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18
O of Ethanol in Wine and Spirits for
Authentication Purposes
Matteo Perini and Federica Camin
Abstract: Since 1986 the European Union has established ofcial isotopic analysis methods for detecting the illegal
addition of sugar and water to wine and to enable geographical traceability. In this paper we investigate the possibility of
using analysis of the
18
O/
16
O stable isotope ratio (expressed as
18
O) of ethanol to improve detection of the watering
of wine and to determine the origin of ethanol. Sixty-nine authentic wine samples from all over Italy, 59 spirits from
fruit and cereals, 5 chemically synthesized ethanols, one concentrated and rectied must, one beet and one cane sugar,
one fresh must, and 6 waters with increasing
18
O values were considered. Ethanol was recovered by distillation, using
a Cadiot spinning band column, following the ofcial OIV methods. The residual water was trapped by storing the
distillate for at least 24 h on a molecular sieve. The
18
O/
16
O ratio was measured using a pyrolyser interfaced with an
isotope ratio mass spectrometer. The
-18
O of ethanol is signicantly related to the
18
O of the fermentation water and
can be considered as a reliable internal reference. The values ranged from+24 to +36 in wine (years 2008 to 2012),
+10 to +26 in fruit and cereal distillates, and from 2 to +12 in synthetic ethanol. The method was shown
to be effective in improving detection of the watering of wine and determining the origin of ethanol (from grapes, other
fruit, or synthesis), but not in detecting the addition of cane or beet sugar to wine.
Keywords: dehydrated ethanol, ethanol origin, TC/EA-IRMS, wine watering
Practical Application: The method can be used to improve the detection of illegal watering of wine.
Introduction
In the last 10 y the demand for alcoholic products (wine and spir-
its) has increased by around 8.6%(source Statistical report on world
vitiviniculture 2012. International Organisation of Vine and Wine
2012, http://www.uibm.gov.it/images/stories/Notizie/sintesi_
impatto_contraf_su_sistema-paese.pdf). The rise in prices and
difcult access to raw materials have encouraged adulteration
in the oenological eld (source: Ufcio italiano brevetti e
marchi. Direzione generale lotta alla contraffazione. Report
2012, http://www.uibm.gov.it/images/stories/Notizie/sintesi_
impatto_contraf_su_sistema-paese.pdf). Since 1986, the European
Union and the Organisation Internationale de la Vigna et du Vin
(OIV) have ofcially established isotopic analytical methods for
detecting the illegal addition of sugar (OIV MA-AS-311-05, OIV
MA-AS-312-06) and water (OIV MA-AS2-12) to wine and to
enable geographical traceability.
With regard to the detection of water addition, this is deter-
mined on the basis of analysis of the isotopic ratio
18
O/
16
O (ex-
pressed as
18
O) of wine water and the comparison of this
value with the reference data dened by the ofcial wine data-
bank (EU Regulation 555/2008). The addition of water causes a
decrease in the original
18
O value of wine and is detectable if
the value falls below the limit dened by the EU wine databank.
This decrease is due to the fact that the vegetal water contained
in wine has a
18
O value that is much higher than tap water as
a consequence of the evapotranspiration processes occurring in
MS 20130240 Submitted 2/20/2013, Accepted 3/25/2013. Authors are with
IASMA Fondazione Edmund Mach, Via E. Mach 1, 38010 San Michele allAdige
(TN), Italy. Direct inquiries to author Camin (E-mail: federica.camin@fmach.it).
plants (Rossmann and others 1999). If the starting wine is orig-
inally characterized by a high
18
O, detection of the addition of
a small percentage of water is not easy. A new isotopic method,
based on analysis of the
18
O of ethanol after fermentation and
distillation, was shown to be very effective in improving the de-
tection of added water in fruit juices (Jamin and others 2003;
Monsallier-Bitea and others 2006). This is based on the relation-
ship existing between the
18
O value of fermentation water and
ethanol (r =0.96, Jamin and others 2003).
18
O analysis of ethanol
can be done using 2 different techniques: isotope ratio mass spec-
trometry (IRMS) combined with high temperature conversion
elemental analyser (TC/EA-IRMS) (Calderone and others 2006)
or gas chromatography-high-temperature conversion (GC-TC)
IRMS in combination with the headspace SPME (solid-phase
microextraction) technique (Aguilar-Cisneros and others 2002;
Yamada and others 2007). Using the SPME procedure, the iso-
topic fractionation varies according to the SPME conditions and is
hard to control (Yamada and others 2007). For this reason, direct
dry-ethanol analysis using TC/EA-IRMS in pyrolysis conditions
appears to be more reliable. An international peer test, which took
place in summer-autumn 2004, demonstrated that a critical aspect
in this type of analysis is dehydration of the sample (Calderone and
others 2006). Indeed, due to the interference of water oxygen, the
sample should be 100% ethanol. To achieve this, Jamin and others
(2003) submitted the distillate obtained from fruit juice according
to the procedure described in the AOAC 995.17 method (content
of ethanol higher than 95%v/v), to trap residual water using a
molecular sieve.
With regard to the detection of added sugar, it is worth re-
membering that according to EC Regulations 479/2008 (Annex
IV, sections 1 and 17) and 123/2007 (Annex XI ter, sections 1
and 17), wine cannot contain ethanol from any sources other than
C
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doi: 10.1111/1750-3841.12143 Vol. 78, Nr. 6, 2013
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Journal of Food Science C839
Further reproduction without permission is prohibited
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18
O of ethanol of wine and spirits . . .
grapes. On the basis of ofcial analysis of the D/H ratio using
2
H-SNIF-NMR (
2
H-Site-specic Natural Isotope Fractionation
Nuclear Magnetic) and the
13
C/
12
C ratio (expressed as
13
C)
using IRMS, it is possible to determine whether ethanol derives
from grapes, cane and beet sugar, or chemical synthesis. On the
other hand, the D/H and
13
C/
12
C values of grapes are generally
similar to those other fruits and cereals. It is therefore important
to nd other isotopic parameters making it possible to determine
the origin of ethanol contained in wine.
In this paper, we present and discuss the
18
O results of ethanol
from wine (N = 69), cereal and fruit spirits (N = 59), beet and
cane sugar, and of synthetic origin (N = 5), obtained for the 1st
time after suitable extraction and/or dehydration. The aim was to
study the relationship between the
18
O of ethanol from grapes
and the
18
O of fermentation water and postfermentation water
in order to verify the ability of ethanol
18
O analysis to improve
detection of the watering of wine (as compared to analysis of the

18
O of wine water). Moreover, we investigated the ability of this
analysis to detect whether the ethanol originated from grapes,
fruit, beet or cane sugar, or chemical synthesis and therefore to
determine the illegal addition of exogenous ethanol to wine (as
compared to ofcial OIV analyses of the D/H and
13
C/
12
C ratios
of ethanol).
Materials and Methods
Samples
A total of 133 fermented samples were considered: 69 authentic
wine samples from all over Italy (2 to 4 samples for each of the
20 regions) belonging to the EU wine databank of years 2008
to 2012, and 59 spirits from fruit and cereals obtained under
controlled conditions in the distillery of our institute or other
certied Italian producers. Moreover, 5 chemically synthesized
ethanols with a purity of 99.9% were provided by Italian Customs.
One sample of concentrated and rectied must (65

Brix), one
of crystallized beet sugar, one of crystallized cane sugar, one of
fresh must (15

Brix), 2 of wine, and 6 of water with different

18
O
values (9.8, 4.0, 1.0, +2.7, +4.9, +7.2) were
considered, to study the effect of the
18
O of fermentation and
postfermentation water and of the addition of beet and cane sugar,
on ethanol
18
O value of grape derivates.
Sample preparation
Ethanol was extracted from the samples of wine, spirit, and fer-
mented must and sugar by distillation, using a Cadiot spinning
band column, following the ofcial methods for spirit and wine
(OIV MA-AS-311-05, OIV-MA-BS-23). The residual water was
trapped by storing the distillate for at least 24 h on a molecular
sieve (3.2 mm pellets, UOP type 3A, Sigma-Aldrich, St. Louis,
Mo., U.S.A.) following the method described by Jamin and oth-
ers (2003). By adding growing percentages of water to absolute
ethanol and measuring the
18
O of ethanol before and after dilu-
tion, we found that the procedure guarantees the expected
18
O
value and therefore is effective for samples containing less than
15% of water. The method is therefore suitable for the distillates
produced from the Cadiot spinning ban column, which guarantees
alcoholic degree higher than 95%. For samples containing more
than 15% of water, a higher amount of molecular sieve and/or a
longer period of storage are required.
Water with a
18
O of +7.2 was obtained by evaporating
deionized water at 100

C for 9 h. Water samples with a
18
O
of 4.0, 1.0, +2.7, +4.9 were obtained by mixing
water with a
18
O of +7.2 with tap water (9.8).
The concentrated and rectied must (65

Brix) and beet sugar

were diluted in the 6 water samples with a growing
18
O value
(from 9.8 to +7.2) and then quantitatively fermented to
ethanol using Saccaromices cerevisie yeast (Lallemand Inc., Montreal,
Canada), following the ofcial method for fruit juice (AOAC
995.17) and for must (OIV MA-AS-311-05).
An increasing amount of beet and cane sugar (7 samples with
growing concentration of sugar from 0gr/L to 180gr/L) was added
to a fresh must (15

Brix) and subjected to fermentation.

y = 0.34x + 31.35
R
2
= 1.00
y = 0.64x + 23.63
R
2
= 1.00
15
17
19
21
23
25
27
29
31
33
35
-11 -9 -7 -5 -3 -1 1 3 5 7 9

18
O water ( vs V-SMOW)

1
8
O

e
t
h
a
n
o
l

(

v
s

V
-
S
M
O
W
)
BEET
CRM
Figure 1Correlation between
18
O values of ethanol and of fermentation water for concentrated and rectied must (CRM) and sugar (BEET).
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18
O of ethanol of wine and spirits . . .
An increasing amount of tap water was added to 2 samples of
wine.
Stable isotope analysis
The D/H and
13
C/
12
C isotope ratios of the alcohol from spirit
and wine were determined using the ofcial methods (OIV MA-
AS-311-05, MA-AS-312-06, OIV-MA-BS-23, OIV-MA-BS-22)
using SNIF-NMR (Site-specic Natural Isotope Fractionation-
Nuclear Magnetic Resonance) (FT-NMR AVANCE III 400,
Bruker BioSpin GmbH, Karlsruhe, Germany) and IRMS (SIRA
II-VGISOGAS, FISIONS, Rodano, Milano, Italy) interfaced with
an Elemental Analyser (NA 1500, Carlo Erba, Milano, Italy). The
D/H values were measured site-specically in the methilic and
methilenic sites of ethanol [(D/H)
I
and (D/H)
II
] and were cor-
rected for the content of methanol and higher alcohols (Bauer-
Christoph and others 1997). They are expressed in parts per mil-
lion (ppm). The
13
C/
12
C values are expressed in scale (see
below) against the international V-PDB standard.
The
18
O/
16
O ratio of wine water was analyzed in CO
2
ac-
cording to the water equilibration method described in the OIV
MA-AS2-12 method, using an IRMS (SIRA II VG ISOGAS,
Rodano, Milano, Italy) interfaced with a CO
2
equilibration sys-
tem (Isoprep 18 VG ISOGAS) and the values are expressed in
scale (see below) against the international V-SMOW standard.
A DeltaPlus-XP isotope ratio mass spectrometer coupled
to a Thermal Conversion/Elemental Analyser (TC/EA) (both
Thermo Scientic, Bremen, Germany) was used for
18
O/
16
O
analysis of ethanol samples. The reactor tube of TC/EA was set to
1450

C (with CG temperature at 80

C) and carrier gas pressure
was at 1.45 bar. One L of alcohol sample was injected 3 times
directly into the pyrolysis furnace using an autosampler (AS 2000
Thermo Scientic) with a micro syringe for liquid. In the pyrol-
ysis tube the oxygen atom of ethanol is quantitatively converted
to carbon monoxide, which is analyzed using IRMS. The ratio
between m/z 30 (
12
C
18
O) and 28 (
12
C
16
O) gives the
18
O/
16
O
ratio in the sample.
The values are expressed on the scale against the in-
ternational V-SMOW standard according to the equation: =
R
sample
R
standard
R
standard
1000 where R is the ratio of the heavy to light
stable isotope in the sample (Rsample) and in the international
reference material (Rstandard).
The
18
O data (the mean values of the 3 injections) were cor-
rected against 2 different ethanol standards, by constructing a lin-
ear equation. The
18
O values of the 2 standards were 17.3
and 36.9 and are currently being validated, through an in-
ternational collaborative study supervised by Eurons Scientic
(Thomas and others 2013). A 3rd ethanol sample named Quality
Control ethanol was used to validate the whole analysis (mea-
surement + correction). The nal value was reported in a control
chart and if it was within the tolerance range the whole session
was validated.
The analytical uncertainty (2s) of the measurements was 0.6
for the
18
O in ethanol, 0.3 for the
18
O in wine water, and
0.3 and 0.8 ppm, respectively, for the
13
C and D/H in ethanol.
The standard deviation of intralaboratory reproducibility for the

18
O of ethanol, obtained by distilling, dehydrating and measuring
the same sample 10 times on different days and using different
operators, was 0.4.
Results and Discussion

18
O of wine ethanol and water
We rst studied the relationship between the
18
O values of
ethanol and fermentation water, fermenting a concentrated and
rectied must diluted with 6 water samples with an increasing

18
O (Figure 1). We found a signicant correlation (r
2
= 1.00)
between the
18
O values of ethanol and the
18
O values of water.
The reason for this is that the oxygen atoms of ethanol also de-
rive from the fermentation water, which is incorporated into sugar
(and then into ethanol) when sucrose is converted to monosaccha-
ride (glucose and fructose) through enzyme-catalyzed hydrolysis
(Monsallier-Bitea and others 2006) and is added to the precursor
y = 0.88x - 22.65
R
2
= 0.57
-4
-2
0
2
4
6
8
10
12
23 25 27 29 31 33 35 37 39

18
O ethanol (, vs V-SMOW)

1
8
O

w
a
t
e
r

(

,

v
s

V
-
S
M
O
W
)
Wine data bank
Min (95% c.i.)
Regression Line
Figure 2Correlation between
18
O values of ethanol and of water of authentic Italian wine samples.
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18
O of ethanol of wine and spirits . . .
Table 1Variation of
18
O values of water and ethanol of 2 wine
samples (A and B) with increasing percentage of tap water.

18
O water
18
O ethanol
% water vs vs
Sample addition V-SMOW V-MOW
A 0 7.1 31.0
A 20 3.7 31.2
A 30 1.6 30.9
A 40 0.0 31.1
B 0 2.8 29.8
B 20 0.3 29.8
B 30 1.3 29.7
B 40 2.5 29.8
of ethanol (acetaldehyde) to form the intermediate acetaldehyde
hydrate.
To investigate this relationship in real wine samples, we then
considered 69 authentic wines from all over Italy. The
18
O values
of wine ethanol varied between +24 and 36: they were
higher than the values found in the literature (Aguilar-Cisneros
and others 2002; Calderone and Guillou 2008), probably because
the wines were produced in different areas and years and because
previous analysis had been performed using different analytical
techniques (SPME and TC/EA respectively, without dehydration
or referring to other standards).
By comparing the
18
O values of ethanol with the
18
O of wine
water, we obtained a signicant (P < 0.001) linear relationship
(
18
O water = 0.88
18
O ethanol 22.65; Figure 2). The linear
equation is similar to that found in the literature for fruit juices
(y = 0.82x 22.08) (Jamin and others 2003). This means that
the correlation is reliable and independent of the matrix and the

18
O values of water, therefore generally effective, regardless of
the origin and the production year of wine. This is very important
because it makes it possible to use this relationship for authenticity
control, avoiding analysis of the
18
O of ethanol for all the samples
in the ofcial wine databank (Reg. CE 555/2008).
We can indeed dene a threshold value for the relationship,
calculating 95% of the condence interval of the regression line
from the following equation:
y = 0.88x 22.65 2

s
where y = 00.88x 22.65 is the linear regression model ob-
tained from the 69 data points, 2 is the Student t and s is the
standard deviation of the residues (difference between calculated
and observed value), which in this case is 1.9.
As the addition of water to wine changes only the
18
O of
water and not that of ethanol, as here demonstrated (Table 1), the
watering of wine changes this relationship, which could go outside
the threshold value, even if the water
18
O is not outside the limit
dened by the wine databank.
To demonstrate the possibility of improving detection of the
watering of wine, we simulated adulteration of the 69 wines by
adding a growing %(from 10% to 50%) of tap water (
18
O =
9) and calculated the number of samples identied as watered
on the basis of the
18
O lower value of wine water and that of the
relationship between the 2
18
Os (Figure 3). In all experimental
cases (from 10% to 50%) the
18
O of ethanol improved detection
of the watering of wine. With an addition of 10% water this new
method made it possible to increase the detection of adulterated
samples from 3 to 10 out of 69. The best results were obtained
with addition of 30% water.

18
O analysis of ethanol can therefore be considered as a reliable
internal reference for improving detection of the watering of wine.

18
O of ethanol from different sources
Table 2 shows the D/H,
13
C, and
18
O values of ethanol from
fruit spirits, vodka, rum, whisky, and of synthetic origin. The D/H
and
13
C values are compatible with the botanical origin of the
0
10
20
30
40
50
60
70
80
10% 20% 30% 40% 50%
% of added water
N
u
m
b
e
r

o
f

a
d
u
l
t
e
r
e
d

s
a
m
p
l
e
s
Only 18O of water
18O of water + ethanol
Figure 3Improvement in wine watering detection by comparing
18
O of water with that of ethanol.
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18
O of ethanol of wine and spirits . . .
Table 2D/H,
13
C, and
18
O values of ethanol from fruit spirits, vodka, rum, whisky, and of synthesis.
(D/H)
I
ppm (D/H)
II
ppm R
13
C vs V-PDB
18
O vs V-SMOW
Apricot (10) Mean 98.9 126.8 2.56 26.5 21.7
Std dev 2.2 3.0 0.03 0.6 1.5
Min 95.1 122.8 2.53 27.3 19.0
Max 102.7 130.2 2.60 25.5 23.9
Grain (1) 98.6 126.9 2.58 26.8 23.6
Cherry (5) Mean 100.0 128.3 2.6 27.0 25.5
Std dev 0.8 1.4 0.0 0.8 1.7
Min 99.2 126.3 2.5 27.8 23.8
Max 101.3 129.7 2.6 25.9 28.1
Arbustus (3) Min 101.8 129.5 2.5 25.0 19.4
Max 103.8 132.4 2.6 24.5 23.4
Raspberry (1) 93.2 127.8 2.74 28.3 21.7
Quince (2) Min 102.5 125.3 2.45 25.1 23.6
Max 102.3 127.3 2.49 26.7 10.3
Pear (20) Mean 100.4 125.4 2.5 27.7 21.1
Std dev 0.8 1.1 0.0 0.6 2.0
Min 98.4 123.6 2.4 28.8 15.9
Max 101.9 127.8 2.6 26.3 24.7
Plum (5) Mean 100.7 124.8 2.5 26.7 19.5
Std dev 1.1 1.6 0.1 0.5 3.1
Min 99.5 122.2 2.4 27.2 13.9
Max 102.3 126.3 2.5 26.0 21.2
Sorb (2) Min 108.5 128.2 2.36 17.7 26.4
Max 112.7 130.2 2.31 16.5 28.8
Rum (5) Mean 110.8 124.2 2.2 12.1 22.1
Std dev 1.1 0.6 0.0 1.3 1.6
Min 109.9 123.6 2.2 13.0 19.6
Max 112.4 125.0 2.3 10.3 23.8
Whisky, oat (1) 98.6 124.2 2.52 26.6 26.7
Vodka (4) Mean 94.8 122.0 2.6 26.0 24.9
Std dev 0.5 0.7 0.0 0.2 2.6
Min 94.3 121.4 2.5 26.2 22.5
Max 95.3 123.0 2.6 25.8 28.5
Synthesis (5) Mean 120.1 139.2 2.3 30.0 1.2
Std dev 3.2 2.7 0.1 1.1 6.3
Min 116.5 137.1 2.3 31.1 2.6
Max 125.1 142.9 2.5 28.4 12.4
90
95
100
105
110
115
120
125
130
-5 0 5 10 15 20 25 30 35 40

18
O ethanol (, vs V-SMOW)
(
D
H
)
I

e
t
h
a
n
o
l

(
p
p
m
)
Wine
Spirit
Synthesis
Figure 4Plot of distribution of
18
O and (DH)
I
values of ethanol samples of different origin.
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O of ethanol of wine and spirits . . .
Table 3Variation of D/H,
13
C, and
18
O of ethanol of one wine sample with increasing percentage of beet and cane sugar.
gr/L sugar addition (D/H)
II
ppm (D/H)
II
ppm R
13
C vs V-PDB
18
O vs V-SMOW
Beet sugar 0 99.5 127.2 2.56 26.8 27.4
30 98.8 129.3 2.62 26.8 27.4
60 97.4 128.3 2.63 26.9 27.5
90 96.7 128.5 2.66 27.1 27.5
120 96.5 128.6 2.67 26.9 27.3
150 95.9 129.2 2.69 27.2 27.4
180 95.3 130.3 2.73 27.0 27.3
Cane sugar 0 100.7 129.4 2.57 28.1 29.1
30 101.9 129.3 2.54 25.8 28.2
60 103.1 129.6 2.51 24.1 27.0
90 103.9 131.2 2.52 22.9 28.4
120 104.8 131.2 2.50 21.8 28.3
150 104.7 131.8 2.52 21.1 28.3
180 105.4 131.4 2.49 20.1 28.4
sugar (for example, the sorb and cane sugar in rum have the char-
acteristically higher values of C4 plants; Martin and others 1996)
and with the values in the literature (Bauer-Christoph and oth-
ers 1997; Ishida-Fujii and others 2005). While the ethanol
18
O
values of cereals overlap with those of wine, the ethanol
18
O
values of most fruit spirits (except cherries) are lower. A possible
explanation is the different
18
O of vegetal water characterizing
these products. All the fruits considered (for example, apples, apri-
cots, or cherries) indeed showed
18
O water values lower than for
grapes (Dunbar and Wilson 1983) and therefore a lower
18
O of
ethanol.
Considering the fruit spirits with (D/H)
I
and
13
C values par-
tially overlapping with those of wine, it is evident that the
18
O of
ethanol is able to improve discrimination between wine ethanol
and ethanol from other fruits (Figure 4).
Moreover, the
18
O of ethanol clearly distinguishes natural
ethanol from synthetic ethanol (Figure 4). The low
18
O value
of synthetic ethanol results from the addition of tap water to ethy-
lene (Schmidt and others 2001).
Due to the possibility of distinguishing the botanical origin for
some species, we investigated the ability of ethanol
18
O analysis
to determine the fraudulent addition of sugar to wine. First we
checked the relationship between the
18
O values of ethanol and
fermentation water, fermenting a sample of beet sugar diluted with
the 6 water samples with an increasing
18
O (Figure 1). In this
case we again found a signicant linear relationship, but the slopes
of the 2 correlation lines were different. The same trend was also
observed for fruit juice (Monsallier-Bitea and others 2006) and
was justied on the basis of the much lower amount of sucrose in
must as compared to beet sugar (which is 100% sucrose). Water
is indeed incorporated into sugar (and then into ethanol) when
sucrose is converted to monosaccharide (Monsallier-Bitea and
others 2006).
Then we added an increasing amount of beet and cane sugar to
a must sample in 7 experiments. In this case, there was no trace
of exogenous water because the sugar was dissolved directly in the
grape must. In both cases, the
18
O of ethanol did not change
with the addition of beet or cane sugar (Table 3).
Ethanol
18
O analysis cannot therefore be used to improve de-
tection of the illegal addition of cane and beet sugar to wine.
Conclusions
The
18
O value of ethanol was shown to be related to the

18
O of the fermentation water and can be considered as a reliable
internal reference.

18
O analysis of ethanol is able to improve detection of the
watering of wine and in some cases of the origin of the ethanol
(for example, from grapes, other fruit, or a synthesis), but not to
detect the addition of cane or beet sugar to wine.
Acknowledgments
We kindly thank dott.sa Filippi and dott. Usseglio Tommaset for
having provided the samples of synthetic ethanol and MIPAAF-
ICQRF for having provided the wine samples.
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