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 consumer 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 significantly 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 commonly used in GC analysis of pesticides. Analysis of ochratoxin A (OTA) in wine by LC/MS and multiple mass spectrometry (MS/MS) permits to confirm the toxin presence without the use of expensive immunoaffinity 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 25:741 774, 2006 Keywords: wine pesticides; wine defects; ethyl carbamate; ochratoxin A; mass spectrometry; lead in wine; illegal additions to the wine I. INTRODUCTION The first 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 identified, so as aroma compounds from the grape (Rapp & Knipser, 1979; Rapp, Knipser, & Engel, *Correspondence to: Riccardo Flamini, CRA, Istituto Sperimentale per la Viticoltura, Viale XXVIII Aprile 26, I-31015 Conegliano (TV), Italy. E-mail: riccardo.flamini@ispervit.it 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 floral aroma classified as aromatic varieties (e.g. Muscats, Malvasie, Riesling, Müller- Thurgau, Gewürztraminer) are characterized from a high monoterpenol content, which increases during the latest stages of ripening (Di Stefano, 1996). A number of monoterpenols has been identified, 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 compounds 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 floral fragrances (structures 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 benefits of a moderate wine consumption. Liquid chromatography-mass spectrometry (LC/MS) permitted to improve the polyphenols characterization (anthocyanins, flavonols, tannins and proanthocyanidins, hydroxycinnamic and hydroxycinnamoyltartaric acids), and to understand several mechanisms involved in the color stability of wine (Flamini, 2003). In the 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 finalized to improve organoleptic characteristics of wine: alcoholic Mass Spectrometry Reviews, 2006, 25, 741 774 # 2006 by Wiley Periodicals, Inc.
& FLAMINI AND PANIGHEL FIGURE 1. The principal monoterpenols that characterize with floral aroma the aromatic grape varieties such as Muscats, Malvasie, Riesling, Müller-Thurgau, and Gewürztraminer. 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. fermentation is promoted by inoculum of 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 flavor and fragrance of product (Flamini, 2003). To guarantee the quality of product, all these steps have to be monitored and verified. 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 certificates are often required, in particular with regard to the presence of pesticides, heavy metals, ethyl carbamate and toxins, for which legal limits are often defined. To prevent frauds and to confirm the product identity, accordance between the real product characteristics and the producer declarations (e.g., variety, geographic origin, quality, vintage) has to be verified. Some maximum limits of grape and wine contaminants are fixed by national and community regulations, and are reported in Table 1. Activity of researchers and organisms of control is devoted to develop new methods to verify the product origin (Ogrinc et al., 2001), to detect illegal additions and adulteration such as sugarbeet, 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 defined. 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, & Calò, 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). 742 Mass Spectrometry Reviews DOI 10.1002/mas
GRAPE AND WINE CHEMISTRY & FIGURE 2. Norisoprenoid compounds that characterize the grape and wine aroma with spice and floral 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. GC/MS and LC/MS have also been applied to develop methods for the legal parameters control finalized 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 (Zöllner 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 3. Sulphur compounds and methoxypyrazines identified 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. Mass Spectrometry Reviews DOI 10.1002/mas 743
& FLAMINI AND PANIGHEL TABLE 1. Maximum limits of some grape and wine contaminants fixed 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. 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 Community (EC) Directives (also adopted by the Italian Legislation) stating maximum residue limits (MRLs) tolerated for each food commodity (Official Gazette of the Italian Republic, 1994, 1995). Wine and grape are included among these commodities, in particular in wine the procymidone MRL has been defined to 0.5 mg/l, such as for cyprodinil and fludioxomil, 0.1 mg/l for myclobutanil, and 2 mg/l for iprodione; MRLs in grape have been fixed to 10 mg/kg for folpet, 5 mg/kg for vinclozolin, and 3 mg/kg for carbaryl (Official 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 final stage of vegetation to prevent grape s attack, which may occurs shortly before the harvest. Among them, vinclozolin and iprodione are currently employed in Italy (Cabras et al., 1983; Matisová et al., 1996). These fungicides show reduced toxicity, but 3,5-dichloroaniline, the probable common final 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 fixed, 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 vinification process lowers the level of pesticides, their contents in wines are significantly lower than in grapes. As a consequence, methods to detect pesticide residues must be very effective and sensitive (Wong & Halverson, 1999). One of the first 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 quantification (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 744 Mass Spectrometry Reviews DOI 10.1002/mas
GRAPE AND WINE CHEMISTRY & FIGURE 4. The principal pesticides used in viticulture: procymidone 37; cyprodinil 38; fludioxonil 39; myclobutanil 40; iprodione 41; folpet 42; vinclozolin 43. carbon stationary phase was performed (Matisová et al., 1996). Analytes were recovered with toluene and analyses performed by GC/MS. Thermal stability, chemical resistance, and stability over a wide ph range 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/N ¼ 3). By performing analysis of a vinclozolin 0.01 mg/l standard solution in MID mode, unambiguous compound identification was obtained, by ITD sensitivity of method for iprodione resulted significantly 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 (Kakalíková, Matisová, & Leško, 1996). Recoveries greater than 92% were reported for metalaxyl standard solutions at concentration 3 100 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, fludioxonil, pyrimethanil, tebuconazole, azoxystrobin, fluazinam, kresoxim-methyl, mepanipyrim, and tetraconazole 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/l for 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 coefficients 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, trifluralin 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 profiling 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). SPME was performed by Mass Spectrometry Reviews DOI 10.1002/mas 745
& FLAMINI AND PANIGHEL TABLE 2. Wine pesticides and corresponding m/z species monitored in SIM mode by automated reverse-phase solidphase-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. a silica fiber coated with polydimethylsiloxane (PDMS), performing extraction under stirring for 30 min and immerging the fiber 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 0.8 5 mg/l levels, procymidone was found in 83% of the positive samples. In 1999, Wong and Halverson performed a SPE and 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 efficiency of 38 compounds, except for allethrin and demeton S-methyl. In 2002, Natangelo et al. compared performances of a quadrupole mass filter (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 herbicide), 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 FIGURE 5. Structures of some herbicides used in viticulture: acetochlor (44) and propanil (45). 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) fiber (65 mm thickness) by direct immersion of the fiber in the sample under stirring and after NaCl addition. Analytes were desorbed by exposing the fiber in the GC injector port 15 min at 2508C. Evaluation of the GC/MS- MS method LODs was carried out by selecting the most abundant daughter ion generated by collisionally induced dissociation (CID) experiment; LODs calculated with S/N ¼ 3 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/l resulted greater than 70% for 116 and 124 analytes in red and white wines, respectively. Identification of compound was confirmed by retention time of target ion and on three qualifier-totarget 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 qualifier 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 746 Mass Spectrometry Reviews DOI 10.1002/mas
GRAPE AND WINE CHEMISTRY & TABLE 3. Comparative data of precision (RSD for three replicate analyses) and limits of detection of pesticides precision (RDS, n=3) LOD (µg/l) 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 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 fiber). Numbers corresponding to structures in Figures 4, 5, and 12 are reported. 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 fibers. 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 verified by SBSE and liquid desorption Atmospheric-Pressure-Chemical- Ionization negative mode (SBSE-LD-LC/APCI-MS) analysis. By liquid liquid 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/MS analysis in SCAN mode, limits of quantification 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 SIM mode, 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 compounds are reported in Figure 7. Vinclozolin and procymidone are easily identified, 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, quantification was performed on (3,5- dichlorophenyl)hydantoin. White wines and sparkling wines from different 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 verified 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 200 350, with a fragmentor voltage 70 V and 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 first application of SBSE with liquid desorption; results obtained by SBSE-TD-GC/MS and SBSE- LD-LC/MS were comparable. The (M þ CH 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 electrospray 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 identified 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 LOD down to low ng/l levels. 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, MBT and DBT are used as heat and light stabilizers for poly(vinyl chloride) (PVC) materials, DBT is 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; Mass Spectrometry Reviews DOI 10.1002/mas 747
& FLAMINI AND PANIGHEL FIGURE 6. 748 Mass Spectrometry Reviews DOI 10.1002/mas
GRAPE AND WINE CHEMISTRY & 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 fiber was used, by performing headspace extraction at 408C for 20 30 min. To perform GC analysis, compounds were thermally desorbed from the fiber 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 influenced by the matrix. In 14% of the 43 table and 14 Port wines analyzed, 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 DBT residues 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 vinification 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 completely. At the end of fermentation, phthalimide, a hydrolysis product that did not show to 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 liquid liquid 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 directinjection analysis using 1,4-dioxan as internal standard; Zimmer et al. performed liquid extraction of sample with 1,1,2- trichlorotrifluoroethane. In 1995, a GC/MS method for detection of both methylisothiocyanate and allylisothiocyanate in wine was 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 classified 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 law fixed 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 perform quantitative 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 fiber 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. HPLC is 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 fluorescence 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. Fenández et al. described a method for analysis of carbamate residues in grape by matrix solid-phase dispersion (MSPD) extraction and LC/MS analysis (Fernández, Picó, & Mañes, 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). Mass Spectrometry Reviews DOI 10.1002/mas 749
& FLAMINI AND PANIGHEL TABLE 4. The 153 wine pesticides analyzed by SPE and GC/MS-SIM with the corresponding target and qualifier 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 ) 750 Mass Spectrometry Reviews DOI 10.1002/mas
GRAPE AND WINE CHEMISTRY & TABLE 4. (Continued ) 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) 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 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. 1151 1153, Copyright 2003, with permission from American Chemical Society. 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, pirimicarb, 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 chromatographic 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 [M þ Na] þ,[mþ H] þ, and [M þ H CH 3 NCO] þ 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 [M þ H] þ showed little fragmentation, and the sensitivity was the highest for all the compounds. N-methylcarbamate insecticides are labile compounds, 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 [M þ H] þ and [M þ Na] þ adducts, whereas the APCI positive mode only produces the [M þ H] þ ion. By performing APCI in negative mode [M CONHCH 3 ] ions are formed for most compounds, and the [M H] 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 confirmation of carbamate with higher levels. The principal species and fragment ions formed from each carbamate recorded by performing APCI analysis in both positive- and negativemode, and by ESI analysis in positive mode, are reported in Table 5. Mass Spectrometry Reviews DOI 10.1002/mas 751
& FLAMINI AND PANIGHEL Carbofuran was found in a grape sample in concentration 0.3 0.5 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 LCelectrospray ionization (C 18 -LC/ESI-MS) (Wu et al., 2002). Six phenylurea pesticides diuron, fluometuron, linuron, monuron, neburon, siduron, and six carbamates barban, carbaryl, chlorpropham, methiocarb, promecarb, propham, were analyzed. Structures of compounds are reported in Figure 13, carbaryl and methiocarb in Figure 12 (structures 52 and 55, respectively). Intube 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 efficiency 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 FIGURE 7. Fragmentation spectra (EI) by stir-bar-sorptive-extraction and thermal-desorption-gc/ms analysis (SBSE-TD-GC/MS) of dicarboximide fungicides vinclozolin, iprodione, and procymidone (structures 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). 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). A Canadian 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 quantification 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 fortified wines) the U.S. Food and Drug Administration published recommendations to minimize EC in wine (Butzke & Bisson, 1997). For detection of EC, a SPE method with elution by methylene chloride and GC/MS-SIM mode analysis has been adopted by the AOAC International (Association of Official Analytical Chemists) for alcoholic beverages (AOAC, 1995). Another GC/MS method for the wine EC analysis by the use of 752 Mass Spectrometry Reviews DOI 10.1002/mas
GRAPE AND WINE CHEMISTRY & 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-barsorptive-extraction applied to the determination of dicarboximide fungicides in wine. p. 125, 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. FIGURE 9. Contaminants used for production of biocides and as polymer stabilizers found in some wines: monobutyltin (46), dibutyltin (47), and tributyltin (48). X ¼ Cl; OH; EHMA (2-ethylhexylmercaptoacetate); 2-MET (2-mercaptoethyltallate). The ethanol content was adjusted at 10%, then samples were saturated with NaCl before to perform methylene chloride liquid liquid extraction. Before analysis extracts were concentrated 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 fortified 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 SIM mode 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 demonstrated (Pound, 1967), and a study on relative rates of carbamates Mass Spectrometry Reviews DOI 10.1002/mas 753