Analysis of ethyl carbamate in Korean soy sauce using high-performance liquid chromatography with fluorescence detection or tandem mass spectrometry and gas chromatography with mass spectrometry

Food Control 18 (2007) 975–982 www.elsevier.com/locate/foodcont

Analysis of ethyl carbamate in Korean soy sauce using high-performance liquid chromatography with Xuorescence detection or tandem mass spectrometry and gas chromatography with mass spectrometry Sung-Kug Park a, Cheong Tae Kim b, Joo-Won Lee c, Ok Hwa Jhee Ju Seop Kang c, Tae Wha Moon b,¤

c,d

, Ae Seon Om d,

a

Center for Food Standard Evaluation, Korea Food and Drug Administration, 231 Jinheungno, Eunpyeong-gu, Seoul 122-704, Republic of Korea b School of Agricultural Biotechnology and Center for Agricultural Biomaterials, Seoul National University, San 56-1, Sillim-dong, Gwanak-gu, Seoul 151-921, Republic of Korea c Department of Pharmacology, College of Medicine, Hanyang University, 17 Heangdang-dong, Sungdong-gu, Seoul 133-791, Republic of Korea d Department of Food and Nutrition, College of Human Ecology and Institute of Biomedical Science, Hanyang University, 17 Heangdang-dong, Sungdong-gu, Seoul 133-791, Republic of Korea Received 19 December 2005; received in revised form 19 May 2006; accepted 26 May 2006

Abstract A speciWc, sensitive procedure involving high-performance liquid chromatography (HPLC) coupled to tandem mass spectrometry (MS/MS) was developed and compared to other analytical methods for quantiWcation of ethyl carbamate (EC) in Korean soy sauce products. HPLC with a Xuorescence detector was not applicable for monitoring trace amounts of EC in soy sauce due to its low detection limit (20 ppb). The use of gas chromatography (GC) coupled to MS could be applied to soy sauce, but it was not as simple and fast as HPLC/ MS/MS. The GC/MS procedure exhibited excellent linearity over the concentration range of 10–200 ppb with a 0.5 ppb limit of detection (LOD) and 82.7 § 3.1% recovery. A procedure involving HPLC/MS/MS with multiple reaction monitoring was developed. The characteristic transitions of m/z 90 ! 62 for EC as well as m/z 104 ! 62 for propyl carbamate (PC) as the internal standard were monitored. Good linearity was obtained both in the range from 10 to 100 ppb and in the range from 0.1 to 20 ppb with a LOD of 0.05 ppb. The average recovery was 92.2 § 1.7%. The applicability of the GC/MS and developed HPLC/MS/MS methods was demonstrated by detection of EC in 12 kinds of commercial Korean soy sauce products at levels of 0.5 and 0.1 ppb, respectively. The new HPLC/MS/MS method provided greater sensitivity with a simpler and shorter conWrmatory analysis than the GC/MS method. © 2006 Elsevier Ltd. All rights reserved. Keywords: Ethyl carbamate; HPLC/MS/MS; Korean soy sauce

1. Introduction Trace amounts of several carcinogens are found in daily foods, and frequent consumption of these foods could lead to considerable exposure, causing concern for public health.

*

Corresponding author. Tel.: +82 2 880 4854; fax: +82 2 873 5095. E-mail address: [email protected] (T.W. Moon).

0956-7135/$ - see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2006.05.013

Ethyl carbamate (EC), which is found in fermented foods and alcoholic beverages, is a by-product of fermentation and storage (Stevens & Ough, 1993; Zimmerli & Schlatter, 1991). EC is a known genetic carcinogen and is metabolized into vinyl carbamate followed by epoxidation, which leads to DNA adduction and mutation in several organs including the lungs, liver, and mammary glands (Mirvish, 1968). The use of diethyl dicarbonate (DEDC) as an antimicrobial food additive in fruit juice has been prohibited in the

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United States (Ough, 1976) because DEDC reacts with ammonia to produce EC (Diachenko, Canas, Joe, & Inovi, 1992). Furthermore, the National Health Institute of Canada has prohibited the use of urea in alcoholic beverages and established an upper limit for the concentration of EC depending on the type of alcoholic beverage (Conacher et al., 1987). Numerous studies have described the presence of EC in alcoholic beverages and fermented foods. Canas et al. (1989) reported that EC levels ranged from 0 to 3 ppb in cheeses, teas, yogurts, and ciders; from 0 to 13 ppb in breads and malt beverages; and from 0 to 84 ppb in soy sauces. Hasegawa et al. (1990) noted the presence of EC in various fermented Japanese foods. They reported that miso, natto, moromi, sake, and soy sauce contained 5, 5, 10, 100, and 50 ppb of EC at the highest level, respectively. It has also been reported that EC is present in several traditional Korean foods, including soy sauce, soybean paste, red pepper paste, kimchi, and fermented rice beverages, among which soy sauce shows the highest concentration (Kim, Kim, Lee, & Noh, 1995; Kim, Koh, Chung, & Kwon, 2000; Koh & Kwon, 1996). To reveal the trace amounts of EC in fermented foods, GC/Xame ionization detection (FID), GC/alkali Xame ionization detection (AFID), and GC/Hall electrolytic conductivity detection (HECD) methods have been applied, but were insuYciently sensitive. Although the modiWed GC method introduced by Walker, Winterlin, Fouda, & Seiber (1974) was able to detect EC at levels as low as 10 ppb (Roach, Roseboro, & Fazio, 1977), additional analysis with GC/MS was needed to conWrm the quality and quantity of the EC. Hence, the GC/MS method established by Ough (1976) has been generally used for determining EC in alcoholic beverages. The high-performance liquid chromatography (HPLC) method, which has a short sample preparation time and high sensitivity, has been employed to detect EC in alcoholic beverages as well (Herbert, Santos, Bastos, Barros, & Alves, 2002). However, these methods may not be suitable for analyzing EC in traditional fermented Korean foods such as soy sauce, soybean paste, and red pepper paste, which have complicated matrix properties diVerent from those of alcoholic beverages. In the present study, a speciWc, sensitive procedure involving HPLC coupled with tandem mass spectrometry (HPLC/MS/MS) was developed and compared to other analytical methods for quantifying EC in Korean soy sauce, which has the highest concentration of EC among traditional fermented Korean foods. 2. Materials and methods 2.1. Materials Seven commercial fermented soy sauce products and six commercial soy sauce products, which are mixtures of fermented and acid-hydrolyzed soy sauce, were obtained from two local markets. EC, butyl carbamate (BC), propyl

carbamate (PC), and aluminum oxide (activated, neutral) were purchased from Sigma Chemicals Co. (St. Louis, MO, USA). The moisture content of the aluminum oxide was adjusted to 10.0% (w/w) for deactivation before use. The 9-xanthydrol was purchased from Fluka Chemicals Co. (Buchs, Switzerland). Celite 545 (not acid-washed) was obtained from Sigma Chemicals Co. and dried at 700 °C for 16 h. Florisil (60–100 mesh) was purchased from Sigma Chemicals Co. and C18 resins (125Å for bulk packing) from Waters Co. (Milford, MA, USA). The Extrelut NT 20 column was obtained from Merck Co. (Darmstadt, Germany). All other reagents were HPLC or analytical grade. 2.2. Stock solutions EC was used as an external standard, and BC and PC were used as internal standards. Each standard solution was prepared by gradual dilution of standard stock solutions (1000 g/ml) with methanol. 2.3. Sample preparation 2.3.1. HPLC with Xuorescence detection (FLD) The sample (20 g) was spiked with PC (250 ppb) and extracted with 20 ml of ethyl acetate twice. The sample extract solution was transferred to a 250 ml separatory funnel containing 7 g of NaCl. After the sample was shaken vigorously for 45 s, the lower aqueous layer was drained oV, and the organic layers were combined and dried by passage through anhydrous sodium sulfate. The dried organic layer was then concentrated in a rotary evaporator (30 °C, 300 mbar). The concentrated residue was dissolved in 500 l of 20% ethanol, and then 100 l of 0.02 M xanthydrol was added followed by 50 l of 1.5 M HCl. The derivatization reaction was conducted for 5 min in the dark, and 20 l was injected into the chromatographic system. 2.3.2. HPLC/MS/MS The sample (10 g) and PC (40 ppb) were transferred into a 100-ml beaker and mixed well. The sample was then placed on an Extrelut NT 20 column. After 10 min, EC and PC were eluted with 300 ml of dichloromethane. The eluate was concentrated in a rotary evaporator (30 °C, 300 mbar), and residual dichloromethane was removed under a nitrogen stream. The residues were dissolved in 2 ml of water, passed through a 0.45 m membrane (Sartorius, Goettingen, Germany), and collected for analysis. The extracts were directly injected into the HPLC/MS/MS system. 2.3.3. GC/MS The sample (15 g), BC (40 ppb), and Celite (15 g) were transferred into a 100 ml beaker and then well mixed. The mixture was loaded onto a glass column (2 £ 40 cm) containing 10 g of deactivated aluminum and 40 g of anhydrous sodium sulfate. The column was eluted with 100 ml of dichloromethane, and the eluate was then concentrated in a rotary evaporator (30 °C, 300 mbar). After a glass column

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(1.5 £ 30 cm) was packed with 10 g of Florisil and washed with 60 ml of dichloromethane, the concentrated sample in 1 ml of methanol was loaded and rewashed with 45 ml of dichloromethane. EC was eluted from the column with 45 ml of 7% methanol/dichloromethane and concentrated in a rotary evaporator (30 °C, 300 mbar). Finally, the concentrated residue was dissolved in 1 ml of methanol and analyzed for EC using the GC/MS system.

210 °C. The mass spectrometer was set for selected ion monitoring (SIM) at m/z 62, 74, and 89 (Fig. 5) as follows: ion source, 250 °C; and ionization mode, electron impact with energy of 70 eV. Peaks containing all three daughter ions (m/z 62, 74, and 89) were used for identiWcation of EC. For quantiWcation of EC in the samples, the signal intensity at m/z 62 for EC was compared with that for the internal standard, BC.

2.4. Equipment and analysis

2.5. Validation studies

EC analysis was conducted with a HPLC system (HP Agilent 1100 series; Hewlett Packard Co., Palo Alto, CA, USA) coupled to a Xuorescence detector (Hewlett Packard Co.) and controlled by Chemstation software (Hewlett Packard Co.). The Zorbax Eclipse AA column (4.6 £ 150 mm, 5 m; Agilent Technologies, Palo Alto, CA, USA) was used, and the injected volume was 20 l. The mobile phases A and B were acetonitrile and a 20 mM sodium acetate solution (pH 7.2), respectively. The elution program was as follows: from 0 to 5 min, the percentage of eluant A increased from 30% to 60%; from 5 to 20 min, the percentage of eluant A increased from 60% to 85% with a constant Xow of 0.75 ml/min; from 20 to 25 min, the percentage of eluant A decreased from 85% to 30% with a constant Xow of 0.75 ml/min; and from 25 to 30 min, initial conditions were restored and maintained for 1 min before a new analysis started. The HPLC/MS/MS system consisted of a HPLC solvent delivery system (Sykam S2100; Sykam GmbH, Eresing, Germany) coupled to MS/MS with an ESI source (Quattro Micro Co., Manchester, UK) and MassLynx 4.0 software (Waters Co.) for operation of the device and spectral analysis. The samples (20 l) were injected onto a Capcell Pak C18 column (2.0 £ 250 mm, 5 m; Shiseido Co., Tokyo, Japan) and separated using a mobile phase consisting of aqueous 0.1% acetic acid and 10% acetonitrile at a Xow rate of 0.2 ml/min for 20 min. The conditions were as follows: capillary voltage, 3.3 kV; source temperature, 120 °C; desolvation gas temperature, 200°C; desolvation gas Xow, 650 L/h; and collision argon gas pressure, 2.5 mbar for MS/ MS. For quantitative analysis the characteristic fragmentations were monitored in multiple reaction monitoring (MRM) mode: m/z 90.0 and m/z 61.8 (daughter ion) for EC, and m/z 104.1 and m/z 61.8 for PC as the internal standard (Fig. 4). For all MRM, the dwell time was about 1 s and the inter-scan delay time was about 0.2 s. The GC/MS system (Hewlett Packard Co.) used for analysis was a GC (HP 6890) with a MS spectrometer (HP 5973) and was controlled by Chemstation software. Substances were separated on a capillary DB-WAX column (0.32 m £ 30 m, 0.25 m coating; J & W ScientiWc Inc., Folsom, CA, USA). Chromatographic conditions were as follows: carrier gas (helium) Xow, 1.8 ml/min; injection mode, splitless; injection volume, 1.0 l; oven temperature program, 70 °C hold for 2 min, 8 °C/min to 150 °C, hold for 0 min, 20–230 °C, hold for 10 min; and injection port at

For validation of the methods, three authentic sample solutions with various concentrations (0.1–1000 ppb) of EC were prepared. The recoveries were calculated as the percentage of EC recovered by each extraction process after spiking the samples with known amounts of EC. The linearity of the standard curves was evaluated in the range from 50 to 1000 ppb for HPLC/FLD, from 0.1 to 100 ppb for HPLC/MS/MS, and from 10 to 200 ppb for GC/MS for each of Wve points (n D 3). LOD was deWned as the lowest EC concentration with estimated peak height greater than three times the noise levels (S/N > 3). 3. Results and discussion 3.1. HPLC/FLD analysis Even though the HPLC/FLD method may be suitable for analyzing EC in alcoholic beverages, there are some limitations, including the limit of detection, simplicity, and reliability, to its use with a large-throughput analysis of complex traditional foods. In a nucleophilic substitution reaction, the derivatization reagent xanthydrol speciWcally reacts with the nucleophilic amine group of EC (Herbert et al., 2002). Based on the formation of Xuorescent xanthyl EC after reacting with xanthydrol in the presence of an acidic catalyst (Fig. 1), EC derivatives can be analyzed by the HPLC/FLD method. The xanthyl EC and PC were eluted at 11.43 and 13.23 min, respectively (Fig. 2), and total run time was about 30 min, without any interference peaks. The LOD was 20 ppb, which was about Wve times higher than the value reported by Herbert et al. (2002). This may have been caused by relatively lower formation of EC derivatives due to interfering components, including pigments in the soy sauce samples. Because of the higher level of LOD, it would be diYcult to apply this method in O

H

NH

OH

O

O HCl

+ O

9-Xanthydrol

NH2

O

Ethyl carbamate

O

Xanthyl ethyl carbamate

Fig. 1. Xanthyl ethyl carbamate formation from 9-xanthydrol and ethyl carbamate under acidic conditions.

S.-K. Park et al. / Food Control 18 (2007) 975–982

a

11.435 Ethylcarbamate

978

LU 90

13.213 IS

60

30

0 0

10

15

20

25

30 min

30

13.237 IS

LU 60

11.459 Ethylcarbamate

b

5

0

0

5

10

15

20

25

30

min

Fig. 2. HPLC/FLD chromatograms of ethyl carbamate and propyl carbamate. ((a) standard, (b) soy sauce).

assessing a trace amount of EC in the sample. The response of EC derivatives showed a good linearity over the range from 50 to 1000 ppb with a regression coeYcient of 0.9990. 3.2. HPLC/MS/MS analysis Because all materials have speciWc mass spectra that can be used to identify them, three to Wve representative ions of the speciWc compound should be selected. It is very important to select ions that are structurally relevant to the target compound (Kim, 2004). In the actual sample, matrix interferences may emerge and contribute to noise. Therefore, when the target compound is present at a low level, it is very diYcult to select suitable ions for identiWcation and quantiWcation. A HPLC/MS/MS method was developed to analyze EC in soy sauce without involving any derivatization procedure of EC. Fig. 4(a) shows the electrospray ionization (ESI+) full and daughter ion spectrum of EC, where the ions of m/z 91, 90, 62, 61, and 60 contained structurally relevant information on EC in the ESI+ full scan mode. The mass spectrum of EC shows a protonated molecule ([M + H]+) at m/z 90. One of the most important aspects when analyzing EC using HPLC/MS or HPLC/MS/MS is minimizing the interface ions that increase the background signal from the mobile phase and/or samples. Therefore, we used MRM for identiWcation and quantiWcation of a trace amount of EC in the sample. The daughter-ion scan spec-

trum was obtained from the characteristic ion at m/z 90 including information on the protonated EC molecule ion ([M + H]+). The daughter ion at m/z 62 resulted from the loss of a C2H4 group in EC, which may be attributable to -rearrangement and -cleavage eVects (MaLaVety & Turecek, 1993). Thus, the daughter ion at m/z 62 was used for the identiWcation and quantiWcation of EC. The protonated molecular ion ([M + H]+) of PC (internal standard) appeared at m/z 104 in HPLC/MS/MS in the ESI+ mode. Fig. 4(b) shows the full and daughter-ion scan spectra of PC. The ions of m/z 105, 104, 83, 62, and 61 contain structurally relevant information about PC in the ESI+ full scan mode. The ion of m/z 62, representing the characteristic peak of PC, resulted from the loss of a C3H6 group due to -rearrangement and -cleavage. The daughter ion scan spectrum was obtained from the characteristic ion at m/z 104, including information on the protonated PC ion ([M + H]+). The daughter ion at m/z 62 produced by the loss of a C3H6 group from PC was used for identiWcation and quantiWcation. The transition ions m/z 90 ! 62 (EC) and m/ z 104 ! 62 (PC) were found to be the most reliable for identiWcation and quantiWcation in MRM, and the chromatograms obtained for EC and PC are shown in Fig. 3. The LOD and LOQ were 0.05 and 0.1 ppb, respectively, which were very sensitive responses compared to those for alcoholic beverages (Benjamin & Frank, 1988; Ough, 1976), suggesting that this method may be useful for detecting

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979

a EC46.4,PC48_5_250_2ML EC04081105

2: MRM of 1 Channel ES+ 104.1 > 61.8 1.07e4

100

ISTD

%

0 EC04081105 100

1: MRM of 1 Channel ES+ 90 > 61.8 8.57e3

STD_EC %

0

Time 2.00

4.00

6.00

8.00

10.00

12.00

b KJ-10_10ML_2ML EC-KCT011

14.00

2: MRM of 1 Channel ES+ 104.1 > 61.8 1.80e3

100

ISTD %

0 EC-KCT011

1: MRM of 1 Channel ES+ 90 > 61.8 2.29e3

100

Sample_EC %

0

Time 2.00

4.00

6.00

8.00

10.00

12.00

14.00

Fig. 3. HPLC/MS/MS chromatograms of ethyl carbamate and propyl carbamate. ((a) standard, (b) soy sauce).

trace amounts of EC in foods such as soy sauce. It showed a good linearity, with a regression coeYcient of 0.9995 and a relative average standard deviation of 0.2% in the range from 10 to 100 ppb and 0.9984 in the range from 0.1 to 20 ppb. The accuracy of the method is summarized in Table 1. The average recovery was 92.2%, with a relative standard deviation (RSD) of 1.7%. 3.3. GC/MS analysis The extraction procedures included dichloromethane extraction combined with alumina and Florisil solid-phase extraction according to the method used by Canas et al. (1989). The retention times of EC and BC (internal standard) were 10.5 and 13.1 min, respectively. As shown in the mass spectrum of EC (Fig. 5), the molecular ions at m/z 89, the ion fragment from the loss of a methyl group at m/z 74, and the ion fragment resulting from McLaVerty rearrangement at m/z 62 were predominantly formed. Consistent with the report by Brumley et al. (1988), the ion at m/z 62 characterizing the carbonyl structure showed the most abundant peak and selectivity, leading to the selection of

this fragment ion at m/z 62 for quantiWcation of EC. The LOD was 0.5 ppb, and the calibration curve showed good linearity (r D 0.9994) in the range from 10 to 200 ppb. The current AOAC oYcial method using GC/MS-SIM, which is applicable to determination of ethyl carbamate levels of 15–70 g/kg in soy sauce, has the LOD of 1 ppb (AOAC, 2000). The accuracy of the method established is summarized in Table 1. Recovery using this GC/MS-SIM method was 82.7% with a RSD of 3.1%. Even though we developed a GC/MS method with improved sensitivity compared to other GC/MS methods, it seemed to be time-consuming for cleanup and to have an inappropriate analytical sensitivity for trace EC analysis in the fermented Korean soy sauce sample. 3.4. Soy sauce analysis Using the HPLC/MS/MS and GC/MS methods established above, we determined the amounts of EC in fermented soy sauce samples (seven kinds) and in samples of fermented soy sauce mixed with acid-hydrolyzed soy sauce (six kinds). As shown in Table 2, EC concentrations in

980

S.-K. Park et al. / Food Control 18 (2007) 975–982

Fig. 4. Full and daughter-ion scan spectra of ethyl carbamate (a) and propyl carbamate (b). Table 1 Recovery of ethyl carbamate from soy sauce by HPLC/MS/MS and GC/MS Spiked EC conc. (ppb]

20.0 40.0 80.0 Average

HPLC/MS/MS

GC/MS

Assayed (ppb)

Recovery (%)

RSD (%)

Assayed (ppb)

Recovery (%)

RSD (%)

18.0 36.7 76.0

90.0 91.7 95.0 92.2

2.2 1.7 1.1 1.7

16.1 32.9 68.0

80.7 82.3 85.0 82.7

5.0 3.1 1.2 3.1

fermented soy sauce and fermented soy sauce mixed with acid-hydrolyzed soy sauce were in the range of 0–99.9 ppb and 0–4.0 ppb, respectively. EC was detected in 11 samples by the HPLC/MS/MS method and in 9 samples by the GC/

MS method. Therefore, the HPLC/MS/MS method was more sensitive and applicable in monitoring the trace EC in soy sauce samples than the GC/MS method. The detection of EC in sample C by only the GC/MS method may have

S.-K. Park et al. / Food Control 18 (2007) 975–982

981

Fig. 5. GC/MS chromatograms of ethyl carbamate and butyl carbamate ((a) standard, (b) soy sauce).

Table 2 Concentration of ethyl carbamate in soy sauce samples collected from local markets (unit: ppb) Soy sauce samples

Fermented soy sauce mixed with acid-hydrolyzed soy sauce

Fermented soy sauce

ND: Not detected.

Analytical method

A B C D E F G H I J K L M

HPLC/MS/MS

GC/MS

0.1 3.5 ND 0.7 0.2 0.8 1.5 ND 88.6 17.3 45.6 2.1 22.0

ND 4.0 2.0 1.0 ND 0.5 ND ND 99.9 19.3 49.8 3.1 17.5

been caused by an overestimation resulting from fragment ions produced by components structurally similar to EC. It has been reported that the fragment ion at m/z 74 is a common ion for all alkyl methyl esters and is susceptible to chemical interference even after extensive sample cleanup (Lachenmeier, Frank, & Kuballa, 2005; Lau, Weber, & Page, 1987). 4. Conclusions A sensitive and speciWc HPLC/MS/MS method was applied for the Wrst time to quantitatively determine EC in traditional Korean soy sauce, which diVers from alcoholic beverages in its complex matrix. The developed method showed satisfactory validation parameters in terms of linearity, limit of detection, and accuracy. It had higher recovery and repeatability than the HPLC/FLD and GC/ MS methods. This new procedure is rapid and sensitive and

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could be utilized for regular monitoring of EC in fermented foods with complex matrices. References AOAC (2000). AOAC OYcial Method 994.07: Ethyl carbonate in alcoholic beverages and soy sauce. In Association of OYcial Analytical Chemists (Ed.), AOAC OYcial Methods of Analysis (17th, pp. 14–15). Gaithersburg, MD: AOAC International. Benjamin, S. C., & Frank, R. (1988). Detection and quantitation of trace levels of ethyl carbamate in alcoholic beverage by selective ion monitoring. Journal of Agricultrual and Food Chemistry, 36, 502–505. Brumley, W. C., Canas, B. J., Perfetti, G. A., Mossoba, M. M., Sphon, J. A., & Corneliussen, P. E. (1988). Quantitation of ethyl carbamate in whisky, sherry, port and wine by gas chromatography/tandem mass spectrometry using a triple quadrupole mass spectrometer. Analytical Chemistry, 60, 975–978. Canas, B. J., Harvery, D. C., Robinson, L. R., Sulivan, M. P., Joe, F. L., Jr., & Diachenko, G. W. (1989). Ethyl carbamate levels in selected foods and beverages. Journal of the Association of OYcial Analytical Chemists, 72, 873–876. Conacher, H. B., Page, B. D., Lau, B. P., Lawrence, J. F., Bailey, R., Calway, P., et al. (1987). Capillary column gas chromatographic determination of ethyl carbamate in alcoholic beverages with conWrmation by gas chromatography/mass spectrometry. Journal of the Association of OYcial Analytical Chemists, 70, 749–751. Diachenko, G. W., Canas, B. J., Joe, F. L., & Inovi, M. (1992). Ethyl carbamate in alcoholic beverages and fermented foods. In J. W. Finely, S. F. Robinson, & D. J. Armstrong (Eds.), Food safety assessment (p. 419). Washington DC: American Chemical Society. Hasegawa, Y., Nakamura, Y., Tonogai, Y., Terasawa, S., Ito, Y., & Uchiyanma, M. (1990). Determination of ethyl carbamate in various fermented foods by selected ion monitoring. Journal of Food Protection, 53, 1058–1061. Herbert, P., Santos, L., Bastos, M., Barros, P., & Alves, A. (2002). New HPLC Method to determine ethyl carbamate in alcoholic beverages using Xuorescence detection. Journal of Food Science, 67, 1616–1620.

Kim, C. T. (2004). Reduction of acrylamide in fried foods by addition of amino acids and vacuum frying. PhD thesis, Seoul National University. Kim, E. J., Kim, D. K., Lee, D. S., & Noh, B. S. (1995). Application of acid urease to prevent ethyl carbamate formation in Takju processing. Foods and Biotechnology, 4, 34–38. Kim, Y. K., Koh, E., Chung, H. J., & Kwon, H. (2000). Determination of ethyl carbamate in some fermented Korean foods and beverages. Food Additives and Contaminants, 17, 469–475. Koh, E., & Kwon, H. (1996). Determination of fermentation speciWc carcinogen, ethyl carbamate, in kimchi. Korean Journal of Food Science and Technology, 28, 421–427. Lachenmeier, D. W., Frank, W., & Kuballa, T. (2005). Application of tandem mass spectrometry combined with gas chromatography to the routine analysis of ethyl carbamate in stone-fruit spirits. Rapid Communications in Mass Spectrometry, 19, 108–112. Lau, B. P. Y., Weber, D., & Page, D. (1987). Gas chromatographic-mass spectrometric determination of ethyl carbamate in alcoholic beverages. Journal of Chromatography, 402, 233–241. Mirvish, S. S. (1968). The carcinogenic action and metabolism of urethane and N-hydroxyurethane. Advances in Cancer Reseaech, 11, 1–42. Ough, C. S. (1976). Ethyl carbamate in fermented beverages and foods. I. Naturally occuring ethyl carbamate. Journal of Agricultural and Food Chemistry, 24, 323–328. Roach, J. A. G., Roseboro, E. L., & Fazio, T. (1977). Determination of urethane in wines by gas-liquid chromatography and its conWrmation by mass spectrometry. Journal of the Association of OYcial Analytical Chemists, 60, 509–516. Stevens, D. F., & Ough, C. S. (1993). Ethyl carbamate formation: reaction of urea and citurulline with ethanol in wine under low to normal temperature conditions. American Journal of Enology and Viticulture, 44, 309–312. Walker, G., Winterlin, W., Fouda, H., & Seiber, J. (1974). Gas chromatographic analysis of urethane in wine. Journal of Agricultural and Food Chemistry, 22, 944–947. Zimmerli, B., & Schlatter, J. (1991). Ethyl carbamate: analytical methodology, occurrence, formation, biological activity and risk assessment. Mutation Research, 259, 325–350.