromatogr. UV spectra obtained from PDA detection assisted in confirming the identities of several compounds.

hop Needles of Norway spruce Medicinal plants Camellia sinensis Tea [74] [279] [280] [281] [282] [283] [284] [285] LC–MS LC–MS Thermospray Ionspray Negative ion mode Lemon peel Orange juice LC–MS–MS Ionspray. flavanones. chlorogenic acids. wheat Soy foods [286] [137] [226] [287] [27] [102] [106] [107] [114] [117] [136] [143] [288] [289] [290] [291] [292] [79] [125] [293] [294] [295] [296] K. ESI APCI APCI APCI APCI APCI Negative (and positive) ion Positive ion Negative ion Isoflavones Resveratrol Flavonol glycosides. beer. rutin. flavone glyosides Flavanones and flavanone glycosides. anthocyanins Procyanidins p-Coumaric acid. tyrosol. flavones. flavanones. chlorogenic acid Flavonol glycosides Catechins Flavanol (glycosides). oleuropein derivatives Tyrosol. hydroxytyrosol derivative Anthocyanins Flavonols Hydroxybenzoic and hydroxycinnamic acids. flavonoids Flavanols.680 Table 7 Applications of LC–MS to the determination of plant phenols Ionization Mode Sample Analyte Ref. flavonol glycosides Flavonols. xanthones Proanthiocyanidins. flavonols and glycosides Isoflavone Flavonoids and glycosides Procyanidins Phenols Hydroxytyrosol and hydroxytyrosol glucoside Phenolic acids Flavonoid glycosides Extensive Phenolic and hydroxycinnamic acids. ligstroside. secoiridoids. A 1000 (2003) 657–691 System . flavonols Oleuropein. LC–MS–MS LC–MS LC–MS LC–MS LC–MS LC–MS LC–MS–MS LC–MS Heated nebulizer interface EI Thermospray Thermospray Thermospray Thermospray Thermospray Plasmaspray Positive ion Positive ion (negative ion for one phenol) Positive ion Positive ion Soybean and soybean products Wines Plant extracts Malt. phenolic acids Isorhamnetin glycosides Steryl ferulate Isoflavones Negative and positive ion mode Negative (and positive) ion Negative ion Negative ion Positive and negative Negative ion Positive and negative Negative ion Negative ion Negative ion Positive and negative Positive and negative ion ion ion ion Soy Sour orange Chocolate Extracts of medicinal plants Olive fruits Cornmeal fibre Tea Apple and pear Orange Table olives. flavones. flavonol glycosides Flavonol glucosides. flavonoids Phenolic acids. Robards / J. FAB Positive ion Olive leaf LC–MS LC–MS LC–MS–MS Ionspray ESI ESI Positive ion ? Positive and negative ion Grape Berries Dried plums LC–MS LC–MS LC–MS ESI ESI ESI Negative ion Cider apple tissues Red beetroot peel Rose hip LC–MS LC–MS LC–MS(-MS) ? LC–MS LC–MS LC–MS LC–MS LC–MS LC–MS–MS LC–MS LC–MS–MS LC–MS LC–MS ESI ESI ESI ESI ESI ESI ESI ESI APCI. chlorogenic acids. ferulic acid. Chromatogr. olive oil Olive mill wastewater Apple Rye.

Wolfender. UV and mass spectra of three related xanthones are shown. 681 . UV and selected ion chromatograms of an extract of Gentianaceae species.K. Phytochemistry 40 (1995) 1265.-L. and O. Copyright 1995 [252]. J. Total ion current. Rodriguez. Hostettmann. A 1000 (2003) 657–691 Fig. Odontuya. Chromatogr. Purev. S. Halenia corniculata using a C 18 reversed-phase and acetonitrile–water gradient. 5. Robards / J. Reprinted from K. G.

From the chromatogram of the extract (Fig.u. The base peak in the negative ion mode was the pseudomolecular ion . Although 2D NMR spectrometry can be used for structural analysis without a reference compound. In other applications. Similar UV spectra supported this assignment. HPLC of a dichloromethane extract of a Gentianaceae from Mongolia using PDA showed in excess of 19 peaks having UV spectra characteristic of xanthones [252]. Limited sensitivity and the need to isolate relatively large quantities of sample are currently the greatest limitations of NMR spectrometry.682 K. In the same study. LC–MS(–MS) was used to tentatively assign two peaks as verbascoside isomers whilst NMR spectrometry was used to distinguish between the isomers (vide infra). Peaks 1 and 2 were identified as glycosides detected as sodium adduct ions at m /z 709 and 679.m. a stationary phase with hydrophilic endcapping specifically developed for separation of very polar analytes contributed to the success of the analysis. All peaks recorded in the UV chromatogram at 254 nm were detected in the total ion current trace whereas a selected ion trace at m /z 363 showed three peaks with a common hexasubstituted xanthone aglycone (labelled 1. Mass spectral data also supported the assignment of a number of peaks in the HPLC chromatograms of phenolic extracts as phthalate esters [35. The following example illustrates the use of stopped-flow LC–NMR in the characterization of the xanthone and flavone constituents from Gentiana ottonis [302]. flavones (6 and 8) and xanthones (7 and 9–11) were identified. The remaining compounds were characterized using similar approaches. the choice of stationary phase is also significant. Such artefacts arise due to the use of plastic equipment for sample storage and extraction. HPLC–APCI-MS in the negative ion mode supported the preliminary identification by loss of 162 a. Apart from the requirement for volatile eluents in API techniques. and unambiguous assignment of the peak by comparison with standard compounds as isorhamnetin-3O-glucoside. 2 and 3 in Fig. Other artefacts have been observed in LC– ESI-MS such as the non-covalent dimer (2M1H)1 of oleuropein [301] with further ions corresponding to the loss of one and two glucose moieties. peaks with UV spectra characteristic of secoiridoids (4). respectively. In this work and elsewhere [292].1. Xanthones with monoamine oxidase inhibitory activity have potential as antidepressive drugs and are useful as chemotaxonomic markers. NMR spectrometry is a powerful complementary technique for structural assignment. the enhanced selectivity of LC–MS enabled detection of co-eluting neoeriocitrin and naringin in grapefruit extracts [35] whilst ligstroside was identified in olive extracts [36] by a consideration of elution order in combination with UV and mass spectral data. together with the molecular mass of 374. Robards / J. Final characterization of compound 8 required NMR spectrometry and MS– MS whilst the secoiridoid glycoside (4) presented NMR signals characteristic for glucose in the 3–4. the technique requires relatively large amounts of the compound. enabled unambiguous identification of 4 as swertiamarin. 6). These ions were generated in the electrospray source. 5. Extracted ion chromatograms at m /z 623 of an olive extract [301] showed two chromatographic peaks with identical mass spectra.36].5 ppm region and typical resonances of the monoterpene moiety in the 5–8 ppm range. These data. Final structural assignment was based on known chemotaxonomy of the Gentianaceae family. CID of the aglycone (m /z 315) in the MS 3 product ion analysis allowed the differentiation of rhamnetin and isorhamnetin. 6 and 8 as C-glycosides. NMR spectra of phenols are frequently complex and identification of the isolated compounds is complicated by the absence of suitable reference standards which requires timeconsuming syntheses of the relevant materials. Nuclear magnetic resonance spectrometry In instances where mass spectral data are insufficient to establish a definitive structure. consistent with the structure of verbascoside (Table 8). A 1000 (2003) 657–691 side. either from the loss of glucose from the dimer or the aggregation of oleuropein molecular ions and fragments within the electrospray source. Molecular masses were assigned to these compounds from LC–MS and fragmentation behaviour indicated compounds 5. 5).2. Chromatogr. (corresponding to a hexose unit) from the pseudomolecular ion (m /z 477). The use of tandem MS as in this instance offers high selectivity but this paper also illustrates the continued need to consider the chromatographic system.

3-dihydro-5. Rodriguez. 6. W. the hydroxytyrosol and the loss of both the rhamnose and the hydroxytyrosol. by analogy with the synthetic phenolic antioxidants and consistent with their free radical chemistry. in the case of some phenolics. Phytochem. However. Wolfender. quinone and hydroquinone derivatives are expected to feature prominently amongst the products.1 H correlations such as COSY or 1 H. 8 (1997) 97. Being . For full structural assignment the compounds were isolated by preparative-HPLC and examined by 1 H 683 NMR whence one of the chromatographic peaks was confirmed as verbascoside by comparison of retention. 6. Reprinted from J.K. Robards / J. As the negative ion mass spectrum showed no structural information. A 1000 (2003) 657–691 Fig. Separation was performed on a C 18 column using an acetonitrile–deuterium oxide gradient. [303]. Thus. The similarity of the tandem mass spectra for the two compounds is strong evidence that the two peaks are due to isomers of verbascoside.13 C correlation experiments such as HMBC or HSQC as applied in the structural assignment of a phenol isolated from green olive pulp [34]. Degradation products of antioxidants Plant phenols are highly reactive species but little attention has been given to determination of their reaction products. mass spectral and 1 H NMR data (Table 8) with that of a pure verbascoside standard. Hiller. The latter required 2D-NMR correlation experiments and also demonstrated that choice of mobile phase was important not only for the chromatography but also for resolution of the LC–NMR signals. These correspond to the loss of the rhamnose sugar. with few other fragment ions. S. Anal. In other cases. Chromatogr. the other was assigned as the ‘‘acteoside isomer’’ identified by Miyase et al. The UV spectrum of this compound was characteristic for a flavanone and from mass spectral data it was most probably a flavanone with one O-prenyl unit and three hydroxyl substituents. Based on previous NMR analyses of verbascoside and its isomers. HPLC chromatogram of a methanol extract of Gentiana ottonis showing the stop-flow 1 NMR spectra of compounds 4. Copyright 1997 [302].7-dihydroxy2-[3-hydroxy . 9 and 11. respectively. information on the 13 C NMR signals is necessary plus 2D correlation experiments involving 1 H. MS and 1 H NMR will provide adequate information for structural elucidation. the combination of UV. and the tandem mass spectra of both compounds were very similar with the only ions of significant intensity at m /z 461 and 161. This hypothesis was confirmed by stopflow LC. whereas the positive ion spectrum showed some structural information with major peaks at m /z 479. dimers and higher oligomers plus. TLC screen of a dichloromethane extract of Monotes engleri demonstrated antifungal activity that was linked to a compound in the HPLC chromatogram of the extract [253].-L. K.1 H NMR but the data were insufficient to identify the compound as 2.4(3-methyl-2-butenyl)oxyphenyl]-4H 1-benzopyran-4-one. 8. 471 and 325. In many instances. Alternatively. it was only in methanol / deuterium oxide eluent that complete structural elucidation of the prenylated flavanone was possible whilst with an acetonitrile / deuterium oxide system the substitution pattern of the B-ring of the flavanone was not fully determined. Hostettmann. tandem mass spectrometric analysis was carried out on the m /z 623 ion of both peaks.

Chromatogr.684 K. Robards / J. A 1000 (2003) 657–691 .

spectrophotometry and HPLC were used to follow the oxidation of several phenols catalysed by apple PPO [305] between pH 4 and 5. The second pathway involved polymerization reactions that were favoured by higher pH values. The usual procedure now encompasses a high-performance separation technique in combination with diode . The design of chemical libraries [309. it is small compared with the total number of known natural products and presumably many phenols remain undiscovered. This will include other phenols.310] is an important component of the search for bioactive materials and various commercially available databases exist but there is regrettably no universal data depository. Two pathways were proposed for the degradation of 4methylcatechol o-quinones. corresponded to an hydroxylation followed by a coupled oxidation of another molecule or oquinone and leading to regeneration of 4methylcatechol. They will react as electrophiles with nucleophilic substances including amino derivatives and water. A 1000 (2003) 657–691 electron-richer than the A-ring of flavonoids. the o-quinones will oxidize any other substances with lower reduction potentials. 7. 685 which increases the nucleophilic character of (1)catechin whereas low pH could favour radical mechanisms by increasing the reactivity of semi-quinone radicals. In the absence of other substrates. The yellow products were identified as dimers and possible mechanisms for their formation were proposed as a Michael-type addition or through a semiquinone radical intermediate. The adoption of particular analytical strategies is related to the purpose of the analysis and the nature of both the sample and analyte. organic peroxyl radicals selectively attack the B-ring [304]. For example. The reactivities of the o-quinone products varied greatly from one phenol to another. less polar than the colourless ones. 8. The products formed in such reactions are pH-dependent [308]. An investigation of phenolic acids and flavonoids in flowerheads of 84 samples of 76 taxa belonging to 66 species resulted in the identification of only three phenolic acids and six flavonoids. The reaction products become quite complex with as few as two phenols (o-quinones) in admixture [307]. Polarography. Several reasons for this interest can be identified but these are as diverse as the phenols themselves. The same pathways were observed with chlorogenic acid although the polymerization reactions seemed to be dominant. The o-quinones of (1)-catechin and (2)-epicatechin are much less stable than that of chlorogenic acid [306] and were not examined by Richard-Forget et al. favoured by acid pH. a consecutive two-electron oxidation reaction produces the flavonoid phenoxyl radical that subsequently scavenges another peroxyl radical to form a quinone. The first. When the number of potential bioactivities is factored in then the acquisition and storage of chemical and related biological data are crucial aspects of an analysis. condensation and polymerization will occur via reaction with the corresponding hydroquinone. Informatics and data handling The range of phenols that have been isolated from fruits and vegetables is vast and yet. Rediscovery of bioactive compounds from previously unstudied sources is a costly and time-consuming problem that is not completely eliminated by database use. one group found that 72% of compounds that reached the structure determination stage were known compounds [311]. predominated at higher pH values. As oxidants. Conclusions There has been a renaissance in the study of bioactive compounds and the interest in plant phenols is intense. [305]. Michael (1→4) addition could be favoured by high pH. the o-quinones will enter along the different pathways according to their oxidative and electrophilic properties. The classification of sample types as plants and foods is not particularly productive in terms of classifying the analytical strategy nor is the distinction between processed and unprocessed products as similar procedures are used for both groups. In summary. In this process the quinones are themselves reduced to the original phenol. Robards / J. Chromatogr. Mostly colourless products were formed during the PPOcatalysed oxidation of (1)-catechin in aqueous buffers at pH below 4 whereas yellow compounds. ascorbic acid and sulfur dioxide.K. In the case of dihydroxyflavonoids.

Food Compos. Heinonen. M. p. M. Nahrstedt. J. Bidlack.I. Hulme (Ed. 1970. Queener. R.A. S.G. Nomenclature API APCI atmospheric pressure ionization atmospheric pressure chemical ionization BHA butylated hydroxyanisole CI chemical ionization CID collisionally-induced dissociation EI electron impact ionization ESI electrospray ionization FAB fast atom bombardment FT(IR) Fourier transform (infrared spectrometry) GC gas chromatography GC–MS gas chromatography–mass spectrometry HPLC high-performance liquid chromatography ISI ionspray ionization LC liquid chromatography LC–MS coupled liquid chromatography–mass spectrometry MALDI–MS matrix assisted laser desorption ionization-mass spectrometry MS mass spectrometry MS–MS tandem mass spectrometry NMR nuclear magnetic resonance spectrometry PDA photodiode array detection PPO polyphenoloxidase RPLC reversed-phase liquid chromatography SFE supercritical fluid extraction SPE solid-phase extraction TBHQ tert. Ciz. London. 44 (2001) 2555. R.P. M. 2000. Sci. Tucker. Technomic. 739–795. Elsevier. M.K.P. Xu. Bidlack.B. P.R. Sci. Sharma. A 935 (2001) 321.686 K. J. A. Sci. Chromatogr. Vol. S. [25] T. 80 [6] F. Phytochemicals as Bioactive Agents. 56 (2001) 973. A. 9. L. Food Chem. W. PA.A. Further instrument sophistication in coupling several systems such as multidimensional chromatography with NMR and MS in series is already occurring. [22] N. [16] Z. Lojek.). Lancaster. in: Atta-ur[8] F. Food Res. Martin-Belloso. Soliva-Fortuny. H. P. (Warsaw) 47 (2002) 21.F. Anal. Daniewski.G.K. [5] K.). p. R.H. S. Phytochemicals as Bioactive Agents. J. P. Wang. ´ ´ M. Analyst 122 (1997) 11R.P. Studies in Natural Product Chemistry. Kingston. ¨ ¨ [26] S. Pandey. Omaye. Agric. Robards. M. O.).R. Clifford. H.O. K. 337. M. Mikkonen. ´ ´ F. Food Agric. in: C. J. 24 (2001) 2033. Laughton. S. S. in: W. Paul Bolwell.W.M. Antolovich. Jurgenliemk. Ren. [7] A. I. 241. L. Maciejewicz. Taylor and Francis. Planta Med. Sahu. M. [18] G. Lancaster. Parr. Heinonen. p.-K. Clifford.W.X. S. Analyst 123 (1998) 31R. S. De Meyer.M. Chromatogr. Ahotupa. Haraguchi. N. Tetrahedron 50 (1994) 9439. Z. Torronen. Van Buren. Topham (Eds. Robards. Roy. Gil. Chem.J. M.S. A. Karjalainen. Meskin. 32 (1999) 345. Torronen. Ryan. Pieters. 2000. [4] M.S. A. K. 1. The Biochemistry of Fruits and their Products. Lien. Vanden Berghe. 77 (1998) 543. Food Agric.N. Li. Sci.H.K. Mykkanen. 15 (2002) 419. R. D.J. Topham (Eds. M. Lin. S.R. Liq. Food Agric. Stevens. in: A. T. Caspi. Med. Johnson. Hakkinen. I.S.R. Ryan. Naturforsch. A. Food Agric. London. Technomic. 49 (2001) 3274. D. A. Tringali (Ed. Food Chem. J. Libman. Hukkanen. [12] A. 66 (1999) 401.O. Gorinstein. J. Zhou. 2000. PA. Chem. pp. [14] B.D. Tomas-Barberan. A.-butylhydroquinone TOF time of flight References [1] W.C. Karenlampi. M. M. Food Agric. Liu. J. [15] G.W. Prenzler. Glover.A. 80 (2001) 985. [20] D. P. 82 (2002) 1166.T.K. D. An increased emphasis on microcapillary columns with nanotechnology ESI systems driven partly by environmental issues seems inevitable. S. A.A.). S. J.R.T. Bioactive Compounds from Natural Sources.P. 21. H.T. Pennington. Mishra. Chan. J. Kokko. Hakkinen. Karenlampi. Pihlaja. J. [2] J.C. Sci. Lavee. ¨ ¨ ¨ A. Y. Scientia Horticulturae 92 (2002) 147. ¨ ¨ ¨ Mykkanen. Dzido. Prenzler. Robards. in: W. p. Li. [23] S. J.F.C. Bidlack. Antolovich. Haemers. 68 (2002) 88. Vlietinck.N.I. Karenlampi. Tomas-Barberan.L. Ferreres. Int. Academic Press. Trakhtenberg. G. Technol. Amsterdam. Anal.R. [10] K. W. 34 (1991) 736. Rahman (Ed. Maatta. Mattern. [13] S. C. Meskin. (2001) 1024. L.M. K. [11] D. Park. ¨ ¨ Ruuskanen. Robards / J. 2001. Torronen. Chromatogr. G. Robards. Saleem. O. S. J. K.R.A. Med. [9] D.M. Bal. [17] W. Swatsitang.T.I. The prediction of the future is dangerous but promising approaches involve the application of HPLC with ESI time of flight mass spectrometers and ESI FT ion cyclotron resonance mass spectrometers. Chem. . [21] H.N. Wen. Mahato. 80 (2001) 1033. A 1000 (2003) 657–691 array detection or mass spectrometry. H. Tetrahedron 57 (2001) 9549. [19] E. Omaye. K.A. S. Relat. ¨ ¨ ¨ [24] S.O. 269. [3] J. D.). Chan.

L. Food Compos. O. B. Agric.D. Middleditch. Commun. Agric.C.-W. Frankel. Lanza. Chromatogr. G.P. G. J. [81] P. Martınez-Valverde. [62] J. S. Herlt. Crystal (Eds. Krewer. Flavonoids and Other Polyphenols. Syst. L. J. Caruso. Chim.C. Garrido. Antolovich. Antolovich. in: L. J. Galli. Am. Tellez. J. Servili. Engelhardt.G.H. in: S. K. Annu. J. Diosady. A 1000 (2003) 657–691 ¨ ¨ [27] S. [28] S. Provan. M. [41] M. Food Chem. Ganguli. Rev.T. Carabias-Martınez.V. [74] L.J. Rodrıguez-Gonzalo. [37] B. D. El Rassi. Asmundo. Murakami. Agric.R. Topham (Eds. Food Res. [29] M. M. Crit. Zuo. Agric. J. Yamaguchi. Man. J. Grayer.R. E. Academic Press.A. Analytical Artifacts: gc. J.E.P. Analyst 126 (2001) 1182. [78] P. Chem.N. Sala. Christensen. Food Chem. 76 (1999) 331.K. Food Chem. J. Cerna. H. Chromatogr. Heinonen. Friedman. Sci. Wrolstad. Agric. Food Chem. 10. Miniati. Omaye. 42 (1994) 1905. Hakkinen. E.N. Chen. Robards. T. J. Technol. Ecol.P. R. [70] P. M. Reunanen. J.E. Elviri. J.H. [47] I. A. L. 26 (1998) 729. [57] A. M. [83] L. Sanchez.V. [59] M. G.M. Food Agric. Andersson. J. Andreasen. Pharm. 10 (1997) 350. K. Bloor. Beecher. Venema.S. Agric. Clifford. Montedoro. Rimando. Vol. E. Ayaz. 11. 278 (2000) 797. K. J. E. S. Agric. Food Agric. H. D.P. Oliveira. Elsevier. G. 48 (2000) 2101. Food Chem. Food Chem. Sci.A. A. pp. R. G. M. [66] I. J.M. A. Agric. Cambridge. Jurgens. Agric. R. J. Med.O. 75 (1997) 87. FitzGerald. 56 (1996) 139. [64] S.H.M. M. K. Hollman. M. [67] H. Kadio)lu. PA. [61] P.S. ´ ´ M. J.S. L. A. X. Paul. Moreno´ ´ ´ ´ Cordero. Mulholland. Boyd. ´ [77] M. Li.B.A. 61 (1972) 1840. L. Kammiovirta.B. 40 (1992) 1571. 43 (1995) 343. [44] M. 50 (2002) 2432. Chromatogr.J. Shahrzad. Thomas. J. [42] P. P. J.N. Meskin. Vol.W. Biochem.G. Y. E. Hollman. [51] L. Food Chem. Food Chem. [55] U. J. 1997. K.A. J. A. P. [33] N. Custer. [73] A. Acta 417 (2000) 231. A 897 (2000) 177. Narala. J. J. Anal.J. Sellappan. J. Fernandez Laespada. K.A. Analyst 127 (2002) 183. M. C. E. Chromatogr. M. Hutabarat. [52] V. D. J. de Souza. A. Anal. J. A Source of New Industrial Products. Ferreira.H.L. Metab. Fallico. Katan. Agric.N. Garcıa. Phytochemical Diversity. Technomic. [53] C. J. E. Food Chem. Merken. [82] M.M.R.M. Rapisarda. Weston. Miniati. Hollman. 14 (2001) 43. Kumpulainen. [49] G. Chromatogr.H. Agric. G. Maccarone. 46 (1998) 4107. Canel. 80 [63] F. 687 [56] A. Liq. Food Chem. S. [79] P. Food Chem. J.C. Bidlack. Periago. B. M. Food Chem.S. A.C. Agric. Bitsch. [58] M. Brenes. P.A. [48] D. Chromatogr. R. F. hplc. S. pp. Food Compos. [39] D. A 913 (2001) 387. ¨ ¨ ¨ Mykkanen. and pc. Kamal-Eldin. R. Mazza. Astola. Rejano. 47 (1999) 2274. Matoba. Karenlampi.A. p. 26 (2000) 1609. Ryan. Viappiani. Nuutila. Camel. [69] H. M.S.M. Am. Ayaz. Lebensm. Seabra. P. Packer (Ed. Hertog. Royal Society of Chemistry. Anal. S. Mattila. 44. Hansen. Lundgren. M. L. Chem. [43] Y. R. Biochem.T. J.H.K. Baldioli. Wrigley. 43 (1995) 2702. Rommel. M. McDonald. [31] M. Baldioli.I. 12 (2001) 243. Dekok.B. de Groot.H. Res.W. Trends Endocrinol. A. Finger. Food Chem. London. van Beek. Ryan. Agric. Agric.N. R. L. Food Chem. Lavee. Rehwald. 57 (1996) 43. Rokach.R.). [68] M. Andersson. Y. H. K. Food Chem. T. [60] A. 35 (1995) 131.O. Marinova. Smillie. Chromatogr. Leitao.M. Robards. B. [84] F. Careri. M.J. Kadioglu. Rousi. Oil Chem. Peerlkamp. S.N. [72] S. M.-Unters. Duke.T. Food Chem. Lancaster. Prenzler. A. [75] M. Anal. Tucker. in: W. 197 (1993) 239. 41–52. J. 1989. Robards. Sticher. Eskilsson.P.A. [65] F. Relat. Linssen. Deng. 335. Free Radic. Visioli. Chromatogr. Food Agric. Wedge. I. Huang. Montedoro. 50 (2002) 1368. Lavee. Agric.B. J.L. P. [76] B. Robards. 20 (1997) 2023. Meyer. Methods in Enzymology. L. J. Agric. Chesson. [34] D. K. A 902 (2000) 251. A.M. Oil Chem.C. Var. Prenzler. Food Chem. C.J. Hayes. C. Garcıa-Pinto. 13 (2002) 8. J. Bjorklund. Hertog. ´ A.K. 44 (1996) 2654.D. Perez-Pavoon. A 902 (2000) 227. E. I.F. Galli. 50 (2002) 6716.L. M. Sci. Jardine. tlc.G. Julkunen-Tiito. 41 (1993) 1237. S.E. M. G. E. Lawson. R. 2000. Meier.K. 1. Schrader. P. [45] E. Atta-ur-Rahman. 72 (1995) 1131. Reunanen. C. M. 76 (2002) 519. J. Phytochemicals as Bioactive Agents. ms. 30 (1997) 571. 67 (2002) 539. 48 (2000) 2837. P. Musci. 33 (2000) S41. Frankel. T. J. Food Chem. Tura.). Chromatogr. A 677 (1994) 677.S. Food Chem.E.M. Agric.N. Tan. Akoh. Robards. A. D. Li. A. Takamura.B. Oksman-Caldentey. Libr. Biophys. Aman. 40 (1992) 1577. Satue. [32] N. Food Sci. A 881 (2000) 449. Pratico. J. Andrade. 17 (1982) 301. C. Soc. Xu. M. 2000. Franke.I. [80] S. J. J. [46] M. M. Rep. M. C. [40] F. J. Katan. Amsterdam. Int. [36] D. Agric. Farnsworth. [35] K. J. T. Duke.J. Food Chem. Chromatogr. A. N. [38] J. S. (2000) 1073. M. J. [30] R. Heinonen. Res.). ´ [71] I. Servili. M. J. E. Gao.S. Food Chem.M. Mangia. ´ ´ [54] R. Evstatieva. J. Antolovich. Soc. D. I. Greenfield. J. Z. Reden. Chromatogr. Laitinen. 76 (2002) 207. A. L. N. Ecol. T. Robards.J. Torronen. [50] G.G. Robards / J.Forsch. Tomas-Barberan. J. Food Chem. C. Hertog. A 975 (2002) 71. Z. 82 (2002) 323.C. Yanishlieva. 40 (1992) 2379. K. Sci. Phytochem.L. . Dayan. 48 (2000) 5834. A 832 (1999) 87.M.A. Swatsitang.N. Johnsson. S. 40 (1992) 1591. R. A 741 (1996) 223. Kuhr. Chromatogr. Salonen. Food Sci. Meyer. Clarkson. 45 (1997) 2539. Koleva. Patsalides. Choudhary. J.

K. M. Vitic. 43 (1995) 1132. [117] L. C. L. A. [111] D. Food Chem. J. Le Floch.B. 47 (1999) 4847. Dourtoglou. 40 Gomez-Cordoves. Relat. J. Akiri. [87] M. Ferreres. 49 (1998) 341. R. Food Chem. J. ´ J.P. Ferreres. Pascual-Martinez. J. S. Hrazdina. Biesenbruch. [130] A. ´ ´ J.M. F. Lin. A. J. Dong. Valentao. Liq.J. [106] P. Fruchier. Food Chem. Agric. Bianco. Mass Spectrom. F. Perez-Ilzarbe. Garcıa-Lidon. Hernandez. Del Valle. N. J. A. Esti.A. Detavernier. A 881 (2000) 439. M. O. 49 (2001) 3606. Chem. K. J. A. C. E. J. Food Chem. Anal. Food Chem. Peterson. Uccella. Lian. F. Food Chem. T. 50 (2002) 3579. ´ Montelongo.B. [123] F. Acta 460 (2002) 61.X.T. 47 (1999) 5044. J. Marnet.C. A. Chromatogr.I.W. Food Agric. A. 47 (1999) 4161. Agric. S. Smith. J. 30 (1997) 585. [95] G. Food Chem. Polidori. P. D. Okogeri. E. Valcarcel. M. Food Compos.M. [113] M. S. I. de Pascual-Teresa. S. Oltz.E. Ferreres. M. J. A. Agric.M. C.L. 38 (1990) 1565. Genovese. [138] P. 42 (1994) 2191. [108] X. M. N. F. 46 (1998) 4209.Y. ´ [135] E. ´ J. J. 21 (1998) 2813. I. Food Agric.A.A. Chromatogr. Bruening. [107] T. Agric. P.M. Nakanishi. 48 (2000) 1657. Lakenbrink. J. 50 (2002) 4861. P. Agric. Wang.H. A.C. R. S. M. Y. Anal. Treutter. ´ ´ I. Seabra. A.K. Food Chem. Food Chem. A 912 Borges. Perez. Phytochem. Food Chem. Valcarcel. Lett. P.G. N. Bernart. M. Cartoni. Chim. Makris. Agric. W.B. ˜ P. Wiss. C. Bois-Dounas. Wrolstad. I. Lee.C. F. Food Chem. Valentao. S. Lopez. 34 (2001) 1033. E. T. Loponen. J. J.J. Torrento. ´ Gomez-Cordoves. Seabra. M. H. J.L. Rios. Food Chem. [119] F. [134] C.J. 33 (2001) 97. Mannerstedt-Fogelfors. Hmamouchi. [105] M. 56 (2001) 343. Ooghe. Drilleau. J. Cadahia. J. Chromatogr. 16 (2002) 655.M. ´ ´ [94] A. Rapid Commun. J. Hogg. J. B. Oleszek. Tacchini. Food Chem. [91] S. Rivas-Gonzalo. Sci. Gil-Izquierdo. Sci.M. Teissedre. J. 43 (1995) 1. Zonas. 46 (1998) 32. J.A. F. 74 (2001) 377. Dekker.M. Morales. Agric. R. Perez. Food Chem. J. Amiot. V.M.J. Varela. Am. Food Chem. Garcıa-Viguera. Lister. L. Keller.C. Robards / J. J.S. A 1000 (2003) 657–691 ˜ F. 50 (2002) 596. [102] N. Malovana. B. Ortuno. J. 47 (1996) 186. M. W. Estrella. Wang. L. S.688 K. Agric. Cutler.L. Agric. J.M.Y. Barbanti. P.T. J.A. [99] Z. [109] S. Bartolome. [141] E. di Giacinto. Pereira. K. Detavernier. Food Chem. Garcıa-Puig. Anal.S. P.M. J. Sutton. Bryngelsson.C. Sporns.J. Cooper. Naturforsch. Food Chem. [110] J. J. R. [139] M. Nair.J. D.T. C. Phytochem. [121] P. Cabanis. U. Agric. Ho. Agric.S. 47 (1999) 4579. Huyghebaert. Perez-Vicente. L. A. Cutler.I. Cinquanta.-Z. J.Q. Chen. ´ P.T. Tena. J. Food Chem. Seabra. Konstantinou. Ho. La Notte. Silva. Andersson. Chromatogr. Lapidot. ´ [129] F. J Cereal Sci. J. A. W. Fuster.K. [98] D. C. Agric. M. Huang. [127] M. Karathanos. van der Sluis.L. Gil-Izquierdo. Lee.K. Wang. Essassi. 47 (1999) 67. Agric. F. O.M. 50 (2002) 5107.I.G. J. J. Sancho. Wang. Anal. [90] S. R. Food Chem. Ma. Bengoechea. Spanos. Food Chem. Bonvehi. ´ ´ J. Agric. 81 (2001) 1034.J. S. Kalt. Agric Food Chem. Vinha. Gil. W. Tena. [114] E. Areias. 48 (2000) 3330. Lajolo. D. Gu. A.H. Schmitz. Kustin. ´ [128] M. Agric. J. Rios. Am. 48 (2000) 6081. M. ˜ D. Food Agric. M.C. L. de Jager. G. ´ ´ M. Strasburg. 47 (1999) 3535. Orte. B. R. Harel. Valentao.B. L. Elviri. Sci. Mangia.L.J. Food Chem. 12 (1999) 227. 13 (1999) 2399.M. Food Chem. Emmons. Taylor.T.M. Carando.C. D. [122] T. Andrade. Agric. Vitic. Anal. Porras. Lebensm. Agric. [103] W. Aubert. Rodis. Chromatogr. Agric. M. J Agric. Enol.Z. [92] W. Brenes.A. ´ ´ [133] A. J. [89] A. 50 (2002) 2308. A.N. A. C. Agric.E. [140] M. J.H. J. ´ ´ J. Gray. [104] M. Granit. J. J. Agric. Garcıa (2001) 249. Rodrıguez-Delgado. F. [85] P. Fresenius J. [125] A. Buiarelli. C. C. J. Molle. S. 110 (1988) 6162. Marshall.E. Soc. P. Food Chem. Agric. 361 (1998) 143. Coccioli. Food Chem. Maiwald. [132] H.G. Seabra. P. J. Garido. M. Ooghe. Kujala. Miro. Escarpa. C. R. [93] A. Tasioula-Margari. Farnell. (1992) 1531. Shi.J. M. Food Chem. Rapid Commun. M. K. Yu. Martinez. C. Agric. A. Silva. Mantzavinou. Oliveira. G. Z. J. Angerosa. He. Garcıa.M. B. 47 (1999) 840. Muzzalupo. Food Chem. Hernandez. 48 (2000) 2848. Q. Technol. Huyghebaert. 8 (1997) 186. Technol.W. R. J. [115] M.S. F. Tsim. Andrade. 43 (1995) 1802. L. N. [97] C.A. Jongen. Lapczynski. Ong. Chem. F. J. Fernandez de Simon.C. J. [86] V. [131] J. Chromatogr. Fang. Liq. Lancaster. Santos-Buelga. 64 (1999) 115. Agric.G. d’Alessandro. 50 (2002) 5987.H. Prior. T. Hibbs. A.B.B. B. Dimberg. Chromatogr.I. K. R. A.P. Areias. Food Chem. R.-G. [137] M. A. J. Ooghe. G. J. Chromatogr. [101] M. ´ [112] A. Careri.T.A. Agric. [116] H. 64 (1994) 155. Hvattum. Rios. W. Andrade. Talanta 46 (1998) 1123. Estrella. Kamal-Eldin. Palma. Anal. 42 (1994) 2183. 25 (2002) 151. Areias. M. Guyot. Agric. Pihlaja. Engelhardt. . Versari.M. [124] A. Duan. Food Chem. Booren. F. ´ [100] M.M. Gu. ˜ P. Agric. Enol. A 922 (2001) 359. [96] B. Garcıa. Food Chem. Lorente.D. J. Agric. 12 (2001) 377. Stach. ´ ´ [120] B.L. [136] X.D. S. Garcıa-Vallejo. Lahrichi. 49 (2001) 5710. Andrade. Sabater. 49 (2001) 1848. Kanner. Conde. L.F. J. Mass Spectrom. T.F.J. 45 (1997) 4071. E. Sporns. Essafi.G. Food Chem. J. A 791 (1997) 127. M.F. M. Ooghe. A 924 (2001) 519. [126] J. Agric. Sanoner. S. K. Gonzalez. [118] M.C. [88] P. del Rıo. 82 (2002) 606. Food Chem. Am.

M. Acta 382 (1999) 39.J. 44 (1996) 2040. Heatherbell.K. Lehtonen. Agric. [185] G. ´ [190] B. M. S. Ewert.C. Barroso. Food Chem. J. Food Chem. Litridou.E. Fernandez-Bolanos. 80 (2000) 1094. Zetta. K. Schuster. O. Phytochemistry 32 (1993) 455. Chromatogr.M. Zemser. R. Chromatogr. Boyles. G. N.S.M. 46 (1998) 3564. F. [192] H. M. de Simon. 24 (2001) 2861.H. Agric. Mazza. Lancaster. A.G. Amakura. C. Liberatore. 8 (1997) 78. 55 (1990) 1011. Anal. M. J. Technol. [178] L.C. Seabra. J. Ital. J. Chim.M. Nutr. Am. Hopia. H. G. J. [196] A. Gao.F. Barroso. Z. [198] C. [169] S. Food Chem. S. Chromatogr. A. M.M. Mulinacci. Consonni. L. Bayman. Lendl. [151] M.M. ´ ´ [148] E. Barton.E. Tsuchiya. Phytochem.L. Perez-Bus´ [188] C. [160] A. Wrolstad. M. Okada. L. Agric. Food Chem.L. G. Gutierrez. Chromatographia 49 (1999) 17. 50 (2002) 1393. Food Chem. Drilleau. J. P. Pinelli. E.T. Mazza. Eur. J. [165] A. 58 (1993) 1135. Food Chem. Espartero. J. Cert. J. Zgorka. Pupin. Unger. Pupin. J. J. Tonogai. ´ E. J. [143] J. J. Solar. Mannino. Pirisi. J.Y. [158] A. Cosio. J. J. H. J. Vahteristo. [176] S. Limiroli. [155] L. [194] S. Toledo. K. [179] A.A. Beecher. S. M. Pihlava. Chromatogr. LeonCamacho. [145] I. Agric. A. [181] E. J. Broennum-Hansen. Fernandez ´ Phytochem.M. Food Chem. J. [144] S. G. Agric. Garau. J. Ruız.A. 47 (1999) 2398.A. 75 (2002) 8. Dawes. Tsimidou. Andersen. Diamandis. Mangas.M. Food Sci. [166] H. B 720 (1998) 225. J. A. J. D. 46 (1998) 1698. B. R. [174] R. ˚ [163] L. Tomono. H. Karumanchiri. M. Bull. [153] G.K. Y.C. Chromatogr. 10 (1997) 49. [161] F.T. Food Chem. Food Chem. Santos-Buelga. Agric. M. Pallaroni. J. Agric.C.M. R. ´ ´ ´ M. 47 (1999) 121. G. Ferreira. [193] R. Piotrowicz. Keene. Guillen. Food Chem. [147] R. A. de Pascual-Teresa. M. P. dos Santos. J. Food Technol. Goldberg. A. Merello. G. Hietaniemi. Agric.P.A. A 1000 (2003) 657–691 [142] H. D. J. Int. 48 (2000) 5331. Angioni. Anal. Food Chem. Edelmann. M. 42 (1994) 118. Molyneux. J. Estienne. G. Dadakova. Delaquis. Arts. Hasegawa. ´ C. Nogata. [150] L. R. Silva. Agric.C. Mouly. Agric. Food Compos. J. M. Tsimidou. Santos-Buelga. Ranalli. Yano. Food Chem.M. [183] B. A. K. Rivas-Gonzalo. A 750 (1996) 209. N.A. Sanoner. J. P. Glowniak. A 891 (2000) 183. Teulada. J. Garcıa-Vallejo. J. J. Gaydou. Y. 49 (2001) 2727. Bianco. d’Alessandro. Wollgast. Mannino. Food Chem. Schols. 213 (2001) 12. 74 (1997) 169.K. J.C. [175] N. Food Chem. M. A. Meyer.A. Anal. Wrolstad. Food Chem. M. [180] R. N. J. Pedersen. Prochazkova. A 736 (1996) 195. Konko. Takeoka. Special Issue (1995) 150.J. Angerosa. Procida. J. Suarez. Kiremire. Folta. A. Food Sci. 46 (1998) 25. J. Goiffon. E. O. A 727 (1996) 203. [167] I. tamante. Yoza. Edenharder. Diewok. Uccella. J. Agric. Chem. Spranger. P.M. Soleas. Bertuccioli. Martin-Belloso. J.P. Linssen. 59 (1994) 1057. 46 (1998) 5156. Food Sci. J. Guillen. Chilla. J. Irelan.T. 46 (1998) 1390. Agric. Walker. Toledo. M. N. Food Agric. Agric. [177] F. Sontag. A 926 (2001) 211. Astola. 67 (2002) 517. Food Agric. Mellenthin. 49 (2001) 952. 63 (1998) 275. Wrolstad. M. R. ´ F. [195] P. Andrade. C. N. J. Electrophoresis 22 (2001) 1573. J. 49 (2001) 1139.M. 21 (1986) 605. Anklam. Hong.J.K. A. J. Riv.L. Sun. Chromatogr. Keller. J. Scalbert. [189] V. Eurola.D. [159] A. Sci. Aksnes. tamante. V. Food Chem. V. A. Agric. D. Ogawa. Sci. Agric. 5 (1993) 363. 49 (2001) 2343. 45 (1997) 373. [182] P. 77 (1998) 45.L. Ricardo da Silva. Merken. J. . [191] M. Guyot. M. 38 (1990) 708. J.F. Posthumus. M. Food Sci. 33 (2000) 475. Scarpati. D. G. R. Lo Scalzo. Food Res. Sostanze Grasse 72 (1995) 163. J. Fukumoto.R.H.S. Ohta. J. A. J. Food Chem. Agric. Food Sci. Food Sci.J. S. M.E. Edwards. [199] F. D. Laraba. Zachwieja. I. Clin. Alcudia. [146] S. P. Swanson. Merken. 689 [173] G. G. Liq.I. M. M. Bianchi. Conde. G. B. Mellerio. P. Agric. Girard.R. Jımenez. Agric. Food Chem. Heredia. P. J. Lewis. V. Chromatogr. Leandro. Tsuji. Gil-Izquierdo. Dennis. E.A. Cabras. J. Trujillo. P. J. 47 (1999) 4009. [184] F. Ital. M. Food Chem. Marnet. [156] C. Nygard.J. A 667 (1994) 59.M. J. R.B. M. [152] A. Chromatogr. Mateos. K.I.L. J. Mahoney. 49 (2001) 2767. J. Liaison-Groupe Polyphenols 16 (2) (1992) 192. J. d’Alessandro.M. J. Bertuccioli. Krizek. K.F. F. Rios. P. Robards / J.M. E. Dao. Platt. R. K. [154] K.E. Rommel. 45 (1997) 403. 47 (1999) 128. Dennis. R. Corana. J. Sutton. Andreoni. Chromatogr. Anal. F. Weisz. Romani. Agric. Katase. 10 (1999) 268. Papadopoulos. Picinelli. [186] Y. M.R. B. E. M. [164] B. M. Agric. D. Mattila. 73 (2001) 119. Agazzi. Fiorentini. L. Albi.. Heinonen. B. Tattini. Ital. M. Agric. Cadahıa.J. Technol. J. 69 (1997) 4405. N.B. Boskou. Food Chem.G. J.J.M. M.H. [162] J. J. Gaydou. [170] G. Anal. M. Hollman. Food Chem. M.T. A 768 (1997) 207. C. Food Chem. [168] Y. Bergmans. De Berrios. Kidoy.F. Piironen. J. C.P. [172] S. L.J. [157] L. Valtonen. R. Food Res.W. 49 (2001) 2185. Mouly. K. N. Food Chem.A. Food Agric. Gao. Vincieri. Cichelli. Breitfellner. 63 (1998) 513. B.E. R. Agric. Trakhtenberg. K. Faure.W. Gorinstein. K. Guillen. [171] A. 38 (1990) 69. J. J. Food Chem. Kumpulainen. Kawaii. Sci. Chromatogr. Bianco. Flinck. Perez-Bus´ [187] D. [149] C. B. Food Chem. Food Chem. Berhow. J. Bandino.E.P. Relat. [197] A.M. Cosio.C. E. Wong. Mazza.

Ferreres. F. [238] G. Martin. [230] S. G. Azzolini. Chromatographia 33 (1992) 369.F.L.H.M. 74 Garcıa-Garcıa.J. 283 (1984) 137. De Nino. Martos. Zidorn. Int. Yost. J. Biochem. Garo. Food [224] I. E. Escarpa. J.-L. K. 48 (2000) 577. Mavi. [244] D. 48 (2000) 1498. P. D’Arcy. Am. Cortesi. Oil Chem. Gaydou. C. S. Pidala. Careri. M. Tomas-Barberan. Food Chem. F. [212] W. Chromatographia 53 (2001) 343. J. S. Talanta 39 (1992) 259. 210 (1999) 109. [206] C. 34 (1999) 265. Perri. [213] D. Weidner. Biomed. Anklam. Quesada. Brenes-Balbuena.H. Giuffreda. 21 (1999) 549. A.K. J. H.G. A. Tomas-Barberan. A. (1993) 15. [243] B. Attaway. Food Agric. A 897 (2000) 161. Appl. M.J. Pace. Riv. Soc. Fuzatti. Procopio. J.690 K. Food Agric. F. O. Purev. [232] N. Caasilit. Geiger. M. 81 (2001) 485. J. Fitzloff. 43 (1995) 1966. [222] F.-L.F. A. Abian. Chromatogr. R. A. J. M.R. J. Technol. Chromatographia 38 (1994) 475. Odontuya. D. Fedeli. K. Food Sci. [210] R. Food Chem. Pieroni. Johnson. [234] H. Lee. 50 (2002) 565. Bocchi. Gutmann.R. M. Mattheis. Anal. Becana. Antus. A. R. Sanchez Gomez. Treutter. Chromatogr. Hiller.V. M. Khokhar. J. 76 (1999) 873. S. Bacteriol. Wen. Santos-Buelga. Nergiz. Chromatogr. Whitecross. Weintraub. [233] P. [255] M. J. Mangia. Avery. Food Chem. R. J. Fan. Plowright. Dabrowski. F. Phytochemistry 40 (1995) 1265. Chromatographia 41 (1995) 94.A. Stuppner. E. N. Rudell. E. Bal.A. D. Robards. ´ [218] A. Pagano. Relat. Rouseff. Heimler. Guedon. F. V.P. in: S.A.). E. A 753 (1996) 157. H. S. [245] D. Beecher. Food Chem. J. E. 1988. Gaydou. Chromatogr. Rejano Navarro. Caffin. 40 (1992) 607. [246] G.R. 634 (1993) 129. Montedoro. Rodriguez. B. 45 (1997) 1185.E. Conde. R. 127 (2002) 435. Scognamiglio. Ameer. Marcel Dekker. [227] J. [258] R. 32 (1997) 533. Ryan. Rovellini. Panzeri. [226] A. C.R. Methods Enzymol. [211] S. 37 (1983) 98. Food Chem.Y. Pharm. [241] P. [209] D. G. Mouly. Seabra. J. Int. [251] A. Mori.K. F. Agric. Pietta. 50 (2002) 1267. R. Gamache. A. D. C. [236] M. 25 (2002) 397. Boskou. J. Agric.L. Chromatogr. Eur. Hostettmann. Phytochemistry 59 (2002) 305. J. Ferreres. Estienne. Hedin.M.N. E. 46 (1998) 968. Agric. Bunzel.L.J. Tsimidou. 81 (2001) 653. Montanari. Estienne. [228] M. G. A 667 (1994) 290. Lavee. [221] D. Moran. Amarowicz. F. Chromatogr. J. Am. Food Technol. Solecka. Baldioli.L.W. C.A. Amarowicz. Manini. 9 (1998) 171. Saleh. Mouly. [257] J. [235] W. J.M. Sindona.-L.C.C. Wolfender.K. [252] K. Rezaaiyan. Carvalho. M. Ralph. Fellman. Agric. K. E. J. J. Singletary. Chromatogr. Technol. L. J. M. J. M. J. Gottschlich. Hatfield. Raffaelli. [237] A. A. Sostanze Grasse 72 (1995) 55. Chem. Escarpa. Phytochemistry 31 (1992) 1173. [256] J. J.A. Tiscornia. [229] R. Cimato. M. [217] M. E. Nelson.E. Phytochem. Martos. [250] K.S. Vandercook. B. J. R. Peng. Karamac. Mass Spectrom. R. Food Chem. J. Chromatogr. Smolensky. B. Robards / J. [219] P. J. J. Byers.S. I. Agric. Marita. Phytochem. 632 (1993) 111. W. A 1000 (2003) 657–691 [200] L. Li.M. Rahman.A.B. Nagy. P. Brolis. F. Chromatogr. [205] S. J. Agric. [242] M. Peterlongo. Helv. Mauri. Park.F. T. Agric. Sci. Sci.M.B. G. Adulteration of Fruit Juice Beverages.L. Anal. Evangelisti. Heimler. 335 (2001) 26. G. [220] P. A. Markowski. Gallardo-Williams. Food Sci. [249] J. M. Wettasinghe. Le Tutour. Nematologica 42 (1996) 564. Ital.B. Tattini.H. Riv. Andrade. 635 (1993) 143. Sostanze Grasse 72 (1995) 241. Soc. 45 (1997) 364. Chen. Cadahia. [215] M. Fong. Askal. Liq. R. Sci. B. Technol. I. Schaufelberger. [253] E. Grayer. Agric. 58 (1992) 157. Chromatogr. Agric. Vlahov. J. G. Tomas-Barberan. A 830 (1999) 301. K. J. Plant Syst. J. Mau-Lastovicka. Jimenez-Diaz.H. Food Sci. [214] D. Gabetta. Ryan. Grasas Aceites (Seville) 36 (1985) 274. Gamache. Pallado. p. P. 231 (2002) 39. M. J.I. [203] R. 28 (1993) 461. Cellerino. R. G. Garcıa-Vallejo. 67 (2002) 155. Chim. 35 (1987) 921.T. Tanner. Ruiz-Barba. [239] G. Chromatogr. Evol. M. 647 (1993) 191. Gonzalez. [207] H. Daniewski. 48 (2000) 4744. Food Chem. [223] I. N. H.H. J. W. Torres. J. Ital. 47 (1982) 2058. J. M. Phillips. Miniati. J.M. J. Chromatogr.A.E. Hortic. Gonzalez. J. Plant Physiol. Chromatographia 41 (1995) 657. Nayudu. Backheet. A. Lombardo. J.V. Hostettmann.F.A. [216] M. ´ ´ A. Agric.A. Gomez-Cordoves.C. Selvaggini.M. Shahidi.B. Evidente. J. P. Boudet. Acworth.W. Scalia. Rhodes (Eds. P. Zunin. Hostettmann. P. Domon. C. Grayer. J. E. Achilli. D. J. Agric. New York. Klucas. J. . G. Food Chem.M. Martos. Aust. Capasso. M. ´ ´ I. Wolfender. S. F.F. [208] A. Groppi. [231] P. 3 (1992) 270. Merken. N. 37 (1999) 491. Food Chem. ´ ´ J. 42 (1994) 70. A. Wrolstad. Food Sci. [247] C. R. J. Food Res. Ferreira. Bartolome. R.P. E. X.M. Servili. [254] P.A. Widmer. Hostettmann. D. ´ [225] E. Steinhart. Food Chem. Harborne. M. W. 24 (1997) 261. Garrido-Fernandez. Acta 81 (1998) 754.L. W. [204] C. H. Arzouyan. J. 44 (1992) 53.M. J. ´ ´ J. B. R. M. K. Anal. Macchioni. C. Technol. J. Ferreres. Pieroni. Plant Physiol. Papadopoulos. Food Chem. C. Magnusdottir. F. T.P.P.D. Hernandez. L. Kacperska. [240] L.A. Agric. A 825 (1998) 9. J.H. Harborne.C. Radovic. Yao. M. [202] D. A. M. Bengoechea.A. J. S. [201] C. G. Estrella. Maciejewicz. 49.R. G. Hostettmann. Food Chem. A. Wang. Sci. Wolfender. J. Kolodziej. Food Agric.A.M. G. F. R. Montano Asquerino. Gill. [248] P. B. J. L.

Manetta. Harnois. Chromatogr. Marsilio. K. Stobiecki. Hostettmann. Keller. Ralambosoa. [276] C. M.J. Jovanovic.M.M. [264] C. Chromatogr. Food Agric. Carle. [262] T. Mangia. Gioacchini. Richard-Forget. 1988. J. Werner. Pietta. Dominguez. F. Marita. Aguero-Aguero.M. Chromatogr. J. Barnes. Steinhart. A 918 (2001) 303. [301] D. Willett.T. Henlin. Baldi.R. C. Chem. Chromatogr. C. B.A. Philippon. P. 30 (1982) 2732. [263] T. Intell. Prenzler. C. U. Stephenson. J. 45 (1997) 3871. Soleas. Agric. C.E. 38 (1990) 1202. I. Wolfender. Isaac. ´ C. Rastrelli. B. J. J. Wang. T. Franzoi. K. M. Pharm. 116 (1994) 4846. Chem. [295] P. [286] A. Lafont. [265] G. Baldi. 42 (1994) 2466. Pestic. da Costa. Fukuda. Gonzalez-Gonzalez. M. W. 45 (1997) 2685. M. J.A. Romero. L.C. A 661 (1994) 121. Mass Spectrom.A. F. P. N. Marinas. [266] D. Pietta. Dam. . 42 (1994) 1697. Lab. Facino. Robards / J.K. Rodriguez. Phytochem. Pietta. A 873 (2000) 29. [284] Y. A 855 (1999) 529. Biol. Maillard. S. J. M.A. Agric. J. Bunzel. R. S. J. I. Takemoto. Carey. Curr. J. Pharm. Agric. Goupy.F. J. ´ V.C. Anal. Nursten. C. Gaydou. Casetta. M. Close. R. I. V. Romani. Rodriguez. P. Akiyama. [309] J. T. H.L. 691 [288] P.U. [260] J. R. Games. B. M. C. P. N. A 1000 (2003) 657–691 [259] R. K. Food Chem. Food Chem. Matthiesen. 7 (1996) 42.Y. Oszmianski. Antolovich.V. Bianchi. Koizumi. A 730 (1996) 31. [287] A. Agric. [268] M. ´ I. 634 (1993) 350. Davies. Musci. J. Food Chem. J. Anal. V. P. Horton. Boutin. 13 (2002) 87. McDowell. [283] P. Chemometr. Urbano. Robards. Baraldini.L.M.-L. Lee. [282] R. Opin. Ng. Sci. Lanza. [299] W. Crosby. Biotechnol. Piironen. J. J. Chromatogr. M. U.J. Herlt.K. A 881 (2000) 403. Food Chem. J. 63 (1997) 2. P. J.V. London. Ralph. Murkovic. [278] D. Phytochemistry 29 (1990) 435. Food Chem. Rouet-Mayer. Simonetti. Colombo. M. [300] K. Wolfender. Souquet. T. Mauri.J. [310] P. J. Aust. Marjanovic. J. Chromatogr. 18 (1999) 362. Moutounet. Agric. Marinello. J. Food Chem. J.M. P. N. Farmaco 48 (1993) 1447. A 910 (2001) 265. H. [305] F. R. K. J. [297] A. K. Borau. ´ A. V. M. R. Berahia. S. D. Chromatogr. Trends Anal. Simic. [285] R. Schmidt.A. C.M.M. 40 (1992) 2114. 50 (2002) 5513. [291] L. Chromatogr. J.M. Agric. Chromatogr. Campestre.E. Bailey. M. Sporns. Agric. Hiller. Robards. J. Food Chem. De Simone. Anal. Creaser. Agric. A. Guillen. [280] J. Corradini. Antolovich. Seitz.S.G.J. S. J.T. Lampi. Chapman and Hall. Schieber. Brenes. J.L. Agric. 47 (1999) 2009. Facino. Syst. The Flavonoids: Advances in Research. D. M. 48 (2000) 5887. Selles. Food Chem. 28 (2001) 269. J. Fauchere. Chromatogr. Steenken. A. ´ [293] M. P. Li. 66 (1994) 203. He. Brauwissen. J.K. 74 (2001) 55. C. [290] A. Wang. J. P. Noro. Garrido. A. Food Chem. Wolfender. E. M. Chassagne. Goldberg. [289] C. Coward. Aramendıa. K. Keller. J. 10 (1996) 1585. [274] J.J. A. Agric. [281] A. [275] M. E. Chromatogr. Carini. Ayers. Kirk. Streker. S. Careri. in: J. A 880 (2000) 203. J. Bramati.J. [270] K. J. J. Rosen.M. [306] M. Analyst 117 (1992) 1105. G. 21 (1992) 271. Martinez. [304] S. Bull. Miyase. Sporns. Letendre. Aldini. Stonard. Harborne (Ed. K.Y. [272] X.G. Lam.C. [269] L. 50 (2002) 3835. Sci. Tosic. Ryan. Chromatogr. [298] W. 8 (1997) 97. J. A. D. Roeder. J. M. Ollilainen. 50 (2002) 5300. [308] S. K. Chromatogr. Food Chem.E. Ryan. Koupai-Abyazani. Chromatogr. Plant Physiol. Food Chem. M. H. Lafontaine.R. Galensa. 5 (1994) 153.). D. Crouzet. Kuroyangi.W. Roda. Phytochem. H. R.M. J. F. Chromatogr. Hakala. 427. Planta Med. M. Wahala. Kucharczyk. Hostettmann. F. Prenzler. Food Chem. J. G. J. Fukushima. Ortuno. Wallet. Klaiber. J. 43 (1998) 43. Hostettmann. L. P. S. D. Jimenez. P. Wolfender. Chem. [271] M. Bocchini. Mauri. Biomed. P.M. Agric. C. Karkola. Nicolas. A. L. J. Barroso.M. p. F. D. Anal. Merfort. Mauri. [292] A. Baumes. Philippon. Glassgen. 50 (2002) 762. Soc. Slimestad.W. [303] T. Castro. Phytochem. Ueno. [273] D. [307] J. Ng. Phytochemistry 54 (2000) 237. Monatsschr. Am. Anal. J. Careri. P. Yang. 48 (1995) 390. Agric. 27 (1989) 221. J. Mulinacci. Metzger. [277] M.C. T.N. [279] C. Krupa. P. J. Carini. Rouet-Mayer. 43 (1995) 2104. Guyot. Food Chem. J. J. J. [296] S. [267] V. Ho. Gardana. Margolis. Jardine. Phytochem. P.M. Garcıa. M. Mauri. A 970 (2002) 3.J. Carle. Minoggio.L. J. J. 647 (1993) 183.L. [294] A. L. Hostettmann. G. P. J. M. Galletti. [302] J.G. Beadle. Hostettmann.C. Schieber. A.L.K. Cheynier. Mass Spectrom. A 794 (1998) 263. Agric. Y. R. Food Chem. 43 (1995) 2458. Vincieri.L. 629 (1993) 389. K. Agric.T. A 718 (1995) 89. 474 (1989) 372. C. [261] J. J.E. Maillard. [311] S. J. Markham. F. E.R. Agric.C. Rapid Commun.G. P. F. Cerrati. Arlandini.T. Naddeo. Garcıa. S.J. Lin. 11 (2000) 85. M. Chromatogr. M. P. G. Food Chem. 48 (2000) 3166. 23 (2000) 61. Food Chem. J. J.G.B.L.A. Pietta. Bayonove.