Sample preparation in the determination of phenolic
compounds 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 2000
Published on the Web 17th April 2000
1
2
2.1
2.2
2.2.1
2.2.2
2.2.3
2.3
2.4
2.5
3
4
5
6
Introduction
Sample preparation
Hydrolysis
Fruit extracts
Juices and related products
Olive oil
By-products
Fruit
Peel and seed
Leaf
Quantification
Future needs—transfer to industry
Acknowledgements
References
1 Introduction
Phenolic compounds occur as secondary metabolites in all
plants.1 They embrace a considerable range of substances
possessing an aromatic ring bearing one or more hydroxy
substituents, although a more precise definition is based on
metabolic origin as those substances derived from the shikimate
pathway and phenylpropanoid metabolism.2 A convenient
classification of the plant phenols distinguishes the number of
constitutive carbon atoms in conjunction with the structure of
the basic phenolic skeleton (Table 1). The range of known
phenolics is thus vast and also includes polymeric lignins and
condensed tannins.
Some plant phenols may be involved in primary metabolism
whereas others have an effect on plant growth or protect the
more vulnerable cell constituents against photooxidation by
ultraviolet light by virtue of their strong UV absorption.3 Plant
phenols also play an important role in disease resistance in the
plant. Intense interest in fruit phenolics is also related to their
physiological activity which depends on their antioxidant
Kevin Robards is Associate
Professor of Chemistry at
Charles Sturt University Riverina. He obtained his PhD in
analytical chemistry from the
University of New South Wales
in 1979. His research interests
are focused on the application
of analytical chemistry to food
science and in particular the
identification and role of naturally occurring phenolic compounds in fruits.
DOI: 10.1039/b000080i
activity, the ability to scavenge both active oxygen species and
electrophiles, the ability to inhibit nitrosation and to chelate
metal ions, the potential for autooxidation and the capability to
modulate certain cellular enzyme activities.4–7 Thus, knowledge of the levels of these compounds in plants is of
considerable interest but is limited by problems of analysis. The
structural diversity of the phenolics and its effect on physicochemical behaviour such as solubility and analyte recovery
presents a challenging analytical problem. Moreover, a number
of phenolic compounds are easily hydrolysed and many are
relatively easily oxidized, which further complicates sample
handling.8,9
This review emphasises the importance of sample preparation in the determination of phenolic compounds in plant
materials particularly fruits. Fruits are an important dietary
source of phenolic substances although interest is also shifting
to other parts of the plant as potential commercial sources of
phenols. Sample preparation is a critical step in analysis and this
is even more significant with real samples where the matrix
components are biologically active and the analytes represent a
diverse spectrum of numerous compounds, many having an
unknown identity. Thus, methods of extraction of phenols from
fruits are generally dependent on several factors while the usual
quantification procedures involve the separation sciences and
are universally applicable. Soleas et al.10 illustrated this point.
They developed a derivatization procedure for determination of
15 phenolic constituents in solid vitaceous plant materials and
concluded that the method ‘should be suitable to measure
polyphenols in fruit, vegetables, and other foods provided that
efficient extraction techniques are employed’. Such statements
are seen frequently in the analytical literature but they tend to
belittle the importance of this step (or perhaps they serve to
underline its critical importance). Rhodes and Price11 observed
that the determination of phenolic species in foods is an
important outstanding problem and reviewed methods for the
extraction and purification of phenolic antioxidants as the
conjugated forms that exist in plant foods.
Knowledge of the extraction of phenolics is also desirable
outside the analytical context for it has important practical
applications in the food industry. For instance, the mechanism
and kinetics of phenolic extraction from wood to wine during
ageing in barrels12 has significant consequences for the
production of quality wines.
2 Sample preparation
Isolation of phenolic compounds from the sample matrix is
generally a prerequisite to any comprehensive analysis scheme.
The ultimate goal is the preparation of a sample extract
uniformly enriched in all components of interest and free from
interfering matrix components. It encompasses a series of steps
Analyst, 2000, 125, 989–1009
This journal is © The Royal Society of Chemistry 2000
989
Table 1 Classification of phenolic compounds with characteristic examples in various fruit.
Basic skeleton
Class
C6
Simple phenols
Benzoquinones
Phenolic acids
Phenylacetic acids
Cinnamic acids
Phenylpropenes
Coumarins
Chromones
Naphthoquinones
Xanthones
Stilbenes
Anthraquinones
Flavonoids
Flavones
C6–C1
C6–C2
C6–C3
C6–C4
C6–C1–C6
C6–C2–C6
C6–C3–C6
Flavonols
Flavonol glycosides
Flavanonols
Flavanones
Flavanone glycosides
Anthocyanins
Common fruit source
Catechol, hydroquinone, resorcinol
Widely distributed
Chalcones
(C6–C3) 2
(C6–C3–C6)2
Citrus
Walnut
Mango
Grape
Citrus
Apple
Pear
Widely distributed
Grape
Usually found in citrus
Tomato
Citrus
Strawberry
Apple
Sweet orange
Pear
Cherry
Peach
Plum
Sweet cherry
Apple
Grape
Pear
Peach
Apple
Pear
Tomato
Lignins
Biflavonoids
ranging from exhaustive solvent extraction and preconcentration procedures to simple liquid–liquid extraction or filtration.
Extraction of the phenolics from the matrix has been a necessary
prerequisite to quantification although enhanced selectivity in
the latter may reduce the need for sample manipulation. This is
not always desirable as, for example, in gas chromatography–
mass spectrometry (GC–MS) where the effects of non-volatile
matrix components on column lifetime are an important
consideration.
The task of recovery is complicated as ‘fruit’ constitutes a
natural matrix with a high enzyme activity, and hence extreme
care must be taken to ensure correct extraction, devoid of
chemical modification, which will invariably result in artefacts.1 Artefactual changes, for example, oxidation and isomerization,13 during the extraction process are a constant
concern. An example is the photochemical isomerization of
trans-resveratrol to the cis isomer.14 Methods of protecting the
compounds from these deteriorative processes have included
the addition of antioxidants (one presumes of higher ‘activity’
than the compounds themselves) during the extraction and the
use of inert atmospheres. The fidelity between the phenolic
profile of the starting material and that of the isolated extract
provides the theoretical basis for judging analytical techniques.
Hence the conditions employed should be as mild as possible to
avoid oxidation, thermal degradation and other chemical and
biochemical changes in the sample.
The precise procedure will depend on the nature of both the
analyte (e.g., total phenols, o-diphenols versus other phenols,
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Analyst, 2000, 125, 989–1009
p-Hydroxybenzoic acid, salicylic acid
p-Hydroxyphenylacetic acid
Caffeic acid, ferulic acid
Eugenol, myristicin
Umbelliferone, aesculetin, scopolin
Eugenin
Juglone
Mangostin, mangiferin
Resveratrol
Emodin
Widely distributed
Grape
Flavanols (catechins)
Examples
Sinensetin, nobiletin, tangeretin, isosinensitin, various
polymethoxylated flavones
Quercetin, kaempferol
Quercetin, kaempferol
Rutin
Dihydroquercetin and dihydrokaempferol glycosides
Hesperitin, naringenin
Naringenin
Hesperidin, neohesperidin, narirutin, naringin, eriocitrin
Naringin
Cyanidin glycosides including acylated derivatives
Glycosides of pelargonidin, peonidin, delphinidin,
petunidin
Glycosides of cyanidin, peonidin, delphinidin, petunidin,
malvidin including acylated forms
Cyanidin glycosides
Cyanidin 3-glucoside and 3-rutinoside
Cyanidin glycosides
Glycosides of cyanidin, peonidin
Cyanidin glycosides
(+)-Catechin, (2)-epicatechin
(+)-Catechin, (2)-epicatechin, (+)-gallocatechin,
(2)-epigallocatechin
(+)-Catechin, (2)-epicatechin
(+)-Catechin, (2)-epicatechin
Phloretin derivatives, notably phloridzin
Arbutin, phloretin glucoside
Chalconaringenin
Pinoresinol
Agathisflavone
specific phenolic classes such as flavonone glycosides or
individual 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 solidphase extraction (SPE) is often involved, although on limited
occasions no sample treatment is necessary. These conventional
methods have limited application to solid and semi-solid
samples because of the long extraction times and precautions
needed to protect the highly reactive phenolic species from
degradation processes. In these instances, supercritical fluid
extraction offers a number of advantages for the recovery and
the extraction behaviour of phenolic compounds has been
modelled using supercritical carbon dioxide and either sand15 or
an inert support as a sample matrix.16 Phenolic compounds were
selected to cover a range of polarities (including benzoic and
cinnamic acids, hydroxybenzaldehydes and catechin). Extraction and collection variables including modifier percentage,
extraction temperature, flow rate, extraction time, trap packing
and rinse solvent were optimized. The latter study revealed that
the use of methanol as modifier was mandatory. Only the less
hydroxylated compounds such as p-coumaric acid, transresveratrol and salicylic acid could be quantitatively recovered
from spiked diatomaceous earth while mean recoveries of more
polar phenolic acids and flavonoids were between 30 and
70%.
Solid-phase microextraction (SPME) is a technique finding
wide acceptance as a sampling method in gas chromatography
(GC). It has been less used for high performance liquid
chromatography (HPLC) and thus its application to analysis of
phenolic compounds in fruits has not been reported. Nevertheless, the application of SPME and HPLC to the determination of hydroxy aromatic compounds in water17 suggests that
the technique warrants closer examination for the determination
of phenols in fruits.
Isolation of phenolic compounds from fruits is further
complicated by their uneven distribution in various forms. For
instance, methanolic extracts from orange peel were rich in
flavones and glycosylated flavanones whereas hydrolysed
extracts comprised mainly phenolic acids and flavonols.18 At
the tissue level, there are significant qualitative and quantitative
differences between the phenolic content of seeds, epidermal
and 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 the
inner tissues (mesocarp and pulp).20 For instance, in many
fruits, flavonol glycosides are chiefly located in the outer
portion or in the epicarp. This is seen in the greater abundance
of glucosides and rutinosides in the peel than the flesh of
passionfruit.21 Anthocyanins are located primarily in the skin of
grapes but are present throughout the fruit in strawberry and
blueberry. The situation with the anthocyanins is further
complicated by pH dependent equilibria22 and in the inner cells
in the skin, anthocyanins are mainly in the neutral quinonoidal
base form, whereas in the outer cell vacuoles, they are found
essentially in the flavylium cationic form.
At the subcellular level, phenolic compounds may accumulate in the vacuoles or in the cell walls. Limited data suggest that
they are located mainly in the vacuoles23 with small amounts in
free space and none in the cytoplasm. The seeming homogeneity of the subcellular distribution is perhaps misleading as
lignin and certain simple molecules (flavonoids and ferulic acid
esters) accumulate in the cell wall whereas soluble phenolic
compounds are stored in the vacuoles. The occurrence of
phenolics in soluble, suspended and colloidal forms and in
covalent combination with cell wall components24 most likely
has a significant impact on their extraction. For instance, during
winemaking mainly soluble phenolic compounds present in the
vacuoles of the grape plant cells are extracted, leaving behind a
large 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 grape
pomace polysaccharides and thus releasing phenols. Total
phenols released ranged from 820 to 6055 mg L21 gallic acid
equivalents (GAE) and varied in response to enzyme type, time
of enzyme treatment, particle size of the pomace and type of
extraction solvent employed. The yield of total phenols was
correlated to the degree of plant cell wall breakdown of grape
pomace (r > 0.6, P < 0.01). These data have important
implications for both the analytical and commercial-scale
recovery of phenols and for studies correlating physiological
activity (e.g., antioxidant potential) with phenol content of the
fruit where dietary intake/availability is of paramount importance.26–28
Phenolic profiles have been reported for various fruits,
generally the edible portions and less commonly with other fruit
parts, although there is an emerging interest in the non-edible
parts of the fruit/plant. This application can be attributed to the
use of phenolic profiles as fingerprints for authentication of
wines, olive oils, citrus juices and other commercial products.
Identification and characterization of phenolic components of
various fruits and assessing the physiological activity of fruit
extracts have also attracted considerable attention. There has
been considerably less ‘interest’ in quantifying the phenolic
components, presumably owing to the limited range of phenols
commercially available as suitable reference compounds.29
This situation is changing and the need to quantify the levels of
phenols is now being addressed.
The extraction procedure is simplified in analyses targetting
a single specific phenolic compound. Here the conflicting
stabilities, solubulities, etc., of the target compounds are not an
issue. For example, trans-resveratrol was determined30 in wine
by LC-MS. Trihydroxyflavanone was added to the wine as an
internal standard and the mixture was centrifuged. Enhanced
selectivity for the separation between trans-resveratrol and
endogenous wine constituents was afforded by sample purification with a tandem SPE method. A limit of detection of 200 pg
(signal-to-noise ratio = 3) was attained in the selected ion
monitoring mode using negative ion electrospray ionization
(ESI) and measuring the deprotonated molecular ion. Hydroxytyrosol has only recently been reported in wine31 using a method
specifically targeting this compound. The analyte was eluted
from a C18 cartridge with ethyl acetate and derivatized with
bis(trimethylsilyl)trifluoroacetamide (BSTFA). Specificity and
sensitivity were achieved by GC-MS using one target and two
qualifying ions. Under these experimental conditions, hydroxytyrosol was detected in all wines analysed at average
concentrations 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 to
structural elucidation and characterization of glycosides. Three
types of hydrolytic treatment are used for this purpose, acidic,
enzymatic and alkaline. Hydrolysis has also been used to
minimize interferences in subsequent chromatography33 and as
an aid to simplifying chromatographic data,34–37 particularly in
instances where appropriate standards are commercially unavailable. In this role, chemical treatment has been more
common because it is less selective and more exhaustive.
Hydrolysis methods when used for purposes other than
characterization/structural elucidation of unknown phenols
result in a reduction in information content. Hence, a sample
extract containing several O-glucosides of a single aglycone
plus the free aglycone will produce after acid hydrolysis a single
HPLC peak. The advantages in terms of simplicity of
interpretation and quantification are apparent as seen in HPLC
of red raspberry juices38 where acid and base hydrolysis
simplified the complex phenolic profiles dramatically. Minor
differences were observed in the profiles resulting from the two
treatments 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, the
nature of the sugar and the position of attachment to the
flavonoid nucleus. For example, glucuronides resist acid
hydrolysis whereas by comparison glucosides are cleaved
rapidly. C-Glycosides generally remain intact although structural rearrangements can occur in presence of hot acids39 owing,
for example, to a Wessely–Moser rearrangement which has the
effect of interconverting 6- and 8-C-glycosides.40 The five
major flavonoid aglycones, quercetin, kaempferol, myricetin,
luteolin and apigenin, were determined41 in freeze-dried fruits
and vegetables after acid hydrolysis of the parent glycosides.
The aglycones were separated by reversed-phase HPLC,
identity of the eluted compounds being confirmed by photodiode array UV detection. Optimum hydrolysis conditions were
presented for flavonol glucuronides, flavonol glucosides and
flavone glycosides. Recoveries of the flavonols quercetin,
kaempferol and myricetin ranged from 77 to 110% and of the
flavones apigenin and luteolin from 99 to 106%.
Alkaline conditions are employed in the isolation of phenols
from certain fruits and fruit products, notably citrus, in order to
determine bound phenols, particularly the phenolic acids. For
instance, orange juice was hydrolysed with sodium hydroxide
Analyst, 2000, 125, 989–1009
991
for 4 h at room temperature under nitrogen42,43 and the total
phenolic acids were recovered by ethyl acetate extraction
followed by silica gel column chromatography. The level of free
acids as determined by direct extraction of the juice was very
low compared with that of bound acids released by hydrolysis.
The content of bound acids was unchanged or slightly elevated
from early to late season fruit while the content of free acids was
reduced during this period.
Phenolic acids, including caffeic, chlorogenic, ferulic and
gallic acids, were also determined in grape and cherry juices44
following recovery by extraction with ethyl acetate from fresh
or hydrolysed juices. Hydrolysis was performed in hydroxide
solution at pH 12.5 and required 48 and 62 h for cherry and
grape juices, respectively. Analysis was performed by reversedphase HPLC using isocratic elution with detection by absorption of UV radiation. The juices contained minor amounts of
phenolic acids in the free state while most were present in
conjugated forms that were liberated by hydrolysis. The
phenolic acids, particularly gallic acid, were unstable in the
alkaline conditions under air and it was necessary to hydrolyse
the juices under argon. Cherry juice contained a high concentration of chlorogenic acid which was hydrolysed rapidly to caffeic
acid. Phenolic acids were recovered from cherry laurel in a
similar fashion45 by extraction of dried mesocarp with light
petroleum. The residue was hydrolysed with sodium hydroxide,
acidified and extracted into ethyl acetate prior to formation of
oxime TMS derivatives that were analysed by GC-MS. Vanillic
acid was present in all cultivars and, based on FID peak areas
and normalization, it was the predominant acid.
Artefacts have been reported with extractions under alkaline
conditions due to degradation of some polymethoxylated
flavones.46 Similarly, flavanones and 3-hydroxyflavanones are
sensitive to alkali under which conditions the dihydro-g-pyrone
ring is broken forming chalcones, which decompose to phenols
and cinnamic acid derivatives.47 Under these circumstances,
hydrolysis has been performed in acidic conditions or using
specific enzymes for known glycosides or technical enzymes
when samples contain a mixture of glycosides.
Similar procedures have been adopted for the analysis of the
fruit. For example, the distribution of free and bound phenolic
acids was determined in orange and grapefruit45 by extraction
with ethyl acetate, silica gel column chromatography clean-up
and HPLC analyses of samples before and after alkaline
hydrolysis (24 h). In all fruit parts (peel, albedo, flavedo, juice
sacs and endocarp), only minor amounts of these acids occurred
in the free state, while most was present in conjugated forms
which were capable of liberation by hydrolysis. The level of
bound acids was generally in the order ferulic acid > sinapinic
acid > coumaric acid > caffeic acid. However, significant
losses of caffeic acid were reported during lengthy hydrolysis
(24 h) due to the reactive nature of the o-dihydroxyphenolic
grouping. The loss of o-diphenols by oxidation via the
corresponding quinones is a constant concern under alkaline
conditions. The remaining acids were relatively stable to
treatment with 2 M sodium hydroxide for 4 h under nitrogen at
room temperature. The peels contained the major quantity of
phenolic acids compared with the endocarp, and the flavedo was
richer in acids than the albedo.
In comparison with citrus fruits, there has been a surprising
lack of interest in the use of alkaline extraction with other fruits.
However, the recovery of phenolics from fruit cuticles of
several varieties of apple, using either cuticular wax scraped
from fruit peel or enzyme-isolated cuticles,48 is an interesting
development. The concentrations of free phenolics in fruit
cuticle ranged from 8 to 45 mg g21 and bound phenolics ranged
from 50 to 110 mg g21 in these cultivars.
Extraction of phenols from freeze-dried olive pulp into
aqueous carbonate solution49 gave acceptable recoveries of
oleuropein and other major phenols. However, extraction of
oleuropein and gallic acid from model solutions using the same
992
Analyst, 2000, 125, 989–1009
conditions but containing lower concentrations of the phenols
showed variable results with total loss of phenol in some
cases.50 The in vitro alkaline hydrolysis of oleuropein produces
several aglycones51 but (elenolic acid glucoside and) hydroxytyrosol alone appear(s) in whole fruits subjected to alkali
treatment during processing.52 The latter, an o-diphenol, is then
easily oxidized.53 There has been considerable interest in the
enzymatic and/or chemical catalysis of olive secoiridoids.
Endogenous hydrolytic enzymes, notably glycosidases, may be
activated during crushing and malaxation54 and catalyse the
hydrolysis of secoiridoids such as oleuropein with the production of oleuropein aglycone.55–57 The latter underwent rapid
isomerization (Fig. 1) via the enolic form II to a dialdehydic
form (III/IV) in aqueous extracts58,59 but was stable when
extracted 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 purification of extracts and this was attributed to catalysis arising
from the acidity of silica. The epimeric phenolic metabolites
(III and IV in Fig. 1) and the precursor enolic form II have
recently been identified60 in samples isolated from methanol–
acetone extracts of freeze-dried green mature olive fruits. These
compounds are confirmed as intermediates in enzymatic
hydrolysis of glucosidic linkage in oleuropein (Fig. 1).
2.2 Fruit extracts
Extraction of phenols from fruit extracts, particularly juices, is
generally simplified by the physical state of the sample, but
complications still arise. The impact of the processing operation
on analytical data for phenols in fruit extracts such as juice, jam
and olive oil must be considered. For example, was the seed or
peel excluded? In the case of peach there is a downy peel which
is removed prior to processing, whereas this is not the case with
many other fruits. Commercially processed products should be
distinguished from the corresponding fresh products recovered
in the laboratory specifically for analysis. Such considerations
are important as they will determine the extent of inclusion of
different parts of the fruit in the nominal portion. For instance,
hand-reamed and commercially squeezed orange juice will
incorporate the albedo and flavedo to different extents. Fruit
maturation is also an important factor and more of the
components of the albedo will be extracted from over-ripe citrus
fruit than from immature fruit. Quantitative data should be
quoted on a dry solids basis or alternatively on juice converted
to a constant Brix value.61,62
2.2.1 Juices and related products. Large differences in the
levels of different phenolic compounds in a juice generally
complicate the simultaneous analysis of the different classes of
phenols. For instance, flavanone glycosides and cinnamoyl-bD-glucopyranoside were isolated63 from the juice of blood
oranges by extraction at 90 °C with dimethylformamide
containing ammonium oxalate solution (to maintain pH)
whereas trans-cinnamic acid, in recognition of the lower
concentration in the juice, was concentrated by reversed-phase
SPE. Hydrolysis of cinnamoyl-b-D-glucopyranoside was
achieved in 4 M HCl by refluxing for 1 h. Flavanone glycosides
(e.g., hesperidin, 100–500 mg L21) are quantitatively the most
important phenolics in orange juice whereas the levels of other
phenolics such as polymethoxylated flavones are much lower
with typical values of 0.1 mg L21,46,64 although higher levels
are found in the peel. For this reason, many methods are
designed for a specific class of phenolic compound,61,62,64–66
although the relative response of the detection system must be
considered and Mouly et al.67 described the simultaneous
separation of flavanone glycosides and polymethoxylated
flavones.
Sample preparation may involve a simple filtration or an
elaborate extraction of the crushed,68 hand-reamed63,69 or
commercially extracted juice.70 Ideally, clear juices require
minimal sample preparation beyond centrifugation and/or
filtration. For instance, apple juices were prepared for analysis
by filtration through polytetrafluoroethylene filters and several
classes of phenolic compounds were identified by HPLC and
quantified in commercial juices71 by absorption at characteristic
wavelengths as hydroxycinnamates (316 nm), anthocyanins
(520 nm), flavan-3-ols (280 nm) and flavonols (365 nm). The
range of concentrations as a percentage of total phenolic
concentration as determined by the Folin–Ciocalteau method
was hydroxymethylfurfural 4–30%, phloridzin 22–36%, cinnamates 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 been
analysed70 by direct injection HPLC following centrifugation
and adjustment to constant Brix. Dihydrochalcones (e.g.,
phloridzin) were characteristic of the apple juices at typical
concentrations of 2–20 mg L21, but the phenolic components of
the remaining juices were not identified. Cloudy juices as
typified by those of citrus fruits are also amenable to direct
Fig. 1 Pathways showing the interconversions observed for one group of plant phenols.
Analyst, 2000, 125, 989–1009
993
analysis following filtration and centrifugation,72–75 although
poor recoveries have been attributed to low solubility of certain
phenolics76 and/or to sorptive losses on the filtration medium.77
In other instances, more extensive sample processing has
been deemed desirable78 and SPE on mini-cartridges has been
employed46,78–80 in an attempt to minimize the effects of the
sample preparation on extract integrity. For instance, interfering
sugars were removed by SPE from dealcoholized berry and fruit
wines and liquors (adjusted to pH 7.0 using sodium hydroxide)
prior to measurement of total phenols81 by the Folin–Ciocalteu
procedure. Suárez et al.82 fractionated phenolics from apple
must and cider into neutral and acidic groups by means of a SPE
method. Extracts were analysed by reversed-phase HPLC using
a phosphate methanol gradient and quantification at 320 nm for
cinnamic acids, 360 nm for flavonols and 280 nm for other
phenols. Recoveries between 84 and 111% were obtained from
spiked samples. The level of phenolic compounds in kiwifruit is
low relative to that of many other fruits. Nevertheless, juice
from kiwifruit was fractionated68 into strongly acidic and
weakly acidic materials by processing on Sep-Pak C18 cartridges. The juice was obtained by treatment of the fruit in a
hammer mill followed by addition of pectolytic enzymes and
ethanol prior to filtration to remove protein. Strongly acidic
compounds were identified as derivatives of coumaric, caffeic
and 3,4-dihydroxybenzoic acids whilst the weakly acidic
fraction contained epicatechin, catechin and procyanidins plus
flavonols present as the glycosides of quercetin and kaempferol.
The use of pectolytic enzymes during commercial processing
of juices may influence the content of phenolic compounds.
Versari et al.83 evaluated the effect of commercial pectolytic
enzymes on the content of phenolic compounds (anthocyanins,
flavonols and ellagic acids) in strawberry and raspberry juices
under enzymatic pectinase treatment. They found that commercial pectinases modified the phenolic composition of the juices
dependent on time and fruit species. Depending on the enzyme
treatment employed, at 6 h, a loss of anthocyanins (220%)
present in raspberry juice was observed, whereas in strawberry
juices the ellagic acid concentration always increased and the
flavonol content decreased (235%).
Flavanone glycosides of citrus juices have also been
recovered by elution with methanol from a Sep-Pak C18
cartridge78 following elution of sugars with aqueous methanol.
Recoveries compared favourably with those achieved by simple
filtration. Preliminary fractionation of citrus juice phenolics has
also been performed84 on polyamide cartridges eluting with
methanol. The extracts were analysed by reversed-phase HPLC
on a cyclodextrin bonded phase to resolve diastereomers and
enantiomers.
The recovery of cinnamic acids, cis- and trans-resveratrol,
flavonoids and flavanols from (grape) wine has been thoroughly
investigated10 and here also SPE has provided a simple means
of 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 problems
that were eliminated by distillation, although matrix dilution
with water was equally effective and a simpler solution. This
also reduced matrix interference by other components and
improved recoveries of phenolic species. The phenolic compounds were eluted from the SPE cartridge with ethyl acetate,
evaporated to dryness by azeotropic distillation and derivatized
with BSTFA prior to quantification by GC-mass selective
detection using an internal standard. SPE was also used to
recover phenolic compounds including phenolic and cinnamic
acids from sherry wine by an initial clean-up on a C18 cartridge
followed by fractionation into acidic and neutral phenolic
fractions using an anionic exchanger cartridge85 or by an on-line
automated robotic system with a polymeric poly(styrene–
divinylbenzene) cartridge using tetrahydrofuran as eluent.86
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Analyst, 2000, 125, 989–1009
Recovery of added phenolic compounds from spiked samples
exceeded 85% in all cases, although other data85 suggest that
much lower recoveries are typical. Nevertheless, SPE was
regarded as superior to liquid–liquid extraction and reduced
analysis times by 50%. In contrast, phenolic compounds
(including hydroxybenzoic acids, hydroxycinnamic acids, phenolic aldehydes, coumarins, flavan-3-ols and flavonol aglycones) have been determined in wood aged fortified wines87 by
direct injection with no sample pre-treatment. The higher levels
of phenolic compounds due to extraction from the wood
probably facilitated direct injection.
In the case of cloudy juices, both filtration and SPE may be
ineffective in recovering phenols located in suspended juice
solids. Under these circumstances, solvent extraction may be a
preferable alternative although even here bound phenols
probably remain intact. Thus, polarity differences in juice
components88,89 have been exploited in a comprehensive
recovery scheme for (carotenoids), polymethoxylated flavones
and flavanone glycosides based on extraction with solvents of
graded polarity. Citrus juice was diluted with methanol,
centrifuged and aqueous sodium chloride added to the supernatant (to minimize the formation of troublesome emulsions).
The solution was then extracted sequentially with hexane and
dichloromethane to isolate the carotenoids and polymethoxylated flavones, respectively, leaving the flavanone glycosides in
solution. Various solvents have been described for the isolation
of flavanone glycosides (ref. 76 and references cited therein),
phenolic acids42 and polymethoxylated flavones61,90,91 from
citrus. Methanol has been deemed76 the most suitable solvent
for extraction of both flavanones and flavanone glycosides,
although difficulties are encountered with specific compounds.
Methanolic extraction has also been favoured for recovery of
phenols from apple, pear and quince purées.92 The isolated
phenolic compounds were quantified by absorption at either 280
or 350 nm. Simple extraction with methanol was compared with
a more detailed procedure involving clean-up of the extract on
an Amberlite XAD-2 column that removed sugars and other
polar compounds. The authors noted that the chromatograms
were ‘somewhat cleaner than those obtained with the simplified
extraction technique and, as a general rule, the amount of each
phenolic compound extracted was higher’. However, the use of
the resin caused low recoveries of arbutin from pear purees.
The efficiencies of several solvents have been compared91 for
the recovery of polymethoxylated flavones from intact citrus
juices and juices treated with sodium hydroxide to eliminate
possible interfering lactones. In terms of total flavones, isobutyl
methyl ketone was only slightly less efficient than benzene but
was more effective for specific flavones. These data demonstrate the need to consider carefully any recovery problem93,94
on an individual basis. Alternatively, polymethoxylated flavones have been isolated from citrus juice by retention on
polystyrene resin followed by elution with ethanol and
acetone.95 The extracts were further purified by silica gel
column chromatography.
The phenolic composition of peach and apple purees and
concentrates,20 intermediate products in the elaboration of
commercial fruit juices, was quantified by homogenizing
samples in acidified methanol and partitioning the phenolic
components into ethyl acetate. Phenols were identified by
HPLC as various benzoic acids and aldehydes, cinnamic acids
and their derivatives, flavan-3-ols, procyanidins, flavonols and
dihydrochalcones. Peach-based products were completely devoid of flavonol and dihydrochalcone derivatives and this was
attributed to the removal of the skin and stone of the fruit in the
manufacturing process. On the other hand, different quercetin
and phloretin glycosides were detected in apple purees and
concentrates.
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 to
sequential extraction with ethoxyethane and ethyl acetate. The
extraction time and temperature were evidently critical. The two
extracts were combined and evaporated to dryness before
analysis. In this way, quantitative data were obtained on the
content of benzoic acids and aldehydes, flavan-3-ols, flavonols,
chalcones, cinnamic acids and their esters, glycosidic derivatives and flavonoids. Differences in levels of flavanols were
attributed to different degrees of pressing of the fruit as these
phenols are found mainly in the skin and seeds.
Specific problems are encountered with some phenols. For
instance, hesperidin, the major flavonoid of sweet orange,
presents difficulties because of its low solubility in aqueous
media. Addition of dimethylformamide to orange juice has been
used67,96 in an effort to improve solubility but in this case some
early-eluting peaks were lost in the chromatogram. This also
results in sample dilution with a decrease in sensitivity. Heating
of the juice has also been used62 to increase hesperidin
solubility. Buffering of the sample in the pH range 4.5–5.0 prior
to extraction has been recommended69 to overcome more
general problems of the pH dependence of flavanone glycoside
recovery. In this instance, oranges were hand-squeezed and the
extract filtered through a stainless steel sieve (1.25 mm) to
remove seed and pulp66,69 although a double layer of cheesecloth has also been used for this purpose.78 The separated juice
was mixed with dimethylformamide and ammonium oxalate (to
maintain pH) and heated for 10 min. The cooled juice was
centrifuged and filtered prior to injection.
Recovery of anthocyanins which comprise a major portion of
the phenolic content of red wine and dark-coloured berry
juices13,97–100 presents some unique challenges. The anthocyanins are glycosides that release the anthocyanidin aglycone by
hydrolysis. The aglycones exist in various forms in pH-
dependent equilibria (Fig. 2) which impacts on their solubility
and extraction behaviour. Six anthocyanidins are widespread
and commonly contribute to the pigmentation of fruits.
Cyanidin is the most common and, in terms of frequency of
occurrence, is followed in decreasing order by delphinidin,
peonidin, pelargonidin, petunidin and malvidin. Glycosylation
of anthocyanidins almost always occurs at the 3-position with
glucose, arabinose and galactose the most common sugar
moieties. For instance, the 3-glucosides and 3-rutinosides of
cyanidin and delphinidin are the dominant species in blackcurrant.101 In addition to glycosylation, acylated anthocyanins
are found fairly often in fruits, the situation being particularly
complex in grapes102–104 where the 3-monoglucosides corresponding to the five aglycones can all be acylated by acetic or
p-coumaric acid.
Anthocyanins are traditionally recovered as the flavylium
cation by extraction with cold methanol containing hydrochloric acid.105 However, the acylated anthocyanins are frequently
labile in solutions containing mineral acid and this is one of the
reasons why the relatively common acylated pigments were
overlooked in earlier studies.106 Replacement of hydrochloric
acid with weaker acids, either formic or acetic acid, permits the
recovery of these compounds.107–109 Care must be exercised to
ensure that acetylated derivatives are in fact natural and not an
artifact of the extraction process.110 With the most labile
anthocyanins, the use of non-acidified solvents is probably a
sensible precaution. Alternatively, SPE on C18 cartridges has
been used.111 For example, anthocyanins were recovered from
diluted fruit juice or wine (after removal of ethanol)112 by
elution from a Sep-Pak C18 cartridge with methanol–formic
acid–water. The extracts were analysed by matrix-assisted laser
desorption/ionization mass spectrometry (MALDI-MS), in
which analytes are usually desorbed and ionized in the source
Fig. 2 Structure of anthocyanins showing the pH-dependent equilibria.101
Analyst, 2000, 125, 989–1009
995
forming protonated or alkali metal adduct ions. However,
anthocyanins exist in the above eluent predominantly in the
aromatic oxonium ion form whence they easily ionize in the
MALDI source to form molecular mass cations in the positive
ion mode.
When the adsorbed anthocyanins are subsequently eluted
from the SPE cartridge with an alkaline borate solution, a class
separation is achieved.111 It appears that those anthocyanins
possessing o-dihydroxy groups (cyanidin, delphinidin, petunidin) form a charged borate complex, resulting in a more
hydrophilic species. This complex is preferentially eluted from
the reversed-phase cartridge whereas those anthocyanins not
containing o-dihydroxy groups (pelargonidin, peonidin, malvidin) are enriched on the cartridge. On the other hand, elution
with hydrochloric acid (0.01%) in methanol produces no
fractionation. A more exhaustive clean-up on polyvinylpyrrolidone was also examined. The relative proportions of the
anthocyanins was different for the two procedures. Thus, for
quantitative analysis the extraction and/or clean-up procedure
should be thoroughly checked.113
Condensed polymeric anthocyanins formed during the winemaking process by interactions between anthocyanins and other
phenols such as flavanols (e.g., catechin) were recovered114
from red wine or apple cider on an ODS column by elution with
methanol. The concentrated lyophilized extracts were then
fractionated by gel permeation chromatography (GPC) using a
mixture of acetone and acidified aqueous urea as eluent.
Anthocyanins and other phenolic compounds were recovered
from the GPC fractions by sorption on a Sep-Pak C18 cartridge
that was washed with water to remove urea. The sorbed
phenolic compounds were eluted with methanol.
2.2.2 Olive oil. Liquid extraction has been widely used for
recovery of phenols from olive oils purchased through retail
outlets115 or obtained directly from commercial processors116,117 or in the laboratory by simulating industrial processing conditions.118,119 In some instances, details of oil production have not been provided120 or it was stated that oils of
different extraction technologies were analysed.121
Phenols have been recovered from olive oil by extraction of
the unsaponifiable matter with aqueous methanol.119 However,
the more usual procedure has involved extraction with methanol120 or aqueous methanol115,116,118,122 of a solution of the oil
in hexane116,118,122–124 or ethoxyethane.120 An internal standard
is included in most procedures. Residual oil must be removed
by overnight storage at subambient temperature,120 by centrifugation118 or by solvent extraction with hexane,122 although
Sephadex column chromatography has also been used8,9 to
effect further clean-up. Direct extraction of the oil with
methanol in an Ultra-Turrax apparatus has also been used for
the recovery of phenols from virgin olive oil dried over sodium
sulfate.125 The methanol was removed and the residue dissolved
in acetonitrile and washed with hexane. After evaporation of the
acetonitrile under vacuum, the residue was dissolved in acetone
prior to derivatization with BSTFA. The extracts were examined by GC-MS and chemical ionization confirmed several
phenolic and secoiridoid derivatives in the extracts. Aglycones
of ligstroside, of decarbomethoxyoleuropein and of oleuropein
were detected. Each aglycone, because of several tautomeric
equilibria involving the ring opening of secoiridoid, showed up
as compounds with four main structures following derivatization. Montedoro et al.8 compared the various methods of
extraction (directly from oil versus a solution of oil) using
different solvent combinations and concluded that aqueous
methanol provided optimum results. Their procedure was used
by Brenes et al.57 to characterize the phenols in Spanish virgin
olive oils. The procedure involved direct aqueous methanol
extraction from oil, partitioning into acetonitrile and washing
with hexane. Despite the extensive use of aqueous methanol as
extractant it has been claimed120 that extraction with neat
996
Analyst, 2000, 125, 989–1009
methanol improved yields of a number of phenolics and
eliminated formation of troublesome emulsions seen with
aqueous methanol.
The work of Litridou et al.126 highlights the need for care in
sample extraction when using SPE where the choice of eluent
and/or eluent volume is critical. Mannino et al.121 reported
gallic acid in olive oil and attributed its appearance to their
extraction procedure involving SPE which eliminated oxidation
prevalent in other procedures. Two approaches have been used
in which olive oil was dissolved in hexane and added directly to
a 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 stored
overnight 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 extract
was then evaporated under nitrogen and fractionated by
reversed-phase SPE using stepwise gradient elution into 40
fractions which were combined ultimately into fraction A
[eluted with methanol–water (20+80 v/v)] or fraction B (eluted
with stronger eluents comprising aqueous methanol and
methanol–chloroform). HPLC analyses of the two fractions
showed that fraction A contained only simple phenols and
phenolic acids, whereas fraction B had a complex nature and
was found to contribute more than fraction A to the oxidative
stability of the oil. Acid and alkaline hydrolysis also yielded
some valuable information and significant changes in the HPLC
profiles were observed, which indicated the presence of ether
and ester bonds. Finally, anion exchange HPLC was used to
determine whether or not monosaccharide residues were
released after acid or alkaline hydrolysis of the given fractions.
Acid hydrolysed extracts showed the presence of small
quantities of glucose and galactose, suggesting that only traces
of glycosides were present in the polar fraction of the oil
investigated.
2.2.3 By-products. Residues from fruit processing have
traditionally presented an economic and environmental problem
as waste products but are becoming increasingly recognized as
valuable commodities for the production of by-products. For
instance, citrus residues remaining from juice extraction can be
a source material from which over 300 valuable by-products can
be produced.127 The whole peel or rind (pericarp) is used for
such products as marmalade, candied peel, bioflavonoids and
peel seasonings. Combined with the pulp residue, it becomes
feed for animals, molasses, alcohols and distilled oils. The
flavedo (exterior yellow peel, pericarp) contains the oil glands
from which cold-pressed and distilled oils and essences are
extracted for the flavouring industries. The albedo (interior
white spongy peel, mesocarp) is rich in pectin and used
extensively as a gelling agent in the food and pharmaceutical
industries. Pulp residue (endocarp) represents the fraction
screened from the pulp, that is, cores, segment walls or
membranes, juice vesicles and seeds. This is usually combined
with the peel residue for the manufacture of stripper oil, citrus
molasses, citric and lactic acids, citrus wine and many other byproducts. The recovery of phenolic compounds from byproducts of fruit processing has attracted considerable attention.
The phenolic content is also of interest in food, pharmaceutical
and cosmetic uses of these by-products where the physiological
activity of the phenols may be important. There are additional
reasons for the interest in the phenolic content of processing
residues. Orange pulpwash128 is obtained during the processing
of oranges for juice and it has been used as an adulterant of the
juice. The phenolic profile provides a fingerprint that is useful
in identifying juice adulteration by the pulpwash.129,130
The waste material produced during refining of cold pressed
citrus peel oils represents a further important source of phenolic
components131 and polymethoxylated flavones have been
determined in peel oils following simple dilution with ethyl
acetate containing an internal standard132 or by extraction and
clean-up using column chromatography.133 Alternatively, the
polymethoxylated flavones were obtained134 directly from the
peel of oranges and tangerines by Soxhlet extraction with
benzene for 4 h. The extracts were concentrated in vacuo and
analysed without further purification by normal-phase HPLC.
Some of these oils are used in perfumes and cosmetics135 and
potentially in the treatment of burnt skin,136 so it is equally
important to be able to establish if they contain phenols with
adverse physiological effects.
Similarly, grape marc resulting from red winemaking is a
valuable source of phenols. Grape marc was extracted137 with a
mixture of ethyl acetate and water in order to recover its
phenolic compounds with a view to their use as food lipid
antioxidants. Crushed and uncrushed marcs were extracted for
various times in order to determine the minimum time required
for ensuring maximum extraction of phenols. The results reveal
a higher extraction of these compounds by the ethyl acetate
acting on the crushed marc. Hence the cost of crushing can be
largely compensated.
Large volumes of water are generated during traditional
olive oil production and subsequently discarded. This requires
treatment138 and the process is not environmentally sustainable. Hence, new extraction technologies have been introduced and there has been considerable interest in comparing the phenol content of oils139 and wastewater140 produced
by the different technologies notably two-phase versus threephase extraction. This is a further instance where the same set
of considerations important in analytical methodology have
important implications for processing technology. The wastewater contains a number of phenols in quantities determined
largely by their partition coefficients and these have been
analysed141 on an uncoated fused-silica capillary electrophoresis column using aqueous ammonium acetate buffer in
methanol and negative ion electrospray mass spectrometric
detection. Quantitative analysis, with p-chlorophenol as the
internal standard, was carried out by single ion monitoring
and limits of detection ranged from 1 pg for 4-hydroxybenzaldehyde and protocatechuic acid to 386 pg for vanillic
acid. Ethyl acetate and butanol extraction have been
used140,142 to recover phenols from fresh olive wastewater.
Wastewaters obtained by employing a benchtop mill were
fractionated143 by liquid–solid extraction (details covered by
patent and not disclosed) and further processed to yield three
extracts. Extract 1 was obtained by fractionation of lyophilized wastewater on an XAD 1180 column and elution with
ethanol. The second extract was obtained by ethyl acetate
extraction of hexane-washed wastewater while the third
fraction was obtained following a fractionation of extract 2
on a Sephadex LH-20 column. Extract 1 contained a complex
mixture of phenolics including many polymers responsible
for a high background absorption at 254 nm. Extract 2
contained mainly low and medium molecular mass phenolics
with elenolic acid as the principal constituent. Extract 3
comprised hydroxytyrosol, tyrosol and an unidentified derivative of the former. Characterization of the wastewater and
particularly its phenolic components is necessary to allow
agricultural uses of the water.144 The biodegradation of the
phenols and investigation of metabolites145 are important
considerations in future work.
2.3 Fruit
The phenolic profile is characteristic of a fruit species and while
there are varietal and seasonal differences these are of
secondary importance. Methods of recovery differ between the
various fruits reflecting these variations. Fruit morphology must
also be considered since the nature and content of phenolics
differs between the various organs of the fruit. Hence extraction
methods can impact significantly on the phenolic content of a
fruit extract depending on which fruit organs are included. For
instance, citrus fruit is particularly complex and comprises the
outer layers collectively termed rind or peel which includes the
flavedo or outer coloured portion with oil glands, the inner
colourless portion, the albedo and the internal structures. The
last part involves the segments surrounded by a continuous
membrane, the endocarp proper with a membrane of mesocarp
tissue extending radially between segments. The interior of a
segment contains the juice (or pulp) vesicles and seeds. The
distribution of phenolic compounds between these organs
differs both qualitatively and quantitatively.
Historically, recovery of phenols by liquid extraction of the
fruit using hot or cold solvents has been common. Suitable
solvents for this purpose are aqueous mixtures with ethanol,
methanol, acetone and dimethylformamide. Extractions have
been performed on freeze-dried ground extracts of the fruit or,
alternatively, by maceration of the fresh, undried fruit with the
extraction solvent.124 In the last case, the required proportion of
water is lower.
Solvent extraction has been widely used to recover phenols
from citrus. Grapefruit portions and peel were dried at 50 °C in
a fan forced air oven146 and the material was ground to a fine
powder, which was extracted with dimethyl sulfoxide. The
extracts 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 using
methanol–dimethyl sulfoxide. The extracts were centrifuged
and subjected to clean-up by SPE using C18 cartridges to
remove polar components. The retained flavonoids were eluted
with methanol–dimethyl sulfoxide, which enhanced the solubility of hesperidin, diosmin and diosmetin. Recoveries of
eriocitrin, naringin, hesperidin and tangeretin from spiked
samples of mesocarp tissue exceeded 96%. Flavones and
flavon-3-ols were relatively abundant in leaves. Extraction with
aqueous ethanol has been used147 to recover flavonoids from a
dried extract of sour orange. The ethanolic extract was filtered
and evaporated to dryness under vacuum prior to analysis by
LC-MS using ESI. Several flavanones, flavanone glycosides
and polymethoxylated flavones were detected and identified in
the extracts. This approach for separation into peel and pulp has
also been applied21 to passion fruit. Clear juice was obtained
from the pulp by filtration through gauze and centrifugation.
Peel was blended with methanol, filtered and evaporated to
dryness. The juice and peel extract were processed on Amberlite
XAD-2 resin to retain selectively phenolic glycosides that were
eluted with methanol.
Interest in the phenolic content of the grape berry has focused
on its anthocyanin22 and catechin contents.148 For instance,
catechins were recovered148 from black grape (and apple) by
extraction of freeze-dried material with aqueous methanol using
a mechanical shaker for 60 min at room temperature. The
extract was filtered and analysed by HPLC using fluorimetric
detection at 310 nm (excitation at 280 nm) for the specific and
sensitive detection of (+)-catechin and (2)-epicatechin. Grape
anthocyanins have also been extracted149 at room temperature
using a mixture of formic acid in aqueous methanol. The acyl
portion of anthocyanins has traditionally been characterized
following mild alkaline hydrolysis since cinnamic acids are not
stable in a hot acid medium. However, anthocyanidins are
unstable in alkaline media. In this instance, the corresponding
anthocyanidins were obtained by hydrolysis of the sample
extract with methanolic HCl whereas acid hydrolysis in aqueous
media completely destroyed cinnamic acids.
Cherries are another fruit in which anthocyanins comprise the
major phenolics particularly in dark-coloured cherry genotypes.13 Mature sweet cherries were pitted and homogenized
with aqueous methanol containing formic acid. The homogenate was filtered and the filtrate analysed by HPLC for the
Analyst, 2000, 125, 989–1009
997
separation and quantification of both anthocyanins and other
phenolic compounds, predominantly neochlorogenic acid and
p-coumaroylquinic acid. A more complex procedure based on
sequential extraction with hexane, ethyl acetate and methanol
has been applied to lyophilized ground tart cherries.150 Further
partitioning of the methanolic extract with ethyl acetate yielded
a fraction containing a mixture of phenolic compounds
including isoflavones, flavanones and flavonol glycosides.
Anthocyanins were recovered during the process in a separate
fraction.
Häkkinen et al.151,152 systematically investigated the recovery of non-anthocyanic phenols from berries other than grape.
Although anthocyanins contributed a significant proportion of
the total phenolic compounds in these berries, the method was
not applicable to these compounds. Three extraction and
hydrolysis procedures were investigated for the recovery of
flavonols (kaempferol, quercetin and myricetin) and phenolic
acids (p-coumaric, caffeic, ferulic, p-hydroxybenzoic, gallic
and ellagic acids) from the frozen berries. The influence of
thawing method (refrigerator, room temperature or microwave)
was examined and showed differential effects on the level of
different flavonols. Microwave thawing produced the most
reliable results and was also the most practical approach for
routine analyses. Flavonols were extracted and hydrolysed to
aglycones by refluxing in aqueous methanol containing hydrochloric acid and tert-butylhydroquinone as an antioxidant.
Recoveries of flavonols were critically dependent on the
concentration of the aqueous methanol extractant. The authors
concluded that it is not an ‘easy task to find a single method
which is adequate for an analysis of a diverse group of phenolics
because of the differing chemical structures and the varying
sensitivity of the compounds to the conditions of hydrolysis and
extraction’.
Mature cider apples153 were sprayed with aqueous formic
acid to avoid oxidation while manually separated into parenchyma 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 low
molecular mass phenols were then extracted with methanol and
polymerized phenols were recovered from the residue with
aqueous acetone. The dry methanol extract and the dry aqueous
acetone extracts were analysed using reversed-phase HPLC
coupled with diode array detection following thiolysis to
quantify phenolic compounds as hydroxycinnamic acid derivatives, flavan-3-ols, flavonols and dihydrochalcones. Procyanidins 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 were
homogenized with aqueous methanol71 using a Waring blender.
The extracts were filtered and the methanol was removed by
rotary evaporation prior to analysis by HPLC. The phenolic
profile of the apple extracts differed from those of juices. The
range of concentrations of phenolic classes in fresh apple
extracts was hydroxymethylfurfural, not detected, phloridzin
11–17%, cinnamates 3–27%, anthocyanins, not detected to
42%, flavan-3-ols, 31–54% and flavonols 1–10%.
Flavanols or catechins are important phenolic components of
apples. Analytical methods for flavanols have generally focused
on the identification of new derivatives or polymeric catechins
(proanthocyanidins) and are not designed for quantification. In
contrast, Arts and Hollman148 optimized the quantification of
flavanols in three model foods: apples, black grapes, and canned
kidney beans. Freeze-dried and fresh samples were examined
and the level of flavanols was not affected by the drying
process. The sample was mixed with aqueous methanol and
shaken in a mechanical shaker at room temperature. The
extracts were filtered and analysed by HPLC without further
processing. Fluorescence detection at 310 nm following
excitation at 280 nm provided selective and sensitive detection
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Analyst, 2000, 125, 989–1009
of (+)-catechin and (2)-epicatechin whereas other phenolics
were detected by their ultraviolet absorption at 270 nm. The
type (ethanol, methanol or acetone) and concentration
(40–100% in water) of extraction solvent influenced flavanol
yield, whereas extraction time (10–60 min) did not. Adequate
extraction was attained with 60–100% methanol for apples and
grapes but recovery decreased to ca. 70% of maximum value
when the percentage of methanol in the extractant was reduced
to 40%. A plausible explanation of this behaviour is the
reduction by methanol of the activity of polyphenol oxidases,
which are widely distributed in plants. This suggests that
extraction with low methanol solvents may not completely
inactivate enzyme activity, resulting in reduced phenol yields.
Recovery of spiked flavanols ranged from 92 to 105%.
Olive contains several distinctive phenolics such as verbascoside, ligstroside and oleuropein. These were recovered154 from
methanolic extracts of olive fruit by partitioning into ethyl
acetate using bioguided fractionation. Such methods are not
common and the extraction procedure developed by Amiot
et al.155 has been widely adopted for the isolation of phenolic
compounds from the fruit.51,53,54,156,157 The details differ but
sample preparation has generally entailed extraction with
aqueous ethanol in the presence of metabisulfite of freeze-dried
olives powdered with the aid of liquid nitrogen. The extracts
were concentrated under reduced pressure, acidified (in some
instances) and washed with hexane to remove lipophilic
compounds.155 The phenolic compounds were partitioned into
ethyl acetate52 in the presence of ammonium sulfite, metaphosphoric acids and methanol.53,54,157 Alternatively, the extracts
have been further processed on a diatomaceous earth Extrelut
cartridge,156 which was sequentially eluted with hexane, ethyl
acetate (non-anthocyanic phenols) and acidic methanol (anthocyanins). Several compounds present in trace amounts were
further fractionated156 by silica phase centrifuge TLC.
Vlahov158 adopted a simpler approach for flavonoid analysis,
in which olive pulp was extracted with aqueous methanol. The
combined extracts were evaporated to dryness, reconstituted in
glacial acetic acid and water followed by centrifugation and
filtration. Bianchi and Pozzi159 have recovered simple phenolic
substances with the basic skeleton C6–C1, C6–C2 and C6–C3
from olives by homogenizing with water in a blender. The
homogenate was evaporated to dryness under reduced pressure,
the residue dissolved in water and the solution partitioned into
ethyl acetate to retrieve the phenolic substances. Extracts have
typically been analysed by HPLC.
Servili et al.140 found higher recoveries of phenolic compounds from olive drupes by SPE than liquid–liquid extraction.
The recovery of the dialdehydic form of elenolic acid linked to
3,4-(dihydroxyphenyl)ethanol and an isomer of oleuropein
aglycon, however, was low. The same group160 developed a
comprehensive scheme for the extraction of phenolic compounds from olive pulp that introduced several precautions
aimed at inhibiting enzyme activity and hence phenolic
modification or destruction. Olives were peeled and destoned,
and the olive pulp was placed in liquid nitrogen and subsequently freeze-dried. The freeze-dried material was stored at
230 °C prior to analysis. Phenolic compounds were recovered
from the olive matrix by extraction with aqueous methanol
containing sodium diethyldithiocarbamate. This mixture was
homogenized for 30 s and filtered using a Büchner funnel. The
methanolic extracts were evaporated under vacuum and nitrogen 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 by
Bianco et al.,161 who extracted phenolic compounds from green
olive fruits by refluxing in boiling methanol for 30 min. The
aqueous extract following removal of methanol was exhaustively extracted with ethyl acetate and purified using
reversed-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 in
phenol recovery. Phenols in the filtered ethanolic extract were
quantified by ultraviolet derivative spectroscopy.
2.4 Peel and seed
The determination of phenols in peel and seed has assumed
increasing importance with the recognition that these fruit parts
are often a source of unique phenols or compounds in much
higher concentration than in the flesh. Free phenolics were
extracted from finely ground citrus peel or seed by refluxing in
methanol.18 After filtration, the methanolic extract was washed
with light petroleum and evaporated to dryness under vacuum.
In contrast, bound phenolics were recovered after alkaline
hydrolysis (4 h) at room temperature and under nitrogen. The
aqueous phase was separated by filtration, extracted with ethyl
acetate and evaporated as before. The dried residues from either
process were dissolved in dimethylformamide for analysis. The
free phenolics were predominantly flavanone glycosides,
glycosylated flavones and polymethoxylated flavones while
bound phenolics comprised largely phenolic acids (caffeic, pcoumaric, ferulic and sinapinic acids), with evidence for the
existence of flavonols bound to cell walls.
Extraction with aqueous ethanol has been used to recover
phenolic compounds from grape seeds and skins. The methods
differ in the use of fresh163 or freeze-dried seeds164,165 and in the
addition of metabisulfite as an antioxidant to the extractant.164,165 Lipids and chlorophyll are eliminated from the
extracts by partitioning into chloroform and the extracts may be
analysed directly or further processed164 by partitioning of the
phenolic compounds into ethyl acetate prior to analysis. The
recovered phenolic compounds were mainly condensed tannins
and anthocyanins from seeds and skin, respectively.
Proanthocyanidins or condensed tannins are oligomeric and
polymeric flavan-3-ols based on various constitutive units. A
method has been devised166 that fractionates grape seed or skin
proanthocyanidins according to their degree of polymerization.
Seeds and skins were recovered from commercially mature
grape berries. Seeds were ground under liquid nitrogen and
extracted with aqueous acetone whereas skins were washed
with methanol to remove organic acids and low molecular mass
phenols before solvent extraction. After a preliminary clean-up
by column chromatography, the proanthocyanidins were precipitated by chloroform–methanol on an inert glass powder
column and recovered by stepwise gradient elution with
increasing proportions of methanol in the solvent. Alternatively,
fractionation has been achieved by gel permeation chromatography164 and elution with methanol or methanol–acetic acid. de
Gaulejac et al.165 provide an interesting comparison of low
pressure chromatograms of seed and wine extracts. The latter
was enriched in simple phenolic compounds such as pcoumaric, gallic and caffeic acids whereas the predominant
phenolic compounds in the seed extract were flavanols and
condensed flavanols.
A number of unusual phenolic compounds have been
identified in olive seeds. Maestro-Durán et al.167 claimed that
salidroside is present in olive seeds whereas nuzhenide was
isolated by Servili et al.160 The latter represents one of the first
dedicated efforts at the characterization of the phenolic content
of the complete olive fruit, in that peel, pulp and seeds were
analysed in three Italian olive cultivars. Nuzhenide was detected
exclusively in the olive seeds of all three varieties and at all
stages of maturation. Similarly, luteolin-7-glucoside and rutin
were detected only in olive peel, whereas verbascoside,
oleuropein and demethyloleuropein were found in all three olive
matrices. The concentration of the last two phenolics was
greatest in olive pulp.
The olive pomace obtained from olive fruit processing
contains seed husk and a small amount of seeds, pulp and peel
which can be separated by industrial methods. Steam explosion
has been examined as a pre-treatment process to increase the
availability of the main components of lignocellulosic biomass.
During steam explosion, lignin is partly depolymerized giving
rise to water-soluble phenolic compounds, which have been
identified168 as vanillic acid, syringic acid, vanillin and
syringaldehyde plus tyrosol and hydroxytyrosol. The results
suggest the presence of hydroxytyrosol as a structural component of the olive stone.
2.5 Leaf
Interest in the phenolic content of plants has recently shifted to
include portions of the plant other than the fruit. The leaves have
attracted particular attention and the phenolic profile of many
medicinal plants has been studied. For instance, the amounts of
both free and bound phenolic acids were determined in Ginkgo
biloba L. leaves169 using a special extraction procedure,
comprising acid and alkaline hydrolyses. Ferulic acid and pcoumaric acid in 14 forbs were fractionated170 after methanol
extraction into four fractions: free phenolic acids extracted into
diethyl ether, ester-bound phenolic acids after alkaline hydrolysis, glycoside-bound phenolic acids after acid hydrolysis and
cell wall-bound phenolic acids after alkaline hydrolysis of the
solid residue remaining from the extraction with methanol. The
cell wall-bound phenols were quantitatively the most important
fraction. Extraction, alkaline and acid hydrolysis have been
combined with purification on a C18 cartridge171 to determine
flavonoids, phenolic acids and coumarins in seven medicinal
species. SPE has also been used172 to isolate phenols from leaf
tissue of Myrtus communis L. The leaf tissue contained small
amounts of phenolic acids (caffeic, ellagic and gallic acids) and
quercetin derivatives (quercetin 3-O-galactoside and quercetin
3-O-rhamnoside), whereas catechin and myricetin derivatives
were present in large amounts.
The isolation and identification of phenolics in olive leaf
have also attracted considerable attention as a source of
phenolic compounds.173 Moreover, the leaf is the primary site
of plant metabolism at the level of both primary and secondary
plant products. In an early report, Gariboldi et al.174 macerated
fresh leaves in methanol for 1 week at room temperature. The
solvent was evaporated under nitrogen and the extract reconstituted in aqueous acetone and successively extracted with
pentane, chloroform and ethyl acetate. The chloroform extract
was fractionated by column chromatography to yield two
secoiridoids. Three flavonoid glycosides, quercitrin, rutin and
luteolin-7-glycoside, one flavonoid aglycone, luteolin, and
chlorogenic acid were identified175 in olive leaves following
extraction (24 h) with aqueous methanol or ethanol to recover
flavonoids and flavonoid glycosides or biflavonoids, respectively.
Alcoholic extraction (methanol or ethanol) of fresh foliage or
freeze-dried material has been the usual approach to the
recovery of phenols from olive leaf. The extract was concentrated176 in a vacuum under a stream of nitrogen, keeping the
temperature below 35 °C until it reached a syrupy consistency
and partitioned in acetonitrile–hexane. Evaporation to dryness
afforded a yellowish foam that dissolved in methanol. Compound identification was achieved using atmospheric pressure
ionization tandem mass spectrometry. Akillioglu and Tanrisever177 used TLC to examine the phenolic profile of olive
shoots in two olive cultivars. Central leaves and axillary buds of
the shoots were studied. Samples were dried (method not
specified) and phenols recovered by extraction with aqueous
ethanol (96%). The phenolic compositions of the leaves and
buds were found to be different, and of the total 59 compounds
identified in the extracts, 30 were specific to leaves, 24 to buds
and the remaining five were common to both organs. Such
differences indicate that the leaves and buds exhibit distinct
Analyst, 2000, 125, 989–1009
999
metabolic functions. A large number of leaf phenolics were
found to be phenylpropanoids, which are known to be
precursors in the lignin biosynthetic pathway, and act as either
promoters or inhibitors of olive growth.
Supercritical fluid extraction (SFE)178,179 has a number of
advantages and has been used in a two-step fractionation of
leaves of rosemary and sage into an essential oil and antioxidant
fraction. Phenols have also been isolated from dried (100 °C),
ground and sieved (@500 mm) olive leaf using supercritical
carbon dioxide modified with methanol.180 The influence of
extraction variables such as modifier content, pressure, temperature, flow rate, extraction time and collection/elution
conditions was studied. The dynamic SFE method produced
clean extracts with higher phenol recoveries (measured as total
phenols by Folin–Ciocalteu method) than sonication in liquid
solvents such as hexane, ethoxyethane and ethyl acetate.
However, the extraction yield obtained was only 45% of that
obtained with liquid methanol. The extracts were screened for
acid compounds such as carboxylic acids and phenols using
electrospray ionization mass spectrometry (ESI-MS) in the
negative ionization mode.
The phenolic content of olive leaf hairs has also been
investigated, and the role of these hairs in plant protection is
diverse. Spectrophotometric analysis of methanolic extracts of
olive leaf hairs indicated the presence of UV-screening
pigments, which have been characterized as phenolics with a
considerable flavonoid contribution.181 Flavonoids including
luteolin, apigenin and quercetin in their glucoside and aglycone
forms were detected and it is believed that such compounds play
an important role in UV-B radiation shielding properties
exhibited by olive leaf hairs. Further investigation has shown
that the UV-B radiation absorptive capacity and the phenolic
content of leaf hairs declines considerably with leaf age.182 The
high UV-B absorptive capacity of the hairs of young leaves thus
indicates a metabolic priority for flavonoid production during
the early phases of leaf development. The number of leaf hairs
was also found to decrease with maturation. Young leaves may
therefore be more prone to damage by UV-B radiation, hence
the greater number of leaf hairs for protection.182
3 Quantification
The need for analyte recovery must be considered in the context
of the quantification procedure as it is ultimately related to the
limited specificity and sensitivity of analytical procedures
(Table 2). Quantification is used here in the broadest sense to
include methods where characterization or identification was
the primary goal.133,224–226 In such instances, measurement of
an amount of substance is often precluded by the number and
diversity of phenolic compounds (and corresponding lack of
reference compounds). Quantification procedures227 are universally applicable to phenolic extracts regardless of species or part
of the plant.
Traditional methods for the determination of total phenols
have relied on direct measurement of absorption of radiation in
the ultraviolet or, more commonly, colorimetric methods using
Folin–Ciocalteau reagent. This reagent, however, is not specific
for phenols and hence other compounds may interfere.228
Moreover, the diversity of phenolics means that selection of a
reagent and/or absorbing wavelength will be a compromise,
although this is less of a problem where a single class of
phenolic predominates. Results are expressed in terms of molar
equivalents of a commonly occurring phenolic, e.g., gallic or
caffeic acid.229
There is generally no correlation104 between data for total
phenols and those obtained by chromatographic techniques,
although the results obtained by colorimetry are usually higher
than the latter. The number and diversity of phenols in a typical
1000
Analyst, 2000, 125, 989–1009
extract mandates a high resolution technique for their separation
and identification. Hence traditional methods based on colorimetry have been replaced in many instances by high resolution
chromatographic analyses to provide profiles and identification
of individual phenolics. Akillioglu and Tanrisever177 used TLC
to characterize the phenolic profile of olive buds and leaves in
two different cultivars. Sample extracts were separated by
cellulose TLC using two-dimensional development with aqueous butanol containing acetic acid followed by aqueous acetic
acid. Phenolics were characterized by RF values and their
fluorescent colours under UV radiation and the variation in
colour when treated with ammonia fumes and Naturstoff
reagent (1% ethanolic solution of diphenylboric acid B-amino
ethyl ester) under both UV radiation and daylight.
Polymethoxylated flavones possess the stability and volatility that makes GC a viable alternative for their analysis. For
this purpose, packed columns are unsuitable,230 whereas high
efficiency open tubular columns are ideal,61,132,231 producing
excellent separations of the flavones extracted from orange peel
oil. Newer stationary phases232 offer improved retention and
selectivity for these compounds but their main advantage is the
low stationary phase bleed that permits operation at elevated
temperatures with minimum interference in the detection
process. This greatly facilitates the use of coupled GC-MS.
For other phenols a derivatization step prior to GC is
generally mandatory. Nevertheless, the excellent resolving
power and detection capabilities of GC and particularly GC-MS
have been exploited for the analysis of phenolic acids and other
phenols159,196 as trimethylsilyl or trifluoroacetate derivatives
(Table 2). Angerosa et al.120 showed GC-MS to be an effective
tool for identification of phenols as their trimethylsilyl derivatives following extraction from olive oil with methanol. Soleas
et al.10 also used this derivatizing agent for the analysis of 15
biologically active components in wine by GC-MS using one
target and two qualifying ions for each compound. Ions were
chosen for each compound on the basis of their abundance,
reproducibility, freedom from interference and specificity to the
particular compound. The molecular ion (M+) was preferred
when found in appreciable abundance. Resolution of all 15
phenolic compounds was excellent and the method should be
appropriate for the determination of phenolics in a range of
fruits.
Other methods have been reported233 but have not found
general acceptance. For instance, capillary zone electrophoresis
(CZE) and micellar electrokinetic capillary chromatography
have been used to separate phenolic compounds (ref. 101 and
references cited therein). The majority of these separations used
buffers at pH 8.0–10.5 that are suitable for the majority of
phenols with pKa values between 8 and 10 but are unsuitable for
pH-sensitive anthocyanins. Anthocyanins were measured in
blackcurrant juice101 by CZE under strongly acidic conditions
favouring the red-coloured flavylium cationic form. Under
these conditions, the anthocyanins were selectively detected by
their absorbance at 520 nm.
In contrast, reversed-phase HPLC avoids the need for
derivatization 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 to
the diversity of phenols in most extracts. Typical mobile phases
include methanol, water and acetic acid combinations that are
used in gradient elution techniques.8,9,116,122 Detection of
phenolics by HPLC is based on measurement of absorption of
radiation in the UV or visible region (anthocyanins). The most
common wavelength for general detection has been
280 nm,116,122,234 although other wavelengths have been used
for the identification of specific phenolics.54,155,158 For example, phenols have been quantified at characteristic wavelengths
as cinnamic acids (320 nm), flavonols (360 nm) and other
phenols (280 nm).82 The paucity of reference compounds
creates difficulties in quantification that have been solved by
Table 2 Conditions used for the determination of phenols in fruits
Sample
Extraction
Quantification
Levels of phenols identified; detection limits
Ref.
Grape
Grape berries protected from
oxidation and crushed but
avoiding damage to seeds,
filtration
Filtration and direct injection
except for procyanidins
where isolation on
Sephadex LH-20 column
HPLC-DADa, 320 nm
Caftaric acid 178–370 mg L21, cis + trans-coutaric acid
66–110 mg L21 and trans-fertaric acid 4–17 mg L21
183
Colorimetry; HPLC-DAD,
280 and 320 nm
Total phenols 99 and 380 mg L21 (by HPLC and
colorimetry, respectively).
Phenolic acids (trans-isomers) and flavonol glycosides
(oxidation of caftaric acid to 2-S-glutathionylcaftaric
acid was evident) (enzymatic clarification caused
hydrolysis of caftaric, coutaric, and quercetin
derivatives)
Identification of anthocyanins (no quantitative data)
104
Grape juice
Grape
Grape skin
Grape
Grapevine leaf
Series of liquid–liquid and
liquid–solid extractions
Aqueous methanol extraction
LC-MS; HPLC-DAD
anthocyanins
Total phenols 10 000–60 000 mg
0–40 000 mg kg21 and flavonols 1000–5000 mg kg21
(fresh mass)
Total phenols 2000–20 000 mg GAEb kg21 (dry mass)
Flavones and flavonols, e.g., kaempferol trace–2000
mg kg21, myricetin trace–2000 mg kg21, quercetin
600–4000 mg kg21 (dry mass)
Spectrophotometry; GC of
TMSc derivatives
Rutin 7.3, hesperidin 112 and naringin 37 mg L21
188,
189
HPLC-DAD, 280 nm
Quantitative data for several catechins, e.g., red wine,
(+)-catechin 115 mg L21, (2)-epicatechin 76
mg L21; white wine, (+)-catechin 10 mg L21,
(2)-epicatechin 5 mg L21
trans-Resveratrol
DLe 8 ng (TICf ); 200 pg (SIMg)
trans-Resveratrol: red wines, 0.4–1.6 mg L21; white
wines, 0.03–0.14 mg L21. DL 0.015 mg L21
Total phenols 70–250 mg L21
Vanillic acid, gentisic acid, m- and p-coumaric acid,
gallic acid, ferulic acid, caffeic acid, cis- and
trans-resveratrol, epicatechin, catechin, morin,
quercetin and cis-and trans-polydatin. DL 24–843
mg L21
Quantitative data for phenolic acids: gentisic, vanillic,
ferulic, m-coumaric, p-coumaric, caffeic, and gallic
acid; flavonoids: catechin, epicatechin, quercetin and
morin
Hydroxytyrosol 2–4 mg L21. DL 15 pg mL21
Quercetin: wine < 0.5–16 mg L21; fruit juice 2.5–13
mg L21. Myricetin: wine < 0.5–9.3 mg L21; fruit
juice < 0.5–6.2 mg L21
Flavonoids
190
Wine
Wine
Tandem SPEd
LC-MS
Wine
None
Wine
Wine
None
Diluted, SPE on C8 cartridge
eluting with ethyl acetate
HPLC, 288 nm (cis-isomer),
308 nm (trans-isomer)
Colorimetry
GC-MS of TMS derivatives
Wine
Dilution and SPE
GC-MS of TMS derivatives
Wine
Wine and fruit
juice
SPE on C18
Hydrolysis in acidic methanol
GC-MS of TMS derivatives
HPLC-DAD
Passionfruit
Acid hydrolysis of methanolic
extract
Pulp filtered, centrifuged to
produce a clear juice;
column chromatography
and enzymatic hydrolysis
See ref. 21
Various, e.g., aqueous
methanol extraction
containing
diethyldithiocarbamate
followed by SPE
Not applied
HPLC
Extraction from powdered
drupes
Purple
passionfruit
Passionfruit
Olive fruit, virgin
olive oil,
vegetation
waters, and
pomace
(Olive mill
wastewater)
Olive
Olive oil
Aqueous methanol extraction
of hexane solution
Olive leaf
Olive oil
Methanol extraction
Methanol extraction
Olive oil
Methanol extraction followed
by partitioning between
acetonitrile and hexane
184
HPLC, 520 nm;
spectrophotometry, 280,
355, 535 nm
Colorimetry, 765 nm
HPLC, 340 nm
Extraction
Light petroleum wash
followed by aqueous
methanol extraction and
fractionation by column
chromatography
Methanol extraction of pulp
and skins; column
chromatography or TLC
Filtration
Grape and citrus
fruit
kg21,
GC-MS of trifluoroacetylated
derivatives
Semi-quantitative data
185
186
187
30
191
12
10
192
31
193
34
21
HPLC-DAD
Cinnamic acid 0.9–3.7 mg kg21
Quantitative data for several phenols recovered by
different methods
194
140
LC-MS
See Table 4
195
HPLC, 280 nm (oleuropein);
340 nm (quercetin and
luteolin glycosides)
Colorimetry, 725 nm (total
phenols), 370 nm (odiphenols)
LC-MS-MS
GC and GC-MS of TMS
derivatives; HPLC, 232 and
278 nm
GC-MS of TMS derivatives
E.g., hydroxytyrosol 200–1100 mg kg21; oleuropein
900–2100 mg kg21 (pulp)
54
Total phenols 150–350 mg kg21 as caffeic acid
equivalents
123
Oleuropein and ligstroside (no quantitative data)
Identification of phenols (no quantitative data)
176
120
Identification of aglycones (no quantitative data)
125
Table 2 continued next page
Analyst, 2000, 125, 989–1009
1001
Table 2 Continued
Sample
Extraction
Quantification
Levels of phenols identified; detection limits
Ref.
Olive oil
Aqueous methanol extraction
from hexane solution of oil
followed by SPE
fractionation; acid and base
hydrolysis
Boiled in 2 M HCl and ethyl
acetate extraction
Aqueous ethanol extraction
with bisulfite; hexane
partitioning and SPE
Colorimetry; HPLC
Total phenols 60–80 mg kg21 as caffeic acid
equivalents; o-diphenols 16–20 mg kg21
126
GC of TMS derivatives
Various phenolic acids 1–100 mg kg21 fresh mass
196
LC-MS; HPLC-DAD
E.g., verbascoside 100–3500 mg kg21; anthocyanic
compounds, and oleuropein derivatives 36–2400
mg kg21; rutin 110–270 mg kg21; vanillic acid 2–6
mg kg21; tyrosol 100–1200 mg kg21; hydroxytyrosol
570–4100 mg kg21 (fresh mass)
Qualitative data on tyrosol, 4-hydroxyphenylethanol
glucoside and oleuropein (halleridone from oxidation,
hydrolysis and cyclization)
156
Olive leaf and
root
Olive
Olive vegetation
water
Fruits
Berries
Chloroform wash to remove
epicuticular waxes,
trituration and maceration
in water. Aqueous solution
filtered
Freeze-dried, acid hydrolysis
containing TBHQ and
liquid extraction
(Enzymatic pectinase
extraction), followed by
aqueous methanol or
aqueous acetone extraction
NMR
HPLC, UV detection
Colorimetry; HPLC, 280 nm
(flavan-3-ols as catechin
equivalents, benzoic acid
derivatives as GAE), 316
nm (hydroxycinnamates as
caffeic acid equivalents),
365 nm (flavonols as rutin
equivalents), 520 nm
(anthocyanins as malvin
equivalents)
HPLC, 260 nm (ellagic and phydroxybenzoic acids), 280
nm (catechins), 320 nm
(hydroxycinnamic acids),
360 nm (flavonols)
Berries
Three extraction and
hydrolysis procedures using
freeze-dried berries
Blueberries
Aqueous acidic methanol
extraction and filtration
HPLC-DAD; GC of
anthocyanidins as TMS
derivatives
Cider apples
Hammer mill, pressed,
clarified and frozen.
Addition of ascorbic acid
and filtration
Hydrolysis in acidified
aqueous methanol
containing TBHQ
Extraction with hot methanol
or, for anthocyanins,
acidified methanol. Extracts
stored at 240 °C
Homogenize, enzyme
treatment, filter
HPLC-DAD, 280 nm
Berries
Berries
Strawberry and
raspberry
juices
Blackcurrant
juice
Berry and fruit
wines and
liquors
Raspberry juice
Raspberry juice
Strawberry
Apple
Commercial powder dissolved
in water
Dealcoholized (wines) and
SPE to remove sugars
SPE, acid and base hydrolysis
Fractionation on Polyamide 6
essential; conventional
systems failed to produce
clean separations
Acetone extraction due to
high pectin content, SPE
Full text not available
Quercetin 1485 mg kg21 dry mass; kaempferol < 20
mg kg21; myricetin 662 mg kg21; luteolin < 10
mg kg21; apigenin < 40 mg kg21
Total phenols 617–4350 mg kg21 GAE (anthocyanin,
blackberries; hydroxycinnamic acid, blueberries and
sweet cherries; flavonol, blueberries; and flavan-3-ol,
red raspberries)
Total phenols 829 and 416 mg kg21 (dry mass) for
strawberry and blackcurrant, respectively. Data for
flavonoids (kaempferol, quercetin, myricetin) and
phenolic acids (p-coumaric, caffeic, ferulic,
p-hydroxybenzoic, gallic and ellagic acids). DL
2–5 ng
Total anthocyanins 1100–2600 mg kg21 (non-acylated
glucosides and galactosides of delphinidin, cyanidin,
petunidin, peonidin, and malvidin); chlorogenic acid
500–1000 mg kg21 (fresh mass)
Total phenols 1.2 g L21 as tannic acid; main phenols,
chlorogenic acid 130 mg L21; (2)-epicatechin 50
mg L21; procyanidin B2 40 mg L21
197
41
198
152,
199
97
200
LC-MS; HPLC-DAD
Quercetin 50–200 mg kg21, myricetin 14–140 mg kg21,
kaempferol 5–20 mg kg21 (fresh mass)
151
Spectrophotometry
Total phenols 5–28 mmol g21 gallic acid, anthocyanins
0.2–4.4 mmol g21 malvidin-3-glucoside (fresh mass)
201
HPLC-DAD; 520 nm
(cyanidin), 370–600 nm
(anthocyanidins), 505 nm
(pelargidin), 280 nm
(flavonol), 355 nm (ellagic
acid)
Capillary zone electrophoresis
Anthocyanins, flavonols and ellagic acids
Colorimetry
Total phenols 91–1820 mg L21 GAE
HPLC (see ref. 104)
HPLC, various systems, 260
nm (ellagic acid), 360 nm
(flavonols)
Data for several phenols as percentage of total peak area
Quercetin 30–210 mg L21; kaempferol 2–6 mg L21
(data quoted for various glycosides)
38
202
HPLC-DAD, 240–550 nm
Anthocyanins 120 mg kg21; quercetin derivatives 40–60
mg kg21; kaempferol derivatives 14–22 mg kg21
(fresh mass)
Total phenols 586–1570 mg kg21 (major phenols in
flesh, catechins including proanthocyanidins)
203
Anthocyanins. DL 25 mg L21
83
101
81
204
Table 2 continued next page
1002
Analyst, 2000, 125, 989–1009
Table 2 Continued
Sample
Extraction
Quantification
Levels of phenols identified; detection limits
Ref.
Apple skin
Extraction of ground apple
peel with acidified
methanol
Quantitative data for quercetin glycosides and
proanthocyanidins
205
Apple
Aqueous methanol extraction
of freeze-dried material (no
pericarp)
Methanol extraction
HPLC, 350 nm (flavonols),
530 nm (anthocyanins), 280
nm (proanthocyanidins),
313 nm (phenolic acids)
HPLC-DAD 280, 350 nm;
200-600 nm post-run scan
Chlorogenic acid, procyanidins/catechin compounds,
rutin and phloridzin
206
Chlorogenic acid, caffeic acid, catechin, epicatechin,
rutin, phloridzin, procyanidin
Total cinnamic acids (by HPLC) 50–160 mg L21.
Chlorogenic acid 20–110 mg L21
29
Apple and pear
Apple juice
Apple and grape
Filtration and direct injection
except for procyanidins
where isolation on
Sephadex LH-20 column
Aqueous methanol extraction
Apple
Ethanol extraction with
metabisulfite and ethyl
acetate partitioning
Apple
Acidified aqueous methanol
extraction of powdered skin
Cider apple
tissues
Freeze-dried, successive
methanol and aqueous
acetone extraction,
thiolysis. Butanol/
hydrochloric acid
hydrolysis for procyanidins
Methanol extraction
Apple peel and
pulp
Apple musts and
ciders
SPE fractionation into neutral
and acidic fractions
HPLC-DAD, 280 nm
Colorimetry; HPLC-DAD,
280 and 320 nm
HPLC, UV (270 nm) or
fluorescence (280/310 nm
excitation/emission)
HPLC, 280 nm (flavan-3-ols,
dihydrochalcones), 320 nm
(hydroxycinnamic
derivatives and flavonols)
HPLC, 280 nm
(proanthocyanidins), 350
nm (flavonols), 530 nm
(anthocyanins)
Colorimetry; HPLC-DAD,
540 nm (procyanidins), 280
nm (other phenols); LC-MS
HPLC-DAD, 280 nm
HPLC-DAD; 280 nm
(polyphenols), 320 nm
(cinnamic acids), 360 nm
(flavonols)
HPLC-DAD, 280, 350 nm
Quince, pear and
apple purees
Pear
Dilution and column
chromatography
Aqueous ethanol extraction of
powdered fruit; clean-up by
liquid–liquid extraction
Peach and apple
purees and
concentrates
Peach
Homogenized in aqueous
methanol, dried and
extracted with ethyl acetate
Full text not available
Peach and
nectarine skin
Methanol extraction
Colorimetry; HPLC
Apricot
Aqueous methanol or ethanol
extraction of pulp
Acid hydrolysis in methanol
Extraction with acidic aqueous
methanol
GC-MS of TMS derivatives
Anthocyanins
Sweet cherry
Orange and
grapefruit
Sour orange
Grapefruit and
pummelo
Citrus
HPLC-DAD, 325 nm
(hydroxycinnamic acids),
280 nm (flavanols), 360 nm
(flavonols)
HPLC-DAD, 210–360 nm
Spectrophotometry and HPLC
HPLC-DAD
HPLC, 280 and 525 nm (and
GC)
Aqueous alcohol extraction
followed by alkaline
hydrolysis
Aqueous ethanol extraction
HPLC, 300 nm
Extraction of dried material
with dimethyl sulfoxide
Extraction with methanol–
dimethyl sulfoxide, SPE
HPLC-DAD
LC-DAD-MS
HPLC-DAD, 285 nm
207
Catechins. DL 0.1–3.9 mg kg21
148
Chlorogenic acid 180–1700 mg kg21 dry mass (many
others reported)
208
Anthocyanins 0–2600 mg kg21, total flavonoids
500–1300 mg kg21 (fresh mass)
209
Quantitative data for various tissues for
hydroxycinnamic acid derivatives, flavan-3-ols,
flavonols, and dihydrochalcones. Total phenols
(summation) (whole apple) 5000 mg kg21
153,
210
Peel: catechins and flavonol glycosides. Pulp:
chlorogenic acid; DL 0.21–0.63 mg L21
Must: chlorogenic acid, catechin, cinnamic ester,
procyanidin, phloridzin, rutin, quercetin
Cider: hydroxycoumaric and hydroxycinnamic acids
211
E.g., rutin 20 mg kg21 (quince), 4.7 mg kg21 (apple)
92
Hydroxycinnamic acids 30–90 mg kg21; flavanols
20–160 mg kg21; flavonols 50–150 mg kg21 (fresh
mass)
212
Quantitative data for cinnamic acids and their
derivatives, flavan-3-ols, procyanidins, flavonols and
dihydrochalcones
Phenolic acids (quinic was the predominant phenolic
acid, followed by gentisic, catechuic, chlorogenic and
syringic acids)
Total soluble phenols 5000–8000 mg kg21; chlorogenic
acid 930–2400 mg kg21; epicatechin 2700–4600
mg kg21; catechin 660–150021 (GAE dry mass);
total anthocyanins 1000–1300 mg kg21 (as
cyanidin-3-glucoside, dry mass)
Caffeoylquinic acid 1800 mg kg21; quinic acid 3000
mg kg21 (dry mass)
Formation of methyl esters following release of acids
Total anthocyanins 20–3000 mg kg21 fresh mass
(3-rutinoside and 3-glucoside of cyanidin as the
major anthocyanins and the same glycosides of
peonidin as minor anthocyanins), neochlorogenic acid
240–1280 mg kg21 and p-coumaroylquinic acid
230–1310 mg kg21
Bound and free sinapic, ferulic, coumaric and caffeic
acids
Identification of various flavonoids (no quantitative
data)
Narirutin 80–2150 mg kg21, naringin 14 000–
21 000mg kg21, neohesperidin 110–220 mg kg21
Data for several flavonoids in various tissues
82
20
213
214
215
149
13
42
147
146
79
Table 2 continued next page
Analyst, 2000, 125, 989–1009
1003
Table 2 Continued
Sample
Extraction
Quantification
Levels of phenols identified; detection limits
Blood orange
HPLC, 280 nm
Narirutin 29–42 mg L21, hesperidin 180–392 mg L21,
didymin 9–31 mg L21, cinnamic derivatives 2–18
mg L21, trans-cinnamic acid 0.1–0.7 mg L21
63
HPLC, 280 nm
Free acids 0.5–5.0 mg L21; total acids 21–43 mg L21
43
Orange juice
Dilution in
dimethylformamide–
ammonium oxalate solution
and centrifugation; SPE
concentration for transcinnamic acid
Free acids: acidify, ethyl
acetate extraction. Total
acids: alkaline hydrolysis in
dark and ethyl acetate
extraction
Squeeze and filtration
SPE C18
Red wine, beer,
apple cider,
and sour
cherry and
blackthorn
fruit liqueurs
Fruit
Filtration
HPLC, UV at 280 nm and
post-column reactor with
absorption at 640 nm
Total phenols: 360–1200 mg L21. Total anthocyanins:
1–280 mg L21
Phenolics present at 1–7 mg L21 (as derivatives of
coumaric, chlorogenic and protocatechuic acid and a
derivative of 3,4-dihydroxybenzoic acid); epicatechin,
catechin, and procyanidins (B3, B2, or B4 and
oligomers). Flavonols as glycosides of quercetin and
kaempferol
Flavanol profiles
216
Kiwifruit juice
Colorimetry, Folin–Ciocalteu;
HPLC, 300 nm
HPLC
Centrifugation and dilution
HPLC, coulometric array
detector
218
Areca fruit
Aqueous acetone extraction
Fruit jams
Acidified aqueous methanol
extraction, SPE
Mixture of standards to
simulate juice
None
Total phenols: colorimetry at
735 nm by Folin–Ciocalteu
method. Condensed tannins:
colorimetry at 500 nm with
vanillin–HCl
HPLC, 520 nm
Data presented for phenolic acids and flavonoids as
number of peaks in chromatogram and total peak
area. DL 0.02–1 ng
Total phenols 580 mg kg21 GAE; condensed tannin
0.85 mg of catechin equiv. g21 (fresh mass)
Quantitative data for anthocyanins in several jam
varieties
Kaempferol-3-rutinoside, rutin, avicularin, quercitrin,
isoquercitrin, isorhamnetin, kaempferol and quercetin
Hydroxycinnamic acids
220
Blood orange
juice
Fruit juices and
wines
Grape must,
apple and
peach
Cherry laurel
Libanotis
dolichostyla
fruit
Diospyros lotus
L. fruit
Light petroleum extraction of
powdered mesocarp,
followed by alkaline
hydrolysis of residue under
nitrogen and ethyl acetate
recovery
SPE
Capillary electrophoresis
HPLC-DAD
Ref.
68
217
219
221
222
GC-MS of TMS derivatives
Data reported as percentages of extract for vanillic,
protocatechuic, p-hydroxybenzoic, caffeic and
p-coumaric acids
45
HPLC
Phenolic acids
80
Light petroleum extraction of
223
GC-MS of TMS derivatives
E.g., Gallic, salicylic, vanillic, p-coumaric and syringic
powdered mesocarp;
acids. Typical levels 300–54 000 mg kg21
alkaline hydrolysis and
ethyl acetate extraction
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 ion
current. g SIM, selected ion monitoring.
‘normalization’,38,45 synthesis or isolation from the sample156
of the relevant phenols or the use of a phenol belonging to the
same class.185,212 For instance, malvidin-3-glucoside was
used185 as the reference compound for the HPLC quantification
of five anthocyanins in grape skins while phloretin glycosides
were quantified20 as phloridzin in apple and peach products.
Similarly, a single reference compound for each phenolic class
was used212 to quantify hydroxycinnamic acids, flavanols and
flavonols in pear by HPLC. In qualitative studies where profiles
are compared, there is often no allowance for variation in
detector response in presenting peak area data. It is not
uncommon that the largest peak is assumed to represent the
phenol with the highest concentration. However, the molar
absorptivities vary greatly between phenols (Table 3) and there
is a need for greater recognition of this variation. The effect of
1004
Analyst, 2000, 125, 989–1009
detection wavelength on the chromatographic profile of olive
leaf phenols is illustrated in Fig. 3.
Flavan-3-ols are of particular interest in beverages where
they are often the cause of instability and turbidity. Their
determination by HPLC with UV detection is prone to
interference by other phenols present at higher concentrations.
The use of a post-column reactor has been employed217 in
which p-dimethylaminocinnamaldehyde condenses with flavanols giving intensely coloured adducts showing maximum
absorption between 632 and 640 nm. The reagent shows both
high specificity and sensitivity for flavanols. Fluorescence72,148
also provides selective and sensitive detection but it has rarely
been used.
GC and HPLC have been compared235 for the determination
of resveratrol in wines. Higher results were obtained by GC
Table 3 Absorption characteristics of selected phenols79,116,152
Phenol
Wavelength/
nm
Molar absorptivitya
(at 280 nm)/
L mol21 cm21
p-Hydroxyphenylacetic acid
275
1 515
Tyrosol
276
1 517
Hydroxytyrosol
281
2 307
3,4-Dihydroxyphenylacetic acid 281
3 109
p-Hydroxybenzoic acid
256
4 143
Protocatechuic acid
260, 295
4 160
Vanillic acid
261, 293
5 210
Caffeic acid
323, 300
10 791
Syringic acid
275
10 891
p-Coumaric acid
306
11 475
o-Coumaric acid
277, 325
17 704
a Measurements collected in solutions of methanol–water + 3% acetic acid
(6+94 v/v).
(0.64–3.00 mg L21) than by HPLC (0.30–2.89 mg L21)
analysis. The GC analysis separated the cis- and trans-isomers
but with longer sample preparation times than required in HPLC
analysis. The cis+trans ratio ranged between 0.5 and 0.9 and
trans-resveratrol underwent a photochemical isomerization
during ripening of the grape berries and also during the process
of winemaking.
Classical ionization techniques have limited application for
the analysis of underivatized plant phenols and will not be
discussed. In contrast, the development of soft ionization
techniques, such as atmospheric pressure ionization (API), for
the investigation of polar, non-volatile and thermolabile
molecules has facilitated the analysis of phenolic compounds by
LC-MS. At this stage, most applications (see Table 2) have
involved qualitative analyses but LC-API-MS can be expected
to revolutionize quantitative determinations as the fundamentals of the technique are more firmly established. Atmospheric pressure chemical ionization (APCI) uses a combination
of a heated capillary and a corona discharge to promote the
formation of ions from the nebulized sample. In coupled mode,
the eluate from the HPLC system is evaporated completely and
the mixture of solvent and sample vapour is then ionized in the
gas phase by ion–molecule reactions and follows the sequence
sample in solution ? sample vapour ? sample ions. Ion
formation is via chemical ionization involving proton transfer,
adduct formation and charge exchange reactions in positive ion
mode or proton abstraction, anion attachment and electron
capture reactions in the negative ion mode.
Aramendía et al.195 reported the LC-API-MS of phenolics
found in olive mill wastewater. Analytes were separated on a
C18 phase by gradient elution with methanol–water containing
formic acid. Mass spectral conditions were optimized by direct
infusion of standards in the flow injection mode into the APCI
source. The study was restricted to the negative ion mode with
detection limits (Table 4) in the total ion current mode ranging
from 0.5 to 500 ng. These detection limits were about 20 times
better when working in the selected ion monitoring mode and
monitoring the [M 2 H]2 ion. Mass spectra were recorded with
soft (215 V) and strong (250 V) voltages applied at the ion
source of the mass spectrometer. With the lower voltages,
deprotonated molecular species [M 2 H]2 were the major ions
observed in the mass spectra with the appearance of very few
fragment ions that were all of low intensity. The presence of
substantial fragmentation from collisionally induced dissociation processes which became evident on increasing the voltage
applied at the source (extraction and cone) voltages gave
structural information about the molecules. Structures were
assigned to major eluent cluster ions from methanol–water–
formic acid mixtures occurring at m/z 91, 113, 137, 159, 181 and
183.
APCI still has the major drawback for polar thermolabile
plant phenols that volatilization of the sample must occur before
ionization. ESI overcomes lack of analyte volatility by direct
formation or emission of ions from the surface of a condensed
phase and sample ions are collected from the condensed phase
inside the ion source and transferred to the mass analyzer.
Hence ESI eliminates the need for neutral molecule volatilization prior to ionization. ESI is used as a generic term that also
covers several variants of the basic technique that differ in the
precise manner in which charged droplets of sample are
produced. The mechanism of ESI remains controversial but in
the meantime these techniques collectively have revolutionized
the field of mass spectrometry and its application to analysis of
plant 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 fragmentation
although fragmentation can often be induced by raising the cone
voltage. Acid (acetic or formic) is often added to mobile phases
in positive ion ESI as a source of protons to assist ionization.
Sensitivity is improved when the organic content in the mobile
phase exceeds 20%. LC-ESI-MS has been used236 to study
flavonol aglycones and glycosides in berries. ESI provided
information on the structures without the need for derivatization. Quercetin was identified in all berries. Our work shows
that the negative ion mode generally provides improved
detection limits but there is a need to establish optimum
conditions for each phenol.
4 Future needs—transfer to industry
Current research on fruit phenolics is driven by three major
forces and these impact on the choice of sample preparation and
the importance attached to this step in the overall analysis. One
is simply to discover new compounds, and as diverse and
numerous as they are now, there is seemingly endless scope for
isolating and identifying new and novel compounds as the
sensitivity of analytical techniques is improved. A second area
concerns the understanding of the role of phenolics as
secondary metabolites within the plant. Third, there is interest in
their antioxidant properties, as alternatives to synthetic antioxidants in the food industry, and as components of the human
diet. In all three areas, sample preparation is rarely critical
although quantitative extraction will enhance the amount of
phenolic available. Of the three areas, only the last has links to
industry, yet even here quantification of naturally occurring
phenolics is not routinely undertaken, in sharp contrast to the
situation with the synthetic antioxidant phenolics.
Macronutrients such as carbohydrates, proteins, and fatty
acids are regularly quantified because they directly impact on
the quality of a finished product. As micronutrients, measurement of phenolics is not seen as important in processing (this is
despite the fact that in many cases food spoilage is linked to
phenolic ‘browning’ reactions). However, the current trend
towards consumer awareness of, and demand for, foods with
beneficial health properties (so-called ‘functional foods’) may
see the quantification of phenolics become increasingly important.
Adulteration represents another area of the food industry
where quantification of phenolic compounds has potential.
Recently Valentão et al.237 reported on the determination of
Vervain flavonoids in the context of quality control. Quantification was carried out with reference to standards and the authors
suggested that because quantification of the flavonoids was
possible it may be applied to quality control. Adulteration of
citrus juices is another area where phenolic quantification may
Analyst, 2000, 125, 989–1009
1005
Fig. 3 HPLC of phenols extracted from leaves of the Manzanillo olive tree showing the effect of detection method on the resulting profile. Samples were
obtained by aqueous methanol (1 + 1) extraction of freeze-dried material. The extract was washed with hexane and injected on to a C18 column using an
aqueous methanol gradient for elution.
be undertaken; however, while the potential is there, it is yet to
come into routine use. As the demand for routine quantification
of phenolics is realized, the emphasis placed on sample
1006
Analyst, 2000, 125, 989–1009
preparation will increase. In the meantime, research studies on
phenols would be well advised to examine this aspect more
closely.
Table 4 Detection limits by LC-APCI-MS using the negative ion mode
(signal-to-noise ratio = 3)195
30
31
Limit of detection/ng
32
Phenol
Scan mode
SIM mode
Gallic acid
Protocatechuic acid
Tyrosol
p-Hydroxybenzoic acid
p-Hydroxybenzaldehyde
Vanillic acid
Syringic acid
p-Coumaric acid
Ferulic acid
40
1
200
200
0.5
100
70
25
80
2
0.05
12
10
0.03
18
3
1
4
33
34
35
36
37
38
5 Acknowledgements
The financial support of Rural Industries and Horticultural
Research and Development Corporations, Australia, is gratefully acknowledged. The assistance of Professor Shimon Lavee,
Israel, in providing the olive leaf sample for Fig. 3 is noted.
39
40
41
42
43
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