Glycosylation of smoke-derived volatile phenols in grapes as a consequence of grapevine exposure to bushfire smoke

Article abstract of DOI:10.1021/jf103045t

The presence of glycosides of smoke-derived volatile phenols in smoke-affected grapes and the resulting wines of Chardonnay and Cabernet Sauvignon was investigated with the aid of highperformance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). All volatile phenols studied (phenol, p-, m-, and o-cresols, methylguaiacol, syringol, and methylsyringol) could be detected as glycosylated metabolites in smoke-affected grapes in a similar fashion to that previously reported for guaiacol. These phenolic glycosides were found in smoke-affected grapes and wines at significantly elevated levels compared to those in non-smoked control grapes and wines. The extraction of these glycosides from grapes into wine was estimated to be 78% for Chardonnay and 67% for Cabernet Sauvignon. After acid hydrolysis, a large proportion of these phenolic glycosides in grapes (50%) and wine (92%) disappeared but the concentrations of volatile phenols determined by gas chromatography-mass spectrometry (GC-MS) were lower than expected. In the case of wine, the majority of the glycosides of phenol, cresols, guaiacol, and methylguaiacol were decomposed upon acid hydrolysis without releasing their respective aglycones, while syringol and methylsyringol were more effectively released. 2010 American Chemical Society.

Full text of DOI:10.1021/jf103045t

J. Agric. Food Chem. 2010, 58, 10989–10998 10989
DOI:10.1021/jf103045t
Glycosylation of Smoke-Derived Volatile Phenols in Grapes as
a Consequence of Grapevine Exposure to Bushfire Smoke
Y
OJI
H
AYASAKA,* GAYLE A. BALDOCK, MANGO
P
ARKER, KEVIN H. PARDON
AVID W. JEFFERY
,
C
ORY A. BLACK, MARKUS J. HERDERICH AND
,
D
The Australian Wine Research Institute (AWRI), Post Office Box 197, Glen Osmond,
South Australia 5064, Australia
The presence of glycosides of smoke-derived volatile phenols in smoke-affected grapes and the
resulting wines of Chardonnay and Cabernet Sauvignon was investigated with the aid of high-
performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). All volatile
phenols studied (phenol, p-, m-, and o-cresols, methylguaiacol, syringol, and methylsyringol) could
be detected as glycosylated metabolites in smoke-affected grapes in a similar fashion to that
previously reported for guaiacol. These phenolic glycosides were found in smoke-affected grapes
and wines at significantly elevated levels compared to those in non-smoked control grapes and
wines. The extraction of these glycosides from grapes into wine was estimated to be 78% for
Chardonnay and 67% for Cabernet Sauvignon. After acid hydrolysis, a large proportion of these
phenolic glycosides in grapes (50%) and wine (92%) disappeared but the concentrations of volatile
phenols determined by gas chromatography-mass spectrometry (GC-MS) were lower than
expected. In the case of wine, the majority of the glycosides of phenol, cresols, guaiacol, and
methylguaiacol were decomposed upon acid hydrolysis without releasing their respective aglycones,
while syringol and methylsyringol were more effectively released.
KEYWORDS: Volatile phenols; glycosides; smoke exposure; smoke taint; grapes; wine; bushfire;
HPLC-MS/MS
INTRODUCTION
including phenol, cresols, methylguaiacol, vinylguaiacol, syrin-
gol, methylsyringol, and vinylsyringol. Some of these com-
pounds, which have not been considered previously for smoke-
affected grape and wine samples, also have distinctive smoke-
related sensory characters (4, 5), which may contribute to the
overall smoke effect on grapes and wine.
In a previous study using a stable isotope tracer technique (1),
we identified seven different glycoconjugates (glycosides) of
guaiacol in grape berries and leaves that had been in direct
contact with mixed d0- and d3-guaiacol solutions. These guaiacol
glycosides were tentatively identified as follows: glucosylgluco-
This study was undertaken to investigate the presence of
glycosides of smoke-derived volatile phenols in grapes exposed
to bushfire smoke and in the wines made from smoke-exposed
grapes, using high-performance liquid chromatography-tandem
mass spectrometry (HPLC-MS/MS). Acid hydrolysis of glyco-
sides in grape and wine samples was also performed to ascertain
the potential extent of volatile phenol release during winemaking
and storage.
side (either gentiobioside or sophoroside, GG), β-
side (glucoside, MG), the glucoside further substituted with a
pentose (diglycoside, DG), such as R- -arabinosyl-β- -glucoside,
β- -apiosyl-β- -glucoside, or β- -xylosyl-β- -glucoside, and R-
rhamnosyl-β- -glucoside (rutinoside, RG). The study also indi-
cated that there was minimal translocation of guaiacol glycosides
between grapevine leaves and berries. The glycosides were present
as low-level natural compounds in control leaves and berries, and
the glycosides were present in significantly elevated amounts in
leaves and berries following exposure of grapevines to smoke
derived from actual bushfires.
D-glucopyrano-
L
D
D
D
D
D
L-
D
MATERIALS AND METHODS
Materials. All chromatographic solvents were HPLC-grade. All
chemicals were analytical-reagent-grade unless otherwise stated. Water
was obtained from a Milli-Q purification system (Millipore, North Ryde,
Australia). All prepared solutions were % v/v, with the balance made up
with Milli-Q water, unless otherwise specified. Merck solvents were
purchased from Rowe Scientific (Lonsdale, Australia). Model wine was
made of water/ethanol (87:13) saturated with potassium hydrogen tartrate
and adjusted to pH 3.2 with the addition of 10% (w/v) tartaric acid.
Guaiacol, 4-methylguaiacol, vinylguaiacol, phenol, o-cresol, m-cresol,
p-cresol, syringol and 4-methylsyringol, and d7-p-cresol were purchased
With regard to smoke taint in grape and wine products, the role
of the guaiacol glycosides remains uncertain; however, it is likely
that they act as precursors to free guaiacol, contributing to the
intensity ofsmoke taint characters after fermentation and in wine,
especially with increasing storage time (2, 3). In addition to
guaiacol, smoke contains a variety of other volatile phenols,
*To whom correspondence should be addressed. Telephone: þ61-8-
8313-6637. Fax: þ61-8-8313-6601. E-mail: yoji.hayasaka@awri.com.au.
© 2010 American Chemical Society
Published on Web 10/05/2010
pubs.acs.org/JAFC

10990 J. Agric. Food Chem., Vol. 58, No. 20, 2010
Hayasaka et al.
Figure 1. Synthesis of syringol-GG (7) via syringol-MG (3).
from Sigma-Aldrich (Castle Hill, Australia). d3-Guaiacol, d3-4-methyl-
guaiacol, and d3-guaiacol-β-D-glucopyranoside (d3-guaiacol-MG) were
previously synthesized in house (1,6). d3-Syringol was prepared according
to the method of Aihara et al. (7), and syringol monoglucoside (syringol-
MG) and syringol gentiobioside (syringol-GG) were prepared on the basis
of modifications to the procedures of Shao et al. (8).
stirred solution of syringol (1) (1.00 g, 6.49 mmol) in 0.3 M NaOH (21 mL,
6.30 mmol) at 14 °C. After 5 h, the reaction mixture was concentrated under
reduced pressure and dried further under high vacuum, yielding a yellow
syrup. Anhydrous pyridine (2.5 mL), acetic anhydride (4 mL), and 4-
dimethylaminopyridine (DMAP) (12.4 mg) were added, and the mixture
was heated under N2 at 90 °C for 1 h and then stirred overnight at room
temperature. The reaction mixture was cooled with an ice-water bath.
Methanol (5 mL) was added, and stirring was continued for 30 min before
removal of the volatiles under reduced pressure. The residue was dissolved in
CH2Cl2 (100 mL) and washed successively with 0.1 M HCl (10 mL), water
(10 mL), and brine (3 ꢀ 10 mL). The organic phase was dried (Na2SO4),
concentrated under reduced pressure, and chromatographed on silica gel
with CH2Cl2 f 4% Et2O/CH2Cl2 (Rf = 0.24 in 10% Et2O/CH2Cl2) to
afford, after solvent removal, syringyl acetate (0.613 g, 3.12 mmol, 50%) and
desired compound 2 (1.08 g), which was recrystallized from ethanol to yield
colorless crystals (0.929 g, 1.92 mmol, 31%; 63% based on recovered syringyl
acetate). mp 140-140.5 °C. [R]D -10.0 (c 0.502, MeOH).
Nuclear Magnetic Resonance (NMR) Analysis. Proton (1H) and
carbon (13C) NMR spectra were recorded with Bruker spectrometers
operating at 400 or 600 MHz for proton and 100 or 150 MHz for carbon
nuclei, respectively. Chemical shifts were recorded as δ values in parts per
million (ppm). Spectra were acquired in chloroform-d or deuterium oxide
(D2O) at ambient temperature, and resonances were assigned by routine
2D correlation experiments. For 1H NMR spectra, the peak as a result of
residual CHCl3 (δ 7.26) orHOD (δ 4.79) was used as the internal reference.
For 13C NMR spectra, the central peak of the CDCl3 triplet (δ 77.16) or
the CH3 peak of acetonitrile (δ 1.47), added when D2O was the solvent,
was used as the internal reference.
High-Resolution Mass Spectrometry (HRMS). Spectra were
obtained on a Bruker microTOF-Q II with electrospray ionization (ESI)
in positive mode or atmospheric pressure chemical ionization (APCI) in
negative mode. Samples dissolved in water or methanol at concentrations
of approximately 1-2 mg/L were analyzed by flow injection.
Optical Rotations. Specific rotations were recorded with a PolAAr 21
polarimeter, referenced to the sodium D line (589 nm) at 20 °C, using the
spectroscopic-grade solvents specified and at the concentrations (c, g/100 mL)
indicated. The measurements were carried out in a cell with a 1 dm path length.
Melting Points. A Buchi Melting Point B-540 unit was used, and
melting points were uncorrected.
2-d3-Methoxy-6-methoxyphenol (d3-Syringol) (d3-1). This com-
pound was prepared as described by Aihara et al. (7). Briefly, 3-methox-
ycatechol (5.00 g, 35.7 mmol) gave the desired 2-benzylated product
(2.96 g, 12.84 mmol, 36%) as a colorless oil after purification on silica
gel with 10% Et2O/20% CH2Cl2/hexane f 10% Et2O/40% CH2Cl2/
hexane (Rf = 0.48) and solvent removal, along with the 1-benzylated
compound (12%) and the 1,2-dibenzylated compound (7%). The 2-ben-
zylated material (2.08 g, 9.03 mmol) was d3-methylated to afford a pale
yellow oil (2.22 g, ca. 100%), which was used crude in the next step.
Hydrogenolysis of the benzyl group, purification on silica gel with 10%
1H NMR (ppm, CDCl3) δ: 7.02 (1H, t, J = 8.4 Hz, H4), 6.56 (2H, d,
0
J = 8.4 Hz, H3,5), 5.31 (1H, dd, J = 8.9, 7.4 Hz, H2 ), 5.27 (1 H, app t, J =
0
0
9.2 Hz, H4 ), 5.25 (1H, app t, J = 9.1 Hz, H3 ), 5.06 (1H, d, J = 7.4 Hz,
0
0
H1 ), 4.25 (1H, dd, J = 12.2, 5.0 Hz, H6a ), 4.11 (1H, dd, J = 12.2, 2.6 Hz,
0
0
H6b ), 3.81 (6H, s, 2 ꢀ ArOCH3), 3.68 (1H, ddd, J = 9.4, 5.0, 2.6 Hz, H5 ),
2.030 (3H, s, COCH3), 2.020 (3H, s, COCH3), 2.018 (3H, s, COCH3), 2.014
(3H, s, COCH3). 13C NMR (ppm, CDCl3) δ: 170.8 (CdO), 170.6 (CdO),
169.6 (CdO), 169.5 (CdO), 153.3 (C2,6), 134.6 (C1), 124.9 (C4), 105.6
0
0
0
0
0
0
(C3,5), 101.5 (C1 ), 73.2 (C3 ), 72.1 (C2 ), 72.0 (C5 ), 68.6 (C4 ), 62.4 (C6 ),
56.4 (2 ꢀ ArOCH3), 20.9 (COCH3), 20.83 (2 ꢀ COCH3), 20.78 (COCH3).
ESI-HRMS (m/z): Calcd for C22H28NaO12 ([M þ Na]þ), 507.1478;
þ
found, 507.1498.
Compound 2 (0.524 g, 1.08 mmol) was dissolved in a mixture of MeOH/
Et3N/H2O (8:1:1, 10.5 mL) and stirred for 16 h at room temperature before
being concentrated to dryness under reduced pressure. Water (2 mL) was
added, and the mixture was concentrated to dryness under reduced pressure.
The addition of water and then concentration process was repeated 2 more
times, yielding a white solid. Recrystallization from ethanol provided
monoglucoside 3 as fluffy white crystals (0.307 g, 0.97 mmol, 90%). mp
167.5-168.5 °C. [R]D -19.1 (c 0.366, H2O).
1H NMR (ppm, D2O) δ: 7.19 (1H, t, J = 8.5 Hz, H4), 6.80 (2H, d, J =
0
Et2O/20% CH2Cl2/hexane f 10% Et2O/40% CH2Cl2/hexane (Rf
=
8.5 Hz, H3,5), 5.03 (1H, d, J = 7.4 Hz, H1 ), 3.86 (6H, s, 2 ꢀ ArOCH3), 3.80
0 0
(1H, dd, J = 12.4, 2.0 Hz, H6b ), 3.71 (1H, dd, J = 12.4, 5.2 Hz, H6a ),
0.28), and solvent removal afforded title compound d3-1 (1.38 g, 8.78
mmol, 98%) as a colorless solid. mp 54.5-55.5 °C [literature mp 55-
55.5 °C (7)]. The spectroscopic data were in full accordance with those
reported by Aihara et al. (7).
3.60-3.47 (3H, m, H2 ,3 ,4 ), 3.34 (1H, ddd, J = 9.2, 5.2, 2.0 Hz, H5 ). 13C
NMR (ppm, D2O) δ: 153.2 (C2,6), 134.1 (C1), 126.3 (C4), 106.9 (C3,5), 103.6
0
0
0
0
0
0
0
0
0
0
(C1 ), 77.0 (C5 ), 76.4 (C3 ), 74.4 (C2 ), 70.0 (C4 ), 61.2 (C6 ), 56.9 (2 ꢀ
ArOCH3). ESI-HRMS (m/z): Calcd for C14H20NaO8 ([M þ Na]þ),
þ
2,6-Dimethoxyphenyl-1-O-β-D-glucopyranoside (Syringol Monoglu-
coside or Syringol-MG) (3). This compound was prepared as detailed
in Figure 1 based on the method of Shao et al. (8). Peracetyl-R-glucopyranosyl
bromide (2.54 g, 6.18 mmol) in acetone (22 mL) was added dropwise to a
339.1056; found, 339.1053.
2,6-Dimethoxyphenyl-1-O-β-
D
-glucopyranosyl-(1f6)-β-D-glucopyr-
anoside (Syringol Gentiobioside or Syringol-GG) (7). This compound

Article
J. Agric. Food Chem., Vol. 58, No. 20, 2010 10991
was prepared as depicted in Figure 1 based on the methods of Shao
et al. (8). A stirred solution of compound 3 (0.360 g, 1.14 mmol),
anhydrous pyridine (1.2 mL), trityl chloride (0.539 g, 1.93 mmol), and
DMAP (14 mg) was heated at 50 °C for 4 h under N2, stirred overnight at
room temperature, and heated for a further 5 h at 50 °C. The mixture was
cooled to room temperature. Acetic anhydride (1.1 mL) was added, and
stirring was continued for 2.25 h at room temperature. The reaction
mixture was cooled with an ice-water bath. Methanol (1 mL) was added,
and stirring was continued for 30 min before removal of the volatiles under
reduced pressure. Toluene (1 mL) was added, and the mixture was
concentrated to dryness under reduced pressure, yielding a white solid.
Chromatography on silica gel with CH2Cl2 f 3% Et2O/CH2Cl2 (Rf =
0.49 in 5% Et2O/CH2Cl2) followed by solvent removal afforded the
desired compound 4 as a white solid (0.505 g, 0.738 mmol, 65%). mp
88.0-91.0 °C. [R]D þ24.3 (c 0.474, CHCl3).
1H NMR (ppm, D2O) δ: 7.19 (1H, t, J = 8.5 Hz, H4), 6.81 (2H, d, J =
0
00
8.5 Hz, H3,5), 5.11 (1H, d, J = 7.5 Hz, H1 ), 4.31 (1H, d, J = 7.9 Hz, H1 ),
0
0
00
4.06 (1H, dd, J = 12.2, 1.4 Hz, H6a ), 3.88-3.84 (m, 2H, H6b ,6a ), 3.86
00
(6H, s, 2 ꢀ ArOCH3), 3.67 (1H, dd, J = 12.2, 6.0 Hz, H6b ), 3.58-3.46
0
0
0
0
00
(4H, m, H2 ,3 ,4 ,5 ), 3.31 (1H, app t, J = 9.4 Hz, H4 ), 3.25 (1H, app t, J =
00
00
9.2 Hz, H3 ), 3.23 (1H, ddd, J = 9.4, 6.0, 2.1 Hz, H5 ), 3.15 (1H, dd, J =
9.2, 7.9 Hz, H2 ). 13C NMR (ppm, D2O) δ: 153.3 (C2,6), 133.6 (C1), 126.2
00
0
00
0
00
(C4), 106.9 (C3,5), 102.83 (C1 ), 102.77 (C1 ), 76.9 (C5 ), 76.5 (C5 ), 76.3
0
00
0
00
00
0
0
(C3 ), 76.1 (C3 ), 74.2 (C2 ), 73.6 (C2 ), 70.2 (C4 ), 69.9 (C4 ), 68.1 (C6 ), 61.þ3
00
(C6 ), 56.8 (2 ꢀ ArOCH3). ESI-HRMS (m/z): Calcd for C20H30NaO13
([M þ Na]þ), 501.1584; found, 501.1588.
Semi-preparative HPLC Purification of Compound 7. An Agilent
1100 HPLC (Agilent, Forest Hill, Australia) equipped with a quaternary
pump and diode array detector (DAD) was used. The column was a 250 ꢀ
˚
10 mm inner diameter, 4 μm, 80 A, Synergi Hydro-RP operated at 25 °C
1H NMR (ppm, CDCl3) δ: 7.38-7.36 (6H, m, ortho CPh3), 7.25-7.23
(6H, m, meta CPh3), 7.15 (3H, tt, J = 7.4, 1.2 Hz, para CPh3), 7.08 (1H, t,
J = 8.4 Hz, H4), 6.61 (2H, d, J = 8.4 Hz, H3,5), 5.38 (1H, dd, J = 8.9, 7.8 Hz,
and protected by a guard column of the same material (Phenomenex, Lane
Cove, Australia). The solvents were water (solvent A) and acetonitrile
(sovent B), with a flow rate of 2.00 mL/min. The linear gradient for solvent
B was as follows: 0 min, 15%; 15 min, 30%; 17 min, 80%; 18 min, 15%;
and 25 min, 15%. A 100 μL injection volume was used. DAD signals were
recorded at 270 and 280 nm, and spectra were stored between 220 and
600 nm. Fractions were collected manually on the basis of retention time
and detector response. Data acquisition and processing were performed
using Agilent ChemStation software (revision B.03.01).
0
0
0
H2 ), 5.21(1 H, app t, J = 9.1Hz, H3 ), 5.18 (1H, app t, J= 9.4Hz, H4 ), 5.16
0
(1H, d, J = 7.8 Hz, H1 ), 3.80 (6H, s, 2 ꢀ ArOCH3), 3.55 (1H, ddd, J = 9.4,
0
0
5.4, 2.7 Hz, H5 ), 3.14(1H, dd, J = 10.3, 5.4 Hz, H6a ), 3.10(1H, dd, J= 10.3,
0
2.7 Hz, H6b ), 2.08 (3H, s, COCH3), 2.00 (3H, s, COCH3), 1.69 (3H, s,
COCH3). 13C NMR (ppm, CDCl3) δ: 170.7 (CdO), 169.7 (CdO), 169.3
(CdO), 153.6 (C2,6), 143.7 (ipso CPh3), 134.6 (C1), 128.8 (ortho CPh3), 127.9
0
(meta CPh3), 127.0 (para CPh3), 124.9 (C4), 105.8 (C3,5), 101.4 (C1 ), 86.5
Smoke Analysis. Smoke Sampling. Smoke was collected from a
prescribed burnoff in the Adelaide Hills in South Australia, Australia, in
June 2009. Active air sampling was carried out using a Gerstel thermal
desorption unit (TDU) desorption tube containing Tenax TA as an
adsorbent (Lasersan, Robina, Australia), connected to a pocket pump
(SKC, Eighty Four, PA). Smoke in the prescribed burnoff site was
introduced into the TDU tube at a rate of 200 mL/min for 40 min. The
TDU tube was then kept at 4 °C until analysis.
0
0
0
0
0
(CPh3), 73.6 (C3 ,5 ), 72.4 (C2 ), 69.1 (C4 ), 61.7 (C6 ), 56.5 (2 ꢀ ArOCH3),
21.0 (COCH3), 20.9 (COCH3), 20.6 (COCH3). APCI-HRMS (m/z): Calcd
for C39H39O11- ([M - H]-), 683.2492; found, 683.2422.
A stirred solution of compound 4 (0.406 g, 0.593 mmol) in 80% acetic
acid (2.6 mL) was heated at 60 °C for 4 h. The mixture was cooled to room
temperature and concentrated under reduced pressure. Toluene (1 mL)
was added, and the mixture was again concentrated under reduced
pressure. The addition of toluene and then concentration process was
repeated 1 more time, yielding a white solid. Chromatography on silica
gel with CH2Cl2 f 30% Et2O/CH2Cl2 (Rf = 0.39) followed by solvent
removal afforded the desired compound 5 as a white solid (0.218 g, 0.493
mmol, 83%). mp 169.5-170.0 °C. [R]D -11.3 (c 0.178, CH2Cl2).
1H NMR (ppm, CDCl3) δ: 7.03 (1H, t, J = 8.4 Hz, H4), 6.57 (2H, d,
Gas Chromatography-Mass Spectrometry (GC-MS) Analysis. Anal-
ysis was carried out with a Hewlett-Packard (HP) 6890 gas chromatograph
and HP 5973 mass selective detector (Agilent Technologies, Forest Hill,
Australia) fitted with a Gerstel autosampler (MPS 2XL), TDU, and
programmed temperature vaporization inlet (CIS-4) (Lasersan). A TDU
tube containing the smoke sample was thermally desorbed by increasing
the TDU temperature from 30 to 280 °C at a rate of 500 °C/min and
holding for 3 min at 280 °C. The thermally desorbed compounds were
trapped in the CIS-4 at -20 °C. Immediately following completion of
desorption, the temperature of the CIS-4 was sharply elevated to 280 °C at
12 °C/s to introduce the desorbed compounds onto a 30 m ꢀ 0.25 mm DB-
Wax with a film thickness of 0.25 μm fused silica capillary column (Agilent
Technologies). The GC oven temperature was started at 50 °C, held at this
temperature for 1 min, ramped to 220 °C at a rate of 10 °C/min, held at this
temperature for 5 min, then ramped to 240 °C at a rate of 20 °C/min, and
held for 6 min. Helium was used as a carrier gas at 1.2 mL/min in constant
flow mode. The transfer line was maintained at 240 °C, and positive
electron impact ion spectra at 70 eV were recorded in the mass range of m/z
35-350 in 1 s. Volatile phenols were identified according to their retention
times and mass spectra by a comparison to those of the respective reference
compounds.
0
J = 8.4 Hz, H3,5), 5.33 (1H, app t, J = 8.3 Hz, H2 ), 5.27 (1 H, app t, J =
9.0 Hz, H3 ), 5.22 (1H, app t, J = 9.2 Hz, H4 ), 5.09 (1H, d, J = 7.0 Hz,
0
0
0
0
H1 ), 3.82 (6H, s, 2 ꢀ ArOCH3), 3.70-3.66 (1H, m, H6a ), 3.65-3.62 (1H,
0
0
m, H6b ), 3.61-3.58 (1H, m, H5 ), 2.62 (1H, app t, J = 6.8 Hz, OH), 2.04
(3H, s, COCH3), 2.034 (3H, s, COCH3), 2.031 (3H, s, COCH3). 13C NMR
(ppm, CDCl3) δ: 170.6 (CdO), 169.9 (CdO), 169.5 (CdO), 153.2 (C2,6),
0
0
0
134.7 (C1), 125.1 (C4), 105.6 (C3,5), 101.7 (C1 ), 74.5 (C5 ), 73.0 (C3 ), 72.2
0
0
0
(C2 ), 68.8 (C4 ), 61.7 (C6 ), 56.4 (2 ꢀ ArOCH3), 20.9 (COCH3), 20.86
þ
(COCH3), 20.82 (COCH3). ESI-HRMS (m/z): Calcd for C20H26NaO11
([M þ Na]þ), 465.1373; found, 465.1375.
This procedure was adapted from the method of Kartha et al. (9).
Peracetyl-R-glucopyranosyl bromide (0.302 g, 0.734 mmol) in CH2Cl2
(2 mL þ 0.5 mL rinse) was added dropwise to a stirred mixture of
compound 5 (0.108 g, 0.244 mmol), Ag2CO3 (0.215 g, 0.779 mmol), and
˚
powdered 4 A molecular sieves (0.56 g) in CH2Cl2 (2.5 mL) under N2 at
0 °C. The mixture was stirred for 3 h at room temperature and filtered
through celite, and the filtrate was concentrated under reduced pressure.
Chromatography on silica gel with 1% EtOH/15% EtOAc/CHCl3 (Rf =
0.26) followed by solvent removal afforded the desired compound 6 as a
white solid (0.356 g), which was a mixture of polyacetylated coupled
products, starting material compound 5, and acetylated glucose, by 1H
NMR and HPLC-MS analyses. The mixture was used in the subsequent steþp
without further purification. ESI-HRMS (m/z): Calcd for C34H44NaO20
([M þ Na]þ), 795.2324; found, 795.2338.
Smoke-Affected and Control Grapes. Smoke-Affected Grapes.
Vitis vinifera L. cv. Chardonnay and Cabernet Sauvignon grapes were
collected from closely located vineyards in Victoria (Australia) in March
2009. The vineyards had been affected by smoke from a series of bushfires
that occurred in the period of February 7-March 14, 2009.
Control Grapes. Three samples each of non-smoked Chardonnay and
Cabernet Sauvignon grapes were collected from various regions of South
Australia.
Winemaking. Smoke-Affected Wines. Wine was made from smoke-
affected Cabernet Sauvignon and Chardonnay grapes that had been
frozen for approximately 6 months at -20 °C prior to winemaking.
Grapes were randomized, thawed at 4 °C overnight, then crushed, and
destemmed. A 5 kg sample of grapes was used for winemaking. Potassium
metabisulfite and tartaric acid were added to the must to achieve a free
sulfur dioxide content of approximately 15 mg/L and pH 3.00-3.10 for
Chardonnay and pH 3.50-3.60 for Cabernet Sauvignon. The musts were
then inoculated with yeast strain AWRI 796 at 20 °C and fermented on
skins for both red and white wines at 25 °C. Ferments were drained and
This procedure was adapted from the method of Pathak (10). Amberlite
IRA-400(Cl) resin was exchanged with 1 M NaOH to IRA-400(OH) resin.
A solution of compound 6 (0.244 g, 0.316 mmol) containing dry IRA-
400(OH) (5.3 g) in MeOH (16 mL) was gently stirred for 24 h at room
temperature. The mixture was filtered, and the filtrate was concentrated
under reduced pressure to yield a white solid, which was purified by semi-
preparative HPLC to yield the title gentiobioside 7 as a fluffy white solid
(0.016 g, 0.033 mmol, 10%) after solvent removal and freeze-drying. mp
195-200 °C (began decomposing from 170 °C). [R]D -29.1 (c 0.216, H2O).

10992 J. Agric. Food Chem., Vol. 58, No. 20, 2010
Hayasaka et al.
Figure 2. GC-MS analysis of smoke from a prescribed burnoff.
pressed after 7 days and fermented to dryness (<1 g/L residual sugar,
confirmed via Clinitest and then enzymatic analysis). Once dry, wines
were racked off gross lees into appropriately sized glass storage vessels,
and potassium metabisulfite was added to achieve a free SO2 level of
30-35 mg/L for Chardonnay and approximately 80 mg/L total for
Cabernet Sauvignon. The wines were cold settled at 4 °C for 3 weeks,
carefully bottled into 375 mL screw cap bottles using dry ice, and stored at
4 °C.
small-lot wines, as well as from control grape and wine samples of the same
grape varieties. A 10 mL aliquot of wine or supernatant obtained from the
grape homogenate was acidified to pH 1.0 with concentrated sulfuric acid
and heated at 100 °C for 1 h (3). Samples were subjected to GC-MS for the
analysis of volatile phenols and HPLC-SRM for the analysis of volatile
phenol glycosides, before and after hydrolysis.
GC-MS Analysis of Volatile Phenols. Sample Preparation. A
5 mL aliquot of grape juice from the homogenate, wine, or hydrolysate
sample was transferred to a 10 mL glass vial with a foil-lined screw cap and
spiked with 100 μL of the labeled internal standard solution detailed
below. After the additionof 2 mL of freshly preparedpentane/ethyl acetate
(1:1), the sample vial was manually shaken for 15 s and left until the sample
mixture separated into two layers. The organic layer was transferred to a
vial ready for analysis.
Control Wines. Three each of commercial Chardonnay and Cabernet
Sauvignon wines were obtained and used as controls. These wines were
made from grapes grown in regions unaffected by bushfire.
HPLC-MS/MS Analysis for Glycosides of Volatile Phenols.
Sample Preparation. Berry extracts were prepared according to the method
described by Hayasaka et al. (1), with a minor modification. Briefly, a 5 g
aliquot of the grape homogenate containing 0.5 mg/kg of d3-guaiacol-MG
as internal standard was centrifuged at 4000 rpm for 5 min to collect the
supernatant. A 2 mL aliquot of the supernatant (or juice hydrolysate for
the hydrolysis experiment described later) was applied to a preconditioned
Extract Clean C18-HF SPE 500 mg/4 mL cartridge (Grace Davison
Discovery Sciences, Australia) to obtain the methanol extract. After
removal of methanol, the residue (extract) was reconstituted with
0.5 mL water, filtered (0.45 μm), and transferred to a vial ready for analysis.
Wine. A 1 mL aliquot of wine containing 1 mg/L of d3-guaiacol-MG as
internal standard was filtered (0.45 μm) and transferred to a vial ready for
analysis. For acid hydrolysis experiments, the hydrolysate was adjusted to
pH 3-4 with 5 M NaOH, then spiked with the internal standard, and
transferred to a vial ready for analysis.
Quantitation. Calibration functions were prepared for the quantitation
of guaiacol, 4-methylguaiacol, phenol, three cresol isomers, syringol, and
4-methylsyringol. The labeled internal standards were d3-guaiacol for
guaiacol, d3-4-methylguaiacol for 4-methylguaiacol, d7-p-cresol for phenol
and cresol isomers, and d3-syringol for syringol and 4-methylsyringol. A
mixed labeled internal standard solution of approximately 10 mg/L was
prepared in ethanol. The respective volatile phenol standards, prepared as
a mixture in ethanol and spiked from 0 to 200 μg/L (and up to 1000 μg/L
for syringol only), and 100 μL of labeled internal standard mixture were
added to 5 mL of model wine. The calibration samples underwent sample
preparation and GC-MS analysis as described.
GC-MS Analysis. Analysis was carried out with a ThermoQuest Trace
GC 2000 gas chromatograph combined with a TSQ mass spectrometer
(Thermo Fisher Scientific, Scoresby, Australia). The GC was equipped
with the same capillary column described under Smoke Analysis. Helium
was used as carrier gas at a flow rate of 1.5 mL/min. The injector was in
splitless mode, and the split vent was opened after 0.5 min, with an injected
sample volume of 2 μL. Temperatures of the injector and transfer line were
200 and 250 °C, respectively. The GC column was maintained at 50 °C for
1 min, ramped at a rate of 8 °C/min to 255 °C, and held for 10 min. Mass
spectra (EI at 70 eV) were recorded in selected ion monitoring (SIM) mode
(Supplementary Table 2 in the Supporting Information). The respective
target ions were used for quantitation of the respective volatile phenols (by
peak area).
HPLC-MS/MS Analysis. HPLC-MS/MS with APCI was carried
out according to the method previously described (1). The tandem mass
spectrometry (MS/MS) parameters were set at -18 V for collision
potential, -5 V for collision cell exit potential, and high for collision gas
pressure. For HPLC-MS/MS in selected reaction monitoring (HPLC-
SRM), the mass transitions from [M - H þ CH3COOH]- ions of the
respective glycosides to the common fragments m/z 323 for glucosylgluco-
side (GG), m/z 161 for monoglucoside (MG), m/z 293 for diglycoside
(DG), and m/z 307 for rutinoside (RG) were monitored with a dwell time
of 50 ms (Supplementary Table 1 in the Supporting Information). For
HPLC-SRM combined with in-source fragmentation, the declustering
potential was increased from -40 to -80 V and the mass transitions from
[M - H þ CH3COOH]- and [M - H]- ions of the respective glycosides to
the common fragment ions for GG, MG, DG, and RG were simultaneously
monitored (Supplementary Table 1 in the Supporting Information).
Quantitative Analysis of the Glycosides. Concentrations of the glyco-
sides were determined using d3-guaiacol-MG (monitoring the mass
transition from m/z 348 f 161) as internal standard using the same
response factor for the internal standard and the respective glycosides.
Acid Hydrolysis. Acid hydrolysates were prepared from smoke-affected
Chardonnay and Cabernet Sauvignon grapes and their corresponding
RESULTS AND DISCUSSION
Smoke Analysis. To investigate major smoke-derived volatile
organic compounds (VOCs), smoke generated from a prescribed
burnoff was trapped in Tenax tubes and analyzed by GC-MS. In
addition to guaiacol, volatile phenols, including syringol, methyl-
syringol, o-, p-, and m-cresols, phenol, and methylguaiacol, were
found to be major components of smoke-derived VOCs
(Figure 2). Vinylguaiacol and vinylsyringol were also present as

Article
J. Agric. Food Chem., Vol. 58, No. 20, 2010 10993
Figure 3. Total ion chromatograms obtained by HPLC-SRM analysis of smoke-affected and control Chardonnay (A) grapes and (B) wines for screening
GG, DG, RG, and MG conjugates of volatile phenols.
minor components. Being derived by the thermal oxidative
decomposition of lignin (5, 11), these volatile phenols have been
commonly found as major components of liquid smoke flavor-
ings (12, 13) and smoke generated from wood stoves (14). The
concentration of the individual volatile phenols can vary signifi-
cantly because of the variation of the fuel source, moisture
content, combustion temperature, and availability of oxygen.
Nevertheless, this experiment demonstrated the possibility that
grapevines can be severely exposed to a number of volatile
phenols from bushfire events in the vicinity of vineyards, as seen
in the case of guaiacol (1, 15, 16).
Glycosides of Volatile Phenols. As first observed in previous
studies (1,3), the glycosides of guaiacol were consistently detected
as their acetic acid adduct ions [M - H þ CH3COOH]- under the
HPLC-MS conditions used. Product ion spectra of the adduct
ions commonly exhibited two distinctive fragment ions, which
were the deprotonated molecular ions [M - H]- resulting from
the neutral loss of 60 Da and glycoside ions representing the mass
of the sugar moiety of guaiacol glycosides, i.e., m/z 161 for
monoglucoside (MG), m/z 323 for glucosylglucoside (GG), m/z
293 for diglycoside (DG), and m/z 307 for rutinoside (RG) (1).
Because of the structural similarity of guaiacol to the other
volatile phenols found from the smoke analysis, any volatile
phenol glycosides present were expected to fragment the same as
the guaiacol glycosides. On the basis of this assumption, a
screening experiment was conducted to detect glycosides of
individual volatile phenols by HPLC-SRM mode, monitoring
the mass transitions from the expected [M - H þ CH3COOH]-
ions to the respective glycoside ions of MG, GG, DG, or RG
(Supplementary Table 1 in the Supporting Information).
wine analyses showed that the smoke-affected samples had
substantial increases in the intensity and number of peaks derived
from volatile phenols conjugated to GG, DG, or RG compared
to those from the control samples (Figure 3). In contrast, there
was no obvious effect on the intensity of MG conjugates in grapes
and the resulting wine from smoke exposure, and MG conjugates
were excluded from further investigation.
In the control grape and wine, only small peaks were recorded
(Figure 3), which were likely to represent volatile phenol glyco-
sides naturally present ingrapes, asseeninthe caseofguaiacol(1).
The presence of precursors to volatile phenols in various grape
varieties has been reported for phenol, guaiacol, 4-vinylguaiacol,
4-vinylphenol (17), cresols, 4-ethylguaiacol, eugenol, 4-ethylphenol,
syringol, and 4-methylsyringol (18). Both studies identified vola-
tile phenols in enzyme and/or acid hydrolysates, indicating that
trace amounts of phenolic glycosides were natural components of
the grapes.
MS/MS Experiments To Confirm the Presence of Individual
Volatile Phenol Glycosides. Volatile phenol glycosides gave pro-
minent [M - H þ CH3COOH]- and [M - H]- ions by in-source
fragmentation with a higher declustering potential, and both
pseudo-molecular ions were dissociated to the same glycoside
fragment ion, m/z 323, 297, and 307 for GG, DG, and RG,
respectively. Therefore, the putative glycosides of volatile phenols
(Supplementary Table 1 in the Supporting Information) were
screened by simultaneously monitoring the mass transitions from
both pseudo-molecular ions to the respective glycoside ions using
HPLC-SRM combined with in-source fragmentation (Figure 4).
Analysis of the smoke-affected Chardonnay grape sample clearly
indicated a single peak derived from syringol-GG, methylsyringol-
GG, guaiacol-RG, or methylguaiacol-RG (panels A, B, E, and F
of Figure 4). Phenol-DG and cresol-DG (panels C and D of
Figure 4) gave multiple peaks derived from the expected mass
transitions because of the presence of multiple DG and cresol
Smoke-affected and control samples of Chardonnay and
Cabernet Sauvignon grapes and wines were analyzed to confirm
whether peaks derived from the putative volatile phenol glyco-
sides increased as a result of smoke exposure. Both grape and

10994 J. Agric. Food Chem., Vol. 58, No. 20, 2010
Hayasaka et al.
Figure 4. HPLC-SRM analysis combined with in-source fragmentation of smoke-affected Chardonnay grapes to confirm the presence of (A) syringol-GG,
(B) methylsyringol-GG, (C) phenol-DG, (D) cresol-DG, (E) guaiacol-RG, and (F) methylguaiacol-RG.
isomers. However, some peaks were characterized by only one
mass transition; therefore, those peaks were ruled out as phenol-
DG or cresol-DG. Furthermore, the product ion spectra of the
respective [M - H þ CH3COOH]- ions of the target phenolic
glycoside peaks (V in Figure 4) were obtained by HPLC-MS/MS
(Supplementary Figure 1 in the Supporting Information). All of
the spectra showed good agreement with the expected fragment
ions (Supplementary Table 1 in the Supporting Information);
therefore, the identification of these glycosides was further
supported. In summary, a broad range of glycosides of volatile
phenols was found in the smoke-affected grape and wine samples
of Chardonnay and Cabernet Sauvignon (Supplementary Table 3
in the Supporting Information). These glycosides were typically
found in both sample types (grape or wine) for both grape
varieties.
For additional confirmation of the presence of syringol glyco-
sides and because syringol was one of the most dominant volatile
phenols (Figure 2), syringol-GG was synthesized via syringol-MG
using a selective protection and coupling regime, as shown in
Figure 1. Pure 1f6-linked, β,β-configured syringol-GG was
obtained after semi-preparative HPLC and fully characterized.
The chromatographic and mass spectrometric properties of the
reference compound were confirmed tobe identical to those ofthe
tentatively identified syringol-GG found in the smoke-affected
grapes by HPLC-MS/MS and HPLC-SRM with co-injection
experiments. Therefore, syringol-GG detected during this study
was determined to be a gentiobioside.
Quantitative Distribution of Glycosides of Volatile Phenols. The
quantitative analysis of the individual glycosides in the grape and
wine samples was carried out by HPLC-SRM, and concentra-
tions were expressed as d3-guaiacol-MG equivalents (Figure 5).
The smoke-affected grape and wine samples contained the glyco-
sides at greatly elevated concentrations compared to those of the
respective control samples. The concentration of total glycosides
for Chardonnay (sum of all phenolic glycosides quantified, but
MG was excluded) was 0.28/21.15 mg/kg in grapes (control/
smoke-affected) and 0.20/16.42 mg/L in wine; for Cabernet
Sauvignon, the concentration was 0.19/21.02 mg/kg in grapes
and 0.21/14.17 mg/L in wine. In both varieties, the distribution
pattern of phenolic glycosides in the smoke-affected grape
samples was similar to that in the resulting wines (Figure 5), but
the concentration of the total glycosides decreased from around
21 mg/kg in the grapes to 14-16 mg/L in the wines. Accordingly,
the apparent extraction rate in the wine was calculated to be 78%
for Chardonnay and 67% for Cabernet Sauvignon.
It should be noted that winemaking was undertaken using
grapes that had been frozen and the Chardonnay wine was made
with the same winemaking procedures used for the Cabernet
Sauvignon wine. Localization of the glycosides in the berries that
we used may be different from that in fresh grapes as a result of
freezing, while the skin contact time for the experimental Char-
donnay wine was considerably longer than employed during
usual winemaking practice for white wine. Such factors could
conceivably have influenced the extraction rate of the glycosides,
particularly for the Chardonnay wine, although plant secondary
metabolites, such as glycosides, are usually highly water-soluble
and should be readily extracted into musts and ferments. Further-
more, because a loss of glycosides resulting from chemical and
enzymatic hydrolysis during fermentation could also have con-
tributed to a reduction in the concentration of glycosides in the
wines, the overall extraction was likely higher than the observed
value.
Upon examination of the glycosylation pattern, it appeared
that only syringol and methylsyringol were dominantly present as
GG conjugates (Figure 5). In fact, syringol-GG was the most
abundant glycoside in all smoke-affected samples. Other GG
conjugates of volatile phenols were either not detected or detected
only in trace amounts. Multiple isomers of DG conjugates were
the most common glycosides of volatile phenols, including
phenol, cresols, guaiacol, methylguaiacol, syringol, and methyl-
syringol. RG conjugates were also commonly found, with the
exception of syringol and methylsyringol. Additionally, vinyl-
guaiacol and vinylsyringol glycosides were present in the smoke-
affected samples in very low amounts (Figure 5).
Acid Hydrolysis and Fermentation. Acid hydrolysis of smoke-
affected grapes and the resulting wines along with control grapes
and wines was carried out to investigate the susceptibility of the
phenolic glycosides to acid. The free volatile phenols were deter-
mined by GC-MS, and the glycosides were determined by
HPLC-SRM, before and after acid hydrolysis. The concentrations

Article
J. Agric. Food Chem., Vol. 58, No. 20, 2010 10995
Figure 5. Quantitation of glycosides of volatile phenols in smoke-affected and control grapes and wines of (A) Chardonnay and (B) Cabernet Sauvignon.
of free volatile phenols in the control grape and wine samples were
unchanged or slightly elevated by acid hydrolysis (data not
shown), further supporting that only small amounts of the phenolic
glycosides were present as natural components of grapes and
wine (Figures 3 and 5). In smoke-affected grapes, the concentration
of GG conjugates decreased markedly after hydrolysis but RG
conjugates remained apparently unchanged (Figure 6A). The
intensity of DG conjugate peaks also declined but to a lesser extent
than GG conjugates. In the wine samples, however, all of the
glycosides disappeared almost completely after acid treatment
(Figure 6B).
After hydrolysis, approximately 50% (grape) and 92% (wine)
of the total phenolic glycosides (MG conjugates were excluded)
had been eliminated from samples of both varieties (Table 1). In
the grape samples in particular, syringol and methylsyringol
glycosides were hydrolyzed to the greatest extent (up to 77%)
compared to the other phenols (up to 49%). Under the hydrolysis
conditions employed, syringol glycosides in wine were cleaved to
the highest extent (up to 98%), although hydrolysis of the other
phenols was also high (up to 94%) (Table 1). The differences
between grapes and wine were likely due to the much higher sugar
content of the grape samples, where juice sugar levels may have
interfered with hydrolysis.
between individual volatile phenols and differed somewhat
between white and red varieties. Syringol and methylsyringol were
found in the highest amounts in grape and wine hydrolysates. The
syringol concentration was over 600 and 700 μg/L (grape juice
and wine, respectively), and the methylsyringol concentration
was up to 177 and 239 μg/L. Of the remaining phenols, guaiacol
was present in concentrations up to 44 and 72 μg/L in the
Cabernet Sauvignon hydrolysates, while phenol, cresols, and
methylguaiacol were in the order of 20 and 50 μg/L or less
(Table 2). In general, the red grape and wine hydrolysates had
higher levels of phenols, although on the whole, the results for red
and white varieties were comparable.
Free volatile phenols in wine can be also released by enzy-
matic activity during fermentation. The release of smoke-derived
phenols through fermentation has been studied by Kennison
et al. (2), using Merlot grapes after experimental exposure of the
grapevines to smoke generated by the combustion of dry straw. In
that study, the concentrations of guaiacol and methylguaiacol
were reported to increase from trace levels in free run juice to 388
and 93 μg/L, respectively, in the finished wine. In the present
study, a significant proportion (up to 73%, depending upon the
analyte and grape variety) of volatile phenols in the wine hydro-
lysates was already present before acid treatment, with the
exception of syringol and methylsyringol (around 2-3%)
(Table 2). These pre-existing phenols in the wines were therefore
considered to be released during fermentation. Although the
smoke-affected grapes contained the highest concentrations of
the glycosides (total glycosides, 21 mg/kg for both varieties;
The greater hydrolysis of phenolic glycosides in the wine
samples wassupported by the observation that the concentrations
of total free volatile phenols in the hydrolysates of both grape
varieties were around 1.2 times higher in the wines than in the
grapes (Table 2). The higher release in the wine samples varied

10996 J. Agric. Food Chem., Vol. 58, No. 20, 2010
Hayasaka et al.
Figure 6. Total ion chromatograms of glycosides (GG, DG, and RG) of volatile phenols obtained by HPLC-SRM analysis of smoke-affected Cabernet
Sauvignon (A) grapes and (B) wines before and after acid hydrolysis.
Table 1. Concentrations of Glycosides of Volatile Phenols in Smoke-Affected
Grapes and Wines before and after Acid Hydrolysis
individual volatile phenols from the corresponding glycosides
following acid treatment of wine was calculated by comparing an
increase of the free phenol to a decrease of the glycosides on a
molar basis (Table 3). The recovery of free phenol, cresols,
guaiacol, and methylguaiacol was estimated to be 3-8%; accord-
ingly, more than 90% of the eliminated glycosides were decom-
posed without releasing aglycones. In contrast, syringol and
methylsyringol were more effectively released, with a recovery
of around 30% (Table 3). It should be emphasized that the
concentrations of the glycosides were expressed as d3-guaiacol
glucoside equivalents for comparison purposes and were not
absolute amounts. Therefore, these recovery values were also
considered to be used for comparison purposes. Sefton (17)
reported that phenol, guaiacol, 4-vinylguaiacol, and 4-vinylphe-
nol were mostly not detectable in acid hydrolysates (pH 3.2 and
45 °C for 4 weeks) of Merlot or Cabernet Sauvignon grapes but
were found in significantly elevated concentrations in enzyme
hydrolysates using Rohapect C. Loscos et al. (18) also reported
that volatile phenols in the extracts of juice from seven different
grape varieties were much more efficiently released by enzymatic
hydrolysis using AR 2000 pectinase enzyme than acid hydrolysis
conducted at pH 2.5 and 100 °C for 1 h. Interestingly, the only
exception to this observation was syringol, which was found in
comparatively similar concentrations in both hydrolysates. This
difference between syringol and other phenols was in accordance
with results from fermentation versus acid hydrolysis in the
present study.
Chardonnay
Cabernet Sauvignon
acid hydrolysis loss
before after amount %
acid hydrolysis
loss
glycosides
beforea aftera amounta
%
Grapes (mg/kg)
phenol
cresols
guaiacol
2.32
3.76
3.03
2.41
7.02
2.62
1.88
0.45 19
1.39 37
1.03 34
0.80 33
4.65 66
1.92 73
1.67
2.27
3.82
1.71
8.46
3.09
0.86
1.68
2.44
1.42
1.95
0.70
0.82 49
0.59 26
1.38 36
0.29 17
6.51 77
2.38 77
2.37
2.00
1.61
2.36
0.70
methylguaiacol
syringol
methylsyringol
total glycosides 21.16 10.92 10.24 48 21.02
9.05 11.97 57
Wines (mg/L)
phenol
cresols
guaiacol
2.40
2.49
2.32
1.63
5.89
1.70
0.14
0.45
0.30
0.35
0.15
0.05
2.26 94
2.04 82
2.02 87
1.28 78
5.74 97
1.65 97
0.94
1.35
2.37
1.23
6.25
2.05
0.05
0.26
0.31
0.25
0.09
0.06
0.88 94
1.08 80
2.06 87
0.97 79
6.16 98
1.99 97
methylguaiacol
syringol
methylsyringol
total glycosides 16.43
1.44 14.99 91 14.19
1.02 13.14 93
a The sum of GG, DG, and RG was expressed as d3-guaiacol-MG equivalents.
Table 1), the concentration of free volatile phenols in the resulting
wines was considerably lower (total phenols, 0.064 mg/L for
Chardonnay and 0.118 mg/L for Cabernet Sauvignon; Table 2).
Accordingly, the release of free volatile phenols from glycosylated
precursors (mainly diglycosides) during the winemaking process
applied was estimated to be low relative to the amounts of grape
phenolic glycosides available.
Considering the substantial loss of the glycosides observed in
wines after acid treatment (Table 1), the amounts of free phenols
released were comparatively low (Table 2). Recovery of the
Unlike enzymatic hydrolysis releasing mainly an intact agly-
cone, acid-catalyzed hydrolysis is less specific and cleaves either
the glycosidic linkage (O-sugar moiety) to release aglycone or the
ether (O-aglycone moiety) to yield the carbocation of the agly-
cone. The carbocation would be further decomposed and/or
readily reacted with some components of grapes (17). Syringol
and methylsyringol glycosides may have a weaker glycosidic
linkage, which makes the release of an aglycone more favorable.

Article
J. Agric. Food Chem., Vol. 58, No. 20, 2010 10997
Table 2. Concentrations of Volatile Phenols in Smoke-Affected Grapes and Wines before and after Acid Hydrolysis
volatile phenols
hydrolysis
phenols
cresols
guaiacol
methylguaiacol
syringol
methylsyringol
total phenols
before after
before
after
before
after
before
after
before
after
before
after
before
after
Chardonnay
grape juice (μg/L)
wine (μg/L)
released by fermentation (%)
released by acid (%)
nda
18
40.9b
59.1c
19
44
5
18
42
3
7
22
36
nd
2
8
nd
20
613
722
nd
2
136
170
8
816
1029
15
15
64
23.8
64.3
11.1
80.6
13.3
86.7
2.8
97.2
1.2
98.8
5.4
93.8
Cabernet Sauvignon
grape juice (μg/L)
wine (μg/L)
nd
35
12
48
4
20
42
8
44
72
nd
4
9
nd
25
634
710
nd
6
177
239
12
118
896
1127
22
26
16
released by fermentation (%)
released by acid (%)
72.9
27.1
42.9
47.6
25.0
63.9
25.0
75.0
3.5
96.5
2.5
97.5
9.4
89.5
a Not detected. b % = (wine concentration - juice concentration)/wine hydrolysate concentration ꢀ 100. c % = (wine hydrolysate concentration - wine concentration)/
wine hydrolysate concentration ꢀ 100.
Table 3. Recovery of Free Phenols from Glycosides in Wine following Acid
Hydrolysis
phenolic glycosides in smoke-affected grapes, there is the potential to
release over 100 μg/L in total of volatile phenols during fermentation
and around 1000 μg/L by strong acid hydrolysis, which may mimic
wine storage conditions to some extent. This knowledge is important
to estimate the effects of winemaking and storage on wine volatile
phenol concentrations resulting from phenolic glycosides present in
grapes following grapevine exposure to bushfire smoke.
loss of
glycosides
(μM)a
gain of free
phenols
(μM)
conversion
(%)
Chardonnay
phenol
cresols
guaiacol
5.610
4.890
4.670
2.857
12.405
3.467
0.277
0.250
0.234
0.094
4.558
1.000
4.9
5.1
ABBREVIATIONS USED
DMAP, 4-dimethylaminopyridine; TDU, thermal desorption
unit; SRM, selected reaction monitoring; VOC, volatile organic
compound.
5.0
3.3
methylguaiacol
syringol
methylsyringol
36.7
28.8
ACKNOWLEDGMENT
Cabernet Sauvignon
We acknowledge Dr. James Kennedy and Con Simos of the
AWRI for valuable discussion. We also thank Dr. Simon Odell of
the AWRI for conducting winemaking and Jeremy Hack of
Metabolomics Australia for HRMS analysis.
phenol
cresols
guaiacol
2.192
2.605
4.763
2.181
13.303
4.181
0.138
0.185
0.371
0.087
4.448
1.387
6.3
7.1
7.8
4.0
methylguaiacol
syringol
methylsyringol
33.4
33.2
Supporting Information Available: Tables of precursor and
product ions of volatile phenol glycosides used for HPLC-MS/
MS analyses, target and qualifier ions of volatile phenols used for
quantitative GC-MS analysis, and retention times of volatile
phenol glycosides detected by HPLC-SRM analysis of smoke-
affected samples and figure displaying product ion spectra of
volatile phenol glycosides obtained from HPLC-MS/MS anal-
ysis of smoke-affected Chardonnay grapes. This material is
available free of charge via the Internet at http://pubs.acs.org.
a Average molecular weight of glycosides (GG, DG, and RG) was 403, 417, 433,
447, 463, and 477 for phenol, cresols, guaiacol, methylguaiacol, syringol, and
methylsyringol, respectively.
All together, we demonstrated that volatile phenols from
bushfire smoke, including phenol, cresols, methylguaiacol, syr-
ingol, and methylsyringol, can be metabolized to glycoconjugate
forms within grapes in a similar fashion to that shown previously
for guaiacol. Using extensive HPLC-MS/MS experiments, these
phenolic glycosides were found in smoke-affected grapes and
wines at significantly elevated levels compared to concentrations
in the non-smoked control grapes and wines. Synthesis and
co-injection of syringol-GG confirmed the presence of this com-
pound in smoke-affected samples. The apparent extraction rate of
these glycosides from grapes to wine was estimated to be 78% for
Chardonnay and 67% for Cabernet Sauvignon. A large proportion
of the total glycosides in grapes (50%) and wine (92%) disappeared
after acid treatment, but the amounts of free phenol volatiles
released were comparatively low. In the case of wine, more than
90% of the eliminated glycosides of phenol, cresols, guaiacol, and
methylguaiacol were decomposed without yielding their known
aglycones, but syringol and methylsyringol were more effectively
released (around 30%) by acid hydrolysis. Nonetheless, these results
provide useful information about the relative contributions of
fermentation and chemical hydrolysis to volatile phenol release into
wine. We have shown that, from a pool of approximately 20 mg/L
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Received for review August 4, 2010. Revised manuscript received
September 16, 2010. Accepted September 18, 2010. The AWRI, a
member of the Wine Innovation Cluster in Adelaide, is supported by
Australia’s grapegrowers and winemakers through their investment
body, the Grape and Wine Research Development Corporation, with
matching funds from the Australian government.