Quantification of cysteine S-conjugate of 3-sulfanylhexan-1-ol in must and wine of petite arvine vine by stable isotope dilution analysis

Article abstract of DOI:10.1021/jf072963o

Making use of a convenient synthetic approach to prepare the deuterated S-3-(hexan-1-ol)-cysteine by a Michael addition reaction, an analytical method was developed to measure the presence of the cysteine S-conjugate, precursor of 3-sulfanylhexan-1-ol (3-mercaptohexan-1-ol), in must and wine from Petite Arvine vine. The method uses a stable isotope dilution assay with a suitable one-step sample preparation and HPLC-MS detection. The method has limits of detection and quantification of 3 and 10 μg/L, respectively. A correlation between the increase of the precursor concentration and the increase of the degree of rot has been established.

Full text of DOI:10.1021/jf072963o

J. Agric. Food Chem. 2008, 56, 2883–2887 2883
Quantification of Cysteine S-Conjugate of
3-Sulfanylhexan-1-ol in Must and Wine of Petite
Arvine Vine by Stable Isotope Dilution Analysis
†
§
†,§
¨
JEAN-LUC LUISIER, HERMANN BUETTNER, SEBASTIAN VOLKER,
THIERRY RAUSIS,† AND URBAN FREY*,†
Institute of Life Technologies, University of Applied Sciences Western Switzerland Valais, Route de
Rawyl 64, CH-1950 Sion 2, Switzerland, and Fachhochschule Muenster, Fachbereich
Chemieingenieurwesen, Stegerwaldstrasse 39, D-48565 Steinfurt, Germany
Making use of a convenient synthetic approach to prepare the deuterated S-3-(hexan-1-ol)-cysteine
by a Michael addition reaction, an analytical method was developed to measure the presence of the
cysteine S-conjugate, precursor of 3-sulfanylhexan-1-ol (3-mercaptohexan-1-ol), in must and wine
from Petite Arvine vine. The method uses a stable isotope dilution assay with a suitable one-step
sample preparation and HPLC-MS detection. The method has limits of detection and quantification
of 3 and 10 µg/L, respectively. A correlation between the increase of the precursor concentration
and the increase of the degree of rot has been established.
KEYWORDS: Cysteine S-conjugate; S-3-(hexan-1-ol)-cysteine; 3-sulfanylhexan-1-ol; 3-mercaptohexan-
1-ol; stable isotope dilution assay; deuterated analogue; Petite Arvine vine
zation, either with an internal standard (7) or with the 15N
analogue used as internal standard (3). Starkenmann et al. used
high-performance liquid chromatography-electrospray ioniza-
tion positive mode mass spectrometry (HPLC-ESI+MS) with
a standard curve of cysteine S-conjugate (11).
INTRODUCTION
Over the past few years, several studies have been published
on the subject of identification and quantification of flavors in
wines. Especially volatile thiols, which are highly odorous
compounds, contribute to the aroma of white wines such as
Sauvignon Blanc (1, 2) or rose´ wines such as Cabernet
Sauvignon and Merlot (3). A typical sulfur-containing aroma
compound, 3-mercaptohexan-1-ol (2), provides the characteristic
aroma of Petite Arvine wine (4, 5) as grapefruit and rhubarb
flavors (6). The present work focuses on this part of aroma of
Petite Arvine.
3-Mercaptohexan-1-ol (3-sulfanylhexan-1-ol) (2) and other
volatile thiols are almost absent from must, but are produced
during alcoholic fermentation from cysteine S-conjugate (7) or
its precursor glutathione S conjugates (8). This explains the
amplification of aroma in Petite Arvine wine. The degradation
of the precursor S-3-(hexan-1-ol)-cysteine (1) by yeasts (Figure
1) releases the volatile thiol 3-mercaptohexan-1-ol (2) by means
of a carbon-sulfur bond cleavage catalyzed by ꢀ-lyase (EC
4.4.1.13) enzymes (9, 10).
Another relevant analytical approach uses a stable isotope
dilution method for the determination of low flavor concentra-
tions (12, 13). Moreover, a stable deuterated analogue has been
found to have physicochemical properties that are very similar
to those of the compound of interest and is also well adapted
to the quantitative determination of aroma in wines (14, 15).
Consequently, isotope dilution analysis can be used for the
determination of cysteine S-conjugate in must and wine.
MATERIALS AND METHODS
Reagents. Commercial starting materials were purchased from
Sigma-Aldrich or Acros Organics. Their purity was checked before
use (melting range, refractive index, or 1H NMR). When a known
compound was prepared according to a literature procedure, pertinent
references are given. S-3-(Hexan-1-ol)-cysteine was synthesized
following the procedure developed recently (16), and the purity was
1
To investigate the biosynthesis of 3-mercaptohexan-1-ol (2)
during the fermentation process of Petite Arvine wine, it is
essential to quantify the cysteine S precursor. Classical thiol-
specificanalyticalmethodsarebasedongaschromatography-mass
spectrometry (GC-MS), after selective extraction and derivati-
checked by H NMR.
* Corresponding author (telephone +41 27 606 86 57; fax +41 27
606 86 15; e-mail urban.frey@hevs.ch).
† University of Applied Sciences Western Switzerland Valais.
§ Fachbereich Chemieingenieurwesen.
Figure 1. Pathway of 3-mercaptohexan-1-ol (2) formation during alcoholic
fermentation (9).
10.1021/jf072963o CCC: $40.75  2008 American Chemical Society
Published on Web 04/17/2008

2884 J. Agric. Food Chem., Vol. 56, No. 9, 2008
Luisier et al.
Table 1. Petite Arvine Samples and Cysteine S-Conjugate Concentrations in the Must
cysteine S-conjugate
rottenc (µg/L)
cysteine S-conjugate
healthyc (µg/L)
clone
rottena (g)
rottenb (% Brix)
healthya (g)
healthyb (% Brix)
rotten berries (%)
PA32
PA87
PA40
PA826
PA859
PA836
PA231
PA208
PA26
PA806
PA804
PA35
PA219
PA825
PA104
PA10
119
145
261
468
578
493
628
409
738
476
357
625
1261
671
1149
848
26.0
25.0
24.5
21.5
22.5
20.5
23.0
21.5
23.0
24.0
25.5
22.5
23.0
23.5
25.0
22.5
25.5
23.5
25.0
23.0
4557
3758
3833
4931
4951
3883
4440
2845
5083
3256
2106
2985
5061
2262
3513
2399
4053
2849
3005
2609
16.0
20.0
19.0
19.0
19.5
19.5
18.0
19.5
18.0
20.5
20.0
18.5
16.0
20.0
18.5
19.5
18.5
17.5
20.0
19.5
2.5
3.7
6.4
8.7
40
43
30
88
75
63
80
63
25
35
53
83
80
55
25
46
45
18
45
34
31
58
51
54
55
44
48
65
57
53
48
74
83
43
73
75
67
84
78
85
10.5
11.3
12.4
12.6
12.7
12.8
14.5
17.3
19.9
22.9
24.6
26.1
31.5
38.3
42.5
47.3
PA235
PA23
PA252
PA1003
1868
1765
2222
2341
a Weight of healthy, respectively rotten, berries in the collected samples. b % Brix measured in the must of healthy, respectively rotten, berries. c Concentrations of
cysteine-S-conjugate of 3-mercaptohexan-1-ol obtained in the must of healthy, respectively rotten, berries.
Figure 2. Reaction pathway to prepare the deuterated S-3-(hexan-1-ol)-cysteine (4).
Samples. The samples of Petite Arvine grapes were taken in 2002
at the experimental Valais State vineyard, where clones of Petite Arvine
grapes are grown. Each sample originates from a different clone. For
each clone, the harvest of three vine stocks was collected at maturity
of the grape, and on the same day, the harvest was manually separated
into healthy and rotten berries. The percent Brix (Table 1) was
determined in each part. It should be noted that no distinction was made
as to the type of rot. The samples were stored at -18 °C until they
were squeezed to give the Petite Arvine must. For microvinification
experiments, must of Petite Arvine of 2001 with a percent Brix of 23.8
and a total acidity expressed in tartaric acid of 7.4 g/L was used.
Microvinification. All of the must used for microvinification
received an addition of sulfur dioxide of 50 mg/L of must. A solution
of yeast was prepared by adding Saccharomyces cereVisiae, strain D96
(1.0 g), in must (5 mL) and water (5 mL). After inoculation of the 500
mL must with 3 mL of the yeast solution, alcoholic fermentation took
place in 900 mL bottles at room temperature.
quantification the natural abundance of the M + 2 peak of the unlabeled
precursor, due to the 34S and two 13C isotopes, was taken into account.
Separation was performed through a Nucleosil 100-5 C18 HD column
(5 µm, 100 Å, 250 × 3.0 mm). The gradient profile was started at 1%
CH3CN (A), 1% aqueous solution of 1% formic acid (B), and 98%
water (C) at a flow rate of 0.60 mL/min. A linear gradient was
applied for 40 min, and the final composition was 40% A, 1% B,
and 59% C.
Synthesis of the Deuterated Cysteine S-Conjugate. Deuterated S-3-
(hexan-1-ol)-cysteine was synthesized (Figure 2) using the method
described by Luisier et al. (16).
(()-2-Amino-3-[1-(2-ethoxy-2-oxoethyl)butyl]sulfanyl[3,3-
2H2]propanoic Acid (3). At 25 °C and under argon, the pH of a solution
of [3,3-2H2]-DL-cysteine (0.20 g, 1.63 mmol) in water (3 mL) was
brought up to 7-8 with a 10% aqueous solution of sodium hydroxide;
this was followed by the addition of ethyl (2E)-2-hexenoate (17, 18)
(460 mg, 3.26 mmol). After 50 h under stirring, the mixture was
acidified to pH 5 with a 15% solution of hydrochloric acid and washed
with diethyl ether (2 × 1.5 mL). After evaporation, trituration of the
residue in diethyl ether afforded a white powder 3: yield ) 0.43 g
(99%); 1H NMR (D2O) δ 0.83 (t, 3, J ) 7.3 Hz, CH2CH2CH3) 1.19 (t,
3, J ) 7.2 Hz, OCH2CH3), 1.4 (m, 2, CH2CH2CH3), 1.5 (m, 2,
CH2CH2CH3), 2.6 (m, 2, CH2CO), 3.1 (m, 1, SCH), 3.82 and 3.86 (s,
1, CHNH2), 4.12 (q, 2, J ) 7.2 Hz, OCH2CH3); APCI+ [M + H]+
266.
(()-2-Amino-3-[1-(2-hydroxyethyl)butyl]sulfanyl[3,3-
2H2]propanoic Acid (4). At 50 °C and under argon, sodium trimethoxy-
borohydride (1.9 g, 15 mmol) was added to a solution of the propanoic
acid derivative (3, 0.43 g, 1.8 mmol) in dimethoxyethane (25 mL).
After 4 h under reflux, methanol (2 mL) was added, followed by water
(2 mL). The solution was acidified to pH 3 with concentrated
hydrochloric acid and evaporated. The crude product was purified using
preparative HPLC with a C18 column to afford a white powder 4: yield
) 0.09 g (25%); 1H NMR (D2O) δ 0.78 (t, 3, J ) 7.3 Hz, CH2CH2CH3)
1H NMR Spectra. Nuclear magnetic resonance spectra of hydrogen
nuclei (1H NMR) were recorded at 400 MHz, with a Bruker Avance
400 spectrometer. Chemical shifts δ are noted relative to the signal of
sodium 2,2-dimethyl-2-silapentane-5-sulfonate (δ 0.00 ppm), and
coupling constants (J) are noted in hertz. Coupling patterns are described
by the following abbreviations: s, singlet; d, doublet; t, triplet; q, quartet;
m, multiplet.
HPLC-MS Analysis. HPLC-MS analyses were performed using an
Agilent 1100 MSD system equipped with a G1311A quaternary pump,
a G1322A degasser, a G1314A UV detector, and a G1947-60101 mass
spectrometer with an atmospheric pressure chemical ionization (APCI)
source negative ion detection mode. Nebulizer pressure was 50 psi,
dry gas temperature was 300 °C, dry gas flow was 4.5 L/min, vaporizer
temperature was 375 °C, corona current was 10 µA, and capillary
voltage was 2000 V. Mass chromatograms, in the single ion monitoring
mode (SIM), of the ions m/z 220 ([M - H]-, unlabeled precursor 1)
and m/z 222 ([M - H]-, labeled precursor) were recorded. For

Quantification of Cysteine S-Conjugate
J. Agric. Food Chem., Vol. 56, No. 9, 2008 2885
Figure 3. HPLC-MS(APCI) SIM mass chromatograms of a typical Petite Arvine must isotopic dilution assay with 100 µg/L of added internal standard
4 with SPE sample concentration step.
1.30 (m, 2, CH2CH2CH3), 1.5 (m, 2, CH2CH2CH3), 1.61 (m, 1,
CH2CH2OH), 1.76 (m, 1, CH2CH2OH), 2.8 (m, 1, SCH), 3.6 (m, 2,
CH2OH), 3.78 (s, 1, CHNH2); APCI+ [M + H]+ 224.
that contained 1 at a concentration of 21 µg/L was analyzed (n
) 5) to check the coefficient of variance (CV) of the method.
The CV was determined to be 3.4%. The linearity was also
determined in wine, where integrated peak area ratios were
plotted against concentration of 1 for a concentration range of
27.8-208.4 µg/L and a 4 concentration of 104.8 µg/L. The
resultant curve was linear [area ratio ) (0.00856 × concentra-
tion) + 0.03876; R2 ) 0.998]. To check the response factor
ratio of labeled 4 versus unlabeled 1, cysteine S-precursor, an
aqueous solution of (20.5 mg/L) and its deuterated analogue
(20.3 mg/L) was used for serial dilutions with water in the
concentration range of 0.203-2.03 mg/L. These solutions were
extracted as described under Sample Preparation. The resultant
curve was linear (R2 ) 0.9999) with a response ratio of
1.0012.
Sample Preparation. At room temperature, the deuterated analogue
4 (100 µL of a 20 mg/L aqueous solution) was added to the must or
wine (20 mL). An aliquot (8 mL) of the samples to be analyzed, either
must or wine, was passed through a SPE cartridge (solid phase
extraction) (Supelclean Envi-18 SPE tube, 3 mL, 0.5 g packing),
previously conditioned with methanol, followed by elution with
methanol (2 mL). After evaporation, the concentrated extract (500 µL)
was ready to be injected into the HPLC-MS system.
RESULTS AND DISCUSSION
Isotopic Dilution Method. Figure 3 shows a typical chro-
matogram of a must of Petite Arvine after adsorption on a SPE
cartridge followed by elution. Under the chromatographic
conditions used, the diastereomers elute with the same retention
time. The limit of detection (LOD), expressed as the concentra-
tion at which the signal-to-noise intensity ratio is 3, and the
limit of quantification (LOQ), expressed as the concentration
at which the signal-to-noise intensity ratio is 10, determined in
must are 3 and 10 µg/L, respectively. These results are similar
to that of the published method (3). The major advantage of
the method presented here is the simplified sample preparation
step, which requires only one preconcentration step on a
commercial SPE column and no derivatization prior to analysis.
The recovery of the SPE preconcentration step was determined
in a must sample with a concentration of 4 of 104 µg/L to be
81%.
Integrated peak area ratios were plotted against concentration
of 4, in must samples, for a concentration range of 10.6-209.6
µg/L and a 1 concentration of 20.7 µg/L. The resultant curve
was linear [area ratio ) (0.0471 × concentration) + 0.0566;
R2 ) 0.998]. In addition, integrated peak area ratios were plotted
against concentration of 1, in must samples, for a concentration
range of 20.7-175.5 µg/L and a 4 concentration of 104.8 µg/
L. The resultant curve was linear [area ratio ) (0.00908 ×
concentration) + 0.02417; R2 ) 0.998]. A typical must sample
Concentration of Precursor in Petite Arvine Must. The
measurement of the concentration of the precursor of 3-mercap-
tohexan-1-ol (2) in the 20 samples of grape juice gave results
between 30 and 90 µg/L (Table 1), which were difficult to interpret.
It is known that this concentration changes considerably and in an
unpredictable way during maturation of the grape (19). We did
find a good correlation between the percentage of rot in the harvest
and the concentration in the healthy berries of the grapes. Figure
4 shows the correlation between the precursor concentrations
in the healthy berries of the grape plotted against the percentage
by weight of rotten berries in the sample. With 20 samples and
a correlation coefficient (R) of 0.745, the confidence level for
this relationship is >99.9%. This means that the formation of
the cysteine S-conjugate is strongly enhanced when the grape
begins to rot. The same analysis was also carried out on the
must obtained from the rotten berries of the grape. The result
shows an inverse correlation between the concentration of
precursor and the degree of rot. However, the confidence level
for this correlation is only 91% (N ) 20; R ) 0.319).
A similar observation was made by the Dubourdieu group
(20) when they found a low level of precursors in healthy grapes,
a high level in “pourri plein”, in which the rotten berries are

2886 J. Agric. Food Chem., Vol. 56, No. 9, 2008
Luisier et al.
cleavage of one of the racemic forms by the yeast, as was found
by Wakabayashi for tryptophanase and E. limosum (9). As the
synthesized cysteine S precursor, according to 13C NMR data
(16), was a 45/55% diastereoisomeric mixture arising from the
reaction of L-cysteine with ethyl hexenoate, we can postulate
that one of the diastereoisomers was transformed preferentially
by yeasts, whereas the second one remained almost unchanged
during the fermentation. In this case one should observe in the
wine an enantiomeric distribution of 3-mercaptohexanol in favor
of one of the enantiomers. In fact, Tominaga et al. observed
that, in Semillon and Sauvignon wines made from botrytized
grapes with a higher precursor concentration, the enantiomeric
distribution of 3-mercaptohexanol was 30:70 in favor of the S
form and that, in dry white wines with a low precursor
concentration, a uniform enantiomeric distribution of 50:50 was
obtained (25). Another possible explanation for the fact that
the total amount of precursor is not converted is an inhibition
of the enzymatic activity (9).
Figure 4. Correlation between the concentration of S-3-(hexan-1-
ol)-cysteine (1) in Petite Arvine must of healthy berries of the grape and
percentage by weight of rotten berries. The error bars represent one
standard deviation of replicates (n ) 2).
ACKNOWLEDGMENT
We are thankful for technical support by Michel Pont, Claudia
Barbara Fretz, Fabrice Micaux, and Pascal Jacquemettaz.
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Received for review October 8, 2007. Revised manuscript received
February 29, 2008. Accepted March 5, 2008. We acknowledge financial
support by the HES-SO//Valais.
JF072963O