330 J. Food Hyg. Soc. Japan Vol. 28, No. 5

Determination of Ethyl Carbamate in Sake by Alkylation and Gas Chromatography with a Flame Ionization Detector

(ReceivedMarch 11, 1987)

Kayoko KOBAYASHI, Masatake TOYODA and Yukio SAITO (NationalInstitute of HygienicSciences: 1-18-1, Kamiyoga, Setagaya-ku,Tokyo, Japan)

A derivatization method for the determination of ethyl carbamate in sake, a Japanese alcoholic beverage, was developed. The ethyl carbamate was extracted with chloroform, purified on a Florisil column and concentrated. The concentratedsample was alkylated with N,N-dimethyl-formamide dimethylacetal, purified on an Extrelut column and concentrated. The ethyl carbamate was determined by capillary gas chromatography with flame ionization detection (GC-FID). The alkylation procedure made it possibleto analyze ethyl carbamate in sake withoutinterference on the gas chromatograms. The recoveries of ethyl carbamate added at a level of 100pg/kg to sake were 73.70 on average. The chemicalstructure of the alkyl derivative of ethyl carbamate was confirmedby GC/MS. This alkylation procedure for determinationof trace amounts of ethyl carbamate in sake is suitablefor routine work. Key words: derivatization; ethyl carbamate; N,N dimethylformamidedimethylacetal; gas chromatographywith flame ionization detector (FID); gas chromatography/massspectro- metry

We have developed a new method for the Introduction determination of ethyl carbamate in sake by The ethyl carbamate was shown to be muta- taking advantage of Ough's approach.4) genic, teratogenic and carcinogenic.1),3) On the other hand, it is recognized that ethyl car- Experimental section bamate occurs naturally at low ppb levels in Samples. Samples were purchased from a fermented foods and alcoholic beverages as a liquor store in Tokyo. result of the reaction of alcohol and urea added Apparatus. A Kuderna-Danish (K-D) eva- to promote the fermentation.4,6) porator with a 5 ml concentrating tube and 5-ball In November, 1985, it was reported that re- Snyder reflux condenser, a 15 mm x 300 mm latively high levels of ethyl carbamate had been chromatography column, a Hewlett Packard gas detected in certain types of wines and other chromatograph (model 5890A) with a flame ioni- alcoholic beverages sold in Ontario. The Cana- zation detector (FID), a Shimadzu Chromatopac dian government laid down a gu deline on the C-R3A recorder and a Hewlett Packard gas levels of ethyl carbamate in table wines (30 ppb), chromatograph (model 5890) with a mass selec- fortified wines (100 ppb), distilled spirits (150 tive detector (model 5790) for ethyl carbamate ppb), fruit brandies and liqueurs (400 ppb).7) derivatives were employed. A JEOL GC/MS In order to determine the level of ethyl car- spectrometer (model JMS-DX 300) was used for bamate in sake, it is necessary to develop a ethyl carbamate. suitable analytical method. Many methods Reagents. Chloroform of the first grade was have been reported so far for the analysis of redistilled. Benzene, diethyl ether, methanol, ethyl carbamate in alcoholic beverages, but they ethyl acetate, acetonitrile and hexane were of are time-consuming.4) special grade. Ethyl carbamate was of reagent October 1987 Determination of Ethyl Carbamate 331 grade from Wako Pure chemical Industries Ltd. with a 5 ml bottom tube. Then 50 ml of aceto- Florisil (60 to 100 mesh) was obtained from nitrile was added, a 5-ball reflux column was Wako Pure Chemical Industries Ltd. N, N-Di- connected to the top of the K-D evaporator, and methylformamide dimethylacetal (N, N-DMF- the solution was concentrated to about 2 ml. DMA) for GC analysis was obtained from The Snyder column and evaporator were rinsed Tokyo Kasei Co. Ltd. Extrelut Refill pack with a small portion of acetonitrile and the (Cat. No. 11738) was purchased from Merck bottom tube was removed. The volume was Ltd. reduced to 1 ml by passing a gentle stream of N2 Procedures. Ethyl carbamate in sake was ex- over the sample in a water bath at 35C. tracted and purified according to the procedure Alkylation: One hundred and fifty micro- proposed by Ough4,5) with some modifications, liters of the solution was transferred to a tube, and determined by GC-FID after alkylation of to which 150l of N, N DMF-DMA was added. ethyl carbamate with N, N-DMF-DMA.10,15) After gentle shaking, the tube with a stopper on Extraction and purification: The pH of 200 ml top was warmed in a water bath at 50C for of the sample was adjusted to 6.5 by addition of 20 min to ensure smooth alkylation. The reac- 1 N sodium hydroxide, and 50 g of sodium chlo- tion mixture (200l) was placed on an Extrelut ride was dissolved in it. Then, the sample was column which was prepared by packing Extrelut transferred into a 500 ml separatory funnel, and (0.54 g) into a glass column (10 cm x 9 mm i.d.). extracted with 180 ml of chloroform for 30 min. After standing for 15 min, the column was The emulsified solution was transferred into two eluted with 10 ml of hexane. The eluate was 300 ml centrifugal tubes and centrifuged at 2, 600 concentrated to dryness and redissolved in 200 rpm for 12 min. The upper layer was reextract- pl of acetonitrile. The solution was subjected to ed twice with 150 ml each of chloroform. The analysis by GC or GC/MS. extracts were combined, dried over anhydrous Conditions of GC and GC/MS sodium sulfate (Na2SO4) and transferred to a (A) Gas-chromatographic conditions for ethyl K-D concentrating flask with a 5 ml bottom carbamate before alkylation tube. A 5-ball Snyder reflux column was con- Column: 0.25 mm x 30 m DURA BOND fused nected to the top of the evaporator. The sam- silica capillary column DB-WAX. Column tem- ple was concentrated to about 5 ml in a hot perature: programmed from 70C (2 min) at the water bath at 60C. The Snyder column and the rate of 10C/min to 110C, at the rate of 4C/ bottom tube were removed. Nine grams of Flori- min to 150C and at 20C/min to 200C (20 min). sil was put into 40 ml of chloroform, and mixed N2 carrier gas: 1.2 ml/min. Injection method: well to make a slurry. The slurry was then splitless (volume, 1: temperature, 200C). added to a 15 mm x 300 mm column filled with Detector temperature: 250C. chloroform. Two grams of anhydrous Na2SO4 (B) Gas-chromatographic conditions for alkyl was placed on the Florisil layer. The column derivative of ethyl carbamate was rinsed with 50 ml of chloroform, leaving a Column: 0.53 mm x 12 m Shimadzu capillary few milliliters covering the surface. The con- column Hi Cap CBP 20. Column temperature: centrate in the tube was applied to the column programmed from 70C (30 sec) to 160C at the and slowly drained down until the surface of the rate of 5C/min, then at 20C/min to 200C (15 solution fell to the top of the Na2SO4 layer. min). N2 carrier gas: 28.5 ml/min. Other con- The tube was rinsed twice with 1.5 ml of chloro- ditions were the same as in (A). form. The rinse was transferred onto the (C) Conditions of GC/MS for ethyl carbamate column. The column was carefully rinsed before alkylation down twice with another 1.5 ml of chloroform Column: 0.25 mm x 30 m DURA BOND fused each time. The ethyl carbamate was eluted silica capillary column DB-WAX. Column tem- from the column with 150 ml of a mixture of perature: 50C (held for 1 min) programmed at benzene, diethyl ether and methanol (108: 32: 1). the rate of 4C/min to 100C, 1C/min to 140C, The first 12 ml was discarded and the remaining and 20C/min to 200C (held for 10 min). N2 eluate was collected in a K-D evaporator fitted carrier gas: 1.2 ml/min. Ionizing voltage: 70 eV. 332 J. Food Hyg. Soc. Japan Vol. 28, No. 5

Ionizing current: 300A. Chamber tempera- perature: programmed from 70C (held for 30 ture: 210C. Fragment ions were monitored at sec) at 10C/min to 240C (held for 10 min). N2 m/z 62, 74, 89, 101 and 129. carrier gas: 1 ml/min. Ionizing voltage: 70 eV. (D) GC/MS conditions for alkyl derivative of Results and Discussion ethyl carbamate Determination of ethyl carbamate Column: 0.25 mm x 30 m DURA BOND fused The sample solution which was extracted from silica capillary column DB-WAX. Column tem- sake and purified chromatographically on the

Fig. 1. Gas chromatogram of ethyl carbamate standard solution Fig. 2. Gas chromatogram of sake extract with- The concentration is 100 pug/ml. For ex- out alkylation perimental procedures see Conditions of The GC-FID conditions were the same as GC and GC/MS (A). in Fig. 1.

Fig. 3. Electron impact mass spectrum of ethyl carbamate For experimental procedures, see Conditions of GC and GC/MS (C). October 1987 Determination of Ethyl Carbamate 333

Fig. 4, Electron impact mass spectrum of the sample peak whose retention time was the same as that of ethyl carbamate The conditions were the same as in Fig. 3.

Fig. 5. Mass chromatogram of the sample at ion current m/z 62, 74 and 89 (ethyl carbamate), and 101 and 129 (other compounds)

Florisil column was analyzed by GC-FID with- Determination of ethyl carbamate after alkylation out derivatization. A chromatogram of stand- Dimethylformamide dialkylacetals18) syn- ard ethyl carbamate is shown in Figure 1. The thesized according to Meerwein1)12) were used substance showing a peak at the same retention as pre-treatment reagents14,15)for GC analysis. time as that of standard ethyl carbamate on the In our procedure, N, N-dimethylformamide-di- chromatogram of sake extract (Figure 2) was methylacetal (N,N-DMF-DMA) reacts with subjected to GC/MS analysis. Figures 3 and 4 ethyl carbamate to yield N, N-dimethylamino- show the spectra of ethyl carbamate and the methylene ethyl carbamate quantitatively, and sample peak. The mass chromatogram (Figure this is detected by GC-FID (Figure 6). 5) based on the fragment ions at 101 and 129 showed that the scan numbers of the peak tops were slightly different from the scan number of a peak top at m/z 62, which indicates that the peak corresponding to ethyl carbamate on GC-FID (Figure 1) consists of at least two components. For precise determination, it is necessary to The extracts from sake were alkylated and obtain a pure peak of ethyl carbamate. subjected to GC after purification by Extrelut 334 J. Food Hyg. Soc. Japan Vol. 28, No. 5

Fig. 6. Gas chromatogram of ethyl carbamate standard solution after alkylation Fig. 7. Gas chromatogram of sake extract after For experimental procedures, see Condi- alkylation and purification on an Extrelut tions of GC and GC/MS (B). column The GC conditions were the same as in Fig. 6.

Fig. 8. Electron impact mass spectrum of ethyl carbamate derivative with N, N-DMF-DMA For experimental procedures, see Conditions of GC and GC/MS (D). column chromatography; the gas chromatogram the analysis of ethyl carbamate in two samples obtained is shown in Figure 7. The recovery of sake at hand; no ethyl carbamate was of ethyl carbamate added at a level of 100 rig/kg detected. to sake was 73.70 on average. The detection Acknowledgment limit was found to be 10 ppb. The structure of the alkyaated product was confirmed by The authors wish to thank Dr. K. Takahashi, GC/MS and found to have a molecular ion of Miss A. Ishigami and Mr. T. Ishizaka for pro- 144, indicating the addition of an N, N-dimethyl- viding the GC/MS data. formyl group to the nitrogen atom of ethyl References carbamate (Figure 8). 1) Schmahl, D., Port, R., Wahrendorf, J.: Int. J. The alkylation procedure proposed herein en- Cancer 19, 77-80 (1977) ables the use of FID for the rapid and precise 2) Toth, B., Boreisha, I.: Europ. J. Cancer 5, 165'S- determination of ethyl carbamate in sake at the 171 (1969). ppb level. This method was applied to 3) Schlatter, J.: "Proceedings of Euro Food Tox. October 1987 Determination of Ethyl Carbamate 335

II" p. 249'-255 (1986), Institute of Toxicology, Zurich. Swiss Federal Institute of Technology & Univer- 8) Dennis, M. T., Howarth, N., Massey, R. C., sity of Zurich, Zurich. Rarker, I.: J. Chromatography 369, 193-498 4) Ough, C. S.: J. Agric. Food Chem. 24, 323 328 (1986). (1976). 9) Bailey, R., North, D., Myatt, D.: ibid. 369, 199 5) Ough, C. S.: ibid. 24, 328-331 (1976). 202 (1986). 6) Zimmeli, B., Bauman, U., Nagell, P., Battaglia, 10) Meerwein, H.: Ber. 89, 2060 (1956). R.: "Proceedings of Euro Food Tox. II" p. 243- 11) Meerwein, H.: Angew. Chem. 71, 530 (1959). 248 (1986), Institute of Toxicology, Swiss Fede- 12) Meerwein, H.: Ann. 641, 1 (1961). ral Institute of Technology & University of Zu- 13) Bredereck, H., Effenberger, F., Hofmann, A.: rich, Zurich. Chemische Berichte 97, 61-73 (1964). 7) Conacher, H. B. S., Page, B. D., : ibid. p. 237N 14) Thenot, J. P.: Anal. Letters 5, 217 (1972). 242 (1986), Institute of Toxicology, Swiss Federal 15) Thenot, J. P.: ibid. 5, 519 (1972). Institute of Technology & University of Zurich,