Determination of Ethyl Carbamate in Alcoholic Beverages and Fermented Foods Sold in Korea

Abstract

Ethyl carbamate (EC) classified as a probable human carcinogen (Group 2A) is naturally formed in alcoholic beverages and fermented foods during fermentation process and/or during storage. The objective of this study was to analyze EC in 34 food items including 14 alcoholic beverages and 20 fermented foods sold in Korea. Each food was collected from 18 supermarkets in 9 metropolitan cities in Korea, and then made into composite. According to food composition and alcohol content, samples were divided into four matrices such as apple juice, milk, Soju (liquor containing about 20% alcohol), and rice porridge. The maximum EC value of 151.06 µg/kg was found in Maesilju (liquor made from Maesil and Soju). Whisky and Bokbunjaju (Korean black raspberry wine) contained 9.90 µg/kg and 6.30 µg/kg, respectively. EC was not detected in other alcoholic beverages. Of 20 fermented foods, Japanese-style soy sauce had highest level of 15.59 µg/kg and traditional one contained 4.18 µg/kg. Soybean paste had 1.18 µg/kg, however, EC was not found in other fermented foods.

INTRODUCTION

Ethyl carbamate (EC, CAS No. 51-79-6), known as urethane, is an ethyl ester of carbamic acid. It was used as a hypnotic in man and an anesthetic in animals in 1940s. However, it was found to be genotoxic and carcinogenic in 1943 (1). EC is rapidly absorbed from the gastrointestinal tract and the skin, and then distributed in the body (2). In liver, up to 90% of EC absorbed is hydrolyzed by microsomal esterase and eliminated as ethanol, carbon dioxide, and ammonia (Fig. 1). About 5% of EC is excreted in the urine after hydroxylation and conjugation. EC is also oxidized to vinyl carbamate (0.5%) by cytochrome P-450 2E1 and further converted into vinyl carbamate epoxide, which can bind covalently to DNA, RNA and proteins. The carcinogenic potential of EC includes gene mutations and DNA damage. Animal studies have shown that EC causes an increase in the incidence of tumors in several tissue sites including lung, liver, and blood vessels (3-5). Consequently, the International Agency for Research on Cancer (IARC) classified EC as a possible human carcinogen (Group 2B) in 1974 (6). Recently, IARC upgraded EC to a probable human carcinogen (Group 2A) in 2007 (7). The concern over the presence of EC and its toxicity in regularly consumed food products and alcoholic beverages has raised global interest to assess the possible risks to human health. The Joint Food and Agriculture Organization/World Health Organization Expert Committee on Food Additive (JECFA) evaluated the exposure to EC in 2005 and concluded that the intake of EC from foods excluding alcoholic beverages would be of low concern (8). However, when both alcoholic beverages and foods were combined, its intake increased about 4-fold and consequently posed potential carcinogenic risks (8).

Fig. 1. Probable activation and inactivation pathways of ethyl carbamate metabolism. * Toxic metabolic pathways are indicated by bold type. * Adducts are responsible for the carcinogenic effect.

EC is naturally formed in fermented foods and alcoholic beverages during the fermentation process and/or during storage. Table 1 gives EC levels in alcoholic beverages from China, EU, Hong Kong, Japan, South Korea, and United States. The contents of EC in alcoholic beverages vary over a wide range. The levels of EC in wine ranged from 8 µg/L in white table wine to 111 µg/L in sake (9), and EC concentrations in stone-fruit brandies ranged from 100 to 22,000 µg/kg (10,11). Chinese rice wine contained nearly twice as much as EC than other Chinese alcoholic beverages (12). About 80% of EC present in spirits was formed during the distillation step and/or within the first 48 hr after distillation (13). In comparison, the contents of EC in beer were lowest, ranging from not-detected (ND) to 5.8 µg/kg. The occurrence of EC in fermented foods is shown in Table 2. The highest level of EC, up to 344 µg/kg, was found in Chinese red sufu, a fermented soybean curd (14). Toasting bread led to increases of between 3- and 8-fold in EC ranging from 3.5 to 33.8 µg/kg on a wet weight basis (15,16). Azodicarbonamide acts as a dough improver by chemically oxidizing thiol groups and rapidly forming the gluten network in the bread-making industry and as a blowing agent in beer. EC has been shown to form from azodicarbonamide breakdown during baking (17). Soy sauce had a wide range of EC from ND to 108 µg/kg. EC was also found in other fermented foods including vinegar, bread, and soybean paste. Generally, the contents of EC in fermented foods were quite lower than those of alcoholic beverages. It seems that the formation of EC is closely related to the content of ethanol.

Table 1. Concentrations (µg/kg) of ethyl carbamate in alcoholic beverages.

Country	Product	No. of samples	Ethyl carbamate	Reference
China	Beer	20	2-3	12
	Wine	30	9-34
	Rice wine	92	8-515
	White spirit	22	12-192
EU members	Beer	13	ND-1	11
	Wine	17	ND-24
	Fortified wine	15	14-60
	Sake	2	81-164
	Stone-fruit brandy	3,244	ND-22,000
	Whisky	210	ND-1,000
Hong Kong	Beer	15	ND-5.8	28
	Wine	20	6.7-47
	Rice wine	21	2.0-330
	Distilled spirits	9	20-66
Japan	Sake	92	ND-202	11
South Korea	Distilled spirits	5	ND-196	32,56
	Wine	30	2.64 ± 3.71
	Rice wine	8	14.11 ± 9.58
	Liquor	2	157-230
	Soju	6	ND
United States	Wine	91	ND-254	54
	Sake/rice wine	34	10-904
	Whisky	212	ND-1,719
	Brandy	26	9-387

Table 2. Concentrations (µg/kg) of ethyl carbamate in fermented foods.

Country	Product	No. of samples	Ethyl carbamate	Reference
Canada	Bread	12	1.4-4.8	15
	Toast	24	1.0-29.2
China	Soy sauce	22	8-108	12
	Vinegar	11	2-51
	Sufu1)	10	12-124
	Red sufu	10	87-344
EU members	Bakery	50	ND-20	11
	Fermented sauce	44	ND-18
	Vinegar	10	ND-33
Hong Kong	Bread/rolls/buns	15	ND-8.6	28
	Soy sauce	5	1.8-17
	Vinegar	18	ND-37
Japan	Soy sauce	26	ND-35.2	38
South Korea	Kimchi	20	ND-16.2	19
	Soy sauce	20	ND-73.3
	Soybean paste	7	ND-7.9
	Vinegar	5	0.3-2.5

¹⁾A cheese-like product that is one of the most popular fermented soybean foods in China.

Although there are currently no harmonized maximum levels for EC, some countries have established their own criteria (Table 3). Canada firstly introduced maximum limits for EC in alcoholic beverages in 1985 after high levels of EC were found in alcoholic beverages. The maximum levels were 30 µg/L for wine and 400 µg/L for fruit brandies. The United States has voluntary limits for domestic alcoholic beverages. Canadian guidelines were adopted by other countries such as Czech Republic, Brazil, France, Germany, and Switzerland. South Korea also set the maximum limit of 30 µg/L only for table wine. There are currently no guidelines governing the presence of EC in fermented foods.

Table 3. Maximum levels of ethyl carbamate for alcoholic beverages.

Country	Wine	Fortified wine	Distilled spirits	Sake	Fruit brandy
Brazil			150
Canada	30	100	150	200	400
Czech Republic	30	100	150	200	400
France			150		1,000
Germany					800
South Korea	30
Switzerland					1,000
United States	15	60

Cited from References 11, 13, and 20.

In general, most of beverages and foods have various components that can hinder the analysis of EC from matrix, which affect EC analysis. Besides, beverages and foods contain low levels of EC, close to µg/kg or below detection limit. Accordingly, a number of analytical methods have been developed in various foods and beverages over the past 30 years (Table 4). Carbamate such as propyl carbamate, butyl carbamate, or ¹³C, ¹⁵N-EC has been used as an internal standard (18-21). However, these standards have different octanol-water partition coefficient from EC. It could lead to the difference in the recoveries between EC and internal standard. Therefore, the deuterated EC (d₅-EC) is recently used to provide more precise recovery correction (12,22-24). The most traditional extraction method is a liquid-liquid extraction. Methylene chloride is commonly used as the organic solvent since EC is a weak polar compound. Solid-phase extraction (SPE) provides considerable advantages and is applied in the AOAC method (25). Extrelut or Chem-Elut has been found to be the most prevailing sorbent (15,18,22,26-28). In fatty food matrix, SPE was combined with a clean-up step such as the elution with pentane or hexane to eliminate nonpolar compounds (22). Another technique for extracting EC is a solid phase microextraction (23,29-31). It has advantages to reduce time and cost as well as to avoid EC losses. The most widespread method of EC determination is gas chromatography (GC) with polar columns. The applied detection systems include flame ionization detection (FID), electrolytic conductivity detection (ECD), and mass spectrometry (MS). The MS is the most useful and authoritative method for the quantification of EC. Recently, GC-MS-MS has been adopted to improve selectivity, sensitivity, and repeatability for EC determination (10,24,32). High performance liquid chromatography (HPLC) coupled with a fluorescence detector was employed after the derivatization with 9-xanthydrol (33). Fourier transform infrared (FTIR) spectroscopy was also used for determining EC without derivatization (10).

Table 4. Summary of the methodology of ethyl carbamate determination in alcoholic beverages and fermented foods.

Product	Internal standard	Sample preparation		Detection
		Liquid-liquid extraction	Solid-phase extraction
Alcoholic beverages	Methyl carbamate	Extraction with CH2Cl2, CHCl3 or ethyl acetate	Chem-Elut or Extrelut	GC-MS
	Propyl carbamate		Alumina	GC-MS/MS
	n-Butyl carbamate	Addition of salts (NaCl, K2CO3 or Na2SO4)	Florisil	GC-ECD
	tert-Butyl carbamate		Headspace solid-phase microextraction	GC-NPD
	13C,15N-Ethyl carbamate	Dilution to 5% or 20% alcohol		GC-TEA
	d5-Ethyl carbamate	Removal of ethanol	Styrenedivinylbenzene copolymer	GC-FID
	Isopropyl carbamate			FTIR-PLS
				HPLC/FLD1)
Fermented foods	Propyl carbamate	Extraction with CH2Cl2, ethyl acetate or petroleum ether	Chem-Elut or Extrelut	GC-MS/MS
	n-Butyl carbamate		Deactivated alumina	GC-MS
	13C,15N-Ethyl carbamate	Removal of nonpolar compounds with n-pentane or hexane	Celite	GC-FID
	d5-Ethyl carbamate		C18
			Florisil

¹⁾It was used after the precolumn derivatization with 9-xanthydrol.

EC is formed through the reaction from ethanol and cyanate, urea, citrulline, or other N-carbamyl compounds (Fig. 2). Diethyl pyrocarbonate used as an antimicrobial agent added in beer, orange juice, and some soft drinks was found to react with ammonia to form EC in 1971 and then banned in the United States (34). Urea is the most important EC precursor in wine and sake (35,36). It is produced from the arginine metabolism through urea cycle in yeast. Carbamyl phosphate generated from arginine metabolism reacts rapidly with ethanol to form EC. In addition, citrulline produced through arginine deiminase (ADI) pathway by lactic acid bacteria is another precursor of EC in wine and soy sauce (37,38). Cyanate is a predominant precursor of EC in stone-fruit spirits and other spirits (13,39,40). Cyanogenic glycosides present mostly in the seed of stone-fruits is enzymatically or thermally degraded to hydrocyanic acid, which is oxidized to cyanate and then reacts with ethanol to form EC (2). A relatively high correlation (R = 0.597) between cyanide and EC was found in Brazilian sugar cane spirits (13). The addition of potassium metabisulfite inhibited the oxidation of hydrocyanic acid to cyanic acid, which in turn reduced the content of EC in ume, Japanese apricot, liqueur (41). On the other hand, no correlation was found between EC and cyanide in German stone-fruit spirits (10). In addition to these precursors, other factors such as the exposure to UV light, storage time, and fermenting temperature have been known to influence the formation of EC (10,12,42,43).

Fig. 2. Mechanism of ethyl carbamate formation in alcoholic beverages and fermented foods. LAB: lactic acid bacteria.

Numerous methods have been developed to reduce EC level in alcoholic beverages (Table 5). These methods include the use of refined materials, antioxidants, and genetically engineered yeasts. The reduction of EC levels has been achieved mostly by either inhibiting the production of its precursors or decomposing the precursors. Earlier preventive technologies include refining raw materials, removing seed from stone-fruits, controlling temperature and pH, filtering a fermentation product, minimizing exposure to light, and/or shortening storage time (10,42-45). Acid urease was added to hydrolyze urea into carbon dioxide and ammonia in grape wine and rice wine (45,46). Copper catalyst and potassium metabisulfite were used as cyanide catalyst and antioxidant, respectively (41,47). Genetic technologies in modifying fermentation strains provides a significant advance in inhibiting the production of urea or enhancing the metabolism of urea in yeast. The arginase encoded by the CAR1 gene in yeast degrades arginine into urea (48), which is transported and metabolized by urea amidolayse (DUR1,2) and urea permease (DUR3) in yeast. Therefore, inhibition of CAR1 expression and enhancement of DUR1,2 and DUR3 expression resulted in the decrease of urea concentrations in sake and grape wine, and consequently reduced the formation of EC (49,50). The utilization of arginine and citrulline via ADI pathway by Lactobaccilus hilgardii X₁B and Oenococcus onei could avoid the possibility of EC formation in wine (51).

Table 5. Approaches to reduce the contents of ethyl carbamate in alcoholic beverages.

Product	Method	Reference
Wine	Acid urease	45
	Addition of diammonium phosphate1)	57
	Lower temperature	43
	Lower pH	51
	Increase of the expression of DUR1,2 (urea amidolyase) and DUR3 (urea permease)	50,57
Stone-fruit spirits	De-stoning prior to mash	10
	Copper catalyst	47
	Deletion of CAR1 (arginase)	48
	Automatic rinsing of the stills/Separation of tailings/non-redistilling tailings	58
Rice wine/sake	Refining process of raw materials	44
	Lower temperature	14
	Knocking out of CAR1 (arginase)	49
	Acid urease	46
Ume liqueurs	Oxygen absorber/Addition of potassium metabisulfite	41
Spirits	Prevention of light exposure/Shorten storage time	42
	Charcoal filtration	59

¹⁾It is added to grape must fermentations to alleviate nitrogen deficiency.

EC is mainly found in fermented foods and alcoholic beverages. Korean diet includes many fermented foods such as Kimchi and soy sauce. Alcoholic beverages made from grains or fruits are also commonly consumed. For these reasons, the quantification of EC in fermented foods and beverages consumed in the Korean population was needed. Therefore, we determined EC in fermented foods and alcoholic beverages collected from 18 supermarkets in 9 metropolitan cities in Korea.

MATERIALS AND METHODS

Chemicals. EC, sodium chloride, and sodium hydroxide were purchased from Sigma-Aldrich (St. Louis, MO, USA). Internal standard d₅-EC was purchased from CDN isotopes (Pointe-Claire, Quebec, Canada). Methylene chloride and hexane were obtained from J.T. Baker (Center Valley, PA, USA). Disposable diatomite Chem Elut SPE column (50 mL) was purchased from Agilent Technology (Milwaukee, WI, USA).

Preparation of standard solution. The stock solution of EC (1000 µg/mL) was prepared by dissolving 0.1 g of EC in acetone. A working solution (400 ng/mL) was made by diluting of stock solution in acetone. The d₅-EC was prepared with the same method above. For spiking in the sample, internal standard was diluted in distilled water instead of acetone.

Samples. Total 34 food items including 14 alcoholic beverages and 20 fermented foods were chosen from 734 foods appeared in the pooled intake data of 2008, 2009, 2010, and 2011 Korea National Health & Nutrition Examination Survey (KNHANES). Samples (500~800 g) were purchased from 18 supermarkets in 9 metropolitan cities according to a scheme devised by the Korea Health Industry Development Institute (KHIDI) to reflect the market share and population. Samples were pooled to make a composite, homogenized by blending, and then kept at −20℃ until analysis.

Determination of ethyl carbamate (EC). EC was determined according to the AOAC official method 994.07 with some modifications (AOAC 1997). The d₅-EC was used as the internal standard instead of n-propyl carbamate. GC/MS conditions were revised to separate the d₅-EC peak from EC peak and shorten a run time. To develop analytical methods depending on food composition, samples were classified into four matrices, which include non-fatty liquid (apple juice), proteinous liquid (milk), alcoholic beverage (Soju, ~20% ethanol), and non-fatty solid (rice porridge).

Apple juice matrix was firstly neutralized using 1 N sodium hydroxide solution and then 10 g of neutralized sample was mixed with 30 mL of distilled water and then 5 g of sodium chloride was dissolved in it. After spiked with 100 ng of d₅-EC, it was loaded into a Chem Elut SPE column. After 4 min of equilibration, EC was eluted with 160 mL of methylene chloride at a rate of 1 drop per second. The eluent was concentrated to about 2~3 mL using a rotary evaporator, transferred into v-vial, and further concentrated to 1 mL under a gentle stream of nitrogen. Each sample was extracted and analyzed by GC-MS in triplicate.

The milk and soju were extracted using the above method with slight modification. Milk was centrifuged to remove proteins before loaded to the SPE column. Soju was diluted into 5% alcohol content to improve chromatographic resolution.

The rice porridge (20 g) was mixed with 40 mL of distilled water and then d₅-EC (50 ng) was added. It was stirred for 20 min and centrifuged at 2000 rpm for 5 min.

The supernatant was loaded into the Chem Elut column after 4 min of equilibration. EC was eluted with 160 mL of methylene chloride. The eluent was concentrated to about 2~3 mL using a rotary evaporator, transferred into v-vial, and further concentrated to 1 mL under a gentle stream of nitrogen.

A 7820A GC-5977E MS (Agilent Technologies, Santa Clara, CA, USA) was used for the quantification and identification of EC. The GC conditions were as follows: capillary column 30 m length × 0.25 mm i.d., 0.25 µm film thickness DB-WAX (J&W, Folsom, CA, USA), helium carrier gas at 1 mL/min, injection volume with 2 µL in splitless mode, and injector 210℃. Oven temperature: 60℃, 10℃/min to 90℃, 2℃/min to 130℃ held for 5 min, 20℃/min to 220℃, and then held for 3 min. The MS was operated in the selected ion monitoring (SIM) with electron impact ionization (70 eV). The MS transfer line and ion source were kept at 240℃ and 230℃, respectively. Mass to charge (m/z) 62 and 64 were major fragment ions of EC and d₅-EC. EC was quantified using calibration curves made from peak area ratios of EC/d₅-EC (m/z 62 vs m/z 64). EC peak was identified by comparing the area ratios of m/z 62 vs m/z 74 that were major fragment ions of EC.

RESULTS AND DISCUSSION

Alcoholic beverages. The concentrations of EC in alcoholic beverages are presented in Table 6. Of 14 alcoholic beverages, the maximum value of 151.06 µg/kg was found in Maesilju, followed by whisky (9.90 µg/kg) and Bokbunjaju (6.30 µg/kg). Maesilju is a traditional Korean liqueur made out of green Maesil (Japanese apricot, a fruit of Prunus mume). The content of EC (151.06 µg/kg) in Maesilju is close to the high level (78.18 ± 63.10 µg/kg) reported previously in 7 Maesilju samples (32). Maesil contains high level of cyanogenic glycoside amygdalin (52), which is degraded to hydrocyanic acid and then oxidized to cyanate. The other ingredient of Maesilju is a Soju that is produced by diluting absolute ethanol to contain about 20% alcohol. Maesil is commonly removed after soaking in Soju for 100 days and the remaining liquid is ripened for several months or longer. It has been known that the concentrations of EC in Maesilju increased up to 216 µg/kg depending on alcohol content, soaking time of Maesil, and fermentation time (32,53). In earlier literatures, high contents of EC have been found in stone-fruit brandies (Table 1). Currently, there is no maximum EC level for Maesilju in Korea. The value of EC in Maesilju exceeds slightly the Canadian limit of 150 µg/kg for fruit brandy. This result indicates that mitigation action to reduce EC levels in Maesilju should be taken.

Table 6. Contents of ethyl carbamate in Korean alcoholic beverages.

Product	Ethyl carbamate (μg/kg)
Beer, canned	ND7)
Beer, PET bottled	ND
Beer, imported	N
Bokbunjaju1)	6.30
Cheongju2)	ND
Hanbangju3)	ND
Makgeolli4)	ND
Maesilju5)	151.06
Red wine	ND
Soju6)	ND
Wine, red	ND
Wine, white	ND
Wine, sparkling	ND
Whisky	9.90

¹⁾Korean black raspberry wine.

²⁾Korean rice wine similar to Japanese sake.

³⁾Liquor made from rice and oriental medicine.

⁴⁾Korean traditional alcoholic beverage made from rice.

⁵⁾Liquor made from Maesil and Soju.

⁶⁾Liquor containing about 20% (v/v) alcohol.

⁷⁾Not detecte.

Whisky has been reported to contain EC ranging from ND to 1,719 µg/kg (11,32,54). The level (9.9 µg/kg) of EC analyzed in this study is in the range of earlier literatures. It is quite lower than maximum permissible limit of 150 µg/kg for distilled spirits established in Canada, Czech Republic, and France. The content (6.3 µg/kg) of EC in Bokbunjaju (Korean black raspberry wine) is comparable to the value (1.66 ± 3.41 µg/kg) reported by Kim et al. (32). In other countries, wine has been known to contain a wide range of EC (ND-549 µg/kg) (11,14,28,54). Korean wines were reported to have an average of 2.64 ± 3.71 µg/kg (32). However, EC was not detected in wine analyzed in this study. Such variations in EC levels in wine are understandable due to differences in the processing conditions, raw materials, yeast, and lactic acid bacteria. EC was also not detected in beer, Soju, Cheongju (Korean rice wine), and Hanbangju (liquor made from rice and oriental medicine), which is close to the values of earlier literatures (22,32).

Fermented foods. The concentrations of EC in fermented foods are shown in Table 7. Of 20 fermented foods analyzed herein, Japanese-style soy sauce had highest content of 15.59 µg/kg and traditional one contained 4.18 µg/kg (Table 7). Soy sauce has been reported to have 8-108 µg/kg in Chinese samples, ND-35.2 µg/kg in Japanese samples, and 1.8-17 µg/kg in Hong Kong samples (Table 2). The content (1.18 µg/kg) of EC in soybean paste was in the range of ND-7.9 reported by Kim et al. (55). The concentrations of EC between 1.4 and 4.8 µg/kg were detected in 12 bread samples (15). Azodicarbonamide used as a flour bleaching agent and a dough conditioner has been reported to increase EC levels during baking (16). However, EC was not detected in 3 different breads analyzed in this study even though azodicarbonamide is allowed to use in wheat flour products with the maximum limit of 45 mg/kg in Korea. EC was not detected in cheese, Chunjang (fermented soybean product colored by caramel), Chengggukjang (soybeans fermented with Bacillus subtilis for 2~3 days), Kimchi, Maesil extract, red pepper paste, Ssamjang (mixture of soybean paste, red pepper paste, garlic, and seasoning), vinegar, and yogurt. These results indicate that most of Korean alcoholic beverages and fermented foods contain EC with low levels or below detection limit. Considering the intakes of Maesilju and soy sauce in the Korean population, preventative actions should be taken to reduce the levels of EC.

Table 7. Contents of ethyl carbamate (µg/kg) in Korean fermented foods.

Product	Ethyl carbamate (μg/kg)
Bread1)	ND6)
Milk bread1)	ND
Corn bread1)	N
Cheonggukjang2)	ND
Cheese	ND
Cheese, mozzarella	ND
Chunjang3)	ND
Kimchi, Chinese cabbage	ND
Maesil extract4)	ND
Red pepper paste, regular	ND
Red pepper paste with vinegar	ND
Ssamjang5)	N
Soybean paste	1.18
Soy sauce, traditional	4.18
Soy sauce, Japanese-style	15.59
Vinegar, brewed	ND
Vinegar, brewed with persimmon	ND
Yogurt, solid type	ND
Yogurt, liquid type	ND
Yogurt, diluted drink	ND

¹⁾Ferment their dough with yeast.

²⁾Korean natto.

³⁾Fermented soybean product colored by caramel.

⁴⁾Fermented liquid made from Japanese apricot and sugar.

⁵⁾Mixture of soybean paste, red pepper paste, garlic, and seasoning.

⁶⁾Not detected.