Journal of Chromatography A, 1217 (2010) 3258–3268

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Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma

Isolation of phenolic compounds from hop extracts using

polyvinylpolypyrrolidone: Characterization by high-performance liquid
chromatography–diode array detection–electrospray tandem mass spectrometry
Paulo J. Magalhães ∗ , Joana S. Vieira, Luís M. Gonçalves, João G. Pacheco, Luís F. Guido, Aquiles A. Barros
REQUIMTE, Departamento de Química da Faculdade de Ciências da Universidade do Porto (FCUP), Rua do Campo Alegre, No. 687, 4169-007 Porto, Portugal

a r t i c l e i n f o a b s t r a c t

Article history: The aim of the present work was the development of a suitable methodology for the separation and
Available online 30 October 2009 determination of phenolic compounds in the hop plant. The developed methodology was based on the
sample purification by adsorption of phenolic compounds from the matrix to polyvinylpolypyrrolidone
Keywords: (PVPP) and subsequent desorption of the adsorbed polyphenols with acetone/water (70:30, v/v). At last,
Hops the extract was analyzed by HPLC–DAD and HPLC–ESI-MS/MS. The first phase of this work consisted
Phenolic compounds
of the study of the adsorption behavior of several classes of phenolic compounds (e.g. phenolic acids,
Polyphenols
flavonols, and flavanols) by PVPP in model solutions. It has been observed that the process of adsorption
Polyvinylpolypyrrolidone (PVPP)
HPLC–DAD
of the different phenolic compounds to PVPP (at low concentrations) is differentiated, depending on the
HPLC–ESI-MS/MS structure of the compound (number of OH groups, aromatic rings, and stereochemistry hindrance). For
example, within the phenolic acids class (benzoic, p-hydroxybenzoic, protocatechuic and gallic acids)
the PVPP adsorption increases with the number of OH groups of the phenolic compound. On the other
hand, the derivatization of OH groups (methylation and glycosylation) resulted in a greatly diminished
binding. The use of PVPP revealed to be very efficient for adsorption of several phenolic compounds such
as catechin, epicatechin, xanthohumol and quercetin, since high adsorption and recovery values were
obtained. The methodology was further applied for the extraction and isolation of phenolic compounds
from hops. With this methodology, it was possible to obtain high adsorption values (≥80%) and recovery
yield values (≥70%) for the most important phenolic compounds from hops such as xanthohumol, cat-
echin, epicatechin, quercetin and kaempferol glycosides, and in addition it allows the identification of
about 30 phenolic compounds by HPLC–DAD and HPLC–ESI-MS/MS.
© 2009 Elsevier B.V. All rights reserved.

1. Introduction and flavonols (e.g. quercetin, kaempferol) which are glycosidically

The hop plant (Humulus lupulus L.) is a dioecious plant of the
Cannabacea family, cultivated in the most temperate zones of
the world for its female inflorescences. Nowadays, the plant is
almost only used in the brewing industry to add bitterness and
aroma to beer [1,2]. For the last 10 years there have been more
and more reports on the beneficial properties of polyphenols in
hops. A considerable amount of effort has been directed toward
determining the flavanoid or polyphenol content in hops because
such is reflected at the end-product and give rise to beer haze [3].
Most of these polyphenols are made up of higher molecular com-
pounds such as the flavonol type tannins. Only about 20% of the
hop polyphenols consist of low molecular substances like cate-
chin or proanthocyanidins, phenolic carbon acids (e.g. ferulic acid)
∗ Corresponding author. Tel.: +351 22 608 2944; fax: +351 22 608 2959.
E-mail address: pauloxenon@gmail.com (P.J. Magalhães).
bound to various sugars [4]. Even resveratrol could be detected in
hops, though at very low concentrations [5]. Combined they repre-
sent about 4–14% (w/w) of the hop dry weight. Furthermore, there
are some polyphenols being almost exclusive to hops. Each plant
has a typical polyphenolic patter and hops have shown to be a very
rich source of prenylated polyphenols (prenylflavonoids) [1,4]. One
of the most important and studied phenolic compounds of this raw
material is the prenylated chalcone xanthohumol (XN) showing
an extraordinary broad spectrum of advantageous activities [4].
This compound is the main prenylflavonoid of hops (0.2–1.1% in
dried hops). In the hop resin, the yellow compound (Greek: xan-
tho = yellow) is accompanied by at least 13 related chalcones, all of
which occur at 10–100 fold lower concentrations relative to XN [6].
Another well-known hop prenylflavonoid is 8-prenylnaringenin
which has been shown to be one of the most potent phytoestro-
gens currently known. However, the content of this compound in
hops is very low (lower than 0.01% in dried hops) [7]. Until nowa-
days, several chemical and biochemical studies have focused on

0021-9673/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.chroma.2009.10.068
 P.J. Magalhães et al. / J. Chromatogr. A 1217 (2010) 3258–3268
Fig. 1. Chemical structure of PVPP – (C6 H9 NO)n = (111.1)n .
Fig. 2. Adsorption of catechin by PVPP through hydrogen bonding between the pro-
ton donor from the polyphenol and the carbonyl group from PVPP, together with
␲-bond overlap (delocalized electrons) polar and hydrophobic interactions between
the aromatic ring of catechin and the PVPP ring.
these prenylflavonoids from hops. Less attention has been paid to
other phenolic antioxidants such as flavanols and phenolic acids
and there is almost no literature data about their identification and
quantification in hops.
It has been known for some time that phenolic compounds
bind to polyvinylpolypyrrolidone (PVPP) (Fig. 1), a commercially
available material produced by cross-linking polyvinylpyrrolidone
(PVP) [8–10]. PVPP is commonly used in beverages (e.g. beer,
wine and juice) production for removal of polyphenols, in order
to prevent the formation of haze in the beverage [10]. This adsor-
bent is biochemically inert and has no known hazards associated
[11]. The adsorption of polyphenols by PVPP is through hydrogen
bonding between the proton donor from the polyphenol and the
carbonyl group from PVPP, together with ␲-bond overlap (delo-
calized electrons) polar and hydrophobic interactions between the
aromatic ring of the polyphenol and the PVPP ring (Fig. 2) [12,13].
The affinity of PVPP increases as the number of phenolic hydroxy
groups increase, as more groups are available for hydrogen bonding
[8,9,12]. The regenerative use of PVPP due to its insolubility in water
is the most important reason for its acceptance in the beverage
processing industry [13].
An important problem that researchers are often faced with
when investigating complex mixtures is the need to isolate the
analytes from natural sources and thoroughly purify the extracts
prior to the analysis. Until nowadays, several techniques for
the extraction, isolation and purification of phenolic compounds
from complex mixtures are often used such as supercritical
fluid extraction (SFE), semi-preparative high-performance liq-
uid chromatography (SP-HPLC), high-speed counter-current liquid
chromatography, precipitation–adsorption and solid-phase extrac-
tion [14–17]. These methods, however, have the disadvantages that
they are time-consuming and only allow small volumes of sam-
ple. In addition, SFE methods require a high investment cost for the
Fig. 3. Chemical structures of phenolic acids.
3259
equipment acquisition and on the other hand, the yield of polyphe-
nols is very low. Since adsorption is a low cost separation technique,
it is preferred for the selective recovery of phenolic compounds
from complex matrices. The use of adsorbents might also be a useful
tool not only to concentrate plant phenolics but also to fractionate
the crude extract or at least to enrich certain compounds. Numerous
of sorbent materials such as cyclodextrin, Sephadex, cellulose triac-
etate, Amberlite XAD and other kind of resins have been applied for
adsorbing valuable polyphenols from plant extracts [14–17]. These
adsorbents, however, have the disadvantage that either have a high
adsorption performance or a high elution performance, but that
do not simultaneously have both a high adsorption performance
and a high elution performance. The main objective of the present
work is to provide a method for extraction and isolation (by adsorp-
tion to PVPP) of phenolic compounds from hop extracts, in order to
facilitate their isolation for subsequent characterization and quan-
tification, removing the prior art drawbacks. The characterization
and quantification of these compounds were reached with high-
performance liquid chromatography with diode array detection
(HPLC–DAD) and tandem mass spectrometry using electrospray
ionization (HPLC–ESI-MS/MS). Mass spectrometry detection is a
powerful tool since it provides molecular weight and structural
information, which can be useful for confirmatory purposes.
2. Materials and methods
2.1. Reagents
The solvents employed for the extraction of the samples were
of HPLC grade: methanol (VWR, Darmstadt, Germany), acetone
(Merck, Darmstadt, Germany) and ethanol (VWR). High-purity
water from a Millipore Simplicity 185 water purification system
(Millipore Iberian S.A., Madrid, Spain) was used for all chemical
analyses and glassware washing.
The Folin–Ciocalteau reagent (Merck) and sodium carbon-
ate (Sigma–Aldrich, Steinheim, Germany) were employed for the
measurement of the Folin–Ciocalteau total phenolic content. The
calibration curve was constructed with gallic acid (Sigma–Aldrich).
For the Vanillin assay, vanillin reagent (Sigma–Aldrich), sulphuric
acid 98% (Merck) and methanol analytical-reagent grade (VWR)
were employed. The calibration curve was constructed with (+)
catechin (Sigma–Aldrich).
For the antioxidant activity assessment, 2,2-diphenyl-1-
picrylhydrazyl (DPPH, Sigma–Aldrich) and methanol analytical-
reagent grade (VWR) were used. The determination of the
reducing power was performed by using potassium ferricyanide
(Sigma–Aldrich), iron (III) chloride hexahydrate (Sigma–Aldrich),
methanol analytical-reagent grade (VWR) and trichloroacetic acid
(TCA, Sigma–Aldrich).
The solvents employed for HPLC and LC–MS analyses were
prepared with methanol and formic acid of HPLC quality (VWR)
and high-purity water (Milli-Q quality). All eluents used were fil-
tered through cellulose filter of 0.45 ␮m pore size (Whattman,
Clifton, USA). Calibration curves were constructed for the fol-
3260 P.J. Magalhães et al. / J. Chromatogr. A 1217 (2010) 3258–3268

Fig. 4. Chemical structures of cinnamic acid and its derivatives.

Fig. 5. Structures and carbon atom numbering system of compounds representing various classes of flavonoids. (+)-catechin and (−)-epicatechin are optical isomers as they
possess two asymmetric carbons.

lowing phenolic compounds: (+)-catechin hydrate (98% purity),
(−)-epicatechin (90% purity), quercetin (98% purity), rutin hydrate
(94% purity), kaempferol (90% purity), benzoic acid (99% purity),
p-hydroxybenzoic acid (97% purity), gallic acid (97% purity), pro-
tocatechuic acid (97% purity), syringic acid (95% purity), cinnamic
acid (99% purity), caffeic acid (99% purity), vanillic acid (97% purity),
p-coumaric acid (98% purity), ferulic acid (99% purity) and sinap-
inic acid (99% purity) from Sigma–Aldrich, xanthohumol (90%
purity) from Hopsteiner (Mainburg, Germany). Structures and triv-
ial names of these compounds which represent various categories
of polyphenols are shown in Figs. 3–5.
PVPP (Polyclar Super R; average particle size = 110 ␮m; specific
surface area = 1.1 m2 /g) was a kind gift from Unicer–Bebidas de
Portugal SGPS S.A. (S. Mamede de Infesta, Portugal) which was
purified by heating (30 min, 100 ◦ C) in an acid solution (12 mol/L
HCl) and further washing with water to pH 7. Surface morphol-
ogy of the polymeric resin was investigated with scanning electron
microscopy (SEM). The sample was submitted to vacuum with
gold/paladium in a JEOL (Tokyo, Japan) JFC 1100 equipment, just
before the examination. The sample was examined with a scan-
ning electron microscope model FEI QUANTA 400 FEG ESEM/EDAX
PEGASUS X4M (Hillsboro, USA). General appearance and surface
morphology of PVPP is demonstrated in Fig. 6.
2.2. Adsorption of phenolic compounds by PVPP in model
solutions
It has been observed in more limited studies [18,19] that binding
is highest in pure water and at pH values sufficiently low to sup-
press ionization of phenolic hydroxyl groups. In these studies, PVPP
was found to bind with each compound more effectively in a water
solution than in solutions containing high content of methanol.
When high concentrations of methanol are used probably there
is a change in the adsorption/desorption of the compounds due

Fig. 6. General appearance and surface morphology of PVPP as viewed under an electron microscope.
 P.J. Magalhães et al. / J. Chromatogr. A 1217 (2010) 3258–3268
to the strong capacity of methanol to remove them from the
PVPP.
Consequently, in this work each phenolic compound was pre-
pared at 0.34 mM in 5% methanol (pH 4.0). However some of the
phenolic compounds, such as xanthohumol, quercetin, rutin and
kaempferol, were insoluble or slightly soluble in pure water, there-
fore in these cases 25% methanol was used. 100 mL of each solution
were mixed with several concentrations of PVPP Polyclar Super R
(0, 0.25, 0.50, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0 and 5.0 mg/mL). The samples
were mixed for 15 min at constant temperature (20 ◦ C) and then
centrifuged at 4500 × g for 5 min. The concentrations of phenolic
compounds in supernatants were further analyzed by spectropho-
tometry. The adsorption efficiency of each phenolic compound by
PVPP was evaluated by determining the percentage decrease in
the absorbance at each specific maximum absorbance wavelength
using the following equation:
A0 − A
Adsorption (%) = × 100 (1)
A0
where A0 is the initial absorbance at specific wavelength and A is
the final absorbance at the same wavelength. Besides the adsorp-
tion process study, another important feature that has been studied
in this methodology was the extraction efficiency of the several
phenolic compounds adsorbed previously by PVPP (desorption).
With this purpose, each compound adsorbed by PVPP was fur-
ther extracted twice with 20 mL of acetone/water (70:30, v/v). The
concentrations of phenolics in supernatants were analyzed by spec-
trophotometry at each specific wavelength. The quantification was
conducted at 280 nm for monomeric flavan-3-ols ((+)-catechin and
(−)-epicatechin), syringic and gallic acid, at 320 nm for the deriva-
tives of cinnamic acid (caffeic, p-coumaric, ferulic and sinapinic
acid), at 250 nm for benzoic, p-hydroxybenzoic, protocatechuic and
vanillic acids and at 350 nm for quercetin, kaempferol, rutin and
xanthohumol. Three independent measurements were made, and
results (recovery yields, %) are presented with means and standard
deviations.
2.3. Extraction of polyphenols from hops
2.3.1. Removal of hydrophobic compounds
Hop cones (30 g) were reduced to powder in a mortar and
consequently extracted with 500 mL of dichloromethane by ultra-
sonication for about 30 min and shaked by vortex for 30 min. The
extract was filtered in a Whatman no.1 filter paper on a Büchner
funnel and the remaining residue was re-extracted with 500 mL of
fresh extraction solvent. The extract was again filtered in a Büchner
filter (Whatman no.1 filter paper). The ground hops (residue) was
kept for further polyphenols extraction.
2.3.2. Extraction of polyphenols from hops
Dilapidated hops were extracted with 500 mL of acetone/water
(70:30, v/v) or methanol/water (70:30, v/v) by sonication for 15 min
and shaking for more 15 min. The extract was filtered in a Büchner
filter (with Whatman no.1 filter paper) and the remaining residue
was re-extracted with 500 mL of fresh solvent for the same period
time. The extract was again filtered in a Büchner filter (Whatman
no.1 filter paper). The combined extracts were evaporated at room
temperature by rotary evaporation to remove the organic phase
(acetone or methanol). After organic solvent evaporation, approx-
imately 300 mL of water extract was obtained. 150 mL of each
hop water extract (sample 1 – initial sample from methanol/water
(70:30, v/v) and sample 2 – initial sample from acetone water
(70:30, v/v)) were lyophilized for further analysis. The other 150 mL
of each hop water extract was kept for further adsorption of phe-
nolic compounds to PVPP.
3261
2.3.3. Adsorption of phenolic compounds to PVPP
150 mL of each hop water extract (adjusted to pH 4.0) was mixed
with 5 g of PVPP (30 mg/mL) for 15 min of shaking. The extract was
filtrated in a Büchner filter (Whatman no.1 filter paper) and the fil-
trate was mixed again with a new portion of PVPP (5 g) for 15 min.
The filtrate was finally lyophilized for further analysis (sample 3
– filtrate from methanol/water (70:30, v/v) initial extraction and
sample 4 – filtrate from acetone/water (70:30, v/v) initial extrac-
tion).
The PVPP collected before in the Büchner filter (Whatman no.1
filter paper) was extracted with 200 mL of acetone/water (70:30,
v/v) by sonication for 15 min and shaking for more 15 min. The
extract was filtrated in a Büchner filter (Whatman no.1 filter
paper). The remaining PVPP in Büchner filter was re-extracted
twice again with 200 mL of fresh extraction solvent for the same
period time that was used before. The combined extracts were
evaporated at room temperature by rotary evaporation to remove
the organic solvent (acetone). After acetone evaporation, approxi-
mately 180 mL of water extract should be obtained. The samples
were lyophilized for further analysis (sample 5 – polyphenols
extract from methanol/water (70:30, v/v) initial extraction and
sample 6 – polyphenols extract from acetone/water (70:30, v/v)
initial extraction).
2.4. Total phenolic content by Folin–Ciocalteau method (FC
method)
The total phenolic content was determined by a modified
Folin–Ciocalteau method, described by Singleton and Rossi [21],
and performed as follows: 1 mL of diluted sample or standard solu-
tion, 4 mL of Folin–Ciocalteau working solution, 5 mL of sodium
carbonate (7.5%, w/v) were introduced into a test tube. This solution
was agitated and left to stand for 2 h for the reaction to take place
and stabilize. The absorbance at 740 nm was determined in a Schi-
madzu UV-3101 spectrophotometer (Kyoto, Japan) and the cells
used were of optical glass with an optical path of 10 mm. The cali-
bration curve was performed with gallic acid (range 50–500 mg/L),
and the results are expressed as mg of gallic acid equivalent (GAE)
per gram of dry weight.
2.5. Total phenolic content by EBC method
The content of total polyphenols in the different samples was
measured as described in Analytica-EBC, section 9, method 9.11,
Nürnberg: Fachverlag Hans Carl, 2000. In this method, 10 mL of
sample and 8 mL of CMC/EDTA reagent (carboxymethyl cellu-
lose/ethylenediaminetetraacetic acid) were transferred to a 25-mL
volumetric flask and thoroughly mixed the content. Then 0.5 mL of
ferric reagent (3.5% ammonium iron citrate) was added to the sam-
ple, which was then thoroughly homogenized. After that, 0.5 mL
of ammonia reagent (ammonia:water, 1:2) was added and thor-
oughly mixed. Finally, the volume was made up to 25 mL with
distilled water and homogenized. The absorbance at 600 nm (opti-
cal glass cells with 10 mm of optical path) was measured after
10 min, for reaction to take place and stabilize. To obtain the content
of polyphenols, the following formula was used: P = A × 820, where
P = polyphenol content (mg/L) and A = absorbance at 600 nm.
2.6. Total flavan-3-ols and proanthocyanidins content (TFC)
The vanillin assay was done as previously described by Sun et
al. [22]. The total volume of reaction was 6 mL, comprising 1 mL
of sample or standard, 2.5 mL of vanillin reagent (1%, w/v), 2.5 mL
of H2 SO4 reagent (20%, v/v) both dissolved in methanol. The cells
used for spectrophotometric measurements were of optical glass
with an optical path of 10 mm and the absorbance was kept at
3262 P.J. Magalhães et al. / J. Chromatogr. A 1217 (2010) 3258–3268
500 nm. The calibration curve was performed with (+)-catechin
(range 50–500 mg/L), and the results are expressed as mg of cat-
echin equivalents (CE) per gram of dry weight.
2.7. Measurement of the antioxidant capacity
The determination of the free radical scavenging activity (DPPH
method) was carried out according to Goupy et al. [23] with minor
modifications. All sample extracts were rediluted 1:4, 1:8, 1:10,
1:20 in methanol in a total volume of 2 mL. A portion of 2.85 mL
of DPPH working solution (0.065 mg/L) was mixed with 0.15 mL of
diluted extract. The absorbance decrease at 515 nm was measured
after 120 min in the dark.
The assessment of the ferricyanide reducing power was car-
ried out as described by Zhao et al. [24]. Briefly, 1 mL of sample
extracts were mixed with 2.5 mL phosphate buffer (0.2 M, pH 6.6)
and 2.5 mL K3 Fe(CN)6 (1%, w/v). The mixture was incubated at 50 ◦ C
for 20 min. Then 2.5 mL of trichloroacetic acid (10%, w/v) were
added to the mixture, which was centrifuged at 1000 × g for 10 min.
The upper layer (2.5 mL) was mixed with 2.5 mL of deionized water
and 0.5 mL FeCl3 ·6H2 O (0.1%, w/v), and the absorbance was mea-
sured at 700 nm. The absorbance increase of the reaction mixture
indicates increasing reducing power.
2.8. HPLC–ESI-MS/MS analysis
The qualitative study of the phenolic compounds in all sam-
ples was performed by HPLC coupled on-line with electrospray
ionization (ESI) mass spectrometry. The HPLC system (Finnigan,
Thermo Electron Corporation, San Jose, USA) consisted of a low-
pressure quaternary pump (model Finnigan Surveyor Plus) and an
auto-sampler (model Finnigan Surveyor Plus with 200-vial capacity
sample). Separations were achieved on a LiChroCart RP-18 column
of 125 mm × 3.0 mm, 3 ␮m particle size (Merck). A guard column
(LiChroCart RP-C18 4.0 mm × 4.0 mm, 5 ␮m) was placed in front of
the analytical column”. The chromatographic conditions were the
following: flow rate 0.3 mL/min, sample injection volume of 20 ␮L
and mobile phase A (100% methanol) and mobile phase B (0.1%
formic acid). A gradient program was used as follows: 90% B in
0 min, from 90% to 0% B in 110 min, followed by 100% A for 20 min
and back to 90% B in 10 min and 10 min of reconditioning before the
next injection. A total of 25 ␮L was injected into the column kept
at room temperature.
A quadropole ion trap mass spectrometer (Finnigan LCQ Deca
XP Plus) equipped with an ESI source in the negative ion mode
and Xcalibur software Version 1.4 (Finnigan) were used for data
acquisition and processing. The interface conditions were applied
as follows: negative mode, capillary temperature, 325 ◦ C; source
voltage, 5.0 kV; capillary voltage, −30.0 V; sheath gas (N2 ) flow at
80 arbitrary units and auxiliary gas (N2 ) flow rate at 30 arbitrary
units. The mass detection was performed in the base peak mode,
for m/z between 100 and 1000. For the LC–MS–MS study energy of
activation of 45% was applied. The negative ion mode was used in
this study due to a better signal-to-noise ratio in comparison with
positive ion mode.
2.9. HPLC–UV analysis
The HPLC system (Jasco Corporation, Tokyo, Japan) consisted
of a low-pressure quaternary gradient unit (model LG-1580-
04) with an in-line DG-1580-54 degasser and a model AS-950
auto-sampler. The system is equipped with a photodiode array
detector (model MD-1510 UV/vis multiwavelength detector). Sep-
arations were achieved on a LiChroCart (Merck) RP-C18 column
(125 mm × 3.0 mm, 3 ␮m). A guard column (LiChroCart RP-C18
4.0 mm × 4.0 mm, 5 ␮m) was placed in front of the analytical col-
umn. The chromatographic conditions were the same as previously
described for HPLC–ESI-MS. The photodiode array detection was
conducted by scanning between 190 and 600 nm. A total of 20 ␮L
was injected into the column kept at room temperature. Analytes
in each sample were identified by comparing their retention times
and UV–vis spectra with those of standard compounds or from lit-
erature. Peak purity was checked to exclude any contribution from
interfering peaks.
3. Results and discussion
3.1. Optimization of polyphenols extraction from hops
According to the literature [20], preliminary removal of hop
lipids and resins is most likely necessary order to recover high
amounts of polyphenols. Moreover, the presence of resins such as
␣-acids or ␤-acids could interfere in the analysis of total polyphe-
nols and antioxidant capacity by colorimetric methods. Diethyl
ether is often chosen for preliminary cleaning because of its abil-
ity to remove hard and soft resins efficiently. Unfortunately, some
polyphenols exhibit significant solubility in this solvent. Therefore,
in this work dichloromethane was used for hops dilapidation.
Preliminary extraction experiments of phenolic compounds
from hops using 70% methanol in water (v/v), 70% ethanol in water
(v/v), 70% acetone in water (v/v) and hot water (50 ◦ C) showed that
acetone and methanol extracts contained more polyphenols, espe-
cially xanthohumol, proanthocyanidins and flavanols (results not
shown). Acetone/water (70:30, v/v) and methanol (70:30, v/v) were
therefore chosen for extraction in the present study.
3.2. Adsorption of phenolic compounds by PVPP in model
solutions
The objective of this part of the work was to study and investi-
gate the adsorption behavior of several phenolic compounds by
PVPP in model solutions. At first, the influence of contact time
between PVPP and each phenolic compound, and the influence of
pH on the adsorption of phenolic compounds by PVPP were inves-
tigated. With this purpose three different pH values were tested
(pH 2, 4 and 6) for adsorption of catechin, epicatechin, gallic acid
and protocatechuic acid. It has been observed that the adsorption of
these compounds was higher when the pH value was 4.0 (data not
shown). These results are in agreement with the study published by
Loomis and Battaile [18] and Andersen and Sowers [19]. It has been
also observed that catechin, epicatechin, gallic acid, and protocate-
chuic acid adsorptions increased with increasing contact time. The
adsorption equilibrium was accomplished within 15 min of con-
tact. No changes in the phenolic concentrations of the supernatant
were observed during the prolonged mixing time (data not shown).
Therefore, 15 min contact duration appears to be sufficient for the
successful reaching of adsorption equilibrium over the entirety of
the system.
The concentrations of PVPP to be used for the adsorption studies
were also tested. Fig. 7 shows the variation of catechin, epicatechin,
gallic acid and protocatechuic acid adsorptions (%) with different
adsorbent concentrations at constant temperature (20 ◦ C). It is eas-
ily observed that adsorption (%) increases as the concentration of
adsorbent increases, as expected, since it depends on the available
adsorption surface. However, for high dosage rates (≥3.0 mg/mL)
of PVPP, there are no significant changes in adsorption capacity for
the studied compounds.
3.2.1. Adsorption isotherm
In the adsorption processes, one or more components of the liq-
uid stream are adsorbed onto the solid adsorbent and separation is
accomplished. This transferring process takes place until dynamic
 P.J. Magalhães et al. / J. Chromatogr. A 1217 (2010) 3258–3268 3263

Fig. 7. The effect of PVPP concentration (mg/mL) on adsorption performance of Fig. 8. The Langmuir adsorption isotherms of catechin and epicatechin onto PVPP
catechin, epicatechin, gallic acid and protocatechuic acid (constant temperature of adsorbent, Q = f(C).
20 ◦ C).

benzoic acid, p-hydroxybenzoic acid and cinnamic acid (Table 1),

equilibrium conditions are reached, which is when the concentra- due to the low amounts of these compounds adsorbed, so high
tion of the solution remains constant. An adsorption isotherm is correlation coefficients do not necessarily imply better fits to the
used to characterize the equilibrium between the amount of solute Langmuir isotherms.
that is accumulated on the adsorbent and the concentration of the As can be seen in Table 1, there was a very obvious increase in the
dissolved adsorbate (fluid) at constant temperature. The Langmuir adsorption equilibrium constant (Kad ) with increasing hydroxyla-
adsorption isotherm and the Freundlich adsorption isotherm are tion of the benzoic acid derivatives. The adsorption value increased
two common isotherms described in the literature for determining 10-fold (p-hydroxybenzoic acid), 27-fold (protocatechuic acid),
the nature of adsorption phenomena. In the present study, Lang- and 44-fold (gallic acid), respectively, with the introduction of
muir isotherm was used for the characterization of adsorption of one, two, and three hydroxyl groups per molecule. The same
phenolic compounds by PVPP. The Langmuir adsorption model is trend was observed for cinnamic acid derivatives. The adsorp-
the most commonly used model in the monocomponent system if tion value increased 7-fold (p-coumaric acid), and 19-fold (caffeic
the solute is retained in only one molecular layer. This isotherm acid), respectively, with the introduction of one and two hydroxyl
model is expressed as groups per molecule. The adsorption values for (+)-catechin and
(−)-epicatechin were considerably greater than would have been
Q Kad · C
= (2) predicted solely from its four aromatic hydroxyls (C5 , C7 , C4 , C5 )
Q0 (1 + Kad · C)
per molecule, which suggested that structural dispositions were of
where Q0 is the maximum concentration retained by the adsor- importance also. As expected, the derivatization of hydroxyl groups
bent surface and Kad is the adsorption equilibrium constant. resulted in greatly diminished binding. The effect of methylation
Both parameters are dependent on the temperature and the can be seen with the pairs protocatechuic acid (Kad = 0.60)/vannilic
adsorbate–adsorbent system. Q and C are equilibrium concentra- acid (Kad = 0.40), gallic acid (Kad = 0.88)/syringic acid (Kad = 0.45) and
tion of adsorbate in the PVPP and in the solution, respectively. The caffeic acid (Kad = 0.75)/ferulic acid (Kad = 0.34). The effect of glyco-
linear form of the equation can be expressed as follows:
1 1 1
= + (3)
Q Q0 Kad · Q0 · C
Adsorption isotherms were established on the amount of solute
adsorbed on the adsorbent (Q, solid-phase absorbance) and resid-
ual absorbance of the supernatant (C, equilibrium liquid-phase
absorbance) at specific wavelength of each phenolic compound.
According to previous study, C represents A/A0 and Q represents
((A0 − A)/A0 )/m where m is the amount of adsorbent used. The anal-
ysis of equilibrium data was done by linear form of the Langmuir
isotherm (Eq. (2)). Plotting 1/Q vs. 1/C it is possible to calculate the
values for the constants Kad (through the slope) and Q0 (through the
y-intercept) for the adsorption of each of the phenolic compounds
by PVPP (e.g. catechin and epicatechin in Figs. 8 and 9). A summary
of the standard binding assay of individual phenolic compounds by
PVPP is listed in Table 1 and is reported as adsorption equilibrium
constant (Kad ). The recovery yields (%) that enable us to evaluate the
efficiency of the extraction/removal (desorption) of the phenolics
adsorbed to PVPP are also depicted in Table 1.
The test for the applicability of the isotherm is the linearity of the Fig. 9. The Langmuir adsorption isotherms of catechin and epicatechin onto PVPP
plot 1/Q vs. 1/C. It must be stated that few points were obtained for adsorbent, 1/Q = f(1/C).
3264 P.J. Magalhães et al. / J. Chromatogr. A 1217 (2010) 3258–3268

Table 1
Adsorption equilibrium constant (Kad ), correlation coefficient (r2 ) and recovery yields (%) for each phenolic compound.

Class Compound Kad r2 Recovery yields (%)

Flavanols (+)-Catechin 2.47 0.9916 (N = 9) 88.3 ± 3.0

(−)-Epicatechin 2.31 0.9918 (N = 9) 95.2 ± 2.0

Benzoic acid Benzoic acid 0.02 0.9691 (N = 5) 89.1 ± 6.0

derivatives p-Hydroxybenzoid acid 0.20 0.9934 (N = 5) 97.1 ± 2.0
Protocatechuic acid 0.60 0.9878 (N = 9) 87.0 ± 3.0
Vanillic acid 0.40 0.9825 (N = 9) 101.6 ± 3.0
Syringic acid 0.45 0.9980 (N = 9) 87.1 ± 6.0
Gallic acid 0.88 0.9719 (N = 9) 90.8 ± 1.0

Cinnamic acid Cinnamic acid 0.04 0.9800 (N = 4) 101.6 ± 8.0

derivatives p-Coumaric acid 0.29 0.9828 (N = 8) 89.9 ± 0.5
Caffeic acid 0.75 0.9775 (N = 9) 86.2 ± 4.7
Ferulic acid 0.34 0.9732 (N = 9) 98.4 ± 2.2
Sinapinic acid 0.41 0.9825 (N = 9) 107.7 ± 10.0

Chalcones Xanthohumol 2.82 0.9995 (N = 9) 101.0 ± 7.5

Flavonols Quercetin 1.94 0.9865 (N = 9) 92.3 ± 0.3

Kaempferol 1.40 0.9990 (N = 9) 98.3 ± 2.3
Rutin 0.60 0.9998 (N = 9) 95.6 ± 0.9

sylation is represented with quercetin/rutin. This effect is especially
severe, since not only is hydrogen bonding to PVPP by a poten-
tial hydroxyl functionality blocked but access to PVPP by other
hydroxyls in the molecule is hindered. For example, rutin is formed
by adding a disaccharide unit, respectively, onto quercetin, and
adsorption constant decreases from 1.94 to 0.60 (Table 1).
Another important feature that can be observed in Table 1 is
the high recovery yield values (>85%) obtained for all the studied
phenolic compounds, confirming that the methodology used for
removal of phenolics previously adsorbed by PVPP (desorption) is
very efficient.
3.3. Application of PVPP for adsorption of phenolic compounds
from hop extracts
After the study and characterization of the adsorption behav-
ior of phenolic compounds by PVPP in model solutions, it was
interesting to understand the methodology efficiency for the
extraction/adsorption of phenolic compounds from hops. All infor-
mation data about the methodology is described in Section 2.3.
It has been reported that, when used in low dosage rates in
beer stabilization, PVPP demonstrated greatly increased affinity
for compounds with higher degrees of hydroxylation. The same
trend was verified in our experiments work as is depicted in
Section 3.1. However, at high dosage rates, removal of phenolic
compounds from beer with PVPP is indiscriminated. Gramshaw
described the situation as follows, “PVPP treatment of beer (high
dosage rates) is always accompanied by decreases in total polyphe-
nols, total flavonols, simple phenolic acids, flavonol glycosides,
catechin, proanthocyanidins, and complexes of polyphenols and
proteins” [11]. When a phenolic compound is slightly adsorbed
by PVPP, even at high PVPP dosage levels the adsorption capac-
ity is not increased. In order to obtain the maximum adsorption of
the phenolics from hop extracts, a high amount of PVPP (approx-
imately 30 mg/mL in two adsorption steps) was used in this work
(see Section 2.3).
The results of the several studied parameters (total polyphenol
content, antioxidant activity and reducing power) for the different
samples are listed in Fig. 10. Two independent measurements were
made for each parameter, and results are presented with means and
standard deviations.
As can be seen in Fig. 10, the phenolic compounds extracted
from original samples 1 (initial sample methanol/water, 70:30, v/v)
and 2 (initial sample acetone/water, 70:30, v/v) were largely recov-

Fig. 10. Analysis of total polyphenols (by FC and EBC methods), antioxidant activity and reducing power of samples (see Section 2.3 for sample preparation information).
Two independent measurements were made for each studied parameter and results are presented with means and standard deviations.
 P.J. Magalhães et al. / J. Chromatogr. A 1217 (2010) 3258–3268 3265

Fig. 11. (A) HPLC/UV (280 nm) chromatogram of sample 6 (polyphenols extract from acetone/water (70:30, v/v) initial extraction); (B) HPLC/UV (370 nm) chromatogram of
sample 6 (polyphenols extract from acetone/water (70:30, v/v) initial extraction); (C) HPLC/UV (280 nm) chromatogram of sample 4 (filtrate from acetone/water (70:30, v/v)
initial extraction); (D) HPLC/UV (370 nm) chromatogram of sample 4 (filtrate from acetone/water (70:30, v/v) initial extraction).

ered in samples 5 (polyphenols extract from methanol/water initial
extraction) and 6 (polyphenols extract from acetone/water initial
extraction). In addition, the polyphenols that were not adsorbed by
PVPP (samples 3 and 4) represent a very small fraction in compari-
son with samples 5 and 6. In this case, the principal disadvantage of
using colorimetric methods for total polyphenol content measure-
ment is the inability of determining which phenolic compounds
were less or not retained in PVPP adsorbent. These methods are
also very susceptible to several interferences such as ascorbic acid,
sugars, and amino acids that can affect the correct quantifica-
tion.
Another important feature that can be observed in Fig. 10 is
the similarity of antioxidant activity and reducing power values
between the original samples (1 and 2) and samples 5 and 6. On the
other hand, it can be also observed that there are no significant dif-
ferences between methanol/water and acetone/water extractions.
The samples were also analyzed by HPLC–DAD in order to obtain
more information about the adsorption behavior of each phenolic
compound by PVPP. Per example, the resulting chromatograms of
samples 4 and 6 are shown in Fig. 11.
The adsorbed amounts of each phenolic compound were cal-
culated via the subtraction of the phenolic compounds amounts
remaining in the filtrate (samples 3 and 4) from the initial amounts
of phenolics present in the initial extracts (samples 1 and 2). The
adsorption capacity (%) of each phenolic compound to the PVPP was
calculated via the division of the adsorbed amounts of each pheno-
lic compound with those of the resin. The recovery yields (%) were
calculated from the amounts of each phenolic compound obtained
via adsorption and desorption process. The results are presented
with means and standard deviations in Fig. 12. The identification
of the major compounds was confirmed by HPLC–ESI-MS/MS (see
Section 3.3).
As can be seen in Fig. 12, it was possible to obtain high recov-
ery yield values (>70%) for the main phenolic compounds from
hops. The effect of glycosylation on the adsorption behavior can be
seen with multifidol/multifidol glucoside. As expected, the adsorp-
tion capacity decreases from ≈94% (multifidol) to ≈67% (multifidol
glucoside) (Fig. 12). In conclusion, these results demonstrate that
PVPP can be a powerful tool to trap flavonoids and other phenolics
extracted from hops.
3.4. Regeneration and reusability of PVPP
Regeneration is an important property of the polymeric adsor-
bent in order to allow it to be used several times in the process.
Regeneration process must be simple and cheap for industrial
application. PVPP grades (such as Polyclar Super R) used for
regeneration are optimized to increase the particle size and
mechanical strength to withstand repeated recycling. Although
this larger particle size (110 ␮m) gives lower polyphenol adsorp-
tion in comparison with single-use PVPP (e.g. Polyclar 10; particle
size = 25 ␮m), higher addition rates can be used, without cost
penalty, due to the very high efficiency of PVPP regeneration. The
principle for regenerating PVPP is to break the bonds between this
adsorbent material and all other adsorbed compounds (e.g. phe-
nolic compounds that were not desorbed in the previous step,
chlorophylls, resins) through washing the material with a caustic
(NaOH) solution. In this work, the PVPP was regenerated very well
using a 4% (w/v) NaOH solution (pH = 13), which was able to remove
the adsorbed polyphenols and other colored compounds. The PVPP
adsorption and subsequent regeneration was tested 5 times. In
these conditions, the adsorbent recovered its original white color
and restored its adsorption capacity completely (data not shown).
3266 P.J. Magalhães et al. / J. Chromatogr. A 1217 (2010) 3258–3268

Fig. 12. Adsorption capacity (%) and recovery yields (%) of the principal phenolic compounds identified in hops.

3.5. Identification of phenolic compounds by
HPLC–DAD–ESI-MS/MS
In this study, a total of 28 compounds were characterized.
Thirteen of them were unambiguously identified by comparing
retention times, UV and MS data with those of the reference
standards. The possible structures of another 15 peaks in the chro-
matogram were tentatively characterized on the basis of literature
data, due to the lack of standard phenolic compounds. The tenta-
tive identification of these compounds is briefly discussed above.
Table 2 summarizes the following information on peaks observed
during HPLC–DAD–ESI-MS/MS analyses: (1) peak labels, (2) UVmax ,
(3) retention times (RT), (4) m/z ratios for the [M−H]− ions and the
major MS/MS fragments, (5) attribution.

Table 2
Identification of phenolic compounds and bitter acids of hops by HPLC–ESI-MS/MS.

Peak label max (nm) RT (min) [M−H]a (intensity) MS/MS fragments (intensity) Attribution

1 275 6.5 866 (100) 713 (100), 695 (100), 575 (40), 407 (30) Proanthocyanidin (trimer)
2 275 9.7 593 (100) 575 (15), 467 (40), 425 (30), 289 (100) Prodelphinidin B3
3 275 13.4 577 (100) 451 (25), 425 (100), 407 (30), 289 (15) Procyanidin B3
4 275 14.8 577 (100) 451 (35), 425 (100), 407 (30), 289 (20) Procyanidin B1
5 275 16.0 866 (100) 739 (35), 695 (100), 577 (55), 289 (30) Proanthocyanidin (trimer)
6 275 17.5 577 (100) 451 (30), 425 (100), 407 (50), 289 (25) Procyanidin B4
7 275 18.5 289 (100) 245 (100), 205 (30), 179 (10) Catechin
8 275 20.4 577 (100) 451 (35), 425 (100), 407 (40), 289 (20) Procyanidin B2
9 275 23.3 866 (100) 739 (45), 695 (100), 577 (60), 289 (30) Proanthocyanidin (trimer)
10 257, 353 26.2 771 (100) 609 (15), 591 (80), 301 (100) Quercetin-3-hexosylrutinoside
11 275 26.8 289 (100) 245 (100), 205 (40), 179 (10) Epicatechin
12 257, 353 32.4 609 (100) 301 (100) Quercetin-3-rutinoside
13 257, 353 35.2 625 (100) 463 (100), 301 (15) Quercetin-3-dihexoside
14 287 36.3 358 (100) 196 (100) 1-[(2-
Methylpropanoyl)phloroglucinyl]-␤-d-
glucopyranoside (multifidol
glucoside)
15 265, 342 42.9 593 (100) 327 (10), 284 (100), 257 (30) Kaempferol-3-rutinoside
16 257, 353 44.0 463 (100), 927 (60) 301 (50), 300 (100), 151 (10), 179 (10) Quercetin-3-hexoside
17 257, 353 46.2 506 (100) 463 (35), 445 (5), 301 (100) Quercetin-3-acetylhexoside
18 265, 342 49.1 447 (100) 327 (15), 285 (100), 284 (50), 257 (10) Kaempferol-3-hexoside
19 265, 342 52.0 490 (100) 327 (5), 285 (100), 284 (20) Kaempferol-3-acetylhexoside
20 287 56.8 196 (100) 177 (5), 151 (100), 125 (10) 2-(2-Methylpropanoyl)-phloroglucinol
21 290 72.9 354 (100) 265 (10), 247 (15), 233 (100), 119 (10) Isoxanthohumol
22 368 82.4 340 (100) 245 (5), 233 (10), 219 (100) Desmethylxanthohumol
23 292 84.4 340 (100) 245 (10), 233 (5), 219 (100) 6-Prenylnaringenin
24 368 88.4 354 (100) 265 (10), 247 (10), 233 (100), 119 (10) Xanthohumol
25 314 98.7 348 (100) 329 (10), 278 (100) Cohumulone
26 314 101.2 361 (100) 343 (5), 292 (100) Humulone
27 314 104.0 400 (100) 330 (70), 287 (100), 276 (10), 262 (15) Colupulone
28 314 106.0 413 (100) 369 (5), 344 (70), 301 (100), 276 (15) Lupulone/adlupulone

Requimte, Departamento de Química, Faculdade de Ciências da Universidade do Porto (FCUP), Rua do Campo Alegre, no. 687, 4169-007 Porto, Portugal.
 P.J. Magalhães et al. / J. Chromatogr. A 1217 (2010) 3258–3268
3.5.1. Chromatographic peaks (14) and (20)
An intense molecular ion [M−H]− with m/z value 358 was
detected for the chromatographic peak (14) (36.3 min). The frag-
mentation, in negative mode, of this peak resulted in a major
fragment with m/z 196 (Table 2), by the loss of 162 m.u. correspon-
dent to the loss of a glucose moiety. According to the literature [25],
this compound is a known constituent of hops and corresponds
to 1-[(2-methylpropanoyl)phloroglucinyl]-␤-d-glucopyranoside
(also known as “multifidol glucoside”. As reported in Table 2,
ESI(−)MS/MS mass spectra of the compound (20) (56.8 min)
was characterized by the presence of the parent ion [M−H]− at
m/z 196 and various fragment ions. Among them, the product
ions at m/z 177, 151 and 125 can be indicated as the main frag-
ments. According to the literature, this compound is probably the
aglycone form of the compound (14): 2-(2-methylpropanoyl)-
phloroglucinol, also known as “multifidol” [25]. It is well known
that glycosides are eluted before aglycones when octadecylsilyl
(RP-18) phase is used in chromatographic analysis. The occur-
rence of this compound has been previously described for hops
[26].
3.5.2. Chromatographic peaks (21) and (24)
The combination of retention time and MS/MS spectral infor-
mation of these compounds (identical molecular ion [M−H]− m/z
354 and identical product ions for both compounds) with those of
pure standards enabled us to identify compounds (21) and (24) as
xanthohumol and isoxanthohumol, respectively.
3.5.3. Quercetin, kaempferol, and their glycosides
Quercetin and kaempferol are not present in hops in a free form
but are glycosidically bound with sugars in position 3 (Fig. 5). Until
nowadays, several glycosides identified in hops were described in
literature [27–29] Due to the unavailability of commercial stan-
dards of several of the studied compounds, the identification of
flavonoid glycosides was done in comparison with literature and
also based on the mass spectrum of kaempferol and quercetin
aglycone standards. In addition, chromatographic and mass spec-
trometric data obtained in this work can be used to obtain
information on the glycoside moiety and the aglycone.
3.5.3.1. Identification of kaempferol glycosides. The chromato-
graphic peaks (15), (18), and (19) were identified as kaempferol
derivatives according to their UV spectra (max at 265 and 342 nm)
and the presence in their MS2 of a major fragment at m/z 285 cor-
responding to kaempferol aglycone. In addition, sugar substitution
on flavanols usually occurs as the O-glycosides, mainly at the 3-
, 7-, and 4 -positions and previous studies indicate that the UV
spectrum is indicative of substitution position [32]. The UV spec-
trum of the compounds (15), (18), and (19) indicates that the sugar
residues are linked to the hydroxyl at the 3-position of the flavonol
molecule, as the UV maximum for band I is always below 355 nm,
which is indicative of substitution of the hydroxyl at the 3-position
[33].
(a) Kaempferol-3-hexoside (glucoside or galactoside) (peak label
18)
The fragmentation, in negative ion mode, of the ion [M−H]−
with m/z 447 detected in the mass spectrum of the peak (ret.
time 49.1 min) resulted in a fragment with m/z 285 (Table 2).
The 162 m.u. difference can correspond to the loss of a glucose
moiety leading us to conclude that the flavonoid detected was
not quercetin-3-rhamnoside but a kaempferol glycoside, since
the ion of the kaempferol aglycone has an m/z value of 285.
Kaempferol-3-hexoside has been reported to be present hops
[30] and has the same retention time and molecular mass as
quercetin-3-rhamnoside. The identification was only possible
3267
by MS/MS experiments, as shown in Table 2. In addition to the
aglycone fragment (m/z 285), a radical aglycone product ion was
also formed (m/z 284) as can be seen in Table 2. In the conditions
used the aglycone fragment was more abundant than the radical
fragment. The same results were obtained by Silva et al. [31].
(b) Kaempferol-3-acetylhexoside (peak label 19)
The fragmentation, in negative mode, of the ion [M−H]−
with m/z 489 detected in the mass spectrum of the peak (ret.
time 52.0 min) resulted in a major fragment with m/z 285
(Table 2). The 205 m.u. difference can correspond to the loss
of an acetylglucose residue (acetyl – m/z 43 and glucose –
m/z 162) leading us to deduce that the flavonoid detected is
kaempferol-3-acetylglucoside. The later elution of kaempferol-
3-acetylglucoside (higher hydrophobicity due to the presence
of acetyl group) in comparison with kaempferol-3-glucoside is
fully consistent with previously reported data in literature [32].
(c) Kaempferol-3-rutinoside (peak label 15)
The peak at 42.9 min was tentatively identified as
kaempferol-3-rutinoside. The [M−H]− with m/z 593 pro-
duced a major MS2 ion at m/z 285 (kaempferol) with the loss
of m/z 308 corresponding to the cleavage of rhamnose–hexose
sugar (Table 2). The UV-max indicated that there was substitu-
tion at the 3-position. The earlier elution of this compound in
comparison with kaempferol-3-glucoside and kaempferol-3-
acetylglucoside is due to the relatively greater hydrophilicity
(higher hydrogen bond interactions between the hydroxyl
groups of the sugar with the eluent water/methanol) of the
rutinoside moiety.
3.5.3.2. Identification of quercetin glycosides. The chromatographic
peaks (10), (12), (13), (16), and (17) were identified as quercetin
derivatives according to their UV spectra (max at 257 and 353 nm)
and the presence in their MS2 of a major fragment at m/z 301 corre-
sponding to quercetin aglycone. As kaempferol glycosides, the UV
spectrum of the compounds (10), (12), (13), (16), and (17) indicates
that the sugar residues are linked to the hydroxyl at the 3-position
of the flavonol molecule.
Another relevant feature that can be seen in Table 2 is that the
more hydrophilic protonated quercetin (301 m/z) glycosides elute
earlier than the protonated kaempferol (m/z 285) glycosides when
the glycosidic residues are the same.
(a) Quercetin-3-hexoside (glucoside or galactoside) (peak label 16)
In the full MS spectrum is detected the molecular deproto-
nated ion, with m/z 463 (higher relative abundance), but also
the ion m/z 927. The fragmentation, in negative mode, of the
ion [M−H]− with m/z 463 detected in the mass spectrum of
the peak (RT = 44.0 min) resulted in two fragments with m/z
300 (higher intensity) and 301 (Table 2). The 162 m.u. dif-
ference can correspond to the loss of a glucose or galactose
moiety leading us to conclude that the flavonoid detected is
quercetin-3-glucoside or quercetin-3-galactoside, respectively.
Unfortunately, in this work mass spectrometry did not allow the
differentiation between glucose and galactose substituents. The
m/z 300 probably corresponds to the radical aglycone quercetin
and m/z 301 corresponds to the quercetin fragment.
(b) Quercetin-3-acetylhexoside (glucoside or galactoside) (peak
label 17)
The fragmentation, in negative mode, of the ion [M−H]− with
m/z 506 detected in the mass spectrum of the peak (ret. time
46.2 min) resulted in several fragments (Table 2). The major
fragments m/z 463 and m/z 301 correspond to the loss of an
acetyl residue (m/z 43) to give quercetin-3-hexoside and to the
loss of an acetylhexoside moiety (m/z 205) to give quercetin
aglycone, respectively. Thus, we suppose that this compound is
quercetin-3-acetylhexoside.
3268 P.J. Magalhães et al. / J. Chromatogr. A 1217 (2010) 3258–3268
(c) Quercetin-3-rutinoside (rutin) (peak label 12)
The MS–MS analysis of the m/z 609 peak (RT = 32.4 min) as
precursor ion gave two base peaks at m/z 300 (radical agly-
cone quercetin) and 301 (quercetin aglycone ion) with the
loss of m/z 308 corresponding to the heterolytic cleavage of
rhamnose–hexose sugar (Table 2). The mass spectrum of this
peak was compared with the mass spectrum data of rutin stan-
dard for further confirmation.
(d) Quercetin-3-dihexoside (peak label 13)
The compound (H) at RT = 35.2 min has two hexosyl residues
on a quercetin molecule ([M−H]− , m/z 625). MS/MS analysis of
the isolated ion m/z 625 showed an intermediate at m/z 463
due to the loss of a hexose (glucose or galactose unit) and the
corresponding quercetin aglycone ion at m/z 301 (second loss
of hexose unit).
(e) Quercetin-3-hexosylrutinoside (peak label 10)
HPLC–MS analysis showed that compound (I) (RT = 26.2 min)
was a quercetin deoxyhexosyldihexoside ([M−H]− , m/z 771).
MS/MS analysis after ion isolation showed that this compound
first lost a hexosyl residue (M-162), indicating that in this com-
pound a hexosyl is the terminal sugar. The occurrence of a
fragment at m/z 609, coincident with quercetin-3-rutinoside
confirmed that this was a quercetin-3-hexosylrutinoside.
3.5.4. Proanthocyanidins trimers
The compounds (1), (5) and (9) have the highest molecular
weight of all the compounds identified by MS analysis in this work.
MS/MS analysis of the isolated ion m/z 866 gave several fragments.
Among them, the product ions at m/z 739, 695, 577 and 289 (cat-
echin monomer) can be indicated as the main fragments. In fact,
the mass spectral peak m/z 866 indicates the presence of proan-
thocyanidins, a catechin or epicatechin trimers.
4. Conclusions
A simple and reliable methodology for extraction and isolation
of polyphenolic antioxidants from hop plant has been described
in this paper. It was possible to obtain high adsorption (≥80%)
and recovery yield (≥70%) values for several polyphenols from
hop plant such as per example xanthohumol, catechin, epicate-
chin, and quercetin and kaempferol glycosides. Moreover, it allows
the identification of about 30 polyphenols by HPLC–DAD–ESI-
MS/MS. It has been also observed that the adsorption of the
studied phenolic compounds by PVPP displayed classical Lang-
muir characteristics over a wide range of PVPP concentrations
(0–5.0 mg/mL), and the isotherms obtained were compared to
determine the extent of selectivity. PVPP preferentially adsorbed
the more highly hydroxylated phenolic compounds in model solu-
tions. The hydroxyl derivatization (methylation and glycosylation)
of the phenolic molecule resulted in greatly diminished binding to
PVPP.
We suggest that the methodology could be used in the future to
other complex matrices in order to obtain polyphenolic-enriched
extracts for application in food, cosmetic and pharmaceutical
industries.
Acknowledgements
P.J.M. (SFRH/BD/27834/2006), L.M.G. (SFRH/BD/36791/2007)
and J.G.P. (SFRH/BD/30279/2006) wish to acknowledge Portuguese
Fundação para a Ciência e a Tecnologia (F.C.T.) for their PhD stu-
dentships. The authors thank UNICER – Bebidas de Portugal SGPS
S.A. for their support, including the supply of hop samples and PVPP.
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