Ethyl Carbamate in Wine

TABLE 8.3. Elution Program for LC/ESI-MS Analysis of Tyramine, Tryptamine, 2-Phenylethylamine, Histamine, Cadaverine, Pntrescine, Spermidine, and Spermine in Wine  

For most amines, the most abundant ions are formed by loss of an ammonia group and for spermine and heptylamine the principal product ion comes from the loss of 1,3-propyl diamine and formation of an adduct with water, respectively. The LODs calculated in a synthetic wine were in the range between 0.5 and 40pg/L. The higher values resulted for phenylethylamine, cadaverine, putrescine, and spermine (10-40 pg/L). 

TABLE 8.4. Data Acquisition Parameters of MRM Transitions Used for LC/ESI-MS Analysis of Biogenic Amines in Wine  

Compound Retention Time (min) Molecular Mass (MW) Precursor Ion [M+H]+ MRM Transition (m/z) Fragmentation Amplitude V Fragmentation Width (m/z) 

EC (15 xg/L or less in table wines 60gg/L or less in fortified wines), the U.S. Food and Drug Administration published recommendations to minimize EC in wine (Butzke and Bisson, 1997). 

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Fauhl, C. and Wittkowski, R. (1992). Determination of ethyl carbamate in wine by GC-SIM-MS after continous extraction with diethyl ether,/. High. Res. Chromatogr., 15,203-205. 

Jagerdeo, E., Dugar, S., Foster, G.D., and Schenck, H. (2002). Analysis of ethyl carbamate in wines using solid-phase extraction and multidimensional gas chromatography/mass spectrometry,/ Agric. Food. Chem., 50(21), 5797-5802. 

Whiton, R.S. and Zoecklein, B.W. (2002). Determination of ethyl carbamate in wine by solid-phase microextraction and gas chromatography/mass spectrometry, Am. J. Enol. Vitic., 53(1), 60-63. 

A great deal of research examining the origins of ethyl carbamate in wine and brandies is described in the literature. Bertrand et al. (1991) concluded that ethyl carbamate concentrations in wine are linked to grape variety, as well as to excessive nitrogen fertilization in the vineyards, but these factors are not highly significant. 

Storage conditions, especially temperature, during distribution and sale may be a decisive factor affecting increases in ethyl carbamate in wine. 

Ethyl carbamate in wine is formed (mostly at the end of fermentation) from urea. The intermediates of its degradation are probably cyanates and cyanic acid (HO-C=N), also known as hydrogen cyanate, which may isomerise to isocyanic acid (H-N=C=0). Iso-cyanic acid can also arise by protonation of the cyanate anion and nucleophilic addition of ethanol to isocyanic acid yields ethyl carbamate. Isocyanic acid also reacts with other nucleophilic reagents, such as water (with formation of ammonia and carbon dioxide), thiols and amino groups of proteins. By catalysis with ornithinecar-bamoyl transferase, citrulline is transformed into ornithine and carbamoyl phosphate, the ethanolysis of which yields ethyl carbamate (Figure 12.39). 

Ethyl Carbamate. In November 1985, the Canadian Government indicated that it had detected ethyl carbamate [51-79-6] (urethane), a suspected carcinogen, in some wines and distilled spirits. Since that time, the U.S. distilled spirits industry has mounted a serious effort to monitor and reduce the amount of ethyl carbamate (EC) in its products. In December 1985, the Canadian Government set limits of 150 ppb in distilled spirits and 400 ppb in fmit brandies, cordials, and Hqueurs. The FDA accepted a plan in 1987 from the Distilled Spirits Council of the United States (DISCUS) to reduce ethyl carbamate in whiskey to 125 ppb or less, beginning with all new production in January 1989. 

Perestrelo, R., Petronilho, S., Camara, J. S., and Rocha, S. M. (2010). Comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry combined with solid phase microextraction as a powerful tool for quantification of ethyl carbamate in fortified wines. The case study of Madeira wine. ]. Chromatogr. A 1217, 3441-3445. 

These studies demonstrate that wine LAB may contribute to ethyl carbamate formation. In the United States, there is a voluntary concentration limit of 15 ng/g for ethyl carbamate in table wines (Canas et al., 1994). One suggested way to achieve this goal is the development of non-arginine-degrading O. oeni strains for the induction of MLF (Mira de Orduna et al., 2001). 

Ethoxyhexadiene 268 Ethyl Carbamate 265 Ethyl Phenols in wines 143... 

Urea is assayed using urease, and ammonium carbonate is formed. Wine contains less than 1 mg/1, and it is certainly of microbial origin. Urea is significant in winemaking as it may be a precursor of ethyl carbamate. In spite of certain reservations, L Office International de la Vigne et du Vin (OIV) authorize the treatment of wines by active urease in an acid medium. This enzyme is extracted from Lactobacillus fermentum. The objective is to reduce excessive urea concentrations in wine to avoid the formation of ethyl carbamate as the wine ages. 

In brandy, ethyl carbamate may also be formed from hydrocyanic acid. Bertsch (1992) followed the development over a two-hour period of hydrocyanic acid and ethyl carbamate in a distillate made from Baco Noir wine in an experimental still. He observed a negative correlation between these two compounds, which is explained by the following reaction sequence, suggested by Bauman and Zim-merli (1988) ... 

Concentrations of ethyl carbamate in bread, yoghurt, soy sauce, beer or wine generally reach tens of (xg/kg. Substantially higher amounts of ethyl carbamate can be encountered in alcohohc beverages, especially in spirits made from stone fruits, which in rare cases can contain thousands of xg/l of ethyl carbamate. The output from the latest EU study is presented in Table 12.20. 

At present, there is no international standard for the maximum allowable level of ethyl carbamate in foods. However, some countries, such as Canada, Korea and some member states of the EU (such as France, Germany and Czech RepubHc) have estabhshed maximum levels of ethyl carbamate in alcohoHc beverages. In the Czech Republic, for example, the limit for the content of ethyl carbamate in fruit spirits is 0.4 mg/1) and for other spirits and wines the maximum level of ethyl carbamate is lower by one order of magnitude. 

It rapidly became apparent that the reaction of ethyl pyrocarbonate in wine was more complex than indicated by the reaction shown above. Ethyl alcohol and carbon dioxide were certainly the main degradation by-products, but small quantities of ethyl carbonate were also formed, and its fruit aroma was perceptible above a certain threshold. Most importantly, ethyl pyrocarbonate is a highly reactive molecnle and combines with certain substances in wine (organic acids, polyphenols, and nitrogen-based componnds) to produce urethanes, e.g. ethyl carbamate, which is toxic and carcinogenic. Quantities never exceeded 2-4 mg/1, significantly below the ofQcial threshold of 30 mg/1 in Canada. However, this risk was sufficient for the product to be completely abandoned. 

Urethane [51-79-6] (ethyl carbamate) occurs as a natural by-product in fermented products such as wine, Hquors, yogurt, beer, bread, oHves, cheeses, and soy sauces. Whereas urethane has a known cancer etiology in experimental animals, no such relationship has yet been proven in humans (108,109). Alcohol may act by blocking the metaboHsm of urethane, and thus exert a protective effect in humans consuming alcohoHc beverages (110). 

Unfortunately, uncontrolled MLF also presents a risk of wine spoilage by compounds that can produce off-flavours (including acetic acid, volatile phenols and mousiness) or that may be hazardous to human health (such as ethyl carbamate and biogenic amines). The most important aspects of the development of LAB and MLF in wines are dealt with in this chapter. 

The metabolism of amino acids does not affect the taste, but is problematic at a toxicological level, because it increases the concentrations of biogenic amines and ethyl carbamate precursors in wine. 

Also, in this case, research results indicate the need for caution in the selection of starter cultures for MLF in wine, since citrulline formation from arginine degradation could result in ethyl carbamate production, even at normal temperatures, during prolonged storage. In addition, spontaneous MLF by undeflned strains should be avoided, as this may lead to formation of ethyl carbamate precursors (Liu etal. 1994). 

Free amino acids in musts are of paramount importance, since they constitute a source of nitrogen for yeasts in alcoholic fermentation, for lactic acid bacteria in malolactic fermentation and can also be a source of aromatic compounds. In certain cases, some amino acids can produce undesirable compounds in wines, such as ethyl carbamate, biogenic amines, ochratoxin A (from 2-phenylalanine) and 3-carbolines (from tryptophane) (Herraiz and Ough 1993 Herraiz et al. 1993). 

Burroughs, L.F. and Sparks, A.H. 1973. Sulphite-binding power of wines and ciders. I. Equilibrium constants for the dissociation of carbonyl bisulphite compounds. J. Sci. Food Agric. 24, 187-198. Canas, B.J., Havery, D.C., Robinson, L.R., Sullivan, M.P., Joe, F.L., Jr., and Diachenko, G.W. 1989. Ethyl carbamate levels in selected fermented foods and beverages. J. Assoc. Off. Anal. Chem. Int. 72, 873-876. 

Monteiro, F.F., Trousdale, E.K., and Bisson, L.F. (1989). Ethyl carbamate formation in wine Use of radioactively labeled precursors to demonstrate the involvement of urea, Am. J. Enol. Vitic., 40,1-8. 

These authors also observed that certain winemaking techniques, such as high temperatures especially during the final maceration, and not removing grape stems, cause increases in the ethyl carbamate content of the wine. Yeasts synthesize ethyl carbamate and also contain a precursor of this compound. The role of lactic bacteria has... 

According to Bertrand (1993), the impact of aging on the ethyl carbamate content of wine varies from one vineyard region to another. Concentrations in Champagne, aged on yeast lees, increase more than those in Bordeaux wines. 

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