Jeffery et al.

Flavonol composition of Australian wines 153

Flavonol composition of Australian red and white wines

determined by high-performance liquid chromatography
D.W. JEFFERY, M. PARKER and P.A. SMITH
The Australian Wine Research Institute, PO Box 197, Glen Osmond, SA 5064, Australia
Corresponding author: Paul A. Smith, fax +61 8 8303 6601, email paul.smith@awri.com.au

Abstract
Background and Aims: Flavonols are present in Vitis vinifera L. wine grapes as glycosides and are found
in wines in both their glycosidic and aglycone forms. Flavonols and their glycosides are important
components in wine because of their impact on colour, taste and health properties. An assessment of the
flavonols and flavonol glycosides present in a wide selection of Australian red and white wines (n = 121
and 44, respectively) was undertaken.
Methods and Results: Analyses were performed using HPLC, with compound identification being
aided by HPLC-MS. In the red wines, quercetin and myricetin dominated the flavonol profile, and
quercetin-3-glucuronide was by far the most abundant glycoside present. White wines had considerably
lower levels of flavonols and their glycosides, in most cases, being below the detection limit of the HPLC
method.
Conclusions: There appeared to be no correlation between the flavonol profile and wine variety or
region in either white or red wines. Contrary to literature reports of rutin in wine, rutin was not found
in any of the wines we analysed, and spiking experiments showed rutin rapidly degraded to the aglycone
quercetin. Furthermore, changes in elution order for some quercetin glycoside standards were observed
depending upon the acid used in the mobile phase.
Significance of the Study: This is the first time the flavonol contents of an extensive cross section of
Australian wines have been reported. The concentrations are consistent with those reported in wines
from other countries. In light of the instability of rutin in wine, critical review should be given to reports
discussing its presence.

Abbreviations
CV coefficient of variation; DAD diode array detection; ESI+ positive electrospray ionisation;
HPLC high-performance liquid chromatography; HPLC-MS high-performance liquid
chromatography-mass spectrometry; LOD limit of detection; LOQ limit of quantitation;
m/z mass-to-charge ratio; NMR nuclear magnetic resonance; R2 coefficient of determination;
SPE solid-phase extraction; UV/Vis ultraviolet/visible.

Keywords: Australian wine, elution order, flavonols, flavonol glycosides, high-performance liquid
chromatography, quantitation, red wine, rutin degradation, white wine

Introduction
Flavonols are a family of polyphenolic compounds found
in Vitis vinifera L. wine grapes and wines. They are largely
localised in the grape skins, where they are found as
flavonol glycosides (flavonols bound to various sugars),
and can be extracted to a small extent upon pressing of
white grapes, and to a larger extent during maceration of
red grapes and wines (Cheynier and Rigaud 1986). Free
flavonol aglycones can be produced from these flavonol
glycosides, as a result of hydrolysis of the glycosidic bond
by enzymes or acidic conditions in the wine. While the
glycosylated flavonols are partially soluble in aqueous
grape juice and more soluble in ethanolic wine, the fla-
doi: 10.1111/j.1755-0238.2008.00017.x
© 2008 The Australian Wine Research Institute
vonol aglycones are poorly soluble in wine and can cause
instability in some red and white wines, observable as
colourless crystals or yellow powdery precipitate. Grape
flavonol concentrations are known to increase with
increased sun exposure in the vineyard (Price et al. 1995,
Spayd et al. 2002, Downey et al. 2004, Giovanelli and
Brenna 2007), although detailed studies on white grapes
are lacking (Crothers 2005).
The presence of flavonols in wine is important because
of their colour, taste and health properties (Williamson
and Manach 2005). They have a pale yellow colour,
evident in their UV/Vis spectrum as an absorbance
maximum at ~350 nm for glycosides and ~370 nm for
154 Flavonol composition of Australian wines
5'
OH OH
1 B
HO O HO 7 O 3' HO
1' OH
A C
OH 5 3 OH
OH O OH O
Kaempferol Quercetin
OH OH
OH OH
HO O HO O HO
OH OMe
OH OH
OH O OH O
Myricetin Laricitrin
aglycones, and are believed to enhance the colour of red
wines, because of their ability to copigment with antho-
cyanins, thus stabilising red colour. Flavonols are also of
interest because of their potential importance to the taste
of wine. Flavonol glycosides have been found to be
important to ‘velvety astringency’ in foods such as black
tea (Scharbert and Hofmann 2005) and Italian Amarone
red wine (Hufnagel and Hofmann 2008).
Chemically, the flavonols consist of six aglycones:
myricetin, quercetin, kaempferol, isorhamnetin, syringe-
tin and laricitrin (Figure 1), which are found in their
glycosylated forms in the grape. Interestingly, different
sugars can be bound to the flavonols, producing the glu-
cosides, glucuronides, galactosides and some diglycosides
(glucosylarabinoside, glucosylgalactoside, glucosylxylo-
side and glucosylrhamnoside) (Monagas et al. 2005a,
Castillo-Muñoz et al. 2007). Because of the similar
chemical nature of these flavonols, similar UV/Vis spectra
and non-availability of reference standards, the correct
identification of compounds by HPLC analysis can be
challenging (Cheynier and Rigaud 1986, Betés-Saura
et al. 1996, Monagas et al. 2005a), especially in the case
of quercetin-3-glucuronide, quercetin-3-glucoside and
quercetin-3-rutinoside (rutin). Rutin, which has been
found in red and white grapes (Cantos et al. 2002,
Giovanelli and Brenna 2007, Gómez-Alonso et al. 2007),
was reported to have a particularly low sensory threshold
in bottled water (Scharbert and Hofmann 2005).
While there are reports in the literature about typical
concentrations of flavonols found in wines (e.g. Makris
et al. 2006), there is little detailed data on concentrations
found in Australian red and white wines. We set out to
identify and quantify the flavonols and their glycosides
present in a selection of Australian red and white wines.
Materials and methods
Chemicals
All chromatographic solvents were HPLC grade; all
chemicals were analytical reagent grade unless otherwise
stated, and water was obtained from a Milli-Q purifica-
tion system (Millipore, North Ryde, NSW, Australia). All
prepared solutions were % v/v with the balance made
up with Milli-Q water, unless otherwise specified.
Sephadex® LH-20, quercetin (ⱖ98% by HPLC, Sigma),
Australian Journal of Grape and Wine Research 14, 153–161, 2008
OH
O
OMe
OH
OH O Figure 1. Structures of
Isorhamnetin flavonol aglycones found in
Vitis vinifera L. grapes (ring
numbering is indicated for
OMe
quercetin). Sugars are attached
OH at the 3-position.
O
OMe
OH
OH O
Syringetin
quercetin-3-O-b-D-glucoside (ⱖ90% by HPLC, Fluka),
quercetin-3-O-b-D-galactoside (ⱖ97% by HPLC,
Fluka), quercetin-3-O-b-D-rutinoside (rutin, ⱖ90% by
HPLC, Fluka), kaempferol (ⱖ90% by HPLC, Sigma),
isorhamnetin (ⱖ95% by HPLC, Fluka), myricetin (ⱖ96%
by HPLC, Sigma) and ellagic acid (~95% by titration,
Sigma) were purchased from Sigma-Aldrich (Castle Hill,
NSW, Australia). Laricitrin (ⱖ99% by HPLC), syringetin-
3-O-galactoside (ⱖ99% by HPLC), syringetin-3-O-
glucoside (ⱖ99% by HPLC) and syringetin (ⱖ99% by
HPLC) were purchased from Extrasynthèse (Genay,
France). Stock solutions of standards were prepared by
dissolving the individual compounds in 5 mL of methanol
in a 10-mL volumetric flask, sonicating the mixtures for
5 min and topping up to the mark with Milli-Q water.
Dilutions were prepared volumetrically by diluting the
stock solution with 50% methanol and sonicating the
mixtures for 5 min before adjusting the final volume.
Wine samples
Freshly opened, commercially available Australian red
and white wines (n = 121 and 44, respectively) from a
cross section of wine regions were assessed over the
course of the study, with vintages ranging from 1991 to
2006. Wine varieties included Cabernet Sauvignon,
Merlot, Shiraz, Pinot Noir, Durif, Chardonnay, Sauvignon
Blanc, Semillon and Riesling. Red wine samples were
centrifuged in a Thermo Electron Corp. IEX Micromax
microcentrifuge (Biolab, Mulgrave, Vic., Australia) at
1500 ¥ g for 5 min prior to analysis.
Extraction of quercetin-3-O-b-D-glucuronide from
coriander seeds
Dried seeds from Coriandrum sativum L. (500 g) were pro-
cessed in a Retsch® Grindomix GM200 (MEP Instruments,
North Ryde, NSW, Australia) at 8000 rpm for two 10-s
pulses. The seed powder was extracted with 1.5 L of 80%
methanol at room temperature with the aid of sonication
for 1 h. The mixture was filtered and the solids were
extracted overnight with a further 400 mL of 80% metha-
nol at room temperature protected from light by alu-
minium foil, while the filtrate was stored at -20°C. The
second extraction was filtered, combined with the first
extract and stored at -20°C for 7 days. The seed extract was
© 2008 The Australian Wine Research Institute
Jeffery et al.
decanted off the solids and concentrated in vacuo on a
rotary evaporator at 40 mbar with a 30°C water bath. The
extract was stored in a refrigerator at 4°C covered in
aluminium foil between manipulations. The aqueous
residue (~200 mL) was extracted with ethyl acetate
(3 ¥ 50 mL) and loaded under gravity onto Varian Nexus
SPE cartridges (6 mL, 200 mg) (Varian, Mulgrave, Vic.,
Australia), which had been conditioned with 3 mL of
methanol followed by 3 mL of water. After loading, the
cartridges were eluted with 6 mL of methanol and recon-
ditioned as above, reusing each cartridge four times, until
all the aqueous extract had been processed. The fractions
were pooled and the methanol was removed in vacuo on a
rotary evaporator as above. The aqueous residue (~25 mL)
was chromatographed on LH-20 (66 g swelled in metha-
nol overnight, slurry-packed 200 ¥ 55 mm column,
120-mm bed height), eluting with methanol into fractions
of 25 mL. Fractions were monitored from 200 to 600 nm
(1-mm quartz cell) with a Cary 300 Scan UV/Vis spectro-
photometer (Varian) and 26 fractions were collected in
total, which were then analysed by HPLC. The following
fractions were combined and concentrated in vacuo, yield-
ing a yellow powder (purity by HPLC given in brackets):
F16-18, 40 mg (90%); F19-21, 102 mg (94%); and F22-
25, 28 mg (90%). Spectral data (NMR, UV/Vis, MS) for the
isolated compound were consistent with the structure of
quercetin-3-O-b-D-glucuronide and in accord with litera-
ture values (Mohle et al. 1985, Day et al. 2000).
HPLC analysis of samples
Analyses were performed on an Agilent 1100 instrument
(Agilent, Forest Hill, Vic., Australia) equipped with a qua-
ternary pump and DAD, using gradient elution based on
the method described by Cozzolino et al. (2004) with a
slight modification as detailed by Mercurio et al. (2007).
The column was a Synergi Hydro-RP (150 ¥ 2 mm, 4 mm,
80 Å) operated at 25°C and protected by a guard column
(4 ¥ 2 mm) of the same material (Phenomenex, Lane
Cove, NSW, Australia). The eluents were acetonitrile/
orthophosphoric acid/water (1:1.5:97.5 v/v, Eluent A)
and Eluent A/acetonitrile (20:80 v/v, Eluent B) with a
flow rate of 0.400 mL/min. The gradient for Eluent B was
as follows: 0 min, 14.5%; 18 min, 27.5%; 24 min, 27.5%;
25 min, 50%; 26 min, 50%; 30 min, 100%; 32 min,
100%; 32.01 min, 14.5%; and 40 min, 14.5%. A 50%
aqueous acetonitrile solution wash was used as the
column wash eluent. A 20 mL injection volume was used
for each sample, and DAD signals were recorded at 280,
320, 353, 370 and 520 nm. Data acquisition and process-
ing were performed using Agilent ChemStation software
(version A.09.03). Compounds in each sample were iden-
tified by the comparison of their retention times and
UV/Vis spectra with those of standards or by HPLC-MS
analysis. Isorhamnetin-3-hexoside was quantified as
quercetin-3-glucoside equivalents.
HPLC-MS analysis of samples
Analyses were performed on an Agilent 1200 instrument
(Agilent) equipped with a binary pump and Agilent 1100
DAD connected in series to a 4000 Q Trap hybrid tandem
© 2008 The Australian Wine Research Institute
Flavonol composition of Australian wines 155
mass spectrometer with a TurboIonSpray source (Applied
Biosystems/MDS Sciex, Concord, Ontario, Canada). The
column was a Synergi Hydro-RP (150 ¥ 2 mm, 4 mm,
80 Å) operated at 25°C and protected by a guard column
(4 ¥ 2 mm) of the same material (Phenomenex). The
eluents were formic acid/water (5:95 v/v, Eluent A) and
formic acid/water/acetonitrile (5:15:80 v/v/v, Eluent B)
with a flow rate of 0.200 mL/min. The linear gradient
for Eluent B was as follows: 0 min, 10%; 35 min, 35%;
50 min, 60%; 55 min, 90%; and 60 min, 90%. The
column was equilibrated with 10% B for 10 min prior to
an injection. A 10 mL injection volume was used for each
sample, and DAD signals were recorded at 280, 370 and
520 nm. Mass spectra were recorded between m/z 200
and 1500 with a scan time of 2 s in positive (IonSpray
5500 V) and negative (IonSpray -4500 V) ion modes
during separate analyses. Other MS parameters were as
follows: source temperature, 500°C; nitrogen as curtain
gas (CUR), 103.4 kPa, nebulising gas (GS1), 344.7 kPa
and drying gas (GS2), 344.7 kPa; entrance potential, 10 V
(-10 V in negative mode); and declustering potential,
60 V (-60 V in negative mode). Data acquisition and pro-
cessing were performed using the Applied Biosystems/
MDS Sciex Analyst software (version 1.4.2).
Spiking experiments involving quercetin glycosides
A mixed quercetin glycoside standard was prepared by
adding quercetin-3-rutinoside (0.53 mg), quercetin-3-
galactoside (0.52 mg), quercetin-3-glucoside (0.70 mg),
quercetin-3-glucuronide (0.64 mg) and 1.2 mL of ethanol
to a 10-mL volumetric flask and sonicating for 5 min. After
the addition of 5 mL of Milli-Q water and sonicating for a
further 5 min, the flask was made up to the mark with
Milli-Q water. A 2007 Riesling wine and a 2006 Shiraz
wine were spiked with the mixed glycoside standard by
adding 500 mL of standard to each wine in a 5-mL volu-
metric flask. For comparison, an additional sample of each
wine was diluted to the same extent by adding 500 mL of
12% v/v ethanol to the wine in a 5-mL volumetric flask.
The spiked wine samples were analysed by HPLC
immediately after preparation (time = 0 h). The white
wine was analysed by HPLC every 40 min for four analy-
ses and then at 4, 6, 8, 10, 12, 14 and 16 h. The red wine
was analysed by HPLC at 4, 6, 8, 24, 27, 48 and 50 h.
Areas in the 370-nm chromatograms at the relevant
retention times for the unspiked, diluted wine samples
were subtracted from the areas of the spiked samples, and
these adjusted areas were converted into concentrations
in mg/L. A first-order rate plot of –ln(rutin in mg/L)
versus time (hours) gave a straight line for each wine
(R2 = 0.999) with a slope equal to the rate constant k.
Half-life in hours for the first-order reactions was then
determined using ln(2)/k. The mixed quercetin glycoside
spiking mixture was also analysed by HPLC over the
course of 5 days to assess its stability.
Statistical analysis
The results reported for the calibration of flavonol stan-
dards were the average of three replicate measurements
for each concentration (at least six levels) of standard.
156 Flavonol composition of Australian wines Australian Journal of Grape and Wine Research 14, 153–161, 2008

The LOD and LOQ for each flavonol was determined by
multiplying the standard deviation of the y-intercept by
3.3 (for LOD) and 10 (for LOQ) and dividing these values
by the slope of the calibration curve for each standard.
The coefficients of variation for HPLC retention time were
determined by dividing standard deviations by the mean
values of replicate injections and expressing the result as
a percentage. Statistical analyses were performed on
Microsoft Excel 2003.
Results and discussion
HPLC methods for compositional analysis
An existing HPLC method (Mercurio et al. 2007) was
used to determine the flavonols present in the various
samples. Although this method was initially developed
for rapid anthocyanin analysis, it has also proven valuable
for analysis of other wine phenolic components, affording
reasonable resolution of many flavonols and their glyco-
sides. An HPLC-MS method was also used to aid in
compound identification, particularly for compounds
where no reference material was available. The HPLC and
HPLC-MS methods utilised the same column but differed,
among other parameters, in the acid used to modify the
eluent; HPLC eluent contained orthosphosphoric acid,
while HPLC-MS eluent contained formic acid.
Calibration of standards
Calibration curves for commercially available standards of
the more common flavonols were obtained in the range
preliminarily identified in commercial Australian red and
white wines. Details derived from these calibrations are
listed in Table 1 and include retention time and % CV, R2,
LOD, LOQ and linear range. These flavonol standards
gave retention time CV values of 0.60% or less and R2
values of 0.9995 or greater, with the exception of
quercetin-3-galactoside, which was 0.9990. Theoretical
LODs ranged from 0.04 mg/L for quercetin-3-glucoside to
0.86 mg/L for myricetin, while theoretical LOQs ranged
from 0.12 mg/L for quercetin-3-glucoside to 2.61 mg/L
for myricetin. Although the HPLC autosampler was
protected from light by aluminium foil, some decompo-
sition of standards was evident, particularly for myricetin,
resulting in slightly inferior calibration data. Laricitrin
also decomposed in the autosampler over the course of
several hours and had to be stored at –20°C until analysis.
Overlaid HPLC chromatograms of the flavonol standards
recorded at 370 nm display the retention times and
elution order of the standards (Figure 2).
Isolation of quercetin-3-glucuronide reference compound
There are a number of literature reports dealing with the
quantitation of quercetin-3-glucuronide in wines (Alonso
et al. 1986, Betés-Saura et al. 1996, Ghiselli et al.
1998, Satué-Gracia et al. 1999, Pellegrini et al. 2000,
Baderschneider and Winterhalter 2001, Gutiérrez et al.
2005, Monagas et al. 2005b, Castillo-Muñoz et al. 2007,
Gómez-Alonso et al. 2007), but there is no commercially
available standard, and generally, relative elution order
and UV/Vis spectra (which are very similar for the quer-
cetin glycosides) are used for confirmation. Correct iden-
tification of the glucuronide was particularly important in
this work because of the close elution of quercetin glyco-
sides using our HPLC and HPLC-MS methods, and in light
of the uncertainty of quercetin glycoside identification in
the literature (Cheynier and Rigaud 1986, Betés-Saura
et al. 1996, Monagas et al. 2005a). Furthermore, it
appeared that the elution order of the glucuronide was
reversed with that of the glucoside when switching from
a mobile phase containing orthophosphoric acid (HPLC)
to one containing formic acid (HPLC-MS). These aspects
required clarification and coriander seeds had been
identified as a rich source of quercetin-3-glucuronide
(Kunzemann and Herrmann 1977). Isolation of the glu-
curonide from coriander seeds involved the extraction of
the milled seed with 80% methanol followed by a brief
clean-up using SPE cartridges, which removed the bulk
of the cinnamic acids, hydrophobic material and water.
Chromatography on LH-20 with methanol and monitor-
ing of fractions by UV/Vis spectroscopy, with fraction
composition confirmed by HPLC, provided over 100 mg
of quercetin-3-O-b-D-glucuronide (identity confirmed by

Table 1. HPLC calibration data for flavonol reference compounds.

Flavonol Retention Retention Coefficient of Detection Quantitation Linear

time* (min) time CV† (%) determination (R2) limit (mg/L) limit (mg/L) range (mg/L)

1 Quercetin-3-galactoside 12.0 0.24 0.9990 0.30 0.92 0.30–10.0

2 Quercetin-3-glucoside 12.7 0.38 0.9999 0.04 0.12 0.04–7.3
3 Quercetin-3-glucuronide 13.0 0.60 0.9997 0.29 0.89 0.29–64.0
4 Syringetin-3-glucoside 16.4 0.10 0.9999 0.12 0.37 0.12–30.8
5 Myricetin 18.1 0.30 0.9996 0.86 2.61 0.86–41.0
6 Quercetin 26.1 0.19 0.9995 0.65 1.96 0.65–45.2
7 Laricitrin 26.6 0.20 0.9999 0.08 0.25 0.08–18.0
8 Kaempferol 29.7 0.07 0.9998 0.08 0.24 0.08–10.0
9 Isorhamnetin 29.9 0.03 0.9996 0.47 1.42 0.47–43.5

* Average retention time for replicate injections.

† Calculated from triplicate injections for at least six calibration levels.

© 2008 The Australian Wine Research Institute

Jeffery et al. Flavonol composition of Australian wines 157

26.089
mAU 4

16.356
5
2 8 9

18.019
12.701

29.717
200 1

29.882
Figure 2. Overlaid and
11.893

expanded HPLC
3
chromatograms of flavonol
150 standards recorded at 370 nm.
13.047

Numbers used for peak

identification are the same as
7 in Table 1. Compound
100

26.685
concentrations are not all the
same.
50

0
12.5 15 17.5 20 22.5 25 27.5 30 min

(a) HPLC 370 nm (b) LC-MS 370 nm (c) LC-MS m/z465*, 479#
1
2 2 3#
21.45

2*
22.21

22.03
12.724

mAU
22.38

280
180 1 3 17
21.90
11.893

260 16 Figure 3. Chromatograms of

160 240 15 the closely eluting quercetin
14 1* glycosides as determined by
220 (a) HPLC at 370 nm with
21.58

140 3 13 orthophosphoric acid in the

200
13.047

12 eluent, and HPLC-MS with

120 180 formic acid in the eluent (b) at
11
370 nm and (c) using extracted
Intensity, cps ( × 106)

160 10 positive ions m/z 465

Absorbance, mAU

100
140 9 (galactoside, glucoside) and
m/z 479 (glucuronide).
8
80 120 Numbers used for peak
7 identification are the same as
100
60 6 in Table 1. Concentrations of
80 compounds were not identical
5
for HPLC and HPLC-MS
40 60 4 analyses.
40 3
20 2
20
1
0
0 0
12 14 min 20.0 22.0 24.0min 20.0 22.0 24.0min

1
H and 13C NMR, UV/Vis and MS data) with a minimum Clearly, the use of HPLC-MS with ESI+ allows for clarifi-
of 90% purity (HPLC) for use as a reference compound. cation of elution order for the glucoside (m/z 465) and
Calibration data for the quercetin-3-glucuronide isolated glucuronide (m/z 479). In the absence of mass spectral
in this manner are included in Table 1. data and reference compounds, however, it would be
The use of authentic quercetin-3-glucuronide allowed unwise to base peak identification on UV/Vis spectra and
us to confirm our concern that the elution order of published retention times, especially if different HPLC
glucoside and glucuronide depended on the acid used in columns or mobile phases are used. This could account
the mobile phase. When orthophosphoric acid was used for some of the discrepancies that appear in the literature
(HPLC), the glucuronide eluted after the glucoside, with regard to the identification of quercetin glycosides.
whereas with formic acid (HPLC-MS), the glucuronide
eluted first. This can be seen in Figure 3, which shows Flavonols in commercial Australian wines
chromatograms of the closely eluting quercetin glycosides Commercially available Australian red wines (n = 121)
as determined by (a) HPLC at 370 nm, (b) HPLC-MS at and white wines (n = 44) were assessed by HPLC to
370 nm and (c) HPLC-MS using extracted positive ions. determine their flavonol profile. HPLC chromatograms
© 2008 The Australian Wine Research Institute
158 Flavonol composition of Australian wines Australian Journal of Grape and Wine Research 14, 153–161, 2008

mAU
a
50

40
6
30
8 9

26.018
20 a b 3 c

29.564
29.767
13.294
12.956

16.338
12.304
10
Figure 4. HPLC chromatogram
0 recorded at 370 nm of a 2004
-10
Semillon wine (a) and a 2006
Shiraz wine (b). Numbers used
5 10 15 20 25 30 min for peak identification are the
9 same as in Table 1. Other
peaks are non-flavonols that

29.722
5
6 have some absorbance at
mAU
b 370 nm, such as cinnamic

26.051
17.904

250 3 acids and polyphenols.

a, ellagic acid; b,
12.935

200 dihydrokaempferol-3-hexoside;
8 c, isorhamnetin-3-hexoside.
4
150 a

29.517
16.540
11.720

1 7
100
12.180

26.737

50

0 5 10 15 20 25 30 min

recorded at 370 nm are displayed in Figure 4 for a 2004
Semillon and a 2006 Shiraz. These chromatograms are
typical of the results obtained using our HPLC method,
and Figure 4b in particular, shows adequate resolution
of the flavonols of interest. The identity of flavonols for
which there were no authentic samples on hand was
confirmed by their HPLC-MS and UV/Vis spectra.
Electrospray in positive ionisation mode generally gave
more intense spectra for the compounds than negative
mode. Thus, tentatively identified isorhamnetin-3-
hexoside (presumably the glucoside) had a molecular ion
([M + H]+) with m/z 479 in positive mode, with a frag-
ment at m/z 317 for the aglycone. Myricetin-3-hexoside
(most likely the glucoside) had a molecular ion ([M + H]+)
with m/z 481 in positive mode, with a fragment at m/z 319
for the aglycone, and laricitrin-3-hexoside (presumably
the glucoside which was identified recently in red wine
for the first time (Castillo-Muñoz et al. 2007)) had a
molecular ion ([M + H]+) with m/z 495 in positive mode,
with a fragment at m/z 333 for the aglycone (data not
shown). Table 2 displays the means, medians and range of
values encountered for the individual flavonols in all the
Australian wines analysed. In the event that standards for
some quercetin glycosides are unavailable, highly accept-
able quantitation results can be obtained by quantifying
these glycosides as quercetin-3-glucoside equivalents, as
the calibration curves are very similar (data not shown).
Flavonol profiles of Australian white wines
Not surprisingly, the flavonol levels in the white wines
were substantially lower than in red wines, and the
ranges of values encountered were somewhat lower than
reported in a recent review (Makris et al. 2006). Gener-
ally, quercetin and isorhamnetin and their glycosides
were the most abundant flavonols detected (Table 2),
although there seemed to be no trend to the flavonols
encountered with respect to wine variety or region
(data not shown). With the exception of isorhamnetin-3-
hexoside and quercetin, which ranged in value from
below the detection limit to 0.14 and 2.05 mg/L, respec-
tively, the remaining flavonols were either below the
detection limit or below the quantitation limit of the
HPLC method. More accurate quantitation may be
achieved with sample clean-up using SPE or by concen-
trating the wine samples, but in the first instance, we
wanted to evaluate the direct analysis of wines.
We were, however, able to determine an unidentified
peak, which was observed eluting at a similar retention
time to quercetin-3-glucuronide in most of the wine
samples. The unassigned compound was identified as the
dihydroflavonol dihydrokaempferol-3-hexoside (presum-
ably the glucoside), based on its HPLC-MS spectrum in
positive mode (m/z 451 with a fragment at m/z 289) and its
UV/Vis spectrum (Figure 5), which was identical to that
reported in the literature (Pozo-Bayón et al. 2003). The
amount of dihydrokaempferol-3-hexoside was not quan-
tified in our work, but the glucoside has been reported in
Riesling wine (Baderschneider and Winterhalter 2001)
and sparkling white wines (Pozo-Bayón et al. 2003). In
addition, an interesting observation was made in relation
to the presence of rutin in white wine, as discussed below.
Flavonol profiles of Australian red wines
The ranges of values obtained for the different flavonols
in red wines were consistent with those reported in a
recent review (Makris et al. 2006), as were the relative
© 2008 The Australian Wine Research Institute
Jeffery et al. Flavonol composition of Australian wines 159

Table 2. Concentrations of flavonol glycosides and aglycones found in Australian red and white
wines expressed as mg/L.

Flavonol Red wine (n = 121) White wine (n = 44)

Range Mean Median Range Mean Median

Quercetin-3-galactoside ND–6.52 1.77 1.31 ND NR –

Quercetin-3-glucoside ND–NQ NR – ND–NQ NR –
Quercetin-3-glucuronide ND–39.67 11.70 10.80 ND–NQ NR –
Isorhamnetin-3-hexoside* ND–NQ NR – ND–0.14 NR –
Syringetin-3-glucoside 1.42–18.40 5.65 5.47 ND NR –
Myricetin 2.70–28.78 11.59 11.71 ND NR –
Quercetin 3.49–37.36 16.18 15.77 ND–2.05 NR –
Laricitrin 0.32–4.77 1.81 1.59 ND NR –
Kaempferol NQ–2.46 0.82 0.76 ND–NQ NR –
Isorhamnetin ND–18.12 3.45 2.50 ND NR –
Total glycosides 1.42–55.69 16.16 13.19 ND–0.80 0.05 –
Total aglycones 7.42–76.51 33.85 32.57 ND–2.26 0.12 –

* Quantified as quercetin-3-glucoside equivalents. ND, not detected (below the detection limit based on calibration data); NQ, not
quantified (below the quantitation limit based on calibration data); NR, not reported (could not be derived from data as values were below
the quantitation limit, or only one sample gave a value above LOQ).

mAU These results suggest that the glucuronide is the most

25
20
15
10
5 those reported by Castillo-Muñoz et al. (2007), whereby
0 B-ring substitution (see Figure 1) and the type of glyco-
300 400 500 600 nm
Figure 5. UV/Vis spectrum obtained from the HPLC chromatogram
peak eluting at ~13 min, which was identified as dihydrokaempferol-
3-hexoside.
abundances of the various flavonols (Table 2). The agly-
cones dominated the flavonol profiles of the wines, with
myricetin and quercetin generally being the most abun-
dant with values ranging from 2.70 to 28.78 mg/L for
myricetin and 3.49 to 37.36 mg/L for quercetin. The
other aglycone values ranged from below the detection
limit to several mg/L, and up to 18.12 mg/L for isorham-
netin. Laricitrin, reported recently in red wine for the first
time (Castillo-Muñoz et al. 2007), had values ranging
from 0.32 to 4.77 mg/L. Interestingly, of the glycosides,
quercetin-3-glucuronide had the highest concentrations,
ranging from below the detection limit to 39.67 mg/L,
and was by far the most dominant quercetin derivative.
© 2008 The Australian Wine Research Institute
stable quercetin glycoside present in red wines.
Syringetin-3-glucoside was also present in considerable
concentrations, with values ranging from 1.42 to
18.40 mg/L (Table 2), whereas the aglycone syringetin
was barely detectable, because of the stability of the
glucoside. The 3-hexosides of myricetin, laricitrin and
isorhamnetin (most likely the 3-glucosides), and the agly-
cone syringetin were identified by HPLC-MS in positive
mode and existed in trace amounts in some wines (data
not shown). These results are generally consistent with
the extent of glycoside hydrolysis of the various wine
flavonols depends on both the nature of the aglycone
side attached.
There seemed to be no trend in flavonol profile with
respect to the variety or regional origin of the wine,
although Pinot Noir wines had either undetectable or
extremely low levels of quercetin glycosides (data not
shown). Interestingly, the Pinot wines had detectable
levels of dihydrokaempferol-3-hexoside (presumably the
glucoside), which eluted at almost the same retention
time as quercetin-3-glucuronide (data not shown). As for
the white wines, the amount of dihydrokaempferol-3-
hexoside was not quantified in red wines, but the
glucoside has been reported in sparkling red wines (Pozo-
Bayón et al. 2003). The occurrence of dihydroflavonols
such as this in Australian red and white wines warrants
further investigation.
Degradation of quercetin-3-rutinoside (rutin) in wine samples
This study uncovered an unexpected finding with regard
to the presence and stability of rutin in wine. While the
presence of rutin in grape skins of V. vinifera L. has been
160 Flavonol composition of Australian wines
well documented, its existence in wine has been debated
amid possible confusion with quercetin-3-glucuronide
(Cheynier and Rigaud 1986, Betés-Saura et al. 1996,
Monagas et al. 2005a). While undertaking this study, we
initially identified a 370-nm absorbing compound eluting
at about 11.4 min as rutin based on the retention time of
the authentic compound. Upon scrutinising the UV/Vis
spectrum of this peak in wines, however, it became clear
that the spectrum was not consistent with that of rutin.
The analysis of some wines by HPLC-MS revealed the
presence of a peak with m/z 303 (positive mode) and m/z
301 (negative mode), which indicated the compound was
either quercetin or ellagic acid (data not shown). Quer-
cetin was ruled out as a candidate as it eluted some
15 min later from the HPLC column. Ellagic acid was
confirmed as the compound of interest after HPLC analy-
sis of an authentic sample, which eluted at 11.4 min and
had a UV/Vis spectrum identical to that reported in the
literature (Boyle and Hsu 1990).
Because of the unexpected absence of rutin in the
wines analysed, experiments were conducted in red and
white wines to determine the degradation kinetics of
rutin. Wines were spiked with a mixture of the rutino-
side, galactoside, glucoside and glucuronide derivatives of
quercetin, and the degradation of rutin was monitored by
HPLC, showing first-order kinetics in both red and white
wines. The curves for rutin degradation and quercetin
formation are shown in Figure 6 for a white wine and a
red wine. Over the course of the experiments, only rutin
was degraded until it was undetectable, with a commen-
surate increase in quercetin, while the other glycoside
levels remained constant (data not shown). The results
indicated that quercetin-3-glucoside was not an interme-
diate in the hydrolysis of rutin, meaning the diglycoside
was potentially directly cleaved from the aglycone.
Rutin degradation was substantially faster in the
white wine, with a half-life of 3 h, while the half-life in
red wine was 9 h. The difference may be attributed to
the copigmentation of rutin with anthocyanins in red
wine providing some protection from degradation. This
finding may well explain the discrepancies that exist in
Rutin (Red)
4.5 Quercetin (Red)
Rutin (White)
4.0
Quercetin (White)
3.5
Flavonol (mg/L)
3.0
2.5
2.0
1.5
1.0
0.5
0.0
0 10 20 30 40
Time (hours)
Figure 6. Degradation of rutin and corresponding formation of quer-
cetin in a 2007 Riesling wine and a 2006 Shiraz wine spiked with
quercetin glycosides and monitored by HPLC.
Australian Journal of Grape and Wine Research 14, 153–161, 2008
the literature with regard to rutin identification in wine,
as our results indicate that rutin does not exist in bottled
white or red wines to any measurable extent. Indeed,
one study has shown that rutin was liberated and
degraded to quercetin during the early stages of the
winemaking process (Rueff et al. 1990). Rutin also
declined to undetectable levels during ageing of Sherry
vinegar (Tesfaye et al. 2002) and was decomposed faster
than other flavonol glycosides in Monastrell wines
during storage (Zafrilla et al. 2003). No reasonable
explanation was apparent to us for the different stabili-
ties of the quercetin glycosides, although rutin differs by
having a diglycoside (incorporating an L-rhamnose)
attached to quercetin, whereas the other derivatives are
monoglycosides (with the D-configuration).
In spite of rutin instability in wine, it is anticipated
that rutin could still serve as a useful external standard for
quantifying quercetin glycosides as ‘rutin equivalents’.
This was concluded because there was minimal degrada-
tion of rutin standards prepared in methanol and water
and stored in the HPLC autosampler for up to 3 days.
Furthermore, the quercetin glycoside spiking mixture
consisting of 12% v/v ethanol was stored in the autosam-
pler and analysed over the course of 5 days with minimal
sample degradation.
Conclusions
An HPLC method, which was originally developed for
anthocyanin analysis, has been successfully applied to the
determination of flavonols and their glycosides in com-
mercially available Australian red and white wines. This
work presents for the first time the flavonol contents of
an extensive cross section of Australian wines, with the
concentrations encountered being consistent with those
reported in wines from other countries. Although my-
ricetin and quercetin generally dominated the flavonol
profile in red wines, there appeared to be no correlation
between the flavonol profile and wine variety or region
in either white or red wines. Interestingly, quercetin-3-
glucuronide was identified as the dominant quercetin
glycoside, while syringetin-3-glucoside was also found
at appreciable levels in red wines. The acid used in the
mobile phase for the HPLC analysis affected the elution
order of some quercetin glycosides, so relying on a rela-
tive elution order garnered from the literature could lead
to erroneous quercetin glycoside identification. Further-
more, we have demonstrated that rutin was not found in
any of the wines examined and was quickly degraded if
spiked into bottled wine. A compound that eluted at the
same retention time as an authentic rutin standard was
identified as ellagic acid, and this result may explain some
of the confusion found in the literature surrounding the
identification of quercetin glycosides.
50
Acknowledgements
The authors thank Yoji Hayasaka and Gayle Baldock,
for assistance with HPLC-MS, and Dimitra Capone, for
selecting and providing the wine samples. This work
was financially supported by Australia’s grapegrowers
and winemakers through their investment body the
© 2008 The Australian Wine Research Institute
Jeffery et al.
Grape and Wine Research and Development
Corporation, with matching funds from the Australian
Government.
References
Alonso, E., Estrella, I. and Revilla, E. (1986) Presence of quercetin-
3-O-glucuronoside in Spanish table wines. Journal of the Science
of Food and Agriculture 37, 1118–1120.
Baderschneider, B. and Winterhalter, P. (2001) Isolation and char-
acterization of novel benzoates, cinnamates, flavonoids, and
lignans from Riesling wine and screening for antioxidant activity.
Journal of Agricultural and Food Chemistry 49, 2788–2798.
Betés-Saura, C., Andrés-Lacueva, C. and Lamuela-Raventós, R.M.
(1996) Phenolics in white free run juices and wines from Penedès
by high-performance liquid chromatography: Changes during
vinification. Journal of Agricultural and Food Chemistry 44,
3040–3046.
Boyle, J.A. and Hsu, L. (1990) Identification and quantitation of
ellagic acid in Muscadine grape juice. American Journal of
Enology and Viticulture 41, 43–47.
Cantos, E., Espín, J.C. and Tomás-Barberán, F.A. (2002) Varietal
differences among the polyphenol profiles of seven table grape
cultivars studied by LC-DAD-MS-MS. Journal of Agricultural and
Food Chemistry 50, 5691–5696.
Castillo-Muñoz, N., Gómez-Alonso, S., García-Romero, E. and
Hermosín-Gutiérrez, I. (2007) Flavonol profiles of Vitis vinifera red
grapes and their single-cultivar wines. Journal of Agricultural and
Food Chemistry 55, 992–1002.
Cheynier, V. and Rigaud, J. (1986) HPLC separation and charac-
terization of flavonols in the skins of Vitis vinifera var. Cinsault.
American Journal of Enology and Viticulture 37, 248–252.
Cozzolino, D., Kwiatkowski, M.J., Parker, M., Cynkar, W.U.,
Dambergs, R.G., Gishen, M. and Herderich, M.J. (2004) Predic-
tion of phenolic compounds in red wine fermentations by visible
and near infrared spectroscopy. Analytica Chimica Acta 513,
73–80.
Crothers, N. (2005) The effects of increased sun exposure and
sun-induced browning on Chardonnay (Vitis vinifera L.) grape and
wine composition and quality. Honours Thesis, Victoria Univer-
sity, Melbourne Victoria, 96 pp.
Day, A.J., Bao, Y.P., Morgan, M.R.A. and Williamson, G. (2000)
Conjugation position of quercetin glucuronides and effect on
biological activity. Free Radical Biology and Medicine 29, 1234–
1243.
Downey, M.O., Harvey, J.S. and Robinson, S.P. (2004) The effect
of bunch shading on berry development and flavonoid accumu-
lation in Shiraz grapes. Australian Journal of Grape and Wine
Research 10, 55–73.
Ghiselli, A., Nardini, M., Baldi, A. and Scaccini, C. (1998) Anti-
oxidant activity of different phenolic fractions separated from an
Italian red wine. Journal of Agricultural and Food Chemistry 46,
361–367.
Giovanelli, G. and Brenna, O. (2007) Evolution of some phenolic
components, carotenoids and chlorophylls during ripening of
three Italian grape varieties. European Food Research and Tech-
nology 225, 145–150.
Gómez-Alonso, S., García-Romero, E. and Hermosín-Gutiérrez, I.
(2007) HPLC analysis of diverse grape and wine phenolics using
direct injection and multidetection by DAD and fluorescence.
Journal of Food Composition and Analysis 20, 618–626.
Gutiérrez, I.H., Lorenzo, E.S.-P. and Espinosa, A.V. (2005) Phe-
nolic composition and magnitude of copigmentation in young
and shortly aged red wines made from the cultivars, Cabernet
Sauvignon, Cencibel, and Syrah. Food Chemistry 92, 269–283.
Hufnagel, J.C. and Hofmann, T. (2008) Orosensory-directed iden-
tification of astringent mouthfeel and bitter-tasting compounds in
red wine. Journal of Agricultural and Food Chemistry 56, 1376–
1386.
Kunzemann, J. and Herrmann, K. (1977) Isolierung und identifi-
zierung der flavon(ol)-O-glykoside in kümmel (Carum carvi L.),
© 2008 The Australian Wine Research Institute
Flavonol composition of Australian wines 161
fenchel (Foeniculum vulgare Mill.), anis (Pimpinella anisum L.) und
koriander (Coriandrum sativum L.) und von flavon-C-glykosiden
im anis. Zeitschrift für Lebensmitteluntersuchung und-Forschung
A 164, 194–200.
Makris, D.P., Kallithraka, S. and Kefalas, P. (2006) Flavonols in
grapes, grape products and wines: Burden, profile and influential
parameters. Journal of Food Composition and Analysis 19, 396–
404.
Mercurio, M.D., Dambergs, R.G., Herderich, M.J. and Smith, P.A.
(2007) High throughput analysis of red wine and grape phenolics-
adaptation and validation of methyl cellulose precipitable tannin
assay and modified Somers color assay to a rapid 96 well plate
format. Journal of Agricultural and Food Chemistry 55, 4651–
4657.
Mohle, B., Heller, W. and Wellmann, E. (1985) UV-induced bio-
synthesis of quercetin 3-O-b-D-glucuronide in dill cell cultures.
Phytochemistry 24, 465–467.
Monagas, M., Bartolomé, B. and Gómez-Cordovés, C. (2005a)
Updated knowledge about the presence of phenolic compounds in
wine. Critical Reviews in Food Science and Nutrition 45, 85–118.
Monagas, M., Suárez, R., Gómez-Cordovés, C. and Bartolomé, B.
(2005b) Simultaneous determination of nonanthocyanin phe-
nolic compounds in red wines by HPLC-DAD/ESI-MS. American
Journal of Enology and Viticulture 56, 139–147.
Pellegrini, N., Simonetti, P., Gardana, C., Brenna, O., Brighenti, F.
and Pietta, P. (2000) Polyphenol content and total antioxidant
activity of vini novelli (young red wines). Journal of Agricultural
and Food Chemistry 48, 732–735.
Pozo-Bayón, M.A., Hernández, M.T., Martín-Álvarez, P.J. and
Polo, M.C. (2003) Study of low molecular weight phenolic com-
pounds during the aging of sparkling wines manufactured with
red and white grape varieties. Journal of Agricultural and Food
Chemistry 51, 2089–2095.
Price, S.F., Breen, P.J., Valladao, M. and Watson, B.T. (1995)
Cluster sun exposure and quercetin in Pinot noir grapes and wine.
American Journal of Enology and Viticulture 46, 187–194.
Rueff, J., Laires, A., Gaspar, J. and Rodrigues, A. (1990)
Mutagenic activity in the wine-making process: Correlations with
rutin and quercetin levels. Mutagenesis 5, 393–396.
Satué-Gracia, M.T., Andrés-Lacueva, C., Lamuela-Raventós, R.M.
and Frankel, E.N. (1999) Spanish sparkling wines (cavas) as
inhibitors of in vitro human low-density lipoprotein oxidation.
Journal of Agricultural and Food Chemistry 47, 2198–2202.
Scharbert, S. and Hofmann, T. (2005) Molecular definition of black
tea taste by means of quantitative studies, taste reconstitution,
and omission experiments. Journal of Agricultural and Food
Chemistry 53, 5377–5384.
Spayd, S.E., Tarara, J.M., Mee, D.L. and Ferguson, J.C. (2002)
Separation of sunlight and temperature effects on the composition
of Vitis vinifera cv. Merlot berries. American Journal of Enology
and Viticulture 53, 171–182.
Tesfaye, W., Morales, M.L., Garcia-Parrilla, M.C. and Troncoso,
A.M. (2002) Evolution of phenolic compounds during an experi-
mental aging in wood of sherry vinegar. Journal of Agricultural
and Food Chemistry 50, 7053–7061.
Williamson, G. and Manach, C. (2005) Bioavailability and bioef-
ficacy of polyphenols in humans. II. Review of 93 intervention
studies. American Journal of Clinical Nutrition 81, 243S–255S.
Zafrilla, P., Morillas, J., Mulero, J., Cayuela, J.M., Martinez-Cacha,
A., Pardo, F. and Nicolas, J.M.L. (2003) Changes during storage
in conventional and ecological wine: Phenolic content and anti-
oxidant activity. Journal of Agricultural and Food Chemistry 51,
4694–4700.
Manuscript received: 6 March 2008
Revised manuscript received: 16 June 2008
Accepted: 23 June 2008