Journal of Agricultural and Food Chemistry
Article
(v
̃
, cm−1): 2961 (w), 1799 (w), 1735 (w), 1682 (m), 1591 (w), 1464
Czech Republic (4), Austria (4), Hungary (2), the United States (2),
New Zealand (2), Australia (1), Bulgaria (1), and France (1). The
wines were stored at 4 °C in glass vials lined with PTFE septa until
analysis.
(w), 1422 (w), 1378 (w), 1244 (w), 1166 (w), 1014 (w), 564 (w),
1
486 (w). H NMR (300 MHz, CDCl3, δ, ppm): 2.12−1.92 (m, 4H,
H-2, H-3), 1.30 (s, 6H, CH-10, CH-11). 13C NMR (75 MHz, CDCl3,
δ, ppm): 180.1 (Cq, C-4), 144.6 (Cq, C-10), 135.0 (Cq, C-6), 125.6
(Cq, C-9), 38.7 (CH2, C-3), 36.9 (Cq, C-1), 30.0 (CH2, C-2), 28.8 (2
× CH3, C-11, C-12).
Automated HS-SPME/GC-MS Analysis for Quantitation of
TDN and Vitispiranes. For sample preparation, automated head-
space−solid-phase microextraction (HS-SPME) was used. Analysis
was performed by gas chromatography−mass spectrometry (GC-MS)
with selected-ion-monitoring (SIM, method A) and selected-reaction-
monitoring (SRM, method B) modes for wines from “Best of Riesling
2015” and “Mundus Vini 2015”, respectively. The parameters for
method B are given in square brackets. The quantitation of free TDN
and vitispiranes was carried out in 20 mL glass vials with 4.50 mL
[4.00 mL] of model wine solution, to which 2 g of sodium chloride,
500 μL [1000 μL] of the wine, and 50 μL of the internal standard
(ISTD target concentrations: method A, 0.43 μg/L TDN-d6 and 0.98
μg/L vitispiranes-d5; method B, 0.41 μg/L TDN-d6 and 0.72 μg/L
vitispiranes-d5) had been added.
1,1-Dimethyl-6-([2H3]-methyl)-1,2-dihydro-([5,7,8-2H3])-naph-
thalene-4-ol (12). Compound 11 (1.90 g, 9.79 mmol) was dissolved
in methanol (100 mL). After addition of cerium(III) chloride
heptahydrate (1.31 g, 3.52 mmol) and sodium borohydride (0.26 g,
6.85 mmol), the reaction mixture was stirred for 30 min at room
temperature. Water (50 mL) was added, and the solution was
saturated with sodium chloride and extracted with dichloromethane
(50 mL). The aqueous phase was extracted with dichloromethane (3
× 30 mL). The pooled extracts were dried with sodium sulfate and
concentrated. The product, 12 (1.74 g, yield 90%, 94% by GC), was
obtained as a colorless oil. IR (ṽ
, cm−1): 3331 (w), 2934 (w), 1456
(w), 1179 (w), 1062 (m), 1035 (m), 996 (m), 950 (m), 685 (w), 486
(m). 1H NMR (300 MHz, CDCl3, δ, ppm): 4.69 (t, 1H, H-4), 2.11−
1.54 (m, 4H, H-2, H-3), 1.31, 1.23 (2x s, 6H, H-11, H-12). 13C NMR
(75 MHz, CDCl3, δ, ppm): 142.6 (Cq, C-9), 137.5 (Cq, C-10), 135.2
(Cq, C-6), 68.9 (CH, C-4), 34.4, 28.9 (2 × CH2, C-2, C-3), 33.6 (Cq,
C-1), 31.5, 31.4 (2 × CH3, C-11, C12).
For the quantitation of the hydrolytically released compounds, 5
mL of the wine sample was hydrolyzed in a 20 mL glass vial lined with
a PTFE septum for 36 h at 100 °C (the headspace within the vial was
air). After cooling, 50 μL [100 μL] of the sample in 4.95 mL [4.9 mL]
of model wine was used for the measurement. Calibration standards
were prepared in 5 mL of model wine. (Calibration ranges are given
in section Calibration and Analytical Validation of GC-MS Methods
A and B.) After incubation of the samples for 5 min at 40 °C, the
samples were extracted for 20 min [30 min] (SPME fiber, Sigma-
Aldrich; polydimethylsiloxane (PDMS), 1 cm, 100 μm df).
Desorption was performed in a programmable-temperature vaporizing
(PTV) inlet at 240 °C for 3 min in splitless mode and purged after 2
min. This injector was equipped with a 2.0 mm (i.d.) metal liner
(ThermoFisher Scientific, Waltham, MA). The fiber penetration
depth was set to 35 mm. Cleaning and conditioning of the PDMS
fiber was conducted at 250 °C for 3 min under a helium flow of 1.2
mL/min in a split−splitless (SSL) inlet equipped with a 2.0 mm (i.d.)
metal liner (ThermoFisher Scientific) with a split of 1:100 before and
after each sampling. The GC analysis was performed with a Trace
1300 gas chromatograph equipped with a TriPlus RSH autosampler
(both ThermoFisher Scientific) on a VF-WAXms column (Agilent,
Waldbronn, Germany, 30 m × 0.25 mm i.d. × 0.25 μm df); helium
was used as the carrier gas with a constant flow of 1.2 mL/min. To
minimize contamination of the analytical column, a polar-deactivated
precolumn (Restek, Bellefonte, PA, 2.5 m × 0.53 mm i.d.) was
coupled via a “T” connector (ThermoFisher Scientific) to the
analytical column, and a backflush was set to 5 min. The oven-
temperature program started isothermally at 50 °C for 1 min, then
increased to 240 °C at 10 °C/min, and was held isothermally for 10
min. The mass-spectrometric (MS) detection was carried out with a
TSQ Duo triple-quadrupole mass spectrometer (ThermoFisher
Scientific). Temperatures for the MS transfer line and ion source
were set to 250 °C. In SIM mode, the ions m/z 157 (TDN), 163
(TDN-d6), 177 (vitispiranes), and 182 (vitispiranes-d5) were used as
quantifier ions. The qualifier ions were m/z 142, 172 (TDN); 148,
178 (TDN-d6); 149, 192 (vitispiranes); and 154, 197 (vitispiranes-
d5), respectively. In SRM mode, argon (purity ≥99.999%) was used as
the collision gas. The mass resolution was set to 1 amu for Q1 and Q3
(cycle time of 200 ms). Mass transitions (SRMs, m/z) and collision
energies are listed hereafter (quantifier SRMs are underlined): 157.1
→ 142.1 (14 V), 157.1 → 115.1 (38 V), and 172.1 → 157.1 (8 V) for
TDN; 163.2 → 148.2 (10 V), 145.1 → 144.1 (15 V), and 178.2 →
163.2 (10 V) for TDN-d6; 177.1 → 93.1 (15 V), 177.1 → 121.1 (10
V), and 192.1 → 177.2 (10 V) for vitispiranes; and 182.2 → 93.1 (15
V), 182.2 → 121.1 (10 V), and 197.2 → 182.1 (10 V) for vitispiranes-
d5. Xcalibur software (version 3.0.63) was used for instrument control
and data acquisition (ThermoFisher Scientific).
1,1-Dimethyl-6-([2H3]-methyl)-1,2-dihydro-([5,7,8-2H3])-naph-
thalene (1.2). Compound 12 (1.69 g, 8.62 mmol) was added to a
mixture of calcium chloride (2 g) and p-toluenesulfonic acid (TsOH,
0.85 mg, 4.49 mmol) in cyclohexane (250 mL). The reaction mixture
was stirred at 50 °C for 2 h. The reaction mixture was then diluted
with dichloromethane (100 mL) and saturated sodium bicarbonate
solution (100 mL). The organic phase was separated, and the aqueous
phase was extracted with dichloromethane (3 × 50 mL). The
combined organic phases were washed with water (50 mL) and
saturated sodium chloride solution (50 mL) and dried over sodium
sulfate. The solvent was removed with a rotary evaporator. The
residue was purified on silica gel by CC (n-hexane, 100%). After
concentration of the fractions, the product, 1.2 (0.73 g, yield 43%,
91% by GC), was obtained as a yellowish oil. IR (ṽ
, cm−1): 3034 (w),
2958 (m), 2820 (w), 2251 (w), 1590 (w), 1456 (m), 1428 (w), 1381
(w), 1359 (m), 1327 (w), 1268 (w), 1092 (w), 1054 (w), 995 (w),
1
850 (m), 808 (w), 766 (w), 725 (w), 694 (w), 679 (s), 481 (s). H
NMR (300 MHz, CDCl3, δ, ppm): 6.40 (dt, J = 9.6/1.8 Hz, 1H, H-
4), 5.90 (dt, J = 9.6/4.5 Hz, 1H, H-3), 2.29 (s, 3H, H-13), 2.20 (dd, J
= 4.4/1.8 Hz, 2H, H-2), 1.25 (s, 6H, H-11, H-12). 13C NMR (75
MHz, CDCl3, δ, ppm): 141.1 (Cq, C-9), 135.2 (Cq, C-6), 132.7 (Cq,
C-10), 127.6 (CH, C-3), 127.2 (CH, C-4), 39.0 (CH2, C-2), 33.1
(Cq, C-1), 28.5 (2 × CH3, C-11, C-12).
Synthesis of Isomeric Vitispiranes and Vitispiranes-d5. The
synthesis was performed according to the methods previously
described by Anderson et al.,27 Nilsson et al.,28 and Eggers et al.29
The MS and NMR data were identical with published data.27−29
Stability of Deuterated Internal Standards at Wine pH
(TDN-d6 and Vitispiranes-d5). The stability of the labeled standards
was investigated as described by Kotseridis et al.30 Internal standards
were dissolved in a 12% ethanol/water mixture, pH-adjusted to 3, and
stirred at room temperature for 24 h. Volatiles were extracted with
diethyl ether and concentrated. Subsequent GC-MS analysis was done
to monitor possible H−D back-exchange.
Model Wine and Wine Samples. For calibration standards and
dilution of the wine samples, a model wine was prepared by dissolving
4 g of tartaric acid and 120 mL of ethanol in deionized water to a
volume of 1 L. The pH value was adjusted to 3.2 by dropwise addition
of a 5 M sodium hydroxide solution.
Riesling wines of different vintages, regions, and wineries were
supplied from the wine competition “Best of Riesling 2015” (131
samples, vintage 1990−2014, mean age 4.7 years), which included
wines originating from Germany (125), Austria (4), the United States
(1), and New Zealand (1), and the wine competition “Mundus Vini
2015” (119 samples, vintage 2012−2014, mean age 1.2 years), which
included wines originating from Germany (94), Luxembourg (8), the
Statistical Analysis. Analyses were performed in duplicate for
each wine. Statistical analyses on volatile concentrations and
determinations of statistical significance were performed using
OriginPro 9.0.0.G (OriginLab Corporation, Northampton, MA).
D
J. Agric. Food Chem. XXXX, XXX, XXX−XXX