Sample preparation in the determination of phenoliccompounds in fruits
Michael Antolovich, Paul Prenzler, Kevin Robards* and Danielle Ryan
School of Science and Technology, Locked Bag 588, Wagga Wagga 2678, Australia.E-mail: [email protected]
Received 6th January 2000, Accepted 8th March 2000Published on the Web 17th April 2000
1 Introduction2 Sample preparation2.1 Hydrolysis2.2 Fruit extracts2.2.1 Juices and related products2.2.2 Olive oil2.2.3 By-products2.3 Fruit2.4 Peel and seed2.5 Leaf3 Quantification4 Future needs—transfer to industry5 Acknowledgements6 References
1 Introduction
Phenolic compounds occur as secondary metabolites in allplants.1 They embrace a considerable range of substancespossessing an aromatic ring bearing one or more hydroxysubstituents, although a more precise definition is based onmetabolic origin as those substances derived from the shikimatepathway and phenylpropanoid metabolism.2 A convenientclassification of the plant phenols distinguishes the number ofconstitutive carbon atoms in conjunction with the structure ofthe basic phenolic skeleton (Table 1). The range of knownphenolics is thus vast and also includes polymeric lignins andcondensed tannins.
Some plant phenols may be involved in primary metabolismwhereas others have an effect on plant growth or protect themore vulnerable cell constituents against photooxidation byultraviolet light by virtue of their strong UV absorption.3 Plantphenols also play an important role in disease resistance in theplant. Intense interest in fruit phenolics is also related to theirphysiological activity which depends on their antioxidant
activity, the ability to scavenge both active oxygen species andelectrophiles, the ability to inhibit nitrosation and to chelatemetal ions, the potential for autooxidation and the capability tomodulate certain cellular enzyme activities.4–7 Thus, knowl-edge of the levels of these compounds in plants is ofconsiderable interest but is limited by problems of analysis. Thestructural diversity of the phenolics and its effect on physico-chemical behaviour such as solubility and analyte recoverypresents a challenging analytical problem. Moreover, a numberof phenolic compounds are easily hydrolysed and many arerelatively easily oxidized, which further complicates samplehandling.8,9
This review emphasises the importance of sample prepara-tion in the determination of phenolic compounds in plantmaterials particularly fruits. Fruits are an important dietarysource of phenolic substances although interest is also shiftingto other parts of the plant as potential commercial sources ofphenols. Sample preparation is a critical step in analysis and thisis even more significant with real samples where the matrixcomponents are biologically active and the analytes represent adiverse spectrum of numerous compounds, many having anunknown identity. Thus, methods of extraction of phenols fromfruits are generally dependent on several factors while the usualquantification procedures involve the separation sciences andare universally applicable. Soleas et al.10 illustrated this point.They developed a derivatization procedure for determination of15 phenolic constituents in solid vitaceous plant materials andconcluded that the method ‘should be suitable to measurepolyphenols in fruit, vegetables, and other foods provided thatefficient extraction techniques are employed’. Such statementsare seen frequently in the analytical literature but they tend tobelittle the importance of this step (or perhaps they serve tounderline its critical importance). Rhodes and Price11 observedthat the determination of phenolic species in foods is animportant outstanding problem and reviewed methods for theextraction and purification of phenolic antioxidants as theconjugated forms that exist in plant foods.
Knowledge of the extraction of phenolics is also desirableoutside the analytical context for it has important practicalapplications in the food industry. For instance, the mechanismand kinetics of phenolic extraction from wood to wine duringageing in barrels12 has significant consequences for theproduction of quality wines.
2 Sample preparation
Isolation of phenolic compounds from the sample matrix isgenerally a prerequisite to any comprehensive analysis scheme.The ultimate goal is the preparation of a sample extractuniformly enriched in all components of interest and free frominterfering matrix components. It encompasses a series of steps
Kevin Robards is AssociateProfessor of Chemistry atCharles Sturt University River-ina. He obtained his PhD inanalytical chemistry from theUniversity of New South Walesin 1979. His research interestsare focused on the applicationof analytical chemistry to foodscience and in particular theidentification and role of natu-rally occurring phenolic com-pounds in fruits.
ranging from exhaustive solvent extraction and preconcentra-tion procedures to simple liquid–liquid extraction or filtration.Extraction of the phenolics from the matrix has been a necessaryprerequisite to quantification although enhanced selectivity inthe latter may reduce the need for sample manipulation. This isnot always desirable as, for example, in gas chromatography–mass spectrometry (GC–MS) where the effects of non-volatilematrix components on column lifetime are an importantconsideration.
The task of recovery is complicated as ‘fruit’ constitutes anatural matrix with a high enzyme activity, and hence extremecare must be taken to ensure correct extraction, devoid ofchemical modification, which will invariably result in arte-facts.1 Artefactual changes, for example, oxidation and iso-merization,13 during the extraction process are a constantconcern. An example is the photochemical isomerization oftrans-resveratrol to the cis isomer.14 Methods of protecting thecompounds from these deteriorative processes have includedthe addition of antioxidants (one presumes of higher ‘activity’than the compounds themselves) during the extraction and theuse of inert atmospheres. The fidelity between the phenolicprofile of the starting material and that of the isolated extractprovides the theoretical basis for judging analytical techniques.Hence the conditions employed should be as mild as possible toavoid oxidation, thermal degradation and other chemical andbiochemical changes in the sample.
The precise procedure will depend on the nature of both theanalyte (e.g., total phenols, o-diphenols versus other phenols,
specific phenolic classes such as flavonone glycosides orindividual compounds) and sample (fruit type, fruit portion—seed/stone, skin, flesh, leaf) and particularly its physical state.In the case of liquid matrices, liquid–liquid extraction or solid-phase extraction (SPE) is often involved, although on limitedoccasions no sample treatment is necessary. These conventionalmethods have limited application to solid and semi-solidsamples because of the long extraction times and precautionsneeded to protect the highly reactive phenolic species fromdegradation processes. In these instances, supercritical fluidextraction offers a number of advantages for the recovery andthe extraction behaviour of phenolic compounds has beenmodelled using supercritical carbon dioxide and either sand15 oran inert support as a sample matrix.16 Phenolic compounds wereselected to cover a range of polarities (including benzoic andcinnamic acids, hydroxybenzaldehydes and catechin). Extrac-tion and collection variables including modifier percentage,extraction temperature, flow rate, extraction time, trap packingand rinse solvent were optimized. The latter study revealed thatthe use of methanol as modifier was mandatory. Only the lesshydroxylated compounds such as p-coumaric acid, trans-resveratrol and salicylic acid could be quantitatively recoveredfrom spiked diatomaceous earth while mean recoveries of morepolar phenolic acids and flavonoids were between 30 and70%.
Solid-phase microextraction (SPME) is a technique findingwide acceptance as a sampling method in gas chromatography(GC). It has been less used for high performance liquid
Table 1 Classification of phenolic compounds with characteristic examples in various fruit.
Flavonols Apple Quercetin, kaempferolPear Quercetin, kaempferol
Flavonol glycosides Widely distributed RutinFlavanonols Grape Dihydroquercetin and dihydrokaempferol glycosidesFlavanones Usually found in citrus Hesperitin, naringenin
chromatography (HPLC) and thus its application to analysis ofphenolic compounds in fruits has not been reported. Never-theless, the application of SPME and HPLC to the determina-tion of hydroxy aromatic compounds in water17 suggests thatthe technique warrants closer examination for the determinationof phenols in fruits.
Isolation of phenolic compounds from fruits is furthercomplicated by their uneven distribution in various forms. Forinstance, methanolic extracts from orange peel were rich inflavones and glycosylated flavanones whereas hydrolysedextracts comprised mainly phenolic acids and flavonols.18 Atthe tissue level, there are significant qualitative and quantitativedifferences between the phenolic content of seeds, epidermaland subepidermal layers (peel) and the internal tissue (cortex).This is easily demonstrated19 using suitable staining reagents.Accumulation of soluble phenolics is greater in the outer tissues(epidermal and subepidermal layers) of the fruit than in theinner tissues (mesocarp and pulp).20 For instance, in manyfruits, flavonol glycosides are chiefly located in the outerportion or in the epicarp. This is seen in the greater abundanceof glucosides and rutinosides in the peel than the flesh ofpassionfruit.21 Anthocyanins are located primarily in the skin ofgrapes but are present throughout the fruit in strawberry andblueberry. The situation with the anthocyanins is furthercomplicated by pH dependent equilibria22 and in the inner cellsin the skin, anthocyanins are mainly in the neutral quinonoidalbase form, whereas in the outer cell vacuoles, they are foundessentially in the flavylium cationic form.
At the subcellular level, phenolic compounds may accumu-late in the vacuoles or in the cell walls. Limited data suggest thatthey are located mainly in the vacuoles23 with small amounts infree space and none in the cytoplasm. The seeming homoge-neity of the subcellular distribution is perhaps misleading aslignin and certain simple molecules (flavonoids and ferulic acidesters) accumulate in the cell wall whereas soluble phenoliccompounds are stored in the vacuoles. The occurrence ofphenolics in soluble, suspended and colloidal forms and incovalent combination with cell wall components24 most likelyhas a significant impact on their extraction. For instance, duringwinemaking mainly soluble phenolic compounds present in thevacuoles of the grape plant cells are extracted, leaving behind alarge amount of phenols associated with the cell walls.25
Enzyme-assisted treatment of the press residue (grape pomace)from wine production was efficient in degrading the grapepomace polysaccharides and thus releasing phenols. Totalphenols released ranged from 820 to 6055 mg L21 gallic acidequivalents (GAE) and varied in response to enzyme type, timeof enzyme treatment, particle size of the pomace and type ofextraction solvent employed. The yield of total phenols wascorrelated to the degree of plant cell wall breakdown of grapepomace (r > 0.6, P < 0.01). These data have importantimplications for both the analytical and commercial-scalerecovery of phenols and for studies correlating physiologicalactivity (e.g., antioxidant potential) with phenol content of thefruit where dietary intake/availability is of paramount im-portance.26–28
Phenolic profiles have been reported for various fruits,generally the edible portions and less commonly with other fruitparts, although there is an emerging interest in the non-edibleparts of the fruit/plant. This application can be attributed to theuse of phenolic profiles as fingerprints for authentication ofwines, olive oils, citrus juices and other commercial products.Identification and characterization of phenolic components ofvarious fruits and assessing the physiological activity of fruitextracts have also attracted considerable attention. There hasbeen considerably less ‘interest’ in quantifying the phenoliccomponents, presumably owing to the limited range of phenolscommercially available as suitable reference compounds.29
This situation is changing and the need to quantify the levels ofphenols is now being addressed.
The extraction procedure is simplified in analyses targettinga single specific phenolic compound. Here the conflictingstabilities, solubulities, etc., of the target compounds are not anissue. For example, trans-resveratrol was determined30 in wineby LC-MS. Trihydroxyflavanone was added to the wine as aninternal standard and the mixture was centrifuged. Enhancedselectivity for the separation between trans-resveratrol andendogenous wine constituents was afforded by sample purifica-tion with a tandem SPE method. A limit of detection of 200 pg(signal-to-noise ratio = 3) was attained in the selected ionmonitoring mode using negative ion electrospray ionization(ESI) and measuring the deprotonated molecular ion. Hydroxy-tyrosol has only recently been reported in wine31 using a methodspecifically targeting this compound. The analyte was elutedfrom a C18 cartridge with ethyl acetate and derivatized withbis(trimethylsilyl)trifluoroacetamide (BSTFA). Specificity andsensitivity were achieved by GC-MS using one target and twoqualifying ions. Under these experimental conditions, hydrox-ytyrosol was detected in all wines analysed at averageconcentrations in red and white wines of 4.0 and 1.9 mg L21,respectively.
2.1 Hydrolysis
Markham32 described the use of hydrolysis as an aid tostructural elucidation and characterization of glycosides. Threetypes of hydrolytic treatment are used for this purpose, acidic,enzymatic and alkaline. Hydrolysis has also been used tominimize interferences in subsequent chromatography33 and asan aid to simplifying chromatographic data,34–37 particularly ininstances where appropriate standards are commercially un-available. In this role, chemical treatment has been morecommon because it is less selective and more exhaustive.Hydrolysis methods when used for purposes other thancharacterization/structural elucidation of unknown phenolsresult in a reduction in information content. Hence, a sampleextract containing several O-glucosides of a single aglyconeplus the free aglycone will produce after acid hydrolysis a singleHPLC peak. The advantages in terms of simplicity ofinterpretation and quantification are apparent as seen in HPLCof red raspberry juices38 where acid and base hydrolysissimplified the complex phenolic profiles dramatically. Minordifferences were observed in the profiles resulting from the twotreatments following sample preparation on Sep-Pak C18
cartridges.There is considerable variation in the lability of the
glycosidic bond under hydrolytic conditions. The rate of acid/base hydrolysis of glycosides depends on acid/base strength, thenature of the sugar and the position of attachment to theflavonoid nucleus. For example, glucuronides resist acidhydrolysis whereas by comparison glucosides are cleavedrapidly. C-Glycosides generally remain intact although struc-tural rearrangements can occur in presence of hot acids39 owing,for example, to a Wessely–Moser rearrangement which has theeffect of interconverting 6- and 8-C-glycosides.40 The fivemajor flavonoid aglycones, quercetin, kaempferol, myricetin,luteolin and apigenin, were determined41 in freeze-dried fruitsand vegetables after acid hydrolysis of the parent glycosides.The aglycones were separated by reversed-phase HPLC,identity of the eluted compounds being confirmed by photo-diode array UV detection. Optimum hydrolysis conditions werepresented for flavonol glucuronides, flavonol glucosides andflavone glycosides. Recoveries of the flavonols quercetin,kaempferol and myricetin ranged from 77 to 110% and of theflavones apigenin and luteolin from 99 to 106%.
Alkaline conditions are employed in the isolation of phenolsfrom certain fruits and fruit products, notably citrus, in order todetermine bound phenols, particularly the phenolic acids. Forinstance, orange juice was hydrolysed with sodium hydroxide
for 4 h at room temperature under nitrogen42,43 and the totalphenolic acids were recovered by ethyl acetate extractionfollowed by silica gel column chromatography. The level of freeacids as determined by direct extraction of the juice was verylow compared with that of bound acids released by hydrolysis.The content of bound acids was unchanged or slightly elevatedfrom early to late season fruit while the content of free acids wasreduced during this period.
Phenolic acids, including caffeic, chlorogenic, ferulic andgallic acids, were also determined in grape and cherry juices44
following recovery by extraction with ethyl acetate from freshor hydrolysed juices. Hydrolysis was performed in hydroxidesolution at pH 12.5 and required 48 and 62 h for cherry andgrape juices, respectively. Analysis was performed by reversed-phase HPLC using isocratic elution with detection by absorp-tion of UV radiation. The juices contained minor amounts ofphenolic acids in the free state while most were present inconjugated forms that were liberated by hydrolysis. Thephenolic acids, particularly gallic acid, were unstable in thealkaline conditions under air and it was necessary to hydrolysethe juices under argon. Cherry juice contained a high concentra-tion of chlorogenic acid which was hydrolysed rapidly to caffeicacid. Phenolic acids were recovered from cherry laurel in asimilar fashion45 by extraction of dried mesocarp with lightpetroleum. The residue was hydrolysed with sodium hydroxide,acidified and extracted into ethyl acetate prior to formation ofoxime TMS derivatives that were analysed by GC-MS. Vanillicacid was present in all cultivars and, based on FID peak areasand normalization, it was the predominant acid.
Artefacts have been reported with extractions under alkalineconditions due to degradation of some polymethoxylatedflavones.46 Similarly, flavanones and 3-hydroxyflavanones aresensitive to alkali under which conditions the dihydro-g-pyronering is broken forming chalcones, which decompose to phenolsand cinnamic acid derivatives.47 Under these circumstances,hydrolysis has been performed in acidic conditions or usingspecific enzymes for known glycosides or technical enzymeswhen samples contain a mixture of glycosides.
Similar procedures have been adopted for the analysis of thefruit. For example, the distribution of free and bound phenolicacids was determined in orange and grapefruit45 by extractionwith ethyl acetate, silica gel column chromatography clean-upand HPLC analyses of samples before and after alkalinehydrolysis (24 h). In all fruit parts (peel, albedo, flavedo, juicesacs and endocarp), only minor amounts of these acids occurredin the free state, while most was present in conjugated formswhich were capable of liberation by hydrolysis. The level ofbound acids was generally in the order ferulic acid > sinapinicacid > coumaric acid > caffeic acid. However, significantlosses of caffeic acid were reported during lengthy hydrolysis(24 h) due to the reactive nature of the o-dihydroxyphenolicgrouping. The loss of o-diphenols by oxidation via thecorresponding quinones is a constant concern under alkalineconditions. The remaining acids were relatively stable totreatment with 2 M sodium hydroxide for 4 h under nitrogen atroom temperature. The peels contained the major quantity ofphenolic acids compared with the endocarp, and the flavedo wasricher in acids than the albedo.
In comparison with citrus fruits, there has been a surprisinglack of interest in the use of alkaline extraction with other fruits.However, the recovery of phenolics from fruit cuticles ofseveral varieties of apple, using either cuticular wax scrapedfrom fruit peel or enzyme-isolated cuticles,48 is an interestingdevelopment. The concentrations of free phenolics in fruitcuticle ranged from 8 to 45 mg g21 and bound phenolics rangedfrom 50 to 110 mg g21 in these cultivars.
Extraction of phenols from freeze-dried olive pulp intoaqueous carbonate solution49 gave acceptable recoveries ofoleuropein and other major phenols. However, extraction ofoleuropein and gallic acid from model solutions using the same
conditions but containing lower concentrations of the phenolsshowed variable results with total loss of phenol in somecases.50 The in vitro alkaline hydrolysis of oleuropein producesseveral aglycones51 but (elenolic acid glucoside and) hydroxy-tyrosol alone appear(s) in whole fruits subjected to alkalitreatment during processing.52 The latter, an o-diphenol, is theneasily oxidized.53 There has been considerable interest in theenzymatic and/or chemical catalysis of olive secoiridoids.Endogenous hydrolytic enzymes, notably glycosidases, may beactivated during crushing and malaxation54 and catalyse thehydrolysis of secoiridoids such as oleuropein with the produc-tion of oleuropein aglycone.55–57 The latter underwent rapidisomerization (Fig. 1) via the enolic form II to a dialdehydicform (III/IV) in aqueous extracts58,59 but was stable whenextracted with aprotic dipolar solvents (e.g., acetone, dioxane)or with protic solvents with pKa values higher than that of water.The oleuropein derivative also disappeared during TLC purifi-cation of extracts and this was attributed to catalysis arisingfrom the acidity of silica. The epimeric phenolic metabolites(III and IV in Fig. 1) and the precursor enolic form II haverecently been identified60 in samples isolated from methanol–acetone extracts of freeze-dried green mature olive fruits. Thesecompounds are confirmed as intermediates in enzymatichydrolysis of glucosidic linkage in oleuropein (Fig. 1).
2.2 Fruit extracts
Extraction of phenols from fruit extracts, particularly juices, isgenerally simplified by the physical state of the sample, butcomplications still arise. The impact of the processing operationon analytical data for phenols in fruit extracts such as juice, jamand olive oil must be considered. For example, was the seed orpeel excluded? In the case of peach there is a downy peel whichis removed prior to processing, whereas this is not the case withmany other fruits. Commercially processed products should bedistinguished from the corresponding fresh products recoveredin the laboratory specifically for analysis. Such considerationsare important as they will determine the extent of inclusion ofdifferent parts of the fruit in the nominal portion. For instance,hand-reamed and commercially squeezed orange juice willincorporate the albedo and flavedo to different extents. Fruitmaturation is also an important factor and more of thecomponents of the albedo will be extracted from over-ripe citrusfruit than from immature fruit. Quantitative data should bequoted on a dry solids basis or alternatively on juice convertedto a constant Brix value.61,62
2.2.1 Juices and related products. Large differences in thelevels of different phenolic compounds in a juice generallycomplicate the simultaneous analysis of the different classes ofphenols. For instance, flavanone glycosides and cinnamoyl-b-D-glucopyranoside were isolated63 from the juice of bloodoranges by extraction at 90 °C with dimethylformamidecontaining ammonium oxalate solution (to maintain pH)whereas trans-cinnamic acid, in recognition of the lowerconcentration in the juice, was concentrated by reversed-phaseSPE. Hydrolysis of cinnamoyl-b-D-glucopyranoside wasachieved in 4 M HCl by refluxing for 1 h. Flavanone glycosides(e.g., hesperidin, 100–500 mg L21) are quantitatively the mostimportant phenolics in orange juice whereas the levels of otherphenolics such as polymethoxylated flavones are much lowerwith typical values of 0.1 mg L21,46,64 although higher levelsare found in the peel. For this reason, many methods aredesigned for a specific class of phenolic compound,61,62,64–66
although the relative response of the detection system must beconsidered and Mouly et al.67 described the simultaneousseparation of flavanone glycosides and polymethoxylatedflavones.
Sample preparation may involve a simple filtration or anelaborate extraction of the crushed,68 hand-reamed63,69 orcommercially extracted juice.70 Ideally, clear juices requireminimal sample preparation beyond centrifugation and/orfiltration. For instance, apple juices were prepared for analysisby filtration through polytetrafluoroethylene filters and severalclasses of phenolic compounds were identified by HPLC andquantified in commercial juices71 by absorption at characteristicwavelengths as hydroxycinnamates (316 nm), anthocyanins(520 nm), flavan-3-ols (280 nm) and flavonols (365 nm). Therange of concentrations as a percentage of total phenolic
concentration as determined by the Folin–Ciocalteau methodwas hydroxymethylfurfural 4–30%, phloridzin 22–36%, cinna-mates 25–36%; anthocyanins not detected, flavan-3-ols 8–27%and flavonols 2–10%. Individual phenols were not identified.Pear, strawberry, raspberry and apple juices have also beenanalysed70 by direct injection HPLC following centrifugationand adjustment to constant Brix. Dihydrochalcones (e.g.,phloridzin) were characteristic of the apple juices at typicalconcentrations of 2–20 mg L21, but the phenolic components ofthe remaining juices were not identified. Cloudy juices astypified by those of citrus fruits are also amenable to direct
Fig. 1 Pathways showing the interconversions observed for one group of plant phenols.
analysis following filtration and centrifugation,72–75 althoughpoor recoveries have been attributed to low solubility of certainphenolics76 and/or to sorptive losses on the filtration me-dium.77
In other instances, more extensive sample processing hasbeen deemed desirable78 and SPE on mini-cartridges has beenemployed46,78–80 in an attempt to minimize the effects of thesample preparation on extract integrity. For instance, interferingsugars were removed by SPE from dealcoholized berry and fruitwines and liquors (adjusted to pH 7.0 using sodium hydroxide)prior to measurement of total phenols81 by the Folin–Ciocalteuprocedure. Suárez et al.82 fractionated phenolics from applemust and cider into neutral and acidic groups by means of a SPEmethod. Extracts were analysed by reversed-phase HPLC usinga phosphate methanol gradient and quantification at 320 nm forcinnamic acids, 360 nm for flavonols and 280 nm for otherphenols. Recoveries between 84 and 111% were obtained fromspiked samples. The level of phenolic compounds in kiwifruit islow relative to that of many other fruits. Nevertheless, juicefrom kiwifruit was fractionated68 into strongly acidic andweakly acidic materials by processing on Sep-Pak C18 car-tridges. The juice was obtained by treatment of the fruit in ahammer mill followed by addition of pectolytic enzymes andethanol prior to filtration to remove protein. Strongly acidiccompounds were identified as derivatives of coumaric, caffeicand 3,4-dihydroxybenzoic acids whilst the weakly acidicfraction contained epicatechin, catechin and procyanidins plusflavonols present as the glycosides of quercetin and kaemp-ferol.
The use of pectolytic enzymes during commercial processingof juices may influence the content of phenolic compounds.Versari et al.83 evaluated the effect of commercial pectolyticenzymes on the content of phenolic compounds (anthocyanins,flavonols and ellagic acids) in strawberry and raspberry juicesunder enzymatic pectinase treatment. They found that commer-cial pectinases modified the phenolic composition of the juicesdependent on time and fruit species. Depending on the enzymetreatment employed, at 6 h, a loss of anthocyanins (220%)present in raspberry juice was observed, whereas in strawberryjuices the ellagic acid concentration always increased and theflavonol content decreased (235%).
Flavanone glycosides of citrus juices have also beenrecovered by elution with methanol from a Sep-Pak C18
cartridge78 following elution of sugars with aqueous methanol.Recoveries compared favourably with those achieved by simplefiltration. Preliminary fractionation of citrus juice phenolics hasalso been performed84 on polyamide cartridges eluting withmethanol. The extracts were analysed by reversed-phase HPLCon a cyclodextrin bonded phase to resolve diastereomers andenantiomers.
The recovery of cinnamic acids, cis- and trans-resveratrol,flavonoids and flavanols from (grape) wine has been thoroughlyinvestigated10 and here also SPE has provided a simple meansof recovery. In comparing diatomaceous earth, C18 and C8
cartridges, the highest recoveries were achieved with the latter.The presence of ethanol in the wine samples presented problemsthat were eliminated by distillation, although matrix dilutionwith water was equally effective and a simpler solution. Thisalso reduced matrix interference by other components andimproved recoveries of phenolic species. The phenolic com-pounds were eluted from the SPE cartridge with ethyl acetate,evaporated to dryness by azeotropic distillation and derivatizedwith BSTFA prior to quantification by GC-mass selectivedetection using an internal standard. SPE was also used torecover phenolic compounds including phenolic and cinnamicacids from sherry wine by an initial clean-up on a C18 cartridgefollowed by fractionation into acidic and neutral phenolicfractions using an anionic exchanger cartridge85 or by an on-lineautomated robotic system with a polymeric poly(styrene–divinylbenzene) cartridge using tetrahydrofuran as eluent.86
Recovery of added phenolic compounds from spiked samplesexceeded 85% in all cases, although other data85 suggest thatmuch lower recoveries are typical. Nevertheless, SPE wasregarded as superior to liquid–liquid extraction and reducedanalysis times by 50%. In contrast, phenolic compounds(including hydroxybenzoic acids, hydroxycinnamic acids, phe-nolic aldehydes, coumarins, flavan-3-ols and flavonol agly-cones) have been determined in wood aged fortified wines87 bydirect injection with no sample pre-treatment. The higher levelsof phenolic compounds due to extraction from the woodprobably facilitated direct injection.
In the case of cloudy juices, both filtration and SPE may beineffective in recovering phenols located in suspended juicesolids. Under these circumstances, solvent extraction may be apreferable alternative although even here bound phenolsprobably remain intact. Thus, polarity differences in juicecomponents88,89 have been exploited in a comprehensiverecovery scheme for (carotenoids), polymethoxylated flavonesand flavanone glycosides based on extraction with solvents ofgraded polarity. Citrus juice was diluted with methanol,centrifuged and aqueous sodium chloride added to the super-natant (to minimize the formation of troublesome emulsions).The solution was then extracted sequentially with hexane anddichloromethane to isolate the carotenoids and polymethoxy-lated flavones, respectively, leaving the flavanone glycosides insolution. Various solvents have been described for the isolationof flavanone glycosides (ref. 76 and references cited therein),phenolic acids42 and polymethoxylated flavones61,90,91 fromcitrus. Methanol has been deemed76 the most suitable solventfor extraction of both flavanones and flavanone glycosides,although difficulties are encountered with specific com-pounds.
Methanolic extraction has also been favoured for recovery ofphenols from apple, pear and quince purees.92 The isolatedphenolic compounds were quantified by absorption at either 280or 350 nm. Simple extraction with methanol was compared witha more detailed procedure involving clean-up of the extract onan Amberlite XAD-2 column that removed sugars and otherpolar compounds. The authors noted that the chromatogramswere ‘somewhat cleaner than those obtained with the simplifiedextraction technique and, as a general rule, the amount of eachphenolic compound extracted was higher’. However, the use ofthe resin caused low recoveries of arbutin from pear purees.
The efficiencies of several solvents have been compared91 forthe recovery of polymethoxylated flavones from intact citrusjuices and juices treated with sodium hydroxide to eliminatepossible interfering lactones. In terms of total flavones, isobutylmethyl ketone was only slightly less efficient than benzene butwas more effective for specific flavones. These data demon-strate the need to consider carefully any recovery problem93,94
on an individual basis. Alternatively, polymethoxylated fla-vones have been isolated from citrus juice by retention onpolystyrene resin followed by elution with ethanol andacetone.95 The extracts were further purified by silica gelcolumn chromatography.
The phenolic composition of peach and apple purees andconcentrates,20 intermediate products in the elaboration ofcommercial fruit juices, was quantified by homogenizingsamples in acidified methanol and partitioning the phenoliccomponents into ethyl acetate. Phenols were identified byHPLC as various benzoic acids and aldehydes, cinnamic acidsand their derivatives, flavan-3-ols, procyanidins, flavonols anddihydrochalcones. Peach-based products were completely de-void of flavonol and dihydrochalcone derivatives and this wasattributed to the removal of the skin and stone of the fruit in themanufacturing process. On the other hand, different quercetinand phloretin glycosides were detected in apple purees andconcentrates.
Commercial juices and nectars of orange, apple, peach,apricot, pear and pineapple93 were concentrated using a rotary
evaporator with a bath temperature below 35 °C prior tosequential extraction with ethoxyethane and ethyl acetate. Theextraction time and temperature were evidently critical. The twoextracts were combined and evaporated to dryness beforeanalysis. In this way, quantitative data were obtained on thecontent of benzoic acids and aldehydes, flavan-3-ols, flavonols,chalcones, cinnamic acids and their esters, glycosidic deriva-tives and flavonoids. Differences in levels of flavanols wereattributed to different degrees of pressing of the fruit as thesephenols are found mainly in the skin and seeds.
Specific problems are encountered with some phenols. Forinstance, hesperidin, the major flavonoid of sweet orange,presents difficulties because of its low solubility in aqueousmedia. Addition of dimethylformamide to orange juice has beenused67,96 in an effort to improve solubility but in this case someearly-eluting peaks were lost in the chromatogram. This alsoresults in sample dilution with a decrease in sensitivity. Heatingof the juice has also been used62 to increase hesperidinsolubility. Buffering of the sample in the pH range 4.5–5.0 priorto extraction has been recommended69 to overcome moregeneral problems of the pH dependence of flavanone glycosiderecovery. In this instance, oranges were hand-squeezed and theextract filtered through a stainless steel sieve (1.25 mm) toremove seed and pulp66,69 although a double layer of cheese-cloth has also been used for this purpose.78 The separated juicewas mixed with dimethylformamide and ammonium oxalate (tomaintain pH) and heated for 10 min. The cooled juice wascentrifuged and filtered prior to injection.
Recovery of anthocyanins which comprise a major portion ofthe phenolic content of red wine and dark-coloured berryjuices13,97–100 presents some unique challenges. The anthocya-nins are glycosides that release the anthocyanidin aglycone byhydrolysis. The aglycones exist in various forms in pH-
dependent equilibria (Fig. 2) which impacts on their solubilityand extraction behaviour. Six anthocyanidins are widespreadand commonly contribute to the pigmentation of fruits.Cyanidin is the most common and, in terms of frequency ofoccurrence, is followed in decreasing order by delphinidin,peonidin, pelargonidin, petunidin and malvidin. Glycosylationof anthocyanidins almost always occurs at the 3-position withglucose, arabinose and galactose the most common sugarmoieties. For instance, the 3-glucosides and 3-rutinosides ofcyanidin and delphinidin are the dominant species in black-currant.101 In addition to glycosylation, acylated anthocyaninsare found fairly often in fruits, the situation being particularlycomplex in grapes102–104 where the 3-monoglucosides corre-sponding to the five aglycones can all be acylated by acetic orp-coumaric acid.
Anthocyanins are traditionally recovered as the flavyliumcation by extraction with cold methanol containing hydrochlo-ric acid.105 However, the acylated anthocyanins are frequentlylabile in solutions containing mineral acid and this is one of thereasons why the relatively common acylated pigments wereoverlooked in earlier studies.106 Replacement of hydrochloricacid with weaker acids, either formic or acetic acid, permits therecovery of these compounds.107–109 Care must be exercised toensure that acetylated derivatives are in fact natural and not anartifact of the extraction process.110 With the most labileanthocyanins, the use of non-acidified solvents is probably asensible precaution. Alternatively, SPE on C18 cartridges hasbeen used.111 For example, anthocyanins were recovered fromdiluted fruit juice or wine (after removal of ethanol)112 byelution from a Sep-Pak C18 cartridge with methanol–formicacid–water. The extracts were analysed by matrix-assisted laserdesorption/ionization mass spectrometry (MALDI-MS), inwhich analytes are usually desorbed and ionized in the source
Fig. 2 Structure of anthocyanins showing the pH-dependent equilibria.101
forming protonated or alkali metal adduct ions. However,anthocyanins exist in the above eluent predominantly in thearomatic oxonium ion form whence they easily ionize in theMALDI source to form molecular mass cations in the positiveion mode.
When the adsorbed anthocyanins are subsequently elutedfrom the SPE cartridge with an alkaline borate solution, a classseparation is achieved.111 It appears that those anthocyaninspossessing o-dihydroxy groups (cyanidin, delphinidin, petuni-din) form a charged borate complex, resulting in a morehydrophilic species. This complex is preferentially eluted fromthe reversed-phase cartridge whereas those anthocyanins notcontaining o-dihydroxy groups (pelargonidin, peonidin, malvi-din) are enriched on the cartridge. On the other hand, elutionwith hydrochloric acid (0.01%) in methanol produces nofractionation. A more exhaustive clean-up on polyvinyl-pyrrolidone was also examined. The relative proportions of theanthocyanins was different for the two procedures. Thus, forquantitative analysis the extraction and/or clean-up procedureshould be thoroughly checked.113
Condensed polymeric anthocyanins formed during the wine-making process by interactions between anthocyanins and otherphenols such as flavanols (e.g., catechin) were recovered114
from red wine or apple cider on an ODS column by elution withmethanol. The concentrated lyophilized extracts were thenfractionated by gel permeation chromatography (GPC) using amixture of acetone and acidified aqueous urea as eluent.Anthocyanins and other phenolic compounds were recoveredfrom the GPC fractions by sorption on a Sep-Pak C18 cartridgethat was washed with water to remove urea. The sorbedphenolic compounds were eluted with methanol.
2.2.2 Olive oil. Liquid extraction has been widely used forrecovery of phenols from olive oils purchased through retailoutlets115 or obtained directly from commercial proces-sors116,117 or in the laboratory by simulating industrial process-ing conditions.118,119 In some instances, details of oil produc-tion have not been provided120 or it was stated that oils ofdifferent extraction technologies were analysed.121
Phenols have been recovered from olive oil by extraction ofthe unsaponifiable matter with aqueous methanol.119 However,the more usual procedure has involved extraction with metha-nol120 or aqueous methanol115,116,118,122 of a solution of the oilin hexane116,118,122–124 or ethoxyethane.120 An internal standardis included in most procedures. Residual oil must be removedby overnight storage at subambient temperature,120 by centrifu-gation118 or by solvent extraction with hexane,122 althoughSephadex column chromatography has also been used8,9 toeffect further clean-up. Direct extraction of the oil withmethanol in an Ultra-Turrax apparatus has also been used forthe recovery of phenols from virgin olive oil dried over sodiumsulfate.125 The methanol was removed and the residue dissolvedin acetonitrile and washed with hexane. After evaporation of theacetonitrile under vacuum, the residue was dissolved in acetoneprior to derivatization with BSTFA. The extracts were exam-ined by GC-MS and chemical ionization confirmed severalphenolic and secoiridoid derivatives in the extracts. Aglyconesof ligstroside, of decarbomethoxyoleuropein and of oleuropeinwere detected. Each aglycone, because of several tautomericequilibria involving the ring opening of secoiridoid, showed upas compounds with four main structures following derivatiza-tion. Montedoro et al.8 compared the various methods ofextraction (directly from oil versus a solution of oil) usingdifferent solvent combinations and concluded that aqueousmethanol provided optimum results. Their procedure was usedby Brenes et al.57 to characterize the phenols in Spanish virginolive oils. The procedure involved direct aqueous methanolextraction from oil, partitioning into acetonitrile and washingwith hexane. Despite the extensive use of aqueous methanol asextractant it has been claimed120 that extraction with neat
methanol improved yields of a number of phenolics andeliminated formation of troublesome emulsions seen withaqueous methanol.
The work of Litridou et al.126 highlights the need for care insample extraction when using SPE where the choice of eluentand/or eluent volume is critical. Mannino et al.121 reportedgallic acid in olive oil and attributed its appearance to theirextraction procedure involving SPE which eliminated oxidationprevalent in other procedures. Two approaches have been usedin which olive oil was dissolved in hexane and added directly toa C8 cartridge58 which was washed under vacuum with hexane–cyclohexane to remove the non-polar fraction of the oil.Phenolic compounds were eluted with acetonitrile and storedovernight at reduced temperature to precipitate the oil droplets.In the second approach, the oil was again dissolved in hexane126
but extracted with aqueous methanol prior to SPE. The extractwas then evaporated under nitrogen and fractionated byreversed-phase SPE using stepwise gradient elution into 40fractions which were combined ultimately into fraction A[eluted with methanol–water (20+80 v/v)] or fraction B (elutedwith stronger eluents comprising aqueous methanol andmethanol–chloroform). HPLC analyses of the two fractionsshowed that fraction A contained only simple phenols andphenolic acids, whereas fraction B had a complex nature andwas found to contribute more than fraction A to the oxidativestability of the oil. Acid and alkaline hydrolysis also yieldedsome valuable information and significant changes in the HPLCprofiles were observed, which indicated the presence of etherand ester bonds. Finally, anion exchange HPLC was used todetermine whether or not monosaccharide residues werereleased after acid or alkaline hydrolysis of the given fractions.Acid hydrolysed extracts showed the presence of smallquantities of glucose and galactose, suggesting that only tracesof glycosides were present in the polar fraction of the oilinvestigated.
2.2.3 By-products. Residues from fruit processing havetraditionally presented an economic and environmental problemas waste products but are becoming increasingly recognized asvaluable commodities for the production of by-products. Forinstance, citrus residues remaining from juice extraction can bea source material from which over 300 valuable by-products canbe produced.127 The whole peel or rind (pericarp) is used forsuch products as marmalade, candied peel, bioflavonoids andpeel seasonings. Combined with the pulp residue, it becomesfeed for animals, molasses, alcohols and distilled oils. Theflavedo (exterior yellow peel, pericarp) contains the oil glandsfrom which cold-pressed and distilled oils and essences areextracted for the flavouring industries. The albedo (interiorwhite spongy peel, mesocarp) is rich in pectin and usedextensively as a gelling agent in the food and pharmaceuticalindustries. Pulp residue (endocarp) represents the fractionscreened from the pulp, that is, cores, segment walls ormembranes, juice vesicles and seeds. This is usually combinedwith the peel residue for the manufacture of stripper oil, citrusmolasses, citric and lactic acids, citrus wine and many other by-products. The recovery of phenolic compounds from by-products of fruit processing has attracted considerable attention.The phenolic content is also of interest in food, pharmaceuticaland cosmetic uses of these by-products where the physiologicalactivity of the phenols may be important. There are additionalreasons for the interest in the phenolic content of processingresidues. Orange pulpwash128 is obtained during the processingof oranges for juice and it has been used as an adulterant of thejuice. The phenolic profile provides a fingerprint that is usefulin identifying juice adulteration by the pulpwash.129,130
The waste material produced during refining of cold pressedcitrus peel oils represents a further important source of phenoliccomponents131 and polymethoxylated flavones have been
determined in peel oils following simple dilution with ethylacetate containing an internal standard132 or by extraction andclean-up using column chromatography.133 Alternatively, thepolymethoxylated flavones were obtained134 directly from thepeel of oranges and tangerines by Soxhlet extraction withbenzene for 4 h. The extracts were concentrated in vacuo andanalysed without further purification by normal-phase HPLC.Some of these oils are used in perfumes and cosmetics135 andpotentially in the treatment of burnt skin,136 so it is equallyimportant to be able to establish if they contain phenols withadverse physiological effects.
Similarly, grape marc resulting from red winemaking is avaluable source of phenols. Grape marc was extracted137 with amixture of ethyl acetate and water in order to recover itsphenolic compounds with a view to their use as food lipidantioxidants. Crushed and uncrushed marcs were extracted forvarious times in order to determine the minimum time requiredfor ensuring maximum extraction of phenols. The results reveala higher extraction of these compounds by the ethyl acetateacting on the crushed marc. Hence the cost of crushing can belargely compensated.
Large volumes of water are generated during traditionalolive oil production and subsequently discarded. This requirestreatment138 and the process is not environmentally sustain-able. Hence, new extraction technologies have been in-troduced and there has been considerable interest in compar-ing the phenol content of oils139 and wastewater140 producedby the different technologies notably two-phase versus three-phase extraction. This is a further instance where the same setof considerations important in analytical methodology haveimportant implications for processing technology. The waste-water contains a number of phenols in quantities determinedlargely by their partition coefficients and these have beenanalysed141 on an uncoated fused-silica capillary electro-phoresis column using aqueous ammonium acetate buffer inmethanol and negative ion electrospray mass spectrometricdetection. Quantitative analysis, with p-chlorophenol as theinternal standard, was carried out by single ion monitoringand limits of detection ranged from 1 pg for 4-hydroxy-benzaldehyde and protocatechuic acid to 386 pg for vanillicacid. Ethyl acetate and butanol extraction have beenused140,142 to recover phenols from fresh olive wastewater.Wastewaters obtained by employing a benchtop mill werefractionated143 by liquid–solid extraction (details covered bypatent and not disclosed) and further processed to yield threeextracts. Extract 1 was obtained by fractionation of lyophi-lized wastewater on an XAD 1180 column and elution withethanol. The second extract was obtained by ethyl acetateextraction of hexane-washed wastewater while the thirdfraction was obtained following a fractionation of extract 2on a Sephadex LH-20 column. Extract 1 contained a complexmixture of phenolics including many polymers responsiblefor a high background absorption at 254 nm. Extract 2contained mainly low and medium molecular mass phenolicswith elenolic acid as the principal constituent. Extract 3comprised hydroxytyrosol, tyrosol and an unidentified deriva-tive of the former. Characterization of the wastewater andparticularly its phenolic components is necessary to allowagricultural uses of the water.144 The biodegradation of thephenols and investigation of metabolites145 are importantconsiderations in future work.
2.3 Fruit
The phenolic profile is characteristic of a fruit species and whilethere are varietal and seasonal differences these are ofsecondary importance. Methods of recovery differ between thevarious fruits reflecting these variations. Fruit morphology mustalso be considered since the nature and content of phenolics
differs between the various organs of the fruit. Hence extractionmethods can impact significantly on the phenolic content of afruit extract depending on which fruit organs are included. Forinstance, citrus fruit is particularly complex and comprises theouter layers collectively termed rind or peel which includes theflavedo or outer coloured portion with oil glands, the innercolourless portion, the albedo and the internal structures. Thelast part involves the segments surrounded by a continuousmembrane, the endocarp proper with a membrane of mesocarptissue extending radially between segments. The interior of asegment contains the juice (or pulp) vesicles and seeds. Thedistribution of phenolic compounds between these organsdiffers both qualitatively and quantitatively.
Historically, recovery of phenols by liquid extraction of thefruit using hot or cold solvents has been common. Suitablesolvents for this purpose are aqueous mixtures with ethanol,methanol, acetone and dimethylformamide. Extractions havebeen performed on freeze-dried ground extracts of the fruit or,alternatively, by maceration of the fresh, undried fruit with theextraction solvent.124 In the last case, the required proportion ofwater is lower.
Solvent extraction has been widely used to recover phenolsfrom citrus. Grapefruit portions and peel were dried at 50 °C ina fan forced air oven146 and the material was ground to a finepowder, which was extracted with dimethyl sulfoxide. Theextracts were filtered before analysis by HPLC. Epicarp,mesocarp, endocarp and leaf tissue of Citrus were lyophilized,ground and extracted79 at ambient temperature for 12 h usingmethanol–dimethyl sulfoxide. The extracts were centrifugedand subjected to clean-up by SPE using C18 cartridges toremove polar components. The retained flavonoids were elutedwith methanol–dimethyl sulfoxide, which enhanced the sol-ubility of hesperidin, diosmin and diosmetin. Recoveries oferiocitrin, naringin, hesperidin and tangeretin from spikedsamples of mesocarp tissue exceeded 96%. Flavones andflavon-3-ols were relatively abundant in leaves. Extraction withaqueous ethanol has been used147 to recover flavonoids from adried extract of sour orange. The ethanolic extract was filteredand evaporated to dryness under vacuum prior to analysis byLC-MS using ESI. Several flavanones, flavanone glycosidesand polymethoxylated flavones were detected and identified inthe extracts. This approach for separation into peel and pulp hasalso been applied21 to passion fruit. Clear juice was obtainedfrom the pulp by filtration through gauze and centrifugation.Peel was blended with methanol, filtered and evaporated todryness. The juice and peel extract were processed on AmberliteXAD-2 resin to retain selectively phenolic glycosides that wereeluted with methanol.
Interest in the phenolic content of the grape berry has focusedon its anthocyanin22 and catechin contents.148 For instance,catechins were recovered148 from black grape (and apple) byextraction of freeze-dried material with aqueous methanol usinga mechanical shaker for 60 min at room temperature. Theextract was filtered and analysed by HPLC using fluorimetricdetection at 310 nm (excitation at 280 nm) for the specific andsensitive detection of (+)-catechin and (2)-epicatechin. Grapeanthocyanins have also been extracted149 at room temperatureusing a mixture of formic acid in aqueous methanol. The acylportion of anthocyanins has traditionally been characterizedfollowing mild alkaline hydrolysis since cinnamic acids are notstable in a hot acid medium. However, anthocyanidins areunstable in alkaline media. In this instance, the correspondinganthocyanidins were obtained by hydrolysis of the sampleextract with methanolic HCl whereas acid hydrolysis in aqueousmedia completely destroyed cinnamic acids.
Cherries are another fruit in which anthocyanins comprise themajor phenolics particularly in dark-coloured cherry geno-types.13 Mature sweet cherries were pitted and homogenizedwith aqueous methanol containing formic acid. The homoge-nate was filtered and the filtrate analysed by HPLC for the
separation and quantification of both anthocyanins and otherphenolic compounds, predominantly neochlorogenic acid andp-coumaroylquinic acid. A more complex procedure based onsequential extraction with hexane, ethyl acetate and methanolhas been applied to lyophilized ground tart cherries.150 Furtherpartitioning of the methanolic extract with ethyl acetate yieldeda fraction containing a mixture of phenolic compoundsincluding isoflavones, flavanones and flavonol glycosides.Anthocyanins were recovered during the process in a separatefraction.
Häkkinen et al.151,152 systematically investigated the recov-ery of non-anthocyanic phenols from berries other than grape.Although anthocyanins contributed a significant proportion ofthe total phenolic compounds in these berries, the method wasnot applicable to these compounds. Three extraction andhydrolysis procedures were investigated for the recovery offlavonols (kaempferol, quercetin and myricetin) and phenolicacids (p-coumaric, caffeic, ferulic, p-hydroxybenzoic, gallicand ellagic acids) from the frozen berries. The influence ofthawing method (refrigerator, room temperature or microwave)was examined and showed differential effects on the level ofdifferent flavonols. Microwave thawing produced the mostreliable results and was also the most practical approach forroutine analyses. Flavonols were extracted and hydrolysed toaglycones by refluxing in aqueous methanol containing hydro-chloric acid and tert-butylhydroquinone as an antioxidant.Recoveries of flavonols were critically dependent on theconcentration of the aqueous methanol extractant. The authorsconcluded that it is not an ‘easy task to find a single methodwhich is adequate for an analysis of a diverse group of phenolicsbecause of the differing chemical structures and the varyingsensitivity of the compounds to the conditions of hydrolysis andextraction’.
Mature cider apples153 were sprayed with aqueous formicacid to avoid oxidation while manually separated into paren-chyma zone (62% by mass), epidermis zone (18%), core zone(11%) and seeds (1%). The tissue samples were then frozen,freeze-dried and extracted with hexane to remove lipids,carotenoids and chlorophyll. Sugars, organic acids and lowmolecular mass phenols were then extracted with methanol andpolymerized phenols were recovered from the residue withaqueous acetone. The dry methanol extract and the dry aqueousacetone extracts were analysed using reversed-phase HPLCcoupled with diode array detection following thiolysis toquantify phenolic compounds as hydroxycinnamic acid deriva-tives, flavan-3-ols, flavonols and dihydrochalcones. Procyani-dins were the predominant phenolic constituents in the fruits,much of them corresponding to highly polymerized structures.In a similar approach, whole apples, peel or flesh werehomogenized with aqueous methanol71 using a Waring blender.The extracts were filtered and the methanol was removed byrotary evaporation prior to analysis by HPLC. The phenolicprofile of the apple extracts differed from those of juices. Therange of concentrations of phenolic classes in fresh appleextracts was hydroxymethylfurfural, not detected, phloridzin11–17%, cinnamates 3–27%, anthocyanins, not detected to42%, flavan-3-ols, 31–54% and flavonols 1–10%.
Flavanols or catechins are important phenolic components ofapples. Analytical methods for flavanols have generally focusedon the identification of new derivatives or polymeric catechins(proanthocyanidins) and are not designed for quantification. Incontrast, Arts and Hollman148 optimized the quantification offlavanols in three model foods: apples, black grapes, and cannedkidney beans. Freeze-dried and fresh samples were examinedand the level of flavanols was not affected by the dryingprocess. The sample was mixed with aqueous methanol andshaken in a mechanical shaker at room temperature. Theextracts were filtered and analysed by HPLC without furtherprocessing. Fluorescence detection at 310 nm followingexcitation at 280 nm provided selective and sensitive detection
of (+)-catechin and (2)-epicatechin whereas other phenolicswere detected by their ultraviolet absorption at 270 nm. Thetype (ethanol, methanol or acetone) and concentration(40–100% in water) of extraction solvent influenced flavanolyield, whereas extraction time (10–60 min) did not. Adequateextraction was attained with 60–100% methanol for apples andgrapes but recovery decreased to ca. 70% of maximum valuewhen the percentage of methanol in the extractant was reducedto 40%. A plausible explanation of this behaviour is thereduction by methanol of the activity of polyphenol oxidases,which are widely distributed in plants. This suggests thatextraction with low methanol solvents may not completelyinactivate enzyme activity, resulting in reduced phenol yields.Recovery of spiked flavanols ranged from 92 to 105%.
Olive contains several distinctive phenolics such as verbasco-side, ligstroside and oleuropein. These were recovered154 frommethanolic extracts of olive fruit by partitioning into ethylacetate using bioguided fractionation. Such methods are notcommon and the extraction procedure developed by Amiotet al.155 has been widely adopted for the isolation of phenoliccompounds from the fruit.51,53,54,156,157 The details differ butsample preparation has generally entailed extraction withaqueous ethanol in the presence of metabisulfite of freeze-driedolives powdered with the aid of liquid nitrogen. The extractswere concentrated under reduced pressure, acidified (in someinstances) and washed with hexane to remove lipophiliccompounds.155 The phenolic compounds were partitioned intoethyl acetate52 in the presence of ammonium sulfite, metaphos-phoric acids and methanol.53,54,157 Alternatively, the extractshave been further processed on a diatomaceous earth Extrelutcartridge,156 which was sequentially eluted with hexane, ethylacetate (non-anthocyanic phenols) and acidic methanol (antho-cyanins). Several compounds present in trace amounts werefurther fractionated156 by silica phase centrifuge TLC.
Vlahov158 adopted a simpler approach for flavonoid analysis,in which olive pulp was extracted with aqueous methanol. Thecombined extracts were evaporated to dryness, reconstituted inglacial acetic acid and water followed by centrifugation andfiltration. Bianchi and Pozzi159 have recovered simple phenolicsubstances with the basic skeleton C6–C1, C6–C2 and C6–C3
from olives by homogenizing with water in a blender. Thehomogenate was evaporated to dryness under reduced pressure,the residue dissolved in water and the solution partitioned intoethyl acetate to retrieve the phenolic substances. Extracts havetypically been analysed by HPLC.
Servili et al.140 found higher recoveries of phenolic com-pounds from olive drupes by SPE than liquid–liquid extraction.The recovery of the dialdehydic form of elenolic acid linked to3,4-(dihydroxyphenyl)ethanol and an isomer of oleuropeinaglycon, however, was low. The same group160 developed acomprehensive scheme for the extraction of phenolic com-pounds from olive pulp that introduced several precautionsaimed at inhibiting enzyme activity and hence phenolicmodification or destruction. Olives were peeled and destoned,and the olive pulp was placed in liquid nitrogen and subse-quently freeze-dried. The freeze-dried material was stored at230 °C prior to analysis. Phenolic compounds were recoveredfrom the olive matrix by extraction with aqueous methanolcontaining sodium diethyldithiocarbamate. This mixture washomogenized for 30 s and filtered using a Buchner funnel. Themethanolic extracts were evaporated under vacuum and ni-trogen flow at 35 °C and purified by SPE using a high-load C18
cartridge, the phenolic compounds being eluted with methanol.The need to inhibit enzymatic activity was also recognized byBianco et al.,161 who extracted phenolic compounds from greenolive fruits by refluxing in boiling methanol for 30 min. Theaqueous extract following removal of methanol was ex-haustively extracted with ethyl acetate and purified usingreversed-phase TLC. Extraction with boiling ethanol (5 min)followed by aqueous ethanol (1 h) has also been applied162 and
the authors noted that boiling inactivated enzymes and aided inphenol recovery. Phenols in the filtered ethanolic extract werequantified by ultraviolet derivative spectroscopy.
2.4 Peel and seed
The determination of phenols in peel and seed has assumedincreasing importance with the recognition that these fruit partsare often a source of unique phenols or compounds in muchhigher concentration than in the flesh. Free phenolics wereextracted from finely ground citrus peel or seed by refluxing inmethanol.18 After filtration, the methanolic extract was washedwith light petroleum and evaporated to dryness under vacuum.In contrast, bound phenolics were recovered after alkalinehydrolysis (4 h) at room temperature and under nitrogen. Theaqueous phase was separated by filtration, extracted with ethylacetate and evaporated as before. The dried residues from eitherprocess were dissolved in dimethylformamide for analysis. Thefree phenolics were predominantly flavanone glycosides,glycosylated flavones and polymethoxylated flavones whilebound phenolics comprised largely phenolic acids (caffeic, p-coumaric, ferulic and sinapinic acids), with evidence for theexistence of flavonols bound to cell walls.
Extraction with aqueous ethanol has been used to recoverphenolic compounds from grape seeds and skins. The methodsdiffer in the use of fresh163 or freeze-dried seeds164,165 and in theaddition of metabisulfite as an antioxidant to the extrac-tant.164,165 Lipids and chlorophyll are eliminated from theextracts by partitioning into chloroform and the extracts may beanalysed directly or further processed164 by partitioning of thephenolic compounds into ethyl acetate prior to analysis. Therecovered phenolic compounds were mainly condensed tanninsand anthocyanins from seeds and skin, respectively.
Proanthocyanidins or condensed tannins are oligomeric andpolymeric flavan-3-ols based on various constitutive units. Amethod has been devised166 that fractionates grape seed or skinproanthocyanidins according to their degree of polymerization.Seeds and skins were recovered from commercially maturegrape berries. Seeds were ground under liquid nitrogen andextracted with aqueous acetone whereas skins were washedwith methanol to remove organic acids and low molecular massphenols before solvent extraction. After a preliminary clean-upby column chromatography, the proanthocyanidins were pre-cipitated by chloroform–methanol on an inert glass powdercolumn and recovered by stepwise gradient elution withincreasing proportions of methanol in the solvent. Alternatively,fractionation has been achieved by gel permeation chromatog-raphy164 and elution with methanol or methanol–acetic acid. deGaulejac et al.165 provide an interesting comparison of lowpressure chromatograms of seed and wine extracts. The latterwas enriched in simple phenolic compounds such as p-coumaric, gallic and caffeic acids whereas the predominantphenolic compounds in the seed extract were flavanols andcondensed flavanols.
A number of unusual phenolic compounds have beenidentified in olive seeds. Maestro-Durán et al.167 claimed thatsalidroside is present in olive seeds whereas nuzhenide wasisolated by Servili et al.160 The latter represents one of the firstdedicated efforts at the characterization of the phenolic contentof the complete olive fruit, in that peel, pulp and seeds wereanalysed in three Italian olive cultivars. Nuzhenide was detectedexclusively in the olive seeds of all three varieties and at allstages of maturation. Similarly, luteolin-7-glucoside and rutinwere detected only in olive peel, whereas verbascoside,oleuropein and demethyloleuropein were found in all three olivematrices. The concentration of the last two phenolics wasgreatest in olive pulp.
The olive pomace obtained from olive fruit processingcontains seed husk and a small amount of seeds, pulp and peel
which can be separated by industrial methods. Steam explosionhas been examined as a pre-treatment process to increase theavailability of the main components of lignocellulosic biomass.During steam explosion, lignin is partly depolymerized givingrise to water-soluble phenolic compounds, which have beenidentified168 as vanillic acid, syringic acid, vanillin andsyringaldehyde plus tyrosol and hydroxytyrosol. The resultssuggest the presence of hydroxytyrosol as a structural compo-nent of the olive stone.
2.5 Leaf
Interest in the phenolic content of plants has recently shifted toinclude portions of the plant other than the fruit. The leaves haveattracted particular attention and the phenolic profile of manymedicinal plants has been studied. For instance, the amounts ofboth free and bound phenolic acids were determined in Ginkgobiloba L. leaves169 using a special extraction procedure,comprising acid and alkaline hydrolyses. Ferulic acid and p-coumaric acid in 14 forbs were fractionated170 after methanolextraction into four fractions: free phenolic acids extracted intodiethyl ether, ester-bound phenolic acids after alkaline hydroly-sis, glycoside-bound phenolic acids after acid hydrolysis andcell wall-bound phenolic acids after alkaline hydrolysis of thesolid residue remaining from the extraction with methanol. Thecell wall-bound phenols were quantitatively the most importantfraction. Extraction, alkaline and acid hydrolysis have beencombined with purification on a C18 cartridge171 to determineflavonoids, phenolic acids and coumarins in seven medicinalspecies. SPE has also been used172 to isolate phenols from leaftissue of Myrtus communis L. The leaf tissue contained smallamounts of phenolic acids (caffeic, ellagic and gallic acids) andquercetin derivatives (quercetin 3-O-galactoside and quercetin3-O-rhamnoside), whereas catechin and myricetin derivativeswere present in large amounts.
The isolation and identification of phenolics in olive leafhave also attracted considerable attention as a source ofphenolic compounds.173 Moreover, the leaf is the primary siteof plant metabolism at the level of both primary and secondaryplant products. In an early report, Gariboldi et al.174 maceratedfresh leaves in methanol for 1 week at room temperature. Thesolvent was evaporated under nitrogen and the extract reconsti-tuted in aqueous acetone and successively extracted withpentane, chloroform and ethyl acetate. The chloroform extractwas fractionated by column chromatography to yield twosecoiridoids. Three flavonoid glycosides, quercitrin, rutin andluteolin-7-glycoside, one flavonoid aglycone, luteolin, andchlorogenic acid were identified175 in olive leaves followingextraction (24 h) with aqueous methanol or ethanol to recoverflavonoids and flavonoid glycosides or biflavonoids, re-spectively.
Alcoholic extraction (methanol or ethanol) of fresh foliage orfreeze-dried material has been the usual approach to therecovery of phenols from olive leaf. The extract was concen-trated176 in a vacuum under a stream of nitrogen, keeping thetemperature below 35 °C until it reached a syrupy consistencyand partitioned in acetonitrile–hexane. Evaporation to drynessafforded a yellowish foam that dissolved in methanol. Com-pound identification was achieved using atmospheric pressureionization tandem mass spectrometry. Akillioglu and Tanris-ever177 used TLC to examine the phenolic profile of oliveshoots in two olive cultivars. Central leaves and axillary buds ofthe shoots were studied. Samples were dried (method notspecified) and phenols recovered by extraction with aqueousethanol (96%). The phenolic compositions of the leaves andbuds were found to be different, and of the total 59 compoundsidentified in the extracts, 30 were specific to leaves, 24 to budsand the remaining five were common to both organs. Suchdifferences indicate that the leaves and buds exhibit distinct
metabolic functions. A large number of leaf phenolics werefound to be phenylpropanoids, which are known to beprecursors in the lignin biosynthetic pathway, and act as eitherpromoters or inhibitors of olive growth.
Supercritical fluid extraction (SFE)178,179 has a number ofadvantages and has been used in a two-step fractionation ofleaves of rosemary and sage into an essential oil and antioxidantfraction. Phenols have also been isolated from dried (100 °C),ground and sieved (@500 mm) olive leaf using supercriticalcarbon dioxide modified with methanol.180 The influence ofextraction variables such as modifier content, pressure, tem-perature, flow rate, extraction time and collection/elutionconditions was studied. The dynamic SFE method producedclean extracts with higher phenol recoveries (measured as totalphenols by Folin–Ciocalteu method) than sonication in liquidsolvents such as hexane, ethoxyethane and ethyl acetate.However, the extraction yield obtained was only 45% of thatobtained with liquid methanol. The extracts were screened foracid compounds such as carboxylic acids and phenols usingelectrospray ionization mass spectrometry (ESI-MS) in thenegative ionization mode.
The phenolic content of olive leaf hairs has also beeninvestigated, and the role of these hairs in plant protection isdiverse. Spectrophotometric analysis of methanolic extracts ofolive leaf hairs indicated the presence of UV-screeningpigments, which have been characterized as phenolics with aconsiderable flavonoid contribution.181 Flavonoids includingluteolin, apigenin and quercetin in their glucoside and aglyconeforms were detected and it is believed that such compounds playan important role in UV-B radiation shielding propertiesexhibited by olive leaf hairs. Further investigation has shownthat the UV-B radiation absorptive capacity and the phenoliccontent of leaf hairs declines considerably with leaf age.182 Thehigh UV-B absorptive capacity of the hairs of young leaves thusindicates a metabolic priority for flavonoid production duringthe early phases of leaf development. The number of leaf hairswas also found to decrease with maturation. Young leaves maytherefore be more prone to damage by UV-B radiation, hencethe greater number of leaf hairs for protection.182
3 Quantification
The need for analyte recovery must be considered in the contextof the quantification procedure as it is ultimately related to thelimited specificity and sensitivity of analytical procedures(Table 2). Quantification is used here in the broadest sense toinclude methods where characterization or identification wasthe primary goal.133,224–226 In such instances, measurement ofan amount of substance is often precluded by the number anddiversity of phenolic compounds (and corresponding lack ofreference compounds). Quantification procedures227 are univer-sally applicable to phenolic extracts regardless of species or partof the plant.
Traditional methods for the determination of total phenolshave relied on direct measurement of absorption of radiation inthe ultraviolet or, more commonly, colorimetric methods usingFolin–Ciocalteau reagent. This reagent, however, is not specificfor phenols and hence other compounds may interfere.228
Moreover, the diversity of phenolics means that selection of areagent and/or absorbing wavelength will be a compromise,although this is less of a problem where a single class ofphenolic predominates. Results are expressed in terms of molarequivalents of a commonly occurring phenolic, e.g., gallic orcaffeic acid.229
There is generally no correlation104 between data for totalphenols and those obtained by chromatographic techniques,although the results obtained by colorimetry are usually higherthan the latter. The number and diversity of phenols in a typical
extract mandates a high resolution technique for their separationand identification. Hence traditional methods based on colori-metry have been replaced in many instances by high resolutionchromatographic analyses to provide profiles and identificationof individual phenolics. Akillioglu and Tanrisever177 used TLCto characterize the phenolic profile of olive buds and leaves intwo different cultivars. Sample extracts were separated bycellulose TLC using two-dimensional development with aque-ous butanol containing acetic acid followed by aqueous aceticacid. Phenolics were characterized by RF values and theirfluorescent colours under UV radiation and the variation incolour when treated with ammonia fumes and Naturstoffreagent (1% ethanolic solution of diphenylboric acid B-aminoethyl ester) under both UV radiation and daylight.
Polymethoxylated flavones possess the stability and vola-tility that makes GC a viable alternative for their analysis. Forthis purpose, packed columns are unsuitable,230 whereas highefficiency open tubular columns are ideal,61,132,231 producingexcellent separations of the flavones extracted from orange peeloil. Newer stationary phases232 offer improved retention andselectivity for these compounds but their main advantage is thelow stationary phase bleed that permits operation at elevatedtemperatures with minimum interference in the detectionprocess. This greatly facilitates the use of coupled GC-MS.
For other phenols a derivatization step prior to GC isgenerally mandatory. Nevertheless, the excellent resolvingpower and detection capabilities of GC and particularly GC-MShave been exploited for the analysis of phenolic acids and otherphenols159,196 as trimethylsilyl or trifluoroacetate derivatives(Table 2). Angerosa et al.120 showed GC-MS to be an effectivetool for identification of phenols as their trimethylsilyl deriva-tives following extraction from olive oil with methanol. Soleaset al.10 also used this derivatizing agent for the analysis of 15biologically active components in wine by GC-MS using onetarget and two qualifying ions for each compound. Ions werechosen for each compound on the basis of their abundance,reproducibility, freedom from interference and specificity to theparticular compound. The molecular ion (M+) was preferredwhen found in appreciable abundance. Resolution of all 15phenolic compounds was excellent and the method should beappropriate for the determination of phenolics in a range offruits.
Other methods have been reported233 but have not foundgeneral acceptance. For instance, capillary zone electrophoresis(CZE) and micellar electrokinetic capillary chromatographyhave been used to separate phenolic compounds (ref. 101 andreferences cited therein). The majority of these separations usedbuffers at pH 8.0–10.5 that are suitable for the majority ofphenols with pKa values between 8 and 10 but are unsuitable forpH-sensitive anthocyanins. Anthocyanins were measured inblackcurrant juice101 by CZE under strongly acidic conditionsfavouring the red-coloured flavylium cationic form. Underthese conditions, the anthocyanins were selectively detected bytheir absorbance at 520 nm.
In contrast, reversed-phase HPLC avoids the need forderivatization and has invariably been the method of choice(Table 2). Isocratic elution has been used in some instances115
but the procedures invariably rely on gradient elution owing tothe diversity of phenols in most extracts. Typical mobile phasesinclude methanol, water and acetic acid combinations that areused in gradient elution techniques.8,9,116,122 Detection ofphenolics by HPLC is based on measurement of absorption ofradiation in the UV or visible region (anthocyanins). The mostcommon wavelength for general detection has been280 nm,116,122,234 although other wavelengths have been usedfor the identification of specific phenolics.54,155,158 For exam-ple, phenols have been quantified at characteristic wavelengthsas cinnamic acids (320 nm), flavonols (360 nm) and otherphenols (280 nm).82 The paucity of reference compoundscreates difficulties in quantification that have been solved by
Grape juice Filtration and direct injectionexcept for procyanidinswhere isolation onSephadex LH-20 column
Colorimetry; HPLC-DAD,280 and 320 nm
Total phenols 99 and 380 mg L21 (by HPLC andcolorimetry, respectively).
Phenolic acids (trans-isomers) and flavonol glycosides(oxidation of caftaric acid to 2-S-glutathionylcaftaricacid was evident) (enzymatic clarification causedhydrolysis of caftaric, coutaric, and quercetinderivatives)
104
Grape Series of liquid–liquid andliquid–solid extractions
LC-MS; HPLC-DAD Identification of anthocyanins (no quantitative data) 184
Wine Dilution and SPE GC-MS of TMS derivatives Quantitative data for phenolic acids: gentisic, vanillic,ferulic, m-coumaric, p-coumaric, caffeic, and gallicacid; flavonoids: catechin, epicatechin, quercetin andmorin
192
Wine SPE on C18 GC-MS of TMS derivatives Hydroxytyrosol 2–4 mg L21. DL 15 pg mL21 31Wine and fruit
juiceHydrolysis in acidic methanol HPLC-DAD Quercetin: wine < 0.5–16 mg L21; fruit juice 2.5–13
Total phenols 829 and 416 mg kg21 (dry mass) forstrawberry and blackcurrant, respectively. Data forflavonoids (kaempferol, quercetin, myricetin) andphenolic acids (p-coumaric, caffeic, ferulic,p-hydroxybenzoic, gallic and ellagic acids). DL2–5 ng
152,199
Blueberries Aqueous acidic methanolextraction and filtration
HPLC-DAD; GC ofanthocyanidins as TMSderivatives
Total anthocyanins 1100–2600 mg kg21 (non-acylatedglucosides and galactosides of delphinidin, cyanidin,petunidin, peonidin, and malvidin); chlorogenic acid500–1000 mg kg21 (fresh mass)
97
Cider apples Hammer mill, pressed,clarified and frozen.Addition of ascorbic acidand filtration
HPLC-DAD, 280 nm Total phenols 1.2 g L21 as tannic acid; main phenols,chlorogenic acid 130 mg L21; (2)-epicatechin 50mg L21; procyanidin B2 40 mg L21
200
Berries Hydrolysis in acidifiedaqueous methanolcontaining TBHQ
Capillary zone electrophoresis Anthocyanins. DL 25 mg L21 101
Berry and fruitwines andliquors
Dealcoholized (wines) andSPE to remove sugars
Colorimetry Total phenols 91–1820 mg L21 GAE 81
Raspberry juice SPE, acid and base hydrolysis HPLC (see ref. 104) Data for several phenols as percentage of total peak area 38Raspberry juice Fractionation on Polyamide 6
essential; conventionalsystems failed to produceclean separations
HPLC, various systems, 260nm (ellagic acid), 360 nm(flavonols)
Quercetin 30–210 mg L21; kaempferol 2–6 mg L21
(data quoted for various glycosides)202
Strawberry Acetone extraction due tohigh pectin content, SPE
Quantitative data for various tissues forhydroxycinnamic acid derivatives, flavan-3-ols,flavonols, and dihydrochalcones. Total phenols(summation) (whole apple) 5000 mg kg21
Homogenized in aqueousmethanol, dried andextracted with ethyl acetate
HPLC-DAD, 210–360 nm Quantitative data for cinnamic acids and theirderivatives, flavan-3-ols, procyanidins, flavonols anddihydrochalcones
20
Peach Full text not available Spectrophotometry and HPLC Phenolic acids (quinic was the predominant phenolicacid, followed by gentisic, catechuic, chlorogenic andsyringic acids)
Anthocyanins Acid hydrolysis in methanol HPLC-DAD Formation of methyl esters following release of acids 149Sweet cherry Extraction with acidic aqueous
methanolHPLC, 280 and 525 nm (and
GC)Total anthocyanins 20–3000 mg kg21 fresh mass
(3-rutinoside and 3-glucoside of cyanidin as themajor anthocyanins and the same glycosides ofpeonidin as minor anthocyanins), neochlorogenic acid240–1280 mg kg21 and p-coumaroylquinic acid230–1310 mg kg21
13
Orange andgrapefruit
Aqueous alcohol extractionfollowed by alkalinehydrolysis
HPLC, 300 nm Bound and free sinapic, ferulic, coumaric and caffeicacids
42
Sour orange Aqueous ethanol extraction LC-DAD-MS Identification of various flavonoids (no quantitativedata)
147
Grapefruit andpummelo
Extraction of dried materialwith dimethyl sulfoxide
‘normalization’,38,45 synthesis or isolation from the sample156
of the relevant phenols or the use of a phenol belonging to thesame class.185,212 For instance, malvidin-3-glucoside wasused185 as the reference compound for the HPLC quantificationof five anthocyanins in grape skins while phloretin glycosideswere quantified20 as phloridzin in apple and peach products.Similarly, a single reference compound for each phenolic classwas used212 to quantify hydroxycinnamic acids, flavanols andflavonols in pear by HPLC. In qualitative studies where profilesare compared, there is often no allowance for variation indetector response in presenting peak area data. It is notuncommon that the largest peak is assumed to represent thephenol with the highest concentration. However, the molarabsorptivities vary greatly between phenols (Table 3) and thereis a need for greater recognition of this variation. The effect of
detection wavelength on the chromatographic profile of oliveleaf phenols is illustrated in Fig. 3.
Flavan-3-ols are of particular interest in beverages wherethey are often the cause of instability and turbidity. Theirdetermination by HPLC with UV detection is prone tointerference by other phenols present at higher concentrations.The use of a post-column reactor has been employed217 inwhich p-dimethylaminocinnamaldehyde condenses with flava-nols giving intensely coloured adducts showing maximumabsorption between 632 and 640 nm. The reagent shows bothhigh specificity and sensitivity for flavanols. Fluorescence72,148
also provides selective and sensitive detection but it has rarelybeen used.
GC and HPLC have been compared235 for the determinationof resveratrol in wines. Higher results were obtained by GC
Table 2 Continued
Sample Extraction Quantification Levels of phenols identified; detection limits Ref.
Orange juice Squeeze and filtration Colorimetry, Folin–Ciocalteu;HPLC, 300 nm
Total phenols: 360–1200 mg L21. Total anthocyanins:1–280 mg L21
216
Kiwifruit juice SPE C18 HPLC Phenolics present at 1–7 mg L21 (as derivatives ofcoumaric, chlorogenic and protocatechuic acid and aderivative of 3,4-dihydroxybenzoic acid); epicatechin,catechin, and procyanidins (B3, B2, or B4 andoligomers). Flavonols as glycosides of quercetin andkaempferol
68
Red wine, beer,apple cider,and sourcherry andblackthornfruit liqueurs
Filtration HPLC, UV at 280 nm andpost-column reactor withabsorption at 640 nm
Flavanol profiles 217
Fruit Centrifugation and dilution HPLC, coulometric arraydetector
Data presented for phenolic acids and flavonoids asnumber of peaks in chromatogram and total peakarea. DL 0.02–1 ng
218
Areca fruit Aqueous acetone extraction Total phenols: colorimetry at735 nm by Folin–Ciocalteumethod. Condensed tannins:colorimetry at 500 nm withvanillin–HCl
Total phenols 580 mg kg21 GAE; condensed tannin0.85 mg of catechin equiv. g21 (fresh mass)
219
Fruit jams Acidified aqueous methanolextraction, SPE
HPLC, 520 nm Quantitative data for anthocyanins in several jamvarieties
220
Fruit juices andwines
Mixture of standards tosimulate juice
Capillary electrophoresis Kaempferol-3-rutinoside, rutin, avicularin, quercitrin,isoquercitrin, isorhamnetin, kaempferol and quercetin
221
Grape must,apple andpeach
None HPLC-DAD Hydroxycinnamic acids 222
Cherry laurel Light petroleum extraction ofpowdered mesocarp,followed by alkalinehydrolysis of residue undernitrogen and ethyl acetaterecovery
GC-MS of TMS derivatives Data reported as percentages of extract for vanillic,protocatechuic, p-hydroxybenzoic, caffeic andp-coumaric acids
GC-MS of TMS derivatives E.g., Gallic, salicylic, vanillic, p-coumaric and syringicacids. Typical levels 300–54 000 mg kg21
223
a DAD, diode array detection. b GAE, gallic acid equivalents. c TMS, trimethylsilyl. d SPE, solid phase extraction. e DL, detection limit. f TIC, total ioncurrent. g SIM, selected ion monitoring.
(0.64–3.00 mg L21) than by HPLC (0.30–2.89 mg L21)analysis. The GC analysis separated the cis- and trans-isomersbut with longer sample preparation times than required in HPLCanalysis. The cis+trans ratio ranged between 0.5 and 0.9 andtrans-resveratrol underwent a photochemical isomerizationduring ripening of the grape berries and also during the processof winemaking.
Classical ionization techniques have limited application forthe analysis of underivatized plant phenols and will not bediscussed. In contrast, the development of soft ionizationtechniques, such as atmospheric pressure ionization (API), forthe investigation of polar, non-volatile and thermolabilemolecules has facilitated the analysis of phenolic compounds byLC-MS. At this stage, most applications (see Table 2) haveinvolved qualitative analyses but LC-API-MS can be expectedto revolutionize quantitative determinations as the funda-mentals of the technique are more firmly established. Atmos-pheric pressure chemical ionization (APCI) uses a combinationof a heated capillary and a corona discharge to promote theformation of ions from the nebulized sample. In coupled mode,the eluate from the HPLC system is evaporated completely andthe mixture of solvent and sample vapour is then ionized in thegas phase by ion–molecule reactions and follows the sequencesample in solution ? sample vapour ? sample ions. Ionformation is via chemical ionization involving proton transfer,adduct formation and charge exchange reactions in positive ionmode or proton abstraction, anion attachment and electroncapture reactions in the negative ion mode.
Aramendía et al.195 reported the LC-API-MS of phenolicsfound in olive mill wastewater. Analytes were separated on aC18 phase by gradient elution with methanol–water containingformic acid. Mass spectral conditions were optimized by directinfusion of standards in the flow injection mode into the APCIsource. The study was restricted to the negative ion mode withdetection limits (Table 4) in the total ion current mode rangingfrom 0.5 to 500 ng. These detection limits were about 20 timesbetter when working in the selected ion monitoring mode andmonitoring the [M 2 H]2 ion. Mass spectra were recorded withsoft (215 V) and strong (250 V) voltages applied at the ionsource of the mass spectrometer. With the lower voltages,deprotonated molecular species [M 2 H]2 were the major ionsobserved in the mass spectra with the appearance of very fewfragment ions that were all of low intensity. The presence ofsubstantial fragmentation from collisionally induced dissocia-tion processes which became evident on increasing the voltageapplied at the source (extraction and cone) voltages gavestructural information about the molecules. Structures wereassigned to major eluent cluster ions from methanol–water–
APCI still has the major drawback for polar thermolabileplant phenols that volatilization of the sample must occur beforeionization. ESI overcomes lack of analyte volatility by directformation or emission of ions from the surface of a condensedphase and sample ions are collected from the condensed phaseinside the ion source and transferred to the mass analyzer.Hence ESI eliminates the need for neutral molecule volatiliza-tion prior to ionization. ESI is used as a generic term that alsocovers several variants of the basic technique that differ in theprecise manner in which charged droplets of sample areproduced. The mechanism of ESI remains controversial but inthe meantime these techniques collectively have revolutionizedthe field of mass spectrometry and its application to analysis ofplant phenols as seen in Table 2.
ESI spectra of phenols typically show a pseudomolecular ion([M + H]+ or [M 2 H]2)236 with minimum fragmentationalthough fragmentation can often be induced by raising the conevoltage. Acid (acetic or formic) is often added to mobile phasesin positive ion ESI as a source of protons to assist ionization.Sensitivity is improved when the organic content in the mobilephase exceeds 20%. LC-ESI-MS has been used236 to studyflavonol aglycones and glycosides in berries. ESI providedinformation on the structures without the need for derivatiza-tion. Quercetin was identified in all berries. Our work showsthat the negative ion mode generally provides improveddetection limits but there is a need to establish optimumconditions for each phenol.
4 Future needs—transfer to industry
Current research on fruit phenolics is driven by three majorforces and these impact on the choice of sample preparation andthe importance attached to this step in the overall analysis. Oneis simply to discover new compounds, and as diverse andnumerous as they are now, there is seemingly endless scope forisolating and identifying new and novel compounds as thesensitivity of analytical techniques is improved. A second areaconcerns the understanding of the role of phenolics assecondary metabolites within the plant. Third, there is interest intheir antioxidant properties, as alternatives to synthetic anti-oxidants in the food industry, and as components of the humandiet. In all three areas, sample preparation is rarely criticalalthough quantitative extraction will enhance the amount ofphenolic available. Of the three areas, only the last has links toindustry, yet even here quantification of naturally occurringphenolics is not routinely undertaken, in sharp contrast to thesituation with the synthetic antioxidant phenolics.
Macronutrients such as carbohydrates, proteins, and fattyacids are regularly quantified because they directly impact onthe quality of a finished product. As micronutrients, measure-ment of phenolics is not seen as important in processing (this isdespite the fact that in many cases food spoilage is linked tophenolic ‘browning’ reactions). However, the current trendtowards consumer awareness of, and demand for, foods withbeneficial health properties (so-called ‘functional foods’) maysee the quantification of phenolics become increasingly im-portant.
Adulteration represents another area of the food industrywhere quantification of phenolic compounds has potential.Recently Valentão et al.237 reported on the determination ofVervain flavonoids in the context of quality control. Quantifica-tion was carried out with reference to standards and the authorssuggested that because quantification of the flavonoids waspossible it may be applied to quality control. Adulteration ofcitrus juices is another area where phenolic quantification may
Table 3 Absorption characteristics of selected phenols79,116,152
be undertaken; however, while the potential is there, it is yet tocome into routine use. As the demand for routine quantificationof phenolics is realized, the emphasis placed on sample
preparation will increase. In the meantime, research studies onphenols would be well advised to examine this aspect moreclosely.
Fig. 3 HPLC of phenols extracted from leaves of the Manzanillo olive tree showing the effect of detection method on the resulting profile. Samples wereobtained by aqueous methanol (1 + 1) extraction of freeze-dried material. The extract was washed with hexane and injected on to a C18 column using anaqueous methanol gradient for elution.
The financial support of Rural Industries and HorticulturalResearch and Development Corporations, Australia, is grate-fully acknowledged. The assistance of Professor Shimon Lavee,Israel, in providing the olive leaf sample for Fig. 3 is noted.
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