2172
J. Sep. Sci. 2014, 37, 2172–2176
Pinggu Wu1 Liqun Zhang2 Liyuan Wang1 Jing Zhang1 Ying Tan1 Jun Tang1 Bingjie Ma1 Xiaodong Pan1 Wei Jiang1
Research Article
Simultaneous determination of ethyl carbamate and 4-(5-)methylimidazole in yellow rice wine and soy sauce by gas chromatography with mass spectrometry
1 Center
for Disease Control and Prevention of Zhejiang Province, Hangzhou, P. R. China 2 Center for Disease Control and Prevention of Hangzhou, Hangzhou, P. R. China Received February 8, 2014 Revised April 30, 2014 Accepted May 15, 2014
We developed a new method, based on alkaline diatomite solid-phase extraction followed by gas chromatography with mass spectrometry, for the simultaneous determination of the toxic contaminants ethyl carbamate (EC) and 4-(5-)methylimidazole (4-MEI) in yellow rice wine and soy sauce. The optimal extraction conditions were defined. With the application of alkaline diatomite solid-phase extraction, damage to the capillary column by organic acids was greatly reduced. With deuterated EC used as the internal standard, the linearity of the calibration curves for EC and 4-MEI was good with correlation coefficient above 0.99. In a spiked experiment with EC and 4-MEI in yellow rice wine and soy sauce, recovery of the added EC was 80.5–102.5% and that of 4-MEI was 78.3–92.8%. The limit of quantification and limit of detection for EC were 6.0 and 2.0 g/kg, respectively, and for 4-MEI were 15.0 and 5.0 g/kg, respectively. The validation results demonstrate that the method is fast, simple, and selective, and therefore is suitable for simultaneously determining the presence of EC and 4-MEI in fermented food. Keywords: Ethyl carbamate / Gas chromatography with mass spectrometry / Methylimidazole / Soy sauce / Yellow rice wine DOI 10.1002/jssc.201400141
1 Introduction Ethyl carbamate (EC, urethane, C2 H5 OCONH2 ) is a genotoxic carcinogen that is present in many fermented foods and beverages [1, 2]. The International Agency for Research on Cancer recently upgraded its classification of EC to group 2A (probably carcinogenic to humans) [3]. The neurotoxic agent 4-(5-)methylimidazole (4-MEI) is generated during the process of caramelization, which is mainly present in class III ammonia caramel (INS No. 150c) or class IV ammonia sulfite caramel (INS No. 150d) [4, 5]. A recent toxicological study showed that 4-MEI can induce alveolar/bronchiolar adenoma and carcinoma in male and female mice [6]. The International Agency for Research on Cancer recently upgraded its classification of 4-MEI to group 2B (possibly carcinogenic to humans) [7]. EC is generated during the fermentation process of some foods and beverages, such as soy sauce and yellow rice wine. Subsequently, caramels are added to parts of the fermented foods in order to stimulate or improve appetite. EC and 4-MEI are hazardous contaminants generated during these Correspondence: Dr. Liqun Zhang, Hangzhou Center for Disease Control and Prevention, Hangzhou, P. R. China E-mail: [email protected] Fax: +86-571-8795-1895
Abbreviations: EC, )methylimidazole
ethyl
carbamate;
4-MEI,
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
4-(5-
processes. Both soy sauce and yellow rice wine are fermented food with caramels as coloring agents, so EC and 4-MEI could be present in these foods simultaneously. Therefore, it is important to establish a method for the simultaneous analysis of EC and 4-MEI in fermented foods and beverages. Many analytical techniques based on GC–MS for determining EC in targeted samples have been reported [8–14]. Sample pretreatment procedures are liquid-liquid extraction (LLE) [8,9,15,16], SPE [11,17,18], and solid-phase microextraction (SPME) [13,19,20]. Several methods have been developed to determine 4-MEI based on TLC [21], fluorimetry [22], capillary electrophoresis [23], HPLC [24], LC–MS [25–27], and GC– MS [28, 29]. The use of GC–MS methods based on ion pairextraction with bis-2-ethyhexilphosphate and isobutylchloroformate derivatization has been used successfully to determine levels of 4-MEI [28, 29]. However, a method to determine the simultaneous presence of EC and 4-MEI has not been reported. Therefore, the objectives of this study were (1) to establish a method for determining the simultaneous presence of EC and 4-MEI in fermented foods by GC–MS without derivatization and (2) to evaluate the optimal extraction conditions and the methodological validations used, including linearity, recovery, LOD, LOQ, precision, and accuracy. Colour Online: See the article online to view Figs. 1B, 2A and 3B in colour.
www.jss-journal.com
Gas Chromatography
J. Sep. Sci. 2014, 37, 2172–2176
2 Materials and methods 2.1 Reagents and materials HPLC grade n-hexane, ether, ethyl acetate, and methanol were obtained from Fisher Chemical, Loughborough, UK. Anhydrous sodium sulfate was heated at 450⬚C for 4 h, then cooled to room temperature and maintained dry. Standard EC and 4-MEI (purity>99.0%) were obtained from Sigma Chemical, St. Louis, MO, USA. Deuterated standard of EC d5 -ethyl carbamate (d5-EC) was purchased from Cerilliant, Round Rock, TX, USA. Alkaline diatomite SPE columns (4000 mg filler per 12 mL) were obtained from Fuyu Tec., Hangzhou, China. 2.2 Preparation of standards Stock standard solutions of EC, 4-MEI, and d5-EC were prepared in methanol at a concentration of 1000 mg/L, and then diluted to 1.0, 2.0, and 2.0 mg/L with methanol. Working standard solutions of EC and 4-MEI at 10, 50, 100, 200, 500, and 1000 g/L were prepared in methanol, with an internal standard at a concentration of 100 g/L. 2.3 Preparation of samples A 2.0 g sample of yellow rice wine or soy sauce and 50 L of 2.0 mg/L d5-EC were added into a centrifuge tube and homogenized. After a 1 min vortex, the mixture was poured slowly into an alkaline diatomite SPE column. After 10 min, the analyte was eluted with 5 mL n-hexane followed by elution with 10 mL of ethyl acetate by vacuum. The collected eluate was dried by anhydrous sodium sulfate (first, wetted by ether, then washed with 2.0 mL ether) and concentrated to 0.5 mL via N2 flow at 30⬚C. Finally, the analyte was diluted to 1 mL with methanol for GC–MS testing.
2173
by monitoring the ion at m/z value of 82, 81, 109, and 182, and m/z 82 was selected as the quantitative ion. The internal standard for d5-EC was detected by monitoring the ion at m/z value of 44, 64, and 76, and m/z 64 was selected as the quantitative ion. EC was quantified with calibration curves constructed from the EC/d5-EC peak area ratios (m/z 62 vs. 64) for standards containing EC of 50, 100, 200, 500, and 1000 g/L. The 4-MEI was quantified with calibration curves constructed from the 4-MEI/d5-EC peak area ratios (m/z 82 vs. 64) for standards containing 4-MEI of 50, 100, 200, 500, and 1000 g/L.
2.5 Spiking sample preparation Yellow rice wine and soy sauce were purchased from local markets. Both of these two different matrices with different blank levels were spiked with three different concentrations in order to determine the recoveries. The spiked levels of EC were 25, 100, 300 g/kg in yellow rice wine, whereas the spiked levels of 4-MEI were 30, 60, and 100 g/kg. The spiked levels of EC were 20, 50, and 100 g/kg in soy sauce, whereas the spiked levels of 4-MEI were 30, 60, and 100 g/kg. Each test was performed six times.
2.6 Validation procedure The proposed analytical method was validated in terms of linearity, extraction recovery precision, accuracy, and selectivity. To demonstrate linearity, the regression coefficient was calculated using d5-EC as internal standard for EC and 4-MEI; the spiked calibrants were analyzed in duplicate during the measurement of the samples. The percentage recovery of EC and 4-MEI was determined by a spiking experiment of two different matrices. Reproducibility was examined by analyzing a positive authentic sample containing EC or 4-MEI. A full set of calibration standards and a blank standard were run with each analysis.
2.4 Chromatographic analysis with GC–MS The GC–MS analysis was completed by use of an Agilent 6890 GC-5973MS instrument. The column was of 30 m × 0.25 mm ID × 0.25 m DB-Innowax capillary column (Agilent Technologies). The oven temperature was programmed from 50⬚C, held for 1 min, increased to 180⬚C at 8⬚C/min, increased again to 230⬚C at 15⬚C/min, and then held for 2 min. Finally, the oven temperature reached 240⬚C and was held for 5 min. The carrier gas was helium (purity ࣙ 99.999%), with constant pressure at a flow rate of 1.0 mL/min at 50⬚C. The sample (1 L) was injected into the split/splitless inlet in splitless mode at 200⬚C. The mass spectrometer was operated in electron impact ionization mode with a source temperature of 230⬚C and an electron multiplier of 70 eV. EC was detected by selective ion mode (SIM) of the major ions at m/z values of 44, 62, 74, and 89, in which m/z 62 was selected as the quantitative ion. The 4-MEI was detected C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
3 Results and discussion 3.1 GC–MS conditions A polar column, such as Innowax (30 m × 250 m × 0.20 m) [30], has often been used in the analysis of EC by GC– MS. For the separation of 4-MEI, either a polar column or nonpolar column, such as DB-5 column (100 m × 320 m × 0.20 m) [29], HP-88 column (100 m × 250 m × 0.20 m) [31], DB-FFAP column (30 m × 320 m × 0.20 m), or Innowax column (30 m × 250 m × 0.20 m), can be used. The efficiency of various capillary columns was compared, and the Innowax column (30 m × 250 m × 0.20 m) was chosen for detection of electron impact source (EI-Source) and 4-MEI. The total ion chromatogram of EC and 4-MEI, in given GC–MS conditions, was shown in Fig. 1, which www.jss-journal.com
2174
J. Sep. Sci. 2014, 37, 2172–2176
P. Wu et al.
Figure 1. (A) GC–MS total ion chromatogram of EC (200 g/L), D5-EC (100 g/L), and 4-MEI (200 g/L) (D5-EC: 10.46 min; EC: 10.53 min; 4-MEI: 14.33 min) and (B) the extracted ion chromatogram of the quantifier ion of D5-EC, EC, and 4-MEI.
illustrates that EC and 4-MEI could be well separated.
3.2 Optimization of sample pretreatment For the clean-up of yellow rice samples, the alkaline diatomite SPE column was applied in this experiment, which was used by us previously in determining the concentration of EC in yellow rice wine [32]. With use of this column, most of the acids were removed. Hence, it was chosen for the simultaneous clean-up of EC and 4-MEI; because it had worked well for the clean-up of EC, this sample pretreatment process would be much simpler. Solvents used in the elution of EC from an alkaline diatomite column include 5% ethyl acetate/ether and ethyl acetate. Although we had used a more polar solvent, 5% ethyl acetate/ether (15 mL), for elution of EC, it was not very efficient for the elution of 4-MEI. Thus, taking into account the volume of solvents used, the recovery, and the concentration of the interferences, the best elution solvent was 10 mL ethyl acetate, which yielded a recovery of EC and 4-MEI above 75%. This method was environment friendly, because less organic solvent was used. Two kinds of matrices were examined. The GC–MS total ion chromatogram of real yellow rice wine sample is shown in Fig. 2, in which the concentration of EC was C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 2. (A) The GC–MS total ion chromatogram of real yellow rice wine and (B) the extracted ion chromatogram of the quantifier ion of D5-EC, EC, and 4-MEI in real yellow rice wine samples.
10.4 g/L, whereas that of 4-MEI was under the LOD. The GC–MS total ion chromatogram of soy sauce spiked with EC and 4-MEI (30 g/kg) is shown in Fig. 3.
3.3 Method performance Calibration curves were constructed by plotting the analyte/internal standard peak area ratios obtained against the concentration values. The results obtained revealed good linearity for EI from 10 to 1000 g/L and for 4-MEI from 50 to 2000 g/L, with correlation coefficients always higher than 0.996 in the two distinct matrices. The linear equation for EI was Y = 2.45X + 1.25, with correlation coefficient of 0.998. (Y is the area ratio of EC and d5-EC, and X is the concentration ratio of EC and d5-EC). The linear equation for 4-MEI was Y = 3.26X−0.75, with correlation coefficient of 0.996 (Y is the area ratio of 4-MEI and d5-EC, and X is the concentration ratio between 4-MEI and d5-EC). www.jss-journal.com
Gas Chromatography
J. Sep. Sci. 2014, 37, 2172–2176
Figure 3. (A) The GC–MS total ion chromatogram of soy sauce spiked with EC and 4-MEI (30 g/kg) and (B) the extracted ion chromatogram of the quantifier ion of D5-EC, EC, and 4-MEI.
2175
For the determination of recovery, two kinds of matrices (yellow rice wine and soy sauce) with different blank levels were spiked with three different concentrations and then measured (each test was performed six times). The results of the spiking experiment with yellow rice wine were shown in Table 1. It can be seen that the average recovery of EC in yellow rice wine was between 82.2 and 102.5% with a RSD from 3.6 to 7.3%, and average recoveries for 4-MEI were 83.3 to 92.8%. The precision measured by the relative standard deviation ranged from 6.6 to 9.3%. This optimized method guarantees that both EC and 4-MEI can be properly quantified. The results of spiking experiment with soy sauce matrix are shown in Table 2. The estimated LOQ and LOD were determined at a S/N of 10:1 and 3:1. The LOQ for EC was 6.0 g/kg, and the LOD was 2.0 g/kg; these values for 4-MEI were 15.0 and 5.0 g/kg. Several countries have set limits of EC in different foods. Canada introduced guidelines and tolerance levels in alcoholic beverages in 1985, which set acceptable limits of 30 g/L in table wines, 100g/L in fortified wines, 150 g/L in distilled spirits, 200 g/L in sakes, and 400 g/L in fruit brandies and liqueurs [33]. The USA set maximal levels for EC of 15 g/L in table wines and 100 g/L in fortified wines. Brazil set the limit of 150 g/L in the sugarcane spirit cachaca [34]. Unfortunately, there are no such limits of EC in China. The Codex Alimentarius of the World Health Organization (WHO) and the European Union (EU) have established a maximum of 250 mg/kg for 4-MeI, for caramels class III and IV [35]. The regulatory limit of caramel pigment is 200 mg/kg in China [36]. Indeed, there is a general agreement that EC and 4-MEI levels should be kept as low as possible. The LOQ of EC in our method is lower than that of AOAC Method 994.07 [17]. The LOQs for EC and 4-MEI are 6.0 and 15.0 g/kg, whereas the LOD for EC and 4-MEI are 2.0 and
Table 1. The average recovery of spiked EC and 4-MEI in yellow rice wine (n = 6)
Compounds
Blank (g/kg)
EC 50.2 4-MEI Undetected
Spiking level (g/kg)
Average recovery (%)
RSD (%)
25 100 300 30 60 100
82.2 92.1 102 83.3 89.7 92.8
7.3 5.4 3.6 9.3 8.2 6.6
Spiking level (g/kg)
Average recovery (%)
RSD (%)
20 50 100 30 60 100
80.5 85.0 96.4 78.3 82.9 90.3
8.3 6.4 4.6 10.5 8.5 7.3
LOD (g/kg)
2.0
5.0
Table 2. The average recovery of spiked EC and 4-MEI in soy sauce (n = 6)
Compounds
Blank (g/kg)
EC 10.6 4-MEI Undetected
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
LOD (g/kg)
2.0
5.0
www.jss-journal.com
2176
P. Wu et al.
5.0 g/kg, respectively. Therefore, our method is suitable for simultaneously determining the presence of EC and 4-MEI in fermented food. GC–MS methods based on ion pair extraction with bis2-ethyhexilphosphate and isobutylchloroformate derivatization have been used successfully to determine levels of 4-MEI [28, 29]. However, a method to determine the simultaneous presence of EC and 4-MEI has not been reported. We believe that the simultaneous measurement of these two toxic substances in food products is feasible and will be a practical and beneficial advance in the assessment of safety of certain foods and beverages.
4 Conclusion A novel method for simultaneously determining EC and 4MEI in fermented foods, based on an alkaline diatomite SPE followed by GC–MS analysis, was successfully developed. The proposed GC–MS method without derivatization is faster, and needs less organic solvent. Two kinds of matrices were applied, and the average spiking recoveries were above 80.5% for EC and 78.3% for 4-MEI. The validation results demonstrated that the method was simple, sensitive, and suitable for the simultaneous determination of EC and 4-MEI in fermented food. This work was supported by the National Food Safety Standard (No. spaq-2011-54). The authors have declared no conflict of interest.
5 References [1] Sen, N. P., Seaman, S. W., Boyle, M., Weber D., Food Chem. 1993, 48, 359–366. [2] Lachenmeier, D.W., Schehl, B., Kuballa, T., Frank, W., Senn, T., Food Addit. Contam. 2005, 22, 397– 405. [3] International Agency for Research on Cancer (IARC), IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, 2010, 96, 1378. [4] Patey, A. L., Shearer, G., Knowles, M. E., Denner, W. H. B., Food Addit. Contam. 1985, 2, 107–112. [5] Commission Directive 2008/128/EC-Official Journal of the European Union L 6/2010.1.2009, 40–42. [6] Chan, P. C., Hills, G. D., Kissling, G. E., Nyska, A., Arch. Toxicol. 2008, 82, 45–53. [7] International Agency for Research on Cancer (IARC), IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, 2012, 101, 447–459.
J. Sep. Sci. 2014, 37, 2172–2176
[11] Hasnip, S., Crews, C., Potter, N., Patel, K. J., Agric. Food Chem. 2007, 55, 2755–2759. [12] Chung, W. C., Kwong, K. P., Chen, L. S., Chromatographia. 2010, 72, 571–575. [13] Zhang, Y., Zhang, J., Anal. Chim. Acta 2008, 627, 212– 218. [14] Xu, X. J., Gao, Y. H., Cao, X. J., Wang X., Song, G. X., Zhao, J. F., Hu, Y. M., J. Sep. Sci. 2012, 35, 804–810. [15] Brumley, W. C., Canas, B. J., Perfetti, G. A., Mossoba, M. M., Sphon, J. A., Corneliussen, P. E., Anal. Chem. 1988, 60, 975–978. [16] Ma, Y. P., Deng, F. Q., Chen, D. Z., Sun, S. W., J. Chromatogr. A. 1995, 695, 259–265. [17] AOAC International, AOAC Method 994.07, Official Methods of Analysis of AOAC International, 16th ed., AOAC, Gaithersburg, MD, 1995. [18] Lachenmeier, D. W., Frank, W., Kuballa, T., Rapid Commun. Mass Spectrom. 2005, 19, 108–112. [19] Whiton, R. S., Zoecklein, B. W., Am. J. Enol. Viticult. 2002, 53, 60–63. [20] Lachenmeier, D. W., Nerlich, U., Kuballa, T., J. Chromatogr A. 2006, 1108, 116–120. [21] Rabe, E., Seibel, W., Wuenscher, K., Getreide-Mehl-Brot. 1988, 42(5), 142–148. [22] Gutierrez M. C., Gomez-Hens, A., Valcarcel, M., Microchem. J. 1986, 34, 332–339. [23] Ong, C. P., Ng, C. L., Lee, H. K., Li, S. F. Y., J. Chromatogr. A. 1994, 686, 319–324. [24] Coffey, J. S., Nursten, H. E., Ames, J. M., Castle, L., Food Chem. 1997, 58, 259–267. ´ J., Lojkova, ´ L., Vacek, J., Kuban, ´ [25] Klejdus, B., Moravcova, V., J. Sep. Sci. 2006, 29, 378–384. ´ L., Klejdusm, B., Moravcova, ´ J., Kuban, ´ V., Food [26] Lojkova, Addit. Contam. 2006, 23, 963–973. [27] Schlee, C., Markova, M., Schrank, J., Laplagne, F., Schneider, R., Lachenmeier, D. W., J. Chromatogr. B. 2013, 927, 223–226. [28] Casal, S., Fernandes, J. O., Oliveira, M. B. P. P., Ferreira, M. A. J. Chromatogr. A. 2002, 976, 285–291. [29] Cunha, S. C., Barrado, A. I., Faria, M. A., Fernandes, J. O., J. Food Compost. Anal. 2011, 24, 609–614. [30] Huang, Z., Pan, X. D., Wu, P. G., Chen, Q., Han, J. L., Shen, X. H., Food Chem. 2013, 141, 4161–4165. [31] Xiong, J., Chen, M. L., Lai, Y. D., Mod. Food Sci. Technol. 2012, 28, 567–570. [32] Wu, P. G, Pan, X. D., Wang, L. Y., Shen, X. H., Yang, D. J., Food Control. 2012, 23, 286–288. [33] Conacher, H. B. S., Page, B. D., Proceedings of the European Society of Toxicology, Zurich, Switzerland, 1986, pp. 237–242.
[8] Fauhl, C., Catsburg, R., Wittkowski, R., Food Chem. 1993, 48, 313–316.
´ [34] Lachenmeier, D. W., Lima, M., Nobrega, I., Pereira, J., ˆ F., Kanteres, F., Rehm, J., BMC Cancer 2010, Kerr-Correa, 10, 266.
[9] Hamlet, C. G., Jayaratne, S. M., Morrison, C., Rapid Commun. Mass Spectrom. 2005, 19, 2235–2243.
[35] WHO, Evaluation of Food Additives, World Health Organization, Rome 1971.
[10] Jagerdeo, E., Dugar, S., Foster, G. D., Schenck, H., J. Agric. Food Chem. 2002, 50, 5797–5802.
[36] Caramel pigment in food additives GB 8817-2001, National Standard of China, 2001.
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.jss-journal.com
J. Sep. Sci. 2014, 37, 2172–2176
Pinggu Wu1 Liqun Zhang2 Liyuan Wang1 Jing Zhang1 Ying Tan1 Jun Tang1 Bingjie Ma1 Xiaodong Pan1 Wei Jiang1
Research Article
Simultaneous determination of ethyl carbamate and 4-(5-)methylimidazole in yellow rice wine and soy sauce by gas chromatography with mass spectrometry
1 Center
for Disease Control and Prevention of Zhejiang Province, Hangzhou, P. R. China 2 Center for Disease Control and Prevention of Hangzhou, Hangzhou, P. R. China Received February 8, 2014 Revised April 30, 2014 Accepted May 15, 2014
We developed a new method, based on alkaline diatomite solid-phase extraction followed by gas chromatography with mass spectrometry, for the simultaneous determination of the toxic contaminants ethyl carbamate (EC) and 4-(5-)methylimidazole (4-MEI) in yellow rice wine and soy sauce. The optimal extraction conditions were defined. With the application of alkaline diatomite solid-phase extraction, damage to the capillary column by organic acids was greatly reduced. With deuterated EC used as the internal standard, the linearity of the calibration curves for EC and 4-MEI was good with correlation coefficient above 0.99. In a spiked experiment with EC and 4-MEI in yellow rice wine and soy sauce, recovery of the added EC was 80.5–102.5% and that of 4-MEI was 78.3–92.8%. The limit of quantification and limit of detection for EC were 6.0 and 2.0 g/kg, respectively, and for 4-MEI were 15.0 and 5.0 g/kg, respectively. The validation results demonstrate that the method is fast, simple, and selective, and therefore is suitable for simultaneously determining the presence of EC and 4-MEI in fermented food. Keywords: Ethyl carbamate / Gas chromatography with mass spectrometry / Methylimidazole / Soy sauce / Yellow rice wine DOI 10.1002/jssc.201400141
1 Introduction Ethyl carbamate (EC, urethane, C2 H5 OCONH2 ) is a genotoxic carcinogen that is present in many fermented foods and beverages [1, 2]. The International Agency for Research on Cancer recently upgraded its classification of EC to group 2A (probably carcinogenic to humans) [3]. The neurotoxic agent 4-(5-)methylimidazole (4-MEI) is generated during the process of caramelization, which is mainly present in class III ammonia caramel (INS No. 150c) or class IV ammonia sulfite caramel (INS No. 150d) [4, 5]. A recent toxicological study showed that 4-MEI can induce alveolar/bronchiolar adenoma and carcinoma in male and female mice [6]. The International Agency for Research on Cancer recently upgraded its classification of 4-MEI to group 2B (possibly carcinogenic to humans) [7]. EC is generated during the fermentation process of some foods and beverages, such as soy sauce and yellow rice wine. Subsequently, caramels are added to parts of the fermented foods in order to stimulate or improve appetite. EC and 4-MEI are hazardous contaminants generated during these Correspondence: Dr. Liqun Zhang, Hangzhou Center for Disease Control and Prevention, Hangzhou, P. R. China E-mail: [email protected] Fax: +86-571-8795-1895
Abbreviations: EC, )methylimidazole
ethyl
carbamate;
4-MEI,
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
4-(5-
processes. Both soy sauce and yellow rice wine are fermented food with caramels as coloring agents, so EC and 4-MEI could be present in these foods simultaneously. Therefore, it is important to establish a method for the simultaneous analysis of EC and 4-MEI in fermented foods and beverages. Many analytical techniques based on GC–MS for determining EC in targeted samples have been reported [8–14]. Sample pretreatment procedures are liquid-liquid extraction (LLE) [8,9,15,16], SPE [11,17,18], and solid-phase microextraction (SPME) [13,19,20]. Several methods have been developed to determine 4-MEI based on TLC [21], fluorimetry [22], capillary electrophoresis [23], HPLC [24], LC–MS [25–27], and GC– MS [28, 29]. The use of GC–MS methods based on ion pairextraction with bis-2-ethyhexilphosphate and isobutylchloroformate derivatization has been used successfully to determine levels of 4-MEI [28, 29]. However, a method to determine the simultaneous presence of EC and 4-MEI has not been reported. Therefore, the objectives of this study were (1) to establish a method for determining the simultaneous presence of EC and 4-MEI in fermented foods by GC–MS without derivatization and (2) to evaluate the optimal extraction conditions and the methodological validations used, including linearity, recovery, LOD, LOQ, precision, and accuracy. Colour Online: See the article online to view Figs. 1B, 2A and 3B in colour.
www.jss-journal.com
Gas Chromatography
J. Sep. Sci. 2014, 37, 2172–2176
2 Materials and methods 2.1 Reagents and materials HPLC grade n-hexane, ether, ethyl acetate, and methanol were obtained from Fisher Chemical, Loughborough, UK. Anhydrous sodium sulfate was heated at 450⬚C for 4 h, then cooled to room temperature and maintained dry. Standard EC and 4-MEI (purity>99.0%) were obtained from Sigma Chemical, St. Louis, MO, USA. Deuterated standard of EC d5 -ethyl carbamate (d5-EC) was purchased from Cerilliant, Round Rock, TX, USA. Alkaline diatomite SPE columns (4000 mg filler per 12 mL) were obtained from Fuyu Tec., Hangzhou, China. 2.2 Preparation of standards Stock standard solutions of EC, 4-MEI, and d5-EC were prepared in methanol at a concentration of 1000 mg/L, and then diluted to 1.0, 2.0, and 2.0 mg/L with methanol. Working standard solutions of EC and 4-MEI at 10, 50, 100, 200, 500, and 1000 g/L were prepared in methanol, with an internal standard at a concentration of 100 g/L. 2.3 Preparation of samples A 2.0 g sample of yellow rice wine or soy sauce and 50 L of 2.0 mg/L d5-EC were added into a centrifuge tube and homogenized. After a 1 min vortex, the mixture was poured slowly into an alkaline diatomite SPE column. After 10 min, the analyte was eluted with 5 mL n-hexane followed by elution with 10 mL of ethyl acetate by vacuum. The collected eluate was dried by anhydrous sodium sulfate (first, wetted by ether, then washed with 2.0 mL ether) and concentrated to 0.5 mL via N2 flow at 30⬚C. Finally, the analyte was diluted to 1 mL with methanol for GC–MS testing.
2173
by monitoring the ion at m/z value of 82, 81, 109, and 182, and m/z 82 was selected as the quantitative ion. The internal standard for d5-EC was detected by monitoring the ion at m/z value of 44, 64, and 76, and m/z 64 was selected as the quantitative ion. EC was quantified with calibration curves constructed from the EC/d5-EC peak area ratios (m/z 62 vs. 64) for standards containing EC of 50, 100, 200, 500, and 1000 g/L. The 4-MEI was quantified with calibration curves constructed from the 4-MEI/d5-EC peak area ratios (m/z 82 vs. 64) for standards containing 4-MEI of 50, 100, 200, 500, and 1000 g/L.
2.5 Spiking sample preparation Yellow rice wine and soy sauce were purchased from local markets. Both of these two different matrices with different blank levels were spiked with three different concentrations in order to determine the recoveries. The spiked levels of EC were 25, 100, 300 g/kg in yellow rice wine, whereas the spiked levels of 4-MEI were 30, 60, and 100 g/kg. The spiked levels of EC were 20, 50, and 100 g/kg in soy sauce, whereas the spiked levels of 4-MEI were 30, 60, and 100 g/kg. Each test was performed six times.
2.6 Validation procedure The proposed analytical method was validated in terms of linearity, extraction recovery precision, accuracy, and selectivity. To demonstrate linearity, the regression coefficient was calculated using d5-EC as internal standard for EC and 4-MEI; the spiked calibrants were analyzed in duplicate during the measurement of the samples. The percentage recovery of EC and 4-MEI was determined by a spiking experiment of two different matrices. Reproducibility was examined by analyzing a positive authentic sample containing EC or 4-MEI. A full set of calibration standards and a blank standard were run with each analysis.
2.4 Chromatographic analysis with GC–MS The GC–MS analysis was completed by use of an Agilent 6890 GC-5973MS instrument. The column was of 30 m × 0.25 mm ID × 0.25 m DB-Innowax capillary column (Agilent Technologies). The oven temperature was programmed from 50⬚C, held for 1 min, increased to 180⬚C at 8⬚C/min, increased again to 230⬚C at 15⬚C/min, and then held for 2 min. Finally, the oven temperature reached 240⬚C and was held for 5 min. The carrier gas was helium (purity ࣙ 99.999%), with constant pressure at a flow rate of 1.0 mL/min at 50⬚C. The sample (1 L) was injected into the split/splitless inlet in splitless mode at 200⬚C. The mass spectrometer was operated in electron impact ionization mode with a source temperature of 230⬚C and an electron multiplier of 70 eV. EC was detected by selective ion mode (SIM) of the major ions at m/z values of 44, 62, 74, and 89, in which m/z 62 was selected as the quantitative ion. The 4-MEI was detected C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
3 Results and discussion 3.1 GC–MS conditions A polar column, such as Innowax (30 m × 250 m × 0.20 m) [30], has often been used in the analysis of EC by GC– MS. For the separation of 4-MEI, either a polar column or nonpolar column, such as DB-5 column (100 m × 320 m × 0.20 m) [29], HP-88 column (100 m × 250 m × 0.20 m) [31], DB-FFAP column (30 m × 320 m × 0.20 m), or Innowax column (30 m × 250 m × 0.20 m), can be used. The efficiency of various capillary columns was compared, and the Innowax column (30 m × 250 m × 0.20 m) was chosen for detection of electron impact source (EI-Source) and 4-MEI. The total ion chromatogram of EC and 4-MEI, in given GC–MS conditions, was shown in Fig. 1, which www.jss-journal.com
2174
J. Sep. Sci. 2014, 37, 2172–2176
P. Wu et al.
Figure 1. (A) GC–MS total ion chromatogram of EC (200 g/L), D5-EC (100 g/L), and 4-MEI (200 g/L) (D5-EC: 10.46 min; EC: 10.53 min; 4-MEI: 14.33 min) and (B) the extracted ion chromatogram of the quantifier ion of D5-EC, EC, and 4-MEI.
illustrates that EC and 4-MEI could be well separated.
3.2 Optimization of sample pretreatment For the clean-up of yellow rice samples, the alkaline diatomite SPE column was applied in this experiment, which was used by us previously in determining the concentration of EC in yellow rice wine [32]. With use of this column, most of the acids were removed. Hence, it was chosen for the simultaneous clean-up of EC and 4-MEI; because it had worked well for the clean-up of EC, this sample pretreatment process would be much simpler. Solvents used in the elution of EC from an alkaline diatomite column include 5% ethyl acetate/ether and ethyl acetate. Although we had used a more polar solvent, 5% ethyl acetate/ether (15 mL), for elution of EC, it was not very efficient for the elution of 4-MEI. Thus, taking into account the volume of solvents used, the recovery, and the concentration of the interferences, the best elution solvent was 10 mL ethyl acetate, which yielded a recovery of EC and 4-MEI above 75%. This method was environment friendly, because less organic solvent was used. Two kinds of matrices were examined. The GC–MS total ion chromatogram of real yellow rice wine sample is shown in Fig. 2, in which the concentration of EC was C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 2. (A) The GC–MS total ion chromatogram of real yellow rice wine and (B) the extracted ion chromatogram of the quantifier ion of D5-EC, EC, and 4-MEI in real yellow rice wine samples.
10.4 g/L, whereas that of 4-MEI was under the LOD. The GC–MS total ion chromatogram of soy sauce spiked with EC and 4-MEI (30 g/kg) is shown in Fig. 3.
3.3 Method performance Calibration curves were constructed by plotting the analyte/internal standard peak area ratios obtained against the concentration values. The results obtained revealed good linearity for EI from 10 to 1000 g/L and for 4-MEI from 50 to 2000 g/L, with correlation coefficients always higher than 0.996 in the two distinct matrices. The linear equation for EI was Y = 2.45X + 1.25, with correlation coefficient of 0.998. (Y is the area ratio of EC and d5-EC, and X is the concentration ratio of EC and d5-EC). The linear equation for 4-MEI was Y = 3.26X−0.75, with correlation coefficient of 0.996 (Y is the area ratio of 4-MEI and d5-EC, and X is the concentration ratio between 4-MEI and d5-EC). www.jss-journal.com
Gas Chromatography
J. Sep. Sci. 2014, 37, 2172–2176
Figure 3. (A) The GC–MS total ion chromatogram of soy sauce spiked with EC and 4-MEI (30 g/kg) and (B) the extracted ion chromatogram of the quantifier ion of D5-EC, EC, and 4-MEI.
2175
For the determination of recovery, two kinds of matrices (yellow rice wine and soy sauce) with different blank levels were spiked with three different concentrations and then measured (each test was performed six times). The results of the spiking experiment with yellow rice wine were shown in Table 1. It can be seen that the average recovery of EC in yellow rice wine was between 82.2 and 102.5% with a RSD from 3.6 to 7.3%, and average recoveries for 4-MEI were 83.3 to 92.8%. The precision measured by the relative standard deviation ranged from 6.6 to 9.3%. This optimized method guarantees that both EC and 4-MEI can be properly quantified. The results of spiking experiment with soy sauce matrix are shown in Table 2. The estimated LOQ and LOD were determined at a S/N of 10:1 and 3:1. The LOQ for EC was 6.0 g/kg, and the LOD was 2.0 g/kg; these values for 4-MEI were 15.0 and 5.0 g/kg. Several countries have set limits of EC in different foods. Canada introduced guidelines and tolerance levels in alcoholic beverages in 1985, which set acceptable limits of 30 g/L in table wines, 100g/L in fortified wines, 150 g/L in distilled spirits, 200 g/L in sakes, and 400 g/L in fruit brandies and liqueurs [33]. The USA set maximal levels for EC of 15 g/L in table wines and 100 g/L in fortified wines. Brazil set the limit of 150 g/L in the sugarcane spirit cachaca [34]. Unfortunately, there are no such limits of EC in China. The Codex Alimentarius of the World Health Organization (WHO) and the European Union (EU) have established a maximum of 250 mg/kg for 4-MeI, for caramels class III and IV [35]. The regulatory limit of caramel pigment is 200 mg/kg in China [36]. Indeed, there is a general agreement that EC and 4-MEI levels should be kept as low as possible. The LOQ of EC in our method is lower than that of AOAC Method 994.07 [17]. The LOQs for EC and 4-MEI are 6.0 and 15.0 g/kg, whereas the LOD for EC and 4-MEI are 2.0 and
Table 1. The average recovery of spiked EC and 4-MEI in yellow rice wine (n = 6)
Compounds
Blank (g/kg)
EC 50.2 4-MEI Undetected
Spiking level (g/kg)
Average recovery (%)
RSD (%)
25 100 300 30 60 100
82.2 92.1 102 83.3 89.7 92.8
7.3 5.4 3.6 9.3 8.2 6.6
Spiking level (g/kg)
Average recovery (%)
RSD (%)
20 50 100 30 60 100
80.5 85.0 96.4 78.3 82.9 90.3
8.3 6.4 4.6 10.5 8.5 7.3
LOD (g/kg)
2.0
5.0
Table 2. The average recovery of spiked EC and 4-MEI in soy sauce (n = 6)
Compounds
Blank (g/kg)
EC 10.6 4-MEI Undetected
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
LOD (g/kg)
2.0
5.0
www.jss-journal.com
2176
P. Wu et al.
5.0 g/kg, respectively. Therefore, our method is suitable for simultaneously determining the presence of EC and 4-MEI in fermented food. GC–MS methods based on ion pair extraction with bis2-ethyhexilphosphate and isobutylchloroformate derivatization have been used successfully to determine levels of 4-MEI [28, 29]. However, a method to determine the simultaneous presence of EC and 4-MEI has not been reported. We believe that the simultaneous measurement of these two toxic substances in food products is feasible and will be a practical and beneficial advance in the assessment of safety of certain foods and beverages.
4 Conclusion A novel method for simultaneously determining EC and 4MEI in fermented foods, based on an alkaline diatomite SPE followed by GC–MS analysis, was successfully developed. The proposed GC–MS method without derivatization is faster, and needs less organic solvent. Two kinds of matrices were applied, and the average spiking recoveries were above 80.5% for EC and 78.3% for 4-MEI. The validation results demonstrated that the method was simple, sensitive, and suitable for the simultaneous determination of EC and 4-MEI in fermented food. This work was supported by the National Food Safety Standard (No. spaq-2011-54). The authors have declared no conflict of interest.
5 References [1] Sen, N. P., Seaman, S. W., Boyle, M., Weber D., Food Chem. 1993, 48, 359–366. [2] Lachenmeier, D.W., Schehl, B., Kuballa, T., Frank, W., Senn, T., Food Addit. Contam. 2005, 22, 397– 405. [3] International Agency for Research on Cancer (IARC), IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, 2010, 96, 1378. [4] Patey, A. L., Shearer, G., Knowles, M. E., Denner, W. H. B., Food Addit. Contam. 1985, 2, 107–112. [5] Commission Directive 2008/128/EC-Official Journal of the European Union L 6/2010.1.2009, 40–42. [6] Chan, P. C., Hills, G. D., Kissling, G. E., Nyska, A., Arch. Toxicol. 2008, 82, 45–53. [7] International Agency for Research on Cancer (IARC), IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, 2012, 101, 447–459.
J. Sep. Sci. 2014, 37, 2172–2176
[11] Hasnip, S., Crews, C., Potter, N., Patel, K. J., Agric. Food Chem. 2007, 55, 2755–2759. [12] Chung, W. C., Kwong, K. P., Chen, L. S., Chromatographia. 2010, 72, 571–575. [13] Zhang, Y., Zhang, J., Anal. Chim. Acta 2008, 627, 212– 218. [14] Xu, X. J., Gao, Y. H., Cao, X. J., Wang X., Song, G. X., Zhao, J. F., Hu, Y. M., J. Sep. Sci. 2012, 35, 804–810. [15] Brumley, W. C., Canas, B. J., Perfetti, G. A., Mossoba, M. M., Sphon, J. A., Corneliussen, P. E., Anal. Chem. 1988, 60, 975–978. [16] Ma, Y. P., Deng, F. Q., Chen, D. Z., Sun, S. W., J. Chromatogr. A. 1995, 695, 259–265. [17] AOAC International, AOAC Method 994.07, Official Methods of Analysis of AOAC International, 16th ed., AOAC, Gaithersburg, MD, 1995. [18] Lachenmeier, D. W., Frank, W., Kuballa, T., Rapid Commun. Mass Spectrom. 2005, 19, 108–112. [19] Whiton, R. S., Zoecklein, B. W., Am. J. Enol. Viticult. 2002, 53, 60–63. [20] Lachenmeier, D. W., Nerlich, U., Kuballa, T., J. Chromatogr A. 2006, 1108, 116–120. [21] Rabe, E., Seibel, W., Wuenscher, K., Getreide-Mehl-Brot. 1988, 42(5), 142–148. [22] Gutierrez M. C., Gomez-Hens, A., Valcarcel, M., Microchem. J. 1986, 34, 332–339. [23] Ong, C. P., Ng, C. L., Lee, H. K., Li, S. F. Y., J. Chromatogr. A. 1994, 686, 319–324. [24] Coffey, J. S., Nursten, H. E., Ames, J. M., Castle, L., Food Chem. 1997, 58, 259–267. ´ J., Lojkova, ´ L., Vacek, J., Kuban, ´ [25] Klejdus, B., Moravcova, V., J. Sep. Sci. 2006, 29, 378–384. ´ L., Klejdusm, B., Moravcova, ´ J., Kuban, ´ V., Food [26] Lojkova, Addit. Contam. 2006, 23, 963–973. [27] Schlee, C., Markova, M., Schrank, J., Laplagne, F., Schneider, R., Lachenmeier, D. W., J. Chromatogr. B. 2013, 927, 223–226. [28] Casal, S., Fernandes, J. O., Oliveira, M. B. P. P., Ferreira, M. A. J. Chromatogr. A. 2002, 976, 285–291. [29] Cunha, S. C., Barrado, A. I., Faria, M. A., Fernandes, J. O., J. Food Compost. Anal. 2011, 24, 609–614. [30] Huang, Z., Pan, X. D., Wu, P. G., Chen, Q., Han, J. L., Shen, X. H., Food Chem. 2013, 141, 4161–4165. [31] Xiong, J., Chen, M. L., Lai, Y. D., Mod. Food Sci. Technol. 2012, 28, 567–570. [32] Wu, P. G, Pan, X. D., Wang, L. Y., Shen, X. H., Yang, D. J., Food Control. 2012, 23, 286–288. [33] Conacher, H. B. S., Page, B. D., Proceedings of the European Society of Toxicology, Zurich, Switzerland, 1986, pp. 237–242.
[8] Fauhl, C., Catsburg, R., Wittkowski, R., Food Chem. 1993, 48, 313–316.
´ [34] Lachenmeier, D. W., Lima, M., Nobrega, I., Pereira, J., ˆ F., Kanteres, F., Rehm, J., BMC Cancer 2010, Kerr-Correa, 10, 266.
[9] Hamlet, C. G., Jayaratne, S. M., Morrison, C., Rapid Commun. Mass Spectrom. 2005, 19, 2235–2243.
[35] WHO, Evaluation of Food Additives, World Health Organization, Rome 1971.
[10] Jagerdeo, E., Dugar, S., Foster, G. D., Schenck, H., J. Agric. Food Chem. 2002, 50, 5797–5802.
[36] Caramel pigment in food additives GB 8817-2001, National Standard of China, 2001.
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.jss-journal.com
