Food Chemistry 141 (2013) 4161–4165
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Food Chemistry
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Analytical Methods Validation (in-house and collaboratory) of the quantification method for ethyl carbamate in alcoholic beverages and soy sauce by GC–MS ⇑ Zhu Huang a, Xiao-Dong Pan b, , Ping-Gu Wu b, Qing Chen b, Jian-Long Han b, Xiang-Hong Shen b a Department of Ophthalmology, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China b Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou 310051, China article info abstract
Article history: A method for ethyl carbamate (EC) determination in alcoholic beverages and soy sauce was developed by Received 28 November 2012 GC–MS. We adopted the diatomaceous earth solid-phase extraction (SPE) column and elution solvent of Received in revised form 20 May 2013 ethyl acetate/diethyl ether (5:95 v/v) for sample cleaning. The in-house validation showed the limit of Accepted 26 June 2013 quantification (LOQ) was 5.0 lg/kg. In the accuracy assay, the total average recovery for was 96.7%. Available online 4 July 2013 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% respec- Keywords: tively. It indicated that the present method performed well in different laboratories. Ethyl carbamate Ó 2013 Elsevier Ltd. All rights reserved. GC-MS Solid-phase extraction Collaboratory study Fermented foods
1. Introduction Page, 1986). The USA set maximal levels for EC of 15 lg/L in table wines and 100 lg/L in fortified wines. Brazil set the limit of 150 lg/ Ethyl carbamate (urethane) is genotoxic and carcinogenic in L in the sugarcane spirit cachaça (Lachenmeier et al., 2010). Unfor- various animal species including mice, rats, hamsters, and mon- tunately, there are no such limits in China. And few studies on EC keys, which suggests a potential carcinogenic risk to human levels in alcoholic beverages and fermented foods were reported (Beland et al., 2005). The World Health Organization’s Interna- (Fu et al., 2010; Wu, Pan, Wang, Shen, & Yang, 2012; Ye et al., tional Agency for Research on Cancer (IARC) classified EC as a 2011; Zhang & Zhang, 2008). Indeed, there is a general agreement group 2A carcinogen (probably carcinogenic to humans) (IARC, that EC levels should be kept as low as possible. 2010). Usually, EC can be found in alcoholic beverages and The technique most widely employed for EC test is GC–MS, and fermented foods. The precursors such as ethanol, urea and hydro- it involves the pretreatment of the sample. The usual method in cyanic acid are able to form EC during the storage or processing of alcoholic beverages adopts a solid-phase extraction (SPE) column certain foods. The mechanism of EC formation in beverages has for extraction, and the analytes are eluted by methylene chloride. been reviewed by Weber and Sharypov (2009). One of the most After evaporation of the extraction, the analysis by GC–MS in common ways in acidic medium was the reaction of urea with eth- SIM mode is performed with monitoring of the m/z 89, 74, 62, anol (Wang, Yang, Zhai, & Zhou, 2007). Another natural way of for- and 44 ions. For example, the AOAC official method 994.07 used mation occurred via cyanide anion. Cyanide anion was produced SPE and GC–MS for EC analysis in alcoholic beverages (AOAC, by decomposing the cyanoglycosides which were found in most 2000). of plants (Lachenmeier, Schehl, Kuballa, Frank, & Senn, 2005). Different sample clean-up procedures are often selected to Several countries have set limits of EC in different foods. Canada avoid interferences from co-extracted matrix components and to introduced guidelines and tolerance levels in alcoholic beverages improve the method sensitivity (Pan et al., 2013). Among them, in 1985, which set acceptable limits of 30 lg/L in table wines, we can mention liquid–liquid extraction (LLE), solid phase extrac- 100 lg/L in fortified wines, 150 lg/L in distilled spirits, 200 lg/L tion (SPE) or solid phase micro-extraction (SPME). SPE columns in sakes, and 400 lg/L in fruit brandies and liqueurs (Conacher & with different types of cartridges, such as diatomaceous earth, alu- mina and Florisil were applied (Hasnip et al., 2007; Kim, Koh, Chung, & Kwon, 2000; Lachenmeier, Kanteres, Kuballa, López, & ⇑ Corresponding author. Address: Zhejiang Provincial Center for Disease Control and Prevention, Bin-Sheng Road No. 3399, Hangzhou City 310051, China. Tel.: +86 Rehm, 2009; Lim & Lee, 2011; Romero, Reguant, Bordons, & Mar- 571 87115274; fax: +86 571 87115261. qué, 2009). However, to our knowledge, the studies on the compar- E-mail address: [email protected] (X.-D. Pan).
0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.06.128 4162 Z. Huang et al. / Food Chemistry 141 (2013) 4161–4165
Table 1 for 1 min, and then to 200 °C at 7.5 °C/min. Splitless injections Wthin-laboratory method performance characteristics for recovery and relative were made with a splitless time of 1.2 min. standard deviation. The mass spectrometer was operated in electron impact ioniza- Spiked Average Average recovery (%) RSD (%) tion mode with a source temperature of 230 °C and an emission (lg/kg) (lg/kg) current of 1506 eV. Ethyl carbamate was detected by SIM of the Rice wine 0.0 24.9 – 2.5 major ions at m/z value of 44, 62 and 89, and m/z 62 was selected 27.2 51.2 97 3.1 as the quantitative iron. The internal standard (d5-EC) was de- 136.0 155.8 96 2.5 tected by monitoring the ion at m/z value of 64 and 76, and m/z 272.0 296.2 100 1.8 Soy sauce 0.0 2.2 – 1.5 64 was selected as the quantitative iron. 25.0 27.6 102 3.9 50 53.6 103 3.6 2.4. In-house study 100.0 104 103 3.6 White spirit 0.0 36.9 – 4.2 34.0 70.4 99 5.8 The optimization of the sample pretreatment was carried out. 136.0 176.1 102 3.0 Briefly, 2.0 g of sample coupled with 100 ul of 1.0 lg/ml d5-EC 408.0 440.3 99 1.8 was added into centrifuge tube. After 1 min vortex, the mixture Wine 0.0 12.0 – 6.8 was poured into solid-phase extraction column. The analyte was 6.0 17.9 98 5.8 24.0 37.75 107 5.5 eluted with 10 ml of solvent after 10 min stationary act. The col- 36.0 47.3 98 4.9 lected elution was dried by anhydrous sodium sulfate and concen-
trated via N2 flow at 30 °C. Finally, the analyte was diluted to 1 ml with methanol for GC/MS test. We compared different types of SPE columns (neutral alumina, diatomite, and Florisil column) and ison of different SPE columns and the validations including inter- three elution solvents, dichloromethane, diethyl ether and ethyl laboratory are few. acetate/diethyl ether (5:95 v/v), for the ability of sample cleaning. The aim of this work was to develop and validate the method of EC analysis in alcoholic beverages and soy sauce by GC–MS. We compared different types of SPE columns (neutral alumina, diato- 2.5. Collaboratory study mite, and Florisil column) and three elution solvents, dichloro- methane, diethyl ether and ethyl acetate/diethyl ether (5:95 v/v) The optimized method in our laboratory was further validated for the ability of sample cleaning. After this, a collaborative study by the inter-laboratory study. The collaborative validation study was designed and carried out according to the harmonized proto- was designed according to the harmonized protocol (blind dupli- col for inter-laboratory method validation. cates, four spiked materials) (Horwitz, 1995). Each laboratory re- ceived a code for identification of the participant together with instruction guidelines, a sample receipt form to check the com- 2. Materials and methods pleteness of the shipment, a method description, a results form, blank material, internal and external standard solutions, and sam- 2.1. Reagents and materials ples (AOAC, 2002). Each sample was required to be tested in six times. The instruments and operating conditions of five laborato- Ethyl acetate, methanol, aether, and anhydrous sodium sulfate ries were showed in Table 2. were obtained from Fisher Chemical Co. (Loughborough, U.K.). Standard ethyl carbamate was obtained from Sigma chemical Co. 2.6. Homogeneity and stability of test materials (St. Louis. Mo.), and d5-ethyl carbamate (d5-EC) was purchased from Cerilliant Co. (Round Rock, TX, USA). Diatomite, neutral alu- The homogeneity and stability of the materials was assessed by mina and florisil SPE columns were obtained from Fuyu Tec. Inc. random sample analyzing before sending the samples, during the (Hangzhou, China). phase of measurement, and after the results had been collected. Each sample was analyzed once. The samples had been stable at 2.2. Spiking and sample preparation room temperature within the time-frame for analysis. The homo- geneity of the samples was checked. The relative standard devia- Rice wine, soy sauce, white spirit and wine were purchased tion of the homogeneity varied between 1.6% and 2.5%. from local markets. The test material used in this study was iden- tical with the material of the validation (including inter-labora- tory) of the GC–MS method (Simon, Palme, & Anklam, 2006). In 2.7. Evaluation of the results submitted brief, EC was added to the four foods at the final concentrations (Tables 1 and 3). Special care was taken that the concentration lev- Five laboratories submitted the corresponding results which els for EC were distributed randomly over the four materials to were statistically evaluated. The values reported were compared achieve individual concentration in each of the four materials. to the values expected. In case of significant deviation from the ex- For each material 20–25 ml were filled in brown glass ampoules. pected value(s), the respective participant was contacted to check Then they were sealed by melting. All samples were coded individ- the results and to find out about potential sources of the error. The ually to avoid back-tracing of numbers and comparison of results. results were statistically evaluated according to the harmonised protocol on collaborative method validation (Horwitz, 1995). 2.3. Instrumental analysis 3. Results and discussion The method of GC–MS was carried out by an Agilent 6890GC- 5973MS. The column was of 30 m  0.25 mm i.d.  1.25 um DB- 3.1. In-house study Innowax capillary. The carrier gas was helium with a flow rate of 1.0 ml/min at 50 °C. The split/splitless injector port and mass spec- 3.1.1. Optimization of sample pretreatment trometer interface line were heated to 200 °C and 250 °C, respec- Some methods on the extraction and clean-up of EC in fer- tively. The oven temperature was programmed from 50 °C, held mented foods were reported in previous studies. Dennis, Howarth, Z. Huang et al. / Food Chemistry 141 (2013) 4161–4165 4163
Table 2 The instruments and operating conditions in five laboratories.
Instrument Shimadzu HP-5890–5971A HP-5890–5971A HP7890–5975CMSD HP-5890–5972 QP-5000 Injection mode Splitless mode, opening Splitless mode, opening Splitless mode, opening Splitless mode, opening Splitless mode, opening splitter 1.2 min splitter 1.2 min splitter 1.2 min splitter 1.2 min splitter 1.2 min after injection after injection after injection after injection after injection Injector temperature 250 °C 260 °C 250 °C 250 °C 260 °C Column model HP-17 DB-5 DB-5 DB-5 DB-5 Column length 30 m 30 m 30 m 30 m 30 m Column diameter 0.25 lm I.D. 0.32 lm I.D. 0.32 lm I.D. 0.32 lm I.D. 0.32 lm I.D. Column film 0.25 lm 0.25 lm 0.25 lm 0.25 lm 0.25 lm Carrier gas He He He He He Column pressure 100 (kPa) 200 (kPa) 200 (kPa) 200 (kPa) 200 (kPa) Ionization mode Electron impact Electron impact Electron impact Electron impact Electron impact Source temperature 180 175 175 175 175 Interface temperature 240 °C 260 260 260 260 Electron energy 70 eV 70 eV 70 eV 70 eV 70 eV
Table 3 Collaboratory study results for determination of ethyl carbamate (EC) in beverages and soy sauce by GC–MS method (five laboratories).
Added, lg/kg Mean, lg/kg Average recovery, % Sr SR RSDr, % RSDR,% Rice wine 0 25 – 1.5 2.4 6.0 9.6 50 76.4 103 2.1 2.5 2.7 3.3 100 127.2 102 2.7 5.3 2.1 4.2 White spirit 0 36.8 – 2.9 3.3 7.8 9.0 50 90.2 107 3.3 3.8 3.7 4.2 100 142.4 106 2.8 3.3 2.0 2.3 Wine 0 12.3 – 0.6 1.1 4.9 9.0 10 23.1 111 0.5 0.8 2.2 3.5 50 65.6 107 0.8 1.8 1.2 2.7 Soy sauce 0 2.5 – 0.12 0.23 4.8 9.2 25 28.2 99 1.4 2.5 5.0 8.9 100 101.5 99 1.5 5.9 1.5 5.8
Key, Pointer, and Massey (1989) reported the usage of Extrelut and EC as internal standard had the potential to improve the method Florisil SPE for the food matrix. Kim et al. (2000) further studied precision in alcoholic beverages and some fermented foods (Dennis the efficiency of different clean-up processes. Jagerdeo, Dugar, Fos- et al., 1989; Canas, Joe, Diachenko, & Burns, 1994; Perestrelo, Pet- ter, and Schenck (2002) reported that SPE clean-up could remove ronilho, Camara, & Rocha, 2010). matrix sufficiently for GC–MS-MS analysis. Hence, in our labora- The good linearity of the method was verified by a 0.9992 cor- tory, three types of columns (neutral alumina, diatomaceous earth, relation coefficient. The limit of quantification (LOQ) was calcu- and Florisil) were compared for EC extraction. As showed in Fig. 1, lated as the concentration of EC in the sample that produced a comparing with the neutral alumina and Florisil column, diatoma- signal equal to 10 times the noise level. The present LOQ value ceous earth obviously reduced the interferences of low mass was 5.0 lg/kg. Comparing with the LOQs obtained by other authors organics. Former study also found that clean-up of fermented foods that ranged between 9.16 lg/kg and 110 lg/kg (Lachenmeier, Ner- with Florisil had the problem with co-elution of interfering low lich, & Kuballa, 2006; Perestrelo et al., 2010), our method was mass compounds (Kim et al., 2000). Furthermore, three eluents, proved to be more sensitive. dichloromethane, diethyl ether and ethyl acetate/diethyl ether (5:95 v/v) were adopted for the recovery of EC. Finally, the diato- 3.2. Collaboratory study maceous earth column and ethyl acetate/diethyl ether (5:95 v/v) were selected due to the high recovery of more than 80%. 3.2.1. Homogeneity and stability of the test material The concentrations of the EC in the samples were shown to be 3.1.2. Analysis stable within the agreed timeframe of 30 days. The homogeneity As shown in Table 1, the range of average recoveries in rice was checked by the RSDs. The RSDs were 1.6–2.5% (n = 10). So, wine, soy sauce, white spirit and wine were 86.6–99.8%, 101.6– the samples were considered sufficiently homogeneous for the 102.7%, 80.9–102.3% and 90.3–107.3%. The total average recovery purpose of the collaborative trial. Furthermore, the participating (Table 1), in the accuracy assays, was 96.7%, which was a satisfying laboratories were requested to store the samples at 4 °C and finish result according to those proposed by AOAC (2000). their measurements after 20 days of receipt of the samples. The precision of the method was evaluated by the relative stan- dard deviation (RSD) in repeated samples (Table 1). Low RSDs 3.2.2. Submitted results (<5%) were observed in rice wine and soy sauce. In white spirit, According to AOAC (2002), the minimum required number of the RSD in samples with spiked level of 34.0 lg/kg was 5.8%. In valid results is eight, but in special cases the study can be con- wine, the RSDs were 6.8% (non-spiked), 5.8% (spiking level of ducted with a minimum of five laboratories. In present study, the 17.4 lg/kg), and 5.5% (spiking level of 37.75 lg/kg). The precision data were submitted by five laboratories. For identifying the outli- of present method was acceptable in different matrices, which ers, Cochran and Grubbs tests were used. Data were considered as might be ascribed to the usage of internal standard. Previous stud- outliers when Grubbs or Cochran showed a statistically significant ies also pointed out that isotope-labelled (deuterium, 13Cor15N) result at a significance level of 1%. No outliers were found in the 4164 Z. Huang et al. / Food Chemistry 141 (2013) 4161–4165
Ion 62.00 (61.70 to 62.70): URETH27.D Abundance Ion 64.00 (63.70 to 64.70): URETH27.D 11000 Ion 60.00 (59.70 to 60.70): URETH27.D 10500 Ion 74.00 (73.70 to 74.70): URETH27.D 10000 (A) 9500 9000 74m/z 8500 60m/z 8000 7500 7000 62m/z 6500 64m/z 6000 5500 5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0 Time--> 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00
Abundance Ion 62.00 (61.70 to 62.70): URETH26.D 12000 Ion 64.00 (63.70 to 64.70): URETH26.D 11500 11000 Ion 60.00 (59.70 to 60.70): URETH26.D 10500 (B) Ion 74.00 (73.70 to 74.70): URETH26.D 10000 9500 9000 8500 8000 7500 7000 6500 6000 5500 64m/z 5000 4500 4000 62m/z 3500 3000 2500 2000 1500 1000 500 0 Time--> 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00
Abundance Ion 62.00 (61.70 to 62.70): URETH25.D Ion 64.00 (63.70 to 64.70): URETH25.D 13000 Ion 60.00 (59.70 to 60.70): URETH25.D 12000 (C) Ion 74.00 (73.70 to 74.70): URETH25.D 11000 10000 9000 8000 7000 6000 64m/z 62m/z 5000 4000 3000 2000 1000 0 Time--> 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00
Fig. 1. Extracted ion chromatograms with treatment of different SPE columns, (A), neutral alumina; (B), diatomaceous earth; (C), Florisil.
data from five laboratories. For assessing the accuracy and preci- dard deviation (Sr), reproducibility standard deviation (SR), sion in inter-laboratories study, submitted data were calculated within-laboratory relative standard deviation (RSDr) and inter-lab- to the mean concentration, average recovery, repeatability stan- oratory relative standard deviation (RSDR). Z. Huang et al. / Food Chemistry 141 (2013) 4161–4165 4165
The mean values of the recoveries found in each laboratory Dennis, M. J., Howarth, N., Key, P. E., Pointer, M., & Massey, R. C. (1989). were calculated for each material (shown in Table 3). The EC recov- Investigation of ethyl carbamate levels in some fermented foods and alcoholic beverages. Food Additives and Contaminants, 6, 383–389. eries in rice wine, white spirit, wine and soy sauce were ranged at Fu, M. L., Liu, J., Che, Q. H., Liu, X. J., He, G. Q., & Chen, J. C. (2010). Determination of 99–111%. The results were similar with the recoveries of our in- ethyl carbamate in Chinese yellow rice wine using high-performance liquid house study (96–107%). It showed that the present method had a chromatography with fluorescence detection. International Journal of Food Science & Technology, 45, 1297–1302. good accuracy in different laboratories. Hasnip, S., Crews, C., Potter, N., Christy, J., Chan, D., Bondu, T., et al. (2007). Survey of The precision of analytical methods is usually characterized for ethyl carbamate in fermented foods sold in the United Kingdom in 2004. Journal repeatability and reproducibility (AOAC, 2002). The repeatability of of Agriculture and Food Chemistry, 55, 2755–2759. Horwitz, W. (1995). Protocol for the design, conduct and interpretation of method- analysis describes the variability of measurements performed on performance studies. Pure and Applied Chemistry, 67, 331–343. one sample within one laboratory under constant conditions. IARC (2010). IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, Reproducibility describes the variability of concentrations between