- Research Notes -

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3a,6a-Dimethylglycoluril, the Product of the Interaction of Urea and Diacetyl, as a Source of Post-bottling Ethyl Carbamate in Wines
by
C.J. Muller  and  K.C. Fugelsang

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cati publication #960502
© copyright May 1996, all rights reserved

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INTRODUCTION

The thorough investigation on the origins of ethyl carbamate in wines by Ingledew, et al. (4), and by Ough (8), Bisson and coworkers at U.C. Davis (5) among others, identified urea, and arginine and citrulline, as the primary sources. Suggestions for control, including the use of acid urease (2), have allowed the wine industry to drastically reduce the levels of ethyl carbamate in table and dessert wines.

However, some wineries continue to experience the puzzling problem by which wines that have tested negative for both ethyl carbamate and urea (and its precursors arginine and citrulline) at bottling, present themselves with relatively high levels of ethyl carbamate, in some cases in excess of 100 µg/L (100 ppb), after those wines are subjected to accelerated 15 storage tests.

The often presented, and perhaps obvious explanation, is that action levels for ethyl carbamate are measured in parts-per- billion (ppb); whereas existing methods to detect urea, including the highly sensitive method by Nagel et al., (7), can detect urea only in the part-per-million (ppm) range. Thus, any residual urea, slightly below the present levels of detection (ca. 1 mg/L, [1 ppm]), might still be enough to generate ethyl carbamate at action levels upon storage by its interaction with the relatively large amounts of ethanol in wines (mass action law).

In the course of our investigations on other ethyl carbamate precursors, in which we have studied the interactions of urea and several carbonyl-containing moieties commonly present in wines, we synthesized 3a,6a-dimethylglycoluril (DMGU)(3), a bicyclic compound formed by the reaction of diacetyl and urea (Figure 1). We had postulated this compound as a source of ethyl carbamate in brandies. Initially, in testing its ability to undergo either acid-catalyzed hydrolysis to produce urea, that could then react with ethanol, or undergo ethanolysis directly, we found that this compound did undergo either process with some difficulty in model solutions. We have reexamined this system and are hereby reporting on the post-bottling formation of ethyl carbamate in model solutions, white and red wines, spiked with 3a,6a dimethylglycoluril.


MATERIALS AND METHODS

Model System

A model wine solution was prepared in distilled water by adding ethanol to 12% (v/v), and adjusting pH to 3.4 with tartaric acid (Aldrich). To this solution, DMGU, synthesized from urea (J.T. Baker) and diacetyl (Aldrich) as per the procedure by Gump and Muller (3), was added as per Table 1.

Wines

French Colombard and Barbera grapes grown on the California State University, Fresno, Farm were vinified using standard procedures at the university's experimental winery. Both wines were made without sulfur dioxide. 3a,6a-Dimethylglycoluril was added to the finished wines as per Table1.

Analytical

Characterization of the solutions and wines (ethanol, pH, TA, etc.) were carried out using standard methodology as described by Zoecklein, et al. (9). Determination of ethyl carbamate was performed by GC/MS using the method of Canas et al. (1) using n-propylcarbamate as internal standard. Potential ethyl carbamate was obtained after baking the samples at 71°C for 48 hrs, followed by GC-MS as above.


RESULTS AND DISCUSSION

Table 1 shows the results of addition of 3a,6a-dimethylglycol- uril to the model solution, white wine, and red wine with respect to both actual and potential ethyl carbamate production.

The higher levels of ethyl carbamate found in both white and red wines (including controls) as compared to the model wine, simply reflect ethyl carbamate formed during the vinification process, or formed from other precursors as postulated by us (6). As it is apparent from the ethyl carbamate analysis, (actual and potential), the model solution, and both white and red wines containing 3a,6a-dimethylglycoluril formed ethyl carbamate only when subjected to accelerated storage conditions (potential); whereas the actual was near the limit of detection for ethyl carbamate at 2 µg/L (2 ppb).

This behavior parallels what is observed in commercial wines. Surely, this compound is not the only source of post-bottling ethyl carbamate in wines; however, the fact that this compound exhibits this behavior, indicates that perhaps other urea-carbonyl adducts might be similarly implicated.


ACKNOWLEDGEMENTS

We wish to express our appreciation to Gordon Burns (ETS Laboratories, St. Helena, CA), and to Art Caputi (E & J Gallo, Modesto, CA), for ethyl carbamate analyses. Our thanks also to American Vineyard Foundation (AVF), and to the California Agricultural Technology Institute (CATI) for their support.


REFERENCES

1. Canas, B.J., Joe, Jr., F.L. Diachenko, G.W., Burns, G. Determination of ethyl carbamate in alcoholic beverages and soy sauce by gas chromatography with mass selective detection: Collaborative study. J. Assoc. Off. Anal. Chem. Internat. 77:1530-1536 (1994).

2. Fumuyiwa, O.O., Ough, C.S. Modification of acid urease activity by fluoride ions and malic acid in wines. Am. J. Enol. Vitic. 42: 79-80 (1991).

3. Gump, E.L., Muller, C.J. A potential source of ethyl carbamate in brandies. CSUF J. Natl. Sci. (Spring):28-29 (1991).

4. Ingledew, W.M., Magnus, C.A., Patterson, J.R. Yeast foods and ethyl carbamate formation in wine. Am. J. Enol. Vitic. 38:322-325 (1987).

5. Monteiro, F.F., Bisson, L.F. Amino acid utilization and urea formation during vinification fermentations. Am. J. Enol. Vitic. 42:199-208 (1991).

6. Muller, C. J., Fugelsang, K.C. Other ethyl carbamate precursors. In: Advances in Enology Seminar, CSUF Viticulture and Enology Research Center, March 18, 1995. CATI Publ. 950301.

7. Nagel, C.W., Weller, K.M. Colorimetric determination of urea in wine. Am. J. Enol. Vitic. 40:143-144 (1989).

8. Ough, C.S. Ethyl carbamate in fermented beverages and foods. I. Naturally occurring ethyl carbamate. J. Agr. Food Chem. 24:323-327 (1976).

9. Zoecklein, B.W., Fugelsang, K.C. Gump. B.H., Nury, F.S. Wine Analysis and Production. Chapman and Hall, NY. (1995).

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