Vin

MASS SPECTROMETRY IN GRAPE AND WINE CHEMISTRY.
PART II: THE CONSUMER PROTECTION

Riccardo Flamini* and Annarita Panighel
CRA, Istituto Sperimentale per la Viticoltura, Viale XXVIII Aprile 26,
I-31015 Conegliano (TV), Italy
Received 25 July 2005; received (revised) 28 November 2005; accepted 28 November 2005
Published online 22 March 2006 in Wiley InterScience (www.interscience.wiley.com) DOI 10.1002/mas.20087
Controls in food industry are fundamental to protect the con-
sumer health. For products of high quality, warranty of origin
and identity is required and analytical control is very important
to prevent frauds. In this article, the state of art of mass
spectrometry in enological chemistry as a consumer safety
contribute is reported. Gas chromatography-mass spectrometry
(GC/MS) and liquid-chromatography-mass spectrometry (LC/
MS) methods have been developed to determine pesticides,
ethyl carbamate, and compounds from the yeast and bacterial
metabolism in wine. The presence of pesticides in wine is
mainly linked to the use of dicarboxyimide fungicides on
vineyard shortly before the harvest to prevent the Botrytis
cinerea attack of grape. Pesticide residues are regulated at
maximum residue limits in grape of low ppm levels, but
signicantly lower levels in wine have to be detected, and mass
spectrometry offers effective and sensitive methods. Moreover,
mass spectrometry represent an advantageous alternative to the
radioactive-source-containing electron capture detector com-
monly used in GC analysis of pesticides. Analysis of ochratoxin
A (OTA) in wine by LC/MS and multiple mass spectrometry
(MS/MS) permits to conrm the toxin presence without the use
of expensive immunoafnity columns, or time and solvent
consuming sample derivatization procedures. Inductively
coupled plasma-mass spectrometry (ICP/MS) is used to control
heavy metals contamination in wine, and to verify the wine
origin and authenticity. Isotopic ratio-mass spectrometry
(IRMS) is applied to reveal wine watering and sugar additions,
and to determine the product origin and traceability. # 2006
Wiley Periodicals, Inc., Mass Spec Rev 25:741774, 2006
Keywords: wine pesticides; wine defects; ethyl carbamate;
ochratoxin A; mass spectrometry; lead in wine; illegal
additions to the wine
I. INTRODUCTION
The rst applications of mass spectrometry in the study of grape
and wine chemistry were performed in the early eighties
by electron impact gas chromatography-mass spectrometry
(GC/MS-EI). Many wine volatile compounds formed with
alcoholic fermentation were identied, so as aroma compounds
from the grape (Rapp & Knipser, 1979; Rapp, Knipser, & Engel,
1980; Williams, Strauss, & Wilson, 1980, 1981; Williams et al.,
1982; Rapp, Mandery, & Ullemeyer, 1983, 1984; Rapp,
Mandery, &Niebergall, 1986; Strauss et al., 1986, 1987; Strauss,
Wilson, & Williams, 1987; Shoseyov et al., 1990; Winterhalter,
Sefton, & Williams, 1990; Humpf, Winterhalter, & Schreier,
1991; Versini et al., 1991; Winterhalter, 1991). It was revealed
that grape varieties with evident oral aroma classied as
aromatic varieties (e.g. Muscats, Malvasie, Riesling, Muller-
Thurgau, Gewurztraminer) are characterized from a high
monoterpenol content, which increases during the latest stages
of ripening (Di Stefano, 1996). A number of monoterpenols has
been identied, the principal are reported in Figure 1. Chemical
transformations involving monoterpenols during fermentation
and wine aging with formation of new monoterpenols in wine,
were also studied (Williams, Strauss, & Wilson, 1980; Di
Stefano, 1989; Di Stefano, Maggiorotto, & Gianotti, 1992).
By GC/MS, was revealed also that norisoprenoid com-
pounds contribute to the forming of grape and wine aroma
(Strauss et al., 1986, 1987; Strauss, Wilson, & Williams, 1987;
Winterhalter, Sefton, &Williams, 1990; Humpf, Winterhalter, &
Schreier, 1991; Winterhalter, 1991). Among them, vitispiranes,
riesling acetal (2,2,6,8-tetramethyl-7,11-dioxatricyclo[6.2.1.0
1,6
]
undec-4-ene), b-damascenone, b-damascone, a- and b-ionol,
a- and b-ionone, 1,1,6-trimethyl-1,2-dihydronaphtalene (TDN),
actinidols, confer particular spice and oral fragrances (struc-
tures reported in Fig. 2).
In the nineties, GC/MS studies of the Sauvignon grapes and
wines revealed sulphur compounds and methoxypyrazines
(grassy note) as the typical aroma compounds of these
varieties (principal structures are reported in Fig. 3) (Harris
et al., 1987; Lacey et al., 1991; Allen, Lacey, & Boyd, 1994,
1995; Tominaga, Darriet, & Dubourdieu, 1996; Bouchilloux,
Darriet, & Dubourdieu, 1998).
Recently, mass spectrometry has been applied to study grape
polyphenols, compounds related to the benets of a moderate
wine consumption. Liquid chromatography-mass spectrometry
(LC/MS) permitted to improve the polyphenols characterization
(anthocyanins, avonols, tannins and proanthocyanidins, hydro-
xycinnamic and hydroxycinnamoyltartaric acids), and to under-
stand several mechanisms involved in the color stability of wine
(Flamini, 2003).
Inthe recent years, viticulture and enology play an important
role for economy of many countries, and considerable efforts are
devoted to improve the product quality and to match the widest
approval of market. Many important industrial processes are nali-
zed to improve organoleptic characteristics of wine: alcoholic
Mass Spectrometry Reviews, 2006, 25, 741 774
# 2006 by Wiley Periodicals, Inc.
*Correspondence to: Riccardo Flamini, CRA, Istituto Sperimentale

per la Viticoltura, Viale XXVIII Aprile 26, I-31015 Conegliano (TV),
Italy. E-mail: riccardo.amini@ispervit.it
fermentation is promoted by inoculumof selected yeast, extraction
of grape components is enhanced by maceration of grape skins
in controlled conditions and by addition of selected enzymes,
malolactic fermentation and barrel- and bottle-aging are performed
to achieve biological stability and to improve avor and fragrance
of product (Flamini, 2003). To guarantee the quality of product, all
these steps have to be monitored and veried.
Community laws, as well as the single Country ones, are
devoted to protect the consumer health, other than the internal
market from introduction of low quality products, by accurate
controls of foods. As a consequence, for exporting of wine and
derivate products, quality certicates are often required, in
particular with regard to the presence of pesticides, heavy metals,
ethyl carbamate and toxins, for which legal limits are often
dened. To prevent frauds and to conrm the product identity,
accordance between the real product characteristics and the
producer declarations (e.g., variety, geographic origin, quality,
vintage) has to be veried. Some maximum limits of grape and
wine contaminants are xed by national and community
regulations, and are reported in Table 1.
Activity of researchers and organisms of control is devoted
todevelop newmethods toverify the product origin (Ogrinc et al.,
2001), to detect illegal additions and adulteration such as sugar-
beet, cane sugar or ethanol addition and watering (Guillou et al.,
2001), to protect consumer health through determination of food
contaminants (Szpunar et al., 1998; MacDonald et al., 1999;
Wong & Halverson, 1999).
On the other hand, to expand the worldwide market,
considerable efforts of the main wine producer countries are
devoted to improve image of product, as a consequence the
product characteristics and origin have to be well dened. The
research in viticulture and enology tries to enhance the typical
characteristics of grape varieties by selection of best clones, and
to identify the more suitable parameters for the product
characterization (Di Stefano, 1996; Flamini, Dalla Vedova, &
Calo`, 2001). For the variety characterization, several parameters
of plant and grape, such as DNA, amphelography, isoenzymes,
chemical compounds of grape (e.g., polyphenols, terpenes and
norisoprenoids, benzenoids, methoxypyrazines), are studied
(Costacurta et al., 2001).
FIGURE 1. The principal monoterpenols that characterize with oral aroma the aromatic grape varieties
such as Muscats, Malvasie, Riesling, Muller-Thurgau, and Gewurztraminer. 1 furan linalool oxide (trans
and cis); 2 linalool; 3 neral (Z) and geranial (E); 4 a-terpineol; 5 trans-ocimenol; 6 pyran linalool oxide
(trans and cis); 7 citronellol; 8 nerol (Z) and geraniol (E); 9 diendiol I; 10 endiol; 11 diendiol II; 12 hydroxy
citronellol; 13 8-hydroxy dihydrolinalool; 14 7-hydroxy nerol (Z) and 7-hydroxy geraniol (E); 15 8-hydroxy
linalool (trans and cis); 16 7-hydroxy-a-terpineol; 17 geranic acid.
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FLAMINI AND PANIGHEL
742 Mass Spectrometry Reviews DOI 10.1002/mas
GC/MS and LC/MS have also been applied to develop
methods for the legal parameters control nalized to the
consumer health protection and to prevent frauds, such as
determination of pesticides in wine, detection of compounds
formed during alcoholic fermentation by yeast and bacteria,
determination of illegal additions to the wine. Also methods for
determination of toxins in the wine have been proposed (Zollner
et al., 2000). Isotopic ratio-mass spectrometry (IRMS) is applied
to determine the product origin and traceability (Guillou et al.,
2001); inductively coupled plasma-mass spectrometry (ICP/MS)
is nowadays a large-used technique to determine heavy metals in
wine.
For these aims the knowledge of the chemical composition
of grape and wine is essential. In the present review, the important
role of mass spectrometry in this frame is discussed, a technique
that in the last years has permitted a rapid increase of enological
chemistry knowledge, also promoted by introduction of new
technologies such as LC/MS and ICP/MS. The state of art of
mass spectrometry in enological chemistry as a consumer safety
contribute is presented.
II. MASS SPECTROMETRY AS THE PRINCIPAL
TOOL FOR THE WINE PESTICIDES AND OTHER
CONTAMINANTS DETECTION
There is a large interest regarding health and safety issues
associated with the fungicides, insecticides and herbicide use,
FIGURE 2. Norisoprenoid compounds that characterize the grape and wine aroma with spice and oral
fragrances. 18 vitispiranes; 19 riesling acetal; 20 b-damascenone; 21 b-damascone; 22 a-ionol; 23 b-ionol;
24 a-ionone; 25 b-ionone; 26 TDN (1,1,6-trimethyl-1,2-dihydronaphtalene); 27 actinidols.
FIGURE 3. Sulphur compounds and methoxypyrazines identied as the typical aroma compounds
of Sauvignon grapes and wines. 28 3-isobutyl-2-methoxypyrazine; 29 3-sec-butyl-2-methoxypyrazine;
30 3-isopropyl-2-methoxypyrazine; 31 3-ethyl-2-methoxypyrazine; 32 3-mercaptohexyl acetate; 33
3-mercaptohexan-1-ol; 34 4-mercapto-4-methylpentan-2-one; 35 4-mercapto-4-methylpentan-2-ol; 36
3-mercapto-3-methylbutan-1-ol.
GRAPE AND WINE CHEMISTRY
&
Mass Spectrometry Reviews DOI 10.1002/mas 743
and the possible presence of their residues in processed foods and
drinks. The high concern about health risks connected with
pesticides, led to the development of several European Commu-
nity (EC) Directives (also adopted by the Italian Legislation)
stating maximum residue limits (MRLs) tolerated for each food
commodity (Ofcial Gazette of the Italian Republic, 1994,
1995). Wine and grape are included among these commodities,
in particular in wine the procymidone MRL has been dened
to 0.5 mg/L, such as for cyprodinil and udioxomil, 0.1 mg/L
for myclobutanil, and 2 mg/L for iprodione; MRLs in grape have
been xed to 10 mg/kg for folpet, 5 mg/kg for vinclozolin, and
3 mg/kg for carbaryl (Ofcial Gazette of the Italian Republic,
2004).
Fungicides, insecticides, and herbicide are commonly used
in viticulture. Structures of principal pesticides are reported in
Figure 4, carbaryl is reported in Figure 12. Dicarboxyimide
fungicides have been widely used against Botrytis cinerea in
vineyards. Vineyards are treated in the nal stage of vegetation to
prevent grapes attack, which may occurs shortly before the
harvest. Among them, vinclozolin and iprodione are currently
employed in Italy (Cabras et al., 1983; Matisova et al., 1996).
These fungicides show reduced toxicity, but 3,5-dichloroaniline,
the probable common nal product of their degradation or
metabolic pathway, seems to be as hazardous as other aromatic
amines.
Although maximum residue limits for most pesticides in
wine have not been xed, several countries have established
guidelines in the authorized use of pesticides and MRLs for the
treatment of vines and grapes used in wine production. Pesticide
residues are regulated at MRLs in grapes at low ppm levels.
Because the vinication process lowers the level of pesticides,
their contents in wines are signicantly lower than in grapes. As a
consequence, methods to detect pesticide residues must be very
effective and sensitive (Wong & Halverson, 1999).
One of the rst MS methods for determination of pesticides
in wine reported in literature was GC/MS-EI by performing
analysis with a diphenil-dimethyl polysiloxane capillary column
and the use of aldrin as internal standard (IS) (Wynn et al., 1993).
Analysis of procymidone was performed by liquid extraction of
sample with hexane, and SIM mode analysis recording signals at
m/z 96 and 283 for procymidone, and m/z 263 and 265 for the IS.
By using both electronic capture detector (ECD) and MS
detector, the same limit of quantication (LOQ) of 2 mg/L was
reported.
In 1996, analysis of vinclozolin and iprodione in wine by
solid phase extraction (SPE) sample preparation with a porous
TABLE 1. Maximum limits of some grape and wine contaminants xed by regulations of single countries, European Union
(EU), United Nations (UN)
contaminant grape wine grape juice Country source
(mg/kg) (ppm) (ppm)
Carbaryl 3 Italy DM 14.12.2004
5 UN Codex Alimentarius
Diuron 0,05 Italy DM 14.12.2004
Fenoxycarb 0,2 Italy DM 14.12.2004
Folpet 10 Italy DM 14.12.2004
2 UN Codex Alimentarius
Iprodione 10 2* UN / Italy Codex Alimentarius / DM 14.12.2004
Myclobutanil 1 0,1* UN / Italy Codex Alimentarius / DM 14.12.2004
Penconazole 0,2 UN / Italy Codex Alimentarius / DM 14.12.2004
Pirimicarb 0,2 Italy DM 14.12.2004
Procymidone 5 0,5* Italy Codex Alimentarius / DM 14.12.2004
Propiconazole 0,5 Italy DM 14.12.2004
Triadimefon 2 Italy DM 14.12.2004
Vinclozolin 5 UN / Italy Codex Alimentarius / DM 14.12.2004
Ochratoxin A 0,002 0,002 0,002 EU CE Regulation n 123/2005
Pb 0,2 0,2 0,05 EU CE Regulation n 466/2001
Histamine 2 Germany recommended (Souza et al., 2005)
5 Belgium recommended (Souza et al., 2005)
8 France recommended (Souza et al., 2005)
10 Switzerland recommended (Souza et al., 2005)
*Only for Italy.
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carbon stationary phase was performed (Matisova et al., 1996).
Analytes were recovered with toluene and analyses performed by
GC/MS. Thermal stability, chemical resistance, and stability over
a wide pHrange of carbon sorbents were evaluated. Recoveries of
two fungicides in both standard solutions and spiked wine
samples ranging between 80% and 97% were reported. MS
analyses were carried out by ion trap detector (ITD) in both
multiple ion detection (MID) and SCAN mode. By selecting the
characteristic ions of each compounds, LOQs of 50 ng/L for
vinclozolin (by recording signals at m/z 178, 180, 198, 200, 212,
215, 285, 287) and of 50 mg/L for iprodione (by recording signals
at m/z 187, 189, 244, 247, 314, 316), were recorded with a signal
to noise ratio of 3 (S/N3). By performing analysis of a
vinclozolin 0.01 mg/L standard solution in MID mode,
unambiguous compound identication was obtained, by ITD
sensitivity of method for iprodione resulted signicantly lower
than for vinclozolin.
In the same year, a GC/MS method for analysis of fungicide
metalaxyl in wine by SPE sample preparation using a carbon
sorbent, was performed (Kakal kova, Matisova, & Lesko, 1996).
Recoveries greater than 92% were reported for metalaxyl
standard solutions at concentration 3100 mg/mL, whereas
recoveries in spiked wines ranged from 80% to 99% depending
on the concentration and the sample matrix. LOQ by GC/MS-IT
was 0.50 mg/L. Metalaxyl residue concentration in wine closely
related to the interval between the last treatment of the vines and
the harvest of the grapes was observed.
Cabras et al. performed two different GC/MS methods by
microextraction with acetone/hexane to determine the fungicides
cyprodinil, udioxonil, pyrimethanil, tebuconazole, azoxystro-
bin, uazinam, kresoxim-methyl, mepanipyrim, and tetracona-
zole in grapes, must, and wine. The methods limits of detection
(LODs) resulted 0.05 mg/L for cyprodinil, pyrimethanil and
kresoxim-methyl, and of 0.10 mg/Lfor the other analytes (Cabras
et al., 1997a, 1998).
To perform the routine monitoring of pesticides, in 1997
Kaufmann developed a fully automated reverse-phase SPE and
GC/MS method for the simultaneous determination of 21
different pesticides in wine (Kaufmann, 1997). By performing
SIM mode analysis and monitoring the m/z species reported in
Table 2, the method showed LODs between 5 and 10 mg/L, and
linearity regression coefcients greater than 0.99 (except for 4,4-
dichloro-benzphenone and dicofol). Recoveries of 17 pesticides
in spiked wine samples ranged from 80% to 115%.
In 1998, Vitali et al. proposed a solid-phase microextraction
(SPME) and GC/MS SIM mode method to determine seven
different insecticides (lindane, parathion, carbaryl, malathion,
endosulfan, methoxychlor, and methidathion), 4 fungicides
(procymidone, vinclozoline, folpet, and captan) and 3 herbicides
(terbuthylazine, triuralin and phosalone) in wine (Vitali et al.,
1998). Authors highlight advantages of SPME as solvent-free
and easy and fast method that requires a very small sample
volume. SPME coupled with GC/MS has been also successfully
applied in several studies on kinetics of fermentation and aroma
proling of wines (Favretto et al., 1998; Vas et al., 1998; Francioli
et al., 1999; Rocha et al., 2001; Vianna & Ebeler, 2001;
Mallouchos et al., 2002; Bonino et al., 2003; Alves, Nascimento,
&Nogueira, 2005; Flamini et al., 2005). SPMEwas performed by
FIGURE 4. The principal pesticides used in viticulture: procymidone 37; cyprodinil 38; udioxonil 39;
myclobutanil 40; iprodione 41; folpet 42; vinclozolin 43.
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a silica ber coated with polydimethylsiloxane (PDMS), per-
forming extraction under stirring for 30 min and immerging the
ber in the wine sample saturated with MgSO
4
. Compounds were
thermically desorbed in the GC injection chamber at 2508C. For
the 14 pesticides investigated, LODs ranging between 0.1 and
6 mg/L were recorded. Pesticide residues contamination was
revealed at detectable levels in 12 of the 21 wines analyzed: 7
pesticides were found at 0.85 mg/L levels, procymidone was
found in 83% of the positive samples.
In 1999, Wong and Halverson performed a SPEand GC/MS-
SIM method to quantify simultaneously 48 different pesticides
in wine (Wong & Halverson, 1999). Sample preparation was
performed by a reverse phase C
18
cartridges with elution of
analytes using ethyl acetate. The Authors observed that addition
of NaCl to the samples prior to extraction increased the extraction
efciency of 38 compounds, except for allethrin and demeton
S-methyl.
In 2002, Natangelo et al. compared performances of a
quadrupole mass lter (Q) MS and an ion trap (IT) MS systems
for the pesticides analysis (Natangelo, Tavazzi, & Benfenati,
2002). Sample preparation was performed by SPME, and the
methods for analysis of propanil (anilide post-emergent herbi-
cide), acetochlor (chloroacetanilide pre-emergent herbicide),
myclobutanil (azole fungicide), and fenoxycarb (carbamate
insecticide) in grape juice and wine, were evaluated. Structures
of propanil and acetochlor are reported in Figure 5 (structure of
myclobutanil is reported in Fig. 4, fenoxycarb in Fig. 12). To
perform the GC/MS-Q analysis, the signals of ions at m/z 161
and 163 (propanil), 146 and 162 (acetochlor), 150 and 179
(myclobutanil), 88 and 116 (fenoxycarb) were recorded in SIM
mode. For the GC/MS-IT analysis, collisionally produced ions
formed by multiple mass spectrometry (MS/MS) at m/z
161 !126; 134 (propanil), m/z 223 !146 (acetochlor), m/z
179 !125; 152 (myclobutanil), m/z 116 !88 (fenoxycarb),
were monitored. SPME was performed with a poly(ethylene
glycol)/divinylbenzene (PEG/DVB) ber (65 mm thickness) by
direct immersion of the ber in the sample under stirring and after
NaCl addition. Analytes were desorbed by exposing the ber in
the GC injector port 15 min at 2508C. Evaluation of the GC/MS-
MSmethod LODs was carried out by selecting the most abundant
daughter ion generated by collisionally induced dissociation
(CID) experiment; LODs calculated with S/N3 and precision
calculated on three replicate analyses, are reported in Table 3.
The two methods showed a comparable sensitivity, suitable to
satisfy the Italian legal limits of myclobulanil and fenoxycarb in
grapes (0.2 mg/kg). Both methods showed good linearity for
samples spiked at concentration below 200 mg/L. The precision
was generally comparable, except in the analysis of myclobutanil
in grape juice where IT showed lower precision.
In 2003, Wong et al. developed an accurate SPE and GC/
MS-SIM method for the multiresidue pesticides detection in
wine by using a 5% diphenyl-95% dimethyl polysiloxane
capillary GC column (Wong et al., 2003). Pesticides were
extracted by a polymeric cartridge, and compounds co-eluted
with analytes were removed by passage through an aminopropyl-
MgSO
4
cartridge. Scheme of sample preparation is reported in
Figure 6. Organohalogen, organonitrogen, organophosphate, and
organosulfur pesticides and residues (in total 153 compounds)
were analyzed performing three different analyses by three
different SIM programs. Recoveries from samples spiked at
10 mg/Lresulted greater than 70%for 116 and 124 analytes in red
and white wines, respectively. Identication of compound was
conrmed by retentiontime of target ion andon three qualier-to-
target ion ratios. LOD for most of the pesticides was less than
5 mg/L. The compounds analyzed are reported in Table 4 together
with the corresponding target and qualier ions and LODs.
Stir-bar-sorptive-extraction (SBSE) uses a stir bar (typically
10-mm length) incorporated in a glass tube and coated with
PDMS. Upon stirring analytes are partitioned between the liquid
matrix of sample and the PDMS phase on the stir bar. Recoveries
increase in according to the volume PDMS to the sample volume
matrix ratio. Subsequently, the stir bar is transferred to a compact
thermal desorption unit mounted on a programmable temperature
TABLE 2. Wine pesticides and corre-
sponding m/z species monitored in SIM
mode by automated reverse-phase solid-
phase-extraction and GC/MS
Pesticide m/z
Ethyl hydrocinnamate (I.S.) 104;178
4,4-Dichloro-benzphenone 111;139
Azinphos-methyl 93;160
Bromopropylate 183;341
Captafol 79;151
Captan 79;149
Chlorpyrifos 314;316
Dichlofluanid 123/224
Dicofol 139;251
Dimethoate 87;125
Endosulfan 241;339
Etrimfos 181;292
Fenamiphos 154;303
Fenamirol 251;330
Folpet (42) 260;295
Iprodione (41) 187;314
Malathion 125;173
Methidathion 125;145
Parathion-methyl 109;263
Procymidone (37) 283;285
Triadimefon (51) 181;208
Vinclozolin (43) 212;285
Numbers corresponding to structures
in Figures 4 and 10 are reported.
FIGURE 5. Structures of some herbicides used inviticulture: acetochlor
(44) and propanil (45).
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vaporization (PTV) injector and analytes are thermally desorbed
into the GC column. The stir bars allow a 500-fold increase in
enrichment, and thus sensitivity, compared to SPME with 100-
mm PDMS bers.
A method to determine the dicarboximide fungicides
vinclozolin, iprodione, and procymidone (structures 43, 41, 37
in Fig. 4, respectively) by SBSE and thermal desorption GC/MS
analysis (SBSE-TD-GC/MS), was proposed (Sandra et al.,
2001). Iprodione was detected as its degradation product (3,5-
dichlorophenyl)hydantoin; the method accuracy was veried by
SBSE and liquid desorption Atmospheric-Pressure-Chemical-
Ionization negative mode (SBSE-LD-LC/APCI-MS) analysis.
By liquidliquid extraction and MS detection, LOD is in the
order of 1 mg/L for vinclozolin. By capillary GC-Ion Trap mass
spectrometry (GC-IT) determinations in the ng/L range for
vinclozolin and mg/L for iprodione, are achieved. By SBSE and
GC/MSanalysis in SCANmode, limits of quantication were 0.5
mg/L for vinclozolin and procymidone, and 5 mg/L for iprodione.
LODs were 0.2 mg/L for vinclozolin and procymidone, and 2 mg/
L for iprodione. By operating in SIMmode, LODs in the order of
2 ng/L for vinclozolin and procymidone, and of 50 ng/L for
iprodione, were achieved. Fragmentation spectra of three com-
pounds are reported in Figure 7. Vinclozolin and procymidone
are easily identied, whereas iprodione showed low abundance.
As a matter of fact, decomposition of iprodione occurs in the GC
column at temperatures above 2008C, the compound is degraded
for 90% to the more stable (3,5-dichlorophenyl)hydantoin.
Decomposition also occurs during thermal desorption of the stir
bar at 3008C and the analyte transfer in the hot transfer line.
Because of the peak area ratio of iprodione and its degradation
product is constant, quantication was performed on (3,5-
dichlorophenyl)hydantoin. White wines and sparkling wines
fromdifferent origin (France, Italy, South Africa) were analyzed,
vinclozolin was found in concentration 2.6 mg/L in a sparkling
wine; procymidone and iprodione resulted more abundant with
concentration between 5 and 65 mg/L.
The accuracy of the SBSE-TD-GC/MS method for the
iprodione detection via degradation product was veried by
SBSE-LD-LC/MS analysis of a sparkling wine. After SBSE
sampling, the stir bar was desorbed in acetonitrile and the LC/
APCI-MS analysis of extract was performed. Analyses were
carried out by a C
18
column using water and 10%tetrahydrofuran
in methanol as mobile phase and gradient elution. APCI was
performed in the negative mode in the mass range at mlz
200350, with a fragmentor voltage 70 Vand capillary voltage
4,000 V. Analyses were performed in SIM mode by recording
signals at mlz 242.9, 245.0, and 246.8 for iprodione. Authors
reported this as the rst application of SBSE with liquid
desorption; results obtained by SBSE-TD-GC/MS and SBSE-
LD-LC/MS were comparable.
The (MCH
3
OH-H)
ion formed for vinclozolin (MW

286) and procymidone (MW 284) was observed; for iprodione
(MW 330) formation of [M-CONHCH(CH
3
)
2
]
ion was due to

the thermolabile character of compound under the chemical
ionization conditions used (spectra are reported in Fig. 8).
Authors reported the negative chemical ionization (NICI) as a
better ionization and robustness method than positive chemical
ionization (PICI), and than both positive and negative electro-
spray ionization (ESI).
In a recent study, SBSE has been applied for the MS
pesticides wine analysis (Hayasaka et al., 2003). On the basis of
fragmentation spectra, 18 different agrochemicals were identi-
ed in wine. Recording signals at m/z 96, 283, and 285 (SIM
mode) procymidone exhibited one of the strongest ion response,
for this compound was estimated a LODdown to lowng/Llevels.
Butyltin compounds, monobutyltin (MBT), dibutyltin
(DBT), and tributyltin (TBT) (structures reported in Fig. 9) are
used for the production of biocides and polymer stabilizers
(Hoch, 2001). In particular, MBTand DBTare used as heat and
light stabilizers for poly(vinyl chloride) (PVC) materials, DBTis
also used as a binder in water-based varnishes (Summary of
Report 6/00, 2000). The main application of TBT is as a biocide
in marine antifouling paints. In vitro studies revealed that
butyltins disrupt the immune response in human, with effect on
the natural killer cells involved in the immune defense against
infections and cancer (De Santiago & Aguilar-Santelises, 1999;
TABLE 3. Comparative data of precision (RSD for three replicate analyses)
and limits of detection of pesticides
grape juice white wine grape juice white wine
GC/MS
Propanil (45) 5.9 5.3 0.1 1
Acetochlor (44) 9,1 7,3 0,2 5
Myclobutanil (40) 11,3 5,1 1,0 8
Fenoxycarb (58) 14,4 4,1 0,3 4
GC/MS-MS
Propanil 9,6 7,2 2 3
Acetochlor 13,0 3,1 5 15
Myclobutanil 17,7 2,2 10 2
Fenoxycarb 11,2 9,1 8 5
LOD (g/L) precision (RDS, n=3)
mg/L based on a signal-to-noise ratio 3:1; obtained by GC/MS-Q SIM-mode
and GC/MS-MS analysis coupled with SPME (PEG/DVB 65 mm thickness ber).
Numbers corresponding to structures in Figures 4, 5, and 12 are reported.
&
FIGURE 6.
&
Whalen, Loganathan, &Kannan, 1999). Butyltin compounds are
widespread contaminants also found in some wines.
Azenha and Vasconcelos studied the presence of these
compounds in Portuguese wines by SPME/GC/MS-IT (Azenha
& Vasconcelos, 2002). Ethyl-derivatized butyltin compounds,
produced by reaction with NaBEt
4
, were extracted and analyzed.
For SPME a PDMS ber was used, by performing headspace
extraction at 408C for 2030 min. To perform GC analysis,
compounds were thermally desorbed from the ber into the liner
in 1.5 min. LODs recorded ranged between 0.05 and 0.2 mg/L as
Sn for MBT, 0.02 and 0.1 mg/L as Sn for DBT, and 0.01 and
0.05 mg/L as Sn for TBT, and showed to be strongly inuenced
by the matrix. In 14% of the 43 table and 14 Port wines analyz-
ed, DBT was found at concentrations ranging between 0.05 and
0.15 mg/L, the presence of MBTwas instead revealed in only one
sample. In order to search the possible sources of DBTresidues in
the wines, a study of some plastic and oak wood materials used in
the process of wine-making and directly in contact with musts
and wines, was performed. The results suggest that high-density
polyethylene containers used to transfer the wine in the vini-
cation process may be the main sources of these contaminants.
Folpet [N-(trichloromethylthio)phthalimide] is a fungicide
used in vineyards (structure 42 reported in Fig. 4) in particular
against downy mildew (Plasmopara viticola), powdery mildew
(Uncinula necator), and gray mold (Botrytis cinerea) (Tomlin,
1994). In the last eighties, laboratory studies indicated a
neoplasm induction in the duodenum of rats. In general, the
presence of fungicides residues in must may inhibit the alcoholic
fermentation. Studies were conducted to assess the natural
hydrolysis of folpet residues in grape musts. Results showed that
folpet residues are fully decomposed by sunlight in grape must,
and that during wine-making the compound is degraded com-
pletely. At the end of fermentation, phthalimide, a hydrolysis
product that did not showto inhibit the alcoholic fermentation, is
present in wine (Hatzidimitriou et al., 1997; Cabras et al., 1997b).
The fungicide may be also added to the wine as illegal
preservative. As a consequence, there is a relevant interest in
the development of methods to determine the folpet residues in
wine.
In 1997, Unterweger et al. described a GC/MS method for
detection of folpet residues in grape juices, fermenting grape
musts and wines (Unterweger, Wacha, & Bandion, 1997). The
sample preparation was performed by liquidliquid extraction
with n-hexane, and analysis by a phenylmethylsiloxane capillary
column using captan as internal standard. The method showed
quantitative recoveries, and LOD of 100 mg/L.
Allyl isothiocyanate is used to protect the wine from the
Candida mycoderma yeast attack and to sterilize the air in wine
storage containers. Illegal additions of methyl isothiocyanate to
wines are made to prevent spontaneous fermentations, and as
a soil fumigant for nematodes, fungi, and other diseases in
vegetables, fruits etc. (Saito et al., 1994; Gandini & Riguzzi,
1997).
Uchiyama et al. performed a method for determination of
methyl isothiocyanate in wine by extraction with ethyl acetate
and GC/MS analysis (Uchiyama et al., 1992). Recoveries from
spiked samples ranged between 83%and 90%in white wine, and
75% and 82% in red wine, and 0.05 mg/L LOD was obtained. In
the same year, were published others GC/MS methods to detect
methyl isothiocyanate in wine (Fostel & Podek, 1992; Zimmer,
Otteneder, & Bierl, 1992). Fostel and Podek proposed a direct-
injection analysis using 1,4-dioxan as internal standard; Zimmer
et al. performed liquid extraction of sample with 1,1,2-
trichlorotriuoroethane. In 1995, a GC/MS method for detection
of both methylisothiocyanate and allylisothiocyanate in winewas
developed, with LODs lower than 1 ng/L (Przyborski, Wacha, &
Bandion, 1995).
Also triazoles are fungicides widely employed in viticulture
to control powdery mildews, rusts, and other fungal pests.
Triazoles are classied as acutely toxic because of may affect the
liver functionality, to decrease kidney weights, altered urinary
bladder structure, to have acute effects on the central nervous
system (Briggs, 1992). Due to their persistence, they can easily
contaminate fruit juices and wines, the Italian lawxed the LODs
of these compounds in wine between 100 and 500 mg/kg.
In 2002, a SPME/GC/MS-EI method for rapid screening of
several triazole residues in wine was developed (Zambonin,
Cilenti, & Palmisano, 2002). The analysis conditions were
optimized for determination of triadimefon, propiconazole,
myclobutanil, and penconazole (structures showed in Fig. 10,
myclobutanil structure 40 reported in Fig. 4).
To performquantitative detection, fragment ions at m/z 128,
210, 293, for triadimefon, m/z 145, 173, 259 for propiconazole,
m/z 179, 206, 288 for myclobutanil, and m/z 159, 161, 248 for
penconazole, were recorded in SIM mode. Mass spectra of
triazoles are showed in Figure 11. SPME was performed by a
silica ber coated with 85 mm thick polyacrylate, operating at
508C under stirring for 45 min; the GC injection port thermal
desorption was performed in 5 min at 2508C. The method LODs
were estimated ranging between 30 ng/Kg for propiconazole, and
100 ng/Kg for triadimefon, lower the maximum residue levels
recommended by the European Legislation in wine and grapes
(e.g., European Directive 90/642/EEC, 1990). Two commercial
wines were analyzed, and 1.0 mg/kg and 1.7 mg/kg of
propiconazole, and 1.1 mg/kg of penconazole, were found in
samples.
HPLCis the most suitable technique to determine polar, low
volatile, and thermally labile pesticides, such as phenylureas and
carbamates. In spite of the high sensitivity of uorescence
detection with post-column derivatization, or the robustness of
UV detection, mass detection has advantages of high sensitivity
and selectivity. Recently, LC/MS methods to perform the
pesticides analysis have been proposed. Fenandez et al. described
a method for analysis of carbamate residues in grape by matrix
solid-phase dispersion (MSPD) extraction and LC/MS analysis
(Fernandez, Pico, & Manes, 2000). The method, with the use of
FIGURE 6. Flow chart of the sample preparation method proposed by Wong et al. (2003) for the SPE/GC/
MS-SIM multiresidue pesticides analysis in wine. (Reprinted from Journal of Agricultural and Food
Chemistry 51, Wong et al., Multiresidue pesticide analysis in wines by solid-phase extraction and capillary
gas chromatography-mass spectrometric detection with selective ion monitoring. p. 1150, Copyright 2003,
with permission from American Chemical Society).
&
TABLE 4. The 153 wine pesticides analyzed by SPE and GC/MS-SIM with the corresponding target and qualier ions, and
limit of detection (LOD)
Pesticide MW target qualifiers LOD Pesticide MW target qualifiers LOD
(T) (Q
1
Q
2
Q
3
) (ppm) (T) (Q
1
Q
2
Q
3
) (ppm)
Acephate 183,2 136 94 95 125 25,0 Fenpropimorph 305,5 128 129 303 117 < 0.5
acenaphthalene-d
10
(I.S.) 164,3 164 162 160 80 Fenson 268,7 77 141 268 51 10,0
Alachlor 269,8 160 188 146 237 1,0 Fenthion 278,3 278 125 109 169 < 1.5
Aldrin 364,9 263 265 261 66 1,5 Fenvalerate I 419,9 167 125 181 152 3,0
Allethrin 302,4 123 79 136 107 3,0 Fenvalerate Il 419,9 167 125 181 169 3,0
Atrazine 215,7 200 215 202 58 1,0 Flucythrinate I 451,4 199 157 181 107 2,5
Azinphos-ethyl 345,4 132 160 77 105 1,0 Flucythrinate Il 451,4 199 158 181 107 2,5
Azinphos-methyl 317,3 160 132 77 105 3,0 Fludioxinil 248,2 248 127 154 182 1,0
Benalaxyl 325,4 148 91 206 204 1,0 Fluvalinate tau-I 502,9 250 252 181 208 0,5
Benfluralin 335,3 292 264 276 293 < 1.0 Fluvalinate tau-II 502,9 250 253 181 208 0,5
BHC-a 290,8 181 183 219 217 1,0 Folpet 296,6 147 104 76 260 15,0
BHC-d 290,8 181 219 183 217 2,0 Fonofos 246,3 109 246 137 110 < 1.0
BHC-g (Lindane) 290,8 181 183 219 111 1,5 Furalaxyl 301,3 95 242 152 146 1,0
Bitertanol I 337,4 170 168 171 57 0,5 Heptachlor 373,3 272 274 100 270 0,5
Bitertanol II 337,4 170 168 171 57 0,5 Heptachlor epoxide 389,3 353 355 351 357 0,5
Bromophos-ethyl 394,1 359 303 357 301 < 1.0 Hexachlorobenzene 284,8 284 286 282 288 < 0.5
Bromophos-methyl 366,0 331 329 333 125 < 1.0 Hexaconazole 352,9 83 214 216 82 1,0
Bromopropylate 428,1 341 183 339 343 0,5 Hexazinone 252,3 171 83 128 71 1,0
Bromoxynil 276,9 277 275 279 88 10,0 Imazalil 297,2 41 215 173 217 6,0
Captafol 349,1 79 80 77 151 25,0 Iprodione 330,2 314 187 189 244 5,0
Captan 300,6 79 80 151 77 10,0 Isofenphos 345,4 213 58 121 255 1,0
Carbaryl 210,2 144 115 116 145 10,0 Malaoxon 314,3 127 99 109 125 3,0
Carbofuran 221,3 164 149 131 123 2,0 Malathion 330,4 173 127 125 93 < 1.5
Carbophenothion 342,9 157 342 121 99 < 1.5 Metalaxyl 279,3 206 45 160 249 1,0
Chlorbenside 269,2 125 127 268 270 1,0 Methidathion 302,3 145 85 93 125 1,0
cis-Chlordane 409,8 373 375 377 371 < 1.0 Methoxychlor 345,7 227 228 152 113 < 1.0
trans-Chlordane 409,8 373 376 377 371 < 1.0 Metolachlor 283,8 162 238 240 146 < 1.0
Chlorfenvinphos 359,6 267 323 269 325 1,0 Mevinphos 224,2 127 192 109 67 < 1.5
Chlorothalonil 265,9 266 264 268 270 1,0 Mirex 545,6 272 274 270 237 < 1.0
Chlorpyrifos 350,6 197 199 314 97 1,0 Monocrotophos 223,2 127 67 192 97 3,0
Chlorpyrifos-methyl 322,5 286 288 125 290 < 1.0 Myclobutanil 280,8 179 150 82 181 1,0
Chlozolinate 332,1 188 259 186 187 1,5 Naled 380,8 109 185 79 145 6,5
chrysene-d
12
(I.S.) 240,4 240 236 241 238 Napropamide 271,4 72 128 100 271 < 1.0
Coumaphos 362,8 362 226 109 210 1,0 Nitralin 345,4 316 274 300 317 0,5
Cyanazine 240,7 212 213 214 68 3,0 Nitrofen 284,1 283 253 283 202 3,0
Cyfluthrin I 434,3 163 206 165 227 1,5 Nitrothal.isopropyl 295,3 236 194 212 254 1,0
Cyfluthrin Il 434,3 163 207 165 227 1,5 Norflurazon 303,7 303 145 102 305 1,0
Cyfluthrin III 434,3 163 208 165 227 2,5 Omethoate 213,2 156 110 79 109 6,0
Cyfluthrin IV 434,3 163 206 199 227 2,5 Oryzalin 346,4 317 275 258 58 100,0
Cyhalothrin 449,9 181 197 208 209 1,5 Oxadiazon 345,2 175 177 258 260 0,6
Cypermethrin I 416,3 181 163 165 209 2,0 Oxadixyl 278,3 105 163 45 132 1,5
Cypermethrin Il 416,3 181 164 165 209 2,0 Oxyfluorfen 361,7 252 361 302 331 1,0
Cypermethrin 111 416,3 163 181 165 209 2,0 Paraoxon 275,2 109 149 275 139 6,0
Cypermethrin IV 416,3 163 182 165 209 2,0 Parathion 291,3 291 109 97 139 1,0
Cyprodinil 225,3 224 225 210 77 < 1.5 Parathion.methyl 263,2 263 109 125 79 1,0
o,p'-DDT 354,5 235 237 165 236 < 0.5 Penconazole 284,2 248 159 161 250 1,0
p,p'-DDT 354,5 235 238 165 236 < 1.0 cis-Permethrin 391,3 183 163 165 184 < 0.5
Deltamethrin 505,2 181 253 251 255 8,0 trans-Permethrin 391,3 183 164 165 184 < 0.5
Demeton-O 230,3 88 60 89 171 2,5 phenanthrene-d
10
(I.S.) 188,3 188 189 184 187
(Continued )
&
APCI or Electrospray (ES) sources in positive mode, showed to
be suitable for detection of carbamate pesticide residues in
fruit and vegetables at the regulatory relevance levels, allowing
the simultaneous determination of 13 different carbamates:
carbaryl, carbofuran, diethofencarb, ethiofencarb, fenobucarb,
fenoxycarb, isoprocarb, methiocarb, metholcarb, oxamyl, pir-
imicarb, propoxur, and thiobencarb. Structures are reported in
Figure 12.
The 13 carbamates were separated by a C
8
column using a
methanol-water gradient. Authors reported the HPLC chromato-
graphic peaks resolution improve by replacing methanol with
acetonitrile, but a rapid contamination of the corona discharge
needle was observed in APCI, probably due to the fact that
acetonitrile is a low-ionizable solvent compared with methanol.
In positive mode, by performing increasing of cone voltage from
10 to 120 V, the signals of three main ions [MNa]
, [MH]
,
and [MH CH
3
NCO]
were observed at 20 V. For both APCI

and ES sources a cone voltage of 20 V provided the molecular
mass information, through the protonated ions [MH]
showed
little fragmentation, and the sensitivity was the highest for all the
compounds. N-methylcarbamate insecticides are labile com-
pounds, and some of them undergo collisionally induced
decomposition even at low cone voltage (e.g. at 20 V the base
peak of the oxamyl APCI-positive spectrum was the ion at m/z
163 formed by the loss of methylisocyanate residue). In positive
mode, ES produces both [MH]
and [MNa]
adducts,
whereas the APCI positive mode only produces the [MH]
ion. By performing APCI in negative mode [MCONHCH

3
]
ions are formed for most compounds, and the [MH]
ion of
diethofencarb and fenoxycarb was observed. Better sensitivity
was achieved operating in positive mode. Authors observed that
the softer ionization of ES with respect to APCI induces lower
fragmentation of oxamyl, and negative fragment ions of
carbamates were obtained by APCI but not by ES. The LC/
APCI-MS negative ion mode approach is proposed as a tool for
conrmation of carbamate with higher levels. The principal
species and fragment ions formed fromeach carbamate recorded
by performing APCI analysis in both positive- and negative-
mode, and by ESI analysis in positive mode, are reported in
Table 5.
TABLE 4. (Continued )
Demeton-5 230,3 88 60 170 89 2,5 Phorate 260,4 75 121 260 97 < 1.0
Desmetryn 213,3 213 198 171 58 < 1.5 Phosalone 367,8 182 367 121 184 < 1.0
Dialifos 393,9 208 173 210 76 1,0 Phosmet 317,3 160 161 77 93 < 1.5
Diallate I 270,2 86 234 236 128 < 0.5 Prochloraz 376,7 180 70 307 310 6,0
Diallate Il 270,2 86 235 236 128 < 0.5 Procymidone 284,1 96 283 285 67 1,0
Diazinon 304,3 179 137 199 152 < 1.0 Profenophos 373,6 208 339 139 206 3,0
Dichlobenil 172,0 171 173 136 100 < 1.5 Prometryn 241,4 241 184 226 105 < 1.5
Dichlofluanid 333,2 123 224 167 226 < 1.5 Propargite 350,5 135 150 231 34 0,5
4,4' -Dichlorobenzophenone 251,1 139 111 141 250 0,5 Propazine 229,7 214 229 172 58 < 1.0
Dichlorvos 221,0 109 185 79 187 < 1.0 Propetamphos 281,3 138 194 236 222 < 1.0
Dicloran 207,0 206 176 178 208 4,0 Propyzamide 256,1 173 175 145 255 1,5
Dicrotophos 237,2 127 67 193 72 3,0 Pyrimethanil 199,3 198 199 77 200 < 1.0
Dieldrin 380,9 79 263 277 279 2,0 Ouinalphos 298,3 146 157 118 156 50,0
Dimethoate 229,3 87 93 125 143 2,5 Ouintozene 295,3 237 249 295 214 < 2.0
Dinoseb 240,2 211 163 147 240 150,0 5imazine 201,7 201 186 173 68 3,0
Dioxathion 456,0 97 125 271 153 5,0 Tebuconazole 307,8 125 250 70 83 1,5
Disulfoton 274,4 88 89 97 142 1,0 Tecnazene 260,9 203 261 215 201 1,0
Endosulfan-a 406,9 241 195 239 237 1,5 Terbufos 288,4 231 57 103 153 < 1.0
Endosulfan-b 406,9 195 237 241 207 3,0 Terbuthylazine 229,7 214 173 216 229 < 1.5
Endrin 380,9 317 263 315 319 3,5 Terbutryn 241,4 226 185 241 170 < 1.0
Endrin aldehyde 380,9 67 345 250 347 2,0 Tetrachlorovinphos 366,0 329 331 109 333 < 1.0
Endrin ketone 380,9 317 67 315 319 < 1.0 Tetradifon 356,1 159 111 229 227 1,0
EPN 323,3 157 169 141 185 < 1.0 Thiometon 246,3 88 125 89 93 1,5
Eptam 189,3 128 43 86 132 1,0 Tolyfluanid 347,3 137 238 106 83 2,6
Ethalfluralin 333,3 276 316 292 333 1,0 Triadimefon 293,8 57 208 85 210 1,0
Ethion 384,5 231 153 97 125 1,0 Triadimenol 295,8 112 168 128 70 4,0
Fenamiphos 303,4 303 154 288 217 < 1.0 Tri-allate 304,7 86 268 270 128 < 1.0
Fenarimol 331,2 139 219 251 107 0,6 Trifluralin 335,3 306 264 290 307 < 1.0
Fenitrothion 277,2 277 125 109 260 1,0 Vinclozolin 286,1 212 198 187 285 1,0
Fenpropathrin 349,4 97 181 125 265 0,6
Pesticide MW target qualifiers LOD Pesticide MW target qualifiers LOD
(T) (Q
1
Q
2
Q
3
) (ppm) (T) (Q
1
Q
2
Q
3
) (ppm)
Reprinted fromJournal of Agricultural and Food Chemistry 51, Wong et al., Multiresidue pesticide analysis in wines by solid-phase
extraction and capillary gas chromatography-mass spectrometric detection with selective ion monitoring. p. 11511153, Copyright 2003,
with permission from American Chemical Society.
&
Carbofuran was found in a grape sample in concentration
0.30.5 mg/kg. By performing analysis of 0.5 g sample, the
authors highlighted that the method is appropriate to detect
carbamate residues at lower levels of MRLs admitted by the
European Union in fruit and vegetables.
Wu et al. proposed a method for analysis of polar pesticides
in wine by automated in-tube SPME coupled with LC-
electrospray ionization (C
18
-LC/ESI-MS) (Wu et al., 2002). Six
phenylurea pesticides diuron, uometuron, linuron, monuron,
neburon, siduron, and six carbamates barban, carbaryl, chlor-
propham, methiocarb, promecarb, propham, were analyzed.
Structures of compounds are reported in Figure 13, carbaryl and
methiocarb in Figure 12 (structures 52 and 55, respectively). In-
tube SPME is a microextraction and pre-concentration technique
that can be coupled online with HPLC, suitable for the analysis of
less volatile and/or thermally labile compounds (Wu et al., 2002).
The technique uses a coated open tubular capillary as the SPME
device, and the extraction process can be automated. Due to the
high extraction efciency of the polypyrrole-capillary coating
toward polar compounds, benzene compounds and anionic
species, and to the high sensitive mass detection, LODs ranging
between 0.01 and 1.2 ng/mL were calculated. Recoveries of
analytes were observed to be affected by the sample ethanol
content. For ESI-MS a capillary voltage of 4,500 V in positive
mode was applied, with a cone voltage depending on the ions
selected (see Table 6). By operating in SIM mode the method
resulted suitable for analysis of carbamate and phenylurea
pesticides in the same run.
III. MASS SPECTROMETRY IN THE ETHYL
CARBAMATE ANALYSIS
The toxicological testing conducted in laboratory on several
animal species indicated that ethyl carbamate (EC) is a potential
human mutagen and carcinogen (IARC Working Group on the
Evaluation of Carcinogenic Risks to Human, 1988). ACanadian
study carried out in 1985, revealed the presence of high levels of
EC (up to several hundred mg/L) in some alcoholic beverages,
especially dessert wines and spirits (Conacher et al., 1987).
During fermentation, with the rapid yeast growth, arginine is
metabolized to form urea, which is by reaction with ethanol, the
EC precursor (Ough, Crowell, & Mooney, 1988; Monteiro,
Trousdale, & Bisson, 1989; Ough et al., 1990). Moreover, the
arginine metabolism of the wine lactic bacteria induces
formation of the EC precursor citrulline (Tegmo-Larsson,
Spittler, & Rodriguez, 1989; Liu & Pilone, 1998). Considerable
efforts have been devoted to develop accurate methods for the
quantication of EC and to clarify the origin of compound
(Ferreira &Fernandes, 1992). The U.S. wine industry established
a voluntary target for EC (15 mg/L or less in table wines; 60 mg/L
or less in fortied wines) the U.S. Food and Drug Administration
published recommendations to minimize EC in wine (Butzke &
Bisson, 1997). For detection of EC, a SPEmethod with elution by
methylene chloride and GC/MS-SIM mode analysis has been
adopted by the AOAC International (Association of Ofcial
Analytical Chemists) for alcoholic beverages (AOAC, 1995).
Another GC/MS method for the wine EC analysis by the use of
FIGURE 7. Fragmentation spectra (EI) by stir-bar-sorptive-extraction
and thermal-desorption-GC/MS analysis (SBSE-TD-GC/MS) of dicar-
boximide fungicides vinclozolin, iprodione, and procymidone (struc-
tures 43, 41, 37 in Fig. 4, respectively) and of the iprodione degradation
product (3,5-dichlorophenyl)hydantoin. (Reprinted from Journal of
Chromatography A, 928, Sandra et al., Stir bar sorptive extraction
applied to the determination of dicarboximide fungicides in wine. p. 121,
Copyright 2001, with permission from Elsevier).
&
SPE styrene/DVB sorbent, was proposed (Jagerdeo et al., 2002).
Recovery of analyte from the cartridge was performed by ethyl
acetate, LOD 0.1 mg/L was reported using
13
C
15
N-labeled EC as
internal standard.
Conacher et al. (1987) developed a GC/MS method suitable
for determination of EC in several different alcoholic beverages.
The ethanol content was adjusted at 10%, then samples were
saturated with NaCl before to perform methylene chloride
liquidliquid extraction. Before analysis extracts were concen-
trated and dissolved in ethyl acetate. The LOD of method was
0.5 mg/kg.
In the earlier 90s, the presence of EC, n-propyl carbamate,
and urea, was investigated in a number of fortied wines (Daudt
et al., 1992). After the sample preparation by liquid extraction
with dichloromethane, GC/MS analyses were performed using
cyclopentyl carbamate as internal standard and by recording in
SIMmode the signal of fragment at m/z 62 for ethyl carbamate, n-
propyl carbamate, and cyclopentyl carbamate. An effect of the
yeast strain and of arginine level on the urea concentration in the
wine was observed. A slight effect on initiating skin cancer in
mice of n-propryl carbamate with respect to EC was demon-
strated (Pound, 1967), and a study on relative rates of carbamates
FIGURE 8. Mass spectra by stir-bar-sorptive-extraction and liquid-desorption-LC/MS negative mode
analysis (SBSE-LD-LC/APCI-MS) of procymidone (1), iprodione (2), and vinclozolin (3) (structures 37,
41, 43 in Fig. 4, respectively). (Reprinted from Journal of Chromatography A, 928, Sandra et al., Stir-bar-
sorptive-extraction applied to the determination of dicarboximide fungicides in wine. p. 125, Copyright
2001, with permission from Elsevier).
FIGURE 9. Contaminants used for production of biocides and as
polymer stabilizers found in some wines: monobutyltin (46), dibutyltin
(47), and tributyltin (48). XCl; OH; EHMA (2-ethylhexylmercapto-
acetate); 2-MET (2-mercaptoethyltallate).
&
formation by reaction of ethyl and n-propyl alcohols with
urea (in model wine solutions) was performed. From the study
resulted that ethanol reacts about 1.34 times as fast as than
propanol.
In another paper of the same year, a GC/MS study on the EC
presence in Madeira wines was reported (Ferreira & Fernandes,
1992). Evolution of EC in the vinication process was followed,
an increase of the compound with fermentation was observed.
Analyses were performed on 10 mL of wine by using n-butyl
carbamate as primary internal standard, and methyl tetradecano-
ate as secondary internal standard. Ions at m/z 44, 62, and 74 were
acquired, quantitative detection of ethyl and butyl carbamates
were performed on the m/z 62 ion.
Fauhl and Wittkowski (1992) proposed a method for
determination of EC in wine by continuous extraction with
diethyl ether, and GC/MS-SIMchemical ionization analysis with
methane as reagent gas. Authors reported as advantage of diethyl
ether extraction the chlorinated solvents replacement, and not
signicant formation of EC during extraction as a result of
thermal treatment. Two characteristic fragments at m/z 62 and 90
(the latter corresponding to [MH]
ion of EC) with 1:1 ratio

were used for identication. Propyl carbamate was added to the
sample as internal standard to perform quantitative analysis
(characteristic fragments at m/z 62, and 104 of [MH]
ion), the
method had LOD 1 mg/L.
Recently a headspace SPME/GC/MS method for EC
detection in wine has been proposed (Whiton & Zoecklein,
2002). In the study different ber were tested with different times
and temperatures of exposition. The method was developed by
sampling a 7 mL of wine sample with propyl carbamate as
internal standard. Sampling conditions were optimized by using a
65 mm PEG/DVB ber exposed to the sample headspace for
30 min at 228C. The analyte was released into GCinjection port at
2508C by direct exposure of ber, signals at m/z 44, 62, and 74
were recorded in SIM mode (the signal at m/z 62 was used for
quantitation, the others for conrmation). For the method a LOD
of 9.6 mg/L was reported.
IV. MASS SPECTROMETRY AND WINE DEFECTS:
COMPOUNDS FROM YEASTS AND BACTERIA
Among the large number of compounds formed by bacteria
metabolism and by the yeast during alcoholic fermentation,
carbonyl compounds play an important role in determining
chemical composition of wine. Because their very low sensory
thresholds, many of these compounds give an important contri-
bute to the organoleptic characteristics of product. Moreover,
FIGURE 10. Structures of triazole residues (fungicides employed in
viticulture) detected in wine: propiconazole (49), penconazole (50),
triadimefon (51).
FIGURE 11. GC/MS-EI mass spectra of triazoles: source temperature
2008C; electron energy: 70 eV; lament current 200 mA; mass range from
m/z 50450; scan time 0.9 s; inter-scan time 0.1 s. (Reprinted from
Journal of Chromatography A, 967, Zambonin et al., Solidphase
microextraction and gas chromatography-mass spectrometry for the
rapid screening of triazole residues in wines and strawberries. p. 258,
&
aldehydes such as formaldehyde, acetaldehyde, acrolein, and
benzaldehyde are reputed to be carcinogens (Nascimento et al.,
1997).
To determine carbonyl compounds at the low level that are
normally present in the wine, GC/MS methods are usually
employed. Among them, the more selective and sensitive method
is by analysis of their O-pentauorobenzyl oximes (O-PFB-
oximes) formed by derivatization with pentauorobenzylhy-
droxylamine (PFBOA) (scheme reported in Fig. 14).
By this approach, glyoxal and methylglyoxal (pyruvic
aldehyde) in wine were studied (de Revel & Bertrand, 1993).
Methylglyoxal is produced by the metabolism of various
microorganisms, such as yeasts Saccharomyces cerevisiae
(Murata et al., 1985), and a bacterium lactobacillus strain
(Baskaran, Prasanna Rajan, & Balasubramanian, 1989). This
compound is reputed to be cytotoxic and to interfere with cell
division in procaryotes and eucaryotes (Murata et al., 1986); as a
consequence the methylglyoxal reduction would be advanta-
geous to the organisms because of the less toxic activity of
products (Inoue et al., 1988). Analysis of O-PFB-oximes was
performed by recording the signal at m/z 181 (the base peak in
mass spectra of PFB-derivatives), in SIM mode. Differentiation
between the saturated and the unsaturated aldehyde derivatives
was achieved by recording signals at m/z 239 and at m/z 250,
respectively. Authors observed that succession of microorgan-
isms, such as Leuconostoc nos in malolactic fermentation, may
increase concentration of glyoxal and methylglyoxal in wine (de
Revel & Bertrand, 1993).
The same year, Vidal et al. identied several methylketones
in Cognac by GC/MS analysis of their O-PFB-oximes (Vidal,
Estreguil, & Cantagrel, 1993). Usually, derivatization with
PFBOA is performed directly in the aqueous samples, but in
these conditions the ketones derivatization requires longer times.
To overcome the problem, a preliminary pentane/ethyl ether
extraction of analytes was performed, derivatization of extracts
was carried out at 658C for 30 min. Because the two geometrical
syn and anti oximes for each aldehyde form, to simplify the
chromatogram the selective reduction of C=N oxime double
bond by pyridine:borane mixture was performed. MS analysis
(SIM mode) showed a better selectivity with respect to GC/
FIGURE 12. Carbamates determined at regulatory relevance by LC atmospheric-pressure-chemical-
ionization (APCI) or electrospray (ES) positive-ion mode (methods proposed by Fernandez, Pico, &Manes,
2000). 52, carbaryl; 53, carbofuran; 54, ethiofencarb; 55, methiocarb; 56, fenobucarb; 57, isoprocarb; 58,
fenoxycarb; 59, diethofencarb; 60, metholcarb; 61, propoxur; 62, pirimicarb; 63, oxamyl; 64, thiobencarb.
&
ECD and close sensitivity. The lowering of electron energy to
35 eV increased sensitivity by lowering LODs of a factor 2.
Methylketones determined in Cognac were 2-heptanone,
2-nonanone, 2-undecanone, 2-tridecanone (formed by oxidation
of volatile fatty acids), with levels sufcient to contribute in the
formation of typical avor of aged Cognac. Authors observed
that concentration of methylketones depended on the spirit age
and on aging conditions (peroxydase concentration, volume of
barrels).
The presence in wine of others a-dicarbonyl compounds
such as 3-hydroxy-2-oxopropanal (a reductone also named
hydroxypropanedial), 2,3-butanedione (diacetyl), and 2,3-pen-
tanedione, was reported. Sauternes winessweet white table
wines produced fromgrapes infected by desirable Botrytis (noble
rot)was revealed to be rich in hydroxypropanedial. If Botrytis
cinerea attack is severe concentration of this compound in wine
may reach to 1 g/L, and it can be used as a marker for estimation
of the grape sanitary condition. Moreover, hydroxypropanedial
and other carbonyl compounds present at high levels in
botrytized wines are important because they play an important
role in the sulfur dioxide combination in winemaking (de Revel
& Bertrand, 1994). It was also reported that reductones in wine
preserve organoleptic qualities, x aromas, and inhibit bacterial
development (Shimohara et al., 1981; Yamanaka & Tsunoda,
1994, 1995). By reaction with amino acids they form browning
compounds such as glyoxal, methylglyoxal, and diacetyl
(Yamagushi, 1969; Shimohara et al., 1974; Velisek & Davidek,
1978).
Guillou et al. (1997) studied the presence of hydroxypro-
panedial in musts and wines. By GC/MS analysis a-dicarbonyl
TABLE 5. The principal species and fragment ions and their relative abundances (R%), recorded by performing positive- and negative-
APCI analysis, and by positive ESI analysis
Compound MW
Positive mode Negative mode Positive mode
m/z and tentative ions R% m/z and tentative ions R% m/z and tentative ions R%
Carbaryl 201
202 [M+H]
+
100 143 [M-H-CH
3
NCO]
-
100
202 [M+H]
+
95
234 [M+H+CH
3
OH]
+
13 145 [M+H-CH
3
NCO]
+
100
224 [M+Na]
+
75
Carbofuran 221
222 [M+H]
+
100 163 [M-H-CH
3
NCO]
-
100
222 [M+H]
+
100
244 [M+Na]
+
20
Diethofencarb 267 268 [M+H]
+
100 266 [M-H]
-
100 268 [M+H]
+
100
182 [M+H-(CH
3
)
2
CH
2
NCO]
+
22
290 [M+Na]
+
20
Ethiofencarb 225
226 [M+H]
+
100 167 [M-H-CH
3
NCO]
-
100
226 [M+H]
+
100
248 [M+Na]
+
62
107 [M-CH
3
CH
2
S-CH
3
NCO]
+
50
164 [M-CH
3
CH
2
S]
+
50
Fenobucarb 207
208 [M+H]
+
100 149 [M-H-CH
3
NCO]
-
100
208 [M+H]
+
100
226 [M+NH
4
]
+
25
Fenoxycarb 301
302 [M+H]
+
100 185 [M-H-(CH
2
)
3
CH
3
NCO
2
]
-
100
302 [M+H]
+
100
230 [M+H-(CH
3
)
2
NCO]
+
28
324 [M+Na]
+
41
Isoprocarb 193
194 [M+H]
+
100 135 [M-H-CH
3
NCO]
-
100
194 [M+H]
+
100
216 [M+Na]
+
15
Methiocarb 225
226 [M+H]
+
100 167 [M-H-CH
3
NCO]
-
100
226 [M+H]
+
100
152 [M-H-CH
3
NCO-CH
3
]
-
60
248 [M+Na]
+
22
Metholcarb 165
166 [M+H]
+
100 107 [M-H-CH
3
NCO]
-
100
166 [M+H]
+
100
188 [M+Na]
+
19
Oxamyl 219 163 [M+H-CH
3
NCO]
+
100 161 [M-H-CH
3
NCO]
-
100
242 [M+Na]
+
100
147 [M-(CH
3
)NCO]
-
40
258 [M+K]
+
40
237 [M+NH
4
]
+
27
251 [M+CH
3
OH]
+
17
Pirimicarb 238 239 [M+H]
+
100 239 [M+H]
+
100
261 [M+Na]
+
29
Propoxur 209
210 [M+H]
+
100 151 [M-H-CH
3
NCO]
-
100
210 [M+H]
+
100
168 [M+H-CH
3
CH=CH
2
]
+
33
153 [M+H-CH
3
NCO]
+
20
Thiobencarb 257
258 [M+H]
+
100 132 [M-CH
2
C
6
H
4
Cl]
-
100
258 [M+H]
+
100
APCI ES
Fragmentor voltages 20 V positive mode, 40 V negative mode. (Reprinted from Journal of Chromatography A, 871, Fernandez et al.,
Determination of carbamate residues in fruits and vegetables by matrix solid-phase dispersion and liquid chromatography-mass spectrometry. p. 47,
&
FIGURE 13. The six phenylurea pesticides (monuron 65, uometuron 66, siduron 67, diuron 68, linuron
69, and neburon 70), and four carbamates (propham 71, chlorpropham 72, barban 73, and promecarb 74;
structures of carbaryl and methiocarb are reported in Fig. 12) detected in wine by automated in-tube SPME
and LC/ESI-MS reverse phase analysis (method proposed by Wu et al., 2002).
TABLE 6. Selected ions and corresponding fragmentator cone voltage (V
f
) used in ESI-MS analysis of wine pesticides
Carbamate
pesticide
MW m/z and ions selected V
f
(V) Phenylurea
pesticide
MW m/z and ions selected V
f
(V)
Carbaryl (52) 201 202 [M+H]
+
30 Monuron (65) 198 199 [M+H]
+
60
145 [M+H-CH
3
NCO]
+
60 221 [M+Na]
+
70
Methiocarb (55) 225 226 [M+H]
+
30 72 [C
3
H
6
NO]
+
100
169 [M+H-CH
3
NCO]
+
60 Fluometuron (66) 232 233 [M+H]
+
60
Propham (71) 179 120 [C
6
H
5
NCO+H]
+
90 72 [C
3
H
6
NO]
+
100
138 [M+H-C
3
H
6
]
+
60 Siduron (67) 232 233 [M+H]
+
60
Promecarb (74) 207 208 [M+H]
+
30 255 [M+Na]
+
120
151 [M+H-CH
3
NCO]
+
60 Diuron (68) 232 233 [M+H]
+
60
Chlorpropham (72) 214 154 [M-C
3
H
7
OH]
+
90 72 [C
3
H
6
NO]
+
100
172 [M-C
3
H
6
]
+
60 Linuron (69) 248 249 [M+H]
+
60
Barban (73) 258 258 [M]
+
30 Neburon (70) 274 275 [M+H]
+
50
178 [M+H-81]
+
60 297 [M+Na]
+
70
Capillary voltage 4,500 V, positive mode (Wu et al., 2002). Structure of compounds are reported in Figures 12 and 13.
&
compounds were detected as quinoxaline derivatives formed by
reaction with 1,2-diaminobenzene (mass spectrumof 3-hydroxy-
2-oxopropanal quinoxaline is reported in Fig. 15).
By analysis of O-PFB-oximes, changes of carbonyl
compounds occurring with malolactic fermentation (MLF) of
wines were studied (Flamini, De Luca, &Di Stefano, 2002). GC/
MS analysis was performed by poly(ethylene glycol) (PEG)
column recording the signals at m/z 181, m/z 239, and m/z 250,
using o-chlorobenzaldehyde as internal standard. Chromato-
grams corresponding to the three signals are reported in
Figure 16. Compounds corresponding to peak number are
reported in Table 7.
Before derivatization, pyruvic acid in the wine was removed
by passage through an ion exchange column. It was necessary
because the large amount of the compound in wine reacts with
PFBOAand forms derivatives, which leave fromthe column as a
broad peak, covering a large range of chromatogram. The study
evidenced marked changes in carbonyl compounds occurring
with MLF, in particular with regard to diacetyl, acetoin and
aliphatic saturated aldehydes, and glyoxal and methylglyoxal
increases were observed. A relevant presence of unsaturated
aldehydes in wine was also found. The dramatic increase of a
peak observed on chromatograms revealed a relevant presence of
glycolaldehyde in wine. Because the higher were the glyoxal
contents, the higher those of glycolaldehyde, a reduction system
involving the two compounds was supposed. To study correlation
between two compounds, and to verify the presence of
glycolaldehyde as promoted by malolactic bacteria, a study on
sterilized synthetic solutions added of glyoxal and inoculated
with a Oenococcus oeni lactic bacteriumwas performed (Flamini
& Dalla Vedova, 2003). PBF-oximes of two compounds were
detected by GC/MS analysis in SIM mode recording signals at
m/z 181, m/z 225 (M
species of glycolaldehyde), m/z 300 (M
species of o-chlorobenzaldehyde), and m/z 448 (M
species of
glyoxal). Concentration of glycolaldehyde turned out to be
associated with the amounts of glyoxal present, and glyoxal was
observed to decrease as glycolaldehyde increased. The reduction
of glyoxal to glycolaldehyde was promoted by the bacterial
activity. A high glycolaldehyde ability to induce browning of
()-catechin in a model wine system was revealed (about
10 times higher than that of ascorbic acid) inferring a possible
role of the compound in the color stability of white wines.
Acetaldehyde is the most abundant carbonyl compound in
wine and distillates. At high concentrations, the compound
confers a strong, pungent note to the beverage (Di Stefano &
Ciol, 1982). In the presence of alcohols, acetaldehyde reacts
with the amino groups in nucleosides to yield compounds
suspected to increase the risk of breast cancer in women
(Nascimento et al., 1997). Quantication of acetaldehyde is also
important for monitoring alcoholic fermentation and to dene the
FIGURE 14. Synthesis of pentauorobenzyl derivatives (O-PFB-oximes) by reaction of carbonyl
compounds with pentauorobenzylhydroxylamine (PFBOA).
FIGURE 15. Mass spectrum of quinoxaline derivative of 3-hydroxy-2-oxopropanal in wine formed by
reaction with 1,2-diaminobenzene (R
1
=H; R
2
=CH
2
OH). (Reprinted from Journal of Agricultural and Food
Chemistry 45, Guillou et al., Occurrence of hydroxypropanedial in certain musts and wines. p. 3383, 3385,
Copyright 1997, with permission from American Chemical Society).
&
enological treatments to the product. Addition of SO
2
to the wine,
made to preserve the product from oxidation and bacterial
attacks, is affected by the level of acetaldehyde. Carbonyl
compounds, by combining with SO
2
and forming bisulte
adducts, subtract the antioxidant to the wine (Di Stefano &Ciol,
1982). Moreover, acetaldehyde present in excess may undergoes
oxidation with consequent acetic acid and ethyl acetate levels
increasing, and acetic sourness of wine.
GC determination of acetaldehyde is usually performed by
direct injection of sample by using packed column, or by analysis
of 2-methylthiazolidin formed by reaction with cysteamine.
These methods usually employ nitrogen phosphorus (NPD) or
ame ionization detectors (FID) (EC Regulation no. 2870/2000,
2000; Miyake & Shibamato, 1993). Recently, a specic GC/MS-
EI method to determine acetaldehyde in wine and hydro-alcohol
matrixes by synthesis of O-PFB-derivatives has been proposed
(Flamini, Tonus, &Dalla Vedova, 2002). Advantage of method is
that GC analysis is performed by using common PEG column;
the compound is quantied on the basis of the sum of two
acetaldehyde O-PFB-oximes signals using 1-octanol as internal
standard. The method showed satisfying linearity and fairly good
reproducibility, with limited costs and times for sample
preparation. Absence of relevant interferences of wine matrix
in the derivatization was veried by comparing results extra-
polated from the calibration curve calculated by addition of
standard acetaldehyde to the sample, with those obtained from
the calibration curve calculated with standard solutions.
Diacetyl is regarded as adding complexity towine avor, but
if present in quite high concentration (higher than 5 mg/L) it can
be overpowering and to confer a distinct butter-like undesirable
note to the wine. Levels of acetoin and diacetyl, produced by
yeasts with alcoholic fermentation, are generally further
increased after MLF (Davis et al., 1985). Reduction of diacetyl
to acetoin (and 2,3-butanediol) is reported to be an advantageous
process for the yeasts because it forms less toxic products.
Moreover, this process increases the levels of the coenzymes
NAD
and NADP
(de Revel & Bertrand, 1994).

Hayasaka & Bartowsky (1999) developed a SPME/GC/MS
method to determine diacetyl in wine, by using deuterated
diacetyl-d6 as internal standard. Diacetyl was quantied as sum
of the both free and sulfur dioxide bounded forms. After NaCl
addition to the wine the SPME ber (coated with 65 mm PEG/
DVB) was exposed to the 3 mL sample headspace at 408C.
Molecular ions of diacetyl (m/z 86) and of diacetyl-d6 (m/z 92)
were scanned in SIM mode, and diacetyl was quantied on the
ratio of the ion response of analyte relative to that of the internal
standard. The use of a deuterated form of diacetyl as internal
standard ensured the robustness of the method in that the
quantitative result would not be affected by changes in the
parameters of the headspace SPME conditions. The method
showed excellent precision and accuracy, to be suitable for
analysis of both white and red wines, linearity concentration
range 0.0110.0 mg/mL, and LOD of 0.01 mg/mL. Authors
suggested that the sensitivity could be signicantly improved by
performing chemical ionization.
Benzothiazoles form part of molecular structure of a large
number of natural products, such as biocides, drugs, avors.
These molecules are used in several industrial products and
processes: 2-mercaptobenzothiazole is a rubber additive chemi-
cal (e.g., vulcanization accelerators), used as corrosion inhibitor,
as fungicide and herbicide (Santodonato et al., 1976).
Furthermore, benzothiazole has been found in the volatile
fraction of oak wood used for aging wines (Perez-Coello, Sanz, &
Cabezudo, 1998). In high level the compound may confer a
rubber or burned negative odor to the wine. A SPME/GC/
MS method has been developed to determine benzothiazole in
wine using a PEG/DVB ber (Bellavia et al., 2000). SPME was
performed by exposing the ber on the headspace of the NaCl
sample added at 508C for 15 min. Analytes were desorbed from
the ber at 2508Cfor 10 min, analysis performed by a 5%phenyl-
methyl-siloxane column. The mass spectrum of benzothiazole
shows the molecular ion at m/z 135 as main peak, and a less
abundant signal at m/z 108 derived by the loss of HCN. SIMmode
analysis was focused on the signal at m/z 135 as target ion, and on
the signal at m/z 108 as qualier ion. Quantitative analysis was
performed by addition of different amounts of standard
benzothiazole to the wine. The method revealed LOD of 45 ng/
L (S/N3), and a wide linearity range, with short analysis time
and low cost. Twelve commercial wines were analyzed by this
method, and the presence of benzothiazole was revealed in all
samples in concentration between 0.24 and 1.09 mg/L. Authors
discarded hypothesis that benzothiazole could derive from oak
wood in that the wines studied were not aged, and formation of
compound during the fermentation was supposed.
Many biogenic amines are reported to be psychoactive and
vasoactive. Some Authors suggested a relationship between the
two diamines putrescine and cadaverine, and histamine respon-
sible of numerous cases of food intoxication (Lovenberg, 1974;
Taylor, 1986; Stratton, Hutkins, & Taylor, 1991). Besides, with
cooking putrescine and cadaverine may be converted into
pyrrolidine and piperidine, respectively (Yamamoto et al.,
1982). These secondary amines, as well as spermidine and
spermine, may undergo nitrosation forming the extremely
carcinogenic nitrosamine. The aromatic amines b-phenylethy-
lamine, tyramine, isopentylamine, and 3-(2-aminoethyl)indole
(tryptamine), are responsible for dietary disturbs, inclusive
hypertension (Stratton, Hutkins, & Taylor, 1991; Anderson
et al., 1993). In wine these compounds are present as odorless
salts, but at the pH value of mouth they may develop repulsive
smells. Moreover, amines may be related to the unsanitary state
of wine. Structures of these amines are reported in Figure 17.
Analysis of biogenic amines is generally performed by LC
with a pre- or post-column derivatization (commonly with o-
phthalaldehyde in presence of mercaptoethanol), and uori-
metric detection of derivatives.
Fernandes and Ferreira by performing derivatization with
heptauorobutyric anhydride (HFBA) and GC/MS analysis
developed a method for simultaneous determination of the
principal diamines, polyamines, and aromatic amines in wine and
grape juices (Fernandes & Ferreira, 2000). The compounds
determined were 1,3-diaminopropane, putrescine, cadaverine,
spermidine, spermine, b-phenylethylamine, and tyramine.
Before derivatization, a sample clean-up was performed by
anionic exchange SPE; GC/MS analysis was performed in SIM
mode by recording one target ion signal, and at least two
qualifying ions, for each HFBA-derivative. Compounds deriva-
tives were quantied on the m/z signal at 104 for b-
phenylethylamine, 480 (putrescine), 494 (cadaverine), 316
&
FIGURE 16.
&
(tyramine), 254 (spermine and spermidine). Amphetamine, d
8
-
putrescine, 1,7-diaminoheptane, norspermidine, and norsper-
mine were used as internal standards. The method showed high
reproducibility with LOD less than 10 mg/L.
In the same year, Ngim et al. put to point a method for
determination of biogenic primary alkylamines in wine by
derivatization with pentauorobenzaldeide (PFB) and GC/MS-
SIM analysis (Ngim et al., 2000). To perform derivatization the
optimal experimental conditions were: pH 12, reaction time
30 min at 248C, concentration of PFB 10 mg/mL. More than
30 biogenic amines, produced by enzymatic degradation or
decarboxylation of aminoacids during fermentation, have been
identied. Analyses were performed by recording signals at m/z
208 and 211, corresponding to a-cleavage products of undeut-
erated PFB-imines and methyl-d
3
-PFB-imine, respectively, and
m/z 213, the molecular ion of pentauoronitrobenzene. Com-
pared with conventional LC analyses, the method showed more
selectivity, and a greater sensibility and resolution. The study was
performed on 12 California wines, alkylamines from methyla-
mine to n-eptylamine with concentration ranging from 0.048 to
91 mg/L were detected, and also 2-phenylethylamine, 1,4-
diaminobutane, and 1,5-diaminopentane were identied in wine.
b-carboline (norharman) and 1-methyl-b-carboline (harman)
are compounds with monoamine oxidase inhibition and benzo-
diazepine receptor binding activities (McIsaac & Estevez, 1966;
Rommelspacher et al., 1981). They have also co-mutagenic
TABLE 7. The compounds corresponding to peak numbers of GC/MS
chromatograms in Figure 16
On the right, identication mode and retention times of syn and anti wine carbonyl
compounds PFB-derivatives recorded by polyethylene glycol capillary column. RT,
retention time; m/z 181, 239, 250: chromatograms registered in SIMmode; MS/EI, mass
fragmentation spectra; lit., data reported in literature. (Reprinted fromVitis 41, Flamini
et al., Changes in carbonyl compounds in Chardonnay and Cabernet Sauvignon wines as
a consequence of malolactic fermentation. p. 109, Copyright 2002, with permission
from Vitis).
FIGURE 16. GC/MS chromatograms obtained by recording the signals at m/z 181, 239, and 250 in SIM
mode in the analysis of carbonyl compounds PFB-derivatives of Cabernet Sauvignon wine. The signal at m/z
181 is the base peak in the mass spectra of derivatives; differentiation between the saturated and the
unsaturated aldehyde is achieved on the signals at m/z 239 and m/z 250, respectively. Compounds
corresponding to peak number are reported in Table 7. (Reprinted from Vitis 41, Flamini et al., Changes in
carbonyl compounds in Chardonnay and Cabernet Sauvignon wines as a consequence of malolactic
fermentation. p. 110, Copyright 2002, with permission from Vitis).
&
activity in that aniline and o-toluidine are mutagenic compounds
when the two b-carbolines are present. Pancreatic or liver tumors
in habitual drinkers, may be caused from small quantity of
substances having activity mutagenic or carcinogenic. In
addition, production of acetaldehyde as ethanol metabolite could
play a role in forming b-carbolines endogenously in alcoholic
patients, promoting the cancer. Adachi et al. studied b-carbolines
in wine and alcoholic drinks by LC-uorescence reverse phase
chromatography, and used LC/MS for identication of com-
pounds (Adachi et al., 1991). By performing thermospray (TSP)
LC/MS in positive ion mode, [MH]
species were observed as

the base peak of mass spectra (see Fig. 18). Mean contents 0.5 mg/
L of norharman and 8.5 mg/L of harman were found in the wines
studied.
There are countries (e.g., Italy) where commercialization of
hybrid grapes and wines is prohibited. Several studies were
performed on volatile compounds considered typical odors of
hybrid grape varieties. Rapp, Versini, & Ullemeyer (1993) by
GC/MS identied o-aminoacetophenone as the cause of the
untypical aging note also called naphthalene note or hybrid
note of V. viniferawines Muller-Thurgau, Riesling, and Silvaner.
In the article, the presence in V. vinifera cvs of the compound
known as foxy smelling component of Labruscana grapes was
reported for the rst time.
Ciol, Garofolo, &Di Stefano (1995) by GC/MS analysis of
fermented synthetic media, observed that o-aminoacetophenone
is a secondary metabolite of Saccharomyces cerevisiae yeasts.
The presence of o-aminopropiophenone and of 3-(o-aminophe-
nyl)-prop-1-en-3-one in fermented synthetic media (the identi-
cations were based on the EI-fragmentation mass spectra) was
also revealed.
Ayear after, a study on the presence of methylanthranilate in
wine by GC/MS was published (Rapp & Versini, 1996). This
compound contributes to the typical hybrid/foxy taint of wines
from American hybrids and wild vines, but the study revealed it
was present also in white wines from V. vinifera with
concentrations higher than 0.3 mg/L.
Heavier containing sulphur compounds have a detrimental
effect on the wine aroma. Odors attributed to them are garlic
(associated with trans-2-methylthiophan-3-ol and 4-methylthio-
butan-l-ol), onion (2-methyltetrahydrothiophenone), burnt, cab-
bage (methionol), and cauliower (2(methylthio)-ethanol).
Other sulphur compounds with low boiling point (less than
908C), often have strong off-avors and very low organoleptic
thresholds (Rapp, Guntert, &Almy, 1985) (structures reported in
Fig. 19). It was observed that addition of 2,2
0
-dithiobis-ethanol
increases the H
2
S concentration in wine, a substance with very
strong odor of rotten eggs and sensory threshold 1 mg/L. The
compound is a precursor of the very unpleasant poultry-like
aroma 2-mercaptoethanol. In a study of 1995, the presence of
bis(2-hydroxyethyl) disulde in wine was reported. The
compound was extracted by ethyl acetate and identied by GC/
MS on the basis of the column retention time and EI mass
spectrum (Anocibar Beloqui, Guedes de Pinho, & Bertrand,
1995). The EI mass spectrumof 2,2
0
-dithiobis-ethanol is reported
in Figure 20. Analysis of several hundred wines revealed a
maximum 2,2
0
-dithiobis-ethanol content of 3.5 mg/L, with
higher abundances in wines from Vitis labrusca or hybrid
varieties.
V. MASS SPECTROMETRY AND WINE DEFECTS:
COMPOUNDS RELEASED FROM OAK WOODS
AND CORKS
Oak woods are commonly used for making barrels used for wine
aging. It may occur that some compounds released fromthe wood
confer defects to the wine. By GC/MS analysis of PFBOA
derivatives, Chatonnet & Dubourdieu (1998) identied several
FIGURE 17. Structures of amines involved in the food intoxication: putrescine (75), cadaverine (76),
histamine (77), isopentylamine (78), spermidine (79), spermine (80), b-phenylethylamine (81), tyramine
(82), triptamine (83).
&
aldehydes as substances responsible of sawdust smell sometimes
occurring in the wine aged in new barriques (225 L oak wood
barrels). Among them (E)-2-nonenal, (E)-2-octenal, 3-octen-1-
one, and 1-decanal, were identied. (E)-2-nonenal is reputed to
be the main responsible of smell sawdust in wine. Authors
observed that (E)-2-nonenal, 3-octen-l-one, (E)-2-octenal, and l-
decanal were correlated to the subtle rancid and the weedy odor
of sawdust detected by smelling the gas efuent from GC.
The toasting process of the wood used to make barrels for
brandy and wine aging, is responsible of production of a great
number of volatile and odoriferous compounds. Some of these
have toasty caramel aroma. Cutzach et al. identied a series of
compounds related to the toasted aroma by performing GC/MS
analysis of wood extracts (Cutzach et al., 1997). In addition to
already knew substances released from tosted wood (2-hydroxy-
3-methyl-2-cyclopenten-1-oneor cyclotene, 3-hydroxy-2-methyl-
4H-pyran-4-one or maltol), 2,3-dihydro-3,5-dihydroxy-6-methyl-
4H-pyran-4-one (DDMP), 4-hydroxy-2,5-dimethyl-3(2H)furan-
3-one (furaneol), 3,5-dihydroxy-2-methyl-4H-pyran-4-one, and
2,3-dihydro-5-hydroxy-6-methyl-4H-pyran-4-one (dihydromal-
tol) were identied on the EI fragmentation spectra (structures
are reported in Fig. 21). These molecules formed by heating in
the presence of proline, inferring that Maillard reactions are
responsible of their presence in the heated oak wood. Also furan
and pyran compounds, such as furfural, 5-(acetoxymethyl)fur-
fural, and 2,5-dimethyl-4-hydroxyfuran-3(H)-one have been
found to be released by wood in the wine.
For the physical properties of cork as an excellent seal for
liquids, cork stoppers are the principal closure for bottled wines,
but tainted corks may cause off-avor or corkiness in wine
(Vasserot, Pitois, & Jeandet, 2001). The compound 2,4,6-
trichloroanisole (TCA), with a ppt odor threshold, has been
identied as the major cause of corkiness in wine. Corks may be
contaminated by chloroanisoles during transport frompackaging
and shipping containers (Lee & Simpson, 1993), by micro-
biological methylation of chlorophenols during the corks
bleaching with hypochlorite (Burttshel et al., 1951), chloroani-
soles may be present as residues of pesticides and insecticides
used in the cork forest (Lee & Simpson, 1993). TCA can be also
formed during microbial contamination of raisins (Aung et al.,
1996). Relationships between trichlorophenol and TCA are
illustrated in Figure 22. A global dollar loss range up to US$
10 billion was estimated for bottled Champagne wines affected
by cork taint (Fuller, 1995).
GC/MS studies on 2,4-dicloroanisole, TCA, 2,3,4,6-tetra-
chloroanisole, pentachloroanisole, 2,4,6-trichlorophenol, 2,3,4,6-
tetrachlorophenol, pentachlorophenol (PCP) and guaiacol in
wines and cork stoppers, revealed a signicant correlation
between TCA in wines and its presence in cork stoppers, as well
as between the TCA level in wine and intensity of cork taint
(Pena-Neira et al., 2000). Irradiation of TCA causes the
formation of 2-chloroanisole, 4-chloroanisole, 2,4-dichloroani-
sole, and 2,6-dichloroanisole (Careri et al., 2001). The GC/MS-
EI fragmentation spectrum of TCA is reported in Figure 23.
Aung et al. (1996) studied the origin of chloroanisoles
causing musty off-avor of raisins. The chloroanisoles of raisin
samples were steam-solvent extracted using a mixture of pentane
and diethyl ether, extracts were concentrated and analyzed by
GC/MS. Authors observed that TCAforms fromraisins sterilized
with propylene oxide or with hydrogen peroxide.
Ayear after, an SPME-GC/MS method to determine TCAin
wine was developed (Evans, Butzke, & Ebeler, 1997). By
performing headspace SPME with a PDMS 100 mm SPME ber,
quantitative recovery of analyte was obtained. Deuterated d
5
-
TCA was used as internal standard. By performing SIM mode
analysis a method LOD 5 ng/L was achieved, with accuracy
8%, and precision (RSD) between 5% and 13%. TCA and
deuterated TCAwere recognized by characteristic double peaks
in the TIC (Fig. 24). Inspection of the extracted ions (Fig. 24,
right) reveals two distinct peaks: the signal at m/z 195 relative to
the TCA [M-15]
fragment ion (which does not occur in the

deuterated compound); the signal at m/z 215 relative to the
deuterated TCA M
species (not observed for TCA). Authors

FIGURE 18. Thermospray LC/MS positive ion mode mass spectra of
b-carboline (norharman, A) and 1-methyl-b-carboline (harman, B).
(Reprinted from Journal of Chromatography, 538, Adachi et al.,
Determination of b-carbolines in foodstuffs by high-performance liquid
chromatography and high-performance liquid chromatography-mass
spectrometry. p. 335, Copyright 1991, with permission from Elsevier).
FIGURE 19. Volatile sulphur compounds with strong off-avors and
very low organoleptic thresholds that may effect the wine aroma: trans-
2-methylthiophan-3-ol (84), 4-(methylthio)-1-butanol (85), 2-methylte-
trahydrothiophen-3-one (86), 3-(methylthio)-1-propanol (methionol)
(87), 2-(methylthio)ethanol (88).
&
proposed the method can be applied for detection of other
cork-related compounds which contribute to cork taint, such as
guaiacol (smoky character), geosmin (mildew), 1-octen-3-ol,
1-octen-3-one (mushroom), and 2-methylisoborneol (earthy).
In 2002, another method to assay trichloro-compounds in
corks and wines was proposed (Soleas et al., 2002). TCA and
trichlorophenol (TCP) were determined by extraction of corks
and wine with a 40%(v/v) aqueous ethanol solution, and extracts
puried by C
18
SPEbefore GC/MS analysis. The TCArecoveries
ranged between 86% and 102%; for TCP between 82% and
103%. LOD and LOQ of TCAwere 0.1 and 2 ng/L, respectively;
for TCP 0.7 and 4 ng/L, respectively. Signicant correlations
were found between the TCA and TCP contents of the wines
and corks, but many wines exhibiting cork taint had low or
undetectable concentrations of TCA.
Recently Hayasaka et al. developed a method for analysis of
TCA in wine based on stir bar sorptive extraction (SBSE) and
GC/MS (Hayasaka et al., 2003). Analysis was performed by a stir
bar 0.5-mm thickness, recording the ions at m/z 167, 169, 195,
197, 210, and 212 in SIM mode for TCA determination. The
SBSE technique was found to be orders of magnitude more
sensitive than conventional analyte enrichment methods; SBSE
often gave better signal-to-noise ratio in SCAN mode other than
methods operating GC/MS in SIM mode.
VI. MASS SPECTROMETRY TO DETERMINE
OCHRATOXIN A IN GRAPE AND WINE
Ochratoxin A (OTA), R-N-[(5-chloro-3,4-dihydro-8,hydroxy-3-
methyl-1-oxo-1H-2-benzopyran-7-yl)carbonyl]phenylalanine
(MW 403.18), is a isocoumarin derivative linked by carboxyl
group to a phenylalanine molecule (structure in Fig. 25). It is a
secondary metabolite of fungi Penicillium verrucosum and
Aspergillus ochraceus, for which the formation is promoted by
factors such as temperature, humidity, and other storage
conditions (Ramos et al., 1998; Blank, Hohler, & Wolffram,
1999). Toxic effects of OTA are renal damage with nephrotoxic
effect (Schwerdt et al., 1999), to interfere with mitochondrial
respiratory function and pH homeostasis (Sauvant, Silbernagel,
&Gekle, 1998), and impaired organic anion transport (Eder et al.,
2000). Moreover, the toxin has inhibition effect on tRNA-
synthetase, accompanied by the reduced protein synthesis, and
FIGURE 20. GC/MS-EI mass spectrum of bis(2-hydroxyethyl) disulde (2,2
0
-dithiobis-ethanol), a
precursor of H
2
S (strong odor of rotten eggs) and of 2-mercaptoethanol (poultry-like aroma) in wine.
(Reprinted by permission of the American Society for Enology and Viticulture. AJEV 46:1, p. 86, 1995).
FIGURE 21. Structures of volatile compounds with a toasty caramel
aroma released in wine from toasted woods during aging: 2-hydroxy-3-
methyl-2-cyclopenten-1-one (89); 3-hydroxy-2-methyl-4H-pyran-4-
one (90); 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one (91);
4-hydroxy-2,5-dimethylfuran-3(2H)-one (92); 2,3-dihydro-5-hydroxy-
6-methyl-4H-pyran-4-one (93); 3,5-dihydroxy-2-methyl-4H-pyran-4-
one (94).
FIGURE 22. Origin of 2,4,6-trichloroanisole and its relationship with
2,4,6-trichlorophenol.
&
enhanced lipid peroxidation via the generation of free radicals
(Hohler, 1998). OTA can also induce the formation of renal
tumors with cytochrome P450-related reactions and DNAadduct
generation as potential contributing mechanisms (Castegnaro
et al., 1998; Pfohl-Leszkowicz et al., 1998). The fungi develop on
grape, in particular in the harvest, in advantageous conditions of
high humidity and temperature; the toxin is then transferred to the
wine in winemaking. OTA is commonly detected in wine by LC,
performing SPE sample preparation by using immunoafnity
column (IAC) (Burdaspal &Legarda, 1999; Visconti, Pascale, &
Centonze, 1999; Castellari et al., 2000). As alternative methods,
capillary electrophoresis with laser induced uorescence detec-
tion and electrospray tandem mass spectrometry methods have
been developed. Also a stable isotope dilution assay (SIDA) and
LC/APCI-MS positive mode method was developed for
quantication of OTA in foods by using d
5
-OTA as internal
standard (Lindenmeier, Schieberle, & Rychlik, 2004).
In 1999, a method to determine OTA in dried vine fruit
(currants, raisins, and sultanas) and conrmation by LC/MS was
proposed (MacDonald et al., 1999). Analyses were performed by
acidic methanolic extraction of samples and clean-up of extracts
by IAC. The quantitative detection of toxin was achieved by
performing reverse-phase LC-uorescence analysis. A LOD of
method of 0.2 mg/kg was reported, with recoveries ranging
between 63%and 77%. But uorescence detection is not absolute
for OTA identication, and toxin presence is usually conrmed
by synthesis of methyl ester, or by OTA enzymatic cleavage to
yield ochratoxin a (7-carboxy-5-chloro-3,4-dihydro-8-hydroxy-
3-methylisocoumarin) (Filali et al., 2001). In this study, to
achieve the LC/MS conrmation an electrospray instrument
operating in positive ionization nebulizer mode, with capillary
voltage 2.6 kV and cone voltage 10 V, was used. Species
monitored were the
35
Cl-containing [MH]
ion at m/z 404, and

the
37
Cl-containing analog at m/z 406 (dwell time 0.1 s). As OTA
FIGURE 23. GC/MS-EI fragmentation spectra of 2,4,6-trichloroani-
sole. (Reprinted from Journal of Agricultural and Food Chemistry 50,
Soleas et al., Method for the gas chromatographic assay with mass
selective detection of trichloro compounds in corks and wines applied to
elucidate the potential cause of cork taint. p. 1034, Copyright 2002, with
permission from American Chemical Society).
FIGURE 24. GC/MSTICof 2,4,6-trichloroanisole (TCA) and deuterated d
5
-TCAused as internal standard
(left). On the right, the GC/MS SIMtraces of extracted ions at m/z 195 of TCAfragment ion, and at m/z 215
of I.S. molecular ion, used for quantication. (Reprinted from Journal of Chromatography A, 786, Evans
et al., Analysis of 2,4,6-trichloroanisole in wines using solid-phase microextraction coupled to gas
chromatography-mass spectrometry. p. 295, Copyright 1997, with permission from Elsevier).
&
contains a chlorine atom, additional conrmation was obtained
by monitoring the two isotopic forms, and by comparing the m/z
404/406 peak area ratios of OTA in samples with standard
solutions. Analysis of IAC extracts showed large amounts of
co-extractives compounds probably responsible of the ion
suppression observed and not occurring in the standard analysis.
By performing analyses of 60 different samples of retail dried
vine fruits, OTA level higher than 0.2 mg/kg was found in 19 of
20 currant, 17 of 20 sultana, and 17 of 20 raisin samples, with
maximum content of 53.6 mg/kg.
Zollner et al. (2000) performed a LC/ESI method to
determine OTA in wine using MS-MS in multiple reaction
monitoring (MRM) with nitrogen as collision gas. Zearalanone
(ZAN) was used as internal standard. The MRM ions selected
were m/z 404 !239 (relative to [MH-Phe]
species), m/z
404 !257, and m/z 406 !241 for OTA, and m/z 321 !123/189
for ZAN. Collisional energy 32.5 eV was applied to have the
highest sensitivity. The method LOD achieved was 0.5 ppb.
In 2001, Soleas et al. published a GC/MS method to assay
OTA in wine and beer (Soleas, Yan, & Goldberg, 2001). After
performing liquid extraction with dichloromethane and deriva-
tization with bis [trimethylsilyl]triuoroacetamide (BSTFA),
detection was performed by monitoring in SIM mode the eight
specic ions at m/z 528, 529, 530, 531, 532, 604, 606, and 619.
Recoveries ranging between 69% and 75% were reported. LOD
and LOQ were estimated at 0.1 and 2 mg/L, respectively. The
method performances are lower than limits achieved by
performing C
18
SPE and reverse-phase LC (gradient) analysis
with photodiode array detection at 333 nm: LOD and LOQ of
0.05 and 0.10 mg/L, respectively. As a consequence, the GC/MS
method is not suitable for routine quantitation, but it is potentially
useful as a conrmatory tool for samples containing OTA in
levels higher 0.1 mg/L.
Leitner et al. (2002) compared different analytical methods
to determine OTA in wine. ZAN was used as internal standard;
the sample clean-up was performed by either IAC or C
18
cartridges. LCanalysis was combined with either ESI/MS-MS or
uorescence detection. An optimized sensitivity was obtained by
splitting the LC eluent in a ratio of 1:50, a 10 mL/min ow was
introduced into the ion source. Detection at electrospray voltage
of 5,600V in MRM mode (collision gas nitrogen) was
performed, the precursor/product ion combinations used were:
m/z 404 !239, m/z 404 !257, and m/z 406 !241 for OTA; m/z
321 !123 and 321 !189 for ZAN (dwell time 400 ms). The
highest sensitivity was obtained using collisional energy 32.5 eV
(as proposed by Zollner et al., 2000), with LOD 0.05 mg/L.
Authors evidenced as advantage of mass spectrometry analysis
the use of the cheaper C
18
SPE cartridge for the sample
preparation, on the contrary the specic IAC sample clean-up
to eliminate the matrix interferences is required for FL detection.
By comparing the selective imunoafnity clean-up coupled with
FL detection, and C
18
SPE coupled with MS-MS detection,
comparable quantitative results were achieved; by coupling IACs
with MS detection no advantages in terms of sensitivity and
accuracy were observed. Finally, LC/MS resulted in general
advantageous as sample preparation, easy automatization, and
unambiguous analyte identication.
In 2003, a study on the occurrence of OTA in 24 wines
commercialized in South Africa and Italy was investigated by
LC-uorescence detection, performing conrmation by LC/MS-
ESI in positive ion mode, was published (Shephard et al., 2003).
The OTAconrmation was performed by CIDexperiments on the
analyte protonated molecular ion [MH]
at m/z 404 and

selected reaction monitoring (SRM) of the resultant product ions
[MH H
2
O CO]
at m/z 358and [MH H

2
O]
at m/z 386.
VII. ICP/MS AND ANALYSIS OF
PB AND AS IN WINE
Sodium arsenite is employed in viticulture as fungicide against
the esca plant disease (Eutypa lata). Arsenic is a suspected
carcinogen and little is known about its chronic sub-lethal effects.
The concentration of As in wines mainly depends on factors such
as soil composition, grape variety, climatic conditions, use of
pesticides, process of vinication, and storage conditions. The
Ofcer Internationale de la Vigne et du Vin (O.I.V.) has set the
maximum limit of As in wines at 200 pg/mL, but in general it is
accepted the presence of only a few pg/mL of As in un-
contaminated wines (Baluja-Santos & Gonzalez-Portal, 1992;
Bruno, Campos, &Curtius, 1994; Pedersen, Mortesen, &Larsen,
1994; Kildahl & Lund, 1996). Traditionally the determining of
As in wine is based on the generation of volatile amines followed
by reaction with silver diethyldithiocarbamate, and measurement
of molecular absorption of the As-containing complex formed
(Handson, 1984). But the method LOD, estimated at 10 pg/mL,
is not sufcient for the wine analysis. Alternatively, hydride
generation atomic absorption spectrometry has been used for the
quantication of several hydride-forming elements in wine,
including As (Baluja-Santos & Gonzalez-Portal, 1992). The
technique normally requires sample decomposition, a time
consuming procedure, which may result in sample contamination
or analyte loss. Unfortunately, both spectral and non-spectral
interferences of As are present, in particular intensity of As
signals are affected by non-spectral interference of carbon-
containing substances. By ICP/MS signals of As may be
enhanced from carbon-containing compounds.
Wangkarn & Pergantis (1999) proposed a method for the
determination of As in wines using microscale ow injection
(mFI) ICP/MS. The use of a microscale owinjection systemwith
a microconcentric nebulizer (MCN) permitted an efcient
sample introduction into the ICP/MS system, and reduced the
matrix effects by a factor of 23 compared with conventional
FI-ICP/MS systems. Sample volumes between 0.2 and 1.0 mL
were injected into the system with a carrier ow rates ranging
from 50 to 200 mL/min. The LOD of method resulted ranging
from 25 to 59 fg As, and in wine resulted of 0.5 pg/mL. The
FIGURE 25. Structure of ochratoxin A (OTA).
&
method was developed on deionised water diluted wine samples
using In as internal standard. By performing external calibration
or standard addition identical results were achieved. Analyses
were performed by quadrupole mass lter in SIMmode recording
the signal at m/z 75 of As
species. By monitoring the signal at

m/z 77 not evidence of
40
Ar
37
Cl
(the species that can interfere

with the signal at m/z 75 of
40
Ar
35
Cl
) was observed. Since wines

contain chloride at about 100 mg/mL it is essential to check for
any chloride interferences. Concentration of As in the samples
studied ranged between 7 and 13 pg/mL.
The presence of lead in wine may be as the consequence of
the airborne particulate matter of atmosphere on grapes, and/or of
the intake by the grapevine fromground water and soil. The metal
may also be released from bronze tanks, taps, pumps and tubing,
containers used in winemaking. Since lead may be a real health
hazard because it affects the hemoglobin biosynthesis and the
nervous system, the maximum permissible lead level in wine is
regulated (International Methods of Wine Analysis, 1990 Paris;
EC Regulation no. 466/2001, 2001). Detection of lead in wine is
usually focused on the determination of the total lead Pb
2
. In
wine, the structurally complex pectic polysaccharide rhamnoga-
lacturonan II (RG-II), an anionic biomolecule released from the
grape berry cell walls during vinication, is present. RG-II is
mainly released as a dimer (dRG-II), which can form coordina-
tion complexes with specic divalent cations as lead.
Szpunar et al. (1998) published a size-exclusion LC-ICP/
MS (SEC-ICP/MS) method for analysis of lead biomolecular
complexes in wine. By this approach, the lead distribution among
biomolecular compounds was determined within 30 min; lead
bound to biomolecules was quantied by comparing the
chromatographic peak area with that of a signal obtained with
ow-injection ICP/MS analysis. The bound lead fraction was
calculated as the ratio of the peak area of the lead signal obtained
by injection of the diluted wine sample on the size-exclusion
column (isocratic elution with a Tris-HCl buffer pH 7.2), to the
peak area of a signal of the same sample obtained by ow-
injection ICP/MS using the chromatographic mobile phase as the
carrier stream (the eluate from the column was introduced
directly into the ICP/MS). Three major lead isotopes
206
Pb,
207
Pb, and
208
Pb (abundance 24.1, 22.1, and 2.4%, respectively)
were monitored. Lead was found to be combined with one major
biomolecular species inwine of ca. 10 kDa and one to three minor
compounds. Between 40%and 95%of lead was found to formthe
complex with the dimer of RG-II, whose average concentration
ranged between 20 and 100 mg/L in red and white wine,
respectively.
In nineties, several applications of ICP/MS for the multi-
element detection in wine were proposed: more than 60 elements
in wine were detected in a single run within 5 min (Williams,
Jarvis, & Willis, 1992); another paper reported analysis of the
major wine elements Mg, Na, K, Ca, and Fe, the minor elements
Al, Cr, Mn, Cu, Zn, Se, Sr, Br, Rh, and trace elements Li, Ti, Ni,
As, I, Ba, Pb, Sc, V, Co, Y, Zr, Mo, Sn, Cs, Ga, Nb, Pd, Cd, Sb, Hf,
W, Hg, Tl, Th, and U (Perez-Jordan et al., 1998).
In 1999, by application of the double-focusing sector eld
ICP/MS (ICP/SMS) the inorganic elements in alcoholic
beverages were detected with fg/mL LODs for high m/z value
elements (e.g., actinide) (Rodushkin, O
dman, & Appelblad,

1999). Measures of the Pb isotopic ratio showed better precision
than that obtained by quadrupole ICP/(Q)MS, in good accor-
dance with as obtained by thermal ionization mass spectrometry
(TIMS).
A year after, the Pb isotopic ratios were determined in 20
different wines from all around the world by using the axial
inductively coupled plasma-time of ight mass spectrometry
(ICP/TOF-MS) (Tian et al., 2000). Data were compared with
results obtained by multicollector ICP/(MC)MS and quadrupole
ICP/(Q)MS instruments. Pb levels between 5 and 150 mg/L were
found in samples, and the ratios
206
Pb/
207
Pb,
208
Pb/
206
Pb, and
206
Pb/
204
Pb were calculated. Ratios
206
Pb/
207
Pb and
208
Pb/
206
Pb
by ICP/TOF-MS and ICP/(MC)MS analysis were similar,
whereas not accordance of the
206
Pb/
204
Pb ratio was observed
between two techniques. The data obtained by ICP/(Q)MS were
not in accordance with those obtained by the two other
techniques.
Almeida and Vasconcelos applied ICP/MS to study the Pb
contamination in Port wine in samples since vintage 1935 until
vintage 1984 (Almeida & Vasconcelos, 1999). By performing
the study on 24 different Port wines Pb contents between 47 and
80 mg/L were found, and a signicant correlation between the
total lead content and the
208
Pb/
206
Pb ratio (with a sensible
decrease in the older wines) was observed.
VIII. MASS SPECTROMETRY FOR DETERMINING
ORIGIN OF WINE AND ILLEGAL ADDITIONS
Nowadays the increasing interest about the foodstuffs prove-
nance is due to the request of consumer of additional warranties
of quality, authenticity, and typicality. The Controlled Origin
Denomination (Appelation dOrigine Controlee, A.O.C.) of
wines represents the unambiguous reference to the geographical
origin and is an important indicator of quality. Some analytical
parameters in wine are detected to evaluate the authenticity of
product.
The
87
Sr/
86
Sr isotope ratio is related to the geographical
origin since it is characteristic of the soil where the vine is grown.
This ratiowas demonstrated not to change during the uptake of an
element from soil by a plant, and in plants it reects the bedrock,
soil, and soil water of growth. In 2002 was proposed a ICP sector
eld multicollector MS (ICP/SF-MC-MS) method for the
determination of the Sr isotope ratios in wine, by using a sector
eld mass analyzer with a multicollector having the precision
required for the discrimination of different wines (<0.005%)
(Barbaste et al., 2002). Because of the
87
Sr measurement by MSis
subject to interference from
87
Rb, a preliminary chromatographic
separation was needed. As a matter of fact, the proximity of the
masses of the two isotopes (86.908882 and 86.909186 for
87
Sr
and
87
Rb, respectively) needs a resolution (m/Dm) of ca. 3 10
5
to discriminate one to another, not achievable by ICP/SF-MS.
Experimental conditions of ICP/SF-MC-MS system were
radio frequency power (Rf) 1,250 W and plasma gas ow rate
0.7 L/min. Precision 100 times better than by ICP/(Q)MS, and
10 times better than by ICP/SF-MS without multicollector was
reported. The study was performed on 11 wines from different
origin and revealed difference in the
87
Sr/
86
Sr ratio between
wines produced on basaltic, mixed, and granitic soils.
&
The same year, another ICP/MS study on the trace and
ultratrace elements contents of 153 wines from the Canary
Islands was published (Perez-Trujillo, Barbaste, & Medina,
2002). Concentration of 20 elements was measured, and the
levels of Te, Pt, Co, Zn, Re, Ti, Sb, Au, Rb were observed to be
linked to the vintage, types of wines, and region of origin.
By ICP/MS, Taylor et al. studied differences in the element
composition of Canadian wines from different region by
performing a simultaneous determination of 34 trace elements
in wine (Li, Be, Mg, Al, P, Cl, Ca, Ti, V, Mn, Fe, Co, Ni, Cu, Zn,
As, Se, Br, Rb, Sr, Mo, Ag, Cd, Sb, I, Cs, Ba, La, Ce, Tl, Pb, Bi,
Th, and U) (Taylor, Longerich, & Greenough, 2003). The
Authors identied several elements useful to distinguish products
from different regions of provenance. Among them, 10 elements
allowed to discriminate two different regions with accuracy of
100%. In particular Sr was conrmed to be a good indicator of
provenance. Because of the presence of ethanol, analysis was
performed using a high Rf (1,500 W) necessary to maintain a
stable plasma (plasma gas ow rate of 14 L/min).
Isotopic techniques in the wine analysis were introduced
nearly 20 years ago from the French Government for detecting
the sucrose enrichment of wines. The site-specic natural iso-
topic fractionation-nuclear magnetic resonance (SNIF-NMR)
was adopted by the O.I.V. as an international method for the wine
analysis (O.I.V., 1990). The European Community Regulation
No. 2676/90 (EC Regulation no. 2676/90, 1990) adopted this
method and also referred to the desirability of isotopic ratio mass
spectrometry (IRMS) of the
13
C/
12
C ratio of ethanol in wine as
additional determination. The European Community proposed
the institution of a data-bank of reference values for wines of the
European wine producing regions (EC Regulation no. 2048/89,
1989).
The
13
C/
12
Cisotope ratio of ethanol and the
18
O/
16
Oratio of
water reveal adulterations such as the cane sugar addition and the
watering of wine (Guillou et al., 2001). During the water
evaporation from the oceans, and with evapo-transpiration of
water from plants, a depletion in heavy isotopes in the vapor
occurs compared with the liquid state. As a consequence, higher
isotope ratios are observed in water from plants with respect to
the ground water. Most of the plants, such as grape and sugar-
beet, x the atmospheric CO
2
by a carboxylation of ribulose
l,5-diphosphate forming two phosphoglycerate molecules. This
process is accompanied by an important depletion in
13
Ccontent.
A second category of plants such as cane and maize, x CO
2
by
carboxylation of phosphoenolpyruvate leading to the oxalacetic
acid, almost without isotope fractionation with respect to the
13
C
content of atmospheric CO
2
. As a consequence, products from
the latter plants have higher
13
Ccontents than analogous products
from grape. The
13
C/
12
C IRMS measurements are carried out on
the CO
2
gas formed by combustion of the sample, the
18
O/
16
O
ratio is determined on a CO
2
gas on the isotopic equilibration
with the water to be analyzed. Intensity of the CO
2
isotopomers
ion currents at m/z 44, 45, and 46 are measured for both the
sample and reference. The isotopic ratio is calculated with
respect to the reference international standard as deviation per
mil (d%):
dX% R
sample
R
reference
=R
reference
1000
where X
2
H/
1
H,
13
C/
12
C,
18
O/
16
O; Risotopic ratios of
sample and the reference international standard.
Martin et al. (1999) reported that by determining the natural
abundance isotopic ratios of H, O, and C, fromwater and ethanol
extracted from the wine of the Bordeaux region in France, it is
possible to distinguish between sub-regions. Authors observed
that SNIF-NMR and IRMS parameters may be subjected to
climatic inuences and are more efcient for characterizing the
year of production of a given appellation than for distinguishing
different appellations.
Ogrinc et al. (2001) investigated the authenticity and
geographical origin of Slovenian wines by combination of IRMS
and SNIF-NMR. Atotal of 102 grape samples were collected in
different wine-growing regions in 1996, 1997, and 1998. The
isotopic ratios determined to discriminate between coastal and
continental regions were
2
H/
1
H of methylene group of ethanol,
d
13
C, and d
18
O values. Authors evidenced that the d
18
O values
were modied by the meteorological events during grape
ripening and harvest.
The year after, authenticity of tequila samples was studied
by performing the headspace SPME sampling with a PDMS/
DVB 65 mm ber and on-line capillary GC/IRMS analysis
(Aguilar-Cisneros et al., 2002). The d
13
C standard values were
similar for 100% agave and mixed tequila samples, whereas the
d
18
O data differed slightly within these categories.
In recent years, D() malic acid has been found by German
authorities in some imported Italian wines. As a result, the
naturalness of products was suspected. While the presence of
large amounts of D() malic acid (some hundreds mg/L) leaves
no doubts about the illegal addition of racemic malic acid, the
occurrence of some tens mg/Lof this acid cannot be always taken
as evidence of illegal acidication. Also if the presence of D()
malic acid has been revealed in musts and wines, as well as in
synthetic media fermented by yeasts, and the formation of D()
malic acid is believed to be the result of side reactions during the
alcoholic fermentation, the assumption that only the L() isomer
occurs in grapes and natural musts and wines has been worldwide
accepted.
In 1995, the occurrence of D() malic acid in 70 Italian
wines was investigated by performing a SPE sample preparation,
derivatization of malic acid into methyl ester and GC/MS
analysis (Gaetano et al., 1995). Analyses were carried out by a
g-cyclodextrin-triuoracetyl chiral column using glutaric acid as
internal standard. For quantitative analysis, SIM of signals at
m/z 100 and 103 (the base peak of mass spectra), relative to the
internal standard and dimethyl malate, respectively, were
registered. An occurrence of D() malic acid in 60% of the
samples inspected was revealed in amounts between 4 and
100 mg/L. The study demonstrated the presence of low amounts
of D() malic acid in wines is not an evidence of illegal
acidication of watered down products.
IX. CONCLUSIONS
Controls in food industry are fundamental to protect the
consumer health. For high quality products, warranty of origin
and identity is required and analytical control is very important to
&
prevent frauds. Common quality control of wine is performed by
spectrophotometric methods, with determination of anthocyanic
and polyphenolic indexes, organic acids, acetaldehyde, after
performing colorimetric or enzymatic reactions; alkaline ele-
ments and heavy metals are determined by atomic spectroscopy;
sugars, glycerine, ethanol, organic acids by LC with UV-Vis and
RI detection; pesticides and volatile compounds by GC using
ECD or FID detector.
In the recent years, a very important contribute of mass
spectrometry has been observed: liquid- and gas-phase methods
for determination of parameters related to illegal additions to the
wine, heavy metals, pesticide residues, compounds released from
microbiological and technological processes, have been pro-
posed. The LC-MS/MS analysis of OTA in wine allows to
conrm the toxin presence without performing time and solvent
consuming sample derivatization procedures, or the use of
expensive IACs columns. The ICP/MS multielement analysis of
wine is a useful tool for the product characterization aimed to
guarantee authenticity, in particular for Appelation dOrigine
Controlee wines. IRMS, coupled with NMR, reveals the wine
watering and sugar addition, and is used to determine the product
origin and traceability. Moreover, the high sensitivity of mass
spectrometry make this technique as an advantageous GC
detector with respect to the traditional radioactive-source-
containing ECDused for pesticides analysis. This is an advantage
also due to the severe regulations and laborious control
procedures required for radioactive sources in laboratory.
In the last 25 years, mass spectrometry provided important
contribution to the grape and wine chemistry knowledge. By the
highversatility and sensitivity of LC/MSand MS/MStechniques,
nowadays present in many laboratories of enological chemistry,
newapproaches to the research are being developed, and the mass
spectrometry role is bounded to increase when new mass spectra
databases will be available.
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&
Riccardo Flamini graduated in Organic Chemistry at Padua University in 1994. After
earning his degree, he specialized in Chemical Synthesis at the Politecnico of Milan. Since
1996 he is a Research Scientist in Organic Chemistry at CRA, Viticulture Research Institute
as Chief of Chemistry Laboratory Staff. Since 2001 he is a Professor by contract of Quality
Control of Wine at the Agricultural Faculty of Padua University.
Annarita Panighel graduated in Chemistry at Padua University. She works as a Research
Scientist at the Chemistry Laboratory of CRA, Viticulture Research Institute.
&

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MASS SPECTROMETRY IN GRAPE AND WINE CHEMISTRY.

PART II: THE CONSUMER PROTECTION

*Correspondence to: Riccardo Flamini, CRA, Istituto Sperimentale

ion formed for vinclozolin (MW

ion was due to

were observed at 20 V. For both APCI

ion. By performing APCI in negative mode [MCONHCH

ions are formed for most compounds, and the [MH]

ion of EC) with 1:1 ratio

species of glycolaldehyde), m/z 300 (M

species of o-chlorobenzaldehyde), and m/z 448 (M

(de Revel & Bertrand, 1994).

species were observed as

fragment ion (which does not occur in the

species (not observed for TCA). Authors

ion at m/z 404, and

at m/z 404 and

at m/z 358and [MH H

species. By monitoring the signal at

(the species that can interfere

) was observed. Since wines

dman, & Appelblad,

dman F, AppelbladPK. 1999. Multielement determination and

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