Food Chemistry 141 (2013) 4161–4165 Contents lists available at SciVerse ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem 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.