Analysis of rotundone at below-threshold levels in red and white wines
a-Guaiene
to SPE. The method used in this study was derived from that
[3]
published by Siebert and co-workers. SPE was performed
on Isolute Envþ (IST) cartridges (1 g, 6 mL volume)
previously conditioned with 10 mL of n-pentane/ethyl
acetate (4:1) then 10 mL of methanol, and finally 20 mL of
model wine (12% ethanol and 4g/L of tartaric acid buffered
to pH 3.2). The wine sample was percolated through
the cartridge, which was then washed with water (10 mL)
and eluted with 20 mL of n-pentane/ethyl acetate (9:1). The
organic solvent was removed under vacuum using an EZ-2
1
H NMR (400 MHz, vinylic protons) d 4.62 (1 H, m), 4.68 (1 H,
13
m); C NMR (100 MHz, vinylic CH carbon, confirmed by
DEPT experiment) d 107.8; GC-MS m/z (rel. inten.) 204 (M , 36),
1
1
2
þ
89 (33), 161 (20), 148 (36), 147 (69), 133 (56), 121 (30), 119 (41),
07 (72), 105 (100), 95 (26), 93 (54), 91 (47), 81 (38), 79 (45), 77 (32).
b-Guaiene
GC/MS m/z (rel. inten.) 204 (M , 53), 189 (27), 161 (75), 149
þ
(
(
18), 148 (9), 147 (22), 133 (38), 121 (18), 119 (64), 107 (36), 105
100), 95 (11), 93 (31), 91 (50), 81 (30), 79 (32), 77 (29).
(
GeneVac, UK) system and the residue dissolved in 1 mL of
ethanol. Then 13 mL of deionised water were added and the
solution was analysed using the SPME-GC/MS/MS method.
Chloro derivative of guaiol
GC/MS m/z (rel. inten.) 242 (M þ 2, 21), 240 (M , 62), 204
þ
þ
(
64), 189 (36), 107 (84), 105 (78), 95 (42), 93 (49), 91 (57), 81 (49),
GC/MS/MS method
7
9 (48), 77 (39).
GC analysis was performed on a Trace GC Ultra gas
chromatograph coupled with a TSQ Quantum Tandem mass
spectrometer and equipped with a Triplus autosampler
Without any purification step, the crude mixture was
directly dissolved in acetonitrile (25 mL) and treated under
magnetic stirring with Co(OAc) O (13 mg, 0.05 mmol)
ꢂ4H
2
2
(
Thermo Electron Corporation, USA). GC separation was
and tert-butyl hydroperoxide (5.0–6.0 M in decane, 5.3 mL).
The resulting mixture was kept at 88C for 4 h and then warmed
to r.t. and kept under stirring for 24 h. After addition of an
aqueous solution of sodium sulfite and extraction with diethyl
ether, the organic phase was separated and dried, and the
solvent was evaporated to give 2.12 g of a complicated mixture
whose main component (GC/MS) was actually rotundone.
Rotundone was then purified from the reaction mixture by
preparative HPLC on a Shimadzu system incorporating two
LC-8A pumps, a SPD-10A UV-vis detector and a FRC-10A
fraction collector. Isocratic elution with 70% methanol over
performed on a 30 m Solgelwax PEG capillary column with
internal diameter of 0.25 mm and a film thickness of 0.25 mm
(
SGE Analytical Science, Australia). For SPME-GC/MS/MS
experiments, a Supelco (Bellafonte, USA) PDMS/DVB 65 mm
fiber for automated holder was immersed in the sample for
6
2
0 min at 408C and then desorbed into the GC liner set at
408C in splitless/surge mode for 4 min. Temperature
programme: 80 8C hold for 4 min after injection, 88C/min
up to 2208C, hold for 1 min, 158C/min up to 2708C, hold for
5
min. Helium was used as the carrier gas at a constant flow
rate of 1.2 mL/min.
The mass spectrometer was operated in electron impact
8
0 min was performed using a Discovery HS C18 5 m column
(
250 ꢀ 21.2 mm, Supelco) at a flow rate of 7.5 mL/min. An
(
EI) ionisation mode at 70 eV. The filament current was 50 mA.
The temperature of the transfer line was 2508C and argon
99.9998% purity) was used as the collision gas with a
aliquot of 2 mL of the reaction mixture was manually injected
each time. Elution of rotundone, occurring at 67 min, was
monitored at 254 nm. Fractions containing the product were
collected, pooled together and diluted 10 times with distilled
water before solid-phase extraction. Fractions of the diluted
sample (100 mL) were loaded onto Isolute ENVþ cartridges
(
collision cell pressure of 1.5 mTorr. The mass spectrometer
was tuned and calibrated using perfluorotributylamine
(
PFTBA). During the quantification studies the mass
spectrometer was calibrated every 48 h. Data acquisition
and analyses were performed using the Xcalibur Workstation
software supplied by the manufacturer.
In order to determine the retention time and the
characteristic mass fragments of rotundone, full scan analysis
(
IST) containing 1 g of resin previously activated with 20 mL
of methanol and equilibrated with 20 mL of water. After
washing the cartridge with 15 mL of water, rotundone was
eluted with 20 mL pentane and the solvent was finally
removed under reduced pressure. The purification process
allowed the recovery of rotundone with more than 90%
(
m/z 50–300) was performed. The retention times were 18.36
1
13
and 18.32 min for rotundone and d -rotundone, respectively.
5
purity (GC/MS). The MS, H (600 MHz) and C NMR
MS/MS experiments were conducted on the precursor ions
(
150 MHz) spectra of the purified rotundone were identical to
þ
[
1]
m/z 218 (M ) and m/z 203 for rotundone and m/z 223 and m/z
those reported in the literature.
2
08 for d -rotundone. The selection of product ions was
5
Despite the low yield (overall yield ca. 10%), this novel
protocol is however quite convenient, since it starts from a
commercially available compound and requires only two
reaction steps without any need for purification of the
intermediate a-guaiene.
performed using the Energy Ramp function from 5 to 25 eV
to optimise the collision energies for the different transitions
and repeating the analysis at different argon collision gas
pressures (1.2 and 1.5 mTorr). For quantitative analysis, we
selected the transitions m/z 218!163 (10 eV) for rotundone
[
3]
d5-Rotundone was synthesised according to the literature
and m/z 223!166 (10 eV) for d -rotundone which were also
5
by treatment of rotundone with sodium ethoxide in
þ
used as qualifiers. In addition, the transition m/z 218!161
(10 eV) was also used as qualifier for rotundone. For
quantification, the chosen transitions were monitored in
multiple reaction monitoring (MRM) mode and the peak
d-ethanol. MS (M 223) and NMR spectra were identical to
[
3]
those previously reported.
Sample preparation for SPME-GC/MS/MS analysis
After the addition of 200 mL of d -rotundone (100 mg/L)
5
area ratios of rotundone to d -rotundone (internal standard)
were calculated as a function of the concentration of the
compounds.
5
as internal standard, 100 mL of each wine were subjected
Rapid Commun. Mass Spectrom. 2011, 25, 483–488
Copyright ß 2011 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/rcm