Ethyl Carbamate In Alcoholic Beverages Research Articles

The High-Throughput Screening of Microorganisms to Eliminate Ethyl Carbamate in Chinese Liquor.

Ethyl carbamate (EC) is a 2A classified carcinogen in Chinese liquor that has raised many problems regarding food safety. Applying microorganisms to control the content of EC precursors in fermented grains has been proven as an effective method to reduce EC in alcoholic beverages. However, the utilization of microorganisms to decrease the precursors of EC (urea and cyanide) is still incomplete in regard to Chinese liquor. Thus, it is necessary to isolate strains with the degradative activities of urea and cyanide. Herein, Bacillus sonorensis F3 and Bacillus licheniformis YA2 strains were isolated from the fermented grains through multiple rounds of high-throughput screening, and the degradative abilities in urea and cyanide reached 95.72% and 75.48%, respectively. In addition, the urease from the B. sonorensis F3 strain and the carbon nitrogen hydrolase from the B. licheniformis YA2 strain were identified by the heterogeneous expression in Escherichia coli. Then, both F3 and YA2 strains were combined at a ratio of 5:1 and applied to eliminate the EC in the simulated fermentation of Chinese liquor; as a result, 51.10% of EC was reduced without affecting the main composition of flavor substances. The obtained strains have great potential in terms of the improvement of quality and safety of Chinese liquor.

Molecularly imprinted bulk and solgel optosensing based on biomass carbon dots derived from watermelon peel for detection of ethyl carbamate in alcoholic beverages.

Biomass carbon dots synthesized by biological waste conform to the trend of ecological environmental protection and the requirements of green chemistry, which show great application potential in practice. In the study, we used watermelon peels as the raw materials to synthesize a novel blue biomass carbon dots (CDs) by a hydrothermal process with high fluorescence quantum yield of 22.8%. Through bulk polymerization and solgel method, two kinds of core-shell nanospheres were developed as fluorescent probes to recognize and detect ethyl carbamate (EC) rapidly without complex samples pretreatment. The obtained CDs@MIPs integrated the high-performance optical characteristics of CDs with excellent selectivity and adsorption of MIPs, which showed ideal linear relationships in the EC concentration range 1-120μg L-1 and low LOD of 0.57μg L-1 and 0.94μg L-1, respectively. Both CDs@MIPs have a short equilibration time which was around 20min, and the imprinting factors (IF) are 4.04 and 2.62. The recoveries of the six spiked samples were satisfying, and the RSD precisions were lower than 5.57%. Gas chromatography-mass spectrometry was seen as a parallel analysis to validate the correctness of the results, which indicated the practicability and reliability of the developed method. This proposal strategy of optical sensors provided an effective channel for trace EC recognition, with numerous advantages, involving eco-friendly, low cost, high sensitivity, separation effect, and good selectivity.

Risk assessment of ethyl carbamate in alcoholic beverages in Korea using the margin of exposure approach and cancer risk assessment

Ethyl carbamate (EC) is a carcinogen mostly found in fermented foods and alcoholic beverages. In Korea, the consumption, as well as domestic production and import of alcoholic beverages, has increased over the past decade, thus increasing the risk of EC exposure. In this study, we estimated the EC content of 145 alcoholic beverages using gas chromatography-mass spectrometry (GC-MS) and optimized the hydromatrix method for solid-phase extraction with improved absolute recovery of EC and better repeatability than the Chem-Elut method of AOAC. The margin of exposure (MOE) approach and cancer risk assessment were performed to assess the risk of EC from alcoholic beverages in Korean. The EC content in some of the alcoholic beverages was not detectable; however, the maximum value was 329.7 μg/kg. Moreover, alcoholic beverages with stone fruit as a component revealed high EC contents. The daily intake of alcoholic beverages by the Korean population of alcohol-drinking adults ranged from 184.2 to 237.1 g/day, depending on gender and age groups. Of the 145 alcoholic beverages, the contributions of soju and beer to the exposure of EC in the Korean population were found to be maximum. MOE and cancer risk for the Korean average drinker were 14,874 and 1.01 × 10−6, respectively, whereas in drinkers of the 95th percentile (P95), they were 2951 and 5.08 × 10−6, respectively. This study indicated that the risk of exposure to EC is of lower concern in average drinkers than in heavy drinkers in the Korean population.

Aspergillus oryzae acetamidase catalyzes degradation of ethyl carbamate

Urethanase (EC 3.5.1.75) catalyzes the hydrolysis of ethyl carbamate (EC) to ethanol, carbon dioxide, and ammonia. From our recent study, we expected that an acetamidase encoded by amdS of Aspergillus oryzae may catalyze the degradation of EC because it is homologous with a Candida parapsilosis urethanase (CPUTNase) recently identified. Urethanase is a prospective candidate to reduce EC in alcoholic beverages, but knowledge of this enzyme is very limited. Recombinant AmdS was expressed to study its enzymatic properties. Purified AmdS was identified as a homo-tetramer consisting of four 60kDa units and exhibited urethanase activity. In a 20% ethanol solution, AmdS had 65% activity compared with a solution without ethanol. Residual activity after 18h indicated that AmdS was stable in 0%-40% ethanol solutions. The optimum temperature of AmdS was 40°C. This enzyme showed urethanase activity at pH 6.4-9.6 and exhibited its highest activity at pH 9.6. The Km value of AmdS for EC was 8.2mM, similar to the Km value (7.6mM) of CPUTNase. AmdS showed activity not only for EC and acetamide but also other amide compounds. In this study, we investigated the enzymatic properties of AmdS that was identified as acetamidase and showed that an amidase can be an enzymatic candidate that degrades EC.

Detection of ethyl carbamate in liquors using surface-enhanced Raman spectroscopy.

Ethyl carbamate (EC), a potential carcinogen, can be formed during the fermentation and storage of alcoholic beverages. In this work, quantitative detection of EC in alcoholic beverages by using surface-enhanced Raman spectroscopy (SERS) is reported. Flower-shaped silver nanostructure substrates and silver nanocube substrates were prepared and employed as SERS platform. Flower-like silver substrates had better Raman enhancement effect on EC and were selected for further EC detection. In EC SERS spectra based on flower-shaped silver substrates, the strongest and reproducible characteristic band at 857 cm−1 was chosen for establishing a linear regression model in the concentrations ranging from 10−5 to 10−9 M, which effectively extended the application scope of the quantitative model for determination EC. Furthermore, a real alcoholic beverage was tested to verify the feasibility and reliability of the method.

Highly sensitive determination of ethyl carbamate in alcoholic beverages by surface-enhanced Raman spectroscopy combined with a molecular imprinting polymer

A simple and reliable method for fast extraction and sensitive detection of ethyl carbamate in alcoholic beverages was developed.

Ethyl carbamate in alcoholic beverages from China: Levels, dietary intake, and risk assessment

A national survey of ethyl carbamate (EC) in alcoholic beverages from the fourth and fifth Chinese total diet study (TDS) performed in 2007 and 2009 was conducted for the first time. Alcoholic beverages samples were collected from 16 provinces representing the average dietary patterns of various provinces of China and covering about 60% of the total Chinese population. The results showed that the average EC level in alcoholic beverages (19.8 μg/kg) in the fifth TDS was higher than that of the fourth TDS (8.5 μg/kg). The dietary intake of EC from the Chinese population was estimated to be 8.27 ng/kg bw per day for average population and 45.67 ng/kg bw per day for high consumers (the 97.5th percentile) in the fifth TDS. The average and high-end estimated daily intakes of EC for the alcoholic beverages were both lower than estimated daily intake (EDI) value (80 ng/kg bw per day) suggested by JECFA, indicating low health risk of EC dietary exposure among Chinese adults at present.The Chinese rice wines, as a kind of traditional fermented alcoholic beverages in China, has a high level of EC and an obvious consumption regional disparity, thus the health risk of EC in Chinese rice wines should be of concern. To estimate the daily intake of EC from Chinese rice wines consumed in China, 890 Chinese rice wines samples including 468 commercial wines and 422 base wines were collected from various regions. The distribution of EC varied significantly among 468 commercial wines, ranging from 6.3 to 775.8 μg/L (mean: 232.9 μg/L). Based on the intake data for Chinese rice wines from the food consumption survey, the daily intake of EC was estimated to be 290.6 ng/kg bw per day for average consumers and 1848.4 ng/kg bw per day for high consumers (the 97.5th percentile). In this study, the estimated daily intake of EC for Chinese rice wines in China was far higher than EDI value suggested by JECFA indicating that Chinese rice wines were the main exposure origin of EC in alcoholic beverages. Health risk assessment of EC using a margin of exposure (MOE) approach recommended by the European Food Safety Authority (EFSA) suggested unlikely health concern with respect to current dietary intake of EC for alcoholic beverages in China. However, a relatively high health risk of EC dietary exposure in Chinese rice wines was observed in some provinces. Therefore, some adapted strategies should be developed and used in industrial scale to control the level of EC for Chinese rice wines.

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

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.

Surface-enhanced Raman scattering for quantitative detection of ethyl carbamate in alcoholic beverages

Ethyl carbamate, a by-product of fermentation and storage with widespread occurrence in fermented food and alcoholic beverages, is a compound potentially toxic to humans. In this work, a new approach for quantitative detection of ethyl carbamate in alcoholic beverages, based on surface-enhanced Raman scattering (SERS), is reported. Individual silver-coated gold nanoparticle colloids are used as SERS amplifiers, yielding high Raman enhancement of ethyl carbamate in three kinds of alcoholic beverages (vodka, Obstler, and white rum). The characteristic band at 1,003 cm(-1), which is the strongest and best reproducible peak in the SERS spectra, was used for quantitative evaluation of ethyl carbamate. The limit of detection, which corresponds to a signal-to-noise ratio of 3, was 9.0 × 10(-9) M (0.8 μg · L(-1)), 1.3 × 10(-7) M (11.6 μg · L(-1)), and 7.8 × 10(-8) M (6.9 μg · L(-1)), respectively. Surface-enhanced Raman spectroscopy offers great practical potential for the in situ assessment and identification of ethyl carbamate in the alcoholic beverage industry.

Validation (in-house and collaboratory) of the quantification method for ethyl carbamate in alcoholic beverages and soy sauce by GC–MS

A method for ethyl carbamate (EC) determination in alcoholic beverages and soy sauce was developed by GC–MS. We adopted the diatomaceous earth solid-phase extraction (SPE) column and elution solvent of ethyl acetate/diethyl ether (5:95 v/v) for sample cleaning. The in-house validation showed the limit of quantification (LOQ) was 5.0μg/kg. In the accuracy assay, the total average recovery for was 96.7%. The relative standard deviations (RSDs) were <5%. Subsequently, a collaborative trial was organized for the further validation. The RSDs for repeatability and reproducibility were 1.2–7.8% and 2.3–9.6% respectively. It indicated that the present method performed well in different laboratories.

Ultrasound-assisted emulsification–microextraction for the sensitive determination of ethyl carbamate in alcoholic beverages

A method based on ultrasound-assisted emulsification-microextraction (USAEME) was proposed in this contribution for the determination of ethyl carbamate (EC) in alcoholic beverages using gas chromatography coupled to triple quadrupole mass spectrometry. To achieve the determination of EC in alcoholic beverages, the influences on the extraction efficiency of type and volume of extraction solvent, temperature, ionic strength, alcohol content, and extraction time were studied, once the extraction solvent had been selected. The optimized conditions were 200.0 μL of chloroform at 30 °C during 5 min with 15% (m/v) sodium chloride addition. The detection limit, relative standard deviations, linear range, and recoveries under the optimized conditions were 0.03 μg L(-1), 4.2-6.1%, 0.1-50.0 μg L(-1), and 80.5-87.9%, respectively. Moreover, the feasibility of the present method was also validated by real samples. To the best of our knowledge, this is the first time that USAEME has been applied to determine a strongly hydrophilic compound in alcoholic beverages.

A survey of levels of ethyl carbamate in alcoholic beverages in 2009–2012, Hebei Province, China

Results of a survey of levels of ethyl carbamate (EC) (urethane) in alcoholic beverages carried out in four successive years from 2009 to 2012 by gas chromatography-mass spectrometry (GC/MS) are presented. The beverages were purchased for sampling from Hebei Province of China, including eight main areas of production. The samples comprised wines (n = 212), grain spirits (n = 143) and wine sauces (n = 164). The data show that the average EC content in these kinds of alcoholic beverages remains nearly constant over the years. The results provide valuable data for food authorities to establish maximum limits for EC in China.

Derivatization followed by gas chromatography‐mass spectrometry for quantification of ethyl carbamate in alcoholic beverages

A sensitive and rapid analytical methodology based on derivatization followed by gas chromatography-mass spectrometry (GC-MS) was developed for the quantitative determination of the toxic contaminant ethyl carbamate (EC, urethane, C(2)H(5)OCONH(2)) in alcoholic samples. EC was extracted using liquid-liquid extraction technique, and then silylated with bis-(trimethylsilyl)trifluoroacetamide, analysed finally by GC-MS. The isopropyl carbamate was used as the internal standard for quantitative analysis of EC in alcoholic samples. In this work, the sample extraction and derivatization reaction conditions were investigated, and the optimal extraction conditions obtained were: pH 9 and solvent of ethyl acetate, and the derivatization conditions were: derivatization reaction temperature of 80°C and time duration of 30 min. With the optimal conditions, the method validations were also studied. In the validation studies, EC exhibited good linearity with a regression coefficient of 0.9999. The limit of detection and limit of quantification were 0.30 and 5.0 μg/kg, respectively. The precision was less than 8.4%. Finally, the proposed technique was successfully applied to the analysis of EC in 35 kinds of alcoholic samples. The experimental results have demonstrated that the proposed technique is a fast, reliable and low-cost method for determination of EC in alcoholic samples.

Development of a novel solid-phase extraction, LC-MS/MS method for the analysis of ethyl carbamate in alcoholic beverages: application to South African wine and spirits

Ethyl carbamate (EC) is a known genotoxic carcinogen that is frequently present in alcoholic beverages and is therefore a public health concern. As a consequence, maximum concentration levels for EC in these commodities are legislated in several countries. Quantitative analytical methods are therefore essential to monitor EC levels in beverages. Most published analytical methods for the determination of EC in alcoholic beverages utilise elaborate sample pre-treatment procedures to obtain injectable samples, or yield low sensitivity, for example where direct injection is used. In addition, these procedures often require large volumes of toxic solvents and are not generally applicable to diverse alcoholic beverages. This paper describes a novel procedure for the determination of EC in wines, fortified wines and spirits. The procedure is based on reversed-phase solid-phase extraction (SPE) sample clean-up combined with normal-phase liquid chromatography-atmospheric pressure chemical ionisation tandem mass spectrometric (NP-LC-APCI-MS/MS) analysis. This method provides a rapid, robust and simple analytical procedure suitable for the analysis of a diverse range of alcoholic beverages. The accuracy of the method (expressed as average recovery from diverse matrices) is 94.5%, with limits of detection (LODs) ranging between 0.25 and 0.63 µg l−1 for different matrices. Benefits such as simplified sample preparation, low detection limits, low solvent consumption and good selectivity render the methodology ideally suited to study the occurrence of EC in diverse commodities. The method was applied to study the occurrence of EC in South African wines, fortified wines and spirits. South African wines, aged 1–9 years, contained 1.8–31 µg l−1 EC (RSD = 69%, n = 106), fortified wines aged 2–34 years contained 2.8–79 µg l−1 EC (RSD = 89%, n = 21), and brandies aged 3–20 years contained 4.4–95 µg l−1 EC (RSD = 105%, n = 26). Factors affecting the formation of EC in these commodities were investigated and storage temperature, alcohol content and pH were found to affect the rate of EC formation. Of these variables storage temperature has by far the greatest effect.

Cancer risk assessment of ethyl carbamate in alcoholic beverages from Brazil with special consideration to the spirits cachaça and tiquira

BackgroundEthyl carbamate (EC) is a multi-site carcinogen in experimental animals and probably carcinogenic to humans (IARC group 2A). Traces of EC below health-relevant ranges naturally occur in several fermented foods and beverages, while higher concentrations above 1 mg/l are regularly detected in only certain spirits derived from cyanogenic plants. In Brazil this concerns the sugarcane spirit cachaça and the manioc (cassava) spirit tiquira, which both regularly exceed the national EC limit of 0.15 mg/l. This study aims to estimate human exposure in Brazil and provide a quantitative risk assessment.MethodsThe human dietary intake of EC via alcoholic beverages was estimated based on WHO alcohol consumption data in combination with own surveys and literature data. This data comprises the EC contents of the different beverage groups cachaça, tiquira, other spirits, beer, wine, and unrecorded alcohol (as defined by the WHO; including alcohol which is not captured in routine government statistics nor taxed). The risk assessment was conducted using the margin of exposure (MOE) approach with benchmark doses obtained from dose-response modelling of animal experiments. Lifetime cancer risk was calculated using the T25 dose descriptor.ResultsConsidering differences between pot-still and column-still cachaça, its average EC content would be 0.38 mg/l. Tiquira contained a considerably higher average EC content of 2.34 mg/l. The whole population exposure from all alcoholic beverages was calculated to be around 100 to 200 ng/kg bw/day, with cachaça and unrecorded alcohol as the major contributing factors. The MOE was calculated to range between 400 and 2,466, with the lifetime cancer risk at approximately 3 cases in 10,000. An even higher risk may exist for binge-drinkers of cachaça and tiquira with MOEs of up to 80 and 15, respectively.ConclusionsAccording to our risk assessment, EC poses a significant cancer risk for the alcohol-drinking population in Brazil, in addition to that of alcohol alone. Model calculations show that the implementation of the 0.15 mg/l limit for cachaça would be beneficial, including an increase of the MOE by a factor between 3 to 6. The implementation of policy measures for tiquira and unrecorded alcohol also appears to be advisable.

Determination of Ethyl Carbamate in Sake Using Headspace Solid Phase Microextraction

An analytical method for the determination of ethyl carbamate in alcoholic beverages has been developed and optimized. A combination of headspace solid phase microextraction (HS-SPME), as the extraction technique, and GC/MS, as the determination technique, was utilized. Analytical grade ethyl carbamate dissolved in ethanol solution was analyzed to determine the optimum analytical conditions. Ethyl carbamate-d5 was added as an internal standard. The following HS-SPME conditions were investigated: type of stationary phase of the fibre, ethanol content, sample volume and extraction time. The optimized procedure showed that the detection limit, relative standard deviation, and recovery were 6.7 μg/L, 0.4–2.0%, and 103.6–107.6%, respectively. The precision of this new method was equivalent to previous analyses. Finally, the developed method was applied to the analysis of ethyl carbamate in sake.

Ethyl carbamate in alcoholic beverages from Mexico (tequila, mezcal, bacanora, sotol) and Guatemala (cuxa): market survey and risk assessment.

Ethyl carbamate (EC) is a recognized genotoxic carcinogen, with widespread occurrence in fermented foods and beverages. No data on its occurrence in alcoholic beverages from Mexico or Central America is available. Samples of agave spirits including tequila, mezcal, bacanora and sotol (n=110), and of the sugarcane spirit cuxa (n=16) were purchased in Mexico and Guatemala, respectively, and analyzed for EC. The incidence of EC contamination was higher in Mexico than in Guatemala, however, concentrations were below international guideline levels (<0.15 mg/L). Risk assessment found the Margin of Exposure (MOE) in line with that of European spirits. It is therefore unlikely that EC plays a role in high rates of liver cirrhosis reported in Mexico.

Optimization of headspace solid-phase microextraction for analysis of ethyl carbamate in alcoholic beverages using a face-centered cube central composite design

The headspace solid-phase microextraction (HS-SPME) of ethyl carbamate from alcoholic beverages was optimized for the first time using a face-centered cube central composite design (CCD). The factors expected to influence the extraction process are discussed. Firstly, some of factors are fixed based on the opinion of expert and previous experiments, which reduce the number of factors and then avoid very complex response models and large variability. Secondly, for three remaining inexplicit factors, sample temperature, pH and %NaCl, a face-centered cube central composite design was performed and a response surface equation was derived. The statistical parameters of the derived model were r = 0.974 and F = 20.183. The optimum conditions were obtained using a grid method. Next, the method was analytically evaluated using the optimum conditions. The detection limit, relative standard deviation, linear range and recovery were 3 μg L −1, 4.3–8.6%, 10–160 μg L −1, and 92.8–97.5%, respectively. Finally, the method was applied to a variety of alcoholic beverages.

Automated determination of ethyl carbamate in stone-fruit spirits using headspace solid-phase microextraction and gas chromatography–tandem mass spectrometry

A fully automated procedure using headspace solid-phase microextraction (HS-SPME) followed by gas chromatographic/tandem mass spectrometric (GC/MS/MS) detection was developed for the determination of the toxic contaminant ethyl carbamate (EC) in stone-fruit spirits. After addition of deuterated internal standard, the optimised HS-SPME extraction with carbowax/divinylbenzene fibres (30 min at 70 °C) was done applying salting out with sodium chloride in the presence of pH 7 buffer solution. For quantitative analysis the characteristic fragmentations of m/ z 74 > 44 and m/ z 62 > 44 for ethyl carbamate as well as m/ z 64 > 44 for ethyl carbamate-d 5 were monitored in the multiple reaction monitoring (MRM) mode using a triple quadrupole instrument. In the validation studies, ethyl carbamate exhibited good linearity with a regression coefficient of 0.998. The limits of detection and quantitation were 0.03 and 0.11 mg/l. The precision never exceeded 4.3% (intraday) and 8.2% (interday) at any of the concentrations examined. A good agreement of analysis results in comparison to conventional sample clean-up over diatomaceous earth columns was found ( R = 0.956, Bias = 0.08 mg/l). The new HS-SPME/GC/MS/MS procedure is suitable for the fast, reliable and inexpensive determination of ethyl carbamate in alcoholic beverages in an automated, and therefore, convenient procedure.

酒類中のカルバミン酸エチルの簡易定量法

We developed a simple method for the determination of ethyl carbamate in alcoholic beverages. Butyl carbamate was added to sake sample as an internal standard and then added to Extrelut column. Ethyl carbamate and butyl carbamate were eluted with hexane-ethyl acetate (1: 1). The eluate was concentrated in a rotary evaporator or Turbo Vap concentrator. The concentrate was analyzed by GC/MS in a selected ion monitoring mode. The detection limit and quantification limit of ethyl carbamate were 2.5 and 7.5μg/l. This method was suitable for the analysis of many sake, Shaoxing rice wine (shokoshu), and plum liquor (umeshu) samples. Applying this extraction and concentration method, we could analyze ethyl carbamate in sake by FID-GC. Twenty times concentrated extract of sake obtained the approximate ethyl carbamate concentration.