Quantitative Analysis and Stability of TPB
J. Agric. Food Chem., Vol. 53, No. 9, 2005 3585
δH (CDCl3): 5.51 (1H, m, H3, 2.49 (1H, s, tCH), 2.01 (2H, m,
H4), 1.70-1.44 (2H, m, H5), 1.89 (3H, m, C2-Me), 1.09 (3H, s, C6-
Me).
δC (CDCl3): 134.8, 125.0, 85.7, 74.8, 74.1, 37.4, 32.1, 24.6, 23.1,
19.7.
A portion of this product (200 mg) in diethyl ether (1 mL) was treated
with n-butyllithium (1.5 mL of 1.6 M) at -78 °C. The solution was
allowed to warm to room temperature and left to stir for 2 h, after
which time [2H4]-acetaldehyde (60 µL) was added and the reaction was
stirred overnight. The mixture was diluted with diethyl ether (1 mL)
and washed with saturated ammonium chloride solution (2 × 3 mL).
The aqueous layer was back-extracted with diethyl ether (2 mL), and
the combined organic extracts were dried and concentrated. The crude
product was chromatographed on silica gel (20% ethyl acetate in
hexanes) to provide recovered alkyne (7) (72 mg) and the desired diol
(8) (114 mg, 69%).
in several key areas, among them the relative polyphenol content
(10). 4 is a highly conjugated molecule and could easily be
protonated, and the resulting cation may then react with
nucleophilic species, such as polyphenols. Adducts between
grape and wine anthocyanins and activated styrene derivatives
have been implicated in the stabilization of red wine color (11-
14). We were thus keen to investigate whether the polyphenol
content of red wines could play a role in the removal of 4 from
the matrix.
In the initial reporting of 4, the levels of this compound were
measured by gas chromatography-mass spectrometry (GC-
MS) using [2H8]-naphthalene as the standard for quantification
(9). This procedure and standard had previously given reliable
results for the quantification of TDN. However, as 4 is much
more potent than TDN, and as submicrogram levels of this
compound would be expected to impact strongly on a wine’s
perceived aroma, it was felt that a stable isotope dilution assay
(SIDA) method specific to the quantification of 4 was required.
This study was undertaken to develop such a method and to
assess its application to wine analysis.
δH (CDCl3): 5.46 (1H, m, H3), 2.76, 2.35 (2H, 2 × s, OH), 1.95
(2H, m, H4), 1.85 (3H, m, C2-Me), 1.60-1.40 (2H, m, H5), 1.03, 1.02
(3H, s, C6-Me). (Both signals were present, but gave a combined
integral of three protons.)
δC (CDCl3): 134.4, 124.2, 87.5, 85.1, 74.3, 57.8, 37.0, 31.6, 24.1,
23.4, 22.7, 22.5, 19.4.
[2H6]-(E)-1-(2,3,6-Trimethylphenyl)buta-1,3-diene [2H6]-(4). To a
solution of (8) (171 mg) in chloroform (12 mL) was added p-
toluenesulfonic acid (500 mg). The reaction was heated to 50-55 °C
for a total of 18 h. The reaction mixture was diluted with diethyl ether
(30 mL) and washed with 10% NaOH solution (2 × 40 mL), water
(2 × 40 mL), and brine (2 × 40 mL). After drying and concentrating,
the residue was chromatographed on silica (hexanes) three times to
afford pure [2H6]-(4) (18 mg, 13%).
MATERIALS AND METHODS
Materials. NMR spectra were recorded as solutions in CDCl3 and
were obtained on a Varian Gemini spectrometer operating at either 300
MHz (1H) or 75.5 MHz (13C). Centrifugation was performed using a
Beckmann L7-55 Ultracentrifuge. All reagents were purchased from
Sigma-Aldrich. All solvents were of the highest commercial grade
available. Diethyl ether and THF were distilled from sodium/benzophe-
none immediately prior to use. All organic solutions were dried over
anhydrous sodium sulfate prior to filtration. Unlabeled 4 was prepared
as indicated in Janusz et al. (9). Details are given below for the labeled
analogue. Buffered model wine solutions were prepared by saturating
a 10% solution of ethanol in water with potassium hydrogen tartrate,
and adjusting the pH to the desired value with 10% aqueous tartaric
acid solution. Mass spectra were recorded as described previously (9).
Methods. [2H3]-2,6,6-Trimethylcyclohex-2-enone (6). n-Butyllithium
(44.0 mL of 1.6 M) was added dropwise to a solution of diisopropyl-
amine (7.24 g) in THF (100 mL) at -30 °C. After being stirred for 30
min, the solution was cooled to -78 °C and 2,6-dimethylcyclohexanone
(5) (9.8 g) was added dropwise. The solution was stirred for 1 h before
[2H3]-methyl iodide (9.4 g) was added, and the mixture was allowed
to warm to room temperature and stirred overnight. Saturated aqueous
ammonium chloride solution (20 mL) was added, and the layers were
separated. The organic layer was dried, the solvent evaporated, and
the residue dissolved in chloroform (50 mL). A solution of bromine
(16 g) in CHCl3 (20 mL) was added dropwise until the red color of
bromine persisted. Residual bromine was removed by washing with
5% thiosulfate, and the organic solution was dried and filtered. Pyridine
(39.2 g) and chloroform (total volume 100 mL) were added, and the
mixture was heated at a gentle reflux for 48 h. After cooling, the solution
was washed with saturated aqueous CuSO4 solution (until no darkening
occurred), water, and brine. After drying and concentrating, the residue
was distilled (90 °C at 31 mm) to give 6 as a clear liquid (4.25 g,
39%).
δH (CDCl3): 6.99 (1H, d J 7.8 Hz, H4′), 6.96 (1H, d J 7.8 Hz, H5′),
6.61 (1H, d J 15.9 Hz, H1), 6.22 (1H, d J 15.9 Hz, H2), 2.28 (3H, s,
C6′-Me), 2.27 (3H, s, C3′-Me), 2.24 (3H, s, C2′-Me). (Both signals
were present, as a consequence of the mechanism of formation of TPB,
but gave a combined integral of three protons.)
MS m/z (%): 178 (40), 177 (51), 176 (11), 163 (71), 162 (100), 161
(40), 160 (45), 159 (51), 157 (7), 148 (31), 147 (51), 146 (44), 145
(73), 144 (89), 143 (47), 142 (15), 132 (16), 131 (22), 130 (20), 118
(13), 117 (15), 116 (9), 93 (7), 79 (8), 65 (5).
Sample Preparation for GC-MS Analysis. 10 mL of wine was
transferred to a 22 mL SPME glass vial containing NaCl (2 g). The
wine was spiked with [2H6]-4 (100 µL of 10 µg/L) (followed by 4 for
standard addition curves), the vial was capped, and the headspace was
extracted and analyzed by GC-MS. For calculating the concentration
of 4 in the wines, replicate standards were prepared at the same time
as the wine samples, by adding the same amount of internal standard
as above to pH 3.2 model wine (10 mL) containing 4 at 0 or 96 ng/L.
These were then used to calculate the relative response factors for 4
and [2H6]-4.
GC-MS Analysis. This was carried out with a Hewlett-Packard
(HP) 6890 gas chromatograph fitted with a Gerstel MPS2 autosampler,
a Gerstel thermal desorption unit (TDU), and Gerstel programmed
temperature vaporization (PTV) inlet (CIS-4). The GC was coupled to
a HP 5973N mass spectrometer. The GC was fitted with a 30 m ×
0.25 mm i.d., 0.25 µm, ZB-Wax fused silica capillary column
(Phenomenex, Sydney, Australia). The carrier gas was helium (BOC
Gases, Ultrahigh Purity), flow rate 1.2 mL/min (constant flow). The
oven was held at an initial temperature of 80 °C for 1 min, then
increased to 170 °C at 4 °C/min, followed by an increase to 250 °C at
50 °C/min, and held at this temperature for 10 min. The Gerstel MPS2
was operated in SPME mode with a 100 µm poly(dimethylsiloxane)
(PDMS, red) fiber fitted (Supelco, USA). The sample was extracted
for 5 min at 30 °C before being desorbed in the injector for 10 min at
220 °C. For quantification of 4, mass spectra were recorded in Selected
Ion Monitoring (SIM) mode. The ions monitored in SIM runs were:
m/z 128, 142, 157, and 172 for 4 and m/z 131, 144, 145, 162, 163,
177, and 178 for [2H6]-4. Selected fragment ions were monitored for
25 ms each (dwell time). The ions at m/z 157 or 172 from the unlabeled
compound, and m/z 178 from the labeled compound, were used for
quantification. The other ions were used as qualifiers.
δH (CDCl3): 6.62 (1H, m, H3), 2.31 (2H, m, H4), 1.80 (2H, t J )
6.1 Hz, H5), 1.75 (3H, m, H9), 1.09 (3H, s, H7,8).
δC (CDCl3): 204.3, 143.2, 133.4, 40.7, 36.3, 23.9, 22.7, 16.1.
[2H7]-2-Hydroxy-4-(1-hydroxy-2,6,6-trimethylcyclohex-2-enyl)-but-
3-yne (8). To a solution of (6) (1.0 g) in THF (20 mL) was added a
solution of ethynylmagnesium chloride (70 mL of 0.5 M) at 0 °C. The
reaction was allowed to warm to room temperature and stirred
overnight. The solution was washed with saturated aqueous ammonium
chloride solution (2 × 50 mL). The aqueous layer was back-extracted
with diethyl ether (50 mL), and the combined organic extracts were
washed with brine (2 × 50 mL) and dried. After concentration, the
residue was distilled (90-100 °C at 19 mm) to give [2H3]-1-hydroxy-
1-ethynyl-2,6,6-trimethylcyclohex-2-ene (7) as a clear pale orange liquid
(0.82 g, 69%).