Hop as an interesting source of resveratrol for brewers: Optimization of the extraction and quantitative study by liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry

Article abstract of DOI:10.1021/jf040179n

Nowadays, hop is used almost exclusively by brewers for bitterness and flavor. Although hop polyphenols have been widely studied in the past decade for their antioxidant activity in the boiling kettle, very little is known about their real impact on healt

Full text of DOI:10.1021/jf040179n

424 J. Agric. Food Chem. 2005, 53, 424−429
Hop as an Interesting Source of Resveratrol for Brewers:
Optimization of the Extraction and Quantitative Study by Liquid
Chromatography/Atmospheric Pressure Chemical Ionization
Tandem Mass Spectrometry
†
†
‡
DELPHINE CALLEMIEN, VESNA JERKOVIC, RAOUL ROZENBERG, AND
,
†
SONIA COLLIN*
Unit e´ de Brasserie et des Industries Alimentaires, Universit e´ Catholique de Louvain,
Croix du Sud, 2/bte 7, B-1348 Louvain-la-Neuve, Belgium, and Laboratoire de Spectrom e´ trie de
Masse, Universit e´ Catholique de Louvain, Place Louis Pasteur 1, B-1348 Louvain-la-Neuve, Belgium
Nowadays, hop is used almost exclusively by brewers for bitterness and flavor. Although hop
polyphenols have been widely studied in the past decade for their antioxidant activity in the boiling
kettle, very little is known about their real impact on health. The discovery of resveratrol in hop pellets
highlights the potential health-promoting effect of moderate beer consumption. Here, we have
optimized a quantitative extraction procedure for resveratrol in hop pellets. Preliminary removal of
hydrophobic bitter compounds with toluene and cyclohexane at room temperature allows 99% trans-
resveratrol recovery by ethanol:water (75:25, v/v) solid/liquid extraction at 60 °C. Reverse phase
liquid chromatography proves an excellent means of separating isomers. In addition, we have
compared two mass spectrometry ionization methodssatmospheric pressure chemical ionization
(APCI) and electrospray ionization (ESI)sin both the positive and the negative modes. On the basis
of standard additions applied with the optimized extraction procedure and reverse phase high-
performance liquid cromatography-APCI(+)-tandem mass spectrometry, it appears that Tomahawk
hop pellets (T90, harvest 2002) contain 0.5 ppm trans-resveratrol, 2 ppm trans-piceid, no cis-
resveratrol, and 0.9 ppm cis-piceid.
KEYWORDS: Hop; extraction; resveratrol; polyphenols; health; HPLC-MS/MS; APCI; ESI
INTRODUCTION
estrogenic activities. Stevens et al. (6) and Lermusieau et al.
7), furthemore, have emphasized the huge antioxidant activity
of hop proanthocyanidins.
(
Hop (Humulus lupulus L.) is a dioecious plant of the
Cannabacea family, cultivated in most temperate zones of the
world for its female inflorescences. For centuries, hop was used
in traditional medicine. Nowadays, the plant is used almost
exclusively by brewers for aroma, bitterness, foam stability, and
antimicrobial protection (1). Hop varieties can be differentiated
on the basis of secondary metabolites (resins, oils, and polyphe-
nols) essentially located in the lupulin glands (2, 3). In wort
and beer, up to 30% of the polyphenols present are derived from
hop, despite the fact that malt is added in much larger amounts
Surprisingly, stilbenes appear never to have been investigated
in this polyphenol-rich matrix before our work in 2003 (8). The
phytoalexin trans-resveratrol is usually synthesized by plants
in response to injury or fungal attack (9, 10). It was first reported
in grapevines (Vitis Vinefera) in 1976 (10) but later discovered
in cranberries (11), blueberries (12), bilberries (12), peanuts (13,
14), and Polygonum cuspidatum (15, 16), a traditional Chinese
medicinal herb. The decrease in coronary heart disease observed
among wine drinkers, known as the famous “French paradox”
(17), has led many groups to investigate this compound in red
wines but also in ros e´ and white wines (18-20). trans-
Resveratrol has also been linked to various health benefits, such
as anticarcinogenic, antioxidant, antiinflammatory, and estro-
genic activities (21).
(nearly 100 times more). Depending on the hop variety, its
geographic origin and freshness, the harvesting procedure, and
the manner in which the dried hop cone is packaged, total
polyphenols account for 3-6% (w/w) (4). They consist mainly
of phenolic acids, oligomeric flavanoids, and prenylflavonoids.
Among them, xanthohumol and hopein have been extensively
investigated by De Keukeleire et al. (4, 5) for their exceptional
Various solid-liquid extraction methods have been applied
to extract trans-resveratrol from a solid matrix, usually with
methanol (9-12, 15, 22, 23), ethanol (13, 16, 24), ethyl acetate
(9-12, 15, 25), diethyl ether (22), acetone (25), acetonitrile (14),
or even a supercritical fluid (26). Quantification has been
*
To whom correspondence should be addressed. Tel: +32(0)10-47 29
1
3. Fax: +32(0)10-47 21 78. E-mail: collin@inbr.ucl.ac.be.
†
Unit e´ de Brasserie et des Industries Alimentaires.
Laboratoire de Spectrom e´ trie de Masse.
‡
1
0.1021/jf040179n CCC: $30.25
© 2005 American Chemical Society
Published on Web 12/17/2004

trans-Resveratrol in Hop Pellets
J. Agric. Food Chem., Vol. 53, No. 2, 2005 425
achieved either with gas chromatography [if the analyte is
prederivatized (9, 10)], centrifugal partition chromatography (15,
25), capillary electrophoresis (16), or reverse phase high-pressure
liquid chromatography (RP-HPLC). The latter is usually hy-
phenated to UV absorbance (13, 14, 24, 26), fluorescence (22),
or mass spectrometry detection techniques such as atmospheric
pressure chemical ionization (APCI) (11) and electrospray
ionization (ESI) (12, 23).
The aim of the present work was to quantify accurately for
the first time trans-resveratrol and analogues in hop pellets.
Procedures for hop resin removal and resveratrol extraction were
optimized. The sensitivity was improved by comparing different
atmospheric pressure ionization techniques for mass spectros-
copy.
Figure 1. Stability of trans-resveratrol under various conditions. Quantifica-
tion by HPLC-UV at 306 nm.
MATERIALS AND METHODS
Chemicals. Ethanol (97%) was obtained from Belgaco (Gent,
Belgium). Acetonitrile (99.99%), toluene (99.97%), and cyclohexane
99.96%) were purchased from Fischer Scientific (United Kingdom).
Acetone (99.9%), n-hexane (99%), and formic acid (pa) were purchased
HPLC-UV Analysis. For the extraction procedure optimization,
separations were carried on a Luna silica column (250 mm × 4.6 mm,
5 µm) (Phenomenex, Holland) eluted with a linear gradient from
dichloromethane to methanol and a constant 4% level of acetic acid:
water mixture (50:50, v/v). The gradient elution was as follows: from
82 to 68% dichloromethane in 30 min, 68 to 46% in 30 min, 46 to
10% in 5 min, and finally isocratic for 5 min at a flow rate of 1 mL/
min. Twenty microliters of sample was injected into the column kept
at 25 °C. trans-Resveratrol was monitored at 306 nm with a UV6000LP
diode array detector.
RP-HPLC-MS/MS Analysis. Quantifications were performed on
a C18 Prevail column (150 mm × 2, 1 mm, 2 µm) (Alltech, Deerfield,
IL) eluted with a linear gradient from water containing 1% acetonitrile
and 0.1% formic acid to acetonitrile. The gradient elution was as
follows: from 95% water to 55% in 23 min, 55 to 0% in 7 min, and
isocratic for 10 min at a flow rate of 200 µL/min. Ten microliters of
sample was injected into the column kept at 30 °C. A SpectraSystem
equipped with an AS3000 autosampler and a P4000 quaternary pump
was used. The system was controlled with the Xcalibur software version
1.2 (Finnigan Mat). Mass spectra were acquired using a LCQ mass
spectrometer equipped with an APCI or ESI source (Finnigan Mat).
APCI inlet conditions were applied as follows: vaporization temper-
ature, 470 °C; capillary voltage, 3 V; capillary temperature, 175 °C;
sheath gas, 40 psi; auxiliary gas, 7 psi; and discharge current, 5 µA.
For the ESI source, conditions were applied as follows: source voltage,
(
from Aldrich (Germany). Methanol (99.9%) and dichloromethane
(
99.9%) were purchased from Romil (Cambridge, United Kingdom).
Acetic acid (99.8%) came from Acros (Geel, Belgium). Petroleum ether
100-140 °C) was obtained from Vel (Darmstadt, Germany). Aqueous
solutions were made with Milli-Q (Millipore, Bedford, MA) water.
trans-Resveratrol (99%), trans-piceid (97%), catechin (98%), epicat-
echin (90%), rutin (95%), myricetin (85%), quercetin (98%), and
kaempferol (90%) were purchased from Sigma-Aldrich (Bornem,
Belgium). cis-Piceid and cis-resveratrol were obtained by irradiating
for 10 min the respective trans forms dissolved in methanol at a
wavelength of 254 nm.
Stability of trans-Resveratrol. A stock solution of 100 ppm trans-
resveratrol was prepared in methanol. The stability of the phytoalexin
was tested under various conditions: (i) 20 °C with exposure to white
light, (ii) 20 °C with exposure to white light under nitrogen, (iii) 20
C with exposure to red light, (iv) 20 °C with exposure to red light
under nitrogen, (v) 4 °C (dark refrigerator), and (vi) -20 °C (dark
freezer). Aliquots of each sample were taken at 0, 1, 3, and 9 days.
(
°
Optimization of trans-Resveratrol Extraction from Hop Pellets.
For these experiments, recovery factors were calculated by standard
addition from normal phase HPLC-UV (λ ) 306 nm) data. Extractions
were carried out on Tomahawk hop pellets (T90, 2002 harvest) under
red light.
4
.5 kV; capillary voltage, 36 V; capillary temperature, 225 °C; sheath
gas, 70 psi; and auxiliary gas, 20 psi. In both methods, collision-induced
dissociation spectra were recorded at 37% relative collision energy.
RemoVal of Hydrophobic Compounds. Hop pellets (2.5 g) were
reduced to powder and introduced into a centrifugal vial. In successive
RESULTS AND DISCUSSION
1
0 min steps, resins and lipids were removed at room temperature under
gentle stirring first with 50 mL of toluene (three times) and then with
0 mL of cyclohexane (three times). At the end of each step, the sample
was centrifuged for 10 min at 3000g. At the last step, hop powder was
also dried under vacuum to get rid of residual solvent. For the
optimization, other procedures were tested as follows: no delipidation
or delipidation with petroleum ether (three times) and n-hexane (three
times) or with petroleum ether alone (six times).
trans-ResVeratrol Extraction. Delipidated hop powder was extracted
three times with 40 mL of ethanol:water (75:25, v/v), each time for 10
min under gentle stirring at 60 °C. This procedure was compared with
extraction at room temperature with either acetone, ethanol, or methanol
mixed with water (75:25, v/v). After each extraction, the sample was
centrifuged for 10 min at 3000g, and the supernatant was collected.
After filtration to remove residual particles, the combined supernatants
were concentrated by rotary evaporation (35 °C) to dryness. The residue
was solubilized in 2 mL of 50:50 (v/v) mixture of ethanol:water.
Assessment of trans-Resveratrol Stability. As depicted in
Figure 1, trans-resveratrol can be stored for 1 week at 20, 4,
or -20 °C without significant deterioration if protected from
white light. In the presence of white light, however, trans-
resveratrol is very quickly degraded at room temperature,
especially if oxygen is also present (40% degradation after 9
days vs 25% if protected with a nitrogen headspace). Accord-
ingly, all subsequent extraction procedures were applied under
red light at room temperature. The extracts obtained were then
stored at -20 °C in the dark until used.
Optimization of trans-Resveratrol Extraction. According
to the literature (6), preliminary removal of hop lipids and resins
is most likely necessary in order to recover high amounts of
polyphenols. Ether is often chosen for preliminary cleaning
because of its ability to remove hard and soft hop resins
efficiently (27). Unfortunately, as indicated by its short retention
time on a polar HPLC column (8), trans-resveratrol is much
more hydrophobic than other hop polyphenols such as catechin,
epicatechin, or procyanidin oligomers. Therefore, it exhibits
significant solubility in diethyl ether (0.11%, w/w or 1.1 mg/
5
Standard Addition Method. Hop was spiked with increasing
amounts of either trans-resveratrol (0, 0.5, 1, and 5 ppm), trans-piceid
0, 5, 10, and 20 ppm), catechin, (0, 75, and 150 ppm), epicatechin
0, 100, and 250 ppm), quercetin (0, 75, 150, and 225 ppm), myricetin
0, 0.5, 1, and 5 ppm), rutin (0, 500, and 1000 ppm), or kaempferol
0, 10, 20, and 30 ppm).
(
(
(
(

426 J. Agric. Food Chem., Vol. 53, No. 2, 2005
Callemien et al.
Figure 2. Comparison of ESI (electrospray ionization) and APCI (atmospheric pressure chemical ionization) in MS/MS analyses. Injection in triplicates
of 5 L trans-resveratrol (10 ppm in methanol) into a 200 L/min RP-HPLC flow.
µ
µ
Figure 3. trans-Resveratrol MS/MS mass spectrum in positive mode APCI.
mL). Four more hydrophobic solvents were tested as follows:
n-hexane, petroleum ether, toluene, and cyclohexane (unfortu-
nately, these solvents removed few hop hard resins). As shown
in Table 1, no delipidation led, as expected, to poor recovery
Three organic solvent:water mixtures (75:25, v/v), usually
used for stilbene extraction, were then compared as regards their
ability to extract resveratrol at room temperature. As shown in
Table 2, ethanol emerged as the most efficient, just above
methanol (about 10% more). Although very often used for
procyanidin recovery (27), the acetone:water mixture proved
inappropriate for extracting trans-resveratrol. Gentle heating at
60 °C, as advised by Romero-P e´ rez (24), improved the recovery
still further (by 8%). When two-step solid/liquid extraction with
toluene and cyclohexane was combined with this last procedure,
99% recovery was achieved. As depicted in Table 3, this method
(50%, standard addition method). A gain of about 20% was
recorded when we applied two-step solid/liquid extraction with
petroleum ether and hexane or one-step solid/liquid extraction
with petroleum ether alone (still 4% better). Two-step solid/
liquid extraction with toluene and cyclohexane proved even
more efficient (30%), probably thanks to the ability of toluene
to remove part of the hard resins.

trans-Resveratrol in Hop Pellets
J. Agric. Food Chem., Vol. 53, No. 2, 2005 427
Figure 4. (a) Separation of eight polyphenolic standards (5 ppm in methanol) by RP-HPLC-APCI(+)/MS-MS. (b) Analyses of Tomahawk hop extract
(
(
T90, harvest 2002). (A) TIC (total ion chromatogram), (B) catechin and epicatechin, (C) rutin, (D) myricetin, (E) trans-resveratrol-1 and cis-resveratrol-2,
F) quercetin, and (G) kaempferol.
Table 1. Recovery Factors of trans-Resveratrol for Various
Hydrophobic Compounds Removal Procedures
Table 3. Recovery Factors Obtained for Nine Hop Polyphenols When
the Optimized Resveratrol Extraction Procedure Is Applied [Toluene
and Cyclohexane for Removing Hydrophobic Compounds and
a
solvent for hydrophobic compounds removal
recovery factor (%)
Ethanol:Water (60
MS-MS Detection]
°C) for Polyphenol Extraction; RP-HPLC-APCI(+)/
a
no delipidation
petroleum ether and n-hexane
petroleum ether
50
68
72
82
recovery
concentration (ppm
toluene and cyclohexane
compound
factor (%)
or mg/kg of hop pellets)
catechin
epicatechin
rutin
myricetin
trans-resveratrol
quercetin
19
7
238
1483
1931
1
0.5
132
16
a
Polyphenols were extracted in all cases with methanol:water (75:25, v/v); UV
λ ) 306 nm) detection.
(
16
80
99
21
22
95
−
Table 2. Recovery Factors of trans-Resveratrol According to the
Solvent Used for Polyphenol Extractiona
kaempferol
trans-piceid
2.1
0.9
solvent for polyphenol extraction
recovery factor (%)
b
cis-piceid
acetone:water (75:25, v/v)
methanol:water (75:25, v/v)
ethanol:water (75:25, v/v)
34
72
82
90
a
Concentrations were found in Tomahawk hop pellets (T90, harvest 2002) by
b
the standard addition method. trans-Piceid equivalent.
b
ethanol:water (75:25, v/v)
a
Hydrophobic compounds were removed in all cases with petroleum ether; UV
Quantitative Analysis of Resveratrol in Hop. As depicted
in Figure 4b, the RP-HPLC-APCI(+)MS/MS (m/z ) 229)
chromatogram obtained for Tomahawk hop pellets (T90, harvest
2002) revealed the presence of 0.5 ppm trans-resveratrol
(standard addition method applied). Although no cis-resveratrol
was found in the sample, two compounds showing higher
polarity (tr ) 16.38 and 17.78 min) and the same mass spectrum
were detected (Figure 5). MS/MS (m/z ) 391) applied to
standards and hop extract enable us to identify these compounds
as trans- and cis-piceid. Standard addition applied with our
aglycone-optimized method indicated concentrations of trans-
and cis-piceid close to 2 and 0.9 ppm, respectively (in both
cases, quantification was done in trans-piceid equivalents, for
which a recovery factor of 95% was calculated).
b
(
λ ) 306 nm) detection. Extraction at 60
°
C.
optimized specifically for resveratrol resulted in a low recovery
of most hop polyphenols (except myricetin).
Optimization of the RP-HPLC-MS/MS Analysis. RP-
HPLC provided higher chromatographic resolution (separation
of trans-resveratrol, tr ) 20.85 min and cis-resveratrol, tr ) 22
min) than normal phase (only one peak at 5.6 min) (see also
Figure 4a). To compare the ESI and APCI ionization methods,
we injected 5 µL of trans-resveratrol solution (10 ppm in
methanol) in triplicate into 200 µL/min RP-HPLC flow. Our
data show that APCI in the positive mode and ESI in the
negative mode gave the highest responses, the former giving a
four-times higher response (Figure 2). APCI in the positive
mode was thus retained for hop sample analysis (monitoring of
the m/z ) 229; see Figure 3).
In conclusion, this study has yielded an optimized quantitative
extraction method for resveratrol analysis in Tomahawk hop

428 J. Agric. Food Chem., Vol. 53, No. 2, 2005
Callemien et al.
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Received for review April 12, 2004. Accepted October 16, 2004. D.C.
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