Eur Food Res Technol (2008) 227:449–461
DOI 10.1007/s00217-007-0741-6
ORIGINAL PAPER
Implementation of headspace solid-phase-microextraction–
GC–MS/MS methodology for determination
of 3-alkyl-2-methoxypyrazines in wine
Rolf Godelmann · Susanne Limmert · Thomas Kuballa
Received: 8 February 2007 / Revised: 10 August 2007 / Accepted: 15 August 2007 / Published online: 17 October 2007
© Springer-Verlag 2007
Abstract A solid phase microextraction (HS-SPME)–
GC–Tandem MS methodology was established for the
analysis of 3-alkyl-2 methoxypyrazines in wine. Due to
their low threshold value (2-methoxy-3-isobutylpyrazine,
MIBP, 1 ng/l white wine, 10 ng/l red wine) these pyrazines
contribute to the typical aroma especially of Sauvignon
Blanc and Cabernet Sauvignon wines. The following Wbers
were tested, varying extraction time (30–80 min),
extraction temperature (30–80 °C), ethanol concentration
(1.25–6.25% vol) and ionic strength of salt added: carbo-
wax/divinylbenzene (CW/DVB), polydimethylsiloxane/
divinylbenzene (PDMS/DVB) and, divinylbenzene/carboxene/
polydimethylsiloxane (DVB/CAR/PDMS) and carboxene/
polydimethylsiloxane (CAR/PDMS). Best results were
obtained with CAR/PDMS. 65 wines (41 Sauvignon Blanc,
24 Cabernet Sauvignon) of worldwide origin were analysed
using the established method. The standard wine parame-
ters (density, alcohol, total extract, pH, total acid, reducing
sugar) were analysed according to FTIR method (Wine-
Scan Foss). MIBP was detectable in 14 Sauvignon Blanc
wines, most of them from New Zealand (concentration 10–
19 ng/l). In one manipulated Sauvignon Blanc wine from
South Africa 173 ng/l MIBP was analysed, more than four
to Wve fold the highest concentration ever found in wine.
There is a correlation between the content of MIBP and the
R. Godelmann (&) · T. Kuballa
Chemisches und Veterinäruntersuchungsamt Karlsruhe,
Weißenburger Straße 3, 76187 Karlsruhe, Germany
e-mail: rolf.godelmann@cvuaka.bwl.de
S. Limmert
Institut für Angewandte Biowissenschaften,
Abteilung für Lebensmittelchemie und Toxikologie,
Universität Karlsruhe (TH), Kaiserstraße 12,
76128 Karlsruhe, Germany
aroma proWle analysis (descriptive analysis) with best
results in a mediate concentration range up to 20 ng/l.
Keywords 3-Alkyl-2 methoxypyrazine · 2-Methoxy-3-
isobutylpyrazine · Wine · Sauvignon Blanc · Cabernet
Sauvignon · Headspace solid phase microextraction · Solid
phase dynamic extraction · GC–MS/MS · Aroma proWle
analysis
Introduction
3-Alkyl-2 methoxypyrazines are aroma substances with
very low threshold values. The most important 3-alkyl-2
methoxypyrazines in grapes and wines are 2-methoxy-3-
isobutyl (MIBP), 2-methoxy-3-isopropyl (MIPP), 2-meth-
oxy-3-sec-butyl (MSBP) and 2 methoxy-3-ethylpyrazine
(MEP) [1]. Due to their low olfactory threshold, these com-
pounds have an extremely high aroma potential (relation of
concentration to olfactory threshold [2]).
The pyrazines were detected in many wine varieties but,
due to their relatively high concentration, contribute to the
typical aroma only in the case of Sauvignon Blanc, Sémil-
lon and Cabernet Sauvignon [3, 4]. The olfactory character-
istics of the 3-alkyl-2-methoxypyrazines are described as
vegetative, herbacious, green pepper [3] and green bell pep-
per [1, 5]. The most important 3-alkyl-2 methoxypyrazine,
3-isobutyl-2-methoxypyrazine (MIBP), was Wrst reported
in green bell pepper [6]. The threshold value of MIBP in
water is 2 ng/l, while 1 ng/l is reported in white wine and
10–15 ng/l in red wine [7, 8]. A high concentration of
MIBP is not an essential condition for good wine quality
[3]. The optimum concentration of MIBP is in the very low
range of 8–15 ng/l. Concentrations higher than 30 ng/l are
associated with disharmonic aromas. MIBP concentrations
123
450
of 3.6–56.3 ng/l were detected in Cabernet Sauvignon
wines [9, 10]. In Sauvignon Blanc wines the concentration
of MIBP was found in a range from 5 to 40 ng/l (France),
10 to 35 ng/l (New Zealand) and 2 to 15 ng/l (Australia)
[3, 11].
2-Methoxy-3-sec-butylpyrazine (MSBP) is the second
most important pyrazine found in wine followed by 2-
methoxy-isopropylpyrazine (MIPP) [1]. Sala et al. [12]
described the threshold value of both compounds, similar to
MIBP, as very low 1–2 ng/l in water and 2 ng/l in red wine.
This is the reason why MIPP could also contribute signiW-
cantly to the aroma of red wine [13].
2-Methoxy-3-ethylpyrazine (MEP) is the most rare of all
pyrazines cited. Its occurrence in wine has been conWrmed.
The odour is earthy and potato-like. At 425 ng/l, its odour
threshold value is much higher than all the other pyrazines
[14] (Table 1).
Due to their low threshold value and associated high
aroma potential there is an incentive to manipulate wine by
adding alkylpyrazines. In spring 2004, for example, there
was a fraud in South Africa where Sauvignon Blanc wine
was manipulated with 3-isobutyl-2-methoxypyrazine.
MIBP was Wrst identiWed in Sauvignon Blanc wine by
GC–MS (SIM) [15] and in Cabernet Sauvignon wine [16].
There were many attempts to improve the analytical proce-
dure by GC–MS methods, for example by stable isotope
dilution mass spectrometry [14]. This technique uses an
isotopically labelled internal standard of the same composi-
tion as the analyte under investigation. Further methods
were solid-phase-micro-extraction (SPME)–GC [17],
Headspace (HS)-SPME–GC–nitrogen-phosphorous-detec-
tion (NPD) [1, 18], HS-SPME–GC–MS (SIM) [10] and
HS-SPME–GC–NPD with time of Xight mass spectrometry
(GC £ GC–TOFMS) [19].
In this article GC parameters, as well as the basic param-
eters for the HS-SPME procedure were optimized, such as
Wber composition, extraction time and temperature, salt and
ethanol concentration. Tandem-MS was used to improve
the determination of pyrazines. Several Sauvignon Blanc
and Cabernet Sauvignon wines from all over the world
were analysed using the established method. Sensory tast-
ing with aroma proWle analysis was carried out with wines
containing high levels of MIBP.
Table 1 Orthonasally determined threshold values of 2-methoxy-3-
alkylpyrazines [5, 8, 12, 14]
2-Methoxy-3-alkylpyrazine Threshold value (ng/l)
2-Methoxy-3-isobutylpyrazine 1–2 (10–15 in red wine)
2-Methoxy-3-sec-butylpyrazine 1–2
2-Methoxy-3-isopropylpyrazine 1–2
2-Methoxy-3-ethylpyrazine 425
123
Eur Food Res Technol (2008) 227:449–461
Materials and methods
Reagents and standards
Reference standard compounds of 2-methoxy-3-isobutyl-
pyrazine (purity 99%), 2-methoxy 3-isopropylpyrazine
(purity 97%), 2-methoxy-3-sec-butylpyrazine (purity 99%),
2-methoxy-3-ethylpyrazine (purity 99%) were obtained
from Sigma-Aldrich, Steinheim, Germany.
The isotopically labelled internal standards [2H2]-MIBP
and [2H2]-MEP were synthesized according the procedure
described by Kotseridis [20]. The purity was checked by
full-scan GC–MS.
Stock solutions of each pyrazine of 2,000
g/ml were
prepared in analytical grade absolute ethyl alcohol and
stored at 4 °C in the dark. The unlabelled standard solutions
were diluted as required for calibration standards (MIBP,
MSBP, MIPP 8–230 ng/l, MEP 15–230 ng/l).
Reagents H2SO4–D2 (in D2O) purity 96%, deuterium
oxide (D2O), ethyl alcohol abs. p.a, sodium carbonate p.a,
sodium chloride puriss. p.a, sodium sulfate p.a, were pur-
chased from VMR (Merck), Darmstadt, Germany.
Sampling
Sodium chloride (2 g) was put in a 20 ml headspace vial
(A-Z Analytik-Zubehör GmbH, Langen, Germany). Five
ml of wine (1:2.5 diluted), 20
l ethyl alcohol and 20
l
of internal standard ISTD MEP were added to the vial.
The vials were closed with magnetic aluminium caps
(silicone/PTFE septum) (WICOM Heppenheim, Ger-
many) and put in the automatic sampler. For matrix cali-
bration, a pyrazine-free wine of 12.5% vol ethyl alcohol
(dilution 1:2.5) was used. All samples were analyzed in
triplicate.
HS-SPME procedure
After sampling, HS-SPME was performed automatically in
a CTC Combi-PAL autosampler. The parameters for
SPME-procedure are:
Pre-incubation time 10 min, incubation temperature
40 °C, velocity of agitator 500 rpm, agitator-on time 5 s,
agitator-oV time 2 s, extraction time 40 min, desorption
time 10 min. Before use, each Wber was conditioned by
inserting it into the GC injector according to the instruc-
tions of the supplier (Table 2).
HS-SPDE procedure
For Headspace solid-phase-dynamic-extraction (HS-SPDE)
a gas-tight syringe with PMDS/AC (polydimethylsiloxane/
activated carbon) was used (Chromtech, Idstein, Germany).
Eur Food Res Technol (2008) 227:449–461 451

Table 2 SPME Wbers (Supelco Bellefonte, Pennsylvania, USA) Table 4 Target mass fragments of 2-methoxy-3-alkylpyrazines
Fiber Film Polarity Type 2-Methoxy-3-alkylpyrazine Mass- Q2 voltage
thickness fragmentation (V)
(
m) (m/z)

Carbowax/divinylbenzene 70 Polar Adsorption 2-Methoxy-3-ethylpyrazine 138 ! 107 ¡14

(CW/DVB) 2-Methoxy-3-isopropylpyrazine 152 ! 137 ¡8
Polydimethylsiloxane/ 65 Bipolar Adsorption 2-Methoxy-3-sec-butylpyrazine 138 ! 107 ¡15
divinylbenzene (PDMS/DVB)
2-Methoxy-3-isobutylpyrazine 124 ! 93 ¡12
Carboxen/polydimethylsiloxane 85 Bipolar Adsorption
ISTD [2H2]-MEP 140 ! 109 ¡14
(CAR/PDMS)
Divinylbenzene/carboxen/PDMS 50/30 Bipolar Adsorption
(DVB/CAR/PDMS)
range of 8–230 ng/l (MIBP, MIPP, MSBP) and 15–230 ng/
l (MEP). A Wve level matrix calibration was carried out in a
GC–MS/MS
concentration range of 7–70 ng/l [21–23] for the calculation
of the detection and quantiWcation limits.
The samples were analyzed with a GC–MS/MS-system
consisting of an Agilent GC modell 6890N and a tandem
Wine parameters
MS system, the triple quadrupole Bear Instruments (now
Varian) Kodiak 1200 equipped with Kodiak software
The following wine ingredients were measured by FTIR
2.1.046.
(Fourier Transform Infrared spectroscopy), WineScan FT
The following GC column was used: VF-1701 ms (Var-
120 Fa. FOSS Hamburg, Germany: density, alcohol
ian, Darmstadt, Germany) (29.5 m, 0.25 mm inner diame-
content, total extract, pH-value, total acid, reducing sugar
ter, 0.25
m Wlm thickness). The injection was done with a
(see Table 7).
split/splitless-injector, 1.5 min splitless with an injector
temperature of 240 °C. The helium carrier Xow was 1.2 ml/
Sensory analysis
min. Oven temperature was 35 °C 1 min, 20 °C/min to
240 °C, 20 min.
The wines were analysed by a sensory panel of six members
The temperature of the transfer line was 250 °C, the tem-
experienced in wine tasting, most of them oYcial members
perature of the ion source 200 °C. Detection of the 3-alkyl-
of the state panel for wine quality certiWcation (oYcial certiW-
2-methoxypyrazines was carried out by the following qual-
cation of wine before marketing). For ranking the aroma pro-
iWer mass fragments (Table 3).
Wle analysis (1–6), reference wine standards were used.
QuantiWcation was carried out by external calibration
The wines were stored at 4 °C in the dark before tasting.
curves with standard solutions in pyrazine-free wine (MIBP,
They were left to stand at room temperature one hour
MSBP, MIPP 8–230 ng/l, MEP 15–230 ng/l, internal stan-
before tasting. The wines were tasted individually and
dard [2H2]-MEP) (Table 3). For quantiWcation, the following
given a ranking of 1–6 for each of six descriptors.
target mass fragments were measured (Table 4, Fig. 1).
For aroma proWle analysis the six most common descrip-
tors of Sauvignon Blanc and Cabernet Sauvignon were
Validation
used: wine fruit, tropical fruit, gooseberry, green bell
pepper, vegetative/grassy/green, spicy/herbaceous for Sau-
A pyrazine-free wine of 12.5% vol ethyl alcohol (dilution
vignon Blanc and berry fruit, blackcurrent, violet/Xowery,
1:2.5) was used (Weißer Burgunder, Baden 2004 Quali-
green bell pepper, spicy/herbaceous, cedar wood/woody for
tätswein trocken 12.5% vol) for matrix calibration. The
Cabernet Sauvignon.
concentration of 2-methoxy-3-alkylpyrazines is in the
Table 3 QualiWer mass fragments of 2-methoxy-3-alkylpyrazines
2-Methoxy-3-alkylpyrazine Mass- Q2 voltage Results and discussion
fragmentation (V)
(m/z) Fiber selection
2-Methoxy-3-ethylpyrazine 138 ! 95 ¡17
The Wbers were tested in a model wine solution consisting
2-Methoxy-3-isopropylpyrazine 152 ! 109 ¡16
of 5 ml distilled water with an ethyl alcohol concentration
2-Methoxy-3-sec-butylpyrazine 151 ! 95 ¡11
of 5% vol. The concentration of pyrazines was 40 ng/vial.
2-Methoxy-3-isobutylpyrazine 151 ! 95 ¡12
To salt out the wine 2 g Na2SO4 was added to each vial.

123
452
Fig. 1 GC–MS/MS chromato-
grams of 2-methoxy-3-alkylpyr-
azines
Ethyl alcohol increases the solubility of the pyrazines in
the aqueous phase, resulting in lower concentration of the
analytes in the headspace. The volatile ethyl alcohol also
competes with the analytes for adsorption on the Wbre. A
higher concentration of ethyl alcohol could damage the
Wber, therefore the concentration was limited to 5% vol, in
line with the dilution step introduced when real wines are
analysed. The following Wbers were tested with varying
extraction time and temperature: carbowax/divinylbenzene
(CW/DVB), polydimethylsiloxane/divinylbenzene (PDMS/
DVB), and divinylbenzene/carboxene/polydimethylsilox-
ane (DVB/CAR/PDMS) (Figs. 2, 3, 4).
Optimum yields of pyrazines were obtained with the ter-
nary Wber DVB/CAR/PDMS with an extraction tempera-
ture of 40 °C and extraction time of 40 min. Extraction
times of more than 40 min (40–240 min) did not increase
the yield signiWcantly. The best recoveries were also found
with the DVB/CAR/PDMS Wber in the optimization proce-
dure reported by Zoecklein [1].
However, yet another Wber, CAR/PDMS (carboxene/poly-
dimethylsiloxane) increased the extraction yield of pyrazines
signiWcantly in comparison with the DVB/CAR/PDMS Wber.
This Wber was exclusively used in further experiments.
123
Eur Food Res Technol (2008) 227:449–461
Ethyl alcohol concentration
Ethyl alcohol concentration in wine is in the range 9–15%
vol. In the experiments with model wine the ethyl alcohol
content was limited to 5% vol by diluting the (12.5% vol)
wine by 1:2.5. Experience has shown that higher ethyl alco-
hol content decreases the yield. In a further experiment the
inXuence of ethyl alcohol content (1.25–6.25% vol) was
studied on the extractability of both 2-methoxy-3-isobutyl-
pyrazine (35–45 ng/l wine) and internal standard [2H2]-
MIBP in the model wine.
Added contents of the pyrazines correspond to 35–45 ng/l
concentration in the original wine. Wine was diluted 1:2,
1:2.5, 1:4, 1:5 and 1:10 resulting in ethyl alcohol concentra-
tions of 1.25, 2.5, 3.1, 5 and 6.25% vol, respectively. The
pyrazine peak area decreases much less than the equivalent
decrease in concentration of the 2-methoxypyrazine MIBP
as the sample is diluted (Fig. 5a). This implies an increase
of 2-methoxypyrazine recovery with lower ethyl alcohol
concentration, in line with the results obtained from the
Wxed concentration deuterated internal standard. Figure 5b
shows the yield of the internal standard. In contrast to the
analytes, the concentration of the internal standard was the
Eur Food Res Technol (2008) 227:449–461 453

MEP MSBP MEP MSBP

ISTD ISTD
MIPP MIBP
A 0,7 MIPP MIBP

A 1,2
0,6
1,1
peak area [ * 1,0e5]

0,5 1,0

p e a k a r e a [ * 1 ,0 e 6 ]
0,9
0,4
0,8
0,7
0,3
0,6
0,2 0,5
0,4
0,1
30 40 50 60 70 80 0,3
extraction time [min] 0,2
30 40 50 60 70 80
B 0,6 extraction time [min]
MEP
MIPP
0,5 MIBP B 1,4
MSBP
peak area [ * 1,0e5]

ISTD 1,2 MEP

0,4 MIPP
peak area [ * 1,0e6] 1,0 MSBP
MIBP
0,3
0,8 ISTD

0,2 0,6

0,1
30 40 50 60
extraction temperature [°C]
Fig. 2 InXuence of extraction time A (n = 4, extraction temperature
40 °C) and extraction temperature B (n = 4, extraction time 80 min) on
adsorption/yield of pyrazines with the CW/DVB Wbre
same in every vial. The yield of the internal standard [2H2]-
MIBP increased if the ethyl alcohol concentration is low-
ered from 6.25 to 3.1% vol (Fig. 5b). Further reduction in
the ethyl alcohol concentration did not increase the yield of
the internal standard [2H2]-MIBP.
Lowering the ethanol concentration in real wine samples
by dilution also decreases the concentration of 2-methoxy-
pyrazines. An optimum concentration of 5% vol of ethanol
was therefore chosen for the experiment. In SPME of 2-
methoxypyrazines in model solutions an increase in ethanol
concentration from 0 to 20% vol dramatically decreased
analyte recovery [1].
Ionic strength
Lower solubilities and therefore higher headspace concen-
trations of polar organic compounds are associated with
higher ionic strength aqueous media (salting out eVect).
0,4
0,2
70 80
0,0
30 40 50 60 70 80
extraction temperature [°C]
Fig. 3 InXuence of extraction time A (n = 4, extraction temperature
40 °C) and extraction temperature B (n = 4, extraction time 50 min) on
adsorption/yield of pyrazines with the PDMS/DVB Wbre
To check the inXuence of ionic strength the salts NaCl,
Na2CO3 and Na2SO4 were tested (each 2 g per sample). The
highest eVect on the yield of pyrazines was achieved with
NaCl, the lowest with Na2CO3 (Fig. 6). There was no sig-
niWcant diVerence between NaCl and Na2SO4 (analysis of
variance, ANOVA, p = 0.00961).
Internal standards
Double deuterated [2H2]-MIBP as internal standard reduced
the sensitivity of MIBP determination. The main mass
fragment m/z 124 occurs in both substances, lowering
sensitivity and selectivity of MIBP determination. Figure 7
shows the detected mass spectrum of [2H2]-MIBP with a
percentage of deuteration of 96% based on the relative peak
intensities. Synthesis of another double deuterated standard
MSBP failed because their is no reactivity at the ternary
carbon atom at the secondary butyl side chain of 2-meth-
oxy-3-sec-butylpyrazine MSBP. According to Kotseridis

123
454 Eur Food Res Technol (2008) 227:449–461

MEP MSBP 1,8

ISTD
MIPP MIBP 1,6

A 1,8 1,4

peak area [ * 1,0e7]

1,6 1,2
peak area [ * 1,0e6]

1,0
1,4
0,8
1,2
0,6
1,0 0,4

0,8 0,2
0,0
0,6
NaCl Na2CO3 Na2SO4
0,4
20 30 40 50 60 70 80
Fig. 6 InXuence of ionic strength on yield of pyrazine (MSBP), n = 3,
extraction time [min] DVB/CAR/PDMS Wber

B 1,8
1,6 Mass fragment m/z 138 of internal standard [2H2]-MEP
MEP
1,4 MIPP did not disturb quantiWcation of MEP. Finally [2H2]-MEP
peak area [ * 1,0e6]

MSBP was used as internal standard for pyrazine determination.

1,2 MIBP
ISTD
1,0 Solid-phase-dynamic-extraction
0,8
0,6 Solid-phase-dynamic-extraction [24, 25] is a more sensitive
0,4
procedure compared to SPME. The analyte is enriched at
the stationary phase of a gas-tight syringe with a capillary
0,2
of stainless steel. This method works according to the same
30 40 50 60 70 80 principle as SPME, with the advantage of larger amounts of
extraction temperature [°C] stationary phase, higher ratio of surface/volume, shorter
extraction time [24].
Fig. 4 InXuence of extraction time A (n = 4, extraction temperature
40 °C) and extraction temperature B (n = 4, extraction time 50 min) on
Experiments with HS-SPDE with the stationary phase of
adsorption/yield of pyrazines with the DVB/CAR/PDMS Wbre PMDS/AC (polydimethylsiloxane/activated carbon) com-
parable to SPME Wber CAR/PDMS were performed. Vary-
ing the parameters extraction temperature, ethyl alcohol
[20] the alkylchain of MIBP is deuterated at CH2. There- concentration, number of strokes and pre-desorption time
fore only the secondary sidechain of MEP could be deuter- did not increase the yield of pyrazines.
ated, the ternary CH of MIPP does not react.
Synthesis of [2H2]-MEP starting from MEP was success- Validation
ful [20]. Full-scan GC-MS showed percentage of deuteria-
tion of 92% based on the relative peak intensities. In Fig. 8 The validation of the method includes the detection and
the detected mass spectrum of [2H2]-MEP is shown. quantiWcation limits as the most important parameters

Fig. 5 InXuence of ethyl A 5 B 2,0

alcohol concentration on yield of 1,8
MIBP (a) and internal standard 4 1,6
peak area [ * 1,0e6]

peak area [ * 1,0e7]

[2H2]-MIBP (b), n = 3, CAR/ 1,4

PDMS Wber 3 1,2
1,0
2 0,8
0,6
1 0,4
0,2
0 0,0
6,25 % 5% 3,1 % 2,5 % 1,25 % 6,25 % 5% 3,1 % 2,5 % 1,25 %
ethyl alcohol concentration ethyl alcohol concentration

123
Eur Food Res Technol (2008) 227:449–461 455

Fig. 7 Detected mass spectrum

of [2H2]-MIBP

Fig. 8 Detected mass spectrum

of [2H2]-MEP

(Table 5). A Wve level matrix calibration was carried out in Analysis of calibration showing no homogenity of
a concentration range from 7 to 70 ng/l to calculate these variances, the detection and quantiWcation limits were
parameters. calculated by weighted linear regression analysis [21–23].

123
456 Eur Food Res Technol (2008) 227:449–461

Table 5 Detection and quantiWcation limits by HS-SPME–GC–MS/ Table 6 Detection limits

MS
Weighted Signal to
2-Methoxy-3-alkylpyrazine Detection QuantiWcation linear regression noise 3:1
limit (ng/l) limit (ng/l)
Detection limit 8.6 0.4
2-Methoxy-3-isobutylpyrazine 8.6 33 of MIBP (ng/l)
2-Methoxy-3-sec-butylpyrazine 7.9 33
2-Methoxy-3-isopropylpyrazine 16 72
2-Methoxy-3-ethylpyrazine 42 189
Analysis of wine samples

Sixty-Wve wines of world-wide origin, 41 Sauvignon Blanc

In Fig. 9a the diagram of the calibration of MIBP and in
Fig. 9b the uncertainness of results of MIBP are plotted.
The advantage of weighted linear regression analysis is
that less exact values are taken into consideration with
lower valuation. The detection limit for MIBP of the estab-
lished method calculated by weighted linear regression
analysis was higher than those only calculated by signal to
noise ratio of 3:1 [10, 18, 19, 26]. The detection limits cal-
culated from the diVerent methods are listed in Table 6.
and 24 Cabernet Sauvignon, were analyzed with the estab-
lished HS-SPME–GC–MS/MS methodology for the deter-
mination of the 2-methoxy-3-alkylpyrazines. The standard
wine parameters (density, alcohol, total extract, pH, total
acid, reducing sugar) were analysed according to the FTIR
method (WineScan Foss). All parameters including concen-
trations of the 2-methoxy-3-alkylpyrazines are listed in
Table 7.
No MSBP, MIPP and MEP could be detected in all
wines. In 14 Sauvignon Blanc wines MIBP was detectable,
most of them from New Zealand (n = 10, MIBP 10–19 ng/l),

Fig. 9 Diagram of calibration A peak area

(a) and uncertainty of results (b) 14000000 Diagram of calibration
of MIBP
12000000

10000000

8000000

6000000

4000000

2000000
pyrazine concentration [ng/l]
0
0 10 20 30 40 50 60 70 80

uncertainness [%] Uncertainness of results [%] subject to concentration

B 140

120

100

80

60

40

20
pyrazine concentration [ng/l]
0
0 10 20 30 40 50 60 70 80

123
 Table 7 Analytical parameters of examined wines including contents of 3-alkyl-2-methoxypyrazines
No. Grape cultivar Country of Smallest Year Density Alcohol Total pH-Value Total Red. MIBP MSBP MIPP MEP
origin geographical % vol extract acid sugar (ng/l) (ng/l) (ng/l) (ng/l)
unit (g/l) (g/l) (g/l)

1 Sauvignon Blanc USA California 2003 0.9933 12 24.4 3.13 6.8 4.5 <10 <10 <10 <40
2 Sauvignon Blanc South Africa 2004 0.9925 12 20.9 3.46 5.8 3.4 10 <10 <10 <40
3 Sauvignon Blanc USA California 2004 0.9921 12.9 23.2 3.18 6.7 3.5 <10 <10 <10 <40
4 Sauvignon Blanc France Pays d‘Oc 2004 0.9922 12.3 21.3 3.57 5.4 2.3 <10 <10 <10 <40
5 Sauvignon Blanc South Africa 2005 0.9917 13.4 21.7 3.41 6.1 1.7 <10 <10 <10 <40
6 Sauvignon Blanc New Zealand Marlborough 2004 0.9947 12.4 27.8 3.28 7.4 7.7 16.5 <10 <10 <40
7 Chardonnay/Sauvignon Blanc South Eastern Australia 2005 0.9922 13.3 25.1 3.31 6.2 5.3 <10 <10 <10 <40
Eur Food Res Technol (2008) 227:449–461

8 Sauvignon Blanc Chile Central Valley 2005 0.9914 12.4 20.2 3.25 5.5 2.5 <10 <10 <10 <40
9 Semillon/Sauvignon South Eastern Australia 2004 0.9940 10.3 20.9 3.27 5.7 3.7 <10 <10 <10 <40
10 Sauvignon Blanc France Pays d’Oc 2004 0.9927 11.9 21.9 3.12 6.7 2.5 <10 <10 <10 <40
11 Sauvignon Blanc South Africa Coastal Region 2005 0.9922 11.6 19.2 3.47 5.5 1.8 <10 <10 <10 <40
12 Sauvignon Blanc USA California 2003 0.9937 12.1 24.8 3.15 6.5 5.3 <10 <10 <10 <40
13 Sauvignon Blanc USA California 2004 0.9920 13 23.5 3.22 6.5 3.6 <10 <10 <10 <40
14 Sauvignon Blanc Australia South Eastern Australia 2005 0.9941 11.6 25 3.24 6.4 6.3 <10 <10 <10 <40
15 Sauvignon Blanc South Africa Stellenbosch 2003 0.9917 12.5 18.7 3.52 5.6 2.4 173 <10 <10 <40
16 Sauvignon Blanc New Zealand East Coast 2004 0.9959 12.2 30.3 3.47 7.1 9.1 <10 <10 <10 <40
17 Sauvignon Blanc France Pays d’Oc 2002 0.9927 11.8 21.4 3.32 5.3 2.4 <10 <10 <10 <40
18 Sauvignon Blanc France Sancerre 2004 0.9925 12.2 22.7 3.25 6.8 3 <10 <10 <10 <40
19 Sauvignon Blanc South Africa Western Cape 2004 0.9907 12.9 20.1 3.3 6.3 2.6 <10 <10 <10 <40
20 Sauvignon Blanc Chile Central Valley 2002 0.9917 12.6 20.8 3.12 5.8 3.5 <10 <10 <10 <40
21 Sauvignon Blanc New Zealand East Coast 2004 0.9955 12.4 31.4 3.42 7 9.2 16.1 <10 <10 <40
22 Sauvignon Blanc France Bordeaux 2004 0.9920 12.3 21 3.15 6.2 4.1 <10 <10 <10 <40
23 Chardonnay/Sauvignon Blanc South East Australia 2004 0.9917 13.3 23.2 3.38 6.1 4.6 9 <10 <10 <40
24 Sauvignon Blanc New Zealand Marlborough 2004 0.9945 12.5 29 3.3 7.6 7.4 17.1 <10 <10 <40
25 Chardonnay/Sauvignon Blanc South East Australia 2004 0.9918 13.3 23.2 3.37 6.1 4.4 <10 <10 <10 <40
26 Sauvignon Blanc New Zealand Marlborough 2004 0.9944 12.4 27.6 3.27 7.4 7.3 17.5 <10 <10 <40
27 Sauvignon Blanc South Africa Coastal Region 2004 0.9917 12.6 21.5 3.12 6.6 3.7 11 <10 <10 <40
28 Sauvignon Blanc South Africa Western Cape 2005 0.9914 13.3 21.8 3.35 6 3.5 10 <10 <10 <40
29 Sauvignon Blanc New Zealand Marlborough 2004 0.9945 12.4 27.3 3.31 7.3 7.1 19 <10 <10 <40
30 Sauvignon Blanc New Zealand Marlborough 2000 0.9916 12.8 21.1 3.27 6.8 2.4 16 <10 <10 <40
31 Sauvignon Blanc South Africa Western Cape 2006 0.9933 11.4 22.1 3.25 6.4 4.8 <10 <10 <10 <40
32 Sauvignon Blanc USA California 2003 0.9938 11.9 24.7 3.19 6.4 5.1 <10 <10 <10 <40
33 Sauvignon Blanc USA California 2003 0.9938 11.9 24.7 3.19 6.4 5.3 <10 <10 <10 <40

123
457
 458

Table 7 continued
No. Grape cultivar Country Smallest Year Density Alcohol Total pH-Value Total Red. MIBP MSBP MIPP MEP

123
of origin geographical % vol extract acid sugar (ng/l) (ng/l) (ng/l) (ng/l)
unit (g/l) (g/l) (g/l)

34 Sauvignon Blanc South Africa Western Cape 2004 0.9936 12.1 24.3 3.41 5.7 6.8 <10 <10 <10 <40
35 Sauvignon Blanc New Zealand Marlborough 2005 0.9941 12.3 26.1 3.33 7 6.3 10 <10 <10 <40
36 Sauvignon Blanc South Africa Robertson 2005 0.9932 12.4 24.7 3.64 8.2 1.1 <10 <10 <10 <40
37 Sauvignon Blanc Chile Valle Central 2005 0.9923 12 20.6 3.15 6.2 2.6 <10 <10 <10 <40
38 Sauvignon Blanc New Zealand Marlborough 2004 0.9944 12.3 27.2 3.29 7.2 7.3 19 <10 <10 <40
39 Sauvignon Blanc USA California 2005 0.9931 12.3 24.2 3.11 6.7 4.2 <10 <10 <10 <40
40 Sauvignon Blanc New Zealand Marlborough 2005 0.9928 12.7 24.3 3.31 6.9 5.5 11 <10 <10 <40
41 Sauvignon Blanc USA California 2004 0.9933 12.3 24.6 3.15 6.9 4.3 <10 <10 <10 <40
42 Cabernet Sauvignon Chile Valley Central 2005 0.9943 13.2 28.9 3.65 4.9 3.4 <10 <10 <10 <40
43 Cabernet Sauvignon Australia South Eastern Australia 2003 0.9966 13.2 35.1 3.54 5.6 8.6 <10 <10 <10 <40
44 Cabernet Sauvignon USA California 2002 0.9947 13.4 31.4 3.54 5.4 6.1 <10 <10 <10 <40
45 Cabernet Sauvignon Argentina Mendoza 2004 0.9953 13.9 33.9 3.81 4.7 5.7 <10 <10 <10 <40
46 Cabernet Sauvignon USA California 2004 0.9951 13.7 32.7 3.78 4.7 6.3 <10 <10 <10 <40
47 Cabernet Sauvignon Chile Central Valley 2004 0.9948 12.9 27.5 3.63 4.5 4.1 19.5 <10 <10 <40
48 Cabernet Sauvignon Marocco Beni M’Ir 2003 0.9958 13.1 33.3 3.83 5.1 5.9 <10 <10 <10 <40
49 Cabernet Sauvignon South Africa Western Cape 2004 0.9955 13.7 34 3.66 5.9 6.6 13.6 <10 <10 <40
50 Cabernet Sauvignon Australia South Eastern Australia 2004 0.9956 13.2 32 3.45 5.8 5.7 <10 <10 <10 <40
51 Cabernet Sauvignon South Africa Stellenbosch 2001 0.9941 14 31.5 3.97 5.1 3 <10 <10 <10 <40
52 Cabernet Sauvignon USA California 2002 0.9955 13.9 34.9 3.43 5.4 7.3 <10 <10 <10 <40
53 Cabernet Sauvignon Chile Valle Central 2003 0.9934 13.5 27.6 3.66 4.5 3.7 <10 <10 <10 <40
54 Cabernet/Shiraz Australia South Eastern Australia 2004 0.9956 13.4 32.9 3.54 5.6 6 <10 <10 <10 <40
55 Cabernet Sauvignon USA California 2002 0.9956 13.2 33.1 3.65 5.7 4.5 <10 <10 <10 <40
56 Cabernet Sauvignon Argentina Uco Valley 2004 0.9935 13.9 28.7 3.74 4.7 3.4 <10 <10 <10 <40
57 Cabernet Sauvignon Chile Valle Central 2005 0.9947 12.3 28.3 3.69 4.5 3.7 <10 <10 <10 <40
58 Cabernet Sauvignon Spain Penedes 2000 0.9944 12.9 29 3.47 5.3 3.6 <10 <10 <10 <40
59 Cabernet Sauvignon Chile Valle Central 2005 0.9949 12 27.3 3.67 5 2.7 <10 <10 <10 <40
60 Cabernet Sauvignon South Africa Paarl 2004 0.9956 13 32.6 3.88 5.2 3.5 <10 <10 <10 <40
61 Cabernet Sauvignon Chile Central Valley 2004 0.9938 13.9 30.4 2.57 5.1 3.8 <10 <10 <10 <40
62 Cabernet Sauvignon South Africa Paarl 2003 0.9931 14.5 30.3 3.57 5.6 4.8 <10 <10 <10 <40
63 Cabernet Sauvignon Californien Central Coast 1998 0.9949 12.8 29.6 3.6 5.6 4.4 <10 <10 <10 <40
64 Cabernet Sauvignon Mexico Parras 2003 0.9951 13.3 32.1 3.75 4.9 4.3 <10 <10 <10 <40
65 Cabernet Sauvignon Chile Central Valley 2003 0.9948 12.5 26.7 3.68 5.1 3.2 <10 <10 <10 <40
Eur Food Res Technol (2008) 227:449–461
Eur Food Res Technol (2008) 227:449–461 459

South Africa (n = 4, MIBP 10–173 ng/l), Australia (n = 1,
MIBP 9 ng/l)
In the manipulated Sauvignon Blanc wine from South
Africa (wine no. 15) 173 ng/l MIBP was detectable
(Fig. 10), more then four to Wve fold higher than the highest
concentration in literature ever cited [3, 11]. In only two
Cabernet Sauvignon wines MIBP was detectable in concen-
trations of 13.6 ng/l (Chile) (wine no. 49) and 19.5 ng/l
(South Africa) (wine no. 47).
Aroma complexity and content of 2-methoxy-3-alkyl-
pyrazines depends on the climatic conditions under which
the grapes are cultivated. Temperature and light are the
most sensitive parameters inXuencing the content of MIBP.
Typical concentration levels of MIBP determined in Sauvi-
gnon Blanc wines ranged from 5 to 40 ng/l in France, from
10 to 35 ng/l in New Zealand and from approximately 2 to
15 ng/l in Australia [11]. South Africa Sauvignon Blanc
wines of the year 2002 (88 wines) varied from <1 to
11.6 ng/l and in the year 2003 from <1 to 14.1 ng/l (115
wines) with highest levels in cooler regions in both years.
The year 2003 was a cooler year in South Africa than 2002
indicating higher levels of MIBP [3].
In Australian wines the MIBP concentration depends on
the mean January temperature (MJT), varying from Hunter
Valley 3.6 ng/l MIBP with MJT 22.7 °C to 56.3 ng/l in
Mornington Peninsula with 18 °C MJT [27].
2-Methoxy-3-alkylpyrazines are products of the amino
acid metabolism starting with the amino acids glycine, leu-
cine, isoleucine and valine and glyoxylate. The piperazine
intermediate is o-methylated after enolisation and dehydra-
tion [28]. A balance between biological formation and
photodegradation may determine the concentration of 2-
methoxy-3-alkylpyrazines in grapes during the ripening
process [12]. Highest concentration of 2-methoxy-3-alkyl-
pyrazines is located in the grape skin. During ripening there
is a decrease of MIBP in the stems and seeds with a
concominant increase in the skins in Cabernet Sauvignon
grapes [29].
During wine processing there is an easy extractability
of MIBP at the beginning of viniWcation while the Wnal
concentration of MIBP in the wine is relatively
unaVected by oenological techniques. The MIBP content
in wine depends mainly on the composition of the
grapes.

Fig. 10 Chromatogram of 5.8

1.241 e 5
South African Sauvignon Blanc (+)138>95 5.3
with added MIBP Smo: 10

5.3 5.5 5.9 6.6 7.861 e 4

(+)138>107
Smo: 10

ISTD
5.9
2.318 e 5
(+)140>109
Smo: 10 5.5

6.1
2.583 e 5
(+)152>109
Smo: 10 6.4

6.1
3.035 e 5
(+)152>137
Smo: 10 6.4

MIBP 6.9 7.1 7.051 e 5

(+)124>93
Smo: 10

Qualifier 6.8
2.636 e 5
(+)151>95
Smo: 10

6.0 7.0 ret.tm.

123
460 Eur Food Res Technol (2008) 227:449–461

Aroma proWle analysis Aroma profile Sauvignon Blanc, MIBP

Fruity
6
5
From all 2-methoxy-3-alkylpyrazines MIBP is responsible 4
for the typical green pepper/herbaceous Xavour of Sauvi- Spicy 3
2
Tropical fruity Wine 2: 9.8 ng/l
Wine 6:16.5 ng/l
1
gnon Blanc and Cabernet Sauvignon grapes and wines 0
Wine 15: 173 ng/l
Wine 21: 16.9 ng/l
because of the low aroma threshold values of 1, 2 and Wine 23: 9 ng/l
Wine 26: 17.5 ng/l
10 ng/l in water, white and red wine, respectively. Vegetativ/grassy, green Gooseberry

Not only 2-methoxy-3-alkylpyrazines play an important

Bell pepper
role in the complexity of the Sauvignon Blanc aroma. Some
mercapto compounds developed under fermentation are Fig. 12 Aroma proWle of Sauvignon Blanc wines, including wine with
responsible for the tropical Xavours of Sauvignon Blanc, a artiWcially added MIBP
number of other chemical compounds also play a lesser or
greater role. Aroma profile Cabernet Sauvignon, MIBP
A high content of MIBP is not necessarily correlated Berryfruit
5
with high wine quality [3]. The MIBP concentration con- 4
3
tributing to an agreeable wine Xavour is in a small range Cedarwood/woody
2
Black current Wine 42: nd
Wine 43: nd
from 8 to 15 ng/l, concentrations higher than 30 ng/l are 1
Wine 45: nd
0
often described as disagreable [4]. Wine 46: nd
Wine 47:19.5 ng/l
In aroma proWle analysis, the Sauvignon Blanc wines are Spicy Violet/flowery
Wine 49:13.5 ng/l
characterised with the descriptors fruit, tropical fruit,
gooseberry, bell pepper, vegetative/grassy/green, and spicy/ Bell pepper
herbaceous. Sauvignon Blanc wines with MIBP in an inter-
Fig. 13 Aroma proWle of Cabernet Sauvignon wines
mediate concentration gave a harmonic range of all descrip-
tors (Fig. 11). Most exotic tropical fruit Xavour was
observed in the wines from New Zealand (Marlborough)
shown in Table 7 with highest content of MIBP in the low Conclusions
to medium range up to 20 ng/l wine.
The aroma proWle of the South African Sauvignon Blanc Due to many advantages the HS-SPME/GC/MS/MS meth-
wine with artiWcially added MIBP (wine no. 15) was dis- odology is able to analyse 2-methoxy-3-alkylpyrazines in
harmonic with dominating green/herbaceous and bell pep- wine in extremely low concentrations. With the detection
per notes (Fig. 12). and quantiWcation limits of 10 and 30 ng/l, respectively,
The following descriptors were used in the aroma proWle calculated by weighted linear regression analysis, manipu-
analysis of Cabernet Sauvignon: berry fruit, blackcurrent, lated wines with added MIBP can be diVerentiated from
violet/Xowery, green bell pepper, spicy/herbaceous, and wines with MIBP in a normal range.
cedarwood/woody. Only two wines produced concentra-
tions of MIBP in a higher range than the threshold value in Acknowledgment The skilful technical assistance in SPME and tan-
red wine of 10 ng/l. All Cabernet Sauvignon wines had dem mass spectrometry of H. Havel is gratefully acknowledged. The
authors wish to thank M. Fuchs and S. Schubert for preparing the wine
equal ratio of the aroma descriptors, especially with regard
analysis by FTIR. The authors also would like to thank Prof. Egmont
to berryfruit and blackcurrent (Fig. 13). R. Rohwer, University of Pretoria, Republic of South Africa, for
reviewing the text in native English.

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1 Wine 38. 19 ng/l
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